Encyclopedia of Respiratory Medicine, Four-Volume Set, Volume 1-4

INTRODUCTION

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espiratory diseases represent one of the largest health problems word wide. Diseases such as asthma and the smoking related diseases are already common and increasing so we urgently need better approaches to treat or cure these diseases. At the same time, new respiratory diseases such as those associated with viruses threaten pandemics that challenge our national health systems. With these continued challenges for new treatment with better patient care, clinical and respiratory researchers have sought better approaches to all aspects of patient care from improved diagnoses to superior therapies. This has lead to an explosion of new research with an increasingly better understanding of how to diagnose diseases and then develop new therapies. Thus, for example ever improving technologies for imaging lung disease have lead to increasingly better diagnoses, although challenges remain as we seek to further improve resolution. At the same time, the revolution in molecular biology, culminating with the publication of the complete human genome, has lead to hopes for finding more precise clues to disease susceptibility pathogenesis in genetic analysis. This is leading to new concepts in pharmacogenomics as we start to use new drugs, including those used for lung cancers, being directed at mutations associated with disease. This is the first Encyclopaedia of Respiratory Medicine. It is our hope that it is comprehensive and captures the key aspects of current patient care, as well as the exciting developments in respiratory science that we all believe will eventually lead to better patient care in the twenty first century. This encyclopaedia is comprehensive in scope and provides clinician and researcher with a snap shot of the current state of knowledge in respiratory medicine. All entries have adhered to a structured layout, starting with an abstract crystallizing the key facts and finishing with reading lists for those who want to delve further into the subject. In addition, most entries have a colour diagram designed to help understanding and provide a valuable aid for undergraduate and post-graduate teaching. These are exciting times for respiratory medicine. We hope this encyclopedia will become a valuble tool for clinicians and researches at all stages of their careers from those beginning their carreers to those established but wanting to update themselves on the new developments. Finally, we would like to thank our Advisory Editorial Board who helped so much in shaping the contents of this works, as well as the authors who wrote the articles and faced the challenge of condensing areas of respiratory medicine, often the subject of entire textbooks, into a short article of 4000 words or less.

GEOFFREY J. LAURENT STEVEN D. SHAPIRO

FOREWORD

Animals live by two principal things, food and breath. Of these, by far the most important is the respiration, for if it is stopped, the man will not endure long, but immediately dies. – Aretaeus the Cappocian (150–200 AD)

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f course, not all medical specialists would agree with this statement, and those who disagree would be quick to posit that it is the failure of ‘‘their’’ particular organ that tends to cause immediate death. However, that is not the issue. The point of this quotation is to illustrate that the proper functioning of the lung has been a subject of great interest for centuries. The Greek physician Aretaeus devoted many of his observations to diabetes, but his manuscript ‘‘On the Causes and Indications of Acute and Chronic Diseases’’ also discussed lung diseases, such as pneumonia. Since his time, great numbers of physicians from all continents and cultures have contributed to our knowledge of respiratory diseases. While acknowledging our rich history of discoveries about pulmonary and respiratory medicine— discoveries that were made by men and women whose names symbolize the great journey of this specialty—one must concede that the field experienced an extraordinary growth spurt beginning in the 1940s. Knowledge of respiratory physiology, which developed very fast during World War II, created a tidal wave of interest that continued for years afterward. The ability to measure and understand respiratory physiology and its alterations became a diagnostic tool, and it opened the door to therapeutic or respiratory support procedures. But, then, in the 1950s and 1960s cell biology and subcellular research entered the scene. The potential of molecular biology and genetics was quickly recognized, and respiratory medicine appreciated that a better understanding of normal and disordered biological respiratory processes hinged on use of these new approaches. Lung and respiratory researchers, impelled in part by the ever-increasing public health burdens of respiratory diseases, seized the opportunity. The stage was set for progress to occur. The architects of this ‘‘revolution’’ in respiratory medicine are well known; it is our good fortune that many have contributed to these four volumes. Four volumes! y Encyclopedia! y Indeed, these four volumes truly constitute an encyclopedia of pulmonary biology and respiratory medicine! Respiratory medicine is still growing. Because it is such a dynamic and exciting field, new investigators will almost surely want to be part of it. However, to do so they will need to know about the established state of knowledge that will be the basis of their work. New investigators in the science of respiratory medicine, whether interested in fundamental research or clinical research or application, will find ideas and inspiration in these volumes. All of the tools of the trade are assembled therein. As noted, respiratory medicine has been a progressive and expanding field but, as is the case with many fields of medicine, the transfer of what we know to the general practice of medicine has been slow and limited. Translation, as it is called, is an emerging discipline in need of assistance; fortunately, the breadth of the knowledge presented in these volumes provides tools to facilitate this translation process. This four-volume encyclopedia is, at once, both a tribute to the centuries of pioneering investigations in the field of respiratory medicine and a foundation for even greater accomplishments in the future. The presentation of all this knowledge in these excellent and comprehensive volumes can only serve to stimulate further work of equal or surpassing significance. The editors and the authors are to be commended for their contributions to this singular effort. Because of their work, respiratory science and medicine will advance faster and patients worldwide will be the beneficiaries. Claude Lenfant, MD Gaithersburg, Maryland

Notes on the Subject Index To save space in the index, the following abbreviations have been used: ALI

acute lung injury

ARDS

acute respiratory distress syndrome

BAL

bronchoalveolar lavage

BPD

bronchopulmonary dysplasia

CAP

community-acquired pneumonia

CFTR

cystic fibrosis transporter regulation

COP

cryptogenic organizing pneumonia

COPD

chronic obstructive pulmonary disease

CWP

coal workers’ pneumoconiosis

G-CSF

granulocyte colony-stimulating factor

GERD

gastroesophageal reflux disease

GM-CSF

granulocyte-macrophage colony-stimulating factor

HUVS

hypocomplementemic urticarial vasculitis syndrome

IL

interleukin

IPF

idiopathic pulmonary fibrosis

IPH

idiopathic pulmonary hemosiderosis

MCP

monocyte chemoattractant protein

M-CSF

macrophage colony-stimulating factor

MIP

macrophage inflammatory protein

MMP

matrix metalloproteinase

NSCLC

non-small cell lung carcinoma

PPAR

peroxisome proliferator-activated receptor

SCLC

small-cell lung carcinoma

SP

surfactant protein

TGF

transforming growth factor

TIMP

tissue inhibitor of metalloproteinases

TNF

tumor necrosis factor

VEGF

vascular endothelial growth factor

Editorial Advisory Board Kenneth B. Adler, North Carolina State University, Raleigh, NC, USA Peter J. Barnes, Imperial College London, UK Paul Borm, Zuyd University, Heerlen, The Netherlands Arnold R. Brody, Tulane Medical School, New Orleans, LA, USA Rachel C. Chambers, University College London, UK Augustine M. K. Choi, University of Pittsburgh, PA, USA Jack A. Elias, Yale University School of Medicine, New Haven, CT, USA Patricia W. Finn, University of California San Diego, La Jolla, CA, USA Stephen T. Holgate, University of Southampton, Southampton, UK Steven Idell, The University of Texas Health Center at Tyler, TX, USA Sebastian L. Johnston, National Heart and Lung Institute, Imperial college London, UK Talmadge E. King, Jr, University of California, San Francisco, CA, USA Stella Kourembanas, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA Y. C. Gary Lee, University College London, UK Richard Marshall, University College London, UK Sadis Matalon, University of Alabama, Birmingham, AL, USA Joel Moss, National Institutes of Health, Bethesda, MD, USA William C. Parks, University of Washington, Seattle, WA, USA Charles G. Plopper, University of California, Davis, CA, USA Bruce W. S. Robinson, The University of Western Australia, Nedlands, Australia Neil Schluger, Columbia University College of Physicians and Surgeons, New York, NY, USA Edwin K. Silverman, Brigham and Women’s Hospital Boston, MA, USA Eric S. Silverman, Brigham and Women’s Hospital, Boston, MA, USA Peter Sly, Institute for Child Health Research, West Perth, Australia Kingman Strohl, Case Western Reserve University, Cleveland, OH, USA Teresa D. Tetley, Imperial College London, UK John B. West, University of California, San Diego, CA, USA

Editors Geoffrey J Laurent, Royal Free and University College Medical School, London, UK Steven D Shapiro, Brigham and Woman’s Hospital, Boston, USA

Dedication To my family, Lal, Guy, David and Gabrielle (GJL). My contribution to this work would not have been possible without the love and support from my wife Nicole and my daughters Calli, Tess, Skylar, and Ellery. I also thank my mentors and trainees for my continual education and the Division of Pulmonary and Critical Care Medicine at Brigham and Women’s Hospital who took care of our patients allowing me the time to undertake this project (SDS)

Permission Acknowledgments The following material is reproduced with kind permission of Lippincott Williams and Wilkins Figure 4 and 8 of ARTERIAL BLOOD GASES Table 1 of ARTERIES AND VEINS Figure 2 and 3 of BREATHING | Breathing in the Newborn Figure 2a, 2b, 3, 4 and 5 of DRUG-INDUCED PULMONARY DISEASE Table 1, 2 and 3 of DRUG-INDUCED PULMONARY DISEASE Figure 2 of ENVIRONMENTAL POLLUTANTS | Diesel exhaust particles Figure 4 of EXERCISE PHYSIOLOGY Figure 2 of FLUID BALANCE IN THE LUNG Figure 2 of GASTROESOPHAGEAL REFLUX Figure 2 of GENE REGULATION Figure 2 of HIGH ALTITUDE, PHYSIOLOGY AND DISEASES Figure 2 and 3 of IDIOPATHIC PULMONARY HEMOSIDEROSIS Table 1 of IDIOPATHIC PULMONARY HEMOSIDEROSIS Figure 1, 2 and 3 of OXYGEN-HEMOGLOBIN DISSOCIATION CURVE Figure 10a, 10b and 11a of SYSTEMIC DISEASE | Eosinophilic Lung Diseases http://www.lww.com

The following material is reproduced with kind permission of Nature Publishing Group Figure 2 of COAGULATION CASCADE | iuPA, tPA, uPAR Figure 1 of COAGULATION CASCADE | Tissue Factor Figure 1a of MATRIX METALLOPROTEINASES Figure 1 of MYOFIBROBLASTS

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Figure 1 of VESICULAR TRAFFICKING http://www.nature.com/nature and http://www.nature.com/reviews

The following material is reproduced with kind permission of Taylor & Francis Ltd Figure 2 of AUTOANTIBODIES Table 1 of BASAL CELLS Figure 1 of NEUROPHYSIOLOGY | Neuroendocrine Cells Table 1 of NEUROPHYSIOLOGY | Neuroendocrine Cells Figure 1 and 2 of SURFACANT | Overview Tables 1, 2, 3 and 4 of SURFACANT | Overview http://www.tandf.co.uk/journals

A ACETYLCHOLINE J Zaagsma and H Meurs, University of Groningen, Groningen, The Netherlands & 2006 Elsevier Ltd. All rights reserved.

Abstract In the airways, acetylcholine is a neurotransmitter in parasympathetic ganglia and in postganglionic parasympathetic nerves, as well as a nonneural paracrine mediator in various cells in the airway wall. Ganglionic transmission by acetylcholine is mediated by nicotinic receptors, which are ligand-gated in channels, whereas postganglionic transmission is through G-protein-coupled muscarinic receptors. Of the five mammalian muscarinic receptor subtypes, mainly M1, M2, and M3 receptors are involved in airway functions. Gq-coupled M1 receptors facilitate ganglionic transmission mediated by nicotinic receptors and modulate surfactant production and fluid resorption in the alveoli. Prejunctional Gi/o-coupled M2 receptors in parasympathetic nerve terminals attenuate acetylcholine release upon nerve stimulation. M2 receptors are also abundantly present in airway smooth muscle; however, the major function of these postjunctional M2 receptors is unknown. Postjunctional Gq-coupled M3 receptors mediate airway smooth muscle contraction and mucus secretion. Dysfunction of the prejunctional M2 autoreceptor induced by allergic airway inflammation has been implied in exaggerated vagal reflex activity and airway hyperresponsiveness in asthma. Inflammation-induced increased M3 receptor stimulation may be involved in airway remodeling in chronic asthma. Possible mechanisms include potentiation of growth factor-induced proliferation of airway smooth muscle cells and induction of a contractile phenotype of these cells. Exaggerated M3 receptor stimulation may also cause reduced responsiveness to b2-adrenoceptor agonists by transductional cross-talk between phosphoinositide metabolism and adenylyl cyclase, which involves protein kinase C-induced uncoupling of the b2adrenoceptor from the effector system. Muscarinic receptor antagonists have been shown to be effective in airway diseases like asthma and, especially, chronic obstructive pulmonary disease. Of these, tiotropium bromide is particularly useful, due to its long duration of action as well as its kinetic selectivity for the M3 receptor.

Introduction Acetylcholine is a neurotransmitter in the central and peripheral nervous system where it plays a major role in the afferent neurons of both the autonomic and somatic (voluntary) branches. As a chemical transmitter, it has been identified as ‘Vagusstoff’ in 1921 by Otto Loewi showing its release from an isolated frog heart following stimulation of the

vagosympathetic trunk; when applied to a second, unstimulated heart, the perfusate slowed its rate, resembling the effect of vagus stimulation. In 1926 Loewi provided evidence for identification of Vagusstoff as acetylcholine. Acetylcholine is the neurotransmitter of all sympathetic and parasympathetic autonomic ganglia and of the postganglionic parasympathetic nerves. In the airways, the parasympathetic ganglia are located near or within the airway wall. Ganglionic transmission mediated by acetylcholine is through nicotinic receptors which belong to the family of ligand-gated ion channels. Postganglionic transmission by acetylcholine, released from parasympathetic nerve terminals, is through muscarinic receptors of which five different subtypes have been identified, all being G-protein-coupled receptors. During periods of airway inflammation vagal release of acetylcholine may be increased by various mechanisms. Hence, both in asthma and (particularly) in chronic obstructive pulmonary disease (COPD) blockade of postjunctional muscarinic receptors is the key to reversing airway obstructions.

Synthesis, Storage, and Release Acetylcholine is synthesized from choline and acetylcoenzyme A (acetyl-CoA) in the cytoplasm of the nerve terminal through the enzyme choline acetyltransferase (ChAT). Choline is taken up by the nerve terminal from the extracellular fluid through a sodium-dependent carrier; this transport is the ratelimiting process in acetylcholine synthesis. Acetyl-CoA is synthesized in mitochondria which are abundantly present in the nerve endings. Most of the synthesized acetylcholine is actively transported from the cytosol into synaptic vesicles by a specific transporter; this vesicular (‘quantal’) package of acetylcholine reaches up to 50 000 molecules per vesicle. Release of acetylcholine is initiated by influx of Ca2 þ ions through voltage-operated N- or P-type calcium channels. The increased intracellular Ca2 þ ions bind to a vesicle-associated protein (synaptotagmin) which favors association of a second vesicle protein (synaptobrevin) with one or more proteins in the plasma membrane of the nerve terminal. Following

2 ACETYLCHOLINE

this vesicle-docking process, fusion between vesicle membrane and plasma membrane occurs, followed by exocytosis. After the expulsion of acetylcholine the empty vesicle is recaptured by endocytosis and can be reused. In the synaptic cleft, the released acetylcholine will associate with post- and prejunctional receptors and is also subject to rapid hydrolysis by the enzyme acetylcholinesterase into choline and acetate. Over 50% of the choline formed will be taken up again by the nerve terminal and reused for neurotransmitter synthesis. Acetylcholine is also present in nonneuronal cells. In recent years it has become clear that in the airways the majority of cells express ChAT and contain acetylcholine, including epithelial cells, smooth muscle cells, mast cells, and migrated immune cells such as alveolar macrophages, granulocytes, and lymphocytes. However, the regulatory role of this nonneuronal acetylcholine in inflammatory airways diseases has yet to be established.

Regulation of Synaptic Transmission and Activity Ganglionic

Preganglionic nerves innervating the parasympathetic ganglia in the airways evoke action potentials during normal breathing with relatively high frequencies, in the range of 1–20 Hz. As a result, basal airway smooth muscle tone in vivo is mediated to a significant extent by cholinergic nerve activity. The pattern of ganglionic action potential bursts coincides with respiration, suggesting that the respiratory centers in the brainstem govern preganglionic nerve activity. However, in addition to this central drive, reflex stimulation through mechanically sensitive afferent nerve terminals in the lungs during respiration is importantly involved as well. The fidelity by which preganglionic impulses are translated into action potentials in the postganglionic neurons is relatively low in parasympathetic airway ganglia, implying a filtering function of these ganglia. This filtering function can be diminished by various inflammatory mediators. Thus, histamine, prostaglandin D2 (PGD2), and bradykinin are able to enhance ganglionic cholinergic transmission and the same is true for tachykinins (substance P, neurokinin A) released by nonmyelinated sensory C-fibers in the airways. Postganglionic

The release of acetylcholine from parasympathetic nerve terminals is regulated by a variety of prejunctional receptors, which may inhibit or facilitate transmitter outflow. In the airways, autoinhibitory

muscarinic M2 receptors, activated by acetylcholine itself, represent an important negative feedback, limiting further release, at higher firing rates in particular. In animal models of allergic airway inflammation and asthma as well as in human asthma, dysfunction of these M2 autoreceptors has been found to contribute to exaggerated acetylcholine release from vagal nerve endings, to increased cholinergic reflex activity in response to inhaled stimuli, and to contribute to airway hyperresponsiveness. Most of this receptor dysfunction is thought to be caused by activated eosinophils that migrate to cholinergic nerves and release major basic protein (MBP) which acts as an allosteric antagonist of muscarinic M2 receptors. Since eosinophilic inflammation is far less prominent in COPD and since M2 autoreceptors are more prominent in larger airways, it is no surprise that these receptors are still functional in patients with stable COPD; however, this does not exclude a dysfunction during acute exacerbations. In addition to M2 autoreceptors, a variety of heteroreceptors modulating acetylcholine release have been identified on cholinergic nerve endings. Catecholamines may inhibit or facilitate acetylcholine overflow through prejunctional a2- and b2-adrenoceptors, respectively. Neurokinins like substance P may enhance cholinergic transmission through facilitatory neurokinin 1 (NK1) and/or 2 (NK2) receptors. Interestingly, substance P may also induce MBP release from eosinophils, causing M2 receptor dysfunction, which could act synergistically to direct facilitation. Allergic inflammation-derived prostanoids, including PGD2, PGF2a, and thromboxane A2, as well as histamine, can also augment acetylcholine release through prejunctional receptors. Taken together, the above observations indicate that parasympathetic acetylcholine release is governed by various regulatory systems, the set-point of which is subject to environmental modulations. During periods of airway inflammation these modulations often result in enhanced cholinergic transmission.

Receptors and Biological Function In the ganglia, acetylcholine interacts with nicotinic receptors. These receptors consist of five polypeptide subunits, together forming a cylindric structure of about 8 nm diameter, which acts as an ion channel. Each subunit passes the membrane four times, so the central pore is surrounded by 20 membranespanning helices. The subunits have been subdivided into five classes, designated a, b, g, d, and e. Of the a and b subunits, 10 and 4 different subtypes have been identified, respectively. Peripheral ganglionic

ACETYLCHOLINE 3

receptors consist of only a and b subunits, the main subtype being (a3)2(b4)3. Each a subunit possesses a binding site for acetylcholine; they need to be occupied both to induce channel opening, which will enhance Na þ and K þ permeability. This results in an inward flux of mainly Na þ ions, causing depolarization and action potential generation in the postganglionic cell (provided the acetylcholine concentration is high enough). Acetylcholine released by postganglionic parasympathetic nerves may choose between five muscarinic receptor subtypes, designated M1 to M5, to interact with. Most organs and tissues express more than one subtype and this is true for many individual cells as well. The five subtypes can be subdivided into two main classes, the odd-numbered receptors (M1, M3, M5), which couple preferentially to heterotrimeric Gqproteins, and the even-numbered (M2, M4) receptors which show selectivity for Gi/o type of G-proteins. The principal signaling route of Gq-coupled receptors is the activation of phospholipase C, mediating hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) (Figure 1, left). IP3 mobilizes Ca2 þ ions from intracellular stores, which generates a rapid and transient rise of the free Ca2 þ concentration in the cytosol. DAG triggers the translocation and activation of protein kinase C (PKC) which is able to phosphorylate a variety of different protein substrates. The main signal transduction of Gi/o-coupled receptors is to inhibit adenylyl cyclase activity, which reduces the intracellular cyclic AMP concentration.

In airway smooth muscle, activation of Gi/o by M2 receptors may also diminish opening of calcium-activated potassium channels (KCa or maxi-K channels) induced by (Gs-coupled) b-adrenoceptors. Thus, K þ efflux, membrane hyperpolarization, and subsequent smooth muscle relaxation, initiated by b-agonist administration, is under restraint of acetylcholine through this mechanism. In the airways of most mammalian species, including human, M1, M2, and M3 receptors are the most important ones. So far, M4 receptors have only been detected convincingly in bronchiolar smooth muscle and alveolar walls of the rabbit, whereas muscarinic M5 receptors, now known to mediate dilatation of cerebral arteries and arterioles, are absent in the lungs. M1 receptors have been found in alveolar walls, parasympathetic ganglia, and submucosal glands. Rat and guinea pig lung studies have indicated their presence in type II alveolar cells, mediating surfactant production and fluid reabsorption, respectively. In parasympathetic airway ganglia of several species, including human, M1 receptor stimulation is able to facilitate ganglionic transmission mediated by nicotinic receptors. Thus, vagal bronchoconstriction, induced by inhalation of SO2, has been found especially sensitive to inhaled pirenzepine, a M1-selective antagonist. In submucosal glands, M1 receptors are not involved in mucus secretion, which appears to be mediated solely by M3 receptors. M2 and M3 receptors represent the major receptor populations, both in intra- and extrapulmonary Epinephrine,
2-agonists

Acetylcholine

PIP2

M3 Gq

PLC

DAG


2 PKC −

IP3

Ca2+

Contraction

AC Gs

+

P

P

−

P

− ATP

P


ARK

cAMP

Relaxation

Figure 1 Cross-talk between M3 muscarinic receptors and b2-adrenoceptors in airway smooth muscle. Generation of 1,2-diacylglycerol (DAG) by M3 receptor-induced phosphoinositide (PIP2) metabolism causes activation of protein kinase C (PKC). PKC may phosphorylate the b2-adrenoceptor as well as Gs, causing uncoupling of the receptor from the effector system. Moreover, PKC may phosphorylate b-adrenoceptor kinase(s) (bARK), which amplifies b-agonist-induced desensitization mediated by bARK-induced phosphorylation of the receptor. AC, adenylyl cyclase; cAMP, cyclic adenosine 30 , 50 -monophosphate; IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C.

4 ACETYLCHOLINE

airways. As already discussed, inhibitory M2 autoreceptors located at parasympathetic nerve terminals have an important regulatory role in limiting acetylcholine release. Postjunctional muscarinic receptor populations in airway smooth muscle are a mixture of M2 and M3 receptors, the M2 subtype being predominant, particularly in the large airways. Contraction, however, is primarily mediated by M3 receptors (even in those smooth muscle preparations where the ratio of M2 : M3 receptors is 90 : 10), the M2 receptor population having at most a minor supporting role. This is confirmed in airway preparations from M2 receptor knockout mice, in which carbachol, a muscarinic agonist, was hardly less potent than in preparations from wildtype mice. Cross-talk between Gi-coupled M2 receptors and Gs-coupled b-adrenoceptors (having opposing effects on cyclic AMP accumulation or maxi-K channel opening) has no major effects in modulating muscarinic agonist induced contraction or b-agonist induced relaxation, at least under physiological circumstances. However, in inflammatory conditions such as asthma, in which Gi-proteins may be upregulated, the situation may change. In contrast to M2 receptors, Gq-coupled M3 receptors, generating IP3 and DAG by stimulating phosphoinositide metabolism, may have a major influence on b2-adrenoceptor function, even in noninflamed airways. This is due to DAG-induced activation of PKC which may (1) phosphorylate the b2-adrenoceptor as well as Gs, causing receptor uncoupling and desensitization, and (2) phosphorylate and activate b-adrenoceptor kinase(s) (bARKs, which are members of the G-protein receptor kinase (GRK) family), amplifying homologous, b-agonist induced desensitization (Figure 1). These processes may explain the well-known attenuation of b-agonist efficacy during episodes of severe bronchoconstriction, for example, during exacerbations. Airway Remodeling

In addition to phosphoinositide metabolism, inhibition of adenylyl cyclase and maxi K-channel activation, stimulation of M3 and/or M2 receptors in airway smooth muscle cells has been shown to activate different promitogenic signaling pathways, including the p42/p44 mitogen-activated protein kinase, Rho/Rho kinase, and PI3 kinase pathways. In vitro studies have revealed that muscarinic agonists do not induce airway smooth muscle cell proliferation by themselves, but enhance the proliferative response induced by peptide growth factors. This effect, which is solely mediated by the M3 receptor,

indicates that acetylcholine may contribute to airway remodeling as observed in asthma and COPD. Indeed, in an animal model of chronic asthma it was recently demonstrated that the long-acting muscarinic antagonist tiotropium bromide inhibited increased airway smooth muscle mass, enhanced airway smooth muscle contractility, and increased expression of contractile proteins in the lung upon repeated allergen challenge. This implies that, in addition to their bronchodilating properties, muscarinic receptor antagonists could be beneficial in the treatment of asthma by preventing chronic airway hyperresponsiveness and decline of lung function. Although not fully established, a more extensive role of acetylcholine in airway remodeling, including airway smooth muscle proliferation, contractile protein expression, promitogenic signaling, and regulation of secretory functions as well as cell migration, has recently been proposed (Figure 2).

Acetylcholine in Respiratory Diseases As indicated above, the parasympathetic nervous system represents a major constrictory pathway of the airways; even basal bronchomotor tone is partly governed by acetylcholine. Both its release and its postjunctional effects, including smooth muscle contraction and mucus secretion, are regulated and mediated, respectively, by muscarinic receptors. So far, no evidence for upregulation of postjunctional M3 and M2 receptors has been found in hyperresponsive airways of patients with asthma and COPD. In contrast, dysfunctional autoreceptors, leading to exaggerated vagal reflexes in the airways, are well established in allergic asthma. M1 receptors, facilitating nicotinic neurotransmission in parasympathetic airway ganglia, do not appear to contribute significantly to bronchomotor tone in humans, either with or without obstructive airway diseases. Hence, the principle therapeutic muscarinic receptor target in asthma and COPD is the M3 receptor. In COPD, muscarinic receptor antagonists like ipratropium, a quarternary nonselective antagonist, are very effective in causing bronchodilatation. A recently introduced antagonist is tiotropium, which acutely occupies both M2 and M3 receptors; however, while it dissociates rapidly from M2 receptors during washout, blockade of M3 receptors persists for hours. This kinetically based M3 receptor subtype selectivity could be of therapeutic advantage since blockade of prejunctional M2 receptors enhances acetylcholine outflow. Both in COPD and, particularly, in allergic asthma, in which M2 autoreceptors are already dysfunctional, this is an unwanted side effect.

ACID–BASE BALANCE 5

ACh Extracellular matrix proteins

Migration

Hypercontractility

Chemotaxis activation

Contraction

Hyperplasia

Figure 2 Proposed mechanisms by which acetylcholine (ACh) could affect airway smooth muscle remodeling. Acetylcholine has been shown to affect airway smooth muscle contractility, contractile protein expression, promitogenic signaling, and proliferation. In addition, like several other G-protein-coupled receptor agonists, acetylcholine could also be involved in airway smooth muscle cell migration, extracellular matrix protein production, and secretion of cytokines and chemokines. Altogether, these effects could contribute to airway remodeling in asthma and COPD. Reproduced from Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans SA, and Meurs H (2004) Acetylcholine: a novel regulator of airway smooth muscle remodelling. European Journal of Pharmacology 500: 193–201, with permission from Elsevier.

See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Neurophysiology: Neural Control of Airway Smooth Muscle; Neuroanatomy; Neurons and Neuromuscular Transmission.

Further Reading Berge RE ten, Santing RE, Hamstra JJ, Roffel AF, and Zaagsma J (1995) Dysfunction of muscarinic M2 receptors after the early allergic reaction: possible contribution to bronchial hyperresponsiveness in allergic guinea-pigs. British Journal of Pharmacology 114: 881–887. Billington CK and Penn RB (2002) M3 muscarinic acetylcholine receptor regulation in the airway. American Journal of Respiratory Cell and Molecular Biology 26: 269–272. Coulson FR and Fryer AD (2003) Muscarinic acetylcholine receptors and airway diseases. Pharmacology and Therapeutics 98: 59–69. Gosens R, Bos ST, Zaagsma J, and Meurs H (2005) Protective effect of tiotropium bromide in the progression of airway

smooth muscle remodeling. American Journal of Respiratory and Critical Care Medicine 171: 1096–1102. Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans SA, and Meurs H (2004) Acetylcholine: a novel regulator of airway smooth muscle remodelling. European Journal of Pharmacology 500: 193–201. Racke´ K and Matthiesen S (2004) The airway cholinergic system: physiology and pharmacology. Pulmonary Pharmacology and Therapeutics 17: 181–198. Wess J (2004) Muscarinic acetylcholine receptor knockout mice: novel phenotypes and clinical implications. Annual Review of Pharmacology and Toxicology 44: 423–450. Wessler I, Kilbinger H, Bittinger F, Unger R, and Kirkpatrick CJ (2003) The nonneural cholinergic system in humans: expression, function and pathophysiology. Life Sciences 72: 2055–2061. Zaagsma J, Meurs H, and Roffel AF (eds.) (2001) Muscarinic Receptors in Airways Diseases. Basel: Birkhauser Verlag. Zaagsma J, Roffel AF, and Meurs H (1997) Muscarinic control of airway function. Life Sciences 60: 1061–1068.

ACID–BASE BALANCE O Siggaard-Andersen, University of Copenhagen, Copenhagen, Denmark & 2006 Elsevier Ltd. All rights reserved.

Abstract The acid–base balance or neutrality regulation maintains a pH around 7.4 in the extracellular fluid by excreting carbon dioxide (carbonic acid anhydride) in the lungs and noncarbonic acid or base in the kidneys. The result is a normal acid–base status in blood and extracellular fluid, i.e., a normal pH, a normal carbon dioxide tension (pCO2 ), and a normal concentration of

titratable hydrogen ion (ctH þ ). A pH, log pCO2 chart illustrates the acid–base status of the arterial blood. The chart shows normal values as well as values to be expected in typical acid– base disturbances, i.e., acute and chronic respiratory acidosis and alkalosis, and acute and chronic nonrespiratory (metabolic) acidosis and alkalosis. The chart allows estimation of the concentration of titratable H þ of the extended extracellular fluid (including erythrocytes), ctH þ Ecf. This quantity is also called standard base deficit but the term base does not directly indicate that the quantity refers to the excess or deficit of hydrogen ions. ctH þ Ecf is the preferred indicator of a nonrespiratory acid–base disturbance being independent of acute changes in pCO2 in vivo. While pH and pCO2 are directly measured, ctH þ Ecf

6 ACID–BASE BALANCE is calculated from pH and pCO2 using the Henderson–Hasselbalch equation and the Van Slyke equation.

Description The acid–base balance or neutrality regulation maintains a pH around 7.4 in the extracellular fluid by excreting carbon dioxide (carbonic acid anhydride) in the lungs and noncarbonic acid or base in the kidneys. The result is a normal acid–base status in blood and extracellular fluid, i.e., a normal pH, a normal carbon dioxide tension (pCO2 ), and a normal concentration of titratable hydrogen ion (ctH þ ). Figure 1 illustrates the acid–base status of the blood, especially the relationships among the three key variables. pH and the Hydrogen Ion Concentration (cH þ )

pH and cH þ of the plasma are both indicated on the abscissa of Figure 1. pH is the negative dacadic logarithm of molal hydrogen ion activity. Concentration of free hydrogen ion (cH þ ) is calculated as 109
pH nmol l
1. pH and pOH are closely related: pH þ pOH ¼ pKw ¼ 13.622 at 371C, where Kw is the ionization constant of water. If H þ is considered a key component of an aqueous solution, then OH
is a derived component. Accounting for H þ and H2O indirectly accounts for OH
as well. It is the author’s conviction that the relevant component is the hydrogen ion, not hydrogen ion binding groups (base) nor hydroxyl ions. The Carbon Dioxide Tension of the Blood (pCO2 )

pCO2 , i.e., the partial pressure of carbon dioxide in a gas phase in equilibrium with the blood, is shown on the ordinate on a logarithmic scale in Figure 1. When pCO2 increases, the concentration of dissolved carbon dioxide and carbonic acid increases, and hence the hydrogen ion concentration increases: CO2 þ H2 O-H2 CO3 -Hþ þHCO
3 The Concentration of Titratable Hydrogen Ion (ctH þ )

ctH þ is indicated on the scale in the upper left corner of Figure 1. ctH þ is a measure of added noncarbonic acid or base. The amount of hydrogen ion added or removed in relation to a reference pH of 7.40 may be determined by titration to pH ¼ 7.40 at pCO2 ¼ 5:33 kPa ( ¼ 40 mmHg) and T ¼ 371C using strong acid or base, depending upon the initial pH. Titratable hydrogen ion is also called base deficit, or with the opposite sign base excess. Unfortunately, the term ‘base’ is ambiguous (it has previously been associated

with cations) and does not directly indicate that the relevant chemical component is the hydrogen ion. The term hydrogen ion excess or acronym HX may also be used. Note: by definition, ctH þ of blood refers to the actual hemoglobin oxygen saturation, not the fully oxygenated blood. Acid and base are defined by the equilibrium: Acidz # Hþ þ Basez
1 where Acidz and Basez
1 is a conjugate acid–base pair. The charge number z may be positive, zero, or negative. A strong acid, e.g., HCl, dissociates completely: HCl-H þ þ Cl
. The anion that follows the hydrogen ion is called an aprote, nonbuffering, or strong anion. At physiological pH, even lactic acid is a strong acid and lactate an aprote anion. A base is a molecule containing a hydrogen ion-binding group. A strong base, e.g. OH
, associates completely with hydrogen ion: OH
þ H þ -H2O. The cation that follows the hydroxyl ion is called an aprote, nonbuffering, or strong cation, e.g., Na þ or K þ . A weak acid (buffer acid) is in equilibrium with its conjugate weak base (buffer base), e.g.: H2 CO3 # Hþ þ HCO
3 hemoglobinz # Hþ þ hemoglobinz
1 The concentration of titratable hydrogen ion may be determined for plasma (P), whole blood (B), or a model of the extended extracellular fluid, i.e., blood plus interstitial fluid (Ecf). The model consists of blood diluted threefold with its own plasma to get a hemoglobin concentration similar to the one obtained if the red cells were evenly distributed in the whole extracellular volume. An acute increase in pCO2 in vivo causes a rise in ctH þ B and a fall in ctH þ P while ctH þ Ecf remains constant. For example, an acute rise in pCO2 from 5.33 to 10.66 kPa (40–80 mmHg) causes a rise in whole blood ctH þ of about 5 mmol l
1, a fall in plasma ctH þ of about 3 mmol l
1, while the extracellular ctH þ remains independent of acute changes in pCO2 . The cause is a redistribution of hydrogen ions within the extended extracellular volume. The hydrogen ion concentration increases more in the poorly buffered interstitial fluid than in the blood plasma, where it increases more than inside the erythrocytes, where hemoglobin binds the hydrogen ions. Hydrogen ions diffuse from the poorly buffered interstitial fluid into the blood plasma and further into the erythrocytes. Very little transfer of hydrogen ions occurs between the intracellular space and the extracellular space, so ctH þ Ecf remains virtually constant during acute

ACID–BASE BALANCE 7

No

rm

–5

0 +5 ex ce ss

pCO2 in arterial blood Concentration of titratable mmHg kPa hydrogen ion in extracellular fluid 20.0 150 mmol l–1 19.0 Siggaard-Andersen 140 0 5 0 5 0 18.0 –1 –1 –2 –2 –3 acid–base chart 130 17.0

H+ deficit

al

120

0 +1

ca ia

ia

pn

pn

60

11.0

9.0 8.0 7.0 6.0

50

40

s ces

5.0

Normal

40

Area

30

20

ex

15

ion

12.0

50

Normal

10 en rog

13.0

10.0 70

it ic defic n ro n Ch n io e g o dr hy

Concentration of in plasma +25 bicarbonate mmol l–1

16.0 15.0 14.0 Hypercapnia

80

er

ca er

0 +2

90

p hy

p hy

ic on

e ut

5 +1

100

r Ch

Ac

H+

110

35

e

ut 3.0

nia

20

onic

2.5

Chr

15

Hypocapnia

ap

oc

p hy

ion

25

Ac

hyd roge n

4.0 3.5

capnia

exc ess

30

Chronic hypo

yd te h

Acu

2.0

+30

6.8

pH in arterial plasma 6.9 7.0

140 120 100 90 Concentration of free hydrogen ion in plasma nmol l–1

7.1

80

7.2

70

7.3

60 50 Acidemia

7.4

40 35 Normal

7.5

7.6

30 25 Alkalemia

7.7

1.5

20

Figure 1 Acid–base chart for arterial blood with normal and pathophysiological reference areas. The acid–base status is shown as a point with three coordinates: pH (abscissa), pCO2 (ordinate), and c tH þ (oblique coordinate). The bands radiating from the normal area (the central ellipse) show reference areas for typical acute and chronic, respiratory and nonrespiratory, acid–base disturbances. Hyper- and hypocapnia are also called respiratory acidosis and alkalosis, respectively. Hydrogen ion excess and deficit, i.e., increased and decreased c tH þ , are also called nonrespiratory (or metabolic) acidosis and alkalosis, respectively. Reproduced from Siggaard-Andersen O (1971) An acid-base chart for arterial blood with normal and pathophysiological reference areas. Scandinavian Journal of Clinical and Laboratory Investigation 27: 239–245. Copyright & 1970, 1974 by Radiometer Copenhagen A/S, A˚kandevej 21, Brønshøj, Denmark.

changes in pCO2 in vivo. ctH þ Ecf is also called standard base deficit (SBD), or with the opposite sign standard base excess (SBE), but the term base is deprecated by the author. It is important to use ctH þ Ecf

rather than ctH þ B (whole blood titratable hydrogen ion) as a measure of a nonrespiratory acid–base disturbance, especially in neonatology where high hemoglobin concentrations and high pCO2 values may be

8 ACID–BASE BALANCE

encountered. The ctH þ B may then be considerably higher than ctH þ Ecf (as much as 4 mmol l
1) causing an erroneous diagnosis of metabolic acidosis, when the situation is merely a redistribution of hydrogen ions within the extended extracellular volume. Projections to the ctH þ scale in the upper left corner of Figure 1 should be made along the slanting so-called vivo-CO2 titration curves, which are virtually straight lines (slightly convex upwards). The slope of the lines depends on the concentration of nonbicarbonate buffers, i.e., mainly hemoglobin. In the chart, the slope corresponds to a hemoglobin concentration of 3 mmol l
1 corresponding to the hemoglobin concentration of the extended extracellular fluid. Variations in the slope due to variations in blood hemoglobin concentration are small and generally without clinical significance. Variations in the concentration of other buffers, e.g., albumin, are even less significant. In summary, the hydrogen ion status of the blood is described by a point in the acid–base chart: the x,y coordinates indicate cH þ and pCO2 , the oblique coordinate is ctH þ Ecf. The Henderson–Hasselbalch Equation

Often a description of acid–base balance is based on the Henderson–Hasselbalch equation, derived from the law of mass action: pH ¼ pK þ log10 ðcHCO
3 =ðaCO2  pCO2 ÞÞ where pK ¼ 6.10 and aCO2 ¼ 0.23 mmol l
1 kPa
1 ¼ 0.0306 mmol l
1 mmHg
1 (solubility coefficient of carbon dioxide in plasma at 371C). aCO2  pCO2 gives the concentration of H2CO3 plus CO2. pH is determined by two variables, pCO2 and cHCO3
,

representing respiratory and metabolic disturbances. cHCO3
is shown in Figure 1 on a horizontal logarithmic scale along the pCO2 ¼ 5:33 kPa line. Projections to the scale should be made at an angle of
451. However, cHCO3
is not independent of pCO2 . For this reason, standard bicarbonate was introduced, i.e., the bicarbonate concentration in plasma of whole blood equilibrated with a gas mixture with a normal pCO2 (5.33 kPa ¼ 40 mmHg) at 371C. However, even the standard bicarbonate is not completely independent of acute changes in pCO2 in vivo, decreasing slightly in acute hypercapnia. To be independent, the equilibration should be performed with a model of the extended extracellular fluid. Projecting from a given point in the chart to the bicarbonate scale along the slanting vivo-CO2 equilibration lines gives the standard bicarbonate concentration of the extended extracellular fluid. The Van Slyke Equation

Blood gas analyzers measure pH with a glass electrode and pCO2 with a membrane-covered glass electrode (Stow-Severinghaus electrode). ctH þ Ecf is calculated from pH, pCO2 , and cHb (concentration of hemoglobin) using a model of the titration curve called the Van Slyke equation (Table 1). The equation calculates the change in buffer base concentration (bicarbonate plus protein anion plus phosphate) from the value at the reference point: pHPy ¼ 7:40; PyCO2 ¼ 5:33 kPa; and T y ¼ 37 C Buffer base (BB) is the difference between the concentrations of buffer anions and buffer cations (the latter being virtually zero at physiological pH). Strong ion difference (SID) is the difference between

Table 1 Van Slyke equation for calculation of the concentration of titratable hydrogen ion in the extended extracellular fluid, ctH þ Ecf þ c tH þ Ecf ¼
ð1
cHbEcf=cHby Þ  ðDcHCO
3 P þ bH Ecf  DpHPÞ c HbEcf ¼ c HbB  V B/V Ecf concentration of hemoglobin in the extended extracellular fluid V B/V Ecf ¼ 1/3 (default value) ratio between the volume of blood and volume of extended extracellular fluid c Hby ¼ 43 mmol l
1 empirical parameter accounting for an unequal distribution of hydrogen ions between plasma and erythrocytes Dc HCO3
P ¼ c HCO3
P
c HCO3
Py y c HCO3
Py ¼ 24.5 mmol l
1 concentration of bicarbonate in plasma at pHPy ¼ 7.40, PCO ¼ 5:33 kPa, T y ¼ 37:0 C 2 DpHP ¼ pHP
pHPy bHþ Ecf ¼ bm Hby  cHbEcf þ bP bm Hby ¼ 2.3 apparent molar buffer capacity of hemoglobin monomer in whole blood bP ¼ 7.7 mmol l
1 (default value) buffer value of nonbicarbonate buffers in plasma for a normal plasma protein (albumin) concentration c HbB ¼ rHbB/MmHb (substance) concentration of hemoglobin in blood (unit: mmol l
1) as a function of the mass concentration, rHbB (unit: g l
1) MmHb ¼ 16 114 g mol
1 molar mass of hemoglobin monomer

Ecf refers to the extended extracellular fluid, B to whole blood, P to plasma. Replacing cHbEcf by cHbB gives ctH þ B; replacing cHbEcf by zero gives ctH þ P. Note: if cHbB ¼ 9.0 mmol l
1 3 rHbB ¼ 14.5 g dl
1, then the Van Slyke equation simplifies to c tH þ Ecf ¼
0.93  (Dc HCO3
P þ DpHP  14.6 mmol l
1).

ACID–BASE BALANCE 9

the concentrations of nonbuffer cations and nonbuffer anions (see Figure 2). According to the law of electroneutrality, the value of BB and SID must be identical. Buffer base is not a suitable indicator of a nonrespiratory acid–base disturbance; although independent of pCO2 , it varies with the albumin and hemoglobin concentrations, which are unrelated to acid–base disturbances.

Normal Acid–Base Balance

Concentration of ions in arterial plasma (mmol l −1)

Acid–base balance refers to the balance between input (intake and production) and output (elimination) of hydrogen ion. The body is an open system in equilibrium with the alveolar air where the partial pressure of carbon dioxide pCO2 is identical to the carbon dioxide tension in the blood. pCO2 is directly proportional to the CO2 production rate (at constant

150

Mg2+ Ca2+ K+

HCO3−

SID+

BB− Pr

−

100

Na+

Cl −

Cations

Anions

HPO42− +H2PO4− SO42− Organic anions

50

Figure 2 Electrolyte balance of arterial plasma showing columns of cations and anions of equal height (law of electroneutrality). The equality of the strong ion difference (SID) and buffer base (BB) is illustrated. The change in concentration of buffer base from normal (at pH ¼ 7.40, pCO2 ¼ 5:3 kPa, and T ¼ 371C) with opposite sign equals the concentration of titratable hydrogen ion.

alveolar ventilation and CO2 free inspired air) and inversely proportional to the alveolar ventilation (at constant CO2 production rate and CO2 free inspired air). CO2 is constantly produced in the oxidative metabolism at a rate of about 10 mmol min
1 ( ¼ 224 ml min
1) and eliminated in the lungs at the same rate so that the pCO2 remains at about 5.33 kPa ( ¼ 40 mmHg). Hydrogen ions associated with any anion other than bicarbonate or exchanging with a cation are eliminated by the kidneys. In the oxidative metabolism of sulfur-containing amino acids, hydrogen ions are produced together with sulfate ions at a rate of about 70 mmol day
1 depending upon the protein intake. Amino acids are oxidized to carbon dioxide and water, and the amino nitrogen, liberated as NH3, combines with carbon dioxide in the liver via the Krebs urea cycle to form neutral urea. Therefore, there is no production of base (ammonia) except in the kidneys, where ammonia formed from glutamine diffuses into the urine where it binds a hydrogen ion (NH3 þ H þ -NH4þ ) thereby preventing an excessively low urine pH. Normal values for the acid–base status of arterial blood are given in Table 2. The lower pCO2 in women than men is probably a progesterone effect on the respiratory center. The values are independent of age except at birth, where babies tend to have higher pCO2 , lower pH, and slightly increased ctH þ Ecf, approaching normal values for adults in the course of a few hours. In the last trimester of pregnancy, the pCO2 is lower (about 1 kPa ¼ 7.5 mmHg), compensated by a slightly increased ctH þ Ecf. A protein-rich diet causes a higher ctH þ Ecf (1–2 mmol l
1) and a slightly lower pH due to production of sulfuric acid from sulfur-containing amino acids. A diet rich in vegetables and fruit causes a lower (negative) ctH þ Ecf and a slightly higher pH due to organic anions binding H þ in the metabolism to carbon dioxide and water. High-altitude hypoxia stimulates ventilation; at 5 km above sea level, pCO2 is decreased to about 3.3 kPa ¼ 25 mmHg. The hypocapnia is compensated by increased ctH þ Ecf, so pH is only slightly elevated. The values fall in the area of chronic hypocapnia in the acid–base chart (Figure 1).

Table 2 Reference values for arterial blood Women þ

cH P c tH þ Ecf pCO2 cHCO3
P

Men
1

36.3–41.7 nmol l (pH: 7.38–7.44)
2.3 to þ 2.7 mmol l
1 4.59–5.76 kPa (33.8–42.4 mmHg) 21.2–27.0 mmol l
1

37.2–42.7 nmol l
1 (pH: 7.37–7.43)
3.2 to þ 1.8 mmol l
1 4.91–6.16 kPa (36.8–46.2 mmHg) 22.2–28.3 mmol l
1

cH þ P: conc. of (free) hydrogen ion in plasma; c tH þ Ecf: conc. of titratable hydrogen ion in extracellular fluid (also called standard base deficit, SBD); pCO2 : tension of carbon dioxide; cHCO3
P: conc. of bicarbonate in plasma.

10

ACID–BASE BALANCE

Acid–Base Disturbances Respiratory Acid–Base Disturbances

Acute respiratory acid–base disturbances are characterized by an acute change in pCO2 associated with an acute change in pH but with unchanged ctH þ Ecf. The relationship between pCO2 and pH is illustrated by the oblique in vivo CO2 equilibration lines in the acid–base chart (Figure 1). Primary increase and decrease in pCO2 are compensated by secondary renal decrease and increase in ctH þ Ecf, respectively. The acid–base chart shows the expected values in chronic hypercapnia and chronic hypocapnia. The effect of the compensation is a return of pH about two-thirds towards normal, slightly more in acute hypocapnia. Nonrespiratory Acid–Base Disturbances

Primary increase and decrease in ctH þ Ecf are compensated by secondary decrease and increase in pCO2 . A very acute rise in ctH þ Ecf, for example, due to anaerobic exercise with lactic acid formation, is only partly compensated because only peripheral chemoreceptors react promptly to a fall in blood pH. It takes about an hour before H þ equilibrium between blood and brain extracellular fluid is achieved and the central chemoreceptors are maximally stimulated. The acid–base values in acute nonrespiratory acidemia are illustrated in Figure 1 by the area labeled ‘acute hydrogen ion excess’. The outline of the area is dotted because it is less well-defined than the other areas of the chart. The compensations in more slowly developing nonrespiratory acidemia or alkalemia are illustrated by the areas labeled ‘chronic hydrogen ion excess’ and ‘deficit’, respectively. The effect of the respiratory compensation is a return of pH one-third to halfway towards normal. Once an increase in ctH þ Ecf has been detected, the question is: what caused the metabolic acidosis? It may be a production of lactic acid due to anaerobic metabolism or acetoacetic acid (ketoacidosis) due to diabetes mellitus or starvation. In both cases the diagnosis may be verified by direct measurement of blood lactate or acetoacetate. When these analyses are unavailable, calculation of the concentration of undetermined anions may be useful, i.e., the sum of the concentrations of measured cations (Na þ and K þ ) minus the sum of the concentrations of measured and calculated anions (Cl
and HCO3
). This equals the sum of the concentrations of unmeasured anions (mainly Protein
, SO24
, HPO24
, fatty carboxylate, lactate, acetoacetate)minus the sum of the concentrations of unmeasured cations (Ca2 þ and Mg2 þ ). A metabolic acidosis with a major increase in undetermined anions usually indicates

organic acidosis. A hyperchloremic acidosis may be a renal acidosis with retention of H þ and Cl
or an intestinal loss of Na þ þ HCO3
with subsequent intake of saline (Na þ þ Cl
). A hypochloremic alkalosis may be due to loss of H þ and Cl
by vomiting. Hypokalemic alkalosis is due to inability of the kidneys to retain hydrogen ions in the presence of potassium depletion. See also: Arterial Blood Gases. Carbon Dioxide. Peripheral Gas Exchange. Ventilation: Overview.

Further Reading Astup P and Severinghaus JW (1986) The History of Blood Gases Acids and Bases. Copenhagen: Munksgaard International Publishers. Davenport HW (1969) The ABC of Acid–Base Chemistry, 5th edn. Chicago: The University of Chicago Press. Grogono AW (1986) Acid–base balance. International Anesthesiology Clinics, Problems and Advances in Respiratory Therapy 24(1). Halperin ML and Goldstein MB (1988) Fluid, Electrolyte, and Acid–Base Emergencies. Philadelphia: Saunders. Hills AG (1973) Acid–Base Balance. Chemistry, Physiology, and Pathophysiology. Baltimore: Williams & Wilkins. International Federation of Clinical Chemistry and International Union of Pure and Applied Chemistry (1987) Approved Recommendation (1984) on Physico-Chemical Quantities and Units in Clinical Chemistry. Journal of Clinical Chemistry and Clinical Biochemistry 25: 369–391. Masoro EJ and Siegel PD (1971) Acid–Base Regulation. Its Physiology and Pathophysiology. Philadelphia: Saunders. Nahas G and Schaefer KE (eds.) (1974) Carbon Dioxide and Metabolic Regulations. New York: Springer. Rooth G (1975) Acid–Base and Electrolyte Balance. Lund: Studentlitteratur. Severinghaus JW and Astrup P (1987) History of Blood Gas Analysis. Boston: Little, Brown and Company. Shapiro BA, Peruzzi WT, and Templin R (1994) Clinical Application of Blood Gases, 5th edn. St Louis: Mosby – Year Book. Siggaard-Andersen O (1971) An acid-base chart for arterial blood with normal and pathophysiological reference areas. Scandinavian Journal of Clinical and Laboratory Investigation 27: 239–245. Siggaard-Andersen O (1974) The Acid–Base Status of the Blood, 4th edn. Copenhagen: Munksgaard and Baltimore: Williams & Wilkins Company. Siggaard-Andersen O (1979) Hydrogen ions and blood gases. In: Brown SS, Mitchell FL, and Young DS (eds.) Chemical Diagnosis of Disease, pp. 181–245. London: Elsevier, NorthHolland Biomedical Press. Siggaard-Andersen O and Fogh-Andersen N (1995) Base excess or buffer base (strong ion difference) as measure of a non-respiratory acid–base disturbance. Acta Anaesthesiologica Scandinavica 39(supplement 107): 123–128. Thomson WST, Adams JF, and Cowan RA (1997) Clinical Acid– Base Balance. New York: Oxford University Press. West JB (1974) Respiratory Physiology, the Essentials. Oxford: Blackwell. West JB (2001) Pulmonary Physiology and Pathophysiology. An Integrated, Case-Based Approach. Philadelphia: Lippincott Williams & Wilkins.

ACUTE RESPIRATORY DISTRESS SYNDROME 11

Acute Exacerbations

see Asthma: Acute Exacerbations. Chronic Obstructive Pulmonary Disease:

Acute Exacerbations.

Acute Lung Injury

see Acute Respiratory Distress Syndrome.

ACUTE RESPIRATORY DISTRESS SYNDROME G Bellingan, University College London, London, UK S J Finney, Imperial College London, London, UK

Table 1 Clinical triggers of ARDS Etiology

Percentage

Sepsis (including pulmonary sepsis) Aspiration of gastric contents Pulmonary contusion Bacteremia Head injury Multiple bony fractures requiring ICU admission Blood transfusion exceeding 10 units in 24 h Cardiopulmonary bypass Burns (including smoke inhalation) Acute pancreatitis Lung reperfusion injury (e.g., posttransplant) Near-drowning

43–52 22–36 8–26 4–12 6–11 5–12 5–8 2 2 1

& 2006 Elsevier Ltd. All rights reserved.

Abstract The acute respiratory distress syndrome (ARDS) is the devastating manifestation of the diffuse pulmonary inflammation that may occur following a wide range of life-threatening systemic illnesses. The rapid onset of inflammation and bilateral nonhydrostatic alveolar edema results in severe hypoxemia and reduced pulmonary compliance often mandating mechanical ventilation. The clinical features, radiology, and pathogenesis are reviewed in this article. The management of patients comprises primarily of ventilatory support while the lung injury resolves. The techniques of ventilatory support can propagate the lung injury and adversely affect outcome; the techniques are discussed in detail here. By contrast, pharmacotherapy has a less clear role in ARDS. Corticosteroids may be beneficial after the acute phases, whilst other anti-inflammatory agents have not proved beneficial. Mortality is determined primarily by the underlying trigger for ARDS, but is approximately 30–40%. Follow-up of survivors has demonstrated that lung function often improves considerably, whereas nonpulmonary morbidities persist even 12 months after discharge from the intensive care unit.

Introduction The acute respiratory distress syndrome (ARDS) and its less extreme manifestation, acute lung injury (ALI), are devastating conditions that result in sudden bilateral nonhydrostatic alveolar edema and severe hypoxemia. Pulmonary failure is usually so severe that patients require mechanical ventilation of their lungs. ARDS and ALI are the consequence of the diffuse pulmonary inflammation that can be triggered by an insult either to the lung itself or more commonly at a distant site. As such, they form part of the spectrum of systemic inflammation that can occur following many life-threatening insults (see Table 1). The constellation of severe respiratory distress, refractory hypoxemia, decreased pulmonary compliance,

The figures in this were based on studies at the university of Washington and Colorado.

and diffuse radiological changes was first appreciated by Ashbaugh and co-workers in 1967. They referred to the clinical scenario as the adult respiratory distress syndrome. Since an identical condition can also occur in children, it is now referred to as the acute respiratory distress syndrome. Subsequently, the clinical syndrome has been increasingly recognized and estimates of the annual incidence of ARDS range from 8 to 70 cases per 100 000 population in developed countries. Overall, mortality in patients with ARDS is approximately 30–40%, although death is usually attributable to the underlying etiology rather than pulmonary failure per se.

Etiology The many possible triggers for ARDS are outlined in Table 1. Sepsis accounts for the majority of cases in general intensive care units (ICUs). Patients with multiple risk factors are more likely to develop ARDS. Intriguingly, ARDS only occurs in a subset of patients who suffer apparently similar insults. It is not clear what explains a particular individual going on

12

ACUTE RESPIRATORY DISTRESS SYNDROME

to develop ARDS. Although environmental factors such as age, sex, smoking history, and intercurrent pulmonary disease may be important, it is widely considered that genetic factors are critical. Candidate genes for which associations have been described include those that encode tumor necrosis factor alpha (TNF-a), angiotensin-converting enzyme, interleukin-6 (IL-6), surfactant protein B, and Toll-like receptors. Nevertheless, it has not been possible to draw definitive conclusions since these associations are complicated by tight linkage disequilibrium to other genes, lack of clear functional effects, and small patient numbers. Moreover, the heterogeneity of ARDS suggests that many genes with varying phenotypes and penetrance may underpin an individual’s susceptibility. Functional genomic approaches may help elucidate these complex genotypes.

After the initial injury, myofibroblasts are observed in the interstitium and then the airspace, and start to produce new matrix substance. Indeed, the lung collagen content can double by 2 weeks. With time, type II alveolar epithelial cells increase in number and may represent a stem cell population of the lung; they differentiate into type I alveolar epithelial cells and repopulate the denuded alveolar basement membrane during healing. Pulmonary function and computed tomography (CT) suggest that many patients subsequently return to have structurally normal lungs although this can take months. Some patients however develop severe fibrosis involving both the alveolar space and interstitium. At its most extreme, the ‘honeycomb’ of advanced fibrotic lung disease may form. Many of these patients succumb to intercurrent infection associated with a prolonged ICU stay. A few can recover with incomplete resolution of pulmonary damage.

Pathology Traditionally, the histological development of ARDS has been described as having three sequential phases which affect the lungs in a diffuse manner: exudative, proliferative, and fibrotic. It is now evident that these stages overlap considerably and fibrotic changes are initiated very early. Within the first 24 h, the lungs appear macroscopically edematous and congested. At this point, light microscopy reveals edema within the airspaces, alveolar walls, and septae. Type I alveolar epithelial cells are swollen, necrotic, and often detached from the underlying basement membrane. Pulmonary endothelial cells may also be swollen, with fibrin thrombi occluding alveolar capillaries. Neutrophils are also initially observed within alveolar capillaries, but inflammatory cells then accumulate rapidly in the edematous alveoli. Over the next few days, the lungs become more uniformly red as alveolar wall and airspace edema increase and red cells leak into the airspaces. Histologically, hyaline membranes form from fibrin-rich edema fluid and line the alveoli. The number of neutrophils increases rapidly as they move via the interstitium into the airspace and there is increased disruption of vascular structures with further neutrophil and fibrin plugs occluding capillaries.

Clinical Features Definition

The diagnosis of ARDS is based on the criteria developed at the 1992 American–European consensus conference and is illustrated in Table 2. Since the definition does not consider current management strategies, the relevance of scenarios in which suboptimal mechanical ventilation and management can influence whether criteria are met or not is not clear. The cutoffs for severity of hypoxemia are arbitrary, the definition of ‘acute’ onset lacks clarity and the use of the chest X-ray opens the way for individual interpretation. Further limitations of the definition include the inclusion of other conditions that probably have different pathological processes (such as severe pneumocystis carinii infection, diffuse alveolar hemorrhage), and exclusion of unilateral disease that may occur following pulmonary lobectomy. Nevertheless, the definition is simple to use and is supported by extensive literature. Post-mortem studies have demonstrated that the definition is 84% sensitive and 94% specific for diffuse alveolar damage; its performance in patients who survive is not clear.

Table 2 American–European consensus criteria for diagnosing ALI and ARDS ALI Chest radiography Clinical scenario Left atrial pressure Oxygenation

Bilateral airspace shadowing Acute onset and associated with a condition known to cause ALI/ARDS No direct or clinical evidence of left atrial hypertension (PAOP o18 mmHg) PaO2/FiO2 o39.9 kPa (300 mmHg)

PAOP, pulmonary artery occlusion pressure.

ARDS

PaO2/FiO2 o26.6 kPa (200 mmHg)

ACUTE RESPIRATORY DISTRESS SYNDROME 13 Natural History

The clinical picture of ARDS is dominated initially by severe hypoxemia due to mismatching of ventilation and perfusion. Indeed, intrapulmonary shunting may result in oxygen saturations that are relatively refractory to increases in the inspired oxygen content. Decreased pulmonary compliance increases the work of breathing and most patients require endotracheal intubation and mechanical ventilation. From a pulmonary perspective, the high oxygen requirements persist for some time. Further increases in the alveolar–arterial oxygen gradient occur with ongoing pulmonary inflammation, particularly in the setting of a positive fluid balance, but may also reflect a superimposed ventilatory pneumonia or pneumothorax. Pneumothoraces tend to occur after the first week and may tension rapidly. Since the inflamed lung may tether to the chest wall, pneumothoraces can be loculated and anterior, and thus easily missed on plain chest radiography. Thoracic CT may be required to locate pneumothoraces accurately. Surprisingly, despite devastating pulmonary failure, oxygenation often slowly improves allowing withdrawal of mechanical ventilation; for many, this may take weeks or months and often necessitate temporary tracheostomy. Although disease can remain compartmentalized with isolated lung failure, ARDS is usually part of the spectrum of systemic inflammation; hence, patients often demonstrate peripheral vasodilation, increased cardiac outputs, and systemic hypotension which may require the administration of vasopressors such as norepinephrine. Secondary pulmonary hypertension can occur and result in acute right ventricular failure. Renal dysfunction is also common as is the need for acute renal support. Other organs can also fail as part of this process and outcome is related to the number of failing organs. The course of ARDS is not a smooth wave of deterioration and recovery. Rather, it is interspersed by episodes of deterioration (commonly linked with intercurrent infection such as ventilator-associated pneumonia or line-related sepsis) and many patients need repeated episodes of inotrope and other organ support prior to final recovery (or demise).

Radiology

The appearance of the plain chest radiograph, although forming part of the consensus definition for ARDS, can be relatively non-specific. Clues distinguishing ARDS from cardiogenic pulmonary edema include normal cardiac dimensions, a normal vascular pedicle width, a peripheral distribution of airspace shadowing, and the absence of septal lines.

Chest CT demonstrates that the lungs are affected in a heterogenous manner. Typically, there is a gradient of opacification from apparently normally aerated lung, through ground-glass appearances, to densely consolidated lung. In the supine patient, this gradient occurs both in ventrodorsal and cephalocaudal directions. These gradients typically reverse within a few minutes if the patient is moved to the prone position. Since alveolar edema would not redistribute so quickly, some of these appearance are not due to increased edema in dependent zones but due to collapse of these areas due to the weight of the overlying lung. Thus ground-glass appearances most likely represent airspace edema, with densely opacified areas representing collapsed edematous lung. Regions of dense opacification in nondependent areas may signify collapse/consolidation due to infection or retained secretions. At later stages, groundglass appearances may be accompanied by bronchial dilatation which persists into recovery, suggesting established fine intralobular fibrosis (Figure 1). Other Pulmonary Investigations

Bronchoalveolar lavage samples are dominated by the granulocytic cell population initially. As the disease evolves, the proportion of granulocytes declines and a greater proportion of macrophages is seen. Persistent neutrophilia often portends a poor prognosis. Since most patients are mechanically ventilated, there are few data about classical pulmonary function tests in patients with ARDS. Re-breathing techniques have been used during positive pressure ventilation and have demonstrated marked reductions in the functional residual capacity, carbon monoxide diffusing capacity (DLCO), and diffusing coefficient (KCO). Lower values of DLCO and KCO tend to be associated with nonsurvival. Disease Severity

There are several scoring systems that evaluate the severity of ARDS. Although these systems have only limited clinical utility in individuals, they describe well the degree of physiological disturbance and act as useful descriptors of disease severity in clinical studies. The scoring systems include the acute physiology and chronic health evaluation II and the lung injury score. The former is used to evaluate all critically ill patients whereas the latter is specifically designed for patients with ARDS.

Pathogenesis Since neutrophils appear early in histological specimens and dominate in bronchoalveolar fluid samples,

14

ACUTE RESPIRATORY DISTRESS SYNDROME

also key initiators of pulmonary inflammation in ARDS. Studies of patient groups at risk of ARDS who do or do not progress to develop refractory hypoxemia suggest compartmentalized intrapulmonary inflammatory changes (e.g., increased IL-8) may precede a systemic inflammatory response. There is widespread activation and/or dysfunction of many cell types within the lung which results in the clinical manifestations of ARDS (Figure 2). Thus endothelial dysfunction and loss of epithelial integrity reduce the barrier function of the alveolar wall and result in alveolar edema. Alveolar edema is further exacerbated by the loss of epithelial cells which normally promote fluid transport out of the alveolus through apical sodium pumps. Surfactant is lost early during ARDS due to reduced production by damaged epithelial cells along with neutralization of preexisting surfactant by the protein-rich edema fluid. Surfactant loss contributes to alveolar collapse, intrapulmonary shunt, and hypoxemia. Endothelial and smooth muscle cell dysfunction result in impaired hypoxic pulmonary vasoconstriction and, along with microthrombi, contribute to the development of secondary pulmonary hypertension and also impacts on outcome.

Animal Models

Figure 1 Typical appearances of ARDS with (a) plain radiology and (b) computed tomography.

it has been considered that they are important in the pathogenesis of ARDS. It is possible that activated and thus rigid 7.5 mm neutrophils may get stuck in pulmonary capillaries where they release a plethora of inflammatory mediators that include chemokines, cytokines, and proteases. Activation may occur at remote sites and/or by circulating cytokines. However, since ARDS can occur in neutropenic patients, neutrophils cannot be absolutely required for the development of ARDS. Circulating inflammatory mediators (e.g., TNF-a, IL-1b, IL-6, IL-8, leukotrienes) along with the changes that occur within the coagulation system during systemic inflammation are

Animal models allow the study of the pathophysiology of ARDS and the effects of interventions whilst tightly controlling both insults and genetic background. However, the multiple models that exist for ARDS/ALI illustrate that none are ideal. Models in which the lung is injured directly include hyperoxia, high tidal-volume mechanical ventilation, and instillation of oleic acid, bleomycin, or thiourea. Alternatively, models mimic systemic sepsis in which the lung is also injured; these include systemic injections of lipopolysaccharide and caecal ligation and puncture. Some authors suggest that two-hit models are more clinically appropriate and combine models of sepsis with hypovolemia, burns, or high tidal-volume ventilation.

Management and Current Therapy The primary management aims for a patient with ARDS are to maintain adequate oxygenation and to support any other failing organs whilst the lung injury resolves. In general, respiratory failure is sufficiently severe to mandate mechanical ventilation, the techniques of which significantly influence mortality. By contrast, few, if any, therapies directed at modifying the pathogenesis and evolution of ARDS, have been demonstrated to effect outcome. A detailed

ACUTE RESPIRATORY DISTRESS SYNDROME 15

Inflammatory trigger

Mechanical ventilation

E.g., bacterial sepsis, massive transfusion Stretch-induced cell signaling

Activated neutrophils

Stretch-induced cell damage

Soluble mediators E.g., cytokines, chemokines, coagulation factors, eicosanoids, ROS

Smooth muscle cells

Endothelial cells

Vascular dysfunction

Reduced HPV Pulmonary hypertension

Epithelial cells

Barrier dysfunction

Production

Surfactant

Alveolar edema Neutralization

Alveolar collapse Figure 2 Pathogenesis of ARDS and ALI. HPV, hypoxic pulmonary vasoconstriction; ROS, reactive oxygen species.

description of optimal mechanical ventilation along with the pharmacotherapeutic approaches that have been explored are outlined below. Conventional Mechanical Ventilation

The manner in which the injured lung is ventilated influences mortality and may perpetuate lung injury. The origins of such ventilator-induced lung injury (VILI) are multifactorial but include the shear stresses exerted on alveoli during overdistension and cyclical collapse/re-inflation, and oxygen toxicity. VILI is illustrated by chest CT of those patients who have survived ARDS: relatively normal lung architecture is often present in previously densely consolidated areas, whereas a reticular pattern of fibrosis is seen in those regions (often nondependent) that were exposed to mechanical ventilation. VILI results in the ongoing release of inflammatory mediators that may spill over into the circulation and propagate systemic inflammation, thereby influencing mortality. Indeed, increased plasma levels of cytokines have been demonstrated in animal models and in patients receiving injuriously large tidal-volume ventilation. This theory has stimulated the development of socalled ‘protective’ strategies for mechanical ventilation which minimize VILI.

Tidal volume Delivery of a normal tidal volume to extensively consolidated lungs inevitably results in overdistension of the remaining lung units. Experimental work demonstrated that this may be a significant trigger for VILI, and resulted in the National Institutes of Health (NIH)-sponsored study of low tidal-volume ventilation in 861 patients. This landmark study showed that mortality could be reduced by the use of lower tidal volumes (4–6 ml kg 1, ideal body mass) or at least the avoidance of more ‘traditional’ tidal volumes (10–12 ml kg 1). The corresponding plateau inspiratory pressures were 25 and 35 cmH2O, respectively. Plasma and bronchoalveolar lavage cytokine and chemokines levels were greater in those patients receiving higher tidal volumes. The study protocol has been adopted by some as the definitive ventilatory strategy. More correctly, it provided excellent evidence for VILI and should form the basis of lung-protective strategies. Indeed, it has been suggested that targeting lower-end expiratory alveolar pressures may be more sensible, since the specific pulmonary compliance (compliance corrected for accessible lung volume) has been reported as normal in patients with ALI/ARDS. Smaller tidal volumes may reduce VILI by virtue of the reduced cyclical volume per se, or through a reduction in the end-inspiratory volume. It is not clear

16

ACUTE RESPIRATORY DISTRESS SYNDROME

which is the important factor although in vitro experiments on mechanically deformed epithelial layers have demonstrated that cyclical changes are more damaging than constant stretch to a high volume. How these results translate to the in vivo scenario is not clear. In the absence of increased respiratory rates, reductions in tidal volume will reduce alveolar ventilation and result in a hypercapnic acidosis. Permissive hypercapnia is generally well tolerated except in the setting of a marked metabolic acidosis and increased intracranial pressure. Hypercapnia may also reduce myocardial contractility, and possibly increase the need for sedation and/or paralysis. The effects on the immune response are still unclear. The degree of hypercapnic acidosis allowable is unclear although many clinicians accept arterial pH values not less than 7.20. When titrating respiratory rate to pCO2 , it must be remembered that increases in respiratory rate will have less influence on alveolar ventilation in ARDS due to the increased dead space, and that reductions in respiratory rate can paradoxically increase CO2 clearance when inspiratory times are particularly prolonged. Tracheal insufflation of oxygen and administration of weak bases are sometimes used to combat the acidosis. Fractional inspired oxygen High fractions of inspired oxygen (FiO2) cause absorption atelectasis and may be cytotoxic to the lung per se. The standard practice is to titrate the FiO2 to arterial saturations of 88–92%, rather than a specific pO2 which may be less relevant to oxygen delivery. Lower targets may be appropriate in those patients whose cardiac output is high or where an improvement of SaO2 would require an unacceptably injurious pattern of ventilation (e.g., through a higher FiO2, positive end expiratory pressure (PEEP), or tidal volume). The benefits of further reductions in FiO2 afforded by the use of inhaled nitric oxide (iNO), prone positioning, and related maneuvers are unknown and still subject to review (vide infra). Intrapulmonary shunting may result in arterial oxygen saturations being relatively independent of FiO2 between 0.8 and 1.0. Positive end expiratory pressure The application of PEEP during mechanical ventilation increases FRC and thus prevents the collapse of alveoli at end expiration. This will reduce intrapulmonary shunt and improve oxygenation. Furthermore, the shear forces required to repeatedly open collapsed alveoli during inspiration are considerable and most likely contribute to VILI. Thus, by keeping these alveoli open throughout the respiratory cycle, PEEP can limit

VILI. Since the lung is heterogeneously affected in ARDS, excessively high levels of PEEP can lead to overexpansion of those regions whose compliance is higher, an effect that may exacerbate VILI. Other detrimental effects of PEEP include reduced cardiac preload and cerebral perfusion. The method to determine the optimal level of PEEP in ARDS is still not established. A randomized multicenter trial in 549 patients, titrating PEEP according to the required FiO2, found no outcome differences between a lower and higher PEEP algorithm. Many clinicians set PEEP by inferring the region of maximal slope on the pressure–volume curve by assessing maximal tidal volume as PEEP is adjusted with a constant pressure control. Mathematical modeling suggests that this is best determined using a decremental rather than an incremental PEEP trial. Ventilatory modes The NIH trial of lower tidal-volume ventilation used a mandatory constant flow, volume control mode of mandatory ventilation. There is considerable interest in the role of descending flow/ pressure control modes since these may promote more even distribution of gas, as flow is slower at the end of expiration and thus more likely to be laminar. Pressure-control modes do not influence peak alveolar pressure, although peak airway pressures may be reduced. The most likely advantage afforded by pressure control ventilation and inverse ratio ventilation is that these may be associated with a greater proportion of the respiratory cycle spent at plateau inspiratory pressure. Greater plateau times promote more homogenous ventilation by allowing slow-filling regions to inter-fill from areas with faster time constants. The recruitment of previously collapsed or partially collapsed slow-filling alveoli reduces shunt and improves oxygenation. Prolongation of inspiration can also improve CO2 clearance by forcing expiration to be delayed to a point where alveolar pCO2 has risen to a maximum. Faster respiratory rates reduce the efficacy of inverse ratio ventilation by shortening the inspiratory time. Excessive prolongation of inspiration may reduce expiratory time to a point at which expiration is not completed. This may result in dynamic hyperinflation and the generation of autoPEEP. Most clinicians consider auto-PEEP undesirable since levels may be unpredictable thus leading to cardiovascular compromise. There are theoretical advantages to modes that allow spontaneous ventilation such as biphasic positive airway pressure and airway pressure release ventilation. These modes allow continued diaphragmatic and intercostal function and the ventilation of dependent, better-perfused regions. Although pressure support modes also allow spontaneous ventilation,

ACUTE RESPIRATORY DISTRESS SYNDROME 17

some argue against them as they preclude any plateau time. No study to date, excluding those examining weaning, has demonstrated any outcome differences according to ventilatory mode. Drainage of pneumothoraces Multiple pneumothoraces may complicate mechanical ventilation of patients with ARDS. Pneumothoraces can be complex and localized as the inflamed lung may tether to the chest wall. Indeed, localization can be difficult by conventional radiography and it may be necessary to insert drains under the guidance of CT. Intercostal drains are generally left in situ until the patient is well established on a spontaneous ventilatory weaning program. Supplemental Techniques for Mechanical Ventilation

Prone positioning Ventilation in the prone position improves oxygenation in approximately 70% of patients. It is not possible to predict which patients will respond, and nonresponders may improve on subsequent turns. Experienced teams can easily turn patients, even with multiple intercostal drains and intravascular catheters, with few complications. Improvements in oxygenation continue in 50% of patients after they are returned to the supine position and are probably the consequence of expansion of previously collapsed lung. More homogenous ventilation may prevent regional overdistension and limit VILI. Improved secretion drainage and altered diaphragmatic mechanics may also contribute to clinical improvement. Despite improvements in oxygenation, prone positioning has not been demonstrated to improve outcome in ARDS. Recruitment maneuvers Sustained and high airway pressures of up to 60 cmH2O may be required to open (or recruit) some regions of collapsed lung. There has been considerable enthusiasm for the use of such recruitment maneuvers which, in combination with the application of PEEP, may keep these newly recruited lung units open. Techniques vary from sophisticated determination of recruitment and derecruitment by stepwise alteration of ventilatory pressures and repeated blood gas sampling through to simply temporarily increasing PEEP to 30 cmH2O for 30 s. Improvements in oxygenation have often been demonstrated, although these may not be sustained. Such high intrathoracic pressures are often accompanied by brief episodes of reduced cardiac output and hypotension, but rarely barotrauma. The role of recruitment maneuvers still has to be clarified in ARDS.

Partial liquid ventilation Partial liquid ventilation has been proposed to improve oxygenation and limit VILI. Perfluorocarbons are dense liquids that have little biological activity but very low surface tension and exceptionally high-solubility coefficients for oxygen and carbon dioxide. The lung is filled (partially or fully) with the perfluorocarbon and then normal mechanical ventilation applied simultaneously. Since the perfluorocarbon is volatile, it evaporates over time through the ventilator circuit. Animal studies suggest that perfluorocarbons reduce atelectasis, improve perfusion matching, and improve secretion clearance. Some studies suggest they may also be anti-inflammatory, reducing permeability rises and neutrophil activation. Human studies have revealed an increase in the incidence of pneumothoraces, mucus plugging, and a disruption of the normal surfactant system. They are ingested by macrophages and their effect on immune function is unclear. A phase II/III study of partial-liquid ventilation is underway in France currently. Extracorporeal gas exchange Traditionally, extracorporeal gas exchange (ECGE) has only been used as a last resort in patients in whom oxygenation cannot be maintained by any other means. Devices vary from those which oxygenate and support the whole cardiac output, to those that provide partial oxygenation, and carbon dioxide removal to supplement conventional ventilation at lower pressures (extracorporeal lung assist, ECLA). Hemorrhage and coagulopathies have been the most significant complications, occurring in about 67% of adult patients. Although no mortality advantage has ever been shown for ECGE, new technology such as heparinbonded circuits, nonocclusive roller pumps, pumpless circuits, and better anticoagulation control, that may reduce complications is leading to a re-evaluation of this modality. Surfactant administration Alveolar surfactant is lost early in ARDS. The administration of exogenous surfactant either via a bronchoscope or nebulizer may reduce surface tension within alveoli and prevent cyclical collapse, improving oxygenation, and ameliorating VILI. Multiple administrations appear necessary due to the neutralization of surfactant by edema fluid. Despite improvements in oxygenation and phase I/II survival benefits, no large studies of surfactant have demonstrated any mortality advantage and thus it is not administered to patients with ARDS. However, there are many outstanding questions regarding the optimal formulation, patient selection, and dosing regimen.

18

ACUTE RESPIRATORY DISTRESS SYNDROME

Inhaled vasodilators iNO and prostacyclin cause selective vasodilatation of the regions of the lung to which they are delivered by ventilation, thus improving ventilation/perfusion matching and oxygenation. Their short half-lives limit systemic vasodilatation and hypotension. Both agents typically improve arterial pO2 by about 20% in responders, and this may be enhanced by the administration of intravenous almitrine bismesylate, an agent that is reported to enhance hypoxic pulmonary vasoconstriction. iNO is usually maximally effective at inspired concentrations of less than 10 parts per million, although the dose–response curve may vary considerably after 24 h. iNO and prostacyclin may also favorably influence inflammation, platelet activity, and vascular permeability. Although frequently used for their beneficial effects on oxygenation, no survival benefit has been shown for either agent in controlled studies. High-frequency ventilation The corollary of the fact that low tidal-volume ventilation is beneficial is that high-frequency ventilation may further protect against VILI. Tidal volumes delivered by jet ventilators and oscillators are less than the anatomical dead space, and respiratory rates are greater than 1 Hz. Gas transport occurs via diffusion rather than bulk flow. To date, no mortality advantage has been demonstrated for these ventilatory techniques in adults. Noninvasive ventilation Noninvasive ventilation (NIV) avoids the need for endotracheal intubation and the associated risks of ventilator-associated pneumonias, sinusitis, and sedation. The use of NIV in hypoxemic respiratory failure is controversial and a consensus conference into the role of NIV in acute respirator failure found little data to support its use in ARDS. Indeed, the natural history of ARDS is often longer than the time for which many patients are able to tolerate tight-fitting masks continuously. It may thus only delay endotracheal intubation. Manipulation of the Alveolar Fluid Balance

High pulmonary capillary pressures increase extravascular lung water and result in worsening oxygenation. Indeed, a persistently positive fluid balance is associated with a worse outcome in ARDS. It is difficult to ascertain whether this association is due to detrimental effects of administered fluids per se or simply a reflection of the severity of the underlying illness requiring fluids for cardiovascular support. Nevertheless, whilst it is critical to maintain organ perfusion with an adequate cardiac output, the administration of intravenous fluids in the absence of

evidence of organ hypoperfusion would seem inappropriate. The careful use of vasopressors may allow the limitation of fluid administration. Apical sodium and probably chloride channels in alveolar epithelial cells play an important role in normal alveoli in maintaining a flux of water into the circulation. A subgroup of the sodium channels can be stimulated by b-agonists such as salbutamol and terbutaline which may be administered either as inhaled preparations or as continuous intravenous infusion. Recent data suggest that salbutamol infusions can reduce extravascular lung water. How this influences outcome in ARDS is not known and thus these agents remain experimental. Manipulation of the Inflammatory Process

Corticosteroids Corticosteroids can reduce inflammation by repressing transcription and destabilizing pro-collagen mRNA. Initially evaluated in large doses in patients considered at risk of ARDS or early in ARDS, they have no beneficial effects on mortality. By contrast, their administration later when repair and remodeling have commenced, may be helpful. Possible negative effects include nosocomial infection, hyperglycemia and an exacerbation of critical illness poly(myo)neuropathy. In a study of only 24 patients, corticosteroids caused a significant improvement in oxygenation and reduced mortality in patients with unresolving ARDS. Methylprednisolone (2 mg kg 1 day 1) was administered from day 8 for the shorter of either the duration of mechanical ventilation or 14 days. Although the study was methodologically limited, many clinicians administer a similar regimen to patients with persistent ARDS after 7 days and in whom there is little evidence for systemic sepsis. The results of the North American Late Steroid Rescue Study (LaSRS) will shortly provide better evidence regarding the role of corticosteroids in ARDS. Other agents Other pharmacological agents that have been assessed in ARDS/ALI are outlined in Table 3. None have been demonstrated to improve mortality despite good rationale for their use and promising preliminary studies.

Outcome Over the last ten years, mortality has fallen from around 60% to around 30% in the current major trials. Although multifactorial in origin, this is in part due to improvement in the whole critical-care process but a greater appreciation that inappropriate mechanical ventilation can further injure the inflamed lung. Most patients succumb to multi-organ failure

ADAMs AND ADAMTSs 19 Table 3 Other Pharmacological agents assessed in ARDS/ALI Agent

Rationale

Effects

Prostaglandin E1

A direct pulmonary vasodilator, inhibitor of platelet aggregation, and inhibitor of neutrophil adhesion

Mortality unaltered More rapid improvement in oxygenation

Dazoxiben

Inhibitor of thomboxane synthase and thus acts as a pulmonary vasodilator and inhibitor of platelet aggregation

Mortality unaltered

Ketoconazole

Inhibitor of thomboxane synthase and 5-lipoxygenase, thus reducing production of leukotrienes, neutrophil chemokines

Mortality unaltered

N-acetyl cysteine

Antioxidant that reduces damage by reactive oxygen species

Mortality unaltered Improved oxygenation and compliance

Lisofylline

Inhibitor of phosphatidic acid which increases cytokine production and activates neutrophils

Mortality unaltered

or sepsis rather than specific pulmonary failure and an inability to maintain adequate oxygenation. Follow-up of patients who survive ARDS has demonstrated progressive improvement in their lung function that continues to improve even one year after discharge from the ICU. Lung volumes seem to improve more rapidly than diffusing capacity and the distance walked in six min. Radiologically, previously densely consolidated areas frequently appear normal, with fibrosis of nondependent regions. By contrast, survivors often have considerable long-term nonrespiratory morbidities, particularly related to physical strength and to psychosocial, functional, and stress indices. Indeed, less than 50% return to work within 12 months. See also: Corticosteroids: Therapy. Extracorporeal Membrane-Gas Exchange. Fluid Balance in the Lung. Genetics: Gene Association Studies. Hypoxia and Hypoxemia. Leukocytes: Neutrophils. Lung Imaging. Nitric Oxide and Nitrogen Oxides. Oxygen Therapy. Oxygen Toxicity. Surfactant: Overview. Ventilation, Mechanical: Positive Pressure Ventilation.

Further Reading Brower RG, Lanken PN, MacIntyre N, et al. (2004) Higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome. New England Journal of Medicine 351: 327–336. Evans TW, Griffiths MJD, and Keogh BF (eds.) (2002) European Respiratory Monograph: ARDS, vol. 70(20). Sheffield: ERS Journals. Herridge MS, Cheung AM, Tansey CM, et al. (2003) One-year outcomes in survivors of the acute respiratory distress syndrome. New England Journal of Medicine 348: 683–693. Meduri GU, Headley AS, Golden E, et al. (1998) Effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial. Journal of the American Medical Association 280: 182–183. Pinhu L, Whitehead T, Evans T, and Griffiths M (2003) Ventilatorassociated lung injury. Lancet 361: 332–340. The Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. New England Journal of Medicine 342: 1301–1308. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 1334–1349.

ADAMs AND ADAMTSs C P Blobel, Cornell University, New York, NY, USA S S Apte, Cleveland Clinic Foundation, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Proteolysis has emerged as a key posttranslational regulator of the function of molecules on the cell surface and in the

extracellular milieu. In principle, proteolysis can activate or inactivate a substrate, or can change its functional properties. ADAM (a disintegrin and metalloprotease) and ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type 1 repeats) proteases are related members of a superfamily of metalloendopeptidases that also includes matrix metalloproteinases (MMPs) and astacins. ADAMs are integral membrane proteins that typically cleave other membrane anchored proteins, whereas ADAMTS proteases lack a membrane anchor, and process both secreted and cell surface molecules.

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ADAMs AND ADAMTSs

ADAMs are implicated in fertilization, neurogenesis, regulation of the function of ligands for the epidermal growth factor receptor, and the release of proteins such as the proinflammatory cytokine tumor necrosis factor alpha (TNF-a) from the plasma membrane. ADAMTS proteases have key roles in the molecular maturation of von Willebrand factor and procollagen and are implicated in the pathogenesis of osteoarthritis. Here, we provide a general overview of the biochemical properties and physiological functions of ADAMs and ADAMTS proteases and describe their relevance to lung and airway disorders.

ADAMs Introduction

ADAMs (a disintegrin and metalloprotease) are a family of membrane anchored glycoproteins that have been implicated in cleaving and releasing proteins from the cell surface. This process, which is referred to as protein ectodomain shedding, affects the function of molecules with key roles in development and disease, including growth factors such as transforming growth factor alpha (TGF-a), heparinbinding epidermal growth factor (HB-EGF), cytokines such as tumor necrosis factor alpha (TNF-a), and receptors such as the tumor necrosis factor receptor 1(TNFR1). The first recognized ADAMs were the two subunits of a sperm protein termed fertilin, which has a critical role in fertilization. Other ADAMs were subsequently identified based on their

sequence homology to fertilin, or based on functional assays, such as their ability to shed the proinflammatory cytokine TNF-a or process and activate the cell surface receptor Notch. Structure

The typical domain organization of an ADAM is shown in Figure 1. Of the over 30 ADAMs that have been identified to date, only about half contain a catalytic site consensus sequence and are therefore predicted to be catalytically active. The remaining ADAMs that are not catalytically active are thought to function mainly in cell–cell or cell–matrix interactions. Regulation of Production and Activity

All catalytically active ADAMs are synthesized with a pro-domain that helps the metalloprotease domain fold in the endoplasmic reticulum (ER), and keeps it inactive until the pro-domain is cleaved, usually just before reaching the trans-Golgi network. Once the pro-domain is removed, additional mechanisms, including phorbol esters, phosphatase inhibitors, calcium ionophores, and activation of G-protein-coupled receptors, regulate the catalytic activity of ADAMs. The disintegrin domain and cysteine-rich region of ADAMs are thought to have a role in cell–cell interaction and potentially also in

Cell

ADAMs Pro

Catalytic

Disintegrin

CRD

EGF-like

TSR

TSR

TM

Cytosolic

(0−14)

ADAMTSs Pro

Catalytic Protease domain

Disintegrin-like

CRD

Spacer

Ancillary domain

Figure 1 All ADAMs are membrane-anchored glycoproteins that are characterized by a conserved domain structure: an N-terminal signal sequence followed by pro (Pro), metalloprotease, and disintegrin domains, a cysteine-rich region (CRD), usually containing an EGF repeat, and finally a transmembrane (TM) domain and cytoplasmic tail. Only about half of the known ADAMs have a catalytic site consensus sequence, and are therefore predicted to be catalytically active. The disintegrin domain was first identified in snake venom toxins that bind to platelet integrins and function as anticoagulants, but is now known to be a characteristic feature of all ADAM and ADAMTS proteins. ADAMTS proteins are similar to ADAMs in the prometalloprotease domain, but unlike ADAMs, all ADAMTSs are catalytically active. ADAMTSs have a functionally critical ancillary domain containing specific modules not found in ADAMs, e.g., thrombospondin type 1 repeats (TSRs) (see text for details). A critical difference between these protease families is the absence of an integral membrane segment in ADAMTS proteases.

ADAMs AND ADAMTSs 21

substrate recognition. Finally, the cytoplasmic domain of catalytically active ADAMs usually contains signaling motifs such as potential phosphorylation sites and proline-rich Src-homology 3 (SH3) ligand domains, and several molecules that interact with the cytoplasmic domains of ADAMs and might regulate their maturation and function have been identified. The catalytic activity and substrate selectivity of ADAMs has been explored using both biochemical and cell biological approaches. One important conclusion from biochemical studies was that individual ADAMs do not have a clear consensus cleavage site in vitro. Since ADAMs and their substrates are both membrane anchored, cell-based assays are critical tools for understanding the substrate selectivity and regulation of ADAMs in the context of the plasma membrane. Studies using cells from ADAM knockout mice, or cells treated with small inhibitory RNA (siRNA) against different ADAMs, have revealed that these enzymes display substrate selectivity in cells, although the mechanism underlying this remains to be established. A common feature, however, is that ADAMs frequently cleave their substrates close to the plasma membrane, resulting in release or ‘ectodomain shedding’ of the substrate’s soluble ectodomain. Biological Function

As noted above, ADAM-dependent ectodomain shedding can profoundly affect the function of the released substrate protein. Ectodomain shedding can enable the released molecule to act at a distance from the cell that it was shed from, which is referred to as paracrine signaling. For example, processing of the EGF receptor (EGFR) ligands TGF-a and HB-EGF by ADAM17 is critical for activation of the EGFR during development, and therefore mice lacking

ADAM17 resemble mice lacking TGF-a or HB-EGF or EGFR. Ectodomain shedding is also a key mediator of the role of TNF-a in autoimmune diseases such as rheumatoid arthritis. Interestingly, receptors can be either inactivated by ectodomain shedding, such as the TNFR1, or activated, such as Notch. Mutations in the cleavage site of the TNFR1 that decrease its shedding cause accumulation of the receptor, leading to increased susceptibility to TNF in patients with TNF-receptor associated periodic febrile syndrome (TRAPS). Function of ADAMs in Lung Development and in Respiratory Diseases

Currently, ADAM17 is the only ADAM with a clearly established role in lung development (Figure 2). Mice lacking ADAM17 are born with respiratory distress, presumably caused by abnormal alveoli with septation defects and thickened mesenchyme, as well as impaired branching morphogenesis and delayed vasculogenesis, and thus reduced surface for gas exchange. Since similar defects are observed in mice lacking HB-EGF or the EGFR, the abnormal lung development in Adam17 / mice is most likely explained by a lack of HB-EGF shedding. With respect to respiratory diseases, smoking has been implicated in the activation of ADAMs and the release of EGFR ligands such as amphiregulin. The resulting activation of the EGFR can presumably contribute to the pathogenesis of lung cancer by stimulating cell proliferation and DNA replication at the same time that mutagens are delivered in smoke. Moreover, Grampositive bacteria stimulate the G-protein-coupled platelet activating receptor (PAR) in patients with cystic fibrosis, which in turn activates ADAMdependent release of HB-EGF, and thus mucin production. Therefore, inhibitors of ADAMs, such as hydroxamic acid type metalloprotease inhibitors,

HB-EGF

HB-EGF ADAM17

Extracellular space

Cell membrane

Cytoplasm Figure 2 Proteolytic processing of heparin-binding EGF-like growth factor (HB-EGF) by ADAM17 releases this growth factor from its membrane tether. The ADAM17-dependent ectodomain shedding of HB-EGF is thought to be critical for the function of this growth factor during lung and heart development.

22

ADAMs AND ADAMTSs

might be useful in the treatment of cystic fibrosis. Finally, mutations in the ADAM33 gene have been linked to asthma susceptibility, although the mechanism underlying the role of ADAM33 in asthma remains to be determined. In light of the key roles of ADAMs in regulating signaling via the EGF receptor and other cell surface signaling pathways, and the critical roles for ADAMs in lung development and in asthma and cystic fibrosis, it appears likely that further studies of this protein family in the context of respiratory disease will uncover novel functions, thus hopefully also providing new targets for drug design.

ADAMTSs Introduction

ADAMTS (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type 1 repeats) comprises a family of 19 secreted metalloproteases that is distinct from the membrane-anchored ADAMs. The founding member of this family, ADAMTS1, was described very recently, in 1997. ADAMTS1 was so named because it resembled the ADAMs in the metalloprotease domain and disintegrin-like module and was thought to be a variant ADAM. Its main point of distinction from ADAMs, apart from the absence of a transmembrane segment, was the presence of three modules resembling thrombospondin type 1 repeats (TSRs). Soon afterwards, it became clear that all 19 ADAMTS proteases shared these major features. Structure

A typical ADAMTS consists of prometalloprotease and ancillary domains. The prometalloprotease domains resemble ADAMs, since the active site sequence is of the reprolysin (snake venom) type. Basic amino acid rich sequences providing sites for removal of the pro-domain by subtilisin-like proprotein convertases (SPCs) are present in the prometalloprotease domain and at its junction with the catalytic domain. The ancillary domain (from N-terminus to C-terminus) consists of a disintegrinlike module, a central TSR, a cysteine-rich module, a cysteine-free spacer, and a variable number of additional TSRs, ranging from 0 (ADAMTS4) to 14 (ADAMTS9 and 20) (Figure 1). An interesting feature of ADAMTS proteases is their clear grouping into distinct subfamilies. Proteases within a subfamily have an identical modular organization, gene structure, and active site sequence, suggesting evolution by gene duplication from a common precursor. Each subfamily has a distinct modular organization,

for example, ADAMTS7 and ADAMTS12, constituting one such subfamily, are the only metalloproteases known to have mucin modules and glycosaminoglycan attachment sites. Regulation of Production and Activity

Transcriptional regulation appears to be very important, since many ADAMTS mRNAs are highly regulated during embryogenesis, for example, ADAMTS2, 3, and 14, or induced in specific circumstances such as inflammation, for example, ADAMTS1. ADAMTS proteases are synthesized as zymogens and undergo removal of the prometalloprotease domain by SPCs either within the secretory pathway or at the cell surface. Subsequent to SPC processing, proteolysis within the ancillary domain appears to be an important posttranslational regulator of activity, resulting in further activation or inactivation of the enzymes. Several ADAMTS proteases bind to the cell surface through unknown mechanisms that suggest activity as operational cell surface proteases, and indicate locations at which posttranslational processing may occur. Unlike ADAMs, the ADAMTSs typically attack specific cleavage sites in their substrates. For instance, ADAMTS4 is a glutamyl endopeptidase that processes Glu–Xaa bonds in many proteoglycans and ADAMTS13 specifically cleaves von Willebrand factor (vWF) at the Tyr1605–Met1606 peptide bond. An important concept in the ADAMTS family is that their catalytic domains alone do not retain activity towards natural substrates. The ancillary domain is critical for the recognition and binding of substrates. In many instances, this requires specific posttranslational modifications in the substrates (such as glycosylation of proteoglycans, triplehelicity in the case of procollagens, and physical unfolding of vWF by fluid shear force). In addition, the minimal substrate binding sites are quite extended in length and difficult to reproduce in synthetic peptides. This makes the development of assays for ADAMTS activity and inhibition quite challenging. The only known ADAMTS inhibitors are tissue inhibitor of metalloprotease 3 (TIMP-3) and a2-macroglobulin. Biological Function

Unexpectedly diverse functions for ADAMTS proteases have been revealed through human genetic disorders and transgenic animals. All ADAMTS genetic disorders are recessive, as is typical of enzyme deficiencies. Inherited thrombocytopenic purpura results from ADAMTS13 mutations, with retention of unusually large polymers of vWF and failure to

ADAMs AND ADAMTSs 23

process these into forms that are optimal for coagulative homeostasis. Acquired thrombocytopenic purpura may result from circulating anti-ADAMTS13 autoantibodies. Ehlers–Danlos syndrome (type VIIC or the dermatosparactic type) is a consequence of ADAMTS2 mutations. In this disorder, tissue (especially skin) fragility results from lack of complete procollagen processing and a decrease in structurally competent collagen fibrils. ADAMTS1 is needed for mouse urinary tract development and fertility, and inhibits angiogenesis in vitro and in vivo by binding to vascular endothelial growth factor (VEGF). ADAMTS4 and ADAMTS5 null mice are developmentally normal, but ADAMTS5 mice are resistant to both mechanically induced and immune arthritis, suggesting that ADAMTS5 is a major cartilage-degrading enzyme. ADAMTS10 mutations cause Weill–Marchesani syndrome (WMS), comprising short stature, brachydactyly, cardiovascular defects, and ectopia lentis. Intriguingly, several aspects of WMS are the opposite of those seen in Marfan syndrome, a fairly common inherited connective tissue disorder. ADAMTS20 mediates melanoblast migration from the neural crest and is deficient in a natural mouse mutant named Belted because of the belt of white fur across the torso. Receptors

Although some ADAMTS proteases can bind to the cell surface, a specific receptor, syndecan-1, has been identified only for ADAMTS4. Nevertheless, because of the affinity of many ADAMTS for heparin and chondroitin sulfate, cell surface proteoglycans may constitute a broad category of receptors for this family. ADAMTS Proteases in Respiratory Diseases

Since thrombocytopenic purpura is a systemic coagulation disorder, patients can develop microthrombi in their lungs and have sometimes manifested with acute respiratory distress syndrome. Pneumothorax has occasionally been reported in Ehlers–Danlos syndrome type VIIC patients and Adamts2 null mice have widening of their distal air spaces. These mice show deficiency of procollagen I as well as procollagen III processing. Both collagens are structurally major components of the lung. Although ADAMTS10 is highly expressed in the lung, WMS cases are not reported to have lung problems.

See also: Asthma: Overview. Epidermal Growth Factors. Matrix Metalloproteinases. Tumor Necrosis Factor Alpha (TNF-a ).

Further Reading Apte SS (2004) A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family. International Journal of Biochemistry and Cell Biology 36: 981–985. Becherer JD and Blobel CP (2003) Biochemical properties and functions of membrane-anchored metalloprotease-disintegrin proteins (ADAMs). Current Topics in Developmental Biology 54: 101–123. Blobel CP (2005) ADAMs: key players in EGFR-signaling, development and disease. Nature Reviews: Molecular Cell Biology 6: 32–43. Dagoneau N, Benoist-Lasselin C, Huber C, et al. (2004) ADAMTS10 mutations in autosomal recessive Weill–Marchesani syndrome. American Journal of Human Genetics 75: 801–806. Gao G, Plaas AH, Thompson VP, et al. (2004) ADAMTS4 (aggrecanase-1) activation on the cell surface involves C-terminal cleavage by GPI-anchored MT4-MMP and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on syndecan-1. Journal of Biological Chemistry 279: 10042–10051. Kheradmand F and Werb Z (2002) Shedding light on sheddases: role in growth and development. BioEssays 24: 8–12. Kuno K, Kanada N, Nakashima E, et al. (1997) Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene. Journal of Biological Chemistry 272: 556–562. Lapiere CM and Nusgens BV (1993) Ehlers–Danlos type VII-C, or human dermatosparaxis: the offspring of a union between basic and clinical research. Archives of Dermatological Research 129: 1316–1319. Lemjabbar H and Basbaum C (2002) Platelet-activating factor receptor and ADAM10 mediate responses to Staphylococcus aureus in epithelial cells. Nature Medicine 8: 41–46. Lemjabbar H, Li D, Gallup M, et al. (2003) Tobacco smoke-induced lung cell proliferation mediated by tumor necrosis factor alpha-converting enzyme and amphiregulin. Journal of Biological Chemistry 278: 26202–26207. Levy GG, Nichols WC, Lian EC, et al. (2001) Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413: 488–494. Porter S, Clark IM, Kevorkian L, and Edwards DR (2005) The ADAMTS metalloproteinases. Biochemical Journal 386: 15–27. Shapiro SD and Owen CA (2002) ADAM-33 surfaces as an asthma gene. New England Journal of Medicine 347: 936–938. Stanton H, Rogerson FM, East CJ, et al. (2005) ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature 434: 648–652. White JM (2003) ADAMs: modulators of cell–cell and cell–matrix interactions. Current Opinion in Cell Biology 15: 598–606. Zhao J, Chen H, Peschon JJ, et al. (2001) Pulmonary hypoplasia in mice lacking tumor necrosis factor-alpha converting enzyme indicates an indispensable role for cell surface protein shedding during embryonic lung branching morphogenesis. Developmental Biology 232: 204–218.

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ADENOSINE AND ADENINE NUCLEOTIDES R Polosa, University of Catania, Catania, Italy D Zeng, CV Therapeutics, Palo Alto, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Adenosine is a purine nucleoside. A growing body of evidence has emerged in support of a proinflammatory and immunomodulatory role for adenosine in the pathogenic mechanisms of chronic inflammatory disorders of the airways such as asthma and chronic obstructive pulmonary disease (COPD). The fact that adenosine enhances mast cell allergen-dependent activation, that elevated levels of adenosine are present in chronically inflamed airways, and that adenosine given by inhalation causes dose-dependent bronchoconstriction in subjects with asthma and COPD emphasizes the importance of adenosine in the initiation, persistence, and progression of these common inflammatory disorders of the airways. These distinctive features of adenosine have been recently exploited in the clinical and research setting to identify innovative diagnostic applications for asthma and COPD. In addition, because adenosine exerts its multiple biological activities by interacting with four adenosine receptor subtypes, selective activation or blockade of these receptors may lead to the development of novel therapies for asthma and COPD. In this article, the evidence for the roles of adenosine receptors in the pathophysiology of chronic inflammatory airway diseases such as asthma and COPD are reviewed.

Introduction The cardiovascular actions of adenosine were first described in a classic publication by Drury and SzentGyorgyi in 1929. It was shown that adenosine causes coronary vasodilation, hypotension, and bradycardia. In 1963, Berne proposed the hypothesis that adenosine mediates the metabolic regulation of coronary blood flow. Since then, a large body of literature has supported the critical role of adenosine in modulating numerous cardiovascular functions. Two well-characterized effects of adenosine have successfully found clinical applications. First, adenosine causes bradycardia, slows A-V nodal conduction, and reduces atrial contractility, and is used as a rapid intravenous bolus for the acute termination of re-entrant supraventricular arrhythmias. Second, adenosine causes coronary vasodilation and increased blood flow, and it is used with radionuclide imaging in the heart to detect underperfused areas of myocardium. The action of adenosine in pulmonary diseases was first discovered in the late 1970s. Holgate and colleagues observed that adenosine and related synthetic analogs were potent agents in augmenting

IgE-dependent mediator release from isolated rodent mast cells. A few years later, adenosine (but not its metabolite inosine or the unrelated nucleoside guanosine) administered by inhalation was shown to be a powerful bronchoconstrictor of asthmatic but, importantly, not of normal airways. Asthma and chronic obstructive pulmonary disease (COPD) are complex syndromes sharing clinical heterogeneity and a number of pathogenic traits, which include variable degrees of airflow obstruction, bronchial hyperresponsiveness (BHR), and chronic airway inflammation. In addition to its known effect as a bronchoconstrictor, a growing body of evidence has emphasized the importance of adenosine in the initiation, progression, and control of chronic inflammation and remodeling of the airways. The key evidence can be summarized as follows: 1. Adenosine is generated in high concentrations at sites of tissue injury (e.g., hypoxia and inflammatory cell activation). 2. Elevated levels of adenosine are present in chronically inflamed airways; they have been observed both in the bronchoalveolar lavage (BAL) fluid and the exhaled breath condensate (EBC) of patients with asthma, and adenosine concentrations are also increased after specific allergen challenge in the plasma of atopic individuals and in the BAL fluid obtained from sensitized rabbits. 3. The progressive accumulation of adenosine in the lungs of mice lacking the purine catabolic enzyme adenosine deaminase (ADA) is strongly associated with development of lung inflammation, tissue eosinophilia, airway hyperresponsiveness mucus metaplasia, airway remodeling, and emphysemalike injury of the lung parenchyma. 4. Adenosine administration by inhalation induces concentration-dependent bronchoconstriction in subjects with asthma and COPD whereas the nucleoside had no discernible effect on airway caliber in normal individuals. 5. Adenosine augments allergen-induced mediator release from human mast cells in vitro, and potentiates the immediate response to allergen in asthmatics. 6. Airway hyperresponsiveness to adenosine better reflects the inflammatory status of the lung in asthma compared with directly acting bronchospasmogens including methacholine; the exquisite sensitivity of adenosine challenge to detect inflammatory changes in human airways has been

ADENOSINE AND ADENINE NUCLEOTIDES 25

beneficially exploited to evaluate modifications in the level of airway inflammation in a prospective and noninvasive manner. 7. Dipyridamole, a blocker of facilitated adenosine uptake, may precipitate asthma. 8. Theophylline, an established asthma therapy, is an adenosine receptor antagonist. Low dose of theophylline blocks AMP-induced bronchoconstriction in asthmatics; this effect is unlikely due to phosphodiesterase inhibition.

In light of these observations, adenosine has been proposed to be an important mediator of asthma. The evidence for the role of adenosine receptor signaling in the pathogenesis of chronic inflammatory disorders of the airways such as asthma and COPD is reviewed below.

Adenosine Metabolic Pathways Adenosine is an endogenous nucleoside consisting of the purine base, adenine, in glycosidic linkage with the sugar ribose (Figure 1). Adenosine is present at low concentrations in the extracellular space and its levels are greatly increased under metabolically stressful conditions as a result of enzymatic cleavage of the nucleotide adenosine 50 -monophosphate (AMP) by the 50 -nucleotidase. This increase in adenosine formation may also occur during inflammation when a large number of infiltrating inflammatory cells are competing for a limited oxygen supply. Intracellular levels of adenosine are kept low principally by its conversion to AMP by the enzyme adenosine kinase. Adenosine may also be degraded to inosine by ADA (Figure 2). It has been proposed that adenosine may function as a ‘retaliatory metabolite’. During injury, the imbalance of O2 supply and demand triggers the increased formation of adenosine. Adenosine may exert its protective effects by decreasing the energy demand of the tissue via a direct inhibitory effect on parenchymal

NH2 N

N

HO

N

N O

OH Figure 1 Structure of adenosine.

OH

cell function and indirectly by providing a more favorable environment for parenchymal cells, the best example of which is adenosine-mediated augmentation of nutrient availability via vasodilatation. Moreover, adenosine helps to maintain tissue integrity by modulating the function of the immune system.

Adenosine Receptors Adenosine elicits its biological activities by interacting with four adenosine receptor subtypes designated as A1, A2A, A2B, and A3 adenosine receptors. The genes for these receptors have been cloned from humans and several animal species. Tissue distributions of these receptors have been determined at the mRNA levels using Northern blot or in situ hybridization techniques or at the protein levels using subtype-selective radioligands or antibodies. In general, these receptors are widely expressed in many tissues. For example, high levels of A1 receptors are found in brain, adrenal gland, and adipose tissue whereas high levels of A2A receptors are found in spleen, thymus, striatum, and blood vessels. In addition, these receptors are often found to co-express in the same tissues or even on the same cells. The relative expression levels of these receptors have been found to be modulated by physiological and/or pathophysiological tissue environments. Although adenosine is the natural agonist for the four adenosine receptors, the ability of adenosine to activate these receptor subtypes varies. In many tissues, A1 and A2A receptors have relatively higher receptor reserves for adenosine, and can be activated by the physiological levels of adenosine and thus mediate the tonic action of adenosine. On the other hand, A2B and A3 receptors appear to have relatively lower affinities and/or receptor reserves for adenosine and require higher concentrations of adenosine for activation. However, the tissue adenosine levels in many pathophysiological conditions can reach significantly high levels to activate the A2B and A3 receptors. Numerous subtype-selective agonists and antagonists of adenosine receptors have been synthesized and are used as pharmacological tools. Although these ligands were classified as selective ligands based on their differential binding affinities for the four adenosine receptors, these compounds are often poorly characterized and not as selective as suggested. There are pharmacological reasons for the lack of functional selectivity. For example, potencies of an agonist for one given receptor could vary from one tissue to another depending on the receptor expression levels and the coupling efficiencies of the cells. As indicated before, adenosine receptors are widely distributed and their expression levels may be modified during disease processes. This

26

ADENOSINE AND ADENINE NUCLEOTIDES Ecto-adenosine deaminase

Ecto-5′-nucleotidase

AMP

Adenosine

Extracellular space

Nucleoside transporter

Adenosine deaminase

5′-nucleotidase

AMP

Inosine

Adenosine

Inosine

Adenosine kinase SAH hydrolase

SAH

Figure 2 Adenosine production and removal occur both intracellularly and extracellularly. Intracellularly, adenosine is formed by the action of 50 -nucleotidase, which dephosphorylates AMP, or by the action of S-adenosyl-homocysteine (SAH) hydrolase. When adenosine concentrations are high, it is phosphorylated to AMP by adenosine kinase (AK) or degraded to inosine by adenosine deaminase (ADA). Both 50 -nucleotidase and ADA are found in the extracellular space where they mediate the formation and degradation of adenosine. The intracellular and extracellular pools of adenosine are kept in equilibrium by the actions of bi-directional nucleoside transporters (NT). Inhibition of AK, ADA, or NT by drugs or genetic deletions raises the tissue adenosine level.

certainly adds complexity in predicting functional selectivity of agonists. In the case of antagonists, the functional blocking effects of competitive antagonists are dependent on the tissue levels of adenosine. If the levels of adenosine are too low to activate a given receptor, an antagonist for this receptor will not have any functional effects regardless of its binding affinity. In addition to these pharmacological issues, there are pharmacokinetic issues. In many cases, the half-life and tissue distributions of adenosine receptor ligands are poorly understood, making it difficult to draw conclusions on the role of receptor subtypes based on the absence of effects of ‘selective ligands’ in animal models. In spite of these limitations, selective agonists and antagonists of adenosine receptors are commonly utilized to establish the functions mediated by adenosine receptor subtypes. The four adenosine receptors also differ in their ability to couple to G-proteins and activate various intracellular signaling pathways. In most cells, A1 and A3 receptors couple to Gi/o and inhibit adenylate cyclase activity whereas A2A and A2B receptors couple to Gs proteins and increase adenylate cyclase activity and intracellular cAMP levels. While the adenylate cyclase–cAMP–protein kinase A axis is the most well-studied second messenger system involved in adenosine receptor function, it is clear that adenosine receptors utilize other signaling pathways as well. These include members of the mitogen-activated protein-kinase family, such as p38, p42/p44 (ERK 1/2), and c-jun terminal kinase, as well as various phospholipases, protein phosphatases, and ion channels.

Adenosine Biological Function Many cell types that play important roles in the pathogenesis of chronic inflammatory airway diseases are known to express adenosine receptors and to exhibit relevant effects through adenosine receptor signaling. These cell types include various inflammatory cells, such as mast cells, eosinophils, lymphocytes, neutrophils, and macrophages, and the structural cells in the lung, such as bronchial epithelial cells, smooth muscle cells, lung fibroblasts, and endothelial cells. The role of the adenosine receptors in these inflammatory and structural cells has been studied extensively using isolated cultured cells in vitro. In addition, numerous animal models have been developed and are extremely useful in determining the potential role of adenosine receptor subtypes in pulmonary functions. A1 Adenosine Receptor

The A1 receptor has been implicated in both pro- and anti-inflammatory aspects of disease processes. It has been shown that activation of the A1 receptor can promote activation of human neutrophils and monocytes and thus, their activation leads to proinflammatory responses. On the other hand, A1 receptors have been reported to be involved in anti-inflammatory and protective pathways in experimental models of injury in the heart, nerves, and kidney. The early evidence suggesting that the A1 receptor plays a role in asthma came from experimental work in rabbits rendered allergic by immunization

ADENOSINE AND ADENINE NUCLEOTIDES 27

protocols with allergen. These animals exhibited a bronchoconstrictor response to adenosine that was attenuated by pretreatment with A1 receptor blockers. In the same model, an antisense that targets the initiation codon of A1 receptor mRNA reduced the bronchoconstrictor response to adenosine and, more importantly, to the early response to allergen. However, the relevance of these observations to human asthma have been questioned due to the fundamental mechanistic difference between adenosine-induced bronchoconstriction in the allergic rabbit, which is due to activation of A1 receptors on the bronchial smooth muscle, and that in man, which appears to be dependent on activation of mast cells that do not express the A1 receptor. Furthermore, it has been shown recently that genetic removal of the A1 receptor gene from ADA-deficient mice resulted in enhanced pulmonary inflammation along with increased mucus metaplasia and alveolar destruction, thus indicating that in this experimental model, the activation of A1 receptors serves an anti-inflammatory and/or protective role in the regulation of pulmonary disorders triggered by elevated adenosine levels. A2A Adenosine Receptor

It is now well established that activation of A2A receptors on lymphoid cells leads to inhibition of an inflammatory response; this is largely due to its ability to induce accumulation of intracellular cyclic AMP in activated immune cells. Perhaps the strongest evidence for the critical role of A2A receptors in the regulation of inflammation in vivo originated from the elegant study of Ohta et al. using mice deficient in A2A receptors. In this model, the absence of the A2A receptor resulted in enhanced tissue inflammation and damage, thus suggesting a negative regulatory role for the A2A adenosine receptor subtype. In the airways, A2A receptors are present on most of the immunoinflammatory cells that have been implicated in asthma. A2A receptors are expressed on mast cells, and their activation results in increases in the intracellular cAMP concentrations, which are known to inhibit the biochemical pathways implicated in the release of histamine and tryptase from human mast cells. Stimulation of A2A receptors on neutrophils inhibits neutrophil adherence to the endothelium, prevents upregulation of integrin expression stimulated with formyl-Met-Leu-Phe and inhibits degranulation of activated neutrophils and monocytes. Activation of T lymphocytes, which plays a key role in the recruitment of leukocytes to the lung in clinical asthma, is also suppressed by A2A receptor activation. Thus, there are a multitude of mechanisms by which activation at A2A receptors

could result in suppression of airway inflammation in asthma and COPD. These findings support the hypothesis that A2A agonists could be potentially useful in controlling the inflammatory processes. A2B Adenosine Receptor

The initial evidence for the role of A2B receptors in asthma and COPD came from selectivity studies of enprofylline, a methylxanthine structurally closely related to theophylline. It was shown that enprofylline is a selective antagonist for the A2B receptors whereas theophylline has similar binding affinities for A1, A2A, and A2B receptors. The therapeutic concentrations of theophylline and enprofylline match their affinities for A2B receptors. Thus, it has been proposed that A2B receptors are possible targets for the long-term clinical benefit achieved with relatively low doses of theophylline and enprofylline. On the other hand, because the anti-inflammatory effects of endogenous adenosine might be mediated by A1 and A2A receptors, blockade of A1 and A2A receptors could be disadvantageous. It has been proposed that a more selective antagonist of the A2B receptors may be more effective and safer than theophylline. Recently, A2B receptors have been shown to have proinflammatory properties in many pulmonary cells. For example, functional human adenosine A2B receptors have been identified in mast cells, endothelial cells, bronchial smooth muscle cells, lung fibroblasts, and bronchial epithelium. Adenosine, via activation of A2B receptors, increases the release of various inflammatory cytokines from human mast cells (HMC-1), from human bronchial smooth muscle cells, human lung fibroblasts, and human airway epithelial cells. These cytokines, in turn, induce IgE synthesis from human B lymphocytes, and promote differentiation of lung fibroblasts into myofibroblasts. These findings provide strong support for the hypothesis that adenosine, via activation of A2B receptors, could enhance airway hyperresponsiveness and the inflammatory responses associated with asthma. Thus, an A2B antagonist could potentially be beneficial in the treatment of asthma and other pulmonary inflammatory diseases. A3 Adenosine Receptor

The functional role of the A3 receptor in the pathogenesis of chronic airway inflammatory diseases remains controversial due in large part to differences in the pharmacology of A3 receptors from different species. For instance, in rodents, mast cell degranulation and/or enhancement of mast degranulation in response to allergen appears to be dependent on A3 receptor activation. In humans, a relatively high density of functionally active A3 receptors is expressed in

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ADENOSINE AND ADENINE NUCLEOTIDES

eosinophils. Transcript levels for the A3 receptor are elevated in lung biopsies of patients with asthma or COPD and appear to be involved in the inhibition of eosinophil chemotaxis when stimulated. Furthermore, inhibition of important proinflammatory functions of human eosinophils by the selective A3 receptor agonist, IBMECA, has been reported. Because asthmatic inflammation is characterized by extensive infiltration of the airways by activated eosinophils, it is possible that the elevated adenosine concentrations associated with asthma could contribute to inhibition of eosinophil activation through stimulation of A3 receptors. In contrast, in their effort of dissecting out specific signaling pathways involved in adenosine-mediated pulmonary inflammation and airway remodeling in ADA-deficient mice, Young and colleagues have recently demonstrated that mice treated with the selective A3 receptor antagonists MRS 1523 resulted in a marked attenuation of pulmonary inflammation, reduced eosinophil infiltration into the airways, and decreased airway mucus production.

Adenosine in Respiratory Diseases Adenosine may play a critical role in the pathogenesis of chronic inflammatory disorders of the airways such as asthma and chronic obstructive pulmonary disease (COPD). Elevated levels of adenosine are present in chronically inflamed airways; they have been observed both in the BAL fluid and the EBC of patients with asthma. Adenosine levels are also raised after allergen exposure in the plasma and during experimental exacerbation of asthmatic symptoms due to exercise in atopic individuals. The observed adenosine elevations indicate that adenosine signaling may regulate aspects of acute and chronic airway disease. Consistent with the hypothesis of adenosine playing a critical role in the pathogenesis of chronic inflammatory disorders, mice deficient in ADA develop features of severe pulmonary inflammation and airway remodeling in association with elevations of adenosine concentrations in the lung. Features of the pulmonary phenotype noted include the accumulation of eosinophils and activated macrophages in the airways, mast cell degranulation, mucus metaplasia in the bronchial airways, and emphysema-like injury of the lung parenchyma. Although the histology seen in ADA-deficient mice fails to accurately resemble that of human asthma, since no epithelial shedding, subepithelial fibrosis, or muscle/submucosal gland hypertrophy were observed in this model, the ADA-deficient mouse is a useful tool to study the pathogenetic role of adenosine in chronic airway inflammation. The role of adenosine in the pathogenesis of asthma is not just limited to its biological effects in

Adenosine

A2B receptor

Mast cell

Cytokines

Prostaglandins and leukotrienes Histamine

Bronchoconstriction Figure 3 Mechanism of adenosine-induced bronchoconstriction. Adenosine, possibly via activation of A2B adenosine receptors, enhances the activation of airway mast cells, leading to increases in the release of potent contractile mediators such as histamine, leukotrienes, prostaglandins, and cytokines. These mediators in turn cause bronchoconstriction in human airways.

airway inflammation and remodeling. Adenosine administration by inhalation is known to elicit concentration-related bronchoconstriction in subjects with asthma and COPD whereas the nucleoside had no discernible effect on airway caliber in normal individuals. Since these initial observations were made, a considerable effort has been directed at revealing the fine mechanisms of adenosine-induced bronchoconstriction, which appear to involve a selective interaction with activated airway mast cells with subsequent release of preformed and newly formed mediators (Figure 3). An important development from adenosine research is the use of an adenosine (or AMP) inhalation challenge as an innovative diagnostic test for asthma and COPD. Compared with other noninvasive surrogate markers of airway inflammation, monitoring of the responsiveness of airways to inhaled adenosine appears to have the selective ability to probe changes in airway inflammation and it has been shown to be very useful when evaluating the effectiveness of different treatment regimens with inhaled corticosteroids. Because the airway response to inhaled adenosine is very sensitive to the effect of inhaled corticosteroids and is a good marker of disease activity, bronchoprovocation with adenosine has been proposed as a convenient and accurate biomarker to monitor corticosteroid requirements in asthma and to establish the appropriate dose needed to control airway inflammation.

ADHESION, CELL–CELL / Vascular 29

Conclusions and Future Direction Over the course of 20 years, the initial observation of the bronchoconstrictive effect of inhaled adenosine has evolved to provide the basis for a new asthma therapy as well as a diagnostic test. Recognition of the potential role of adenosine receptor signaling in the pathogenesis of chronic airway inflammatory diseases advocates the principle that modulating adenosine receptor signaling is likely to constitute a considerable advance in the management of asthma and COPD. The clinical evaluation of selective agonists or antagonists for the adenosine receptors should help elucidate the multiple roles of adenosine and its receptors in the pathophysiology of asthma and COPD. See also: Asthma: Overview. Bronchoalveolar Lavage. Chronic Obstructive Pulmonary Disease: Overview.

Further Reading Blackburn MR, Lee CG, Young HWJ, et al. (2003) Adenosine mediates IL-13-induced inflammation and remodeling in the lung and interacts in an IL-13-adenosine amplification pathway. Journal of Clinical Investigation 112(3): 332–344. Blackburn MR, Volmer JB, Thrasher JL, et al. (2000) Metabolic consequences of adenosine deaminase deficiency in mice are associated with defects in alveogenesis, pulmonary inflammation and airway obstruction. Journal of Experimental Medicine 192(2): 159–170. Cronstein BN (1994) Adenosine, an endogenous anti-inflammatory agent. Journal of Applied Physiology 76: 5–13. Cushley MJ, Tattersfield AE, and Holgate ST (1983) Inhaled adenosine and guanosine on airway resistance in normal and asthmatic subjects. British Journal of Clinical Pharmacology 15(2): 161–165.

Feoktistov I and Biaggioni I (1995) Adenosine A2b receptors evoke interleukin-8 secretion in human mast cells. An enprofyllinesensitive mechanism with implications for asthma. Journal of Clinical Investigation 96(4): 1979–1986. Feoktistov I, Polosa R, Holgate ST, and Biaggioni I (1998) Adenosine A2B receptors: a novel therapeutic target in asthma? Trends in Pharmacological Sciences 19: 148–153. Fozard JR (2003) The case for a role for adenosine in asthma: almost convincing? Current Opinion in Pharmacology 3(3): 264–269. Fredholm BB, Ijzerman AP, Jacobson KA, Klotz KN, and Linden J (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacological Reviews 53: 527–552. Ohta A and Sitkovsky M (2001) Role of G protein-coupled adenosine receptors in down-regulation of inflammation and protection from tissue damage. Nature 414: 916–920. Polosa R, Ng WH, Crimi N, et al. (1995) Release of mast cellderived mediators after endobronchial adenosine challenge in asthma. American Journal of Respiratory and Critical Care Medicine 151: 624–629. Rorke S, Jennison S, Jeffs JA, et al. (2002) Role of cysteinyl leukotrienes in adenosine 50 -monophosphate induced bronchoconstriction in asthma. Thorax 57(4): 323–327. Ryzhov S, Goldstein AE, Matafonov A, et al. (2004) Adenosineactivated mast cells induce IgE synthesis by B lymphocytes: an A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma. Journal of Immunology 172(12): 7726–7733. Shryock JC and Belardinelli L (1997) Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology and pharmacology. American Journal of Cardiology 79(12A): 2–10. Spicuzza L, Bonfiglio C, and Polosa R (2003) Research applications and implications of adenosine in diseased airways. Trends in Pharmacological Sciences 24(8): 409–413. Zhong H, Belardinelli L, Maa T, and Zeng D (2005) Synergy between A2B adenosine receptors and hypoxia in activating human lung fibroblasts. American Journal of Respiratory Cell and Molecular Biology 32(1): 2–8.

ADHESION, CELL–CELL Contents

Vascular Epithelial

Vascular H M DeLisser, University of Pennsylvania School of Medicine, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Four distinct families of adhesion molecules (cadherins, immunoglobulin superfamily members, selectins, and integrins) mediate

vascular cell–cell adhesion. These molecules are required for the formation of the junctional complexes that enable the assembly of endothelial cells into functional vascular networks; they mediate the leukocyte–endothelial adhesive interactions involved in the trafficking of leukocytes out of the circulation; and they contribute to contacts between pericytes and the endothelium that are important to the regulation of endothelial cell proliferation. In the lung these vascular cell–cell interactions are the bases of the host defense to lung infection or injury, and the regulation of lung vascular permeability, but are disturbed or dysregulated in diseases such as asthma or the acute respiratory distress syndrome.

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ADHESION, CELL–CELL / Vascular

Proper cadherin expression and function are essential for normal cell segregation during embryonic development and are required for the initiation and maintenance of normal tissue architecture. The extracellular domain has a variable number of homologous repeats of B110 amino acids with putative Ca2 þ binding sequences (cadherin-repeats), while the cytoplasmic tail forms complexes with cytoplasmic proteins of the catenin family (a-catenin, b-catenin, g-catenin/plakoglobin, and p120) that mediate association with the actin cytoskeleton. Endothelial cells principally express two cadherins: vascular endothelial-cadherin (VE-cadherin), a molecule expressed almost exclusively by endothelial cells, as well as the more widely distributed N-cadherin. They appear to be functionally different, with VE-cadherin mediating endothelial cell–cell adhesion, while N-cadherin may promote endothelial cell adhesion to pericytes and smooth cells. Other nonclassical cadherins, such as T-cadherin and VE-cadherin-2 (protocadherin 12), have been described on

Description Families of Adhesion Molecules

A diverse group of protein molecules mediate cell– cell as well as cell–matrix adhesion. These cell adhesion molecules are grouped into four major families: cadherins, immunoglobulin (Ig) superfamily members, selectins, and integrins (Figure 1).

Cadherins Cadherins were initially identified as a family of single-pass, transmembrane proteins responsible for mediating calcium-dependent, homophilic, cell–cell adhesion. These ‘classical cadherins’ are now recognized to be part of a larger cadherin superfamily that also includes the structural and functionally related desmosomal cadherins as well as other subfamilies with cytoplasmic domains that are unrelated to those of the classical cadherins. (Unless otherwise specified, the term cadherin will refer to the classical protein.)

Cadherins

Ig superfamily

Selectins

Integrins

P-selectin

VE-cadherin

E-selectin

PECAM-1 MAdCAM-1

L-selectin JAM-A C C

C C C C C C

C C C C C C C C C






Extracellular repeating elements

lg-like domain

Lectin-like domain




Integrin
subunit

Ca2+ binding sites

Mucin-like domain

EGF-like domain



Integrin  subunit

Transmembrane domain Anchoring domains

Transmembrane and cytoplasmic domains

Cytoplasmic domain

PDZ binding motif

C

Short consensus repeat

Transmembrane and cytoplasmic domains

Transmembrane and cytoplasmic domains

Figure 1 Families of cell adhesion molecules. Ilustrated are structural features that distinguish the members of each of the four major families of cell adhesion molecules. The extracellular domains of cadherins are composed of a variable number of extracellular repeats with putative Ca2 þ binding sites, and conserved sequences in the cytoplasmic domain. Immunoglobulin superfamily members share a common structure in which a variable number of Ig-like homology domains are present in the extracellular domains of the molecule. The presence of other structural motifs such as mucin-like domains contribute to the structural diversity of these receptors. The selectins are composed of an NH2-terminal lectin-like binding domain, followed by an EGF-like motif, a variable number of short consensus repeats, a single-pass transmembrane domain, and a short cytoplasmic tail. Integrins are heterodimeric proteins composed of two noncovalently linked a and b subunits, each with large extracellular domains and smaller but functionally important cytoplasmic tails.

ADHESION, CELL–CELL / Vascular 31

the endothelium, but their functions are currently not known. Immunoglobulin superfamily The Ig superfamily represents a very diverse group of receptors that function in a variety of cell types and in a number of biological processes. Members of this family are defined by the presence of a variable number of Iglike homology domains in the extracellular domains of these molecules. The Ig-like motif represents a sandwich-like structure of two b-sheets each consisting of antiparallel b strands containing 5–10 amino acids. Appropriately positioned cysteine residues mediate the formation of intrachain disulfide bonds and stabilization of each Ig-like domain. The presence of additional structural domains such as mucinlike regions (e.g., mucosal adressin cell adhesion molecule-1 (MAdCAM-1)) contribute to the structural diversity of this family. Several Ig superfamily members, including intercellular cell adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), MAdCAM-1, vascular cell adhesion molecule-1 (VCAM-1), and the junctional adhesion molecules (JAMs), are expressed on the endothelium and play important roles in leukocyte– endothelial adhesion. Ligand binding for these endothelial Ig superfamily molecules may be homophilic (PECAM-1 and the JAMs) or involve interactions with b2 (ICAM-1, JAM-A, and JAM-C) or a4 integrins (MAdCAM-1, JAM-B, and VCAM-1). Selectins The selectins are a small family of receptors found only on vascular-related cells that mediate the initial adhesive interactions of circulating leukocytes with the endothelium at sites of inflammation or in lymphoid tissues. Selectin structure includes an N2-terminal lectin-like binding domain, followed by an epidermal growth factor (EGF)-like motif, a variable number of short consensus repeats, a single-pass transmembrane domain, and a short cytoplasmic tail. Three selectins (P-selectin, E-selectin, and L-selectin) have been identified, each differing in their patterns of expression and regulation. P-selectin is stored in cytoplasmic granules in platelets and endothelial cells, but is rapidly mobilized to the cell surface, where it is transiently expressed (minutes), in response to agonists such as histamine or thrombin. E-selectin is not present under resting conditions, but is synthesized and expressed briefly (hours) on vascular endothelial cells following cytokine stimulation. In contrast, L-selectin is constitutitively expressed on most leukocytes but is proteolytically cleaved from the surface following cellular activation. Selectins recognize sialylated and fucosylated oligosaccharides (e.g., sialyl Lewis x (sLex) and related

tetrasaccharides), as well as sulfated molecules that lack sialic acid or fucose, such as sulfatides and heparin glycosaminoglycans. Thus, in vitro, selectins are able to bind to a variety of glycoproteins on leukocytes and endothelial cells bearing these structural motifs. Several of these are mucin-like glycoproteins with many serine or threonine residues that are potential sites for attachment of O-linked glycans. In vivo, however, P-selectin glycoprotein ligand-1 (PSGL-1) is the dominant ligand for P- and L-selectin in inflammatory settings and may also be physiologically relevant for E-selectin. Recent reports also suggest that for neutrophils, CD44 may be also be a physiological E-selectin ligand. Additional L-selectin ligands have been identified on high endothelial venules (HEV) of lymphatic tissues and on some activated endothelial cells. These include CD34, glycosylated cell adhesion molecule (GlyCAM-1), and podocalyxin. Integrins The integrins are a family of heterodimeric membrane glycoproteins composed of noncovalently associated a and b subunits. To date 18 a and 8 b subunits have been identified and more than 24 a/b pairs have been described in vertebrate tissue. The combination of a and b subunits determines the ligand specificity. Although most integrins bind ligands that are components of the extracellular matrix, certain integrins (b2 and a4 integrins) bind receptors of the immunoglobulin superfamily (e.g., ICAM-1, MAdCAM-1, VCAM-1, and the JAMs). Significant redundancy, however, exists in that most integrins are capable of binding several different matrix or cell surface proteins and many of these ligands bind to multiple integrins. Through their cytoplasmic domains integrins are able to bind to a complex of cytoplasmic proteins, including a-actinin, vinculin, talin, and paxillin, linked to actin filaments. These integrin–cytoskeletal assemblies in turn form the foundation for intracellular signaling cascades. Endothelial Cell–Cell Adhesion

Endothelial cells adhere to one another through two morphologically distinct junctional structures, adherens junctions (AJs) and tight junctions (TJs), each composed of characteristic molecules (Figure 2). Although very similar structures are present in epithelial cells, their distribution within intercellular junction differs in the two cell types. In epithelial junctions TJs are located toward the apical region of the intercellular contact with the AJs positioned below the TJs, while AJs and TJs are intermingled with each other along the endothelial intercellular junction. Gap junctions represent a third type of junctional complex formed by endothelial cells.

32

ADHESION, CELL–CELL / Vascular Apical/luminal surface Junctional complexes

Associated transmembrane proteins

Tight junctions

Occludin, claudins, JAMs

Adherens junctions

Cadherins

Gap junctions

Connexins

Nonjunctional intercellular proteins

PECAM-1, endoglin

Basal lamina Figure 2 Adhesion structures found in epithelial and endothelial cells. Shown are intercellular junctional complexes (TJs and AJs) and their associated transmembrane proteins. Gap junctions mediate intercellular communication and other proteins such as PECAM-1 and endoglin mediate cell adhesion but are not present in specific junctional complexes.

However, unlike AJs and TJs, these structures mediate cell–cell communication rather than cell–cell adhesion. Epithelial cells also express another type of adhesive complex, the desmosone, but these structures are absent from endothelial cells. Adherens junctions These junctions are formed by clusters of dimerized cadherin molecules that bind to cadherin dimers on adjacent cells. Cadherins localize at AJs only when cells contact one another. The short cytoplasmic domain of cadherins interacts through a C-terminal domain with b-catenin and plakoglobin (g-catenin). b-catenin and plakoglobin associate with a-catenin which in turn binds to actin microfilaments. This association with the catenins is required for the adhesive function of cadherins and thus is essential to the assembly of AJs. An additional binding partner is p120, an src substrate that is homologous to b-catenin and plakoglobin. In contrast to b-catenin and plakoglobin, p120 binds loosely to a membrane proximal region of the cadherin cytoplasmic tail, associating with a-catenin or actin cytoskeleton. It appears that p120 is involved in the regulation of the cadherin turnover and stability. The formation of AJs provides the cell–cell attachments that are required for the assembly of the endothelium into patent tubular networks. AJs also contribute to maintenance of endothelial barrier function, in addition to the regulation provided by TJs (see below). Anti-VE-cadherin antibodies, both in vitro and in vivo are found to increase vascular permeability by disrupting VE-cadherin clustering at the junctions while leaving other junctional structures

intact. Further, AJs are required for the organization and/or maintenance of TJs and gap junctions and thus are critical to the integrity of the entire intercellular junctional complex. In addition, at least two other nonadhesive functions have been ascribed to AJs. First, b-catenin, plakoglobin, and p120 are all able to translocate to the nucleus, where in conjunction with other transcription factors they are able to modulate gene expression. Consequently, the association of these catenins with VE-cadherin in AJs may maintain them at the membrane and thus prevent their nuclear translocation. Second, VE-cadherin engagement promotes endothelial cell quiescence by modulating vascular endothelial growth factor (VEGF) receptor-mediated signaling – inhibiting the receptor-dependent proliferative responses, while activating receptor-mediated survival signals. Tight junctions These junctions were originally identified in epithelial cells by electron microscopy as dense regions in which membrane leaflets between the adjacent cells are closely opposed such that the membrane bilayers at the junctions are indistinguishable. In freeze-fracture preparations, TJs have the appearance of fibrillar strands within the membrane. They form a continuous seal around the apical region of the lateral membranes of adjoining cells, subdividing it into apical and basolateral domains. This is somewhat less so for endothelial cells, where the position of TJs relative to other intercellular junctional complexes can vary. Further, the level and extent of TJs depend on the vessel type and location. Endothelial cell TJs are well developed in arteries

ADHESION, CELL–CELL / Vascular 33

and arterioles, but significantly less so in veins and postcapillary venules, the principal site for the extravasation of fluid and inflammatory leukocytes. TJs are also well developed in the vessels of the brain where they contribute to the blood–brain barrier, but are much less organized in the vasculature of other organs. Like other junctional structures, TJs are composed of both transmembrane and intracellular molecules. Three types of TJ-associated integral membrane proteins have been identified. These are occludin, the claudins, and several Ig superfamily members, including the JAMs, endothelial cell-selective adhesion molecule (ESAM), and coxsackie- and adenovirus receptor (CAR). Occludin and the claudins (of which more than 20 have been identified) define the presence of this junction and constitute the molecular basis of the TJ strands, while JAM-A may serve accessory functions such as the recruitment of TJ-associated proteins. The intracellular domains of occludin, the claudins, and the JAMs interact with zonula occludens-1 (ZO-1), a member of a family of membrane-associated, PDZ, and guanylate kinase domain-containing proteins. ZO-1 can bind to actin filaments and thus anchor TJs to the cell’s cytoskeleton. In addition to ZO-1, other PDZ-containing and/or actin binding proteins are recruited that contribute to the formation and function of TJs. Tight junctions restrict both the diffusion of solutes through intercellular spaces (‘barrier function’) and the movement of molecules between the apical and basolateral domains of the plasma membrane (‘fence function’). As such, TJs are the major regulator of cell permeability via the paracellular pathway (i.e., the extracellular space between the lateral membranes of neighboring cells) and are important to the maintenance of cell polarity. TJs are also likely involved in leukocyte transendothelial migration, possibly through interactions mediated by the JAMs. Gap junctions Gap junctions constitute another junctional complex that bridges the intercellular space between adjacent endothelial cells. However, unlike the other junctional structures described above, which mediate cell–cell adhesion, gap junctions promote intercellular communication. Metabolites, ions, and second messengers, including Ca2 þ , cAMP, and inositol triphosphate, are able to pass through gap junction channels from one cell to another, enabling the coordination of multicellular responses. Gap junction channels are dodecameric structures made up of the connexin family of proteins. Six individual connexin proteins oligomerize in the plasma membrane of one cell to form a hemichannel or connexon, and the docking of two connexons, one from each

opposing cell, results in a complete gap junction channel. Other intercellular junction proteins Endothelial cells express several other adhesive proteins that are concentrated in intercellular junctions but are not specifically located in either AJs or TJs. These include (1) PECAM-1, the loss of which compromises vascular barrier function and angiogenesis; (2) S-dndo 1, an Ig superfamily member and a mediator of homophilic adhesion; and (3) endoglin, a regulator of transforming growth factor beta (TGF-b) signaling, the absence of which leads to a phenotype reminiscent of VE-cadherin-null mice. Endothelial Cell–Leukocyte Adhesion

The recruitment of leukocytes to sites of infection or injury begins with a well-defined set of sequential adhesive interactions between the circulating leukocyte and the activated endothelium (Figure 3). A similar cascade of events is believed to also mediate the trafficking of lymphocytes across high endothelial venules in lymphoid tissues. Endothelial activation Endothelial activation is characterized by the enhanced expression of endothelial selections (P- and E-selectin) and members of the Ig superfamiy (ICAM-1, MAdCAM-1, and VCAM-1). This typically involves de novo synthesis, with peak expression occurring over several hours: 4–6 h for E-selectin and 12–24 h for VCAM-1 and ICAM-1. For P-selectin, however, endothelial surface expression occurs within minutes following stimulation with noncytokine mediators such as thrombin and histamine as preformed P-selectin is mobilized from Weibel–Palade bodies. The expression of other molecules such as PECAM-1 and the JAMs that have been implicated in leukocyte trafficking do not appear to be affected by inflammatory mediators. Leukocyte rolling The initial leukocyte–endothelial adhesive event is one in which the circulating leukocyte rolls along the surface of the inflamed endothelium (and on already adherent leukocytes). This process involves an initial capture and fast rolling of the leukocyte, followed by a period of slow rolling and deceleration prior to leukocyte arrest. This step has long been recognized as principally mediated by selectins, with each L- and P-selectin mediating the capture and fast rolling, while E-selectin is preferentially involved in the slow rolling. Interestingly, L-selectin-mediated capture and rolling requires a critical threshold of shear stress to occur, while rolling adhesion mediated P- and E-selectin

34

ADHESION, CELL–CELL / Vascular

Leukocyte rolling

Capture

Fast rolling

Firm adhesion

Diapedesis

Slow rolling

L-selectin P-selectin E-selectin 2 integrins/ICAM-1/2
41/IVCAM-1 or
47/MAdCAM-1 PECAM-1 JAMs CD99 Figure 3 Leukocyte–endothelial interactions. The sequence of adhesive interactions involved in the emigration of leukocytes from the circulation across the endothelium to extravascular sites is illustrated. The steps of these interactions, in order, are leukocyte rolling, firm adhesion, and diapedesis (transendothelial migration). Each step is mediated, depending on the cell type and context, by the interactions of specific cell adhesion molecules and their ligands.

demonstrates weak or no dependence on a shear threshold. The basis for this appears to reside in the fact that L-selectin engages its ligands through exceptionally labile adhesive bonds that are only significantly stabilized above a critical shear threshold. Although originally thought to mediate only firm adhesion and transendothelial migration (see below), b2 integrins are now recognized to cooperate with Eselectin in the deceleration of the rolling leukocyte. Depending on the cell type, there is also evidence that a4b1, a4b7, and CD44 are able to respectively promote rolling on VCAM-1, MAdCAM-1, and hyaluronate-covered surfaces. Activation–firm adhesion As the leukocyte rolls, interactions between selectins and their ligands and between chemokines and chemoattractants associated with the endothelium and G-protein-coupled receptors on the leukocyte surface, activate b2 (and probably a4) integrins on the leukocyte surface. This activation, which involves upregulation of integrin expression and adhesive activity and the shedding of L-selectin, enables the arrest and firm adhesion of the leukocyte to the endothelium. This phase of leukocyte recruitment is mediated by the binding of endothelial Ig superfamily members to (activated)

leukocyte integrins (ICAM-1/aMb2, ICAM-1, -2/a1, b2, VCAM-1/a4b1, and MAdCAM-1/a4b7). Transendothelial migration (diapedesis) The adherent leukocyte then ‘crawls’ over the luminal surface, a process that involves the cyclic modulation of integrin receptor avidity. Once a junction has been located, the emigrating leukocyte squeezes between the closely opposed endothelial cells, crossing the basement membrane to enter the extravascular tissue. Significantly, this occurs without disrupting the integrity of the endothelium. This process of leukocyte transendothelial migration or diapedesis is mediated by at least five cell–cell adhesion molecules: Ig superfamily members PECAM-1 and JAM-A, -B and -C, as well as CD99, a molecule with a unique structure. With the exception of JAM-B, all of these molecules are expressed on both leukocytes and endothelial cells and thus are capable of homophilic adhesion. The JAMs also bind leukocyte integrins, heterophilic interactions that have also been implicated in their activity in leukocyte trafficking. PECAM-1 appears to be required for the initial entrance or penetration of the junction, while CD99 appears to be involved more distally in the passage through the intercellular junction. While traversing the junction, homophilic

ADHESION, CELL–CELL / Vascular 35

interactions of leukocyte and endothelial PECAM-1 also upregulate a6b1 on transmigrating leukocytes, an integrin that is required for the passage of the leukocyte through the matrix of the basement membrane. Although animal studies have implicated the JAMs in leukocyte diapedesis, their specific roles remain to be determined. Pericyte–Endothelial Cell Adhesion

Pericytes are perivascular cells imbedded within the basement membrane of the endothelium of capillaries and postcapillary venules. Contact between the endothelial cell and the pericyte is made by cytoplasmic processes of the pericyte indenting the endothelial cell, and vice versa. This results in the so-called ‘peg and socket’ contact. The normally intervening basal lamina is absent at these pericyte–endothelial cell interdigitations and growth factors (e.g., epidermal growth) factor may concentrate at these contacts. In some tissues, TJs between pericytes and endothelial cells have been reported and ‘adhesion plaques’ have been described in the pericyte membrane. An inverse correlation exists between endothelial cell proliferation and the extent of pericyte coverage, with tissues demonstrating the slowest rate of endothelial cell turnover having the greatest pericyte coverage. This suggests that pericytes are a major negative modulator of capillary growth and hence a promoter of vessel quiescence. This may be mediated by the pericyte-derived TGF-b, an inhibitor of endothelial cell growth and migration, the activation of which is dependent on pericyte–endothelial cell contact. Pericytes are also contractile, and thus points of cell–cell contact may permit contractions of the pericyte to be transmitted to the endothelial cell to reduce the caliber of the vessel or alter vascular permeability.

Vascular Cell–Cell Adhesion in Normal Lung Function Lung Defenses

The recruitment of leukocytes is an essential component of the lung’s defense against infection or inflammatory insults. Although data certainly indicate that the leukocyte–endothelial interactions described above may operate in the lung to regulate the emigration of circulating leukocytes, there is also evidence that leukocyte recruitment in the pulmonary circulation may differ, depending on the stimulus, from that which occurs in postcapillary venules at extrapulmonary sites. Although the b2 integrins have been shown to be required for neutrophil emigration in various models of acute inflammation involving

the systemic vascular bed, including neutrophil recruitment mediated Streptococcus pneumoniae, HCl, and C5a, these stimuli mediate neutrophil extravasation from the pulmonary circulation that is independent of the b2 integrins. An even more complex picture is suggested by studies of mice deficient in both P-selectin and E-selectin or P-selectin and ICAM-1. In these mutant animals, neutrophil emigration into the peritoneal cavity was completely inhibited during S. pneumoniae-induced peritonitis. In contrast, neutrophil extravasation into the alveolar space during S. pneumoniae-induced pneumonia was intact. Together these data suggest that leukocyte recruitment from the pulmonary circulation can occur through pathways that do not require selectins, b2 integrins, or ICAM-1. These observations may be due in part to the fact that primary site for leukocyte extravasation is in the pulmonary capillaries and not in postcapillary venules as occurs in the systemic vasculature. This is significant because the average diameter of the leukocyte (particularly the neutrophil) approaches or exceeds that of the capillaries. As a result, intravascular leukocytes must deform in order to transit through the pulmonary microvasculature and thus are in close apposition with the pulmonary endothelium. Consequently, processes that change the physical properties of the leukocyte or compromise their ability to deform may be sufficient to slow and arrest the leukocyte in the microcirculation of the lung. Thus, it should not be surprising that in the lung there may be less stringent requirements for molecular-meditated processes to capture and attach the intravascular leukocyte to inflamed pulmonary endothelium. Lung Permeability

In the lung, as in other vascular beds, endothelial cell permeability depends largely on the regulation of paracellular fluid and solute transport through intercellular junctions. The degree of ‘openness’ of this pathway is governed by the balance of centrally directed contractile forces and opposing forces generated by cell–cell and cell–matrix adhesive complexes that tether endothelial cells to each other and to the basement membrane. Of the various junctional complexes, TJs are recognized as the primary determinants of the endothelial barrier function. However, disruption of VE-cadherin function results in interstitial edema in the lung and there is evidence that in the setting of hyperosmolarity, E-cadherin expression may be upregulated on the endothelium of the lung microvasculature where it acts to promote barrier function. These data suggest that cadherin-dependent

36

ADHESION, CELL–CELL / Vascular

activity mediated through AJs may also contribute to the regulation of vascular permeability in the lung. Barrier integrity varies with vessel type, as the endothelium of the pulmonary microcirculation is much more restrictive than that of the pulmonary arteries or veins. This suggests that there may well be site-specific differences in junctional architecture and/or cytoskeletal machinery in the pulmonary vasculature.

Vascular Cell–Cell Adhesion in Respiratory Diseases Asthma

Asthma is an inflammatory disease characterized by increased infiltration of various inflammatory cells in the bronchial mucosa and airways. In allergic asthma, antigen exposure results in the activation of mast cells and TH2 lymphocytes and their subsequent release of a number of cytokines and lymphocyte or eosinophil chemoattractants. These inflammatory mediators alter the expression and activity of cell adhesion molecules on these asthma-associated leukocytes and on the bronchial microvasculature in specific patterns that target their recruitment out of the circulation into the bronchial tissues. Analyses of bronchial tissue from allergic and other asthmatics, as well as studies of murine models of asthma indicate that interactions between E-selectin and its ligands, 1CAM-1 and aLb2 and VCAM-1 and a4b1, are involved in the recruitment of eosinophils in the allergic airway response. It should be noted however, that there are reports in which acute antibody inhibition of a4b1, b2 integrin, or ICAM-1 may reduce airway hyperresponsiveness without reducing eosinophil or lymphocyte accumulation. This suggests that these molecules may mediate processes other than leukocyte–endothelial interactions, such as leukocyte activation or antigen presentation, that contribute to the development of the asthmatic phenotype. Acute Lung Injury

A large and diverse group of microbial and inflammatory insults may acutely injure the lung culminating in the acute respiratory distress syndrome (ARDS) and respiratory failure. A key feature of this syndrome is endothelial cell injury and subsequent dysfunction manifested by impaired barrier function and increased vascular permeability. This dysfunction must necessarily involve destabilization of intercellular junctions, but the exact molecular basis is still being defined. A variety of inflammatory agents including histamine, lipopolysaccharide, leukotriene B4, platelet activating factor, nitric oxide thrombin,

as well as cytokines (TNF-a, IL-1, and g-IFN) increase transendothelial permeability, at least in part, by inducing endothelial cell contraction and the resultant formation of interendothelial gaps. These alterations in endothelial morphology require the recruitment and activation of calcium-dependent cytoskeletal proteins (e.g., actin and myosin) that alter endothelial cell contour and reorganize intercellular junctional complexes. The alterations in endothelial permeability induced by inflammatory mediators may involve changes in the concentration of VE-cadherin and PECAM-1 in endothelial intercellular junctions as cytokines have been shown to redistribute these molecules out of endothelial cell–cell contacts with a concomitant increase in permeability. An important contributor to the pathogenesis of endothelial dysfunction in ARDS appears to be activated neutrophils sequestered in and adherent to the pulmonary capillaries. These trapped leukocytes release reactive oxygen species and granular constituents that undermine the normal barrier function of the endothelium. These products may also contribute the inflammatory cascade by activating monocytes and macrophages leading to release of additional proinflammatory mediators. Inhibition of b2 integrins, ICAM-1, and selectins blocks neutrophil recruitment and permeability injury in animal models of acute lung injury or infection although there do appear to be alternative pathways as noted above. This has made these molecules attractive therapeutic targets for the treatment of ARDS. However, the potential for inhibition of host defenses and reparative responses continues currently to be an obstacle to the widespread application of this approach.

Acknowledgments This work was supported by the Department of Defense (PR043482), National Institutes of Health (HL079090), and the Philadelphia Veterans Medical Center. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Cell: Epithelial. Adhesion, Cell– Matrix: Integrins. Asthma: Overview. Endothelial Cells and Endothelium. Leukocytes: Neutrophils; Monocytes; T Cells.

Further Reading Allt G and Lawrenson JG (2001) Pericytes: cell biology and pathology. Cells Tissues Organs 169: 1–11. Angst BD, Marcozzi C, and Magee AI (2001) The cadherin superfamily. Journal of Cell Science 114: 629–641.

ADHESION, CELL–CELL / Epithelial 37 Bazzoni G and Dejana E (2004) Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiological Reviews 84: 869–901. Boitano S, Safdar Z, Welsh DG, Bhattacharya J, and Koval M (2004) Cell–cell interactions in regulating lung function. American Journal Physiology (Lung Cell Molecular Physiology) 287: L455–L459. Dudek SM and Garcia JG (2001) Cytoskeletal regulation of pulmonary vascular permeability. Journal of Applied Physiology 91: 1487–1500. Hogg JC and Doerschuk CM (1995) Leukocyte traffic in the lung. Annual Review of Physiology 57: 97–114. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110: 673–687. Juliano RL (2002) Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members. Annual Review of Pharmacology and Toxicology 42: 283–323. Ley K (2003) The role of selectins in inflammation and disease. Trends in Molecular Medicine 9: 263–268. Mandell KJ and Parkos CA (2005) The JAM family of proteins. Advanced Drug Delivery Reviews 57(6): 857–867. Muller WA (2003) Leukocyte–endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends in Immunology 24: 327–334. Sohl G and Willecke K (2004) Gap junctions and the connexin protein family. Cardiovascular Research 62: 228–232. Vincent PA, Xiao K, Buckley KM, and Kowalczyk AP (2004) VEcadherin: adhesion at arm’s length. American Journal Physiology Cell Physiology 286: C987–C997. Vorbrodt AW and Dobrogowska DH (2003) Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers: electron microscopist’s view. Brain Research Reviews 42: 221–242.

Epithelial J K McGuire, Children’s Hospital and Regional Medical Center, Seattle, WA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract A continuous epithelial sheet lines the respiratory tract to provide a barrier to the outside world and protect against invasion by microorganisms. Central to these functions are the complex junctions that regulate adhesion between adjacent epithelial cells. These junctions create epithelial polarity, regulate paracellular transfer of molecules across the epithelial barrier, and maintain the integrity of the epithelial layer. Far more than just ‘cellular glue’, the major junctional complexes that regulate cell–cell adhesion, i.e., tight junctions, adherens junctions, and desmosomes, are critical in tissue patterning and differentiation during development, epithelial cell sensing of the pericellular environment, and modulation of intercellular signaling. Dozens of different proteins have been associated with cell–cell adhesion functions in mammalian epithelia and only recently have the structure and function of specific proteins expressed at cell–cell junctions in the lung and airways been identified. For example, the claudins are major transmembrane proteins regulating extracellular interaction at tight junctions, and the E-cadherin/b-catenin complexes that form adherens junctions

are critical in cell motility and differentiation and their loss may be associated with epithelial carcinogenesis. However, little is known about how specific changes in cell–cell adhesion contribute to respiratory disease, and this remains an area of intense study.

Description From the upper airway to distal alveoli, an essentially continuous epithelial sheet comprising of several specialized cell types lines the respiratory tract to create a semipermeable barrier to the outside world and provide protection from invading microorganisms. Essential to these functions are the complex structures that produce cell–cell adhesion between individual epithelial cells. Far more than just ‘cellular glue’ holding epithelial cells together, cell–cell adhesion complexes organize development and differentiation of the lung and respiratory tract, promote and maintain structural and functional integrity of the epithelial surface, and regulate cellular responses to injury and disease. Integral to the consideration of cell–cell adhesion in respiratory epithelia are the specific structural and functional characteristics of these cells. Respiratory epithelial cells are polarized, with distinct apical and basolateral domains, with the apical domain facing the external environment or lumen and the basolateral domain forming contacts with the substratum or neighboring cells. In addition to regulating cell shape, polarization contributes to the asymmetric distribution of organelles and molecules within epithelial cells and to the arrangement of cytoskeletal networks. Cell–cell adhesion complexes are key determinants of epithelial cell polarity and are essential in major respiratory epithelial functions such as maintenance of the barrier to the external environment and other critical epithelial functions including secretion and repair. A high degree of similarity exists across different species in the structure of cell–cell junctions and as in most vertebrate epithelia, three major types of junctions mediate intercellular adhesion in respiratory epithelial cells: tight junctions, adherens junctions, and desmosomes. Additionally, all epithelia studied thus far have an adhesive belt called the zonula adherens that encircles the cell just below the apical surface and that is characterized by an electron-dense cytoplasmic actin plaque (Figure 1). Along with the tight junction, which is just apical to the zonula adherens, this adhesive belt delineates the apical and basolateral epithelial compartments and is an important contributor to regulation of epithelial permeability and barrier function. Although the various epithelial cell–cell junctions are composed of unique proteins and serve distinct functions, in general, they

38

ADHESION, CELL–CELL / Epithelial

Tight junction

Adherens junction

Desmosome

Figure 1 Cell–cell junctions in respiratory epithelia. Schematic diagram of the three major types of junctions mediating cell adhesion in epithelial cells. Tight junction transmembrane components occludin (in red above), claudin, and JAM (blue) interact in the extracellular space and cytosolic plaque proteins (green) interact with the actin cytoskeleton (pink). Adherens junctions are formed by transmembrane cadherins (orange) that bind the actin cytoskeleton (pink) via the catenins (yellow and blue). Desmosomes are formed by the desmosomal cadherins (blue and pink) and cytosolic plaque proteins (aqua) that interact with intermediate filaments (green). Electron micrograph of mouse tracheal epithelium shows ultrastructure of zonula adherens near the apical/luminal epithelial surface.

Table 1 Major cell–cell junction proteins Junction

Transmembrane proteins

Cytosolic proteins

Cytoskeletal linker proteins

Tight junction

Occludin Claudins Junctional adhesion molecule (JAM) Cadherins Desmosomal cadherins

Plaque proteins

PDZ domain proteins (zonula occludens, PAR)

b-Catenin Plakoglobin, plakophilins, p0071

a-Catenin Plakins

Adherens junction Desmosome

share common structural features; principal among these are transmembrane receptors, usually glycoproteins that bind proteins on the extracellular surface and determine the specificity of intercellular interactions. The cytosolic domains of these receptors associate with a variety of cytoplasmic proteins that link them structurally to the cytoskeleton and regulate interaction with intracellular signaling pathways (Table 1).

Epithelial Cell–Cell Adhesion in Normal Lung Function Tight Junctions

Early electron microscopic assessment of the contacts between epithelial and endothelial cells identified a

series of apparent fusions between the outer plasma membranes of adjacent cells where the intercellular space disappeared. These initial ultrastructural studies suggested and subsequent studies have confirmed that tight junctions are a central component of the barrier to the paracellular passage of solutes, molecules, and inflammatory cells. Thus, an understanding of tight junction structure is important in considering the regulation of lung barrier function. Over 40 different proteins have been localized to the tight junction in epithelial, endothelial, and myelinated cells that can be further classified as tight junction integral transmembrane proteins and cytoplasmic plaque proteins. The transmembrane proteins include the tetraspan protein occludin and members of the claudin family, which contain four transmembrane domains, two extracellular domains,

ADHESION, CELL–CELL / Epithelial 39

and are oriented with both the N- and C-terminal ends towards the cytoplasm, and the single transmembrane Ig-domain-containing, junctional adhesion molecule (JAM). All of these interact directly with cytoplasmic PDZ domain-containing plaque proteins such as the zonula occludens (ZO) and PAR proteins, which function as adapters to recruit cytoskeletal or signaling molecules. The tetraspan extracellular domains interact in the paracellular space with extracellular domains of tight junction proteins on neighboring cells and occludin, the first integral membrane protein to be found in tight junctions of many cell types, also directly binds F-actin via the last 150 amino acids of its carboxyl tail. The ‘tightness’ of tight junctions appears to be regulated by changes in the combination and expression ratios of different claudin family members, which can engage in homotypic or heterotypic interactions between several of the 24 distinct claudin gene products thus far identified in humans. One defined role of claudins in regulating permeability is the formation of ionselective pores, and different types of claudins appear to determine the specificity of the pores for individual ions such as Na þ , Cl
, or Ca2 þ . Whereas tight junctions can form in the absence of occludin, claudins appear to be essential for tight junction formation. It is likely that JAM has an important role in tight junction assembly and resealing after disassembly in epithelia by engaging in homophilic interactions in the paracellular space. Tight junction plaque proteins have important roles in coordinating intracellular signaling by recruiting cytosolic proteins to the tight junction during assembly, and recent reports of ZO proteins localizing to the nucleus, in addition to their tetraspanbinding at tight junction plaques, suggest that they transduce signals from the cell membrane to the nucleus where they may regulate gene expression. Recruitment of plaque proteins to the tight junction may also have a role in regulating cell motility and proliferation. Consequently, although tight junctions perform key functions in regulating epithelial permeability, it is becoming increasingly clear that tight junctions are multifunctional complexes involved in many vital epithelial cell functions. Only recently have specific patterns of tight junction protein expression in the respiratory tract been characterized, and much work remains to further define how tight junctions are regulated in normal and abnormal lung function. Adherens Junctions

Adherens junctions are ancient structures present across eukaryotes and even in related structures in

single-celled organisms such as yeast, and as such, adherens junctions have critical functions in coordinating cell polarity, cytoskeletal dynamics, cell sorting, and differentiation. In addition to regulating organization within epithelia, adherens junctions are also important in transmitting information from the environment to the interior of cells. At the core of adherens junctions are the calcium-dependent transmembrane glycoprotein cadherins, and the major epithelial cadherin is E-cadherin, the best characterized member of the cadherin family and the primary cadherin expressed in respiratory epithelium. The extracellular domain of E-cadherin, like other classical cadherins, is comprised of five calcium-binding cadherin repeats that participate in homotypic interactions with neighboring cells. There is a single-pass transmembrane domain and the cadherin cytosolic domain is linked to cytoskeletal structures via the catenins. Cadherin adhesive activity is regulated in several ways including the level of cadherin gene expression, which correlates with the strength of adhesion, and the type of cadherin expressed, which affects the specificity of cell interaction and plays a role in embryo patterning and determination of cell fate. Posttranscriptional mechanisms regulating cadherin adhesion include modulation of cadherin clustering at the cell surface and changes in cadherin interaction with the catenins. Proteolysis of cadherin domains is now recognized as an important means of posttranslational cadherin modification with extracellular domains being cleaved by metalloproteinases, the transmembrane domains targeted by g-secretases, and intracellular domains degraded by caspases. Cadherins are key mediators of morphogenesis, epithelial sheet integrity, growth control, and maintenance of the terminally differentiated phenotype. Differential expression of different cadherin classes is likely to be important in cell sorting and embryo patterning in development and specific cadherins may be associated with the determination of cell fate. For example, E-cadherin is the primary cadherin expressed in lung epithelia, whereas N-cadherin is expressed at cell– cell junctions in pleural mesothelial cells. The cytoplasmic E-cadherin domain binds the armadillo family member protein b-catenin, and this binding induces a tertiary structure that in turn binds a-catenin, which nucleates the assembly of a multimeric complex that links E-cadherin/b-catenin complexes to the actin cytoskeleton. This process both stabilizes the intercellular junctions and allows for coordination of cytoskeletal dynamics with changes in cell–cell adhesion. These protein interactions are regulated by phosphorylation events of both the E-cadherin cytoplasmic tail (increase interaction) and

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b-catenin (decrease interaction) and by the recruitment of various other potential adherens junctionassociated proteins. Therefore, this complexity allows for many levels of regulation of adherens junctionbased cell adhesion. In addition to its structural role in adherens junctions, b-catenin also functions as a noncadherin-dependent signaling factor. In concert with activation of the Wnt signaling pathway, free cytoplasmic b-catenin translocates to the nucleus and binds to transcription factors of the lymphocyte enhancer-binding factor-1/T-cell factor (LEF-TCF) pathway to regulate expression of downstream target genes associated with cell migration and proliferation including matrilysin (MMP-7), c-myc, WISP, and cyclin D1. A comprehensive consideration of b-catenin in cell signaling is beyond the focus of this discussion of cell adhesion, but recent studies have suggested a role for b-catenin in terminal differentiation of alveolar epithelium. Adherens junctions at the cell surface associate with several other types of intercellular junctions and membrane receptors. Ultrastructural studies of epithelial sheets reveal closely apposed membranes at sites of cell–cell contact where adherens junctions alternate with desmosomes, and formation of adherens junctions has been shown to be necessary for desmosome and tight junction formation. Adherens junction-specific cadherins also associate with other cell surface receptors that participate in signaling such as connexins, Notch, receptor tyrosine kinases, and phosphatases, suggesting that adherens junctions can be regulated by extracellular cues. Desmosomes

Desmosomes are specialized junctions that anchor stress-bearing intermediate filaments at sites of strong intercellular adhesion. This provides a scaffold that gives integrity to tissues such as skin, heart, and respiratory epithelium that is subject to mechanical stress. At the core of desmosomes are proteins of the cadherin (the desmogleins and desmocollins) and armadillo (plakoglobin, the plakophilins, and p0071) families, which form membrane-associated complexes that are tethered to intermediate filaments via the plakins. Analogous to adherens junction cadherins, the single-pass transmembrane desmosomal cadherin extracellular domains interact in the extracellular space; however, in contrast to adherens junction cadherins, desmosomal cadherins interact in a heterotypic manner as each desmosome must contain at least one desmoglein and one desmocollin. Desmocollins and desmogleins are expressed in a tissueand differentiation-specific manner, and thus it has been proposed that desmosomal cadherins may

function in directing epithelial differentiation. The cytoplasmic cadherin tails directly bind an armadillo protein, usually plakoglobin (g-catenin), and this complex in turn associates with a cytoskeletal linking protein, e.g., desmoplakin, that mediates attachment to the intermediate filaments. The result is a series of connections between adjacent cells that maintains tissue tensile strength. Similarities exist between the regulation of adherens junction and desmosomal adhesion; as for adherens junctions, desmosomal adhesion during development is regulated by the timing of and type of desmosomal cadherin or armadillo protein expressed, and as discussed above, desmosome assembly appears to be downstream of and dependent on adherens junction assembly. Posttranslational mechanisms regulating desmosomal adhesion include proteolytic processing of intracellular cadherin domains by caspases and cleavage of extracellular domains by metalloproteinases. Although desmosomes are present throughout the airway and alveolar epithelium, little is known about the specific regulation of desmosome-based cell adhesion in the respiratory tract.

Cell–Cell Adhesion in Respiratory Diseases Although the net permeability of the lung and airways is jointly regulated by the epithelium and endothelium, disruption of paracellular permeability and leakage of fluid and proteins across the airway or alveolar epithelium are hallmarks of a variety of respiratory diseases including asthma, allergic rhinitis, acute lung injury, and pneumonia among others. Furthermore, pathogens may directly target junctional proteins to facilitate entry into the host. However, the specific mechanisms by which changes in tight and adherens junctions and the proteins that regulate these junctions in diseases of the airways and lung remain largely undefined. It has been known for a few years that some types of lung cancer, like other carcinomas, show altered staining patterns for several adherens junction proteins, and an increasing amount of evidence suggests that inactivation of E-cadherincatenin complexes plays a significant role in carcinogenesis. Additionally, altered b-catenin signaling has been implicated in idiopathic pulmonary fibrosis. Thus, these pathways may be attractive targets for therapy in these historically difficult to treat diseases. The available knowledge on the structure, assembly, and function of intercellular junctions in normal and abnormal processes is rapidly increasing. These complexes clearly have more than a structural role and perform critical functions in tissue development,

ADHESION, CELL–MATRIX / Focal Contacts and Signaling 41

homeostasis, and response to injury. However, our understanding of the regulation of epithelial cell–cell adhesion in respiratory tract development and maintenance of normal lung function, and in injury and disease remains limited. Thus, this remains an area of intense investigation. See also: Adhesion, Cell–Cell: Vascular. Asthma: Overview. Basal Cells. Bronchiectasis. Bronchiolitis. Connexins, Tissue Expression. Contractile Proteins. Defense Systems. Epithelial Cells: Type I Cells; Type II Cells. Gene Regulation. Lung Development: Overview. Matrix Metalloproteinases. Panbronchiolitis. Signal Transduction. Transcription Factors: Overview. Tumors, Malignant: Overview; Metastases from Lung Cancer.

Further Reading Braga VMM (2002) Cell–cell adhesion and signalling. Current Opinion in Cell Biology 14: 546–556.

Bremnes RM, Veve R, Hirsch FR, and Franklin WA (2002) The E-cadherin cell–cell adhesion complex and lung cancer invasion, metastasis, and prognosis. Lung Cancer 36: 115–124. Godfrey RWA (1997) Human airway epithelial tight junctions. Microscopy Research and Technique 38: 488–499. Gonzalez-Mariscal L, Betanzos A, Nava P, and Jaramillo BE (2003) Tight junction proteins. Progress in Biophysics and Molecular Biology 81: 1–44. Perez-Moreno M, Jamora C, and Fuchs E (2003) Sticky business: orchestrating cellular signals at adherens junctions. Cell 112: 535–548. Schneeberger EE and Lynch RD (2004) The tight junction: a multifunctional complex. American Journal of Physiology. Cell Physiology 286: C1213–C1228. Wheelock MJ and Johnson KR (2003) Cadherins as modulators of cellular phenotype. Annual Review of Cell and Developmental Biology 19: 207–235. Yin T and Green KJ (2004) Regulation of desmosome assembly and adhesion. Seminars in Cell and Developmental Biology 15: 665–677. Zhurinsky J, Shtutman M, and Ben-Ze’ev A (2000) Plakoglobin and b-catenin: protein interactions, regulation and biological roles. Journal of Cell Science 113: 3127–3139.

ADHESION, CELL–MATRIX Contents

Focal Contacts and Signaling Integrins

Focal Contacts and Signaling G D Rosen and D S Dube, Stanford University Medical Center, Stanford, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Focal adhesions (FAs) are contact points for the cell with the extracellular matrix. These complex structures regulate communication of the cell with the surrounding extracellular environment and signaling through these FAs regulates diverse cellular processes, including proliferation, migration, apoptosis, spreading, and differentiation. The principal components of the FAs are integrins, which are ab heterodimers that regulate cell– matrix and cell–cell interactions. Focal adhesion kinase (FAK) is a kinase that localizes to FAs and regulates signaling in FAs. FAK communicates signals between integrins and intracellular proteins, which regulate diverse cellular processes such as cell polarity, migration, and invasion. FAs play a central role in regulating normal developmental processes such as blood vessel morphogenesis but increased activity of FA can incite aberrant events such as tumor cell invasion and pulmonary fibrosis. A further exploration of the role of FA in health and disease will provide greater insight into the regulation of normal developmental processes and into the pathogenesis of lung diseases such

as lung cancer, acute respiratory distress syndrome, and pulmonary fibrosis.

Introduction This article explores the physiology of focal contacts, and cell signaling in normal and pathological states. Focal adhesions (FAs) are specialized multimolecular structures that exist at the points of contact between the cell membrane and the extracellular matrix. Our current understanding of the physiological roles of FAs suggests that in addition to mediating cell adhesion, they also function as a physiological signaling link between the cytoplasm and the extracellular milieu by promoting the bidirectional transmission of biochemical signals. Integrins are the core components of FAs whose orderly function underlies the diverse roles associated with FAs. Integrins are a family of a/b heterodimeric glycoprotein receptors that span the cell membrane. Morphologically, these receptors exhibit an intracellular domain, a transmembrane domain, and an ectodomain that is in association with the cell matrix or other cells. The

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intracytoplasmic domains of integrins can associate with actin, adaptor proteins, and enzymatic components of signaling cascades and their substrates. This complex association of the cell matrix, integrins, and cytosolic proteins forms the molecular conglomerates that are termed FAs and mediates the bidirectional flow of signals between the cell matrix and the cell. The binding of ligands to integrins in either the extracellular or intracellular domains induces conformational changes within the integrins that affect the binding of ligands on the reciprocal end of the integrin molecule. The activation of FAs by paracrine and endocrine stimuli culminates in the activation of specific intracytoplasmic signaling cascades, including tyrosine phosphorylation, focal adhesion kinase (FAK)-mediated signaling, mitogen-activated protein-kinase (MAPK), and the generation of phosphatidylinositol-4, 5-bisphosphate. These intracellular signals result in a multitude of cytoplasmic responses that regulate, for example, cell proliferation, motility, and differentiation. Based on the multiplicity of functions attributed to FAs, the strict regulation of their function forms the basis of orderly organ structure and tissue function. A better understanding of the mechanisms that are involved in the regulation of FAs has led to the development of potent antithrombosis and anti-inflammatory therapies. Moreover, the multiple roles of FAs further promise to aid the elucidation of diverse pathological processes associated with FAs. Some of these processes include tumor growth, pathological organ remodeling, cell barrier defects, and embryogenesis as well as many other pathological entities that are regulated by cell–matrix and cell–cell contact.

Focal Contacts: Structure and Function Interference reflection microscopy first identified FAs as dark areas at the interface between the ventral cell surface and the extracellular matrix. Diverse types of focal adhesions have since been described. Subsequently, advances in gene cloning have led to the determination of the primary structures of many of the constituents of FAs. Early analysis of FA structure identified integrins as core glycoproteins intimately associated with the structure of FAs. The elucidation of the primary structure of integrins revealed three domains: an extracellular domain, a transmembrane domain, and an intracellular domain as well as sites for tyrosine phosphorylation. In parallel with the elucidation of the primary structure, the solution of the crystal structures of the components of FAs further advanced testable hypotheses on structure– function relationships of the individual protein components of FAs. Studies of the conformational

rearrangements within integrin molecules using diverse tools such as negative stain microscopy, nuclear magnetic resonance, and spectroscopy have further aided the understanding of the function of focal adhesion molecules. These studies have allowed the evaluation of conformational changes within integrins using ligand analogs that bind to ligand-induced binding sites (LIBSs). Site-directed mutagenesis has also aided the characterization of the roles of submolecular components of protein components of FAs, while in vivo studies of these mutagenesis studies have led to the discovery of unique phenotypes, thus further allowing the determination of the functional roles of individual components of FAs. Most recently, the use of fluorescent green protein (FGP) tagged proteins has allowed the determination of stoichiometric relationships of the various components of FAs.

Components of Focal Adhesions: Integrins and Extracellular Matrix Integrins are a large family of heterodimeric transmembrane glycoprotein receptors uniquely found among metazoans whose quaternary structure is characterized by a noncovalent union between a- and b-subunits. There are 18 a-subunits (MW 120– 180 kDa) and 8 b-subunits (MW 90–110 kDa) that assemble in various permutations to generate 24 distinct integrin heterodimers. These various subunit combinations expand the repertoire of intracellular and extracellular ligands for integrins. In turn, the expanded receptor capability created by the many combinations of a- and b-subunits accounts for the diverse functional roles of integrins. Integrins are central to the structure of FAs. The significance of integrins in metazoan cell structure and tissue organization is emphasized by the observation that the number of integrins parallels the evolutionary complexity of an organism. It has been proposed that the evolution of metazoans as multicellular organisms hinged upon the parallel evolution of integrins as anchor apparatus to the cell matrix and intercellular molecular adhesives. Figure 1 shows the subclassification of the integrin receptor family. Integrins have a large extracellular domain, a transmembrane domain, and an intracytoplasmic domain. The ectodomains of integrins exist in three major conformational states. The first of these states is a bent conformation that is similar to the crystal structure of avb3. This conformation is considered to be a low-affinity state. The second conformation has been referred to as a ‘closed head piece’ conformation and is considered to represent an intermediate affinity conformational state. Finally, there is the

ADHESION, CELL–MATRIX / Focal Contacts and Signaling 43


ΙΙb

Collagen receptors
1

3∗

1∗


9


8 8

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V 6

Leukocyte-specific receptors


10


5

5

RGD receptors


2


D


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M


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Figure 1 The integrin receptor family. Integrins are ab heterodimers that span the cell membrane. This figure shows the mammalian subunits and their ab associations. Asterisks denote alternatively spliced cytoplasmic domains. Reproduced from Hynes RD (2002) Integrins: bi-directional, allosteric signaling machines. Cell 110: 673–687, with permission from Elsevier.

high-affinity ‘open head piece’ conformation, which is induced by ligand binding. The high-affinity conformation is thought to be acquired by the extension of the angle between the b- and hybrid domains. A detailed discussion of the crystal structures of the various integrins and their interacting partners is outside the scope of this article. The evidence so far supports a model whereby the binding of a ligand to the ectodomain mediates the extension and simultaneous separation of the a- and b-subunits of the integrin heterodimers within the transmembrane and intracytoplasmic domains. This model also suggests that the binding of a ligand in the intracellular domain of integrins by adaptor proteins such as talin also mediates counter conformational alterations that cause the exposure of LIBS in the ectodomain. It has further been proposed that these conformational changes are dynamic and dependent on regulatory factors such as growth factors.

Regulation of Integrins Intracytoplasmic Association between the a- and b-Chains

A considerable body of experimental data reveals that the interaction between the a- and b-subunits in the cytoplasm regulates the activation states of the integrin receptors. Studies utilizing the platelet integrin aIIbb3, for example, have revealed that the aIIb short intracytoplasmic domain negatively regulates the activation of the integrin receptors. The experimental deletion of the intracytoplasmic aIIb domain or the conserved GFFKR sequence leads to

constitutively activated integrin receptors. Similarly, deletions of conserved intracytoplasmic b3 sequences also culminate in constitutively activated integrin receptors. These observations have led to the conclusion that the interaction between these two cytoplasmic domains negatively controls the activation of integrins. This interaction seems to occur through a salt bridge between the R995 of the aIIb and the D723 in the b3. In support of this idea, the experimental mutation of either amino acid to the charge neutral alanine leads to a constitutively active integrin. Nuclear magnetic resonance (NMR) studies have demonstrated that the intracytoplasmic portions of the a- and b-domains interact with each other. Furthermore, it has been shown that point mutations in F992A or R995D impair the interaction of the a- and b-domains. The Role of Talin in Integrin Activation

Studies support the conclusion that talin, a cytoskeletal actin-binding protein that co-localizes with active forms of integrins, is a crucial element in the activation of integrins. Talin is a homodimer consisting of two antiparallel subunits, each approximately 270 kDa. Each of the talin subunits is comprised of a 220 kDa C-terminal and a 70 kDa globular head, which is thought to contain the binding sites for integrins. Talin has been demonstrated to bind the b1D, b2, b3, b5, and b7 intracytoplasmic integrins at their N-terminus. Structural analysis of the PTB-like 96 amino acid subdomain of the talin FERM (4.1, ezrin, radixin, moesin) domain shows a major integrin-binding site. Besides the ubiquitous interaction

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of talin with diverse integrins, the overexpression of a talin fragment containing the head domain leads to the activation of integrin molecules. The role of talin in integrin activation is further supported by the observation that talin knockdown abrogates aIIb3 activation by endogenous agonists. This observation suggests that talin is a critical common downstream target in the activation of at least some integrins. Molecular mechanisms of the role of talin in the regulation of integrins are complex and still being explored. Vonogradova and colleagues have demonstrated that the talin head domain alters the NMR spectrum for the interaction between the b3 and aIIb domains. Other investigators have shown that the talin head domain attenuates the fluorescence resonance energy transfer (FRET) between fluorophoretagged a- and b-integrins within viable cells. These observations support a model whereby the binding of the talin phosphotyrosine-binding (PTB) domain to integrin disrupts the inhibitory interaction between a- and b-membrane proximal domains.

Regulation of Talin–Integrin Interactions The preceding section considers that the interaction of talin with intracytoplasmic domains of integrin is pivotal to the regulation of integrin function and hence FAs. A variety of mechanisms have been advanced to account for how these interactions are regulated. The talin head has a sixfold higher affinity in comparison to the intact talin molecule. This observation has led to the hypothesis that in the whole talin molecule, integrin binding sites are obscured. Therefore, a potential mechanism whereby talin regulates integrin activation is through the modulation of its conformations by physiological factors, which then reveal or mask integrin binding sites. Evidence supporting this possibility accrues from observations made from the function of the ERM (ezrin, radixin, moesin) family of proteins. The major integrin-binding site in talin lies within the talin FERM domain, and binding occurs via a variant of the classical PTB domain–NPxY interaction. In the ERM family, the intramolecular interaction of the N-terminal FERM domain with the C-terminal obscures ligandbinding sites. Intriguingly, ERM phosphorylation, PIP2 (phosphatidyl inositol 4,5-bis-phosphate), and rho-dependent pathways abrogate the native inhibition in the unstimulated ERM molecules presumably by unmasking the interaction of the FERM domain. Although the role of phosphorylation in the activation of integrins is still debated, it is notable that the binding of PIP2 to talin has been shown to mediate a conformational change within the talin molecule that culminates in the exposure of integrin-binding

sites in the FERM domain. The role of PIP in the regulation of talin is complicated by the observation that talin directly stimulates a splice variant of PIP2producing enzyme PIPKIg-90 (phosphatidylinositol phosphate kinase type Ig-90) thereby increasing PIP2 production. Interestingly, PIPKIg-90 and integrin b tails have overlapping binding sites on the talin F3 subdomain. This suggests a novel control strategy whereby PIPKIg-90 and talin may displace each other from integrin-binding sites following activation. Another regulatory mechanism for talin–integrin interaction is thought to involve calpain-mediated proteolysis of integrin b tails at sites straddling the NPXY/NXXY motifs that are necessary for the anchorage of integrins to the cytoskeleton and FAs. Calpains are calcium-dependent thiol proteases that have a diverse substrate spectrum that includes constituents of the cytoskeletal apparatus and cell-signaling proteins. Structurally, they are composed of an 80 kDa catalytic subunit and a 30 kDa regulatory subunit. Although calpain was historically considered to be involved in the detachment of migrating cells, accumulating evidence suggests that calpain also functions by regulating actin remodeling during cell migration. Supportive data have been obtained through experimental overexpression of calpain in aortic endothelial cells. These experiments reveal that overexpression of calpain leads to formation of focal adhesions. In contrast, when calpain is inhibited in fibroblasts and endothelial cells, adherence is maintained while cell dispersion is attenuated. Additional evidence points to the fact that the regulation of actin filament formation in endothelial cells is dependent on calpain-mediated proteolysis. The capacity of calpain to cleave integrins with diverse molecular structures suggests that calpains recognize higherorder protein structure rather than primary protein sequence. Calpains also cleave tyrosine kinases (PTK) such as focal adhesion kinase (FAK) and src and phosphatases (PTP) PTP-1B, Shp-1, and PTP-MEG. Other investigators have reported that calpain cleaves the N-terminal 47 kDa of talin, which is known to be hyperphosphorylated by protein kinase A.

Focal Adhesion Structure and Signaling FAs exhibit dynamic spatial–temporal variations in function. A large group of proteins are core to the structural behavior of focal adhesions. This is in keeping with the diversity of roles that are associated with FAs. The structural integrity of FA molecules relies on adaptor molecules that perform core scaffolding functions. Multiple other molecules also interact with the FAs and perform both structural and enzymatic chores of FAs. The overall function

ADHESION, CELL–MATRIX / Focal Contacts and Signaling 45

and net effect of these scaffolds is to juxtapose kinases with their substrates and thereby facilitate FA-mediated signaling. The full repertoire of proteins that have been observed to interact with FAs is large and continuously expanding. In general, the relative expression or activation of different scaffolding proteins depends on the tissue of origin and cellular stimulus. Therefore, scaffolding proteins not only confer on FA structural integrity but are also responsible for the functional diversity associated with FAs. A core component of the FA complex is the p130Cas protein. The structure of p130Cas includes an SH3 domain, a proline-rich motif, and a very complex substrate-binding region that has the Src-SH2 binding region. The p130Cas SH3 domain interacts with the FAK possibly through interactions with the YDYV motif. This same YDYV motif also binds c-Src. It is thought that c-Src mediates the phosphorylation of the substrate domain of p130Cas. It is intriguing to also note that FAK has the capability to phosphorylate an Src binding site within p130Cas. The phosphorylation of the substrate-binding site within p130Cas achieves two major roles. First it allows the localization of p130Cas to the FA and second it allows p130Cas to interact with other proteins containing the SH2 domain such as protein tyrosine phosphatase (PTP)-PEST, NcK, and CrK. Crk/CrkL is an adaptor protein that interacts with phosphorylated p130Cas via an SH2 domain. The interaction of Crk with P130Cas leads to the formation of a complex that activates Dock 180, which is a novel exchange factor. Ultimately, the binding of Dock 180 to Crk and ELMO culminates in the activation of Rac, which augments the activation of p130Cas phosphorylation. In general, the association of p130Cas with Crk activates GTPases that are involved in the maintenance and reorganization of the actin cytoskeleton, which are important for cell motility and are crucial to establishing cellular structure. Paxillin is another adaptor molecule that interacts with a diverse range of proteins such as FAK, PKL, PTP-PEST, and b1 and a4 integrin intracytoplasmic tails. Similar to P130Cas, paxillin also complexes with a wide range of distinct exchange factors in a phosphorylation-dependent and tissue-specific manner. In summary, scaffold proteins bring into close proximity protein moieties important for the function of the FAs. The functional diversity conferred by the combinations afforded through these proteins results in tissue-specific function. Kinases

Tyrosine phosphorylation of focal adhesion components is considered to be the core event in the

generation of focal adhesion-mediated cell signaling. Tyrosine phosphorylation creates sites for binding of Src-homology domain (SH2-containing proteins). Moreover, tyrosine phosphorylation directly regulates a variety of kinases and phosphatases. The principal kinases that regulate focal adhesions are FAK and Src. Multiple other kinases (ser/thr kinases, Abl, PYK2,Csk) also probably play a role but their precise role in regulation of focal adhesions has not been fully elucidated.

Focal adhesion kinase FAK is a 125 kDa protein kinase that was initially shown to localize to FAs. Moreover, it was observed to be phosphorylated in relation to Src-mediated transformation. FAK is an FERM domain protein and is known to bind to many different signaling proteins. In general, it is considered that phosphorylation of FAK is the sentinel event in FA-mediated cell signaling. In response to integrin association with a ligand, FAK is activated by autophosphorylation at tyrosine Y397. One model proposes that subsequent to this autophosphorylation, Y397 acts as a docking site for Src that in turn mediates the phosphorylation of FAK at Y576 and Y577 and potentially at other sites. The phosphorylation of these tyrosines creates molecular contact points for proteins containing SH2 domains. As a result of this, the Ras-regulated MAPK pathway is activated (Figure 2). In support of this model, it has been observed that phosphorylation of Y926 in FAK creates an interaction site (pYNQV) for the SH2 domain of Grb2, an adapter protein that regulates growth factor signaling through Ras/SOS through binding of its SH2 and SH3 domains. This event is considered to be critical for the activation of the MAPK pathway. Grb2 can be phosphorylated at the Y926 position only when FAK is dissociated from the focal adhesion complex. This observation suggests that the phosphorylation of FAK causes it to dissociate from the FA complex and then phosphorylates its target proteins and in turn initiates signaling cascades. The interaction of FAK with FAs occurs through the FA-targeting domain (FAT) of FAK. Indeed, when the FAT domain in FAK is mutated, the FAK molecules are incapable of autophosphorylation and cannot phosphorylate known endogenous substrates. Although the role of FAK as a regulatory kinase is widely accepted, it is intriguing that FAK molecules lacking the kinase domain are still functional in some cell-signaling pathways. This has led to the proposal that FAK may predominantly perform a structural role by acting as a scaffold for diverse constituents of FAs. However, these same data have been interpreted

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F

K

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 Integrin

Graf SH3

Paxillin

COOH Pro Pro Y925 712–715 878–881 –P

Pro Y397 371–374 –P SH3-SH2 Src/Fyn

SH2 PI3 kinase

SH2 PI3 kinase

SH2 GRB-2

Paxillin Grb2 Spreading

Sow/Ras Erk (MAPK)

p130Cas Crk/Nek?

Akt?

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Proliferation Migration

Figure 2 Signaling through focal adhesion kinase (FAK). Association of FAK with integrins activates signaling pathways that regulate cell proliferation, apoptosis, spreading, and migration. Reproduced from Laboratory of Molecular Biophysics http://biop.ox.ac.uk, with permission.

to imply that other kinases have the ability to duplicate the kinase functions of FAK. Src tyrosine kinase The c-Src tyrosine kinase performs a pivotal role in the regulation of the function of FAs and the cytoskeleton. In transformed cells, vSrc is localized at three distinct locations: perinuclear, the actin cytoskeleton, and focal adhesions. Studies utilizing vSrc mutants in LA 29 rat fibroblasts, which express a temperature-sensitive (ts) v-src mutant, with D1025 rat fibroblasts, transfected with a ts mutant of v-fps, have demonstrated that at restrictive temperatures vSrc is inactive and localizes to the perinuclear regions where it is intimately associated with the microtubular cytoskeleton. In contrast, at more permissive temperatures v-Src localizes to focal adhesions at the cell periphery where it interacts with RhoA-induced actin fibers. An interesting observation is that targeting of v-Src is independent of its own tyrosine kinase function; rather it depends on its SH3 domain. However, this localization is dependent on the binding of the p85 regulatory subunit of phosphatidylinositol kinase (P13 K) to the v-Src SH3 domain as well as on P13 K kinase function. Although not completely analogous to v-Src, overexpressed c-Src in fibroblasts localizes to a perinuclear location and associates with microtubules and organelles such as endosomes and the trans-Golgi network. The localization of c-Src is dependent on

GTPse activity, which directs c-Src to diverse cellular locations. The extrapolation of the function of c-Src from the observations of v-Src is limited by the observation that c-Src does not associate with p85 of p13 K. Thus, the role of c-Src still needs to be fully defined.

Focal Adhesion Molecules in Respiratory Disease A characterization of the role of focal adhesion kinases in normal cellular function and in disease is an area of active investigation. Focal adhesion molecules such as integrins and FAK play key roles in the regulation of cell–cell communications, structure, and remodeling during development of the lung and other tissues. Perturbations of the tightly controlled functions of focal adhesions have been shown to affect tumorigenesis and metastasis but the role of focal adhesions in diseases of the lung and other organs is less well defined. However, studies have begun to reveal a role for focal adhesions in cellular function and disease states. For example, focal adhesions have been shown to be important in lymphocyte homing to sites of inflammation as b7 integrins have been shown to be crucial in the localization of lymphocytes to sites of inflammation in the gastrointestinal tract. Focal adhesions are also important

ADHESION, CELL–MATRIX / Integrins 47

in hemostasis as GII3b integrins are crucial in platelet aggregation and their blockade has given rise to a potent class of antagonists that are used to prevent platelet aggregation following interventional procedures in cardiology. Also, FAK is required for blood vessel morphogenesis and migration of vascular smooth muscle cells. FAK has been shown to be required for microtubule organization, nuclear movement, and neuronal migration during brain development and to be a negative regulator of axonal branching and synapse formation. In the acute respiratory distress syndrome, perturbation of the cell surface barrier is thought to be mediated through impaired focal adhesion function thus leading to increased permeability. Additionally, TGF-b-induced activation of FAK has been shown to mediate the TGF-b-mediated suppression of apoptosis in lung fibroblasts, which likely plays a role in the pathogenesis of idiopathic pulmonary fibrosis. We among others have shown that FAK is cleaved during apoptosis and a recent study revealed that FAK interacts with p53 and inhibits p53-mediated apoptosis. Further studies will elucidate the precise role for focal adhesions in normal cellular function and in disease. See also: Adhesion, Cell–Cell: Vascular ; Epithelial. Adhesion, Cell–Matrix: Integrins. Apoptosis. Cell Cycle and Cell-Cycle Checkpoints. Cytoskeletal Proteins. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils. Platelets.

Further Reading Calderwood DA (2004) Integrin activation. Journal of Cell Science 117(5): 657–666. Calderwood DA (2004) Talin controls integrin activation. Biochemical Society Transactions 32(3): 434–437. (Review: PMID: 15157154: PubMed – indexed for MEDLINE.) Calvete JJ (2004) Structures of integrin domains and concerted conformational changes in the bidirectional signaling mechanism of alphaIIbbeta3. Experimental Biology and Medicine (Maywood) 229(8): 732–744. Diagne I, Hall SM, Kogaki S, Kielty CM, and Haworth SG (2003) Paxillin-associated focal adhesion involvement in perinatal pulmonary arterial remodelling. Matrix Biology 22(2): 193–205. Frame MC, Fincham VJ, Carragher NO, and Wyke JA (2002) vSrc’s hold over actin and cell adhesions. Nature Reviews: Molecular Cell Biology 3(4): 233–245. Hynes RD (2002) Integrins: bi-directional, allosteric signaling machines. Cell 110: 673–687. Kanda S, Miyata Y, and Kanetake H (2004) Role of focal adhesion formation in migration and morphogenesis of endothelial cells. Cellular Signalling 16(11): 1273–1281.

Mould AP and Humphries MJ (2004) Cell biology: adhesion articulated. Nature 432(7013): 27–28. Petit V and Thiery JP (2000) Focal adhesions: structure and dynamics. Biology of the Cell 92(7): 477–494. Schlaepfer DD, Mitra SK, and Ilic D (2004) Control of motile and invasive cell phenotypes by focal adhesion kinase. Biochimica et Biophysica Acta 1692(2–3): 77–102. Schoenwaelder SM and Burridge K (1999) Bidirectional signaling between the cytoskeleton and integrins. Current Opinion in Cell Biology 11(2): 274–286. Takagi J, Petre BM, Walz T, and Springer TA (2002) Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110(5): 599–611. Vinogradova O, Velyvis A, Velyviene A, et al. (2002) A structural mechanism of integrin alpha(IIb)beta(3) ‘‘inside-out’’ activation as regulated by its cytoplasmic face. Cell 110(5): 587–597. Wen LP, Fahrni JA, Troie S, et al. (1997) Cleavage of focal adhesion kinase by caspases during apoptosis. Journal of Biological Chemistry 272(41): 26056–26061. Wozniak MA, Modzelewska K, Kwong L, and Keely PJ (2004) Focal adhesion regulation of cell behavior. Biochimica et Biophysica Acta 1692(2–3): 103–119.

Integrins L M Schnapp, University of Washington, Seattle, WA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Integrins are a large family of cell adhesion receptors that mediate cell–cell and cell–extracellular matrix adhesion. Integrin members are expressed on virtually every cell and provide essential links between the extracellular environment and intracellular signaling pathways. In doing so, integrins play a role in most essential cell behaviors, including cell survival, apoptosis, differentiation, and transcriptional regulation. Phenotypes of integrin knockout mice illustrate important roles of integrins in lung development, pulmonary fibrosis, and emphysema. Integrins contribute to immune response in the lung by mediating trafficking of leukocytes to areas of inflammation. In addition, they function as receptors for a variety of pulmonary pathogens. Because of their central role in different disease processes, therapeutic agents are being developed to target integrin–ligand interactions.

Integrins are a major family of cell adhesion receptors. The term integrin was first used in the 1980s to describe cell surface receptors that ‘integrated’ the cytoskeleton of one cell with that of another cell or extracellular matrix protein. Since the first description, it is apparent that integrins are essential for many important biological processes including, but not limited to, development, cell proliferation, cell survival, migration, immune function, and wound healing. Integrins are found in all metazoa, vertebrates, and invertebrates. No homologs have been

48

ADHESION, CELL–MATRIX / Integrins

Platelet integrin


1


IIb

Leukocyte integrins
E


L

3


3


M
X


2

2


4
5
6

7


D

4

1
V


7

8

5 6


8
9
10
11

Figure 1 Integrin family. a/b associations of mammalian subunits are illustrated. Subunits in red recognize the RGD motif, subunits in blue are collagen receptors, and subunits in purple are laminin receptors. Some integrins have restricted cell type expression (i.e., leukocyte integrins and platelet integrin).

Table 1 Nomenclature of b2 leukocyte integrins Leukocyte integrin

Names

aLb2

Lymphocyte function-associated antigen (LFA)-1, CD11a/CD18 Macrophage adhesion molecule (Mac)-1, CD11b/CD18, CR3 p150,95, CD11c/CD18 CD11d/CD18

aMb2 aXb2 aDb2

identified to date in prokaryotes, plants, or fungi. The number of integrin members roughly correlates with the complexity of the organisms. Currently, there are 18 known a subunits and 8 known b subunits, which combine to form 24 different heterodimers in mammals (Figure 1). Different a/b combinations alter the ligand specificity of the receptor. Additional complexity arises due to alternatively spliced variants of several integrin subunits. Integrins can be roughly divided into subfamilies according to their shared b subunit, although a subunits can sometimes bind to more than one b subunit, thus blurring the original subfamilies. The receptors are generally named according to the subunit composition (i.e., a8b1). However, older nomenclature still exists (Table 1). For example, the b1 subfamily of integrins was originally termed VLA (very late activation) antigens because they were originally detected on T cells ‘very late after’ mitogen stimulation (i.e., a4b1 is also referred to as VLA-4). All mammalian cells express some member of the integrin family. In addition, some integrins have cell-restricted expression, most notably the

b2-containing integrins, which are found exclusively on leukocytes, and the integrin aIIbb3 (GPIIb/IIIa), found exclusively on megokaryocytes and platelets.

Structure Integrins are heterodimeric glycoproteins that are composed of two noncovalently associated subunits, a and b (Figure 2). Each a and b subunit contains a large extracellular domain, a single pass transmembrane domain, and a short cytoplasmic domain (20–60 amino acids, except for the b4 subunit cytoplasmic domain, which contains 1000 amino acids). The cytoplasmic domain interacts with the cytoskeleton and/or cytoplasmic-signaling molecules. A subset of a subunits contains an inserted (I) domain that is homologous to the A domain of von Willebrand factor and is important in ligand binding. The extracellular domains of both subunits form the ligandbinding site. Divalent cations such as Ca2 þ or Mg2 þ are required for functional activity. Divalent cations can promote or suppress ligand binding, change ligand specificity, or stabilize integrin structure. In general, Mg2 þ promotes cell adhesion, Ca2 þ decreases it, and Mn2 þ increases ligand affinity. Insight into the structure of the extracellular domain was obtained from X-ray crystal structure analysis. The extracellular domain resembles a globular head atop two legs, one from each subunit (Figure 2). The globular heads form the main contact between the subunits and the ligand. The head of the a subunit is composed of a seven-bladed b-propeller

ADHESION, CELL–MATRIX / Integrins 49


 A domain

-propeller Hybrid domain PSI domain Thigh domain

EGF repeats

Calf domain

Calf domain Transmembrane domains

domain, followed by an immunoglobulin (Ig)-like ‘thigh’ domain and two ‘calf’ domains. The b subunit head has a bA domain (similar to the I domain of a subunits) followed by an Ig-like hybrid domain, PSI domain (plexin, semaphorin, and integrin), and four EGF-like domains. Recently, the crystal structure of the extracellular domain of avb3 bound to a protypical Arg–Gly–Asp (RGD) ligand was determined. The RGD peptide is inserted in a crevice between the heads of the a subunit and the b subunit. Amino acid residues previously predicted to be important in ligand binding by mutational analysis were confirmed to participate in ligand binding. Furthermore, occupation by ligand resulted in a conformational change of the extracellular domain that is likely to be transmitted to the cytoplasmic domain and contribute to outside-in signaling.

Regulation of Activity

Cytoplasmic domains Figure 2 Schematic representation of integrin based on structure of avb3. Ligand-binding pocket is formed by the b-propeller and bA domain of a and b subunits, respectively, and is indicated by the arrow.

Integrins can exist in different conformational states, and many integrins exist at rest in a low-affinity state (Figure 3). However, in response to agonists, signaling pathways are activated that cause a conformational change in the integrins to allow high-affinity binding (‘affinity modulation’). This inside-out signaling refers to the rapid reversible change in integrin affinity in response to external agonists. The classic

ECM





















Extracellular

Intracellular

Actin cytoskeleton

Inactive low affinity

Conformational change/ high affinity

Clustering/ increased avidity

Figure 3 Schematic representation of integrin affinity and avidity modulation in response to inside-out signaling. In the resting (inactive) state, interaction of cytoplasmic tails via salt bridge (black bar) inhibits interaction with cytoskeletal and signaling partners. After agonist-induced signaling, the salt bridge is broken by cytoplasmic proteins such as talin. This results in a conformational change in the extracellular domain, allowing high-affinity binding to ligand. Diffusion of integrin within the plasma membrane leads to clustering and increased avidity.

50

ADHESION, CELL–MATRIX / Integrins

example of inside-out signaling occurs in platelets. On a resting platelet, the integrin aIIbb3 exists in a low-affinity state and is unable to bind soluble fibrinogen. After platelet activation (by agonists such as thrombin, collagen, ADP, or epinephrine), aIIbb3 undergoes a conformational change to a high-affinity state and binds soluble fibrinogen, causing platelet aggregation. Regulation of integrin activation is also critical for leukocyte trafficking. Leukocytes circulate in a nonadhesive state and require activation (i.e., by cytokines or chemokines) to allow targeted integrinmediated adhesion to the vascular endothelial cells and subsequent transmigration. Activation of b2 integrins is also critical for phagocytosis, antigen presentation, and T-cell killer function. Integrin cytoplasmic domains regulate the activation state of the integrin. Truncation of the a or b integrin cytoplasmic domain results in a constitutively active receptor. In the resting (inactive) state, the cytoplasmic tails of a and b subunits are thought to be in close proximity to each other, connected by a salt bridge formed between several highly conserved residues within the cytoplasmic domain. When the salt bridge is broken, the integrin becomes activated. Several cytoplasmic proteins can bind to cytoplasmic tails and activate integrins, including talin, calcium and integrin binding protein (CIB), and b3 endonexin. Ligand binding can also be strengthened by increased lateral mobility of the integrin within the plasma membrane. This results in clustering of the integrin and increased ligand binding, referred to as avidity modulation.

as mechanotransducers. Integrins also coordinate signaling with many receptor tyrosine kinases and therefore determine cellular responses to soluble growth factors and cytokines. In addition, integrins can associate with other transmembrane or membrane-associated proteins (e.g., PDGF-bR, uPAR, and TM4SF) to influence cell signaling.

Integrin Ligands: More Than Just Glue Originally described as receptors that mediated adhesion to extracellular matrix proteins such as fibronectin, collagen, and laminin, it is increasingly evident the integrins play a broader role in homeostasis, and their ligand repertoire is much larger than originally appreciated. Many integrins recognize the tripeptide sequence RGD found in many matrix proteins, including fibronectin, vitronectin, and fibrinogen, and other extracellular proteins, including the latency-associated peptide (LAP)-TGF-b1. Integrins bind to a variety of counterreceptors (i.e., VCAM, ICAM, and E-cadherin), illustrating the importance of integrins in cell–cell adhesion. An increasing number of different substrates have been identified as biologically relevant ligands for integrins, including VEGF, matrix metalloproteinase (MMP)-2, sperm fertilin, and the endogenous angiogenesis inhibitors angiostatin, endostatin, and tumstatin. Identification of novel ligands has expanded the spectrum of biological processes that integrins regulate.

Integrins in Respiratory Diseases Biological Function Outside-in signaling occurs after ligand binding to integrins. The cytoplasmic tails do not contain any intrinsic catalytic activity but act as organizing centers and scaffolding for other signaling components and cytoskeletal proteins. Most integrins activate focal adhesion kinase, which subsequently activates a number of classic signaling pathways, such as mitogen-activated protein kinase, PI3-kinase, protein kinase C, and c-JUN kinase. These pathways regulate diverse cell behaviors, including survival, proliferation, migration, and transcriptional activity. Integrins are important links to the actin cytoskeleton through binding of the cytoplasmic tails to actinbinding proteins such as talin, filamin, integrin-linked kinase, and a-actinin. Integrin-mediated adhesion can activate the Rho family of small GTPases that are involved in actin cytoskeleton rearrangement. Integrin activation of RhoA, Rac-1, or CDC42 results in the formation of stress fibers, lamellopodia, and filopodia, respectively. Thus, integrins are situated to act

The nonredundant, important function of the different integrins is illustrated by the distinct phenotypes of the knockouts. In addition, several human genetic diseases illustrate the clinical importance of the integrin family. Leukocyte adhesion deficiency (LAD1) is a rare autosomal dominant disorder in which patients suffer from recurrent life-threatening bacterial infections, despite normal numbers of circulating leukocytes. LAD-1 is caused by lack of functional b2 integrins on leukocytes, which results in the inability of leukocytes to migrate to tissue sites of inflammation and infection. Glanzmann’s thrombasthenia is an autosomal recessive disorder in which patients suffer from severe mucocutaneous bleeding due to a lack of functional aIIbb3 on platelets. Mutations in a4b6, normally expressed on the basal surface of keratinocytes, cause some cases of the skin blistering disease epidermolysis bullosa. Mutation of the a7 integrin subunit, expressed primarily on skeletal and cardiac muscle, is responsible for some cases of congenital myopathy.

ADHESION, CELL–MATRIX / Integrins 51

Because of their diverse roles in many processes, integrins are attractive targets in many diseases. Drugs aimed at directly blocking receptor–ligand interactions have been developed and include monoclonal antibodies and small molecule ligand mimetics (based on RGD). The platelet integrin aIIbb3 antibody, abciximab, was the first anti-integrin drug to be approved for clinical use. Several agents targeting integrins are being tested for use in asthma, cancer, and inflammatory diseases. Integrins in Lung Development

Early studies using in vitro models of lung branching showed that administration of RGD peptides decreased branching morphogenesis. These studies suggested a critical role of cell–matrix interactions, mediated through integrins in lung development. Further studies on knockout mice have confirmed the importance of several integrins in lung development. Mice lacking the a3 integrin subunit exhibit decreased branching morphogenesis and immature bronchiolar epithelium, in addition to abnormal kidney development. The underlying epithelial basement membrane was severely disrupted, demonstrating a critical role of a3 in basement membrane assembly. Mice lacking the a9 subunit die soon after birth due to bilateral chylothorax, suggesting abnormalities in lymphatic development. Recently, VEGF-C and VEGF-D, key effector proteins in lymphatic development, were identified as novel ligands for a9b1. a9b1–VEGF interactions mediated endothelial cell adhesion and migration, suggesting a mechanism for phenotype found in a9-deficient mice. The absence of lung phenotype in other integrin knockouts does not necessarily imply the absence of a role in lung development. In mice, lung development begins at approximately E12.5 and continues until approximately 4 weeks after birth. Knockouts of several potentially important integrins for lung development were embryonic lethal before lung development was completed (i.e., b1, a5); thus, determining their contribution to lung development is difficult. In addition, upregulation of other integrins or related proteins may compensate for the loss of an integrin subunit during development. Integrins and Fibrosis

The integrin avb6 is normally expressed at low levels on airway epithelium. Mice that lack the integrin subunit b6 develop exaggerated inflammation in the lung at baseline. Interestingly, following bleomycin administration, which typically results in lung injury and fibrosis, the b6-null mice were protected from development of pulmonary edema and fibrosis. This

was due to a defect in the activation of TGF-b1, an important mediator of pulmonary injury and fibrosis. TGF-b1 is secreted in a latent form, complexed with LAP, and is unable to signal through its receptor. LAP contains an RGD site that binds to avb6 and results in activation of TGF-b1. The activation of TGF-b1 by avb6 requires contact between avb6-expressing cells (i.e., epithelial cells) and TGF-b receptor-expressing cells (i.e., macrophages); it also requires an intact integrin cytoplasmic tail. Like avb6, avb8 also binds to the RGD sequence in LAP–TGF-b1 and activates TGF-b1. However, in contrast to avb6, avb8 activation requires metalloproteinase activity and results in free active TGF-b. Thus, integrin-mediated regulation of TGF-b activity can occur by several distinct mechanisms. Another interesting finding in the b6-null mice is the development of emphysema over time. Backcrossing the b6-null mice onto a MMP-12-null background eliminates the development of emphysema, suggesting that the development of emphysema is due to increased expression of MMP-12, a macrophage elastase. The findings suggest a model wherein active TGF-b1 normally suppresses MMP-12 expression by macrophages. In the absence of avb6-mediated TGF-b activation, MMP-12 expression is no longer suppressed and ultimately results in a matrixdegrading phenotype in the lung. Integrins and Asthma

In asthma, integrins, including aLb2 and a4b1, contribute to trafficking of eosinophils and other inflammatory cells to the lung during an asthma exacerbation. Therefore, integrins are attractive targets for novel anti-inflammatory therapies in asthma. Efalizumab, a humanized anti-aL monoclonal antibody, inhibits leukocyte migration to areas of inflammation and is approved for use in moderate to severe psoriasis. Efalizumab was tested in patients with mild asthma. Despite a significant decrease in activated eosinophils in the lung, there was no significant difference in pulmonary function after allergen challenge. There has been much interest in targeting the a4 integrin subunit in asthma and other chronic inflammatory diseases, such as Crohn’s disease, rheumatoid arthritis, and multiple sclerosis. In multiple sclerosis, treatment with the monoclonal a4 antibody natalizumab resulted in a significant reduction in relapses, leading to Food and Drug Administration approval of natalizumab. However, since approval, several unexpected cases of progressive multifocal leukoencelphalopathy (PML) have been reported in patients receiving natalizumab. PML is a rare, fatal demyelinating disease caused

52

ADHESION, CELL–MATRIX / Integrins

by reactivation of human polyoma virus JC. The contribution of natalizumab to the development of PML is not entirely clear, but it likely relates to preventing migration of immune cells that normally repress latent virus into the central nervous system. These findings have raised serious concerns regarding the safety of a4 antagonists and put into question any future work targeting the a4 subunit. Integrins and Acute Lung Injury

The general paradigm for leukocyte trafficking occurs in several steps: 1. selectin-mediated rolling of leukocytes on endothelium, 2. activation of leukocyte b2 integrins by chemoattractants, 3. integrin-mediated firm adhesion of leukocytes to endothelium, and 4. integrin-mediated transmigration of leukocytes into the interstitium. In the lung, leukocyte trafficking differs from the general model in several aspects: the classical selectinmediated rolling does not occur; transmigration of leukocytes occurs at pulmonary capillaries, not postcapillary venules; and transmigration of leukocytes into interstitium and alveoli may follow a b2-dependent or b2-independent pathway. In the lung, involvement of b2 integrins for leukocyte migration depends on the stimulus. For example, leukocyte trafficking in response to Escherichia coli, Pseudomonas aeruginosa, lipopolysaccharide (LPS), and IL-1 is dependent on b2 integrins, whereas leukocyte trafficking in response to Streptococcus pneumoniae, group B streptococcus, Staphylococcus aureus, and hydrochloric acid is independent of b2 integrins. Integrins and Pathogens

Many pathogens utilize host integrins to facilitate cell attachment and infection (Table 2). Many viruses have RGD motifs in capsid proteins, which facilitates attachment and internalization of organisms. Hantavirus Table 2 Virus–integrin interactions Virus

Integrin receptor

Hantavirus Echovirus Rotavirus Papillomavirus Coxsackievirus Foot and mouth disease virus Adenovirus HHV-8 (KSHV)

avb3, aIIbb3 a2b1 a2b1, axb2, a4b1 a6b1 avb3, avb6 avb3, avb6 avb3, avb5, avb1 a3b1

causes severe pulmonary disease and thrombocytopenia and infects pulmonary epithelial cells and platelets. Pathogenic strains of hantaviruses attach to cells via b3-containing integrins. Another pathogen, foot and mouth disease virus (FMDV), causes a devastating disease of cattle. The primary route of infection by FMDV is through the epithelial cells and associated lymphoid tissues in the upper respiratory tract, followed by dissemination to other epithelial tissues. The outer capsid of FMDV contains a highly conserved RGD tripeptide motif on an exposed loop that interacts with av-containing integrins and facilitates viral attachment and infection of cells. Adenovirus is important both for the diseases it causes in humans, including respiratory infections, and because replicationdefective variants of adenovirus are being used as vectors for gene therapy. Adenovirus contains a conserved RGD sequence within a highly variable region in the penton base protein. In contrast to the viruses just mentioned, adenoviruses do not use integrins for initial attachment. Instead, adenovirus attaches to cells through a common coxsackievirus adenovirus receptor and then the RGD site interacts with av-containing integrins to facilitate internalization. Likewise, the causative virus of Kaposi’s sarcoma, human herpes virus 8 (HHV-8), interacts with a3b1 on target cells via RGD sequence on an outer envelope to facilitate viral entry and uptake. Thus, viruses and other pathogens have exploited integrins for their advantage. Integrins and Malignancy

Most adherent cells are anchorage dependent; that is, they require integrin-mediated signaling to stimulate cell-cycle progression, promote cell survival, and respond to soluble growth factors such as EGF and PDGF. Loss of integrin-mediated signaling causes a subtype of apoptosis, termed ‘anoikoisis’ from the Greek word meaning ‘homelessness’. Following malignant transformation, the requirement of integrin ligation is often bypassed by mutations in oncogenes or tumor suppressor genes, thus allowing anchorage-independent growth. However, integrin signaling still contributes to tumorigenesis and tumor progression. Depending on the integrin, cell type, and signaling pathway, activated integrins may facilitate tumor growth, metastasis, and attachment at distant sites or suppress tumor activity. In general, tumor cells, either through direct signaling mechanisms or through selection process, increase expression of integrins that promote cell proliferation, survival, and migration and decrease expression of integrins that promote a quiescent, differentiated state. Although it is difficult to make generalizations, a2b1 and a3b1 expression is associated with a tumor

ADRENERGIC RECEPTORS 53

suppressor phenotype, whereas avb3, avb6, and a6b4 expression is associated with tumor progression. The development of blood supply is essential for tumor survival and growth, and it is a potential target of antitumor agents. The endothelial integrin avb3 is upregulated in tumor neovasculature. In vitro and animal studies showed that avb3 antagonists (antibodies or peptides) caused tumor regression, inhibited neovascularization, and induced apoptosis of endothelial cells. Several anti-avb3 agents show promise as antiangiogenic therapy for cancer treatment. See also: Adhesion, Cell–Cell: Vascular; Epithelial. Adhesion, Cell–Matrix: Focal Contacts and Signaling. Asthma: Overview. CD11/18. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fibroblasts. Matrix Metalloproteinases.

Further Reading Calderwood DA (2004) Integrin activation. Journal of Cell Science 117: 657. Doerschuk CM (2000) Leukocyte trafficking in alveoli and airway passages. Respiration Research 1: 136. Guo W and Giancotti FG (2004) Integrin signalling during tumour progression. Nature Reviews: Molecular Cell Biology 5: 816. Humphries MJ, McEwan PA, Barton SJ, et al. (2003) Integrin structure: heady advances in ligand binding, but activation still makes the knees wobble. Trends in Biochemical Sciences 28: 313. Hynes R (2002) Integrins. Bidirectional, allosteric signaling machines. Cell 110: 673. Plow EF, Haas TA, Zhang L, Loftus J, and Smith JW (2000) Ligand binding to integrins. Journal of Biological Chemistry 275: 21785. Schwartz MA (2001) Integrin signaling revisited. Trends in Cell Biology 11: 466. Sheppard D (2000) In vivo functions of integrins: lessons from null mutations in mice. Matrix Biology 19: 203. Sheppard D (2003) Functions of pulmonary epithelial integrins: from development to disease. Physiological Reviews 83: 673. Sheppard D (2004) Roles of alphav integrins in vascular biology and pulmonary pathology. Current Opinion in Cell Biology 16: 552.

ADRENERGIC RECEPTORS A E Tattersfield, Nottingham University, Nottingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Adrenergic receptors, which includes a, b and dopamine receptors, belong to the large family of G-protein-coupled, seven transmembrane domain receptors. b-Adrenoceptors are the best characterized and predominant adrenoceptors in the lung, with both b1 and b2 receptors being widely distributed. b2Adrenoceptors are an important therapeutic target and their polymorphisms may influence the response to b2 agonist treatment. Their numbers and functions are regulated by b-agonist stimulation and by drugs, such as corticosteroids, and cytokines. a-Adrenoceptors are found on vascular smooth muscle, presynaptic nerve endings, airways, and submucus glands, and they may help to condition inspired air. There is evidence for D1 dopamine expression on alveolar cells, where they help to clear lung edema, and for D2 receptors on sensory nerves in the lung, where they may modulate neurogenic inflammation and reflexmediated symptoms.

Introduction Adrenergic receptors include a, b, and dopamine receptors; they are part of the large family of G-proteincoupled, seven transmembrane domain receptors. b-Adrenoceptors are the best characterized and predominant adrenoceptors in the lung; the role of a-adrenoceptors and dopamine receptors is less well established.

b-Adrenoceptors b1 and b2 receptors were initially identified on the basis of tissue selectivity to a range of agonists. Both b-adrenoceptors were subsequently characterized and cloned, as was a third (b3) receptor and there are putative claims for a b4 receptor in the heart. There is 54% homology between b1 and b2 receptors. Structure

The b2-adrenoceptor contains 413 amino acids with seven transmembrane-spanning domains, three intraand three extracellular loops, an intracellular C-terminus, and an extracellular N-terminus (Figure 1). The third intracellular loop is important for linking to G-proteins. Site-directed mutagenesis studies show that b-agonists bind to residues on the hydrophobic region within the cell membrane between the third and sixth transmembrane domains as shown in Figure 1. The salmeterol molecule anchors to an exosite on the fourth transmembrane domain, allowing the molecule to stimulate the receptor repetitively. The intracellular third loop and tail of the b-adrenoceptor contains phosphorylation sites that are involved in receptor downregulation. Polymorphisms

Nine single-base mutations or polymorphisms in the coding region of the b2-adrenoceptor gene on

54

ADRENERGIC RECEPTORS

Y

Y


2-Agonist

NH2

Cell membrane I

II

III

IV

V

VI VII P

s

P

cAMP PKA PKC PTK

P = Phosphorylation sites P P

H COO

P

+


arrestin

P
ARK (GRK)

Inhibits receptor function

Figure 1 Schematic representation of the human beta 2-adrenoceptor showing the 7 trans membrane domains and the site on the third, fifth, and sixth domains that are required for beta-agonist binding. P marks the phosphorylation sites concerned with receptor ancapling. The blue circles indicate the amino acids that are essential for b-agonist binding. Adapted from ‘‘b-adrenergic receptors and their regulation’’ by PJ Barnes.

chromosome 5Q have been identified, though only four of these cause amino acid substitutions – at codons 16, 27, 34, and 167. The two most common polymorphisms are at codon 16, where glycine is substituted for arginine, and codon 27 where glutamic acid is substituted for glutamine. Both polymorphisms are common, with 37% and 23% of subjects being homozygous for the gly 16 and glu 27 polymorphisms, respectively, in a large general population study. There is linkage disequilibrium between polymorphisms at codons 16 and 27, that is, they are more likely to occur together, and this can cause difficulties when trying to determine the functional effects of a specific polymorphism. b-Adrenoceptor Activation

When a b-agonist binds to the b-adrenoceptor/Gs complex guanosine diphosphate (GDP) is released, allowing guanosine triphosphate (GTP) to bind and activate the a-subunit of Gs, which then dissociates from the b-receptor and is free to stimulate adenylate cyclase. Adenylate cyclase catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (AMP), which in turn activates protein kinase A. Cyclic AMP has a range of actions that promote smooth muscle relaxation in airways and other activities such as inhibition of mediator release in inflammatory cells (see Figure 2 and Table 1). Most of the effects of b-agonists are mediated via cyclic AMP and activation of protein kinase A, but some, such as the opening of maxi K þ channels, may, in part at least, be due to direct activation by the Gs a-subunit.

Protein kinase A is also able to phosphorylate and activate a transcription factor, CREB or cyclic AMP response element binding protein. By binding to the cyclic AMP response element (CRE) on the promoter region of target genes, CREB is able to cause transcription of various genes including the b-receptor gene. b2-Receptor expression may therefore be increased transiently by b-agonist stimulation. CREB also interacts with other transcription factors within the nucleus including some proinflammatory cytokines such as activating protein (AP-1) and nuclear factor kappa B (NF-kB). b-Adrenoceptor Regulation and Activity

Until recently, it was thought that b-adrenoceptors existed in high- or low-affinity states, the high-affinity state being increased in the presence of b-agonists and associated with an increased binding affinity for b-agonists. The affinity for b-adrenoceptor antagonists did not differ between high- and low-affinity states. b-Adrenoceptor activation with this model only occurred when an agonist was present. However, recent work suggests that receptors may have constitutive activity, that is, activity when no agonist is present. Some b-adrenoceptor antagonists are now thought to act by stabilizing this constitutive activity rather than as a competitive antagonist and hence they can be described as inverse agonists. The number of b-adrenoceptors in the airways and other tissues depends on the rates at which they are synthesized and degraded; their function also depends on the extent to which they are coupled to the catalytic unit. Both receptor numbers and functions

ADRENERGIC RECEPTORS 55

K+
 
-Agonist

Gs


-Adrenoceptor
 

Gs

ATP

Phosphorylation of myosin light chain kinase

AC

Inhibition of PI hydrolysis

cAMP

Cell membrane

Inactive PKA

Active PKA

Ca2+ extrusion and sequestration Stimulation of Na+/K+ ATPase Membrane hyperpolarization

Smooth muscle relaxation

Inhibition of mediator release

Activation of Ca2+ gated potassium channels Figure 2 Schematic representation of the signaling pathway following stimulation of the b-receptor; this causes coupling of the receptor to the Gs protein and stimulation of adenylate cyclase (AC). This in turn causes increased accumulation of cyclic-AMP (cAMP) and protein kinase A (PKA) and a fall in intracellular calcium. The most important cellular effects for asthma are smooth muscle relaxation and inhibition of mediator release from inflammatory cells.

Table 1 Main actions of b-adrenergic agonists at different receptor subtypes

underlie this development:

Tissue

Receptor

Response

Airways

b2 mainly

Heart Blood vessels

b1/b2 b2

Uterus Metabolic

b2 b2/b3

Muscle

b2

Bronchodilatation, reduction in mediator release from mast cells, increased mucus production, increased ciliary activity Tachycardia, inotropic action Dilatation, fall in blood pressure, compensatory reflex increase in heart rate Relaxation Increase in glucose, insulin, lactate, pyruvate, nonesterified fatty acids, glycerol, and ketone bodies; decrease in potassium, phosphate, calcium, and magnesium Tremor

1. Receptor uncoupling. Exposure of a b-receptor to a b-agonist causes phosphorylation of sites on the 3rd intracellular loop and tail of the b-adrenoceptor, causing the receptor to uncouple from its Gs protein and catalytic unit. Receptor uncoupling occurs within minutes and is rapidly reversible. At least two kinases are involved: the cyclic AMP-dependent protein kinase A and the G-protein-dependent b-adrenergic receptor kinase (bARK); protein kinase G may also contribute. Protein kinase A is activated by fairly low concentrations of b-agonist, as seen with therapeutic doses, whereas activation of bARK requires higher agonist concentrations and the cofactor b-arrestin. bARK causes homologous b-receptor desensitization, that is, to b-agonists only, whereas protein kinase A causes heterologous desensitization, so that the response to other agonists that use the same catalytic unit, for example, forskolin, is reduced. 2. Sequestration of receptors. Following exposure to a b-agonist, b-adrenoceptors may be internalized within the cell, and sequestrated into vesicles. This can also occur within minutes and is again reversible, the receptors being recycled to the cell surface. 3. Downregulation. With longer term exposure to bagonists, however, b-receptors may be internalized and degraded and hence are not available for recycling. Polymorphisms at codon 16 and 27 have been shown to affect b-adrenoceptor downregulation

are modified by b-agonists and by other factors, of which cytokines and corticosteroids are particularly relevant to asthma.

Regulation by b-agonists With repeated or continuous exposure to a b-agonist there is a reduction in the response, a phenomenon known as desensitization or tolerance. Several mechanisms have been shown to

56

ADRENERGIC RECEPTORS

in vitro. Receptors with the gly 16 polymorphism have shown increased downregulation whilst those with the glu 27 polymorphism were protected against downregulation. 4. Reduced synthesis. b-Adrenoreceptor numbers may also be lower as a result of a reduction in receptor mRNA, following continuous exposure. Regulation by other factors 1. Corticosteroids. Corticosteroids have a number of effects in vitro that may increase or decrease the response to a b-agonist. They are able to increase b-receptor numbers by inhibiting downregulation of b-receptors, and by increasing b-receptor gene transcription. However, the activity of CREB can be inhibited by glucocorticoid transcription factors, thus providing a possible mechanism whereby glucocorticoids might reduce the efficacy of a b-agonist. 2. Cytokines. The proinflammatory cytokine interleukin 1b has been shown to attenuate b-receptor responsiveness by uncoupling the receptor from its catalytic unit; this appears to be due to induction of the inducible form of cyclooxygenase (COX 2) and release of inhibitory prostanoids. Distribution of b-Adrenoceptors

The distribution and type of b-receptor within the lung has been determined by receptor-binding studies; airway epithelium and alveoli have the highest receptor density. Some 70% of the b-adrenoceptors in the lung are of the b2-receptor subtype, the remainder being b1-receptors. Only b2-receptors are found on airway and vascular smooth muscle whereas both b1- and b2-receptors are found on submucosal glands and alveoli. b-Receptors are distributed widely throughout the body and some of the more important sites are shown in Table 1.

patients with asthma who are homozygous for arginine at codon 16.

a-Adrenoceptors Classifying a-adrenoceptors has been less straightforward than classifying b-adrenoceptors. The two main classes are the a1-adrenoceptors, classic postsynaptic excitatory receptors, and a2-adrenoceptors, which are usually, though not invariably, presynaptic, where they inhibit neuronal mediator release. There are three fully defined a1-adrenoceptor subtypes (a1A, a1B, a1D, and a putative a1L) and four subtypes of the a2-adrenoceptor (a2A, a2B, a2C, and a2D). Most of the subtypes have now been cloned. a-Adrenoceptor Activation

The postsynaptic a1-adrenoceptor is coupled to a Gq protein and produces physiological effects via activation of phospholipase C, which mobilizes intracellular calcium by increasing inositol 1, 4, 5-trisphosphate concentrations, and diacylglycerol, which activates protein kinase C. a1-Adrenoceptors also activate other pathways such as the mitogen-activated protein kinase pathway. A single base polymorphism has been described in a1A but no effect on receptor function has been identified as yet. Activation of a2 receptors usually causes inhibition of adenylate cyclase through coupling with membrane-linked pertussis toxin-sensitive Gi/o protein. Distribution

Alpha-adrenoceptors are widely distributed on neuronal and non-neuronal tissues. Within the lung, aadrenoceptors have been located predominantly in smaller airways, on serous cells in submucus glands, and on pulmonary blood vessels. a1-Adrenoceptors are thought to be present on bronchial blood vessels and a2-adrenoceptors on airway ganglia and presynaptic cholinergic nerve endings.

b-Receptor Numbers and Function in Disease

Positron emission tomography studies indicate that there are normal numbers of b-receptors in the lungs of people with mild asthma, and they may even increase in patients dying from asthma. The function of b-receptors in the lungs of patients with asthma also appears to be normal, although it may be affected by previous b-agonist exposure and receptor polymorphisms. There is no evidence to suggest that the presence of asthma is associated with b2-receptor polymorphisms or haplotypes. However, there is some evidence that the response to b2-agonists is reduced in

Function

The role of a-adrenoceptors in the lung is not entirely clear and work is hampered by lack of specificity of many of the ligands used to investigate a-adrenoceptor subtypes. a1-Adrenoceptors classically cause smooth muscle contraction and could therefore help control bronchial blood flow and conditioning of inspired air. Other possible roles include inhibition of histamine release from mast cells and acetylcholine release from cholinergic nerve endings, and modulating the content of airway secretions through serous cell stimulation.

ADRENERGIC RECEPTORS 57 Function in Disease

The possibility that a-adrenergic mechanisms might contribute to asthma was explored several years ago, particularly when the density of a-receptors was found to be higher in patients with asthma. a-Adrenoceptor agonists caused bronchoconstriction in some but not all studies although whether this is a specific or non-specific effect is not clear. Similarly nonselective a-adrenoceptor antagonists such as indoramin caused some degree of bronchodilatation or protected against induced bronchoconstriction in some studies, but all the drugs studied had additional pharmacological effects that could have been responsible. None of the a-adrenoceptor antagonists available caused worthwhile bronchodilatation in asthma when given regularly or by inhalation. These data and the fact that norepinephrine when infused causes bronchodilatation rather than bronchoconstriction suggest that a-adrenergic mechanisms are not making an important contribution to the bronchoconstriction seen in asthma.

Dopamine Receptors Of the five subtypes of dopamine receptors identified (D1–D5), D1 and D5 receptors have similar homology and both stimulate adenylate cyclase. D2, D3, and D4 receptors are also homologous but they inhibit adenylate cyclase. Most work relates to the D1 and D2 subtypes. Distribution and Function

Dopamine receptors are found in the central and peripheral nervous system. They have been studied in the brain predominantly and in the context of the lung there is interest in the role that central dopaminergic pathways might play in the development of nicotine addiction. Within the lung there is evidence for D1 receptors on alveoli in the rat, which, when stimulated, increase the clearance of lung edema. D2 receptor mRNA and protein are expressed in sensory ganglia in the airways and dopamine receptor activation has been shown to inhibit depolarization of the vagus in animals and man, and neuropeptide release from nerve endings. D2 dopamine receptors may therefore have a role in modulating neurogenic inflammation and reflex-mediated symptoms such as cough. D2 receptor agonists have reduced cough, mucus production, and tachypnea in animal models and there is interest in whether they might reduce symptoms such as cough and

breathlessness in patients with chronic obstructive pulmonary disease. Dopamine in low doses stimulates dopamine receptors but as the dose is increased it stimulates b1adrenoceptors followed by a1- and a1-adrenoceptors. See also: Asthma: Overview. Bronchodilators: Beta Agonists. G-Protein-Coupled Receptors.

Further Reading Barnard ML, Ridge KM, Saldias F, et al. (1999) Stimulation of the dopamine 1 receptor increases lung edema clearance. American Journal of Respiratory and Critical Care Medicine 160: 982– 986. Barnes PJ (1986) Neural control of human airways in health and disease. American Review of Respiratory Diseases 134: 1289– 1314. Barnes PJ (1995) Beta-adrenergic receptors and their regulation. American Journal of Respiratory and Critical Care Medicine 152: 838–860. Birrell MA, Crispino N, Hele DJ, et al. (2002) Effect of dopamine receptor agonists on sensory nerve activity: possible therapeutic targets for the treatment of asthma and COPD. British Journal of Pharmacology 136: 620–628. Carswell H and Nahorski SR (1983) b-Adrenoceptor heterogeneity in guinea pig airways: comparison of functional and receptor labelling studies. British Journal of Pharmacology 79: 965–971. Goidie RG, Papidimitriou SM, Paterson SW, et al. (1986) Autoradiographic localisation of b-adrenoceptors in pig lung using [125I]-iodocyanopindolol. British Journal of Pharmacology 88: 621–628. Green SA, Spasoff AP, Coleman RA, Johnson M, and Liggett SB (1996) Sustained activation of a G protein coupled receptor via ‘anchored’ agonist binding. Molecular localization of the salmeterol exosite within the b-adrenergic receptor. Journal of Biological Chemistry 39: 24029–24035. Green SA, Turki J, Innis M, and Liggett SB (1994) Amino-terminal polymorphisms of the human b-adrenergic receptor impart distinct agonist-promoted regulatory properties. Biochemistry 33: 9414–9419. Hall IP and Tattersfield AE (1998) b-Adrenoceptor agonists. In: Barnes PJ, Rodger IW, and Thomson NC (eds.) Asthma. Basic Mechanisms and Clinical Management, 3rd edn, pp. 651–676. London: Academic Press. Koshimizu T, Tanoue A, Hirasawa A, Yamauchi J, and Tsujimoto G (2003) Recent advances in a1-adrenoceptor pharmacology. Pharmacology and Therapeutics 98: 235–244. Lands AM, Arnold A, McAuliff JP, et al. (1967) Differentiation of receptor systems activated by sympathomimetic amines. Nature 214: 597–598. Reihsaus E, Innis M, MacIntyre N, and Liggett SB (1993) Mutations in the gene encoding for the b2-adrenergic receptor in normal and asthmatic subjects. American Journal of Respiratory Cell and Molecular Biology 8: 334–339. Schwartz J, Carlsson A, Caron M, et al. (1998) The IUPHAR compendium of receptor characterisation and classification: dopamine receptors. IUPHAR Media (London) 142–151. Strader CD, Sigal IS, and Dixon AF (1989) Mapping the functional domains of the b-adrenergic receptor. American Journal of Respiratory Cell and Molecular Biology 1: 81–86.

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AEROSOLS S P Newman, Nottingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The inhaled route is used to deliver drugs as aerosols for the maintenance therapy of asthma, chronic obstructive pulmonary disease, and other conditions. The deposition of aerosol particles in the respiratory tract is an important prerequisite to obtaining a good clinical effect. Generally, inhaler devices should deliver particles smaller than approximately 5 mm in diameter in order to enter the lungs. A variety of inhaler devices are available for inhalation therapy. Pressurized metered dose inhalers (pMDIs) have been widely used for 50 years, but many patients have problems using them correctly. They are currently being reformulated with ozone-friendly propellants. Breath-actuated inhalers and spacer attachments may be useful supplements to pMDIs for some patients. Dry powder inhalers (DPIs) are easier to use correctly than pMDIs, and they do not require propellants. Many pharmaceutical companies seem to be prioritizing DPIs above pMDI reformulation, and they are also preferred by many patients. Nebulizers continue to be used widely, but the limitations of jet and ultrasonic nebulizers have led to the development of novel systems, sometimes involving vibrating meshes. Finally, a new class of inhalers (soft mist inhalers) is emerging, composed of multidose devices containing liquid formulations, some of which could challenge pMDIs and DPIs in the portable inhaler market.

Inhaled Drug Delivery The pulmonary route may be used to deliver drugs for the maintenance therapy of some lung diseases, most notably asthma and chronic obstructive pulmonary disease (COPD). Drugs are also given by inhalation to treat other chest problems, including respiratory tract infections in cystic fibrosis. In addition, it is hoped that inhaled drugs intended to have a systemic action in the body (e.g., insulin) will soon be marketed. The potential benefits of the inhaled route have long been recognized, but the importance of good quality inhaler devices that deliver drugs reliably to the lungs has only been appreciated during the past 25 years.

Aerosol Properties An understanding of aerosol properties and aerosol deposition is an important prerequisite for optimizing inhalation therapy. Drugs are given by inhalation as aerosols of solid particles or liquid droplets, but for simplicity the term ‘particle’ may be used to describe both solid and liquid dispersions. The most important property of an aerosol particle is its

size, and this is best expressed as the aerodynamic diameter, which also takes into account particle density and shape. For spherical particles, aerodynamic diameter (Da) and physical diameter (Dp) are related by the formula Da ¼ DpOr, where r is the specific gravity of the material from which the particles are made. In practice, aerosol particles are seldom spherical; for instance, micronized drug particles are often highly irregular in shape. Aerosol systems found in medicine are usually heterodisperse, indicating that the particles in a particular spray or cloud have a wide range of sizes. Monodisperse aerosols, in which all the particles have approximately the same size, are not normally found in pharmaceutical products, although they can be made using specialized equipment. It is preferable to describe the mass or volume distribution of an aerosol rather than the distribution of particles by number since many small particles may contain much less drug than a few large particles. In practice, particle size spectra from inhaler devices often approximate to log-normal distributions. The mass median aerodynamic diameter (MMAD) may be used to express the average aerosol size. This diameter is such that half the aerosol mass is contained in larger particles and half in smaller particles. The spread of particle sizes may be expressed as a geometric standard deviation (GSD), a dimensionless quantity. A perfectly monodisperse aerosol has a GSD of 1. A typical pharmaceutical aerosol may contain particles ranging in size from o0.5 to 410 mm, with an MMAD of 3–4 mm and a GSD of 2.0–2.5. As explained later, deposition of aerosols depends critically on particle size. The fraction of the aerosol mass contained in particles o5 mm in diameter is usually termed the respirable fraction or fine particle fraction (FPF). These are the particles with the greatest likelihood of reaching the lungs in adults, although even smaller particles may be needed for drug therapies in small children. In adults, particles o3 mm in diameter are needed in order to deliver drugs to the alveolated regions – for instance, to deliver inhaled a1 antitrypsin to the alveoli of patients with emphysema. Particle size distributions of aerosols intended for pulmonary delivery may be quantified by several methods. The approach favored within the pharmaceutical industry is the cascade impactor, through which the aerosol is drawn by a vacuum pump, and particles of different sizes are collected on a series of stages. Each stage can be washed out with a solvent

AEROSOLS 59

so that the amount of drug associated with different size bands may be quantified by an analytical technique. Supplementary particle size data may be provided by optical methods, the best known of which is laser diffraction. This involves passing the aerosol cloud through a laser beam, and the angle of diffraction of the laser light is inversely proportional to particle size. It is important to remember that these in vitro measurements are undertaken primarily for purposes of quality control and product release, and they may not predict accurately drug delivery to the lungs in vivo.

Deposition of Pharmaceutical Aerosols Several mechanisms cause aerosol particles to deposit in the respiratory tract, but the two most important ones relating to pharmaceutical aerosols are inertial impaction and gravitational sedimentation. Inertial impaction takes place mainly in the oropharynx and at the bifurcations between major airways, when the aerosol particle has too much inertia to follow the air stream as it changes direction. The probability of inertial impaction occurring is proportional to D2a Q, where Q is the inhaled flow rate. Deposition in central lung regions may be enhanced by the effects of air turbulence, especially at fast inhaled flow rates. Gravitational sedimentation takes place mainly in smaller conducting airways and in the alveoli, when particles settle onto the airway surface under gravity either during slow steady breathing or during breath-holding. The probability of gravitational sedimentation occurring is proportional to D2a T, where T is the residence time of the particle in the airways. A third deposition mechanism (Brownian diffusion) is also important for aerosol particles o1 mm in diameter, which may be pushed in a random direction toward airway walls by collisions with gas molecules. Some particles (especially those o1 mm in diameter) are not deposited, and after inhalation they are simply exhaled. In addition to particle size, the patient’s inhalation also plays a major part in determining the site of aerosol deposition. The inhaled flow rate is particularly important, with slow inhalation usually being recommended in order to reduce impaction losses in the oropharynx. Deep inhalation and a period of breathholding help to increase gravitational sedimentation in the peripheral parts of the lungs. For most pharmaceutical aerosols, lung deposition is enhanced by a combination of aerosol particles o5 mm in diameter and a slow inhaled flow rate (20–30 l min
1). As will be explained later, there is an exception to this rule for dry powder inhalers, where faster inhalation

may preferable. Particles are filtered efficiently from the inhaled air by the nasal passages, so wherever practicable it is better to deliver an inhaled aerosol via a mouthpiece (with mouth breathing) than via a face mask (with nose breathing). The airways of the patient who inhales the aerosol particles also determine the site and extent of deposition in two major ways. First, random variations in airway geometry between different individuals will lead to random variations in the deposition pattern. Hence, for aerosols delivered from any inhaler device, considerable intersubject variability of deposition is to be expected. Second, in patients with asthma, COPD, and other obstructive conditions, the airways may be narrowed by bronchospasm, inflammation, and mucus hypersecretion so that aerosol particles may deposit preferentially in the larger airways of the lungs, with less deposition in the peripheral airways. Both electrostatic charge and humidity affect aerosol deposition in a variety of ways. The most striking effect of humidity is that dry particles composed of water-soluble materials are likely to absorb water when they enter the respiratory tract and, hence, to increase in size. The deposition of pharmaceutical aerosols may be quantified by radionuclide imaging (gamma scintigraphy, single photon emission computed tomography (SPECT), and positron emission tomography (PET)). SPECT and PET are three-dimensional imaging methods and provide information about the distribution pattern within the lungs. However, PET is relatively complex and is probably not practical for use on a regular basis. Certain pharmacokinetic methods are also useful for assessing delivery of some drugs to the lungs. For instance, the plasma or urinary concentrations of albuterol in the first 30 min after inhalation are considered to result solely from pulmonary absorption.

Pressurized Metered Dose Inhalers The pressurized metered dose inhaler (pMDI) has been the backbone of inhalation therapy for asthma for approximately 50 years, since its introduction by 3 M Riker Laboratories in 1956. Patients and physicians recognized the convenience of the pMDI, which contains 100–200 doses in a small portable device that is immediately ready for use (Figure 1). The pMDI consists of an aluminum can mounted in a plastic actuator. Individual doses (25–100 ml) are delivered as a spray via a sophisticated metering valve. The drug is usually a micronized suspension of drug particles but may be a solution dissolved in propellants, ethanol, or another excipient as a co-solvent.

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AEROSOLS Patient presses canister while breathing in Canister Actuator

Drug formulation in propellants

Spray plume

Metering valve Actuator nozzle

Figure 1 Design and operation of a typical pressurized metered dose inhaler.

The best known pMDI therapies include the b-agonists albuterol, terbutaline, and salmeterol and the glucocorticosteroids beclomethasone dipropionate, budesonide, and fluticasone propionate. Successful pMDI therapy is highly dependent on the patient’s inhalation technique, and patient education about their use is essential. In most pMDI products, it is necessary for the patient to press the pMDI at the same time as inhaling. Failure to do this is sometimes described as poor coordination or hand–lung dyscoordination, and it is probably the most important problem patients have with pMDIs. A second major problem using pMDIs is the so-called cold Freon effect, where the patient stops inhaling when the cold propellant spray is felt on the back of the throat. Freon is one of the trade names of chlorofluorocarbon (CFC) propellants. In order to optimize lung deposition from pMDIs, patients also need to inhale slowly and deeply and to hold the breath for several seconds. Even with perfect inhalation technique, no more than 10–20% of the dose from a CFC pMDI is deposited in the lungs, with the majority of the dose being deposited in the oropharynx. However, the lung dose will vary from product to product according to the nature of the formulation and the diameter of the actuator orifice. Until recently, all pMDIs were formulated in CFC propellants, giving the pMDI an internal pressure of approximately 300 kPa (3 atm) and a spray velocity at the nozzle exceeding 30 m s
1. However, it is possible to reduce the spray velocity by modifications to the actuator design, for instance, in the Spacehaler device (formerly known as Gentlehaler). During the past few years, the pharmaceutical industry has been forced to start reformulating pMDIs in non-CFC propellants, consisting of one of two hydrofluoroalkanes (HFA-134a or HFA-227). This challenge arose

following the discovery that the degradation of CFCs damages stratospheric ozone and has proved to be a major stimulus to the development of novel inhaler technologies. The switch to HFA-powered pMDIs is in progress and will take several more years to complete. In the meantime, CFCs have been granted an essential-use exemption in pMDIs under the Montreal Protocol of 1987, reflecting their importance to the well-being of society. HFAs are greenhouse gases, and despite the fact that their contribution to global warming is small, this issue could restrict their future use. The development of novel HFA pMDI formulations has not been a simple manner, owing to a range of technical factors and the need to demonstrate clinical efficacy and safety for the reformulated products. Individual companies have adopted one of two strategies. One strategy involves making a product that is bioequivalent with the CFC pMDI that is to be replaced so that the HFA pMDI can be used in exactly the same doses as the CFC pMDI. The alternative strategy is to make a product that deposits drug in the lungs more efficiently than a CFC pMDI. This usually involves formulating a corticosteroid product as a solution, enabling a very small particle size to be achieved as the propellant evaporates. With such a product, it is also possible to reduce the spray velocity and to deposit up to half the dose in the patient’s lung, with greatly reduced oropharyngeal deposition, so that asthma control may be achieved using only a fraction of the CFC pMDI dose. A formulation of beclomethasone dipropionate (Qvar) was the first of these products to reach the market, and several similar products are either already marketed or in development. Breath-actuated pMDIs may be helpful in patients with poor coordination, who cannot actuate the pMDI at the same time as inhaling. These devices contain triggering mechanisms that are operated by the patient’s inhalation via the mouthpiece. However, it is unlikely that breath-actuated pMDIs confer any additional benefit on patients who can use a conventional pMDI successfully.

pMDIs with Spacer Devices Spacer devices are widely used with pMDIs. These vary greatly in size and shape, with volumes of commercially available models ranging from 50 to 750 ml. The concept of a spacer is to place some distance between the point at which the aerosol is generated and the patient’s mouth, allowing the propellant to evaporate and the rapidly moving aerosol cloud to slow down before it is inhaled (Figure 2). The most successful spacers have a one-way valve in

AEROSOLS 61

Formulation: ordered mixture of drug and carrier

DPI device

Powder de-aggregated by patient’s inhalation

Figure 3 Principle of operation of a dry powder inhaler (DPI). The formulation most frequently consists of an ordered mixture of micronized drug and carrier lactose, which is de-aggregated by the patient’s inhalation through the device.

Figure 2 pMDI connected to a large volume spacer device.

the mouthpiece, which allows the pMDI to be actuated into the spacer, with a brief pause before the patient inhales so that it is not necessary to actuate and inhale simultaneously. Some spacers function effectively if the patient takes a series of relaxed tidal breaths from the device immediately after actuating a dose. Spacers reduce oropharyngeal deposition of drug and may increase lung deposition, but the majority of the dose is often deposited on the walls of the spacer. This may allow the reduction of the total body burden of inhaled corticosteroids compared with a standard pMDI. Large volume spacers, such as the Volumatic and Nebuhaler, have a well-accepted role in hospital emergency rooms for treating acute asthmatic attacks. Specially designed spacers with a volume of 200–300 ml are available for treating young children. Most spacer devices are made of plastic, which may acquire a static charge during handling. This results in a suspended aerosol cloud being attracted to the spacer walls, with a marked reduction in the dose available for inhalation. Specific handling and washing techniques are usually recommended, and at least one lightweight metal spacer is available that is not susceptible to the effects of static charge. With correct use, including control over electrostatic charge effects, large volume (4500 ml) spacer devices may deposit more than 30% of the dose from a CFC pMDI in the patient’s lungs.

Dry Powder Inhalers Dry powder inhalers (DPIs) have been available commercially since approximately 1970, although the earliest prototypes were described several decades earlier. DPIs contain a powder formulation, which most frequently consists of an ordered mixture of micronized drug (o5 mm in diameter) and larger

carrier lactose particles that are required to improve powder flow properties. The patient’s inhalation through the device is used to disperse the powder and to ensure that some of the dose is carried into the lungs (Figure 3). An alternative type of formulation used in some DPIs consists either of micronized drug particles alone loosely aggregated into small spherules or of cospheronized drug and lactose. DPIs are basically of three types: (1) unit-dose devices, in which an individual dose in a gelatin capsule or blister is loaded by the patient immediately before use; (2) multiple unit-dose devices, which contain a series of blisters or capsules; and (3) reservoir devices, in which powder is metered from a storage unit by the patient before inhalation. Unit-dose devices, including Spinhaler and Rotahaler, were the only DPIs available until the mid-1980s. Patients generally find multiple unit-dose devices, such as the Diskus (Accuhaler), and reservoir DPIs, such as the Turbuhaler, to be more convenient than unit-dose DPIs since they provide several weeks’ treatment. DPIs tend to deposit a greater fraction of the dose in the lungs compared with CFC pMDIs, but in practice lung deposition varies widely between devices (Figure 4). Powder formulations are susceptible to the effects of moisture, and protecting the formulation against these effects is an important part of DPI design. By the end of 2004, at least 16 DPIs had been marketed in different areas of the world for asthma and COPD therapy, involving a range of unit-dose, multiple unit-dose, and reservoir systems. A further 20–30 DPIs were also known to be in development. The anticipated expansion of the generics market for inhaled asthma and COPD drugs is likely to result in a number of these novel DPIs reaching the market. It is interesting to note that the major pharmaceutical companies with an interest in inhaled asthma and COPD drugs appear to be prioritizing the DPI over reformulated HFA pMDIs products. In particular,

62

AEROSOLS 100 90

Percentage of dose

80 70 60 50 40 30 20 10 D is kh U ale ltr ah r al Pu er lv Sp inal in ha Ae ler ro liz e Ai r rm M AG ax Tu hal rb er uh Ea aler sy ha C lic ler kh al e N ov r ol iz e Ta r ifu Fl n ow ca p Ec s lip se

0

Figure 4 Mean percentage of the dose deposited in the lungs from 14 dry powder inhalers (DPIs), obtained in scintigraphic studies. The high lung deposition from the Flowcaps and Eclipse DPIs probably reflects the properties of the formulation as much as the DPI.

combination products in DPIs containing a longacting b-agonist and a corticosteroid (e.g., Advair Diskus and Symbicort Turbuhaler) have been very successful. However, DPIs tend to be more expensive than pMDIs, and this may limit their use, especially in developing countries. DPIs have two major advantages over pMDIs. First, they do not contain propellants. Second, all currently marketed models are breath-actuated, and patients find them easier to use correctly than pMDIs. However, this second advantage is closely linked to a disadvantage. In order to disperse the powder as efficiently as possible, and hence to maximize lung deposition, it may be necessary for patients to inhale as forcefully as possible via the DPI, and some patients may be either unable or unwilling to do this. All DPIs exhibit some degree of inhaled flow rate dependence, with forceful (fast) inhalation tending to give higher lung deposition than more gentle (slow) inhalation. For instance, in the Turbuhaler DPI, a reduction in peak inhaled flow rate from 60 to 30 l min
1 was shown to result in a reduction in lung deposition from 27% to 14% of the dose. In this respect, DPIs present a paradox since fast inhalation per se is generally associated with enhanced deposition in the oropharynx, as described previously. Low inspiratory effort through a DPI may result in a reduced emitted dose and poor particle deaggregation. The actual magnitude of the peak inhaled flow rate associated with forceful inhalation will vary between devices from o30 to 4100 l min
1, according to the resistance to airflow of each device. Not only the peak inhaled flow rate achieved through the DPI but

also the time taken to reach the peak flow will determine how efficiently particles are deaggregated. In practice, it seems that almost all patients with stable asthma or COPD can inhale sufficiently well via DPIs to benefit from them. Several so-called active DPIs have been developed, in which the powder is dispersed by some mechanism other than the patient’s inhalation – for instance, by an internal source of compressed air or by a fan driven by an electric motor. These active DPIs are generally more complex than breath-actuated DPIs and may come to be used primarily for therapies that require very efficient and reproducible targeting of drugs to specific lung regions, such as inhaled peptides for systemic therapy. Sophisticated formulations for use in DPIs are also in development. These include drug/lactose blends, in which the surface of the lactose particles has been smoothed in order to aid dispersion, or particles made by processes other than micronization. For instance, a spray-dried formulation of the antibiotic tobramycin is under development for the treatment and prevention of respiratory tract infections in patients with cystic fibrosis, consisting of low-density spherical particles that disperse efficiently with minimal inspiratory effort. An advantage of these sophisticated formulations is that often they can be delivered efficiently to the lungs using very simple and inexpensive DPI devices.

Nebulizers Drugs may often be formulated as solutions in water or ethanol, and they may be delivered by nebulizers

AEROSOLS 63 Signal from piezoelectric crystal

Drug formulation in reservoir

Mouthpiece Baffle Venturi

Mesh

Drug formulation in nebulizer cup

Compressed air

To mouthpiece

Figure 5 Design and operation of a typical jet nebulizer.

Figure 6 Principle of operation of a mesh-based nebulizer system. A mesh or grid is vibrated by a piezoelectric crystal, and a dispersion of micron-sized liquid droplets is formed.

that convert the solution into a spray. A variety of devices may be used to form the spray, and the three most common are jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. An important advantage of nebulizers is that they can be used with relaxed tidal breathing. This makes them attractive for delivering inhaled drugs to children, the elderly, and those undergoing acute asthmatic attacks, who may not be able to use pMDIs or DPIs successfully. Currently, nebulizers represent the most practical way to deliver very large drug doses (4100 mg) that are occasionally needed for some inhaled antibiotics. Jet nebulizers are operated by compressed air passing through a narrow constriction (a venturi). A single dose contained in a volume of typically 2–4 ml in a cup within the nebulizer is drawn up a feed tube and is fragmented into droplets (Figure 5). Only the smallest droplets are delivered directly to the patient; larger droplets impact on baffle structures situated close to the nozzle and are returned to the cup to be nebulized again. Several minutes are required to nebulize the entire dose, and even at completion of treatment the majority of the dose remains within the device as large droplets on internal walls. There are major differences in performance between different commercially available nebulizers, with lung deposition ranging from o2% to 20% of the dose. Jet nebulizers can also be used to aerosolize micronized suspensions of corticosteroids. Recent developments in technology have included breath-enhanced nebulizers, in which passage of inhaled air through the device is used to increase aerosol output, and adaptive aerosol delivery systems, in which aerosol generation is synchronized to coincide with the first part of the patient’s inhalation. Adaptive aerosol delivery systems seem to be

able to reduce the intersubject variability of aerosol delivery. Ultrasonic nebulizers have many properties similar to jet nebulizers, but the aerosol is formed in a different way. A piezoelectric crystal is located beneath the cup, and a fountain of droplets is generated. Ultrasonic nebulizers are less popular now than a few years ago, possibly for several reasons. They may not handle either suspensions or viscous solutions well, and there is evidence that they damage some drug molecules, probably by heat generated during the nebulization process. Jet and ultrasonic nebulizers cannot compete with pMDIs and DPIs in the portable inhaler market, partly because they are singledose devices and partly because they generally need either a compressor or a power source in order to function. Several novel nebulizers are available in which the spray is formed by the passage of drug solution through a vibrating mesh or grid of micron-sized holes (Figure 6). The mesh is usually vibrated by a piezoelectric crystal, but unlike ultrasonic nebulizers, there is no evidence that this process damages drug molecules. Mesh-based systems deliver a higher proportion of the dose, and achieve higher lung deposition, compared to jet or ultrasonic nebulizers. A smaller percentage of the dose is retained in the device at the end of treatment, and this can result in less wastage for expensive drug substances. Nebulization time is short compared to that of jet and ultrasonic nebulizers, which should improve patient compliance. Some vibrating mesh nebulizers are small, compact, and battery operated, giving them practical advantages over jet and ultrasonic nebulizers. Careful cleaning of all nebulizers is essential in order to avoid bacterial contamination and to ensure that the working parts (particularly narrow nozzles) function correctly.

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Soft Mist Inhalers A recent development in inhaler technology has been the development of low-velocity sprays known as soft mist inhalers. These devices represent a new class of multidose inhaler devices and contain liquid formulations similar to those in nebulizers. A variety of principles are utilized, including forcing liquid under pressure through a nozzle array, ultrasonics, vibrating meshes, and several novel approaches, such as condensation of vapors to form particle dispersions. Many of these devices are able to achieve extremely high lung deposition (450% of the dose), and they are capable of delivering drugs to the deepest parts of the lungs. This may allow them to play a major future role in inhalation therapy, particularly in situations in which precise aerosol targeting is needed. In 2004, one soft mist inhaler (Respimat) was launched in Europe for asthma and COPD therapy as a direct replacement for the same drugs given either in a CFC pMDI or in a DPI. The spray is formed by passing a metered dose (typically 15 ml) via a sophisticated nozzle system under pressure. The velocity of the spray is only a fraction of that found in a CFC pMDI. This device deposits a greater percentage of the drug in the lungs compared to a CFC pMDI (Figure 7), and it is clinically effective

Exhaled

100%

Device

80%

Oropharynx 60%

Lungs

40% 20% 0% CFC pMDI

Respimat

Figure 7 Fractionation of the dose from a novel Respimat soft mist inhaler compared to that from a pMDI formulated with chlorofluorocarbon (CFC) propellants. Data from Newman SP et al. (1998) Lung deposition of fenoterol and flunisolide delivered using a novel device for inhaled medicines. Chest 113: 957–963.

using smaller doses. It is probable that other soft mist inhalers will be marketed in the relatively near future, and some could mount a significant challenge to pMDIs and DPIs in the portable inhaler market. See also: Asthma: Overview. Bronchodilators: Anticholinergic Agents; Beta Agonists. Chronic Obstructive Pulmonary Disease: Overview: Emphysema, Alpha-1Antitrypsin Deficiency. Corticosteroids: Therapy. Cystic Fibrosis: Overview. Particle Deposition in the Lung.

Further Reading Adjei AL and Gupta PK (1997) Inhalation Delivery of Therapeutic Peptides and Proteins. New York: Dekker. Bisgaard H, O’Callaghan C, and Smaldone GC (eds.) (2003) Drug Delivery to the Lung. New York: Dekker. Dalby RN, Byron PR, Peart J, and Farr SJ (eds.) (2002) Respiratory Drug Delivery VIII. Raleigh, NC: Davis Horwood. Dalby RN, Byron PR, Peart J, Suman JD, and Farr SJ (eds.) (2004) Respiratory Drug Delivery IX. River Grove, IL: Davis Healthcare. Dolovich M, MacIntyre NR, Dhand R, et al. (2000) Consensus conference on aerosols and delivery devices. Respiratory Care 45: 588–776. Hickey AJ (ed.) 2003. Aerosol delivery and asthma therapy (theme issue). Advanced Drug Delivery Reviews 55, 777–928. Mitchell JP and Nagel MW (2003) Cascade impactors for size characterization of aerosols from medical inhalers: their uses and limitations. Journal of Aerosol Medicine 16: 341–377. More´n F, Dolovich MB, Newhouse MT, and Newman SP (eds.) (1993) Aerosols in Medicine: Principles, Diagnosis and Therapy. Amsterdam: Elsevier. Newman SP, et al. (1998) Lung deposition of fenoterol and flunisolide delivered using a novel device for inhaled medicines. Chest 113: 957–963. Newman SP and Newhouse MT (1996) Effect of add-on devices for aerosol drug delivery: deposition studies and clinical aspects. Journal of Aerosol Medicine 9: 55–70. O’Callaghan C and Barry PW (1997) The science of nebulised drug delivery. Thorax 52(supplement 2): S31–S44. Pauwels R, Newman SP, and Borgstro¨m L (1997) Airway deposition and airway effects of antiasthma drugs delivered from metered dose inhalers. European Respiratory Journal 10: 2127– 2138. Smith IJ and Parry-Billings M (2003) The inhalers of the future? A review of dry powder devices on the market today. Pulmonary Pharmacology and Therapeutics 16: 79–95.

Allergic Bronchopulmonary Aspergillosis see Asthma: Allergic Bronchopulmonary Aspergillosis.

ALLERGY / Overview 65

ALLERGY Contents

Overview Allergic Reactions Allergic Rhinitis

Overview J A Grant and C C Horner, University of Texas Medical Branch, Galveston, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Allergy is the term used for a collection of diseases mediated by immunologic mechanisms. Allergic disorders include allergic rhinitis, conjunctivitis, asthma, urticaria, angioedema, food allergy, drug allergy, and anaphylaxis. The prevalence of allergic diseases has been increasing in recent times. They are a major cause of morbidity and decreased quality of life. The development of allergic diseases is influenced by heritable and environmental factors. Diagnosis often involves documenting responses to allergen such as in skin testing, radioallergosorbant allergen testing, or bronchial provocation testing. Allergic disorders share the common pathology of inflammation of affected tissues. Allergy requires sensitization to an allergen and response on reexposure to that same allergen. Pathogenesis involves production and release of cytokines, chemokines, and lipid mediators which cause tissue damage and recruit inflammatory cells. Current therapies include allergen avoidance, antihistamines, leukotriene modifiers, corticosteroids, phosphodiesterase inhibitors, humanized monoclonal anti-IgE, and immunotherapy.

Introduction Allergy encompasses a wide variety of disorders that share immunologic mechanisms. For centuries, patients had allergic symptoms but the causative agent for sensitivity to allergens was unknown. Allergic symptoms were first described by Leonardo Bottallo in sixteenth century Europe. Wyman identified ragweed as the trigger of the ‘autumnal catarrh’ and Blackely identified hay fever as being initiated by grass pollen in the 1870s. Twenty years later Behring described the adverse skin reactions to tubercle bacillus as ‘hypersensitivity’. Portier and Richet attempted to confer immunity to sea anemone toxin by injecting dogs with subsequent doses of toxin. They employed the term ‘anaphylaxis’ (denoting antiprotection) in 1902 to describe a lethal reaction after the dogs received the second dose of the toxin. Von Pirquet used the term ‘allergy’ in 1906 to describe the skin reaction to cowpox vaccine at 24 h post-vaccination. His definition of allergy described an organism’s alteration

by contact with an organic agent. Four years later, Noon observed reactions to pollen extracts on abraded skin and began immunotherapy with these extracts. Dale and Laidlaw produced respiratory distress and anaphylaxis in animals by histamine infusion in 1919. Another major discovery was the identification of cytokines. This class includes chemokines that are important in the recruitment of inflammatory cells. In 1921, Prausnitz and Kustner demonstrated that a factor could be transferred to a nonsensitized person’s skin and confer sensitivity. This factor was called ‘reagin’ by Coca and Cooke. In 1966, Ishizaka’s discovery of IgE established a scientific basis for the specific reactions. Concurrently, S G O Johannson independently identified a protein in multiple myeloma that was also elevated in allergic patients. Subsequently, the WHO determined that both proteins were IgE. In 1968, Gell and Coomb described four classes of immunologic reactions. These classes are IgE-mediated immediate hypersensitivity (e.g., anaphylactic shock), IgG- or IgM-mediated cytotoxic reactions (e.g., immune hemolytic anemia), immune complexmediated reactions (e.g., serum sickness), and delayed hypersensitivity (e.g., contact dermatitis). Allergic diseases can be mediated by any of these mechanisms. Allergic disorders include allergic rhinitis, conjunctivitis, asthma, urticaria, angioedema, food allergy, drug allergy, and anaphylaxis.

Etiology Atopy refers to the tendency of patients to be sensitive to allergens. Atopic diseases include eczema, allergic rhinitis, and asthma. Over the past century, genetics has been thought to affect allergic disease occurrence. The incidence of allergic disease in patients with an allergic family history is higher than in those without one. Atopy is thought to be autosomally transmitted and multigenic. It is probably a complex trait influenced by both heritable and environmental factors. Environmental exposures likely have effects on gene expression and the development of allergic disease. The hygiene hypothesis proposes

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that in Western countries the developing immune system is deprived of environmental microbial antigens that stimulate Th1 cells. This lack of stimulation increases the presence of atopic disease. Other factors such as diet and exposure to high pollen counts at birth may also increase allergic disease. Linkage studies for allergy and asthma have produced 420 distinct chromosomal regions with linkage to asthma or related traits. The lack of distinct phenotypes, the inexact definitions of allergic diseases, and the presumed influence of numerous genes have made positional cloning with linkage studies problematic. The linkages reproduced most frequently are on 6p, 5q, 12q, and 13q. Linkage on 14q and 7p was identified in founder populations in Iceland and Finland. Chromosome 2q14–2q32, which includes the IL-1 gene, has been linked to asthma. Candidate gene analysis is a promising technique. Candidate genes encode biochemical markers that affect allergic diseases. Many of these genes control IgE and cytokine production. Candidate genes include one loci on 5q near the gene cluster for IL-4, IL-5, IL-9, and IL-13. Polymorphisms of the IL-9 gene are associated with human asthma. 11q13, which encodes the b chain of the high-affinity IgE receptor has been linked to asthma. Polymorphisms of the IL-4 receptor a chain are associated with atopic asthma. PHF11, present in the locus for total IgE on 13q14, is expressed in many immune-related tissues. It is associated with total IgE and has been linked with asthma in multiple genome screens. The ADAM33 gene on 20p13 encodes a disintegrin and metalloprotease. This gene was mapped in 2002 as an asthma and airway hyperresponsiveness gene by Van Eerdewegh and co-workers. A cluster of single nucleotide polymorphisms (SNPs) was identified in the ADAM33 gene and demonstrated significant associations with asthma. Howard and co-workers reproduced the association seen by Van Eerdewegh’s group and lent support to the potential role of ADAM33 in asthma susceptibility (see Genetics: Gene Association Studies).

Pathology Allergic inflammation is present in all allergic diseases. Most tissues exhibit vasodilation and increased vascular permeability. Eosinophils, neutrophils, CD4 þ T cells, and basophils eventually infiltrate the site of allergy. Asthma is a disorder which also involves an increase in mucous glands, mucus hypersecretion, smooth muscle hypertrophy, and airway remodeling. Recruitment of eosinophils is a prominent feature of allergic diseases (see Leukocytes: Eosinophils). Eosinophils migrate across blood vessels into tissues

by binding endothelial cell adhesion molecules. Major basic protein, lipid mediators, and cytokines enable the eosinophil proinflammatory effects. Eosinophils may also repair damage to mucosal surfaces with fibrogenic growth factor and matrix metalloprotease. This repair mechanism may result in remodeling of airway tissue as seen in asthma. Mast cells are present in tissues throughout the body. Features of an allergic reaction vary with the anatomic site. The site of allergen contact determines the tissue or organ involved. The concentration of mast cells at the site determines the severity. The wheal and flare is the typical cutaneous allergic response. When allergen binds to specific IgE on mast cells, mast cell mediators are released and cause local blood vessel dilation. These vessels leak fluid and macromolecules and produce redness and swelling (known as the wheal). Dilation of vessels on the edge of the swelling causes redness (known as the flare).

Clinical Features Patients with allergic rhinitis typically experience rhinorrhea, congestion, sneezing, and itchy nose. Patients also may report postnasal drip and associated eye, ear, and throat symptoms. Patients commonly exhibit reactions to dust mite, animal dander, molds, and pollens. Small molecular weight chemicals can also react with self-proteins and become allergens. Reactivity to allergens can be determined by immediate hypersensitivity skin testing. Drops of allergen are placed into the dermis by various methods. Most skin testing employs a prick device composed of metal or plastic to introduce allergen into the skin. Allergen binds IgE and the mast cells in the skin release histamine to produce the wheal and flare response. The skin response can be compared to controls of saline (negative control) and histamine (positive control). Radioallergosorbant allergen testing (RAST) quantitates allergenspecific IgE in a patient’s serum. RAST is usually reserved for patients who have a contraindication to skin testing or are taking medications that either interfere with testing (antihistamines) or interfere with treatment should a reaction occur (beta blockers or ace inhibitors). Nasal cytology can reveal the presence or absence of inflammatory cells, especially eosinophils. Adjunct tests such as rhinoscopy and rhinomanometry can provide further characterization of the nasal airway (see Allergy: Allergic Rhinitis). Patients with asthma report symptoms of wheezing, shortness of breath, cough, and chest tightness. These patients’ airways show hyperresponsiveness

ALLERGY / Overview 67

with reversibility. Patients may experience asthmatic symptoms in response to allergens, infections, exercise, nonsteroidal anti-inflammatory drugs, gastroesophageal reflux disease, stress, and irritants. Lung function can be assessed by spirometry before and after treatment with bronchodilators. Bronchial hyperresponsiveness can be investigated with bronchial provocation testing such as with methacholine or exercise challenge testing (see Asthma: Overview). Urticaria is localized edema in skin or mucous membranes. Patients have pale to pink wheals that are extremely pruritic. These lesions are transient and usually resolve within 24 h. Angioedema involves local edema in deeper areas of skin or mucous membranes. These lesions are often painful. Urticaria/angioedema can be triggered by temperature, sun, direct pressure, medication, infections, foods, or systemic diseases. Atopic eczema is a skin disorder with pruritis, erythematous macules or papules, and xerosis. Lesions may become excoriated with crust and exudates. Skin tends to be dry and more permeable to allergens and bacteria. Chronic irritation may cause lichenification of the skin and scaling patches or papules. Young children typically have lesions on the face, scalp, and extensor surfaces. Older children and adults have flexor surface involvement. Food allergy is most frequent in young children. Symptoms may include urticaria, angioedema, rash, flushing, rhinitis, wheezing, or anaphylaxis after ingestion of the allergenic food. Some patients experience the oral allergy syndrome which is usually confined to the oropharynx. Symptoms may consist of pruritis and angioedema of the tongue, lips, palate, and throat. Patients with birch pollen-induced rhinitis may develop oral allergy symptoms after eating raw potato, carrot, apple, celery, or hazelnut. Patients with ragweed pollen-induced rhinitis may develop symptoms after eating melons or bananas. Major food allergens in children are milk, egg, peanuts, soybeans, wheat, fish, and tree nuts. Major food allergens in adults are peanuts, tree nuts, fish, and shellfish. Conformational and sequential food epitopes are responsible for food allergy. Patients with IgE to sequential epitopes react to all forms of a food and tend to have persistent allergy, whereas those with IgE to conformational epitopes tolerate small amounts of food after heating or partial hydrolysis because these conformational epitopes are destroyed. These patients tend to have clinical tolerance. Prick skin testing for foods can be performed as described previously. Negative prick skin tests have a high negative predictive value. Positive skin tests are not conclusive. RAST may also be performed to aid in diagnosis.

Drug allergy reactions most commonly consist of urticaria, morbilliform rash, or anaphylaxis. Skin testing for drug allergy is not standardized and the predictive value of this technique is unclear. Anaphylaxis is an immediate generalized systemic allergic reaction in response to an allergen. Symptoms may include flushing, pruritis, urticaria, angioedema, wheezing, shortness of breath, chest tightness, abdominal pain, nausea, vomiting, diarrhea, laryngeal edema, arrhythmia, myocardial infarction, and hypotension. Serum tryptase can be measured within 6 h of a reaction to determine if mast cell degranulation has occurred during the reaction. Serum histamine and urinary LTE4 may be quantitated to provide evidence of anaphylaxis.

Pathogenesis Immunology plays a large role in allergic diseases. T lymphocytes are the major effector cells of these immune responses (see Leukocytes: T cells). CD4 þ lymphocytes are present in two predominant types, Th1 and Th2 cells. Th1 are helper cells that produce IL-2 and interferon gamma (IFN-g) and promote cellmediated reactions. Th2 are helper cells that produce IL-4, IL-5, IL-6, IL-10, and IL-13 and are involved in humoral immunity and allergic inflammation. Th2 cytokines augment antibody production (especially IgE), enhance eosinophil production, and are associated with allergic and antibody-driven responses (Figure 1). Th1 and Th2 cells suppress each other. Patients with an allergic phenotype generate responses to allergens that favor Th2 responses. Another type of T cell is the regulatory T cell (T reg). Thymus-derived CD4 þ CD25 þ regulatory cells are termed naturally occurring T regs. Adaptive T regs are T-cell populations induced by in vitro or in vivo manipulation. Active T-cell suppression by T regs promotes immunologic tolerance, but the mechanism of this suppression is controversial. There is a potential role for T regs in the control or prevention of Th2 responses. CD4 þ CD25 þ T cells and IL-10producing T regs have been shown in humans to prevent T-cell activation by allergens and are possibly deficient in atopic patients. T regs may also secrete the immunosuppressive cytokines IL-10 and TGF-b. IL-10 inhibits macrophage activation and antagonizes IFN-g while TGF-b inhibits T- and B-cell proliferation. IgE-mediated allergic responses occur through numerous steps. First, a patient is exposed to an allergen. Then antigen-presenting cells internalize the allergen which is then processed and presented to Th2 cells by class II MHC molecules. T-cell receptors bind the presented allergen thus activating the Th2

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ALLERGY / Overview APC

Th2 cell

Allergen IL-4

Specific IgE

Re-exposure to allergen B cell Arachidonic acid

Prostaglandins, leukotrienes

Mast cell Preformed mediators (histamine, TNF-
)

Vascular leak, bronchoconstriction, inflammation, tissue damage, late response

Early response

Figure 1 Allergen is processed by antigen presenting cells (APCs) and presented to Th2 cells. Stimulated by IL-4, B cells produce allergen-specific IgE. Upon re-exposure to allergen, allergen cross-links specific IgE on mast cells and activates allergic responses.

cells. The activated cells produce IL-4. Atopic patients have more allergen-specific IL-4 secretory T cells in circulation than nonatopics and produce more IL-4 per cell. B cells, in turn, are stimulated by IL-4 to class-switch to produce IgE, specific to the allergen. Approximately 20% of an exposed population will generate specific IgE to an allergen. This IgE binds to receptors on mast cells. When the patient is subsequently exposed to the allergen, the allergen binds the IgE already present on the mast cell surfaces and cross-links the antibodies. Antibody cross-linking of mast cells activates a variety of responses. The early phase of immediate hypersensitivity occurs within minutes and involves the release of preformed mediators from granules. These mediators include biogenic amines (histamine), enzymes (tryptase, chymase), carboxypeptidase, cathepsin G, acid hydrolases, and tumor necrosis factor alpha (TNF-a) (see Leukocytes: Mast Cells and Basophils). Histamine causes endothelial cells to contract and allow plasma to leak into tissue. Endothelial cells also produce prostacyclin and nitric oxide which promote vascular smooth muscle relaxation and lead to vasodilation. Bronchial smooth muscle constriction is also caused by histamine. Antibody cross-linking also triggers the initiation of several pathways which lead to cytokine, prostaglandin, and leukotriene production. Arachadonic acid is converted to prostaglandin D2 and the cysteinyl leukotrienes (LTC4, LTD4, LTE4) (see Lipid Mediators: Leukotrienes). These mediators are responsible for vascular leak, bronchoconstriction, inflammation, and tissue damage. These substances lead to inflammatory changes hours after exposure, referred to as the late-phase response. The late phase involves recruitment and infiltration of the mucosa

with neutrophils, eosinophils, basophils, and Th2 cells. TNF-a activates endothelial expression of adhesion molecules E-selectin and ICAM-1 and promotes cell infiltration. IL-3 promotes mast cell proliferation. IL-5 stimulates eosinophil production and activation (see Interleukins: IL-5). The chemokines eotaxin and monocyte chemotactic protein-5 from epithelial cells recruit eosinophils. IL-4 and IL13 induce Th2 differentiation. While mast cells are responsible for the majority of leukotriene production in the early response, basophils and eosinophils produce most of the leukotrienes in the late-phase response.

Animal Models Mouse models have been used in the study of allergy because mice are readily available, have a well-characterized immune system, and strains are genetically characterized. Knockout mice can be used to evaluate the role of a cell type or mediator. Many allergists propose that allergic rhinitis and asthma may represent a continuum of inflammation and should be considered as a united allergy airway disorder. Mouse models have been used to investigate this concept. Mice were systemically sensitized with allergen (most often ovalbumin) and then challenged with airway allergen. The inflammatory response in the mouse nose resembles human allergic rhinitis. Nasal mucosal thickening can be seen on imaging. Experimental asthma in mice also mimics human asthma. Bronchial hyperresponsiveness can be documented by plethysmography. Most of the inflammation in mice is seen in the lower airways where the minority of allergen is deposited. This may indicate increased sensitivity in the lower airways. Inhaled

ALLERGY / Overview 69

allergen causes an increase in allergen-specific IgE and eosinophils in the blood and increases bone marrow eosinopoiesis. Currently, it is unclear why sensitization causes symptoms in the nose, the lower airway, or both. There may be a genetic cause so studying different strains of mice may be useful. Mouse models have also been used in the study of atopic eczema. Mice have been sensitized epicutaneously with ovalbumin on shaved skin. Elevated serum total and specific IgE and IgG1 and increased dermal IL-4, IL-5, and IFN-g were observed. Deficiencies of these cytokines decreased the eczematous phenotype. For example, IL-4-deficient mice showed Th1biased skin inflammation with decreased eosinophils and eotaxin mRNA in the dermal infiltrate. IL-5deficient mice similarly had less pronounced epidermal and dermal thickening and the dermal infiltrate lacked eosinophils. IFN-g-deficient mice showed only slight dermal thickening. The necessity of certain lymphocytes in eczema was demonstrated. ab T cells are essential since T-cell receptor a-chain-deficient mice did not develop a dermal infiltrate or induction of IL-4 or IgE. Mice that lack gd T cells showed no change in infiltrate. Likewise, mice that lack B cells still develop an infiltrate and elevated IL-4. Numerous animal models have been utilized in food allergy. Rats and mice have been used to assess foodinduced anaphylaxis. Animals ingested ovalbumin by gavage or in drinking water and were subsequently challenged intraperitoneally. The route, dose, and age of the animal were shown to influence sensitization. Rats and swine sensitized to allergens and subsequently challenged orally, demonstrated alterations in small intestinal pathology. Tolerance is dose-dependent for specific antigens. Mice fed ovalbumin or peanuts required a 50-fold higher dose of peanuts to develop tolerance; low doses of peanuts were more likely to induce sensitization. The importance of an intact mucosal barrier was shown when mice, fed a novel dietary protein while their gastrointestinal tracts were inflamed, developed sensitization and high serum IgE. Allergic conjunctivitis has been studied in guinea pigs, rats, and mice. Mice exposed to ragweed by topical contact with conjunctival and nasal mucosa developed signs of allergic conjunctivitis and ragweedspecific IgE. Regulators of vascular permeability are important in allergic conjunctivitis. Substance P has been shown to be a mediator of allergic conjunctivitis and acts through NK1 receptors on blood vessels to produce conjunctival hyperpermeability. Nitric oxide has been shown to play a major role in regulating vascular permeability and stimulating prostaglandin E2 production. T-cell adhesion molecules are integral in allergic conjunctivitis. Guinea pig models with ovalbumin have shown that the integrin very

late activation antigen-4 (VLA-4) plays a critical role in eosinophil infiltration. Other mice studies showed that antibodies against the integrin intercellular adhesion molecule-1 (ICAM-1) and its ligand leukocyte function-associated antigen-1 (LFA-1) inhibited clinical and histological signs of conjunctivitis. The significance of IL-1 for inflammatory changes in conjunctivitis was demonstrated using an IL-1 receptor antagonist in mice exposed to cat dander antigens. Studies in rats using ovalbumin showed that IFN-g suppresses the development of allergic conjunctivitis during the induction phase.

Management and Current Therapy One of the cornerstones of allergy treatment is avoidance. Avoiding or reducing allergen exposure prevents or minimizes the body’s response to allergen. Environmental control measures help one decrease exposure. For example, patients with house dust-mite allergy can encase their mattresses and pillows in special covers to minimize exposure to dust mites while asleep. One study showed that in inner-city children with atopic asthma, a comprehensive environmental intervention decreased exposure to indoor allergens and reduced asthma-associated morbidity. Avoiding allergic foods or drugs can prevent reactions (Figure 2). Depending on one’s sensitivities, allergen avoidance may range from simple to extremely difficult. Other therapies available to treat allergic disorders include antihistamines, leukotriene modifiers, corticosteroids, phosphodiesterase inhibitors, humanized monoclonal anti-IgE, and immunotherapy. H1-antihistamines have been used for decades for the relief or prevention of allergic symptoms. Antihistamines have recently received the designation of inverse agonists because they stabilize the inactive form of the H1 histamine receptor. First-generation antihistamines have marked sedation; second-generation antihistamines that are relatively nonsedating Therapy algorithm Allergy diagnosis

Asthma diagnosis

Avoidance of allergen

Avoidance of triggers, allergens

Pharmacotherapy Pharmacotherapy Immunotherapy Immunotherapy Figure 2 Therapy for both allergic rhinitis and asthma starts with avoidance of allergens. Pharmacotherapy can be added. Some patients with allergic rhinitis and asthma benefit considerably from immunotherapy.

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have also been identified. Antiallergic activities include inhibiting the release of mast cell mediators probably through direct inhibition of Caþ channels. Anti-inflammatory effects include inhibiting cell adhesion molecule expression and inhibiting inflammatory cell chemotaxis (e.g., eosinophil chemotaxis). These inhibitions probably involve the downregulation of NF-kB, a transcription factor that regulates adhesion proteins and proinflammatory cytokines. Antihistamines have established roles in the treatment of allergic rhinitis and urticaria. A potential role exists for the treatment of anaphylaxis or asthma. The cysteinyl leukotrienes and LTB4 are products of arachadonic acid metabolism by 5-lipoxygenase. Leukotrienes cause airway inflammation and obstruction by affecting mucus production, smooth muscle contraction, and vascular permeability. They may also affect airway remodeling. The effects of leukotrienes are most likely mediated through the CysLT1 receptor. The CysLT1 receptor antagonists montelukast, pranlukast, and zafirlukast block the actions of LTC4, LTD4, and LTE4. A nonselective antagonist of the CysLT1 and CysLT2 receptors is not yet clinically available. Zileuton is a 5-lipoxygenase inhibitor and decreases the production of leukotrienes. Leukotriene modifiers are useful in allergic rhinitis and asthma. Leukotriene modifiers inhibit an important part of the inflammatory cascade and decrease eosinophil survival, goblet cell hyperplasia, mucus release, collagen deposition, and airway smooth muscle proliferation. Corticosteroids block the production of inflammatory cytokines (see Corticosteroids: Therapy). They may be given topically at the site of inflammation (nose, lungs, or skin) or delivered systemically. Glucocorticoids are the most potent therapy for treating all allergic disorders. They are liposoluble

hormones that enter the cell and bind a cytoplasmic glucocorticoid receptor (see Corticosteroids: Glucocorticoid Receptors). This receptor translocates to the nucleus and binds a glucocorticoid-response element in the promoter region of target genes. The glucocorticoid receptor can also bind transcription factors like NF-kB and AP-1 and prevent these factors from binding their DNA-response elements. Glucocorticoids control airway inflammation by inhibiting transcriptional activity of genes encoding proinflammatory molecules such as cytokines, chemokines, adhesion molecules, and mediator-synthesizing enzymes. They may suppress histone acetylation and stimulate histone deacetylation. They may also interfere with signal transduction pathways, such as MAP kinase enzymatic cascades involved in the regulation of transcription factors. Theophylline is a nonselective phosphodiesterase inhibitor with a narrow therapeutic ratio and significant drug interactions and has been used exclusively in asthma (see Bronchodilators: Theophylline). Inhibitors of phosphodiesterase type 4 have recently been developed. These inhibitors increase the intracellular concentration of CAMP and exhibit a broad range of anti-inflammatory effects on effector cells. Blocking the PDE4B receptor subtype appears responsible for anti-inflammatory properties of these agents. Cilomast and roflumilast are PDE4 inhibitors that are in late phase III clinical trials. Roflumilast has demonstrated more selectivity and a superior therapeutic ratio (Figure 3). The humanized monoclonal anti-IgE omalizumab is a new therapy which decreases the amount of IgE available for reactions and downregulates the number of IgE receptors on mast cells. This therapy is currently available for the treatment of severe asthma. Its use in food allergy is being investigated.

Clinical features • Allergic rhinitis • Asthma • Urticaria • Angioedema • Atopic eczema • Food allergy • Oral allergy syndrome • Drug allergy • Anaphylaxis

Rhinorrhea, congestion, sneezing, itchy nose Wheezing, shortness of breath, cough, chest tightness Pruritic wheals Deep local swelling of skin or mucus membranes Pruritis, erythematous maculesor papules, xerosis, lichenification Urticaria/angioedema, rash, flushing, rhinitis, wheezing, anaphylaxis Pruritis/angioedema of tongue, lips, palate, throat Uticaria, morbilliform rash, anaphylaxis Flushing, pruritis, urticaria/angioedema, wheezing, shortness of breath, abdominal pain, nausea, vomiting, diarrhea, laryngeal edema, arrhythmia, myocardial infarction, hypotension

Figure 3 Clinical features associated with various allergic disorders.

ALLERGY / Overview 71 Diagnostic workup Allergic rhinitis

Asthma

History and physical examination

History and physical examination

Immediate hypersensitivity skin testing or RAST

Spirometry

Medication trial Nasal cytology

Rhinoscopy or rhinomanometry

Bronchial provocation testing

Figure 4 Diagnostic workups start with the history and physical exam. Initial testing for allergic rhinitis consists of skin testing or RAST. Adjunct tests include nasal cytology, rhinoscopy, and rhinomanometry. Lung function in asthma is assessed by spirometry and a medication trial is begun. Bronchial provocation testing can assess bronchial hyperresponsiveness if needed.

Patients with allergic rhinitis and/or asthma who are (Figure 4) poorly controlled on medications may find immunotherapy (allergy shots) a feasible alternative. Immunotherapy involves administering injections of allergens to which a patient is sensitive. Increasing doses of allergen are given weekly until the patient is at a maintenance dose. This maintenance dose is continued monthly for 3 to 5 years. Patients receive relief from nasal allergy symptoms and their asthma may improve. The mechanisms of immunotherapy are not well defined. Immunotherapy may induce specific T-cell tolerance or a shift from a Th2 to a Th1 phenotype. Possibly, the Th2 response is inhibited, Th1 response is upregulated, or both. Immunotherapy also increases the production of IL-10 and TGF-b by T cells including T regs. IgG is also produced in response to antigen instead of the typical IgE. Allergen-specific IgG or blocking antibodies may compete with IgE for allergen and inhibit IgE activation of mast cells. IgG can also bind epitopes on allergen that are not recognized by IgE. This IgG binding may prevent cross-linking of IgE. Allergen-IgG complexes on antigen-presenting cells might impair antigen processing or the co-stimulation of T cells and render patients anergic. Immunotherapy is a type of desensitization, where small amounts of allergen are given until the patient tolerates the allergen. The same theory is used in treatment of drug allergies by giving increasing doses of drug until a therapeutic dose is tolerated. Unmethylated CG dinucleotides, or CpG motifs, are responsible for the immunostimulatory effect of

bacterial DNA and induce a Th1 type response in humans. Synthetic oligodeoxynucleotides mimic the bacterial DNA immunostimulatory sequences. These synthetic nucleotides can be conjugated to allergen to produce an allergen vaccine that is more immunogenic but less allergenic than allergen alone. These vaccines are currently under clinical trials. Recently, there has been an interest in pharmacogenetics. This field recognizes that medications may work more efficaciously in certain patients because of their genetic makeup. Single nucleotide polymorphisms (SNPs) may signal a change in proteins or amino acids in an individual. These changes may alter a drug’s target, uptake, metabolism, or excretion. A polymorphism in Gly 16 promotes bronchodilator resistance while the Arg 16 polymorphism potentiates a greater response to bronchodilators. An alternatively spliced form of glucocorticoid receptor b is present with higher frequency in corticosteroid-resistant patients. A polymorphism in the 5-lipoxygenase promoter decreases the response to the 5-lipoxygenase inhibitor zileuton. Pharmacogenetics is an exciting area that may shape the future of treatment for allergic diseases. See also: Allergy: Allergic Reactions; Allergic Rhinitis. Asthma: Overview. Bronchodilators: Theophylline. Chemokines. Corticosteroids: Glucocorticoid Receptors; Therapy. Genetics: Gene Association Studies. Immunoglobulins. Interleukins: IL-5. Leukocytes: Mast Cells and Basophils; Eosinophils; T cells. Lipid Mediators: Leukotrienes; Prostanoids. Matrix Metalloproteinases.

Further Reading Abbas AK and Lichtman AH (2003) Cellular and Molecular Immunology, 5th edn. Philadelphia: Saunders. Adelman DC, Casale TB, and Corren J (2002) Manual of Allergy and Immunology, 4th edn. Philadelphia: Lippincott Williams & Wilkins. Broide DH (2001) Molecular and cellular mechanisms of allergic disease. Journal of Allergy and Clinical Immunology 108: S65–S71. Cakebread JA, et al. (2004) The role of ADAM33 in the pathogenesis of asthma. Springer Seminars in Immunopathology 25: 361–375. Creticos PS, Chen Y, and Schroeder JT (2004) New approaches in immunotherapy: allergen vaccination with immunostimulatory DNA. Immunology and Allergy Clinics North America 24: 569–581. Groneberg DA, et al. (2003) Animal models of allergic and inflammatory conjunctivitis. Allergy 58: 1101–1113. Gutermuth J, et al. (2004) Mouse models of atopic eczema critically evaluated. International Archives of Allergy and Immunology 135: 262–276. Hallstrand TS and Henderson WR (2002) Leukotriene modifiers. Medical Clinics of North America 86: 1009–1033. Hellings PW and Ceuppens JL (2004) Mouse models of global airway allergy: what have we learned and what should we do next? Allergy 59: 914–919. Helm RM and Burks AW (2002) Animal models of food allergy. Current Opinion in Allergy and Clinical Immunology 2: 541–546.

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Holgate ST (2004) Pharmacogenetics: the new science of personalizing treatment. Current Opinion in Allergy and Clinical Immunology 4: 37–38. Joad J and Casale TB (1988) Histamine and airway caliber. Annals of Allergy 61: 1–7. Kay AB (2001) Allergy and allergic diseases: first of two parts. New England Journal of Medicine 344(1): 30–37. Kim DS and Drake-Lee AB (2003) Allergen immunotherapy in ENT: historical perspective. Journal of Laryngology & Otology 117: 940–945. Lipworth BJ (2005) Phosphodiesterase-4 inhibitors for asthma and chronic obstructive pulmonary disease. Lancet 365: 167–175. Morgan WJ, et al. (2004) Results of a home-based environmental intervention among urban children with asthma. New England Journal of Medicine 351: 1068–1080. Pelaia G, et al. (2002) Molecular mechanisms of corticosteroid action in chronic inflammatory airway diseases. Life Sciences 72: 1549–1561. Robinson DS, Larche M, and Durham SR (2004) Tregs and allergic disease. Journal of Clinical Investigation 114: 1389–1397. Sampson HA (2004) Update on food allergy. Journal of Allergy and Clinical Immunology 113(5): 805–819. Shearer WT and Li JT (2003) Primer on allergic and immunologic diseases. Journal of Allergy and Clinical Immunology 111(2): S441–S778. Simons FER (2004) Advances in H1-antihistamines. New England Journal of Medicine 351: 2203–2217. Till SJ, et al. (2004) Mechanisms of immunotherapy. Journal of Allergy and Clinical Immunology 113(6): 1025–1034. Weiss ST and Raby BA (2004) Asthma genetics 2003. Human Molecular Genetics 13: R83–R89.

Allergic Reactions S H Arshad, University Hospital of North Staffordshire, Stoke-on-Trent, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Allergy is a harmful response to an otherwise innocuous substance. Allergic reactions are immunologically mediated reactions to external substances, usually proteins. These are often, but not always, mediated by immunoglobulin E (IgE) antibody. Initial exposure to an allergen results in sensitization with the production of allergen-specific IgE antibodies. These antibodies circulate in the blood but are largely bound to high-affinity receptors on the surface of basophils and mast cells and lowaffinity receptors on eosinophils, macrophages, and platelets. On further exposure, the allergen reacts with the IgE bound to mast cells and basophils, causing degranulation and release of preformed mediators such as histamine and tryptase. These mediators cause the immediate phase of the type I reaction, which occurs within a few minutes (immediate hypersensitivity). Other mediators and cytokines are released and eosinophils are attracted to the site of activity, precipitating the late phase of the type I reaction, which starts 4–6 h after exposure. Immediate hypersensitivity reaction occurs in asthma, rhinitis, and anaphylaxis. In a sensitized asthmatic, early and late phase asthmatic reaction can be observed following bronchial allergen challenge. Repeated or continued exposure to allergen results in chronic airway inflammation, which is characteristic of asthma.

Systemic allergic reactions vary in severity from mild (such as generalized urticaria) to severe and life-threatening reactions with cardiovascular collapse and death. Anaphylaxis is the clinical syndrome that represents the life-threatening systemic allergic reaction. It results from the immunologically induced release of mast cell and basophil mediators after exposure to a specific antigen in previously sensitized individuals. Clinically indistinguishable reactions, caused by non-IgE-mediated immune mechanisms, are termed anaphylactoid reactions. Common causes of anaphylaxis are foods, drugs, insect stings, latex, and allergen extracts used for immunotherapy. The symptoms of anaphylaxis occur within a few minutes of exposure and are generally related to the skin, gastrointestinal tract, respiratory tract, and cardiovascular systems. Common manifestations include generalized urticaria/angioedema, nausea, vomiting, stridor, wheezing, hypotension, and syncope. Anaphylaxis requires immediate treatment with epinephrine, given intramuscularly. The dosage for adults is 0.3–0.5 ml, and for children is 0.01 ml kg
1, of a 1:1000 solution. The dose can be repeated at 5–15 min intervals. Supportive treatment includes cardiopulmonary resuscitation (if required), intravenous fluids, oxygen, antihistamine, and corticosteroids. Following the first episode, an assessment by an allergist is essential to establish the cause and for appropriate advice on preventive measures including avoidance of the offending agent and self-injectable epinephrine.

Immune Responses Atopy is defined as the genetic predisposition to form immunoglobulin (IgE) antibodies on exposure to allergens. The production of IgE is central to the induction of allergic diseases. Allergens are proteins with the capability to react to the immune system through their antigenic determinants. Initial exposure to the antigen results in sensitization. Antigens enter the body through the respiratory and gastrointestinal mucosa and the skin. Allergenic proteins are engulfed by antigen-presenting cells (APCs) such as monocytes, macrophages, and dendritic cells inducing primary immune response. The antigen is broken down to reveal the specific part of the molecule called antigenic determinant or epitope. Once processed in this way, the antigen is bound to the MHC class II molecules on the surface of these cells and the complex is presented to the T lymphocyte cell receptor. Bacterial antigens favor the production of Th1 cells with the secretion of its profile of cytokines, particularly interferon gamma (IFN-g). In atopic individuals, and in the presence of co-stimulatory signals, naı¨ve T cells are converted to activated CD4 þ T-helper-2 (Th2) cells. T lymphocytes play a central role in orchestrating the allergic reaction. Th2 cells produce cytokines, such as interleukin-4 (IL-4) and IL-13. These cytokines cause proliferation and switching of B cells to IgE producing B and plasma cells, specific to the antigen (Figure 1). Some of these cells have a long life and are called memory cells. IL-4 is the most

ALLERGY / Allergic Reactions 73 Antigen

IgE

Antigen-presenting cell

Naive T cell

Th2 cell

B cell

Antigen

Release of mediators such as histamine and tryptase Mast cell

important pro-allergy cytokine. Apart from switching of B cells to IgE production, it also stimulates T cells and macrophages, and enhances the expression of low-affinity IgE receptor (FceR2) on B cells and adhesion molecules on endothelial cells. This latter action promotes the movement of cells out of the blood vessels. It also inhibits other types of immune reaction, such as antibody-dependent cell-mediated cytotoxicity. IL-13 has similar but weaker biological activity though it lasts longer than IL-4. IFN-g inhibits allergic responses and its effects are opposite to IL-4 and IL-13. Therefore, it is crucial in the regulation of IgE production. Other cytokines, which inhibit allergic responses, are IL10, IL-12, TGF-b, and IL-8. The direction of immune response on exposure to allergen depends on the balance of Th1 and Th2 reactivity and ensuing cytokine milieu. In atopic individuals the balance is tilted towards the production of Th2 type cytokines (IL-4 and IL-13) as opposed to Th1 type, such as IFN-g. The Th2 differentiation and production of IgE is also suppressed by regulatory T cells (CD4 þ CD25 þ ). Thus, an inappropriately weak T regulatory mechanism would facilitate Th2 dominance and production of IgE and allergic disease. It is likely that allergen exposure in early childhood results in a lifelong T cell memory pool. In atopic individuals, the immunological memory is dominated by Th2 type cells leading to allergic reactivity, whereas in nonatopic subjects, Th1 cells dominate the memory pool. In addition to genetic predisposition, environmental factors (infections, diet, etc.) may also influence the outcome of these initial responses by altering the cellular and cytokine milieu within the lymph nodes. In atopic subjects with their Th2 skew, IgE is formed specific to the antigenic protein following initial exposure (sensitization). The IgE circulates in the blood in small quantities but is mostly present in the tissues bound to high-affinity receptors (FceR1) on

FEV1

Figure 1 A schematic representation of immediate (IgE-mediated) hypersensitivity reaction.

4.5 4 3.5 3 2.5 2 1.5 1 0

1

2

3

4

5

6

7

8

9

10 11 12

Hours (postallergen exposure) Figure 2 Early and late-phase asthmatic reaction following allergen inhalation challenge. FEV1, forced expiratory volume.

the surface of mast cells and basophils and FceR2 on eosinophils, macrophages, and platelets. This IgE can be detected in the serum by immune assays or in the skin by allergy skin tests. On further exposure, multivalent antigens bind and cross-link IgE bound to FceR1 on cell surface leading to the signaling cascade that causes rapid release of preformed mediators such as histamine, tryptase, and heparin (Figure 1). These mediators cause the immediate phase of the type I hypersensitivity reaction, which occurs within a few minutes (hence immediate hypersensitivity). There is also induction of rapid synthesis of arachidonic acid metabolites such as prostaglandins and leukotrienes and expression of cytokines (IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, and tumor necrosis factor alpha (TNF-a)) and chemokines. Eosinophils are attracted to the site of activity, precipitating the late phase of the type I reaction, which starts 4
6 h after exposure (Figure 2). Immediate hypersensitivity is central to all IgEmediated allergic reactions and occurs in asthma, rhinitis, and anaphylaxis. During acute allergic reactions, the process is acute with the release of huge quantities of histamine causing typical symptoms and signs, such as acute bronchospasm or anaphylaxis. However, the process may be subacute or chronic and localized to one site such as lung or nose.

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Repeated exposure to allergens leads to the induction of a more chronic inflammatory process with the influx of inflammatory cells including T lymphocytes and eosinophils. Cytokines, produced by a variety of inflammatory cells, including T cells, regulate the inflammatory process. Proliferation of Th2 subsets producing predominantly IL-4 and IL-5 results in differentiation and isotype switching of the naı¨ve B cells to IgE-producing plasma cells as well as activation and influx of inflammatory effector cells such as eosinophils. Eosinophils are potentially tissue damaging, particularly after priming with IL-5. Various cytokines upregulate adhesion molecules on endothelial and epithelial cells, thereby enhancing migration of eosinophils into the mucosa.

Allergic Reaction in the Lung Allergic reaction in the lung results in airway inflammation. Exposure to allergen is recognized as important in initiating and maintaining allergic airway inflammation in atopic asthmatics. In the appropriate setting of repeated allergen exposure and Th2 type immune responses, a cytokine milieu is created with upregulation of adhesion molecules and continuous recruitment and activation of inflammatory cells from the bloodstream towards the bronchial mucosa. The release of cytokines and inflammatory mediators by activated cells causes amplification and persistence of the inflammatory process. However, structural cells such as epithelial cells and smooth muscle cells are not merely passive recipients of immune-related tissue damage but are active participants of the complex inflammatory cascade, which may well have initiated at the epithelial/ mesenchymal level within the airways. Nonatopics show similar inflammation in their airways and thus IgE-mediated allergy in not a prerequisite for airway inflammation in asthma or rhinitis. Early Asthmatic Reaction

The effect of allergen exposure can be observed and studied in a controlled fashion during bronchial allergen challenge. In an atopic asthmatic, inhalation of allergen to which the patient is sensitized, results in an immediate hypersensitivity reaction with the release of mast cell mediators in the bronchial mucosa. These mediators enhance vascular dilatation, increase permeability of the venule, and increase mucus secretion, resulting in edema and congestion, typical of an acute phase reaction. Histamine and leukotrienes are potent bronchoconstrictors. Histamine stimulates local type c neurones leading to the release of several neuropeptides, including substance

P, which further increase vascular permeability and cause stimulation of parasympathetic reflexes augmenting mucous secretion and bronchoconstriction. These changes manifest clinically in cough, wheeze, and dyspnea. Late Asthmatic Response

Clinically, the effect of early asthmatic reaction diminishes after 30 min (Figure 2). This is followed by a relatively asymptomatic period during which a plethora of cytokines and mediators draw leukocytes to the tissues. Events initiated during the early response result in vascular dilatation and increased permeability, edema formation, and the accumulation of cells. IL-5, secreted from mast cells, lymphocytes, and eosinophils is the most important cytokine for eosinophils. Besides attracting them to the site of inflammation, it also causes their proliferation, activation, and increased survival. Other eosinophilic cytokines are IL3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and chemokines. Upon activation, eosinophils release mediators such as eosinophilic cationic proteins, major basic proteins, leukotrienes, and prostaglandins. These and other mediators enhance inflammation and cause epithelial damage. This results in bronchoconstriction clinically 4–12 h later and prolonged bronchial hyperresponsiveness, mucus secretion, and edema formation. Further secretion of a host of cytokines including IL-3, IL-4 and IL-5, contribute to an ongoing inflammation. Chronic Inflammation

With continued or repeated exposure to allergen, a state of chronic inflammation develops with increased numbers of activated Th2 cells, expressing mRNA for the secretion of IL-3, IL-4, IL-5, and GMCSF. These cytokines are important in the continuation of inflammation and the attraction of mast cells and eosinophils. These cells cause further increase in histamine, prostaglandins, and eosinophilic toxic products, causing epithelial damage. There is upregulation of intercellular adhesion molecules in the blood vessels promoting stickiness of the endothelium to leukocytes and facilitating their passage across, into the tissues. Increased permeability and cellular infiltration causes mucosal edema. Even in patients with mild, intermittent asthma, a state of low-grade inflammation persists, in the absence of symptoms. It is hypothesized that almost continuous exposure to very small amounts of allergens, such as house dust-mite, or pollen during summer, contributes to this ongoing allergic reaction without causing symptoms. Under the influence of IL-4 from mast cells, more B cells are switched to the

ALLERGY / Allergic Reactions 75

production of IgE antibodies, thus maintaining allergic reaction. Bronchoscopy studies reveal increased numbers of activated inflammatory cells and cytokines in the respiratory mucosa and secretions. Clinical Effects

The clinical features of asthma are due to the airway narrowing causing obstruction to airflow. This airway obstruction has three elements: 1. Excessive bronchial smooth muscle contraction. Inflammatory mediators such as histamine, bradykinin, prostaglandins, and leukotrienes act directly on their specific receptors to cause bronchoconstriction. In asthma, the smooth muscles contract easily and excessively following exposure to inflammatory mediators perhaps due to heightened sensitivity of their receptors. On the other hand, b2-receptors may have a diminished response. This feature is called bronchial hyperresponsiveness. 2. Thickening of bronchial wall. Bronchial wall thickening is due to inflammatory and fibrotic changes. Increased capillary permeability allows plasma exudation into the mucosa causing edema and cellular infiltration. Proliferation of fibroblast and myofibroblast leads to thickening of the basement membrane with deposition of collagen and hypertrophy of bronchial smooth muscles (airway remodeling). This leads to irreversible airway obstruction in chronic asthma. 3. Excessive luminal secretions and cellular debris. There is excess mucus secretion due to glandular hyperplasia. The epithelium is fragile and damaged epithelial cells are found in the sputum. Impaired ciliary function encourages retention of thick mucus in the lumen. During severe exacerbation, the lumen of the airway is blocked by thick mucus, plasma proteins, and cell debris. Allergic reactions in the nose follow a similar process with inflammation of the nasal mucosa, resulting in rhinorrhea, sneezing, and nasal blockage.

Systemic Allergic Reactions and Anaphylaxis Allergic reactions vary widely in severity from mild pruritis and urticaria to circulatory collapse and death. An acute systemic allergic reaction with one or more life-threatening features, such as stridor or hypotension, is termed anaphylaxis. Allergic reactions with troublesome but not life-threatening reactions, such as generalized urticaria/angioedema and

bronchospasm of mild to moderate severity, may be called severe allergic reactions. Traditionally, the term anaphylaxis is used for IgE-mediated reactions. Systemic reactions that clinically resemble anaphylaxis but are caused by non-IgE-mediated mediator release from mast cells and basophils are referred to as anaphylactoid reactions. Anaphylaxis occurs in 30/100 000 population/year with a mortality of 1–2%. Offending agents include foods, drugs, insect stings, and exercise but in 20% of cases no cause can be found (idiopathic). Pathogenesis

Systemic allergic reaction occurs as a result of degranulation of mast cells and basophils. Mast cells in respiratory and gastrointestinal tract, skin, and perivascular tissues are involved in both IgE and non-IgEmediated allergic reaction. IgE-mediated release is caused by antigen-specific cross-linking of IgE molecules on the surface of tissue mast cells and peripheral blood basophils. Non-IgE-mediated release may be due to direct stimulation of mast cells. Mast cells produce both histamine and tryptase while basophils secrete histamine but not tryptase. Histamine is a primary mediator of anaphylaxis and signs and symptoms of anaphylaxis can be reproduced by histamine infusion. Clinical Features

Anaphylaxis is a rapidly developing generalized reaction that involves respiratory, cardiovascular, cutaneous, and gastrointestinal systems. Clinical manifestations vary depending on the cause of anaphylaxis, route of entry, host factors (such as degree of sensitization, associated factors such as exercise, comorbidity, etc.) and the amount of allergen exposure. The initial symptoms, such as nasal congestion or pruritis, can quickly progress to collapse or death. Laryngeal edema, cardiovascular collapse, and severe bronchospasm are life-threatening features. In one large series of fatal anaphylactic reactions, 70% of the deaths were from respiratory causes, and 24% were from cardiovascular causes. In 10–20% of cases, skin may not be involved. Anaphylaxis can be confused with septic or other forms of shock, asthma, airway foreign body, and panic attacks (Table 1). Symptoms commonly occur within a few seconds or minutes of exposure and death may occur within minutes. Speed of onset of symptoms is indicative of the severity. Occasionally, the onset of symptoms may be delayed for 2 h or more. In general, the later the symptoms begin after exposure to a causative agent, the less severe the reaction. Food allergens may have slower onset or slow progression and gastrointestinal

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ALLERGY / Allergic Reactions

Table 1 Differential diagnosis of anaphylaxis Acute cardiac event Vasovagal syncope Acute angioedema Acute severe asthma Pulmonary embolism Foreign body inhalation Carcinoid syndromes Pheochromocytoma Seizure Systemic mastocytosis Panic attack Vocal cord dysfunction

symptoms are more common. Onset of anaphylaxis to insect stings or allergen injections is usually rapid: 70% begin in o20 min and 90% in o40 min.

Table 2 Clinical features of anaphylaxis Symptoms

Physical examination

Cardiovascular

Faintness, syncope, palpitations

Throat

Throat tightness, hoarseness, inspiratory stridor, difficulty in swallowing Nasal congestion, rhinorrhea, sneezing Pruritis, lacrimation Generalized warmth, tingling, pruritis, rash Chest tightness, wheezing, cough, shortness of breath Nausea, vomiting, abdominal pain, bloating, cramps, diarrhea Dizziness, a sense of impending doom, lightheadedness

Hypotension, tachycardia, arrhythmias Laryngeal edema

Upper respiratory tract Ocular Skin

Chest

Management Gastrointestinal

A quick initial assessment should determine the nature and progression of the clinical event (Table 2). Continuous monitoring is essential as progression from a mild to a severe episode may occur rapidly. Epinephrine injected intramuscularly into the thigh provides the most efficient absorption (Figure 3). If there is no response to several doses of intramuscular epinephrine, intravenous administration may be needed, by using a formulation of 1:10 000 (0.1 mg ml
1) at 1 mg min
1, which can be increased to 2–10 mg min
1. If the response is still inadequate, transfer the patient to an intensive care unit for close monitoring and endotracheal intubation, if required. Specific treatment for coexisting medical conditions (e.g., coronary artery disease) may be necessary. There may be complete resolution of the reaction. However, if there are concerns, continued monitoring for remaining or recurring symptoms is essential. A short course of corticosteroids may reduce the risk of recurring or protracted symptoms of a biphasic reaction but this is not proven. Patients receiving badrenergic blocking agents may not respond adequately to epinephrine. They require continued fluid replacement and may respond to glucagon. Patients receiving angiotensin-converting enzyme inhibitors may also be at increased risk of anaphylaxis and be more refractory to treatment with epinephrine. If there is any doubt regarding the diagnosis, blood should be taken for plasma histamine or serum tryptase levels within the first 4 h after the onset of symptoms. Elevated serum levels of b-tryptase indicate mast cell activation and degranulation in both IgE-mediated (anaphylaxis) and non-IgE-mediated (anaphylactoid) reactions. b-tryptase is useful in differentiating anaphylaxis from other events having

Neurologic

Conjunctival injection Flushing, urticaria, swelling of the lips, tongue or uvula Wheezing, tachypnea, cyanosis, respiratory arrest

Loss of consciousness, seizures

similar clinical manifestations, particularly if hypotension is present. Blood for plasma histamine needs to be processed immediately to avoid detecting artificially high levels due to spontaneous basophil histamine release. If this is not possible, urinary histamine (or metabolites) levels can be checked for up to 24 h. After initial treatment of acute anaphylaxis, the patient should be followed-up closely for the possibility of recurrent episodes. For mild to moderate episodes and good response to treatment, further monitoring can be done at home. However, following a severe episode, in-patient monitoring may be required for late-phase reactions. Subsequent Assessment

All individuals who have had a known or suspected anaphylactic episode require a careful allergy evaluation. The aims are to review the diagnosis of anaphylaxis and prevent or minimize the risk of future anaphylactic episodes by identifying the cause, educate the patient and/or family members regarding avoidance of the offending agent, education and training to deal with future inadvertent exposures, and consideration of desensitization, if appropriate. The level of confidence in the diagnosis of the original episode should be reviewed with details of the

ALLERGY / Allergic Reactions 77

Evaluate: breathing status, pulse, blood pressure, and level of consciousness Recognize life-threatening features such as stridor, shock, arrhythmia, seizure, and loss of consciousness

• Institute CPR if there is loss of circulation or respiration • Maintain airway with an airway device or tracheotomy • Oxygen, if there is circulatory or respiratory compromise

• Epinephrine (1:1000) 0.3−0.5 ml i.m. (children: 0.15 ml), repeat every 10−15 min, as needed • Chlorpheniramine 10 mg i.v. (then 6 hourly) • Hydrocortisone 200 mg i.v.

Improve

Problems persist

Late-phase symptoms

Monitor

Discharge home

• Hypotension: intravenous fluids (colloids) and, if needed, vasopressor agents (e.g., dopamine) • Bronchospasm: nebulized
2-agonist, consider use of i.v. aminophylline. Mechanical ventilation may be required • Urticaria/angioedema: oral/i.v. antihistamines and steroids Figure 3 Emergency management of anaphylactic and anaphylactoid reactions.

events before and during the episode. Results of any laboratory tests (e.g., serum tryptase or urine histamine) may be helpful in supporting the diagnosis of anaphylaxis and differentiating it from other entities. However, a careful and comprehensive history is the most useful part of the assessment. Information on any previous similar episodes, known food or drug allergy, and medication record should be sought. The history might suggest a specific cause such as insect sting or peanut consumption just before the episode. However, the cause may not be obvious from history and sometimes no cause can be found despite thorough searching (idiopathic anaphylaxis). Diagnostic Tests

Skin prick tests (SPTs) or determination of specific IgE in serum (in vitro test) is helpful in identifying a

specific cause of anaphylaxis in cases of food, insect, and some cases of drug (penicillin, insulin) allergy. An SPT is more sensitive than in vitro testing and is the diagnostic procedure of choice. When possible, standardized extracts should be used. If skin tests or in vitro tests do not provide a cause, then challenge to the suspected agent/agents should be considered. Challenge procedures are helpful in IgE-mediated allergic reaction where standardized test material is not available and in non-IgE anaphylactic reactions (such as to aspirin). Prevention

Once the offending agent has been identified (e.g., food, medication, or insect sting), patients should be educated regarding the specific exposures and be counseled on avoidance measures (Table 3). All those

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ALLERGY / Allergic Reactions

Table 3 Specific measures to reduce allergic reactions to common provoking agents Provoking agents

Specific preventive methods

Foods

Patients should be taught to read and interpret food labels They should be encouraged to ask about ingredients in restaurant meals They should be provided with a list of alternative foods School personnel should be fully informed of the pupil’s allergy history Patients should carry a medical identification bracelet or card Use alternatives, when possible, but avoid crossreactive antibiotics If penicillin is essential, desensitization could be performed Remove any hives or nests in the garden Wear long shoes and full trousers when walking in the fields Be alert to the presence of insects when outdoors Discontinue exercise at the earliest symptom Avoid any exacerbating factors such as aspirin or NSAIDs Avoid exercise for 4–6 h after eating if there is a history of anaphylaxis after food ingestion Patients should carry a medical identification bracelet or card All procedures should be conducted in a latex-free environment Food with known crossreactivity to latex should be avoided Consider an alternative procedure that does not require RCM Use low osmolar RCM Consider pretreatment with steroids, antihistamine, and ephedrine Allergen immunotherapy should only be administered under the supervision of a trained allergist Consider alternative therapy in those who are at higher risk (Table 4) Care should be taken to avoid dosing errors Observe patients for at least 20 min after the injection Consider reducing the dose, if interval between injections had been longer than planned or on opening of new vials Adjust dose if large local reaction occurs Reduce dose in highly sensitive patients or if there is concomitant high allergen exposure

Penicillin

Insect sting

Exercise

Latex

Radiocontrast material (RCM) Allergen extracts

with a risk of future anaphylaxis outside the medical settings should carry and be educated in the use of self-injectable epinephrine and antihistamines. Self-injectable epinephrine is available in two different strengths (for adults, 0.3 ml of 1:1000 solution and for children, 0.3 ml of 1:2000 solution) in readyto-use syringes. Antihistamine (such as chlorpheniramine 4–8 mg orally) may be sufficient for a mild episode but epinephrine should not be held back if symptoms are severe from the outset or response to antihistamine is inadequate. Humanized, monoclonal anti-IgE antibody has shown protection against peanut-induced anaphylaxis.

Allergic Reactions Foods

Food allergic reactions are common in children and presents clinically with systemic involvement (as described above), although oral (itching, numbness and tingling of lips and mouth) and gastrointestinal symptoms may be more prominent. Although any food can cause a reaction, commonly implicated foods are milk, egg, peanuts, tree nuts, fish, and shellfish. Symptoms often occur within minutes of ingestion and certainly within 2 h. Assessment of specific IgE to

suspected food, either by skin prick test or in vitro test, in the presence of a suggestive history, is sufficient to make a definitive diagnosis. However, food challenges (single or double blind) may be required. Strict avoidance of the offending food is essential. Patients should also carry, and be trained, in the use of epinephrine in an emergency following inadvertent exposure. Penicillin

Allergic reaction to penicillin is the most common cause of anaphylaxis. Severe reactions are usually attributable to parenteral administration. Atopy and family history of penicillin allergy does not increase the risk of a reaction. A history of penicillin allergy is not reliable as nearly 80% of these individuals tolerate penicillin without ill effects. However, these subjects should have a skin test to major and minor determinants before penicillin is administered. The risk of a reaction following a negative test is less than 2%. Skin tests do not resensitize a patient to penicillin. A positive skin test in the presence of a history of reaction to penicillin indicates a more than 50% risk of an allergic reaction and penicillin should be avoided, or if penicillin is mandatory, desensitization should be considered. Cross-reactivity exists between

ALLERGY / Allergic Reactions 79

cephalosporins and penicillins due to the common b-lactam ring structure. Aspirin and Nonsteroidal Anti-Inflammatory Drugs

Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) can induce life-threatening systemic, nonIgE-mediated reaction causing rhinoconjunctivitis, bronchospasm, urticaria, and laryngeal edema. In vitro or skin tests are not available and oral challenges are required for confirmation. If the diagnosis is confirmed, aspirin and NSAIDs should be strictly avoided. Desensitization may be performed if these drugs are considered essential. Insect Sting

Stinging insects belong to the order Hymenoptera. Common stinging insects include honeybees, wasps, yellow jackets, hornets, and fire ants. The self-reported prevalence of insect sting allergy is approximately 1%. Insect venoms contain several well-characterized allergens that can trigger anaphylactic reactions. Localized reaction may occur at the site of the sting and a large local reaction may involve, for example, the whole limb. However, these do not predispose to systemic allergic reaction. Urticaria and angioedema are key features of systemic allergic reactions caused by insect sting. Skin test is the preferred method of confirming allergy and identifying the responsible insect species. However, careful interpretation is needed, as falsepositive reactions are common. Following a systemic allergic reaction, only about half of the patients will react to a future sting. Patients should carry self-injectable epinephrine for early treatment of a sting, and allergen immunotherapy should be considered, where risk of future sting is substantial. The immunotherapy is 490% effective but optimal duration is not known. Intraoperative Drugs

Common agents responsible for intraoperative anaphylaxis are neuromuscular blocking agents, latex, antibiotics, anesthesia induction agents, radiocontrast material, and opioids. Neuromuscular blocking agents and thiopental are responsible for most anaphylactic reactions during general anesthesia. Both IgE-mediated (muscle relaxants, latex) and direct stimulation of mast cell (opioids, radiocontrast material) occurs. Clinical manifestations of intraoperative reactions differ from anaphylactic reactions due to other causes as cardiovascular collapse, airway obstruction, and flushing is prominent. It may be difficult to differentiate allergic reaction from the pharmacologic effects of a variety of medications

administered during general anesthesia. Plasma tryptase is useful in differentiating allergic reaction (due to mast cell release of mediators) from pharmacologic or other causes. Skin testing is used for diagnosis where IgE-mediated mechanism is suspected. Otherwise, graded challenge may be required. Latex

IgE-mediated allergic reactions to natural rubber latex became common during the 1990s due to a sudden increase in the use of rubber gloves. Risk factors for latex allergy include atopy and previous repeated exposure to latex (e.g., multiple surgical procedures, healthcare workers). Skin tests are indicated for investigation of latex allergy in those who have a history of possible latex allergic reaction and for screening those who are at high risk. However, in the last few years, the use of latex-free gloves and other products have been effective in reducing the occurrence of latex allergic reactions. Blood Transfusions

Allergic reactions may complicate 1–3% of blood transfusions. Most reactions are mild, are associated with cutaneous manifestations such as pruritis, maculopapular, or urticarial rash and flushing, and require no specific treatment except discontinuation of transfusion and perhaps antihistamine. However, severe or life-threatening reactions with hypotension and bronchoconstriction may occur occasionally. Most of these reactions are due to IgE or IgM antibodies to serum proteins (e.g., albumin, complement components, IgG, and IgA). Other mechanisms include transfusion of allergen, IgE antibodies, bacterial components or inflammatory substances such as cytokines, histamine, or bradykinin. Blood components containing large amounts of plasma, such as fresh frozen plasma, may be associated with more severe allergic reactions. Serum tryptase, measured soon after the reaction, may differentiate allergic from transfusion-related reaction. Allergen Extract

Allergen extracts are injected during skin test and allergen-specific immunotherapy. The risk of allergic reaction is extremely low with skin prick test but not insignificant when these extracts are injected for treatment. In a survey between 1985 and 1989 in the US, there were 17 deaths from allergen immunotherapy but none from skin testing. Mild, localized reactions are relatively common and respond to antihistamine. Factors that increase the risk of an allergic reaction should be kept in mind by those involved

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Table 4 Risk factors for systemic reactions during allergen immunotherapy Severe asthma Highly sensitive patient Too rapid increase in the dose of allergenic extract Starting a new vial of extract A history of previous systemic reactions to allergen immunotherapy Asthmatic symptoms present immediately before receiving an injection Administration of pollen extracts during high environmental pollen exposure Concomitant treatment with b-adrenergic blocking agents Fever or an upper respiratory tract infection at the time of administration of allergenic extracts

Lieberman P (2002) Anaphylactic reactions during surgical and medical procedures. Journal of Allergy and Clinical Immunology 110(supplement 2): S64–S69. Moffitt JE (2003) Allergic reactions to insect stings and bites. Southern Medical Journal 96(11): 1073–1079. Moneret-Vautrin DA and Kanny G (2002) Anaphylaxis to muscle relaxants: rational for skin tests. Allergic Immunology (Paris) 34(7): 233–240. Noone MC and Osguthorpe JD (2003) Anaphylaxis. Otolaryngologic Clinics of North America 36(5): 1009–1020. Sampson HA (2003) Anaphylaxis and emergency treatment. Pediatrics 111(6): 1601–1608. Sicherer SH and Leung D (2004) Advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insect stings. Journal of Allergy and Clinical Immunology 114(1): 118–124. Tang AW (2003) A practical guide to anaphylaxis. American Family Physician 68(7): 1325–1332.

in administration of allergen-specific immunotherapy (Table 4). Exercise

Allergic Rhinitis

Exercise-induced anaphylaxis is a physical form of allergy that may occur in isolation or in combination with ingestion of food or drug, such as aspirin or NSAIDs. The episode resembles typical anaphylaxis and emergency treatment is the same as for anaphylaxis due to other causes. Patients should carry self-injectable epinephrine. Exercise may also cause urticaria or bronchospasm without anaphylaxis.

P van Cauwenberge, J-B Watelet, T Van Zele, and H Van Hoecke, Ghent University Hospital, Ghent, Belgium

See also: Allergy: Overview. Asthma: Overview. Chemokines, CXC: IL-8. Immunoglobulins. Interferons. Interleukins: IL-4; IL-10; IL-12; IL-13. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumor Necrosis Factor Alpha (TNF-a ).

Further Reading Cockcroft DW (1998) Airway responses to inhaled allergens. Canadian Respiratory Journal 5(supplement A): 14A–17A. El Biaze M, Boniface S, Koscher V, et al. (2003) T cell activation, from atopy to asthma: more a paradox than a paradigm. Allergy 58(9): 844–853. Gould HJ, Sutton BJ, Beavil AJ, et al. (2003) The biology of IgE and the basis of allergic disease. Annual Review of Immunology 21: 579–633. Holgate ST, Davies DE, Puddicombe S, et al. (2003) Mechanisms of airway epithelial damage: epithelial–mesenchymal interactions in the pathogenesis of asthma. European Respiratory Journal 44(supplement): 24s–29s. Joint Task Force on Practice Parameters, American Academy of Allergy, Asthma and Immunology, American College of Allergy, Asthma and Immunology, and the Joint Council of Allergy, Asthma and Immunology (1998) The diagnosis and management of anaphylaxis. Journal of Allergy and Clinical Immunology 101(6 Pt 2): S465–S528. Kemp SF and Lockey RF (2002) Anaphylaxis: a review of causes and mechanisms. Journal of Allergy and Clinical Immunology 110(3): 341–348.

& 2006 Elsevier Ltd. All rights reserved.

Abstract Over the last decades, the prevalence of allergic rhinitis has risen to epidemic proportions. Nasal symptoms involve sneezing, nasal itch, rhinorrhea, and nasal congestion. These symptoms result from an immunologically mediated (usually IgE-mediated) inflammation of the nasal mucosa, following allergen exposure in sensitized patients. Although allergic rhinitis is often trivialized, it has become clear that the disease can cause serious morbidity beyond the nasal manifestations, that it has a significant impact on quality of life and substantial socioeconomic consequences, and that it is associated with multiple comorbidities, including asthma, conjunctivitis, sinusitis, and otitis media. Recently, the Allergic Rhinitis and its Impact on Asthma (ARIA) Working Group proposed a new classification for allergic rhinitis, based on the duration of symptoms, rather than on the type of exposure. The severity of the disease is categorized based on the impact of symptoms on quality of life parameters. Early and correct diagnosis is the basis for the management of allergic rhinitis and starts with a thorough clinical history and physical examination. To confirm the allergic origin of rhinitis symptoms, allergy tests are performed. The test of first choice is the skin prick test, which has a good sensitivity and specificity. Environmental control measures (allergen avoidance), pharmacological treatment, immunotherapy, and education are the cornerstones of therapeutic management of allergic rhinitis. Nowadays, many effective pharmacological agents are available and new potential targets for pharmacotherapy, new routes of administration, and alterations in treatment dosages and schedules are continuously being investigated. To facilitate and standardize the management of allergic rhinitis and to improve the patient care, satisfaction, and compliance, several clinical practice guidelines have been developed. Among those, the ARIA guidelines provide stepwise treatment recommendations, based on the best available evidence from research.

ALLERGY / Allergic Rhinitis 81

Introduction Allergic rhinitis (AR) is defined as a nasal disease with the presence of immunologically mediated hypersensitivity symptoms of the nose, for example, itching, sneezing, increased secretion, and blockage. The great majority of cases are immunoglobulin E (IgE) antibody-mediated. In the recent Allergic Rhinitis and its Impact on Asthma (ARIA) report, AR is considered as a major chronic respiratory disease because of its high prevalence in all countries, its significant impact on quality of life or work performance, its considerable economic burden, and its association with multiple comorbidities, asthma in particular. Several important epidemiological surveys (e.g., European Community Respiratory Health Survey, International Study of Asthma and Allergies in Childhood (ISAAC), and Swiss Study on Air Pollution and Lung Diseases in Adults) have recently improved our knowledge about the prevalence of rhinitis. The majority of monocentric studies have reported a prevalence of seasonal AR ranging from 1% to 40% and a prevalence of perennial rhinitis ranging from 1% to 18%. Furthermore, the ISAAC study phase 1 confirmed this large variation in the prevalence of rhinitis symptoms throughout the world. Over the last 40 years the prevalence of AR, like other allergic disorders, has risen to truly epidemic proportions. Despite the increasing insight into the pathophysiology of allergy and the availability of more effective treatment options, this upward trend in the incidence of AR continues, being most prominent in countries with a Western lifestyle, especially among children and adolescents. As a consequence, great attention is being paid to the identification of the factors responsible for this disease. It is well established that allergic diseases tend to occur within families and have a genetic basis. However, numerous generations are needed before changes in the gene pool occur. Therefore, the recent increase in prevalence of AR and allergy in general cannot be explained by genetic factors and is largely attributed to alterations in the environment. Diverse environmental and lifestyle factors (prenatal maternal influences, allergen exposure, active and passive smoking, viral and other respiratory infections, early life microbial exposure, indoor air quality/house dampness, outdoor air pollution, urban vs farming living environment, socioeconomic status, dietary factors, etc.) have been identified as possible risk factors or as protective factors in the pathogenesis of allergy, although the evidence supporting their involvement varies widely. In order to allow the introduction of individualized primary and secondary

prevention strategies and to meet the challenge of the growing impact of allergy, further assessment of the complex interactions within and between genetic and environmental determinants is required.

Etiology Allergens are antigens that induce and react through an IgE-mediated inflammation. The number of identified allergens has expanded enormously. They originate from animals, insects, plants, or fungi. In AR, airborne allergens are the most common provoking agents and the increase in time spent indoors in recent years is thought to be responsible for the rise in incidence of allergic diseases. The most common indoor allergens are derived from mites, domestic animals, insects, or plants. Mites, such as Dermatophagoides pteronyssinus or D. farinae, usually induce asthma or perennial AR. They feed on human skin and multiply under hot and humid conditions. Other types of mite, such as Tyrophagus putrecentiae and Acarus siro, are present in flour. The dander and secretions of many animals contain powerful allergens capable of inducing severe reactions. They can remain airborne for prolonged periods of time. Specific allergens have been identified in cat, dog, horse, cattle, rabbit, and other rodents. Outdoor allergens include pollen and molds. Pollen can be categorized into two groups based on their mode of transport: anemophilous pollen are carried by the wind and can be transported over long distances, while entemophilous pollen are carried by insects. The nature and number of pollen vary with geography, temperature, and climate. Fungi, molds, and yeasts can liberate large quantities of allergenic spores into the atmosphere. Their development and growth are faster in hot and humid conditions. The main atmospheric molds are Cladosporium and Alternaria. Finally, it has been noted that inhalation of insect waste or some bacteria seems to be able to induce an IgE-mediated reaction. AR can also be triggered by food and occupational allergens. Many allergens have enzymatic activity. Simultaneous exposure to both allergenic and proteolytic activity probably allows greater access to cells of the immune system and enhances sensitization and allergic inflammatory reactions.

Pathology Classification of Allergic Rhinitis

Before the publication of the ARIA report, AR was subdivided into seasonal AR, perennial AR, and by extension occupational AR, based on the time of

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exposure to the offending allergen(s). Seasonal AR is related to a wide variety of outdoor allergens such as pollens and molds. Perennial AR is most frequently caused by indoor allergens such as house dust mite, molds, cockroaches, and animal dander. Occupational AR occurs in response to airborne allegens in the workplace. Common causes are laboratory animals, wood dust (particularly hard woods), chemicals, and solvents. The distinction between seasonal and perennial AR, however, is not applicable in all patients and in all countries because *

*

*

*

symptoms of perennial rhinitis may not be present all year round; pollen and molds are perennial allergens in some parts of the world; many patients are sensitized to multiple allergens and present with symptoms during a number of periods in the year or even throughout the year; and symptoms of seasonal AR do not always occur strictly within the defined allergen season, due to a ‘priming effect’ and the concept of ‘minimal persistent inflammation’.

Therefore, this classification was considered to be inaccurate and the ARIA Working Group proposed a major change in the classification of AR. The ARIA classification for AR uses the terms ‘intermittent’ and ‘persistent’ to describe the duration of symptoms. Based on the impact of symptoms on quality-of-life parameters, the severity of the disease is classified as ‘mild’ or ‘moderate–severe’ (Table 1).

Histopathology of Allergic Rhinitis

Pollen-induced rhinitis is the most characteristic IgE-mediated allergic disease and is triggered by the interaction of mediators released by cells that are implicated in both allergic inflammation and nonspecific hyperreactivity. AR is characterized by a huge inflammatory reaction. The resulting inflammatory cell infiltrate is made up of different cells. The cellular response begins with chemotaxis, selective recruitment, and transendothelial migration of cells. These cells can be localized within the different compartments of the nasal mucosa and represent an activation state. Their survival is prolonged and they release a large amount of inflammatory mediators. They also participate in the regulation of IgE synthesis and communicate with the immune system. Biopsies from allergic nasal tissue demonstrate a thicker basement membrane and a greater number of intraepithelial monocytes, subepithelial eosinophils, and neutrophils (Figure 1).

Clinical Features Symptoms of rhinitis include rhinorrhea, nasal obstruction, nasal itch, and sneezing; often, patients with rhinitis are subdivided into ‘sneezers and runners’ and ‘blockers’, based on the main symptom(s).

Table 1 ARIA classification for allergic rhinitis ‘Intermittent’ rhinitis Symptoms are present: p4 days a week or p4 consecutive weeks

‘Persistent’ rhinitis Symptoms are present: 44 days a week and 44 consecutive weeks

‘Mild rhinitis’

‘Moderate/severe’ rhinitis X1 of following items are present:

None of following items are present: sleep disturbance impairment of daily activities, leisure, and/or sport impairment of work or school work troublesome symptoms In untreated patients

Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108(5 supplement): S147–S334.

Figure 1 Hematoxylin-eosin staining (magnification
40) of normal nasal mucosa demonstrating epithelial cells, seromucous glands, and subepithelial lymphoid layer.

ALLERGY / Allergic Rhinitis 83

These rhinitis symptoms, however, do not necessarily have an allergic origin. In the differential diagnosis, AR must be differentiated from several types of nonallergic rhinitis and other nasal inflammatory conditions (Tables 2 and 3). The clinical history remains the most essential step for establishment of the diagnosis. Apart from the classical rhinitis symptoms, the patient must be questioned about the presence of other symptoms commonly associated with rhinitis, such as loss of smell, snoring, sleep disturbance, postnasal drip, cough, sedation, conjunctivitis, and lower respiratory symptoms. The history should also contain an evaluation of the severity and duration of the problem, the impact on daily life, and the response to treatment, and should document potential allergic and nonallergic triggers; it must also include a family and occupational history.

Clinical examination starts with a general inspection of the nose, ears, and throat. A complete and systematic nasal examination is required, especially in patients with persistent rhinitis. However, anterior rhinoscopy gives only limited information. Nasal endoscopy is therefore an essential complementary investigation, not to confirm AR, but to exclude other conditions, such as polyps, foreign bodies, tumors, and septal deformations. During allergen exposure, the sinonasal mucosa of patients with AR can demonstrate a bilateral, but not always symmetrical, swelling. Often, mucosal changes in color are seen, from a purplish to a more common pale coloration. An increase in vascularity is also commonly noticed. In the absence of allergen exposure, the nasal mucosa may appear completely normal, but in patients who have suffered from rhinitis for several years, irreversible mucosal hyperplasia and/or viscous secretions may also occur (Figures 2–6).

Table 2 Classification of rhinitis Allergic rhinitis Infectious rhinitis: viruses, bacteria, fungi Occupational rhinitis: allergic and nonallergic Drug-induced rhinitis, e.g., aspirin Hormonal rhinitis: puberty, pregnancy, menstruation, endocrine disorders Emotional rhinitis Atrophic rhinitis Irritant-induced rhinitis Food-induced rhinitis, e.g., red pepper NARES: nonallergic rhinitis with eosinophilic syndrome Rhinitis associated with gastroesophagel reflux Idiopathic rhinitis Adapted from International Rhinitis Management Working Group (1994) International Consensus Report on the Diagnosis and Management of Rhinitis. Allergy 49 (supplement 9): 5–34.

Diagnostic Evaluation

To confirm the allergic origin of rhinitis symptoms, the ARIA Working Group states that allergy tests should be performed. In vivo and in vitro tests for the diagnosis of allergic diseases are directed towards the detection of free or cell-bound IgE. Immediate hypersensitivity skin tests are a major diagnostic tool to demonstrate IgE-mediated allergic reactions. If properly performed, skin tests are the best available method for detecting the presence of allergen-specific IgE. The first choice of test is the skin prick test, which has a good sensitivity and specificity (Figures 7 and 8). It is very important that skin tests are performed carefully and interpreted correctly. Therefore,

Table 3 Differential diagnosis of rhinitis Mechanical factors Deviated septum Adenoidal hypertrophy Hypertrophic turbinates Foreign bodies Choanal atresia Tumors Benign Malignant Granulomas Wegener’s granulomatosis Sarcoid Infectious (tuberculosis, leprosy) Malignant – midline destructive granuloma Ciliary defects Cerebrospinal rhinorrhea Adapted from International Rhinitis Management Working Group (1994) International Consensus Report on the Diagnosis and Management of Rhinitis. Allergy 49(supplement 9): 5–34.

Figure 2 Profuse watery anterior rhinorrhea in a child with allergic rhinitis.

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Figure 3 The ‘nasal salute’ refers to habitual rubbing of the nose due to constant irritation. It usually signifies an underlying allergic phenomenon of producing a profuse rhinorrhea requiring frequent wiping. It is most marked in children.

Figure 4 Habitual rubbing of the nose usually produces a horizontal ‘nasal crease’ below the bridge of the nose.

Before allergen challenge

After allergen challenge

Figure 5 Schematic presentation of sinonasal mucosa swelling before and after allergen challenge.

it is recommended that trained healthcare professionals carry them out. The skin reaction can be affected by the quality of the allergen extract, the patient’s age, the use of some pharmacological agents (e.g., oral antihistamines and topical skin corticosteroids), and can also demonstrate seasonal variations. In addition, the possibility of false-positive and false-negative results must be considered (Table 4). Measurement of total serum IgE lacks specificity and is of little predictive value in allergy screening in rhinitis. On the contrary, serum-specific IgE is as valuable as skin testing. Skin tests, however, are less expensive, have a greater sensitivity, allow a wide allergen selection, and give results in less than half an hour. Serum-specific IgE is indicated in young children, in patients with dermographism or widespread dermatitis, in patients who are noncompliant for skin testing, or in those who did not continue with antihistamine treatment (short-acting antihistamines for 36–48 h, long-acting antihistamines for 4–6 weeks), as this can result in false-negative skin test results. Serum-specific IgE measurement is also a safer option in patients who are very allergic and where an anaphylactic reaction to skin testing is a possible risk. It is important to remember that positive in vivo or in vitro tests for allergen-specific IgE must always be interpreted in relation to the entire clinical presentation. The presence of allergen-specific IgE antibodies is not sufficient for the diagnosis of allergic disease, as patients can be sensitized in the absence of (or prior to) the development of any symptoms. Nasal challenge tests are used in particular for research purposes and are important in the diagnosis of occupational rhinitis. The International Committee on Objective Assessment of Nasal Airways has set up guidelines concerning the indications, techniques, and evaluation of nasal challenge tests. In addition to allergen provocations, nasal challenge tests with aspirin, non-specific agents (histamine, metacholine), and occupational agents can be performed. Imaging (sinus plain radiographs, computed tomography, and magnetic resonance imaging) is not indicated for the diagnosis of AR, but may be necessary to exclude other conditions or complications. Other diagnostic tests that are employed to assess the nasal airways include nasal peak flow, rhinomanometry, and acoustic rhinometry, but these are rarely used in the diagnosis of AR. Objective testing of a patient’s ability to smell can be performed by olfactory testing. Mucociliary function can be measured by nasomucociliary clearance, ciliary beat frequency, or electron microscopy, but these tests are of little relevance in the diagnosis of AR.

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Figure 6 Nasoendoscopic visualization (right nasal cavity: Hopkins 301) of nasal mucosa swelling in an allergic patient before (a) and (b) after allergen challenge. Table 4 Causes of false-positive and false-negative skin tests Causes of false-positive skin tests: * Dermographism * Irritant reactions * Non-specific enhancement of a nearby strong reaction * Improper technique/material Causes of false-negative skin tests: * Poor initial potency or loss of potency of extracts * Use of drugs modulating allergic reaction * Diseases attenuating skin response * Decreased activity of the skin (infants and elderly patients) * Improper technique/material

Figure 7 Kit with standardized allergen extracts and positive and negative control solutions used for allergy skin tests.

Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108(5 supplement): S147–S334.

Comorbidities

Figure 8 The technique used for skin prick testing involves introducing a drop of diluted allergen followed by puncturing the skin with a calibrated lancet (1 mm) at an angle of 451. The drops should be placed 2 cm apart. All patients undergoing skin prick testing should also have a positive (histamine) and negative diluent (saline) control test included. Skin tests should be read at the peak of their reaction by measuring (in mm) the wheal and the flare around 15 min after pricking. The relevance of skin prick testing should always be interpreted in the context of the patient’s history. Positive results can occur in people without symptoms and, similarly, false-negative results may also occur.

It must be emphasized that AR should be evaluated as a global systemic disease. Allergic inflammation does not necessarily limit itself to the nasal airway, but the treating physician must also be aware of the possible comorbidities of AR, including asthma, conjunctivitis, sinusitis, and otitis media. The link between asthma and rhinitis in particular has gained much interest. Epidemiological studies have shown that up to 80% of patients with asthma demonstrate symptoms of rhinitis, while approximately 20–40% of patients with AR have clinical asthma. There is growing evidence that rhinitis is a risk factor for the development of asthma, independent of atopy. Additionally, the airway mucosa of nose and bronchi have many similarities and the clinical and pathophysiological changes in asthma and AR are often very comparable. Although there are still some differences that should be highlighted, the strong relationship between rhinitis and asthma has introduced the concept of ‘the united airway disease’.

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Based on these findings the ARIA guidelines recommend that patients with persistent AR should be evaluated for asthma by history, chest examination, and, if possible and where necessary, by the assessment of airflow obstruction before and after using a bronchodilator, whereas patients with asthma should be evaluated for rhinitis by history and physical examination. Another frequent comorbidity of AR is conjunctivitis. It is estimated that 42% of patients with AR experience symptoms of allergic conjunctivitis and that 33–56% of the cases of allergic conjunctivitis occur in association with AR. This coexistence, referred to as ‘rhinoconjunctivitis’, seems to be a typical feature in patients with seasonal pollen allergy. As eye symptoms substantially contribute to the burden of allergic rhinoconjunctivitis, adequate assessment and treatment of conjunctivitis should be part of the overall management. AR is also considered as a contributing factor in acute and chronic rhinosinusitis. Up to 54% of adults with chronic rhinosinusitis have symptoms of AR. Similarly, a high concordance (between 25% and 75%) of these disorders is found in children. Conversely, there is a high prevalence of sinus disease in patients with AR: abnormal sinus radiographs occur in over 50% of adults and children with perennial AR and acute rhinosinusitis occurs often during the allergy season. There is still some controversy regarding the etiological role of AR in otitis media with effusion. Most epidemiological data suggest an association between these two diseases. However, the available evidence is compromised by a possible referral bias and by the lack of prospective, controlled studies. It is still not clear whether AR predisposes to the development of otitis or whether nasal dysfunction worsens otitis. It can be concluded that although the exact pathophysiological links between AR and its several comorbidities still need to be elucidated, AR is not an isolated disorder but is part of a systemic disease process. Hence, the ARIA Working Group recommends a coordinated diagnostic and therapeutic approach instead of a fragmented, organ-based management.

Pathogenesis Symptoms of AR develop upon inhalation of allergens in individuals previously exposed to such allergens and against which they have made IgE antibodies. The pathophysiological process of AR can be subdivided in two phases: the initial sensitization phase during which allergen presentation results in primary allergen-specific IgE antibody formation

by B lymphocyte, and the clinical disease phase during which symptoms in response to subsequent antigen exposure become manifest. The clinical disease phase, in turn, can be subdivided into two distinct phases: an early phase largely mediated through mast cells; and a late phase, which involves cellular infiltration and mediator release. Sensitization Phase

The development of sensitivity to an allergen requires IgE antibody production directed at the epitope. After allergen exposure, antigen-presenting cells present the allergens to CD4 þ cells. A subset of these CD4 þ cells, the T-helper type 2 (Th2) lymphocytes, generate Th2 cytokines, including IL-4 and IL-13, which stimulate IgE synthesis in combination with B-cell–T-cell ligand–receptor interactions that are pivotal in the B-cell isotype switching toward IgE synthesis. These B-cell–T-cell interactions include a major histocompatibility complex class II and T cell receptor/CD3 interaction and a binding of the CD40L on T lymphocytes and CD40 expressed on B cells. As a result, allergen-specific IgE antibodies are produced and these sensitize mast cells and other IgE receptor-bearing cells. There is now increasing evidence that IgE is produced locally in the nasal mucosa since nasal B cells, in the presence of IL-4 and CD40L positive mast cells, are able to produce IgE locally. Clinical Disease Phase

The clinical disease phase consists of two phases: an early phase and a late phase. The early phase is largely mediated through mast cells. In sensitized patients, allergen re-exposure mediates cross-linkage, of adjacent IgE molecules bound to mast cell surfaces. If a mast cell is activated by IgE cross-linking, it releases granule products containing histamine, tryptase, chymase, and cytokines such as interleukin-4 (IL-4), IL-5, IL-8, IL-13, and tumor necrosis factor alpha (TNF-a) into the extracellular environment. Second upon activation mast cells generate arachidonic acid products including cysteinyl-leukotrienes (LTC4, LTD4, LTE4) and prostaglandin D2 (PGD2) from the phospholipid cell membrane. Nasal challenge with allergens shows the local release within 10– 15 min of histamine, tryptase, PGD2, LTB4, and LTC4. These mediators cause the characteristic watery rhinorrhea by stimulating gland and globet cell secretion, vasodilation, and blood vessel leakage, which is characteristic for the early phase. Histamine is the most important mediator in AR and induces symptoms of nasal itching, sneezing, discharge, and transient nasal blockage whereas leukotrienes appear to be relatively more important than histamine in

ALLERGY / Allergic Rhinitis 87

inducing nasal blockage. In addition, the mast cell is thought to contribute to other features in rhinitis, namely the eosinophilic mucosal inflammation. Eosinophils are present in nasal mucosal biopsies within the submucosa and epithelium in active rhinitis. They generate vasoactive mediators and have the capacity to produce cytotoxic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin, and eosinophil cationic protein. Although the eosinophils feature heavily in the late-phase allergic response, their role in the early-phase allergic response is not clear. The late phase starts 4–8 h after allergen exposure. Clinically, it can be similar to the early phase but, in general, nasal congestion is more prominent. The late response involves a process of cellular accumulation. The expression of adhesion molecules and the presence of cytokines IL-3, IL-4, IL-5, IL-8, granulocytemacrophage colony-stimulating factor, and TNF-a enhances cell activation, accumulation of neutrophils, eosinophils, and T lymphocytes, and prolongs the survival of eosinophils within the nasal mucosal tissue. There is evidence showing that, in persistent rhinitis, there is an upregulation of the adhesion mechanisms with increased expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. During this late phase, activated basophils are responsible for histamine release. All these events amplify the allergic inflammatory response, leading to a real cascade of reactions (Figures 9 and 10).

Although the inflammatory reaction in AR is triggered by allergen exposure, it has been demonstrated that even in cases of subliminal exposure to the allergen(s), in the absence of symptoms, a certain degree of inflammatory infiltration at the mucosal level persists. This is called the ‘minimal persistent inflammation’. The Priming Effect

The ‘priming effect’ refers to the phenomenon where the amount of allergens necessary to evoke an immediate response decreases with repeated allergen challenges or exposures. Nasal challenge induces an immediate clinical response in allergic subjects and a concomitant appearance of an inflammatory infiltrate. The mucosal inflammation may persist 48–72 h after allergen exposure. If the subjects are rechallenged within this period the response is more pronounced: the so-called priming effect. This effect is hypothesized to be a result of the influx and subsiding activity of inflammatory cells during the latephase allergic response. Clinically, this explains the observation that decreasing allergen quantities are required to elicit symptoms as the pollen season progresses. In patients allergic to tree and grass pollen, the tree pollen season has a priming effect on the subsequent grass pollen season and these patients can often develop symptoms early in the grass pollen season when pollen counts are still very low.

Histamine Concentration

Leukotrienes Tachykinins

Early phase

Late phase

Time

Blockage Rhinorrhea Sneezing Itching Figure 9 Nasal allergen provocation results in a significant increase in mediators (histamine, leukotrienes, and tachykinins) and immediate nasal symptoms (blockage, rhinorrhea, sneezing, and itching) in allergic rhinitis patients. Between 2 and 24 h after allergen provocation, late-phase nasal symptoms, especially blockage, are observed in allergic rhinitis patients.

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IL-4 IL-13

Allergen

IgE Mast cell

IL-5 IL-4

Th2

IL-6 Th0

Eosinophil

IL-4

APC IL-12

Th1

IFN-
IL-2

IL-18 Figure 10 Allergic inflammation is characterized by a preponderance of T-helper type 2 (Th2) lymphocytes over T-helper type 1 (Th1) cells. APC, antigen-presenting cell; IL, interleukin; IFN-g, interferon gamma; IgE, immunoglobulin e; Th0, precursor T cell.

Systemic Component in the Allergic Rhinitis Response

In addition to the local events in the nose, there is a systemic element to the inflammatory process of AR. A variety of mechanisms have been proposed to explain the pathophysiological link between the upper and lower airways, including the loss of nasal protective function, altered breathing pattern, postnasal drip causing pulmonary aspiration of nasal contents, the presence of a nasal–bronchial reflex, and the progression of systemic inflammation. Recent data suggests that bidirectional systemic inflammation involving the bone marrow is likely to be important. Local allergen provocation (in the nose or bronchi) leads to upregulation and release from the bone marrow of hemopoietic eosinophil/basophil progenitor cells, which migrate to both nose and lungs where they can undergo differentiation and activation in situ.

Animal Models Besides clinical and in vitro studies, animal models of allergic airway disease may provide useful information concerning upper airway inflammation and serve as a model for relatively invasive experimental procedures. Although AR in animals is rare, chronic rhinitis associated with Aspergillus infection has been reported in rodents, poultry, dogs, and horses. In the absence of a common naturally occurring model of AR, sensitivity to allergens must be induced in healthy animals. A number of research groups are currently applying different experimental protocols to

induce experimental airway allergy. In guinea pigs, ovalbumin (OVA) is the most common sensitizing agent. To induce sensitization OVA is repeatedly injected into the peritoneum over the course of several weeks; this is followed by exposure to aerosolized OVA, which leads to allergen-specific IgE production. In addition to systemic sensitization, intranasal applications of OVA may induce allergen-specific IgE production but this exclusive local sensitization has been described in only a small number of studies. The upper airways of mice are less attractive for research compared to the lower airways, and this is for several reasons. First, mice are obligatory nose breathers, resulting in a significant baseline inflammation. Second, eosinophilic inflammation present in the nose of mice with experimental airway inflammation is rather limited compared to the inflammatory response in the lower airways. Several animal models of AR have shed light on the cellular and cytokine profiles associated with airway inflammation. They demonstrate that the relative importance of mast cells, eosinophils, IgE, and the cytokines IL-4 and IL-5 in the development of allergic inflammation varies with the sensitization protocol used and with the animal strain.

Management and Current Therapy Environmental control measures (allergen avoidance), pharmacological treatment, immunotherapy, and education are the cornerstones of therapeutic management of AR. In select cases, nasal surgery may be recommended as an adjunctive intervention.

ALLERGY / Allergic Rhinitis 89 Prevention

The treatment of patients with AR starts with the identification of possible allergens and subsequent prevention. If possible, allergen avoidance is the first step in the management of AR. The most common indoor allergens are dust mites and dog and cat dander. The use of simple measures to avoid the allergens can relieve the symptoms but there is still a paucity of data relating to the effectiveness of avoidance. Clinical improvement can be expected within weeks. However, some allergens require a longer period before they are effectively removed. Pharmacological Agents

Intranasal steroids Intranasal steroids are a potent and highly effective treatment for patients with AR. Their effect is based on localized repression of the inflammatory response. They reduce the number of eosinophils, the amount of eosinophilic cationic protein present, and the number of mast cell progenitors. Their multiple sites of action may account for their extreme potency. Clinically their efficacy exceeds that of antihistamines, decongestants, and cromoglycin. Compared to antihistamines they are more effective in reducing nasal blockage. However, they have a limited effect on associated eye symptoms and a relatively slow onset of action. There is some evidence that intranasal steroids have a beneficial effect on asthma symptoms. At the recommended doses, intranasal steroids cause few side effects. Systemic side effects have not been observed, and the risk of systemic absorption and possible effect on growth in children has been extensively studied but has shown no effect at the recommended doses. Despite these findings, nasal steroids should be used at the lowest possible dose in children. To date, there is no convincing evidence that doses greater than the recommended maximum increase efficacy. Antihistamines Antihistamines typically reduce itching, sneezing, and rhinorrhea but have no or little effect on nasal congestion associated with the late-phase reaction. The currently used antihistamines are clearly less effective than topical nasal steroids. They are subdivided into first- and second-generation histamines. First- and second-generation antihistamines differ in their side effects. First-generation antihistamines produce sedation and other central nervous system symptoms in X20% of patients and may cause drying of the mouth and urinary hesitancy. Secondgeneration antihistamines also have varying degrees of anticholinergic, antimuscarinic, and antiadrenergic effects, but to a lesser extent than first-generation antihistamines.

Cromoglycate and nedocromil Sodium cromoglycate and nedocromil sodium are both drugs that have been demonstrated to influence mast cell degranulation in in vitro studies. However, no inhibitory effect on histamine release has been demonstrated in mast cells recovered from nasal tissue. These drugs also inhibit the intermediate conductance pathways of mast cells, eosinophils, epithelial cells, fibroblasts, and sensory neurons. Cromoglycate blocks symptoms of the immediate and late phase and is effective even when used shortly before allergen inhalation. Cromoglycate is more effective than placebo; however, most studies show it to be less effective than nasal steroids. Both drugs do not induce any serious side effects. Ipratropium Ipratropium bromide blocks the activity of atropine and therefore reduces rhinorrhea when used intranasally. It does not block sneezing, pruritus, or nasal obstruction and is thus of greatest use in nonallergic rhinitis with predominant rhinorrhea. Vasoconstrictors Local vasoconstrictors reduce nasal blockage and are therefore indicated to relieve the symptoms of AR. However, they create a tachyphylaxis with rebound rhinitis as a symptom that develops after 3–7 days. Excessive consumption of nasal decongestants can cause rhinitis medicamentosa. Therefore, nasal decongestants are not recommended in the treatment of AR and should preferably be used for a short period when symptomatic vascular decongestion is indicated for the alleviation of nasal obstruction. Oral pseudoephedrine as a sustained release preparation has been shown to decrease both the nasal congestion and nasal airway resistance in patients with hay fever but only at doses where there is a high risk of systemic side effects. Leukotriene modifiers While sneezing and nasal itching correlate best with histamine levels in experimental AR, nasal congestion correlates with LTC4 levels. Leukotriene receptor antagonists provide some benefits in patients with AR. However, as demonstrated by some recent studies, leukotriene modifiers may be less effective than intranasal steroids. In children, leukotriene receptor antagonists have been shown to reduce exercise-induced asthma especially in combination with inhaled steroids. Allergen-specific immunotherapy Allergen-specific immunotherapy is the practise of administering gradually increasing doses of therapeutic vaccines of standardized allergen extracts until reaching an arbitrary dose that is maintained for several years. Traditionally, allergen-specific immunotherapy

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is administered subcutaneously. There is now good evidence that immunotherapy with seasonal and perennial allergens is clinically effective for the treatment of AR, but it should only be considered in patients with severe symptoms of AR, when allergen avoidance and pharmacotherapy have failed to reduce symptoms or when pharmacotherapy has been associated with unacceptable side effects. In addition, specific immunotherapy for AR, when administered early in the disease process, has been demonstrated to modify the long-term progress of the allergic inflammation and disease, by preventing the development of new sensitizations and by preventing the development of asthma. The exact mechanisms at the basis of the beneficial effects of allergen-specific immunotherapy are complex and are still not completely understood. Recent evidence suggests both a shift away from a Th2-type response as well as the generation of regulatory T cells. As immunotherapy is not free of risk and may provoke systemic reactions (severe asthma attacks and anaphylaxis in particular), it can only be carried out by or under the supervision of trained specialists, with direct access to the necessary rescue medication. Because of these risks, patients must be closely observed for 20–30 min after injection. More recently, other administration routes for allergen-specific immunotherapy have been investigated, including nasal, sublingual-swallow, and oral immunotherapy. Sublingual administration has shown to be effective. Novel treatments Increasing insights into the pathophysiology of AR, the roles of diverse cells and their cytokine products, and receptors and mediators involved in allergic inflammation has provided new (potential) targets for pharmacotherapy. Modulation of allergic response through antimediators and antireceptors has gained much interest (e.g., anti-IgE, anti-IL-5, anti-CCR3). Role of Surgery in the Management of Allergic Rhinitis

diagnostic tests have been developed, many effective pharmacological agents are currently available, and new potential targets for pharmacotherapy, new routes of administration, and alterations in treatment dosages and schedules are continuously being investigated. To provide and disseminate this knowledge from research into practice, to facilitate and standardize the management of AR, and to improve the patient care and, consequently, the patient satisfaction and compliance several clinical guidelines have been developed. Before 1998, European and American guidelines for the management of AR were developed, based on expert opinion. The European guidelines are in many respects very similar to the American guidelines, but a Table 5 Classification schemes of statements of evidence Category of evidence Ia: Evidence from meta-analysis of randomized controlled trials Ib: Evidence from at least one randomized controlled trial IIa: Evidence from at least one controlled study without randomization IIb: Evidence from at least one other type of quasi-experimental study III: Evidence from nonexperimental descriptive studies, such as comparative studies, correlation studies, and case–control studies IV: Evidence from expert committee reports or opinions or clinical experience of respected authorities or both Strength of evidence of recommendations A: Directly based on category I evidence B: Directly based on category II evidence or extrapolated recommendation from category I evidence C: Directly based on category III evidence or extrapolated recommendation from category I or II evidence D: Directly based on category IV evidence or extrapolated recommendation from category I, II, or III evidence Adapted form Shekelle PG, Woolf SH, Eccles M, and Grimshaw J (1999) Developing guidelines. British Medical Journal 318: 593– 596.

Table 6 Strength of evidence for the treatment of rhinitis

Surgery does not relieve allergic inflammation and should only be used in case of turbinate hypertrophy or cartilaginous or bony obstruction, contributing to or aggravating rhinitis symptoms, especially nasal obstruction. In these cases, a conchotomy and/or septo(rhino)plasty is recommended. In cases of secondary and independent sinus disease, functional endoscopic sinus surgery can be performed.

Intervention

Clinical Guidelines for the Management of Allergic Rhinitis

SIT, allergen-specific immunotherapy. Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108(5 supplement): S147–S334.

Our insight into the pathophysiology of AR has increased in recent years. Highly sensitive and specific

Oral anti-H1 Intranasal anti-H1 Cromones Antileukotrienes Subcutaneous SIT Sublingual/nasal SIT Allergen avoidance

Seasonal AR

Perennial AR

Adults

Children

Adults

Children

A A A A A A D

A A A A A A D

A A A

A A A

A A D

A A

ALLERGY / Allergic Rhinitis 91

prominent feature of the former is the stepwise approach recommended for the treatment of rhinitis, which is similar to the Global Initiative for Asthma guidelines for the treatment of asthma. In 1999, the ARIA Working Group was founded, under the initiative of the World Health Organization. Unlike the European and American guidelines,

the ARIA guidelines are formulated on evidencebased medicine, categorized by Shekelle. Based on these categories of evidence, the strength of evidence for a certain recommendation is graded from A to D (Table 5). The evidence for the recommended pharmacotherapy and immunotherapy for AR treatment is particularly strong (category A evidence strength).

Diagnosis of allergic rhinitis Allergen avoidance

Intermittent symptoms

Mild

Moderate/ severe

Persistent symptoms

Moderate/severe

Mild

Intranasal corticosteroids Not in preferred order: • oral H1-blocker • intranasal H1-blocker • and/or decongestant

Review patient after 2− 4 weeks Not in preferred order: • oral H1-blocker • intranasal H1-blocker • and/or decongestant • intranasal corticosteroid • (cromones)

In persistent rhinitis: review the patient after 2−4 weeks

If failure: step-up If improved: continue for 1 month

failure

improved

Step-down and continue treatment for 1 month

Review diagnosis, compliance, query infections or other infections, or other causes

↑ Intranasal CS dose

Itch/sneeze: +H1 blocker

Rhinorrhea: + ipratropium Blockage: + decongestant or oral CS (short term)

Failure Surgical referral If conjunctivitis add : Oral H1-blocker or intraocular H1-blocker or intraocular cromones (or saline) Consider specific immunotherapy Figure 11 Stepwise treatment algorithm for allergic rhinitis in adolescents and adults, as recommended by ARIA. CS, corticosteroids. Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108 (5 supplement): S147–S334.

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The evidence for allergen avoidance, on the other hand, is limited (category D evidence strength) (Table 6). Similar to the European guidelines, a stepwise approach for the treatment of AR is recommended by ARIA with the following first-line treatment approaches (Figure 11): oral or intranasal H1-antihistamines, with limited use of decongestants, for mild intermittent rhinitis; oral or intranasal H1-antihistamines or intranasal corticosteroids, with limited use of decongestants and cromones, for moderate to severe intermittent and mild persistent rhinitis; and intranasal corticosteroids, with step-down and step-up options, in conjunction with H1-antihistamines, decongestants, ipratropium, and eventually oral corticosteroids, for moderate to severe persistent rhinitis. Additionally, specific immunotherapy should be considered in persistent disease and when the symptoms are moderate to severe and do not respond to conventional treatment. When conjunctivitis is present, oral or intraocular H1-antihistamines should be used. Whereas the European guidelines did not take into account the costs and availability of the treatment strategies in different countries, the ARIA guidelines are developed for the whole world and recognize that the outcome of disease management largely depends on compliance with the suggested treatment, which in turn is influenced by the availability and affordability of the specific interventions. Therefore, a flexible stepwise approach is recommended, based on the four cornerstones of patient education, allergen avoidance, pharmacotherapy, and immunotherapy, but modifiable in low-income countries. See also: Allergy: Overview; Allergic Reactions. Asthma: Overview. Chemokines. Chymase and Tryptase. Histamine. Immunoglobulins. Interleukins: IL-4; IL-5; IL-10; IL-13. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; T cells. Lipid Mediators: Leukotrienes; Prostanoids.

Further Reading Aalberse RC (2000) Molecular mechanisms in allergy and clinical immunology. Structural biology of allergens. Journal of Allergy and Clinical Immunology 106: 228–238. Barnes PJ (2003) Pathophysiology of allergic inflammation. In: Adkinson F Jr, Yunginger JW, Busse WW, et al. (eds.) Middleton’s Allergy Principles and Practice, pp. 483–499. Pennsylvania: Mosby. Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108 (5 supplement): S147–S334. Howarth PH (2003) Allergic and nonallergic rhinitis. In: Adkinson F Jr, Yunginger JW, Busse WW, et al. (eds.) Middleton’s Allergy Principles and Practice, pp. 1391–1410. Pennsylvania: Mosby. International Rhinitis Management Working Group (1994) International Consensus Report on the Diagnosis and Management of Rhinitis. Allergy 49(supplement 9): 5–34. Johansson SG, Bieber T, Dahl R, et al. (2004) A revised nomenclature for allergy for global use: Report of the Nomenclature Review Committee of the World Allergy Organization. Journal of Allergy and Clinical Immunology 113: 823–826. Malm L, Gerth van Wijk R, and Bachert C (1999) Guidelines for nasal provocations with aspects on nasal patency, airflow and airflow resistance. Rhinology 37: 133–135. Salib RJ and Howarth PH (2003) Remodelling of the upper airways in allergic rhinitis: is it a feature of the disease? Clinical and Experimental Allergy 33: 1629–1633. Shekelle PG, Woolf SH, Eccles M, and Grimshaw J (1999) Developing guidelines. British Medical Journal 318: 593–596. Van Cauwenberge P, Bachert C, Passalacqua G, et al. (2000) Consensus statement on the treatment of allergic rhinitis. Allergy 55: 116–134. Wheatley LM and Platts-Mills TAE (1999) Allergens. In: Naclerio RM, Durham SR, and Mygind N (eds.) Rhinitis, pp. 45–58. New York: Marcel Dekker, Inc. Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema (1998) ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee. Lancet 351: 1225–1232.

Relevant Website http://www.ginasthma.com – Global Initiative for Asthma. Global strategy for asthma management and prevention (2003).

ALVEOLAR HEMORRHAGE O C Ioachimescu, Cleveland Clinic Foundation, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Bleeding into the alveoli characterizes the syndrome of diffuse alveolar hemorrhage (DAH) and represents a potential

life-threatening condition. There are many causes of DAH, including vasculitides, such as Wegener’s granulomatosis, microscopic polyangiitis, Good pasture’s syndrome, connective tissue disorders, and other conditions. Pathologically, the syndrome is due to pulmonary vasculitis, to a ‘bland’ alveolar hemorrhage, or it represents the nondominant pathology, as seen in diffuse alveolar damage from acute respiratory distress syndrome (ARDS). Most patients present with dyspnea, cough, hemoptysis (the latter in only 66% of the cases), anemia, and

ALVEOLAR HEMORRHAGE 93 new pulmonary infiltrates. Urgent bronchoscopy and bronchoalveolar lavage is generally required to confirm the diagnosis, with a superior yield when it is performed in the first 48 h. In patients with evidence of DAH and renal involvement (pulmonary–renal syndrome), kidney biopsy may be considered to identify the etiology and direct the therapy. This chapter will describe the mean features of the DAH syndrome and review in short the main causes of alveolar bleeding.

Introduction Hemoptysis is most commonly due to disruption of the bronchial circulation of various (endo)bronchial conditions such as bronchitis, bronchiectasis, neoplastic disorders, or (rarely) disorders of the pulmonary circulation. Hemorrhage from a bronchial source can be rapidly fatal, since a brisk bleeding can lead to a large amount of blood to occupy anatomic and functional dead space of the respiratory tract; in this setting the alveoli can also be flooded quickly, mimicking the true alveolar hemorrhage. Anatomical injury at the alveolar–capillary basement membrane level of the pulmonary microcirculation may cause hemoptysis from arteriolar, venular, or capillary source. Bleeding into the alveoli characterizes the syndrome of diffuse alveolar hemorrhage (DAH). Although alveolar hemorrhage may be focal, generally there are multiple areas affected, hence the term DAH. Diffuse alveolar hemorrhage should always be considered a potentially life-threatening condition; it requires early recognition, care in stabilizing the patient (airway protection, sometimes selective intubation, mechanical ventilation, etc.) and specific treatment once the etiology is established. Synonyms with DAH that can be found in the literature are: (intra)pulmonary hemorrhage, pulmonary alveolar hemorrhage, pulmonary capillary hemorrhage, alveolar bleeding, and microvascular pulmonary hemorrhage. The DAH syndrome is relatively rare, although no studies addressed its specific epidemiology. Currently, our understanding of the etiopathogenesis and the appropriate management relies mainly on case reports or case series of specific disorders leading to DAH. Unfortunately, the therapeutic approach is rather non-specific, with a few notable exceptions (Wegener’s granulomatosis (WG), Goodpasture’s syndrome, systemic lupus erythematosus (SLE), etc.)

Etiology Various diseases can lead to DAH syndrome (Table 1). To date, no prospective studies of DAH have estimated the relative frequency of various etiologies. A wellknown retrospective review of 34 cases of DAH suggested that the most common cause was WG, which

Table 1 Causes of diffuse alveolar hemorrhage (DAH) DAH associated with vasculitis Wegener’s granulomatosis Microscopic polyangiitis Goodpasture’s syndrome Isolated pauci-immune pulmonary capillaritis Connective tissue disorders Antiphospholipid antibody syndrome Mixed cryoglobulinemia Henoch–Scho¨nlein purpura, IgA nephropathy Pauci-immune or immune complex-associated glomerulonephritis Behc¸et’s syndrome Acute lung graft rejection Thrombotic thrombocytopenic purpura and idiopathic thrombocytopenic purpura ‘Bland’ DAH Mitral stenosis and mitral regurgitation Anticoagulants, antiplatelet agents, or thrombolytics; disseminated intravascular coagulation Pulmonary venoocclusive disease Infections: human immunodeficiency virus (HIV) and infective endocarditis Toxins: trimellitic anhydride, isocyanates, crack cocaine Drugs: propylthiouracil, diphenylhydantoin, amiodarone, mitomycin, penicillamine, sirolimus, methotrexate, nitrofurantoin, gold, all-trans retinoic acid (ATRA), bleomycin (especially with high-flow O2), montelukast, zafirlukast, infliximab Idiopathic pulmonary hemosiderosis DAH as nondominant pathology Diffuse alveolar damage (DAD) Malignant conditions (e.g., pulmonary angiosarcoma) Lymphangioleiomyomatosis/tuberous sclerosis Pulmonary capillary hemangiomatosis Lymphangiography

accounted for one-third of cases, followed by Goodpasture’s syndrome (13%), idiopathic pulmonary hemosiderosis (IPH) (13%), collagen vascular disease (13%), and microscopic polyangiitis (9%). A review of 29 cases of DAH associated with capillaritis found that the most common cause was isolated pulmonary capillaritis. Other conditions associated with DAH are briefly discussed below. Overall, three different histologic patterns may be seen. DAH Associated with Vasculitis

This pathologic pattern is characterized by neutrophilic infiltration of the interalveolar and peribronchiolar septal vessels (pulmonary interstitium). This sequentially leads to anatomic disruption of the capillaries, and red blood cell extravasation into the alveoli and interstitium. This leads to neutrophil fragmentation and apoptosis, with subsequent release of the intracellular proteolytic enzymes and reactive oxygen species, which begets more inflammation,

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intra-alveolar neutrophilic nuclear dust, fibrin and inflammatory exudate, and fibrinoid necrosis of the interstitium. DAH Associated with ‘Bland’ Pulmonary Hemorrhage

This pattern is characterized by intra-alveolar red cell extravasation without any evidence of inflammation or destruction of the alveolar capillaries, venules, and arterioles; the epithelial lesions are usually discrete. DAH Associated with Other Conditions (Nondominant Pathology)

Here, the DAH is secondary to diffuse alveolar damage (DAD), lymphangioleiomyomatosis (LAM), drug-induced lung injury, metastatic neoplasia to the lungs, mitral stenosis, etc. DAD is the main underlying lesion of the acute respiratory distress syndrome (ARDS), and is characterized by intra-alveolar hyaline membranes, interstitial edema with minimal inflammation, and at times by ‘secondary’ DAH.

Clinical Presentation The syndrome of DAH may present with a constellation of symptoms, signs, and laboratory results that may suggest the underlying etiology (e.g., WG, Goodpasture’s syndrome, drug-related vasculitis, etc.) or only establish the diagnosis of the syndrome without a specific etiology. Symptoms

The onset of DAH is most often acute or subacute (less than 1 week). Dyspnea, cough, and fever are the common initial symptoms. Some patients, however, present with ARDS requiring mechanical ventilation. Hemoptysis may be absent at the time of presentation in up to one-third of patients with DAH syndrome. When present, it ranges from fulminant and life-threatening to mild and intermittent in nature. Physical Examination

The lung examination is usually non-specific, unless there are physical signs of an underlying systemic vasculitis or collagen vascular disorder (rashes, purpura, eye lesions, hepatosplenomegaly, clubbing, etc.).

The presence of Kerley B lines points toward mitral valve disease or pulmonary venoocclusive disease. Computed tomographic imaging studies may show areas of consolidation alternating with areas of ground-glass attenuation and preserved, normal areas. Gallium scan, rarely ordered today for this purpose, used to be performed in the past to reveal areas of active vasculitis or inflammatory activity (rather non-specific and with unclear sensitivity, too). Other nuclear studies, geared to reveal breakdown of the microcirculatory integrity and extravasation of red blood cell out of the vessels, have not been confirmed by the ultimate test of time. Pulmonary Function Tests

The DAH leads to various degrees of oxygen transfer impairment and hypoxemia, sometimes severe enough to require ventilatory support. A sensitive marker for DAH is a sequential increase in diffusing lung capacity for carbon monoxide (DLCO). In this setting, due to an increased availability of intraalveolar hemoglobin, capable of binding with highaffinity carbon monoxide, the DLCO is generally normal or elevated. Unfortunately, the severe condition of these patients and the associated hemoptysis generally preclude any DLCO measurements. An obstructive lung disease associated with recurrent DAH may be due to lymphangioleiomatosis, histiocytosis X, or pulmonary capillaritis in the setting of microscopic polyangiitis or WG. Recurrent episodes of DAH generally lead to interstitial fibrosis and ventilatory restrictive defects, as seen in idiopathic pulmonary hemosiderosis. Laboratory Abnormalities

The blood work generally reveals acute and/or chronic anemia, ‘stress’ leukocytosis, elevated erythrocyte sedimentation rate, and C-reactive protein (particularly in the cases of DAH caused by systemic diseases or associated with a pattern of vasculitis). Since several causes of DAH may present as pulmonary–renal syndromes (i.e., association of pulmonary hemorrhage with different types of glomerulonephritis), blood urea nitrogen (BUN) and creatinine concentration elevations, and abnormal urine sediments (red blood cells/casts, white blood cells/casts, proteinuria of glomerular origin) can be seen.

Imaging Studies

The radiographic findings are generally non-specific and show new and/or old, patchy or diffuse alveolar opacities. Recurrent episodes of DAH lead to reticular interstitial opacities due to pulmonary fibrosis, usually with minimal honeycombing (if any).

Diagnosis DAH represents a medical emergency, since it represents a potentially fatal condition. Despite recent advances and refinements of the diagnostic and therapeutic tools in DAH, it remains a highly morbid

ALVEOLAR HEMORRHAGE 95

condition, with substantial fatality. One must have a low threshold to entertain the diagnosis, to confirm it and to thoroughly look for the underlying etiology. Methodically, two separate steps are to be followed. Establish the Diagnosis of DAH Syndrome

The triad of elevated DLCO, hypoxemia, and new pulmonary infiltrates in the setting of hemoptysis and dyspnea suggests the diagnosis of DAH. Patients who present with hemoptysis need to be screened for focal sources of pulmonary hemorrhage (i.e., bronchitis, bronchiectasis, infection, neoplastic processes, etc.); upper airway and gastrointestinal sources must also be excluded carefully. It is important to remember that most disorders manifested with severe hemoptysis can cause alveolar infiltrates. In patients without hemoptysis, the clinical evaluation needs also to screen for evidence of congestive heart failure, pneumonia, inhalational or drug-related lung injury, and other causes of bleeding. Most patients suspected of having DAH need a bronchoscopic examination, which serves two purposes: documentation of alveolar hemorrhage by visual inspection and bronchoalveolar lavage (BAL; especially if there is no hemoptysis) and exclusion of an associated infection. This procedure has a higher yield if it is performed early (within 48 h) rather than later. An increasing hematocrit or progressively bloodier aspect of three sequential BAL aliquots from an affected area is diagnostic of DAH. In subacute or recurrent episodes of DAH, counting the hemosiderin-laden macrophages (siderophages) as demonstrated by Prussian Blue staining on a pooled BAL specimen centrifugate may be useful for diagnosis (generally more than 30% siderophages). BAL specimens should be sent for routine bacterial, mycobacterial, fungal, viral, and Pneumocystis carinii microscopic studies and cultures. The use of transbronchial biopsy in patients with suspected DAH is of unclear value due to the small size of the specimens and possible sampling bias. Unless another clinical condition is suspected, transbronchial biopsy is generally not useful clinically. Identify the Specific Etiology

Identifying the specific etiology is generally equivalent to the process of differential diagnosis. Serologic studies need to be performed early during the course of the disease, although the results are generally not available in a timely manner for immediate management of the disease. Complement fractions C3 and C4 and anti-dsDNA, anti-glomerular basement membrane (GBM), antineutrophil cytoplasmic antibodies (ANCA), and antiphospholipid antibodies

represent the basic panel to be checked in DAH patients (see Autoantibodies). If the diagnosis of DAH is still not clear or the underlying etiology is still not known after a thorough clinical evaluation, imaging studies, serologies, and bronchoscopy, surgical biopsy should be entertained. Besides, the results of a surgical biopsy may become available faster than the serologic tests. The ‘ideal’ site to biopsy is dependent on the pretest probability of the underlying disorder: for WG a nasal biopsy may suffice, while a kidney biopsy (with immunofluorescent studies) may be less invasive than others in diagnosing Goodpasture’s syndrome, microscopic polyangiitis, or systemic lupus erythematosus. In isolated pulmonary disease or pauci-immune pulmonary capillaritis a lung biopsy is a mandatory test. Immunofluorescence reveals linear deposition of immunoglobulins and immune complexes along the basement membrane in Goodpasture’s syndrome and granular deposits in SLE, whereas the systemic vasculitides appears pauci-immune. In cases of pulmonary–renal syndrome, the kidney biopsy shows focal segmental necrotizing glomerulonephritis. Additionally, skin biopsy of a lesion can demonstrate leukocytoclastic vasculitis or Henoch–Scho¨nlein purpura (in the latter case, immunoglobulin A (IgA) deposits are suggestive of the diagnostic).

Current Therapy Corticosteroids are currently the backbone of DAH syndrome therapy, especially if associated with systemic or pulmonary vasculitis, Goodpasture’s syndrome, and connective tissue disorders. Most authors recommend intravenous methylpredisolone (up to 500 mg every 6 h, although lower doses seem to have similar efficacy) for approximately 4–5 days, followed by a gradual taper to maintain doses of oral steroids. In the setting of pulmonary–renal syndrome, the therapy needs to be initiated as soon as possible, since the potential of reversibility is lost exponentially in the first week of disease activity. Other immunosuppressive drugs can be used in DAH (e.g., cyclophosphamide, azathioprine, mycophenolate mofetil, etanercept, etc.) depending on the disease severity, failures to respond to corticosteroids and the underlying disease (e.g., WG, Goodpasture’s syndrome). Intravenously administered cyclophosphamide (2 mg kg 1 day 1, adjustable to renal function) is generally the preferred adjunctive immunosuppressive drug in the initiation phase of the treatment, and continued several weeks until blood marrow suppression, infections, or other limiting effects require discontinuation and thereafter switch to consolidative

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and/or maintenance therapy with methotrexate or other agents. Plasmapheresis is indicated in the treatment of DAH associated with Goodpasture’s syndrome or other vasculitis processes when the titers of pathogenetic immunoglobulins and immune complexes are very high (e.g., plasmapheresis in the setting of ANCA-associated vasculitis with overwhelming endothelial injury and/or hypercoagulable state). However, its utility in DAH syndromes other than Goodpasture’s syndrome has not been evaluated in prospective studies. If intravenous immunoglobulin (IVIG) therapy adds anything to the treatment of DAH due to vasculitis or other connective tissue disease is yet unclear. Besides the treatment of vasculitis and underlying disorder, stabilization of the patients with moderate and severe hemoptysis entails supplemental oxygen, bronchodilators, reversal of any coagulopathy, red blood cell transfusion, intubation with bronchial tamponade/protective strategies for the less involved lung, mechanical ventilation, etc. Several case reports showed success in treating alveolar hemorrhage due to allogeneic hemaopoietic stem cell transplantation, ANCA-associated vasculitis, systemic lupus erythematosus, and antiphospholipid syndrome with recombinant-activated human factor VIIa, which may become a new tool in the (otherwise poor) armamentarium available for this condition.

Prognosis Recurrent episodes of DAH may lead to various degrees of interstitial fibrosis, especially in patients with underlying WG, mitral stenosis, long-standing and severe mitral regurgitation, and idiopathic pulmonary hemosiderosis. A post-DAH syndrome has also been described, particularly in microscopic polyangiitis, and idiopathic pulmonary hemosiderosis and consists of progressive obstructive ventilatory defects and anatomic emphysema. Mortality

The disease fatality varies with the underlying cause of DAH; patients with systemic lupus erythematosus, anti-GBM antibody disease and several forms of ANCA-associated vasculitis can have a high mortality (up to 50%) due to the disease activity, infections, and cardiovascular morbidity (e.g., Churg–Strauss syndrome).

Experimental Models Experimental models of different conditions (e.g., microscopic polyangiitis, anti-GBM antibody disease,

etc.) have allowed the indirect study of DAH syndrome and its close interrelation with iron metabolism. Iron is of crucial importance in the inflammatory and anti-infectious defense of the respiratory tract and lung parenchymal homeostasis. Local macrophages have a major contribution to iron recycling from red blood cells, through the phagocytosis of the intra-alveolar erythrocytes and eventual return of the electrolyte to the bone marrow erythron, where iron is incorporated in the structure of heme in maturing red blood cells. The understanding of alveolar pathology in DAH has been enriched lately by a literature explosion in the field of ferroproteins and genetics of the regulatory factors involved in the iron metabolism. It is beyond the scope of this section to review exhaustively the respiratory iron metabolism, hence mention will only be made of the main facts. The free iron’s presence in the extracellular milieu together with local hypoxia induces reactive oxygen species and other free radical production, with subsequent peroxidation of the cellular membranes, cell apoptosis, and preinflammatory cytokine release. The phagocytosis capacity of the alveolar macrophages seems to be easily exhaustible, becoming hemosiderin-laden macrophages (siderophages), which in turn leads to more free iron and heme available in alveolar spaces and pulmonary interstitium. Recently, J774 macrophages have been used among other experimental models to investigate the presence of different ferroproteins and their role. A recently discovered peptide, hepcidin, seems to have a major ferrostat role in local and general iron overload syndromes; its role (if any) is yet unclear in the DAH syndromes. Another interesting protein, ferroportin, seems to be involved in macrophage iron recycling from engulfed erythrocytes. It is possible that macrophage’s iron retentive mechanism is abnormal, similar to what is seen in human hereditary hemochromatosis type 4, or ferroportin disease.

Specific Causes of DAH Wegener’s Granulomatosis

WG is a systemic disease characterized by necrotizing granulomatous inflammation of the small vessels, involving predominantly the respiratory tract and kidneys. The organ involvements can be recalled using the mnemotechnic JERKS: (1) Joint disease % (arthralgias and arthritis); (2) ENT (rhinosinusitis) % (both upper and and eye disease; (3) Respiratory % about 70–95% of cases); lower respiratory tracts, (4) Kidney involvement (lesions of focal segmental % necrotizing glomerulonephritis); (5) Skin and other % of the lung organs (systemic). The gross appearance

ALVEOLAR HEMORRHAGE 97

ANCA-associated small vessel vasculitis who were treated with plasma exchange in conjunction with induction immunosuppressive regimens had a remission of the DAH. Other therapies, such as trimethoprim– sulfamethoxazole, tumor necrosis factor inhibitors, and anti-CD20 monoclonal antibodies have been tried with unproven efficacy or are under scrutiny. Mortality is considerable and most often caused by infections, respiratory failure, and renal failure. Microscopic Polyangiitis

Figure 1 Diffuse alveolar hemorrhage associated with, necrotizing pulmonary vasculitis (shown) in a patient with Wegener’s granulomatosis.

in WG with DAH is dark red, sometimes associated with multiple nodules and cavitary lesions (exceptionally solitary pulmonary nodules), and much more frequently with areas of consolidations and focal or geographic necrosis (Figure 1). The chest radiograms show fluffy alveolar and/or interstitial infiltrates reflecting the diffuse microvasculature disorder. The diagnosis of WG is established based on clinical presentation and serologic confirmation, that is, positive ANCA antibodies (c- or p-ANCA). In organlimited WG there is ANCA positivity in 60% of the cases, while in generalized disease in 90–95% of cases. c-ANCA (directed against proteinase-3) is present in 85–90% of generalized forms of the disease. If diagnosis is still in doubt, surgical biopsy of affected organs (kidneys, lungs, nasal mucosa) may be useful for diagnosis. In WG, the DAH due to pulmonary capillaritis may mark the disease onset or occur later during the course, in a subclinical and/or recurrent fashion. The progressively bloodier BAL aliquots with large numbers of red blood cells and hemosiderin-laden macrophages in the absence of an infectious etiology confirm the diagnosis of DAH. The standard inductive therapy of DAH is high-dose corticosteroids (e.g., 1 g methylprednisolone daily for 3 days) and daily intravenous cyclophosphamide. Azathioprine generally substitutes cyclophosphamide in about 6 months or after remission in an inductivemaintenance approach and/or after a cumulative high dose of cyclophosphamide is reached. IVIG or therapeutic plasma exchange may be used for persistent disease. In one study, 20 out of 20 patients with

Microscopic polyangiitis (MP), a small-vessel variant of polyarteritis nodosa, is sometimes difficult to differentiate from WG due to similar clinical presentations, serologic and pathologic findings. The typical pathologic feature in microscopic pauci-immune neutrophilic polyangiitis (found in virtually 100% of cases) is a focal and segmental necrotizing glomerulonephritis, renal lesion also seen in WG, Goodpasture’s syndrome, and other connective tissue disorders. The lack of granulomatous inflammation differentiates pathologically between WG and MP. A positive serum perinuclear ANCA (p-ANCA), directed against myeloperoxidase’s epitopes, strongly supports the diagnosis, but it can be also seen in 20–30% of WG cases. Microscopic polyangiitis causes relatively frequently a syndrome of severe DAH. Treatment with corticosteroids and cyclophosphamide followed by azathioprine is similar to WG therapy. Plasmapheresis and IVIG may be also useful in difficult-to-treat cases. As in other hemorrhagic conditions, factor VIIa has also been used with various success. The short-term and long-term (5-year) mortality rates from DAH in MP are approximately 25% and 40%, respectively. Goodpasture’s Syndrome

Goodpasture’s syndrome is a form of anti-basement membrane antibody disease and is characterized by a combination of DAH and glomerulonephritis. Anti-GBM antibody, the pathognomonic immunoglobulin, is directed against alpha-3 (IV) collagen from GBM and is found in the serum of more than 90% of patients and part of the linear immunofluorescent deposits in the glomerular membrane noted in the disease. The syndrome typically involves DAH in a smoker (usually a young male, although older patients, women, and nonsmokers can also have it). Isolated, renal-sparing DAH, a rare occurrence, may have anti-GBM both in the serum and in the glomerular membrane in a typical linear antibody deposition. The treatment of Goodpasture’s syndrome includes urgent plasmapheresis and corticosteroids,

98

ALVEOLAR HEMORRHAGE

cyclophosphamide, and/or azathioprine. The isolated DAH seems to respond well to corticosteroids alone. Alveolar bleeding secondary to Goodpasture’s syndrome has a 2-year survival rate close to 50%, while patients presenting with renal insufficiency have a worse outcome. Churg–Strauss Syndrome

Churg–Strauss syndrome (allergic and granulomatous angiitis) is a systemic disorder characterized by asthma, peripheral blood eosinophilia, and systemic vasculitis of small-caliber vessels with extravascular necrotizing granulomas. It involves mainly the upper respiratory tract, the lungs, and the peripheral nerves. Tissue eosinophilia may involve the lungs or the gastrointestinal tract. Pulmonary hemorrhage is rare in this condition, while radiographic abnormal findings are described in 50–90% of the cases. p-ANCA positivity occurs in approximately 35–40%. Histologically, it is distinguished by small- and medium-vessel involvement and eosinophil-rich infiltrates (sometimes presenting with an aspect of chronic eosinophilic pneumonia). Of note, a Churg–Strauss-like syndrome can occur due to chronic ingestion of carbamazepine, quinine, or macrolides, while potential risk of aggravation of the disorder can occur on cysteinil leukotriene receptor blockers. Isolated Pauci-Immune Pulmonary Capillaritis

An interesting case of a DAH syndrome is represented by pauci-immune pulmonary alveolar capillaritis in the absence of any other systemic involvement or abnormalities. Several cases have been shown to have elevated serum titers of p-ANCA, but this finding may reveal in fact a fruste form of MP. Isolated, pauci-immune pulmonary capillaritis has been also seen in association with the drug called all-trans retinoic acid (ATRA). Available literature on pauciimmune pulmonary alveolar capillaritis shows that respiratory failure necessitating mechanical ventilation is frequent in this condition and the response to corticosteroids and immunosuppressive agents is favorable. Connective Tissue Disorders

Diffuse alveolar bleeding has been described in collagen vascular diseases such as SLE, scleroderma, rheumatoid arthritis, polymyositis/dermatomyositis, Henoch–Scho¨nlein syndrome, Behc¸et disease, and mixed connective tissue disorder. In SLE, pulmonary complications occur in more than 50% of cases, while DAH can occur in up to 5% of the patients

(although it is the most frequent disorder in this category presenting with alveolar bleeding); DAH has generally a poor prognostic connotation (50% fatality rate) and is due to a process of pulmonary capillaritis. When DAH occurs in SLE, contrary to WG, glomerulonephritis is generally absent. Immunofluorescent studies show granular deposits of IgG and complement (C3) in the pulmonary interstitium, alveolar blood vessels, and the GBM. Of note, the syndrome of DAH is clinically and pathologically distinct from acute lupus pneumonitis, which presents similarly and may be the inaugural presentation of SLE. Therapy consists of corticosteroids and cyclophosphamide and/or azathioprine. Plasmapheresis has no proven benefit. In rheumatoid arthritis, the syndrome of DAH secondary to pulmonary vasculitis generally occurs late, in ‘burnout’ disease. Hematologic Conditions

DAD seems to be the dominant lesion in the lungs of the patients who undergo chemotherapy for leukemia or stem cell transplantation; this lesion can be accompanied by DAH, sometimes fulminant and fatal, even in the absence of hemoptoic sputa. The offending factors seem to be the chemotherapeutic agents associated with actinic lesions, thrombocytopenia, and superimposed infections. The therapy includes platelet transfusions, reversal of coagulation abnormalities, and high-dose corticosteroids. DAH of the Immunocompromised Patient

Alveolar bleeding can occur in immunocompromised hosts due to a myriad of factors (infectious, chemotherapeutic, and immunosuppressive drugs, radiation therapy, thrombocytopenia, pulmonary edema, lung malignancy, other lung comorbidities, etc.) that have a common denominator: the endothelial injury. The exact frequency of the syndrome in the immunocompromised patients is currently unknown, partly because the hemorrhage can be subclinical, and because empirical therapy is instituted early, since the risk of invasive evaluation outweighs the benefits. While subclinical DAH in this setting may have minimal impact on survival, the alveolar hemorrhage associated with ‘serious’ causes like Kaposi’s sarcoma, invasive fungal infections (Aspergillus, Pseudoallescheria boydii, etc.), Mycobacterium, Legionella, or other invasive bacterial species may have catastrophic consequences. In human immunodeficiency virus (HIV) infection or acquired immunodeficiency syndrome (AIDS) patients, the cytomegalovirus (CMV) and Kaposi’s sarcoma represent the main risk factors for developing DAH. In a study of

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HIV-infected patients with radiographic infiltrates, in up to 44% of them more than 20% of the BAL cells were hemosiderin-laden macrophages. Drug-Induced DAH

Many drugs have been associated with DAH, including anticoagulants (warfarin, heparin, etc.), thrombolytic agents, and platelet antiaggregant agents; as a rule, in order to produce DAH, a ‘second hit’ (infectious, inflammatory, inhalatory, etc.) is required. Several drugs can produce lung injury and DAD, with secondary DAH (e.g., sirolimus, methotrexate, nitrofurantoin, etc.). Other drugs causing DAH trigger a process of pulmonary capillaritis (e.g., propylthyouracil, phenytoin, mitomycin, and ATRA). Penicillamine can cause pulmonary capillaritis associated with glomerulonephritis with granular immunofluorescent deposits (pulmonary– renal syndrome). Interestingly, these agents are also responsible for p-ANCA generation. The standard therapy is discontinuation of the presumed offending drug and (rarely) plasma exchange. Other Causes of DAH

Other conditions associated with alveolar bleeding are: different coagulopathies, toxic exposures (isocyantes, trimellitic anhydride, crack cocaine, etc.) primary antiphospholipid antibody syndrome, mixed cryoglobulinemia, Behc¸et’s syndrome, Henoch– Scho¨nlein purpura, lung transplant acute rejection, mitral stenosis and regurgitation, pulmonary venoocclusive disease, pulmonary capillary hemangiomatosis, LAM, and tuberous sclerosis Bourneville. In patients with primary antiphospholipid syndrome (APS), the thromboembolic complications occur in up to 15% of cases, pulmonary hypertension in about 2% of the cases, interstitial lung disease in up to 1% of patients, with DAH in less than 1% of cases. Idiopathic Pulmonary Hemosiderosis

Idiopathic pulmonary hemosiderosis (IPH) is a diagnosis of exclusion (see Idiopathic Pulmonary Hemosiderosis); it is a rare disease characterized by recurrent episodes of DAH and ‘bland’ alveolar hemorrhage (Figure 2). IPH occurs most frequently in children (80% of cases), but adult cases with onset up to the eighth decade of life have been reported (20% of cases). The pathogenesis of IPH is largely unknown. Some cases have been linked to fungi (Stachybotrys atra), others to environmental insecticides; it seems that several cases have initially been called IPH, when in fact coagulopathies such as van Willebrand’s disease have been found upon

Figure 2 Diffuse alveolar hemorrhage without any evidence of pulmonary vasculitis (‘bland’ alveolar hemorrhage) in a patient with idiopathic pulmonary hemosiderosis.

further scrutiny. Corticosteroids, hydroxychloquine, azathyoprine, and other immunosuppressive agents have been used with favorable effects. Lung transplantation has been reported as rather unsuccessful in a couple of patients with progressive disease and significant pulmonary fibrosis, due to recurrent pulmonary bleeding. Survival with IPH varies widely, although recent data has suggested better outcomes, most likely due to more aggressive immunosuppressive therapies.

Summary of Therapeutic Options DAH syndromes represent a serious condition with possible catastrophic consequences, caused by a myriad of conditions, associated or not with pulmonary capillaritis. Dyspnea, cough, hemoptysis and new alveolar, fluffy infiltrates in conjunction with bronchoscopic findings of bloody BAL, with numerous erythrocytes and siderophages make the syndrome diagnosis evident. Rarely, a surgical biopsy from the lung or another organ involved by the underlying condition may be necessary. The advent of ANCA has revolutionarized the diagnosis of WG, microscopic polyangiitis, Churg–Strauss syndrome, and other ANCA-associated conditions. The therapy in DAH targets both the autoimmune destruction of the alveolar capillary membrane and the underlying condition; corticosteroids and immunosuppressive agents are still the ‘gold standard’ of therapy in the majority of cases. Factor VIIa seems to be a promising new therapy for DAH, although further evaluation is needed.

100 ALVEOLAR HEMORRHAGE See also: Acute Respiratory Distress Syndrome. Autoantibodies. Bronchoalveolar Lavage. Granulomatosis: Wegener’s Disease. Idiopathic Pulmonary Hemosiderosis. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome.

Further Reading Afessa B, Cowart RG, and Koenig SM (1997) Alveolar hemorrhage in IgA nephropathy treated with plasmapheresis. Southern Medical Journal 90: 237–239. Afessa B, Tefferi A, Litzow MR, and Peters SG (2002) Outcome of diffuse alveolar hemorrhage in hematopoietic stem cell transplant recipients. American Journal of Respiratory and Critical Care Medicine 166: 1364–1368. Agusti C, Ramirez J, Picado C, et al. (1995) Diffuse alveolar hemorrhage in allogeneic bone marrow transplantation: a postmortem study. American Journal of Respiratory and Critical Care Medicine 151: 1006–1010. Bar J, Ehrenfeld M, Rozenman J, et al. (2001) Pulmonary–renal syndrome in systemic sclerosis. Seminars in Arthritis and Rheumatism 30: 403–410. Collard HR and Schwarz MI (2004) Diffuse alveolar hemorrhage. Clinics in Chest Medicine 25: 583–592. vii. Dhillon SS, Singh D, Doe N, et al. (1999) Diffuse alveolar hemorrhage and pulmonary capillaritis due to propylthiouracil. Chest 116: 1485–1488. Dweik RA, Arroliga AC, and Cash JM (1997) Alveolar hemorrhage in patients with rheumatic disease. Rheumatic Diseases Clinics of North America 23: 395–410. Dweik RA and Stoller JK (1999) Role of bronchoscopy in massive hemoptysis. Clinics in Chest Medicine 20: 89–105. Franks TJ and Koss MN (2000) Pulmonary capillaritis. Current Opinion Pulmonary Medicine 6: 430–435. Gertner E (1999) Diffuse alveolar hemorrhage in the antiphospholipid syndrome: spectrum of disease and treatment. Journal of Rheumatology 26: 805–807. Green RJ, Ruoss SJ, Kraft SA, et al. (1996) Pulmonary capillaritis and alveolar hemorrhage: update on diagnosis and management. Chest 110: 1305–1316. Henke D, Falk RJ, and Gabriel DA (2004) Successful treatment of diffuse alveolar hemorrhage with activated factor VII. Annals of Internal Medicine 140: 493–494. Ioachimescu O (2003) Idiopathic pulmonary hemosiderosis in adults. Pneumologia 52: 38–43. Ioachimescu OC, Kotch A, and Stoller JK (2005) Idiopathic pulmonary hemosiderosis in adults. Clinical Pulmonary Medicine 12: 16–25. Ioachimescu OC, Sieber S, and Kotch A (2004) Idiopathic pulmonary hemosiderosis revisited. European Respiratory Journal 24: 162–170. Jennings CA, King TE Jr, Tuder R, Cherniack RM, and Schwarz MI (1997) Diffuse alveolar hemorrhage with underlying

Alveolar Proteinosis

isolated, pauciimmune pulmonary capillaritis. American Journal of Respiratory and Critical Care Medicine 155: 1101– 1109. Klemmer PJ, Chalermskulrat W, Reif MS, et al. (2003) Plasmapheresis therapy for diffuse alveolar hemorrhage in patients with small-vessel vasculitis. American Journal of Kidney Diseases 42: 1149–1153. Lai RS, Lin SL, Lai NS, and Lee PC (1998) Churg–Strauss syndrome presenting with pulmonary capillaritis and diffuse alveolar hemorrhage. Scandinavian Journal of Rheumatology 27: 230–232. Lauque D, Cadranel J, Lazor R, et al. (2000) Microscopic polyangiitis with alveolar hemorrhage: a study of 29 cases and review of the literature-Groupe d’Etudes et de Recherche sur les Maladies ‘‘Orphelines’’ Pulmonaires (GERM ‘‘O’’ P). Medicine (Baltimore) 79: 222–233. Leatherman JW (1988) The lung in systemic vasculitis. Seminars in Respiratory Infections 3: 274–288. Murray RJ, Albin RJ, Mergner W, and Criner GJ (1988) Diffuse alveolar hemorrhage temporally related to cocaine smoking. Chest 93: 427–429. Nadrous HF, Yu AC, Specks U, and Ryu JH (2004) Pulmonary involvement in Henoch–Scho¨nlein purpura. Mayo Clinic Proceedings 79: 1151–1157. Pastores SM, Papadopoulos E, Voigt L, and Halpern NA (2003) Diffuse alveolar hemorrhage after allogeneic hematopoietic stem-cell transplantation: treatment with recombinant factor VIIa. Chest 124: 2400–2403. Schwarz MI and Brown KK (2000) Small vessel vasculitis of the lung. Thorax 55: 502–510. Schwarz MI and Fontenot AP (2004) Drug-induced diffuse alveolar hemorrhage syndromes and vasculitis. Clinics in Chest Medicine 25: 133–140. Schwarz MI, Mortenson RL, Colby TV, et al. (1993) Pulmonary capillaritis: the association with progressive irreversible airflow limitation and hyperinflation. American Review of Respiratory Diseases 148: 507–511. Schwarz MI, Zamora MR, Hodges TN, et al. (1998) Isolated pulmonary capillaritis and diffuse alveolar hemorrhage in rheumatoid arthritis and mixed connective tissue disease. Chest 113: 1609–1615. Segal SL, Lenchner GS, Cichelli AV, et al. (1988) Angiosarcoma presenting as diffuse alveolar hemorrhage. Chest 94: 214–216. Specks U (2001) Diffuse alveolar hemorrhage syndromes. Current Opinion in Rheumatology 13: 12–17. Travis WD, Colby TV, Lombard C, and Carpenter HA (1990) A clinicopathologic study of 34 cases of diffuse pulmonary hemorrhage with lung biopsy confirmation. American Journal of Surgical Pathology 14: 1112–1125. Zamora MR, Warner ML, Tuder R, and Schwarz MI (1997) Diffuse alveolar hemorrhage and systemic lupus erythematosus: clinical presentation, histology, survival, and outcome. Medicine (Baltimore) 76: 192–202.

see Interstitial Lung Disease: Alveolar Proteinosis.

ALVEOLAR SURFACE MECHANICS 101

ALVEOLAR SURFACE MECHANICS S B Hall and S Rugonyi, Oregon Health & Science University, Portland, OR, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The major component of the recoil forces that tend to deflate the lungs is the surface tension of a thin liquid layer that lines the alveoli. This surface tension is well below the value for a clean air/water interface, indicating the presence of a surfactant. The surface tensions in situ specify that the surfactant films must have certain characteristics. Following large expansions of the air/water interface during deep inhalations, the surfactant must adsorb rapidly to form the interfacial film. When compressed by the shrinking surface area during exhalation, the films must be sufficiently rigid to resist the tendency to collapse from the interface. Materials washed from the lungs show that pulmonary surfactant is a mixture containing mostly lipids with some proteins that is synthesized and secreted by the type II pneumocyte. The disorder in which an abnormality of pulmonary surfactant most clearly plays a role is the respiratory distress syndrome of premature babies, although altered surfactant function may also contribute to the acute respiratory distress syndrome that occurs in patients of all ages.

Description The importance of alveolar surface mechanics is readily evident from the pressure–volume (P–V) characteristics of the lungs. Pressures required to maintain any given volume are substantially higher for lungs inflated with air than with saline. The fundamental difference between the two procedures is that saline eliminates an air/water interface, the surface tension of which contributes to the inward recoil forces for lungs inflated with air. The presence of

surface tension implies that the curved surfaces of the pulmonary airspaces are lined by a layer of liquid. Electron microscopy has demonstrated that such a layer coats the alveoli, and that it is thin, with an average thickness of 0.2 mm, and continuous. The surface tension resulting from the air/water interface of this alveolar lining represents the major component of contractile forces in the lungs. Surface tension results from an imbalance of forces on molecules close to an interface between two separated phases. For molecules deep within a substance, attractive forces towards neighboring constituents are equal in all directions (Figure 1). Within a few molecular diameters of the interface, however, components experience a reduced attraction towards constituents in the adjoining phase, resulting in a net inward pull and a force per unit length, or surface tension, that tends to contract the interfacial area. The force along a curved surface, such as in the alveoli, translates directly into a difference in pressures across the interface. For a spherical interface with radius R, mechanical equilibrium between surface tension, s, and the difference in pressures, p
p0, occurs (Figure 2) when pR2  ðp
p0 Þ ¼ 2pRs which leads directly to the law of Young and Laplace for a sphere: Dp ¼ 2s=R Inflation of lungs with air therefore requires higher pressure to overcome interfacial forces that are absent during inflation with saline.

Air Water

Figure 1 Origin of surface tension: molecules in the bulk water experience an attractive force towards neighboring molecules that is equal in all directions, resulting in no net force. Within a few molecular diameters of the interface, however, the absence of neighbors towards the surface results in a net inward pull that tends to shrink the interfacial area. A film of surfactant at the interface tends to spread, resulting in an opposing force that lowers surface tension.

102 ALVEOLAR SURFACE MECHANICS p0 p

R




Figure 2 Relationship of surface tension to hydrostatic pressure across a spherical surface. Mechanical equilibrium occurs when the inward recoil force of surface tension (s) and the opposing force of hydrostatic pressure (p
p0) are equal. At the midsection of a spherical bubble with radius R, surface tension will produce a force (length  s) ¼ 2pRs, and the force from hydrostatic pressure will be [area  (p
p0)] ¼ pR2(p
p0). The equality leads directly to the law of Young and Laplace, Dp ¼ 2s/R.

Several methods have been used to determine surface tension in the lungs. The difference in P–V curves between air- and saline-filled lungs can provide surface tensions, either with simple assumptions concerning alveolar geometries and tissue forces, or with an energetic analysis that makes those assumptions unnecessary. Rinsing the lungs with a series of detergents or liquids provides an air/liquid interface with constant known surface tensions, and the intersection of P–V curves from these and normal lungs indicates points of common surface tension. Fluorocarbon droplets deposited on the interfacial film in peripheral alveoli spread according to the relative surface tensions of the fluorocarbon and film, and the shape of droplets containing different materials has provided the most direct estimates of surface tensions in situ. These different approaches have produced remarkably consistent results. During excursions through large volumes, surface tension shows a major hysteresis between inflation and deflation that explains the hysteresis of the P–V mechanics. Deflation from total lung capacity to functional residual capacity lowers surface tension from B30 mN m
1 to o5 mN m
1. Over tidal volumes, surface tension varies between 10 mN m
1 and values as low as 1 mN m
1. These surface tensions are well below the values for a clean air/water interface, which would be 70 mN m
1, and indicate the presence of a surfactant. Surfactants are the general class of compounds that have higher concentrations at the surface than in the aqueous medium. They are amphipathic, with distinct hydrophilic and hydrophobic regions of the molecule, and best satisfy the energetics of both portions by orientation across an interface. Once trapped within the two-dimensional surface, surfactants tend to spread, resulting in a force that expands the interface and opposes surface tension. Surfactant films with higher densities produce larger reductions in surface

tension. The very low surface tensions in the lungs indicate films with particularly high densities. Surfactants can lower surface tension by adsorbing to an interface only to a limited extent. Adsorbed surfactants reach a maximum density, above which further constituents form a three-dimensional bulk phase at the interface rather than adding to the twodimensional film. Surface tensions in the lungs reach values well below this minimum equilibrium value, and they also vary with volume. These observations indicate that the low surface tensions and high densities of the films result not from insertion of more constituents, but from a decrease in surface area during deflation. The low surface tensions reached during deflation are impressively stable in static lungs for prolonged periods. The low magnitude and particularly the stability of the surface tensions indicate that the compressed films have specific characteristics. When compressed above the maximum equilibrium density, films that can flow from the surface will collapse to form the three-dimensional bulk phase, thereby reestablishing equilibrium surface tensions. Only films that are solid, defined by their inability to flow, can resist the tendency to collapse and demonstrate the behavior observed in the lungs. When compressed sufficiently, even solid films collapse. An interface without surface tension has no basis for existence, and at sufficiently high densities, solid films must also rupture. The hysteresis of surface tension, and of hydrostatic pressures, between deflation and inflation over large volumes reflects at least partially material lost from the interface during collapse. Lavaging the lungs produces the increase in recoil pressures expected from removal of a surfactant and a resulting increase in surface tension. Material recovered from the lungs can form films with characteristics indicated by the in situ measurements. Material obtained from alveolar foam, when suspended in saline, can form small bubbles that persist for prolonged periods, indicating that the pressuredifference across their surface, which would tend to dissolve the gas in the surrounding liquid, must be low, and that according to the law of Young and Laplace, surface tension must also be small. When compressed in vitro by changing their surface area, films formed from lavaged material can reach and sustain surface tension below the minimum equilibrium value. Material purified from lavaged material can also restore the P–V mechanics of the original lungs. These observations provide the basis for the identification of the surfactant in the lungs. Material recovered by lavage contains two sets of phospholipid vesicles that differ in size. The larger

ALVEOLAR SURFACE MECHANICS 103

Air

Liquid layer

Type I cell

Molecular view

Lamellar body Tubular myelin

Small vesicles

Surfactant film

Type II cell

Figure 3 Schematic of pulmonary surfactant in the alveolus. Constituents of pulmonary surfactant are synthesized in type II pneumocytes and assembled into lamellar bodies, which are secreted into a thin liquid layer that lines the alveolus. The vesicles unravel and first form tubular myelin, which appears to represent the immediate precursor of a film at the air/water interface. Lavage of the lungs recovers both the large multilamellar vesicles, which contain mostly phospholipids with small amounts of cholesterol and proteins, and small unilamellar vesicles, which contain only the lipids.

form has the same morphological appearance as a subcellular organelle, the lamellar body, of the type II pneumocytes (Figure 3). Organic extracts of these larger particles can restore the P–V mechanics of lavaged lungs, which perhaps best defines these vesicles as ‘pulmonary surfactant’. The smaller particles may represent material excluded from the interface during compression to very low surface tensions. Vesicles of both sizes contain the same set of phospholipids with small amounts of cholesterol. The larger particles are much more capable of lowering surface tension in vitro than the smaller vesicles. Four proteins copurify with the larger particles but are absent from the smaller forms. The organic extracts of the large particles, which function well in lavaged lungs, lack surfactant proteins SP-A and SP-D, and based on a variety of assays, their primary function appears related to processes other than the lowering of surface tension. SP-B and SP-C, which are sufficiently hydrophobic to extract with the lipids into organic solvents, determine the difference in surface activity between the large and small particles.

Normal Physiological Processes To achieve the surface tensions observed in the lungs, pulmonary surfactant must satisfy conflicting constraints. Vesicles must first adsorb rapidly to form the interfacial film. Pulmonary mechanics become normal during the first few breaths following the initial air-inflation of fluid-filled lungs, suggesting that adsorption to the newly created air/water interface forms a film having the equilibrium density within seconds. The low surface tensions reached during deflation indicate that the compressed film avoids the reverse process of desorption to re-establish the equilibrium density. Replicating in vitro the full

performance of films in the lungs has been difficult, and the mechanisms by which pulmonary surfactant functions in the alveoli remain the subject of active investigation. Adsorption

The adsorption of pulmonary surfactant is fundamentally different from the process for other more common surfactants, which insert into the interface as individual molecules. Surfactants share the general characteristic that they exist in solution as individual monomers only up to a certain concentration, above which they aggregate into structures such as micelles and bilayers (Figure 4), the nature of which depends on the effective shape of the particular molecule. The ‘critical micelle concentration’ at which phospholipids aggregate is less than 10
9 M. Constituents of pulmonary surfactant therefore exist in the alveolar lining exclusively as vesicles, which insert as collective units into the interface. Because the vesicles themselves are stable in aqueous medium, an energy barrier limits adsorption, which occurs quite slowly for lipid vesicles without the hydrophobic proteins. In the alveolar lining, lamellar bodies secreted by the type II pneumocytes unravel to form a distinct intermediate structure known as tubular myelin that apparently represents the direct precursor of the interfacial film (Figure 3). SP-A is required in vitro for the reconstruction of tubular myelin, which is absent from extracted surfactants and transgenic animals that lack SP-A. Extracted surfactants, however, function well in surfactant-deficient lungs, and mice without SP-A have normal pulmonary mechanics. The functional significance of tubular myelin for adsorption is therefore unclear. The absence of SP-B, whether in transgenic animals or in patients with

104 ALVEOLAR SURFACE MECHANICS

Figure 4 Aggregation of surfactants. Above a certain concentration, the hydrophobic portion of surfactants makes them insoluble as individual molecules, and causes their aggregation into structures that expose their hydrophilic components to the aqueous medium while sequestering the hydrophobic segments. The particular form of the aggregate depends on the effective shape of the specific compounds. Single chain surfactants tend to form micelles. Biological phospholipids form bilayers that may stack into the concentric layers of a multilamellar vesicle.

genetic abnormalities, does produce abnormal pulmonary function, consistent with the crucial role suggested by in vitro studies of this protein for adsorption. Stability of Compressed Film

Under equilibrium conditions, attempts to increase the density of surfactant monolayers above a maximum density, either by adding more constituents or by decreasing interfacial area, instead forms a threedimensional bulk phase that coexists with the twodimensional film. The bulk phase of phospholipids is a liquid-crystal, in which layers of material stack in the regularly repeating manner characteristic of a crystal, but with each layer having the disordered structure that is characteristic of liquids. Slowly compressed monolayers of pulmonary surfactant in vitro flow into these stacked structures (Figure 5), and their ability to flow indicates that the films are fluid. In the lungs, the prolonged low surface tensions, well below the minimum equilibrium values, indicate films that resist flow, and that therefore have the defining characteristic of a solid. The structure of two-dimensional solid films could be either highly ordered, analogous to a three-dimensional crystal, or amorphous, like a glass. At physiological temperatures, a single constituent of pulmonary surfactant, dipalmitoyl phosphatidylcholine (DPPC), can form highly ordered films that approach the structure of a two-dimensional crystal. DPPC has the unusual characteristic relative to other biological phospholipids that both acyl chains are fully saturated and that it constitutes an unusually large amount (30–50%) of pulmonary

surfactant. A widely held view contends that the functional film in the lung consists of essentially pure DPPC. The difference in composition between the secreted vesicles and the hypothetical functional film of DPPC could result either from selective adsorption of DPPC or from selective collapse of other constituents. Both processes are difficult to reconcile with current understandings of how adsorption and collapse occur. One prediction of the model is that P–V curves should change abruptly over a narrow range of temperatures. Films of DPPC, like three-dimensional crystals, melt from solid to fluid structures at specific temperatures. At surface tensions below the minimum equilibrium value, rates of collapse increase when solid films melt, resulting in increased surface tensions that would produce higher recoil pressures. Measurements of the temperature dependence for P–V curves have yielded conflicting results, and the presence of a highly ordered film remains unconfirmed. Although films containing only DPPC would explain the stability of low surface tensions in the lungs, experimental evidence to support that possibility is limited. The solid films that sustain low surface tensions in situ could also have a structure that resembles a two-dimensional glass. Three-dimensional liquids, if cooled fast enough and far enough below their freezing temperatures, retain their disordered structure but become frozen in place, forming amorphous solids, or glasses. Two-dimensional fluid films, defined by their ability to flow into collapsed structures, similarly become jammed into a form that resists collapse if supercompressed to sufficiently high

ALVEOLAR SURFACE MECHANICS 105

Figure 5 Liquid-crystalline collapse. Under equilibrium conditions, surfactant films reach a minimum surface tension at which they undergo a phase transition to form a three-dimensional bulk phase. Phospholipids form bulk smectic liquid crystals, and compression of fluid phospholipid films at the minimum equilibrium surface tension can cause the film to flow into stacked structures. Solid films, which can resist flow and remain at the interface below the minimum equilibrium surface tension, eventually also collapse from the interface at very low surface tensions, although probably by a process more like fracture.

densities and low surface tensions. These supercompressed films retain their solid behavior and slow rates of collapse when expanded, even when returned to the surface tensions at which they originally collapsed. If they reach low surface tensions in the lungs during a single exhalation, the films would be transformed, and their ability to avoid collapse could persist through multiple cycles of tidal breathing. To achieve low surface tensions, however, the initially fluid films must be compressed faster than they can collapse. The required rates may occur during normal breathing, but they are faster than rates in quasi-static experiments with excised lungs. The supercompressed films, which would require no compositional change, could explain surface tensions observed in the lungs, but like the films of pure DPPC, the process by which they would form remains unclear. Original views concerning surface tension in the lungs considered an interfacial film with the thickness of one molecule. Although perhaps difficult to explain how they might form, monolayers that have the characteristics of films in the lungs are well described, and more complicated structures were unnecessary to explain the observed behavior. Electron microscopy, however, has demonstrated that in situ, at least parts of the interface are occupied by films that are multilayered. Whether these structures are formed during adsorption or collapse, and the extent to which the additional material might affect the mechanical characteristics of the film, are both unknown.

Physiological Processes in Respiratory Diseases The disorder in which an abnormality of pulmonary surfactant most clearly represents a major pathogenic factor is the respiratory distress syndrome (RDS) that occurs in premature babies. Ventilation of immature lungs that lack adequate amounts of surfactant injures the alveolocapillary barrier, resulting in pulmonary edema and respiratory failure. Two

mechanisms, both involving shear stresses, could explain how a deficiency of pulmonary surfactant would produce the injury. First, elevated surface tension would produce an increased tendency for small alveoli to collapse, and the shear stresses involved in reopening the closed airspaces could produce the injury. Second, the meniscus of any fluid column in the small airways would have an increased surface tension, and the greater pressure-difference across the interface could rupture the epithelial cells over which it passes. Elevated surface tensions would also lower interstitial and pericapillary pressures, resulting in a greater transmural pressure-difference that would increase the flow of fluid across the alveolocapillary membrane. The most direct evidence that deficient surfactant causes RDS comes from manipulation of surfactant levels. Subsequent to removing surfactant by lavage, ventilation of animals produces an injury that replicates RDS. Conversely, giving exogenous surfactant to premature babies at risk for RDS prevents or reverses the disorder. The acute respiratory distress syndrome (ARDS) was originally called adult RDS to point out the clinical similarities between adults with injured lungs and the infants with RDS, and to suggest that the common presentation resulting from a variety of insults might reflect an abnormality of surfactant acting as the final common pathway. Although the primary defect in ARDS is an inflammatory process, abnormal surfactant might perpetuate the initial injury by the same processes that result from an elevated surface tension in RDS. Surfactant function could be altered in ARDS by either deficiency or inhibition. Injured lungs have reduced levels of the large active surfactant vesicles, suggesting a deficiency. Pulmonary surfactant could also be inhibited by the large number of extraneous compounds, such as plasma proteins and membrane lipids, that reach the alveolus in injured lungs and that can act as surfactants. Direct evidence for elevated surface tensions in ARDS, however, is limited. The presence of edema complicates the interpretation

106 ALVEOLAR WALL MICROMECHANICS

of P–V mechanics, and the distinction of small lungs, caused by fluid-filled airspaces that occur with any pulmonary edema, from stiff lungs, caused by increased surface tension, has been difficult. Evidence that exogenous surfactants can mitigate ARDS has also been lacking. The larger doses required to treat adults with ARDS relative to premature babies with RDS has limited the therapeutic agents that can be used. Initial trials with surfactants that lack SP-B have produced no improvement, just as early attempts to treat babies with RDS using aerosolized DPPC had no benefit. The role of therapeutic surfactants in ARDS, and of abnormal surfactant in its pathogenesis, therefore remains unresolved. See also: Acute Respiratory Distress Syndrome. Alveolar Hemorrhage. Alveolar Wall Micromechanics. Breathing: Breathing in the Newborn; Fetal Lung Liquid; First Breath. Bronchoalveolar Lavage. Drug-Induced Pulmonary Disease. Epithelial Cells: Type I Cells; Type II Cells. Fluid Balance in the Lung. Infant Respiratory Distress Syndrome. Lung Anatomy (Including the Aging Lung). Lung Imaging. Surfactant: Overview; Surfactant Protein A (SP-A); Surfactant Proteins B and C (SP-B and SP-C); Surfactant Protein D (SP-D).

Further Reading Bastacky J, Lee CY, Goerke J, et al. (1995) Alveolar lining layer is thin and continuous: low-temperature scanning electron

microscopy of rat lung. Journal of Applied Physiology 79: 1615–1628. Goerke J and Clements JA (1985) Alveolar surface tension and lung surfactant. In: Macklem PT and Mead J (eds.) Handbook of Physiology The Respiratory System, vol. III, part 1, pp. 247– 261. Washington, DC: American Physiological Society. Hoppin J, Frederic G, Joseph C, et al. (1986) Lung recoil: elastic and rheological properties. In: Fishman AE (ed.) Handbook of Physiology: A Critical, Comprehensive Presentation of Physiological Knowledge and Concepts, pp. 195–215. Bethesda, MD: American Physiological Society. Keough KMW (1992) Physical chemistry of pulmonary surfactant in the terminal air spaces. In: Robertson B, van Golde LMG, and Batenburg JJ (eds.) Pulmonary Surfactant: from Molecular Biology to Clinical Practice, pp. 109–164. Amsterdam, New York: Elsevier. Lewis JF and Jobe AH (1993) Surfactant and the adult respiratory distress syndrome. American Review of Respiratory Diseases 147: 218–233. Piknova B, Schram V, and Hall SB (2002) Pulmonary surfactant: phase behavior and function. Current Opinion in Structural Biology 12: 487–494. Robertson B (1984) Pathology and pathophysiology of neonatal surfactant deficiency (‘‘respiratory distress syndrome,’’ ‘‘hyaline membrane disease’’). In: Robertson B, Van Golde LMG, and Batenburg JJ (eds.) Pulmonary Surfactant, pp. 383–418. Amsterdam: Elsevier. Schu¨rch S, Green FH, and Bachofen H (1998) Formation and structure of surface films: captive bubble surfactometry. Biochimica et Biophysica Acta 1408: 180–202. Stamenovic D (1990) Micromechanical foundations of pulmonary elasticity. Physiological Reviews 70: 1117–1134. Wilson TA (1981) Mechanics of the pressure-volume curve of the lung. Annals of Biomedical Engineering 9: 439–449.

ALVEOLAR WALL MICROMECHANICS F G Hoppin Jr, Brown University, Providence, RI, USA

Introduction

& 2006 Elsevier Ltd. All rights reserved.

The alveolar wall is the site of gas exchange by passive diffusion between inspired gas and the pulmonary capillary blood. Its tiny scale supports passive diffusion by providing an immense net-diffusing area and a short path length. Because it is elastically extensible, the gas-exchanging lung units can readily expand and contract during breathing. How does this diaphanous structure meet these functional needs and at the same time avoid structural weakness, configurational instability, and fluid accumulation?

Abstract The site of gas exchange in the lung is the alveolar wall. Its structure supports passive diffusion of the respiratory gases by spreading the pulmonary capillary bed over an immense surface area and by having an extremely thin air/blood barrier. It enables the alveolar gas spaces to inflate and deflate by its great extensibility. Yet it remains stiff enough to withstand the severe collapsing forces of surface tension acting in tiny confines and to transmit elastic tensions throughout the network of alveolar walls, maintaining by those tensions the fine and gross configuration of the lung and a reasonable distribution of ventilation within the lung. It can modulate local ventilation by activation of its smooth muscle. The mechanical properties that meet these exacting requirements can be understood in terms of the behavior of elastic networks and the elastic properties of the wall’s solid components and of pulmonary surfactant. Dysfunction at the level of the alveolar wall lies at the core of most disorders of the lung by impairing diffusion, ventilation, and perfusion.

Normal Mechanics The lung, like a parachute or a spinnaker, is a tensed structure (Figure 1). Patency of the airspaces and a relatively even distribution of expansion and contraction during breathing depend on satisfactory

ALVEOLAR WALL MICROMECHANICS 107

50 µm E J B

Figure 1 Photomicrograph of inflated lung, showing the network of alveolar walls, partitioning the lung into alveolar gas spaces. The predominant termination of the profile of the alveolar wall is a triple junction with two other walls (J). Where alveolar gas spaces open into an alveolar duct (right top), the profiles either appear bent (B) or simply end (E), and these edges of the wall are invariably reinforced with connective tissue cables, often accompanied by smooth muscle. Reproduced from Butler JP, Oldmixon EH, and Hoppin FG Jr (1996) Dihedral angles of septal ‘bend’ structures in lung parenchyma. Journal of Applied Physiology 81: 1800–1806, used with permission of The American Physiological Society.

transmission of tensions (lung elastic recoil) throughout. The evidence for the network of alveolar walls being the major tension-bearing structure in the lung include the following: *

*

*

*

It contains the majority of the lung’s tension-bearing material. Its tension-bearing structures are continuous throughout the lung. Distortions of the inflated lung whether by gravity, local indentation of the surface, or local internal expansions or contractions behave as expected for a diffuse, isotropic elastic network. Estimates of lung elastic recoil, based on stereological data and elastic properties of the tensionbearing components of the alveolar wall are close to observed lung-distending pressure differences over a range of inflation volumes.

The other tension-bearing structures, acting mechanically in parallel, include the lung’s outer rind (pleura), which has been thought to account for only B20% of the work of inflation, and the fibrous connective tissue systems of the airway and bronchovascular trees, which have been estimated to bear B10%. The network of alveolar walls is a cable/membrane structure. The membrane portion of the alveolar wall is tensed at its edges either by other walls or by an embedded cable of relatively heavy connective tissue. It is thin, polygonal, and pseudoplanar. It has three

main tension-bearing components: the fine, isotropic fibrous network; the basement membranes of the epithelium and capillary endothelium; and the air-liquid interfaces on either side (Figure 2). Along most of its perimeter (B66%), the wall joins two others (J in Figure 1). At this junction, there is no cable; the fine fibrous network, basement membranes, and air-liquid interfaces are directly continuous from one wall to the adjacent walls. The tensions of the walls at such junctions are resolved by the balance of the three walls pulling in three directions. Another mechanism is needed where the alveolar spaces open into the alveolar duct. At these locations, the wall either meets one other wall (B24% of its perimeter, B in Figure 1) or simply ends (B9% of the perimeter, E in Figure 1) and it requires some ancillary support. This is provided by relatively heavy connective tissue cables that are curved so as to oppose the wall’s tensions. The fine fibrous network of the walls connects directly with these cables. The cables of adjoining walls form a lattice that frames the alveolar duct. Centrally, their connective tissues merge with the still heavier connective tissues of the terminal bronchioles. The difference between the E and B configurations, incidentally, probably does not reflect a functional difference beyond the geometric complexity of space packing of alveoli and of the branching airway system. The cables and membranes, being continuous structures throughout the network, are capable of transmitting tensions from membrane to membrane or cable to cable across the lung in any direction. However, there is also a distinct serial connection between the cables and membranes at the level of the alveolar duct and its surrounding alveoli, where the membranes pull radially against the cables. This is dramatically apparent as dilation of the ducts of lungs that have been washed with detergent to increase the wall’s interfacial tension. Normally, however, the elastic properties of these very different systems are sufficiently matched so that expansion and contraction of the duct and its surrounding alveoli are relatively symmetrical during breathing. Lying alongside most of the cables are bundles of smooth muscle. Its function relates to its characteristic stiffening when it is activated and passively length-cycled at typical breathing frequencies. As a result of the series relationship of the alveolar duct and its surrounding alveoli (above), stiffening of the cables reduces the compliance of the respiratory unit. Indeed, the lung is substantially stiffened in response to low levels of carbon dioxide (hypocapnia). This suggests a homeostatic mechanism. Relatively under’ lung units are characteristically ’ Q) perfused (high V= hypocapnic. A local response that stiffens the lung

108 ALVEOLAR WALL MICROMECHANICS

Alveolar lining liquid Epithelial cell Basement membrane Interstitium Basement membrane Endothelial cell

Epithelial cell nucleus Fine fiber network Endothelial cell nucleus Red blood cell Plasma

Endothelial cell Fused basement membrane Epithelial cell Alveolar lining liquid Figure 2 Schematic depiction of the structure of an alveolar wall. It is pseudoplanar and has dimensions on the order of 100  100  10 mm. In the middle is the interstitium, containing the supporting fine fiber network, the marsh-like pulmonary capillary bed, and small amounts of interstitial fluid. Outside the interstitium on both sides of the wall is a basement membrane supporting the single layer of thin alveolar epithelial cells and, outside that, a fluid alveolar lining. The capillary itself is a single thin endothelial cell and its supporting basement membrane, fused on one side of the capillary with the basement membrane of the epithelium.

locally reduces the ventilation of such units. This hypocapnic pneumoconstriction improves the overall efficiency of gas exchange, much as hypoxic pulmonary vasoconstriction reduces the perfusion of un’ units. ’ Q) derventilated (low V= Tensions in adjoining walls are quite uniform. This conclusion is drawn from a vector analysis of the tensions at J and B junctions, the assumption that the walls are free to rotate relative to each other, and data for the configuration of fixed inflated specimens. At J junctions, the distribution of dihedral angles between walls, extremely narrow around 1201, is consistent with B2% variation of wall tensions. At B junctions, the inferred distribution is somewhat wider, but has a notably sharp cutoff below 1201. Open soap films supported on complex wire frames show these several features at their J and B junctions, where they are well understood to reflect (1) locally uniform film tensions and (2) a mechanism for adopting the least energy configuration, that is, minimal surface area. This finding suggests, intriguingly, that lung structure during growth and development (perhaps even during remodeling of the adult lung?) responded to these same physical principles. Local uniformity of wall

tensions, of course, does not imply global uniformity, which varies systematically over large distances in the lung, for example, vertically in the gravity field. The wall is highly extensible, undergoing up to about twofold change in linear dimensions for the deepest breaths, that is, about eightfold change of volume. (Birds followed a different evolutionary route by having unidirectional air flow through a relatively stiff gas exchange apparatus.) The risks inherent in such extensibility include instability, collapse, and gross unevenness of ventilation among the lung’s different air compartments. These risks are limited by the wall’s positive compliance and by the effects of network geometry. A given distortion of the elastic network stretches the walls on the side away from the displacement and realigns them closer to the direction of the displacement. As the walls are positively compliant, the effect is to increase tension on that side, and the effect of that increase is enhanced by the realignment of the walls to better oppose the displacement. The converse occurs on the side towards the displacement. This mechanism acts to stabilize configuration at the fine (alveolus) and gross (regional) levels.

ALVEOLAR WALL MICROMECHANICS 109

The wall’s positive compliance is conferred independently by both the air–liquid interface and the solid components (fine fibrous network and basement membranes). Interfacial tension modulated by pulmonary surfactant varies monotonically from near 0 to B30 dynes cm 1 over a fourfold change of surface area. The fine fibrous connective tissues are collagen fibers (stiff as steel in isolated fibers) and elastin (the prototypical rubber). They are substantially entangled and cross-linked, and the compressibility of a proteoglycan matrix modifies the extent to which collagen fibers fold and stretch. The resulting solid components are capable of more than the about twofold linear extension, that is, of maintaining tension over the full operating range of lung volume. The tensions in the interface interact with those of the solid elements of the wall through curvature. There is no reason to postulate shear forces between them. The interface tenses the cables at E and B junctions by curvature that is convex to the airspace, that is by draping over the cable region like washing on a clothes line. At the inner corners of the alveolus (J junctions and the inner side of the B junctions), by contrast, the curvature of the interface is sharply concave to the airspace, and therefore interfacial tension reduces the pressure in (i.e., sucks out on) the junctional tissues, transmitting tension to the cable and/or other septae at that junction. In lungs prepared in protocols that permit collapse, the freedom to shear permits the solid elements of the wall to pleat or fold beneath a smoothly curved interface in the corners of alveoli. This is probably not the configuration in vivo. Transiently low distending pressures (o2–3 cmH2O) permit folding. High distending pressures (416– 20 cmH2O) are required to resolve the folds. With the appropriate protocols, the wall’s fine fiber network remains quite planar right into the corners of the alveoli even down to 2–3 cmH2O. This is consistent with the capability of the fine fiber network to bear tension over the full feasible range of lung inflation, and with its roles of supporting the capillary network and of maintaining the configuration of the alveolus, particularly at low inflation volumes. The pulmonary capillaries are notably affected by the mechanical state of the wall. The majority of the capillary bed lies within the flat portions of the wall. There, the capillary distending pressure (the difference between intracapillary and alveolar gas space pressures) is in equilibrium with the combined opposing forces of intervening structures, namely the basement membranes of the capillary and alveolar epithelium, the fine fiber network, and the air–liquid interface. The effects are evident in the prominent bulging of the pulmonary capillaries into the alveolar space when hydrostatic capillary pressure increases

or interfacial tension is reduced (e.g., in the salinefilled lung). Conversely, the planar forces of the wall’s solid structures and interfacial tension flatten the capillaries when the wall is stretched at high lung volumes. By contrast, the vessels in the corners, for example, within the J junctions, where the curvatures of the solid structures and interface are concave to the alveolar space, are distended at high lung volumes, and may remain open even when the vessels within the flat portions are compressed. Under those conditions, blood–flow in such regions is not completely eliminated and there is gas exchange evidence compatible with very low perfusion units. Fluid balance is also impacted by mechanical factors, again with different mechanisms in the flat portions of the wall and the corners. Throughout the body, the volume of fluid in the interstitial spaces is controlled by the balance between hydrostatic and osmotic factors across the capillary walls (Starling equilibrium). The lung, however, is a special case because of the powerful forces that develop when interfacial tension acts on very tight curvatures. In particular, the tissue pressure in the alveolar corners (J junctions) is lowered. This may be beneficial by helping to draw interstitial fluid from the flat portions of the alveolar wall into the corners, whence it may be cleared by the lymphatics. But it is also counterproductive insofar as it opposes the fluid-absorptive forces across the microvascular walls and inhibits lymphatic drainage. Of greatest concern, it can lead to fluid accumulation within the alveolus. Ordinarily, a simple mechanical negative-feedback loop limits the accumulation of fluid any increase of alveolar fluid initially decreases the curvature of the interface, reducing the pressure-lowering effect of interfacial tension and thereby favoring the return of fluid to the capillaries and lymph. If, however, the fluid gathers to the point that the corners of the alveolar space are filled, the feedback loop reverses; any further gathering of fluid increases the curvature, leading to further alveolar filling or collapse. This problem is substantially mitigated by the interfacial tension-lowering effect of pulmonary surfactant, which has enabled evolution of alveolar walls at a much smaller scale than would otherwise have been possible, providing an immense surface area for gas exchange within a reasonably sized chest.

The Mechanical Role of the Alveolar Wall in Respiratory Diseases Mechanical dysfunction of the alveolar wall is at the core of most major acute and chronic lung diseases, as readily predictable from the above discussion and seen in the following examples.

110 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS

The essence of emphysema is loss of alveolar walls. This impairs gas exchange by reducing surface area of the pulmonary capillary bed and increasing the path lengths for passive diffusion. It also relaxes the network of alveolar walls. This has the effect of reducing the dilating effects of the network on the embedded and extrapulmonary airways and blood vessels. Narrowing of the airways increases their airflow resistance and forces the subject to breathe at disadvantageously high chest wall volumes in order to mount sufficient elastic tensions to hold the airways open. Inhomogeneity of damage to the network of alveolar walls and its effects on airway and blood vessel calibers cause substantial maldistribution of ventilation and perfusion, itself a major cause of inefficiency of gas exchange. A range of mechanical dysfunctions at the level of the alveolar wall permits blood to shunt through the lung without exchanging gas. Fluid can gather (pulmonary edema) secondary to the high hydrostatic pressures of heart failure, or even in normal individuals at very high altitudes. Toxic, infectious, or immunological entities can allow excessive fluid or protein leakage from damaged capillaries. Airspaces can collapse (atelectasis) from local underinflation and gas absorption, from dysfunctional surfactant, from trauma, or from chest wall dysfunction. High distending forces (e.g., deep breaths) are required for reopening. Repeated opening and closing of airspaces in a patient breathing with ventilator support can have its own direct physical and inflammatory consequences. Pulmonary fibrosis due to a wide range of causes can thicken and stiffen the alveolar wall, impairing

Amyloidosis

gas exchange and placing a mechanical load on breathing. See also: Alveolar Surface Mechanics. Atelectasis. Chronic Obstructive Pulmonary Disease: Overview; Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General. Diffusion of Gases. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matrix Proteoglycans. Fluid Balance in the Lung. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Lung Development: Overview. Myofibroblasts. Occupational Diseases: Overview. Pulmonary Fibrosis. Smooth Muscle Cells: Airway. Stress Distribution in the Lung. Surfactant: Overview. Ventilation: Uneven.

Further Reading Butler JP, Oldmixon EH, and Hoppin FG Jr (1996) Dihedral angles of septal ‘bend’ structures in lung parenchyma. Journal of Applied Physiology 81: 1800–1806. Greaves IA, Hildebrandt J, Hoppin FG Jr (1986) Micromechanics of the lung. In: Fishman AP, Macklem PT, Mead J, and Geiger S (eds.) Handbook of Physiology. The Respiratory System, vol. III(1), pp. 217–231. Bethesda: American Physiological Society. Hoppin FG Jr, Stothert JC Jr, Greaves IA, Lai Y-L, Hildebrandt J (1986) Lung recoil: elastic and rheological properties. In: Fishman AP, Macklem PT, Mead J, and Geiger S (eds.) Handbook of Physiology. The Respiratory System, vol. III(1), pp. 195–215. Bethesda: American Physiological Society. Mead J (1961) Mechanical properties of lungs. Physiological Reviews 41: 281–330. Stamenovic´ D (1990) Micromechanical foundations of pulmonary elasticity. Physiological Reviews 70: 1117–1134. West JB (2003) Thoughts on the pulmonary blood–gas barrier. American Journal of Physiology. Lung Cellular and Molecular Physiology 285: L501–L513.

see Interstitial Lung Disease: Amyloidosis.

ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS P A D’Amore, Harvard Medical School, Boston, MA, USA M K Sakurai, Boston Children’s Hospital, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Angiogenesis is crucial in pulmonary development to establish the groundwork structure for the lungs’ main function of

gas exchange across a thin blood gas barrier. This network of blood and air conduits begins to form in early gestation and continues through early childhood, requiring intricate epithelial–mesenchymal interactions that are regulated by various angiogenic and growth factors. Disruptions in this complex system lead to inadequate gas exchange and diseases of respiratory distress. At this time, the precise roles of these growth factors and interactions between the epithelial and mesenchymal cells are poorly understood. A better understanding of these processes may lead to improved therapies for various respiratory diseases.

ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS 111

Introduction The basic function of the lung is gas exchange, and efficient gas exchange requires a thin air–blood interface. Hence, epithelial–mesenchymal interactions are crucial to the formation of a blood gas. Lung development, which begins in early gestation and continues into early childhood, requires complex interactions that are regulated by a number of angiogenic and growth factors. Although it is not whether mesenchymal development drives epithelial growth or vice versa, several factors have been identified as important for the development of a functional blood gas barrier. Any disruption to this intricate and highly orchestrated process can cause ineffective gas exchange with resulting respiratory distress. Further knowledge of the factors influencing pulmonary development may lead to therapeutic interventions for various respiratory diseases.

General Embryology Lung development is divided into five stages, starting in early gestation and ending postnatally in early childhood. Basic organogenesis occurs during the initial embryonic phase. The next three phases – pseudoglandular, canalicular, and saccular – describe the morphogenesis of primitive lung parenchyma during those stages. The final alveolar stage extends into childhood. Concurrent with mesenchymal development, the lung vasculature develops from mesodermal cells by a combination of vasculogenesis and angiogenesis. Vasculogenesis is the process of de novo blood vessel development from mesenchymal precursor cells, whereas angiogenesis involves new vessels sprouting from existing vasculature. Initially, the blood lakes, formed by vasculogenesis, create a capillary plexus and connect to drain into the pulmonary veins. Eventually, the pulmonary arteries bud off the aortic arches by angiogenesis and grow into the mesenchyme where they anastamose with this capillary plexus. This vascular plexus surrounds the developing airways. The time frame of these phases overlaps, given the independent development of each lobe. During the embryonic stage, the initial lung buds originate from ventral diverticuli of the primitive foregut and proliferate into the surrounding mesenchyme. These buds undergo branching morphogenesis to create a complex network of airways that are lined with columnar epithelium. Simultaneously, vascular lakes of hematopoietic cells that have formed by vasculogenesis appear in the mesenchyme. Neither the pulmonary artery nor veins have formed at this time. At first the capillaries only drain into

systemic veins but eventually connect to the pulmonary veins that arise from the primitive cardiac atrium during the pseudoglandular phase. Bronchial tree formation and acinar structure development at approximately 5–17 weeks into gestation mark the pseudoglandular stage. Epithelial– mesenchymal interactions determine and regulate the branching patterns. Transplantation experiments have demonstrated that the mesenchyme directs the growth and branching of epithelial tubules forming the conductive airways. In vitro experiments have shown that removing the mesenchyme from the tip of a budding epithelial tube prevents further branching and that transplanted mesenchyme can induce new budding. During the pseudoglandular stage, differentiation of the cells comprising the airway walls occurs proximal to distal. These cells become ciliated, nonciliated, goblet, and basal epithelial cells. At the distal ends, cuboidal epithelial cells line the forming acini, the mesenchyme begins to thin, and a network of capillaries develops. During this stage, the venous system connects to the heart. Later in the pseudoglandular phase, the pulmonary artery buds from the aorta and primitive arterial branches begin to develop alongside the airways. At this point, the basic airway pattern has been established. The canalicular stage at gestational weeks 16–26 encompasses acini and pulmonary vasculature development, as well as the initiation of surfactant synthesis. At the crucial gas-exchange region, acini development involves widening of the peripheral tubules, further thinning of the mesenchyme and increased vascularization. The cuboidal epithelium thins to form the blood gas barrier and differentiates into type I and type II pneumocytes. The type II pneumocytes initiate surfactant production. During the saccular stage, which occurs during 24–38 weeks of gestation, continued expansion of the air spaces occurs and is accompanied by increased vasculature and decreased interstitial tissue as the lung prepares for its main function of gas exchange. At this phase, the airways end in thin-walled terminal sacs, hence the name saccular stage. The vessels continue to grow and elongate. With the lessening of interstitial tissue, the airspaces begin to approach one another and a capillary bilayer is formed in the intersaccular septa. The surfactant system continues to mature in preparation for the function of gas exchange. The alveolar stage starts at the end of gestation and continues for 1–2 years postnatally. Less than 10% of alveoli are present at birth and a majority of alveolar formation occurs via the process of septation after birth. The walls of the airspaces are called primary septa and contain a distinct double capillary

112 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS

network. Secondary septa form to partition the saccules and form alveoli. This process increases the alveolar surface area and thus the gas-exchange area. These thick septa transform into a thin epithelial– endothelial cell lining via a process that is not well understood. The exact mechanisms that underlie the five stages of lung development have yet to be elucidated; however, various angiogenic growth factors and developmental factors have been demonstrated to play important roles in this process.

Angiogenic Growth Factors, Developmental Factors, and Their Roles Epithelial–mesenchymal interactions are crucial in the formation of a thin blood gas interface during lung development. Multiple angiogenic and growth factors interact throughout the five stages of pulmonary development. A variety of in vivo and in vitro techniques have been used to elucidate the role of particular growth factors in this process. These studies point to the importance of maintaining a delicate balance of growth factors during the process of pulmonary development. Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is a wellestablished angiogenic factor. Differential splicing of a single gene generates three distinct VEGF isoforms in the mouse and at least five in humans. VEGF acts to stimulate the proliferation and migration of vascular endothelium as well as to increase vascular permeability. In the lung, VEGF is expressed by type II pulmonary epithelial cells and localizes to the basement membrane. An analysis of the various VEGF isoforms in the mouse revealed that the lung produces primarily VEGF188. This isoform includes two domains, encoded by exons 6 and 7, which are highly charged and bind with high affinity to the heparan sulfate, thus accounting for the basement membrane localization. This specific localization is postulated to be important for inducing capillary development. VEGF interacts with tyrosine kinase receptors to exert its cellular functions. These receptors (VEGFR1 or Flt-1; VEGFR2 or Flt-2 and VEGFR3) are localized on endothelial cells. Recent studies have revealed that they are also expressed by a variety of nonendothelial cell types. VEGF expressed by the pulmonary epithelium induces vascular development via the VEGFR2 on the endothelial precursor cells. Alterations in VEGF expression lead not only to abnormal vasculature but also to abnormal lung morphology. Additional evidence indicates that VEGF is important for maintenance of newly formed blood vessels as well.

The tight regulation of VEGF presumably contributes to the formation and maintenance of a normal blood gas barrier. Experiments involving transgenic mice have revealed the importance of precise regulation of VEGF expression. Targeted disruption of VEGF expression results in embryonic mortality, and inactivation of just a single VEGF allele causes abnormal blood vessel formation, leading to early embryonic lethality. Furthermore, overexpression of VEGF by the respiratory epithelium produces abnormal vascular formation, which also leads to disruption of lung morphology and embryonic lethality. Hypoxia increases VEGF production by lung epithelial cells, whereas hyperoxia depresses the expression of VEGF as well as its receptors. Platelet-Derived Growth Factors

Platelet derived growth factor (PDGF) is composed of dimers of A and/or B chains and acts via two tyrosine kinase receptors PDGFR-a and -b. Early in gestation, PDGF protein localizes to airway epithelial cells as well as to mesenchymal cells. PDGF is produced by the epithelium and interacts with PDGF receptors (PDGFRs) on interstitial mesenchymal cells. Expression of PDGF appears to change throughout gestation, increasing during the pseudoglandular phase with minimal production by the saccular stage. The localization of PDGF as well as the gestation-dependent expression pattern indicates an important role during lung development. PDGF is also involved in the formation of the vessel wall. PDGF B produced by immature endothelial cells acts as a mitogen and chemoattractant for undifferentiated mesenchymal, recruiting them to the nascent vessel. Upon contact with the endothelium, latent transforming growth factor beta (TGF-b) is activated. The activated TGF-b induces the differentiation of the mesenchymal to become smooth muscle cells/pericytes. It also acts upon the endothelial cells to inhibit their proliferation and migration, and it induces the production of basement membrane. Taken together, these factors lead to the formation of a stable, mature vasculature. PDGF appears to regulate the generation of myofibroblasts. The development and analysis of PDGF A and PDGF B knockout mice has revealed a role for PDGF in lung development. Homozygous PDGF A null mutants do not develop alveolar myofibroblasts and therefore have reduced elastin deposition, resulting in abnormal alveolarization. Failure of alveolar septation leads to emphysema in the surviving mutant mice. PDGF B-deficient mice are embryonic lethal, due to generalized hemorrhage and edema, most likely secondary to defective blood wall formation.

ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS 113

Precise regulation of PDGF levels during each phase of development is also important. Overexpression of PDGF A by the respiratory epithelium causes overgrowth of the mesenchyme and results in death from abnormal lung architecture. Transforming Growth Factor Beta

The TGF-b family of cytokines regulates cell proliferation, differentiation, recognition, and death via serine/threonine kinase activity receptors. The TGF-b1, TGF-b2, and TGF-b3 have been identified in the developing lung where they have been shown to stimulate extracellular matrix deposition by lung fibroblasts and inhibit epithelial cell proliferation. Spatial and temporal patterns of expression during lung development suggest a role for TGF-b in branch morphogenesis. Both mesenchymal and epithelial cells of the primitive lung express TGF-b1. TGF-b2 expression localizes to the tips of developing bronchioles, while TGF-b3 has been demonstrated in the proximal respiratory tract epithelium as well as the terminal growing buds. Signaling via the TGF-b type I receptor (TGFbRI) directs the formation of branch points while activation of the TGF-b type II receptor (TGFbRII) exhibits an inhibitory effect. The inhibitory effect of TGF-b on lung branching is mediated by the stimulation of the production and deposition of extracellular matrix proteins and inhibition of production of collagenase and proteolytic enzymes. Control over these molecules allows TGF-b to modulate lung branching. In culture, TGF-b1 and TGF-b2 decrease lung explant size and inhibit branching. TGF-b3 null mice, which die at birth, have delayed lung development and exhibit cleft palate as well as pulmonary epithelial, mesenchymal, and vascular dysplasia. In contrast to TGF-b3-deficient mice, TGF-b1 knockout mice have normal lung development but die by 1 month of age from fulminant pulmonary inflammatory infiltrates. The normal lung development has been attributed either to functional redundancy provided by the other TGF-b isoforms or to rescue via transplancental transfer of maternal TGF-b. Overexpression of TGF-b1 results in neonatal lethality and hypoplastic lungs, with decreased saccular formation and epithelial differentiation. Conversely, inhibition of TGFbRIIs stimulates lung development. Insulin-Like Growth Factors

Insulin-like growth factor-I ( IGF-I) and Insulin-like growth factor-II (IGF-II) are peptides similar in structure to proinsulin. These growth factors modulate cell proliferation and differentiation via two receptors,

IGF-IR and IGF-IIR. IGF-IR is a tyrosine kinase receptor, whereas IGF-IIR is a cation-independent mannose 6-phosphate receptor. In addition, IGF binding proteins (IGFBPs), which modulate IGF function, have been identified. IGF-I, IGF-II, and IGF-IR have been localized during early gestation to endothelial cells lining the primary vascular plexus, suggesting a role for these factors in pulmonary vascular development. Studies suggest that IGFs may function as survival factors during pulmonary vascular development. In vitro experiments involving the treatment of fetal lung explants with IGF-IR inhibitors exhibit not only a reduction in endothelial cell number but also an increase in mesenchymal cell apoptosis. In transgenic mice, disruption of the IGF-IR gene is lethal postnatally from respiratory failure. Lungs in these mutants appear hypoplastic although no gross defect in lung architecture has been observed. Other studies have suggested that VEGF may act downstream of IGF-1 in vascular development. IGF is important in pulmonary development as a survival factor perhaps via signaling through other growth factors. Epidermal Growth Factor and Transforming Growth Factor Alpha

Epidermal growth factor (EGF) and TGF-a are members of the EGF family that act through the EGF receptor (EGFR). EGF and TGF-a are produced by the mesenchyme and appear to act on the EGFR located on pulmonary epithelial cells as well as in neighboring mesenchyme. The source of these factors and the localization of EGFR indicate an important role for EGF and TGF-a in epithelial– mesenchymal interactions. EGF is also expressed by alveolar epithelial cells and regulates type 2 cell proliferation in an autocrine manner. Both EGF and TGF-a affect growth and branching of the pulmonary tree. In vitro experiments demonstrate that administration of exogenous EGF increases cellular proliferation as well as branching, whereas inhibition of EGF leads to decreased branching in explants. Targeted disruption of TGF-a demonstrates no adverse effects, most likely due to the functional redundancy provided by EGF. However, disruption of EGFR results in respiratory distress, leading to neonatal mortality. The lungs of these mutants exhibit septal thickening, decreased branching, deficient alveolarization, and reduced epithelial differentiation. Overexpression in the lung of TGF-a disrupts alveolar development and causes fibrotic lesions. Clearly the IGFs have an important role in epithelial–mesenchymal interactions during lung development.

114 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS Fibroblast Growth Factors

The fibroblast growth factors (FGFs) are a family of proteins that mediate a variety of processes, including cell proliferation, differentiation, cell angiogenesis, and development. These growth factors interact with specific tyrosine kinase receptors to mediate their effects. Binding to heparin sulfate proteoglycans on cell surfaces and within the extracellular matrix potentiates the effects of some FGFs. Specific FGFs and FGF receptors (FGFRs) regulated temporally and spatially during lung development implicate them in epithelial–mesenchymal interactions. Abnormal FGF expression and signaling during pulmonary development lead to anomalous epithelial branching and differentiation. FGF-10 and keratinocyte growth factor (KGF or FGF-7) induce pulmonary epithelial proliferation and branching. FGF-10 regulates patterning by chemotaxis, whereas KGF influences patterning by promoting epithelial growth. FGF-10 null mice display perinatal mortality from aberrant lung development. Disruption of FGFR2 expression results in pulmonary atresia distal to the main stem bronchi. Interruption of FGFR3 and FGFR4 signaling leads to excess elastin deposition, aberrant alveolization, and postnatal lethality. Transgenic overexpression of KGF results in pulmonary malformations that resemble cystic adenomatoid malformations. The function of KGF appears to overlap with other factors since KGF knockout mice have no gross pulmonary abnormalities. FGF signaling is critical not only for lung development but for postnatal repair following lung injury as well.

Diseases and Therapeutic Considerations Pulmonary development is a complicated system involving multiple growth factors and extensive epithelial–mesenchymal signaling. Any disruptions in this system can lead to inadequate gas exchange and diseases of respiratory distress. Both the vascular and airway development are affected in diseases with abnormal lung formation such as pulmonary hypoplasia. A better understanding of these developmental processes may elucidate the mechanisms underlying various respiratory diseases and thus lead to improved therapies. Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is a condition that affects approximately 1 in every 3000–5000 infants and is one of the most common causes of fetal hypoplastic lungs. In CDH, improper diaphragmatic development leads to abdominal content herniation into the thoracic cavity. This results in impaired lung

growth due to the loss of opposition to the lung’s inherent tendency to collapse. Intrusion of the abdominal viscera into the thoracic cavity is a secondary event that may further hinder lung growth and development. In addition to being small in size, the lungs in CDH are also immature. Despite timely repair of the diaphragmatic defect, many infants suffer postnatally from respiratory compromise due to underdeveloped lungs. Survival and long-term outcome is dependent on the severity of pulmonary hypoplasia, the extent of bronchopulmonary dysplasia from ventilatory injury, and other associated anomalies. Lungs from patients with pulmonary hypoplasia associated with CDH exhibit significantly decreased number of airways, decreased vessel density, and more muscularized arteries. A murine model phenotypically similar to CDH has been developed using nitrofen exposure. Lungs from nitrofen-treated mice have decreased levels of VEGF, which may contribute to their immature state. Nitric oxide, important in the regulation of smooth muscle cell proliferation, and nitric oxide synthase are also reduced. The decreased production and diffusion of nitric oxide may contribute to the muscularized arteries, which in turn lead to pulmonary hypertension. The possibility of growth-factor-induced pulmonary growth is currently under investigation in hopes of a therapeutic intervention for CDH. Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD), also known as neonatal chronic lung disease, is an important cause of respiratory illness, especially in preterm infants. In BPD, acute injury of the lung may be caused by multiple factors, including pulmonary oxygen toxicity, barotrauma from mechanical ventilation and cellular immaturity. BPD is more common among premature infants because of higher susceptibility of the immature lungs to injury. The acute injury causes an acute inflammatory reaction, which leads to interstitial fibrosis and emphysema with associated histologic features including airway mucosal metaplasia, smooth muscle hyperplasia, and atelectasis. Long-term histologic changes include decreased alveolar numbers and surface area. Airway lavage samples from patients with BPD contain increased levels of TGF-b, which are associated with an adverse prognosis. As described above, in vivo experimental models have shown that excessive expression of TGF-b inhibits lung morphogenesis and induces pulmonary fibrosis; EGF and PDGF are also thought to be involved in the development of pulmonary fibrosis in BPD. Decreased levels of VEGF in the presence of alveolar damage are also

ANTICOAGULANTS 115

likely to contribute to the abnormal pulmonary vasculature associated with lung injury. Manipulation of TGF-b may lead to therapeutic interventions to prevent the development of chronic pulmonary fibrosis associated with BPD.

Acknowledgments The authors were supported by CA45548 (PD’A) and EY05318 (PD’A). See also: Bronchopulmonary Dysplasia. Epidermal Growth Factors. Fibroblast Growth Factors. Insulin-Like Growth Factors. Keratinocyte Growth Factor. Platelet-Derived Growth Factor. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Vascular Endothelial Growth Factor.

Further Reading Alescio T and Cassini A (1962) Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium. Journal of Experimental Zoology 150: b83–b94. Burri PH (1984) Fetal and postnatal development of the lung. Annual Review of Physiology 46: 617–628.

Carmeliet P, Ferreira V, et al. (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380: 435–439. Chinoy MR (2003) Lung growth and development. Frontiers in Bioscience 8: 392–415. deMello DE, Sawyer D, et al. (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568–581. Gaultier C, Bourbon J, and Post M (eds.) (1999) Lung Development. New York: Oxford University Press. Han RN, Post M, et al. (2003) Insulin-like growth factor-I receptor-mediated vasculogenesis/angiogenesis in human lung development. American Journal of Respiratory Cell and Molecular Biology 28: 159–169. Jankov RP and Keith A (2004) Tanswell. Growth factors, postnatal lung growth and bronchopulmonary dysplasia. Paediatric Respiratory Reviews 5(supplement A): S265–S275. Kumar VH and Ryan RM (2004) Growth factors in the fetal and neonatal lung. Frontiers in Bioscience 9: 464–480. Shannon JM (1997) Epithelial–mesenchymal interactions in lung development. In: McDonald JA (ed.) Lung Growth and Development, pp. 81–118. New York: Dekker. Warburton D and Bellusci S (2004) The molecular genetics of lung morphogenesis and injury repair. Paediatric Respiratory Reviews 5(supplement A): S283–S287. Warburton D, Schwarz M, et al. (2000) The molecular basis of lung morphogenesis. Mechanisms of Development 92: 55–81.

ANTICOAGULANTS A Gu¨nther and C Ruppert, University of Giessen Lung center (UGLC), Giessen, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract Anticoagulants are widely used for the prevention and treatment of venous and/or arterial thrombosis. Anticoagulants comprise a chemically heterogeneous group of drugs acting at different steps within the coagulation cascade. Heparin and heparin-based anticoagulants are indirect anticoagulants that bind to antithrombin and enhance the inhibitory capacity of this natural anticoagulant. Coumarin derivatives (e.g., warfarin) interfere with the hepatic synthesis of coagulation factors (vitamin K antagonists). A third class comprises direct inhibitors of enzymes of the clotting cascade, primarily thrombin. Classic anticoagulants such as unfractionated heparin and coumarins have some clinical drawbacks. Heparin requires parenteral application, has serious adverse effects, and is difficult to dose and monitor due to variable and unpredictable pharmacokinetics. The orally active coumarin derivatives have a narrow therapeutic window and multiple interactions with food and drugs, necessitating individualized dosing and monitoring. During the past 10 years, the search for safer and more effective antithrombotic agents resulted in the development or discovery of new molecules, including fondaparinux and idraparinux (both selective inhibitors of factor Xa), thrombin inhibitors for parenteral use such as recombinant hirudin and hirulogs, and the orally active ximelagatran.

The first effective agent for therapy of thromboembolism was unfractionated (UF) heparin. It has become the anticoagulant of choice for the treatment and prevention of arterial and venous thrombotic diseases, including pulmonary embolism. Due to the problems accompanying heparin therapy (unpredictable pharmacokinetics, low bioavailability at low doses, unpredictable dose response, need for careful laboratory monitoring, bleeding, and thrombocytopenia), the search for improved anticoagulants resulted in the introduction of low-molecular-weight (LMW) heparins (Table 1). These heterogeneous compounds have been shown to produce a more predictable anticoagulant response and fewer adverse effects than UF heparin, reflecting the improved pharmacokinetic properties (greater bioavailability after subcutaneous injection (B30%), longer half-life, and dose-independent clearance). The expectation, however, that these new compounds might eliminate the risk of bleeding was not confirmed. Another newer anticoagulant is danaparoid sodium, a heparinoid that is often used for treatment of heparin-induced thrombocytopenia.

116 ANTICOAGULANTS Table 1 Heparins and heparinoids INN name

Brand name

Manufacturer

Unfractionated heparin Heparin-sodium Generic (e.g., Liquemin) Low-molecular-weight heparins Ardeparin Normiflo

Wyeth–Ayerst

Certoparin

Mono-Embolex

Novartis

Dalteparin

Fragmin

Pharmacia

Enoxaparin

Clexane

Aventis-Pharma

Nadroparin

Fraxiparine

Reviparin

Clivarine

Sanofi– Synthelabo Abbott

Tinzaparin

Innohep

LeoLabs

Heparinoids Danaparoid

Orgaran

Celltech Pharma

Pentosanpolysulfate

Elmiron

Ortho-McNeal

Hemoclar Fibrezym

Sanofi Aventis Bene Arzneimittel

Oral anticoagulants (4-hydroxycoumarin derivatives), the second major class of traditional anticoagulants, were developed in parallel to heparin, based on a serendipitous discovery. Today, several coumarins are used as agents of choice for longterm anticoagulant therapy (Table 2). For historical and marketing reasons, some countries use more warfarin (e.g., United States, Canada, and United Kingdom), others phenprocoumon (e.g., Germany), and yet others acenocoumarol. Another class of drugs, called indaniones, are similar to coumarins in terms of pharmacodynamics and are mostly used in France. The traditional anticoagulants UF heparin and coumarin derivatives are highly effective agents and relatively safe when administered at appropriate dosages and carefully monitored. LMW heparins improved and simplified anticoagulant therapy by obviating the need for routine coagulation monitoring. Newer agents, including fondaparinux and

Method of preparation

Mean molecular weight

Anti-Fxa: anti-FIIa ratio

Extraction from porcine mucosa

14 000 (range: 5 000–40 000)

1.0

Peroxidative depolymerization of UF heparin Isoamylnitrite depolymerization of UF heparin Nitrous acid depolymerization of UF heparin Benzylation and alkaline depolymerization of UF heparin Nitrous acid depolymerization of UF heparin Nitrous acid depolymerization of UF heparin and size exclusion chromatography Haparinase digestion of UF heparin

5 300

2.0

5 200

2.2

6 000

1.9–3.2

4 500

3.3–5.3

4 300

2.5–4.0

3 900

3.6–6.1

6 500

1.5–2.5

6 500

Anti-FXa 4 anti-FIIa n.a.

Extraction from porine mucosa Sulfation of pentosan derived from beech tree bark

6 000

idraparinux, represent the first agents of a new class of FXa inhibitors. Both are fully synthetic analogs of the unique antithrombin-binding pentasaccharide sequence found in UF and LMW heparin. Advances in molecular biology and biotechnology have led to the recombinant production of hirudin, the most potent naturally occurring direct thrombin inhibitor. Hirudin also serves as a standard for the development of LMW inhibitors of thrombin. Argatroban was the first synthetic thrombin inhibitor approved for prophylaxis and treatment of various thrombotic disorders mainly in patients with heparin-induced thrombocytopenia. The major drawback of argatroban and hirudins is their requirement for parenteral administration. Ximelagatran and its active metabolite, melagatran, are members of a class of newly developed oral direct thrombin inhibitors. These agents have been approved for anticoagulant treatment very recently and expand the treatment options for thrombotic disorders. Whether these new agents

ANTICOAGULANTS 117 Table 2 Coumarin and indandione derivatives INN

Brand name

Manufacturer

Pharmacokinetics t 12 (h)

Duration of action (days)

Acenocoumarol (Nicoumalone) Ethylbiscoumacetate Dicumarol Phenprocoumon Tioclomarol Warfarin

Sintrom Tromexane Generic Marcumar Apegmone Coumadin Warfilone

Novartis Ciba-Geigy Generic Roche Merck Lipha Bristol-Myers Squibb Merck Frosst

9 2.5 n.a. 150 24 35–45

2–4 1–2 n.a. 7–10 2–3 4–5

Anisindione Fluindione Phenindione

Miradon Previscan Dindevan Pindione

Schering Procter & Gamble Goldshield Merck Lipha

9 6 6

3–4 2–3 2–3

will be more effective and safer than traditional anticoagulants remains to be determined.

Chemical Structure Heparin and Heparinoids

Heparin is a naturally occurring, highly sulfated glycosaminoglycan that has been found in mast cells in a large number of mammalian and nonmammalian vertebrates. Material for clinical use is derived from bovine lungs or pig intestinal mucosa and is prepared either as UF heparin or depolymerized LMW heparin. Heparin is a heterogeneous mixture of unbranched polysaccharide chains composed of 15–100 alternating 1-4-linked mucosaccharide units of D-glucosamine and L-iduronic acid or D-glucuronic acid (Figure 1(a)). Heparin is the most negatively charged molecule in the human body. The molecular weight of UF heparin ranges from 5 to 40 kDa with an average of B14 kDa. LMW heparins are much smaller (mean MW, B5 kDa) and are produced from UF heparin by chemical or enzymatic depolymerization. The chemical composition is similar but not identical. The anticoagulant effect is mediated by a unique pentasaccharide unit (Figure 1(b)) that binds antithrombin with high affinity and activates the inhibitor by approximately 1000-fold. This pentasaccharide sequence is found in B30% of the chains of UF heparin (known as high-affinity heparin) but in only 15–25% of the chains of LMW heparin. Danaparoid sodium is an LMW heparinoid isolated from porcine mucosa with a mean MW of 6 kDa (range, 4–12 kDa). It consists of a mixture of 84% heparan sulfate (of which 4% has high antithrombin activity), 12% dermatan sulfate, and 4% chondroitin sulfate. The characteristic disaccharide

repeat units of these glycosaminoglycans are depicted in Figure 1(c). Pentosanpolysulfate (PPS) is a highly sulfated, semisynthetic polysaccharide derived from beech tree bark with a mean molecular weight of 6 kDa (range, 2–9 kDa). The degree of sulfation is higher than in heparin, resulting in a higher negative charge density. Pentosan consists of b-1-4-linked D-xylose units branched with 4-O-methyl-D-glucuronic acid in a ratio of 1 uronic to 9 xylose units (Figure 1(d)). In contrast to heparin and other glycosaminoglycans, PPS is orally bioavailable. FXa Inhibitors

Fondaparinux is a fully synthetic analog of the unique pentasaccharide sequence found in heparins. Idraparinux represents the second generation of this class. The O-methylation and O-sulfation result in a higher affinity for antithrombin and a higher negative charge, which in turn accounts for the tight binding of antithrombin and the prolonged half-life of idraparinux (80 h vs. 17 h for fondaparinux) (Figure 2). Vitamin K Antagonists

The clinically used vitamin K antagonists are derivatives of either 4-hydroxycoumarin or indan-1,3dione (Figure 3). The substituent at the 3 position of the coumarin backbone or side-chain variations influence pharmacokinetic and pharmacodynamic properties (Table 2). Pharmaceutical preparations of warfarin, acenocoumarol, and phenprocoumon contain a racemic mixture of R- and S-enantiomers. The stereoisomers exhibit different pharmacodynamics and pharmacokinetics since they undergo stereoselective biotransformation in the liver.

118 ANTICOAGULANTS k

CH2OSO3 O

O

COOk 4

1

4

O

OH

1 OH

O

k

NHSO3k

OSO3

(a)

n

Heparin CH2OSO3k O

CH2OSO3k O

COOk O

O OH

O

OH

O

k

OSO3

COOk

O

OH

O

OH OSO3k

NHSO3k

OH

NHCOCH3

CH2OSO3k O

O

O NHSO3k

Heparin pentasaccharide

(b)

COOk O

CH2OSO3k O

O

O 4

1

4

OH

O

COO

1

4

OH

1

n

O

NHCOCH3

n

Dermatan sulfate

R1O 4

1

1 3

OH

Heparan sulfate COOk O

O

O3SO 4

OH OH

4

CH2OH O

k

k

OH

CH2OR2 O

Chondroitin sulfate O

A: R1 = SO3k R2 = H C: R1 = H R2 = SO3k

1 3

O NHCOCH3

OH

(c)

O O

OSOk 3 OSO3k

O

O O

n

O

OSOk 3

O O

OSOk 3

OSO3k COOk O

OSOk 3 OSO3k

O O

OSOk 3

O

OSO3k

O

H3CO OSO3 OSO3k

(d)

Pentosanpolysulfate

Figure 1 Chemical structures of heparin and other members of the glycosaminoglycan family. (a) The major repeat unit of heparin is depicted, which is found in up to 90% in heparin from beef lung and up to 70% in heparin from pig mucosa. The disaccharide consists of 1-4 linked sulfated L-iduronic acid and sulfated D-glucosamine. (b) The specific pentasaccharide sequence that is responsible for highaffinity binding to antithrombin is depicted. (c) The major repeat units of heparan sulfate, dermatan sulfate, and chondroitin sulfate A are depicted. (d) A typical sequence of pentosanpolysulfate is shown.

Hirudin and Hirulogs

Hirudin is a naturally occurring inhibitor of thrombin that is a single-chain polypeptide of 65 amino acid residues with an apparent molecular weight of 7 kDa. The structure is stabilized by three intramolecular disulfide bridges. In 1986, recombinant hirudin (r-hirudin) became available. Desirudin

([Val1, Val2]-63-desulfohirudin) and lepirudin ([Leu12]-63-desulfohirudin) are therapeutically used recombinant hirudins produced in yeast (Table 3). They are not sulfated and differ only in the first two amino acids of the N terminus. Both natural and recombinant hirudins show almost identical pharmacodynamic and pharmacokinetic properties.

ANTICOAGULANTS 119 COOk O

CH2OSO3k O

CH2OSO3k O

CH2OSO3k O

O COOk

HO OH

O

OH

O NHSO3k

O

OSO3k

O

NHSO3k

OH

OH

OH

OCH3

OSO3k

NHSO3k

Fondaparinux

COOk

CH2OSO3k O

CH2OSO3k O

O

CH2OSO3k O

O COOk

O H3CO OCH3

OSO3k

OCH3

O OCH3

OSO3k

OCH3

O O

OCH3

OSO3 OCH3k

OCH3 OSO3k

Idraparinux

Figure 2 Chemical structure of FXa inhibitors. The structures of fondaparinux and idraparinux are given.

OH 4 12 O

O 1 2 3

3

O

O

4-Hydroxycoumarin

Indan-1, 3-dione

O O

OH

O

O

O OH

Warfarin

OH

Phenindione

O O O

O

OH

O

O

F

Dicumarol NO2

O

OH

O

OH

Fluindione Acenocoumarol O

O O

O

OH

O Ethylbiscoumacetate

O

O

OCH3 O Anisindione

Phenprocoumon

Figure 3 Chemical structures of coumarin and indandione derivatives. The lead structures and derivatives are given. Clinically used coumarin derivatives are substituted at the 3-position, and indandiones are substituted at the 2-position. Asymmetric carbons are indicated by asterisks.

120 ANTICOAGULANTS Table 3 Direct thrombin inhibitors INN name

Brand name

Manufacturer

Route of administration

Half-life

Desirudin Lepirudin Bivalirudin Argatroban Melagatran Ximelagatran

Revasc Refludan Angiomax Novastan Melagatran Exanta

Aventis/Novartis Schering, Berlex The Medicine Company Texas Biotechnology AstraZeneca AstraZeneca

Parenteral (i.v., s.c.) Parenteral (i.v.) Parenteral (i.v.) Parenteral Parenteral Oral

2–3 h 1.3 h 25 min 45 min 3–5 h 3–5 h

1 H2N

Val Val Tyr Thr Asp Cys Thr Glu Ser

20

Gly 10 Val Asn Ser Gly Glu Cys Leu Cys Leu Asn Gln Cys

40 Thr Val Cys Gly

Gly Gln Gly Asn Lys Cys IIe Leu 30

Gln Asn Lys Glu Gly Asp Ser Gly

Glu Gly

65 60 50 Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Gln

COOH

SO3−

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Gln Hirudin

D−Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Bivalirudin Figure 4 Structure of hirudin and hirulogs. The amino acid sequences of natural hirudin and the hirulog bivalirudin are given. The C-terminal sequences blocking the anion-binding exosite (amino acids 54–65 of hirudin) are depicted in blue, and the sequences binding to the active site of thrombin (amino acids 45–48 of hirudin) are depicted in light blue.

The discovery of the bifunctional inhibition mechanism of hirudin resulted in the development of a new type of bivalent oligopeptide inhibitors, termed hirulogs. Bivalirudin, also termed hirulog-1, is a semisynthetic 20-amino acid peptide consisting of the N-terminal and the C-terminal active peptide domains from hirudin joined by a glycine linker (Figure 4).

mimicking the cleavage site in fibrinogen. The arginine-derived structures are shown in Figure 5. The pharmaceutical preparation consists of a mixture of R and S stereoisomers at a ratio of approximately 65:35. Ximelagatran has a molecular weight of 473.6 Da and is a pro-drug without intrinsic anticoagulant activity. Following resorption, it is rapidly converted into its active metabolite, melagatran (MW, 429.5 Da).

Synthetic Active Site Inhibitors of Thrombin

Argatroban and the orally active ximelagatran are LMW active site inhibitors of thrombin. They are the result of structure-based drug design and belong to the peptidomimetic (arginominetic) group of inhibitors

Mode of Action The classical and new anticoagulants interact with different clotting factors at different levels of blood

ANTICOAGULANTS 121 O H2N

OH

NH H2N

NH

Arginine

H3C

NH O

OH

O S O

NH

N O O

NH

HN

N

O CH3

O HO

O

N

NH2

NH H2N

CH3

NH

Ximelagatran Argatroban

Hydrolysis

Reduction

NH N O O

HN O OH

HN

NH2

Melagatran Figure 5 Chemical structures of synthetic thrombin inhibitors. The chemical structures of the arginomimetics argatroban, ximelagatran, and melagatran are shown. Bioconversion of ximelagatran into its active metabolite, melagatran, involves hydrolysis of the carboxyl ester and reduction of the hydroxyamidino group.

coagulation. A summary of their targets within the coagulation cascade is shown in Figure 6. Heparin and Heparinoids

Heparin acts as an accelerator of antithrombin (formerly called antithrombin III). The pentasaccharide sequence of heparin binds to the lysine site in antithrombin inducing a conformational change of

the antithrombin molecule, which facilitates binding to specific clotting factors and accelerates the rate at which antithrombin inhibits these factors by approximately 1000 times. UF heparin inhibits factors Xa, IIa (thrombin), IXa, XIa, and XIIa, with FXa and thrombin representing the most responsive and most critical factors within the clotting cascade. Heparin, antithrombin, and thrombin form a ternary complex in which thrombin initially binds to the

122 ANTICOAGULANTS Extrinsic pathway Vascular injury

Coumarins, indandiones

Tissue factor (TF) FVII

Intrinsic pathway

HMW kininogen Prekallikrein FXIIa FXI

FVII TF FVIIa

FIX

TFPI, FVIIai Coumarins, indandiones

Ca2+

FX

FXIa

UF heparin, LMW heparin Danaparoid, fondaparinux

Surface

FIXa

FVIII

Ca2+, PL

PL, Ca2+ Tenase complex

FIXa, thrombin

Antithrombin Hirudin, hirulogs Argatroban, melagatran

FVIIIa FXa 2+

APC

Prothrombin FV

Coumarins, indandiones

PL, Ca Prothrombinase complex

2+ Ca , PL, FXa

Thrombin FXIII

FVa

FXIIIa Heparin cofactor II

APC

Fibrinogen Danaparoid Dermatan sulfate

Fibrin

Ca2+

Crosslinked fibrin

Figure 6 Overview of the coagulation cascade and targets of anticoagulants. The targets of anticoagulants within the clotting cascade are given.

heparin–antithrombin complex in a non-specific manner to any site of the heparin molecule and then slides along the surface until it binds to the inhibitor. The affinity of heparin for the antithrombin–thrombin complex is much lower than that for unreacted antithrombin. Thus, heparin can dissociate from the complex and bind to additional unreacted antithrombin molecules, resulting in a continuing anticoagulant effect. The complex, however, is not effective in inhibiting fibrin-bound thrombin. UF heparin also blocks the thrombin-induced feedback activation of factors V and VIII. The sliding mechanism requires heparin chain lengths consisting of at least 18 saccharide units. The lower content of glycosaminoglycan chains 418 saccharide units in LMW heparin accounts for the greater relative activity against factor Xa. This difference is described in an activity ratio (anti-FXa:anti-FIIa ratio), which is 1:1 for UF heparin and 1.5:1 to 6:1 for LMW heparin (see Table 1). The anticoagulant activity of UF heparin is expressed in relation to the fourth international standard: Pharmaceutical preparations have specific activities of 150–190 U mg
1. The

anticoagulant activity of LMW heparin is expressed relative to the first international standard for LMW heparin; the specific activity ranges between 80 and 120 anti-FXa U mg
1 and between 35 and 45 antithrombin U mg
1. In addition to the interaction with antithrombin, both UF and LMW heparin enhance the release of tissue factor pathway inhibitor (TFPI), which forms a complex and inactivates FXa and subsequently FVIIa. UF and LMW heparins also bind to platelet factor 4, which is the predicate for heparin-induced thrombocytopenia (HIT), a severe complication of heparin therapy that is associated with paradoxic clotting, including deep vein thrombosis, pulmonary embolism, or occasional arterial thromboses. In HIT, antibodies form against the heparin–platelet factor 4 complex, leading to platelet activation and aggregation. LMW heparins bind less extensively to platelet factor 4 as well as to plasma proteins, endothelial cells, and macrophages. However, LMW heparins should not be used in patients with established HIT since HIT antibodies have an almost 100% cross-reactivity with LMW heparin.

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The heparinoid danaparoid sodium exerts its pharmacological effects in a similar manner via binding and activation of the serpin inhibitor heparin cofactor II. Danaparoid has little effect on platelet function and exhibits low cross-reactivity with heparin-induced antibodies, making it an option for the treatment of HIT. Similar to LMW heparins, danaparoid also exerts a higher inhibitory activity against FXa. The overall antithrombotic activity is much lower compared to that of UF heparin (anti-FXa activity B10% and antithrombin activity B1% of that of UF heparin). PPS may act via an antithrombin or heparin cofactor II-independent pathway, but the exact mechanism of action is not known. It has been used since the 1960s as an anticoagulant but does not seem to play a major role in today’s clinical medicine with regard to anticoagulation. In the United States, PPS is approved for pain relief in the management of interstitial cystitis. Fondaparinux and Idraparinux

Fonaparinux and idraparinux are selective indirect inhibitors of FXa. Their activity also requires the presence of antithrombin, but inhibition is achieved solely through binding of the pentasaccharide sequence to antithrombin and induction of a conformational change. Vitamin K Antagonists

Coumarin and indandione derivatives act as vitamin K antagonists and exert their anticoagulatory effect by interfering with the hepatic synthesis of vitamin K-dependent clotting factors. Synthesis of factors II (prothrombin), VII, IX, and X involves carboxylation of glutamate residues to g-carboxyglutamates by a specific carboxylase, which is required for their biological activity. These g-carboxyglutamate residues promote binding of the clotting factors to phospholipids, thereby accelerating coagulation. Coumarin and indandione derivatives interfere with the vitamin K cycle by inhibiting vitamin K epoxide reductase. By this mechanism, coumarins decrease the synthesis of coagulation factors by 30–50%. The anticoagulant response to coumarins is delayed by 2 or 3 days since previously formed coagulation factors circulate with a long half-life (4–6 h for FVII, 20–50 h for factors IX and X, and 50–70 h for thrombin). A major disadvantage of vitamin K antagonists is the narrow therapeutic index and the need for intensive monitoring. The anticoagulatory effect is assessed by the international normalized ratio (INR), which is defined as the ratio of the patient prothrombin time (PT) compared to the mean PT of normal donors normalized to the international sensitivity index, a correction factor for

the response of different thromboplastin reagents. The therapeutic INR range is 2.0–3.0. An INR o2.0 may be ineffective in preventing thrombotic events, and an INR 43.0 is associated with increased bleeding. The manifold interaction of coumarins with food and drugs has an important impact on the anticoagulatory response by either increasing or decreasing anticoagulant activity. Direct Thrombin Inhibitors (Hirudin, Hirulogs, and Synthetic Inhibitors)

Hirudin is a highly potent (Ki ¼ 22 fM) inhibitor specific for the serine protease thrombin. It forms a stable noncovalent 1:1 stoichiometric complex with the B-chain of a-thrombin, thereby abolishing its ability to cleave fibrinogen (Figure 7). Hirudin inhibits both free and fibrin-bound thrombin. During inhibition, hirudin displaces fibrin from thrombin. Bivalirudin is a bivalent inhibitor (Ki ¼ 1:9 nM) blocking both the active site and the substrate recognition site (anion-binding exosite). Argatroban (Ki ¼ 39 nM) and melagatran (Ki ¼ 2 nM) are peptidomimetics of the sequence of fibrinopeptide A that interact with the active site of thrombin. They are reversible, noncovalent, competitive inhibitors blocking the enzyme’s interaction with its substrate. The pro-drug ximelagatran has no intrinsic anticoagulant activity but is rapidly converted into melagatran following gastrointestinal resorption. The direct thrombin inhibitors are recommended agents for prevention and treatment of HIT. Lepirudin and argatroban are approved for the treatment of HIT in the United States. Bivalirudin, which is approved for anticoagulation during percutaneous coronary intervention, appears to be promising in patients with HIT. Although displaying pharmacologic advantages, its use for this indication is only weakly recommended due to the limited data available. Novel Anticoagulants

In addition to the previously described anticoagulants, novel agents that directly target specific steps within the coagulation cascade have been developed and tested in clinical phase II or phase III studies or are currently undergoing preclinical and clinical testing. They include LMW inhibitors of FXa and recombinant forms of naturally occurring anticoagulants. Recombinant tissue factor pathway inhibitor (tifacogin) Tifacogin is a recombinant form of the endogenous extrinsic pathway inhibitor TFPI, also referred to as anticonvertin or lipoprotein-associated coagulation inhibitor. TFPI is a 276-amino acid residue, high-affinity Kunitz-type serine protease inhibitor.

+ + +

+ + +

Fibrin

+ + +

Hirudin

Heparin recognition site = anion-binding exosite II

+ + +

Fibrin

124 ANTICOAGULANTS

Bivalirudin

Thrombin + + +

+ + +

Substrate recognition site = anion-binding exosite I

Binding of fibrin(ogen), PAR-1, heparin cofactor III thrombomodulin

Binding of heparin, prothrombin 2 fragment (F2) platelet gp Ib

+ + +

Active site

+ + +

Fibrin

Specificity pocket

Synthetic inhibitors (Argatroban, melagatran) Figure 7 Mechanism of action of thrombin inhibitors. The interaction of hirudin, hirologs, and synthetic thrombin inhibitors with thrombin is shown.

Inhibition of the tissue factor-mediated extrinsic coagulation pathway occurs in a two-step manner: TFPI directly binds to FXa at or near its active site via the second Kunitz domain and produces a FXadependent feedback inhibition of the TF–FVIIa catalytic complex by formation of a quaternary complex (FXa–TFPI–TF/FVIIa), in which the second Kunitz domain binds to FXa and the first Kunitz domain binds FVIIa (Figure 8). Despite showing promising results in animal thrombosis models, a phase III trial failed to demonstrate significant benefit in outcome in sepsis patients. Recombinant nematode anticoagulant protein c2 Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent 85-amino acid residue anticoagulant protein originally identified in the hookworm Ancylostoma caninum. Like TFPI, rNAPc2 inhibits initiation of the extrinsic coagulation pathway by binding to a noncatalytic exosite on FX or

FXa prior to the formation of the quaternary inhibitory complex with tissue factor–FVIIa. In a phase II study on patients who underwent knee replacement surgery, a low dose of rNAPc2 decreased the rate of deep vein thrombosis and major bleeding.

Active site inhibited factor VII Active site inhibited factor VII (FVIIai; ASIS) is a recombinant variant of activated factor VII in which the catalytic function is irreversibly blocked. The resulting molecule retains its TF binding capacity but is enzymatically inactive. FVIIai exerts its effects by competing with plasma FVII(a) for tissue factor binding on cell surfaces. The formation of an inactive binary FVIIai–TF complex attenuates the initiation of coagulation. However, although recombinant FVIIai was shown to inhibit TF-mediated injury in animal models, it failed to elicit a beneficial response in coronary patients in a phase II trial.

ANTICOAGULANTS 125 3 TFPI 1

2 FXa

Ca2+

TFPI

FXa

Ca2+

TFPI

FVIIa

FVIIa Ca2+

Ca2+

TF

FXa

TF Ca2+

Figure 8 Mechanism of action of tissue factor pathway inhibitor (TFPI). TFPI binds to FXa in solution forming a binary complex. Subsequent binding of FXa–TFPI to the TF–FVIIa complex results in the final quaternary complex inhibiting initiation of coagulation.

Direct FXa inhibitors DX-9065a (Daiichi) is a synthetic, LMW, nonpeptidic, direct reversible and parenteral inhibitor of factor Xa. BAY 59-7939 (Bayer), razaxaban (Bristol-Myers Squibb), LY 517717 (Eli Lilly), and YM-60828 (Yamanouchi Pharmaceutical) are orally active synthetic FXa inhibitors. Activated protein C (drotrecogin alpha (activated)) The protein C system also regulates coagulation by modulation of the activity of the two clotting factors, FVa and FVIIIa. Protein C, the key component of this system, is a vitamin K-dependent zymogen of an anticoagulant protease, which is activated on the surface of intact endothelial cells by thrombin that has bound to the integral membrane protein thrombomodulin. Activated protein C (APC) can cleave the phospholipid membrane-bound (activated) clotting factors FVa and FVIIIa, which blocks the function of the prothrombinase and tenase

complexes and thus inhibits the propagation of coagulation (Figure 9). Activation of protein C is augmented by the endothelial cell protein C receptor, and the anticoagulant activity of APC is supported by protein S, a vitamin K-dependent cofactor. The protein C system also has anti-inflammatory and antiapoptotic properties. Drotrecogin alpha (activated) is a recombinant version of APC. Drotrecogin alpha was approved for treatment of severe sepsis in adults based on a phase III trial in which 28-day mortality was reduced. Thrombomodulin Thrombomodulin is a complex multifunctional endothelial cell surface glycoprotein receptor. High-affinity binding to thrombin involving EGF domains 4–6 converts thrombin from a procoagulant into an anticoagulant state that can activate protein C. Thrombomodulin not only regulates hemostasis but also plays an important role in the

126 ANTICOAGULANTS Protein C APC TM

FV

Protein S

FVIIIi FVIIIa A P C

EPCR

Protein S

Th

FVi FVa A P C

Figure 9 Protein C pathway: mechanism of action of protein C and thrombomodulin. (Top) Thrombin (Th) generated in the vicinity of intact endothelial cells binds to thrombomodulin (TM) and activates protein C. This process is enhanced by the endothelial cell protein C receptor (EPCR). (Bottom) Activated protein C (APC) and protein S form a complex on the membrane of endothelial cells that cleaves and inactivates FVIIIa (-FVIIIi) and FVa (-FVi) and thus inhibits coagulation. In the case of FVIIIa, this process is further enhanced by FV, which in this context functions as an anticoagulant cofactor protein.

modulation of inflammation. The EGF 3–6 domains are involved in activation of thrombin-activatable fibrinolysis inhibitor, a natural anti-inflammatory molecule that inhibits vasoactive peptides such as the complement anaphalotoxin C5a. Soluble thrombomodulin is a recombinant variant that is undergoing clinical testing.

Role of Anticoagulants in Respiratory Medicine Thromboembolic events, including deep vein thrombosis and pulmonary embolism, are commonly encountered in clinical practice. In addition to the occlusion of a vessel, ‘periembolic’ processes, such as activation of circulating platelets and inflammatory cells at the surface of thrombi and the liberation of vasoactive mediators (thromboxan, serotonin, and leukotriene B4) and fibrin(ogen)-derived split products, markedly contribute to cardiopulmonary dysfunction, with increased pulmonary vascular resistance, elevated right heart pressure, and gas exchange disturbances. Anticoagulants are used for prevention and treatment of a variety of indications, including prevention of deep vein thrombosis and pulmonary embolism postoperatively after general or orthopedic surgery (e.g., hip replacement/fracture), in patients with acute spinal cord injuries, multiple trauma, ischemic stroke, after coronary angioplasty, heart valve replacement, and in many other medical conditions as well as for treatment of established deep vein thrombosis, unstable angina, and ischemic stroke. In a meta-analysis,

subcutaneous LMW heparin was shown to be as effective and safe as intravenous UF heparin for the initial treatment of nonmassive pulmonary embolism. Intravascular clot formation due to predominance of a procoagulant and suppression of fibrinolytic factors is a key event in a variety of inflammatory and infectious diseases, such as septic organ failure. Microthrombosis/microembolism is commonly found in patients with acute respiratory distress syndrome (ARDS), of which sepsis is the major underlying cause, and in patients with chronic pulmonary hypertension. The lung microvascular compartment represents the largest surface of the pulmonary vascular bed and may be of crucial importance in the dissolution of microthrombi. Under physiological conditions, patency of capillaries is provided by a high concentration of antithrombin that is ‘activated’ by glycosaminoglycans (e.g., heparan sulfate) and thrombomodulin on the surface of endothelial cells. In addition, clearance of thrombi is achieved through the production and secretion of tPA and uPA by microvascular endothelial cells. Under inflammatory or infectious conditions (e.g., sepsis and ARDS), this balance between anticoagulation and fibrinolysis regulation is shifted toward coagulation with a decrement in fibrinolysis, mainly due to activation of the extrinsic tissue factor/FVII-dependent coagulation pathway and secretion of plasminogen activator inhibitor-1 (PAI-1). In experimental models of acute lung injury (ALI), APC prevented intravascular coagulation, edema formation, and inflammatory cell recruitment in the lung. Although administration of APC has been shown to decrease mortality in patients

ANTICOAGULANTS 127

with severe sepsis, clinical studies demonstrating its efficacy in ARDS are lacking. In ALI/ARDS, fibrin deposition occurs in both intravascular and extravascular locations. In patients with ALI/ARDS, the alveolar hemostatic balance is shifted toward predominance of procoagulant activity, which is mainly attributable to the extrinsic pathway enzymes tissue factor and FVII. In contrast, the fibrinolytic capacity of the alveolar space is markedly reduced. In lavage fluid from patients with ARDS, concentrations of urokinase, representing the predominant plasminogen activator in this compartment, were markedly decreased, whereas elevated activities of PAI-1 and a2-antiplasmin were consistently encountered. Under these conditions, fibrinogen leaking into the alveolar space due to an impaired barrier function is rapidly converted into fibrin. The function of fibrin formation in the alveolar space is largely unsettled. It may well exert beneficial effects by preventing pulmonary hemorrhage and serve as a primary matrix of wound repair. On the other hand, alveolar fibrin is a potent inhibitor of surfactant function, thus promoting atelectasis formation and contributing to the severe impairment of gas exchange properties. In vitro studies have demonstrated an incorporation of hydrophobic surfactant compounds into nascent fibrin strands, resulting in an almost complete loss of the surface tension-lowering properties. Alveolar/interstitial fibrin deposition and aberrant local fibrin turnover have likewise been shown in patients with chronic interstitial lung disease, including idiopathic pulmonary fibrosis, sarcoidosis, and hypersensitivity pneumonitis. Alveolar fibrin appears to be a key factor in triggering the fibroproliferative process in the lung. Delayed clearance of fibrin from the lung may promote fibroblast activation, proliferation, and tissue remodeling (i.e., replacement of the primary fibrin matrix by a secondary collagenous matrix associated with scarring and honeycombing). Thrombin also acts as a chemoattractant for inflammatory cells and fibroblasts and stimulates the release of proinflammatory cytokines. Moreover, it induces the release of growth factors and fibrogenic mediators, thereby stimulating fibroblast proliferation and connective tissue synthesis. Although the use of anticoagulants is not part of the standard clinical practice for these conditions, blockade of the extrinsic coagulation pathway and prevention of extravascular fibrin formation may be a promising approach for acute inflammatory and chronic interstitial lung disease. Experimental studies addressing anticoagulant intervention (e.g., by administration of APC, heparin, and direct thrombin inhibitor) in animal models of ALI and pulmonary fibrosis have been shown to

protect the lung and to reduce the fibroproliferative response. In addition, different anticoagulants are being tested in ongoing experimental and clinical studies. A phase II study of recombinant TFPI in patients with severe sepsis showed improvement in lung function and a trend toward improved survival in the subset of patients with ARDS. A follow-up phase III study, however, failed to demonstrate improved survival. Similarly, the beneficial effects of recombinant antithrombin observed in animal models could not be confirmed in humans. Heparin is being evaluated in humans with idiopathic pulmonary fibrosis. In a similar vein, heparin is considered an alternate agent in the treatment of allergic asthma. Glycosaminoglycans such as heparan sulfate are expressed as part of a proteoglycan on cell surfaces and are thought to play a role in the regulation of the inflammatory response (e.g., by binding chemokines). Heparin, which is synthesized by and stored exclusively in mast cells, has been found to exert anti-inflammatory effects in animal models and in human disease. Heparin binds and inhibits a variety of cytotoxic and inflammatory mediators, including eosinophilic cation protein and peroxidase. Furthermore, heparin has been associated with the inhibition of lymphocyte activation, neutrophil chemotaxis, smooth muscle growth, and vascular tone. The first clinical trial with inhaled heparin was performed in 1969, but further human studies using inhaled heparin were not published until the early 1990s. Three smaller studies of patients with exercise-induced asthma showed that inhaled heparin preserved specific airway conductance better than inhaled cromolyn or placebo following exercise. Studies investigating the effects of inhaled heparin on bronchial reactivity following inhaled allergen challenge have yielded mixed results.

Side Effects and Contraindications The most devastating complication of anticoagulant therapy is bleeding. The occurrence of bleeding, primarily in the gastrointestinal tract and the central nervous system, is a major cause of morbidity and mortality. Consequently, anticoagulants are contraindicated in any case in which the risk of hemorrhage may be greater than the potential clinical benefit of anticoagulation. Risk factors for bleeding include advanced age, serious illness (cerebral, cardiac, kidney, or liver disease), cerebrovascular or peripheral vascular disease, and an unstable anticoagulant effect. Whereas the action of unfractionated heparin may be antagonized by protamine sulfate, no specific antidote is available for LMW heparins, danaparoid, fondaparinux, or the direct thrombin inhibitors

128 ANTICOAGULANTS

hirudin and ximelagatran. The anticoagulant effect of coumarin derivatives may be antagonized by low doses of vitamin K and substitution with fresh frozen plasma. Other adverse effects of heparins are heparin-induced thrombocytopenia and osteoporosis, which can occur after long-term treatment and increase the risk for fractures. An absolute contraindication for coumarins is pregnancy because they cause fetal bone abnormalities by interfering with g-carboxylation of proteins synthesized in the bone. Heparins do not cross the placental barrier and do not cause these problems. Long-term use of ximelagatran carries the risk of severe liver injury. Administration in patients with hepatic disease is not recommended. As outlined previously, HIT is an antibody-mediated adverse effect of heparin associated with an increased thrombotic risk. According to the seventh American College of Clinical Pharmacology consensus conference guidelines, alternative, nonheparin anticoagulants should be used in patients with strongly suspected or confirmed HIT. Recommendations include direct thrombin inhibitors as well as danaparoid. Although withdrawn from the US market, danaparoid remains approved and available for prevention and treatment of HIT in Canada, Europe, Australia, and Japan. Because fondaparinux does not cross-react with HIT antibodies, it may also be useful for this indication. A general recommendation, however, cannot be made due to the minimal data available. Antihirudin antibodies are commonly generated during treatment with lepirudin. Although they are usually not clinically significant, the European Agency for the Evaluation of Medical Products recommended the use of nonhirudin anticoagulants in patients who have previously been exposed to lepirudin. Coumarins should not be used in patients with confirmed or strongly suspected HIT because they can contribute to skin necrosis and venous limb gangrene. For patients receiving coumarins at the time of diagnosis of HIT, the use of vitamin K for reversal of coumarin-induced anticoagulation is recommended. See also: Acute Respiratory Distress Syndrome. Coagulation Cascade: Overview; Antithrombin III;

Antioxidants

Factor X; Protein C and Protein S; Thrombin. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Thrombolytic Therapy.

Further Reading Ansell J, Hirsh J, Poller L, et al. (2004) The pharmacology and management of the vitamin K antagonists: the seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 204S–233S. Bussey H, Francis JL, and the Heparin Consensus Group (2004) Heparin overview and issues. Pharmacotherapy 24: 103S–107S. Desai UR (2004) New antithrombin-based anticoagulants. Medical Research Reviews 24: 151–181. Ginsberg JS, Greer I, and Hirsh J (2001) Use of antithrombotic agents during pregnancy. Chest 119: 122S–131S. Happel KI, Nelson S, and Summer W (2004) The lung in sepsis: Fueling the fire. American Journal of the Medical Sciences 328: 230–237. Hirsh J and Raschke R (2004) Heparin and low-molecular-weight heparin: The seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 188S–203S. Hirsh J, Fuster V, Ansell J, and Halperin JL (2003) American Heart Association/American College of Cardiology Foundation guide to warfarin therapy. Circulation 107: 1692–1711. Idell S (2001) Anticoagulants for acute respiratory distress syndrome. Can they work? American Journal of Respiratory and Critical Care Medicine 164: 517–520. Idell S (2002) Adult respiratory distress syndrome: Do selective anticoagulants help? American Journal of Respiratory Medicine 1: 383–391. Laterre PF, Wittebole X, and Dhainaut JF (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31(supplement): S329–S336. Nowak G (2002) Pharmacology of recombinant hirudin. Seminars in Thrombosis and Hemostasis 28: 415–423. Srivastava S, Goswami LN, and Dikshit DK (2005) Progress in the design of low molecular weight thrombin inhibitors. Medical Research Reviews 25: 66–92. Warkentin TE and Greinacher A (2004) Heparin-induced thrombocytopenia: Recognition, treatment, and prevention. The seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 311–337. Weitz JI (1997) Low-molecular weight heparins. New England Journal of Medicine 337: 688–698. Weitz JI, Hirsh J, and Samama MM (2004) New anticoagulant drugs. The seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 265S–286S. Wittkowsky AK (2003) Warfarin and other coumarin derivatives: Pharmacokinetics, pharmacodynamics, and drug interactions. Seminars in Vascular Medicine 3: 221–230.

see Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic.

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ANTIVIRAL AGENTS M A Parniak, E N Vergis, and M E Abram, University of Pittsburgh, Pittsburgh, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Most respiratory tract infections (RTIs) are viral in origin; numerous different viruses cause RTIs, many of which can be quite serious. However, only five drugs are approved for their treatment, and these drugs are directed at only two viruses. Four drugs – amantadine, rimantadine, zanamivir, and oseltamivir – are used for the prophylaxis and treatment of influenza virus infection. These drugs target two different stages of influenza replication. Orally administered amantadine and rimantadine act only against influenza A and block the ion channel formed by the viral M2 protein, thereby preventing viral ribonucleoprotein release. Zanamivir and oseltamivir are active against influenza A and B, and they are competitive inhibitors of the viral neuraminidase. These drugs block nascent viral release from the infected cell and thus viral spread by preventing neuraminidase-catalyzed removal of sialic acid residues from membrane glycoproteins. Oseltamivir is administered orally, whereas zanamivir must be delivered topically by inhalation of the dry powder. The fifth approved drug, the ribonucleoside analog ribavirin, is administered by nebulization and is approved for the treatment of pediatric respiratory syncytial virus infection. Its mechanism of action is uncertain, but it may involve alteration of cellular nucleotide pools and inhibition of viral RNA synthesis.

Introduction Viruses are the cause of most respiratory tract infections (RTIs); numerous different viruses can infect the respiratory tract. Many of these infections can be quite serious, especially in the young, the elderly, chronically ill, and immunocompromised individuals. However, there are only five drugs approved for the treatment of viral RTIs, and these drugs are directed at only two viruses. Four of the approved drugs are used for the treatment or prophylaxis of influenza, and one is used for the treatment of pediatric respiratory syncytial virus infection.

Drugs for the Treatment of Influenza Influenza virus mainly infects the upper respiratory tract, and most individuals recover within approximately 1 week without medical treatment. Nevertheless, RTI due to influenza is a major cause of morbidity and mortality worldwide. The World Health Organization estimates that influenza can account for up to 15% of worldwide upper RTIs

annually, with up to 500 000 deaths. Influenza viruses that cause human disease are classified into groups A–C. Influenza A is the most serious pathogen since it leads to large recurrent epidemics with significant morbidity and mortality. Four drugs (Figure 1) – the adamantane derivatives amantadine and rimantadine and the neuraminidase inhibitors zanamivir and oseltamivir – are used for the treatment or prophylaxis of influenza virus infection. These drugs target different stages of influenza virus replication (Figure 2). Amantadine and rimantadine inhibit an early stage involved in the uncoating of the viral ribonucleoprotein, whereas zanamivir and oseltamivir inhibit a viral enzyme needed to allow nascent virus release from the infected cell. Amantadine and Rimantadine

The inhibitory activity of amantadine (adamantan-1ylamine hydrochloride) against influenza A was first identified in 1964, and this drug was first marketed in 1966. The close analog, rimantadine (1-adamantan-1-yl-ethylamine hydrochloride), was developed subsequently due to the adverse neurological side effects associated with the use of amantadine. These structurally similar compounds are used primarily for prophylaxis of influenza A infection, particularly for individuals with high risk of exposure such as healthcare workers, and prevent influenza-like illness in up to 80% of treated individuals. Both drugs can also reduce the duration of symptoms of established influenza A infection if therapy is initiated within 48 h of discernible symptoms. The adamantanes are effective only against influenza A. However, since influenza A is the source of most serious cases of influenza-related RTI, the adamantanes are valuable drugs for the treatment and especially prophylaxis of influenza infections. Amantadine and rimantadine are administered orally, once or twice daily with adult dosages of 100–200 mg per day. For prophylaxis, the drugs are administered for up to 7 days during high-risk periods of an epidemic. The two drugs have very different pharmacokinetic profiles despite their similarity in structure. Amantadine is rapidly absorbed after oral delivery, has a half-life of approximately 15 h, is not metabolized, and is excreted almost entirely in the urine. In contrast, rimantadine is more slowly absorbed, has a half-life twice that of amantadine, and undergoes extensive hepatic metabolism. Although peak plasma levels of rimantadine are less than those

130 ANTIVIRAL AGENTS H3C

NH2 HCl

NH2 HCl

Rimantadine

Amantadine

O

OH

O

OH

NH

O H

H

HO N H

NH2

NH2

O HN

HN OH OH

O

Zanamivir

O

Oseltamivir

Figure 1 Drugs used for prophylaxis or treatment of influenza virus infection. The drug oseltamivir is shown as the active caboxylic acid form, although it is administered as the ethyl ester pro-drug.

of amantadine, the former is more highly concentrated in respiratory secretions than amantadine, thereby providing better access to the viral target. Mechanism of action Amantadine and rimantadine target the influenza A virus M2 membrane protein. The influenza virus enters the cell via endocytosis, and fusion of the virus membrane envelope with the endosome membrane provides a mechanism for the release of the viral ribonucleoprotein (RNP) into the cytoplasm and thus to the nucleus, where replication of the viral genomic RNA can take place. The RNP interacts with the viral M1 protein that underlies the viral envelope. Furthermore, M1 masks a nuclear localization signal on the RNP. Unless the M1–RNP interactions are disrupted, the RNP can neither be released into the cytoplasm nor imported into the nucleus. Disassembly of the complex may also be important for activation of the viral RNA polymerase. The influenza M2 membrane protein is critical in enabling disruption of the M1–RNP complex. The M2 protein is a homotetramer that is considered to form an ion channel in the viral membrane envelope (Figure 3). During acidification of the viruscontaining endosome, the M2 protein enables protons to enter the virus particle. Acidification of

the interior of the virus particle results in dissociation of the RNP from the M1 protein. This allows release of the RNP into the cytoplasm and subsequent import into the nucleus. Amantadine and rimantadine bind to the viral M2 membrane protein, blocking the channel and preventing proton transport into the viral particle. The M2 protein is not found in influenza B virus, which accounts for the lack of antiviral activity of amantadine and rimantadine against influenza B. Contraindications Central nervous system side effects are noted in significant numbers (up to 10%) of amantadine-treated individuals. Symptoms include dizziness, anxiety, difficulty in concentrating, and insomnia, all of which are problematic for healthcare workers, a major target group for prophylactic treatment during influenza epidemics. Central nervous system side effects reverse once treatment is stopped. Severe toxic reactions or death can occur in patients with renal insufficiency and include serious neurotoxicity (mental status changes, hallucinations, tremor, myoclonus, seizures, and coma) or cardiac arrhythmias. Dosages of the drugs, especially amantadine, must be lowered and carefully monitored in such patients. Rimantadine is significantly better tolerated than amantadine.

ANTIVIRAL AGENTS 131

Zanamivir, oseltamivir

Amantadine, rimantadine

Figure 2 Influenza virus replication cycle showing stages inhibited by approved therapeutic drugs. Following attachment, endocytosis, and acidification of the virion-containing endosome, the virion core is also acidified in a process mediated by the viral M2 membrane proton channel protein. This enables a pH-dependent release of viral ribonucleoprotein (RNP). Adamantane drugs target the virus M2 protein, preventing virion acidification and release of RNP. Nascent virus release from an infected cell requires cleavage of terminal sialic acid residues from cell surface and virion envelope proteins. Neuraminidase inhibitors target this stage of replication, preventing spread of virus to uninfected cells.

Neuraminidase Inhibitors

The viral neuraminidase inhibitors zanamivir (5-acetylamino-4-gunadino-6-(1,2,3-trihydroxypropyl)-5,6dihydro-4H-pyran-2-carboxylic acid) and oseltamivir (4-acetylamino-5-amino-3-(1-ethylpropoxy)-cyclohex1-ene carboxylic acid) were approved for therapeutic use in 1999. Both drugs are reversible competitive inhibitors of influenza virus neuraminidase. The drugs are effective against both influenza A and influenza B and also against strains of influenza A virus resistant to

the adamantane drugs. Zanamivir and oseltamivir have virtually no activity against human cell neuraminidases at levels that provide potent antiviral activity. Viral resistance to zanamivir or oseltamivir is uncommon. Both drugs are approved for treatment of influenza in adults and children (1 year or older for oseltamivir and 7 years or older for zanamivir) within 48 h of onset of discernible symptoms. Oseltamivir can also be used for prophylaxis in adults. Zanamivir has low oral bioavailability and is administered topically by dry powder inhalation, whereas oseltamivir can be

132 ANTIVIRAL AGENTS H+

NH2HCl V

V H

A S H

Viral membrane envelope

H

G

A S G

M2 protein subunits Figure 3 Schematic of the mechanism of adamantane drug inhibition. The drugs interact with specific residues of the influenza A virus M2 transmembrane protein. This protein forms a channel for the influx of protons into the virion. Only two of the four M2 subunits that form the channel are shown. Binding of amantadine or rimantadine blocks the channel, preventing intravirion acidification.

administered orally (bioavailability 460%, with a plasma half-life of approximately 8 h). Oseltamivir is not metabolized and is excreted almost entirely in the urine. Oseltamivir is administered as the ethyl ester pro-drug that lacks antiviral activity. This is hydrolyzed to the active oseltamivir carboxylate by esterases in the intestinal mucosal epithelia and in the liver. Mechanism of action Zanamivir and oseltamivir target the viral enzyme neuraminidase. Neuraminidase, also called sialidase, is one of the two major influenza viral proteins (the other is hemagglutinin, which is important for binding and fusion of the virus envelope with the host cell). Neuraminidase is essential for release of newly formed virus from infected cells and for virus spread throughout the respiratory tract of the infected host. Nascent virions bud off from the infected cell encased in an envelope composed of a lipid bilayer derived from the cell plasma membrane with associated viral envelope proteins. Many cell surface glycoproteins of mammalian cells possess sialic acid as the terminal sugar, a residue added during glycosylation events in subcellular Golgi organelles. Influenza virus envelope proteins are formed in the same subcellular compartment and are therefore also sialylated. However, the cell surface receptor for influenza virus attachment is sialic acid. Removal of the cell surface and viral envelope protein sialic acid residues is essential to prevent reattachment of the nascent virions

to the same cell and to prevent aggregation of the virus particles. Neuraminidase cleaves terminal sialic acid residues from cellular and viral membrane glycoproteins, thus destroying the cellular receptors recognized by the viral hemagglutinin. Influenza neuraminidase is a tetramer of identical subunits (Figure 4). The active site in each subunit is a deep groove on the protein surface that comprises residues that are apparently identical in all strains of influenza A and B. The inhibitors zanamivir and oseltamivir result from rational drug design based on the neuraminidase sialic acid-binding site determined by crystallography. Both drugs bind with high affinity to the active site of viral neuraminidase (Figure 4), thus preventing binding of the normal sialic acid glycoprotein substrate. However, the inhibition mechanisms of the two drugs differ. Zanamivir is an analog of sialic acid and acts as a classical competitive inhibitor to prevent substrate binding to the unliganded enzyme. Oseltamivir is a transition-state analog of sialic acid cleavage and thus interferes with neuraminidase conformational changes needed to allow substrate binding. Nonetheless, the result is the same, and both drugs have similar antiviral potency. Contraindications Few serious clinical toxicities have been reported with zanamivir or oseltamivir. Inhaled zanamivir is generally well tolerated, although exacerbations of asthma may occur. Orally administered oseltamivir may cause nausea and

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Figure 4 Structure of influenza B virus neuraminidase bound with the inhibitor zanamivir (in red). The structure was based on pdb1A4G. Adapted from Taylor NR, Cleasby A, Singh O et al. (1998) Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of influenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran-6-carboxamides and sialidase from influenza virus types A and B. Journal of Medicinal Chemistry 41: 798–807.

gastrointestinal discomfort, although these are transient and less likely if the drug is administered with food.

NH2 O N

Drug for the Treatment of Respiratory Syncytial Virus Infection

N N O

Ribavirin HO

The synthetic nucleoside ribavirin (1-(3,4-dihydroxy5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-[1,2,4]triazole-3-carboxylic acid amide) is a guanosine analog that is approved for the treatment of pediatric respiratory syncytial virus (RSV) infection (Figure 5). RSV is the most common cause of severe lower respiratory tract disease in infants, causing up to 90% of bronchiolitis and 40% of bronchopneumonia cases. Disease symptoms are much milder in older children and adults. Although orally administered ribavirin is rapidly absorbed with good bioavailability (approximately 45%), treatment of pediatric RSV infection requires the drug to be administered by continuous aerosol (nebulizer) for 12– 18 h daily for 3–6 days. This therapy is expensive and requires specialized equipment and monitoring. Its use is therefore generally reserved for treatment of RSV infection in high-risk infants (e.g., premature infants, immunocompromised children, or infants with congenital heart disease).

antiviral activity of ribavirin is unclear and may differ for different types of viruses. However, it is generally thought that ribavirin therapy alters cellular nucleotide pools, thereby affecting viral mRNA synthesis. It is likely that ribavirin must be phosphorylated by cellular kinases to provide antiviral activity. Ribavirin-50 -monophosphate inhibits the cellular enzyme inosine-50 -phosphate dehydrogenase, blocking the conversion of inosine-50 -monophosphate to xanthosine-50 -monophosphate. This decreases levels of guanosine triphosphate (GTP), which impacts on both RNA and DNA metabolism. Ribavirin-50 triphosphate may also be a competitive inhibitor of GTP-dependent 50 -capping of viral mRNA.

Mechanism of action Ribavirin is in fact a broadspectrum antiviral agent that inhibits a wide range of RNA and DNA viruses. The mechanism for the

Contraindications Aerosolized ribavirin may cause bronchospasm or conjunctival irritation. It requires close supervision, especially with mechanical

HO

OH

Figure 5 Structure of ribavirin, which is used for the treatment of high-risk pediatric respiratory syncytial virus infection.

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ventilation, because precipitation of the drug may occur. Healthcare workers involved in the administration and monitoring of aerosol ribavirin treatment may occasionally experience irritation of the eyes and respiratory tract. Ribavirin is mutagenic, teratogenic, and embryotoxic; thus, aerosolized ribavirin is a risk to pregnant healthcare workers. See also: Aerosols. Bronchiolitis. Pneumonia: Viral. Upper Respiratory Tract Infection. Vaccinations: Viral. Viruses of the Lung.

Further Reading Abdel-Magid AF, Maryanoff CA, and Mehrman SJ (2001) Synthesis of influenza neuraminidase inhibitors. Current Opinion in Drug Discovery and Development 4: 776–791. Broughton S and Greenough A (2004) Drugs for the management of respiratory virus infection. Current Opinion in Investigative Drugs 5: 862–865. De Clercq E (2004) Antiviral drugs in current clinical use. Journal of Clinical Virology 30: 115–133. Dreitlein WB, Maratos J, and Brocavich J (2001) Zanamivir and oseltamivir: two new options for the treatment and prevention of influenza. Clinical Therapeutics 23: 327–355.

Garman E and Laver G (2004) Controlling influenza by inhibiting the virus’s neuraminidase. Current Drug Targets 5: 119–136. Johnston SL (2002) Anti-influenza therapies. Virus Research 82: 147–152. Kandel R and Hartshorn KL (2001) Prophylaxis and treatment of influenza virus infection. BioDrugs 15: 303–323. Lew W, Chen X, and Kim CU (2000) Discovery and development of GS4104 (oseltamivir): an orally active influenza neuraminidase inhibitor. Current Medical Chemistry 7: 663–672. Oxford JS, Bossuyt S, Balasingam S, et al. (2003) Treatment of epidemic and pandemic influenza with neuraminidase and M2 proton channel inhibitors. Clinical Microbiology and Infection 9: 1–14. Snell NJC (2001) New treatments for viral respiratory tract infections – opportunities and problems. Journal of Antimicrobial Chemotherapy 47: 251–259. Tam RC, Lau JY, and Hong Z (2001) Mechanisms of action of ribavirin in antiviral therapies. Antiviral Chemistry and Chemotherapy 12: 261–272. Taylor NR, Cleasby A, Singh O, et al. (1998) Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of influenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran6-carboxamides and sialidase from influenza virus types A and B. Journal of Medicinal Chemistry 41: 798–807. Torrence PF and Powell LD (2002) The quest for an efficacious antiviral for respiratory syncytial virus. Antiviral Chemistry and Chemotherapy 13: 325–344.

APOPTOSIS A M K Choi and X Wang, University of Pittsburgh, Pittsburgh, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The phenomenon of programmed cell-death, which is more commonly known as apoptosis, was first described by C Vogt in 1842. Apoptosis, the term was proposed by Kerr and colleagues in 1972, is an active process of cellular self-destruction with distinctive morphological and biochemical features . It is indispensable in the development and maintenance of homeostasis within all multicellular organisms. The molecular machinery of apoptosis is also important for the regulation of homeostasis during adulthood, which is important for the control of neoplasia and autoimmunity. Understanding how apoptosis occurs in different situations will help to understand the pathogenesis of a number of human diseases and therefore provide clues to the treatment.

Apoptotic Pathways Caspases are involved in various programmed celldeath pathways reported in the literature. They are a

family of cysteine proteases, and many of them are implicated as important initiators or effectors of the apoptosis process. To date, at least 14 members of this family have been identified, but only a subset of them are partially or fully characterized. Like many enzymes, caspases are synthesized in the cell as inactive precursors and can be cleaved to active form comprised of two associated heterodimers. They are activated or inactivated through a series of intracellular steps, or pathways, in response to death or survival signals, which are subject to multiple regulations. Clearly, the discovery of diverse apoptosis pathways involving signals primarily via the death receptors (extrinsic pathway) or the mitochondria (intrinsic pathway) using caspases as effector molecules has dominated the field. Lately, a more sophisticated view of signaling pathways has arisen which leads to the observation that cell death can occur even in the absence of caspases. The death receptor pathway. The extrinsic pathway is initiated at the cell surface through the

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and caspase-8 are the key components of the Fas DISC. Once caspase-8 associates with FADD, the high local concentration of caspase-8 is believed to lead to its autoproteolytic cleavage to p43/41 and p20, and activation. Studies of knockout mice and mutant cell lines established that the DISC is essential for Fas apoptosis signaling. DISC assembly differs between cell types in ways that influence the efficiency of Fas signaling. In ‘type I’ cell lines and re-stimulated primary T cells, the DISC forms efficiently, and apoptosis can be induced with bivalent anti-Fas stimuli without additional cross-linking. The activated caspase-8 directly cleaves caspase-3 in its downstream. However, in ‘type II’ cell lines and recently activated primary T cells, the DISC is formed inefficiently, hypercrosslinking of Fas is necessary to induce apoptosis, and caspase-8 does not activate caspase-3 and cleaves Bid directly instead. The truncated Bid (tBid) translocates to mitochondria (Figure 1). TNF receptor type I (TNFR1) is another important member in the death receptor family. TNF is a highly pleiotropic cytokine that elicits diverse cellular responses ranging from proliferation and differentiation to activation of apoptosis. The different biological activities are mediated by two distinct cell surface receptors: TNFR1 and TNFR2. TNFR1 appears to be the key mediator of TNF signaling. Upon binding of its ligand, TNFR1 recruits the adaptor protein TRADD directly to its cytoplasmic DD. In turn, TRADD serves as an assembly platform to diverge TNFR1 signaling from the DD: interaction of

Fas/TNF-R1 family protein. Ligation of Fas either by its ligand, FasL, or by its agonistic antibodies triggers the homotrimeric association of the receptors. The clustering of the death domains (DDs) in the intracellular portion of the receptors recruits the adapter molecule, Fas-associated DD containing protein (FADD), which then recruits procaspase-8. Activation of procaspase-8 through self-cleavage leads to a series of downstream events, including activation of procaspase 3, cleavage of multiple caspase substrates, and induction of mitochondrial damages. Fas (CD95/APO-1) is the best-characterized member of the tumor necrosis factor (TNF) superfamily of receptors. Its main and best-known function in signaling is the induction of apoptosis. Fas receptors are expressed on the surface of cells as preassociated homotrimers. Fas is a prototype death receptor characterized by the presence of an 80 amino acid DD in its cytoplasm tail. This domain is essential for the recruitment of a number of signaling components upon activation by either agonistic anti-Fas antibodies or the cognate Fas ligand that initiate apoptosis. The complex of proteins that form upon triggering of Fas is called the death-inducing signaling complex (DISC). The DISC consists of an adaptor protein and initiator caspases and is essential for induction of apoptosis. A number of proteins have been reported to regulate formation or activity of the DISC. In the DISC, the adaptor molecule FADD is bound to Fas. FADD has been shown to interact with several proteins through its death effector domain (DED), including caspase-8. These two molecules, FADD

FasL Fas Membrane

}

FADD

Caspase-8 Activated caspase-8

DISC

Bid

Bax

tBid Activated-Bax Mitochondria

Cytochrome c Caspase-9 Caspase-3 Cell death Figure 1 A schematic diagram outlining the cell death pathways.

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TRADD with RIP-1 and TRAF-2 leads to nuclear factor kappa B (NF-kB) activation. Alternatively, TRADD can recruit FADD and procaspase-8, which is subsequently activated to initiate apoptosis. Under native conditions, a controversy has been reported about the formation of a TNFR1-associated DISC at the plasma membrane. It was reported that TNFR1-induced apoptosis involves two sequential signaling complexes. The initial plasma membrane bound complex (complex I) consists of TNFR1, the adaptor TRADD, the kinase RIP1, and TRAF2 and rapidly signals activation of NF-kB. In a second step, TRADD and RIP1 associate with FADD and caspase-8, forming a cytoplasmic complex (complex II) in human fibrosarcoma cells, HT1080. Recently, it has been reported that the DISC forms at the plasma membrane in several cell lines. We agree with this report in that the difference between the results of Micheau and co-workers and Harper and co-workers may be based on the different compositions of the lysis buffers used and the fact that different labeling and precipitation protocols are employed; we also discovered that the DISC formed at the plasma membrane in primary cultured hepatocytes (our unpublished results), which is consistent with the results of Schneider-Brachert and co-workers. The mitochondrial pathway. Many death stimuli do not seem to depend on the death receptor pathway. Instead, the death signals are transmitted to mitochondria through unique intracellular signaling pathways, where release of cytochrome c is induced. Cytochrome c activates Apaf-1, in the presence of dATP, which in turn activates procaspase-9. Activated caspase-9 can then cleave downstream effector caspases. Mitochondria apoptosis pathway is involved in many types of cell death induced by stress signals, such as irradiation, DNA-damaging drugs, hormone, or growth factor withdrawal. It has to be pointed out that cytochrome c release and caspase activation may not be the only effects caused by the various insults on mitochondria. Others include mitochondrial depolarization and free-radical generation. Death stimuli transmitted through the Fas/TNFR1 death receptor family are mainly mediated directly by caspase cascades in cytosol. However, in certain types of cells, such as hepatocytes, the effector caspases may not be efficiently activated by caspase-8 and thus the mitochondria pathway mediated by Bid becomes critical. As mentioned above, Bid is cleaved by caspase-8 and translocated to mitochondria to induce cytochrome c release. The mitochondria pathway is subject to regulation by Bcl-2 family proteins. This family of proteins consists of both death antagonists (Bcl-2, Bcl-XL, Bcl-W, Bfl-1, and Mcf-1) and death agonists (Bax, Bak, Bid,

Bim, Bnip3, Bnip3L (Nix), Bad, Bik, and Bok). They share structural homology in Bcl-2 homology (BH)1, 2, 3, and 4 domains, although not all members have all domains. The BH1 and BH2 domains of the antagonists are required to heterodimerize with the death agonists and repress cell death. Conversely, the BH3 domain of the death agonists is required for them to heterodimerize with the death antagonists and to promote cell death. Bcl-2 family proteins can regulate caspase activation through the regulation of cytochrome c release from mitochondria, which is inhibited by the death antagonists (Bcl-2 or Bcl-XL), and promoted by the death agonists (Bax or Bid). However, the exact mechanisms by which Bcl-2 proteins modulate apoptosis are still subject to much debate and controversy. One hypothesis is that both proapoptotic and antiapoptotic Bcl-2 proteins bind directly to components of the mitochondrial pore, leading to either its opening or closure, respectively. A second hypothesis is that upon activation, proapoptotic members such as Bax and Bak insert into the outer mitochondrial membrane where they oligomerize to form a protein-permeant pore of their own. Regulation of Bcl-2 proteins can occur at multiple levels. For example, Bid is cleaved by caspase-8 to form tBid, which then translocates to the mitochondrion and induces permeability transition, suggesting a linkage between the extrinsic and intrinsic pathways. In the context of signal transduction, phosphorylation also plays a critical role in regulating Bcl-2 proteins. In general, serine phosphorylation of Bad by multiple kinases causes its sequestration and hence inactivation by 14-3-3 proteins, and phosphorylation of Bim can target it for proteosomal degradation (13). The other apoptosis is the endoplasmic reticulum (ER)-involved pathway. As mentioned above, two major apoptotic cascades are triggered by specific initiator caspases: the death receptor pathway and the mitochondrial pathway. These are activated by caspase-8 and caspase-9, respectively. The key caspase in the ER pathway is caspase-12. Proper folding of polypeptide into a three-dimensional structure is essential for cellular function, and protein malfolding can threaten cell survival. Various conditions can perturb the protein folding in the ER, which is collectively called ER stress. In order to adapt ER stress conditions, the cells respond in three distinct ways such as transcriptional induction of ER chaperones, translational attenuation, and ER-associated degradation. After ER functions are severely impaired, the cell is eliminated by apoptosis via transcriptional induction of CHOP/GADD153, the activation of c-Jun NH2-terminal kinase, and/or the activation of caspase-12. This type of cellular stress is receiving increased attention because it is considered a cause of

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pathologically relevant apoptosis and is especially implicated in neurodegenerative disorders. ER stress activates caspase-12 on the surface of the ER, and caspase-12-deficient cells are resistant to ER stress inducers, indicating that caspase-12 is significant in ER stress-induced apoptosis. However, the mechanism responsible for caspase-12 activation is largely unknown, unlike in the case of both caspase-8 and caspase-9 whose activation mechanisms have been revealed at the molecular level.

Organelle Dysfunction Mitochondria. As the main energy source for all cellular processes, the proper function of the mitochondrion is important to the survival of the cell. The dysfunction of this organelle can usually be attributed to depolarization of the membrane, resulting in the loss of its property of selective mitochondrial membrane permeability (MMP), usually resulting in cell death. Many proapoptotic signals and antiapoptotic defenses converge in the mitochondrion. The congregation of proteins like Bax on the outer membrane cause alteration in the membrane permeability and compromise the integrity of the mitochondrial membrane, where MMP ensue. On the other hand, the mitochondrion can mount a defense through the antideath members of the Bcl-2 family, namely Bcl-2 and Bcl-XL. For example, these proteins usually reside at the mitochondrial membrane and can inhibit the function of Bax and consequently prevent the onset of MMP. It has been reported that caspase-8 resides in mitochondria. In the human breast carcinoma cell line MCF7, caspase-8 predominantly colocalizes with and binds to mitochondria. After induction of apoptosis through Fas or TNFRI, active caspase-8 translocate to plectin, a major cross-linking protein of the three main cytoplasmic filament systems, whereas the caspase-8 prodomain remain bound to mitochondria. Golgi apparatus. To date, there is no direct evidence suggesting that the Golgi apparatus is directly involved in the initiation of any apoptotic pathway. However, it has been reported that many apoptosissignaling proteins are enriched at the Golgi membrane. Among them are caspase-2, TNFR, Fas, and TNF-related apoptosis-inducing ligand receptor 1. Recently, we have found that the DISC (Fas/FADD/ caspase-8) forms first in Golgi complex, and then translocates to plasma membrane. The blockage of the DISC transport dramatically decreases the DISC level in plasma membrane. Plasma membrane. The plasma membrane is the first line of defense for the cell against extracellular

stimuli. With the right type of stimulus, the corresponding receptor will instigate a signal cascade that results in cell death. To date, three different types of death receptors have been identified: the TNFR, the Fas, and the APO-3. There was a report that Fas in type I lymphocytes associates with glycosphingolipid-enriched detergent-resistant membrane microdomains termed lipid rafts. Although there is a controversy if the DISC formation is involved in lipid raft, we have found that the DISC formation localizes in the lipid raft of lung endothelial cells exposed to hypoxia/reoxygenation. The lipid-based machinery may be involved in the formation of carriers trafficking from the Golgi complex to the cell surface.

Bcl-XL It is well known that Bcl-XL protects cells from apoptosis mediated by a variety of stimuli, but the mechanisms are not fully understood. Overexpression of Bcl-XL reportedly confers protection upon mitochondria, making it more difficult for numerous stimuli to induce permeability transition pore opening; or Bcl-XL tends to form small channels that assume a mostly closed conformation, preferring cations; Bcl-XL sequesters BH3 domain-only molecules in stable mitochondria complexes, preventing the activation of Bax. Bcl-XL was reported to block Bax translocation. Studies by others reported that Bcl-XL could not block activation of Fas by FasLinduced apoptosis, especially caspase-8 activation. A role for caspase-8 mediating Bid cleavage accompanied by cytochrome c release was observed in focal cerebral ischemia endothelial cells and focal cerebral ischemia in rats. It has been established that Bcl-XL blocks Bid cleavage and functions downstream of caspase-8 to inhibit Fas-induced apoptosis of MCF7 breast carcinoma cells. Recently, we have found that Bcl-XL inactivates caspase-8 by disrupting DISC formation in the plasma membrane, Golgi complex, and nucleus. Bcl-XL retains the DISC in mitochondria where caspase-8 is inactivated. Bcl-XL breaks the physical association of Fas and caspase-8 with GRASP65, a Golgi-apparatus-related protein. This indicates that Bcl-XL downregulates the transfer of DISC to the plasma membrane by the Golgi component, at the same time diverting the DISC formation to the mitochondria. FLIP (FLICE (Fas-associated death-domain-like IL-1beta-converting enzyme)-inhibitory protein)) was identified as an inhibitor of Fas signals. At least four splice variants have been identified (31–34). The largest variant FLIP long (FLIPL), a protein of 440 amino acids, is highly homologous to caspase-8. In fact, FLIPL contains a

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caspase-like domain at the carboxy-terminus but, as indicated by the name, was originally characterized as a molecule with inhibitory activity on caspase-8, in which FLIPL inhibits the final cleavage between the prodomain and the p20 subunit of the p43/41 intermediate. In addition to inhibition of receptor-mediated apoptosis, more recently, it has been reported that FLIP also promotes activation of NF-kB and Erk signaling pathway. Therefore, FLIP is not simply an inhibitor of death-receptor-induced apoptosis but also mediates the activation of NF-kB. The death effector domain (DED) of FLIP binds to Fas/FADD complexes and inhibits the recruitment and activation of procaspase-8 and therefore acts as antiapoptotic molecule. We have reported that, in addition to inhibiting the recruitment of caspase-8 into the DISC by decreasing DISC formation in the plasma membrane, FLIP blocks the transfer of the DISC formed in the Golgi to the plasma membrane. FLIP expression also inhibits Bax activation and Bax-induced apoptotic cell death by promoting the association of the inactive form of PKC to Bax, which inactivates Bax.

The Effect of Tyrosine Kinases on Apoptosis Tyrosine kinases are important mediators of the signaling cascade, determining key roles in diverse biological processes such as growth, differentiation, metabolism, and apoptosis in response to external and internal stimuli. Tyrosine kinases are a family of enzymes, which catalyze phosphorylation of select tyrosine residues in target proteins, using ATP. Tyrosine kinases are primarily classified as receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR) and c-Met, and nonreceptor tyrosine kinases, such as SRC, ABL, FAK, and Janus kinase. The receptor tyrosine kinases are not only cell surface transmembrane receptors, but also enzymes having kinase activity. The structural organization of the receptor tyrosine kinase exhibits a multidomain extracellular ligand for conveying ligand specificity, a single pass transmembrane hydrophobic helix, and a cytoplasmic portion containing a tyrosine kinase domain. The kinase domain has regulatory sequence both on the N- and C-terminal end. Nonreceptor tyrosine kinases are cytoplasmic proteins, exhibiting considerable structural variability. The nonreceptor tyrosine kinase has a kinase domain and often possesses several additional signaling or protein–protein interacting domains such as Src homology (SH)2, SH3, and the phosphotyrosine homology (PH)

domain. Generally, receptor tyrosine kinases activate many signaling pathways to inhibit apoptosis by regulating Bcl-2 family member protein expression and their phosphorylation. For example, receptor tyrosine kinases activate the serine/threonine kinase Akt, which inhibits apoptosis through the Bad phosphorylation. Here, we focus on recent novel findings that tyrosine kinases are involved in apoptotic regulation. The initiation of Fas-mediated apoptotic pathway is to stimulate Fas activities through Fas phosphorylation. The phosphorylation at the Fas tyrosine is thought to be prerequisite for Fas membrane trafficking and DISC formation in hepatocytes exposed to hyperosmolarity and Fas ligand in endothelial cells of the lung exposed to hypoxia/reoxygenation (our unpublished results). EGFR is reported to associate with Fas and induce Fas phosphorylation at the tyrosine site through EGFR tyrosine kinase activity. Hepatocyte growth factor (HGF) receptor, c-Met, can inhibit Fas-mediated apoptosis by physically binding to Fas, which may block the Fas conformation change required for trafficking and DISC formation in hepatocytes. HGF can upregulate the c-Met/Fas association through c-Met-signaling pathway in lung epithelial and endothelial cells (our unpublished results). HGF also inhibits Bax and Bid activation by activating p38 MAPK that induces Bax and Bid phosphorylation, which decreases their conformation change and activation. The Src kinase family comprises three ubiquitously expressed members (Src, Fyn, Yes), which share functional domains such as an amino-terminal myristoylation sequence for membrane targeting, SH2 and SH3 domains, a family member-specific unique region, and a kinase domain and a carboxy-terminal noncatalytic domain. Recent studies on vascular smooth muscle cells and endothelial cells indicated an involvement of c-Src, but not of Fyn, in the c-Jun NH2-terminal kinase (JNK) activation in response to oxidative stress. Also, oxidative stress-induced activation of Erk-5 in fibroblasts was shown to be Src-dependent, but not Fyn-dependent, whereas Fyn, but not Src, was required for activation of p90 ribosomal S6 kinase by reactive oxygen species. It has been reported that hydrophobic bile acids rapidly activate Yes but not Fyn, Src, or Lck. Activated Yes associates with the EGFR in a protein kinase A-sensitive way and acts as an EGFR-activating kinase, thereby triggering a decisive event in bile acid-induced apoptosis. Phosphorylation of the p53 tumor suppressor protein is a critical event in the upregulation and activation of p53 during cellular stress. It was demonstrated that the signaling pathway linking oxidative stress to p53 through PDGFb receptor. Hydrogen peroxide-induced phosphorylation of the

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PDGFb receptor and p53 activation was inhibited by kinase-inactive forms of the PDGFb receptor.

Concluding Remarks Apoptosis plays an important role in cell growth, differentiation, and homeostasis. It is obvious that our current knowledge in terms of the mechanism of apoptosis and its regulation is far from complete. For example, we do not have conclusive answers to questions such as: how does the DISC formation transport from Golgi complex to plasma membrane? What is the importance/significance of the DISC formed first in Golgi complex? How does FLIP affect the Bax/mitochondria apoptotic pathway? What is the mechanism of the dual function (antiapoptosis and proapoptosis) of tyrosine kinases? Much more effort is required to dissect and document these pathways. Studies of the apoptotic regulation will help understand the mechanisms of cell death or cancer development. We must better characterize the novel pathways of cell death and further our understanding of the pathologies underlying a variety of human health problems. See also: CD14. Cysteine Proteases, Cathepsins. DNA: Repair. Extracellular Matrix: Collagens. Hepatocyte Growth (Scatter) Factor. Ion Transport: Overview. Myofibroblasts. NADPH and NADPH Oxidase. Oncogenes and Proto-Oncogenes: jun Oncogenes. Transcription Factors: AP-1; NF-kB and Ikb. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Anto RJ, Mukhopadhyay A, Denning K, and Aggarwal BB (2002) Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl. Carcinogenesis 23: 143–150. Bin L, Li X, Xu LG, and Shu HB (2002) The short splice form of casperc-FLIP is a major cellular inhibitor of TRAIL-induced apoptosis. FEBS Letters 510: 37–40. Cao XX, Mohuiddin I, Chada S, et al. (2002) Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL. Molecular Medicine 8: 869–876. Chen K, Albano A, Ho A, and Keaney JF Jr (2003) Activation of p53 by oxidative stress involves platelet-derived growth factorbeta receptor-mediated ataxia telangiectasia mutated (ATM) kinase activation. Journal of Biological Chemistry 278: 39527– 39533. Cheng EH, Wei MC, Weiler S, et al. (2001) BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAXand BAK-mediated mitochondrial apoptosis. Molecular Cell 8: 705–711. Creagh EM, Conroy H, and Martin SJ (2003) Caspase-activation pathways in apoptosis and immunity. Immunological Reviews 193: 10–21.

Eramo A, Sargiacomo M, Ricci-Vitiani L, et al. (2004) CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells. European Journal of Immunology 34: 1930–1940. Gniadecki R (2004) Depletion of membrane cholesterol causes ligand-independent activation of Fas and apoptosis. Biochemical and Biophysical Research Communications 320: 165–169. Harper N, Hughes M, MacFarlane M, and Cohen GM (2003) Fasassociated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis. Journal of Biological Chemistry 278: 25534–25541. Hsu H, Shu HB, Pan MG, and Goeddel DV (1996) TRADDTRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84: 299–308. Huang DC, Hahne M, Schroeter M, et al. (1999) Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-x (L). Proceedings of the National Academy of Sciences, USA 96: 14871–14876. Kataoka T, Budd RC, Holler N, et al. (2000) The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways. Current Biology 10: 640–648. Kerr JF, Wyllie AH, and Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26: 239–257. Kroemer G and Reed JC (2000) Mitochondrial control of cell death. Nature Medicine 6: 513–519. Krueger A, Schmitz I, Baumann S, Krammer PH, and Kirchhoff S (2001) Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex. Journal of Biological Chemistry 276: 20633–20640. Liou AK, Clark RS, Henshall DC, Yin XM, and Chen J (2003) To die or not to die for neurons in ischemia, traumatic brain injury and epilepsy: a review on the stress-activated signaling pathways and apoptotic pathways. Progress in Neurobiology 69: 103–142. Los M, Wesselborg S, and Schulze-Osthoff K (1999) The role of caspases in development, immunity, and apoptotic signal transduction: lessons from knockout mice. Immunity 10: 629–639. Micheau O and Tschopp J (2003) Induction of TNF receptor Imediated apoptosis via two sequential signaling complexes. Cell 114: 181–190. Momoi T (2004) Caspases involved in ER stress-mediated cell death. Journal of Chemical Neuroanatomy 28: 101–105. Muppidi JR and Siegel RM (2004) Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death. Nature Immunology 5: 182–189. Paul MK and Mukhopadhyay AK (2004) Tyrosine kinase – role and significance in Cancer. International Journal of Medical Science 1: 101–115. Peter ME and Krammer PH (2003) The CD95(APO-1/Fas) DISC and beyond. Cell Death and Differentiation 10: 26–35. Plesnila N, Zinkel S, Amin-Hanjani S, Qiu J, Korsmeyer SJ, and Moskowitz MA (2002) Function of BID – a molecule of the bcl2 family – in ischemic cell death in the brain. European Surgical Research 34: 37–41. Reed JC (1998) The Bcl-XL family proteins. Oncogene 17: 3225– 3226. Reinehr R, Schliess F, and Haussinger D (2003) Hyperosmolarity and CD95L trigger CD95/EGF receptor association and tyrosine phosphorylation of CD95 as prerequisites for CD95 membrane trafficking and DISC formation. FASEB Journal 17: 731–733. Scaffidi C, Fulda S, Srinivasan A, et al. (1998) Two CD95 (APO-1/ Fas) signaling pathways. The EMBO Journal 17: 1675–1687.

140 AQUAPORINS Scaffidi C, Schmitz I, Krammer PH, and Peter ME (1999) The role of c-FLIP in modulation of CD95-induced apoptosis. Journal of Biological Chemistry 274: 1541–1548. Schneider-Brachert W, Tchikov V, Neumeyer J, et al. (2004) Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles. Immunity 21: 415–428. Srinivasan A, Li F, Wong A, et al. (1998) Bcl-xL functions downstream of caspase-8 to inhibit Fas- and tumor necrosis factor receptor 1-induced apoptosis of MCF7 breast carcinoma cells. Journal of Biological Chemistry 273: 4523–4529. Stegh AH, Barnhart BC, Volkland J, et al. (2002) Inactivation of caspase-8 on mitochondria of Bcl-xL-expressing MCF7-Fas cells: role for the bifunctional apoptosis regulator protein. Journal of Biological Chemistry 277: 4351–4360. Stegh AH, Herrmann H, Lampel S, et al. (2000) Identification of the cytolinker plectin as a major early in vivo substrate for caspase 8 during CD95- and tumor necrosis factor receptor-mediated apoptosis. Molecular and Cellular Biology 20: 5665–5679. Szegezdi E, Fitzgerald U, and Samali A (2003) Caspase-12 and ERstress-mediated apoptosis: the story so far. Annals of the New York Academy of Sciences 1010: 186–194. Tschopp J, Irmler M, and Thome M (1998) Inhibition of fas death signals by FLIPs. Current Opinion in Immunology 10: 552–558. Wajant H, Pfizenmaier K, and Scheurich P (2003) Tumor necrosis factor signaling. Cell Death and Differentiation 10: 45–65.

Wang X, DeFrances MC, Dai Y, et al. (2002) A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Molecular Cell 9: 411–421. Wang X, Wang Y, Zhang J, et al. (2005) FLIP protects against hypoxia/reoxygenation-induced endothelial cell apoptosis by inhibiting Bax activation. Molecular and Cellular Biology 25: 4742–4751. Wang X, Zhang J, Kim HP, et al. (2004) Bcl-XL disrupts deathinducing signal complex formation in plasma membrane induced by hypoxia/reoxygenation. FASEB Journal 18: 1826–1833. Wang X, Zhou Y, Kim HP, et al. (2004) Hepatocyte growth factor protects against hypoxia/reoxygenation-induced apoptosis in endothelial cells. Journal of Biological Chemistry 279: 5237– 5243. Xiao C, Yang BF, Asadi N, Beguinot F, and Hao C (2002) Tumor necrosis factor-related apoptosis-inducing ligand-induced deathinducing signaling complex and its modulation by c-FLIP and PEDPEA-15 in glioma cells. Journal of Biological Chemistry 277: 25020–25025. Yin XM (2000) Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways. Cell Research 10: 161–167. Yin XM and Ding WX (2003) Death receptor activation-induced hepatocyte apoptosis and liver injury. Current Molecular Medicine 3: 491–508.

AQUAPORINS A S Verkman and Y Song, University of California, San Francisco, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Aquaporins (AQP) are a family of water transporting proteins expressed in many epithelial, endothelial, and other tissues. AQP1 is expressed in microvascular endothelia throughout the lung/airways, AQP3 in basal cells in large airways, AQP4 at the basolateral membrane of epithelia throughout the airways, and AQP5 at the apical membrane of type I alveolar epithelial cells and submucosal gland acinar cells. The expression of some of these aquaporins increases near the time of birth and appears to be regulated by growth factors, inflammation, and osmotic stress, suggesting a role of aquaporins in lung physiology. However, studies in transgenic mouse models of AQP deficiency have provided evidence against an important physiological role for aquaporins in many lung functions. Although AQP1 and AQP5 provide the principal route for osmotically driven alveolar water transport, alveolar fluid clearance in the neonatal and adult lung is not affected by AQP deletion, nor is lung CO2 transport or fluid accumulation in experimental models of lung injury. In the airways, AQP3 and AQP4 facilitate water transport; however, airway hydration, airway surface liquid layer volume and composition, and airway fluid absorption are not impaired by AQP3/AQP4 deletion. In airway submucosal glands, AQP5 deletion significantly reduced the rate and increased the protein content of fluid secretions. Thus, although AQPs have many important extrapulmonary physiological functions, in the lung/ airways AQPs appear to be important mainly in airway submucosal gland function. The substantially slower rates of

fluid transport in airways, pleura, and lung compared to renal and some secretory epithelia may account for the apparent lack of functional significance of AQPs at these sites. However, the possibility remains that AQPs may play a role in lung physiology under conditions of stress/injury not yet tested or in functions unrelated to transepithelial fluid transport.

Description Fluid movement between the distal airspace and interstitial and vascular compartments of the lung/airways is important in the maintenance of airspace hydration, the absorption of airspace fluid near the time of birth and in pulmonary edema, and the secretion of fluid onto the airway surface by submucosal glands. Aquaporins (AQPs), a family of small (B30 kDa monomer) integral membrane water channel proteins, are expressed in many cell types involved in fluid transport. There are more than 10 aquaporins in mammals, at least four of which are expressed in the respiratory system (Figure 1). AQP1 is expressed in microvascular endothelia near airways and alveoli, as well as in microvessels and mesothelial cells of visceral and parietal pleura. AQP3 is expressed in the basolateral membrane of basal epithelial cells lining the trachea and large airways, and at the basal membrane in human (but not rodent) alveoli. AQP4 is expressed in the basolateral membrane

AQUAPORINS 141 AQP3 AQP4 AQP5 Nasopharnyx (upper airways) Submucosal gland

Airway surface liquid

Microvessels

AQP1 Trachea

AQP4 AQP3

Pleura Lung Distal airway

AQP1 Alveolus

Type I

Microvessels

Type II

AQP5

AQP1

Figure 1 Aquaporin expression in lung and airways. AQP1 is expressed in microvascular endothelial and pleural membranes; AQP3 in large airway epithelia; AQP4 at the basolateral membrane in epithelia throughout the airways; and AQP5 at the apical membrane of alveolar type I cells and of serous acinar cells in submucosal glands.

High osmolality

Low osmolality

Water

Figure 2 Tetrameric structure of AQPs. Each AQP tetramer consists of four B30 kDa monomers, each of which contains a narrow water-transporting pore. Water moves across AQP pores in response to osmotic gradients.

of ciliated columnar cells in bronchial, tracheal, and nasopharyngeal epithelia. AQP5 is expressed in the apical membrane of type I alveolar epithelial cells and of acinar cells in submucosal glands. AQP5 has also been reported in human lung to be expressed in bronchial and nasopharyngeal acinar and ciliated duct columnar cells. This expression pattern in fluid

transporting cells provides indirect evidence for a role of aquaporins in lung/airway physiology. AQPs 1, 4, and 5 are water selective, and consist of tetramers of four monomeric B30 kDa subunits, each of which contains an independent narrow pore pathway for water transport (Figure 2), whereas AQP3 (an ‘aquaglyceroporin’) transports both water and glycerol.

142 AQUAPORINS

Normal Physiological Processes Developmental Regulation of Aquaporin Expression

Rodent studies have shown developmental regulation of lung aquaporin expression with distinct patterns for each aquaporin. AQP1 is detectable just before birth in rodents, increasing several-fold perinatally and into adulthood. Functional measurements in rabbit showed significantly increased lung water permeability in the perinatal period in parallel to increasing AQP1 expression. AQP1 expression is also upregulated by treatment with corticosteroids. In contrast, little AQP5 is expressed at birth and gradually increases until adulthood, whereas AQP4 expression strongly increases just after birth and is upregulated by -agonists and glucocorticoids. Regulation of Aquaporin Expression in Adult Lung

Regulated aquaporin expression is also seen in the adult lung. As in prenatal lung, AQP1 expression can be upregulated by corticosteroids. AQP1 and AQP5 expression are reduced in rodent lung following adenoviral infection, and AQP5 expression is increased after bleomycin exposure. Reduced AQP5 expression was found after exposure of a mouse lung epithelial cell line (MLE-12) to tumor necrosis factor alpha (TNF-a), suggesting a possible mechanism for its downregulation in viral infection in vivo. AQP5 expression was increased in MLE-15 and alveolar epithelial cells exposed to hypertonicity. Although potentially interesting, the physiological relevance of many of these observations is unclear, as the airway/ lung is probably not exposed to significant hypertonicity, and regulated AQP expression is a general phenomenon not specific to the lung/airways. Role of Aquaporins from Functional Studies in Knockout Mice

Transgenic mice lacking each of the lung aquaporins (AQP1, AQP3, AQP4, and AQP5) were generated in our laboratory both individually and in combinations. Comparative studies were done in wild-type and AQPdeficient mice to investigate the physiological role of aquaporins in the lung/airways. These mice have been quite informative in elucidating important roles of various aquaporins outside the lung. For example, mice lacking AQPs 1–4 are defective in their ability to concentrate urine, mice lacking AQP1 have impaired angiogenesis and cell migration, mice lacking AQP3 have reduced epidermal hydration, mice lacking AQP4 have altered brain water balance and impaired neural signal transduction, mice lacking AQP5 have impaired saliva secretion, and mice lacking AQP7 manifest progressive fat accumulation and adipocyte hypertrophy.

Fluid Transport in Distal Lung

The proposed aquaporin functions in distal lung include alveolar fluid absorption at the time of birth and in the adult lung, gas (CO2, O2) exchange, and formation/resolution of lung edema in response to acute and subacute lung injury. AQP5 is expressed in type I alveolar epithelial cells and AQP1 in microvascular endothelial cells. Osmotic water permeability between alveolus and capillary was B10-fold reduced in lungs of AQP1 and AQP5 null mice compared to wild-type mice, and 30-fold reduced by AQP1/AQP5 codeletion (in double knockout mice). Interestingly, AQP1 deletion mildly reduced hydrostatic lung edema in an isolated perfused lung preparation, and computerized tomographic analysis of two humans lacking AQP1 showed a blunted increase in airway wall thickness following saline infusion compared to control subjects. Reduced hydrostatic lung edema in AQP1 deficiency may be related to an abnormality in microvasculature deficiency, since on theoretical grounds hydrostatic driving forces should be unable to produce significant net fluid movement across a water-only pathway. Alveolar fluid clearance is an important function of the alveolar epithelium. Fluid absorption is the result of sodium absorption through the epithelial sodium channel (ENaC) in response to the electrochemical driving force created by the basolateral membrane Na-K-ATPase of type II alveolar epithelial cells. The consequent osmotic imbalance drives water absorption primarily through the type I cells. Alveolar fluid clearance is measured in fluid-filled lung models from the kinetics of increasing concentration of an airspace volume marker such as radiolabeled albumin. Remarkably, even with maximal stimulation of alveolar fluid absorption with betaagonists and pretreatment with keratinocyte growth factor (to increase the number of type II cells), AQP1 or AQP5 deletion did not reduce alveolar fluid clearance. Further, the rapid absorption of fluid from the airspace just after birth was not impaired by aquaporin deletion, nor was lung edema following acidinduced epithelial cell injury, thiourea-induced endothelial cell injury, or hyperoxic subacute lung injury. The much slower rate of maximal alveolar fluid absorption (0.016 ml min 1cm 2) compared to fluid absorption in kidney proximal tubule or saliva secretion in salivary gland (410 ml min 1 cm 2) may account for the lack of effect of AQP1 and AQP5 deletion on alveolar fluid clearance. Because rates of fluid transport are relatively low in lung, the low intrinsic (aquaporin independent) water permeability of the alveolar epithelial and capillary membranes appears to be adequate to allow fluid

AQUAPORINS 143

transport to occur without impairment under normal physiological conditions and in response to clinically relevant stresses. Fluid Transport in the Pleura

Fluid is continuously secreted into and reabsorbed from the pleural space. Little fluid is present in the pleural space (0.2 ml kg 1) despite its large surface area (4000 cm2 in man, 10 cm2 in mouse). Fluid entry into the pleural space involves filtration across microvascular endothelia near the pleural surface, and movement across a mesothelial barrier lining the pleural space, whereas fluid clearance is thought to occur primarily by lymphatic drainage. Pleural fluid can accumulate in pathological conditions such as congestive heart failure, lung infection, lung tumor, and the acute respiratory distress syndrome. AQP1 is expressed in microvascular endothelia near the visceral and parietal pleura and in mesothelial cells in visceral pleura. Osmotic water permeability across the pleural barrier, measured from the kinetics of pleural fluid osmolality changes after instillation of hypertonic or hypotonic fluid into the pleural space, was rapid in wild-type mice (50% osmotic equilibration in 2 min), and slowed by fourfold in AQP1 knockout mice. However, the clearance of saline instilled in the pleural space was not affected by AQP1 deletion, nor was the accumulation of pleural fluid in a fluid overload model produced by intraperitoneal saline administration or in a thiourea model of acute endothelial injury. Thus, although rapid osmotic equilibration across the pleural surface is facilitated by AQP1, as found in distal lung, AQP1 does not appear to play a major role in physiologically important mechanisms of pleural fluid accumulation or clearance. Fluid Transport in the Airways

Potential functions of aquaporins in the airways include humidification of inspired air, regulation of airway surface liquid (ASL) volume and composition, and absorption of fluid from the airways. Evaporative water loss in the airways is thought to drive water influx from capillaries and interstitium into the ASL by the generation of an osmotic gradient. The depth and ionic composition of the ASL should depend theoretically on the ion transporting properties of the airway epithelium and the rate of evaporative water loss, as well as the water permeability of the airway-capillary barrier. Osmotic water permeability in upper airways, measured by dilution of an airway volume marker in response to an osmotic gradient, was reduced in mice lacking AQP3 and/or AQP4. However, there was little effect of AQP3/AQP4 deletion on humidification of lower airways, as measured from the moisture content of expired air during mechanical ventilation with dry

air through a tracheotomy, or of upper airways, as measured from the moisture content of dry air passed through the upper airways in mice breathing through a tracheotomy. Also, the depth and salt concentration of the ASL in the trachea, as measured in vivo using fluorescent probes and confocal microscopy, was not altered by AQP3/AQP4 deficiency. Finally, active isosmolar fluid absorption, measured in nasopharyngeal airways (using a volume marker as done for alveolar fluid clearance) was not impaired by aquaporin deletion. Thus, although AQP3/AQP4 facilitate osmotic water transport in the airways, they play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption. Interestingly, one study showed increased airway reactivity in response to bronchoconstricting agents in AQP5 null mice. The mechanism of this phenotype was not established, but may be related to indirect effects of AQP5 deletion on agonist-induced fluid secretion from submucosal glands as described below. Fluid Secretion by Airway Submucosal Glands

Submucosal glands in mammalian airways secrete a mixture of water, ions, and macromolecules onto the airway surface. Glandular secretions are important in establishing ASL fluid composition and volume, and in antimicrobial defense mechanisms. Abnormally viscous gland secretions in cystic fibrosis have been proposed to promote bacterial adhesion and inhibit bacterial clearance. Submucosal glands contain serous tubules, where active salt secretion into the gland lumen creates a small osmotic gradient driving water transport across a water-permeable epithelium, as well as mucous cells and tubules, where viscous glycoproteins are secreted. AQP5 is expressed at the luminal membrane of the serous epithelial cells. Pilocarpinestimulated fluid secretion was found to be reduced by twofold in AQP5 null mice, as determined by nasopharyngeal fluid collections and video imaging of fluid droplets (covered with mineral oil) secreted by individual submucosal glands. Analysis of secreted fluid showed a twofold increase of total protein concentration in AQP5 null mice, suggesting intact protein and salt secretion across a relatively water-impermeable epithelial barrier. There was no significant difference of submucosal gland morphology or density in wildtype versus AQP5 knockout mice. AQP5 thus facilitates fluid secretion in submucosal glands, indicating that the luminal membrane of serous epithelial cells is the rate-limiting barrier to water movement. CO2 Transport by Aquaporins in Lung

AQP1-dependent CO2 transport has been proposed based on measurements in AQP1-overexpressing

144 ARTERIAL BLOOD GASES

Xenopus oocytes; however, subsequent studies showed unimpaired CO2 permeability in AQP1-deficient erythrocytes, where rapid CO2 transport occurs by a passive membrane solubility-diffusion mechanism. Measurements of CO2 movement in the perfused and in vivo mouse lung showed no effect of AQP1 deletion, providing direct evidence against the role of AQP1 in lung CO2 transport.

Aquaporins and Respiratory Disease A small number of AQP1-deficient humans have been identified. Although initially reported to have no phenotype, subsequent studies showed that they manifest a urine concentrating defect that is qualitatively similar to that found in AQP1-deficient mice. As mentioned above, AQP1-deficient subjects have also been found to have a small reduction in the increase in bronchiolar wall thickness following intravenous volume overload compared to normal controls, though other lung phenotypes have not been reported. The significance of this observation is unclear. Together with a considerable body of data in transgenic mice, the functional studies suggest that aquaporins play at most a minor role in normal lung physiology and clinically relevant states of lung injury, with the possible exception of AQP5 in submucosal fluid secretion. It remains unresolved why aquaporins are expressed at multiple sites of fluid movement in the lung/airways, and why the expression of lung aquaporins appears to be altered in states of stress. When available, nontoxic aquaporin-selective inhibitors will be useful to examine effects of acute aquaporin inhibition, which may reveal lung/ airway phenotypes that might not be manifest in mice or humans with chronic aquaporin deficiency. If significant aquaporin-dependent phenotypes are found, then pharmacological modulation of aquaporin function may have clinical applications, such as AQP5 inhibition in reducing glandular fluid secretions in allergic and infectious rhinitis, or increasing AQP5

expression/function to reduce secretion/ASL viscosity in cystic fibrosis. See also: Basal Cells. Fluid Balance in the Lung.

Further Reading Agre P, King LS, Yasui M, et al. (2002) Aquaporin water channels
from atomic structure to clinical medicine. Journal of Physiology 542: 3–16. Bai C, Fukuda N, Song Y, et al. (1999) Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice. Journal of Clinical Investigation 103: 555–561. Borok Z and Verkman AS (2002) Lung edema clearance: 20 years of progress. Invited review: role of aquaporin water channels in fluid transport in lung and airways. Journal of Applied Physiology 93: 2199–2206. Dobbs L, Gonzalez R, Matthay MA, et al. (1998) Highly waterpermeable type I alveolar epithelial cells confer high water permeability between the airspace and vasculature in rat lung. Proceedings of the National Academy of Sciences, USA 95: 2991–2996. Folkesson H, Matthay MA, Frigeri A, and Verkman AS (1996) High transepithelial water permeability in microperfused distal airways: evidence for channel-mediated water transport. Journal of Clinical Investigation 97: 664–671. King LS, Nielsen S, and Agre P (1996) Aquaporin-1 water channel protein in lung-ontogeny, steroid-induced expression, and distribution in rat. Journal of Clinical Investigation 97: 2183– 2191. King LS, Nielsen S, Agre P, and Brown RH (2002) Decreased pulmonary vascular permeability in aquaporin-1-null humans. Proceedings of the National Academy of Sciences USA 99: 1059–1063. Krane CM, Fortner CN, Hand AR, et al. (2001) Aquaporin-5 deficient mouse lungs are hyperresponsive to cholinergic stimulation. Proceedings of the National Academy of Sciences, USA 98: 14114–14119. Ma T, Fukuda N, Song Y, Matthay MA, and Verkman AS (2000) Lung fluid transport in aquaporin-5 knockout mice. Journal of Clinical Investigation 105: 93–100. Saadoun S, Papadopoulos M, Hara-Chikuma M, and Verkman AS (2005) Targeted AQP1 gene deletion impairs angiogenesis and cell migration. Nature 434: 786–792. Song Y and Verkman AS (2001) Aquaporin-5 dependent fluid secretion in airway submucosal glands. Journal of Biological Chemistry 276: 41288–41292. Verkman AS (2002) Physiological importance of aquaporin water channels. Annals of Medicine 34: 192–200.

ARTERIAL BLOOD GASES J W Severinghaus, UCSF, San Francisco, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Blood gas analyzers consist of three electrodes measuring pH, PCO2 , and PO2 at 371C. They were introduced in about 1960

following inventions by R Stow (CO2) and L Clark (PO2) both dating from 1954. From these outputs, internal computers calculate O2 saturation, base excess, bicarbonate, and other derived variables such as the compensation by the body for acid–base abnormalities. Arterial PO2 and PCO2 can be approximated using heated skin surface ‘transcutaneous’ electrodes, which are commonly used in premature infants and nurseries. Hemoglobin oxygen saturation, SO2%, is also directly measured by

ARTERIAL BLOOD GASES 145 multiwavelength blood oximeters. Arterial SO2 is approximated by pulse oximeters, which detect the arterial pulsatile variations in red and infrared light penetrating a finger, ear, or other tissue, a method invented by T Aoyagi in Tokyo in 1973 that became commercially available in 1983. Interpretation of blood gases and acid–base balance is briefly discussed. Figures include schema of the three electrodes, a pulse oximeter probe, an acid–base compensation diagram, and photographs of the first three-function blood gas analyzer, a combined PO2 PCO2 transcutaneous electrode in use on a child, and a pulse oximeter probe on a finger.

Henderson–Hasselbalch (HH) equation: pH ¼ pK0 þ log½HCO
3 =SPCO2  In plasma, pK0 ¼ 6.1, the effective dissociation constant of H2CO3 (carbonic acid), calculated as S PCO2 . S is CO2 solubility, 0.031 mM l
1 Torr
1. HCO3
is plasma bicarbonate content calculated as plasma [CO2 content – SPCO2 ].

Introduction

Electrodes

Arterial blood gas analyzers directly measure pH, PCO2 and PO2 , (mmHg or Torr) and calculate standard base excess (SBE), bicarbonate (HCO3
), oxygen saturation (SO2), and other variables useful in diagnosis and clinical management of patients in emergencies, anesthesia, surgery, recovery, and intensive care. These tests were rarely done until the 1950s when electrodes were invented and developed. Understanding of acid–base and blood gas theory depended on discoveries in physical chemistry.

pH Electrode

Ionic Theory

Electrochemistry and physical chemistry of solutions of acids, alkalis, metals, and salts were transformed from empiricism to theory by the 1884 thesis of Svante Arrhenius in Uppsala, Sweden. Wilhelm Ostwald then used a platinum electrode to measure hydrogen ion strength electrically. In 1893, Ostwald’s student Walther Nernst applied the longestablished laws of gases to ions in solution to calculate the electrical potential of batteries or cells. Buffers

Shortly after 1900, Lawrence J Henderson at Harvard adapted the mass action law to relate [H þ ] to PCO2 and HCO3
:

In 1905, Max Cremer noted that hydrogen ions permeated some kinds of very thin glass, developing electrical potential gradients across the glass. Fritz Haber and Z Klemensiewicz made the first glass pH electrode in 1909. Its potential was a linear function of pH, not H þ ion concentration. In 1925, the first glass cup-shaped blood pH electrode was produced by Phyllis T Courage. By 1933, capillary blood pH electrodes were being made commercially. Blood pH was corrected to 371C with the Rosenthal factor (
0.0147 1C
1) until thermostatted blood pH electrodes became available in the 1950s. A pH and reference electrode is schematically shown in Figure 1. Details Special glass compositions permit hydrogen ions to diffuse through imperfectly annealed submicroscopic cracks, probably exchanging loci with loosely bound alkali cations, especially lithium. Some pH electrodes are sensitive to very high Na þ concentrations. At 371C, the electromotive force (EMF) across the glass measured with reference electrodes (usually silver–silver chloride) is 61.5 mV per pH unit change (a 10-fold change in H þ ion concentration). Sample

K ¼ ½Hþ ½HCO
3 =H2 CO3 Glass body

He demonstrated how respiration could buffer metabolic acids by reducing PCO2 . The kidneys can also change blood and extracellular fluid buffer base to partially normalize pH in respiratory acidosis or alkalosis.

AgCl internal reference H+ permeable glass Liquid junction Saturated KCl

Origin of pH

In order to define H þ ion concentrations such as 0.000 000 04 moles liter
1 (e.g., in blood) more elegantly, in 1907, S P L Sørensen suggested defining pH as the negative log of hydrogen ion concentration (or activity). In 1915, K A Hasselbalch converted Henderson’s equation to log form, later dubbed the

AgCl reference

Sample Figure 1 Schema of a blood pH electrode with a liquid junction to a reference electrode in a thermostatted cuvette.

146 ARTERIAL BLOOD GASES

For use in blood, the reference electrode usually contacts blood via a liquid junction containing saturated KCl. Rapid diffusion of K þ into red cells causes an often-ignored error of
0.01 pH at the liquid junction when calibration is done with aqueous buffers. PCO2 Measurement

Until the worldwide polio epidemics of 1950–52, PCO2 was measured by the HH equation. This required measuring plasma CO2 content by acidification and extraction in a manometric Van Slyke apparatus and measuring pH and correcting it to 371C. In Copenhagen’s communicable disease hospital in 1950–51, sometimes up to 100 patients at a time were manually ventilated by volunteers using a bag and mask with O2. The laboratory director Poul Astrup, needing a faster analytic method than the HH equation, devised a new simple method. He measured pH before and after equilibrating the sample with known PCO2 . He then computed patient PCO2 by extrapolation. His method became the standard for the rest of the decade. PCO2 Electrode

At Ohio State University in 1954, while also trying to resolve the polio problem, Richard Stow invented a PCO2 electrode. He covered a glass bulb-shaped pH electrode with a rubber glove (through which CO2 can diffuse but H þ cannot), over a film of distilled water. However, Stow’s electrode drifted because various cations in pH glass altered the distilled water pH. This electrode was stabilized by John Severinghaus at the National Institute of Health (NIH) by adding bicarbonate and salt, and was manufactured by many firms from 1959 onwards. A blood PCO2 electrode is illustrated in Figure 2.

Details An internal, nearly flat, glass pH electrode is separated from the sample by a membrane permeable to CO2 but not ions (e.g., Teflon or silastic). Under the membrane, a spacer (e.g., lens-cleaning tissue paper) holds a film of electrolyte containing about 10 mM NaHCO3 and usually 0.1 M salt or KCl. Thermostatted to 37oC, the output signal is a log function of PCO2 , about 30 mV per decade in distilled water (Stow’s design), doubling to 61 mV per decade with bicarbonate electrolyte. Oxygen Electrode

In 1950, Leland Clark at Antioch College, Ohio used perfused isolated liver to study steroid metabolism. He needed oxygenated blood so he built a bubble oxygenator and discovered how to defoam the blood using silicone oil on glass wool. The journal Science rejected his paper on the basis that he hadn’t measured the PO2 in the oxygenated blood; to this end, Clark invented a polarographic oxygen electrode. He covered a platinum disc cathode sealed in glass with cellophane to keep blood protein from poisoning the cathode. It served his purpose but was not accurate, requiring very high flow past the sensor due to the depletion of oxygen at the membrane surface due to its consumption by the cathode. In October 1954, a sudden inspiration led Clark to substitute a less O2-permeable and electrically insulating polyethylene membrane for cellophane, by mounting a reference electrode and cathode in electrolyte in a sealed probe (Figure 3). His invention was presented at a meeting of the FASEB in April 1956. Details In a polarographic oxygen electrode, a negatively biased platinum cathode donates electrons to

Stow–Severinghaus PCO2 electrode Sample

AgCl external reference electrode AgCl internal reference H+ permeable glass 0.1M KCl + 0.01M KHCO3

Glass body

Teflon or silastic

membrane

Electrolyte in paper spacer Sample Figure 2 Schema of a blood PCO2 electrode with Teflon CO2 permeable membrane and spacer to contain electrolyte for pH measurement.

ARTERIAL BLOOD GASES 147 Clark-type oxygen electrode 25 µm polypropylene membrane

Sample

0.1 m KCl electrolyte 10 µm Platinum wire

Solid glass AgCl reference Sample Figure 3 Schema of Clark’s polarographic PO2 electrode, after cathode size was greatly reduced to avoid ‘stirring’ effect.

dissolved oxygen: O2 þ 4e
þ 2H2 O ) 4OH
The only major functional change since Clark’s original invention has been to reduce the cathode diameter from 2 mm to about 10 mm, requiring far more sensitive current analysis, which was not available 50 years ago. This almost eliminated the need for the sample to be rapidly stirred. The electrolyte usually contains KCl, and may have added agents for viscosity. No separator is needed between cathode and membrane. The cathode is biased to about
0.65 V at which all oxygen molecules reaching the cathode are reduced. Cathode current is a linear function of the membrane surface PO2 .

Figure 4 The first blood gas analyzer containing three electrodes in a water bath at 371C with tonometer for preparing blood for calibration of PO2 electrode. Reproduced from Severinghaus JW (2002) The invention and development of blood gas apparatus. Anaesthesiology 97: 253–256, with permission from Lippincott Williams & Wilkins.

Blood Gas Analyzers

In 1958, Severinghaus and Bradley created the first three-function blood gas analyzer by mounting a Clark PO2 electrode with a tiny stirring paddle, a Stow–Severinghaus PCO2 electrode, and a commercial pH electrode in a water bath at 371C (Figure 4). A small tonometer was included in which blood could be equilibrated with air or a known gas to calibrate the Clark electrode. Modern blood gas analyzers compute many variables from the three measured values. Transcutaneous PO2

PaO2 can be estimated transcutaneously using a flat Clark type PO2 electrode, typically internally heated to about 431C. Heating causes sufficient dermal vasodilation to raise skin capillary PO2 to nearly equal arterial PO2 . Heating also raises blood PO2 by about 7% per degree, while skin oxygen consumption reduces surface PO2 , these two factors approximately canceling each other out. No correction factors for

Figure 5 Transcutaneous combined PO2 and PCO2 electrode monitoring a patient recovering from anesthesia.

temperature or skin metabolism are thus needed. Introduced in the mid-1970s, these devices are widely used on premature and term infants to help control oxygen therapy and prevent blindness following retinal vascular growth interference (Figure 5). Transcutaneous PCO2

Arterial PCO2 can also be estimated transcutaneously using flat CO2 electrodes heated (e.g., to 431C) to increase skin capillary blood flow. The signal must be corrected
4.7% per degree to 371C, and reduced about 4 Torr to compensate for skin metabolism and electrode surface cooling.

148 ARTERIAL BLOOD GASES Hemoglobin Oxygen Saturation

Prior to about 1970, this was measured by vacuum extraction of oxygen from blood, before and after equilibrating a sample with air. Multiwavelength Oximetry

Compared with oxygenated blood, desaturated blood strongly absorbs red light (at about 660 nM wavelength). At the isobestic point, 805 nM (near infrared), absorption is unaffected by oxygenation. Multiwavelength oximeters use the ratio of optical density of a thin film of hemolyzed blood at red and infrared wavelengths to calculate saturation and hemoglobin concentration, with small corrections for other pigments such as bilirubin, detected at other wavelengths. A typical laboratory ‘bench’ oximeter using at least five wavelengths can be precise to at least 0.1% saturation. Some oximeters use filters to select wavelengths while others use more stable and precisely defined diffusion-grating monochromators, avoiding the need for user calibration. Confirmatory testing with dyes is recommended. Some blood gas analyzers include a multiwavelength oximeter (often termed CO-oximeter because it also measures the fraction of hemoglobin bound to carbon monoxide). Pulse Oximetry

Light passing through a finger or ear is partly absorbed by the blood in its path. Arteries expand with each pulse, absorbing a bit more light. Pulse oximeters measure the amplitude of the pulsatile variation of light as a fraction of total transmitted red (e.g., 660 nM) and infrared (e.g., 900–950 nM) light (Figures 6 and 7). The ratio of these two ratios was shown by T Aoyagi in 1973 (Nihon Kohden Co, Tokyo) to be a unique function of arterial oxygen saturation, theoretically independent of venous or capillary saturation, or skin color, tissue thickness, or other pigments. In the late 1940s, Earl Wood (Mayo Clinic) modified G Millikan’s 1942 original ear oximeter by adding a pneumatic pressure capsule. Wood showed that when blood was readmitted to a pressure-blanched ear, the ratios of the decreases in red and infrared light passing through the ear were unique functions of oxygen saturation.

Indications Blood gas analysis has become so commonplace that its use is nearly universal in diagnosis during admission, in emergencies, trauma, intensive care, anesthesia, and surgery. It is considered by physicians to

Figure 6 Pulse oximeter probe taped on fingertip with red and infrared LEDs on nail side and a photo diode on the dorsal side.

Photodiode

Finger

Red and infrared LEDs

Figure 7 Schema of pulse oximeter probe on a finger.

be the most useful and important diagnostic procedure available. Its indications are global and will not be listed here.

Common Patterns of Results and Interpretation Diagnostic Terminology

A pH of less than 7.35 is called acidemia, while that over 7.45 is termed alkalemia. A PCO2 over 45 Torr indicates respiratory acidosis or hypercapnia, while values under 35 (males) or 30 (females) indicate respiratory alkalosis or hypocapnia. A standard base excess (SBE) more negative than
5 mM is metabolic acidosis and over þ 5 mM is metabolic alkalosis. Presence of compensatory responses to chronic acid– base respiratory or metabolic imbalances can be predicted and used in diagnosis (Figure 8). Normal PO2 at sea level in young adults is 90– 100 mmHg. It falls with age to 60–70 mmHg at age 80. There is no consensus on what PO2 level is defined as ‘hypoxia’. Arterial oxyhemoglobin ‘functional’ saturation (100 x HbO2/[HbO2 þ HHb]) is normally 97–98% (i.e., 2–3% deoxyhemoglobin,

ARTERIAL BLOOD GASES 149 30

7.7

7.6

7.5

7.4

Metabolic SBE (mM)

M

20 CR

10 0 −10 −20

AR

AR

7.3 Metabolic alkalosis 7.2

Metabolic acidosis 7.1

CR

7.0 pH

M

−30 10

20

Respiratory alkalosis

Respiratory acidosis

30 40 50 60 70 Respiratory PaCO2 (Torr)

80

90

Figure 8 Acid–base compensation diagram predicting in vivo compensation for respiratory and metabolic acid–base imbalance. AR and CR, acute and chronic respiratory, respectively; M, metabolic. Reproduced from Schlichtig R, Grogono AW, and Severinghaus JW (1998) Human PaCO2 and standard base excess compensation of blood gas apparatus. Critical Care Medicine 26: 1173–1179, with permission from Lippincott Williams & Wilkins.

HHb). Carboxyhemoglobin or methemoglobin or other abnormal forms, if present, reduces ‘fractional’ saturation (or % oxyhemoglobin) computed as 100 x HbO2/total Hb but is not counted in ‘functional’ saturation. The terms ‘hypoxia’ or ‘hypoxemia’ generally imply that SaO2 is at least 5% lower than expected (at that age and altitude). Temperature Correction

Clinicians often ask whether they should instruct the laboratory to correct blood gas values to patient temperature. In general, this is not necessary. The appropriate pH and PCO2 for optimal physiologic function is the same function of temperature as are the in vitro temperature correction factors. At 301C in a hypothermic patient, the appropriate pH is 7.4 measured at 371C, or 7.55 corrected to 301C. Animals with a normal body temperature of 301C have a pH of 7.55. Fish in Antarctic waters at 01C have a pH of 8.0, as does normal human blood cooled in vitro to 01C. Blood at 90% SaO2 with PO2 ¼ 60 Torr will have PO2 ¼ 30 Torr at 251C, but delivers oxygen at least as effectively to tissues in hypothermia (as shown by decreased tissue lactate). However, physiologists who wish to study pulmonary gas transport and compare alveolar and arterial blood gas tension gradients must correct blood values from the laboratory (at 371C) to the body temperature under study. SID or SBE?

The interpretation of acid–base balance divides clinicians into two ‘schools of thought’. Strong ion

difference (SID) can identify the causes of many metabolic abnormalities in addition to obtaining an approximation of the degree of plasma metabolic acid– base abnormality, provided that all anions and cations are measured. This unfortunately is not a measure of the whole body extracellular fluid acid–base condition. For that one needs the SBE, which is computed from directly measured arterial pH, PCO2 , and PO2 without separate ion measurements. SBE is thus a quantitative analysis of imbalance while SID is an approximation of imbalance with additional causative suggestions. Hydrogen Ion Concentration or pH?

pH is used widely both for convenience and because chemical activities and potentials are log functions of concentrations. All animals no matter what their normal body temperature maintain their pH 0.6 unit above neutrality, i.e., a ratio of [OH
]/[H þ ] of about 16 whereas [H þ ] varies by a factor of more than 4 between 01C (fish) and 401C (hummingbirds). Buffering is a linear function of pH, an exponential function of [H þ ] making straight pH lines on acid– base compensation plots. Some clinicians prefer to interpret acid–base abnormalities using an approximation of Hendersen’s equation: Hþ ¼ 24PCO2 =HCO
3 using nM l
1 for H þ , mmHg for PCO2 and mM l
1 for HCO3
. This requires a constant 24 combining the dissociation constant K, CO2 solubility, equating carbonic acid with dissolved CO2, and a factor for the three different units.

Oxygen Dissociation Curve Computations and Corrections Blood gas analytic apparatus commonly provides a computed value of SO2 (oxygen saturation). The observed PO2 is first corrected from observed pH to pH ¼ 7.4 by obs obs ÞÞ logP7:4 O2 ¼ logPO2
ð0:48ð7:4
pH

At pH ¼ 7.40, 371C, the relationship of SO2 to PO2 is most simply and accurately expressed by SO2 % ¼ 100ð23; 400ðP3O2 þ 150PO2 Þ
1 þ 1Þ
1 No temperature correction (e.g., to patient temperature) is needed, all measurements being at 371C.

150 ARTERIES AND VEINS

SO2 in a blood sample does not vary with sample temperature.

State of the Art Electrodes versus Optical Sensors

Optodes can measure pH, PCO2 and PO2 using dyes, fluorescence, quenching, and other optically responsive material, permitting the use of extremely small sensors on optical fiber tips in blood at catheter tips or inside tissue cells. They rival electrodes in accuracy and cost, in particular providing portable and disposable sensors (e.g., for cardiac bypass oxygenator control, bedside and field blood gas analysis). See also: Acid–Base Balance. Diffusion of Gases. Diving. Erythrocytes. High Altitude, Physiology and Diseases. Oxygen–Hemoglobin Dissociation Curve. Permeability of the Blood–Gas Barrier. Ventilation: Control.

Further Reading Geha DG (1990) Blood gas monitoring. In: Blitt CDK (ed.) Monitoring in Anesthesia and Critical Care Medicine. New York: Churchill-Livingston. Nunn JF (1993) Nunn’s Applied Respiratory Physiology, 4th edn. Oxford: Butterworth-Heinemann. Schlichtig R, Grogono AW, and Severinghaus JW (1998) Human PaCO2 and standard base excess compensation for acid-base imbalance. Critical Care Medicine 26: 1173–1179. Severinghaus JW (1979) Simple, accurate equations for human blood O2 dissociation computations. Journal of Applied Physiology 46: 599–602. Severinghaus JW (2002) The invention and development of blood gas apparatus. Anaesthesiology 97: 253–256. Severinghaus JW and Astrup PB (1987) History of Blood Gas Analysis. International Anesthesiology Clinics, vol. 25(4). Boston: Little Brown. Severinghaus JW, Astrup P, and Murray J (1998) Blood gas analysis and critical care medicine. American Journal of Respiratory Critical Care Medicine 157: S114–S122. Severinghaus JW and Bradley AF Jr (1958) Electrodes for blood PO2 and PCO2 determination. Journal of Applied Physiology 13: 515–520.

ARTERIES AND VEINS D E deMello, Saint Louis University Health Sciences Center, St Louis, MO, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The lung’s vasculature is different from that of most other organs because it has a double arterial supply and a double venous drainage system. The pulmonary artery which carries deoxygenated blood to the lungs branches alongside the airways into conventional and supernumerary arteries before it empties into the vast capillary network in the alveolar walls where gas exchange occurs across air–blood barriers. Conventional and supernumerary pulmonary veins drain oxygenated blood to the left atrium of the heart. The smaller (intra-acinar) vessels develop at the same time as do the acini of the lung and a major component of acinar lung development occurs postnatally. Alveoli increase from about 20 million at birth to about 300 million by the time lung growth is complete in adolescence. Consequently, a large component of intra-acinar vessels develop postnatally and during this critical growth phase are susceptible to local influences such as increased blood flow from intracardiac left to right shunts which can curtail vessel growth. Arterial wall structure is composed of endothelial cells that line the lumen, smooth muscle cells that make up the media, and fibroblasts that contribute the adventitial fibrous sheath. The wall structure changes from proximal to distal vessels in the lung, and alterations in the normal structure may occur during intrauterine life or postnatally in response to a variety of stimuli. Altered wall structure results in functional changes reflected in pulmonary artery pressure and resistance.

Blood vessel assembly begins in primitive mesenchymal cells that undergo a complex series of steps before the mature vessel structure and function is attained. These stages in vessel development are under the control of a large number of transcriptional and growth factors. The timing and dose of some of these ‘angiogenic’ factors is critical to normal embryonic and fetal development; absence or even reduced expression of some factors is lethal for the developing embryo.

Anatomy, Histology, and Structure Unlike most other organs, the lung, because of its gas exchange function, has a double arterial supply and double venous drainage (Figure 1). One of the arterial systems, the pulmonary arterial tree, serves as a conduit for deoxygenated blood from the body to the alveoli where it drains into a vast capillary network within which gas exchange and oxygenation occur. From the alveoli, oxygenated blood is transported to the left atrium of the heart via the pulmonary veins. The other arterial system is the bronchial arterial tree which serves a nutrient function to the airways and perihilar structures. The arterial supply to the pleura, except at the hilar region, is from the pulmonary artery. For the intrapulmonary structures there are no bronchial veins, so all intrapulmonary structures drain to the pulmonary vein, resulting in a small amount of venous admixture in the left atrium. The hilar structures, however, drain to true bronchial

ARTERIES AND VEINS 151

Pulmonary artery

Alveoli Respiratory bronchiolus

Pulmonary vein

Bronchioli Bronchial artery Bronchi

Precapillary shunts

To left atrium

Capillary bed

To right atrium

Azygos vein

Figure 1 The lung’s vasculature consists of a double arterial and a double venous system. The pulmonary artery supplies the alveoli whereas the bronchial artery supplies the airways, the pulmonary artery, and the gas exchange region. Both systems drain via the pulmonary veins to the left atrium, except for the ‘true’ bronchial veins at the hilum which drain via the azygos veins to the right atrium. Reproduced from Reid L, Fried R, Geggel R, and Langleben D (1986) Anatomy of pulmonary hypertensive states. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, vol. 4, pp. 221–263. New York: Churchill Livingstone, with permission from Elsevier.

veins and then to the azygos system and the right atrium.

Segmental hilum

Lateral pathway

Conventional and Supernumerary Arteries

The main pulmonary artery that forms the outflow path of the right ventricle divides into the right and left pulmonary arteries which enter the lung at the hilum. Within the lung the pulmonary arteries travel alongside the airway branches within a connective tissue sheath. The arterial branching pattern is similar to that of the airway it accompanies, but dissection has revealed many more branches from the pulmonary artery than from its accompanying airway. Two types of arterial branches exist. Those that branch dichotomously next to the airways are conventional branches, the longest of which are the axial pathways running the longest possible course from hilum to distal pleura. The lateral branches of the conventional pulmonary arteries supply the alveoli near the axial pathways (Figure 2). The other type of branch is the supernumerary artery which is short, arises at right angles to the axis of the pulmonary artery, and supplies the alveoli in the immediate vicinity of the artery (Figure 3). Supernumerary arteries are more numerous toward the periphery of any arterial pathway. Over the preacinar length of an axial artery, the ratio of the number of supernumerary to conventional

Axial pathways

Pleura

Figure 2 The path of axial and lateral airways in the human lung. The pulmonary arteries are alongside the airways within the bronchoarterial sheath and follow the same path. Reproduced from Davies P, deMello DE, and Reid LM (1990) Structural methods in the study of development of the lung. In Gil J (ed.) Models of Lung Disease: Microscopy and Structural Methods, vol. 47 of Lung Biology in Health and Disease, pp. 409–472. New York: Dekker, with permission.

branches is of the order of 3 : 1 and the lumen crosssection area of the supernumerary side branches is about one-third of the total cross-sectional area of all side branches (Figure 4). Supernumerary arteries have

152 ARTERIES AND VEINS 19-week fetus Hilum

Conventional artery Supernumerary Airway

1

3

1 2 2

3

4 5 4 6 5 6

7 8 7 8

9 10 10

Airways Conventional arteries Supernumerary arteries Figure 3 The anatomy of the pulmonary arterial tree. Conventional arteries divide alongside the airways at acute angles to the main axis and supply the respiratory region at the end of the axial pathway. Supernumerary arteries are short, arise at right angles to the main axis, and supply the airspaces adjacent to the axial vessel. Modified from deMello DE and Reid L (2002) Vascular development of lung. In: Tomanek R (ed.) Assembly of the Vasculature and its Regulation, vol. 2, pp. 211–237. Boston: Birkhauser, with permission.

a special sphincter at their point of origin which probably serves a functional purpose in recruitment. This valve is responsive to vasoconstrictive agents suggesting that it may be responsible for regulation of blood flow into the supernumerary artery. Because the diameter of the supernumerary vessel is smaller than that of the conventional artery from which it arises, it has a higher critical opening pressure, providing a built-in mechanism for recruitment as pulmonary artery pressure rises. It is noteworthy that the two types of pulmonary artery have different pharmacological profiles; for example, supernumerary arteries are 30 times more sensitive in their response to a vasoconstrictor such as 5-hydroxytryptamine (5-HT) compared to conventional arteries, and endogenous nitric oxide selectively attenuates the vasoconstrictor response to 5-HT in the supernumerary but not in the conventional artery. In pathological conditions, these short vessels provide an alternate route for blood in the conventional artery obstructed by thromboemboli. The capillary bed between an artery proximal to the block can open to conduct blood to an arterial bed distal to the block. Blood flow can produce remodeling of a vessel whether artery or vein, and injection techniques have demonstrated that such compensatory or collateral flow produces arcades between axial pulmonary arteries in a number of pathological states (Figure 5).

11 11

12 12

13 14 14

18 19 21.23 21 23 25

9

15 15 16 17 17 20 20 22 24 Terminal bronchiolus

Figure 4 Posterior basal lung segment in a 19-week fetus. Airway and accompanying conventional artery branches are numbered. The more numerous supernumerary arteries (58) are also shown. Reproduced from Hislop A and Reid L (1972) Intrapulmonary arterial development in fetal life: branching pattern and structure. Journal of Anatomy 113: 35–48, with permission from Blackwell Publishing Ltd.

Conventional and Supernumerary Veins

The venous drainage resembles the arterial pattern in that there are more venous tributaries than airway branches. The ‘conventional’ veins arise from the points of division of an airway and pass to the periphery of the unit, combining as tributaries to form progressively larger venous conduits. The supernumerary veins are short and drain small regions around the conventional veins. Additional tributaries to the pulmonary veins arise from pleura and connective tissue septa within the lung. Lung development is unique in that there is a set timetable for the growth and maturation of its major tissue components, airways, alveoli, and blood vessels, and that a large portion of its development continues postnatally until about 12 years of age. Laws of Lung Development

The template for the growth pattern of the lung is reflected in the three ‘laws of lung development’.

ARTERIES AND VEINS 153 Airway

Artery

Hilum

Vein

m Preacinar Resistance artery TB Intraacinar

RB AD A

pm Precapillary unit

Postcapillary unit

nm Capillary

Figure 5 Lung arteriogram from a premature infant who died at 9 months of age with pulmonary hypertension and vascular hypo plasia. Note dilated, tortuous vessels and interpulmonary artery arcades (arrows). Reproduced from Rendas A et al. (1980) American Review of Respiratory Disease 121: 873–880, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

Figure 6 Human lung arteriograms. Top, newborn; bottom left, 18 months; right, adult. Increase in radiodensity with age reflects progressive postnatal increase in number of intra-acinar arteries. Reproduced from Reid L (1978) The pulmonary circulation: remodeling in growth and disease. The 1978 J Burns Amberson Lecture. American Review of Respiratory Disease 119: 531–546, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

These generalizations are also predictive of the effect of perturbation at different times during development and they offer a framework against which to assess vascular growth patterns and to interpret anomalous development. Law I – Airways. The airways (i.e., bronchi and bronchioli) are present by the 16th week of intrauterine life.

Figure 7 The arteriovenous loop between the right and left side of the heart. The acinus is a unit of lung and includes that portion of lung that is subtended by the terminal bronchiole, i.e., respiratory bronchioles, alveolar ducts, and alveoli. The pulmonary artery that accompanies the airways is shown, including the resistance segment that is immediately precapillary in the newborn child when the acinus is 1 mm in diameter. In the adult the acinus is 1 cm in diameter and the resistance vessels are further downstream at the level of the alveolar ducts and alveoli. m, muscular; pm, partially muscular; nm, nonmuscular; TB, terminal bronchiole; RB, respiratory bronchiole; AD, alveolar duct; A, alveolus. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, pp. 767–777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

Law II – Alveoli. At birth the ‘alveolar’ spaces are more primitive than in the adult. They have been described as ‘primitive saccules’. Of these saccules about 20
106 are present at birth. After birth the alveoli multiply, so that by the age of 8 the number is about 300
106 and within the adult range. Law III – Vascular. The ‘vascular law’ reflects the first two. The preacinar branches of the pulmonary artery (i.e., those that accompany bronchi or bronchioli), as well as the preacinar venous tributaries, appear at virtually the same time as do the accompanying airways. The intra-acinar vessels appear as alveoli grow (Figure 6). Preacinar Arteries: Structure and Size

The cellular structure of any vessel is composed of three cell types that make up the three coats of a vessel: the intima is constituted by endothelial cells, the media is composed of smooth muscle cells or their precursors, intermediate cells or pericytes, and the adventitia is made up of fibroblasts. Intercellular products also contribute to the composition of each coat. Regional and organ specificity dictate functional differences. A number of mediators, cytokines, growth factors, or hormones determine and modulate these differences.

154 ARTERIES AND VEINS Conventional branches Diameter of axial artery and side branches (µm)

Supernumerary branches 1000

External diameter of axial pathway 500

Acinus Lobule Figure 8 The relative sizes and numbers of conventional and supernumerary pulmonary arteries. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, pp. 767–777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

Partially muscular

Muscular

Non muscular

Capillary (a)

M

I P E Artery

(b)

Lumen

M

(c) Figure 9 (a) Diagram depicting the light microscopic appearance of the structure of a pulmonary artery. In its path from the hilum to the periphery, the muscle coat gradually disappears so that the most peripheral segment still precapillary, is nonmuscular. (b) Diagrammatic illustration of the ultrastructural features of the pulmonary artery. From the hilum toward the periphery, muscle cells (M) are replaced by intermediate cells (I), and in the immediate precapillary segment, pericytes (P) are present. (c) Same as (a), in cross-section. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, pp. 767–777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

ARTERIES AND VEINS 155 Fetus 100 60

Percentage arterial population

20

Child 100 60 NM

M

PM

20

Adult 100 60 20 100

200

300

400

Arterial size (ED, µm) Figure 10 The structure of vessels of different sizes at different ages in the human. The distribution of muscular (M), partially muscular (PM), and nonmuscular (NM) arteries is similar in the fetus and adult; however, in the child, larger arteries are nonmuscular and partially muscular. ED, external diameter. Reproduced with permission from the BMJ Publishing Group Davies G and Reid L (1970) Thorax 25: 669–681.

In the lung, the vessels proximal to the capillary bed are arteries and those distal to the capillary bed are veins. The immediate pre- and postcapillary vascular segments are functionally specialized and serve to protect the capillary bed (Figure 7). They are the key reactive site for remodeling in disease. Preacinar artery structure is established early in fetal life as elastic, transitional, or muscular and the program for structure is a regional or topographic one. In the adult along an axial pathway, the first seven generations which are vessels down to a diameter of 3000 mm (distended) are elastic. Vessels from 3000 to 2000 mm in diameter representing generations seven to nine have a transitional structure and smaller vessels down to a diameter of 30 mm have a muscular structure. The largest partially muscular and nonmuscular arteries are found in diameters up to 150 and 130 mm respectively (Figure 8). Along any arterial pathway, a complete muscular coat gives way to an incomplete coat which at first consists of a spiral before disappearing from vessels, still larger than capillaries, to produce a nonmuscular artery (Figure 9). On the arterial side of the capillary bed, nonmuscular, partially muscular, and muscular small arteries are identified. On the venous side a similar arrangement occurs. By

electron microscopy intermediate cells and pericytes, each a precursor of the smooth muscle cell, are identified: the intermediate cell in the nonmuscular part of the partially muscular artery and the pericyte in the nonmuscular wall. Whereas the pericyte lies within the basement membrane of the endothelial cell, the intermediate cell, like a smooth muscle cell, has its own basement membrane, but, unlike the latter does not have dense bodies. Characteristics of an artery include size, structure, and the thickness of the media as well as its position within the branching pattern. Because vessel remodeling occurs with growth and in disease, it is best to characterize a pulmonary artery by its position in the branching pattern defined by the structure of the accompanying airway, a landmark that remains relatively constant from fetal life. In arteries, the relative circumferences of the internal and external elastic laminae appear to influence the size and morphology of the endothelial cell and its microenvironment. For example, in vessels larger than 200 mm, the external lamina is shorter than the internal suggesting that it is the former that restricts distension and that the internal lamina may never be smoothed out. In the smaller arteries the situation is

156 ARTERIES AND VEINS

reversed suggesting that the internal lamina is the limiting structure. The balance between these is changed in disease. The functional significance of this, in pulsatile flow and propagation of the pulse wave, has not been established. Postnatally, the length and diameter of the entire pulmonary vascular tree changes, but structural changes occur mainly at the sites of alveolarization in the acinar region, that is, in vessels distal to the level of the terminal bronchiole. In the newborn lung the resistance arteries, that is, vessels with a smooth muscle wall, are upstream from the alveolar wall. Normally before birth pulmonary arterial muscularization stops at the beginning of the acinus, so that the peripheral vessels are muscularized later (Figures 10 and 11). In conditions such as idiopathic pulmonary hypertension of the newborn, congenital heart lesions associated with increased pulmonary artery flow or pressure before birth, and pulmonary hypoplasia, a well-developed muscle coat is found in more peripheral and smaller arteries than is normal. The functional correlate of this structural change is an elevation in pulmonary artery pressure and resistance. AD

Species differences in structure and rate of lung growth occur and need to be considered when interpreting experimental results. In general, animals like the sheep that can walk or run within hours of birth have a prenatal burst in lung development, preparing the animal as it were for considerable, immediate postnatal activity. In mammals like the rat, mouse, and pig a similar burst in lung growth occurs postnatally, and in the human, postnatal lung growth continues for several years until adolescence. Postacinar Veins: Structure and Size

The veins are present at the periphery of an acinus within their own connective tissue sheath. As additional tributaries are added in their course toward the hilum, they increase progressively in size. Conventional axial veins enter the larger veins at an acute angle and are the main pathways from periphery to hilum. Their numbers are equivalent to the airway generations and to conventional arteries. The supernumerary tributaries are shorter, drain the lung immediately around the axial vein, and connect with RB

TB

Pleura

Alv

Species Variation

Normal Fetus 3 days 10 months 3 years 10 years 19 years Cases PPHN Mec Asp (fatal) HLHS IDTAPVR Figure 11 Top: diagram of an acinus of the lung which includes the terminal bronchiole (TB), respiratory bronchioles (RB), alveolar ducts (AD), and alveoli (alv). Below: the open bars indicate the level beyond which at different ages the arteries are nonmuscular. The blue bars indicate the extent of muscularization (‘precocious muscularization’) in different disease processes: PPHN, persistent pulmonary hypertension of the newborn; Mec Asp (fatal), fatal cases of PPHN with meconium aspiration; HLHS, hypoplastic left heart syndrome; IDTAPVR, infradiaphragmatic total anomalous venous return. Reproduced from Reid L, Fried R, Geggel R, and Langleben D (1986) Anatomy of pulmonary hypertensive stages. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, vol. 4, pp. 221–263. New York: Churchill Livingstone, with permission from Elsevier.

ARTERIES AND VEINS 157 Pathway 1

1200

∗

800

External diameter of axial pathway

Diameter of axial vein and tributaries (µm)

400

Hilum Pathway 2 1200

800 + + +

400

Hilum Pathway 3 Conventional veins 400 Supernumerary veins

Figure 12 Diagrammatic reconstruction of conventional and supernumerary venous tributaries in a 20 week fetus. Pathway 2 is 9.31 mm in length. *Point where pathway 2 joins pathway 1. þ Point where pathway 1 joins pathway 2. z Point where pathway 3 joins pathway 2. There is a higher density of venous tributaries at the periphery. Reproduced from Hislop A (1971) The fetal and childhood development of the pulmonary circulation and its disturbance in certain types of congenital heart disease. PhD Thesis, London University, with permission from A A Hislop.

the conventional veins at right angles (Figure 12). The preacinar tributaries are developed by 20 weeks gestation, and subsequent tributaries arise within the acinus. Supernumerary tributaries exceed conventional, their ratio being 3.5 : 1, and more vessels drain from the capillary bed than enter. Postcapillary veins have an endothelial lining, but no muscle coat. Larger veins contain an internal elastic lamina with only occasional medial smooth muscle fibers. The medial thickness increases in yet larger veins, but even the largest veins do not have a well-defined external elastic lamina. Elastin and

collagen are present between the smooth muscle cells. The largest nonmuscular veins are approximately 150 mm in diameter and as with the arteries, overlap in size groups occurring between muscular, partially muscular, and nonmuscular veins. At 20 weeks gestation, the vein structure consists of endothelial cells resting on collagen mixed with an occasional elastic fiber. Even the largest veins have no muscle in their wall. Scattered muscle fibers appear by 28 weeks, but a continuous layer is only developed at term, and even then, no elastic lamina is present. Postnatally, the thickness of the muscle

158 ARTERIES AND VEINS

increases but not as much as in arteries. At birth the smallest muscular vein measures about 105 mm in diameter, at 3 years the smallest is 130 mm, and at 10 years 70 mm. No significant structural change occurs after this time. Intra-Acinar Arteries and Veins: Structure and Size

By 5 years of age, axial pathways have finished development and future growth occurs in the more peripheral and smaller arteries that appear as alveoli are formed, but in these vessels, muscularization is slower, so that during childhood the transition from muscular to partially to nonmuscular occurs in vessels of a larger size than in the fetus or adult (Figure 10). Therefore, in the child, the structure of intra-acinar arteries cannot be predicted by their size. Bronchial Arteries

Early in gestation, primitive bronchial arteries arise from the dorsal aorta in the neck region near the celiac axis and are distributed with the early branches of the airways (Table 1). When lobar or segmental airway branches are present at about the 6th week of embryonic life, the central bronchial artery branches disappear. Between the 9th and 12th weeks, definitive bronchial arteries arise from the aorta, pass along the superior surface of the airways, and communicate with the pre-existing capillary bed in the distal airways. Sometimes the primitive bronchial arteries persist and migrate with their point of origin, to a site below the diaphragm, still near the Table 1 The development of the bronchial arteries Week of gestation

Airway

Blood vessels

4th

Main

5th

Lobar

6th 9th to 12th

Segmental

Primitive ventral aorta Pulmonary vein links to heart 6th arch supplies lung Paired systemic arteries from dorsal aorta Only blood from right ventricle to pulmonary artery Systemic arteries disappear Bronchial arteries enter peribronchial plexus

The primitive paired bronchial arteries arise from the dorsal aorta near the celiac axis in the neck. These have usually disappeared before the 5th week; if they persist, they then migrate with the celiac axis to below the diaphragm as seen in certain abnormal conditions (e.g., sequestered segment). Reproduced from deMello DE and Reid LM (1997) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 1117–1127. Philadelphia: Lippincott-Raven Press, with permission from Lippincott Williams & Wilkins.

celiac axis. A portion of the lung bud may remain attached and manifests later as a lung sequestration.

Arteries and Veins in Normal Lung Function Blood Vessel Assembly

Two processes are involved in the development of blood vessels: (1) angiogenesis, the branching of new vessels from pre-existing ones, and (2) vasculogenesis, the formation of blood lakes that undergo transformation into vessels. In the lung, blood vessel development must be coordinated temporally and spatially with airway and alveolar development. Angioblasts are the precursor cells from which blood vessels are derived and they are of mesodermal origin. In the development of the pulmonary vasculature, angioblasts must interact with epithelial and mesenchymal components of the lung. Central events determine for a given vessel, the direction of blood flow, its distribution or drainage and its central connections, and local or peripheral events serve as modifiers. These determine the fine structure of the vessels as they supply the ultimate functioning unit of the lung. Vessel branching occurs at the end of a pathway and lengthening or widening results from cell multiplication. Chimeric experiments have shown that angioblasts migrate widely and to considerable distances from their sites of origin. Biologic differences in vessel cells, for example, endothelial cells from different sites in the vascular tree, exist, reflecting the influence of the local milieu. The transcription factor TAL1/SCL is a marker of angioblasts. In the presence of extracellular matrix, endothelial cell precursors form protrusions that result in the formation of vascular cords and then vessels. This process requires integrin-mediated adhesions. Vascular endothelial growth factor (VEGF) plays a role as a mitogen and as a vascular morphogen in vasculogenesis. Whereas vascular smooth muscle cells arise through progenitors within the mesoderm, they are also derived from endothelial cells. In the fetal mouse, transmission electron microscopy shows that between 9 and 10 days, primitive angioblast precursors within the mesenchyme surrounding the lung bud, form vascular lakes that have hematopoietic cells in their lumen (Figures 13(a)– 13(c)). Mercox pulmonary vascular injections combined with scanning electron microscopy of the vascular casts indicate that conventional and supernumerary branches from the main pulmonary artery are derived by angiogenesis and that the more distal vessels, those of the future alveolar region, arise by vasculogenesis, that is, from a lumen that appears

ARTERIES AND VEINS 159

Figure 13 Transmission electron micrographs of fetal mouse thoraces. (a) 9 day fetus: a space between densely packed mesenchymal cells contains membrane-bound vesicles. Magnification
8750. (b) 10 day fetus: mesenchymal cells around intercellular spaces appear thin and endothelial-like. Magnification
5250. (c) 10 day fetus: some intercellular spaces contain hematopoietic precursor cells. Magnification
5250. Reproduced from deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568–581, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

locally within the mesenchyme: at 12 days, there are four generations of central arterial branches but no luminal connection is seen between these vessels and the lakes in the peripheral lung mesenchyme, where already a dense collection of lakes containing hematopoietic cells is present. By 14 days, five to seven generations of central artery branches, supernumerary and conventional, are present. There is now a connection between the central vessels and the peripheral system so that casts of the peripheral vessels are obtained by central injection (Figures 14(a)– 14(c)). Between 15 days and term, there is increasing complexity of the peripheral vascular casts reflecting an increase in the connections between the central and peripheral systems (Figure 14(d)). Whereas the processes of angiogenesis and vasculogenesis occur separately but concurrently, a third process, fusion, between these two systems is necessary for the circulation to be established.

In the human, examination of serial sections of embryos and fetuses of different ages in the Carnegie Collection of Human Embryos housed in the Carnegie Institute of Washington, DC (now located in the Museum of Human Development in the Armed Forces Institute of Pathology in Washington, DC) revealed that the blood lakes are the first to form and are present in the primitive mesenchyme around the lung bud in the neck between stages 14 and 18 (32– 44 days of gestation). As gestation progresses, abundant lakes appear in the subpleural mesenchyme. At stage 23 (56.5 days), pulmonary artery branches accompany the airways but lag behind the airway by two to three generations. The thick-walled arteries end blindly in a solid cord of cells. Between 12 and 16 weeks, an extensive capillary network is present in the subpleural mesenchyme surrounding the most distal airway buds but separated from them by mesenchyme. By 22 to 23 weeks, the capillary network

160 ARTERIES AND VEINS

S

S

R

L

R

PA

PA L

PA

PA

Aorta Aorta

13 days

12 days

(a)

(b)

PA

S

L

R

PA L

PV R

PV

CL

15 days (c)

16 days (d)

Figure 14 Photomicrographs of pulmonary arterial mercox casts of mouse fetuses. (a) 12 day fetus: up to four generations of arterial branches are present. (b) 13 day fetus: isolated patches of peripheral vessels are filled with mercox. (c) 15 day fetus: mercox filling of more peripheral vessels results in visualization of several generations of arterial branches with an increase in diameter. (d) 16 day fetus: mercox filling of the expanded peripheral vascular network results in a markedly dense cast that obscures the proximal vessels. S, systemic; R, right; L, left; PA, pulmonary artery; PV, pulmonary vein; CL, cardiac lobe. Reproduced from deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568–581, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

approaches the airway epithelium and bulges into the air space indicating that blood barriers for future gas exchange have formed. At this time, the pulmonary artery accompanies even the most distal airway branch just beneath the pleura. So from this study, it appears that in the human also, the three processes of vascular development, that is, angiogenesis, vasculogenesis, and fusion, contribute to the establishment of the pulmonary circulation. Other studies using markers for endothelial cell precursors in

mouse embryos and three-dimensional reconstruction of serial sections of human embryos suggest that the intrapulmonary vascular tree is predominantly developed by the process of vasculogenesis. Controlling Mechanisms: Genes and Factors Involved in Vessel Assembly

The path from angioblast precursor within primitive mesenchyme to mature blood vessel is complex and

ARTERIES AND VEINS 161

Primitive mesenchyme

Hemangioblasts

Endothelial cell commitment ('blood island')

Migration and tube formation

Smooth muscle recruitment and differentiation

Mature blood vessel

Proteases

SCL/tal-1

Ets-1 Fra1 Vezf1

ARNT ELF-1 EPAS Fli-1 GATA2 GATA3 HIF-1
HOXD3 NERF-2

Ets-1

AML-1 COUP-TFII HESR1 HOXB3 PPAR-

dHAND MEF2C SMAD5 SmLIM

LKLF

Transcription factors Growth factors and receptors bFGF Flk-1 Flt-1 integrin
V3 P/GH TGF- TIE2 VEGF

Angiopoietin-1 TIE2

Figure 15 The multiple steps involved in vessel assembly from primitive mesenchyme requires the sequential action of numerous factors. Reproduced from Pediatric and Developmental Pathology vol. 7, 2004, pp. 422–424, A matter of life and breath: context article, deMello DE, figure 1, with kind permission of Springer Science and Business Media.

involves a number of steps including commitment to differentiate into an endothelial cell, the action of proteases, migration and tube formation, recruitment and differentiation of smooth muscle cells, and acquisition of mature vessel structure and function. This complex process is under the coordinated control and influence of a large number of genes and growth factors (Figure 15), which have been identified by experiments involving overexpression or knockout of growth factors or genes. A fine-tuned and delicate balance in the temporal and spatial expression of a variety of genes and factors is essential for both normal intrauterine vessel development and postnatal vessel maintenance. For example, knockout of the VEGF gene or even its reduced expression in heterozygosity results in lethal defects in vessel formation in the mouse embryo, whereas overexpression of VEGF in the lung results in the formation of oversized vessels and disordered airway morphogenesis. Even the relative ratios of VEGF isoforms is critical for normal vessel and airway development. This was demonstrated in the VEGF 120 mouse in which the other VEGF isoforms, VEGF 164 and VEGF 188, are not expressed. Homozygous VEGF 120 mice have a severe reduction in the number of intra-acinar arteries and air–blood barriers and a

delay in lung development resulting in hypoplastic lungs (Figures 16 and 17). Knockout of the Flt-1 (VEGF receptor) gene produces a lethal defect in angiogenesis and knockout of the Flk-1 (VEGF receptor) gene results in a lethal failure of vasculogenesis. Transforming growth factor beta (TGF-b) is important for the eventual wall structure of the developing vasculature and influences growth, migration, and differentiation of endothelial, smooth muscle, and mesenchymal cells and pericytes. The receptor tyrosine kinase gene (tie-1) and receptor (Tie -2) are involved in the regulation of vasculogenesis and angiogenesis. Transitional Circulation and Perinatal Adaptation in Humans

At birth, as the lung expands with air, structural changes occur within the resistance segment of the pulmonary arteries producing an increase in compliance, and drop in resistance and pulmonary artery pressure as a result of an increase in external diameter and decrease in wall thickness. By 4 months the resistance arteries lose their fetal wall thickness, but in vessels less than 200 mm in diameter, wall thickness gradually increases.

162 ARTERIES AND VEINS

1 mm

E13.5

1 mm

E15.5

1 mm

1 mm

1 mm

E18.5

E18.5

E18.5

Figure 16 Photographs of fetal mouse lungs from three gestational ages: top, E13.5; middle, E15.5; bottom, E18.5. Lungs of wild-type (VEGF þ / þ , left) and heterozygous (VEGF 120/ þ , middle) fetuses do not differ in size. At all gestational ages, the lungs of homozygous fetuses (VEGF 120/120, right) are smaller than the lungs of wild-type and heterozygous littermates. Reproduced from Galambos C, Ng Y-S, Ali A, et al. (2002) Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194–203, with permission.

Structure

The distribution of preacinar elastic and transitional arteries is unchanged during childhood, although the size range varies with age. Intra-acinar arteries which develop as new alveoli are formed postnatally, are nonmuscular initially, and acquire muscle coats gradually with time. By 10 years of age, even alveolar wall level arteries have muscle coats.

Arteries and Veins in Respiratory Diseases Abnormalities of pulmonary arteries or veins can result from disorders of vessel assembly or from postnatal events that interfere with postnatal vascular growth or cause structural remodeling of the existing vasculature.

Veins

Disorders of Vessel Assembly

During childhood, the veins increase in size and peripheral veins increase in number. Within an acinus, the number of veins exceeds that of the arteries presumably to facilitate blood flow through the capillary bed.

These disorders are the consequence of aberrations in the processes involved in vessel assembly, that is, angiogenesis or vasculogenesis and result in failure of growth, overgrowth, or structural/functional abnormalities.

ARTERIES AND VEINS 163

1 mm (a)

1 mm (b)

1 mm (a)

1 mm (c)

1 mm (b)

1 mm (c)

Figure 17 Lung mercox vascular casts of VEGF 120/120 fetal mouse littermates. Genotypes: (a), wild-type; (b), heterozygous; (c), homozygous. Gestational ages: top, E17; bottom, E18. The homozygous fetal casts are smaller, and fewer peripheral vessels make the casts less dense than those of the wild type or heterozygous littermates. Reproduced from Galambos C, Ng Y-S, Ali A, et al. (2002) Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194–203, with permission.

Aberrant angiogenesis Absence of the main pulmonary artery or its branches The main pulmonary artery or one of its branches fails to grow and the lung is supplied instead by collateral vessels from the systemic circulation. Usually the pattern of intrapulmonary artery branching is unaffected so it can be presumed that central sprouting or angiogenesis fails, but that peripheral vasculogenesis is normal and the systemic collaterals assume the role of the intrapulmonary arterial tree. Misalignment of blood vessels This disorder often accompanies another serious condition, alveolar capillary dysplasia (Figure 18). The central pulmonary arteries and veins are ‘misaligned’ so that the veins are displaced from their normal location at the periphery of a lung lobule and instead share the bronchoarterial sheath. When present in all lobes of

the lung and in association with alveolar capillary dysplasia, the condition is fatal. Hypoplastic vascular tree: dwarfism and congenital diaphragmatic hernia The small thoracic cage in most forms of dwarfism or skeletal dysplasia is associated with small lungs. The main structural components of the lung, airways, alveoli, and vessels are hypoplastic but additional abnormalities suggest a direct metabolic effect as well. In congenital diaphragmatic hernia, the restricted space available for fetal lung growth produces hypoplastic lungs with a reduced number of airways and alveoli. Because the pulmonary arteries travel and branch with the airways, their number is also reduced and the size is small but appropriate for the smaller lung volume. Arterial structure is also altered so that the walls are thicker and muscle extends into smaller, more peripheral vessels.

164 ARTERIES AND VEINS

(a)

(b)

(c) Figure 18 (a) Post-mortem lung barium angiograms. (left) Preterm neonatal lung reveals filling of preacinar and intra-acinar arteries. (right) Lung of a term infant with alveolar capillary dysplasia; the angiogram has the look of a ‘pruned tree’ because of a reduced number of intra-acinar arteries. (b) In the normal lung (left), the pulmonary artery (long arrow) is present within the bronchovascular sheath and the vein (short arrow) lies within the pulmonary septum at the periphery of the lung lobule. In misalignment of the pulmonary vessels (right), the pulmonary arteries (long arrows) and veins (short arrows) lie within the same bronchovascular sheath. The pulmonary arteries are filled with barium (post-mortem injection) and the veins are empty. Movat pentachrome stain. Magnification
100. (c) The normal term lung (left) has numerous air–blood barriers (arrows) within alveolar walls. In alveolar capillary dysplasia (right) air–blood barriers are absent and vessels larger than normal capillaries are present in the middle of thickened alveolar septa (arrows). Movat pentachrome stain. Magnification
200. Reproduced from deMello DE (2004) Pulmonary pathology. Seminars in Neonatology 9: 311– 329, with permission.

Disordered vasculogenesis Alveolar capillary dysplasia In this rare and fatal disorder, air–blood barriers which are critical for gas exchange fail to form within alveolar walls. When the entire lung is affected, the condition is incompatible with independent air-breathing existence (Figure 18). Sometimes, however, a single lobe is

affected suggesting failure of a local inductive mechanism for triggering capillary growth. Fusion Intrapulmonary arteriovenous malformations point to aberrant connections between the central and peripheral pulmonary vasculature during fetal development. This is usually a circumscribed

ARTERIES AND VEINS 165

lesion suggesting that while overall angiogenesis and vasculogenesis has occurred normally, the mechanism that regulates fusion (? via chemotaxis) is faulty and results in communications between arteries and veins. Rarely, the process may involve an entire lobe. Postnatal Disorders Idiopathic (persistent) pulmonary hypertension of the newborn (PPHN), with or without meconium aspiration (also known as meconium aspiration syndrome) In both instances, the overall pattern of arterial growth is normal, but significant functional abnormalities at birth result from structural alterations in preacinar and intra-acinar arteries. The vessels are often small and have thick muscle walls and adventitial coats. There is precocious muscularization of intra-acinar arteries so muscle coats are present within smaller, normally nonmuscularized arteries. Congenital heart disease with left-to-right shunts In many types of congenital heart disease, the pulmonary circulation develops normally in utero, but adaptational changes occur after birth. An increase in pulmonary blood flow from left to right shunts interferes with postnatal intra-acinar artery growth and if left uncorrected will result in a reduction in intra-acinar artery number, increased pulmonary vascular resistance, and pulmonary hypertension. Systemic arteriovenous anastomoses in which high pulmonary blood flow occurs before birth will result in an abnormal vascular structure at birth. See also: Acute Respiratory Distress Syndrome. Alveolar Hemorrhage. Angiogenesis, Angiogenic Growth Factors and Development Factors. Arterial Blood Gases. Bronchial Circulation. Bronchopulmonary Dysplasia. Chronic Obstructive Pulmonary Disease: Acute Exacerbations. Coagulation Cascade: Factor VII. Diffusion of Gases. Epithelial Cells: Type I Cells; Type II Cells. Hypoxia and Hypoxemia. Infant Respiratory Distress Syndrome. Lung Anatomy (Including the Aging Lung). Lung Development: Overview; Congenital Parenchymal Disorders; Congenital Vascular Disorders. Lymphatic System. Neonatal Circulation. Nitric Oxide and Nitrogen Oxides. Oxygen Therapy. Oxygen Toxicity. Pediatric Pulmonary Diseases. Permeability of the Blood–Gas Barrier. Pulmonary Circulation. Pulmonary Edema. Pulmonary Vascular Remodeling. Smooth Muscle Cells: Vascular. Surgery: Transplantation. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome. Vascular Disease. Vascular Endothelial Growth Factor. Vasculitis: Overview; Microscopic Polyangiitis.

Further Reading Davies G and Reid L (1970) Thorax 25: 669–681. Davies P, deMello DE, and Reid LM (1990) Methods in experimental pathology of pulmonary vasculature. In: Gil J (ed.) Models of Lung Disease: Microscopy and Structural Methods, vol. 47 of Lung Biology in Health and Disease, pp. 843–904. New York: Dekker. deMello DE (1999) Structural elements of human fetal and neonatal lung vascular development. In: Control Mechanisms in the Fetal and Neonatal Pulmonary Circulation, Proceedings of the Ninth Pulmonary Circulation Conference, Sedalia, CO, Oct 1999, pp. 37–64. deMello DE (2004) A matter of life and breath: context article. Pediatric and Developmental Pathology 7: 422–424. deMello DE (2004) Pulmonary pathology. Seminars in Neonatology 9: 311–329. deMello DE and Reid L (2000) Embryonic and early fetal development of human lung vasculature and its functional implications. Pediatric and Developmental Pathology 3: 439–449. deMello DE and Reid L (2004) Pre- and post natal development of the pulmonary circulation. In: Haddad GG, Abman SH, and Chernick V (eds.) Basic Mechanisms of Pediatric Respiratory Disease, pp. 77–101. Ontario: BC Decker. deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, pp. 767–777. New York: Raven Press. deMello DE and Reid LM (1991) Pre and post-natal development of the pulmonary circulation. In: Chernick V and Mellins RB (eds.) Basic Mechanisms of Pediatric Respiratory Disease: Cellular and Integrative, pp. 36–54. Philadelphia: BC Decker. deMello DE and Reid LM (1997) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 1117–1127. Philadelphia: Lippincott-Raven. deMello DE and Reid LM (2002) Vascular development of lung. In: Tomanek R (ed.) Assembly of the Vasculature, vol. 2, pp. 211–237. Boston: Birkhauser. deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568–581. Galambos G, Ng YS, Ali A, et al. (2002) Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194–203. Hall SM, Hislop AA, Pierce CM, and Haworth SG (2000) Prenatal origins of human intrapulmonary arteries. American Journal of Respiratory Cell and Molecular Biology 23: 194–203. Hislop A (1971) The fetal and childhood development of the pulmonary circulation and its disturbance in certain types of congenital heart disease. PhD Thesis, London University. Hislop A and Reid L (1972) Intra-pulmonary arterial development during fetal life: branching pattern and structure. Journal of Anatomy 113: 35–48. Hislop A and Reid L (1973) Fetal and childhood development of the intrapulmonary veins in man: branching pattern and structure. Thorax 28: 313–319. Hislop A and Reid L (1973) Pulmonary arterial development during childhood: branching pattern and structure. Thorax 28: 129–135. Oettgen P (2001) Transcriptional regulation of vascular development. Circulation Research 89: 380–388.

166 ASTHMA / Overview Reid L (1978) The pulmonary circulation: remodeling in growth and disease. The 1978 J Burns Amberson Lecture. American Review of Respiratory Disease 119: 531–546. Reid L, Fried R, Geggle R, and Langleben D (1986) Anatomy of pulmonary hypertensive states. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, Vol. 4, pp. 221–263. New York: Churchill Livingstone.

Asbestos

Rendas A, et al. (1980) American Review of Respiratory Disease 121: 873–880. Schachtner SK, Wang YQ, and Baldwin SH (2000) Quantitative analysis of embryonic pulmonary vessel formation. American Journal of Respiratory Cell and Molecular Biology 22: 157–165.

see Occupational Diseases: Asbestos-Related Lung Disease.

ASTHMA Contents

Overview Allergic Bronchopulmonary Aspergillosis Aspirin-Intolerant Occupational Asthma (Including Byssinosis) Acute Exacerbations Exercise-Induced Extrinsic/Intrinsic

Overview P Chanez, Hoˆpital Arnaud de Villeneuve, Montpellier, France & 2006 Elsevier Ltd. All rights reserved.

Asthma is one of the most frequent chronic diseases. It is responsible for absenteeism from school and work, thereby handicapping daily life. Its management is based on drug therapy, control of the environment, therapeutic education, and management of triggering factors.

Abstract Asthma is one of the most prevalent chronic diseases in most of the countries in the world. Its continuous increase is clearly described in most places, confirming the potential importance of the environment. These environmental factors interact in a susceptible individual with some complex polygenic background, to reveal the asthma phenotype. Several triggers including inhaled allergens, viruses and some indoor and outdoor pollutants have been pointed as potential culprits to induce, perpetuate, or exacerbate asthma. Inflammation and structural changes are hallmarks of asthmatic airways occurring from the nose to the distal part of the lung. The relationships between those structural changes and clinical and functional abnormalities clearly deserve further investigations. Those findings led to the reinforcement of the use of inhaled corticosteroids as the pivotal treatment for asthma. The adjunction of long-acting b2 agonists has been shown to be the next logical step of pharmacology, in case of poor control when using inhaled steroids alone. The long-term management should include some tailor-made environment control and educational measures leading to a better partnership with the patients. In some occasions, the addition of leukotriene receptor antagonists, specific immunotherapy, and more recently, subcutaneous anti-IgE may offer better control for a subset of patients. A better understanding of the mechanisms, especially in the most severe forms of the disease, is paramount to develop better preventive strategies and innovative therapies.

Physiopathology Asthma is an inflammatory disorder accompanied by remodeling of the airways. This inflammation is secondary to polymorphic inflammatory infiltration, rich in mast cells, and eosinophils. In genetically predisposed subjects, this inflammation may cause symptoms which, in general, are related to diffuse variable bronchial obstruction that is spontaneously reversible or subsides under the influence of treatment. This inflammation is also associated with bronchial hyperresponsiveness to a wide variety of stimuli. This definition of asthma is supported by the physiopathological and clinical findings on the disease. The variability and reversibility of the airflow impairment, under the influence of bronchodilators and glucocorticoids, distinguish asthma from other bronchial disorders. Chronic rhinosinusitis is very often associated with asthma (approximately 80% of cases) and must be investigated, not only by questioning, but also by careful nasal examination.

ASTHMA / Overview 167

Several arguments support a link between persistent rhinosinusitis and asthma: the common characteristics of inflammation and tissue reorganization, similar epidemiology and chronicity, higher risk of asthma in cases of rhinitis, higher risk of bronchial hyperreactivity in patients suffering from rhinitis, occurrence of bronchial inflammation after nasal challenge with an allergen, and the same aggravating and triggering factors. The efficacy of local corticoid therapy constitutes another link. On the other hand, there is no solid evidence in favor of the bronchial efficacy of nasal treatment. Systemic treatments also require additional evaluation of their joint efficacy in rhinosinusitis and asthma.

Diagnosis Positive Diagnosis

This must be based on the definition of asthma. The two characteristic elements are clinical (chronicity, variability, and reversibility of symptoms), and functional. Tests of respiratory function clearly confirm the diagnosis provided that: 1. spirometry shows a reversible obstructive ventilatory disorder from 12% to 15% in comparison with theoretical values, or at least 180 ml in absolute value, with short-acting b2-mimetics; 20% or 250 ml during a 10-day test with glucocorticoids; 2. peak expiratory flow (PEF) has a variability X20%; and 3. bronchial hyperreactivity is another feature of the diagnosis. There have been few studies on the sensitivity and specificity of the functional signs of asthma and their predictive values seem insufficient. Those maintained in the international recommendations are cough, dyspnea, sibilant rhonchi (wheezing), chest tightness, and expectoration. There may be one or several symptoms. Symptoms may be absent at the time of the examination. Definition of reversibility or its significance is only obtained from expert opinions or by consensus. The same is true for the usefulness of the corticosteroid test to identify asthmatic patients; a short course of oral corticosteroid is used generally. The dose and duration of steroid treatment given in the literature are highly variable. However, these criteria are generally considered to be sufficient to propose the diagnosis of asthma and to potentially qualify nonresponding asthmatic patients as steroidresistant.

Table 1 Differential diagnosis of asthma in the child and adult In the child

In adults

Obliterating bronchiolitis Cystic fibrosis Foreign body Tracheobronchomalacia Bronchial inhalations Vocal cord dysfunction Upper airway abnormalities Immunodeficiency (IgA, IgG2, IgG4) Ciliary dyskinesia Abnormal aortic arch

Bronchiectasis Cystic fibrosis Foreign body Tracheobronchomalacia Bronchial inhalations Vocal cord dysfunction COPD Heart failure Bronchial amyloidosis Abnormal aortic arch Tracheobronchial cancer Bronchiolitis

Alternative Diagnosis

The main differential diagnoses are summarized in Table 1.

Clinical Definitions From the point of view of the terminology, three essential terms must be used and seem to be operational in clinical medicine and for the follow-up of asthmatics – the seriousness of the asthma which refers to the current state of the patient (serious acute asthma), the control of the asthma which refers to recent events (symptoms of brief duration and exacerbations), and the severity of the asthma which is mainly evaluated over the past year. Exacerbations

The definition of an exacerbation is variable and it is based on the unanticipated use of drugs, the persistent symptoms (repetition of short-lasting symptoms generally on two consecutive days), the increased bronchial obstruction, and the requirement for substantial change in treatment with oral corticoids being the most frequent. Exacerbations form part of poor control of the asthma, but differ from it. The permanence of shortlasting symptoms with return to the baseline state between these symptoms defines simple poor control, whereas an exacerbation is characterized by a persistent worsening without a return to the baseline state. Control

Several asthma evaluation control questionnaires have been developed and concern the events occurring during the 1–4 weeks before the visit. Some even cover a period of 3 months, or even the time since the previous visit. There are no data in the literature pointing to one or the other of these periods.

168 ASTHMA / Overview

Juniper showed that her questionnaire (ACQ) was more discriminating than a daily diary. The authors clearly specified that this comparison concerned clinical trials and no conclusion could be deduced for daily clinical practice. These indexes are often correlated with generic and specific quality of life questionnaires: AQLQ, SF 36, Saint Georges questionnaire. The definition of optimal (or excellent) and suboptimal (or acceptable) control is based on experts’ agreement and their clinical experience.

Table 2 Factors aggravating controlled asthma

Severity

Table 3 Examples of candidate genes implicated in the development of asthma

The notion of severity was initially based on the same parameters as control, though these were assessed retrospectively over a longer period, often including the number of successful anti-inflammatory treatments and the best level of respiratory function. At present, the assessment of asthma severity must be made more objectively, according to the control and the quantity of drugs required to obtain and maintain it. Difficult Asthma

Difficult asthma may only be defined after answering questions about its diagnostic reality, patient adherence to treatment, and the existence of major comorbidity which may interfere with management, making it impossible to obtain an acceptable control. Positive answers to these questions aid in diagnosing severe asthma. Severe asthma is characterized by frequent and serious exacerbations (stays in intensive care units), the persistence of airway obstruction, resorting to high doses of inhaled corticosteroids or even oral steroid dependence, and in some cases, by lack of steroid sensitivity. It should be pointed out that patients, nursing staff (nurses, educators in asthma schools etc.) , general practitioners, and even respiratory physicians are not always familiar with these notions of seriousness, control, exacerbations, and severity. They, therefore, often differ in their assessment of the same clinical situation. Efforts to provide information, education, and coherence are therefore required more than ever. Precipitating Factors

The risk factors of loss of control of asthma are summarized in Table 2. Serious Acute Asthma

From the patient’s point of view, the seriousness of an attack may be defined by the following characters: * *

it is unusual and a doctor must be called; it leads to the discontinuation of current activity (work, school, or play); and

Interruption of anti-inflammatory treatment Viral infections Administration of aspirin or a betablocker Hormonal factors with premenstrual recurrence in woman Contact with allergen Atmospheric pollution Domestic pollution Meteorological factors Stress

Chromosome

Candidate gene

5q 6q

Th2 related cytokines TNF-a

11q

Clara cell protein CC10 FceRIb: IgE highaffinity receptor Interferon g

12q 14q 16q

T-lymphocytes receptor IL-4 receptor a

20q

ADAM 33

*

Potential relation with asthma Airway inflammation Airway inflammation, treatment Airway inflammation, treatment

Airway inflammation Airway inflammation, treatment Airway remodeling

Certain authors note that an attack leading to a change in primary therapy must be considered to be potentially serious.

For the doctor, it is always a potentially fatal medical emergency. Table 3 presents the clinical signs of serious acute asthma (SAA). Various items have been recognized as essential to management of SAA: 1. Value of evaluation protocols. 2. Requirement to measure respiratory function (PEF) and blood gases. 3. Efficacy of treatment with inhaled short-acting b2mimetics, systemic glucocorticoids, inhaled anticholinergics (48 h mainly in the child), and oxygen therapy. 4. Value of second-line therapy with intravenous b2mimetics, nebulization using helium oxygen as vector (heliox), intravenous magnesium. 5. Requirement for regular clinical re-evaluation. 6. The criteria for hospitalization are not validated, though they are widely used and consist of PEF o60% of the predicted value or o100 l min 1, no clinical improvement, severe underlying asthma,

ASTHMA / Overview 169 Table 4 Clinical signs of serious acute asthma

Table 6 Why new therapies are needed in asthma?

Threatening syndrome Worsening in a few days Increase in the frequency of attacks Increase of severity of attacks Resistance to treatment Increase in drug consumption Disease-free intervals less and less frequent Progressive decrease in PEF

Therapy

Intermittent mild persistent

Moderate persistent

Severe persistent

Side effects

None

Few potential

Efficiency Compliance

High Low

Variable Variable

Highest frequency Low Usually better

Signs of immediate seriousness Unusual and/or progressive dyspnea Difficulty in speaking or coughing Agitation Sweats and/or cyanosis SCM muscle permanently tensed RR 430 min 1 HR 4120 min 1 Paradoxical pulse 420 mmHg PEF o150 l min 1 Gain in PEF under treatment o60 l min PaCO2 440 mmHg

Table 7 Goals for asthma treatment Minimize symptoms Achieve best lung function Prevent asthma exacerbations Obtain treatment with the best therapeutic balance Educate patients for best partnership Avoid decline in lung function 1

Signs of distress Consciousness disorders Collapsus Pause in breathing Respiratory silence PEF, peak expiratory flow; HR, heart rate; RR, respiratory flow; SCM, sternocleidomastoid.

Table 8 ICS side effects usually not clinically significant if low doses are used Frequent and mild Oral candidosis Hoarseness Potential and mild Cough Skin bruising

Table 5 Risk factors of mortality in serious acute asthma Severe asthma (bronchial obstruction during intercritical period or frequent exacerbations) Patients presenting major rapid variations in bronchial obstruction (variation in PEF 430%) Major reversibility under bronchodilators Psychosocial instability Use of three drugs (or more) for asthma Frequent resort to ER for SAA, with hospitalization and/or intensive care Poor perception of symptoms Elderly patients Patients with hypereosinophilia Poor compliance and denial of disease

and psychosocial problems. The same is true for criteria concerning the return home (PEF 4 60% or 300 l min 1). After an episode of SAA, oral glucocorticoid therapy must be prescribed for from 7 to 14 days, combined with inhaled glucocorticoid therapy and follow-up with therapeutic education. The risk factors for SAA mortality are given in Tables 4 and 5. Suissa showed that the death rate due to asthma decreased by 21% per additional vial of inhaled corticoids received during the previous year. The death rate due to SAA therefore decreased if low doses of

Rare and potentially severe Surrenal insufficiency Biologically proven but long-term clinical significance unknown Osteoporosis Impaired growth velocity Cataract

inhaled corticoids were given as primary therapy. These data validate a fundamental aspect of the longterm therapeutic management of asthma (Tables 6, 7, and 8). Asthma Associations

Bronchopulmonary allergic aspergillosis This entity is defined by the following major symptoms: 1. recurrent lung infiltrates with eosinophils, sensitive to corticoids, 2. highly characteristic proximal bronchiectasis, 3. sometimes high blood eosinophilia (41000), 4. increased total IgE and specific IgE (measured by RAST) and IgG (precipitins) immune reaction against Aspergillus fumigatus, and 5. Aspergillus can sometimes be present in sputum.

170 ASTHMA / Overview

These point to severe asthma, usually justifying long-term oral corticoid therapy and, in certain circumstances, antifungal treatment. Churg–Strauss syndrome This is a granulomatous and necrosing vasculitis. It is a rare form of asthma characterized by the severity of the respiratory symptoms, the levels of blood eosinophilia (generally above 1500 mm3), and the existence of extrarespiratory signs (neurological and cutaneous). It is often oral steroid-dependent and sometimes requires immunosuppressive agents.

Triggering and Aggravating Factors Asthma is a multifactorial syndrome. It is more appropriate to speak of triggering factors rather than etiological factors. The diagnostic procedure should take place in two stages – first, by investigating for the presence of such a factor in a patient and second, by evaluating its role in the global evaluation of the asthma (seriousness, control, and severity). Genetic Component

The genetic component is indisputable. It is well known that asthma runs in families. Conventionally, this hereditary constituent seems to be associated with atopy. However, recent work has demonstrated that even asthmatics with no demonstrable personal or family allergic factor (intrinsic asthma) had asthmatics in their family. Asthma is almost certainly polygenic in origin. Association with numerous polymorphisms have been reported at different loci in relation with or not with the known physiopathological data for the disease. The finding that different patients have different responses to antiasthmatic treatments has made it possible to develop potentially promising strategies for pharmacogenetic studies. For instance, data concerning the polymorphism of b2-mimetic receptor and the potential severity of the disease represent an interesting approach. Some examples of candidate genes implicated in the development of asthma are reported in Table 3. Environmental Component

Inhaled allergens These are allergens present in the ambient air. When inhaled in small quantities, they are capable of sensitizing subjects and triggering symptoms when they reach the level of the respiratory mucosa. Dermatophagoides pteronissimus. These form one of the major allergens of household dust. Animal proteins. Proteins produced by pets, laboratory experiments, or leisure activities.

Arthropods. They are insects such as locusts or cockroaches, which can cause asthma. Atmospheric molds and yeasts. They are an important source of allergens. Allergens present in insalubrious habitats are associated with more severe asthma. Food allergens Food and drinks may cause respiratory reactions after allergic sensitization, but nonallergic reactions or toxic reactions may also occur due to non-specific histamine release. Drugs Very often, drugs behave as haptens. Atopic subjects and asthmatics do not have any predisposition to hypersensitivity to drug products but when these occur, they are more violent. Aspirin intolerance This sometimes involves severe asthma, often with a late-onset with pan-rhino sinusitis with polyposis and systemic reactions, often with anaphylactic shock after ingestion of aspirin or nonsteroidal anti-inflammatory drugs. Occupational sensitizers They may be allergenic, but they may also act by toxic, irritative, or pharmacological mechanisms. The most important directly allergic allergen is wheat flour, responsible for bakers’ asthma. Another classical example of occupational asthma is due to work in contact with isocyanates. Demonstration that the asthma is caused by an occupational agent requires the use of a coherent, clinical, and functional diagnostic procedure, sometimes including bronchial challenge tests in the laboratory. Other factors Importance of viruses This is variable as a function of age in the triggering of asthma exacerbations. In the infant, wheezing bronchitis is usually caused by viruses though the possibility of asthma must not be over- or underestimated. Certain studies show that more than 60% of asthma exacerbations are related to a viral respiratory infection. Viral infections can be involved at various levels: * * * *

occurrence of exacerbations, prevention of the occurrence of asthma, induction of asthma, and persistence and development of chronic asthma due to chronic infections.

Bacterial infection This plays a secondary role in the physiopathology of asthma and as a triggering factor in exacerbations. The presence of endotoxins in the environment may be involved in the triggering

ASTHMA / Overview 171

of exacerbations in asthmatic patients, though it may also potentially protect an individual from the development of asthma symptoms (hygiene theory). Pollution SO2, NO2, and ozone act in synergy to trigger an attack. These different pollutants are responsible for exacerbations, sometimes serious or even very serious, especially in a child. The main sources of pollution are boiler plants (using fuel-oil and coal), household and industrial refuse incinerators, and motor vehicles (diesel engine). Particulate pollution has been shown to be associated in industrialized areas with an increase in asthma exacerbations. Diesel exhaust particles worsen allergic symptoms by amplifying the release of inflammatory mediators. Indoor pollution should not be forgotten and in particular that produced by home boilers. There are several evidences indicating that indoor pollution indicating a potential role for endotoxins, cockroaches, and parents’ smoking are major contributors for asthma exacerbations and lack of control in children with asthma. There is no definitive argument proving the involvement of atmospheric pollution in the acquisition of the asthma phenotype of a given subject. Gastroesophageal reflux Gastroesophageal reflux (GER) is more frequent in asthmatics than in a normal population. The acidity of the lower esophagus induces reflex bronchoconstriction. Contamination of the bronchi may also occur following incompetence of the cardia. Controlled studies have not shown the value of systematic treatment of GER to improve the asthma, even in the most severely affected patients. Psychological and/or psychiatric factors These are certainly involved, both in the expression of symptoms and beliefs on treatment, and therefore therapeutic compliance. They are, however, always difficult to evaluate. Influence of endocrine factors This is probable and the role of sex hormones is always mentioned first (effect of puberty, episodes of genital life in woman). Premenstrual exacerbations are rare events which can contribute to the severity of the disease. It has been shown recently that severe asthma is more frequent in women than in men. Menopause is an important period for asthma revival, poor control, or late-onset, but the exact sustaining mechanisms are yet to be understood. Sexual female hormonal replacement has been shown in one epidemiological study to be associated with an increase in emergency room attendance for asthma.

Exercise Induced Asthma

This is characterized by the occurrence of bronchial obstruction at the end of exercise. In certain cases, the asthma occurs during exercise though it is then possible to ‘run through the asthma’ if the exercise is continued. This is practically always present in children and adolescents and it may constitute a marker of bronchial hyperreactivity and inflammation. It is essential to control this form of asthma to allow normal physical activity and a harmonious physical and psychological development. Intercritical bronchial obstruction may be responsible for exertional dyspnea though this is not exertional asthma in the strict sense of the term. Smoking

Tobacco smoke (active and passive) by itself induces inflammation of the airways with hypersecretion, paralysis, and ciliary destruction, and infiltration of the distal aerial spaces by neutrophils. Active smoking is associated with a more severe asthma which responds less well to corticosteroids. It is not always easy to distinguish between asthma in the smoker and chronic obstructive pulmonary disease (COPD) with a certain degree of variability of the respiratory function. Maternal smoking may induce more asthma or atopy-associated diseases in children. Passive smoking by children is clearly responsible for uncontrollable asthma and this factor must be always looked for in the child. Many asthmatic adolescents are smokers and they have a high risk of loss of control and SAA. Adapted educational strategies must always be proposed.

Treatment Asthma treatment is based on three main axes: drug therapy, control of aggravating and triggering factors, and education of patients and health professionals. The objectives must be discussed with the patient, though the main aim is to obtain an acceptable control of the disease. Pharmacology

Corticocorticosteroids The efficacy of treatment with inhaled corticocorticosteroids is well established. It decreases functional signs, exacerbations, deaths, and costs, and improves the level of respiratory function and bronchial reactivity. All these effects have been demonstrated in comparison with placebo. Inhaled corticoids considerably improve the risk–benefit ratio of long-term treatment (Figure 1). They are indicated in persistent, mild, moderate, or

172 ASTHMA / Overview

severe asthma. The local side effects of inhaled corticoids (candidiasis, hoarse voice) are effectively prevented by rinsing the mouth after inhalation or by the use of a spacer. At recommended dosages, in persistent mild to moderate asthma (and up to 1500 mg day 1 of beclomethasone equivalents), the risks of systematic adverse effects (adrenal insufficiency, osteoporosis) are low. The main adverse effects are susceptibility to bruises and skin atrophy. In the child, the mean dosage of 400 mg day 1 of budesonide does not modify growth. However, questions still remain about inhaled corticosteroids their efficacy in modifying the severity of asthma, their interaction with the natural history of the disease, or the long-term systemic side effects are still a topic of debate.

100

75

Side effects

Efficacy

50

25

0 Mild

Moderate

Severe

Figure 1 Risk–benefit ratio potential for drugs in the treatment of persistent asthma.

Bronchodilators b2-mimetics are very potent bronchodilators. They stimulate bronchial smooth muscle b2-receptors. Short-acting b2-agonists (from 4 to 6 h) may be distinguished from long-acting b2-agonists (12 h and more). This class of drug has no or very few anti-inflammatory effects, and causes little tachyphylaxis. Side effects are minor with mainly tremors, which disappear during treatment, and usually nonserious tachycardia-type palpitations. In persistent asthma, on-demand treatment with short-acting b2-agonists must be combined with continuous anti-inflammatory treatment. In case of suboptimal or unacceptable control in patients requiring daily doses of these short-acting b2-agonists and/or if symptoms occur at night, a long-acting b2-agonist may be prescribed. The use of shortacting b2-agonists is then reserved for the treatment of episodes of dyspnea occurring in spite of wellconducted treatment. A high consumption is then associated with loss of control. It is better to give them on demand, rather than systematically, and they are not associated with a risk of an increased death rate. The value of long-acting b2-agonists is demonstrated by a high level of evidence. These agents improve the control of asthma when inhaled corticoid therapy is insufficient alone. Their combination with inhaled corticoid therapy is better than a double dose of the latter drug. This strategy improves the control of the asthma, decreases the incidence and seriousness of exacerbations, and improves patient quality of life. At present, LABAs should never be considered as the sole treatment for asthma (Figures 2 and 3).

BHR

FEV1

Hours

Days

Weeks

Symptoms

∆% DEP

Months

Exacerbations

Figure 2 Improvement of control components after first treatment initiation in asthma.

…Years

ASTHMA / Overview 173 + High-dose ICS ± OCS ± Theophylline ± AntiIGE ± Steroid sparing ± Ipratropium ± Leukotriene antagonists

+ LA
2 agonists + Moderate-dose ICS ± Leukotriene antagonists

+ Low-dose ICS SA
2 agonists as required

Mild controlled

Severe uncontrolled

Education Environment control ± specific immunotherapy Comorbidities treatment Figure 3 Treatment of asthma according to control.

Figure 4 Pathology of asthma.

Inhaled corticoid–long-acting b2-agonist combination Combination in a same inhaler of a corticoid and a long-acting b2-agonist follows recommendations for the treatment of persistent, moderate to severe asthma, with facilitated administration, and the objective of improving compliance, notably of corticoid therapy. Two combinations have been developed: fluticasone–salmeterol and formoterol– budesonide. The benefit of these fixed combinations in comparison with separate combination of longacting b2-agonists and inhaled corticocorticosteroids

is not completely established and nor is the value of a fixed combination administered systematically or modulated on demand (Figure 4). Cysteinyl–leukotriene receptor antagonists The cysteinyl–leukotriene receptor antagonists used at present are montelukast and zafirlukast. They are used orally and potentially can treat asthma and rhinosinusitis. These agents have been shown to be useful in exercise induced-asthma, as well as a singleagent therapy of mild or moderate asthma. They lead

174 ASTHMA / Overview

to bronchodilation and have been able to improve asthma control in long-term placebo-controlled studies. Their equivalence with low-dose inhaled corticoid therapy (400 mg day 1 of beclomethasone) is suspected but not fully demonstrated. The equivalence of a combination of an antileukotriene in comparison with a long-acting b2-agonist in noncontrolled patients treated with inhaled corticoid therapy alone is not proven. Their role in severe asthma is uncertain; it should be interesting, though, to follow their benefit in aspirin-induced asthma, which was recently demonstrated in some clinical studies. Theophylline This is a less potent bronchodilator than the b2-mimetics. It also has numerous other actions and in particular an anti-inflammatory activity. Its effects are strictly dependent on its serum concentration, which must be maintained in a relatively narrow range between 10 and 20 mg l 1. Side effects are also dose-dependent. At present it is not used much for primary treatment of asthma, except in the most severely affected patients. Anticholinergics Ipratropium and oxitropium are antagonists of muscarinic receptors. They are bronchodilators but are less potent and slower to act than b2-agonists. They are indicated in the treatment of SAA and exacerbations of chronic asthma as a complement to short-acting b2-agonists. Their efficacy beyond the first 48 h of the treatment is unknown. Proof of their efficacy exists especially for treatment in children. There are few adverse effects. Their efficacy in the management of chronic asthma has not been evaluated. Tioptropium (Spiriva) is a long acting anticholinergic drug, which has been developed for the treatment of COPD. Its potential interest in asthma and specifically severe asthma deserves further investigations. Control of the Environment

Although environmental control seems useful, there is no complete evidence of its efficacy. It comprises the reduction of exposure to allergens and non-specific irritants and the prevention of active and passive smoking. Allergy is one of the major factors associated with asthma and the most important for children of school age. Prevention of exposure to allergens is logically the first measure to be taken. This obviously concerns pets though this is not necessarily well accepted by patients or close relatives and friends. It is possible to try to eradicate house-dust mites though this is rarely complete. Diagnosis of occupational

asthma with a maximum of evidence may force a patient to change jobs. It is more difficult to adopt outdoor pollution-control measures which mainly concern the authorities. Immunotherapy

Specific immunotherapy is of some help in allergic asthmatic patients if simple rules are applied. Patients with persistent severe rhinitis and persistent mild asthma benefit most from this treatment. It is contraindicated in uncontrolled and severe asthma. On the other hand, the development of anti-IgE strategies may give hope to better control in severely allergic patients. Education

Education of patients is indicated in all asthmatics, though it should be adapted to the severity, importance of triggering factors, and specific personality of each patient. It must be based on a precise educational diagnosis. Objectives must be defined in partnership with the patient. The educational methods used depend on these objectives. This therapeutic education may be individual or may be provided in institutions such as asthma schools.

Conclusion Asthma is a bronchial inflammatory disease that alters the structure of the airways including the upper airways, which may sometimes have systemic repercussions. It is chronic, variable, and reversible and also a multifactorial, polygenic, and environmental disease. Management must be based on a long-term strategy. The major objective is to obtain satisfactory control to allow an optimal quality of life. Treatment involves use of a potent and well-tolerated pharmacopoeia, but may only be implemented in partnership with the patient. See also: Asthma: Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; Exercise-Induced; Extrinsic/Intrinsic. Bronchodilators: Anticholinergic Agents. Capsaicin. Carbon Monoxide. CD14. Chemokines. Chymase and Tryptase. Cilia and Mucociliary Clearance. Cyclic Nucleotide Phosphodiesterases. DNA: Repair. Dust Mite. Environmental Pollutants: Oxidant Gases. Eotaxins. Epidermal Growth Factors. Extracellular Matrix: Surface Proteoglycans. Fibroblasts. Gastroesophageal Reflux. G-Protein-Coupled Receptors. Histamine. Immunoglobulins. Interferons. Kinins and Neuropeptides: Vasoactive Intestinal Peptide; Other Important Neuropeptides. Leukocytes: Eosinophils. Lipid Mediators: Overview; Leukotrienes; Prostanoids; Platelet-Activating Factors. Oxidants and Antioxidants: Oxidants. Pediatric Pulmonary

ASTHMA / Overview 175 Diseases. Proteinase-Activated Receptors. Signs of Respiratory Disease: Lung Sounds. Symptoms of Respiratory Disease: Dyspnea. Tumor Necrosis Factor Alpha (TNF-a ).

Further Reading Abramson MJ, Puy RM, and Weiner JM (2003) Allergen immunotherapy for asthma. Cochrane Database of Systematic Reviews 4: CD001186. Agertoft L and Pedersen S (2000) Effect of long-term treatment with inhaled budesonide on adult height in children with asthma. New England Journal of Medicine 343: 1064–1069. American Thoracic Society (1995) Standardization of spirometry, 1994 update. American Journal of Respiratory and Critical Care Medicine 152: 1107–1136. ANAES (2004) Recommandations pour le suivi me´dical des patients asthmatiques adultes et adolescents. Paris 169pp (with an english translation on site www.anaes.fr). Anderson SD and Holzer K (2000) Exercise-induced asthma: is it the right diagnosis in elite athletes? Journal of Allergy and Clinical Immunology 106: 419–428. Barnes PJ (1995) Inhaled glucocorticoids for asthma. New England Journal of Medicine 332: 868–875. Barnes PJ (2004) Corticoresistance in airway disease. Proceedings of the American Thoracic Society 1: 264–269. Bateman ED, Boushey HA, Bousquet J, et al. (2004) GOAL Investigators Group. Can guideline-defined asthma control be achieved? The gaining optimal asthma control study. American Journal of Respiratory and Critical Care Medicine 170: 836–844. British Thoracic Society (2003) British guideline on the management of asthma. Thorax 58(supplement 1): S1–S83. Chung KF, Godard P, Adelroth E, et al. (1999) Difficult/therapyresistant asthma: the need for an integrated approach to define clinical phenotypes, evaluate risk factors, understand pathophysiology and find novel therapies. European Respiratory Journal 13: 1198–1208. Cookson W (1999) The alliance of genes and environment in asthma and allergy. Nature 402(supplement 6760): B5–B11. Cottin V and Cordier JF (1999) Churg–Strauss syndrome. Allergy 54: 535–551. D’Amato G, Liccardi G, D’Amato M, and Cazzola M (2002) Outdoor air pollution, climatic changes and allergic bronchial asthma. European Respiratory Monograph 21: 30–51. De Blay F, Casel S, Pauli G, and Bessot JC (2000) Respiratory allergies and household allergenic environment. Revue des Maladies Respiratories 17: 167–176. Ducharme FM (2003) Inhaled glucocorticoids versus leukotriene receptor antagonists as single agent asthma treatment: systematic review of current evidence. British Medical Journal 326: 621. Eggleston PA, Bush RK, and American Academy of Asthma, Allergy and Immunology (2001) Environmental allergen avoidance: an overview. Journal of Allergy and Clinical Immunology 107(supplement 3): S403–S405. Everard ML, Bara A, Kurian M, Elliott TM, and Ducharme F (2002) Anticholinergic drugs for wheeze in children under the age of two years. Cochrane Database of Systematic Reviews 1: CD001279. Gern JE and Busse WW (2000) The role of viral infections in the natural history of asthma. Journal of Allergy and Clinical Immunology 106: 201–212. Gibson PG, Coughlan J, and Wilson AJ (1998) The effects of self management and asthma education and regular practioner

review in adults with asthma. Cochrane Database of Systematic Reviews 3: CD001279. Gibson PG, Henry RL, and Coughlan JL (2003) Gastro-oesophageal reflux treatment for asthma in adults and children. Cochrane Database of System Review 2: CD001496. Holgate ST (1999) Genetic and environmental interaction in allergy and asthma. Journal of Allergy and Clinical Immunology 104: 1139–1146. Juniper EF, O’Byrne PM, Ferrie PJ, King DR, and Roberts JN (2000) Measuring asthma control. Clinic questionnaire or daily diary? American Journal of Respiratory and Critical Care Medicine 162: 1330–1334. Juniper EF, O’Byrne PM, Guyatt GH, Ferrie PJ, and King DR (1999) Development and validation of a questionnaire to measure asthma control. European Respiratory Journal 14: 902–907. Kips JC, O’Connor BJ, Inman MD, et al. (2000) A long-term study of the anti-inflammatory effect of low-dose budesonide plus formoterol versus high-dose budesonide in asthma. American Journal of Respiratory and Critical Care Medicine 161: 996– 1001. Kips JC, Peleman RA, and Pauwels RA (1999) The role of theophylline in asthma management. Current Opinion in Pulmonary Medicine 5: 88–92. Lemiere C, Bai T, Balter M, et al. (2004) Adult asthma consensus guidelines update 2003. Canadian Respiratory Journal 11(supplement A): A9–A18. Meslier N, Racineux JL, Six P, and Lockhart A (1989) Diagnostic value of reversibility of chronic airway obstruction to separate asthma from chronic bronchitis: a statistical approach. European Respiratory Journal 2: 497–505. O’Hollaren MT, Yunginger JW, Offord KP, et al. (1991) Exposure to an aeroallergen as a possible precipitating factor in respiratory arrest in young patients with asthma. New England Journal of Medicine 324: 359–363. Parker JM and Guerrero ML (2004) Airway function in women: bronchial hyperesponsiveness, cough and vocal cord dysfunction. Clinics in Chest Medicine 25: 321–330. Pauwels RA, Lofdahl CG, Postma DS, et al. (1997) Effect of inhaled formoterol and budesonide on exacerbations of asthma. New England Journal of Medicine 337: 1405–1411. Reddel H, Ware S, Marks G, et al. (1999) Differences between asthma exacerbations and poor asthma control. Lancet 353: 364–369. Rosenstreich DL, Eggleston P, Kattan M, et al. (1997) The role of cockroach allergy and exposure to cockroach allergen in causing morbidity among inner-city children with asthma. New England Journal of Medicine 336: 1356–1363. Salmeron S, Liard R, Elkharrat D, et al. (2001) Asthma severity and adequacy of management in accident and emergency departments in France: a prospective study. Lancet 358: 629–635. Shrewsbury S, Pyke S, and Britton M (2000) Meta-analysis of increased dose of inhaled steroid or addition of salmeterol in symptomatic asthma (MIASMA). British Medical Journal 320: 1368–1373. Sistek D, Tschopp JM, Schindler C, et al. (2001) Clinical diagnosis of current asthma: predictive value of respiratory symptoms in the SAPALDIA study. Swiss study on air pollution and lung diseases in adults. European Respiratory Journal 17: 214–219. Suissa S, Ernst P, Benayoun S, Baltzan M, and Cai B (2000) Lowdose inhaled corticocorticosteroids and the prevention of death from asthma. New England Journal of Medicine 343: 332–336. ten Brinke A, Ouwerkerk ME, Zwinderman AH, Spinhoven P, and Bel EH (2001) Psychopathology in patients with severe asthma is associated with increased health care utilization. American Journal of Respiratory and Critical Care Medicine 163: 1093–1096.

176 ASTHMA / Allergic Bronchopulmonary Aspergillosis Thomas NS, Wilkinson J, Lio P, et al. (2000) Genetic factors involved in asthma and atopy. Studies in British families. Revue des Maladies Respiratories 17: 177–182. Thomson NC, Chaudhuri R, and Livingston E (2004) Asthma and cigarette smoking. European Respiratory Journal 24: 734–739. Venables KM and Chan-Yeung M (1997) Occupational asthma. Lancet 349: 1465–1469. Vignola AM, Chanez P, Godard P, and Bousquet J (1998) Relationships between rhinitis and asthma. Allergy 53: 833–839. Vollmer WM, Markson LE, O’Connor E, et al. (1999) Association of asthma control with health care utilization and quality of life. American Journal of Respiratory and Critical Care Medicine 160: 1647–1652. Wark PA and Gibson PG (2001) Allergic bronchopulmonary aspergillosis: new concepts of pathogenesis and treatment. Respirology 6: 1–7. WHO/NHLBI Workshop Report (1995) Global Strategy for Asthma Management and Prevention. National Institutes of Health, National Heart, Lung and Blood Institute, Publication Number 95-3659 (revised 2002). Wilson AJ, Gibson PG, and Coughlan J (2003) Long acting beta-agonists versus theophylline for maintenance treatment of asthma. Cochrane Database of Systematic Reviews 3: CD001281. Woolcock AJ and Peat JK (1997) Evidence for the increase in asthma worldwide. Ciba Foundation Symposium 206: 122–134.

Allergic Bronchopulmonary Aspergillosis P Wark, Southampton University, Southampton, UK & 2006 Elsevier Ltd. All rights reserved.

Introduction Allergic bronchopulmonary aspergillosis (ABPA) is a complex condition that results from hypersensitivity to the fungus Aspergillus fumigatus (Af). ABPA was first described in the UK in 1952, and has been estimated to occur in 1–2% of chronic asthmatics and up to 15% of patients with cystic fibrosis. It is unclear whether the prevalence of ABPA has declined with the widespread use of inhaled corticosteroids to treat asthma. There is an excessively high prevalence of ABPA in cystic fibrosis where evidence suggests that ABPA may cause a faster decline in lung function. In chronic asthma, ABPA follows a more variable course with recurrent exacerbations and, at least in some patients, it leads to proximal bronchiectasis and irreversible fibrotic lung disease. Exposure to Af can cause a wide range of pulmonary diseases, which are summarized in Figure 1, including: severe life-threatening pneumonia in the immunosuppressed; subacute infections such as the

formation of aspergilloma in those with pre-existing pulmonary cavities; a form of extrinsic allergic alveolitis; and hypersensitivity diseases. Subjects with asthma or cystic fibrosis (CF) may become sensitized to Af following inhalation of spores; once sensitized this results in a type I, IgE-mediated reaction and a spectrum of clinical responses from acute exacerbations of asthma to a more sustained and intense inflammatory response that shows features of both type I and type III hypersensitivity and leads to ABPA. ABPA is unique in these disorders as there is evidence of persistence of the organism within the airways resulting in an intense local and systemic immune response that is associated with mucus impaction and may lead to bronchiectasis and pulmonary fibrosis.

Etiology Fungi belonging to the genus Aspergillus are ubiquitous spore-forming filamentous fungi and while any member of the group can cause allergic sensitization most disease is attributable to Af. The organism is present in outdoor environments where it readily grows in decomposing organic matter, such as decaying vegetation in soil, mulches, wood chips, freshly mown grass, and sewage treatment debris. Indoors, Af can be found in damp areas especially on walls or ceilings where water damage has occurred; in addition, spores can be found in very large quantities in bird excreta. Acute inhalation of spores is known to trigger acute asthma in sensitized asthmatics, while in subjects with ABPA inhalation of Af antigens leads to a dramatic worsening of the acute and late-phase response with falls in FEV1 of 50–79%. Exacerbations of clinical disease have been reported to occur coincident with high outdoor spore counts but a direct temporal relationship to inhalation and an acute exacerbation of ABPA remains unclear. A genetic predisposition to ABPA is also possible, with reports of familial occurrence. Two small studies have shown a higher carriage rate of at least one mutation of the cystic fibrosis transmembrane regulator (CFTR) gene in subjects with ABPA, compared to subjects who are skin-test positive to Af and those with asthma alone. The alleles HLA-DR2 and -DR5 have also been linked to severity of disease in ABPA. Most recently, a polymorphism of surfactant proteins has been shown to be associated with increased serum IgE and blood eosinophilia in patients with ABPA. These findings suggest there may be a link between ABPA and an inability to effectively clear the organism in a group whose immune response is likely to be one associated with atopy and a T-helper (TH)-2 response.

ASTHMA / Allergic Bronchopulmonary Aspergillosis 177

No previous lung disease

Asthma

Cystic fibrosis

Cavitatary lung disease

Immunocompromised

Inhale Af spores

Acute asthma (those sensitized up to 30%)

No immune response

Colonization with Af

Mucoid impaction extrinsic allergic alveolitis (all extremely rare)

ABPA (1− 2%)

ABPA (10 −15%)

Aspergilloma

Invasive pulmonary aspergillosis

Figure 1 A summary of the wide range of pulmonary diseases caused by exposure to Aspergillus fumigatus. Aspergillus fumigatus is a ubiquitous environmental fungus and spores are regularly inhaled. For most individuals with no previous lung disease, this appears to cause no immune response and no symptoms. High-dose exposure or exposure of susceptible individuals may trigger extrinsic allergic alveolitis or, more rarely, colonization leading to an intense immune response as in ABPA with areas of mucoid impaction and inflammation. Up to 30% of subjects with asthma are sensitized to Af. In those individuals exposure to Af may initiate an acute asthma response. A proportion of asthmatics seem to be particularly prone to the effects of Af and are unable to clear the organism. It persists in the lower airways and initiates the intense immune response resulting in ABPA, which occurs in 1–2% of asthmatics. In CF, particularly in those with atopy or a history of asthma-like symptoms, sensitization to Af occurs. In at least 10–15% of CF individuals the organism is not cleared and colonization ensues. This initiates an immune response like that seen in asthma but is associated also with the endobronchial inflammation and chronic infection already present in CF. An aspergilloma (fungal ball) consists of masses of fungal mycelia, inflammatory cells, fibrin, mucus, and tissue debris, these usually developing in a preformed lung cavity. In a study of 549 subjects with pulmonary cavities due to old tuberculosis, aspergillomas were present in 11%. Subjects with immunosuppression subsequent to chemotherapy or the use of immunosuppressives or with disorders such as chronic granulomatous disease are unable to clear Af. In these cases the immune response is so ineffective that direct fungal invasion occurs, leading to a severe acute pneumonia, sepsis, and often death.

Pathology In the early stages of ABPA, pulmonary lesions consist of bronchial mucoid impaction, and areas of eosinophilic pneumonia. In mucoid impaction, bronchi are dilated and filled with mucus that contains necrotic eosinophils, Curschman’s spirals, Charcot-Leyden crystals, and occasionally fungal hyphae. Eosinophilic pneumonia is synonymous with radiographic infiltrates, composed of focal areas of alveoli filled with eosinophils and macrophages. As the disease progresses, endobronchial inflammation increases, which begins as a mixed eosinophilic/neutrophilic obliterative bronchiolitis. This then progresses to the complete replacement of bronchial structures by inflammatory cells (histiocytes, lymphocytes, and plasma cells),

known as bronchocentric granulomatosis. At the center of these areas are necrotic inflammatory cells and Af hyphae. Destruction of the bronchial wall matrix and subsequent scarring and repair are thought to account for the development of bronchiectasis and fibrosis. The intensity of the inflammatory infiltrate present in the airways of subjects with ABPA certainly correlates with the severity of bronchiectasis present. Pathological features of ABPA are demonstrated in Figure 2.

Clinical Features Diagnosis and Disease Staging

The criteria for diagnosis were standardized in 1977 by Patterson and co-workers and are summarized in

178 ASTHMA / Allergic Bronchopulmonary Aspergillosis Table 1 Diagnostic criteria for ABPA Essential criteria Asthma Immediate skin-prick test (SPT) positive to Af Total serum IgE 4 1000 ng ml
1 (400 IU ml
1) Serum specific IgG and IgE antibodies to Af (or positive precipitins) Presence of bronchiectasis Yes, define as ABPA-CB No, define as ABPA-S

(a)

(b)

(c) Figure 2 (a) Hyphae of Aspergillus fumigatus grown in vitro; (b) mucoid impaction. The dilated bronchus is filled with mucus that contains eosinophils, cell debris, and the products of eosinophil degranulation (Charcot-Leyden crystals). The bronchial epithelium is heavily infiltrated by inflammatory cells. (c) Bronchocentric granulomatosis: the epithelium of the bronchus becomes completely replaced by a granulomatous infiltrate of histiocytes, lymphocytes, plasma cells, and eosinophils. In addition, there are necrotic inflammatory cells and mucus within the dilated airway and marked generalized bronchial wall thickening. Slides Courtesy of Dr B Addis, Southampton General Hospital.

Table 1. They require the patient to have a pre-existing diagnosis of asthma (or cystic fibrosis), immediate-type skin reactivity to Af, and, at least during exacerbations or in the absence of treatment,

Nonessential criteria Transient pulmonary infiltrates on chest radiograph Blood eosinophilia Precipitating antibodies to Af Expectoration of mucus plugs

peripheral blood eosinophilia, precipitating antibodies to Af antigen, elevated serum IgE, and IgG antibodies against Af. Radiological evidence of proximal bronchiectasis is frequently found with ABPA, but is no longer felt to be a prerequisite for diagnosis. However, the presence of bronchiectasis along with skin-test reactivity and eosinophilia is quite specific for ABPA. ABPA has been subdivided into five stages. Stage I is the initial acute presentation, with eosinophilia, immediate-type skin reactivity to Af, total serum IgE greater than 2500 ng ml
1, and pulmonary infiltrates on a chest radiograph. Stage II is the disease in remission, where there is persistent immediate-type skin reactivity and precipitating antibodies to Af antigens. In stage III there is an exacerbation of symptoms with all the characteristics of stage I but with a twofold rise in serum IgE and new pulmonary infiltrates. Stage IV patients have asthma where control of symptoms is dependent on chronic use of highdose corticosteroids. In stage V, chronic disease has progressed to predominately fixed airflow obstruction with extensive bronchiectasis and fibrosis. Skin-prick testing is a useful screening test to identify potential patients with ABPA. It is highly sensitive but not specific. Consequently, a negative skin-prick test for Af can rule out ABPA whereas a positive test warrants further investigation, particularly in the case of an asthmatic with frequent exacerbations or parenteral corticosteroid dependence. Patients with a positive skin test should be evaluated with serology including measurement of total serum IgE, specific IgG, and IgE antibodies to Af and precipitins. Patients with an IgE 4 1000 ng ml
1 (400 IU ml
1) and positive IgE Af and IgG Af are likely to have ABPA and warrant further investigation with a high-resolution computed tomography (CT) scan of the chest to determine the presence of bronchiectasis. Patients with central bronchiectasis are classed as

ASTHMA / Allergic Bronchopulmonary Aspergillosis 179

ABPA-CB (ABPA with central bronchiectasis) and those without are classed as ABPA-S (ABPA serology). In milder disease, a high index of suspicion is required, with the criteria fulfilled only during an exacerbation or when off parenteral corticosteroids. Radiology

During acute disease flares chest radiographs may demonstrate transient pulmonary infiltrates that occur as a result of eosinophilic pneumonitis and areas of mucoid impaction that appear as areas of atelectasis with toothpaste shadows and the finger-in-glove appearance of bronchi filled with inspissated mucus. Permanent radiographic changes are a feature of more advanced disease with the development of central bronchiectasis and in some areas of parenchymal fibrosis. While bronchial wall thickening and minimal bronchiectasis (limited to 1–2 segments) has been described in asthma, the presence of widespread central bronchiectasis, which is best seen on high-resolution CT scans, is characteristic of ABPA. In addition, areas of bronchial wall thickening, mucoid impaction, circular opacities, and areas of atelectasis are common. ABPA and Cystic Fibrosis

Diagnosis of ABPA in CF is more difficult though the criteria have recently been reviewed and standardized by a consensus conference; these criteria are summarized in Table 2. The diagnosis in CF is complicated by the pre-existing presence of bronchiectasis and chronic endobronchial infection. A multiple regression analysis of over 14 000 individuals with CF determined that wheezing, a diagnosis of bronchial asthma, and colonization with Pseudomonas aeruginosa were independent risk factors for ABPA. The development of ABPA in someone with cystic fibrosis heralds a more complicated clinical course and is associated with more bacterial colonization, lower lung function, and an increase in pulmonary complications.

Pathogenesis Af is an effective pathogen in humans as intrinsic qualities of the organism enhance its ability to infect Table 2 Diagnostic criteria for ABPA in cystic fibrosis An acute or subacute deterioration in clinical symptoms (cough, wheeze, exercise capacity, change in pulmonary function, or increase in sputum) not attributable to another cause Immediate skin test reactivity to Af A total serum IgE 4 400 IU ml
1 Along with one of the following: precipitins to Af or specific IgG to Af new or recent infiltrates, mucus plugging, or proximal bronchiectasis on either chest radiograph or CT scan

the lungs and cause disease. The spores of Af are 2– 5 mm in size with a hydrophobic coat that allows them to be inspired into the lungs. Once inspired, the conidia of Af are able to bind to surfactant molecules in the distal airway lumen, as well as complement (C3) and fibrinogen. The conidia germinate within the airways and form focal areas of mycelia. The mature organisms are then capable of releasing allergens, virulence factors, and proteases. These factors contribute to: (1) impaired mucociliary clearance; (2) impaired action of fungicidal proteins and complement in the airway lining fluid; and (3) inhibition of phagocytosis and the killing capacity of phagocytic cells (macrophages, neutrophils). As the organism is ubiquitous in the environment, sensitized individuals are likely to regularly inhale spores and this will always represent a fresh source of antigenic stimulus. However, Af is unique as an aeroallergen and inhalation alone is insufficient to lead to ABPA. Persistence of viable Af within the airways appears to be an important factor in determining the development of ABPA. Viable Af has been found growing on and between bronchial epithelial cells, despite an intense inflammatory cell infiltrate while Af proteases lead to the release of proinflammatory mediators from epithelial cells. These proteases also have the ability to detach epithelial cells from their basement membrane and are particularly potent. This loss of epithelial integrity may lead to exposure of the underlying matrix, to which Af can also adhere allowing direct damage of the airways and the development of bronchiectasis; in addition, the disruption of mucosal integrity enhances antigen uptake and activation of T cells. In addition to its direct effect on the airway epithelium, Af appears to be able to elicit a powerful TH2 immune response. The presence of Af infection in mice induces a TH2 lymphocyte response, while the extracts from Af when co-cultured with B cells elicit the release of IgE and subjects with ABPA develop specific TH2 CD4 þ cells in response to exposure. Epithelial cell activation and a TH2 immune response favors recruitment and activation of eosinophils. Eosinophil activation and release of toxic granular proteins are likely to contribute to damage to both the epithelium and airway matrix. In asthma TH2 cells also release IL-4 and IL-13, which induces the release of TGF-b from epithelial cells, leading to a repair response with myofibroblast activation, fibrosis, and airway wall remodeling. This intense recruitment of inflammatory cells to the airway lumen and the development of progressive airway wall damage is in keeping with airway inflammation found in patients with ABPA and the development of radiographic bronchiectasis.

180 ASTHMA / Allergic Bronchopulmonary Aspergillosis

1

Mucoid impaction

Af conidia

Tenacious mucus

Alveolar macrophage 3

2

Proteases

Epithelial detachment

Mycelia

Fibroblast 8 Allergens

Inflammatory cytokines/chemokines

4

6 5

IgE IL-5

B cell

TH2 lymphocyte

IL-4 & IL-13

Eosinophil

7

Figure 3 A model for the pathogenesis of ABPA. 1: The conidia of Af are just 2–5 mm in diameter and are easily respired into the distal airways. 2: The conidia are trapped within the tenacious mucus bilayer, binding to surfactants, complement, and fibrinogen. The conidia attach to bronchial epithelial cells. Here they germinate and mature, forming mycelia or small fungal balls. 3: Mature Af produces proteases and allergens. Aspergillus proteases are extremely potent. They activate epithelial cells, releasing proinflammatory cytokines and chemokines, leading to the hypersecretion of mucus, thus damaging epithelial cells, which results in widespread epithelial detachment and the loss of mucosal integrity. This allows the proteases to damage the airway matrix also. In addition, proteases can block the ability of alveolar macrophages to ingest Af, the net effect being impaired mucociliary clearance and facilitated colonization. 4: Activated epithelial cells release proinflammatory mediators, such as interleukin-8 (IL-8), IL-6, RANTES, and MCP-1, activating and attracting lymphocytes, eosinophils, and neutrophils to the airways. 5: Aspergillus allergens are potent activators of TH2 type immune response from TH cells. These cells release IL-5, which recruits and activates eosinophils. 6: Eosinophils migrate to the airways and infiltrate. Once activated, they release toxic granular proteins that further damage the epithelium and airway matrix. 7: Activated TH2 lymphocytes produce IL-4 and IL-13, which induce isotype switching of bells to produce IgE antibodies. In addition, IL-4 and IL-13 have been shown to induce epithelial cells to secrete the anti-inflammatory but pro-fibrotic mediator TGF-b. 8: Epithelial and airway matrix damage elicits a wound-repair response in the airways. Release of TGF-b has been shown to induce fibroblasts to release growth factors and collagen. This is thought, at least in part, to induce airway wall remodeling in asthma.

A model for the pathogenesis of ABPA is proposed in Figure 3. ABPA appears to develop in susceptible individuals who are sensitized to Af; they are unable to clear the organism, which then adheres to the bronchial epithelium releasing allergens and proteases. This activates an intense TH2 immune response and leads to progressive airway destruction and remodeling.

Animal Models A number of investigators have employed animal models to determine the role of Af antigens in triggering the immune response and in determining the

constitutive components responsible for this response. Most investigators have used crude Af extract to sensitize animals, often followed by ongoing exposure to viable conidia or Af spores. Uniquely, sensitization with Af extract does not require an adjunct; it is thought that Af proteases effectively disrupt the mucosal barrier enhancing antigen presentation. Recently, recombinant antigen components of Af extract have been used; identifying Asp f 1, 3, and 4 as independent inducers of airway inflammation and bronchial hyperresponsiveness (BHR). The most widely employed animal model has been murine. As in humans, sensitization to Af leads to a

ASTHMA / Aspirin-Intolerant Table 3 Components of the immune response to Aspergillus elucidated by murine models of ABPA Immune mediator

Response observed in the model

Block IL-13

Reduced pulmonary eosinophilia and goblet cell hyperplasia No change to subepithelial fibrosis Reduced IgE and pulmonary eosinophila More pronounced TH1 response and consequent pulmonary inflammation Inhibit all aspects of the TH2 inflammatory response to Af Reduced pulmonary eosinophils Airway inflammation occurs with monocyte infiltrate and BHR still increased (a) Treated at time of Af sensitization, increased airway inflammation, and BHR (b) Treated at 14–30 postsensitization, reduced airway inflammation, and BHR Increased TH2-mediated inflammation Increased IgE production Reduced TH1 response with lower levels of interferon-g and reduced pulmonary neutrophils Reduced clearance of Af

Block IL-4

Addition of IL-10 Block IL-5

Inhibit MCP-1 or inactivate receptor CCR2 CCR2 knockout mice

potent response with production of IgE, IgG1, pulmonary eosinophilia, and TH2 activation and their recruitment to the airways. In addition, such models have identified Af as potent inducers of BHR, airway remodeling, goblet cell hyperplasia, and mucus hypersecretion. The ability to use knockout animals and block specific cytokines, chemokines, and their receptors in murine models has allowed a greater understanding of the specific components of the immune response to Af; the most important of these are summarized in Table 3. The profound TH2 immune response to Af has been well demonstrated in these models along with the suggestion that such a response is relatively ineffective in clearing the organism. See also: Chemokines, CXC: IL-8. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Interleukins: IL-4; IL-13. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Bateman ED (1994) A new look at the natural history of aspergillus hypersensitivity in asthmatics. Respiratory Medicine 88: 325. Bosken CH, Myers JL, et al. (1988) Pathological features of allergic bronchopulmonary aspergillosis. American Journal of Surgical Pathology 12(3): 216–222. Chetty A (2003) Pathology of allergic bronchopulmonary aspergillosis. Frontiers in Bioscience 8: e110–e114. Greenberger P, Miller T, et al. (1993) Allergic bronchopulmonary aspergillosis in patients with and without evidence of bronchiectasis. Annals of Allergy 70: 333–338.

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Grunig G and Kurup VP (2003) Animal models of allergic bronchopulmonary aspergillosis. Frontiers in Bioscience 8: e157–e171. Kauffman HF (2003) Immunopathogenesis of allergic bronchopulmonary aspergillosis and airway remodelling. Frontiers in Bioscience 8: e190–e196. Kauffman HF and Tomee JF (1999) Inflammatory cells and airway defense against Aspergillus fumigatus. Immunology and Allergy Clinics of North America 18: 619–640. Kurup VP and Banerjee B (1996) Allergic aspergillosis: antigens and immunodiagnosis. Advances in Medical Mycology 2: 133–154. Patterson R, Greenberger P, et al. (1982) Allergic bronchopulmonary aspergillosis: staging as an aid to management. Annals of Internal Medicine 96: 286–291. Soubani AO and Chandrasekar PH (2002) The clinical spectrum of pulmonary aspergillosis. Chest 121: 1988–1999. Stevens D, Moss RB, et al. (2003) Allergic bronchopulmonary aspergillosis in cystic fibrosis state of the art: Cystic Fibrosis Foundation Consensus Conference. Clinical Infectious Diseases 37(supplement 3): S225–S264. Varkey B (1998) Allergic bronchopulmonary aspergillosis: clinical perspectives. Immunology and Allergy Clinics of North America 18(3): 479–501. Wark PAB and Gibson PG (2001) Allergic bronchopulmonary aspergillosis: new concepts of pathogenesis and treatment. Respirology 6: 1–7. Wark PAB, Gibson PG, et al. (2003) Azoles for allergic bronchopulmonary aspergillosis associated with asthma. Cochrane Database System Review 4(CD001108).

Aspirin-Intolerant A P Sampson, University of Southampton School of Medicine, Southampton, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Aspirin-intolerant asthma (AIA) is a phenotype experienced by 10–20% of persistent asthmatics, in whom acute bronchoconstriction is induced by ingestion of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs share the ability to inhibit synthesis of prostanoids by blockade of cyclooxygenase (COX). Acute reactions to NSAIDs can be life threatening and may be associated with rhinoconjunctival and dermal symptoms. Drugs that selectively inhibit COX-2 appear to be better tolerated than nonselective inhibitors of COX-1 and COX-2. Patients with AIA usually have persistent underlying asthma, often associated with nasal polyposis. Pathologically, the bronchial and nasal airways of AIA subjects show chronic eosinophilia, with evidence of activation of eosinophils and mast cells during acute reactions. The etiology of AIA is unclear, but the proposed mechanism focuses on the inhibition by NSAIDs of the synthesis of a prostanoid, putatively prostaglandin E2, that would normally suppress local inflammatory reactions. The consequent synthesis of cysteinyl-leukotrienes and other leukocyte-derived mediators contributes to bronchoconstriction and other acute features. Treatment of AIA involves avoidance of NSAIDs combined with conventional management of underlying asthma, with 75% of AIA patients requiring corticosteroids. Controlled desensitization with regular doses of an NSAID can provide protection against acute reactions.

182 ASTHMA / Aspirin-Intolerant

Introduction The classical aspirin-intolerant asthma (AIA) syndrome was described by Samter and Beers in 1968 as a triad of rhinosinusitis (often with nasal polyps), asthma, and aspirin sensitivity. Patients with fullblown AIA are about twice as likely to be female as male, but no more likely to be atopic than the general population. Patients typically present in early middle age with nasal congestion, anosmia, and rhinorrhea, and many develop nasal polyps, often with secondary infection of the paranasal sinuses. Bronchoconstriction and airway inflammation usually emerge later, and this becomes severe, perennial asthma, with about 75% of patients needing oral or inhaled corticosteroids to maintain control of their symptoms. Acute respiratory reactions to nonsteroidal anti-inflammatory drugs (NSAIDs) may be accompanied by rhinoconjunctival symptoms and by dermal symptoms such as facial flushing and exacerbation of preexisting urticaria. Aspirin challenges reveal that the tendency to bronchoconstrict in response to therapeutic doses of NSAIDs is more prevalent than the full-blown ‘aspirin triad’ recognized clinically. Based on patient history alone, the prevalence of NSAID intolerance in adult asthmatics is 3–5%, but it rises to 19% when consecutive asthmatic patients are challenged with oral aspirin. Prevalence in asthmatic children is less than 2% based on history alone, but 13–16% of postpubertal asthmatic children respond adversely when aspirin challenged. NSAID intolerance is overrepresented in the severe asthmatic population. Among patients who have experienced a near-fatal asthma exacerbation requiring treatment in the intensive care unit, 24% are aspirin-sensitive based on history alone. Most life-threatening acute exacerbations in AIA patients are directly due to inadvertent use of NSAIDs, and in a few cases the precipitating NSAID was prescribed by the patient’s own physician. However, over 40% of life-threatening asthma exacerbations in AIA patients cannot be attributed to NSAID ingestion, illustrating the severity of the underlying chronic asthma in these patients.

Etiology Aspirin (acetylsalicylic acid) and other members of the family of NSAIDs inhibit formation of the prostanoid family of lipid mediators by blocking isozymes of cyclooxygenase (COX). They are used therapeutically for their mild analgesic, antipyretic, and anti-inflammatory actions. NSAIDs are variably associated with adverse reactions including prolonged bleeding, nephritis, and gastric ulceration.

Table 1 Chemical classes of some nonsteroidal anti-inflammatory drugs (NSAIDs) Acetic acids Indomethacin Sulindac Diclofenac

Fenamates Mefenamic acid

Oxicams Piroxicam

Propionic acids Ibuprofen

Pyrazoles Phenylbutazone

Benoxaprofen

Oxyphenbutazone

Salicylates Aspirin (acetylsalicylic acid) Sodium salicylate

In 1919, Cooke recognized that in some patients with asthma, aspirin may also precipitate life-threatening acute exacerbations. Such reactions have now been recognized in response to a wide variety of NSAIDs of diverse chemical classes (Table 1). A family history of AIA is reported by only 6% of AIA patients, suggesting that any genetic influences are subtle and expressed phenotypically only in the presence of relevant environmental factors. Leukotriene C4 synthase is the terminal enzyme for the synthesis of the bronchoconstrictor cysteinylleukotrienes postulated to contribute to airway narrowing in AIA. A biallelic polymorphism in the promoter region of the LTC4 synthase gene has been reported, involving an A to C transversion at a position 444 bp upstream of the transcription start site. The variant 444C allele may lead to enhanced transcription of the LTC4 synthase gene in relevant cells. The prevalence of the variant allele has been reported to be significantly elevated in AIA patients from a Polish population, but this has not been replicated in US Caucasian or Japanese populations.

Pathology A classification system for allergic and pseudoallergic reactions to NSAIDs was proposed by Stevenson, Sanchez-Borges, and Szczeklik in 2001. AIA is classified as a type 1 pseudoallergic reaction in which asthmatic patients with a high frequency of sinusitis and nasal polyps experience lower respiratory tract reactions and/or rhinoconjunctival symptoms after exposure to therapeutic doses of aspirin and other NSAIDs. Reactions are dose-dependent and may be life threatening. The type 2 category describes urticarial reactions or angioedema induced by NSAIDs in patients with pre-existing chronic urticaria, and types 3 and 4 describe urticarial or sporadic reactions in patients who are otherwise normal. In contrast to types 1–4 above, in which patients show extensive cross-reactivity to many NSAIDs, patients classified in types 5–8 react adversely only to a single

ASTHMA / Aspirin-Intolerant

drug. These reactions include urticaria/angioedema, anaphylaxis, aseptic meningitis, and hypersensitivity pneumonitis. A consistent, although not diagnostic, pathological finding in AIA patients is chronic eosinophilia in the blood, nasal polyps, and bronchoalveolar lavage (BAL) fluid. Immunohistochemical studies in bronchial biopsies have confirmed a marked bronchial mucosal eosinophilia in AIA patients, with eosinophil counts three- to fourfold higher than in aspirintolerant asthmatics and 10- to 15-fold higher than in normal subjects. After NSAID challenge, further increases in eosinophils, their activation marker ECP (eosinophil cationic protein), and histamine have been described in the nasal airways, BAL fluid, and plasma of AIA patients, suggesting activation of eosinophils and mast cells. In the nasal airway, aspirin challenge of AIA patients is associated with increments in nasal tryptase and histamine, strongly suggesting mast cell activation. Tryptase and histamine levels also rise in the serum of patients experiencing systemic reactions to oral aspirin, but not in those with localized respiratory reactions. Release of tryptase is a recognized marker of mast cell activation in the pulmonary airway, and occurs within 5 min of allergen challenge in allergic asthmatics. However, tryptase did not rise in the BAL fluid of AIA patients challenged with inhaled or endobronchial lysine-aspirin. In contrast, a rise in urinary levels of the PGD2 metabolite 9a, 11b-PGF2 following endobronchial lysine-aspirin challenge has been interpreted as evidence for mast cell activation in AIA. Together, the evidence suggests that inflammatory mediators released both from mast cells and from eosinophils contribute to the pulmonary and rhinoconjunctival symptoms following NSAID exposure in AIA patients.

Clinical Features As there are no acceptable in vitro tests for AIA, confirmation of aspirin intolerance can only be obtained by NSAID challenge under controlled conditions. Lung function is monitored while patients ingest incremental oral doses of aspirin or inhaled doses of a lysine-aspirin conjugate or sulpyrine. Concomitant use of b2-adrenergic agonists, cromones, and inhaled corticosteroids may mask responses to NSAID challenge, leading to a high rate of false-negative results. In a 3-day oral challenge protocol, incremental doses of aspirin are given at 3-hourly intervals up to a maximum of 650 mg. The challenge is terminated when forced expiratory volume in 1 s (FEV1) falls by at least 20%. Reactions begin around

183

50 min after oral aspirin ingestion, ranging from 20 to 120 min. A shorter protocol involves the inhalation of incremental aerosolized doses of lysine-aspirin, a soluble and nonirritant form (this is not available in the US). Respiratory reactions to inhaled lysineaspirin often occur within 1 min, so shorter dosing intervals of 30–60 min allow the entire challenge to be completed within 1 day. Inhaled lysine-aspirin challenges are safer than oral challenges as reactions are localized to the airways and are easily reversible with inhaled b2-agonists. The sensitivity of inhaled lysine-aspirin challenge is similar to oral aspirin challenge.

Pathogenesis The Cyclooxygenase Theory

The most successful model to explain acute respiratory reactions to NSAIDs is the cyclooxygenase theory promulgated by Szczeklik in 1975. This postulates that reactions are related directly to the pharmacological activity of NSAIDs in inhibiting isozymes of COX, the key enzymes in the synthesis of the prostanoid family of lipid-inflammatory mediators. Intolerance to an individual NSAID in vivo was shown to be predictable by its potency in inhibiting COX in vitro, with strong inhibitors (including aspirin, indomethacin, mefenamic acid, ibuprofen, and piroxicam) being common precipitants of adverse reactions, while weak inhibitors (such as sodium salicylate) precipitated reactions rarely or only at high doses. The concept that inhibition of prostanoid synthesis is the trigger for NSAID-induced reactions is recognized as a key advance. The Role of Cysteinyl-Leukotrienes: The Shunting Hypothesis and the PGE2 Brake Hypothesis

The second key advance was the elucidation in 1979 of the structure of slow-reacting substance of anaphylaxis (SRS-A) as a mixture of potent bronchoconstrictor products of a related family of lipid mediators, the cysteinyl-leukotrienes (cysteinyl-LTs). The cysteinyl-LTs – LTC4, LTD4, and LTE4 – are now recognized to have important bronchoconstrictor and proinflammatory roles in many phenotypes of asthma. Their particular relevance to AIA emerged from the recognition that their biosynthetic pathway, the 5lipoxygenase (5-LO) pathway, shares arachidonic acid as a common precursor with the prostanoid pathway (Figure 1). The leukotriene pathway is not inhibited by NSAIDs. Specific blockade of the prostanoid pathway by NSAIDs was therefore proposed to shunt arachidonate away from conversion into

184 ASTHMA / Aspirin-Intolerant

NSAIDs

Membrane phospholipids Phospholipase A2

− Cyclooxygenase-1

Arachidonic acid −

Zileuton

5-lipoxygenase FLAP LTA4

PGG2 PGH2

Cyclooxygenase-2

Prostanoid synthases & isomerases

LTC4 synthase LTC4 LTD4 LTE4 (Cysteinyl-leukotrienes)

− Montelukast Pranlukast Zafirlukast

CysLT1 receptors

PGE2

TXA2

PGD2 PGF2


Prostanoid receptors (EP1−4, TP, DP, CRTH2, FP)

Bronchoconstriction Vascular permeability Edema Mucus secretion Eosinophil migration

Figure 1 The 5-lipoxygenase and cyclooxygenase pathways of eicosanoid metabolism.

prostanoids, which have relatively little bronchoconstrictor activity, towards the formation of cysteinylLTs, which are highly potent bronchoconstrictors. This ‘shunting’ hypothesis is superficially attractive but measurements of lipid mediators in isolated leukocytes treated with NSAIDs argues against such a simple mechanism. It has therefore been superseded by an alternative notion, the ‘PGE2 brake’ hypothesis. This proposes that NSAIDs block the formation of an anti-inflammatory prostanoid, PGE2, which is otherwise known to suppress leukotriene synthesis by leukocytes. Exposure to NSAIDs may thus liberate the 5-LO pathway from suppression by endogenous PGE2 in vivo, at least in susceptible individuals (Figure 2). There are three main lines of experimental evidence supporting this model: 1. The triggering effect of NSAIDs on LT synthesis can be mimicked in vitro in a number of inflammatory leukocyte subtypes. Endogenous PGE2 suppresses, and NSAIDs consequently enhance, leukotriene synthesis in eosinophils, neutrophils, basophils, and macrophages, but apparently not in human lung mast cells. Eosinophils themselves generate sufficient PGE2 to suppress their own LTC4 synthesis by about 90%. Treatment of eosinophils with indomethacin inhibits endogenous PGE2 synthesis and stimulates LTC4 release. Replacement experiments showed that

exogenous PGE2 restores the braking effect, returning LTC4 synthesis to the levels seen before indomethacin treatment. PGE2 may act in an autocrine or paracrine manner at EP2 receptors on the cell surface, followed by an increase in intracellular cAMP and activation of protein kinase A, but the subsequent steps by which 5-LO activity is inhibited are unknown. 2. Eicosanoids can be measured in biological fluids, including bronchoalveolar lavage (BAL) fluid, and urine. Endoscopic challenge with lysine-aspirin reduces PGE2 and thromboxane A2 levels in the BAL fluid of AIA patients and aspirin-tolerant asthmatics, but a dramatic rise in BAL fluid cysteinyl-LTs is seen only in the AIA group. Following challenge with oral aspirin or inhaled lysine-aspirin, urinary LTE4 levels, used as a marker of whole-body cysteinyl-LT production, rise three to sevenfold in AIA patients, but not in aspirin-tolerant asthmatics; this response is not seen after methacholine-induced bronchoconstriction or placebo challenge. At the same time, there is a fall in urinary markers of prostanoid synthesis, such as 11-dehydro-thromboxane A2. In AIA patients, preinhalation of PGE2 before challenge with inhaled lysine-aspirin completely ablates the rise in urinary LTE4 and prevents the consequent bronchoconstriction, providing strong evidence that cysteinyl-LT synthesis has a functional role in the NSAID-induced airway narrowing. The

ASTHMA / Aspirin-Intolerant

185

Mechanism of acute NSAID reactions Mast cell / eosinophil

NSAID COX-1

FLAP 5-LO

LTC4 synthase

LTC4

cAMP

ECP

Histamine Tryptase IL-5

EP2

PGE2

Bronchoconstriction Eosinophillia Vasodilation / edema Remodeling / BHR

Figure 2 In this model, PGE2 derived from constitutive cyclooxygenase-1 (COX-1) normally acts via cell surface EP2 receptors to suppress the activity of mast cells and eosinophils, including the activity of the 5-lipoxygenase pathway. However, when an NSAID ingested by a susceptible individual inhibits COX-1, the reduced synthesis of PGE2 is insufficient to suppress the leukocyte effectively. The result is a surge in the synthesis and release of LTC4 and other mediators, leading to bronchoconstriction and inflammations, AIA patients may be susceptible to this response because they have exaggerated LTC4 synthase expression, and/or a defect in the PGE2 brake, based perhaps on insufficient COX-1 activity or more likely on impaired signaling from EP2 receptors.

protective effect of inhaled PGE2 does not correlate with its relatively weak bronchodilator activity, confirming that PGE2 preinhalation protects by restoring the suppression of cysteinyl-LT synthesis, not by dilating airways directly. 3. The effector role of cysteinyl-LTs in adverse respiratory and rhinitic reactions to NSAIDs in most AIA patients has been confirmed with placebocontrolled clinical trials of specific leukotriene modifier drugs. The 5-LO inhibitors zileuton and ZD-2138 markedly blocked the rise in urinary LTE4 and the fall in FEV1 following oral aspirin challenge of AIA. Rhinoconjunctival and dermal reactions to oral aspirin are also blocked by zileuton. Antagonists of CysLT1 receptors block oral NSAID-induced respiratory reactions in AIA patients. The PGE2 Brake is Derived from COX-1

The cyclooxygenase theory and the PGE2 brake hypothesis are thus crucial in understanding how NSAIDs trigger LT synthesis and bronchoconstriction, but they do not explain why only some asthmatics are susceptible to these responses. AIA patients tolerate selective COX-2 inhibitors including nimesulide, meloxicam, and rofecoxib, and respond adversely most often to NSAIDs with a greater selectivity for COX-1, such as aspirin and indomethacin. This suggests that cytoprotective/anti-inflammatory PGE2 in the lung is produced by constitutive COX-1. COX-1 is expressed in a large number of cell types,

including mast cells, eosinophils, macrophages, vascular endothelial cells, bronchial epithelium, and bronchial smooth muscle. The exact cellular sources of the putative PGE2 brake remain unclear. AIA patients may overproduce cysteinyl-LTs chronically and acutely after NSAID exposure because of a defect in endogenous PGE2 synthesis. However, inhaled lysineaspirin equieffectively inhibits airway PGE2 synthesis both in AIA patients and in aspirin-tolerant asthmatics. Baseline levels of PGE2 and other prostanoids are not consistently different in the BAL fluid and urine of AIA patients and control groups. Immunohistochemical studies of AIA bronchial biopsies have found little evidence for a meaningful anomaly in the cellular expression of COX isozymes. One possibility is that a defective PGE2 brake may derive not from a failure of PGE2 synthesis, but of leukocytes to be suppressed by PGE2. An anomaly in EP2 receptor structure, expression, or signaling might be one explanation, and this would also be consistent with the lack of clinical benefit shown in AIA patients treated with the stable PGE1 analog misoprostol. Suggestive evidence of an anomaly within the cysteinyl-LT biosynthetic pathway itself is that baseline cysteinyl-LT synthesis appears to be two- to sevenfold higher in AIA patients than in control groups, even in the absence of exposure to NSAIDs, as demonstrated by measurements of cysteinyl-LTs in BAL fluid, induced sputum, and urine (Figure 3). The increases seen after NSAID exposure are superimposed upon this chronically elevated baseline. Immunohistochemical analysis of bronchial biopsies from

Cys-LT production

186 ASTHMA / Aspirin-Intolerant

NSAID AIA

ATA N 0

1

2

3

Time (h) Figure 3 Schematic diagram showing that cysteinyl-leukotriene production is chronically elevated in patients with aspirin-intolerant asthma (AIA) compared to those with aspirin-tolerant asthma (ATA) and normal subjects (N). A further rise in cysteinyl-LT production after exposure to a nonsteroidal anti-inflammatory drug (NSAID) occurs only in the AIA group.

AIA and aspirin-tolerant asthmatic subjects shows that counts of cells immunostaining for LTC4 synthase, the terminal enzyme for cysteinyl-LT synthesis, were fivefold higher in AIA biopsies than in aspirintolerant asthma biopsies and 18-fold higher than in normal biopsies. The numbers of LTC4 synthasepositive cells in the bronchial mucosa correlated with elevated levels of cysteinyl-LTs in the BAL fluid and with bronchial responsiveness to inhaled lysine-aspirin. Persistent overproduction of cysteinyl-LTs in steady-state AIA may therefore be related to overexpression of LTC4 synthase in the bronchial wall, much of it within eosinophils and mast cells. This may also contribute to the further surge in cysteinylLT synthesis when PGE2 suppression is removed by NSAIDs.

Animal Models Although the anti-inflammatory actions of NSAIDs and their effects of prolonged bleeding, nephritis, and gastric ulceration can be replicated in animal models, there has been relatively little interest in developing animal models of AIA. This may reflect, at least in part, the difficulty of replicating the diverse immunopathological features of asthma, especially chronic remodeling of structural airway tissues, and also of replicating the typically high degree of disease severity and glucocorticoid dependency observed in AIA patients.

Management and Current Therapy National and international management guidelines for asthma (e.g., GINA guidelines) are based on disease severity and make no therapeutic distinctions between AIA and other asthma phenotypes. Asthmatics should

avoid using NSAIDs either prescribed inadvertently or purchased over the counter. NSAID-induced acute reactions in susceptible asthmatics can be treated with nebulized b-2 adrenergic agonists, repeated frequently over several hours where necessary. Decongestants and antihistamines (topical or oral) can be used for associated rhinoconjunctival symptoms. In the most severe cases, intubation and mechanical ventilation in the intensive treatment unit may be required. Treatment of chronic AIA focuses on anti-inflammatory therapy of the upper and lower airways using topical and inhaled/insufflated corticosteroids. Antibiotics may be required when purulent nasal secretions indicate infection, and many AIA patients require repeated polypectomies. Some AIA patients may require NSAIDs for concomitant inflammatory disease such as arthritis, and in such cases, aspirin desensitization may be an option. Acute respiratory reactions to NSAIDs in AIA patients are always followed by a refractory period lasting 2–5 days during which further reactions to NSAIDs cannot be induced. Sensitivity to NSAIDs re-emerges within a week, but regular low-dose NSAIDs can maintain the refractory state indefinitely. Daily or alternate-day dosing of NSAIDs is therefore used clinically to desensitize AIA patients to inadvertent ingestion of NSAIDs and to allow NSAID therapy of concomitant diseases such as arthritis. Cross-desensitization occurs such that repeated dosing with one NSAID provides protection against adverse reactions to other NSAIDs. Chronic desensitization reduced the numbers of acute exacerbations and hospital admissions in AIA patients compared to a control group of AIA patients who avoided NSAIDs, associated with reductions in corticosteroid use. The mechanism of desensitization is unknown, but is tentatively linked to reductions both in the quantity of cysteinyl-LTs synthesized following NSAID exposure, and with reduced bronchial responsiveness to cysteinyl-LTs. In the nasal airway, desensitization has been linked to a reduction in expression of the CysLT1 receptor on infiltrating leukocytes. In small studies in AIA patients, the antiallergic cromone sodium cromoglycate, the antiviral agent acyclovir, the antibiotic roxithromycin, and the longacting b2-agonist salmeterol have all been reported to block not only the bronchial responses to inhaled NSAIDs, but also the associated rise in urinary LTE4 excretion. The mechanism of these drugs is difficult to understand in this context, but may involve prevention of mast cell degranulation. On the basis of the pathophysiological evidence of a central role of cysteinyl-LTs in AIA, clinical trials of 5-LO inhibitors and CysLT1 receptor antagonists have been reported. Zileuton improved lung function and rescue beta-2

ASTHMA / Occupational Asthma (Including Byssinosis) 187

agonist use and restored the sense of smell in a 6week study of 40 AIA patients. In an 8-week crossover trial of montelukast in 80 AIA patients, there were significant improvements in lung function, use of rescue therapy, symptom scores, night-time awakenings, and asthma quality-of-life (QOL) scores compared with placebo. In the clinic, treatment response to leukotriene modifiers is variable, but a treatment trial is advised in patients with AIA uncontrolled by topical corticosteroids. See also: Allergy: Overview. Asthma: Overview. Bronchoalveolar Lavage. Bronchoscopy, General and Interventional. Chymase and Tryptase. Genetics: Overview. Histamine. Immunoglobulins. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils. Pulmonary Function Testing in Infants.

Further Reading Drazen JM, Israel E, and O’Byrne PM (1999) Treatment of asthma with drugs modifying the leukotriene pathway. New England Journal of Medicine 340: 197–206. Pavord ID and Tattersfield AE (1994) Bronchoprotective role for endogenous prostaglandin E2. Lancet 345: 436–438. Samter M and Beers RF (1968) Intolerance to aspirin: clinical studies and consideration of its pathogenesis. Annals of Internal Medicine 68: 975–983. Sanak M and Szczeklik A (2000) Genetics of aspirin-induced asthma. Thorax 55(supplement 2): S45–S47. Stevenson D, Sanchez-Borges M, and Szczeklik A (2001) A classification of allergic and pseudoallergic reactions to drugs that inhibit cyclooxygenase enzymes. Annals of Allergy, Asthma and Immunology 87: 1–4. Stevenson DD, Simon RA, and Zuraw BL (2003) Sensitivity to aspirin and nonsteroidal antiinflammatory drugs. In: Adkinson NF, Bochner BS, Yunginger JW, et al. (eds.) Middleton’s Allergy: Principle and Practice, 6th edn., pp. 1695–1710. St Louis, MO: Mosby. Szczeklik A, Gryglewski RJ, and Czerniawska-Mysik G (1975) Relationship of inhibition of prostaglandin biosynthesis of analogues to asthma attacks in aspirin sensitive patients. British Medical Journal 1: 66–69. Szczeklik A and Stevenson DD (2003) Aspirin-induced asthma: advances in pathogenesis, diagnosis, and management. Journal of Allergy and Clinical Immunology 111: 913–921.

Occupational Asthma (Including Byssinosis) D J Hendrick, Royal Victoria Infirmary, Newcastle upon Tyne, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Respirable agents in the workplace are responsible for about 10% of asthma arising in adult life, and for an annual incidence

of occupational asthma (OA) of 25–100 per million employed in industrially developed countries. For 5–10% of cases, toxic mechanisms are responsible, and asthma is the result of accidents that release agents such as chlorine, sulfur dioxide, and acetic acid into occupational environments. For the remaining 90–95%, asthma appears to arise through hypersensitivity mechanisms. Many of the several hundred causal ‘asthmagens’ are reactive chemicals of low molecular weight, though some are naturally occurring allergens of high molecular weight. Some agents (e.g., di-isocyanates, epoxy resins, flour) have such sensitizing potency that at current exposure levels in some occupations (e.g., spray painting and baking) the risk of OA exceeds that of asthma arising spontaneously. OA is thus important in an epidemiologic sense, and it serves as a useful model of asthma in general. Asthma of occupational origin is like asthma resulting from any other cause. However, when due to hypersensitivity mechanisms, it has one unusual characteristic. If the diagnosis is recognized within 6–24 months and exposure ceases, there is a meaningful possibility that active disease will resolve. Therefore, the onus is on physicians to consider the possibility of an occupational cause in every adult presenting with asthma.

Introduction It was not until the twentieth century that investigatory techniques acquired the sophistication to distinguish airway disorders from those of the lung parenchyma/interstitium. The definition of asthma, the elucidation of its clinical characteristics and pathogenic mechanisms, and the evolution of strategies for its recognition, management, and prevention have consequently been comparatively recent events. Nevertheless, it is likely that asthma was the explanation for many of the respiratory disorders of antiquity and the middle ages that were recognized to afflict (and sometimes devastate) workers in certain trades and industries. The realization that asthma may arise as a direct consequence of inhaled occupational agents has been the focus of particular attention over the last three to four decades, and our understanding of occupational asthma (OA) has largely arisen during this period. OA has no recognized differences from asthma in general with respect to its pathology and genetic etiology, and so this article will focus on its features of special interest. Definitions and Classification

Asthma is a disease of the intrathoracic airways that is characterized, and often defined, by its means of clinical expression (diffuse airway obstruction that varies in degree over time) and its underlying pathogenic basis (a state of enhanced airway responsiveness). OA is caused by exposure to agents, almost exclusively airborne, that are encountered primarily in the workplace. Such exposure in susceptible individuals causes the level of airway responsiveness to

188 ASTHMA / Occupational Asthma (Including Byssinosis)

increase into the asthmatic range. Two distinct mechanisms are recognized. First, hypersensitivity mechanisms appear responsible since there is a latency period of presumed sensitization between exposure onset and symptom onset; this accounts for 90–95% of cases. Second, acute toxicity is the initiating event; this is generally a consequence of inhalation of toxic agents during an industrial accident. Asthma following the latter is sometimes identified as the reactive airways dysfunction syndrome (RADS) or, more simply, irritant asthma. Neither term is fully satisfactory. The first may be interpreted, incorrectly, to indicate a disorder other than asthma, while the second may be mistaken for pre-existing asthma that is exacerbated non-specifically at work (e.g., because of exertion in cold air); this is ‘work-aggravated’ not ‘occupational’ asthma, and is not associated with an increase in airway responsiveness. It is currently unclear whether repeated exposures to toxic agents at dose levels insufficient to provoke clinical reactions might nevertheless cause minor increases in airway responsiveness. If so, the cumulative effect might elevate airway responsiveness to a level at which asthma eventually becomes inevitable (‘low-level RADS’). This would simulate the latency period association with presumed hypersensitivity mechanisms, and ongoing exposure might then provoke acute symptoms in a non-specific fashion. This would appear to simulate hypersensitivity mechanisms also, but such exposure should provoke reactions in any asthmatic individual with a sufficient level of airway responsiveness. Key Clinical Features

When hypersensitivity mechanisms are responsible, further exposures to the particular inducing asthmagen may provoke specific asthmatic reactions. Symptoms are dependent on the degree of hypersensitivity, the magnitude of the provoking stimulus, the level of airway responsiveness, and the ease with which the affected individual perceives changing respiratory sensations. Airway responsiveness varies in level from subject to subject over a considerable range, and can be quantified throughout the population at large. Its distribution (like that of most biologic parameters) is unimodal, and it follows the common biologic pattern of a ‘bell-shaped’ curve. The tail in which responsiveness is most marked gives rise to asthma, but the adjacent segment implies vulnerability, and the opposite tail implies comparative impunity. It is misleading to think of asthmatic and nonasthmatic subjects being fundamentally different because of the presence or absence of airway hyperresponsiveness;

the issue, rather, is whether an individual’s level of airway responsiveness is sufficiently high to make meaningful bronchoconstriction likely when there are appropriate provoking stimuli. Whether the resulting degree of bronchoconstriction is perceived to be distressing (or is perceived at all), is very dependent on psychological factors. This adds an important further level of complexity and variability, since at all degrees of asthmatic severity as defined physiologically, there will be a wide spectrum of perceived disability. This is particularly so in OA because of resentment, even anger, over the possible liability of a third party (the employer), and the possibility of compensation. Longstanding OA, like asthma generally, is commonly associated with airway obstruction that has a reduced capability for reversal. It may come to simulate chronic obstructive pulmonary disease (COPD). Epidemiology

Over recent decades, OA has proved consistently to be the commonest type of newly diagnosed occupational lung disease in industrially developed countries, though the various disorders attributable to asbestos have an equal cumulative incidence. Between them, asthma and asbestos account for 65–70% of all incident respiratory disorders of occupational origin. In most outbreaks of OA no more than a few per cent of exposed workers become affected, but there are examples with both pathogenic pathways of prevalences approaching 50%. The reported incidence of OA varies widely at a global level, depending on local employment patterns and diagnostic criteria. Estimates from statutory notification schemes, compensation registers, voluntary surveillance schemes, and general population surveys, suggest that 5–15% (median 9–10%) of asthma beginning in adult life is occupational. When asthma arises for the first time in a working adult, the background odds favoring an occupational cause over a nonoccupational cause are consequently of the order 1 in 10 only. This is consistent with the estimate from SWORD (Surveillance of Work-Related and Occupational Respiratory Disease) data that about 40 cases of OA per million employed workers arise in the UK each year. SWORD has usefully considered the incidence of new cases within specific working groups for which there are particular occupational exposures. For example, among spray painters, who may use asthmagenic di-isocyanate, epoxy resin, and acrylic paints, the average annual incidence of OA from 1992 to 1997 was 1464 per million – more than threefold the UK national average for all cases of incident asthma.

ASTHMA / Occupational Asthma (Including Byssinosis) 189

Thus, in the case of spray painters developing asthma in adult life, the background odds favor an occupational cause over a coincidental cause. Other settings associated similarly with greater than even odds include baking, metal treatment, chemical processing, and plastics manufacture. Most national estimates range from 25 to 100 million per year.

Table 1 Commonly reported causes of occupational asthma Animal sources Arthopods/ insects Birds Mammals Marine species

Bees, locusts, mites, silkworms, weevils Feather bloom, excreta Rodent urinary protein, pancreatic enzyme supplements Corals, crabs, fishmeal, oysters, prawns, sponges

Etiology Many occupational agents (some 400) have been reported to be definite or probable sensitizers capable of inducing asthma. Some of the most prominent are classified in Table 1, and the most common reports to SWORD over a 9-year period are listed in Table 2. Notable agents reported to cause RADS over recent years have been acetic acid, chlorine/chlorine dioxide/hydrochloric acid, di-isocyanates, dinitrogen tetroxide, endotoxin, fire smoke, freons, hydrobromic acid, Iraq/Iran war gases, pentamidine, phosphoric acid, silo and swine confinement gases, sulfur dioxide/sulfuric acid, tear gas, and welding fume.

Clinical Features Once a diagnosis of asthma is suspected from the clinical history or physical examination and confirmed objectively (by the demonstration of reversible airway obstruction or the measurement of airway responsiveness), the diagnostic issue turns to whether it has arisen occupationally. If the toxicity pathway has provided the mechanism, the diagnosis is clear and needs no further investigation. Asthma becomes evident during the recovery phase from what is usually a combination of conjunctivitis, rhinitis, pharyngolaryngitis, tracheobronchitis, and (possibly) pneumonitis induced by a major exposure to a toxic chemical or organic dust. In survivors, full recovery is the rule. If asthma arises, it is generally the only persisting respiratory problem, though occasionally bronchiectasis or an obliterative bronchiolitis is also detectable. If hypersensitivity has provided the mechanism, the diagnosis is more challenging and may be extremely difficult. This is partly because the latency period during which sensitization occurs (usually 3–24 months) may be very short (days or weeks) or very prolonged (several years), and partly because in most working environments associated with OA, most cases of incident asthma are coincidental and quite unrelated to occupation. What follows addresses this diagnostically more challenging variety of OA. Clinical History

The history provides an obvious starting point, but may be importantly distorted. Affected workers

Vegetable sources Beans Castor, coffee, soy Colophony Pine resin solder Enzymes Bromelain (pineapple), papain (papaw) Grain/flour Gums Acacia, arabic, karaya Mushrooms Oil mists Tea Tobacco Wood Iroko, latex, redwood, western red cedar Microbial sources Endotoxin Gram-negative organisms Enzymes Bacillus subtilis (alcalase, maxatase, subtilisin) Spores Chemical sources Inorganic Salts/oxides of aluminum, chromium, cobalt, nickel, platinum, vanadium Stainless-steel welding fume Organic Acid anhydrides/phthalates, acrylates, amines, azodicarbonamide, benzalkonium, chloramine, di-isocyanates, fluxes, formaldehyde, furfuryl alcohol, glutaraldehyde, isothiazolinones, organophosphate pesticides, plexiglass, polyvinyl pyrolysis fume, reactive dyes, styrene, sulphone chloramides, tannic acid Pharmaceutical sources Intermediates 6-amino penicillanic acid Drugs Cimetidine, cephalosporins, ispaghula (psyllium), penicillins, piperazine, sulphonamides, tetracycline

Table 2 Causes of occupational asthma reported to SWORD over 9 years Agent Di-isocyanates Laboratory animals/insects Flour/grain Fluxes/glues/resins/solders Aldehydes Wood dust Welding fume Enzymes Latex Others Total

1990–98 (%) 16 9 8 7 5 5 2

47 100

The figures are rounded to the nearest whole number.

1998 (%) 13 12 7 9 5

14 6 34 100

190 ASTHMA / Occupational Asthma (Including Byssinosis)

anxious to remain employed may deny or minimize relevant symptoms; they may also exaggerate or falsify critical aspects. An overwhelming belief that occupational exposure (and/or employer negligence) is responsible may, curiously, make a diagnosis of OA the ‘independent variable’ on which the symptoms depend: ‘‘if improvement during holidays/vacations is a cardinal diagnostic feature of OA then, yes, this must be so in my case since I know I have OA.’’ For a classical case, there is exposure to a known asthmagen and other exposed workers are affected. For the affected individual, there is recognition that symptoms arise or worsen after a minimum of 1–2 hours and a maximum of 8 h from the onset of each sufficiently strong exposure, and persist for hours or days. Such ‘late’ asthmatic reactions are characteristically associated with increases in airway responsiveness and so are much more definitive of OA than ‘immediate’ reactions, which may simply result non-specifically through ‘irritant’ mechanisms. Mild late reactions may resolve within hours so that there is full recovery by the following day, but more commonly the rise in airway responsiveness is sufficient to worsen asthmatic severity for several days. A weekend away from work may therefore be insufficient to allow full recovery, and the association between occupational exposure and symptoms may not be recognized until there is a 2-week period of vacation (or sick leave). Even then gradual recovery, particularly cessation of disturbed sleep, may not become obvious until the second week, only to be reversed within a matter of days of returning to work. Serologic Investigations

Laboratory investigation for diagnostic IgE antibodies to relevant asthmagenic agents has proved disappointing for two reasons. First, many occupational asthmagens are reactive chemicals of low molecular weight. They are not thought to act as sensitizers until coupled with appropriate haptogenic body proteins, and it has proved difficult to produce suitable complexes for antibody detection. Second, exposed individuals may develop IgE responses without any apparent ill effect. Antibodies appear to correlate more closely with exposure than disease. Nevertheless, when radioallergosorbant allergen testing to relevant asthmagens is available, positive tests increase the probability of OA, even if sensitivity and specificity are limited. Peak Expiratory Flow

More popular and more readily available is peak expiratory flow (PEF) monitoring. Test subjects take measurements on several occasions each day

for periods of several weeks, so that any differences in pattern between work days and rest days can be detected. Statistical software can aid interpretation. The unsupervised recordings may lack reliability and precision, and work-related changes may reflect nonspecific ‘irritant’ reactions rather than specific hypersensitivity responses. There is consequently some difference of opinion over the value of PEF monitoring. In practice, however, it provides the most widely used diagnostic tool for OA. Figure 1 illustrates a typical positive outcome. Inhalation Challenge Tests

The greatest diagnostic confidence comes from laboratory inhalation challenge tests that are monitored by both supervised serial measurements of spirometry and repeated measurements of airway responsiveness. When there is deteriorating ventilatory function and increasing airway responsiveness, and the changes can be evaluated statistically, a diagnosis of OA can be considered ‘confirmed’ with considerable confidence, especially if the outcome is shown to be repeatable and the tests are carried out in a double-blind fashion. Thus, neither test subject nor the immediately supervising physician knows whether the challenge exposure involved the suspected asthmagen or a dummy ‘placebo’. These principles, with two methods of statistical evaluation, are illustrated in Figures 2 and 3. In practice, such tests require sophisticated equipment, are very time-consuming, pose potential risks, and are inevitably restricted to a few centers. They involve a fraction of 1% of all cases, but are particularly valuable (arguably indispensable) when hitherto unrecognized occupational asthmagens are first investigated. Figure 3 illustrates one such case involving a novel asthmagen. Return-to-Work Studies

A useful, and practical, compromise is the ‘return-towork’ challenge test. For this, the test subject is kept from work (or at least from exposure to the suspected asthmagen) for a period of 2–3 weeks, during which time any asthmatic medication is reduced to a minimum (ideally discontinued). In true OA, some improvement is likely (or there is no deterioration with treatment reduction), and can be demonstrated by serial measurements of spirometry and airway responsiveness. Hourly spirometric monitoring over the 3 days prior to the return-to-work generates confidence limits, and so allows the detection of any statistically significant deterioration subsequently (usually within a few days only if the asthma is occupational). If airway responsiveness too increases significantly, OA in the individual is reasonably ‘confirmed’. The method

Diurnal variation (%)

ASTHMA / Occupational Asthma (Including Byssinosis) 191 50 20 460 440 420

PEF (l min–1)

400 380 360 340 320 300 280

M T W T F S S M T W T F S S M T W T F S S M T W T F S S M T W T F S S M T W T F S S 18 19 20 21 22 23 24 25 26 27 28 29 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 11 12 Readings 9 9 9 8 9 8 7 9 8 7 9 8 8 7 5 9 9 8 8 8 8 9 8 3 6 8 6 7 8 9 9 7 7 9 8 6 9 7 8 8 7 6 W W W W W Comments

Figure 1 Serial PEF measurements in a laboratory technician sensitized to a floor cleaning material. Serial 2-hourly PEF measurements, waking to sleeping, for 6 weeks. Top panel: diurnal variation expressed as % predicted. Middle panel: daily maximum, mean, and minimum PEF. Bottom panel: date and number of PEF readings per day. W, days without waking measurement. Dotted line: PEF ¼ 359 represents the predicted PEF. The shaded columns represent work days. Reproduced from Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 54. London: Saunders, with permission from Elsevier.

does not confirm the identity of the asthmagenic agent, and is only suitable if the test subject is still employed and the employer cooperates fully. In many cases the possibility of an occupational cause does not arise until after the affected individual has ceased employment or the work environment has changed. Figure 4 illustrates this methodology in a newly appointed factory safety officer who found himself working with di-isocyanates.

FEV1 (l)

4

3

2

0 −40 0

Animal Models 80 2 Min

4

6

8 10 12

24 H

Figure 2 Inhalation challenge test with nebulized ceftazidime (3.2 mg) in a production worker. Upper plot: mean FEV1 at each hourly measurement point from 3 control days. Middle plot: lower 95% confidence band for the hourly control FEV1 derived from the pooled variance. Lower plot: FEV1 following nebulized ceftazidime administered in a double-blind fashion. The lower confidence band is breached for well over an hour, indicating a significant decrement consistent with a late asthmatic reaction. Reproduced from Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 64. London: Saunders, with permission from Elsevier.

Several animal species have provided invaluable insight as to how asthma arises following exposure to occupational agents. ‘Sensitization’ has been achieved by inhaled, dermal, and/or peritoneal routes for many occupational asthmagens (notably acid anhydrides, colophony, di-isocyanates, latex, plicatic acid), and both immediate and late asthmatic reactions have been provoked by subsequent inhalation challenge. The airways are then characterized by inflammation, eosinophil infiltration, mucus hypersecretion, and hyperresponsiveness. Hypersensitivity mechanisms have been confirmed by transferring lymphocytes or serum from affected to unaffected animals, which then respond to inhaled challenge in a similar fashion

192 ASTHMA / Occupational Asthma (Including Byssinosis) 5.0 4.0 FEV1 (l)

3.0 2.0 1.0 0 −40

(a)

0

60

Min

2

4

6

8

10 12

24

H

FEV1 area decrement 2−12 h (l × h)

9 8

Confidence limits

7 6 5

99%

4 3

95%

2 1 0 1

(b)

2 Control days

3 SINOS (32 µg)

Figure 3 Inhalation challenge test with nebulized SINOS (32 mg) in a research and development technician. (a) The FEV1-time plot following challenge with a newly developed low-temperature bleach-activating agent, SINOS. The shaded zone defines the 2–12 h area decrement (2–12 hAD). It provides a summary measure to quantify a late asthmatic reaction. The line demarcating the upper boundary represents the mean of the measurements during the 40 min before challenge. (b) Comparison of the 2–12 hAD following the SINOS challenge with those following three double-blind control challenges with nebulized solvent alone. It exceeds their 95% and 99% confidence limits, confirming that a significant late asthmatic reaction has occurred. The outcome was reproduced when the procedure was repeated. Reproduced from Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 64. London: Saunders, with permission from Elsevier.

to the donor animals. Specific IgE antibodies to the inducing agents (or hapten conjugates) have been evident commonly, but not invariably. Occasionally specific IgG antibodies are reported. There is involvement of both CD4 þ and CD8 þ T cells, with a dominant T-helper-2 cells (Th2) response. The mechanisms include deposition of excess extracellular matrix (and increased activity of matrix metalloprotease, MMP) and activation of the vascular endothelium associated with the release of vascular endothelial growth factor (VEGF). Experiments with inhibitors of MMP and VEGF have shown substantial reductions of the markers of asthmatic activity, possibly pointing a way to novel strategies for future management. Animal models have additionally confirmed that airway inflammation induced by exposure to toxic levels of certain reactive chemicals may also induce airway hyperresponsiveness. This simulates the RADS pathway. Ozone and chlorine exert their effects

through oxidative stress, which is associated with increased expression of inducible nitric oxide synthase and an increase in airway responsiveness. Ozone has also been shown to suppress Th1 responses (possibly thereby enhancing Th2 responses), and acute exposure to ozone or nitrogen dioxide amplifies asthmatic responses to allergenic agents in already-sensitized animals. Animal models have also been used to investigate the exposure threshold levels at which airway inflammation and hyperresponsiveness first develop. The importance of this for occupationally induced asthma in humans is readily evident, though extrapolation from animals is necessarily fraught with uncertainty. The threshold levels of exposure that trigger meaningful responses once sensitization has occurred may differ critically from those that are responsible for initial sensitization, and are likely to differ appreciably from individual to individual.

ASTHMA / Occupational Asthma (Including Byssinosis) 193 3.00

FEV1 (l s–1)

2.00

Test day 3

1.00

Mean of three control days Lower boundary for control days Work

0.00 07:30 08:30 09:30 10:30 11:30 12:30 13:30 14:30 15:30 16:30 17:30 18:30 19:30 20:30 21:30 22:30 23:30 Time (h) Figure 4 Return-to-work study of a safety officer employed by a factory using di-isocyanates. He developed asthma within months of employment onset. He was kept from work for several weeks during which airway responsiveness improved steadily (PD20.methacholine; 3.2-25.0 mg). An increase in PD20 of twofold or more indicates a significant change. The figure illustrates the third successive day of the return-to-work study. The lowered initial FEV1 measurement reflects an equivocal late reaction from the preceding day. On Day 4, following the 3 days at work, the PD20 was significantly decreased to 9.0 mg (i.e., to less than half the pretest value of 25.0 mg).

Management and Current Therapy Drug and ancillary therapies for OA are those of asthma of any cause, but OA arising by the hypersensitivity route offers an additional and critical means of management. If the relevance of an occupational sensitizer is recognized within 6–24 months of symptom onset, and if exposure then ceases, there is a meaningful probability of the asthmatic state resolving entirely (i.e., the level of airway responsiveness falls into the nonasthmatic range). This is almost unknown for adults with nonoccupational asthma, unless it is drug induced (beta-blockers, nonsteroidal anti-inflammatory drugs (NSAID). There is inevitably much variability from individual to individual, but if exposure ceases within as short a period as 6 months, the probability of complete resolution may exceed 50%. If the period exceeds 24 months, it is more likely that active asthma will continue, even in the absence of ongoing exposure. Most individuals developing OA have unskilled or semiskilled jobs. Their susceptibility to develop asthma with exposure to sensitizing agents will inevitably put them at a disadvantage in the labor market,

and this will be enhanced if active asthma persists. It is unfortunate that most lose their current jobs and never become gainfully employed again. This poses several management challenges. A correct diagnosis is the cornerstone, since mild asthma rarely leads to job loss of itself. A false-positive diagnosis may have devastating but unnecessary economic consequences for both the affected individual and the employer. A dilemma arises when asthma is occupational, but is mild, has been active for several years, and does not improve much after an experimental period away from the workplace. If the affected individual cannot find (or does not wish for) alternative employment, the risk of continuing exposure needs to be assessed. It will certainly diminish the long-term probability of regaining a nonasthmatic level of airway responsiveness, but this may be minimal anyway. More importantly it may cause a further and permanent increase in airway responsiveness, and hence worsening asthma with the possibility of remodeling and fixed airway obstruction. A period of close surveillance, with objective serial measurements of spirometry and airway responsiveness, may help the affected individual and his medical advisors to judge whether the

194 ASTHMA / Occupational Asthma (Including Byssinosis)

obvious benefits justify the uncertain risks. There is anecdotal evidence that tolerance develops in some affected individuals and that they have little to lose by continued exposure. Complete cessation of exposure offers the best outcome, especially if this can be achieved by using an alternative, nonsensitizing agent for the particular industrial process. If this is not possible or practical, it may be that exposure levels can be reduced by modifications to job plans, task sharing/exchanging, improved industrial hygiene (ventilation/extraction), or the use of respiratory protection equipment. Prevention

Prevention offers the best means of control. When a risk of OA is identified, surveillance programs may identify emergent cases at a point when exposure cessation is likely to produce cure. This assumes such programs are efficient. Some employers rely primarily on environment measurements, and reassure themselves (inappropriately) that if they have complied with regulatory limits, any emergent cases of asthma must be nonoccupational. When a clear risk of OA is recognized, the onus should lie with disproving the diagnosis when new cases of asthma arise, rather than the converse. It is the very nature of ‘allergy’ that some affected individuals become sensitized at exposure levels that are harmless to the majority, and that lie within regulatory limits. Compensation

Compensation is inevitably an issue for affected workers, particularly those who become disabled and unemployed. Equally inevitably, compensation systems differ from country to country. Regretfully, few include provision for retraining and re-placement. The physician managing OA needs to be familiar with local procedures in order to offer proper advice. At a time of increasing litigation, the physician too may find him- or herself the focus of a legal (negligence) suit if he or she has failed to provide correct advice.

Byssinosis The respiratory effects of dust from cotton and other biologic fibers became prominent in the nineteenth and early/mid-twentieth centuries. They appeared initially to be specific to the cloth manufacturing industry and the appellation, byssinosis, arose to identify the disorder characterized by them. This has had the misleading consequence of segregating cotton dust asthma from the many other types of OA that

were identified later, and of disguising the other respiratory disorders that may result from cotton dust. A very characteristic feature of reported byssinosis has been a prominence of symptoms on the first work day following periods without exposure. These tend to diminish or even cease on subsequent days, at least in some individuals, despite similar levels of ongoing exposure. While chest tightness and breathlessness may be a consequence of asthma, some affected individuals describe an influenza-like reaction with fever and malaise (‘Monday fever’). The latter may occur on the very first employment day, that is, before any possibility of sensitization. Such symptoms have also been generated in experimental conditions when naı¨ve volunteers are exposed. The same febrile reaction may occur in subjects heavily exposed to metal fume or the fume arising from certain polymers. It is closely related to neutrophil inflammation and the release into plasma of pyrogenic interleukin6 (IL-6). These non-specific systemic symptoms are features of ‘inhalation fevers’ generally. They occur widely with exposure to a variety of organic dusts, and in these contexts are now known more commonly by the term ‘organic dust toxic syndrome (ODTS)’. Products from microbial overgrowth appear to be responsible, and high airborne concentrations of respirable spores and endotoxins are characteristically seen. This is consistent with the recognition that byssinosis is most prevalent among those who harvest, transport, store, sort, process, and clean cotton before its ‘decontaminated’ fibers are woven into cloth. The matter is complex since heavy exposure of naı¨ve subjects to organic dust may also provoke cough, chest tightness, and bronchoconstriction. These symptoms are rarely severe, and rarely last for more than a day or two. Nevertheless, this response simulates RADS, and no clear sensitizer has been incriminated in the pathogenesis of byssinosis. Some individuals affected by other forms of OA may also have more prominent symptoms on Mondays that peter out as the working week progresses. A further ‘characteristic’ of byssinosis has also proved to be non-specific, namely the development of COPD. Early studies of byssinosis were necessarily cross-sectional, and it was assumed that subjects with COPD had initially experienced the typical acute and reversible features (i.e., asthma and/or inhalation fever), which had progressed to produce ‘chronic byssinosis’. It seems more likely now that COPD and asthma (and inhalation fever/ODTS) are independent disorders arising from cotton dust exposure, which result from different levels of susceptibility within exposed workers and different patterns of cellular response. Even so, it is to be expected that COPD might develop

ASTHMA / Acute Exacerbations 195

because of asthma as well as independently of it, and there is convincing evidence to support this from recent longitudinal studies.

Acute Exacerbations S L Johnston, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

See also: Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-6. Occupational Diseases: Asbestos-Related Lung Disease.

Further Reading Beach JR, Young CL, Avery AJ, et al. (1993) Measurement of airway responsiveness to methacholine: relative importance of the precision of drug delivery and the method of assessing response. Thorax 48: 239–243. Brooks SM, Weiss MA, and Bernstein IL (1985) Reactive airways dysfunction syndrome (RADS). Persistent asthma syndrome after high level irritant exposures. Chest 88: 376–384. Burge PS, Pantin CFA, Newton DT, et al. (1999) Development of an expert system for the interpretation of serial peak expiratory flow measurements in the diagnosis of occupational asthma. Occupational and Environmental Medicine 56: 758–764. Cote J, Kennedy S, and Chan-Yeung M (1990) Outcome of patients with cedar asthma with continuous exposure. American Review of Respiratory Diseases 141: 373–376. Glindmeyer HW, Lefante JJ, Jones RN, et al. (1994) Cotton dust and across-shift change in FEV1 as predictors of annual change in FEV1. American Journal of Respiratory and Critical Care Medicine 149: 584–590. Hendrick DJ and Burge PS (2002) Occupational asthma. In: Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) Occupational Disorders of the Lung – Recognition, Management, and Prevention, pp. 33–76. London: Saunders. Herrick CA, Xu L, Wisnewski AV, et al. (2002) A novel mouse model of diisocyanate-induced asthma showing allergic-type inflammation in the lung after inhaled antigen challenge. Journal of Allergy and Clinical Immunology 109: 873–878. Lee KS, Jin SM, Kim SS, and Lee YC (2004) Doxycycline reduces airway inflammation and hyperresponsiveness in a murine model of toluene diisocyanate-induced asthma. Journal of Allergy and Clinical Immunology 113: 902–909. Lee YC, Kwak YG, and Song CH (2002) Contribution of vascular endothelial growth factor to airway hyperresponsiveness and inflammation in a murine model of toluene diisocyanate-induced asthma. Journal of Immunology 168: 595–600. Meyer JD, Holt DL, Cherry NM, and McDonald JC (1999) SWORD ’98: surveillance of work-related and occupational respiratory disease in the UK. Occupational Medicine 47: 485–489. Newman Taylor A (2000) Asthma. In: McDonald JC (ed.) Epidemiology of Work Related Diseases, 2nd edn., ch. 8, pp. 149– 174. London: BMJ Books. Schilling RSF, Hughes JPW, Dingwall-Fordyce I, et al. (1995) An epidemiological study of byssinosis among Lancashire cotton workers. British Journal of Industrial Medicine 12: 217–227. Stenton SC, Avery AJ, Walters EH, and Hendrick DJ (1994) Technical note: statistical approaches to the identification of late asthmatic reactions. European Respiratory Journal 7: 806–812. Stenton SC, Dennis JH, Walters EH, and Hendrick DJ (1990) The asthmagenic properties of a newly developed detergent ingredient – sodium iso-nonanoyl oxybenzene sulphonate. British Journal of Industrial Medicine 47: 405–410. Venables KM, Topping MD, Howe W, et al. (1985) Interaction of smoking and atopy in producing specific IgE antibody against a hapten protein conjugate. British Medical Journal 290: 201–204.

Abstract Acute exacerbations of asthma are acute worsenings of asthma symptoms accompanied by reductions in lung function, normally provoked by some external event or combination of events. Exacerbations may be relatively mild or severe. The most severe may lead to asthma death. Symptoms include increased breathlessness, wheeze, cough, or chest tightness. Severity is graded on a combination of symptoms, clinical signs, and lung function. The majority of asthma exacerbations, particularly in children, are precipitated by acute respiratory tract viral infections. These may interact with a number of other cofactors such as allergen exposure, air pollution, and exercise. Exacerbations are more likely to occur on a background of poorly controlled rather than well-controlled underlying disease. Pathology involves increased airway inflammation with most inflammatory cell types implicated in pathogenesis. Most prominent, however, are neutrophils, lymphocytes, and eosinophils. A wide variety of acute inflammatory mediators are increased during exacerbations. Severity of virus infection is the major determinant of severity of exacerbation. Asthmatics have increased susceptibility to viral and bacterial infection. Treatment includes optimal control of underlying disease, inhaled/ oral steroids, and bronchodilators. The role of anti-infective therapy is under investigation.

Introduction Asthma is itself a heterogeneous disease. Asthma exacerbations also, therefore, are by definition heterogeneous as exacerbations are defined as a worsening of a pre-existing state. Given that the etiology (see below) of exacerbations is also heterogeneous, heterogeneity of both the underlying cause and of the etiology of the exacerbation makes the exacerbation itself very varied in its presentation. One of the difficulties in clinical practice and clinical research is defining exacerbations accurately and differentiating them from poor control of the underlying disease. There is no agreed definition of an exacerbation, but most clinicians and clinical researchers would agree on something like ‘episodes of relatively sudden onset and rapidly progressive increase in symptoms of shortness of breath, cough, wheeze, or chest tightness accompanied by reduction in lung function and normally provoked by some external event or combination of events’. Exacerbations of asthma are very important as they are the major cause of morbidity and mortality in asthma and are also responsible for the greater part of healthcare costs associated with asthma treatment. Currently available treatments for asthma exacerbations include supportive care, oxygen if required, bronchodilators, and oral/inhaled corticosteroids.

196 ASTHMA / Acute Exacerbations

This treatment is at best only partially effective. Understanding the causes and mechanisms of asthma exacerbations is therefore of great importance as there is an urgent need for more effective preventive and interventional therapy.

Etiology In infants and young children the vast majority (approaching 100%) of exacerbations are precipitated by acute respiratory tract virus infections. Respiratory syncytial virus (RSV) is the major cause of acute wheezing illness severe enough to require hospitalization in infants and 1–2-year-old children. There is, however, increasing evidence that rhinoviruses also play an important role and most recent data suggest RSV is implicated in 70–80% of hospitalized wheezing lower respiratory tract infections and rhinoviruses in around 40%. The incidence of dual infection with these two viruses and with a variety of other respiratory viruses is likely to be as high as 20–30%. There is much less data available on community lower respiratory wheezing illness but birth cohort studies have now reported that rhinoviruses are the dominant cause of acute attacks of wheezing in this age group, perhaps causing around three times as many episodes of wheezing illness as RSV and other respiratory viruses. In school-age children and teenagers, viruses precipitate at least 80% of acute asthma exacerbations. Rhinoviruses again are by far the most common virus implicated accounting for around two-thirds of viruses detected. In adults, viruses likely cause around two-thirds to three-quarters of asthma exacerbations. In adults in particular where virus loads tend to be much lower than in children, the evidence is less strong than it is in children and further work is required to clarify the role of viruses. In all these age groups, viruses can interact with a number of other factors in provoking asthma exacerbations. There are good data in both adults and children that virus infection and exposure to an allergen to which the patient is sensitized interact in a synergistic manner in increasing the risk of exacerbation. There is also evidence that air pollution interacts with virus infection in increasing the risk of lower respiratory illness when infected and it is possible that a number of other cofactors also play a role. Poor control of the underlying disease is a major risk factor for asthma exacerbation and treatment with prophylactic therapy, principally with inhaled corticosteroids, but also with leukotriene receptor antagonists and long-acting beta-agonists is a major protective factor against exacerbation.

Asthma exacerbations show marked seasonality in all age groups, peaking 1–2 weeks after school return, most especially in the autumn and most especially in school-age children. This seasonal pattern is important as it emphasizes the times at which prophylactic therapy is most needed. Asthmatics have recently been shown to have increased susceptibility to virus infection through impaired innate immunity and there is a biological rationale that they are also likely to have impaired acquired antiviral immunity. Clinical studies confirm that asthmatics are more susceptible to virus infection and bacterial infection, having a greater risk of invasive pneumococcal disease. To date, very little is known about susceptibility to infection in asthma but this is an important area for future research. Genetic susceptibility is also an underresearched area and very little is known in this regard. Finally, there is increasing evidence that chronic infection with the atypical bacteria Chlamydophila pneumoniae and Mycoplasma pneumoniae may play a role in exacerbations of asthma and a recent study confirms therapeutic benefit of treating asthma exacerbations with an antibiotic active against these organisms. Further studies are required to confirm these findings and to shed further light on the role of chlamydia and mycoplasma.

Pathology Relatively little is known about the pathology of asthma exacerbations for an obvious reason: sampling the lower respiratory tract during an exacerbation is extremely difficult. In one study asthmatic patients were bronchoscoped during naturally occurring colds, and eosinophilic, neutrophilic, and CD8 þ T-cell inflammatory responses were found. Studies of post-mortem samples from patients dying of asthma exacerbation also reveal CD8 þ T cells, activated CD8 þ T cells, increased perforin expression, and impaired ratios of interferon gamma (IFN-g) to interleukin-4 (IL-4), possibly implicating impaired antiviral immunity in asthma death. Noninvasive methods of sampling the lower airway include measurement of exhaled breath condensate or exhaled nitric oxide, and sputum sampling. Studies with sputum again show both eosinophilic and neutrophilic inflammation with increased levels of IL-8 and fibrinogen. Markers of both eosinophil and neutrophil activation are also increased and sputum lactate dehydrogenase (LDH) as a marker of virus-induced cytotoxicity is also markedly elevated. Levels of sputum LDH and eosinophil cationic protein were associated with longer hospital stay, indicating that virus-induced cell damage was the major predictor of

ASTHMA / Acute Exacerbations 197

the severity of asthma exacerbation and that eosinophilia as a consequence of either viral and/or allergen exposure was also an important contributor.

Clinical Features Asthma exacerbations present with a sudden worsening of wheeze, cough, shortness of breath, and chest tightness with a reduction in lung function. Exacerbations can range from mild to severe, the most severe leading to asthma death. Diagnosis is normally made on the clinical history with the assistance of peak expiratory flow measurement and/or spirometry. Clinical examination will normally reveal a distressed, anxious patient with tachycardia, tachypnea, and audible wheeze on auscultation (though in the most severe exacerbations the chest can be almost silent). Patients will have prolonged expiration, and signs of severe exacerbation include inability to talk in complete sentences, tachycardia above 110 beats min
1, a respiratory rate above 25 breaths min
1, and a peak expiratory flow below 50% of predicted or best. Presence of pulsus paradoxus also indicates a severe attack and life-threatening attacks are suggested by a silent chest, presence of cyanosis, bradycardia, or hypotension, and a peak expiratory flow below 30% of predicted or best. Measurement of blood gases should be carried out. Hypoxia is usual. In milder exacerbations, hypocapnia is common due to hyperventilation while in more severe and life-threatening exacerbations, PCO2 starts to rise. A raised PCO2 or rising PCO2 is an indication for intensive care. Chest X-rays should be performed to exclude pneumonia and pneumothorax. The response to therapy and the progress of the exacerbation are monitored with serial peak expiratory flow or spirometry testing. Routine hematology and biochemistry are indicated but other blood testing is not normally required.

Pathogenesis Asthma exacerbations are always mixed in their pathogenesis. They involve a mixture of acute and chronic inflammation provoked by virus infection and other stimuli including allergen exposure, air pollution, tobacco smoke, etc. Much of the information we have gained regarding the pathogenesis of asthma exacerbations has come from experimental studies of rhinovirus infection in asthmatic and normal subjects. These studies have implicated neutrophils, CD4 þ and CD8 þ lymphocytes, and eosinophils, but in addition mast cells, macrophages, and mediators of acute inflammation are also shown to play important roles, including leukotrienes. Most cytokines/chemokines

that have been investigated have been found to be elevated in exacerbations. Perhaps the most prominent are interleukin-8, interleukin-1b, tumor necrosis factor alpha (TNF-a), the regulated upon activation normally T-cell expressed and secreted factor (RANTES), and IFN-g. The mechanisms of induction of lower airway inflammation in the context of respiratory virus infection are of great interest as these represent potential targets for interventional therapy. Nuclear factor kappa B (NF-kB) is very strongly implicated in virus-induced inflammation, as is activating protein 1 (AP-1). However, a number of other transcription factors are also implicated. Mechanisms of induction of mucus secretion are also of great interest – complex pathways are involved, but NF-kB, Sp1 transcription factors, and the epidermal growth factor receptor signaling pathway are all implicated. Airway obstruction is a result of acute smooth muscle contraction, chronic smooth muscle hypertrophy, mucus secretion, acute tissue edema, and chronic tissue inflammation with airway fibrosis/remodeling. An important aspect of pathogenesis is host resistance to infection as virus infection is the major precipitant to exacerbation. Recent studies have confirmed that asthmatic bronchial epithelial cells mount defective apoptotic and IFN-b innate immune responses to rhinovirus infection. In consequence, rhinovirus infection is robust while in normal epithelial cells infection is largely abortive. The clinical studies indicating increased susceptibility to virus infection in asthma are consistent with this biological evidence, and more recent evidence that asthmatics are also susceptible to invasive pneumococcal disease possibly indicates a more generalized immune deficit. There is an urgent need to increase our understanding of host immunity to both viral and bacterial infection in asthma.

Experimental Models There are no small-animal models of rhinovirus infection as rhinoviruses only infect humans and nonhuman primates. There are animal models of other virus infections including influenza, RSV, and parainfluenza, and these models have been combined in some instances with allergen exposure to try to increase our understanding of the pathogenesis of virus-induced asthma exacerbations. These studies indicate that impaired T helper (Th)-1 immune responses increase susceptibility to virus infection and that airway inflammation, airway obstruction, and bronchial hyperreactivity are increased when virus infection occurs in the presence of ongoing allergic inflammation. Other studies have confirmed an increased risk of developing allergen sensitization if

198 ASTHMA / Acute Exacerbations

allergen exposure occurs in the context of an acute respiratory virus infection. Studies with RSV indicate that the major protective immune responses are production of neutralizing antibody, natural killer (NK) cell, and CD8 þ T-cell IFN-g responses. A great deal of further work is needed to increase our understanding of the interaction between virus infection and allergen exposure in the context of asthma exacerbations. In the absence of an animal model, investigators have carried out human rhinovirus experimental infections in asthmatic and normal subjects, and have demonstrated bronchial hyperactivity and airway obstruction in asthmatic volunteers. These studies have also implicated Th1 responses in increased susceptibility to virus infection and further work in these models is ongoing to try to increase our understanding of the pathogenesis of virus-induced asthma. In vitro models of host immunity to virus infection and of virus-induced lower airway inflammation include infection of airway epithelial cells and macrophages with a variety of respiratory viruses including rhinoviruses, RSV, and influenza. These studies demonstrate that virus infection induces many proinflammatory cytokines and chemokines, dependent upon transcription factor activation such as NF-kB, AP-1, and NF-IL-6. In turn, much of the activation of transcription factors is dependent upon activation of oxidant pathways. Production of nitric oxide appears to exert some degree of protection against virus infection.

Management and Current Therapy Perhaps the most important aspect to management of asthma exacerbations is prevention of asthma exacerbations. Optimal control of underlying disease with optimal therapy including inhaled corticosteroids, leukotriene modifiers, and long-acting betaagonists has been shown to reduce exacerbation frequency by approximately 50%. Once exacerbations occur, the initial response is to give short-acting bronchodilators, if mild via inhalation through a spacer, and if more severe by nebulization. Short-acting beta-agonists are initial therapy. The addition of anticholinergic bronchodilators has been shown to further improve lung function and to reduce the need for hospitalization. In mild exacerbations, increasing the dose of inhaled corticosteroids can reduce the severity of the exacerbation and speed recovery. However, evidence indicates that doubling the dose is usually not adequate and that quadrupling the dose or giving high-dose therapy may give better responses. In moderate to

severe exacerbations, oral/systemic corticosteroid therapy is indicated. Intravenous magnesium has been shown to be of some benefit in more severe exacerbations failing to respond to initial therapy. Oxygen and supportive care should be given to all exacerbations where hypoxia is a feature. Leukotriene modifiers have been shown to be more beneficial in exacerbations in infants and young children. Their intravenous use in the acute setting has also been shown to produce some benefit, though there is not much evidence available as yet. Use of standard antibiotics in two placebo-controlled studies has produced no evidence of benefit. However, a recent study with an antibiotic active against atypical bacteria and with anti-inflammatory properties has shown clinically significant benefit over placebo. The most severe exacerbations require high dependency unit or intensive care monitoring and some require invasive ventilation. Hospitalization with acute exacerbation is a major risk factor for asthma mortality, and preventive therapy including inhaled corticosterioids and self-management plans are strongly indicated in such patients. See also: Chemokines. Leukocytes: Eosinophils; Neutrophils. Viruses of the Lung.

Further Reading British Thoracic Society, Scottish Intercollegiate Guidelines Network (2003) British guidelines on the management of asthma. Thorax 58(Supplement 1): 11–94. Corne JM, Marshall C, Smith S, et al. (2002) Frequency, severity, and duration of rhinovirus infections in asthmatic and nonasthmatic individuals: a longitudinal cohort study. Lancet 359(9309): 831–834. Gern JE (2002) Rhinovirus respiratory infections and asthma. American Journal of Medicine 112(Supplement 6A): 19S–27S. Gern JE and Busse WW (2002) Relationship of viral infections to wheezing illnesses and asthma. Nature Reviews: Immunology 2(2): 132–138. Gern JE and Lemanske RF Jr. (2003) Infectious triggers of pediatric asthma. Pediatric Clinics of North America 50(3): 555–575. Green RM, Custovic A, Sanderson G, et al. (2002) Synergism between allergens and viruses and risk of hospital admission with asthma: case-control study. British Medical Journal 324(7340): 763. (Erratum British Medical Journal 324 (7346): 1131.) Johnston SL and Martin RJ (2005) Chlamydophila pneumoniae and Mycoplasma pneumoniae: a role in asthma pathogenesis? American Journal of Respiratory and Critical Care Medicine, doi: 10.1164/rccm.200412–1743pp. Message SD and Johnston SL (2002) Viruses in asthma. British Medical Bulletin 61: 29–43. Message SD and Johnston SL (2004) Host defense function of the airway epithelium in health and disease: clinical background. Journal of Leukocyte Biology 75(1): 5–17. Talbot TR, Hartert TV, Mitchel E, et al. (2005) Asthma as a risk factor for invasive pneumococcal disease. New England Journal of Medicine 352(20): 2082–2090.

ASTHMA / Exercise-Induced 199 Wark PA, Johnston SL, Bucchieri F, et al. (2005) Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus. Journal of Experimental Medicine 201(6): 937–947. Wark PA, Johnston SL, Moric I, et al. (2002) Neutrophil degranulation and cell lysis is associated with clinical severity in virusinduced asthma. European Respiratory Journal 19(1): 68–75.

Exercise-Induced N C Thomson and M Shepherd, University of Glasgow, Glasgow, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Exercise-induced asthma (EIA) is described in asthmatics, elite athletes, and the general population alike. Difficulties reconciling the variety of presentations complicate our understanding of the disease process and prevalence. Exercise-induced bronchospasm (EIB) can be measured during and after exercise in susceptible individuals, but whether this signifies EIA is unclear. Exercise can cause an inflammatory response in the airway that appears to be dose dependent and exaggerated by breathing cold dry air. Classical understanding of the pathogenesis of EIA suggests that drying of the airway mucosa leading to an osmotic gradient across the epithelium and basement membrane combined with cooling of the bronchial wall triggers bronchospasm in EIA. Modern investigation suggests that airway surface stress can be communicated to the bronchial wall by chemical signals arising from the epithelium. Managing a patient with EIA requires careful investigation to ensure the correct diagnosis and consideration of alternative disease processes should be made. Pharmacological intervention can both treat and limit the extent of EIB, but behavioral modifications can also be introduced. Finally, clinicians managing EIA should be aware of the conflicts that arise when regular asthma medications are used by patients engaging in competitive sport.

Introduction Increased airway resistance triggered by vigorous exercise is variously called exercise-induced asthma (EIA) or exercise-induced bronchospasm (EIB). Although a consensus exists on the criteria for making a diagnosis of EIB, the range of circumstances in which it has been recognized mean a precise clinical definition of EIA is difficult to achieve. EIB has been described in asthmatics and those with no history of asthma and subjects ranging from school children to elite athletes. It is not clear whether the disease is the same in all cases. While it may be more accurate to reserve the term EIA for exercise-induced, symptomatic bronchial narrowing occurring in previously diagnosed asthma we will use the term broadly to describe asthmatic symptoms or physiological changes consistent with asthma developing during or immediately after exercise.

Epidemiology EIA is common in known asthmatic subjects and exercise has been recognized as a trigger for ‘asthma’ since the seventeenth century. At least 90% of asthma sufferers will experience a fall in forced expiratory volume (FEV1) or peak flow during or shortly after an appropriate exercise challenge. Some authorities believe that all asthmatics will have demonstrable bronchospasm following sufficient exercise challenge. In atopic patients who suffer only from allergic rhinitis prevalence drops to 40%. Studies of other groups are complicated by the definition of EIA used. For example, comparing the prevalence of EIB, hypersensitivity to methacholine, and self-reported symptoms of EIA consistently identify different groups within a given population. Most of the prevalence data provided are therefore relatively generalized (Table 1). EIB is a useful model for clinical asthma and it has been used as a screening tool. These cohorts are frequently drawn from populations engaged in regular exercise so may not represent a truly unselected population. Based on these studies the prevalence of EIA in the general adult population is estimated at between 6% and 13% and this varies geographically with higher levels in the UK and Australia and lower levels in developing countries. In cohorts of normal school children challenged with exercise then tested by spirometry up to 12% are shown to have EIA. The identification of exercise-induced bronchial narrowing is often a surprise to both child and adult subjects, who may not have been aware of any such problem. A poor perception of airflow restriction is generally recognized in asthma but also casts some doubt on the definition of EIA based on spirometry alone. It is not known if otherwise normal subjects who experience a fall in FEV1 with exercise will benefit from asthma therapy or will go on to develop clinical asthma later in life. Failure to recognize potentially reversible EIA may not be a trivial matter. Children who suffer distressing

Table 1 Estimated prevalence (percent study population) of exercise induced asthma based on a number of studies using different methodology Study population

Children (o18 years) (%)

Adults (%)

General population Asthma Atopic nonasthmatic Athletes Elilte Recreational

9 45–80 15

4–20 70–90 40

19

4.2–20 11–50

EIA prevalence has been reported using physiological based parameters and self-reporting questionnaires.

200 ASTHMA / Exercise-Induced

symptoms will tend to avoid exercise leading to possible psychosocial and neurological development problems. Similarly, the importance of correct identification of EIA is highlighted by studies demonstrating lower self-esteem scores in children who perceive themselves to be socially disadvantaged by asthma. A surprisingly high prevalence of EIA is reported in both recreational and elite athletes (Table 2). Anecdotally, this association has been explained by the focus of some asthmatic children on ventilatory control and on athletic pursuit to achieve this. Whatever the cause, around 23% of recreational athletes report asthma induced by exercise while within the elite ranks prevalence rates of between 16% in summer outdoor events and 50% in winter events such as cross-country skiing or ice skating are reported. While asthmatics frequently state that swimming tends to cause less bronchospasm than other sports, a particularly high prevalence of EIA has been observed in elite swimmers in whom up to 79% have been reported to have demonstrable bronchial hyperresponsiveness to methacholine and 33% have asthma. The prevalence of EIA reported in elite athletes has raised concern among sporting governing bodies regarding the potential for inappropriate use of performance enhancing medications. As a result a considerable body of research has focused on EIA developing in elite athletes. However, elite athletes are subject to a variety of additional possible triggers of EIB including the stress of competition. Thus, whether this research is equally applicable to recreational athletes or nonathletic sufferers from EIA is not known. Therefore, it appears that EIA, defined by a fall in FEV1, occurs in a relatively high proportion of the general population. This prevalence is increased by underlying atopy, asthma, and athletic pursuit,

Table 2 The prevalence of EIA based on sporting pursuit Athletes

Season

Olympians

Summer Winter

Elite/events

Summer Winter

All seasons a

Activity

Asthmaa (%)

EIB (%)

23 22 Track/field Indoor Nordic skiing Skating Nonendurance Swimming

19 12 60 6.4 1 44–50

12 50 35

Definition of asthma varies among reports and includes selfreporting of ever diagnosed asthma and current or ever used asthma medications.

particularly outdoor winter endurance events. Accurate assessments of prevalence are hampered by difficulty in definition stemming from the range of possible criteria for diagnosis and the variety of presenting complaints, which will be outlined later. Further difficulties stem from poor perception and variable self-reporting. This difficulty in precise definition has also limited understanding of the pathology underlying EIA.

Pathogenesis Pathophysiology

The normal respiratory responses to exercise result in an increase in ventilation of up to 200 l min
1 . In order to facilitate the increased airflow, mild bronchodilation occurs early during an exercise event and this can be measured using spirometry. Classically, EIA was described as taking place after the exercise challenge was completed, but it is clear that airway narrowing can occur during exercise lasting more than 12 min and in the postexercise period. The ‘stop–start’ nature of some sporting events means that symptoms developing during exercise are entirely consistent with a diagnosis of EIA. A variable period of protection from further exercise-induced bronchial narrowing known as the refractory period is well described. A refractory period may last from 30 min to 2 h and may be due in part to the bronchodilator properties of prostaglandin E2 (PGE2). The cause of EIA and the refractory period are not fully understood; however, considerable evidence points to a cellular response to increased ventilation triggering an inflammatory reaction in the airway. EIA may therefore be considered as a subset of chronic asthma in which exercise is the trigger to inflammation. While this hypothesis is attractive, a variety of studies have produced conflicting data regarding the inflammatory character of EIA. The three-stage model proposed by R Gotshall serves as a useful framework to consider the pathogenesis of EIA. In this model an exercise challenge serves as a trigger sensed in the airway that ultimately signals to the cells of the airway controlling caliber. Hyperventilation alone can cause bronchoconstriction in human and canine subjects and has been identified as the key element of exercise that triggers EIA. How this trigger leads to bronchoconstriction is a matter of controversy. Airway Cooling and Hyperosmolarity

In 1864, H H Slater noted that cold air triggered asthma and offered pulmonary vascular congestion as a possible explanation. This theory has been

ASTHMA / Exercise-Induced 201

developed by McFadden and others. Airway cooling in response to inspired cold air may trigger vasoconstriction of the bronchial vasculature. Subsequent reflex vasodilation and hyperemia with extravasated fluid leading to mucosal edema could lead to airway narrowing. Although attractive, this theory fails to acknowledge the capacity of the upper airways to warm inspired air such that it is unlikely that cold air is delivered to medium-sized airways, the main site of narrowing in asthma. However, cooling of the airways could take place if evaporation of mucosal water exceeded the replacement capacity of the airway. Exercise is associated with mouth breathing, which bypasses the humidifying effect of the nasal mucosa, and dry air has been shown to increase the capacity of an exercise challenge to trigger EIB. Evaporation of water from the upper airways might take place as a result of a large increase in ventilation of relatively dry air. This would cause an increase in the osmolarity of the airway mucosa. This concept of airway hyperosmolarity has been developed by Anderson and colleagues and has become popular in current literature. Inhaling hyperosmolar mannitol or saline solutions can trigger bronchospasm in the absence of an exercise stimulus. It has been difficult to separate the specific elements of these two processes and it may be that both are partly responsible for the effect of hyperventilation on the airway in EIA. Since cold air holds less moisture than warm air, this may add to the drying of the airway. Alternative mechanisms for transducing the proasthmatic stimulus to a bronchoconstrictive response can be postulated. These include stretching of the airway wall cells and altered pressure dynamics of the airway lumen. Ultimately, a signal to alter the diameter of the airway lumen is generated that results in a fall in FEV1. The nature of this signal is also controversial but biochemical mediators associated with inflammation offer a possible explanation. Inflammation in EIA

It has become clear that an inflammatory process in the airway leads to bronchial hyperresponsiveness and chronic asthma. Exacerbations of asthma can be

caused by a variety of inflammatory triggers such as allergen exposure or viral infection. While less intuitive it seems possible that EIA may also have an inflammatory basis. Table 3 details the variety of inflammation markers that have been studied with reference to EIA. Biological markers of inflammation A variety of studies have demonstrated elevated inflammatory cells in the airway lumen and bronchial wall from athletes engaged in a wide spectrum of athletic pursuits. These cells include eosinophils, T lymphocytes, macrophages, and mast cells all of which are recognized as key cellular elements of the asthmatic inflammatory response. Changes in airway inflammatory cell number correlate with changes in bronchial reactivity and some groups have demonstrated that the degree of bronchospasm in response to exercise challenge more closely reflects the level of airway eosinophilia than the prechallenge methacholine sensitivity. Bronchial biopsies taken from resting cross-country skiers demonstrated increased inflammatory cells in the bronchial wall compared to nonathlete controls. Regular exercise may therefore lead to a persistent airway inflammation beyond the acute effects of exercise challenge. Cell-based studies appear to support the role of exercise as a trigger for airway inflammation. The study of winter athletes described above demonstrated that airway wall inflammation occurred even in the absence of symptomatic EIB. This suggests that airway inflammation is not sufficient to cause EIA and that exercise per se can induce inflammation in normal subjects. Unfortunately, none of these studies offer an explanation for why some athletes are susceptible to the inflammatory effects of exercise while others are not. Inflammatory cells in the airway are presumed to cause bronchial hyperresponsiveness by the production of chemical mediators of inflammation. A variety of such mediators have been studied in asthma and EIA (Table 3). Early studies investigating the role of the mast cell stabilizers such as nedcromil and sodium cromoglycate identified histamine as a likely

Table 3 Inflammatory mediators in EIA Mediator

Role in asthma

Induced by exercise

Efficacy of inhibitor

Histamine Leukotrienes Prostaglandin D2

Bronchoconstrictor Bronchoconstriction, smooth muscle proliferation, chemoattractant


þ þ

? þþ Variable

Various inflammatory mediators have been investigated for their capacity to induce or maintain airway inflammation in EIA. Evidence for induction during exercise comes from studies looking at plasma, urine, or sputum levels of the mediator or product. The role of histamine has recently been challenged due to the overriding effect of leukotriene antagonists with no additional effect of antihistamine and a consistent difficulty in demonstrating exercise induction.

202 ASTHMA / Exercise-Induced

trigger for EIA although this has been challenged recently. The role of other chemical mediators of inflammation including cysteinyl leukotrienes (LTD4 and LTC4) and prostaglandins (PGD2 and PGF2) have been supported by the discovery of increased levels in postexercise plasma and urine and by the effects of specific antagonists. The role of inflammatory cells and their products in the development of EIA is supported by studies demonstrating the capacity of exercise to increase their availability to airway cells. However, not all investigators have found increased levels of proasthmatic mediators in response to exercise, and the presence of a particular agent does not imply a causal link. Further evidence is available from interventions aimed at reducing airway inflammation. Do Anti-Inflammatory Therapies Prevent EIA?

Inhaled corticosteroids (ICs) are the most widely used airway-delivered anti-inflammatory agents. Among well-controlled asthmatic subjects who use regular ICs to abolish normal symptoms 50% will still experience EIB on testing. This suggests that EIA is relatively resistant to standard doses of IC therapy. This hypothesis is supported by early studies demonstrating only a 50% reduction in exercise-induced fall of FEV1 when ICs were introduced. When a short-acting b-adrenergic receptor agonist was added to the IC therapy, this further reduced EIB by 30%, hinting that increased airway resistance in EIA is a complex phenomenon. The introduction of modern anti-inflammatories has reinforced the theory that EIA is an inflammation-mediated phenomenon. Leukotrienes and in particular LTD4 are regarded as among the most potent proasthmatic inflammatory mediators. Leukotriene receptor antagonists (LTRAs) inhibit the effect of these mediators at their specific receptors. The introduction of LTRAs to common practice suggested that they might have efficacy in treating EIA, a suggestion supported by subsequent examination. LTRA therapy provides a dose-dependent protection from EIB and shortens the time to recovery of FEV1. In contrast antihistamine therapy does not appear to add any further protection over LTRA medication. Several lines of evidence support the role of inflammatory processes in EIA. Exercise can trigger and maintain airway inflammation that is associated with bronchial hyperresponsiveness. Intervention studies show that combating inflammation can improve EIA. What is unclear, however, is what predisposes an individual to develop exercise-induced airway inflammation and why otherwise potent antiinflammatory agents are only partially effective in

preventing EIA despite improved control in underlying asthma. Finally, various chemical triggers of asthma have been investigated for their ability to trigger or enhance bronchoconstriction on exercise challenge. Of these adenosine, the product of ATP breakdown, seems worthy of further investigation. Exercise increases circulating adenosine levels suggesting that any proasthmatic activity of this chemical could then be enhanced by exercise. A correlation between the fall in FEV1 following exercise challenge in asthmatic subjects and plasma adenosine levels has been reported. Adenosine is believed to trigger inflammatory cell degranulation, and in the context of asthma this may enhance a mild asthmatic response making it considerably more severe. Such studies may ultimately explain why exercise can be the only trigger for symptomatic EIA in otherwise nonasthmatic subjects. Integrating the Two Pathogenic Theories

It can be concluded that exercise-induced hyperventilation can trigger physical changes in the airway that are subsequently transduced to an inflammatory signal in the bronchial wall, which can be assumed to lead to bronchoconstriction and possibly chronic inflammation in susceptible people. Can physical changes at the surface of the airway communicate to the bronchial wall to cause inflammation and bronchial hypersensitivity? Evidence for pathways communicating signals between different airway compartments has been accumulating over the last decade, offering possible mechanisms to complete the model. As yet these pathways are untested in the context of EIA but do offer intriguing possibilities. It is becoming clear that airway epithelial stress can cause the release of inflammatory mediators that promote airway inflammation. Recent research supports the development of an integrated epithelium– mesenchyme complex (the epithelial mesenchymal trophic unit) reminiscent of the embryonic lung. Epithelial monolayers in vitro can trigger inflammatory signals in fibroblasts and smooth muscle cells of the airway supporting such a communication. Inflammatory cells found superficially in the airway, such as eosinophils, can be activated by osmolar stress and release mediators capable of eliciting an asthmatic response offering a more direct line of communication. Thus, drying and cooling of surface epithelium could in theory at least trigger generalized airway inflammation as seen in chronic asthma. The hyperemia following airway rewarming would support an inflammatory process by supplying exudates and inflammatory cells (Figure 1).

ASTHMA / Exercise-Induced 203

Hyperventilation/exercise

Cooling of the airway & bronchial vasoconstriction

Reactive vasodilitation & mucosal hyperemia

Hyperosmolarity of airway mucous layer

Mucosal congestion & exudate formation

Accumulation of inflammatory cells in the airway lumen

Osmotic stress communicated to superficial cells of the airway

Degranulation Degranulation of inflammatory cells – release of inflammatory mediators LTD4 and PGE2

Release of proinflammatory cytokines from epithelial cells

Bronchial wall inflammation, airway smooth muscle contraction, mucosaledema, possible epithelial cell shedding Figure 1 Summary diagram of the proposed pathogenesis of EIA. A series of pathological events following increased ventilation in exercise are proposed that trigger an asthmatic response in susceptible individuals.

Clinical Presentation Symptoms associated with exercise can arise from a variety of sources including the lungs, heart, and gastrointestinal tract. A careful history can help to identify the most likely source, but EIA may present with classical symptoms such as dyspnea, wheeze, and cough or with more obscure symptoms making the diagnosis difficult. Complaints such as headache, abdominal pain, chest pain, cramps, and severe fatigue may all respond to treatment for EIA. Symptoms may develop early during an exercise event or following its completion. The association with exercise should

alert the clinician to the possibility of EIA although the complaint may seem unrelated to the lungs. Frequently patients believe they are simply ‘out of condition’ and even well-controlled asthmatics may not associate their exercise-induced symptoms with asthma. EIA can be the only manifestation of hyperresponsive airways so the absence of a formal diagnosis of asthma does not exclude the possibility of EIA.

Differential Diagnosis Since the diagnosis of EIA is not always straightforward to make a high index of clinical suspicion, it

204 ASTHMA / Exercise-Induced Table 4 Differential diagnosis of the patient presenting with exercise-induced breathlessness System

Disease

Respiratory

EIA COPD Pulmonary fibrosis Pulmonary vascular hypertension Angina Ventricular dysfunction Dysrhythmia Poor fitness

Cardiovascular

General

COPD, chronic obstructive pulmonary disease; EIA, exerciseinduced asthma.

should be combined with an open mind regarding the variety of other potential sources of symptoms. In particular, true vocal cord dysfunction can be difficult to distinguish from EIA when symptoms predominantly present with exercise. A list of alternative diagnoses for exercise-induced dyspnea is presented (Table 4). It is not yet clear if patients who have demonstrable exercise-induced bronchoconstriction but no symptoms will benefit from pharmacological treatment. Examination of the lungs of a subject with exerciseinduced dyspnea will usually be normal in the context of asthma or lone EIB. The presence of wheeze or hyperinflation might point to chronic airflow obstruction that has gone unrecognized. A full examination might point to an alternative diagnosis as listed. Again quiescent asthma or lone EIB will usually be associated with normal pulmonary function tests while abnormalities may point to alternative pulmonary diagnoses or chronic airflow obstruction.

Investigation The need for further investigation will be directed by the clinical findings but may fall into two groups. In the first a pragmatic trial of pharmacological intervention prior to a predictable exercise trigger of symptoms can be the most useful and easiest test. In some cases, however, detailed pulmonary function in response to exercise is desirable either to monitor treatment, to make a difficult diagnosis, or if the presence of EIA would limit the performance of essential life-saving work (American Thoracic Society (ATS) Guideline). Other uses for detailed exercise testing include the diagnosis of asthma in elite athletes for drug monitoring or performance testing. In a pragmatic trial a patient should be prescribed either a preventative or a reliever inhaled therapy and advised to use this prior to or during exercise that would normally trigger the reported symptoms. Antiinflammatory drugs such as inhaled corticosteroids

or cromoglycate have been shown to have some preventative efficacy in this setting. b2-adrenergic agonists have been shown to relieve exercise-induced bronchoconstriction but their efficacy is reduced in EIB. Patients should be able to record the effect of treatment on symptoms or on peak flow with a small degree of training. Where it is desirable to direct investigations towards a specific diagnosis of EIA, a variety of alternative procedures, ranging from exercise provocation to bronchial challenge, are available. Various procedures have been described that are capable of triggering airway narrowing in asthma consistent with a diagnosis of EIA. The principal element of all these tests is to raise minute ventilation as occurs with exercise. It is possible to trigger bronchial narrowing by hyperventilation and this has been recommended as a surrogate for more formal exercise challenge. More exercise specific tests have been developed for both field and laboratory. While testing in the field has the advantage of replicating the conditions that normally trigger asthma in the subject, the equipment required to make useful measurements has to be portable. This has led to the development of protocols designed to measure pulmonary responses to exercise under laboratory conditions that replicate the essential features of outdoor activity. Guidelines recommending the best practice for performing these tests are available, the essential features of which are summarized below. Patient Instructions

Subjects should avoid bronchoprotective or bronchoreliever medication for 48 h and have eaten only a light meal. Antihistamines and caffeine should also be avoided. Exercise should be kept to a minimum prior to the test, as around 50% of EIA sufferers will experience a refractory period of up to 4 h after vigorous exercise. Any medical or orthopedic contraindications to exercise should be considered and the ability of the patient to fulfill the physiological requirements should be ensured. Exercise

Exercise can be performed on an electronic treadmill or stationary bicycle as both methods have been validated. The desired level of exercise is based on 80–90% of maximal heart rate (based on an HR 220 – age) or 50–60% of maximal voluntary ventilation. The degree of exercise required to achieve this level of exercise response will vary considerably among subjects and it is advised that a period of rapid progressive increase in workload is used to achieve these targets and then maintained for at least 4 min. The

ASTHMA / Exercise-Induced 205

total exercise time for adults should be around 6– 8 min. During the exercise, heart rate and where possible minute ventilation should be measured to ensure that an adequate stimulus to the airway is being achieved. Climatic Considerations

Outdoor exercises are generally better than indoor exercises at triggering bronchospasm. This is believed to be due to lower humidity and temperature and cool dry air is known to trigger asthma in exercise sensitive subjects better than indoor room air. Maintaining the ambient temperature at 20–251C and relative humidity at 50% is satisfactory. Exercise should be performed with a nose clip to prevent nasal humidification of inspired air. Severe responses to exercise have been described in susceptible people and it is advisable to have medical supervision available to monitor patients’ responses and administer bronchodilator therapies as required. Measurements

FEV1 (% fall)

FEV1 is the most useful and convenient measurement to make in the laboratory. Where an alternative diagnosis is suspected full flow-volume loops can offer additional information, while peak expiratory flow might be used if field testing is performed. FEV1 should be measured before exercise and following the exercise challenge. Intervals of 5, 10, 15, 20, and 30 min are recommended by the ATS; however, additional measurements can be taken and should be in the event of severe symptoms of bronchial obstruction. Changes in FEV1 can then be observed by plotting percent resting FEV1 against time (Figure 2).

20 18 16 14 12 10 8 6 4 2 0

What constitutes a ‘positive’ exercise test is controversial due to the variety of techniques described for measurement. Most authorities consider a fall of more than 10% of resting FEV1 as abnormal especially as the ‘normal’ response to exercise is bronchodilation. Some groups require a greater than 15% fall in FEV1 to diagnose EIA and this appears to be the most frequently quoted value. Plotting the FEV1 against time allows an area under the graph calculation to be made that improves the reliability of the test. Alternative Testing Procedures

Other techniques to mimic the airway response to exercise have been described. These are generally thought to be more convenient than full exercise challenge, but are necessarily less specific. Osmotic drying of the airway has been achieved by inhalation of mannitol and this method has received some attention from testers from the field of elite sport. Eucapnic hyperventilation achieves a ventilation rate that mimics that achieved by exercise and has also been used as an outpatient measure of the airway responses. Management

The efficacy of asthma medications for exerciseinduced symptoms will be dealt with elsewhere. Patients should be encouraged to manage their symptoms rather than stop the exercise activity that triggers it. Occasionally, altering the exercise environment is helpful, for example, changing to indoor from outdoor activity. Some elite athletes have learned to manage their asthma by introducing a targeted warm up to trigger a mild episode of EIA and the resultant refractory period elite may allow the completion of the competitive element of the activity. Sporting authorities restrict the use of most inhaled therapeutic agents for asthma. It is sensible for athletes and their coaches to be aware of these restrictions, which can be accessed easily. A range of websites for agencies involved in regulating drugs in sport is given at the end of this article.

Conclusion 0

10 20 30 40 50 60 70 Time from start of exercise (min)

Figure 2 A plot of change in FEV1 as percent baseline with time exercised. Exercise begins at time 0 and ceases at the point marked by the arrow. FEV1 is measured and the percent change is calculated. , change in FEV1; , the effect of an agent , the effect of an agent that changes maximum fall in FEV1; that reduces the duration of EIB. As can be seen it is frequently more accurate to represent changes to EIB by describing the area under the curve than any single element of the plot.

EIA may be a specific disease entity in some circumstances or may be the only manifestation of latent asthma. It is common in asthma sufferers and in athletes of all capabilities. The basis for the development and persistence of exercise triggered airway pathology is not fully understood but probably reflects the ability of hyperventilation to trigger airway inflammation. Diagnosis can be complicated by the variety of presentations and detailed investigations

206 ASTHMA / Extrinsic/Intrinsic

are sometimes required. Asthma management is not always successful at controlling symptoms and behavioral changes may have to be made to achieve control of the disease. See also: Exercise Physiology. Lipid Mediators: Leukotrienes.

Further Reading Anderson SD and Brannan JD (2002) Exercise-induced asthma: is there still a case for histamine? Journal of Allergy and Clinical Immunology 109(5): 771–773. Anderson SD and Daviskas E (2000) The mechanism of exerciseinduced asthma is. Journal of Allergy and Clinical Immunology 106(3): 453–459. Gotshall RW (2002) Exercise-induced bronchoconstriction. Drugs 62(12): 1725–1739. Milgrom H (2004) Exercise-induced asthma: ways to wise exercise. Current Opinion in Allergy and Clinical Immunology 4: 147–153. Seale JP (2003) Science and physicianly practice: are they compatible? Clinical and Experimental Pharmacology and Physiology 30(11): 833–835. Storms WW (2003) Review of exercise-induced asthma. Medicine and Science in Sports and Exercise 35(9): 1464–1470. Tan RA and Spector SL (2002) Exercise-induced asthma: diagnosis and management. Annals of Allergy, Asthma, and Immunology 89(3): 226–235.

which many cells and cellular elements play a role, in particular, eosinophils, mast cells, T lymphocytes, neutrophils, and epithelial cells. Some patients develop structural changes of the airway, a process known as remodeling, possibly due to ongoing inflammation and abnormal repair processes. Susceptible individuals experience recurrent episodes of wheezing, breathlessness, chest tightness, and cough, particularly at night and in the early morning. These episodes are usually associated with widespread but variable airflow obstruction, which is often reversible, and bronchial hyperresponsiveness to a variety of stimuli. Acute asthma is a common medical emergency and requires prompt assessment and treatment. Advances in the understanding of the genetic and environmental factors that account for asthma and its pathogenesis should lead to improved management strategies.

Introduction Historical Perspective

The symptoms of asthma were described by Aretaeus over 2000 years ago. However, despite significant progress in our understanding of its pathogenesis and considerable improvements in pharmacological treatment, we have been unable to halt the relentless increase in prevalence that has taken place over the last 30 years.

Definition Relevant Websites http://www.olympic.org – Home page of the International Olympic Movement, offering insight into the role of antidoping authorities in elite sport. It includes useful links to national Olympic committees detailing specific national guidelines for athletes. http://www.wada-ama.org Homepage of the world antidoping authority. Including details of the use of prescribed proscribed therapeutics in sport. http://www.asthma.org.uk Asthma UK link to exercise induced asthma with tips for patients and some general information regarding school and preschool sporting activities for asthma sufferers.

Extrinsic/Intrinsic N C Thomson and G Vallance, University of Glasgow, Glasgow, UK & 2006 Elsevier Ltd. All rights reserved.

Several different definitions have been devised that describe the asthma phenotype. In 1997 the National Asthma Education and Prevention Program Expert Panel Report defined asthma as: ‘‘A chronic inflammatory disorder of the airways in which many cells and cellular elements play a role, in particular, mast cells, eosinophils, T lymphocytes, neutrophils, and epithelial cells. In susceptible individuals, this inflammation causes recurrent episodes of wheezing, breathlessness, chest tightness, and cough, particularly at night and in the early morning. These episodes are usually associated with widespread but variable airflow obstruction that is often reversible either spontaneously or with treatment. The inflammation also causes an associated increase in the existing bronchial hyperresponsiveness to a variety of stimuli.’’

Epidemiology Prevalence of Asthma and Atopy

Abstract Asthma is one of the most common chronic diseases, affecting 300 million people worldwide. There has been a significant increase in prevalence over the last 30 years, particularly in the West. Complex relationships between genetic and environmental factors, such as viral infections, allergens, and occupational agents, influence the origin and progression of the disease. Asthma is a chronic inflammatory disorder of the airway in

Asthma is one of the most common chronic conditions affecting 300 million people worldwide. The Global Initiative for Asthma (GINA) estimates that one in 20 people in the world now have asthma, with a significant increase in the prevalence of disease over the last 30 years. This is in parallel with an increase in other atopic diseases, such as allergic rhinitis and

Country

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207

Scotland Jersey Guernsey Wales Isle of Man England New Zealand Australia Republic of Ireland Canada Peru Trinidad & Tobago Costa Rica Brazil United States of America Fiji Paraguay Uruguay Israel Barbados Panama Kuwait Ukraine Ecuador South Africa Finland Malta Czech Republic Ivory Coast Colombia Turkey Lebanon Kenya Germany France Japan Norway Thailand Sweden Hong Kong United Arab Emirates Philippines Belgium Austria Saudi Arabia Argentina Iran Estonia Nigeria Spain Chile Singapore Malaysia Portugal Uzbekistan FYR Macedonia Italy Oman Pakistan Tunisia Latvia Cape Verde Poland Algeria South Korea Bangladesh Morocco Occupied Territory of Palestine Mexico Ethiopia Denmark India Taiwan Cyprus Switzerland Russia China Greece Georgia Romania Nepal Albania Indonesia Macau

0

5

10

15

20

25

30

35

40

Prevalence of asthma symptoms (%) Figure 1 Ranking of the prevalence of current asthma symptoms in childhood by country: written questionnaire. Reproduced with permission from GINA (2004) Self-reported wheezing in the previous 12 month period in 13- to 14-year-old children. Global Burden of Asthma, p. 6.

atopic dermatitis. These increases have been most noticeable in affluent countries with a mild climate such as the UK, New Zealand, Australia, and North America (Figure 1) and correlate with urbanization and the adoption of a westernized life style. It is more common in children than in adults. The clearest risk factor for the development of asthma is atopy. Atopy is the genetic predisposition for the development of an IgE-mediated response to common aeroallergens. Complex relationships between atopy and

environmental factors such as viral infections, allergens, and occupational agents influence the origin and progression of the disease. Morbidity

The morbidity from asthma is considerable. Surveys of patients with asthma indicate that many have poorly controlled symptoms, impaired indices of quality of life, and are often receiving inadequate

208 ASTHMA / Extrinsic/Intrinsic

treatment. Hospital admission rates for asthma, particularly in children, increased from the 1970s until the mid 1980s and have since then remained stable. In the US during 2002 there were 13.9 million outpatient visits, 1.9 million emergency room visits, and 484 000 hospitalizations. Many children and adults with asthma lose time from school and work, respectively. The financial impact of asthma is considerable; in the US the estimated total cost exceeds $6 billion per annum, with hospitalization and emergency room visits making up 50% of that figure.

Classification Asthma can be classified on the basis of severity and etiology. Severity

The severity of asthma can be graded by assessing the frequency and severity of symptoms and measurements of lung function before treatment is started or by the level of treatment required to achieve asthma control (Table 1). Etiology

Mortality

International mortality figures for asthma are often unreliable due to misclassification of the cause of death. In Western countries, where studies were restricted to the 5–34 years age group, the mortality from asthma increased steadily from the mid-1970s to the late 1980s. More recent studies suggest a plateau or decline in deaths from asthma. Risk factors for increased morbidity and potential mortality include socioeconomic deprivation, ethnicity, urban dwelling, and comorbid issues such as drug abuse. The vast majority of deaths occur among those with chronic severe asthma; few deaths occur among those with previously mild disease. Deaths are associated with inadequate treatment with inhaled or oral steroid and with poor follow-up and monitoring. Natural History

The findings of the Tucson Children’s Respiratory Study suggested three clinical phenotypes of childhood asthma. Transient infant wheezing occurs during infancy, but not after the age of three years. These children have no family history of atopy and have a good prognosis. The second phenotype is the nonatopic wheeze of the toddler and early school years, after an early lower respiratory tract infection. The third phenotype is persistent atopic wheeze, which describes children who continue to wheeze at age 10 and have associated atopy and airway hyperresponsiveness. Many children have a favorable outcome with spontaneous remission in their adolescence. Risk factors for progression into adulthood include early onset with severe symptoms, poor lung function, and airway hyperresponsiveness. It occurs more commonly in girls with associated atopy. Most adults with mild-to-moderate asthma appear to continue to have symptoms of a similar severity. Remission of adult asthma is rare. Irreversible airflow obstruction can develop in nonsmokers with asthma particularly in those individuals with severe symptoms and mucus hypersecretion. Smokers with asthma have an accelerated decline in lung function.

In 1947, Rackeman was the first to subdivide asthma into intrinsic and extrinsic asthma. He noted that extrinsic asthma, now described as allergic asthma, started before the age of 30 years and was associated with atopy. Intrinsic or nonallergic asthma was noted to begin in middle age and was not associated with allergy, but with nasal polyps. It is more common in women.

Etiology Genetic

Twin and family studies have demonstrated that atopic diseases cluster in families and have a genetic basis. Genome-wide scans have shown that many genes determine the risk of asthma. The region of chromosome 5q31-33 controls the production of interleukin (IL)-4 and IL-13 and has been linked to atopy. Linkage studies have implicated other candidate genes on chromosomes 2, 3, 4, 6, 7, 11, 12, 13, 17, and 19. Classical positional cloning approaches have led to the identification of new genes of potential significance such as ADAM33, IL4RA, and CD14. This may contribute to our understanding of the mechanism of disease, such as the role of ADAM33 in airway hyperresponsiveness. They may also cast light on different individual responses to therapy, as there are polymorphisms of a number of common drug targets. The delineation of the precise relationships between these genetic factors and environmental agents is now required. Environment

Hygiene hypothesis In 1989, Strachan noted an inverse relationship between family size and hay fever, with children with more siblings having a lower risk of atopy. It has been suggested that childhood exposure to infection may protect against risk of atopy. Low levels of infection in infancy, associated with improvements in public health, may deprive the immune system of the Th1 stimulus that normally balances the Th2 predominance of the neonate. This

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209

Table 1 Classification of asthma severity by daily medication regimen and response to treatment Patient symptoms and lung function on current therapy

Current treatment step Step 1: intermittent

Step 2: mild persistent

Step 3: moderate persistent

Level of severity Step 1: intermittent Symptoms less than once a week Brief exacerbations Nocturnal symptoms not more than twice a month Normal lung function between episodes

Intermittent

Mild persistent

Moderate persistent

Step 2: mild persistent Symptoms more than once a week but less than once a day Nocturnal symptoms more than twice a month but less than once a week Normal lung function between episodes

Mild persistent

Moderate persistent

Severe persistent

Step 3: moderate persistent Symptoms daily Exacerbations may affect activity and sleep Nocturnal symptoms at least once a week 60%oFEV1 o80% predicted OR 60%oPEFo80% of personal best

Moderate persistent

Severe persistent

Severe persistent

Step 4: severe persistent Symptoms daily Frequent exacerbations Frequent nocturnal asthma symptoms FEV1 p60% predicted OR PEFp60% of personal best

Severe persistent

Severe persistent

Severe persistent

Reproduced with permission from GINA (2004) Global Strategy for Asthma Management and Prevention, Chapter 7, Part 4A, Figures 5–7, p. 7.

unrestrained Th2 response is postulated to predispose to allergic disease. European studies showing lower levels of atopic disease amongst children raised in rural communities have suggested that exposure to bacterial endotoxin may play a role in the development of tolerance to common allergens. However, the high levels of asthma associated with cockroach allergy in inner-city areas of the US, and the simultaneous increase in Th1 diseases, such as type 1 diabetes, illustrate that our understanding of the relationship between environmental exposure and disease is not yet complete. Allergen exposure Sensitization to the house dust mite Dermataphagoides pteronnysinus is the most common risk factor for the development of asthma in adults and children. Early exposure to the dust mite antigen Der p1 has been shown to increase the risk of asthma fivefold. Peat elegantly demonstrated the doseresponse relationship between dust mite level and severity of asthma symptoms across six different regions

of Australia with increasing humidity levels. Dust mites thrive in a humid environment, and improved westernized building construction techniques, favoring colonization by house dust mites, may have contributed to the increase in asthma. Studies at altitudes where the levels of house dust mite are extremely low have suggested that a low allergen environment does improve symptoms of asthma. However, simple measures such as mattress covers and carpet cleaning have shown little effect on reducing the domestic burden of dust mite and asthma symptoms. Exposure to other indoor allergens from animal dander and cockroach are also important risk factors for asthma. Removal of the pet from the family is advised to reduce exposure to the allergen. However, early exposure to cat allergen has recently been demonstrated to protect against development of asthma and the full intricacies of the relationship remain to be elucidated. Cockroach infestation has become rampant in inner-city areas of the US and sensitization to the

210 ASTHMA / Extrinsic/Intrinsic

German cockroach Blatella germanica has become an important risk factor for asthma. Outdoor air environment Sensitization to the fungus Alternaria is a risk factor for asthma. It has been suggested that outbreaks of asthma after thunderstorms are related to release of fungal spores of Cladosporium. The relationship between air pollution and asthma is complex as there has been considerable reduction in air pollution over the time period when asthma prevalence has increased. Extremely high levels of asthma have been noted in the rural Highlands of Scotland. It has been suggested that particulate pollution may protect against incidence but exacerbate symptoms in those sensitized. Diet Changes in the modern processed diet have been linked with an increased risk of asthma. High levels of omega-3 oils associated with eating fresh fish have been shown to reduce the risk of asthma. Breast-feeding to 3 months has also been associated with lower levels of asthma. Cigarette smoking There is much evidence to suggest that exposure to tobacco smoke in utero and in early childhood increases the risk of allergy and wheeze. Nevertheless, as the total numbers of smokers in the UK have fallen during the last 30 years, it is not the whole answer to the rise in asthma prevalence.

Pathology Fatal Asthma

In cases of fatal asthma the lungs are hyperinflated due to air trapping caused by plugging of the medium

to small airways with mucus and inflammatory cells, particularly eosinophils. Histologically, the airways show characteristic changes: an intense infiltration by inflammatory cells, particularly eosinophils and T lymphocytes, sloughing of the surface epithelium, thickening of the reticular basement, increase in the airway smooth muscle mass, increased numbers of epithelial goblet cells, vasodilatation, and edema (Figure 2). Neutrophils are found also in those who die suddenly from acute asthma. Chronic Asthma

Information on the pathology of asthma has been obtained from bronchial biopsies obtained from patients with mainly mild asthma. The histological changes are similar although less pronounced than those obtained from cases of fatal asthma. The similarity of the histological changes in allergic or extrinsic and nonallergic or intrinsic asthma suggests a final common pathogenic mechanism in both types of asthma. Neutrophils are found more commonly in patients with severe asthma.

Clinical Features Acute Asthma

Acute asthma is a common medical emergency. It is characterized by a progressive increase in dyspnea, cough, or wheeze. The decrease in expiratory airflow can be quantified by a fall in peak expiratory flow (PEF) or forced expiratory volume in 1 s (FEV1). Deterioration usually progresses over hours to days, although in some cases it may be more sudden and require rapid treatment within minutes. The severity of exacerbation is highly variable. Respiratory tract

Mucus Epithelial cells and goblet-cell hyperplasia Thickening of sub-basement membrane Cellular infiltrate

Hypertrophy of smooth muscle

Vascular congestion Figure 2 Autopsy specimen of airway from a subject who died from acute asthma showing characteristic histological changes. Hematoxylin and Eosin,  40. Photograph courtesy of Dr F Roberts, Department of Pathology, Western Infirmary Glasgow.

ASTHMA / Extrinsic/Intrinsic

infections are thought frequently to precipitate attacks of asthma, particularly in children. Infections are mainly viral, especially human rhinoviruses but also respiratory syncytial virus, adenoviruses, parainfluenza, and influenza viruses. The role of infection in provoking asthma attacks in adults is less certain. Clinical assessment Clinical features A short history of the features of an exacerbation should be elicited. The aims are to ascertain duration and severity of symptoms, with the perspective of current medication and prior admissions. Assessment must be rapid and accurate to permit prompt treatment. The history is usually one of increasing breathlessness and wheeze. Patients often have difficulty speaking and sleep is disturbed by the severity of these symptoms. There is increasing need for bronchodilator treatment, which becomes less effective. The patient may show signs of exhaustion and reduced conscious level. There is invariably an associated tachycardia, increase in respiratory rate, and auscultation of the chest may reveal severe wheeze or absent breath sounds that indicates very severe airflow obstruction. The chest becomes hyperinflated and patients may use accessory respiratory muscles. Investigations Measurement of pulse oximetry is necessary in acute asthma to evaluate oxygen saturation. The aim is to maintain SpO2492%. Arterial blood gas analysis is necessary if SpO2o92%. If oxygenation remains inadequate despite supplemental oxygen, additional complications should be considered, particularly pneumonia. The earliest abnormality is respiratory alkalosis and hypocarbia, but normal oxygen tension. As airflow obstruction increases there is uneven distribution of inspired air and changes in the normal ventilation-to-perfusion ratio. As severity increases, hypoxemia develops. The presence of normal levels of arterial carbon dioxide tension is ominous as it indicates the patient is becoming exhausted. Monitoring response to treatment should be on the basis of PEF and clinical examination. A chest radiograph may show evidence of hyperinflation, mucus plugging, and atelectasis in an acute exacerbation, but these findings may add little to management. A chest radiograph should be performed if pneumothorax is suspected. Levels of severity Asthma guidelines have been developed to ensure prompt, systematic history and examination, and ensure accurate assessment of severity. The British Guideline on the Management of Asthma defines a moderate exacerbation as one presenting with increasing symptoms of wheeze,

211

dyspnea, or breathlessness and a fall in PEF to 50–75% of the best or predicted. However, if the PEF falls to 33–55% best or predicted and is accompanied by an inability to speak in complete sentences, tachypnea of 425 breaths per min, or tachycardia of 110 beats per min, the exacerbation is classified as severe. Life-threatening features are a PEF less than one-third of best or predicted or hypoxia demonstrated by arterial oxygen saturations of less than 92% on air or arterial partial pressure of less than 8 kPA. Normal levels of CO2, a silent chest on examination, or feeble respiratory effort, cyanosis, bradycardia, dysrhythmia, hypotension, exhaustion, confusion, or coma are all signs of a near fatal episode. Chronic Asthma

The diagnosis based on a history of episodic respiratory symptoms especially after exercise or during the night is usually not difficult. Demonstration of reversible airflow obstruction gives a simple, reliable, and objective diagnosis of asthma. Evidence of reversibility can be found through the history of symptoms of episodic cough, wheeze, chest tightness, or dyspnea, measurement of PEF, or spirometry, and trials of therapy. Conditions to be considered in the differential diagnosis are listed in Table 2. Clinical assessment Clinical features Asthma may present with wheeze, shortness of breath, cough, or chest tightness. The hallmark of asthma is that these symptoms tend to be variable and intermittent. They are often worse at night and early morning and provoked by triggers such as allergens or exercise (Table 3). Less common factors are rhinitis, bacterial sinusitis, menstruation, gastroesophageal reflux, and pregnancy. When cough is the predominant symptom without wheeze, this is Table 2 Differential diagnosis of asthma Disease

Children

Adults

Cystic fibrosis Gastroesophageal reflux Bronchiectasis Ciliary dyskinesia Developmental disorder of the airway Inhaled foreign body Chronic obstructive pulmonary disease Left ventricular function Pulmonary thromboembolism Vocal cord dysfunction Upper airway obstruction Pulmonary eosinophilia Bronchial carcinoid

O O O O O O

O O O

O, Diagnosis should be considered.

O O O O

O O O O O O O O

212 ASTHMA / Extrinsic/Intrinsic Table 3 Triggers of asthma Infections, particularly viral Allergens, e.g., house dust mite, pollens, animals Occupational agents, e.g., isocyanate-containing paints, flour Environmental pollutants, e.g., cigarette smoke, sulfur dioxide Drugs, e.g., beta-blockers Exercise Cold air Hyperventilation Foods Psychological factors

referred to as cough-variant asthma. The physical sign of wheezing (usually expiratory, bilateral, polyphonic, and diffuse) is associated with asthma but has low sensitivity and specificity, and in many patients examination will be normal. Investigation A simple measure of pulmonary function by PEF is helpful, not only for initial assessment, but also to monitor symptoms, alert to deterioration in airflow obstruction, and evaluate response to treatment. Twice-daily recording of PEF for two weeks is a simple and cheap method of demonstrating variation in airflow obstruction, with a diurnal variation of greater than 15% confirming the diagnosis. However, in patients with mild asthma, the PEF may show normal variability. Spirometry demonstrates an obstructive pattern with reduction of the ratio of FEV1 to forced vital capacity (FVC). The key feature is that this airway obstruction may be reversible. Administration of a bronchodilator typically causes an increase in FEV1 of 12–15%. However, failure to demonstrate reversibility does not exclude asthma, or prove irreversible disease. Airway hyperresponsiveness is a characteristic feature of asthma and can be demonstrated by bronchial provocation techniques. The most common methods are provocation by inhalation of methacholine or histamine and exercise challenge. Fall in FEV1 is measured by serial spirometry after inhalation of increasing concentrations of methacholine. Results are expressed as the concentration of the agent that elicits a fall of 20% in FEV1. This concentration defines the degree of bronchial responsiveness and severity of disease. Skin prick testing, measurement of total and specific IgE levels, and blood eosinophilia are difficult to interpret in asthma because they have variable sensitivity and specificity. Routine chest radiographs in asthma may yield no new information and may be normal in chronic asthma. Assessment of control Asthma control can be determined by assessing symptoms, inhaled b2 adrenoceptor agonists use, lung function as well as rate

of exacerbations, number of emergency consultations for asthma, and hospital admissions. Good control is described as the presence of minimal symptoms during day and night, minimal need for reliever medication, no exacerbations, no limitation of physical activity, and normal lung function. It may not be possible to achieve good asthma control in patients with moderate or severe persistent asthma (Table 1). Specific clinical problems Asthma during pregnancy The course of asthma during pregnancy varies, with a similar proportion of women improving, remaining stable, or worsening. The risk of an exacerbation of asthma is high immediately postpartum, but the severity of asthma usually returns to preconception level after delivery. Changes in b2-adrenoceptor responsiveness and changes in airway inflammation induced by high levels of circulating progesterone have been proposed as possible explanations for the effects of pregnancy on asthma. Gastroesophageal reflux Gastroesophageal reflux can trigger attacks of asthma although the incidence is unclear. The mechanism is unknown; possibilities include aspiration or an esophagio-bronchial reflux triggered by acid irritation of the esophageal mucosa.

Pathogenesis The pathogenesis of asthma involves acute and chronic inflammation as well as remodeling (Figure 3). The underlying process is one of inflammation involving eosinophils and T lymphocytes, with the release of various mediators and cytokines, although recent evidence indicates a role for other cells including mast cells, neutrophils, macrophages, and epithelial cells. Some patients develop structural changes of the airway, a process known as remodeling. The mechanisms involved in remodeling remain to be clarified but probably consist of ongoing inflammation and abnormal repair processes. Remodeling occurs in many asthmatic patients, although the extent varies. It is thought that remodeling may play an important role in causing symptoms and loss of lung function in severe asthma, although this hypothesis remains to be established. Airway Inflammation

There are two distinct responses to inhalation of an allergen. The immediate hypersensitivity reaction, in which wheeze occurs within minutes, is comparable to the wheal-and-flare response of skin. Further wheeze is caused by the late phase response, mounted between 6 and 9 h after allergen provocation.

ASTHMA / Extrinsic/Intrinsic

Inducers of asthma

213

Inflammatory mediators and asthma

Allergen Antigen presenting cell Dendritic cell Lymph node Acute airway inflammation

Chronic airway inflammation

Th2 lymphocyte

Airway remodeling

Cytokines (e.g., IL-4, IL-5, IL-13)

Symptoms Exacerbations Disability Figure 3 In susceptible individuals allergens, occupational agents, and other known and unknown inducers of asthma cause airway inflammation. The inflammation may be acute and resolved, but in most individuals the inflammation is chronic and may be associated with airway remodeling. Airway inflammation and remodeling cause asthma symptoms, exacerbations, and disability.

The immediate reaction involves the activation of mast cells by allergen cross-linking two IgE antibodies (Figure 4). IgE antibodies are produced by B cells in response to processed antigen, which is presented by airway dendritic cells in the draining lymph node. The IgE antibodies bind mast cells by their high-affinity receptors (FceRI). Cross-linking by allergen of IgE antibodies initiates signal transduction by the FceRI effecting degranulation of the mast cell. This promotes release of mediators such as histamine, tryptase, eicosanoids, and reactive oxygen species. These spasmogens cause secretion of mucus, smooth muscle constriction, and vasodilation. Leakage of plasma protein causes edema of the airway wall, impedes clearance of mucus, and causes formation of plugs; all result in reduced airway conductance. The late phase reaction includes the accumulation of activated eosinophils, lymphocytes, macrophages, neutrophils, and basophils. The ability of cytokines to induce the expression of adhesion molecules provides a mechanism for cell migration from the circulation to the airway. Eosinophils are thought to play a central role in the pathogenesis of chronic asthma. IL-5 controls the production of eosinophils by the bone marrow and their subsequent release into the circulation. They migrate from circulation to the airway under the influence of chemokines and release toxic granule proteins, including major basic proteins, eosinophil peroxidase and eosinophil cationic protein, Th2

Mast cell (e.g., leukotrienes, histamine, tryptase)

Epithelium

Eosinophil

(e.g., prostaglandins, IL-6, IL-8)

(e.g., leukotrienes, major basic proteins)

Inflammatory mediators

BronchoMucosal constriction edema

Mucus hypersecretion

Bronchial reactivity

Airway remodeling

Symptoms of asthma Figure 4 Possible pathways in the development of airway inflammation in asthma following exposure to allergen.

cytokines, and leukotrienes. Major basic proteins causes direct airway damage with epithelial shedding. Leukotrienes increase vascular permeability and constrict smooth muscles. Challenge of the airway with allergen increases the local levels of IL-5, which correlates directly with the degree of airway eosinophilia. Recently, doubts have arisen about the role of eosinophils in causing airway hyperresponsiveness in asthma. Treatment of patients with allergic asthma using anti-interleukin-5 monoclonal antibody does not prevent allergen-induced bronchoconstriction or airway hyperresponsiveness, despite markedly suppressing eosinophil numbers within the airways. T cells are found in abundance in the inflamed airways of asthma patients and it is widely held that the Th2 subset is a driving force in allergic inflammation. The T helper subsets were characterized by Mosman on the basis of their signature cytokines. Th2 cell cytokines include IL-4, IL-5, and IL-13. IL-5 is involved in eosinophil maturation and activation, whereas IL-4 and IL-13 control synthesis of IgE. However, the Th2 paradigm for allergic asthma is now thought to be too simplistic and an additional role for Th1 cells has been postulated.

214 ASTHMA / Extrinsic/Intrinsic

The mast cell degranulation is crucial to the acute response to allergen, with release of histamine and synthesis of mediators effecting early recruitment, adhesion, and proliferation of cells. It may also contribute to remodeling as it contains proteoglycans with various functions, including support structures for remodeling. The role of other inflammatory cells in the pathogenesis of asthma including neutrophils and macrophages is less clearly characterized. There are also complex interactions between neural control of airways and inflammation. Airway Remodeling

There has been considerable evidence that inflammation alone may not explain the full pathophysiology of asthma. Two large longitudinal studies of inhaled corticosteroids have shown little effect on the natural history of asthma. Acute airway inflammation should be resolved to permit resumption of normal function. In chronic asthma, a state of increased injury and repair is thought to lead to remodeling. It is coordinated by inflammatory cells, such as eosinophils, mast cells and T cells, and structural cells such as fibroblasts. Growth factors are secreted in response to epithelial damage and cause thickening of the smooth muscle and basement membrane, resulting in airway narrowing. It is thought that a primary abnormality of the epithelium predisposes to such damage by toxins. The interaction between the thickened basement membrane and altered submucosa, known as the epithelial-mesenchymal trophic unit, is postulated to be important early in the origins of disease. This hypothesis is supported by the recent identification of ADAM33 as an asthma susceptibility gene expressed abundantly by the smooth muscle and fibroblasts, but not by inflammatory cells.

Animal Models Animal models have been used to study both the pathogenesis and treatment of asthma. Asthma does not occur spontaneously in animals although both horses and the Berenji greyhound develop a respiratory condition that has some features of asthma. Most animal models involve sensitization to an allergen such as ovalbumin or house dust mite allergen challenge. The species commonly used include mice, guinea pigs, sheep, rats, monkeys, and dogs. The mouse is often used in these models, mainly because this species allows for the application in vivo of gene deletion technology as well as the low cost and availability of inbred species with known characteristics. Animal models of allergen-induced airway

inflammation and hyperresponsiveness have provided important information on the acute inflammatory response to allergen exposure but have been less relevant to the study of mechanisms of chronic asthma and airway remodeling. The interpretation of data from experiments in animal models is influenced by the protocol used to sensitize and challenge the animal and the strain of animal used. See also: ADAMs and ADAMTSs. Allergy: Overview. Angiogenesis, Angiogenic Growth Factors and Development Factors. Arterial Blood Gases. Asthma: Overview; Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; Exercise-Induced. Bronchoalveolar Lavage. Bronchodilators: Anticholinergic Agents; Beta Agonists. Carbon Dioxide. Chymase and Tryptase. Corticosteroids: Therapy. Dendritic Cells. Dust Mite. Endothelial Cells and Endothelium. Environmental Pollutants: Overview. Epidermal Growth Factors. Genetics: Overview; Gene Association Studies. Histamine. Immunoglobulins. Leukocytes: Eosinophils; Neutrophils; Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Overview. Matrix Metalloproteinases. Neurophysiology: Neural Control of Airway Smooth Muscle. Oxygen Therapy. Pneumothorax. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Signs of Respiratory Disease: Breathing Patterns; General Examination; Lung Sounds. Smooth Muscle Cells: Airway. Symptoms of Respiratory Disease: Cough and Other Symptoms. Tumor Necrosis Factor Alpha (TNF-a ). Upper Airway Obstruction. Upper Respiratory Tract Infection.

Further Reading Barnes P, Drazen J, Rennard S, and Thomson NC (eds.) (2002) Asthma and COPD – Basic Mechanisms and Clinical Management. London: Academic Press. Bel E (2004) Clinical phenotypes of asthma. Current Opinion in Pulmonary Medicine 10(1): 44–50. Bousquet JP, Jeffery Busse W, Johnson M, and Vignola A (2000) Asthma – from bronchoconstriction to airways inflammation and remodeling. American Journal of Respiratory and Critical Care Medicine 161: 1720–1745. British Thoracic Society (2003) British guideline on the management of asthma. Thorax 58: i1–i94. Busse W (2001) Asthma. New England Journal of Medicine 5: 350–362. GINA (2004) Self-reported wheezing in the previous 12 month period in 13- to 14-year-old children. Global Burden of Asthma, p. 6. GINA (2004) Global Strategy for Asthma Management and Prevention, Chapter 7, Part 4A, Figure 5–7, p.7. Kay AB (2001) Allergy and allergic diseases. New England Journal of Medicine 344: 30–37. Kips JC, Anderson GP, Fredberg JJ, et al. (2003) Murine models of asthma. European Respiratory Journal 22(2): 374–382. Larche M, Robinson R, and Kay AB (2003) The role of T lymphocytes in asthma. Journal of Allergy and Clinical Immunology 111: 450–459.

ATELECTASIS 215 McFadden E (2003) Acute severe asthma. American Journal of Respiratory and Critical Care Medicine 168: 740–759. National Asthma Education and Prevention Program Expert Panel Report: guidelines for the diagnosis and management of asthma, Update on Selected Topics – 2002. Journal of Allergy and clinical Immunology 110(5 pt 2): S141–S219. NHLBI/WHO (2004) Global initiative for asthma: global strategy for asthma management and prevention. NHLBI/WHO Workshop Report NIH 02-3659. Bethesda: NIH.

O’Byrne PM and Thomson NC (eds.) (2001) Manual of Asthma Management, 2nd edn. London: W B Saunders. Rodrigo G, Rodrigo M, and Jesse B (2004) Acute asthma in adults: a review. Chest 125(3): 1081–1102. Tattersfield A, Knox A, Britton J, and Hall I (2002) Asthma. Lancet 360: 1313–1322. Thomson NC, Chaudhuri R, and Livingston E (2004) Asthma and cigarette smoking. European Respiratory Journal 24: 822–833.

ATELECTASIS P A Kritek, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Atelectasis is the loss of volume resulting from decreased gas in a given portion of lung. The mechanisms that cause atelectasis can be divided into three categories: passive, adhesive, and resorptive. Passive atelectasis results from space-occupying lesions in either the pleural space or the parenchyma compressing adjacent normal lung tissue. Adhesive atelectasis is caused by a decrease in the level or activity of surfactant leading to an increase in surface tension in the alveolus and subsequent collapse. Resorptive atelectasis ensues when there is partial or complete occlusion of flow of gas between alveoli and the trachea. Oxygen, carbon dioxide, and nitrogen will diffuse from the alveolus into the capillary until all gas is removed from the alveolar space. Small areas of atelectasis can be found in normal lungs due to the effects of gravity. Pneumothoraces or large bullae can result in passive atelectasis. Adhesive atelectasis is a feature of respiratory distress syndrome of the neonate as well as acute respiratory distress syndrome in adults. Resorptive atelectasis is found distal to an obstructive lesion, such as a tumor or mucus plug. Atelectasis associated with anesthesia is complex and caused by a combination of these mechanisms.

thereby creating a smaller space in which to maintain the inflated lung (Figure 1). As a result, adjacent lung tissue will lose gas and subsequently collapse. This form of atelectasis is referred to as passive because the collapsed lung is not inherently abnormal but is being affected by an adjacent pathologic process. If the inciting cause of the atelectasis is resolved (e.g., a pneumothorax is evacuated), the underlying atelectatic lung should reexpand and return to normal function. It should be noted, however, that after a segment of lung has collapsed, there are local changes in permeability, inflammatory markers, alveolar macrophage function, and surfactant associated with all types of atelectasis that may predispose to abnormal function upon reinflation. Adhesive Atelectasis

Atelectasis is the loss of volume resulting from decreased gas in a given portion of lung. These changes can be small, affecting only subsegmental regions, or more dramatic, leading to collapse of an entire lung. The mechanisms that result in atelectasis can be divided into three categories: passive, adhesive, and resorptive. Each of these is discussed individually. Note that although discussions of radiographic descriptions of atelectasis are included, the discussion is grounded in these pathophysiologic aspects of atelectasis.

Adhesive atelectasis results from the absence, loss, or decreased activity of surfactant within the alveoli (Figure 2). Surfactant, produced by type II alveolar cells, decreases surface tension as alveolar surface area decreases and balances the retraction forces of the lung in order to avoid end-expiratory alveolar collapse. When surfactant is decreased or inactivated, the balance is upset and atelectasis ensues. In this situation, there is loss of gas volume based on mechanical forces within the alveolus as opposed to external compression. This form of atelectasis can occur in the neonatal infant in the setting of immature type II alveolar cells and decreased surfactant production. Alternatively, certain adult disease states, including ventilator-associated pneumonia and acute respiratory distress syndrome (ARDS), are associated with decreases in absolute surfactant levels or its activity.

Passive Atelectasis

Resorptive Atelectasis

Passive atelectasis results from a space-occupying lesion within the pleural space or the parenchyma

Resorptive atelectasis results when there is partial or complete occlusion of flow of gas between alveoli

Description

216 ATELECTASIS

Pneumothorax

Pleural space

Normal alveolus

Passive atelectasis

Figure 1 Normal alveolus and passive atelectasis.

Edema and decreased surfactant

Normal alveolus

Adhesive atelectasis

Figure 2 Normal alveolus and adhesive atelectasis.

and the trachea (Figure 3). As the section of obstructed lung continues to be perfused, the partial pressure of oxygen in the alveolus equilibrates with that in the alveolar capillary. The loss of oxygen in the alveolus leads to increased concentrations of nitrogen and carbon dioxide in the alveolus, and subsequent gradients result in movement of both gases from the alveolus into the capillary. This process will continue until all gas has been removed from the airspace, a phenomenon that takes 18–24 h in normal volunteers with a completely occluded lobe. Resorptive atelectasis can result from any cause of obstruction to airflow. Common etiologies include

tumor, mucus plug, and foreign body. The process of resorptive atelectasis is thought to occur more quickly in the setting of oxygen-rich gas because the first step of oxygen absorption is more rapid and there is a larger portion of gas that is absorbed initially.

Atelectasis in Normal Lung Function By definition, atelectasis is caused by loss of gas from a normally gas-filled section of lung. However, there is a form of passive atelectasis that is commonly seen in ‘normal’ lungs that is referred to as dependent atelectasis. This is the loss of volume in alveoli and

ATELECTASIS 217

Tumor

Normal alveolus

Resorptive atelectasis

Figure 3 Normal alveolus and resorptive atelectasis.

small airways in the lower lung zones based on gravity-related decreases in transpulmonary pressures. Although these areas of atelectasis may have physiologic consequences in patients with underlying lung disease (e.g., ARDS), the small loss of volume most likely has no physiologic consequence in normal subjects. The increased use of computed tomography (CT) has led to a greater awareness of these changes. Dependent atelectasis will resolve with position change, as demonstrated by repeated tomography with the patient in the prone position.

Atelectasis in Respiratory Diseases

reserved for collapse of lung associated with pleural thickening, most commonly found in association with asbestos exposure. The etiology of rounded atelectasis is not well understood but is thought to result from pleural fibrosis contracting and causing adjacent lung to curl upon itself and collapse. The radiographic findings are best characterized on chest tomography. The lesions are classically peripheral, subpleural, uniform in density, and mass-like in appearance. They often have a curvilinear opacity of bronchi and vessels (termed a comet tail) extending toward the hilum. Care needs to be taken in diagnosing a lung nodule as rounded atelectasis purely on CT findings because there are many reports of malignancy masquerading as rounded atelectasis.

Passive Atelectasis

Passive atelectasis can occur in a generalized or localized manner. In the setting of a large pneumothorax, the majority of the parenchyma of the ipsilateral lung will undergo passive atelectasis. In contrast, lung tissue adjacent to a bleb or a parenchymal cyst can collapse in a more localized form of passive atelectasis. Both examples illustrate a space-occupying phenomenon that causes subsequent loss of gas and collapse. With relief of a large space-occupying lesion, such as a pneumothorax, the underlying lung may develop reexpansion pulmonary edema. This process is thought to result from changes in pulmonary capillary permeability but is not well understood. The pulmonary edema is usually transient and resolves with supportive care. There is a unique form of passive atelectasis termed rounded atelectasis. This description is

Adhesive Atelectasis

The classic example of absorptive atelectasis is that of respiratory distress syndrome (RDS) of the neonate. As discussed previously, type II alveolar cells are often immature and not fully functional in the preterm infant, predisposing the infant to RDS. There have been significant decreases in morbidity and mortality in RDS with the initiation of surfactant (natural or synthetic) replacement in the immediate neonatal period. This improvement is in part due to the marked decrease in atelectasis and resulting improved gas exchange with surfactant therapy. Many studies demonstrate decreased surfactant levels in adults with ARDS. There is also experimental evidence for decreased surfactant activity in the setting of leakage of plasma proteins into the alveolar space. At the same time, studies using CT have

218 ATELECTASIS

demonstrated areas of atelectasis in ARDS. The etiology of this atelectasis is likely multifactorial. There are increased changes in dependent lung zones suggesting a component of passive atelectasis accompanying the adhesive atelectasis, the latter presumably from decreased quantity or activity of surfactant. Some believe that the recurrent opening and closing of small, atelectatic lung units contributes to ventilator-induced lung injury (termed ‘atelectrauma’). In response to this, there has been increasing research on how to avoid or overcome the atelectasis associated with ARDS. Efforts to replace surfactant through a variety of modalities have not demonstrated a mortality benefit, although some studies have shown improved gas exchange. Lung ventilation strategies aimed at maintaining an ‘open lung’, such as recruitment maneuvers, high-frequency jet ventilation, and conventional ventilation with high positive end expiratory pressure (PEEP), have shown transient rises in oxygenation but no improvement in mortality. There is no conclusive evidence supporting any specific treatment of atelectasis associated with ARDS. Resorptive Atelectasis

Endobronchial tumors are a common cause of resorptive atelectasis, often resulting in segmental or lobar collapse. One retrospective review reported an incidence of atelectasis in one of five patients with small cell lung cancer. Because these are gradual processes occurring over weeks to months, the atelectatic lung often does not completely reexpand or function normally if the obstruction is relieved. However, because there is a risk for postobstructive pneumonitis and there is evidence for increased bacterial growth in atelectatic lung, clinicians will often attempt to minimize the obstruction. In contrast to the more gradual development of atelectasis with tumor growth, resorptive atelectasis can occur rapidly with acute occlusions of large airways. Mucus plugs, in both patients with asthma and those with altered secretion clearance, have been described as causes of resorptive atelectasis. Resorptive atelectasis has also been reported with malpositioned endotracheal tubes that selectively intubate the right lung. In a short period of time, the entire left lung can undergo atelectasis that will resolve with repositioning of the endotracheal tube.

by CT) and is often clinically significant. Lobar atelectasis can result in parenchymal shunt and be manifest as marked hypoxemia. There is evidence of a positive correlation between the amount of atelectatic lung and the degree of hypoxemia. The atelectasis associated with anesthesia is caused by multiple mechanisms. The first contributor is dependent atelectasis (a form of passive atelectasis) from prolonged recumbent positioning with exaggerated effects of gravity. There is also evidence of changes in diaphragm position and shape with supine positioning for anesthesia contributing to dependent atelectasis. These effects are more pronounced when neuromuscular blockade is used in conjunction with anesthesia. All these changes are especially pronounced in morbidly obese patients. Some anesthesiologists advocate for intermittent use of large tidal volume breaths to overcome atelectasis intraoperatively, whereas others advocate for elevated levels of PEEP with ventilation. Administration of supplemental oxygen as part of mechanical ventilation contributes to resorptive atelectasis. Results are mixed with regard to whether higher inspired fractions (80–100%) during induction and maintenance of anesthesia increase the likelihood of atelectasis compared to lower oxygen/ nitrogen mixtures. There is also evidence of early airway closure during anesthesia contributing to the resorptive mechanism of atelectasis. It has been suggested that repeated episodes of atelectasis, from the previously mentioned means, can also lead to impaired surfactant function resulting in a component of adhesive atelectasis as well. The issues of atelectasis often persist into the postoperative period, resulting in persistent hypoxemia. The resorptive atelectasis associated with higher inspired fractions of oxygen persists after extubation despite attempts at recruitment at the end of the operation. Dependent atelectasis, which in the normal subject will resolve with changes in position and deep breathing, often persists due to decreased mobility, pain, and sedation in the postoperative period. These conditions also commonly lead to impaired clearance of secretions predisposing to obstruction of airways and resorptive atelectasis. Deep breathing, incentive spirometry, vibration beds, chest physiotherapy, noninvasive ventilation, and therapeutic bronchoscopy all seem to have similar results in terms of resolution of postoperative atelectasis.

Atelectasis Associated with General Anesthesia

Atelectasis associated with general anesthesia deserves special mention because it occurs at rates as high as 90% of anesthetized patients (as detected

See also: Alveolar Surface Mechanics. Breathing: Breathing in the Newborn. Diffusion of Gases. Lung Imaging. Mucus. Oxygen Therapy. Peripheral Gas Exchange. Physiotherapy. Signs of Respiratory

AUTOANTIBODIES 219 Disease: Lung Sounds. Surfactant: Overview. Ventilation, Mechanical: Positive Pressure Ventilation.

Further Reading Brower RG, et al. (2003) Effects of recruitment maneuvers in patients with acute lung injury and acute respiratory distress syndrome ventilated with high positive end-expiratory pressure. Critical Care Medicine 31(11): 2592–2597. Fraser RS and Paraˆe PD (1999) Fraser and Paraˆe’s Diagnosis of Diseases of the Chest, 4th edn. Philadelphia: Saunders. Gunther A, et al. (2001) Surfactant alteration and replacement in acute respiratory distress syndrome. Respiration Research 2(6): 353–364. Hallman M, Glumoff V, and Ramet M (2001) Surfactant in respiratory distress syndrome and lung injury. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 129(1): 287–294. Hedenstierna G and Rothen HU (2000) Atelectasis formation during anesthesia: causes and measures to prevent it. Journal of Clinical Monitoring and Computing 16(5–6): 329–335.

Kreider ME and Lipson DA (2003) Bronchoscopy for atelectasis in the ICU: a case report and review of the literature. Chest 124(1): 344–350. Magnusson L and Spahn DR (2003) New concepts of atelectasis during general anaesthesia. British Journal of Anaesthesia 91(1): 61–72. Peroni DG and Boner AL (2000) Atelectasis: mechanisms, diagnosis and management. Paediatric Respiratory Reviews 1(3): 274–278. Rouby JJ, et al. (2003) Acute respiratory distress syndrome: lessons from computed tomography of the whole lung. Critical Care Medicine 31(4 supplement): S285–S295. Spragg RG, et al. (2004) Effect of recombinant surfactant protein C-based surfactant on the acute respiratory distress syndrome. New England Journal of Medicine 351(9): 884– 892. Vaaler AK, et al. (1997) Obstructive atelectasis in patients with small cell lung cancer. Incidence and response to treatment. Chest 111(1): 115–120. Woodring JH and Reed JC (1996) Types and mechanisms of pulmonary atelectasis. Journal of Thoracic Imaging 11(2): 92–108.

AUTOANTIBODIES O C Ioachimescu, Cleveland Clinic Foundation, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Autoimmunity represents an immune response directed against self-antigens, which may be (artificially) separated into T-cell and B-cell responses. The importance of the B cells in autoimmunity is correlated to the production of several autoantibodies, which have a role in diagnosis, pathogenesis, guiding therapy, and predicting outcome of the patients with these conditions. Among these autoantibodies, antineutrophil cytoplasm antibodies (ANCAs) have been distinguished as very important in several conditions, such as Wegener’s granulomatosis, microscopic polyangiitis, Goodpasture’s syndrome, drug-related vasculitides, etc. We review in this article the importance of different classes of autoantibodies and their relationship to underlying disorders.

Introduction: Autoimmunity and Lung Disorders The phenomenon of autoimmunity has been the object of exploration for over a century. Recent technical and methodological advances in the study of cellular and biochemical processes of autoimmunity have led to a publication ‘big bang’. Nevertheless, the etiopathogenesis of most autoimmune disorders remains, to date, largely unknown. In this article, we discuss the basic mechanisms leading to autoimmunity, as we understand them today, and then the

relevance of several classes of autoantibodies in various lung conditions. An autoimmune disorder is a pathologic condition caused by an autoimmune response, which is critically dependent upon antigen quality, concentration, and persistence, and the magnitude of the interaction between self and foreign components. The autoimmune response leads to end-organ organ-damage through cellular or humoral mechanisms. The humoral response is represented by antibody-mediated injury, which could be differentiated in direct, cytotoxic antibody damage or immune complex-related damage. Despite their diverse etiology, there are several pathogenetic mechanisms which are common to all autoimmune conditions. With few exceptions, they require the presence of self-reactive CD4 positive lymphocytes. The immune system is naturally endowed with myriad mechanisms responsible for recognition of and defense against foreign assaults. Direct and rapid responses can be mediated by a set of germline-encoded receptors, called Toll-like receptors (TLRs), which recognize specifically the molecular determinants of various pathogens. Activation of the innate immune defense mechanisms leads to a response from different cell types (local dentritic cells, natural killer lymphocytes, neutrophils, monocytes, macrophages, basophils, eosinophils, and mastocytes), ranging from production of chemokines, cytokines, adhesion molecules, antimicrobial, pro- and antiapoptoic factors. This response is reproducible (acts

220 AUTOANTIBODIES

by the same intensity at each antigenic exposure), is not antigen specific, and creates a pool of memory cells for long-term immunity. The adaptive immunity requires contributions from cells whose receptors are generated by VDJ recombination, the T and B lymphocytes. Furthermore, inducible expression of TLRs in B cells may provide a link between the innate and adaptive branches of the immune system. Genetic susceptibility to autoimmune diseases may occur at several levels: the major histocompatibility complex (MHC) haplotype and polymorphisms of genes involved in establishing self-tolerance and immune regulation, for example, the autoimmune regulator (AIRE), the T-cell immunoglobulin and mucin-domain-containing (TIM) family, and cytolytic T-lymphocyte-associated antigen 4 (CTLA-4). There are several major pathways that can initiate or modulate autoimmunity: 1. molecular mimicry of self-proteins by viral agents can activate specific T cells that attack both the specific virus and the host; 2. bacterial infections can cause intense inflammation, with secondary polyclonal activation of ‘bystander’ T cells, which will act in concert in intensifying the local injury; 3. epitope exposure and maintenance in the local milieu as a result of self local damage may perpetuate the immune response; and 4. drug metabolism or infection can cause the formation of proteins which are seen as ‘foreign’ and thus result in a neo-antigen-specific response to the modified self. There is also a range of possible posttranslational modifications (PTMs) of proteins that can allow immune recognition of neo-self epitopes. The most direct way the posttranslational changes can influence T-cell reactivity is to generate a new peptide–MHC (pMHC) complex that can stimulate stronger binding to T lymphocytes through T-cell receptors. In general, the immune recognition can be modified by posttranslational changes of the antigens in a number of ways: 1. Different additions (glycosylation, methylation, phosphorilation, etc.), as in collagen-induced arthritis. 2. Enzymatic or spontaneous conversions (deamidation or citrullination) as in celiac disease or rheumatoid arthritis (RA). In the latter, multiple autoantibodies have been described: antiperinuclear factor, antifilaggrin, and antikeratin antibodies. The target antigens are the result of PTM, namely deimination of the natural aminoacid

arginine to the amino acid citrulline by the activity of peptidylarginine deiminase (PAD). This discovery led to the development of a new serologic test for RA, that is, antibody against cyclic citrullinated peptide (CCP), which has a high specificity (497%) for the disease. Anti-CCP binding also seems predictive of progression to RA in recent-onset arthritis, or retrospectively in blood samples positive for rheumatoid factor (RF), donated years before onset of symptoms of the disease. There is also evidence that citrullination may play a role in T-cell autoreactivity in RA by enhancing the strength of the bonds to MHC II molecules. Therefore, expression of PAD4 and citrullinated proteins, presence of anticitrulline antibody-secreting plasma cells in the inflamed synovium, the strong MHC–peptide bonds, and the predictive strength of anti-CCP testing, all prove that this autoimmune cascade is involved in the progression of RA. 3. Extracellular modifications by proteolysis or antibody binding. The current understanding is that differential PTMs might therefore provide the means of provoking a local autoaggressive immune response as a consequence of an infection. This would be an alternative explanation of the autoimmunity by self-mimicking microbial antigens, for which definitive proof remains elusive in humans as yet.

ANCAs and Associated Lung Disorders In 1982, antineutrophil cytoplasm antibodies (ANCAs) were first described in patients with pauci-immune glomerulonephritis. By 1985, ANCA had already been linked to Wegener’s granulomatosis (WG); within a few more years, a link with microscopic polyangiitis (MP), and ‘renal-limited’ vasculitis has been made. As of today, ANCAs have become very important factors in the diagnosis, pathogenesis, and classification of vasculitides. ANCAs can be determined in two different ways: indirect immunofluorescence assay (IIA) and enzyme-linked immunosorbent assay (ELISA). While IIA is more sensitive than ELISA, the latter is clearly more specific; hence, in clinical testing for ANCA it is recommended to screen with IIA, and to confirm all positive results with ELISA directed against the specific antigens, if possible in a reference laboratory. Furthermore, and unfortunately, there is a significant subjective component to the interpretation of the immunofluorescence tests, so confirmation by other tests is needed before releasing the definitive ‘positive test’. The main target antigens for ANCAs are

AUTOANTIBODIES 221

proteinase 3 or PR3 (PR3–ANCA) and myeloperoxidase or MPO (MPO–ANCA). PR3 and MPO are located in the azurophilic granules of neutrophils, and the peroxidase-positive lysosomes of monocytes, respectively. Other azurophilic granule proteins can cause p-ANCA autoantibodies: lactoferrin, elastase, cathepsin G, bactericidal permeability inhibitor, catalase, lysozyme, azurocidin, beta-glucuronidase, etc.

*

specimens, because formalin-fixed neutrophils do not cluster charged cellular components around the nucleus. Mixed or atypical pattern, seen mostly in patients with other immune-mediated conditions than systemic vasculitis (e.g., connective tissue disorders, inflammatory bowel disease, and autoimmune hepatitis). Such patterns may be confused with p-ANCA patterns.

Immunofluorescence Assay

When the sera of patients with ANCA-associated vasculitis (AAV) or other conditions are incubated with ethanol-fixed human neutrophils, three major IIA patterns are observed (Figure 1): *

*

c-ANCA pattern (cytoplasmic), with diffuse staining of the cytoplasm. In most cases, the responsible antibodies are PR3–ANCA in the setting of WG; rarely, MPO–ANCA can present in the cytolasmic, uniform immunofluorescence. p-ANCA pattern (perinuclear), which results from staining around the nucleus, which in fact is fixation artifact when ethanol is used. With ethanol fixation, positively charged granule constituents cluster around the negatively charged nuclear membrane, leading to perinuclear fluorescence. The usual antibody responsible for this pattern is MPO– ANCA (rarely PR3–ANCA), generally in the setting of MP. A positive p-ANCA immunofluorescence staining pattern may also be detected in a wide variety of inflammatory illnesses; it has a low specificity for vasculitis. The p-ANCA pattern on IIA can be similar to that caused by antinuclear antibodies (ANAs), the reason why individuals with ANA frequently have ‘false-positive’ results on ANCA testing by immunofluorescence. Rigorous IIA testing procedures for ANCA (especially when a diagnosis of vasculitis is entertained) entails the use of both formalin- and ethanol-fixed neutrophil

Enzyme-Linked Immunosorbent Assay

Specific ELISA kits for antibodies directed against PR3, MPO, and other azurophilic granule components are commercially available. PR3–ANCA and MPO–ANCA should be part of any standardized approach to the testing for ANCA; they are associated with substantially higher specificities and positive predictive values than their corresponding IIA patterns (c- and p-ANCA, respectively). Antineutrophil cytoplasm antibodies are mainly associated with Wegener’s granulomatosis, microscopic polyangiitis, Churg–Strauss syndrome (CSS), ‘renal-limited’ vasculitis (pauci-immune glomerulonephritis without evidence of extrarenal disease), and drug-induced vasculitides. In these conditions, there is either PR3–ANCA or MPO–ANCA, but almost never both. ANCA with different antigen specificities may be detected in various other rheumatologic and gastrointestinal disorders. Wegener’s Granulomatosis

The histopathologic hallmark of WG is necrotizing granulomatous inflammation that involves the respiratory tract, kidneys, skin, and/or the joints. Nearly 90% of patients with generalized, active WG are ANCA-positive, while in limited disease (restricted to upper or lower respiratory tract disease and no renal involvement) only 40–60% of patients may be ANCA-positive. Thus, the absence of ANCA does

Figure 1 Indirect immunofluorescence assay patterns on ethanol-fixed human neutrophils. (a) Diffuse, cytoplasmic cANCA; (b) perinuclear pANCA; (c) antinuclear (ANA) illustrated as control.

222 AUTOANTIBODIES

not exclude the diagnosis; among WG patients with ANCA positivity, 80–95% have PR3–ANCA by ELISA, the rest nearly always have MPO–ANCA. The diagnostic performance of PR3–ANCA for WG (positive and negative predictive values) is related mainly to disease prevalence in the population searched, and the disease activity. Persistently, high or rising titers of ANCAs are often associated with disease relapses. However, this association may not occur in 10–30% or more of those with such ANCA profiles during one or more years of follow-up. Microscopic Polyangiitis

Approximately 70% of patients with MP are ANCA positive. Most ANCA-positive MP patients have MPO–ANCA, with a minority having PR3–ANCA. ANCA serologies are useful in distinguishing MP from classic polyarteritis nodosa (PAN), a vasculitis of medium-sized muscular arteries. In general, PAN is associated with neither PR3–ANCA, nor MPO– ANCA. Since PR3–ANCA or MPO–ANCA may occur in both WG and MP, it is important to know that these diseases cannot be distinguished solely on ANCA tests. However, distinction between MP and WG is not clinically that important, since their treatment and prognosis are similar. Churg–Strauss Syndrome

Both PR3–ANCA and MPO–ANCA have been detected in patients with CSS; overall, 50% of CSS patients are ANCA positive, whereas in those with active, untreated disease the percentage is even higher. So far, no identification has been made of any consistent clinical differences with therapeutic or prognostic implications between ANCA-positive CSS and ANCA-negative CSS. Renal-Limited Vasculitis

Pauci-immune vasculitis limited to the kidney is characterized by focal and segmental glomerular inflammation and necrosis with little or no deposition of immunoreactants (IgG, IgA, IgM, and complement fractions). Almost all patients are ANCA positive, and up to 80% of them have MPO–ANCA. Some consider this disorder as part of the WG/MP spectrum because the renal histologic findings are indistinguishable, and because some patients with renal-limited vasculitis eventually develop extrarenal manifestations of either WG or MP.

positive. One study, for example, found that 38 of 100 sera with anti-GBM antibodies also had ANCA; of these, 25 had MPO–ANCA, 12 had PR3–ANCA and 1 both types of ANCAs. Almost all such patients have both ANCAs and anti-GBM antibodies at the first serum examination. The clinical significance of combined ANCA and anti-GBM serologies is unclear. In some, the titers of ANCAs are low and there are no clinical manifestations of vasculitis. In others, however, there are disease features of anti-GBM antibody disease, but quite typical of systemic vasculitis, including purpura, arthralgias, and granulomatous inflammation, suggesting the concurrence of two disease processes. The incriminated self-antigen for the anti-GBM antibodies is the same as in patients with anti-GBM antibody disease alone, suggesting that the inciting epitopes are the same. Alternatively, the production of ANCA could precede that of anti-GBM antibodies, with pulmonary or renal damage caused by ANCA leading to secondary anti-GBM antibody formation. Other Rheumatologic Disorders

ANCAs have been reported in virtually all rheumatic diseases, including RA, systemic lupus erythematosus (SLE), Sjogren’s syndrome (SS), scleroderma, inflammatory myopathies, dermatomyositis, relapsing polychondritis, and the antiphospholipid syndrome. In most cases, the IIA pattern is p-ANCA. Many reports of ANCAs in these diseases preceded the era of reliable ELISA assays for PR3– and MPO–ANCA, and used only IIA. Various target antigens have been described in these disorders, such as lactoferrin, elastase, lysozyme, cathepsin G, and others. In other cases, the specific target antigens have not been identified yet. Cystic Fibrosis

Non-MPO p-ANCAs are common in patients with cystic fibrosis (CF), particularly among those with bacterial airway infections. The ANCA is generally directed against BPI (bactericidal/permeability-increasing) protein. In one series of 66 patients with CF, BPI-IgG and BPI-IgA ANCAs were found in 91% and 83%, respectively. Anti-BPI titers were directly related to the severity of airway destruction. It is unclear whether this relationship represents an epiphenomenon or a response to overwhelming infection, with major release of endotoxins.

Anti-GBM Antibody Disease (Goodpasture’s Syndrome)

Others

Up to 40% of patients with antiglomerular basement membrane (anti-GBM), antibody disease are ANCA

ANCA has also been observed in isolated patients with autoimmune hepatitis, Bu¨rger’s disease,

AUTOANTIBODIES 223

(pre)eclampsia, subacute bacterial endocarditis, leprosy, malaria, and chronic graft-versus-host disease. ANCA positivity is seen in 60–80% of patients with ulcerative colitis and in primary sclerosing cholangitis. It can be observed in only 10–27% of patients with Crohn’s disease, in whom only low titers are present. The p-ANCA is the predominant appearance, and is directed against a myeloid cell-specific 50 kDa nuclear envelope protein. Other reported antigens include BPI, lactoferrin, cathepsin G, elastase, lysozyme, and PR3. The pathogenetic significance of these antibodies is unclear. The titers of ANCAs do not vary with the activity or severity of the disease and, in ulcerative colitis, do not fall even after colectomy. Drug-Induced ANCA-Associated Vasculitis

Several medications can induce various forms of AAV. Most patients diagnosed with drug-induced AAV have high titers of MPO–ANCA. In addition, most have also antibodies to elastase or lactoferrin. Relatively few have PR3–ANCA positivity. Many cases of drug-induced AAV are associated with constitutional symptoms, arthralgias/arthritis, and cutaneous vasculitis. However, the full range of clinical features associated with ANCAs, including crescentic glomerulonephritis and alveolar hemorrhage, can also occur. Discontinuation of the offending agent may be the only intervention necessary for mild cases of AAV induced by medications; such cases have in general only constitutional symptoms, arthralgias/arthritis, and/or cutaneous vasculitis. Some patients, however, require high doses of corticosteroids and even cyclophosphamide because of more severe manifestations. The most renowned medications capable of causing AAV are propylthiouracil (PTU), carbimazole, thiamazole, hydralazine, procainamide, minocycline, penicillamine, allopurinol, phenytoin, and clozapine. Drug-induced AAV is quite rare; consequently, the above medications should not be incriminated until other etiologies have been thoroughly excluded. Propylthiouracil

PTU may be the most common offending agent causing drug-induced AAV. Generally, the medication is taken for long periods of time before the complication occurs (months to years). Vasculitis is a rare complication, whereas a much higher percentage develops serological evidence of ANCA. In a cross-sectional study, 27% of patients receiving long-term treatment with PTU developed MPO–ANCA. The postulated mechanism by which PTU leads to AAV stems from the observation that PTU accumulates in

neutrophils and then it binds to MPO altering its structure, which could lead to ANCA production in susceptible individuals. Discontinuation of the offending drug may be the only intervention necessary in mild cases. Some patients, however, require high doses of corticosteroids and even cyclophosphamide, while others require maintenance therapy. ANCA titers usually persist at low levels, even after active vasculitis goes into resolution, which points out that previous ingestion of PTU is an important piece of information in the history of patients with other pathologies and prior PTU-induced, ‘by-stander’ ANCA. Hydralazine

Hydralazine may cause drug-induced lupus and drug-induced AAV. Unlike hydralazine-induced lupus syndrome, hydralazine-induced AAV is frequently associated with a pauci-immune glomerulonephritis, antibodies to double stranded DNA (dsDNA), high titers of MPO–ANCA, and is a more ‘serious’ condition. Minocycline

Minocycline had produced fever, livedo reticularis, arthritis, and ANCA in a seven-patient case series. The p-ANCA IIA pattern associated with this disorder is usually directed against ‘minor’ antigens such as cathepsin G, elastase, and bactericidal/permeability increasing (BPI) protein, rather than against ‘major’ antigens, such as MPO. Symptoms typically resolve after minocycline discontinuation, and recur with drug rechallenge. Some patients require treatment with corticosteroids for short periods of time. More serious manifestations of minocyclineinduced AAV are crescentic glomerulonephritis, lupus-like syndrome, and cutaneous ‘classic’ PAN. Pathogenesis of ANCA-Associated Disorders

Substantial evidence in animal models and human observations supports a significant pathogenetic role of ANCA in producing widespread tissue damage. The hypothesis is that antibodies produce a necrotizing vasculitis by inciting a respiratory burst with diffuse endothelial damage, chemotaxis, and degranulation of neutrophils and monocytes. If autoantibody (ANCA) generation is secondary to a cryptic epitope exposure or a primary event, it is still unclear. After a cryptic antigen exposure, the epitope spread may occur, leading to a more generalized, systemic reaction (Figure 2). The number of activated B lymphocytes seems to correlate with the activity score of the disease (e.g., Birminham vasculitis score), which supports the hypothesis that B lymphocytes

224 AUTOANTIBODIES AAT ANCA

Insult

PR3 or MPO Macrophage

MMP-12 Neutrophil

TNF-
IL-8 Monocyte

Endothelial cell Figure 2 A simplified model of ANCA pathogenesis and neutrophil/monocyte priming. Of note, alpha-1 antitrypsin (AAT) is a natural tissue inhibitor of proteinase 3 (PR3), myeloperoxidase (MPO), or other lysosomal enzymes. In addition, binding ANCA to the cell surface is mediated by specific FcgR receptors for immunoglobulins. IL-8, interleukin-8; MMP-12, matrix metalloproteinase 12; TNF-a, tumor necrosis factor alpha. Copyright (2005) from Journal of COPD by Ioachimescu O and Stoller J. Reproduced by permission of Taylor & Francis Group, LLC., http://www.taylorandfrancis.com.

are involved in the disease pathogenesis. Furthermore, while in normal conditions MPO and PR3 mRNA transcripts are found almost exclusively in early promyelocytes, it was noted that in AAV disorders (and not in SLE or other conditions), both MPO and PR3 mRNA are found in high concentrations in the peripheral neutrophils, and this correlates well with neutrophil total number and disease activity, but not with ANCA titer, ‘left shift’, or cytokine levels, including tumor necrosis factor alpha (TNF-a). Cell membrane PR3 and MPO expression has been found in the affected glomeruli of the ANCA-associated diseases (e.g., WG, MP), but not in normal glomeruli, suggesting that local factors also play an important role in gauging the organ involvement. In CSS, it is still unclear what the eosinophil’s contribution to the disease pathogenesis is.

Autoantibodies in Connective Tissue Disorders Laboratory screening is commonly used for evaluation of connective tissue disorders (CTDs), although there are rare tests which are sensitive or specific enough to establish the diagnosis. For example, Westergren sedimentation rate and C-reactive protein are commonly elevated in infections, malignant, or inflammatory conditions; therefore, a high value does not add too much towards the diagnosis, while a low titer may make an active CTD seem unlikely. Table 1 illustrates a synopsis of autoantibodies found in different immune conditions.

Table 1 The main pathogenic autoantibodies in different lung conditions Antibody

Lung disease

ANCA ANA Anti-Sm, Anti-dsDNA Anti-U1-RNP RF CCP Anticentromere Anti-Scl Anti-Jo1 Cryoglobulins Anti-GM-CSF

WG, MP, CSS, drug-related AAV SLE, SS, scleroderma, RA, MCTD, UCTD SLE MCTD RA, SLE, MCTD, UCTD RA CREST syndrome Scleroderma DM/PM – ILD Essential and secondary cryoglobulinemia PAP

ANAs are found positive in most of CTD patients, with different frequencies (from 30% in RA, to 95% in SLE and scleroderma). On the other hand, other lung conditions such as idiopathic pulmonary fibrosis and coal worker’s pneumoconiosis may have positive ANAs in titers above 1:40 in up to 33% of the cases, as opposed to 2–3% of the general population. An extractable nuclear antigen (ENA) panel is available in most of the reference laboratories, which will guide the testing against common antigens if ANA is positive. Anti-dsDNA and anti-Smith (Sm) antibodies are relatively specific for SLE: 95–97%, and 50–99%, respectively. While anti-dsDNA antibodies seem to correlate with the disease activity, anti-Sm antibodies tend to persist after normalization of anti-dsDNA titers. An interstitial lung disease (ILD)

AUTOANTIBODIES 225

identical to usual interstitial pneumonia (UIP) or non-specific interstitial pneumonia (NSIP) can be seen in SLE, RA, scleroderma, or dermatomyositis, while bronchiolitis obliterans with organizing pneumonia (BOOP) can be frequently seen in RA. In SLE, the most frequent pulmonary complications are pleurisy and pleuritis, lupus pneumonitis, shrinking lung syndrome, bacterial pneumonia, diffuse alveolar hemorrhage (DAH), thromboembolic disease, and pulmonary arterial hypertension. Traditionally, RF is found with higher frequency in patients with RA and long-standing disease, with multiple extra-articular manifestations. In other lung conditions, such as hypersensitivity pneumonitis (bird fancier’s lung) or idiopathic pulmonary fibrosis (IPF), RF can be positive in up to half of the cases. Since part of the workup for interstitial lung disorders is exclusion of associated rheumatic conditions, reliance on more specific testing is generally required to diagnose a systemic disease like RA. This can be achieved by anticyclic citrullinated peptide (CCP) antibodies, which have a much higher specificity, of 91–98%. In RA, the lung involvement can take the form of ILD (UIP, NSIP, BOOP), DAH, pleuritis and pleural effusion, rheumatoid nodules (follicular, constrictive, or obliterative) bronchiolitis, cricoarythenoid arthritis with upper airway obstruction or drug-induced pneumonitis (methotrexate, gold, cyclophosphamide, rituximab etc.). In the workup of disorders associated with myopathy or myositis, the determination of muscle creatinphosphokinase (or aldolase) is important. Polymyositis (PM) and amyotrophic dermatomyositis (ADM) can both present with lung disease in more than 65% of cases, as one study found at the time of diagnosis; it is important to know that, contrary to rheumatoid lung disease, in PM or ADM, the pulmonary involvement may be inaugural. In DM/PM, ANA is positive in up to 30% of the patients; one in three patients will also have antibodies against an ENA called histidyl tRNA synthetase, or Jo-1 antibodies. Other antiaminoacyl tRNA synthetases have been identified to date: anti-PL7 or anti-treonyl tRNA synthetase, anti-PL12 or anti-alanyl tRNA synthetase, anti-OJ or anti-isoleucyl tRNA synthetase, anti-EJ or anti-glycyl tRNA synthetase, anti-KS or anti-asparaginyl tRNA synthetase, and anti-Wa directed against a 48 kDa protein yet uncharacterized, but known to be bound to acetylated tRNA. It has been noted that patients with anti-synthetase syndrome (arthritis, Raynaud’s syndrome, mechanic’s hands, and anti-Jo-1 antibodies) have a much higher incidence of pulmonary disease. Pathologic pictures of NSIP, UIP, or BOOP can be seen in these settings.

The interstitial lung disease in scleroderma can occur with both limited and diffuse disease. ANA is generally positive in both forms of the disease, anticentromere or anti-TO/TH antibodies found in limited disease, while anti-Scl antibodies are more often found in the diffuse variants of scleroderma. The cellular and fibrotic form of NSIP can together account for 70% of patients with scleroderma, suggesting a high rate of pulmonary involvement. Sjogren’s syndrome is an autoimmune exocrinopathy and disorder of the epithelia characterized by lymphocytic infiltration of the glandular and nonglandular subepithelial tissue. It presents with xerostomia (dry mouth), xerophtalmia (dry eyes) and keratoconjunctivitis, xerotrachea (dry trachea, presenting as dry cough and frequent infections), and arthritis. The pulmonary involvement in SS manifests as lymphocytic bronchitis, lymphocytic follicular bronchiolitis, bronchial-associated lymphoid tissue lymphoma, lymphocytic interstitial pneumonia (LIP) or cystic lung disease. The serologic markers of this condition (either primary or secondary SS) are represented by SS-A (anti-Ro) and SS-B (anti-La) antibodies. Mixed connective tissue disorder (MCTD) is an overlap syndrome with features of RA, SLE, scleroderma, and DM/PM, which do not meet the criteria for the individual disorders, in the presence of antiribonucleoprotein (anti-RNP) antibodies on ENA testing, directed against U1 RNP, which is rich in uridine. Pulmonary involvement is common in patients with MCTD, and has feature of UIP or NSIP, with significant septal thickening, less ground-glass attenuation and honeycombing on CT scans. ‘Incomplete’ rheumatic conditions can occur and are usually called undifferentiated connective tissue disorder (UCTD) and may involve the respiratory tract in a fashion similar to MCTD, although without any evidence of anti-RNP antibodies. In cryoglobulinemia, a condition characterized by cryoglobulins in the serum, there is a systemic inflammatory response with involvement of small and medium-size vessels, and generated by cryoglobulincontaining immune responses. By definition, cryoprecipitation is a phenomenon of protein precipitation at temperatures lower than 371C, and it can be present when serum proteins or plasma proteins precipitate (cryofibrinogens and cryoglobulins respectively). Cryoglobulins are a mixture of immunoglobulins and complement components, which generally precipitate upon refrigeration of the serum. Three types of cryoglobulinemic conditions have been described (Brouet’s classification): type I, characterized by isolated monoclonal immunoglobulins (Ig) and seen mostly in hematologic conditions; type II with polyclonal Ig seen in hepatitis C, HIV

226 AUTOANTIBODIES

and other viral conditions; and type III, with mixed cryoglobulins, encountered in CTDs. Pulmonary involvement may be commonly seen in type III cryoglobulinemia and, rarely, in type I, and is mostly subclinical. In up to 50% of cases, there may be cough, pleuritic chest pain, and/or dyspnea. Pulmonary function tests usually show evidence of reactive small airway disease, and occasionally a decreased diffusing lung capacity for CO. Pulmonary vasculitis, DAH, or BOOP can also be found (though rarely). Another rare condition is hypocomplementemic urticarial vasculitis syndrome, which presents with urticarial vasculitis, arthritis, glomerulonephritis, and obstructive lung disease; it is thought to be caused by serum IgG against C1q fraction of the complement, which will decrease significantly (similar to SLE, but more commonly, with angioedema and eye inflammation).

Autoantibodies in Other Conditions Lung Cancer

The antibodies responsible for the paraneoplastic syndromes are discussed in articles Tumors, Malignant: Overview; Bronchogenic Carcinoma. Sarcoidosis

A possible relationship between sarcoidosis and autoimmunity was described more than a century ago, although is still not accepted to be an autoimmune condition. Good preliminary results of the CD20 targeting in several autoimmune conditions (sarcoidosis, SLE, RA, type II cryoglobulinemia, neuropathies, WG, Goodpasture’s syndrome, etc.) have opened important avenues for research, conceivably capable of improving our understanding of the pathogenesis of these conditions and the effectiveness of pathogenesis-directed therapy. Pulmonary Alveolar Proteinosis

Animal and human studies have confirmed a pivotal role played by granulocyte-macrophage colony-stimulating factor (GM-CSF) in pulmonary alveolar proteinosis (PAP) pathogenesis. A decreased GM-CSF pathway activity seems to be the common pathogenic pathway. It was shown that a neutralizing (or blocking) anti-GM-CSF IgG antibody can be found in bronchoalveolar lavage fluid and sera of patients with idiopathic PAP. The sensitivity of the serum anti-GM-CSF assay is close to 100% and the specificity too is close to 100% when using a cutoff titer of 1:400. Furthermore, anti-GM-CSF antibodies are increasingly used as a diagnostic tool in

PAP. To what degree a high end-titer of anti-GM-CSF represents an indication to treat more aggressively, or with a larger dose of GM-CSF, remains to be proven.

Conclusions During the past century, much has been accomplished in defining, understanding, and treatment of autoimmune conditions, yet more is to be learned. Autoantibodies play an important role in these immune processes, and of particular importance are ANCAs, involved in the pathogenesis of the so-called ANCA-associated vasculitides. The importance of the autoantibodies stems not only from their contribution to the diagnosis of different conditions, but also from their role in pathogenesis and the importance in monitoring the disease progression. See also: Cryoglobulinemia. Cystic Fibrosis: Overview. Granulomatosis: Wegener’s Disease. Interstitial Lung Disease: Overview. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome. Toll-Like Receptors. Tumors, Malignant: Overview. Vasculitis: Overview.

Further Reading Bonfield TL, Russell D, Burgess S, et al. (2002) Autoantibodies against granulocyte macrophage colony-stimulating factor are diagnostic for pulmonary alveolar proteinosis. American Journal of Respiratory Cell and Molecular Biology 27: 481–486. Csernok E (2003) Anti-neutrophil cytoplasmic antibodies and pathogenesis of small vessel vasculitides. Autoimmunity Reviews 2: 158–164. Imbert-Masseau A, Hamidou M, Agard C, Grolleau JY, and Cherin P (2003) Antisynthetase syndrome. Joint, Bone, Spine 70: 161–168. Ioachimescu O and Stoller J (2005) A review of alpha-1 antitrypsin deficiency. Journal of COPD 2(2): 263–275. Mahadeva R, Dunn AC, Westerbeek RC, et al. (1999) Anti-neutrophil cytoplasmic antibodies (ANCA) against bactericidal/ permeability-increasing protein (BPI) and cystic fibrosis lung disease. Clinical and Experimental Immunology 117: 561–567. Miescher PA, Zavota L, Ossandon A, and Lagana B (2003) Autoimmune disorders: a concept of treatment based on mechanisms of disease. Springer Seminars in Immunopathology 25(supplement 1): S5–S60. Paran D, Fireman E, and Elkayam O (2004) Pulmonary disease in systemic lupus erythematosus and the antiphospholpid syndrome. Autoimmunity Reviews 3: 70–75. Quintana FJ and Cohen IR (2004) The natural autoantibody repertoire and autoimmune disease. Biomedicine & Pharmacotherapy 58: 276–281. Schmitt WH (2004) Newer insights into the aetiology and pathogenesis of myeloperoxidase associated autoimmunity. Japanese Journal of Infectious Diseases 57: S7–S8. Seo P and Stone JH (2004) The antineutrophil cytoplasmic antibody-associated vasculitides. American Journal of Medicine 117: 39–50. Sharma OP (2002) Sarcoidosis and other autoimmune disorders. Current Opinion in Pulmonary Medicine 8: 452–456.

AUTOANTIBODIES 227 Strange C and Highland KB (2004) Interstitial lung disease in the patient who has connective tissue disease. Clinics in Chest Medicine 25: 549–559. Uchida K, Nakata K, Trapnell BC, et al. (2004) High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis. Blood 103: 1089–1098.

Wiik A (2003) Autoantibodies in vasculitis. Arthritis Research & Therapy 5: 147–152. Wisnieski JJ, Baer AN, Christensen J, et al. (1995) Hypocomplementemic urticarial vasculitis syndrome. Clinical and serologic findings in 18 patients. Medicine (Baltimore) 74: 24–41.

B BASAL CELLS M J Evans, University of California, Davis, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Despite differences in basal cell distribution, the unifying feature in all animal species is that the number of basal cells present is related to the height of the columnar epithelium. This relationship is associated

Abstract Basal cells are an integral part of pulmonary airway epithelium. They exist as a separate layer of cells covering most of the basement membrane zone. In this central position, they can interact with columnar epithelium, neurons, the basement membrane zone, and underlying mesenchymal cells. In addition, they interact with inflammatory cells, lymphocytes, and dendritic cells. The interactions with trafficking leukocytes and neurons take place in the lateral intercellular space between basal cells. In this central position basal cells become a very important part of the epithelial–mesenchymal trophic unit of larger airways. Structurally, basal cells function in attachment of columnar epithelium with the basement membrane zone. Failure of attachment between columnar and basal cells is thought to be responsible for sloughing of the columnar epithelium in asthmatics. They also function in regulation of fibroblast growth factor-2 signaling from the basement membrane zone, neurogenic inflammation, the inflammatory response, transepithelial water movement, and oxidant defense of the tissue and formation of the lateral intercellular space for airway epithelium. A subpopulation of basal cells (parabasal cells) has the potential to function as progenitor cells. Clinically, basal cells are probably also involved with the formation of squamous cell carcinoma and, possibly, the progression to organized carcinoma.

LIS LIS

*

*

LIS BC

BC

PCB

*

BC BMZ (a)

Introduction Basal cells are derived from undifferentiated columnar epithelium in the developing airway. They are characterized by their basal position in the columnar epithelium, the presence of hemidesmosomes (characterized by alpha 6 beta 4 integrins), cytokeratins 5 and 14, and the nuclear protein p63 (Figure 1(a)). The distribution of basal cells varies by airway level and animal species. Airways that are larger in diameter have more basal cells than airways with smaller diameters. For example, the largest numbers of basal cells are found in the trachea. As the airway decreases in diameter, the number of basal cells also decreases, and none are present in the terminal bronchioles. As basal cells increase in number, they displace columnar cells on the basement membrane zone (BMZ). In human airways, 90–95% of the BMZ is covered by basal cells from the trachea to bronchioles 1–3 mm in diameter (Figure 1(b)).

(b) Figure 1 (a) Electron micrograph of tracheal epithelium from a young rhesus monkey demonstrating basal cells (BC), parabasal cells (PCB), the lateral intercellular space (LIS), and the basement membrane zone (BMZ). The lateral intercellular space is the open area between cells (asterisks). The lateral intercellular space is reduced or absent between ciliated cells and ciliated cells next to secretory cells (arrowheads). Magnification 1500. Copyright 2001 from Cellular and molecular characteristics of basal cells in airway epithelium. Experimental Lung Research 27: 401–415 by Evans MJ, Van Winkle LS, Fanucchi MV, et al. Reproduced by permission of Taylor & Francis, Inc. (b) Scanning electron micrograph of a sheep tracheal whole mount treated with EDTA. The columnar epithelium has been released leaving a layer of basal cells attached to the BMZ. The basal cells cover between 90% and 95% of the BMZ. Magnification 1100.

230 BASAL CELLS

with the role of basal cells in attachment of columnar epithelium to the BMZ. Functionally, large airway epithelium is stratified, with a layer of basal cells attached to the BMZ and a layer of columnar epithelium attached to the basal cells. Thus, basal cells act as a separate layer of cells in a central position, that can interact with columnar epithelium, neurons, BMZ, and the underlying mesenchymal cells. In addition, they can interact with inflammatory cells, lymphocytes, and dendritic cells in the epithelium. These interactions take place in the lateral intercellular space. The lateral intercellular space is a distinct space between basal cells, basal and adjacent secretory cells, and secretory cells (Figure 1(a)). The lateral intercellular space is hydrated with the aid of the proteoglycan hyaluronan, which is bound to CD44 adhesion molecules on the surface of basal cells. In this central position, basal cells become a very important and integral part of the epithelial–mesenchymal trophic unit of large airways. The large number of receptors found on basal cells that bind growth-regulating proteins and trafficking leukocytes (Table 1) supports this concept.

Basal Cell Functions in the Normal Lung Junctional Adhesion

The structural role of basal cells in the airways is for attachment of columnar epithelium to the BMZ. Epithelial cells are attached to the BMZ by hemidesmosomes and cell adhesion molecules. Cytokeratins 5 and 14 link anchoring junctions of basal cells with the cytoskeletons of adjacent cells through desmosomes and to the BMZ with hemidesmosomes. This arrangement of junctional adhesion provides mechanical stability to a group of cells or tissue. In airway epithelium, basal cells are the only cells that form hemidesmosome junctions with the BMZ. Columnar cells are attached to the BMZ via desmosome attachment with basal cells. The significance of basal cells in junctional adhesion can be demonstrated by treating the tissue with ethylenediaminetetraacetic acid (EDTA). Desmosome junctions are dependent on calcium. When the tissue is treated with EDTA, the calcium is removed from the desmosome, and the columnar epithelium is released leaving the basal cells attached to the BMZ (Figure 1(b)). The number of basal cells present at a particular airway level and their morphology is related to their role in junctional adhesion. When the columnar epithelium increases in height, there is an increase in the size and shape of basal cells along with a corresponding increase in desmosome attachment with the columnar epithelium and hemidesmosome attachment with the BMZ. These changes maintain a

Table 1 Cellular and molecular characteristics of basal cells Cell surface characteristics Alkaline phosphatase Aquaporin 3 transmembrane water channels b-adrenergic receptors CD44 transmembrane glycoproteins Epidermal growth factor receptor Fibroblast growth factor receptor-1 Fas receptors and ligand ICAM-1 IgE receptor Integrins (a6b4) Lectins LEEP-CAM MRP transmembrane transporters MUC 1,4,8 Neurokinin-1 receptor Neutral endopeptidase Syndecan-4 4-1 BB receptor Intracellular characteristics Adrenomedullin receptor (mRNA) Autotaxin (mRNA) Annexin II Bcl-2 protein Extracellular SOD (mRNA) Leukemic inhibitory factor P63 nuclear protein Reproduced from Evans MJ, Van Winkle LS, Fanucchi MV, et al. (2001) Cellular and molecular characteristics of basal cells in airway epithelium. Experimental Lung Research 27: 401–415.

constant amount of junctional adhesion between the columnar epithelium and the BMZ. Thus, the relationship between basal cell junctional adhesions and height of the epithelium is constant and not related to airway level or animal species. Fibroblast Growth Factor-2 Signaling

Fibroblast growth factor-2 is stored in the BMZ of the airways where it binds with perlecan, a heparan sulfate proteoglycan that is an intrinsic constituent of the BMZ. Fibroblast growth factor-2 is released from perlecan in response to various conditions and becomes an important cytokine within the local microenvironment of the epithelial–mesenchymal trophic unit. In airway epithelium, basal cells are the only cell type involved with fibroblast growth factor-2 signaling. Fibroblast growth factor-2 signals by forming a ternary complex with fibroblast growth factor receptor-1 (FGFR-1) and syndecan-4. When the fibroblast growth factor-2 ternary complex is formed, it initiates tyrosine kinase signaling associated with cell proliferation, migration, and differentiation. Basal cells express the cell surface receptors FGFR-1 and syndecan-4 whereas columnar cells do not (Figure 2). Presumably, fibroblast growth factor-2 is stored in airway BMZ as an intact growth factor to aid in rapid cellular responses to changes in local environmental

BASAL CELLS 231 Syndecan-4 FGFR-1

Basal cell FGF-BP FGF-2

Perlecan

Collagen I, III, & V

Perlecan

Basement membrane zone Figure 2 Ternary signaling complex in airway epithelium. In this illustration, BMZ-bound FGF-2 is released, and formation of the FGF-2 ternary complex with basal cells occurs via diffusion or binding with FGF-binding protein (FGF-BP). This is an example of how basal cells may function in growth factor signaling, neurogenic inflammation, and the inflammatory response through interactions with substances in the lateral intercellular space. Reproduced from Evans MJ, Fanucchi MV, Baker GL, et al. (2003) Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen. American Journal of Physiology: Lung, Cellular and Molecular Physiology 285: L931–L939, with permission from The American Physiological Society.

conditions such as sloughing of damaged columnar epithelium or damage to the BMZ by leukocyte trafficking. Neurogenic Inflammation

Basal cells are involved with regulation of neurogenic inflammation. In response to various inhaled foreign materials, axons in the airway epithelium release neuropeptides into the lateral intercellular space, initiating the process of neurogenic inflammation (increased vascular permeability, neutrophil adhesion, vasodilatation, gland secretion, ion transport, smooth muscle contraction, increased cholinergic transmission, and cough). Basal cells contain the protein leukemic inhibitory factor. Leukemic inhibitory factor is thought to function in neurogenic inflammation by stimulating the release of neuropeptides (tachykinins) from axons and the formation of neurokinin receptors. Neutral endopeptidase is a cell surface enzyme also associated with the process of neurogenic inflammation. The enzyme neutral endopeptidase is expressed mainly on the surface of basal cells. The enzyme neutral endopeptidase cleaves neuropeptides in the lateral intercellular space. Cleavage of neuropeptides by neutral endopeptidase modulates the neurogenic inflammatory responses in the airways. Inflammation

Basal cells participate in the inflammatory response by upregulating expression of receptors for migratory

inflammatory cells and lymphocytes. Human basal cells upregulate intercellular adhesion molecule-1. Human basal cells can also upregulate expression of IgE receptors indicating that they may be involved with allergic responses of the airway. An unusual cell adhesion molecule, lymphocyte endothelial–epithelial cell adhesion molecule, is expressed in the basal cell layer of human bronchial epithelium. Lymphocyte adhesion to epithelia and endothelia is mediated by lymphocyte endothelial–epithelial cell adhesion molecule. Human basal cells also express the receptor 4-1BB. The receptor 4-1BB is a member of the tumor necrosis factor receptor super-family and is associated with T cell activation. Human basal cells express the Fas receptor and its ligand FasL. Ligation of the Fas receptor by migratory inflammatory cells can lead to their apoptosis. Expression of these molecules by basal cells may play an important role in regulation of the inflammatory response. They interact with inflammatory cells when the latter are moving through the lateral intercellular space of airway epithelium. Transepithelial Water Movement

Basal cells have the aquaporin water channel AQP3, which is not found in the columnar epithelial cells. Instead, columnar epithelial cells have the aquaporin water channel AQP4. Water transfer between cells and the matrix occurs through these water channels. AQP3 is found in a basolateral position in the airways and in other tissues, implying movement of water between the extracellular matrix and epithelium. The presence of AQP3 water channels in the membranes of basal cells of normal airway epithelium demonstrates a unique role for the basal cell in fluid modulation of airway surface liquids and also in the lateral intercellular space. The cellular distribution of AQPs 3 and 4 in airway epithelium implies the presence of cell-specific pathways for transcellular water movement between the extracellular matrix and epithelium. The cystic fibrosis transmembrane conductance regulator protein is a regulator of AQP3 water channels in basal cells suggesting a role in this disease. Downregulation of AQP 3 is thought to play a role in the pathogenesis of bronchiectasis. Oxidant Defense of the Tissue

Basal cells in normal human airway epithelium express extracellular superoxide dismutase mRNA. Extracellular superoxide dismutase is a secreted protein found in the extracellular matrix responsible for metabolizing superoxide free radicals. The physiologic functions are not fully defined but it is thought to be critical for the protection of extracellular matrix elements against oxidative damage. The multidrug resistance-associated protein transmembrane transporter is

232 BASAL CELLS

found in bronchial epithelium and plays a major role in cell detoxification and defense against oxidant stress via efflux of glutathione conjugates into the lateral intercellular space. The pattern of multidrug resistance-associated protein transmembrane transporter expression differs markedly according to cell type. In basal cells it is distributed over the entire circumference of the cell whereas in ciliated cells it is restricted to the basolateral surface. Expression of extracellular superoxide dismutase and multidrug resistance-associated protein transmembrane transporter by basal cells in normal subjects indicates that basal cells participate in defense of the tissue against oxidative stress. Progenitor Cells

The basal cell has the capacity to be the progenitor of columnar airway epithelium. This has been demonstrated in studies where denuded airways were repopulated with enriched populations of basal cells, in biphasic organotypic cultures, and in vivo following loss of the columnar epithelium. Under these conditions, basal cells dedifferentiate into a highly proliferative cell phenotype from which a mucociliary epithelium redifferentiates. In vivo there are two populations of proliferating basal cells (basal and parabasal cells). Basal cells have their nuclei next to the basement membrane. Parabasal cells are taller, and their nuclei are above the layer of basal cell nuclei. Basal cells make up 31% of the cell population in large airways and the taller parabasal cells make up 7%. However, parabasal cells have a proliferative fraction 4 to 5 times greater than basal cells. The higher proliferative fraction in parabasal cells suggests they may be more active in reparative proliferation following injury when compared with basal cells. Parabasal cells probably are the intermediate cells seen with electron microscopy. Intermediate cells are known to be the primary proliferating cells following injury to the columnar epithelium. During growth of the airway, the basal cell has a high rate of proliferation. The purpose of such proliferation in the growing airway is for the formation of new basal cells. The increase in basal cells per millimeter is related to their role in attaching columnar epithelium to the BMZ. Increased proliferation of parabasal cells during development is probably associated with formation of the columnar epithelium. In the normal adult airway epithelium, the rate of basal cell proliferation is low. Such proliferation may be for replacement of dying basal cells. When basal cells are lost due to injury or apoptosis, proliferation of surviving basal cells occurs, and they are replaced. Proliferation of parabasal cells is most likely associated with normal turnover of the columnar epithelium.

Basal Cells in Respiratory Disease Asthma

Defining the mechanism of columnar cell attachment to the BMZ was critical to the understanding of asthma and other disease conditions associated with sloughing of epithelium. In conditions where columnar cells are sloughed from the epithelium of larger airways, basal cells remain attached to the basal lamina. Desmosomal attachment between the columnar epithelium and basal cells represents a plane of cleavage between the two cell populations. Failure of desmosomal attachment between columnar and basal cells is thought to be responsible for sloughing of the columnar epithelium in asthmatics. The mechanism of desmosomal failure between airway cells is not known. However, it may not be failure per se but rather a specific protective function of basal cells. Shortly after the columnar epithelium has been sloughed, basal cells flatten out and form a protective barrier. This is considered to be an important protective function of the basal cell along with the ability to quickly release desmosomal attachments to damaged columnar epithelial cells. It has also been speculated that stimulation of the epithelial neural system causes edema of the lateral intercellular space with a subsequent local sloughing of the epithelium. In a similar manner, an influx of inflammatory cells could overwhelm the lateral intercellular space and cause local sloughing of the epithelium. However, these scenarios are speculations and the reasons for or mechanisms of epithelial sloughing are not known at this time. Bronchiogenic Cancer

The p63 nuclear protein is specific for basal cells. Its functions are not clear, however, it has been used as a marker for tumors of basal cell origin in several tissues. In the lung, p63 was shown to be expressed in basal cells of normal tissue, in areas of squamous metaplasia, and in squamous cell carcinomas of the bronchi. In poorly organized carcinomas, p63 is expressed in most of the cells. However, it is only found in the basalar external cells of organized carcinomas. In adenocarcinomas, p63 is present occasionally but is not present in small cell carcinomas. These findings indicate that basal cells are probably involved with the formation of squamous cell carcinoma and, possibly, the progression to organized carcinoma. Basal cells have also been implicated in abnormal epithelium found in idiopathic pulmonary fibrosis when p63 was used as a marker. See also: Adhesion, Cell–Cell: Vascular; Epithelial. Adhesion, Cell–Matrix: Focal Contacts and Signaling; Integrins. Aquaporins. Cell Cycle and Cell-Cycle

BREATHING / Breathing in the Newborn 233 Checkpoints. Epidermal Growth Factors. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fibroblast Growth Factors. Stem Cells.

Further Reading Boers JE, Ambergen AW, and Thunnissen FB (1998) Number and proliferation of basal and parabasal cells in normal human airway epithelium. American Journal of Respiratory Critical Care and Medicine 157(6 Pt 1): 2000–2006. Evans MJ, Fanucchi MV, Baker GL, et al. (2003) Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen. American Journal of Physiology: Lung, Cellular and Molecular Physiology 285: L931–L939. Evans MJ and Moller PC (1991) Biology of airway basal cells. Experimental Lung Research 17: 513–531. Evans MJ and Shami SG (1989) Lung cell kinetics. In: Lenfant C and Massaro M (eds.) Lung Cell Biology, vol. I. of the series Lung Biology in Health and Disease, pp. 1–36. New York: Marcel Dekker, Inc.

Basement Membranes

Evans MJ, Van Winkle LS, Fanucchi MV, and Plopper CG (1999) The attenuated fibroblast sheath of the respiratory tract epithelial-mesenchymal trophic unit. American Journal of Respiratory Cell and Molecular Biology 21: 655–657. Evans MJ, Van Winkle LS, Fanucchi MV, et al. (2001) Cellular and molecular characteristics of basal cells in airway epithelium. Experimental Lung Research 27: 401–415. Ford JR and Terzaghi-Howe M (1992) Basal cells are the progenitors of primary tracheal cultures. Experimental Cell Research 198: 69–77. Inayama Y, Hook GE, Brody AR, et al. (1988) The differentiation potential of basal cells. Laboratory Investigation 58: 706–717. Johnson NF and Hubbs AF (1990) Epithelial progenitor cells in rat trachea. American Journal of Respiratory Cell and Molecular Biology 3: 579–585. Mercer RR, Russell ML, Roggli VL, and Crapo JD (1994) Cell number and distribution in human and rat airways. American Journal of Respiratory Cell and Molecular Biology 10: 613–624. Persson CGA and Erjefalt JS (1997) Airway epithelial restitution after shedding and denudation. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung, pp. 2611–2627. Philadelphia: Lippincott-Raven.

see Extracellular Matrix: Basement Membranes.

Berylliosis

see Occupational Diseases: Hard Metal Diseases – Berylliosis and Others.

Bradykinin

see Kinins and Neuropeptides: Bradykinin.

BREATHING Contents

Breathing in the Newborn Fetal Breathing Fetal Lung Liquid First Breath

Breathing in the Newborn J A Adams, Mount Sinai Medical Center, Miami Beach, FL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The complex physiology of breathing in the newborn has important developmental and maturational aspects. Early in fetal

life the respiratory system and controllers respond to intrauterine stimuli (PaCO2 , PaO2 , and lung inflation). The complex network of control of breathing can be summarized into three basic components: (1) controllers, (2) effectors, and (3) sensors. At birth a multitude of inputs are responsible for initiation of rhythmic ventilation, and adaptation of the pulmonary circulation to extrauterine life. The normal physiology of breathing in the newborn is affected by posture, sleep state, and gestational age. Pathophysiological aspects of breathing in the newborn can be divided into: (1) fetal
pathologies that occur during fetal life and ultimately result in abnormal lung development,

234 BREATHING / Breathing in the Newborn (2) immediate postnatal
events that occur primarily as a result of poor or abnormal transition to extrauterine life, and (3) neonatal
events occurring in the neonatal period that lead to intrinsic lung disease or deranged control of breathing such as apnea. This article emphasizes the general importance of understanding the influence of age, posture, sleep state, and maturation on breathing in the newborn, and provides a general overview of these. Paramount to the interpretation of normative and study data is the understanding of the influence of these factors.

Description The undertaking of a complex vital physiological function such as breathing in the newborn is a marvelous feat. Initiation of breathing in the newborn requires a multitude of appropriately synchronized events and signaling pathways. In preparation for extrauterine life and in order to assume the responsibility for extrauterine gas exchange, an anatomical, maturational, and functional process must occur. The alveoli are developed by the 25th week of gestation and by the 35th week of gestation, adequate quantities of surfactant (surface active material that keeps alveoli open and maintains alveoli stability) are present. Pulmonary circulation parallels alveolar development, and thus by the 25th week of gestation alveolar gas exchange can take place, but requires assisted ventilation. During fetal life, respiration is present in human fetuses from 10 weeks onward. These fetal breathing movements are not as well synchronized as those occurring in the newborn period. In fetal life, breathing is discontinuous and becomes continuous after birth. The fetus spends nearly 30% of its time engaged in discoordinate form of breathing associated with rapid irregular electrocortical activity, as seen in active sleep state. In addition, since the entire airways are filled with amniotic fluid, at term almost 600 ml of amniotic fluid is ‘inhaled’ per day. Maintenance of normal amniotic fluid volume, and respiratory activity, are crucial in the development of the airway, and maturation of lung structure and function. The fetus is also capable of modulating breathing movements in response to: (1) PaCO2 (hypercarbia increases fetal breathing), (2) PaO2 (hypoxia abolishes fetal breathing in sleep, and (3) pulmonary reflexes (inflation reflex of Herring–Bauer lung distension with saline infusion decreases frequency of breathing). The overall framework for the control of breathing can be schematically viewed as: (1) controller (central nervous system and brainstem); (2) effectors (respiratory muscles and airway); and (3) feedback (chemoreceptor, mechanoreceptors, i.e., stretch receptors) (Figure 1). Functional maturity of the controller, effectors, and feedback mechanism allows for extremely premature newborns to survive, albeit requiring ventilatory assistance. The maturational process continues during gestation until term at 38–40

weeks of gestation, but even after birth the continual adaptation to air-breathing is an ongoing process. The First Breaths

During vaginal birth but less so during Cesarean section delivery, the thoracic cage is compressed to as much as 160 cmH2O pressure; this produces an ejection of tracheal fluid via the airways. The recoil of the chest wall causes a passive inspiration and establishes an air–liquid interface. The first active breath is made slightly easier by the fact that some fetal lung fluid is retained in the alveoli and smaller airway, thus requiring less distending pressure than a totally collapsed lung. In addition, in near-term and term newborns, surfactant produced by type II pneumocytes decreases alveolar surface tension, which prevents the lungs from total collapse at the lower transpulmonary pressures that occur in the subsequent breaths (Figure 2). The stimuli for the first active inspiration is debatable but is likely to be a multifactorial set of events with which the newborn is confronted including change in temperature, light, noise, gravity, hypercapnea, sudden change in PaO2 , etc. A complete discussion on the control of breathing during the first breaths is beyond the scope of this article, but suffice it to say that environmental nonrespiratory stimuli will enhance or facilitate the general tone of the respiratory neurons. In addition to the mechanical effects of the first breath, the pulmonary circulation that parallels lung development and is maintained at high pulmonary vascular resistance during fetal life must also transition from a fetal to a newborn circulation. The latter is characterized by a gradual lowering of the pulmonary vascular resistance and eventual closure and redirection of blood flow via the foramen ovale and ductus arteriosus, and a change from a parallel circulation to one in series (Figure 3). The resultant effect is ultimately to match ventilation to perfusion for the most efficient oxygen extraction and delivery and carbon dioxide removal. Sleep state modulates control of breathing. The sleep and waking cycles begin to develop during fetal life. In the premature newborn at 24 weeks’ gestation, rapid eye movement (REM) periods account for nearly 80% of the total sleep time. At term, REM sleep is 60–65% of sleep time, while in adulthood it accounts for only 20–25%. There are major differences in the control of breathing between awake and sleep, which are independent of age. During sleep the brain is controlled more by stimulation related to gas exchange than any other stimuli. When comparing the breathing patterns and pulmonary mechanics

BREATHING / Breathing in the Newborn 235

Controllers Effectors

Cerebrum Cerebellum Midbrain

Sensors

Brainstem

Chemoreceptors

Lung receptors

Upper airway

Spinal cord Proprioreceptors

Vagal resp. motor efferents

Upper airway receptors

Resp. muscles

Lung

Figure 1 Schematic representation of the control of breathing. Adapted from Givan DC (2003) Physiology of breathing and related pathological processes in infants. Seminars in Pediatric Neurology 10: 271–280, with permission from Elsevier.

among newborns, measurements should always be made in the same sleep state and in the same posture. Noninvasive methods to quantify these breathing patterns over various sleep states can serve as a basis for comparison. Breathing is affected by gestational age. Maturation of ventilatory response to chemical and mechanical stimuli is also influenced by gestational age. The ventilatory response to hypoxia in newborns is biphasic. It is characterized by an initial phase of hyperventilation followed by hypoventilation below baseline levels. The etiology of such biphasic response is unclear, and may be related to metabolic demands or neurotransmitters (Figure 4). This response is clearly different to that which occurs in older infancy and adults. The ventilatory response to carbon dioxide in premature infants is also different from that in term newborns. In premature infants the ventilatory response to hypercapnea is quantitatively and qualitatively different from that in older term neonates and adults. Term infants and adults increase ventilation through an increase in tidal volume

and frequency; premature infants do not increase frequency in response to hypercarbia, and have a prolonged expiration. This response appears to be centrally mediated at the level of the brainstem and probably involves the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Furthermore, the ventilatory response to hypercapnea is influenced by sleep state in term newborns. There is a greater response in minute ventilation during quiet state compared to REM (Figure 5). Breathing is affected by posture in newborns. The characteristics of a very compliant chest wall in newborns have a significant mechanical effect on breathing as efficiency of ventilation is reduced due to increased chest wall compliance. In full term infants, a change from supine to prone posture increases minute ventilation and respiratory drive, with a concomitant decrease in thoracoabdominal asynchrony. In premature infants (o35 weeks’ gestation) supine posture is associated with higher respiratory rate, lower arterial oxygen saturation, lower ventilatory response to hypercapnea, and increased thoracoabdominal

236 BREATHING / Breathing in the Newborn Saline

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0 0

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Figure 2 (a) Pressure–volume curves after saline and air expansion of the lung. Lower pressures are required to expand a saline-filled lung compared to air. The deflation curve in air is not superimposable on the inflation curve (hysteresis) due to mobilization and orientation of surfactant during deflation decreasing alveolar surface tension as the alveolar surface contracts. Adapted from Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology & Management of the Newborn, pp. 257–278. Philadelphia: Lippincott Williams & Wilkins, with permission from Lippincott Williams & Wilkins. (b) Pressure– volume loops of a normal lung (red) and surfactant-deficient lung. Note that the slope of the loop is decreased in the surfactant-deficient lung. A greater amount of pressure is required for a lower change in lung volume. (Compliance ¼ D volume/D pressure.)

synchrony. Thus, posture appears to play a role in pulmonary mechanics, primarily in the efficiency of ventilation. Whether these changes occur as a result of conferring greater chest wall stability or diaphragmatic mechanical advantage in the prone posture in both full term and preterm infants remains to be clearly elucidated. In spite of this, conferring a mechanical advantage in the prone posture for full term infants does not justify its use in the care of the full term newborns upon hospital discharge. Sudden infant death rates have clearly decreased in the US as a result of the ‘Back to Sleep Campaign’.

Pathophysiology In order to understand the effects of various pathological states on breathing in the newborn it is useful to describe three distinct time periods for occurrence of pulmonary pathology: (1) fetal, (2) immediate postnatal, and (3) neonatal.

Fetal

To a large extent, lung growth is dependent on amniotic fluid production and volume, and the mechanical

BREATHING / Breathing in the Newborn 237

Pulmonary vascular resistance

Pulmonary venous ret

Left atrial pressure

PaO2 (>30)

Ventilation

Closed ductus arteriosus

Closed ductus venosus

Closed foramen ovale

L–R shunt ductus Right atrial pressure

Umbilical venous ret

Peripheral vascular resistance Figure 3 Schematic of the transition to neonatal circulation once ventilation is established. L–R, left to right; ret, return. Multiple factors play a role in the conversion of this circulation, including prostaglandins, endothelin, and endothelial-derived nitric oxide. Adapted from Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology & Management of the Newborn, pp. 257–278. Philadelphia: Lippincott Williams & Wilkins, with permission from Lippincott Williams & Wilkins.

% Change in baseline

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−30 Time (s) Figure 4 The biphasic ventilatory response to hypoxia. Note the increase in ventilation, followed by a marked decrease in ventilation below baseline values. Adapted from Martin RJ, Di Fiore JM, Jana L, et al. (1998) Persistence of the biphase ventilatory response to hypoxia in preterm infants. Journal of Pediatrics 132: 960–964, with permission from Elsevier.

shear it exerts on the primitive airways and alveolar ducts. Thus, a spectrum of pulmonary hypoplasia due to multiple etiologies is the major pathological problem in the fetal period.

Immediate Postnatal

Failure to transition from a fluid-filled to a gas-filled pulmonary tree and to reabsorb the fetal lung fluid results in a transient condition called transient

238 BREATHING / Breathing in the Newborn

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pulmonary hypertension with increased pulmonary vascular resistance and right to left shunting via the patent ductus arteriosus and foramen ovale. This condition has several underlying etiologies including intrauterine hypoxia, pneumonia, and other pulmonary vascular pathologies.

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Figure 5 Ventilatory response to hypercapnea. Note the difference in slope of the line between active state (REM) and quiet state in full term infants. End tidal CO2 ¼ PET CO2. Reproduced from Cohen G, Xu C, and Henderson-Smart D (1991) Ventilatory response of the sleeping newborn to CO2 during normoxic rebreathing. Journal of Applied Physiology 71(1): 168–174, with permission from The American Physiological Society.

tachypnea of the newborn (TTN or respiratory distress syndrome (RDS) type II). This condition is characterized by the early appearance of tachypnea and a variable degree of respiratory distress in an otherwise ‘healthy appearing’ newborn. The greater occurrence of TTN in newborns delivered by Cesarean section make this condition rather common. Onset of respiratory distress after the initial newborn transitional period (about 4–6 h) usually signifies intraparenchymal lung pathology due to pneumonia (viral or bacterial). Medications administered during labor and delivery can also have an effect on breathing; these include narcotics or magnesium sulfate, both of which can depress respiratory drive. In the premature infant, failure to transition from fetal to extrauterine life can be due to pulmonary insufficiency secondary to RDS type I (surfactant deficiency). Surfactant administration has markedly improved mortality and morbidity of this disease process and has radically changed the outcomes for premature newborns. In both premature and term newborns, failure of the pulmonary circulation to transition from intrauterine fetal circulation to that of the newborn results in the well known persistent pulmonary hypertension of the newborn (PPHN) or persistent fetal circulation (PFC) characterized by

In premature infants, particularly those born at less than 34 weeks’ gestation who have immature lungs or an intrinsic pulmonary pathology, a frequent respiratory problem is apnea. Apnea is typically defined as cessation of breathing for more than 15 s; however, the literature is replete with various definitions for the duration of cessation of breathing. Apnea is most commonly seen in premature infants, and the younger the gestational age the more common its occurrence (Figure 6). Apnea in the premature infant can be caused by intracranial pathology, metabolic derangements, and infection, among others. The diagnosis of apnea of prematurity is one of exclusion and thus an exhaustive undertaking of diagnostic tests should be done prior to categorizing apnea as such (Figure 7). In addition to cessation of breathing or ineffective ventilation, clinically relevant apneas decrease arterial oxygen saturation and cause bradycardia. With the advent of computerized monitoring and noninvasive technologies to measure breathing and arterial oxygen saturation, long-term studies in newborns have become possible in the home environment. It has been found that apneas can occur at any gestational age and that their severity can only be detected if various physiological parameters are simultaneously monitored. Apnea has been typically classified into three types: (1) central (no respiratory efforts and thus no airflow), (2) obstructive (respiratory efforts against a partially or totally occluded airway), and (3) mixed (combination of central and obstructive within the same event). With the advent of more refined monitoring techniques, it is evident that purely obstructive apneas in the newborn are rare, and that most apneas in premature infants have a mixed/obstructive characteristic. Cessation of airflow is present followed by obstructive breaths or obstructive breaths are followed by cessation of respiratory effort. Perhaps the most clinically relevant and important aspect of apnea relates to the changes in arterial oxygen saturation that it can cause and bradycardia (Figures 8(a)–8(c)). Using respiratory inductive plethysmography that measures both ribcage and abdominal excursions and can be calibrated in the newborn to provide a relative estimate of tidal volume, we have been able to study apneas in premature newborns. In

BREATHING / Breathing in the Newborn 239

Relative incidence of apnea

Apnea of prematurity

Prematurity

Term 1 mo 2 mo Term 1 mo 2 mo 6 mo Age Figure 6 Relative incidence of apnea as a function of maturation. Note the dramatic decrease in apnea after term gestation. Reproduced with permission from NeoReviews, Vol. 3, pages e66–e70, Copyright 2002.

Immaturity

Hypercapnic response

Inhibitory reflexes

Hypoxic depression

Apnea

Seizures

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Hypoxemia or anemia

Drugs

Reflux Head & body position

Figure 7 A schematic representation of the etiology of apnea in premature newborns. Examples of cause of apneas are highlighted in yellow. IVH, intraventicular hemorrhage. Adapted with permission from NeoReviews, Vol. 3, page(s) e66–e70 and NeoReviews, Vol. 3, page(s) e59–e65, Copyright 2002.

addition to apneas, newborns also experience hypopnea (decreased tidal volumes below 25% of the unimpeded baseline value). Hypopnea is a commonly recognized problem in sleep disorder breathing in adults but not commonly appreciated in newborns due to the lack of ability to measure changes in tidal volume over prolonged periods. The end result of both apneas and hypopneas is to cause decreased functional residual capacity (FRC). Since newborns typically have relatively low FRC and oxygen stores, the concomitant decrease in arterial oxygen saturation during apneas or hypopneas is not surprising. Additionally, we have found using noninvasive methods that during periods of apnea in the premature newborn the cardiac output can decrease by as much as 50%. Cerebral blood flow fluctuations have also been shown to occur to a greater degree during periodic breathing, apnea, and REM sleep. Whether cardiac output and episodic arterial oxygen desaturation and changes in cerebral blood flow will have untoward long-term neurological effects needs to be determined.

Conclusion Disordered breathing in the premature and term infant can be ascribed to a multitude of causes, which

240 BREATHING / Breathing in the Newborn 30.5 mm s–1

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(b) Figure 8 Examples of respiratory inductive plethysmographic recordings: (a) normal tidal breathing in the newborn; (b) central apnea of 14 s duration; and (c) mixed/obstructive apnea of 21 s duration (note a decrease in HR to 90 bpm, followed by a decrease in arterial oxygen saturation to a nadir of 80%). Vt, the tidal volume from calibrated respiratory inductive plethysmography derived as a percent of the calibration value. RC, the volume contribution of the ribcage to tidal volume. AB, the abdominal volume contribution to tidal volume. EPANG, the phase angle between the RC and AB, obtained from a plot of RC vs. AB; values of 180o denote complete paradoxical motion of the RC and AB in opposite directions. ECG, electrocardiogram. HR, heart rate derived from the electrocardiogram. OxiP, the arterial pulse obtained from the pulse oximeter. SAT, percent arterial oxygen saturation.

BREATHING / Breathing in the Newborn 241 5.6 mm s–1

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have their basis in pulmonary developmental problems, intrinsic pulmonary disease, or deranged control of breathing. The influence of sleep state, posture, and gestational age are paramount in the interpretation of both normative and study data. See also: Breathing: Fetal Breathing; Fetal Lung Liquid; First Breath. Infant Respiratory Distress Syndrome. Sudden Infant Death Syndrome.

Further Reading Adams JA, Zabaleta IA, and Sackner MA (1994) Comparison of supine and prone noninvasive measurements of breathing patterns in fullterm newborns. Pediatric Pulmonology 18: 8–12. Adams JA, Zabaleta IA, and Sackner MA (1997) Hypoxemic events in spontaneously breathing premature infants: etiologic basis. Pediatric Research 42: 463–471. Adams JA, Zabaleta IA, Stroh D, Johnson P, and Sackner MA (1993) Tidal volume measurements in newborns using respiratory inductive plethysmography. American Review of Respiratory Diseases 148: 585–588. Avery GB, Fletcher MA, and McDonald MG (1999) Neonatology Pathophysiology & Management of the Newborn. Philadelphia: Lippincott Williams & Wilkins. Baird TM, Martin RJ, and Abu-Shaweesh JM (2002) Clinical associations, treatment, and outcome of apnea of prematurity. NeoReviews 3(4): e66–e70. Campbell AJ, Bolton DP, Taylor BJ, and Sayers RM (1998) Responses to an increasing asphyxia in infants: effects of age and sleep state. Respiration Physiology 112: 51–58.

Chernick V (1981) The fetus and the newborn. In: Hornbein T (ed.) Regulation of Breathing, Part II, pp. 1141–1179. New York: Dekker. Cohen G, Xu C, and Henderson-Smart D (1991) Ventilatory response of the sleeping newborn to CO2 during normoxic rebreathing. Journal of Applied Physiology 71(1): 168–174. Curzi-Dascalova L (1992) Physiological correlates of sleep development in premature and full-term neonates. Neurophysiology Clinincs 22: 151–166. Fanaroff AA and Martin RJ (2002) Neonatal–Perinatal Medicine Disease of the Fetus and Infant. St Louis: Mosby. Givan DC (2003) Physiology of breathing and related pathological processes in infants. Seminars in Pediatric Neurology 10: 271–280. Guilleminault C and Robinson A (1996) Developmental aspects of sleep and breathing. Current Opinion in Pulmonary Medicine 2: 492–499. Martin RJ, Abu-Shaweesh JM, and Baird T (2002) Pathophysiologic mechanisms underlying apnea of prematurity. NeoReviews 3: e59–e65. Martin RJ, Di Fiore JM, Jana L, et al. (1998) Persistence of the biphasic ventilatory response to hypoxia in preterm infants. Journal of Pediatrics 132: 960–964. Mortola JP and Saiki C (1996) Ventilatory response to hypoxia in rats: gender differences. Respiration Physiology 106: 21–34. Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology & Management of the Newborn, pp. 257–278. Philadelphia: Lippincott Williams & Wilkins. Polin RA and Fox WW (1992) Fetal and Neonatal Physiology. Philadelphia: Saunders. Rigatto H (1992) Maturation of breathing. Clinical Perinatology 19: 739–756. Stocks J, Sly PD, Teppers RS, and Morgan WJ (1996) Infant Respiratory Function Testing. New York: Wiley-Liss.

242 BREATHING / Fetal Breathing

Fetal Breathing R Harding and S B Hooper, Monash University, Melbourne, VIC, Australia C A Albuquerque, Santa Clara Valley Medical Center, San Jose, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Fetal breathing movements (FBMs) are breathing-like movements that occur episodically in healthy mammalian fetuses. As with postnatal breathing, FBMs are centrally organized rhythmic contractions of the diaphragm, but may also involve other skeletal muscles such as those of the chest wall and upper respiratory tract. Owing to the airways being filled with liquid, FBMs cause only minor changes in lung volume but typically lower intrathoracic pressure by up to 5 mmHg and alter the shape of the fetal chest. By altering intrathoracic pressure they affect blood flow within the fetus and fluid movement within the fluid-filled fetal airways. FBMs are characteristically highly variable in frequency and amplitude, but become more organized with increasing gestational age and relate to fetal behavioral states. They are inhibited by fetal hypoxia and hence can be used in the diagnosis of fetal compromise. FBMs are critical for normal in utero lung growth and development as they maintain the fetal lung in an expanded state by opposing lung recoil: in the absence of FBMs the fetal lungs tend to ‘deflate’ leading to lung hypoplasia. At birth, breathing becomes continuous, possibly by removal of inhibitory substances produced by the placenta or fetal brain and by increased carbon dioxide production.

Introduction Fetal breathing movements (FBMs) are breathinglike movements that occur episodically in healthy fetuses during much of gestation. FBMs have been observed in many mammalian species, including man, as well as in birds and reptiles. As with postnatal breathing, FBMs are centrally organized rhythmic contractions of the diaphragm, but may also involve other skeletal muscles such as those of the chest wall and upper respiratory tract. They play no role in fetal gas exchange, as the fetal ‘airways’ are filled with liquid; in sheep, they typically lower intrathoracic pressure by up to 5 mmHg. Most of the available information on FBMs has been obtained from two species, humans and sheep. In humans, FBMs can be detected by ultrasonography from about 10 weeks of gestation. They are observed as rhythmic descending movements of the diaphragm and are usually coincident with inward movement of the chest wall and outward movement of the abdominal wall. In chronically catheterized fetal sheep, FBMs can be detected as electrical activity of the diaphragm muscle, rhythmic reductions in intratracheal or intrathoracic pressure, or fluid movement within the trachea.

Two other types of inspiratory efforts have been recognized in the fetus; isolated deep inspiratory efforts and asphyxial gasping. Isolated deep inspiratory efforts, often termed hiccups, are commonly observed in healthy fetal humans, pigs, and sheep; typically, these occur in low-frequency bouts. Asphyxial gasps are strong inspiratory efforts initiated by fetal asphyxia and involve intense activation of many respiratory muscles.

Central and Peripheral Control of FBMs FBMs are an expression of rhythmic activation of neurons in the fetal brainstem. These brainstem neurons generate rhythmic bursts of activity in phrenic motoneurons and in vagal preganglionic fibers destined for dilator muscles of the upper respiratory tract. With development, episodes of FBMs become temporally associated with fetal behavioral states involving body movements, rapid eye movement (REM) sleep, or arousal. During the last third of gestation, FBMs are usually infrequent or absent in association with the fetal state resembling quiet, or non-REM, sleep (low activity state). The periods of FBM/REM sleep/activity and the intervening periods of apnea/non-REM sleep/inactivity are of similar duration, both occupying B50% of the time. Compared to postnatal life, respiratory drive in the fetus is relatively low and is regulated mainly by fetal CO2 levels, as the incidence and amplitude of FBMs are increased by elevated fetal PaCO2 (partial pressure of carbon dioxide in arterial blood) levels and decreased by low fetal PaCO2 levels. It is assumed that this CO2 drive is mediated by acidification of the fluid surrounding the central chemoreceptors, as altering the pH of fetal blood or cerebrospinal fluid (CSF) can have effects similar to those of alterations in PaCO2 levels. FBMs are inhibited by moderate to severe hypoxia (Figure 1), by what is thought to be a central inhibitory mechanism that also inhibits other forms of fetal skeletal muscle activity and alters fetal behavioral state favoring reduced activity. This inhibitory mechanism may override the stimulatory effects of mild hypoxia. Although peripheral chemoreceptors are active in the fetus and do respond to hypoxia, they do not apparently play a role in the hypoxic inhibition of FBMs. Recent studies suggest that central adenosine receptors play a major role in the central inhibition of FBMs and alteration in fetal behavior by hypoxia.

Effects of FBMs on Airway Fluid Movement and Lung Volume As FBMs alter fetal intrathoracic pressure, it is to be expected that they affect fluid movement within the

BREATHING / Fetal Breathing 243 50

Incidence of FBMs (min h–1)

40

30

20

∗ ∗

10 ∗

∗

∗

∗ ∗ ∗ ∗

∗ 24 h normoxia

∗ ∗

24 h hypoxia

0 −4

0

4

8

12

16

20

24

28

Period of hypoxia (h) Figure 1 Effects of 24 h of fetal hypoxia on the incidence of FBMs in a late gestational fetal sheep. Hypoxia causes a profound but transient inhibition of FBMs, with normal values returning after 14–18 h of hypoxia. Asterisks show values that differ significantly from control values. Reproduced from Hooper SB and Harding R (1990) Changes in lung liquid dynamics induced by prolonged fetal hypoxemia. Journal of Applied Physiology 69: 127–135, used with permission from the American Physiological Society.

respiratory tree. Oscillatory fluid flows synchronous with diaphragm movements have been detected in the fetal trachea and at the nose in humans. Although there is some controversy as to the volume of fluid moved with each fetal ‘breath’, it is clear that ‘tidal volume’ in the fetus is much smaller than after birth, which can be attributed to the much greater viscosity of liquid relative to air. In fetal sheep, for example, ‘tidal volumes’ measured by flow meters in the trachea are much less than 1 ml, whereas after birth, in the same species, tidal volumes are typically 30– 50 ml. Thus, FBMs are essentially isovolumic, causing only very small changes in thoracic volume with each breath. However, in human fetuses, ‘tidal volumes’ of about 2 ml have been estimated by ultrasound, in the trachea and at the nose. The reason for the larger ‘tidal volume’ in humans is unknown but may be due to the slower breathing rates of human fetuses compared to fetal sheep. Although FBMs individually may have little effect on volume flow within the airways, it is clear that episodes of FBM can result in the movement of much greater volumes, relative to total lung fluid volume. This effect on fluid movement can be largely attributed to FBM-related changes in transpulmonary pressure and upper airway resistance. Studies in sheep have shown that net fluid movement to and from the fetal lungs is affected by FBM episodes such that most of the efflux occurs during these episodes; during episodes of FBM the laryngeal dilator muscles

are rhythmically active (in phase with the diaphragm) and laryngeal adductor muscles are largely quiescent. Hence, the resistance to tracheal fluid movement offered by the upper airway is lowered during FBMs, allowing fluid that has accumulated within the airways to flow into the pharynx, from where it is either swallowed or flows into the amniotic sac. During periods of fetal apnea, the laryngeal dilator muscles become quiescent and adductor tone is usually present, resulting in raised resistance of the upper airway. Together with a lack of inspiratory muscle activity, this results in low rates of fluid efflux from the trachea, although increased efflux may occur with fetal postural adjustments or other activities, including those resembling straining movements or Valsalva maneuvers that are common in the fetus. The fetal upper airway plays a critical role in regulating the flow of fluid to and from the lungs, thereby preserving lung volume and the composition of liquid within the airways. Removal of the influence of the upper airway by creating a tracheo-amniotic bypass results in a major loss of lung liquid during periods of apnea and a large ingress of amniotic fluid during FBM episodes. This loss of upper airway function not only allows near total lung deflation during periods of fetal apnea, it also allows large influxes of amniotic fluid during FBM episodes. This brings the alveolar epithelium into contact with undiluted amniotic fluid with potentially harmful effects, especially if the amniotic fluid contains meconium.

Although the net flow of liquid within the fetal trachea is away from the lungs, primarily due to continuous liquid secretion, periods of influx may occur, especially in association with periods of vigorous FBMs. This may explain the entry of substances into the lungs following their deposition in the amniotic fluid.

Effects of FBMs on Fetal Blood Flows As FBMs alter intrathoracic pressure, it is to be expected that they will affect blood flows within the fetus. FBM-related changes in blood flows have been observed in the umbilical vein, fetal vena cava, foramen ovale and, more recently, in the pulmonary artery. In the pulmonary artery, episodes of vigorous FBMs are associated with increased blood flow, likely to be a consequence of reduced resistance in the pulmonary capillaries as a result of an altered transmural pressure.

Functional Importance of FBMs A major function of FBMs is to maintain lung liquid volume, and hence lung expansion, which is known to be essential for normal growth and structural maturation of the fetal lung. This role of FBMs has been demonstrated in experimental models that have eliminated FBMs while preserving the integrity of the diaphragm. The long-term abolition or suppression of normal FBM results in lung hypoplasia and structural immaturity of the lungs, which is probably due to a chronic reduction in lung expansion rather than abolition of the small phasic movements of the chest wall. A chronic reduction in fetal lung expansion leads to a reduction in lung tissue growth and alterations in the structure of the alveolar wall and epithelium; similar changes in lung development are caused by the abolition of FBMs. Abolishing the diaphragmatic movements that cause FBM is thought to reduce the force that normally opposes the inherent elastic recoil of the lungs, thereby allowing the fetal lungs to ‘deflate’; a further reduction occurs if the resistance offered by the upper airway is removed (Figure 2).

Use of FBMs in Clinical Assessment In the healthy human fetus, FBMs occur at an average frequency of 60 per min and are accompanied by increased body movements and heart rate variability. FBMs are first detectable at about 10–12 weeks of gestation when they are usually irregular and sporadic. From about 28 weeks of gestation onwards, FBMs become more regular and organized

Lung volume (% of intact fetus)

244 BREATHING / Fetal Breathing

100 80 60 40 20 0

Intact fetus

No No FBMs Collapsed FRC FBMs & lung newborn no UA

Figure 2 Lung luminal volumes in fetal and neonatal sheep, expressed in relation to values in the intact, late gestation ovine fetus in utero. Lung liquid volume, and hence lung expansion, is reduced by the prolonged absence of FBMs (no FBMs) induced by phrenic nerve blockade or a high section of cervical spinal cord. A further reduction occurs if the fetal upper airway is bypassed, allowing direct continuity between the fetal lungs and the amniotic sac (no FBMs & no UA). Lung luminal volume is reduced further when the lungs are removed from the fetus (collapsed lung) due to unopposed recoil of the fluid-filled lung. Functional residual capacity (FRC) in the air-breathing newborn is shown for comparison. Data from Harding R and Hooper SB (1996) Regulation of lung expansion and lung growth before birth. Journal of Applied Physiology 81: 209–224.

into discrete episodes. Using color Doppler and spectral ultrasonography analyses, the breath-to-breath interval and the inspiratory phase of the respiratory cycle have been shown to increase from 22 to 35 weeks gestation, but then decrease towards term. FBMs are used as one component of the ‘fetal biophysical profile’ which is widely used to assess fetal health; the occurrence of FBMs is observed, together with assessments of fetal heart rate, amniotic fluid volume, fetal body movements, and fetal body dimensions. If FBMs are not detected, the fetus may be hypoxic, may have a neural disorder affecting the brainstem and phrenic motor nerves, or it may have an abnormality of skeletal muscle function. However, it must be recognized that the inhibition of FBMs by chronic fetal hypoxia may be only transient, as has been shown in sheep (Figure 1). FBMs have also proven to be useful for the diagnosis of intrauterine infection in patients with preterm rupture of membranes as they have a high negative predictive value for intra-amniotic infection; a depression in FBM incidence is non-specific but is suggestive of infection. Studies of patients with premature rupture of membranes have shown a decrease in FBM incidence in those patients positive for amniotic fluid infection, clinical chorioamnionitis, or neonatal sepsis. This may be related to inflammatory cytokines affecting fetal behavioral states.

BREATHING / Fetal Breathing 245

Fetal Breathing, Amniotic Fluid Volume, and Lung Growth There is conflicting evidence on the role of FBMs in the development of pulmonary hypoplasia in human fetuses with preterm rupture of membranes and reduced amniotic fluid volume (oligohydramnios). Studies examining the role of FBM in the lung hypoplasia associated with premature rupture of membranes have shown either no change or a reduction in the incidence of FBM. However it is likely that both the oligohydramnios following membrane rupture and a reduced incidence of FBMs may contribute to the associated fetal lung hypoplasia. In humans, it has been shown that a prolonged reduction in FBMs at critical periods of development (i.e., o24 weeks of gestation) can lead to pulmonary hypoplasia; similarly, pulmonary hypoplasia can develop in human fetuses after prolonged (more than 6 days) membrane rupture. It is likely that oligohydramnios leads to lung hypoplasia due to increased flexion of the fetal trunk, which compresses the fetal lungs causing their deflation and reduced growth rates.

Effects of Labor on FBMs At term, the incidence of FBMs decreases. In sheep, the incidence of FBMs decreases 2–3 days before the onset of labor and remains reduced until delivery. This may be related to increased circulating concentrations of prostaglandin E2, which is known to inhibit FBMs. Similarly, in humans, fetal apnea of 20–60 min duration is considered to be a reliable indicator of premature labor with delivery within 48–72 h. During spontaneous or induced labor, the incidence of FBMs is decreased to less than 10% of the time during the latent phase and is further decreased during the active phase of labor. In human pregnancy, the rupture of fetal membranes at term does not appear to affect FBMs. However, a study before term showed a significant reduction in FBMs for the first 2 weeks of membrane rupture, compared to controls, with a return to near normal by the third week. With increased uterine contractility there is a decrease in the incidence of FBMs.

Maternal Smoking and Fetal Breathing As with most drugs taken by the mother, tobacco smoking has been shown to affect fetal behavior. Nicotine readily crosses the placenta, and is thought to reduce utero-placental and/or umbilico-placental blood flow, contributing to fetal hypoxemia; furthermore, the formation of carboxyhemoglobin as a result

of maternal smoking reduces the oxygen carrying capacity of fetal blood. FBMs are inhibited by maternal tobacco smoking, and the inhibition may continue for up to an hour following a single cigarette. It is thought that this effect is due mostly to fetal hypoxemia, or more specifically to reduced cerebral oxygen delivery. Maternal smoking has recently been shown to increase the resistance in the fetal cerebral artery, and this may also contribute to reduced cerebral oxygen delivery. The inhibition of FBMs by maternal smoking may contribute to the known adverse effects of smoking on fetal lung development. The same may apply to other drugs taken by the mother, such as narcotics, sedatives, or analgesics.

Respiratory Transition at Birth The physiological mechanisms underlying the transition from discontinuous fetal breathing to continuous postnatal breathing are complex and not fully understood. During parturition and when the umbilical cord is cut at birth, the neonate may become profoundly hypoxemic, hypercapnic, and acidemic. It is also exposed to lower environmental temperatures, leading to increased heat loss, and to a greatly increased degree of external sensory stimuli, thereby changing the behavioral state to one of arousal. It is also possible that the removal of circulating inhibitory or suppressive substances that originate in the placenta (e.g., prostaglandin E2, adenosine, neuroactive progesterone metabolites) may contribute to the onset of continuous breathing. For example, their removal may lead to an increase in metabolic activity (e.g., by stimulating thermogenesis), and hence an increase in the rate of CO2 production, or to an increase in the sensitivity of central chemoreceptors to CO2. It has also been suggested that endocrine changes at birth, such as a large increase in circulating catecholamines, lead to respiratory stimulation, possibly via an increase in fetal metabolism and hence CO2 production. Studies of fetal sheep maintained ex utero by extracorporeal oxygenation also support the notion that CO2 plays a crucial role in the maintenance of continuous breathing after birth. The integrity of the vagus nerves has been shown to be essential for the onset of adequate breathing at birth. Although the critical pathways have not yet been identified, it is likely that volume receptive feedback from the lungs is involved. Studies in neonatal lambs have shown that a reduction in end-expiratory lung volume (functional residual capacity, FRC), which reduces vagal sensory traffic from pulmonary stretch receptors, results in profound hypoventilation, periodic breathing, and active glottic adduction during periods of apnea. This indicates

246 BREATHING / Fetal Lung Liquid

that receptive vagal feedback of lung volume at endexpiration, which is normally maintained by an adequate FRC, is essential for continuous breathing in the newborn, and explains, at least in part, the benefits of positive end-expiratory pressure (PEEP) in the treatment of infantile apnea. See also: Breathing: Fetal Lung Liquid. Lung Development: Overview.

stretch stimulus that is essential for normal fetal lung growth. If the distending influence of fetal lung liquid is absent, the lungs fail to grow, which is the primary mechanism for fetal lung hypoplasia in humans. At birth, the airways are cleared of liquid to allow the entry of air and the onset of air breathing, due to a reversal of the transepithelial ionic gradient that promotes reabsorption into the lung parenchyma; this is stimulated by the release of stressrelated hormones during labor. Increases in transpulmonary pressure, due to changes in fetal posture, are likely to also contribute to the clearance of fetal lung liquid at birth.

Further Reading

Description

Bissonnette JM (2000) Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 278: R1391–R1400. Bocking AD (2003) Assessment of fetal heart rate and fetal movements in detecting oxygen deprivation in-utero. European Journal of Obstetrics and Gynecology 110: S108–S112. Champagnat J and Fortin G (1997) Primordial respiratory-like rhythm generation in the vertebrate embryo. Trends in Neuroscience 20: 119–124. Cosmi EV, Anceschi MM, Cosmi E, et al. (2003) Ultrasonographic patterns of fetal breathing movements in normal pregnancy. International Journal of Gynaecology and Obstetrics 80: 285–290. Harding R (1997) Fetal breathing movements. In: Crystal RG, West JB, Weibel ER, et al. (eds.) The Lung, Scientific Foundations, pp. 2093–2103. Philadelphia: Lippincott-Raven. Harding R and Bocking AD (2001) Fetal Growth and Development. Cambridge: Cambridge University Press. Harding R and Hooper SB (1996) Regulation of lung expansion and lung growth before birth. Journal of Applied Physiology 81: 209–224. Hooper SB and Harding R (1990) Changes in lung liquid dynamics induced by prolonged fetal hypoxemia. Journal of Applied Physiology 69: 127–135. Laudy JA and Wladimiroff JW (2000) The fetal lung. 1: Developmental aspects. Ultrasound in Obstetrics and Gynecology 16: 284–290. Rigatto H (1996) Regulation of fetal breathing. Reproduction, Fertility and Development 8: 23–33.

Throughout embryonic and fetal development, the future airways of the lung are filled with liquid and the lungs take no part in gas exchange. This liquid (fetal lung liquid) is a unique secretory product of the lung and is quite unlike fetal plasma or amniotic fluid in composition; it has a low pH, a high Cl
concentration, and low protein content. Fetal lung liquid is secreted across the pulmonary epithelium and leaves the lungs by flowing out of the trachea.

Fetal Lung Liquid S B Hooper and R Harding, Monash University, Melbourne, VIC, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Fetal lung liquid is secreted across the pulmonary epithelium and enters the future airspaces due to an osmotic gradient created by the transepithelial flux of ions into the lung lumen. This liquid leaves the lungs by flowing out of the trachea, whereby it is either swallowed or contributed to amniotic fluid volume. The high resistance to liquid movement through the fetal upper airway promotes the retention of lung liquid within the future airways, which provides a small (1 or 2 mmHg) internal distending pressure on the lungs. This acts as an internal hydrostatic splint that maintains the fetal lungs in a distended state and provides a

Control of Fetal Lung Liquid Secretion

Fetal lung liquid is formed by the net movement of Cl
and Na þ across the epithelium into the lumen, which provides an osmotic gradient for the movement of water in the same direction. This mechanism is thought to be driven by Na þ /K þ ATPase, which creates an electrochemical gradient for Na þ to enter epithelial cells via the Na þ /K þ /2Cl
cotransporter. The resultant Na þ -linked Cl
entry across the basolateral membrane increases intracellular Cl
concentrations that passively exit the cell, down its electrochemical gradient, via selective channels located in the apical surface. The net movement of Cl
into the lung lumen generates a small transepithelial potential difference (lumen negative) that also promotes the movement of Na þ . As a result, water moves down an osmotic gradient generated by the net movement of Na þ and Cl
(Figure 1). However, water movement across the epithelium must also depend on the intraluminal hydrostatic pressure existing, which will oppose its movement into the lung lumen. At rest, a small hydrostatic distending pressure (1 or 2 mmHg above ambient pressure) is present within the lung lumen and, therefore, the secretion of lung liquid must normally occur against a small hydrostatic pressure. When intraluminal pressures are increased above 5 or 6 mmHg, lung liquid secretion ceases, indicating that the osmotic pressure driving lung liquid can be counterbalanced by a hydrostatic pressure of 5 or 6 mmHg. On the other hand, when the fetal lungs are partially deflated and the intraluminal hydrostatic pressure is reduced, fetal lung liquid secretion rates increase.

BREATHING / Fetal Lung Liquid 247

Interstitial space

Epithelial cell

Lung lumen

H2O

Na+

K+

2Cl− Cl−

2K+ 3Na+ Na+

Figure 1 The proposed mechanism for fetal lung secretion across the pulmonary epithelium. Na þ /K þ ATPase, located on the basolateral surface of pulmonary epithelial cells, provides the free energy for Na þ to enter the cell via the Na þ /K þ /2Cl
cotransporter, which promotes the entry of Cl
against its electrochemical gradient. Cl
exits the cell across the apical membrane down its electrochemical gradient, which causes a transepithelial potential difference (lumen negative) that promotes the movement of Na þ into the lung lumen. The combined net flux of Na þ and Cl
into the lung lumen provides an osmotic gradient for water to flow in the same direction.

Although the mechanisms for fetal lung liquid secretion are relatively clear, the precise cellular origin is less clear. Early in gestation most epithelial cells are likely to contribute, but later in gestation the most likely candidates are the epithelial cells residing in the smaller, terminal airways because they constitute the vast majority (o90%) of the internal surface area of the lung. This is further supported by the finding that fetal bronchial epithelial cells predominantly absorb Na þ , whereas cultured fetal type II epithelial cells exhibit similar ionic fluxes as that described in the intact fetal lung. However, the relative contribution of type I and type II cells to fetal lung liquid secretion is unknown. Control of Fetal Lung Liquid Volume

Fetal lung liquid exits the lung by flowing out of the trachea, whereby it is either swallowed or enters the amniotic sac. The factors regulating liquid movement within the fetal airways, particularly during late gestation, are complex but primarily involve the

transpulmonary pressure gradient, the degree of lung recoil, and the resistance to liquid efflux offered by the fetal glottis and upper airway. During apnea, when fetal breathing movements (FBMs) are absent, active adduction of the glottis increases the resistance to lung liquid efflux and promotes its retention within the future airways. This promotes lung expansion, and the resultant increase in lung recoil generates the small intraluminal hydrostatic pressure (1 or 2 mmHg above ambient pressure) that characterizes fetal apneic periods. During FBMs, phasic abduction of the glottis greatly reduces the resistance to lung liquid efflux via the trachea and, therefore, the liquid leaves the lungs at a greater rate; usually two or three times the rate observed during apnea (Figure 2). Individual FBMs are essentially isovolumetric because the chest wall partially collapses when the diaphragm contracts, giving rise to small changes in thoracic volume with each movement. This is mainly due to the high viscosity of lung liquid, compared with air, which greatly increases the resistance to fluid movement through the fetal airways, particularly at high flow rates. Because the fetal chest wall is very compliant, it is unable to sustain the transpulmonary pressure gradients required to move liquid at flow rates equivalent to the postnatal flow of air. As a result, the tidal volume in the fetus is very small (o0.3 ml kg
1) compared to that of the newborn (7–10 ml kg
1). The transpulmonary pressure gradient is a major determinant of fetal lung liquid volume and is influenced by a number of factors, particularly fetal posture. For example, when intrauterine space is limiting, which can occur in the absence of amniotic fluid (oligohydramnios), the fetus is forced into a flexed position that greatly increases the curvature of the thoracoabdominal spine. This chronically increases abdominal pressure, elevates the diaphragm, and increases the transpulmonary pressure gradient leading to a decrease in lung liquid volume. The resultant decrease in lung expansion is considered to be the primary mechanism for the lung hypoplasia induced by oligohydramnios. Other changes in fetal posture (e.g., stretching), resulting in a decrease in spinal curvature, explain the influxes of lung liquid that are occasionally observed during periods of FBMs. Because fetal body movements most commonly occur during FBMs, reductions in transpulmonary pressure associated with these movements would promote the influx of lung liquid when upper airway resistance is low. Fetal Lung Liquid Clearance at Birth

Fetal lung liquid must be removed rapidly at birth to allow the onset of air breathing, and failure to do so

248 BREATHING / Fetal Lung Liquid Glottis adducted High resistance to liquid efflux

Dilated glottis Low resistance to liquid efflux

0 mmHg

Intraluminal distending pressure 1–2 mmHg

Lung liquid

Intraluminal pressure ∼ 0 mmHg

Lung liquid

Figure 2 Control of fetal lung liquid volumes during periods of apnea and FBMs. During apnea, the glottis is actively adducted, which restricts the efflux of lung liquid and promotes the accumulation within the future airways, thereby maintaining an intraluminal distending pressure of 1 or 2 mmHg above ambient pressure (amniotic sac pressure). During periods of FBMs, the glottis phasically dilates, which greatly reduces the resistance to lung liquid efflux. As a result, the liquid leaves the lungs at a higher rate, causing a reduction in lung liquid volume and the distending pressure (at end-expiration) reduces to ambient pressure.

is a major cause of respiratory morbidity in newborn infants. In particular, it is relatively common in preterm infants and near-term infants delivered by cesarean section in the absence of labor. Thus, the processes responsible for lung liquid clearance at birth mature relatively late in gestation, and labor plays a major role in activating and/or facilitating these processes. One mechanism for lung liquid clearance at birth results from a reversal of the osmotic gradient driving liquid secretion. This process is initiated by a cAMP-mediated activation of amiloride-inhibitable Na þ channels, located on the apical surface of lung epithelial cells, resulting in an increase in Na þ entry into the cell. This increases Na/K ATPase activity, located within the basolateral membrane, resulting in increased Na þ and Na þ -linked Cl
flux across the epithelium, from lung lumen to interstitium. This reverses the osmotic gradient and promotes the uptake of lung liquid into the interstitial tissue, from where it is gradually cleared via the circulation and lymphatics. The increase in intracellular cAMP levels is thought to be due to an epinephrine-mediated increase in b-adrenoceptor activity because epinephrine is a potent stimulator of lung liquid reabsorption in late gestation. The rigors of labor, particularly during delivery of the head, induce a major stress

response within the fetus, leading to a large increase in a number of stress-related hormones, including epinephrine and vasopressin. Because vasopressin can also stimulate lung liquid reabsorption, it is likely that a number of stress-related hormones act through the same pathway. The ability of epinephrine to induce lung liquid reabsorption matures late in gestation and is mediated by the prepartum increase in circulating glucocorticoids (perhaps in synergy with thyroid hormones), which also mature many other aspects of the lung. This explains why preterm infants commonly suffer from liquid retention within the pulmonary airways after birth and explains one of the many beneficial effects that antenatal glucocorticoids, administered to women at risk of preterm labor, have on neonatal respiratory outcome in preterm infants. The maturational effect of glucocorticoids on lung liquid reabsorption involves an increase in the response of epithelial cells to epinephrine as well as an increase in the intracellular machinery responsible for lung liquid secretion/ reabsorption; this includes Na/K ATPase activity as well as Na channel expression. Although liquid uptake across the epithelium is a major mechanism for clearing liquid from the terminal airways, it is unlikely to be the only mechanism.

BREATHING / Fetal Lung Liquid 249

The maximum reabsorption rates (8–10 ml h
1 kg
1) demonstrated experimentally cannot account for the large volume of liquid that must be cleared, particularly because this process does not begin until delivery of the head. Similarly, the measured increase in lung tissue water content at birth cannot account for the total volume of liquid that is cleared from the airways. Although it was once assumed that lung liquid was forced from the airways due to compression of the chest as it passed through the birth canal, this theory has been discounted because the head and shoulders offer the widest obstacle for human delivery. However, because transpulmonary pressure is a major factor regulating the volume of fetal lung liquid, the effects of labor on fetal posture are likely to significantly contribute to liquid loss from the airways. Reduced amniotic fluid volumes, particularly following rupture of the membranes, as well as uterine contractions during labor, are known to cause posture-related increases in transpulmonary pressure and lung liquid loss. This may explain the large loss of liquid that occurs after labor has begun but before (by many hours) second-stage labor commences and delivery of the head begins (i.e., when stress-related hormones are released).

The Role of Fetal Lung Liquid in Development and Disease Fetal lung liquid plays a major role in the growth and development of the fetal lung by acting as an internal splint that maintains the lungs in an expanded state. The luminal volume of the fetal lung is higher than the end-expiratory lung volume (functional residual capacity) of the postnatal air-filled lung. The decrease in resting lung volume at birth (by 5–15 ml kg
1) is due to the removal of the distending influence of lung liquid in addition to the entry of air into the airways. The latter causes an air/liquid interface to form, which creates surface tension and, despite the presence of surfactant, increases lung recoil. This causes partial collapse of the lung and the formation of a subatmospheric intrapleural pressure after birth, which does not exist before birth. During fetal life, the distending influence of the lung liquid provides a stretch stimulus that is essential for lung growth and determines the three-dimensional tissue structure of the lung as well as the differentiated state of alveolar epithelial cells. If the distending influence of lung liquid is removed, growth and structural development of the lung ceases, particularly that of the terminal air sacs, and alveolar epithelial cells differentiate into the surfactant secreting type II cell phenotype. On the

other hand, overdistension of the fetal lungs induces a rapid increase in lung growth that can result in a near doubling in lung cell number within 7 days. Similarly, structural development accelerates and alveolar epithelial cells differentiate into type I cells following prolonged overdistension, which can reduce the proportion of type II alveolar epithelial cells to o2% of all alveolar epithelial cells. Because lung growth and development are very sensitive to the degree of fetal lung expansion, it is not surprising that most instances of fetal lung hypoplasia in humans are caused by conditions that reduce fetal lung expansion. These conditions include oligohydraminos, whether it is induced by premature rupture of the membranes or urinary tract abnormalities (which reduce or prevent the flow of fetal urine into the amniotic sac), congenital diaphragmatic hernia, or other space-occupying thoracic lesions (e.g., tumors and cysts). As indicated previously, oligohydramnios causes an increase in fetal trunk flexion, which increases the transpulmonary pressure gradient and reduces the degree of lung expansion. Herniation of the diaphragm allows abdominal contents to migrate into the fetal thorax, which acts as a space-occupying lesion that prevents lung expansion. In both conditions, the reduction in lung growth can be so severe that, after birth, the resulting respiratory insufficiency is fatal. The presence of lung liquid within the fetal airways may also affect the pulmonary circulation. Studies indicate that the distending influence of lung liquid, and the small hydrostatic pressure it generates, may contribute to the high pulmonary vascular resistance (PVR) in the fetus. As a result of the high PVR, B88% of right ventricular output bypasses the fetal lungs and enters the systemic circulation via the ductus arteriosus, which connects the main pulmonary artery with the descending aorta. With the clearance of lung liquid and the onset of ventilation at birth, PVR markedly decreases, allowing a rapid increase in pulmonary blood flow, to accept the entire output of the right ventricle, and the ductus arteriosus to close. The mechanisms responsible for the high PVR near term and the sudden decrease at birth are multifactorial. However, evidence indicates that the small hydrostatic distending pressure, caused by the retention of liquid within the future airways, may be a major contributing factor by causing compression and closure of perialveolar capillaries. Loss of this distending pressure at birth and the increase in lung recoil may contribute to the rapid decline in PVR when air first enters the lungs. See also: Breathing: Breathing in the Newborn; Fetal Breathing; First Breath. Lung Development: Overview.

250 BREATHING / First Breath

Further Reading Barker PM and Olver RE (2002) Invited review: clearance of lung liquid during the perinatal period. Journal of Applied Physiology 93: 1542–1548. Bland RD and Nielson DW (1992) Developmental changes in lung epithelial ion transport and liquid movement. Annual Review of Physiology 54: 373–394. Harding R and Hooper SB (1996) Regulation of lung expansion and lung growth before birth. Journal of Applied Physiology 81: 209–224. Harding R and Hooper SB (2004) Physiologic mechanisms of normal and altered lung growth. In: Polin RA, Fox WW, and Abman SH (eds.) Fetal and Neonatal Physiology, pp. 802–811. Philadelphia: Saunders. Hooper SB and Harding R (1995) Fetal lung liquid: a major determinant of the growth and functional development of the fetal lung. Clinical and Experimental Pharmacology and Physiology 22: 235–247. Olver RE, Walters DV, and Wilson M (2004) Developmental regulation of lung liquid transport. Annual Review of Physiology 66: 77–101.

attempts to separate the first breath from the breathing-like activities preceding birth. In fact, it is wellknown that the fetus presents intermittent breathing from at least the second trimester of gestation (see Breathing: Fetal Breathing). Breathing-like movements in utero are not for gas exchange, which is provided by the placenta, but are important in controlling the pulmonary fluid and, with it, fetal lung growth. In egg-laying animals such as birds, the definition of first breath is blurry because, even before hatching, breathing contributes to gas exchange, in conjunction with that provided by the chorioallantoic membrane. Small marsupials are another special case. In fact, these newborn mammals do not need to ventilate the lungs at birth because gas exchange occurs by diffusion through their skin. Hence, in these neonates, breathing acts occur sparsely, seemingly randomly, for several days after birth.

Physiological Processes

First Breath

Mechanics of the Onset of Breathing

J P Mortola, McGill University, Montreal, QC, Canada

The fetal lung is filled by fluid of its own production. At birth, some of the fluid is squeezed out of the upper airways during the passage through the pelvic canal. However, this mechanism is not crucial because differences in the mode of delivery (by breech or head, by cesarean section or vaginal) have no major impact on the cardiopulmonary adjustments at birth. Indeed, in many species with large litters, the mode of delivery alternates among pups, and in marine mammals delivery by tail first is the norm. What is important, though, is the occurrence of labor because the hormonal surge accompanying this phase of delivery is responsible for the switch from the prenatal production of the fetal pulmonary fluid to its postnatal absorption. Quantitative analyses of the mechanical events accompanying the first breath are based on observations and measurements performed in infants and, to a lesser extent, in lambs; very little information is available on other species. The first inspiration does not accomplish an immediate and homogeneous aeration of the lungs. In fact, although some alveolar areas pop open, many others will be ventilated only several minutes or even hours later. The work that the inspiratory muscles need to produce to expand the lungs with air is quite high, probably 8–10 times more than the respiratory work required during quiet breathing at older ages or in adults. The distortion of the thorax during inspiration, favored by the high compliance of the chest wall, is an additional factor that increases the total work of the inspiratory muscles.

& 2006 Elsevier Ltd. All rights reserved.

Abstract In mammals and other vertebrates, intermittent breathing activities originate early during development; however, the term ‘first breath’ is used to indicate the first breathing act of a new independent organism. Usually, this coincides with the establishment of continuous pulmonary ventilation, although this is not always the case. What triggers the onset of regular breathing at birth remains an unsolved question. The general state of stress, created by all the new stimuli and events surrounding the time of birth, and the sudden increase in oxygenation, raises metabolic rate. The gaseous component of metabolism could be the common mechanism for the establishment of a sustained ventilation after birth. The respiratory work of the first inspiration is high because of the friction of the column of pulmonary fluid as it is displaced toward the peripheral airways. The formation of the air–liquid interface generates surface pressure, which is the primary determinant of the recoil pressure of the lungs. Lung expansion with air and the rise in oxygenation are the main factors responsible for the changes in pulmonary circulation at birth. The early breathing pattern is irregular, and the expiratory flows are briefly interrupted by closure of the vocal folds. This mechanism raises intra-airway pressure and contributes to the clearing of the pulmonary fluid.

Description First breath indicates the first breathing act of a new independent organism. When applied to mammals, it refers to the first active muscle effort aiming to pump air into the lungs at birth. The specifications ‘independent organism’ and ‘at birth’ are semantic

BREATHING / First Breath 251

Lung volume (ml) A few days

P ∝
/r

100

· P ∝ V ·R

50 First breath

−40

−30

−20

−10

0

10

20

30

40

Pleural pressure (cmH2O) Figure 1 Schematic representation of the changes in pleural pressure and lung volume during the first breath and during some of the following breaths (dashed lines) until a few days. The arrows indicate the direction of the loop, and the areas of the loop are an approximate indication of the external work of the respiratory muscles. (Left) The silhouette of the lungs indicates that during the first breath (bottom) of the total pressure (P ) required to generate flow (V ), the largest fraction is to overcome airflow resistance (R ). Later (top), the largest component of the inspiratory pressure is due to surface tension (g ) and the radius of curvature (r ) of the air–liquid interface.

The total inspiratory pressure is mainly contributed by the airflow resistance and by the surface pressure (Figure 1). This latter is determined by the product of the surface tension at the air–liquid interface and the radius of curvature of the interface. In the liquid-filled lung before the first breath, because there is no interface, the surface pressure is nil. The interface forms with the entrance of air during the first inspiration, and as the air progresses toward the small peripheral airways, the surface pressure continues to rise because the curvature of the interface continues to decrease. Eventually, after the first few breaths, the pressure determined by surface forces will be the primary reason for the tendency of the lungs to deflate. Surfactants produced by specialized cells of the alveolar epithelium play a fundamental role in lowering the surface tension and the propensity of the lungs to deflate. This reduction in lung recoil pressure is important for the progressive establishment of the end expiratory reserve of air, or functional residual capacity (see Surfactant: Overview). Indeed, the first breath contributes 10–20% to what will be the functional residual capacity in an infant a few days old. The airflow resistance of the first inspiration is high because of the viscosity of the column of pulmonary fluid. The incoming air displaces this fluid toward the lung peripheral airways. Eventually, the fluid will pass in the pulmonary interstitium, where it will be cleared by the pulmonary circulation and, to

a lesser extent, the lymphatics. The process of clearing the airways from the fluid takes a few hours in human infants and probably a longer period of time in larger species. During this time, air and fluid mix with the formation of foam, a process favored by the presence of surfactant. The air trapped in the pulmonary liquid in the form of foam contributes to gas exchange, and this explains why blood oxygenation rises rapidly in the minutes after birth, despite the much longer time required for the clearance of the fluid. Once the airways are liquid-free, the pressure to overcome airflow resistance is small. This is the main reason for the large decrease in the work of breathing of the following breaths compared to the first breath. Theoretically, at birth several mechanisms may help the newborn’s respiratory muscles in generating the large pressures required for the first breathing acts. For example, the contraction of the upper airway muscles, such as during sucking, or the outward recoil of the chest during recovery after the squeeze through the pelvic passage can produce subatmospheric pressures. Also, the erection of the pulmonary capillaries with the rise in pulmonary blood flow may contribute some negative (and therefore inspiratory) pressure within the airways. In reality, when subjected to experimental scrutiny, none of these mechanisms have demonstrated an appreciable contribution to the pressure required for the first inspirations.

252 BREATHING / First Breath Changes in Pulmonary Circulation

Birth is accompanied by dramatic vascular changes. The lung shifts from receiving less than 10% of the cardiac output during fetal life to receiving practically all of it after birth. To a great extent, this change is due to the closure of the ductus arteriosus, which in fetal life permits a large fraction of the blood of the pulmonary trunk to bypass the pulmonary circulation, and to the drop in resistance of the pulmonary vessels (Figure 2). Because the ductus arteriosus enters the aortic arch, its postnatal closure reduces blood-flow to the descending aorta, in favor of the upper body and brain. The first breath and the onset of continuous ventilation play a crucial role in the cardiovascular changes accompanying birth, mostly because of two mechanisms – lung expansion and oxygenation. The increase in lung volume, by itself and independent from the rise in oxygen pressure, increases pulmonary blood-flow approximately fivefold, according to animal experiments. This may seem astonishing given that lung volume does not differ much before and after the first breath; in fact, the volume of the fetal liquid-filled lung is not lower, and most probably larger, than that of the air-filled postnatal lung. However, after birth, the surface tension created at the air–liquid interface reduces the pressure in the

lung interstitial tissue, which promotes vascular expansion and a decrease in pulmonary vascular resistance. Lung ventilation also promotes the secretion of pulmonary prostaglandins, with vasodilating effects. The importance of the rise in oxygen levels in lowering pulmonary vascular resistance has been appreciated for a long time. Sustained hypoxia delays this process, leading to right ventricular hypertrophy. It also seems that the respiratory alkalosis accompanying the onset and establishment of pulmonary ventilation contributes to the vascular adjustments at birth. The Early Breathing Pattern

Despite the mechanical constraints, in infants the first inspiration is approximately 40 ml deep – one of the deepest of the first few days (Figure 1). It is not clear why this is the case. Among the various possibilities is a change in the threshold of the mechanisms normally controlling tidal volume, notably the airway slowly adapting stretch receptors and the central integration of their inputs. Although the activity of these receptors is related to changes in lung volume, their stimulus is transpulmonary pressure. In utero, the lungs are liquid-filled; hence, the transpulmonary pressure is low because of the absence of surface forces. At birth, with the entrance of air,

Fetus

Newborn

Aorta

Aorta

Ductus arteriosus × 0.05 Pulmonary trunk

Right ventricle

Pulmonary ×7

Left ventricle

Right ventricle

Lungs

Lungs

Left atrium Right atrium Foramen ovale

Left ventricle

Left atrium

Descending aorta × 0.5

Figure 2 Schematic representations of the main features of cardiopulmonary circulation before birth (left) and after birth (right). Numbers indicate the approximate changes in blood flow occurring at birth as the result of lung expansion and oxygenation.

surface forces immediately increase transpulmonary pressure. Therefore, intrapulmonary airway receptors may have to reset to the new mechanical condition, and in the process, the control of tidal volume may not be as accurate as it will be in the following breaths. Peripheral chemoreceptors are also known to undergo resetting with the abrupt change in stimulus. In the case of peripheral chemoreceptors, the abrupt change at birth is caused by the rise in arterial oxygenation with the onset of ventilation. Another set of airway receptors, the rapidly adapting ones, may be involved in the deep inspiration of the first breath. These receptors are normally activated by a sudden lung stretch, and their reflex effect (called Head’s reflex) is that of promoting the activity of the inspiratory muscles. Hence, the increase in transpulmonary pressure with the first air expansion may provoke this reflex, resulting in further activation of the inspiratory muscles. During the minutes following birth, the breathing pattern is very irregular, and short apneas are interspersed with bursts of high-frequency and shallow breathing. Tachypnea, with average rates between 70 and 90 breaths per minute, has been observed after both vaginal and cesarean section deliveries, and it usually subsides within a few hours. In infants who have difficulty clearing pulmonary fluid, transient tachypnea can last much longer. It seems likely that the high-frequency pattern is the reflex response resulting from the stimulation of a group of receptors located in the pulmonary interstitium. These receptors are known to cause rapid and shallow breathing when activated by an increase in pulmonary interstitial pressure, such as during lung congestion. After the first breath, and for much of the following hours, during expiration the flow is decreased, and often briefly interrupted, by the narrowing of the vocal folds. Some widening and narrowing of the glottis with inspiration and expiration, respectively, are normal events during breathing. However, in the neonatal period narrowing is exaggerated, and in the first minutes after birth interruptions of expiration at various times are common (Figure 3). This expiratory pattern, or grunting, is controlled by neural information from the lungs and serves important purposes. By delaying expiration, it maintains the average air volume within the lungs higher than it would be with unobstructed expiration, and it raises the end expiratory level. Because it increases the intra-airway pressure, grunting contributes to drainage of pulmonary fluid from the peripheral airways into the lung interstitium, where it is picked up by the lymphatic circulation. This mechanism can be effective because the rise in intra-airway pressure is

Tidal volume inspiration

BREATHING / First Breath 253

Obstructed expirations

Unobstructed expiration

End-expiratory volume

Figure 3 Schematic representation of a breath with unobstructed expiration (blue) and of several types of obstructed expirations (red). In the unobstructed condition, the lung volume decreases during expiration along a quasi-exponential trajectory. In case of obstruction, the expiratory trajectory is distorted, lung volume takes longer to decrease, and the end expiratory volume rises.

small. If airway pressure reached higher values, as can occur during crying or other forced expiratory maneuvers or during assisted positive airway pressure ventilation, it would hinder the pulmonary circulation and aggravate gas exchange. What Triggers the Onset of Regular Breathing at Birth?

This question is still unanswered. Once outside the shielded intrauterine environment, the newborn is bombarded by many stimuli – visual, acoustic, thermal, tactile, and pressure – either totally new or of unusual intensity. Also, neural and chemical information present before birth assumes a new dimension after birth. For example, as air enters the lungs, the increase in transpulmonary pressure brings the airway slowly adapting receptors to a new level of activity. The arterial chemoreceptors, sensing a major increase in the partial pressure of oxygen, decrease or cease their prenatal activity; it will take a few days before they reset to the new oxygen level. The prenatal surge of many antioxidant enzymes testifies to the uniqueness of birth as a hyperoxic event. The relative contribution of so many stimuli and new conditions to the onset of ventilation and the establishment of a steady pattern is difficult to quantify. Various experimental approaches have emphasized or dismissed one or the other mechanism. It is worth noting that at birth, as a result of the multiple stimuli and of the general state of stress, the newborn’s metabolic rate rises much higher than the fetal value. Also, the level of the metabolically produced carbon dioxide increases, which may play a fundamental role in the maintenance of pulmonary ventilation. In animal fetuses exteriorized and kept alive with extracorporeal circulation to provide for their gas exchange, continuous breathing did not

254 BREATHING / First Breath

Birth

2nd

1st breath

3rd 4th 5th 6th

Metabolic level

Thermal, mechanical and sensory stimuli, neural and chemical inputs

Figure 4 Many new stimuli are produced during the events surrounding birth. Either individually or by creating a general state of stress, they increase the metabolic level, which may represent the common mechanism for the maintenance of a continuous breathing pattern. Sensory stimuli include tactile, pressure, and pain stimuli. Neural inputs refer to the facilitation originating from the airway rapidly adapting receptors, as well as to neurochemical substances that may be increased within the brainstem as a result of air-breathing. Chemical inputs refer to the putative role of the chemoreceptors and to the state of relative hyperoxia. The spirometric recording at the top is from a newborn infant delivered by cesarean section. Time bars of 1 s are plotted along the zero volume line. Starting from the first breath, the amount of air exhaled is less than the amount inhaled – a surfactant-dependent process that forms the functional residual capacity.

start even after umbilical cord occlusion as long as carbon dioxide was not allowed to rise. Hence, it is possible that the many stimuli thought to be involved in the onset of breathing do so, ultimately, by favoring the increase in metabolic rate (Figure 4). Also, the relative hyperoxic condition accompanying birth may cause increased metabolism since hyperoxia has a hypermetabolic effect in young animals. Metabolic rate plays a key role in controlling the level of pulmonary ventilation in many circumstances, ranging from muscle exercise to responses to cold or hypoxia. Therefore, its crucial involvement in the maintenance of continuous respiration at birth would not be a special case. Contrary to the rather abrupt onset of continuous breathing observed at birth in the majority of mammals, in birds and other egg-laying animals breathing commences gradually. At first, breathing is intermittent, and it becomes more regular and continuous during the many hours of the hatching process, in parallel with the progressive decline of the gas exchange function of the chorioallantoic membrane. This process is similar to that observed in some small marsupials, in which pulmonary ventilation becomes continuous over a period of several days, whereas gas exchange through the skin gradually regresses. These examples support the view that the establishment of continuous ventilation at birth is not necessarily an event timed with a particular stage of development. Rather, it may be so closely linked to gaseous

metabolism that as long as extrapulmonary organs take care of gas exchange, pulmonary ventilation does not need to become a continuous process. See also: Breathing: Breathing in the Newborn; Fetal Breathing. Lung Development: Overview. Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants. Pulmonary Circulation. Signs of Respiratory Disease: Breathing Patterns. Surfactant: Overview.

Further Reading Bland RD (1997) Fetal lung liquid and its removal near birth. In: Crystals RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 2115–2127. Philadelphia: Lippincott-Raven. Hodson WA (1997) The first breath. In: Crystals RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 2105–2114. Philadelphia: LippincottRaven. Lagercrantz H and Slotkin TA (1986) The ‘stress’ of being born. Scientific American 254: 100–107. Mortola JP (2001) Respiratory Physiology of Newborn Mammals. A Comparative Perspective. Baltimore: Johns Hopkins University Press. Strang LB (1991) Fetal lung liquid: secretion and reabsorption. Physiology Review 71: 991–1016. Tod ML and Cassin S (1997) Fetal and neonatal pulmonary circulation. In: Crystals RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 2129– 2139. Philadelphia: Lippincott-Raven.

BRONCHIAL CIRCULATION 255

BRONCHIAL CIRCULATION E M Wagner, Johns Hopkins Asthma and Allergy Center, Baltimore, MD, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The bronchial circulation is the systemic vascular supply to the lung, and it supplies blood to conducting airways down to the level of the terminal bronchioles as well as nerves, lymph nodes, visceral pleura, and the walls of large pulmonary vessels. Within the airway wall, the circulation is composed of parallel vascular plexuses adjacent to airway smooth muscle. The density of this vascular network predicts its role in the clearance of aerosols delivered to the airway mucosa. As in other systemic vascular beds, inflammatory cells are recruited to the airway wall through postcapillary venules. Inflammatory proteins are largely vasodilatory in their effect on bronchial vascular smooth muscle tone. Most vasodilatory action is mediated at least partially through endothelial cell-derived nitric oxide. Inflammatory proteins play a major role in the loss of bronchial endothelial barrier function causing airway wall edema, as demonstrated in conditions of asthma and allergy. The most prominent pathologic feature of the bronchial circulation is its proliferative capacity. Unlike the pulmonary vasculature, the bronchial circulation is pro-angiogenic in asthma, chronic pulmonary thromboembolism, interstitial pulmonary fibrosis, cystic fibrosis, and other inflammatory conditions. Although the recruitment of new bronchial vessels to ischemic lung parenchyma may prove beneficial, hemoptysis resulting from rupture of abnormal, bronchial vessels can be life-threatening.

Anatomy, Histology, and Structure The variability in origin and number of systemic arteries perfusing the airways both among and within species has contributed to the difficulty in study and the relative paucity of information concerning the physiological function of the bronchial vasculature. Leonardo da Vinci is frequently credited with providing the first anatomical drawings of the bronchial circulation. Although he understood the need for this circulation as described in his notebooks that ‘‘nature gave a vein and an artery to the trachea which would be sufficient for its life and nourishment,’’ reproduction of his efforts demonstrated that his drawings were those of pulmonary veins. The first complete anatomical drawings of the bronchial circulation in 1721 should be credited to the Dutch anatomist Frederich Ruysch. Since that time, the anatomical arrangement of the bronchial circulation has been described for humans, sheep, pigs, dogs, opossum, rabbits, and several small rodents. The bronchial vasculature originates from the aorta, subclavian, or

intercostal arteries (Figure 1). The bronchial artery courses to the dorsal aspect of the carina, where it bifurcates and sends branches down the mainstem bronchi. This vascular bed perfuses the airways from the level of the carina to the terminal bronchioles. The bronchial arteries send arterioles throughout the airway adventitia that perfuse capillaries that are prominent in both the adventitia and the mucosa of the airway wall. Thus, the bronchial vasculature forms parallel vascular plexuses, situated on either side of the airway smooth muscle. In extraparenchymal airways, bronchial venules drain into bronchial veins that subsequently drain into the right heart. In the intraparenchymal airways, postcapillary venules collect bronchial venous drainage into pulmonary venules and/or alveolar capillaries that drain into pulmonary veins and the left atrium. Bronchial anastomoses with pulmonary precapillary vessels have also been reported. Additionally, the bronchial artery sends branches to large pulmonary vessels as vasa vasorum, nerves, lymph nodes, and the visceral pleura. Values reported for absolute bronchial blood flow vary according to the method used to measure flow and the species evaluated. However, all reports demonstrate that flow through the bronchial vasculature represents less than 3% of cardiac output. The vasculature of the airway wall, which constitutes the major perfusion pathway, is tortuous and forms a dense network of vessels. Morphometry of postmortem or resected lung specimens from normal human subjects showed that blood vessels of the mucosa comprise o1% of the airway wall area and within the advential area approximately 8% of the wall area. Subjects with inflammatory airways disease show a significant increase in the overall number of airway vessels. However, several studies have suggested that the increase is proportionate to the increase in wall area. Few studies of the embryonic development of the bronchial vasculature exist. In humans, between weeks 9 and 12 of gestation, the bronchial artery arises as an outgrowth from the aorta. This process occurs later than the development of the pulmonary vasculature. One or two vessels extend from the dorsal aorta and form along the cartilaginous plates of the large airways. The vessels develop longitudinally along the airways to the lung periphery as far as the terminal bronchioles. However, the process by which the parallel vascular plexuses interdigitate and proliferate on either side of airway smooth

256 BRONCHIAL CIRCULATION

Aortic arch Bronchial artery

Carina

Descending aorta

Figure 1 The bronchial artery originates from the aorta and courses to the dorsal aspect of the carina, where it bifurcates and sends branches down the mainstem bronchi. This vascular bed perfuses the airways from the level of the carina to the terminal bronchioles. In extraparenchymal airways, bronchial venules drain into bronchial veins that subsequently drain into the right heart. In the intraparenchymal airways, postcapillary venules collect bronchial venous drainage into pulmonary venules and/or alveolar capillaries that drain into pulmonary veins and the left atrium. Additionally, the bronchial artery sends branches to the pulmonary artery as vasa vasorum, nerves, lymph nodes, and the visceral pleura.

muscle has not been studied. Whether the fine capillary networks within the airway wall, the vasa vasorum within large pulmonary vessel walls, or vessels anastomosing with pulmonary vessels form as a continual outgrowth of arterial sprouting or as de novo formation from mesoderm is unknown.

Table 1 Methods to measure bronchial blood flow Method

Species

Flow probe Microspheres Artery cannulated/perfused Isolated vascular pouch Anastomotic blood collection Inert gas

Sheep Dog, sheep Sheep Sheep Dog, sheep Human

Bronchial Circulation in Normal Lung Function Given the separate structures within the lung that are perfused by branches of the bronchial vasculature, it is likely that the bronchial circulation regulates and supports diverse physiologic functions. However, due to the difficulty in accessing this vascular bed in most species, its complex anatomy within the lung, and its small proportion of blood flow relative to pulmonary flow, the role of the bronchial circulation in supporting airway and lung function is incompletely described. Most published studies have focused on characterizing regulators of bronchial blood flow, with few directed at understanding function

(Tables 1 and 2). However, several specific physiological functions have been defined and supported by experimental evidence, including that of airway cell nutrition, clearance of inhaled substances, recruitment of inflammatory cells, and conditioning inspired air. The evidence for each of these functions is presented in the following sections. Airway Cell Nutrition

Although all vascular beds exist to support the metabolic needs of the tissue perfused, assessment of

BRONCHIAL CIRCULATION 257 Table 2 Bronchial blood flow responses Vasoconstrictors

Vasodilators

a-Adrenergic agonists Endothelin Glucocorticoids Increased airway pressure Increased left atrial pressure Vasopressin

b-Adrenergic agonists Adenosine Antigen Bradykinin Histamine Hypercarbia Hypoxia Nitric oxide Prostanoids

cellular function during acute changes in perfusion can provide suggestive evidence for the importance of maintaining a critical level of blood flow. Within the airway wall, it is questionable whether oxygen delivery by the blood is necessary given the proximity to airway luminal oxygen. In animal models, bronchial vascular smooth muscle demonstrated significant reduction in tone when exposed to hypoxemia and hypercarbia. Additionally, when the sheep bronchial artery was perfused with an artificial perfusate without calcium, a prompt reduction in airway smooth muscle tone was noted. In an effort to determine the importance of bronchial perfusion on mucociliary function in sheep, clearance of an insoluble tracer (99mTc-sulfur colloid) was determined. When bronchial blood flow was substantially reduced from a normal level, mucociliary activity was significantly slowed. Whether this response was due to changes in mucus gland production, ciliary function, or neural control mechanisms, each could be related to alterations in substrate delivery to specific cells of the airway wall supplied by the bronchial artery. These studies demonstrate the importance of normal bronchial perfusion to maintain diverse cell function within the airway. Clearance

The normal function of any vascular bed is to support the removal of excess metabolites from a tissue. In addition, the bronchial circulation is juxtaposed between the airway epithelial barrier to the external environment of inhaled substances and the internal environment of the bloodstream. This circulation could be important in the systemic uptake of soluble, inhaled substances. Studies have shown that both large increases or decreases in bronchial blood flow decreased the blood uptake of a soluble tracer (99mTc-DTPA) deposited on airway epithelial surface. Subsequent work demonstrated that increased flow accompanied by increased pressures caused a hydrostatic edema that limited soluble tracer uptake.

These studies demonstrate the complexity of the interaction between aerosol uptake and the underlying airway vasculature. Vasomotor and endothelial barrier changes of the airway vasculature can impact aerosol uptake of both therapeutic and toxicological substances. Several studies have shown that the level of blood flow through the bronchial circulation can affect both the magnitude and the time course of agonistinduced airway smooth muscle constriction by contributing to the passive washout of delivered agonist. Furthermore, it is interesting to note that most airway smooth muscle agonists cause bronchial vasodilation, thus suggesting a homeostatic mechanism for preserving normal airway smooth muscle tone. Another aspect of clearance by this vasculature is confirmed by the study of the metabolic capacity of the bronchial endothelium. Angiotensin-converting enzyme (ACE) inhibitor significantly depressed metabolism of a synthetic peptide substrate for ACE in an in situ perfused bronchial preparation. This study concluded that the bronchial circulation is pharmacokinetically and metabolically active with respect to bradykinin and that the enzymes responsible for this metabolic activity line the vascular lumen. Recent work confirmed the importance of ACE activity in vivo by demonstrating inhibition of bradykinin vasodilation after treatment with an ACE inhibitor. This result further suggests an important regulatory role for bronchial endothelial ACE in the metabolism of kinin peptides known to contribute to airway pathology. Each aspect of clearance, from inhaled substances removed from an active site causing physiologic changes to endothelial metabolism, suggests an important, albeit incompletely characterized, function. Recruitment of Inflammatory Cells

Recruitment of leukocytes to the airway provides an important defense function due to their antimicrobial, secretory, and phagocytic functions. Leukocyte recruitment in systemic organs involves an orchestrated series of molecular events between rolling leukocytes and postcapillary venular endothelium. Since the tracheal/bronchial circulations are systemic circulatory beds, the same endothelial cell adhesion molecules likely are responsible for the welldocumented leukocyte recruitment of inflammatory airways diseases. Although this is generally assumed, only recently have studies using intravital microscopy to document specific molecules and mechanisms of recruitment in vivo been performed in airways. Research suggests that ventilatory stresses imposed on airway endothelium may exert

258 BRONCHIAL CIRCULATION

additional cell stimulation that is not predicted by static endothelial cell culture conditions, thus confirming the molecules and mechanisms responsible for specific leukocyte recruitment in models that are relevant for airways disease appear to be essential. Conditioning Inspired Air

Typically ascribed to the airway circulation is the function of heating and humidifying inspired air. Both the tracheal and the bronchial circulations have been shown to dilate when healthy human subjects or animals are challenged with dry air hyperventilation. However, whether the airway circulation contributes significantly to heating and humidification has been debated because the pulmonary circulation provides a huge heat sink. In experimental systems in which bronchial perfusion could be limited, little effect on exhaled temperatures or on airway obstruction was observed. However, measurements of water exchange demonstrated that the bronchial vasculature served an important role in hydrating the airway mucosal surface and interstitial compartments of peribronchial tissues and limited the degree of airway mucosal injury after hyperpnea. Thus, it appears that the airway circulation is important in preserving normal humidification of airway cells.

Bronchial Circulation in Respiratory Diseases Proliferation

The unique proliferative capacity of the bronchial circulation compared to the pulmonary vasculature in a variety of lung diseases has long been recognized. Chronic inflammatory conditions and chronic pulmonary thromboembolism are the two most cited conditions leading to bronchial vascular proliferation. Inflammation can contribute significantly to bronchial vascular remodeling and life-threatening hemoptysis. Hemoptysis in the vast majority of patients originates from the systemic rather than the pulmonary vasculature, and the bronchial vessels are almost universally involved. Techniques for identifying and embolizing bronchial vessels have been well described. However, the etiology of hemoptysis requiring therapeutic embolization is variable, and the pathology of bronchial vessels leading to this acute condition is poorly understood. Several studies have focused on the chronic inflammation of asthma and airway vascular remodeling. Li and Wilson demonstrated increased vascular density in biopsy specimens of subjects with mild asthma compared to normal control volunteers. However, others have shown that only biopsy specimens from

asthmatic subjects with concurrent Mycoplasma pneumoniae infections demonstrated significantly increased vessel numbers. In studies of alterations in airway vascularity in models of inflammation, rodent models have shown increased vessel numbers, size, and permeability characteristics. Airway remodeling following Mycoplasma pulmonis infection showed sustained alterations in the tracheal vasculature that could be reversed with corticosteroid treatment. Additionally, tracheal vessels of rats with chronic M. pulmonis infection demonstrated significantly increased permeability when challenged with substance P. These results suggest abnormal endothelial cell barrier function after inflammationinduced proliferation. Perhaps the most extensively studied form of systemic vascular proliferation within the lung is that which occurs after pulmonary artery embolization. In 1847, Virchow recognized that the bronchial circulation could proliferate and sustain lung tissue distal to a pulmonary embolism. Bronchial arteriograms in patients with chronic thromboembolic disease demonstrate the unique capacity of systemic vessels to proliferate and to invade the ischemic lung parenchyma. Both a dilated bronchial artery and a fine meshwork of vessels distal to the pulmonary occlusion can be seen. Neovascularization of the systemic circulation into the lung after pulmonary artery obstruction has been confirmed and studied in humans, sheep, dog, pig, guinea pig, rat, and mouse. Systemic blood flow to the lung has been shown to increase as much as 30% compared to the original pulmonary blood flow after pulmonary artery occlusion. Although precise mechanisms of vascular proliferation are still under investigation, in a model of chronic pulmonary thromboembolism in the mouse, the pro-angiogenic CXC chemokines appear to play a major role in vascular proliferation. This observation further supports a growing body of evidence, reported by Strieter and colleagues, from studies of non-small cell lung cancer and idiopathic pulmonary fibrosis that the ELR þ CXC chemokines are important pro-angiogenic factors within the lung. Loss of Endothelial Barrier Function

Numerous studies on a variety of models, as well as autopsy specimens of human lungs, demonstrate the propensity for the systemic airway circulation to contribute to fluid accumulation within and around airways. In particular, inflammatory cytokines have been shown to cause the postcapillary venular endothelium to form gaps and allow transudation of plasma and protein into the interstitium. Changes in airway wall geometry due to fluid and inflammatory

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cell accumulation and vasodilation of the bronchial vasculature have contributed to a geometric theory of asthma. Whether mechanisms are related to physical encroachment on small airway lumen or the loss of parenchymal tethering, inflammationinduced changes involving bronchial vascular fluid exudates likely contribute to the pathology of asthma. Another means by which the bronchial circulation can alter barrier function within the lung is through a portal-like response. Several studies have demonstrated that inhalation of injurious chemicals can result in loss of pulmonary endothelial barrier function. Pulmonary vascular leak due to smoke inhalation has been shown to be attenuated when the bronchial vasculature is occluded. Authors of separate studies concluded that inflammatory proteins released within the large airways after smoke inhalation are carried by the bronchial vasculature to pulmonary tissue, where they impact pulmonary endothelial barrier function. However, these results are in contrast to those of Pearse and colleagues, who showed that bronchial perfusion attenuated the loss of pulmonary endothelial barrier function in ischemia–reperfusion. These authors suggested that bronchial endothelial-derived nitric oxide helps to preserve pulmonary barrier function. However, in each of these models, the bronchial vasculature serves as a conduit to transport substances to the pulmonary circulation where function is altered.

Bronchial Infections

See also: Angiogenesis, Angiogenic Growth Factors and Development Factors. Asthma: Exercise-Induced. Bronchodilators: Beta Agonists. Chemokines, CXC: IL-8. Cilia and Mucociliary Clearance. Endothelial Cells and Endothelium. Fluid Balance in the Lung. Leukocytes: Neutrophils. Lung Development: Overview. Neonatal Circulation. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts.

Further Reading Butler J (ed.) (1992) The bronchial circulation. In: Lung Biology in Health and Disease. New York: Dekker. Cudkowicz L (1968) The Human Bronchial Circulation in Health and Disease. Baltimore: Williams & Wilkins. Deffebach ME, Charan NB, Lakshminarayan S, and Butler J (1987) The bronchial circulation – small, but a vital attribute of the lung. American Review of Respiratory Disease 135: 463–481. Wagner EM (1995) The role of the tracheobronchial circulation in aerosol clearance. Journal of Aerosol Medicine 8(1): 1–5. Wagner EM (1997) Bronchial circulation. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundations, pp. 1093–1105. New York: Lippincott–Raven. Wagner EM (1997) Bronchial vessels. In: Barnes PJ, Grunstein MM, Leff AR, and Woolcock AJ (eds.) Asthma. New York: Lippincott–Raven. Wanner A (1989) Circulation of the airway mucosa. Journal of Applied Physiology 67: 917–925. Widdicombe J (ed.) (2003) Archives of Physiology and Biochemistry. Eighth International Meeting of the DaVinci Society, Nimes, France, pp. 287–368.

see Bronchiectasis. Bronchiolitis. Panbronchiolitis.

BRONCHIECTASIS R Wilson, Royal Brompton and Harefield NHS Trust, London, UK R Boyton, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Bronchiectasis is abnormal chronic dilatation of one or more bronchi due to loss of structural elements in the bronchial wall. There are a number of known causes and several associated conditions, but the underlying cause is not known in many cases. The prevalence is unknown, but thin-section computed tomography has increased its recognition. Symptoms are of chronic productive cough, breathlessness, recurrent exacerbations usually provoked by infection, and tiredness. There may be crackles over affected areas but not in all cases, and there may be wheezes due to airflow obstruction that is largely irreversible. Subsegmental

airways are permanently dilated, tortuous, and contain excess secretions. Characteristically, the elastin layer of the wall is deficient, and muscle and cartilage show signs of destruction. Increased mucus production and impaired clearance predispose to bacterial infection, which stimulates chronic inflammation in which lymphocytes predominate within the wall and neutrophils in the lumen. The inflammation causes more damage, which in turn further impairs the lung defenses, leading to more infection and a vicious cycle of events. Treatment involves daily physiotherapy to drain affected areas and appropriate antibiotics to treat infection and thus reduce inflammation.

Introduction Bronchiectasis is defined as abnormal chronic dilatation of one or more bronchi. This structural abnormality predisposes the bronchi to bacterial

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infection. The most common symptoms are chronic cough and sputum production. Recurrent bronchial infections occur that may become chronic, causing daily purulent sputum production. Cases of cystic or saccular bronchiectasis, in which severe loss of bronchial wall structure leads to large balloon-like dilatations, are now infrequently seen in developed countries. This type of bronchiectasis often followed severe lung infections, usually in childhood, and was characterized by production of very large volumes of sputum and finger clubbing. Improved socioeconomic conditions, vaccination programs, and ready availability of antibiotics are responsible for the reduced incidence of cystic or saccular bronchiectasis. Much more common today is a cylindrical form in which the damage to the bronchial wall is less severe. This type of bronchiectasis, which is usually bilateral and may be diffuse, although the lower lobes are usually worst affected, has been called ‘modern’ bronchiectasis. In varicose bronchiectasis, there are localized constrictions caused by scarring superimposed on cylindrical changes. Traction bronchiectasis occurs in fibrotic lung conditions such as fibrosing alveolitis, in which the airway walls are pulled apart by the fibrotic process. Interestingly, in these cases the bronchiectasis is usually asymptomatic, probably because the mucosa is unaffected. The current prevalence of bronchiectasis is unknown. Published data from older studies were based on chest radiographs, which have been shown to be very insensitive for detecting bronchiectasis. The availability of thin-section, high-resolution computed tomography (CT), which is a much easier investigation to perform than the old ‘gold standard’ of bronchography, has led to increased recognition in patients who might otherwise have been undiagnosed or classified as having chronic bronchitis or asthma. CT has enabled a diagnosis of bronchiectasis to be made in much milder cases, and this must be kept in mind when comparing older studies to those published recently. Indeed, this can cause confusion in terminology because some patients with asthma or smoking-related chronic bronchitis have bronchiectatic airways by CT criteria. A high prevalence of bronchiectasis has been reported in specific ethnic groups (e.g., Native Americans in North America, New Zealand Maoris, and Western Samoans). However, it is unclear whether genetic predisposition, environmental factors, or social practices are responsible. Most patients with bronchiectasis are nonsmokers. This may be because a history of childhood respiratory problems is common, and consequently starting smoking is less likely, but it could also be influenced by referral practice because a smoker presenting with a chronic

productive cough is more likely to be advised on smoking cessation rather than be referred for investigation.

Etiology There are a number of known causes of bronchiectasis (Table 1) and several other conditions that have been associated with bronchiectasis (Table 2) without a clear understanding of the pathological link. Congenital

Several types of congenital bronchiectasis occur because of the absence or deficiency of elements of the bronchial wall that are crucial to the retention of its normal architecture. In Williams–Campbell syndrome, there is no or much reduced cartilage in the bronchi, generally extending beyond the first division. Congenital tracheobronchomegaly (Munier– Kuhn syndrome) is another example of this type, and bronchiectasis can occur in Marfan’s and Ehlers– Danlos syndromes. Patients with intralobar pulmonary sequestration have blind-ending bronchi that Table 1 Causes of bronchiectasis Congenital (e.g., deficiency of structural elements of the bronchial wall) Infective (e.g., tuberculosis, whooping cough, or nontuberculous mycobacteria) Mechanical obstruction within lumen (e.g., inhaled foreign body) or external compression (e.g., tuberculous lymph node) Deficient immune response (e.g., common variable immune deficiency or human immunodeficiency virus) Inflammatory pneumonitis (e.g., aspiration of gastric contents or inhalation of toxic gases) Excessive immune response (e.g., allergic bronchopulmonary aspergillosis or lung transplant rejection) Abnormal mucus clearance (e.g., primary ciliary dyskinesia, cystic fibrosis, or young’s syndrome) Fibrosis (e.g., fibrosing alveolitis, sarcoidosis, or radiation pneumonitis)

Table 2 Conditions associated with bronchiectasis Infertility (e.g., primary ciliary dyskinesia, cystic fibrosis, or Young’s syndrome) Inflammatory bowel disease (e.g., ulcerative colitis, Crohn’s disease, or coeliac disease) Connective tissue disorders (e.g., rheumatoid arthritis, systemic lupus erythematosus, or Sjogren’s disease) Malignancy (e.g., acute or chronic lymphatic leukemia) Diffuse panbronchiolitis, predominantly reported in Japan Yellow nail syndrome I dystrophic and colored (usually yellow) nails, lymphedema, and pleural effusions a1-Antiproteinase deficiency, usually causes emphysema Mercury poisoning may cause Young’s syndrome (obstructive azoospermia, sinusitis, and bronchiectasis)

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trap mucus and are prone to repeated infections that lead to bronchiectasis. Postinfective

A severe pneumonia of any sort can damage the bronchial wall sufficiently to leave residual bronchiectasis. Mycobacterium tuberculosis, Bordetella pertussis, and measles virus are particularly likely to cause damage, but the diagnosis can be difficult to make because the patient’s history of the illness is vague. Chronic cough and sputum production should follow the severe lung infection, and bronchiectasis should be confined to the affected area. Some patients give a history of childhood pneumonia when they present in adult life with bronchiectasis, but they have diffuse disease on CT and have had an asymptomatic period in between. The importance of the childhood history in such cases is uncertain, but it may be that the earlier event provides a starting point for later problems if a bronchial infection occurs and is not cleared from the damaged area. Bronchiectasis is a common but not universal finding in Sawyer–James (Macleod’s) syndrome. This condition is a form of obliterative bronchiolitis that most commonly follows an infectious insult to the developing lung during the first 8 years of life. There is unilateral hyperlucent, hypovascular lung with marked air trapping. Nontuberculous mycobacteria, particularly Mycobacterium avium complex, can cause bronchiectasis. This type of infection seems to occur particularly in middle-aged females who present with a long history of dry cough. Mechanical Obstruction

Localized bronchiectasis can occur as a result of bronchial obstruction from any cause, and the obstruction can either be in the lumen of the airway (e.g., inhaled foreign body) or due to compression from outside (e.g., tuberculous lymph node at the origin of the right middle lobe bronchus). The obstruction leads sequentially to impaired mucus clearance, distal infection, chronic inflammation, damage to the bronchial wall, and bronchiectasis. Middle lobe syndrome, which involves recurrent acute infections at this site, has been described in cases without evidence of obstruction. It may be that the anatomy of the right middle lobe, which is long and acutely angulated with a collar of lymph nodes at its origin, is the reason. Immune Deficiency

Children with severe immune deficiencies do not usually survive, although a proportion develop

bronchiectasis before they die. Bronchiectasis is a frequent complication of the rare X-linked hypogammaglobulinemia, in which there is complete absence of the B-lymphocyte system. Common variable hypogammaglobulinemia, in which there is a reduction in the amount of antibody present in one or more of the major classes, is much more commonly found when investigating an adult population with bronchiectasis. These patients will often give a history of acute severe respiratory infections (pneumonia) in addition to their chronic symptoms. Solitary IgA deficiency occurs in approximately 0.2% of the population and can be present without any clinical problems, although some patients suffer frequent viral-like illnesses. IgG subclass deficiency may be transient and can also occur in healthy subjects. More important is functional antibody deficiency, which involves failure to mount an antibody response to polysaccharide antigens (e.g., Streptococcus pneumoniae capsular antigen). This is associated with low IgG-2 levels, low pre-immunization pneumococcal antibody levels, and failure to mount a sustained antibody response to pneumococcal vaccination. An acquired immunodeficient state can occur secondary to multiple myeloma, chronic lymphatic leukemia, protein-losing enteropathy, nephrotic syndrome, or lymphoma. Bronchiectasis can also complicate HIV infection, and this may be seen more frequently now that patients are surviving much longer with antiretroviral therapy. Inflammatory Pneumonitis

Inhalation of toxic gases, such as ammonia or smoke by victims caught in fires, and aspiration of gastric contents can cause sufficient damage to lead to bronchiectasis. Acid reflux in patients with hiatus hernia can cause cough and wheeze, and may aggravate bronchiectasis symptoms, but is unlikely to be the sole cause of the disease. Excessive Immune Response

Bronchiectasis in patients with allergic bronchopulmonary aspergillosis (ABPA) typically affects the proximal bronchial tree and the upper lobes, although more generalized disease is not uncommon. It is caused by an immune reaction involving eosinophils and fungus colonizing the airways. Atelectasis occurs because of obstruction by plugs of inspissated secretions containing fungal hyphae. Acute episodes of fever, wheeze, expectoration of viscid sputum plugs, and pleuritic pain, sometimes associated with consolidation in different areas on the chest radiograph, merge insidiously over time as bronchiectasis develops into chronic purulent sputum production,

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where exacerbations of ABPA are difficult to distinguish from infective exacerbations of bronchiectasis. Some disorders causing constrictive obliterative bronchiolitis also damage the large airways. This pathology is seen in lung transplant rejection, chronic graft versus host disease, rheumatoid arthritis, and after inhalation of toxic gases. Abnormal Mucociliary Clearance

Three different forms of impaired mucociliary clearance result in bronchiectasis: primary ciliary dyskinesia (Kartagener’s syndrome), cystic fibrosis (CF), and Young’s syndrome. Forms of infertility are associated with all three. Primary ciliary dyskinesia is rare and thought to be an autosomal recessive condition with incomplete penetrance involving numerous genes. There are usually, but not always, ultrastructural abnormalities in the cilia that make them move in a slow, disordered manner or the cilia may be completely static. Most commonly, there is the absence of one or both dynein arms (Figure 1), but abnormalities of the microtubules or radial spokes have been described. In cases with normal ultrastructure, the abnormality may be beyond the resolution of the electron microscope or may involve the orientation of the cilia on the cell surface. Cilia

line the nose, paranasal sinuses, and bronchi as far as the respiratory bronchioles and also form the tails of spermatozoa. Impaired ciliary function occurs at all these sites, and diffuse bronchiectasis is usually associated with chronic sinusitis, middle-ear disease, and often, but not invariably, male infertility. Females may have some reduction in fertility, and an increased risk of ectopic pregnancy, due to abnormal ciliary movement in the fallopian tubes. The condition may present in the neonatal period with pneumonia or segmental collapse due to mucus impaction or in childhood with recurrent infections. Patients cough continuously because this is their only form of mucus clearance. Approximately 50% of cases have dextracardia and a smaller proportion full situs inversus, which is thought to be due to abnormal cellular microtubules that are involved in rotation of organs in the embryo. Kartagener’s syndrome is bronchiectasis, chronic sinusitis, and dextracardia named after the German pediatrician who first described it. Young’s syndrome is bronchiectasis, chronic sinusitis, and azoospermia due to a functional blockage of the sperm in the caput epididymis, which is usually enlarged and palpable in the scrotum. The cause is unknown, and it may occur later in life after successful parenthood. The respiratory secretions are viscid, making ciliary clearance inefficient. The Outer arm Inner arm

Normal cilium

}

Dynein

Nexin link

Radial spoke

Cell membrane

Figure 1 (Top) Diagram of normal ultrastructure of a cilium and (bottom) electron micrograph of an abnormal cilium from a patient with primary ciliary dyskinesia that has no dynein arms.

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syndrome has been linked to mercury poisoning in childhood (pink disease), and its incidence has decreased since calomel (mercurous chloride) was removed from teething powders and worm medication. A Young’s syndrome equivalent is recognized in women who had mercury poisoning in childhood. CF is caused by mutation of a gene on the long arm of chromosome 7 that codes for the CF transmembrane conductance regulator (CFTR), a cyclic-AMPdependent chloride channel that has wide ranging effects, including hydration of the airways. Most cases present in childhood and are associated with severe disease. However, late presentation has been described with atypical CF alleles, some of which are associated with milder bronchiectasis, normal pancreatic function, normal sweat electrolytes, and male fertility. Upper lobe bronchiectasis and culture of Staphylococcus aureus are clues in this type of patient, and CF genotyping should be performed in suspected cases. Fibrosis

Traction bronchiectasis is common in the CT scans of many patients with all forms of pulmonary fibrosis, but this is rarely symptomatic. When symptomatic bronchiectasis does occur, it is most common in association with rheumatoid arthritis. In these cases, there may be an immunological explanation, or bacterial infection may establish itself within damaged lung, particularly if patients are immunosuppressed or if bronchial architecture is distorted. Infection causes neutrophilic airway inflammation and mucus hypersecretion that can become self-perpetuating if the infection is not eradicated. Associated Conditions

Pulmonary involvement is a frequent extraarticular manifestation of rheumatoid arthritis, and bronchiectasis may occur in the absence of interstitial lung disease. Airflow obstruction and constrictive obliterative bronchiolitis may be associated with the bronchiectasis. Other connective tissue diseases, including Sjogren’s syndrome and systemic lupus erythematosus, have varied pulmonary manifestations, including bronchiectasis. Bronchiectasis is associated with ulcerative colitis, Crohn’s disease, and celiac disease. The association of bowel and lung disease may be related to an underlying immune system dysregulation, with cells migrating between both sites and exposure of both epithelia to common environmental antigens and shared epithelial antigens. The association with ulcerative colitis is best established and two presentations are recognized. First, patients with a history

of severe colitis eventually undergo colectomy and then develop abrupt onset of cough and sputum production soon afterwards. Second, patients with one condition develop the other several years later. In the latter patients, flare ups of their bowel disease may or may not be associated with exacerbations in their lungs. Bronchiectasis associated with ulcerative colitis is varied in severity, but in some cases it is associated with florid diffuse neutrophilic airway inflammation and very large volumes of purulent sputum, which is often sterile. Another feature is that bronchiectasis regresses with anti-inflammatory medication, which contradicts the concept that bronchiectasis is always an irreversible condition. Onset of chronic cough and sputum production has also been linked to bowel resection in Crohn’s disease. Diffuse panbronchiolitis is a chronic airway condition first described in Japan, where it seems to be much more common. Patients present in middle age with a productive cough and breathlessness. The condition has subsequently been found in Korea and China, and occasional Caucasian patients with a similar syndrome have been described. There is inflammatory hypertrophy of the walls of the respiratory bronchioles, which involves infiltration with plasma cells, lymphocytes, and foamy cells. If the condition is untreated, it progresses to bronchiectasis. There is a remarkable response to treatment with long-term low-dose erythromycin, which has led to the investigation of macrolide antibiotic treatment for other forms of bronchiectasis. Alpha-1-antiproteinase deficiency predisposes to emphysema, particularly in cigarette smokers. Sometimes, bronchiectasis is also present, and occasionally it is the predominant pathology. There is a proteinase–antiproteinase imbalance in the airways of all patients with bronchiectasis, and possibly these patients were predisposed to airway as well as alveolar problems following an infection that caused initial airway damage. Yellow nail syndrome is a rare condition involving yellow discoloration of dystrophic nails, primary lymphedema, and pleural effusions. Chronic rhinosinusitis is common, and a significant proportion of patients have bronchiectasis. Idiopathic Bronchiectasis

Even at centers that perform a full set of investigations into possible causes of bronchiectasis, approximately half of patients remain idiopathic. There is a predominance of females in this group and also in many cases a history of wheezy bronchitis in childhood. This is followed by a period of good health before the onset of chronic cough and sputum

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production in adult life, often in their 20s or 30s. Patients may describe the onset of their problems as a severe ‘viral-like’ illness that went into their chest and did not resolve. Patients usually have chronic rhinosinusitis, which suggests that whatever abnormality is predisposing to the illness is present throughout the respiratory tract. These patients suffer from profound tiredness and difficulty concentrating, much more so than patients with bronchiectasis due to a postinfective cause or primary ciliary dyskinesia. This condition may involve a dysregulated innate and/or adaptive immune response.

Pathology In parts of the lung with bronchiectasis, subsegmental airways are permanently dilated, tortuous, and partially or totally obstructed by copious amounts of secretions. Side branches of the airways are frequently obliterated. Structural proteins are lost from the bronchial wall, and there is a variable amount of fibrosis. Characteristically, the elastin layer of the wall is deficient or absent, and the muscle and cartilage layers show signs of destruction. These changes weaken the wall and lead to the distortion of the normal architecture. The process often involves bronchioles, and long-standing obstruction may result in complete fibrosis of small airways. The airway epithelium is damaged and ciliated cells are lost. There is goblet cell hyperplasia and mucus gland hypertrophy. There may be peribronchial pneumonic changes with evidence of parenchymal damage. The pulmonary arteries may thrombose and can recanalize. With long-standing disease there is hypertrophy of the bronchial arteries with anastamosis and sometimes considerable shunting of blood to the pulmonary arteries. There is usually chronic inflammation, in which lymphocytes predominate in the bronchial wall and neutrophils in the lumen. The walls of bronchi and bronchioles contain lymphoid follicles and nodes containing B lymphocytes, CD4 þ and CD8 þ T lymphocytes, and mature macrophages.

Bacteriology The bacterial species isolated in bronchiectasis are listed in Table 3. Mixed infections are common. Bronchiectatic airways are often chronically colonized, and for long periods a balance may be struck in which the bacteria colonize the mucosal surface without stimulating a systemic inflammatory response. Patients carry the same strain for long periods, and acquisition of a new strain is not necessarily associated with an exacerbation. The species

Table 3 Bacteria isolated from sputum of patients with bronchiectasis Common Hemophilus influenzae Hemophilus parainfluenzae Pseudomonas aeruginosa Less common Streptococcus pneumoniae Moraxella catarrhalis Stenotrophomonas maltophilia Staphylococcus aureus Mycobacterium species Other Gram-negative bacilli

of bacteria and their concentration in the lumen are both important determinants of the level of inflammation. Host factors and the presence of additional viral infection will also influence when an exacerbation occurs, and it may be a combination of events that upsets the balance and provokes an exacerbation. This may lead to invasion of the mucosa and peribronchial pneumonia. The etiology and severity of the bronchiectasis influence the type of bacterial infection. For example, S. aureus is associated with CF and ABPA, and Pseudomonas aeruginosa is associated with extensive lung disease and severe airflow obstruction. The initial infection with P. aeruginosa is usually a nonmucoid strain, which becomes a mucoid phenotype when chronic infection occurs. More than any other species, P. aeruginosa is likely to establish chronic infection, and once this has occurred it usually persists for the rest of the patient’s life. However, compared to CF, there is less evidence that this is associated with more rapid disease progression. Patients with chronic P. aeruginosa infection do have worse quality of life, and this may be due to several factors: their underlying disease is worse; treatment is more often required in hospital for intravenous antibiotics because ciprofloxacin is the only active oral antibiotic; because of antibiotic resistance, treatment is less effective and therefore exacerbations occur more frequently; and long-term antibiotic prophylaxis is used more frequently. Tuberculosis is a rare complication of bronchiectasis, but when it does occur the classic clinical and radiological features may not be present. A high index of suspicion should be maintained in patients presenting with persistent low-grade fever, weight loss, or hemoptysis. Environmental nontuberculous mycobacterial species are sometimes isolated from bronchiectatic sputum. This may be a ‘one-off’ isolate indicating environmental exposure just before the sputum sample was obtained. The mycobacterium may establish itself in the airway without apparently affecting the patients’ condition. However,

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long-term follow-up is required to ensure this is true colonization and not low-grade infection. Some species are more pathogenic and therefore more likely to require treatment (e.g., M. avium complex and M. kansasii). These species can cause infection in patients without pre-existing lung disease or demonstrable immune deficiency. The infection may cause bronchiectasis, and in such cases it can be difficult to determine if the infection is primary or secondary. When there is a combination in a CT scan of mild to moderate cylindrical bronchiectasis and peripheral nodules, some of which may be cavitating, sputum samples should be sent for mycobacterial microscopy and culture.

Clinical Features Symptoms

The most common symptoms are chronic cough and sputum production. Exacerbations are caused by bronchial infections, which may be viral or bacterial or both, or there may be chronic bacterial infection causing daily purulent sputum production. Usually, an exacerbation is associated with increased sputum volume and purulence, although the volume can decrease because the sputum becomes more viscous and therefore difficult to clear. Low-grade temperature is common, whereas a high temperature is suspicious of pneumonia. Chronic rhinosinusitis is very common, and most patients have expiratory airflow obstruction, the severity of which correlates with the severity of bronchiectasis. This is usually largely irreversible, although some patients do have an asthmatic component. Chest pains are common and are usually described as an ache, but they may be pleuritic during exacerbations. Hemoptysis is usually minor and complicates an exacerbation; serious hemoptysis requiring selective embolization or surgery is rare, probably because of the ready availability of antibiotics and because cystic bronchiectasis is less common. Undue tiredness and difficulty concentrating occur in poorly controlled disease. Physical Signs

There may be coarse inspiratory crackles heard over the site of bronchiectasis, but sometimes there are no signs to suggest the diagnosis. Airflow obstruction can cause wheezes, and there may be late inspiratory squeaks indicating small airway disease. Clubbing is unusual in ‘modern’ bronchiectasis because it is associated with more severe disease. A 24 h sputum collection can be very informative because patients tend to be inaccurate in their description of the color and volume of what they produce.

Investigations

These are listed in Table 4. Suspicion of bronchiectasis should lead to a high-resolution thin-section (e.g., fast scan time of 1 s or less, 1 mm sections, and 10-mm spacing) CT scan. Investigation may discover a treatable cause of bronchiectasis. Younger patients, those with associated conditions, and those in whom respiratory function is deteriorating and/or infective exacerbations are becoming more frequent or prolonged should be seen by a respiratory physician with a special interest in bronchiectasis who has access to all the investigations listed in Table 4. Chest radiographs are an insensitive test for bronchiectasis; in one study, they detected less than half of patients who subsequently had positive bronchography. The CT findings are related to the presence of dilated air-filled bronchi, dilated secretion-filled bronchi, and loss of volume resulting from parenchymal loss, which leads to crowding of the bronchi. The appearance depends on the orientation of the bronchi to the scanning plane. When they lie in the same plane, they appear as tram lines that do not decrease (taper) in diameter in the usual way as they progress to the outside of the lung (Figure 2(a)). The easiest way to determine whether a bronchus is dilated is to compare it to the adjacent pulmonary artery. Dilated bronchi that are perpendicular to the Table 4 Investigation of bronchiectasis All patients Chest radiograph PA and lateral Sinus radiograph Lung function tests High-resolution thin-section CT scan Sputum microscopy to determine cell type (neutrophils or eosinophils) Sputum culture and sensitivities Sputum smear and culture for acid fast bacilli Skin tests (atopy, aspergillus) Sweat test, nasal potentials, CF genotyping Cilia studies (if nasal mucociliary clearance is prolonged or nasal nitric oxide is low, proceed to light microscopy of ciliary beat frequency and then electron microscopy) Blood investigationsa Selected patients Fiberoptic bronchoscopy Videofluoroscopy of swallowing to detect aspiration Ph study for acid reflux Semen analysis Tests for associated conditions Blood tests for rarer immune deficiencies a Full blood count with differential white cell count; C-reactive protein; erythrocyte sedimentation rate; total immunoglobulin (Ig) levels of IgG, IgM, IgA, and IgE and specific antibodies to pneumococcus and tetanus antigens; aspergillus radioallergosorbent test (IgE) and precipitins (IgG); rheumatoid factor; protein electrophoretic strip; and a1-antiproteinase.

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Figure 2 High-resolution thin-section computed tomography of bronchiectasis demonstrating (a) a nontapering bronchus, (b) the signet ring sign, (c) tree-in-bud pattern, and (d) mosaic perfusion.

scanning plane have a circular appearance, and then the smaller pulmonary artery gives a ‘signet ring’ appearance (Figure 2(b)). Mucus-filled bronchi appear as branching tubes or nodules, and many filled small airways (exudative bronchiolitis) give a ‘treein-bud’ appearance (Figure 2(c)). End-expiratory scans exaggerate increased transradiency in areas where gas trapping has occurred because of obstruction of small airways. Since gas trapping is usually patchy, this gives a pattern of mosaic perfusion (Figure 2(d)). Peripheral blood inflammatory markers (neutrophil count and C-reactive protein) and sputum appearance can be used to monitor the response of an exacerbation to treatment. Most patients with bronchiectasis require long-term follow-up because disease progression can occur insidiously or in a stepwise rather than gradual manner. A history of more frequent and prolonged exacerbations, not responding well to treatment, is usually associated with an increase in sputum volume and purulence, increased breathlessness, as well as increased tiredness. CT scan features associated with subsequent disease progression are bronchial wall thickening, mucus plugging, and tree-in-bud pattern. Lung function may show a decrease in FEV1 and gas transfer. These changes are reversible, but mosaic perfusion is not. Health status questionnaires, such as the St George’s Respiratory Questionnaire, and measures of exercise capacity, such as the shuttle walking test, can also be used to monitor the patient’s progress. A

deterioration should lead to a review of sputum bacteriology (including mycobacteria) and aspergillus serology, as well as physiotherapy practice, before considering a change to the management regimen. P. aeruginosa is often isolated for the first time in these circumstances, but it may reflect the patient’s poor condition rather than be the cause of it.

Pathogenesis Excess mucus is produced in the dilated, tortuous bronchiectatic airways. The mucus is poorly cleared partly because of the abnormal anatomy but also because cilia are lost when the epithelium is damaged, and the mucus is less elastic and more viscous largely due to its inflammatory cell DNA content. Bacteria adhere avidly to the stationary mucus and multiply so that they are often present in very high concentrations. Large numbers of neutrophils are attracted into the bronchial lumen from the circulation by chemotactic products of the bacteria and also mediators released by host cells (e.g., interleukin (IL)-8, C5a, and leukotriene B4). Serum levels of adhesion molecules are elevated, suggesting that endothelial activation also occurs in the lung. Activated neutrophils spill proteolytic enzymes and reactive oxygen species while trafficking through the lung and during phagocytosis. These may overwhelm the body’s ability to neutralize them and cause tissue damage in the affected area. The exuberant inflammation contains infection within the

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Impaired host defences

Tissue damage

Microbial infection

agar beads to establish chronic infection after they are instilled into the airway, but a considerable amount of pneumonia also occurs. Another model achieved the same result by partially ligating a bronchus and instilling bacteria distally, but this was technically difficult. Future work may utilize candidate genes to generate inducible transgenic animal models that mimic the abnormal inflammatory responses seen in the lung in bronchiectasis.

Management and Therapy Surgical Treatment Inflammation

Figure 3 A vicious cycle of bacterial infection-driven, host inflammation-mediated lung tissue damage in bronchiectasis.

lung, so bacteremia and spread of infection outside the lung are very rare. However, the inflammation may fail to eradicate the infection due to impaired local host defenses, so the infection becomes chronic, and it may slowly spread to involve adjacent normal bystander airways. Immune complexes form between bacterial antigens and antibodies that are produced locally and arrive by transudation. These stimulate other inflammatory processes. Epithelial cells, lymphocytes, and macrophages release cytokines and other factors that orchestrate and perpetuate the inflammation. Bronchiectatic secretions contain large amounts of IL-8, IL-1a, IL-1b, tumor necrosis factor-a, IL-6, and granulocyte colony-stimulating factor. The lung defenses are impaired by the damage caused directly by bacterial products and by inflammation, which in turn promotes continued infection. This has been termed a ‘vicious cycle’ of events (Figure 3). Bronchiectasis may occur because of an acute insult to the bronchial wall (e.g., whooping cough in a young child or inhalation of a toxic gas), and the damaged area may subsequently become infected. In other cases, there may be a recognized deficiency of the host defenses that renders the patient prone to infection (e.g., primary ciliary dyskinesia or common variable immune deficiency). However, in many cases the starting point of the vicious cycle may be obscure. It seems more likely that as yet unknown deficiencies in the host’s immune defense system, or an abnormality in the body’s ability to control the inflammatory response, will explain these cases, and cases in which bronchiectasis progresses rapidly, rather than the direct effect of a virulent infectious process. There are no satisfactory animal models of bronchiectasis. A rat model uses bacteria embedded in

The only curative treatment of bronchiectasis is surgical resection. Surgery is only appropriate when the bronchiectasis is localized and there is no underlying condition that predisposes to generalized bronchiectasis, such as primary ciliary dyskinesia. Good results are obtained from resection of severe localized bronchiectasis when the bronchiectatic area contributes little to lung function, which is otherwise reasonably preserved. Modern cylindrical bronchiectasis is usually bilateral, and surgery is rarely considered for this reason. Palliative surgical resection may be considered if a localized area of severe bronchiectasis defies medical management and acts as a sump for infection of other areas, even if less severe bronchiectasis is present elsewhere. Lung transplantation (usually two lungs or heart–lung) is used successfully to treat respiratory failure due to bronchiectasis. Medical Treatment

Patients with daily sputum production must perform postural drainage at least once daily and do this two or three times daily during exacerbations. They are taught to adopt the correct position to drain affected areas and to clear mucus by controlled breathing techniques sometimes aided by chest percussion. Patients need to be reminded about and encouraged to perform physiotherapy, and their technique should be regularly reviewed. Physical exercise should also be encouraged because it aids mucus clearance. A number of conditions that are recognized as causes or associations of bronchiectasis may require specific treatment (e.g., hypogammaglobulinemia and allergic bronchopulmonary aspergillosis). In patients with an asthmatic component, inhaled bronchodilators and corticosteroids are used in the usual way, and beta-agonists are often used to improve mucociliary clearance. However, there is little evidence to support the widespread use of inhaled corticosteroids in the absence of asthma. If this approach is used, then careful measure of objective benefit should be sought (e.g., lung function). Short

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courses of systemic corticosteroids may be used during severe exacerbations. Patients with chronic respiratory failure require long-term oxygen treatment. Carbon dioxide retention is sometimes a problem. Nasal intermittent positive-pressure ventilation is effective in these cases and surprisingly well tolerated despite a history of sinusitis. This approach is also effective in assisting mucus clearance. Antibiotics are given during infective exacerbations associated with purulent sputum, breathlessness, and malaise. The choice of antibiotic is influenced by the likely bacterial species (Table 3), (previous) sputum culture results, and the presence or absence of P. aeruginosa. As a general rule, because of the damaged airways and the level of infection, the antibiotic dosage has to be higher and the course longer than usual. The amount of bacterial resistance is also high due to previous antibiotic prescription (e.g., b-lactamase production in Hemophilus influenzae). Intravenous antibiotics are used for severe exacerbations, resistant species, and when oral antibiotics have failed. Continuous antibiotic treatment can be given as prophylaxis by the oral or nebulized route or as regular planned courses of intravenous treatment. This improves patient well-being, probably by reducing the frequency of exacerbations and the number of bacteria in the lung (and thus the level of inflammation). However, there is no evidence that it alters disease progression, and there is a definite risk that it will drive the lung flora toward more resistant strains and species, such as P. aeruginosa and Stenotrophomonas maltophilia. This approach should be reserved for patients who, despite optimum medical management, continue to have very frequent (more than six per year) exacerbations, some of which involve admission to the hospital. There has been great interest in the past few years in macrolide antibiotic prophylaxis. The benefit is thought not to be due to their action of killing bacteria but via effects on inflammatory cells and possibly mucus production. Several studies have

provided encouraging results, and this is a promising area of future research. See also: Bronchiolitis. Bronchomalacia and Tracheomalacia. Cilia and Mucociliary Clearance. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Defense Systems. Immunoglobulins. Panbronchiolitis. Pneumonia: Mycobacterial. Primary Ciliary Dyskinesia.

Further Reading Amsden GW (2005) Anti-inflammatory effects of macrolides – an underappreciated benefit in the treatment of community-acquired respiratory tract infections and chronic inflammatory pulmonary conditions? Journal of Antimicrobial Chemotherapy 55: 10–21. Barker AF (2002) Bronchiectasis. New England Journal of Medicine 346: 1383–1393. Bush A, Cole PJ, Hariri M, et al. (1998) Primary ciliary dyskinesia – diagnosis and standards of care. European Respiratory Journal 12: 982–988. Evans D, Bara AI, and Greenstone M (2005) Prolonged Antibiotics for Purulent Bronchiectasis, the Cochrane Collaboration. New York: Wiley. Hansell DM (1998) Imaging of obstructive pulmonary disease. Bronchiectasis Radiology Clinics of North America 36: 107–128. Hermaszewski RA and Webster ADB (1993) Primary hypogammaglobulinaemia: a survey of clinical manifestations and complications. Quarterly Journal of Medicine 86: 31–42. Homma H, Yamanake A, Tanimoto S, et al. (1983) Diffuse panbronchiolitis. A disease of the transitional zone of the lung. Chest 83: 63–69. Mukhopadhyay S, Singh M, Cater JI, et al. (1996) Nebulised antipseudomonal antibiotic therapy in cystic fibrosis: a metaanalysis of benefits and risks. Thorax 51: 364–368. Pasteur MC, Helliwell SM, Houghton SJ, et al. (2000) An investigation into causative factors in patients with bronchiectasis. American Journal of Respiratory and Critical Care Medicine 162: 1277–1284. Wilson R (2003) Bronchiectasis. In: Gibson GJ, Geddes DM, Costabel U, Sterk PJ, and Corrin B (eds.) Respiratory Medicine, pp. 1445–1464. London: Saunders/Elsevier. Wilson R and Abdallah S (1997) Pulmonary disease caused by non-tuberculous mycobacteria in immunocompetent patients. European Respiratory Monograph 12: 247–272.

BRONCHIOLITIS R L Smyth and S P Brearey, Alder Hey Children’s Hospital, Liverpool, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Bronchiolitis is a common respiratory tract infection usually affecting infants and young children during annual epidemics. It

is characterized by wheeze, respiratory distress, and poor feeding. Respiratory syncytial virus (RSV) is the most common cause for bronchiolitis and is amongst the most important pathogens causing respiratory infection in infants worldwide. The healthcare burden of bronchiolitis is large, due to large numbers of hospitalized infants and the high risk of nosocomial spread during epidemics. Most children will suffer only mild, short-lived symptoms. A small proportion will need admission to hospital, where treatment is generally supportive until the

BRONCHIOLITIS 269 illness resolves. Some will require ventilatory support for which mortality can be up to 10%. Infants at high risk of severe disease include those born prematurely, those with chronic lung disease, and immunocompromised infants. RSV infects ciliated epithelial cells, causing sloughing of the epithelium, cytokine and inflammatory mediator release, increases in mucus production, and interstitial edema. Clinical manifestations of bronchiolitis are a combined result of viral toxicity and the immune response to infection. Innate immune responses are important to the pathogenesis of bronchiolitis, as severe infection tends to occur after maternal antibody protection has waned and before the infant’s adaptive immune responses have matured. Immunoprophylaxis, in the form of intramuscular anti-RSV IgG1, is effective in reducing rates of hospitalization for high-risk infants.

Introduction Bronchiolitis, meaning inflammation of the bronchioles, is a clinical complex usually affecting children less than 2 years old. It is characterized by wheezing, dyspnea, tachypnea, and poor feeding. The clinical characteristics, originally termed ‘congestive catarrhal fever’, have been recognized for over 150 years. It was not until the late 1950s that the epidemiology and viral etiology of the illness were described. Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and is amongst the most important pathogens causing respiratory infection in infants worldwide. Epidemics occur during the winter months in temperate climates and during the rainy season in tropical climates (Figure 1). In the US, more than 120 000 infants are hospitalized annually with RSV infection, with more than 200 deaths

attributed to RSV lower respiratory tract disease. Total hospital charges for RSV-coded primary diagnoses over the 4-year period from 1997 to 2000 have been estimated at US$2.6 billion. Within hospitals, there is a high risk of nosocomial spread of RSV during epidemics, which can put vulnerable infants at risk of severe disease. Infection in infancy also predisposes children to the development of recurrent wheeze, thus increasing the long-term healthcare burden of this disease. Prophylaxis is available as a recombinant monoclonal antibody, but is expensive and currently limited to infants at highest risk of severe disease. Development of an effective vaccine would have a dramatic effect on morbidity and healthcare costs, but this is unlikely to occur in the near future. Treatment is generally supportive until the infection runs its natural course.

Etiology RSV is the primary cause of bronchiolitis. Seroepidemiological studies show that over 90% of children are infected with RSV by 2 years of age. Infection is spread by droplet exposure or direct contact with secretions. Fomites are infectious outside the body for up to 12 h. RSV was first isolated from chimpanzees in 1955 and was originally called chimpanzee coryza agent (CCA). Subsequently, RSV has been shown to cause 75–85% of cases of bronchiolitis. Other pathogens known to cause bronchiolitis are shown in Table 1. Historically, in over 10% of cases of bronchiolitis, no pathogen is detected. More

5000 4500 4000

Number of reports

3500 3000 2500 2000 1500 1000 500 0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 Year Figure 1 Laboratory reports to Communicable Disease Surveillance Centre, Health Protection Agency, UK of infections due to respiratory syncytial virus in England and Wales, 1990–2004 (4 weekly). Reproduced with permission from HPA.

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recently, polymerase chain reaction (PCR) techniques have identified other pathogens, such as human metapneumovirus (hMPV) and coronavirus, which may have gone undetected previously. Co-infection with Table 1 Bronchiolitis pathogens Respiratory syncytial virus Rhinovirus Parainfluenza viruses (1,2,3) Influenza viruses (A and B) Human metapneumovirus Coronavirus (NL-63) Adenovirus Bordatella pertussis Mycoplasma pneumoniae

hMPV and RSV may be associated with a higher risk of severe disease. RSV is a negative-sense single-stranded RNA pneumovirus from the Paramyxoviridae family (Figures 2 and 3). The viral genome contains only 10 genes that encode for 11 viral proteins. Nine of these are structural proteins and glycoproteins that form the viral coat and bring about attachment to host cells, whilst the other two direct viral replication within the host cell. Table 2 describes the functions of each protein. Based on immunological techniques, two different strains of the virus have been identified: A and B. Subsequently, it has been demonstrated that the two strains are also genetically distinct. Most variability between strains is due to differences in the amino acid sequence of the G protein on the viral coat. Strain A is seen more commonly in the UK and North America. There is some evidence that strain A results in more severe infections. The F protein, responsible for fusion of infected cells with adjacent uninfected cells, facilitates cell-to-cell transmission of the virus and results in epithelial cell syncytia (appearance of apparently large multinucleate cells), which give the virus its name.

Pathology

Figure 2 Negative stain electron micrograph of RSV. Scale ¼ 100 nm. Reproduced with permission from Professor C A Hart.

Envelope proteins

G SH F

Nucleocapsid complex (N, P, and L proteins)

Matrix M1 proteins M2 Negative-sense RNA genome Lipid envelope

Figure 3 A schematic diagram of the RSV virion.

RSV initially causes inflammation of the bronchioles and destruction of ciliated epithelial cells. The submucosa becomes edematous, whilst cellular debris and mucus form plugs within the bronchioles. Neutrophils and alveolar macrophages are the

BRONCHIOLITIS 271 Table 2 RSV proteins and their functions Protein Surface glycoproteins F

Function

G SH

Virus penetration and fusion of infected cells with uninfected cells Virus attachment Unknown

Nucleocapsid-associated proteins N P L M2-1 M2-2

Encapsidates genome Phosphoprotein RNA polymerase Transcription elongation factor Regulation of transcription

Matrix protein M1

Non structural proteins NS1 NS2

May mediate association of nucleocapsid with envelope

Antagonize interferon-induced antiviral response Unknown

predominant inflammatory cells in the small airways. There is a peribronchiolar infiltration with lymphocytes that is associated with edema fluid accumulating within the alveoli. Severe disease involves destruction of the respiratory epithelium, parenchymal necrosis, and formation of hyaline membranes. The bronchiolar epithelium regenerates after 3–4 days, but cilia do not reappear until up to 15 days after the illness has resolved.

Clinical Features The spectrum of severity for infected children is wide. After an incubation period of 1–2 days, most infants exhibit predominantly upper respiratory tract symptoms. These include rhinorrhea, cough, and low-grade fever that often persists for several weeks, before resolving, without any other symptoms. Thirty to fifty percent of infants will progress to develop lower respiratory tract signs and symptoms within 2–3 days of infection. Infants may develop a rapid respiratory rate, wheeze, and other signs of respiratory distress (subcostal and intercostal recession, tracheal tug, and nasal flaring). Apnea at presentation is common, especially in infants less than 6 weeks old, those with a history of apnea of prematurity, and those with congenital heart disease. Approximately 2–3% of infected children require admission to hospital. Reasons for admission include hypoxia, inadequate fluid intake, apnea, and signs of imminent respiratory failure. A more severe cough,

pharyngitis, conjunctivitis, and otitis media may also be present. Irritability, signs of cardiovascular compromise, lethargy, and exhaustion are late signs and may be followed by respiratory arrest if no clinical interventions are made. One to two percent of infants hospitalized with bronchiolitis require ventilatory support and mortality in this group may be up to 10%. Premature birth, chronic lung disease of prematurity, congenital heart disease, cystic fibrosis, and immunodeficiency all predispose infants to a high risk of severe disease. In addition to this, boys are more likely to be hospitalized than girls. Infants exposed to high levels of particulate air pollution or cigarette smoke are also more likely to suffer from severe bronchiolitis. A chest radiograph will typically show hyperinflation, a flattened diaphragm, and patchy peribronchial infiltration. The chest radiograph can be normal even in severe cases. A reduction in oxygen saturations is associated with increased respiratory rate and is an accurate indicator for reduced gas exchange that is seen in severe disease. Arterial or capillary blood gas analysis is helpful in assessing changes in the clinical status, particularly if ventilatory support is being considered. Most infants, given adequate supportive care, improve clinically in 3–4 days. Within 2 weeks of the height of their illness most infants will have a normal respiratory rate and any radiological abnormalities will have cleared. However, up to 20% of infants may suffer from persistent wheezing and airway obstruction for several months, especially those that required hospital admission. In the longer term, airway reactivity is increased in children after infection and persists for at least 5–8 years. It is still uncertain whether RSV causes asthma in later life. RSV infection does not confer lasting immunity to reinfection. However, subsequent infections are usually less severe and are more likely to be limited to the upper respiratory tract.

Pathogenesis RSV first infects the ciliated epithelial cells of the respiratory tract. Subsequent disease manifestations are caused by a combination of viral cytotoxicity and the immune response to infection. These effects are summarized in Figure 4. On contact with epithelial cells, the G protein attaches the virus to epithelial cells and allows viral RNA and enzymes to enter the cell and initiate production of new viral RNA and proteins. The F protein mediates the formation of syncytia that enable the virus to spread rapidly. Multiple new viruses are assembled within cells, which are ultimately

272 BRONCHIOLITIS Soluble mediators: surfactant protein, immunoglobulin, sCD14

RSV

Opsonization Virus aggregation Increased phagocytosis

Epithelial cell sloughing

Airway inflammation Bronchoconstriction Mucus accumulation Surfactant inhibition

Macrophage

Epithelial cell

Inflammatory cytokines and chemokines

Neutrophil Eosinophil

Macrophage

NK cell

Dendritic cell

+ B lymphocyte CD4 T-helper cell

CD8+ cytotoxic T cell

Th2 response Th1 response

Figure 4 Pathogenesis of RSV bronchiolitis. RSV first infects ciliated epithelial cells of the respiratory tract. Inflammatory cytokines and chemokines, secreted by infected epithelial cells and macrophages, attract inflammatory cells and cause inflammation and bronchoconstriction in the airway. The predominant inflammatory cells in the airway are neutrophils and alveolar macrophages. Dendritic cells are the main antigen-presenting cells that stimulate T-lymphocyte function. Neutrophil survival is prolonged and IL-9 secretion promotes mucus production. CD4 þ T-helper lymphocytes may be skewed to produce proinflammatory (Th2) cytokines. The combination of epithelial cell sloughing, mucus plugging, and airway inflammation leads to the clinical presentation of acute bronchiolitis.

destroyed, leading to sloughing of the epithelium. As the cells are destroyed they release proinflammatory substances and cytokines that increase capillary permeability and attract inflammatory cells such as neutrophils (interleukin-8 (IL-8) secretion), macrophages (IL-1b, MIP-1a), eosinophils (RANTES, MIP1a), and natural killer cells (interferon (IFN)-a/b). Some secreted mediators, such as IL-9 stimulate mucus production, whilst others, such as leukotrienes, cause bronchoconstriction. Pulmonary surfactant function is also inhibited. Increased production and impaired clearance of cellular debris and mucus results in plugging of the small airways, air trapping, and atelectasis. For infants with preexisting poor lung function, such as those with chronic lung disease, this airway obstruction is likely to have more clinical significance and cause severe disease. The immune response to RSV includes innate and adaptive components. Innate immune responses are now recognized as more important than previously thought and are capable of influencing the subsequent adaptive immune response.

Innate immunity is important in defense against RSV and other viruses that cause bronchiolitis as the adaptive immune responses of infants are relatively immature. The innate immune response is rapid and recruits effector molecules and phagocytic cells to the site of infection through the release of cytokines. Pulmonary surfactant provides early defense from viral infection. Surfactant proteins A and D are members of the collectin family that can bind to the surface of a number of pathogens including RSV. The virus is thus opsonized and capable of binding to complement and receptors on phagocytic cells (neutrophils and macrophages), eosinophils, and natural killer cells. Toll-like receptors (TLRs) are the most important of these receptors. They are present on epithelial cells and phagocytic cells and recognize distinctive nonhuman pathogen-associated molecular patterns (PAMPs). Although their precise role is still being elucidated, TLRs are important receptors to the innate and adaptive immune responses to RSV. TLR2 and TLR4 are receptors for surfactant proteins A and D, both of which bind to RSV. TLR4 and CD14 have been shown to be co-receptors for the

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RSV F protein. Genetic polymorphisms in the TLR4 gene are associated with a risk of severe disease. TLR3 recognizes double-stranded RNA in endosomes of the cell and initiates an immune response to RSV. Pulmonary dendritic cells also express TLRs and are the predominant antigen-presenting cells in RSV infection. Circulating dendritic cells are recruited to sites of viral replication in the lungs. They produce large amounts of type I interferon (IFN-a and -b) and stimulate subsequent T-lymphocyte responses to RSV infection. Regulatory T lymphocytes (Treg) are known to dampen inflammatory responses and could be stimulated by RSV either via TLRs expressed on their cell surface or via pulmonary dendritic cells. Therefore, they provide a way by which the T-lymphocyte response to RSV can be influenced by both innate and adaptive immunity. Alveolar macrophages have phagocytic functions, but act mainly as antigen-presenting cells, interacting with helper and cytotoxic T lymphocytes. Along with respiratory epithelial cells they respond, via TLR and other signaling pathways, by secreting cytokines that increase tissue permeability as well as attracting and activating additional inflammatory cells. Neutrophils are the predominant airway leukocytes in RSV bronchiolitis, representing about 90% of cells in the upper airway and 80% of cells in the lower airway. Neutrophil chemotaxis is dependent on the production of the potent chemotractant, IL-8, by airway epithelial cells and macrophages. IL-8 is secreted very soon after infection, leading to an almost immediate inflammatory response before the infection is established. A later peak in IL-8 secretion is dependent on viral replication. Genetic polymorphisms in the IL-8 gene are associated with a risk of severe disease. Neutrophil recruitment and adherence to epithelial cells is increased by persistence of RSV infection. Neutrophils, whose survival is prolonged, secrete products such as myeloperoxidase and neutrophil elastase, which amplify viral cytotoxicity. Neutrophils have also been shown to secrete IL-9 in large quantities in the lungs of severely infected infants. IL-9 is a potent proinflammatory cytokine that is known to cause eosinophilic inflammation, bronchial hyperresponsiveness, and increased mucus production. Neutrophil function therefore plays an important role in the pathological changes that occur in bronchiolitis. Eosinophils might be expected to have an important role in the pathogenesis of bronchiolitis. They have antiviral activity and eosinophil chemoattractants are secreted by infected respiratory epithelial cells. Following the clinical trials of the formalininactivated vaccine, postmortem examinations revealed massive eosinophilic infiltrates. However,

subsequent clinical studies have not reported significant numbers of eosinophils in the airways of infants with RSV bronchiolitis. Adaptive immunity to RSV is generally composed of protective humoral immunity (B cells and antibodies) and viral clearance (T cells). The humoral response to RSV infection results in the production of IgG, IgM, and IgA antibodies in both blood and airway secretions. Protective benefits of these antibodies are demonstrated by the reduced likelihood of infection in the first month of life in term babies, due to the carriage of maternal IgG antibodies. Prophylaxis with monoclonal anti-RSV IgG (palivizumab), which has been shown to reduce the incidence and frequency of infections in high-risk infants, also demonstrates the role of the humoral response. Evidence from animal models confirms that antibody protects against RSV disease, but once infection is established, the T-cell response promotes viral clearance. The role of cell-mediated immunity is demonstrated in children with deficient cellular immunity who shed the virus for many months after initial infection, compared to 2 weeks for immunocompetent infants. The cell-mediated response is composed of the CD8 þ cytotoxic T lymphocytes and CD4 þ T-helper lymphocytes. Cytotoxic lymphocytes are important in both recovery from and the pathogenesis of RSV bronchiolitis. They promote clearance of RSV from the lungs but also cause pulmonary injury. Functional studies in T-lymphocyte-depleted mice demonstrated prolonged RSV replication, yet no overt evidence of illness. CD4 þ T-helper cells have traditionally been subdivided further according to the cytokine profiles they secrete: T-helper-1 (Th1) cells produce interferon gamma (IFN-g) and other antiviral cytokines. Th2 cells produce cytokines that induce eosinophil proliferation, and the release of leukotrienes and IgE antibodies leading to an enhanced inflammatory response. The idea that RSV bronchiolitis might be a Th2-type disease, and that this may explain airway obstruction and postbronchiolitic wheeze, has somewhat limited evidence to support it. Multiple factors are more likely to influence the T-lymphocyte response to RSV infection.

Animal Models Several experimental animal models have been used to improve our understanding of the pathophysiology of RSV bronchiolitis. The variety of animal models used reflects the diversity of clinical manifestations of RSV disease, dependent on an individual’s age, genotype, phenotype, immune status, and concurrent disease. RSV has been shown to replicate in

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animal models: primates, cotton rats, mice, calves, guinea pigs, ferrets, and hamsters have been used. The need to investigate immunological responses to RSV, using good animal models, was highlighted after the unsuccessful development of the first formalin-inactivated (FI) RSV vaccine in the 1960s. When natural RSV infection occurred in children given the vaccine, 80% of these children were hospitalized and two children died. Interpretation of the trial results was hampered by the lack of a small animal model, in which vaccine-enhanced disease could be studied. It is now apparent that other vaccine formulations are also capable of stimulating inappropriate immune responses to RSV. Therefore, comprehensive studies using animal models are essential before any further human vaccine trials are undertaken and are key to our understanding of the pathogenesis of bronchiolitis. Cotton rats have proved to be useful animal models, as they are uniformly susceptible to pulmonary infection through adulthood and are more immunologically responsive than mice to RSV. Studies using the cotton rat model were largely responsible for the development of RSV-neutralizing IgG in preventing pulmonary infection and treatment with ribavarin. Despite this, cotton rats have no congenic, transgenic, or knockout strains and there is only limited availability of reagents used to characterize immunological responses to RSV. Mice have been the most widely used model, due to a wide array of inbred, congenic, transgenic, and knockout strains. They also have the most reagents available, specific to RSV immunology, and have relatively low maintenance costs. Many insights have been gained into the immune response to RSV, which could not have been achieved using other models. The BALB/c mouse is particularly important, as it develops similar airway inflammatory responses to humans after intranasal RSV infection. Mice studies have been useful in investigating the immunology of RSV infection, especially the cytokine responses to the virus and for experimental vaccines.

Management and Current Therapy After admission to hospital, infants should be monitored and assessed regularly. Pulse oximetry, fluid balance, and respiratory rate are useful in assessing changes in the infant’s condition. Barrier nursing and stringent hand-washing policies have been shown to reduce nosocomial spread of RSV. Rapid RSV antigen testing, by either immunofluorescence or enzyme immunoassay, of nasopharyngeal secretions is useful in determining RSV status in hospital.

Despite considerable efforts to develop effective treatments for RSV bronchiolitis, no effective measures exist other than supportive care. The cornerstones of supportive care are supplemental oxygen to correct hypoxia and adequate fluid administration. Infants with inadequate fluid intake may need nasogastric feeding or, if this is unsuccessful, intravenous fluids. Intravenous fluids should be given with care due to the possibility of worsening lung fluid accumulation and left ventricular overload. A large number of trials and meta-analyses have investigated the efficacy of b2 agonists and ipratropium for patients with bronchiolitis. There is no compelling evidence that bronchodilators are useful in the treatment of bronchiolitis. There may be a subgroup of patients for which bronchodilators are safe and efficacious. However, no criteria exist to identify this subgroup and a significant number of studies found that patients deteriorated after receiving bronchodilators. The inconsistency of these results can be explained by our knowledge of the causes of wheeze in bronchiolitis. Bronchodilators will have no effect on increased mucus production, sloughed epithelial cells in the airway, or interstitial edema. Ribavarin is an antiviral preparation administered by aerosol and designed to inhibit the synthesis of viral structural proteins. Although early trials of ribavarin supported its use, later trials found no significant positive effect. In addition, it is potentially teratogenic and labor intensive to administer. Other treatments that have failed to demonstrate consistent benefits in the treatment of bronchiolitis include epinephrine, inhaled or systemic corticosteroids, recombinant human DNase, interferon-a, vitamin A, and antibiotics.

Prevention Two immunoprophylactic agents are currently available that are recommended for infants at risk of severe infection. RSV immune globulin intravenous (RSV-IGIV) is a polyclonal hyperimmune globulin, prepared from donors selected for having high serum titers of RSV neutralizing antibody. Palivizumab is a humanized murine monoclonal IgG1 antibody against the RSV F-glycoprotein and is given as a monthly intramuscular injection during the RSV season. RSV-IGIV was the first immunoprophylactic agent to be licensed for prevention of severe RSV bronchiolitis and clinical trials demonstrated a 41–63% reduction in hospital admissions attributable to RSV lower respiratory tract infections. However, RSV-IGIV has a number of disadvantages that have resulted in a decline in its use in favor of palivizumab. Early trials reported an increase in postoperative mortality in patients with cyanotic

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congenital heart disease who received RSV-IGIV. Administration required intravenous access and a 4-h infusion every month. There are concerns regarding volume overload, interference with live vaccines, and theoretical risks of infection from human donors. Trials of palivizumab have also demonstrated benefits to high-risk infants, with early trials demonstrating a 45–55% reduction in hospital admissions attributable to RSV lower respiratory tract infections. It is free from potential risk of infection from human donors, does not interfere with immune response to vaccines, and can be administered easily, without the need for hospital admission. Clinical trials also demonstrated its safety for infants with hemodynamically significant congenital heart disease. However, no trial has demonstrated a significant reduction in mortality due to RSV infection. Although palivizumab was shown to reduce rates of hospitalization and intensive care admissions in large double-blind randomized control trials, several studies have questioned its cost-effectiveness. The American Academy of Pediatrics recommends its use for certain high-risk groups: children under 2 years with chronic lung disease requiring medical therapy; infants born before 28 weeks’ gestation in the first year of life; and infants born at 29–32 weeks’ gestation up to 6 months of age. Infants born at 32–35 weeks’ gestation have a lower risk of hospitalization and palivizumab is only recommended for these infants if they have two other risk factors such as child care attendance, school age siblings, parental smoking, or congenital abnormalities of the airways. Infants with hemodynamically significant congenital heart disease are also likely to benefit and palivizumab is recommended in this group, particularly those with cyanotic heart disease, congestive heart failure, or pulmonary hypertension. There is little knowledge regarding its benefits for immunocompromised infants, children with cystic fibrosis, or prophylaxis in the second year of life.

Bronchiolitis Obliterans

See also: Antiviral Agents. Bronchodilators: Beta Agonists. Chemokines. Cilia and Mucociliary Clearance. Epithelial Cells: Type I Cells; Type II Cells. Genetics: Gene Association Studies. Immunoglobulins. Infant Respiratory Distress Syndrome. Leukocytes: Eosinophils; Pulmonary Macrophages. Mucus. Pediatric Pulmonary Diseases. Surfactant: Overview. Toll-Like Receptors. Vaccinations: Viral. Viruses of the Lung.

Further Reading American Academy of Pediatrics Committee on Infectious Diseases and Committee of Fetus and Newborn (2003) Revised indications for the use of palivizumab and respiratory syncytial virus immune globulin intravenous for the prevention of respiratory syncytial virus infections. Pediatrics 112(6): 1442–1446. Arden KE, Nissen MD, Sloots TP, and Mackay IM (2005) New human coronavirus, HCoV-NL63, associated with severe lower respiratory tract disease in Australia. Journal of Medical Virology 75: 455–462. Black CP (2003) Systematic review of the biology and medical management of respiratory syncytial virus infection. Respiratory Care 48(3): 209–233. Byrd LG and Prince GA (1997) Animal models of respiratory syncytial virus infection. Clinical Infectious Diseases 25: 1363–1368. McNamara PS and Smyth RL (2002) The pathogenesis of respiratory syncytial virus disease in childhood. British Medical Bulletin 61: 13–28. Semple MG, Cowell A, Dove W, et al. (2005) Dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis. Journal of Infectious Diseases 191: 382–386. Welliver RC (2003) The burden of respiratory syncytial virus (RSV) and the value of prevention. Journal of Pediatrics 143(5): S111–S162. Welliver RC (2004) Bronchiolitis and infectious asthma. In: Feigin RD, Demmier GJ, Cherry JD, and Kaplan SL (eds.) Textbook of Pediatric Infectious Diseases, 5th edn., pp. 273–285. Philadelphia: Elsevier. Wohl MB (1998) Bronchiolitis. In: Chernick V, Boat TF, and Kendig EL (eds.) Kendig’s Disorders of the Respiratory Tract in Children, 6th edn, pp. 473–485. Philadelphia: Harcourt. Wright JR (2005) Immunoregulatory functions of surfactant proteins. Nature Reviews: Immunology 5: 58–68.

see Interstitial Lung Disease: Cryptogenic Organizing Pneumonia.

BRONCHOALVEOLAR LAVAGE M Drent and J A Jacobs, University Hospital Maastricht, Maastricht, The Netherlands & 2006 Elsevier Ltd. All rights reserved.

Abstract Provided its limitations are kept in mind, there is a place for bronchoalveolar lavage (BAL) in the evaluation of diffuse lung

diseases. The analysis of cells obtained by BAL may be helpful in narrowing the differential diagnosis. The cellular analysis of the BAL fluid (BALF) relies on an accurate identification and differential counting of the cells obtained. Universal standards are not available; consequently, each institution should ensure that the various readers of BALF samples have been properly trained in cellular analysis. Using a standard technique for performing the BAL and handling the sample will also reduce variability. To explore the possible role for BALF analysis in the

BRONCHODILATORS / Anticholinergic Agents 285

Bronchocentric Granulomatosis

see Systemic Disease: Sarcoidosis.

BRONCHODILATORS Contents

Anticholinergic Agents Beta Agonists Theophylline

Anticholinergic Agents N J Gross, Hines VA Hospital, Chicago, IL, USA

the routine treatment of airways obstruction particularly for chronic obstructive pulmonary disease (COPD) but also for asthma in certain circumstances.

Published by Elsevier Inc.

Chemistry Abstract Anticholinergic agents have bronchodilator effects on the human airways and have a role in the treatment of obstructive airways diseases, particularly chronic obstructive pulmonary disease (COPD). Those in approved clinical use are synthetic quaternary ammonium congeners of atropine, and include ipratropium bromide and tiotropium bromide, each of which is very poorly absorbed when given by inhalation. Ipratropium has a relatively short duration of action and has been widely used for many years, either alone or in combination with the short-acting b-adrenergic agent albuterol for the maintenance treatment of stable COPD. Tiotropium, which was introduced in the early 2000s, has a duration of action of at least 1–2 days making it suitable for once-daily maintenance treatment of COPD. Both agents have a wide therapeutic margin and are very well tolerated by patients.

Introduction Naturally occurring anticholinergic agents such as atropine and scopolamine which are present in many plants, for example, belladonna spp. have been used in traditional medical cultures to relieve breathlessness for millennia. These agents, which are all tertiary ammonium alkaloids, and which were smoked or chewed, are well absorbed into the systemic circulation and thus have multiple systemic side effects that limited their clinical usefulness as bronchodilators. In the last century it was discovered that synthetic quaternary ammonium analogs of atropine were very poorly absorbed but retained topical anticholinergic activity. This enabled the development of quaternary ammonium analogs of atropine such as ipratropium, oxytropium, and tiotropium bromides for clinical use as inhaled bronchodilators. In the last two decades, these agents have become important components in

The structure of some natural and synthetic anticholinergic agents is shown in Figure 1. The parent compound, atropine, is a tertiary ammonium alkaloid due to a 3-valent nitrogen atom on the tropine ring. Although widely used for respiratory purposes in previous eras, its rapid absorption and widespread systemic distribution resulted in unacceptable systemic effects. Atropine and other natural tertiary agents are no longer used for respiratory diseases and will not be further considered. Synthetic analogs such as ipratropium bromide and tiotropium bromide are quaternary ammonium compounds and carry a charge on the 5-valent nitrogen atom which renders them less able to cross tissue barriers. However, they are able to penetrate mucosal surfaces sufficiently to exert pharmacologic actions on local structures such as smooth muscle.

Mode of Action Anticholinergic agents compete with acetylcholine for muscarinic receptors, inhibiting the numerous

N

Br −•H2O + C H CH3 N 3 7

CH3

O CH2OH O C CH

Atropine

O CH2OH O C CH

Ipratropium bromide

Figure 1 The molecular structure of tertiary and quaternary ammonium compounds.

286 BRONCHODILATORS / Anticholinergic Agents

‘housekeeping’ actions of the parasympathetic branch of the autonomic nervous system. When taken by the inhalational route, the poorly absorbed synthetic quaternary ammonium agents have actions that are limited to the sites of their deposition, namely the mouth where they inhibit salivary gland secretions, and the airways where they relax bronchial smooth muscle. For reasons that are not well understood, they tend not to inhibit respiratory gland secretions nor mucociliary clearance. Unlike other bronchodilators such as b-adrenergic agents or methyl xanthines, inhaled anticholinergic agents have no demonstrated effects other than as airway smooth muscle relaxants. They do not have clinically significant anti-inflammatory effects. They do not have significant cardiac, or pulmonary vascular actions, nor systemic effects. They have not been shown to have long-term effects on disease progression (although trials with tiotropium that explore such an effect are underway at present). Cholinergic Activity in the Lungs

In humans, airway caliber is largely controlled by the cholinergic parasympathetic system via branches of the vagus nerve, which stimulates smooth muscle contraction. At rest, parasympathetic activity provides bronchomotor tone, a low level of bronchial smooth muscle activity. There is evidence that resting bronchomotor tone is increased in both asthma and COPD. In addition to supplying bronchomotor tone, cholinergic activity can also be reflexly increased by numerous stimuli such as inhalation of cold dry air, mechanical factors, irritant particles, aerosols and gases, and specific mediators such as histamine and bronchoconstricting eicosanoids. Clinically, the use of an anticholinergic agent aims both to abolish cholinergic tone of airway smooth muscle and to inhibit the phasic increases in reflex bronchoconstriction.

Three muscarinic receptor subtypes, called Ml, M2, and M3, are expressed in human lungs (Figure 2). M1 receptors are found in peribronchial ganglia, and M3

Preganglionic nerve

Ganglion

M2

Postganglionic nerve

Available Agents

In the US, ipratropium bromide is available as a metered-dose inhaler (MDI) and as a nubilizable solution. Combinations with albuterol are also available in both forms. Its duration of action, both as monotherapy and as a combination, is 4–6 h. Tiotropium bromide is available as a dry powder inhaler; its duration of action is greater than 24 h, probably at least 48 h. Oxitropium bromide is available in several countries outside the US, and has a duration of action of 6–8 h. All of these agents are functionally selective for M1 and M3 muscarinic receptor subtypes, and tend to spare M2 receptors by dissociating rapidly from the latter. The half-life of the tiotropium– M3 receptor complex is 35 h compared with 0.3 h for ipratropium. Apart from tiotropium’s somewhat slower onset of action, much longer duration of action, and possibly slightly greater bronchodilator action at peak effect, the actions and side effects of each of these agents are very similar.

Clinical Role and Uses

Muscarinic Receptors in Lungs

M1

receptors are present on bronchial smooth muscle cells. Both are believed to mediate bronchomotor tone and reflex bronchoconstriction. M2 receptors, in contrast, are located on postganglionic autonomic fibers and are believed to be autoreceptors whose stimulation provides feedback inhibition of further acetylcholine release from terminals of these nerves, tending to limit vagally mediated bronchoconstriction. An implication of this scheme is that M1 and M3 receptors would be appropriate targets for anticholinergic bronchodilators, while M2 receptors should be spared. Indeed, there is evidence that M2 receptors are selectively damaged by certain viruses as well as eosinophil products, perhaps accounting for the bronchospasm associated with viral infections and asthma.

M3

Smooth muscle

Figure 2 The presumed location of muscarinic receptors in the parasympathetic pathway of human lungs.

Stable COPD

The major clinical use of inhaled anticholinergic agents is for the routine treatment of stable COPD. Both ipratropium, given three to four times daily, and tiotropium given once daily qam, are approved and recommended by current guidelines as options for first-line therapy for stable COPD of all degrees of severity. In practice, tiotropium is generally preferred because its prolonged duration of action provides more consistent bronchodilation around the clock, and its once-daily use is more convenient for patients. The onset of bronchodilation is slower than with inhaled short-acting b-adrenergic agents, peak effects being reached at 30–60 min with ipratropium

BRONCHODILATORS / Anticholinergic Agents 287

and at about 3–4 h with tiotropium. The magnitude of bronchodilation, measured as the peak of FEV1 improvement, is very approximately 0.15–0.25 l following a single dose of 40 mg ipratropium by MDI, 0.30 l following 400 mg of ipratropium by nebulization, and 0.30 l following 18 mg of tiotropium dry powder inhaler (DPI). Uniquely, regular use of tiotropium results in a rise in the prebronchodilator (trough) FEV1 of 0.1–0.15 l after about 1 week of once-daily use, and a peak FEV1 level (3 h after the morning dose) that is about 0.05 l higher than that after the first day of use, indicating a further benefit with regular use. These improvements with tiotropium compare favorably with those obtained with long-acting b-adrenergic bronchodilators. In parallel with improvements in FEV1, a decrease in functional residual capacity (a marker of hyperinflation) occurs by 4 weeks of regular treatment with tiotropium. No tachyphylaxis has been observed with long-term use of anticholinergic agents. In addition to these effects on lung function, measurable symptomatic improvements have been consistently reported with regular use of tiotropium such as clinically significant improvements in dyspnea, exercise capacity, health status (quality of life), and most importantly, an approximately 20–25% reduction in the frequency of acute exacerbations of COPD. The mechanism of the latter, which is shared by some other treatments for COPD, is not well understood. Acute Exacerbations of COPD

Studies comparing bronchodilator treatments for acute exacerbations of COPD do not show consistent benefit for treatments that include anticholinergic agents. Nevertheless, most current guidelines recommend the addition of ipratropium to a short-acting b-agonist in the initial treatment regimen for these serious events. Tiotropium is not appropriate as monotherapy for these events; however, it should be continued through the exacerbation when patients have been receiving it already.

agent. It has not been possible to predict reliably which asthmatics will benefit from ipratropium other than by an individual trial. Ipratropium may also be a useful alternative bronchodilator for those rare asthmatic patients who cannot tolerate the palpitations or tachycardia that a b-adrenergic agent may produce. The role of tiotropium has not been studied in asthma. Acute Severe Asthma

Although short-acting b-adrenergic agents are the bronchodilators of choice in the initial management of acute severe asthma, several studies and a metaanalysis suggest that the inclusion of ipratropium in the initial treatment of these serious events provides more rapid improvement in lung function and may avoid prolonged emergency room treatments and hospitalization. Current guidelines recommend that ipratropium (usually by nebulization) be added when the response to initial treatments with a short-acting b-agonist is ‘less than complete’ or ‘poor’. In practice, it is very common for the combination of albuterol and ipratropium to be given routinely as initial treatment for episodes of acute severe asthma, at least for the first few treatments. Pediatric Airways Disease

For pediatric asthma, both stable and acute severe forms, the role of ipratropium is similar to that in adults. Although studies with adequate statistical power or long duration in children are lacking, adult doses have been used without adverse effects in children down to 4 years of age. As in adults, anticholinergic agents have a very limited role in asthma. The role of tiotropium has not been studied in pediatric asthma. There are occasional reports of the use of ipratropium in other pediatric diseases such as cystic fibrosis, viral bronchiolitis, exercise-induced asthma, and bronchopulmonary dysplasia. Although these sometimes suggest a benefit from the use of ipratropium, the body of evidence is insufficient to make any recommendation in these conditions.

Stable Asthma

Numerous studies have compared ipratropium with a variety of short-acting b-adrenergic agents for the regular treatment of stable asthma. Almost uniformly, these reports show that ipratropium is a less potent bronchodilator than a short-acting b-adrenergic agent in asthmatic patients. No anticholinergic agent has been approved for use in asthma in the US. However, a few patients with otherwise typical asthma respond well to it, and it may occasionally add to the bronchodilation obtained with an adrenergic

Side Effects and Adverse Events Being very poorly absorbed, inhaled anticholinergic agents have a very wide therapeutic margin and are very well tolerated, even in rare instances when massive doses have accidentally been given. In normal clinical use, dryness of the mouth is common to all agents in this class but is rarely sufficiently severe for the patient to discontinue use. Bad taste is an occasional complaint, as is a brief coughing spell shortly

288 BRONCHODILATORS / Beta Agonists

after inhalation. One serious but rare idiosyncratic effect is paradoxical bronchospasm whose mechanism is unknown. It has been reported to occur in perhaps 0.3% of patients following use of any of the above anticholinergic bronchodilators. The drug should be discontinued in patients who experience wheezing, chest tightness, and dyspnea within an hour of the inhalation. Ipratropium was extensively studied for possible adverse effects on mucociliary clearance from the lungs, urinary outflow, and increased intraocular pressure (well-known side effects of atropine). These were found not to be problems with inhaled ipratropium, nor have they been found with oxitropium or tiotropium. However, each of these agents can cause dilatation of the pupil and, possibly, precipitate acute glaucoma, if they are placed directly onto the eye. Care should be taken that neither the mist from a nebulized anticholinergic treatment nor even minute amounts of tiotropium dry powder accidentally find their way into the eye. See also: Acetylcholine. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview.

Further Reading Cattapan SE and Gross NJ (2002) Anticholinergic agents. In: Barnes PJ, Drazen JM, Rennard S, and Thomson NC (eds.) Asthma and COPD: Basic Mechanisms and Clinical Management, pp. 527–534. New York: Academic Press. Celli BR and MacNee W (2004) Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. European Respiratory Journal 23: 932–946. Coulson FR and Fryer AD (2003) Muscarinic acetylcholine receptors and airway diseases. Pharmacology & Therapeutics 98: 59–69. Global Initiative for Chronic Obstructive Lung Disease (2003) Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: Update. Bethesda: National Institutes of Health, National Heart, Lung, and Blood Institute. Gross NJ (2004) Tiotropium bromide. Chest 126: 1946–1953.

airway epithelial cells. Drugs targeted at b2-adrenoceptors fall into two main classes: short-acting b-agonists (SABAs) such as salbutamol and terbutaline and long-acting b2-agonists (LABAs) such as salmeterol and formoterol. These agents are in general composed of a benzene ring with a chain of two carbon atoms and either an amine head group or a substituted amine head group. Beta2-agonists interact selectively at the b2-adrenoceptor to stimulate elevation in cell cyclic AMP content which is responsible for the majority of downstream signaling events following from receptor stimulation. The major clinical effect of b2-agonists is airway smooth muscle relaxation which produces the acute bronchodilator effects of these drugs. In addition, b2adrenoceptor agonists are bronchoprotective agents, that is, they inhibit bronchoconstriction induced by irritant challenge, for example, with histamine or methacholine. The b2-adrenoceptor is highly polymorphic and potential functional effects which may be of clinical relevance have been described for some of these polymorphisms. SABAs are first-line bronchodilators agents used in the management of asthma: LABAs should be used in the management of moderately severe asthma in combination with inhaled steroids. Side effects of these agents include tremor, tachycardia, and hypokalemia: there have also been concerns that regular use of some b2-adrenoceptor agonists (e.g., fenoterol) may be associated with asthma exacerbation and very rarely death.

Introduction Beta-adrenoceptors were initially separated into b1- and b2-subtypes based upon differences in physiological response profiles in the heart, blood vessels, and airways. Subsequently a b3-subtype was identified and shown to be present in a number of tissues, most notably adipocytes. The main consequences of activation of these three subtypes are shown in Table 1. Given the profile of receptor mediated effects shown in Table 1, the most effective b-agonists in the management of airway disease can be seen to be selective b2-adrenoceptor agonists. These fall into two main groups: the short-acting b2-agonists (SABAs) and long-acting b2-agonists (LABAs). Frequently used SABAs include salbutamol (albuterol) and terbutaline, whilst the most frequently used LABAs

Table 1 Effects of the three subtypes of b-adrenoceptor

Beta Agonists I P Hall, Nottingham University, Nottingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Beta2-adrenoceptor agonists are the mainstay bronchodilator agents used in the treatment of asthma and chronic obstructive pulmonary disease. Three b-adrenoceptors have been characterized using molecular pharmacology and molecular physiological approaches: b2-adrenoceptors are the major receptor subclass mediating both bronchodilatation and effects on

b1-Adrenoceptor

b2-Adrenoceptor

b3-Adrenoceptor

Tachycardia Positive inotropy

Bronchodilatation Inhibition of mediator release from mast cells Mucus secretion Increased ciliary beat frequency Relaxation of uterine smooth muscle Venous and arterial dilatation

Lipolysis

BRONCHODILATORS / Beta Agonists 289

are formoterol and salmeterol. In the 1960s–70s other drugs were used, including isoprenaline and fenoterol. However, because of concerns over an increase in asthma deaths associated with use of the nonselective b-agonist isoprenaline, this is no longer in general use in clinical practice: similar concerns regarding asthma deaths associated with fenoterol led to its withdrawal from the market in New Zealand and some other countries in the 1990s. Beta2-adrenoceptor agonists can also be classified according to whether they are partial agonists or full agonists. In most assay systems salbutamol and salmeterol act as partial agonists whereas terbutaline and formoterol are generally full or near full agonists. A number of novel b-adrenoceptor agonists are currently in development. In general these are full agonists with prolonged duration of action.

kinase A activation. In airway smooth muscle, these effects include phosphorylation of key targets, including myosin light chain kinase and calcium activated potassium channels, increased calcium sequestration and/or removal and inhibition of contractile signaling pathways (Figure 2). However, the correlation between whole cell cyclic AMP content and physiological effects (e.g., smooth muscle relaxation) is not precise which has led to the suggestion that some effects of receptor stimulation are cyclic AMP independent. These effects could potentially be mediated directly by the a-subunit of Gs or by non-Gs coupled pathways. This may be relevant to some of the longer-term effects of b2-adrenoceptor stimulation on gene transcription.

Chemical Structure

Beta2-adrenoceptor agonists have two main clinically relevant effects. First, they provide the mainstay bronchodilator therapy used in the management of asthma and other airway diseases where reversible airflow obstruction may be present, for example, chronic obstructive pulmonary disease (COPD). As required SABAs are first-line agents for the management of mild asthma: most guidelines (e.g., those produced by the British Thoracic Society) suggest ‘as required’ usage is appropriate as monotherapy as long as patients are not requiring more than an average of one dose per day. For those patients requiring frequent doses (i.e., more than one daily of SABAs), an inhaled steroid should be added to the treatment. In general, the LABAs should be used for those patients who are inadequately controlled on moderate doses of inhaled steroid (typically 400– 800 mg of inhaled beclomethasone or equivalent) where control is inadequate. These drugs should not be used as monotherapy. Salbutamol, terbutaline, salmeterol, and formoterol are all available in both aerosol form administered via metered dose inhalers (MDIs) and also as dry powder formulations in most countries. Beta2-agonists are also used in the management of acute asthma where they are generally given by nebulizer (salbutamol or terbutaline) or in severe acute asthma by intravenous infusion. In the past, oral preparations of both salbutamol and terbutaline were used in the management of asthma but these have been superseded by long-acting inhaled formulations. A very small number of patients have been treated with subcutaneous b2-agonists (usually terbutaline): whilst there are anecdotal reports of benefit there are no high-quality randomized control trials utilizing this

The chemical structure of the four most frequently used b-agonists is shown in Figure 1. Most are based on the naturally occurring catecholamine epinephrine. The structure activity relationship of these drugs depends on the presence or substitution of hydroxyl (OH) groups on the benzene ring and on the substituted head group. Substitution of the hydroxyl groups at position 3 and 4 on the benzene ring generally results in production of a less potent catecholamine: however, these drugs are more resistant to breakdown by catechol-O-methyl transferase (COMT). Substitutions on the a carbon atom prevent oxidation by monoamine oxidase (MAO). The action of b-agonists within tissue is terminated as a consequence of uptake into sympathetic nerve endings (uptake 1) or uptake into other innervated tissues such as smooth muscle (uptake 2) and subsequent degradation by COMT and MAO. Beta-adrenoceptor agonists can also be conjugated to sulfates or glucuronides in the liver, gut wall, and lung.

Mode of Action Beta2-adrenoceptor agonists mediate clinical effects by stimulation of the b2-adrenoceptor. This receptor is one of the superfamilies of G-protein-coupled receptors (GPCRs). The b2-adrenoceptor is coupled via the stimulatory G protein Gs to adenylate cyclase. Increases in adenylate cyclase activity result in elevation of intracellular cyclic AMP content and subsequently activation of protein kinase A. The majority of the downstream events stimulated by b2-agonists are therefore a consequence of protein

Role of b2-Adrenoceptor Agonists in Respiratory Medicine

290 BRONCHODILATORS / Beta Agonists HO OH HO

CHCH2NHCH3

Epinephrine H H CHOH−CH2−NH–C–CH2

HO

OCH3

CH3 OHCHN Formoterol HOH2C OH HO

CH3

CHCH2NH–C–CH3 CH3

Salbutamol CH(OH)CH2NH(CH2)6O(CH2)4Ph

CH2OH OH Salmeterol HO OH

CH3

CHCH2NH–C–CH3 CH3 HO Terbutaline Figure 1 Structure of a range of b-agonists.

approach which should only be initiated by specialist asthma clinics dealing with severe patients. The use of SABAs and LABAs is also recommended in most COPD guidelines: again SABAs should be used for rapid relief of symptoms and

LABAs reserved for patients with more severe disease. In general, the therapeutic effect in terms of both bronchodilatation and symptom relief is much smaller than in true asthma. Some patients with severe COPD have been treated with regular (four

BRONCHODILATORS / Beta Agonists 291
2AR

Gs

AC

ATP

PKA activation

cAMP

Downstream effects (e.g., relaxation of airway smooth muscle)

5′AMP Phosphodiesterases Figure 2 Activity of b2-agonists. AC, adenylate cyclase; AMP, adenosine monophosphate; ATP, adenosine triphosphate; b2AR, b2-adrenoceptor; Gs, stimulatory G protein; PKA, protein kinase A.

times daily) nebulized b2-agonists (sometimes in combination with an anticholinergic); this approach should be reserved for patients with severe disease and where there is objective evidence of benefit. In addition to the bronchodilator effects of b2-agonists, this class of drugs also protects against the actions of bronchoconstrictor stimuli; this may be part of the basis for the beneficial effects of LABAs seen when used regularly. These bronchoprotective effects can be readily demonstrated in the lung function laboratory by the administration of b2-agonists before bronchial provocation tests. The degree of protection seen is maximal within the first 12–24 h when regular dosing is instituted with both SABAs and LABAs; subsequently, the degree of bronchoprotection afforded falls somewhat although still remains above placebo levels. Contraindications

In general, b2-agonists are safe and effective drugs in the management of reversible airflow obstruction. However, there have been a number of concerns over the last 50 years regarding their usage. Arrhythmias have been reported following both intravenous and inhaled administration of b2-agonists. These are probably due to a direct effect of b2-adrenoceptor agonist stimulation in the heart and in addition hypokalemia which occurs as a consequence of stimulation of sodium potassium ATPase. These effects are likely to be related to relative selectivity for b2- over b1-receptors and dose; it is possible that arrhythmias were related to the excess of asthma deaths seen following the introduction of isoprenaline and fenoterol in the 1960s–70s. Troublesome side effects with b2-agonists are relatively rare. The main limiting factor is tremor which again is dose related. There have been persistent concerns regarding regular usage of SABAs and LABAs in terms of over all

asthma control. These concerns arose from a number of studies where regular (generally four times a day) SABAs were used: in these studies overall asthma control and/or lung function deteriorated in groups taking regular as opposed to as required treatment. These studies were in part responsible for the advice to use SABAs on an as-required rather than regular basis in the management of asthma. Very recently these concerns have extended to LABAs resulting in a black box warning from the FDA. One possible explanation underlying this effect is the presence of polymorphic variation within the b2-adrenoceptor. The receptor is highly polymorphic, with nine known single nucleotide polymorphisms (SNPs) in its coding region. Four of these code for amino acid substitutions of which three may have functional relevance (Arg/Gly16, Gln/ Glu27, Thr/Ile164). The Thr164 polymorphism alters agonist-binding properties of the receptor but is rare (allelic frequency 3% in Caucasian populations). The Arg16 polymorphism has been associated in several studies with worse overall asthma control and in a recent prospective trial individuals given regular salbutamol who were Arg16 homozygous showed markedly reduced responses compared with individuals homozygous for the Gly16 form of the receptor. Arg16 homozygous individuals account for roughly 15% of the Caucasian population. See also: Adrenergic Receptors. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. G-Protein-Coupled Receptors. Leukocytes: Mast Cells and Basophils.

Further Reading British Thoracic Society, Scottish Intercollegiate Guidelines Network (2003) British guideline on the management of asthma. Thorax 58(supplement I): 1–94. Drazen JM, Israel E, Boushey HA, et al. (1996) Comparison of regularly scheduled with as-needed use of albuterol in mild asthma. New England Journal of Medicine 335: 841–847.

292 BRONCHODILATORS / Theophylline Fenech A and Hall IP (2002) Pharmacogenetics of Asthma. British Journal of Clinical Pharmacology 53(1): 3–15. Hall IP (2004) The beta-agonist controversy revisited. Lancet 363(9404): 183–184. Hall IP and Tattersfield AE (1992) b-agonists. In: Clark TJ, Godfrey S, and Lee T (eds.) Asthma, 3rd edn., pp. 341–366. London: Chapman and Hall. Israel E, Chinchilli VM, Ford JG, et al. (2004) Use of regularly scheduled albuterol treatment in asthma: genotype-stratified, randomised, placebo-controlled cross-over trial. Lancet 364: 1505–1512. Kobilka BK, Dixon RAF, Frielle T, et al. (1987) cDNA for the human b2 adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proceedings of the National Academy of Sciences of the United States of America 84: 46–50. National Collaborating Centre for Chronic Conditions (2004) Chronic obstructive pulmonary disease: national clinical guideline on management of chronic obstructive pulmonary disease in adults in primary and secondary care. Thorax 59(supplement I): 1–232. Reihsaus E, Innis M, MacIntyre N, and Liggett SB (1993) Mutations in the gene encoding for the beta 2-adrenergic receptor in normal and asthmatic subjects. American Journal of Respiratory Cell and Molecular Biology 8: 334–339. Sears MR, Taylor DR, Print CG, et al. (1990) Regular inhaled bagonist treatment in bronchial asthma. Lancet 336: 1391–1396. Tattersfield AE, Knox AJ, Britton JR, and Hall IP (2002) Asthma. Lancet 360: 1313–1322. van Schayck CP, Dompeling E, van Herwaarden CLA, et al. (1991) Bronchodilator treatment in moderate asthma or chronic bronchitis: continuous or on demand? A randomized controlled study. British Medical Journal 303: 1426–1431. Vathenen AS, Higgins BG, Knox AJ, et al. (1988) Rebound increase in bronchial responsiveness after treatment with inhaled terbutaline. Lancet 1: 554–558.

Theophylline P J Barnes, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

not controlled by bronchodilator therapy. Side effects are related to plasma concentrations and include nausea, vomiting, and headaches due to phosphodiesterase inhibition and, at higher concentrations, to cardiac arrhythmias and seizures due to adenosine A1 receptor antagonism.

Theophylline has been used in the treatment of airway disease since 1930. Indeed, theophylline is still the most widely used antiasthma therapy worldwide because it is inexpensive. Theophylline became more useful with the availability of rapid plasma assays and the introduction of reliable slow-release preparations. However, the frequency of side effects and the relatively low efficacy of theophylline have recently led to reduced usage since inhaled b2 agonists are far more effective as bronchodilators and inhaled steroids have a greater anti-inflammatory effect. However, in patients with severe asthma and chronic obstructive pulmonary disease (COPD), it remains a very useful drug. There is increasing evidence that theophylline has an anti-inflammatory or immunomodulatory effect.

Chemical Structure Theophylline is a methylxanthine similar in structure to the common dietary xanthines, caffeine and theobromine (Figure 1). Several substituted derivatives have been synthesized but none has any advantage over theophylline, apart from the 3-propyl derivative enprofylline, which is more potent as a bronchodilator and may have fewer toxic effects. Many salts of theophylline have also been marketed, with the most common being aminophylline, which is the ethylenediamine salt used to increase solubility at neutral pH. Salts such as choline theophyllinate do not have any advantage and others, such as acepifylline, are virtually inactive so that theophylline remains the only methylxanthine in clinical use.

Mode of Action Abstract Theophylline is a methylxanthine that has been used to treat airway diseases for more than 70 years. It was originally used as a bronchodilator, but the relatively high doses required were associated with frequent side effects, so its use declined as inhaled b2 agonists became more widely used. Recently, it has been shown to have anti-inflammatory effects in asthma and chronic obstructive pulmonary disease (COPD) at lower concentrations. The molecular mechanism of bronchodilatation is non-specific phosphodiesterase inhibition, but the anti-inflammatory effect may be due to histone deacetylase activation, resulting in switching off of activated inflammatory genes. Theophylline is given systemically (orally as slow-release preparations for chronic treatment and intravenously for acute exacerbations of asthma), and blood concentrations are related to hepatic metabolism. This may be increased or decreased in several diseases and by concomitant drug therapy. Theophylline is usually used as an add-on therapy in asthma patients not well controlled on inhaled corticosteroids and in COPD patients with severe disease

Although theophylline has been in clinical use for more than 70 years, its mechanism of action is still uncertain. In addition to its bronchodilator action, theophylline has many other actions that may be relevant to its antiasthma effect (Figure 2). Many of these effects are seen only at high concentrations that far exceed the therapeutic range. There is increasing evidence that theophylline has anti-inflammatory effects, with a reduction in eosinophils and T lymphocytes in asthma and of neutrophils in COPD. Conversely, withdrawal of theophylline results in clinical deterioration and increased inflammation in patients with severe asthma and COPD. Several molecular mechanisms of action have been proposed for theophylline (Table 1). Phosphodiesterases, which break down cyclic nucleotides (cyclic

BRONCHODILATORS / Theophylline 293 O H3C

N N

O

O

H N

H3C

N

O

N N

CH3

O

CH3 N

H

N

O

CH3

Theophylline

CH3 N

N

N

N CH3

Caffeine

Theobromine

Figure 1 Structures of naturally occurring methylxanthines.

Agonist

Agonist

R

GC G

AC Theophylline

PDE3,4,7 ATP

PDE5

3′,5′cGMP

3′,5′cAMP +

AMP

Bronchodilatation

GMP

GTP

+

↓ Inflammatory cells

Figure 2 The inhibitory effect of theophylline on phosphodiesterases (PDE) may result in bronchodilatation and inhibition of inflammatory cells. ATP, adenosine triphosphate; AMP, adenosine monophosphate; PKA, protein kinase A; GTP, guanosine triphosphate; GMP, guanosine monophosphate; PKG, protein kinase G.

Table 1 Mechanisms of action of theophylline Phosphodiesterase inhibition (nonselective) Adenosine receptor antagonism (A1, A2A, and A2B receptors) Increased interleukin-10 release Stimulation of catecholamine (epinephrine) release Mediator inhibition (prostaglandins, tumor necrosis factor-a) Inhibition of intracellular calcium release Inhibition of NF-kB (decreased nuclear translocation) Increased apoptosis Increased histone deacetylase activity (increased efficacy of corticosteroids)

AMP and cyclic GMP) in the cell, are weakly inhibited by theophylline, and this accounts for the bronchodilator effect of theophylline (predominantly phosphodiesterase-3 (PDE3) and PDE5) (Figure 2). However, it is unlikely to account for the nonbronchodilator effects of theophylline that are seen at subbronchodilator doses. Theophylline inhibits adenosine receptors at therapeutic concentrations and an inhibitory action on A2B receptors may account for its inhibitory effect on mast cells. Adenosine antagonism is unlikely to account for the anti-inflammatory effects

of theophylline but may be responsible for serious side effects, including cardiac arrhythmias and seizures. Theophylline enhances the release of interleukin-10, which has anti-inflammatory effects from inflammatory cells, but this is secondary to PDE inhibition and requires high concentrations. It inhibits the proinflammatory transcription factor nuclear factor kappa B (NF-kB) but this also requires concentrations above the therapeutic range. Theophylline promotes apoptosis in eosinophils, neutrophils, and T lymphocytes associated with a reduction in the antiapoptotic protein Bcl-2. Recruitment of histone deacetylase-2 (HDAC2) by glucocorticoid receptors switches off inflammatory genes. Theophylline is an activator of HDAC at therapeutic concentrations, thus enhancing the anti-inflammatory effects of corticosteroids (Figure 3). This mechanism is independent of PDE inhibition or adenosine antagonism, and the antiinflammatory effects of theophylline are inhibited by an HDAC inhibitor trichostatin A. Low doses of theophylline increase HDAC activity in bronchial biopsies of asthmatic patients and correlate with the reduction in eosinophil numbers in the biopsy.

294 BRONCHODILATORS / Theophylline Inflammatory stimuli (e.g., IL-1
, TNF-)

NF-B

Corticosteroids

p65 p50

GR

Inflammatory protein (e.g., GM-CSF, IL-8)

+

Co-activators p65 p50

CBP

HAT Acetylation

↓ Inflammatory gene transcription

Theophylline

Gene activation

GR

HDAC Deacetylation

Gene repression

↓ Inflammatory gene transcription

Figure 3 Theophylline directly activates histone deacetylases (HDACs), which deacetylate core histones that have been acetylated by the histone acetyltransferase (HAT) activity of coactivators such as CREB-binding protein (CBP). This results in suppression of inflammatory genes and proteins, such as granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-8 (IL-8), that have been switched on by proinflammatory transcription factors such as nuclear factor kappa B (NF-kB). Corticosteroids also activate HDACs, but they do so through a different mechanism resulting in the recruitment of HDACs to the activated transcriptional complex via activation of the glucocorticoid receptors (GR), which function as a molecular bridge. This predicts that theophylline and corticosteroids may have a synergistic effect in repressing inflammatory gene expression.

Pharmacokinetics

Table 2 Factors affecting clearance of theophylline

There is a close relationship between improvement in airway function and serum theophylline concentration. Below 10 mg l 1 bronchodilatation is minimal and above 25 mg l 1 additional benefits are outweighed by side effects, so the therapeutic range for bronchodilatation was usually taken as 10– 20 mg l 1. It is now clear that theophylline has antiasthma effects other than bronchodilatation and that these may be seen below 10 mg l 1. A more useful therapeutic range is 5–15 mg l 1. The dose of theophylline required to give these therapeutic concentrations varies between subjects, largely because of differences in clearance. In addition, there may be differences in bronchodilator response to theophylline and, with acute bronchoconstriction, higher concentrations may be required to produce bronchodilatation. Theophylline is rapidly and completely absorbed, but there are large interindividual variations in clearance due to differences in hepatic metabolism (Table 2). Theophylline is metabolized in the liver by the cytochrome P450 microsomal enzyme system (mainly CYP1A2), and a large number of factors may influence hepatic metabolism. Increased clearance is seen in children (1–16 years old) and in cigarette and marijuana smokers. Concurrent administration of phenytoin and phenobarbitone increases activity of P450, resulting in increased metabolic breakdown so that higher doses may be required.

Increased clearance Enzyme induction (rifampicin, phenobarbitone, and ethanol) Smoking (tobacco and marijuana) High-protein, low-carbohydrate diet Barbecued meat Childhood Decreased clearance Enzyme inhibition (cimetidine, erythromycin, ciprofloxacin, allopurinol, zileuton, and zafirlukast) Congestive heart failure Liver disease Pneumonia Viral infection and vaccination High-carbohydrate diet Old age

Reduced clearance is found in liver disease, pneumonia, and heart failure, and doses need to be reduced to half and plasma levels monitored carefully. Increased clearance is also seen with certain drugs, including erythromycin, certain quinolone antibiotics (ciprofloxacin but not ofloxacin), allopurinol, cimetidine (but not ranitidine), fluoxamine, and zafirlukast, which interfere with cytochrome P450 function. Thus, if a patient on maintenance theophylline requires a course of erythromycin, the dose of theophylline should be halved. Viral infections and vaccination may also reduce clearance, and this may be particularly important in children. Because of these variations in clearance, individualization of

BRONCHODILATORS / Theophylline 295

theophylline dosage is required and plasma concentrations should be measured 4 h after the last dose with slow-release preparations when steady state has usually been achieved. There is no significant circadian variation in theophylline metabolism, although there may be delayed absorption at night, which may relate to the supine posture.

Role of Theophylline in Respiratory Medicine Acute Severe Asthma

In patients with acute severe asthma, intravenous aminophylline is less effective than nebulized b2 agonists and should therefore be reserved for those patients who fail to respond to b agonists. Theophylline should not be added routinely to nebulized b2 agonists since it does not increase the bronchodilator response and may only increase their side effects. It is of little proven benefit in exacerbations of COPD.

Theophylline is a less preferred option than inhaled corticosteroids and is recommended as a second-line choice of controller in the management of asthmatic patients. Although LABAs are more effective as an add-on therapy, theophylline is considerably less expensive and may be the only affordable add-on treatment when the costs of medication are limiting. Chronic Obstructive Pulmonary Disease

Theophylline is still used as a bronchodilator in COPD, but inhaled anticholinergics and b2 agonists are preferred. Theophylline tends to be added to these inhaled bronchodilators in more severe patients and has been shown to give additional clinical improvement when added to a LABA. A theoretical advantage of theophylline is that its systemic administration may have effects on small airways, resulting in a reduction of hyperinflation and thus a reduction in dyspnea. Sleep Apnea

Asthma

Theophylline has little or no effect on bronchomotor tone in normal airways but reverses bronchoconstriction in asthmatic patients, although it is less effective than inhaled b2 agonists and is more likely to have unwanted effects. Theophylline and b2 agonists have additive effects, even if true synergy is not seen, and there is evidence that theophylline may provide an additional bronchodilator effect even when maximally effective doses of b2 agonist have been given. This means that if adequate bronchodilatation is not achieved by a b agonist alone, theophylline may be added to the maintenance therapy with benefit. Addition of low-dose theophylline to either high- or low-dose inhaled corticosteroids in patients who are not adequately controlled provides better symptom control and lung function than doubling the dose of inhaled steroid, although it is less effective as an add-on therapy than a long-acting b2 agonist (LABA). Theophylline may be useful in patients with nocturnal asthma since slow-release preparations are able to provide therapeutic concentrations overnight, although it is less effective than a LABA. Studies have also documented steroid-sparing effects of theophylline. Although theophylline is less effective than a b2 agonist and corticosteroids, a minority of asthmatic patients appear to derive unexpected benefit, and even patients on oral steroids may show a deterioration in lung function when theophylline is withdrawn. Theophylline has been used as a controller in the management of mild persistent asthma, although it is usually found to be less effective than low doses of inhaled corticosteroids.

Theophylline has been used to treat sleep apnea in adults and apnea in neonates because it increases ventilation rate. However, there is no convincing evidence that it is effective for these indications, and it cannot be routinely recommended.

Adverse Effects and Contraindications Unwanted effects of theophylline are usually related to plasma concentration and tend to occur when plasma levels exceed 20 mg l 1. However, some patients develop side effects even at low plasma concentrations. To some extent, side effects may be reduced by gradually increasing the dose until therapeutic concentrations are achieved. The most common side effects are headache, nausea, and vomiting (due to inhibition of PDE4), abdominal discomfort, and restlessness (Table 3). There may also be increased acid secretion and diuresis (due to inhibition of adenosine A1 receptors). There was concern that theophylline, even at therapeutic concentrations, may lead to behavioral disturbance and learning difficulties in schoolchildren, although

Table 3 Side effects of theophylline Nausea and vomiting Headaches Gastric discomfort Diuresis Behavioral disturbance (?) Cardiac arrhythmias Epileptic seizures

296 BRONCHOMALACIA AND TRACHEOMALACIA

it is difficult to design adequate controls for such studies. At high concentrations, cardiac arrhythmias may occur as a consequence of PDE3 inhibition and adenosine A1 receptor antagonism, and at very high concentrations seizures may occur (due to central A1 receptor antagonism). Use of low doses of theophylline that give plasma concentrations of 5–10 mg l 1 largely avoids side effects and drug interactions and makes it unnecessary to monitor plasma concentrations (unless checking for compliance). Care must be taken when using theophylline in children, elderly patients, those taking drugs that inhibit cytochrome P450, and those with heart and liver disease because of the pharmacokinetic considerations discussed previously.

Future Directions The use of theophylline has been declining, partly because of the problems with side effects but mainly because more effective therapy with inhaled corticosteroids has been introduced. Oral theophylline is still a useful treatment in some patients with difficult asthma and appears to have effects beyond those provided by steroids. Rapid-release theophylline preparations are cheap and are the only affordable antiasthma medication in some developing countries. Theophylline has anti-inflammatory effects at doses that are lower than those needed for bronchodilatation, and plasma levels of 5–10 mg l 1 are now recommended instead of the previously recommended 10–20 mg l 1. Because the molecular mechanisms for the anti-inflammatory effects of theophylline are better understood (HDAC activation), there is a strong scientific rationale for combining low-dose theophylline with inhaled corticosteroids, particularly in patients with more severe asthma. The synergistic effect of low-dose theophylline and corticosteroids on inflammatory gene expression may

account for the add-on benefits of theophylline in asthma, whereas in COPD theophylline appears to overcome corticosteroid resistance in vitro and therefore may be a useful anti-inflammatory therapy in the future. New drugs based on this molecular action of theophylline may also be developed in the future. See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Cyclic Nucleotide Phosphodiesterases. Gene Regulation.

Further Reading Barnes PJ (1996) The role of theophylline in severe asthma. European Respiratory Review 6: 154S–159S. Barnes PJ (2003) Theophylline: new perspectives on an old drug. American Journal of Respiratory and Critical Care Medicine 167: 813–818. Barr RG, Rowe BH, and Camargo CA Jr (2003) Methylxanthines for exacerbations of chronic obstructive pulmonary disease: meta-analysis of randomised trials. British Medical Journal 327: 643. Cosio BG, Tsaprouni L, Ito K, et al. (2004) Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages. Journal of Experimental Medicine 200: 689–695. Hansel TT, Tennant RC, Tan AJ, et al. (2004) Theophylline: mechanism of action and use in asthma and chronic obstructive pulmonary disease. Drugs of Today (Barcelona) 40: 55–69. Kankaanranta H, Lahdensuo A, Moilanen E, and Barnes PJ (2004) Add-on therapy options in asthma not adequately controlled by inhaled corticosteroids: a comprehensive review. Respiratory Research 5: 17. Rabe K, Magnussen H, and Dent G (1996) Theophylline and selective PDE inhibitors as bronchodilators and smooth muscle relaxants. European Respiratory Journal 8: 637–642. Ram FS, Jones PW, Castro AA, et al. (2002) Oral theophylline for chronic obstructive pulmonary disease. Cochrane Database of Systematic Reviews CD003902. Shah L, Wilson AJ, Gibson PG, and Coughlan J (2003) Long acting beta-agonists versus theophylline for maintenance treatment of asthma. Cochrane Database of Systematic Reviews CD001281. Weinberger M and Hendeles L (1996) Theophylline in asthma. New England Journal of Medicine 334: 1380–1388.

BRONCHOMALACIA AND TRACHEOMALACIA P N Mathur and J R Ladwig, Indiana University Hospital, Indianapolis, IN, USA & 2006 Elsevier Ltd. All rights reserved.

tracheobronchomalacia. Diagnosis is best performed by bronchoscopy but improvements in computed tomography has made radiographic diagnosis easier. Treatment varies from various means of stenting the airway to surgical correction.

Abstract Tracheomalacia and bronchomalacia are a loss of cartilaginous support of the trachea and large airways. This can occur as a congenital defect or as a result of acquired disease processes. The loss of cartilaginous support leads to airway obstruction upon expiration. Symptoms of dyspnea and cough result from

Introduction Tracheomalacia is a condition of weakened cartilaginous support of the trachea. The term ‘bronchomalacia’ refers to loss of supportive structure for

BRONCHOMALACIA AND TRACHEOMALACIA 297

the large airways. With the loss of structural support, the trachea does not maintain its diameter and the membranous portion of the trachea collapses anteriorly, causing obstruction of airflow during expiration.

Etiology Tracheomalacia and bronchomalacia can be the result of multiple etiologies. In infants, the cause is often a congenital defect in cartilaginous development which usually resolves spontaneously as the child reaches 6 months of age and older. It may also be the result of congenital vascular rings causing compression of the airways resulting in abnormal development or erosion of cartilaginous rings. In adults, tracheobronchomalacia may also be the result of previously unrecognized congenital abnormalities, or acquired anatomic or pathologic processes. Substernal goiter may present with cough in tracheomalacia. The enlargement of thyroid tissue can lead to compressive erosion of tracheal rings. Neoplasm may also exert pressure on the tracheal rings or cause destruction by local invasion resulting in tracheobronchomalacia. Inflammatory processes may also be a cause of tracheomalacia. Relapsing polychondritis, an autoimmune disease directed against cartilage, may affect the larynx and cartilaginous rings of the large airways. It is most often seen in the fourth decade; airway symptoms are more common amongst women with the disease. Patients with severe chronic obstructive pulmonary disease (COPD) may develop atrophy of cartilage in the airway, leading to easy collapsibility of airways. The diagnosis of tracheomalacia in a patient with COPD may go unrecognized until progression of disease prompts bronchoscopy or other imaging. Patients who have had prolonged intubation or require tracheostomy may also develop tracheomalacia. The local pressure effect of the inflated balloon at the distal end of the tube can cause local inflammation, overdistention, or local tissue ischemia resulting in erosion of the cartilaginous rings.

Clinical Features Diagnosis of tracheobronchomalacia includes a thorough history and physical examination to exclude other conditions or elicit clinical features typical of tracheobronchomalacia. Clinical findings are nonspecific and often manifest in dyspnea on exertion and cough. The cough of tracheobronchomalacia has been characterized as seal-like. Diagnosis of tracheobronchomalacia is aided by pulmonary function testing, imaging, and direct visualization via bronchoscopy. Pulmonary Function Testing

Patients with tracheomalacia show reduced expiratory flow measurements. Flow–volume curves often demonstrate expiratory flow limitation (Figure 1). Inspiratory flow is preserved while the expiratory loop is similar to that of COPD. Diagnostic Imaging of the Trachea

As a general rule, plain chest radiographs are not useful for diagnosis of tracheomalacia. In extreme cases of airway narrowing, a large air column above the obstruction may be seen. A simple computed tomography (CT) scan of the chest may not be useful for tracheobronchomalacia. However, it is very useful for identification of

16.00

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Pathology Congenital tracheobronchomalacia represents a failure or incomplete development of the cartilaginous rings of the airway. In adults, tracheobronchomalacia is acquired more commonly as described above. The acquired insult often leads to compression and local destruction of the tracheal rings.

−16.00

Volume (l) Pre

Post

Figure 1 Expiratory flow measurements. Courtesy of C Mayer, University of Cincinnati.

298 BRONCHOMALACIA AND TRACHEOMALACIA

extrinsic causes of tracheal compression which may result in tracheobronchomalacia such as substernal goiter, vascular abnormalities, and neoplasm. One method of making CT more beneficial in diagnosing tracheobronchomalacia is to compare inspiratory and expiratory images. This aids in demonstrating the dynamic narrowing of the trachea and it may then be more obvious on the CT scan (Figures 2(a) and 2(b)). Newer advances in CT imaging show significant sensitivity and specificity for identifying airway stenosis. A ‘virtual bronchoscopy’ can be performed with the images acquired from a routine CT scan that is then reconstructed through computer software (Figures 2(c) and 2(d)). The imaging is enhanced when a multidetector CT image is used instead of a single detector one. There are different methods to render the data received by the CT scan. Data may be surface or volume rendered. Surface rendering is faster and requires less computer power. However, volume rendering provides more

detail of the mucosal-airway interface allowing more detail of the mucosa. Virtual bronchoscopy has been found to have high sensitivity (90%) and specificity (96.6%) for central airway stenosis. The virtual bronchoscopy provides the benefit of detailed imaging in patients that might not be candidates for routine bronchoscopy. It also provides the bronchoscopist a noninvasive method to acquire information allowing for planning prior to an interventional procedure or foregoing a procedure for lesions not amenable to repair. The gold standard diagnostic tool for tracheobronchomalacia is bronchoscopy. If a patient is able to tolerate bronchoscopy with minimal sedation, various respiratory maneuvers can be performed (forced expiration, cough) to exaggerated or elicit airway obstruction. Once the diagnosis of tracheobronchomalacia is made, consideration of therapeutic options is undertaken. Correction of underlying extrinsic processes (substernal goiter, vascular rings, and neoplasm),

Figure 2 (a) Inspiration, (b) expiration, (c) virtual bronchoscopy inspiration, (d) virtual bronchoscopy expiration. Courtesy of C Mayer, University of Cincinnati.

BRONCHOMALACIA AND TRACHEOMALACIA 299

systemic illness (relapsing polychondritis), and airway disease (COPD) should be treated appropriately. If the causative process cannot be eliminated or when the tracheomalacia already results in airway compromise, several options exist to palliate the obstruction.

airway pressure (CPAP) to maintain a patent airway. In one case of relapsing polychondritis, it provided a temporary measure, delaying the time to invasive procedures. If further investigation identifies the type of patient most likely to benefit from CPAP, it may prove a useful bridge prior to a more invasive therapy.

Management and Current Therapy Balloon Bronchoplasty

Surgery

Airways may be dilated by rigid or inflatable balloon dilators. The modality is best used for extrinsic or intrinsic compression of the airways. Balloon bronchoplasty is easily performed through a flexible bronchoscope. Alone, the modality does not provide sustained benefit. However, it has been described as a useful tool in optimizing the airway caliber prior to stent placement.

Multiple surgical approaches to tracheomalacia have been described. One surgical procedure used for treatment of tracheobroncomalacia is a membranous-wall tracheoplasty. This is done through a right thoracotomy. The procedure involves sewing a polypropylene mesh to the membranous wall of the trachea which then provides external support of the tracheal wall. Another external wall stent procedure has been investigated in an animal model using reabsorbable stents. Although still experimental, this may provide another alternative in the right patient population.

Airway Stents

Tracheobronchomalacia is one of the indications for airway stenting, as described by the ERS/ATS statement on interventional pulmonology. There are a number of different types of airway stents, each with pros and cons. Generally stents can be categorized as silicone, metal, or a hybrid of the two materials. Silicone stents have the benefit of being cheaper and more easily removed. However, they require knowledge of rigid bronchoscopy for placement and have higher rates of migration. Currently, the most widely used stent is the Dummon stent, which is a silicone stent that has studs on the outer surface to retard migration. Metal stents have the advantage of being placed via flexible bronchoscopy and are less prone to migration. Metal stents may be uncovered or covered by a silastic or polyurethane coating. However, uncovered stents should not be used in benign disease such as tracheobronchomalacia, as they allow ingrowth of malignant or granulation tissue. Metal stents are very difficult to remove, may fracture, and pose a risk if future interventional bronchoscopy is needed (laser, APC) because of the potential for conduction of current/heat or airway fire. In choosing a stent, the airway should be evaluated by bronchoscopy and/or CT scan to determine the extent of the obstruction. The optimal stent is one that exceeds the margins of the stenosis and whose diameter is larger than that of the airway. Placement of a stent is expected to bridge the extent of the abnormal airway. Inadequate stenting may only result in moving the ‘choke point’, the area of airflow obstruction, distal to the stent. One method of noninterventional stenting still under investigation is the use of continuous positive

See also: Bronchiectasis. Bronchoscopy, General and Interventional. Relapsing Polychondritis. Trauma, Chest: Postpneumonectomy Syndrome. Upper Airway Obstruction.

Further Reading Adliff M, et al. (1997) Treatment of diffuse tracheomalacia secondary to relapsing polychondritis with continuous positive airway pressure. Chest 112: 1701–1703. Aquino SL, Shepard J-AO, Ginns LC, et al. (2001) Acquired tracheomalacia: detection by expiratory ct scan. Journal of Computer Assisted Tomography 25(3): 394–399. Bolliger CT and Mathur PN (2002) ERS/ATS statement on interventional pulmonology. European Respiratory Journal 19: 356–373. Davis S, et al. (1998) Effect of continuous positive airway pressure on forced expiratory flows in infants with tracheomalacia. American Journal of Respiratory and Critical Care Medicine 158: 148–152. Ernst A, et al. (2004) Central airway obstruction. American Journal of Respiratory and Critical Care Medicine 169: 1278–1297. Hoppe H, et al. (2004) Grading airway stenosis down to the segmental level using virtual bronchoscopy. Chest 125: 704–711. Hopper KD, et al. (2000) Mucosal detail at CT virtual reality: surface versus volume rendering. Radiology 214: 517–522. Miyazawa T, et al. (2004) Stenting at the flow-limiting segment in tracheobronchial stenosis due to lung cancer. American Journal of Respiratory and Critical Care Medicine 169: 1096–1102. Murray JF and Nadel JA (eds.) Textbook of Respiratory Medicine, 3rd edn., pp. 1368–1373. Sewall A, Gregory K, et al. (2003) Comparison of resorbable poly-L-lactic acid – polyglycolic acid and internal palmaz stents for the surgical correction of severe tracheomalacia. The Annals of Otology, Rhinology, and Laryngology 112: 515–521. Wright CD (2003) Tracheomalacia. Chest Surgery Clinics of North America 13: 349–357.

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BRONCHOPULMONARY DYSPLASIA E Bancalari, University of Miami School of Medicine, Miami, FL, USA

90 80

& 2006 Elsevier Ltd. All rights reserved.

Oxygen at 28 d Oxygen at 36 wks PMA

70

Bronchopulmonary dysplasia (BPD) is one of the most common sequelae in premature infants who survive after prolonged mechanical ventilation. Its pathogenesis is multifactorial and includes prematurity, perinatal infections, pulmonary volutrauma, oxygen toxicity, and increased pulmonary blood flow due to patent ductus arteriosus. It is characterized by chronic respiratory failure with abnormalities in lung function that can persist into adulthood. This is due to severe alterations in lung development characterized by decreased alveolar and capillary formation plus emphysema, fibrosis, and airway obstruction in more severe cases. The prevention of BPD is based on the avoidance of all the factors that are implicated in its pathogenesis.

Bronchopulmonary dysplasia (BPD) was first described by Northway and co-workers in 1967 and has become a major complication in premature infants who require prolonged mechanical ventilation. The natural course of severe respiratory distress syndrome (RDS) in the premature infant was altered in the 1960s by the introduction of mechanical ventilation. As a result, smaller and sicker infants survive, but many of these survivors are left with chronic lung damage. These infants frequently remain oxygen- and ventilator-dependent for prolonged periods of time, and the mortality rate can be as high as 30–40%. In surviving infants, pulmonary function may remain abnormal for years, and they have increased risk of neurodevelopmental sequelae and impaired growth curves. The incidence of BPD is closely and inversely related to both, birth weight and gestational age (Figure 1). For this reason, with increasing survival of extremely premature infants, the number of patients at risk for developing BPD has increased.

Pathology Most of the pathologic descriptions of BPD represent the most severe form of chronic lung damage since those infants with milder forms of BPD rarely die. Macroscopically, the lungs are firm, heavy, and dark in color, with a grossly abnormal appearance showing emphysematous areas alternating with areas of collapse. Histologically, the lungs show areas of emphysema, with abnormal alveoli that have coalesced into larger cystic areas, surrounded by areas of atelectasis (Figure 2). Widespread bronchial and

60 BPD (%)

Abstract

50 40 30 20 10 0

501−750

751−1000 1001−1250 1251−1500 501−1500

Birth weight (g) Figure 1 Incidence of BPD (% of total births) in the NICHD Neonatal Research Network, Jan. 1995 to Dec. 1996. PMA, post menstrual age. Reproduced from Lemons J, Bauer CR, Oh W, et al. (2001) Very low birth weight outcomes of the National Institute of Child Health and Human Development Research Network January 1995 through December 1996. Pediatrics 107: 1–8.

Figure 2 Low-magnification view from a lung with BPD showing areas of emphysema alternating with areas of partial collapse. Reproduced from Kluwer Academic Publishers Advances in Perinatal Medicine, 1982, p. 181, Barotrauma to the lung, Bancalari E and Goldman SL, figure 29, with kind permission of Springer Science and Business Media.

bronchiolar mucosal hyperplasia and metaplasia reduce the lumen in many small airways. In addition, there is interstitial edema and an increase in fibrous tissue with focal thickening of the interstitial spaces. Lymphatics may be dilated and torturous. There may be vascular evidence of pulmonary hypertension, such as medial muscle hypertrophy, elastic degeneration, and reduction in the branching of the pulmonary vascular bed. The heart frequently shows evidence of biventricular hypertrophy. Infants dying with severe BPD show a marked reduction in the number of alveoli and capillaries, with a reduction in the gas exchange surface area. Infants who have mild

BRONCHOPULMONARY DYSPLASIA 301

BPD at the time of death have only mild or moderate alveolar septal fibrosis, lack of severe airway epithelial lesions, and normal-appearing pulmonary vessels. Nonetheless, they show evidence of inhibition of acinar and capillary development.

Clinical Features The diagnosis of BPD is based on clinical and radiographic manifestations, which are not specific. With rare exceptions, BPD occurs in preterm infants and is preceded by the use of prolonged mechanical ventilation. Radiographic findings include hyperinflation and nonhomogeneity of pulmonary tissues, with fine or coarser densities extending to the periphery in the more severe forms (Figure 3). In milder forms, the radiographic changes are also milder, revealing mainly diffuse haziness. Once lung damage has occurred, these infants continue to require mechanical ventilation and increased oxygen concentration for months or sometimes years. Because of the increased use of antenatal steroids and exogenous surfactant, many small infants have mild initial respiratory disease and require ventilation with low pressures and oxygen concentration. After a few days or weeks of mechanical ventilation, these infants frequently show progressive deterioration in lung function, an increase in their ventilatory and oxygen requirements accompanied by signs of respiratory failure, and ultimately develop BPD.

This deterioration is frequently triggered by bacterial or viral infections or respiratory failure secondary to a patent ductus arteriosus (PDA). Infants with BPD who survive show a slow but steady improvement in lung function and roentgenographic changes, with gradual weaning from ventilator and oxygen therapy. Signs of respiratory distress, such as chest retractions and tachypnea, frequently persist long after extubation. More severe BPD may evolve into progressive respiratory failure, and even death, as a result of severe lung damage and pulmonary hypertension or intercurrent infections. Some of these infants may also develop anastomoses between systemic and pulmonary circulations, which may aggravate their pulmonary hypertension. Acute pulmonary infection, either bacterial or viral, frequently complicates the course of BPD and, in many cases, is the precipitating cause of death. Because of chronic hypoxia and higher energy expenditure required by their increased work of breathing, weight gain in infants with BPD is usually below normal, even when receiving appropriate caloric intake for their age. In addition, poor feeding tolerance and a tendency toward fluid retention and pulmonary edema often requires fluid restriction as well as diuretic therapy, further limiting the calories that can be provided.

Pathogenesis Because BPD occurs almost exclusively in premature infants who have received mechanical ventilation and increased oxygen concentrations, prematurity, baro/volutrauma, and oxygen toxicity have been considered crucial factors in BPD pathogenesis. Other factors that may play an important role in BPD pathogenesis include perinatal infections, pulmonary edema resulting from a PDA or excessive fluid administration, and nutritional deficiencies. Prematurity

BPD occurs almost exclusively in the extremely premature infant. This suggests that the vulnerability of the very preterm infant may be related to the immature state of lung development. It is likely that premature birth followed by therapeutic interventions disrupts lung development during this crucial period, producing an interruption and/or disruption in the development of alveoli, capillaries, and lung matrix. Barotrauma/Volutrauma Figure 3 Chest radiograph from an infant with bronchopulmonary dysplasia, showing areas of mild hyperinflation alternating with coarse densities.

Almost all infants who develop BPD have received prolonged mechanical ventilation; therefore, it is

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likely that mechanical trauma also plays an important role in the pathogenesis of BPD. The presence of an endotracheal tube may also contribute to BPD pathogenesis by increasing the risk of pulmonary infections. Studies on preterm experimental animals have demonstrated that only a few breaths with excessive tidal volumes given prior to surfactant replacement produce lung damage with a decrease in lung compliance. Oxygen Toxicity

Pulmonary oxygen toxicity is also considered an important factor in the pathogenesis of BPD. High inspired oxygen concentrations can increase the production of cytotoxic oxygen free radicals, which overwhelm the antioxidant defenses in the capillary endothelial cells and the alveolar epithelial cells of the premature lung because of incomplete development of the pulmonary antioxidant enzyme system. The precise concentration of oxygen that is toxic to the premature lung is unknown, but it is possible that any concentration in excess of room air may increase the risk of lung damage when administered over prolonged periods of time. Infection and Inflammation

Evidence has been mounting to support a role for infection and inflammation in the pathogenesis of BPD, particularly in very small infants who develop BPD after receiving prolonged mechanical ventilation for poor respiratory effort rather than because of severe underlying lung disease. The evidence of an association between ureaplasma urealyticum tracheal colonization and the development of BPD has been inconsistent. However, maternal infections, specifically chorioamnionitis, are associated with an increased risk of BPD. Several inflammatory cytokines are present in higher concentrations in fetal cord blood and in the amniotic fluid of mothers who deliver infants who subsequently develop BPD. An inflammatory response in the lung can also be triggered by other factors, including oxygen free radicals, ventilation with excessive tidal volumes, and increased pulmonary blood flow due to a PDA. Among the markers of inflammation found in high concentrations in tracheobronchial secretions in infants who develop BPD are neutrophils, macrophages, leukotrienes, platelet-activating factor (PAF), interleukin (IL)-6, IL-8, and tumor necrosis factor. The increased concentration of inflammatory mediators may be responsible for the bronchoconstriction, vasoconstriction, and increased vascular permeability characteristic of these infants. The potential role of inflammation in the pathogenesis of

BPD is supported by the documented beneficial effects of steroids in these infants. Patent Ductus Arteriosus and Pulmonary Edema

Infants with RDS who receive greater fluid intake or do not have a diuretic phase in early life have a higher incidence of BPD. High fluid intake increases the incidence of PDA, which then produces increased pulmonary blood flow, an increase in interstitial lung fluid, and causes a decrease in pulmonary compliance and increased resistance. This deterioration in lung mechanics may prolong the requirement for mechanical ventilation and high inspired oxygen concentrations, thereby increasing the risk of BPD. In addition, the increased pulmonary blood flow can induce endothelial damage with neutrophil margination and activation in the lung and contribute to the progression of the inflammatory cascade. These interrelated factors may explain the strong association between the duration of the PDA and the increased risk of BPD. Infants with established BPD have a predisposition for fluid accumulation in their lungs. Capillary permeability may be increased due to the oxygen toxicity, volutrauma, or infection. Infants with BPD can also have increased plasma levels of vasopressin with a reduced urine output and decrease in free water clearance. With excessive accumulation of lung fluid, lung function is further compromised, perpetuating a cycle in which more aggressive respiratory support is required, thereby resulting in additional lung injury. Figure 4 shows the relative influence of different factors in the pathogenesis of BPD. Increased Airway Resistance

Infants with severe BPD frequently have a marked increase in airway resistance. This can impair distribution of the inspired gas and thereby favor uneven lung expansion. The airway obstruction may be secondary to bronchiolar epithelial hyperplasia, metaplasia, and mucosal edema, and it may also relate to pulmonary edema secondary to PDA or fluid overload. These infants may also have bronchoconstriction resulting from smooth muscle hypertrophy. Other possible factors producing increased airway resistance in infants with BPD include inflammatory mediators such as leukotrienes and PAF, which have been found in high concentrations in the airways of infants with BPD. Finally, tracheobronchomalacia, which may be present in some infants with severe BPD, can produce marked airway obstruction, particularly during periods of agitation and increased intrathoracic pressure.

BRONCHOPULMONARY DYSPLASIA 303 Other Factors

OR for BPD

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Figure 4 Perinatal and postnatal risk factors for the development of BPD defined as X28 days’ duration of oxygen-dependency during hospitalization. Obtained by logistic regression analysis from all extremely premature infants born at UM/JMH during the period 1995–2000 (N ¼ 505 alive at 28 days). Birth weight (Bw), 500–1000 g; gestational age (GA), 23–32 weeks; PDA, patent ductus arteriousus; RDS, respiratory distress syndrome; OR, odds ratio. Reproduced from Bancalari E, Claure N, and Sosenko IRS (2003) Bronchopulmonary dysplasia: changes in pathogenesis, epidemiology and definition. Seminars in Neonatology 8: 63, with permission from Elsevier.

Other factors have been proposed as having a pathogenetic role in BPD. One of these factors is a genetic predisposition to abnormal airway reactivity since there have been reports of a stronger family history of asthma in infants with BPD. Vitamin A deficiency may also play a role because infants who develop BPD have lower vitamin A levels than those who recover without BPD. This possible association is supported by the similarities between some of the airway epithelial changes observed both in BPD and in vitamin A deficiency and by clinical evidence that vitamin A administration in the first weeks of life reduces the incidence of BPD. Another factor suggested to play a role in the development of BPD is early adrenal insufficiency because infants with BPD have lower cortisol levels in the first week of life. Figure 5 summarizes the most important factors that contribute to the pathogenesis of BPD.

Prematurity-respiratory failure mechanical ventilation

Excessive tidal volume Decreased lung compliance Volutrauma

Increased inspired oxygen Deficient antioxidant systems Nutritional deficiencies Oxygen toxicity

Pre/postnatal infections PMN’s activation Inflammatory mediators elastase/proteinase inhibitors imbalance

Patent ductus arteriosus Excessive fluid intake

Increased pulmonary blood flow-lung edema

Acute lung injury inflammatory response

Airway damage Metaplasia Smooth muscle hypertrophy ↑ Mucus secretion

Airway obstruction Emphysema-atelectasis

Vascular injury Increased permeability Smooth muscle hypertrophy

Pulmonary edema Pulmonary hypertension

Bronchopulmonary dysplasia Figure 5 Factors contributing to the pathogenesis of bronchopulmonary dysplasia.

Interstitial damage ↑ Fibronectin ↑ Elastase ↓ Alveolar septation ↓ Vascular development

Matrix damage ↑ Fibrosis Decreased number of alveoli and capillaries

304 BRONCHOPULMONARY DYSPLASIA

Animal Models Because BPD is a chronic process with multiple factors involved in its pathogenesis, it has been extremely difficult to develop suitable animal models. However, there are two models that closely resemble human BPD. The first model was developed by Drs Robert de Lemos and Jacqueline Coalson in preterm baboons. When these animals are delivered at approximately 75% gestation and are subsequently ventilated over several weeks with positive pressure and high oxygen concentrations, they develop physiologic and morphologic changes that closely resemble those of human BPD. This is a very labor-intensive and expensive model that requires a full intensive care setting for these animals to survive. A similar model has been developed in preterm lambs that, after several weeks of mechanical ventilation, also develop histopathological changes very similar to human BPD. These models are used extensively by investigators who are exploring various aspects of lung development, injury, and repair as well as possible strategies to prevent BPD.

Prevention and Management Strategies to prevent BPD focus on eliminating or reducing the multiplicity of factors known to contribute to lung injury (Figure 5). Since lung immaturity is the main predisposing factor in the pathogenesis of BPD, prevention of BPD should start prenatally by attempting to avoid premature birth. When preterm birth is imminent, administration of antenatal steroids reduces the incidence and severity of RDS and the subsequent development of severe BPD. Minimizing volutrauma and exposure to high inspired oxygen concentrations is also critical to reduce BPD. Surfactant replacement therapy in infants with RDS improves their respiratory course, enabling mechanical ventilation with lower inspiratory pressures and oxygen concentrations. The use of positive end expiratory pressure (PEEP) is critical because insufficient PEEP is associated with increased ventilatorassociated lung damage. Ventilation with high frequency has not been conclusively shown to reduce BPD, although data from experimental animals suggest that this ventilation modality may reduce lung injury. Data demonstrating an increased risk of BPD in infants exposed to prenatal and postnatal infections suggest that prevention and aggressive management of these infections should also play a preventive role in BPD. Avoidance of excessive fluid intake is also important in the prevention of BPD. Diuretics, specifically loop diuretics, may be indicated to improve pulmonary fluid balance and reduce interstitial lung

water. Prompt closure of a PDA using prostaglandin inhibitors or by surgical ligation is important in reducing the risk and/or severity of BPD. Bronchodilators, most of them b-agonists, administered by inhalation have been shown to reduce airway resistance in infants with BPD. Because their effect is short-lived and often associated with cardiovascular side effects (e.g., tachycardia, hypertension, and possible arrhythmias), their use tends to be limited to acute exacerbations of airway obstruction. Adequate nutrition is important in BPD prevention. General undernutrition, particularly an insufficiency in dietary protein, may increase the vulnerability of the preterm infant to oxidant lung injury and the development of BPD. Additional nutrients, including those that may increase intracellular glutathione (e.g., sulfur-containing amino acids), inositol to serve as substrate for surfactant, and selenium and other trace minerals to function as essential cofactors for the pulmonary antioxidant enzymes, may provide added protection to the premature infant against the development of BPD. Clinical trials in preterm infants with severe RDS have shown that supplemental vitamin A administration reduces the incidence and severity of BPD. Although the use of corticosteroids clearly has short-term benefits in lung function in infants with BPD, the optimal age of treatment, dose schedule, and duration of therapy have not been established. Most alarming are recent follow-up data demonstrating that infants who received short or prolonged steroid therapy had worse neurological outcome compared to controls. Table 1 summarizes some of the strategies to prevent BPD. Future directions in BPD-prevention may include exogenous administration of specific antioxidants, genetic manipulation, and strategies for maturing the preterm lung that have greater selectivity and fewer adverse effects than the use of glucocorticoids. Table 1 Potential strategies to prevent BPD Avoid premature birth Antenatal steroids Surfactant replacement Minimize volutrauma and duration of mechanical ventilation Minimize oxygen exposure Prevention/aggressive management of pre- and postnatal infections Avoid excessive fluid administration Prompt patent ductus asteriosus closure Maintain normal serum vitamin A levels Investigational Corticosteroids and other anti-inflammatory drugs Exogenous administration or induction of antioxidant enzymes Gene therapy/genetic manipulation

BRONCHOSCOPY, GENERAL AND INTERVENTIONAL 305 See also: Epithelial Cells: Type II Cells. Fluid Balance in the Lung. Infant Respiratory Distress Syndrome. Lung Development: Overview. Pediatric Pulmonary Diseases. Peripheral Gas Exchange.

Further Reading Bancalari E (2004) Pathophysiology of chronic lung disease. In: Polin R, Fox W, and Abman S (eds.) Fetal and Neonatal Physiology, 3rd edn., vol. 1, pp. 954–961. Philadelphia: Saunders. Bancalari E (2005) Neonatal chronic lung disease. In: Fanaroff AA and Martin RJ (eds.) Neonatal–Perinatal Medicine Diseases of the Fetus and Infant, 8th edn. St. Louis: Mosby. Bancalari E, Claure N, and Sosenko IRS (2003) Bronchopulmonary dysplasia: changes in pathogenesis, epidemiology and definition. Seminars in Neonatology 8: 63–71. Bancalari E and Goldman SL (1982) Barotrauma to the lung. Advances in Perinatal Medicine 181. Bancalari E, Wilson-Costello D, and Iben SC (2005) Management of bronchopulmonary dysplasia. In: Maalouf E (ed.) Best Practice Guidelines, Early Human Development. Oxford: Elsevier. Bland RD and Coalson JJ (2000) Chronic Lung Disease in Early Infancy. New York: Dekker. Coalson JJ, Winter VT, Gerstmann DR, et al. (1992) Pathophysiologic, morphologic, and biochemical studies of the premature baboon with bronchopulmonary dysplasia. American Review of Respiratory Disease 145: 872–881. D’Angio CT and Maniscalco WM (2004) Bronchopulmonary dysplasia in preterm infants: pathophysiology and management strategies. Paediatric Drugs 6: 303–330. Gonzalez A, Sosenko IRS, Chandar J, et al. (1996) Influence of infection on patent ductus arteriosus and chronic lung disease in premature infants weighting 1000 grams or less. Journal of Pediatrics 128: 470–478. Grier DG and Halliday HL (2003) Corticosteroids in the prevention and management of BPD. Seminars in Neonatology 8: 83–91.

Husain AN, Siddiqui NH, and Stocker JT (1998) Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia. Human Pathology 29: 710–717. Jobe AH and Bancalari E (2001) Bronchopulmonary dysplasia. American Journal of Respiratory and Critical Care Medicine 163: 1723–1729. Lemons J, Bauer CR, Oh W, et al. (2001) Very low birth weight outcomes of the National Institute of Child Health and Human Development Research Network January 1995 through December 1996. Pediatrics 107: 1–8. Northway WH, Moss RB, Carlisle KB, et al. (1990) Late pulmonary sequelae of bronchopulmonary dysplasia. New England Journal of Medicine 323: 1793–1799. Northway WH Jr, Rosen RC, and Porter DY (1967) Pulmonary disease following respirator therapy of hyaline membrane disease: bronchopulmonary dysplasia. New England Journal of Medicine 276: 357–368. Pierce MR and Bancalari E (1995) The role of inflammation in the pathogenesis of bronchopulmonary dysplasia. Pediatric Pulmonology 19: 371–378. Rojas MA, Gonzalez A, Bancalari E, et al. (1995) Changing trends in the epidemiology and pathogenesis of neonatal chronic lung disease. Journal of Pediatrics 126: 605–610. Sosenko I and Bancalari E (2003) Bronchopulmonary dysplasia (BPD). In: Greenough A and Milner A (eds.) Neonatal Respiratory Disorders, 2nd edn., pp. 399–422. London: Arnold. Watterberg KL, Demers LM, Scott SM, et al. (1996) Chorioamniotis and early lung inflammation in infants in whom bronchopulmonary dysplasia develops. Pediatrics 97: 210–215. Watterberg KL, Scott SM, Backstrom C, et al. (2000) Links between early adrenal function and respiratory outcome in preterm infants: airway inflammation and patent ductus arteriosus. Pediatrics 150: 320–324. Yoon BH, Romero R, Jun JK, et al. (1997) Amniotic fluid cytokines (interleukin-6, tumor necrosis factor-a, interleukin-1b, and interleukin-8) and the risk for the development of bronchopulmonary dysplasia. American Journal of Obstetrics and Gynecology 177: 825–830.

BRONCHOSCOPY, GENERAL AND INTERVENTIONAL D J Feller-Kopman and R Bechara, Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Bronchoscopy refers to examination of the tracheobronchial tree via a rigid or flexible bronchoscope. This chapter will review both diagnostic and therapeutic bronchoscopy, with a focus on the available technology and procedural techniques used to diagnose and treat a variety of lung diseases. As ‘Bronchoscopy: Regular and Interventional’ is a topic for which textbooks are available, the reader is encouraged to explore the relevant literature, with some pertinent references listed in the further reading section.

Introduction Gustav Killian has been described as the ‘Father of Bronchoscopy’. With the vision of using a metallic

tube, electric light, and topical cocaine to prevent glottic closure, Killian removed a pork bone from a farmer’s airway in 1897. Over the subsequent years, Killian went on to develop bronchoscopes, laryngoscopes, and endoscopes, as well as describe techniques such as using fluoroscopy and X-ray to define endobronchial anatomy. Over the next 150 years, bronchoscopic techniques and instruments continued to be refined. In 1966, Shigeto Ikeda, presented the first prototype flexible fiberoptic bronchoscope at the 9th International Congress on Diseases of the Chest in Copenhagen. In 1968 Machita and Olympus introduced the first commercially available fiberoptic bronchoscopes. In 1980, Dumon presented his use of the neodymium:yttrium aluminum garnet (Nd:YAG) laser via the fiberoptic bronchoscope, and since that time the flexible bronchoscope has been widely utilized as both a diagnostic and therapeutic tool for both diseases of the parenchyma and central airways.

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With the miniaturization of electronic devices, Ikeda was able to incorporate the video chip into the bronchoscope, and Pentax introduced the first video bronchoscope in 1987. Suddenly, endoscopic pictures could be printed out and shared, and the physicians no longer needed to look through an eyepiece, but instead the endoscopic image could be projected onto monitors, allowing everyone in the room to visualize what was happening in the airway.

Modern Video Bronchoscopes Little has changed in the appearance of bronchoscopes since 1968. The external diameter of the flexible bronchoscope varies from 2.7 to 6.3 mm diameter. The diameter of the working channel ranges from 1.2 to 3.2 mm. A working channel X2:8 mm is recommended for more therapeutic flexible bronchoscopy, as well as endobronchial ultrasound (EBUS), as it allows for better suction and the passage of larger instruments. Most flexible bronchoscopes can flex 1801 up and 1301 down. It is important to note the relative anatomy at the tip of the bronchoscope. By convention, as viewed from the operator’s perspective, the camera is at 9:00, suction at 6:00 and the working channel at 3:00. These landmarks play a role when navigating the airways as the bronchoscope may need to be rotated in order to visualize the intended target or guide a tool to its intended target.

Airway Anatomy It is crucial that the bronchoscopist become an expert in airway anatomy. Anatomical knowledge should include the naso and oropharynx, as well as the larynx as these structures are visualized, yet often overlooked, by the pulmonologist, who is typically more concerned about lower airway/parenchymal disease. Additionally, the segmental anatomy of the lungs, from both an external and internal perspective, is required, and we recommend knowledge of both the name and number system. One must also be familiar with the anatomy external to the airway, primarily the intrathoracic vessels and lymph nodes, as these will serve as reference points for transbronchial needle aspiration and EBUS, and will help avoid injury should the bronchoscopist use therapy such as the Nd:YAG laser or brachytherapy. The use of endoscopic simulators has been associated with a more rapid acquisition of bronchoscopic expertise and we recommend the novice bronchoscopist use a simulator as much as possible.

Preparation Prior to the procedure, it is crucial that the bronchoscopist review the patient’s relevant history,

physical examination, and imaging studies. A distinct plan should be in place regarding the sequence of sampling techniques, and everyone participating in the procedure should be familiar with both this plan and also their particular roles during the procedure. Topical anesthesia is crucial, and we typically use 1% lidocaine, keeping the total dosage o400 mg. Stronger concentrations do not provide additional sensory anesthesia and will only limit the volume of lidocaine one can apply to the airways. Most procedures are performed under conscious sedation with a narcotic and a benzodiazepine. All practitioners involved in the administration of conscious sedation are required to undergo formal training by their institution and should have a thorough understanding of the effects, side effects, and typical dosages, as well as antagonist drugs. The physician should also be comfortable in airway management including the use of bag-valve mask, oral airways, and endotracheal intubation. If one plans to perform bronchoscopy through an endotracheal tube (or tracheostomy), the internal diameter should ideally be 47.5 mm in order to accommodate the bronchoscope and maintain patient ventilation. Depending on the length of the procedure, it may be necessary to remove the bronchoscope intermittently to ensure adequate ventilation.

Diagnostic Bronchoscopy There are many indications for diagnostic bronchoscopy. The most common indication is for the diagnosis and staging of suspected lung cancer. Other indications include evaluation of diffuse lung disease, infiltrates in the immunocompromised host, hemoptysis, and cough. Additionally, bronchoscopy with bronchoalveolar lavage (BAL) and/or brushing is useful for the diagnosis of community and healthcare associated pneumonia. Cancer Diagnosis and Staging

Lung cancer is the leading cause of cancer deaths in the US, and the incidence and number of lung cancer deaths continues to increase amongst women in the US. Bronchoscopy provides a minimally invasive approach for the diagnosis of tumors in the central airways. Though the yield is somewhat lower for solitary parenchymal lesions, advances in navigation with techniques such as computed tomography (CT) fluoroscopy and electromagnetic guidance (to be discussed later) have significantly improved the yield for peripheral tumors. The bronchoscopic evaluation of patients with suspected malignancy is guided by clinical symptoms as well as radiographic findings. Depending upon the

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history and chest CT findings, bronchoscopy may be the diagnostic modality of choice as it can both make the diagnosis and stage the patient at the same time. If appropriate staging is to be performed, biopsy of the lesion that will place the patient in the highest clinical stage should occur prior to other biopsies. For example, if a patient presents with a left lower lobe mass and a right paratracheal lymph node (4R), the appropriate procedure would be a bronchoscopy with transbronchial needle aspiration (TBNA) of the 4R node, with attention then turned to the left lower lobe lesion, as a positive TBNA would stage the patient as IIIb and therefore change the treatment plan. Initial biopsy of the mass could contaminate the working channel and make the TBNA a false positive, precluding the patient from curative surgery. There are several available tools by which to obtain specimens during bronchoscopy including forceps biopsy, brushing, bronchial wash/lavage, and TBNA. Electrocautery snare forceps removal of a pedunculated airway lesion can also provide excellent tissue for the pathologist. The choice of the above modalities is primarily determined by the location of the pathology; however, data support the use of the combination of techniques to improve diagnostic yield, as opposed to using them in isolation. Endobronchial needle aspiration should be used with all visible lesions, as its use in combination with conventional techniques has been associated with an improvement in the diagnostic yield. If the lesion is not visible endoscopically, BAL can be performed. Briefly, the bronchoscope is wedged in the target segmental or subsegmental bronchus leading to the lesion. Two or three aliquots of 40–60 ml of normal saline solution are instilled, and then aspirated. Ideally, the return should be between 40% and 60% of the instilled volume, however this is dependent upon the segment lavaged, with better return coming from less dependant locations. BAL is thought to sample approximately 1 million alveoli and the cellular and noncellular contents of the lavage fluid have been shown to closely correlate with the inflammatory nature of the entire lower respiratory tract. Transbronchial biopsy, brushing, and TBNA can also be performed for peripheral lesions. The diagnostic yield of bronchoalveolar lavage for peripheral cancer ranges from 4% to 68%. Without advanced guidance (discussed below), the yield for transbronchial biopsy ranges from 49% to 77%. The yield from brushing alone is 26–57%. The combination of all the three techniques results in a combined yield of upto 68%. TBNA of peripheral nodules has been shown to have a higher diagnostic yield than other sampling techniques, and should be used to biopsy

peripheral lesions, as well as mediastinal and hilar lymph nodes. Despite TBNA being introduced more than 25 years ago, it remains an underutilized technique, with only 12% of pulmonologists reporting its routine use for the diagnosis and staging of lung cancer. The technique, however, is incredibly useful, and may preclude further invasive surgery in 29% of patients. The yield with TBNA has been associated with tumor cell type (small cell 4 non-small cell 4 lymphoma), lymph node size, and lymph node location. The use of CT fluoroscopy to guide TBNA and transbronchial biopsy has several advantages over standard fluoroscopy. As opposed to standard fluoroscopy, which is typically used only in two dimensions, CT provides the ability to visualize the target in three dimensions. With standard fluoro, either the patient or the C-arm needs to be rotated to confirm the biopsy tool is not anterior or posterior to the target. CT fluoro provides real-time three dimensional confirmation of the appropriate (Figure 1) and inappropriate (Figure 2) biopsy sites. The use of CT fluoro to guide TBNA has been associated with an accuracy of 88%, including patients who have previously undergone a nondiagnostic bronchoscopy with standard TBNA. Endobronchial ultrasound is another important modality used to help improve accuracy of TBNA. With the development of a miniaturized 20 MHz transducer that can be inserted via a 2.8 mm working channel, lymph nodes and masses adjacent to the airway can now be visualized (Figure 3). Like CT fluoro, EBUS has also been shown to improve the yield of TBNA. Recently, a bronchoscope with a dedicated ultrasound probe and distinct working TM channel has been developed (Puncturescope , Olympus Corporation, Tokyo, Japan), and has the benefit

Figure 1 CT fluoroscopy showing the biopsy forceps in the target.

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Several studies have shown that the use of autofluorescence increases the detection of early stage lung cancer by up to sixfold when compared to white light bronchoscopy. The definitive role of AF bronchoscopy in the early detection of lung cancer remains to be defined. Diffuse Parenchymal Lung Disease

Figure 2 CT fluoroscopy showing the biopsy forceps anterior to the target, near the pleura. Note that without rotation of the patient/C-arm, the biopsy forceps may appear to be within the target on standard fluoroscopy.

Figure 3 Endobronchial ultrasound image showing tumor invading airway wall from the 12:00–2:00 position.

of providing real-time guidance for TBNA of mediastinal and hilar lymph nodes, with excellent results. EBUS has been shown to better differentiate airway invasion versus compression by adjacent tumor when compared to CT, and can suggest the histology of a solitary pulmonary nodule based on the ultrasound morphology. Autofluorescence (AF) bronchoscopy is an increasingly popular tool used for the early detection of cancer in the central airways, primarily carcinoma in situ (CIS), and squamous cell carcinoma. When exposed to light in the violet–blue spectrum (400–450 nm), the normal airway fluoresces green. As submucosal disease progresses from normal, to metaplasia, to dysplasia, to CIS, there is a progressive loss of the green AF, causing a red–brown appearance of the airway wall.

Diffuse parenchymal lung disease describes a group of infectious, inflammatory, and fibrotic disorders which may involve the interstitial, alveolar, bronchial, and vascular structures of the tracheobronchial tree. The most commonly used sampling techniques for patients with diffuse disease include BAL, bronchial brushing, transbronchial biopsy (TBBx), and occasionally endobronchial biopsy (EBBx) and TBNA. Though associated with a low morbidity, transbronchial biopsy should be used only when the potential results will impact on treatment decisions. Diseases in which transbronchial biopsy can prove diagnostic or has been shown to significantly increase the diagnostic yield as compared to less invasive means include lymphangitic carcinomatosus, sarcoidosis, rejection after lung transplantation, hypersensitivity pneumonitis, and sometimes, invasive fungal infection. The overall diagnostic yield for transbronchial biopsy in this category depends on the disease entity. For example, the yield for sarcoidosis can approach 90%, but is much lower for patients with vasculitis or cryptogenic organizing pneumonia. Aside from providing a specific diagnosis in cases of cancer or infection, the results of BAL can serve to limit the differential diagnosis considerably. For example, a BAL with lymphocyte predominance suggests granulomatous disease such as sarcoidosis, berylliosis, or a lymphoproliferative disorder. Neutrophil predominance suggests bacterial infection, acute interstitial pneumonia, and can be seen in patients with asbestosis or usual interstitial pneumonitis. Eosinophils are seen in patients with eosinophilic pneumonias, hypereosinophilic syndromes, or Churg–Strauss syndrome. Patients with pulmonary alveolar proteinosis (PAP), have a unique appearance to the BAL fluid that is described as milky or opaque. The alveolar macrophages are filled with PAS-positive material, and lamellar bodies can be seen under electron microscopy. Infectious Diseases

Community acquired pneumonia The role of bronchoscopy in community acquired pneumonia (CAP) remains controversial. When used, BAL and protected brush are the main diagnostic procedures, and the specimens should ideally be sent for

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respiratory tract culture prior to the initiation of antibiotics, the use of semiquantitative or quantitative culture data, and the use of negative culture data to discontinue antibiotics in patients who have not had changes in their antibiotic regimen within the last 72 h. Additionally, the use of a bronchoscopic strategy was supported as a way to reduce 14-day mortality.

Figure 4 (a) Protected specimen brush; (b) protected specimen brush with the protective wax cap expelled.

quantitative culture, with a threshold of 104 colonyforming units (CFU) for the BAL and 103 CFU for the protected brush (Figure 4). In addition to providing a microbiologic diagnosis, another important indication for bronchoscopy in patients with CAP is to rule out an obstructing endobronchial lesion in the right clinical setting. Obviously, it is crucial to avoid contamination with upper airway secretions when performing bronchoscopy in patients with pneumonia. Key procedural aspects include minimizing suctioning, as well as minimizing the instillation of lidocaine, as secretions in the working channel will be flushed back into the airways, and high concentrations can be bacteriostatic. Healthcare and ventilator associated pneumonia The American Thoracic Society and Infectious Disease Society have recently reviewed these topics in great detail, and used the techniques of evidence based medicine to guide their recommendations. Some major points included the collection of a lower

Immunocompromised host The early diagnosis and initiation of the appropriate antibiotic is the cornerstone of successful treatment of the immunocompromised patient with pneumonia. Additionally, it is important to note that multiple diagnoses can often be present simultaneously in these patients, and noninfectious conditions may have a similar presentation of cough, dyspnea, fever, and an infiltrate on imaging. Bronchoscopy is an excellent method of evaluating these patients as less invasive techniques can miss the diagnosis in approximately 30% of patients, and earlier diagnosis may improve mortality. BAL is the most commonly used bronchoscopic technique used to obtain a diagnosis in immunocompromised patients, with an overall diagnostic yield of approximately 60–70%. In comparative studies, the sensitivity of TBBx has been shown to be roughly the same, though the combined use of both techniques may increase the yield. The results of BAL have been shown to change management in up to 84% of cases. Even in the immunocompromised host, bronchoscopy with BAL remains a safe procedure. Brushing and TBBx, however, have been associated with a higher incidence of bleeding complications in patients who are thrombocytopenic. The ‘gold standard’ for tissue diagnosis remains open lung biopsy, which can yield a specific diagnosis in 62% of patients, and result in a significant increase in survival. There are no data suggesting that bronchoscopy reduces mortality in this patient population. Hemoptysis There are many causes of hemoptysis including infectious, inflammatory, vascular, and neoplastic processes. Though one would think that bronchoscopy can often make the diagnosis of a radiographic occult neoplasm in a patient with hemoptysis, a bronchoscopic diagnosis of malignancy is made in o5% of patients. Indications for bronchoscopy in patients with normal chest imaging include age 440 years, male gender, and a 440 pack-year smoking history. Though the appropriate timing for bronchoscopy is controversial, there is a greater likelihood of identifying the bleeding source when performed within the first 48 h of symptoms. The combined use of bronchoscopy and CT is also recommended. If patients are clinically stable, we prefer

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to obtain the CT first, as it can serve as a ‘road map’ to guide bronchoscopy.

Therapeutic Bronchoscopy Bronchoscopy in Hemoptysis

The definition of massive hemoptysis has ranged from 100–1000 cm3 expectorated in a 24 h period. As the majority of patients with massive hemoptysis die from asphyxia, and not exanguination, and the anatomic dead-space is approximately 150 cm3, we consider any amount over 100 cm3 in 24 h as massive. In addition to identifying the source and cause of bleeding, bronchoscopy clearly plays an important therapeutic role in patients with massive hemoptysis. If available, we strongly recommend rigid bronchoscopy as the procedure of choice in these patients. In addition to securing an airway, and providing oxygenation and ventilation, the rigid bronchoscope allows the passage of large-bore suction catheters as well as a variety of other tools that can help stop the bleeding including cryotherapy, electrocautery, argon plasma coagulation (APC), and Nd:YAG laser, as well as bronchial blockers. If rigid bronchoscopy is not available, we recommend tracheal intubation with the largest endotrachcal tube available, with selective right or left-mainstem intubation to protect the non-bleeding lung as needed. Double-lumen endotracheal tubes, or specialized tubes that come with an endobronchial blocker (e.g., Univent, Vitaid, Williamsville, NY), can be more difficult to place, especially in the setting of massive hemoptysis. The main role of flexible bronchoscopy in the patient with massive hemoptysis lies in obtaining lung isolation, and ‘protecting the good lung’, as the suction channel of a flexible scope is relatively small. All bronchoscopists should become familiar with bronchial blockers (e.g., Arndt Bronchial Blocker, Cook Critical Care, Bloomington, IN), which can be passed in parallel to the flexible scope, and some, even inserted through the working channel. These catheters are guided to the culprit segmental, lobar, or mainstem bronchus and the balloon is inflated to the recommended volume/pressure. After a maximum of 24 h, the balloon should be deflated under bronchoscopic visualization. Other bronchoscopic techniques used to control hemoptysis include the topical application of iced saline, epinephrine (1 : 20 000), thrombin/thrombinfibrinogen, or cyanoacrylate solutions. Other Therapeutic Bronchoscopic Techniques

Argon plasma coagulation Argon plasma coagulation (APC) is a noncontact method using ionized

argon gas (plasma) to achieve tissue coagulation and hemostasis. As the plasma is directed to the closest grounded source, APC has the ability to treat lesions lateral to the probe, or around a bend, that would not be suitable for laser therapy. The depth of penetration for APC is approximately 2–3 mm, and hence the risk of airway perforation is also less when compared to lasers. Laser therapy The Nd:YAG laser is the most widely used laser in the lower respiratory system, and has been used for both benign and malignant disease. The primary advantage of laser photoresection includes rapid destruction/vaporization of tissue. Lesions most amenable to laser therapy are central, intrinsic, and short (o4 cm), with a visible distal endobronchial lumen. When lesions meet these criteria, patency can be re-established in more than 90% of cases. Care must be taken however, as the depth of penetration can approach 10 mm and airway perforation with resultant pneumothorax, pneumomediastinum, and vascular injury have been reported. In view of this, we encourage its use only by experienced interventional bronchoscopists. Nevertheless, the safety record of laser bronchoscopy is excellent, with an overall complication rate of o1%. Cryotherapy Cryotherapy is a safe and effective tool for a variety of airway problems. By releasing nitrous oxide stored under pressure, the tip of the cryoprobe rapidly cools to 891C. We primarily use cryotherapy for the removal of organic foreign bodies with high water content such as grapes and vegetable matter, in addition to facilitating the removal of tenacious mucus or blood clots when performing flexible bronchoscopy. Compared to the other techniques described in this chapter, cryotherapy results in delayed tumor destruction, requiring a repeat bronchoscopy to remove the necrotic tumor. The distinct advantage of cryotherapy lies in the fact that the normal cartilage and fibrous tissue of the airway are relatively cryoresistant, in addition to the lack of risk of airway fires. Electrocautery Electrocautery uses alternating current at high frequency to generate heat, which cuts, vaporizes, or coagulates tissue depending on the power. Electrocautery is a contact mode of tissue destruction and a variety of cautery probes are available including blunt tip probes, knifes, and snares. We favor the use of the cautery snare for pedunculated lesions of the airway as the stalk can be cut and coagulated while preserving the majority of the tissue for pathologic interpretation. As with laser therapy,

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the risks of electrocautery include airway perforation, airway fires, and damage to the bronchoscope. Photodynamic therapy Photodynamic therapy (PDT) involves the intravenous injection of a photosensitizing agent, then activating the drug with a nonthermal laser to produce a phototoxic reaction and cell death. As tumor cells retain the drug longer than other tissues, waiting approximately 48 h after drug injection will lead to preferential tumor cell death as compared to normal tissue injury. As with cryotherapy, maximal effects are delayed, and a repeat, ‘clean-out’ bronchoscopy should be performed 24–48 h after drug activation. The primary side effect from PDT is systemic phototoxicity, which can last up to 6 weeks after injection. Newer drugs are being developed with the hopes of increasing tumor selectivity and reducing the duration of skin phototoxicity. As laser activation uses a nonthermal laser source, airway fires are not an issue. PDT has been shown to be curative for early stage lung cancer of the airways and is an especially attractive option for patients with endobronchial CIS who are not surgical candidates due to other comorbidities. Brachytherapy Brachytherapy refers to endobronchial radiation, primarily used for the treatment of malignant airway obstruction. The most commonly used source of radiation is iridium-192 (192Ir), which is inserted bronchoscopically via a catheter. Brachytherapy may be delivered by either low-dose rate (LDR), intermediate-dose rate (IDR), or high-dose rate (HDR) methods, with most authors currently recommending the afterloading HDR technique. This allows the bronchoscopist to place the catheter in the desired location and the radiation oncologist to deliver the radiation in a protected environment. The main advantage of HDR is patient convenience, as each session lasts o30 min; however, multiple bronchoscopies are required to achieve the total 1500 cGy dose that is currently recommended. The LDR technique may be appropriate for patients who live far from the hospital or who are otherwise hospitalized as it only requires one bronchoscopy, but the catheter has to stay in place for 20–60 h. The main advantage of brachytherapy as compared with external-beam radiation is the fact that less normal tissue is exposed to the toxic effects of radiation. The most common side effects include intolerance of the catheter, radiation bronchitis, airway perforation, and, occasionally, massive hemorrhage. Treatment of tumors in the right and left upper lobes has the highest incidence of hemorrhage, as these are located near the great vessels.

Airway stents Montgomery is credited as initiating the widespread use of airway stents after his development of a silicone T-tube in 1965 for use in patients with tracheal stenosis. Dumon, however, introduced the first completely endoluminal airway stent in 1990. Airway stents are the only technology that can alleviate extrinsic airway compression. They are commonly used in conjunction with the other modalities for patients with intrinsic or mixed disease. Over the last 15 years, there has been an explosion in both stent design and the number of endoscopists who place airway stents. As with any procedure, it is crucial to understand the indications and contraindications of the procedure as well as be able to anticipate, prevent and manage the associated complications. Unfortunately, the ideal stent has

Figure 5 Metal stent in the trachea.

Figure 6 Some available silicone stents ex vivo.

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not yet been developed. This stent would be easy to insert and remove, yet would not migrate; would be of sufficient strength to support the airway, yet be flexible enough to mimic normal airway physiology and promote secretion clearance; biologically inert to minimize the formation of granulation tissue; and available in a variety of sizes. There are currently two main types of stents: metal, generally Nitinol (Figure 5), and silicone (Figure 6). Though metal stents are easily placed, they can be extremely difficult to extract, and may cause excessive granulation tissue formation (Figure 7). They are

Figure 7 Metal stent removal.

available in covered and uncovered varieties. For malignant airway obstruction, the only appropriate metal stents are covered models, which minimize tumor ingrowth. Some authors feel that there is no indication for an uncovered metal stent. The main advantage of metal stents is their larger internal : external diameter ratio as compared to that of silicone stents. Though silicone stents require rigid bronchoscopy for placement, they are more easily removed and are significantly less expensive. The future of airway stenting likely lies in the creation of bioabsorbable stents, made out of materials such as vicryl filaments or poly-L-lactic acid (SR-PLLA). In addition to malignant airway obstruction, airway stents can be helpful in patients with tracheobronchomalacia and tracheoesophageal fistula. In patients with tracheoesophageal fistula, doublestenting of the esophagus and airway is recommended to maximally prevent aspiration.

Powered instrumentation The microdebrider is a tool consisting of a hollow metal tube with a rotating blade coupled with suction. We have had excellent results with this technology in the treatment of both benign and malignant central airway obstruction. A primary advantage of the microdebrider is the rapidity of obtaining a patent airway and the lack of risk of airway fires as compared to modalities using heat.

Figure 8 Super dimension bronchoscopy showing the locatable guide in the target in the axial, coronal, and sagittal planes on CT, as well as the ‘fighter-pilot’ view in the lower right, with the target 0.2 cm directly ahead.

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Future Directions in Bronchoscopy In the appropriate patient, lung volume reduction surgery has been shown to improve both quality and quantity of life. The major drawback to this surgery is the associated morbidity and cost of the procedure. Recent studies have suggested that lung volume reduction can be obtained bronchoscopically, either by creating channels in the distal airways/parenchyma, or by the placement of one-way endobronchial valves to promote deflation of hyperinflated lung that does not significantly contribute to gas exchange. The results of large-scale, multicenter trials will become available within the next several years, but the preliminary data are promising. The use of electromagnetic navigation is a novel technology that has been shown to allow accurate sampling of peripheral solitary pulmonary nodules o1 cm in diameter. Briefly, a virtual bronchoscopy is generated from the patients CT scan. Anatomic landmarks that are easily identified, such as the carinae, are marked, as is the target. At the time of bronchoscopy, the patient lies in an electromagnetic field and a steerable, ‘locatable guide’ is placed through an ‘extended working channel’ of the bronchoscope. The location of the guide in the electromagnetic field is accurate to o5 mm in the x, y, and z axes, as well as yaw, pitch, and roll. The points previously identified in the virtual bronchoscopy are then marked with the locatable guide, which in essence, marries the CT scan with the bronchoscopic image. Navigation is then performed by looking at the CT in the axial, sagittal, and coronal planes, and the guide is steered toward the target (Figure 8). Once found, the guide is removed, and standard bronchoscopic tools such as TBNA needles and forceps are placed through the extended working channel. This technology not only has the potential to revolutionize diagnostic bronchoscopy,

but therapeutic bronchoscopy as well. For example, if a patient with a 2.5 cm, stage Ia, nonsmall cell cancer is not an operative candidate, this technology may allow for bronchoscopic treatment by either radiofrequency ablation or the implantation of fiducials to allow stereotactic radiosurgery. See also: Alveolar Hemorrhage. Bronchiectasis. Bronchiolitis. Bronchomalacia and Tracheomalacia. Drug-Induced Pulmonary Disease. Interstitial Lung Disease: Overview. Lung Anatomy (Including the Aging Lung). Mediastinal Masses. Panbronchiolitis. Pneumonia: Overview and Epidemiology; The Immunocompromised Host. Tumors, Malignant: Overview. Upper Airway Obstruction. Upper Respiratory Tract Infection.

Further Reading Becker HD (1991) Atlas of Bronchoscopy: Technique, Diagnosis, Differential Diagnosis, Therapy. Philadelphia: BC Decker. Bolliger CT and Mathur PN (2000) Interventional Bronchoscopy. Basel: Karger. Detterbeck FC, DeCamp MM Jr, Kohman LJ, and Silvestri GA (2003) Invasive staging: the guidelines. Chest 123(90010): 167S–175S. Ernst A, Feller-Kopman D, Becker HD, and Mehta AC (2004) Central airway obstruction. American Journal of Respiratory and Critical Care Medicine 169(12): 1278–1297. Ernst A, Silvestri GA, and Johnstone D (2003) Interventional pulmonary procedures: guidelines from the American College of Chest Physicians. Chest 123(5): 1693. Fagon JY, Chastre J, Wolff M, et al. (2000) Invasive and noninvasive strategies for management of suspected ventilator-associated pneumonia: a randomized trial. Annals of Internal Medicine 132(8): 621–630. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. (2005) American Journal of Respiratory and Critical Care Medicine 171(4): 388–416. Prakash USB (1994) Bronchoscopy. New York: Raven Press.

C Calcium Channels

see Ion Transport: Calcium Channels.

CAPSAICIN M G Belvisi and D J Hele, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The pungent ingredient in red pepper fruits of the genus Capsicum, which includes paprika, jalapen˜o, and cayenne, is called capsaicin. Capsaicin is known to stimulate sensory nerves leading to the activation of nociceptive and protective reflex responses (e.g., cough, bronchospasm) and the release of neurotransmitters from both peripheral and central nerve endings. This latter effect causes a collection of inflammatory responses often referred to as ‘neurogenic inflammation’, which in the airways results in bronchoconstriction, plasma extravasation, and mucus hypersecretion. The capsaicin receptor has recently been identified and has been named the type 1 vanilloid receptor (VR1; TRPV1). It has previously been suggested that there is an upregulation of TRPV1 expression in inflammatory diseases and that inappropriate activation of this receptor may lead to sensory nerve hyperresponsiveness. Thus, it would appear that airway inflammatory diseases (e.g., asthma and chronic obstructive pulmonary disease) may respond to treatment with an effective and selective inhibitor of TRPV1 and to this end much work is being carried out to develop novel inhibitors.

via the peripheral release of neuropeptides, a phenomenon known as ‘neurogenic inflammation’. A characteristic feature of many nociceptive sensory fibers is their sensitivity to capsaicin. However, until recently the molecular mechanisms involved in activation of sensory nociceptive fibers were unknown. Pharmacological evidence for the presence of a ‘capsaicin receptor’ in sensory nerves was provided by the discovery of two capsaicin analogs, resiniferatoxin (potent agonist) and capsazepine (an antagonist). First, specific binding sites for resiniferatoxin were demonstrated on dorsal root ganglion membranes and second, capsazepine has been found to inhibit numerous capsaicin-evoked neuronal responses including those in the airways. The capsaicin receptor has recently been identified and has been named the type 1 vanilloid receptor (VR1; transient receptor potential vanilloid 1 (TRPV1)).

Capsaicin and Functional Responses in the Airways Cough

Introduction Sensory nerves in the airways regulate central and local reflex events such as bronchoconstriction, airway plasma leakage, and cough. Sensory nerve activity may be enhanced during inflammation such that these protective reflexes become exacerbated and deleterious. Sensory nerve reflexes are under the control of at least two different classes of sensory fiber, the myelinated rapidly adapting stretch receptors (RARs) and nonmyelinated capsaicin-sensitive C fibers. In the airways, activation of RARs and C fibers elicits cough, bronchoconstriction, and mucus secretion via an afferent central reflex pathway. Activation of C fibers in the airways also mediates efferent excitatory nonadrenergic noncholinergic (e-NANC) responses such as bronchoconstriction, mucus secretion, plasma exudation, and vasodilatation

Inhalation challenge with capsaicin and low pH (e.g., citric acid) has been shown to evoke cough and has been used as a model for the last 50 years to investigate the action of potential antitussive therapies in clinical trials. In fact, agonists at TRPV1 such as capsaicin and resiniferatoxin are the most potent stimulants of the cough reflex so far described in man. Therefore, it has been suggested that TRPV1 activation may be one of the primary sensory mechanisms in cough. However, if activation of TRPV1 is an important initiating factor for the cough reflex then an endogenous capsaicin-like ligand must be present. A number of putative endogenous ligands that are known to activate TRPV1 have been demonstrated to cause cough. TRPV1 has been shown to be sensitive to a fall in the extracellular pH, and H þ ions not only stimulate the receptor directly but also

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increase the sensitivity of the receptor to capsaicin and low pH solutions, which are known to elicit cough in animals and humans. Interestingly, the TRPV1 antagonists capsazepine and iodo-resiniferatoxin have been shown to inhibit capsaicin, citric acid, and anandamide-induced cough in conscious guinea pigs suggesting that these agents are inducing cough via a common mechanism, that is, activation of TRPV1 (Figure 1). However, it is unlikely that TRPV1 activation is the only stimulus for cough since some tussigenic agents such as hypertonic saline are not inhibited by TRPV1 antagonism.

Neurogenic Inflammation Capsaicin activates sensory nerves leading to the activation of nociceptive and protective reflex responses and the release of neurotransmitters from both peripheral and central nerve endings. This latter effect causes a collection of inflammatory responses 2

Coughs min–1

Capsazepine (10 µM) 1.5

Control

1

0.5

0

30 µM

80 µM

Capsaicin

Citric acid (0.25 M)

Figure 1 Representation of data demonstrating that the TRPV1 antagonist capsazepine inhibits capsaicin and citric acid-induced cough in conscious guinea pigs suggesting that these agents are inducing cough via a common mechanism, i.e., activation of TRPV1.

often referred to as ‘neurogenic inflammation’, which in the airways results in bronchoconstriction, plasma extravasation, and mucus hypersecretion (Figure 2). Bronchoconstriction

Early experiments using the relatively weak TRPV1 antagonist capsazepine provided pharmacological validation that capsaicin-induced bronchospasm in guinea pig bronchi did indeed involve the activation of TRPV1. Contractile responses to resiniferatoxin and capsaicin were unaffected by the neurokinin (NK)-1 antagonist CP 96345, partially inhibited by the NK-2 antagonist SR 48968, but nearly abolished by a combination of the antagonists. These data suggest that resiniferatoxin and capsaicin both release tachykinins that act on both NK-1 and NK-2 receptor subtypes in a TRPV1-dependent manner. More recently, a more potent and selective agent has been used to confirm these observations. Interestingly, contractile and relaxant responses to capsaicin and resiniferatoxin have also been examined in human isolated bronchus (5–12 mm outside diameter). Bronchi isolated from 10 of 16 lungs contracted in response to capsaicin. The capsaicin-induced contractions were mimicked by resiniferatoxin and inhibited by capsazepine. The contractile response to capsaicin was not affected by the potent NK-2 selective antagonist SR 48968, whereas responses to concentrations of NKA, NKB, substance P, neuropeptide g, and neuropeptide K, which produced contractions of a similar size, were almost abolished by SR 48968. These results suggest that capsaicin and resiniferatoxin can alter smooth muscle tone in a TRPV1-dependent manner, but this response does not appear to involve substance P or related neurokinins. Airway hyperresponsiveness (AHR) to bronchoconstrictor agents is recognized as a critical feature of

Capsaicin

Vasodilatation

Mucus secretion

Plasma exudation

C fibers

Central reflexes, e.g., cough

Neuropeptide release SP, NKA, CGRP…

Bronchoconstriction Figure 2 Activation of sensory nerves by capsaicin. SP, substance P; CGRP, calcitonin gene-related peptide; NKA, neurokinin A. Adapted from Barnes PJ (2001) Neurogenic inflammation in the airways. Respiration Physiology 125: 145–154.

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bronchial asthma and sensory nerves in the airway are strongly implicated in the hyperresponsiveness. Several studies have demonstrated that pretreatment with capsaicin (which depletes sensory neuropeptides) significantly inhibited the late bronchial response that was observed after ovalbumin inhalation, AHR, and eosinophil accumulation in an allergic guinea pig model, and AHR to histamine in a rabbit model. It remains to be seen whether TRPV1 antagonists are effective in this regard. Plasma Extravasation

Activation of C fibers in the airways also mediates efferent excitatory nonadrenergic noncholinergic (e-NANC) responses such as plasma exudation and vasodilatation via the peripheral release of neuropeptides. Plasma extravasation has been shown to be induced in rats or guinea pigs by intravenous injections of substance P (SP) and capsaicin. The effect of intravenous capsaicin was absent in capsaicin-desensitized animals and in those pretreated with capsazepine. Capsaicin-induced plasma extravasation was also markedly inhibited by CP 96345, a nonpeptide antagonist of tachykinin NK-1 receptors. These data suggest that capsaicin-induced plasma leakage is mediated by the release of neuropeptides and the activation of NK-1 receptors via a TRPV1dependent mechanism. Mucus Secretion

When stimulated capsaicin-sensitive C fibers (afferents) containing the neuropeptides SP, NKA, and calcitonin gene-related peptide have also been shown to evoke neurogenic secretion from airway mucussecreting cells. However, although capsaicin has been shown to elicit mucus secretion, there is no data available describing the effect of TRPV1 antagonists on this response.

Molecular Mechanism of Action of Capsaicin

Capsazepine Airway C fiber

Capsaicin, RTx − H+ (citric acid, low pH), heat +

Cholinergic reflex activation

TRPV1

Cough Chest tightness

AHR

Mucus secretion

Figure 3 Events mediated via TRPV1 following the activation of nociceptive airway C fibers by noxious stimuli.

(PMg/PNaB5). TRPV1 is activated by heat (4431C), but is effectively dormant at normal body temperature and low pH (o5.9) and may act as an integrator of chemical and physical pain-eliciting stimuli. When activated, TRPV1 produces depolarization through the influx of Na þ , but the high Ca2 þ permeability of the channel is also important for mediating the response to pain. Gating by heat is direct but the receptor can be opened by ligands or stimuli such as mild acidosis, which also reduces the threshold for temperature activation and potentiates the response to capsaicin. To summarize, TRPV1 mediates nociception and contributes to the detection and integration of diverse chemical and thermal stimuli. The expected role for TRPV1 is in pain pathways and recent data from a study with TRPV1 knockouts showed impaired inflammatory thermal hyperalgesia. This has led to a growing interest in developing small molecule antagonists for this target. Recent preclinical data demonstrating efficacy in rodent models of both thermal and mechanical neuropathic or inflammatory pain has fuelled this enthusiasm. The established role of sensory nerve activation in the cough reflex and the role of TRPV1 in inflammatory pain has also alerted the respiratory community to the therapeutic potential of TRPV1 antagonists as antitussives and as therapy for airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD).

The Type 1 Vanilloid Receptor

TRPV1 is a membrane-associated vanilloid receptor. It is a nociceptor-specific ligand-gated ion channel expressed on the neuronal plasma membrane of nociceptive C fibers and is required for the activation of sensory nerves by vanilloids such as capsaicin, the pungent extract from plants in the genus Capsicum (e.g., hot chilli peppers) (Figure 3). TRPV1 also mediates the response to painful heat, extracellular acidosis, protons, and tissue injury. TRPV1 is an outwardly rectifying cation-selective ion channel with a preference for calcium (PCa/PNaB10) and magnesium

Ligands for TRPV1

Exogenous agonists of TRPV1 include capsaicin and resiniferatoxin. Endogenous agonists include the cannabinoid receptor agonist anandamide, N-arachidonoyl-dopamine, inflammatory mediators (bradykinin, 5-HT, and prostaglandin E2 (PGE2)), hydrogen ions, heat, arachidonic acid, lipoxin A4, prostacyclin, ethanol, and several eicosanoid products of lipoxygenases including 12-(S)- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5-(S)-hydroxyeicosatetraenoic acid and leukotriene B4. Interestingly, the effect of bradykinin

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may be indirect given that data exists suggesting that it appears to activate bradykinin B2 receptors on afferent neurons leading to the generation of lipoxygenase metabolites that have agonist activity at TRPV1. The evidence to support the suggestion that TRPV1 can be gated not only by vanilloids such as capsaicin, but also by protons, heat, agonists at certain G-protein-coupled receptors, ethanol, cannabinoids, and lipoxygenase products of arachidonic acid has come from electrophysiological patch-clamp recording studies on the cell bodies of TRPV1-expressing cells. Furthermore, pharmacological antagonism of TRPV1 has been shown to inhibit action potential discharge evoked by each of these stimuli. However, due to the nonselective effects of several of these tool compounds, this pharmacological approach has its limitations. Furthermore, if the antagonist inhibits but does not completely block a given response, it is not clear whether activation of the TRPV1 is obligatory for the response or merely contributes to the end response. In studies using TRPV1–/– mice it was concluded that whereas TRPV1 is required for action potential discharge of C fiber terminals evoked by capsaicin and anandamide, it plays a contributory role in responses evoked by low pH of bradykinin. These data suggest that TRPV1 is one of multiple ion channels responsible for the bradykinin-evoked generator potentials (i.e., membrane depolarization) in C fiber terminals. TRPV1 Receptor Antagonists

Antagonists of TRPV1 include ruthenium red, a dye that exhibits properties of noncompetitive antagonism for the TRPV1, and has a poorly defined mechanism of action and limited selectivity. Capsazepine is another agent characterized as a weak but relatively selective, competitive TRPV1 receptor antagonist (over other TRPVs). However, at the concentrations required to antagonize the TRPV1 (approximately 10 mM), capsazepine has demonstrated non-specific effects such as inhibition of voltage-gated calcium channels and nicotinic receptors. Furthermore, this ligand has poor metabolic and pharmacokinetic properties in rodents, where it undergoes extensive firstpass metabolism when given orally. Recently, a number of antagonists with improved potency and/or selectivity have been described and these include antagonists that are structurally related to agonists such as iodo-resiniferatoxin, which is 100-fold more potent than capsazepine. Previous evidence has indicated that iodo-resiniferatoxin is a potent antagonist at the TRPV1 both in vitro and in vivo. Since then several studies have used this

antagonist as a pharmacological tool in order to explore the role for TRPV1 in various physiological settings. Recently, however, although the in vitro antagonistic activity of iodo-resiniferatoxin has been confirmed, in vivo studies have demonstrated some agonist activity of this agent at high doses. Therefore, there is a possibility that this agent retains some agonistic activity and that an agonist-dependent desensitization effect on sensory nerves may well contribute to some of the antagonistic activity observed. The GlaxoSmithKline lead antagonist SB-366791, which has a good selectivity profile in a series of assays, and potent vanilloid agonist AM-404 should provide useful tools for probing the physiology and pharmacology of TRPV1. Several TRPV1 receptor antagonists that share no obvious structural resemblance to any class of vanilloid agonist have also been developed. These compounds have been discovered by high-throughput random screening projects. At present this group of antagonists includes: (1) N-(haloanilino)carbonyl-N-alkyl-N-arylethylendiamines discovered at SKB; (2) N-diphenyl-Nnapthylureas discovered at Bayer; and (3) pyridol [2,3-d]-pyrimidin-4-ones discovered at Novartis.

Localization of TRPV1 The capsaicin-sensitive vanilloid receptor is expressed mainly in sensory nerves including those emanating from the dorsal root ganglia and afferent fibers that innervate the airway, which originate from the vagal ganglia. In the dorsal root and trigeminal ganglion, TRPV1 is localized to small and mediumsized neurons. Somatic sensory neurons of the vagus nerve are located in the jugular ganglion, whereas visceral sensory neurons of the nerve are located in the nodose ganglion. Previous studies have demonstrated that TRPV1-containing neurons are abundant in these ganglia. The traditional view that TRPV1 is simply a marker of primary sensory nerves is now being challenged. The TRPV1 receptor-has been detected in guinea pig and human airways by receptor-binding assays and has been identified in nonneuronal cell types such as mast cells, fibroblasts, and smooth muscle.

Role of TRPV1 in Airway Inflammatory Disease A role for TRPV1 in airway inflammatory disease would depend on the presence of endogenous activators of this channel under physiological and pathophysiological conditions. In fact, it is quite possible that this situation does, in fact, exist in the ‘disease’ scenario given that TRPV1 activation can be initiated

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and responses to other activators potentiated in an acidic environment, which has been shown to be present in diseases such as asthma and COPD. Interestingly, it has previously been established that the airway response to TRPV1 activation is enhanced in certain disease conditions. In particular, it has been reported that the cough response elicited in response to capsaicin is exaggerated in diseases such as asthma and COPD. Anandamide is also an endogenous ligand known to be produced in central neurons. However, more recent studies have suggested that anandamide is synthesized in lung tissue as well as a result of calcium stimulation suggesting that this mediator could also be involved in the activation of TRPV1 under normal and disease conditions. Inflammatory agents (e.g., bradykinin, ATP, PGE2, and nerve growth factor (NGF)) can indirectly sensitize TRPV1 to cause hyperalgesia. Thus, bradykinin and NGF, which activate phospholipase C (PLC), release TRPV1 from phosphatidylinositol 4,5-biphosphate (PIP2)-mediated inhibition. However, PLC also regulates TRPV1 by diacylglycerol (DAG) formation and subsequent activation of protein kinase C (PKC), which in turn phosphorylates and sensitizes TRPV1. Inflammatory stimuli, including prostaglandins and NGF among others, have also been shown to upregulate the expression and function of TRPV1 via the activation of p38 mitogen-activated protein-kinase (MAPK)- and protein kinase A (PKA)-dependent pathways. Previous data has suggested that there is an upregulation of TRPV1 in inflammatory diseases. For example, TRPV1 immunoreactivity is greatly increased in colonic nerve fibers of patients with active inflammatory bowel disease. Furthermore, a recent study has found an increase in TRPV1 expression in sensory nerves found in the airway epithelial layer in biopsy specimens from patients with chronic cough compared to noncoughing, healthy volunteers. There was a significant correlation between the tussive response to capsaicin and the number of TRPV1positive nerves in the patients with cough. Therefore, it has been postulated that TRPV1 expression may be one of the determinants of the enhanced cough reflex found in patients with chronic cough and that the recently described TRPV1 antagonists could be effective in the treatment of chronic persistent cough due to diverse causes.

Conclusions Thus, it would appear that airway inflammatory diseases (e.g., asthma and COPD) may respond to treatment with an effective and selective inhibitor of the ‘capsaicin receptor’ TRPV1 and to this end much

work is being carried out to develop novel inhibitors. Interestingly, capsaicin-sensitive nerve stimulation in subjects with active allergic rhinitis produces reproducible and dose-dependent leukocyte influx, albumin leakage, and glandular secretion. These results provide in vivo evidence for the occurrence of neurogenic inflammation in the human upper airway with active allergic disease and may therefore suggest the therapeutic utility of TRPV1 antagonists in the management of this disease. In addition, the treatment of persistent cough is a facet of airway diseases that is sorely in need of effective treatment and a TRPV1 inhibitor may prove extremely effective against cough induced by gastroesophageal acid reflux, for example, as well as that associated with asthma and other diseases of the airways described above. See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Kinins and Neuropeptides: Bradykinin; Neuropeptides and Neurotransmission; Tachykinins. Neurophysiology: Neural Control of Airway Smooth Muscle; Neuroanatomy.

Further Reading Barnes PJ (2001) Neurogenic inflammation in the airways. Respiratory Physiology 125: 145–154. Belvisi MG (2003) Airway sensory innervation as a target for novel therapies: an outdated concept? Current Opinion in Pharmacology 3: 239–243. Belvisi MG (2003) Sensory nerves and airway inflammation: role of A delta and C-fibres. Pulmonary Pharmacology and Therapeutics 16: 1–7. Belvisi MG and Geppetti P (2004) Cough * 7: current and future drugs for the treatment of chronic cough. Thorax 59: 438–440. Caterina MJ and Julius D (2001) The vanilloid receptor: a molecular gateway to the pain pathway. Annual Review of Neuroscience 24: 487–517. Coleridge HM and Coleridge JC (1994) Pulmonary reflexes: neural mechanisms of pulmonary defence. Annual Review of Physiology 56: 69–91. Holzer P (1991) Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacological Reviews 43: 143–201. Hwang SW and Oh U (2002) Hot channels in airways: pharmacology of the vanilloid receptor. Current Opinion in Pharmacology 2: 235–242. Karlsson JA (1993) A role for capsaicin sensitive, tachykinin containing nerves in chronic coughing and sneezing but not in asthma: a hypothesis. Thorax 48: 396–400. Maggi CA and Meli A (1988) The sensory-efferent function of capsaicin-sensitive sensory neurons. General Pharmacology 19: 1–43. Morice AH and Geppetti P (2004) Cough 5: the type 1 vanilloid receptor: a sensory receptor for cough. Thorax 59: 257–258. Rogers DF (2002) Pharmacological regulation of the neuronal control of airway mucus secretion. Current Opinion in Pharmacology 2: 249–255. Szallasi A and Blumberg PM (1999) Vanilloid (Capsaicin) receptors and mechanisms. Pharmacological Reviews 51: 159–212. Szolcsanyi J (2004) Forty years in capsaicin research for sensory pharmacology and physiology. Neuropeptides 38(6): 377–384.

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CARBON DIOXIDE R A Klocke, University at Buffalo, Buffalo, NY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Large quantities of CO2 produced in tissues must be efficiently transferred into blood and transported to the lungs where CO2 is excreted. Transport of CO2 dissolved in solution alone is not adequate to meet these requirements. Carbon dioxide is transported as three different, but interrelated, entities in blood. A small portion of total CO2 content in blood exists as dissolved carbon dioxide. Although small in absolute quantity, dissolved CO2 has a key role in exchange because it is the only form that rapidly crosses membranes separating blood from tissues and alveolar gas. The largest quantity of CO2 exists in blood as bicarbonate ion. This ion is a product of the dissociation of carbonic acid, a compound formed when CO2 combines with water. A modest amount of carbon dioxide is transported as carbamate, a compound formed by binding of CO2 to amino groups of the hemoglobin molecule. Exchange of oxygen augments simultaneous CO2 exchange through changes in the molecular configuration of hemoglobin. This structural change increases buffering of hydrogen ions and binding of CO2 as carbamate. The time required for completion of these interrelated processes is critical because blood remains in the pulmonary capillaries for less than 1 s. Two processes overcome this limitation. The natural rate of conversion between CO2 and carbonic acid is quite slow, but is catalyzed in vivo by carbonic anhydrase. In addition, a specialized transporter protein facilitates bicarbonate transfer across the erythrocyte membrane. Reactions of CO2 play a major compensatory role in diseases that cause metabolic acidosis. Hydrogen ions combine with bicarbonate ions and are converted into CO2, which is subsequently excreted in the lungs. This process reduces circulating hydrogen ions and is an important factor in defense of acid–base homeostasis. Although the rare congenital absence of isomers of carbonic anhydrase have been previously associated with abnormalities in the brain, bones, and kidneys, recent evidence raises the possibility that CO2 excretion may also be impaired in these patients.

Introduction Carbon dioxide (CO2) and water are the end products of oxidative metabolism that generates the energy required for maintenance of body homeostasis. Large quantities of CO2 produced in tissues must be efficiently transferred into blood and transported to the lungs where it is excreted. Transport of CO2 dissolved in solution alone is not adequate to meet these requirements.

CO2 Transport in Blood Carbon dioxide is transported in three forms: as dissolved CO2, as bicarbonate ion, and bound to hemoglobin as a carbamate compound.

Table 1 Relative contributions of dissolved CO2, bicarbonate ion, and carbamate compounds to carbon dioxide transport in arterial blood and excretion in the lungs

Dissolved CO2 Bicarbonate ion Carbamate compounds

Transport (%)

Excretion (%)

5 88 7

8 79 13

Dissolved CO2

At normal CO2 tensions present in blood, dissolved CO2 has an approximate concentration of 1.2 mM. The difference between arterial and venous blood is small and accounts for only 8% of the total CO2 excreted in the lungs (Table 1). However, dissolved CO2 is crucial to gas exchange since it is the only form that can rapidly traverse the capillary membranes separating circulating blood from tissues and alveolar gas. Bicarbonate Ion

Carbon dioxide combines with water to form carbonic acid, a weak acid with a pH of 3.5. At the pH range present in the body, this acid almost completely dissociates into hydrogen ions and bicarbonate ions: CA

H2 O þ CO2 2 H2 CO3 2Hþ þ HCO
3

½1

The natural rate of formation of carbonic acid from CO2 is very slow and requires 60–90 s to reach completion. However, erythrocytes contain large quantities of carbonic anhydrase (CA), an enzyme that catalyzes this reaction. The hydrogen ions formed in this reaction are buffered effectively by hemoglobin, which is the dominant nonbicarbonate buffer in blood. Release of oxygen from the hemoglobin molecule alters the quaternary structure of hemoglobin, thereby increasing the affinity of binding sites for hydrogen ions (the Bohr effect). This increases the buffering capacity of deoxygenated hemoglobin. Figure 1 illustrates the buffering curves of oxygenated and reduced hemoglobin. As hemoglobin is reduced in the tissues, its buffering curve shifts upward and significant amounts of hydrogen ion can be buffered without any change in pH. It is estimated that approximately one half of the hydrogen ions released in aerobic metabolism are buffered in this manner. The majority of CO2 in blood is carried as bicarbonate (Table 1).

CARBON DIOXIDE 321

H+ added (mEq)

3.0

Ar

Ve

ter

no

ial

us

Actual ∆pH

2.0 ∆H+ from CO2

1.0 0.0 ∆pH without H+ binding 7.30

7.35 pH

7.40

Figure 1 Buffering curves of hemoglobin in arterial and venous blood. With release of oxygen bound to hemoglobin, buffering sites on hemoglobin are able to bind more hydrogen ions (the Bohr effect). The increased binding of hydrogen ions occurs as blood–acid base status shifts from the arterial to venous buffering curve. Hydrogen ions generated from CO2 exchange in the tissues are buffered with less change in pH than would occur if buffering took place along the arterial curve.

Carbamate Compounds

Amino groups of proteins can reversibly bind hydrogen ions: þ R-NHþ 3 2H þ R-NH2

½2

where R represents the remainder of the protein moiety. Carbon dioxide can bind to the uncharged amino groups: R-NH2 þ CO2 2R-NHCOOH 2R-NHCOO
þ Hþ

½3

to form carbamic acids that dissociate into carbamate and hydrogen ions at normal blood pH. The quantity of CO2 bound to serum proteins and the e-amino groups of hemoglobin is minimal and does not change significantly between arterial and venous CO2 tensions. However, the a-amino groups of the N-termini of the hemoglobin molecule contribute substantially to CO2 transport. The changes in molecular configuration accompanying reduction of hemoglobin alter the equilibrium constants of reactions [2] and [3], favoring binding of CO2 as carbamate. As a result, the relative contribution of carbamate to the exchange of CO2 is approximately twice as great as its relative concentration in blood (Table 1). The a-amino groups of the b-chains of hemoglobin are affected to a greater extent by conformational changes of the molecule and are responsible for three-quarters of the carbamate contribution to exchange. The concentration of 2,3-diphosphoglycerate (2,3DPG) within the erythrocyte influences the contribution of carbamate compounds to CO2 exchange. The

two N-terminal portions of the b-chains on the surface of hemoglobin are more widely separated in the reduced state as a result of the accompanying change in the quaternary structure. Positive charges on the b-chains induce the negatively charged 2,3DPG molecule to enter the enlarged cavity between the two separated b-chains of reduced hemoglobin. The positioning of 2,3-DPG near the N-terminal a-amino groups alters the equilibrium constants of reactions [2] and [3], and reduces the quantity of the uncharged form of the a-amino groups that binds CO2. Haldane Effect

The carbon dioxide dissociation curve of normal human blood is illustrated in Figure 2. The total concentration of CO2 in all forms is plotted as a function of the carbon dioxide partial pressure in blood. As seen in the figure, at any given PCO2 the total CO2 content is greater in blood with a reduced oxygen content compared to fully oxygenated blood. This is known as the Haldane effect, named after one of the investigators who first described this phenomenon. The difference in CO2 content between the two curves at the same PCO2 is termed oxylabile CO2. It is produced by the changes in the quaternary structure of hemoglobin that accompany oxygenation and reduction. Oxylabile CO2 has two components. The first is an increase in bicarbonate concentration resulting from greater buffering of hydrogen ions by reduced hemoglobin. The second component is the result of increased carbamate formation associated with reduced hemoglobin. The Haldane effect enhances the transport of carbon dioxide. The shift of the CO2 dissociation curve caused by release of oxygen allows for transport of CO2 with a lower CO2 tension in venous blood than would occur if there were no shift in the position of the dissociation curve (Figure 2). The synergism between oxygen and carbon dioxide exchange facilitates transport of CO2 to a much greater extent than the transport of O2.

Time-Dependent Processes in CO2 Exchange The actual exchange of CO2 in the pulmonary capillaries involves a series of complicated processes. Exchange of each form in which CO2 is transported is discussed separately, but the processes occur simultaneously (Figure 3). The reactions involved in conversion of bound CO2 to dissolved CO2 are complex and require finite time. These reactions probably reach completion in 0.3–0.4 s, which is

322 CARBON DIOXIDE 60

70% HbO2

CO2 content (ml dl−1)

40

54

∆PCO2

v

v′

52 20 97.5% HbO2

50 a

∆PCO2 without Haldane effect

48 40

45

50

0 0

20

40

60

PCO (mmHg) 2 Figure 2 Carbon dioxide dissociation curves of blood. The two curves represent the relationship between CO2 content and PCO2 of arterial (oxygen saturation of 97.5%) and venous (oxygen saturation of 70%) blood. Binding of oxygen to hemoglobin causes a shift from the venous to arterial CO2 dissociation curve. The inset in the figure illustrates the effect of this shift on blood PCO2 . The difference in PCO2 between arterial (a) and venous blood (v ) is substantially less than would occur if the Haldane effect were not operative and CO2 exchange took place along a single dissociation curve (a and v 0 ). Data used to construct the dissociation curves taken from Forster RE, DuBois AB, Briscoe WA, and Fisher AB (1986) The Lung, p. 238. Chicago: Yearbook Medical Publishers.

sufficient for excretion of CO2 to occur during the time the blood remains in the pulmonary capillary (0.7 s at rest, 0.5 s during exercise). Although the process of diffusion of CO2 across the alveolar–capillary membrane is quite rapid (B0.01 s), the chemical reactions and other transport processes necessary for CO2 exchange appreciably slow the actual rate of exchange. In peripheral tissues, these same processes occur in an opposite manner.

Dissolved CO2

When blood reaches the pulmonary capillaries, dissolved CO2 diffuses from the plasma and the interior of the erythrocyte across the alveolar–capillary membrane into the alveoli. The reduction in dissolved CO2 rapidly lowers capillary PCO2, and disturbs the equilibrium of carbamate and bicarbonate reactions. This promotes conversion of the latter two compounds into CO2 which, in turn, diffuses out of the capillary into the alveoli.

Bicarbonate Ion

As blood PCO2 falls, equation [1] is reversed and bicarbonate is converted first into carbonic acid and then into free CO2. Carbonic anhydrase inside the erythrocyte catalyzes this reaction by a factor of 13 000–15 000. Hydrogen ions required for conversion of bicarbonate to CO2 are largely supplied by hemoglobin. The simultaneous binding of oxygen to hemoglobin causes the release of hydrogen ions. In the plasma, the conversion of bicarbonate ion to CO2 occurs at a much slower rate. Plasma proteins are much less effective buffers than hemoglobin and are not affected by simultaneous oxygen transfer. Therefore, fewer hydrogen ions are available for bicarbonate conversion. There is a modest amount of carbonic anhydrase attached to the capillary endothelium, but this is sufficient to provide catalysis that is only 1/100 of that inside the red cell. As a result of the differences in buffering power and catalysis, the concentration of bicarbonate inside the erythrocyte decreases much more rapidly than that in plasma.

CARBON DIOXIDE 323 Carbonic Anhydrase

CO2 Alveolus CA

CA

Plasma

CA

Cl − HCO− + H+ 3

CA

CA

CA

CA

CO2 + H2O

H2CO3

3 Cl−

HCO3− + H+

HHb

Hb− + H+ R−NHCOO− + H+

CA

CO2 + H2O + H+ + R−NH2 R−NH+3

H2CO3

R−NHCOOH

Figure 3 Carbon dioxide exchange in the lung. Solid lines indicate reactions that take place rapidly. The dashed line represents the slower reaction of formation of CO2 from H2CO3 as catalyzed by carbonic anhydrase (CA) localized to the capillary endothelium. The dotted lines indicate buffering reactions of hemoglobin. The number 3 indicates the band 3 anion transporter protein that exchanges bicarbonate and chloride ions between the erythrocyte and plasma. Reproduced from Klocke RA (1997) Carbon dioxide transport. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 1633–1642. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

This difference in bicarbonate concentration leads to transfer of bicarbonate from the plasma into the erythrocyte. Bicarbonate ions cannot enter the red cell independently because they are negatively charged. They exchange simultaneously in the opposite direction with intracellular chloride ions. This paired bicarbonate–chloride exchange is accomplished in an electrically neutral fashion by a transmembrane protein known as the band 3 anion transporter protein. Through this process of anionic exchange, more than one-half of the total bicarbonate converted to CO2 in the lung originates in plasma, but enters the erythrocyte to be converted into CO2 before being excreted. Carbamate Compounds

As PCO2 decreases within the erythrocyte, through reactions [2] and [3] carbon dioxide bound to the a-amino groups is released as free CO2 and diffuses into the alveoli. The hydrogen ions required for these reactions are supplied through the buffering reactions of hemoglobin. Simultaneous binding of oxygen facilitates the release of CO2 bound to hemoglobin. Oxygenation reduces the affinity of the amino groups for CO2 and promotes CO2 release. Because this portion of CO2 exchange is dependent on oxygenation of hemoglobin, this reaction is delayed until oxygen is first bound to hemoglobin.

Five of the more than one dozen identified isomers of the enzyme carbonic anhydrase (CA) are potentially involved in CO2 exchange in the lungs and tissues. Both CA I and II are present in large quantities within the erythrocytes of humans and many mammals. This enzymatic activity is much greater than that thought necessary for efficient CO2 exchange, but the reason for the excess enzyme is not apparent. CA I is a less potent enzyme and its activity is partially inhibited by chloride ion. Although CA I comprises 85–90% of the total enzyme inside human erythrocytes, CA II is responsible for the majority of total enzymatic activity. Some erythrocytic CA II is bound to intracellular portions of the band 3 anion transporter protein, providing a highly efficient complex for interconversion of CO2 and HCO3
and transmembrane exchange of the bicarbonate ion. CA IV is a high-potency isomer that is localized to membranes, especially the sarcolemma of muscle tissue and the capillary endothelium of most tissues. Its impact on gas exchange in the lung is probably limited by its low concentration and the lack of substantial plasma buffering compared to that inside the red cell. Mathematical models of CO2 exchange in muscle suggest sarcolemmal CA IV may play a role in maintaining acid-base balance when large quantities of lactic acid are produced during exercise. CA III and V isomers also are present in some striated muscle, but their role, if any, in CO2 exchange is unclear.

Carbon Dioxide in Disease Carbon dioxide elimination can be impaired in multiple disease states. However, this derangement is rarely caused by abnormal transport, but is usually the result of reduced ventilation or mismatching of ventilation and blood flow within the lung. Genetic deficiencies of CA II are accompanied by renal tubular acidosis, osteopetrosis, and cerebral calcifications. Most studies have failed to detect abnormalities in CO2 exchange in patients at rest, presumably because CA I present in erythrocytes is sufficient to support CO2 exchange. However, studies have not been conducted during exercise when abnormalities would be more likely to occur. A recent description of patients with carbonic anhydrase deficiencies suggests that even CO2 exchange at rest may not be completely normal. The difference between end-tidal gas (a surrogate for alveolar gas) and arterial blood CO2 tensions was increased, implying that CO2 exchange did not reach equilibrium during transit through the pulmonary capillary bed. Further studies are required to validate this possible impairment.

324 CARBON MONOXIDE

The carbon dioxide dissociation curve of blood is markedly affected by acid–base alterations. Abnormalities that increase blood bicarbonate concentration, i.e., metabolic alkalosis and compensated respiratory acidosis, increase the total quantity of carbon dioxide in all forms at any given CO2 tension. The opposite is true in metabolic acidosis, which is characterized by a marked reduction in bicarbonate, and therefore total CO2 content, at any give PCO2 . The ratio of the arterial–venous difference in CO2 content divided by the arterial–venous difference in PCO2 provides an index of the efficiency of carbon dioxide transport. Although this ratio has varied in studies of patients with acid–base abnormalities, it is significantly reduced only in metabolic acidosis. This decreased efficiency of CO2 transport is caused by a reduction in the Haldane effect in metabolic acidosis. This results in elevation of venous, and therefore tissue, PCO2 . However, it is important to note that there are few studies of CO2 transport in clinical circumstances and much more data is needed to characterize fully the effects of acid–base disturbances on CO2 exchange. Reactions of carbon dioxide have a prominent role in acid–base balance that is particularly emphasized in metabolic acidosis. This common condition occurs most frequently when oxidation of glucose is impaired and energy is generated via biochemical pathways that produce strong acids. Two common conditions are impairment of oxygen delivery to tissues leading to production of lactic acid and diabetic ketoacidosis that results in generation of acetoacetic and b-hydroxybutyric acids. In both situations, these strong acids ionize completely and release large quantities of hydrogen ions. With the resultant increase in hydrogen ion concentration, bicarbonate ion binds to hydrogen ion to form carbonic acid, which is further converted into CO2 and water. The CO2 formed in this buffering reaction is excreted in

the lungs, completing the removal of free hydrogen ions from the circulating blood. Thus, the reactions of carbon dioxide play a major role in defending acid–base balance in these circumstances. See also: Acid–Base Balance. Hemoglobin. Ventilation, Perfusion Matching. Ventilation: Overview.

Further Reading Forster RE, DuBois AB, Briscoe WA, and Fisher AB (1986) The Lung, pp. 235–242. Chicago: Yearbook Medical Publishers. Geers C and Gros G (2000) Carbon dioxide transport and carbonic anhydrase in blood and muscle. Physiological Reviews 80(2): 681–715. Henry RP and Swenson ER (2000) The distribution and physiological significance of carbonic anhydrase in vertebrate gas exchange organs. Respiratory Physiology 121(1): 1–12. Klocke RA (1987) Carbon dioxide transport. In: Farhi LE and Tenney SM (eds.) Handbook of Physiology: The Respiratory System, pp. 173–197. Bethesda: American Physiological Society. Klocke RA (1997) Carbon dioxide transport. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 1633–1642. New York: Raven Press. Lindskog S and Silberman DN (2000) The catalytic mechanism of mammalian carbonic anhydrases. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 175–195. Basel: Birkhauser. Swenson ER (2000) Respiratory and renal roles of carbonic anhydrase in gas exchange and acid–base regulation. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 281–341. Basel: Birkhauser. Tanner MJA (2002) Band 3 anion exchanger and its involvement in erythrocyte and kidney disorders. Current Opinion in Hematology 9(2): 133–139. Venta PJ (2000) Inherited deficiencies and activity variants of the mammalian carbonic anhydrases. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 403–412. Basel: Birkhauser. Wetzel P and Gros G (2000) Carbonic anhydrases in striated muscle. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 375–399. Basel: Birkhauser.

CARBON MONOXIDE D Morse, University of Pittsburgh, Pittsburgh, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Carbon monoxide (CO) is generated in the human body by the catabolism of heme. This reaction is catalyzed by an enzyme known as heme oxygenase, which has both an inducible (heme oxygenase-1) and constitutive (heme oxygenase-2) form. CO was once believed to be a mere byproduct of heme breakdown, but is now known to modulate a number of cellular functions

including inflammation, cellular proliferation, and apoptotic cell death. This article offers a broad overview of our current understanding of the role of CO in respiratory disease.

Introduction Carbon monoxide (CO) is known to most pulmonologists as an air pollutant arising from the partial combustion of organic molecules and as a potentially lethal gas when inhaled in high concentrations. The avid binding of CO to heme iron results

CARBON MONOXIDE 325 CO

Heme

Heme oxygenase

Biliverdin

Biliverdin reductase

Bilirubin

Fe2+ Ferritin Figure 1 The enzymatic reaction catalyzed by heme oxygenase results in the release of CO.

in displacement of oxygen from hemoglobin, thus reducing the oxygen carrying capacity of blood. This phenomenon in turn leads to tissue ischemia and the familiar symptoms of CO poisoning. It is less widely appreciated that CO, in addition to being an environmental pollutant, is a biological product of ordinary metabolism. CO is generated in the human body by the catabolism of heme. This endogenously produced CO results in the normal baseline human carboxyhemoglobin level of 0.4–1%, and CO can be measured in the breath as it is excreted. The enzyme that releases CO from the breakdown of heme is known as heme oxygenase. There is a constitutively expressed form of this enzyme (heme oxygenase-2 (HO-2)) and an inducible form (heme oxygenase-1 (HO-1)). As one of the three byproducts of heme degradation (Figure 1), CO was initially considered a catabolic waste product. In fact, it was suggested in 1969 that CO production from humankind could contribute significantly to air pollution. We now know that this concern was misplaced, and it has more recently become evident that CO production serves a number of biological purposes. This article focuses on the functions of CO that relate most closely to pulmonary medicine, but it should be noted that a large body of literature exists describing functions for CO such as neurotransmission and vasodilation that cannot be discussed in detail here.

Structure CO is a colorless, odorless diatomic gas composed of a single carbon atom that is triply bonded to a single oxygen atom. It is an inert gas that does not readily engage in chemical reactions, but its affinity for heme is approximately 200 times that of oxygen. CO is slightly soluble in water (and therefore plasma).

Regulation of Activity The rate-limiting step in the generation of CO is heme catalysis by heme oxygenase. The inducible

form of heme oxygenase, HO-1, is widely distributed throughout the body and is highly evolutionarily conserved. Its activity is transcriptionally regulated, and it is induced by more stimuli than any other known gene. The inducers of HO-1 include mainly conditions that would cause stress to cells, such as increased oxidant burden, radiation, heavy metals, and hypoxia. Not surprisingly, there are a number of cis-acting DNA sequence elements that serve as potential binding sites for transcription factors in the proximal promoter region and at distal enhancer sites. The dominant sequence element in the distal enhancer regions of mouse and human ho-1 is the stress-responsive element (StRE). The StREs represent targets of multiple dimeric proteins generated by intrafamily homodimerization or intra- and interfamily heterodimerization of individual members of the Jun, Fos, CREB, ATF, Maf, and the Cap’n’collar/ basic-leucine zipper subclasses of the basic-leucine zipper superfamily of transcription factors.

Biological Functions Anti-Inflammatory Effects

Several lines of evidence suggest that one of the reasons CO may be generated under conditions of stress is for its ability to dampen inflammation. There has been one reported case of human HO-1 deficiency, and the affected child exhibited signs of chronic inflammation and was highly vulnerable to oxidative stress. Experimental studies have shown that when mice or macrophages are stimulated with lipopolysaccharide, a component of bacterial cell walls, the production of proinflammatory cytokines (such as tumor necrosis factor alpha, interleukin-6 and interleukin-1b) is greatly increased. This model is frequently used to simulate human sepsis, a condition that is associated with variable elevation of these same cytokines. When these same mice or cells are administered low concentrations of CO (250 ppm) along with lipopolysaccharide, the production of the proinflammatory cytokines is inhibited. Furthermore, the production of the anti-inflammatory cytokine interleukin-10 is augmented by CO treatment. CO has also been shown to affect the expression of granulocytemacrophage colony-stimulating factor (GM-CSF), a glycoprotein that promotes the proliferation and the differentiation of hematopoietic progenitor cells into neutrophils and macrophages. A number of chronic inflammatory pulmonary diseases such as chronic obstructive pulmonary disease (COPD), asthma, and sarcoidosis are associated with elevated levels of GM-CSF. The anti-inflammatory effects of CO are mediated primarily via the mitogen-activated proteinkinase (MAPK) signaling pathways.

326 CARBON MONOXIDE Antiapoptotic Effects

Programmed cell death, or apoptosis, must be finely balanced for the maintenance of health and resolution of disease. Under conditions of stress, excessive apoptosis may lead to worsening organ dysfunction, and one could postulate that a stress-induced antiapoptotic molecule could provide protection. This thinking led to the examination of an antiapoptotic role for CO. The first experiments performed in vitro demonstrated that applying CO to fibroblasts or endothelial cells could prevent cell death in response to tumor necrosis factor alpha. This effect was confirmed in animal models of disease, including ischemia/reperfusion injury and transplantation. The antiapoptotic effect of CO is not universal, however. In Jurkat T cells, CO has been shown to increase Fas/ CD95-induced apoptosis. Additionally, high concentrations of CO lead to apoptosis of tissue in rodents, associated with CO poisoning and tissue injury. As with the anti-inflammatory effects, the antiapoptotic effects of CO are primarily mediated by the MAPK signaling pathways. Antiproliferative Effects

Regulation of cell proliferation is important not only for the maintenance of homeostasis, but in the body’s response to disease. It appears likely that stress-induced production of CO modulates cell growth in disease states, thereby potentially altering the progression of a variety of illnesses. The antiproliferative function of CO has been established in vitro in a number of different cell types. CO applied directly to smooth muscle cells causes growth arrest, and endogenous CO released from vascular smooth muscle cells as a consequence of hypoxiainduced HO-1 expression has the same effect. This endogenously produced CO also results in increased production of guanosine 30 ,50 -(cyclic)phosphate (cyclic GMP) in co-cultured endothelial cells, which in turn leads to downregulation of the expression of endothelial-derived mitogens such as platelet-derived growth factor and endothelin-1. The antiproliferative effect of CO has been implicated in the protection conferred by CO against vascular stenosis associated with balloon catheter injury. CO has also been shown to suppress the proliferation of human airway smooth muscle cells, which may be relevant to the pathogenesis of asthma, where inflammatory cell-derived mediators stimulate smooth muscle proliferation. CO has been further shown to arrest the growth of cultured T lymphocytes, macrophages, and fibroblasts, which may account in part for the influence of CO on the progression of immune-mediated and fibrotic lung diseases. The

antiproliferative effects of CO are generally mediated by increased intracellular cyclic GMP levels, although in some cell types the MAPK pathways may participate as well.

Carbon Monoxide in Respiratory Diseases The lung is an organ with a particular vulnerability to oxidative injury as the interface between the atmosphere and the rest of the body. Oxidative stress may also be caused by pathologic conditions such as infection, inflammation, and ischemia reperfusion. Lungs therefore require potent defense mechanisms and possess high levels of antioxidant enzymes. HO-1 is one of the few inducible molecules that can protect the lungs from an increased oxidant burden under stressful circumstances. In the lungs, HO-1 is highly expressed in alveolar macrophages, but is also found in epithelial cells, fibroblasts, and endothelial cells. The CO produced by HO-1 activity is primarily excreted in the breath, and the level of exhaled CO has been shown to increase in disease states and varies with therapy or exacerbations. Our current knowledge about the role of CO in lung disease is derived mainly from preclinical animal and cell culture studies; highlights of this work are summarized below and in Figure 2. Increasingly, the focus of CO research has been directed at potential therapeutic applications, such as using inhaled CO to limit lung injury due to a variety of insults. The CO animal and cell studies alluded to in the sections that follow generally use a concentration range of 50–250 ppm. For reference, this inhaled concentration would result in carboxyhemoglobin levels ranging from 8% to 25% at equilibrium (after 8 h of exposure) in humans. Acute Lung Injury

Many of the initial studies done to characterize the location and function of HO-1 in the lung used rodent models of acute lung injury such as hyperoxia. In rodents, high concentrations of inhaled oxygen result in lung edema and death within several days. This oxidant injury results in increased expression of HO-1 in the lung, and overexpression of HO-1 in the bronchiolar epithelium by adenoviral gene transfer results in enhanced protection from the lethal effects of hyperoxia. Inhaled CO confers similar protection against hyperoxic lung injury. In vitro studies suggest that a part of the mechanism of protection by CO is due to prevention of cell death in response to increased oxygen tension. Ventilator-induced lung injury may complicate acute lung injury in humans, and recent rodent studies suggest that inhaled CO also exerts anti-inflammatory effects in the setting of high tidal volume ventilation.

ell p

roli fe

rati o

n

CARBON MONOXIDE 327

le c

CO

Bron

onst

rictio

n

Sm

oot hm

us c

choc

Pulmonary Fibrosis

CO

Apoptosis

CO

Inflammation

thought to be major contributors to the pathogenesis of COPD. Patients with COPD have lower HO-1 expression in alveolar macrophages than controls, and a microsatellite polymorphism in the promoter for HO-1 has been associated with the development of emphysema. This suggests that an abnormal response of HO-1 to oxidative stress may be linked to the development of COPD.

CO

Immunohistochemical analysis of lung tissue from patients with various forms of interstitial lung disease reveals increased expression of HO-1, primarily in alveolar macrophages. Increased expression of HO-1 by adenoviral transfer has been shown to suppress lung fibrosis in a murine bleomycin model, and inhaled CO has similar effects. The mechanism by which HO-1 and CO confer protection against fibrosis has not been fully elucidated, but CO has been shown to alter the cytokine milieu, decrease matrix production by fibroblasts, slow fibroblast proliferation, and inhibit epithelial apoptosis, any of which could contribute to a modulatory effect on fibrosis.

CO

Pulmonary Vascular Disease

Fibroproliferation

Figure 2 Schematic summary of CO effects in lung disease. CO has been shown to suppress cell proliferation, bronchoconstriction, apoptosis, inflammation, and fibroproliferation.

Obstructive Lung Disease

Asthma is a disease characterized by inflammation, and so it is not surprising that exhaled CO levels have been shown to increase during asthma exacerbations. Studies in mice and cell culture suggest that the CO generated in the lung of asthmatics may affect the course of disease. Low-concentration inhaled CO has been shown to attenuate aeroallergeninduced inflammation in mice, resulting in decreased interleukin-5 and eicosanoid mediator levels, as well as decreased bronchoalveolar lavage inflammatory cell counts. Exogenously administered CO has also been shown to decrease airway hyperresponsiveness in mice, and as previously noted, CO may modulate the lung remodeling in asthma due to its inhibitory effect on airway smooth muscle cell proliferation. Exposure to reactive oxygen species and an imbalance in oxidant/antioxidant status are also

Following the discovery that nitric oxide could mediate vascular relaxation by increasing intracellular levels of cyclic GMP, it was natural to postulate that CO would have a similar effect, as CO was also known to induce cyclic GMP. We now know that CO causes vasodilation directly via activation of soluble guanylate cyclase in smooth muscle cells and indirectly through inhibition of the vasoconstrictors endothelin-1 and platelet-derived growth factor-B. In addition to these vasodilatory effects, CO may also prevent vascular remodeling by inhibiting smooth muscle cell proliferation. Enhanced activity of HO-1 in the lung has been shown to prevent the development of hypoxic pulmonary hypertension and inhibit structural remodeling of the pulmonary vessels in rats. Inhaled CO has also been shown to attenuate experimental pulmonary hypertension in rats. CO has also been strongly implicated in the development of hepatopulmonary syndrome in experimental models, and patients with hepatopulmonary syndrome have been shown to have higher carboxyhemoglobin levels than control subjects. In summary, CO production under conditions of physiological stress results in a wide range of adaptive responses that are relevant to pulmonary disease. Improving our understanding of the biological activities of CO will continue to enhance our knowledge of pulmonary pathophysiology. The goals of current research include such things as monitoring disease

328 CAVEOLINS

activity via measurement of exhaled CO, or even manipulating levels of CO in the body therapeutically. If successful, such research could bring CO fully into the day-to-day practice of pulmonary medicine. See also: Acute Respiratory Distress Syndrome. Apoptosis. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Pulmonary Fibrosis.

Further Reading Ameredes BT, Otterbein LE, Kohut LK, et al. (2003) Low-dose carbon monoxide reduces airway hyperresponsiveness in mice. American Journal of Physiology: Lung Cellular and Molecular Physiology 285(6): L1270–L1276. Brouard S, Otterbein LE, Anrather J, et al. (2000) Carbon monoxide generated by heme oxygenase-1 suppresses endothelial cell apoptosis. The Journal of Experimental Medicine 192(7): 1015– 1026. Dolinay T, Szilasi M, Liu M, and Choi AM (2004) Inhaled carbon monoxide confers antiinflammatory effects against ventilatorinduced lung injury. American Journal of Respiratory and Critical Care Medicine 170(6): 613–620. Fujita T, Toda K, Karimova A, et al. (2001) Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nature Medicine 7(5): 598–604. Horvath I, Donnelly LE, Kiss A, et al. (1998) Raised levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1 in airway macrophages in asthma: a new marker of oxidative stress. Thorax 53: 668–672.

Carcinoma

Maines MD (1993) Carbon monoxide: an emerging regulator of cGMP in the brain. Molecular and Cellular Neurosciences 4: 389–397. Morita T and Kourembanas S (1995) Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide. The Journal of Clinical Investigation 96: 2676–2682. Morita T, Mitsialis SA, Hoike H, Liu Y, and Kourembanas S (1997) Carbon monoxide controls the proliferation of hypoxic smooth muscle cells. Journal of Biological Chemistry 272: 32804–32809. Otterbein LE, Bach FH, Alam J, et al. (2000) Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nature Medicine 6: 422–428. Otterbein LE, Haga M, Zuckerbaun BS, et al. (2003) Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nature Medicine 9: 183–190. Otterbein LE, Mantell LL, and Choi AM (1999) Carbon monoxide provides protection against hyperoxic lung injury. American Journal of Physiology: Lung Cellular and Molecular Physiology 276: L688–L694. Yachie A, Niida Y, Wada T, et al. (1999) Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. The Journal of Clinical Investigation 103: 129–135. Yet SF, Perrella MA, Layne MD, et al. (1999) Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice. The Journal of Clinical Investigation 103: R23–R29. Zhou Z, Song R, Fattman CL, et al. (2005) Carbon monoxide suppresses bleomycin-induced lung fibrosis. American Journal of Pathology 166(1): 27–37.

see Tumors, Malignant: Bronchogenic Carcinoma; Bronchial Gland and Carcinoid Tumor;

Carcinoma, Lymph Node Involvement.

CAVEOLINS M P Lisanti and J-F Jasmin, Albert Einstein College of Medicine, New York, NY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Caveolae are small invaginations of the plasma membrane that are implicated in endocytosis, vesicular trafficking, and signal transduction. Caveolin proteins (Cav), the principal structural proteins of caveolae, consist of three distinct genes, namely Cav1, Cav-2, and Cav-3. Cav-1 and Cav-2 are usually coexpressed and are particularly abundant in endothelial cells, fibroblasts, smooth muscle cells, and epithelial cells. In contrast, Cav-3 is muscle-specific and is solely expressed in smooth, cardiac, and skeletal muscle cells. Caveolin proteins appear to play a role as key regulators of pulmonary structure and function. Homozygous deletion of the Cav-1 gene (Cav-1 (
/
)) results in numerous pulmonary abnormalities, characterized by a loss

of Cav-1 and Cav-2 protein expression, a loss of caveolae formation, thickened alveolar septa, lung hypercellularity, pulmonary endothelial cell proliferation, fibrosis, and exercise intolerance. Cav-2 (
/
) mice show similar pulmonary abnormalities to the Cav-1 (
/
) mice, but without a loss of Cav-1 expression and normal caveolae formation. Furthermore, Cav-1 (
/
) mice develop pulmonary hypertension and right ventricular hypertrophy. Interestingly, the endogenous levels of caveolin proteins appear to be altered in numerous pulmonary disorders, such as pulmonary hypertension, lung cancers, pulmonary fibrosis, and pulmonary interstitial edema.

Introduction Caveolae, which were first identified in the 1950s as ‘smooth’ uncoated pits, are now defined as 50– 100 mm flask-shaped invaginations of the plasma

CAVEOLINS 329

membrane; they are implicated in endocytosis, vesicular trafficking, and signal transduction (Figure 1). Caveolae can either be observed as curved U-shaped invaginations of the plasma membrane, fully invaginated caveolae, grape-like clusters of interconnected caveolae (caveosome), or as a transcellular channel (as a consequence of the fusion of individual caveolae) (Figure 2). These caveolar domains are a subset of lipid rafts that contain the essential coat protein, named caveolin (Cav). As shown in Figure 3, caveolins appear as membrane-bound proteins that form homo-oligomers and show an unusual topology, with both their NH2 and COOH termini facing the cytoplasm. The caveolin gene family consists of

three distinct genes, namely Cav-1, Cav-2, and Cav3. Cav-1 and Cav-2 are usually coexpressed and are particularly abundant in endothelial cells, adipocytes, fibroblasts, and smooth muscle cells. On the other hand, Cav-3 appears to be muscle-specific and is thus solely expressed in smooth, cardiac, and skeletal muscle cells. Cav-1 further encodes two isoforms, namely Cav-1a and Cav-1b. These two Cav-1 isoforms derive from two distinct Cav-1 mRNAs generated by alternative transcription initiation sites.

Expression of Caveolin Proteins in the Lungs Although all three caveolin isoforms are expressed in the lungs, Cav-1 still remains the most extensively studied isoform, with wide expression in endothelial cells, fibroblasts, smooth muscle cells, bronchial epithelial cells, and alveolar type I cells. However, Cav-1 is poorly expressed in alveolar type II cells. Cav-1 is also present in the developing lung, suggesting important roles for caveolin proteins in lung development. Interestingly, in the developing lung, the expression of the two Cav-1 isoforms appears to be cell-type specific, with Cav-1a being expressed in endothelial cells and Cav-1b being expressed in alveolar type I cells. This cell-type specific expression could be ascribed to differential transcriptional regulation of Cav-1 in lung epithelial and endothelial cells. Whether this cell-type specific expression of Cav-1 isoforms also occurs in adult mouse lung remains to be clarified.

RBC

Figure 1 Electron micrograph (EM) depicting endothelial cell caveolae (arrows) from a mouse lung endothelial cell. RBC, red blood cell. Scale ¼ 200 nm. Adapted from Razani B, Wang XB, Engelman JA, et al. (2002) Caveolin-2-deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae. Molecular and Cellular Biology 22(7): 2329–2344, with permission of the American Society for Microbiology.

6

Caveolin Proteins and Pulmonary Endocytosis and Transcytosis Based on their appearance and membrane localization, caveolae were initially proposed as transport vesicles. These vesicles were then thought to either

1

2 5

3 4

Nucleus

Figure 2 Schematic representation of a cell illustrating morphological variants of caveolae. These include: 1, traditional caveolae; 2, fully invaginated caveolae; 3, cavicles (internalized caveolae not associated with the plasma membrane); 4, caveosome or rosette-like structures; 5, grape-like clusters of interconnected caveolae; 6, transcellular channels.

330 CAVEOLINS

internalize macromolecules (endocytosis) or transport them across the cell (transcytosis). The presence of several proteins implicated in vesicle formation, docking, and fusion in lung caveolae strongly supports a role for caveolae in endocytosis and transcytosis. In accordance with this idea, several viruses, bacteria, and toxins, such as simian virus 40 and cholera toxin, are thought to be preferentially internalized by caveolae. Furthermore, transcytosis of gold-conjugated albumin appears to be restricted to caveolae in the lungs. The caveolar localization of gp60, a 60 kDa albumin-binding protein, supports the role of caveolar domains in the transcytosis of albumin. The recent generation of caveolin-deficient mice elegantly confirmed the implications of lung caveolae in albumin endocytosis. Indeed, lung endothelial cells of Cav-1 (
/
)-deficient mice show defects in the uptake and transport of gold-conjugated albumin.

Caveolin Proteins and Pulmonary Signal Transduction Besides their roles as transport vesicles, caveolae microdomains also function in signal transduction via their compartmentalization of signal transduction

C-terminal

cascades. Indeed, lung caveolae are known to compartmentalize several signaling molecules, such as protein kinase C (PKC), phosphatidylinositol-3 (PI3) kinase, heterotrimeric G-protein subunits, extracellular signal-related kinase (ERK), endothelial nitric oxide synthase (eNOS), signal transducer and activator of transcription-3 (STAT3), and Ras-related GTPases. In accordance with a role in signal transduction, lung caveolae are also enriched in the calcium ATPase and inositol triphosphate receptors. Most of the proteins sequestered within caveolar domains possess caveolin-binding motifs and consequently interact with Cav-1. These protein–protein interactions are mediated by the interaction of a caveolin-binding motif (within specific target proteins) with a specific region of Cav-1 (residues 82–101), named the scaffolding domain (Figure 3). Interestingly, Cav-1 also functions as a negative regulator of many of these signaling molecules (Figure 4). The generation of Cav-1 (
/
)-deficient mice supports the Cav-1mediated ‘negative regulation’ of several proteins, such as eNOS, ERK1/2, and STAT3. For example, lungs of Cav-1 (
/
)-deficient mice show increased NO production, as well as microvascular hyperpermeability. Interestingly, administration of an NOS inhibitor

Transmembrane domain Scaffolding domain Oligomerization domain

N-terminal Figure 3 Schematic representation of the topology of caveolins as the principal structural proteins of caveolae. Some important domains of Cav-1, such as the oligomerization, scaffolding, and transmembrane domains, are illustrated.

CAVEOLINS 331 Caveolin-1

82

1

101

178

DGIWKASFTTFTVTKYWFYR

(Scaffolding domain of Cav-1)

ΦXΦXXXXΦXXΦ

(Caveolin-binding sequence motif)

Inactivation

PI3 Kinase

eNOS

STAT3

ERK1/2

G-protein subunits

PKC

Modulation of pulmonary signal transduction Figure 4 Schematic representation of the interaction of the Cav-1 scaffolding domain with the caveolin-binding sequence motifs of PI3 kinase, eNOS, STAT3, ERK1/2, G-protein subunits, and PKC. Reproduced from Okamoto T, Schlegel A, Scherer PE, and Lisanti MP (1998) Caveolins, a family of scaffolding proteins for organizing ‘preassembled signaling complexes’ at the plasma membrane. Journal of Biological Chemistry 273: 5419–5422, copyright & 1998 by the American Society for Biochemistry and Molecular Biology.

(L-NAME) to Cav-1 (
/
)-deficient mice reverses their microvascular hyperpermeability, thus supporting Cav-1-mediated inhibition of eNOS activity. Similarly, Cav-1 (
/
)-deficient mice show hyperactivation of cardiac ERK1/2 and pulmonary STAT3 signaling.

Pulmonary Phenotypes of Caveolin-Deficient Mice Surprisingly, mice with homozygous deletion of the different caveolin genes are viable and fertile. Yet, Cav-1 (
/
)-deficient mice show numerous abnormal phenotypes, such as a reduction in life span, loss of caveolae formation in tissues that usually express Cav-1, loss of Cav-2 expression, resistance to dietinduced obesity, decreased vascular tone secondary to eNOS hyperactivation, and the development of cardiomyopathy. On the other hand, Cav-3 (
/
)deficient mice show a loss of caveolae formation in their cardiac and skeletal muscles and develop progressive cardiomyopathy. Importantly, the generation of caveolin-deficient mice identified caveolin proteins as key regulators of pulmonary structure and function. Indeed, Cav-1 (
/
)-deficient mice demonstrate pulmonary abnormalities, characterized by thickened alveolar septa, hypercellularity, fibrosis, endothelial cell

proliferation, and exercise intolerance. Importantly, selective homozygous deletion of the Cav-2 gene results in similar thickening of the alveolar septa, hypercellularity, and endothelial cell proliferation as in Cav-1 (
/
)-deficient mice, but without any defects in caveolae formation or Cav-1 expression. These findings suggest a selective role for Cav-2 in lung structure and function. Interestingly, Cav-1 (
/
)deficient mice also develop pulmonary hypertension and right ventricular hypertrophy, as shown by increases in the right ventricular systolic pressures and the ratio of the right ventricular weight over the left ventricular weight, respectively. Whether Cav-2 (
/
)-deficient mice also develop pulmonary hypertension and right ventricular hypertrophy still remains to be determined. Similarly, although Cav-3 is highly expressed in the smooth muscle cells of the lungs, the potential pulmonary phenotypes of Cav-3 (
/
)-deficient mice still remain to be assessed.

Pathological Implications of Caveolin Proteins The endogenous expression of caveolin proteins appears to be altered in numerous lung diseases, such as pulmonary hypertension, lung cancers, pulmonary fibrosis, and pulmonary interstitial edema.

332 CAVEOLINS Pulmonary Hypertension

Given the pulmonary phenotype of caveolin-deficient mice and their roles as negative regulators of several pro-proliferative signaling pathways, caveolin proteins could be important regulators of pulmonary hypertension development. Accordingly, both Cav-1 and Cav-2 protein levels appear to be decreased in the lungs of rats subjected to myocardial infarction (MI)-induced pulmonary hypertension. The downmodulation of Cav-1 and Cav-2 expression is associated with the increased tyrosine-phosphorylation of STAT3, as well as the upregulation of Cyclin D1 and D3 in the lungs of MI rats. The decreased expression of caveolin proteins could thus represent an initiating mechanism leading to the activation of several proproliferative pathways, such as the STAT3/cyclinsignaling cascades and consequently leading to the development of pulmonary hypertension. This hypothesis is supported by the findings of increased STAT3 phosphorylation and increased cyclin D1/D3 expression in the lungs of Cav-1 (
/
)- and Cav-2 (
/
)-deficient mice. Furthermore, pulmonary Cav-1a expression was shown to be decreased in rats with monocrotaline-induced pulmonary hypertension as early as 48 h after monocrotaline treatment. Accordingly, monocrotaline treatment of cultured pulmonary arterial endothelial cells also results in decreased Cav-1a expression, increased STAT3, and ERK1/2 phosphorylation, and the stimulation of DNA synthesis within 48 h. Lung Cancers

Caveolin proteins are often perceived as tumor-suppressor genes. Accordingly, the Cav-1 gene is localized to a suspected tumor-suppressor locus on human-chromosome 7 (7q31.1/D7S522). Caveolin1’s well-known negative regulation of several proproliferative proteins, such as ERK1/2, H-Ras, c-Src, and cyclin D1, strongly supports the tumor-suppressor role of Cav-1. Accordingly, a reduction of Cav-1 expression was reported in numerous lung carcinoma cell lines. Caveolin-1 gene transcriptional activity also appears to be downregulated in human lung adenocarcinomas. The generation of caveolin-deficient mice strongly supports the tumor-suppressor roles of Cav-1. Indeed, Cav-1 (
/
)-deficient mice interbred with tumor-prone transgenic mice show accelerated development of multifocal dysplastic mammary lesions, as well as enhanced lung metastasis. Although Cav-1 definitely appears to act as a tumor-suppressor gene in numerous cancers, its precise role in the development of metastasis remains obscure. Indeed, the expression of Cav-1 conversely increases in ipsilateral hilar/peribronchial lymph node

metastases isolated from patients with lung carcinoma. The upregulation of Cav-1 even correlates with the cancer stage and mortality rate in patients with lung adenocarcinoma and pulmonary squamous cell carcinoma. Although the increased expression of Cav-1 might promote the inhibition of apoptosis, the induction of filopodia formation, or even the stimulation of angiogenic processes, the exact mechanisms underlying the prometastatic effects of Cav-1 remain puzzling and may well be cell-type specific. Pulmonary Fibrosis

Given the pulmonary phenotype of caveolin-deficient mice, caveolin proteins could also be implicated in the development of lung fibrosis. Accordingly, the expression of Cav-1 is altered in several models of lung fibrosis, such as irradiation, CdCl2/TGF-b1, and bleomycin. Western blot analysis of total lung homogenates indicates an overall decrease in Cav-1 expression in mice with irradiation- and bleomycininduced fibrosis. This decrease in pulmonary Cav-1 expression is associated with hyperactivation of the Ras-p42/44 MAP kinase pathway (ERK signaling), which is thought to favor collagen deposition. Importantly, lung fibroblasts isolated from scleroderma patients with pulmonary fibrosis also show a marked decrease in Cav-1 expression and hyperactivation of ERK signaling. Interestingly, the expression of Cav-1 appears to be differentially regulated in alveolar type I cells and endothelial cells. Indeed, although the expression of Cav-1 appears to decrease in lung epithelial cells, it conversely might increase in lung endothelial cells. Whereas an upregulation of endothelial Cav-1 expression could indicate an increase in endothelial caveolar transcytosis, the downregulation of epithelial Cav-1 expression might impair the metabolic function and reduce the signal transduction capacity of alveolar type I cells. Thus, the loss of lung epithelial Cav-1 expression appears as an indicator of subcellular alterations in lung fibrogenesis. Pulmonary Interstitial Edema

Caveolin proteins might also be implicated in the development of pulmonary interstitial edema. Indeed, slight increases (B5%) in extravascular water are sufficient to drive the recruitment of endothelial caveolae and the upregulation of Cav-1 protein levels at the air–blood barrier plasma membrane. These effects could be subsequent to increased hydraulic interstitial pressures and shear stress. The recruitment of endothelial caveolae might thus represent an important mechanotransduction response to flow increases.

CD1 333

Conclusions Caveolin proteins represent novel regulators of pulmonary structure and function, which may play essential roles in the development of a number of pulmonary diseases. Indeed, caveolin-deficient mice show numerous abnormalities in pulmonary structure and function. Furthermore, caveolin protein expression appears to be altered in several pulmonary disorders such as pulmonary hypertension, lung cancers, pulmonary fibrosis, and pulmonary interstitial edema. Thus, tight regulation of caveolin protein expression appears fundamental for normal pulmonary functioning. Whether in vivo modulation of the caveolin protein levels could have therapeutic effects on pulmonary diseases remains to be determined. Interestingly, in vivo administration of a cell-permeable Cav-1 mimetic peptide was previously shown to reduce microvascular hyperpermeability, inflammation, and tumor progression in mice. Whether the in vivo administration of such a cell-permeable Cav-1 peptide could complement the decreased expression of endogenous Cav-1 and prevent the development of pulmonary hypertension, pulmonary fibrosis, and some lung cancers remains to be investigated. See also: Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Pulmonary Edema. Signal Transduction. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumors, Malignant: Overview. Vascular Disease.

Further Reading Cohen AW, Hnasko R, Schubert W, and Lisanti MP (2004) Role of caveolae and caveolins in health and disease. Physiological Reviews 84: 1341–1379. Gratton JP, Lin MI, Yu J, et al. (2003) Selective inhibition of tumor microvascular permeability by cavtratin blocks tumor progression in mice. Cancer Cell 4: 31–39. Ho CC, Huang PH, Huang HY, et al. (2002) Up-regulated caveolin-1 accentuates the metastasis capability of lung

adenocarcinoma by inducing filopodia formation. American Journal of Pathology 161: 1647–1656. Jasmin JF, Frank PG, and Lisanti MP (2005) Caveolin proteins in cardiopulmonary disease and lung cancers. Advances in Molecular and Cell Biology 36: 211–233. Jasmin JF, Mercier I, Hnasko R, et al. (2004) Lung remodeling and pulmonary hypertension after myocardial infarction: pathogenic role of reduced caveolin expression. Cardiovascular Research 63: 747–755. Mathew R, Huang J, Shah M, Patel K, Gewitz M, and Sehgal PB (2004) Disruption of endothelial-cell caveolin-1alpha/raft scaffolding during development of monocrotaline-induced pulmonary hypertension. Circulation 110: 1499–1506. Okamoto T, Schlegel A, Scherer PE, and Lisanti MP (1998) Caveolins, a family of scaffolding proteins for organizing ‘‘preassembled signaling complexes’’ at the plasma membrane. Journal of Biological Chemistry 273: 5419–5422. Razani B, Engelman JA, Wang XB, et al. (2001) Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. Journal of Biological Chemistry 276: 38121–38138. Razani B, Wang XB, Engelman JA, et al. (2002) Caveolin-2deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae. Molecular and Cellular Biology 22: 2329–2344. Razani B, Woodman SE, and Lisanti MP (2002) Caveolae: from cell biology to animal physiology. Pharmacological Reviews 54: 431–467. Schubert W, Frank PG, Razani B, et al. (2001) Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. Journal of Biological Chemistry 276: 48619– 48622. Schubert W, Frank PG, Woodman SE, et al. (2002) Microvascular hyperpermeability in caveolin 1 (
/
) knock-out mice. Journal of Biological Chemistry 277: 40091–40098. Tourkina E, Gooz P, Pannu J, et al. (2005) Opposing effects of protein kinase C alpha and protein kinase C epsilon on collagen expression by human lung fibroblasts are mediated via MEK/ ERK and caveolin-1 signaling. Journal of Biological Chemistry 280: 13879–13887. Williams TM, Medina F, Badano I, et al. (2004) Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo. Role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion. Journal of Biological Chemistry 279: 51630–51646. Zhao YY, Liu Y, Stan RV, et al. (2002) Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice. Proceedings of the National Academy of Sciences, USA 99: 11375–11380.

CD1 S M Behar, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract CD1 proteins are a distinct lineage of antigen-presenting molecules that evolved to present lipid and glycolipid antigens to T

cells. The human CD1 locus contains five distinct genes, CD1A– E, which have an overall structure similar to class I major histocompatibility complex (MHC) including their association with b2 microglobulin. However, unlike class I MHC, the CD1 antigen-binding groove is larger, deeper, and lined almost exclusively with hydrophobic amino acids. The ability of CD1 to present lipid antigens to T cells significantly expands the universe of antigens that can be recognized by T cells. CD1 proteins are divided into two groups: group 1 CD1 (CD1a, -b, and -c) and

334 CD1 group 2 CD1 (CD1d). Group 1 CD1-restricted human T cells can recognize mycobacterial cell wall lipids and are elicited as part of the immune response to Mycobacterium tuberculosis. Some CD1d-restricted T cells, also known as natural killer T (NKT) cells, recognize endogenous self-antigens while others recognize foreign microbial antigens. CD1d-restricted NKT cells rapidly secrete cytokines following activation and are thought to be an important link between innate and adaptive immunity. NKT cells appear to enhance pulmonary immunity against several pulmonary pathogens. Conversely, NKT cells are required for the development of airway hyperreactivity and inflammation in rodent models of allergic pulmonary hypersensitivity. Thus, CD1-restricted T cells are an important component of the T cell response to infection and tumors, and have an important role in the regulation of many diverse immune responses.

Introduction CD1 was first recognized as a human cell surface antigen frequently expressed by thymocytes and certain neoplasms of T cell origin. Later, it was found to be highly expressed by Langerhans’ cells and dendritic cells (DCs). Even today, CD1 remains a useful phenotypic marker of these cells. However, the finding that CD1 was a target of certain autoreactive human T cells led to the insight that CD1 may be an antigen-presenting molecule. We now understand that while certain CD1-restricted T cell clones are autoreactive, presumably because they recognize endogenous self-lipid antigens, other CD1-restricted T cells recognize foreign antigens. In a series of landmark studies, Porcelli, Beckman, and Brenner demonstrated that CD1b and CD1c present microbial antigens to human T cells. Eventually, the antigens presented by CD1 began to be identified. The first antigen to be purified was mycolic acid, a unique lipid molecule found in the cell wall of Mycobacterium tuberculosis, which was recognized by a CD1brestricted T cell clone. Mycolic acid, which is responsible for the acid-fastness of M. tuberculosis and is thought to be a bacterial virulence factor, belongs to a family of a-branched b-hydroxy fatty acids and its two acyl chains are approximately 90 carbons long. This discovery shattered the paradigm that T cells could only recognize short peptide sequences and was the first evidence that CD1 presented microbial lipid antigens to T cells. These findings have generated considerable interest in determining whether CD1-restricted T cells contribute to host defense against infection, as the lipid antigens recognized by them may be potential vaccine candidates. Microbial pathogens are unable to rapidly vary lipid antigen structure when subjected to immune selective pressure because lipids are products of multistep biosynthetic pathways. CD1-presented antigens are also attractive vaccine candidates because the limited degree of polymorphisms in the

CD1 locus makes it more likely that such antigens would elicit a consistent immune response, even in genetically diverse human populations.

Structure The human CD1 genetic locus is located on chromosome 1 and is unlinked to the major histocompatibility complex (MHC) locus, which is found on chromosome 6. Five distinct genes are encoded within the human CD1 locus, CD1A, -B, -C, -D, and -E, and in contrast to MHC, there is only limited polymorphism between unrelated individuals. CD1A, -B, and -C have been designated group I CD1 based on their primary sequence homology. Genes encoding CD1 proteins have been detected in all mammals studied to date, and these studies have observed tremendous variation in the number and type of CD1 genes found in different species. Importantly, mice and rats only have orthologs of CD1D (group II CD1). The murine CD1 genes, known as CD1d1 and CD1d2, are more than 95% homologous to one another and may have arisen by gene duplication. The homology between human and mouse CD1d is striking as human CD1d-restricted T cells recognize murine CD1d, and vice versa. Thus, rodents are a useful experimental model to ascertain the function of CD1d and CD1d-restricted T cells. Small animal models that are suitable for the study of group I CD1 also exist and include the rabbit and the guinea pig. Thus, the CD1 genetic locus is evolutionarily conserved in mammals and is distinct from the MHC locus. While CD1 and MHC may share a common ancestor, CD1 has evolved into a distinct lineage of antigen-presenting molecules specialized for the presentation of lipid antigens to T cells. Despite only limited amino acid sequence homology to MHC, the CD1a, -b, -c, -d, and -e polypeptides all associate with b2 microglobulin and form a tertiary structure similar to class I MHC (Figure 1). However, unlike class I MHC, the CD1 antigenbinding groove is larger, deeper, and lined almost exclusively with hydrophobic amino acids. These properties make CD1 particularly well suited to bind lipid and glycolipid molecules. Nearly all of the lipid antigens presented by CD1 have a polar head group and one or two lipid tails. Combining crystallographic and structure-function data, a model emerges in which the hydrophobic acyl chains of the lipid antigen are buried deep within the antigenbinding groove, while the polar head group is closer to the molecular surface and just emerges from the antigen-binding groove. The T cell receptor (TCR) recognizes the combination of the polar head group and the surface of the CD1 protein. The crystal structures of human CD1a and CD1b, and murine

CD1 335

(a)

(b)

(c)

Figure 1 Crystal structure of CD1b. In 2002, Gadola et al. solved the three-dimensional structure of CD1b with phosphatidylinositol in the antigen-binding groove. The CD1b heavy chain is displayed in blue and b2-microglobulin is displayed in yellow. The phosphatidylinositol phospholipid ligand is colored according to the CPK scheme. In (a) and (b), CD1b is observed as it might appear on the cell surface, looking either parallel (a) or perpendicular (b) to the two a-helices formed by the a1 and a2 domains that create the antigen-binding pocket. In (c), the perspective is looking down at the ‘top’ of CD1b, which is the portion of the molecule that interacts with the T cell receptor. The view in (c) is enlarged compared to (a) and (b), and the a3 domain and b2 microglobulin have been removed for clarity.

CD1d, have all been solved and reveal some interesting differences. The antigen-binding groove of CD1a is shallower and less complex than that observed for CD1b or CD1d. CD1b has the most complicated antigen-binding pocket consisting of a maze of channels, giving it the capacity to bind lipid antigens with very long acyl chains. Thus, the antigenbinding groove of the different CD1 molecules may affect the binding and selection of the antigens that are presented to CD1-restricted T cells.

Regulation of Production and Activity Group I CD1 is strongly expressed on cortical thymocytes and on a variety of APC subtypes in several different tissues. For example, Langerhans’ cells in the skin express very high levels of CD1a, and other DCs, particularly in inflamed tissue, express CD1a, -b, and -c. In addition, CD1c and CD1d are found on certain B cell subsets. Murine CD1d is more widely expressed, and is detected on nearly all lymphocytes and myeloid-lineage cells. Although little is known about CD1 regulation, cell surface expression of group I CD1 increases during cytokine-induced differentiation and maturation of monocyte-derived dendritic cells. Similarly, CD1d is upregulated during macrophage activation by cytokines and microbial products. Modulation of CD1 surface expression may be one way that the immune system regulates activation of CD1-restricted T cells. This could be particularly important for the regulation of immune responses by CD1d-restricted natural killer (NK) cells. Following protein translation, CD1 associates with b2m in the endoplasmic reticulum. Processing and loading of lipid antigens requires a unique set of accessory molecules, which are beginning to be

delineated. These include glycosidases to trim sugars from glycolipid antigens and microsomal triglyceride transfer protein and saponins that facilitate lipid transfer between membranes and CD1. Following transportation to the cell surface, CD1 proteins undergo extensive recycling through the endocytic pathway, which is dependent on a tyrosine-containing motif found in the cytoplasmic tail of CD1. For example, CD1a, which has a truncated cytoplasmic tail lacking the tyrosine-based motif, is primarily expressed on the cell surface although it can also be detected in the recycling endosome. In contrast, CD1b is expressed on the cell surface but also has an extensive distribution in the endocytic compartment and colocalizes with class II MHC. Such differences are likely to affect the source and type of lipid each CD1 molecule encounters. For example, CD1b may have evolved to sample lipids from the late endocytic compartment while CD1a may have evolved to sample lipids from the extracellular milieu. Thus, each CD1 protein follows a unique recycling pathway and has a different steady state distribution.

Biological Function The main function of both group I and group II CD1 is antigen presentation of lipids and glycolipids to T cells. Although there are likely to be certain constraints on the type of lipids presented by CD1, a surprisingly diverse number of lipids have been shown to bind or be presented by CD1 to T cells. Group I CD1 presents a number of lipids derived from the cell wall of M. tuberculosis to human T cells including the lipopeptide didehydroxymycobactin (CD1a), mycolic acid and glucose monomycolate (both CD1b), and isoprenoid glycolipids (CD1c).

336 CD1

Both self and foreign antigens have been identified that are presented by CD1d. CD1d-restricted T cells can recognize phospholipids (including phosphatidylinositol and phosphatidylethanolamine) and certain ceramides, although whether these antigens are important in immunoregulation or autoimmune disease is not yet certain. In addition, certain lipids from Leishmania donovani and M. tuberculosis bind to CD1d and activate NKT cells. Finally, synthetic lipid antigens are presented by CD1d and activate CD1drestricted T cells. The prototype of these antigens is a-galactosylceramide. Although their physiological relevance is unknown, these synthetic antigens have been extremely useful tools. Because these antigens are potent and specific activators of CD1d-restricted T cells, they have been used in vitro and in vivo to study the function of CD1d-restricted T cells, particularly in whole animal models, and may ultimately be useful pharmacologically as a way to exploit the therapeutic potential of these T cells. As more CD1presented antigens are discovered, their apparent diversity substantiates the hypothesis that antigen presentation by CD1 to T cells significantly expands the number of microbial antigens that can be recognized by T cells and enhances their capacity to mediate antimicrobial immunity.

CD1-Restricted T Cells Similar to the MHC/peptide complex, recognition of the CD1/lipid antigen complex is mediated by the T cell receptors (TCRs) (Figure 2). Both the ab and the gd TCRs can recognize the CD1/antigen complex, and although the initial descriptions of CD1-restricted T cells were distinguished by their lack of CD4 or CD8 accessory molecules, examples of CD4 þ and CD8 þ CD1-restricted T cells have been reported. Functional differences among these various CD1-restricted T cell subsets exist. For example, human CD4 þ and CD4
8
CD1d-restricted T cells differ in the spectrum of cytokines produced following activation. Another difference is that while CD1restricted M. tuberculosis antigen-specific T cells can kill infected cells, CD8 þ T cells accomplish this using cytotoxic granules, whereas killing by CD4
8
T cell-mediated killing is CD95/CD95L dependent (Figure 2). Like other T cells, CD1-restricted T cells require costimulation for their proliferation. However, they are typically CD28 negative and their costimulatory requirement appears to be independent of the CD28-CD80/CD86 axis. The V, D, and J gene segments encoding group 1 CD1-restricted TCRs are similar to those used by MHC-restricted T cells. Interestingly, many CD1-restricted TCRs have an increased frequency of basic residues in the

CDR3 region. These positively charged residues are hypothesized to interact with the polar head group of lipid antigens, which usually carry a negative charge. An important advance in the biology of CD1d-restricted T cells was the finding that many NKT cells are CD1d-restricted. NKT cells are a unique subset of lymphocytes found in humans and other species that not only express the T cell antigen receptor (i.e., TCR), but also cell surface antigens that are characteristic of NK cells. NKT cells are not abundant in lymphoid organs but are increased in frequency in the bone marrow and certain peripheral organs such as the liver. Two distinct subsets of CD1d-restricted NKT cells have been described. The best-described subset is referred to as invariant NKT cells (iNKT cells), because they express an invariant TCR alpha (a) chain. Murine iNKT cells use a canonical Va14Ja281 TCRa chain, which preferentially pairs with TCR beta (b) chains encoded by Vb2, -7, or -9. Human iNKT cells are similar and are encoded by an invariant Va24-JaQ TCRa chain. Thus, the TCR repertoire of iNKT cells has only limited diversity. A second subset of CD1d-restricted T cells uses a more diverse repertoire, although it may be less varied than MHC-restricted T cells. Although NKT cells have become synonymous with CD1d-restricted T cells, caution is required as conventional T cells can also express NK cell markers, particularly when activated. Thus, more precise methods are required to identify CD1-restricted T cells. The specificity of most iNKT cells for the synthetic antigen a-galactosylceramide provides one way to identify these T cells. Thus, flow cytometry has been used to detect binding of CD1d multimers loaded with a-galactosylceramide to iNKT cells. When identified this way, the phenotype of iNKT cells resembles memory T cells. Even their pattern of chemokine receptor expression is typical of T cells that home to peripheral tissues rather than lymphoid organs. Consistent with their activated phenotype, CD1d-restricted iNKT cells rapidly produce a burst of IL-4 within hours of stimulation, followed secretion of large amounts of interferon gamma (IFN-g). Not surprisingly, iNKT cells can influence whether CD4 þ T cell responses acquire a Th1- or Th2-like phenotype. Thus, iNKT cells are increasingly viewed as a link between innate and adaptive immunity, because of the kinetics of their response, as well as their ability to influence conventional T cell immunity (Figure 2).

CD1 in Respiratory Diseases How CD1 and CD1-restricted T cells affect the expression of respiratory diseases is still being defined. As antigen-presenting molecules, CD1 and

CD1 337 Perforin Granzyme Granulysin

IL - 12 T cell Activation of adaptive immunity

Fas - FasL T cell

CD1b TCR

IFN -
TNF -

(a)

T cell

APC TCR CD1b

IFN -
TNF - 

NK cell

Activation of innate immunity

IFN -
TNF - 

Effector functions

M

Immunoregulation

Perforin Granzyme Granulysin

IL - 12 T cell

Fas - FasL NKT cell

NKT cell

APC TCR CD1d

IFN -
TNF -

(b)

Activation of adaptive immunity

CD40–CD40L

IFN -
TNF- 

CD1d TCR

M

Activation of innate immunity

IFN -


Effector functions

NK cell

Immunoregulation

Figure 2 The effector and immunoregulatory functions of CD1-restricted T cells. (a) Presentation of microbial or self-lipid antigens by CD1a, -b, or -c expressed on DCs leads to activation of group I CD1-restricted T cells. When stimulated, these T cells secrete IFN-g and TNF-a, which can lead to activation of macrophages (Mf). CD1-restricted T cells are also potent cytolytic T cells and kill target cells either by granule-dependent mechanisms or by Fas-mediated killing. CD1-restricted T cells also stimulate DC maturation and IL-12 production. Thus, by inducing DC maturation and secreting cytokines, group 1 CD1-restricted T cells can modulate other innate and adaptive immune responses. (b) CD1d presentation of exogenous antigens such as aGalCer or endogenous ligands leads to the activation of CD1d-restricted NKT cells. IL-12 is an important costimulatory signal for NKT cell activation, which is produced by DCs after CD40–CD40L interaction or after TLR activation by microbial ligands. Activated NKT cells secrete IFN-g, TNF-a, and IL-4, which can stimulate other cell types and influence the polarization of CD4 þ T cells. In addition, NKT cells are potent cytolytic cells, and when activated can kill target cells by the mechanisms shown. Adapted from Sko¨ld M and Behar SM (2005) The role of group 1 and group 2 CD1-restricted T cells in microbial immunity. Microbes and Infection 7: 544–551, with permission from Elsevier.

CD1-restricted T cells potentially play a role in immunity to pathogens, tumors, and a variety of immunologically mediated diseases. Lower numbers of circulating CD1d-restricted iNKT cells are found in patients with several types of cancers, including multiple melanomas and prostate cancer, and in patients with autoimmune conditions such as systemic sclerosis and rheumatoid arthritis. Patients with sarcoidosis and autoimmune diabetes not only have fewer iNKT cells, but those that are present are dysfunctional. These findings support a role for NKT cells in natural tumor immunosurveillance and in

preventing the emergence of autoimmune disease, but do not delineate how NKT cells exert their protective role. The ability of CD1 to present microbial antigens to T cells has strongly implicated CD1restricted T cells in microbial immunity. Not only can CD1-restricted T cells recognize mycobacterial lipids, but these lipid antigens are processed and presented by M. tuberculosis infected cells, demonstrating that the CD1 antigen-presentation pathway is physiologically relevant during infection. Furthermore, recognition of infected cells leads to activation of CD1-restricted T cells, which induces IFN-g

338 CD11/18

and tumor necrosis factor alpha secretion and target cell lysis. Thus, CD1-restricted T cells express effector functions that are beneficial during infection. Finally, to address the relevance of these observations during human tuberculosis, it should be noted that CD1-restricted responses to mycobacterial lipid antigens are detected in the peripheral blood of people infected with M. tuberculosis, indicating that CD1restricted T cell responses are physiologically primed following infection. Mouse models for human diseases are powerful tools to identify the mechanisms by which the immune system combats disease. As CD1d orthologs exist in mice, more information has been obtained on the role of CD1d and CD1d-restricted NKT cells in murine models of human disease. For example, it has been shown that iNKT cells contribute to natural tumor immunosurveillance. Although NKT cells prevent the induction of certain autoimmune diseases, they are an important source of Th2 cytokines during the induction of allergen-induced airway hyperreactivity. Lastly, CD1d-restricted NKT cells are important for host protection against pathogenic viruses, bacteria, fungi, and parasites. Mice lacking CD1d are more susceptible to certain pulmonary infections including Pseudomonas aeruginosa, Pneumococcus pneumoniae, and Cryptococcus neoformans. In other models, specific activation of CD1d-restricted NKT cells can enhance immunity and prolong survival of mice infected with virulent M. tuberculosis. NKT cells are thought to make an early contribution to host immunity, which is not surprising given their rapid activation and production of various cytokines. Less is known about the in vivo role of group 1 CD1-restricted T cells but more information is becoming available with the development of alternative small animal models such as the guinea pig and the

potential to make transgenic mice expressing human group I CD1 genes. See also: Interferons. Leukocytes: T cells; Pulmonary Macrophages. Systemic Disease: Sarcoidosis.

Further Reading Bollyky PL and Wilson SB (2004) CD1d-restricted T-cell subsets and dendritic cell function in autoimmunity. Immunology and Cell Biology 82: 307–314. Brigl M and Brenner MB (2004) CD1: antigen presentation and T cell function. Annual Review of Immunology 22: 817–890. Dascher CC and Brenner MB (2003) Evolutionary constraints on CD1 structure: insights from comparative genomic analysis. Trends in Immunology 24: 412–418. Gadola SD, Zaccai NR, Harlos K, et al. (2002) Structure of human CD1b with bound ligands at 2.3 A, a maze for alkyl chains. Nature Immunology 3: 721–726. Godfrey DI and Kronenberg M (2004) Going both ways: immune regulation via CD1d-dependent NKT cells. Journal of Clinical Investigation 114: 1379–1388. Kronenberg M and Gapin L (2002) The unconventional lifestyle of NKT cells. Nature Reviews: Immunology 2: 557–568. Moody DB and Porcelli SA (2003) Intracellular pathways of CD1 antigen presentation. Nature Reviews Immunology 3: 11–22. Porcelli SA (1995) The CD1 family: a third lineage of antigen presenting molecules. Advances in Immunology 59: 1–98. Shinkai K and Locksley RM (2000) CD1, tuberculosis, and the evolution of major histocompatibility complex molecules. Journal of Experimental Medicine 191: 907–914. Sko¨ld M and Behar SM (2003) Role of CD1d-restricted NKT cells in microbial immunity. Infection and Immunity 71: 5447–5455. Sko¨ld M and Behar SM (2005) The role of group 1 and group 2 CD1-restricted T cells in microbial immunity. Microbes and Infection 7: 544–551. van der Vliet HJ, Molling JW, von Blomberg BM, et al. (2004) The immunoregulatory role of CD1d-restricted natural killer T cells in disease. Clinical Immunology 112: 8–23. Watts C (2004) The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules. Nature Immunology 5: 685–692.

CD11/18 D S Wilkes and T J Webb, Indiana University School of Medicine, Indianapolis, IN, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Integrins are cell membrane receptors that assemble as heterodimers by the noncovalent association of the a and b subunits. The b2 family of leukocyte integrins comprises four a subunits (CD11a–d) with a common b2 subunit (CD18). These adhesion molecules have been shown to play an important role in host defense. The critical role of the CD11/CD18 receptors in

leukocyte adherence has been shown using mAb blocking experiments, knockout animal models, and studies of individuals with a deficiency in these glycoproteins. The use of mAbs against these adhesion receptors results in defective adherence, which suggests that this approach may have therapeutic potential in treating certain pulmonary diseases.

Introduction Lymphocyte migration from the bloodstream into secondary lymphoid organs is essential for maintaining homeostasis and for providing efficient defense

CD11/18 339

globular head of an integrin. Inserted into the b propeller fold is the ‘inserted’ or I domain. The I domain is present in 9 out of 18 a subunits and is homologous to the A domain of the von Willebrand factor protein. Consequently, this region is frequently referred to as the I/A domain. A key feature of the I/A domain is the metal ion-dependent adhesion site or the MIDAS motif, which is composed of D-X-S-X-S. It is the MIDAS motif plus surrounding residues that form the actual ligand binding site. The b2 subunit (CD18) also contains a MIDAS motif, but it has not been directly shown to bind metal ions. The short and highly conserved cytoplasmic region of CD18 contains tyrosine and several serine and threonine residues, which account for stimulus-induced phosphorylation (Figure 2). An interesting feature of the extracellular region of CD18 is a highly conserved cysteine-rich region (CRR) consisting of four tandem repeats of an eight-cysteine motif. Notably, a point mutation in this region prevents cell surface expression of CD18. The a subunits of CD11/CD18 are highly homologous but distinct from the common b subunit. Mac-1 and p150, 95 are more related to each other (63% identity at the amino acid level) than to LFA-1 (36% homology), and this is reflected by their close functional similarity. Polymorphisms in LFA-1 and Mac-1 have been reported. Accordingly, monoclonal antibodies (mAbs) have been shown to recognize specific activation or conformation states of CD11/CD18 receptors.

against pathogens. This migration occurs mostly through high endothelial venules and is partially regulated by signaling cascades involving adhesion and activation. Integrins mediate the firm adhesion of leukocytes to the endothelium that allows for the transmigration of cells into tissue. The CD11/CD18 leukocyte adhesion molecules consist of four cell surface membrane glycoproteins, namely LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150, 95 (CD11c/CD18), and adb2 (CD11d/CD18). The a subunits form a noncovalent association with a common b subunit (CD18) of 94 kDa to form a1b2 heterodimers. The divalent cations Ca2 þ and Mg2 þ are essential for the stabilization and function of the a1b2 complex. Signaling events through the b2 integrins lead to full activation status of the molecules. The active form of integrin is usually characterized by lateral clustering of the proteins on the cell membrane and by a change in structural conformation.

Cellular Location and Structure The four distinct genes encoding for the a subunits occur in a cluster on chromosome 16, band p11– p13.1. This region also contains the gene encoding sialophorin (CD43), a leukocyte adhesion receptor, and the b isoform protein kinase C (PKC), suggesting the presence of a gene cluster involved in leukocyte adhesion and activation. The gene encoding CD18 is on chromosome 21, band q22. The major structural features of CD11/CD18 are illustrated in Figure 1. CD11a–d and CD18 are transmembrane proteins spanning the plasma membrane once, with a short C-terminal cytoplasmic region and a large N-terminal extracellular domain. In the a subunit there are seven repeating homologous sequences at the N terminus. This part of the a subunit has been modeled as a b propeller fold and is a major contributor to the

Biosynthesis The a and b subunits of CD11/CD18 are synthesized in leukocytes from independent precursors. The two subunits associate as precursors prior to further carbohydrate processing in the Golgi. Association of ab

Signal sequence Cysteine rich repeats

I/A domain MIDAS motif D-X-S-X-S


2 1 22



1

2

MIDAS motif D-X-S-X-S

3

4

110

1 361

M

M

M

5

6

7

449

2

3

4 628 700 723 769 TM C

Figure 1 Schematic diagram of the primary structure of CD11/CD18. The main structural features of the a (CD11) and b (CD18) subunits are illustrated. In the b subunit, the red regions 1–4 outline the four cysteine-rich repeats. The yellow region represents the transmembrane (TM) site, and C represents the cytoplasmic end. In the a subunit, the seven tandem repeats in the extracellular portion are shown in teal, and repeats 5–7 contain a metal binding site (M).

340 CD11/18

CD18
2-integrins

Mac-1

LPS

CD11b

Tyrosine kinases Cytoskeleton rearrangement & cell motility

Ca2+ influx

Monocyte Lymphocyte CD11a LFA-1

CD18

ICAM-1

Endothelial cells Figure 2 Overview of b2 integrin signaling. b2 integrin engagement induces phosphorylation of tyrosine kinases. This can result in cytoskeletal rearrangement, an event that controls cell motility. Also, Ca2 þ signals are generated as described in detail in the legend to Figure 3.

Table 1 b2 adhesion molecules and their ligands Name

Structure

Ligand

Cell distribution

CD11a/CD18 (LFA-1) CD11b/CD18 (Mac-1)

aLb2 aMb2

ICAM-1 (CD54), ICAM-2 (CD106) iC3b, factor w, fibrinogen, ICAM-1?, LPS, Leishmania gp63

CD11c/CD18 (p150,95)

axb2

iC3b

CD11d/CD18

adb2

All leukocytes Monocytes, macrophages, some lymphocytes, natural killer cells, neutrophils, granulocytes Monocytes, granulocytes, macrophages, natural killer cells, certain lymphocytic tumor cell lines Monocytes, macrophage foam cells, splenic red pulp macrophages, lymphocytes

LFA-1, lymphocyte function-associated antigen; Mac-1, macrophage-1 antigen; ICAM, intercellular adhesion molecules; iC3b, proteolytic fragment of complement protein C3; factor w, coagulation factor w.

precursors appears to be important for further carbohydrate processing and cell surface expression of the heterodimers. Following association, the CD11 and CD18 subunit precursors undergo an increase in their molecular mass that is accompanied by a conversion on N-linked carbohydrates to their complex form. As expected, more CD18 precursors are synthesized in lymphocytes and monocytes than each of the three a subunits. Interestingly, the type of a subunit affects the glycosylation pattern of CD18.

Expression and Biological Function The CD11/CD18 glycoproteins are differentially expressed (Table 1). The level of CD11/CD18

expression is dependent on the cell type and the state of cell activation and differentiation. The CD11/ CD18 glycoproteins are only expressed in leukocytes. Whereas CD11a/CD18 is present on all leukocytes, CD11b, c/CD18 is normally restricted to expression on monocytes, macrophages, polymorphonuclear lymphocytes (PMNs), and natural killer (NK) cells. However, p150, 95 and the recently described adb2 are mainly expressed by tissue macrophages. In B and T cells, all CD11/CD18-dependent functions – such as mitogen, antigen, and alloantigeninduced proliferation, T cell-mediated cytotoxicity, B cell aggregation, and Ig production – are inhibited by anti-LFA-1 mAbs. This is consistent with LFA-1 being the only heterodimer normally expressed on these

CD11/18 341

Whereas MAC-1 is a phagocytic receptor, used by neutrophils to engulf bacteria, yeast, and other microorganisms, the roles of p150, 95 and adb2 have not been well documented. All CD11/CD18 receptors contribute to chemotaxis and adhesion to cytokine-activated endothelium by neutrophils and monocytes and to antibody-dependent cellular cytotoxicity (ADCC).

cells. Granulocytes, monocytes, and NK cells express all three CD11/CD18 heterodimers, which accounts for the wider range of CD11/CD18-dependent functions mediated by these cells. LFA-1 seems to mediate an early Mg2 þ -dependent adhesion step in cell–cell interaction. Studies have shown that LFA-1 is primarily responsible for adhesion and signaling at the immunological synapse, involved in the clustering of proliferating lymphocytes, and found between CTLs and their targets. Anti-LFA-1 mAbs inhibit T-cell proliferation in response to stimuli that require cell–cell contact. Notably, APC-independent T-cell responses do not require LFA-1. Also, at high effector:target ratios or during secondary stimulation, the role of LFA-1 is significantly diminished, perhaps as a result of increased expression of several other adhesion molecules. Mac-1 is essential for binding iC3b and other ligands, spreading, homotypic adhesion, and phagocytosis of opsonized particles in neutrophils, a role that may also be fulfilled by p150, 95 in resident macrophages. Following activation, Mac-1 can initiate signaling via its linkage to the actin cytoskeleton and associated signaling proteins. Also, Mac-1 is thought to act as a signaling partner for other leukocyte receptors, including lipopolysaccharide (LPS)/LPS binding protein receptors (CD14), formyl-methionylleucyl-phenylalanine (FMLP) receptors, urokinase plasminogen activator receptors (CD87), and FcRs.

Ligands Several ligands have been shown to interact with CD11/CD18 molecules in a divalent cation-dependent manner, as shown in Table 1. The first wellcharacterized ligand was iC3b, a component of the complement system. iC3b binds to Mac-1 and p150, 95 at 37oC in resting cells, but it is greatly enhanced in cells activated by phorbol esters. LFA-1 binds to the two N-terminal domains of intracellular adhesion molecules (ICAM)-1 and -2. ICAM-1 is inducible and expressed on leukocytes and endothelial and epithelial cells in response to inflammatory cytokines (Figure 3). In contrast to ICAM-1, ICAM-2 is constitutively expressed on endothelial cells and a variety of B and T lymphoblastoid cell lines, and its expression is not enhanced by inflammatory stimuli. However, unlike ICAM-2, ICAM-1 was uniquely found to be involved in migration of lymphocytes into inflamed tissue and in trapping lymphocytes in the lung. Concurring with

Endothelial cells

ICAM-1 CD18

CD11a

Lymphocyte

Tyrosine kinases

PLC-  1/2

PLC- 1/2

PIP2 DAG PKC

Ins(1,4,5)P3 Ca2+ release

Figure 3 b2 integrins can induce calcium signaling in leukocytes. Ligand binding of b2 integrins causes activation of tyrosine kinases. The tyrosine kinases are phosphorylated, which increases the catalytic activity of PLC-g. Then, PLC-g catalyzes the hydrolysis of phosphatidylinositol-4,5-biphosphate (PIP2) into both diacylglycerol (DAG) and inositol triphosphate (Ins(1,4,5)P3). Next, Ins(1,4,5)P3 stimulates the release of Ca2 þ from internal stores, which is followed by an influx of Ca2 þ .

342 CD11/18

those data, studies using a murine model of asthma found that ICAM-1 is important in the migration of lymphocytes to the lungs during an allergic inflammatory response. Also, in another study examining the homing mechanisms of cytotoxic T cells, LFA-1 was shown to be critical for the retention of effector CD8 T cells in the lungs. Moreover, in an adoptive transfer model involving alloreactive CD4 T cells, adherence of T cells in the lungs was shown to be partially dependent on LFA-1 and its receptor ICAM-1. Conflicting data exist regarding whether Mac-1 also binds to ICAM-1; however, binding of two other ligands, coagulation factor w and fibrinogen, to an activated form of Mac-1 has been shown. All three heterodimers (CD11a–c/CD18) bind to LPS, as determined by blocking studies using mAbs, suggesting that binding is mediated by homologous regions in the CD11 subunits or by the common CD18 subunit.

CD11/CD18 in Respiratory Disease Leukocyte adhesion deficiency (LAD-1) is a disorder caused by the lack of surface expression of CD11/ CD18, which is due to heterogeneous defects in the CD18 subunit. The classical disease is characterized by recurring necrotic soft tissue infections, impaired wound healing with a lack of pus, and severe gingivitis. Also, phagocytes from patients with this disorder have functional defects in aggregation, chemotaxis, phagocytosis, endothelial cells, and complement iC3b binding and ADCC. Two phenotypes of LAD-1 have been reported based on the level of CD18 expressed. In the severe form, CD11/CD18 is not detectable, and death from bacterial infections usually occurs during the first few years of life. However, in the moderate phenotype cells express 2–5% of the normal level of CD18 and the clinical course is much milder. In CD18deficient mice, as with LAD-1 patients, marked neutrophilia was found. However, in contrast to LAD-1 or CD18-deficient mice, CD11b-deficient mice do not exhibit any leukocytosis or display a significant increase in bacterial infections. Nevertheless, in vivo studies have shown that these mice have defective firm adhesion, and in vitro studies have shown that neutrophils from these mice have defects in adhesion, iC3b-mediated phagocytosis, phagocytosis-induced respiratory burst, and homotypic aggregation. Remarkably, although CD11a-deficient mice displayed normal CTL responses to a systemic virus infection, they failed to develop a DTH response to 2,4-dinitrofluorobenzene sensitization and challenge. Because ICAM-1 is a major ligand for LFA-1 and perhaps Mac-1, ICAM-1-deficient mice are

particularly interesting. Two groups have reported ICAM-1 knockout mice. Although both ICAM-1deficient murine lines develop normally and have moderate leukocytosis, the mice displayed multiple abnormalities in their inflammatory responses, specifically impaired neutrophil emigration in response to chemical peritonitis, resistance to septic shock, and decreased contact hypersensitivity reaction. Intriguingly, it has been shown that during allergic lung inflammation, there is a delayed increase in eosinophils in the airway lumen and a prolonged presence of eosinophil infiltrates in lung tissue in ICAM-2deficient mice. In contrast, the importance of ICAM2 in binding T cells has been demonstrated in frozen section assays of chronically inflamed human airway epithelium; however, inflammation of the lungs in ICAM-2-deficient mice was due to an accumulation of eosinophils, not lymphocytes. Overall, it has been suggested that ICAM-2 expressed in vascular endothelium, alveolar walls, or large airway epithelium participates in the traffic of eosinophils from the bloodstream to the airway lumen.

Conclusion Lymphocyte homing is tightly regulated; however, the molecular basis of lymphocyte migration to the lungs is not well understood. Because the lung is the portal of entry for airborne pathogens, lymphocytes must constantly recirculate. Circulating lymphocytes become transiently trapped within the lung. This has been shown for normal lymphocytes and even more so for activated/memory lymphocytes. Notably, retention is mediated, at least in part, by LFA-1. Interestingly, studies have shown that effector and memory cytotoxic T lymphocytes traverse to the lungs and reside in the lung parenchyma for extended periods. In addition, it has been suggested that normal lung microvessels can constitutively support the retention of activated CD8 T cells. Moreover, murine studies investigating the role of LFA-1 in an acute RSV infection showed that treatment of mice with neutralizing Abs to LFA-1 resulted in diminished illness and delayed viral clearance. These data further support the hypothesis that using blocking mAbs against b2 integrins could be an immunotherapeutic strategy. Many diseases that affect the lungs involve inflammation. Following sequestration and initial adhesion, neutrophils migrate along the capillary endothelium and into the interstitium and alveoli. This emigration can occur through either a CD11/CD18-dependent or -independent pathway. The adhesion pathway used depends on the stimulus. Stimuli that have been shown to induce CD18-dependent emigration include Escherichia coli, E. coli lipopolysaccharide,

CD14 343

Pseudomonas aeruginosa, IgG immune complexes, interleukin-1, and phorbol myristate acetate. These stimuli appear to act by inducing the translocation of NF-kB, resulting in the production of inflammatory cytokines and ICAM-1 on the pulmonary capillary endothelial cells. Stimuli that have been shown to induce the CD11/CD18-independent neutrophil emigration include Streptococcus pneumoniae, group B Streptococcus, Staphylococcus aureus, hydrochloric acid, hyperoxia, and C5a. Previous studies have shown that the absence of CD11/CD18 in granulocytes and monocytes attenuates the acute cellular inflammatory responses in vivo; therefore, these data suggest that blocking CD11/CD18 may ablate the tissue damage induced by these cells. Consequently, this was shown to be the case in several ischemia–reperfusion injury animal models. In fact, giving anti-Mac-1 mAbs before or soon after the induction of ischemia–reperfusion injury significantly reduced tissue injury, microvascular permeability, and acidosis. However, the lack of protection of the lung tissue in some of these models may be an indication of the importance of other adhesion pathways mediating PMN migration. Lastly, detailed investigations of the LAD-1 syndrome in humans and studies involving adhesion molecule-deficient mice have provided novel insights into the cell and molecular biology of leukocyte emigration. However, further studies must be done to address the discrepancies observed between deficient animals and wild-type animals treated with blocking mAbs.

Acknowledgments This work was supported by National Institutes of Health grants HL60797 and HL/AI67177 to D S Wilkes. See also: Adhesion, Cell–Matrix: Integrins. Coagulation Cascade: Fibrinogen and Fibrin. Complement. Dendritic Cells. Leukocytes: Eosinophils; Neutrophils; Monocytes; Pulmonary Macrophages.

Further Reading Arnaout MA (1990) Structure and function of the leukocyte adhesion molecules CD11/CD18. Blood 75: 1037–1050. Buyon JP, Slade SG, Reibman J, et al. (1990) Constitutive and induced phosphorylation of the a- and b-chains of the CD11/CD18 leukocyte integrin family: relationship to adhesion-dependent functions. Journal of Immunology 144: 191–197. Dayer JM, Isler P, and Nicod LP (1993) Adhesion molecules and cytokine production. American Review of Respiratory Disease 148: S70–S74. Doerschuk CM (2000) leukocyte trafficking in alveoli and airway passages. Respiratory Research 1: 136–140. Doerschuk CM, Mizgerd JP, Kubo K, et al. (1999) Adhesion molecules and cellular biomechanical changes in acute lung injury. Chest 116: 37S–43S. Etzioni A, Doerschuk CM, and Harlan JM (1999) Of man and mouse: leukocyte and endothelial adhesion molecule deficiencies. Blood 94: 3281–3288. Graham IL, Gresham HD, and Brown EJ (1989) An immobile subset of plasma membrane CD11b/CD18 (Mac-1) is involved in phagocytosis of targets recognized by multiple receptors. Journal of Immunology 142: 2352–2358. Hogg N and Bates PA (2000) Genetic analysis of integrin function in man: LAD-1 and other syndromes. Matrix Biology 19: 211– 222. Lehmann JCU, Jablonski-Westrich D, Haubold U, et al. (2003) Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking. Journal of Immunology 171: 2588–2593. Pilewski JM and Albelda SM (1993) Adhesion molecules in the lung: an overview. American Review of Respiratory Disease 148: 531–537. Thatte J, Dabak V, Williams MD, et al. (2003) LFA-1 is required for retention of effector CD8 T cells in mouse lungs. Blood 101: 4916–4922. van Spriel AB, Keusen JHW, van Egmond M, et al. (2001) Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation. Blood 97: 2478–2486. van Spriel AB, van Ojik HH, Bakker A, et al. (2003) Mac-1 (CD11b/CD18) is crucial for effective Fc-receptor mediated immunity to melanoma. Blood 101: 253–258. Xu B, Wagner N, Pham LN, et al. (2003) Lymphocyte homing to bronchus-associated lymphoid tissue (BALT) is mediated by L-selectin/PNAd, a4b1 integrin/VCAM, and LFA-1 adhesion pathways. Journal of Experimental Medicine 197: 1255– 1267.

CD14 Y Tesfaigzi and M Daheshia, Lovelace Respiratory Research Institute, Albuquerque, NM, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract CD14 is a glycolipid-anchored membrane glycoprotein expressed on cells of the myelomonocyte lineage including

monocytes, macrophages, and some granulocytes. CD14 is a key molecule in the activation of innate immune cells and exists as a membrane-anchored or soluble form. It is encoded by a gene on human chromosome 5q31.1, a region where several genes implicated in asthma pathogenesis are localized. CD14 is expressed in a variety of hematopoietic and parenchymal cells and it has a range of biological activity including cell differentiation, immune response, and host-pathogen interactions. While CD14 is an essential part of the lipopolysaccharide

344 CD14 (LPS) receptor complex, it requires interaction with long-terminal repeat 4 to successfully transmit LPS-induced signals to the cell. In addition, CD14 is an integral part of the mechanism by which macrophages interact and engulf apoptotic cells. A functional single nucleotide polymorphism within the promoter region of CD14 that affects expression levels is associated with a higher risk of developing atopy. The interactions of CD14 with other receptors are important for the normal signaling of LPS and host-pathogen interactions and affect the susceptibility or resistance to a variety of diseases including atopy and septic shock.

Introduction CD14 was originally discovered through reactivity of monoclonal antibodies on human peripheral blood monocytes and was cloned in 1988. Subsequently, the protein was purified, and our understanding of its role in inflammatory responses has been increasing steadily. While this protein is best known for its ability to act as a receptor for bacterial endotoxin, lipopolysaccharides (LPS), and to elicit proinflammatory host responses, other functions for CD14 such as those related to the recognition of apoptotic cells and clearance by phagocytosis have also gained importance. This latter role involves a process that suppresses inflammation and appears to contradict its proinflammatory function, making this protein highly interesting to study.

Structure The CD14 protein is encoded by a gene localized on the human chromosome 5q31.1 or on the syntenic mouse chromosome 18. In humans and mice, CD14 has an amino acid length of 375 and 366, respectively, that is transcribed by a 1367-nucleotide mRNA. CD14 exists in two different forms: a membrane-anchored and a soluble (sCD14) form. The membranebound CD14 is a glycoprotein with a molecular weight of 53–55 kDa, attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The sCD14 is generated when the membrane-bound CD14 is shed and released into the bloodstream.

levels of sCD14 have been associated with inflammatory/infectious diseases such as dermatitis, septic shock, malaria infection, human immunodeficiency virus (HIV), and severe burns.

Biological Functions CD14 and LPS Binding

While LPS is the main ligand for CD14, several other molecules such as peptidoglycans, phospholipids, and dextrans, can interact with CD14. Several lines of evidence suggest a crucial role for CD14 in the biological effects of LPS: (1) The neutralizing antibody to CD14 inhibits the interaction of LPS with the receptor and protects primates from endotoxininduced shock; (2) CD14-deficient mice are 10–100 times less sensitive to LPS than their wild-type counterparts because they produce very low amounts of proinflammatory cytokines and are resistant to a lethal dose of Gram-negative bacteria; (3) overexpression of CD14 renders mice more susceptible to LPS-induced septic shock or to Gram-negative bacteria-induced death. The susceptibility of CD14-deficient mice is specific to Gram-negative bacteria because responses to infection by Gram-positive bacteria such as Staphylococcus aureus are not affected by the absence of CD14. The interaction between LPS and CD14 is facilitated by the soluble LPS-binding protein (LBP), which increases the affinity of LPS to CD14 by more than 100- to 1000-fold. Therefore, LBP-deficient mice are more resistant to LPS than wild-type mice and are defective in their ability to mount appropriate inflammatory responses to bacterial infection. For example, LBP-deficient mice are incapable of controlling infection by Gram-negative bacteria such as Salmonella and succumb to a dose of pathogen, which is well tolerated by their heterozygous or wild-type littermates. It has also been reported that LBP may be incorporated as a transmembrane protein in the cytoplasmic membrane of mononuclear cells to mediate activation of these cells by LPS, revealing that LBP has a different mode of action in LPS signaling.

Regulation of Expression

CD14 as a Component of LPS Receptor Complex

The regulatory sequence of CD14 contains multiple consensus-binding sites for CAAT/enhancing and binding protein (C/EBP) and Sp transcription factors. The gene expression is induced by transforming growth factor beta (TGF-b) and vitamin D and is downregulated by interleukin (IL)-4. Membranebound CD14 is expressed by a variety of cells, including monocytes/macrophages, neutrophils, granulocytes, and hepatocytes. Increased serum

Although CD14 is the major membrane receptor for binding LPS, it is incapable of inducing intracellular signaling because it lacks an intracellular domain. CD14 must form a complex with Toll-like receptor 4 (TLR4), which is bound to myeloid differentiation protein-2 (MD-2) to transmit intracellular signaling in response to LPS binding (Figure 1). The mature human MD-2 and TLR4 proteins consist of 160 and 839 amino acid residues, respectively. After LPS

CD14 345

LPS LPS LBP

MD-2

LBP

TLR4

LPS

CD14

TIR

of NF-kB, but it does so via an alternative pathway that does not require MyD88 and TRAF6. Additionally, TRIF/TICAM can activate the interferon related factor 3 (IRF3) pathway and increase interferon beta (IFN-b) to turn on the JAK/STAT pathway. LPS signaling is decreased in the absence of CD14 or TLR4, revealing the primordial importance of these molecules for LPS effects. Although MyD88 ( / ) mice showed a significant reduction in tumor necrosis factor alpha (TNF-a) production, these mice still exhibit NF-kB and MAPK activation and IFN-b release in response to LPS. Additionally, TRIF/TICAM-1 ( / ) mice exhibit suppressed expression of IFN-b as well as lower IFN-g production; however, the MyD88-dependent activation of IRAK-1, NF-kB, and MAPK are not affected. Complete loss of LPS-induced responses are only observed in mice deficient for both MyD88 and IRIF/TICAM-1 genes, suggesting that LPS acts entirely through these two signaling pathways. CD14 and Phagocytosis

Figure 1 Interaction of CD14 with TLR-4 for signaling through the intracellular TIR domain. LPS, lipopolysaccharide; LBP, LPSbinding protein; TLR4, Toll-like receptor 4; MD-2, myeloid differentiation protein-2.

interacts with the LBP, the heterodimer binds to CD14, which subsequently activates TLR4/MD-2 and initiates signaling through the Toll/IL-1R (TIR) intracellular domain. However, airway epithelial, endothelial, and dendritic cells respond to LPS even though they lack the membrane-bound CD14 because the soluble CD14 is believed to form the LPS complex and activate TLR4. Activation of LPS receptor complex (LPSRC) causes many cellular changes, including increased transcription of multiple genes, release of a variety of cytokines, cell proliferation, and cell death. LPSRC activation results in the recruitment of at least two major cytoplasmic proteins to TLR4: the myeloid differentiation factor 88 (MyD88) and the TIR-containing adapter molecule (TRIF, also known as TICAM). The MyD88-dependent pathway leads to the recruitment of the MyD88 adaptor-like protein (Mal, also known as TIRAP), then the IL-1R-associated kinase (IRAK) and TRAF6, which can either activate mitogen-activated protein kinase kinase (MAPKK) with the ultimate activation of activation protein 1 (AP-1), or can activate IKK and phosphorylate IKba, causing the rapid ubiquitin-dependent proteolysis of the IKK and translocation of free nuclear factor kappa B (NF-kB) to the nucleus. The TRIF/ TICAM pathway can also activate IKK and induction

Cells undergoing apoptosis are cleared rapidly by phagocytosing macrophages, thus preventing the release of intracellular material from dying cells, which would cause inflammation and tissue damage. The removal of apoptotic cells in the body involves a complex array of macrophage receptors, including CD14. CD14 is upregulated in migratory macrophages infiltrating Burkitt’s lymphoma tumors to engage in clearance of apoptotic cells. The monoclonal antibody 61D3 that binds to CD14 on the surface of human macrophages markedly inhibits the capacity of macrophages to interact with and engulf cells undergoing apoptosis. The CD14-mediated interaction of cells with macrophages is specific for apoptotic cells because interaction of viable cells with macrophages is not affected by the monoclonal antibody 61D3. When transiently expressed on the surface of COS cells (African green monkey kidney fibroblast-like cell line), CD14 was found to impart an increase in both of apoptotic lymphocytes by these cells, and both were inhibited by the monoclonal antibody against CD14. Quantitative histological studies indicated that CD14-deficient mice have defective clearance capacity leading to persistence of apoptotic cells in all tissues studied. Additionally, TL-9 mouse macrophages deficient in CD14 expression were completely unable to phagocytose apoptotic thymocytes, and removal of CD14 from J774 macrophages significantly reduced the phagocytic ability of these cells. These studies show that CD14 is an integral part of the phagocytosis process. There is growing evidence that CD14 is a crucial component of the mechanism by which apoptotic

346 CD14

cells are recognized. CD14 interacts with phosphatidylserine, whose exposure to the outer leaflet of the cell membrane is a universal feature of apoptotic cells. CD14 acts as a tethering receptor for apoptotic cells, and when CD14 was removed from macrophages by cleaving its glycosylphosphatidylinositol tether, the phagocytosis of apoptotic lymphocytes by macrophages was substantially reduced. The intracellular adhesion molecule-3 (ICAM-3) is one of many molecules on the surface of apoptotic cells that supports the essential recognition by macrophages. Interestingly, the ICAM-3-mediated interaction of apoptotic leukocytes involves CD14 but is independent of macrophage lymphocyte function-associated antigen-1 (LFA-1), a2b2, and avb3. Several models have suggested that the engagement of CD14 and apoptotic cells would not induce generation of inflammatory responses, as is the case following CD14 and LPS interaction. The size and structural differences between the two ligands could explain why the distinct biological outcome of CD14 activation and the interaction of CD14 and the apoptotic cells do not need the presence of LBP. It is possible that when CD14 binds to apoptotic cells, it does not engage TLR4 or if TLR4 is activated, CD14 interaction with the dying cells, may interact with yet unknown receptor(s) to block the TLR4 responses. CD14 and Respiratory Disease

Several lines of evidence have led to the hypothesis that the CD14 gene may be associated with asthma. Engagement of CD14 by LPS and other bacterial cell wall components enhances IL-12 secretion from dendritic cells, which is believed to be an obligatory signal for the differentiation of naive T cells into Th1-like cells (Figure 2). Activation of CD14 by LPS leads to the release of IL-12, IL-18, and IFN-g, which can deviate the Th2 immune response and decrease the production of immunoglobulin E (IgE) by the B cells. It is also conceivable that upregulation of sCD14 could induce apoptosis in the pathogenic cells involved in asthma and/or induce the induction of suppressive factors such as IL-10 or TGF-b, leading to a decreased severity of the disease. These hypotheses are supported by reports in several British cohorts that reduced sCD14 levels in amniotic fluid and breast milk are associated with the subsequent development of atopy and that high levels of sCD14 are protective against development of recurrent wheezing in hospitalized children with respiratory syncytial virus infection. Furthermore, rhinovirus infection, the most common trigger of

T

sCD14

Regulatory sequence

Coding sequence

CD14 locus

+ DC

+ Macrophage

IL-12 Th0

IFN-
B cells

+ Th1

− −

IgE

IFN-


Figure 2 A T allele within the regulator promoter region of CD14 is associated with increased sCD14 levels, which is known to mediate interleukin-12 (IL-12) and interferon gamma (IFN-g) production by dendritic cells (DC) and macrophages, respectively. These cytokines cause a Th1 differentiation of T lymphocytes and suppress the production of IgE by B cells.

acute asthma exacerbations, significantly reduces CD14 levels. The CD14 gene is located in a chromosomal locus proximal to several genes implicated in the pathogenesis of asthma, including IL-3, IL-4, IL-5, IL-9, IL-13, and granulocyte-macrophage colonystimulating factor (GM-CSF). C-159 T, a common single nucleotide polymorphism (SNP) in the proximal promoter region of CD14, has been associated with different levels of sCD14 expression, and homozygous carriers of the T allele have significantly increased sCD14 and decreased total serum IgE. Increased sCD14 levels are associated with low IL-4 and high IFN-g production. The underlying mechanism is believed to be the decreased binding of transcription factor Sp3 to the GC box that includes the polymorphic nucleotide, resulting in enhanced transcriptional activity. Many population-based studies have found associations of the functional promoter polymorphism C159 T with a severity or susceptibility to atopy and serum IgE levels. However, different studies have reported conflicting results as to whether the C or the T allele is associated with atopy, and two studies in Germany involving a large cohort of children found no association with asthma. It is still unclear whether these differences are caused by other SNPs within the same gene or differences in LPS concentrations within various studies. A role for conferring increased sensitivity of this SNP with LPS exposure was supported by the finding in a study of farmers that those homozygous for the -159 T allele have significantly lower lung function and an increased prevalence of wheezing compared to those with the C allele.

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 347 See also: Apoptosis. Asthma: Overview. Dendritic Cells. Endotoxins. Interferons. Leukocytes: Monocytes; Pulmonary Macrophages. Toll-Like Receptors.

Further Reading Baldini M, Vercelli D, and Martinez FD (2002) CD14: an example of gene by environment interaction in allergic disease. Allergy 57(3): 188–192. Cook DN, Pisetsky DS, and Schwartz DA (2004) Toll-like receptors in the pathogenesis of human disease. Nature Immunology 5(10): 975–979. Gregory CD (2000) CD14-dependent clearance of apoptotic cells: relevance to the immune system. Current Opinion in Immunology 12(1): 27–34. Koppelman GH and Postma DS (2003) The genetics of CD14 in allergic disease. Current Opinion in Allergy and Clinical Immunology 3(5): 347–352. Savill J, Dransfield I, Gregory C, and Haslett C (2002) A blast from the past: clearance of apoptotic cells regulates

immune responses. National Review of Immunology 2(12): 965–975. Schmitz G and Orso E (2002) CD14 signalling in lipid rafts: new ligands and co-receptors. Current Opinion in Lipidology 13(5): 513–521. Takeda K and Akira S (2004) TLR signaling pathways. Seminars in Immunology 16(1): 3–9. Triantafilou M and Triantafilou K (2002) Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends in Immunology 23(6): 301–304. Ulevitch RJ, Mathison JC, and da Silva Correia J (2004) Innate immune responses during infection. Nature Immunology 5(10): 987–995. Ulevitch RJ and Tobias PS (1999) Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Current Opinion in Immunology 11(1): 19–22. Wright SD (1995) CD14 and innate recognition of bacteria. Journal of Immunology 155(1): 6–8. Zhang G and Ghosh S (2001) Toll-like receptor-mediated NFkappaB activation: a phylogenetically conserved paradigm in innate immunity. Journal of Clinical Investigation 107(1): 13–19.

CELL CYCLE AND CELL-CYCLE CHECKPOINTS A Carnero, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain & 2006 Elsevier Ltd. All rights reserved.

Abstract Completion of the cell cycle requires the precise coordination of a variety of macromolecular synthesis, assembly, and movement events. The DNA must be replicated and the chromosomes condensed, segregated, and decondensed. The spindle pole must duplicate, separate, and migrate, and changes in the cell and nuclear membrane must ensure a complete and correct duplication. Checkpoint controls are superimposed to ensure correct cell-cycle transition by monitoring the execution of specific events and coupling them to further progression. Cell-cycle checkpoints ensure that the system only proceeds to the next stage when the previous stage has been completed. Correct control of the cell cycle results from the coordinated and sequential activation–inactivation of a family of key regulators known as cyclin-dependent kinases. In this article, the mechanisms that ultimately regulate the checkpoints and their contribution to the cell cycle are discussed.

Checkpoints and the Cell Cycle In 1970, Lee Hartwell and colleagues recognized the first cell division cycle (cdc) mutants among a large collection of temperature-sensitive lethal mutants of Saccharomyces cerevisiae. These clones were further identified as mutants that arrested division at a unique stage of the cell cycle regardless of their stage at the time they were shifted to restrictive temperature. The

phenotypes of the cdc mutants revealed that the execution of late events in the cell cycle depended on the prior completion of early events. Cell division occurs by an ordered series of metabolic and morphogenic changes that are collectively called the cell cycle. The cell cycle is divided into four distinct phases (Figure 1): *

*

*

*

Synthetic (S) phase, in which the replication of DNA takes place Mitotic (M) phase, in which equal distribution of duplicated DNA and cellular division take place Gap 1 (G1) phase: the gap between completion of the M phase and the beginning of the S phase Gap 2 (G2) phase: the gap between completion of the S phase and the beginning of the M phase

Completion of the cell cycle requires the precise coordination of a variety of macromolecular synthesis, assembly, and movement events. The DNA must be replicated and the chromosomes condensed, segregated, and decondensed. The spindle pole must duplicate, separate, and migrate, and changes in cell and nuclear membrane must ensure a complete and correct duplication. Checkpoint controls are superimposed to ensure correct cell-cycle transition by monitoring the execution of specific events and coupling them to further progression. Cell cycle checkpoints ensure that the system only proceeds to the next stage when the

348 CELL CYCLE AND CELL-CYCLE CHECKPOINTS Spindle checkpoint

G2/M checkpoint

G2

CDK4

CDK2 CDK2

Cyclin D

G1 CDK1

Cyc lin E

Cyc

lin B

M

G1 checkpoint

S Cyclin A DNA damage checkpoint Figure 1 Cell cycle and cell-cycle checkpoints.

previous stage has been completed. The key checkpoints appear to be as follows: * *

*

*

G2/M: No entry to M without completion of S. Spindle checkpoint: Prevents anaphase onset until completion of mitotic spindle assembly. Start or restriction (R) point: Blocks progress for nonproliferating or differentiated cells; integrates external signals to proceed with the cell cycle. DNA damage: monitoring of DNA damage and block of cell-cycle progress; allows repair mechanisms to proceed; if DNA repair fails, the cell may be directed into apoptosis.

The coordination of these complex processes is achieved by a series of coordinated and sequential phase transitions of key regulators known as cyclin-dependent kinases (CDKs), which are one of the major targets detected in the cdc mutants (Figure 1).

Mammalian Cell Cycle Regulators The CDKs are a family of closely related Ser/Thr kinases that are activated by association with a regulatory subunit called cyclin (Table 1). The typical catalytic subunit contains a 300-amino acid catalytic core that is inactive when monomeric and unphosphorylated at certain positions. Cyclins are members of structurally related proteins whose levels usually oscillate during the cell cycle. The homology among cyclins is mostly limited to the cyclin box, the domain responsible for

CDK binding and activation. Mutations in this region inhibit both binding and activation. Each CDK interacts with a specific subset of cyclins, although the size of the subset varies (Table 1). The cyclin function is primarily controlled by changes in cyclin levels. In mammalian cells, sequential oscillation in the levels of major cyclins (E, A, and D) reflects levels of messenger RNA and specific proteolysis. Whereas cyclins are essential to activate CDKs, the specific destruction of cyclins is central to the proper regulation of the cell cycle. In terms of proteolysis, the cyclins can be roughly divided into two classes – those that are constitutively unstable through the cell cycle and whose level is therefore determined by the ratio of transcription (cyclins D and E) and those that are unstable in only one phase of the cell cycle. The G2 cyclins (A and B type) are stable throughout interphase and specifically destroyed at mitosis. Cyclin degradation involves the ubiquitin-dependent degradation machinery. However, the details of the conjugation cyclin–proteasome differ for the different cyclins. E and D cyclins are ubiquitinated by the SCF complex, whereas G2 cyclins are ubiquitinated by the anaphase-promoting complex (APC). The SCF complex is active throughout the cell cycle, and the destruction of its substrates depends on their phosphorylation, with different phosphate-binding proteins (F box proteins) guiding different sets of substrates to destruction. The APC is activated at the onset of anaphase and degrades its substrates as the cell exits mitosis.

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 349 Table 1 Complexes of cyclin-dependent kinases (CDKs) Kinase

Regulatory subunit

Substrate

Function

CDK1 CDK2 CDK3 CDK4 CDK5 CDK6 CDK7 CDK8 CDK9 CDK10 CDK11

Cyclin A and B Cyclin A, E, and D Cyclin E Cyclin D p35, cyclin D Cyclin D Cyclin H Cyclin C Cyclin T

pRb, NF, Histona H1 pRb, p27 E2F1/DP1 pRb, Smad3 NF, Tau pRb CDK1/2/4/6 RNA pol II pRb, MBP Ets2 RNA pol II

G2/M G1/S, S G1/S G1/S Neuronal differentiation G1/S CAK Transcriptional regulation G1/S Transcription Transcription

Cyclin L

Degradation Cyclin synthesis

P P P pRb

CKIs Cyclin

CDK

Inactive

Active

Cyclin

Cyclin

CDK

CDK

thr161

thr14

P P P pRb E2F

tyr15 thr161

E2F

thr161 thr14 tyr15

Cyclin H

CDC25 WEE 1

CDK7

DPI

E2F

Transcription

thr161 Figure 2 CDK regulation at the G1 checkpoint.

In addition to cyclin-binding, complete CDK activation requires phosphorylation at a conserved 160/ 161 threonine residue. This phosphorylation may affect the cyclin-binding site because it enhances the binding of some CDK/cyclin complexes. This activating phosphorylation is performed by another CDK/ cyclin complex termed CAK, which consists of CDK7 bound to cyclin H. Mammalian CDK7/cyclin H complexes can phosphorylate CDK1 and CDK2 complexed with various cyclins as well as CDK4 (Figure 2). CDK/cyclin complexes can also be inhibited by phosphorylation of the CDK subunit at the amino terminus (Thr14 and Tyr15 residues in human CDK1 and CDK2). Inactivation via tyrosine 15 phosphorylation is important in the control of the initiation of mitosis (Figure 2). The phosphate in tyrosine 15 is removed by a specific phosphatase, CDC25. There are at least three CDC25 family members in

mammalian cells. CDC25C is more likely to activate cyclin B/CDC2 at mitosis, whereas CDC25A is phosphorylated and activated by cyclin E/CDK2 at the initiation of DNA synthesis. Finally, numerous studies have identified additional regulatory subunits for the CDKs, the cyclindependent kinase inhibitory proteins (CKIs). These proteins bind and inhibit CDK activity. There are two families of CKIs: the CIP/KIP family and the INK4 family (Table 2). The CIP/KIP family includes p21waf1/cip1/sdi1(p21waf1), p27kip1, and p57kip2 and seems to be central in differentiation processes. The members of this family are general inhibitors of all the cyclin/CDK complexes and accumulate in response to a broad range of antiproliferative stimuli. These proteins preferentially associate with the cyclin/CDK complex rather than with the individual CDK subunits. The overexpression of these proteins

350 CELL CYCLE AND CELL-CYCLE CHECKPOINTS Table 2 Involvement of cell-cycle regulatory elements in human cancer Protein

Alteration

Tumor

CDK4

Mutation

Melanoma

Overexpression Overexpression Overexpression Overexpression

Breast, prostate, gastric, parathyroid carcinoma, lung cancer Colorectal carcinoma Breast, ovarian, and gastric carcinoma Hepatocellular carcinoma, lung cancer

CDC25A CDC25B p27KIP1 p57KIP2

Activation Activation Inactivation/degradation Deletion/inactivation

Head and neck cancer, lung cancer Breast cancer, lymphoma, head and neck cancer, lung cancer Breast, prostate, and colorectal carcinoma, lung cancer Beekwith–Wiedemann syndrome

p16INK4a p15INK4b

Deletion/inactivation, mutation Deletion/inactivation

Melanoma, lymphoma, lung cancer, pancreatic carcinoma Leukemia, lymphoma

Plk1 Aurora A Aurora B

Overexpression Overexpression Overexpression

Lung cancer, head and neck squamous carcinoma Several tumors, lung cancer Several tumors, lung cancer

pRb

Deletion/inactivation

Retinoblastoma, lung cancer

p53

Mutation, deletion

Many tumors, lung cancer

Cyclin Cyclin Cyclin Cyclin

D1 D2 E A

arrests cells in different states of the cell cycle according to their biochemical function. The INK4 family of CKIs consists of four related proteins: p16ink4a, p15ink4b, p18ink4c, and p19ink4d. These proteins specifically bind and inactivate CDK4 and CDK6 kinases, inducing G1 arrest. These proteins appear to bind the CDK subunit alone, competing with their activating partner, cyclin D. It is possible that these proteins inhibit CDK activity by reducing the affinity for cyclin D or by competitively preventing the binding of cyclin D to the CDK subunit.

The Cell Cycle Progression through the cell cycle results from the coordinated and sequential activation–inactivation of the previously described elements. Restriction Point

After the proliferative stimuli (growth factors and oncogenes) induce cell proliferation, a first checkpoint (the R point) at late G1 integrates both positive and negative external and internal signals before the cell commits to another round of DNA replication (Figure 1). In mammalian cells the R point is regulated mainly by the CDKs bound to the D type of cyclins, and its activation is mainly driven by transcriptional activation of cyclin D synthesis. Most growth factors and oncogenes trigger cyclin D transcription. There are three types of D cyclins, and they are in part cell-type specific, with most cells expressing D3 and either D1 or D2. The D type of cyclins

associate and activate CDK4 and, in some cells, also CDK6. These cyclins have a very short half-life and their expression is highly growth factor inducible. Dtype cyclins are fundamental in cell-cycle regulation of the retinoblastoma protein (pRb; the major regulator of the R checkpoint). The phosphorylation of pRb is controlled by two opposing reactions: phosphorylation by CDKs in G1, which converts pRb into its inactive form in terms of ligand-binding activity, and dephosphorylation in late M by phosphatases, which reconstitute active pRb. As a result of this interplay of enzymatic reactions, the pRb phosphorylation state fluctuates as the cell passes through the cell cycle. In cycling cells, pRb is found in its active, underphosphorylated form only during the early period of the G1 phase. Although changes in the array of phosphorylation events may occur at other phases and inactivating phosphorylation of newly synthesized pRb may occur throughout the cycle, pRb phosphorylation in late G1 phase and its dephosphorylation in late M phase are the two critical events regulating pRb growth-restraining activities. In vitro, cyclin E- and cyclin D-activated kinases phosphorylate pRb differentially producing an overlapping subset of sites. This has led to the development of three different models of how different phosphorylation events may contribute to pRb control in cells. The first possibility is that both CDK complexes have identical effects, inactivating pRb by phosphorylating in different positions. Alternatively, phosphorylation by different complexes could have different effects by regulating the binding to a different subset of effectors. Finally, combined phosphorylation of both kinases may be necessary to

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 351

completely inactivate pRb. It is difficult to determine which model is correct. Cyclin D1 and pRb functions seem to be interdependent. When cyclin D1 is ectopically expressed in G1 phase, pRb is phosphorylated earlier than in the normal cycle and G1 progression is accelerated. Antibodies to cyclin D1 arrest most cell types before S phase; however, they do not arrest if they lack functional pRb. The effect of the INK4 family of CKIs also seems to be dependent on the presence of functional pRb since the absence of pRb makes the cells insensitive to INK4induced G1 arrest. Unlike cyclin D, cyclin E is both rate-limiting and required for the G1/S transition in both pRb-positive and -negative cells. It has been shown that combined expression of c-myc and oncogenic ras allows entry of growth factor-deprived cells into S phase without pRb phosphorylation. Activation of cyclin E, but not cyclin D, was seen in these cells. A possible target of this pRb-independent pathway is the other members of the pRb family of pocket proteins, p107 and p130, that form stable complexes with cyclin E/CDK2 and cyclin A/CDK2 and whose expression is dramatically regulated at the end of G1 or S phase, respectively. Sequential phosphorylation of particular sites may result in the orchestrated disruption of pocket protein complexes containing different transcription factors, leading to the sequential succession of events required to promote progression through the cell cycle. However, CDK4 is dispensable in most cell types, probably due to a compensatory role by CDK6. Both CDK4 and CDK6 null mice develop normally with minor problems, such as diabetes or some impaired hematopoiesis, respectively. Double CDK4/CDK6 null mice die in early stages of embryogenesis due to severe anemia, but they show normal organogenesis. Double null MEFS proliferate normally and become immortal upon serial passage. These results indicate that in certain circumstances, D-type CDKs are not essential for cell-cycle entry. However, it still has to be confirmed whether a true redundancy for the CDK4/6 activity exists or whether it is an effect of molecular plasticity in early embryogenesis that will never take place in normal developing embryos. One of the consequences (in addition to chromatin remodeling) of pRb phosphorylation is the release of sequestered E2F proteins. The E2F proteins heterodimerize with DP1, forming the active transcription factor. The activity of E2F/DP1 induces the transcription of a number of genes involved in DNA synthesis and cell proliferation. High levels of INK4 proteins lead to inhibition of CDK4/6 kinases and keep pRb unphosphorylated and bound to the E2F transcription factor. The sequestered factor is thus inactivated, inducing a block in G1. This scheme is

much more complex in its details. First, there are six members of the E2F transcription factor family (five activators and a negative regulator, E2F6), and only three of them (E2F1, -2, and -3) are under direct control of pRb. Hypophosphorylated pRb binds the other two members only weakly. E2F4 and E2F5 appear to bind preferentially to p107 and p130, which have a weak affinity for the other three. This suggests the possible existence of two parallel pathways: pRb/E2F1, -2, or -3 and p107/p130/E2F4 or -5. Also, pRb may regulate a number of downstream effectors besides E2Fs, including Elf-1, Myo-D, PU.1, ATF-2, and c-Abl proteins; however, the effector functions of most of these are not well-known. S Phase

Once external signals have been integrated at the R checkpoint and the cells are committed to duplication, the cells initiate the S phase, during which other CDKs take control of DNA replication. CDK2 is considered to be most directly involved in DNA replication. Cyclins E and A sequentially activate CDK2, and both are thought to be essential for initiating DNA replication. Ectopic overexpression of cyclin E in mammalian fibroblasts causes premature entry into S phase, and microinjection of antibodies against cyclin E prevents S phase initiation. Cyclin A activates CDK2 soon after cyclin E does, concomitant with DNA synthesis. Ablation of cyclin A synthesis blocks entry into S phase. Cyclin A co-localizes with sites of DNA replication in S phase nuclei, suggesting that it may be directly involved in the assembly, activation, or regulation of replication structures. In vitro studies using SV40 as a model system have demonstrated that CDKs are necessary for the assembly of replication origin initiation complexes containing unwound DNA. Multiple experiments demonstrate the essential role of CDK2 in the cell cycle. Overexpression of p27 or dominant-negative CDK2 mutants inhibit proliferation. Furthermore, injection of antibodies against CDK2 subunit or treatment of the cells with CDK2 inhibitors also block proliferation. Moreover, a central role for CDK2/cyclin E has been indicated from observations of mice engineered to express cyclin E in place of cyclin D1. Animals lacking cyclin D1 have proliferative defects and fail to activate CDK2 in a subset of tissues. These phenotypes were suppressed in a knock-in animal that expresses cyclin E from the cyclin D1 promoter, suggesting that loss of CDK/cyclin D phosphorylation of pRb has no effect if cyclin E is no longer dependent on pRb inactivation.

352 CELL CYCLE AND CELL-CYCLE CHECKPOINTS

However, recent findings challenge this view. The inhibition of CDK2 by several ways has no effect on proliferation in some cell lines. High levels of CDK4 may compensate for the absence of CDK2. Moreover, both CDK4 and CDK2 activities may be unnecessary for proliferation in pRb-negative cells. Similar conclusions were derived using CDK2 null mice, which develop normally except for some defects in meiosis. DNA Damage Checkpoint

The cells will arrest at the G1/S checkpoint in response to DNA damage to prevent the replication of mutated DNA. p53 tumor suppressor plays a critical role during this damage-induced checkpoint because p53-deficient cells fail to undergo G1/S arrest after genotoxic stress. After exposure to genotoxic insults, p53 is activated and transcriptionally regulates many targets (e.g., p21 waf1, GADD45, reprimo, and 143-3 d) that directly provoke cell-cycle arrest. G2/M Checkpoint and Early Mitosis

The orchestration of mitotic events requires exquisite regulation, and the penalty for errors in chromosome segregation is severe. Chromosomal imbalances are responsible for many cases of abortions, birth defects, and cancer. Once S phase is complete, the cell duplicates cell structures and separates the chromosomes. For this process, the cell must establish another checkpoint at the onset of G2, before the initiation of mitosis. The key component is a mitotic regulator composed of CDK1 and cyclin B. Together, these proteins form the mitosis-promoting factor (MPF), the activity of which initiates mitosis. MPF is regulated by periodic accumulation and destruction of cyclins, although additional controls regulate the complex. The mitotic phase can be defined by three transitions that involve cyclin B. First, cyclin B activates MPF and initiates prophase. CDK1 bound to cyclin B must also be phosphorylated by CAK in threonine 161 and dephosphorylated in tyrosine 15 and threonine 14. This activation will induce the specific phosphorylation of mitotic targets triggering downstream events. Nuclear lamins, kinesin-related motors and other microtubule-binding proteins, condensins, and Golgi matrix components are substrates of CDK1/cyclin B important for nuclear envelope breakdown (NEBD), chromosome separation, spindle assembly, chromosome condensation, and Golgi fragmentation. Second, MPF activates a ubiquitin–proteolytic system that degrades cyclin B and initiates anaphase. Finally, the cyclin B destruction machinery is turned off and the cycle is reset.

Centrosomes are the major microtubule-organizing center of animal cells. In most cell types, duplicated centrosomes remain closely paired and continue functioning as single microtubule organizing centers during G2. After G2, however, they separate and migrate apart. The separation of centrosomes seems to be regulated by several kinases. Nek2, a member of the NIMA family, is thought to phosphorylate the centrosomal protein c-Nap1 causing the dissolution of a dynamic structure that tethers duplicate centrosomes to each other. An abrupt increase in c-Nap1 phosphorylation at the G2/M transition is caused by the cell-cycle-regulated inhibition of a type 1 phosphatase. At a later step, several kinesinrelated motor proteins and cytoplasmic dynein are required for centrosome separation. In organisms undergoing open mitosis, NEBD occurs soon after centrosome separation. During interphase, the nuclear envelope is stabilized by the nuclear lamina, but at the onset of mitosis, this structure disassembles as a consequence of lamin hyperphosphorylation. The predominant kinase triggering mitotic lamina depolymerization in vivo is CDK1/cyclin B. PLKs are key regulators of mitosis and cytokinesis because they are subject to complex temporal and spatial control. PLK1 seems to be involved in mitotic entry, centrosome maturation, spindle-assembly, and APC regulation. PLK1 is involved in the recruitment of a-tubulin during centrosome maturation, a process by which centrosomes increase their microtubule nucleation activity required for spindle formation. The association of PLKs with the centrosome also has implications for the regulation of mitotic entry. The initial activation of CDK1/cyclin B occurs at the centrosome, probably through phosphorylation of both CDC25 and Myt1. In mitotic cells, PLK1 also associates with the kinetochores. These structures play an important role in the spindle checkpoint. This checkpoint operates to ensure that all chromosomes have undergone bipolar attachment to the spindle before sister chromatids are separated. Another family of kinases that plays a critical role in chromosomal segregation and cell division is the Aurora family. Aurora kinases are implicated in the centrosome cycle, spindle assembly, chromosome condensation, chromosome–microtubule attachment, the spindle checkpoint, and cytokinesis. Aurora kinases are regulated through phosphorylation, the binding to specific partners, and proteolysis. In mammals, there are three Aurora members that differ in expression patterns, localization, and timing of activity. Aurora A is upregulated at the onset of mitosis. It localizes at centrosomes during interphase and at both spindle poles and microtubules during early mitosis. Aurora B

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 353

reaches maximal levels later in mitosis, first associated with the centromeres/kinetochores, then relocalizes to the midzone of the central spindle and finally concentrates at the midbody between dividing cells. Aurora A is implicated primarily in centrosome maturation and spindle assembly, whereas Aurora B is thought to regulate chromosome condensation and cohesion, kinetochore assembly, the spindle checkpoint, and the coordination between chromosome segregation and cytokinesis. Aurora B constitutes a key link between structural aspects of the microtubule–kinetochore interactions and checkpoint signaling. The third member of the family, Aurora C, has a more restricted expression pattern and its functions are mostly unknown. Spindle Checkpoint

Proper distribution of chromosomes between daughter cells is controlled by the spindle-assembly checkpoint that separates metaphase from anaphase, two clearly defined stages within mitosis. At this stage (metaphase), sister chromatids (pairs of identical chromosomes to be segregated between the daughter cells) are aligned at the midzone of the mitotic spindle. The spindle checkpoint, a safeguard mechanism that prevents defects in chromosome segregation, inhibits anaphase onset until the mitotic spindle is correctly assembled. Metaphase–anaphase transition

At the start of anaphase, cohesins, the molecules responsible for sister chromatid cohesion, are degraded by a protease known as separase. In turn, separase is activated by degradation of a specific inhibitor, securin, by APC, a ubiquitin–ligase multiprotein complex (Figure 3). APC is the ultimate gatekeeper of the spindle-assembly checkpoint, ensuring that sister chromatids do not segregate unless an identical set of chromosomes are properly aligned at the spindle midzone. APC activity is regulated primarily by Mad2, a sensor of microtubule attachment to the kinetochores that sequesters Cdc20, a molecule required for APC activation. Mad2 binds to Bub3 and BubR1. The Mad/Bub complex is recruited to unattached kinetochores and inhibits the activation of the APC/Cdc20 complex, which is necessary for sister chromatid separation. This results in metaphase arrest until all kinetochores are correctly aligned. Completion of kinetochore attachment leads to the dissociation of Mad2 from Cdc20 (Figure 3). The spindle checkpoint requires mitotic CDK activity. Inhibiting CDK activity overrides checkpointdependent arrest. Following inhibition, the interaction between APC and Cdc20 transiently increases while the inhibitory checkpoint protein Mad2 dissociates from Cdc20. CDK inhibition also overcomes Mad2-induced mitotic arrest. In addition, in vitro

Improper spindle Aur Plk

Normal spindle

Bub

Cdc20

Cdc20

APC

APC Separase Cohesin cleaved Sister chromatid separation

Securin

UbUbUbUb

Cohesin intact

Metaphase arrest

Figure 3 Spindle checkpoint.

Mad2

354 CELL CYCLE AND CELL-CYCLE CHECKPOINTS

CDK1-phosphorylated Cdc20 interacts with Mad2 rather than APC. Thus, CDK1 activity seems to be required to restrain APC/Cdc20 activation until completion of spindle assembly. In addition, evidence indicates that CDK1 activity is required for the efficient function of the spindle checkpoint protein Bub1. Phosphorylation of key elements may play a role in the timing of the events that lead to mitosis exit and in regulating the spindle checkpoint function. Phosphorylation of APC subunits by PLKs appears to help APC activity. PLK-1 recruits spindle checkpoint proteins to centromeres and creates the tension-sensing phosphoepitope on mitotic kinetochores and APC. Aurora B, Survivin, and INCENP physically interact and form a complex that localizes to the kinetochores in prometaphase, to the cell equator during metaphase, and to the midbody during cytokinesis. The absence of function of these proteins has revealed their involvement in the spindle checkpoint. These proteins are also required to establish bipolar attachment of chromosomes, probably by destabilizing kinetochore–microtubule attachments that lack tension. Aurora communicates with the other downstream components probably through direct phosphorylation of BubR1 and Mps1.

Cell Cycle and Cell-Cycle Checkpoints in Respiratory Diseases Hanahan and Weinberg suggested that all cancer genotypes are the manifestation of six essential physiological alterations shown by most, and perhaps all, types of human tumors. Although breaching of these six physiological barriers seems to be necessary for most tumors to achieve full malignancy, cancer is increasingly viewed as a cell-cycle disease. This view reflects evidence that the vast majority of tumors, and likely all tumors, have suffered one or more defects that derail the cell-cycle machinery (Table 2). Such defects can target either components of the cell cycle, including checkpoint mechanisms, or elements of the upstream signaling cascades, whose events eventually converge to trigger cell-cycle events. Lung cancer remains a worldwide major health challenge. Despite improvements in treatment, the 5year survival rate for individuals with lung cancer is only approximately 15%. Histologically, 80% of lung cancers are non-small cell lung cancer (NSCLC), whereas the remaining 20% of cases are small cell lung cancer (SCLC). On the basis of cell morphology, adenocarcinoma and squamous cell carcinoma are the most common types of NSCLC.

The pRb protein is inactivated in more than 90% of SCLCs as a result of different mechanisms, including point mutations and abnormal mRNA expression. Changes in the other Rb family members (p107 and p130) have been detected in only a few cases. In contrast to SCLC, the majority of NSCLC cases exhibit abnormalities in the upstream regulators of the pRb pathway, including inactivation of p16ink4a, reduced levels of p27Kip, and enhanced expression of cyclin D1. p16ink4a abnormalities are frequently found in NSCLCs but are rare in SCLCs. Perhaps 30–50% of early stage primary NSCLCs do not express p16ink4a (Table 2). The overexpression of cyclin A has been consistently shown to be a negative prognostic indicator of lung cancer. Also, cdc25A, one of the rate-limiting mechanisms for G1 progression into S phase, is frequently overexpressed in NSCLC. The gene cdk10 is essential for cellular proliferation, and its effect is also exerted in the G2/M transition. Only recently was this gene found to be overexpressed in lung adenocarcinomas. Also, cyclin B1 and CDK1, regulators of the G2/M transition, are overexpressed in lung cancer. Following DNA damage, a series of events is initiated that ends with p53 activation and the transcriptional regulation of many cell-cycle regulators. The fundamental importance of p53 in lung cancer is highlighted by the frequency of its mutations—80% in SCLC and 50% in NSCLC. Once the p53 gene is deleted or mutated, cells become susceptible to DNA damage and deregulated cell growth.

Ceramide and the Cell Cycle The sphingolipid ceramide is generated from the membrane-associated sphingomyelin by the activation of neutral sphingomyelinase in response to a variety of extracellular inducers. Ceramide acts as a second messenger to mediate many of the effects of these inducers. Ceramide is an important molecule that regulates diverse signaling pathways involving apoptosis, cell senescence, cell cycle, and differentiation. For the most part, ceramide’s effects are antagonistic to growth and survival. Interestingly, ceramide and the progrowth agonist, diacylglycerol (DAG), appear to be regulated simultaneously but in opposite directions in the sphingomyelin cycle. Whereas ceramide stimulates signal transduction pathways that are associated with cell death or at least are inhibitory to cell growth (e.g., stress-activated protein kinase or mitogen-activated protein kinase pathways), DAG activates the classical and novel isoforms of the protein kinase C (PKC) family. These PKC isoforms are associated with cell growth

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 355

and cell survival. Thus, ceramide and DAG generation may serve to monitor cellular homeostasis by inducing prodeath or progrowth pathways, respectively. Ceramide levels are elevated in response to diverse stress challenges, including chemotherapeutic drug treatment, irradiation, or treatment with prodeath ligands such as tumor necrosis factor-a. Consistent with this notion, ceramide is a potent apoptogenic agent. Ceramide activates stress-activated protein kinases such as c-jun N-terminal kinase and thus affects transcription pathways involving c-jun. Ceramide activates protein phosphatases such as protein phosphatase-1 and protein phosphatase-2. Ceramide activation of protein phosphatases has been shown to promote inactivation of a number of progrowth cellular regulators, including the kinases PKC-a and Akt, Bcl-2, and the retinoblastoma protein. Ceramide levels remain relatively constant throughout the life span of fibroblasts but increase with the onset of cellular senescence (a terminally arrested state that somatic cells acquire after a limited round of replication), increasing severalfold compared to the levels of young cells. Whereas in differentiation and apoptosis ceramide levels increase transiently, in senescence the increase in ceramide levels is permanent. The addition of a cell-permeable ceramide to young fibroblasts to concentrations usually seen in senescent cells mimics the senescent phenotype, with many of its markers. Higher concentrations of ceramide induce apoptosis. Cells treated with ceramide are unable to produce the AP-1 transcription factor and progressively inhibit pRb phosphorylation. It has been found that ceramide levels increase as cells exit mitosis to G1 phase. This increase precedes the dephosphorylation of pRb that occurs as the cells exit G2/M. In contrast, no changes in ceramide have been observed during other cell-cycle phase transitions, suggesting a role for the endogenous ceramide in the transition from G2/M to G1. In agreement with this, microinjection of ceramide or sphingosine was sufficient to reinitiate the cell cycle by reactivating G2/M transition in Xenopus oocytes. Ceramide also inhibits phospholipase D and PKC. Phospholipase D and PKC transduce mitogenic signals, and their progressive inactivation has been observed during cellular senescence, probably due to the increased in ceramide.

Acknowledgments This work was supported by grant BIO-01-0069 from the Spanish Ministry of Science and Technology and grant PI020126 from the Spanish Ministry of Health. See also: DNA: Repair; Structure and Function. Tumor Necrosis Factor Alpha (TNF-a). Tumors, Malignant: Overview.

Further Reading Adams PD (2001) Regulation of the retinoblastoma tumor suppressor protein by cyclin/cdks. Biochimia et Biophysica Acta 1471(3): M123–M133. Bell SP and Dutta A (2002) DNA replication in eukaryotic cells. Annual Review of Biochemistry 71: 333–374. Hanahan D and Weinberg RA (2000) The hallmarks of cancer. Cell 100: 57–70. Harris SL and Levine AJ (2005) The p53 pathway: positive and negative feedback loops. Oncogene 24: 2899–2908. Hartwell LH, Culotty J, Pringle JR, and Reid BJ (1974) Genetic control of the cell division cycle in yeast. V. Genetic analysis of mutants. Genetics 74: 267–286. Hartwell LH, Culotty J, and Reid BJ (1970) Genetic control of the cell division cycle in yeast. I. Detection of mutants. Proceedings of the National Academy of Sciences USA 66: 352–359. Hartwell LH and Kastan MB (1994) Cell cycle control and cancer. Science 266: 1821–1828. Jeffrey PD, Russo AA, Polyak K, et al. (1995) Mechanism of CDK activation revealed by the structure of a cyclin A–CDK2 complex. Nature 376: 313–320. Kelly TJ and Brown GW (2000) Regulation of chromosome replication. Annual Review of Biochemistry 69: 829–880. Malumbres M and Carnero A (2003) Cell cycle deregulation: a common motif in cancer. Progress in Cell Cycle Research 5: 5–18. Meijer L, Jezequel A, and Ducommun B (2000) Progress in Cell Cycle Research, vol. 4. Norwell, MA: Kluwer. Murray AW (2004) Recycling the cell cycle: cyclins revisited. Cell 116: 221–234. Murray AW and Hunt T (1993) The Cell Cycle: An Introduction. Oxford: Oxford University Press. Osada H and Takahasi T (2002) Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer. Oncogene 21: 7421–7434. Peters JM (2002) The anaphase-promoting complex: proteolysis in mitosis and beyond. Molecular Cell 9: 931–943. Sherr CJ (2004) Principles of tumor suppression. Cell 116(2): 235–246. Sherr CJ and Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes & Development 13: 1501–1512. Stein GS, Baserga R, Giordano A, and Denhardt DT (eds.) (1999) The Molecular Basis of Cell Cycle and Growth Control. New York: Wiley–Liss.

356 CHEMOKINES

CHEMOKINES O Morteau, Children’s Hospital, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Chemokines are an extended family of small size chemoattractant proteins that mediate leukocyte migration and positioning. They have been classified into the C, CC, CXC, and CX3C subfamilies based on the position of a cysteine residue in their primary structure. Chemokines interact with structurally related G-protein-coupled receptors located on the surface of leukocytes, and trigger a cascade of intracellular events involving several distinct signaling pathways. The biological functions of chemokines include chemoattraction, integrin activation, leukocyte degranulation and mediator release, and angiogenesis, or angiostasis. They are also involved in various ways in the establishment and maintenance of the innate and adaptive immune systems. The importance of chemokines and their receptors in the pathogenesis of respiratory diseases is exemplified in conditions as diverse as airway allergy (asthma), bacterial and viral infections (tuberculosis, respiratory syncytial virus infection), allograft complications (acute lung allograft rejection, graft-versus-host disease), lung cancer, and other pathologies including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and acute respiratory distress syndrome.

1996, some chemokine receptors were found to act as co-receptors for human immunodeficiency virus (HIV)-1, and in 1998, viral chemokines were identified and characterized. The role of chemokines and their receptors (including CCR4, CCR9, and CCR10) in tissue-specific homing was confirmed in 1999. The fast-expanding chemokine family was designated by various successive terms, including ‘the platelet factor-4 family’, ‘the small inducible cytokine family’, and ‘the intercrines’. The standard term ‘chemokines’ (a neologism for ‘chemotactic cytokines’) was eventually coined in 1992 at the Third International Symposium on Chemotactic Cytokines in Baden. In 1995, the International Union of Pharmacology Committee on Receptor Nomenclature and Classification (NC-IUPHAR) created a subcommittee on chemokine receptor nomenclature. In 1999, a chemokine nomenclature system that parallels the chemokine receptor nomenclature was proposed at the Keystone Symposium on Chemokines and Chemokine Receptors.

Introduction

Chemokine Structure and Nomenclature

Chemokines are an extended family of small size chemoattractant cytokines that facilitate leukocyte migration and positioning, and exert their effects via binding to receptors located at the target cell surface. To date, 43 chemokines have been identified, binding to 19 different receptors. The first chemokine was discovered by Walz and co-workers in 1977. It was a procoagulant and angiostatic factor called platelet factor 4, now renamed CXCL4. From 1984 to 1989, several investigators cloned the cDNAs for structurally related proteins, including IP-10 (IP10 – 10 kDa IFN (interferon)-ginducible protein), JE, Mig, RANTES (RANTES – regulated on activation normal T cell expressed and secreted), I-309, KC, and MIP-1 a (MIP – macrophage inflammatory protein). At the time a function had yet to be attributed to this growing gene family, and it is only with the characterization of the neutrophil chemoattractant interleukin 8 (IL-8), also called CXCL8, that chemokines were recognized as chemotactic molecules. Between 1989 and 1994, interest in the chemokine field grew dramatically as MCP-1 (MCP – monocyte chemoattractant protein), RANTES, and eotaxin were reported to target monocytes, T cells, and eosinophils, respectively. In

Most chemokines are secreted proteins of 67 to 127 amino acids (only CXCL16 and CX3CL1 are membrane-bound), and contain a conserved tetra-cysteine motif located at the NH2 terminus. Chemokine nomenclature is based on the relative position of the first two of these four consensus cysteines (Table 1). The separation of the cysteines by a nonconserved amino acid defines the CXC chemokine subclass, while the presence of two adjacent cysteines defines the CC chemokine subclass. Two minor chemokine subclasses (C and CX3C) either lack two out of four canonical cysteines (XCL1 and XCL2 chemokines), or contain three intercalated nonconsensus amino acids (CX3CL1 chemokine). CXC chemokines can be further discriminated into two structural subgroups (ELR þ and ELR–), based on the presence or absence of the tripeptide motif glutamic acid–leucine–arginine (ELR) N-terminal to the first cysteine. One structure/function correlate is the specificity of ELR þ CXC chemokines for neutrophils. Another one is the ability of ELR þ CXC chemokines to promote the neoformation of blood vessels (angiogenesis), while ELR
CXC chemokines exert the opposite effect (angiostasis), as we will see later. The CXC, CC, CX3C, and C chemokine subclasses are

CHEMOKINES 357 Table 1 Chemokines and their receptors Class

Systemic name

Common synonyms

Receptor

CXC (a)

CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL15 CXCL16

GROa, MGSA, KC (mouse), MIP-2 (mouse) GROb, MIP-2a GROg, MIP-2b Platelet factor 4 ENA-78 GCP-2 PBP, CTAP-III, b-TG, NAP-2 IL-8, MDNCF, GCP, LIF, NAP-1, LYNAP Mig, CGR-10 (mouse) IP-10, CGR2 (mouse) I-TAC, b-R1, IP9, H174 SDF-1a, SDF-1b, PBSF, TLSF, TPAR1 BCA-1, BLC BRAK, Bolekine

CXCR2 CXCR2 CXCR2 Unknown CXCR2 CXCR1, CXCR2 CXCR2 CXCR1, CXCR2 CXCR3 CXCR3 CXCR3 CXCR4 CXCR5 Unknown Unknown CXCR6

CC(b)

CCL1 CCL2 CCL3 CCL4 CCL5 CCL6 CCL7 CCL8 CCL9 CCL10 CCL11 CCL12 CCL13 CCL14 CCL15 CCL16 CCL17 CCL18 CCL19 CCL20 CCL21 CCL22 CCL23 CCL24 CCL25 CCL26 CCL27 CCL28

1-309, TCA-3 (mouse), SIS-f (mouse) MCP-1, MCAF, HC11, JE (mouse) MIP-1a, LD-78a, PAT464, GOS19, hSISa MIP-1b, ACT-2, HC21, MAD-5, LAG-1 RANTES, SIS-d C10 (mouse), MRP-1 (mouse) MCP-3, NC28, FIC, MARC (mouse) MCP-2, HC14 MRP-2 (mouse), MIP-1g (mouse) Eotaxin MCP-5 (mouse) MCP-4, ckb10, NCC-1 CC-1, HCC-1, NCC-2, CCCK-1, Ckb1 HCC-2, Lkn-1, MIP-5, CC-2, NCC-3 HCC-4, LEC, NCC-4, LMC, Mtn-1, LCC-1 TARC DC-CK-1, PARC, MIP-4, AMAC-1, ckb7 MIP-3b, ELC, exodus-3, ckb11 MIP-3a, LARC, exodus-1, ST38 (mouse) 6Ckine, SLC, exodus-2, TCA4, ckb9 MDC, STCP-1, dc/bck (mouse) MPIF-1, MIP-3, ckb8-1 Eotaxin-2, MPIF-2, ckb6 TECK, ckb15 Eotaxin-3, MIP-4a ESkine, CTACK, skinkine, ILC (mouse) MEC

CCR8 CCR2 CCR1, CCR5 CCR5 CCR1, CCR3, CCR5 Unknown CCR1, CCR2, CCR3 CCR3 Unknown Unknown CCR3 CCR2 CCR2, CCR3 CCR1 CCR1, CCR3 CCR1 CCR4 Unknown CCR7 CCR6 CCR7 CCR4 CCR1 CCR3 CCR9 CCR3 CCR10 CCR10

C (g)

XCL1 XCL2

Lymphotactin a, SCM-1a Lymphotactin b, SCM-1b

XCR1 XCR2

CX3C (d)

CX3CL1

Fractalkine, neurotactin (mouse)

CX3CR1

sometimes referred to as a, b, g, and d chemokines, respectively. Aside from the official nomenclature, chemokines can be divided on the basis of their biological functions into inflammatory chemokines, homeostatic chemokines, and dual chemokines (Table 2). Inflammatory/inducible chemokines are expressed in inflamed tissues in response to proinflammatory

cytokines or pathogens. They orchestrate innate and adaptive immune responses via recruitment of effector leukocytes in infection, inflammation, tissue injury, and tumors. By contrast, homeostatic/ constitutive chemokines are involved in immune surveillance through regulation of lymphocyte and dendritic cell trafficking. They guide leukocytes during hematopoiesis in the bone marrow and thymus,

358 CHEMOKINES Table 2 Inflammatory, homeostatic, and dual chemokines Chemokines

Receptors

Functions

Inflammatory CXCL6 CXCL8 CXCL1 CXCL2 CXCL3 CXCL5 CX3CL1 CCL2 CCL3 CCL5 CCL7 CCL8 CCL11 CCL13 CCL14 CCL15 CCL24 CCL26 CCL27 CCL28 XCL1 XCL2

CXCR1, CXCR2 CXCR1, CXCR2 CXCR2 CXCR2 CXCR2 CXCR2 CX3CR1 CCR2 CCR1, CCR5 CCR1, CCR3, CCR5 CCR1, CCR2, CCR3 CCR3 CCR3 CCR2, CCR3 CCR1 CCR1, CCR3 CCR3 CCR3 CCR10 CCR10 XCR1 XCR1

Innate immunity

Homeostatic CXCL12 CXCL13 CXCL14 CCL18 CCL19 CCL21

CXCR4 CXCR5 Unknown Unknown CCR7 CCR7

Hematopoiesis Homeostasis of leukocytes Development of antigen-presenting cells T cell–dendritic cell interaction (spleen, lymph node) T lymphopoiesis Spleen and lymph node T cell homing

Dual function CXCL9 CXCL10 CXCL11 CXCL16 CCL1 CCL17 CCL20 CCL22 CCL25

CXCR3 CXCR3 CXCR3 CXCR6 CCR8 CCR4 CCR6 CCR4 CCR9

T lymphopoiesis Adaptive immunity (Th1 responses)

Extravasation Innate and adaptive immunity

T lymphopoiesis, extravasation T lymphopoiesis, adaptive immunity T lymphopoiesis Development of dendritic cells, adaptive immunity Adaptive immunity (cutaneous T cells) T lymphopoiesis, adaptive immunity, T cell and B cell trafficking in small intestine

Adapted from Moser B, Wolf M, Walz A, and Loetscher P (2004) Chemokines: multiple levels of leukocyte migration control. Trends in Immunology 25: 75–84.

during initiation of adaptative immune responses in the spleen, lymph nodes, and Peyer’s patches, and in immune surveillance of healthy peripheral tissues. Sharing both functions are the ‘dual chemokines’, which participate in immune defense functions and also target noneffector leukocytes, such as precursor and resting mature leukocytes, at sites of leukocyte development and immune surveillance. Genes encoding inflammatory chemokines are found in two major clusters on human chromosomes 4 (CXC chemokines) and 17 (CC chemokines), while genes for homeostatic chemokines are located alone

or in small clusters on chromosomes 1, 2, 5, 7, 9, 10, and 16.

Chemokine Receptors Human formyl-peptide receptor was the first chemokine receptor cloned in 1990. Two human receptors for CXCL8 (IL-8) were subsequently cloned in 1991: CXC chemokine receptor 1 (CXCR1) and CXCR2. As of today, 19 receptors have been identified (Table 1). The protein sequences of chemokine receptors share 25% to 80% amino acid identity,

CHEMOKINES 359

which suggests a common ancestor. All chemokine receptors are G-protein-coupled receptors (GPCRs) with seven transmembrane domains. However, chemokine receptors share a few features that are less commonly found in other GPCRs. They include a length of 340 to 370 amino acids; an acidic N-terminal segment; the sequence DRYLAIVHA (or a variation of it) in the second intracellular loop; a short basic third intracellular loop; and a cysteine in each of the four extracellular domains. An additional characteristic is the presence of a tyrosine sulfation in the N-terminus, which is critical for CCR5 activity as an HIV co-receptor. Even though the three-dimensional structure of chemokine receptors is unknown, the presence of seven transmembrane domains was inferred from the structure of rhodopsine. However, the presence of extracellular and intracellular loops is more speculative. It is commonly accepted that chemokine receptors contain seven transmembrane domains separated by amino acid loops directed toward the outside (extracellular loops) and inside (intracellular loops) of the cell (Figure 1). While extracellular domains serve as binding sites for the chemokine ligands, intracellular loops interact with G proteins and scaffolding proteins to trigger a cascade of intracellular changes (see chemokine–chemokine receptor interactions). The ligand-binding site of chemokine receptors is highly complex. It is composed of multiple noncontiguous domains and at least two distinct subtypes: one for docking and the other for triggering.

Extracellular space NH2

Cell membrane

IV

III II

Cytoplasm

V

I

VI VII

The classification systems of chemokines and chemokine receptors parallel each other. As illustrated in Table 1, human CC and CXC chemokine receptor names consist of the CCR and CXCR roots (where R stands for receptor) followed by numbers. Similarly, CC and CXC chemokine names consist of the CCL and CXCL roots (where L stands for ligand) followed by numbers. The lymphotactin and fractalkine receptors are named XCR1 and CX3CR1, respectively. A chemokine receptor has a distinct chemokine and leukocyte specificity, but may bind multiple chemokines. Reciprocally, a chemokine may bind multiple receptor subtypes. However, a number of chemokine–chemokine receptor relationships are monogamous (SDF-1/CXCR4, TECK/CCR9, BCA1/CXCR5, MIP-3a/CCR6, lymphotactin/XCR1, fractalkine/CX3CR1) (where SDF represents stromal cell-derived factor, TECK represents thymus-expressed chemokine, BCA represents B-cell attracting chemokine). Importantly, distinct receptor subtypes specific for the same chemokine and same function can be coexpressed on the same cell. In addition, some nonchemokine ligands (including several HIV proteins) can bind chemokine receptors as well. For example, CCR3 and mainly CCR5 and CXCR4, serve as co-receptors for HIV infection of CD4 þ cells.

Chemokine–Chemokine Receptor Interaction Chemokine Binding

Chemokine receptors are allosteric molecules, which means that their conformation can be modified by the interaction with another molecule (ligand), allowing further binding to a third molecule (G-protein). More specifically, chemokine binding to the extracellular domains of the chemokine receptor triggers a change in tertiary structure of the receptor. This modification allows the intracellular domains to bind and activate heterotrimeric G-proteins. In response, the activated G-proteins exchange guanosine diphosphate for guanosine triphosphate (GTP), and dissociate into a- and bg-subunits. Chemokine receptors can couple to several Ga isotypes. For example, chemotaxis, the main function of most chemokines, requires coupling of the receptor to Gai, a Ga isotype sensitive to pertussis toxin. This was demonstrated by the complete inhibition of cell migration following cell treatment with pertussis toxin.

COOH

Figure 1 Schematic structure of a chemokine receptor. The seven transmembrane domains and the extracellular and intracellular loops are represented.

Intracellular Signaling

The bg-subunits that dissociate from Gai mediate chemokine-induced signals by activating the

360 CHEMOKINES

phosphoinositide-3 kinases (PI3K), which produce phosphoinositol-3, 4, 5-triphosphate (PIP3). Chemokine stimulation triggers a change in leukocyte shape associated with the formation of an elongated edge, or pseudopod. PIP3K and its product PIP3 translocate to the pseudopod, where they colocalize with GTPase Rac. PIP3 not only activates Rac via specific guanine nucleotide exchange factors (GEFs), but also serves as a docking site for protein kinase B, which translocates to the pseudopod and can induce actin polymerization. Rac, a protein required for leukocyte migration, activates downstream effectors (p21-activated kinase, and WAVE), which stimulate actin-related protein (Arp) 2/3. The actin polymerization induced by Arp allows further pseudopod extension. In moving leukocytes, formyl peptide activates a different set of mediators. Parallel to the sequence of events triggering Rac activation, another pathway leads from the pertussis toxin-sensitive Ga12/13 to the activation of Rho by Rho GEFs at the uropod (extended pole of the leukocyte opposite to the pseudopod). Activated Rho induces focal activation of myosin II and contraction of actin–myosin complexes via its effector p160ROCK, a serine/threonine kinase. This process triggers the retraction of the pseudopod. The Rac and Rho pathways, which occur at opposite edges of the leukocyte, inhibit each other. This mutual inhibition is critical to induce and maintain functional and morphological cell polarity, and drive locomotion. At least one additional signaling pathway remains to be characterized. It is the one leading to the rapid integrin activation involved in leukocyte–endothelial cell adhesion. Chemokine Receptor Silencing

Chemokine receptor signaling is a transient process, due to the presence of GTPase activity in the Ga subunits. GTP hydrolyzation catalyzed by GTPase induces the reunion of Ga with Gbg, allowing the return to the initial conformation of inactive heterotrimers. Molecules called regulators of G-protein signaling can, depending on the Ga isotype, either stimulate or inhibit the GTPase activity of the Ga subunits. Chemokine receptor silencing can also be mediated by desensitization triggered by receptor phosphorylation by G-protein-coupled receptor kinases, and by downregulation induced by receptor sequestration and internalization. Chemokine Receptor-Independent Pathways

Alternative GPCR-independent (and chemokine receptor-independent) signaling pathways result from

the binding of sulfated sugars of the glycosamine family to a few chemokines, such as CCL5 and CXCL4.

Biological Functions of Chemokines At least four major biological functions can be attributed to chemokines: leukocyte chemoattraction, integrin activation, leukocyte degranulation, and angiogenesis or angiostasis. Chemoattraction

Chemokines attract leukocytes that bear the appropriate chemokine receptors via formation of a chemokine gradient. This gradient acts as a road map for the migrating cells, guiding them toward their final destination. As described earlier (see chemokine– chemokine receptor interactions), the signaling of cell movement by chemokine receptors is a complex phenomenon and is not yet fully elucidated. Integrin Activation

Leukocyte migration involves passage from the tissues to the blood and lymphatic vessels and from the vessels to the tissues (extravasation). Cells undergo a multistep process to bind the vessel endothelium. They tether to vessel walls via activation of selectins, then bind the endothelium via activation of integrins, which are able to regulate their affinity through rapid conformational change. This conformational change in the integrin structure leads to cell arrest, a mechanism that allows cells to subsequently cross the endothelium. Chemokines can activate integrins, and are involved in the multistep process for neutrophils, T cells, eosinophils, and monocytes. Leukocyte Degranulation and Mediator Release

Chemokines can stimulate leukocyte degranulation or release of inflammatory mediators, which can be features of allergic reactions. For example, CCL2 (MCP-1) stimulates histamine release by basophils and mast cells, and CXCL8 (IL-8) stimulates neutrophil granule exocytosis. In addition, chemokines stimulate the macrophage respiratory burst leading to the release of reactive oxygen intermediates. Angiogenesis or Angiostasis

The formation of new blood vessels (angiogenesis) is a physiological feature associated with embryonic development, wound healing, chronic inflammation, and the growth of malignant tumors. Some chemokines (mostly CXC chemokines) promote angiogenesis, while others mediate its inhibition (angiostasis).

CHEMOKINES 361

As stated earlier (see section ‘Chemokine structure and nomenclature’), the angiogenic property of the CXC chemokines correlates with the presence of the tripeptide motif glutamic acid–leucine–arginine (ELR). Proangiogenic chemokines include the ELR þ CXC chemokines CXCL1, 2, 3, 5, 6, 7, 8, and 12, and CCL2 (MCP-1). Antiangiogenic chemokines include the ELR– CXC chemokines CXCL 4, 9, 10, 11, and 13, and CCL21 (SLC)(SLC – secondary lymphoid-tissue chemokine). As we will see later, the angiogenic and angiostatic properties of chemokines are of major relevance in lung cancer and inflammation. The four biological functions reviewed above are used in combination for various biological responses. For example, tumor rejection involves leukocyte chemotaxis to the tumor and angiostasis; allergic reactions involve leukocyte migration and degranulation.

Chemokines in Respiratory Diseases Asthma and Airway Allergy

Asthma is characterized by airway hyperresponsiveness (AHR) and T-helper-2 (Th2) cell, eosinophil, and mast cell infiltration (see Asthma: Overview; Extrinsic/Intrinsic). A number of chemokines and receptors are associated with the development of asthma (Table 3). It is important to bear in mind that most studies on chemokines in asthma are based on the use of mouse models. Although these murine models are useful tools, their relevance to the human disease is not absolute, due partly to species differences. Moreover, mouse models are characterized by acute allergic reactions, whereas human asthma is a chronic disease. Finally, differences may arise between the various murine models, in terms of the cell populations and chemokines involved in the allergic response, and the relevance of a clearance mechanism. Eotaxins are a family of chemokines that attract eosinophils. Three forms (CCL11 or eotaxin, CCL24 or eotaxin-2, and CCL26 or eotaxin-3) were identified in humans, whereas two forms only were identified in mice. CCL11 (eotaxin) is a potent chemoattractant of eosinophils. It binds exclusively to the CCR3 receptor, which is expressed on the surface of eosinophils, mast cells, and basophils. Neutralization of CCL11 reduces both airways inflammation and AHR, via reduction of eosinophil and Th2 trafficking. However, mice genetically deficient in CCL11 were only partially protected in models of allergic airway inflammation, which supports the idea of functional redundancy between chemokines. Although the CCL11 receptor CCR3 has been regarded as a potential therapeutic target, a model of

CCR3-deficient (knockout) mouse gave new insights on the complexity of the mechanisms involved. CCR3 knockouts exhibited a dramatic reduction of eosinophil recruitment to the lung following allergen challenge. Unexpectedly though, AHR was increased in these mice, which was later attributed to an increased accumulation of mast cells in the trachea. An additional study showed a protection against allergen-induced AHR in CCR3-deficient mice in a model that did not involve mast cell recruitment. CCL17 (TARC) (TARC – thymus- and activationregulated chemokine) (see Chemokines, CC: TARC (CCL17)) CCL22 are chemokines that induce the selective migration of Th2 cells via binding to CCR4. Neutralization of either CCL17 or CCL22 leads to abrogation of lung eosinophilia and AHR in mice. Additional studies have confirmed that CCL22 and CCR4 contribute to the recruitment of Th2 cells to the lung during allergen-driven inflammation in mice and humans. CCL1 is the ligand to the CCR8 chemokine receptor expressed on Th2 cells, and the CCL1/CCR8 tandem has been suspected to mediate the asthmatic reaction. However, studies using CCL1 and CCR8 knockout mice gave controversial results. It is suggested that CCL1/CCR8 might be involved in eosinophil recruitment in some models, but might not be critical to Th2 recruitment to the lung in vivo. CXCR4 expression on leukocytes is widespread, and this receptor is involved in B and T lymphopoiesis. In addition, CXCR4 mediates Th2-cell trafficking during allergic reactions in mice, as shown in studies where CXCR4 blockade with either an antibody or a receptor antagonist resulted in abrogation of both airway inflammation and AHR. Increased levels of CCL2 (MCP-1), CCL7 (MCP3), and CCL13 (MCP-4) were observed in bronchoalveolar lavage (BAL) cells, and bronchial biopsies of asthmatic patients, but the role of these chemokines in asthma is still controversial (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)). Allergic diseases caused by the fungus Aspergillus fumigatus are collectively referred to as allergic bronchopulmonary aspergillosis (ABPA) in humans, and allergic aspergillosis or fungal asthma in rodents. ABPA is characterized by severe lung eosinophilia, increased mucous secretion, enhanced serum IgE production, and chronic lung fibrosis. Specific chemokines are associated with ABPA. CCR2 and its major ligand CCL2 (MCP-1) (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)) are the key factors in the clearance of A. fumigatus spores in mice. Genetic deficiency in either CCR2 or CCL12 leads to exacerbated allergic disease in mice. By contrast, the absence of the CXCL8 (IL-8) receptor

362 CHEMOKINES Table 3 Chemokines and chemokine receptors in respiratory diseases Clinical condition

Chemokines potentially involved

Chemokine receptors potentially involved

Asthma

CCL11 (eotaxin) CCL17 (TARC) CCL22 CCL1 CCL2 (MCP-1) CCL7 (MCP-3) CCL13 (MCP-4) CCL5 (RANTES) CXCL8 (IL-8)

CCR3 CCR4 CCR4 CCR8 CXCR4 CCR2 CCR2 CCR2 CCR5 CXCR2

Chronic obstructive pulmonary disease

CXCL8 (IL-8) CCL2 (MCP-1) CXCL10 (IP-10)

CXCR2 CCR2 CXCR3

Acute respiratory distress syndrome

CCL2 (MCP-1) CXCL8 (IL-8) CXCL1 (GROa) CXCL2 (GROb) CXCL5 (ENA-78) KC (mouse CXCL1) CCL5 (RANTES)

CCR2 CXCR2 CXCR2 CXCR2 CXCR2 CXCR2 CCR1, CCR3, CCR5

Idiopathic pulmonary fibrosis

CCL2 (MCP-1) CCL3 (MIP-1a) CCL4 (MIP-1b) CXCL2 (GROb)

CCR2 CCR5 CCR5 CXCR2

Acute lung allograft rejection

CCL5 (RANTES) CCL2 (MCP-1) CXCL9 (Mig)

CCR1, CCR3, CCR5 CCR2 CXCR3

Graft-versus-host disease

CCL2 (MCP-1) CCL3 (MIP-1a) CCL5 (RANTES) CXCL9 (Mig) CXCL10 (IP-10)

CCR2 CCR5 CCR5 CXCR3 CXCR3

Tuberculosis

CCL2 (MCP-1) CCL3 (MIP-1a) CCL5 (RANTES) CCL7 (MCP-3) CCL12 (MCP-5, mouse) CXCL5 (ENA-78) CXCL8 (IL-8) CXCL10 (IP-10)

CCR2 CCR1, CCR5 CCR1, CCR3, CCR5 CCR1, CCR2, CCR3 CCR2 CXCR2 CXCR1, CXCR2 CXCR3

Respiratory syncytial virus infection

CCL5 (RANTES) CCL2 (MCP-1) CCL3 (MIP-1a) CCL4 (MIP-1b) CXCL8 (IL-8) CX3CL1 (fractalkine)

CCR1, CCR3, CCR5 CCR2 CCR1, CCR5 CCR5 CXCR1, CXCR2 CXCR1

Lung cancer

CXCL5 (ENA-78) CXCL9 (Mig) CXCL10 (IP-10) CXCL11 (I-TAC) CXCL12 (SDF-1)

CXCR2 CXCR3 CXCR3 CXCR3 CXCR4

CHEMOKINES 363

CXCR2 or of CCR4 in mice enhances fungal clearance dramatically. CCL5 (RANTES) and its receptor CCR5 inhibit the innate response of alveolar macrophages against A. fumigatus. Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) (see Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General; and Smoking Cessation) is characterized by neutrophil, macrophage, and CD8 þ T-cell infiltration. CXCL8 is found in very high concentrations in the sputum of COPD patients, and these levels correlate with disease severity. In addition, CXCL8 neutralization by blocking antibodies reduces the chemotactic response of neutrophils to sputum from COPD patients. CXCL8 activates neutrophils via the CXCR1 and CXCR2 receptors. CXCR2 is also expressed on monocytes, which makes it a more attractive therapeutic target than CXCR1. Smallmolecule inhibitors of CXCR2 are currently being tested in clinical trials. The expressions of CCL2 (MCP-1) and its receptor CCR2 are increased in macrophages and epithelial cells of COPD patients. The CCL2/CCR2 couple might play a role in the recruitment of blood monocytes to the lung. Similarly, the expressions of some CXCR3 ligands, such as CXCL10 (IP-10), are elevated in CD8 þ T cells in peripheral airways of COPD patients.

levels correlate with neutrophil concentration in BAL, but not with the severity of lung injury or subsequent clinical course. CCL2 is detectable in BAL of ARDS patients at the onset of ARDS, and persists in the lungs of patients with sustained ARDS. CCL2 concentrations are increased in ARDS patients with sepsis and shock, and this augmentation correlates with increased survival. No correlation was found between CCL2 levels and leukocyte numbers, which suggests that CCL2 may play another function than promoting monocyte recruitment, in ARDS. A number of animal models have been used to mimic ARDS, resulting in a short list of chemokines potentially involved in the disease. In a model of ischemia–reperfusion injury, CXCL5 production correlated with the occurrence of neutrophil-dependent lung injury, and treatment with anti-CXCL5 antibodies were able to attenuate lung damage. In a model of bacterial pneumonia, depletion of CXCL2 was associated with a higher mortality, suggesting a protective role for that chemokine in ARDS. Similarly, lung-specific transgenic expression of KC, the rodent homolog of human CXCL1, was shown to enhance resistance to Klebsiella-induced pneumonia in mice. CCL5 (RANTES) mediates T cell and monocyte chemotaxis via the CCR1, CCR3, and CCR5 receptors (see Chemokines, CC: RANTES (CCL5) for review). In a murine model of pancreatitis-associated lung injury, treatment with the CCL5 (RANTES) receptor antagonist Met-RANTES protected against lung damage but provided little or no protection against pancreatitis.

Acute Respiratory Distress Syndrome

Acute (or adult) respiratory distress syndrome (ARDS) is characterized by the rapid onset of severe respiratory failure due to injury of the lung alveoli and the surrounding capillaries. ARDS can be triggered by clinical events as diverse as major surgery, trauma, multiple transfusions, severe burns, pancreatitis, and sepsis. Several chemokines have been found in increased concentrations in the BAL fluid from patients with established, or at risk for, ARDS. They include CCL2 (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)), and the CXCR2 ligands CXCL8 (which also bind CXCR1), CXCL1 (GROa) (GRO – growth-related oncogene), CXCL2 (GROb) (see Chemometrics, CXC: CXCL1 (GRO1)–CXCL3 (GRO3)) and CXCL5 (ENA-78) (ENA – epithelial neutrophil-activating protein). Levels in CXCL8 were reportedly higher in patients with ARDS plus pneumonia, than in those with ARDS or pneumonia alone. In patients with established ARDS, CXCL8

Idiopathic Pulmonary Fibrosis

The BAL levels of CCL2 (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)), CCL3, and CCL4 were increased in patients with idiopathic pulmonary fibrosis (IPF), although the expression of CCR5 (which binds CCL3 and CCL4) was markedly reduced in lymphocytes from IPF patients. This is in agreement with the hypothesis of a downregulation of the Th1 response in IPF. CXCL2 concentration was also elevated in the BAL of IPF patients. Acute Lung Allograft Rejection

Acute lung allograft rejection is a major complication of lung transplantation and can lead to bronchiolitis obliterans syndrome (BOS), which is characterized by the progressive obliteration of the small airways. Several chemokines were detected in elevated concentrations in the BAL of patients with acute lung

364 CHEMOKINES

allograft rejection or BOS. They include CCL5 (RANTES), CCL2, CXCL9 (see Chemokines, CXC: CXCL9 (MIG)), and CXCL8. In a rat model, in vivo neutralization of either CCL5 or CXCR9 attenuated acute lung allograft rejection via a decrease in mononuclear cell recruitment, and (in the case of CXCL9) a reduction in the expression of the CXCL9 receptor CXCR3. In a murine model, loss of CCL2/CCR2 signaling reduced the recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)). Graft-versus-Host Disease

Graft-versus-host disease (GVHD) is a complication of allogeneic bone marrow transplantation (allo-BMT), in which the immune cells of the donor’s bone marrow attack the recipient’s organs and tissues, particularly the skin, eyes, stomach, intestines, liver, and lung. In the lung, GVHD can be associated with idiopathic pneumonia syndrome (IPS), an inflammatory disease characterized by diffuse interstitial pneumonitis and alveolitis leading to interstitial fibrosis. Host macrophages are recruited to the lung during the peritransplantation period, and thought to contribute to alloantigen presentation to donor T cells infiltrating the lung, and to lung damage via cytokine production. Several studies using mouse models have focused on the role of chemokines in the recruitment of effector cells to the lung during GVHD and IPS. The chemokines expressed in the lung after allo-BMT include CCL2, CCL3, CCL4, CCL5, CCL11, and the CXCR3 ligands CXCL9 (see Chemokines, CXC: CXCL9 (MIG)), CXCL10, and CXCL11. The roles of these chemokines differ, depending on when they are evaluated after allo-BMT. For example, CCL2, CXCL10, and CXCL11 are expressed prior to CCL3 and CCL4. Neutralization of CXCL9 and CXCL10 with antibodies reduced IPS severity and CD8 þ T-cell infiltrates. Further inhibition occurred with the elimination of CXCR3 expression from the donor T cells, showing that CXCR3 is a key component of T-cell recruitment to the lung after allo-BMT. The CCL3/CCR5 couple also influences the recruitment of donor T cells to the lung, and a differential role was established for CCR5, depending on the conditioning (lethal irradiation versus no irradiation) of the recipient. The elimination of CCL5 expression from donor T cells inhibited their recruitment to the lung at late time points after transplantation, probably via a decrease in CCR5 expression.

The production of the CCR2 ligand CCL2 (MCP1) in the lung correlated with host macrophage recruitment, and elimination of CCR2 but not of CCL2 expression decreased IPS severity. Tuberculosis

Mycobacterium tuberculosis is a strong inducer of chemokine expression. Human macrophages produce CCL2, CCL3, CCL4, and CCL5 in response to virulent strains of M. tuberculosis. Additional in vitro studies suggest that the chemokines interacting with CCR1, CCR2, and CCR5 may mediate leukocyte recruitment to the site of M. tuberculosis infection. However, direct evidence for a role of these chemokines in tuberculosis is still missing. Expression of CXCL8 and CCL2, but not CCL3, CCL4, and CCL5 was detected in a human alveolar epithelial cell line in response to virulent strains of M. tuberculosis. Granulocytes from the blood of tuberculosis patients produced CXCL8 and CXCL1 in response to a M. tuberculosis antigen. CXCL10 concentration was elevated in the bronchial epithelium of tuberculosis patients, and BAL levels of CCL2, CCL5, CCL7, CCL12, CXCL8, and CXCL10 were increased in tuberculosis patients (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)). It was hypothesized that the increase in expression of the HIV co-receptors CCR5 and CXCL4 on M. tuberculosis-infected monocytes may result in increased productivity of HIV infection. Expression of CCL2, CCL3, CCL7, CCL12, CXCL5, and CXCL10 was observed in response to M. tuberculosis infection in mice. However, CCL2deficient mice were able to clear M. tuberculosis infection, despite an initial increase in bacterial burdens in the lung and spleen. Yet, mice deficient in the CCL2 receptor CCR2 were unable to control infection induced by high M. tuberculosis doses and succumbed. CCR5-deficient mice controlled M. tuberculosis aerosol and intravenous infection and formed granulomas. It is possible that CCR1 compensates for the absence of CCR5 in those mice. Finally, expression of chemokines by macrophages in response to M. tuberculosis appears to be influenced by the cytokine tumor necrosis factor (TNF)-a, which is a major product of M. tuberculosis infection in macrophages. Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age. Some RSV-infected lung epithelial cells were shown to express increased amounts of CC and

CHEMOKINES 365

CXC chemokines, including CCL1 (I-309), CCL20 (Exodus-1), CCL17 (TARC), CCL5 (RANTES), CCL2 (MCP-1), CCL22 (MDC) (MDC – macrophage-derived chemokine), CCL3 (MIP-1a), CCL4 (MIP-1b), CXCL1 (GROa), CXCL2 (GROb), CXCL3 (GROg), CXCL5 (ENA-78), CXCL8, CXCL11 (I-TAC) (I-TAC – IFN-g-inducible T-cell a-chemoattractant), and CX3CL1 (fractalkine). The expression of CCL5, which mediates eosinophil and monocyte recruitment to the lung, is frequently enhanced during RSV infection in patients and murine models. Productions of CCL2, CCL3, and CCL4 are elevated in monocytes and eosinophils exposed to RSV. Infection of eosinophils with RSV leads to the release of CCL5, and CCL3, which is a chemoattractant for human blood eosinophils. CCL3 levels were found highly elevated in the respiratory tract of children with RSV-associated severe bronchiolitis. CXCL8 is a powerful chemoattractant for neutrophils, the main inflammatory cell type to migrate to the lung during RSV infection. CXCL8 expression is markedly increased during RSV infection. A surface protein of RSV exhibits mimicry with the chemokine CX3CL1, and binds the CX3CR1 receptor, which induces leukocyte migration and facilitates RSV infection of cells. Lung Cancer

Cell transformation from preneoplastic to neoplastic, tumor growth, invasion, and metastases are events that depend on the formation of new blood vessels (angiogenesis). Angiogenesis is the product of an imbalance in the expression of angiogenic versus angiostatic factors. As noted earlier, ELR þ CXC chemokines promote angiogenesis, while ELR
CXC chemokines mediate angiostasis. ELR þ CXC chemokines are important mediators of angiogenesis during tumorigenesis of non-small cell lung cancer (NSCLC), and their presence correlates with patient prognosis. In a murine model, CXCL5 showed a high degree of correlation with NSCLC-derived angiogenesis. By contrast, the ELR– CXC chemokines CXCL4, CXCL9, CXCL10, and CXCL11 are angiostatic factors. The angiostatic action of CXCL9, CXCL10, and CXCL11 in NSCLC is mediated via CXCR3. In addition, CXCL12 plays important roles in lung cancer invasion and metastasis, and in the spread of

lung metastases to other organs (see Chemokines, CXC: CXCL12 (SDF-1)). See also: Asthma: Overview; Extrinsic/Intrinsic. Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12); RANTES (CCL5); TARC (CCL17). Chemokines, CXC: CXCL12 (SDF-1); CXCL9 (MIG); CXCL1 (GRO1)–CXCL3 (GRO3). Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General; Smoking Cessation.

Further Reading Algood HMS, Chan J, and Flynn JAL (2003) Chemokines and tuberculosis. Cytokine & Growth Factor Reviews 14: 467–477. Gerard C and Rollins BJ (2001) Chemokines and disease. Nature Immunology 2: 108–115. Hoffman SJ, Laham FR, and Polack FP (2004) Mechanisms of illness during respiratory syncytial virus infection: the lungs, the virus and the immune response. Microbes and Infection 6: 767– 772. Humbles AA, Lu B, Friend DS, et al. (2002) The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inflammation and hyperresponsiveness. Proceedings of the National Academy of Science, USA 99: 1479–1484. Lloyd CM and Rankin SM (2003) Chemokines in allergic airway disease. Current Opinion in Pharmacology 3: 443–448. Mackay CR (2001) Chemokines: immunology’s high impact factor. Nature Immunology 2: 95–101. Moser B, Wolf M, Walz A, and Loetscher P (2004) Chemokines: multiple levels of leukocyte migration control. Trends in Immunology 25: 75–84. Murphy PM (2002) International union of pharmacology. XXX. Update on chemokine receptor nomenclature. Pharmacological Reviews 54: 227–229. Puneet P, Moochhala S, and Bathia M (2005) Chemokines in acute respiratory distress syndrome. American Journal of Physiology Lung Cellular and Molecular Physiology 288: L3–L15. Rosenkilde ME and Schwartz TW (2004) The chemokine system – a major regulator of angiogenesis in health and disease. APMIS 112: 481–495. Rot A and von Andrian UH (2004) Chemokines in innate and adaptive host defense: basic chemokine grammar for immune cells. Annual Reviews in Immunology 22: 891–928. Schuh JM, Blease K, Kunkel SL, and Hogaboam CM (2003) Chemokines and cytokines: axis and allies in asthma and allergy 14: 503–510. Strieter RM, Belperio JA, Burdick MD, et al. (2004) CXC chemokines: angiogenesis, immunoangiostasis and metastases in lung cancer. Annals of the New York Academy of Science 1028: 351–360. Thelen M (2001) Dancing to the tune of chemokines. Nature Immunology 2: 129–134. Wysocki CA, Panoskaltsis-Mortari A, Blazar BR, and Serody JS (2005) Leukocyte migration and graft-versus-host disease. Blood 105: 4191–4199.

366 CHEMOKINES, CC / C10 (CCL6)

CHEMOKINES, CC Contents

C10 (CCL6) MCP-1 (CCL2)–MCP-5 (CCL12) RANTES (CCL5) TARC (CCL17) TECK (CCL25)

C10 (CCL6) R M Strieter and M P Keane, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

IL-3, IL-4, IL-13, GM-CSF, SCF Pulmonary fibrosis +

& 2006 Elsevier Ltd. All rights reserved.

CCL6

Abstract CCL6 is a CC chemokine that was cloned in the mouse in 1991. Although a human homolog has not been described to date, this novel chemokine shares significant homology with the human CC chemokine, CCL15. In contrast to the other members of the CC chemokine family, CCL6 has four instead of three exons. CCL6 is produced by macrophages, fibroblasts, keratinocytes, vascular smooth muscle cells, and skeletal muscle. CCL6 is chemotactic for monocytes, macrophages, and T cells. CCL6 has been shown to be present in a variety of chronic inflammatory disorders. High expression of CCL6 has been found in the cerebellum and spinal cord of mice with experimental autoimmune encephalitis. Furthermore, intracerebroventricular injection of CCL6 protein promotes the recruitment of large number of macrophages. CCL6 has an important role in the development of pulmonary fibrosis and it mediates some of the pro-fibrotic effects of interleukin-13 (IL-13). CCL6 also has an important role in the airway remodeling seen in animal models of asthma. Interestingly, CCL6 has a protective effect in animal models of peritoneal sepsis and it improves macrophage phagocytic functions. Furthermore, CCL6 has been shown to have a role in tumor growth and invasion.

M
, T cells

− IFN-

Airway remodeling Bronchial hyperreactivity

Figure 1 Regulation and function of CCL6 in respiratory disease.

CCL6 is a CC chemokine that was cloned in the mouse in 1991. It was identified along with a series of other hematopoietic specific mRNAs in mouse bone marrow cultures stimulated with granulocytemacrophase colon-stimulating factor (GM-CSF). Although a human homolog has not been described to date, this novel chemokine shares significant homology with the human CC chemokine, CCL15. Both of these chemokines are chemotactic for monocytes and T cells.

heparin-binding proteins. The chemokines display four highly conserved cysteine amino acid residues. The CC chemokine family has the first two NH2-terminal cysteines in juxtaposition, and designated the CC cysteine motif. In contrast to the other members of the CC chemokine family, CCL6 has a novel second exon. With the other members of the CC family, the second exon contains the first three of four highly conserved cysteine residues and the third exon contains the fourth. In contrast, exons 3 and 4 of CCL6 contain the four conserved cysteines distributed in a similar manner to exons 2 and 3 of the other CC chemokines. The novel second exon of CCL6 codes for a large number of charged amino acids. The CCL6 gene (Scya6) is closely lined to the CCL2 gene (Scya2) locus on mouse chromosome 11. The genes for all of the other murine CC chemokines are also located on chromosome 11, suggesting that they all evolved from the same ancestral gene. The primary translation product of CCL6 contains 116 amino acids including an amino terminal signal peptide. CCL6 has an amino terminal region of 28 amino acids, which is in contrast to the other CC chemokines that have an amino terminal region length of 9–10 amino acids.

Structure

Regulation of Production and Activity

Chemokines in their monomeric form range from 7 to 10 kDa and are characteristically basic

CCL6 is expressed in hematopoietic cells, fibroblasts, keratinocytes, vascular smooth muscle cells, and

Introduction

CHEMOKINES, CC / C10 (CCL6) 367

skeletal muscle and is stimulated by exposure to interleukin-3 (IL-3), IL-4, IL-13, and GM-CSF (see Figure 1). Furthermore, C10 is differentially regulated by T-helper-1 (Th1) and T-helper-2 (Th2) cytokines. Bone-marrow-derived macrophages produce CCL6 in response to IL-4, IL-10, and IL-13 in a dose-dependent manner. In contrast, interferon gamma (IFN-g) inhibits IL-3- and GM-CSF-induced expression of CCL6. Stem cell factor has been shown to induce CCL6 from eosinophils.

Biological Function CCL6 is chemotactic for monocytes, macrophages, and T cells. CCL6 has been shown to be present in a variety of chronic inflammatory disorders. High expression of CCL6 has been found in the cerebellum and spinal cord of mice with experimental autoimmune encephalitis. Furthermore, intra-cerebroventricular injection of CCL6 protein promotes the recruitment of large number of macrophages. In a model of chronic peritoneal inflammation, there is delayed (24 h) but sustained elevation in CCL6 levels in peritoneal fluid. These findings demonstrate an important role for CCL6 in chronic inflammation and specifically macrophage recruitment. Interestingly, despite its proinflammatory actions, CCL6 has been shown to improve survival in a murine model of septic peritonitis. Following cecal ligation and puncture, there is a significant increase in levels of CCL6 over baseline, and neutralization of CCL6 leads to increased mortality. In contrast, administration of systemic CCL6 immediately after surgery leads to a significant improvement in survival. This is associated with an increase in TNF-a and CCL2. CCL6 and IL-1 can enhance TNF-a secretion from peritoneal macrophages and IL-13 induces CCL6 from these peritoneal macrophages. CCL6 also enhances the bacterial phagocytic capability of peritoneal macrophages and leads to a significant decrease in bacteremia. In similar murine models of septic peritonitis Stat6
/
mice are protected from lethality, and this is associated with increased peritoneal lavage levels of CCL6, CCL22, IL-12, and TNF-a. This in turn leads to enhanced bacterial clearance. Further support for the important role in mononuclear cell recruitment is seen in a model of skin healing. CCL6 has been shown to be strongly expressed in full-thickness excisional skin wounds as early as day 1 and to be localized to macrophages of the granulation tissue and the keratinocytes at the wound edge, suggesting an important role in the strong macrophage influx that is seen in healing skin. Furthermore, CCL6 has been shown to be expressed

in skeletal muscle and may contribute to the mononuclear cell influx in muscle tissue that is associated with muscular dystrophy. In addition to its chemotactic properties, CCL6 also promotes local tissue apoptosis, thereby facilitating tumor invasion in a murine cancer model. CCL6 also directly stimulates tumor growth and the leukemogenic phenotype in 32D cells.

Receptors The receptor for CCL6 is not known. Interestingly, deletion of CCR1 leads to a similar effect on IL-13induced inflammation as neutralization of CCL6, suggesting that CCL6 may signal through CCR1.

CCL6 in Respiratory Disease CCL6 is elevated in the pathogenesis of bleomycininduced pulmonary fibrosis. Similarly both IL-4 and IL-13, which are potent inducers of CCL6, are elevated during the pathogenesis of bleomycin-induced pulmonary fibrosis. Neutralization of IL-13, but not IL-4, attenuates the development of fibrosis and levels of CCL6. This is consistent with the known important role of IL-13 in the development of pulmonary fibrosis. Furthermore, neutralization of CCL6 also attenuates bleomycin-induced pulmonary fibrosis and intrapulmonary macrophage numbers. This suggests that in addition to its direct effect on fibroblasts, IL-13 has a role in the development of pulmonary fibrosis via the induction of CCL6 and the subsequent recruitment of macrophages. Further support for the role of CCL6 in lung remodeling and repair is seen in studies using IL-13 transgenic mice. The treatment of IL-13 þ / þ mice with anti-CCL6 leads to decreased levels of mRNA for matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-4 (TIMP-4). Furthermore, neutralization of CCL6 leads to a decrease in the ability of IL-13 to stimulate production of CCL2, CCL3, MMP-2, MMP-9, and cathepsins K, L, and S. Interestingly, deletion of CCR1 leads to a similar effect on IL-13-induced inflammation as neutralization of CCL6 suggesting that CCL6 may signal through CCR1. In a murine model of fungal-induced asthma, there is a significant increase in CCL6 in bronchoalveolar lavage (BAL), and this is significantly higher than levels of CCL2, CCL3, or CCL11. In this model the major source of CCL6 is alveolar macrophages, fibroblasts, and vascular smooth muscle cells. Neutralization of CCL6 prior to intratracheal challenge with Aspergillus fumigates leads to decreased airway inflammation and bronchial hyperresponsiveness although it had no effect on IL-10 or

368 CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12)

IgE levels. In a chronic model of fungal allergic airway disease, CCR1
/
mice have significantly lower levels of CCL6, CCL11, CCL22, and IL-4 and IL-13 at 30 days as compared to CCR1 þ / þ mice. This is associated with a decrease in subepithelial fibrosis and fewer numbers of goblets cells. Interestingly, there is no difference in airway hyperresponsiveness at 30 days, suggesting that the Th2-inducible chemokines are most important in the airway remodeling of asthma rather than the bronchial hyperreactivity. See also: Asthma: Overview; Allergic Bronchopulmonary Aspergillosis. Chemokines. Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12). Extracellular Matrix: Basement Membranes; Degradation by Proteases. Interleukins: IL-6. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Monocytes; T cells. Matrix Metalloproteinases. Pulmonary Fibrosis. Transgenic Models. Tumors, Malignant: Overview.

Further Reading

Orlofsky A, Wu Y, and Prystowsky MB (2000) Divergent regulation of the murine CC chemokine C10 by Th(1) and Th(2) cytokines. Cytokine 12: 220. Steinhauser ML, Hogaboam CM, Matsukawa A, et al. (2000) Chemokine C10 promotes disease resolution and survival in an experimental model of bacterial sepsis. Infection and Immunity 68: 6108. Wang W, Bacon KB, Oldham ER, and Schall TJ (1998) Molecular cloning and functional characterization of human MIP-1 delta, a new C–C chemokine related to mouse CCF-18 and C10. Journal of Clinical Immunology 18: 214. Yi F, Jaffe R, and Prochownik EV (2003) The CCL6 chemokine is differentially regulated by c-Myc and L-Myc, and promotes tumorigenesis and metastasis. Cancer Research 63: 2923. Zhu Z, Ma B, Zheng T, et al. (2002) IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13induced inflammation and remodeling. Journal of Immunology 168: 2953.

MCP-1 (CCL2)–MCP-5 (CCL12) A D Luster, W K Hart, and A M Tager, Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Asensio VC, Lassmann S, Pagenstecher A, et al. (1999) C10 is a novel chemokine expressed in experimental inflammatory demyelinating disorders that promotes recruitment of macrophages to the central nervous system. American Journal of Pathology 154: 1181. Berger MS, Kozak CA, Gabriel A, and Prystowsky MB (1993) The gene for C10, a member of the beta-chemokine family, is located on mouse chromosome 11 and contains a novel second exon not found in other chemokines. DNA and Cell Biology 12: 839. Berger MS, Taub DD, Orlofsky A, et al. (1996) The chemokine C10: immunological and functional analysis of the sequence encoded by the novel second exon. Cytokine 8: 439. Hogaboam CM, Gallinat CS, Taub DD, et al. (1999) Immunomodulatory role of C10 chemokine in a murine model of allergic bronchopulmonary aspergillosis. Journal of Immunology 162: 6071. Keane MP, Belperio JA, Henson PM, and Strieter RM (2005) Inflammation, injury and repair. In: Nadel M, et al. (eds.) Textbook of Respiratory Medicine, 4th edn. Philadelphia: Elsevier/ Saunders. Keane MP, Belperio JA, and Strieter RM (2003) Chemokines in pulmonary fibrosis. In: Strieter RM, Kunkel SL, Standiford TJ, and Lenfant C (eds.) Chemokines in the Lung, Lung Biology in Health and Disease, vol. 172. New York: Decker. Keane MP, Belperio JA, and Strieter RM (2003) Cytokine biology and the pathogenesis of interstitial lung disease. In: Schwarz MI and King TE (eds.) Interstitial Lung Disease, 4th edn. Hamilton, ON: BC Decker. Ma B, Zhu Z, Homer RJ, et al. (2004) The C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesis of IL-13-induced inflammation and remodeling. Journal of Immunology 172: 1872. Orlofsky A, Berger MS, and Prystowsky MB (1991) Novel expression pattern of a new member of the MIP-1 family of cytokine-like genes. Cell Regulation 2: 403. Orlofsky A, Lin EY, and Prystowsky MB (1994) Selective induction of the beta chemokine C10 by IL-4 in mouse macrophages. Journal of Immunology 152: 5084.

Abstract The attraction of leukocytes into tissues is essential for inflammation and host response to infection. This process is controlled by chemotactic cytokines known as chemokines. The monocyte chemoattractant proteins (MCP) are an important subfamily of chemokines that share structural as well as functional features. By activating various G-protein-coupled chemokine receptors, the MCPs induce the recruitment and activation of multiple subsets of leukocytes, including monocytes and macrophages, effector CD4 þ and CD8 þ T cells, B cells, dendritic cells, basophils, mast cells, natural killer cells, and eosinophils. Increased expression of MCP chemokines has been detected in a variety of respiratory diseases involving the recruitment of these leukocytes into the lung, including both inflammatory diseases such as asthma, in which leukocyte recruitment participates in disease pathogenesis, as well as infectious diseases such as tuberculosis, in which leukocyte recruitment participates in host immune responses. Functional roles for the MCP chemokines have been demonstrated in several rodent models of respiratory diseases, in which disease severity can be modulated by inhibition of MCP signaling. The MCPs and their receptors therefore are attractive targets for rational development of drugs that will have therapeutic potential in multiple respiratory diseases.

Introduction The monocyte chemoattractant proteins (MCP) constitute an important subfamily of chemotactic cytokine (CC) chemokines that share structural features and have overlapping functions. By activating G-protein-coupled CC chemokine receptors present on the surface of different classes of leukocytes, the MCPs participate in inflammatory responses by

CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12) 369

directing the recruitment and activation of these cells. Four human MCP (hMCP) proteins, hMCP-1 (CCL2), hMCP-2 (CCL8), hMCP-3 (CCL7), and hMCP-4 (CCL13), and four mouse MCP (mMCP) proteins, mMCP-1, mMCP-2, mMCP-3, and mMCP5 (CCL12), have been identified that share substantial amino acid identity. The cross-species assignments of orthologs among these genes is not entirely clear, but it is generally accepted that mMCP-1, mMCP-2, and mMCP-3 are orthologs of hMCP-1, hMCP-2, and hMCP-3, respectively. However, no mouse ortholog has been described for human MCP-4 to date, and mMCP-5 has no ortholog in the human genome, likely reflecting divergence of the human and mouse MCP gene clusters after speciation.

The secondary and tertiary structure of MCP-1 has been solved by both nuclear magnetic resonance (NMR) and crystal analyses. As is characteristic of the chemokine family, the N-terminal cysteines are followed by a long loop that leads into three

hMCP-2

hMCP-1

hMCP-3

hMCP-4

Structure As is characteristic of chemokines, the MCPs are small basic proteins. All the MCPs are members of the CC, or b-chemokine family, whose primary structures contain four conserved cysteine residues, of which the first two are adjacent to each other. Substantial homology exists between all members of the MCP family, with amino acid identity ranging from 74% between hMCP-1 and hMCP-3, to 38% between mMCP-1 (excluding a unique C-terminal extension) and mMCP-2 (Figure 1). Phylogenetic analysis of the protein sequences of the MCP family (Figure 2) suggests that the human and mouse MCP gene clusters diverge by gene duplication after speciation.

-23

Hu Hu Hu Hu Mu Mu Mu Mu

MCP-1 MCP-2 MCP-3 MCP-4 MCP-1 MCP-2 MCP-3 MCP-5

Hu Hu Hu Hu Mu Mu Mu Mu

MCP-1 MCP-2 MCP-3 MCP-4 MCP-1 MCP-2 MCP-3 MCP-5

mMCP-1

mMCP-3

mMCP-2

mMCP-5 Figure 2 Phylogenetic analysis of protein sequences of human and mouse MCPs. Analysis was performed using the neighborjoining method of ClustalW.

+1

+25

MK VSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLAS MK VSAALLCLLLMAATFSPQGLAQPDSVSIPITCCFNVINRKIPIQRLES MK ASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQRLES MK VSAVLLCLLLMTAAFNPQGLAQPDALNVPSTCCFTFSSKKISLQRLKS MQ VPVMLLGLLFTVAGWSIHVLAQPDAVNAPLTCCYSFTSKMIPMSRLES MK IYAVLLCLLLIAVPVSPEKLTGPD--KAPVTCCFHVLKLKIPLRVLKS MR ISATLLCLLLIAAAFSIQVWAQPDGPNA-STCCY-VKKQKIPKRNLKS MK IS-TLLCLLLIATTISPQVLAGPDAVSTPVTCCYNVVKQKIHVRKLKS +26

+75

YRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT YT RITNIQCPKEAVIFKTQRGKEVCADPKERWVRDSMKHLDQIFQNLKP YRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL YV-ITTSRCPQKAVIFRTKLGKEICADPKEKWVQNYMKHLGRKAHTLKT Y K R I T S S R C P K E A V V F V T K L K R E V C A D P K K E W V Q T Y I K N L D R N Q M R S E P>> YE R I N N I Q C P M E A V V F Q T K Q G M S L C V D P T Q K W V S E Y M E I L D Q K S Q I L Q P YR R I T S S R C P W E A V I F K T K K G M E V C A E A H Q K W V E E A I A Y L D M K T P T P K P YR R I T S S Q C P R E A V I F R T I L D K E I C A D P K E K W V K N S I N H L D K T S Q T F I L>>

Figure 1 Alignment of protein sequences of human and mouse MCPs. For comparison to the other MCPs, the unique C-terminal extension of mMCP-1 is not included, and the 98 amino acid N-terminal sequence is presented. Amino acids conserved between different MCPs are boxed.

370 CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12)

antiparallel b-pleated sheets in what is referred to as a ‘Greek key’ motif. The protein then terminates in an a-helix that overlies the b-pleated sheets. With regard to quaternary structure, all MCPs exist as dimers under conditions required for NMR and Xray crystallographic structural analyses. However, the physiologic quaternary structures of the MCPs, for example, whether they function physiologically as monomers, dimers, or higher-order multimers, remains to be determined.

members of the CC chemokine family, therefore only bind CC chemokine receptors (CCRs). All of the MCP proteins described to date, with the exception of mMCP-2, activate CCR2 to varying degrees. In addition, hMCP-2, hMCP-3, hMCP-4, and mMCP-3 activate CCR3, hMCP-2 and hMCP-3 activate CCR1, and hMCP-2 activates CCR5 (Figure 3). The CC chemokine receptor(s) activated by mMCP-2 have not yet been determined.

Biological Function Regulation of Production and Activity MCPs are generally considered to be ‘inflammatory’ chemokines, whose inducible expression directs the migration of leukocytes participating in inflammatory responses, as opposed to ‘homeostatic’ chemokines, whose constitutive expression directs the positioning of leukocytes under basal conditions. During inflammatory responses, the MCPs are highly induced in multiple cell types and tissues by a variety of stimuli, such as lipopolysaccharide, tumor necrosis factor alpha, interleukin (IL)-1b, interferon gamma (IFN-g), and platelet-derived growth factor (Table 1). Conversely, expression of MCP family members can be suppressed by glucocorticoids or immunusuppressive cytokines such as transforming growth factor beta and IL-10. In addition to transcriptional regulation of MCP expression, MCP activity can also be regulated by posttranslational modification. For example, glycosylation of hMCP-1 reduces its ability to direct the migration of monocytes in vitro. Posttranslational truncation of the first four or five N-terminal amino acids of hMCP-1, or the first five amino acids of hMCP-2, abrogates their chemoattractant activity. Additionally, the truncated form of MCP-2 acts as an inhibitor of other chemokines, including MCP-1, MCP-2, MCP-3, and RANTES (regulated upon activation normally T cell expressed and secreted).

Receptors The MCPs stimulate cells by binding to specific surface chemokine receptors, which belong to the G-protein-coupled seven transmembrane domain receptor superfamily. Binding of MCPs to these receptors activates multiple signaling pathways that regulate the cellular machinery that propels cells during MCP-directed migration. Each of the major chemokine families has its own specific set of chemokine receptors. Although many chemokines bind to more than one chemokine receptor, the members of each chemokine family bind only to the chemokine receptors specific for that family. The MCPs, as

MCPs stimulate the directed migration, or chemotaxis, and activation of leukocytes that express receptors specific for these chemokines. The induction of MCP expression in tissue sites of inflammation consequently directs the recruitment of activated leukocytes into these sites as a critical feature of host inflammatory responses to infection, injury, or allergy. The classes of leukocytes recruited by the MCPs can be predicted by the chemokine receptors expressed by these cells (Figure 3). As noted, all of the MCP proteins except mMCP-2 are active on CCR2, which is expressed on monocytes and macrophages, antigen-experienced (CD45RO þ ) T cells, B cells, immature dendritic cells, basophils, mast cells, and natural killer (NK) cells. hMCP-2, hMCP-3, hMCP-4, and mMCP-3 are active on CCR3, which is expressed by eosinophils, basophils, and TH2polarized CD4 þ T cells. hMCP-2 and hMCP-3 are active on CCR1, which is expressed by monocytes, T cells, dendritic cells, and eosinophils. hMCP-2 is also active on CCR5, which is expressed by TH1-polarized CD4 þ T cells and antigen-experienced (CD45RO þ ) CD8 þ T cells, monocytes and macrophages, and dendritic cells. Mice transgenically overexpressing MCP-1 driven by various different tissue-specific promoters have been generated. Consistent with MCP-1 directing the migration of monocytes and macrophages, these transgenic mice generally have increased accumulation of these cells specifically in those tissues in which there is increased MCP-1 expression. For example, mice overexpressing MCP-1 driven by the surfactant protein C promoter, in which MCP-1 is constitutively secreted into the airspaces, have increased numbers of monocytes, as well as lymphocytes, recovered in bronchoalveolar lavage (BAL) fluid. In contrast, normal numbers of monocytes and lymphocytes are present in the lung parenchyma of these mice. Mice genetically deficient for MCP-1 have also been generated. These mice are viable, develop normally, and have normal numbers of monocytes in their circulation and macrophages in their tissues. These mice have reduced monocyte recruitment in models of

CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12) 371 Table 1 MCP production MCP

Cell type/organ

Inducer

MCP-1

Fibroblasts Endothelial cells Vascular smooth muscle cells Monocytes/macrophages (including cell lines HL60, U937, THP1) Neutrophils Keratinocytes Synovial cells Type II pneumocyte cell line Mesangial cells Retinal pigmented epithelial cells Malignant cell lines (glioma, sarcoma, melanoma, hepatoma) Luteal cells Secondary lymphoid organs Lung (epithelium, alveolar macrophages) Brain (astrocytes) Spinal cord Seminal vesicles Kidney

PDGF / IL-1 / TNF-a / viruses / dsRNA / LPS / cholera toxin IL-1 / TNF / IFN-g / IL-4 / MM-LDL / stretch PDGF / MM-LDL / stretch LPS / IFN-g / PMA

MCP-2

MCP-3

MCP-4

MCP-5

Fibroblasts Neutrophils Osteosarcoma cell line Astrocytes Organs: small intestine, peripheral blood, heart, placenta, lung, skeletal muscle, ovary, colon, spinal cord, pancreas, thymus Porcine luteal cells Fibroblasts Monocytes Platelets Bronchial epithelium Kidney Astrocytes Skin Endothelial cells Dermal fibroblasts Bronchoalveolar lavage cells Bronchial epithelial cell lines (A549, BEAS-2B) PBMC Nasal epithelium Arterial plaques (endothelial cells/macrophages) Organs: small intestine, thymus, colon, heart, placenta Macrophage cell line (RAW 264.7) Lung (alveolar macrophages/smooth muscle cells) Spinal cord Lymph node stromal cells Thymic stromal cells

TNF IFN-g IL-1 IL-1 / TNF IL-1 / TNF / IFN-g / basic FGF / LIF / IL-6 IL-1 / TNF / LPS

Asthma and granuolma models EAE models of multiple sclerosis / seizure Contusion injury Inflammation (e.g., glomerulonephritis)/hypoxia/transplant rejection IL-1 / IFN-g / dsRNA / measles virus IL-1 / IFN-g EAE / multiple sclerosis

PDGF TNF / IL-1 / IFN-g / LPS / lipoarabinomannan Asthma models glomerulonephritis EAE / multiple sclerosis Atopy IL-1 / TNF Asthma IL-1 / TNF / IFN-g PHA / IL-2 Sinusitis Atherosclerosis

IFN-g / LPS Asthma models Spinal cord contusion injury

dsRNA, double stranded RNA; EAE, experimental allergic encephalomyelitis; IFN-g, interferon gamma; IL, interleukin; LIF, leukemia inhibitory factor; LPS, lipopolysaccharide; MM-LDL, minimally modified-low density lipoprotein, PDGF, platelet-derived growth factor; PHA, phytohemagglutanin; PMA, phorbol myristate acetate; PBMC, peripheral blood mononuclear cells. Reproduced from Rollins BJ (2000) MCP-1, MCP-2, MCP-3, MCP-4, and MCP-5. In: Oppenheim JJ and Feldman M (eds.) Cytokine Reference, pp. 1145–1160. San Diego: Academic Press, with permission from Elsevier.

372 CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12) MCPs activating CCR2:

hMCP-1 mMCP-1 hMCP-2 mMCP-3 hMCP-3 mMCP-5 hMCP-4

Cells expressing CCR2: Monocytes/ TH1 CD4+ macrophages T cells

TH2 CD4+ T cells

Biologic activity:

CD8+ T cells

B cells

Dendritic cells

Basophils/ mast cells

NK cells

Cell recruitment and activation

(a) MCPs activating CCR3:

hMCP-2 hMCP-3

MCPs activating CCR1:

hMCP-4 mMCP-3

Cells expressing CCR3:

Cells expressing CCR1: TH2 CD4+ T cells

Biologic activity:

hMCP-2 hMCP-3

Monocytes/ macrophages

Basophils/ Eosinophils mast cells

Cell recruitment and activation

(b)

Biologic activity:

Dendritic cells

Eosinophils

Cell recruitment and activation

(c)

MCPs activating CCR5:

hMCP-2

Cells expressing CCR5: Monocytes/ macrophages

Biologic activity:

TH1 CD4+ T cells

CD8+ T cells

Dendritic cells

Cell recruitment and activation

(d) Figure 3 Key biological functions of MCPs. By signaling through the chemokine receptors (a) CCR2, (b) CCR3, (c) CCR1, and (d) CCR5, the MCPs induce the recruitment and activation of multiple classes of leukocytes, including monocytes and macrophages, effector CD4þ and CD8þ T cells, B cells, dendritic cells, basophils and mast cells, NK cells, and eosinophils.

tissue inflammation, however, including models of peritonitis and delayed hypersensitivity reactions, indicating that MCP-1 has a nonredundant role in directing monocytes into inflamed tissues.

Role of MCPs in Respiratory Diseases As would be expected from their biologic activity, increased expression of MCP chemokines has been

CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12) 373

detected in a variety of respiratory diseases involving the recruitment of leukocytes into the lung, including inflammatory diseases such as asthma, in which leukocyte recruitment participates in disease pathogenesis, as well as infectious diseases such as tuberculosis, in which leukocyte recruitment participates in host immune responses. Functional roles for the MCP chemokines have been demonstrated in a variety of respiratory disease models in rodents, in which disease severity has been modulated by targeting MCP/CCR2 signaling. Asthma

Increased levels of MCP-1, MCP-3, and MCP-4 have been noted in the airways of asthmatic subjects, and gene polymorphisms of MCP-1 have been associated both with asthma susceptibility and severity. Further, treatment resulting in improvements in forced expiratory volume has been associated with reductions in airway expression of MCP-3 and MCP4. Increased airway expression of the MCPs, including MCP-1, MCP-3, and MCP-5, has also been noted in mouse models of allergic pulmonary inflammation. Inhibition or deletion of the MCPs, or one of their receptors, CCR2, has produced conflicting results in these models, however, and consequently the role of the MCPs in asthma remains to be definitively established.

Other Pulmonary Infections

Elevated expression of MCP-1 in the lung has been demonstrated in mouse models of infection with Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae, influenza A, Aspergillus fumigatus, Cryptococcus neoformans, and Pneumocystis carinii. MCP-1 has been demonstrated to participate in the recruitment of both macrophages and NK cells in a mouse model of pneumococcal pneumonia, and to direct the recruitment of macrophages responsible for the phagocytosis and removal of dying neutrophils in a mouse model of pseudomonas pneumonia. In so doing, MCP-1 expression is thought to attenuate the development of lung injury in the pseudomonas model. In a model of influenza pneumonia, CCR2-deficient mice are protected from the early pathological manifestations of infection due to reduced macrophage recruitment. In this model, however, the delay in macrophage accumulation in the CCR2-deficient mice caused a subsequent delay in T cell recruitment, and an increase in pulmonary viral titers at early time points following infection. Finally, in a model of invasive aspergillus pneumonia performed in neutropenic mice, treatment of infected mice with neutralizing antiMCP-1 antibody reduced NK cell recruitment at early time points, and led to a greater than threefold increase in pathogen burden in lungs and twofold greater mortality.

Tuberculosis Lung Allograft Rejection

Increased levels of MCP-1, MCP-3, and MCP-4 have been noted in the lungs of patients with tuberculosis. A requirement for CCR2, and presumably its MCP ligands, for control of Mycobacterium tuberculosis infection has been dramatically demonstrated in mouse models. Mice genetically deficient for CCR2 have a rapidly progressive and fatal course following high- or moderate-dose M. tuberculosis infection, developing lung mycobacterial burdens 100-fold greater than wild-type mice controls, which control infection and survive. CCR2-deficient mice demonstrated multiple defects in leukocyte trafficking following infection, including defects in early macrophage recruitment to the lung and subsequent defects in macrophage and dendritic cell recruitment to the mediastinal lymph nodes. T cell activation in the mediastinal lymph nodes was consequently delayed, resulting in reduced accumulation of T cells primed to produce IFN-g in the lungs of infected CCR2-deficient mice. Though this remains to be demonstrated in humans, the cellular responses mediated by the activation of CCR2 by its MCP ligands appear essential for immune control of M. tuberculosis infection.

MCP-1 levels are elevated in the lungs of transplant recipients experiencing acute rejection, as well as those experiencing chronic rejection, that is, bronchiolitis obliterans syndrome (BOS), compared with healthy transplant recipients. Elevated BAL levels of MCP-1 further have been shown to predict the development of BOS. Additionally, in allograft recipients shifted from cyclosporine- to tracrolimus-based immunosuppression due to refractory acute rejection, clinical and functional stabilization was accompanied by significant and sustained reductions in lavage MCP-1 levels. Induction of MCP-1 expression has also been noted in mouse and rat models of allograft rejection and BOS. In a mouse model of tracheal transplantation, both the recruitment of mononuclear phagocytes and the severity of BOS were significantly reduced by the interruption of MCP-1/CCR2 signaling. Idiopathic Pulmonary Fibrosis

Elevated levels of MCP-1 have been noted in the lungs of idiopathic pulmonary fibrosis patients. Similarly, the expression of MCP-1 is increased in the

374 CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12)

lungs in rodent models of pulmonary fibrosis. In the mouse model of pulmonary fibrosis induced by bleomycin, inhibition of endogenous MCP-1 function by the transgenic expression of a dominant negative N-terminal deletion MCP-1 mutant reduced pulmonary fibrosis, although interestingly, without affecting macrophage or lymphocyte recruitment into the lung. A similar result has been reported in experiments using CCR2-deficient mice, which were protected from fibrosis despite having leukocyte recruitment in the lung equivalent to that of wild-type controls. However, another group of investigators has recently reported that in their experiments performing the bleomycin model of pulmonary fibrosis in CCR2-deficient mice, lung macrophage recruitment was reduced. MCP-1/CCR2 signaling thus may contribute to the development of pulmonary fibrosis through both leukocyte-dependent and nonleukocyte-dependent effects. Acute Lung Injury/Acute Respiratory Distress Syndrome

Elevated MCP-1 levels are present in BAL fluid of acute respiratory distress syndrome patients, and have been correlated both with alveolar monocyte recruitment and lung injury score. Elevated MCP-1 expression has also been noted in several rodent models of acute lung injury, including a model of gastric aspiration-induced pneumonitis. In this model, MCP-1-deficient mice had decreased survival compared with wild-type controls. Wild-type mice in this model demonstrated areas of compartmentalized inflammation in the lung with prominent granuloma formation following acid aspiration, whereas MCP1-deficient mice demonstrated severe diffuse pneumonitis without granulomas, suggesting that MCP-1 protects uninjured lung regions by promoting the isolation and compartmentalization of tissue with active inflammation. Pulmonary Hypertension

Elevated levels of MCP-1 have been noted in the circulation of patients with both primary pulmonary hypertension (PPH) and chronic thromboembolic pulmonary hypertension (CTEPH). In patients with PPH, treatment with epoprostenol has been associated with significant reductions in circulating MCP-1 levels. In patients with CTEPH, circulating MCP-1 levels were significantly correlated with pulmonary vascular resistance. Elevated levels of MCP-1 in the circulation as well as in BAL fluid have also been demonstrated in a rat model of pulmonary hypertension induced by monocrotaline. Inhibition of MCP-1 signaling in this model reduced mononuclear cell

infiltration into the lung, as well as right ventricular systolic pressure, right ventricular hypertrophy, and medial hypertrophy of the pulmonary arterioles. Other Respiratory Diseases

Elevated expression of the MCPs has also been noted in several other respiratory diseases, including chronic obstructive pulmonary disease, pulmonary alveolar proteinosis, sarcoidosis, hypersensitivity pneumonitis, eosinophilic pneumonia, and coalworker’s pneumoconiosis.

Concluding Remarks By virtue of their ability to direct the migration of multiple classes of leukocytes into the lungs, the MCPs are importantly involved in multiple respiratory diseases. In inflammatory pathologies in which leukocyte recruitment participates in disease pathogenesis, such as asthma, allograft rejection, or ARDS, inhibition of MCP signaling is an attractive strategy for therapeutic intervention. In infectious pathologies in which leukocyte recruitment participates in host immune responses, such as tuberculosis or other pneumonias, augmentation of MCP signaling may be beneficial. The MCPs and their receptors therefore represent attractive targets for rational development of drugs that will have therapeutic potential in multiple respiratory diseases. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Dendritic Cells. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Monocytes; T cells; Pulmonary Macrophages. Vascular Disease.

Further Reading Belperio JA, Keane MP, Burdick MD, et al. (2001) Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome. Journal of Clinical Investigation 108: 547–556. Goodman RB, Strieter RM, Martin DP, et al. (1996) Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 154: 602–611. Gu L, Tseng S, Horner RM, et al. (2000) Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 404: 407–411. Gunn MD, Nelken NA, Liao X, and Williams LT (1997) Monocyte chemoattractant protein-1 is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation. Journal of Immunology 158: 376–383. Ikeda Y, Yonemitsu Y, Kataoka C, et al. (2002) Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats. American Journal of Physiology: Heart and Circulatory Physiology 283: H2021–H2028.

CHEMOKINES, CC / RANTES (CCL5) 375 Katsushi H, Kazufumi N, Hideki F, et al. (2004) Epoprostenol therapy decreases elevated circulating levels of monocyte chemoattractant protein-1 in patients with primary pulmonary hypertension. Circulation Journal 68: 227–231. Lu B, Rutledge BJ, Gu L, et al. (1998) Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. Journal of Experimental Medicine 187: 601–608. Miotto D, Christodoulopoulos P, Olivenstein R, et al. (2001) Expression of IFN-gamma-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in TH1- and TH2-mediated lung diseases. Journal of Allergy and Clinical Immunology 107: 664–670. Moore BB, Paine R III, Christensen PJ, et al. (2001) Protection from pulmonary fibrosis in the absence of CCR2 signaling. Journal of Immunology 167: 4368–4377. Nomiyama H, Egami K, Tanase S, et al. (2003) Comparative DNA sequence analysis of mouse and human CC chemokine gene clusters. Journal of Interferon Cytokine Research 23: 37–45. Peters W, Scott HM, Chambers HF, et al. (2001) Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America 98: 7958–7963. Reynaud-Gaubert M, Marin V, Thirion X, et al. (2002) Upregulation of chemokines in bronchoalveolar lavage fluid as a predictive marker of post-transplant airway obliteration. Journal of Heart and Lung Transplantation 21: 712–730. Rollins BJ (2000) MCP-1, MCP-2, MCP-3, MCP-4, and MCP-5. In: Openheim JJ and Feldman M (eds.) Cytokine Reference, pp. 1145–1160. San Diego: Academic Press. Rosseau S, Hammerl P, Maus U, et al. (2000) Phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome. American Journal of Physiology: Lung Cellular and Molecular Physiology 279: L25–L35. Szalai C, Kozma GT, Nagy A, et al. (2001) Polymorphism in the gene regulatory region of MCP-1 is associated with asthma susceptibility and severity. Journal of Allergy and Clinical Immunology 108: 375–381.

RANTES (CCL5) A E I Proudfoot, C A Power, and Z Johnson, Serono Pharmaceutical Research Institute, Geneva, Switzerland & 2006 Elsevier Ltd. All rights reserved.

Abstract RANTES (regulated upon activation normally T cell expressed and secreted) is a member of the subfamily of cytokines called chemokines, the name given to chemoattractant cytokines in 1994. The family consists of small basic proteins whose role is to provide the directional signal for leukocyte migration. The chemokine family are distinct from other cytokines in that they act on G-protein-coupled heptahelical transmembrane spanning receptors. Initially, chemokines were named according to their function but due to the confusion in the nomenclature which arose from the simultaneous identification by different laboratories of many chemokines in the mid to late 1990s, a systematic nomenclature was introduced in 2001. In the new nomenclature the chemokines were named according to their genomic localization, and RANTES was renamed CCL5. RANTES probably

has the broadest range of target cells of known chemokines due to its multiple receptor usage and thus is implicated in the recruitment of memory T cells, monocytes, eosinophils, and basophils, thus making it one of the key players in respiratory disease.

Introduction Chemokines are structurally classified into subfamilies based on their pattern of disulfide bridging, which allows their division into four subclasses as defined by the distribution of the four highly conserved cysteines. Thus the a-chemokines have the first Cys pair close to the N-terminus and separated by an amino acid (CXC chemokines) whereas the b-chemokines have the first two Cys residues adjacent (CC chemokines). These two subclasses comprise the majority of chemokines, but there are also two minor subclasses, which each have only one or two members: CX3C chemokines have three amino acids separating the amino terminal Cys residues and C chemokines possess only one disulfide bridge. Regulated upon activation normally T-cell expressed and secreted (RANTES)/CCL5 belongs to the b-chemokine or CC-chemokine subclass. RANTES was initially isolated from a T cell cDNA library although it was subsequently found to be induced in other cell types upon induction with proinflammatory cytokines.

Structure RANTES has the canonical monomeric fold of the chemokine family (Figure 1(c)) and the canonical dimeric form of the CC subfamily (Figure 1(b)) consisting of a flexible N-terminal loop, followed by three antiparallel b-sheets, which are connected by short flexible turns called the 30s and 50s loop and an a-helical C-terminus (Figure 1(a)). RANTES has an extremely high molecular weight, 4600 kDa, as determined by techniques such as size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC). This high-molecular-weight quaternary structure is not only found in vitro, but the protein has been observed to be secreted from granules of T cells as a complex with soluble heparin, also greater than 600 kDa. Such a higher-order quaternary structure is not limited to RANTES but has also been described for MIP-1a and MIP-1b, both of which share receptor usage with RANTES. Since RANTES interacts with several receptors, mutagenesis has been used to identify the epitopes conferring receptor specificity. One common feature has emerged in that the N-terminus is critical for receptor activation. Both truncation by total protein synthesis, and extension by either retention of the initiating methionine of the recombinant protein in

376 CHEMOKINES, CC / RANTES (CCL5)

(a)

(b)

Arg47 Lys45

Arg44

(c)

(d)

Figure 1 Structure of RANTES. (a) Monomer; (b) dimer. (c) RANTES (red) is overlaid with six other chemokines (gray) from the CC (MCP-1 and MIP-1b), CXC (IL-8, NAP-2, and PF4) and CX3C (fractalkine) classes. (d) The BBXB motif forming the GAG binding site of RANTES on the 40s loop.

procaryotic expression systems, or by chemical coupling techniques, have shown that this significantly affects receptor activation capacity. Truncation of the first eight amino acids produced a protein that had acquired the ability to bind to CCR2, and extension of the N-terminal produced antagonists (see below). The one exception was the deletion of the first two amino acids producing the variant 3-68-RANTES; this molecule had enhanced activity for CCR5 whilst inhibiting CCR1-mediated events. RANTES has a second essential interaction, which is low-affinity binding to glycosaminoglycans (GAGs), a common feature amongst the chemokine family. The binding site for GAG has been identified for RANTES as being a BBXB motif on the 40s loop (Figure 1(d)). Furthermore the variant in which the basic residues in this motif were replaced with Ala residues was shown to have lost its ability to recruit cells in vivo, thereby proving conclusively that GAG binding is essential for chemokine activity in vivo.

Regulation of Production and Activity Genomic Localization

The RANTES locus has been mapped to chromosome 17q11.2–q12 using somatic cell hybrids and by in situ hybridization of the cDNA probe. Several other CC chemokine genes have also been mapped to this locus.

The RANTES gene spans 7.1 kb and comprises three exons of 133, 112, and 1075 bases respectively and two introns of approximately 1.4 and 4.4 kb. This exon–intron organization is similar to that reported for other CC chemokine family members. The locus has been sequenced from nine mammalian species which code for proteins with striking homology, where the identity ranges from 71% to 100%, with similarities ranging from 79% to 100% (Figure 1). Expression Pattern

RANTES expression was initially described as being restricted to antigen- or mitogen-activated T cells. However it turns out that RANTES is also expressed by a broad range of cell types after cellular activation by tumor necrosis factor alpha (TNF-a) and interleukin (IL)-1b including epithelial cells and fibroblasts. In addition RANTES is very strongly expressed by platelets. RANTES expression is a hallmark of inflammatory diseases and RANTES mRNA or protein has been detected in pathological specimens (disease tissue or fluids) from bronchoalveolar lavage (BAL) from asthmatics as well as many other inflammatory diseases (Table 1).

Receptors RANTES is one of the most promiscuous chemokines in that it binds to several receptors (Figure 2). It

CHEMOKINES, CC / RANTES (CCL5) 377

binds to three CC receptors with high affinity (CCR1, CCR3, and CCR5 through which it induces cellular recruitment) and to CCR4 with low affinity. In addition, it binds to the promiscuous nonsignaling receptors DARC and D6, which are thought to act as chemokine sinks. RANTES has the highest affinity for CCR1 and CCR5, binding with low nanomolar affinity, whilst the affinity for CCR3 has been reported to range from 1 to 100 nM, and that for CCR4 is micromolar. CCR1 and CCR5 are expressed

Table 1 Disease association for RANTES receptors Receptor

Disease indication

CCR1

Multiple sclerosis Rheutmatoid arthritis Organ transplant Asthma, rhinitis Nephritis

CCR3

Asthma Allergy

CCR5

Multiple sclerosis Rheumatoid arthritis Transplant Asthma Nephritis IBD Acquired immune deficiency syndrome (AIDS)

on T cells, immature dendritic cells, and monocyte/ macrophages; in the case of the latter, the expression of CCR1 is predominant on circulating monocytes, whereas CCR5 is upregulated when monocytes acquire the macrophage phenotype. Using specific small molecule inhibitors of CCR1 and CCR5, it has been shown that whilst both receptors can mediate transendothelial migration induced by RANTES, RANTES activation of CCR1 mediates cellular arrest whilst activation of CCR5 mediates cellular spreading. Its role on CCR3 and CCR4 is less clear, although high levels of RANTES expression in samples from asthmatic patients (see below) could imply that it assists in the recruitment of eosinophils through CCR3. RANTES signals through the classical chemoattractant receptor signaling pathway initiated by ligand-induced receptor activation, which results in the formation of a heterotrimeric G-protein complex at its cytoplasmic tail by recruitment of a membraneanchored Gbg heterodimer to the receptor-bound Ga subunit. The Gbg complex regulates the activation of phosphatidyl inositol-phospholipase Cb, catalyzing the breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG), which act synergistically in the mobilization of intracellular Ca2 þ . DAG and Ca2 þ activate various isoforms of protein kinase C, which

CXCL16 CXC13 CXCL12

D6

CCR6 XCL1 XCL2 CX3CL1

Constitutive: basal trafficking/homing

DARC

CCL27 CCL28

CXCL20

XCR1

CCL11 CCL7 CCL13

CCR8

CXCR5

CXCR4

CCL5 CCL5

CXCR6

CCL1

CX3CR1 CXCR1

CCR10

CCL25

Inducible: inflammatory

CCR9 CCR7

CCL19 CCL21

CXCR3

CCR5 CCL3 CCL4

CCL5 CCL8

CXCR2

CCR4 CCL17 CCL22

CCR1 CCR3 CCL5 CCL7 CCL11 CCL13

CCR2 CCL2 CCL8 CCL7 CCL13

CCL5

CXCL8 CXCL6 CXCL8 CXCL5 CXCL1 CXCL2 CXCL3 CXCL7

CXCL9 CXCL10 CXCL11

CCL3 CCL7

Figure 2 Chemokine receptors and their ligands. The high-affinity receptors to which RANTES/CCL5 binds are indicated as those that signal (red) and those that do not signal (black). Since RANTES has only been reported to interact with CCR4 with low (micromolar) activity it is not depicted as a CCR4 ligand in this figure.

378 CHEMOKINES, CC / RANTES (CCL5)

can phosphorylate many other downstream targets. The two major signaling pathways activated through the binding of chemokines are: (1) the phosphoinositide 3 kinase (PI3K) pathway, and studies with PI3Kg
/
mice have demonstrated that RANTES activity is severely hampered in the absence of this enzyme and (2) the mitogen-activated protein-kinase (MAPK) pathway, which is responsible for phosphorylating and activating downstream transcription factors that in turn regulate the synthesis of other inflammatory proteins, including chemokines.

Biological Function Since their identification, the importance of chemokines in cell migration has been demonstrated in both basal cell trafficking and in inflammation. An interesting contrast in the chemokine system exists between the receptors involved in basal trafficking (controlled by homeostatic chemokines), which tend to be specific, whilst those involved in inflammatory cell trafficking are shared by several chemokines (Figure 2). In inflammatory situations, expression of inducible or inflammatory chemokines is upregulated. Control of expression of these chemokines is under the temporal control of proinflammatory cytokines. Proinflammatory cytokines such as IL-1b, TNF-a, and interferon gamma (IFN-g) alone or in combination induce chemokine expression at sites of inflammation in nonlymphoid tissue. RANTES is an example of an inducible/inflammatory chemokine. Inflammatory chemokine receptors (including those

to which RANTES binds, that is, CCR1, CCR3, and CCR5) are upregulated on inflammatory cells following priming, which are then attracted to sites of inflammation. RANTES may potentially mediate the activation and trafficking of a range of immune cells including T cells, monocytes, basophils, eosinophils, natural killer cells, dendritic cells, and mast cells, all of which play a role in respiratory diseases (Figure 3). RANTES is often associated with inflammatory exudates and is predominantly secreted by CD8 þ T cells, epithelial cells, fibroblasts, and platelets, in which it is stored in alpha granules as densely packed aggregates. Much understanding of the chemokine system has come from studies using knockout mice. Despite its being one of the earliest identified and most widely studied chemokines, there are few reports in the literature describing the RANTES-deficient mouse. Published work indicates a role for RANTES in both normal T-cell function and in monocyte and T-cell recruitment in the contact hypersensitivity response. However no data with the RANTES
/
mice in airway inflammation are available. On the other hand there are numerous publications on studies of knockout mice for the various RANTES receptors, but again, none for CCR1
/
or CCR5
/
mice in airways inflammation. A role for CCR1 has been demonstrated in the murine model of multiple sclerosis, experimental allergic encephalomyelitis (EAE), CCR5-deleted mice are fully susceptible to EAE, surprisingly, as high levels of CCR5 expression are seen in multiple sclerosis patients. However, deletion of

Resting T cell

B cell

Activated T cell CCR1,3,(4),5

Eosinophil CCR1,3

NK cell CCR5

Basophil mast CCR3,(4)

Dendritic cell CCR1,5

Monocyte CCR1(5) Macrophage CCR5(1)

Neutrophil Figure 3 Leukocyte subsets. Leukocytes recruited by RANTES that play a role in respiratory disease are shown in red whilst those that are not recruited by RANTES are shown in black. The RANTES receptors found on each cell type are indicated.

CHEMOKINES, CC / RANTES (CCL5) 379

CCR3, which is the predominant chemokine receptor expressed by eosinophils, results in a 50–70% reduction in eosinophil accumulation in the airway lumen and the lung tissue after challenge. These data confirm the role of CCR3 in eosinophil recruitment to the lung during allergic airways inflammation. However, no protection was seen in the bronchial hyperreactivity to methacholine, which could be explained by the increase in lung tissue mast cells seen in this model and highlights the controversial role of the eosinophil in respiratory disease.

Role of RANTES in Respiratory Disease Increased RANTES expression, in combination with the presence of RANTES receptor expressing cells, has been associated with a variety of human inflammatory diseases. RANTES produced by lung epithelial cells has been identified as a major eosinophil chemoattractant in the BAL fluid of asthmatics. Additionally bronchial biopsies from patients with asthma show elevated RANTES mRNA expression, and levels of RANTES have been shown to reach a peak expression level approximately 4 h after allergen challenge in human subjects. Furthermore RANTES is a potent lymphocyte and monocyte chemoattractant acting via CCR1 and CCR5, and therefore contributes toward the recruitment of T cells and macrophages into the lungs of asthmatic patients. In line with its activity as a chemoattractant for T cells and eosinophils, RANTES has been observed at high levels in polyps from patients suffering from nasal polyposis in chronic hyperplastic sinusitis. Therapeutic Potential

The therapeutic potential of RANTES as an anti-inflammatory and anti-infective target has been shown by several approaches in animal models of disease. The use of neutralizing antibodies has clearly demonstrated the importance of this chemokine in several pulmonary pathologies. Airway obstruction in an experimental model of obliterative bronchiolitis and airway hyperreactivity in a respiratory syncytial viral infection model were both considerably ameliorated by treatment with neutralizing RANTES antibodies. Beneficial effects were also observed with anti-RANTES antibodies in acute lung allograft rejection and allograft transplant-induced fibrous airway obliteration as well as in cardiac allograft rejection. As mentioned above, the N-terminal region of RANTES is critical for receptor activation. In fact modification of this region has produced several variants with receptor antagonist properties. The most

widely studied is Met-RANTES, which is produced by the extension of the N-terminus by the initiating methionine when the human protein is produced recombinantly in Escherichia coli. The protein retains high affinity for human CCR1, CCR3, and CCR5 but is unable to cause receptor activation except in the case of CCR5, on which it has partial agonist activity. Pharmacological studies on the murine receptors surprisingly showed that whilst it also retains high affinity for murine CCR1 and CCR5, it is no longer able to compete for binding of Eotaxin/ CCL11 from murine CCR3. This variant has shown the beneficial effects of inhibiting RANTES receptors in many models, in the lung as well as other organs. In fact Met-RANTES was shown to be more effective than individual antichemokine antibody treatment in the ovalbumininduced airway inflammation model. This was interesting since it does not bind to murine CCR3 and yet still reduces eosinophil accumulation in the lungs, indicating that blockade of CCR1 and/or CCR5 may also be potential targets for asthma. In an extensive dose–response study in this model, the inhibition observed was inversely bell-shaped, for which a definitive explanation remains to be elucidated. However the lack of efficacy at high doses could be one of the reasons why it has not been developed for the clinic, in addition to the residual partial agonist activity, which was shown not to be the reason for the inverse bell-shaped dose–response. CCR1 has been shown to be crucial in the inflammatory response to the murine pneumonia virus, which has been used as a model for severe human respiratory syncytial virus disease. This model has highlighted the importance of inflammation to the pathogenesis of chronic disease, demonstrating that the inflammatory response remains active and acute even when virus replication ceases in response to appropriate antiviral therapy. Met-RANTES treatment prevented the inflammatory response to the virus and resulted in reduced morbidity and mortality when administered in conjunction with the antiviral agent ribavirin. These results highlight the interesting possibility of using chemokine antagonists in dual therapies. This had been previously demonstrated by using Met-RANTES in synergy with cyclosporin to prevent organ transplant rejection, at suboptimal doses of both agents. Another N-terminally modified RANTES variant, AOP-RANTES, provided several important insights into the inhibition of human immunodeficiency virus (HIV) infectivity. This variant was produced by chemical modification of the N-terminal in an attempt to improve the characteristics of Met-RANTES. Unexpectedly, this variant was even more potent on CCR5 than the parent WT-RANTES protein,

380 CHEMOKINES, CC / TARC (CCL17)

whilst retaining antagonistic properties on CCR1. It was found to be an extremely potent inhibitor of HIV infectivity, not due to its antagonistic properties on CCR5 but on the contrary due to its ability to drive internalization of CCR5, and moreover, to prevent the recycling of functional receptors to the cell surface, thereby removing the essential co-receptor. Furthermore, it provided evidence that infection of primary macrophages could be prevented, an issue that was much debated in the chemokine–HIV field since there were reports both of inhibition and enhancement of infection by R5 HIV strains (those that require CCR5 for cell entry). Despite a different mode of action, AOP-RANTES showed the same profile of inhibition of airways inflammation in mice. Yet another RANTES variant has provided novel insights into strategies that can effectively inhibit inflammation. As discussed above, mutation of the heparin binding site in RANTES abrogates its ability to recruit cells in vivo, but what was unexpected is that it was capable of inhibiting RANTES-mediated recruitment, even by the non-specific inflammatory agent thioglycollate. This inhibitory effect translated into a potent inhibitor of clinical symptoms in the murine model of multiple sclerosis, experimental autoimmune encephalomyelitis, EAE. Whilst heparin has been shown to reduce inflammatory symptoms both in human and in rodent disease models, including pulmonary inflammation, the effect was attributed to an effect on proinflammatory cytokines. Given the fact that the GAG mutant 44AANA47RANTES had anti-inflammatory properties, together with the fact that heparin could inhibit both RANTES- and thioglycollate-mediated cellular recruitment in vivo, its anti-inflammatory effect could also be attributed to the interference with the establishment of chemokine gradients. Again this variant was able to reduce the cellular infiltrate into inflamed lungs in mice. In summary, this single chemokine provides many therapeutic targets since it acts with high affinity on three receptors, all implicated in respiratory disease. Moreover, in view of the extensive knowledge of the protein, it provides several different approaches to inhibit its activity. See also: Chemokines. Interleukins: IL-5; IL-16. Platelets.

Further Reading Chvatchko Y, Proudfoot AE, Buser R, et al. (2003) Inhibition of airway inflammation by amino-terminally modified RANTES/ CC chemokine ligand 5 analogues is not mediated through CCR3. Journal of Immunology 171: 5498–5506.

Gerard C and Rollins BJ (2001) Chemokines and disease. Nature Immunology 2: 108–115. Handel TM, Johnson Z, Crown SE, Lau EK, and Proudfoot AEI (2005) Regulation of protein function by glycosaminoglycans as exemplified by chemokines. Annual Review of Biochemistry 74: 385–410. Hebert CA (ed.) (1999) Chemokines in Disease. Totowa, NJ: Humana Press. Johnson Z, Kosco-Vilbois MH, Herren S, et al. (2004) Interference with heparin binding and oligomerization creates a novel antiinflammatory strategy targeting the chemokine system. Journal of Immunology 173: 5776–5785. Johnson Z, Schwarz M, Power CA, Wells TN, and Proudfoot AEI (2005) Multi-faceted strategies to combat disease by interference with the chemokine system. Trends in Immunology 26: 268–274. Luster AD (1998) Chemokines: chemotactic cytokines that mediate inflammation. New England Journal of Medicine 338: 436–445. Mack M, Luckow B, Nelson PJ, et al. (1998) AminooxypentaneRANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity. Journal of Experimental Medicine 187: 1215–1224. Power CA (2002) Knock out models to dissect chemokine receptor function in vivo. Journal of Immunological Methods. Proudfoot AEI (2002) Chemokine receptors: multifaceted therapeutic targets. Nature Reviews: Immunology 2: 106–115. Proudfoot AEI, Handel TM, Johnson Z, et al. (2003) Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proceedings of the National Academy of Sciences, USA 100: 1885–1890. Rothenberg ME (ed.) (2000) Chemokines in Allergic Disease. New York: Dekker. Schall TJ, Bacon K, Toy KJ, and Goeddel DV (1990) Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 347: 669–671. Schall TJ, Jongstra J, Dyer BJ, et al. (1988) A human T cell-specific molecule is a member of a new gene family. Journal of Immunology 141: 1018–1025. Simmons G, Clapham PR, Picard L, et al. (1997) Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 276: 276–279.

TARC (CCL17) T L Ness, C M Hogaboam, and S L Kunkel, University of Michigan, Ann Arbor, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Thymus and activation-regulated chemokine (TARC) was the first CC chemokine identified as a T-cell chemoattractant. It is known as CC chemokine ligand 17 (CCL17) according to the new nomenclature and shares most amino acid identity with CCL22. CCL17 expression is highest in the thymus, but can also be detected in other tissues such as the small intestine, colon, and lung. CCL17 is completely absent from the spleen. Dendritic cells are the predominant source of constitutive CCL17 production, whereas most immune cells and structural

CHEMOKINES, CC / TARC (CCL17) 381 cells are capable of inducible expression. CCL17 primarily acts through its receptor CCR4 as a chemoattractant of T-helper-2 (Th2) cells. However, CCR4 is also found on Th1 cells, regulatory T cells, natural killer (NK) T cells, NK cells, platelets, and macrophages. CCL17 inhibits classical macrophage activation and type 1 responses, which further drive type 2 immune responses. CCL17 is associated with several Th2-mediated respiratory diseases such as bronchial asthma, allergic rhinitis, pulmonary fibrosis, and eosinophilic pneumonia, and represents a potential target for treatment of these pathologic disorders.

Introduction Thymus and activation-regulated chemokine (TARC) was first identified and cloned from phytohemagglutinin-stimulated peripheral blood mononuclear cells by Imai and colleagues in 1996. TARC was one of four novel sequences isolated using an Epstein-Barr virus vector signal sequence trap. As its name indicates, TARC is constitutively produced in the thymus, but can also be detected in other tissues such as the colon, small intestine, and lung. It was the first CC chemokine identified that specifically interacted with a high-affinity receptor found predominantly on T cells, to a lesser degree on monocytes, but not on granulocytes. TARC acts as a powerful T cell chemoattractant and has been implicated in the pathogenesis of several Th2-mediated diseases. TARC was renamed CC chemokine ligand 17 (CCL17) according to the new nomenclature system for chemokines and chemokine receptors.

Structure While most CC chemokines are found on human chromosome 17 (mouse chromosome 11), CCL17 is

tightly linked to CX3CL1 and CCL22 on human chromosome 16q13 (mouse chromosome 8q). The CCL17 gene (2176 bp) comprises 4 exons and encodes a 94 amino acid preprotein with a 23 amino acid signal sequence. The CCL17 protein contains the four cysteine residues characteristic of all CC chemokines and shares the highest degree of homology with CCL22 (32% amino acid identity) (Figure 1). The secreted CCL17 protein is approximately 8 kDa and has a theoretical isoelectric point of 9.7. The tertiary monomeric structure of CCL17 is a threestranded antiparallel b-sheet flanked by a C-terminal helix, like other CC chemokines, although dimerization results in a more compact molecule than other CC chemokines. CCL17 has a greater distribution of basic charge on its surface, allowing it to bind more tightly to glycosoaminoglycans than most CC chemokines. This contributes to its ability to form a gradient within the extracellular matrix of endothelial cells to attract and immobilize receptorexpressing cells. Polymorphism screening of the human CCL17 gene identified two rare single nucleotide changes within exon 3 (2134C4T and 2037G4A) and a polymorphism in the promoter region (–431C4T). The promoter region contains nuclear factor kappa B (NF-kB) and signal transducer and activator of transcription 6 (STAT-6) recognition sites as well as the regulatory elements required for interleukin (IL)-4 and tumor necrosis factor alpha (TNF-a)-mediated induction. The –431C4T change alters the putative binding site for the transcription factor activator protein 4 and results in increased promoter activity in vitro. Individuals homozygous for the –431C4T

2176 bp

C>T

SP

(a)

CC

CX3CL1

CCL17

FracTARC (b) Figure 1 Sequence structure of CCL17. (a) The genomic sequence of human CCL17 (2176 bp) is comprised of four exons. Untranslated regions are depicted as hatched boxes. The locations of the signal peptide (SP), CC motif, and
431C4T polymorphism are indicated. (b) In the mouse, tissue-specific (brain and kidney) alternative splicing results in production of fracTARC, a transcript containing the signal peptide sequence of CX3CL1/fractalkine (red) and the entire coding region of CCL17 (blue) minus the first three N-terminal amino acids.

382 CHEMOKINES, CC / TARC (CCL17)

polymorphism have elevated systemic concentrations of CCL17 as well as an increased incidence of asthma. Murine CCL17 is unique in that its transcription is regulated not only from its own promoter, but also from the promoter of the closely linked CX3CL1 (fractalkine) gene. Alternative splicing results in expression of murine fracTARC, a protein containing the CX3CL1 signal sequence and the mature CCL17 protein. Processing of the signal sequence results in loss of the first three amino acids from the N-terminus, normally found in CCL17. Transcriptional regulation of CCL17 is tissue specific, as CCL17 is expressed in the thymus and lung while fracTARC can only be found in the brain and kidney. fracTARC has not been detected in human tissues thus far.

Regulation of Production and Activity Dendritic cells (DCs), specifically myeloid-related DCs (CD11c þ CD11b þ ), are responsible for constitutive production of CCL17 in most peripheral lymphoid and nonlymphoid organs. Immature monocyte-derived DCs (moDCs) express high levels of CCL17 that are increased several-fold upon maturation. CCL17 is inducible in most leukocytes including DCs, macrophages, mast cells, B cells, and naive T cells after exposure to various cytokines (IL-3, IL-4, IL-13, and granulocyte-macrophage colonystimulating factor (GM-CSF)), phytohemagglutinin, ovalbumin, or stimulation with Toll-like receptor agonists (i.e., lipopolysaccharide). Transforming growth factor beta (TGF-b) has been shown to act as a negative regulator of CCL17 production. Structural cells such as bronchial, epithelial, and endothelial cells, keratinocytes, fibroblasts, and smooth muscle cells are also a significant source of inducible CCL17. CCL17 is not found in the spleen, even after systemic bacterial infection or in vitro stimulation of cells from the spleen. The expression pattern of this chemokine accentuates its importance in mediating the attraction of effector cells to sites commonly exposed to infection and/or stimulation by foreign antigens, i.e., mucosa and lymph nodes (LNs).

Biological Function DCs fulfill a fundamental regulatory role linking both innate and adaptive immune responses. DCs act as professional antigen-presenting cells, transporting antigens to the draining lymph nodes (LNs), where they prime T-cells and initiate antigen-specific responses. DCs produce several constitutive and inducible cytokines/chemokines that are responsible for activating and recruiting specific T-cell

populations. CCL18 and CCL19 direct naive T cells into the T-cell zone, while CCL2, CCL3, CCL17, and CCL22 attract activated and memory T cells. CCL17 is a highly selective chemoattractant for Th2 cells and CLA þ skin-homing memory T cells (Figure 2). CCL17 expression is upregulated in the skin of patients with psoriasis, cutaneous delayed type hypersensitivity, and atopic dermatitis (AD) as well as in the epithelium of mice with AD-like disease. Serum levels of CCL17 are increased in AD patients and correlate with disease severity, eosinophil number, and soluble E-selectin. Intradermal injection of CCL17 in mice results in dose-dependent recruitment of CD4 þ lymphocytes into the skin. CCL17 regulates its own production from keratinocytes and stimulates transcription of IL-4 mRNA, further highlighting its importance in driving Th2 responses. When CCL17 is neutralized (genetic deletion or antibody neutralization), mice are more resistant to allograft rejection, contact hypersensitivity, bacteriainduced fulminant hepatitis, and antigen-specific asthmatic reactions. All improvements were associated with reduced effector T-cell recruitment and decreased Th2 cytokine production. Although CCL17 is foremost recognized as a Th2 cell chemoattractant, its receptor is also found on DCs, Th1 cells, CD25 þ T cells, natural killer (NK) cells, NK T cells, platelets, and macrophages. Th1 cells and CD4 þ NK T cells migrate in response to CCL17. Receptor expression on CD25 þ regulatory T cells suggests that CCL17 may serve as an important immunomodulatory mediator of innate and acquired immune responses. In vitro treatment with CCL17 induces Ca2 þ mobilization, aggregation, and granule release from platelets. Recently, the effects of CCL17 on macrophages has been the subject of increasing interest. Macrophages undergo differentiation comparable to T-cell maturation (Th1/Tc1 vs. Th2/Tc2). Resident macrophages that are classically activated (caMf or M1 macrophages) by stimulation with microbial products exhibit strong antimicrobial innate immune responses such as production of inflammatory cytokines/chemokines, expression of inducible nitric oxide synthase, efficient killing activity, and induction of a Th1 response. In contrast, IL-4 and IL-13 drive the development of alternatively activated macrophages (aaMf), characterized by expression of anti-inflammatory/immunoregulatory cytokines/ chemokines, increased mannose receptor expression, arginase production, and decreased killing activity. Individuals with a predominance of aaMf are more susceptible to infection. AaMf inhibit the generation of caMf via production of IL-10 and CCL17. This

CHEMOKINES, CC / TARC (CCL17) 383 Adaptive immune response Memory T cells

Innate immune response NK cells NK T cells

CD45RO

CCR8

Regulatory T cells Th2 cells

CCL17

CD25 CCR4

CLA

Skin homing T cells

DCs

Platelets

caM /M1

Figure 2 CCL17 regulates both innate and adaptive immune responses. CCL17 is centrally involved in the adaptive immune response (shown in blue circle) as a potent chemoattractant of CD4 þ Th2 cells (CLA þ skin homing, CD25 þ regulatory, and CD45RO þ memory T cells). CCR4 is the receptor for CCL17. CCR4 is also expressed on several innate immune cells (shown in yellow circle). Natural killer (NK) cells and dendritic cells (DCs) chemotax in response to CCL17. Evidence suggests that CCL17 utilizes both CCR4 and CCR8 on NK cells. CCL17 triggers granule release and aggregation of platelets in a CCR4-dependent manner. CCL17 inhibits classical activation of macrophages (caMf), thereby suppressing type 1 innate immune responses. Natural killer T cells (NK T cells) and DCs play a fundamental role linking both innate and adaptive immune responses (shown in the overlapping green region). DCs produce several cytokines/chemokines that activate and attract innate cells while also serving as professional antigen-presenting cells priming antigenspecific responses.

has been demonstrated in murine models of thermal injury and sepsis. Thus, CCL17 functions as an effective promoter of type 2 responses primarily through its chemoattraction of Th2 cells while modulating type 1 responses by suppressing caMf.

Receptors Initial studies demonstrated high-affinity binding of radiolabeled CCL17 to Jurkat cells; CXCL8 or other CC chemokines could not compete with this binding. CCL17-induced T-cell chemotaxis was pertussis toxin sensitive suggesting involvement of a G-protein-coupled receptor. In a series of transfection studies, CCR4 was identified as the primary receptor for CCL17. CC chemokine receptor 4 (CCR4) is found on a variety of cells (Figure 2). CCL17 and CCR4 have been implicated in the pathogenesis of many allergic diseases as they are central mediators involved in the homing of T cells to the skin. In addition to CCL17, CCL22 is the only other ligand known to bind CCR4. Despite the fact that they bind the same receptor, the different binding affinities, expression patterns, and kinetics of these two chemokines suggests important nonredundant roles in vivo. CCL22 binds CCR4 with higher affinity and is a more potent inducer of integrin-dependent

T-cell adhesion. CCR4 internalization is rapid following CCL22 binding, but delayed after exposure to CCL17. CCL22 desensitizes CCR4 þ T cells to CCL17, while CCL17 has no effect on CCL22 signaling. CCL22 is subject to serine protease degradation whereas CCL17 is a much more stable ligand. Considering the abundant expression of CCL17 (and not CCL22) by structural cells, it is very likely that CCL17 acts to specifically arrest CCR4high cells (i.e., Th2 cells) in circulation while CCL22 subsequently directs these cells within the affected tissue. CCR8 plays a role in activation, migration, and proliferation of lymphoid cells. Early binding and chemotaxis studies also identified CCR8 as a potential receptor for CCL1, CCL4, and CCL17. However, no intracellular Ca2 þ changes were induced upon ligation of CCL4 or CCL17. CCL17-induced chemotaxis was attributed to low levels of CCR4 expression, as at physiologically relevant concentrations, CCL17 was unable to bind, inhibit CCL1-binding, or chemoattract CCR8high-expressing cells. However, the receptor usage of CCL17 may be cell type dependent as CCL17 is able to compete with CCL1 for binding in IL-2-activated adherent NK cells. CCL1 and CCL22 only partially inhibit CCL17-induced responses suggesting that CCL17 is able to act through both CCR4 and CCR8 in these cells.

384 CHEMOKINES, CC / TARC (CCL17)

CCL17 also interacts with both chemokine clearance receptors, the Duffy antigen/DARC and the D6 receptor. The Duffy antigen, found on red blood cells, binds both CC and CXC chemokines, while D6 is expressed on lymphatic endothelial cells and is specific for inflammatory CC chemokines. While ligand binding does not produce a functional signal, it does induce rapid internalization of these ligand/receptor complexes, facilitating regulation of inflammatory responses and return to homeostatic levels.

CCL17 in Th2-Type Respiratory Diseases CD4 þ T cells play a fundamental role in the pathogenesis of bronchial asthma and other allergic respiratory diseases. Cytokines/chemokines are pivotal mediators of inflammation, Th2 cytokine production, eosinophil and T-cell recruitment, and airway hyperresponsiveness (AHR) observed in asthmatic patients. When moDCs are treated in vitro, cells from allergic patients upregulate CCL17 expression compared to similarly treated cells from nonallergic patients. CCR4 is detected on virtually all T cells from endobronchial biopsies of asthmatic patients following allergen challenge. CCL17 concentrations are higher in the serum, airway epithelium, and bronchoalveolar lavage (BAL) fluid from asthmatics after antigen challenge. Children suffering from acute and chronic asthma exhibit high levels of plasma CCL17, which directly correlate with the severity of the disease. Individuals homozygous for the – 431C4T CCL17 polymorphism have more peripheral eosinophilia, are more susceptible to antigen challenge, produce higher levels of allergen-specific immunoglobulin E (IgE), and have a higher incidence of atopic asthma. Blocking CCL17 with a neutralizing antibody attenuates several aspects of pulmonary inflammation (eosinophilia, T-cell recruitment, and Th2 cytokine production) and AHR in a murine model of ovalbumin-induced asthma. While mice deficient for the CCL17 receptor showed no protection in this model, the CCR4
/
mice were protected against allergic airway inflammation initiated by chronic fungal challenge. These data clearly indicate that CCL17 plays a significant role in the inflammation and pathogenesis observed in asthmatic patients. In addition to asthma, CCL17 is centrally involved in the pathogenesis of several respiratory diseases. Allergic rhinitis is a condition characterized by eosinophil, mast cell, and Th2 cellular infiltration into the nasal mucosa. The epithelium and mononuclear cells in the nasal mucosa of allergic patients express increased levels of CCL17, compared to nonallergy patients. CCL17 is significantly increased in BAL

fluid of patients with eosinophilic pneumonia, an inflammatory lung disorder often caused by fungal or helminth infections or drug reactions. Concentrations of CCL17 directly parallel expression of the Th2 cytokines IL-5 and IL-13 and the number of CCR4 þ CD4 þ T cells in the BAL fluid. Inflammatory cell recruitment is central to the pathogenesis of interstitial lung disease. In murine bleomycin-induced pulmonary fibrosis and rat radiation pneumonitis models, CCL17 and CCR4 are upregulated in the lung during the disease. CCL17 was localized to epithelial cells (bleomycin model) or alveolar macrophages (radiation model) in the lung and CCR4 was predominantly found on lung macrophages as well as lymphocytes (only in the radiation model). Epithelial cells were also a prominent source of CCL17 in idiopathic pulmonary fibrosis patients. Neutralization of CCL17 resulted in significantly fewer T cells, macrophages, NK cells, and neutrophils recruited into the BAL fluid, as well as reduced fibrosis. In a murine Th2-mediated lung granuloma model, CCL17 neutralization decreased granuloma size and CCR4 þ cell recruitment and increased overall cytokine/ chemokine production. No CCL17- or CCR4-specific compounds have been developed for use in human trials. In Th2mediated respiratory diseases, anti-allergic and antiinflammatory agents result in decreased CCL17 expression, Th2 cell recruitment, CCR4 expression, eosinophilia, IgE production, and type 2 cytokine production, further highlighting the importance of CCL17 in Th2-mediated pathogenesis. See also: Allergy: Overview; Allergic Reactions; Allergic Rhinitis. Asthma: Overview; Acute Exacerbations. Bronchoalveolar Lavage. Chemokines. Dendritic Cells. Dust Mite. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Fibroblasts. G-Protein-Coupled Receptors. Interleukins: IL-4; IL-10; IL-13. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Monocytes; T cells; Pulmonary Macrophages. Platelets. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Alferink J, Lieberam I, Reindl W, et al. (2003) Compartmentalized production of CCL17 in vivo: strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen. Journal of Experimental Medicine 197(5): 585–599. Belperio JA, Dy M, Murray L, et al. (2004) The role of the Th2 CC chemokine ligand CCL17 in pulmonary fibrosis. Journal of Immunology 173(7): 4692–4698. Chvatchko Y, Hoogewerf AJ, Meyer A, et al. (2000) A key role for CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock. Journal of Experimental Medicine 191(10): 1755– 1764.

CHEMOKINES, CC / TECK (CCL25) 385 D’Ambrosio D, Albanesi C, Lang R, et al. (2002) Quantitative differences in chemokine receptor engagement generate diversity in integrin-dependent lymphocyte adhesion. Journal of Immunology 169(5): 2303–2312. Imai T, Yoshida T, Baba M, et al. (1996) Molecular cloning of a novel T cell-directed CC chemokine expressed in thymus by signal sequence trap using Epstein–Barr virus vector. Journal of Biological Chemistry 271(35): 21514–21521. Inngjerdingen M, Damaj B, and Maghazachi AA (2000) Human NK cells express CC chemokine receptors 4 and 8 and respond to thymus and activation-regulated chemokine, macrophage-derived chemokine, and I-309. Journal of Immunology 164(8): 4048–4054. Jakubzick C, Wen H, Matsukawa A, et al. (2004) Role of CCR4 ligands, CCL17 and CCL22, during Schistosoma mansoni egginduced pulmonary granuloma formation in mice. American Journal of Pathology 165(4): 1211–1221. Katakura T, Miyazaki M, Kobayashi M, Herndon DN, and Suzuki F (2004) CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages. Journal of Immunology 172(3): 1407– 1413. Katoh S, Fukushima K, Matsumoto N, et al. (2003) Accumulation of CCR4-expressing CD4 þ T cells and high concentration of its ligands (TARC and MDC) in bronchoalveolar lavage fluid of patients with eosinophilic pneumonia. Allergy 58(6): 518–523. Kawasaki S, Takizawa H, Yoneyama H, et al. (2001) Intervention of thymus and activation-regulated chemokine attenuates the development of allergic airway inflammation and hyperresponsiveness in mice. Journal of Immunology 166(3): 2055–2062. Mariani M, Lang R, Binda E, Panina-Bordignon P, and D’Ambrosio D (2004) Dominance of CCL22 over CCL17 in induction of chemokine receptor CCR4 desensitization and internalization on human Th2 cells. European Journal of Immunology 34(1): 231–240. Schuh JM, Power CA, Proudfoot AE, et al. (2002) Airway hyperresponsiveness, but not airway remodeling, is attenuated during chronic pulmonary allergic responses to Aspergillus in CCR4
/
mice. FASEB Journal 16(10): 1313–1315. Vestergaard C, Deleuran M, Gesser B, and Larsen CG (2004) Thymus- and activation-regulated chemokine (TARC/CCL17) induces a Th2-dominated inflammatory reaction on intradermal injection in mice. Experimental Dermatology 13(4): 265–271.

TECK (CCL25) K F Buckland and C M Hogaboam, University of Michigan, Ann Arbor, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Thymus-expressed chemokine (TECK) is a CC chemokine first identified in 1997. The sequence of TECK/CCL25 was selected by comparison of random sequencing products to previously described molecular sequences of murine CC chemokines. CCL25 is expressed predominantly in the thymus and its function is associated with T-cell development. In addition, CCL25 is highly expressed in the small intestine and has a pivotal role in the selective recruitment of lymphocytes to the epithelial lining of the small intestine. The function of CCL25 is comparable to

that of the lymphoid chemokines, CCL17, CCL19, CCL21, and CXCL12. CCL25 mRNA was also detected in murine pro-Tcells, a population of noncommitted intrathymic T-cell progenitor cells. Detection of mCCL25 mRNA in murine fetal thymus further supported early hypotheses that the role of CCL25 was early in thymic development. This lymphoid tissue chemokine is important in the migration, localization, and maturation of immature double positive thymocytes. In addition, CCL25 participates in the development and gut-homing of T-cell subsets and localization of immunoglobulin A (IgA)-secreting plasma cells within the gut associated lymphoid tissue. CCL25 exerts these functions primarily via binding of the CC chemokine receptor CCR9.

Introduction Thymus-expressed chemokine (TECK) is a CC chemokine that was first identified by Vicari and colleagues in 1997. This discovery was achieved by analysis of a murine cDNA library from RAG-1 deficient mice in experiments designed to identify novel genes involved in T-lymphocyte development. Initial analysis of the expression of the murine TECK transcript revealed expression predominantly in the thymus, hence thymus-expressed chemokine or TECK. It has since been designated as CCL25 according to the novel chemokine and chemokine receptor nomenclature.

Structure Following identification of murine CCL25, homologous genes were identified in human, rat, and hamster genomes. The nucleotide homology between human (hCCL25) and murine (mCCL25) transcripts of CCL25 is 71%. The amino acid sequence of hCCL25 is described in Figure 1. The unprocessed precursor is 150 amino acids long and has a molecular weight of 16 339 Da. Alternative splicing can yield two isoforms of hCCL25. Isoform 2 is an antagonist of isoform 1. Although CCL25 contains the conserved cysteine motif, which grants membership of the CC chemokine family, the structural relationship of CCL25 to other CC chemokines is distant and its homology low. Greatest amino acid identity is shared with CCL17. The percentage protein identity shared between CCL25 and some of the most closely related chemokines is outlined in Table 1. CCL25 displays differences from other chemokines even at the gene level. The hCCL25 gene is located on chromosome 19p13.2, not chromosome 17 as would be expected. This region is syntenic with mouse chromosome 8, where the mCCL25 gene has been mapped, while the genes encoding most chemokines are clustered on mouse chromosome 11.

386 CHEMOKINES, CC / TECK (CCL25) MNLWLLACLVAGFLGAWAPAVHTQ∗GVFEDCCLAYHYPIGWAVLRRAWTY IQEVSGSCNLPAAIFYLPKRHRKVCGNPKSREVQRAMKLLDARNKVFAKLHH NMQTFQAGPHAVKKLSSGNSKLSSSKFSNPISSSKRNVSLLISANSGL Figure 1 The amino acid sequence structure of CCL25 is given using the accepted standard lettering for amino acids. The asterisk ( * ) denotes the putative cleavage site for the signal peptide. Conserved double cysteines are present at positions 30 and 31. Disulfide bonds are predicted between cysteines at positions 30–58 and 31–75, highlighted in blue. Alternative splicing can occur at position 65 or 150, highlighted in red. Amino acids 1–23, highlighted in green, from a potential FT chain, a site for posttranslational modification. The sequence consisting of amino acids 24–150 is known to yield small inducible protein A25, black text.

Table 1 Percentage amino acid identity between CCL25 and other chemokines mCCL25 hCCL25 hCCL17 mCCL3 mCCL4 mCCL7 mCCL12 mCCL11

hCCL25

49.3 28.7 23.9 23.9 22.7 23.1 22.7

Regulation of Production and Activity CCL25 synthesis is highly regulated, as only a few specific cell populations are able to secrete this chemokine. Murine CCL25 mRNA is expressed at significant levels only in the thymus and small intestine, although low levels of mRNA were also detected in liver, brain, testis, and pro-T cells. The high thymic expression was revealed to be care of major histocompatibility complex (MHC) II þ CD11c þ thymic dendritic cells (DCs). In contrast, CCL25 expression is absent from CD11c
thymic DCs and neither bone marrow nor naive splenic DCs express CCL25. Thymic endothelium also expresses CCL25 at low levels, while endothelial cells are a major site of CCL25 expression in the small intestine. Both the production and the responses to CCL25 can be enhanced by antigen stimulation. Lipopolysaccharide stimulation can induce CCL25 expression in the spleen in vivo, while responsiveness of thymocytes to CCL25 is enhanced following T-cell receptor (TCR) stimulation and in CD69 þ thymocyte subsets. Thus costimulatory conditions may be required for CD8 þ T cells to retain CCL25 receptor expression and effectively migrate to the intestinal lamina propria.

Biological Function Chemokines such as CCL25 are a key component of the system of tissue-restricted recirculation of memory and effector lymphocytes. CCL25 expression in

human and mouse thymus was localized to thymic DCs. In contrast, stimulated and unstimulated human monocyte-derived DCs and bone marrowderived cells all lacked CCL25 expression. CCL25 mRNA was also detected in murine pro-T-cells, a population of noncommitted intrathymic T-cell progenitor cells. Alternatively these cells can deviate from T-cell lineage to become natural killer (NK) cells or DCs. Detection of mCCL25 mRNA in murine fetal thymus supported early hypotheses that the role of CCL25 was early in thymic development. mCCL25 is chemotactic for murine activated monocytes, DCs, thymocytes, and a human lymphocyte cell line, THP-1, following their activation with interferon gamma (IFN-g). In contrast CCL25 was inactive on neutrophils, bone marrow cells, splenic, and peripheral blood lymphocytes or NK cell-enriched populations in vitro. Further none of these was recruited following intraperitoneal injection of CCL25 in vivo. Chemokines such as CXCL12, CCL3, CCL21, and CCL25 are involved in the selection processes allowing T-cell progenitors (pro-T cells) to progress into functional single positive T cells. In the developing thymus maturing thymocytes migrate from the cortex to the medulla. Early in embryogenesis CCL25, CXCL12, and CCL21 are expressed but not the related chemokines CCL17, CCL19, or CCL22. CCL25 expression has been localized to MHC II þ N418 þ DCs in the thymic medullary stroma. This region of the thymus is where single positive thymocytes emigrate from, into the bloodstream. CCL25 induces chemotaxis of immature CD4 þ CD8 þ double positive and mature single positive CD4 þ or CD8 þ human thymocytes. Thus CCL25 is thought to induce chemotaxis of double positive thymocytes from the cortex to the medullary region of the thymus and also to regulate localization of single positive thymocytes within the medulla. Through the chemoattraction of thymocytes and macrophages, CCL25 produced by thymic DCs may be the driving chemokine in achieving the colocalization of DCs with thymocytes and macrophages required for clonal deletion of self-reactive thymocytes. However, CCL25 is not solely responsible

CHEMOKINES, CC / TECK (CCL25) 387

for chemotaxis of thymocytes, which can also respond to other chemokines including CXCL12. The specificity of CCL25 for particular thymocyte subsets is of great interest in further elucidating its function. Lymphocyte recirculation is tightly regulated by the coordinated expression of chemoattractant receptors and adhesion molecules. This ensures that lymphocytes, which have previously encountered antigen, home to the appropriate site in which antigen exposure may reoccur. CCL25 is highly expressed in the epithelial lining of the small intestine and strongly associated with intestinal homing of particular lymphocyte populations to this antigenrich site. Importantly, CCL25 expression is weak or absent in other segments of the intestinal tract including colon and stomach, which supports the high specificity of lymphocyte trafficking. This restricted expression profile also separates the function of CCL25 from CCL17, another CC chemokine that is highly expressed in the thymus and small intestine but in contrast to CCL25, is also expressed in lung, colon, and activated mononuclear leukocytes. The CCL25 receptor is expressed only by discreet subsets of memory CD4 þ and CD8 þ lymphocytes that also express the intestinal homing adhesion molecule a4b7 but not by other systemic memory populations. In the intestine CCL25 is expressed in CD11c þ dendritic stromal cells, and c-kit þ Lin
bone marrowderived cells chemotax toward CCL25. Thus CCL25 has a role in the formation of gut cryptopatches, an essential process in the generation of thymus-independent intraepithelial lymphocytes. CCL25 also has an important role in B-cell homing to the gut. CCL25 is abundantly expressed in epithelial cells lining the small intestine and is a potent and selective chemoattractant for immunoglobulin A (IgA)-secreting plasma cells. Oral antigens induce effector/memory cells that express essential receptors for intestinal homing, namely the integrin a4b7 and CCR9, the receptor for CCL25. This imprinting of gut tropism is mediated by DCs from Peyer’s patches in a positive feedback mechanism. Similar to T-cell development, bone marrow pre-pro B cells also migrate toward CCL25 while more mature bone marrow-derived B cells do not. Further, CCR9
/
mice had a reduction in pre-pro B-cell numbers. CCR9/CCL25 interaction provides a cell survival signal in certain cell populations. CCL25 selectively rescues T cells but not thymocytes from tumor necrosis factor alpha (TNF-a)-induced apoptosis via activation of livin, a member of the inhibitor of apoptosis (IAP) family. However, antibody neutralization of CCL25 did not prevent repopulation of fetal thymus with T-cell precursors. Anti-CCL25

antibody treatment in 2–4-week-old mice led to a significant reduction in the total number of CD8 þ gd and ab in the intraepithelial lymphocyte compartment demonstrating an important role for CCL25 in the development of these cell populations. Further, a reduction in TCRgd þ numbers in the intestinal mucosal epithelium correlated with an increased number present in lymph nodes and spleen in CCR9
/
mice supporting the requirement for CCL25/CCL25 receptor for homing of TCRgd þ intraepithelial lymphocytes. Emergence of double positive thymocytes and TCRab þ cells during embryogenesis was also delayed by approximately 1 day and pre-pro B-lymphocyte numbers were reduced. Therefore CCL25 has differential functions across separate cell types. Immature double positive thymocytes use CCL25 for differentiation, homing, development, maturation, and selection. Mature single positive thymocytes, T and B cells, use CCL25 for localization and cell homeostasis, and to resist apoptosis. In addition malignant cells have been shown to use CCL25 for inappropriate proliferation and resistance to apoptosis as well as infiltration.

Receptors CC chemokine receptor 9 (CCR9) is a receptor for CCL25. This G-protein-coupled receptor was additionally identified and named as CCR10, D6, and GPR-9-6 but these are in fact the same receptor and are henceforth known as CCR9. CCL25 selectively and efficaciously binds CCR9 but CCR9 is not selective for CCL25 but also binds CCL2–5, CCL7–8, and CCL12–13. CCR9 is highly expressed in the thymus and by T-lymphocyte subsets found in the small intestine. Expression has been localized to thymocytes and DCs in the thymus, and intraepithelial lymphocytes and lamina propria lymphocytes in the small intestine. Upregulation of CCR9 occurs during intraepithelial lymphocyte precursor differentiation allowing responsiveness to CCL25. CCR9 expression is at least several-fold greater in thymus than in spleen, bone marrow, lymph node, liver, or peripheral blood leukocytes. Activation of CCR9 leads to phosphorylation of glycogen synthase kinase 3b and forkhead transcription factor. This provides a cell survival signal. CCR9 also signals via activation of pkb/Akt, PI3-K, and mitogen-activated protein kinase (MAPK) but does not require MAPK for activation of chemotaxis. CCR9 is highly expressed by intestinal melanoma cells and abhorrent expression of CCR9 is associated with metastasis to the small intestine. CCR9/CCL25 play an important role in T-cell progenitor migration within the thymus. Immature

388 CHEMOKINES, CC / TECK (CCL25) CCR9 DN3

= GPCR

Subcapsular zone

DN4 pDP

= Thymocyte

DN2 DP CCL25 Thymocyte generation

DN1

Cortex 69+ SP

DC

Medulla SP

SP

Thymus Migration/ circulation

CCR9

CCX-CKR?

CCR11 TCR

Homing and expansion

EC CCL17

CD4 SP

CCR4

Immunity balance & regulation

DC

CCL25 CCR9 DC

Lung


 SP

TCR CD8 SP

TCR
 SP

Small intestine

Figure 2 CCL25 is important in the migration, localization, and maturation of. immature CD4 þ CD8 þ double positive thymocytes and TCRgd þ T cells. In addition, CCL25/CCR9 participate in the migration and development of intraepithelial lymphocytes of the small intestine. The role of an additional receptor for CCL25, CCX-CKR, is as yet unknown but it is highly expressed in the lung. DN, double negative thymocyte; DP, double positive thymocyte; GPCR, G-protein-coupled receptor; SP, single positive thymocyte; TCR, T-cell receptor.

double negative thymocytes do not express CCR9 but CCR9 is highly expressed in double positive thymocytes. Single positive CD8 þ thymocytes continue to express CCR9 as they mature into naive T cells and migrate out of the thymus. CCL25 induces migration of immature double positive and mature single positive thymocytes via CCR9. CCL25 is also involved in the development of ab and gd T-cell lineages. Approximately 50% of TCRgd þ thymocytes express CCR9 and are responsive to CCL25 compared to 3% of TCRgd
double negative cells. In TCRgd þ cells CCL25 response predominates over CXCL12. Circulating TCR gd þ T cells and CD8 þ T cells from lymph node, spleen, Peyer’s patches, and small intestine express CCR9. Conversely CD4 þ thymocytes have lost expression of CCR9 and this correlates with a lack of CCR9 on CD4 þ T cells in secondary lymphoid organs. The expression of chemokine receptors selective for intestinal trafficking is further supported by

upregulation of expression of a4b7, an intestinal homing integrin. Only Peyer’s patch DCs are able to induce high-level expression of a4b7 and response to CCL25 sufficient for gut homing of T cells. In correlation, CCR9 expression is maintained following activation of CD8 þ ab lymphocytes, with ovalbumin and lipopolysaccharide, in mesenteric lymph nodes but is rapidly downregulated on the same lymphocyte subsets if activated in the peripheral lymph nodes. Circulating CCR9 þ T cells that lack a4b7 are likely to have only recently exited the thymus. Surprisingly, gene deletion of CCR9 had no major effect on intrathymic T-cell development. Functional redundancy revealed by CCR9
/
mice may be due to CCL25 binding by CCX-CKR.

CCL25 in Respiratory Diseases CCL25 expression is high in the thymus where its function is associated with T-cell development, and

CHEMOKINES, CC / TECK (CCL25) 389

in the small intestine where it has a pivotal role in the selective recruitment of lymphocytes to the epithelial lining. The functional requisite for CCL25/CCR9 in T-cell trafficking is not fully understood as mCCL25 also binds mCCX-CKR, a receptor expressed throughout the body and abundantly in the lung. The affinity of this receptor for CCL25 is high but the efficacy of this interaction is low. As in the mouse, binding of human CCL25 to CCX-CKR also fails to elicit functional responses. However significant diversity between human and murine CCX-CKR biology has hampered investigation of the in vivo function of this ligand–receptor interaction. CCL25 mRNA has been detected in the tonsils of humans, mice, and pigs. Mucosal sites such as the intestinal and respiratory epithelium present highly specialized challenges to the immune system as they are constantly exposed to potential pathogens. The palatine and nasopharangeal tonsils produce IgA and may play a similar role in aerodigestive tract as the Peyer’s patches of the gastrointestinal tract. Leukocytes are central in the pathophysiology of infectious and allergic respiratory diseases. Chemokines mediate the migration and activation of lymphocytes in normal and inflammatory conditions. CCL25 is known to have roles in both primary and secondary lymphoid organs, the thymus and small intestine, respectively. These different lymphoid organs and other immunological sites such as the thymus, spleen, small intestine, lymph nodes, and intestinal and respiratory epithelium can be discussed as separate compartments. Indeed, each has a specific role in lymphocyte trafficking. However, therapeutic strategies must consider that the immune system functions as a highly integrated system in vivo. Any imbalance or dysregulation, of for instance a chemokine, for example, CCL25, in one apparently isolated tissue site can be cause or effect (or exacerbation) of disease at a distant site. Despite the lack of expression of CCL25 in the lung this chemokine may still have an important contribution to the immunological component of certain respiratory diseases due to its pivotal role in thymocyte development and trafficking of these T-lymphocyte progenitor cells (Figure 2). CCL25- or CCR9-selective compounds are unlikely to be useful acute therapy in respiratory

diseases. However, CCR9/CCL25 expression or responsiveness may prove a good phenotypic marker perhaps for postimmunological therapy assessment of thymic involution and atrophy. Indeed immunodeficient states caused by chemotherapy or following respiratory infection may be managed better for the long term by stimulating thymic reactivation, perhaps involving increased CCL25/CCR9 activity. See also: Chemokines. Chemokines, CC: TARC (CCL17). Chemokines, CXC: CXCL12 (SDF-1). Dendritic Cells. Endothelial Cells and Endothelium. G-Protein-Coupled Receptors. Leukocytes: T cells.

Further Reading Bowman EP, Kuklin NA, Youngman KR, et al. (2002) The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells. Journal of Experimental Medicine 195: 269–275. Gosling J, Dairaghi DJ, Wang Y, et al. (2000) Cutting edge: identification of a novel chemokine receptor that binds dendritic celland T cell-active chemokines including ELC, SLC, and TECK. Journal of Immunology 164: 2851–2856. IUIS/WHO Subcommittee on Chemokine Nomenclature (2003) Chemokine/chemokine receptor nomenclature. Cytokine 21(1): 48–49. Kunkel EJ, Campbell DJ, and Butcher EC (2003) Chemokines in lymphocyte trafficking and intestinal immunity. Microcirculation 10: 313–323. Mora JR, Bono MR, Manjunath N, et al. (2003) Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells. Nature 424: 88–93. Nomiyama H, Amano K, Kusuda J, et al. (1998) The human CC chemokine TECK (SCYA25) maps to chromosome 19p13.2. Genomics 51: 311–312. Uehara S, Song K, Farber JM, et al. (2002) Characterization of CCR9 expression and CCL25/thymus-expressed chemokine responsiveness during T cell development: CD3(high)CD69 þ thymocytes and gammadeltaTCR þ thymocytes preferentially respond to CCL25. Journal of Immunology 168: 134–142. Vicari AP, Figueroa DJ, Hedrick JA, et al. (1997) TECK: a novel CC chemokine specifically expressed by thymic dendritic cells and potentially involved in T cell development. Immunity 7: 291–301. Youn BS, Kim CH, Smith FO, et al. (1999) TECK an efficacious chemoattractant for human thymocytes, uses GPR-9-6/CCR9 as a specific receptor. Blood 94: 2533–2536. Zaballos A, Gutierrez J, Varona R, et al. (1999) Cutting edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for the chemokine TECK. Journal of Immunology 162: 5671–5675.

390 CHEMOKINES, CXC / CXCL12 (SDF-1)

CHEMOKINES, CXC Contents

CXCL12 (SDF-1) IL-8 CXCL9 (MIG) CXCL10 (IP-10) CXCL1 (GRO1)–CXCL3 (GRO3)

CXCL12 (SDF-1) R M Strieter and B N Gomperts, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

sequence is highly conserved among species with 99% homology between mouse and humans. This highlights the importance of this molecule in development and regeneration.

& 2006 Elsevier Ltd. All rights reserved.

Abstract SDF-1 (stromal cell-derived factor-1) is also known as CXCL12, and is a CXC chemokine that binds to the CXC chemokine receptor, CXCR4. It is widely expressed and highly conserved having 99% homology with lower vertebrates, suggesting that it is important in primordial development. Two splice variants, SDF-1a and SDF-1b, have been identified and determined to have different regulation and functions. The SDF-1/CXCR4 complex results in activation of a variety of signal transduction pathways. SDF-1 plays a role in several functions, including trafficking of hematopoietic and nonhematopoietic adult stem cells, development of the central nervous system, vasculature and hematopoietic cells, cell motility, chemotaxis and adhesion, and cell proliferation and survival. It also inhibits lymphotropic strains of HIV-1 to enter into CXCR4 þ cell lines. SDF-1 plays a critical role in lung repair after injury through recruitment of circulating mesenchymal progenitor cells such as fibrocytes and progenitor epithelial cells. SDF-1 is also an important molecule in invasion and organ-specific metastasis of cancer; hence, SDF-1 is one of the most important known chemokines.

Introduction Stromal cell-derived factor-1 (SDF-1) was discovered independently by three different groups and has been known as pre-B-cell growth-stimulating factor/ stromal cell-derived factor-1 (PBSF/SDF-1) or TPA repressed gene 1 (TPAR1). SDF-1 is referred to as CXCL12 according to the consensus chemokine nomenclature. There are two alternatively spliced variants of CXCL12, CXCL12-a (68 residues) and CXCL12-b (72 residues), which have been shown to have different functions. A new isoform, CXCL12-g, was recently described. Subsequent studies show CXCL12 to be a very efficient chemoattractant for lymphocytes and monocytes. CXCL12 is constitutively expressed in a wide range of tissues and the CXCL12

Structure Although genes encoding other members of the chemokine family are localized on chromosome 4q or 17q, the human CXCL12 gene maps to chromosome 10q. cDNA of CXCL12-a and CXCL12-b encode proteins comprised of 89 and 93 amino acids, respectively. The nucleotide sequence contains a single open reading frame of 267 nucleotides encoding the 89 amino acid polypeptide. CXCL12 contains four conserved cysteine residues of the chemokine family. It does not contain the ELR motif, although its role in angiogenesis is controversial. The genomic structure of the CXCL12 gene reveals that human CXCL12-a and CXCL12-b are encoded by a single gene that arises by alternative splicing. The strong evolutionary conservation and unique chromosomal localization of the CXCL12 gene suggest that CXCL12-a and CXCL12-b may have important functions distinct from those of other members of the chemokine family. The N-terminus appears to be critical for the activity of CXCL12, in that peptides corresponding to the N-terminal 9 residues of CXCL12 have been shown to bind to CXCR4 and to induce intracellular calcium and chemotaxis in T lymphocytes. The individual peptides have similar activities to CXCL12 but are less potent. A dimer of CXCL12 amino acid residues 1–9 demonstrates enhanced activity. The Nterminal peptides can act as CXCR4 agonists or antagonists. Nuclear magnetic resonance spectroscopy demonstrates that CXCL12 is a monomer with a disordered N-terminal region (residues 1
8), and differs from other chemokines in the packing of the hydrophobic core and surface charge distribution (Figure 1).

CHEMOKINES, CXC / CXCL12 (SDF-1) 391

5′ flanking region

Exon 1

Exon 2

Exon 3

Exon 4

GCGCG 5′

3′ CXCL12-
CXCL12-

(a)

∗∗

Residues 123456789

89 amino acids

Isoform splice site

93 amino acids

Refresh motif 12-17 C-terminus

N-terminus

9 amino acid binding site

Loop region docking site

Serum cleavage 67

Residues 3 N-terminus

C-terminus

CXCL12-


72

Residues 3 N-terminus

CXCL12-

C-terminus

(b) Figure 1 (a) Schematic cartoon of the CXCL12 gene demonstrating the approximate splice site for CXCL12-a and CXCL12-b. (b) Line diagram of CXCL12 structure with binding and docking sites and sites of cleavage to create CXCL12-a and CXCL12-b isoforms. *Only Lys-1 and Pro-2 are directly involved in receptor activation.

Regulation of Production and Activity Synthesis

CXCL12 is produced by a wide variety of cell types. mRNA for CXCL12 has been found in lung, brain, heart, liver, kidney, spleen, lymph nodes, stomach, gastrointestinal tract, muscle, lung-derived fibroblasts, and especially high levels are found in bone marrow stromal cells, especially those that support B cell lymphopoiesis. CXCL12 is regulated through complicated interactions among cytokines/growth factors, chemokines, and other regulatory factors. For example, CXCL12 gene expression is regulated by the transcription factor hypoxia-inducible factor1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of CXCL12 in ischemic tissues in direct proportion to reduced oxygen tension. Interferon gamma significantly reduces CXCR4 expression and SDF-1-induced cell migration and proliferation of CXCR4-positive cells. Processing

CXCL12-a and CXCL12-b are secreted as full-length molecules. When exposed to human serum, fulllength CXCL12-a (1–68) undergoes processing first at the C-terminus to produce CXCL12-a (1–67) and

then at the N-terminus to produce CXCL12-a (3–67). By contrast, full-length CXCL12-b (1–72) is processed only at the N-terminus to produce CXCL12-b (3–72). Serum processing of CXCL12-a at the C-terminus reduces the ability of the polypeptide to bind to heparin and to stimulate B cell proliferation and chemotaxis. The additional processing at the N-terminus renders both forms of CXCL12 unable to bind to heparin and activate cells. The differential processing of CXCL12-a and CXCL12-b allows precise regulation of CXCL12’s biologic activity (Figure 1). The processing of CXCL12-g has not been determined. Activation

The binding of CXCL12 to its receptor, CXCR4, results in the activation of several signaling pathways. It has been suggested that after binding to CXCR4, CXCL12 initially triggers dimerization of the receptor. In addition, there is evidence that CXCR4 has to be included in membrane lipid rafts in order to more efficiently interact with other surface receptors, downstream proteins of signal transduction pathways, and therefore to induce chemotaxis. CXCR4 activation leads to dissociation of the heterotrimeric protein complex (Gabg) to a and bg subunits that

392 CHEMOKINES, CXC / CXCL12 (SDF-1)

CXCL12 Plasma membrane Integrins

CXCR4 Src ptk

JAKS P

Gi
Gi








Fak

P13Ky



Pax

src PDK1

PIP3

Ras mTOR

Akt

PTEN SHIP

Rho

Rac

cdc42

Raf 1B PKC

NF-B

Actin polymerization Cytoskeletal rearrangements Chemotaxis

mek Erk1/2

Transcriptional activation Cell growth/survival Figure 2 Simplified version of signal transduction pathways activated by the CXCL12/CXCR4 axis that are relevant to cell growth, proliferation, and chemotaxis. Activation of these pathways varies among cell types.

results in phosphorylation of the tyrosine residues of the C-terminus via JAK2 and JAK3 kinases. The phosphorylated CXCR4/CXCL12 complex is then rapidly internalized through a mechanism involving G-protein-coupled receptor kinases followed by the binding of b-arrestin. This process terminates CXCR4 receptor signaling; and activates a number of signaling pathways, including calcium flux, and focal adhesion components, such as proline-rich kinase-2 (Pyk-2), p130Cas, focal adhesion kinase, paxilin, Crk, Crk-L, protein kinase C, phospholipase C-g, mitogen-activated protein kinase (MAPK) p42/ 44-ELK-1, PI3-kinase, AKT, and nuclear factor kappa B (NF-kB) cascades. PI3-kinase ultimately signals through Rho/Rac/cdc42 to upregulate downstream effector proteins involved in actin polymerization and cytoskeletal rearrangements resulting in chemotaxis and adhesion molecule upregulation. CXCL12 activation of CXCR4 under conditions in which the receptor is stimulated with less than saturable concentrations results in movement of CXCR4 into clathrin-coated pits and from there into endosomes, for trafficking back to the plasma membrane to be re-expressed on the cell surface. However, if CXCR4 is exposed to prolonged saturating concentrations of CXCL12 a significant proportion of CXCR4 from endosomes moves to the lysosome for degradation.

Hematopoietic cells lacking protein tyrosine phosphatases (SHIP1 and SHIP2) have altered chemotaxis to CXCL12, implying that they are important in modulating activation by CXCR4 and the absence of membrane-expressed hematopoietic phosphatase, CD45, also results in reduced chemotaxis in lymphocytes. After CXCL12 stimulation, CD45 has also been shown to interact with CXCR4 in the membrane lipid rafts (Figure 2).

Biological Functions Development

Targeted disruption of the CXCL12 gene in mice results in perinatal mortality at around e15.5. The numbers of B cell progenitors in mutant embryos are severely reduced in fetal liver and bone marrow, but myeloid progenitors are only reduced in the bone marrow and not in the fetal liver, indicating that CXCL12 is responsible for B cell lymphopoiesis and bone marrow myelopoiesis. Similarly, mice deficient for CXCR4 die perinatally and display profound defects in the hematopoietic and nervous systems. CXCR4-deficient mice have severely reduced B cell lymphopoiesis, reduced myelopoiesis in fetal liver, and a virtual absence of myelopoiesis in bone marrow. However, T-cell lymphopoiesis is unaffected.

CHEMOKINES, CXC / CXCL12 (SDF-1) 393

Furthermore, CXCR4 is expressed in developing vascular endothelial cells, and mice lacking CXCR4 or CXCL12 have defective formation of the large vessels supplying the gastrointestinal tract. They are also defective in cardiogenesis and have abnormal cerebellar development. The similarities between the CXCL12- and CXCR4-deficient mice indicate that CXCR4 is the primary physiological receptor for CXCL12, suggesting a monogamous relationship between CXCR4 and CXCL12. This receptor ligand selectivity is unusual among chemokines and their receptors, as it is common to have several ligands that bind to one receptor.

Trafficking

Varying SDF-1 expression levels in different cells and tissues create a chemokine gradient that regulates trafficking of cells that express the CXCR4 receptor namely: hematopoietic stem/progenitor cells such as CD34 þ hematopoietic stem cells, pre-B cells, and T cells (in particular naive T cells). In addition, more recently, CXCR4 has been found on primordial germ cells, neural stem cells, liver stem cells, muscle stem cells, retinal pigment epithelial progenitor cells, intestinal epithelial cells, fibrocytes/circulating mesenchymal stem cells, and circulating progenitor epithelial cells in regeneration of tissue after injury, implying that the CXCL12/CXCR4 biological axis is also important in trafficking of nonhematopoietic stem/progenitor cells.

Cell Motility and Chemotaxis

CXCL12 has been shown to induce cell motility through rearrangement of F-actin bundles. CXCL12 may accumulate in tissues after binding to heparin sulfate proteoglycans, which then provides a directional CXCL12 gradient for chemotaxis. CXCL12induced cell movement can be inhibited by blocking the PI3Kinase-AKT pathway in many cell lines, including A549 and H157 non-small cell lung cancer (NSCLC) cell lines. Interestingly, metastases of human NSCLC cells in severe combined immunodeficiency (SCID) mice are 99% positive for CXCR4 expression, whereas significantly fewer cells of the primary tumor express CXCR4. Under hypoxic conditions, nuclear HIF-1a induces increased levels of CXCR4 expression on these cancer cell lines, which leads to increased ability of these tumor cells to invade and metastasize. PI3-kinase inhibitors and overexpressing PTEN inhibit activation of HIF-1a and CXCR4 expression.

Cell Adhesion

CXCL12 via CXCR4 increases adhesion of cells to fibrinogen, fibronectin, stroma, and endothelial cells, through activation of adhesion molecules, such as integrins. Cell Secretion

After stimulation by CXCL12, cells secrete more matrix metalloproteinases (e.g., MMP-9), nitric oxide, and vascular endothelial growth factor (VEGF), probably through activation of the NF-kB pathway. Cell Proliferation and Survival

In some experimental conditions, CXCL12 was found to stimulate proliferation and survival of hematopoietic cells, astrocytes, and some tumor cell lines through activation of the PI3-kinase-AKT and MAPK p42/44 pathways. CXCL12 has also been found to be a survival factor for glioblastoma cells and induces proliferation in a dose-dependent manner. This proliferation correlates with phosphorylation and activation of both ERK 1/2 and AKT, and these kinases are independently involved in glioblastoma cell proliferation.

Receptor CXCR4 is a G-protein-coupled seven transmembrane receptor that was originally cloned as an orphan chemokine receptor and was known as LESTR or fusin. CXCR4 is expressed on the cell surface of most leukocytes, including all B cells, and monocytes and most T lymphocyte subsets, but just weakly on NK cells. It is also expressed on nonhematopoietic cells such as endothelial cells and epithelial cells, and adult stem cells such as fibrocytes and circulating progenitor epithelial cells. CXCR4 is also an essential cofactor for T-tropic HIV-1 and HIV-2 env-mediated fusion and entry into CD4 þ lymphocytes. Recently, a second alternatively spliced CXCR4 receptor was cloned and named CXCR4-Lo. CXCR4Lo has lower gene expression in most tissues than CXCR4 except in the spleen and lung; its function is not yet known.

CXCL12 in Respiratory Diseases Lung Injury and Repair

Circulating mesenchymal progenitor cells, fibrocytes, express CXCR4 and traffic to the lungs in response to CXCL12 and mediate fibrosis in a murine model of bleomycin-induced pulmonary fibrosis. Specific

394 CHEMOKINES, CXC / CXCL12 (SDF-1)

neutralizing antibodies to CXCL12 inhibit pulmonary recruitment of these circulating fibrocytes and attenuate lung fibrosis. In addition, CXCL12 expression is increased in the submucosal glands, ducts, and airway epithelium in a mouse tracheal transplant model, which provides a chemokine gradient for CXCR4 þ circulating progenitor epithelial cells to traffick and contribute to repair of the injured airway. The mucus in the submucosal gland lumen provides proteoglycans to depot CXCL12 and create the CXCL12 gradient for chemotaxis. The CXCL12 expression in epithelial cells moves apically to the airway surface during regeneration of the airway. Moreover, CXCL12 can induce neovascularization, which is critical in acute and chronic lung injury and repair. For example, bronchial biopsy specimens from patients with asthma show a significant increase in the number of blood vessels and colocalization of CXCL12-positive endothelial cells together with CXCL12-positive macrophages and T lymphocytes in the submucosa compared with control subjects. These findings suggest that CXCL12 may play a role in remodeling of asthmatic airways via angiogenesis. Lung Cancer

CXCL12 is an important molecule in lung cancer invasion and metastasis and is also important in the development of lung metastases from tumors from other organs (e.g., breast, renal cell carcinoma, rhabdomyosarcoma, etc.). Both NSCLC tumor specimens resected from patients and NSCLC cell lines express CXCR4, but not CXCL12. NSCLC cell lines undergo chemotaxis in response to CXCL12. CXCL12–CXCR4 activation of NSCLC cell lines show PI3 kinase activation, intracellular calcium mobilization and MAPK activation with enhanced ERK-1/2 phosphorylation without change in either proliferation or apoptosis. Target organs in a murine model that are the preferred destination of human NSCLC metastases produce higher levels of CXCL12 than the primary tumor, thereby creating a chemotactic gradient. The administration of specific neutralizing anti-CXCL12 antibodies to SCID mice with human NSCLC abrogates organ metastases, without affecting primary tumor-derived angiogenesis. The CXCL12–CXCR4 biological axis is therefore involved in regulating the metastasis of NSCLC. In addition, the ability of NSCLC cells to metastasize correlates with their expression of CXCR4, which is upregulated under conditions of hypoxia through HIF-1a. Inhibition of PI3-kinase or overexpression of PTEN reduces CXCR4 expression and their metastatic potential.

Small cell lung cancer (SCLC) cells express high levels of functional CXCR4 receptors for CXCL12. CXCL12 induces integrin activation, which results in increased adhesion of SCLC cells to fibronectin and collagen. This is mediated by activation of integrins and CXCR4, and is inhibited by CXCR4 antagonists. Stromal cells protect SCLC cells from chemotherapy-induced apoptosis, and this protection can also be antagonized by CXCR4 inhibitors. Renal cell carcinoma commonly metastasizes to other organs such as the adrenal gland and lung, both of which express high levels of CXCL12. Both heterotopic and orthotopic mouse models of tumors from a renal carcinoma cell line develop lung metastases, which can be abrogated by treating the mice with neutralizing antibodies to CXCL12. In summary, CXCL12 is a pleiotropic chemokine with a variety of functions beyond that of leukocyte trafficking that are relevant to the lung. See also: Adhesion, Cell–Cell: Epithelial. Angiogenesis, Angiogenic Growth Factors and Development Factors. Chemokines. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells. Extracellular Matrix: Basement Membranes; Collagens; Matrix Proteoglycans; Surface Proteoglycans. Fibroblasts. G-Protein-Coupled Receptors. Human Immunodeficiency Virus. Hypoxia and Hypoxemia. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Neutrophils; T cells. Matrix Metalloproteinases. Myofibroblasts. Pulmonary Fibrosis. Signal Transduction. Stem Cells. Vesicular Trafficking.

Further Reading Gazitt Y (2004) Homing and mobilization of hematopoietic stem cells and hematopoietic cancer cells are mirror image processes, utilizing similar signaling pathways and occurring concurrently: circulating cancer cells constitute an ideal target for concurrent treatment with chemotherapy and antilineage-specific antibodies. Leukemia 18: 1–10. Kucia M, Jankowski K, Reca R, et al. (2004) CXCR4-SDF-1 signaling, locomotion, chemotaxis and adhesion. Journal of Molecular Histology 35: 233–245. Luster AD (1998) Chemokines–chemotactic cytokines that mediate inflammation. New England Journal of Medicine 338: 436– 445. Mackay CR (2001) SDF-1. In: Oppenheim JJ and Feldman M (eds.) Cytokine Reference, pp. 1119–1127. San Diego: Academic Press. Murdoch C (2000) CXCR4: chemokine receptor extraordinaire. Immunology Reviews 177: 175–184. Murphy PM (2002) International Union of Pharmacology. XXX. Update on chemokine receptor nomenclature. Pharmacological Reviews 54: 227–229. Phillips RJ, Burdick MD, Hong K, et al. (2004) Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. Journal of Clinical Investigation 114: 438–446.

CHEMOKINES, CXC / IL-8

IL-8 R M Strieter, M P Keane, and J A Belperio, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract CXCL8/IL-8 is a member of the CXC chemokine family. The CXC chemokines can be further divided into two groups on the basis of a structure/function domain consisting of the presence or absence of three amino acid residues (Glu–Leu–Arg; ‘ELR’ motif) that precede the first cysteine amino acid residue in the primary structure of these cytokines. CXCL8/IL-8 and the other ELR þ CXC chemokines are chemoattractants for neutrophils and act as potent angiogenic factors. CXCL8/IL-8 binds to the CXCR1 and CXCR2 receptors, which are found on neutrophils, T lymphocytes, monocytes/macrophages, eosinophils, basophils, keratinocytes, and mast cells and endothelial cells. CXCL8/IL-8 has been shown to have an important role in the recruitment of neutrophils in bacterial pneumonia and to correlate with the development and mortality of adult respiratory distress syndrome. Furthermore, CXCL8/IL-8 also has a key role in the vascular remodeling that is seen in pulmonary fibrosis.

Introduction CXCL8 is a member of the CXC chemokine family and is also known as IL-8. The CXC chemokines can further be divided into two groups on the basis of a structure/function domain consisting of the presence or absence of three amino acid residues (Glu–Leu– Arg; ‘ELR’ motif) that precede the first cysteine amino acid residue in the primary structure of these cytokines. CXCL8/IL-8 and the other ELR þ CXC chemokines are chemoattractants for neutrophils and act as potent angiogenic factors.

Structure The precursor molecule of CXCL8/IL-8 consists of 99 amino acids with an associated 20 amino acid signal sequence. Several mature forms of CXCL8/IL8 have been identified, that are a result of repeated NH2-terminal amino acid cleavage, including the major 72 amino acid mature form. The 72 amino acid form of CXCL8/IL-8 binds to neutrophils twofold more than the 77 amino acid form, and is twoto threefold more potent in inducing cytochalasin B-treated neutrophil degranulation. Interestingly, the 77 amino acid form can induce apoptosis in leukemic cells, whereas the 72 amino acid form does not. The NH2-terminus of CXCL8/IL-8, similar to other CXC chemokines that bind and activate neutrophils, contains three highly conserved amino acid residues consisting of Glu–Leu–Arg (the ELR motif). The COOH-terminus of CXCL8/IL-8 contains a

395

heparin-binding domain, which may be important in binding to glycosaminoglycans.

Regulation of Production and Activity The gene for CXCL8/IL-8 is found on human chromosome 4, q12–21, and consists of four exons and three introns. The 50 -flanking region of CXCL8/IL-8 contains the usual ‘CCAAT’ and ‘TATA’ box-like structures. In addition, this region has a number of potential binding sites for several nuclear factors. The CXCL8/IL-8 promoter region is regulated in a cell-specific fashion requiring a nuclear factor kappa B (NF-kB) element plus either activating protein 1 (AP-1) or a C/EBP (NF-IL-6) element under conditions of transcriptional induction with tumor necrosis factor alpha (TNF-a) or interleukin-1 (IL-1). Although the CXCL8/IL-8 promoter has two C/EBP (50 and 30 ) cis-elements with NF-kB nested between them, the 50 -C/EBP element appears to be the only C/ EBP element involved in transcriptional regulation of CXCL8/IL-8. In specific cell lines, the AP-1 along with NF-kB or C/EBP and NF-kB cis-elements is sufficient for full transcriptional activation of the CXCL8/IL-8 promoter. Members of the NF-kB/rel and C/EBP families of transcriptional factors can interact on a protein:protein level via the Rel:basic leucine zipper (bZIP) domain of these proteins, respectively. In order for full transcriptional activation of the promoter, there is a cooperative interaction between all three major cis-elements (AP-1, 50 -C/EBP and NF-kB). While the promoter region of CXCL8/ IL-8 gene has been extensively studied, the mechanisms related to signal transduction and optimal transactivation of the CXCL8/IL-8 gene have been less studied. Mitogen-activated protein (MAP) kinases are necessary to optimally induce the gene expression and protein production of CXCL8/IL-8 from airway epithelial cells in response to TNF-a. TNF-a activation of the MAP kinases is able to achieve the induction of CXCL8/IL-8 gene expression and protein production by NF-kB-dependent, -independent, and posttranscriptional mechanisms. Direct inhibition of the MAP kinases (extracellular regulated kinase (ERK) and c-jun N-terminal protein kinase (JNK)) and NF-kB, but not p38, decreases TNF-a-induced transcription of the CXCL8/IL-8 promoter. Inhibition of JNK signaling reduces TNFa-induced transcription of the CXCL8/IL-8 promoter in an NF-kB-dependent manner, whereas inhibition of ERK impairs TNF-a-induced transcription of the CXCL8/IL-8 promoter in an NF-kB-independent and AP-1-dependent manner. Activation of the p38 MAP kinase is important for promoting TNFa-induced CXCL8/IL-8 protein production in a

396 CHEMOKINES, CXC / IL-8

posttranscriptional manner. These findings would also be relevant to downstream signal transduction events related to activation of Toll-like receptors and IL-1 type I receptor for the induction of CXCL8/IL-8 gene expression and protein production.

Biological Function CXCL8/IL-8 is a potent recruiter of neutrophils and induces angiogenesis. It is also chemotactic for eosinophils. CXCL8/IL-8 has dose-dependent effects on basophils, which can lead to either stimulation or inhibition of release of both LTB4 and histamine. CXCL8/IL-8 also leads to activation of basophils with enhanced binding of basophils to endothelial cells. CXCL8/IL-8 is not chemotactic for monocytes; however, stimulation of monocytes with CXCL8/ IL-8 leads to a rise in cytosolic calcium and generation of reactive oxygen species. CXCL8/IL-8 can selectively inhibit the IL-4-induced IgE and IgG4 production from B cells. CXCL8/IL-8 can induce migration and proliferation of keratinocytes (Figure 1).

Receptors Chemokine activities are mediated through pertussissensitive G-protein-coupled receptors. CXCL8/IL-8 binds to the CXCR1 and CXCR2 receptors, which are found on neutrophils, T lymphocytes, monocytes/macrophages, eosinophils, basophils, keratinocytes, and mast cells and endothelial cells. While the transmembrane and the second and third intracellular/cytoplasmic domains of these receptors are well conserved, the NH2- and COOH-terminal ends of these receptors are variable. The intracellular COOH-terminus of these receptors is rich in serine and threonine amino

acid residues that may be important in phosphorylation and signal coupling via G proteins.

CXCL8/IL-8 in Respiratory Disease CXC chemokines have been found to play a significant role in mediating neutrophil infiltration in the lung parenchyma and pleural space in response to endotoxin and bacterial challenge. Passive immunization with neutralizing CXCL8/IL-8 antibodies blocked 77% of endotoxin-induced neutrophil influx in the pleura of rabbits. However, in the context of microorganism invasion, depletion of a CXC chemokine and reduction of infiltrating neutrophils may have a major impact on the host (Figure 2). CXCL8/IL-8 has been implicated in mediating neutrophil sequestration in the lungs of patients with pneumonia. CXCL8/IL-8 has been found in the bronchoalveolar lavage of patients with community acquired pneumonia and nosocomial pneumonia. While there is no murine structural homolog for CXCL8/IL-8, both CXCL1 and CXCL2/3 have been found in murine models of Klebsiella pneumoniae, Pseudomonas aeruginosa, Nocardia asteroides, and Host defense/pneumonia Acute lung injury/ARDS CXCL8

Angiogenesis Tumor growth Figure 2 Role of CXCL8/IL-8 in respiratory disease.

Monocyte/macrophage Lymphocytes

Epithelial cells

CXCL8

Chemotaxis

Endothelial cells

Neutrophils Eosinophils

Fibroblasts Neutrophils

Chemotaxis

Keratinocytes Basophils

Endothelial cells

Angiogenesis Figure 1 Source and biological functions of CXCL8/IL-8.

Pulmonary fibrosis

CHEMOKINES, CXC / IL-8

Aspergillus fumigatus pneumonia. In a model of A. fumigatus pneumonia, neutralization of TNF resulted in marked attenuation of the expression of CXCL1 and CXCL2/3 that was paralleled by a reduction in the infiltration of neutrophils and associated with increased mortality. In addition, Laichalk and associates administered a TNF agonist peptide consisting of the 11 amino acid TNF binding site (TNF70–80) to animals intratracheally inoculated with K. pneumoniae and found markedly elevated levels of CXCL2/3 associated with increased neutrophil infiltration. Depletion of CXCL2/3 during the pathogenesis of murine K. pneumoniae pneumonia resulted in a marked reduction in the recruitment of neutrophils to the lung that was paralleled by increased bacteremia and reduced bacterial clearance in the lung. Since ELR þ CXC chemokine ligands in the mouse use the CXC chemokine receptor, CXCR2, Standiford and associates used specific neutralizing antibodies to CXCR2 and demonstrated that blocking CXCR2 results in markedly reduced neutrophil infiltration in response to P. aeruginosa, N. asteroides, and A. fumigatus pneumonias. The reduction in neutrophil elicitation was directly related to reduced clearance of the microorganisms and increased mortality in these model systems. These studies have established the critical importance of ELR þ CXC chemokines in acute inflammation and the innate immune response to a variety of microorganisms. Sekido and associates demonstrated that CXCL8/ IL-8 significantly contributed to reperfusion lung injury in a rabbit model of lung ischemia-reperfusion injury. Reperfusion of the ischemic lung resulted in the production of CXCL8/IL-8, which correlated with maximal pulmonary neutrophil infiltration. Passive immunization of the animals with neutralizing antibodies to CXCL8/IL-8 prior to reperfusion of the ischemic lung prevented neutrophil extravasation and tissue injury, suggesting a causal role for CXCL8/IL-8 in this model. Ventilator-induced lung injury in a murine model is associated with increased expression of CXCL1 and CXCL2 that parallels lung injury and neutrophil recruitment. Furthermore, these levels correlated with NF-kB activation. CXCR2–/– mice were protected from ventilatorinduced lung injury. Several studies have demonstrated that CXCL8/ IL-8 levels correlate with the development and mortality of the acute respiratory distress syndrome. Of particular interest is the study of Donnelly and colleagues, which correlated early increases in CXCL8/IL-8 in bronchoalveolar lavage fluid (BALF) with an increased risk of subsequent development of adult respiratory distress syndrome (ARDS), and also

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demonstrated that alveolar macrophages were an important source of CXCL8/IL-8 prior to neutrophil influx. High concentrations of CXCL8/IL-8 were found in BALF from trauma patients, some within 1 h of injury and prior to any evidence of significant neutrophil influx. Patients who progressed to ARDS had significantly greater BALF levels of CXCL8/IL-8 than those who failed to develop this condition. Levels of CXCL8/IL-8 in plasma, as opposed to lavage, were not found to be significantly different between patients who did or did not develop ARDS. Furthermore, there is an imbalance in the expression of ELR þ (including CXCL8/IL-8) as compared to ELR– CXC chemokines from BALF of patients with ARDS as compared to controls. This imbalance correlated with angiogenic activity and both procollagen I and procollagen III levels in BALF. These findings suggest that CXCL8/IL-8 and other CXC chemokines have an important role in the fibroproliferative phase of ARDS via the regulation of angiogenesis. Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology that is characterized by the accumulation of neutrophils within the airspace and mononuclear cells within the interstitium, followed by the progressive deposition of collagen within the interstitium and subsequent destruction of lung tissue. While the mechanisms of cellular injury and the role of classic inflammatory cells remain unclear, increases in neutrophils in BALF and in lung tissue have been demonstrated from patients with IPF. While the number or proportion of neutrophils in BALF does not correlate with activity of alveolitis and has limited prognostic value, declines in BALF neutrophils typically occur among patients exhibiting favorable responses to therapy. Neutrophilic alveolitis has been described in humans with IPF, collagen vascular diseases with associated ILD, as well as diverse animal models of pulmonary fibrosis. CXCL8/IL-8 is significantly elevated in IPF, as compared to either normal or sarcoidosis patients, and correlates with BALF presence of neutrophils. The alveolar macrophage is an important cellular source of CXCL8/IL-8 in IPF. In addition, these studies have suggested that levels of CXCL8/IL-8 in IPF may correlate with a worse prognosis. While studies have suggested an important role for CXCL8/IL-8 in mediating neutrophil recruitment, CXC chemokines have been found to exert disparate effects in regulating angiogenesis. This latter issue is relevant to IPF, as the pathology of IPF demonstrates features of dysregulated and abnormal repair with exaggerated angiogenesis, fibroproliferation, and deposition of extracellular matrix, leading to progressive fibrosis and loss of lung function. There have been limited investigations to delineate factors that

398 CHEMOKINES, CXC / CXCL9 (MIG)

may be involved in the regulation of this angiogenic activity during pulmonary fibrosis. In IPF lung tissue there is an imbalance in the presence of CXC chemokines that behave as either promoters of angiogenesis CXCL8/IL-8 or inhibitors of angiogenesis CXCL10. This imbalance favors augmented net angiogenic activity. Lung tissue from IPF patients has elevated levels of CXCL8/IL-8, as compared to control lung tissue, and demonstrates in vivo angiogenic activity that can be significantly attributed to CXCL8/IL-8. Immunolocalization of CXCL8/IL-8 demonstrated that the pulmonary fibroblast was the predominant interstitial cellular source of this chemokine, and areas of CXCL8/IL-8 expression were essentially devoid of neutrophil infiltration. This supports an alternative biological role for CXCL8/IL-8 or other ELR þ CXC chemokines in the interstitium of IPF lung tissue. The pulmonary fibroblast is the predominant cellular source of CXCL8/IL-8 in the interstitium of IPF, supporting the notion that the pulmonary fibroblast has a pivotal role in mediating the angiogenic activity during the pathogenesis of IPF. Indeed, the pulmonary fibroblast has received increasing attention as a pivotal cell in the pathogenesis of IPF. Relative levels of CXCL8/IL-8 and CXCL10 from IPF pulmonary fibroblast conditioned media demonstrated a significant imbalance favoring CXCL8/IL-8-induced angiogenic activity. In contrast, normal pulmonary fibroblasts had greater levels of CXCL10 that favored a net inhibition of angiogenesis. The difference in expression of CXCL8/IL-8 and CXCL10 between IPF and control pulmonary fibroblasts lends further support to the notion of a phenotypic difference between IPF and normal pulmonary fibroblasts, which has been well described. See also: Acute Respiratory Distress Syndrome. Angiogenesis, Angiogenic Growth Factors and Development Factors. Chemokines. Chemokines, CXC: CXCL1 (GRO1)–CXCL3 (GRO3). Extracellular Matrix: Basement Membranes. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Neutrophils; Monocytes; T cells. Pneumonia: Overview and Epidemiology. Pulmonary Fibrosis. Transgenic Models. Tumors, Malignant: Overview.

pneumonia and adult respiratory distress syndrome. Infection and Immunity 61: 4553. Donnelly SC, Strieter RM, Kunkel SL, et al. (1993) Interleukin-8 and development of adult respiratory distress syndrome in atrisk patient groups. Lancet 341: 643–647. Greenberger MJ, Strieter RM, Kunkel SL, et al. (1996) Neutralization of macrophage inflammatory protein-2 attenuates neutrophil recruitment and bacterial clearance in murine Klebsiella pneumonia. Journal of Infectious Disease 173: 159. Jordana M, Schulman J, McSharry C, et al. (1988) Heterogeneous proliferative characteristics of human adult lung fibroblast lines and clonally derived fibroblasts from control and fibrotic tissue. American Review of Respiratory Disease 137: 579. Keane MP, Arenberg DA, Lynch JP, et al. (1997) The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. Journal of Immunology 159: 1437. Keane MP, Donnelly SC, Belperio JA, et al. (2002) Imbalance in the expression of CXC chemokines correlates with bronchoalveolar lavage fluid angiogenic activity and procollagen levels in acute respiratory distress syndrome. Journal of Immunology 169: 6515. Koch AE, Polverini PJ, Kunkel SL, et al. (1992) Interleukin-8 (IL8) as a macrophage-derived mediator of angiogenesis. Science 258: 1798. Laichalk LL, Bucknell KA, Huffnagle GB, et al. (1998) Intrapulmonary delivery of tumor necrosis factor agonist peptide augments host defense in murine Gram-negative bacterial pneumonia. Infection and Immunity 66: 2822. Lynch JP, Standiford TJ, Kunkel SL, Rolfe MW, and Strieter RM (1992) Neutrophilic alveolitis in idiopathic pulmonary fibrosis: the role of interleukin-8. American Review of Respiratory Disease 145: 1433. Sheppard D (2001) Pulmonary fibrosis: a cellular overreaction or a failure of communication? Journal of Clinical Investigation 107: 1501. Southcott AM, Jones KP, Li D, et al. (1995) Interleukin-8, differential expression in lone fibrosing alveolitis and systemic sclerosis. American Journal of Respiratory and Critical Care Medicine 151: 1604. Strieter RM (2002) Interleukin-8: a very important chemokine of the human airway epithelium. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L688. Strieter RM, Kunkel SL, Elner VM, et al. (1992) Interleukin-8: a corneal factor that induces neovascularization. American Journal of Pathology 141: 1279. Strieter RM, Kunkel SL, Showell H, et al. (1989) Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-a, LPS, and IL-1b. Science 243: 1467. Strieter RM, Polverini PJ, Kunkel SL, et al. (1995) The functional role of the ‘ELR’ motif in CXC chemokine-mediated angiogenesis. Journal of Biological Chemistry 270: 27348.

CXCL9 (MIG) Further Reading Belperio JA, Keane MP, Burdick MD, et al. (2002) Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilatorinduced lung injury. Journal of Clinical Investigation 110: 1703. Broaddus VC, Boylan AM, Hoeffel JM, et al. (1994) Neutralization of IL-8 inhibits neutrophil influx in a rabbit model of endotoxin-induced pleurisy. Journal of Immunology 152: 2960. Chollet-Martin S, Montravers P, Gibert C, et al. (1993) High levels of interleukin-8 in the blood and alveolar spaces of patients with

P C Fulkerson and M E Rothenberg, Cincinnati Children’s Hospital, Cincinnati, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Human CXCL9 was identified in 1993 by a differential hybridization screening technique, comparing cDNA libraries

CHEMOKINES, CXC / CXCL9 (MIG) 399 obtained from an unstimulated and interferon gamma (IFN-g)activated macrophage cell line. CXCL9 is a member of the alpha or CXC subfamily of chemokines. The CXC chemokines can be subdivided based on the presence or absence of a tripeptide motif Glu–Leu–Arg (ELR) N-terminal to the conserved CXC region. CXCL9, a non-ELR-containing CXC chemokine, mediates most of its biological function through binding to CXCR3, a seven-transmembrane-domain receptor coupled to G proteins. CXCL9 primarily attracts activated T lymphocytes, preferentially of the Th1 phenotype, which express high levels of CXCR3. The expression of CXCL9 is induced primarily by the Th1-associated cytokine IFN-g and correlates with tissue infiltration of T lymphocytes in a number of Th1-associated diseases, suggesting that CXCL9 plays an important role in the regulation of effector cell recruitment to sites of inflammation. Remarkably, CXCL9 also regulates eosinophil accumulation in experimental asthma, a Th2-associated lung disease. In addition, CXCL9 has potent angiostatic activity, inhibiting blood vessel growth in wound repair and inhibiting tumor growth and tumor-associated vessel expansion.

preferentially of the Th1 phenotype, which express high levels of CXCR3.

Structure CXCL9 is a member of the alpha or CXC subfamily of chemokines. CXC chemokines have a single amino acid that separates invariant cysteines one and two (of four). The CXC chemokines can be subdivided based on the presence or absence of a tripeptide motif Glu–Leu–Arg (ELR) N-terminal to the conserved CXC region. The gene coding for CXCL9, a nonELR-containing CXC chemokine, maps to the CXC gene cluster on human chromosome 4 (4q21) and murine chromosome 5 (5 53 cM). The gene is organized into four exons and the arrangement is highly conserved between humans and mice (Figure 1). The 2.5 kb human mRNA transcript and 1.3 kb murine transcript is translated into a 125 and 126 amino acid protein, respectively. The human and murine CXCL9 proteins are 68% homologous. Despite differences in primary sequences, chemokines have a remarkably similar three-dimensional structure, comprised of a short N-terminal region, a large core, which is stabilized by disulfide bonds and hydrophobic interactions, and a C-terminal a-helix. The most highly conserved sequence among chemokines is the four cysteine residues. The first and third cysteines and the second and fourth cysteines are linked by conserved disulfide bonds. While chemokines share four common secondary structural elements, three b-strands arranged in a single antiparallel b-sheet and one a-helix, most of the variation in chemokine amino acid sequence occurs in the Nand C-terminal regions. Structure–function studies have shown that chemokines have two main sites of interaction with their receptors, one in the N-terminus and the other in the N-loop region after the second cysteine. Deletion of just a few of the N-terminal

Introduction Cytokines play a central role in macrophage physiology, both as macrophage activators (e.g., interferons (IFN)) and mediators of macrophage activity (e.g., chemokines). Monokine induced by interferon gamma (IFN-g) (MIG, CXCL9) was discovered in an effort to identify genes induced during macrophage activation. A differential hybridization screening technique, comparing cDNA libraries obtained from an unstimulated and IFN-g-activated mouse macrophage cell line, was utilized to initially identify the murine CXCL9 gene. The murine CXCL9 cDNA was then used to screen a cDNA library made from an IFN-g-activated human macrophage cell line. This led to the identification of a new member of the chemokine gene family, human MIG/CXCL9, in 1993. CXCL9 mediates most of its biological function through binding to CXCR3, a seven-transmembrane-domain receptor coupled to G proteins. CXCL9 primarily attracts activated T lymphocytes, Human CXCL9/SCYB9 gene Exon 1

Exon 2

Exon 3

Exon 4

(a)

Murine CXCL9/Scyb9 gene Exon 1

Exon 2

Exon 3

Exon 4

(b) CDS Figure 1 Human and murine CXCL9 gene organization. The gene coding for CXCL9 maps to the CXC gene cluster on human chromosome 4 (4q21) and murine chromosome 5 (5 53 cM). The gene is organized into four exons and the arrangement is highly conserved between humans and mice. CDS, coding sequence.

400 CHEMOKINES, CXC / CXCL9 (MIG)

residues of CXCL9 results in a loss of receptor binding capacity. In addition, the non-ELR CXC chemokines contain a C-terminal segment rich in positively charged amino acids. Notably, CXCL9 has a unique extended basic C-terminal region, which when truncated results in a dramatic decrease in its biological activity.

Regulation of CXCL9 Production and Activity The IFNs are an important group of cytokines that mediate some of their biological effects through regulation of specific RNA and protein expression in a responding cell. The expression of CXCL9 is primarily dependent on the Th1 cytokine IFN-g, but in its absence, IFN-a or IFN-b is able to induce CXCL9 expression. The IFN-g-induced transcriptional activation of CXCL9 depends upon responsive elements in the promoter region of the gene, which are recognized by signal transducers and activators of transcription (STAT)1 or STAT1-containing factors. The promoter of CXCL9 also contains regions that mediate transcriptional activation in response to tumor necrosis factor alpha (TNF-a). IFN-g and TNF-a synergize to induce the expression of several genes, including CXCL9. In addition to inducing expression, transcription factors modulate inflammatory responses by negatively regulating chemokine expression. The Th2associated transcription factor STAT6 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR-g) have been shown to inhibit IFN-g-induced expression of CXCL9. The N-terminal region of most chemokines is crucial for receptor binding and signaling activities.

Amino peptidases or endopeptidases that process chemokines at the N-terminus play an important role in the regulation of chemokine activity. The membrane-associated protease dipeptidyl peptidase IV/CD26 cleaves two amino acids from polypeptides with a proline, alanine, or hydroxyproline at the second position. The removal of the N-terminal dipeptide from CXCL9 results in decreased receptor binding capacity and a dramatic decrease in chemotactic activity. In addition, matrix metalloproteinase (MMP) 9, another class of chemokine-processing proteases, cleaves CXCL9 within its extended C-terminus. C-terminal truncation of CXCL9 results in a significant decrease in its biological activity. Since the highly basic C-terminal segment of CXCL9 may associate with extracellular matrixpoteoglycans, localization and establishment of chemotactic gradients may also be affected by protease processing of CXCL9.

Biological Function Chemokines are chemotactic cytokines that orchestrate the migration and activation of leukocyte populations under baseline (homeostatic) and inflammatory conditions. The predominant function of CXCL9 is the recruitment of primed T lymphocytes to the sites of inflammation (Figure 2). CXCL9 has been associated with the infiltration and retention of activated T lymphocytes in inflammatory diseases of the skin, joints, and central nervous system. Furthermore, studies have implicated CXCL9 as a critical mediator of primed T-lymphocyte trafficking in transplant models. In addition to promoting leukocyte recruitment, chemokines can also function as natural

Angiostasis inhibits capillary growth R3

CXC

CXCR3 Promotes T and B lymphocyte recruitment and function

CCR3 Inhibits eosinophil recruitment

Figure 2 CXCL9 regulates lymphocyte and eosinophil chemoattraction and inhibits angiogenesis. The predominant function of CXCL9 is the recruitment of primed T lymphocytes expressing high levels of CXCR3 to the sites of inflammation. In addition to promoting leukocyte recruitment, chemokines can also function as natural antagonists to prevent the recruitment or activation of a different cellular population. In animal models, CXCL9 has been shown to function as a potent inhibitor of eosinophil recruitment toward diverse stimuli. In addition, CXCL9 inhibits blood vessel growth in wound repair and tumor models.

CHEMOKINES, CXC / CXCL9 (MIG) 401

antagonists to prevent the recruitment or activation of a different cellular population. In animal models, CXCL9 has been shown to function as a potent inhibitor of eosinophil recruitment toward diverse stimuli. Although cell trafficking is considered the central task of chemokines, there is increasing evidence that a variety of other leukocyte functions may also be modulated by chemokines. CXCL9 has also been shown to stimulate effector functions, such as cytokine production (by recruited T lymphocytes) and promote T-cell proliferation. In a host defense model using gene-targeted mice, murine CXCL9 contributed to the humoral response to a bacterial pathogen, suggesting that CXCL9 not only recruits T cells to sites of inflammation, but also maximizes interactions among activated T cells, B cells, and dendritic cells within lymphoid organs. Angiogenesis, the growth of new capillaries from either pre-existing vessels or from progenitor endothelial cells, is an important biological event that is essential to several physiologic and pathologic processes, including development, wound repair, and tumor growth. CXC chemokines have pivotal, yet opposing, roles in the control of inflammation and angiogenesis, as a result of the shared expression of their specific receptors by both leukocytes and endothelial cells. The ELR-containing CXC chemokines are potent promoters of angiogenesis. In contrast, members that lack the ELR motif, such as CXCL9, are potent inhibitors of angiogenesis. It is proposed that the angiostatic properties of CXCL9 are important to prevent unlimited vessel growth in wound repair.

Receptors Chemokines mediate their functions through binding to seven-transmembrane-domain receptors coupled to G proteins. The receptors often recognize more than one chemokine, and alternatively, several chemokines can bind to multiple receptors. CXCL9 shares its primary receptor, CXCR3, with two other non-ELR-containing CXC chemokines, CXCL10 and CXCL11. CXCR3 is expressed on a small subset of circulating blood T cells, B cells, and natural killer (NK) cells, but T-cell activation greatly enhances CXCR3 expression. CXCR3 is preferentially expressed on Th1 lymphocytes, suggesting that CXCR3 and its ligands are more active in the setting of Th1driven inflammatory responses. Besides being an agonist for CXCR3, CXCL9 has been shown to act as a natural antagonist for CCR3, a Th2-associated chemokine receptor highly expressed on eosinophils. Studies have demonstrated that CXCL9 inhibits CCR3-mediated functional responses in both human

and mouse eosinophils. This suggests that the polarization of an inflammatory response may be enhanced with the expression of CXCR3 ligands, including CXCL9, that attract Th1 cells, but also concomitantly block the migration of Th2 cells in response to CCR3 ligands.

CXCL9 in Respiratory Diseases Lung transplantation is a therapeutic option for many patients with end-stage pulmonary disorders. Acute and chronic lung rejection is a common complication. Acute rejection is characterized by perivascular and peribronchiolar leukocyte infiltration and is the major risk factor for the development of chronic lung allograft rejection, or bronchiolitis obliterans syndrome (BOS), the leading cause of morbidity and mortality post-lung transplantation. BOS is a chronic inflammatory process characterized by persistent peribronchiolar leukocyte infiltration that eventually invades and disrupts the basement membrane, submucosa, and airway epithelium. The persistent leukocyte infiltration is followed by an aberrant repair process, with increased matrix deposition and granulation tissue formation, ultimately leading to obliteration of airways. Elevated levels of CXCL9 (and the other CXCR3 ligands) have been found in the bronchoalveolar lavage (BAL) fluid from patients with acute and chronic lung allograft rejection, as compared with healthy lung transplant recipients. In a murine model of BOS, neutralization of CXCL9, or its receptor CXCR3, resulted in inhibition in the recruitment of mononuclear cells and a marked reduction in extracellular matrix deposition and airway obliteration. Similarly, CXCL9 neutralization resulted in attenuation of intragraft mononuclear cells and decreased parameters in an animal model of acute lung allograft rejection. These studies demonstrate the importance of CXCL9/CXCR3 interactions during both acute and chronic allograft rejection, suggesting that CXCL9 may be an important ligand in promoting the continuum of acute to chronic rejection. Diffuse lung injury is a frequent complication of allogeneic stem cell transplantation (SCT). Idiopathic pneumonia syndrome (IPS), a noninfectious diffuse lung injury, is associated with a high degree of mortality. The pathophysiology of IPS involves toxic damage due to irradiation and chemotherapy, a donor T-cell response, and the production of inflammatory cytokines. IPS is characterized by a diffuse pneumonitis involving both alveolar and interstitial space, as well as mononuclear cell infiltrates around vascular and bronchial structures. These pathologic changes are further associated with decreased

402 CHEMOKINES, CXC / CXCL10 (IP-10)

pulmonary function. In a murine SCT model, increased BAL fluid level of CXCL9 is associated with recruitment of CXCR3 expressing donor CD8 þ T cells to the lung after SCT. Neutralization of CXCL9 reduces the IPS severity and the recruitment of donor T cells to the lung after SCT. Together with the lung rejection studies, the significant decrease in lung injury and pathology in these models has been predominantly attributed to reduced effector cell recruitment and underscores the importance of the CXCL9/CXCR3 interaction in inflammatory diseases in the lung. In addition to being associated with the chemoattraction of Th1-associated effector cells into the lung, CXCL9 has been shown to inhibit the accumulation of cells in Th2-mediated asthma models. One of the hallmarks of allergic airway disease is the accumulation of an abnormally large number of inflammatory cells, such as eosinophils and T lymphocytes, into the lung. In experimental asthma models, CXCL9 has been shown to negatively regulate eosinophil recruitment into the lung. With no detectable CXCR3 on the surface of eosinophils, CXCL9 inhibited eosinophil chemoattraction in vitro. Moreover, intravenous administration of low doses of CXCL9 (B10– 30 mg kg
1) induced strong and specific inhibition of eosinophil recruitment into the lung in response to a variety of stimuli. Importantly, CXCL9 also inhibited a CCR3-mediated functional response, superoxide anion formation, in eosinophils. Regulation of eosinophil accumulation and activation in the lung is important since recent studies have demonstrated dramatic protection from experimental asthma in eosinophil-deficient animal models. Tumor cells have evolved the ability to regulate the angiogenic process through an imbalance in the regulatory factors resulting in an environment favoring angiogenesis. Studies have shown that the balance in expression between the angiogenic and the angiostatic CXC chemokines is correlated with the aggressiveness and metastic potential of human nonsmall cell lung carcinoma (NSCLC). Overexpression of CXCL9, an angiostatic CXC chemokine, at the tumor site resulted in inhibition of NSCLC tumor growth and metastasis via a decrease in tumor-derived vessel density. These findings support the importance of CXCL9 in inhibiting NSCLC tumor growth by attenuation of tumor-derived angiogenesis and demonstrate the potential of delivery of potent angiostatic CXC chemokines as a therapeutic option for NSCLC. See also: Allergy: Overview. Asthma: Overview. Chemokines, CXC: CXCL10 (IP-10). G-Protein-Coupled Receptors. Leukocytes: Eosinophils; T cells.

Further Reading Baggiolini M (1998) Chemokines and leukocyte traffic. Nature 392: 565–568. Baggiolini M, Dewald B, and Moser B (1997) Human chemokines: an update. Annual Review of Immunology 15: 675–705. Cascieri MA and Springer MS (2000) The chemokine/chemokinereceptor family: potential and progress for therapeutic intervention. Current Opinion in Chemical Biology 4: 420–427. Chada S, Ramesh R, and Mhashilkar AM (2003) Cytokine- and chemokine-based gene therapy for cancer. Current Opinion in Molecular Therapeutics 5: 463–474. Farber JM (1997) Mig and IP-10: CXC chemokines that target lymphocytes. Journal of Leukocyte Biology 61: 246–257. Liao F, Rabin RL, Yannelli JR, et al. (1995) Human mig chemokine: biochemical and functional characterization. Journal of Experimental Medicine 182: 1301–1314. Luster AD (1998) Chemokines: chemotactic cytokines that mediate inflammation. New England Journal of Medicine 338: 436– 445. Moser B and Loetscher P (2001) Lymphocyte traffic control by chemokines. Nature Immunology 2: 123–128. Moser B, Wolf M, Walz A, and Loetscher P (2004) Chemokines: multiple levels of leukocyte migration control. Trends in Immunology 25: 75–84. Park MK, Amichay D, Love P, et al. (2002) The CXC chemokine murine monokine induced by IFN-g (CXC chemokine ligand 9) is made by APCs, targets lymphocytes including activated B cells, and supports antibody responses to a bacterial pathogen in vivo. Journal of Immunology 169: 1433–1443. Romagnani P, Lasagni L, Annunziato F, Serio M, and Romagnani S (2004) CXC chemokines: the regulatory link between inflammation and angiogenesis. Trends in Immunology 25: 201–209. Rothenberg ME (2000) Basic science of chemokines and their receptors. In: Rothenberg ME (ed.) Chemokines in Allergic Disease, pp. 1–67. New York: Dekker. Schwarz MK and Wells TN (2002) New therapeutics that modulate chemokine networks. Nature Reviews: Drug Discovery 1: 347–358. Zlotnik A and Yoshie O (2000) Chemokines: a new classification system and their role in immunity. Immunity 12: 121–127.

CXCL10 (IP-10) F Kheradmand and D B Corry, Baylor College of Medicine, Houston, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Interferon-inducible protein of 10 kDa (IP-10, CXCL10) is the tenth member of the CXC family of small chemotactic cytokines and plays an essential role in the recruitment of T-helper-1 (Th1) cells, natural killer (NK) cells, macrophages, and dendritic cells into sites of tissue inflammation. In addition to leukocyte trafficking, CXCL10 binds to several G-protein-coupled receptors and induces a variety of cellular effects such as inhibition of endothelial cell proliferation, inhibition of growth factor-dependent hematopoiesis, and tumor necrosis. In humans, diverse cellular responses to CXCL10 are mediated through its two cell surface receptor isoforms that have distinct tissue expression patterns. CXCL10 acts as an antagonist of CCR3, a T-helper-2

CHEMOKINES, CXC / CXCL10 (IP-10) (Th2) cell-specific chemokine receptor through competition with eotaxin (CCL11), a functional ligand for this receptor. Modulation of CXCL10 is implicated as a mediator of several pathological conditions of the lung such as sarcoidosis, pulmonary fibrosis, emphysema, and asthma. In mice, overexpression of CXCL10 results in exaggerated Th2 immune responses to antigen in a murine model of allergic lung disease, whereas deletion of this gene results in worsening of pulmonary fibrosis in a bleomycin-based model of acute lung injury. Excessive production of CXCL10 by human lung T cells is associated with smoking-induced emphysema. Activated human macrophages obtained from the lungs of patients with emphysema showed high expression of CXCR3, a functional CXCL10 receptor, ligation of which induced secretion of macrophage metalloelastase (matrix metalloproteinase 12; MMP-12), an elastolytic proteinase implicated in lung destruction in emphysema.

Introduction As part of a directed search for cytokine-responsive genes, a gene with a predicted polypeptide size of 10 kDa (interferon-inducible protein of 10 kDa, IP10, CXCL10) was discovered. Subsequently, a variety of cells including T cells, endothelial cells, monocytes, fibroblasts, and keratinocytes were shown to release CXCL10 in response to interferon gamma (IFN-g) and lipopolysaccharide (LPS). CXCL10 peptide shows approximately 40% homology to the other members of the CXC family, in particular interleukin-8 (IL-8) and platelet factor 4 (PF4). Further characterization of the amino acid sequence of this protein showed that the first two of four conserved cysteines are separated by one amino acid (CXC, where X could be any amino acid). Thus, the common name IP-10 was changed to CXCL10 using the chemokine systematic nomenclature. Consistent with its anti-angiogenic effects, this cytokine is shown to play a prominent role in T-cell-dependent tumor immunity and, most recently, upregulation of this chemokine is reported in chronic and acute inflammatory diseases such as acute allograft rejection, central nervous system inflammation in multiple sclerosis, psoriasis, and emphysema (see Figure 1).

Structure The human CXCL10 gene is located on chromosome 4 (q12–21) in a cluster that contains many other members of the CXC family such as IL-8 and PF4. Mouse and human CXCL10 have 67% sequence identity while the two other mouse chemokines that bind to the same CXCR3 receptor, CXCL9, and CXCL11, are less than 30% identical to CXCL10. Nuclear magnetic resonance (NMR) spectroscopy further showed that CXCL10 and CCL11 bind in a similar manner to both CCR3 and CXCR3. A ‘twostep’ model describes binding of CXCL10 to its receptor, where in the first step, the docking domain or the region between the first pair of conserved cysteines and the first b-strand binds to a region of the N-terminus of CXCR3. In the second or activation step, residues near the N-terminus of CXCL10 bind to a second region on the receptor that can, only in the case of CXCL10, induce an active conformation. This second step is not fulfilled upon binding of CXCL10 to CCR3, providing an atomic basis for the inhibition of this receptor by CXCL10.

Regulation of Production and Activity Transient and rapid increase in CXCL10 mRNA and protein synthesis in response to IFN-g and LPS was first reported in a monocytic cell line, but was later reported in a variety of primary cells. Additionally, stimulation of cells with double-stranded RNA has been shown to induce the transcription of CXCL10 in a manner dependent on intact interferon-stimulated response element (ISRE) and nuclear factor kappa B (NF-kB) binding sites within its promoter. Thus, in addition to IFN-g, multiple Toll-like receptors (TLRs) such as TLR-3 ligand or double-stranded RNAs can also induce upregulation of this chemokine. Immunohistochemical analysis has shown that bronchial epithelium is an important source of

Protective functions

Tumor immunity Lung fibrosis T-cell host defense

403

Pathological diseases

CXCL10

Emphysema Allograft rejection Sarcoidosis Asthma

LPS Figure 1 Biological functions of CXCL10. Many cells of the lung including epithelial cells, macrophages, and T cells express CXCL10 upon activation by IFN-g or LPS. CXCL10 plays a protective role in T-cell-dependent tumor immunity and host defense. Under pathological conditions, CXCL10 is associated with emphysema, allograft rejection, sarcoidosis, and exaggerated allergic lung inflammation.

404 CHEMOKINES, CXC / CXCL10 (IP-10)

CXCL10, which serves to recruit activated T-helper1 (Th1) cells for normal host defense against intracellular pathogens of the lung. Induction of CXCL10 mRNA in response to IFN-g is seen as early as 30 min, with peak levels seen after 5 h. Signaling intermediates requisite to CXCL10 gene expression include Jak-2 and STAT1. Interestingly, prostaglandin E2, histamine, and vasoactive intestinal peptide, which all inhibit Jak-2 and STAT1, also inhibit IFNg-dependent induction of CXCL10. Peroxisome proliferator-activated receptor-g (PPAR-g), a member of the nuclear hormone receptor superfamily, originally shown to play an important role in adipocyte differentiation and glucose homeostasis, also regulates inflammatory responses by inhibiting IFN-g-induced mRNA and protein expression of CXCL10 in endothelial cells, although the molecular mechanism for this inhibition is not understood.

Biological Function The most notable biological activity of CXCL10 is its ability to direct the recruitment of Th1 and related effector cells in acute and chronic inflammatory conditions. Along with the other CXCL chemokine members, CXCL10 must bind to cell surface heparan sulfate proteoglycans to establish the necessary chemokine gradients required for extravasation of these critical cellular mediators of host defense and tissue transplant rejection. As with other chemokines, the functions of CXCL10 are redundant with respect to other class-specific chemokines. In experimental models of transplantation, anti-CXCL10 monoclonal antibodies prolonged allograft survival but, surprisingly, CXCL10-deficient mice acutely rejected the same allografts. This can be partly explained by redundancy in the activity of two other members of the CXC family, CXCL9 (Mig) and CXCL11 (I-TAC), which share the same receptor as CXCL10. Nonetheless, in support of an important role for CXCL10 in allograft rejection, compared with wild-type donors, use of CXCL10 null donor hearts grafted into wild-type mice reduced intragraft expression of cytokines, chemokines, and their receptors, and associated leukocyte infiltration, and graft injury. These findings support the notion that tissue-specific generation of CXCL10 in response to inflammation is sufficient for the progressive infiltration and amplification of multiple effector pathways, and that targeting of CXCL10 can prevent acute rejection under some circumstances. Further, CXCL10 blockade impeded the expansion and migration of peripheral antigen-specific T cells in a mouse model of virus-induced organ-specific autoimmune disease. Finally, the role that CXCL10 plays

in many pathological conditions is complicated by its dual regulatory activity since it can act as an agonist for Th1 responses by binding to its own receptor, and as an antagonist for CCR3 by competing with CCR3 ligands for binding to this receptor.

Receptors CXCL10 binds to and activates two distinct receptors, CXCR3 and CXCR3B, as do monokine induced by IFN-g (Mig/CXCL9) and IFN-inducible T-cell alpha chemoattractant (ITAC/CXCL11). These receptors were cloned from a human CD4 þ T-cell library and were found to have significant sequence homology to the two IL-8 receptors. Both the human and mouse CXCR3 genes were mapped to the X chromosome. Receptor structure function analysis revealed that similar to the other chemokine receptor family members, CXCR3 is a seven transmembrane G-protein-coupled receptor, which mediates calcium mobilization and chemotaxis in response to the ligands CXCL10 and CXCL9. Initially, expression of CXCR3 was thought to be restricted to IL-2-activated T lymphocytes and not in resting T and B lymphocytes, monocytes, or granulocytes. Subsequently, CXCR3 was found on other activated cells of the immune system including natural killer (NK) cells, granulocytes, and macrophages. In addition to chemotaxis, the known CXCR3 ligands, CXCL9, CXCL10, and CXCL11, induce secretion of macrophage metalloelastase (matrix metalloproteinase 12; MMP-12) from activated human lung macrophages. Mutational analysis showed that binding of CXCL10 to its receptor is through the N-terminal residue Arg-8, preceding the first cysteine. CXCR3expressing, GAG-deficient Chinese hamster ovary cells remained responsive to CXCL10, suggesting that the CXCR3 and heparin binding sites of CXCL10 are only partially overlapping an interaction thought to be important for its sequestration on endothelial and other cells.

CXCL10 in Respiratory Diseases CXCL10 has been associated with a wide spectrum of lung inflammatory diseases through both pro- and antifibrotic effects and its ability to recruit cells that secrete or respond to IFN-g. For example, CXCL10 mRNA and protein are markedly increased in the lungs of mice challenged with bleomycin, a potent inducer of lung fibrosis. However, CXCL10 is thought to be protective in this setting as mice deficient in CXCL10 showed increased susceptibility to fibrosis while transgenic CXCL10 overexpressing mice showed reduced mortality and less severe

CHEMOKINES, CXC / CXCL10 (IP-10)

can contribute to Th2-type inflammation in experimental asthma. Contrary to this study, adenovirusmediated overexpression of CXCL10 in the airways in a mouse model of asthma resulted in significant inhibition of eosinophils in the BAL fluid accompanied by enhanced IFN-g, and reduced IL-4 secretion that was dependent on IFN-g. These findings are more in line with the above-mentioned multifunctional activity of CXCL10 regarding its binding to CXCR3 and acting as a potent antagonist of many Th2 chemokines. Collectively, these contradictory data illustrate that local expression of the chemokine CXCL10 can exert diverse effector responses depending on the exact immunological context. Smoking-induced emphysema is associated with chronic activation of Th1 type inflammation and, in particular, upregulation of CXCL9 and CXCL10, and their shared receptor, CXCR3 on lung airway epithelial cells, T cells, and macrophages. Expression of CXCL10 has also been demonstrated in the lung macrophages and in T cells isolated in the lungs of patients with smoking-induced emphysema. The accumulation of T cells and monocytes at sites of ongoing inflammation represents the earliest step in a series of events that lead to granuloma formation in sarcoidosis and destruction of lung elastin fibers in emphysema. High levels of a CXCL10 protein have been demonstrated in the BAL fluid of patients with pulmonary sarcoidosis and lymphocytic alveolitis as compared with patients with inactive disease or control subjects. Similarly, lung CD4 þ T cells secreted a higher concentration of CXCL10 in emphysema subjects as compared to controls. In addition, more recently it has been shown that beyond their historic role as chemoattractants, members of the CXCL

bleomycin-induced pulmonary fibrosis. The mechanism for CXCL10-dependent survival in this model was inhibition of recruitment of lung fibroblasts, which are largely responsible for synthesis of the matrix proteins that underlie most fibrosing conditions. Paradoxically, however, elevated levels of CXCL10 have been found in bronchoalveolar lavage (BAL) fluids of humans who have undergone lung transplantation with acute and chronic rejection, a complication that usually leads to widespread fibrosis of the transplanted lung. However, in vivo neutralization of CXCR3 or CXCL10 decreased intragraft recruitment of CXCR3-expressing mononuclear cells and attenuated chronic lung rejection and bronchiolitis obliterans syndrome in mice. Furthermore, overexpression of CXCL10 was associated with worsening of lung fibrosis in the same transplantation model. Thus, CXCL10 appears to suppress lung fibrosis due to bleomycin, but is a primary mediator of lung transplant rejection. Although CXCL10 is primarily associated with Th1, or type I, immunity, its upregulation in type II inflammatory conditions such as asthma has also been reported. Mice that overexpress CXCL10 in the lung were shown to exhibit significantly increased eosinophilia, IL-4 levels, and CD8 ( þ ) lymphocyte recruitment compared with wild-type controls that received the same allergen. In addition, there was an increase in the percentage of IL-4-secreting T lymphocytes in the lungs of CXCL10 transgenic mice. In contrast, mice deficient in CXCL10 demonstrated the opposite results compared with wild-type controls, with a significant reduction in these measures of Th2-type allergic airway inflammation. These findings suggest that CXCL10, a Th1-type chemokine, CXCR3

405

Neutrophil Neutrophil elastase

A1AT IFN-


Elastin degradation Airway obstruction Emphysema

Th1 cell CXCL10

MMP-12

CXCR3 Macrophages Figure 2 CXCL10 in emphysema. Th1 effector cells of the lung secrete CXCL10, which, through its receptor CXCR3, recruits additional lung T cells, thus amplifying the inflammatory response to tobacco smoke. CXCL10 also recruits activated macrophages and neutrophils and induces macrophages to secrete MMP-12, an elastolytic enzyme that inhibits a1-antiproteinase (A1AT), which in turn is required for protection against human emphysema.

406 CHEMOKINES, CXC / CXCL10 (IP-10)

family (in particular CXCL10) actively induce production of MMP-12; this is a potent inhibitor of a1-antitrypsin, which is linked to the development of smoke-induced emphysema in humans (see Figure 2). Taken together, these data suggest that CXCL10 plays an important role in regulating recruitment of Th1 inflammation and may play a role in lung destruction that is associated with the chronic inflammatory lung diseases such as emphysema and sarcoidosis. See also: Chemokines. Chemokines, CXC: CXCL9 (MIG). Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General; Smoking Cessation. Drug-Induced Pulmonary Disease. Eotaxins. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. G-Protein-Coupled Receptors. Leukocytes: Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Leukotrienes. Matrix Metalloproteinases. Serine Proteinases.

Luster AD, Greenberg SM, Leder P, et al. (1985) Gamma-interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315: 672–676. Luster AD and Leder P (1993) IP-10, a -C-X-C- chemokine, elicits a potent thymus-dependent antitumor response in vivo. Journal of Experimental Medicine 178: 1057–1065. Marx N, Mach F, Sauty A, et al. (2000) Peroxisome proliferatoractivated receptor-gamma activators inhibit IFN-gamma-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells. Journal of Immunology 164: 6503–6508. Medoff BD, Sauty A, Tager AM, et al. (2002) IFN-gamma-inducible protein 10 (CXCL10) contributes to airway hyperreactivity and airway inflammation in a mouse model of asthma. Journal of Immunology 168: 5278–5286. Moser B and Willimann K (2004) Chemokines: role in inflammation and immune surveillance. Annals of the Rheumatic Diseases 63: ii84–ii89. Saetta M, Mariani M, Panina-Bordignon P, et al. (2002) Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 165: 1404–1409. Wiley R, Palmer K, Gajewska B, et al. (2001) Expression of the Th1 chemokine IFN-gamma-inducible protein 10 in the airway alters mucosal allergic sensitization in mice. Journal of Immunology 166: 2750–2759.

Glossary Further Reading Agostini C, Cassatella M, Zambello R, et al. (1998) Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. Journal of Immunology 161: 6413–6420. Belperio JA, Keane MP, Burdick MD, et al. (2003) Role of CXCL9/CXCR3 chemokine biology during pathogenesis of acute lung allograft rejection. Journal of Immunology 171: 4844–4852. Campanella GS, Lee EM, Sun J, and Luster AD (2003) CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10). Journal of Biological Chemistry 278: 17066–17074. Dufour JH, Dziejman M, Liu MT, et al. (2002) IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. Journal of Immunology 168: 3195–3204. Gasperini S, Perin A, Piazza F, et al. (1995) The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation. Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. Journal of Experimental Medicine 182: 219–231. Grumelli S, Corry D, Song L, et al. (2004) An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema. Public Library of Science Medicine 1: 74–83. Hancock WW, Gao W, Csizmadia V, et al. (2001) Donor-derived IP-10 initiates development of acute allograft rejection. Journal of Experimental Medicine 193: 975–980. Loetscher M, Gerber B, Loetscher P, et al. (1996) Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes. Journal of Experimental Medicine 184: 963–969. Loetscher P, Pellegrino A, Gong JH, et al. (2001) The ligands of CXC chemokine receptor 3, I-TAC, Mig, and IP10, are natural antagonists for CCR3. Journal of Biological Chemistry 276: 2986–2991.

Adipocytes – fat cells Allograft – graft of tissue from non-self donor of the same species Bleomycin – a chemotheraputic agent Chemokine – small chemoattractant proteins that stimulate the migration and activation of cells Cytokines – proteins made by cells that affect the function of another cell Dendritic cells – cells found in T-cell regions of lymph node and the most potent stimulators of T-cell responses Eotaxins – CC chemokines that act predominantly on eosinophils Jak-2 – a Janus-family tyrosine kinase (JAK) signaling molecules that are activated by aggregation of cytokine receptors Lipopolysaccharide – a cell wall antigen present in Gram-negative bacteria Matrix metalloproteinases – a large family of zincdependent endopeptidases Multiple sclerosis – an autoimmune-mediated neurodegenerative disorder Proteoglycans – cell surface proteins with carbohydrate side chains

CHEMOKINES, CXC / CXCL1 (GRO1)–CXCL3 (GRO3) 407

Psoriasis – an inflammatory skin disorder STAT1 – signal transducer and activator of transcription, a transcription factor, that is phosphorylated by JAKs T-helper cell type 1 – a subset of CD4 T cells that are characterized by the cytokines they produce. They are mainly involved in activating macrophages Toll-like receptors – cell surface receptors that are initiated by Toll pathway that activates the transcription factor NF-kB by degrading its inhibitor IkB

nuclear factor kappa B (NF-kB) transcription factor dependent. Probing against leukocyte gene library revealed two additional GRO-like molecules, designated GROb and GROg; these had respective 90% and 86% amino acid identity with GROa. Compared to GROa, there are 11 amino acid substitutions in GROb and 15 in GROg of which some confer conformational changes in protein structure. The GROb and GROg molecules were found to be identical to independently identified human MIP-2a and MIP-2b which are chemokine genes homologous to the MIP-2 genes of mice. It is now accepted that the GROa, GROb, and GROg are members of the CXCL groups of chemokines and in current standardized nomenclature are designated CXCL1–3.

CXCL1 (GRO1)–CXCL3 (GRO3)

Structure

S W Chensue, VA Ann Arbor Healthcare System, Ann Arbor, MI, USA

Genes consisting of approximately 1100 nucleotide base pairs for CXCL1–3 are encoded on long arm of chromosome 4 in or near the CXC chemokine locus (4q21). The protein structure of CXCL1 closely resembles other members of the CXCL group. In solution, CXCL1 is a dimeric chemokine consisting of monomers of 73 amino acid residues. The dimer contains a six-stranded antiparallel b-sheet packed against two C-terminal antiparallel a-helices (Figure 1). The CXC designation derives from characteristic conserved double-cysteine motif separated by single amino acid in the N-terminal portions of the molecule. CXCL1–3 are also classed as ELR chemokines based on the presence of a glutamate–leucine–arginine sequence adjacent and N-terminal to the CXC motif.

& 2006 Elsevier Ltd. All rights reserved.

Abstract CXCL1–3 are members of the CXCL class of chemokines with neutrophil chemotactic and angiogenic biologic properties. These molecules exist as dimers and interact principally with the guanosine nucleotide-protein-coupled, CXCR2 chemokine receptor expressed by neutrophils as well as by other cell types. CXCL1–3 are elicited by microbial products and cytokines and likely contribute to inflammatory and repair responses as part of a broad spectrum of chemotactic mediators. Evidence to date indicates that these molecules participate in multiple pulmonary diseases including infections, adult respiratory distress syndrome, chronic obstructive pulmonary disease, hyperoxia-induced lung injury, fibrosis, and neoplasm.

Introduction The GROa molecule was first identified in the 1980s as an early response gene produced by cultured melanoma cells with autocrine growth promoting function. GROa also known as melanoma growth stimulating activity (MGSA) was expressed constitutively by serum-cultured transformed fibroblasts and could be induced in normal cells by the inflammatory cytokines, interleukin-1 (IL-1), and tumor necrosis factor alpha (TNF-a). While initially thought to be a growth-related oncogene due to its growthpromoting effects on melanoma cells, it was quickly apparent that GROa was structurally related to the supergene family of small 8–15 kDa inflammatory neutrophil chemotactic proteins or chemokines that included IL-8, neutrophil activating protein-2, and macrophage inflammatory protein-2 (MIP-2). Like these molecules, GROa was mapped to the chemokine locus on chromosome 4 and its expression was

Figure 1 Secondary molecular structure of CXCL1 (GROa) dimer. Note six antiparallel b-pleated sheets (red) positioned against two antiparallel a-helices (blue).

408 CHEMOKINES, CXC / CXCL1 (GRO1)–CXCL3 (GRO3)

Regulation of Production While CXCL1 is produced constitutively in some transformed cell lines, under physiologic conditions it is likely induced in normal cells by exogenous stimuli such as microbial products and/or inflammatory cytokines. In particular, bacterial endotoxin, IL-1, and TNF-a are potent inducers of CXCL chemokines in mononuclear phagocytes, epithelial cells, and structural mesenchymal cells. However, it should be noted that numerous infectious agents have been described as inducers of CXCL gene expression. The gene loci for CXCL1–3 have NF-kB binding sites and as such, the NF-kB transcription pathway is considered critical in regulating the expression of these genes. It is likely that during infection microbial products interact with Toll-like receptors on host cells to initiate phosphorylation events leading to release of active NF-kB which translocates to the nucleus to bind to gene promoter sites. The CXCL genes represent only a portion of the expressed gene profile. IL-1 can be among those induced genes and there is evidence indicating that IL-1 may regulate CXCL1–3 gene expressions by stabilizing transcript levels through inhibition of transcript degradation. The restoration of transcription regulatory pathways results in eventual arrest of gene expression. Neoplastic cells can display dysregulated CXCL gene expression which may allow them to function as autocrine growth or oncogenic factors.

Biological Function Originally, CXCL1 (GROa) was considered to be an oncogenic growth factor since it appeared to have the capacity to promote growth and malignant transformation of cultured melanocytes. Since that time a number of other functions have been attributed to this group of molecules. These include regulation of tissue repair-related mesenchymal cell function. Two examples should be mentioned. First, CXCL1 caused a dose-dependent decrease in the expression of interstitial collagens by cultured rheumatoid synovial fibroblasts. The effect was specific, as there was no demonstrable effect on other products. Unlike its mitogenic effect on melanoma cells, it had no effect on the proliferation rate of fibroblasts. Thus, CXCL1 may limit or temper the scarring process following injury. Second, similar to other ELRþ chemokines CXCL1 is implicated as a proangiogenic factor, that is, stimulating the growth of new small vessels at sites of injury or tumor growth. This contrasts with ELR-CXCL chemokines which inhibit angiogenesis. These opposing activities suggest the presence of

carefully orchestrated signals that are required to shape events involved in the tissue repair response. The best-characterized function of CXCL1–3 is their chemoattractant activity. Like other ELRþ CXCL chemokines, these molecules have potent effects on neutrophil migration and activation. This activity appears to be further amplified by natural enzymatic truncation of the amino terminal portion of the molecules; this modification causes a 30-fold increase in biologic potency. Thus, under conditions of inflammation, proteolytic enzymes may boost the chemotactic function of these molecules. Numerous studies have provided circumstantial evidence for an in vivo association of neutrophil accumulation with production of CXCL1–3. However, it has been difficult to provide direct evidence by neutralization approaches due to the redundancy of CXC ligands. Some direct evidence for in vivo biologic activity has been derived from CXCR2 receptor knockout mice which display impaired neutrophil recruitment and delayed wound healing. In addition, engineered chemical antagonists of the CXCR1 and CXCR2 receptors likewise cause impaired neutrophil mobilization. Taken together, the functional activities of CXCL1–3 would appear to be teleologically appropriate for the acute inflammatory response to infection. Following epithelial injury and infection, these molecules would simultaneously promote epithelial healing, leukocyte recruitment, and neovascularization, representing a coordinated effort to prevent further infestation, initiate microbial elimination, and optimize delivery of additional leukocytes and antimicrobial humoral factors.

Receptors The functional high-affinity receptor for CXCL1–3 is well characterized and is designated by standardized nomenclature as CXCR2, formerly known as IL-8B or IL-8 type 2 receptor. CXCR2 is promiscuous and binds multiple ELRþ CXC ligands. CXCR2 is an archetypical guanosine nucleotide-protein-coupled receptor (GPCR) with seven transmembrane hydrophobic domains having three intracellular and three extracellular hydrophilic loops. Ligation of the extracellular amino-terminal portion of the receptors triggers calcium influx and intracellular activation of transduction factors such as protein kinase C and MAP kinase. These events stimulate downstream functions such as cell movement, degranulation, adhesion, and respiratory burst. CXCR2 receptors have been detected on neutrophils, monocytes, basophils, mast cells, endothelial cells, T lymphocytes, natural killer, and neuroendocrine cells. However, neutrophils appear to be the dominant expressing cells.

CHEMOKINES, CXC / CXCL1 (GRO1)–CXCL3 (GRO3) 409

Due to the promiscuity of the receptor, it is difficult to assess the contribution of individual CXC ligands in experimental animals with targeted deletion of CXCR2. Another promiscuous receptor for CXCL1– 3 is the Duffy antigen/chemokine receptor. Duffy binds both CXC and CC chemokines and is expressed by erythrocytes in Duffy-positive individuals, endothelial cells of postcapillary venules, and Purkinje cells of the cerebellum. The normal physiological function of Duffy remains unclear. The transduction pathways activated by Duffy are also unknown and the receptor has not been shown to act through a G protein. Reportedly, Duffy facilitates CXCL1 distribution and enhances neutrophil movement across endothelial cell monolayers.

Role of CXCL1–3 in Respiratory Diseases In addition to beneficial functions, CXCL1–3 have been implicated to participate in multiple pulmonary diseases ranging from infectious to neoplastic and usually are part of a broader spectrum of chemokine and cytokine mediators (Figure 2). In the lung, alveolar macrophages are critical sentinel cells and are likely an important source of CXCL1–3 but type II alveolar epithelial cells are known to be a potential source. The roles of these chemokines in different lung diseases are discussed separately below.

Infections In view of their neutrophil chemotactic properties, it might be expected that CXCL1, 2, or 3 would be detected during lung bacterial infections that elicit

Inflammation and repair Neutrophil recruitment

significant neutrophil influx. Indeed, these chemokines are readily detectable in bronchoalveolar lavage fluids of patients with infectious and inflammatory lung disease but at low levels in those of normal volunteers. Experimental studies of Pseudomonas aeruginosa, an important Gram-negative bacterium causing fatal necrotizing pneumonias, have shown this agent to be capable of inducing CXCL chemokines in alveolar macrophages and epithelial cells. Depletion of alveolar macrophages impairs both the production of CXCL1 and the recruitment of neutrophils. Other Gram-negative bacterial causes of pneumonia such as Escherichia coli are similarly capable of eliciting CXCL chemokines and innate recognition of bacterial endotoxin appears to be the common stimulatory molecule. However, Grampositive bacteria and Pneumocystis are also capable of eliciting CXCL1. In general, viruses such as respiratory syncitial virus (RSV) appear to elicit strong CC chemokine responses in the lung, but epithelial damage and secondary infection will elicit CXCL chemokines.

Adult Respiratory Distress Syndrome It is well known that critically ill patients are at increased risk for acute lung injury which is clinically manifest as adult respiratory distress syndrome (ARDS). CXCL1 is notably elevated in lung lavage fluids obtained from ARDS patients. Accordingly, neutrophil influx correlates well with levels of CXCL chemokines. The alveolar epithelial damage observed in ARDS appears to be related to the inflammation and as such the CXCL chemokines might be contributing to the disease. Due to extensive redundancy in neutrophil recruitment, CXCL1–3 likely only contribute partially and there is evidence to indicate that other chemokines such as CXCL8 may be more important.

Tissue repair

Angiogenesis

Chronic Obstructive Pulmonary Disease

CXCL1,2,3

Tumor growth

ARDS Hyperoxia injury

Fibrosis COPD Lung diseases

Figure 2 Potential areas of functional involvement of CXCL1–3 in physiology and lung diseases.

There is strong evidence to suggest that inflammatory leukocytes including neutrophils contribute to the destruction of structural matrix observed in chronic obstructive pulmonary disease (COPD). Circumstantial evidence suggests that CXCL chemokines might contribute to neutrophil recruitment and activation during COPD. CXCL chemokines including CXCL1 are present in the sputum and lung lavage fluid of COPD patients. Cigarette smoke, the major cause of COPD, has been shown to elicit CXCL1 and neutrophil influx in rat lungs. Moreover, the neutrophil mobilization could be blocked with CXCL2 receptor antagonists. This receptor also appears to show

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enhanced expression on monocytes from smokers. These cells likewise migrate to lungs where they can potentially produce matrix destructive enzymes.

Fibrosing Alveolitis Cryptogenic fibrosing alveolitis (CFA) is a progressive fibrotic disease of the lung characterized by significant local recruitment of neutrophils which can comprise 10–20% of the cells in the lung lavage of affected patients. These cells are thought to contribute to the disease. Patients with CFA have increased levels of CXCL1 in their plasma and their neutrophils are hyperresponsive to the chemokine. Thus, chemokine-targeted therapies may have potential benefit in controlling the disease.

Hyperoxia-Induced Lung Injury Hyperoxia-induced lung injury is a potential serious side effect of oxygen therapy in hospitalized patients. The disease is characterized by infiltration of neutrophils with associated endothelial and epithelial cell injury, followed by interstitial fibrosis. CXCL1–3 may be important contributors to this condition. Animal experiments demonstrate that oxygen treatment induces expression of CXCL1–3, which correlates with the influx of neutrophils and mortality. Genetic deletion of the CXCR2 receptor blocked neutrophil recruitment and resulted in reduced oxygen-induced mortality.

Lung Cancer The role of CXCL1–3 in neoplastic disease of the lung remains controversial and studies are very limited. Due to their angiogenic and mitogenic properties, these chemokines are generally considered to support the growth of neoplastic tissue. Growth can potentially be enhanced by direct stimulation of chemokine receptors expressed by tumor cells especially melanocytic and neuroendocrine types. In addition, angiogenic activity would help to provide vascular support to growing tumors. In contrast to this notion, at least one study has provided evidence that CXCL2 is angiostatic and could inhibit the growth of Lewis lung carcinoma in mice. Clearly,

more studies would be needed to help clarify the contribution of CXCL1–3 in lung carcinoma. See also: Acute Respiratory Distress Syndrome. Chemokines. Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-1 and IL-18. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens. Tumor Necrosis Factor Alpha (TNF-a). Tumors, Malignant: Overview.

Further Reading Belperio JA, Keane MP, Arenberg DA, et al. (2000) CXC chemokines in angiogenesis. Journal of Leukocyte Biology 68: 1–8. Cao Y, Chen C, Weatherbee JA, Tsang M, and Folkman J (1995) gro-beta, a –C–X–C– chemokine, is an angiogenesis inhibitor that suppresses the growth of Lewis lung carcinoma in mice. Journal of Experimental Medicine 182: 2069–2077. Glynn PC, Henney EM, and Hall IP (2001) Peripheral blood neutrophils are hyperresponsive to IL-8 and Gro-alpha in cryptogenic fibrosing alveolitis. European Respiratory Journal 18: 522–529. Goodman RB, Pugin J, Lee JS, and Matthay MA (2003) Cytokinemediated inflammation in acute lung injury. Cytokine & Growth Factor Reviews 14: 523–535. Hashimoto S, Pittet JF, Hong K, et al. (1996) Depletion of alveolar macrophages decreases neutrophil chemotaxis to Pseudomonas airspace infections. American Journal of Physiology 270: L819– L828. Sager R, Haskill S, Anisowicz A, Trask D, and Pike MC (1991) GRO: a novel chemotactic cytokine. Advances in Experimental Medicine and Biology 305: 73–77. Stoeckle MY (1991) Post-transcriptional regulation of gro alpha, beta, gamma, and IL-8 mRNAS by IL-1 beta. Nucleic Acids Research 19: 917–920. Sue RD, Belperio JA, Burdick MD, et al. (2004) CXCR2 is critical to hyperoxia-induced lung injury. Journal of Immunology 172: 3860–3868. Traves SL, Smith SJ, Barnes PJ, and Donnelly LE (2004) Specific CXC but not CC chemokines cause elevated monocyte migration in COPD: a role for CXCR2. Journal of Leukocyte Biology 76: 441–450. Vanderbilt JN, Mager EM, Allen L, et al. (2003) CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury. American Journal of Respiratory Cell and Molecular Biology 29: 661–668. Villard J, Dayer-Pastore F, Hamacher J, Aubert JD, Schlegel-Haueter S, and Nicod LP (1995) GRO alpha and interleukin-8 in pneumocystis carinii or bacterial pneumonia and adult respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 152: 1549–1554. Wuyts A, Govaerts C, Struyf S, et al. (1999) Isolation of the CXC chemokines ENA-78 GRO alpha and GRO gamma from tumor cells and leukocytes reveals NH2-terminal heterogeneity. Functional comparison of different natural isoforms. European Journal of Biochemistry 260: 421–429.

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CHEMORECEPTORS Contents

Central Arterial

Central

PaCO2

N S Cherniack, UMDNJ-New Jersey Medical School, Newark, NJ, USA M D Altose, Case Western Reserve University School of Medicine, Cleveland, OH, USA

Chemoreceptors Chemical drives

& 2006 Elsevier Ltd. All rights reserved.

Controller Ventilatory demand

Abstract The powerful stimulation of ventilation by carbon dioxide inhalation and the near-constancy of arterial PCO2 during rest and exercise indicate the importance of CO2/H þ receptors in the regulation of breathing. There are both peripheral and central chemoreceptors with the central ones accounting for about 70% of the CO2 effect. These sensors operate in feedback control systems that help maintain levels of CO2 in the body within narrow limits. Techniques that allow individual cells to be studied have provided much new information on central chemoreceptors. Groups of neurons that increase their activity in response to changes in acidity in their external environment are widely scattered in the medulla. It is generally believed that the proximal stimulus to these central receptors is intracellular acidity. These neuronal groups when stimulated by the changes in local hydrogen ion concentration excite breathing, but each group separately accounts for a rather small part of the increase in ventilation that can be elicited by CO2 inhalation. There are no data yet on whether the various sensory groups have different thresholds though some seem to be excited only during wakefulness, which might imply a high threshold, and others only during sleep, which is not easily explained by threshold differences. Furthermore, it is not known whether the various groups differ in their operative ranges or sensitivity. It is also possible that some of these groups are parts of other functional systems involved, for example, in producing arousal. Otherwise healthy humans who do not respond to breathing CO2 can maintain normal levels of arterial CO2 when awake. But in patients with thoracic diseases, low sensitivity to CO2 may contribute to CO2 retention. During sleep, a threshold level of CO2/H þ is required to prevent apnea. In the absence of a response to CO2, abnormal elevation of PCO2 occurs during sleep. Depressed chemosensitivity may be involved in the sudden infant death syndrome. In addition, depressed chemosensitivity may predispose to respiratory failure in patents with thoracic diseases.

Introduction Description of Central Chemoreceptor Effects on Ventilation

Respiration is regulated by a negative feedback control system that serves to hold PCO2 in the arterial

PaO2 PbrainCO2

Respiratory muscles, lung

Ventilation O2, CO2 body stores PaCO2

PaO2 PbrainCO2

Figure 1 Block diagram showing components and pathways involved in the chemical control of breathing.

blood and in the brain within narrow limits and to maintain acid–base homeostasis (see Figure 1). This system consists of a controller made up of respiratory neurons and chemoreceptors and a plant or controlled system consisting of the body tissues in which CO2 is generated by metabolic processes. The system operates to maintain PCO2 (or H þ ) at some desired level called the operating point. The two components are linked via the circulation, which carries information about levels of PCO2 and PO2 in the blood. When CO2 levels in the body rise as a result of increases in metabolic CO2 production or disturbances that decrease ventilation, central chemoreceptors in the brain and peripheral chemoreceptors in the carotid and aortic bodies are stimulated. Impulses from these chemoreceptors, in turn, act on respiratory motor neurons in the medulla to produce compensatory increases in ventilation to return PCO2 to its normal level. Similarly, disturbances that increase ventilation and reduce levels of PCO2 and H þ ion cause compensatory decreases in breathing to help restore PCO2 /H þ levels. When CO2 is breathed, the chemoreceptors augment ventilation, minimizing the rise in PCO2 that would otherwise occur. The increases in ventilation

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PCO

2

A

B

Ventilation Figure 2 Effect of ventilation on PCO2 . At low ventilation and high PCO2 (A, slope highlighted in red) the effect of ventilation on PCO2 is greater than at (B) where ventilation is higher and PCO2 is lower. Slope is an index of plant gain.

are proportional to the deviation in PCO2 when CO2 is inhaled but the compensation is never sufficient to restore PCO2 to its original levels. The sensitivity or gain of the chemical controller is usually expressed as the change in ventilation produced by a given change in PCO2 in the arterial blood and averages about 2 l for each mmHg change in PCO2 in adult humans. In general, the higher the gain the less is the rise in PCO2 . However, the efficacy of the control system also depends on the effect of ventilation on PCO2 . The ratio of the change in PCO2 to the change in ventilation is sometimes called plant gain. As shown in Figure 2, because the effects of ventilation on PCO2 are less at lower than at higher levels of PCO2 , plant gain increases as resting arterial PCO2 rises (when breathing CO2 free gas). Increases in plant gain and controller gain (more precisely their product or loop gain) will shorten the time needed for the control system to reach equilibrium after a disturbance. However, too high a loop gain can lead to control-system instability and oscillations in minute ventilation. All animals that extract oxygen from the atmosphere have CO2/H þ responsive chemoreceptors. CO2 receptors have been found on the ventral surface of the caudal and rostral medulla of the isolated bullfrog brainstem. Fish have CO2 levels of only a few mmHg and receptors for CO2/H þ sample the ambient water rather than changes within the body. In anuran amphibians, CO2 chemoreceptors account for 70% of the ventilatory response to CO2. In cold-blooded species the normal PCO2 varies considerably with body temperature becoming lower as temperature drops. In mammals, on the other hand, there is relatively little direct effect of temperature on PCO2 levels that roughly average 40 mmHg in the arterial blood of humans at rest. Temperature elevations, if anything, increase ventilation, particularly in panting animals.

Carbon dioxide chemoreceptors have been most extensively studied in mammals. Approximately 70% of the ventilatory response to CO2 results from the activation of central chemoreceptors in the brain. Only about 30% of the increase in ventilation following the inhalation of a CO2 enriched gas mixture is attributable to the activity of peripheral chemoreceptors, mainly the carotid body chemoreceptors. Because of the rapidity of the effects of CO2 inhalation on the arterial blood as compared to the brain, the peripheral chemoreceptors respond far more quickly to changes in CO2 than do the central ones. Both central and peripheral chemoreceptors respond to PCO2 only when it reaches a threshold value. The peripheral chemoreceptors are believed to respond to lower levels of PCO2 than the central chemoreceptors. Drives from central and peripheral chemoreceptors have an additive effect. While the peripheral chemoreceptors respond to changes in arterial PCO2 , the central chemoreceptors respond to changes in PCO2 in the brain. Levels of CO2 in the brain depend on rates of cerebral perfusion in relation to metabolism as well as ventilation. Since changes in PCO2 occurring naturally are always associated with changes in H þ , it is now generally considered that the proximate stimulus for central chemoreception is intra- and extracellular brain H þ , rather than the H þ concentration of the cerebrospinal fluid (CSF), previously thought to be the main factor determining ventilation. The importance of chemical control in mammals in determining ventilation depends on behavioral states and higher brain center influences may modify the characteristics of the chemical controller. It is generally agreed that chemical stimuli are the main factors regulating breathing during nonrapid eye movement (nonREM) sleep when decreases in PCO2 of only a few mmHg can produce apnea. In the waking state, neural drives emanating from sensory receptors in the body and from the environment constitute a greater portion of the ventilatory drive as shown in Figure 3. As a result breathing can be maintained at near-normal levels in the absence of any discernible response to inhaled CO2. A discernible response to CO2 is also not necessary for normal PCO2 levels to be maintained during moderate exercise, but in its absence PCO2 rises considerably rather than decreasing when the anaerobic threshold is exceeded.

Structure and Function Location and Morphology of Central Chemoreceptors

The precise location and distribution of central respiratory-related chemoreceptors in the brain has

Ventilation

CHEMORECEPTORS / Central 413

Awake

Asleep

0 PaCO2

Figure 3 Effect of changes in arterial PCO2 on ventilation during sleep and wakefulness. As PaCO2 is decreased, apnea occurs during sleep but not in the awake state.

been a matter for investigation for decades and has not yet been fully established. It is generally thought that the central chemoreceptors are grouped in clusters in several different locations in the medulla and even in other areas of the brain. The characteristics of the different clusters in terms of their thresholds, sensitivities, and mechanisms of sensing CO2 seem to vary. The study of central chemoreceptors is further complicated because CO2 centrally affects more than ventilation. It increases sympathetic activity, causes the release of catecholamines, helps produce arousal, increases cerebral blood flow, increases anxiety, and can produce a sense of air hunger. Thus, it is possible that different central chemoreceptors may also differ in their functions. In studies in intact animals, central chemoreceptors were defined as cells that, when stimulated by acid or CO2, lead to an increase in ventilation or respiratory motor activity and/or when inhibited, decrease the respiratory stimulating actions of acid or CO2 without affecting respiratory activity excited by other types of respiratory stimuli. For some time it was believed that respiratory motor neurons themselves had properties of chemosensitivity. Animal studies by Loeschke and by Mitchell and others, using the direct application of acidic or basic fluids, demonstrated discrete clusters of chemoresponsive neurons on the ventrolateral surface of the medulla. These include a rostral chemosensitive area (Mitchell’s area) and a caudal chemosensitive area (Loeschke’s area). Stimulation of the Loeschke and Mitchell areas on the ventral surface of the medulla increases breathing in anesthetized animals. Correspondingly, ablation by cooling of the intermediate area between them, presumably by blocking afferent nerve fibers from the rostal and caudal areas, produces apnea. It has been observed that patients with brain lesions around

Mitchell’s area show depressed ventilatory responses to CO2 during sleep. This supports the presumption that the ventrolateral medulla is an important site of central chemoreceptors. With time, more precise ways of stimulating or blocking specific areas of the brain using microinjection or dialysis with various agents such as carbonic anhydrase inhibitors, solutions equilibrated with CO2, neuronal stimulants and inhibitors, and neurotoxins were developed. Using these techniques, additional chemoreceptive areas were identified farther from the ventral surface and in the dorsal areas of the medulla, in the pons, in the midbrain, and even in higher brain structures. However, stimulation and ablation interventions in vivo are imperfect techniques for identifying the precise location of central chemoreceptors because the penetration of a local intervention to deeper and more distant structures cannot be totally controlled. Even quite localized stimulation and ablation can produce reactions in more distant areas through neural connections that can be either excitatory or inhibitory to breathing. These interventions can affect systems other than respiration, such as circulation, metabolism, autonomic nervous system, and arousal, which may indirectly alter breathing. Techniques that use fluorescent voltage-sensitive dyes and those that measure increased activity of early immediate genes such as c-fos and c-jun to signal neural responses following exposure to high CO2 levels have identified neurons activated by CO2. However, such responses may also not be specific and lead to increases in ventilation. Studies of isolated brain and spinal cord preparations of neonatal animals and in brain slices and cell cultures have identified additional chemoreceptive areas and more importantly have elucidated critical cellular and molecular mechanisms that are involved in sensing changes in CO2/H þ . However, in these studies the criteria for chemoresponsiveness have been broadened to include changes in transmembrane potentials. The relationship of these membrane changes to respiratory activity is indirect. Groups of cells that depolarize and increase their excitability with hypercapnia and with local changes in acidity have also been found in nonrespiratory areas of the brain but the magnitude of the responses is less than in cells from areas involved with respiration. It is currently believed that central chemoreceptors away from sites near the ventral medullary surface (like the retrotrapezoid nucleus) are located in the dorsal medullary regions including the nucleus of the solitary tract, the locus ceruleus, the median raphe, the pre-Boetzinger complex, and the fastigial nucleus

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of the cerebellum. Chemosensitive cells that respond to CO2 by increasing respiratory frequency have also been found in the spinal cord of brainstem–spinal cord preparations from newborn mice. Almost all of the areas in which CO2/H þ responsive cells have been found are concerned with other physiological functions besides respiration. For example, neurons in the raphe are also involved in blood pressure control, thermoregulation, and the sleep– waking cycle making it difficult to distinguish direct from indirect effects on respiration. The effect of CO2 in producing arousal seem to be mediated in particular by receptors in the cerebellum, midbrain, posterior thalamus, and basal ganglia, in areas where chemosensitive cells have been identified. How the different chemoreceptors contribute to the overall ventilatory response to CO2/H þ is not clear. One possibility is that there is a hierarchical arrangement of central chemoreceptors with various groups differing in their threshold and sensitivity to CO2/H þ . This is suggested by the observation that direct stimulation of a single group of chemoreceptors produces only 20–30% of the overall ventilatory response to the inhalation of 7% CO2. Furthermore, the threshold and sensitivity of some chemoreceptors may be influenced by higher brain centers. For example, when artificial CSF equilibrated with 25% CO2 is used to focally stimulate chemoreceptors in different locations in the rat brain, the results vary during naturally occurring cycles of sleep and wakefulness. Stimulation of the median raphe increased breathing 15–20% (by increasing frequency) in sleeping rats but had no effect during wakefulness. On the other hand, stimulation of the retrotrapezoid nucleus increased tidal volume and ventilation by 24% but only during wakefulness. Stimulation with the same fluid in the nucleus of the solitary tract increased breathing both during sleep and wakefulness. Other factors that can affect the responsiveness of any one group of central chemoreceptors include the blood flow to the site and excitatory or inhibitory inputs from other chemosensitive areas. Moreover, since few experiments report effects of focal stimulations on ventilation/CO2/H þ response slopes it is difficult to distinguish effects on the chemoreceptive process itself from effects that add to or subtract from ventilation without necessarily affecting the sensing of CO2. There are no anatomical features that distinguish chemosensitive cells that are excited by acid changes from nonchemosensitive cells. In fact, not all such cells have the same morphology. In general, they are located in close proximity to blood vessels in the brain and are situated so as to sense changes in cerebrospinal H þ concentration.

Chemoreceptor cells on the ventral surface of the medulla are located in the marginal glial layer and are surrounded by fine blood vessels. The technique of c-fos immunohistochemical staining has identified CO2-responsive chemoreceptor cells in both the ventral and dorsal medulla with dendrites directed toward the surface of the brain, suggesting that the dendrite may in fact be the chemosensitive portion of the cell. Cellular Mechanisms of Chemoreception

It has been observed that for a given change in extracellular fluid pH at the medullary surface, the ventilatory response is greater with CO2 inhalation than with the intravenous infusion of a fixed acid. One possible explanation is that CO2 and H þ have separate and distinct stimulatory effects on ventilation. Alternatively, since CO2 penetrates the cell membrane much more easily than H þ it may be that hydrogen ion changes at some site like the cell interior, more accessible to the freely diffusible CO2 than to fixed acid, is the true stimulus and that the change in internal cellular pH is the proximate signal to the central chemoreceptors. Nonchemosensitive cells rapidly restore internal pH after hypercapnic acidosis but this process is slowed in chemosensitive cells allowing them to signal H þ changes. Internal pH is restored through an Na þ /H þ exchange mechanism in response to acidosis and by Cl–/HCO–3 exchange following an alkaline challenge. An inhibition or an absence of the Na þ /H þ subtype 3 exchanger may be important in allowing chemoreceptor cells to respond to cellular acidity. Calcium channels may also be involved in the cellular mechanisms of chemoreception. L-type calcium channels in chemosensitive cells in the locus ceruleus are activated by intracellular acidity leading to membrane depolarization. There is the possibility that some chemoreceptors are stimulated by changes in the intracellular–extracellular pH gradient rather than by intracellular pH alone. Some central chemosensitive cells are also thought to contain pH-sensitive TASK-1 potassium channels as well as calcium-activated potassium channels. These potassium channels are inhibited by extracellular acidosis resulting in a reduction in the intracellular–extracellular potassium gradient and a lowering of the depolarization threshold. It may be that different types of central chemoreceptors utilize different sensing processes. In ectotherms, extracellular fluid H þ varies inversely with temperature without any effect on ventilation. This indicates that at least in cold-blooded animals H þ per se may not be the sole or main stimulus to the chemoreceptors. Rather, it has been

CHEMORECEPTORS / Central 415

proposed that ventilation is controlled so as to maintain a constant ratio of hydroxyl to hydrogen ion (H þ /OH–) by an amino acid whose dissociation with temperature is like that of water. The fractional dissociation of imidazole (alpha-imidazole) like water remains constant with changes in temperature and temperature-related changes in H þ . However, such a protein would be affected by changes in pH under isothermal conditions. Based on these findings, the alpha-stat theory proposes that central chemoreceptors regulate ventilation as a function of the fractional dissociation (alpha) of the imidazole group of histadine, part of a protein considered to be the pH-sensitive molecule in the central chemoreceptor. This mechanism in cold-blooded animals may in turn regulate the patency of membrane potassium or calcium channels so that intracellular pH also remains constant as the ambient temperature changes. Neurotransmitters are also considered to play a role in mediating chemoresponses. There is considerable evidence that acetylcholine through a muscarinic receptor type is involved in the transmission of both peripheral and central chemoreceptor signals. Other neurotransmitters have also been implicated in central chemoreceptor function. Suramin, a P2 purinoceptor antagonist, when injected into the retrofacial area of the medulla (Boetzinger complex) of anesthetized rats decreases phrenic nerve discharge and reduces the respiratory response to CO2. Cells in the rat medullary raphe that are stimulated by acidosis are serotonergic and contained tryptophan hydroxylase, the rate-limiting enzyme for serotonin synthesis. On the other hand, cells that are inhibited by acidosis lack this enzyme. Other studies indicate that glutamate receptors, neurokinin, and gamma-aminobutyric acid (GABA) receptors may also play a role in mediating central chemoreceptor responses. The transmission of chemoreceptor signal to respiratory motor pathways is only partially affected by synaptic blockade. This suggests the signal transmission also involves metabolic and/or electrical coupling via intracellular channels that form gap junctions. Gap junctions have been demonstrated in CO2-sensitive cells in the nucleus of the solitary tract/ dorsal motor nucleus of the vagus, the medullary raphe, and locus ceruleus. While these gap junctions seem to be involved in the sensing of CO2, how they operate to facilitate or modify chemoresponses is unknown. Cardiovascular Effects of Central Chemoreceptors

Hypercapnia in addition to its effect on respiration also stimulates the autonomic nervous system.

Increased levels of CO2 heighten sympathetic discharge. About half of this increase is attributable to central and the rest to peripheral chemoreceptors. Neurons that mediate increases in sympathetic activity are found on the rostal ventrolateral medulla, in the same vicinity as respiratory chemoreceptors. However, it is not clear whether the same neurons excite ventilation and stimulate sympathetic activity. The increased sympathetic activity that is produced by both central and peripheral chemoreceptors is offset by vagal stretch receptor activity that increases as ventilation is stimulated by hypercapnia. Increases in blood pressure have been reported with cyanide injections into the ventral medulla suggesting that there are oxygen-responsive cells that can stimulate sympathetic activity. Depolarization occurs when cyanide is injected into cells cultured from the rostral ventral medullary surface but this response is absent when heme-oxygenase activity is blocked. There are also thought to be hypoxia receptors in the pre-Boetzinger complex that have a respiratory stimulating action. It has been suggested that these oxygen-sensitive neurons are involved in gasping. The effects of hypoxia on the brain are complex since hypoxia also exerts an important depressive effect on ventilation perhaps by increasing levels of GABA. Hypoxia can also increase lactate levels simulating central CO2 receptors and augment cerebral blood flow.

Central Chemoreceptors in Health and Disease Measurement of Chemoreceptor Sensitivity

As CO2 increases, ventilation rises linearly until it reaches about two-thirds of the maximum breathing capacity. Tidal volume and frequency also increase linearly until tidal volumes of 1.5–2.0 l are attained. Tidal volumes thereafter tend to plateau and ventilation increases primarily through increases in breathing frequency. The frequency of breathing increases more as a result of a shortening of expiratory rather than inspiratory duration. CO2 sensitivity is measured by steady state and rebreathing techniques. Steady-state methods require long periods for PCO2 in both the arterial blood and the brain to reach equilibrium. In addition, hypercapnia produces a near-linear increase in cerebral blood flow so that brain PCO2 rises less than arterial PCO2 as higher concentrations of CO2 are inhaled. Consequently, changes in brain PCO2 are not simply related to arterial PCO2 . The rebreathing technique is a simpler and more rapid method for assessing chemosensitivity. After

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30–45 s of rebreathing a gas mixture with an initial concentration of 7% CO2 and 30% or more oxygen, differences in alveolar, arterial, and cerebral venous PCO2 narrow greatly and with continued rebreathing increase with time at essentially the same rate. Steady-state and rebreathing methods for measuring ventilatory responses to CO2 provide comparable results in normal humans. It has been suggested that a period of hyperventilation preceding rebreathing to lower starting levels of PCO2 allows threshold levels of PCO2 to be discerned. The large contribution of neural drives from higher brain centers, independent of blood gas levels in the wakefulness state, complicates the interpretation of measurements of CO2 response in conscious subjects. Probably the most accurate measurements of CO2 sensitivity may be obtained in nonREM sleep, when breathing is almost exclusively under chemical control, provided that the test can be carried out without affecting sleep stage. The more rapid response to CO2 of peripheral versus central chemoreceptors has been used to separate peripheral from central receptor contributions. Techniques have included devolution of the steady response or by measuring the effects of a single breath of gas containing high concentrations of CO2. In animals the direct effects of CO2 or acids on peripheral chemoreceptor activity can be assessed but there is no direct way of measuring central chemoreceptor neural responses. Since diseases of the lung or thorax, by changing their mechanical properties, can alter the ventilatory response to CO2 even if chemoreceptor function is normal, more direct ways of assessing respiratory output have been proposed. These include measuring the force generated by the inspiratory muscles contracting nearly isometrically against an occluded airway (occlusion pressure) and measurement of the electrical activity of the diaphragm via surface or esophageal electrodes. Factors Affecting CO2 Responsiveness

Ventilatory responses to CO2 are quite variable in humans. Unlike the response to hypoxia, which increases substantially soon after birth, the central response to CO2 is nearly fully developed in the newborn. The ventilatory effect of CO2 on peripheral chemoreceptors does increase somewhat in the early newborn period as evidenced by an enhanced transient response. The ventilatory responses to CO2 increase as a function of body size but otherwise do not change appreciably with advancing age. Even accounting for body size the ventilatory response to CO2 in women is less than in men. Hypercapnic

ventilatory responses in women also vary through the menstrual cycle and are greatest in the luteal phase as a result of the respiratory stimulant effect of the higher progesterone levels. High progesterone/ estrodiol levels seem to explain the hyperventilation of pregnancy. A variety of other endocrine and metabolic factors can influence CO2 responsiveness. Hyperthyroidism increases hypercapnic ventilatory responses and the augmentation of chemosensitivity is closely linked to the increase in metabolic rate. Conversely, hypothyroidism blunts chemosensitivity and when severe, can lead to hypoventilation and hypercapnia. The acid–base status of the body also has a significant effect on CO2 responsiveness. Elevated bicarbonate ion concentration increases the buffering of H þ and decreases the ventilatory response to CO2. There are important behavioral influences on breathing and on the ventilatory responses to CO2. The removal of wakefulness effects during sleep and anesthesia blunts CO2 responsiveness. Relaxation techniques such as yoga can also reduce the ventilatory response to CO2. In contrast, emotional states of fear and anxiety increase resting breathing and lower the resting PCO2 level and may also increase CO2 sensitivity. It is still not clear whether these behavioral effects originating from higher brain centers act to enhance the sensitivity of brainstem chemical control mechanisms or whether they constitute a separate parallel pathway. The hyperventilation syndrome is a condition characterized by persistent hyperventilation and hypocapnia and a variety of symptoms including shortness of breath, chest pain, palpitations, dizziness, and parasthesias. This syndrome is often associated with agoraphobia and panic attacks. The observation that panic attack can be precipitated in these patients by breathing CO2-enriched gas mixtures has led to the suggestion that the disorder is the result of chemoreceptor hyperactivity. However, ventilatory responses to CO2 are generally no greater than normal in the hyperventilation syndrome. The marked variability in respiratory chemosensitivity is due in part to genetic influences. There is a familial correspondence in ventilatory responsiveness and the correspondence is closer in identical as compared to fraternal twins. Genetic influences have also been shown in animal studies: mutant mice with abnormalities in RET, MASH-1, and PHOX2B genes have depressed CO2 responses. Disorders of Central Respiratory Chemical Control

During sleep and with the loss of wakefulness drives, central chemoreceptors play a particularly important

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role in maintaining regular breathing. Normally during sleep, PCO2 rises by a few mmHg and ventilatory responses to CO2 are somewhat reduced. If CO2 sensitivity during sleep is heightened, loop gain is increased. Under these conditions a reduction in PCO2 of only a few mmHg during nonREM sleep may destabilize respiratory control and lead to the development of periodic apneas. This is the basis for Cheyne–Stokes respiration, commonly seen in patients with heart failure, particularly during sleep. An accompanying decrease in CO2 drive to upper airway muscles can result in a loss of upper airway patency in individuals with predisposing anatomic abnormalities, setting the stage for obstructive sleep apnea. As PCO2 rises during the course of an apneic period, ventilatory drive increases or there may be a direct arousal effect so that the apnea terminates and breathing resumes only to begin the entire cycle over again. Persistent and severe periodic apneas during sleep can lead to chronic CO2 retention and hypercapnia even during wakefulness. This can be reversed by treatment of the sleep apnea with continuous positive airway pressure that serves to tether open the upper airways. Chronic hypercapnia often accompanies severe respiratory diseases that markedly impair the function of the ventilatory pump. However, not all individuals with comparable levels of ventilatory dysfunction develop hypercapnia. The propensity for hypercapnia is thought to result only when severe diseases of the ventilatory pump are associated with low CO2 responsiveness. That some individuals are intrinsically predisposed to the development of hypercapnia is suggested by the observations that normal relatives of hypercapnic patients with chronic obstructive pulmonary disease have lower CO2 responses than do relatives of normocapnic patients with chronic obstructive pulmonary disease. Other studies have failed to find abnormal chemoresponses in patients with lung disease and hypercapnia and have attributed the elevated PCO2 levels to disordered gas exchange in the lungs and a rapid-shallow breathing pattern that minimizes alveolar ventilation. Impaired chemosensitivity is also the critical underpinning of the obesity-hypoventilation (Pickwickian) syndrome. These patients who suffer from severe obesity that imposes a mass load on the chest wall also demonstrate chronic hypercapnia and experience obstructive sleep apnea leading to severe daytime somnolence. If the impairment in chemosensitivity is sufficiently severe, hypercapnia can result even in the absence of diseases of the ventilatory pump. Central alveolar hypoventilation is characterized by absent ventilatory responses to hypercapnia and hypoxia. Often

near-adequate levels of ventilation can be maintained during wakefulness but hypoventilation can become profound during sleep. It is thought that congenital central alveolar hypoventilation, also termed Ondine’s curse, is caused by maldevelopment of neural crest cells; it is often associated with Hirschsprung’s disease in which varying lengths of the colon are aganglionic. Patients often have a family history of the disorder that is associated with several different gene mutations especially of the PHOX2B gene, the receptor tyrosine kinase, the RET gene and endothelin1 and 3 genes, and brain-derived neurotrophic factor. Artificial mechanical ventilatory support is often required to maintain adequate levels of ventilation and a normal PCO2 . See also: Hypoxia and Hypoxemia. Kinins and Neuropeptides: Neuropeptides and Neurotransmission; Other Important Neuropeptides.

Further Reading Bradley SR, Pieribone VA, Wang W, et al. (2002) Chemosensitive serotonergic neurons are closely associated with large medullary arteries. Nature Neuroscience 5: 401–402. Bruce EN and Cherniack NS (1987) Central chemoreceptors. Journal of Applied Physiology 62: 389–402. Cherniack NS, Dempsey J, Fencl V, et al. (1977) Workshop on assessment of respiratory control in humans. I. Methods of measurement of ventilatory responses to hypoxia and hypercapnia. American Review of Respiratory Diseases 115: 177– 181. Dean JB, Ballantyne D, Cardone DL, Erlichman JS, and Solomon IC (2002) Role of gap junctions in CO2 chemoreception and respiratory control. American Journal of Physiology. Lung Cellular and Molecular Physiology 283: L665–L670. Dreshaj IA, Haxhiu MA, and Martin RJ (1998) Role of the medullary raphe nuclei in the respiratory response to CO2. Respiratory Physiology 111: 15–23. Erlichman JS, Cook A, Schwab MC, Budd TW, and Leiter JC (2004) Heterogeneous patterns of pH regulation in glial cells in the dorsal and ventral medulla. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 286: R289–R302. Kiwull-Schone H, Wiemann M, Frede S, Bingmann D, and Kiwull P (2003) Tentative role of the Na þ /H þ exchanger type 3 in central chemosensitivity of respiration. Advances in Experimental and Medical Biology 536: 415–421. Morrell MJ, Heywood P, Moosavi SH, Guz A, and Stevens J (1999) Unilateral focal lesions in the rostrolateral medulla influence chemosensitivity and breathing measured during wakefulness, sleep and exercise. Journal of Neurology, Neurosurgery and Psychiatry 67: 637–645. Nattie EE (1999) CO2, brainstem chemoreceptors and breathing. Progress in Neurobiology 59: 299–331. Nattie EE (2001) Central chemosensitivity, sleep, and wakefulness. Respiratory Physiology 129: 257–268. Neubauer JA and Sunderram J (2004) Oxygen-sensing neurons in the central nervous system. Journal of Applied Physiology 96: 367–374.

418 CHEMORECEPTORS / Arterial

F L Powell, University of California – San Diego, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Arterial chemoreceptors include the carotid and aortic bodies, which are small organs with a very high blood flow. Glomus cells in the arterial chemoreceptors sense changes in arterial PO2 , PCO2 , and pH by mechanisms involving ion channels and hemecontaining molecules. Decreased PO2 and pH, and increased PCO2 , depolarize glomus cells and trigger the release of neurotransmitters. An unidentified neurotransmitter depolarizes sensory nerve endings in the carotid and aortic body so the intensity of the arterial stimulus is coded as the frequency of action potentials in the afferent nerves going to respiratory and cardiovascular centers in the medulla. Hypoxia and hypercapnia potentiate the effect of each other on the carotid body and the effect of PCO2 is mediated by changes in intracellular pH. The response to changes in arterial blood gases is extremely rapid (in seconds). Arterial chemoreceptors exhibit plasticity during chronic hypoxia by increasing their O2-sensitivity.

Introduction Arterial chemoreceptor is a generic term for both the carotid body chemoreceptors and aortic body chemoreceptors. Arterial chemoreceptors respond to changes in arterial PO2 , PCO2 , and pH and evoke negative feedback reflexes in the respiratory and cardiovascular systems to maintain blood gas homeostasis. These are the most important chemoreceptors that respond to PO2 , making them essential for a normal hypoxic ventilatory response. Most of our knowledge about arterial chemoreceptors is based on studies of the carotid body, and the aortic bodies will be considered similar unless noted otherwise.

Structure The carotid bodies are small (E2 mm diameter in humans) sensory organs located near the carotid

Cell Types

Carotid bodies include several different types of cells (Figure 1). Glomus, or type I, cells are round or ovoid, 10–12 mm in diameter, and thought to be the primary chemoreceptor cell in the carotid body. These cells develop from the neural crest and are neurosecretory. The ultrastructure of glomus cells is typical of cells that actively synthesize and secrete O2, CO2, H+ Blood SN

Arterial

sinus at the bifurcation of the common carotid artery at the base of the skull. The aortic bodies are on the aortic arch near the aortic arch baroreceptors. Afferent nerves travel from the carotid bodies to the central nervous system (CNS) in the glossopharyngeal (IX cranial) nerve, and from the aortic bodies to the CNS in the vagus (X cranial) nerve. Efferent nerves to the carotid bodies include sympathetic and parasympathetic innervation of blood vessels, as well as sympathetic innervation of chemoreceptor cells. The carotid body is organized into lobules or glomoids that are surrounded by dense capillary networks and penetrated by the branches of the carotid sinus nerve. Blood supply to the carotid body is from the 1– 2 mm long carotid body artery, which branches off the external carotid artery and continues through the carotid body to perfuse the superior cervical and nodose ganglia. Blood supply to the aortic bodies is from short vessels branching off the aortic arch. Carotid bodies have an extremely high blood flow for their size, for example, 1.5 l per 100 g per min in a cat or E15 times blood flow to the brain tissue. There may be some shunt pathways for blood flow past the capillary networks in carotid body glomoids.

C

Okada Y, Chen Z, and Kuwana S (2001) Cytoarchitecture of central chemoreceptors in the mammalian ventral medulla. Respiratory Physiology 129: 13–23. Patrick W, Webster K, Puddy A, Sanii R, and Younes M (1995) Respiratory response to CO2 in the hypocapnic range in awake humans. Journal of Applied Physiology 79: 2058–2068. Sun MK and Spyer KM (1991) Responses of rostroventrolateral medulla spinal vasomotor neurones to chemoreceptor stimulation in rats. Journal of the Autonomic Nervous System 33: 79–84. Wiemann M and Bingmann D (2001) Ventrolateral neurons of medullary organotypic cultures: intracellular pH regulation and bioelectric activity. Respiratory Physiology 129: 57–70.

GC DA NE ACh SP

SC

Figure 1 Schematic of carotid body. PO2 , PCO2 , and pH in arterial blood stimulate glomus cells (GC) to release an unidentified neurotransmitter that excites the carotid sinus nerve (CSN) and sends action potentials to the medulla. Clear- and dense-cored vesicles contain dopamine (DA), norepinephrine (NE), acetylcholine (ACh), and substance P (SP). Sheath cells (SC) surround glomus cells.

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substances, including both clear and dense-cored vesicles. Glomus cells are connected to each other by gap junctions and make presynaptic and postsynaptic connections with afferent nerves in the carotid sinus nerve. Glomus cells contain several neurotransmitters and neuromodulators, as well as the enzymes to synthesize these substances (Figure 1). Opiates (e.g., met-enkephaline) and catecholamines are packaged in dense-cored vesicles in glomus cells. Dopamine is the most abundant catecholamine in the carotid body but norepinephrine is found as well. Clear-cored vesicles are thought to contain acetylcholine. Other neuroactive agents in glomus cells include serotonin (5-hydroxytryptamine), substance P, neurokinin A, bombesin, and atrial natriuretic peptide (ANP). Sheath cells, also known as type II or sustentacular cells, resemble glial or Schwann cells. Sheath cell bodies are located near the periphery of the carotid body and their processes reach into the organ to cover the surfaces of glomus cells that are not in contact with nerves or other glomus cells. Sheath cells generally prevent direct connections between glomus cells and capillary endothelial cells; however, they do not act as a diffusion barrier for relatively large (40 000 molecular weight) substances from the blood. Carotid bodies also include capillary endothelial cells and autonomic ganglia cells.

potassium channel conductance, which depolarizes glomus cells. Similarly, heme-oxygenase (HO) may act as an O2-sensor because hypoxia inhibits HO-2 and the production of carbon monoxide (CO); glomus cells are inhibited by low concentrations of CO. Nitric oxide synthases (NOS-1 and NOS-3) may sense O2 because hypoxia inhibits NOS and nitric oxide (NO) inhibits carotid body activity. However, NOS is only in the carotid sinus nerve afferents so it is not essential for O2-sensing by glomus cells. Finally, there is evidence for mitochondrial cytochromes with low oxygen affinity in glomus cells that may act as O2-sensors, although it is not clear how these depolarize glomus cells. Several kinds of potassium (K þ ) channels also contribute to O2-sensing in glomus cells. Background K þ channels (also known as leak currents) are sensitive to physiological levels of hypoxia, which decreases their open probability and depolarizes the glomus cell. Glomus cells also have a unique inward rectifying K þ channel responsible for an HERG-like K þ current that is sensitive to O2. However, it is not known if this channel responds to physiological levels of hypoxia. Glomus cells also have large conductance Ca2 þ -activated K þ channels (BK channels) that may be physically linked with HO-2 in the glomus cell membrane. BK channels are O2-sensitive and opened by CO so they represent a mechanism of oxygen sensing that involves oxidases and K þ channels.

Oxygen Sensing

CO2 and H þ Sensitivity

Oxygen sensing in the carotid body has been localized to the glomus cells but the precise mechanism is still unknown. Decreased PaO2 depolarizes the glomus cell, which increases intracellular calcium Ca2 þ to cause exocytosis of a neurotransmitter and excitation of the carotid sinus nerve. In carotid bodies in vivo, PO2 at the glomus cells is less than the arterial PO2 but it is greater than tissue PO2 in most other organs. This is because blood flow is relatively high and diffusion distances are small in carotid bodies. Hence, molecular mechanisms of O2-sensing in the carotid bodies are sensitive to PO2 in the physiological range. Candidates for molecular sensors providing the initial event in oxygen sensing fall into two general classes: heme-containing enzymes and O2sensitive ion channels. Multiple O2-sensors that are sensitive to different ranges of PO2 may be involved in glomus cell chemotransduction. Heme-containing enzymes that could sense O2 include NADP(H) oxidase in glomus cells. NADP(H) oxidase generates reactive oxygen species (ROS) such as H2O2 in the presence of oxygen. ROS increases potassium channel conductance so hypoxia decreases

The mechanism of PCO2 and pH chemoreception in glomus cells appears to be a common response to changes in intracellular pH. Intracellular acidity inhibits Ca2 þ -activated K þ currents in glomus cells, leading to depolarization and increased current through voltage-gated Ca2 þ channels. The increase in intracellular Ca2 þ promotes exocytosis of an excitatory neurotransmitter that stimulates the carotid sinus nerve. The increase in intracellular Ca2 þ from hypoxia is potentiated by hypercapnia, contributing to the multiplicative effect of O2 and CO2 on carotid body activity. PCO2 can diffuse into glomus cells and cause large changes in intracellular pH. Extracellular pH changes in blood cause smaller changes in intracellular pH of glomus cells. Hence, carotid body chemoreceptors are less sensitive to metabolic changes than respiratory changes in pH.

Sensory Coding Figure 2 shows the effect of arterial stimuli on the frequency of action potentials in the carotid sinus

420 CHEMORECEPTORS / Arterial P aCO

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carotid sinus nerve. Arterial chemoreceptor activity increases when PaO2 falls below 100 mmHg if PaCO2 is maintained at normal levels. However, if PaCO2 decreases, then PaO2 must decrease further to stimulate chemoreceptor activity to the same level. This latter scenario would occur, for example, when hypoxia stimulates ventilation and decreases PaCO2 . Conversely, carotid body chemoreceptors can be stimulated at higher PO2 levels if PaCO2 is increased. Hence, CO2 potentiates carotid body O2-sensitivity and vice versa. This synergistic, or multiplicative, effect of hypoxia and hypercapnia on carotid body chemoreceptors is important because it explains the multiplicative effect of PaO2 and PaCO2 on ventilation. CO2/pH

10 7.45 5 0 0 (b)

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Figure 2 Carotid body chemoreceptor response (action potential frequency) to PaO2 and PaCO2 (a) and PaCO2 and pHa (b) for two different fibers. Increasing PCO2 increases the response to PO2 , and the effect of the two stimuli together is more than the sum of the individual effects, as explained in the text. Decreasing pH increases the response at any PCO2 , but PCO2 still has an effect when pH is held constant. Adapted from Biscoe TJ, Purves MJ, and Sampson SR (1970) Journal of Physiology 208: 121 and Hornbein TF (1968) In: Torrance RW (ed.) Arterial Chemoreceptors. Blackwell Scientific Publications.

nerve. The response to O2 is hyperbolic while the response to CO2 is essentially linear. As explained above, hypoxia, hypercapnia, and acidity cause glomus cells to depolarize, increase intracellular Ca2 þ , and release an excitatory neurotransmitter. These are graded responses to stimuli so the frequency of action potentials in the carotid sinus nerve codes the stimulus intensity. Hypoxia

The physiological stimulus for carotid bodies is O2 partial pressure; they do not respond to changes in O2 content or hemoglobin saturation if PO2 remains constant. This explains the lack of ventilatory response in clinical conditions such as anemia or carbon monoxide poisoning. There is some evidence that aortic bodies may respond to changes in O2 content. Carotid bodies are sensitive to PO2 over the entire physiological range, although they are more sensitive to hypoxia. At normal PaO2 (100 mmHg) and PaCO2 (40 mmHg), there is a low, tonic level of activity in the

Figure 2(b) shows the effect of pH and PCO2 on carotid body chemoreceptors. PaCO2 changes can affect chemoreceptor activity even if pHa is held constant, and vice versa. Aortic bodies in humans do not respond to pHa changes, and this is one exception to remember about the aortic bodies being qualitatively different from the carotid bodies. Therefore, the carotid bodies are the only chemoreceptors that respond to metabolic changes in pHa when PaCO2 is constant in humans. Not shown in Figure 2 is the increase of slope of the carotid body response to PCO2 caused by hypoxia. This results from the multiplicative effect of O2 and CO2 on the carotid body described above. Pattern and Speed of Response

Arterial chemoreceptors respond rapidly (within seconds) to changes in PaO2 , PaCO2 , and pHa. Changes in arterial blood gases that occur in phase with breathing, especially during conditions such as exercise, can be sensed by arterial chemoreceptors and may stimulate ventilation. This rapid response explains how ventilation can be altered within a single breath when arterial blood gases change. Carotid body chemoreceptors contain carbonic anhydrase, which will increase the speed of response to PaCO2 according to the intracellular pH-sensing mechanisms described above. Although different patterns of action potential frequency (e.g., bursting) can occur with different stimuli, the CNS only responds to the average discharge frequency from carotid bodies. There is no evidence that stimulating carotid body chemoreceptors with O2 versus CO2, for example, can result in different reflex effects. Neurotransmitters

Despite the identification of several neuroactive substances being released from carotid bodies during

CHEMORECEPTORS / Arterial

stimulation, the excitatory neurotransmitter between glomus cells and the carotid sinus nerve is not known. Acetylcholine is the most likely candidate. Acetylcholine stimulates carotid body activity via nicotinic receptors although chemoreception is not blocked by nicotinic or muscarinic receptor blockers. Substance P stimulates carotid body activity but it is not present in the glomus cells of all species. Norepinephrine and serotonin excite carotid sinus nerve activity but they do not appear to be the primary neurotransmitters from glomus cells and act as neuromodulators. Hypoxia stimulates release of dopamine from glomus cells in a graded fashion but dopamine is mainly inhibitory in the carotid body. Dopamine acts primarily on dopamine-2 autoreceptors on glomus cells to limit excitability by negative feedback. Met- and leuenkephalins also depress carotid sinus nerve activity. Multiple inhibitory neurotransmitter systems suggest that limiting and modulating chemoreceptor activity is physiologically significant. Autonomic Modulation

Stimulating the peripheral end of the transected carotid sinus nerve inhibits carotid body chemoreceptor activity in experimental animals. This involves at least two mechanisms. Parasympathetic nerves release NO to cause vasodilation, which increases local PO2 and decreases hypoxic stimulation. Sensory fibers may also release NO, which increases cyclic guanosine monophosphate (GMP) in glomus cells and inhibits their activity. It is not known if the autonomic effect is physiologically significant in vivo.

Arterial Chemoreceptors and Respiratory Disease Diseases of the arterial chemoreceptors per se are relatively rare in populations living at sea level but chemodectomas (carotid body tumors) are more common in populations found at high altitude. This may contribute to the blunted hypoxic ventilatory response observed in some high-altitude natives and people suffering from chronic mountain sickness. The carotid bodies and their innervation may also be damaged during surgical procedures in the region, such as carotid endarterectomy. However, the consequences of this for respiration are relatively unstudied. More commonly, arterial chemoreceptors are important in respiratory disease in terms of the physiological responses they elicit with, for example, hypoxemia or CO2 retention. The basic responses to changes in arterial blood gases have been described above. It is known that these responses can be altered by chronic hypoxia in healthy subjects, for example

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during acclimatization to hypoxia at high altitude. It is reasonable to suspect that similar adaptations may take place in patients with chronic hypoxemia from respiratory disease, although arterial CO2 levels are higher than in normal subjects acclimatized to altitude. However, it has been impossible to study the arterial chemoreceptors in patients with respiratory disease to date so it is not clear if the adaptations described for normal subjects occur in patients. Similarly, it is not clear if some of the difference in the progress of respiratory diseases in different patients depends on individual differences in the premorbid arterial chemoreceptor sensitivities and reflex response, or in the ability of these processes to adapt to chronic hypoxia. The effects of chronic hypoxia on arterial chemoreceptors in healthy subjects are described next. Plasticity in Chronic Hypoxia

Chronic hypoxia (days to weeks) increases O2-sensitivity of carotid body chemoreceptors. This increases afferent input from carotid bodies to respiratory centers in the brain and stimulates ventilation. This contributes to the time-dependent increase in ventilation during acclimatization to hypoxia. The effect is specific to hypoxia because it occurs even when arterial pH and PCO2 are held at normal levels during hypoxia and it does not occur with chronic application of other stimuli such as CO2. Changes in ion channels with chronic hypoxia tend to depolarize glomus cells and increase O2-sensitivity. Chronic hypoxia downregulates K þ conductance, increases the density of Na þ and Ca2 þ channels, and preferentially increases Ca2 þ influx through L-type Ca2 þ channels in glomus cells. Chronic hypoxia also increases the density of O2-sensitive Ca2 þ -activated K þ channels on glomus cells. Chronic hypoxia also affects neurotransmitters in the carotid body, although it does not cause a coordinated increase in excitatory, or decrease in inhibitory neurotransmitters. Substance P, an excitatory neurotransmitter in the carotid body, decreases in carotid bodies during chronic hypoxia. Dopamine, an inhibitory neurotransmitter in the carotid body, increases in carotid bodies during chronic hypoxia. Chronic hypoxia increases nicotinic receptors on afferent nerve endings in the carotid body but the effects of acetylcholine on O2-sensitivity do not change. The expression of new neuromodulators in the carotid body can increase O2-sensitivity. Endothelin1 (ET1) is not observed in carotid bodies under normoxic control conditions but it stimulates carotid bodies. Chronic hypoxia increases ET1 and blocking the ETA receptor for ET1 eliminates the increase in

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O2-sensitivity with chronic hypoxia in experimental animals. Chronic hypoxia also causes significant morphological changes in the carotid body but these cannot explain the functional changes. Carotid bodies show hypertrophy, hyperplasia of glomus cells, and increased capillarity after exposure to 2 or more weeks of hypoxia. These changes are reversible. Neovascularization is stimulated by endothelial growth factor (VEGF), which is controlled by hypoxic inducible factor 1a (HIF-1a), and endothelin-1 (ET1). See also: Acid–Base Balance. Arterial Blood Gases. Chemoreceptors: Central. High Altitude, Physiology and Diseases. Ventilation: Control.

Further Reading Biscoe TJ, Purves MJ, and Sampson SR (1970) Journal of Physiology 208: 121.

Dinger B, He L, Chen J, Stensaas L, and Fidone S (2003) Mechanisms of morphological and functional plasticity in the chronically hypoxic carotid body. In: Lahiri S, Semenza G, and Prabhakar N (eds.) Oxygen Sensing: Responses and Adaptation to Hypoxia, pp. 439–465. New York: Dekker. Fidone S, Gonzales C, Almaraz L, and Dinger B (1997) Cellular mechanisms of peripheral chemoreceptor function. In: Crystal RG and West JB (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 1725–1737. Philadelphia: Lippincott-Raven. Gonzalez C, Almaraz L, Obeso A, and Rigual R (1994) Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiological Reviews 74: 829–898. Heath D and Williams DR (1995) High-Altitude Medicine and Pathology, 4th edn. Oxford: Oxford University Press. Hornbein TF (1968) In: Torrance RW (ed.) Arterial Chemoreceptors. Blackwell Scientific Publications. Prabhakar N (2000) Oxygen sensing by the carotid body chemoreceptors. Journal of Applied Physiology 88: 2287– 2295. Zak FG and Lawson W (1982) The Paraganglionic Chemoreceptor System: Physiology, Pathology, and Clinical Medicine. New York: Springer.

CHEST WALL ABNORMALITIES J L Allen and O H Mayer, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA K W Gripp, A.I. DuPont Hospital for Children, Wilmington, DE, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Chest wall abnormalities can substantially impact the quality of breathing. Chest wall dysfunction can occur as a primary disorder due to congenital (e.g., VATER syndrome), genetic (e.g., Jarcho–Levin syndrome), or acquired (e.g., scoliosis) causes, or can occur as a secondary disorder due to conditions such as obesity, neuromuscular disease, or chronic obstructive pulmonary disease. Recently, much has been learned about the genetic mechanisms producing chest wall disorders. These mechanisms include dominant negative action (e.g., Marfan syndrome), gain-of-function mutation (e.g., achondroplasia), haploinsufficiency (e.g., Campomelic dysplasia), and loss-of-function mutations (e.g., Jarcho–Levin syndrome). Clinical features common to all these disorders include, in severe cases, respiratory insufficiency due to pulmonary hypoplasia or respiratory pump failure. There are a variety of noninvasive interventions to help counteract the effects of chest wall dysfunction; these improve airway clearance (mechanical percussion devices and cough assist devices) and improve ventilation (noninvasive or invasive nocturnal or continuous mechanical ventilation). Surgical interventions to correct or prevent further progression of the disorder include bracing, spinal fusion, and, more recently, introduction of the vertical expandable prosthetic titanium rib (VEPTR), which has the advantage of allowing for future spinal growth.

Introduction The chest wall, including the ribcage and respiratory muscles, comprises the ‘pump’ component of the respiratory system. Respiratory loads, primarily elastic and resistive, arise from the lung and the chest wall itself. Disorders of the thoracic cage can affect both the pump and the load components. This chapter describes some of the basic mechanisms by which congenital abnormalities of the chest wall are produced, and the evaluation and treatment of chest wall disorders.

Etiology Chest wall abnormalities can be categorized as primary or secondary. Primary chest wall disorders can be congenital or acquired. There are two types of congenital abnormalities. Those with a genetic basis such as achondroplasia or Jarcho–Levin syndrome have a predictable recurrence risk, while those that arise embryologically or secondary to intrauterine environmental causes, such as VATER (vertebral, anal, tracheo-esophageal fistula, radial, and renal anomalies) syndrome, are without a known genetic basis or recurrence risk. Acquired chest wall disorders, such as idiopathic scoliosis or pectus excavatum become apparent later in childhood or adolescence; while they may be familial, there is no certain genetic or environmental cause.

CHEST WALL ABNORMALITIES 423

Secondary chest wall disorders lead to dysfunction caused by other illnesses or mechanical limitations. Examples include chest wall muscle weakness due to neuromuscular disorders and malnutrition, restrictive chest wall disease due to pleural effusions or obesity, and mechanical inefficiencies of the chest wall imposed by diaphragmatic flattening and loss of the area of apposition seen in the pulmonary hyperinflation of obstructive lung diseases such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease. Only primary chest wall disorders are discussed in this article. Secondary chest wall disorders are discussed in the articles listed in ‘See also’.

Pathology Skeletal dysplasias may lead to severe pulmonary complications, due either to pulmonary hypoplasia or to respiratory pump failure. The underlying defect in skeletal dysplasia may affect either the extracellular matrix or the intracellular metabolism of cells forming skeletal structures. Though the ribs and cartilage provide the bulk of the support of the thoracic cavity, muscular abnormalities can also have substantial impact on thoracic mechanics and function. Congenital Disorders, Genetic Basis Known

Intrinsic skeletal dysplasias vary greatly in severity and in their effects on the respiratory system, ranging from lethality due to pulmonary hypoplasia to respiratory pump failure and restrictive lung disease. The most common form of skeletal dysplasia (frequency 1/10 000) is achondroplasia, characterized by

rhizomelic short stature and macrocephaly. Patients with achondroplasia have short flared ribs, forming a shallow thorax with decreased chest wall circumference. In addition, patients often have severe midfacial hypoplasia causing obstructive apnea, or central apnea due to brainstem compression secondary to a small foramen magnum and mild hydrocephalus. The rare patients with homozygous achondroplasia show a much more severe phenotype and die in early infancy, often from respiratory complications. In Jarcho–Levin syndrome, or spondylothoracic dysplasia, rib and spinal abnormalities produce a short thorax with a fan-like configuration of ribs (Figure 1) due to their fusion at the costovertebral junctions. This also presents in infancy and causes severe lung restriction and respiratory insufficiency for which some patients require mechanical ventilation. The characteristic findings of cleidocranial dysplasia are wide, late-closing fontanels, hypoplastic or absent clavicles, and supernumerary teeth. More subtle abnormalities include moderate short stature, a narrow thorax with short ribs, hypoplastic pubic bones, and brachydactyly. Respiratory distress may occur but is relatively rare. Other clinical malformation syndromes affecting the thorax and their genetic bases (where known) are listed in Table 1. Congenital Disorders, Environmental or Genetic Basis Unknown

Jeune syndrome, or asphyxiating thoracic dystrophy, is a disorder of rib development in which the cross-sectional area of the ribcage does not increase

Figure 1 10-year-old girl with Jarcho–Levin syndrome and the fan-like arrangement of ribs with bilateral vertical expandable prosthetic ribs in place.

Table 1 Malformation syndromes affecting the thorax Disorder

Physical findings

Thoracic involvement

Respiratory complications

Inheritance mode

Gene

Pathomechanism

VATER association

Anal atresia; TE fistula; radial and renal anomalies

None

Embryologic in origin

Hemifacial microsomia with microtia and small mandible; may be bilateral; preauricular skin tags; renal anomalies Aortic dilatation; lens dislocation; arachnodactyly; joint laxity Multiple fractures, joint laxity; blue sclera; dentinogenesis imperfecta Macrocephaly, short limb dwarfism; narrow foramen magnum Bowed long bones; male to female sex reversal; cleft palate

Rare, due to small thoracic cage and/or scoliosis Rare, due to small thoracic cage and/or scoliosis

Sporadic

Facioauriculovertebral spectrum; Goldenhar syndrome Marfan syndrome

Vertebral segmentation anomalies; structural rib anomalies; scoliosis Hemivertebrae; structural rib anomalies; scoliosis

Sporadic

None

Embryologic in origin

Scoliosis; pectus excavatum or carinatum; pneumothorax Rib fractures

Rarely due to abnormal thorax or pneumothorax Unstable thorax with multiple rib fractures

Autosomal dominant

FBN1

Dominant negative action

Autosomal dominant

COLIA1 or COLIA2

Dominant negative action

Small thoracic cage

Restrictive lung disease; central and obstructive apnea Respiratory insufficiency may lead to death in infancy Rare

Autosomal dominant

FGFR3

Gain-of-function

Autosomal dominant

SOX9

Haploinsufficiency

Autosomal dominant

CBFA1

Haploinsufficiency

Autosomal recessive (most cases of Puerto Rican ancestry)

DLL3

Loss of function

Autosomal recessive

DTDST

Loss of function

Osteogenesis imperfecta Achondroplasia

Campomelic dysplasia

Cleidocranial dysplasia Spondylothoracic dysplasia; Jarcho–Levin syndrome Diastrophic dysplasia

Wide anterior fontanel; supernumerary teeth; short stature Vertebral and rib segmentation defects; short neck; long fingers

Short limb dwarfism, clubfeet; cleft palate; cervical kyphosis

Small thoracic cage; kyphocoliosis

Partially or completely absent clavicle Short thorax due to vertebral and rib defects

Progressive kyphoscoliosis

Respiratory insufficiency due to small thoracic volume, may be lethal Respiratory compromise due to kyphosis

Category refers to the presumed genetic mechanism by which the mutations cause the phenotype (see text). Mutations in the listed genes have been identified in the respective disorders. In some disorders, there is a strict correlation between the genotype (i.e., a specific mutation or a mutation in a specific gene), while in others, heterogeneity is suspected or proven (e.g., not all patients with Jarcho–Levin syndrome have mutations in DLL3). The genetic basis of the disorders are discussed in detail in ‘Pathogenesis’ section.

CHEST WALL ABNORMALITIES 425

Figure 2 Anterior computed tomography (CT) reconstruction of Jeune syndrome with the narrow and long thorax also showing the short ribs.

Figure 3 CT image of Jeune syndrome at the carina that demonstrates the short ribs and anterior chest wall deformity.

normally and the thorax becomes long but narrow or constricted (Figures 2 and 3). It often presents in early infancy and results in severe pulmonary compromise, often requiring invasive mechanical ventilation and aggressive airway clearance. Acquired Disorders

The most frequent chest wall disorder in children is pectus excavatum. Though pectus excavatum and

pectus carinatum may be cosmetically disturbing, they usually have little or no impact on cardiopulmonary function. Pectus excavatum, while typically acquired as an isolated deformation, can occur with connective tissue disorders like Marfan syndrome, or in subjects with substantial respiratory muscle weakness like spinal muscular atrophy type 1. Scoliosis is most commonly idiopathic, but can also be congenital or secondary. Idiopathic scoliosis is the most common form of scoliosis, accounting for 80–85% of all lateral spinal curvature. The frequency of onset increases with age, with the most common age of onset being adolescence (11 and older); idiopathic scoliosis is very uncommon under 4 years of age. Though it commonly presents with the relatively benign finding of spinal asymmetry on forward bending, if the curve is greater than 351, there is a greater than 40% chance of significant lung restriction and of moderate to severe gas trapping likely due to airway compression. The risk for progression is related to both the degree of curve and the age of the patient, with curves of 301 rarely progressing in adulthood. Scoliosis is much more likely to progress in prepubertal children. Congenital scoliosis is less common than idiopathic scoliosis and differs in having a causative spinal abnormality. Abnormalities include hemivertebrae, as in Goldenhar syndrome or the facioauriculovertebral spectrum, abnormal vertebral segmentation, as in the VATER association, and inadequate ossification. In congenital scoliosis the curve may not be clinically apparent at birth, but with longitudinal growth the spine will grow asymmetrically creating a curve. Because of the many different causes, the rate of progression and prognosis is very variable. Secondary scoliosis can occur due to inadequate truncal support, such as in neuromuscular disease like Duchenne muscular dystrophy and spinal muscular atrophy (Figure 4). The curve occurs after progressive loss of muscle tone and truncal support with the spine curving to one side and, in some situations, rotating. This can be further worsened with loss of ambulation and the different forces that act on the ribcage when a child is in the sitting position as opposed to upright. Isolated rib fusion can occur and can also lead to progressive scoliosis as the lateral edge of the thorax is fixed while the spine continues to grow, leading to a poorly compliant chest wall. Conversely, absence of ribs can also cause progressive scoliosis due to inadequate support with a highly compliant or flaccid thoracic cavity.

426 CHEST WALL ABNORMALITIES

embryologic anomaly without a known genetic cause and no significant recurrence risk, presents with structural vertebral and rib anomalies. A second example is the facioauriculovertebral spectrum, also known as Goldenhar syndrome or hemifacial microsomia. In addition to structural vertebral and rib anomalies, eye and ear findings and a small jaw may affect one side of the face, or rarely, both sides. Gene Mutations: Autosomal Dominant, Dominant Negative Action

Figure 4 Chest radiograph in a 2-year-old male with spinal muscular atrophy type-1 demonstrating significant scoliosis.

Clinical Features Though there are many different causes of chest wall abnormalities, the clinical manifestations are often quite similar. They can vary from primarily cosmetic disorders, as in pectus excavatum, carinatum, and mild idiopathic scoliosis, to severe restrictive lung disease as in the severe genetic skeletal dysplasias, progressive congenital scoliosis, and neuromuscular disease. With an increasing scoliotic curve, the lung on the concave side becomes more compressed and the range of motion of the diaphragm will decrease. This decreases vital capacity and, with further worsening, can lead to significant nocturnal hypoventilation or to more continuous hypoventilation and the need for noninvasive ventilation due to eventual respiratory muscle fatigue and failure. Pulmonary compression may also lead to poor airway clearance in the affected lung due to inability to fully expand the lung and airway narrowing that may occur from the lung compression or airway bending. In this environment, poor airway clearance can lead to retained secretions or ‘mucus plugging’ and segmental atelectasis, persistent or chronic pneumonia, and bronchiectasis.

Pathogenesis Embryologic Abnormalities

Congenital anomalies of the chest wall can occur due to embryological anomalies, or due to underlying gene mutations. The VATER association, an

In contrast to embryologic abnormalities, disorders caused by gene mutations may have a recurrence risk, and may be present in other family members in addition to the patient. Autosomal dominant conditions include the connective tissue disorders, Marfan syndrome and osteogenesis imperfecta (Table 1). Marfan syndrome is caused by heterozygous mutations in the fibrillin 1 gene (FBN1) resulting in an abnormal gene product. Fibrillin is the major constitutive element of extracellular microfibrils. Incorporation of a structurally abnormal protein disrupts the macromolecular structure of the extracellular microfibrils, despite the fact that the patient has one wild-type allele encoding structurally and functionally normal fibrillin. This mechanism of action, by which the structurally abnormal gene product is disease-causing regardless of the presence of normal gene product, is called dominant negative. Another example for this mechanism is osteogenesis imperfecta, resulting from a single mutation in one of the genes encoding a procollagen chain. Mature collagen molecules consist of three procollagen chains forming a triple helical structure. Incorporation of a single structurally abnormal component disrupts this complex structure and leads to the abnormal connective tissue properties including brittle bones. Autosomal Dominant, Gain-of-Function Mutations

Achondroplasia is caused by a heterozygous point mutation in the fibroblast growth factor receptor 3 gene (FGFR3), resulting in a glycine to arginine amino acid substitution at position 380 of the protein product. This membrane-bound receptor has an intracellular tyrosine kinase domain that is activated upon ligand binding and ultimately regulates cell proliferation. The function of the normal receptor is negative regulation of enchondral growth, as demonstrated by the skeletal overgrowth seen in mice lacking both functional copies of the gene. The pathogenesis of the mutation involves constitutive activation of FGFR3, inhibiting proliferation of growth plate chondrocytes and thus causing short limb dwarfism. This gain-of-function mechanism is

CHEST WALL ABNORMALITIES 427

further enhanced by diversion of the abnormal gene product from the normal lysosomal degradation process to increased recycling with prolonged survival. Patients whose parents both have achondroplasia are at 25% risk for homozygosity of the mutation, resulting in extreme short limb dwarfism and lethal respiratory insufficiency. A second lethal skeletal dysplasia, thanatophoric dysplasia, is also caused by mutations in FGFR3. Autosomal Dominant, Haploinsufficiency

A third pathogenic mechanism causing autosomal dominant skeletal disorders is haploinsufficiency for the functional gene product. This mechanism most often affects protein products acting as transcription factor regulating the expression of other genes. A 50% decrease of the functional protein is diseasecausing in dosage sensitive pathways. Campomelic dysplasia and cleidocranial dysostosis are examples of skeletal dysplasias due to gene mutations resulting in haploinsufficiency for the respective transcription factors (see Table 1). Autosomal Recessive Conditions

Diastrophic dwarfism (achondrogenesis type IB) and Jarcho–Levin syndrome are due to mutations in both alleles of the respective gene, causing loss-of-function of protein products with enzymatic or transport function (Table 1). The pattern of malformation seen in Jarcho–Levin syndrome is suggestive of abnormal segmentation in early embryological development. In contrast to haploinsufficiency, loss-of-function in both gene alleles is necessary to cause disease. Achondrogenesis type IB causes a clinical syndrome of extreme short stature, poor ossification of the skull and vertebral bodies, severe micromelia of the limbs, extremely short ribs, and stellate long bones. The mutation resides in the diastrophic dysplasia sulfate transporter (DTDST) gene, leading to decreased or absent sulfate transport and abnormalities in cartilage proteoglycans sulfation.

Management and Current Therapy Nonsurgical

For subjects with hypoventilation or respiratory failure, intermittent nocturnal noninvasive ventilation can be initiated using a nasal interface. Continuous mechanical ventilation that allows both mobility and full use of both the mouth and nose can be achieved using rhythmic abdominal compression using a Pneumo-Belts or having a tracheostomy placed to allow for invasive mechanical ventilation. In situations in which mobility is not necessary, patients may also use negative pressure ventilation, using a device that applies negative pressure directly to the thorax (cuirass or ‘chest shell’) or to the body below the neck (PortaLungs or poncho/jacket). Maximizing airway clearance with manual or mechanical chest percussion and postural drainage augmented with nebulized b2-adrenergic agonists is also very important. For subjects with segmental or lobar compression, the application of distending pressure via nasal mask or tracheostomy can help to open the airway and maintain patency during breathing and coughing. Applying cyclic positive pressure to inflate the lungs rapidly followed by negative pressure using

Other Disorders

Other disorders with structural abnormalities of the chest wall are genetic in nature but the abnormal genes have not yet been identified. Examples include Jeune’s syndrome, the cerebrocostomandibular syndrome (gaps in the posterior aspects of the ribs), and the Fryns syndrome (diaphragmatic hernias in combination with multiple other anomalies). Fryns syndrome is inherited as an autosomal recessive trait, and the multiple affected organ systems are suggestive of underlying mutations in a gene widely expressed during early development.

Figure 5 Postoperative lateral view of chest CT reconstruction in a 4-year-old girl with scoliosis and rib fusion, with VEPTR in place between the 4th and 5th ribs (black arrow) and between the iliac crest and 5th rib posteriorly (white arrow). Note the expanded intercostal space between the 4th and 5th ribs.

428 CHEST WALL ABNORMALITIES

Figure 6 Postoperative posterior view of chest CT reconstruction in a 4-year-old girl with scoliosis and rib fusion, with VEPTR in place between the 4th and 5th ribs (white arrow) and between the iliac crest and 5th rib (black arrow) posteriorly.

a device such as the Cough Assists device can replace or augment coughing, especially in patients with significant muscle weakness or cognitive disability. Surgical

Though it is not necessary to correct pectus excavatum in many situations, a ratio of the transverse to the anterioposterior diameter of greater than 3.5 is felt by many to be an indication for surgery. Severe pectus excavatum can impair cardiac output in the upright position and increase the work of breathing at high workloads. The Nuss procedure can correct an abnormality by applying internal, anterior pressure at the apex of the pectus curve. A rigid curved rod is inserted along the inner anterior chest wall and is then rotated up and sutured in place. The correction can be quite substantial and often the bar can be removed after a year without the defect returning. Correction for scoliosis has traditionally involved straightening the spine either with traction alone or permanently with posterior spinal fusion. For idiopathic scoliosis with curves of 451, surgical repair is often performed after a trial of bracing. While scoliosis surgery may not result in an immediate gain in lung volume, it helps minimize the future loss of vital capacity. However, spinal fusion reduces spinal growth in the fused segments and limits future thoracic growth. Alternatives include spinal growing

rods or the vertical expandable prosthetic titanium rib (VEPTR), both of which allow thoracic expansion. For more substantial chest wall abnormalities such as congenital scoliosis with fused ribs, absent ribs, or chest wall constriction from a variety of causes, reconstruction using the VEPTR allows for both lateral stabilization and vertical expansion of the thorax with growth (Figures 5 and 6). The devices can be arranged in a variety of orientations with expansion of the ribs laterally, or from the spine or iliac crest. See also: Chronic Obstructive Pulmonary Disease: Overview. Cystic Fibrosis: Overview. Lung Development: Overview; Congenital Parenchymal Disorders; Congenital Vascular Disorders. Nutrition in Respiratory Disease. Obesity. Pectus Excavatum. Pediatric Pulmonary Diseases. Pleural Effusions: Overview. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Trauma, Chest: Bronchopleural Fistula; Postpneumonectomy Syndrome; Subcutaneous Emphysema. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation; Ventilator-Associated Pneumonia.

Further Reading Beiser G, Epstein S, Stampfer M, et al. (1972) Impairment of cardiac function in patients with pectus excavatum, with improvement after operative correction. New England Journal of Medicine 287(6): 267–272.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 429 Boyer J, Amin N, Taddonio R, and Dozor A (1996) Evidence of airway obstruction in children with idiopathic scoliosis. Chest 109: 1532–1535. Byers P and Steiner R (1992) Osteogenesis imperfecta. Annual Review of Medicine 43: 269–282. Campbell R and Hell-Vocke A (2003) Growth of the thoracic spine in congenital scoliosis after expansion thoracoplasty. Journal of Bone and Joint Surgery America 85-A(3): 409–420. Castile R, Staats B, and Westbrook P (1982) Symptomatic pectus deformities of the chest. American Review of Respiratory Diseases 126(3): 564–568. Cho J, Guo C, Torello M, et al. (2004) Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia. Proceedings of the National Academy of Sciences, USA 101(2): 609–614. Cornier A, Ramirez N, Arroyo S, et al. (2004) Phenotype characterization and natural history of spondylothoracic dysplasia syndrome: a series of 27 new cases. American Journal of Medical Genetics 128A: 120–126. Francomano C (1995) The genetic basis of dwarfism. New England Journal of Medicine 332: 58–59.

Chloride Channels

Hunter A, Reid C, Pauli R, and Scott C (1996) Standard curves of chest circumference in achondroplasia and the relationship of chest circumference to respiratory problems. American Journal of Medical Genetics 62: 91–97. Kose N and Campbell R (2004) Congenital scoliosis. Medical Science Monitor 10(5): RA104–RA110. Lyons-Jones K (1988) Smith’s Recognizable Patterns of Human Malformation. Philadelphia: Saunders. McMaster M (2001) Congenital scoliosis. In: Weinstein S (ed.) The Pediatric Spine: Principles and Practice, 2nd edn, pp. 161–177. Philadelphia: Lipincott Williams & Wilkins. Park H, Lee S, Lee C, et al. (2004) The Nuss procedure for pectus excavatum: evolution of techniques and early results on 322 patients. Annals of Thoracic Surgery 77: 289–295. Stacey A, Bateman J, Choi T, et al. (1988) Perinatal lethal osteogenesis imperfecta in transgenic mice bearing engineered mutant pro-alpha 1(I) collagen. Nature 332: 131–136. Tosi L (1997) Osteogenesis imperfecta. Current Opinion in Pediatrics 9(1): 94–99. Wong K-S, Hung I, Wang C, and Lien R (2004) Thoracic wall lesions in children. Pediatric Pulmonology 37: 257–263.

see Ion Transport: Chloride Channels.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE Contents

Overview Acute Exacerbations Emphysema, Alpha-1-Antitrypsin Deficiency Emphysema, General Smoking Cessation

Overview P J Barnes, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Chronic obstructive pulmonary disease (COPD) is characterized by progressive and largely irreversible airflow limitation due to narrowing and fibrosis of small airways and loss of airway alveolar attachments as a result of emphysema. Cigarette smoking is the most important risk factor, but other noxious gases are important too in developing countries. Genes may determine which smokers are susceptible to the development of airflow obstruction. There is a specific pattern of inflammation characterized by increased numbers of macrophages, neutrophils, and T lymphocytes (particularly CD8 þ cells), which is an amplification of the inflammation seen in normal cigarette smokers and increases with disease progression. Proteinases, particularly MMP-9, result in degradation of alveolar wall elastin resulting in emphysema. The major symptom is exertional dyspnea

caused by air trapping and hyperinflation as small airways close, but cough and sputum production are common due to mucus hypersecretion. Management of COPD involves smoking cessation. Bronchodilator therapy reduces hyperinflation; regular long-acting inhaled b2-agonists and anticholinergics are preferred to short-acting drugs. Inhaled corticosteroids do not reduce inflammation but reduce exacerbations and are usually given to patients with severe disease with frequent exacerbations. Long-term oxygen is indicated for patients with respiratory failure who have evidence of pulmonary hypertension. Nonpharmacological treatments include pulmonary rehabilitation, noninvasive ventilation, and lung volume reduction.

Introduction Chronic obstructive pulmonary disease (COPD) is characterized by progressive development of airflow limitation that is not fully reversible. The term COPD encompasses chronic obstructive bronchiolitis with obstruction of small airways and emphysema

430 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview

with enlargement of airspaces and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways. Chronic bronchitis, by contrast, is defined by a productive cough of more than 3 months duration for more than 2 successive years; this reflects mucus hypersecretion and is not necessarily associated with airflow limitation. COPD is common and is increasing globally. It is now the fourth leading cause of death in the US and the only common cause of death that is increasing; this is thought to be an underestimate as COPD is likely to be contributory to other common causes of death. It is predicted to become the third commonest cause of death and the fifth commonest cause of disability worldwide in the next few years. It currently affects more than 5% of the adult population and is underdiagnosed in the community. COPD consumes an increasing proportion of healthcare resources, which currently exceed those devoted to asthma by threefold.

Etiology Several environmental and endogenous factors, including genes, increase the risk of developing COPD (Table 1). Environmental Factors

In industrialized countries, cigarette smoking accounts for most cases of COPD, but in developing countries, other environmental pollutants, such as particulates associated with cooking in confined spaces, are important causes. It is likely that there are important interactions between environmental factors and a genetic predisposition to develop the disease. Air pollution (particularly sulfur dioxide and particulates), exposure to certain occupational chemicals such as cadmium, and passive smoking may all Table 1 Risk factors for COPD Environmental factors

Endogenous (host) factors

Cigarette smoking Active Passive Maternal

a1-Antitrypsin deficiency Other genetic factors Ethnic factors Airway hyperresponsiveness? Low birth weight

Air pollution Outdoor Indoor: biomass fuels Occupational exposure Dietary factors High salt Low antioxidant vitamins Low unsaturated fatty acids Infections

be additional risk factors. The role of airway hyperresponsiveness and allergy as risk factors for COPD is still uncertain. Atopy, serum IgE, and blood eosinophilia are not important risk factors. However, this is not necessarily the same type of abnormal airway responsiveness that is seen in asthma. Low birth weight is also a risk factor for COPD, probably because poor nutrition in fetal life results in small lungs, so that decline in lung function with age starts from a lower peak value. Genetic Factors

Longitudinal monitoring of lung function in cigarette smokers reveals that only a minority (15–40% depending on definition) develop significant airflow obstruction due to an accelerated decline in lung function (two- to fivefold higher than the normal decline of 15–30 ml forced expiratory volume in 1s (FEV1) year
1) compared to the normal population and the remainder of smokers who have consumed an equivalent number of cigarettes (Figure 1). This strongly suggests that genetic factors may determine which smokers are susceptible and develop airflow limitation. Further evidence that genetic factors are important is the familial clustering of patients with early onset COPD and the differences in COPD prevalence among different ethnic groups. Patients with a1-antitrypsin deficiency (proteinase inhibitor (Pi)ZZ phenotype with a1-antitrypsin levels o10% of normal values) develop early emphysema that is exacerbated by smoking, indicating a clear genetic predisposition to COPD. However, a1-antitrypsin deficiency accounts for o1% of patients with COPD and many other genetic variants of a1-antitrypsin that are associated with lower than normal serum levels of this Pi have not been clearly associated with an increased risk of COPD. This has led to a search for associations between COPD and polymorphisms of other genes that may be involved in its pathophysiology. So far, few significant associations have been detected and even those reported have not been replicated in other studies. A 10-fold increased risk of COPD in individuals who have a polymorphism in the promoter region of the gene for tumor necrosis factor alpha (TNF-a) that is associated with increased TNF-a production has been reported in a Taiwanese population but not confirmed in Caucasian populations. Several other genes have been implicated in COPD, but few have been replicated in different populations (Table 2). Techniques such as DNA microarray (gene chips) to detect single nucleotide polymorphisms, proteomics to detect novel proteins, and gene expression profiling to measure which known and novel genes are expressed are now

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 431

FEV1 (% predicted at age 25)

100 Nonsmoker or nonsusceptible smoker 75 Susceptible smoker Stopped smoking aged 50 years

50 Disability 25

Stopped smoking aged 60 years

Death 0 25

50 Age (years)

75

Figure 1 Natural history of COPD. Annual decline in airway function showing accelerated decline in susceptible smokers and effects of smoking cessation. Patients with COPD usually show an accelerated annual decline in forced expiratory volume in 1 s (FEV1), often greater than 50 ml year
1, compared to the normal decline of approximately 20 ml year
1, although this is variable between patients. The classic studies of Fletcher and Peto established that 10–20% of cigarette smokers are susceptible to this rapid decline. However, with longer follow-up more smokers may develop COPD. The propensity to develop COPD amongst smokers is only weakly related to the amount of cigarettes smoked and this suggests that other factors play an important role in determining susceptibility. Most evidence points towards genetic factors, although the genes determining susceptibility have not yet been determined.

Table 2 Some of the genes associated with COPD susceptibility Candidate genes

Risk

a1-Antitrypsin

ZZ genotype high risk MZ, SZ genotypes small risk Associated in some populations Associated in some studies

a1-Chymptrypsin Matrix metalloproteinase-1, -2, -9, -12 Microsomal epoxide hydrolase Glutathione S-transferase Heme oxygenase-1 Vitamin D binding protein TNF-a promoter

Increased risk Increased risk Small risk Inconsistent Inconsistent

Interleukin-13

Small risk

being used to study cigarette smokers who develop COPD compared with matched smokers who do not. This may identify markers of risk, but may also reveal novel molecular targets for the development of treatments of the future.

Pathology The term COPD includes chronic obstructive bronchiolitis with fibrosis and obstruction of small airways and emphysema with enlargement of airspaces and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways. Chronic bronchitis, by contrast, is defined by a productive cough of more than 3 months duration for more than 2

successive years; this reflects hypersecretion of mucus and is not necessarily associated with airflow limitation. Most patients with COPD have all three pathological mechanisms (chronic obstructive bronchitis, emphysema, and mucus plugging) as all are induced by smoking, but may differ in the proportion of emphysema and obstructive bronchitis (Figure 2). There has been debate about the predominant mechanism of progressive airflow limitation and recent pathological studies suggest that it is closely related to the degree of inflammation, narrowing, and fibrosis in small airways. Emphysema may contribute to the airway narrowing in the more advanced stages of the disease. There is inflammation in small airways with an increase in macrophages and neutrophils in early stages of the disease indicating innate immune response, but in more advanced stages of the disease there is an increase in lymphocytes (particularly cytotoxic CD8 þ T cells) including lymphoid follicles, indicating acquired immunity. Emphysema describes loss of alveolar walls due to destruction of matrix proteins (predominantly elastin) and loss of type 1 pneumocytes as a result of apoptosis. Several patterns of emphysema are recognized: centriacinar emphysema radiates from the terminal bronchiole, panacinar emphysema involves more widespread destruction, and bullae are large airspaces. Emphysema results in airway obstruction by loss of elastic recoil so that intrapulmonary airways close more readily during expiration. Chronic hypoxia may lead to hypoxic vasoconstriction, with structural changes in pulmonary

432 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview COPD

Normal

Disrupted alveolar attachments (emphysema) Mucosal inflammation, fibrosis

Emphysema + small airway obstruction

↑

Lung hyperinflation trapped gas Dyspnea

Mucus hypersecretion Airway held open by alveolar attachments

Airway obstructed by

Mucus hypersecretion

Cough and sputum

↓ Exercise tolerance

• Loss of attachments • Mucosal inflammation + fibrosis

Deconditioning

• Obstruction of lumen by mucus

Figure 2 Mechanisms of airflow limitation in COPD. The airway in normal subjects is distended by alveolar attachments during expiration, allowing alveolar emptying and lung deflation. In COPD, these attachments are disrupted because of emphysema thus contributing to airway closure during expiration, trapping gas in the alveoli, and resulting in hyperinflation. Peripheral airway are also obstructed and distorted by airway inflammation and fibrosis (chronic obstructive bronchiolitis) and by occlusion of the airway lumen by mucus secretions which may be trapped in the airway because of poor mucociliary clearance.

Chronic hypoxia

Pulmonary vasoconstriction Muscularization Intimal hyperplasia

Pulmonary hypertension

Fibrosis Obliteration

Cor pulmonale Edema Death

Figure 3 Vascular changes in COPD. Chronic hypoxia results in hypoxic pulmonary vasoconstriction and leads to structural changes, resulting in secondary pulmonary hypertension. Over time, this may lead to right heart failure (cor pulmonale), which has a poor prognosis, most patients only surviving for 6–12 months.

vessels that eventually lead to secondary pulmonary hypertension (Figure 3). Inflammatory changes similar to those seen in small airways are also seen in pulmonary arterioles.

Clinical Features The clinical features of COPD are usually straightforward and care should be taken to evaluate those features of the illness that are not typical. Symptoms

The symptoms of COPD are slowly progressive over many years, in contrast to the episodic and variable

Poor health status Figure 4 Symptoms of COPD. The most prominent symptom of COPD is dyspnea, which is largely due to hyperinflation of the lungs as a result of small airways’ collapse due to emphysema and narrowing due to fibrosis, so that the alveoli are not able to empty. Hyperinflation induces an uncomfortable sensation and reduces exercise tolerance. This leads to immobility and deconditioning and results in poor health status. Other common symptoms of COPD are cough and sputum production as a result of mucus hypersecretion, but not all patients have these symptoms and many smokers with these symptoms do not have airflow obstruction (simple chronic bronchitis).

symptoms of asthma. Patients have usually lost a considerable amount of their lung volume by the time they present to a doctor, with FEV1 values of o50%. There is usually a history of heavy smoking for many years, often 425 pack years (1 pack year ¼ 20 cigarettes daily for 1 year). Progressive shortness of breath on exertion is the predominant symptom and compensatory behavior of the patient may delay diagnosis. Dyspnea results from the hyperinflation secondary to small airway narrowing resulting in poor quality of life which is further exacerbated by the deconditioning due to reduced physical activity (Figure 4). Cough and sputum production are common symptoms but are also found in cigarette smokers without airflow limitation (chronic bronchitis). A change in the character of the cough may indicate lung carcinoma. Wheezing may occur during exacerbations and periods of breathlessness. Ankle swelling may occur when there is cor pulmonale. Weight loss often occurs in advanced disease, but the mechanism is uncertain. Loss of skeletal muscle bulk may be a response to systemic inflammation. Signs

When FEV1450% predicted, there may be no abnormal signs. The typical patient with more severe COPD shows a large, barrel-shaped chest due to hyperinflation, diminished breath sounds, and

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 433

distant heart sounds due to emphysema, and prolonged expiration with generalized wheezing on expiration. Diagnosis

Diagnosis is commonly made from the history of progressive dyspnea in a chronic smoker and is confirmed by spirometry, which shows an FEV1/vital capacity (VC) ratio of o70% and FEV1 o80% predicted. Staging of severity is made on the basis of FEV1 (Table 3), but exercise capacity and the presence of systemic features may be more important determinants of clinical outcome. Measurement of lung volumes by body plethysmography shows an increase in total lung capacity, residual volume, and functional residual capacity, with consequent reduction in inspiratory capacity, representing hyperinflation as a result of small airway closure (Table 4). This results in dyspnea which may be measured by Table 3 Staging of COPD by severity of airflow limitation Stage 0 (at risk) Stage I (mild)

Stage II (moderate)

Stage III (severe)

Stage IV (very severe)

Chronic symptoms (cough, sputum) Normal spirometry 7Chronic symptoms (cough, sputum) FEV1/FVC o70%, FEV1 480% predicted Chronic symptoms (cough, sputum, dyspnea) FEV1/FVC o70%, FEV1 o80–50% predicted Chronic symptoms (cough, sputum, dyspnea) FEV1/FVC o70%, FEV1 450–30% predicted Chronic symptoms (cough, sputum, dyspnea) FEV1 o30% predicted or respiratory insufficiency/right heart failure

dyspnea scales and reduced exercise tolerance, which may be measured by a 6 min or shuttle walking test. Carbon monoxide diffusion is reduced in proportion to the extent of emphysema. A chest X-ray is rarely useful but may show hyperinflation of the lungs and the presence of bullae. High-resolution computerized tomography demonstrates emphysema but is not used as a routing diagnostic test. Blood tests are rarely useful; a normocytic normochromic anemia is more commonly seen in patients with severe disease than polycythemia due to chronic hypoxia. Arterial blood gases demonstrate hypoxia and in some patients hypercapnia. Natural History

COPD is slowly progressive with an accelerated decline in FEV1, leading to slowly increasing symptoms, decreasing lung function, and eventually respiratory failure (Figure 1). The only strategy to reduce disease progression is smoking cessation, although this is relatively ineffective once FEV1 has fallen below 50% predicted. Patients with more severe COPD develop acute exacerbations, which have a prolonged effect on quality of life for many months. Most of the medical costs associated with COPD are linked to acute exacerbations that lead to hospital admission. There is still debate about the role of acute exacerbations in disease progression, but the decline in lung function may be accelerated further following an acute exacerbation.

Pathogenesis Recently, there have been important new insights into the pathogenesis of COPD. Chronic Inflammatory Mechanisms

Table 4 Lung function test abnormalities in COPD Lung function test

Result

Forced expiratory volume in 1 s (FEV1, liters) Forced vital capacity (FVC, liters) FEV1/FVC (%) Peak expiratory flow (PEF, liters/min) Total lung capacity (TLC, liters) Inspiratory capacity (IC, liters) Functional residual capacity (FRC, liters) Residual volume (RV, liters) Specific airway conductance (sGaw, cmH2O
1 s
1) Transfer factor for carbon monoxide (TLCO, ml/min/ mmHg) Transfer coefficient corrected for alveolar volume (KCO (TLCO/VA), ml/min/mmHg/l)

k k k k m k m m k k k

There is a specific pattern of chronic inflammation, particularly affecting small airways and lung parenchyma. This leads to fibrosis and narrowing of small airways and to destruction of the lung parenchyma, resulting in abnormalities in gas exchange and collapse of small airways on expiration. The inflammation of COPD is characterized by increased numbers of neutrophils, macrophages, and T lymphocytes (particularly CD8 þ cytotoxic T cells) and the degree of inflammation increases with disease severity (Figure 5). The pattern of inflammation is markedly different from that of asthma (Table 5). Many inflammatory mediators are now implicated in COPD, including lipid mediators (such as leukotriene B4), chemokines (such as interleukin (IL)-8), and proinflammatory cytokines (such as tumor necrosis factor

434 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview

Cigarette smoke (and other irritants) Alveolar macrophage

Epithelial cells TGF-
CTG

Fibroblast

Chemotactic factors IL-8, CXC chemokines LTB4

CD 8+ lymphocyte

Neutrophil

Proteases

Emphysema

Fibrosis

Mucus hypersecretion

Figure 5 Inflammatory mechanisms in COPD. Cigarette smoke (and other irritants) activate macrophages in the respiratory tract that release neutrophil chemotactic factors, including interleukin-8 (IL-8) and leukotriene B4 (LTB4). These cells then release proteases that break down connective tissue in the lung parenchyma, resulting in emphysema, and also stimulate mucus hypersecretion. These enzymes are normally counteracted by protease inhibitors, including a1-antitrypsin, secretory leukoprotease inhibitor (SLPI), and tissue inhibitor of matrix metalloproteinases (TIMP). Cytotoxic T cells (CD8 þ ) may also be recruited and involved in alveolar wall destruction.

Table 5 Differences between inflammation in COPD and asthma Inflammation

COPD

Asthma

Inflammatory cells

Neutrophils CD8 þ T cells þþþ CD4 þ cells þ Macrophages þþþ LTB4 TNF-a IL-8, GRO-a Oxidative stress þþþ Epithelial metaplasia Fibrosis þþ Mucus secretion þþþ AHR 7 Peripheral airways Predominantly parenchymal destruction 7

Eosinophils Mast cells CD4 þ T cells Macrophages þ LTD4, histamine IL-4, IL-5, IL-13 Eotaxin Oxidative stress þ Epithelial shedding Fibrosis þ Mucus secretion þ AHR þþþþ All airways No parenchymal effects

Inflammatory mediators

Inflammatory effects

Location

Response to corticosteroids

þþþ

LT, leukotriene; TNF, tumor necrosis factor; IL, interleukin; GRO, growth-related oncogene; AHR, airway hyperresponsiveness.

alpha (TNF-a) and IL-6. Oxidative stress is markedly increased in COPD and increases with disease severity; it is due to both the effects of cigarette smoke and the release of reactive oxygen species from activated inflammatory cells. There is also an increase in proteases, particularly enzymes that degrade elastin, such as neutrophil elastase (derived predominantly from neutrophils) and matrix metalloproteinase-9 (MMP-9, derived predominantly from macrophages). MMP-9 may be the predominantly elastolytic enzyme causing emphysema; it also activates transforming growth factor beta (TGF-b), a cytokine that

is expressed particularly in small airways that may result in the characteristic peribronchiolar fibrosis (Figure 6). MMP-12 may also contribute to elastolysis and is prominent in smoke exposure models of COPD in mice, where it plays a critical role in activating TNF-a. Amplifying Mechanisms

In normal cigarette smokers, there is a similar inflammatory response to that seen in COPD patients, but COPD represents an amplification of the normal

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 435

Chemotactic peptides

Macrophage

Neutrophils Neutrophil elastase

↓1-AT Pro-MMP-9

↑MMP-9 Elastolysis

Latent TGF-


Emphysema

Active TGF-


Small airway fibrosis (chronic obstructive bronchiolitis) Figure 6 Possible interrelationship between small airway fibrosis and emphysema in COPD. Transforming growth factor (TGF)-b is activated by matrix metalloproteinase-9 (MMP-9) and is in turn activated by MMP-9.

inflammatory response of the respiratory tract to inhaled noxious agents. Acute exacerbations represent a further amplification of the inflammatory response. The molecular and genetic mechanisms of this amplification remain to be determined. However, recently it has been shown that histone deacetylase activity (particularly HDAC2) is markedly reduced in the lungs, airways, and macrophages of COPD patients and that this is correlated with increased expression of inflammatory genes, such as IL-8. This amplifying mechanism may be induced by oxidative stress, which impairs the function of HDAC2 in particular. This also accounts for the steroid resistance of COPD since HDAC2 is required for switching off of activated inflammatory genes. Adenovirus infection also amplifies inflammation in cells in vitro and in experimental animals in vivo and its effects may also be mediated through a reduction in HDAC activity in lungs. In COPD patients, there is evidence for latent adenovirus infection in lungs of many COPD patients. Systemic Effects

There is increasing evidence for systemic (nonpulmonary) effects in patients with COPD, particularly in patients with severe disease and these might have an important negative impact on the quality of life (Table 6). The commonest systemic effect is loss of lean body mass and atrophy of skeletal muscles. This may reflect poor mobility, but may also be due to systemic effects of inflammatory mediators such as TNF-a and IL-6, which may induce apoptosis of skeletal muscles. Other systemic effects include osteoporosis, depression, and normocytic normochromic anemia. Circulating levels of acute phase proteins, such as

Table 6 Systemic abnormalities in COPD Systemic feature

Possible mechanisms

Cachexia Muscle wasting

TNF-a, IL-6, leptin Apoptosis of skeletal muscle due to TNF-a? Inactivity Chronic hypoxia, erythropoetin TNF-a? TNF-a, IL-6 CRP, fibrinogen?

Polycythemia Normocytic anemia Depression Cardiovascular abnormalities Osteoporosis

Inactivity, corticosteroids, cytokines?

TNF, tumor necrosis factor; IL, interleukin; CRP, C-reactive protein.

C-reactive protein and IL-6, are increased even in the stable state, but are further increased during exacerbations and this may be a factor predisposing to the markedly increased incidence of ischemic heart disease.

Management COPD is managed according to the severity of the disease, with a progressive escalation of therapy as the disease progresses (Figure 7). Antismoking Measures

Smoking cessation is the only measure so far shown to slow the progression of COPD, but in advanced disease, stopping smoking has little effect and the chronic inflammation persists. Nicotine replacement therapy (gum, transdermal patch, inhaler) helps in quitting smoking, but bupropion, a noradrenergic antidepressant, is more effective. More effective

436 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview

0: at risk FEV1 ≥80%

I: mild FEV1 ≥80%

II: moderate FEV1 79–50%

III: severe FEV1 49−30%

IV: very severe FEV1 <30%

Avoidance of risk factor(s); vaccination Add short-acting bronchodilator when needed Add regular treatment with one or more long-acting bronchodilators Add rehabilitation Add inhaled corticosteroids if repeated exacerbations Add long-term O2 if chronic respiratory failure Consider surgery Figure 7 Stepwise approach to COPD therapy. Management of COPD involves increasing the intensity of treatment depending on increasing severity of the disease. Long-acting bronchodilators are indicated in patients with moderate disease who are symptomatic, together with pulmonary rehabilitation. In severe disease an inhaled steroid may be added if there are frequent exacerbations and this is conveniently given as a combination inhaler containing a long-acting b2-agonist and corticosteroid. Long-term oxygen therapy and surgery are considerations in patients with very severe disease.

antismoking therapies, including partial nicotinic agonists and cannabinoid receptor antagonists, are currently being evaluated.

Establish diagnosis Assess symptoms

Stop smoking Healthy lifestyle Immunization

Bronchodilators

Bronchodilators are the mainstays of current drug therapy for COPD (Figure 8). The bronchodilator response measured by an increase in FEV1 is limited in COPD, but bronchodilators may improve symptoms by reducing hyperinflation and therefore dyspnea, and may improve exercise tolerance, despite the fact that there is little improvement in spirometric measurements. Previously short-acting bronchodilators, including b2-agonists and anticholinergics, were most widely used, but more recently, long-acting bronchodilators have been introduced. These include the long-acting inhaled b2-agonists salmeterol and formoterol and the once-daily inhaled anticholinergic tiotropium bromide. In patients with more severe disease, these therapies appear to be additive. Theophylline is also used as an add-on bronchodilator in patients with very severe disease, but systemic side effects may limit its value. Antibiotics

Acute exacerbations of COPD are commonly assumed to be due to bacterial infections, since they may be associated with increased volume and purulence of the sputum. However, it is increasingly recognized that exacerbations may be due to upper respiratory tract viral infections or may be noninfective, questioning the place of antibiotic treatment in many patients. Controlled trials of antibiotics in

Bronchodilators

Treat obstruction

Assess for hypoxia

Long-term oxygen therapy

Pulmonary rehabilitation program Figure 8 Treatment strategy in COPD. All patients should be encouraged to stop smoking and should also receive influenza and pneumococcal vaccination. The mainstays of treatment are bronchodilators to reduce symptoms. Bronchodilators may improve symptoms by deflating the lungs, even without any significant change in spirometry. In more severe patients, pulmonary rehabilitation may be very helpful in improving health status and reducing hospitalization. For patients with respiratory failure due to right heart failure, long-term oxygen therapy is indicated.

COPD show a relatively minor benefit of antibiotics in terms of clinical outcomes and lung function. Although antibiotics are still widely used in exacerbations of COPD, methods that can reliably diagnose bacterial infection in the respiratory tract are needed so that antibiotics are not used inappropriately. There is no evidence that prophylactic antibiotics prevent acute exacerbations. Oxygen

Home oxygen accounts for a large proportion (over 30% in the US) of healthcare spending on COPD.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 437

Long-term oxygen therapy was justified by two large trials showing reduced mortality and improvement in quality of life in patients with severe COPD and chronic hypoxemia (PaO2 o55 mmHg). More recent studies have demonstrated that patients with less severe hypoxemia do not appear to benefit in terms of increased survival, so that selection of patients is important in prescribing this expensive therapy. Similarly, nocturnal treatment with oxygen does not appear to be beneficial in terms of survival or delaying the prescription of long-term oxygen therapy in patients with COPD who have nocturnal hypoxemia. Corticosteroids

Inhaled corticosteroids are now the mainstay of chronic asthma therapy and the recognition that chronic inflammation is also present in COPD provided a rationale for their use. Indeed, inhaled corticosteroids are now widely prescribed in the treatment of COPD. However, the inflammation in COPD shows little or no suppression even by high doses of inhaled or oral corticosteroids. This may reflect the fact that neutrophilic inflammation is not suppressible by corticosteroids as neutrophil survival is prolonged by steroids. There is also evidence for an active cellular resistance to corticosteroids, with no evidence that even high doses of corticosteroids suppress the synthesis of inflammatory mediators or enzymes. This is related to a decreased activity and expression of histone deacetylase 2, which is required for corticosteroids to switch off activated inflammatory genes, such as TNF-a, IL-8, and MMP-9. Approximately 10% of patients with stable COPD show some symptomatic and objective improvement with oral corticosteroids and it is likely that these patients have concomitant asthma, as both diseases are very common. Furthermore, these patients have elevated sputum eosinophils and exhaled nitric oxide, which are features of asthmatic inflammation. Long-term treatment with high doses of inhaled corticosteroids fails to reduce disease progression, even at the early stages of the disease. However, there is a small protective effect against acute exacerbations (approximately 20% reduction) in patients with severe disease. In view of the risk of systemic side effects in this susceptible population, inhaled corticosteroids are only recommended in patients with FEV1o50% predicted who have two or more severe exacerbations a year. There is a small beneficial effect of systemic corticosteroids in treating acute exacerbations of COPD, with improved clinical outcome and reduced length of hospital admission. The reasons for this discrepancy between steroid responses in acute versus

chronic COPD may relate to differences in the inflammatory response (increased eosinophils) or airway edema in exacerbations. Other Drug Therapies

Systematic reviews show that mucolytic therapies reduce exacerbation by about 20%, but most of the benefit appears to derive from N-acetyl cysteine, which is also an antioxidant. This treatment should be considered in patients who have frequent exacerbations. There is no evidence that cysteinyl leukotriene antagonists, such as montelukast, are beneficial in COPD, although they are commonly used. Opiates, such as codeine, may reduce dyspnea in COPD patients but the minimal benefit is outweighed by side effects. Noninvasive Ventilation

Noninvasive positive pressure ventilation, using a simple nasal mask and avoiding the need for endotracheal intubation, reduces the need for mechanical ventilation in acute exacerbations of COPD in hospital, although some studies have shown less benefit. Domiciliary noninvasive positive pressure ventilation may improve oxygenation and reduce hospital admissions in patients with severe COPD with hypercapnia and may improve long-term survival, although large controlled trials are needed. Pulmonary Rehabilitation

Pulmonary rehabilitation consists of a structured program of education, exercises, and physiotherapy and has been shown in controlled trials to improve the exercise capacity and quality of life of patients with severe COPD, with a reduction in healthcare utilization. Pulmonary rehabilitation is now an important part of the management plan in patients with severe COPD. There is debate about the duration and frequency of pulmonary rehabilitation. Most of the benefits appear to relate to exercise so that modified simplified programs are now often used. Lung Volume Reduction

Surgical removal of emphysematous lung improves ventilatory function in carefully selected patients. The reduction in hyperinflation improves the mechanical efficiency of the inspiratory muscles. Careful patient selection after a period of pulmonary rehabilitation is essential. Patients with localized upper lobe emphysema with poor exercise capacity do best, but there is a relatively high operative mortality, particularly with patients who have a low diffusing capacity. Significant functional improvements include increased FEV1, reduced total lung capacity

438 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview

and functional residual capacity, improved function of respiratory muscles, improved exercise capacity, and improved quality of life. Benefits persist for at least a year in most patients, but careful long-term follow-up is needed in order to evaluate the longterm benefits of this therapy. More recently, nonsurgical bronchoscopic lung volume reduction has been achieved by insertion of one-way valves by fiber optic bronchoscopy. This gives significant improvement in some patients and appears to be safe, but collateral ventilation reduces the efficacy of this treatment so that significant deflation of affected lung may not be achieved. Management of Acute Exacerbations

Acute exacerbations of COPD should be managed by supplementary oxygen therapy, initially 24% oxygen, and checking that there is no depression of ventilation. Antibiotics should be given if the sputum is purulent or there are other signs of bacterial infection. High doses of corticosteroids reduce hospital stay and are routinely given. Chest physiotherapy is usually given, but there is little objective evidence for benefit. Noninvasive ventilation is indicated for incipient respiratory failure and reduces the need for intubation. New Treatments

Apart from quitting smoking, no other treatments, including corticosteroids, slow the progression of COPD. Yet this disease is associated with an active inflammatory process and progressive proteolytic injury of lung tissues even in its most advanced stages, suggesting that pharmacological intervention may be possible. A better understanding of the cellular and molecular mechanisms involved in COPD provides new molecular targets for the development of new drugs and several classes of new drugs are now in development (Table 7). Several inflammatory mediators are implicated in COPD, providing logical targets for the development of synthesis inhibitors or receptor antagonists. These include 50 -lipoxygenase inhibitors that prevent the synthesis of leukotriene B4 and specific leukotriene B4 antagonists, several of which are now being evaluated in COPD. Specific antagonists of CXCR2, one of the receptors on neutrophils that is activated by IL-8, have been developed and humanized antibodies and soluble receptors that block TNF-a have already been developed for use in other chronic inflammatory diseases. More potent and stable antioxidants are also in development. However, it is not certain that antagonizing a single mediator will have a major

Table 7 New treatments for COPD Mediator antagonists Leukotriene B4 antagonists (e.g., LY 29311, SB 201 146, BIIL284) 50 -lipoxygenase inhibitors (e.g., Bay x1005) Interleukin-8 antagonists (SB 225 002: CXCR2 antagonists) TNF inhibitors (monoclonal antibodies, soluble receptors, TNF convertase inhibitors) Antioxidants (stable glutathione analogs, superoxide dismutase analogs) Protease inhibitors Neutrophil elastase inhibitors (e.g., ONO-5046) Cathepsin inhibitors Matrix metalloproteinase inhibitors (marimastat, selective MMP inhibitors) a1-Antitrypsin (purified, human recombinant, gene transfer) Secretory leukoprotease inhibitor (human recombinant, gene transfer) Elafin (human recombinant, gene transfer) New anti-inflammatory drugs Phosphodiesterase-4 inhibitors (e.g., cilomilast, roflumilast) p38 mitogen-activated protein-kinase inhibitors (e.g., SB 220 025) Nuclear factor kappa B inhibitors (IkB kinase-2 inhibitors) Adhesion molecule inhibitors (e.g., E-selectin inhibitors, glycoprotein selectin inhibitors)

clinical effect, since many mediators with overlapping actions are involved in COPD. Several inhibitors of neutrophil elastase are now in clinical development. To date, only one has been reported in a clinical study and this showed no benefit, but this may reflect the fact that several proteases are involved or the fact that it may be difficult to monitor efficacy in such a slowly progressive disease. Several nonselective matrix metalloproteinase inhibitors have been developed and there is now a search for more selective inhibitors in order to avoid the musculoskeletal side effects that have hampered the development of this class of drug. Another approach is supplementation of endogenous antiproteinases using human recombinant a1-antitrypsin, secretory leukoprotease inhibitor or elafin, or even gene therapy, although there are doubts that sufficient protein can be delivered by either approach. While corticosteroids are ineffective, other anti-inflammatory treatments, particularly those that inhibit neutrophilic inflammation, might be effective. The most promising amongst these are phosphodiesterase (PDE)4 inhibitors, which have an inhibitory effect on key inflammatory cells involved in COPD, including macrophages, neutrophils, and cytotoxic T lymphocytes. Several PDE4 inhibitors are in development for treatment of COPD, although a limitation of drugs in this class is the common side effect of nausea and headaches, also caused by PDE4 inhibition. Other novel anti-inflammatory approaches in development include inhibitors of nuclear factor kappa

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B (NF-kB) and inhibitors of p38 mitogen-activated protein-kinase, although none of these have reached clinical trials. Drug Delivery

Current inhaler devices have been developed to deliver drugs to the conducting airways in patients with asthma. However, COPD predominantly affects peripheral airways and lung parenchyma, which may not be optimally targeted by current inhalers. It is likely that new inhaler devices delivering aerosols of smaller particle size or systemic drugs are more likely to be useful in COPD. In the future, targeted delivery to specific cells, such as macrophages, might be possible, particularly if new treatments have systemic toxicity. There is also a rationale for giving systemic therapies to reach the lung periphery and to treat systemic features of the disease. See also: Bronchodilators: Anticholinergic Agents; Beta Agonists. Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General. Corticosteroids: Therapy. Leukocytes: Pulmonary Macrophages. Oxygen Therapy.

Further Reading Agusti AG, Noguera A, Sauleda J, et al. (2003) Systemic effects of chronic obstructive pulmonary disease. European Respiratory Journal 21: 347–360. Barnes PJ (2000) Chronic obstructive pulmonary disease. New England Journal of Medicine 343: 269–280. Barnes PJ (2003) New concepts in COPD. Annual Review of Medicine 54: 113–129. Barnes PJ (2004) Mediators of chronic obstructive pulmonary disease. Pharmacological Reviews 56: 515–548. Barnes PJ and Hansel TT (2004) Prospects for new drugs for chronic obstructive pulmonary disease. Lancet 364: 985–996. Barnes PJ, Ito K, and Adcock IM (2004) A mechanism of corticosteroid resistance in COPD: inactivation of histone deacetylase. Lancet 363: 731–733. Barnes PJ, Shapiro SD, and Pauwels RA (2003) Chronic obstructive pulmonary disease: molecular and cellular mechanisms. European Respiratory Journal 22: 672–688. Calverley PM and Walker P (2003) Chronic obstructive pulmonary disease. Lancet 362: 1053–1061. Hogg JC (2004) Pathophysiology of airflow limitation in chronic obstructive pulmonary disease. Lancet 364: 709–721. Hogg JC, Chu F, Utokaparch S, et al. (2004) The nature of smallairway obstruction in chronic obstructive pulmonary disease. New England Journal of Medicine 350: 2645–2653. Hurst JR and Wedzicha JA (2004) Chronic obstructive pulmonary disease: the clinical management of an acute exacerbation. Postgraduate Medical Journal 80: 497–505. Mannino DM (2002) COPD: epidemiology, prevalence, morbidity and mortality, and disease heterogeneity. Chest 121: 121S–126S. Pauwels RA and Rabe KF (2004) Burden and clinical features of chronic obstructive pulmonary disease (COPD). Lancet 364: 613–620.

Rennard SI (2004) Treatment of stable chronic obstructive pulmonary disease. Lancet 364: 791–802. Sutherland ER and Cherniack RM (2004) Management of chronic obstructive pulmonary disease. New England Journal of Medicine 350: 2689–2697. Wouters EF (2004) Management of severe COPD. Lancet 364: 883–895.

Acute Exacerbations J A Wedzicha and J R Hurst, Royal Free and University College Medical School, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Chronic obstructive pulmonary disease (COPD) is a condition characterized by lower respiratory tract symptoms, airflow obstruction, and airway inflammation. The natural history of COPD is punctuated by episodes of acute deterioration in respiratory health termed exacerbations. Exacerbations are responsible for much of the morbidity, mortality, and healthcare costs associated with COPD, and also affect the rate of decline in lung function. Most exacerbations are caused by episodes of tracheobronchial infection. The clinical features comprise a worsening in symptoms beyond usual day-to-day variation. Examination may reveal tachypnea and signs of respiratory failure. Pathologically, exacerbations are associated with an increase in airway and systemic inflammation which, at least in more severe disease, is predominantly neutrophilic. Simple investigations are indicated to exclude differential diagnoses and assess exacerbation severity. Treatment aims to support respiratory function whilst drugs aimed at the underlying cause are able to act. The cornerstone of therapy is inhaled bronchodilators, with oral corticosteroids in all but the mildest exacerbations. Antibiotics are indicated if there has been a change in the character of the sputum. Some patients with respiratory failure will require supplemental oxygen or assisted ventilation. Strategies to prevent exacerbations include inhaled corticosteroids, long acting bronchodilators, and influenza vaccination.

Introduction It would be difficult to exaggerate the importance of chronic obstructive pulmonary disease (COPD) as a global health problem for the twenty-first century. COPD is the fifth commonest cause of death in the world and, unlike other prevalent diseases, death rates from COPD continue to rise. Much of this mortality is related to episodes of acute deterioration in respiratory health termed exacerbations. COPD is defined by the presence of lower respiratory tract symptoms, poorly reversible airflow obstruction, and inflammation of the lung. The natural history of COPD comprises progression of this airflow obstruction, associated with worsening symptoms and increasing limitation to daily activities. However, patients are also prone to periodic

440 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Acute Exacerbations

deteriorations in respiratory health termed exacerbations. Exacerbations are important events that cause much of the morbidity, mortality, hospital admissions, and therefore healthcare costs associated with this disease. In the UK, around 10% of acute medical admissions to hospital may be attributed to exacerbations of COPD, the in-hospital mortality is 10%, and exacerbations account for 70% of the direct medical costs attributable to COPD. Exacerbations become more frequent and more severe as the severity of the underlying disease increases. In patients with moderately severe COPD, the median annual exacerbation frequency is between two and three events per year. However, this masks important differences between individual patients, some of whom are more susceptible to exacerbation than others. The factors determining a greater susceptibility to exacerbation are poorly understood but are thought to include the presence of daily bronchitic symptoms, greater severity of underlying disease, greater lower airway inflammation, and the presence of lower airway bacteria in the stable state. Exacerbations are extremely heterogeneous events responsible, at one extreme, for life-threatening episodes of respiratory failure in patients with severe COPD, but to no more than a troublesome and transient increase in symptoms in patients with milder disease. This heterogeneity has caused debate about exactly how an exacerbation should be defined, but for practical purposes an exacerbation is a sustained episode of increased symptoms, acute in onset, that is beyond the patient’s usual day-to-day variation. This may or may not necessitate a change in therapy. In addition to direct effects on morbidity and mortality, exacerbation frequency also affects the natural history of disease. Patients susceptible to frequent exacerbations experience an accelerated decline in forced expiratory volume in 1 s (FEV1), and have a poorer quality of life. The importance of exacerbations are summarized in Figure 1.

Etiology It is generally accepted that exacerbations are caused by episodes of tracheobronchial infection, and perhaps pollution. Evidence for the former is derived from reports demonstrating a higher prevalence of pathogens in samples taken from the lower airways at the time of exacerbation than in the stable state. Evidence that pollution causes exacerbations arises from relationships between the degree of air pollution and hospital admissions for COPD. PM10

Morbidity

Healthcare costs

Exacerbation of COPD

Mortality

Disease progression

Figure 1 The importance of exacerbations in COPD. Table 1 The most important bacterial and viral pathogens isolated from lower airway samples in patients with exacerbations of COPD Bacteria

Viruses

Haemophilus influenzae Moraxella catarrhalis Streptococcus pneumoniae Pseudomonas aeruginosa

Rhinovirus Coronavirus Influenza Parainfluenza Adenovirus Respiratory syncytial virus

Bold type indicates the most common isolates.

(particulate matter up to 10 mm in size), largely produced by diesel exhaust, appear particularly important. In a proportion of exacerbations, the cause remains obscure. A number of studies have investigated the isolation frequency of individual pathogens at exacerbation of COPD. The relative prevalence of specific agents varies by the methodology of the study, and the severity of both the patient and exacerbation. In patients with moderate to severe underlying disease, bacterial and viral pathogens may each be isolated from lower airway samples in approximately 50% of exacerbations. The most important bacterial and viral pathogens are listed in Table 1. The identification of organisms in lower airway samples at the time of exacerbation has been taken to imply causation, but the issue is complex because pathogenic microorganisms may also be found in the lower airways of patients with stable COPD. This is especially true for bacteria where up to 50% of patients with severe disease have the same bacterial species present in the stable state, a phenomenon

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Acute Exacerbations 441

termed lower airway bacterial colonization. More recently, it has been suggested that a change in the colonizing strain may result in exacerbation, and further evidence that bacteria do indeed cause exacerbations may be drawn from the clinical benefit observed in trials of antibiotics during such events. There is ongoing controversy regarding the role of atypical organisms such as Chlamydia and Mycoplasma in the etiology of exacerbations. The most important viral pathogen is rhinovirus. Exacerbations in which a virus is isolated, or those associated with coryzal symptoms, are more severe as assessed by changes in symptoms and lung function. Reflecting the circulation of respiratory viruses, exacerbations are more common in the winter than the summer months.

Pathology COPD is defined as a disease state characterized by lower respiratory tract symptoms and airflow obstruction in association with an abnormal inflammatory response within the lung. A number of studies using endobronchial biopsy, bronchoalveolar lavage, sputum, and exhaled markers have demonstrated that airway inflammation is increased at exacerbation compared to the baseline state. In studies examining endobronchial biopsies, exacerbations in mild COPD were associated with an infiltration of eosinophils, whilst in more severe disease the infiltrate was characterized by neutrophils. Regarding soluble mediators, increased concentrations of various neutrophilic indices have been reported including interleukin-8, leukotriene-B4, myeloperoxidase, and elastase.

Clinical Features Exacerbations are defined as an increase in respiratory symptoms, acute in onset, that is beyond the patient’s usual day-to-day variation. These symptoms typically include increased dyspnea, sputum volume and sputum purulence, with or without increased cough, wheeze, chest tightness, coryzal symptoms, fatigue, edema, and confusion. Findings on examination are non-specific, but may include tachypnea and use of the accessory muscles of respiration, pursed-lip breathing, polyphonic expiratory wheeze, and, in severe exacerbations, signs of CO2 retention such as flap and bounding pulse.

Pathogenesis It is generally assumed (based on observations that pathogens are more frequently isolated at the time of

exacerbation and that airway inflammatory markers are increased at exacerbation) that exacerbations result from increased airway inflammation following acquisition of a new pathogen. However, a direct relationship between the clinical severity of exacerbation and degree of airway inflammation has never been demonstrated, and the mechanisms by which airway inflammation results in worsening symptoms and lung function remain largely obscure. It has also been reported that systemic inflammatory markers such as interleukin-6, fibrinogen, and C-reactive protein are higher at exacerbation than in stable COPD. This is important because of the association between systemic inflammatory markers and cardiovascular events, cardiovascular disease being a common cause of death in COPD. The origin of the systemic inflammatory response is unclear, though a ‘spillover’ effect from the lung is thought most likely.

Animal Models There is no animal model of exacerbation of COPD.

Management and Current Therapy A summary of the clinical management of an exacerbation of COPD is presented in Table 2. Initial management is directed at excluding differential diagnoses and assessing exacerbation severity. The diagnosis of exacerbation is symptomatic, there is no confirmatory test, and follows exclusion of differential diagnoses such as pneumonia, pneumothorax, pleural effusion, lung carcinoma, pulmonary embolus, or cardiac disease which may also occur in a patient with COPD and mimic the presence of exacerbation. Such diagnoses may generally be excluded on the basis of a clinical history, examination, and simple investigations such as the chest radiograph and electrocardiogram. Assessment of severity is important for guiding decisions on initial treatment strategy and the need for hospital admission. However, there is no widely accepted severity scoring system. pH, more useful than PaCO2 as a marker of acute changes in alveolar ventilation, and PaO2 are important indicators. A pH o7.30 suggests a severe exacerbation. It should be remarked that measuring lung function is usually unhelpful as changes at exacerbation may be small, absolute values may be misleading in the absence of a baseline, and the tests may be difficult to perform in patients who are acutely unwell. The principles of treatment are to support respiratory function whilst therapies directed against the underlying pathophysiology, such as antimicrobial and anti-inflammatory agents, are able to act.

442 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Acute Exacerbations Table 2 A summary of the management of an acute exacerbation of COPD in the hospital setting Principle

Details

Assess severity Give oxygen Give bronchodilators Give corticosteroids Give antibiotics Consider need for ventilatory support Also consider

Clinical findings and pH/arterial blood gas tensions Controlled therapy, aiming for arterial saturations 90–92%. Reassess blood gas tensions after 30 min Increased dose or frequency of b2-agonists and/or anticholinergics. Nebulised or inhaled via spacer Oral, or with initial intravenous dose When there has been a change in the character of sputum. Usually oral In respiratory failure not responding to controlled oxygen therapy Fluid balance, anticoagulation, comorbidities, and need for additional therapies, e.g., intravenous theophylline

The cornerstone of therapy, all that may be required in the most mild exacerbations, is an increase in the dose or frequency of inhaled bronchodilators. Although the airflow obstruction in COPD is, by definition, largely irreversible, there may be additional bronchoconstriction at exacerbation and these drugs can achieve a dramatic symptomatic response through effects on dynamic hyperinflation. Shortacting b2-adrenoceptor agonists such as salbutamol have a more rapid onset of action and are preferred to anticholinergics such as ipratropium. Combinations of b2-agonists and anticholinergics are often recommended in exacerbations not responding to b2-agonists alone. Inhaled therapy via a large volume spacer is as effective as wet nebulization. Exacerbations not responding to an increase in bronchodilators alone should be treated with systemic corticosteroids. The rationale is that these drugs may act on the increased inflammation observed at exacerbation, though specific evidence for this is lacking. Systemic corticosteroids result in a more rapid improvement in FEV1, though at the expense of more frequent adverse events, particularly hyperglycemia. A dose of 30–40 mg prednisolone equivalent for 10–14 days is appropriate. Longer courses have no additional effect and there is no benefit from dose tapering. Nebulized budesonide is an alternative. The evidence of benefit from antibiotics is restricted to those exacerbations characterized by a change in the character of sputum. Sputum purulence is a reliable predictor of the presence of bacteria. Parenteral antibiotics are rarely required and given that coverage of Haemophilus influenza, Moraxella catarrhalis, and Streptococcus pneumoniae is required, an oral aminopenicillin, macrolide, or tetracycline agent is most commonly used first line. Severe exacerbations, or exacerbations in patients with more severe underlying disease, may result in arterial hypoxemia, primarily through ventilation–perfusion imbalance. Oxygen should be administered to correct this respiratory failure. The risk of

hypercapnia in a proportion of patients with COPD necessitates that oxygen be administered in a controlled manner. There is little risk of hypercapnia if oxygen is titrated to achieve saturations between 90% and 92%. Achieving adequate oxygenation only at the expense of rising PaCO2 or decreasing pH suggests the need for noninasive ventilation (NIV). NIV is clearly superior to respiratory stimulant drugs such as doxapram in the management of acute hypercapnic respiratory failure, with improvements in mortality, length of hospital stay, and the need for tracheal intubation. NIV is most commonly administered as pressure-cycled bi-level positive airway pressure (BiPAP), and is thought to be effective by off-loading fatigued respiratory muscles. Patients failing to improve on NIV, or those with a particularly severe exacerbation, may require endotracheal intubation and a period of mechanical ventilation. The decision to institute such therapy in COPD is complex and should consider the wishes and prior functional status of the patient, comorbidities, and the degree of reversibility. However, mortality in the intensive-care unit for patients with COPD, around 20%, is not greater than that seen in respiratory failure from other causes. Regarding other therapies, there is no evidence that the routine use of theophyllines, heliox, antitussives, mucolytics, or respiratory physiotherapy is beneficial. Other supportive measures such as fluid balance should be attended to, and comorbidities managed. Patients failing maximal therapy or in whom escalation of therapy is inappropriate should be considered for a range of palliative drugs. Given the importance of exacerbations, considerable benefit may be anticipated from therapies which prevent these events. Large trials have demonstrated evidence for benefit of high-dose inhaled corticosteroids (fluticasone 1000 mg day 1) in patients with moderate to severe underlying disease (FEV1o50% predicted). Long-acting bronchodilators, both b2agonists and anticholinergics, have also been associated with decreased exacerbation frequency, as has

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, Alpha-1-Antitrypsin Deficiency 443

influenza vaccination. Initial trials of antibiotic prophylaxis were carried out many years ago, often in patients with simple chronic bronchitis, and current interest in this area, with more modern drugs, is high. Mucolytics may reduce exacerbation frequency in patients with milder disease. Finally, appropriate use of long-term oxygen therapy and domiciliary NIV in patients with respiratory failure is associated with reduced hospitalization, though a specific effect on exacerbations has not been demonstrated. See also: Bronchoalveolar Lavage. Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-6. Viruses of the Lung.

Further Reading Anthonisen NR, Manfreda J, Warren CP, et al. (1987) Antibiotic therapy in exacerbations of chronic obstructive pulmonary disease. Annals of Internal Medicine 106: 196–204. Burge PS, Calverley PM, Jones PW, et al. (2000) Randomised, double blind, placebo controlled study of fluticasone propionate in patients with moderate to severe chronic obstructive pulmonary disease: the ISOLDE trial. British Medical Journal 320: 1297–1303. Calverley PMA (2004) Reducing the frequency and severity of exacerbations of chronic obstructive pulmonary disease. Proceedings of the American Thoracic Society 1: 121–124. Hurst JR and Wedzicha JA (2004) Chronic obstructive pulmonary disease: the clinical management of an acute exacerbation. Postgraduate Medical Journal 80: 497–505. Lightowler JV, Wedzicha JA, Elliott MW, and Ram FSF (2003) Non-invasive positive pressure ventilation to treat respiratory failure resulting from exacerbations of chronic obstructive pulmonary disease: cochrane systematic review and meta-analysis. British Medical Journal 326: 185–187. National Institute for Clinical Excellence (NICE) (2004) Chronic obstructive pulmonary disease: national clinical guideline for management of chronic obstructive pulmonary disease in adults in primary and secondary care. Thorax 59(supplement I). Pauwels R, Anthonisen N, Bailey WC, et al. (2001) Global Initiative for Chronic Obstructive Lung Disease. National Heart, Lung and Blood Institute, National Institutes of Health, Bethesday, MD. Saint S, Bent S, Vittinghoff E, and Grady D (1995) Antibiotics in chronic obstructive pulmonary disease exacerbations. A metaanalysis. Journal of the American Medical Association 273: 957–960. Sethi S (2004) Bacteria in exacerbations of chronic obstructive pulmonary disease. Phenomenon or epiphenomenon? Proceedings of the American Thoracic Society 1: 109–114. Siafakas NM, Anthonisen NR, and Georgopoulos D (eds.) (2003) Lung Biology in Health and Disease Volume 183: Acute Exacerbations of Chronic Obstructive Pulmonary Disease. New York: Dekker. Wedzicha JA (2004) Role of viruses in exacerbation of chronic obstructive pulmonary disease. Proceedings of the American Thoracic Society 1: 115–120. Wedzicha JA and Donaldson GC (2003) Exacerbations of chronic obstructive pulmonary disease. Respiratory Care 48: 1204–1213. Wood-Baker RR, Gibson PG, Hannay M, Walters EH, and Walters JAE (2005) Systemic corticosteroids for acute exacerbations of

chronic obstructive pulmonary disease. Cochrane Database of Systemic Reviews, Issue 4.

Relevant Website http://www.cochrane.org – The Cochrane Database of Systematic Reviews.

Emphysema, Alpha-1Antitrypsin Deficiency R A Stockley, Queen Elizabeth Hospital, Birmingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Serum a1-antitrypsin (AAT) deficiency is the most common genetic condition recognized in a Caucasian population of Northern European origin. It predisposes to several clinical entities, although the susceptibility to the development of emphysema predominates. The classical clinical presentation (although not universal) is of early onset basal panacinar emphysema associated with neutrophilic inflammation in the lower airways. Proteinases released from these neutrophils remain active in the lung due to the deficiency of AAT and damage many of the structural proteins (particularly elastin), leading to alveolar destruction and coalescence. The progression is generally more rapid than in nondeficient subjects, leading to respiratory failure and cor pulmonale. Therapy is predominantly along conventional lines for chronic obstructive pulmonary disease, although AAT-deficient patients are more likely to be accepted as potential lung transplant recipients because of their younger age and general lack of comorbidity. Augmentation therapy is available in some countries, and circumstantial evidence suggests that it is effective in disease modification. However, no suitably powered controlled clinical trial has been undertaken to prove this hypothesis.

Serum deficiency of a1-antitrypsin (AAT) was first noted and described by Laurell and Eriksson in 1963. The authors identified five serum samples with an absence of a protein band in the a1 region on paper electrophoresis. Review of the patients indicated that three had emphysema at an early age (younger than 45 years old) and a fourth individual had a family history of emphysema. In subsequent studies, it became apparent that all individuals possessed two genes for AAT that were expressed in a codominant manner. However, some genes led to decreased serum levels, and whereas heterozygotes did not appear to be excessively at risk for developing emphysema, homozygotes did show such a risk. This led to the apparent autosomal recessive nature of inheritance of the disease trait (Figure 1).

444 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, Alpha-1-Antitrypsin Deficiency

MM

ZZ

1

2

MZ

MZ

3

MM

4

MZ

ZZ

MZ

5

MZ

MZ

MZ

MZ

ZZ

MM

ZZ

Figure 1 Idealized five-generation inheritance of the common Z gene. In generation 1, an affected homozygote individual marries a ‘normal’ homozygote M individual. All children would be MZ heterozygotes and not at risk. In generation 2, the heterozygote marries another heterozygote, resulting in a one in four chance of a homozygote Z (at-risk) individual. In generation 3, all children are heterozygote, and at-risk individuals only recur in generation 5, if marriage to a further heterozygote occurs, thereby ‘skipping’ a generation.

The patients identified initially were young, and subsequent studies suggested that the archetypal patient was an individual with early onset panacinar emphysema, predominantly basal in distribution. The highest incidence of the deficiency was in Scandinavia, and the condition was primarily of Northern European origin with decreasing prevalence further away from Scandinavia. The highest incidence of serum deficiency is approximately 1:1600 in Denmark, and the lowest is approximately 1:5000 in the United States.

Etiology Although smoking is recognized as the major etiological agent in the development of emphysema, it is recognized that not all smokers develop the condition. This suggests coenvironmental factors that amplify the detrimental effects of cigarette smoking and/ or genetic factors. Indeed, studies have indicated that emphysema runs in families, often irrespective of smoking history. Serum deficiency of AAT is the major recognized genetic risk factor for the development of emphysema. Epidemiological studies have indicated that life expectancy in such individuals is reduced, particularly if they smoke, and most patients with serum deficiency of AAT who present to healthcare systems have a current or past history of cigarette smoking. Despite this, it is well recognized that even

nonsmokers with AAT deficiency can develop airflow obstruction, although in such individuals the onset of physiological abnormality and symptoms is generally much later than in current or ex-smokers. There have been very few other studies of factors that influence the development of airflow obstruction, although a past history of wheezing and exposure to kerosene heaters have also been implicated.

Pathology There have been few pathological studies of the lung in patients with serum AAT. In the limited studies that have been carried out, the presence of panacinar emphysema has been well recognized. In addition, evidence of bronchitis and bronchiectasis has also been noted. Recent studies have shown that the lungs of affected individuals have evidence of neutrophilic inflammation and the presence of polymerized AAT. Patients with serum deficiency of AAT show accumulation of AAT in hepatocytes (Figure 2) and progressive evidence of fibrosis and cirrhosis. The condition often results in neonatal jaundice, which usually settles, although childhood liver failure and the development of hepatocellular carcinoma in adult patients with cirrhosis are also known to occur. Finally, other rare pathological conditions are known to occur in patients with serum deficiency of AAT, including necrotizing panniculitis and vasculitis.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, Alpha-1-Antitrypsin Deficiency 445

Figure 2 A histological section of liver is shown demonstrating the PAS-positive inclusion bodies typical of AATD (arrow).

Clinical Features The majority of patients with serum deficiency of AAT are identified through investigation of respiratory symptoms, although approximately 20% of patients are identified through family screening. There is often a delay in the diagnosis, which may be up to 7 years from the presentation of symptoms to establishment of the cause. The typical patient often presents in the third or fourth decade with symptoms of breathlessness on exertion, which is gradually progressive. Because of the young age of onset, an initial diagnosis of asthma is often made, although lung physiology usually indicates poor reversibility and effort-dependent airways collapse. Direct questioning also indicates that the patients often have other classic symptoms of chronic obstructive pulmonary disease (COPD), including cough, sputum production, wheeze (with and without infections), and fatigue. The condition, and hence symptomatology, is progressive, especially in subjects who continue to smoke, and it generally follows a more rapid path yet broadly similar to that of patients who have normal AAT and more standard COPD. As indicated previously, nonsmokers can develop airflow obstruction, although this occurs approximately 10–20 years later, affecting patients in their 50s and 60s. This archetypal presentation has been shown to have some relationship to selective testing. Because the original patients who were identified in 1963 were young with extensive panlobular basal emphysema (Figure 3), there has been a general tendency to test such patients for AAT deficiency and not test patients who are older with a more diffuse and centrilobular pattern of emphysema. Recent studies using high-resolution computed tomography (CT) scan have shown that approximately one-third of patients with serum deficiency of AAT who are symptomatic have a more centrilobular pattern of emphysema and

Figure 3 A three-dimensional CT scan of the lungs of a patient with AATD. Emphysematous areas are dark, demonstrating the predominantly basal distribution of the disease.

typical distribution toward the apices of the lungs rather than the classical basal panacinar emphysema. In addition, approximately 30% of patients demonstrate evidence of bronchiectasis on high-resolution CT, and patients can present much later in life even with relatively mild disease when they are smokers. The average age of patients presenting to the UK ADAPT registry is 50 years (Figure 4). Patients with AAT deficiency are susceptible to recurrent infections presenting as exacerbations of their condition. Studies have suggested that such episodes are associated with more inflammation in the lung and a greater degree of neutrophilic recruitment than in patients who have normal serum AAT. In addition, the episodes may be longer in duration and show a relationship with the decline in lung function. This probably reflects the low levels of AAT in the blood and the failure of an acute phase response during infections, leading to reduced protection of the lung and a reduced ability to dampen and switch off inflammation.

Pathogenesis AAT is a proteinase inhibitor that is the major serum inhibitor and one of the major lung inhibitors of serine proteinases. These proteinases, and particularly human neutrophil elastase, have a broad spectrum of activity and have been shown to induce many of the features of chronic lung disease in animal models, particularly the development of emphysema. Serum deficiency of AAT is reflected in an

446 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, Alpha-1-Antitrypsin Deficiency 120

Number of patients

100 n = 535

80 60 40 20 0 15

20

25

30

35

40

45 Age

50

55

60

65

70

75

Figure 4 Frequency histogram of the age of patients referred for the first time to the UK registry.

associated deficiency of the same protein in the lung since the majority of lung AAT is derived from serum by simple passive diffusion. A variety of gene defects can result in reduced or no secretion of AAT from the hepatocytes. The most common deficiency, referred to as AAT PIZ, arises from a single point mutation in the AAT gene that leads to a glutamic acid-to-lysine amino acid switch at position 342. This single amino acid change results in alteration of the tertiary structure of the protein, such that the b-sheets open and the reactive loop becomes more mobile. The net result is that the reactive loop of one protein molecule can insert into the b-sheets of a second molecule, forming a dimer. This subsequently links to a third molecule, etc., forming long polymer chains. These polymers accumulate within the hepatocyte such that less than 10% of the protein made by the gene follows the normal secretion pathway into the serum. A variety of other gene defects occur, including the complete absence of the genes, point mutations, and frame shift mutations that result in premature stop codon, and amino acid changes that markedly reduce the antiproteolytic activity of the molecule. Reduced AAT within the lung leads to an associated defect in the ability of the lung to inactivate proteinases (particularly neutrophil elastase) once they are released from the activated neutrophil. This enables the enzyme to retain its activity, causing damage to lung connective tissues and resulting in the development of the pathological changes of chronic lung disease and emphysema. The process may become self-perpetuating for several reasons. Early studies suggested that neutrophil elastase could stimulate alveolar macrophages to release neutrophil chemoattractants, particularly leukotriene B4. This would in turn lead to further neutrophil activation and recruitment into the lung,

releasing further elastase activity during the activation process and hence perpetuating the cycle of events (Figure 3). Recent studies have suggested that the AAT polymers, which are also present in the lung, may be proinflammatory. The polymers have the ability to stimulate epithelial cells to release neutrophil chemoattractants, leading to an amplification of the neutrophilic inflammation by an alternative route. Animal Models

No naturally occurring animal model similar to patients with human AAT deficiency exists. However, because AAT is thought to be predominantly an inhibitor of neutrophil elastase in the airways, circumstantial evidence of its role does exist. Installation of human neutrophil elastase into the airways of experimental animals reproducibly induces the development of airspace enlargement typical of emphysema. In addition, the enzyme also generates pathological changes in the airway consistent with human chronic bronchitis and mucus secretion. Similarly, strategies that lead to neutrophil accumulation in the lungs (e.g., the installation of endotoxin) also result in the development of airspace enlargement. Finally, animals exposed chronically to cigarette smoke also develop changes consistent with emphysema, and at least in the early phase of the condition, this is associated with neutrophilic influx. AAT augmentation can prevent both the early inflammatory changes and the subsequent connective tissue degradation, indicating a key role for this protein. The pallid mouse has partially reduced concentrations of AAT in the plasma. This mouse is particularly susceptible to the development of emphysema. Although it is known that the mouse also has a

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, Alpha-1-Antitrypsin Deficiency 447

further genetic mutation affecting the cell membrane protein called syntaxin, the mice gradually develop progressive changes consistent with emphysema. Furthermore, compared to controls, the exposure to cigarette smoke leads to the much more rapid development of emphysematous change in these mice. In addition, the pattern of emphysema in the pallid mouse is more consistent with human panlobular emphysema, whereas the control mouse tends to develop centrilobular changes. Since the classical type of emphysema in human subjects with AAT deficiency is lower zone panacinar emphysema, the partially deficient pallid mouse provides an animal model that may be more relevant to the human disease. The development of a ‘knockout’ mouse for the AAT gene has proved to be difficult. The major reason is that the mouse has a sequential sequence of five AAT-like genes on the same chromosome. There are many repeated sequences, making individual gene knockout a major technological problem. Recently, however, a gene disruption model of the PIZ gene was reported in the literature, but the authors did not report how this affects the response to airways inflammation. Furthermore, gene disruption models have the problem of potentially reflecting developmental abnormalities rather than subsequent responses to injury or insult. It is therefore imperative to develop an appropriate animal model with late (postdevelopmental) knockout of the appropriate AAT gene followed by replacement with the normal or abnormal AAT genes from the human to mimic human serum deficiency. The development of such a model has major implications for future studies in AAT deficiency because recurrent augmentation studies could be undertaken without concern of immune reaction to a foreign pattern.

Management The most important management strategy is prevention of smoking or cessation of smoking as soon as the diagnosis has been made. The patient should be assessed in detail, as for all COPD individuals. Physiological testing should be carried out to determine the presence of reversible airflow obstruction and response to the appropriate class of agent. In addition, subjects who have recurrent exacerbations, particularly those with more severe disease, should also be treated with inhaled corticosteroids, again according to the general guidelines of COPD patients. As indicated previously, acute exacerbations of the lung are associated with a greater degree of inflammation and more rapid progression of lung disease. Thus, such episodes should be identified and treated rapidly with appropriate antibiotic therapy.

For more than 15 years, augmentation therapy with human AAT purified from plasma has been available in many countries (particularly the United States). This form of therapy seems to be logical because it increases the serum AAT and hence lung AAT levels to normal, or at least to a putative protective level. However, no sufficiently powered controlled clinical trial has been undertaken to demonstrate the efficacy of such a strategy. Indirect studies have suggested that inflammation in the lung can be reduced by augmentation therapy, thus modulating the potentially damaging process. In addition, cohort studies from several countries have suggested that individuals receiving augmentation therapy show a slower decline in lung function than those not receiving such therapy. These studies are clearly suggestive, but because there is a failure to match for socioeconomic status and country of origin there is a high degree of uncertainty regarding the efficacy of such therapy. The only controlled study was performed several years ago in Denmark and Holland, and it suggested that augmentation therapy may lead to a reduction in the loss of lung tissue as assessed by CT scan. The implication is that such a strategy reduces the progression of emphysema. Other conventional approaches that have been used in patients with serum deficiency of AAT include lung transplantation. Patients often do well because they are younger and have less comorbidity than nondeficient patients with a similar degree of physiological abnormality. Lung volume reduction surgery may be appropriate in some individuals, although the basal nature of the disease makes this less amenable to a surgical approach, and evidence suggests that any benefits are relatively short lived. As our understanding of the pathogenic processes in AAT deficiency and the generation of emphysema increases, exciting new strategies may be developed that may be applicable. Because neutrophilic inflammation is thought to be central, strategies to reduce neutrophil traffic are therefore potentially beneficial. A reduction or absence of neutrophilic traffic would reduce the proteinase burden on the lung, making the deficiency in AAT less critical, as indicated previously. The chemoattractants in such patients are being characterized and leukotriene B4 may play a key role (Figure 5). Leukotriene B4 receptor antagonists have been developed and are highly effective in vitro. If leukotriene B4 is central to neutrophil traffic in AAT deficiency, such a strategy may be highly effective. Patients develop emphysema, and in more than 60% of patients this tends to be panlobular and basal in origin. Studies have shown that all-trans retinoic acid can reverse emphysema in animal models in

448 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General AAT Elastase release

Macrophages

Emphysema

Neutrophil recruitment

LTB4 Figure 5 The cycle of events in AATD leading to emphysema. Neutrophils in the lung release elastase. This stimulates macrophages to release the chemoattractant LTB4 because AATD results in levels of the protein too low to inactivate the enzyme. LTB4 recruits more neutrophils, causing more elastase release. Lung damage leading to emphysema is a collateral effect of the enzyme release.

which cigarette smoke or elastase have been used to induce the pathological change. It has been proved that the retinoic acid gamma receptor is the key to this repair process, and specific agonists have been developed. Preliminary studies are under way in man, specifically regarding AAT deficiency. In summary, serum deficiency of AAT has provided an experiment of nature in which deep understanding of the pathogenic processes in the development of emphysema has been obtained. Such patients tend to be young with little comorbidity and rapidly progressive lung disease that is associated with excessive inflammation. Thus, they represent an ideal model for understanding and monitoring the pathogenic processes involved and specifically for developing and assessing new agents and strategies to retard or reverse the progressive loss of lung function. See also: Bronchiectasis. Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations; Emphysema, General. Lipid Mediators: Platelet-Activating Factors. Retinol and Retinoic Acid. Surgery: Lung Volume Reduction Surgery.

Further Reading Alpha-1-Antitrypsin Deficiency Registry Study Group (1998) Survival and FEV1 decline in individuals with severe deficiency of alpha-1-antitrypsin. American Journal of Respiratory and Critical Care Medicine 158: 49–59.

Dawkins PA and Stockley RA (2001) Animal models of chronic obstructive pulmonary disease. Thorax 56: 972–977. Dowson LJ, Guest PJ, and Stockley RA (2001) Longitudinal changes in physiological, radiological and health status measurements in alpha-1-antitrypsin deficiency and effect as associated with decline. American Journal of Respiratory and Critical Care Medicine 164: 1805–1809. Eriksson S (1963) Pulmonary emphysema and alpha-1-antitrypsin deficiency. Acta Medica Scandinavia 175: 197–205. Hill AT, Campbell EJ, Bayley DL, et al. (1999) Evidence for excessive bronchial inflammation during an acute exacerbation of chronic obstructive pulmonary disease in patients with alpha-1antitrypsin deficiency (piZ). American Journal of Respiratory and Critical Care Medicine 160: 1968–1975. Lomas DA and Parfrey H (2004) a1-Antitrypsin deficiency 4: molecular pathophysiology. Thorax 59: 529–535. Luisetti M and Seersholm N (2004) a1-Antitrypsin deficiency 1: epidemiology of a1-antitrypsin deficiency. Thorax 59: 164–169. Needham M and Stockley RA (2004) a1-Antitrypsin deficiency 3: clinical manifestations of natural history. Thorax 59: 441–445. Piitulainen E, Tomling G, and Eriksson S (1998) Environmental correlates of impaired lung function in non-smokers with severe alpha-1-antitrypsin deficiency (piZZ). Thorax 53: 939–943. Sandhouse RA (2004) a1-Antitrypsin deficiency 6: new and emerging treatments for a1-antitrypsin deficiency. Thorax 59: 904– 989. Silverman EK, Pierce JA, Province MA, et al. (1989) Variability of pulmonary function in alpha-1-antitrypsin: clinical correlates. Annals of Internal Medicine 111: 982–991. Stockley RA (2002) a1-Antitrypsin deficiency. In: Voelkel NF and MacNee W (eds.) Chronic Obstructive Lung Disease, pp. 80–89. New York: Decker. Stoller JK and Aboussouan LS (2004) a1-Antitrypsin deficiency 5: intravenous augmentation therapy: current understanding. Thorax 59: 708–712.

Emphysema, General A G N Agustı´ and B G Cosı´o, Hospital Son Dureta, Palma de Mallorca, Spain & 2006 Elsevier Ltd. All rights reserved.

Abstract Emphysema has been defined as abnormal permanent enlargement of airspaces distal to terminal bronchioles accompanied by destruction of their walls that lead to loss of lung elasticity and closure of small airways. Emphysema is recognized as one of the main components of chronic obstructive pulmonary disease, the second one being inflammation of the airways, lung parenchyma, and pulmonary vasculature. This leads to small airway closure and air trapping with hyperinflation, which are responsible for the clinical features of exertional dyspnea, cough, and sputum production. Different patterns of emphysema are recognized, however, suggesting possible different pathogenic processes within the lung. The centrilobular pattern of emphysema is most closely associated to cigarette smoking, whereas the panacinar pattern, which is characterized by a more even involvement of the acinus, is often associated with a1-antitrypsin deficiency. Assessment of emphysema by high-resolution computed tomography scan has been shown to correlate with histologic and functional abnormalities. Smoking cessation,

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General 449 bronchodilators, and inhaled corticosteroids are the mainstay of therapy, although pulmonary rehabilitation and, in selected cases, lung volume reduction may improve pulmonary function and quality of life of these patients.

Introduction The Ciba Guest Symposium, later modified by Snider and others, defined emphysema as ‘‘abnormal permanent enlargement of airspaces distal to terminal bronchioles, accompanied by destruction of their walls without obvious fibrosis.’’ This definition emphasizes the destruction of the alveolar surface with a minimal reparative response in the lung matrix and the ability of this destructive process to reduce the gas-exchanging surface of the lung (Figure 1). Pulmonary emphysema was first described by Laennec in 1834 from observations of the cut surface of postmortem human lungs that had been fixed by inflation. He postulated that lesions were due to overinflation of the lung that compressed capillaries,

leading to atrophy of lung tissue. However, the observation more than a century later that emphysema could be produced experimentally by depositing the enzyme papain in the lung and the description of emphysema in patients with a1-antitrypsin (AAT) deficiency led naturally to the hypothesis that emphysema results from a proteolytic imbalance caused by cigarette smoking. It is now recognized that emphysema is a part of a global disease known as chronic obstructive pulmonary disease (COPD). Many previous definitions of COPD emphasized the terms ‘emphysema’ and ‘chronic bronchitis’, but the recognition that COPD has both airway and airspace characteristics led to the common definition of the disease. The pathological hallmarks of COPD are inflammation of the large (bronchitis) and small airways (bronchiolitis), the lung parenchyma, and the pulmonary circulation, as well as the destruction of lung parenchyma (emphysema). The functional consequence of bronchiolitis and emphysema is airflow limitation. These lesions are associated with a chronic inflammatory response to a lifetime exposure to inhaled toxic gases and particles, mostly tobacco smoke, that involves cells of both the innate and the adaptive immune response.

Etiology In the 1950s, air pollution and airway infections were thought to be the major etiologic factors responsible for COPD. However, the prevalence of cigarette smoking in the United States peaked by the 1960s, and by then adequate cross-sectional epidemiologic evidence had accumulated for the Surgeon General’s advisory committee to state that ‘‘cigarette smoking is the most important of the causes of chronic bronchitis and increases the risk of dying from chronic bronchitis and emphysema.’’ The relation between cigarette smoking and emphysema shows a rough dose-response curve between packs per year and the presence of emphysema, but only approximately 40% of heavy smokers develop substantial lung destruction. However, this observation should not be confused with the fact that only 15–20% of people who smoke develop COPD, because emphysema is sometimes found in people who maintain normal lung function. Several endogenous (genetic background) and environmental factors increase the risk of COPD (Table 1).

Pathology Figure 1 Giant microtome section of human lung with extensive emphysema.

Current descriptions of COPD pathology include changes in large airways, small airways (bronchiolitis), alveolar space (emphysema), and pulmonary

450 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General

vasculature (Table 2). The various lesions are a result of chronic inflammation in the lung, which in turn is initiated by the inhalation of noxious particles and gases such as those present in cigarette smoke. Table 1 Risk factors for development of COPD in smokers Smoking history Quantity Age of onset of smoking Other environmental exposures Various host characteristics Impaired lung growth, shortened stable phase of lung function, or enhanced age-related decline in function Airway responsiveness Antiprotease deficiency (function-modifying polymorphisms) Enhanced adaptive immunological ‘reactivity’

Table 2 Sites of inflammatory cell increases in COPD Large airways Macrophages T lymphocytes (especially CD8 þ ) Neutrophils (severe disease only) Eosinophils (in some patients) Small airways Macrophages T lymphocytes (especially CD8 þ ) Eosinophils (in some patients) Parenchyma Macrophages T lymphocytes (especially CD8 þ ) Neutrophils Pulmonary arteries T lymphocytes (especially CD8 þ ) Neutrophils

(a)

Large airway changes consist of mucous gland enlargement and goblet cell hyperplasia. They are not believed to contribute directly to the airflow limitation in these patients because their contribution to the increased airway resistance that occurs in COPD is marginal. In contrast, small airways (o2 mm) are the sites of increased airway resistance in COPD. Changes that predispose to narrowing of the small airways include goblet cell hyperplasia, smooth muscle hypertrophy, excess mucus, edema, and inflammatory cellular infiltration (Figure 2). Subepithelial fibrosis with collagen deposition in the small airways appears to be the most critical factor in airway narrowing. Respiratory bronchiolitis may be of particular importance since mononuclear inflammatory cells may cause proteolytic destruction of elastic fibers in the respiratory bronchioles and alveolar ducts. The resulting narrowing of this structure seems to be involved in the early airflow obstruction in cigarette smokingrelated COPD. In addition, small airway patency is maintained by the surrounding lung parenchyma, which provides radial traction on bronchioles at points where alveolar septa attach. The loss of bronchiolar and alveolar attachments due to extracellular matrix destruction causes airway distortion and narrowing in COPD. Alveolar space changes constitute the cornerstone of the term emphysema. They are due to chronic inflammation of alveolar walls and destruction with coalescence into larger alveolar spaces. Emphysema is therefore a structural abnormality of the lungs that affects the gas-exchanging airspaces (i.e., the respiratory bronchioles, alveolar ducts, and alveoli). Of note, the definition excludes enlarged airspaces caused by fibrosis to acknowledge that primary fibrotic processes can abnormally tether and enlarge airspaces.

(b)

Figure 2 (a) A normal small airway or membranous bronchiole o2 mm in diameter. (b) An abnormal membranous bronchiole in a smoker with abnormal FEV1. All the layers of the wall are thickened, there is abundant inflammatory cellular infiltrate, the lumen is narrowed, and the epithelium is abnormal with goblet cells. Reproduced with permission of Professor M G Cosı´o, McGill University, Montreal, QC, Canada.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General 451

However, recent data indicate that small airway fibrosis may be a major manifestation of COPD. Emphysema is often classified into distinct pathological types, with centriacinar and panacinar being the most important (Figure 3). Centriacinar emphysema is characterized by enlarged airspaces found initially in association with

respiratory bronchioles, although in more severe cases virtually the whole acinar unit may be involved. In this pattern of emphysema, the focal nature of the lesions stands out against often apparently normal lung, and quite small lesions can be identified. This type of emphysema is most closely associated with tobacco smoking and is most often found

(a)

(b)

(c) Figure 3 (a) A normal lung parenchyma macroscopic and microscopic. (b) Macroscopic and microscopic sections of centrilobular emphysema. Lobules are well defined by fibrous walls. By microscopy, the emphysematous area is surrounded by normal alveolar structures. (c) Macroscopic and microscopic sections of panlobular emphysema. All airspaces are enlarged in the lobule or acinus. Reproduced with permission of Professor M G Cosı´o, McGill University, Montreal, QC, Canada.

452 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General

in upper lobes of the lung, where separate lesions may coalesce to produce larger cavities. Panacinar emphysema is characterized by abnormally large airspaces found evenly distributed across the acinar unit. Adjacent acinar units are usually involved to a similar degree, giving a confluent appearance to the cut surface of the lung, with extensive areas being involved. This type of emphysema is usually associated with AAT deficiency and is more common in the lower lobes. The following are other types of emphysema: *

*

*

Paraseptal emphysema, in which the abnormal airspaces run along the edge of the acinar unit but only where it abuts against a fixed structure, such as the pleura, a vessel, or a septum. Scar or irregular emphysema, in which the emphysematous spaces are found around the margins of a scar. Because the scar may not be related to the anatomy of the acinar unit, this type of emphysema is not classified in relationship to the acinus. Bullae are areas of emphysema that locally overdistend to produce a lesion that, if superficial, protrude on the pleural surface. By convention, only lesions more than 1 cm in diameter justify the description of bullae.

Pulmonary vasculature changes in COPD include perivascular inflammation, intimal thickening, muscularization of arterioles, in situ thrombosis, loss of capillaries and precapillary arterioles, and vascular congestion and stasis.

Structure–Function Correlations Many studies have attempted to address the pathological changes of the small airways in smokers and their relationship to the flow limitation found in COPD (Table 3). Of special interest are studies comparing the airway changes in smokers with various degrees of emphysema and COPD to nonsmokers Table 3 Causes of airflow limitation in COPD Irreversible Fibrosis and narrowing of airways Loss of elastic recoil due to alveolar destruction Destruction of alveolar support that maintains patency of small airways Reversible Accumulation of inflammatory cells, mucus, and plasma exudates in bronchi Smooth muscle contraction in peripheral and central airways Dynamic hyperinflation during exercise

since they give an indication not only of the effects of smoking and emphysema but also of the effect of aging in the airways. By studying smokers who had tests of pulmonary function done, including those reflecting the small airways, before undergoing resection for lung tumors, investigators at McGill University developed a pathological score to describe the microscopic changes in the small airways that allowed them to study the correlations between morphology and function. Specifically, they scored luminal occlusion, goblet cell metaplasia, squamous cell metaplasia, mucosal ulcers, muscle hypertrophy, inflammatory cell infiltrate, fibrosis, and pigment deposition of the airway wall in airways smaller than 2 mm in diameter. The first abnormalities that could be seen in older smokers were changes in the epithelium, with squamous and goblet cell metaplasia and a chronic inflammatory infiltrate, and a slight increase in the connective tissue in the walls of the small airways. As the pathologic and functional abnormalities progressed, the cellular inflammatory infiltrate changed little, but there was a progressive increase in the connective tissue pigment and muscle in the airway wall. When the physiological measurements reflecting small airway abnormalities, such as nitrogen washout test, volume of isoflow, and Vmax50, and other function tests, such as the percentage of forced vital capacity in 1 s (FEV1/FVC), midflow rate, and residual volume, were compared to the pathological score, all measurements showed a progressive deterioration as the score for the morphological abnormalities increased, but only the group with the most severe small airway score demonstrated a substantial amount of emphysema. The striking correlation between the progression of physiological impairment and the degree of small airway disease suggested that inflammatory changes of the small airways made an important contribution to the functional deterioration seen in COPD even in the presence of emphysema. Furthermore, in subjects with a normal FEV1/ FVC ratio, two tests of small airway function, the slope of phase III of the nitrogen washout and the volume of isoflow of the air–helium flow volume loops, were able to detect mild abnormalities of the small airways when spirometric tests were normal. Hogg and colleagues confirmed these findings by studying lung tissue from patients with different degrees of airflow limitation according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification. They showed that progression of COPD is associated with the accumulation of inflammatory mucous exudates in the lumen and infiltration of the wall by innate and adaptive inflammatory immune cells that form lymphoid follicles.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General 453

These changes are coupled to a repair or remodeling process that thickens the walls of these airways.

Clinical Features Symptoms, signs, and the diagnosis of COPD are described elsewhere. However, the radiologic assessment of emphysema deserves special consideration. In 1978, Thurlbeck and Simon described criteria for the diagnosis of emphysema using the chest radiograph. These were based on indications of hyperinflation and vascular pruning and showed only a moderate correlation with macroscopic emphysema as assessed by the picture matching technique. When computed tomography (CT) scans became available, it became possible to extract quantitative data on lung density. When quantitative histologic assessment is compared with quantitative CT assessment,

a good correlation is found even within the range of those showing normal age change and early emphysema. However, attempts to use qualitative grading, scoring, or picture matching techniques have shown poorer correlation with lung function than actual quantitative measurements. The CT scan, particularly high-resolution CT scan (HRCT), can detect the extent and severity of emphysematous lung by directly visualizing areas of destroyed lung, identified as areas of low attenuation without visible walls (Figure 4). HRCT scans also allow reliable distinction of centrilobular emphysema from other causes of airflow obstruction, such as bronchiectasis and constrictive bronchiolitis.

Pathogenesis COPD is characterized by chronic inflammation throughout the airways, parenchyma, and pulmonary vasculature. The intensity and cellular and molecular characteristics vary as the disease progresses. Over time, inflammation damages the lungs and leads to the pathologic changes characteristic of COPD. In addition to inflammation, two other processes thought to be important in the pathogenesis of COPD are an imbalance of proteinases and antiproteinases in the lung and oxidative stress (Figure 5). Inflammation

Figure 4 Specimen radiograph (left) and HRCT scan (right) show the typical, poorly defined, low-attenuation lesions of emphysema (A). Note the absence of definable walls around the areas of low attenuation.

The recognition that inflammation plays a key role in the pathogenesis of COPD is now so widespread and considered so important that it has led to the inclusion of the term ‘abnormal inflammatory response’ in the disease definition. Thus, the GOLD guidelines define COPD as a ‘‘disease state characterized by not fully reversible airflow limitation that is usually both

Noxious particles and gases Host factors Antioxidants Lung inflammation

Antiproteases Proteases

Oxidative stress

Repair mechanisms COPD pathology Figure 5 Pathogenesis of COPD noxious particles and gases, especially tobacco smoke, in susceptible subjects triggers an inflammatory response in the lungs. In addition to inflammation, two other processes thought to be important in the pathogenesis of COPD are an imbalance of proteinases and antiproteinases in the lung and oxidative stress.

454 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General

progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases.’’ It is now believed that the establishment of COPD is associated with further enhancement of the inflammatory response already present in the small airways of normal smokers, which is paralleled by the development of structural abnormalities. These events are reflected in the progressive deterioration of pulmonary function. The overall cellular inflammatory infiltrate in smokers’ airways was described by Finkelstein and coworkers, who carefully differentiated and quantitated the number of cells per millimeter of airway wall in smokers, categorized according to the type of emphysema, and nonsmokers’ lungs obtained at surgery. They found a large number and a wide variation in the number of cells between cases and also within the airways of the same patients. Patients with centrilobular emphysema tended to have more inflammatory cells than patients with panlobular emphysema. It can be concluded based on their results that the peripheral airways of cigarette smokers exhibit a large number and variety of inflammatory cells and that the extent of the cellular infiltration shows a wide variability, indicating that inflammation is unevenly distributed throughout the small airways. Other authors phenotyped the T lymphocytes found in airway biopsies and lung specimens and characterized them as predominantly CD8 þ T cells. Interestingly, CD8 þ T cells not only were increased in these subjects but also correlated negatively with the degree of airflow limitation. This increase in lymphocytes is also associated with an increase in the socalled bronchial-associated lymphoid tissue (BALT), which is rarely found in healthy nonsmokers, is more frequent in cigarette smokers, and shows a further sharp increase in patients with severe (GOLD 3) and very severe (GOLD 4) COPD. This increase in lymphocyte subtypes and the appearance of BALT at this stage of COPD suggest the development of an adaptive immune response that may be driven by microbial colonization and infection, among others. The finding of increased numbers of T lymphocytes and especially CD8 þ T cells only in smokers who develop COPD is intriguing and supports the notion that T-cell inflammation in the lungs is important and may be essential for the development of COPD, raising the hypothesis of the role of autoimmunity in COPD. Elastase–Antielastase Imbalance

In 1963, Laurell and Eriksson first described the association between emphysema and AAT deficiency, the primary inhibitor of the neutral serine proteinase

neutrophil elastase (NE). With respect to AAT, we now know that patients with the deficiency have mutations in the AAT gene. The most common mutation is the Z mutation, which converts Glu to Lys. These mutations impair secretion of the protein from hepatocytes, resulting in markedly decreased circulating levels of this serine proteinase inhibitor. PiZ AAT is slightly less effective as an inhibitor due to its slower rate of association with NE than normal PiM AAT. These genetic changes allow NE to act relatively unopposed, thereby shifting the balance in favor of elastolysis. Other proteases, such as metalloproteinase-9 and -12 (MMP-9 and -12, respectively), have been shown to be related to the development of emphysema throughout transforming growth factor beta activation and fibrosis. Tissue inhibitor of metalloproteinase-3 (TIMP-3) deficiency leads to a combination of developmental airspace enlargement and progressive destructive emphysema in adults, supporting the role of MMPs in COPD. Oxidative Stress

Cigarette smoke and inflammatory cells have the capacity to produce reactive oxygen species, and they have been postulated to play a variety of roles in the pathogenesis of emphysema. One intriguing finding was that cigarette smoke can oxidize a methionine residue in the reactive center of a1PI, inactivating it and thus altering the elastase–antielastase balance. Oxidants cannot degrade extracellular matrix but may modify elastin, making it more susceptible to proteolytic cleavage. MacNee and others have found that intracellular oxidation of IK-kb promotes its ubiquitination and the release of free nuclear factor kappa B (NF-kB), which translocates to the nucleus, resulting in transcription of proinflammatory genes such as interleukin-8 and tumor necrosis factor alpha. Barnes and colleagues found that cigarette smoke oxidizes and inactivates histone deacetylase 2 (HDAC2), which acts to counter histone acetylase. Acetylation of histone unwinds chromatin, allowing transcriptional complexes to bind to DNA. Thus, in the absence of HDAC2, RNA polymerase II and NFkB form a proinflammatory transcription complex. This may explain steroid resistance in COPD because the activated glucocorticoid receptor acts to translocate HDAC2 to the nucleus, which keeps the chromatin wound inactive. Voelkel and colleagues hypothesized that oxidative stress has a central role in alveolar septal cell apoptosis and emphysema induced by vascular endothelial growth factor (VEGF) receptor blockade. Compared to control animals, rats treated with the

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General 455

VEGF receptor blocker SU5416 showed increased alveolar enlargement, alveolar septal cell apoptosis, and expression of markers of oxidative stress, all of which were prevented by the superoxide dismutase mimetic M40419.

Animal Models of Emphysema Many investigators have tried to produce an animal model of emphysema by destroying the elastic fibers in the lung of different animals using a variety of proteinases, following the first description by Gross in 1965, who instilled papain into the lungs of rodents and found emphysema. However, none of them fully reproduce the full picture of pathologic changes seen in patients with COPD. For hypothesis testing in relation to pathogenic mechanisms of human COPD, animal models representing the various patterns of lung destruction and function observed in humans are needed. Because the major environmental factor that predisposes patients to COPD is long-term cigarette smoking, a cigarette smoke exposure model would be advantageous. A variety of animals have been exposed to cigarette smoke, including dogs, rabbits, guinea pigs, and rodents. The results in terms of lung pathology have been variable. To take advantage of the defined genome and the well-established methods of modulating gene expression, murine models of cigarette smoke exposure have been evaluated. Although airspace enlargement has been observed in these murine models, little attention has been paid to how well this simulates the varied pathologic features in smokingassociated emphysema of humans. Recent trends in molecular genetics and advances in molecular physiology make the mouse an ideal investigative model for determining genomic variants affecting susceptibility to COPD. Mice are susceptible to the development of emphysema, and several strains have been described that develop emphysema after prolonged cigarette smoke exposure. Guerassimov and colleagues characterized different phenotypes of mice in response to cigarette smoke in different strains with and without reduced AAT levels and serum elastase inhibitory capacity. Interestingly, Voelkel and colleagues described an animal model of autoimmune emphysema by injecting intraperitoneally xenogeneic endothelial cells in rats, which may help to sustain the role of autoimmunity in the pathogenesis of emphysema.

Management COPD is managed according to the severity of the disease in a stepwise approach, with a progressive

escalation of therapy as the disease progresses. Smoking cessation, pharmacological treatment with bronchodilators and inhaled corticosteroids, and oxygen therapy are the mainstay of treatment for COPD. Nonpharmacologic management of emphysema involves pulmonary rehabilitation, lung volume reduction, and, in selected severe cases, lung transplantation. Pulmonary Rehabilitation

The goals of rehabilitation in COPD are multifactorial, including decrease and control of respiratory symptoms, increase in physical capacity, improvement in health status, reduction of the psychological influence of physical impairment and disability, prevention of complications and exacerbations, and, ultimately, prolongation of life. There is much evidence that rehabilitation improves dyspnea on exertion and that associated with daily activities in COPD. Favorable outcomes have been reported after pulmonary rehabilitation in maximum exercise tolerance, peak oxygen uptake, endurance time during submaximal testing, functional walking distance, and strength of peripheral and respiratory muscles. Pulmonary rehabilitation also results in a clinically significant improvement in disease-specific and general measures of quality of life. The effect size of pulmonary rehabilitation largely exceeds what can be achieved by the best pharmacological therapy. This intervention is therefore judged to be evidence based with respect to both exercise tolerance and symptoms of dyspnea and fatigue. These effects are longlasting (41 year) and not necessarily related to improvements in exercise ability. Lung Volume Reduction

Lung volume reduction surgery (LVRS) consists of the removal of the most damaged parts of the lungs in selected patients with emphysema, particularly those with upper lobe emphysema and poor exercise capacity after a full rehabilitation program (Table 4). Initial reports showed that LVRS appeared to produce functional benefits, including increased FEV1, reduced total lung capacity and functional residual capacity, and improved function of respiratory muscles. However, uncertainty about morbidity and mortality, the occurrence, magnitude, and duration of benefits, and preoperative predictors of benefit led to multicenter, randomized clinical trials. The National Emphysema Treatment Trial used mortality and maximum exercise capacity 2 years after randomization as the primary outcomes. Overall mortality did not differ in this study between patients undergoing LVRS

456 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General Table 4 Indications and contraindications for lung volume reduction surgery Features

Indications

Contraindications

Clinical

Age o75 years Disability despite optimal medical treatment, including pulmonary rehabilitation Ex-smoker (43–6 months)

Age 475–85 years Comorbid illness with 5-year mortality 450% Severe coronary artery disease Pulmonary hypertension Severe obesity or cachexia Surgical constraints: previous thoracic procedure, pleurodesis, chest wall deformity

Physiological

FEV1 post BDo35–40% Hyperinflation Increased RV/TLC RV4200–250% TLC4120% DLCOo50%

FEV1 post BD435%

Imaging

RVo150% TLCo100%

CXR: hyperinflation CT: marked emphysema with heterogeneity (upper lobe predominance is ideal) Isotope scan: target areas for resection

CXR: no hyperinflation CT: minimal emphysema; homogeneous, severe emphysema Isotope scan: absence of target zones

FEV1, forced expiratory volume in 1 s BD, bronchodilator; TLC, total lung capacity; DLCO, carbon monoxide diffusing capacity; RV, residual volume; CXR, chest X-ray; CT, computed tumography. Adapted from Flaherly KR and Martinez FJ (2000) Lung volume reduction surgery. Clinics in Chest Medicine 21(4): 835, with permission.

and those assigned medical therapy only. Exercise capacity after 24 months had improved by more than 10 W in 16% of patients in the surgery group compared to 3% of patients in the medical therapy group. Patients assigned surgery were significantly more likely than those assigned medical therapy to have improvements in the distance walked in 6 min, the percentage predicted values for FEV1, general and health-related quality of life, and degree of dyspnea. After interim analyses, patients with FEV1 20% or less of the predicted value and either homogeneous emphysema or a diffusing capacity of carbon monoxide that was 20% or less of the predicted value had a high risk of death after LVRS, with little chance of functional benefit. The National Emphysema Treatment Trial found that patients with predominantly upper lobe emphysema and low exercise capacity had lower mortality after LVRS than the corresponding medical therapy group. Cost-effectiveness analysis shows that LVRS is costly compared to medical therapy. Currently, the use of endobronchial valve placement to reduce lung volume endoscopically is being investigated. Preliminary results indicate that it can improve lung volumes and gas transfer in patients with COPD and prolong exercise time by reducing dynamic hyperinflation. Lung Transplantation

Pulmonary emphysema was initially thought to be a contraindication to lung transplantation, but

smoking-related and AAT deficiency emphysema have become the most common indication for pulmonary transplantation. Patients with diffuse disease, low FEV1 (o20% predicted), hypercapnia (PaCO247.3 kPa), and associated pulmonary hypertension are directed toward lung transplantation. The advantages of this approach have to be carefully balanced against the well-known disadvantages. Complete replacement of the diseased and nonfunctioning lungs can contribute to a striking improvement in pulmonary function and exercise tolerance, but the paucity of available donor lungs has created a long waiting list, the operation is associated with initial morbidity and mortality, and there is a risk of chronic allograft dysfunction or bronchiolitis obliterans syndrome. Furthermore, with strict adherence to selection criteria, very few patients with emphysema are candidates for any surgical therapy. See also: Chronic Obstructive Pulmonary Disease: Overview; Emphysema, Alpha-1-Antitrypsin Deficiency. Matrix Metalloproteinase Inhibitors. Matrix Metalloproteinases. Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants.

Further Reading Agusti A, MacNee W, Donaldson K, and Cosio M (2003) Hypothesis: does COPD have an autoimmune component? Thorax 58(10): 832–834. Barnes PJ (2000) Chronic obstructive pulmonary disease. New England Journal of Medicine 343(4): 269–280.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Smoking Cessation 457 Barnes PJ and Stockley RA (2005) COPD: current therapeutic interventions and future approaches. European Respiratory Journal 25: 1084–1106. Calverley PM and Walker P (2003) Chronic obstructive pulmonary disease. Lancet 362(9389): 1053–1061. Cosio MG and Majo J (2002) Inflammation of the airways and lung parenchyma in COPD: role of T Cells. Chest 121(supplement 5): 160S–165S. Fishman AP (2005) One hundred years of chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 171(9): 941–948. Fishman A, Martinez F, Naunheim K, et al. (2003) A randomized trial comparing lung-volume-reduction surgery with medical therapy for severe emphysema. New England Journal of Medicine 348(21): 2059–2073. Flaherly KR and Martinez FJ (2000) Lung volume reduction surgery. Clinics in Chest Medicine 21(4): 835. Geddes D, Davies M, Koyama H, et al. (2000) Effect of lungvolume-reduction surgery in patients with severe emphysema. New England Journal of Medicine 343(4): 239–245. Hogg JC (2004) Pathophysiology of airflow limitation in chronic obstructive pulmonary disease. Lancet 364(9435): 709–721. Hogg JC, Chu F, Utokaparch S, et al. (2004) The nature of smallairway obstruction in chronic obstructive pulmonary disease. New England Journal of Medicine 350(26): 2645–2653. Mahadeva R and Shapiro SD (2002) Chronic obstructive pulmonary disease: experimental animal models of pulmonary emphysema. Thorax 57(10): 908–914. Pauwels RA, Buist AS, Calverley PM, Jenkins CR, and Hurd SS (2001) Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/ WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) workshop summary. American Journal of Respiratory Critical Care Medicine 163(5): 1256–1276. Shapiro SD and Ingenito EP (2005) The pathogenesis of chronic obstructive pulmonary disease: advances in the past 100 years. American Journal of Respiratory Cell and Molecular Biology 32(5): 367–372. Sutherland ER and Cherniack RM (2004) Management of chronic obstructive pulmonary disease. New England Journal of Medicine 350(26): 2689–2697. Wouters EF (2004) Management of severe COPD. Lancet 364(9437): 883–895.

Smoking Cessation M C McCormack and C S Rand, Johns Hopkins Univerisity, Baltimore, MD, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Smoking is a well established cause of chronic lung disease and is the leading preventable behavior contributing to patient morbidity and mortality. Smoking cessation has been shown to have a strong beneficial effect on the course of chronic obstructive pulmonary disease (COPD). Research has clearly established that physician advice regarding smoking cessation will significantly increase the likelihood that a patient will quit smoking. There are five steps recommended as a starting point for the clinician addressing smoking cessation. The 5 A’s include ‘asking’ the patient about smoking, ‘advising’ cessation, ‘assessing’ readiness to quit, ‘assisting’ in the process, and ‘arranging’ follow-up. If the patient

is unwilling to quit smoking, the 5 R’s can be employed: emphasizing ‘relevance’ of cessation, identifying ‘risks’ of smoking, focusing on ‘rewards’, discussing ‘roadblocks’, and using ‘repetition’. Pharmacologic therapy is recommended for most patients seriously considering smoking cessation with few exceptions. Nicotine replacement therapy and bupropion are considered first-line agents. Combination therapy may be used in patients who are not successful in quitting while using single-drug therapy. Despite evidence that clinician advice to quit smoking increases abstinence rates, clinicians inconsistently intervene. System-based programs provide opportunities to further improve rates of smoking cessation.

Smoking and COPD Smoking is a well-established cause of chronic lung disease and is the leading preventable behavior contributing to increased patient morbidity and mortality. In the normal aging process, the lung loses elasticity and pulmonary mechanics deteriorate with slowing of forced expiration by approximately 20–30 ml per year. Smokers experience an accelerated decline in lung function, which is particularly severe in a susceptible subset of the smoking population. When forced expiratory volume in 1 s (FEV1) reaches 50% predicted, individuals become symptomatic and activity is limited by dyspnea. When FEV1 reaches 30– 40% predicted, exercise limitation becomes disabling. The mean survival after the onset of disabling chronic obstructive pulmonary disease (COPD) is 5 years. Smoking cessation has a strong beneficial effect on the course of COPD. In 1983, the National Heart, Lung, and Blood Institute initiated The Lung Health Study, a multicenter trial investigating the effect of smoking cessation and ipratropium bromide on the decline in lung function in adults with mild COPD. Patients who were randomized to smoking cessation received intensive counseling and pharmacotherapy. The study found that among those who quit, the accelerated decline in lung function was actually reversed during the first year of cessation and smokers who quit had an average increase in FEV1 of 47 ml during the first year of cessation. This was followed by a subsequent decline characterized by that of the normal aging population of 31 ml per year. This was half the average rate of decline compared to individuals who continued smoking. Among smokers who quit, improvement in FEV1 was more pronounced among heavier smokers, women, those with worse lung function, and those with greater bronchial responsiveness.

Clinical Strategies to Promote Smoking Cessation Physician Advice

Research has clearly established that physician advice to stop smoking significantly increases the likelihood

458 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Smoking Cessation

that a patient will quit smoking. The Agency for Healthcare Research promotes widely used evidencebased guidelines to assist healthcare providers in brief smoking cessation counseling. These guidelines propose five basic steps to intervention in the primary care setting, which can be summarized by the ‘5 A’s’. The first step is to ‘ask’ the patient about their tobacco use. This can be consistently achieved by routinely updating smoking status while reviewing the vital signs. Next, ‘advise’ the patient of the benefits of smoking cessation. Here, the message should be clear, strong, and personal. This is an opportunity to relate the specific benefits as they may relate to the patient. For example, ‘‘As your doctor I’m very concerned about the effect your smoking is having on your breathing and your overall health. If you quit smoking now, there is an excellent chance that your lungs will begin to heal and your risk of COPD, heart disease, and cancer will be significantly reduced. For these reasons, I strongly advise you to quit smoking.’’ Physician advice may include not only discussion of health benefits but also economic factors (e.g., cost of smoking) and family concerns (e.g., passive smoking, role modeling). The third ‘A’ entails ‘assess’ing the patient’s readiness to quit smoking. This includes assessing the willingness to stop smoking in the short term (the next 30 days) as well as identifying longerterm goals. For example, one might ask, ‘‘On a scale of 1–10 how ready are you to quit smoking in the next 30 days?’’ Once the practitioner has ascertained that the individual is ready to quit smoking, he/she can then ‘assist’ the patient in preparing for a successful quit attempt. The hallmarks for adequate preparation can be remembered by the pneumonic STAR: setting a quit date, telling family, friends, and co-workers, anticipating challenges, and removing tobacco products from the environment. The healthcare provider should also provide practical advice. This may include reviewing pitfalls in prior smoking cessation attempts and avoiding triggers. Identifying support individuals and discouraging smoking on the part of others in the home is also important. The practitioner’s office should also be identified as a source of support. Supplemental materials may be useful and pharmacotherapy should be addressed at this stage. Once a smoking cessation plan is in place, followup is essential. The final step is to ‘arrange’ a visit. The first office visit should occur shortly after the quit date, preferably within 1 week, and a subsequent visit within 1 month is recommended. A more intensive, group or individual, smoking cessation program is an alternative to outpatient follow-up. If the patient is unwilling to quit after the second step ‘assess’, the clinician should employ motivation

enhancing strategies. Here, the ‘5 R’s’ may be employed. The first is to address ‘relevance’ by encouraging the patient to identify how tobacco cessation is personally relevant. Identifying ‘risks’ can also be a motivating factor and the clinician can assist the patient in recognizing personal health risks. The patient should be encouraged to focus on the ‘rewards’ of smoking cessation and these may include not only improved health but also economic and social benefits, as well as potential improvement in personal appearance and self-esteem. The clinician should ask the patient to identify ‘roadblocks’ and help troubleshoot potential barriers to successful cessation. Finally, ‘repetition’ is essential in patients who are not yet ready to begin an attempt to quit as this increases the likelihood of future attempts and future success. Research suggests that the consistency of the provider message is a critical component in a patient’s decision to quit smoking. Addressing Patient Barriers to Smoking Cessation

Tobacco dependence is an addictive disorder and for this reason few patients quit with their initial attempt. Tobacco dependence includes not only physical addiction (for most smokers) but also includes significant behavioral and psychological dependence on tobacco, which can create barriers to successful cessation. For these reasons, many smokers cycle in a relapse-and-remission pattern for years before successfully quitting. According to the 2000 Surgeon General’s Report, 70% of the 50 million smokers in the US have made at least one prior quit attempt. While the tendency to relapse can be very discouraging, it is important to recognize this pattern as part of the normal process of quitting as repeated interventions are often required to attain long-term abstinence. Despite the complexities of this addiction, there has been significant success in achieving smoking cessation at a national level over the past several decades. Between 1965 and 1993, the prevalence of adult smoking in the US decreased from 42% to 25% while quit rates increased from 25% to 50%. Although cigarettes contain as many as 600 additives, the nicotine in tobacco has been identified as the primary substance associated with addiction. Nicotine absorption is pH dependent and depends on the route of exposure. Nicotine is absorbed transdermally, through the gastrointestinal mucosa, and is rapidly absorbed across the huge surface area of the alveoli of the lungs. The half-life of nicotine is 2 h and this substance undergoes a significant first-pass effect in the liver, where 70–80% of the metabolism occurs via the cytochrome P-450 system. The effect of nicotine is mediated by binding

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Smoking Cessation 459

to the nicotinic cholinergic receptors, a subset of acetylcholine receptors. Several additives in tobacco products are thought to influence the metabolism of nicotine, including ammonia and menthol. While ‘light’ or low-tar cigarettes have been promoted as less toxic, evidence suggests that coverage of the filter perforations with the fingertips and compensatory behavior to achieve equivalent nicotine levels, including deeper inhalation, shorter time between puffs, and smoking more cigarettes, negate the potential benefit of ‘light’ cigarettes over regular cigarettes. Nicotine withdrawal symptoms are an important early barrier to smoking cessation. The nicotine withdrawal syndrome is well characterized and includes craving the use of nicotine, irritability, difficulty concentrating, restlessness, increased appetite, and anxiety. Some of the warning signs that a patient may have high dependence on nicotine include withdrawal symptoms during previous quit attempts, smoking more than one pack per day, smoking the first cigarette within 30 minutes of awakening, and smoking during times of illness. Preparing the patient to deal with symptoms of nicotine withdrawal is an important aspect of counseling patients about smoking cessation. A history of withdrawal symptoms or evidence of nicotine dependence should prompt strong encouragement of nicotine replacement therapy. Social and environmental triggers comprise another potential barrier to effective long-term cessation. Smokers have generally created a world in which smoking is an important part of their social and leisure activities. Patients should be encouraged to anticipate this change by creating an alternative nonsmoking support network and avoiding activities where smoking is common (e.g., bars, clubs). Social activities and settings where alcohol is served can be risky for patients trying to quit smoking because one of the more common triggers for relapse is the use of alcohol. For this reason, abstinence from alcohol use should be encouraged during smoking cessation attempts. Weight gain after smoking cessation is a common concern among smokers considering quitting. Weight gain is likely caused by decreased metabolic activity and increased caloric intake. While women and adolescent smokers are frequently fearful of weight gain if they quit smoking, this concern is shared by many smokers and has been shown to persist in the older, medically ill population. The majority of smokers who quit will gain some weight but most will gain greater than 10 pounds (4.5 kg). AfricanAmericans, those under age 55, and heavy smokers are at greatest risk for major weight gain. There is evidence that attempts to prevent weight gain may undermine the success of cessation

attempts. However, studies suggest that increased exercise during smoking cessation can decrease and delay weight gain. Pharmacologic therapy with nicotine replacement and bupropion has also been shown to delay weight gain but not to decrease weight gain in the long run. It is important for clinicians to address the possibility of weight gain. Clinicians should stress the overall importance of quitting smoking for improved health and should help define this as the primary goal. The clinician should also offer to help the individual address weight gain once the patient has achieved success with cessation. There is evidence that a sequential approach to cessation and weight control is superior to a simultaneous approach. Pharmacologic Therapy

There have been many advances in the pharmacologic treatment of tobacco dependence in the recent past. It is recommended that all individuals seriously attempting smoking cessation receive pharmacologic therapy with a few exceptions. In addition to individuals with medical contraindications, special consideration of the appropriateness of pharmacotherapy should be given to individuals who smoke less than 10 cigarettes per day, pregnant women or nursing mothers, and adolescents. Pharmacologic therapy should be utilized in a coordinated approach by the healthcare provider that incorporates motivated behavioral change efforts. A list of the various smoking cessation products that are available is given in Table 1. Nicotine replacement therapy and bupropion are the recommended first-line therapies for tobacco cessation. There is no clear distinction as to which of these therapies is superior. Therefore, the practitioner must weigh the decision and consider the circumstances specific to the patient. Bupropion and nicotine replacement therapy may be used simultaneously and there is some evidence that this may confer additional benefit when compared to monotherapy. Nicotine replacement therapy Nicotine replacement therapy (NRT) is available in many forms, including the transdermal patch, chewing gum, inhaled, nasal spray, and lozenges. Nicotine substitution is effective in reducing symptoms of nicotine withdrawal. The nicotine patch is a widely used method of delivery of NRT. The patch is available as an over-thecounter or prescription medication. There is both a 24 h formulation as well as a 16 h formulation. If sleeplessness is a significant side effect, the 24 h patch can be removed prior to bedtime or the 16 h patch can be substituted. The patch should be placed in a

460 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Smoking Cessation Table 1 Summary of smoking cessation products Product

Dosage

Package

Price (US$)a

Comments

Over-the-counter products Nicotine gum Nicorette

2 mg
9–24 days 4 mg
9–24 days

170 pieces 110 pieces 50 pieces

59.99 49.99 31.99

Emphasize importance of adequate use. Target is 9–24 per day for 4–6 weeks, then gradual tapering. Special instructions about ‘chew-and-park’ technique

21 mg/24 h 14 mg/24 h 7 mg/24 h 15 mg/16 h 22 mg/24 h

14 14 14 14 14

44.99 44.99 44.99

Target is maintenance for 4–10 weeks, with gradual taper following. If sleeplessness occurs, the 24 h patch may be removed at bedtime or the 16 h patch may be used

2 mg 4 mg

72 pieces 72 pieces

39.99 39.99

1 mg
8–10 mg/day 2 sprays ¼ 1 mg

100 mg

55.99

Provides rapid onset of nicotine. Tapering recommended at 6 weeks. Nasal irritation is a frequent side effect

10 mg/cartridge (delivers 4 mg)

168 cartridges

153.49

Absorption is through the oral mucosa, not the lungs. May simulate behavioral aspect of smoking

150 mg 1 per day
3 days, then 2/day 150 mg 1 per day
3 days, then 2/day

60 tablets

136.99

60 tablets

105.49

Begin treatment 1 week prior to quit date. No tapering needed. May be combined with nicotine replacement therapy

Nicotine patch Nicoderm CQ

Nicotrol Generic/store brand Nicotine lozenge Nicorette

Prescription products Nicotine nasal spray Nicotrol NS

Nicotine vapor inhaler Nicotrol inhaler Bupropion SR Zyban Generic

a

patches patches patches patches patches

34.99

Dissolves in 10–30 min. More than 9 lozenges per day recommended as initial therapy. Tapering recommended at 6 weeks

Based on pricing at a large pharmacy chain in Baltimore, MD, US, 2004.

hairless area between the neck and the torso. The most common side effect is a local skin reaction, which occurs in approximately half of the patients who receive this therapy. The nicotine patch has been studied in patients with cardiovascular disease and has not been associated with an increase in cardiovascular events. While the patch offers passive dosing that provides steady-state levels of nicotine, this system does not allow the user to adjust as needed in response to craving or situational urges to smoke. Patients may benefit from the addition of a shortacting nicotine replacement therapy, such as nicotine gum, if they are unable to quit with the patch alone. Nicotine chewing gum is another form of nicotine replacement therapy. This is available in a 2 mg per piece formulation, which is recommended for smokers of less than 25 cigarettes per day and a 4 mg per piece formulation for individuals who smoke 25 or more cigarettes per day. One advantage of the nicotine chewing gum is that it can be used in an ad lib fashion as cravings or symptoms of withdrawal occur. However, adherence to this therapy can be a

barrier to effectiveness. Often patients do not utilize enough of the chewing gum. This can be addressed by employing scheduled dosing intervals of 1–2 h. Furthermore, technique is sometimes problematic. The gum should be chewed until a mint taste is experienced. At this time, the chewing gum should be parked between the cheek and the gum to allow buccal mucosa absorption of the nicotine. This chewing and parking behavior is repeated for approximately 30 min at which time the gum is discarded. The individual should refrain from eating or drinking for 15 min before or after use to maximize absorption. While less commonly used, nicotine inhalers and nicotine nasal spray are alternative methods of nicotine delivery. They are both available by prescription only. Nicotine inhalers are dispensed as canisters containing 4 mg of nicotine delivered over 80 breaths. The recommended dose is between 6 and 16 canisters per day. The patient should refrain from eating or drinking 15 min before or after using the inhaler. The most common side effects are local irritation in the mouth and throat as well as cough and rhinitis.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Smoking Cessation 461

Nicotine nasal sprays have an aerosolized dose of 0.5 mg per nare with a combined total dose of 1.0 mg. Initial dosing should be 1–2 doses per hour for a total of 8–40 doses per day. Each bottle contains about 100 doses. The side effects of this treatment include a high frequency of nasal irritation. Nicotine nasal spray also has a greater dependence potential than other nicotine replacement therapies although less than that of cigarette smoking. Utilizing a combination of nicotine replacement strategies has been attempted. The goal is to provide a steady-state level of nicotine accompanied by ad lib use during times of cravings or abstinence symptoms. There is a suggestion that this may yield additional benefit but long-term safety has not been proven. Bupropion Bupropion (Zybans) is the first nonnicotine medication shown to be effective for smoking cessation. This is available exclusively as a prescription medication indicated for smoking cessation (Zybans) and for depression (Wellbutrins). Although the precise mechanism of action is unknown, it is presumed to be mediated by its capacity to block neural reuptake of dopamine and norepinephrine. The effectiveness of this agent for smoking cessation does not appear to be mediated through an antidepressant effect. In fact, bupropion has been found to be equally efficacious in improving smoking cessation rates in patients without depression as in those with depression. However, when choosing an agent for a depressed smoker, bupropion should be seriously considered as it may provide multiple benefits. The medication should be initiated 1 week prior to quitting at a dose of 150 mg orally for 3 days, followed by 150 mg twice daily for 7–12 weeks. Treatment for up to 6 months can be considered for maintenance. The most commonly reported side effects are insomnia and dry mouth. Bupropion is contraindicated in individuals with a seizure disorder, with current or previous bulimia or anorexia nervosa, or in those who have taken a monoamine oxidase inhibitor within the last 14 days or are currently taking another medication containing bupropion. Other medications There are two second-line agents that have been employed for tobacco cessation when first-line treatments are contraindicated or have failed. These include clonidine, an antihypertensive, and nortriptylene, an antidepressant. Limited research suggests that these medications may increase abstinence compared with placebo but these products are not approved by the US Food and Drug Administration for the indication of smoking cessation. The use of other antidepressants in the treatment of smoking cessation is an area of continuing

investigation as depression is more common among smokers than the general population. However, currently there are no other antidepressants recommended for smoking cessation.

Smoking Cessation Programs Smoking cessation programs are effective in initiating and maintaining smoking cessation. In the Lung Health Study, an intensive intervention program resulted in sustained smoking cessation in 22% of patients compared to 5% of those who received usual care. A significant dose–response relationship has been shown between the intensity of counseling and the effectiveness in achieving cessation. Intense programs typically involve interactions that are longer than 10 min, with greater than 4 sessions, for a total of more than 30 min of counseling. These sessions might include some strategies that have been found to be particularly effective, such as practical counseling, establishing social support within the program, and helping to secure social support beyond the extent of the program. Although more intense counseling is preferable, interventions of less than 3 min have still been shown to have a positive effect. Person-to-person interaction has been found to be effective and can take the form of telephone, individual, or group counseling. Local hospitals and branches of national organizations may offer organized treatment programs. The American Lung Association offers low-cost smoking cessation programs, as well as an internet-based multistep program.

Complementary Medicine Many patients inquire about the benefits of acupuncture, hypnosis, or other complementary and alternative medicine strategies for smoking cessation. Acupuncture has been proposed to be an effective aid to cessation because it may release neurotransmitters, such as serotonin, noradrenaline, and cholecystokinin, which may affect nicotine craving and withdrawal. However, meta-analytic studies have not found acupuncture to be superior to sham acupuncture, suggesting that any potential benefits are derived from positive beliefs about the utility of the procedure. Hypnotherapy has been utilized with the intent of strengthening the will to stop smoking and decreasing cravings. A meta-analysis of nine randomized trials did not confirm significant benefit from hypnotherapy compared to other interventions. While the use of complementary medicine may be helpful on an individual basis, no alternative or complementary smoking cessation techniques or products have been substantiated by evidence in the medical literature.

462 CHYMASE AND TRYPTASE

System-Based Support for Patient Smoking Cessation Despite strong evidence that physician advice regarding smoking is an effective cessation tool, national data show that over one-third of US smokers are not advised to quit in a given office visit. Systemwide programs have been shown to increase the frequency with which smokers are identified. Identification of smoking status by stickers on patient charts or by recording smoking status with the vital signs are useful strategies. This also provides an opportunity for nonphysician healthcare workers to participate. The involvement of nurses, in addition to physicians, in the process of tobacco cessation intervention has been associated with increased long-term abstinence rates. System-wide programs have also been shown to increase the number of patients who receive counseling during office visits. Despite concerns that patients may be annoyed by increased frequency of smoking cessation interventions, follow-up studies have found enhanced patient satisfaction. Many successful system-based models have focused on removing barriers, such as access and cost. The addition of telephone-based programs has been shown to increase participation. Full coverage of the costs of patient smoking cessation services and pharmacotherapy has been associated with increased participation in cessation programs and decreased prevalence of smoking. The most successful systemwide approaches treat tobacco cessation as a key indicator of performance. These systems incorporate individuals from all levels of the organization. Successful programs solicit feedback and make interim adjustments. This results in the creation of an infrastructure capable of maintaining a smoking cessation program over time. See also: Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations. Particle Deposition in the Lung.

Further Reading Anthonisen NR, Connett JE, Kiley JP, et al. (1994) Effects of smoking intervention and the use of an inhaled anticholinergic bronchodilator on the rate of decline of FEV1. Journal of American Medical Association 272: 1497–1505. Curry SJ, Grothaus LC, McAfee T, et al. (1998) Use and cost effectiveness of smoking cessation services under four insurance plans in a health maintenance organization. New England Journal of Medicine 339: 673–679. Fiore MC, Bailey WC, Cohen SJ, et al. (2000) Treating Tobacco Use and Dependence: Clinical Practice Guideline. Rockville, MD: US Department of Health and Human Services, Public Health Service. Hollis JF, Bills R, Whitlock E, et al. (2000) Implementing tobacco interventions in the real world of managed care. Tobacco Control 9(supplement I): i18–i24. Rigotti NA (2002) Treatment of tobacco use and dependence. New England Journal of Medicine 7: 506–512. Scanlon PD, Connett JE, Waller LA, et al. (2000) Smoking cessation and lung function in mild-to-moderate chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 161: 381–390. Wise RA (1997) Changing smoking patterns and mortality from chronic obstructive pulmonary disease. Preventive Medicine 26: 418–421. Wise RA, Kanner RE, Lindgren P, et al. (2003) The effect of smoking intervention and an inhaled bronchodilator on airways reactivity in COPD: The Lung Health Study. Chest 124: 449–458. Zang ZA and Wynder EL (1998) Smoking trends in the United States between 1969 and 1995 based on patients hospitalized with non-smoking-related diseases. Preventive Medicine 27: 854–861.

Relevant Websites http://www.ash.org – provided by a non-profit organization, Action on Smoking and Health, and offers information about smoking risks, quitting smoking, and legal issues related to smoking. http://www.lungusa.org – provided by the American Lung Association and offers an online smoking cessation program, practical smoking cessation advice, and information about smoking-related lung disease. http://www.surgeongeneral.gov/tobacco/ – provided by the US Department of Health and Human Services and contains the most recent Surgeon General’s Report on the Health Consequences of Smoking, as well as information for physicians and consumers.

CHYMASE AND TRYPTASE P Peachell, University of Sheffield, Sheffield, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The serine proteases, tryptase and chymase, contribute to the pathophysiology of respiratory disease. The principal site of

synthesis of these proteases is the lung mast cell and proteases are stored within secretory granules, complexed to heparin. Activation of mast cells, by allergens and other stimuli, leads to the release of a wide variety of mediators, including proteases. The released proteases may cause bronchoconstriction, promote inflammation, modulate neuronal activity, and participate in airway remodeling. These effects of proteases may be mediated by direct hydrolysis of substrates and by activation of

CHYMASE AND TRYPTASE 463 protease-activated receptors. These receptors are upregulated in respiratory disease. Inhibitors of these proteases, which are in development, could have a role in the therapeutic management of respiratory disease.

Introduction The lung mast cell has long been recognized as central to the mediation of asthma, especially asthma that has an allergic basis. Activation of the mast cell, by allergens or other stimuli, leads to the generation of autacoids such as histamine, cysteinyl-leukotrienes, prostaglandin D2, cytokines, and prominent levels of proteases. These generated mediators act synergistically and can have profound effects on the airways and blood vessels causing bronchoconstriction and promoting inflammation. In addition to these more immediate effects, it is likely that over a longer timeframe, mast cells contribute to airway remodeling. Mast cell-derived products can cause changes in the architecture of the airway and this could have important consequences as far as the behavior of the asthmatic airway is concerned. The importance of histamine as a mediator of allergic disorders has been known for over 70 years. Moreover, the fact that slow-reacting substance of anaphylaxis (SRS-A), later identified as a mixture of cysteinyl-leukotrienes, is a very potent bronchoconstrictor and that prostanoids can act similarly, has been known for some considerable time. However, although studies some 50 years ago demonstrated that mast cells contain proteases, a fuller appreciation of how generated proteases might influence the development of allergic disease has only been realized more recently. A major reason for this is the identification, a little over 10 years ago, of a novel group of receptors that are activated by proteases. The widespread expression of protease-activated receptors (PARs) within the lung and the potential activation of PARs by mast cell proteases has substantially enlarged the scope of protease-mediated effects within the lung. Thus, proteases are no longer considered simply as enzymes whose egress from mast cells may cause some modest structural modifications to the extracellular matrix. Indeed, a large body of evidence has now accumulated showing that mast cell proteases may contribute significantly to the pathophysiology of asthma and other respiratory disorders. In human mast cells, tryptase is the most abundant protease and it follows that most of the available literature has focused on this protease. Other proteases variably expressed by mast cells include chymase, cathepsin, and carboxypeptidase. In fact, the expression of proteases by mast cells has led to an

important classification of mast cell populations. Mast cells that express tryptase, but not chymase, are often classified as MCT mast cells whereas those that contain tryptase and chymase (and other proteases) are referred to as MCTC mast cells. Connective tissue mast cells, such as those found in skin, are more or less exclusively MCTC mast cells. At mucosal surfaces, such as the lung, MCT mast cells appear to predominate although, within the bronchi, MCTC mast cells are more prominent. Interestingly, the heterogeneity in protease content displayed by different mast cell populations is associated with heterogeneity in function. For example, chemokine receptor expression between the two subsets differs. In addition, MCTC mast cells, but not MCT mast cells, are responsive to a wider array of activators such as neuropeptides, compound 48/80, and C5a, and the spectrum and quantities of mediators generated, quite apart from the differences in the complement of proteases that can be generated, also differ.

Structure Mammalian mast cell chymases fall into two phylogenetic groups: alpha and beta. In primates, however, only the alpha form appears to be expressed and it is possible that beta-chymases are rodent specific. The gene for human alpha-chymase is found on chromosome 14 within a cluster encoding for a number of additional proteases. The human tryptase gene locus, found on chromosome 16, is more complex than that for alpha-chymase due to the existence of multiple tryptase genes and allelic variants. The most important mast cell tryptase is beta-tryptase, although isoforms of this type exist that may display differences in activity. In addition, alpha-tryptase has also been detected but not always, as about one-fourth of individuals lack this isoform. That interindividual differences exist in protease content could potentially have an influence on disease susceptibility. Of particular interest are reports demonstrating that mast cell tryptase forms tetramers and that the tetrameric form is associated with optimal catalytic activity. Within the tetramer, the active sites of each of the monomers are orientated centrally creating a central pore. The geometry of this pore is such that larger substrates cannot be accommodated and, as a consequence, smaller peptidergic substrates are preferentially hydrolyzed. Although small molecule inhibitors of serine proteases are effective at attenuating the catalytic activity of tryptase, larger proteinaceous inhibitors that are effective at reducing the activity of alternative serine proteases, such as chymase, are more or less without exception ineffective. Again the reason for this resistance to inhibition

464 CHYMASE AND TRYPTASE

probably lies in the restricted access of larger moieties to the catalytic site.

capable of hydrolyzing substrates that the tetrameric form does not cleave.

Biological Function Regulation of Production and Activity The regulation of protease synthesis by mast cells has not been clearly delineated. However, recent studies using mast cell progenitors, CD34þ cells, isolated from either cord blood or peripheral blood suggest that cytokines could influence protease expression. Culture of progenitors with the mast cell growth factor, stem cell factor (SCF), and alternative cytokines led to the development of mast cells whose tryptase content was fairly constant but where chymase levels could be detected in practically all cells but where the content was variable. The suggestion was made that MCT cells serve as precursors for MCTC cells and that exposure to appropriate stimuli can drive these cells to an MCTC phenotype. However, this concept is not supported by a more recent study in which MCT mast cells isolated from human lung were exposed to SCF and alternative cytokines, as phenotypic conversion was not observed. Data from this later study suggest parallel rather than sequential development of MCT and MCTC cells. Once synthesized, mast cell proteases are packaged within the secretory granules associated with heparin. Heparin is a heavily sulfated proteoglycan and the anionic nature serves to complex and to stabilize the proteases. Unusually, both chymase and tryptase are packaged within the granules as fully active forms and not as proforms. The mechanism of this process is unclear but the possibility that dipeptidyl peptidase 1 (DPP1), which liberates dipeptides from substrates which in turn activate proteases, might be involved has been suggested by studies utilizing DPPl-null mice. Mast cells are thus primed to liberate chymase and tryptase in active forms upon appropriate activation. Following mast cell activation by allergens and other stimuli, degranulation takes place and the granule contents are disgorged extracellularly. A wide array of granule-associated autacoids, such as histamine, is released along with proteases and heparin. Heparin appears to play a crucial role in ensuring that the active tetrameric form of tryptase is maintained. In the absence of heparin, the tetramer dissociates into monomers that are largely thought of as being relatively inactive. The tetrameric form of tryptase shows optimal catalytic activity with restricted substrate specificity governed by the size constraints of the central pore, but recent data suggest that the monomers are far from inert and may be

Tryptase cleaves polypeptides on the C-side of arginine and lysine residues whereas chymase acts on the C-side of phenylalanine, tyrosine, and tryptophan. The biological effects of these proteases are a consequence of proteolysis and can be due to direct effects on substrates or those that are mediated indirectly following activation of PARs. Given the widespread distribution of PARs within the lung it is probable that PAR-mediated effects are more important and far-reaching. Potential processes that mast cell proteases may mediate in the lung are (1) bronchoconstriction, (2) inflammation, (3) remodeling, and (4) neuronal effects. There appears to be some discordance in the literature as to whether mast cell proteases cause direct bronchoconstriction. Certainly, differences appear to exist among species and among the type of airway studied. Reports exist that human bronchi constrict in response to proteases, in a PAR-mediated fashion, but it is probably fair to say that alternative mast cell mediators, histamine and the cysteinyl-leukotrienes, display a greater facility to induce smooth muscle contraction than proteases. For the large part, mast cell proteases appear to display proinflammatory effects. Effects on the vasculature include vasodilation and increases in permeability, these effects being PAR mediated. In addition, proteases have been shown to be chemotactic for inflammatory cells, promoting cell recruitment and, additionally, a whole host of airway cells have been reported to be activated by proteases to release a variety of proinflammatory autacoids. That said, conflicting data have shown anti-inflammatory effects of PAR activation in which agonists of PARs have been shown to attenuate inflammation in various animal models. The mechanism of this effect has not been delineated but the suggestion exists that the cytoprotective and anti-inflammatory prostanoid prostaglandin E2 (PGE2) may be involved. The release of proteases following mast cell activation can lead to changes in the extracellular matrix. Tryptase, for example, has been shown to hydrolyze fibronectin and collagen, if presented in an appropriately denatured conformation. Alternatively, the suggestion has been made that the PARdependent release of matrix metalloproteases from airway cells may play a more prominent role in matrix protein degradation. PAR-mediated mitogenesis of cells, including airway smooth muscle cells, has also been reported. Collectively, these events are

CHYMASE AND TRYPTASE 465

likely to be important in mediating airway remodeling. Proteases may also be involved in modulating neuronal pathways. PARs have been identified on sensory nerve terminal endings and receptor activation is associated with the release of neuropeptides such as substance P and calcitonin gene-related peptide (CGRP), peptides that have been implicated in the development of neurogenic inflammation. That mast cells are known to be located close to nerve endings provides support for such a mechanism. Additionally, certain neuropeptides, such as vasoactive intestinal peptide (VIP), are substrates for tryptase. VIP is a bronchorelaxant and tryptase inactivates the neuropeptide suggesting a greater tendency for airway smooth muscle to constrict as a consequence of this action. Thus, proteases may play an important role in modulating levels of neuropeptides in the lung.

Receptors Four PARs have been identified: PAR1, PAR2, PAR3, and PAR4. All are 7-transmembrane, G-proteincoupled receptors. Tryptase acts at PAR2 whereas chymase acts at PAR1. The mechanism of PAR activation is intriguing. PARs express an extended extracellular N-terminus and, following exposure to a relevant protease, the N-terminus is hydrolyzed and truncated exposing a peptide sequence (SLIGKV for PAR2 and SFLLRN for PAR1) that is still tethered to the receptor and that, autointeractively, engages the second extracellular domain of the receptor. This induces a conformational change in the receptor and activation results.

Mast Cell Proteases in Respiratory Diseases The lung mast cell has long been recognized as central to the mediation of asthma. Mast cell numbers increase in asthma such that the potential for heightened generation of mediators, such as proteases, in response to allergens, exists. This, allied to an increase in the expression of PARs in asthma, suggests exaggerated responses to proteases in the pathophysiological context. An involvement of mast cell proteases in alternative respiratory diseases, however, is less clearly defined. For example, chronic obstructive pulmonary disease (COPD) is a disease characterized by neutrophil involvement, and the extent to which mast cells contribute is uncertain. However, since mast cells subserve a sentinel function within the lung, being found in high numbers at the mucosal surface, they are anatomically predisposed to react to

any airborne triggers. This suggests that mast cells could potentially be involved in a wide variety of respiratory disorders and, as a corollary, proteases may be important too. In asthma, the consequences of mast cell protease activity are likely to be largely proinflammatory (Table 1). For example, proteases are known to cause vasodilation and increase vascular permeability as well as being chemotactic for inflammatory cells, such as eosinophils. An important role for proteases in allergic inflammation is supported by studies using PAR2-deficient mice that demonstrated attenuated inflammatory responses in a model of allergy. An additional effect of proteases in asthma is likely to involve bronchoconstriction but how important this contribution might be is uncertain. Proteases are also known to modulate levels of neuropeptides in the lung and this activity is likely to influence the severity of both bronchoconstrictor and inflammatory events in the lung. Over the longer term, mast cell proteases may be involved in airway remodeling since proteases are known to stimulate the proliferation of a variety of pulmonary cells and are also known to cause degradation of the extracellular matrix. Because proteases have the potential to mediate a very wide-ranging number of effects, there has been significant interest in developing inhibitors of proteases for the treatment of asthma. This is especially true for tryptase and a recent study has shown that the tryptase inhibitor APC 366 partly attenuates allergen-induced late-phase airway obstruction (assessed by monitoring forced expiratory volume in 1 s) in asthma but has no effect on the early phase. Whether drugs that inhibit tryptase will show any benefit in the treatment of asthma remains to be seen. However, it might be argued that strategies aimed at targeting the mast cell directly, since this would prevent the release of all mast cell mediators, might constitute a more effective approach to treat asthma. Table 1 Main effects that the mast cell proteases, tryptase and chymase, can mediate in the lung Process

Protease-mediated effect

Inflammation

Increased vascular permeability Vasodilation Chemotaxis Airway smooth muscle contraction Increased release of neuropeptides promoting neurogenic inflammation Inactivation of bronchorelaxant neuropeptides Degradation of extracellular matrix either directly or by stimulating metalloprotease release Stimulation of pulmonary cell proliferation

Bronchoconstriction Neuromodulation

Remodeling

466 CILIA AND MUCOCILIARY CLEARANCE See also: Allergy: Allergic Reactions. Asthma: Overview; Extrinsic/Intrinsic. Cysteine Proteases, Cathepsins. Extracellular Matrix: Degradation by Proteases. Kinins and Neuropeptides: Neuropeptides and Neurotransmission; Tachykinins; Vasoactive Intestinal Peptide. Leukocytes: Mast Cells and Basophils. Lipid Mediators: Overview; Leukotrienes; Prostanoids. Serine Proteinases. Smooth Muscle Cells: Airway. Systemic Disease: Sarcoidosis.

Further Reading Cairns JA (2005) Inhibitors of mast cell tryptase beta as therapeutics for the treatment of asthma and inflammatory disorders. Pulmonary Pharmacology & Therapeutics 18: 55–66. Caughey GH (2001) New developments in the genetics and activation of mast cell proteases. Molecular Immunology 38: 1353– 1357.

Cocks TM and Moffatt JD (2001) Protease-activated receptor-2 (PAR2) in the airways. Pulmonary Pharmacology & Therapeutics 14: 183–191. Lan RS, Stewart GA, and Henry PJ (2002) Role of proteaseactivated receptors in airway function: a target for therapeutic intervention? Pharmacology & Therapeutics 95: 239– 257. Levi-Schaffer F and Piliponsky AM (2003) Tryptase, a novel link between allergic inflammation and fibrosis. Trends in Immunology 24: 158–161. Reed CE and Kita H (2004) The role of protease activation of inflammation in allergic respiratory diseases. Journal of Allergy and Clinical Immunology 114: 997–1008. Schmidlin F and Bunnett NW (2001) Protease-activated receptors: how proteases signal to cells. Current Opinion in Pharmacology 1: 575–582. Sommerhoff CP, Bode W, Matschiner G, Bergner A, and Fritz H (2000) The human mast cell tryptase tetramer: a fascinating riddle solved by structure. Biochimica et Biophysica Acta 1477: 75–89.

CILIA AND MUCOCILIARY CLEARANCE L E Ostrowski and W D Bennett, University of North Carolina, Chapel Hill, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Mucociliary clearance is an innate defense mechanism that protects the pulmonary system from the harmful consequences of inhaled agents, including those of biological, chemical, and physical nature. Ciliated cells, which line the surface epithelium of the airways, provide the force necessary for mucociliary clearance by the coordinated beating of their cilia. These highly specialized cells are therefore critical to the health and function of the pulmonary system. In this article, the basic biology of ciliated cells and the structure of the cilium are described. Their function and interaction with other components of the mucociliary system and the importance of mucociliary clearance to respiratory disease are discussed.

Introduction Mucociliary clearance is the process whereby the pulmonary system is cleared of inhaled foreign material by the continuous beating of ciliated cells to transport mucus and any entrapped material to the pharynx, where it is subsequently removed by swallowing. This well-coordinated system consists of mucous and serous cells in the submucosal glands and secretory goblet cells in the airway epithelium that secrete water, mucus, and other proteins to produce a fluid layer on the airway surface and also the ciliated cells that propel the fluid out of the lung toward the mouth (Figure 1). The fluid layer

consists of a low-viscosity sol phase near the cells’ apical surface and a more viscous gel (or mucus) phase near the tips of the cilia. Whether the two phases are entirely distinct or a viscosity gradient exists through the depth of the entire airway fluid is not clear. The efficiency of the mucociliary clearance (MCC) system at removing airway secretions and associated trapped substances depends on three primary factors: the beat frequency and coordination of the cilia, the quantity and rheology of airway secretions derived from surface goblet cells and submucosal glands, and the periciliary fluid depth that is modulated by ion transport of the airway epithelium. In health, this system is very effective at clearing mucus and associated bacteria and toxins from the lung. However, in a variety of airway diseases (e.g., chronic bronchitis, asthma, and cystic fibrosis (CF)) this apparatus becomes dysfunctional, leading to further exacerbation of airway inflammation and obstruction. Patients with primary ciliary dyskinesia (PCD), a genetic disease characterized by abnormal ciliary ultrastructure and function, have impaired MCC that results in chronic lung, sinus, and middle ear disease, demonstrating the critical importance of properly functioning cilia to the health of the pulmonary system.

Ciliated Cells and Cilia Ciliated cells, which provide the force necessary for MCC, are found throughout the trachea and large

CILIA AND MUCOCILIARY CLEARANCE 467

Ion channels

Mucin granules

Cilia

Mucus

Periciliary liquid Cl−

Na+

Ciliated cell

Goblet cell

Basal cell Basement membrane Figure 1 Schematic of airway epithelium illustrating the two major cell types, ciliated and goblet. The epithelium is covered by a fluid layer whose depth is regulated by ion transport across the cell membranes and within which mucins are secreted to trap foreign matter for mucociliary transport out of the lung.

airways and are present distally as far as the terminal bronchioles. Figure 2 shows a cross section of a normal human large airway illustrating the heavily ciliated surface. The percentage of ciliated cells in the human airway epithelium decreases from approximately 50% in the tracheal epithelium to o20% in the bronchiolar region. Ciliated cells are also present in areas of the upper respiratory tract, including the nasal sinuses and nasopharynx. On the apical surface of each ciliated cell is an organized array of up to 200 individual cilia; the adult lung has been estimated to contain approximately 3
1012 total cilia. A typical cilium from an adult tracheal cell is approximately 7 mm long and 0.1 mm in diameter. Ciliated cells in the more peripheral airways have fewer cilia, and the cilia are shorter as well. Each cilium demonstrates the highly conserved axonemal structure, consisting of a central pair of microtubules surrounded by a ring of nine microtubule doublets (Figure 3). Each of the outer doublets consists of a complete tubule (the A tubule, containing 13 tubulin subunits) and a partial tubule (the B tubule, containing 11 subunits). Attached to each complete tubule are two rows of large multiprotein complexes, the inner and outer dynein arms. Each dynein arm contains at least one catalytic dynein heavy chain that converts the energy of ATP hydrolysis into the bending motion of the ciliary axoneme by attaching to the adjacent microtubule and undergoing a conformational change, causing the microtubules to move relative to each other. The detailed structure of the human cilium is only beginning to be unraveled, but the realization that defects in ciliary function are

Figure 2 Ciliated cells of the human airway epithelia. Photomicrograph of a large airway showing the epithelial surface covered by numerous cilia extending from the tall, columnar ciliated cells. The paraffin-embedded large airway (bronchus) was stained with Richardson’s to visualize the cilia.

responsible for a much wider spectrum of disease than previously thought has stimulated studies on the assembly, structure, and function of this fascinating organelle.

468 CILIA AND MUCOCILIARY CLEARANCE

Radial spoke Radial spoke Inner dynein arm Central pair microtubule

Outer dynein arm

A microtubule B microtubule

Outer dynein arm

Inner dynein arm

Nexin link Figure 3 (Left) Schematic of a prototypical axoneme illustrating some of the structural features that are highly conserved among motile 9 þ 2 axonemes. (Right) Transmission electron micrograph showing a normal human cilium in cross section. Many of the conserved structures are easily visible.

Regulation of Ciliary Beat Frequency In the airway, cilia typically beat with a frequency of approximately 10–15 Hz, although reported values vary slightly depending on the conditions used. Analysis of ciliary waveform from nasal samples by digital high-speed video has revealed a mostly planar pattern, with the cilium being fully extended in the forward power stroke. During the recovery stroke, the cilium curls backwards in the same plane. Figure 4 shows an image from a video recording of an actively beating ciliated cell. During the recovery stroke, the cilium curls backwards in the same plane. The regulation of ciliary beat frequency (CBF) has been an area of research interest for many years, and many different agents have been reported to stimulate or inhibit CBF. For example, increases in the intracellular concentration of cAMP consistently stimulate CBF in airway epithelial cells. This increase is presumed to be due to the phosphorylation of axonemal proteins by the action of cAMP-dependent protein kinase (PKA). PKA has been demonstrated to be an integral component of the ciliary axoneme; however, the regulatory protein responsible for the increased CBF has yet to be identified. Agents that increase intracellular calcium also elevate CBF in mammalian airway cells, although, again, the detailed molecular pathway has not been elucidated. Other regulatory molecules, including cGMP, PKG, protein kinase C, calmodulin, phospholipase C, and nitric oxide, have all been reported to have effects on ciliary beating, although the different experimental systems used have sometimes produced conflicting results. It seems likely that the regulation of CBF occurs through the interaction of several pathways, and more research is required to determine the individual contributions of each to the overall regulation of CBF. For effective MCC to occur, ciliary beating must also be coordinated. Cilia in the airway

Figure 4 Image of an actively beating human ciliated cell. Ciliated cells isolated from human bronchial tissue were visualized using differential interference contrast optics and high-speed video images were recorded.

beat in a metachronal wave, and, again, the mechanisms that initiate and maintain the proper orientation and coordination between the ciliated cells are unknown.

Measurement of Mucociliary Clearance MCC rates can be measured in vivo by assuming that a nonpermeating, inhaled marker depositing on the airway surface moves out of the lung at the same rate as the airway secretions in which it is immersed. The most common technique is to use inhaled, radiolabeled particles, aqueous or dry, that upon deposition in the lung can be followed by a gamma camera or scintillation detectors to determine their rate of egress from the lung. Using these techniques, scientists have been able to study MCC in healthy subjects and in patients with airways disease and to assess

CILIA AND MUCOCILIARY CLEARANCE 469

the effects of various therapies on MCC. The rate of tracheal mucus velocity in healthy controls has been estimated to be 4 or 5 mm min  1 using this technique, with slower rates reported in the more distal airways. Increases in CBF have been shown to translate into increased rates of clearance, and it has been reported that a 16% increase in CBF resulted in a 450% increase in tracheal mucus velocity. Thus, agents that increase CBF increase MCC and can provide benefits to patients with a range of airway diseases.

Mucociliary Clearance in Pulmonary Disease A number of short-acting b-adrenergic agonists (salbutamol, isoproterenol, and albuterol) intended to ease bronchoconstriction in patients with airways disease have also been shown to enhance the rate of MCC from the lung. Figure 5 illustrates this effect in a healthy subject. Figures 5(a) and 5(c) show the

gamma camera images of a healthy person’s lungs immediately after inhalation of such radiolabeled particles. Figure 5(b) shows the gamma camera image 30 min after depositing the radiolabeled particles and no intervention (i.e., a control measure of MCC), illustrating a mild loss of image intensity during the 30-min period (i.e., in comparison to Figure 5(a)). On the other hand, Figure 5(d) illustrates a 30-min posttreatment gamma camera image in the same individual after inhaled albuterol treatment, showing more rapid movement of radiolabeled particles out of the lung compared to the control condition (compare Figures 5(b) and 5(d)). This enhancement in MCC is likely due to increases in CBF induced by the b-adrenergic agonist and consequent increases in intracellular cAMP discussed previously, although it is clear that these agents have pleiotropic effects. Table 1 provides a list of other pharmacological agents that can stimulate or depress in vivo MCC. Although the mucociliary apparatus in the normal lung responds quite well to pharmacological stimulation, it is less responsive in patients

Figure 5 Stimulation of mucociliary clearance by a b-adrenergic in a healthy subject. Top panels illustrate clearance associated with no intervention. (a) A gamma camera image of the lungs immediately after inhalation of radiolabeled particles (i.e., particle deposition image). (b) Thirty-minute postdeposition image. Bottom panels illustrate clearance in the same subject associated with inhaled albuterol treatment. (c) Particle deposition image that was immediately followed by inhaled albuterol treatment. (d) Thirty-minute postdeposition (and treatment) image showing increased clearance of the radiolabeled particles.

470 CILIA AND MUCOCILIARY CLEARANCE Table 1 Factors affecting mucociliary clearance

Conclusions

Factor

MCC is a remarkably effective mechanism for maintaining pulmonary health. Impaired MCC is a common feature of many airway diseases, and agents that improve MCC are beneficial in the treatment of these diseases. Many of these agents have a direct effect on ciliated cells and increase CBF. However, further research is needed to understand the complex interactions between cilia, airway surface liquid, mucus, and MCC so that more effective treatments may be developed.

Increase

Decrease

Pharmacological Adrenergic agonists Cholinergic agonists Histamine ATP Amiloride Hyperosmolar solutions

Anesthetics Atropine Antigen

Pathological

Cystic fibrosis Primary ciliary dyskinesia Chronic bronchitis Asthma Respiratory infections

Environmental

Pseudohypoaldosteronism Chronic cough

Acute exposure to Sulfur dioxide Cigarette smoke Ozone

Chronic exposure to Sulfur dioxide Cigarette smoke Ozone NO2

with airways disease. This may be due in part to the loss of ciliated epithelium associated with chronic inflammation (e.g., patients with chronic obstructive airway disease who have smoked cigarettes most of their lives). Other pathological conditions for which MCC is known to be depressed are also listed in Table 1. In support of the need to hydrate mucus for effective MCC, patients with pseudohypoaldosteronism, a rare inherited disease characterized by poor Na þ absorption from the airway surface, have MCC rates three or four times higher than normal. The increased rate of clearance in these patients may be due to enhanced ciliary beating in response to an increased low-viscosity load on the cilia. In contrast, the airway surfaces of CF patients are poorly hydrated, leading to decreased MCC and promoting the formation of adherent mucus plaques on CF airway surfaces, ultimately causing airflow obstruction and promoting chronic infection. Finally, a number of environmental pollutants can influence the rate of MCC, either acutely or chronically, and are also listed in Table 1. For example, although sulfur dioxide may acutely stimulate MCC, chronic exposure to it causes a depression in MCC. This is likely due to a remodeling of the airway epithelium (i.e., a loss of ciliated cells and increase in mucus-producing cells) similar to that seen with chronic cigarette exposure.

Acknowledgments The authors thank R Wonsetler, Dr P Spears, Dr M Chua, and the Hooker Microscopy Facility for preparing the ciliated cell video; K Burns and the histology core for excellent technical services; L Brown for assistance in preparing the figures; Dr S H Randell and the Cell Culture Facility for samples; and the individuals who donated tissue specimens. See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Cystic Fibrosis: Overview. Environmental Pollutants: Overview. Mucins. Mucus. Primary Ciliary Dyskinesia.

Further Reading Bennett WD (2002) Effect of b-adrenergic agonists on mucociliary clearance. Journal of Allergy and Clinical Immunology 110: S291–S297. Bennett WD, Noone PG, Knowles MR, and RC Boucher (2001) Regulation of mucociliary clearance by purinergic receptors. In: Salathe M, et al. (eds.) Cilia and Mucus: From Development to Respiratory Defense. New York: Dekker. Brody SL (2004) Genetic regulation of cilia assembly and the relationship to human disease. American Journal of Respiratory Cell and Molecular Biology 30(4): 435–437. Knowles MR and Boucher RC (2002) Mucus clearance as a primary innate defense mechanism for mammalian airways. Journal of Clinical Investigation 109(5): 571–577. Noone PG, Leigh MW, Sannuti A, et al. (2004) Primary ciliary dyskinesia: diagnostic and phenotypic features. American Journal of Respiratory and Critical Care Medicine 169(4): 459–467. Ostrowski LE, Blackburn K, Radde KM, et al. (2002) A proteomic analysis of human cilia: identification of novel components. Molecular & Cellular Proteomics 1(6): 451–465. Robinson M and Bye PT (2002) Mucociliary clearance in cystic fibrosis. Pediatric Pulmonology 33(4): 293–306. Snell WJ, Pan J, and Wang Q (2004) Cilia and flagella revealed: from flagellar assembly in chlamydomonas to human obesity disorders. Cell 117(6): 693–697. Wanner A, Salathe M, and O’Riordan TG (1996) Mucociliary clearance in the airways (state of the art). American Journal of Respiratory and Critical Care Medicine 154: 1868–1902.

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CLARA CELLS B R Stripp and S D Reynolds, University of Pittsburgh, Pittsburgh, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Clara cells are an abundant population of nonciliated secretory cells present within the bronchiolar epithelium of the mammalian lung. Their differentiation during lung development coincides with changes occurring within airways in preparation for adaptation to an air-breathing environment. Clara cells fulfill three major functions in the adult lung. First, they represent the major secretory cell of the bronchiolar epithelium where they contribute nonmucinous secretory proteins to the extracellular lining fluid. Second, Clara cells represent the principal site of xenobiotic metabolism within the lung. Finally, Clara cells represent an abundant progenitor cell pool for maintenance of the epithelium in the steady-state and for regeneration of the injured bronchiolar epithelium. Altered Clara cell function is likely to be a significant factor contributing to declining lung function in airway disease. Remodeling of the conducting airway epithelium in the setting of chronic lung disease involves changes in Clara cell function that have been demonstrated through measurement of reduced production of the most abundant secretory product of Clara cells, CCSP. Roles for Clara cells and their secretions in airway homeostasis and responses to environmental stress have been demonstrated in studies involving genetically modified mouse models. Clara cells have been shown to play a central role in cytokine-mediated initiation of airway remodeling, and animal models of reduced Clara cell secretion have been shown to phenocopy pathophysiological outcomes observed in patients with chronic lung disease. New insights into functions for Clara cells in airway homeostasis point to central roles for this cell type in the regulation of airway inflammation and protection from environmental agents.

Introduction Conducting airways of the lung function as the primary interface between the environment and the vast mucosal surface of the alveolus and as such are a critical component of homeostatic mechanisms aimed at maintenance of a relatively clean and sterile intrapulmonary compartment. Functions of the airway include establishment of a physical barrier to environmental agents, filtering and conditioning of inhaled air, metabolism of reactive chemicals, as well as clearance of deposited material and cellular debris. Although these activities are shared by the airway as a whole, regional specialization has evolved to account for differences in deposition of dusts and particulates, soluble and reactive oxidant gases and microorganisms, and to allow for rapid adaptation to changes in environmental conditions.

Structurally and functionally distinct regions within the conducting airway epithelium are recognized as epithelial–mesenchymal trophic units. Each of these units is composed of regionally specific ‘epithelial’ (surface epithelium and submucosal glands), ‘interstitial’ (basement membrane zone, fibroblasts, smooth muscle, cartilage and vasculature), ‘neural’ (afferent and efferent nerves), and ‘immunological’ (static and migratory immune system cells) tissues. Epithelial cell differentiation is likely to be influenced by tissue interactions within the trophic unit as well as by interactions between adjacent trophic units. Therefore, the epithelium is heterogeneous in the steady state and exhibits a degree of functional plasticity in response to acute or chronic perturbation. As indicated by the title of this article, the following discussion will focus on the principal secretory cell of the bronchiolar epithelium, the Clara cell, and its role in distal airway homeostasis. Similarities and distinctions between the Clara cell and the dominant secretory cell subsets of more proximal airways will be discussed and the terminology describing the classical bronchiolar Clara cell and Clara-like cells of proximal airways will be refined.

Secretory Cells of Conducting Airways The structural and functional distinctions that define regions within the conducting airways are reflected in the phenotypic properties of the dominant secretory cell type within each region. Accordingly, three categories of secretory cells were originally described in terms of their ultrastructural characteristics: serous cells, neuroendocrine cells (also termed dense-core granulated cells, endocrine cells, or Kultchitshy cells), and nonciliated secretory cells. Functional differences were inferred from these morphological observations and assessed in vitro using partially purified cell populations. This body of work refined suspected distinctions among the three subpopulations of secretory cells and provided support for subdivision of the nonciliated secretory cell population into bronchiolar Clara cells and tracheobronchial Clara-like and mucus cells. Serous Cells

Serous cells are characterized by an electron-dense cytoplasm, discrete electron-dense granules that are 600 nm in diameter, and an abundance of both rough and smooth endoplasmic reticulum. These

472 CLARA CELLS

cells secrete neutral mucin and have been consistently described in submucosal glands, small bronchi, and bronchioles of the human airway and rat, and variably in cat and hamster. Although mice lack morphologically distinct serous cells, expression of the serous cell marker SPURT (also termed PLuNK) in tracheobronchial secretory cells suggests functional, if not phenotypic, redundancy between the human serous cell and mouse proximal secretory cell types. Neuroendocrine Cells

Neuroendoreine cells are an extremely rare cell type distinguished by a large number of small spherical dense core granules each of which is surrounded by an electron-lucent halo. These cells are found singly or in clusters (termed neuroepithelial bodies), are often innervated, secrete neuropeptides, and have been implicated as oxygen sensors and as a niche for maintenance of bronchiolar tissue-specific stem cells in the mouse. Hyperplasia of neuroendocrine cells and neuroepithelial bodies is a common pathological finding of neonatal and chronic lung disease and is mimicked following acute injury of mouse airways. Bronchiolar Clara Cells

Secretory cells of the bronchiolar epithelium are referred to as Clara cells, named after Max Clara who provided a detailed description of their morphological properties in 1937. In the light of microscopic study, the Clara cell was portrayed as a cuboidal, nonciliated cell, of the human and rabbit terminal bronchiole that contained a basally situated nucleus, an apical dome that extended a variable distance into the airway lumen, and discrete, oval, densely staining granules. Subsequent ultrastructural analysis detected electrondense, 500–600 nm diameter granules in humans. More detailed ultrastructural analysis of nonciliated bronchiolar epithelial cells of the mouse and rat lung detected abundant smooth endoplasmic reticulum and secretory granules located within the apical domain of the cell. Mouse Clara cells were further distinguished by their large round mitochondria that contained few poorly defined cristae and a relatively electron dense mitochondrial matrix. Comprehensive analysis of nonciliated bronchiolar cells in 15 mammals, including man, identified the presence of smooth endoplasmic reticulum (except nonhuman primates) and ovoid secretory granules (except rhesus monkey and cat) as unifying characteristics of Clara cells from multiple species. Although morphometric analysis demonstrated heterogeneity both within and among species, the organellar composition of nonciliated bronchiolar cells clearly distinguished these cells from mucus and serous cells of the more proximal

conducting airway and from neuroendocrine cells. These studies indicated that the term Clara cell was a useful collective term to describe nonciliated bronchiolar secretory cells. Tracheobronchial Clara-Like Cells

Morphological analysis of the tracheal epithelium from multiple species demonstrated that 56–90% of nonciliated cells exhibited ultrastructural characteristics of Clara cells. Subsequent use of the molecular marker, Clara cell secretory protein (CCSP), to define secretory cell populations at all airway levels led to the perception that nonciliated secretory cells of the tracheobronchial and bronchiolar epithelium were synonymous. However, the finding that CCSPexpressing cells were a subset of nonciliated/nonmucus cells in the human tracheobronchial epithelium and subsequent identification of secreted proteins unique to tracheobronchial nonciliated/nonmucus cells of both humans and mice suggested that structurally similar cells within the two compartments were functionally divergent. These molecular distinctions in combination with differential responsiveness of tracheobronchial and bronchiolar compartments to various stimuli, early segregation of the tracheobronchial and bronchiolar lineages during lung development and identification of separate hierarchies for maintenance of these two populations in the mature epithelium, led to use of the term ‘Clara-like’ to describe nonciliated/nonmucus cells of the tracheobronchial epithelium. As summarized by Plopper and colleagues, the original description of the Clara cell defined a morphological entity (nonciliated cell) in a precise location (the terminal bronchiole) of two species (human and rabbit). The term Clara-like cell was developed to highlight the similarities between the bronchiolar and tracheobronchial nonciliated/nonsecretory cell and the potential for distinct functions of these populations in airway homeostasis. Mucus Cells

Mucus cells are found primarily in the proximal airways of healthy humans and are characterized by an electron-dense cytoplasm that contains a heterogeneous population of electron-lucent confluent granules that are 800 nm in diameter, numerous Golgi complexes, and granular endoplasmic reticulum (rough endoplasmic reticulum). These cells secrete high-molecular-weight glycoprotein that is acidic in nature due to sialic acid or sulfate groups. The number of mucus cells increases with acute lung infection or injury and these cells become hyperplastic and are distributed to more distal compartments of

CLARA CELLS 473

the chronically diseased human lung. Comparative analysis of secretory cell populations in six mammalian species demonstrated that representation of mucus cells was highly variable among the species studied (0–20%). Few mucus cells were found in airways of rodents maintained under specific pathogen-free conditions but were a prominent component of the acute response to irritants and environmental pollutants in rats. Interestingly, mouse proximal airway secretory cells are refractory to such stimuli. However, metaplastic transition to a mucus-producing phenotype was associated with overexpression of Th2 cytokines. This analysis suggested that differential responsiveness of tracheobronchial cells to immune system factors could account for the high degree of variation in mucus cell numbers and may represent a critical adaptive response of proximal airway secretory cells to changing environmental conditions. Analysis of allergic inflammation in mice also supported the idea that mucus cells were derived from a nonmucus producing cell and that expression of high-molecular-weight glycoconjugate was a gainof-function phenotype. The potential for phenotypic plasticity identified the need for molecular markers that could distinguish functionally distinct secretory cell types at the light microscopic level.

Molecular Markers for Clara and Clara-Like Cells Products of airway secretory cells were initially identified through immunization of rabbits with lavagable proteins from adult rat and correlation of immunoreactive antigens with cellular morphology in bronchioles and bronchi. Subsequent metabolic labeling of cell-associated and released proteins from partially purified rabbit Clara cells identified proteins of 6, 48, and 180 kDa as major products of this cell type and demonstrated that the 6 kDa protein represented 30% of total released radioactivity. A similar preparation of the low-molecular-weight protein was used to generate an affinity-purified antibody preparation and immunogold methods demonstrated that this antigen localized to secretory granules within rat Clara cells. These methods were also applied to identification of markers for human Clara cells and resulted in discovery of a homodimeric 10 kDa protein with a pI of 4.8 that was antigenically similar in human, dog, and rat. This protein, which is termed CCSP, CC10, CC16, uteroglobin, or SCGB1A1, has since been identified as the human ortholog of rabbit uteroglobin and is the founding member of the secretoglobin family of low-molecular-weight, acidic, secretory proteins.

Antibodies specific for CCSP have been used to determine the number and distribution of CCSPimmunoreactive (CCSP-IR) cells in humans and commonly used animal models. Studies in humans, rats, and mice demonstrated that the majority of Clara cells expressed CCSP and that these cells were negative for periodic acid Schiff (PAS)-reactive material in normal individuals. These data suggested that CCSP was a faithful marker for human bronchiolar Clara cells and similar immunological profiles of distal conducting airway secretory cells from multiple species indicated that expression of CCSP was a common molecular characteristic of secretory cells of this type. In contrast, the frequency of CCSPIR cells in the tracheobronchial epithelium of human, rat, and mouse airways was less than that of Claralike cells. Identification of subsets of tracheobronchial Clara-like cells provided further support for the notion that distinct populations of nonciliated secretory cells populate the proximal and distal airways and identified a need to further delineate these distinctions at the molecular level.

Properties of Clara Cells in the Normal Adult Lung Early studies describing the ultrastructural properties of Clara cells provided a number of clues into their potential roles in airway biology. Abundant mitochondria and smooth endoplasmic reticulum suggested that they were a metabolically active cell, and the presence of membrane bound granules suggested a secretory function. Since then, a combination of in vivo and in vitro models has been employed to further explore these and other functions of Clara cells. Secretion

In rodents, secretions from Clara cells account for approximately 10% of the protein content of total airway surface fluid recovered by bronchoalveolar lavage and represent a much larger percentage of protein within the surface fluid of conducting airways. This changes both qualitatively and quantitatively the setting of lung injury not only due, in part, to altered lung permeability resulting in the infiltration of serum proteins, but also as a result of injuryrelated changes in the rate at which the secretory proteins synthesized and secreted. Even though it is clear that Clara cells constitutively secrete low-molecular-weight proteins into the lumen of airways, it is less clear whether proteins loaded into secretory granules are part, of this constitutive secretory pathway or represent a distinct pool whose secretion is regulated by extracellular cues. Evidence supporting

474 CLARA CELLS

a regulated exocytic process comes from studies investigating ultrastructural changes occurring to Clara cells following either infection with pneumotropic viruses or injury resulting from exposure to oxidant gases. In each case, the ensuing perturbations to airways result in rapid loss of Clara cell secretory granules suggesting the rapid exocytosis of stored pools of secretory proteins. Clara cells secrete a number of nonmucinous secretory proteins including surfactant proteins A and D, proteolytic enzymes, antimicrobial peptides, secretoglobins, and a variety of growth factors, chemokines and cytokines. CCSP is the most abundant secreted product of the Clara cell, accounting for up to 40% of the secreted protein product. Clara cells of CCSP-deficient mice (CCSP / ) exhibit ultrastructural changes including loss of secretory granules and a dramatic reduction in the abundance of rough endoplasmic reticulum, that are associated with defects in airway secretion. The phenotype of CCSP / mice has been extensively investigated using a variety of challenge models and these studies have associated CCSP deficiency with alterations in the pulmonary response to inflammatory stimuli, changes in mucosal immunity, and elevated susceptibility to inhaled oxidant pollutants. The finding that the response of CCSP / mice following challenge with environmentally relevant agents phenocopies aspects of the respiratory complications seen in human patients suffering from chronic lung disease highlights a critical role for Clara cell secretion in lung homeostasis and disease. Metabolism

Both the metabolic capacity of Clara cells and their location within the bronchiolar epithelium of distal conducting airways are central to their role in protection from the adverse effects of environmental lung pollutants. As is the case with hepatocytes, which are responsible for detoxification of lipophilic xenobiotic compounds including furans and aromatic hydrocarbons in the liver, Clara cells are rich in smooth endoplasmic reticulum (sER) and mitochondria and are the principal site of expression for cytochrome P450 monooxygenase family. Such enzymes are referred to as phase I metabolizing enzymes and localize to the sER fraction of intracellular membranes. Phase I metabolism of lipophilic compounds results in the generation of highly reactive intermediates that undergo secondary reactions catalyzed by the phase II class of xenobiotic metabolizing enzymes. Phase II reactions within Clara cells are principally catalyzed by enzymes belonging to the family of glutathione transferases, resulting in the

generation of water-soluble glutathione adducts that can be efficiently effluxed from the cell through the action of plasma-membrane-associated glutathione transporters. Clara cells are well situated to effectively clear lipophilic xenobiotic compounds delivered to the lung either through inhalation or systemically via the extensive parenchymal capillary network. The physico-chemical properties of inhaled lipid-soluble xenobiotic compounds result in deposition deep within airways of the lung. Generally, these compounds are present within inspired air at relatively low concentrations and can be effectively metabolized and cleared without overt lung injury. In contrast, systemically delivered agents, either xenobiotic pollutants or drugs, can be delivered to the lung at sufficiently high concentrations to overcome the capacity of the phase II metabolizing system leading to Clara cell injury from excessive production of bioactivated products of phase I metabolism. Clara cell injury resulting from exposure to high doses of lipophilic compounds has been extensively characterized in the laboratory of Plopper and colleagues and involves dilation of the sER, formation of large cytoplasmic vacuoles and the generation of cytoplasmic blebs at the apical membrane. Differences are observed in the regio-selectivity of this injury response, with Clara cells of the bronchiolar epithelium exhibiting the greatest susceptibility. Significant species differences in susceptibility are also observed, the principal mechanism for which is variability in the substrate specificity and expression levels of phase I metabolizing enzymes. Mechanisms of repair following Clara cell injury by lipophilic compounds will be discussed in more detail below. Proliferation

The bronchiolar epithelium is relatively quiescent in the steady state but has the capacity for rapid and complete regeneration following acute injury. Careful pulse/chase studies coupled with autoradiography and ultrastructural analysis demonstrated that normally quiescent Clara cells responded to oxidant-induced ciliated cell injury through transition to a cell type referred to as the type A Clara cell, which was distinguished from the Clara cell by virtue of its lack of secretory granules and smooth endoplasmic reticulum. These morphologically distinct cells entered the cell cycle and produced nascent Clara and ciliated cells at a ratio of 2:1. Re-differentiation of this transitional epithelium was associated with the appearance of a type B cell intermediate, distinguished from the type A cell by the presence of granules and from Clara cells by an absence of sER, which most

CLARA CELLS 475

likely represented an intermediate cell type in the reestablishment of the mature Clara cells. Subsequent pulse-labeling studies demonstrated tight correlation between DNA synthesis and expression of CCSP in cells of the steady-state and regenerating bronchiolar epithelium. Thus, mature Clara cells, which fulfill a range of differentiated functions that are critical to aspects of airway homeostasis (detailed above), serve as a reservoir of abundant bipotential progenitors that mediate bronchiolar maintenance and regeneration through proliferation and differentiation.

Bronchiolar Stem Cell Hierarchy Clara cells are part of a stem cell hierarchy that contributes to maintenance and renewal of the bronchiolar epithelium. Stem cell hierarchies have been described that contribute to maintenance of epithelia within a number of different tissues such as the small intestine and colon, skin, cornea, and hematopoietic system. Components of such hierarchies include a tissue-specific stem cell, one or more types of transitamplifying progeny, and terminally differentiated cells. Tissue-specific stem cells are a rare population of cells that are maintained within ‘protective’ (either physically or biologically) microenvironments referred to as niches, and have properties of unlimited capacity for self-renewal, infrequent proliferation in the steady-state, and a relatively undifferentiated phenotype. Tissue-specific stem cells give rise to transit-amplifying progeny, frequently referred to as progenitor cells, the properties of which can vary significantly according to tissue. Among tissues that maintain ‘classical’ stem cell hierarchies, such as the intestine, transit-amplifying cells represent a continuously proliferating population that is maintained in close proximity to the stem cell niche. In such cell hierarchies, transit-amplifying cells have a finite capacity for self-renewal and eventually expend their mitotic potential. As transit-amplifying cells of classical stem cell hierarchies exit the proliferative compartment, they assume differentiated characteristics necessary for epithelial functions. Such differentiated cells have a short life span and as a result the transitamplifying cell compartment is continually activated. The existence of lung tissue-specific stem cells has been revealed using mouse models of airway injury in which proliferative cells have been selectively ablated using either chemical or conditional transgenic approaches. Parenteral administration of high does of lipophilic agents such as naphthalene causes selective injury to Clara cells as a result of xenobiotic metabolism. In contrast with epithelial regeneration following oxidant-mediated injury to ciliated cells, repair following Clara cell depletion originates from

two types of regenerative foci within bronchioles: one defined by virtue of clusters of pulmonary neuroendocrine cells that form neuroepithelial bodies (NEBs) and the other localized to the broncho-alveolar duct junction (BADJ) of terminal bronchioles. Pulse/chase studies demonstrated that a subpopulation of mitotic cells within these microenvironments exhibited characteristics of tissue stem cells, including infrequent proliferation and capacity for multipotent differentiation. The molecular phenotype of the bronchiolar tissue stem cell was revealed through conditional ablation of CCSP-expressing cells in transgenic mice. These experiments demonstrated that the CCSP-expressing component of the NEB and BADJ microenvironments, the variant CCSP-expressing cell (vCE), constituted the bronchiolar tissue-specific stem cell. A role for vCE cells in bronchiolar regeneration as well as secondary functions relating to interactions between the bronchiolar and alveolar epithelium was demonstrated by the finding that irreparable bronchiolar injury resulted in death due to compromised parenchymal function. The combination of Clara cell depletion and ablation approaches demonstrated that regeneration of the bronchiolar epithelium was entirely dependent on CCSP-expressing cells and that all proliferative cells of the bronchiolar stem cell hierarchy, including the stem cell and progenitor cell pools, share the common molecular characteristic of CCSP expression. As such, CCSP-expressing cells represent at least two functionally distinct populations within bronchioles of the mouse airway, each of which is linked as part of a common lineage. The presence of the large, uniformly distributed Clara cell population that fulfills both differentiated and proliferative functions is of considerable practical importance in airways of the lung due to the need for efficient epithelial regeneration in the setting of acute airway injury. Incorporation of both maintenance and repair functions in a single cell type (the Clara cell) distinguishes the bronchiolar epithelium from other epithelia (such as the intestine and epidermis) which rely on distinct populations of cells to fulfill these activities. Furthermore, the ability of the Clara cell to transition between the quiescent and proliferative states distinguished this cell from other differentiated bronchiolar cell subsets that lack this capacity. Recognition of dual roles for Clara cells in bronchiolar homeostasis and regeneration, and distinctions between the functional capacity of Clara cells and progenitor cells of other epithelial hierarchies, has led to use of the term ‘conditionally differentiated’ to describe bronchiolar Clara cells. In contrast, the term ‘terminally differentiated’ has been used to describe differentiated cells, such as ciliated cells, that lack

476 CLARA CELLS

mitotic potential. Mechanisms that regulate maintenance and provisional activation of the dispersed conditionally differentiated Clara cell population are likely to be distinct from those governing the activity of spatially distinct progenitor and differentiated cell pools of the gut and skin. The presence of the conditionally differentiated Clara cell population results in limited participation of the bronchiolar tissuespecific stem cell in routine epithelial maintenance.

Clara cells in Airway Disease Histopathology and Biomarkers

Injury and remodeling of the conducting airway is a characteristic pathological feature of many types of chronic lung disease including asthma, chronic obstructive pulmonary disease (COPD), obliterative bronchiolitis, idiopathic pulmonary fibrosis, and cystic fibrosis. Changes to airways are multicompartmental, with variable contribution made by tracheobronchial and bronchiolar airways, and within these regions affecting both epithelial and mesenchymal components. Epithelial involvement has been demonstrated upon histopathological examination of lung tissue from affected patients. Contributions of the bronchiolar epithelium to the pathophysiology of airway disease are further supported by studies characterizing changes in the abundance of cell type-specific biomarkers such as CCSP within airway lining fluid and serum. Due to its low molecular weight, CCSP penetrates both the epithelial and endothelial barrier of the lung resulting in serum levels that are predictive of changes to the lung. Factors that influence serum levels of such ‘noninvasive’ biomarkers include their rate of synthesis and secretion within the epithelium, integrity of both the epithelial and endothelial barrier, and the glomerular filtration rate which serves to clear and catabolize these proteins. Serum levels of CCSP increase dramatically immediately following acute lung injury due to changes in the permeability of the epithelial/endothelial barrier. However, serum CCSP content decreases in the setting of chronic lung disease due to reduced synthesis and/or secretion that results from Clara cell depletion or injury. Although changes in CCSP levels certainly reflect the status of Clara cells and the bronchiolar region, changes in the integrity of more proximal airways may also influence intrapulmonary and serum levels of this biomarker. Functional Significance in Disease Pathophysiology

Roles for changes to the bronchiolar epithelium in the pathophysiology of chronic lung disease have been

implicated from both the histopathology and studies performed using primary cultures of airway epithelium, but have been unequivocally demonstrated in studies involving use of genetically modified mouse models. Primary cultures of human bronchial epithelium can be prepared from cells recovered by bronchial brushing. Studies comparing such cultures derived from normal subjects versus subjects with chronic lung disease have demonstrated diseaserelated differences in their functional properties that are intrinsic to the epithelium. However, even though cultures of bronchial epithelium may be reflective of events occurring within bronchiolar airways, the inability to sample epithelium from smaller caliber bronchiolar airways for establishment of representative cultures makes direct analysis impossible. Direct demonstration of a role for Clara cell injury and/or loss in chronic lung disease can be demonstrated in mouse models in which Clara cells can be either selectively ablated or genetically modified. Conditional ablation of CCSP-expressing cells using a regulable transgenic mouse model results in progressive lung inflammation and increased lung permeability similar to that observed with acute respiratory distress syndrome. Direct roles for altered Clara cell secretion in modulation of airway disease are suggested from studies using CCSP as a biomarker and further supported by the spectrum of pulmonary phenotypes discussed above for CCSP / mice. However, genetic manipulation of epithelial responsiveness to T-cell cytokines such as IL-13 demonstrates a direct role for CCSP-expressing cells in mediating influences of these cytokines on outcomes such as airway hyperreactivity and mucus cell metaplasia in airways. As such, CCSP-expressing cells, of which Clara cells represent the most abundant component, are sufficient to mediate cytokine-induced airway remodeling for initiation of the asthmatic phenotype, and represent a critical cell type whose altered function contributes to perpetuation of chronic lung disease through further immunoregulatory imbalances and the altered susceptibility to environmental pathogens and pollutants.

Ontogeny of Clara Cells Identification of specialized subsets of secretory cells in the proximal and distal airway implied segregation of the two compartments during embryonic development. Several studies have used morphological and molecular markers as well as lineage tagging methods to assess the temporal and spatial characteristics of this process. Standard embryological approaches have demonstrated that the lung is derived from the foregut endoderm 22–26 days after

CLARA CELLS 477

fertilization in humans. The trachea forms initially as a tube and is separated from the presumptive esophagus at 30 weeks gestation. In contrast, the intrapulmonary airways are formed through a budding process in which the endodermally derived epithelium invades the splanchnic mesenchyme and undergoes dichotomous branching directed by complex epithelial–mesenchymal interactions involving multiple interacting signal transduction pathways. In utero lineage tagging studies in mice clearly demonstrated specification of proximal (trachea and bronchi) and distal (bronchioles, acini, and alveoli) prior to formation of the definitive lung buds on embryonic day 9.5 and tissue transplantation experiments demonstrated determination of these lineages by embryonic day 16. Within the intrapulmonary airway epithelium, cytodifferentiation occurs in a proximal to distal wave and the first morphologically distinct phenotype to emerge is the neuroepithelial cell followed by the ciliated cell, the Clara cell, and finally the basal cell. Immunohistochemical analysis of epithelial differentiation in humans detected foci of CCSP-IR cells located predominantly at branchpoints from the 15th week of gestation onward. Localization of CCSP-IR to neuroepithelial bodies at branchpoints suggested that communication between these two cell types initiated during human lung development. Analysis of proliferation in hamster bronchiolar airways correlated regions of high epithelial proliferation with neuroepithelial bodies suggested that trophic units in the developing airway were synonymous with airway segments. Analysis of intrapulmonary airway epithelial differentiation in mice demonstrated that undifferentiated embryonic epithelial cells co-express multiple lineage markers during the embryonic stage of lung development and that this mixed pattern of gene expression resolves to the adult cell type-specific pattern as cells differentiate during the pseudograndular to canalicular transition. Studies in rabbits demonstrated reciprocal changes in cytoplasmic glycogen content (decrease) and granule content (increase) indicative of a transition from embryonic to mature secretory products. Similarly, the volume density of smooth endoplasmic reticulum and mitochondria underwent a rapid increase suggestive of maturation in metabolic capacity that reached adult levels by 10 weeks of age. Studies in rats further supported the idea that the terminal bronchiole was the last airway compartment to differentiate and that Clara cells in this region matured as a cohort. These studies support the argument that Clara cells of the adult airway are derived from immature polymorphic precursors, but did not distinguish between stochastic processes regulating differentiation of a

field of cells and derivation from a specific progenitor cell pool.

Relationship between Clara and Clara-Like Cells The aforementioned studies highlighted several important distinctions between the proximal and distal Clara-like and Clara cell populations. First, these data suggested that CCSP was a faithful marker for human bronchiolar Clara cells and similar immunological profiles of distal conducting airway secretory cells from multiple species indicated that expression of CCSP was a common molecular characteristic of secretory cells in this region. However, representation of CCSP-immunoreactive cells in human bronchi was too low to account for all morphologically defined Clara cells in this region and led to the suggestion that proximal human Clara cells were antigenically distinct from those of the distal conducting airway. Dissimilarity between proximal and distal secretory cells was further substantiated by the identification of messenger RNAs that encoded low-molecular-weight secretory proteins specific to proximal airway secretory cells of the human and mouse. Second, early segregation of the tracheobronchial and bronchiolar domains during lung development and identification of distinct proximal and distal secretory lineages in mice suggested that the unique functional characteristics of the proximal and distal epithelial–mesenchymal trophic units were based on discrete secretory cell subsets. Third, colocalization of CCSP with neutral glycoconjugate suggested a previously unrecognized precursor/progeny relationship between Clara-like and mucus cells of the upper airway. Such metaplastic transitions have been substantiated in proximal secretory cells of the mouse and have suggested that mucus production was a gain-of-function phenotype induced by proinflammatory conditions. These molecular studies dovetailed nicely with ultrastructural identification of transitional cells in the upper airway. Collectively, these studies suggested that proximal secretory cell phenotype was plastic and implied that function was influenced by both intrinsic factors and environmental conditions. Although additional studies are needed to clarify extrinsic factors regulating proximal and distal secretory cell function, existing studies suggest that the Clara-like and Clara cell populations play unique roles in airway homeostasis and that these distinctions are partially due to differences in the origin of these two cell types. See also: Mucus. Neurophysiology: Neuroendocrine Cells. Notch. Proteinase Inhibitors: Secretory

478 COAGULATION CASCADE / Overview Leukoprotease Inhibitor and Elafin. Stem Surfactant: Surfactant Protein D (SP-D).

Cells.

Further Reading Broeckaert F, Clippe A, et al. (2000) Clara cell secretory protein (CC16): features as a peripheral lung biomarker. Annals New York Academy Science 923: 68–77. Bucchieri F, Puddicombe SM, et al. (2002) Asthmatic bronchial epithelium is more susceptible to oxidant-induced apoptosis. American Journal Respiratory Cell Molecular Biology 27(2): 179–185. Cardoso WV (2001) Molecular regulation of lung development. Annual Review Physiology 63: 471–494. Evans MJ, Cabral-Anderson LJ, et al. (1978) Role of the Clara cell in renewal of the bronchiolar epithelium. Laboratory Investigation 38(6): 648–653. Hong KU, Reynolds SD, et al. (2001) Clara cell secretory proteinexpressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. American Journal of Respiratory Cell and Molecular Biology 24(6): 671–681.

Kuperman DA, Huang X, et al. (2002) Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nature Medicine 8(8): 885–889. Plopper CG (1983) Comparative morphologic features of bronchiolar epithelial cells. The Clara cell. American Review of Respiratory Disease 128(2 Pt 2): S37–S41. Plopper CG, Hill LH, et al. (1980) Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung. III. A study of man with comparison of 15 mammalian species. Experimental Lung Research 1(2): 171–180. Plopper CG, Suverkropp C, et al. (1992) Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of naphthalene. Journal of Pharmacology and Experimental Therapeutics 261(1): 353–363. Reynolds SD, Giangreco A, et al. (2004) Airway injury in lung disease pathophysiology: selective depletion of airway stem and progenitor cell pools potentiates lung inflammation and alveolar dysfunction. American Journal of Physiology: Lung Cellular and Molecular Physiology 287(6): L1256–L1265.

COAGULATION CASCADE Contents

Overview Antithrombin III Factor V Factor VII Factor X Fibrinogen and Fibrin Intrinsic Factors iuPA, tPA, uPAR Protein C and Protein S Thrombin Tissue Factor

Overview P F Neuenschwander, University of Texas Health Center, Tyler, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Blood coagulation is necessary for maintaining vascular integrity, and thus for maintaining life. Nature has developed an elaborate and elegant series of controlled proteolytic events that lead to the rapid formation of a stable platelet-fibrin plug at the site of vascular damage. Tissue factor (TF), the trigger for coagulation, is normally restricted from contact with the circulation by being expressed largely in adventitial fibroblasts. Once

the vessel is compromised, the exposed TF binds escaping factor VIIa (fVIIa) molecules with high affinity to form a potent procoagulant complex on local phospholipid surfaces. This enzyme–cofactor complex activates both fIX and fX to initiate the coagulant response, which is subsequently rapidly amplified through numerous positive feedback reactions. This burst of coagulant activity is dampened and finally quenched by specific protease inhibitors as well as the self-regulating nature of the coagulation complexes, thus preventing the potential lethal consequences of widespread thrombosis and allowing the complex process of wound healing to begin. Vascular integrity and maintenance of hemostasis is critical to normal lung function to allow efficient and unrestricted gas exchange. Lung injury or insult can lead to exposure or expression of TF in various cell types in the lung and can circumvent many of the normal coagulation control mechanisms. The resulting unregulated or

COAGULATION CASCADE / Overview 479 dysfunctional coagulation can lead to pulmonary fibrosis and the progression of an inflammatory response such as that observed in acute lung injury and acute respiratory distress syndrome.

Introduction In 1935, W H Howell wrote: ‘‘nature had a difficult problem to solve in creating a circulating medium for the animal body that would remain entirely liquid while in the blood-vessels, but would promptly set into a gel as soon as the vessels were wounded, and the blood was in danger of being lost.’’ Indeed, while this problem alone seems quite daunting, designing a fluid that can alter its response in proportion to the insult, limit the clot to the site of damage, and terminate the process once bleeding has subsided is even more remarkable. To accomplish this, nature has developed an elaborate and highly efficient sequence of enzyme-driven proteolytic reactions that utilize numerous feedback reactions, nonenzymatic cofactors, and specific inhibitors to regulate this explosive and critical, yet potentially lethal, process.

History Perhaps one of the earliest recorded instances of research into blood coagulation is the observation in 1666 by the Italian physician Marcello Malpighi of the existence of strands of fiber in the substance of a washed blood clot. Following this initial observation, more than one century passed before the next major advancement. In the mid-1800s, the concept of an enzymatic reaction that could lead to the deposition of fibrin was put forth, and it was not until 1904 that the first real scientifically based theory of blood coagulation was proposed. This theory consisted of four clotting factors (tissue thromboplastin, ionic calcium, prothrombin, and fibrinogen) and stood for three decades. Starting in the mid-1930s, a flurry of descriptions of novel clotting factors appeared in the literature, and in 1954 the International Committee for the Standardization of the Nomenclature of Blood Clotting Factors adopted the use of Roman numerals to designate the various clotting factors, with the activated form of each indicated by the addition of a lowercase ‘a’ suffix. The coagulation cascade as it is known today consists of more than 10 different clotting factors organized into extrinsic, intrinsic, and common pathways (Figure 1). Although traditionally it is still referred to as a cascade, this scheme can more accurately be described as a network, as it involves numerous interlinked reactions and feedback reactions that are all intimately involved in the tight regulation of this complex physiologic process.

Extrinsic Pathway The extrinsic pathway is the major initiator of blood coagulation in vivo. Clotting by this pathway is triggered when circulating blood comes in contact with tissue factor (TF) (Figure 2). TF is a cell-bound transmembrane glycoprotein and nonenzymatic protein cofactor that is constitutively expressed in adventitial cells of the vasculature and is not normally in contact with the circulation. Exposure of blood to TF occurs as a result of vascular damage. Circulating molecules of the plasma serine protease factor VIIa (fVIIa) (which is always present at B1% of the total fVII) then escape the circulation and bind avidly to the exposed TF generating a potent procoagulant complex. The generated fVIIa–TF complex propagates and amplifies coagulation via the proteolytic activation of fIX (intrinsic pathway) and fX (common pathway). Factor VII (fVII) is a member of the vitamin Kdependent coagulation protein family, which also includes fIX, fX, prothrombin, protein C, and protein S. With the exception of protein S, which is a nonenzymatic cofactor for protein C, these proteins are synthesized in an inactive zymogen form and require activation via limited proteolysis to produce an active serine protease. Each of these vitamin K-dependent proteins contains a g-carboxyglutamic acid (Gla) domain at the N-terminus followed by two epidermal growth factor-like repeats and the protease (or catalytic) domain. The Gla domain is generated through posttranslational g-carboxylation of specific glutamic acid residues by a cellular carboxylase that uses vitamin K as a cofactor. This domain is responsible for the anionic-phospholipid-binding properties of these proteins in the presence of ionic calcium. The epidermal growth factor (EGF)-like repeats are required for cofactor binding and to some extent substrate recognition, while the protease domain is responsible for the catalytic activity of the resulting enzyme.

Intrinsic Pathway It has long been known that plasma will spontaneously clot upon contact with a glass surface. This reaction is triggered intrinsically by contact autoactivation of fXII (Hageman factor). The generated fXIIa in complex with high-molecular-weight kininogen then activates fXI to fXIa and coagulation ensues (Figure 1). Since deficiency of fXII does not result in a hemorrhagic condition the contact activation pathway is generally not considered part of the coagulation system per se but serves as a link between coagulation, inflammation, and the

480 COAGULATION CASCADE / Overview Intrinsic pathway (amplification) XI XIa Extrinisic pathway (initiation)

TF / PL Ca2+

VII

IX VIIa

IXa Ca2+

VIIIa / PL

Ca2+

TF / PL

X

X Xa Va / PL

Ca2+ Prothrombin (II)

Thrombin (IIa) Common pathway

Fibrinogen (I) Fibrin monomer (Ia)

Fibrin clot XIII XIIIa

Stable clot

Figure 1 The modern coagulation scheme. The modern coagulation scheme is divided into the extrinsic, intrinsic, and common pathways (indicated). Although typically thought of as separate entities, these three pathways are all interlinked by numerous feedback reactions (not shown; refer to Figure 3) and cross-reactions resulting in a complex coagulation network. In this scheme, green arrows represent proteolytic processes and blue arrows represent enzymatic conversions. Required cofactors are indicated by green ovals and are juxtaposed next to their associated reaction. PL represents anionic phospholipid and the requirements for ionic calcium are indicated by Ca2 þ .

complement system. In contrast, deficiency of fXI results in a bleeding diathesis (albeit mild), as does deficiency in either fVIII or fIX (hemophilias A & B, respectively). Thus, these three proteins comprise the contemporary intrinsic pathway.

During coagulation, fIX can be activated by either of two enzymes: fXIa or the fVIIa–TF complex. Since fXIa is not present initially, it is the fVIIa–TF complex from the extrinsic system that is likely responsible for the initial activation of fIX. The level of fIXa

COAGULATION CASCADE / Overview 481

Figure 2 The initiation of coagulation. Blood coagulation is triggered at the site of vascular damage when escaping molecules of fVIIa bind to TF to form the procoagulant fVIIa–TF complex. TF is constitutively expressed in adventitial fibroblasts and does not normally come in contact with the circulation. The fVIIa–TF complex generated at the site of vascular injury triggers coagulation via the proteolytic activation of fX to form fXa (arrow). Platelets (small ovals) that come into contact with the exposed collagen in the basement membrane become activated (spiked ovals). These activated platelets adhere to each other to form an initial platelet plug as well as provide additional clotting factors and an effective phospholipid membrane to support coagulation reactions. This results in the formation of a stable platelet–fibrin plug at the site of injury to stop the outflow of blood. The large curved ovals represent red blood cells, the graycolored cells lining the blood vessel represent endothelial cells, and the brown-colored cells outside the vasculature represent TF-containing adventitial fibroblasts.

is subsequently amplified by fXIa that is generated later via a positive feedback reaction involving thrombin (see below). fXI is one of two coagulation enzymes that is not a member of the vitamin Kdependent protein family (the other being fXIII). Although fXI is synthesized as a zymogen and requires proteolytic activation, it neither contains a Gla domain nor EGF-like repeats. As such, fXIa does not require calcium ions or a phospholipid surface for activity. fIX is a vitamin-K dependent protein with similar structure to factors VII and X. The activation of fIX requires proteolytic cleavage at two sites to produce the serine protease form of fIXa. In order to express nominal procoagulant activity fIXa must bind to its nonenzymatic protein cofactor fVIIIa, which can be considered an essential activator. It is the fIXa–fVIIIa complex that is the true procoagulant enzyme. The binding of fIXa to fVIIIa occurs in the presence of calcium ions and an anionic phospholipid surface. Similar to the fVIIa–TF complex in the extrinsic system, the fIXa–fVIIIa complex also activates fX to propagate the coagulant response. FVIII is a large glycoprotein homologous to fV (the cofactor for fX; see below). FVIII is extremely labile and highly susceptible to proteolysis. It circulates in

plasma in a noncovalent complex with von Willebrand’s factor, which protects it from degradation and stabilizes its activity. Although not an enzyme, fVIII requires proteolytic processing at three sites to produce the active cofactor fVIIIa. The primary activator of fVIII in vivo is thrombin, although fXa can also catalyze this reaction. fVIII activation substantially reduces its size and releases it from von Willebrand’s factor to produce the active cofactor as a calcium-linked heterotrimer.

Common Pathway The activation of fX can be by either the extrinsic or intrinsic pathway and produces the identical product, fXa. Once generated, fXa binds to its nonenzymatic protein cofactor (fVa) in a calciumdependent fashion on a phospholipid surface. This complex proteolytically activates prothrombin to generate a-thrombin. As with fVIII, fV requires proteolytic activation to express nominal cofactor activity. The primary activator of fV is thrombin, although fXa can also activate fV. Both fX and prothrombin are vitamin-K dependent proteins with structural characteristics similar to other members of this family. While activation of fX

482 COAGULATION CASCADE / Overview XI XIa TF / PL + Ca2+ VII

IX VIIa

IXa VIII

VIIIa X PL +

X Ca2+

V

Xa

PL + Ca2+

Va Prothrombin (II)

Amplified feedbacks

requires a single proteolytic cleavage, activation of prothrombin requires two cleavages resulting in the release of the N-terminal Gla domain from the molecule. Thus, although prothrombin contains a Gla domain and requires phospholipid binding for activation, thrombin lacks a Gla domain and does not exhibit a phospholipid requirement. Once generated, thrombin proteolytically processes fibrinogen to release fibrinopeptides A and B and produce insoluble fibrin monomer, which polymerizes into a fibrin mesh (clot). Thrombin also proteolytically activates fXIII whose product, fXIIIa, is a calcium-dependent transglutaminase that cross-links the initially unstable fibrin clot to stabilize it.

Thrombin

Platelets Blood platelets play a pivotal role in hemostasis through formation of a platelet plug at the site of vascular damage. The platelet plug is relatively friable and can be readily dislodged if left unaided. Thus, platelet activation and function are closely linked to coagulation, which welds the friable platelet plug in place. The unactivated platelet is nonthrombogenic with a neutral phospholipid surface. Upon activation, initially by exposure to collagen in the basement membrane and later by thrombin, platelet phospholipids become scrambled to expose procoagulant anionic phospholipids. Activated platelets also release fVa and expose binding sites for fIXa and fVIIIa as well as proteins involved in platelet adhesion and aggregation.

Coagulation Control Positive Feedback Reactions and Control Mechanisms

Signal amplification is an inherent characteristic of biological enzymatic cascades. The coagulation cascade builds on this using numerous positive feedback reactions. The number of feedback reactions that clotting enzymes participate in increases as one progresses down the cascade, with thrombin involved in the most (Figure 3). The result is an explosive amplification of the initial stimulus resulting in rapid formation of a clot at the site of damage. The primary feedback reaction during coagulation is the feedback activation of fVII. This occurs via feedback of fVIIa–TF or fXa and rapidly amplifies the number of active fVIIa–TF complexes present. The activation of fVII by fVIIa is TF-dependent and can occur on any phospholipid membrane containing TF. In contrast, the activation of fVII by fXa requires an anionic phospholipid surface and does not require TF.

Platelet activation Figure 3 Positive feedback reactions. There are five major positive feedback reactions in coagulation that serve to greatly amplify the initial coagulant stimulus: the activation of fVII, fVIII, fV, fXI, and platelets. The number of feedback reactions in which an enzyme is involved increases as one progresses down the coagulation cascade (red arrows): fVIIa is involved in one feedback, fXa is involved in two, and thrombin is involved in four. Required cofactors for each feedback reaction are indicated in the figure.

The feedback activation of fVIII is largely catalyzed by thrombin, although fXa can also activate fVIII in the presence of anionic phospholipid. This latter pathway may serve to generate the critical initial molecules of fVIIIa before significant thrombin is generated. Once generated, however, thrombin is a very potent feedback activator of fVIII, fV, fXI, and platelets. Thrombin’s feedback activation of fV serves to amplify its own formation directly, while activation of fXI serves to amplify the whole of coagulation. The former provides a smaller but rapid amplification, while the latter provides a larger and more controlled amplification due to the numerous subsequent steps encountered before clot formation. The activation of platelets by thrombin can be considered a feedback reaction due to the prior activation of platelets by extracellular matrix proteins (i.e., collagen). Thus, thrombin activation of platelets can be envisioned as a secondary activation that results in amplified platelet plug formation progressing away from the basement membrane towards the lumen of the vessel. Cofactors and Self-Damping Reactions

One of the critical control features in coagulation is the requirement for cofactors. FVIIa, fIXa, and fXa

COAGULATION CASCADE / Overview 483

all require protein cofactors for nominal activity. These cofactors are all nonenzymatic and act strictly as regulatory molecules. Thus, the procoagulant activity of the enzyme is largely, if not entirely, dictated by the availability and activation status of the cofactor. In the case of coagulation initiation, circulating molecules of fVIIa remain essentially inert in the absence of TF. The increase in TF availability brought about by vascular damage is the triggering event, thus linking the intensity of the response to that of the insult; large ruptures expose more TF than smaller ones. Similarly, fXa has limited activity towards prothrombin in the absence of fV. Binding of fXa to fV increases its activity to a small extent, but optimal fV cofactor activity is only observed upon its proteolytic activation. This activation is largely catalyzed by thrombin. Thus, unlike fVIIa, fXa exhibits three potential levels of procoagulant activity during coagulation: fXa o fXa–fV o fXa–fVa. The activity of fIXa is also regulated by a cofactor, fVIII. Formation of the fIXa–fVIIIa complex on a phospholipid surface in the presence of ionic calcium increases the procoagulant activity of fIXa by a factor of B109. Thus, fIXa is an inactive catalytic subunit that becomes active only in the context of the enzyme–cofactor complex. FVIII does not express any measurable cofactor activity until it is proteolytically activated, unlike fV. Thus, regulation of fIXa is entirely governed by the activation status of fVIII and the level of fVIIIa. During coagulation initiation, generation of fIXa by the fVIIa–TF complex will be of no immediate consequence other than to prime the system for explosive amplification immediately upon generation of small amounts of fVIIIa. This design places a critical importance on fVIII activating enzymes and their control of fVIII activation and activity. As such, nature has placed heavy restrictions on fVIII levels and fVIIIa activity: (1) fVIII levels in plasma are remarkably low (B300 pM; the lowest level of any of the clotting factors); (2) FVIII is highly susceptible to proteolytic degradation and in fact has to be protected from degradation by circulating in a noncovalent complex with a carrier protein, von Willebrand’s factor; (3) once proteolytically activated, fVIIIa is extremely labile and exhibits a half-life of only 1– 3 min; and (4) several enzymes (largely activated protein C) can proteolytically inactivate fVIIIa. Inhibition and Negative Control Mechanisms

A second critical control feature of coagulation is the presence of multiple coagulation inhibitors in plasma, which provides an additional level of safety and regulation (Figure 4). Of the numerous enzyme

α1-PI XIa TF / PL AT Heparin IXa

IX

APC

VII VIIa

VIIIa / PL

TFPI TF / PL

X

AT

X

Xa APC

Va / PL Prothrombin (II)

AT

Thrombin (IIa)

HCII Heparin

Figure 4 Inhibitors of coagulation. Inhibitors of various coagulation reactions are indicated in red with associated targets encircled. a1-PI, a1-protease inhibitor; AT, antithrombin; TFPI, tissue factor pathway inhibitor; HCII, heparin cofactor II; APC, activated protein C. Heparin is required for inhibition of fIXa by AT and thrombin by HCII.

inhibitors in plasma, the five major coagulation inhibitors are antithrombin (formerly called antithrombin III or heparin cofactor I), heparin cofactor II, a2macroglobulin, a1-protease inhibitor (formerly called a1-antitrypsin), and TF pathway inhibitor (TFPI). Antithrombin Deficiency of antithrombin results in extensive venous thrombosis, suggesting that this serpin (serine protease inhibitor) is a primary coagulation inhibitor. Owing to its unfortunate designation as antithrombin, it is often thought that its target is solely thrombin. In fact, antithrombin inhibits fXa as well as thrombin. Heparin greatly augments the ability of antithrombin to inhibit both of these proteases. While antithrombin also inhibits fIXa and fVIIa, this occurs only in the presence of heparin. In the case of fVIIa, exceedingly high, and possibly not physiologically relevant, levels of heparin are required to observe this inhibition. Heparin cofactor II In contrast to antithrombin, heparin cofactor II is a specific serpin with thrombin as its sole target. The activity of this inhibitor is not only enhanced by heparin but also by other polysaccharides such as dermatan sulfate. a2-Macroglobulin Deficiency of a2-macroglobulin has yet to be described, suggesting a critical function

484 COAGULATION CASCADE / Overview

of this inhibitor. However, only a small fraction of fXa inhibition can be attributed to a2-macroglobulin and at most 25% of thrombin inhibition under certain conditions. This, along with the propensity of this inhibitor to react with numerous other proteases, supports the notion of a2-macroglobulin being an essential general-maintenance inhibitor and protease scavenger rather than a coagulation regulatory inhibitor per se. a1-Protease inhibitor The major plasma inhibitor of fXIa is a1-protease inhibitor, which is also a weak inhibitor of fXa. Despite this, the deficiency of a1proteinase inhibitor results in emphysema and not coagulopathies. Thus, the major role of this inhibitor is probably inhibition of neutrophil elastase. TF pathway inhibitor The major plasma inhibitor of fVIIa is TFPI. TFPI is a multivalent inhibitor containing three reactive sites, each of which possesses a Kunitz-type structural fold homologous to bovine pancreatic trypsin inhibitor but with very specific and distinct specificities. The first Kunitz domain of TFPI reacts specifically with the fVIIa–TF complex while the second domain reacts with fXa and reactivity of the third Kunitz domain is as yet unclear. A unique feature of TFPI is that although it is a fairly strong inhibitor of the fVIIa–TF complex it does not inhibit free fVIIa. This is significant in that it allows low levels of fVIIa to circulate freely under normal conditions to serve as a primer for immediate triggering of coagulation upon vascular injury (see above). An additional unique feature of TFPI is its ability to react strongly with the direct product of fVIIa–TF activity, i.e., fXa. TFPI binds to both the fVIIa–TF complex and fXa to lock these proteins together in a quaternary complex and prevent further reactions from taking place. This elegant mechanism of inhibition is different from traditional modes of enzyme inhibition and allows coagulation to be triggered while subsequently rapidly shutting down the initiation complex to prevent ongoing triggering and potential excessive thrombosis. The overall affinity of TFPI for the fVIIa–TF–fXa tertiary complex is quite tight and in the picomolar range, thus making TFPI by far the most specific and potent coagulation inhibitor in plasma. Activated protein C (APC) Although not an enzyme inhibitor in the classical sense, APC controls coagulation by downregulating the activity of the fIXa– fVIIIa and fXa–fVa complexes. Instead of targeting the catalytic portion of these complexes, APC regulates their activity by proteolytic inactivation of their cofactors, fVIIIa and fVa (Figures 1 and 4). This

anticoagulant pathway involves the proteolytic activation of protein C, the precursor of APC, by thrombin bound to the endothelial cell receptor thrombomodulin. Thrombomodulin serves to alter the substrate specificity of thrombin and essentially converts it from a procoagulant to an anticoagulant. In this way, thrombin can act to stop its own generation as well as that of fXa.

Coagulation in Respiratory Diseases In normal lung function, coagulation serves the critical purpose of maintaining the hemostatic balance as well as preserving the integrity of the pulmonary vasculature to allow efficient gas exchange. Dysfunctional coagulation in the lung can lead to the onset of pulmonary fibrosis, which is typically followed by respiratory failure. In addition, dysfunctional coagulation within the pulmonary artery and pulmonary microvasculature can lead to thrombosis with concomitant pulmonary injury and infarction. The delicate hemostatic balance in the pulmonary vasculature can be tipped towards a more procoagulant state by a variety of acute pulmonary insults resulting from exposure to numerous toxic chemicals as well as from the development of sepsis or pneumonia. Chronic pulmonary insults such as those observed in interstitial lung diseases can also promote a procoagulant state. The elevated procoagulant state observed in acute lung injury (ALI) and interstitial lung disease is characterized by a general increase in the levels of clotting factors and decrease in the levels of anticoagulant and fibrinolytic factors. These factors have been measured in bronchoalveolar lavage (BAL) and/or pleural exudates from distressed lungs, indicating a concomitant increase in vascular permeability and accumulation of plasma components in the alveolar and interstitial spaces. The activation of coagulation by TF-expressing cells in these exudates ultimately results in the observed deposition of alveolar and interstitial fibrin. Dysfunctional activation of the coagulation cascade in ALI is spearheaded by the increased expression of TF in intravascular and extravascular cells. While TF is normally expressed in mesothelial cells and various other lung cells, alveolar macrophages, lung epithelial cells, and lung fibroblasts all show increased TF expression in a bleomycin-induced lung injury model. In addition, monocytes, neutrophils, and endothelial cells can all be induced to express TF under pathophysiologic conditions in response to endotoxin and various cytokines such as TNF-a and TGF-b. This increased expression of TF produces an environment conducive to constitutive lowlevel initiation of coagulation. Although the intrinsic

COAGULATION CASCADE / Overview 485

pathway of coagulation is activated to some degree, the extrinsic pathway is accepted to be the driving force in this dysfunctional clotting resulting in elevated levels of activated factor X and thrombin, and ongoing fibrin deposition. While extravascular fibrin deposition in the alveolar and interstitial spaces is a major result of dysfunctional pulmonary coagulation, several of the clotting factors are also involved in propagating an inflammatory response via cell signaling through protease-activated receptors (PARs). There are currently four identified PARs, each of which is differentially expressed on various cell types in the lung: PAR-1 is expressed in platelets, mononuclear cells, and endothelial cells; PAR-2 is expressed in mononuclear cells, endothelial cells, and neutrophils; and PAR-3 and -4 are expressed in platelets. Activation of each of these receptors occurs through proteolytic cleavage of an activation site within the extracellular domain of the receptor. The newly formed N-terminus functions as a tethered ligand, which subsequently binds to an intramolecular site to induce cell signaling through coupled heterotrimeric G-proteins. Thrombin can activate cells via PAR-1, PAR-3, and PAR-4, while the fVIIa–TF complex can activate cells via PAR-2 and fXa can activate cells via PAR-1, PAR2, and the nonproteolytically activated endothelial protease receptor-1 (EPR-1). As a result of cell activation and cytokine release, TF expression is further stimulated causing a proliferation of the coagulant response. Concomitant with this increase in procoagulant potential is a reduction in anticoagulant machinery: the expression of TFPI is reduced and the inhibition potential of AT is severely restricted by cleavage of heparin-like glycosaminoglycan chains from cell-surface proteoglycans (syndecans and glypicans). In addition, thrombomodulin (a cell-surface thrombin receptor required for protein C activation) is downregulated resulting in lowered APC generation. Several activated cell types also secrete elevated levels of plasminogen activator inhibitor-1 (PAI-1), thus lowering the activity of uPA and greatly attenuating fibrinolysis. Anticoagulant Treatment

The use of various coagulation inhibitors as therapeutic treatments for pulmonary fibrosis has not been clearly established and remains controversial. While experiments in animal models have demonstrated beneficial reductions in ALI upon treatment with anticoagulants, these results have not been borne out consistently in human trials. Although successful in phase II trials, the use of TFPI in phase III trials (OPTIMIST) failed to show any beneficial effect

above placebo in overall mortality, despite a reduction in procoagulant potential. The use of APC as a potential therapy for pulmonary fibrosis remains encouraging since the PROWESS trials (examining the use of APC in sepsis) have demonstrated a significant reduction in mortality in cases of severe sepsis, which is a major cause of ALI and ARDS. Taken together, these studies suggest more complex interactions and place greater import on roles of clotting factors other than in coagulation per se that may involve their cell signaling functions and the linkage between coagulation and inflammation. See also: Acute Respiratory Distress Syndrome. Anticoagulants. Bronchoalveolar Lavage. Chemokines. Coagulation Cascade: Antithrombin III; Factor V; Factor VII; Factor X; Fibrinogen and Fibrin; Intrinsic Factors; iuPA, tPA, uPAR; Protein C and Protein S; Thrombin; Tissue Factor. Complement. Endothelial Cells and Endothelium. Endothelins. Extracellular Matrix: Basement Membranes. Fibrinolysis: Overview; Plasminogen Activator and Plasmin. Fibroblasts. GProtein-Coupled Receptors. Interstitial Lung Disease: Overview. Leukocytes: Monocytes. Lipid Mediators: Platelet-Activating Factors. Myofibroblasts. Platelets. Pleural Effusions: Pleural Fluid, Transudate and Exudate. Pneumonia: Overview and Epidemiology. Proteinase-Activated Receptors. Pulmonary Circulation. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Thrombolytic Therapy.

Further Reading Abraham E (2000) Coagulation abnormalities in acute lung injury and sepsis. American Journal of Respiratory Cell and Molecular Biology 22: 401–404. Beutler E, Lichtman MA, Coller BS, and Kipps TJ (eds.) (1995) Williams Hematology, 5th edn., Part X, Chs. 118–147, pp. 1149–1591. New York: McGraw-Hill. Chapman HA (2004) Disorders of lung matrix remodeling. Journal of Clinical Investigation 113: 148–157. Colman RW, Hirsh J, Marder VJ, and Salzman EW (eds.) (1994) Hemostasis and Thrombosis: basic principles and clinical practice, 3rd edn., p. 1713. Philadelphia: J.B. Lippincott Company. Davie EW and Ratnoff OD (1964) Waterfall sequence for intrinsic blood clotting. Science 145: 1310–1312. Dugina TN, Kiseleva EV, Chistov IV, Umarova BA, and Strukova SM (2002) Receptors of the PAR family as a link between blood coagulation and inflammation. Biochemistry (Moscow) 67: 74–75. Howell WH (1935) Theories of blood coagulation. Physiological Reviews 15: 435–470. Idell S (2001) Anticoagulants for acute respiratory distress syndrome: can they work? American Journal of Respiratory and Critical Care Medicine 164: 517–520. Laterre P-F, Wittebole X, and Dhainaut J-F (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31: S329–S336. Levi M, Schultz MJ, Rijneveld AW, and van der Poll T (2003) Bronchoalveolar coagulation and fibrinolysis in endotoxemia and pneumonia. Critical Care Medicine 31: S238–S242.

486 COAGULATION CASCADE / Antithrombin III Macfarlane RG (1964) An enzyme cascade in the blood clotting mechanism, and its function as a biochemical amplifier. Nature 202: 498–499. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 1334–1349. Welty-Wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inflammation in acute lung injury. Thrombosis and Haemostasis 88: 17–25.

Antithrombin III A Gu¨nther and C Ruppert, University of Giessen Lung Center (UGLC), Giessen, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract Antithrombin, formerly referred to as antithrombin III, is a heparin-binding protein and the major plasma inhibitor of coagulation proteases, primarily thrombin and factor Xa. In addition to its function in controlling the process of coagulation, antithrombin has potent anti-inflammatory properties. This, together with the observations that coagulation contributes to inflammation and that antithrombin levels are significantly reduced in patients with sepsis, provided a strong rational for antithrombin replacement therapy. In numerous animal models of experimental sepsis and several small-scale studies involving patients with sepsis, administration of antithrombin has been shown to be effective. A large prospective, randomized, doubleblind, placebo-controlled phase III trial, however, failed to show a significant improvement in overall survival. More studies are needed to answer open questions, such as those that regard the concomitant use of heparin, dosing, and the patient population that may benefit from antithrombin therapy.

Introduction Antithrombin, also referred to as antithrombin III (ATIII), heparin cofactor I, and thrombin inhibitor I, is a circulating plasma protein and the major inhibitor of thrombin, FIXa, FXa, and other serine proteases, accounting for B80% of the thrombin inhibitory activity in plasma. Antithrombin functions as a key regulator of blood coagulation. It was discovered in 1905 by the German physician Paul Oskar Morawitz, who described an inhibitory substance in plasma and serum that ‘lowered’ thrombin activity. In a 1918 paper, William Henry Howell and L Emmett Holt described two new factors in blood coagulation. They coined the name heparin for a new anticoagulant originally isolated from dog liver by Howell’s graduate student Jay McLean in 1916, and they identified an ‘antecedent substance’ in plasma and serum ‘which is converted into antithrombin by a reaction with heparin’ and named it pro-antithrombin. In 1939, Brinkhouse et al. showed that the anticoagulant effect of heparin was mediated by an

endogenous plasma component that they termed heparin cofactor. Fourteen years later, Monkhouse et al. isolated a protein component from plasma displaying both heparin cofactor and antithrombin activity. They suggested that both activities may be functions of the same protein. This hypothesis was substantiated in 1968, when Ulrich Abildgaard purified ATIII from plasma and showed that this protein has heparin cofactor activity and is the main thrombin inhibitor in normal plasma. It was Abildgaard who first showed that thrombin inhibition occurs via formation of an inactive, stable complex (1969) and thus proved older theories suggesting that the inactivation occurs enzymatically (Gerendas, 1960), with antithrombin being a substrate for thrombin (Seegers et al. 1960). In 1973, Rosenberg and Damus purified a sufficient amount of ATIII for biochemical characterization. They confirmed the formation of an irreversible equimolar complex between thrombin and antithrombin and postulated that heparin specifically accelerated this complex formation by binding to ATIII and causing a conformational change in the antithrombin molecule. The term ‘antithrombin III’ has largely been replaced by ‘antithrombin’ alone. The designation arose from a historical classification of mechanisms responsible for thrombin inactivation made in the 1950s: ATI was defined as the ability of fibrin to adsorb thrombin, ATII designated heparin cofactor activity, ATIII was used to describe plasma/serum-derived inhibitors causing a slowly progressive irreversible inhibition of thrombin, ATIV designated an intermediate acting inhibitor arising during coagulation, ATV designated an inhibitor found in pathological conditions, and ATVI was defined as the anticoagulant action of elevated fibrin degradation products. In recent years, other functions of antithrombin independent of its effect on coagulation have become evident, including anti-inflammatory and antiangiogenic activities.

Structure Antithrombin is a single-chain glycoprotein consisting of 432 amino acids with a molecular weight of 58 kDa that belongs to the serine proteinase inhibitor (serpin) superfamily of proteins (Figure 1). Two isoforms have been isolated from normal plasma. The a form is glycosylated at four asparagine residues (Asn96, Asn135, Asn155, and Asn192) and accounts for 90–95% of the circulating protein. The b form comprises a small fraction (5–10%) that lacks glycosylation at Asn135. This glycosylation heterogeneity is of potential functional importance since the b isoform binds heparin with B10-fold higher

COAGULATION CASCADE / Antithrombin III 487

RCL C
-sheet B
-sheet D -helix

Heparin pentasaccharide

A
-sheet

Figure 1 Structure of antithrombin and antithrombin in complex with heparin pentasaccharide. On both structures, the A b-sheet is shown in red, the B b-sheet in green, the C b-sheet in yellow, and the D a-helix in blue. The reactive center loop (RCL) is shown in purple. Reproduced from Quinsey NS, Greedy AL, Bottomley SP, Whisstock JC, and Pike RN (2004) Antithrombin: in control of coagulation. International Journal of Biochemistry and Cell Biology 36: 386–389, with permission from Elsevier.

affinity. Like other serpins (antithrombin shares approximately 30% homology in amino acid sequence with other serpins), antithrombin folds into a highly conserved secondary structure with three b-sheets (A–C) and nine a-helices (A–I). Disulfide bonds are formed between Cys8–Cys128, Cys21–Cys95, and Cys247–Cys430, of which the first is required for heparin cofactor activity. The reactive center loop (RCL), a flexible stretch of B17 amino acids, protrudes the main body of the molecule and contains the reactive site bond Arg393–Ser394 (P1–P10 bond), which is cleaved by the target protease (Figure 2). A high-affinity binding site (heparin binding site) is located on the D a-helix and involves positively charged amino acids (Arg47, Lys125, Arg129, and Arg132) that are able to interact with negatively charged sulfate groups in glycosaminoglycan chains of, for example, heparin.

Regulation of Production and Activity Antithrombin is synthesized in the liver and circulates in plasma at a concentration of B150 mg ml
1, with a biological half-life of 50–70 h. The gene for antithrombin (SERPINC1) is located on chromosome 1 at 1q23–25.1, comprises 13.37 kb, and contains seven exons and six exons. The primary translation product is a precursor protein containing a 32 amino acid signal sequence that is cleaved prior to secretion. Posttranslational modifications include disulfide bond formation and glycosylation. Little is known about the regulation of biosynthesis.

Cell culture experiments employing isolated rat hepatocytes showed that antithrombin synthesis is not altered in the presence of antithrombin–protease complexes but is stimulated by the supernatant medium of LPS-treated macrophages. Following reaction with the target protease and complex formation, the protease–antithrombin complexes are rapidly cleared from the circulation ðt1=2 E2
3 minÞ, possibly mediated by a specific receptor on hepatocytes.

Biological Function Antithrombin is the major inhibitor of thrombin, FIXa, and FXa in plasma, but it also inactivates other serine proteases, such as FXIa, FXIIa, plasmin, kallikrein, and the complement factor C1. Inhibition by antithrombin occurs via an irreversible suicide substrate mechanism and involves the formation of a stable 1:1 stoichiometric complex between the active site of the target protease and the reactive site of antithrombin. The protease initially recognizes antithrombin as a substrate. Cleavage of the reactive site P1–P10 bond (Arg393–Ser394) in antithrombin triggers a striking conformational change. The RCL inserts into the A b-sheet and transports the covalently bound protease to the opposite pole of the inhibitor. This translocation results in a deformation and thus inactivation of the protease. As a result, the protease is trapped and the attempted cleavage is stopped at the stage of an acyl intermediate.

488 COAGULATION CASCADE / Antithrombin III Vascular injury

Kallikreinkinin-system

Cellular effects

Coagulation

Prekallikrein FXII

FXIIa

Kallikrein HMWkininogen

FXI

Prostacyclin release (endothelial cells) LPS-induced release of IL-6, TF Leukocyte rolling + adhesion leukocyte recruitment

Tissue factor (TF) FVII FVII TF FIX FVIIa

Platelet aggregation SMC, fibroblast proliferation ECM production Inflammatory cell recruitment Proinflammatory mediators Profibrotic growth factors

PAR

FX

FXIa

Antithrombin FIXa

Kinin

PL, Ca2+

Bradykinin

FVIII

FVIIIa

Vascular permeability contraction of SMC vasodilation

FXa

Prothrombin

PL, Ca2+

FV

FVa

Thrombin

FXIII FXIIIa

Immune complexes + C1 (C1q, C1r, C1s)

Activating surfaces + C3b

+C4 C1qrs +C2

C4a C2b

Fibrinogen

Fibrin split products Plasmin

+fB +fD

Complement system

Crosslinked fibrin

Fibrin

tcuPA

Two chain tPA

uPAR

C3bBb

C4b2a

Plasminogen C3b C3b attachment

C3

C3a C4b2a3b (C5 convertase)

C3a

C3b C3b attachment

Fibrinolysis/ MMP-system Pro-MMP

C3bBb3b (C5 convertase)

MMP Degradation of ECM

C5 C5a C5b +C7 +C8 +C9 +C6

C5b-C9

Killing of pathogens Opsonozation of pathogens Recruitment of inflammatory cells

Membrane attack complex (MAC)

Figure 2 Overview of antithrombin targets. Plasma enzyme systems and their interaction as well as the targets of antithrombin therein and on cells are shown. Components of the coagulation system are shown in black, the kallikrein–kinin system is shown in green, the fibrinolytic and MMP system in blue, and the complement system in magenta. The cellular response is depicted in brown. Although not being the primary target, some components of plasmatic systems may not be able to interact with other systems (dotted lines). The effects of antithrombin are indicated in red by - (activation/stimulation) or (blockade). F, coagulation factor; C, complement factor; PL, phospholipids; teu-PA, two-chain urokinase; UPAR, urokinase receptor; ECM, extracellular matrix; HMW, high molecular weight; SMC, smooth muscle cell; PAR, protease activated receptor.

Although plasma concentration is quite high (2–3 mM), antithrombin is a relatively inefficient inhibitor in vivo. Inhibition increases B1000 times after interaction with glycosaminoglycans, such as heparin. These glycosaminoglycans bind antithrombin via its high-affinity binding site and induce a conformational change in antithrombin, making the reactive site more accessible to the protease attack. The heparin-induced allosteric activation is mediated by a unique pentasaccharide unit in the glycosaminoglycan chain. Heparin can also function as a template to which both antithrombin and the target protease bind and that brings the reactive center of antithrombin and the catalytic center of thrombin into close proximity (so-called approximation mechanism). This complex formation involves binding to

a secondary (low-affinity) heparin binding site and requires heparin molecules containing at least 18 saccharide units. The interaction between antithrombin and heparin is of high clinical and therapeutic importance, but its physiological importance is questioned since heparin is not released from mast cells into the circulation and is not detectable in plasma. Instead, a closely related glycosaminoglycan, heparan sulfate, which is located on the extracellular matrix of the endothelial lining layer of vessels, is believed to precisely control coagulation in the vasculature. In a large number of studies, anti-inflammatory properties of antithrombin have been reported that are independent from its anticoagulant activity. Proposed mechanisms include the promotion of

COAGULATION CASCADE / Antithrombin III 489

prostacyclin (prostaglandin I2) production and release from endothelial cells and the inhibition of the proinflammatory signaling pathway of nuclear factor-kB, thereby inhibiting tumor necrosis factor-amediated expression of cytokines, chemokines, and adhesion molecules. A modified antithrombin (inactive form with low heparin affinity in which the cleaved reactive bond loop is inserted into the A sheet in a similar manner as in the proteinase complexes) has been shown to exert potent anti-angiogenic and antitumor activity. The underlying molecular mechanism is obscure, however. Studies with a similar latent form of antithrombin (heat-denatured antithrombin with low heparin affinity) and prelatent antithrombin (i.e., another latent form with retained heparin binding) showed that the anti-angiogenic effect was mediated by induction of apoptosis by disrupting cell–matrix interactions through inhibition of focal adhesion kinase activity and focal adhesion formation. Targeted disruption of the antithrombin gene in mice resulted in an embryonic lethal phenotype. The embryos died during mid- to late embryogenesis (approximately 15.5 gestational days) and showed extensive fibrin(ogen) deposition in myocardium and liver as well as subcutaneous hemorrhagic lesions, possibly due to consumptive coagulopathy and/or liver dysfunction.

Receptors There is accumulating evidence that binding of antithrombin to the cell surface is critical to exert its anti-inflammatory activity. Syndecan-4, a G-proteincoupled transmembrane proteoglycan with extracellular heparan sulfate chains, may be one cellular receptor for antithrombin.

Antithrombin in Respiratory Diseases Antithrombin deficiency may be inherited or acquired due to decreased synthesis, increased loss, or increased consumption. Inherited antithrombin deficiency was the first recognized inherited thrombophilia and is heterogeneous with respect to its genetic origin and clinical manifestation. Based on results from functional and immunological assays, deficiency may be type I or type II. Type I deficiency is characterized by a heterozygous mutation resulting in a 50% reduction in both antigen levels and functional activity and an increased risk for venous thrombosis. In type II deficiency, antigen levels are normal, but function is reduced. This group includes mostly single amino acid mutations affecting the reactive site (variant ‘RS’, with a high risk of

thrombosis) or the heparin binding site (variant ‘HBS’, with a low risk of thrombosis). A third subgroup (‘PE’) of dysfunctional variants has pleiotropic (multiple) effects, altering both functional activity and plasma antigen levels (B70% of control). Intravascular clot formation due to the predominance of procoagulant and suppression of fibrinolytic factors is a key manifestation in a variety of diseases, including atherosclerosis and septic organ failure. With regard to the pulmonary vasculature, microthrombosis is a consistent finding in patients with acute lung injury (ALI), acute respiratory distress syndrome (ARDS) of both septic or nonseptic origin, and in patients with chronic pulmonary hypertension. The microvascular bed, which represents by far the largest surface in the pulmonary vasculature and plays a crucial role in dissolution of microthrombi, is particularly affected. Activation of the coagulation system with microthrombi formation, as well as uncontrolled and overwhelming inflammation and damage to the microvascular bed, contributes substantially to organ dysfunction in ARDS or septic shock. Accordingly, in patients with severe sepsis, low plasma levels of antithrombin resulting from a decreased synthesis and/or enhanced clearance by thrombin–antithrombin complex formation have been found. This correlates with the development of disseminated intravascular coagulation (DIC), ARDS, and poor outcome, suggesting a possible therapeutic role for antithrombin in these patients. In various animal models of ALI, sepsis, septic shock, and DIC, administration of antithrombin attenuated the inflammatory response and displayed positive effects in preventing organ dysfunction and improving survival. However, the beneficial effects could not be confirmed in humans. Clinical studies investigating the efficacy of antithrombin in patients with severe sepsis demonstrated a reduction in mortality and improvement of organ dysfunction. However, the findings did not reach statistical significance due to the limited number of patients enrolled. In the large phase III multicenter KyberSept trial, which included more than 2300 patients with sepsis, no effect on 28-day mortality was achieved. Instead, evidence of an adverse interaction with heparin was observed. Patients in the antithrombin group who did not receive concomitant low-dose heparin prophylaxis exhibited a significant improvement in 90-day mortality. Possible explanations for these adverse results are the achieved antithrombin plasma levels, which were lower than expected, and the blockade of anti-inflammatory actions of antithrombin by interaction with heparin (thereby preventing interaction with cellular glycosaminoglycan receptor molecules).

490 COAGULATION CASCADE / Factor V

The influence of antithrombin on the clinical course of ARDS or ALI has not been addressed in detail. In studies investigating patients with severe sepsis, different effects on respiratory function were observed. Whereas one study showed an apparent but nonsignificant improvement in respiratory function, others showed no reduction in lung dysfunction. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Matrix: Integrins. Anticoagulants. Coagulation Cascade: Overview; Factor X; Protein C and Protein S; Thrombin. Thrombolytic Therapy.

Further Reading Bayston TA and Lane DA (1997) Antithrombin: molecular basis of deficiency. Thrombosis and Haemostasis 78: 339–343. Esmon CT (2001) Role of coagulation inhibitors in inflammation. Thrombosis and Haemostasis 86: 51–56. Laterre PF, Wittebole X, and Dhainaut JF (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31(supplement): S329–S336. Opal SM (2000) Therapeutic rationale for antithrombin III in sepsis. Critical Care Medicine 28(supplement): S34–S37. Perry DJ (1994) Antithrombin and its inherited deficiencies. Blood Reviews 8: 37–55. Quinsey NS, Greedy AL, Bottomley SP, Whisstock JC, and Pike RN (2004) Antithrombin: in control of coagulation. International Journal of Biochemistry and Cell Biology 36: 386–389. Roemisch J, Gray E, Hoffmann JN, and Wiedermann CJ (2002) Antithrombin: a new look at the actions of a serine protease inhibitor. Blood Coagulation and Fibrinolysis 13: 657–670. Silverman GA, Bird PI, Carrell RW, et al. (2001) The Serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Journal of Biological Chemistry 276: 33293–33296.

Factor V P F Neuenschwander, University of Texas Health Center, Tyler, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Blood coagulation factor V (fV) is a large plasma glycoprotein similar in structure to fVIII. It is synthesized as a single-chain inactive precursor in the liver and present in the circulation at a concentration of roughly 10 mg ml
1. Activation of fV occurs via ordered proteolysis at three sites by thrombin or fXa. The proteolytically activated form of fV (fVa) binds tightly to fXa in the presence of ionic calcium and an anionic phospholipid surface to produce a potent procoagulant. The fully assembled complex subsequently generates a-thrombin with a catalytic efficiency 300 000-fold greater than fXa alone. FV is also secreted from activated platelets, thus helping to localize fXa activity to the site of vascular damage. As with fXa, unregulated activation or activity of fV/fVa in the lung is associated with the development of pulmonary fibrosis and the inflammatory response

observed in acute lung injury and acute respiratory distress syndrome.

Introduction In 1944, the Norwegian hematologist Dr Paul Owren discovered factor V (also known as proaccelerin or labile factor) and its deficiency was termed parahemophilia. In 1954, those studying coagulation adopted the Roman numeral convention and this factor was officially named factor V (fV) with its activated form named factor Va (fVa). Although fV was known to be important, it was not until 1959 that the role of fV as a cofactor for activated factor X (fXa) in the common pathway of blood coagulation began to emerge.

Structure Gene

The gene for human fV is on chromosome 1 at position q21–25. It is 72.3 kb in length and contains 25 exons and 24 introns, resulting in an mRNA of 6.9 kb (Figure 1). Exons 1–7 encode the A1 domain while exons 8–12 encode the A2 domain. These are followed by the largest exon in the gene (exon 13), which encodes the entire B domain. The A3 domain is encoded by exons 14–18, and exons 19–22 and 23–25 encode the C1 and C2 domains, respectively. Protein

fV is a very large glycoprotein (13% carbohydrate) that is the nonenzymatic cofactor for fXa. It is synthesized as a preprotein of 2224 amino acids containing 19 half cystines (Mr ¼ 249 000). Although the liver is the major source of plasma fV, endothelial cells and megakaryocytes have also been shown to synthesize fV and the latter may contribute to the presence of fV in platelets. The structure of fV is highly homologous to that of fVIII and consists of three A domains (homologous to ceruloplasmin), a single large B domain (no known homologies), and two C domains as depicted in Figure 2. fV undergoes several posttranslational modifications including glycosylation at 37 potential N-linked sites. The B domain is also unique in that it contains two conserved repeats of 17 amino acids and a large region containing 35 repeats of 9 amino acids with a consensus of [Thr, Asn, Pro]–Leu–Ser–Pro–Asp–Leu– Ser–Gln–Thr. Despite these features, there is no known function of the B domain other than to act as a spacer (activation peptide) to separate the heavy and light chains of fVa. Other posttranslational modifications of fV include the sulfation of seven

COAGULATION CASCADE / Factor V

1

2

3

4 5 6 7 8 9 10 11 12

A1

13

14 15 16 17

18 19 20 21 22

B

A2

A3

23

C1

491

24 25

C2

Figure 1 Organization of the fV gene. The organization of the fV gene depicted with corresponding encoded regions of each protein (not to scale). Exons (coding regions) are shown as black boxes and are numbered sequentially from left to right (50 to 30 ). Introns (noncoding regions) are shown as horizontal lines between exons. The region encoded by each exon is depicted below the gene as a colored bar. The fV gene is 72.3 kb in length and results in an mRNA of 6.9 kb. Exons 1–7 encode the A1 domain, exons 8–12 encode the A2 domain, exon 13 encodes the B domain, exons 14–18 encode the A3 domain, and exons 19–22 and 23–25 encode the C1 and C2 domains, respectively.

A1

A2

B

A3

C1

C2 Factor V -COOH polypeptide

NH2-

Processing (a) Activation cleavages 709 S−S

S−S

S−S

1018

1545 S−S

S−S

S−S

S−S

NH2SH

SO4

SO4 SO4 SO4

SH SH

SO4 SO4 SO4

-COOH SH

Processed factor V

SH

(b) APC inactivation cleavages 306 NH2-

506

A1

679 -COOH Heavy chain

A2

Ca2+ COOH-

C2

C1

(c)

-NH2

A3

Light chain

Factor Va

Figure 2 Organization of the fV protein. (a) The immature fV polypeptide containing the prepeptide followed by the A1, A2, B, A3, C1, and C2 domains. The 37 potential N-linked glycosylation sites are indicated as tree structures. The hatched region in the B domain reflects the location of the 35 repeats of 9 amino acids each and the two gray regions represent acidic regions within the B domain. The proteolytic processing site to remove the signal peptide is indicated by an arrow. (b) The fully processed and mature form of fV secreted into plasma. Predicted disulfide bonds are indicated above the bar by S–S and free sulfhydrils are indicated below the bar by –SH. The positions of the seven potential sulfated Tyr residues are indicated below the bar (SO4) and the positions of the three phosphorylated Ser residues are indicated above the bar as solid blue ovals. The three activation cleavage sites (Arg709, Arg1018, and Arg1545) are indicated by numbered arrows. (c) Activation of fV by thrombin or fXa releases the B domain in two fragments to produce an fVa heavy chain (A1– A2 fragment) and light chain (A3–C1–C2 fragment) as a heterodimer held together by a single calcium atom. The APC catalyzed inactivation cleavages (Arg306, Arg506, and Arg679) are indicated by numbered arrows (see also Figure 3).

tyrosine residues (Tyr665, Tyr696, Tyr698, Tyr1494, Tyr1510, Tyr1515, Tyr1565) and the phosphorylation of three serine residues (Ser692, Ser804, Ser1506). Subsequent to fV synthesis, a 28 amino acid signal peptide is removed to produce the mature single-chain form of fV that is secreted into plasma (2196 amino acids).

Regulation of Activation and Activity Activation

Plasma fV requires sequential proteolytic activation at three sites (Arg709, Arg1018, Arg1545) to produce the active cofactor (Figure 2). The primary activator of fV

492 COAGULATION CASCADE / Factor V

is thrombin, although fXa can also catalyze this reaction in the presence of ionic calcium and an anionic phospholipid surface. While plasmin has been reported to be capable of activating fV, this activation is transient as plasmin also proteolytically inactivates fVa (see below). The activation of fV releases the B domain as two fragments (Mr ¼ 71 000 and Mr ¼ 150 000) and produces the active fVa molecule as a noncovalent calcium-linked heterodimer composed of a heavy chain (A1–A2 fragment; Mr ¼ 105 000) and a light chain (A3–C1–C2 fragment; Mr ¼ 74 000). Activity

While fXa exhibits reactivity towards prothrombin in the absence of fV, this activity is very limited. The binding of unactivated fV to fXa is fairly weak and increases the activity of fXa only to a small extent. In contrast, fVa binds to fXa tightly and produces optimal fXa activity in the presence of ionic calcium and an anionic phospholipid surface. Thus, fV has the ability to directly control the activity of fXa producing three levels of procoagulant activity during coagulation: fXa with no cofactor, fXa bound to fV, and fXa bound to fVa. The latter form of the complex is the major procoagulant complex during coagulation. The membrane-binding portion of fVa resides in the light chain C1 and C2 domains (Figures 2 and 3). Although fXa can bind to anionic phospholipid in the absence of fVa, the high affinity of fVa for both phospholipid and fXa results in enhanced retention of fXa onto the lipid surface. Unlike fVIIIa, fVa is extremely stable in the presence of ionic calcium. Thus, inactivation of fVa occurs largely through proteolysis by activated protein C (APC) at Arg506 followed by Arg306 and Arg679 (Figures 2 and 3). Since both APC and fXa compete for binding to the fVa light chain, high levels of fXa can protect fVa from proteolytic inactivation by APC. However, as the ratio of fXa to APC is reduced when APC levels are increased as a result of the formation of the thrombin–thrombomodulin complex, the equilibrium would be expected to shift towards fVa inactivation. Additionally, in the presence of protein S (a cofactor for APC) the protective effect of fXa is abolished. The first cleavage by APC at Arg506 in fVa reduces the procoagulant activity of fVa by roughly 40%. Subsequent proteolysis at Arg306 (enhanced by protein S) results in complete abrogation of fVa cofactor activity. The importance of APC inactivation of fVa is evidenced in fVLeiden where Arg506 is mutated to Gln, resulting in persistent fVa activity and thrombophilia. Thrombin can partially inactivate fVa via cleavage at Arg643. Plasmin also inactivates fVa in a

phospholipid-dependent manner by proteolysis at numerous sites including Arg313 and Arg306 in the heavy chain and Arg1765 in the light chain. Since fVa is protected from inactivation by plasmin when bound to fXa, the significance of this reaction is unclear. Neutrophil elastase has also been shown to proteolytically inactivate fVa after partially activating it, similar to plasmin. It has been suggested that this may play a role during sepsis and other conditions of increased inflammation when elastase levels are elevated. Interestingly, cleavage at Arg506 in unactivated fV by APC has been shown to produce an alternate form of fV (fV506) that can serve as an anticoagulant cofactor in support of the APC-dependent inactivation of fVIIIa. The in vivo significance of this reaction has been questioned. Nonetheless, in certain pathological situations, this anticoagulant role of fV506 may prove to be of import.

Biological Function The concentration of fV in plasma is roughly 10 mg ml–1 with a half-life of 12 h. Plasma fV comprises about 80% of the total fV with the remaining 20% of the fV present in platelet a granules that can be secreted upon platelet activation. Although the majority of the fV in plasma is synthesized in the liver, endothelial cells and megakaryocytes have also been shown to be capable of synthesizing fV and may be a partial source of platelet fV. Deficiency of plasma fV results in the bleeding diathesis known as parahemophilia, a rare autosomal recessive trait. While causing only mild bleeding problems in humans, deletion of the fV gene in transgenic mice leads to either midembryogenic lethality or fatal perinatal hemorrhage. Deficiency of platelet fV also results in a bleeding diathesis in humans (fVQuebec), although this disorder has been shown to be due to the general degradation of platelet a-granule proteins and is not solely due to deficiency of platelet fV. Interestingly, while platelet fV in humans has been shown to be largely derived from uptake of plasma fV, murine transgenic models have demonstrated a distinct difference between plasma and platelet pools of fV in mice. When fV was expressed solely in murine platelets it was capable of rescuing the lethal phenotype of fV knockout mice, suggesting a critical role of platelet fV in hemostasis.

FV in Respiratory Diseases Normal lung function requires effective hemostasis. However, if left unregulated, excessive procoagulant

COAGULATION CASCADE / Factor V

493

A2

506

A3

306 A1 C1

C2

Figure 3 Three-dimensional model of the activated fV molecule. A three-dimensional model of fVa (Protein Databank file 1FV4) depicting the relation of fVa to the phospholipid surface (along the bottom; not shown) and the organization of the fVa heavy and light chains. The various domains are labeled and color coded (A1 ¼ purple; A2 ¼ orange; A3 ¼ red; C1 ¼ cyan; C2 ¼ blue). The proposed copper-binding site is shown with a Cu2 þ ion as an orange sphere between the A1 and A3 domains. Two potential sites for calcium binding are possible and are indicated by a Ca2 þ ion as a green sphere. The free sulfhydril residues are indicated as green sticks (two in the A2 domain and one each in the C1 and C2 domains) and the two major APC inactivation sites (Arg506 and Arg306) are indicated in yellow by arrows.

activity can lead to pulmonary fibrosis and potentially pulmonary emboli, both of which can be followed by respiratory failure. Excessive fibrin deposition can occur as a direct result of septic shock as well as pneumonia and other acute pulmonary events. These situations can result in acute lung injury (ALI), which is manifested most severely in acute respiratory distress syndrome (ARDS). A hypercoagulable state is observed in these cases as increased levels of clotting factors in pulmonary fluids. As with the other coagulation factors, the levels of fV are increased in bronchoalveolar lavage and pleural fluids in patients with ALI. Interestingly, the fV in these fluids has been reported to be largely inactive. It is unclear what this inactive fV signifies, but considering the high levels of fXa and thrombin that are found in these fluids and the requirement of fXa for fVa to generate a-thrombin, this may be

indicative of excessive, persistent procoagulant activity resulting in accumulation of APC-inactivated fVa. This inactive fV may also represent degradation products of plasmin and/or elastase activity. Interestingly, the therapeutic administration of APC has demonstrated beneficial reductions in ALI in various animal models as well as demonstrated a significant reduction in mortality in phase III studies of sepsis (PROWESS). Thus, inactivation of fVa may be important in the control of fibrin deposition in cases of severe sepsis. Since sepsis is the most common cause of ARDS, this also implies an equal importance of fVa in ALI. See also: Acute Respiratory Distress Syndrome. Anticoagulants. Chemokines. Coagulation Cascade: Overview; Antithrombin III; Factor VII; Factor X; Fibrinogen and Fibrin; Intrinsic Factors; Protein C

494 COAGULATION CASCADE / Factor VII and Protein S; Thrombin; Tissue Factor. Complement. Endothelial Cells and Endothelium. G-Protein-Coupled Receptors. Leukocytes: Monocytes. Platelets. Proteinase-Activated Receptors. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis.

Factor VII L V M Rao, University of Texas Health Center, Tyler, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Further Reading Abraham E (2000) Coagulation abnormalities in acute lung injury and sepsis. American Journal of Respiratory Cell and Molecular Biology 22: 401–404. Brodsky SV (2002) Coagulation, fibrinolysis and angiogenesis: new insights from knockout mice. Experimental Nephrology 10(5–6): 299–306. Chapman HA (2004) Disorders of lung matrix remodeling. Journal of Clinical Investigation 113: 148–157. Duga S, Asselta R, and Tenchini ML (2004) Coagulation factor V. International Journal of Biochemistry and Cell Biology 36: 1393–1399. Hoyer LW, Wyshock EG, and Colman RW (1994) Coagulation factors: factors V and VIII. In: Colman RW, Hirsh J, Marder VJ, and Salzman EW (eds.) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn., pp. 109–133. Philadelphia: JB Lippincott Company. Ichinose A and Davie EW (1994) The blood coagulation factors: their cDNAs, genes, and expression. In: Colman RW, Hirsh J, Marder VJ, and Salzman EW (eds.) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn., pp. 19–54. Philadelphia: JB Lippincott Company. Laterre P-F, Wittebole X, and Dhainaut J-F (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31: S329–S336. Levi M, Schultz MJ, Rijneveld AW, and van der Poll T (2003) Bronchoalveolar coagulation and fibrinolysis in endotoxemia and pneumonia. Critical Care Medicine 31: S238– S242. Mann KG, Gaffney D, and Bovill EG (1995) Molecular biology, biochemistry, and lifespan of plasma coagulation factors. In: Beutler E, Lichtman MA, Coller BS, and Kipps TJ (eds.) Williams Hematology, 5th edn., pp. 1206–1226. New York: McGraw-Hill. Mann KG and Kalafatis M (2003) Factor V: a combination of Dr Jekyll and Mr Hyde. Blood 101(1): 20–30. Pellequer J-L, Gale AJ, Getzoff ED, and Griffin JH (2000) Three-dimensional model of coagulation factor Va bound to activated protein C. Thrombosis and Haemostasis 84: 849–857. Sun H, Yang TL, Yang A, Wang X, and Ginsburg D (2003) The murine platelet and plasma factor V pools are biosynthetically distinct and sufficient for minimal hemostasis. Blood 102(8): 2856–2861. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 1334– 1349. Welty-Wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inflammation in acute lung injury. Thrombosis and Haemostasis 88: 17–25. Yang TL, Cui J, Taylor JM, Yang A, Gruber SB, and Ginsburg D (2000) Rescue of fatal neonatal hemorrhage in factor V deficient mice by low level transgene expression. Thrombosis and Haemostasis 83: 70–77.

Factor VII(a) is responsible for triggering the clotting cascade that eventually leads to thrombin generation, fibrin deposition, and platelet activation. Factor VIIa (FVIIa) is enzymatically active only after complex formation with its cellular receptor, tissue factor (TF). Factor VIIa–TF activity in vivo is tightly regulated by plasma inhibitors, particularly tissue factor pathway inhibitor. In addition to maintaining hemostasis, FVIIa may also influence various cellular processes by transmitting cellular signals via activation of G-protein-coupled proteinase-activated receptors. Since TF is constitutively expressed in the lung on airway epithelium and populations of alveolar macrophages, transudation of FVII along other plasma proteins across the injured alveolar capillary barrier leads to activation of coagulation in lung, leading to fibrin formation. Further, ligation of TF by FVIIa induces expression of several immunoregulatory genes in the lung. These responses play a crucial role in ordered repair and restoration of lung structure and function. However, increased production of TF in the lung following an inflammatory injury would lead to an enhanced procoagulant response that is excessive relative to that needed for resolution and repair. This would lead to fibrin deposition in the alveoli, interstitium, and capillaries. This, coupled with increased cytokine production and cell migration and activation that may occur through FVIIa–TF-induced cell signaling, independent of coagulant response, would lead to acute lung injury, such as acute respiratory distress syndrome (ARDS). Thus, blockade of FVIIa binding to TF or inhibition of FVIIa–TF proteolytic activity may have therapeutic benefit in patients with ARDS.

Introduction After exposure of blood to an injured vessel wall, a native factor VII (FVII)–tissue factor (TF) complex is formed as the first step of TF-dependent blood coagulation. Tissue factor is a transmembrane cellular receptor for FVII. In health, TF is constitutively expressed in many cells, including fibroblasts and pericytes in and surrounding blood vessel walls, but is absent from blood cells and endothelial cells that line blood vessels. However, under certain pathophysiological conditions, monocytes and endothelial cells express TF. Thus, either vessel wall injury or a specific disease process allows FVII to come in contact with TF. Because the single-chain native FVII is a zymogen, i.e., functionally inert, there has been wide interest in elucidating how formation of the FVII–TF complex triggers the coagulation process. Initially, it was believed that native FVII possessed minimal enzymatic activity, sufficient to initiate the processes. However,

COAGULATION CASCADE / Factor VII

this was found not to be physiologically relevant. Rapid preferential activation of FVII bound to TF by trace amounts of downstream clotting proteases, factor IXa, and factor Xa, and/or autoactivation of FVII bound to TF by FVIIa–TF are now thought to be responsible for the conversion of FVII–TF complexes to FVIIa–TF complexes. In blood, about 1% of FVII circulates as FVIIa. It is still unclear how circulating FVIIa originates. Once traces of the FVIIa–TF complex are formed at the injury site, it readily activates its substrates factor X and factor IX to active serine proteases, factor Xa and factor IXa, respectively. Factor IXa generated by FVIIa–TF, in the presence of its cofactor factor VIIIa, efficiently activates factor X. Factor Xa, in turn, cleaves prothrombin to thrombin, which converts fibrinogen to fibrin. Factors Xa and IXa also activate FVII bound to TF to FVIIa–TF, thus amplifying the initiation stimulus. Thrombin activates the cofactors, factor VIII and factor V, which serve as membrane-bound receptors/cofactors for clotting factors IXa and Xa, respectively. The need for factor IXa/factor VIIIa-induced activation of factor X for hemostasis stems from the effective inhibition of FVIIa–TF complexes by a plasma inhibitor, tissue factor pathway inhibitor (TFPI) following the initial generation of factor Xa.

Structure Human FVII is a multidomain, single-chain glycoprotein, consisting of 406 amino acid residues with a molecular weight of 50 000. FVII is synthesized primarily in the liver and is secreted into the blood as a zymogen of a serine proteinase. Prior to secretion from the liver, FVII undergoes several posttranslational modifications, including g-carboxylation of 10 glutamic acid residues located in its N-terminus, incomplete b-hydroxylation of aspartic acid residue 63, N-glycosylation of two asparagine residues (residues 145 and 322), and O-glycosylation of serine 52 and serine 60 (Figure 1). The activation of FVII to FVIIa involves the hydrolysis of a single peptide bond between arginine 152 and isoleucine 153, resulting in a two-chain molecule, consisting of a light chain of 152 amino acid residues and a heavy chain of 254 amino acid residues. The light chain of FVIIa contains the g-carboxyglutamic acid (GLA) domain (located in residues 1–45), followed by an aromatic stack-domain and two epidermal growth factor (EGF)-like domains. The heavy chain of FVIIa contains the catalytic domain featuring the active site catalytic triad (His-193, Asp-242, and Ser344). The catalytic domain of FVIIa exhibits a high

GLA AS EGF

EGF

495

Catalytic domain


Figure 1 A schematic representation of FVII structure. FVII is a multidomain protein consisting of an N-terminal domain containing 10 g-carboxy glutamic acid residues (GLA), followed by an aromatic stack (AS), two epidermal growth factor-like domains (EGF), and a catalytic domain at the C-terminal with Oglycosylation of serine 52 and 60 in the first EGF domain and N-glycosylation of asparagines 145 and 322. b represents potential b-hydroxylation of aspartic acid 63. The arrowhead points to the activational cleavage site at Arg152-Ile153.

degree of sequence and structural homology with trypsin.

Regulation of Production and Activity Factor VII concentration in plasma, compared to other vitamin K-dependent clotting factors, is extremely low (0.5 mg ml
1 or 10 nM). Limited steadystate FVII mRNA levels in the liver are responsible for the low mean plasma concentration of FVII. The liver is believed to be the entire source for plasma FVII as FVII mRNA expression is restricted to the liver. FVII may be synthesized locally (extrahepatically) in a limited number of cells under special circumstances, such as alveolar macrophages in interstitial lung diseases and smooth muscle cells in human atherosclerotic vessels. The human FVII gene spans 13 kb and is located in chromosome 13, just 2.8 kb 50 to the factor X gene. As in promoters of many other clotting factor genes, the FVII promoter lacks a TATA box, a sequence present in about 80% of RNA polymerase II eukaryotic promoters. A major transcription start site is identified at
51. The first 185 bp 50 of the transcription start site are sufficient to confer maximal promoter activity. A liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4), and a ubiquitous transcription factor, Sp1, which are shown to bind within the first 108 bp of the promoter region of FVII, play a critical role in FVII promoter activity. ARP1, an orphan nuclear hormone receptor, interacts with two regions of the FVII 50 flanking region, the HNF-4 binding region (
77 to
47), and the nuclear hormone response region (
237 to
200). ARP1 binding represses the transcriptional activation of the FVII gene. Expression of FVII may also be modulated by interactions taking place outside of the HNF-4 and Sp1 binding region of the promoter. A decanucleotide insert polymorphism at
323 is shown to reduce FVII gene expression.

496 COAGULATION CASCADE / Factor VII

The decanucleotide insert polymorphism correlates with both a lower FVII antigen and coagulant activity. In contrast to the decanucleotide insertion, a base substitution at
402 (G-A) is associated with the increased FVII expression. Once FVII is secreted from the liver to circulating blood, its activity is regulated by a variety of mechanisms; among them activation of FVII by cleavage of the peptide bond between Arg 152 and Ile 153, and allosteric influences exerted by cofactor TF, substrates, and inhibitors play predominant roles. Activation of zymogen FVII to enzyme FVIIa is largely TF-dependent. Upon activation, FVII structure undergoes conformational changes that form the substrate-binding cleft. However, the conformational change in FVIIa is incomplete until it binds to TF. Thus, FVIIa alone exists only in a partially active form, and is driven to the active enzyme under the influence of TF. Among the clotting factors, FVII has the shortest half-life – approximately 2–3 h. In contrast to other activated clotting proteins, which are cleared rapidly from the circulation (within seconds to a few minutes), FVIIa exhibits a long circulatory half-life (t1/2 ¼ B2 h), about the same as the zymogen. Pharmacokinetic studies in rats suggested that the liver was responsible for a major proportion of the FVIIa clearance. At present, the receptors and proteins that are responsible for the clearance of FVII or FVIIa in the liver are unknown.

Biological Function The principal biological function of FVII is to maintain hemostasis. Vascular damage that permits blood to leave the vasculature can be fatal unless the blood loss is stopped fast. FVII is responsible for initiating the cascade of highly sensitive and fast-acting proteolytic events that ultimately lead to the clot that seals the injury. Vessel wall injury disrupts the endothelial cell barrier that normally separates TFexpressing cells from the circulating blood, thus allowing circulating FVII to come in contact with TF. Complex formation with TF facilitates the cleavage of FVII zymogen to its active form, FVIIa. FVIIa, in concert with TF, then activates factor IX and X leading to localized thrombin generation, which subsequently leads to fibrin deposition, platelet activation, and clot formation, thereby stopping blood loss. However, FVII action can also lead to a deleterious initiation of the cascade within the vasculature in pathological conditions. For example, FVIIa-induced clotting upon the rupture of an atherosclerotic plaque would lead to a major blood clot (thrombus formation) that could become life-threatening.

Initiation of FVIIa-induced clotting in sepsis will lead to disseminated intravascular coagulation and the bleeding disorder. It is important to note that aberrant expression of TF in pathological conditions, not changes in circulating FVII, is primarily responsible for thrombotic disorders associated with the initiation of the coagulation cascade. The FVIIa–TF-induced coagulation pathway not only leads to clot formation but also contributes to vascular remodeling, which is caused by growth factors secreted by activated platelets as well as the intermediary products, factor Xa and thrombin, that promote vascular smooth cell proliferation and alter endothelium. In addition, FVIIa protease may also have other biological functions that are not related to its coagulant activity. Recent studies suggest that FVIIa interaction with TF on a variety of cells, including fibroblasts, epithelial cells, and endothelial cells, activates multiple signaling pathways leading to altered gene expression, ultimately affecting various cellular processes, such as cell survival, proliferation, and migration. FVIIa–TF is shown to activate cell signaling by activating G-protein-coupled proteinaseactivated receptors (PARs), particularly PAR2. FVIIa–TF-induced cell signaling could contribute to various pathophysiological processes, such as development, inflammation, angiogenesis, tumor metastasis, wound healing, and vascular remodeling. However, one should note here that, at present, the evidence for noncoagulant biological functions for FVIIa is mainly derived from data from cell model systems. This needs to be confirmed using relevant in vivo model systems.

Receptors Tissue factor is the only known cellular receptor for FVII and FVIIa. Tissue factor is a transmembrane glycoprotein with a 219-amino acid extracellular domain, a 23-residue transmembrane region, and a 21residue intracellular domain. The lungs and central nervous system are known to contain high levels of TF activity. In the lungs, the bronchial mucosa and alveolar epithelial cells constitutively express high levels of TF. Tissue factor is constitutively expressed on the surface of many extravascular cells, such as fibroblasts and pericytes within the blood vessel wall, but not in cells within vasculature that contact blood, such as monocytes and endothelial cells. However, under certain pathological conditions, such as sepsis, monocytes/macrophages express TF. Although TF can be readily induced in cultured endothelial cells by a variety of stimuli, such as inflammatory cytokines, bacterial lipopolysaccharides, and growth factors, its expression in vivo is somewhat

COAGULATION CASCADE / Factor VII

controversial. Vessel wall injury that disrupts the endothelial cell barrier or pathological expression of TF in monocytes and endothelial cells results in circulating blood coming into contact with TF on cell surfaces, allowing FVII to interact with TF, and subsequent activation of the coagulation pathway. Recent studies suggest that, in addition to the presence of TF in the vessel wall, there is a pool of TF in circulating blood that contributes to the propagation of thrombosis at the site of vascular injury. At present, the source of blood-borne TF and the physiological significance of blood-borne TF to hemostasis are unclear.

Factor VII in Respiratory Diseases Fibrin is a conspicuous part of acute and chronic inflammatory disorders of the lung. Extravascular fibrin is shown to play an important role in pathogenesis of both acute lung injury and repair. Coagulation proteases and extravascular fibrin may liberate cleavage products that are capable of potentiating the acute inflammatory response via effects on vascular permeability, chemotaxis, and immunomodulation. Further, fibrin deposits influence the reparative response to acute lung injury by promoting collagen deposition. Persistent fibrin deposition in areas of pulmonary scarring will lead to fibrosis. In addition to fibrin, other coagulation proteases, particularly thrombin, may also play a role in pathogenesis of lung diseases by promoting inflammation, fibroblast proliferation, and collagen deposition through activation of proteinase-activated receptors. In acute lung injury, alveolar fibrin deposition is potentiated by consistent changes in endogenous coagulation and fibrinolytic pathways. The only known physiological pathway that leads to thrombin generation and fibrin formation is the FVIIa–TF pathway of blood coagulation. Thus, FVII/FVIIa, through generation of thrombin and fibrin, plays a critical role in respiratory diseases. Consistent with this, increased FVII activity levels were observed in bronchoalveolar lavage fluids of patients with a variety of lung diseases, such as sarcoidosis, idiopathic pulmonary fibrosis, adult respiratory distress syndrome (ARDS), and interstitial lung diseases. Acute lung injury also leads to the upregulation of TF in resident and infiltrating macrophages, as well as in other constitutive cell populations within the lung. Local overexpression of TF in association with increased FVII levels and the presence of other downstream clotting proteins in alveolar lining fluids predispose the patients with respiratory diseases to alveolar fibrin deposition. Transudation of plasma proteins across the injured alveolar capillary is

497

primarily responsible for the increased FVII levels in the alveolar compartment. However, alveolar macrophages were also shown to synthesize FVII locally. Although TFPI levels are also increased in alveolar lining fluid in response to acute lung injury, the levels are not sufficient to block the FVIIa–TF-induced coagulation. In addition to the alveolar compartment, the disordered fibrin is also observed in the injured pleural space. Increased coagulation and concurrently depressed fibrinolysis was found in the pleural fluids of patients with exudative pleuritis. Increased levels of FVII and TF in the pleural fluid in response to injury are responsible for the increased procoagulant activity. Although the overall pattern of increased coagulation in pleural exudates is similar to those observed in alveolar injury, the procoagulant activity expressed in exudative pleural fluids was much lower than that observed in bronchoalveolar lavage (BAL). This could be due to increased concentration of coagulation inhibitors in pleural fluids. Although fibrin deposition in the alveolar and interstitial spaces of the lung is a well-defined feature of ARDS and plays an important role in lung injury and repair, FVIIa–TF complexes may also exert direct effects on the pathogenesis of lung injury (Figure 2). FVIIa binding to TF is shown to induce the expression of several immunoregulatory genes that may be involved in the pathogenesis of ARDS. FVIIa
TF is shown to stimulate IL-1b, IL-8, and other chemokines and growth factors, which could regulate cell migration, activation, and collagen production. FVIIa interaction with TF on lung fibroblasts upregulates the expression of profibrotic factors, connective tissue growth factor (CCN2), and Cyr61 (CCN1). In addition to its effects on cytokine and growth factor production, FVIIa is also shown to promote the production of reactive oxygen species by macrophages and to increase the expression of MHC class II and b integrin expression in macrophages. More importantly, serine proteases generated by FVIIa–TF activation of the coagulation pathway, i.e., factor Xa and thrombin, also have independent inflammatory signaling functions (Figure 2). The importance of FVII in ARDS is well illustrated in the baboon model system. Blockade of FVII interaction with TF by administering active site inactivated human FVIIa (ASIS) or by inhibiting the proteolytic function of FVIIa–TF complex with TFPI, prevented respiratory failure in sepsis-induced acute lung injury. In the lung, ASIS prevented pulmonary hypertension and edema, preserved gas exchange and lung compliance, and decreased fibrin deposition in the alveolar space. Consistent with data obtained in experimental models of severe sepsis, phase II clinical

498 COAGULATION CASCADE / Factor VII Coagulation proteins

Signaling receptor

TF VIIa PAR2

X

Inflammatory and other effects IL-1, IL-6, IL-8, chemokines, growth factors, ROS, VEGF, CCN1, CCN2, collagenases, pulmonary fibrosis

Xa PAR1

TF VIIa Xa Va Prothrombin

PAR2

Thrombin PAR1

PAR3

Fibrinogen

Mostly same as above, MCP-1, edema

Fibrin PAR4

Platelet activation, secretion vascular tone, cell motility, cell permeability, PMN chemotaxis, inflammatory cytokines, adhesion molecules, growth factors, pulmonary fibrosis

Figure 2 Noncoagulant functions of FVIIa and downstream clotting proteases generated by FVIIa. FVIIa–TF, directly or through elaboration of factor Xa and thrombin, upregulates the expression of various proinflammatory cytokines and promotes cell migration and activation in lung via activation of proteinase-activated receptors. These responses contribute to ordered repair and pathogenesis of acute lung injury through multilevel interactions among them. TF, tissue factor; PAR, proteinase-activated receptor; IL, interleukin; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor; MCP-1, monocyte chemoattractant protein-1; PMN, polymorphonuclear cells.

trials showed that TFPI is effective in reducing mortality in patients with severe sepsis. However, a larger phase III trial failed to show a significant difference in 28-day all-cause mortality between the TFPI treatment and placebo groups. In summary, FVIIa–TF-mediated coagulation plays a major role in the development of lung injury in sepsis. FVIIa, TF, and downstream products, factor Xa, thrombin, and fibrin all contribute to the lung injury in ARDS. Blockade of FVIIa–TF function by natural plasma inhibitor or modified FVIIa is shown to prevent lung injury, suggesting that the blockade of FVIIa–TF may be a feasible therapeutic strategy in patients with ARDS. See also: Acute Respiratory Distress Syndrome. Coagulation Cascade: Factor X; Thrombin; Tissue Factor. G-Protein-Coupled Receptors.

Further Reading Broze GJ Jr (1995) Tissue factor pathway inhibitor and the current concept of blood coagulation. Blood Coagulation and Fibrinolysis 6: S7–S13. Edgington T, Dickinson CD, and Ruf W (1997) The structural basis of function of the TF-VIIa complex in the cellular initiation of coagulation. Thrombosis and Haemostasis 78: 401–405. Eigenbrot C (2002) Structure, function, and activation of coagulation factor VII. Current Protein and Peptide Science 3: 287–299.

Hagen FS, Gray CL, O’Hara P, et al. (1986) Characterization of a cDNA coding for human factor VII. Proceedings of the National Academy of Sciences, USA 83: 2412–2416. Idell S (2003) Coagulation and fibrinolysis, and fibrin deposition in acute lung injury. Critical Care Medicine 31(supplement 4): S213–S220. Idell S, James KK, Levin EG, et al. (1989) Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. Journal of Clinical Investigation 84: 695–705. Konigsberg W, Kirchhofer D, Riederer MA, and Nemerson Y (2001) The TF:VIIa complex: clinical significance, structure– function relationships and its role in signaling and metastasis. Thrombosis and Haemostasis 86: 757–771. Monroe DM, Hoffman M, and Roberts HR (2002) Platelets and thrombin generation. Arteriosclerosis, Thrombosis, and Vascular Biology 22: 1381–1389. Pollak ES, Hung HL, Godin W, Overton GC, and High KA (1996) Functional characterization of the human factor VII 50 -flanking region. Journal of Biological Chemistry 271: 1738–1747. Rao LVM and Pendurthi UR (2005) Tissue factor
factor VIIa signaling. Arteriosclerosis, Thrombosis, and Vascular Biology 25: 47–56. Rapaport SI and Rao LVM (1995) The tissue factor pathway: how it has become a ‘prima ballerina’. Thrombosis and Haemostasis 74: 7–17. Thim L, Bjoern S, Christensen M, et al. (1988) Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells. Biochemistry 27: 7785–7793. Welty-Wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inflammation in acute lung injury. Thrombosis and Haemostasis 88: 17–25.

COAGULATION CASCADE / Factor X

renamed fX based on the Roman numeral naming convention adopted by the International Committee for the Standardization of the Nomenclature of Blood Clotting Factors in 1959. The function of fX in coagulation was initially unclear since fX acted as a substrate for some reactions but as an enzyme in others. The view of fX as a zymogen that is activated to an enzyme was not accepted until the early 1960s when the coagulant action of Russell’s viper venom was found to be directed towards fX whose product, fXa, was capable of activating prothrombin. This discovery, along with the realization that fX was activated by the intrinsic and extrinsic pathways, led in 1964 to fX being given a central position in the coagulation cascade.

Factor X P F Neuenschwander, University of Texas Health Center, Tyler, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Blood coagulation factor X (fX) is a vitamin-K dependent serine protease zymogen synthesized in the liver and present in the circulation as a glycosylated, two-chain, disulfide-linked molecule. Activation of fX occurs via limited proteolysis at a single site to release a small 52 amino acid-activation peptide. This reaction is catalyzed by either the intrinsic or extrinsic pathways of blood coagulation and requires ionic calcium as well as anionic phospholipid exposed on activated platelets or damaged cell surfaces. The activated fX that is generated, fXa, is an active serine protease and displays high homology to the trypsin family of serine proteases. FXa is involved in propagation of the coagulant response as well as in cell signaling pathways linked with the inflammatory response. The procoagulant activity of fXa requires calcium and anionic phospholipid, enabling fXa to bind to its nonenzymatic protein cofactor fVa (activated fV) and activate prothrombin to generate a-thrombin. FXa can also enhance its own generation and activity via positive feedback activation of fVII and fVIII, as well as activation of fV. The cellsignaling function of fXa does not require fVa but requires a viable enzyme active site for binding to effector cell protease receptor-1 (EPR-1) or activation of protease-activated receptors PAR-1 and PAR-2. Unregulated activation or activity of fX/fXa in the lung is associated with the development of pulmonary fibrosis and the inflammatory response observed in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS).

Structure Gene

The gene for human fX is on chromosome 13 at position q32-qter. It is 27 kb long and contains eight exons (I–VIII) and seven introns (A–G), resulting in an mRNA of 1.5 kb (Figure 1). Exon I encodes a prepeptide and exon II encodes a propeptide and g-carboxyglutamic acid-containing domain (Gla). These are separated from each other by intron A (6.5 kb) and from the other exons by intron B (8.8 kb). Exon III is the smallest and encodes an 8 amino acid aromatic stack region separating the Gla domain from two epidermal growth factor (EGF)like repeats encoded by exons IV and V. Intron C (0.9 kb), the smallest in the gene, separates exons III and IV while intron D (1.4 kb) separates exons IV and V. Exons encoding the light chain of fX (I–V) are separated from those encoding the heavy chain (VI– VIII) by intron E (2.8 kb). Exon VI encodes the activation peptide region and a small portion of the protease domain. This is followed by intron F (3.2 kb), exon VII, and intron G (1.4 kb). Exon VIII

Introduction Factor X (fX), also called Stuart factor, is a vitamin-K dependent serine protease zymogen that is activated in the first common step of the intrinsic and extrinsic pathways of blood coagulation. It was serendipitously discovered in 1956 through a patient exhibiting a hemorrhagic disorder not attributable to a deficiency of any of the known clotting factors of the time. This new factor was named after the patient first described with this deficiency (Mr. Stuart). Stuart factor was

I

II A

pre

pro

B

gla

as egf-1

499

III IV V VI C D E F

egf-2

ap

VII VIII G

Protease domain

Figure 1 Organization of the fX gene. Exons (coding regions) are shown as blue boxes and are numbered sequentially from I to VIII. Introns A–G (noncoding regions) are shown as horizontal lines between exons. The regions of fX encoded by each exon is depicted below the gene as a shaded bar: pre, prepeptide coding region; pro, propeptide coding region; gla, g-carboxyglutamic acid domain coding region; as, aromatic stack coding region; egf-1 and egf-2, epidermal growth factor-like domain coding regions; ap, activation peptide coding region; protease domain, catalytic domain coding region.

500 COAGULATION CASCADE / Factor X

encodes the majority of the protease domain and at 0.6 kb is the longest uninterrupted coding sequence in the gene.

(Mr) of fX from 58 200 to 58 900. Additional processing of fX occurs by an unknown mechanism to release the basic tripeptide Arg140–Lys–Arg142 from the center of the molecule, dividing fX into two chains. The mature form of fX in plasma is a glycosylated two-chain zymogen linked by a disulfide bond between Cys132 and Cys302 (Figure 2(b)). The N-terminally derived light chain (Mr ¼ 16 200) contains the Gla- and EGF-like domains, and the C-terminally derived heavy chain (Mr ¼ 42 000) contains the activation peptide and the latent serine protease domain. The activated form of fX, fXa, is an active serine protease sharing structural homology to the trypsin family and containing the canonical catalytic triad His42, Asp88, and Ser185 (His57, Asp102, and Ser195 by chymotrypsinogen numbering convention). fXa exhibits procoagulant and esterase activities. The latter is dependent exclusively on the heavy chain whereas the former requires the additional presence

Protein

The fX protein is synthesized in the liver as a singlechain pre-proprotein of 488 amino acids with 24 half cystines (Figure 2(a)). The first 40 amino acids of the polypeptide constitute a leader sequence containing the pre- and propeptides. The prepeptide signal sequence is cleaved off by a signal peptidase while the propeptide directs posttranslational g-carboxylation of the first 11 Glu residues of the N-terminus (Gladomain) and is subsequently removed by a processing protease. fX also undergoes b-hydroxylation of Asp63 in the first EGF-like domain and N-linked glycosylation at Asn157 and Asn167 within the activation peptide region. The high degree of glycosylation (15% carbohydrate) increases the relative mass

N -linked carbohydrate

Factor X polypeptide

NH2−













Processing






Pre-pro leader Gla domain sequence Processing



− COOH

EGF1

EGF2

Protease (catalytic) domain

(a)

Activation cleavage Activation peptide

Processed factor X NH2−




















H

− COO NH 2 − 

− COOH Heavy chain

Light chain

Activated factor X (factor Xa) NH2−




















−COOH

NH

2−

(b)

 Light chain

− COOH Heavy chain

(c) Figure 2 Structural elements of the fX protein. (a) The immature fX polypeptide containing the pre-propeptides, g-carboxyglutamic acid (Gla) domain, EGF-like repeats, activation peptide, and protease domain. The 11 Gla residues are indicated (g) as is the bhydroxylated Asp63 (b) and glycosylation sites in the activation peptide region. Disulfide bonds are indicated by thin lines connecting looped regions of the main chain. Sites of proteolytic processing are indicated by the three arrows. (b) The fully processed and mature form of fX found in plasma. The tripeptide Arg–Lys–Arg has been removed to produce the two-chain form. The light and heavy chains (indicated) remain held together by a single disulfide bond in the center of the amino acid sequence. The activation cleavage site and the glycosylated activation peptide are indicated by arrows. The activation peptide is released upon proteolysis to produce fXa (c).

COAGULATION CASCADE / Factor X 221-loop 184-loop

124-loop

145-loop

501

its activity towards fX; and (3) calcium binding at the junction of the Gla- and EGF-1 domains of the activating enzyme and fX correctly alters their conformation and topography on the phospholipid surface. The binding to phospholipid surfaces is regulated by the anionic character of the membrane on activated platelets or damaged cell surfaces and increases the local concentration of enzyme and substrate at the surface to substantially augment catalysis. Activity

60-loop

74-loop

243-loop 36-loop

Figure 3 Insertion loops in the fXa catalytic (serine protease) domain. The crystal structure of the fXa heavy chain or protease domain (Protein Databank code 1FAX) showing the insertion regions in comparison to chymotrypsin (labeled colored regions). The canonical serine protease catalytic triad residues (Ser, His, Asp) are indicated as orange stick structures in the center of the molecule. The orange sphere in the lower left of the structure is a bound calcium ion required for the procoagulant and esterase activities of fXa.

of the light chain. Despite the high level of structural homology between fXa and the trypsin family, fXa differs from this family by the insertion of varioussized peptide sequences on the external surface of the molecule (Figure 3). These regions, or insertion loops, aid in defining the physiological behavior of fX/Xa and impart to fXa its individual specificity for substrates and inhibitors.

Regulation of Activation and Activity

fXa activates prothrombin via proteolysis of two bonds to produce a-thrombin and an activation peptide (fragment 1–2). While this reaction can be slowly catalyzed by fXa alone, the true procoagulant in this step is a complex of fXa and fVa, the latter being a nonenzymatic protein cofactor. The calcium- and phospholipid-dependent formation of the fXa–fVa complex increases the proteolysis of prothrombin roughly 3
105-fold and alters the bondcleavage order to produce a different intermediate than that observed with fXa alone: fXa produces an inactive prethrombin-2 intermediate while the fXa–fVa complex produces a partially active meizothrombin intermediate. FXa is inhibited in plasma principally by the serine protease inhibitor (serpin) antithrombin (antithrombin III). Heparin, or heparan sulfate on cell surfaces, mediates this inhibition by binding antithrombin and inducing a conformational change in the inhibitor. fXa can also be inhibited by tissue factor pathway inhibitor (TFPI) and, to a lesser extent, a2-macroglobulin. Attenuation of fXa activity also occurs via proteolytic inactivation of fVa by activated protein C (APC), resulting in reduced enhancement of fXa activity.

Activation

fX zymogen is activated to fXa via hydrolysis of a peptide bond in the heavy chain at Arg194. This releases the heavily glycosylated activation peptide (52 amino acids; Mr ¼ 8000) from the N-terminus of the heavy chain (Figure 2(c)). Physiologically, this reaction is catalyzed by either of two enzyme–cofactor complexes, fIXa–fVIIIa or fVIIa–tissue factor (TF), each of which generates the identical fXa product. Both reactions have an absolute requirement for calcium ions and anionic phospholipid. The calcium requirement is threefold: (1) calcium binding to the Gla-domains of the activating enzyme and fX refolds these domains to promote their interaction with phospholipid surfaces; (2) calcium binding to the protease domain of the activating enzyme enhances

Biological Function Human fX zymogen exists in plasma at a concentration of 140–170 nM with a half life of B1.5 days. While the major procoagulant role of fXa is to generate a-thrombin, fXa can also activate fVII in a fVaindependent positive feedback loop, as well as fV and fVIII (Figure 4). Although the latter two reactions are inefficient, they may be important in early stages of coagulation to generate initial small amounts of these active cofactors. The importance of fXa in coagulation is manifested by the bleeding diathesis observed in patients deficient in fX, the severity of which is proportional to the level of deficiency. Complete deletion of the fX gene in transgenic knockout mice leads to partial

502 COAGULATION CASCADE / Factor X

Intrinsic VIII

Extrinsic VII

IXa VIIa

VIIIa

PL, Ca2+

TF X PL,

TFPI

Ca2+

Xa

V Va

Antithrombin

PL, Ca2+

Heparin

Prothrombin Thrombin

Fibrinogen Fibrin clot

Figure 4 Biological procoagulant activities and inhibition of fXa. fX is activated to fXa by the intrinsic or extrinsic pathway. The fXa generated subsequently activates prothrombin to a-thrombin leading to fibrin deposition. FXa can augment its own generation by positive feedback activation of fVII and fVIII. Both of these reactions occur independent of fVa and require phospholipid (PL) and ionic calcium (Ca2 þ ). FXa can also enhance its own activity towards prothrombin by activation of fV to generate fVa. Solid arrows represent proteolytic conversions and dotted arrows represent enzymatic actions. Enzymes are indicated by boldface text compared to cofactors and zymogens. A lowercase ‘a’ following the numerical designation designates an activated molecule. Regulation of fXa activity by the inhibitors antithrombin and TFPI are also indicated. TF, tissue factor.

embryonic lethality due to excessive bleeding as well as fatal bleeding in neonates. In addition to its procoagulant role, fXa is involved in cell signaling and induces mitosis in smooth muscle cells, nitric oxide release, and expression of adhesion molecules and TF in endothelium. Cytokine and chemokine production is also differentially affected by fXa in various cell types. FXa induces monocyte chemotactic protein-1 (MCP-1) and interleukins IL-6 and IL-8 in endothelium, and cytokines vascular endothelial growth factor (VEGF) and platelet-derived growth factor-A (PDGF-A) in lung fibroblasts. These signaling activities all require a functional fXa active site and either binding to effector cell protease receptor-1(EPR-1) or proteolytic activity toward protease-activated receptor-1 (PAR-1) or PAR-2.

Receptors The activity of fXa as a cell-signaling factor involves the cellular protease-activated receptors PAR-1 and PAR-2. These G-protein-coupled receptors are

integral membrane proteins containing seven membrane-spanning helices with an intracellular C-terminus and an extracellular N-terminus. The four currently identified PARs are differentially expressed on various cell types and are activated via proteolytic cleavage rather than ligand binding. PAR-1 is expressed on the surface of platelets, mononuclear cells, and endothelial cells while PAR-2 is expressed on the surface of mononuclear cells, endothelial cells, and neutrophils. Although each PAR is slightly different and exhibits a unique combination of specificities for various proteases, their general activation scheme remains the same: cleavage of an activation site within the extracellular domain of the receptor produces a new N-terminus. This new N-terminus functions as a tethered ligand that binds intramolecularly to induce an intracellular signal via activation of heterotrimeric G-proteins. Although the procoagulant action of fXa does not per se require a cellular receptor, activated platelets express the receptor EPR-1 that is involved in expression of fXa procoagulant activity. EPR-1 is also expressed in leukocyte subpopulations, human brain microvascular pericytes, smooth muscle cells, endothelial cells, and human lung fibroblasts. Unlike the PARs, EPR-1 does not require proteolysis to function. Although the active site of fXa is required for the cell signaling function of EPR-1, it is not required for binding to EPR-1. It is likely that the signaling function of EPR-1 on endothelial cells is mediated through PAR activation by the fXa–EPR-1 complex.

fX in Respiratory Diseases Effective hemostasis is paramount for the maintenance of normal lung function. Left unregulated, excessive procoagulant activity in the lung can lead to the onset of pulmonary fibrosis followed by respiratory failure. This circumstance can be brought about by sepsis, pneumonia, or other acute catastrophic events and is archetypical of the most severe manifestation of acute lung injury (ALI), acute respiratory distress syndrome (ARDS). The increased procoagulant state in ARDS is evidenced by increased levels of clotting factors in bronchoalveolar lavage and pleural fluids of afflicted patients. fXa plays a dual role in this setting by also increasing the proinflammatory response via receptor-based signaling on platelets, mononuclear cells, endothelial cells, and neutrophils. Therapeutic strategies for treatment of pulmonary fibrosis have included the use of coagulation inhibitors such as antithrombin, heparin, TFPI, and APC to attenuate coagulation. Each of these inhibitors targets numerous enzymes including fXa: antithrombin

COAGULATION CASCADE / Fibrinogen and Fibrin 503

is a broad-spectrum serine protease inhibitor whose reactivity is enhanced by heparin. TFPI targets the fVIIa-TF activation of fX, but is also a potent fXa inhibitor in the absence of fVIIa-TF. APC inactivates fVa thus attenuating the procoagulant activity of fXa. Each of these inhibitors has demonstrated beneficial reductions in ALI in various animal models. Unfortunately, these results have not been reproduced in clinical studies. While TFPI was successful in phase II clinical trials, the results in phase III trials (OPTIMIST study) were disappointing and showed no beneficial effect above placebo. APC, however, has demonstrated a significant reduction in mortality in cases of severe sepsis (PROWESS study), which is the most common cause of ALI and ARDS. See also: Acute Respiratory Distress Syndrome. Anticoagulants. Chemokines. Coagulation Cascade: Overview; Antithrombin III; Factor V; Factor VII; Fibrinogen and Fibrin; Intrinsic Factors; Protein C and Protein S; Thrombin; Tissue Factor. Complement. Endothelial Cells and Endothelium. G-Protein-Coupled Receptors. Interleukins: IL-1 and IL-18; lL-4; lL-5; lL-6; lL-7; lL-9; IL-10; IL-12; IL-13; IL-15; IL-16; IL-17; IL-23 and IL-27. Leukocytes: Monocytes. Platelets. ProteinaseActivated Receptors. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis.

Further Reading Abraham E (2000) Coagulation abnormalities in acute lung injury and sepsis. American Journal of Respiratory Cell and Molecular Biology 22: 401–404. Altieri DC (1995) Proteases and protease receptors in modulation of leukocyte effector functions. Journal of Leukocyte Biology 58: 120–127. Altieri DC (1995) Xa receptor EPR-1. FASEB Journal 9: 860–865. Bretschneider E and Schror K (2001) Cellular effects of factor Xa on vascular smooth muscle cells – inhibition by heparins? Seminars in Thrombosis and Hemostasis. 27: 489–493. Chapman HA (2004) Disorders of lung matrix remodeling. Journal of Clinical Investigation 113: 148–157. Dugina TN, Kiseleva EV, Chistov IV, Umarova BA, and Strukova SM (2002) Receptors of the PAR family as a link between blood coagulation and inflammation. Biochemistry (Moscow) 67: 74–75. Ichinose A and Davie EW (1994) The blood coagulation factors: Their cDNAs, genes, and expression. In: Colman RW, Hirsh J, Marder VJ, and Salzman EW (eds.) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn., pp. 19–54. Philadelphia: J.B. Lippincott Company. Idell S (2001) Anticoagulants for acute respiratory distress syndrome: can they work? American Journal of Respiratory and Critical Care Medicine 164: 517–520. Laterre P-F, Wittebole X, and Dhainaut J-F (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31: S329– S336. Levi M, Schultz MJ, Rijneveld AW, and van der Poll T (2003) Bronchoalveolar coagulation and fibrinolysis in endotoxemia and pneumonia. Critical Care Medicine 31: S238–S242.

Mann KG, Gaffney D, and Bovill EG (1995) Molecular biology, biochemistry, and lifespan of plasma coagulation factors. In: Beutler E, Lichtman MA, Coller BS, and Kipps TJ (eds.) Williams Hematology, 5th edn., pp. 1206–1226. New York: McGraw-Hill. Riewald M and Ruf W (2002) Orchestration of coagulation protease signaling by tissue factor. Trends in Cardiovascular Medicine 12: 149–154. Ruf W, Dorfleutner A, and Riewald M (2003) Specificity of coagulation factor signaling. Journal of Thrombosis and Haemostasis 1: 1495–1503. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 1334–1349. Welty-Wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inflammation in acute lung injury. Thrombosis and Haemostasis 88: 17–25.

Fibrinogen and Fibrin A Gu¨nther and C Ruppert, University of Giessen Lung Center (UGLC), Giessen, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract Fibrinogen is a complex multifunctional glycoprotein that plays a key role during blood clotting. Thrombin-mediated conversion of fibrinogen into fibrin and the subsequent cross-linking by factor XIII results in the formation of the three-dimensional framework of the thrombus. In addition, fibrinogen promotes platelet adhesion and aggregation via interaction with a specific integrin receptor. Next to its essential function in normal hemostasis and wound repair fibrin(ogen) also exerts multiple regulatory effects and is involved in pathological processes such as thrombosis and atherosclerosis. Abnormal fibrin turnover has also been implicated in a number of respiratory diseases including acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and chronic interstitial lung diseases such as pulmonary fibrosis.

Introduction In 1666 the Italian anatomist Marcello Malphigi described fibrin as a white fibrous substance in a hemostatic plug and thus confirmed the much earlier observation of the Roman physician Claudius Galenos (AD 129–199) who proposed the presence of ‘fibers’ (Latin: fibrae) in circulating and clotted blood. In 1832 the German anatomist and physiologist Johannes Mu¨ller provided evidence that blood contains ‘soluble fibrin’, and in 1859 Denis de Commercy proposed the existence of a soluble fibrin precursor that could be salted out of plasma. In the second half of the nineteenth century Rudolf Virchow, a German pathologist, identified fibrinogen as being the precursor of fibrin, and Alexander Schmidt, a German physiologist, identified the so-called ‘fibrin-ferment’ (i.e., thrombin) as fibrinogen-converting factor. At the

504 COAGULATION CASCADE / Fibrinogen and Fibrin

same time the Swedish physiologist Olof Hammarsten advanced Schmidt’s theory and suggested that preceding fibrin formation, activation of fibrinogen by thrombin occurred by limited proteolysis. These observations led to Paul Morawitz’s classical four-factor theory of blood coagulation according to which coagulation is initiated when the clot-promoting factor thrombokinase (today referred to as thromboplastin or tissue factor) is released by destroyed tissues or liberated by platelets and leukocytes. Thrombokinase in the presence of calcium converts prothrombin into thrombin, which in turn converts fibrinogen into fibrin. In subsequent years identification of numerous clotting factors resulted in the proposal of the ‘waterfall’ or ‘cascade’ theory of blood clotting. In the final step of this classical paradigm of blood coagulation soluble fibrinogen is cleaved by thrombin forming soluble fibrin monomers, which then polymerize to make an insoluble fibrin clot. It took several decades to elucidate the exact activation mechanism of fibrinogen. In the early 1950s Bailey found that thrombin removed small peptides from the N-terminus of fibrinogen and hypothesized that the newly exposed N-termini must be the contact sites for fibrin polymerization. As new analysis tools became available in the second half of the twentieth century, new insights in fibrin(ogen) structure were given. Fibrinogen and fibrin are multifunctional proteins. Fibrinogen binds via a glycoprotein receptor (GPIIb/ IIIa) to platelets mediating platelet adhesion and aggregation during hemostasis. Fibrin is an essential matrix of wound repair by stabilizing wound fields and supporting local cell proliferation and migration.

chromosome 4 (4q28). For the g-chain an elongated splice variant (427 amino acid residues) exists in human plasma accounting for B8% of the total fibrinogen g pool. The major form of fibrinogen circulating in human plasma is that of a homodimeric g/g molecule (‘fibrinogen 1’), whereas heterodimeric g/g0 molecules (‘fibrinogen 2’) and homodimeric g0 /g0 molecules account for B15% and less than 1%, respectively. The central E-domain contains the fibrinopeptides (FP)-A and -B, which are released from the N-terminus of the Aa (Aa 1–16) and the Bb (Bb 1–14) chains upon enzymatic cleavage by thrombin. The C-terminal ends of the g-chain are important for interaction with GP-IIb/IIIa receptors on platelets, polymerization of fibrin, and cross-linking by FXIIIa. They also contain two self-association sites (gXL and D:D, see Figure 1), which participate in the fibrin(ogen) assembly and cross-linking process, and a high specific binding site for tPA. The C-terminal Aa-chain protuberances contain sites for interaction with platelets (integrin binding to arginine–glycine–aspartic acid (RGD) sequences). Two sets of high-affinity binding sites for both tPA and plasminogen, which play an important role in the initiation of fibrinolysis, are located on the a-C-domain and the distal D-domain. Calcium binding sites are integral parts of the fibrinogen molecule. Among them there are three high-affinity binding sites, two of which are associated with the D-domain and one located at the E-domain. Ca2 þ is important for stabilizing fibrinogen against heat denaturation and proteolytic digestion by trypsin or plasmin, and for fibrin monomer polymerization.

Structure

Fibrinogen Biosynthesis, Fibrin Assembly, Cross-Linking, and Fibrin(ogen)olysis

Fibrinogen is a trinodular disulfide-bridged molecule, composed of two symmetrical half molecules, each half consisting of three different polypeptide chains, termed Aa (610 amino acids, 67 kDa), Bb (461 amino acids, 56 kDa), and g (411 amino acids, 47 kDa) (Figure 1). The two halves are covalently joined in an antiparallel orientation in the central Edomain by five disulfide bridges. At the two ends of the cylindrical fibrinogen molecule, the globular Bband g-C-termini and the Aa-C-terminal random coil form the distal D-domains. The three polypetide ˚ -long coiled-coil rechains interweave forming 160 A gions, which connect the central E-domain to the outer D-domains. Inter-chain disulfide bonds further stabilize the structure. The overall molecular weight of fibrinogen is 340 kDa and its overall length is B450 A˚ (45 nm). Each of the polypeptide chains is a product of a single-copy gene located on the long arm of

Fibrinogen is constitutively produced in the liver and circulates in human plasma at a concentration of 150–400 mg dl
1, and has a half-life of 3–5 days. About 75% of the total fibrinogen pool circulates in plasma whereas the rest distributes to tissues, interstitial fluids, and lymph. Plasma fibrinogen levels are regulated by genetic (e.g., Bb-chain polymorphisms) as well as environmental factors. Each gene is separately transcribed and translated into a precursor polypeptide, which is then processed and assembled into the mature protein. The rate-limiting step in the fibrinogen biosynthesis is the synthesis of the fibrinogen Bb-chain. In addition to the liver, fibrinogen biosynthesis has been found in lung epithelial cells under inflammatory conditions. The thrombin-catalyzed release of the fibrinopeptides FP-A and FP-B exposes two types of binding site in the N-terminal regions of the fibrin molecules,

COAGULATION CASCADE / Fibrinogen and Fibrin 505

28 36 65

C C

8 9

C

N

N

N

N

N

N

28

C

36 65 9 8

Coiled-coil region

A
67 kDa B 56 kDa  47 kDa

C C

Coiled-coil region E

D D-domain

D

E-domain

D-domain

° 450 A

Db

Db


c FPB

D:D 

D:D



 FPA

Da ′

XL

 Da

XL

A

Fibrinogen Thrombin

FPA FPB

Fibrin
c Db

Db

D:D

EB 



 EA

Da XL

D:D

′

 Da

XL A

Figure 1 Structural model and domains of fibrin(ogen). The major domains (E- and D-domains) of fibrin(ogen) and the constitutive Ddomain association sites participating in fibrin polymerization and cross-linking are shown. EA/EB – polymerization sites that are exposed upon cleavage of the fibrinopeptides FPA and FPB. Da/Db – association sites that interact with available EA/EB sites in fibrin. gA/g0 – normal (gA) and elongated (g0 ) variant of the g-chains with FXIIIa-cross-linking site in the C-terminal region. gXL/D:D – self-association sites. ac – domain in the C-terminal region of the Aa-chain, which dissociates from its noncovalent association with the E-domain upon thrombin cleavage of FPB. Adapted from Mosesson MW (1998) Fibrinogen structure and fibrin clot assembly. Seminars in Thrombosis and Hemostasis 24(2): 169–174, with permission from Thieme New York.

termed EA and EB, which combine with constitutive complementary binding pockets in the D-domains (Da, Db) of neighboring molecules. The initial release of FP-A from the Aa-chain and (noncovalent) assembly of Da:EA associations results in the formation of double-stranded, twisted fibrils. The molecules within a strand are joined by longitudinal contacts between the D-domains (D:D interaction) (Figure 2). Subsequently and concomitantly, fibrils

undergo branching and lateral fibril association resulting in a network of fibers with increased thickness. The EB:Db interaction is not absolutely required for lateral fibril/fiber association, but it contributes to this process through cooperative interactions resulting from alignment of D-domains forming so-called trimolecular and tetramolecular branch points in the fibrin polymer (Figure 2). The fibrin clot, initially held together only by noncovalent

506 COAGULATION CASCADE / Fibrinogen and Fibrin

interactions, becomes further stabilized by the incorporation of covalent bonds between C-terminal gXL sites of fibrin units. FXIIIa (plasma transglutaminase) catalyzes the formation of covalent e-(g-glutamyl)lysine isopeptide bonds in the presence of Ca2 þ resulting in the formation of g-dimers and higherorder forms of cross-linked g-chains (g-trimers, g-tetramers). Plasmin-catalyzed degradation of fibrinogen and fibrin occurs similarly; however, degradation of cross-linked fibrin results in somewhat different macromolecular intermediates and end product fragments due to its g-g cross-links. When cross-linked fibrin is cleaved, the a-chain polymers are removed first and degraded to low-molecular-weight fragments. Next the coiled-coil region joining the D- and E-domains are cleaved resulting in macromolecular intermediates such as fragment DD/E, fragment YD/ DY, or fragment YX/XD. Terminal split products of cross-linked fibrin are FsE, D-dimer, as well as D-trimer and D-tetramer derived from tri- and tetramolecular branch points (Figure 2).

Fibrinogen Thrombin FPA Fibril assembly, branching and lateral fibril associations

D:E

Tetramolecular branch point Trimolecular branch point

D:D

FXIII

Thrombin

FXIIIa FPB Cross-linking and fiber growth by lateral fibril associations - tetramer - trimer D:D D:E

Biological Function


c -dimer

Fibrinolysis E

D D

E

D

E

D

D D

E

D

D D

E

D E

E

D

D D

D

E

D

D D

E

D D

E

E

D

D D

E

D

D

Two-stranded cross-linked protofibril

DD/E

YD/DY

Lysis intermediate fragments

YX / XD

Lysis terminal split products D D E

D D D

D

FsE D-dimer

D D

D-tetramer

D

D-trimer

Figure 2 Schematic model of fibrin assembly, cross-linking, and plasmin-catalyzed degradation. D:E – noncovalent interactions between EA and Da sites that form end-to-middle staggered overlapping double-stranded fibrils. D:D – longitudinal interaction between two D-domains within a strand. ac – C-terminal a-chain protuberances, which become untethered from its noncovalent association with the E domain upon cleavage of FP-B and promote lateral fibril associations by self-association with other acdomains. g-dimer, g-trimer, g-tetramer – FXIIIa-mediated crosslinks between C-terminal gXL sites of D-domains. DD/E, YD/DY, YX/XD – intermediate lysis fragments. FsE, D-dimer, D-trimer, Dtetramer – terminal lysis fragments of plasmin-catalyzed fibrinolysis. Adapted from Mosesson MW (1998) Fibrinogen structure and fibrin clot assembly. Seminars in Thrombosis and Hemostasis 24(2): 169–174, with permission from Thieme New York.

The key role of fibrin(ogen) during hemostasis is to form the framework for thrombus formation. Cellular and proteinaceous components of a thrombus are stabilized by the covalent cross-linking of fibrin resulting in a stable hemostatic plug of the platelets. Thus, fibrin stabilizes damaged tissues and serves as a primary matrix of wound repair. However, this function is not solely limited to hemostasis and wound healing but may also play a role in developmental processes. Fibrin also exerts regulatory effects including the initiation of fibrinolysis and the regulation of thrombin activity. Fibrin, but not fibrinogen, accelerates the activation of plasminogen by tPA. This is mediated through formation of a tenary complex between tPA and plasminogen on the surface of fibrin involving the above-mentioned specific binding sites (Aa148–160, g312–324, Aa392–610), which are cryptic in the fibrinogen molecule and become exposed during fibrin formation. Next to the initiation of fibrinolysis, fibrin is also involved in the modulation of prolonged fibrinolysis. The most important plasmin inhibitor a2-antiplasmin becomes cross-linked to the Aa-chain of fibrinogen and fibrin by activated factor XIII. This cross-linking process is reversible and a2-antiplasmin can also be released from degraded fibrin. By this, fibrin restricts plasmin activity to sites where it is required and protects fibrinogen and other plasma proteins from degradation.

COAGULATION CASCADE / Fibrinogen and Fibrin 507

Fibrin contains nonsubstrate thrombin-binding sites (two low-affinity sites in the E-domain and one high-affinity site in the g0 -chain) which mediate an antithrombin activity (referred to as antithrombin I). Experimental evidences and clinical observation suggest that antithrombin I is an important factor for downregulating thrombin activity. By this mechanism diffusion of thrombin and the extent of clot propagation is limited. Fibrinogen and fibrin can interact with cells via integrin binding to RGD motifs in the Aa- and g-chain. Binding of fibrinogen to the platelet integrin aIIbb3 (also referred to as GPIIb/IIIa-receptor, CD41a) is of central importance during primary hemostasis. Fibrinogen acts as a bridging molecule and thus mediates platelet adhesion, aggregation, and formation of platelet-rich thrombi. Recently, a regulatory role for fibrinogen in inflammatory cell function was discovered. The finding that leukocyte engagement of fibrin(ogen) via the integrin receptor aMb2 is critical in the inflammatory response suggested a physiologically relevant role for fibrin(ogen) as an inflammatory mediator in innate immunity. Targeted deletion of the gene encoding the Aachain or g-chain in mice resulted in animals without detectable plasma fibrinogen levels. About 30% of Fbg
/
mice developed bleeding events shortly after birth; however, most of these animals showed a resolution of the bleeding events and survived the neonatal period. In addition, adult mice showed an increased risk for fatal abdominal hemorrhage, and pregnancy resulting in fatal uterine bleeding. A study investigating wound-healing defects in fibrinogennull mice showed no differences with respect to the time required to overtly heal wounds; however, a role for fibrin(ogen) in cellular migration and organization of wound fields could be demonstrated. Fibrinogen knockout mice crossed with atherosclerosissusceptible apolipoprotein E-deficient mice did not show a decreased extent of atherosclerosis despite the absence of fibrinogen.

Receptors The fibrinogen receptor on platelets has been identified as a membrane-bound heterodimeric glycoprotein complex, termed GPIIb/IIIa. The receptor belongs to the b3 subfamily of integrins (aIIbb3) and binds fibrinogen via a specific recognition site g400–441. Two other potential binding sites are located at Aa95–98 (RGDF) and at Aa572–575 (RGDS). The latter sequence is also found in other proteins that interact with the integrin receptor such as von Willebrand’s factor, vitronectin and fibronectin.

The fibrinogen receptor represents a target for pharmacological agents that specifically inhibit platelet aggregation. Three intravenous GPIIb/IIIa antagonists are currently marketed for the prevention of myocardial infarction in patients undergoing angioplasty or stenting: the monoclonal antibody Abciximab (mouse-human chimeric Fab fragments), and eptifibatide and tirofiban, two low molecular mass inhibitors. The leukocyte integrin aMb2 (CD11b/CD18, Mac1) is a further high-affinity receptor for fibrinogen on stimulated macrophages and neutrophils. This receptor belongs to the b2 (CD18) subfamily of integrins, which play a pivotal role in leukocyte function. Multiple binding sites, including g190–202 and g377–395, are implicated in this interaction.

Fibrin(ogen) in Respiratory Diseases A large number of congenital or acquired fibrin(ogen) disorders are described as being associated with a bleeding diathesis or thrombophilia. They include afibrinogenemia (essentially absent fibrinogen), hypofibrinogenemia (plasma levels less than 100 mg dl
1), or dysfibrinogenemia (structurally abnormal fibrin molecules). Elevated fibrinogen plasma levels were found to be strongly and independently related to cardiovascular disease such as coronary heart disease and acute myocardial infarction. The use of fibrinogen plasma levels as a marker predicting the cardiovascular risk has been widely accepted. In contrast, the role of fibrin(ogen) in respiratory disease is still not fully settled. However, there is increasing evidence that fibrin(ogen) plays a central role in the pathogenesis of inflammatory and chronic interstitial lung disease, including the acute respiratory distress syndrome (ARDS), severe pneumonia, and idiopathic pulmonary fibrosis (IPF). Hyaline membranes, that is, the accumulation of fibrin-rich material in the alveolar space, are commonly found in ARDS and other acute or chronic interstitial lung diseases. Such kind of extravascular fibrin deposition results from imbalanced coagulation and fibrinolysis. Analysis of bronchoalveolar lavage fluids from patients with ARDS demonstrated a prominent procoagulant (tissue factor and FVII) and antifibrinolytic (plasminogen activator inhibitor (PAI)-1, a2-antiplasmin) activity, while the fibrinolytic capacity (urokinase) was found to be markedly depressed. Thus, in concert with fibrinogen leakage into the alveolar space, rapid and pronounced fibrin formation is to be expected under these conditions (Figure 3). Furthermore, experimental data suggest a far-reaching incorporation of hydrophobic surfactant compounds into the growing fibrin matrix. As a

508 COAGULATION CASCADE / Fibrinogen and Fibrin

Phospholipids SP-B SP-C

Alveolar hemostatic balance:

Normal

Inflammation fibrinogen leakage

Anticoagulation fibrinolysis Procoagulation antifibrinolysis

Surfactant inhibition: Fibrinogen + Fibrin oligomer + + Fibrin polymer + + +

Fibrinogen

Polymerizing fibrin = Surfactant-trap Alveolar coagulation atelectasis formation

Fibrin oligomer

Specialized alveolar fibrin polymer: • Incorporation of pulmonary surfactant with promotion of alveolar collapse • Lower susceptibility to fibrinolytic enzymes • Altered mechanical properties

Fibrin oligomer

Persistent fibrin deposition Theory: Collapse induration: Persistent atelectasis/ fibrin deposition Alveolar wall apposition

Fibroblasts

Fibroblast activation Fibrosis

Deposition of fibrous tissue Fibrosis, honeycombing

Figure 3 Diagram illustrating the potential mechanisms by which fibrin(ogen) contributes to respiratory disease. Under physiological conditions the phospholipid lining layer at the air–water interface reduces the surface tension and thereby promotes lung inflation upon inspiration and prevents lung collapse during expiration. Under inflammatory conditions, fibrinogen, leaking into the alveoli, is rapidly converted into fibrin due to a high procoagulant and antifibrinolytic activity in the alveolar compartment. Surfactant function is greatly inhibited by incorporation of hydrophobic surfactant compounds (PL, SP-B/C) into polymerizing fibrin. Persistence or delayed clearance of this ‘specialized’ fibrin matrix promotes fibroproliferative processes (‘collapse induration’) finally resulting in a structural remodeling of the lung with loss of compliance and gas exchange properties.

result, such incorporation may yield a further increase in alveolar surface tension, alveolar instability, and finally gas exchange disturbances (Figure 3). Indeed, pronounced impairment of gas exchange could

be provoked by formation of fibrin in the distal lung in otherwise healthy lungs. In addition, fibrin clots embedding natural surfactant display markedly altered mechanical properties

COAGULATION CASCADE / Intrinsic Factors

and reduced susceptibility to proteolytic degradation. These properties, persistent alveolar collapse, reduced susceptibility to proteolysis, and sustained suppression of fibrinolytic enzymes, may prevent rapid clearance of fibrin from the lungs. Thus, persistent deposition of fibrin in the distal lung may promote fibroblast invasion and replacement of the primary fibrin matrix by a secondary collagenous matrix (Figure 3). Additionally, the fibrinopeptides A/B and fibrin(ogen) scission products have, next to thrombin itself, been shown to serve as potent fibroblast mitogens. However, it has to be stated that some fibrotic response was also encountered in fibrinogen null mice, suggesting that at least other extracellular matric (ECM) proteins may replace fibrin to some extent. Consequently, targeting abnormalities of fibrin turnover by anticoagulant and fibrinolytic interventions has been shown to protect the lung in animal models of acute lung injury and reduced the fibroproliferative response in bleomycin-induced lung fibrosis. Currently, anticoagulants such as heparin and fibrinolysins are being tested in ongoing experimental and clinical studies. Finally, it should be mentioned that in recent studies in a mouse model of allergic asthma evidence was provided that fibrin accumulation contributes to airway hyperresponsiveness. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Matrix: Integrins. Anticoagulants. Coagulation Cascade: Overview; Antithrombin III; iuPA, tPA, uPAR; Thrombin. Fibrinolysis: Overview; Plasminogen Activator and Plasmin. Interstitial Lung Disease: Overview; Alveolar Proteinosis; Amyloidosis; Cryptogenic Organizing Pneumonia; Hypersensitivity Pneumonitis; Idiopathic Pulmonary Fibrosis. Surfactant: Overview. Thrombolytic Therapy.

Further Reading Bennet JS (2001) Platelet–fibrinogen interactions. Annals of the New York Academy of Sciences 936: 340–354. Budzynski AZ (1998) Fibrinogen and fibrin: biochemistry and pathophysiology. Critical Reviews in Oncology/Hematology 6: 97–146. Everse SJ (2002) New insights into fibrin(ogen) structure and function. Vox Sanguinis 83(supplement 1): 375–382. Gu¨nther A, Ruppert C, Schmidt R, et al. (2001) Surfactant alteration and replacement in acute respiratory distress syndrome. Respiratory Research 2: 353–364. Idell S (2002) Endothelium and disordered fibrin turnover in the injured lung: newly recognized pathways. Critical Care Medicine 30(supplement): S274–S280. Idell S (2003) Coagulation, fibrinolysis, and fibrin deposition in acute lung injury. Critical Care Medicine 31(supplement): S213–S220. Koenig W (2003) Fibrin(ogen) in cardiovascular disease: an update. Thrombosis and Haemostasis 89: 601–609.

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Levi M, Schultz MJ, Rijneveld AW, and van der Poll T (2003) Bronchoalveolar coagulation and fibrinolysis in endotoxemia and pneumonia. Critical Care Medicine 31(supplement): S238–S242. Medved L and Nieuwenhuizen W (2003) Molecular mechanisms of initiation of fibrinolysis by fibrin. Thrombosis and Haemostasis 89: 409–419. Mosesson MW (1998) Fibrinogen structure and fibrin clot assembly. Seminars in Thrombosis and Hemostasis 24(2): 169–174. Mosesson MW (2003) Fibrinogen gamma chain functions. Journal of Thrombosis and Haemostasis 1: 231–238. Ploplis VA and Castellino FJ (2002) Gene targeting of components of the fibrinolytic system. Thrombosis and Haemostasis 87: 22–31. Wilberding JA, Ploplis VA, McLennan L, et al. (2001) Development of pulmonary fibrosis in fibrinogen-deficient mice. Annals of the New York Academy of Sciences 936: 542–548.

Intrinsic Factors P F Neuenschwander, University of Texas Health Center, Tyler, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The intrinsic coagulation pathway consists of factors XI, IX, and VIII (fXI, fIX, fVIII). FXI and fIX are serine protease zymogens requiring proteolytic activation to express protease and procoagulant activity. FVIII is a non-enzymatic cofactor that also requires proteolytic activation. FXI can be activated by either fXIIa (contact activation) or by thrombin (positive feedback). The activated fXI (fXIa) subsequently activates fIX to generate fIXa that binds to thrombin-activated fVIII (fVIIIa) to generate a potent procoagulant complex on an anionic phospholipid surface. This complex activates fX to propagate and amplify the coagulant response. The activity of the intrinsic pathway is regulated by the activation status of fVIII, defined by the spontaneous decay of fVIIIa activity. The rapid activation and decay of fVIII/fVIIIa provides a pulse of activity allowing controlled fibrin deposition to occur without excessive clot formation. This pathway is activated during pathological coagulation in the lung in acute lung injury models. If left unregulated it can lead to excessive generation of fXa, resulting in the deposition of extravascular alveolar fibrin followed by respiratory failure.

Introduction The intrinsic pathway of blood coagulation is so named due to the presence of all the required reactants in the circulation, with no external protein source required. It is evidenced most clearly in vitro by the ability of blood or plasma to spontaneously clot upon its collection into a glass vessel. The trigger for this mode of coagulation is the autoactivation of factor XII (fXII; Hageman factor), which auto-hydrolyzes on anionic surfaces (contact activation) to form fXIIa. In the presence of high-molecular-weight kininogen the fXIIa proteolytically activates fXI to generate fXIa, which subsequently activates fIX

510 COAGULATION CASCADE / Intrinsic Factors Co nt XII activ act atio n

XIIa

Intrinsic pathway

XI XIa Thrombin feedback Ca2+

VIIa–TF Extrinsic system

Ca2+ IX IXa VIIIa/PL Ca2+ X Xa

Figure 1 Intrinsic pathway of coagulation. Consisting of fXI, fIX, and fVIII, the intrinsic pathway of blood coagulation can be initiated by fXIIa via contact activation, by thrombin via feedback activation of fXI, or by the fVIIa–TF complex from the extrinsic pathway via crossover activation of fIX. In all cases, fIXa is generated that can form a calcium and phospholipid dependent complex with fVIIIa. This complex subsequently activates fX to amplify and propagate the coagulant response. Green arrows indicate enzymatic actions and blue arrows indicate zymogen to enzyme conversions. Requirements for ionic calcium are indicated.

(Christmas factor). The fIXa that is formed then binds to fVIIIa and this complex activates fX to fXa as part of the common pathway (Figure 1). Deficiency of fXII does not result in a hemorrhagic condition. Thus, contact activation is not considered part of the coagulation system per se but rather serves to connect coagulation with inflammation and the complement system. In contrast, deficiency of factors VIII or IX results in a bleeding diathesis (hemophilias A & B, respectively) as does deficiency of fXI (Rosenthal syndrome or hemophilia C), albeit mildly. Thus, the formal contemporary intrinsic pathway of coagulation consists of these three factors. During in vivo coagulation, fIX can be activated by either of two enzymes: fXIa or the fVIIa–tissue factor (TF) complex from the extrinsic pathway. Since fXIa is not normally present in the circulation, it is the fVIIa–TF complex that is likely responsible for the initial activation of fIX. This low level of fIXa is subsequently amplified by fXIa that is generated later in the pathway via a positive feedback reaction involving thrombin.

Structure of the Intrinsic Factors Genes

The gene for human fXI is on chromosome 4 at position q35. It is 23 kb in length and contains 15 exons

and 14 introns resulting in an mRNA transcript of 2.2 kb (Figure 2, top). Exon 1 encodes the 50 untranslated region and exon 2 encodes the prepeptide at the N-terminus of the polypeptide. The exon pairs 3–4, 5–6, 7–8, and 9–10 each encode an apple domain (Apple 1–4, respectively). Exon 11 encodes the activation region and the remaining exons (12–15) encode the protease domain. The fIX gene is located on the X chromosome at position q26-27.3. It is 34 kb in length and consists of 8 exons and 7 introns resulting in a transcript of 2.8 kb (Figure 2, middle). The structure of the fIX gene is very similar to that of fVII, fX, and protein C. Exon 1 encodes the prepeptide, exon 2 encodes the propeptide and Gla domain, exon 3 encodes a small aromatic stack region, exons 4 and 5 each encode an epidermal growth factor (EGF)-like repeat, exon 6 encodes the activation peptide region, and exons 7 and 8 together encode the protease domain. The gene for fVIII is also located on the X chromosome in the region of q28. It is roughly 186 kb in size and consists of 26 exons and 25 introns that produce a transcript of 8.8 kb (Figure 2, bottom). Exon 1 encodes the prepeptide region while exons 2– 7 encode most of the A1 domain. The small acidic a3 region is encoded by exon 8 while exons 9–13 encode the A2 domain. These are followed by the largest exon in the gene (exon 14; 3.1 kb), which encodes the a2, B, and a3 domains. The A3 domain is encoded by exons 15–19, and exons 20–23 and 24–26 encode the C1 and C2 domains, respectively. Proteins

FXI is one of two coagulation enzymes that is not a member of the vitamin-K-dependent protein family. It is synthesized in the liver as a precursor that undergoes proteolytic processing to remove an N-terminal signal sequence of 18 amino acids. The domainal organization of fXI consists of four apple domains, the activation region, and the latent catalytic (protease) domain. FXI is unique among the coagulation proteases in that it exists in plasma as a homodimer linked by a disulfide bond between the fourth apple domains (Cys321) of each monomer. FIX is a vitamin-K-dependent protein with a structure similar to that of fVII, fX, and protein C. The pre-pro-fIX polypeptide is synthesized in the liver and is proteolytically processed to remove the leader sequence and propeptide (46 amino acids) before secretion. FIX also undergoes several other posttranslational modifications including vitamin-K-dependent g-carboxylation of the first 12 Glu residues (Gla domain), b-hydroxylation of Asp64, partial sulfation of Tyr155, phosphorylation of Ser158, addition

COAGULATION CASCADE / Intrinsic Factors 1

2

3

4

6 7

5

8 9 10

11

12

13

14

511

15

Factor XI

Apple 1

Pre

Apple 2

Apple 3

Apple 4

Ap

Processing 1

2 3

Protease domain

Activation 5 6

4

7

8

Factor IX Pre

Pro

Gla

EGF1 EGF2 AP
Activation

Processing 1

2 34

56

7 8 9 10 12 13 14

15

Protease domain

19 21 22

23 24 25

26

B

a3

A2

a2

A1

a1

Factor VIII

A3

C1

C2

Figure 2 Gene organization of the intrinsic factors. The organization of the genes for fXI (top), fIX (middle), and fVIII (bottom) are depicted with corresponding encoded regions of each protein (not to scale). Exons (coding regions) are shown as black boxes and are numbered sequentially from left to right (50 to 30 ). Introns (noncoding regions) are shown as horizontal lines between exons. The region encoded by each exon is depicted below the gene as a colored bar. The fXI gene is 23 kb in length and encompasses 15 exons (1.5 kb) and 14 introns (21.5 kb). The gene encodes a prepeptide (Pre), four apple domains (Apple1–4), an activation peptide (AP) region, and the latent protease domain. The fIX gene is 34 kb in length and encompasses 8 exons (2.8 kb) and 7 introns (31.2 kb). The gene encodes a prepeptide (Pre), propeptide (Pro), g-carboxyglutamic acid domain (Gla), aromatic stack region (black), two epidermal growth factorlike domains (EGF1–2), an activation peptide (AP) region, and the latent protease domain. Enzymatic processing and activation sites in fXI and fIX are indicated by pointers; N-linked glycosylation sites are indicated as tree structures; b-hydroxylated Asp64 in fIX is indicated by b; and the di- and tetrasaccharide on Ser53 and Ser61 in fIX are indicated by black diamonds. The fVIII gene is the largest of the three and is 186 kb in length with 26 exons (9 kb) and 25 introns (177 kb). The gene encodes a prepeptide (red), the A1, a1, A2, a2, B, a3, A3, C1 and C2 domains. Processing and posttranslational modifications of fVIII are indicated in Figure 3.

of N-linked carbohydrate at Asn157 and Asn167, and additions of disaccharide (xylose–glucose) to Ser53 and tetrasaccharide to Ser61. Thus, the mature secreted form of the fIX zymogen is a single-chain molecule of 415 amino acids (Mr ¼ 55 000) consisting of a Gla domain, two EGF-like repeats, the activation peptide region, and the latent catalytic domain. FVIII is a very large glycoprotein that is the nonenzymatic cofactor for fIXa and is synthesized in the liver. The domainal organization of fVIII (Figure 3) consists of two A domains (homologous to ceruloplasmin), a single large B domain, a third A domain, and two C domains. Each A domain is juxtaposed by a small highly acidic region designated as a1, a2, and a3. The secreted form of fVIII consists of a heavy chain (MrB200 000) and a light chain (MrB80 000) held together through a divalent metal

ion-dependent (Cu2 þ ) interaction. Further proteolysis within the B domain (likely by plasma proteases) produces a variably sized heavy chain. FVIII is protected from further degradation by circulating in plasma in a noncovalent complex with von Willebrand’s factor, which stabilizes its activity.

Regulation of the Intrinsic Factors Activation

FXI is activated by proteolysis at a single site (Arg369) to produce fXIa and express procoagulant activity. This activation occurs via fXIIa or thrombin. Activation by fXIIa occurs when fXI zymogen binds to an anionic surface via high-molecularweight kininogen, whereas activation by thrombin does not have this requirement. In contrast, the

512 COAGULATION CASCADE / Intrinsic Factors A1

A2

a1

B

a2

A3

a3

C1

C2

NH2−

−COOH

Processing

Factor VIII polypeptide

Processing

(a)

s-s

s-s

s-s

SO4 SO4

SO4 SO4 SO4

SO4 SO4 SO4

??

s-s

s-s

s-s

s-s

s-s

s-s

s-s

−COOH Processed factor VIII

NH2−

SH

SH

SH Heavy chain

Light chain

(b) Activation cleavages NH2−

A1

A2

− COOH

B

Me2+ COOH−

C2

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A3

APC inactivation cleavages

–NH2

Activation cleavages

Plasma factor VIII

Ac

tiva

tio

n

A1

A2 Me2+

(c)

C2

C1

A3

Factor VIIIa

Figure 3 Organization of factor VIII. (a) The immature fVIII polypeptide containing the prepeptide followed by the A1, a1, A2, a2, B, a3, A3, C1, and C2 domains. The 25 potential N-linked glycosylation sites are indicated as tree structures. The two proteolytic processing sites are indicated by arrows. (b) The fully processed and mature form of fVIII, consisting of a heavy chain and a light chain. The variable size of the heavy chain is due to proteolysis of the B domain after processing (indicated by a fading color pattern). Predicted disulfide bonds are indicated above the bar by S–S and free sulfhydrils are indicated below the bar by -SH. The positions of the eight known/ proposed sulfated Tyr residues are also indicated (SO4). (c) The heterodimeric nature of plasma fVIII held together by a metal bridge requiring Cu2 þ . The three activation cleavage sites are indicated by arrows. Activation releases the B and a3 domains and separates the remaining heavy chain into A1–a1 and A2–a2 subunits. The resulting active fVIIIa heterotrimer is depicted in the bottom right. The A2–a2 subunit is held in place solely through electrostatic interactions and readily dissociates to inactivate fVIIIa. The inactivation cleavages catalyzed by activated protein C (APC) are indicated on the fVIIIa molecule by arrows.

activation of fIX requires proteolytic cleavage at two sites (Arg145 and Arg180) to produce the serine protease. This activation is catalyzed by either fXIa or the fVIIa–TF complex to produce fIXa. Plasma fVIII requires proteolytic activation at three sites (Arg372, Arg740, Arg1689) to produce the active cofactor (Figure 3). The primary activator of fVIII in vivo is thrombin, although fXa can also catalyze this reaction in the presence of anionic phospholipid and ionic calcium. The resulting active fVIIIa molecule has a substantially reduced size and is a metal-linked (Cu2 þ ) heterotrimer composed of the A1–a1, A2–a2, and A3–C1–C2 fragments. Activity

Unlike the other coagulation factors, fXIa does not require a cofactor or a phospholipid surface for activity, although ionic calcium is required. This is different from fIXa, which requires binding to its nonenzymatic protein cofactor (fVIIIa) in the

presence of calcium ions and an anionic phospholipid surface to express nominal procoagulant activity. When in this complex, the activity of fIXa is increased by a factor of 109. Thus, it is only when in this complex that fIXa activates fX to propagate and amplify the coagulant response. The activity of the fIXa–fVIIIa complex is almost entirely regulated by fVIIIa. Once activated, fVIIIa is extremely labile and exhibits a half-life on the order of several minutes. This is largely due to the spontaneous dissociation of the A2–a2 subunit domain from the activated heterotrimer (Figure 3). Although the binding of fIXa to fVIIIa somewhat stabilizes this interaction via fIXa binding sites located in the A2 domain, the dissociation of the A2–a2 subunit domain remains the limiting factor in the activity of the fIXa–fVIIIa complex. Once dissociated, the A2–a2 subunit domain is susceptible to proteolysis by activated protein C (APC) at Arg562, preventing its re-association. APC also cleaves at Arg336 in the A1–a1 subunit, releasing the a1 domain and further

COAGULATION CASCADE / Intrinsic Factors

inactivating the cofactor. The inactivation of fVIII by APC is accelerated by protein S, a nonenzymatic cofactor for APC.

Biological Function The fXI and fIX zymogens exist in plasma at a level of 5 mg ml
1 with half-lives of 60–80 h for fXI and 24 h for fIX. In contrast, the plasma level of fVIII is 50 times lower (B0.1 mg ml
1). The half-life of fVIII in plasma is ambiguous, but is probably around 8–12 h. The clearance of fVIII from plasma involves the low-density lipoprotein receptor-related protein (LRP). This is supported by a murine LRP knockout model that shows an increase in circulating fVIII levels. The major roles of the intrinsic factors are to amplify the coagulant response upon initiation by the fVIIa–TF complex (Figure 1). This necessarily involves the activations of fIX by fXIa and fX by the fIXa–fVIIIa complex. These proteins are also all intimately involved in coagulation regulation. As centralized players in the coagulation cascade, the activity of these proteins has a major impact on the level of coagulation attained. This is due to a combination of the positive feedback activations of fXI and fVIII by thrombin, as well as the rapid decay of fVIIIa activity. The combination of these events results in an intense but brief pulse of procoagulant activity. This allows for both the rapid formation of a fibrin clot as well as the prevention of excessive clot formation. The importance of the intrinsic factors is evidenced in the bleeding diatheses observed in patients that are deficient in these factors as well as in murine knockout models, which all show prolonged bleeding consistent with the hemophilias. Interestingly, the combined knockout of fXI and protein C partially corrects the hypercoagulable state observed in protein C knockouts, which is associated with severe protein C deficiency in humans. This suggests an additional potential role of fXI (and by inference the intrinsic pathway) in certain thrombotic states.

Receptors Unlike other coagulation factors (fVIIa, fXa, and thrombin) none of the intrinsic factors possess cell signaling activity. Platelets have been shown to have specific receptors for fIXa, fVIIIa, and fXIa that may play a role in modulation of their activities. Similarly, endothelial cells and collagen IV have been reported to express fIXa receptors that may influence fIXa plasma concentrations and/or activity. None of these receptors are currently well described.

513

Intrinsic Factors in Respiratory Diseases While pulmonary hemostasis is critical for maintaining lung function, it has been rigorously demonstrated that unregulated and/or ongoing procoagulant activity leads to deposition of extravascular fibrin. This is observed in instances of sepsis, pneumonia, and other acute events that can all lead to acute respiratory distress syndrome (ARDS). The levels of most clotting factors in these instances are increased in both the alveolar space and pleural cavity. Since the trigger for coagulation is the expression of TF, whose levels are elevated under these circumstances, the extrinsic pathway has been the major focus of investigation in lung injury models. However, the intrinsic factors have also been reported to be present in bronchoalveolar lavage of patients with ARDS, and are also likely to play a role in the propagation of pulmonary fibrosis. Studies investigating this inference directly have not yet been reported. See also: Acute Respiratory Distress Syndrome. Anticoagulants. Chemokines. Coagulation Cascade: Antithrombin III; Factor VII; Factor X; Fibrinogen and Fibrin; Protein C and Protein S; Thrombin; Tissue Factor. Complement. Endothelial Cells and Endothelium. G-Protein-Coupled Receptors. Leukocytes: Monocytes. Platelets. Proteinase-Activated Receptors. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis.

Further Reading Abraham E (2000) Coagulation abnormalities in acute lung injury and sepsis. American Journal of Respiratory Cell and Molecular Biology 22: 401–404. Chapman HA (2004) Disorders of lung matrix remodeling. Journal of Clinical Investigation 113: 148–157. Fay PJ and Jenkins PV (2005) Mutating factor VIII: lessons from structure to function. Blood Reviews 19: 15–27. Gui T, Lin HF, Jin DY, et al. (2002) Circulating and binding characteristics of wild-type factor IX and certain Gla domain mutants in vivo. Blood 101(1): 153–158. Ichinose A and Davie EW (1994) The blood coagulation factors: their cDNAs, genes, and expression. In: Colman RW, Hirsh J, Marder VJ, and Salzman EW (eds.) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn., pp. 19–54. Philadelphia: J.B. Lippincott Company. Idell S (2003) Coagulation, fibrinolysis, and fibrin deposition in acute lung injury. Critical Care Medicine 31: S213–S220. Laterre P-F, Wittebole X, and Dhainaut J-F (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31: S329– S336. Levi M, Schultz MJ, Rijneveld AW, and van der Poll T (2003) Bronchoalveolar coagulation and fibrinolysis in endotoxemia and pneumonia. Critical Care Medicine 31: S238–S242. Mann KG, Gaffney D, and Bovill EG (1995) Molecular biology, biochemistry, and lifespan of plasma coagulation factors. In: Beutler E, Lichtman MA, Coller BS, and Kipps TJ (eds.)

514 COAGULATION CASCADE / iuPA, tPA, uPAR Williams Hematology, 5th edn., pp. 1206–1226. New York: McGraw-Hill. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 1334–1349. Welty-Wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inflammation in acute lung injury. Thrombosis and Haemostasis 88: 17–25.

iuPA, tPA, uPAR M A Olman, University of Alabama, Birmingham, AL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The fibrinolytic system is a cascade of enzymes, and their inhibitors and cell surface receptors which were initially characterized based on their capacity to proteolytically degrade fibrin. The effector proenzyme plasminogen is widely distributed in tissue as an inactive zymogen, and its activity is regulated largely at the level of activation by the plasminogen activators, urokinase and tissue-type plasminogen activator (tPA). Studies in vitro and in transgenic/gene deleted mice amply demonstrate that the role of this enzyme system extends far beyond the dissolution of fibrin, to tissue remodeling, growth factor modulation, and cell migration. Not surprisingly, plasminogen activators, their naturally occurring inhibitor, plasminogen activator inhibitor type I (PAI-1), and plasminogen activator receptors have been implicated in a number of respiratory disorders including acute lung injury, pulmonary fibrosis, airway remodeling in asthma and infectious pneumonia. In some disorders, the proximate consequences of the plasminogen activators/inhibitors on disease pathogenesis are independent of their known mediation of the dissolution of fibrin.

Introduction The concept of fibrinolysis was first proposed in the 1890s following observation of spontaneous dissolution of a blood clot, and it was recognized as a proteolytic process a decade later. Prior to World War II, it was discovered that filtrates from hemolytic streptococci (subsequently named streptokinase) required a human plasma-derived component(s) (subsequently termed plasminogen) to induce fibrinolysis. The recognition by Astrup and colleagues in 1947 that animal tissues contain a factor that can activate plasminogen and dissolve fibrin clots jump-started modern investigations in the field. Tissue-type plasminogen activator (tPA) was purified from human uterine tissue and the general molecular mechanistic model of tPA-dependent fibrinolysis was developed in the late 1970s, followed by the purification and cloning of the fibrinolytic inhibitor, plasminogen activator inhibitor type I (PAI-1) a decade later. Structure–function studies on the plasminogen activators

themselves have yielded a number of therapeutic agents that are improved over the naturally occurring molecules in their enhanced fibrin selectivity, prolonged half-life, and greater resistance to inhibition by PAI-1. Since then, in vitro and transgenic mice studies have extended the influence of these serine protease/inhibitors to other biological processes including tissue repair/remodeling, development, angiogenesis, and malignancy. More recently, it has been recognized that the lung exhibits compartmentalized plasminogen activator (PA) activity, with tPA being the prime vascular compartment PA, while urokinase plasminogen activator (uPA) is the prime alveolar and interstitial PA. In contrast, plasminogen and PAI-1 are detected in all lung compartments under various circumstances.

Structure The proteases of the fibrinolytic cascade are trypsinlike proteases that are produced by many cell types and tissues as inactive single-chain zymogens. They are activated through proteolytic cleavage at an Arg or Lys residue, to generate a disulfide bonded, active two-chain form. The mature proteins share certain structural features including an EGF-like domain, a variable number of kringle domains, and the key serine in their protease catalytic domain in the B chain (see Table 1). The exception to this rule is tPA which is active in its single-chain form. tPA also has an N-terminal fibronectin homology domain, which functions to bind to fibrin, while plasminogen has an N-terminal autoactivation peptide that changes its fibrin-binding kinetics and susceptibility to activation by uPA/tPA. A kringle-like structure, kringle 2 of tPA, binds to fibrin, lysine binding sites, and o amino acids (EACA). The major naturally occurring inhibitor of PA, PAI-1, is a 52 kDa glycoprotein member of the serine protease inhibitor (serpin) family that also includes the plasmin inhibitor, a-2 antiplasmin. PAI-1 is the predominant inhibitor for both tPA and uPA, and has a second order rate constant for inhibition of 107–108 M
1 s
1. PAI-1 functions as a ‘pseudosubstrate’ that forms sodium dodecyl sulfate (SDS)stable, PA–PAI-1 complexes of 1:1 stoichiometry, followed by cleavage of the peptide bond at Arg346– Met347 (P1–P10 ) bond in the PAI-1 reactive center loop. PAI-1’s activity is highly dependent on the conformation of this reactive center loop. PAI-1 is secreted in an active form with an exposed reactive center loop, while spontaneous/induced insertion of the loop into the b-sheet structure results in latency (plasma half-life ðt1=2 Þ 90 min at 371C). In a third conformation, PAI-1 is instead cleaved by PAs at the

COAGULATION CASCADE / iuPA, tPA, uPAR

515

Table 1 Key features of fibrinolytic system molecules Name

Chromosome

Mr (kDa)

AA (#)

Conc.

t1=2

Key domains

uPA; PLAU

10q24

54

411

40 pM

7 min

tPA; PLAT

8p12-p11

68

530

70 pM

4 min

Plasminogen; PLG

6q26-q27

92

791

2 mM

50 h

PAI-1

q22.1-22.3

50

379

300 pM

10 min

uPAR; PLAUR

19q13.1-q13.2

55

313

EGF–AA 4-43 – binds uPAR kringle, Lys 158–Ile159 – activation site, B chain – catalytic domain FN-repeat – binds fibrin, EGF, kringle 1, kringle 2 – binds fibrin, Arg275–Ile276 – activation site, B chain – catalytic domain N-term activation peptide – Lys78 – enhanced PA activation and fibrin binding; kringle 1–5 – bind fibrin, a-2-antiplasmin, extracellular matrix; Arg561–Val562 – activation site B chain – catalytic domain AAs – 55,109,110,116,123 – together bind vitronectin; reactive center – Arg346–Met 347, reactive center loop – Ser 331– Arg346 D1 – binds uPA, D2/3 – enhance uPA binding, C-term GPI link

Adapted from Colman RW, Hirsh J, Marder VJ, Clowes AW, and George JN (eds.) (2001) Thrombosis and Hemostasis, 4th edn. Philadelphia, PA: Lippincott Williams and Wilkins.

Arg346–Met347 bond, or other nontarget proteases in the reactive center loop (i.e., elastase, thrombin). Binding of PAI-1 to vitronectin in the blood or the extracellular matrix stabilizes PAI-1 in the active conformation, alters its protease specificity, and promotes its clearance through the low-density lipoprotein receptor-related protein (LRP).

Regulation of Production and Activation Serine proteases of the fibrinolytic system are synthesized in several cell types and circulate largely as inactive (zymogen) forms. The coagulation/fibrinolytic proteases are in the trypsin-like protease family and cleave their target proteins at arginine or lysine bonds. Key biochemical features of this cleavage include the formation of an ester bond between the Ser residue oxygen of the protease and the acyl group of the substrate, resulting in the formation of a tetrahedral complex. This is followed by the loss of the C-terminal portion of the substrate to yield an acylenzyme intermediate. The Ser–acyl bond is then catalytically replaced by water to release the N-terminal substrate fragment and regenerate the active site serine in the enzyme. The main site of synthesis of tPA is in endothelial cells, where tPA is stored in granules, but other mesenchymal cells can produce tPA including monocytes, smooth muscle cells, and fibroblasts. Regulated synthesis and release of tPA are induced by multiple stimuli including stress, adrenergic stimulation, DDAVP, histamine, and thrombin. tPA activity in plasma also exhibits diurnal variation. uPA is

present widely throughout most connective tissues, and is expressed in fibroblasts, epithelial, and mononuclear lineage cells. While uPA is present at high concentration in urine (50 mg ml
1) and the kidney, it is detected at low concentration (2 ng ml
1) in plasma where it is found as a free, single chain, inactive form (scuPA), with a t1=2 similar to that of tPA. Plasma levels of uPA are less volatile than those of tPA and PAI-1. ScuPA is converted into an active form through proteolytic cleavage by a number of different proteases, and through autoactivation. These include kallikrein, mast cell tryptase, cathepsins, leukocyte elastase, and some metalloproteinases. By virtue of uPA binding to its cognate receptor, uPAR, uPA-dependent plasminogen activation at the cell surface is enhanced, and uPA–PAI-1 complexes bound to uPAR are internalized, with recycling of unbound uPAR to the cell surface. The serpin inhibitor, PAI-1, is produced in an active form by many cell types in a highly regulated fashion. PAI-1 expression is regulated by such varied factors and events as the cell cycle, thrombin, insulin, very-low-density lipoprotein (VLDL), fibrin fragments, interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha (TNF-a), and transforming growth factor beta (TGF-b). It also exhibits diurnal variation, which may account for the diurnal variation in thrombotic events. Plasma PAI-1 levels in normal subjects are highly variable ranging from 2 to 4100 ng ml
1, and it has a circulating t1=2 exponential a phase of 8 min, and b phase of 30 min. A second major pool of blood PAI-1 is contained within platelets, in an inactive form.

516 COAGULATION CASCADE / iuPA, tPA, uPAR

Biological Functions Transgenic mice studies indicate that tPA and uPA are redundant for plasminogen activation and thrombolysis, whereas plasminogen is uniquely important for thrombolysis. tPA-dependent fibrinolysis is relatively fibrin localized, by virtue of the formation of a ternary complex of fibrin, plasminogen, and tPA. Mice deficient in tPA exhibit a mild thrombophilic phenotype, and abnormal neurological function. The enzymatic activity of the single chain uPA is a matter of some controversy; nonetheless, in vivo, even a small amount of active plasmin or uPA can activate single chain uPA. Active uPA cleaves the Arg561–Val562 bond of plasminogen to generate plasmin; and scuPA and plasminogen can undergo reciprocal activation, as well as autoactivation. Despite the absence of direct binding of scuPA to fibrin, it exhibits some significant fibrin specificity. Furthermore, the binding of scuPA to its receptor, uPAR, increases its catalytic efficiency on the cell surface by two orders of magnitude. Other biologically relevant proteolytic functions of uPA are the activation of other proenzymes (i.e., MMP-2), progrowth factors (HGF, bFGF, FGF2), and cleavage of matrix fibronectin (Figure 1). uPA can act as a mitogen and its N-terminal fragment binds to some integrins in a uPAR-independent manner. Numerous signaling events occur as a consequence of uPA binding to its cognate receptor (Kd ¼ 1 nM), uPAR, through uPA’s

Prourokinase (scuPA)

N-terminal region (see below). As with tPA deletion, mice devoid of uPA have a mild thrombophilic phenotype. In contrast to tPA deletion, uPA deletion in mice significantly affects tissue remodeling/repair processes in multiple organs including the lung, kidney, and vasculature, sometimes in a manner that is independent of uPAR. uPA knockout mice have impaired inflammatory cell recruitment, and are more susceptible to infection from several Gram-positive and Gram-negative organisms, Cryptococcus neoformans, and Pneumocystis carinii, in part through mediating T-lymphocyte proliferation.

Receptors Receptors for tPA can be divided into those that contribute to activation and those that participate in the clearance of tPA and tPA/PAI-1 complexes. The urokinase receptor (uPAR; CD-87) is a 50–60 kDa heavily glycosylated cell surface receptor for the serine protease urokinase (uPA; Kd ¼ 1 nM). uPAR is linked to the exoplasmic leaflet of the plasma membrane through a C-terminal glycosylphosphatidylinositol (GPI) chain (Figure 2). Despite the absence of a transmembrane or intracytoplasmic domain, intracellular signaling through uPAR is well documented, and it is largely dependent on its ligation with uPA. Signaling through uPAR can occur directly, through poorly characterized mechanisms, or by the association of uPAR with other signaling partners such as

tcuPA

PAI-1

Fibrin PAI-1


-2-antiplasmin

Plasmin

Plasminogen

ProMMPs ECM glycoproteins Growth factors

Receptor-mediated events

FDPs Active MMPs ECM fragments Activate, INH, ECM release Proliferation, adhesion/migration, survival, ECM synthesis

Figure 1 Fibrinolytic cascade in tissue repair and remodeling. The zymogen (scuPA) is activated by plasmin or auto-activated into active two-chain uPA (tcuPA). tcuPA will activate the millimolar concentrations of plasminogen present in plasma and in tissue to form the broad spectrum serine protease plasmin. The consequences of plasmin protease action include fibrinolysis, activation of proforms of matrix metalloproteinases (MMPs), direct cleavage of matrix glycoproteins such as laminin and helicase cleaved collagen, activation/ inactivation (INH) of growth factors and/or their release from the extracellular matrix (ECM). The serpin PAI-1 will bind and inactivate tcuPA, while a-2 antiplasmin will bind and inactivate plasmin, thereby effectively downregulating plasmin action. Through binding of scuPA and plasminogen to their cell surface receptors, plasminogen activation is more favorable, and its inhibition by a-2-antiplasmin is less favorable. Cell-localized uPA/PAI-1 may also regulate inflammatory and mesenchymal cell adhesion/migration through uPARmediated events.

COAGULATION CASCADE / iuPA, tPA, uPAR

517

• Inhibits uPA I-1

PA

• ECM degradation • Induction of cell migration • Activation of growth factors Plasmin

• Adhesion • Signal transduction ECM

uPA

Vitronectin

Plasminogen uPAR Extracellular space

Integrins Cell membrane

• Intracellular signaling cascade • Induction of cell proliferation

Intracellular space

Src FAK Figure 2 uPAR operates independently and cooperatively as a multifunctional receptor. uPA binding to uPAR enhances its interaction with vitronectin, enhances local cell surface plasmin generation, directly initiates intracellular signal cascades, and enhances uPAR interactions with other transmembrane receptors, such as integrins. PAI-1 binds to solution phase or receptor-bound uPA, leading to internalization of PAI-1–uPA–uPAR complexes by LRP and recycling of uPAR to the cell surface. Under some circumstances, integrin receptors are internalized as well leading to cell detachment. uPAR is thus capable of modulating cell migration, proliferation, cell survival, as well as cell surface localized proteolysis. FAK, focal adhesion kinase. Adapted from Wilex AG and from Sperl S, Mueller MM, Wilhelm OG, et al. (2001) The uPA/uPA-receptor system as a target for tumor therapy. Drug News & Perspectives 14: 401–411.

integrins and G-protein-coupled receptors. uPAR-initiated signals utilize a number of intracellular downstream pathways. uPA/uPAR may modulate cell motility/migration through protease-related and nonprotease-related mechanisms. Studies of pulmonary disease in transgenic mice support an important role for uPAR in modulating cell migration that is largely independent of its role in modulating proteolysis. For example, neutrophil recruitment in response to a murine interleukin-8 homolog is inhibited by nonproteolytically active uPA. Leukocyte recruitment in vivo is dependent on uPAR-integrin (aMb2, Mac-1) interactions as demonstrated in Pseudomonas aeruginosa infection of the lung or recruitment into the inflamed peritoneum. uPAR also is important for leukocyte recruitment in the Mac-1-independent pulmonary infection due to Streptococcus pneumoniae, in a uPA-independent manner in antigen-inflamed lung, or in bleomycin-induced lung injury. On balance, one can say that uPAR-b2 interactions mediate leukocyte recruitment, but other uPAR nonproteolytic functions (i.e., signaling, interactions with vitronectin, etc.) may also condition the response. The best evidence for the role of uPAR in tissue repair and

remodeling is in a ureteral obstruction model of renal fibrosis, where uPAR-deficient mice have increased total collagen and increased fibrotic interstitial area.

Fibrinolytic System in Respiratory Diseases Given the protean nature of the biological processes that depend on the fibrinolytic system it is not surprising that it plays a role in infectious, inflammatory, allergic, fibrotic, and vascular diseases of the lung. The normal fibrinolytic activity of the lung is compartmentalized with uPA, the predominant alveolar/parenchymal PA, and tPA, the predominant endovascular PA. With an excess of PAs over PAI-1 activity, and ample tissue plasminogen, the normal lung possesses a net profibrinolytic activity. Acute Lung Injury

Most studies of acute lung injury (ALI) in both patients and experimental animal models demonstrate an early (begins hours after injury), transient (approximately 7–10 days), large increase in alveolar/ parenchymal PAI-1 and a-2 antiplasmin, which is

518 COAGULATION CASCADE / iuPA, tPA, uPAR

greater than the increases observed in PAs. This incurs a net inhibition of fibrinolysis that, along with influx of fibrinogen and increased tissue factor procoagulant activity, induces an alveolar and parenchymal deposition of fibrin. Under some circumstances, plasminogen availability may be the rate-limiting step. Despite the influx of plasmaderived PAs and PAI-1, resident/inflammatory lung cells locally produce a significant fraction of the increased PAI-1. The alveolar fibrin persists and contributes to the formation of hyaline membranes that are one of the hallmarks of diffuse alveolar damage, the characteristic pathology of the ALI syndrome. The increased susceptibility of mice with deletion of PAI-1 to hyperoxic lung injury supports the biological importance of this pathway. Recent data in ALI patients suggest that the level of PAI-1 in pulmonary edema fluid may not only discern those with ALI from those with hydrostatic pulmonary edema, but may also predict outcome in ALI. Although PAI-1 induction appears to exert a dominant influence over the pathobiology of ALI, uPA also plays a role. For example, uPA knockout mice are also protected from endotoxin-induced lung injury, and uPA participates in LPS-induced neutrophil activation in vitro. Hypoxia itself may contribute significantly to the induction of PAI-1 seen in ALI as hypoxic exposure of mice induces macrophage PAI-1 expression in lung. This PAI-1 increase may contribute to the excess intravascular fibrin observed in the hypoxia-exposed wild-type mouse as compared with the PAI-1 deficient mouse. Pulmonary Fibrosis

Pulmonary fibrosis is characterized by the deposition of disordered extracellular matrix in the alveolar and parenchymal compartments of the lung. Histologically, usual interstitial pneumonia (UIP) demonstrates a spatial heterogeneity. Areas of normalappearing tissue are adjacent to areas of relatively acellular, fibrin-rich, fibrotic tissue, which are adjacent to areas of fibroblast/myofibroblast-rich foci, that are actively remodeling. These fibroblastic foci have recently been shown to correlate with prognosis in UIP. The bronchoalveolar lavage (BAL) from patients with pulmonary fibrosis have increased levels of PAI-1 and the procoagulant, tissue factor. Furthermore, primary fibroblasts isolated from patients with idiopathic pulmonary fibrosis express high levels of uPAR, which along with uPA stain positive in fibrotic lesions. Most of the observations implicating the fibrinolytic cascade in fibroproliferative lung disease have been derived from experimental animal models of bleomycin-induced fibrosis. This model

shares some of the histologic features of the human disease; however, it is not progressive and is highly inflammatory, and when bleomycin is administered intratracheally, the fibrosis is bronchocentric. In the bleomycin model of fibroproliferative lung disease, PAI-1 is required, while plasminogen and/or tPA- or uPA-deficient mice have an enhanced fibrotic response compared to wild-type mice, indicating a net fibrinolytic activity is protective against fibrosis in this model. Interestingly, PAI-1’s effect appears to be independent of its capacity to mediate fibrinolysis, as mice devoid of fibrinogen have a fibrotic response to bleomycin comparable to that of wild-type mice. Other considerations may be activation and/or matrix release of growth factors by plasmin and/or uPA. Attempts at inducing a net profibrinolytic state through intratracheal exogenous urokinase, or through lung-specific or adenovirally mediated uPA overexpression, have been uniquely successful in reducing established fibrosis. Furthermore, aerosolized uPA can reduce both the physiological and histological and biochemical derangements seen in rabbits after bleomycin instillation, when begun during the active fibrotic phase. These observations provide therapeutic impetus, as patients with UIP usually present after the fibrotic process is well established. Asthma

Mast cells activated by phorbol ester/calcium ionophore upregulate their PAI-1 expression, and lung mast cells from patients with asthma exacerbations express PAI-1. Experimental evidence for a pathogenic role for PAI-1 in airway remodeling is available. High levels of PAI-1 and TGF-b are expressed in the airway epithelium in the ovalbumin-sensitized, murine asthma model. Further, ovalbumin-sensitized PAI-1-deficient mice exhibit increased airway collagen and fibrin deposition after ovalbumin challenge, as compared with wild-type mice, but exhibit no difference in airway inflammation. Infectious Lung Disease

In patients with moderate to severe bacterial pneumonia, excess fibrin degradation products and increased PAI-1 in the alveolar compartment occur, albeit less dramatically than that seen in ALI. Furthermore, these changes are anatomically restricted to the consolidated lobes. However, it is unlikely that a PAI-1-mediated inhibition of fibrinolysis is critical to the pathogenesis of the bacterial pneumonitis. Studies of murine pneumococcal pneumonia in PAI-1 or plasminogen-deficient mice demonstrate an inflammatory cell recruitment, bacterial outgrowth, and survival comparable to that in wild-type mice.

COAGULATION CASCADE / Protein C and Protein S

These observations support a nonfibrinolytic role for uPA and uPAR in the host response to bacterial pulmonary infection, as described above. There is a role for uPA in immune-mediated lung diseases. uPA is important for T-lymphocyte proliferation, Th1-type immune response to Cryptococcus neoformans, and Th2-type cytokine response to schistosomal antigen challenge in mice. In contrast, uPAR, independent of uPA, plays a major role in pulmonary recruitment of lymphocytes after experimental intratracheal antigen challenge. Pleural Disease

Exogenous scuPA administration has shown promise as a therapeutic agent that will prevent pleural adhesions in tetracycline-instilled rabbits. However, a recent double-blind trial of 454 patients with pleural space infection, treated with intrapleural streptokinase or placebo plus routine care, indicates no difference in the mortality rate, the rate of surgery, the radiographic outcome, or the length of hospital stay. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Matrix: Focal Contacts and Signaling; Integrins. Anticoagulants. Arteries and Veins. Asthma: Overview. Caveolins. CD11/18. Coagulation Cascade: Overview. Extracellular Matrix: Degradation by Proteases. Fibrinolysis: Overview. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Matrix Metalloproteinases. Pneumonia: Overview and Epidemiology. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts.

519

matrices by inactivating integrins. Journal of Cell Biology 160: 781–791. Eitzman DT, McCoy RD, Zheng X, et al. (1996) Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. Journal of Clinical Investigation 1: 232–237. Gunther A, Lubke N, Ermert M, et al. (2003) Prevention of bleomycin-induced lung fibrosis by aerosolization of heparin or urokinase in rabbits. American Journal of Respiration and Critical Care Medicine 168: 1358–1365. Gyetko MR, Sud S, Sonstein J, et al. (2001) Cutting edge: antigendriven lymphocyte recruitment to the lung is diminished in the absence of urokinase-type plasminogen activator (uPA) receptor, but is independent of uPA. Journal of Immunology 167: 5539–5542. Hattori N, Degen J, Sisson T, et al. (2000) Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice. Journal of Clinical Investigation 106: 1441–1443. Idell S, James KK, Levin EG, et al. (1989) Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome. Journal of Clinical Investigation 84: 695–705. Maskell NA, Davies CW, Nunn AJ, et al. (2005) U.K. controlled trial of intrapleural streptokinase for pleural infection. New England Journal of Medicine 352: 865–874. Olman MA (2003) Mechanisms of fibroproliferation in acute lung injury. In: Matthay MA and Lenfant C (eds.) Acute Respiratory Distress Syndrome, pp. 313–354. New York: Dekker. Prabhakaran P, Ware LB, White KE, et al. (2003) Elevated levels of plasminogen activator inhibitor-1 in pulmonary edema fluid are associated with mortality in acute lung injury. American Journal of Physiology: Lung Cellular and Molecular Physiology 285: L20–L28. Sperl S, Mueller MM, Wilhelm OG, et al. (2001) The uPA/uPAreceptor system as a target for tumor therapy. Drug News & Perspectives 14: 401–411.

Protein C and Protein S Further Reading Bertozzi P, Astedt B, Zenzius L, et al. (1990) Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. New England Journal of Medicine 322: 890–897. Blasi F and Carmeliet P (2002) UPAR: a versatile signalling orchestrator. Nature Reviews Molecular Cell Biology 3: 932– 943. Blasi F, Vassalli J-D, and Dano K (1987) Urokinase-type plasminogen activator: proenzyme, receptor, and inhibitors. Journal of Cell Physiology 104: 801–804. Chapman HA (1997) Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration. Current Opinion in Cell Biology 9: 714–724. Cho SH, Tam SW, Demissie-Sanders S, Filler SA, and Oh CK (2000) Production of plasminogen activator inhibitor-1 by human mast cells and its possible role in asthma. Journal of Immunology 165: 3154–3161. Colman RW, Hirsh J, Marder VJ, Clowes AW, and George JN, (eds.) (2001) Thrombosis and Hemostasis, 4th edn. Philadelphia, PA: Lippincott Williams and Wilkins. Czekay R-P, Aertgeerts K, Curriden S, and Loskutoff D (2003) Plasminogen activator inhibitor-1 detaches cells from extracellular

E C Gabazza, O Taguchi, and K Suzuki, Mie University Graduate School of Medicine, Mie, Japan & 2006 Elsevier Ltd. All rights reserved.

Abstract Protein C and protein S are essential factors of the protein C pathway, which plays a fundamental role in the regulation of the coagulation/fibrinolysis system, inflammation, and cell survival. The conversion of protein C to its active form, activated protein C, occurs on the phospholipid-rich surface of cell membranes in the presence of calcium, thrombomodulin, and its receptor endothelial protein C receptor. Protein S acts as a cofactor by enhancing the function of activated protein C. Activated protein C suppresses coagulation activation by inhibiting activated factors V and VIII, suppresses the inflammatory response by inhibiting cytokine secretion, and improves cell survival by inhibiting apoptosis. Chronic inflammatory disorders of the lung are associated with decreased activation of protein C. Treatment with activated protein C improves outcome in patients with sepsis. Activated protein C is also effective for the treatment of lung fibrosis and bronchial asthma in animal models. Activated protein C therefore represents a promising

520 COAGULATION CASCADE / Protein C and Protein S therapeutic approach for incurable inflammatory disorders of the lung.

Protein C (PC) and protein S (PS) are vitamin Kdependent glycoproteins and essential components of the PC pathway. The circulating level of PC is approximately 2–4 mg l 1 and that of total PS is on average 20–25 mg l 1. PC is the zymogen precursor of activated PC (APC), which is the effector enzyme of the PC pathway. PC is converted to its active form, APC, by thrombin bound to thrombomodulin (TM) on the phospholipid surface of endothelial cells, epithelial cells, monocytes, and platelets. PS acts as a cofactor of APC by enhancing the proteolysis of coagulation factors Va and VIIIa. Endothelial protein C receptor (EPCR) is another factor required for optimal activation of PC. EPCR is the receptor of both PC and APC that was originally described on the cell surface of vascular endothelial cells but later found to be also present in bronchial and alveolar epithelial cells. EPCR enhances PC activation by presenting the PC substrate to the thrombin–TM activation complex. The PC pathway is involved in multiple biological functions in the lung. It plays a protective role in diverse acute and chronic inflammatory diseases of the lung, including acute respiratory distress syndrome, lung fibrosis, and bronchial inflammation, by virtue of its inhibitory activity on coagulation, inflammation, extracellular matrix accumulation, and its profibrinolytic effect.

Structure PC is synthesized in the liver and endothelial and lung cells. The molecular weight of human PC is 62 kDa, and its half-life in the systemic circulation is 8 h. The gene structure of PC shows high sequence homology with other vitamin K-dependent factors. The human PC gene is located on chromosome 2, position q13–q14, and spans approximately 11.2 kilobases of genomic DNA; it contains nine exons and eight introns. The molecular structure of PC consists of two polypeptide (a light and a heavy) chains of unequal length linked by a single disulfide bond. The light chain contains the vitamin K-dependent g-carboxy glutamic acid (Gla) domain and two epidermal growth factor-like domains (Figure 1). Binding of the Gla domain to negatively charged phospholipids is fundamental for the anticoagulant activity of APC. The Gla domain has high affinity for calcium ions, which appear to be essential for PC binding to phospholipids on cell membrane. Calcium binding to the Gla domain also changes the conformation of the PC molecule, making its structure more suitable for interacting with the thrombin–TM complex. EPCR, which influences the rate of PC activation, also interacts with the Gla domain of PC. The two epidermal growth factor (EGF)-like domains appear to be important for changing the conformation of the PC molecule to facilitate its interaction with PS. The heavy chain of PC contains a short-activation peptide COOH

Activation peptide

NH2

S H

D

Gla domain

EGF-like domain

Catalytic domain

Figure 1 Structure of human protein C. The light chain of protein C is composed of the g-carboxy glutamic acid (Gla) domain that binds to negatively charged phospholipids, an aromatic region, and two epidermal growth factor-like domains. The heavy chain consists of the serine protease domain and the activation peptide, which is released after cleavage by the thrombin/thrombomodulin complex. Purple cycles indicate activated peptide. Red cycles indicate active residues: His211 (H), Asp257 (D), and Ser360 (S).

COAGULATION CASCADE / Protein C and Protein S

and a serine protease or catalytic domain. The activation peptide is released during PC cleavage by the thrombin–TM complex. PS is another vitamin K-dependent anticoagulant protein whose major function is to facilitate the inhibition of factor Va and factor VIIIa by APC. PS circulates in plasma in both a free, active form and an inactive form bound to C-4b binding protein (C4BP) of the complement system. Expression of PS has been described in the liver, lung, lymphocytes, brain, megakaryocytic cell lines, and osteoblasts. Two genes of PS have been identified on chromosome 3, in position p11.1–11.2; one is the active gene that contains 15 exons and 14 introns, and the other (pseudogene) has high sequence homology with the active gene but lacks exon 1. The molecular weight of PS is 69 kDa, and its half-life in circulation is 30 h. The mature PS is a multidomain glycoprotein containing a phospholipid-binding Gla domain, a thrombin-sensitive region, four EGF-like domains, and a sex hormone-binding globulin-like domain region containing two laminin G-type repeats (Figure 2). As in other vitamin K-dependent proteins, the Gla domain is necessary for PS binding to negatively charged phospholipid and for supporting APC binding to the membrane. The thrombin-sensitive region and the first EGF-like domain appear to help in the orientation and localization of APC during its interaction with factor Va and factor VIIIa. The second

521

EGF-like domain and the laminin G-type domains are also indispensable for full expression of APC cofactor function of PS. The laminin G-type domain contains the binding site for C4BP.

Activation of PC Several factors, including negatively charged phospholipids, calcium ions, thrombin, the two cell membrane-bound proteins TM and EPCR, and platelet factor-4, are required for optimal generation of APC. PC activation occurs in a stepwise manner. In the first step, thrombin binds to TM, and this binding alters the substrate specificity of thrombin so that it is no longer able to convert fibrinogen to fibrin, activate factor V, or induce aggregation of platelets. In the second step, PC interacts with both components of the complex through calcium bridges. The thrombin–TM complex then activates PC by the cleavage of an internal peptide bond and releases an activation peptide. EPCR favors the interaction of PC with the thrombin–TM complex and thereby enhances generation of APC (Figure 3). Platelet factor-4 may modulate PC and TM by interacting with the Gla domain of PC and with the O-linked carbohydrate region of TM, respectively, resulting in enhanced APC generation. PC inhibitor (PCI) may also regulate the activation of PC by the thrombin–TM

NH2

COOH

Gla Thrombindomain sensitive domain

EGF-like domains

Sex hormone-binding globulin-like domain

Figure 2 Structure of human protein S. The mature protein S contains a g-carboxy glutamic acid (Gla) domain, an aromatic region, a thrombin-sensitive region, four epidermal growth factor-like domains, and the sex hormone-binding globulin-like domain.

522 COAGULATION CASCADE / Protein C and Protein S TF/VIIa IX

PS-C4BP

IXa PL, Ca2+, Mg2+, VIIIa

APC-PCI

–

PCI PS APC

X

Xa PL, Ca2+, Va

Prothrombin

EPCR

– PAR-1

Thrombin TM

Thrombin

Fibrinogen

Fibrin

EPCR

PC

Figure 3 Coagulation cascade and anticoagulant protein C pathway (dotted arrows indicate inhibition). PC, protein C; TM, thrombomodulin; EPCR, endothelial protein C receptor; APC, activated protein C; PS, protein S; PCI, protein C inhibitor; C4BP, C4b binding protein; PAR-1, protease-activated receptor-1; TF, tissue factor; PL, phospholipids.

complex on negatively charged proteoglycan and phosphotidylethanolamine of cell membrane.

Biological Functions of APC

thrombin, which is necessary for activation of the zymogen, thrombin-activatable fibrinolysis inhibitor. Vitronectin accelerates the inhibition of PAI-1 by APC. APC may also suppress the action of other fibrinolytic inhibitors, such as PCI and a1 antitrypsin.

Inhibition of Coagulation

APC inhibits activation of the coagulation cascade by proteolytic degradation of factor Va and factor VIIIa in the presence of phospholipids, calcium, and PS. Factor V and factor VIII circulate in plasma in inactive forms and are converted into their active forms, factor Va and factor VIIIa, by activated factor X (factor Xa) and thrombin during the initiation of the coagulation cascade (Figure 3). The clinical importance of the PC pathway in the inhibition of coagulation is illustrated by the frequent occurrence of thromboembolic complications in subjects with deficiency of PC or resistance to APC. The phospholipid surface of various cells, including vascular endothelial cells, platelets, monocytes, alveolar macrophages, and bronchoalveolar epithelial cells, may support activation of PC. Thus, generation and function of APC may take place in the intravascular and extravascular spaces of a wide variety of organs, including those of the respiratory system (Figure 4).

Inhibition of Inflammation

APC may inhibit the inflammatory response by blocking the cellular secretion of inflammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b, monocyte chemoattractant protein-1, and macrophage inflammatory protein 1-a, and by downregulating the cell-surface expression of adhesion molecules (e.g., intercellular adhesion molecule-1) required for transendothelial migration of inflammatory cells to the sites of tissue injury. APC may also protect against inflammation by decreasing endothelial cell permeability and migration of inflammatory cells. APC enhances lung endothelial barrier by binding to EPCR and transactivation of the sphingosine 1-phosphate receptor. APC also suppresses the nuclear translocation of transcription factors, including those from the nuclear factor kappa B (NF-kB) family. The cellular effect of APC appears to be mediated by EPCR and, interestingly, by protease-activated receptor-1 (PAR-1), the thrombin receptor.

Stimulation of Fibrinolytic Activity

APC may directly stimulate fibrinolytic activity by inactivating plasminogen activator inhibitor-1 (PAI1) or PCI, another inhibitor of plasminogen activator, and indirectly by decreasing the formation of

Regulation of Tissue Remodeling

APC may suppress excessive extracellular matrix deposition by stimulating the expression and activation of premetalloproteinases and by blocking the

Stimulation of fibrinolysis

Inhibition of coagulation

TAFIa APC • PCI APC • PAI-1

TAFI

TB

Regulation of remodeling

MMP

Inhibition of apoptosis

Inhibition of inflammation

Permeability Cytokines

PDGF

P53 Bax Bcl-2

Pro-MMP C4BP

PTB

PS

Prothrombinase APC C4BP APC EPCR

FX Tenase

Va

APC

VIIIa

PS

SP1R

PS TM

APC

?

TB

PC EPCR

PAR-1

Cell membrane of endothelial cells, monocytes/macrophages, platelets or epithelial cells Figure 4 Activation and biological functions of the protein C pathway. Protein C (PC) is converted to activated protein C (APC) on the phospholipid-rich surface of endothelial cells, monocytes/macrophages, platelets, and epithelial cells by the thrombin (TB)–thrombomodulin (TM) complex in the presence of calcium. The endothelial protein C receptor (EPCR) enhances PC activation. APC blocks prothrombinase formation and activation of coagulation by inactivating activated factor V (Va) and activated factor VIII (VIIIa). APC stimulates fibrinolysis directly by forming a complex with plasminogen activator inhibitor-1 (PAI-1) and protein C inhibitor (PCI) or indirectly by preventing thrombin-induced activation of thrombin-activatable fibrinolysis inhibitor. APC exerts anti-inflammatory activity by reducing endothelial permeability and by suppressing the secretion of cytokines. APC affects cell survival by regulating the expression of pro- and antiapoptotic factors. The anti-inflammatory and antiapoptotic activity of APC appears to be mediated by EPCR, the protease-activated receptor-1 (PAR-1), or the sphingosine 1phosphate receptor (SP1R).

524 COAGULATION CASCADE / Protein C and Protein S

secretion of platelet-derived growth factor from a variety of cells, including monocytes/macrophages and endothelial and epithelial cells. APC may also affect tissue repair by promoting angiogenesis. Regulation of Cell Survival

free PS level induced by injection of C4BP, which blocks the APC cofactor activity of PS, exacerbates the systemic levels of cytokines induced by Escherichia coli infection, with this response being suppressed by PS supplementation.

APC may promote cell survival by upregulating the expression of antiapoptotic factors. APC protects from neural and brain endothelium apoptosis by inhibiting the expression of the tumor suppressor protein p53 and the proapoptotic protein Bax and by stimulating the expression of Bcl-4, an antiapoptotic protein. This protective action of APC is also mediated by both EPCR and PAR-1 receptors.

Miscellaneous Functions

Biological Functions of PS

PC and PS in Respiratory Diseases

Inhibition of Coagulation

Thromboembolism with secondary pulmonary hypertension may occur in patients with hereditary deficiency of PC or PS, suggesting the clinical importance of the anticoagulant PC pathway. Impairment of PC function may also occur in acquired conditions. Activation of PC is impaired in the lungs of patients with several inflammatory diseases, such as sarcoidosis and bronchial asthma, and of patients with idiopathic pulmonary diseases and collagen vascular disease-associated pulmonary fibrosis. Impairment of APC generation has also been demonstrated in experimental animal models of lung injury and bronchial allergic inflammation. The mechanism of this low APC generation is not completely understood. There is increased concentration of PAI-1 and PCI in the intracellular space in patients with inflammatory lung disorders, and several inflammatory mediators such as TNF-a may decrease in vitro the expression of PC, TM, and EPCR in endothelial cells or bronchial epithelial cells. Proteolytic cleavage of membrane-bound TM and EPCR may also occur during lung injury. Thus, the subnormal level of APC in the lung may result in part from its accelerated inhibition by the serpin family of proteases, by excessive consumption or downregulation of PC and PS, or by low availability of cell surface TM and EPCR. Dysfunction of the PC pathway may perpetuate the activation of mechanisms known to play crucial roles in the pathogenesis of lung injury, pulmonary fibrosis, and bronchial asthma. In experimental animal models, administration of exogenous APC protects from acute lung injury by limiting excessive activation of these host defense mechanisms. Therapy with APC also improves clinical outcomes in patients with sepsis, some of them also with acute respiratory distress syndrome. Administration of exogenous APC blocks development of pulmonary

PS exerts anticoagulant activity mainly by acting as a cofactor to APC in the inactivation of factors Va and VIIIa. The high affinity of PS for negatively charged phospholipids helps in APC binding to the membrane. This function is lost when PS binds to the b chain of C4BP; thus, only free PS supports the anticoagulant activity of APC in vivo. This function has clinical importance because a deficiency of PS is associated with severe thrombotic complications. PS also exhibits in vitro APC-independent anticoagulant activity by directly inhibiting the prothrombinase complex and the factor X activating complex. PS blocks the binding of prothrombin to factor Va and the amidolytic activity of factor Xa. Both the free form and PS in complex with C4BP can display this direct anticoagulant property. Stimulation of Fibrinolysis

PS may accelerate fibrinolysis by suppressing activation of thrombin-activatable fibrinolysis inhibitor or by supporting the APC-mediated inhibition of PAI-1. However, the physiological significance of this profibrinolytic function of PS is unknown. Regulation of the Inflammatory Response

Removal of dying cells is crucial for avoiding inflammation induced by autoantigens. PS may regulate the inflammatory response by helping in the clearance of apoptotic cells. PS binds to negatively charged phospholipids exposed on apoptotic cells and enhances severalfold their phagocytosis by macrophages. PS also serves to localize C4BP to cell membrane to inhibit activation of the complement system. Indirect observations also indicate that PS supports host defense mechanisms. For example, a decreased

PS is also a mitogen of smooth muscle cells and thus it may play a role in vascular repair. The receptor that mediates this action is still elusive, but it may be similar to a tyrosine kinase receptor of the Tyro3/Axl family. PS is synthesized by osteoblasts and thus it may participate in bone formation.

COAGULATION CASCADE / Thrombin 525

fibrosis in the mouse. The beneficial effect of APC may depend on its ability to inhibit generation and the profibrotic activity of thrombin and to decrease the secretion of the potent fibroblast mitogen platelet-derived growth factor and the expression of various cytokines with fibrogenetic activity. The stimulatory activity of APC on fibrinolysis and prometalloproteinases may also explain its antifibrotic effect. APC has the potential to inhibit allergic bronchial inflammation, as demonstrated in animal models of bronchial asthma. Th2 cytokines, including IL-4, IL-5, and IL-13, play a key role in the pathogenesis of asthmatic inflammation. APC may block Th2 cytokine expression from lymphocytes, leading to restoration of Th2/Th1 balance, less eosinophil infiltration and IgE concentration in the lung, and reduced airway hyperresponsiveness. APC appears to inhibit Th2 cytokine expression by decreasing nuclear translocation of NF-kB and the signal transducer and activator of transcription. APC may also block allergic inflammation in the lung by inhibiting recruitment of eosinophils.

Future Perspectives Studies have demonstrated that dysfunction of the PC pathway due to low availability of some of its components plays a pivotal role in the pathogenesis of lung injury and bronchial inflammation. Correction of this dysfunction by exogenous administration of APC may help cure these disorders. The success of APC in the treatment of patients with sepsis supports this assumption. APC also holds promise for the treatment of chronic inflammatory diseases of the lung. Studies using appropriate delivery systems such as inhalation therapy are needed to prove its effectiveness.

Gabazza EC, Taguchi O, Kamada H, et al. (2004) Progress in the understanding of protease-activated receptors. International Journal of Hematology 79: 117–122. Kobayashi H, Gabazza EC, Taguchi O, et al. (1998) Protein C anticoagulant system in patients with interstitial lung disease. American Journal of Respiratory and Critical Care Medicine 157: 1850–1854. Rezende SM, Simmonds RE, and Lane DA (2004) Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S–C4b binding protein complex. Blood 103: 1192–1201. Shimizu S, Gabazza EC, Taguchi O, et al. (2003) Activated protein C inhibits the expression of platelet-derived growth factor in the lung. American Journal of Respiratory and Critical Care Medicine 167: 1416–1426. Suzuki K (2005) Protein C. In: High KA and Roberts HR (eds.) Molecular Basis of Thrombosis and Hemostasis, pp. 393–423. New York: Dekker. Suzuki K (2005) Protein S. In: High KA and Roberts HR (eds.) Molecular Basis of Thrombosis and Hemostasis, pp. 459–478. New York: Dekker. Suzuki K, Gabazza EC, Hayashi T, et al. (2004) Protective role of activated protein C in lung and airway remodeling. Critical Care Medicine 32: S262–S265. Van de Wouwer M, Collen D, and Conway EM (2004) Thrombomodulin–protein C–EPCR system: integrated to regulate coagulation and inflammation. Arteriosclerosis, Thrombosis and Vascular Biology 24: 1374–1383. Yasui H, Gabazza EC, Tamaki S, et al. (2001) Intratracheal administration of activated protein C inhibits bleomycin-induced lung fibrosis in the mouse. American Journal of Respiratory and Critical Care Medicine 163: 1660–1668. Yuda H, Adachi Y, Taguchi O, et al. (2004) Activated protein C inhibits bronchial hyperresponsiveness and Th2 cytokine expression in mice. Blood 103: 2196–2204.

Thrombin R C Chambers, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract See also: Coagulation Cascade: Overview; Factor V; Factor X; Fibrinogen and Fibrin; Thrombin; Tissue Factor. Complement. Fibrinolysis: Overview; Plasminogen Activator and Plasmin. Interstitial Lung Disease: Overview. Serine Proteinases.

Further Reading Bernard GR (2003) Drotrecogin alfa (activated) (recombinant human activated protein C) for the treatment of severe sepsis. Critical Care Medicine 31: S85–S93. Castellino FJ (2001) Gene targeting in hemostasis: protein C. Frontiers in Bioscience 6: D807–D819. Dahlback B and Villoutreix BO (2003) Molecular recognition in the protein C anticoagulant pathway. Journal of Thrombosis and Haemostasis 1: 1525–1534. Esmon CT (2003) The protein C pathway. Chest 124: 26S–32S. Esmon CT, Fukudome K, Mather T, et al. (1999) Inflammation, sepsis, and coagulation. Haematologica 84: 254–259.

Thrombin is a serine protease that plays a central role in hemostasis by promoting blood coagulation. It is formed from its precursor, prothrombin, following tissue injury and converts fibrinogen to fibrin in the final step of the coagulation cascade. Thrombin also exerts numerous cellular effects, including promoting platelet aggregation, inflammatory cell recruitment, endothelial cell activation, and proliferation of mesenchymal cells. These effects are mediated via the proteolytic cleavage and activation of a novel family of cell-surface receptors known as proteinase-activated receptors. Thrombin activity is under tight regulatory control, and excessive or persistent generation of thrombin has been implicated in a number of respiratory diseases, including acute lung injury, interstitial lung disease, asthma, and chronic lung disease of prematurity.

Introduction At the end of the nineteenth century, Buchanan, a Scottish physiologist, suggested the presence of a

526 COAGULATION CASCADE / Thrombin

blood-borne clotting agent, which he named thrombin. In 1951, the protein was purified, and since then there have been rapid developments in our understanding of the structure–function relationships of this proteinase. Thrombin is activated during the final stages of the coagulation cascade and promotes the cleavage of soluble fibrinogen to form an insoluble fibrin clot. Besides its critical role in fibrin formation, thrombin exerts numerous cellular effects via the proteolytic activation of a novel family of cell surface receptors, the proteinase-activated receptors (PARs).

Structure The prothrombin gene is located on chromosome 11 (cytoband p11–q12) and contains 14 exons and 13 introns. Prothrombin is synthesized in the liver as a pre-propeptide that undergoes extensive posttranslational modification prior to secretion. This includes the carboxylation of glutamyl residues by the microsomal enzymes, the vitamin K-dependent carboxylases. The resultant g-carboxy glutamyl residues are necessary for calcium-dependent interaction with a negatively charged phospholipid surface, which is essential for the conversion of prothrombin to thrombin by the prothrombinase complex. The resultant 39 kDa globular protein comprises two chains cross-linked by four disulfide bonds that house a deep, narrow groove that contains the catalytic triad consisting of His43, Asp99, and Ser205. This groove is hydrophobic and exhibits a preference for apolar amino acids preceding arginine at a thrombin susceptible bond, such as Leu Asp Pro

Arg/Ser (where ‘/’ represents the cleavage site). The high specificity of thrombin toward its substrates and receptors is conferred by the depth and narrowness of the active site cleft, as well as by its unique anionbinding exosite (basic patch). In addition, thrombin possesses three specificity pockets that interact with inhibitors.

Regulation of Production and Activity Thrombin is the major effector of the coagulation cascade and is generated following the stepwise activation of coagulation proteinases (Figure 1). In vivo, the activation of the coagulation cascade is initiated via the extrinsic pathway. Upon tissue injury, plasma is exposed to tissue factor expressed on nonvascular cells or activated endothelial cells, allowing a complex between factor VII and tissue factor to form. Factor VII is then activated to factor VIIa. The tissue factor–factor VIIa complex subsequently catalyzes the initial activation of factor IX (to factor IXa) and factor X (to factor Xa). Factor Xa in association with activated factor V catalyzes the conversion of prothrombin to thrombin, which in turn catalyzes the conversion of fibrinogen to fibrin, the main constituent of a clot. This mechanism is believed to generate only limited amounts of thrombin. Sustained coagulation is achieved when thrombin synthesized through the initial factor VII–tissue factor complex pathway catalyzes the activation of factors XI, IX, VIII, and X. In this manner, the intrinsic pathway is activated, leading to the sustained generation of thrombin and blood coagulation. The extrinsic pathway is therefore paramount in initiating

Extrinsic pathway

Intrinsic pathway

Tissue factor

Factor VII

Thrombin

Factor VIIa

Factor XIa

Factor IXa

Factor X

Factor X

Factor Xa

Prothrombin

Thrombin

Fibrinogen Figure 1 The coagulation cascade.

Factor IX

Fibrin

Factor XI

COAGULATION CASCADE / Thrombin 527

coagulation via the activation of a limited amount of thrombin, whereas the intrinsic pathway maintains coagulation by the dramatic amplification of the initial signal. The coagulation cascade is tightly controlled by both negative feedback mechanisms, as well as by circulating and locally produced inhibitors. The extrinsic pathway is mainly controlled by tissue factor pathway inhibitor (TFPI). This protein forms a complex with factor Xa generated at the initiation of coagulation. This complex then inactivates factor VIIa–tissue factor complexes, thus inhibiting further synthesis of factors IXa and Xa. The intrinsic pathway is controlled by antithrombin III, which inhibits thrombin and other serine proteases of the coagulation cascade in the presence of heparin. Other important physiological inhibitors include heparin cofactor II and protease nexin-1, which inhibit thrombin, and a2-macroglobulin and a1-antitrypsin (also known as a1 proteinase inhibitor), which inhibit thrombin and factors IXa, Xa, and XIa. Finally, blood coagulation is also regulated when thrombin binds to the endothelial cell surface receptor, thrombomodulin, and is converted from a procoagulant to an anticoagulant by activating protein C. Protein C, in conjunction with protein S, inactivates factors Va and VIIIa and thereby prevents further thrombin generation.

Biological Function Thrombin plays a critical role in blood coagulation by converting fibrinogen to fibrin and by promoting platelet aggregation. In addition, thrombin exerts a range of other cellular effects that may play important roles in subsequent inflammatory and tissue repair responses, as part of the normal response to injury, but that may also lead to lung pathology. In addition to being one of the most potent physiological agonists of platelet aggregation, thrombin also promotes the release of thromboxane A2 and platelet factor-4 and the fibrogenic mediators, platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-b), involved in early tissue repair responses. Thrombin exerts dramatic effects on vascular tone and permeability via both direct effects on endothelial cell contraction and indirect mechanisms involving the release of nitric oxide and serotonin from endothelial cells and histamine from mast cells. In terms of influencing inflammatory responses, thrombin is a chemoattractant for inflammatory cells and stimulates the release of a host of proinflammatory cytokines, including monocyte chemotactic protein-1, interleukin-6 (IL-6), and IL-8, by a number of cell types. Thrombin further

influences inflammatory cell trafficking by inducing the expression of cell surface adhesion molecules, such as P-selectin and intercellular adhesion molecule-1. Finally, thrombin is a potent fibroblast and smooth muscle cell mitogen and induces extracellular matrix synthesis via the production of fibrogenic mediators, including PDGF, TGF-b, and connective tissue growth factor, which act via both autocrine and paracrine mechanisms.

Receptors Most of thrombin’s cellular effects are mediated via the proteolytic activation of ubiquitously expressed cell surface receptors, termed PARs (Figure 2). These receptors belong to the family of G-protein-coupled seven-transmembrane domain receptors and currently comprise four members, of which three (PAR1, PAR3, and PAR4) are thrombin receptors. These receptors display a unique mechanism of activation involving limited proteolysis of the N-terminus, resulting in the unmasking of a cryptic ligand. This ligand binds to the second extracellular loop of the receptor and thereby triggers signaling via heterotrimeric G-proteins. Synthetic peptides corresponding to the tethered ligand sequences of PAR1 and PAR4 are capable of activating these receptors independently of receptor cleavage and are widely used as experimental tools to invoke the involvement of these receptors in mediating thrombin cellular responses. In contrast, PAR3 is not capable of initiating cell signaling by thrombin but, rather, is thought to act as an accessory receptor in concert with other PARs.

Thrombin in Respiratory Diseases Activation of the coagulation cascade is a feature of a number of respiratory conditions associated with inflammation and excessive deposition of connective tissue proteins. Intra-alveolar accumulation of fibrin occurs in the lungs of patients with pulmonary fibrosis, in acute lung injury (ALI), and in acute respiratory distress syndrome (ARDS), in which rapid fibroproliferation and matrix synthesis can lead to the development of extensive fibrotic lesions. Bronchoalveolar lavage fluid obtained from patients with ARDS has also been reported to contain tissue factor–factor VII/VIIa complexes that can trigger activation of the extrinsic coagulation cascade. Levels of active thrombin are increased in the lungs of patients with pulmonary fibrosis associated with systemic sclerosis, in pulmonary fibrosis associated with chronic lung disease of prematurity, and in asthma. Increased procoagulant activity and/or tissue factor

528 COAGULATION CASCADE / Thrombin

Thrombin

PAR1 activation

Fibrin deposition

Inhibition of surfactant function Fibroblast & inflammatory cell recruitment

Microvascular permeability Production of proinflammatory cytokines Inflammatory cell trafficking and mitogenesis

Production of fibrogenic mediators Fibroblast chemotaxis & proliferation Fibroblast to myofibroblast transformation and ECM synthesis

Lung inflammation and fibrosis Figure 2 Proposed mechanisms for the role of thrombin in lung inflammation and fibrosis.

levels have also been identified in patients with idiopathic bronchiolitis obliterans-organizing pneumonia, sarcoidosis, hypersensitivity pneumonitis, and pleural disease. The pathological importance of excessive or smoldering activation of the coagulation cascade in both acute and chronic lung injury has been demonstrated in extensive studies employing experimental animal models using direct coagulation proteinase inhibitors and genetically modified mice. Evidence suggests that both fibrin persistence and thrombin signaling contribute to the pathogenesis of ALI/ARDS and pulmonary fibrosis. In terms of the receptor by which thrombin acts in these disease settings, studies of PAR1-deficient mice suggest that PAR1 plays an important role in both the acute inflammatory phase (inflammatory cell recruitment and microvascular injury) and the chronic fibrotic phase (lung collagen accumulation) in experimentally induced lung injury. Several agents aimed at targeting the coagulation cascade, including antithrombin III and TFPI, have undergone trials in humans with ALI/ARDS, but the results have been largely disappointing. The results of the phase III, randomized, double-blind, placebocontrolled multicenter PROWESS trial of intravenous infusion of the anticoagulant activated protein C in severe sepsis (which included patients with ARDS) and subsequent reduction in mortality resulted in approval of the use of this drug in severe sepsis. ARDS subgroup analysis was not presented so

that the ability of this agent to protect the lung in ARDS remains to be determined. Future studies of this agent in this and other respiratory disease settings associated with uncontrolled thrombin generation are eagerly awaited. See also: Acute Respiratory Distress Syndrome. Alveolar Hemorrhage. Asthma: Overview. Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12). Coagulation Cascade: Overview; Factor VII; Factor X; Thrombin. Connective Tissue Growth Factor. G-ProteinCoupled Receptors. Interstitial Lung Disease: Overview. Pulmonary Fibrosis. Signal Transduction. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Bernard GR, Vincent JL, Laterre PF, et al. (2001) Recombinant Human Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group. Efficacy and safety of recombinant human activated protein C for severe sepsis. New England Journal of Medicine 344: 699–709. Chambers RC (2003) Proteinase-activated receptors and the pathophysiology of pulmonary fibrosis. Drug Development Research 60: 29–35. Cocks TM and Moffatt JD (2000) Protease-activated receptors: sentries for inflammation? Trends in Pharmacological Science 21: 103–108. Coughlin SR (2000) Thrombin signalling and protease-activated receptors. Nature 407: 258–264. Dahlback B (2000) Blood coagulation. Lancet 355: 1627–1632. Gunther A, Lubke N, Ermert M, et al. (2003) Prevention of bleomycin-induced lung fibrosis by aerosolization of heparin or

COAGULATION CASCADE / Tissue Factor 529 urokinase in rabbits. American Journal of Respiratory and Critical Care Medicine 168: 1358–1365. Howell DCJ, Goldsack NR, Marshall RP, et al. (2001) Direct thrombin inhibition reduces lung collagen accumulation and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis. American Journal of Pathology 159: 1383–1395. Howell DC, Johns RH, Lasky JA, et al. (2005) Absence of proteinase-activated receptor-1 signaling affords protection from bleomycin-induced lung inflammation and fibrosis. American Journal of Pathology 166: 1353–1365. Idell S (2003) Coagulation, fibrinolysis, and fibrin deposition in acute lung injury. Critical Care Medicine 31: S213–S220. Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, and Plevin R (2001) Proteinase-activated receptors. Pharmacology Reviews 53: 245–282. Ploplis VA, Wilberding J, McLennan L, et al. (2000) A total fibrinogen deficiency is compatible with the development of pulmonary fibrosis in mice. American Journal of Pathology 157: 703–708. Ruf W (2004) Protease-activated receptor signaling in the regulation of inflammation. Critical Care Medicine 32: S287–S292. Suzuki K, Gabazza EC, Hayashi T, et al. (2004) Protective role of activated protein C in lung and airway remodeling. Critical Care Medicine 32(5 supplement): S262–S265. van der Poll T, Levi M, Nick JA and Abraham E (2005) Activated protein C inhibits local coagulation after intrapulmonary delivery of endotoxin in humans. American Journal of Respiratory and Critical Care Medicine 171: 1125–1128. Wagers SS, Norton RJ, Rinaldi LM, et al. (2004) Extravascular fibrin, plasminogen activator, plasminogen activator inhibitors, and airway hyperresponsiveness. Journal of Clinical Investigation 114: 104–111.

Tissue Factor L V M Rao and U Pendurthi, University of Texas Health Center, Tyler, TX, USA & 2006 Elsevier Ltd. All rights reserved.

organ dysfunction. Blockade of TF attenuates acute lung injury associated with Gram-negative sepsis in experimental animal model systems.

Introduction The main function of tissue factor (TF) in vivo is to trigger the blood coagulation process. After vascular injury, circulating plasma clotting factor VII (FVII) comes in contact with TF. Tissue factor binds and activates FVII. The resultant TF–FVIIa complexes initiate coagulation cascade by activating both factors IX and X, which ultimately leads to thrombin generation, platelet activation, and fibrin deposition. The primary role of TF in the complex is to induce allosteric changes in FVIIa, which dramatically increase FVIIa activity, and thus making it an efficient catalyst of factor IX and factor X activation. Tissue factor is constitutively expressed in mesenchymal cells residing in the adventitial lining of blood vessels but not in intravascular cells. Bacterial infection or certain other pathological conditions induce TF expression in monocytes, and probably in endothelial cells. Thus, FVII comes in contact with TF only upon disruption of blood vessel barriers or in a disease state. However, recent studies suggest TF presence in plasma, and the blood-borne TF may contribute to thrombosis. At present, the source of blood-borne TF and its pathophysiological significance are unclear. In addition to its role in coagulation, TF may also have a nonhemostatic function. Tissue factor-FVIIa, and the coagulation proteases generated by TF–FVIIa, i.e., factor Xa and thrombin, are shown to activate proteinase-activated receptors (PARs). The TF-dependent signaling pathways contribute to a variety of pathophysiological processes, including pathogenesis of acute lung injury (ALI) and organ dysfunction.

Abstract Tissue factor (TF) is a cellular receptor for coagulation factor VII (FVII), and its activated form, factor VIIa (FVIIa). Exposure of TF to blood upon vascular injury leads to TF-FVIIa complex formation, which triggers blood coagulation cascade. In health, TF is constitutively expressed in many extravascular cells, including fibroblasts and pericytes in and surrounding blood vessel walls and lung epithelial cells, but absent from blood cells and endothelial cells that line blood vessel wall. Certain pathological conditions, such as sepsis, induce TF expression in monocytes, and increase the expression in lung, kidney, and other tissues. Such increased TF activity contributes to the procoagulant environment in both intravascular and extravascular compartments. Increased TF expression in parenchymal and recruited lung cells, coupled with transendothelial leakage of plasma proteins, leads to alveolar fibrin deposition. In addition, TF-FVIIa, and downstream coagulant proteases generated by TF-FVIIa amplify multiple steps of the inflammatory response through activation of proteinase-activated receptors (PARs). Both the TF pathway-induced fibrin deposition and cell signaling contribute to the pathogenesis of acute lung injury and

Structure Tissue factor is an integral membrane glycoprotein. The mature protein, following cleavage of a 32-residue leader sequence from the primary translational product, consists of a single polypeptide of 263 amino acids, with the predicted mol wt. of 29 593. The glycosylated TF migrates as a B45 000 Da protein on SDS polyacrylamide gels. The majority of the molecule resides on the cell surface except for 23 residues in the transmembrane region and a 21-residue C-terminal region in the cytoplasm. The extracellular domain of TF contains three potential N-glycosylation sites (NXT/S) at residues Asn11, Asn124, and Asn135. However, the carbohydrate chains are not required for the procoagulant function of TF. There are five half-cystine residues; four

530 COAGULATION CASCADE / Tissue Factor

half-cystines in the extracellular domain of TF form two disulfide bonds and the half-cystine in the cytoplasmic region (Cys245) is acylated by palmitic acid and stearic acid. Analysis of the human TF protein sequence revealed the presence of two putative phosphorylation sites (Ser253 and Ser258) in the cytoplasmic domain. Protein kinase C-dependent phosphorylation of Ser253 enhances subsequent Ser258 phosphorylation by a Pro-directed kinase. Palmitoylation of the cytoplasmic Cys245 negatively regulates Ser258 phosphorylation. Tissue factor exhibits a distant sequence homology with members of the class 2 cytokine receptor superfamily. Similar to cytokine receptors, the extracellular domain of TF is composed of two immunoglobulin-like domains. The crystal structure of TF revealed that the two Ig-like domains are tightly packed with loops from both modules interdigitating to make a ‘dovetailed’ joint at the interface (Figure 1). The binding site for factor VII lies at the interface region and involves residues from both the domains. TF does not undergo major structural changes upon FVIIa binding. In contrast, FVIIa loses its segmental flexibility upon binding to TF. This positions the FVIIa active site at an appropriate distance above the membrane surface for efficient cleavage of its substrates, factors IX and X.

Regulation of Production and Activity Tissue factor is expressed in a cell-specific manner. The TF gene is constitutively expressed in many extravascular cell types, such as fibroblasts in adventitia, epithelium of skin and lung, astrocytes in brain, Bowman’s capsule of glomeruli in the kidney, and stromal cells in endometrium. Although the TF gene is not expressed in luminal vascular cells in health, it is inducibly expressed in several vascular cell types, including monocytes, vascular endothelial cells, and smooth muscle cells. Detailed studies on the regulation of the TF promoter in various cell types indicate that Sp1 controls the basal (constitutive) TF gene expression whereas a distal enhancer ( 227 to 172) containing two AP-1 sites and a nuclear factor kappa B (NF-kB) site mediates the induction of the human TF gene in monocytes and endothelial cells (Figure 2). Proximal enhancers ( 109 to 59) containing overlapping Egr-1 and Sp1 sites mediate TF induction in epithelial-like cells and vascular smooth muscle cells, respectively. Various exogenous and physiological agents can induce TF expression in vascular endothelial cells, epithelial cells and cells of the monocyte/macrophage lineage. The agents that induce TF synthesis that are relevant to pathophysiology include inflammatory

Domain 1


1


2

Domain 2

Figure 1 Structure of tissue factor extracellular domain. Factor VII binding sites are shown in yellow and the region important for factor X activation is shown in red. Reproduced with minor modifications from Harlos K, Martin DMA, O’Brien DP, et al. (1994) Crystal structure of the extracellular region of human tissue factor. Nature 370: 662–666, with permission from Nature Publishing Group.

cytokines (interleukin-1 a and b, interleukin-2, and tumor necrosis factor alpha (TNF-a)), growth factors (fibroblast growth factor, platelet-derived growth factor, and vascular endothelial cell growth factor), thrombin, C-reactive protein, endotoxins, virus infections, and immune complexes. The level of TF induction and expression varies significantly from one stimulus to another, and from one cell type to another. Functional expression of TF is tightly regulated at the cell surface by multiple mechanisms. Cellular localization of TF dictates its functional activity. TF residing in the anionic phospholipid milieu of the plasma membrane is more active than the TF present within neutral phospholipids. Similarly, TF association with caveolae limits its activity, and the disruption of caveolae leads to increased TF functional

COAGULATION CASCADE / Tissue Factor 531 −250

−200 AP-1

Inducible

c-fos/c-jun

NF-B

−150 Sp1

−100

−50

Sp1/Egr-1

+1 bp TATA

Egr-1

c-rel/p65

Sp1

Sp1

Constitutive

Figure 2 The promoter region of tissue factor gene. Transcription factor binding sites in the human TF promoter are shown by boxes. The binding sites consist of (left to right): two activator protein-1 (AP-1, beige), one nuclear factor kappa B (NF-kB, red), two Sp1 (turquoise), and three overlapping Sp1 (turquoise)/early growth response gene product (Egr-1, magenta). A summary of constitutive (c-fos/c-jun (oblong) and Sp1 (round))and inducible transcription factors (c-Rel/P65 (diamond) and Erg-1 (pointed cylinder)) that bind to the promoter of TF gene is also shown.

activity. Although it is not well established, transformation of TF from dimers to monomers is also shown to regulate TF activity. Tissue factor pathway inhibitor-1 (TFPI-1) regulates TF–FVIIa activity by dual mechanisms, forming the inactive ternary complex TF–FVIIa–FXa–TFPI and promoting the endocytosis (and subsequent degradation) of TF–FVIIa complexes.

Biological Function The absence of TF-deficient humans and the embryonic lethality of TF / mice indicate that TF is essential for life. The primary function of TF is to initiate blood coagulation cascade following the vascular injury that ultimately leads to platelet activation and fibrin formation, which are vital in sealing the injured vessels to protect against hemorrhage after tissue injury. Aberrant TF expression in the vessel wall and/or circulating cells initiates life-threatening intravascular thrombosis in various diseases, such as atherosclerosis, cancer, and sepsis. In addition, TF plays a nonhemostatic role in many biological processes, such as vascular development, inflammation, angiogenesis, and tumor metastasis.

Receptors Tissue factor acts as a cellular receptor for FVIIa. The resultant TF–FVIIa complexes not only activate the coagulation pathway but also activate, either directly or through generation of factor Xa and thrombin, PAR-mediated cell signaling. The TF–FVIIa complex activates PAR-2, whereas the TF–FVIIa– FXa complex activates both PAR-1 and PAR-2. Factor Xa, although not efficiently, also activates both

PAR-1 and PAR-2, whereas thrombin activates PAR1, PAR-3, and PAR-4. PARs are members of a large family of seven transmembrane domain cell surface receptors that transmit extracellular signals through G-proteins. Activation of PARs involves proteolytic cleavage of a specific susceptible bond in the extracellular N-terminal portion of the receptors, which uncovers a new N-terminus that acts as a tethered ligand capable of activating the self or an adjacent PAR. PAR signaling influences cytoskeletal functions and gene expression patterns that regulate diverse cellular responses including cell migration, proliferation, and apoptosis. TF-dependent PAR-mediated cell signaling is thought to be in part responsible for pathogenesis of ALI and fibrotic lung disease.

Tissue Factor in Respiratory Diseases Tissue factor plays a critical role in intra-alveolar and intravascular fibrin deposition, which is frequently associated with ALI or ARDS (Figure 3). In lung, the microvascular endothelium and the alveolar epithelium form the alveolar-capillary barrier. The disruption of the alveolar-capillary barrier, which often occurs in acute injury or ARDS, results in leakage of plasma into the alveolus. This allows FVII to come in contact with TF on alveolar macrophages and lung epithelial cells. TF–FVIIa complexes thus formed trigger the coagulation cascade, with subsequent thrombin formation and fibrin deposition. Systemic or pulmonary bacterial infections activate inflammation, which in turn increases TF expression in alveolar macrophages and lung epithelial cells. Clinical studies in patients with ARDS or evolving pneumonia show an increased TF and FVIIa activity in bronchoalveolar lavage fluid (BALF). Although TFPI-1, the inhibitor of TF–FVIIa, also increases in

532 COAGULATION CASCADE / Tissue Factor

Alveolar space

Microorganisms endotoxins

Plasma exudation

TF Macrophages epithelial cells fibroblasts

TF

PAR-1/PAR-2

Thrombin Inflammatory cytokines

TF–FVIIa TF–FVIIa–FXa

FXa

PAR-2, PAR-1

FXa

Fibrin

DIC, Microthrombosis

IL-1, IL-6, IL-8

Thrombin

PAR-1 ALI Vascular space

Fibrin Cell recruitment, surfactant dysfunction etc.

CTGF/Procollagen

Fibrosis Figure 3 Role of tissue factor in acute lung injury. Microorganisms/endotoxin induce TF expression in monocytes and endothelial cells in vascular space and upregulate TF expression in alveolar macrophages and lung epithelial cells in the alveolar compartment. Damage to alveolar capillary barrier allows plasma-clotting factors to come in contact with TF expressed macrophages, alveolar epithelial cells, or fibroblasts in the alveolar compartment. Formation of TF–FVIIa complex on these cells triggers the activation of coagulation cascade leading to thrombin generation and fibrin formation. TF–FVIIa, either directly or through generation of thrombin, activates PAR-mediated cell signaling that could promote lung fibrosis through elaboration of connective tissue growth factor (CTGF) and procollagen synthesis in fibroblasts. PAR signaling upregulates cytokine production and TF in endothelial cells and macrophages. All these processes contribute to acute lung injury (ALI) and lung fibrosis. DIC, disseminated intravascular coagulation.

BALF in ARDS patients, the increase is much smaller than the increase in TF expression. Thus, TFPI-1 levels are not adequate to inhibit TF-induced coagulation in the alveolar space. The imbalance between TF and TFPI-1, coupled with impaired fibrin removal caused by depression of fibrinolysis in ARDS patients, leads to intrapulmonary fibrin deposition. Although fibrin provides the critical matrix for cell migration and collagen deposition that are necessary for wound healing and repair mechanisms, excessive fibrin accumulation in the lung contribute to injury in multiple ways. Fibrin deposits enhance inflammatory response by increasing vascular permeability, activating endothelial cells to produce proinflammatory cytokines and chemokines, inducing the accumulation of activated neutrophils, and inactivating lung surfactant function. Excess fibrin deposition in the lung can also be profibrotic. Tissue factor and proteases generated by the TF pathway of coagulation can also exert direct effects on both intravascular and extravascular cells in amplifying the inflammatory cascade. In lethal Escherichia coli sepsis animal models, inhibition of the

TF–FVIIa complex significantly reduced the inflammatory response, as measured by a decrease in the plasma levels of IL-6 and IL-8, and protected the animals against the lethal effects of septic shock. Blockade of TF–FVIIa assembly or its function prevented sepsis-induced hypoxemia, pulmonary hypertension, lung edema, and loss of pulmonary system compliance. In contrast, efficient inhibition of fibrin formation by blocking the coagulation cascade at the downstream of TF–FVIIa neither attenuated IL-6 and IL-8 increase nor reversed the lethal outcome of sepsis. Genetically modified mice expressing low levels of TF exhibited reduced IL-6 expression and increased survival in a mouse model of endotoxemia. These data suggest that TF–FVIIa contributes directly to ALI in sepsis in part through selective stimulation of proinflammatory cytokines. Recent in vitro cell studies have established that TF-dependent initiation of coagulation provides an efficient signaling mechanism via PARs. TF–FVIIa stimulates a number of proinflammatory cytokines and chemokines that are relevant to ALI through PAR2-mediated cell signaling. Nascent factor Xa in the transient

COAGULATION CASCADE / Tissue Factor 533

ternary TF–FVIIa–FXa complex also efficiently activates both PAR-1 and PAR-2. PAR signaling in endothelial cells and respiratory epithelial cells induces proinflammatory cytokines, such as IL-6 and IL-8. TF–FVIIa, FXa, and thrombin activation of PARs also upregulate the expression of connective tissue growth factor (CTGF) in lung fibroblasts. CTGF is a potent fibroblast mitogen and chemoattractant, and plays an important role in promoting connective tissue formation after tissue injury. However, overexpression of CTGF, which may occur in excessive and/ or persistent activation of the TF pathway of coagulation, would lead to pulmonary fibrosis. The role of TF in ALI is not limited to the alveolar compartment or lung interstitium. Aberrant expression of TF may promote thrombi in the pulmonary artery, resulting in pulmonary dysfunction. Tissue factor-induced diffused intravascular thrombosis and disseminated intravascular coagulation could also cause acute edematous pulmonary injury. Further, activation PAR signaling in endothelial cells upregulates IL-6 and TF, which provides a self-amplifying loop to sustain the inflammation. The coagulation abnormalities observed in ALI are similar to those present in sepsis, which is a major risk factor for ALI. Therefore, selective anticoagulants used as adjunctive therapy for the treatment of sepsis are expected to influence the clinical course of ALI. Although animals studies have demonstrated a consistent reduction in lung injury with the administration of anticoagulants that selectively block TF– FVIIa function, the information on their potential benefits in humans are either lacking or, where available, less convincing. A phase II study evaluating TFPI-1 in the treatment of severe sepsis suggested that lung function in ARDS patients was improved. However, a phase III trial failed to demonstrate a survival benefit. Active site-inhibited FVIIa (ASIS), which blocks TF–FVIIa complex formation by competing with endogenously generated FVIIa for limited TF sites, prevented lung injury after E. coli sepsis in baboons. The outcome of phase II clinical trails with ASIS in ARDS is yet to be reported.

Summary Tissue factor, a cellular receptor that occupies a central position in hemostasis and thrombosis, plays a key role in ALI. Tissue factor contributes to the pathogenesis of ALI by generating extravascular

Collagens

see Extracellular Matrix: Collagens.

fibrin deposition and mediating cell signaling via PARs. Selective blockade of TF–FVIIa complex formation or its activity may have therapeutic benefits in patients with ARDS. See also: Coagulation Cascade: Overview; Factor VII; Factor X; Thrombin. Connective Tissue Growth Factor. G-Protein-Coupled Receptors. ProteinaseActivated Receptors. Pulmonary Fibrosis. Signal Transduction.

Further Reading Camerer E, Kolsto AB, and Prydz H (1996) Cell biology of tissue factor, the principal initiator of blood coagulation. Thrombosis Research 81: 1–41. Eilertsen KE and Osterud B (2004) Tissue factor: (patho)physiology and cellular biology. Blood Coagulation and Fibrinolysis 15: 521–538. Giesen P, Rauch U, Bohrmann B, et al. (1999) Blood–borne tissue factor: another view of thrombosis. Proceedings of the National Academy of Sciences, USA 96: 2311–2315. Harlos K, Martin DMA, O’Brien DP, et al. (1994) Crystal structure of the extracellular region of human tissue factor. Nature 370: 662–666. Idell S (2003) Coagulation and fibrinolysis, and fibrin deposition in acute lung injury. Critical Care Medicine 31(supplement 4): S213–S220. Idell S, Gonzalez K, Bradford H, et al. (1987) Procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome – contribution of tissue factor associated with factor VII. American Review of Respiratory Diseases 136: 1466–1474. Konigsberg W, Kirchhofer D, Riederer MA, and Nemerson Y (2001) The TF: VIIa complex: clinical significance, structurefunction relationships and its role in signaling and metastasis. Thrombosis and Haemostasis 86: 757–771. Mackman N (1997) Regulation of the tissue factor gene. Thrombosis and Haemostasis 78: 747–754. Mackman N (2004) Role of tissue factor in hemostasis, thrombosis, and vascular development. Arteriosclerosis Thrombosis and Vascular Biology 24: 1015–1022. Morrissey JH (2001) Tissue factor: an enzyme cofactor and a true receptor. Thrombosis and Haemostasis 86: 66–74. Rao LVM and Pendurthi UR (2003) Regulation of tissue factorfactor VIIa expression on cell surfaces: a role for tissue factorfactor VIIa endocytosis. Molecular and Cellular Biochemistry 253: 131–140. Rao LVM and Pendurthi UR (2005) Tissue factor-factor VIIa signaling. Arteriosclerosis Thrombosis and Vascular Biology. 25: 47–56. Rapaport SI and Rao LVM (1995) The tissue factor pathway: how it has become a ‘prima ballerina’. Thrombosis and Haemostasis 74: 7–17. Ruf W (2004) Protease-activated receptor signaling in the regulation of inflammation. Critical Care Medicine 32: S287–S292. Welty-wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inflammation in acute lung injury. Thrombosis and Haemostasis 88: 17–25.

534 COLLECTINS

COLLECTINS J A Whitsett and P S Kingma, Cincinnati Children’s Hospital, Cincinnati, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Surfactant protein A (SP-A) and surfactant protein D (SP-D) are members of the collectin family of mammalian lectins that are expressed in the lung. Although SP-A and SP-D influence pulmonary surfactant structure and homeostasis in the alveoli, both are essential components of the innate immune system that recognize, bind, and clear invading microbes from the lung. SP-A and SP-D bind polysaccharides, phospholipids, and glycolipids expressed on the surface of viral, fungal, and bacterial pathogens. Both proteins directly kill pathogens or enhance their uptake by phagocytes. As a consequence of their physiologic function, SP-A and SP-D are targets for developing clinical therapies designed to limit the proliferation of microorganisms and the resulting inflammatory damage from persistent infection.

Each day, the human lung exchanges approximately 15 000 liters of air that contains a multitude of microorganisms and fine particles. Despite the absence of a rigid protective physical barrier at the air–tissue interface, the lung is infrequently subjected to infection or injury by toxicants. Pulmonary homeostasis is maintained in large part by multilayered innate and acquired immune systems that simultaneously defend B150 m2 of lung surface area from microbial invasion and maintain the integrity of the alveoli. Most infectious agents that enter the respiratory system are trapped in nasal passages and proximal conducting airways and removed by mucociliary clearance. Microbes that evade this first level of defense are deposited in the distal airways or alveolar compartments, where they interact with host defense proteins, respiratory epithelial cells, and marrowderived cells, including macrophages, leukocytes, and lymphocytes. Whereas resident macrophages respond rapidly, subsequent activation of the acquired immune system and recruitment of other immune cells takes time, during which pathogen invasion and proliferation may occur. The collectin family of host defense proteins has evolved to quickly recognize pathogens and instruct innate inflammatory responses to clear invading pathogens from the lung. This article focuses on the role of the pulmonary collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), which are recognized as critical components of the innate host defense of the lung that link innate and acquired immunity.

Collectin Family of Polypeptides The collectins are a family of collagenous proteins that belong to the C-type lectin superfamily. The collectin family includes the pulmonary collectins, SP-A and SP-D, as well as six other collectins that were identified in mammals: mannose-binding protein (MBP), collectin liver-1 (CL-1), collectin placenta 1, conglutinin, collectin of 43 kDa (CL-43), and collectin of 46 kDa (CL-46). Collectins are defined by four structural domains shared by all family members: a short amino-terminal cross-linking domain, a triple-helical collagenous domain, a neck domain, and a carbohydrate recognition domain (CRD) (Figure 1). Three neck domains combine to form a triple coiled-coil structure and subsequently orchestrate the ‘zipper-like’ assembly of the remaining domains into a trimer that serves as the minimal functional unit of each collectin. Trimers further combine into multimeric proteins through disulfide bonds among the cysteine-rich amino-terminal domain. The collagen domain consists of repeating triplets of Gly–X–Y, where X and Y can be any amino acid but are frequently proline or hydroxyproline. In SP-D, conglutinin, CL-1, CL-46, and CL-43, the collagen domain preferentially associates to form cruciform tetramers of 12 monomeric proteins. In contrast, the collagenous sequences of both SP-A and MBP are relatively short and interrupted in the Gly– X–Y repeat, which inserts a bend in the collagenous domain that produces regions of flexibility that enable the collagen domains of SP-A and MBP to angle away from the protein core and form an octameric bouquet-like structure of 18 monomers. Whether in cruciform or bouquet forms, the collagenous domains provide proper spacing between the separate trimeric subunits, which may allow crosslinking of carbohydrate structures on the surface of microorganisms facilitating their aggregation and neutralization. The CRD of all known collectins is composed of 115–130 amino acid residues. Comparisons of multiple collectins reveal that 22 amino acids of the CRDs are highly conserved. The majority of these residues preserve the C-type lectin fold within the CRD; however, the remaining sequences diverge significantly and probably dictate saccharide binding specificity. Although several calcium-binding sites have been identified, crystallographic analysis of the CRD of MBP and SP-D showed the presence of

COLLECTINS 535 SP-A

CHO-

CRD Neck domain Collagenous domain

cys-

N-terminal domain Monomer

Trimer

Octadecamer

SP- D

CRD CHO-

Neck domain Collagenous domain

cys-

N-terminal domain

Monomer

Trimer

Dodecamer

Figure 1 Structure of pulmonary collectins. Each pulmonary collectin contains a carbohydrate recognition domain (CRD), a neck domain, a collagenous domain, and a cysteine (cys)-rich amino-terminal cross-linking domain. The primary sites of N-linked glycosylation (CHO) are shown. Three neck domains combine to form a triple coiled-coil structure that leads to the formation of a collagen-like triple helix and a functional trimer. Trimeric subunits further combine into higher oligomeric forms containing 12 (SP-D) or 18 (SP-A) monomers.

between two and four calcium ions in most collectins. One calcium ion is known to be part of a troughlike pocket that facilitates saccharide binding. Within this pocket, binding occurs through direct coordination of the calcium ion, as well as hydrogen bond interactions with amino acid side chains. Another calcium ion, which is found only in SP-D, is located close to the neck domain and may explain why the neck domain of SP-D is able to bind lipopolysaccharide (LPS) and phospholipids. The role of the remaining calcium ions and the exact number of calcium ions required for collectin function remain unclear.

Synthesis of Pulmonary Collectins Surfactant Protein A

SP-A was the first pulmonary collectin identified and was named a surfactant protein on the basis of its close association with lipids in pulmonary surfactant. SP-A is encoded by two genes located within a ‘collectin locus’ on the long arm of chromosome 10 that also includes the genes for SP-D and MBP.

In humans, SP-A synthesis begins during the second trimester of gestation and occurs primarily in the alveolar type II epithelial cells, Clara cells, and in cells of tracheobronchial glands. Although the SP-A protein was initially recognized in the lung, it also has been detected in extrapulmonary tissues; however, the reported tissue distribution of SP-A in the literature is inconsistent. This is due primarily to non-specific binding of anti-SP-A antibodies as well as tissue-specific differences in mRNA versus protein expression. For example, reverse transcriptase polymerase chain reaction analysis has shown low amounts of SP-A mRNA in human prostate, pancreas, thymus, colon, and salivary gland. This expression pattern, however, varies among species, and when tested by either immunohistochemistry or Western analysis, the SP-A protein was not detected in extrapulmonary tissues in humans. Therefore, whereas SP-A gene expression may occur at low levels in multiple tissues, the SP-A protein seems to be primarily restricted to the respiratory system. Expression of SP-A increases with advancing gestation and in response to multiple forms of lung injury. Increased protein levels can be detected within

536 COLLECTINS

hours of exposure to bacterial endotoxin or after challenging mice with inhaled Pseudomonas aeruginosa. In addition, SP-A content and mRNA expression increase following toxin-mediated lung injury including hyperoxia. The mechanisms that control SP-A gene expression are complex and occur at the levels of gene transcription and mRNA stability. In studies using alveolar type II cells, as little as 400 bp of the 50 -flanking region of the SP-A gene was required for appropriate cell-specific, developmental, and inflammatory expression. Comparisons across multiple species revealed that several response elements are present within this region. These include a cAMP response element, an E box that binds upstream stimulatory factor-1 and -2, a GT box that binds Sp1, a thyroid transcription factor-1 (TTF-1) binding element, a GATA binding site, and a reversed nuclear factor kappa B (NF-kB) binding site. Transcription factor activation and binding to these response elements was increased in human fetal lung following exposure to epidermal growth factor, cAMP, g-interferon, and interleukin (IL)-1b and inhibited by glucocorticoid. cAMP, which increases in response to hyperoxia, activates protein kinase A, which in turn causes increased phosphorylation and activation of TTF-1. IL-1, which is increased as part of the response to infection, activates NF-kB. TTF-1 and NFkB subsequently act in an additive manner to bind the SP-A promoter and initiate transcription.

Unlike SP-A, the tissue distribution of SP-D requires that the SP-D promoter respond to relatively ubiquitous transcription factors. To facilitate this, the mouse SP-D promoter contains consensus transcription factor-binding sequences for the AP-1 family, forkhead transcription factors FoxA1 and FoxA2, TTF-1, nuclear factor of activated T cells (NFAT), and multiple sites for CCAAT enhancer-binding proteins (C/EBPs). NFATs, C/EBPs, and members of the AP-1 family activate the transcription of immune response genes in several organs. Interestingly, members of the C/EBP family are also prominent factors in the regulation of the hepatic acute phase response to systemic injury, suggesting that the regulation of SP-D by C/EBP may mediate an analogous pulmonary acute phase response to microbial- or toxinmediated damage within the lung. Developmentally regulated expression of SP-D is increased by glucocorticoid treatment in utero. Steroids likely increase SP-D mRNA levels through activation of C/EBP binding. In addition, as part of the innate immune system, SP-D protein levels may increase up to 70-fold following exposure to IL-4 or IL-13 in vivo. IL-4 and IL-13 induce calcineurin-mediated dephosphorylation of NFAT. Once activated, NFAT works cooperatively with TTF-1, AP-1 family members, and C/EBPs to form a multiprotein transcriptional complex that induces SP-D expression.

Surfactant Protein D

Relationship between Collectin Structure and Function

Human SP-D was named on the basis of sequence similarity to SP-A, expression in alveolar epithelial cells, and association with pulmonary surfactant. SP-D is encoded by a single gene located in close proximity to the SP-A gene on human chromosome 10. Although SP-D was first recognized in the lung and is expressed primarily by type II and other nonciliated bronchiolar respiratory epithelial cells, SP-D mRNA and protein were detected in many nonpulmonary tissues. SP-D immunostaining was detected in the epithelial cells of parotid glands, sweat glands, lachrymal glands, skin, gall bladder, bile ducts, pancreas, stomach, esophagus, small intestine, kidney, adrenal cortex, anterior pituitary, endocervical glands, seminal vesicles, and urinary tract. Extrapulmonary levels of SP-D mRNA and protein, however, are severalfold lower than those detected in the lung. SP-D mRNA is first detected in the mouse or rat lung at midgestation and increases prior to birth and during the neonatal period. SP-D mRNA increases following lung injury caused by bacterial endotoxin, inhaled microorganisms, and hyperoxia.

Pulmonary collectins are pattern-recognition molecules that bind to polysaccharides, phospholipids, and glycolipids on the surface of a variety of microorganisms (Table 1). In order to recognize the large variety of microorganisms that enter the lung, SP-A and SP-D must have a broad binding specificity. This is achieved by an open through-like pocket that facilitates binding interactions between the terminal carbohydrate of polysaccharides on the surface of microbes and the amino acid side chains within the CRD. Despite the required broad binding specificity, SP-A and SP-D display a monosaccharide binding preference for mannose or glucose over galactose. C-type lectins can be divided into two distinct subtypes with monosaccharide binding preference for either mannose/glucose or galactose. Oligosaccharide binding preference is determined by a Glu–Pro–Asn or Glu–Pro–Arg motif that is present within the binding pocket of SP-D and SP-A, respectively. Computational docking studies have shown that additional binding specificity arises through hydrogen binding between saccharide residues flanking the terminal

COLLECTINS 537 Table 1 Binding interactions of SP-A and SP-Da Organism Gram-negative bacteria Pseudomonas aeruginosa Klebsiella pneumoniae Escherichia coli 0111 Escherichia coli K12 Escherichia coli J5 Hemophilus influenzae, type A Hemophilus influenzae, type B Hemophilus influenzae, nontyped Salmonella minnesota

þ

than 10 000-fold when all CRDs are simultaneously bound to an organism. The collagenous domain of collectins is thought to have functional roles beyond the positioning of the CRD. For example, a specific GEKGEP motif within the collagen domain of MBP is involved in binding to the C1q receptor. This same sequence is present in SP-A but absent in SP-D, which does not interact with the C1q receptor. In addition, the collagen domain of MBP binds two serum proteases (MBP-associated serum protease-1 and -2) and consequently activates the complement cascade. Collectin function is also influenced by the distribution of attached carbohydrates. In particular, collectins vary in the location and number of asparagine-linked glycosylation sites across species. Examination of SP-A and SP-D across multiple species reveals at least one conserved site of glycosylation in each protein. The conserved sugar in SP-A is linked to the CRD, whereas the conserved sugar in SP-D is linked to the collagen domain. Mutant SP-A proteins lacking the glycosylation sequence are defective in interaction with the influenza A virus. However, functional differences between wild-type SP-D and a SP-D mutant lacking the conserved glycosylation site were not detected.

Collectins Modulate the Inflammatory Response in the Lung

SP-A

SP-D

þ þ

þ þ


þ
þ þ þ

þ þ

Gram-positive bacteria Staphylococcus aureus Streptococcus pneumonieae Group B streptococci

þ þ þ

þ þ þ

Fungi Aspergillus fumigatus Cryptococcus neoformans Candida albicans Pneumocystis carinii

þ þ þ þ

þ þ þ þ

þ þ þ þ

þ þ

Viruses Influenza A Respiratory syncytial virus Herpes simplex type I Cytomegalovirus Rotavirus Other Mycobacterium tuberculosis Mycoplasma pneumoniae

þ þ

þ þ

Lipopolysaccharide Rough Smooth Lipid A

þ
þ

þ



a

The presence ( þ ) or absence (
) of binding is shown. Where no symbols are given, no consistent data are available.

carbohydrate and amino acid side chains outside the CRD binding pocket. The majority of collectin–polysaccharide binding interactions occur within the CRD; however, the amino terminus, collagenous domain, and neck domain determine the degree of multimerization and spacing of the CRD, which in turn influences collectin function. For example, dodecameric SPD can bind, aggregate, and enhance neutrophil inactivation of influenza A virus, whereas trimeric SPD binds and weakly aggregates the virus. The monomeric CRD is defective in polysaccharide binding. This cooperative nature of polysaccharide binding by the CRD allows multiple CRDs from a single fully assembled collectin to bind clusters of glycoproteins or glycolipids on the surface of a microorganism. The interaction of multiple CRDs can increase binding affinity more

SP-A and SP-D modulate the host defense response to a variety of pulmonary pathogens (Figure 2). Several studies support both anti- and proinflammatory mechanisms for collectin-mediated control of immune cells. SP-A and SP-D indirectly decrease inflammatory cell activation by neutralizing noxious stimuli within the respiratory environment. When in the fully assembled multimeric form, a single collectin binds the saccharide ligands present on multiple organisms and consequently forms protein bridges between microbes. These bridges induce extensive microbial aggregation, which enhances clearance of organisms by mucociliary cells lining the respiratory tract. In addition, microbial binding of both proteins increases cell wall permeability and inhibits growth of both Gram-negative bacteria and fungal pathogens. Finally, studies of aerosolized LPS and SP-D demonstrated that SP-D scavenges inhaled LPS and limits the inflammatory effects of this molecule. The pulmonary collectins stimulate immune cellmediated recognition and clearance of pulmonary pathogens through three mechanisms. First, SP-A and SP-D act as chemoattractants and recruit alveolar neutrophils and macrophages to sites of infection. Second, collectins coat the surface of microorganisms

538 COLLECTINS Alveolar macrophage Phagocytosis

Phagocytosis

Oxygen radicals

Oxygen radicals

Cytokines

Cytokines

SP-D

SP-A

Bacteria, viral, and fungal pathogens Aggregation

Aggregation

Clearance

Clearance

Figure 2 Pulmonary collectins in host defense. In vitro and genetically engineered animal models suggest that SP-A and SP-D play multiple roles in pulmonary host defense. The pulmonary collectins bind and aggregate viral, bacterial, and fungal pathogens. SP-A and SP-D enhance phagocytosis of microorganisms and regulate the inflammatory response of alveolar macrophages.

and act as opsonins. With the CRD bound to the pathogen, the exposed domain of the collectin interacts with specific receptors on the surface of phagocytic cells and induces uptake and killing of the microorganism. Third, SP-A and SP-D have a direct, nonopsonic stimulatory effect on phagocytosis by immune cells. This is likely due to binding and upregulation of the mannose receptor, a pattern-recognition receptor involved in phagocytosis. The most compelling evidence for the role of SP-A and SP-D in modulating the inflammatory response comes from animal models of collectin deficiency, which show that the anti-inflammatory effects of SP-A and SP-D predominate in vivo. Mice with targeted deletions of the surfactant protein A (Sftp-a) gene (Sftp-a
/
) survived normally and had no differences in pulmonary surfactant composition, secretion, clearance, or pool size. However, Sftp-a
/
mice had defects in pulmonary host defense. Clearance of group B Streptococcus, Hemophilus influenza, respiratory syncytial virus (RSV) and P. aeruginosa was delayed in Spa
/
mice. Decreased bacterial clearance in Sftp-a
/
mice was associated with defects in alveolar macrophage-mediated recognition and uptake of bacteria. In addition, oxygen radical production and killing of engulfed microorganisms by Sftp-a
/
macrophages was markedly reduced. Although alveolar macrophage

phagocytosis and oxygen radical production were decreased, markers of lung inflammation were increased in Sftp-a
/
mice. Sftp-a
/
mice had an exaggerated inflammatory response and produced increased levels of proinflammatory cytokines TNFa, IL-6, and MIP2 when challenged with an infectious organism. Unlike Sftp-a
/
mice, which had relatively normal surfactant function, deletion of the Sftp-d gene generated defects in both pulmonary host defense and surfactant homeostasis. Sftp-d
/
mice survived normally but developed a gradually worsening pulmonary inflammation and surfactant lipid accumulations. Inflammatory lesions consisted of increased numbers of enlarged, foamy alveolar macrophages. Progressive emphysema developed in Sftp-d
/
mice, probably as a result of the reactive oxygen species and metalloproteinases that were released by the abnormally activated alveolar macrophages. Uptake and clearance of viral pathogens, including influenza A and RSV, were deficient in Sftp-d
/
mice. In contrast, clearance of group B Streptococcus and H. influenza was unchanged. When exposed to various infectious pathogens, oxygen radical release and production of the proinflammatory mediators, TNF-a, IL-1, and IL-6, were increased in Sftp-d
/
mice. SP-A and SP-D play both anti- and proinflammatory roles in pulmonary host defense. Evidence

COLLECTINS 539

suggests a mechanism by which SP-A and SP-D can simultaneously mediate anti- and proinflammatory processes in the lung. In the unbound state, the CRDs of SP-A or SP-D bind to signal-regulating protein-a (SIRPa), a transmembrane protein involved in signal transduction, which initiates a signaling cascade that inhibits proinflammatory mediator production by alveolar macrophages. In contrast, if the CRDs of SP-A or SP-D are occupied by a microbial ligand, the collectin reverses orientation and binds to calreticulin/ CD91 via the collagenous tail. Calreticulin/CD91 subsequently initiates a pathway that releases proinflammatory mediators and stimulates phagocytosis. Therefore, depending on the presence of infectious particles and subsequent binding orientation of the CRD, SP-A and SP-D can either enhance or suppress immune cell function.

SP-A and SP-D Receptors SP-A and SP-D interact with several receptors or binding molecules on host cells. These include glycoproteins and glycolipids, which bind the CRD, as well as membrane proteins, which interact with other regions of the collectin. Both SP-A and SP-D bind the LPS receptor CD14; however, the mechanism of binding differs between each protein. SP-D binds CD14 via interactions between the CRD and N-linked oligosaccharides on CD14, whereas SP-A binds directly to the protein backbone. As mentioned previously, SP-A and SP-D bind the LPS signaling protein SIRPa. SP-A also inhibits peptidoglycan-induced cytokine release by the Toll-like receptor-2. The fact that both collectins bind LPS and multiple proteins involved in LPS signaling suggests a possible mechanism for controlling the cellular response to LPS; however, the precise steps of this pathway remain to be determined. SPR-210 is a receptor found on alveolar type II cells, macrophages, and lymphocytes. SP-A binding to SPR-210 inhibits lipid secretion by type II cells, suppresses T lymphocyte proliferation, and enhances uptake of mycobacterium by macrophages. Evidence indicates that SPR-210, through the activation of cellular myosins, mediates high-affinity binding and uptake specifically of SP-A-coated particles and bacteria. SP-A and SP-D show calcium-dependent but CRDindependent binding to gp340, a member of the scavenger receptor superfamily. Although gp340 is located on alveolar macrophages and colocalizes with SP-D in multiple tissues, there is no evidence that gp340 represents a physiologically relevant receptor for either protein.

Collectins in Clinical Disease Human disease resulting from a mutation or deletion in SP-A or SP-D has not been identified. However, the apparent immune modulatory activities of these proteins, combined with the results of microbial challenge experiments in SP-A- and SP-D-deficient mice, suggest that altered function or decreased levels of either protein would result in clinical disease that would reflect a loss of either the pro- or anti-inflammatory properties of each protein. A variety of clinical conditions characterized by increased inflammation or lung parenchyma damage, including adult respiratory distress syndrome, bronchopulmonary dysplasia, asthma, chronic obstructive pulmonary disease, and cystic fibrosis, are associated with decreased levels of SP-A or SP-D in broncheolar lavage fluid. Altered collectin function is also associated with increased susceptibility to infection. The severity of RSV infections correlates with genetic polymorphism in SP-D. Specifically, infants hospitalized with RSV were more likely than controls to be homozygous for the allele coding methionine at amino acid 11 of SP-D. Furthermore, in a population of children who have increased susceptibility to infection, mutations were identified in the serum collectin, MBP, that interfered with appropriate protein folding and secretion.

Summary Collectins are an essential component of the innate immune system and modulate the inflammatory response of immune cells. In the absence of infection, SP-A and SP-D can inhibit inflammatory responses within the lung. However, in the presence of a microbial challenge, SP-A and SP-D recognize invading pathogens and orchestrate immune cell activation and phagocytosis of microbes. Therefore, the pulmonary collectins can provide a critical dual-role mechanism for initiating the critical host defense response to infection while limiting the potential damaging effects of inflammation and microbial invasion on the lung parenchyma. See also: Clara Cells. Endotoxins. Epithelial Cells: Type II Cells. Leukocytes: Pulmonary Macrophages. Surfactant: Overview; Surfactant Protein A (SP-A); Surfactant Protein D (SP-D). Toll-Like Receptors. Transcription Factors: Overview.

Further Reading Clark H and Reid KB (2002) Structural requirements for SP-D function in vitro and in vivo: therapeutic potential of recombinant SP-D. Immunobiology 205: 619–631.

540 COLONY STIMULATING FACTORS Crouch E and Whitsett JA (2003) Diverse roles of lung collectins in pulmonary innate immunity. In: Ezekowitz RAB and Hoffman JA (eds.) Innate Immunity. Totowa, NJ: Humana Press. Crouch E and Wright JR (2001) Surfactant proteins A and D and pulmonary host defense. Annual Review of Physiology 63: 521– 524. Gardai SJ, Xiao YQ, Dickinson M, et al. (2003) By binding SIRPa or clareticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation. Cell 115: 13–23. Hawgood S and Poulain FR (2004) The pulmonary collectins and surfactant metabolism. Annual Review of Physiology 63: 495– 519. Hickman-Davis JM, Fang FC, Nathan C, et al. (2001) Lung surfactant and reactive oxygen–nitrogen species: antimicrobial activity and host–pathogen interactions. American Journal of Physiology: Lung Cellular and Molecular Physiology 281: L517–L523.

Korfhagen TR, Bruno MD, Ross GF, et al. (1996) Altered surfactant function and structure in SP-A gene targeted mice. Proceedings of the National Academy of Sciences, USA 93: 9594–9599. Korfhagen TR, Sheftelyevich V, Burhans MS, et al. (1998) Surfactant protein D regulates surfactant phospholipid homeostasis in vivo. Journal of Biological Chemistry 273: 28438–28443. Kuroki Y and Sano H (1999) Functional roles and structural analysis of lung collectins SP-A and SP-D. Biology of the Neonate 76(supplement 1): 19–21. Kuroki Y, Takahashi H, Chiba H, and Akino T (1998) Surfactant proteins A and D: disease markers. Biochimica Biophysica Acta 19: 334–345. Palaniyar N, Nadesalingam J, and Reid KB (2002) Pulmonary innate immune proteins and receptors that interact with Grampositive bacterial ligands. Immunobiology 205: 575–594. Van de Wetering JK, van Golde LMG, and Bateenburg JJ (2004) Collectins: players of the innate immune system. European Journal of Biochemistry 271: 1229–1249.

COLONY STIMULATING FACTORS B C Trapnell and S Abe, Cincinnati Children’s Hospital, Cincinnati, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The colony stimulating factors (CSFs) comprise a small family of homodimeric cytokines central to blood formation, leukocyte function, and overall immune competence. Family members include macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF), and interleukin-3 (IL-3). M-CSF and G-CSF are relatively lineage-specific and affect the survival, growth, differentiation, and function of mononuclear phagocytes (monocytes/macrophages) and neutrophils, respectively. GM-CSF is less restricted and stimulates growth of myeloid progenitors as well as the survival and function of both macrophages and neutrophils. IL-3 is pluripotent and affects all hematopoietic lineages. CSF production and clearance are strictly regulated to maintain homeostasis and are modulated during conditions of stress such as the increased turnover of leukocytes in intensely inflammatory lung diseases like cystic fibrosis. The CSFs function within a complex yet precisely regulated signaling network involving local, paracrine, and endocrine pathways, the effects of which extend beyond hematopoiesis. For example, a critical role has been established for GM-CSF in the regulation of pulmonary surfactant homeostasis and innate immunity in the lung. Deficiency of endogenous GMCSF bioactivity due to neutralizing anti-GM-CSF antibodies is central to the pathogenesis of idiopathic pulmonary alveolar proteinosis.

Introduction In the mid-1960s, Donald Metcalf identified colony stimulating factor (CSF) in mice and several years later in humans. Over the next decade, four CSF

family members were identified, purified, and studied intensely. The genes for each CSF have been isolated from multiple species, cloned, and characterized in detail, and both human and murine recombinant CSF molecules are commercially available. Their respective receptors have also been identified, cloned, and characterized. M-CSF and G-CSF are relatively lineage-specific and stimulate formation of macrophage and granulocyte (neutrophil) colonies from bone marrow precursors, respectively. GM-CSF gives rise to both macrophage and neutrophils and IL-3 (also called multi-CSF) is a pluripotent factor stimulating formation of macrophages, neutrophils, eosinophils, basophils, mast cells, megakaryocytes, and erythroid cells.

M-CSF Structure

Human M-CSF (also known as CSF-1) is a complex cytokine that exists in three isoforms, each of which is composed of disulfide-linked homodimers derived from alternatively spliced mRNA transcripts. M-CSFb is comprised of two 522 amino acid (AA) polypeptides representing the full-length M-CSF coding sequence. M-CSFa is composed of two 224 AA polypeptides that lack residues 150–447. M-CSFg is composed of two 406 AA polypeptides and lacks residues 332–447. The molecular weight of M-CSFb (45–90 kDa) exceeds the predicted value (60 kDa) due to glycosylation. The crystallographic structure of M-CSF has been determined at 2.5 A˚ resolution

COLONY STIMULATING FACTORS 541

C

S C S S N C N M-CSF

G-CSF

C

S S

N

N

GM-CSF

IL-3

Figure 1 Crystal structures of cytokine growth factor monomers. All structures are depicted in worm format using the Cn3D viewer from the National Center for Biotechnology Information’s Molecular Modeling DataBase (MMDB) structural database. The structures were derived from the following NCBI Protein DataBase (PDB) files: Human MCSFa–PDB 1HMC A; Human G-CSF–PDB 1PGR A; Human GM-CSF–PDB 1CSG; Human IL-3–PDB IJLI. The amino terminal (N) and carboxy terminal (C) end of each polypeptide is indicated. Disulfide bonds are marked (S).

and M-CSF shares structural similarity to GM-CSF and G-CSF (Figure 1). Mature murine and human M-CSF are similar except that the former is 519 AA residues in length. Production and Activity

Human M-CSF is derived from a single gene at 1p13– 21; its predicted length is 554 AA including a 32 AA signal peptide. M-CSF mRNA is alternatively spliced within exon 6 resulting in three major transcripts encoding the three protein isoforms. All isoforms contain the 149 N-terminal residues required for biological activity. M-CSF is present as an integral cellsurface protein and several soluble forms. An active soluble form is released from the cell surface by proteolytic cleavage. Murine and human M-CSF are highly homologous within the N-terminal (bioactive) but not the C-terminal region of the molecule. M-CSF is produced in a wide variety of cells and normal blood levels (3–8 ng ml
1) are tightly controlled. More than 95% of M-CSF present in the blood is cleared by intravascular monocytes and macrophages, which internalize, degrade M-CSF, and proliferate in response. M-CSF pools in blood and tissues appear to be compartmentalized. Superimposed upon this ‘coarse’ control of M-CSF levels is a ‘fine’ control which can be recruited under stress conditions such as infection. At such times, local M-CSF release further stimulates local mononuclear phagocytes. Other factors stimulating M-CSF production include lipopolysaccharide (LPS), IL-2, phorbol esters, tumor necrosis factor alpha (TNF-a) and interferon gamma (IFN-g). Receptors

The M-CSF receptor (CD115) belongs to the protein tyrosine kinase receptor superfamily and is identical to the cfms proto-oncogene. It consists of one

140–150 kDa (972 AA) polypeptide with extracellular, membrane-spanning, and cytoplasmic domains. The cytoplasmic region includes an autophosphorylation domain that associates with phosphoinositol-3 OH kinase (PI3K) and is required for signaling. The M-CSF receptor gene is located at 5q33-3. It is normally expressed on macrophage lineage cells, osteoclasts, and placental trophoblasts. It is also expressed on mammary epithelial cells during lactogenic differentiation and other cell types in certain pathological conditions. The half-life is short (2–3 h) in the absence of ligand and M-CSF receptors are internalized and rapidly degraded upon ligand binding. Receptor expression is modulated by endotoxin and various cytokines. Ligand binding causes receptor dimerization, auto- and intermolecular phosphorylation of intracytoplasmic residues, activation of PI3K, Srcfamily kinases, and Raf-1 and other downstream signaling events. Human M-CSF is able to activate the murine M-CSF receptor but not vice versa. Biological Functions

M-CSF stimulates bone marrow progenitors to form macrophage colonies in soft agar as well as promoting the survival, growth, differentiation, and activation of macrophage lineage cells. In mature macrophages it stimulates production of cytokines (IL-1b, TNF-a, IFN, and G-CSF), plasminogen activator, prostaglandin E, and reactive oxygen species and regulates other metabolic processes. M-CSF stimulates the antimicrobial activity of macrophages and is important in bone metabolism, atherogenesis, lipoprotein clearance, and uterine function.

G-CSF Structure

Human G-CSF (also known as CSFb and pluripoietin) is expressed in two isoforms (177 and 174 AA)

542 COLONY STIMULATING FACTORS

due to alternative splicing. Its molecular weight (21 kDa) is larger than predicted (19 kDa) due to Olinked glycosylation at Thr133. There are two interchain disulfide bonds (Cys36–Cys42 and Cys64– Cys74) important for solubility and stability. The crystallographic structure of G-CSF has been deter˚ resolution (Figure 1). Mature murine mined at 2.5 A and human G-CSF are similar except that the former is 178 AA residues in length. Production and Activity

Human G-CSF is derived from a single gene at 17q21–22; its predicted length is 207 AA including a 30 AA signal peptide. G-CSF mRNA is polyadenylated and alternatively spliced in humans (but not mice); the 174 AA isoform is the dominant variant and is 20-fold more active than the 177 AA isoform. It is biologically active as a soluble homodimer. G-CSF is produced by activated macrophages/ monocytes as well as various other cells; its production is under tight control and blood levels are normally very low. G-CSF is cleared by granulocytes, which bind, internalize, degrade, and respond to it. G-CSF production is under both ‘coarse’ and ‘fine’ regulatory control. Factors stimulating its production include other CSFs (IL-3, GM-CSF, M-CSF), IL-1, IL-4, TNF-a, IFN-g, LPS, and phorbol esters. G-CSF expression is regulated at transcriptional and posttranscriptional levels. Receptors

The G-CSF receptor (CD114) belongs to the hematopoietic receptor superfamily and comprises a single 120–150 kDa polypeptide with extracellular, membrane-spanning, and cytoplasmic domains. It does not have intrinsic tyrosine kinase activity but is thought to bind and mediate phosphorylation of the Jak2 kinase upon ligand-mediated dimerization. The human G-CSF receptor gene is located at 1p35–34.3 and expression is strictly regulated transcriptionally. It is normally expressed on neutrophils, neutrophil progenitors, and platelets, but can also be found on endothelial and other cells. Expression of the G-CSF receptor is stimulated by all-trans retinoic acid and downregulated by GM-CSF, TNF, LPS, C5a, formylMet-Leu-Phe (fMLP), and phorbol esters. Ligand binding causes receptor dimerization, phosphorylation by Jak2 kinase, and downstream signaling events. Human G-CSF binds and stimulates the murine G-CSF receptor and vice versa. Biological Functions

G-CSF stimulates bone marrow progenitors to form neutrophil colonies in soft agar as well as promoting

the survival, proliferation, and differentiation of neutrophil lineage cells. G-CSF stimulation of mature neutrophils results in increased production of reactive oxygen intermediates, expression of C3b receptors, and release of arachidonic acid and myeloperoxidase. Neutrophil functions stimulated by G-CSF include phagocytosis, chemotaxis, and antibody-dependent cellular cytotoxicity (ADCC) against tumor cells. G-CSF promotes the proliferation and migration of endothelial cells in vitro, the secondary platelet aggregation induced by adenosine diphosphate, and angiogenic activity in rabbit cornea, and stimulates growth of some small cell lung cancer cell lines. It also stimulates production of acute phase response proteins.

GM-CSF Structure

GM-CSF (also known as CSF-2, CSFa or pluripoietin-a) is a 22 kDa homodimeric polypeptide cytokine in humans and mice that exists as a single soluble isoform. The mature human polypeptide is 127 AA in length (124 AA for mouse) and contains two intrachain disulfide bonds (Cys54–Cys96 and Cys88– Cys121 in human, Cys51–Cys93 and Cys85–Cys118 in mouse) and two potential N-linked glycosylation sites (Asn27 and Asn37 in human, Asn66 and Asn75 in mouse). The crystallographic structure of GM-CSF has been determined at 2.5 A˚ resolution (Figure 1). Production and Activity

Human GM-CSF is derived from a single gene at 5q23–31; its predicted length is 144 AA including a 17 AA signal peptide. Glycosylation is not required for biological activity, but nonglycosylated GM-CSF is 10-fold more active than the glycosylated form. Murine and human GM-CSF share approximately 56% homology but do not cross-react immunologically or functionally. GM-CSF is produced by T lymphocytes, macrophages, epithelial cells, endothelial cells, and various other cells. Production is tightly regulated. GM-CSF levels are normally only B5–45 pg ml
1 in serum but increase during infection. The levels are maintained by dual ‘coarse’ and ‘fine’ regulatory control. Factors stimulating GM-CSF production include LPS, IL-2, IL-1, and TNF-a. Production by macrophages is also stimulated by adherence and by phagocytosis. GM-CSF pools in the lungs and blood are compartmentalized and expression is regulated transcriptionally and posttranscriptionally.

COLONY STIMULATING FACTORS 543 Receptors

The GM-CSF receptor belongs to the hematopoietic receptor superfamily and is composed of heterodimeric a and b chains. The human a chain (CDw116) is an 80 kDa transmembrane protein with an extracellular region, a membrane-spanning region, and a short cytoplasmic tail and has 11 potential N-linked glycosylation sites. The human b chain (KH97) is a 130 kDa transmembrane protein with an extracellular domain, a membrane-spanning region, and a large cytoplasmic domain, and three potential N-linked glycosylation sites. The b chain is shared with the receptors for IL-3 and IL-5. The GM-CSF receptor is normally expressed on macrophages, neutrophils, and eosinophil lineage cells, and has been also demonstrated on CD34 þ progenitor cells, erythrocyte and megakaryocyte precursors, B and T fetal lymphocytes, vascular endothelial cells, fibroblasts, osteoblasts, and uterine cells. Expression of the GM-CSF receptor is stimulated by IFN-g, TNF-a, IL-3, erythropoietin (EPO), and stem cell factor (SCF). The a chain binds GM-CSF with low affinity (3.23  10
9 M). The b chain does not bind GMCSF, but associates with the ligand-bound a chain increasing the ligand-binding affinity (1.203  10
10 M). The b chain constitutively associates with Jak2 kinase and association with the ligand-bound a chain results in tyrosine phosphorylation of the receptor and Jak2, and signaling through multiple pathways. Biological Functions

GM-CSF stimulates bone marrow progenitors to form neutrophil and macrophage colonies in soft agar and has pleiotropic effects on hematopoietic and other cell types. It is a survival and growth factor for hematopoietic progenitors and stimulates differentiation and activation of granulocytes and monocytic cells, and serves as a growth factor for endothelial and epithelial cells, erythroid cells megakaryocytes, and T cells. GM-CSF exhibits overlapping activities on hematopoietic progenitors with other cytokines including M-CSF, G-CSF, IL-3, IL-6, and SCF. GM-CSF stimulates a broad range of innate immune functions in macrophage and neutrophil lineage cells including phagocytosis, chemotaxis, cytokine release, superoxide radical production, antigen presentation, and others, and contributes to host defense against bacterial, viral, fungal, and parasitic infections and to antitumor surveillance. GM-CSF has critical roles in the lung in mice and humans where it stimulates terminal differentiation of alveolar macrophages and is required for

surfactant homeostasis and innate immunity in the lung. Many of the effects of GM-CSF on alveolar macrophage innate immune and other functions are mediated by the transcription factor PU.1 (Figure 2).

IL-3 Structure

lnterleukin-3 (IL-3) (also known as multi-CSF, hematopoietic cell growth factor, burst-promoting activity, P-cell stimulating factor activity, thy-1 inducing factor) is a 14–30 kDa homodimeric polypeptide cytokine in humans (28 kDa in mice) that exists as a single isoform. Mature human IL-3 is 133 AA in length and contains one intrachain disulfide bond (Cys16–Cys84) and two potential N-linked glycosylation sites (Asn15 and Asn70). Mature mouse IL-3 is 140 AA in length and contains two disulfide bonds (Cys17–Cys80 and Cys89–Cys140). It has four potential N-linked glycosylation sites, but is glycosylated only at Asn16 and Asn86. The crystallographic structure of a variant of human IL-3 has been determined (Figure 1). Production and Activity

Human IL-3 is derived from a single gene at 5q23; its predicted length is 152 AA including a 19 AA signal peptide. Glycosylation is not required for biological activity. Human and murine IL-3 share only 29% homology and do not cross-react immunologically or functionally. IL-3 is produced by activated T lymphocytes, and various other cells. IL-3 gene expression is regulated transcriptionally and posttranscriptionally. Receptors

The IL-3 receptor belongs to the hematopoietic receptor superfamily and is composed of heterodimeric a and b chains. The human a chain (CD123) is a 70 kDa transmembrane protein with an extracellular region, a membrane-spanning region, and a short cytoplasmic tail with six potential N-linked glycosylation sites. The extracellular domain of the a chain shares tertiary structural similarity with the GM-CSF receptor a chain. The human b chain (KH97) is shared with the receptors for GM-CSF and IL-5 and has been described above. Mice have two b chains: AIC2B, which corresponds to the human b chain (KH97), and another, AIC2A, which associates only with the IL-3a chain. The IL-3 receptor is normally expressed on hematopoietic progenitor cells, monocytes, and B lymphocytes. Expression of the a chain is tightly regulated at the level of transcription.

544 COLONY STIMULATING FACTORS Surfactant film

Large surfactant aggregates Tubular myelin

SP-A SP-D

Alveolar stabilization

Host defense Surfactant pool size

Small surfactant aggregates

Alveolar macrophage

Secretion Recycling

ABCA3 transporter Lipids

Lamellar body SP-B SP-C

G Processing

Secretory vesicle SP-A SP-D SP-A Pro-SP-B Pro-SP-C SP-D

Uptake L PU.1 GM-CSF Catabolism

ER N

Type II alveolar epithelial cell

Type I alveolar epithelial cell

Figure 2 Regulation of surfactant homeostasis by GM-CSF. Surfactant proteins (SP)(SP-A, SP-B, SP-C, and SP-D) and surfactant lipids are produced in pulmonary type II alveolar epithelial cells and released as large aggregates into the alveolar space (green arrows). A thin surfactant film stabilizes the alveolus by reducing the tremendous force of surface tension at the air–liquid–tissue surface. Small surfactant aggregates derived from this film are either recycled in type II cells (yellow arrow) or taken up into alveolar macrophages. GMCSF, also produced by type II cells, stimulates increased levels of the transcription factor PU.1 in alveolar macrophages. PU.1, in turn, is required to stimulate catabolism of surfactant lipids and proteins in these cells (white arrows in macrophage). In the absence of GM-CSF signaling (human idiopathic pulmonary alveolar proteinosis, GM-CSF or GM-CSF receptor knockout mice), surfactant production proceeds as usual, but catabolism in alveolar macrophages is impaired resulting in intra-alveolar and intracellular surfactant accumulation. GM-CSF, via PU.1, stimulates alveolar macrophage terminal differentiation. ER, endoplasmic reticulum; G, Golgi body; N, nucleus.

In humans, only the a chain binds IL-3 and does so with low affinity (10
7 M). Association of the ligand-bound a chain with the b chain, which does not bind IL-3, converts ligand binding to high affinity (10
10 M). In mice, the same pattern is observed for binding to the a chain (53  10
8 M) and conversion to high-affinity binding by association with AIC2B, which increases a chain binding affinity (33  10
10 M). The mouse a/b (AIC2B) receptor binds IL-3 with high affinity (43  10
10 M). Ligand binding results in phosphorylation of the b chain and multiple other cellular proteins. Signal transduction requires the presence of the b subunit, which apparently does not have intrinsic kinase activity. Biological Function

IL-3 stimulates bone marrow progenitors to form erythroid, granulocyte, and macrophage colonies in soft agar. It serves as a growth factor stimulating progenitor to form erythroid, megakaryocyte, neutrophil, eosinophil, basophil, mast cell, and monocytic lineages. IL-3 is capable of activating

monocytes and may have an immunomodulatory role.

CSF in Respiratory Diseases Each of the CSFs discussed above have immunomodulatory properties on mature myeloid cells in mice and humans. Targeted gene ablation studies in mice have been particularly helpful in delineating the roles of CSFs in the lung. CSF in Murine Lung Diseases

Naturally occurring osteopetrotic (Op/Op) mice are deficient in M-CSF due to a null-mutation in the M-CSF gene. At birth, there are reduced numbers of macrophage lineage cells including pulmonary alveolar macrophages. Alveolar macrophage numbers correct with age in association with increased pulmonary IL-3 levels, alveolar macrophage metalloprotease release, and development of emphysema. G-CSF knockout mice have neutropenia and do not develop neutrophilia during infection. They have

COLONY STIMULATING FACTORS 545

increased incidence of spontaneous lung infections, which contribute significantly to increased mortality. Recently, a homeostatic mechanism regulating blood neutrophil counts has been identified involving IL-23, IL-17, and G-CSF. GM-CSF knockout mice do not have gross hematological abnormalities other than reduced eosinophil counts, but develop a lung phenotype characterized by the accumulation of surfactant lipids and surfactant proteins. The lung abnormality is due to decreased surfactant catabolism in alveolar macrophages. GM-CSF, via PU.1, regulates surfactant catabolism and a number of innate immune functions in macrophages. GM-CSF also regulates innate immune functions in mature neutrophils. GMCSF receptor b chain (AIC2B) knockout mice have a pulmonary phenotype similar to GM-CSF knockout mice. IL-3 knockout mice have impaired delayed-type hypersensitivity. In contrast, gene targeting of either AIC2B or A1C2A did not result in hematologic abnormalities (other than decreased numbers of eosinophils in AIC2B-deficient mice), apparently due to the presence of redundant IL-3 receptors in mice. CSF in Human Lung Diseases

All of the CSFs are capable of modulating functions of myeloid lineage cells in humans and pulmonary CSF levels are increased during lung inflammation. For example, GM-CSF and IL-3 levels are elevated in asthma; GM-CSF is elevated in cystic fibrosis; and GM-CSF and MCSF are elevated in smokers. CSF levels may participate in the pathogenesis of these and other lung disorders by modulating macrophage and neutrophil functions and the inflammatory response. Genetic disorders of CSF deficiency corresponding to each of the CSF knockout models discussed above have not been identified in humans. However, idiopathic pulmonary alveolar proteinosis is specifically associated with high levels of neutralizing, anti-GMCSF autoantibodies, which completely eliminate GM-CSF bioactivity and result in a ‘functional deficiency’ of GM-CSF. Clinically, this disease is characterized by surfactant accumulation in the lungs and an increased rate of secondary infections, similar to that in GM-CSF-deficient mice. Together, data in mice and humans strongly suggest that GM-CSF, via PU.1, regulates pulmonary surfactant homeostasis and innate immunity in the lung by modulating alveolar macrophage function. See also: CD14. Croup. Hypoxia and Hypoxemia. Inhibition of Differentiation (ID) Proteins. Interleukins: IL-12. Interstitial Lung Disease: Alveolar

Proteinosis. Leukocytes: Neutrophils; Pulmonary Macrophages. Stem Cells. Surfactant: Overview; Surfactant Protein A (SP-A); Surfactant Proteins B and C (SP-B and SP-C); Surfactant Protein D (SP-D). TollLike Receptors. Transcription Factors: PU.1. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Dranoff G, Crawford AD, Sadelain M, et al. (1994) Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 264(5159): 713–716. Fitzgerald KA, O’Neill LAJ, Gearing AJH, and Callard RE (2001) The Cytokine Facts Book, 2nd edn. San Diego: Academic Press. Gaviria JM, Garrido SM, and Root RK (2001) Clinical use of granulocyte colony-stimulating factor in infectious diseases. Current Clinical Topics in Infectious Diseases 21: 302–322. Hubel K, Dale DC, and Liles WC (2002) Therapeutic use of cytokines to modulate phagocyte function for the treatment of infectious diseases: current status of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and interferon-gamma. Journal of Infectious Diseases 185: 1490–1501. Kitamura T, Tanaka N, Watanabe J, et al. (1999) Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colonystimulating factor. Journal of Experimental Medicine 190(6): 875–880. LeVine AM, Reed JA, Kurak KE, Cianciolo E, and Whitsett JA (1999) GM-CSF-deficient mice are susceptible to pulmonary group B streptococcal infection. Journal of Clinical Investigation 103(4): 563–569. Lieschke GJ and Burgess AW (1992) Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (1). New England Journal of Medicine 327: 28–35. Lieschke GJ and Burgess AW (1992) Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (2). New England Journal of Medicine 327: 99–106. Nishinakamura R, Wiler R, Dirksen U, et al. (1996) The pulmonary alveolar proteinosis in granulocyte macrophage colonystimulating factor/interleukins 3/5 beta c receptor-deficient mice is reversed by bone marrow transplantation. Journal of Experimental Medicine 183(6): 2657–2662. Roilides E and Farmaki E (2001) Granulocyte colony-stimulating factor and other cytokines in antifungal therapy. Clinical Microbiology and Infection 7(supplement 2): 62–67. Roilides E, Lyman CA, Mertins SD, et al. (1996) Ex vivo effects of macrophage colony-stimulating factor on human monocyte activity against fungal and bacterial pathogens. Cytokine 8: 42–48. Rosen SG, Castleman B, and Liebow AA (1958) Pulmonary alveolar proteinosis. New England Journal of Medicine 258: 1123–1142. Seymour JF, Presneill JJ, Schoch OD, et al. (2001) Therapeutic efficacy of granulocyte-macrophage colony-stimulating factor in patients with idiopathic acquired alveolar proteinosis. American Journal of Respiratory and Critical Care Medicine 163(2): 524– 531. Shibata Y, Berclaz P-Y, Chroneos Z, et al. (2001) GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU. 1. Immunity 15: 557–567. Shibata Y, Otake K, Zsengeller Z, Plalaniyar N, and Trapnell BC (2001) Alveolar macrophage deficiency in M-CSF-deficient,

546 COMPLEMENT osteopetrotic mice spontaneously corrects with age and is associated with matrix metalloproteinase expression and emphysema. Blood 98: 2845–2852. Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349(26): 2527–2539.

Relevant Websites http://www.ncbi.nlm.nih.gov – National Center for Biotechnology Information: Molecular Modeling DataBase. http://rarediseasesnetwork.epi.usf.edu – Rare Lung Diseases Consortium.

COMPLEMENT N Rawal, University of Texas, Tyler, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Complement is an important part of the host’s immune system that fights infections. The complement system consists of about 35 different proteins; most of these are soluble plasma proteins, but some are membrane bound. The complement system recognizes, opsonizes, kills, and clears bacteria, parasites, and other pathogens. It also aids in the clearance of immune complexes and altered host cells, for example, apoptotic or necrotic cells. Bacteria or immune complexes activate complement to initiate a cascade in which one complement component activates another. Products generated during the activation process elicit cellular responses such as phagocytosis and inflammation that involve innate and adaptive immune systems. Complement activation ultimately results in assembly of the cytolytic membrane attack complex, which then lyses targeted bacteria. Activation of complement is under the strict control of complement regulatory proteins, which include about one third of the total complement proteins. Excessive or inappropriate activation of complement contributes to the pathogenesis of several immuno-inflammatory diseases and conditions, including glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, hemolyticuremic syndrome, multiple sclerosis, tissue graft rejection, and reperfusion injury. Complement activation products are implicated in lung inflammation and injury from various causes, and respiratory diseases, such as pneumonia, acute respiratory distress syndrome, pulmonary fibrosis, smoke-induced damage and asthma, may involve complement activation.

Introduction Complement was first described in the 1890s as a heat-labile bactericidal activity in the bloodstream that ‘complemented’ heat-stable antibodies in the killing of microorganisms. Bordet was awarded the Nobel Prize in 1919 for discovering this factor and defining its actions. He showed that the bactericidal factor present in normal serum had no activity by itself, but that it became lytic in the presence of the heat-stable antibody (i.e., antibody-mediated immunity). Since then, three distinct pathways of complement activation have been recognized (Figure 1). Although the antibody-dependent classical pathway

of complement activation was the first to be discovered, components involved in its activation were purified and characterized only in the 1960s and 1970s from work done in the laboratories of Nelson and Mu¨eller-Eberhard. In the 1950s, Pillemer suggested an alternative pathway to the classical pathway. This second pathway of complement activation did not require an antibody, and its existence was not accepted until later in the 1970s. More recently (1996), Matsushita and Fujita discovered a third pathway of complement activation. The lectin pathway involves recognition of sugar residues present on the cell surface of microbes by mannan-binding lectin (MBL) and ficolins.

Structure Complement proteins can be divided into two general groups based on their structure. Components C3, C4, C5, C1 inhibitor, C3a receptor (C3aR), and C5a receptor (C5aR) form one group, whose primary structure resembles members of other protein families almost across their entire length. The second group includes most of the remaining complement components. These are modular proteins with just short sequences of amino acids in common with other proteins. C3, C4, and C5 belong to the same family as a2macroglobulin. Except for C5, these proteins have an internal thioester bond, which becomes exposed when activated by proteolysis. Then the metastable thioester can be hydrolyzed, or it can react with hydroxyl or amino groups on cells or proteins to form ester (Figure 2) or amide bonds. C1 inhibitor belongs to the serpin superfamily of serine protease inhibitors, while C3aR and C5aR belong to the family of 7 membrane domain G-protein-coupled receptors. C1r, C1s, factor D, factor B, mannan-binding lectin-associated serine proteases (MASPs), C2, and factor I belong to the second group. These are enzymes of the complement system that generate biologically active complement fragments during

COMPLEMENT 547

Classical pathway (IgM- or IgG-containing immune complexes)

Lectin pathway (carbohydrates)

Alternative pathway (bacteria, viruses, yeast)

MBL/ficolins

C1

C3(H2O) B

MASPs C4

D

C4 C2

C2

P

C3

Opsonization

C3 convertase C3a C5

Inflammation

C5 convertase C5a C5b C6 C7

Terminal pathway

C8 C9 Membrane attack complex

Lysis

Figure 1 The complement system. Activation of complement occurs via three pathways: the classical pathway, the lectin pathway, and the alternative pathway. The established nomenclature and symbols for complement proteins have been used. C1, C4, C2, C3, C5, C6, C7, C8, C9 are complement components that directly participate in the activation process. C3(H2O), an inactive form of C3; MBL, mannan-binding lectin; MASPs, mannan-binding lectin-associated serine proteases; B, complement factor B; D, complement factor D; P, complement protein, properdin; C3a, one of the cleavage products of C3; C5b and C5a, the cleavage products of C5; C3 convertase and C5 convertase, serine proteases that are transiently generated up on the activation of complement and cleave C3 and C5, respectively.

C3a or C4a C3 or C4 O C

C3b or C4b

C3 convertase

SH O

C+

OH

Cell surface

H O H

C3b or C4b S−

O

C

SH

O

have similar structures, and they interact with C5b and each other in a sequential manner to form the cytolytic membrane attack complex that lyses bacteria. Complement receptors, CR1 and CR2, and complement regulatory proteins, C4b-binding protein (C4bp), factor H, decay accelerating factor (DAF), and membrane cofactor protein (MCP) also belong to the second group. These proteins are composed of repeats of complement control protein (CCP) modules.

Surface-bound C3b or C4b

Figure 2 Activation and deposition of C3 and C4. C3 and C4 have an internal thioester bond that is exposed when they are cleaved. The reactive thioester bond reacts with hydroxyl groups (shown in the figure) or amino groups found on a wide range of proteins and cell membranes of the target surface via an ester or an amide bond.

complement activation. All have a catalytic domain that is structurally similar to that found in serine proteases of the trypsin subfamily. C2 and factor B are structurally very similar. C6, C7, C8, and C9

Functions of Complement Complement proteins circulate in blood as inactive molecules. When activated, complement components are cleaved into two products that have biological activity (Figure 3). The larger fragment called ‘b’ usually remains attached to the surface of the microorganism while the smaller fragment called ‘a’ may diffuse away. Activation of the complement via any of the three pathways has two phases: opsonization and formation of membrane attack complex

548 COMPLEMENT

(Figure 1). In the first phase, when an activator is recognized, the complement system is activated, and a series of protein–protein interactions and proteolytic cleavages culminate in the assembly of transient complexes with protease activity. These complexes are called C3 convertases. Although the three pathways are activated by different mechanisms, formation of C3 convertase results in cleavage of the same peptide bond in C3. Cleavage of C3 into C3b and C3a is central to the process of complement activation. The larger fragment, C3b, covalently attaches to (Figure 2) and coats (opsonizes) the cell surface so that phagocytic cells recognize the particle, while the smaller C3a fragment promotes an inflammatory response by attracting eosinophils and phagocytes to the site. The second part of complement activation is the effector phase. C3b attached to a foreign surface is cleaved again forming smaller fragments iC3b and C3d. These fragments are ligands that present the particle to the adaptive immune system through the CD21/CD19 receptor system; this antigen presentation then prompts production of antibodies by the CD58 B cell population. In this phase of complement activation, cleavage of C5 by C5 convertase initiates the formation of a membrane attack complex (Figure 1). This last enzymatic step in the complement cascade constitutes the terminal activation common to all three pathways. The products of C5 cleavage, C5b and C5a, play an important role in killing microorganisms (Figure 3). The larger fragment, C5b, binds with other complement proteins C6, C7, C8, and C9 to form the membrane attack complex and lyses cells to which the complex is

Activation products C3b, C4b, iC3b C3b

Complement activation

C3a, C5a

attached. The smaller fragment, C5a, is one of the most potent proinflammatory mediators known. It has many diverse activities, including the release of mediators from mast cells, chemoattraction of neutrophils and eosinophils, upregulation of b2 integrins, and it causes L-selectin shedding from eosinophils along with release of granules and an oxidative burst.

Alternative Pathway of Complement The alternative pathway is one of the host’s innate immune responses to infection. Six complement proteins, C3, properdin (P), and factors B, D, H and I are involved in activation, amplification, and regulation of this pathway. The alternative pathway can operate in the absence of antibodies. It is activated in response to the activating surfaces of certain bacteria, viruses, fungi, and yeast within few minutes after they come in contact with plasma. It is also spontaneously activated in the fluid phase at a very low rate. Initiation of complement activation via the alternative pathway involves spontaneous activation of C3 in the fluid phase. C3 has a thioester bond that reacts with any exposed hydroxyl or amino group. The slow hydrolysis of the thioester converts inactive C3 to C3(H2O) (Figure 4). C3(H2O) is like a functionally active C3b-like molecule; it binds factor B in a Mg2 þ dependent fashion to form the complex C3(H2O),B. Factor B in complex with C3(H2O) is then cleaved by factor D to form a C3 convertase, C3(H2O),Bb. The C3 convertase cleaves C3 to generate C3b and C3a. C3b generated by C3(H2O),Bb has an active thioester through which it can bind to

Activity Opsonization of bacteria and immune complexes

Function Phagocytosis

Solubilization and clearance of circulating immune complexes Activation of lymphocytes and phagocytes

Lymphocytosis and phagocytosis

Increased vascular permeability smooth muscle contraction Activation and degranulation of mast cells and basophils

Inflammation and degranulation

C5a

Activation and chemotaxis of neutrophils

Chemotaxis

C5b-9 complex

Lysis of bacteria and foreign cells

Lysis

Figure 3 Functions of complement. Products generated during complement activation possess biological functions, which elicit cellular responses of the innate and adaptive immune systems to help fight infections. C3b and C3a, cleavage products of C3; C5b and C5a, the cleavage products of C5; C4b, one of the cleavage products of C4; iC3b, inactivated C3b; C5b-9 complex, the membrane attack complex.

COMPLEMENT 549 C3

Initiation

C3 convertase (fluid phase)

Metastable C3b

C3

Deposition of C3b Surface-bound C3b Amplification Recognition and regulation

Nonactivator

Activator

C3 convertase

iC3b

C5 convertase Lysis Membrane attack complex Figure 4 Activation of the alternative pathway of complement. Activation of the alternative pathway of complement involves initiation, deposition of C3b, recognition of an activator, amplification, regulation, and lysis. C3, the third component of complement that directly participates in the activation process; C3b, the proteolytically activated form of C3; iC3b, inactivated C3b; C3 convertase and C5 convertase, serine proteases that are transiently generated up on the activation of complement and cleave C3 and C5, respectively. The dashed lines represent the amplification loop.

a surface. Deposition of C3b is continuous and occurs on host cells (self) as well as foreign particles (nonself). The regulatory proteins, factors H and I, control discrimination between self and nonself surfaces. They readily inactivate C3b that deposits on host cells, while they are much less effective on C3b deposited on an activating surface such as bacteria, fungi, and viruses (Figure 4). Other complement regulatory proteins, such as DAF and MCP, are found on host cell surfaces where they can inactivate the C3 convertase, thereby preventing autologous complement activation on host tissue. A special feature of the alternative pathway is the amplification of the number of C3b molecules deposited on the surface (Figure 4). This occurs by C3b-dependent positive feedback. C3b is the product of C3 cleavage catalyzed by C3 convertase. It forms the noncatalytic subunit of the C3 convertase (C3b,Bb). Thus, every C3b deposited can form a new C3 convertase with factors B and D in the presence of Mg2 þ . This mechanism produces more C3b and consequently more enzymes. Factors H and I control the amplification process. Factor H enhances the dissociation of C3b,Bb, while C3b in complex with factor H is inactivated to iC3b by factor I. Properdin is a positive regulator that enhances amplification of surface-bound C3b by binding to C3b,Bb and stabilizing it. The C3 convertase, C3b,Bb, also cleaves C5, but does so inefficiently. However, as C3b density

increases due to amplification of the number of C3b molecules deposited by C3b,Bb, C3b complexes form. These C3b complexes form C3 convertases that exhibit higher affinities for C5 and cleave C5 at a velocity approaching Vmax thereby switching the enzyme from C3 to C5 cleavage.

Classical Pathway of Complement Seven proteins, C1, C2, C4, C3, C4b-binding protein, C1 inhibitor and factor I, are responsible for recognition, activation, and regulation of the classical pathway of the complement. The pathway is primarily activated by the interaction of C1 with immune complexes or aggregates containing IgG or IgM. The C1 complex consists of three components: C1q, proenzyme C1r, and proenzyme C1s in the ratio 1:2:2. C1q is the recognition unit of the C1 complex and binds to the Fc region of the antibody. The C1q molecule consists of six globular heads, each connected by a central fibril region. When two or more heads of C1q interact with an immune complex or an activator, a conformational change occurs that results in the autoactivation of proenzyme C1r, thereby converting it to the active protease C1r. Activated C1r in turn cleaves proenzyme C1s converting it to the active protease C1s. C1 inhibitor is the only plasma inhibitor that inactivates C1r and C1s enzymes. The mechanism of inactivation involves C1 inhibitor binding to active sites on activated C1s and C1r.

550 COMPLEMENT

Activated C1s cleaves C4 into two products, C4a and C4b. The smaller fragment, C4a, is released into the fluid phase, while the larger fragment, C4b, may attach to the surface of an activator. Freshly activated C4b has a reactive acyl group through which it binds covalently to hydroxyl (Figure 2) or amino groups close to the C1 molecule. C2 forms an Mg-dependent complex with C4b. If binding of C2 to C4b occurs close to activated C1s, then C2 is cleaved by C1s to C2b and C2a. The larger fragment C2a remains attached to C4b to form a bimolecular complex (C4b,C2a), which is the C3 convertase of the classical pathway of the complement. The C3 convertase, C4b,C2a, essentially cleaves C3 to C3b and C3a. Attachment of C3b results in opsonization of the target surface for phagocytosis. C3b can also attach to C4b, the noncatalytic unit of the C3 convertase enzyme, to form the complex, C3bC4b,C2a. This complex is also a C3 convertase, but because it has a much higher affinity for C5 than for C3, the enzyme primarily cleaves C5 and is referred to as a C3/C5 convertase. C3/C5 convertases of the classical pathway are inactivated in three ways. First, the soluble plasma complement regulatory protein, C4b-binding protein, binds to C4b and prevents assembly of C3/C5 convertase. Second, C4b-binding protein and DAF dissociate the catalytic subunit, C2a, from bound C4b causing the enzyme to lose activity. Third, C4b is inactivated to iC4b by the proteolytic activity of factor I, for which C4b-binding protein and the red cell complement receptor, CR1, act as cofactors.

Lectin Pathway of Complement The lectin pathway of the complement is activated when MBL binds to hexoses with carbon 3 and 4 OH groups such as N-acetyl-D-glucosamine, glucose, fucose, and mannose. These sugars are expressed as repetitive O-polysaccharide structures on surfaces of bacteria, yeast, parasites, mycobacteria, and certain viruses. Because MBL does not recognize the OH configuration present in galactose and sialic acid, the sugars commonly found on mammalian glycoproteins, the body’s innate immune system can readily distinguish nonself structures from self. The lectin pathway plays a major protective role during the vulnerability window experienced by infants between decay of maternal antibody and establishment of an effective adaptive immune system. Activation of the complement via the lectin pathway occurs when the proenzyme forms of MASPs are activated. MASPs resemble the proteolytic enzymes, C1r and C1s, of the classical pathway. A complex of MBL, MASP-1, and MASP-2

(MBL–MASP1–MASP2) appears to be analogous in overall organization and function to the C1 complex (C1q–C1r2–C1s2) of the classical pathway. The proenzyme forms of MASPs are activated when MBL binds to the cell-surface carbohydrates of microbes. Activated MASP-1 and MASP-2 are regulated by C1 inhibitor in a manner similar to that observed for C1s and C1r enzymes. Activated MASP-2 cleaves C4 and C2 to form the classical pathway C3/C5 convertases (C4b,C2a), which then generate the membrane attack complex.

Complement Receptors There are four complement receptors for C3 fragments: CR1, CR2, CR3, and CR4. CR1 (CD35) is found on erythrocytes, lymphocytes, neutrophils, dendritic cells, and others. The main functions of CR1 are phagocytosis and clearance of immune complexes. Erythrocytes bearing CR1 bind to circulating immune complexes or particles that have complement ligands C3b, iC3b, and C4b deposited on them. The erythrocytes then transport the complexes to liver and splenic macrophages for destruction. CR1 also regulates complement activation. It acts as a cofactor for factor I to cleave surface-bound iC3b or iC4b to smaller fragments: surface-bound components C3dg or C4d and soluble components C3c or C4c. Immune complexes with fixed iC3b attach to neutrophils via CR1 and CR3. Subsequent release of cytotoxic enzymes, oxygen metabolites, and leukotrines by the activated neutrophils kills the bacteria. However, in autoimmune diseases (e.g., systemic lupus erythematosus) where immune complexes with fixed iC3b are trapped in normal tissue, the same mechanism turns against the host to cause neutrophil-mediated tissue damage. CR2 (CD21) is a receptor for C3 cleavage fragments C3b, iC3b, C3d, and C3dg. CR2 enhances recognition of antigens by B cell activation and proliferation. CR3 (CD11b/CD18) is expressed mainly on mononuclear phagocytes and natural killer cells; it helps to clear opsonized bacteria. This complex is a receptor for iC3b and is used by macrophages to adhere to an extracellular matrix. CR3, along with CR4, mediates adhesion of neutrophils to endothelial cells at inflammation sites via ICAM-1. CR4 (CD11c/CD18) is primarily expressed on myeloid cells, dendritic cells, natural killer cells, platelets, B lymphocytes, and alveolar macrophages. Binding of CR4 to iC3b initiates ingestion of particles that are deposited on the membrane via CR3. The C5a (CD88) and C3a receptors (C3aR) are G-protein-coupled receptors. Both C3a and C5a

COMPLEMENT 551

receptors are found on circulating white cells such as neutrophils, monocytes, basophils, and eosinophils. In addition, they are also found on endothelial cells, lung epithelial cells, hepatocytes, astrocytes, and microglial cells. Binding of C3a or C5a to their respective receptors invokes functions such as increased vascular permeability, edema, and degranulation of mast cells. C1qRp, C1qRo2, cC1qR, and CR1 are thought to be C1q receptors. Binding of C1q to these receptors on various cells initiates cellular responses such as phagocytosis, chemotaxis, clearance of apoptotic cells, and increase in superoxide metabolism by neutrophils.

damage. Complement activation also contributes to transfusion-related acute lung injury, which has features similar to early ARDS. The development of transfusion-related acute lung injury in most cases is attributed to the presence of antigranulocyte antibodies and other substances in blood-derived products, which activate granulocytes and the complement. Complement activation may also cause vascular endothelium damage during reperfusion of ischemic tissues. Specific inhibition of C5a or inhibition of the complement cascade by general depletion of the complement confers protection in animal models with acute lung injury.

Immune Complex Disease Complement in Respiratory Diseases Various cells of the lung, including alveolar macrophages, lung fibroblasts, and type II alveolar cells, produce most of the complement components. Thus, although the complement system in the lung is well poised for killing and clearance of pulmonary infections, unwanted activation of the complement system can lead to lung injury. Immune injury to the lung is classified according to type of hypersensitivity (I-IV), depending upon the type of immunological reaction responsible for injury. Complement is involved in types II and III hypersensitivity reactions, which include certain drug allergies and immune complex diseases, but the complement and its products may also contribute to anaphylaxis and asthma, which are type I hypersensitivities.

Acute Respiratory Distress Syndrome Acute respiratory distress syndrome (ARDS) is a pulmonary disorder resulting from a variety of severe injuries. The injury may be directed at the lungs, as with pulmonary infection, aspiration of gastric contents, severe thoracic trauma, toxic gas (smoke) inhalation, or it may be indirect as in the acute systemic inflammatory response to severe sepsis, acute pancreatitis, drug overdose, reperfusion injury, and others. Although the pathogenesis of ARDS is not yet fully defined, it is clear that activation of the complement cascade and proinflammatory cells injures the lung. The inadvertent activation of the complement system results in C5a generation, which increases capillary permeability. Clinical studies correlate the extent of complement activation (indicated by increased plasma levels of activation products, C3a and C5a) with the development of ARDS in patients. C5a attracts neutrophils and macrophages to the lungs where they release oxygen free radicals to cause inflammation and alveolar epithelial

Immune complexes form in the body when an antibody recognizes a specific antigen. The complement system prevents the immune complex from precipitating in the serum by making it more soluble and then removing it through mechanisms involving IgG, C3b, iC3b, and complement receptors CR1 and CR3. Various insults may lead to immune complex diseases. For example, immune complexes can form the following: persistent infection (e.g., streptococcus infection); production of antibody to self-antigen (e.g., systemic lupus erythematosus); or inhalation of antigenic materials from molds, plants, or animals (e.g., Farmer’s lung and Pigeon fancier’s lung). The continued production of antibodies leads to extended formation of immune complexes that can overwhelm the complement system. Because the soluble immune complexes are poorly cleared from circulation, they often become embedded in various tissues where they trigger inflammatory reactions. Complement activation in the lung induced by immune complexes leads to neutrophil accumulation and lung injury. The role of the complement was defined in experiments with mice where treatment with a C5a blocking antibody attenuated the immune complex-induced permeability and neutrophil infiltration into the lung. In addition, mice lacking the C5aR were completely protected from immune complex alveolitis, and treatment with a C5aR antagonist reduced edema formation in normal mice. These studies suggest C5a and C5aR are important mediators of lung injury in immune complex disease.

Asthma The role of the complement in asthma is controversial, but recent studies of allergen-induced asthma in C3- or C3aR-deficient mice showed that lack of these complement components significantly reduced airway hyperresponsiveness, lung eosinophilia, and

552 COMPLEMENT

IL-4-producing cells. In addition, attenuation of Agspecific IgE and IgG1 responses was also observed. These findings suggest that the complement may modulate susceptibility to asthma. Interaction of C3a with epithelial cells can regulate lung inflammatory responses such as synthesis of mucus, while increased production of C3a in asthmatic patients during exercise implies a role for complement in exercise-induced asthma. In addition, complement depletion by cobra venom factor, a protein found in snake venom that consumes C3 and C5, blunted the asthmatic response in a guinea pig model of occupational asthma, indicating a role for complement in cellular infiltration and lung injury.

Anaphylaxis Complement activation does not occur in systemic anaphylaxis because IgE immune complexes do not activate complement. In this immediate hypersensitivity reaction, the antigen binds to an IgE antibody on mast cells or basophils, causing release of mediators that produce life-threatening symptoms. However, studies with mice that lack IgE suggest that generation of C3a and C5a via complement activation contributes to the bronchoconstriction and hypotension of anaphylaxis. Indeed, additional animal studies implicate C3a and C5a as putative mediators of anaphylactic shock. Both C3a and C5a peptides contract airway smooth muscle in the guinea pig independent of histamine, and treatment with soluble CR1 (sCR1), which inhibits complement activation, eliminates the antigen-induced hypotensive response in these animals.

Pleural Disease Pleura effusions accompany lung injury resulting from various insults, including immune mechanisms that occur in diseases such as rheumatoid arthritis and lupus erythematosus. Circulating immune complexes localize in the pleural capillaries and activate the complement, thereby causing endothelial injury. Also, immune complexes and complement can be generated locally, further adding to the injury. Measurement of the complement in posttraumatic pleural effusions shows that levels of C3 and C4 are decreased relative to normal serum, suggesting local consumption of the complement via the classical pathway. The fact that levels of SC5b-9 are higher in pleural effusions from patients with tuberculosis than in those with malignant effusions suggests that measuring SC5b-9 levels might differentiate the mechanism and origin of the effusion.

Therapeutic Considerations There is no single inhibitor of all three pathways of complement activation. Since activation products C3a and C5a mediate inflammatory reactions leading to tissue damage, attempts at inhibition focused on blocking complement activation at the C3/C5 level. Studies with mice either deficient in C3/C5 or transgenically lacking C3aR/C5aR indicate that selective control of complement activation products reduces lung injury, yet maintains opsonization and lysis of bacteria. Small antagonist molecules that inhibit C5a generation or its binding to C5aR can reduce proinflammatory activities of C5a in rats, while recombinant sCR1 not only reduces inflammation and tissue injury by injected immune complexes but also protects against lung injury in different models of complement-dependent acute inflammation. These animal studies, together with data from ongoing human clinical trials of small molecular antagonists for C3a and C5a receptors, will facilitate development of clinically useful inhibitors to control complement-mediated inflammation. See also: Acute Respiratory Distress Syndrome. Allergy: Overview; Allergic Reactions; Allergic Rhinitis. Asthma: Overview; Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; ExerciseInduced; Extrinsic/Intrinsic. G-Protein-Coupled Receptors. Pleural Effusions: Overview.

Further Reading Czermak BJ, Lentsch AB, Bless NM, et al. (1998) Role of complement in in vitro and in vivo lung inflammatory reactions. Journal of Leukocyte Biology 64: 40–48. Davies KA and Walport MJ (1998) Processing and clearance of immune complexes by complement and the role of complement in immune complex diseases. In: Volanakis JE and Frank MM (eds.) The Human Complement System in Health and Disease, pp. 423–454. New York: Dekker. Guo RF and Ward PA (2002) Mediators and regulation of neutrophil accumulation in inflammatory responses in lung: Insights from the IgG immune complex model. Development of C5a receptor antagonists. Free Radical Biology and Medicine 33: 303–310. Joseph J and Sahn S (1993) Connective tissue diseases and the pleura. Chest 104: 262–270. Ko¨hl J (2001) Anaphylatoxins and infectious and non-infectious inflammatory diseases. Molecular Immunology 38: 175–187. Prince JE, Kheradmand F, and Corry DB (2003) Immunologic lung disease. Journal of Allergy and Clinical Immunology 111: S613– S623. Regal JF (1997) Role of the complement system in pulmonary disorders. Anaphylatoxins and infectious and non-infectious inflammatory diseases. Immunopharmacology 38: 17–25. Tsokos GC and Tolnay M (2004) Complement and autoimmunity. In: Szebeni J (ed.) The Complement System: Novel Roles in Health and Disease, pp. 307–314. New York: Kluwer Academic Publishers.

CONNECTIVE TISSUE GROWTH FACTOR 553

CONNECTIVE TISSUE GROWTH FACTOR J A Lasky, Tulane University Health Science Center, New Orleans, LA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Connective tissue growth factor (CTGF) is a peptide growth factor that is upregulated during fibrogenesis. CTGF was originally found to promote fibroblast proliferation, migration, adhesion, and extracellular matrix formation, and subsequently has been implicated in fibrogenesis, wound healing, chondrogenesis, angiogenesis, transdifferentiation, and tumor growth. CTGF has numerous cellular profibrotic activities, which include increasing synthesis of fibrillar collagen and fibronectin, enhancing secretion of factors that impede matrix degradation, and stimulating the transformation of fibroblasts into the more fibrogenic myofibroblast phenotype. Increased CTGF expression is observed during lung fibrogenesis in animal models, as well as in diseases that cause lung fibrosis in humans. Overexpression of CTGF has been reported to cause lung fibrosis in rats, as well as to render lung fibrosis-resistant mouse strains more susceptible to fibrogenic agents. These observations have raised interest in developing CTGF inhibitors for employment in trials to arrest lung fibrogenesis in humans.

Introduction Connective tissue growth factor (CTGF), also termed fisp12, bIG-M2, and IGF-BP8, is a secreted cysteinerich growth factor that belongs to the CCN (cyr61, CTGF, nov) family. It was first isolated in 1991 from serum-stimulated NIH3T3 cells and named fisp12 (fibroblast-inducible secreted protein-12). The term ‘connective tissue growth factor’ was introduced the same year by others to describe a cysteine-rich mitogen secreted by human vascular endothelial cells.

Since then the CTGF cDNAs for pig, rat, cow, and frog have been sequenced. CTGF is a highly conserved protein. Compared to human CTGF, there is 94% homology displayed in murine CTGF, 96% homology in rat CTGF, and 92% homology in pig CTGF. CTGF was originally found to promote fibroblast proliferation, migration, adhesion, and extracellular matrix formation as its name suggested. It has also been reported that CTGF is transcriptionally activated by transforming growth factor beta (TGF-b) and is an important downstream mediator of TGFb’s profibrotic activities. In light of these findings, CTGF is proposed as a profibrotic factor that plays an important role in the pathways leading to fibrosis. However, a host of evidence has shown that the physiological functions of CTGF are far more than what its name suggests. Other than fibroblasts, expression of CTGF has been reported in diverse cell types such as epithelial cells, neuronal cells, endothelial cells, smooth muscle cells, and chondrocytes. In contrast, under certain circumstances CTGF has been shown to act as an antimitogenic factor or apoptosis inducer instead of as a mitogen. CTGF has thus been implicated in fibrogenesis, wound healing, chondrogenesis, angiogenesis, transdifferentiation, and tumor growth.

Gene and Protein Structure Both human and murine CTGF genes span about 3 kb of DNA and are composed of five exons and four introns (Figure 1). The hCTGF gene resides on

CTGF gene structure

lntron 1 Exon 1

Intron 2 Exon 2

Intron 3 Exon 3

Intron 4 Exon 5

Exon 4

3′

5′

1 N′

21 SP

27

97 I IGFBP

101

167

199

243

330

256

II

III

IV

VWC

TSP1

CT

3498 C′

CTGF protein structure Figure 1 Gene and protein structure of CTGF. The CTGF gene is composed of five exons and four introns. The dark blue boxes in the gene structure map represent open reading frames. CTGF protein consists of a signal peptide (SP) at the N-terminal and four modules, shown as light blue boxes. The numbers shown on top of each region are the amino acid numbers at the beginning or end of the region. IGFBP, I-insulin growth factor binding protein; VWC, von Willebrand factor type C repeat; TSP1, thrombospondin type 1; CT, C-terminal module.

554 CONNECTIVE TISSUE GROWTH FACTOR

of CTGF has been shown recently to bind to lowdensity lipoprotein receptor-related protein (LRP). Several lines of evidence suggest that module 4, which harbors the cystine knot of CTGF, might be the functional domain for the majority of CTGF’s activities. For example, module 4 accounts for the heparin-binding capacity of CTGF. More compelling evidence arises from studies on some smaller CTGF variants existing in various cell types and body fluids. These 10–20 kDa proteolytic fragments are composed of either module III and IV or module IV only. Similar to the full-length CTGF, these CTGF fragments are able to bind to heparin as well as to stimulate mitosis and cell adhesion.

chromosome 6q23.1 and the mCTGF gene maps to the (10A3–10B1) region of the murine genome. The 50 untranslated region of the CTGF gene contains a variety of transcriptional regulatory elements such as AP-1, TATA, M-CAT, and SP-1 as well as a unique TGF-b response element (TbRE), which accounts for induction of CTGF expression by TGF-b. hCTGF also contains a cis-acting repressive element in the 30 untranslated region. A single 2.4 kb mRNA transcript of CTGF has been reported in most studies and encodes for a 349 amino acid hCTGF peptide (348 amino acids for mCTGF). Two larger CTGF transcripts (3.5 and 7 kb) are found in glioblastoma cells, and the functional significance of these elongated transcripts is presently unclear. CTGF is a 36–38 kDa peptide that is highly conserved among species. The most frequent variations occur in the N-terminal signal peptide region. The secreted form of the protein consists of 323 amino acids containing 38 conserved cysteine residues that form disulfide bridges within the peptide. As a member of the CCN family, CTGF protein has four modules that are shared with all other CCN family members. The four domains are: an I-insulin growth factor binding module (module I, IGFBP), a von Willebrand factor type C repeat (module II, VWC), a thrombospondin type 1 module (module III, TSP1), and a C-terminal module (module IV, CT), which contains a cystine knot (Figure 1). Interestingly, each module is encoded by one exon and is separated by an intron; however, the exact biological function of these four modules remains unknown. Module 3

Fibroblasts Epithelial cells Endothelial cells Airway smooth muscle cells Tumor cells

CTGF Receptors There is very limited information regarding the CTGF receptor/s (Figure 2). CTGF binding was evaluated in a human chondrocytic cell line using radiolabeled CTGF. Scatchard analysis demonstrated two classes of CTGF binding sites, but only one band at approximately 280 kDa was detected using a crosslinking study. As of yet, the bona fide CTGF receptor/s has not been isolated, and the receptor gene has not been cloned or sequenced. A study using affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis has revealed CTGF binding to a membrane protein of a mass of approximately 620 kDa which is characterized as the multiligand receptor LRP. Consistent with this observation, CTGF induces tryrosine phosphorylation of the cytoplasmic

CTGF

Integrin
v3

LRP

FAK(?)

Receptor (?)

(?)

ERK1/2

(?) CRMP-1 (inhibit metastasis)

Target gene

Gene

↑
5 Integrin (cell adhesion) Extracellular matrix

↓ Bcl-2 (apoptosis)

↑
1-Type I collagen ↑ Fibronectin (fibrosis and airway remodeling)

Figure 2 CTGF receptor binding, signal transduction, and biological activities. CRMP-1, collapsin response mediator protein 1; ERK, extracellular signal regulated kinase; FAK, focal adhesion kinase; LRP, low-density lipoprotein receptor-associated protein.

CONNECTIVE TISSUE GROWTH FACTOR 555

domain of LRP in fibroblasts, and the LRP antagonist, receptor-associated protein (RAP) inhibited CTGF-induced tryrosine phosphorylation of LRP. The biological significance of CTGF binding to LRP is highlighted by the fact that inhibition of LRP signaling reduced CTGF- mediated synergistic induction of alpha-smooth muscle actin (SMA) protein. Another CTGF receptor candidate is integrin avb3 which functions as a co-receptor with heparan sulfate proteoglycans (HSPG) for CTGF-mediated hepatic stellate cell (HSC) adhesion. Integrin avb3 is also involved in the angiogenic activity of CTGF.

Modulators of CTGF Expression There are multiple factors and stimuli that regulate the expression of CTGF. TGF-b1 is perhaps the bestcharacterized factor capable of increasing CTGF expression. CTGF expression is upregulated in response to TGF-b1 through a novel TGF-b1 response element in the CTGF promoter. Inhibition of extracellular signal regulated kinase (ERK) and c-jun N-terminal kinase (JNK), but not of p38 mitogen-activated protein kinase (MAPK) and phosphoinositol-3 kinase (PI3 K), blocks the effect of TGF-b1 on CTGF mRNA and protein expression. Although IL-4 was found to have no effect on basal CTGF expression in cultured human lung fibroblasts, it decreases TGF-b-induced CTGF expression through a transcriptional mechanism that involves modulation of Smad and ERK signaling. Notably, IL-4 did not affect fibronectin or alpha1(I) collagen mRNA expression induced by TGF-b in human lung fibroblasts. There are other peptide factors that have the capacity to induce CTGF expression, such as VEGF and thrombin. Similarly, angiotensin II, alpha-tocopherol, shear stress, cell stretch and static pressure, H2O2, and O2 increase the expression of CTGF. The potent vasodilator NO decreases CTGF expression. Thus, CTGF is thought to play a role in vascular wall remodeling. Conversely, there are several proinflammatory cytokines, for example, tumor necrosis factor alpha (TNF-a), IL-1, and IL-4, which decrease CTGF expression. The effect of any given factor on CTGF expression under the complex milieu that occurs in injury and repair may not correlate closely with the in vitro findings. For example, TNF decreases CTGF mRNA and peptide expression in cultured bovine artery endothelial cells, 3T3 fibroblasts, and smooth muscle cells. TNF and IL-1b reduce TGF-b1-stimulated CTGF mRNA expression, but unlike IL-4, do not inhibit TGF-b-induced Smad2/3 phosphorylation. In comparison, CTGF expression is actually upregulated at times when TNF expression is increased in the bleomycin model of murine pulmonary

fibrosis. In addition, dexamethasone has been reported to increase CTGF expression in BALB/c 3T3 fibroblasts in vitro, which is of concern since corticosteroids are frequently employed in an attempt to arrest inflammation in subjects afflicted with fibrotic diseases. However, dexamethasone did not increase the expression of CTGF in a murine model of wound healing. In yet another example, the 3-hydroxy3-methylglutaryl (HMG) CoA reductase inhibitor, simvastatin, inhibits TGF-b-induced CTGF mRNA and protein expression in lung fibroblasts isolated from patients with interstitial pulmonary fibrosis (IPF). In addition, simvastatin was found to reduce TGF-b-induced alpha smooth muscle action and collagen gel contraction through a Rho-dependent signaling mechanism. A large retrospective review did not demonstrate any survival advantage between IPF patients that were taking HMG CoA reductase inhibitors and those patients that were not. However, by nature of the retrospective design, it is not known whether the IPF patients taking statins had lower expression of CTGF compared with the IPF population who were not taking statins. These examples demonstrate the necessity of evaluating factors that regulate CTGF expression in vivo as well as in vitro.

CTGF and Fibrogenesis There are now numerous publications showing that CTGF expression is enhanced during fibrogenesis in multiple organs, including lung, skin, kidney, liver, bowel, and heart, as well as within desmoplasia surrounding tumors. This is not unexpected since TGFb1 is increased in fibroproliferative pathology, and because TGF-b1, -b2, and -b3 upregulate CTGF expression in vitro. Importantly, subcutaneous injection of TGF-b1 results in increased expression of CTGF within dermal fibroblasts and subsequent formation of a fibrotic lesion. A rat skin wound model has revealed an upregulation of CTGF mRNA expression that follows an increase in TGF-b1 mRNA expression, which suggests that CTGF is induced by the increased expression of TGF-b1 in vivo. Because dermal fibroblasts synthesize CTGF and proliferate in response to CTGF, investigators have proposed that CTGF may act as an autocrine mitogen induced by TGF-b1. In addition, many manuscripts demonstrate that CTGF expression is reduced in mice that are protected from fibrosis. For example, proteinaseactivated receptor 1 (PAR-1) knockout mice are protected from bleomycin-induced lung injury and express less TGF-b1 and CTGF mRNA compared with wild-type littermates. Urinary CTGF excretion is reduced in patients with diabetic nephropathy

556 CONNECTIVE TISSUE GROWTH FACTOR

treated with Losartan, an angiotensin converting enzyme inhibitor. In sum, these studies clearly show that there is a correlation between CTGF expression and fibrogenesis, but do not prove a cause-and-effect relationship between CTGF and fibrosis. The majority of publications regarding CTGF expression and fibrogenesis have been correlative; however, there are a few manuscripts that attempt to define the role of CTGF in fibrogenesis. A study using a model of carbon tetrachloride-induced liver fibrosis demonstrated that CTGF antisense oligonucleotides (ODN) decreased liver CTGF mRNA expression as well as that of type I collagen mRNA, but did not affect tissue inhibitor of metalloproteinase-1 (TIMP-1) expression or the degree of fibrosis. In contrast, two models of renal fibrosis demonstrate that treatment with a CTGF antisense ODN attenuates the induction of CTGF, fibronectin, alpha1(I) collagen, plasminogen activator inhibitor 1, and TIMP-1, and suppresses renal interstitial fibrogenesis, without affecting TGF-b mRNA expression. Repair of cartilage is another area strongly supporting a healing role for CTGF. A rat model of osteoarthritis revealed CTGF expression at the sites of wound repair, and the administration of rCTGF enhances chondrogenesis in vivo. It is difficult to reconcile these disparate findings regarding the importance of CTGF in fibrogenesis, although species variances and magnitude of CTGF inhibition could be entertained as possible explanations. CTGF and Dermal Fibrogenesis

Dermal fibrogenesis is addressed here because of the high frequency of pulmonary fibrosis in association with scleroderma, and because there is increasing evidence that CTGF is involved in the pathogenesis of scleroderma. This is not unexpected since TGF-b1 is increased in fibroproliferative dermal pathology, such as scleroderma, and because TGF-b1, -b2 and -b3 upregulate CTGF expression in vitro. Importantly, subcutaneous injection of TGF-b1 results in increased expression of CTGF within dermal fibroblasts and subsequent formation of a fibrotic lesion. In addition, a positive correlation between CTGF expression and skin sclerosis has been reported. A rat skin wound model has revealed an upregulation of CTGF mRNA expression that follows an increase in TGF-b1 mRNA expression, which suggests that CTGF is induced by the increased expression of TGF-b1 in vivo. Administration of rCTGF alone did not result in dermal fibrosis; however, rCTGF did prolong the stability of fibrosis caused by administration of rTGF-b. An explanation for these findings may be related to the fact that collagen accumulation

also depends upon factors that degrade the matrix. Administration of recombinant CTGF reduced matrix degradation in human mesangial cells exposed to high concentrations of glucose. Furthermore, a neutralizing anti-CTGF antibody reduced TGF-b1mediated inhibition of matrix degradation. CTGF is heavily implicated in the pathogenesis of scleroderma. Indeed, serum CTGF levels correlate with the extent of skin and lung fibrosis in patients afflicted with scleroderma. Normal unperturbed skin fibroblasts do not express CTGF, whereas fibroblasts in areas of wound repair or isolated from the skin of people afflicted with scleroderma express abundant CTGF. The previously mentioned profibrotic activities of CTGF in vitro, namely fibroblast mitogen, stimulator of collagen expression, and enhancer of cell adhesion, are considered as support for the role of CTGF in the pathobiology of scleroderma. Interestingly, in contrast to normal wound repair wherein CTGF promoter activity is under control of a TGF-b response element, scleroderma fibroblast CTGF expression is resistant to SMAD3 inhibition of TGF-b signal transduction, but rather is dependent upon Sp1. CTGF and Lung Fibrogenesis

Considerable attention has also been given to defining the role of CTGF in lung fibrogenesis. CTGF mRNA expression is increased in the lungs of mice that develop pulmonary fibrosis following administration of bleomycin as well as mice treated with an adenoviral vector expressing TGF-b1. Moreover, CTGF expression is elevated in lavage specimens derived from fibrotic human lung. The cellular immunoreactivity for CTGF was markedly increased in the lung tissue of patients with IPF compared to normal lungs, and immunolocalization of CTGF was confined predominantly to proliferating type II alveolar epithelial cells and activated fibroblasts. Interestingly, TGF-b mRNA expression decreased in a population of patients with IPF who improved clinically following treatment with interferon gamma (IFN-g), but changes in CTGF expression were not reported in a larger study of IPF patients who underwent lung biopsy before and 6 months after administration of IFN-g versus placebo. The ambiguity regarding the biological significance of enhanced CTGF expression in lung fibrogenesis mirrors that discussed above for renal and liver fibrosis. Others have clearly demonstrated that adenoviral gene transfer of CTGF into rat lung induces transient fibrosis. Moreover, administration of a hCTGF adenoviral vector intranasally 3 days prior to intratracheal administration of bleomycin caused

CONNECTIVE TISSUE GROWTH FACTOR 557

the normally bleomycin-resistant strain of BALB/c mice to develop lung fibrosis. In contrast, another study found normal lung histopathology in three lines of transgenic mice that overexpress CTGF using the lung-specific surfactant protein-C promoter. Moreover, the CTGF transgenic mice did not demonstrate a significant change in the severity of bleomycin-induced lung fibrosis in comparison to their wild-type littermates. In another experiment, the degree of bleomycin-induced lung fibrosis in mice that were heterozygous for a CTGF null allele was evaluated. Although lung fibroblasts from heterozygous CTGF knockout lungs express 50% less CTGF mRNA compared with those from wild-type littermates (pre or post in vitro treatment with TGFb1), there was no difference observed in the severity of bleomycin-induced lung fibrosis in heterozygous CTGF knockout mice compared with their wild-type littermates. In total, these findings suggest that small variances in CTGF expression have at best small and transient effects on the severity of lung fibrosis. The data also indicate that if anti-CTGF therapy is to be effective in limiting the progression of lung fibrosis, then the degree of inhibition will need to be much greater than 50%.

Modulation of Cancer Biology Early studies demonstrated significant associations between CTGF expression and advanced features of breast cancer, which implicated CTGF in the progression of breast cancer. CTGF functions as a survival and differentiation factor in human rhabdomyosarcoma. However, conflicting results arose from studies pertaining to the role of CTGF with other types of carcinomas. CTGF impedes the growth of oral squamous cell carcinoma cells and inhibits metastasis and invasion of human lung adenocarcinoma in a collapsin response mediator protein (CRMP)1-dependent mechanism. CTGF regulates normal endothelial homeostasis, as well as the angiogenic process during embryonic development, placentation, tumor formation, fibrosis, and wound healing. CTGF knockout mice exhibit vascular defects during embryogenesis and fetal development. CTGF modulates tumor-associated angiogenesis through both intrinsic angiogenic activity and induction of other angiogenic molecules (e.g., fibroblast growth factor (bFGF), vascular endothetial growth factor (VEGF)). CTGF released by human breast cancer cells exposed to hypoxia promotes angiogenesis through modulating the balance of extracellular matrix synthesis and degradation via matrix metalloproteinases (MMPs)

secreted by endothelial cells. The release of CTGF from tumor cells appears to be essential for the angiogenesis in chicken chorioallantoic membrane angiogenesis assays. In summary, CTGF is a peptide growth factor that affects an array of biological processes, and is clearly necessary for normal development. CTGF expression is mediated by factors implicated in fibrogenesis and CTGF has profibrogenic activity in vitro and in vivo. It remains to be seen whether or not inhibition of CTGF expression will result in a significant improvement in the course of patients suffering from fibroproliferative diseases. See also: Interleukins: IL-1 and IL-18; IL-4. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Blom IE, Goldschmeding R, and Leask A (2002) Gene regulation of connective tissue growth factor: new targets for antifibrotic therapy? Matrix Biology 21(6): 473–482. Bonniaud P, Martin G, Margetts PJ, et al. (2004) Connective tissue growth factor is crucial to inducing a profibrotic environment in ‘fibrosis-resistant’ BALB/c mouse lungs. American Journal of Respiratory Cell and Molecular Biology 31(5): 510–516. Chang CC, Shih JY, Jeng YM, et al. (2004) Connective tissue growth factor and its role in lung adenocarcinoma invasion and metastasis. Journal of the National Cancer Institute 96(5): 364– 375. Grotendorst GR, Okochi H, and Hayashi N (1996) A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene. Cell Growth Differentiation 7: 469–480. Ivkovic S, Yoon BS, Popoff SN, et al. (2003) Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130(12): 2779–2791. Leask A and Abraham DJ (2003) The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology. Biochemistry and Cell Biology 81(6): 355–363. Nishida T, Kubota S, Kojima S, et al. (2004) Regeneration of defects in articular cartilage in rat knee joints by CCN2 (connective tissue growth factor). Journal of Bone and Mineral Research 19(8): 1308–1319. Okada H, Kikuta T, Kobayashi T, et al. (2005) Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis. Journal of the American Society of Nephrology 16(1): 133–143. Uchio K, Graham M, Dean NM, Rosenbaum J, and Desmouliere A (2004) Down-regulation of connective tissue growth factor and type I collagen mRNA expression by connective tissue growth factor antisense oligonucleotide during experimental liver fibrosis. Wound Repair and Regeneration 12(1): 60–66. Yokoi H, Mukoyama M, Nagae T, et al. (2004) Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis. Journal of the American Society of Nephrology 15(6): 1430–1440.

558 CONNEXINS, TISSUE EXPRESSION

CONNEXINS, TISSUE EXPRESSION in close contact were separated by a uniform 2–3 nm ‘gap’, hence the term ‘gap junction’. Perfection of techniques to isolate junctions from rat liver and heart enabled the identification and subsequent cloning of the channel forming proteins of gap junctions, known as connexins.

M Koval, Emory University School of Medicine, Atlanta, GA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Connexins are transmembrane proteins that form gap junction channels. Gap junctions interconnect cells to allow the diffusion of signaling molecules and metabolites between cells. Cells in the respiratory system exhibit unique patterns of connexin expression which determine whether they are interconnected with neighboring cells and the types of signals transmitted between them. Connexin expression in the lung is altered in response to injury as a compensatory mechanism to alter the extent of cell– cell coupling. Gap junctional communication between type II and type I cells plays a critical role in integrating the alveolus to enable type I cells to act as mechanical sensors to regulate pulmonary surfactant secretion by type II cells. By regulating intercellular signaling, controlling the flow of metabolites, and restricting the flow of toxic agents, connexins enable the cells of the lung to act as an integrated system.

Structure Connexins are transmembrane proteins that span the membrane bilayer four times with both the N- and C-terminus oriented towards the cytoplasm (Figure 1). Unlike most transmembrane proteins, connexins are not glycosylated. Based on the human and murine genomes, there are almost two dozen mammalian connexins which show cell- and tissue-specific expression patterns. By protein homology, connexins fall into two subgroups, alpha and beta, which are defined by characteristic amino acids in the first and third transmembrane domains and second extracellular loop. The cytoplasmic loop and C-terminal tail domains show the greatest level of sequence divergence. A functional gap junction channel is composed of two connexin hexamers (or hemichannels) in two adjacent cells which dock to form a complete channel. Gap junction channels are typically arranged in semicrystalline arrays, known as plaques, at sites of cell–cell contact where intercellular communication occurs. However, free connexin hemichannels dispersed throughout the plasma membrane can also act

Introduction Gap junction channels interconnect cells by forming a direct link to enable the diffusion of small aqueous molecules and ions from one cell to its nearest neighbor. This enables both the flow of specific intercellular signals and metabolic cooperation between communicating cells in a tissue. Intercellular communication was initially correlated with sites identified by electron microscopy where two adjacent cells

Lumenal/extracellular

1

2

3

N C

Cytosolic (a)

4

Cx37 Cx43 Cx46 Cx40 Cx50 Cx59 Cx62 Cx40.1 Cx31.9 Cx45 Cx47 Cx36 (b)






Cx25 Cx26 Cx30 Cx32 Cx31.1 Cx30.3 Cx31 (c)

− Connexin hexamer

Figure 1 Connexins. (a) Connexins are transmembrane proteins that span the bilayer four times, with N- and C-terminus oriented towards the cytoplasm. (b) Dendrogram showing connexins aligned by protein sequence homology. Based on sequence homology, connexins form two subgroups, alpha and beta. (c) Gap junction channels are composed of two hexamers, one in each cell, that dock to form a complete channel. Dashed lines indicate the path of diffusion for aqueous molecules and ions through gap junctions from one cell to another.

CONNEXINS, TISSUE EXPRESSION 559

as bona fide plasma membrane channels, enabling the exchange of aqueous molecules between the cytoplasm to the extracellular environment.

Regulation of Production and Activity Connexins have a relatively rapid half-life of 1–5 h, suggesting that gap junction turnover is a constant process. Connexins are cotranslationally inserted into the endoplasmic reticulum membrane and then assembled into hemichannels. The site of hemichannel assembly depends on connexin subtype, where beta connexins form hemichannels in the endoplasmic reticulum/Golgi while alpha connexins form hemichannels in intermediate compartment aspect of the Golgi apparatus. Hemichannels are then transported to the plasma membrane where they dock with hemichannels on neighboring cells to form complete gap junction channels. Gap junctions are turned over by a unique endocytic mechanism shared by tight junctions where one cell engulfs a portion of the other cell to internalize a piece of a gap junction plaque. Internalized plaques are degraded by a combination of proteosomal and lysosomal hydrolysis. Cells that express multiple connexins have the potential to form heteromeric or mixed gap junction channels. The rules that govern connexin intermixing are a combination of innate incompatibility and regulated assembly. For instance, alpha and beta connexins are innately incompatible; an alpha connexin such as Cx43 cannot form a mixed gap junction channel with a beta connexin such as Cx32. However, cells also regulate the formation of mixed gap junctions by compatible connexins. For instance, two of the compatible connexins expressed by alveolar epithelial cells are Cx43 and Cx46. Type I alveolar epithelial cells form mixed gap junctions composed of Cx43 and Cx46, whereas type II cells prevent Cx43 and Cx46 from intermixing. Other examples of cells which regulate connexin assembly include endothelial cells which restrict formation of mixed gap junctions containing Cx37 and Cx43. The mechanisms that regulate connexin intermixing are not known at present. However, one ramification of differential connexin expression and assembly is that cells within a given tissue can create distinct cell–cell interfaces to preferentially allow or restrict intercellular communication. Connexin expression is regulated at the transcriptional level. In the normal lung, most epithelial cells express Cx32 and Cx43, whereas endothelial cells express predominantly Cx37, Cx40, and Cx43. Type I and type II alveolar epithelial cells have been studied in considerable detail. The major connexins expressed by alveolar epithelial cells are Cx26, Cx32,

Cx43, and Cx46. Others are expressed at low levels, such as Cx30.3, Cx37, and Cx40. Based on immunohistochemistry of whole lung sections, Cx32 is expressed exclusively by type II alveolar epithelial cells in normal adult rat lung. Cx43 is fairly ubiquitous and is the major connexin functionally interconnecting type II and type I cells. Expression of Cx37 by alveolar epithelial cells in situ is low, but consistently detectable by immunohistochemistry. Considerably more Cx37 is expressed by bronchiolar epithelium, but this is still less than the level observed for pulmonary endothelial cells.

Biological Function The primary function of gap junction channels is to interconnect cells. Gap junction channels have relative selectivity, that is, gap junction channels composed of different connexins show different rates of diffusion for a given molecule. For instance, diffusion of ATP is B10-fold faster through Cx43 channels as compared to Cx32. Conversely, glutathione flux through Cx32 and Cx43 channels is roughly comparable. One consequence of gap junction formation by two or more connexins is that cells form gap junction channels with unique permeability characteristics. For instance, Cx26 has a fairly restrictive permeability and forms heteromeric gap junction channels with Cx32. Cx32 is fully permeable to cAMP and cGMP. However, channels containing both Cx26 and Cx32 are preferentially permeable to cGMP as compared to cAMP. Other examples of mixed gap junction channels which have unique permeability include Cx37/Cx43, Cx40/Cx43, and Cx43/Cx45. In addition to metabolic coupling, gap junction channels also transmit signals from one cell to another. Most typically, this is observed by the transmission of transient increases in cytosolic calcium (calcium ‘waves’) throughout a tissue. For instance, transmission of calcium signals through gap junctions regulates vascular tone, particularly in small vessels where gap junctions interconnect endothelial cells to smooth muscle cells. In airway epithelium, intercellular transmission of inositol trisphosphate creates oscillatory waves that synchronize ciliary beat frequencies in order to coordinate the outward flow of mucus. In the alveolus, gap junctions between type I and type II cells enable these two classes of alveolar epithelial cells to act in concert to regulate surfactant secretion. During normal breathing the changes in lung volume generally correspond to relatively small changes (B5%) in alveolar surface area, although during sighs or yawns, alveolar surface area may

560 CONNEXINS, TISSUE EXPRESSION

increase by about 10–15%. Stress analysis of alveoli indicates that type I cells stretch to a level equivalent to the overall level of alveolar stretch. However, type II cells are more or less shielded from stretch. In response to stretch, type I cells increase cytosolic calcium, which is subsequently transmitted to type II cells through gap junctions. The rise in type II cell cytosolic calcium is sufficient to trigger lamellar body fusion with the plasma membrane and stimulates surfactant secretion. Thus, gap junctions enable type I cells to act as mechanosensors and to actively contribute to the regulation of pulmonary surfactant release. Interestingly, a role for disrupted cell–cell communication in regulating surfactant release is potentially underscored by surfactant abnormalities associated with diseases such as acute respiratory distress syndrome (ARDS) and ventilator-induced injury, where cell–cell contacts are disrupted. To date, there are few direct data from transgenic mouse models related to respiratory function. This is due, in part, to the difficulty of working with some connexin-deficient models. For instance, Cx43-deficient mice have a neonatal lethal phenotype due to cardiac malformations of the outflow tract which, in turn, lead to pulmonary edema. Whether Cx43 has a role in control of lung fluid balance through regulation of epithelial barrier function is not known at present. However, mice doubly deficient in Cx37 and Cx40, two prominent blood vessel connexins, show permeability defects and localized hemorrhages, consistent with a role for endothelial gap junctions in regulating vessel barrier function. Note, however, that any role for connexins in altering barrier function will be regulatory rather than structural, since the structural component of endothelial or epithelial barrier function is due to claudin-family tight junction proteins.

Connexin Binding Proteins The extracellular portion of connexins interacts exclusively with other connexins. However, the cytoplasmic C-terminus interacts with a number of different protein cofactors, which interlink them with the cytoskeleton and regulate connexin function. Since there is considerable variability in the C-terminus, different connexins interact with different proteins. For instance, the zona occludens-1 (ZO-1) scaffold protein, which links junction proteins to the actin cytoskeleton, binds to Cx43, Cx45, and Cx46, but not Cx32. As another example of a connexin–cytoskeleton interaction, tubulin binds to Cx43 and provides a direct link to microtubules. In addition, connexin binding proteins can regulate connexin open probability, which relates to the

mean time that channels are open. For instance, calmodulin binds to Cx32-containing gap junction channels and increases their sensitivity to calciuminduced closure. Connexins are also regulated by phosphorylation on the C-terminus, which typically decreases connexin open probability. Phosphorylation of plasma membrane connexins also acts as a signal for gap junction disassembly. Several kinases are known to phosphorylate connexins, including sarcoma (src) kinase and mitogen-activated protein kinases, which help decrease the extent of gap junctional communication during cell division and proliferation.

Connexins in Respiratory Diseases Human diseases directly attributable to mutant connexins include peripheral neuropathy (Charcot– Marie–Tooth disease; Cx32), premature cataract (Cx46, Cx50), and musculoskeletal disorders (oculodentodigital dysplasia; Cx43). Interestingly, different mutations in the same connexin can cause different diseases. For instance, distinct mutations in Cx26 are associated with either deafness or skin disease (keratoderma). To date, no human respiratory disease has been directly attributable to a connexin deficiency or mutation; however, potential roles for connexins in lung disease can be deduced from animal and in vitro models. In the injured lung, type II cell hyperplasia increases the frequency of type II cells in direct contact with other type II cells, both of which express Cx32. Since type I–type II cell communication is mediated through Cx43-compatible connexins and is not mediated by Cx32, this provides type II cells with an independent pathway for communication that does not involve type I cells. During the acute phase of acute lung injury, connexin expression in the alveolus is altered, where both Cx43 and Cx46 expression is elevated. Cx46expressing alveolar epithelial cells do not express typical type II cell markers and thus may represent a distinct subtype of cells proliferating in response to injury. Interestingly, Cx46 has relatively limited permeability as compared to Cx32 and Cx43, suggesting a possible role for Cx46 in limiting metabolic depletion or intercellular transmission of toxic agents. Transmission of toxic agents through gap junctions is very efficient, a phenomenon known as the ‘bystander effect’. Even radiation levels on the order of one alpha particle per five cells can create cytotoxic substances transmitted through Cx43 gap junction channels. Although deleterious in the setting of lung injury, bystander effects have been explored as a

CONTRACTILE PROTEINS 561

chemotherapeutic approach, most notably in the use of viruses expressing thymidine kinase combined with ganciclovir as a potential treatment of asbestosinduced mesothelioma. A limitation in the use of bystander effects as an anticancer therapy is that tumor cells typically have relatively low levels of connexin expression and gap junctional communication, which limit their susceptibility to cell–cell transfer of toxic agents. However, this underscores the notion that gap junctional communication plays a role in the control of cell growth. Although the precise role for gap junctions in regulating cell growth is largely unknown, mice deficient in Cx32 exhibit increased lung tumor load in response to carcinogens such as urethane and radiation, suggesting that Cx32 might be a lung tumor suppressor. There are also clues that connexins may play a role in the pathology of cystic fibrosis (CF). Cells expressing the DF508 mutant of the cystic fibrosis transmembrane conductance regulator (CFTR) associated with CF lack efficient connexin assembly and show abnormally low levels of gap junctional communication. Further work is required to define a functional link between CFTR and connexin function. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Cell: Vascular; Epithelial. Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene.

Further Reading Abraham V, Chou ML, George P, et al. (2001) Heterocellular gap junctional communication between alveolar epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 280: L1085–L1093. Ashino Y, Ying X, Dobbs LG, and Bhattacharya J (2000) [Ca2 þ ]i oscillations regulate type II cell exocytosis in the pulmonary alveolus. American Journal of Physiology 279: L5–L13. Boitano S, Safdar Z, Welsh DG, Bhattacharya J, and Koval M (2004) Cell–cell interactions in regulating lung function. American Journal of Physiology: Lung Cellular and Molecular Physiology 287: L455–L459. Chanson M, Berclaz PY, Scerri I, et al. (2001) Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells. American Journal of Pathology 158: 1775–1784. Chipman JK, Mally A, and Edwards GO (2003) Disruption of gap junctions in toxicity and carcinogenicity. Toxicological Sciences 71: 146–153. Goldberg GS, Valiunas V, and Brink PR (2004) Selective permeability of gap junction channels. Biochima et Biophysica Acta 1662: 96–101. Goodenough DA and Paul DL (2003) Beyond the gap: functions of unpaired connexon channels. Nature Reviews: Molecular Cell Biology 4: 285–294. Harris AL (2001) Emerging issues of connexin channels: biophysics fills the gap. Quarterly Reviews of Biophysics 34: 325–472. Kelsell DP, Dunlop J, and Hodgins MB (2001) Human diseases: clues to cracking the connexin code? Trends in Cell Biology 11: 2–6. Koval M (2002) Sharing signals: connecting lung epithelial cells with gap junction channels. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L875–L893. Saez JC, Berthoud VM, Branes MC, Martinez AD, and Beyer EC (2003) Plasma membrane channels formed by connexins: their regulation and functions. Physiological Reviews 83: 1359–1400.

CONTRACTILE PROTEINS J R Sellers, National Institutes of Health, Bethesda, MD, USA

discussed. During many types of respiratory disease smooth muscle proliferates, hypertrophies, and/or upregulates its contractile protein complement in response to the stress.

Published by Elsevier Ltd.

Abstract

Introduction

All cells must have machinery in them to move vesicles, proteins, and mRNA, to carry out membrane trafficking of receptors and synthesized proteins, and to promote adhesion with other cells or their substrate. In addition, most cells undergo cell division in which a single cell is divided in half by a cytokinetic ring. Many types of cells can secrete vesicularized contents or undergo endocytosis or phagocytosis. The machinery responsible for this plethora of functions is loosely termed contractile proteins and consists of actin, myosin, and associated regulatory or structural proteins. While the function of these proteins is well documented in smooth and striated muscle, less is known about their roles in nonmuscle cells. In this article the function of these proteins in both muscle and nonmuscle cells will be

The respiratory system contains a multitude of different cell types. Smooth muscle cells line the trachea, the bronchi, and the bronchioles and are present in the vascular system. Ciliated columnar epithelium, cuboidal epithelium, and Clara cells are present at different locations. Endothelial cells are present in the vasculature and elsewhere. Mast cells are involved in the immunologic response and motile macrophages phagocytose extracellular debris. A common feature of all these cells is that they contain the cellular machinery to move intracellular vesicles that

562 CONTRACTILE PROTEINS

maintain a cytoskeleton. The term ‘contractile proteins’ will be used to define the proteins used in cell motility, cytokinesis, intracellular vesicular trafficking, and maintaining cell morphology and cell contacts.

Role of Muscle in Respiration The diaphragm muscle is of the skeletal or striated type and is the major muscle of ventilation. Accessory muscles of ventilation include the scalene, the sternocleidomastoid, the pectoralis major, the trapezius, and the external intercostals. Smooth muscle is found in the trachea and in the pulmonary arteries and smaller vessels. Striated muscles contain regular arrays of thick and thin filaments that make up the sarcomere, the basic contractile unit (Figure 1). Bipolar thick filaments containing the motor protein myosin lie at the center of the sarcomere. Thin filaments lie on either side of the thick filament and are attached to Z-lines that are built of many proteins including a-actinin, an important structural protein, and CapZ which binds to the end of an actin filament and helps anchor it to the Z-line. The thin filament is composed of polymerized G-actin subunits and contain the regulatory proteins troponin and tropomyosin as well as the large protein nebulin. Troponin consists of three subunits: TN-C, a small calmodulin-like calcium-binding protein; TN-I, an inhibitory subunit; and TN-T, a tropomyosin-binding subunit. Troponin and tropomyosin are each present in a stoichiometry of one per seven actin molecules (Figure 1). Tropomyosin is an elongated coiled-coil a-helical protein whose length matches the distance spanned by seven actin monomers. It lies in the groove of the thin filament.

Regulation of Striated Muscle Contraction In resting muscle (in the absence of calcium), troponin holds tropomyosin in a position on the actin filament that blocks myosin from binding in a productive manner. Contraction is initiated following nerve stimulation, which releases calcium from internal stores called the sarcoplasmic reticulum. Calcium binds to the TN-C subunit, which induces a rearrangement of the other troponin subunits, resulting in a movement of tropomyosin from its blocking position to one that allows myosin to interect with actin filaments and pull them toward the center of the thick filament, thus shortening the sarcomere and the muscle. When calcium is sequestered in relaxation, troponin and tropomyosin move back to their inhibitory positions, myosin no longer interacts productively with actin, and the giant elastic protein titin restores the sarcomere to its resting length. Titin, the largest protein in the genome, stretches from the Z-line to the center of the myosin thick filament. Portions of titin consist of repeating elastic elements that can be unfolded to allow for lengthening during muscle contraction. Upon relaxation of the muscle fiber, the titin elastic elements begin to refold, thus shortening the molecule and aiding return of the muscle fiber to resting length. The fact that muscle shortens without either of its two filament systems undergoing a decrease in length is due to the fact that these two filaments slide past one another during contraction. The function and localization of nebulin on the thin filament is not well known. It may be template to determine the length of the actin filament and/or may play a role in the calcium-dependent regulation. The individual myosin molecules in a thick filament interact asynchronously with actin in a manner

Titin

Thick filament

Thin filament

Z-lines

Nebulin

CapZ

Figure 1 Schematic of a striated muscle sarcomere. The schematic shows the manner in which the thin filaments and thick filaments interdigitate. When the muscle shortens, the myosin molecules in the thick filament actively pull the thin filaments toward the center of the thick filament. Other important proteins are shown as described. An expanded view of a thin filament is given showing the positions of tropomyosin and troponin. The position of nebulin on the thin filament is not shown since its localization has not been firmly established.

CONTRACTILE PROTEINS 563

that is tightly coupled to the hydrolysis of ATP. To illustrate the coupling of this process to ATP, a single powerstroke of myosin is shown in Figure 2. We start with myosin at the end of its powerstroke where the products of ATP hydrolysis have just been released.

Myosin head AM

Actin ATP

In this state (often referred to as the rigor state), the myosin head is bound to actin at a 451 angle with respect to actin. Upon binding ATP the myosin head dissociates from actin, and hydrolyzes ATP to give the products ADP and Pi which are still bound to the active site. While dissociated from actin, the myosin head recocks to assume the prepowerstroke conformation. Myosin then rebinds to actin at a 901 angle and subsequently undergoes a tilting conformational change which moves the actin filament incrementally toward the center of the thick filament. This process is associated with the ordered release of Pi and ADP from the myosin active site to restore the starting rigor conformation.

ATP

Smooth Muscle Contractility

(A)M.ATP

M.ATP

ADP.Pi M.ADP.Pi

ADP.Pi

(A)M.ADP.Pi

Pi

AM.ADP

ADP

AM Figure 2 Myosin cross-bridge cycle. See text for description of events. A, actin; M, myosin; AM, myosin bound strongly to actin; (A)M, myosin bound weakly to actin.

Smooth muscle is found in tissues such as the stomach, the intestines, the bladder, arteries, the trachea, and the uterus. Smooth muscle contains interdigitating thick and thin filaments, but their order is not nearly as precise as exists in striated muscle, giving rise to a ‘smooth’ versus striated appearance in the light microscope. The difference in microscopic appearance between smooth muscle and skeletal muscle is but one of the profound differences between the two systems. The activation of skeletal muscle tissue is directly driven by electrical activity at the neuromuscular junction. Smooth muscle on the other hand can also be activated by adrenergic and cholinergic receptors on the cell surface. In general, smooth muscles contract more slowly than do skeletal muscle fibers and maintain the contraction for longer periods of time. Smooth muscles can be divided into phasic muscles which contract more rapidly and tonic muscles which contract slowly and maintain tension for long periods of time. At least part of the difference in contractile speed of phasic and tonic smooth muscles derives from the expression of different, alternatively spliced isoforms of the smooth muscle myosin gene. The mechanism of calcium-dependent regulation of smooth muscle is profoundly different from that of skeletal muscle. The dominant site of regulation of smooth muscle is at the thick filament. The enzymatic activity of smooth muscle myosin is greatly activated by phosphorylation of its regulatory light chain subunit at Ser19 by myosin light chain kinase (MLCK) which, in turn, is active when bound to the calcium-bound form of calmodulin (Figure 3). Myosin is inactivated by dephosphorylation of Ser19 by a myosin phosphatase (MYPT1). The dephosphorylated myosin is essentially completely inactive. Signal transduction pathways impinge on both MLCK and MYPT1 affecting their respective

564 CONTRACTILE PROTEINS RhoA signaling pathway Other signaling pathways ???

CaM kinase II

ROK active

+Ca2+ Myosin-P active

P-MYPT1 less active

MYPT1 fully active

Myosin inactive

MLCK inactive

MLCK.Ca.CaM active −Ca2+

Phosphatase Figure 3 Regulation of smooth and nonmuscle myosin.

activities. Since the steady state level of myosin phosphorylation and, thus its activity, is primarily a function of the ratio of the activity of these two important regulatory proteins, pathways such as the RhoA pathway or those regulated by heterotrimeric G-proteins can profoundly affect the contractile state of smooth muscle and also the activity of myosin in nonmuscle cells. RhoA signaling results in the activation of Rho kinase (ROK) which phosphorylates MYPT1 and inhibits its activity. MLCK can be phosphorylated by protein kinase A and CaM kinase II resulting in a reduction of its activity. Some kinases in various signal transduction pathways can directly phosphorylate myosin at Ser19 of the regulatory light chain. Smooth muscle contains tropomyosin, but lacks troponin on its thin filaments. Instead, smooth muscle thin filaments bind another potential regulatory protein, termed caldesmon. Caldesmon is an elongated protein whose C-terminal end binds to actin and the small calcium-binding protein calmodulin, while its N-terminal end binds to myosin. In vitro experiments have shown that in the absence of calcium, caldesmon can inhibit the enzymatic and mechanical activities of myosin and that this inhibition can be reversed by the addition of calcium and calmodulin. Caldesmon might also play a structural role by tethering thick and thin filaments via its spatially separated binding sites for actin and myosin. Many of the striated muscle contractile proteins have homologous isoforms that are expressed in smooth muscle tissue. There is a smooth muscle-specific myosin heavy chain gene and the genes for the light chains of smooth muscle myosin differ from those of skeletal muscles. In place of well-defined Zlines, smooth muscles have dense bodies which contain a smooth muscle-specific isoform of a-actinin.

Contractile Systems in Nonmuscle Cells Most of the cells of the respiratory tissue are not muscle and will be called ‘nonmuscle’ cells for this

review. Nevertheless, these cells contain ‘contractile’ proteins, including actin, myosin, tropomyosin, caldesmon, MLCK, and a myosin-specific phosphatase that are very similar to those found in smooth muscles. These proteins are used in cells for a variety of purposes including maintaining cell–cell and cell– substrate adhesion, cell division, cell motility, and cell morphology. The clearest role for contractile proteins in nonmuscle cells is the constriction of the cytokinetic ring during cell division. In another example, a dense cortical bundle of actin and myosin filaments is found in the periphery of resting basophil and mast cells. Upon stimulation this cortical network is disassembled to allow the secretory vesicles access to the plasma membrane. In addition, directed cell motility requires the coordinated action of both the actin filament network and myosin. The contractile states of various cells are similarly affected by the RhoA signaling pathway described above and also by other signal transduction networks, such as the Rac pathway. Here, one of the downstream kinases from these pathways, such as ROK and, possibly PAK, can directly phosphorylate Ser19 of the nonmuscle myosin regulatory light chain. Discussed below are more detailed descriptions of actin and myosin and roles that they play. Actin

Actin is often the most abundant protein in cells. G-actin, the globular monomeric form of actin, has a molecular weight of about 42 kDa. Many G-actin subunits polymerize to create a filamentous form of actin, F-actin, which has the appearance of a twisted string of pearls (Figure 2). The pseudo repeat of the F-actin helix is about 37 nm and contains 14 actin monomers. Actin monomers preferentially bind at the ‘barbed’ end of the actin filament and preferentially dissociate from the ‘pointed’ end giving the structure polarity. Unlike in striated muscle tissue where actin plays predominantly a single role as the

CONTRACTILE PROTEINS 565

structural element upon which myosin pulls, in nonmuscle cells actin does many chores. It forms a structural cytoskeleton which plays important roles in cell–cell and cell–substrate adhesion. Motile cells have an actin-rich lamillipodial region at the leading edge. Many proteins are involved in this process including a complex called Arp2/3 which binds to the side of an existing actin filament and nucleates the polymerization of another branching actin filament at a 701 angle to create a dendritic network. Since most actin filaments have their barbed ends at the plasma membrane, the polymerization of actin becomes the driving force for membrane protrusion. In cells with sterocelia or microvilli, multiple actin filaments are held in closely spaced, parallel bundles via cross-linking proteins such as fimbrin and villin to create membrane-bound spikes which effectively increase the surface area. Dynamic filopodial extensions of the cell membrane are formed when fascin bundles actin filaments that are actively polymerizing. Actin-binding proteins such as vinculin, a-actinin, and talin link actin to focal adhesions that promote cell–substrate adhesions. There are many actin-binding proteins that control the polymerization state of actin filaments. Some proteins bind to and sequester G-actin subunits to prevent polymerization, whereas other proteins facilitate polymerization by increasing the rate of addition of actin to the barbed end of the actin filaments. There are capping proteins such as CapZ that bind to the barbed end of the actin filament and prevent actin monomer addition. Proteins such as gelsolin or cofilin sever F-action filaments and then remain bound as a cap to the barbed end to expedite depolymerization from the pointed ends of filaments. In some cases, actin polymerization can drive the movement of vesicles inside the cell and some bacterial pathogens such as Listeria and viruses such as Vaccinia hijack this process by recruiting polymerizing actin networks to drive their movements within the cytoplasm of infected cells. Infection to other cells occurs when the pathogen drives into the cell membrane of one cell and is engulfed by a neighboring cell. Myosins

Myosin was first isolated by Kuhne from skeletal muscle tissue in the nineteenth century. In addition to the striated and smooth muscle myosins already discussed which function in muscle contraction, it is now clear that there is a large superfamily of myosins that perform a variety of other functions within cells. These include functions such as phagocyotis, endocytosis, providing structural stability for stereocelia

and microvilli, and movement of intracellular cargo such as vesicles, melanosomes and mRNA. There at least 19 classes of myosins based on the sequence of the heavy chain gene, but no species contains representatives of each class. The human genome contains 39–40 possible myosin heavy chain genes representing 11 of the different classes of myosins (Figure 4). The myosins discussed in muscle cells and the nonmuscle myosin isoforms described above are all class II myosins which are also called ‘conventional’ myosins since these were the first myosins to be discovered and studied. Myosins from the other classes are commonly referred to as ‘unconventional’ myosins. Myosins are typically composed of one or two heavy chains and one to twelve associated light chain subunits. There are three major functional domains in myosins, a motor, a neck, and a tail. The motor domain, which is usually located at or near the N-terminus of the protein, is about 700 amino acids in length. It interacts with actin and contains the nucleotide-binding site. Some myosins have N-terminal extensions that range in size from a few amino acids to over 1000 amino acids in length although their functions have not been clearly elucidated. The neck domain which follows the motor is composed of an a-helical sequence of the heavy chain which interacts with lower-molecular-weight proteins termed light chains. The light chains are either calmodulin or calmodulin-family member proteins. The light chains provide rigid support for the extended heavy chain helix. There is evidence that the neck region may function as a lever arm during actin translocation. Light chains are critical for regulation of the enzymatic activity of the smooth and nonmuscle myosin II, as seen above, but may also play regulatory roles in other myosin classes as well. Together the motor domain and neck region are sometimes termed the ‘head’. The third functional domain of myosin is the tail, located at the C-terminus of the molecule. The tail is at least partially responsible for the targeting of myosin to its proper cellular localization or cargo and may be involved in the regulation of enzymatic activity. The tail regions of different myosin classes are very diverse and may contain different functional domains such as FERM (band 4.1, ezrin, radixin, moesin), PH (pleckstrin homology), MyTH4 (myosin tail homology-4), SH3 (src homology-3), or zincbinding domains. Myosin IX molecules have a functional GTPase activating (GAP) domain in their tails. The tails of many classes of myosins have coiled-coil forming domains that may allow two heavy chains to dimerize, creating molecules with two heads. Others have phosphorylation sites regulating their self-association or their association with cargo.

566 CONTRACTILE PROTEINS VII

XVI III

MYO3A

XV

O1

MYO18A

MY

MYO3B MYO18B

0

X MYO16

MYO 7A MY O 7B

XVIII

) 5B ene 1 YO og (M eud ps

15

Y0

M

Nonmuscle myosin-IIc MYH14 Smooth muscle myosin-II MYH11 Nonmuscle myosin-IIb MYH10 Nonmuscle myosin-IIa MYH 9

MYO9A

IX

MYO9B MYO1A MYO1B MYO1D MYO1G

[KIAA1000] MYH15

I

[KIAA1512] {MYH?} MYO1C

II

Cardiac-
MYH7 Cardiac- MYH6 Cmbryonic MYH3 Extraocular MYH13 IIx/d MYH1 IIb MYH4 Perinatal MYH8 lla MYH2

MYO1H MYO1E MYO1F

MYO5A

MYO5C

MYO5B

MYO6 VI MYO19 XIX

V Figure 4 Human myosin family tree. The various classes of myosin present in humans are shown. Myosin 15B is a pseudogene and is thus indicated with a dashed line.

Myosin classes Some of the interesting myosin classes that exist in vertebrates will be discussed next, starting with the class II, conventional myosins. Myosin II molecules have long C-terminal coiledcoil regions which dimerize to form two-headed structures. Filaments are formed by a self-association of the tail regions. The structure of filaments formed by different isoforms of myosin II vary in both their packing and in their size. Vertebrate striated muscle myosins form bipolar thick filaments of around 1.2 mm in length. The bipolar nature allows a single thick filament to pull actin filaments from adjacent Z-lines toward its center. Analysis of the human genome predicts 14 myosin II heavy chain genes. Eight of these are found in either cardiac or skeletal muscle tissue where they are expressed in a muscle type and, sometimes, developmentally controlled manner. For example, some myosin heavy chain genes are expressed in fast versus slow muscle or in the cardiac ventricle as opposed to the atria. Others are found in fetal muscle, but not

in adult. There are three genes for nonmuscle myosin II (also called ‘cytoplasmic’ myosins II) and one for smooth muscle myosin II. Most cells contain more than one nonmuscle myosin II isoform, but the individual functions of these myosins are still not well known. Humans have eight genes for myosin I. These proteins are characterized by a motor domain with little or no N-terminal extension. The neck region contains one or more IQ motifs. Their short tails do not have any coiled-coil forming sequences and do not appear to dimerize. The study of myosin I in various organisms has been complicated by the number of isoforms present. Some myosin I isoforms are expressed fairly ubiquitously in many tissues while others are more cell or tissue specific. For example, myosin Ie is abundant in the enterocyte brush border epithelium where it is associated with the actin bundle via its motor domain and a protein constituent of a lipid raft through its tail domain. In contrast myosin Ic is

CONTRACTILE PROTEINS 567

expressed in most tissues examined. It is found in the stereocilia of the hair cells of the inner ear and may play a role in sensory adaptation in hearing. In other cells, it likely plays a more generalized function. Other myosin I isoforms play roles in vesicle transport, endocytosis, and recycling of endosomes. The human genome has three myosin V heavy chain genes. These myosins have a long neck region with six IQ motifs that bind calmodulin and a coiledcoil segment of the tail that dimerizes to produce a two-headed structure. Myosin Va is a cargo transporting protein. Its role in transporting the pigmented melansome granules in melanocytes is well studied. It has also been implicated in the transport of vesicles in neuronal cells. The two other mammalian myosin V isoforms, Vb and Vc, also participate in intracellular vesicle trafficking. Two problems must be overcome to efficiently move cargo along cytoskeletal actin filaments in cells. First, it is necessary that the cargo remain attached to the actin filament at all times via the myosin–actin interaction. Otherwise, the cargo would rapidly diffuse away from actin filament. To overcome this, myosin V has evolved its kinetics such that a single molecule can move processively on actin filaments. In other words, the two motor domains of a single myosin V molecule are capable of taking many alternating ‘steps’ in which one of the two heads remains bound to actin while the other head is moving forward searching for a new binding site. The second problem is related to the helical structure of an Factin filament. If the myosin merely stepped from one actin monomer to the next closest actin monomer, it would have to follow the helical pitch of the actin filament which would have the effect of running the cargo into the cytoskeleton after taking a few steps. To overcome this, myosin V has evolved a long neck which allows the molecule to take 36 nm steps (the distance of about 13 actin monomers) coinciding with the half helical pitch of the actin filaments. Thus, myosin V is able to literally step across the ‘top’ on an actin filament while keeping its cargo positioned above the cytoskeleton. Myosins Va, Vb, and Vc associate either directly or indirectly with Rab proteins which are G-protein family members associated with membrane vesicles. Myosin Va has been shown to indirectly interact with Rab27a via a linker protein, termed melanophilin. Myosin Vb interacts with Rab11a on perinuclear vesicles and myosin Vc with Rab8. Most myosins move toward the barbed end of actin filaments, but myosin VI moves in the opposite direction. Mutations in the single myosin VI gene result in deafness in humans. However, the protein has a broad expression distribution in other cells and

tissues. Mice lacking myosin VI are deaf and show circling behavior, but appear to be otherwise healthy, although there are defects in membrane trafficking. Myosin VI is found on clathrin-coated vesicles in polarized cells and is thought to play a role in endocytosis. In some cells, it is also found associated with the Golgi complex and myosin VI mutant mice have smaller Golgi complexes than do wild-type mice. A single myosin X gene is found in humans. The tail contains coiled-coil, PH, FERM, and MyTH4 domains. It is widely distributed in cells and tissues, but present in low abundance mainly at the tips of filopodia. In macrophages, it localizes to areas of phagocytosis. In frog oocytes, myosin X interacts with microtubules and is involved in spindle assembly and nuclear anchoring. For more details on the other classes of myosin, the reader is directed toward the ‘Further reading’ section.

Role of Contractile Proteins in Respiratory Disease No mutations involving contractile proteins have been shown to be responsible for hereditary respiratory diseases, but they play important roles in the motility of macrophages, mast cells, and fibroblasts that lead to invasion of these cells during lung injury and disease states. The role of airway smooth muscle in the pathophysiology of asthma has been well studied, but there is considerable disagreement as to whether smooth muscles increase in size, number, or content of contractile proteins in asthma. See also: Cytoskeletal Proteins. Leukocytes: Mast Cells and Basophils; Pulmonary Macrophages. Platelets. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Signal Transduction. Smooth Muscle Cells: Airway. Vesicular Trafficking.

Further Reading Barany M (ed.) (1996) Biochemistry of Smooth Muscle Contraction. New York: Academic Press. Bresnick AR (1999) Current Opinion in Cell Biology 11: 26–33. Buss F, Luzio JP, and Kendrick-Jones J (2002) Traffic 3: 851–858. Pollard TD (2003) Nature 422: 741–745. Pollard TD and Borisy GG (2003) Cell 112: 453–465. Reck-Peterson SL, Provance DW, Mooseker MS, and Mercer JA (2000) Biochimica et Biophysica Acta 1496: 36–51. Ridley AJ, Schwartz MA, Burridge K, et al. (2003) Science 302: 1704–1709. Schiwa M (ed.) (2003) Molecular Motors. New York: Wiley-VCH. Sellers JR (1999) Myosins, 2nd edn. Oxford: Oxford University Press. Sellers JR (2000) Biochimica et Biophysica Acta 1496: 3–22. Somlyo AP and Somlyo AV (2003) Physiological Reviews 83: 1325–1358. Stephens NL (2001) Lung 179: 333–373.

568 CORTICOSTEROIDS / Glucocorticoid Receptors

CORTICOSTEROIDS Contents

Glucocorticoid Receptors Therapy

& 2006 Elsevier Ltd. All rights reserved.

led to debilitating side effects, much work has been performed in an attempt to understand the mechanism of corticosteroid action. It is hoped that by understanding how corticosteroids function at the cell and molecular level it will be possible to develop new safer drugs in the future.

Abstract

The Glucocorticoid Receptor

Corticosteroids bind to and activate a cytoplasmic glucocorticoid receptor (GR) which exists as several isoforms derived from a single gene product by alternative splicing. The activated glucocorticoid receptor translocates into the nucleus and binds to specific response elements in the promoter regions of antiinflammatory genes such as lipocortin-1 and secretory leukocyte protease inhibitor. However, the major anti-inflammatory effects of glucocorticoids appear to be due largely to interaction between the activated glucocorticoid receptor and transcription factors, notably nuclear factor kappa B (NF-kB) and activator protein-1, that mediate the expression of inflammatory genes. NF-kB switches on inflammatory genes via a process involving recruitment of transcriptional coactivator proteins and changes in chromatin modifications such as histone acetylation. The interactions between NF-kB and the glucocorticoid receptor result in differing effects on histone modifications and subsequent chromatin remodeling. GR is subjected to posttranslational modifications and these may influence hormone binding and nuclear translocation, alter glucocorticoid receptor interactions and protein half-life. Therapeutically, drugs that enhance glucocorticoid receptor nuclear translocation (long-acting b-agonists) and GR-associated histone deacetylase activity (theophylline) have been shown to be effective add-on therapies. In addition, dissociated glucocorticoids that target NF-kB preferentially have also been successful in the treatment of allergic disease in the skin.

Classically, corticosteroids exert their effects by binding to a single 777 amino acid glucocorticoid receptor (GR) that is localized to the cytoplasm of target cells. GRs are expressed in almost all cell types and their density varies from 200 to 30 000 per cell, with an affinity for cortisol of B30 nM, which falls within the normal range for plasma concentrations of free hormone. GR has several functional domains (Figure 1). The corticosteroid ligand-binding domain (LBD) is at the C-terminus of the molecule and is separated from the DNA-binding domain (DBD) by a hinge region. There is an N-terminal transactivation domain that is involved in activation of genes once binding to DNA has occurred. This region may also be involved in binding to other transcription factors. The inactive GR is part of a large protein complex (B300 kDa) that includes two subunits of the heat shock protein hsp90, which blocks the nuclear localization of GR and one molecule of the immunophilin p59 (FKBP4). In 1985, GR was the first transcription factor to be cloned and two isoforms were described: the classical 94 kDa a isoform and a C-terminal truncated isoform, GRb, which lacked the N-terminal 9b exon and consequently the LBD. GRb is localized to the nucleus and due to its inability to bind ligands it does not switch on gene expression. However, enhanced expression of GRb can have a dominant negative effect on GRa via the formation of GRa/GRb heterodimers. The physiological role of abnormal GRb expression has been suggested to be important in reducing corticosteroid responses in severe asthma but this is controversial. Interestingly, the highest expression of GRb is seen in neutrophils, which are relatively corticosteroid insensitive. The human GR gene covers a region more than 80 kb in length within chromosome 5 and contains 8

Glucocorticoid Receptors I M Adcock, R Hayashi, and K Ito, Imperial College London, London, UK G Caramori, University of Ferrara, Ferrara, Italy

Introduction Corticosteroids are vitally important steroid hormones that control a number of key physiological functions in man including glucose homeostasis, after which they are named, and the regulation of metabolism, cell survival/death, development, and neurological function. Corticosteroids are also one of the most effective anti-inflammatory agents available and are used to treat a number of chronic diseases including inflammatory bowel disease, psoriasis, rheumatoid arthritis, and asthma. Since the elucidation of their clinical effectiveness in the early 1950s and the realization that high doses of corticosteroids

CORTICOSTEROIDS / Glucocorticoid Receptors 569

1

1

2

2

3 4 5 6 78

3

4

5

9

6

7

8

1

9

2

9


34 5 6 78

9


GR


GR Transactivation/repression

Dominant negative 0

Figure 1 The GR is comprised of nine exons. Alternative splicing of exon 9 at the 5 end of the coding region leads to the formation of the classic GRa isoform and the dominant negative GRb isoform.

coding exons (exons 2–9) and alternative 50 -noncoding exon 1s. The promoter region does not contain either a TATA or a CCAAT box but is characterized by the presence of multiple GC boxes. The promoter contains DNA-binding sites for a number of transcription factors including AP-1, NF-kB, Sp1, and YY1 and also GR itself, although the regulation of GR expression in response to physiological and inflammatory stimuli is unclear. There are at least three distinct promoters for the human GR, although correlation with the rat GR suggests that more may exist. It is clear that each GR initiation site has its own promoter region and this leads to the idea that multiple independent promoter regions producing the same protein by splicing upstream of the translation initiation site may partially explain tissue-specific GR expression (Figure 2). Furthermore, a single GR mRNA species can produce up to eight functional N-terminal GR isoforms via alternative translation initiation mechanisms including leaky ribosomal scanning and ribosomal shunting. Importantly, these GR isoforms can display diverse cytoplasm-to-nucleus trafficking patterns and distinct transcriptional activities and each GR isoform regulates both a common and a unique set of genes. The levels of these GR isoforms differ significantly among tissues and it is possible that cell-specific GR isoforms can generate specificity in glucocorticoid control of transcription in different tissues.

Mechanism of GR Action Corticosteroids are thought to freely diffuse from the circulation into cells across the cell membrane and bind to cytoplasmic GR (Figure 3). Once the corticosteroid binds to GR, hsp90 dissociates, allowing the nuclear localization of the activated GR–corticosteroid complex and its binding to DNA.

GR combines with another GR to form a dimer at consensus DNA sites termed glucocorticoid response elements (GREs, GGTACAnnnTGTTCT) in the regulating regions of corticosteroid-responsive genes. This interaction allows GR to associate with a complex of DNA–protein modifying and remodeling proteins, including steroid receptor coactivator-1 (SRC-1) and cAMP response element binding protein (CREB) binding protein (CBP), which produce a DNA–protein structure that allows enhanced gene transcription. The particular ligand, the number of GREs, and the position of the GREs relative to the transcriptional start site may be important determinants of the magnitude and direction of the transcriptional response to corticosteroids.

Nuclear Localization of GR Control of nuclear protein import allows regulation of transcription factor activity and gene regulation. Nuclear importation of proteins is an active process for proteins 440 kDa that contain a nonconsensus basic targeting sequence or nuclear localization sequence (NLS). GR contains a classical basic NLS (NLS1, residing in the hinge region) and a second poorly characterized NLS (NLS2, residing across the LBD); nuclear import via NLS1 proceeds more rapidly than that seen under NLS2 control. Recent studies have shown that nuclear import of GR is mediated through its NLS and interaction with importins, with importin-a selectively binding to NLS1 and importins 7 and 8 binding to both NLS1 and NLS2. GR nuclear export is also tightly regulated; however, the role of the exportin-1 (CRM1) pathway is currently unclear. Importantly, the NLS–importin-a interaction is often influenced directly by the phosphorylation status of the imported proteins.

570 CORTICOSTEROIDS / Glucocorticoid Receptors 1

421

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1 27 86 90 98 316 331 336 A B C1 C2 C3 D1 D2 D3

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1 27 86 90 98 316 331 336 A B C1 C2 C3 D1 D2 D3

GR-A

DBD

GR
-A

DBD

LBD

Possible phosphorylation/ nitration sites

Figure 2 The GR can produce eight distinct products (A, B, C1, C2, C3, D1, D2, and D3) by use of alternative translation initiation sites corresponding to methionine residues at 1, 27, 86, 90, 98, 316, 331, and 336. These alternative proteins may be produced from both GRa and GRb transcripts. Posttranslational modification of GR particularly by phosphorylation and nitration alters GR function and contributes to the potential for diverse function in distinct tissues.

Histone Modifications and Chromatin Remodeling GR, like other transcription factors, increases gene transcription through its action on histone modifications, chromatin remodeling, and recruitment of RNA polymerase II to the site of local DNA unwinding. DNA is tightly compacted around a protein core. This chromatin structure is composed of nucleosomes, consisting of an octamer of two molecules each of core histone proteins (H2A, H2B, H3, and H4) surrounded by B146 bp DNA. Expression and repression of genes is associated with alterations in chromatin structure by enzymatic modification of core histones. Specific residues (lysines, arginines, and serines) within the N-terminal tails of core histones are capable of being posttranslationally modified by acetylation, methylation, ubiquitination, or phosphorylation, all of which have been implicated in the regulation of gene expression. The ‘histone code’ refers to these modifications, which are set and maintained by histone-modifying enzymes and contribute to coactivator recruitment and subsequent increases in transcription. Transcriptional coactivators such as CBP have intrinsic histone acetyltransferase (HAT) activity. This activity is recruited to the site of active gene transcription by the binding of transcription factors to DNA, thus suggesting an amplifying mechanism for histone acetylation. Increased gene transcription is

therefore associated with an increase in histone acetylation, whereas hypoacetylation induced by histone deacetylases (HDACs) is correlated with reduced transcription or gene silencing. Histone acetylation is an active process whereby small changes in the activity of HATs or HDACs can markedly affect the overall histone acetylase activity associated with inflammatory genes. Importantly, these changes in histone acetylation appear to be targeted toward regions of DNA associated with specific activator sites within the regulatory regions of induced inflammatory genes, although a global loosening of histone structure has also been proposed. However, this regulatory system is more complex. Under resting conditions, just under half of the potential lysine residues available for acetylation are in fact acetylated, and these residues have a rapid turnover. Upon stimulation small increases in the total number of acetylated lysines occur at specific loci. This situation suggests that even small changes above or below the resting level are enough to lead to an activated chromatin state. Acetylation of lysines has two effects: (1) a loss of the electrostatic attraction between charged histones and DNA; and (2) formation of bromodomains by the acetylated lysine residues. This allows loosening of the chromatin structure, recruitment of chromatin remodeling complexes such as switch/sucrose nonfermentable (SWI/SNF), and subsequent recruitment of further large transcriptional coactivator complexes and RNA polymerase II.

CORTICOSTEROIDS / Glucocorticoid Receptors 571 Corticosteroid

GR

Cofactors SRC-1 CBP GR GR GRE

RNAP II Ac Ac Ac Histone acetylation

SWI/SNF

SLPI, MKP-1,
-receptor, CD163 Figure 3 Mechanism of gene activation by the GR. Corticosteroids can freely diffuse across the plasma membrane where it associates with the inactive cytosolic GR. Upon ligand binding GR is activated and can translocate to the nucleus where it binds to a glucocorticoid response element (GRE) within the controlling region for glucocorticoid-responsive target genes. These GREs may be 50 or 30 to the start site for transcription. Once bound to DNA, GR recruits a complex containing basal transcription factors, coactivators such as CBP and SRC-1, chromatin modifiers such as SWI/SNF and RNA polymerase II (RNAP II), which together induce histone modifications including acetylation (Ac) and chromatin remodeling, and subsequent production of mRNAs encoding various genes including SLPI, MKP-1, CD163, and the b2-adrenoceptor.

Recruitment of Transcriptional Coactivators by GR GR interacts with CBP and other transcriptional coactivator proteins, including SRC-1 and glucocorticoid receptor interacting protein-1 (GRIP-1), that enhance local HAT activity. In addition, correct association of GR with DNA and other proteins determines the assembly of coactivator complexes on GR target promoters, resulting in differential acetylation of histones on distinct lysine residues. Steroid receptor coactivators recruited by GR can, in turn, recruit other coactivators and remodeling complexes including SWI/SNF and mediator complexes in a coordinated manner through bromodomain interactions. Histone H1 phosphorylation may also play a role in gene expression activated by the GR. Only the phosphorylated form of histone H1 can be displaced from the mouse mammary tumor virus (MMTV) promoter by the GR. Furthermore, long exposure to corticosteroids leads to H1 dephosphorylation. This

may explain the previously puzzling ‘refractory’ state of the MMTV promoter obtained on long exposure to corticosteroids. In vitro, GR has a ‘hit-and-run’ mechanism of action rather than a stable association with the GRE. GR resides on DNA for less than 10 s before being ejected and replaced by another GR. This ejection may allow binding of additional regulatory factors that enhance gene transcription, such as HAT-containing complexes, and may also play a role in feedback regulation. Interestingly, in the absence of adenosine triphosphate and chromatin remodeling factors, the GR stably interacts with the corticosteroid-responsive MMTV long terminal repeat promoter.

Gene Repression by GR In spite of the ability of corticosteroids to induce gene transcription, the major anti-inflammatory effects of corticosteroids are through repression of inflammatory and immune genes. GR binding to a more variable negative GRE (nGRE, ATYACnnTnTGATCn)

572 CORTICOSTEROIDS / Glucocorticoid Receptors

The interaction between proinflammatory transcription factors and the GR may result in differing effects on histone modifications such as acetylation/deacetylation through a number of different mechanisms including GR binding to, or recruiting, nuclear receptor co-repressors such as NCoR and HDACs or possibly by direct repression of NF-kB-associated HAT activity. Secondly, the GR dimer can bind to a GRE that overlaps the DNA-binding site for a proinflammatory transcription factor or the start site of transcription, thus blocking gene expression. Thirdly, the GR dimer can induce the expression of the NF-kB inhibitor IkB-a in certain cell types. Similarly, induction of GILZ (glucocorticoid inducible leucine zipper) can prevent AP-1 DNA binding and activity in some cells. Fourthly, corticosteroids can increase the levels of cell ribonucleases and mRNA destabilizing proteins, thereby reducing the levels of mRNA (Figure 3). Finally, GR may affect RNA polymerase II phosphorylation status (see below). As mentioned above, the context in which the GRE and kB sites are found within a promoter of specific genes may drastically affect the final effect on gene expression. For example, dexamethasone can enhance cytokine-inducible expression of TLR2 via a GR-p65 association on the promoter where the GRE overlaps with the kB site. This does not absolutely

was originally proposed as a possible mechanism for corticosteroid-mediated gene repression; however, most glucocorticoid repressible inflammatory genes do not generally possess nGREs in their promoters, suggesting that other mechanisms of inhibition are more important. The inhibitory effect of corticosteroids appears to be due largely to interaction between the activated GR and the transcription factors, such as NF-kB and AP-1, which mediate the expression of inflammatory genes. Full inflammatory gene expression probably requires that a number of transcription factors act together in a coordinated manner, and repression of a single transcription factor may only partially modify the full response. Glucocorticoids may be able to reduce inflammatory gene expression by repressing downstream targets of transcription factor activation, irrespective of the precise activated transcription factors involved. GR, acting as a monomer, binds only to specific coactivator/modulator complexes that are activated by proinflammatory transcription factors (Figure 4). The GR complex can regulate gene products in at least five other ways (Figure 4). Firstly, GR acting as a monomer can bind directly or indirectly with the transcription factors AP-1 and NF-kB, which are upregulated during inflammation, thereby inhibiting the proinflammatory effects of a variety of cytokines.

X

Nongenomic effect on membrane or kinases (ERK, Akt, PI3K)

GR GR

1

↑ IB, GILZ

6

GR GR

GRGR

5 GR GR

2

GR

/

GR

X

↓ mRNA stability

GRGR GR

3 GR

AP-1/ NF-B

GR ?

4

↓ RNA pol II phosphorylation

X RNAP II

P

Figure 4 Mechanisms of gene repression by the GR. Activated GR acting as a homodimer can bind to a GRE that overlaps the DNAbinding site for a proinflammatory transcription factor or the start site of transcription to prevent inflammatory gene expression 1 . Activated GR can repress AP-1/NF-kB-mediated gene expression by either inducing the expression of the NF-kB inhibitor IkB-a or the AP-1 inhibitor GILZ via classic gene induction mechanisms 2 . Acting as a monomer, GR can directly or indirectly suppress AP-1/NF-kB activity by 3 altering cofactor activity or by 4 dephosphorylating RNA polymerase II (RNA pol II). GR can also reduce the levels of mRNA by either inducing the dual specificity MAPK phosphatase-1 (MPK-1) that in turn regulates p38 MAPK-mediated mRNA stability or increasing the levels of cell ribonucleases and mRNA destabilizing proteins 5 . Finally, GR can induce rapid nongenomic effects either through a membrane receptor or affecting activation of kinases such as ERK, Akt, or PI3K 6 . Mechanisms that have been reported to be reduced under conditions of oxidative stress are shown by #.

CORTICOSTEROIDS / Glucocorticoid Receptors 573

require GRE and NF-kB linear association as similar effects of p65 and GR can be seen during the induction of stem cell factor (SCF) in human fibroblasts despite the GRE and kB-RE being separated by B1700 bp implying a more complex interaction between GR and NF-kB, for example, than previously thought. An alternative mechanism for GR-p65 cross-talk has been proposed by Yamamoto who clearly showed that 100 nM dexamethasone acts by repressing the phosphorylation of RNA polymerase II CTD induced by tumor necrosis factor alpha (TNF-a), thereby altering the ability of RNA polymerase II to elongate. Explanations for the differences between the models may involve concentration and timing of dexamethasone treatment of cells along with the precise inflammatory stimulus used. Evidence for cross-talk between GR and AP-1/NFkB has been described in a series of elegant experiments using mice expressing mutated GRs that are unable to dimerize and subsequently bind to DNA. In this model, Schutz and colleagues have confirmed a role for GR DNA binding as a dimer in the control of pro-opiomelanocortin expression and T-cell apoptosis but not in that of inflammatory genes regulated by AP-1 or NF-kB. This suggests that it will be possible to develop corticosteroids with a greater therapeutic window. In addition, corticosteroids may play a role in repressing the action of mitogen-activated proteinkinases (MAPKs), such as the extracellular signalregulated kinase (ERK) and c-jun N-terminal kinase (JNK). More recently, it has been shown that dexamethasone can rapidly induce the dual specificity MAPK phosphatase-1 (MKP-1) and thereby attenuate p38 MAPK activation. Rogatsky and colleagues have in turn shown reciprocal inhibition of rat GR reporter gene activity by JNKs by a direct phosphorylation of Ser-246, whereas ERK can inhibit GR action by an indirect effect, possibly through phosphorylation of a cofactor. In addition, we and others have shown that enhanced MAPK activity is associated with attenuated GR function in severe asthma.

Regulation of mRNA Stability Adenylate-uridylate-rich elements (AREs) are found within the 30 -UTR of many inflammatory genes and control the stability of mRNA. These sequences are very heterogeneous and include both AUUUA pentamers and AT-rich stretches. Binding of mRNA to ARE-binding proteins results in the formation of messenger ribonucleoprotein (mRNP) complexes, which control mRNA decay. Several ARE-binding proteins have been reported and include tristetrapolin

(TTP), which promotes mRNA decay, and HuR family members, which are associated with mRNA stability. Importantly, HuR binding to AREs is dependent upon p38 MAPK. In several cell types dexamethasone has been reported to alter the expression of either TTP and/or HuR, thereby affecting cyclooxygenase-2 (COX-2) and CC chemokine ligand 11 (CCL11) mRNA half-life possibly through induction of MKP-1. However, this does not appear to be the case for dexamethasone actions on granulocytemacrophage colony-stimulating factor mRNA stability in lung epithelial cells.

Nongenomic Actions of Glucocorticoids The traditional genomic theory of glucocorticoid action, whether directly interacting with DNA or involving cross-talk with other transcription factors, does not fully explain the rapid effects of glucocorticoids, and it is thought that some nongenomic actions are mediated by a distinct membrane receptor. Initially described in amphibians this receptor has been described in mammalian cells and has distinctive hormone-binding properties compared to the well-characterized cytoplasmic receptor and is probably linked to a number of intracellular signaling pathways acting through G-protein-coupled receptors and a number of kinase pathways including MAPKs, Akt, and PI3K. There are a number of reviews in the literature providing a summary of the evidence for the rapid effects seen and, of these, one important in respiratory disease may include changes in bronchial blood flow induced by inhaled glucocorticoids during treatment of asthmatic exacerbations. In addition, the classical receptor is associated with a number of kinases and phosphatases within the inactive GR/hsp90 complex. These enzymes are released upon hormone binding and may also account for the rapid induction of tyrosine kinases by glucocorticoids seen in some cell types.

Posttranslational Modifications of GR GR is a phosphoprotein containing numerous potential phosphorylation sites, including those for ERK, p38 MAPK, protein kinase C, and protein kinase A. Evidence obtained during the past 10 years clearly suggests that altered GR phosphorylation status can affect GR-ligand binding, hsp90 interactions, subcellular localization, nuclear-cytoplasmic shuttling, and transactivation potential, possibly through association with coactivator molecules. Ligand binding induces GR hyperphosphorylation at seven sites, which regulates transactivation and reduces nonspecific DNA binding, although this response varies

574 CORTICOSTEROIDS / Glucocorticoid Receptors

during the cell cycle, with cells being less sensitive to corticosteroids during G2/M. Thus, global changes in GR charge may affect its function as well as specific phosphorylation events. MAPK activation or overexpression can also target specific serine/threonine residues in GR, decreasing GR-mediated transactivation (Figure 2). In addition, recent evidence suggests that GR phosphorylation is involved in receptor turnover and that phosphorylation can target the receptor for hormone-mediated degradation. As such, phosphorylation-induced targeting of GR for ubiquitination and proteosomal degradation may play an important role in overall GR responsiveness. GR sumoylation appears to have the opposite effect of ubiquitination and results in increased GR activity. In addition to phosphorylation, nitrosylation of GR at an hsp90 interaction site induced by the nitric oxide (NO) donor S-nitroso-DL-penicillamine has also been shown to prevent GR dissociation from the hsp90 complex and a reduction in ligand binding. It has become clear that histones are not the only targets for histone acetylases and recent evidence has suggested that acetylation of transcription factors can modify their activity. For example, the p65 component of NF-kB can also be acetylated thus modifying its transcriptional activity. Recent evidence suggests that GR is also acetylated upon ligand binding and that deacetylation is critical for interaction with p65 at least at low dexamethasone concentrations.

GR LBD Crystal Structure The crystal structure of the GR LBD has been determined in a ternary complex with dexamethasone and a TIF2 coactivator peptide following point mutation of Phe602. The overall structure is similar to that of other nuclear hormone receptor (NHR) LBDs but contains a unique dimerization interface and a second charge clamp that may be important for cofactor selectivity. Unlike other NHR LBDs, the GR LBD also has a distinct binding pocket that may explain ligand selectivity and lead to rational-based design of selective dissociated GR agonists. Modification of the dimerization interface by mutation of I628A resulted in reduced transactivation ability without affecting the ability to repress an NF-kB-driven reporter gene. The contrasting effects of this mutant suggests that the monomer and dimer forms of GR may regulate distinct signaling pathways confirming data obtained from the GRdim(
/
) mouse. The most intriguing aspect of the ligand-binding pocket compared to other NHRs is the presence of an additional branch extending from the center. This pocket is opposite C17 of dexamethasone and it is possible to speculate that altered transactivation or

transrepression properties of GR ligands may result from glucocorticoid side chains filling this pocket. The crystal structure of GR antagonists and dissociated ligands in this model will confirm this view.

Therapeutic Implications Inhaled glucocorticoids are now used as first-line therapy for the treatment of persistent asthma in adults and children in many countries, as they are the most effective treatments for asthma currently available. However, at high doses systemic absorption of inhaled glucocorticoids may have deleterious effects, so there has been a search for safer steroids for inhalation and even for oral administration.

Effects of Long-Acting b-Agonists on GR Function Long-acting b2-adrenoceptor agonists (LABAs) and glucocorticoids are the two most effective treatments for asthma and are more potent in combination than either drug alone. Whereas glucocorticoids are used to treat airway inflammation, LABAs are used as bronchodilatory agents to bring rapid relief of airway bronchoconstriction. LABAs do not have major anti-inflammatory actions in themselves in vivo as opposed to their potent effects in vitro, suggesting that the added benefit of combination therapy probably relates to cross-talk between the two drugs. In several lung cell types salmeterol and formoterol can induce GR nuclear translocation and enhance GR– glucocorticoid response element binding in the absence of ligand. Translocation of GR by b2-agonists was less effective than that seen with dexamethasone and was protein kinase A (PKA) dependent. This study has since been confirmed in vivo, which has indicated that salmeterol and formoterol can induce GR nuclear translocation and that this may prime the receptor to be more responsive to a concomitant or subsequent challenge with glucocorticoid.

Dissociated Glucocorticoids Whilst the major anti-inflammatory effects of corticosteroids are almost certainly due to transrepression, the underlying molecular mechanisms for the side effects of glucocorticoids are complex and not fully understood. Certain side effects such as diabetes and glaucoma are due to transactivation events whilst others are due to transrepression (hypothalamic-preoptic area-adrenal (HPA) suppression). In addition, the precise molecular events underlying glucocorticoid induction of osteoporosis is unclear but probably requires both gene induction and gene repression. In

CORTICOSTEROIDS / Glucocorticoid Receptors 575

mice with GRs that do not dimerize there is no transactivation, but transrepression appears to be normal. Despite this uncertainty there has been a search for ‘dissociated’ glucocorticoids that selectively transrepress without significant transactivation, thus potentially reducing the risk of systemic side effects. Several nonsteroidal ‘selective glucocorticoid receptor agonists’ (SEGRA) have recently been reported that show dissociated properties in human cells. These are now in clinical development where they show good separation between transrepression and transactivation actions in the skin. This suggests that the development of glucocorticoids and SEGRA with a greater margin of safety is possible and may even lead to the development of oral compounds that have reduced adverse effects. Furthermore, the newer topical glucocorticoids used today, such as fluticasone, mometasone and budesonide, appear to have more potent transrepression than transactivation effects, which may account, at least in part, for their selection as potent anti-inflammatory agents. This suggests that the development of glucocorticoids with a greater margin of safety is possible and may even lead to the development of oral glucocorticoids that do not have significant adverse effects. The recent resolution of the crystal structure of the glucocorticoid receptor may help in better design of dissociated glucocorticoids.

Other Approaches to Anti-Inflammatory Therapy The elucidation of the molecular mechanisms of glucocorticoids raises the possibility that novel nonsteroidal anti-inflammatory treatments might be developed that mimic the actions of glucocorticoids on inflammatory gene regulation. Inhibition of specific HATs activated by NF-kB may prove to be useful targets, especially if they also repress the action of other proinflammatory transcription factors. Alternatively, activation of HDACs may have therapeutic potential, and theophylline has been shown to have this property, resulting in marked potentiation of the anti-inflammatory effects of glucocorticoids both in vitro and in vivo. This action of theophylline is not mediated via phosphodiesterase inhibition or adenosine receptor antagonism and, therefore, appears to be a novel action of theophylline. It may be possible to discover similar drugs that could form the basis of a new class of anti-inflammatory drugs without the side effects that limit the use of theophylline. Many of the anti-inflammatory effects of glucocorticoids appear to be mediated via inhibition of the transcriptional effects of NF-kB, and small-molecule inhibitors of kappa B kinase 2 (IKK-2), which activate NF-kB, are in development. However,

glucocorticoids have additional effects, so it is uncertain whether IKK-2 inhibitors will parallel the clinical effectiveness of glucocorticoids. They may have side effects, such as increased susceptibility to infections; however, as a corollary to this, if glucocorticoids were discovered today, it is unlikely that they would be used in humans because of the low therapeutic ratio and their side effect profile (Figure 4).

Conclusion Advances in delineating the fundamental mechanisms of gene transcription, especially recruitment of histone-modifying cofactors, have resulted in better understanding of the molecular mechanisms whereby glucocorticoids suppress inflammation. The challenge is to see if these mechanisms hold true in primary cells in vivo. This will undoubtedly lead to the development of drugs that target novel aspects of GR function and potentially restore glucocorticoid sensitivity to diseases that are unresponsive to current therapeutic strategies.

Acknowledgments We would like to thank other members of the Cell & Molecular Biology Group for their helpful discussions. Work in our groups is supported by Associazione per la Ricerca e la Cura dell’Asma (ARCA, Padua, Italy), Asthma UK, The British Lung Foundation, The Clinical Research Committee (Brompton Hospital), The Medical Research Council (UK), The National Institutes of Health (USA), The Wellcome Trust, AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline (UK), Mitsubishi Pharma (Japan), Novartis, and Pfizer. See also: Allergy: Overview. Corticosteroids: Therapy. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia; Hypersensitivity Pneumonitis. Laryngitis and Pharyngitis. Lipid Mediators: Prostanoids. Transcription Factors: Overview. Vasculitis: Microscopic Polyangiitis.

Further Reading Barnes PJ (2002) Scientific rationale for inhaled combination therapy with long-acting beta2-agonists and corticosteroids. European Respiratory Journal 19: 182–191. Barnes PJ and Adcock IM (2003) How do corticosteroids work in asthma? Annals of Internal Medicine 139: 359–370. Bledsoe RK, Montana VG, Stanley TB, et al. (2002) Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell 110: 93–105. Dean JL, Sully G, Clark AR, and Saklatvala J (2004) The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilisation. Cell Signal 16: 1113–1121.

576 CORTICOSTEROIDS / Therapy De Ruijter AJ, Van Gennip AH, Caron HN, Kemp S, and Van Kuilenburg AB (2003) Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochemical Journal 370: 737–749. Hermanson O, Glass CK, and Rosenfeld MG (2002) Nuclear receptor coregulators: multiple modes of modification. Trends in Endocrinology and Metabolism 13: 55–60. McNally JG, Muller WG, Walker D, Wolford R, and Hager GL (2000) The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 287: 1262–1265. Norman AW, Mizwicki MJ, and Norman DP (2004) Steroid– hormone rapid actions, membrane receptors and a conformational ensemble model. Nature Reviews: Drug Discovery 3: 27–41. Reichardt HM, Kaestner KH, Tuckermann J, et al. (1998) DNA binding of the glucocorticoid receptor is not essential for survival. Cell 93: 531–541. Schacke H, Schottelius A, Docke WD, et al. (2004) Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects. Proceedings of the National Academy of Sciences, USA 101: 227–232. Urnov FD and Wolffe AP (2001) Chromatin remodeling and transcriptional activation: the cast (in order of appearance). Oncogene 20: 2991–3006. Yudt MR and Cidlowski JA (2002) The glucocorticoid receptor: coding a diversity of proteins and responses through a single gene. Molecular Endocrinology 16: 1719–1726.

Therapy P J Barnes, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Corticosteroids are by far the most effective anti-inflammatory treatment for asthma and switch off multiple activated inflammatory genes through reversing histone acetylation. They suppress inflammation in the airways and reduce airway hyperresponsiveness. Inhaled corticosteroids are now first-line therapy for all patients with persistent asthma, controlling asthma symptoms, and preventing exacerbations. Long-acting inhaled b2-agonists improve control and are commonly given as combination inhalers with the corticosteroid, which improves compliance and controls asthma at a lower dose of corticosteroid. Oral steroids have numerous metabolic and endocrine side effects, and the lowest dose needed to control the disease should be used. Inhaled corticosteroids, which are absorbed from the lungs into the systemic circulation, have negligible systemic side effects at the doses most patients require. In contrast, inflammation in chronic obstructive pulmonary disease is corticosteroid resistant due to inactivation of histone deacetylase, and steroids are only indicated in reducing exacerbations in patients with severe disease. Systemic corticosteroids are also used as antiinflammatory treatments in pulmonary eosinophilic syndromes and pulmonary vasculitides and in interstitial lung disease.

Corticosteroids are used in the treatment of several lung diseases. They were introduced for the treatment of asthma soon after their discovery in the

1950s and remain the most effective therapy available for this condition. The introduction of inhaled corticosteroids, initially as a way of reducing the requirement for oral steroids, has revolutionized the treatment of chronic asthma, and they are now used as first-line therapy for all patients with persistent symptoms. In contrast, inhaled corticosteroids are much less effective in chronic obstructive pulmonary disease (COPD) and should only be considered in patients with severe disease. Oral steroids are indicated in the treatment of several other pulmonary diseases, such as sarcoidosis, interstitial lung diseases, and pulmonary eosinophilic syndromes.

Chemical Structures The adrenal cortex secretes cortisol (hydrocortisone) and, by modification of its structure, it was possible to develop derivatives, such as prednisolone and dexamethasone, with enhanced corticosteroid effects but with reduced minerallocorticoid activity. These derivatives with potent glucocorticoid actions were effective in asthma when given systemically but had no antiasthmatic activity when given by inhalation. Further substitution in the 17a ester position resulted in steroids with high topical activity, such as beclomethasone dipropionate (BDP), triamcinolone, flunisolide, budesonide, and fluticasone propionate, which are potent in the skin (dermal blanching test) and were later found to have significant antiasthma effects when given by inhalation (Figure 1).

Mode of Action Corticosteroids enter target cells and bind to glucocorticoid receptors in the cytoplasm. The steroid–receptor complex is transported to the nucleus, where it binds to specific sequences on the upstream regulatory element of certain target genes, resulting in increased (or rarely decreased) transcription of the gene that leads to increased (or decreased) protein synthesis. Glucocorticoid receptors may also interact with transcription factors and coactivator molecules in the nucleus and thereby influence the synthesis of certain proteins independently of any direct interaction with DNA. The repression of transcription factors, such as activator protein-1 and nuclear factor kappa B (NF-kB), is likely to account for many of the anti-inflammatory effects of steroids in asthma. In particular, corticosteroids reverse the activating effect of these proinflammatory transcription factors on histone acetylation by recruiting histone deacetylase-2 (HDAC2), which reverses the acetylation of activated inflammatory genes (Figure 2).

CORTICOSTEROIDS / Therapy 577 CH2 OH HO

H3C C O 17

CH2OCOC2H5

CH2 OH

C O

OH

OCOC2H5 CH3

HO

H3C

C O O O

HO

C

H CH2CH2CH3

Cl O

O

Hydrocortisone

O Budesonide

Beclomethasone dipropionate

SCH2F

CH2 OH

C O

C O

HO

OCOC2H5 CH3

O O

HO

CH2 OH C

CH3 CH3

F

C O HO

O O

C

CH3 CH3

F O

O

O

F

F

Fluticasone propionate

Flunisolide

Triamcinolone acetonide

Figure 1 Chemical structures of inhaled corticosteroids.

Inflammatory stimuli e.g., IL-1, TNF- 

Corticosteroids (low dose)

IKK-2 p65

GR

NF-
B p50

AF

pC
B Inflammatory genes cytokines, chemokines adhesion molecules inflammatory receptors, enzymes, proteins

CBP

p65

HDAC2

p50

HAT

Acetylation

Deacetylation

↑ Gene transcription

Gene repression

Figure 2 Corticosteroids suppression of activated inflammatory genes. Inflammatory genes are activated by inflammatory stimuli, such as interleukin-1b (IL-1b) or tumor necrosis factor alpha (TNF-a), resulting in activation of IKK-2 (inhibitor of IkB kinase-2), which activates the transcription factor NF-kB. A dimer of p50 and p65 NF-kB proteins translocates to the nucleus and binds to specific kB recognition sites and also to coactivators, such as CREB-binding protein (CBP) or p300/CBP-activating factor (pCAF), which have intrinsic histone acetyltransferase (HAT) activity. This results in acetylation of core histone H4, resulting in increased expression of genes encoding multiple inflammatory proteins. Glucocorticoid receptors (GR) after activation by corticosteroids translocate to the nucleus and bind to coactivators to inhibit HAT activity directly and recruiting HDAC2, which reverses histone acetylation leading to suppression of these activated inflammatory genes.

The mechanisms of the action of corticosteroids in asthma are poorly understood but are most likely related to their anti-inflammatory properties. Corticosteroids have widespread effects on gene transcription,

increasing the transcription of anti-inflammatory genes and suppressing transcription of inflammatory genes (Table 1). Steroids have inhibitory effects on many inflammatory and structural cells that are

578 CORTICOSTEROIDS / Therapy

activated in asthma (Figure 3). Inhaled steroids reduce the number and activation of inflammatory cells in the epithelium and submucosa, with a healing of the damaged epithelium. Steroids potently inhibit the formation of proinflammatory cytokines and decrease eosinophil survival by inducing apoptosis. They also inhibit the expression of multiple inflammatory genes in airway epithelial cells; indeed, this may be the most important action of inhaled corticosteroids in suppressing asthmatic inflammation (Figure 4). Inhaled steroids reduce airway hyperresponsiveness, but this effect may take several weeks or months and

Table 1 Effect of corticosteroids on gene transcription Increased transcription Lipocortin-1 b2-Adrenoceptor Secretory leukocyte inhibitory protein IkB-a (inhibitor of NF-kB) Anti-inflammatory or inhibitory cytokines IL-10, IL-12, IL-1 receptor antagonist Mitogen-activated protein kinase phosphatase-1 (inhibits MAP kinase pathways) Decreased transcription Inflammatory cytokines IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, IL-13, IL-15, TNF-a, GM-CSF, SCF Chemokines IL-8, RANTES, MIP-1a, eotaxin Inducible nitric oxide synthase (iNOS) Inducible cyclooxygenase (COX-2) Inducible phospholipase A2 (cPLA2) Endothelin-1 NK1 receptors Adhesion molecules (ICAM-1 and VCAM-1)

Inflammatory cells

presumably reflects the slow healing of the damaged inflamed airway. It is important to recognize that steroids suppress inflammation in the airways but do not cure the underlying disease. When steroids are withdrawn, there is a recurrence of the same degree of airway hyperresponsiveness, although in patients with mild asthma it may take several months to return. Steroids increase the expression of b2-adrenoceptors in the lung, and this may prevent the loss of nonbronchodilator response to b2-agonists that occurs after regular b2-agonist use.

Pharmacokinetics Prednisolone is readily and consistently absorbed after oral administration with little interindividual variation. Enteric coatings to reduce the incidence of dyspepsia delay absorption but not the total amount of drug absorbed. Prednisolone is metabolized in the liver, and drugs such as rifampicin, phenobarbitone, or phenytoin, which induce P-450 enzymes, lower the plasma half-life of prednisolone. The plasma half-life is 2 or 3 h, although its biological half-life is approximately 24 h, so it is suitable for daily dosing. There is no evidence that previous exposure to steroids changes their subsequent metabolism. Prednisolone is approximately 92% protein bound, the majority to a specific protein transcortin and the remainder to albumin; it is the unbound fraction that is biologically active. The pharmacokinetics of inhaled corticosteroids is important in relation to systemic effects. The fraction of steroid that is inhaled into the lungs acts locally on

Structural cells

Eosinophil Numbers ↓ (apoptosis)

Epithelial cell ↓

T lymphocyte ↓ Cytokines

Cytokines mediators

Endothelial cell Mast cell

↓ Numbers

↓ Leak

Corticosteroids

Airway smooth muscle ↑ 2-Receptors

Macrophage ↓ Cytokines

↓ Cytokines Mucus gland

Dendritic cell ↓ Numbers Figure 3 Effect of corticosteroids on inflammatory and structural cells in the airways.

Mucus ↓ secretion

CORTICOSTEROIDS / Therapy 579

the airway mucosa but may be absorbed from the airway and alveolar surface. This fraction therefore reaches the systemic circulation. The fraction of inhaled steroid that is deposited in the oropharynx is swallowed and absorbed from the gut. The absorbed fraction may be metabolized in the liver before reaching the systemic circulation (first-pass metabolism) (Figure 5). Budesonide and fluticasone propionate have a greater first-pass metabolism than BDP and are therefore less likely to produce systemic effects at high inhaled doses. The use of a large-volume spacer chamber reduces oropharyngeal deposition and therefore reduces systemic absorption of corticosteroids, although this effect is minimal in corticosteroids with a high first-pass metabolism. Inhaled corticosteroids

Epithelial cells Cytokines IL-1 IL-6 GM-CSF RANTES Eotaxin MIP-1

Enzymes iNOS COX-2 cPLA2

Peptides ET-1

Adhesion molecules ICAM-1

Mouth rinsing and discarding the rinse has a similar effect, and this procedure should be used with highdose dry powder steroid inhalers since spacer chambers cannot be used with these devices.

Role of Corticosteroids in Respiratory Medicine Acute Severe Asthma

Although the value of corticosteroids in acute severe asthma has been questioned, others have found that they speed the resolution of attacks. Hydrocortisone is given intravenously in acute severe asthma. There is no apparent advantage in giving very high doses of intravenous steroids (e.g., 1 g methylprednisolone). Intravenous steroids are indicated in acute asthma if lung function is o30% predicted and for patients in whom there is no significant improvement with nebulized b2-agonist. Oral prednisolone (40–60 mg) has a similar effect as intravenous hydrocortisone and is easier to administer. Asthma

↓ Inflammation

Figure 4 Inhaled corticosteroids may inhibit the transcription of several ‘inflammatory’ genes in airway epithelial cells and thus reduce inflammation in the airway wall. NF-kB, nuclear factor kB; AP-1, activator protein-1; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-1, interleukin-1; iNOS, inducible nitric oxide synthase; NO, nitric oxide; COX-2, inducible cyclooxygenase; cPLA2, cytoplasmic phospholipase A2; PG, prostaglandin; ET, endothelin; ICAM, intercellular adhesion molecule.

Inhaled steroids are recommended as first-line therapy for all patients with persistent asthma. Inhaled steroids should be started in any patient who needs to use a b2-agonist inhaler for symptom control more than three times a week. They are effective in mild, moderate, and severe asthma in children as well as adults. Although it was recommended that inhaled corticosteroids should be initiated at a relatively high dose and then the dose reduced once control was achieved, there is no evidence that this is more effective than starting with the maintenance dose. Dose–response

MDI ~10–20% inhaled Mouth and pharynx Lungs Systemic circulation

~80–90% swallowed (↓ spacer/mouthwash) Absorption from GI tract Liver GI tract

Inactivation in liver 'first pass'

Systemic side effects

Figure 5 Pharmacokinetics of inhaled corticosteroids. GI, gastrointestinal; MDI, metered dose inhaler.

580 CORTICOSTEROIDS / Therapy

studies for inhaled corticosteroids are relatively flat, with most of the benefit derived from doses o400 mg BDP or equivalent. Oral steroids are reserved for patients who cannot be controlled on other therapy, with the dose being titrated to the lowest dose that provides acceptable control of symptoms. Short courses of oral steroids (30–40 mg prednisolone daily for 1 or 2 weeks) are indicated for exacerbations of asthma, and the dose may be tailed off over 1 week once the exacerbation is resolved; although the tail-off period is not strictly necessary, patients find it reassuring. For most patients, inhaled steroids should be used twice daily, which improves compliance once control of asthma has been achieved (which may require 4-times daily dosing initially or a course of oral steroids if symptoms are severe). If a dose of more than 800 mg via a metered dose inhaler is used daily, a spacer device should be used because this reduces the risk of orpharyngeal side effects. Inhaled steroids may be used in children in the same way as in adults, and at doses of 400 mg daily or less there is no evidence of growth suppression. The dose of inhaled corticosteroid should be the minimal dose that controls asthma, and once control is achieved the dose should be slowly reduced. There is convincing evidence that addition of an inhaled long-acting b2-agonist (salmeterol or formoterol) improves asthma control and compliance with therapy, leading to the widespread use of fixed combination inhalers. Combination inhalers provide better control of asthma at lower doses of inhaled corticosteroids. COPD

COPD patients occasionally respond to steroids, and these patients are likely to also have asthma. Steroids have no objective short-term benefit of airway function in patients with true COPD, although they may often produce subjective benefit because of their euphoric effect. Corticosteroids do not appear to have any significant anti-inflammatory effect in COPD, and there appears to be an active resistance mechanism that may be explained by impaired activity of HDAC2. Inhaled corticosteroids have no effect on the progression of COPD, even when given to patients with presymptomatic disease. Inhaled corticosteroids reduce the number of exacerbations in patients with severe COPD (FEV1 o50% predicted) who have frequent exacerbations, and they are recommended in these patients. Oral corticosteroids are used to treat acute exacerbations of COPD and reduce the rate of recovery, although the effect is small.

Other Diseases

Systemic corticosteroids are indicated in selected patients with severe interstitial lung disease, sarcoidosis, and pulmonary vasculitis. They are not effective in bronchiectasis or cystic fibrosis, although inhaled corticosteroids are commonly used in these diseases.

Adverse Effects and Contraindications Corticosteroids inhibit adrenocorticotrophic hormone (ACTH) and cortisol secretion by a negative feedback effect on the pituitary gland. Hypothalamopituitary-adrenal (HPA) axis suppression is dependent on dose and usually only occurs when a dose of prednisolone 47.5–10 mg daily is used. Significant suppression after short courses of steroid therapy is not usually a problem, but prolonged suppression may occur after several months or years. Steroid doses after prolonged oral therapy must therefore be reduced slowly. Symptoms of ‘steroid withdrawal syndrome’ include lassitude, musculoskeletal pains, and, occasionally, fever. HPA suppression with inhaled steroids is seen only when the daily inhaled dose exceeds 2000 mg BDP or equivalent daily. Side effects of long-term oral corticosteroid therapy are well described and include fluid retention, increased appetite, weight gain, osteoporosis, capillary fragility, hypertension, peptic ulceration, diabetes, cataracts, and psychosis. Their frequency tends to increase with age. The incidence of systemic side effects after inhaled steroids is an important consideration (Table 2). Initial studies suggested that adrenal suppression only occurred when inhaled doses of more than 1500–2000 mg daily were used. More sensitive measurements of systemic effects include indices of bone metabolism, such as serum osteocalcin and urinary pyridinium cross-links, and, in children, knemometry, which may be increased with Table 2 Side effects of inhaled corticosteroids Local side effects Dysphonia Oropharyngeal candidiasis Cough Systemic side effects Adrenal suppression and insufficiency Growth suppression Bruising Osteoporosis Cataracts Glaucoma Metabolic abnormalities (glucose, insulin, and triglycerides) Psychiatric disturbances (euphoria and depression)

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inhaled doses as low as 400 mg in some patients.The clinical relevance of these measurements is not clear, however. Nevertheless, it is important to reduce the likelihood of systemic effects by using the lowest dose of inhaled steroid needed to control the asthma, by using add-on therapies such as long-acting b2-agonists, and by using a large-volume spacer to reduce oropharyngeal deposition. Several systemic effects of inhaled steroids have been described and include dermal thinning and skin capillary fragility, which is relatively common in elderly patients after high-dose inhaled steroids. Other side effects, such as cataract formation and osteoporosis, are reported, but often in patients who are also receiving courses of oral steroids. There has been particular concern about the use of inhaled steroids in children because of growth suppression. Most studies have been reassuring in that doses of 400 mg or less have not been associated with impaired growth, and there may even be a growth spurt as asthma is better controlled. There is evidence that use of high-dose inhaled corticosteroids is associated with cataract and glaucoma, but it is difficult to dissociate the effects of inhaled corticosteroids from the effects of courses of oral steroids that these patients usually require. Inhaled steroids may have local side effects due to the deposition of inhaled steroid in the oropharynx. The most common problem is hoarseness and weakness of the voice (dysphonia), which is due to laryngeal deposition and may be due to atrophy of the vocal cords. Oropharyngeal candidiasis occurs in 5% of patients. The incidence of local side effects may be related to the local concentrations of steroid deposited and may be reduced by the use of largevolume spacers, which markedly reduce oropharyngeal deposition. Local side effects are also less likely when inhaled steroids are used twice daily rather than four times daily. There is no evidence of atrophy of the lining of the airway or of an increase in lung infections (including tuberculosis) after inhaled steroids. It is difficult to extrapolate systemic side effects of corticosteroids from studies in normal subjects. In asthmatic patients, systemic absorption from the lung is reduced, presumably because of the reduced and more central deposit of the inhaled drug,

Cough

particularly in more severe cases. Another important issue is the distribution of inhaled corticosteroids in patients with asthma since most of the drug deposits in larger airways. Because inflammation is also found in small airways, particularly in severe cases, the inhaled corticosteroids may not adequately suppress inflammation in these airways. See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Corticosteroids: Glucocorticoid Receptors. Gene Regulation. Systemic Disease: Sarcoidosis.

Further Reading Adcock IM and Lane SJ (2003) Corticosteroid-insensitive asthma: molecular mechanisms. Journal of Endocrinology 178: 347– 355. Allen DB (2004) Systemic effects of inhaled corticosteroids in children. Current Opinion in Pediatrics 16: 440–444. Barnes PJ (2004) Corticosteroid resistance in airway disease. Proceedings of the American Thoracic Society 1: 264–268. Barnes PJ and Adcock IM (2003) How do corticosteroids work in asthma? Annals of Internal Medicine 139: 359–370. Barnes PJ, Ito K, and Adcock IM (2004) A mechanism of corticosteroid resistance in COPD: inactivation of histone deacetylase. Lancet 363: 731–733. Barnes PJ, Pedersen S, and Busse WW (1998) Efficacy and safety of inhaled corticosteroids: an update. American Journal of Respiratory and Critical Care Medicine 157: S1–S53. Efthimou J and Barnes PJ (1998) Effect of inhaled corticosteroids on bone and growth. European Respiratory Journal 11: 1167– 1177. Kankaanranta H, Lahdensuo A, Moilanen E, and Barnes PJ (2004) Add-on therapy options in asthma not adequately controlled by inhaled corticosteroids: a comprehensive review. Respiratory Research 5: 17. Leone FT, Fish JE, Szefler SJ, and West SL (2003) Systematic review of the evidence regarding potential complications of inhaled corticosteroid use in asthma. Chest 124: 2329–2340. Leung DY and Bloom JW (2003) Update on glucocorticoid action and resistance. Journal of Allergy and Clinical Immunology 111: 3–22. Lipworth BJ (1999) Systemic adverse effects of inhaled corticosteroid therapy: a systematic review and meta-analysis. Archives of Internal Medicine 159: 941–955. Pedersen S (2001) Do inhaled corticosteroids inhibit growth in children? American Journal of Respiratory and Critical Care Medicine 164: 521–535. Rowe BH, Edmonds ML, Spooner CH, Diner B, and Camargo CA Jr (2004) Corticosteroid therapy for acute asthma. Respiratory Medicine 98: 275–284.

see Symptoms of Respiratory Disease: Cough and Other Symptoms.

582 CROUP

CROUP G Geelhoed, Princess Margaret Hospital, Perth, WA, Australia & 2006 Elsevier Ltd. All rights reserved.

in children under 8 years of age. Any swelling in this area due to inflammation results in significant impingement on the airway. The younger the child is, the greater the need to monitor them closely.

Abstract Croup is a common childhood problem with a peak incidence of 60 per 1000 child years in those aged between 1 and 2 years. All children who present to emergency departments with croup should be treated with steroids, oral dexamethasone 0.15 mg kg 1 or prednisolone 1 mg kg 1 being the drugs of choice. A compromised but functioning airway should never be made worse by upsetting the child. Children who require epinephrine may be sent home safely provided they have also received steroids and have improved sufficiently to have no stridor at rest over a number of hours.

Introduction The term croup describes an acute clinical syndrome of hoarse voice, barking cough, and stridor; it is usually seen in young children. Croup results from swelling of the upper airway, in and around the larynx, usually as a result of a viral infection. Parainfluenza virus type 1 accounts for around half the cases during winter, and parainfluenza type 2, influenza type A, adenoviruses, respiratory syncytial virus, enteroviruses, and possibly mycoplasma pneumonia cause most of the other cases. Croup is a common childhood problem with a peak incidence of 60 per 1000 child years in those aged between 1 and 2 years, although it may be seen up to the teenage years. As such, it is by far the most common cause of acute upper airways obstruction likely to present to emergency departments.

Pathogenesis/Pathology Unless otherwise qualified, the term ‘croup’ in this article refers to viral laryngotracheobronchitis. In viral laryngotracheobronchitis the causative virus is transmitted via the respiratory route with the port of entry being the nose and nasopharynx. Viral replication occurs, and as the infection spreads, the walls of the larynx and trachea become edematous with a fibrinous exudate partially occluding the lumen of the trachea. In addition to luminal narrowing, edema of the vocal cords and subglottic larynx leads to stridor, hoarseness, and a characteristic bark-like cough. Distress caused by obstruction tends to be most marked in younger children due to the small size of their larynx, the presence of loose submucous tissues, and the tight encirclement of the subglottic area by the cricoid cartilage. This is the narrowest airway point

History The typical presentation of croup is in a preschool child with a history of a recently acquired upper respiratory tract infection. The child develops a barking or seal-like cough, a hoarse voice, and, if obstruction is severe enough, stridor. If stridor is inspiratory this indicates obstruction at the laryngeal level or above while expiratory stridor or biphasic stridor indicates problems in and around the trachea and more severe obstruction. Less commonly, older children may present with recurrent croup with no viral prodrome. They and their families tend to be atopic and suffer from asthma more than the general population. They should, however, be treated in the same manner as those with ‘viral’ croup. In the smaller child especially, problems with feeding, swallowing difficulties, and whether the child has been cyanosed should be ascertained. It is important to know whether or not the child has had croup in the past and specifically whether the child has had mild stridor in between acute attacks. This is important as any child who has a pre-existing narrowing of the airway is much more likely to proceed to dangerous obstruction with an acute obstruction superimposed on it. Immunization history is important to obtain to exclude the diagnosis of diphtheria, which is very rare, and to check whether the child has been given the Hib vaccine should epiglottitis be suspected.

Examination Most children with croup are not distressed and have only a barking cough with no stridor at rest or stridor that is audible only with physical activity. Signs due to viral illness such as mild fever and nasal discharge are often present. In more severe cases, the child may have a more pronounced stridor at rest. If obstruction progresses, the child may exhibit increasing substernal, intercostal, and subcostal retractions. A worrying sign is that of altered consciousness reflected as anxiety, restlessness, and obvious fatigue. Decreased air entry and respiratory effort, extreme pallor, and cyanosis require immediate intervention. Patients with mild croup should have their throats examined but

CROUP 583

this should be deferred in more severe cases. A compromised but functioning airway should never be made worse or compromised by upsetting the child. The child’s preferred position may also give clues as to the severity of obstruction and to a diagnosis other than croup. Hyperextension or other abnormal positioning of the neck may suggest epiglottitis or a retropharyngeal abscess.

Diagnostic Tests Oximetry is of limited value as children may maintain oxygen saturations in the high nineties even when badly obstructed. In difficult diagnostic cases, a lateral soft tissue X-ray of the neck may help; however, the possible benefits should be weighed against the risks of moving or disturbing the child if obstruction is more than mild and expert advice should be sought. Croup, however, is usually an easy ‘spot diagnosis’ requiring no diagnostic tests.

Differential Diagnosis Although rare, it is important to establish that other more sinister causes of acute upper respiratory tract obstruction masquerading as croup are not present. Especially in the younger child one should inquire regarding long-term symptoms preceding the present episode, such as low-grade stridor. This might suggest underlying congenital airway or a vascular anomaly (e.g., tracheomalacia, congenital subglottic stenosis, congenital bilateral cord paralysis, laryngeal web, or vascular ring compression of the trachea). One should also inquire as to possible trauma, toxic ingestions, dysphagia, and drooling. Drooling may suggest epiglottitis, peritonsil abscess, or foreign body in the airway or esophagus. Classic croup and epiglottitis are hard to confuse as the latter usually presents as a pale, toxic, drooling child with a short history who does not cough much. Early on, however, the distinction may be more difficult to make. A child with severe croup with a high fever who does not respond to epinephrine and steroids may have tracheitis and will need a more aggressive approach. The possibility of a foreign body should be kept in mind for children who do not respond to treatment or have a prolonged course. While usually parents will volunteer a history of an acute obstruction or a sudden coughing fit the history may not always be known to them. A definitive diagnosis may be made by directly viewing the upper airway but this should only be done by an experienced pediatric anaesthesiologist, intensivist, or emergency physician in an appropriate clinical setting.

Treatment and Disposition All children who present to emergency departments with croup should be treated with steroids. Prior to the regular use of steroids a general rule of thumb was to admit children with stridor at rest to hospital for observation while allowing those with occasional stridor and barking cough only to be managed at home. As many children will improve within a few hours of taking steroids they may be discharged home after a short stay in an emergency department or an observation ward. Other factors such as the distance of home from medical care, the availability of transport, the child’s past history with regard to severe airway obstruction, and parental concern and attitude all need to be taken into account when making the decision to admit. Oral dexamethasone in a one-off dose of 0.15 mg kg 1 (or an equivalent dose of prednisolone of 0.75 mg kg 1) is recommended. Most children with croup will require only one dose but if croup (as opposed to viral) symptoms persist, a further dose may be given 24 h later. It is often more convenient to use prednisolone (rounded off to a simple 1 mg kg 1) in the community as it is more readily available. Unpublished work suggests that children treated with prednisolone may present more commonly than those treated with dexamethasone and would benefit from a second dose. Steroids may be administered intramuscularly or intravenously (in injectable form) in the rare case of severe obstruction where there is concern that the child may aspirate given their respiratory difficulties. Oral dexamethasone has been found to be as effective as inhaled steroids such as budesonide and to work as fast at a fraction of the cost. Combining dexamethasone and budesonide is no more effective than dexamethasone alone. There is no place for antibiotics in a typical case of croup. The effectiveness of ‘steam’ or humidified air is largely unproven despite its once common usage. Where obstruction is judged to be severe, the use of nebulized epinephrine should be considered. It is generally considered that epinephrine does not change the natural history of croup, such as length of stay in hospital or need to intubate, due to its short-lasting effects. However, it will buy time while waiting for steroids to take effect or waiting for an anaesthesiologist to arrive in a worst case scenario. Five milliliters of 1 in a 1000 epinephrine nebulized with oxygen can be used for all children and may be repeated after 10 min if needed. While in the past it was recommended that any child who received epinephrine for croup should be admitted, a number of studies have now shown that children may be sent home safely provided they have also received steroids

584 CRYOGLOBULINEMIA

and have improved sufficiently to have no stridor at rest over a number of hours. The universal use of steroids for croup in emergency departments has resulted in a fall in the relapse rate of those sent home, a fall in the average length of stay in hospital, and a dramatic reduction in the number of children needing intensive care and intubation.

Prognosis Most children with croup have mild symptoms and do not need hospitalization, and will recover within a few days. Their symptoms will be shortened even further with the use of steroids. Despite the substantial impact of steroids an occasional child will still follow a prolonged course with cough and marked stridor for many days. While other diagnoses such as foreign body need to be considered, most children will settle with time.

Prevention For most children, croup is a one-off episode and well tolerated especially if steroids are used. Children who suffer repeated episodes of recurrent croup as described above may benefit from steroid use at home at the first hint of croup symptoms. Although no trials have evaluated this approach, anecdotal evidence suggests this practice appears to have benefit.

Controversies/Future Research Although once controversial, use of steroids is now generally accepted for all children who present to emergency departments with croup. While it is clear that both prednisolone and dexamethasone are effective in the treatment of croup, direct comparisons have not been published as yet. Unpublished work suggests the shorter half-life of prednisolone may result in a greater number of children returning to medical care. While a once-only dose of dexamethasone is sufficient for the vast majority of children

with croup, a second dose of prednisolone 24 h later may be needed in some cases. See also: Corticosteroids: Therapy. Laryngitis and Pharyngitis. Lung Anatomy (Including the Aging Lung). Lung Development: Congenital Parenchymal Disorders. Pediatric Pulmonary Diseases. Signs of Respiratory Disease: Breathing Patterns; General Examination. Upper Airway Obstruction. Vaccinations: Bacterial, for Pneumonia.

Further Reading Bouchier D, Dawson KP, and Fergusson DM (1984) Humidification in viral croup: a controlled trial. Australian Paediatric Journal 20: 289–291. Denny FW and Clyde WA Jr (1986) Acute lower respiratory tract infections in nonhospitalized children. Journal of Pediatrics 108: 635–645. Geelhoed GC (1996) Sixteen years of croup in a Western Australian Teaching Hospital: the impact of routine steroid treatment. Annals of Emergency Medicine 621–626. Geelhoed GC (1997) Croup. Pediatric Pulmonology 23(5): 370– 374. Geelhoed GC and Macdonald WGB (1995) Oral dexamethasone in the treatment of croup: 0.15 mg/kg is as effective as 0.3 mg/kg or 0.6 mg/kg. Pediatric Pulmonology 20: 362–367. Geelhoed GC, Turner J, and Macdonald WB (1996) Efficacy of a small single dose of oral dexamethasone for outpatient croup: a double blind placebo controlled clinical trial. British Medical Journal 313(7050): 140–142. Kelley PB and Simon JE (1992) Racemic epinephrine use in croup and disposition. American Journal of Emergency Medicine 10(3): 181–183. Klassen TP, Craig WR, Moher D, et al. (1998) Nebulized budesonide and oral dexamethasone for treatment of croup: a randomized controlled trial. JAMA 279(20): 1629–1632. Neto GM, Kentab O, Klassen TP, and Osmond MH (2002) A randomized controlled trial of mist in the acute treatment of moderate croup. Academic Emergency Medicine 9: 873–879. Prendergast M, Jones JS, and Hartman D (1994) Racemic epinephrine in the treatment of laryngotracheitis: can we identify children for outpatient therapy. American Journal of Emergency Medicine 12(6): 613–616. Stoney PJ and Chakrabarti MK (1991) Experience of pulse oximetry in children presenting with croup. Journal of Laryngology and Otology 105: 295–298.

CRYOGLOBULINEMIA A Pesci, Universita` degli Studi di Parma, Parma, Italy P Manganelli, Azienda Ospedaliero – Universitaria di Parma, Parma, Italy & 2006 Elsevier Ltd. All rights reserved.

Abstract Mixed cryoglobulinemia is a disease characterized by the clinical triad of palpable purpura, arthralgia, and weakness. It is

associated with the presence of circulating cryoglobulins. It may be secondary to infectious, neoplastic, and autoimmune diseases. A striking association between hepatitis C virus (HCV) infection and mixed cryoglobulinemia has been documented in the majority of patients (75–95%). In mixed cryoglobulinemia, mild to moderate lung involvement may be shown, but severe lung involvement is very rare. Chest X-rays and high-resolution computed tomography may show interstitial lung involvement ranging from mild ground glass and/or consolidation appearance to honeycombing pattern. Most frequently, lung involvement is

CRYOGLOBULINEMIA 585 subclinical. In nonsmoking patients with HCV-associated mixed cryoglobulinemia, free of pulmonary symptoms and with normal chest radiographs, bronchoalveolar lavage demonstrated a T lymphocyte alveolitis not predictive of a significant later deterioration in lung function. Patients without respiratory symptoms do not usually need treatment, even in the presence of high levels of cryoglobulins, whereas in those with progressive pulmonary disease, anti-inflammatory/immunosuppressive treatments should be considered.

Mixed cryoglobulinemia is a distinct clinical entity that belongs to the small vessel systemic vasculitides, according to the Chapel Hill Consensus Conference nomenclature. It is a systemic inflammatory syndrome due to deposition of both soluble and cryoprecitable circulating immune complexes within the wall of small-sized vessels (i.e., capillaries, venules, and arterioles), resulting in activation of complement and vascular injury. Cryoglobulinemia refers to the presence in serum of one or more immunoglobulins (Igs) that precipitate at temperatures below 371C and redissolve on rewarming. Cryoglobulins are usually classified into three subgroups according to the Ig composition: (1) single or type I, composed of single monoclonal Ig, usually IgM or, less frequently, IgG; (2) mixed, type II; and (3) mixed, type III. Mixed cryoglobulins are immune complexes composed of monoclonal IgM (type II) or polyclonal IgM (type III) that have rheumatoid factor (RF) activity and bind to polyclonal IgG. The analysis of cryoprecipitates is generally performed by means of immunoelectrophoresis or immunofixation. However, using more sensitive methodologies, such as immunoblotting or two-dimensional polyacrylamide gel electrophoresis, type II mixed cryoglobulins may result composed of oligoclonal IgM or a mixture of polyclonal and monoclonal IgM associated with polyclonal IgG (type II–III mixed cryoglobulins), indicating a possible transition from type III to type II mixed cryoglobulins. Type I cryoglobulins are nearly always found in patients with myelo- and lymphoproliferative diseases. Type II and III mixed cryoglobulins can be associated with various infectious, neoplastic, or autoimmune diseases.

Etiology Because chronic hepatitis is frequently observed during the clinical course of mixed cryoglobulinemia, a possible role for hepatotropic viruses in the pathogenesis of the disease has long been suggested. It is estimated that hepatitis B virus is a causative factor in mixed cryoglobulinemia in less than 5% of patients. Since the discovery of hepatitis C virus (HCV)

as the major etiological agent of non-A/non-B chronic hepatitis, many epidemiological studies have suggested an important role for HCV in the pathogenesis of mixed cryoglobulinemia. Indeed, 75–95% of patients with mixed cryoglobulinemia have signs of HCV infection, and examination of the soluble and cryoprecipitating immune complexes suggests that the IgG component, to which the IgM–RF fraction binds, is directed against the HCV proteins. Indeed, the cryoprecipitate contains most of the known HCV antigens (core, E1, E2, NS3, NS4, and NS5) and their corresponding antibodies. Moreover, the concentration of HCV RNA in the cryoprecipitate is up to 1000-fold greater than that in the supernatant. Finally, a direct involvement of HCV in immune complex-mediated cryoglobulinemic vasculitis has been suggested by immunohistochemical and molecular biology studies, including HCV RNA detection by in situ hybridization. Therefore, because of the striking association between HCV infection and mixed cryoglobulinemia, the term ‘essential mixed cryoglobulinemia’ should be reserved for only a small number of patients without a known cause of the disease.

Pathology The information on the lung pathology in mixed cryoglobulinemia is scanty. Cellular inflammation and fibrosis are the most common lung manifestations. Widespread vasculitis involving small and medium-sized vessels in the lung, as well as in other organs, has been reported in postmortem examination of cases of mixed cryoglobulinemia. The pattern of organizing pneumonia has also been reported from surgical lung biopsies in patients with cryoglobulinemia.

Clinical Features The major clinical manifestations of mixed cryoglobulinemia include palpable purpura (which primarily appears on the lower limbs but less frequently on the buttocks and trunk), arthralgia, and weakness (Meltzer’s triad). Cryoglobulinemic purpura is typically intermittent. Other clinical manifestations are due to the involvement of the liver (mild to moderate chronic hepatitis and, less frequently, overt cirrhosis), kidneys (type I membranoproliferative glomerulonephritis), and the peripheral nervous system (sensory or sensorimotor peripheral neuropathy and, less frequently, multiplex mononeuropathy). The clinical picture of mixed cryoglobulinemia may also include arthritis (generally mild and nonerosive oligoarthritis), leg ulcers, Raynaud’s phenomenon, sicca syndrome,

586 CRYOGLOBULINEMIA

and, less frequently, the central nervous system, cardiac and gastrointestinal involvement, and hyperviscosity syndrome. It should be emphasized that low levels of circulating mixed cryoglobulins can be detected in more than 50% of HCV-infected individuals, whereas overt cryoglobulinemic vasculitis develops in approximately 5%. Pulmonary Manifestations

Pulmonary symptoms are infrequent. However, mixed cryoglobulinemia patients may complain of

exertional dyspnea, wheezing, and pleuritic pain. Severe lung involvement due to alveolar hemorrhage, adult respiratory distress syndrome, or acute lung injury is a very rare complication of mixed cryoglobulinemia. Chest X-rays and high-resolution computed tomography (HRCT) may show interstitial lung involvement ranging from mild ground glass (Figure 1) and/or consolidation appearance to a honeycombing pattern (Figure 2). In a large series of mixed cryoglobulinemia patients, clinically evident interstitial lung disorder was observed in only four of 206 patients (2%) during a long-term follow-up.

Figure 1 High-resolution computed tomography demonstrating bilateral ground glass appearance in a patient with mixed cryoglobulinemia.

Figure 2 High-resolution computed tomography demonstrating bilateral honeycombing pattern in a patient with mixed cryoglobulinemia.

CRYOGLOBULINEMIA 587 p<0.001 100

75

%

p<0.001 50

25

N.S.

0 Macrophages

Lymphocytes

Neutrophils

Figure 3 Bronchoalveolar lavage cell count of 16 patients with mixed cryoglobulinemia (solid triangles) and 13 healthy control subjects (open triangles). Lines are means. Data from Manganelli P, Salaffi F, Subiaco S, et al. (1996) Bronchoalveolar lavage in mixed cryoglobulinemia associated with hepatitis C virus. British Journal of Rheumatology 35: 978–982, by permission of Oxford University Press. p<0.004

N.S.

100

75 N.S. 50

25

subclinical T lymphocyte alveolitis) (Figure 3). The percentage of CD3þ lymphocytes was significantly higher in mixed cryoglobulinemia patients than in controls, whereas no significant differences were found in the percentage of CD4þ , CD8þ , and CD19þ lymphocytes, neutrophils, and eosinophils (Figure 4). The 5-year follow-up of these patients did not demonstrate deterioration in lung function. Interestingly, treatment of five patients with recombinant interferon-a for a mean period of 12 months resulted in a significant decrease in the percentage of BAL lymphocytes without changes in the T cell subsets.

0 CD3+

CD4+

CD8+

Figure 4 T cell subsets in bronchoalveolar lavage of 16 patients with mixed cryoglobulinemia (solid triangles) and 13 healthy control subjects (open triangles). Data from Manganelli P, Salaffi F, Subiaco S, et al. (1996) Bronchoalveolar lavage in mixed cryoglobulinemia associated with hepatitis C virus. British Journal of Rheumatology 35: 978–982, by permission of Oxford University Press.

Chest radiographs may also demonstrate pulmonary infiltrates, cavitary lesions, and pleural thickening. Lung function tests often reveal airflow obstruction and impairment of gas exchange with reduced DLCO. 67-Gallium scintigraphy may demonstrate hilar and/or parenchymal uptake of the radionuclide. Bronchoalveolar Lavage

Bronchoalveolar lavage (BAL) performed on nonsmoking patients with HCV-associated mixed cryoglobulinemia, free of pulmonary symptoms and with normal chest radiographs, demonstrated a lower percentage of alveolar macrophages and higher percentage of lymphocytes than in healthy controls (i.e.,

Diagnosis

There are no available classification schemes or diagnostic criteria for mixed cryoglobulinemia. In clinical practice, the main diagnostic parameters are serum mixed cryoglobulins with RF activity, low C4 serum levels, skin purpura, and leukocytoclastic vasculitis of small-sized blood vessels due to the deposition of circulating immune complexes and activation of complement. Diagnostic features may also include the involvement of one or more organs (i.e., peripheral nerves, renal, and/or liver). Although cryoglobulin detection and immunologic characterization are necessary for a correct classification and diagnosis, the amount of serum cryoglobulins does not generally correlate with the severity, activity, and prognosis of the disease. When pulmonary involvement is suspected, complete lung function testing, resting room air, arterial blood gases, and chest X-rays should be obtained. If any abnormality is observed, HRCT of the thorax should be performed. Fiberoptic bronchoscopy with transbronchial biopsy

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and BAL should be reserved for patients with dubious diagnosis or severe pulmonary involvement. Outcome

The most common clinical pattern in patients with mixed cryoglobulinemia is a mild, slowly progressive disorder with good prognosis and survival. However, in one-third of the cases, a moderate to severe clinical course is observed; in these patients, the prognosis can be affected by renal failure. Mixed cryoglobulinemia may also be complicated, generally after a long-lasting benign clinical course, by malignancies such as B cell non-Hodgkin’s lymphoma and hepatocellular carcinoma. Lung involvement is usually mild and does not affect survival, but it may predispose the lung to infectious complications and rarely lead to respiratory insufficiency from clinically evident pulmonary fibrosis.

Pathogenesis The mechanism(s) of cryoprecipitation remains unknown. However, different hypotheses have been suggested to explain this in vitro phenomenon, namely (1) structural modification of the variable proportions of Ig heavy and light chains, (2) a reduced concentration of sialic acid, (3) reduced amounts of galactose in the Fc portion of the Ig, and (4) the presence of N-linked glycosylation sites in the CH3 domains as a result of somatic Ig mutations during autoimmune responses. Moreover, this phenomenon can be caused by specific interactions among single components of the cryoprecipitate, such as the IgM cryoprecipitable RF and the Fc portion of IgG, the corresponding autoantigen. Relationship to HCV Infection and Lung Diseases

Since HCV is the main etiological agent suspected in mixed cryoglobulinemia, the possible role of this viral agent in some lung diseases deserves consideration. Indeed, it has been suggested that HCV may play a role in the pathogenesis of pulmonary fibrosis due to an increased incidence of serologic markers of HCV infection in Japanese and Italian patients with lung fibrosis compared to healthy controls. This observation, however, was not confirmed in a British series of patients with pulmonary fibrosis. The discrepancy among these results may depend on differences in both the geographical prevalence of HCV infection and serological assays for its detection. However, lung function tests and HRCT may show findings compatible with diffuse parenchymal lung disease in patients with chronic HCV infection, even in the absence of respiratory symptoms. In addition,

HCV RNA has been detected in pulmonary tissue and BAL fluid from HCV þ patients with diffuse parenchymal lung disease. Furthermore, BAL from patients with both chronic hepatitis C and HCV-associated mixed cryoglobulinemia, without clinical symptoms and radiological findings of diffuse parenchymal lung disease, demonstrated a neutrophilic or lymphocyte alveolitis. Unfortunately, it is not clear if this finding may contribute to the process that leads to pulmonary fibrosis seen in a minority of cases of chronic hepatitis C with or without mixed cryoglobulinemia. Finally, HCV may affect lung function in patients with obstructive lung diseases. Indeed, it has been found that chronic HCV infection is associated with an accelerated decline in lung function in both patients with chronic obstructive pulmonary disease and patients with bronchial asthma. Chronic HCV infection also impairs responses to inhaled corticosteroid therapy, as well as reversibility with salbutamol in bronchial asthmatic patients who do not respond to interferon-a therapy.

Management and Current Therapy Since HCV plays a crucial role in the etiology/pathogenesis of mixed cryoglobulinemia, HCV eradication with interferon-a and ribavirin should be attempted in the majority of patients. In other patients, or in those who have failed to respond to the antiviral therapy, anti-inflammatory/immunosuppressive treatments, including corticosteroids, low antigen content diet, plasma exchange, and cytotoxic drugs (mainly cyclophosphamide), should be considered. Patients without respiratory symptoms do not usually need treatment, even in the presence of high levels of cryoglobulins, whereas in symptomatic patients with progressive pulmonary disease, anti-inflammatory/immunosuppressive treatment should be taken into account. See also: Bronchoalveolar Lavage. Interferons. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Pulmonary Fibrosis. Vasculitis: Overview.

Further Reading Bombardieri S, Paoletti P, Ferri C, et al. (1979) Lung involvement in essential mixed cryoglobulinemia. American Journal of Medicine 66: 748–756. Dammacco F, Sansonno D, Piccoli C, Tucci FA, and Ravanelli V (2001) The cryoglobulins: an overview. European Journal of Clinical Investigation 31: 628–638. Ferri C, Giaggioli D, Cazzato M, et al. (2003) HCV-related cryoglobulinemic vasculitis: an update on its etiopathogenesis and therapeutic strategies (review). Clinical and Experimental Rheumatology 21(supplement 31): S78–S84.

CYCLIC NUCLEOTIDE PHOSPHODIESTERASES 589 Ferri C, La Civita L, Fazzi P, et al. (1997) Interstitial lung fibrosis and rheumatic disorders in patients with hepatitis C virus infection. British Journal of Rheumatology 36: 360–365. Ferri C, Sebastiani M, Giuggioli D, et al. (2004) Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Seminars in Arthritis and Rheumatism 33: 355–374. Irving WL, Day S, and Johnston ID (1993) Idiopathic pulmonary fibrosis and hepatitis C virus infection. American Review of Respiratory Disease 148: 1683–1684. Jennette JC, Falk RJ, Andrassy K, et al. (1994) Nomenclature of systemic vasculitides. Proposal of an International Consensus Conference. Arthritis & Rheumatism 37: 187–192. Lamprecht P, Gause A, and Gross W (1999) Cryoglobulinemic vasculitis (review). Arthritis & Rheumatism 42: 2507–2516. Manganelli P, Salaffi F, and Pesci A (1997) Clinical and subclinical alveolitis in connective tissue diseases assessed by bronchoalveolar lavage. Seminars in Arthritis and Rheumatism 26: 740–754.

Manganelli P, Salaffi F, Subiaco S, et al. (1996) Bronchoalveolar lavage in mixed cryoglobulinemia associated with hepatitis C virus. British Journal of Rheumatology 35: 978–982. Meliconi R, Andreone P, Fasano L, et al. (1996) Incidence of hepatitis C virus infection in Italian patients with idiopathic pulmonary fibrosis. Thorax 51: 315–317. Okutan O, Kartaloglu Z, Ilvan A, et al. (2004) Evaluation of highresolution computed tomography and pulmonary function tests in patients with chronic hepatitis C virus infection. World Journal of Gastroenterology 10: 381–384. Salaffi F, Manganelli P, Carotti M, et al. (1996) Mixed cryoglobulinemia: effect of alpha-interferon on subclinical lymphocyte alveolitis. Clinical and Experimental Rheumatology 14: 219–220. Ueda T, Ohta K, Suzuki N, et al. (1992) Idiopathic pulmonary fibrosis and high prevalence of serum antibodies to hepatitis C virus. American Review of Respiratory Disease 146: 266–268. Viegi G, Fornai E, Ferri C, et al. (1989) Lung function in essential mixed cryoglobulinemia: a short-term follow-up. Clinical Rheumatology 8: 331–338.

CYCLIC NUCLEOTIDE PHOSPHODIESTERASES V C Manganiello and J Fontana, National Institutes of Health, Bethesda, MD, USA E Degerman, Lund University, Lund, Sweden F Ahmad, National Institutes of Health, Bethesda, MD, USA Published by Elsevier Ltd.

Abstract The cyclic nucleotides, cAMP and cGMP, are important intracellular messengers that regulate multiple critical signaling pathways important in normal physiology, as well as pathogenesis of disease. Their intracellular concentrations are regulated by two enzyme systems: cyclases, which catalyze formation of cAMP and cGMP from ATP and GTP, respectively, and cyclic nucleotide phosphodiesterases (PDEs), which cleave the high-energy 30 50 cyclic phosphate bond of cAMP and cGMP to yield 50 AMP and 50 GMP, respectively. PDEs are divided into two major classes: I and II. Class I PDEs contain a conserved catalytic core (B250–300 amino acids) and include the majority of presently identified PDEs. The 11 structurally related, highly regulated, and functionally distinct mammalian PDE gene families (PDEs 1–11) are class I PDEs, as are PDEs found in Drosophilia, nematodes, yeast, sponges, and parasites such as Trypanasoma brucei and T. cruzei. Class II PDEs are fewer in number and structurally unrelated to class I PDEs; no mammalian class II PDEs have been identified. Mammalian PDEs possess a conserved modular molecular architecture, with a conserved catalytic domain containing a signature sequence motif proximal to the C-terminal tail and downstream from a regulatory region in the N-terminal portion of the molecule. It is becoming clear that PDEs are important components of signaling modules and microdomains that are critical to establishing and maintaining intracellular cyclic nucleotide gradients, and compartmentalization of cyclic nucleotide pathways and responses. At present, mutations in the PDE6 gene represent the

only known association of PDE mutations with human disease, for example, certain subclasses of retinitis pigmentosa. The mammalian PDE superfamily is a major potential target for drug discovery, that is, PDE3 inhibitors for certain types of cardiac failure and peripheral vascular disease, PDE4 inhibitors for inflammatory disorders such as chronic obstructive pulmonary disease and asthma, and PDE5 inhibitors for erectile dysfunction and, more recently, for pulmonary hypertension.

Introduction After Sutherland and co-workers discovered cAMP and cGMP in the late 1950s, these intracellular second messengers became rapidly recognized as important regulators of myriad critical cellular processes, many of which are central to pulmonary physiology in health and disease, that is, cell proliferation and differentiation, metabolism, secretion, vascular and airway smooth muscle relaxation, production of inflammatory mediators, cytokines, chemokines, adhesion molecules, extracellular matrix, collagen, etc. Although signaling induced by cAMP and cGMP primarily involves their activation of cAMP- and cGMP-dependent protein kinases (PKAs and PKGs), with subsequent phosphorylation of critical effectors, direct interactions of cyclic nucleotides with binding proteins reflect alternative mechanisms for transduction of their signals. These binding proteins include cyclic nucleotide-gated channels, cAMPactivated guanine nucleotide exchange factors (GEFs) that regulate Rap1, and several cyclic nucleotide phosphodiesterases (PDEs), which contain allosteric, noncatalytic cyclic nucleotide binding sites (Figure 1).

590 CYCLIC NUCLEOTIDE PHOSPHODIESTERASES

PDEs

Rap1

cAMP GEF

PKA

CNG channels

PDE4 PDE7 PDE8

cAMP

PDE1 PDE2 PDE3 PDE10 PDE11 5′AMP

5′GMP

PDE5 PDE6 PDE9

cGMP

PDEs 2,5,6

PKG

CNG channels ATP Cyclases

GTP

Figure 1 Role of cyclic nucleotide phosphodiesterases (PDEs) in regulation of signaling by cyclic nucleotides. GEF, guanine nucleotide exchange factor; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; CNG channels, cyclic nucleotide-gated channels.

The Sutherland group discovered PDEs almost simultaneously with cAMP and cGMP. PDEs catalyze hydrolysis of cAMP and cGMP, and thereby regulate their intracellular concentrations, and, consequently, their signaling pathways and effects. In tissue extracts and cells, maximal rates of cyclic nucleotide hydrolysis (via PDEs) exceed rates of synthesis, and PDE inhibitors are required to produce measurable increases in cyclic nucleotides during incubations of cells and tissue preparations with hormones and agents that increase cyclic nucleotide synthesis. Molecular genetics has occasioned a virtual explosion of information concerning diversity and complexity of the mammalian PDEs, and the organization of the large mammalian PDE superfamily into 11 structurally related, highly regulated, and functionally distinct gene families (PDE1–11). We are only beginning to understand the extent and complexity of regulation of PDE expression and activity, as well as the complexity of regulation of cyclic nucleotide signaling by PDEs. Not only are PDEs important regulators of the generation, amplitude, and duration of intracellular cyclic nucleotide signals, but their specific subcellular locations, their association with molecular scaffolds and interacting partners, and their contribution to regulation of signaling compartmentalization, specificity, and functions within signaling microdomains are becoming a common theme in cell biology.

PDE Structure Catalytic Domain

The 11 PDE gene families differ in their primary amino acid sequences, substrate specificities, kinetic

characteristics, sensitivities to effectors and pharmacological agents, physiological roles, and mechanisms by which they are regulated. Most families contain more than one gene; within the same family and subfamily, multiple homologous isoforms are generated from the same or different genes via utilization of different promoters/transcriptional initiation sites, alternative mRNA splicing, or regulation of translation. Twenty PDE genes encode more than 50 proteins. PDE nomenclature includes, in order, two letters to indicate species, followed by a number identifying the specific gene family and letters that represent individual subfamilies and splice variants. For example, the PDE4 family is comprised of four subfamilies, PDE4A–D: HSPDE4D3 indicates the human PDE4 gene family, PDE4D gene, splice variant 3. PDEs share a common structural organization, with a conserved catalytic domain (B250–300 amino acids) in the C-terminal portion, followed by a short hydrophilic C-terminal tail and preceded by an N-terminal regulatory region (Figure 2). The conserved catalytic core contains histidine-containing signature motifs, H–D–X2–H–X4–N, and consensus metal-binding domains. Catalytic domains contain family-specific sequences that determine differences in substrate affinities, catalytic properties, and sensitivities to specific effectors and inhibitors, as well as common structural determinants involved in cleavage of cyclic phosphate bonds. Some families are relatively specific for cAMP as substrate (PDEs 4, 7, 8); others, cGMP (PDEs 5, 6, 9); some exhibit high affinities for both cAMP and cGMP (PDEs 1, 2, 3, 10, 11) (Figure 1). Some of the latter hydrolyze cAMP and cGMP in a mutually competitive manner. Although methylxanthines inhibit almost all PDEs,

CYCLIC NUCLEOTIDE PHOSPHODIESTERASES 591 Regulatory domain

• Targeting domains • Autoinhibitory domains • Phosphorylation sites • Allosteric ligand-binding sites, i.e., GAF domains • Effector interaction sites, i.e., calmodulin-binding, PAS domains • Dimerization domains

Conserved catalytic domain

C-terminal domain

• PDE signature motif • Substrate specificity • Inhibitor sensitivity •
-Arrestin interaction sites • ERK phosphorylation sites

Figure 2 Common structural pattern for different PDE gene families.

family-selective inhibitors, that is, pharmacological agents that inhibit specific families with 10–100-fold greater potency than other families, have been discovered for several families, for example, PDEs 1, 2, 3, 4, 5, 6, 7. The crystal structure of the catalytic cores of several PDEs has been solved. This information should aid in the design of drugs with greater selectivity, specificity, and potency for individual PDE isoforms.

Although cGMP binding is not the primary function of GAF domains, PDEs 2, 5, and 6 contain homologous GAF domains that bind cGMP with high affinity and selectivity, but with different functional sequellae.

PDE Functions

Regulatory Domain

PDEs: Critical Components of Spatially Organized Signaling Modules and Microdomains

PDEs are modular enzymes with differences in cyclic nucleotide substrate specificities and inhibitor sensitivities, and with isoform-specific regulatory domains/cassettes (Figure 2). These characteristics allow cells to utilize PDEs with subtle differences in their catalytic and regulatory properties at different subcellular locations to achieve specificity of cAMP signaling and function. Divergent N-terminal portions of PDE molecules contain structural determinants involved in selective responses of individual PDEs to specific signals that regulate catalytic activity, protein–protein interactions, or targeting to specific subcellular locations. These regulatory domains contain autoinhibitory modules, dimerization domains, GAF domains (an acronym for proteins, i.e., cGMP-binding PDEs, Arabena adenylyl cylase, and fhlA (an Escherichia coli transcriptional activator) that contain these regions), subcellular localization signals, sites for phosphorylation by PKA, PKG, Ca2 þ /calmodulindependent sensitive protein kinases, protein kinase B (PKB/Akt), and protein kinase C, etc. PDE1 proteins are activated by binding of calmodulin to Ca2 þ /calmodulin-binding regions; PDE8 proteins are activated by interactions of regulatory PAS (acronym for Per, ARNT, and Sim proteins) domains with inhibitor kappa B (IkB). PDEs 2, 5, 6, 10, and 11 contain N-terminal region GAF domains; they function as conserved nucleotide switches that regulate adjacent catalytic domains.

Regulation of signaling specificity and specific functional readouts in intracellular microdomains is achieved by using small, discrete subsets of related proteins with subtle differences in their structural, regulatory, and catalytic/functional properties. From a ‘traditional’ perspective, PDEs are important for stringent regulation of the generation, amplitude, duration, and termination of intracellular cAMP and cGMP signals and their physiological effects. Newer techniques, for example, use of fluorescence-resonance energy transfer (FRET) and cyclic nucleotide biosensors (cyclic nucleotide-gated channels, whole cell patch clamps, etc.), indicate that, in different types of cells, localized activation of cyclases produces localized increases in cyclic nucleotides, and that these intracellular pools of cAMP and cGMP are temporally, spatially, and functionally compartmentalized. Specific PDE isoforms clearly play important roles in these spatially constricted and temporally regulated microdomains, especially in establishing cyclic nucleotide gradients and regulating their turnover and diffusion. Inhibition of specific PDEs allows diffusion of cAMP from localized microdomains to different subcellular compartments. Although differential localization of specific PDE isoforms supports the concept that localization is related to function, the most salient example of a specifically localized PDE serving as a critical effector and regulator of a unique cellular function is the

592 CYCLIC NUCLEOTIDE PHOSPHODIESTERASES

photoreceptor PDE6. Light-induced activation of PDE6, whose expression is almost virtually confined to photoreceptor rod and cone cells, results in cGMP hydrolysis, closure of cGMP-gated cation channels, hyperpolarization of cell membranes, and initiation of visual signal transduction. Recent studies, including RNA silencing of specific PDE4 variants or PDE4B and D knockout (KO) mice, as well as real-time dynamic imaging and FRET analysis of cAMP gradients, indicate that PDE4D, not PDE3, is primarily responsible for terminating the increase in cAMP induced by activation of the b-receptor in rodent cardiomyocytes. Conversely, accumulation of cAMP in response to forskolin, which activated the entire pool of myocyte adenylyl cyclase, was markedly potentiated by specific PDE3, not PDE4, inhibitors. Confocal microscopy suggested localization of PDE3 to internal membranes, and PDE4 isoforms, along with PKA, to sarcomeric M and Z lines. Different targeting domains in PDE4 variants allow them to be spatially compartmentalized in different subcellular regions. In Calu-3 airway epithelial cells, PDE4D may be localized in a microdomain of the apical membrane, in proximity to adenosine A2B receptors and the cystic fibrosis transmembrane conductance regulator (CFTR). Thus, PDE4D may form a barrier to diffusion of cAMP and regulation of CFTR by PKA at the apical membrane. Historically, in cells containing multiple PDEs, family-specific PDE inhibitors have been utilized to pharmacologically link specific PDEs to regulation of specific intracellular cyclic nucleotide pools, their specific signaling pathways, and physiological effects. For example, in cultured mammalian oocytes, inhibitory effects of cAMP on meiotic progression and oocyte maturation are enhanced or mimicked by PDE3, not PDE4 or 5, inhibitors, suggesting that PDE3 regulates a cAMP pool that controls maturation. (In fact, female PDE3A-deficient mice are sterile.) In addition, PDE KO mice exhibit distinct phenotypes, with disruption of specific signaling pathways, indicating that other PDEs cannot compensate for targeted PDE isoforms. Production of tumor necrosis factor alpha (TNF-a) in response to lipopolysaccharide (LPS) is dramatically inhibited in PDE4B KO mice. Studies with PDE4B and 4D KO mice indicate that they play complimentary, but not redundant, roles in control of neutrophil function and that one gene cannot completely compensate for the other. PDE4D KO mice do not exhibit acetylcholine-induced contraction and hyperreactivity of airway smooth muscle. The molecular basis for compartmentalization of cAMP signaling involves, in large part, the targeting/

anchoring of PKA isoforms at specific subcellular locations via A-kinase anchoring proteins (AKAPs). PKA catalytic subunits bind to AKAPs via PKA regulatory subunits. AKAPs contain targeting domains, which specify their intracellular addresses, and scaffolding domains, which organize formation of localized signaling modules/microdomains consisting of PKA, other kinases, kinase substrates, phosphatases, PDEs (especially PDE4 isoforms), and other signaling molecules. Different AKAPs are associated with different constellations of signaling effectors. These spatially segregated modules sense intracellular cAMP gradients and effectively compartmentalize activation of effectors, transduction of signals, and propagation of specific physiological responses. In some functional microdomains, cAMP activation of PKA results in phosphorylation of substrates as well as phosphorylation/activation of PDE4, resulting in modulation and termination of cAMP signals and return of PKA to its basal state. PDE4D isoforms, together with PKA and other signaling effectors, interact/associate with b-arrestins, which are scaffolding proteins that interact with activated b-receptors and regulate b-receptor signaling, trafficking, and internalization. Internalization of b-receptor/b-arrestin complexes reduces generation of cAMP by adenylyl cyclase, and targeting of PKA and PDE4D to activated b-receptors increases localized degradation of cAMP, thus ‘desensitizing’ the complex to future signaling via cAMP. In T lymphocytes, recruitment of PDE4 (in complex with b-arrestin) to lipid rafts potentiates T-cell receptor (TCR) signaling during T-cell activation induced by ligation of the TCR and co-receptor CD28. PDEs: Signal Integrators

Because of their intrinsic regulatory properties, different responses to regulatory signals, and different interacting partners, PDEs serve as a ‘locus’ for crosstalk between different signaling pathways. For example, PDE2, which is allosterically activated by binding of cGMP to noncatalytic sites in GAF domains, is highly concentrated in adrenal glomerulosa cells. In these cells, atrial natriuretic factor activates guanylyl cyclase and increases cGMP, which in turn activates PDE2, reduces cAMP, and inhibits aldosterone production. On the other hand, in other cells, nitric oxide (NO) activates soluble guanylyl cyclase and increases cGMP, which inhibits PDE3 and increases cAMP, resulting, for example, in smooth muscle relaxation, inhibition of platelet aggregation, or stimulation of renin secretion from the renal juxtaglomerular apparatus. In cultured vascular smooth muscle cells, agents that increase cGMP

CYCLIC NUCLEOTIDE PHOSPHODIESTERASES 593

inhibit PDE3, resulting in activation of PKA and inhibition of nuclear factor kappa B (NF-kB)-dependent regulation of chemokine and adhesion molecule expression. In platelets, effects of NO, cAMP, and cGMP seem to be regulated by the interplay of PDEs 2, 3, and 5. PDEs: Homeostatic Regulators

To ensure tight regulation of intracellular concentration of cAMP and cGMP, cyclic nucleotide signaling pathways contain intrinsic mechanisms for negative feedback control. cAMP-induced activation of PKA can result in phosphorylation/activation of PDE3 and PDE4 isoforms, enhanced destruction of cAMP, and termination or dampening of cAMP signals. Chronic, long-term elevation of cAMP also initiates negative feedback control, by increasing transcription of PDE3 and PDE4 genes, resulting in increased enzyme protein and activities. In some cells, this phenomenon upregulates cAMP hydrolysis and is related to ligand-induced downregulation of cAMP signaling and cellular responses, such as tachyphylaxis or desensitization. In cultured rat and human vascular myocytes, cAMP increases expression of membrane-associated PDE3B, without any effect on PDE3A. Thus, cell-specific adaptive mechanisms to prolonged elevation of cAMP have specific functional consequences. Negative feedback regulation by increased intracellular cGMP involves binding of cGMP to GAF domains, resulting in allosterically induced conformational changes that facilitate PKG-induced phosphorylation of the N-terminal domain and increases

affinity of the catalytic site for cGMP and catalytic activity.

Clinical Applications: Specific PDE Inhibitors Nonselective PDE inhibitors such as theophylline are used under certain circumstances to treat asthma. Despite considerable effort to develop family-specific inhibitors to replace theophylline, no PDE inhibitors are currently used as therapy for major pulmonary diseases. PDE3 inhibitors, which inhibit platelet activation/aggregation, relax airway and vascular smooth muscle, and enhance myocardial contractility, failed in long-term trials of chronic cardiac failure. One PDE3 inhibitor, milrinone, is used for acute treatment of adults with decompensated and refractory cardiac failure, while another, cilostazol (Pletaal), is used for treatment of intermittent claudication. PDE4 enzymes are relatively highly expressed in inflammatory/immune cells, and specific PDE4 inhibitors exhibit potent anti-inflammatory actions in preclinical studies and model systems. Roflumilast, a PDE4 inhibitor, is currently in phase 3 trials for treatment of chronic obstructive pulmonary disease (COPD) and asthma. On the other hand, the PDE5 inhibitors – Viagra, Cialis, and Levitra – have achieved remarkable success, with very favorable side-effect profiles, in the treatment of erectile dysfunction. The success of these drugs may be related to their inhibition of PDE5, at a time and locus where NO, produced in response to sexual arousal, activates guanylyl cyclase. PDE5 inhibitors protect cGMP from hydrolytic destruction; cGMP activates

Neuronal (n)NOS Endothelial (e)NOS GTP

NO

Soluble guanylyl cyclase

Proteins ATP cGMP

PKG ADP

PDE5 inhibitors

Ca2+

Protein-P

PDE5

Lower Ca2+

5′ GMP

Ca2+

Relax vascular smooth muscle

Penile erection

Figure 3 Mechanism of action of PDE5 inhibitors. NOS, nitric oxide synthase.

Pulmonary vasodilation

Ca2+

594 CYSTEINE PROTEASES, CATHEPSINS

PKG, which phosphorylates critical effectors that lower intracellular Ca2 þ and induce vascular relaxation, leading to penile erection (Figure 3). These drugs are beginning to show progress as potential therapy for pulmonary hypertension (PHT); whether their effectiveness in PHT is related to NO physiology in the pulmonary vascular bed, as in the corpus cavernosum, is not certain.

Conclusions Compartmentalization of cyclic nucleotide signaling requires the successful confluence of multiple factors. Scaffolding molecules, at different intracellular addresses, spatially organize different subsets of signaling molecules, including PDEs, kinases, phosphatases, substrates, and other signaling and effector molecules, and thus generate discrete functional microdomains. By virtue of their distinct intrinsic characteristics, their differential expression and regulation, and their targeting to different subcellular locations and microdomains where they interact with cellular structural elements, regulatory partners and molecular scaffolds such as AKAPs and b-arrestin, different PDEs respond to and regulate multiple inputs, and modulate the intracellular diffusion of cyclic nucleotide gradients and functional compartmentalization of cyclic nucleotide signals. Integration of the myriad combinations of signaling molecules provides a variety of networks involved in generation, transduction, modulation, and termination of cyclic nucleotide-gated signals and actions, and establishes unique cyclic nucleotide phenotypes that characterize individual cells. See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Cystic Fibrosis. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Transcription Factors: NF-kB and Ikb.

Further Reading Beavo JA and Brunton LL (2002) Cyclic nucleotide research
still expanding after half a century. Nature Reviews, Molecular and Cellular Biology 3: 710–717. Conti M (2004) A view into the catalytic pocket of cyclic nucleotide phosphodiesterases. Nature Structural and Molecular Biology 11: 809–811. Corbin JD and Francis SH (1999) Cyclic GMP phosphodiesterase 5: target of sildenafil and related compounds. Journal of Biological Chemistry 274: 13729–13733. Degerman E, Rahn Landstrom T, Stenson Holst L, et al. (2004) Role for phosphodiesterase 3B in regulation of lipolysis and insulin secretion. In: LeRoth D, Taylor SI, Olefsky JM (eds.) Diabetes Mellitus: A Fundamental and Clinical Text, 3rd edn., pp. 373–383. Philadelphia: Lippincott Raven. Francis SH, Turko IV, and Corbin JD (2001) Cyclic nucleotide phosphodiesterases: relating structure and function. Progress in Nucleic Acid Research and Molecular Biology 65: 1–52. Houslay MD and Adams DR (2003) PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signaling cross-talk desensitization, and compartmentalization. Biochemical Journal 370: 1–18. Jin SL, Lan L, Zoudilova M, et al. (2005) Specific role of phosphodiesterase 4B in lipopolysaccharide-induced signaling in mouse macrophages. Journal of Immunology 175(3): 1523– 1531. Masciarelli S, Horner K, Liu C, et al. (2004) Cyclic nucleotide phosphodiesterase 3A-deficient mice as a model of female infertility. Journal of Clinical Investigations 114(2): 196–205. Mongillo M, McSorley T, Evellin S, et al. (2004) Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. American Heart Association Journal 95(1): 67–75. Rich T and Karpen JW (2002) Cyclic AMP sensors in living cells: what signals can they actually measure? Annals of Biomedical Engineering 30: 1088–1099. Spina D (2004) The potential of PDE4 inhibitors in respiratory disease. Current Drug Targets
Inflammation and Allergy 3: 221–226. Torphy T (1998) Phosphodiesterase isoenzymes: molecular targets for novel antiasthma agents. American Journal of Respiratory and Critical Care Medicine 157: 351–370. Wong W and Scott JD (2004) AKAP signaling complexes: focal points in space and time. Nature Reviews, Molecular and Cellular Biology 5: 959–970.

CYSTEINE PROTEASES, CATHEPSINS F Bu¨hling, Otto-von-Guericke University of Magdeburg, Magdeburg, Germany & 2006 Elsevier Ltd. All rights reserved.

mice, and selective protease inhibitors have added new insight into the specific functions of single proteases. This article discusses some of these functions in relation to the lung, especially the role of lysosomal cysteine proteases in matrix remodeling, immunoregulation, surfactant protein processing, and apoptosis.

Abstract Lysosomal cysteine proteases or cathepsins are a family of more than 10 enzymes. The description of new lysosomal cysteine proteases with restricted expression and outstanding enzymatic activity and also the use of molecular biologic methods, knockout

Introduction The term ‘cathepsin’ was introduced in 1920 and it stands for ‘lysosomal proteolytic enzyme’, regardless

CYSTEINE PROTEASES, CATHEPSINS 595

of the enzyme class. Therefore, in addition to cysteine proteases, this term also includes enzymes using other catalytic mechanisms, such as serine proteases (cathepsins A and G) and aspartic proteases (cathepsins D and E). This article focuses on cysteine proteases, which are part of the papain family. These proteases are localized predominantly in the lysosomes of cells. Other nonlysosomal cysteine proteases are caspases, calpains, and hemoglobinases. Since the discovery of cathepsin B in 1941, more than 12 cathepsins belonging to the cysteine proteases have been described. Until the 1980s, classical protein biochemical methods were used to characterize cathepsins B, C, H, L, and S. The importance of these enzymes became clear when it was shown that up to 40% of the cellular protein turnover could be suppressed by cysteine protease inhibitors. Later, molecular biology was used to discover, for example, cathepsins F, K, O, V, W, and X. Sequence homologies of the cysteine proteases cathepsins B, C, H, L, and S indicate that these enzymes diverged early during eukaryotic evolution. Some other genes encoding novel enzymes, such as cathepsins F, K, V, W, and X, may be the result of relatively recent gene duplication events. In addition, rodents differ from humans with respect to cathepsin expression; that is, in contrast to humans, rodents do not express cathepsin V, but they do express a group of homologous cathepsins in the placenta.

distinguished. Some of the genes may be the result of relatively recent gene duplication events, as suggested by three gene pairs with common chromosomal localization and high sequence homologies: cathepsins S and K, F and W, and L and V. Active cathepsins are mostly monomeric proteins. Several three-dimensional structures of cysteine proteases are available, and they demonstrate that enzymes of the papain family share a common fold. The main structural features of a papain-like twodomain catalytic platform are depicted in Figure 1 using a model of mature cathepsin H; the active site is located at the interface between the two domains. It contains cysteine and histidine, which form the nucleophile thiolate–imidazole ionic pair. Most cysteine proteases of the papain family are endopeptidases ‘by design’. Exopeptidases, such as cathepsins B, C, H, and X, are likely to have evolved from the endopeptidase template by addition of structural elements capable of interacting with the C or N terminus of a substrate.

Structure Cathepsin genes are localized on different chromosomes (Table 1). Based on their amino acid sequence homology and specific sequence motifs, cathepsin Blike (B, O, C, and Z), cathepsin L-like (L, V, K, S, and H), and cathepsin F-like (W and F) enzymes may be

Figure 1 Structural model of mature cathepsin H. Courtesy of Dr W Brand, Martin-Luther-University Halle, Halle, Germany.

Table 1 General characteristics of cathepsins Cathepsin (synonyms)

Chromosomal localization (human/mouse)

Expression in respiratory system

Comments; disease association

B

8/14

Ubiquitous macrophages

C (DPPI)

11/7

Mast cells

Carboxypeptidase; aberrant expression in lung tumors and numerous inflammatory diseases Dipeptidyl peptidase I; Papillon– Lefevre syndrome

F H

11/19 15/9

Ubiquitous Ubiquitous, type II pneumocytes

K (O2)

1/3

L O S V (L2, U) W (lymphopain) X (Z, B2)

9/13 4/3 1/3 9/– 11/19 20/2

Bronchial epithelial cells, activated macrophages and fibroblasts Ubiquitous, macrophages

Aminopeptidase, surfactant processing Collagenase, elastase; pyknodysostosis, lung fibrosis Pulmonary emphysema

Macrophages ? Cytotoxic lymphocytes Ubiquitous, granulocytes

Elastase, stable at neutral pH Elastase Activity unknown Carboxypeptidase

596 CYSTEINE PROTEASES, CATHEPSINS

Regulation of Production and Activity Cathepsins are synthesized as inactive pre-proenzymes and undergo posttranslational glycosylation. The prepart of the molecule is necessary for intracellular trafficking toward the lysosomal compartment, which is realized using mannose 6-phosphate receptors. Truncated forms lacking the pre-protein are misdirected and therefore secreted or accumulated in the cytoplasm of cells; these forms have been found in some tumor cells. The pro-parts of the cathepsins serve as competitive inhibitors by occupying the active center of the enzymes. They are cleaved under acid conditions within the endosomal/lysosomal compartment. The activity of cathepsins is regulated at different levels. First, the transcription of cathepsin genes is controlled by cell-type-specific mechanisms and exogenous factors. Thus, different cell types do express different cathepsins. Some cathepsins, such as cathepsins B, H, F, L, and X, are ubiquitously expressed in almost all cells but differ in their expression level. These cathepsins are found in high concentrations in alveolar macrophages and at lower levels in almost all other cell types. Cathepsin H is increased in type II pneumocytes. Other cathepsins show a more specific expression level, such as cathepsins K and S. Cathepsin S is predominantly expressed in macrophages, whereas cathepsin K has been found in bronchial epithelial cells, activated macrophages (epithelioid cells and multinucleated giant cells), and activated lung fibroblasts. In addition, cytokines may regulate cathepsin expression in a cell-type-specific way. Proinflammatory cytokines (i.e., interleukin (IL)-6 and IL-1b) induce cathepsin expression. Transforming growth factor beta may suppress the expression. Microbial components also increase cathepsin expression in several cells. Second, the maturation and posttranslational processing is regulated by other proteases and the pH in the endosomal/lysosomal compartment. Intracellular microorganisms, which are able to prevent the acidification of the lysosomal compartment, prevent the maturation and activation of cathepsins and, thus, their proteolytic degradation. Third, the activity of mature enzymes depends on the pH of the environment. Neutral or alkaline pH induces the irreversible inactivation of most cathepsins except cathepsins S and K. Furthermore, because of the exceptional proteolytic activity and the high concentration of cathepsins (up to 1 mM in lysosomes), their activity is controlled by a network of endogenous inhibitors. These inhibitors are cystatins, which are expressed intracellularly (cystatins A and B) or extracellularly (cystatins C, D, and F), and other endogenous cathepsin inhibitors, including kininogens, serpins, and a2-macroglobulin.

Cathepsins cleave a variety of proteins and polypeptides. On the one hand, they are involved in lysosomal bulk protein degradation. On the other hand, single cathepsins specifically degrade certain proteins. For example, cathepsins S, L, and V are involved in the processing of the invariant chain of MHC class II molecules, cathepsin K degrades collagen with a unique specificity, and cathepsins K and V are very efficient elastases. Other proteins that are cleaved by cathepsins include surfactant proteins; kinins; thyroid hormones; other enzymes or their precursors, such as granzymes, cathepsin D, neutrophil elastase, cathepsin G, and proteinase 3; trypsinogen; neurotransmitters; and amyloid proteins.

Biological Functions Related to the Respiratory System During recent years, several knockout and transgenic mouse models have been generated. The published phenotypes and the functional associations found in these animals are summarized in Table 2. The biological functions directly linked to the respiratory system are antigen presentation, matrix remodeling, surfactant processing, and apoptosis (Figure 2). Antigen Presentation

The process depends on proteolytic processing at different levels. First, extracellular antigens, which are taken up by antigen presenting cells, are mostly degraded in the endosomal/lysosomal compartment. The resulting peptides are displayed on the cell surface after formation of MHC class II peptide complexes. Studies have shown that the pattern of peptides to be presented depends on distinct proteases. For example, positive selection in the thymus depends on the antigen peptides presented on epithelial cells and negative selection of peptides on dendritic cells; in mice, the former depends on cathepsin L and the latter on cathepsin S. Removal of one of these two enzymes by generating chimeric knockout mice leads to a failure of T cell maturation. Second, during the early stages of biosynthesis, MHC class II ab heterodimers associate with a type II membrane protein, the invariant chain (Ii), to form a nonameric complex of abIi. Degradation of Ii is a precondition for binding of antigenic peptides to class II molecules. It has been shown that the cathepsins F, L, S, and V are involved in Ii processing. In thymic epithelial cells of mice, cathepsin L is critical for Ii processing. In human thymus, this function is likely performed by cathepsin V. Cathepsin S may be involved in Ii processing in other human epithelial cells. Cathepsin S is also the critical enzyme in dendritic

CYSTEINE PROTEASES, CATHEPSINS 597 Table 2 Characteristics of cathepsin-deficient and -overexpressing mice Cathepsin

Generated by

Functional association

B C

Peters C et al. Ley TJ et al.

Thyroglobulin processing, trypsinogen processing, apoptosis Proteolytoic activation of granzyme A.B, cathepsin G, proteinase 3, neutrophil elastase, mast cell chymase

H K

L

Peters C et al. Saftig P et al. Kola I et al. Kiviranta R et al. (transgen) Peters C et al.

S W

Chapman HA et al. Peters C et al.

Antigen processing

Bone resorption, lung matrix degradation fibrosis

Antigen presentation, hair growth, myocardial remodeling, neurotransmitter processing, cathepsin D processing apoptosis Antigen presentation, angiogenesis, atherogenesis

Lysosomal protein breakdown

Invariant chain processing

Matrix remodeling

Surfactant protein processing

Cathepsins Prohormone processing

Protease activation/inactivation

Regulation of apopt osis

Defensin inactivation

Protease inhibitor cleavage

Figure 2 Biological functions of cathepsins. Functions that are discussed in this article are shown in dark blue.

cells and B lymphocytes. Mouse macrophages express large amounts of cathepsin F, which seems to be responsible for Ii processing in these cells. The suppression of cathepsin F in mice prevents eosinophilia and IgE response after ovalbumin challenge, which illustrates the potential role of defined cathepsins as targets for new immunomodulatory drugs. Further studies should characterize the expression and function of additional cysteine proteases (e.g., cathepsins B, K, and L) in antigen presenting cells of the lung and also explore in more detail the effects of selective inhibition of these enzymes on the development of allergic reactions. Matrix Remodeling

Cathepsins are involved in intracellular and extracellular matrix (ECM) degradation. Within the lysosomes of macrophages and fibroblasts, cathepsins

degrade phagocytozed fragments of ECM proteins. It has been shown that macrophages, mast cells, smooth muscle cells, fibroblasts, and various tumor cells release cathepsins. Macrophages can acidify their environment upon release of lysosomal proteases and thus contain their activity. In addition, cathepsins C, K, S, and V are characterized by relatively high pH stability, and they retain some proteolytic activity at neutral pH. Cathepsin K is characterized by a unique collagenolytic activity. The enzyme cleaves collagen fibers at multiple sites, whereas mammalian proteases, such as collagenases, gelatinases, and stromelysin, cleave collagen helices at a defined site, leaving 2/3 and 1/3 fragments. Other cathepsins remove only teleopeptide fragments. The enzyme seems to be important in the development of lung fibrosis. In a mouse model of bleomycin-induced lung fibrosis, cathepsin K plays

598 CYSTEINE PROTEASES, CATHEPSINS

a critical role in the degradation of surplus ECM proteins; therefore, cathepsin K-deficient mice develop more lung fibrosis than do wild-type mice. Cathepsins V, K, and S are the most efficient elastases described so far. These enzymes are coexpressed in activated macrophages of artherosclerotic plaques, in which 60% of the elastinolytic activity may be attributed to cysteine proteases. Therefore, it has been suggested that they play a crucial role in degradation of the elastin matrix. Further studies will show whether the same enzymes are involved in regulation of elastin turnover in the lung. In transgenic murine models employing the inducible expression of cytokines along the alveolar surface, the overproduction of IL-13 or interferon gamma caused a phenotype that mirrors human chronic obstructive pulmonary disease, with emphysema, enlarged lungs, and enhanced pulmonary compliance. These changes were accompanied by increased expression of matrix metalloproteinases and cathepsins in the lung. When mice were given the cysteine protease inhibitor E64, they showed markedly attenuated emphysematous changes, implying that cysteine proteases are important in the development of cytokine-induced emphysema. Lung cancer cells can secrete cathepsins L and B in vitro, and anticathepsin L antibodies protect against the formation of solid tumors by implanted myeloma cells in vivo. Therefore, it has been suggested that cathepsins may play a critical role in tumor invasion and progression. In addition, in a number of in vitro models using cells of different origin, it was shown that inhibition of cathepsin B or cathepsin L by specific inhibitors, or suppression of protease expression using antisense technologies, decreases the invasion of tumor cells into the extracellular matrix. Processing of Surfactant Proteins by Cysteine Proteases

The surfactant proteins SP-B and SP-C play an essential role in the metabolism and dynamics of the pulmonary surfactant lipids. Hereditary SP-B deficiency in infants or mice leads to respiratory failure at birth. Mutations in the SP-C gene and SP-C deficiencies are associated with interstitial lung disease. In addition, both hereditary alveolar proteinosis in infants without detectable mutations in the SP-B or the SP-C gene and acquired pulmonary alveolar proteinosis in children and adults are characterized by intraalveolar accumulation of mature surfactant proteins and abnormal SP-B precursors. Therefore, insufficient processing of proSP-B or proSP-C due to a lack or dysfunction of one or more proteases involved in surfactant protein processing may be another,

undiscovered, cause of surfactant dysfunction in pulmonary diseases in infants, children, and adults. The primary translation products of SP-B and SP-C (i.e., proproteins (proSP-B and proSP-C) with molecular masses of 42 and 21 kDa, respectively) undergo extensive C- and N-terminal processing in multivesicular bodies and lamellar bodies. The lysosomal protease cathepsin H is localized in lamellar bodies of type II pneumocytes. Immunoelectron microscopic investigations have shown that cathepsin H, proSP-B, and proSP-C are colocalized in multivesicular and lamellar bodies. In human fetal type II pneumocytes, the expression and enzymatic cathepsin H activity is upregulated during in vitro differentiation in parallel with surfactant protein secretion. Incubation of differentiated type II pneumocytes with E64, a potent inhibitor of cysteine proteases, interrupts SP-B processing and leads to additional features of SP-B deficiency, including abnormal SP-C processing and aberrant lamellar bodies. In addition, experiments using recombinant proSP-B have shown that cathepsin H may be involved in N- and C-terminal proSP-B processing. The SP-C processing by cathepsin H has been investigated in more detail in vitro, and it has been suggested that cathepsin H is involved in the N-terminal processing of proSP-C in electron-dense multivesicular bodies. In general, published results indicate that in vitro processing of proSP-B and proSP-C by cathepsin H alone does not result in the mature surfactant proteins; therefore, additional enzymes also seem to be involved in this complex processing cascade. Studies using knockout mice for different combinations of lysosomal proteases have been initiated to determine the in vivo relevance of cathepsin H and to characterize other enzymes involved in surfactant protein processing. Apoptosis

Evidence has accumulated that shows that lysosomal proteases play an important role in the regulation of apoptosis. Destabilization of the lysosomal membrane and release of proteases (cathepsins) trigger or support the process of apoptosis in various systems. The process was described to be the third pathway of apoptosis, in addition to the Fas/CD95- and the mitochondrial pathways. Cysteine proteases and cathepsin D play the most important roles in apoptosis. For example, it was shown in vitro and in vivo that cathepsin B is an important execution protease involved in apoptosis of tumor cells and hepatocytes. Cathepsin B/L double-knockout mice died from apoptotic neuronal loss.

CYSTIC FIBROSIS / Overview 599

Cathepsins in Respiratory Disease Cathepsins are specifically involved in processing and degradation of a variety of protein substrates and therefore regulate different cellular functions. Future studies involving genetically modified animals and specific inhibitors will determine more specifically the function of defined enzymes in vivo. Data suggest that cathepsins contribute to the pathogenesis of the following respiratory diseases, which may be influenced by the modification of cathepsin activity: *

*

*

Lung fibrosis and emphysema have been shown to be associated with insufficient or excessive cathepsin activity, respectively. Adequate regulation of cathepsin expression or activity may prevent tissue remodeling via the regulation of matrix remodeling and apoptosis of fibroblasts. Allergic immunoreactions may be suppressed by the selective inhibition of cathepsin activity via the suppression of antigen presentation and/or the shift of the antigenic epitopes that are presented by the accessory cells. Alveolar proteinosis and similar diseases may in part be caused by insufficient surfactant protein processing due to cathepsin insufficiency that leads to accumulation of surfactant proteins in the alveolar space.

See also: ADAMs and ADAMTSs. Apoptosis. Chronic Obstructive Pulmonary Disease: Emphysema, General. Interstitial Lung Disease: Alveolar Proteinosis; Amyloidosis; Idiopathic Pulmonary Fibrosis.

Proteasomes and Ubiquitin. Proteinase Inhibitors: Alpha-2 Antiplasmin; Antichymotrypsin; Cystatins; Secretory Leukoprotease Inhibitor and Elafin. ProteinaseActivated Receptors. Pulmonary Fibrosis. Serine Proteinases. Surfactant: Overview; Surfactant Proteins B and C (SP-B and SP-C). Transgenic Models.

Further Reading Barrett AJ, Rawlings ND, and Woessner JF (1998) Handbook of Proteolytic Enzymes. New York: Academic Press. Bromme D and Kaleta J (2002) Thiol-dependent cathepsins: pathophysiological implications and recent advances in inhibitor design. Current Pharmaceutical Design 8: 1639–1658. Bu¨hling F, Ro¨cken C, Brasch F, et al. (2004) Pivotal role of cathepsin K in lung fibrosis. American Journal of Pathology 164: 2203–2216. Bu¨hling F, Waldburg N, Reisenauer A, et al. (2004) Lysosomal cysteine proteases in the lung. Role in protein processing and immunoregulation. European Respiratory Journal 23: 620–628. Chapman HA, Riese RJ, and Shi GP (1997) Emerging roles for cysteine proteases in human biology. Annual Review of Physiology 59: 63–88. Lah TT and Kos J (1998) Cysteine proteinases in cancer progression and their clinical relevance for prognosis. Biological Chemistry Hoppe–Seyler 379: 125–130. Riese RJ and Chapman HA (2000) Cathepsins and compartmentalization in antigen presentation. Current Opinion in Immunology 12: 107–113. Turk V, Turk B, and Turk D (2001) Lysosomal cysteine proteases: facts and opportunities. EMBO Journal 20: 4629–4633. Wolters PJ and Chapman HA (2000) Importance of lysosomal cysteine proteases in lung disease. Respiratory Research 1: 170–177. Yan SQ, Sameni M, and Sloane BF (1998) Cathepsin B and human tumor progression. Biological Chemistry Hoppe–Seyler 379: 113–123.

CYSTIC FIBROSIS Contents

Overview Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

Overview + and J F Collawn, University of Alabama, Z Bebok Birmingham, AL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Cystic fibrosis (CF) or mucoviscidosis is the most frequent autosomal recessive genetic disorder in people with European ancestry. The classic form of CF results from loss of function

mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The gene is localized to the long arm of chromosome 7 and encodes a protein that acts both as a chloride channel and as a regulator of other transporters. Loss of CFTR activity results in a plethora of pathological consequences, suggesting a central role for CFTR in epithelial physiology. The disease is characterized by viscous secretions of the exocrine glands in multiple organs and elevated levels of sweat chloride. The most life-threatening clinical features of CF include chronic bacterial infections of the upper and lower airways, airway obstruction, bronchiectasis, respiratory failure, and cor pulmonale. The gastrointestinal manifestations are

600 CYSTIC FIBROSIS / Overview primarily caused by inefficient exocrine pancreatic function and include meconium ileus, maldigestion, and failure to thrive. Male infertility and reduced fertility in females are also important symptoms in adults with CF. Current therapy focuses on ameliorating the symptoms of malnutrition, airway obstruction, and inflammation. As a result of these symptomatic treatments, the survival improved dramatically. The new therapeutic approaches fall into two broad categories, gene replacement and targeted drug development.

Introduction Cystic fibrosis (CF) is a monogenic disorder with a complex phenotype and clinical variability. It is observed in approximately 1 in 2500 births among Caucasians, and has a carrier frequency of 1 in 25 individuals, making it the most common lethal monogenetic disease within this demographic group. CF is caused by mutations in a 180 kb gene localized to the long arm of chromosome 7. The gene encodes the 1480 amino acid cystic fibrosis transmembrane conductance regulator (CFTR) protein. CF cases present with complex phenotypes and clinical variability. Defects in CFTR lead to altered exocrine secretion and pathological changes in multiple organ systems. Therefore, CF provides an excellent example of the ways in which a single gene defect confers a wide variety of clinical symptoms. The most commonly affected organs include the airways, gastrointestinal (GI) tract, pancreas, sweat glands, hepatobiliary system, and genital tract. The most life-threatening clinical features of CF are recurrent and persistent airway infections leading to bronchiectasis, respiratory failure, and cor pulmonale. Pancreatic insufficiency is present in 90% of patients due to obstruction of the pancreatic ducts and subsequent fibrosis. Exocrine pancreatic dysfunction begins in utero and causes postnatal steatorrhea and failure to thrive. Meconium ileus is present in B10% of newborns with CF. CF patients also present abnormally high levels of chloride (Cl
) and sodium (Na þ ) in the sweat, which is considered as a diagnostic standard of the disease before genetic testing. In 95% of adult CF males, infertility is caused by azoospermia as a result of the complete bilateral absence of vas deferens (CBAVD). In females, reduced fertility may result from abnormally viscous intrauterine mucus. Other manifestations of the disease include liver cirrhosis and diabetes mellitus. Early references to CF describe a serious infant disorder characterized by unusual saltiness of skin. Detailed studies have been performed only since the beginning of the twentieth century, prior to which most infants died soon after birth. Landsteiner, who also defined the basic human blood groups, described cases of meconium ileus associated with defective

pancreas function. However, CF was first recognized as a distinct pathological entity in the 1930s by pathologist Dorothy Anderson, who defined one of the most important signs of the disease, the ‘‘cystic fibrosis of the pancreas.’’ After this report, Farber described CF as ‘‘generalized state of thickened mucus’’ and named it mucoviscidosis, a term that is still widely used in Europe. Anderson and Hodges discovered the autosomal recessive inheritance pattern of the disease in 1946, and suggested that CF was caused by a single gene defect. In the 1950s, Di Sant’Agnese and colleagues observed that levels of Cl
and Naþ were increased in the sweat of children with CF. This led to the development (in 1959) of the first accurate diagnostic test, the sweat test for cystic fibrosis. In the 1980s, two critical observations led to a better understanding of the basic pathophysiology of the disease. First, Knowles and colleagues found that respiratory epithelium in CF has defective Naþ and Cl
transport. Second, Paul Quinton described the sweat ducts of CF patients as impermeable to Cl
. The gene responsible for the disease was identified in 1989. This opened exciting possibilities for understanding the molecular basis of CF and provided new hope for potential therapies. Fifteen years later, this advance was further reinforced by the successful completion of the lowresolution structure of the CFTR protein, along with the high-resolution structures of mouse and human wild-type and DF508 NBD1 domains. Although there is no cure for CF to date, the life expectancy of CF patients has increased significantly. In the 1950s, few children with CF lived to school age; whereas today, patient survival extends to the mid-30s.

Etiology CF is caused by mutations of the CFTR gene encoding the CFTR protein. Mutations result in decreased levels or inefficient function of CFTR and subsequent pathological changes (Figure 1). CFTR is a multidomain, integral membrane glycoprotein consisting of two homologous halves. Each half contains six predicted transmembrane segments (TMD1 and TMD2) and a nucleotide-binding domain (NBD1 and NBD2). A regulatory domain (R domain) is interposed between the two halves of the protein. CFTR is part of a multiprotein assembly in the apical plasma membrane of epithelial cells (Figure 2). Although more than 1400 mutations (listed in the CFTR Mutation Database, see ‘Relevant websites’) are known in CFTR, deletion of phenylalaline at the 508 position (DF508) is the most frequent cause of the disease and contributes to over 90% of CF cases. Other mutations are often linked to certain

CYSTIC FIBROSIS / Overview 601

geographical regions or are unique. Only four of them (G542X, N1303K, G551D, and W1282X) have a frequency above 1%. CFTR gene mutations are classified into six broad groups based on their impact on CFTR synthesis and

CFTR gene mutations

Absent or defective CFTR protein

Altered secretion in exocrine glands

Impaired mucosal defense

Infections Figure 1 Etiology and pathogenesis of cystic fibrosis. Summary of how mutations in the CFTR gene lead to infections.

intracellular trafficking (Figures 3(a) and 3(b)). Class I, nonsense, and frameshift mutations abolish CFTR synthesis. Examples include the G542X and R1162X nonsense mutations that encode premature stopcodons. These mutations result in truncated mRNA transcripts that are unstable. Mutations that result in low CFTR levels (Class V) include missense defects such as A455E, and promoter and splicing abnormalities. Class I and V mutations result in little or no protein product. Class II mutations include the most frequent mutation, DF508. Deletion of phenylalanine at the 508 position leads to the synthesis of a protein that is recognized by the ER quality control system as misfolded and degraded by the proteasome after retrotranslocation from the ER. Class III mutations lead to the production of CFTR that reaches the plasma membrane, but has defective regulation and function. G551D, a missense mutation within NBD1, is an example of this class and the third most common disease-associated mutation. The G551D mutant is unable to conduct chloride in response to cAMP. Other mutants in this category exhibit ineffective nucleotide binding or hydrolysis (S1255P, G551S, G1244E, and G1349D). Class IV mutants, such as the R117H and R347P mutants, result in the synthesis of

Glycan chains

COOH Syntaxin 1A

EC

TMD1

TMD2

Cell membrane

IC YSDI

H3 H2

NBD1

NH 2

NBD2

AP-2

COPII NH2 Blocked by Munc-18

R domain Syntaxin 8

DTRL

EBP50

H1

E3KARP CAL CAP70

Ezrin PKA Actin

Figure 2 Model of CFTR and interacting proteins. The CFTR molecule is an integral membrane glycoprotein composed of 12 membrane-spanning segments forming two transmembrane domains (TMD1 and TMD2), two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain (R domain). Syntaxin 1A binds to the N-terminal tail and inhibits CFTR function. This interaction can be blocked by Munc-18. N-linked glycosylation sites are located on the fourth extracellular loop. CFTR C-terminal tail associates with PDZ domain containing proteins (ezrin-radixin-moesin (ERM)-binding phosphoprotein, EBP50; Na þ /H þ exchanger 3 kinase A regulatory protein, E3KARP; CFTR-associated ligand, CAL; CFTR-associated protein of 70 kDa, CAP70) through the DTRL sequence. These interactions influence function or trafficking, or both. Sequential interactions through EBP50, ezrin, and protein kinase A (PKA) anchor CFTR to the actin cytoskeleton. The adaptor complex II (AP-2) interacts with CFTR’s internalization signal, YSDI, to promote CFTR endocytosis. COPII binds to NBD1 and facilitates CFTR exit from the ER. Syntaxin-8, a t-SNARE protein that associates with CFTR through the R-domain, facilitates CFTR recycling. EC, extracellular space; IC, intracellular space.

602 CYSTIC FIBROSIS / Overview −

Cl

Cl − TJ

6 4

5

7

ES

Endosome

Carrier vesicle 8

LS Lysosome

Golgi

ER

3

A

AA

Proteasome

2

1

Nucleus (a)

−

Cl

Class IV: altered conductance

Class III: altered regulation

TJ

Class VI: inefficient recycling

Golgi

ER

Class II: block in processing

Class V: reduced synthesis Class I: no synthesis Nucleus (b) Figure 3 (a) Model for CFTR synthesis, trafficking, and degradation. 1, Synthesis and co-translational insertion of CFTR into the endoplasmic reticulum (ER) membrane; 2, proteasomal degradation of misfolded CFTR; 3, ER–Golgi processing, glycosylation; 4, vesicular trafficking to the cell surface; 5, endocytosis through clathrin-coated pits; 6, recycling to the cell surface; 7, lysosomal; and 8, proteasomal degradation. (b) Classes of CFTR mutations. Class I: defective protein production (G542X and R1162X); Class II: defective protein processing (DF508); Class III: defective regulation (G551D); Class IV: defective conduction (R117H, R347P); Class V: decreased levels of CFTR (A455E); and Class VI: unstable CFTR. TJ, tight junction; ES, endosome.

CYSTIC FIBROSIS / Overview 603

a CFTR protein with defective chloride conductance. The most recently identified class of CFTR mutants (Class VI) maintain normal biosynthetic processing and chloride channel function of the truncated CFTR, but the biological stability of the fully processed protein is dramatically reduced.

Pathology Respiratory Tract

Histological signs of CF lung disease have been observed as early as the third trimester, with the obstruction of submucosal gland ducts by thick mucus. Recurring pulmonary exacerbations, due to repeated cycles of infection and inflammation, result in lung damage and compromise pulmonary function. Inflammation is present at an early age and remains active even in clinically stable patients. As a result, pulmonary fibrosis, bronchiectasis, respiratory insufficiency, and cor pulmonale remain the hallmarks of CF pathology and the primary cause of morbidity. Gastrointestinal Tract

The pathological features of the gastrointestinal tract are linked to reduced exocrine pancreatic functions and inadequate intestinal absorption. Neonatal meconium ileus, a diagnostic feature of CF, occurs only in 10% of cases. Distal intestinal obstruction syndrome (DIOS), constipation, megacolon, rectal prolapse, and pancreatic fibrosis develop later during adolescence. Pancreatic insufficiency is present in approximately 90% of CF subjects. Pancreatic fibrosis leads to an increased incidence of diabetes mellitus. Diabetes affects B1% of children and 15% of adults with CF. Hepatobiliary System

Hepatic dysfunction increases with age and 10–15% of adolescents and adults with CF have liver cirrhosis. Genital Tract

Azoospermia is present in 95% of adult CF males as a result of CBAVD and consequential dilated or absent seminal vesicles. In women, there is reduced fertility that is caused by abnormally viscous intrauterine mucus.

Clinical Features Diagnosis

CF cases represent a great variety of clinical symptoms that vary from patient to patient and by age groups. In the majority of cases, CF is diagnosed

before adolescence, but some remain asymptomatic until adult age. In 10% of newborns with CF, meconium ileus is the first diagnostic sign. Later, the symptoms are related mainly to respiratory infections and intestinal malabsorption. The classical diagnostic sweat test for CF involves the biochemical detection of high levels (4100 mg ml
1) of sodium and chloride in a sweat sample collected following pilocarpine iontophoresis. The excessive loss of electrolytes may result in heat exhaustion in hot climates. Diagnosis can be confirmed by genetic testing of blood or buccal cells. The classic clinical symptoms of CF are summarized in Table 1. Airway Infection and Inflammation

At birth, typically there are no significant signs of lung disorder in infants with CF. During early childhood, however, cough and thick mucus production, impaired mucociliary clearance, and high susceptibility to infections begin to manifest themselves. Chronic upper airway infections and the production of thick, viscous mucus result in sinus obstruction and nasal polyposis. Pulmonary infection and inflammation in the CF lung are caused by surprisingly few bacterial pathogens. Pseudomonas aeruginosa is the most common isolate (80%), followed by Staphylococcus aureus (51%), Haemophilus influenzae (17%), Methicillin-resistant Staphylococcusaureus (MRSA) (12%), and Stenotrophomonas maltophilia (11%). Viral infections with respiratory syncytial virus (RSV), Table 1 Clinical features of cystic fibrosis Respiratory tract Chronic respiratory tract infection Recurrent wheeze Bronchiectasis Nasal polyposis Chronic sinusitis Cor pulmonale Hepatobiliary system Liver cirrhosis Genital tract Male infertility (90%, CBAVD, azoospermia) Reduced fertility in females (20%) Gastrointestinal tract Meconium ileus Steatorrhea Distal intestinal obstruction syndrome Vitamin deficiency Failure to thrive Rectal prolapse Diabetes mellitus Electrolyte imbalance High sweat sodium and chloride Excessive salt loss in sweat Systemic electrolyte imbalance

604 CYSTIC FIBROSIS / Overview

influenza, and adenovirus are also frequent in children with CF. Therefore, CF should be considered in any young child with persistent symptoms following infection with these pathogens. Chronic lower airway infections leading to fibrosis, bronchiectasis, respiratory insufficiency, and cor pulmonale remain the hallmarks of morbidity in CF. Gastrointestinal and Pancreatic Manifestations

CF patients suffer from gastrointestinal problems related to inadequately controlled intestinal absorption and pancreatic insufficiency. These include meconium ileus in newborns, steatorrhea, DIOS, constipation and acquired megacolon, rectal prolapse, pancreatitis, and failure to thrive during adolescence. Genital Manifestations, Fertility

Ninety-five per cent of adult CF males are infertile as a result of CBAVD and consequential dilated or absent seminal vesicles. Although B80% of adult female CF patients are fertile, reduced fertility may result from abnormally viscous intrauterine mucus, that is, o80% water in contrast to the normal 93– 96% hydration that appears to be necessary for sperm migration.

Pathogenesis Ion channels selectively expressed in the apical or basolateral membrane domains of epithelial cells regulate ion composition and hydration of secreted material. Discrepancies in the expression of these ion transporters engender impaired salt composition and aberrant hydration of the glandular fluid. CFTR is a cAMP-regulated, bidirectional Cl
channel primarily expressed in the apical membranes of epithelial cells in a variety of organs including the respiratory tract, gastrointestinal tract, exocrine secretory glands, kidneys, and bile ducts. Tissue specificity is known to be acquired both at the level of transcription and by expression of CFTR splice variants. Interestingly, CFTR mRNA, protein, and functional activity have also been demonstrated in T and B lymphocytes, red blood cells, and cardiomyocytes. Based on its broad expression pattern, it is apparent that CFTR has a central role in electrolyte transport regulation in a number of tissues and cell types. The current concepts regarding CF pathogenesis can be summarized based on the primary functions of CFTR. CFTR as Ion Transporter

Pathogenesis of sweat abnormalities in CF The direction of chloride movement through CFTR

depends on the function of the epithelia in which CFTR is expressed. In the apical surface of the secretory coils of sweat glands, CFTR controls Cl
efflux. Movement of Cl
promotes H2O movement and hydration of the secretum. In contrast, in the ducts of the sweat glands, Cl
is reabsorbed through CFTR. Because the secretory coil cells express either CFTR or other (non-CFTR, Ca2 þ -activated) anion channels, in the absence of CFTR, Cl
can still be secreted in the secretory coils. However, Cl
(and consequently Na þ ) reabsorption are deficient because in the duct cells, CFTR is the only chloride channel able to reabsorb Cl
(Figure 4). Pathogenesis of airway abnormalities in CF Because of the multiple physiological functions of CFTR, several models have been proposed to link CFTR mutations to the development of airway infection. Results from CF mice, raised under germfree conditions and from infants with CF studied by polymerase chain reaction (PCR) or other sensitive pathogen detection methods, suggest that inflammation precedes infection. Not all studies, however, are in agreement with this hypothesis and support the idea that infection precedes inflammation. Alterations in salt and fluid secretion The development of infection in the CF lung may result from alterations in salt and fluid secretion. Two models have been put forward to explain how fluid and electrolyte secretion alterations may contribute to lung infections. Under physiological conditions, the function of CFTR in the apical membrane of airway epithelial cells and serous secretory cells is to secrete or reabsorb Cl
(Figure 5(a)). Intracellular ion conditions are maintained mainly through the action of the basolateral sodium–potassium pump and other ion transporters. Under basal conditions, both serous acinus cells and surface epithelial cells secrete Cl
, Naþ , and H2O to hydrate the airway surface liquid (ASL). Physiological ASL volume and salt concentration is maintained through regulated Naþ and Cl
reabsorption through ENaC and CFTR. The first hypothesis, referred to as the ‘low-volume hypothesis’, suggests that in the absence of CFTR, ENaC is hyperactive, resulting in increased Na þ absorption from the periciliary fluid layer. This causes increased water absorption from the airways and leads to isotonic, but diminished airway surface fluid. As a result, the airway fluid is poorly hydrated (Figure 5(b)). Infections follow because the bacteria are not cleared and become trapped in the viscous surface fluid. A second model termed the ‘high-salt hypothesis’ is based on the assumption that under normal

CYSTIC FIBROSIS / Overview 605 Skin surface TJ

Cl− Na+

Na+

Na+

cAMP

K+

S

Cl−

Cl−

Duct (absorption)

K+

TJ Duct cell – electrolyte reabsorption Cl− Na+

H2O

TJ

TJ Cl−

Cl−

K+

Ca2+

cAMP

S

Cl− Na+ K+

CFTR

Na+

Na+ Na+

TJ

TJ

Secretory cells – fluid and electrolyte secretion

S

Secretory unit

Cl− Na+ K+ K+

Figure 4 CFTR function in sweat glands. A predominant function of CFTR in the secretory coils of sweat glands is to secrete Cl
. In contrast, CFTR in the apical membrane of the sweat gland ducts primarily reabsorbs Cl
. The secretory coils of sweat glands secrete electrolytes and fluid that is regulated mainly through the action of basolateral (sodium–potassium pump, Cl
and K þ transporters) and apical (ENaC, CFTR, Ca2 þ activated) ion channels. Naþ and Cl
are reabsorbed from the ducts of the sweat glands. Because the secretory coil cells express either CFTR or other (non-CFTR, Ca2 þ -activated) anion channels, in the absence of CFTR, Cl
can still be secreted in the secretory coils. However, Cl
(and consequently Naþ ) reabsorption is deficient because in the duct cells, CFTR would be the only chloride channel able to reabsorb Cl
. TJ, tight junction.

conditions, airway and submucosal gland epithelial cells behave in a similar way to sweat glands and absorb more Cl
and Naþ than water, resulting in hypotonic airway surface liquid. In CF, CFTR is missing or functionally defective, and therefore Cl
absorption is decreased, resulting in higher luminal Cl
and Naþ concentrations than in healthy individuals (Figure 5(c)). This model is supported by the finding that defensins are inactivated under high salt conditions, and loss of defensin activity may predispose patients to bacterial infections. Distinguishing between these two possible models continues as an ongoing debate.

CFTR as Receptor

Loss of the CFTR protein has also been linked to altered cell surface protein sialysation, resulting in increased asialo-GM1 molecule expression. This result has significance because asialo-GM1 is a receptor for a number of respiratory pathogens including P. aeruginosa and S. aureus. CFTR itself has been proposed as receptor for P. aeruginosa, suggesting that epithelial cells without CFTR are unable to bind, internalize, and clear this pathogen.

CFTR as Regulator of Other Transporters

CFTR plays a central role not only in secretion of chloride and bicarbonate ions, but also regulates a number of other ion channels. These regulatory functions appear to be tissue-and cell type-specific, and reflect the ion channel expression profiles of the specific cells. CFTR has been shown to regulate Naþ reabsorption by inhibiting the activity of the epithelial Naþ channel, ENaC, suggesting that CFTR defects may lead to disturbances of both Na þ secretion and absorption. It has been proposed that the lung pathology in CF is primarily due to disregulation of sodium transport through ENaC. Supporting this hypothesis, transgenic mice with airway-specific overexpression of ENaC (i.e., increased Naþ reabsorption), but normal CFTR levels, have recently been shown to develop mucus obstruction, goblet cell metaplasia, neutrophilic infiltration, and poor bacterial clearance, that are the hallmarks of CF lung disease. In addition to the evidence that CFTR conducts HCO3
, it also regulates HCO3
transport through the HCO3
/Cl
exchanger. In the intestinal lumen and pancreatic ducts, secretion of fluid containing high concentrations (100–140 mM) of HCO3
is

606 CYSTIC FIBROSIS / Overview

Na +

Cl −

H2O Na +

Normal airway submucosal gland ASL

Cl − Cl − cAMP

Ciliated, surface cells

Ca 2+ K+

Na+

S

Mucussecreting cells

2Cl − K + Na + Serous secretory cells

(a)

K+

CFTR

Na +

Na + CF airway submucosal gland and surface epithelial cells

H2O H2O

X

Ciliated, surface cells

Cl −

Cl −

Low ASL

Ca 2+

cAMP Na+

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Mucussecreting cells

2Cl − K + Na +

Serous secretory cells

K+

(b) Cl −

Na + CF airway submucosal gland and surface epithelial cells NaCl

NaCl

NaCl

ASL

Na +

X

H2O Cl −

Cl −

NaCl

Ca 2+

cAMP NaCl

(c)

Na+

K+

S

Mucussecreting cells

Ciliated, surface cells

Serous secretory cells

2Cl − K + Na +

K+

Figure 5 (a) CFTR function in the airways. The function of CFTR in the apical membrane of airway epithelial cells and serous secretory cells is to secrete or reabsorb Cl
. Intracellular ion conditions are maintained mainly through the action of the basolateral sodium– potassium pump and other ion transporters. Under basal conditions, both serous acinus cells and surface epithelial cells secrete Cl
, Naþ , and H2O to hydrate the airway surface liquid (ASL). Physiological ASL volume and salt concentration is maintained through regulated Naþ and Cl
reabsorption through ENaC and CFTR. The ASL is cleared by mucociliary clearance and evaporated. (b) Lowvolume hypothesis. The low-volume airway surface liquid (ASL) hypothesis argues that both the airway surface cells and serous secretory cells of the acini secrete Cl
. In the absence of CFTR, Cl
secretion is diminished; ENaC is hyperactive and absorbs more Naþ than under physiological conditions. This results in increased salt and water reabsorption and loss of ASL fluid volume. In CF, the surface epithelial cells are flattened and the ducts of the submucosal glands are plugged with viscous mucus. (c) High-salt hypothesis. The highsalt hypothesis argues that CFTR is the predominant pathway for Cl
reabsorption from the airways and in the absence of CFTR, Cl
cannot be absorbed from the airways. However, Cl
can be secreted through alternative Cl
channels resulting in hyperpolarization of the luminal surface. As a consequence, Naþ reabsorption is inefficient without a counter ion and NaCl accumulates in the ASL.

CYSTIC FIBROSIS / Overview 607

necessary to maintain the solubility of mucins and the inactive state of digestive enzymes. The majority of bicarbonate transport in these organs is achieved through an epithelial Cl
/HCO3
exchanger, regulated by CFTR. This particular CFTR function is therefore likely to have important physiological implications, and may help explain the severely impaired HCO3
secretion in CF. Chloride channels other than CFTR, including Ca2 þ, activated and outwardly rectified anion channels require the presence of CFTR in the plasma membrane. Loss of CFTR has profound effects on chloride transport both directly and indirectly through these pathways, compounding the defects in epithelial chloride transport that result from loss of CFTR. Another important cellular effect of CFTR is volume regulation through the Ca2 þ -dependent potassium channel, KCNN4. During regulatory volume decrease (RVD), anion and cation channels are activated, permitting the passive loss of inorganic ions and osmotically obliged water. In a CFTR knockout mouse model, cell volume regulation in jejunal crypts is deficient as a consequence of dysfunctional KCNN4. The activity of other K þ channels such as ROMK2 and KvLQT1 have also been shown to be modulated by CFTR expression. To make matters worse, proper cell volume through aquaporin 3 also appears to be dependent on CFTR. Finally, the activity of gap junction channels is affected by functional CFTR expression, indicating that CFTR plays a significant role in maintaining electrolyte transport not only across individual cells, but also across the epithelial monolayer as a whole. CFTR and Gene Expression Regulation

Because of the difficulties that have been experienced in attempting to correlate CF genotype and pulmonary disease severity, alternative functions of CFTR remain of considerable interest. One of the hallmarks of CF is enhanced expression of proinflammatory mediators in CF lungs. For example, reduced Smad3 expression has been reported to selectively alter TGFb1-mediated signaling in CF epithelium, and CF epithelial cells may be functionally deficient in IL10, a key anti-inflammatory cytokine that controls expression of the inhibitory subunit, IkB. The presence of cell surface CFTR has also been shown to be necessary for RANTES (regulated upon activation, normal T-cell expressed, and presumably secreted) expression, a chemokine that selectively influences the migration of monocytes, CD45 RO þ memory T lymphocytes, and eosinophils. Furthermore, CF airway epithelial cells fail to express RANTES in response to P. aeruginosa, the predominant bacterium

observed in CF patients. Although the complete list of inflammatory genes affected by CFTR expression in different tissues remains incomplete, this area of research may explain the multifaceted nature of CF lung disease and will remains an important aspect for future studies.

Animal Models Important structural and functional characteristics of CFTR have been discovered using cell culture models. Despite the progress made, animal models are necessary to investigate the pathogenesis of CF and evaluate novel therapeutic strategies. After cloning the CF gene in 1989, a novel approach was applied to generate mouse strains with mutations in the mouse Cftr gene. Mutations to specific sites in the mouse genome were targeted using homologous recombination in mouse embryonic stem cells. The mutant embryonic stem cells were then injected into blastocysts to create chimeric mice. Chimeric mice were bred to obtain individuals homozygous for the null mutation. Several different CF mouse strains were developed using this approach. A comprehensive list of models can be found in the Virtual Repository of Cystic Fibrosis European Network. Gene Targeted (‘Knockout’ or KO) and Residual Function CF Mouse Models

Gene targeted (KO) mouse models were created using a method called ‘replacement strategy’. An interruption that was introduced to the mouse Cftr gene resulted in complete loss of CFTR mRNA and protein. Residual function models were created using the ‘insertion strategy’. These models, despite the interruption in the Cftr genome may produce some CFTR mRNA and demonstrate minimal residual CFTR activity. Although these models represented an important step in CF research, it was apparent that the artificial mutations would not accurately model naturally occurring mutations. Mouse Models with Naturally Occurring, DiseaseCausing Mutations in the Mouse Cftr Genome

New mouse strains with naturally occurring mutations, most importantly those with DF508, G480C, (Class II), and G551D (Class III) mutations, have been developed. These models were created either by the ‘exon 10 replacement’ strategy or by the ‘hit and run’ procedure. The ‘exon replacement’ strategy uses a selection marker inserted into one of the introns that regulates the activity of the transcription of the mutant gene. The ‘hit and run’ method results in a

608 CYSTIC FIBROSIS / Overview

mutant exon only and the transcription and activity of the mutant allele is therefore identical to the normal allele. It has been shown that the DF508 mutation in the mouse Cftr sequence caused a similar processing defect as in the human protein. Transgenic Mouse Models Expressing Human CFTR

Transgenic mouse models were constructed on a ‘knockout’ background with a null mutation in the endogenous CFTR locus (Cftr
/
). First, the human wild-type CFTR transgene was expressed in the intestinal tract under the control of the fatty-acidbinding protein (FABP) promoter from rat. These mice have an improved survival rate compared to the null mutants. Using a similar approach, a second transgenic mouse model was developed expressing human CFTR carrying the G542X mutation. These mice have been used to study the effects of different compounds on the suppression of this premature stop mutation. Differences in the Phenotypic Properties of CF Mouse Models and Human CF

Because a number of laboratories have created CF mouse models, it is important to note that although the phenotypic hallmarks are the same, important differences between models with the same mutation have been observed. These variations relate to the genetic background and a number of other factors. In general, intestinal disease is the most prominent feature of CF mice and the symptoms are comparable to CF in humans. Because of the severity of intestinal obstruction, CF mice are kept on a liquid diet to improve survival. The pancreatic involvement in CF mice is less severe than in humans. The most striking differences regarding the respiratory defect between the mouse models and human CF were described in studies. Initial characterization of CF mice showed no significant lung disease. Later, pulmonary abnormalities were described in mice bred on a different genetic background, suggesting the presence of genetic modifiers. However, despite extensive studies, an appropriate model for CF lung disease in CF mice has proved challenging. Pathological changes in the lungs of CF mice resemble pulmonary fibrosis rather than abnormalities seen in human patients with CF. It appears that there are additional Cl
transporters in mouse lungs that make modeling of the human disease incomplete. Interestingly, infection of CF mice with Pseudomonas promotes some changes consistent with CF. Although the suitability of mouse models for CF lung disease remains controversial, some of the models

have already been used to test new therapeutic interventions. Other Animal Models

Sheep, pig, and ferret models are being considered because the structure of their lungs resembles the human lung more. These models may become more important in the future for testing new therapeutic interventions.

Therapy Traditionally, CF treatments have focused on ameliorating symptoms of intestinal obstruction, malnutrition, airway obstruction, infection, and inflammation. Pancreatic enzyme replacement decreased the severity of these symptoms significantly, resulting in improved survival, and increased the importance of ambulatory care. Increased understanding of CF pathophysiology and genetic defects provide a conceptual basis for targeted drug development. New therapeutic approaches fall into two broad categories: gene replacement and drug development (CFTR repair). In theory, the gene replacement therapies could be applied to all mutations, whereas drugs target specific mutations. Compounds intended for CFTR repair are being screened to rescue DF508 from the ER quality control (Class II mutants), to stimulate defective channels at the cell surface such as G551D (Class III mutants), or to promote readthrough of premature stopcodons for mutants such as W1282X (Class I mutants).

Preventive and Symptomatic Treatments The increased life expectancy of CF patients is a direct result of the improved preventive care and antibiotic treatments against respiratory pathogens. A variety of general and transmission-based, CFspecific infection control guidelines are now being implemented. General precautions focus on the potentially infectious nature of body fluids, particularly respiratory tract secretions. Transmission-based precautions, on the other hand, prevent transmission of multidrug-resistant organisms such as Burkholderia cepacia, P. aeruginosa, or viruses such as RSV, influenza, and adenovirus by employing procedures such as maintaining sterilized respiratory equipment, segregating patients, and vaccinating young CF patients. These measures have decreased the severity of lung disease and resulted in a dramatic improvement in the patients’ health status. Antibiotic Treatments

S. aureus and H. influenzae are often the first agents to infect patients with CF. In the short term,

CYSTIC FIBROSIS / Overview 609

treatment of these pathogens with oral antibiotics is typically effective. Later in the course of the disease, P. aeruginosa (particularly the mucoid strains) become predominant. Mucoid P. aeruginosa is remarkably resistant to even intensive antibiotic regimens in part because antibiotics fail to penetrate bacterial biofilms. A specifically formulated tobramycin (TOBI) solution developed for use in inhalers provides a high-dose aminoglycoside antibiotic in the lungs that is effective against P. aeruginosa. TOBI has been shown to increase lung function in CF patients, with less toxicity compared to systemic aminoglycoside treatments. Macrolide antibiotics are well tolerated by CF patients. One member of this class, azithromycin, exhibits broad antibacterial activity and has also been shown to decrease neutrophil chemotaxis, inhibit the expression of proinflammatory cytokines, and interfere with biofilm formation by P. aeruginosa. It is clear that the use of combined antibiotic therapy during acute exacerbations significantly reduces the development of resistant organisms and chronic airway infections. To date, however, no consensus has been reached regarding the possible benefit of periodic antibiotic administration in the absence of overt pulmonary exacerbation. No significant advantage of this type of treatment (compared to symptomatic antibiotic therapy) has been shown for this type of therapy.

intrapulmonary percussive ventilator (IPV) to help loosen thick, mucous plugs within the airways. Similarly, aerosolized medicines such as albuterol, ipratropium bromide, and Pulmozymes (dornase alfa) are used to solubilize and break up CF airway secretions. Lung Transplantation

Lung or heart–lung transplantation is an important option for CF patients with severe lung disease. Based on present criteria, patients are considered for transplant waiting lists when the forced expiratory volume in s (forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC)) falls below 30%. While precedence should be given to patients with the most severe symptoms, due to the long waiting period for a transplant, patients should be evaluated well before FEV1/FVC falls below 30%. Contraindications for transplantation include ventilator dependence, drugresistant bacterial infection, and poor overall health/ nutritional status. Based on recent reports, this treatment increases the 5-year survival rate from o30% to 66% and perhaps more importantly, dramatically improves quality of life.

Treating the Fundamental CF Defect – Prospects for CF Interventions Gene Therapy

Anti-Inflammatory Therapy

Both steroid and nonsteroid anti-inflammatory drugs have been used in CF treatments with limited benefits. Although treatment with steroids may improve function, they also have a number of deleterious side effects (growth retardation, glucose intolerance, and cataract development). High doses of nonsteroidal anti-inflammatory drugs such as ibuprofen have also been used to treat CF lung disease and have been shown to reduce the decline in lung function. Based on altered pharmacokinetics of ibuprofen in CF patients and the potential adverse effects, however, the widespread use of anti-inflammatory agents of this sort has been limited. Physiotherapy and Mucolytic Treatment

A major goal regarding CF therapy is to prevent the development of pulmonary hypertension and consequent cor pulmonale. Daily chest physiotherapy and aerosol inhalation are often used to improve oxygen delivery. A typical physiotherapy session involves either manual chest percussion (pounding), or use of a device such as the ThAIRapy vest or the

Since the discovery of the CFTR gene in 1989, gene replacement strategies have been sought using both nonviral and viral vectors. Proof-of-principle in the lung has been established and progress achieved in understanding the barriers preventing efficient gene transfer to surface airway epithelium. A number of gene therapy clinical trials have been completed, although none with positive, long-term effects. Newly developed adeno-associated and lenti-viral vectors provide continuing hope for gene therapy-based CF interventions in the future. Translational Readthrough, Rescue Agents, and Channel Modulators

Treatment of premature stop mutations with aminoglycoside antibiotics is based on the finding that these agents promote translational readthrough. Although clinical results from using this strategy have been promising, mutations that are treatable with this approach (such as W1284X) are rare. Agents that ‘rescue’ the common DF508 mutant from the ER quality control are of particular interest. A number of laboratories and pharmaceutical and academic centers

610 CYSTIC FIBROSIS / Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

have launched high throughput screening programs to identify compounds that correct CFTR folding. Similar studies concentrating on modulators of dysfunctional CFTR mutants such as G551D are being tested in preclinical and clinical trials. It is likely that a combination of several of these approaches will be required to overcome the CFTR defects. For example, treatment of patients with DF508 CFTR may require both, rescue of the protein from ERAD and methods or agents that activate the channel and stabilize the protein at the apical surface. The past few years have provided significant advancements in our understanding of the CFTR protein, and future prospects for alleviating many of the symptoms of CF are at hand. Establishing new interventions based on molecular understanding of CF defects will require further insights regarding CFTR regulation, processing, and protein–protein interactions. Having said this, the rapid progress over the past few years regarding CFTR structure and function suggests the possibility of a number of promising therapies in the near future. See also: Bronchiectasis. Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Defensins. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia. Mucins. Mucus. Oxygen Therapy.

Further Reading Kirk KL and Dawson DC (2003) The Cystic Fibrosis Transmembrane Conductance Regulator. New York: Kluwer Academic/ Plenum. Riordan JR (2005) Assembly of functional CFTR chloride channels. Annual Review of Physiology 67: 29.1–29.18. Welsh MJ, Ramsey B, Accurso FJ, and Cutting GR (2001) Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, et al. (eds.) The Metabolic and Molecular Bases of Inherited Disease, 8th edn., pp. 5121–5188. New York: McGraw-Hill. Yankaskas JR, Marshall BC, Sufian B, Simon RH, and Rodman D (2004) Cystic Fibrosis Adult Care, Consensus Conference Report. Chest 125: 1S–39S.

Relevant Websites http://www.genet.sickkids.on.ca – This is a database of mutations in the CFTR gene and is currently maintained by the laboratory of Lap-Chee Tsui. http://central.igc.gulbenkian.pt – This is a database maintained by the laboratory of Dr Margarida Amaral that provides information on CFTR expression, function, and detection. http://www.cff.org – This is a patient registry that was started by the CF Foundation to track the condition of patients with CF in the US. The registry is updated yearly by the CF Foundation. http://www.sciencedirect.com – The cited issue of the Journal of Cystic Fibrosis summarizes the consensus work of the European Cystic Fibrosis Network and provides detailed information about CFTR structure, function, genetics, and disease models.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene R G Crystal and R G Pergolizzi, Weill Medical College of Cornell University, New York, NY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Cystic fibrosis (CF), the most common autosomal recessive disorder in Caucasians, primarily manifests in epithelial cells in the gastrointestinal system, airways, reproductive system, and sweat glands. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a 230 kb, 29 exon gene on chromosome 7q31.2. The 480 amino acid singlechain CFTR protein functions as a cyclic AMP-regulated chloride channel on the surface epithelium. Over 1000 different CFTR mutations have been identified and although there has been some success correlating CFTR genotype and the severity of CF disease, especially with regard to pancreatic disease, many questions remain unresolved. CF pulmonary disease is highly variable among individuals with the same genotype, most likely arising from the presence of modifier genes and environmental factors. This article summarizes the structure and function of the CFTR gene and the protein, the current classification of CFTR mutations, and the relationship of those classes to CF disease phenotype.

Introduction Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasians, with a frequency of about 1 in 2500 live births. The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel in the apical membrane of epithelial cells. The CF phenotype is characterized by pulmonary, pancreatic, gastrointestinal, and reproductive abnormalities. The greatest impact of mutations of the CFTR gene is on pulmonary disease, accounting for most morbidity and deaths. The relationship between specific mutations in CFTR and severity of CF is very complex, leading to broad variability in symptoms of disease. Improved treatment modalities for CF have improved life expectancy for patients over the last two decades. Data from the US registry show that median age at death in patients with CF has doubled in the last 20 years to mid-30s in 2005. The major clinical phenotype of CF is poor mucociliary clearance with chronic airway bacterial infection, excessive mucus secretion, and obstructive lung disease. Most affected individuals (495%) have pancreatic insufficiency, and a high level of male infertility results from the absence or obstruction of the vas deferens. Infants with CF are likely to present with meconium ileus, obstruction of the bowel with inspissated secretions.

CYSTIC FIBROSIS / Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene 611

In this article we summarize the available information regarding the advances in understanding the CFTR gene and its mutations, and the effects these have on CFTR function and the clinical CF phenotype.

of CFTR channels is linked to ATP hydrolysis. CFTR gating activity is acutely modified by phosphorylation. Modulation of gating activity of the CFTR chloride channel is regulated by kinases, especially protein kinase A (PKA), and phosphatases. However, the impact of PKA on CFTR gating activity is not an allor-nothing process, since biochemical measurements show that PKA readily phosphorylates the CFTR to a stoichiometry of at least five ATPs per CFTR molecule, and incremental changes in gating appear with each additional event. Most of the residues in CFTR that become phosphorylated are located in the large cytoplasmic R domain. Deletion of the R domain renders CFTR constitutively active in the absence of phosphorylation. Therefore, it is likely that the R domain plays a role in regulating the gating activity of CFTR and that this regulation depends on the phosphorylation state of this domain. Potential phosphorylation sites outside the R domain have also been identified, and one in NBD1 at G551 has been linked to particularly severe symptoms of CF. Adenine nucleotides, magnesium ion concentration, and inorganic phosphate also impact the rate and duration of opening of the chloride channel.

CFTR Structure and Organization The human CFTR gene occupies 230 kb on the long (q) arm of human chromosome 7 at region q31.2, encoding an mRNA transcript of 6129 bp and a single-chain 480 amino acid polypeptide, which functions as a chloride channel in membranes of epithelial cells of the lung, liver, pancreas, intestines, reproductive tract, and sweat glands. The CFTR protein is made up of five domains: two transmembrane domains (MSD1 and MSD2) that form the chloride ion channel, two nucleotide-binding domains containing several phosphorylation sites (NBD1 and NBD2) that bind and hydrolyze ATP, and a regulatory (R) domain (Figure 1). Expression of the CFTR protein confers cyclic adenosine monophosphate (cAMP)-regulated chloride conductance in nonepithelial cells, and reconstitution of purified CFTR protein into lipid bilayers generates cAMPactivated chloride channels with properties similar to those in CFTR-expressing cells.

CFTR Mutations Over 1000 mutations in the CFTR gene have been described. There is variability in why these mutations cause CF, but all result in a loss of chloride ion transport, which upsets the sodium and chloride ion

CFTR Activity and Regulation The CFTR protein itself comprises a metabolically gated chloride ion pore, and the opening and closing

MSD1

MSD2

Cell membrane

P

P

N P

R domain

P

P NBD2 NBD1 ATP ADP

ATP

C

ADP Figure 1 Schematic of the CFTR protein with respect to the epithelial cell membrane. The major domains of CFTR are shown, including the membrane-spanning domains (MSD1 and 2), nucleotide-binding domains 1 and 2 (NBD1 and 2), and the regulatory (R) domain. The N-terminal and C-terminal regions are indicated. MSD2 has asparagine N-glycosylation sites. The NBDs utilize adenosine triphosphate (ATP), converting it to adenosine diphosphate (ADP). The R domain has several phosphorylation sites. The membranespanning domains form the chloride channel in the cell membrane.

612 CYSTIC FIBROSIS / Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene Table 1 Cellular phenotype of CFTR mutations Class

Affected

Location in CFTR protein

Examples

I II III IV V VI

Biosynthesis Maturation Cl
gating Cl
conductance Splicing Protein stability

Various Various NBDs Intramembrane domains Exons 8 and 12 C-terminus

G542X, R553X F508del, S945L, H949Y, D979A G551D R117H, G622D, M1137V, I1139V, DM1140, D1152H, and D1154G A455E, Exon 8 Tn, 1717-9T, C-D565G Q1412X, 4326delTC, 4279insA, 4271delC

balance needed to maintain the normal mucus layer lining the lung and other organs. In the lung, the ion imbalance leads to the formation of a thick mucus layer that traps bacteria, resulting in chronic infections. In the gastrointestinal and reproductive tracts, the thick mucus blocks organ function, while in the skin, the sweat glands produce sweat with high sodium ion concentrations. The mutations of the CFTR gene are grouped into functional classes; five classes are universally agreed upon, and a proposed sixth class relating to the possible impact of the mutation on biosynthesis is controversial (Table 1). Class I relates to protein synthesis, class II to protein maturation, class III to chloride channel regulation and gating, class IV to chloride conductance, class V to CFTR mRNA splicing, class VI to protein stability, and class VII to effects on the regulation of other channels. Although attempts to correlate specific CFTR mutations with severity of CF disease have not been completely successful, mutants defined as classes I to III tend to have severe CF symptoms, whereas classes IV and V are linked to milder manifestations of CF disease. The most common CFTR mutation worldwide is a deletion of phenylalanine at position 508 (F508del) causing problems in maturation and trafficking of the protein to the cell surface. There is variability in the occurrence of this mutation among ethnic groups. F508del occurs in the DNA sequence that codes for the first nucleotide-binding domain (NBD1). Homozygotes for F508del tend to have the most severe symptoms of CF. Other mutations are found throughout the entire promoter and coding regions of the CFTR gene. Mutations are particularly common in the NBD1, NBD2, and R domains, which is probably related to the importance of proper function of these domains. Twelve CFTR mutations, including 1717–1G4A, G542X, W1282X, N1303K, F508del, 3849 þ 10kbC4T, 621 þ 1G4T, R553X, G551D, R117H, R1162X, and R334W account for over 90% of all CF patients. There is also a genetic element modulating posttranscriptional processing of CFTR mRNA

transcripts. There is a polymorphic string of thymidines at the end of intron 8 of the CFTR gene, with three different alleles identified based on the number of thymidines (T5, T7, or T9) present at this locus. It has been shown that the shorter the poly(T) tract, the greater the relative amount of exon 9-CFTR mRNA transcripts present in respiratory epithelium. Since exon 9 encodes part of the important first nucleotidebinding domain, the resulting CFTR protein is inactive. A polymorphic TG repeat adjacent to the thymidine repeat region also influences the CF phenotype, with individuals carrying 12 or 13 TG repeats being more likely to exhibit an abnormal phenotype than those with 11 repeats. Class I Mutations Affecting Biosynthesis (No CFTR Synthesis)

Class I mutations lead to reduced production of CFTR mRNA. This class includes the most severe CF phenotypes due to the total lack of CFTR protein. In those cases where truncated proteins are produced, they are usually unstable, and are rapidly degraded in the endoplasmic reticulum (ER). The most common examples of class I mutations are G542X and R553X. Class II Mutations Affecting Protein Maturation (Block in CFTR Processing)

In class II mutations, such as F508del, CFTR is trapped in the endoplasmic reticulum and subsequently proteolytically degraded. Small amounts of F508del CFTR might reach the apical domain of respiratory epithelial cells in patients with cystic fibrosis, suggesting that defective CFTR maturation might still be rescued after passage through the endoplasmic reticulum. CFTR chaperones such as phenylbutyrate, 8-cyclopentyl-1,3-dipropylxanthine (CPX), and genistein that can force functional protein to the cell membrane, increase the efficiency of protein folding, or suppress protein degradation could provide therapy for class II mutants. Other examples of class II mutations are S945L, H949Y, and D979A.

CYSTIC FIBROSIS / Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene 613 Class III Mutations Affecting Chloride Channel Regulation/Gating (Block in Regulation)

Class III mutants produce a CFTR protein that gets transported to the cell membrane but does not respond to cAMP stimulation. All mutations so far attributed to this class are located within the NBDs, and probably act by preventing conformational change. Examples of class III mutations include G551D. Class IV Mutations Affecting Chloride Conductance (Altered Conductance)

This group includes cases where the CFTR protein is correctly trafficked to the cell membrane and responds to cAMP stimulation but the resulting chloride ion current is reduced. Examples of class IV mutations are R117H, M1137V, I1139V, M1140del, D1152H, and D1154G. Many of the class IV mutations are located within the intramembrane regions of the CFTR protein, particularly within transmembrane helix 12. Class IV mutants tend to have milder forms of CF. Class V Mutations Affecting CFTR Splicing

Class V mutations affect splicing of the mRNA resulting in exon skipping or extra-cryptic exons. Both mRNA and protein stability may be affected by these mutations. Examples of this class include the exon 8 thymidine polymorphism (discussed above) and 1717-9 T C-D565G in exon 12. Other CFTR Mutations

It has been suggested that mutations affecting protein stability that result in a short lifetime for the CFTR protein should be placed in ‘class VI’. Examples of class VI mutants involve frameshifts leading to terminal truncations, including Q1412X 4326delTC, 4279insA, and 4271delC. Although the CFTR C-terminal domain is not critical for essential CFTR functions, it appears to be required for maintaining the stability of the protein.

Regulation of CFTR by Other Proteins CFTR is associated with other membrane proteins in a regulatory complex, which includes other transport proteins such as the epithelial sodium channel. The N-terminus of CFTR binds to the N-terminus of syntaxin 1, a vesicle fusion protein found at neuronal synapses and airway epithelium. Binding of CFTR with syntaxin 1 interferes with CFTR channel activity. The DTRL motif at the C-terminus of CFTR binds to scaffolding/adaptor proteins such as EBP50 (a sodium hydrogen exchange regulatory factor),

CFTR-associated ligand, CAP70, and Shank2. These interactions regulate CFTR maturation, oligomeric state, channel gating, and phosphorylation state. Phosphorylation of the R domain stimulates CFTR channel activity and is regulated by protein kinases A and C (PKA and PKC).

Genotype/Phenotype Correlation The presence of particular CFTR genotypes (classes I–III) has been shown to correlate with pancreatic insufficiency, liver disease, early onset, high sweat chloride levels, and meconium ileus. However, it has not been possible to closely correlate specific CFTR mutations with the severity of lung disease, although the fraction of normal CFTR mRNA detected in splicing mutants Exon 8-5T and 3849 þ 10kbC4T correlates directly with the penetrance and severity of lung disease. Unlike lung disease, the severity of pancreatic insufficiency in CF can be correlated with specific CFTR mutations. R117H, R334W, and R347P are associated with mild pancreatic disease, and individuals carrying two ‘severe’ alleles will present with pancreatic insufficiency. The environment and modifier genes may significantly alter the progression and severity of CF. Secretion of electrolytes and water by the epididymal epithelium is critical for sperm maturation and transport. Only 2–3% of male CF patients are fertile, with the absence of vas deferens occurring in essentially all patients. There is also a reduction in fertility in female CF patients, although not to the extent seen in males. Menstrual irregularity and oligomenorrhea coupled with the presence of a plug of thick mucus in the cervix contribute to reduced fertility in female CF patients. Congenital bilateral absence of the vas deferens (CBAVD) patients may carry CFTR mutations without exhibiting other aspects of the CF phenotype. CBAVD prevents transport of spermatozoa from the testicles or epididymis to the vas deferens, resulting in infertility. It has been suggested that a combination of particular alleles at several polymorphic loci affects the quantity/quality of CFTR, resulting in a partial phenotype like CBAVD in which 70% of patients carry one CFTR mutation and 10% carry two. A mild CFTR mutation may then become a diseasecausing mutation in the vas deferens of CBAVD patients due to the overall decrease in full-length CFTR mRNA. However, CBAVD patients show no evidence of pulmonary disease, suggesting a possible CFTR mRNA transcript ‘threshold’ level for appearance of pulmonary manifestations of CF. It is clear that CFTR genotype alone does not account for the diverse spectrum of disease observed

614 CYSTIC FIBROSIS / Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

for the CF pulmonary phenotype. Secondary genetic factors distinct from CFTR have been identified that may influence the severity of CF lung symptoms. These proposed ‘modifier genes’ include inflammatory and anti-inflammatory molecules, antioxidants, molecules that influence airway reactivity, molecules capable of inducing alterations in CFTR trafficking, and alternative ion channels. It has been suggested that particular alleles at several polymorphic loci might cooperate, resulting in an overall decrease in the function of the CFTR protein. The subtle contribution of polymorphic alleles could explain why apparently individually normal CFTR alleles cause disease and might be responsible for variation in the phenotypic expression of CFTR. An example of this can be seen in the cooperation of two mild mutations, R347H and D979A. R347H alone leads to somewhat decreased Cl
channel activity, and D979A alone causes misprocessing of CFTR. However, an individual carrying both mutations showed a severe decrease in Cl
channel activity.

Associated Disorders Disseminated bronchiectasis is an obstructive pulmonary disorder involving structural abnormalities often associated with childhood lung infections and immunodeficiencies. CFTR mutations may contribute to the phenotype in non-CF obstructive pulmonary diseases such as disseminated bronchiectasis. In two studies of 16 and 32 patients, respectively, the frequency of the T5 allele was significantly increased over controls and 13 CFTR mutations were detected. Two recent studies of 39 and 20 idiopathic chronic pancreatitis patients showed that at least 30% of patients carried CFTR mutations, demonstrating that CFTR mutations are associated with this disorder. Chronic rhinosinusitis, a chronic inflammation of the sinus epithelium, is also associated with CF. Individuals with chronic rhinosinusitis are three times more likely to carry CFTR mutations than controls. Asthma has been suggested to be more common in association with CFTR mutations, but these observations have not been found to be statistically significant. Pancreatitis and atrophy of pancreatic acinar tissue has also been correlated with an increased incidence of CFTR mutations.

New Therapies Although there have been improvements in the understanding of the genetic basis and management of CF in the last decade, there is as yet no cure. However, additional study of the structure and function of CFTR and the development of new drugs to

circumvent the abnormalities caused by mutations in the CFTR gene promise to yield major improvements in the treatment of CF. One of the most attractive approaches to CF is the augmentation of the existing genetic complement of airway epithelial cells with a normal CFTR transgene. The possibility for CFTR gene transfer was quickly established in vitro and in animal models after cloning of the gene in 1989. The first gene therapy clinical trials in CF patients were carried out in 1993, and 29 clinical trials have been published as of 2004. Repeat administration of CFTR adenoviruses, adeno-associated virus, and nonviral strategies have been shown to be safe to humans. Progress has been made in overcoming the problems associated with gene transfer to airway epithelial cells but difficulties remain in the establishment of long-term expression of CFTR in vivo.

Acknowledgments We thank N Mohamed for help in preparing this manuscript. These studies were supported in part by NIH P01 HL51746 and the Will Rogers Memorial Fund, Los Angeles, CA, USA. See also: Bronchiectasis. Cystic Fibrosis: Overview. Ion Transport: Calcium Channels; Chloride Channels.

Further Reading Chiba-Falek O, Kerem E, Shoshani T, et al. (1998) The molecular basis of disease variability among cystic fibrosis patients carrying the 3849 þ 10kb c4 T mutation. Genomics 53: 276–283. Chu CS, Trapnell BC, Curristin S, Cutting GR, and Crystal RG (1993) Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nature Genetics 3: 151–156. Crystal RG, McElvaney NG, Rosenfeld MA, et al. (1994) Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nature Genetics 8: 42–51. Dalemans W, Barbry P, Champigny G, et al. (1991) Altered C1channel kinetics associated with the major (F508) cystic fibrosis mutation. Nature 354: 526–528. Dorwart M, Thibodeau P, and Thomas P (2004) Cystic fibrosis: recent structural insights. Journal of Cystic Fibrosis 3(supplement 2): 91–94. Harvey BG, Leopold PL, Hackett NR, et al. (1999) Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus. Journal of Clinical Investigation 104(9): 1245–1255. Kerem B and Kerem E (1996) The molecular basis for disease variability in cystic fibrosis. European Journal of Human Genetics 4: 65–73. McKone EF, Emerson SS, Edwards KL, and Aitken ML (2003) Effect of genotype on phenotype and mortality in cystic fibrosis: a retrospective cohort study. The Lancet 361(17): 1671–1676. Ratjen F and Do¨ring G (2003) Cystic fibrosis. The Lancet 361: 681–689.

CYTOSKELETAL PROTEINS 615 Rave-Harel N, Kerem E, Nissim-Rafinia M, et al. (1997) The molecular basis of partial penetrance of splicing mutations in cystic fibrosis. American Journal of Human Genetics 60: 87–94. Riordan JR, Rommens JM, Kerem B, et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066–1073. Vankeerberghen A, Cuppens H, and Cassiman JJ (2002) The cystic fibrosis transmembrane conductance regulator: an intriguing

protein with pleiotropic functions. Journal of Cystic Fibrosis 1: 13–29. Welsh MJ, Ramrey BW, Accurso F, et al. (2001) Cystic fibrosis. In: Scriver CR, Sly WS, Childs B, et al. (eds.) The Metabolic and Molecular Bases of Inherited Disease, 8th edn, pp. 5121–5189. New York: McGraw Hill.

CYTOSKELETAL PROTEINS A D Verin, Johns Hopkins University School of Medicine, Baltimore, MD, USA N V Bogatcheva, Baylor College of Medicine, Houston, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The cytoskeleton represents the intricate inner carcass that stabilizes the cell by a network of three major components: microfilaments, microtubules, and intermediate filaments. This network is anchored to the membrane by highly specialized protein complexes, providing both cell attachment to other cells or extracellular matrices, as well as stabilization and dynamic rearrangement of cell-surface structures. Not only does the cytoskeleton maintain cell shape but it also manages compartmentalization and proper orientation of many biochemical processes including signal transduction. Cytoskeletal remodeling is critical for cell contraction, motility, and morphogenesis; in addition, the cytoskeleton serves as a cellular mechanosensor and mechanotransducer. Signals originating from cytoskeletal elements control gene expression, cell differentiation, proliferation, and survival. The vast majority of physiological responses, such as leukocyte migration and phagocytic activity, the ability of fibroblasts to heal wounds, angiogenesis, control over vascular/bronchial tone, and regulation of endothelial/epithelial barrier function depend upon cytoskeletal proteins, involved in the organization of intercellular contacts, and contractile proteins, inducing tension. Maintenance of barrier function is of particular importance in lung physiology, since an increase in alveolar capillary permeability and resultant edema represent the underlying causes of lung damage characterized as acute lung injury or acute respiratory distress syndrome. Different therapeutic strategies counteracting hyperpermeability are currently under development; some of which target signaling pathways, leading to cytoskeletal rearrangement.

Introduction The dynamic cytoskeletal network composed of microfilaments, microtubules, and intermediate filaments is arranged into a functional system with the help of multiple associated proteins. These proteins regulate filament polymerization/depolymerization processes and define overall cytoskeleton organization,

providing bonds between filaments of the same or different types and attachment to the nuclear or plasma membranes and cell organelles. The current hypothesis holds that living cells organize their cytoskeleton by the principle of ‘tensional integrity’ architecture. Such structure gains stability from continuous inward-directed tension counterbalanced by the resistance of elements, which either provide peripheral attachment or cannot be compressed. Thus, cell shape is determined by the net effect of the pull of contractile microfilaments and the adhesive tethering and rigidity of microtubules. The lattice of intermediate filaments stiffens the whole cell including the cell nuclear compartment to render protection from both centrifugal and centripetal forces. This interplay between cytoskeletal elements may result in cell spreading or contraction, development of asymmetrical shape, or cell migration. Organelle and vesicle intracellular motility is also governed by cytoskeletal rearrangement. Remodeling of specific cytoskeletal structures, especially those associated with the membrane, initiates a series of signaling events, consequently the cytoskeleton provides not only the structural basis for cell shape maintenance and movement, but also mediates cell physiological responses serving as an important signal transducer.

Microfilaments (Actin Cytoskeleton) The actin cytoskeleton plays a central role in cell contractility, motility, vesicle and organelle movement, and overall organization of cell architecture. Two processes are of particular importance for these cell functions: actin polymerization and the interaction of actin with molecular motor myosin. In the cell, the complex actin network is assembled from linear protein complexes called microfilaments, or thin filaments (Figure 1). The microfilament core consists of fibrillar actin (F-actin), formed from globular actin (G-actin) in the process of polymerization. Actin monomers and

Vessel lumen

Centrosome

ZO proteins

ZO proteins

Centrosome

Tight junctions Catenins

Nucleus













Catenins



Nucleus

Adherent junctions Complexus adherent junctions

FAK

FAK

Focal adhesion

FAK

FAK

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Extracellular matrix Focal adhesions Hemidesmosomes

Hemidesmosome

Figure 1 Schematic representation of the cytoskeletal organization of endothelial cells. In a functional monolayer, endothelial cells are attached to each other via several junctional complexes (tight junctions, adherent junctions, complexus adherent junctions). Cell adhesion to the extracellular matrix is provided by integrins (shown in green), the transmembrane elements of focal adhesions and hemidesmosomes. All junctional adhesion complexes are linked to microfilaments (red), microtubules (black), or intermediate filaments (purple) via numerous specific proteins. An important regulatory role is attributed to focal adhesion kinase (FAK), which controls signaling cascades, governing overall cytoskeletal organization. In an agonist-challenged monolayer, the development of the actomyosin-driven inward-directed tension, along with the disruption of interendothelial cell contacts, results in endothelial barrier dysfunction, a prerequisite of pulmonary edema.

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newly formed actin nuclei are known to possess structural polarity; it is not surprising then, that the polymerization rates at the two ends of microfilaments are different. Polymerization and depolymerization processes are regulated by numerous actin-binding proteins, including severing and capping proteins (ADF/cofilin, gelsolin, severin, fragmin), and proteins controlling actin nucleation and elongation (ARP2/3, profilin). Some of these proteins associate with the filament in a way that may enable filament branching (cofilin, ARP2/3). Single actin filaments may be organized into bundles or gels by the numerous bundling or gelating proteins (a-actinin, villin, synapsin, fimbrin, filamin). In nonmuscle cells, actin structures are very dynamic, being in constant transition between polymerized actin and monomers. In muscle cells, the equilibrium is shifted toward the microfilament formation; nevertheless, relaxed smooth muscle cells still retain detectable amounts of nonpolymerized actin. In the quiescent cell, the actin cytoskeleton might consist of peripheral bundles (so-called cortical actin) or cell-crossing bundles (stress fibers). In spreading or migrating cells, actin polymerization accounts for the appearance of surface protrusions such as lamellipodia and filopodia. Several proteins help to organize cortical actin and actin of cellsurface structures. Peripheral actin bundles contain the actin-binding protein cortactin, known to regulate both actin nucleation and actin crosslinking. Branching activity of ARP2/3 and anticapping, antibranching, and profilin-recruiting activities of the actin-binding protein VASP seem to control the dynamics and architecture of the orthogonal (lamellipodia) and parallel (filopodia) actin network. Depending on the cell type, actin filaments may be aligned with different structural and regulatory proteins (troponins, tropomyosin, caldesmon, calponin), affecting F-actin stability and interaction with myosins. Myosins are molecular motors, with diverse structure and function; however, all of them are able to move along actin filaments in an ATP-consuming fashion. Cell contractile activity depends mainly on the unique capacity of conventional myosin II to form filaments (thick filaments). Myosin II filaments bundle actin threads into stress fibers or tension fibers; tension is generated by the sliding of actin and myosin filaments along each other. Whereas most myosin molecules move toward faster growing (plus) ends of actin filaments, some nonconventional myosins (myosin VI and IX) are able to move toward the minus ends of actin filaments, a unique function of these cellular motors. So far, nonconventional myosins have been shown to be important primarily for the vesicular transport

and maintenance of specific structures on the cell surface. The force generated by actin polymerization or actomyosin interaction is usually directed towards the membrane, providing either vesicle movement or cell shape change. Actin association with the membrane might be rendered by several proteins or protein complexes, dictating the specific contacts within the membrane compartment. For instance, myosin V, important for vesicular transport, is recruited to different kinds of cellular vesicles by specific proteins of the Rab family. Cleavage furrows, microvilli, filopodia, and lamellipodia contain proteins of the ezrinradixin-moesin (ERM) family, linking the actin cytoskeleton to the plasma membrane. Cortical actin fibers are attached to the membrane via cell junction complexes, whereas tensile fibers associate with the membrane through the focal contacts, integrincontaining complexes anchoring the cell surface to the substrate (Figure 1). The temporal and spatial organization of the actin cytoskeleton and the contractile state of the actomyosin system depends upon several factors, which include intracellular calcium levels, activity of small GTPases of the Rho family (Rho, Rac, and Cdc42), and phosphatidylinositol (4,5) bisphosphate (PIP2) concentration. In smooth muscle and nonmuscle cells, the calcium–calmodulin complex activates myosin light chain kinase, which phosphorylates myosin light chains (MLC). MLC phosphorylation induces myosin ATPase activity, myosin polymerization, and stress fiber formation. Rho, via activation of its downstream effector Rho kinase, also increases MLC phosphorylation levels and induces stress fiber formation. In addition, Rho signaling elevates the level of PIP2, which affects actin polymerization via the regulation of ARP2/3, gelsolin and cofilin/ADF activity, and actin linkage to the membrane through a-actinin, filamin, ERM, talin, and vinculin. Other GTPases of the Rho family, namely Rac and Cdc42, induce formation of filopodia and lamellipodia, respectively. Rac has been shown to be involved in cortical actin formation and endothelial barrier enhancement via p21-activated protein kinase (PAK)/LIM kinase/cofilin phosphorylation pathway.

Microtubules (Tubulin Cytoskeleton) The tubulin cytoskeleton is critical for cell migration, chromosome, and vesicle movement and the organization of some cell-surface structures such as flagella and cilia. Tubulin-associated motor activity as well as tensile force produced by microtubule

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depolymerization is involved in diverse cellular events including cilia and flagella movement, organelle transport, and mitotic and premeiotic cell rearrangement. Within the cell, heterodimers of a- and b-tubulin are polymerized in a head-to-tail manner to form a linear protofilament. Protofilaments are subsequently joined through lateral interactions to form the wall of the cylindrical microtubule. Microtubules are polymerized from microtubule organizing centers, such as centrosomes (Figure 1) or flagella basal bodies. In some cell types, including epithelium, microtubules are able to disconnect and move from the centrosome after nucleation. Microtubules are highly dynamic, constantly switching between phases of growth and shrinkage mediated by the addition or loss of tubulin dimers from the ends of the microtubule. This property of microtubules is known as dynamic instability and is dependent on the binding and hydrolysis of GTP by tubulin subunits. Rapidly growing and shrinking ( þ ) ends of microtubules are oriented toward the cell periphery while the slower (–) ends are closer to the nucleus. The assembly and stability of microtubules is regulated not only by the nucleotide state of tubulin, but also by their interaction with cellular factors like microtubule-associated proteins (MAPs). The most studied MAPs are tau protein, dynein, and kinesin. As is the case with many other MAPs, tau controls the assembly of microtubules in a phosphorylation-dependent manner: in the unphosphorylated form tau promotes assembly of microtubules and inhibits the rate of depolymerization. Similar to myosin, dynein and kinesin function as ATP-consuming microtubule motors. The role of dynein is to transport cargo towards the minus end of microtubules. Dynein motor domains (heads) have microtubule-activated ATPase activity. The tail of dynein connects to the large dynactin protein complex, which targets and tethers dynein to diverse intracellular structures such as Golgi membranes, chromosomes, and microtubules. In contrast to dynein, most kinesins (N-kinesins) move towards the plus end of microtubules. Remaining kinesins (C-kinesins) move in the opposite direction. Similar to dynein, kinesin has an N-terminal motor domain (head) and C-terminal tail, which determines the cargo specificity for each individual kinesin. The interaction between the catalytic domain (head) and the adjacent linker region determines the direction of movement for each kinesin isoform. There has been accumulating evidence of coordination and interaction between microfilaments and microtubules. For instance, myosin V, involved in vesicular transport, is known to associate with kinesin. The myosin V/kinesin heteromotor complex allows long-range movement of vesicles along

microtubules, followed by the short-range movement on actin filaments. Microtubules have also been shown to be essential for the regulation of cell migration. Whereas this process is largely driven by actin polymerization and subsequent actomyosin contraction, the interaction between microtubules and microfilaments is necessary to guide the cell. In migrating fibroblasts, the growing ends of microtubules were shown to be targeted and attached to newly formed cortical actin foci. The actin-binding proteins capturing microtubules remain unknown, but several proteins have been shown to bind both polymerized actin and tubulin (ezrin, Mip-90, plakins, mDia, caldesmon, tau) or MAP (cortactin). Microtubules are also known to suppress cell contractility, although the mechanism of such suppression is not completely understood. According to the tensegrity model, microtubules mechanically resist actomyosin-driven tension; however, there is evidence that some microtubule-associated molecules may regulate the state of contractile proteins. For example, the disruption of microtubules by microtubule inhibitors, like vinblastine or nocodazole, activates the Rho/Rho kinase complex, inhibits MLC phosphatase activity thereby increasing MLC phosphorylation and induces stress fiber formation.

Intermediate Filaments The function of intermediate filaments (IFs) is less well understood than that of microfilaments and microtubules. The prevailing hypothesis holds that IFs provide stability to individual cells and cell monolayers, helping to organize the latter into an integral system. In some cell types, IFs have been shown to be important for contractile and motile activity. Several studies have demonstrated interdependence between the IF network organization and the state of microfilaments and microtubules in the cell. IFs are formed by groups of proteins exhibiting characteristic cell-expression profiles (e.g., keratins are expressed in epithelia, neurofilament proteins in neuronal cells, desmin in myogenic cells, and vimentin in mesenchymal cells) and ubiquitous nuclear lamins. Whereas keratins, desmin, and vimentin form the cytoplasmic IF network, nuclear lamins assemble the nuclear envelope. IFs represent dynamic polymers maintaining an equilibrium with the IF protein tetramers. IF organization is likely controlled by phosphorylation and by the association with proteins affecting IF assembly, such as a-crystallin. IF-associated proteins help to organize IFs into a network by linking them either together or to other cytoskeletal elements. For example, plectin has been shown to cross-link IFs to microfilaments,

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microtubules, and membrane adhesions. IF attachment to the plasma and nuclear membranes might be provided by integral or membrane-associated proteins (including ankyrin and spectrin).

Integrins and Focal Adhesions Cell attachment to the surrounding environment is provided by several surface proteins, with the most studied group consisting of transmembrane proteins called integrins. Some integrins act as substrate adhesion molecules, whereas others mediate cell–cell interactions. The establishment of integrin-containing contacts is critical for the processes of cell spreading, migration, contractility, and barrier function maintenance. Integrins are composed of separate a and b subunits, noncovalently linked through their extracellular regions. Subunit combination determines integrin specificity toward the ligands. Integrins, which are involved in extracellular matrix binding, are usually organized into complexes called focal adhesions (Figure 1). These complexes consist of an intricate combination of cytoskeletal proteins, linking the cytoplasmic domain of b integrin to actin filaments through a-actinin, vinculin, talin, and tensin. Some types of integrins may interact with intermediate filaments, creating basal membrane hemidesmosomes (Figure 1). While integrins on migrating cells are dispersed and form small adhesion sites, clusters of integrins forming larger focal adhesion plaques may be found at sites of cell attachment to the extracellular matrix. Initial adhesion sites and mature adhesion plaques appear to be associated with different pools of actin filaments, since protruding lamellipodia establish contacts via ARP2/3-branched actin meshwork, whereas mature adhesion plaques are linked to the parallel actin filament bundles, forming stress fibers. Integrin binding to ligands is known to promote the attachment of integrins to the actin cytoskeleton while the size of focal adhesion sites depends on the actomyosin-driven tension applied to the point of contact. Additionally, the tubulin network regulates focal adhesion dynamics. Extensions of microtubules targeted to newly formed adhesion sites arrest focal adhesion growth and trigger their disassembly. Such control may be important for the coordination of the process of cell migration, since stabilization of cell– substrate contacts in the cell’s leading edge would interfere with polarized movement. Several signaling molecules regulate the establishment of integrin-containing contacts. Rho GTPase affects integrin association with the cytoskeleton via phosphatydylinositol 4-phosphate 5-kinase/PIP2/

vinculin and talin conformational changes. Focal adhesion-specific protease, calpain, controls both turnover and activation of focal adhesion components. Small GTPases of the Ras family have been shown to alter integrin clustering or change integrin affinity to ligands. Focal adhesions are known to serve as sensors and external signal transducers as they coordinate several signaling cascades involving numerous proteins including tyrosine kinases (focal adhesion kinase (FAK), members of Src family, and C-terminal Src kinase (CSK)), serine/threonine kinases (PKC, PAK, and ILK), tyrosine phosphatases, and modulators of small G proteins. A predominant role in the assembling of focal adhesion protein complex belongs to paxillin and zyxin, which function as adapters for an array of signaling and cytoskeletal molecules. The phosphorylation of FAK and paxilin appears to be a prerequisite of external stimuli-induced cytoskeletal reorganization. One molecule that couples to phosphorylated paxillin is adapter protein CrkII, and is implicated in actin cytoskeleton remodeling. An emerging body of evidence indicates that, upon dissociation from focal adhesions, paxillin and zixin may be shuttled to the nucleus to regulate gene transcription.

Cell Adhesion Molecules Several types of molecule are specifically expressed on the cell surface to provide cell–cell recognition and cell adhesion to surrounding cells. Under quiescent conditions, adhesion molecules participate in maintenance of endothelial barrier function, enforcing contacts between cells in a monolayer. Inflammation changes the pattern of adhesion molecules exposed on the cell surface, inducing the interaction of activated mobile cells from the blood vessel lumen with activated endothelial cells. Adhesion molecules are structurally diverse and include selectins, selectin ligands, integrins, and adhesion molecules of the IgG superfamily, such as intercellular adhesion molecules (ICAMs), vascular adhesion molecule (VCAM), platelet-endothelial adhesion molecule (PECAM), and junction adhesion molecule (JAM). These last two proteins are involved in homophilic contact (JAM–JAM and PECAM–PECAM) organization, whereas ICAMs and VCAM serve as ligands for the specific integrins. Adhesion proteins can associate with the actin cytoskeleton through actin-binding proteins such as cortactin or ERM, or comprise parts of large junctional protein complexes, characterized as tight or adherent junctions. In addition to their cytoskeletal functions, some adhesion molecules (for instance,

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PECAM-1) might serve as scaffolding molecules in several signaling pathways. As mentioned before, control over cell adhesion mostly occurs via alteration of adhesion protein expression on the cell surface. Function of some adhesion proteins, in turn, is regulated by their phosphorylation.

Tight Junctions Tight junctions, or zonula occludens (ZO), are characteristic of epithelial and endothelial cells (Figure 1). Located at the border between apical and lateral membranes, tight junctions regulate the passage of proteins and liquids across the cell monolayer. Tight junctions include occludin, claudin family members, JAMs 1–3, cingulin, and linker proteins from the ZO family, which serve to bind the former proteins to each other or to the actin cytoskeleton. In both endothelial and epithelial cells, VASP has been found in the complex with zonula occludens-1 (ZO-1) protein. The regulation of tight junction assembly is achieved via phosphorylation by several protein kinases. Thus, activation of the MAP kinase pathways controls ZO-1-occludin interaction with plasma membrane and VASP association with tight junctions is regulated by PKA-dependent phosphorylation.

Adherent Junctions Adherent junctions, or zonula adherens, are also widely represented in both endothelial and epithelial cells although located more basolaterally relative to tight junctions (Figure 1). Adherence junctions are known to play a role in monolayer barrier maintenance, vascular morphogenesis, and cell migration. The core of adherent junctions is formed by cellspecific transmembrane proteins called cadherins. Extracellular domains of cadherins from adjacent cells undergo calcium-dependent homophilic binding, while cytoplasmic domains associate with b-catenin or g-catenin (placoglobin), linking the complex via a-catenin/vinculin to the actin cytoskeleton. p120 catenin binds the juxtamembrane domain of cadherins and controls their lateral dimerization. Newly formed cadherin-containing contacts have been found to contain VASP. This protein appears to be important in the establishment of monolayers, since the disruption of VASP function interferes with epithelial sheet formation. PECAM-1 is also found within adherent junctions, but is not exclusive to them. PECAM-1 is able to act as a scaffold to recruit b- and g-catenins to cell contacts in a phosphorylation-dependent manner.

Junctional organization and stability of junctional proteins within cells is tightly controlled by PKA-, PKC-, and tyrosine kinase-dependent phosphorylation as well as the balance between Rho and Rac activity. Moreover, several signaling proteins associate with adherent junctions, including receptor tyrosine kinase VEGFR-2, Src homology phosphatase 2 (SHP2), protein tyrosine phosphatase m (PTPm), and IQ GTPase activating protein (IQGAP). The establishment or disruption of cell junctions is now thought to affect gene expression patterns and overall cell fate via nuclear translocation of bcatenin. g- and p120 catenins, members of the same armadillo family of transcriptional regulators, may also be involved in regulating gene expression.

Desmosomes Desmosomes are basolaterally located junctions restricted mostly to epithelial and myocardial cells. They are critical for tissue integrity, play an important role in epithelial morphogenesis and may act as signaling centers. Desmosomes are formed by the transmembrane glycoproteins of the cadherin superfamily (desmogleins and desmocollins), associated with placoglobin and desmoplakins, which, in turn, anchor keratin- or desmin-containing IFs. Endothelial cells do not have desmosomes but they assemble unique structures called complexus adherence junctions (Figure 1), in which a network of vimentin is linked to classical cadherin via the desmosomal proteins plakoglobin and desmoplakin. These latter proteins may recruit PECAM-1, driving its interaction with intermediate filaments.

Gap Junctions Gap junctions are membrane structures, serving to provide electrical and metabolic coupling of adjacent cells, such as capillary endothelial cells and alveolar epithelial cells, or type I (gas exchanging) and type II (surfactant-producing) epithelial cells. Cell interconnection is thought to coordinate microcompartment responses to the external stimuli; however, it could also result in the transmission of toxins, for instance, in the setting of oxidative stress, and therefore affect cell survival. Adjacent cells form gap junctions by the association of two hemichannels, each consisting of six tissue-specific connexins. On the cytoplasmic side, connexins may associate with ZO-1 and tubulin, which have the potential to modulate their function. The regulation of gap junctions can also be achieved by their interaction with the calcium-binding protein

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calmodulin, and PKA-, PKC-, or Src-dependent phosphorylation.

Cytoskeleton in Mechanotransduction Airway smooth muscles, airway and alveolar epithelial cells, lung fibroblasts, and adherent inflammatory cells upon infiltration into the lung are continuously subjected to mechanical forces caused by changes in lung volume during the respiratory cycle. While endothelial cells also experience such forces, endothelial cells of larger vessels are subjected to constant changes in blood flow or shear stress. Cells are known to sense their mechanical environment and respond to it with cytoskeletal rearrangement and alterations in gene expression. Changes in local tension, generated during exercise, and in diseases such as asthma, emphysema, and cystic fibrosis activate several signaling pathways affecting cell contractility, migration, proliferation, monolayer integrity, synthesis of proinflammatory mediators, and/or surfactant exocytosis. Excessive forces, like those applied during mechanical ventilation, may trigger mechanosensitive signaling cascades leading to catastrophic consequences manifested as ventilator-induced lung injury. While the cytoskeleton represents the target of the mechanical stress-induced cell response; mechanotransduction per se relies upon cytoskeletal structures associated with plasma membrane anchors. Mechanical strain, administered to extracellular domains of integrins, results in increased tyrosine phosphorylation of proteins, associated with focal adhesion sites. This event activates multiple signaling cascades, leading to alterations in cell metabolism relative to the changing environment. Propagation of mechanical deformation-induced signals also depends upon cytoskeletal structures. Calcium elevation waves are known to diffuse via gap junctions from the stretchinjured alveolar epithelial cells to affect uninjured cells in monolayer. Further research will be needed to understand the major pathways responsible for mechanical deformation-triggered alterations in cell metabolism, in order ultimately to prevent the detrimental consequences of such alterations.

Cytoskeleton in Lung Hyperpermeability Reorganization of the endothelial cytoskeleton leads to alteration in cell shape and provides a structural basis for increase of vascular permeability, which has been implicated in the pathogenesis of many diseases including asthma, sepsis, and acute lung injury. In perfused rabbit lungs, selective disruption of actin microfilaments leads to a significant increase in

vascular permeability and marked interstitial edema formation, implicating the direct involvement of microfilaments in the regulation of permeability in vivo. While the importance of the microtubule component in the maintenance of endothelial cell barrier function remains largely unexplored, intravenous administration of anticancer drugs, which specifically target microtubules, can lead to the sudden development of pulmonary edema in breast cancer patients suggesting the potential importance of the microtubule network for the regulation of lung permeability. Proinflammatory mediators, released by activated cells at the site of inflammation (reactive oxygen species, tumor necrosis factor (TNF-a), interleukins, platelet activator factor (PAF), endothelin, prostanoids and leukotrienes), may significantly affect endothelial barrier function. Two parameters are of particular importance for the maintenance of barrier function: the contractile state of the actin cytoskeleton and intercellular junctional integrity. Edemagenic factors are known to induce stress fiber formation and increase endothelial cell contractility, while altering tight junction and adherent junction organization. Notably, those oxidants and other inflammatory mediators, while increasing endothelial permeability, enhance cell–substrate adhesion. Such strengthening of focal contacts is likely to be a prerequisite of activation of integrin-mediated signal transduction pathways but also can serve to prevent detachment of endothelial cells from the monolayer. Because several proinflammatory mediators lead to cytoskeletal changes via the same signal transduction pathways, direct interference of intracellular signaling may have profound therapeutic implications. One potential strategy would be to block key signaling events leading to cytoskeletal rearrangement, namely calcium entry, Rho kinase, PKC or Src family kinase activation. The clinical significance and safety of several pharmacological inhibitors, including PKC inhibitor LY333531 and Rho kinase inhibitor Y27632, are currently under investigation. See also: Adhesion, Cell–Matrix: Integrins.

Further Reading Alexander JS and Elrod JW (2002) Extracellular matrix, junctional integrity and matrix metalloproteinase interactions in endothelial permeability regulation. Journal of Anatomy 200: 561–574. Bershadsky AD, Balaban NQ, and Geiger B (2003) Adhesion-dependent cell mechanosensitivity. Annual Review of Cell and Developmental Biology 19: 677–695. Chidgey M (2002) Desmosomes and disease: an update. Histology and Histopathology 17: 1179–1192.

622 CYTOSKELETAL PROTEINS Dos Santos CC and Slutsky AS (2000) Mechanism of ventilatorinduced lung injury: a perspective. Journal of Applied Physiology 89: 1645–1655. Dudek SM and Garcia JGN (2001) Cytoskeletal regulation of pulmonary vascular permeability. Journal of Applied Physiology 91: 1487–1500. Ilan N and Madri JA (2003) PECAM-1: old friend, new partners. Current Opinion in Cell Biology 15: 515–524. Johnson-Leger C, Aurrand-Lions M, and Imhof BA (2000) The parting of endothelium: miracle, or simply a junctional affair? Journal of Cell Science 113: 921–933. Koval M (2002) Sharing signals: connecting lung epithelial cells with gap junction channels. American Journal of Physiology 283: L875–L893. Krause M, Dent EW, Bear JE, Loureiro JJ, and Gertler FB (2003) Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annual Review of Cell and Developmental Biology 19: 541–564.

Lee TJ and Gotlieb AI (2003) Microfilaments and microtubules maintain endothelial integrity. Microscopy Research and Technique 60: 115–127. Sellers JR (2000) Myosins: a diverse superfamily. Biochimica et Biophysica Acta 1496: 3–22. Van Nieuw Amerongen GP and van Hinsbergh VWM (2003) Targets for pharmacological intervention of endothelial hyperpermeability and barrier function. Vascular Pharmacology 39: 257–272. Wang Y and Gilmore TD (2003) Zyxin and paxillin proteins: focal adhesion plaque LIM domain proteins go nuclear. Biochimica et Biophysica Acta 1593: 115–120. Wojciak-Stothard B and Ridley AJ (2003) Rho GTPases and the regulation of endothelial permeability. Vascular Pharmacology 39: 187–199. Yin HL and Janmey PA (2003) Phosphoinositide regulation of the actin cytoskeleton. Annual Review in Physiology 65: 761–789.

D Deep Venous Thrombosis (DVT)

see Pulmonary Thromboembolism: Deep Venous Thrombosis.

DEFENSE SYSTEMS R Boyton, Imperial College London, London, UK R Wilson, Royal Brompton and Harefield NHS Trust, London, UK D M Altmann, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The lung is the anatomical site not only for gaseous exchange, but also for high-turnover sampling of pathogens and other antigenic and allergenic material. It constitutes a vast mucosal surface area, constantly exposed to inhaled substances that, allowed free ingress, would cause substantial damage or death. Any defense mechanism must accommodate the fact that the respiratory tract is made up of delicate tissues required for gas exchange. The lung, therefore, requires a multilayered system of protection that allows a fast, efficient, and effective response able to deal with infectious pathogens and other foreign particles without damaging the lung itself in the process. This encompasses physical barriers, innate immune mechanisms, and the cellular and antibody adaptive immune response. The price of excessive or inappropriate inflammatory responses can be high and may lead to lung damage. The first line of defense against entry to the lung is from physical barriers such as mucus and cilia, followed by the innate immune response that includes a battery of mediators such as lactoferrin, lysozyme, collectins, and defensins. Release of these molecules leads directly to lysis of pathogens, or destruction through opsonization or the recruitment of inflammatory cells. Rapid activation of the innate immune response also comes through the triggering of Toll-like receptors (TLRs) and the chemotactic recruitment and activation of neutrophils. Natural killer (NK) cells provide a fast and effective immune surveillance response against infectious pathogens. Type I interferons exert a direct antiviral effect. The adaptive immune response contributes by making neutralizing antibodies and T lymphocytes orchestrate antigen-specific immune responses through their ability to differentiate self from nonself and provide memory. T lymphocyte populations of different cytokine phenotypes – so-called T-helper-1 (Th1) and T-helper-2 (Th2) – dramatically alter the balance between pathogen clearance and tissue damage.

Introduction Most pathogens gain entry to the body through mucosal surfaces. The lung, being the largest epithelial

surface in the body, constitutes the major site of entry. The airways are continually challenged by airborne microorganisms and ingress of environmental particles and employ robust mechanisms to combat infection, starting with physical barriers to entry, and failing this, mounting innate or specific adaptive immune responses. Lung diseases such as cystic fibrosis (CF) and the acquired immunodeficiency syndrome (AIDS) constitute illustrative examples of how certain types of respiratory infections thrive when elements of the innate response in the first case, or the specific immune response in the second, are impaired. The specific part of the protective response that is missing or suboptimal dictates, both qualitatively and quantitatively, the nature of the infecting organism and lung damage. It is possible that some chronic lung diseases such as bronchiectasis or chronic obstructive pulmonary disease (COPD) may result from a dysregulated immune response to infectious pathogens or inhaled foreign particles driven in part by underlying differences in the individual’s immune response repertoire and also by levels of exposure. The specific profile of cytokines made by activated T lymphocytes must be highly regulated and appropriate; the appropriateness of T-helper-1 (Th1) and T-helper-2 (Th2) cytokine profiles in the respiratory immune response is critical to maintaining the balance between protective immunity and excessive inflammation. The balance between cytokines that promote, for example, bronchial infiltrates (primarily either neutrophilic or eosinophilic) will have a major impact on the type of disease. Immunity in the lung faces the particular problem that, at the same time as requiring the capacity for rapid and potent clearance of infection, an overzealous or inappropriate cellular or cytokine response, either due to immune dysregulation or failure to clear antigen, may cause inflammatory changes that can permanently alter the function and architecture of the airways.

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Immune mechanisms in the lung must deal with a wide range of pathogens, including bacteria, mycobacteria, fungi, and viruses. While specific mechanisms for dealing with pathogens may differ to some extent (e.g., the importance of type I interferons and cytotoxic T cells for antiviral responses), there are features common to the immune mechanisms. These consist of a multilayered defense that may involve several modes of innate mediators, Tolllike receptor (TLR) activation, leukocyte infiltration, antibodies, natural killer (NK) cells, and activation of specific T lymphocytes.

Physical Barriers and Soluble Mediators Mechanical barriers offer the first line of defense to microorganisms entering the respiratory tract. Mucins of the mucociliary blanket lining the surface of the airways trap microorganisms which can then be cleared by ciliary movement. Cells of the respiratory tract produce a range of soluble mediators, either constitutively or following activation in response to those particles that pass this barrier. Airway surface fluid (ASF) contains several proteins with antimicrobial activity. These mechanisms are sensitive to local salt concentrations, and are therefore believed to be impaired in CF. Furthermore, the CF transmembrane conductance regulator (CFTR), mutation of which is the cause of the disease, has itself been shown to act as a receptor for internalization and clearance of some microorganisms, particularly pseudomonas. Airway epithelial secretory proteins can be divided into mucins and nonmucins. The nonmucin secretory proteins include lactoferrin, peroxidase, lysozyme, and antileukoprotease as well as type I interferons. Most of the nonmucin secretory proteins are secreted by the serous cells contained within submucosal glands. Among proteins known to bind bacteria in the host innate response are lipopolysaccharide (LPS)binding protein (LBP) and bactericidal/permeabilityincreasing protein (BPI). Genes with sequence homology to LBP and BPI have been identified and shown to be expressed in the upper respiratory tract. The genes include plunc (palate, lung, and nasal epithelial clone), lunx, and spurt. The role of these related gene family members in innate immunity is largely inferred from their relationship to the LBP and BPI proteins, their distribution in the respiratory tract, and findings in disease, such as the observation that SPLUNC1 is increased in the sputum of patients with COPD. There are at least 12 mucin genes. The mucin genes MUC1, MUC2, MUC4, MUC5AC, and MUC5B are abundantly expressed in the respiratory

tract. Their gene products contribute directly or indirectly to the pulmonary defense mechanism against infectious pathogens. Respiratory tract mucus is not only essential for lubricating the tissue surface and protecting from injury but also has the function of trapping inhaled particles. These can then be excluded from entry to the airways by the beating action of the cilia. Goblet cells in the airway epithelium and secretory cells in the submucosal glands are the main contributors to mucus secretion. However, overproduction of mucus, as seen in inflammatory contexts such as asthma, chronic bronchitis, bronchiectasis, and CF, causes airway narrowing or obstruction, impeding airflow. Lysozyme – made by glandular serous cells, surface epithelial cells, and macrophages – is a major constituent of lavage fluid and sputum. It represents an important antimicrobial defense, particularly against Gram-positive bacteria. In transgenic mice overexpressing lysozyme in the lung, elimination of pulmonary pseudomonas infection as well as group B streptococci is dramatically enhanced, leading to a decrease in systemic dissemination, enhanced recruitment of neutrophils and protection from death. Furthermore, clinical study of susceptibility and resistance to acute bronchitis show a correlation of protection with levels of macrophage-derived lysozyme. Lactoferrin, another arm of the constitutive defense in the airways, is produced by serous cells and neutrophils. It is able to kill and agglutinate bacteria which it recognizes on the basis of carbohydrate motifs, and also stimulates superoxide production by neutrophils. As with lysozyme, the concentration of lactoferrin is markedly increased in the lower respiratory tract in subjects with chronic bronchitis. The a- and b-defensins have broad microbicidal activity against mycobacteria, Gram-negative and -positive bacteria, fungi, and some viruses. They act by inducing permeabilization and show upregulation in the lung in response to interleukin-1 (IL-1). Binding of defensins to complement components also leads to triggering of the alternative complement cascade. The function of the defensins is highly dependent on salt concentration and likely to be impaired in CF. One of the members of the b-defensin family is tracheal antimicrobial peptide (TAP), which is strongly expressed in the ciliated airway epithelium and upregulated in response to bacterial LPS. The collectins are a family of pre-immune opsonins, able to recognize carbohydrates on the surface of a broad range of pathogens, including bacteria and viruses. This, in turn, leads to recruitment of other cells and defense mechanisms, including macrophage chemotaxis, phagocytosis, immune cell proliferation,

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and activation of the alternate complement cascade. Members of the collectin family include surfactant proteins A and D (SP-A and SP-D), conglutinin, collectin-43, and mannose-binding lectin (MBL). Collectins can directly affect activation of inflammatory cells, including macrophages and lymphocytes. In SP-A knockout mice there is enhanced susceptibility to group B streptococcal pneumonia and sepsis after intratracheal infection. Also, following infection with pseudomonas there is an inability to destroy bacteria and decreased phagocytosis by alveolar macrophages. SP-A function is dependent on binding to the TLR recognition system (see below) as a functional TLR4 molecule is required for induction of leucocyte activation. Human defense pathways share the use of many highly conserved genes and functions with invertebrate species that use these mechanisms in the absence of any adaptive immune response. A feature common to induction of many of the protective factors that contribute to the rapid, innate response is that these mechanisms are often evolved to recognize and respond to conserved structures of microorganisms. The family of Toll receptors were initially described in fruit flies as transmembrane receptors, stimulated by the secreted Spatzle factor, and responsible for the developmental establishment of dorsoventral polarity. However, using mutant strains for several molecules in this pathway, it was subsequently appreciated that there was a functional role also in innate immunity, through activation of the antifungal peptide drosomycin, to fungal infection and of a response to Gram-positive bacterial infection. Furthermore, a role in a conserved pathway of innate immunity was suggested by homology to the mammalian IL-1 receptor gene. Mammalian TLRs were subsequently cloned and shown to constitute the receptors for a large, evolutionarily conserved system of pattern recognition, leading both to innate immune mechanisms and to priming for antigen presentation to the adaptive immune system. Analysis of the natural agonists of TLRs is an area of intense research activity. Generic structures for recognition can include bacterial carbohydrates, LPSs, or forms of bacterial DNA (unmethylated cysteine– guanosine-rich areas known as CpG sequences) that are not seen in human genes. Known ligands for TLR1 include triacyl lipopeptides of bacteria and mycobacteria. TLR2 is activated by diverse ligands (including fungal zymosan and bacterial peptidoglycan), TLR3 by double-stranded RNA during viral infection, TLR4 by several ligands (including LPS), and TLR9 by bacterial CpG DNA sequences. Singlestranded RNA viruses are recognized by TLR8 in humans and TLR7 in mice.

The downstream inflammatory consequences of TLR binding and activation are multiple and diverse. A common downstream signaling pathway acts through activation of nuclear factor kappa B (NFkB). The impact of this activation can encompass release of cytokines, chemotaxis for inflammatory cell types, and priming of the adaptive immune response through the activation of dendritic cells. TLRs are widely expressed and are further inducible during inflammation. TLR expression in the lung encompasses both the expression patterns on inflammatory cells of hematopoietic origin and the expression by lung tissue itself. Alveolar epithelial cells express TLR2 and TLR3, as do alveolar macrophages. TLR4 and TLR6 expression is also found in the lung. Information on TLR expression and susceptibility to lung infection has come mainly from studies of mice carrying null mutations for TLR genes or common TLR signaling molecules such as MyD88. From such studies TLR4 is important for the control of chronic Mycobacterium tuberculosis infection. Granulomas from patients with active tuberculosis show expression of TLR1–5 and TLR9. TLR4 is also implicated in defense against respiratory syncytial virus (RSV) from studies in knockout mice. TLR4null mice infected with RSV show impaired NK cell trafficking, reduced cytokine expression, and reduced virus clearance. For virus recognition, there also exist other non-TLR pathways such as PKR and RIG1. Immunoglobulin A (IgA) is considered the most important immunoglobulin for defense in the respiratory tract. This is borne out by the susceptibility to chronic bronchial infections in patients with IgA immunodeficiencies. Although, like other immunoglobulins, it is secreted by B lymphocytes, unlike the other immunoglobulins such as IgG and IgE found in the lung, synthesis is not dependent on specific T lymphocyte help. Thus, CD4 T cell knockout mice have normal IgA responses. It is thought that IgAsecreting B cells are stimulated locally in the lung. The IgA response is an important component of rapid, local lung immune responses to infection by viruses including influenza and RSV. An important component of the direct response of cells in the respiratory tract, including epithelial cells and macrophages, to viral infection comprises the type I interferons, a and b. Following type I interferon release, several factors are released with the ability to interfere with viral replication. Knockout mice carrying null mutations for the receptor to these interferons show enhanced viral replication in the lung. Interestingly, protection may nevertheless be maintained through an augmented neutralizing antibody response. Furthermore, resolution of infection in

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these circumstances can be associated with an altered inflammatory response, with the appearance of granulocytic pulmonary inflammatory cells.

Cells of the Innate Immune Response Activation of many of the pathways described above can, in turn, lead to release of mediators that increase neutrophil migration to the lung. Within hours of experimental infection or LPS injection, there is massive neutrophil recruitment until these cells constitute 60–80% of bronchial alveolar lavage (BAL) cells. This can occur through the stimulation of factors including IL-8, complement activation, or the release of chemokines. Activated neutrophils have considerable capacity to phagocytose and neutralize bacteria, and can also secrete various factors including defensins, tumor necrosis factor alpha (TNF-a), IL-1b, and IL-6. For example, experimental infection with Haemophilus influenzae causes respiratory epithelial cells to both release IL-8 and upregulate expression of a molecule called intercellular adhesion molecule-1 (ICAM-1). The IL-8 leads to neutrophil recruitment and the enhanced ICAM-1 to increased neutrophil adherence, both of these contributing to clearance of the infection. Furthermore, neutrophils utilize a range of microbicidal products. These include oxidants, microbicidal peptides, and lytic enzymes. Neutrophil generation of microbicidal oxidants results from the activation of a multiprotein enzyme complex known as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, responsible for transferring electrons from NADPH to O2, and thus leading to formation of superoxide anion. Indeed, a wide array of functionally overlapping cytotoxic pathways has so far been elucidated in neutrophils, and it remains uncertain which are the most significant in host defense. Knockout mouse studies have tended to place more emphasis on the pathways associated with the neutrophil granule proteins than the direct effect of free radical reactions. Evidence also comes from the analysis of patients with chronic granulomatous disease (CGD), an immunodeficiency most commonly associated with a mutation in the gene encoding the b subunit of cytochrome b558 or gp91, called CYBB on the X chromosome and associated with defects in the neutrophil oxidative respiratory burst. While CGD is a profound immunodeficiency in terms of susceptibility to bacterial and fungal infections, the patients show considerably greater resistance to infection than individuals with severe neutropenia (and so deficient in the full gamut of neutrophil-dependent host defense mechanisms).

Alveolar macrophages respond to signals from microbes both through TLR recognition and through a number of other specific, cell-surface receptors. The repertoire of receptors expressed on the alveolar macrophage surface is vital for binding, uptake, and response to microbes and inhaled particles. An important family of receptors is the class A macrophage scavenger receptors (SRAs), a group of pattern recognition receptors comprising three members: SRA-I/ II, macrophage receptor with collagenous structure (MARCO), and scavenger receptor with C-type lectin (SRCL). In a murine model of pneumococcal pneumonia, MARCO knockout mice show impaired clearance of bacteria from the lungs, increased pulmonary inflammation and cytokine release, and diminished survival. NK cells pose a further barrier to infectious agents entering the lung. NK cells derive from the same hematopoietic lineage as T lymphocytes, but, unlike classical T cells, do not mature in the thymus and do not express rearranged antigen receptors. In view of the relationship between the role of receptors expressed by NK cells in recognition of alterations in self-proteins and that of T cells, NK cells may be regarded as occupying the boundary between evolution of the innate and adaptive immune systems. NK cells use what is now appreciated to be a large number of families of cellular receptors to survey the body – most notably the large, polymorphic family of killer cell immunoglobulin-like receptors (KIRs) – to screen for cells that, for reasons of infection or transformation, have altered expression of human leukocyte antigen (HLA) class I tissue antigens. The rules governing NK cell activation are complex, not the least because different members of the receptor families can be either inhibitory with respect to the intracellular signal they transmit on ligand engagement, or activatory. Thus, while NK cells were until recently defined simply as cells that patrol for HLA class I expression that has been diminished as a consequence of infection or cellular transformation, the ‘missing self’ hypothesis, they are now considered cells that survey for targets that either lack proper class I expression or overexpress ligands for activatory NK cell receptors. The KIR family of receptors is particularly focused on recognition of alterations in HLA-C expression. If the NK cells fail to receive a cellular signal that normal HLA class I has been recognized, they can enter a program of activation leading to lysis of the infected cell and release of interferon gamma (IFN-g). This can, in turn, lead to recruitment of other cells. In experimental RSV infection, there is an extremely rapid antiviral NK cell IFN-g response that precedes and leads to recruitment of virus-specific, cytotoxic T lymphocytes.

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Local release of IL-12 is an important early event leading to stimulation of NK cells for rapid antiviral immunity in the lung. Recent research in sarcoidosis has focused on a specific population of NK cells, termed NKT cells, which are considered to have regulatory properties in many diseases. It has been observed that patients with sarcoidosis show a significantly reduced population of CD1d-restricted NKT cells, a finding that is considered consistent with the excessive Th1 inflammatory activation associated with the disease.

Adaptive Immunity The specific, adaptive immune response to infection in the lung depends on the specific recognition of foreign, microbial antigens by the clonotypic receptors of B and T lymphocytes. The antibodies against a given pathogen may be directed against several different binding sites, termed epitopes. Some antibodies are able to neutralize infections through interference with a vital, structural component of the microbe. Others may clear infections through opsonization for phagocytosis and through activation of the complement system. After initial exposure, a state of B cell memory is established, leading to the ability to mount a more rapid IgG antibody response on any subsequent re-exposure. Furthermore, the B-lymphocyte-derived IgG response is unique in the immune response in showing affinity maturation. This is the ability to introduce small numbers of random mutations into the genes for the antibody, somatically mutating the sequence so that receptors with better affinity for the epitope are selected at each generation of cell division. This antibody response and its affinity maturation are absolutely dependent on cytokine-mediated help ensuing from antigen recognition by CD4 T cells. The antibody response to infection mounted by B lymphocytes acts alongside the T lymphocyte response. T lymphocytes carry the surface markers CD3 along with either CD4 or CD8, mature in the thymus, and express an antigen receptor that must recognize peptide fragments of microbial antigen presented by host HLA molecules. T lymphocytes mediate their effector function either through the release of a wide range of soluble mediators called cytokines or, in the case of CD8 cytotoxic T cells, by direct killing of target cells. Early studies aimed at cloning the genes for the antigen receptor used by T lymphocytes identified two separate systems of receptors now known to delineate separate lineages of thymus-differentiated T cells. One receptor heterodimer was the ab receptor, the other the gd receptor. The cells expressing the ab T cell receptor are the

conventional CD4 and CD8 populations with the properties of mounting cytolytic responses to infected cells, making cytokines, and stimulating B cells. This involves recognition of peptide fragments of micorobial antigens loaded through the biosynthetic pathway of classical HLA class I or class II molecules into the peptide-binding groove. The T cells expressing the gd receptor are particularly associated with defense at epithelial surfaces, have a different mode of recognizing foreign antigens, but are also involved in lung immunity. They do not need antigen processing or major histocompatibility complex (MHC) restriction for antigen recognition. The types of antigen they recognize can be categorized into those that are widely distributed and are constitutively expressed in host cells and by microbial pathogens, and those that are inducibly expressed and might be restricted to specific cell types. Presentation may be by classical MHC molecules or by related molecules such as CD1d. Examples of the families of recognized antigens include the nonprotein pyrophosphates and alkylamines that are found in bacteria, plant or animal cells, and bacterial and mammalian homologs of the stress protein Hsp60/GroEL. CD4-positive, TCR ab expressing T lymphocytes are termed Th cells. Their effector function is through the release of cytokines that stimulate or activate some other cell type: for example, IL-4, which stimulates B cells and attracts eosinophils; IL-2, which drives other T lymphocytes, including CD8 cells to divide; and IFN-g, which causes other T lymphocytes to differentiate at the same time as it activates macrophages and dendritic cells. CD4 cells can themselves kill target cells through the release of TNF-a. CD8-positive, TCR ab T lymphocytes are termed cytotoxic cells (or sometimes, killer cells). On recognition of microbial peptides presented by HLA class I molecules, they function by lysing infected cells, particularly by release of granules of perforin.

Responses by T-Cell Subpopulations The functional distinction between Th1 and Th2 subpopulations was initially made in experimental mouse models in which antigens were given under rather polarizing conditions, although the categorization, while often less clear-cut in naturally induced T cell immune responses of humans, appears to have meaning in this context also. Th1 cells are driven to differentiate primarily by the presence of the cytokines IL-12 and IL-18, and, in response to antigen, can make IFN-g as well as IL-2 and TNF-a. These are the cells historically described as those mediating delayed hypersensitivity responses to local viral or

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bacterial infection, stimulating local macrophage activation, neutrophil recruitment, and specific cytotoxic T-cell responses. In the respiratory immunology sphere, sarcoidosis represents an overtly Th1 environment in which the majority of CD4 T cells express receptors for IL-12 and make Th1 cytokines. Th2 cells are driven to differentiate primarily by the presence of the cytokine, IL-4. They can respond to antigen challenge by making more IL-4 as well as IL-5 and IL-13. IL-10 is considered a regulatory cytokine that may be associated with Th1, Th2, or regulatory T cell (T reg) responses (see below). The Th2 cytokine response profile is important for driving B lymphocytes to make antibody responses and in classswitching of antibody production to make IgG1a and IgE. Th2 cytokines can also lead to recruitment and activation of basophils and eosinophils. Thus, in the respiratory context, a prototypic Th2 response is that seen in atopic asthma or in aspergillosis. The choice of differentiation pathway from the Th0 precursor to Th1 to Th2 is made by mature, post-thymic T cells in the peripheral immune system and is influenced by many variables including the local cytokine milieu, the nature of the antigen-presenting cell and available co-stimulatory molecules, the avidity of the interaction, and the nature of the antigen. Cytolytic CD8 cells can similarly be divided into Tc1, and Tc2. This distinction follows the same split as for T-helper cells: Tc1 cells are primarily characterized by release of IFN-g, and Tc2 cells primarily by release of IL-4. The various T cell subsets described here – Th1, Th2, Tc1, and Tc2 – can each be associated either with protection from pathogens or (in the case of either an overzealous, dysregulated immune response or the need to mount a continued response in the face of an inability to cope with antigen load) with damaging immune pathology. A number of specific observations have been made about mechanisms regulating Th1/Th2 balance in the lung. It remains to be seen whether these observations are unique to pulmonary immune responses or also have relevance to other sites. Pulmonary dendritic cells (DCs) from mice exposed to respiratory antigen transiently produced IL-10. These pulmonary DCs can stimulate the development of CD4 þ Treg cells that also produce high amounts of IL-10 and have the ability to transfer specific immunological tolerance. Many observations have been made about the possible role of Treg cells in modulating potentially immunopathogenic immune responses in the lung, particularly in asthma. A particularly novel Treg population has been described in the studies of pulmonary immunity. A population of Treg cells develops in vivo from naive CD4 þ CD25
T cells during

Th1-polarized responses, distinct from CD25 þ TR cells. In terms of transcription factor expression, they are positive both for Foxp3 and T-bet, thus showing features intermediate between Th1 and Treg cells. These antigen-specific Treg cells are induced by CD8 þ DCs, produce IL-10 and INF-g, and inhibit development of airway hyperreactivity. The contributions of different T cell populations to protection and tissue damage during lung infection have been modeled in a wide variety of transgenic and knockout mouse strains. Protection of mice from experimental influenza infection is particularly related to the transfer of IFN-g secreting CD8 cells, although CD4 cells also offer protection. While Th1 CD4 cells protect against lethal challenge, transfer of Th2 cells exacerbates disease. Absolute numbers of systemic antiviral T cells can be enumerated using fluorescent peptide/MHC tetramers as a probe. From these studies, it is clear that there exists robust, long-lasting CD8 immunity following viral infection, with cytotoxic precursor cells detectable at more than 20-fold increased levels for life. Thus, immune individuals have potent memory such that virus can be cleared considerably faster than in immunologically naive individuals. The ability of CD8 cells to clear a pulmonary viral infection is not simply related either to the number of cells or to their cytolytic capacity, as Tc1 cells are able to isolate and clear influenza infections far more rapidly than equally cytolytic Tc2 cells. Thus, besides the cytolytic activity, the ability to migrate appropriately to the site of infection and attract other appropriate cell types is also crucial. Mice expressing transgenic IL-4 targeted to the lung show delayed clearance of virus, although this, in turn, leads to an enhanced neutralizing antibody response. This is in line with the view that there is considerable redundancy in mechanisms for clearing infection from the lung, although these may be associated with different inflammatory consequences. In experimental infection with RSV, Th2-dependent eosinophilia can be reversed by treatment with the Th1-polarizing cytokine IL-12. However, despite reversing the eosinophilia, the clinical outcome is relatively unchanged. A number of studies show inability to clear infection in knockout mice lacking IFN-g or IFN-g receptors. The role of gd T cells in protection from respiratory pathogens is less well characterized than TCR ab CD4 and CD8 cells. Experiments have compared susceptibility to infection in knockout mice lacking TCR ab cells compared with those lacking TCR gd cells. The TCR gd knockout mice are extremely susceptible to mycobacterial and nocardia infections as well as to lethal bacterial pneumonia following infection with Klebsiella.

DEFENSINS 7 See also: Cilia and Mucociliary Clearance. Collectins. Complement. Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Defensins. Immunoglobulins. Interferons. Interleukins: IL-1 and IL-18; IL-4; IL-5; IL-6; IL-7; IL-9; IL-10; IL-12; IL-13; IL-15; IL-16; IL-17; IL-23 and IL-27. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; Monocytes; T cells; Pulmonary Macrophages. Mucins. Mucus. Surfactant: Surfactant Protein A (SPA); Surfactant Protein D (SP-D). Toll-Like Receptors.

Further Reading Basu S and Fenton MJ (2003) Toll-like recpeptors: function and role in lung disease. American Journal of Physiology. Lung Cellular and Molecular Physiology 286: L887–L892. Crouch E, Hartshorn K, and Ofek I (2000) Collectins and pulmonary innate immunity. Immunological Reviews 173: 52–65. Diamond G, Legarda D, and Ryan LK (2000) The innate response of the respiratory epithelium. Immunological Reviews 173: 27–38. Doherty PC, Hou S, and Tripp RA (1994) CD8 þ T cell memory to viruses. Current Opinion in Immunology 6: 545–552. Kronenberg M (2005) Toward an understanding of NKT cell biology: progress and paradoxes. Annual Review of Immunology 23: 877–900. Lanier L (2005) NK cell recognition. Annual Review of Immunology 23: 225–274.

Moore TA, Moore BB, Newstead MW, and Standiford TJ (2000) Gamma delta T cells are critical for survival and early proinflammatory cytokine gene expression during murine Klebsiella pneumonia. Journal of Immunology 165: 2643–2650. Rogge L, Papi A, Presky DH, et al. (1999) Antibodies to the IL-12 receptor beta 2 chain mark human Th1 but not Th2 cells in vitro and in vivo. Journal of Immunology 162: 3926–3932. Rowe SM, Miller S, and Sorscher EJ (2005) Cystic fibrosis. New England Journal of Medicine 352: 1992–2001. Sallusto F, Geginat J, and Lanzavecchia A (2004) Central memory and effector memory T cell subsets: function, generation, and maintenance. Annual Review of Immunology 22: 745–763. Segal A (2005) How neutrophils kill microbes. Annual Review of Immunology 23: 197–224. Takeda K, Kaisho T, and Akira S (2003) Toll-like receptors. Annual Review of Immunology 21: 335–376. Turner SJ, Kedzierska K, La Gruta NL, Webby R, and Doherty P (2004) Characterization of CD8 þ T cell repertoire diversity and persistence in the influenza A virus model of localized, transient infection. Seminars in Immunology 16: 179–184. Underhill DM (2004) Toll-like receptors and microbes take aim at each other. Current Opinion in Immunology 16: 483–487. Woodland DL (2003) Cell-mediated immunity to respiratory virus infections. Current Opinion in Immunology 15: 430–435. Zangh P, Summer WR, Bagby GJ, and Nelson S (2000) Innate immunity and pulmonary host defence. Immunological Reviews 173: 39–51.

DEFENSINS P S Hiemstra, Leiden University Medical Center, Leiden, The Netherlands & 2006 Elsevier Ltd. All rights reserved.

Abstract Defensins are members of a large family of cationic antimicrobial peptides that form an essential element of innate immunity. They are key effector molecules in host defense against infection due to their broad-spectrum antimicrobial activity. Defensins are small, cationic peptides that contribute to the antimicrobial activity of phagocytes, the skin, and the mucosa (including that of the lung). Neutrophils and epithelial cells are the main cellular sources of these peptides, but monocytes, macrophages, dendritic cells, and lymphocytes also produce defensins. The two families of antimicrobial defensins expressed in the human lung are the a- and the b-defensins, mainly produced by neutrophils and epithelial cells, respectively. The members of these families are structurally different, but display partly overlapping activities. Recent studies show that defensins not only act as endogenous antibiotics, but also display a range of activities involved in inflammation, immunity, and wound repair. Studies in animal models and human disease strongly suggest an important role of these molecules in vivo. In addition, genetic studies show associations between polymorphisms in defensin genes and inflammatory lung disease.

Introduction The host is equipped with a variety of antimicrobial mechanisms, including the action of antimicrobial reactive oxygen and nitrogen intermediates, and antimicrobial peptides and proteins. These antimicrobial polypeptides have been the subject of intense research in the last decade and are considered interesting templates for the pharmaceutical development of efficient broad-spectrum antimicrobial peptides. Mammalian defensins are small (3–5 kDa) cationic peptides that have been identified in humans, monkeys, cows, and rodents. These peptides kill a wide range of Gram-negative and Gram-positive bacteria, fungi, and viruses. Following the discovery of arginine-rich antimicrobial components in rabbit and guinea pig neutrophils in the 1960s and 1970s, the first human defensins were described and named in 1985. Three neutrophil defensins were isolated from human neutrophils and named human neutrophil peptides 1–3 (HNP1–3). Soon afterwards, the fourth much less abundant neutrophil defensin, HNP-4, was identified. Neutrophil defensins are highly abundant in neutrophils: they make up 5% of the total protein

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content of the neutrophil, and are the predominant component of myeloperoxidase-containing neutrophil azurophilic granules. Like all defensins that have been characterized at the peptide level to date, neutrophil defensins display broad-spectrum antimicrobial activity against a range of microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and (enveloped) viruses. Based on their structure, neutrophil defensins belong to the a-family of defensins. The Paneth cells in the human small intestine express other a-defensins, named human defensin (HD) 1 and 2. The first mammalian b-defensin was discovered in the bovine respiratory tract in 1991. The first human b-defensin (human b-defensin 1; hBD-1) was isolated from hemofiltrate and characterized in 1995. Subsequently, several human b-defensins were characterized at the protein and gene level. j-Defensins are the last family of defensins that have been identified. These circular mini-defensins were isolated from rhesus monkeys in 1999. They appeared to have evolved in primates, but the human y-defensin genes are pseudogenes that are not expressed.

Table 1 Human defensin families Peptide family a-Defensins Neutrophil defensins HNP-1, HNP-2, HNP-3, HNP-4 Paneth cell defensins HD-5, HD-6

b-Defensins Epithelial defensins hBD-1, hBD-2, hBD-3, hBD-4

Distribution Neutrophils; also NK-cells and CD8 cells Paneth cells of the small intestine; also nasal and bronchial epithelial cells, female reproductive tract

Epithelial cells; also various leukocyte subsets

In addition to this cluster on chromosome 8, four additional clusters have been identified encoding defensin genes that are transcribed. However, the gene products of these defensin genes have not yet been explored. The gene for HNP-2 has not been found; for this reason and based on the amino acid sequence of the peptide, it is likely that HNP-2 is generated from HNP-1 and/or HNP-3 by proteolytic processing (Table 1).

Structure Defensins are small (3–5 kDa), nonglycosylated cationic peptides with a b-sheet structure that contain six cysteine residues that form three intramolecular disulfide bridges. Defensins are divided into the three families (a, b, and y) based on the spacing and connectivity of the cysteines in the disulfide bridges, and on the overall molecular structure. a-Defensins are arginine-rich peptides containing 29–35 amino acid residues, with the following disulfide alignment: 1–6, 2–4, and 3–5. b-Defensins are 36–42 amino acids in length and are characterized by an 1–5, 2–4, 3–6 disulfide alignment. y-Defensins are circular minidefensins that arise from two a-defensin mRNA precursors, and are expressed in rhesus monkeys. The genes encoding these defensins are present in humans, but not expressed due to the presence of a premature stopcodon. The genes encoding the human a-defensins and the human b-defensins (hBD) 1–6 are located in a cluster on the short arm of chromosome 8. The HE2 (human epididimys secretory protein) gene is located in close proximity of the hBD-3 gene. A splice variant of this HE2 gene, HE2b1, is expressed in bronchial epithelium and encodes a cationic peptide containing the six-cysteine-motif that is characteristic of b-defensins. Multiple copies and polymorphisms for several a- and b-defensin genes have been demonstrated, and complicate the fine mapping of this region.

Regulation of Defensin Production Defensins are produced as precursor polypeptides containing a signal peptide, a propeptide, and the mature peptide. The propiece is removed by proteolytic processing, resulting in the release of the mature peptide. For neutrophil a-defensins, this processing occurs in the developing neutrophils in the bone marrow, and the mature peptide is stored in the azurophilic granules of neutrophils. However, circulating prodefensin levels are increased in patients with inflammatory disorders. Paneth cell a-defensins are stored in the secretory granules of these cells, and following secretion into the intestinal lumen processing occurs extracellularly. Paneth cell trypsin has been identified as the major processing enzyme for HD-5. Phagocytosis of microorganisms by neutrophils leads to release of the defensins in the phagolysosome, where they contribute to intracellular killing. Neutrophil stimulation may also lead to release of the granular content into the environment of the cell, thus explaining the high amounts of neutrophil defensins that are observed in inflammatory exudates. HD-5 and HD-6 are primarily expressed in the Paneth cells. All human b-defensins that have been identified in some detail (hBD1-4; HE2b1) are expressed in human lung tissue. These b-defensins are mainly

DEFENSINS 9

produced by epithelial cells, but hBD-1 and hBD-2 are also expressed in monocytes, macrophages, dendritic cells, and lymphocytes. Inflamed epithelia are a rich source of antimicrobial peptides such as b-defensins. Indeed, production of b-defensins by epithelial cells is mainly inducible under inflammatory conditions, although production of hBD-1 is constitutive. Expression of b-defensins is increased by a variety of stimuli. Microbial products increase b-defensin expression through the action of Toll-like receptors (TLRs) such as TLR2, TLR4, and TLR9. TLRs are pattern-recognition receptors for conserved molecular patterns present on microbial products that allow the innate immune system to recognize microbial presence and respond accordingly. In addition, proinflammatory cytokines (such as IL-1b, TNF-a, and IL-17), growth factors (such as TGF-a), and activators of protease-activated receptors increase b-defensin expression. Selected microorganisms have been shown to downregulate expression of b-defensins, thus increasing susceptibility of the host to infection.

Biological Function Most defensins that have been characterized at the peptide level were identified based on their antimicrobial activity. Defensins exert this activity by interfering with the integrity of the microbial membrane. Defensins kill a range of Gram-positive and Gram-negative bacteria, fungi, and viruses. Selected defensins display anti-HIV activity. Differences exist with respect to the selective activity against specific microorganisms for the different defensins. The main sites of antimicrobial action of defensins appear to be the phagolysosome of neutrophils, where ingested microorganisms are killed, and the surface of the skin and mucosa. Defensins in these compartments form part of a cocktail of antimicrobial molecules, and may act in synergy with these molecules. Pathogens and commensals have developed a variety of resistance mechanisms that serve to protect them against the action of antimicrobial peptides such as defensins. This bacterial resistance is determined by a variety of mechanisms, including changes in the charge or structure of the bacterial cell membrane. Also the ability of certain bacteria to decrease epithelial bdefensin expression may contribute to pathogen survival. In addition to their antimicrobial activity, defensins display a range of other activities (Figure 1). Defensins display chemotactic activity for a range of cell types, are involved in adaptive immunity through their effect on T cells and dendritic cells, and activate mast cells, epithelial cells, and fibroblasts leading to

-Defensins (HNP1– 4)

Activation of epithelial cells and fibroblasts


-Defensins (hBD1–6)

• Antimicrobial activity • Chemoattractant for T cells, dendritic cells, monocytes, and macrophages • Mast cell activation

Figure 1 Defensin production and activities in the lung. Neutrophils infiltrating the airways release neutrophil a-defensins (HNP1–4), whereas airway epithelial cells are the main cell type producing b-defensins. However, other cell types such as lymphocytes, macrophages, and dendritic cells also contribute to b-defensin production in human lung tissue. a- and b-defensins display a range of overlapping and distinct activities. See text for details.

release of proinflammatory mediators, proteinase inhibitors, and extracellular matrix components. They also modulate fibroblast and epithelial cell proliferation and aid in mucin production and wound repair in airway epithelium. The role of human defensins in animal models has been demonstrated in a number of studies. Intratracheal instillation of neutrophil defensins into mice induced inflammation, and increased lung permeability and dysfunction. HD-5 transgenic mice that express the human transgene in the intestinal Paneth cells were found to be markedly resistant to oral challenge with Salmonella typhimurium. Further support for the in vivo contribution of defensins to host defense against infection comes from studies in knockout mice lacking murine defensins or the metalloprotease involved in processing of Paneth cell a-defensins. The observation that mice deficient in mouse b-defensin 1 (mBD-1) clear Haemophilus influenzae inefficiently from their lungs further supports a role for defensins in defense against respiratory infection. Although various in vivo studies have shown that defensins contribute to host defense against infection, it is likely that many of these observations are not solely explained by direct antimicrobial activity of defensins. The ability of defensins to promote chemotaxis, cause mast cell degranulation, and their other activities (Figure 1) likely contributes markedly to their in vivo antimicrobial action.

10

DENDRITIC CELLS

Receptors The ability of defensins to activate eukaryotic cells has prompted the search for cellular receptors that bind defensins. This led to the discovery that b-defensins bind to the chemokine receptor CCR6 on immature dendritic cells, and murine b-defensin-2 (mBD-2) activates cells via TLR4. The receptor involved in recognition of a-defensins has not yet been identified.

Defensins in Respiratory Disease Variations in gene-copy numbers and single nucleotide polymorphisms have been described for various a- and b-defensin genes. These polymorphisms may contribute to inflammatory lung disease, as demonstrated by the association of single nucleotide polymorphisms in b-defensins with chronic obstructive pulmonary disease (COPD). The contribution of variable copy numbers of defensin genes to disease susceptibility or severity has not yet been established. An increasing number of studies have explored the concentrations of defensins in lung diseases. Defensin levels are increased in airway secretions and plasma in a range of respiratory disorders, which is probably explained by the fact that lung inflammation is often characterized by neutrophil accumulation and epithelial cell activation. Lung disorders, in which increased defensin levels are found, include cystic fibrosis, diffuse panbronchiolitis, acute respiratory distress syndrome, a1-antitrypsin deficiency, mycobacterial infections, pneumonia, pulmonary fibrosis, and sarcoidosis. Whereas increased levels may be a mere reflection of inflammation or may contribute to disease, functional impairment of defensin activity may also play a role in inflammatory lung disease. Defensins show optimal activity at the low-salt conditions that appear to be characteristic for mucosal secretions of the lung. It has been suggested that these salt concentrations are higher in the epithelial lining fluid of patients with cystic fibrosis, and that this may

cause inactivation of defensins. However, these increased salt concentrations in epithelial lining fluid of the airways in cystic fibrosis were not confirmed in all studies. Conversely, because a1-antitrypsin is an inhibitor of the proinflammatory activity of neutrophil a-defensins, its deficiency may facilitate this activity. Taken together, these data demonstrate that defensins play a role in a variety of lung diseases. See also: Cystic Fibrosis: Overview. Defense Systems. Leukocytes: Neutrophils. Matrix Metalloproteinases. Mucus. Panbronchiolitis. Pneumonia: Overview and Epidemiology. Proteinase Inhibitors: Secretory Leukoprotease Inhibitor and Elafin. Thrombolytic Therapy.

Further Reading Bals R and Hiemstra PS (2004) Innate immunity in the lung: how epithelial cells fight against respiratory pathogens. European Respiratory Journal 23: 327–333. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews: Microbiology 3(3): 238–250. Devine DA (2003) Antimicrobial peptides in defence of the oral and respiratory tracts. Molecular Immunology 40(7): 431–443. Devine DA and Hancock REW (eds.) (2005) Mammalian Host Defense Peptides. Cambridge: Cambridge University Press. Gallo RL (ed.) (2005) Antimicrobial Peptides in Human Health and Disease. Norfolk, UK: Horizon Bioscience. Ganz T (2002) Antimicrobial polypeptides in host defense of the respiratory tract. Journal of Clinical Investigation 6: 693–697. Ganz T (2003) Defensins: antimicrobial peptides of innate immunity. Nature Reviews: Immunology 3(9): 710–720. Schutte BC and McCray PB Jr (2002) b-Defensins in lung host defense. Annual Review of Physiology 64: 709–748. Van Wetering S, Tjabringa GS, and Hiemstra PS (2005) Interactions between neutrophil-derived antimicrobial peptides and airway epithelial cells. Journal of Leukocyte Biology 77: 444–450. Yang D, Biragyn A, Hoover DM, Lubkowski J, and Oppenheim JJ (2004) Multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neurotoxin in host defense. Annual Review of Immunology 22(1): 181–215. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415(6870): 389–395.

DENDRITIC CELLS J W Upham and T L Hughes, Telethon Institute for Child Health Research, Perth, WA, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Dendritic cells (DCs) are specialized, migratory antigen presenting cells which are able to regulate immune reactions. DCs

are especially sensitive to signals derived from microbes, allergens, and the airway tissue microenvironment. They can polarize naı¨ve T cells into either Th1 and Th2 effector cells, and are increasingly recognized as having a central role in the establishment of long-lasting adaptive immunity as well as the maintenance of tolerance to inhaled antigens. DCs form a closely meshed network within the respiratory mucosa, and are rapidly recruited from the circulation in response to a variety of proinflammatory stimuli.

DENDRITIC CELLS 11 DCs also have a crucial role in the initiation and maintenance of the dysregulated immune responses seen in many respiratory diseases. In allergic asthma, for example, allergens are captured by DCs in the airways, subsequently priming a Th2 immune response, with the subsequent development of allergic airway inflammation. Increased numbers of DCs also infiltrate the airways and lung parenchyma in a variety of other inflammatory diseases. This chapter summarizes the current literature on the origin and function of DCs, as well as their contribution to the pathology seen in respiratory disease. The future is likely to witness a concerted effort to develop a range of new therapies targeted at DCs, including DC-based vaccines and DC-based immunomodulation.

the respiratory intraepithelial tissue, at a density of 500–1000 cells mm
2. Under experimental conditions, they can be derived from either lymphoid or myeloid progenitor cells; however, the differentiation pathways that generate DCs in vivo are largely unknown. Over 30 years ago, Ralph Steinman discovered DCs in murine lymphoid tissues. He described them as phagocytic cells with unusual shapes that were distinct from macrophages and had a highly potent stimulatory role in immune function.

Dendritic Cell Subpopulations Introduction The lung mucosa together with the skin and gastrointestinal tract comprise the extensive interface that exists between the host and the external environment. This faces a continuous threat from foreign proteins and pathogens and thus requires a highly proficient immune surveillance system. Dendritic cells (DCs) provide a crucial role in this process. Their migratory nature allows them to sample environmental proteins, process them, and subsequently present them in regional lymph nodes to T lymphocytes, initiating the adaptive immune response. The term ‘dendritic cell’ arises from their characteristic morphology (Figure 1). These cells extend long, irregular dendrites in order to maximize their surface area and thus their exposure to environmental antigens. They exist as a high-density network in

Dendritic cells can be divided into subsets, partly determined by their origin and function but also by their location. Myeloid DCs can be recognized by the expression of the myeloid markers CD11c, CD33, and CD13. They have a lineage relationship with macrophages and monocytes and require granulocyte-macrophage colony-stimulating factor (GM-CSF) for survival. Myeloid DCs can also be differentiated from monocytes in vitro using cytokines such as GM-CSF and interleukin-4 (IL-4) or IL-13. Differentiation of monocytes into DCs is likely to be a normal phenomenon. There seem to be some monocytes (identified by CD16 expression) that are committed to differentiating into DCs. However, in the presence of inflammatory signals all monocytes are thought to be able to differentiate into either macrophages or DCs.

Figure 1 (a) Confocal microscopic image of DCs within the epithelium of a normal mouse trachea. Longitudinal section, magnification 40. The extensive branching processes are thought to facilitate antigen capture. These processes retract when DCs migrate to regional lymph nodes. Photograph courtesy of Phil Stumbles and Roz Wealthall. (b) Transmission electron micrograph of myeloid DCs isolated by enzymatic digestion from normal mouse lung. Scale ¼ 2 mm. Image courtesy of Phil Stumbles, Christoph von Garnier, and Luis Filgueira.

12

DENDRITIC CELLS

The origin of the plasmacytoid subset of DCs has not been established. They are long-lived cells, which morphologically resemble plasma cells and do not express myeloid markers. They express the IL-3 receptor a chain CD123, and respond to IL-3 for their growth and survival. Producing large amounts of ainterferon, it is thought that their primary role is in the host defense against viral infections and in the development of peripheral tolerance. Langerhans cells (LCs) represent one subpopulation of DCs that are located in epithelial tissues. These cells have been most extensively characterized in the epidermis of the skin where they express a distinctive array of molecules including CD1a, Ecadherin, Langerin, and Birbeck granules. Less is known about LCs in the lung, though it appears that many DCs in the airway epithelium express LC-specific markers. Dendritic Cells in the Lung

Dendritic cells form a tightly meshed network within antigen-exposed tissues of the upper airway, bronchial mucosa, lung interstitium, and pleura. The density of DCs in the airways is related to the extent of antigen exposure, being greatest in the proximal airways, and diminishing towards the periphery. A variety of proinflammatory stimuli induce a marked increase in the density of lung DCs, including exposure to bacterial and viral infection.

Dendritic Cell Function in the Normal Lung Dendritic cells at mucosal surfaces display a distinct phenotype in the steady state, being adept at antigen uptake, and having a poor capacity to present antigen to T cells. They are able to respond immediately and efficiently to many microbial and inflammatory stimuli via Toll-like and cytokine receptors. Subsequent to antigen uptake and environmental signals, the DC morphology and cell surface marker expression is altered. This facilitates migration to regional lymph nodes, where antigens are presented and T cell responses effected (see Figure 2). Antigen Uptake

Antigen uptake occurs via several mechanisms. Constitutive, low-level uptake of small soluble particles is mediated by micropinocytosis, with larger particles also being taken up in the fluid phase by the cytoskeleton-dependent process of macropinocytosis. Receptor-mediated endocytosis, however, is 100-fold more efficient at antigen uptake. There are various specialized receptors on the dendritic cell surface that

specifically target foreign proteins. These receptors include C-type lectin receptors, such as the mannose receptor, Langerin, and DEC205, as well as receptors such as Fcg and complement receptors, which recognize opsonized bacteria. Specific receptors for the uptake of apoptotic bodies are also expressed. The high expression of the high-affinity IgE receptor FceRI by human asthmatic airway DCs indicates a role for these receptors in the uptake of the inhaled allergens. Migration and Maturation

In response to pathogens, tissue damage, or proinflammatory cytokines, DCs migrate to regional lymph nodes and acquire the ability to prime naı¨ve T cells. The process of DC maturation involves changes in the expression of specific chemokine receptors, with concomitant downregulation of tissue-homing receptors and upregulation of lymph node-homing chemokine receptors. In addition to chemokines, other mechanisms are likely to regulate DC migration, including integrin and cadherin expression, and extracellular matrix-degrading enzymes, although these have not been well characterized in relation to respiratory tract DCs. Spontaneous migration of DCs to draining lymph nodes is also observed under healthy steady-state conditions in the absence of tissue inflammation, although the mechanisms involved are not well characterized. These spontaneously migrating DCs retain a somewhat ‘immature’ phenotype, even after moving to the lymph nodes, and this is likely to be critical for the development of immune tolerance. A number of mechanisms exist to regulate DC function within the lungs. Freshly isolated lung DCs exhibit a poor capacity for antigen presentation and express only low levels of costimulatory molecules. However, culturing purified lung DCs for short periods in the absence of exogenous stimuli is sufficient to elicit spontaneous DC activation, suggesting that the tissue environment in the lung provides inhibitory signals that counteract DC activation in vivo. Alveolar macrophages are known to maintain lung DCs in an immature state through the release of nitric oxide. Furthermore, airway epithelial cells may also have the ability to regulate DC function. Antigen Presentation

Dendritic cells not only contribute to the expansion and differentiation of T lymphocytes, but also the qualitative nature of the T cell response. The role of DCs in shaping the differentiation pathway of a naı¨ve T cell is potentially powerful because DCs provide the precursor T cell with its first activation

DENDRITIC CELLS 13 Antigen capture Dendritic cells at the environmental interface. Antigen uptake is facilitated by receptors specific for opsonins, pathogen-associated molecular patterns and apoptotic bodies

Migration Following antigen uptake and environmental signals, dendritic cells migrate through the lymphatics to regional lymph nodes

Antigen presentation Dendritic cells present the processed antigenic peptide−MHC complex. Surface costimulatory molecules facilitate the dendritic cell-T cell interaction

T cell response In response to antigen presentation, costimulatory molecule ligation and cytokine production by the DCs, T cells proliferate and differentiate along one of several pathways

Th1

Th2

Regulatory

Figure 2 The key concepts of DC function. Upon capturing antigen at the environmental interface, DCs migrate via lymphatics to the secondary lymphoid organs. Here they act as potent antigen-presenting cells, influencing subsequent T lymphocyte responses.

signals. The mechanisms by which DCs differentially regulate Th1 and Th2 immune responses is incompletely understood, but is likely to be due to several factors, such as antigen dose, the spectrum of costimulatory molecule expression, and the influence of microbial stimuli that induce Th1-polarizing cytokines (IL-12, IL-23, IFN-a). Airway DCs are also able to influence the induction of peripheral T cell tolerance. Exposure to inhaled innocuous antigen under normal conditions induces a state of immunological tolerance. In this regard, it has been well established that soluble antigen delivered via the respiratory mucosa, in the absence of inflammatory signals or sensitized T cells, induces a profound state of T cell tolerance

following systemic antigen challenge. Recent studies have confirmed a role for airway DCs in mediating this tolerogenic process. This mechanism is governed by the expression of IL-10 by pulmonary DCs that drives the generation of a population of IL-10-producing regulatory T cells (T reg) in draining lymph nodes capable of suppressing subsequent responses to antigenic challenge. T reg induction is critically dependent on inducible costimulatory (ICOS)–inducible costimulatory ligand (ICOSL) interactions, suggesting the requirement for specialized costimulatory pathways in this process. Thus, although the mechanisms governing the development of T cell tolerance or sensitization are not entirely clear, it seems likely that this will involve the coordinated

14

DENDRITIC CELLS

regulation of DC maturational state together with DC-derived cytokines.

fungi, the extent to which deficiencies in lung DC function contributes to particular human pulmonary infections has not been well defined.

Dendritic Cells in Respiratory Diseases

Therapeutic Considerations

Dendritic Cells in Asthma and Allergic Rhinitis

Because DCs are extremely responsive to external stimuli, and because they are thought to play a role in many disorders of the respiratory tract, there is considerable interest in the development of new therapeutic strategies targeted at DCs. In inflammatory lung diseases such as asthma, the aim is to inhibit DC function in order to reduce inflammation. By contrast, in pulmonary infection and malignancy, the aim is to augment DC function in order to enhance adaptive immunity directed against microbial pathogens or tumor antigens. Many well-known anti-inflammatory drugs are known to inhibit DC activation, including corticosteroids, cyclosporin, and tacrolimus, though these agents clearly affect other cells such as lymphocytes and other molecules involved in the inflammatory cascade. Corticosteroids inhibit DC maturation and antigen uptake and induce DC apoptosis, while agents such as vitamin D analogs and N-acetylcysteine block DC maturation via inhibition of the transcription factor (i.e., nuclear factor kappa B (NFkB)). Imidazoquinolines (e.g., imiquimod and resiquimod) and specific immunostimulatory sequences derived from bacterial DNA (e.g., CpG motifs) enhance the ability of DCs to release Th1-polarizing cytokines such as IL-12 and IFN-a. In the search for new vaccines against pulmonary infections, researchers are increasingly seeking to target DCs in order to develop protective immune memory. DC-based therapies for lung cancer have been tested in phase 1 and II clinical trials, using the patient’s own DCs pulsed with tumor antigens. As more is learnt about DC biology and its role in a variety of lung diseases, it is likely that new molecules will be developed that specifically target DCs.

In both asthma and allergic rhinitis, increased numbers of dendritic cells are present within the airway mucosa of the lung and nose, respectively. Many of these cells express the high-affinity IgE receptor, and treatment with inhaled corticosteroids leads to a decrease in the numbers of DCs within the airway mucosa. Following allergen inhalation challenge in allergic asthma there is a rapid reduction in the numbers of myeloid DCs within the circulation as myeloid DCs are recruited to the lung under the influence of chemokines and leukotrienes. In contrast, while short-term allergen inhalation does not appear to alter plasmacytoid DC numbers in the airway, repeated low-dose allergen exposure over several days induces the accumulation of plasmacytoid DCs in the nasal mucosa. Increasing evidence points to changes in the status of blood DCs in asthma. Alterations in the relative proportions of plasmacytoid versus myeloid DC have been reported in asthma. Various allergens and microbial stimuli have the ability to directly alter the function of DCs, inducing both DC activation and secretion of inflammatory mediators such as IL-1b, IL-6, TNF-a, IL-10, IL-12, and PGE2. Importantly, the pattern of mediator release differs between atopic and nonatopic individuals, and this may be one mechanism by which DCs contribute to Th1/Th2 polarization and the pathogenesis of atopy and allergic airway inflammation. Dendritic Cells in Other Respiratory Diseases

Various other diseases of the respiratory tract are associated with changes in the number of dendritic cells in the airway mucosa or lung parenchyma, including idiopathic non-specific interstitial pneumonia, hypersensitivity pneumonitis, obliterative bronchiolitis following lung transplantation, diffuse panbronchiolitis, Langerhans cell histiocytosis, and cigarette smoking. In each of these conditions it is not clear whether the observed accumulation of DCs in the lung plays a role in the pathogenesis of the disease, or has developed as a non-specific or even protective response to lung injury or inflammation, so there is a clear need for further longitudinal studies of DC function in each of these conditions. While it is clear from various animal models that DCs play a key role in the development of protective immunity to viruses, bacteria (including Mycobacteria), and

See also: Allergy: Overview. Asthma: Overview. Chemokines. Dust Mite. Epithelial Cells: Type I Cells; Type II Cells. Interferons. Interleukins: IL-1 and IL-18; IL-4; IL-5; IL-6; IL-7; IL-9; IL-10; IL-12; IL-13; IL-15; IL-16; IL-17; IL-23 and IL-27. Interstitial Lung Disease: Overview. Leukocytes: Monocytes; Pulmonary Macrophages.

Further Reading Akbari O, Freeman GJ, Meyer EH, et al. (2002) Antigen-specific regulatory T cells develop via ICOS-ICOS ligand pathway and inhibit allergen-induced airway hyper reactivity. Nature Medicine 8: 1024–1032. Banchereau J and Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392(6673): 245–252.

DIFFUSION OF GASES 15 Cochand L, Isler P, Songeon F, and Nicod LP (1999) Human lung dendritic cells have an immature phenotype with efficient mannose receptors. American Journal of Respiratory Cell and Molecular Biology 21: 547–554. Engleman EG (2003) Dendritic cell-based cancer immunotherapy. Seminars in Oncology 30(3 supplement 8): 23–29. Hammad H, Charbonnier AS, Duez C, et al. (2001) Th2 polarization by Der p 1-pulsed monocyte-derived dendritic cells is due to the allergic status of the donors. Blood 98: 1135–1141. Holt PG and Upham JW (2004) The role of dendritic cells in asthma. Current Opinion in Allergy and Clinical Immunology 4: 39–44. Lambrecht BN, De Veerman M, Coyle AJ, et al. (2000) Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation. Journal of Clinical Investigation 106: 551–559. Lambrecht BN and Hammad H (2003) Taking our breath away: dendritic cells in the pathogenesis of asthma. Nature Reviews: Immunology 3(12): 994–1003. Long J, Fogel M, Thompson PJ, and Upham JW (2004) Higher prostaglandin E2 production by dentritic cells from subjects with asthma compared with normal subjects. American Journal of Respiratory and Critical Care Medicine 170: 485–491.

Mellman I and Steinman RM (2001) Dendritic cells: specialized and regulated antigen processing machines. Cell 106(3): 255–258. Moser M and Murphy KM (2000) Dendritic cell regulation of Th1–Th2 development. Nature Immunology 1(3): 199–205. Tailleux L, Schwartz O, Herrmann JL, et al. (2003) DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells. Journal of Experimental Medicine 197: 121–127. Todate A, Chida K, Suda T, et al. (2000) Increased numbers of dendritic cells in the bronchiolar tissues of diffuse panbronchiolitis. American Journal of Respiratory and Critical Care Medicine 162: 148–153. Tunon-de-lara JM, Redington AE, Bradding P, et al. (1996) Dendritic cells in normal and asthmatic airways: expression of the a subunit of the high affinity immunoglobulin E receptor (FceRIa). Clinical and Experimental Allergy 26: 648–655. Upham JW, Denburg JA, and O’Byrne PM (2002) Rapid response of circulating myeloid dendritic cells to inhaled allergen in asthmatic subjects. Clinical and Experimental Allergy 32: 818–823. Upham JW and Stumbles PA (2003) Why are dendritic cells important in allergic diseases of the respiratory tract? Pharmacology and Therapeutics 100: 75–87.

DIFFUSION OF GASES R E Forster, University of Pennsylvania, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract This article summarizes different diffusion processes that transport respiratory gases in the body, their theoretical bases and pertinent equations. Gases move within cells by diffusion alone, so it is essential to maintain life. Gases that are neither metabolized nor produced (inert gases), such as nitrogen and anesthetic gases, equilibrate between capillary blood and alveolar gas or peripheral tissues in one capillary transmit time. Metabolism of O2 in a cell simultaneously with diffusion causes a marked drop in PO2 with distance from a capillary which limits the size of a cell, even under steady state conditions. Chemical reaction simultaneous with diffusion also severely reduces the transient rate of uptake of O2, CO, and NO by red cells; the more rapid the chemical reaction, the greater the relative reduction. The rate of CO uptake by red cells limits the rate of its uptake in the lungs. Gas diffusion can be facilitated by reacting reversibly with a carrier molecule which also diffuses, adding to the net flux. Cell membranes have been considered highly permeable to gases, because the gases dissolve in membrane lipid but recently some have been found to be relatively impermeable and some to contain membrane proteins which transport gas.

Basic Concepts Diffusion is the statistical movement of molecules from a region of higher to one of lower concentration

produced by the random thermal motion of the molecules. Diffusion can be considered a mixing process in which a particular molecular species moves under its concentration gradient independent of other molecules. It is the ultimate transport mechanism for respiratory gases as it alone carries them between capillary blood and cell cytoplasm. The rate of diffusion (dq/dt in mol s
1) in a steady state is proportional to the concentration gradient (@c/ @x in mol cm
3 cm
1) times the area (A in cm2), times a constant, the diffusion coefficient (d in cm2 s
1): dq=dt ¼ Ad@c=@x

½1

This equation is known as Fick’s first law of diffusion. (Note: respiratory physiologists and clinicians have been measuring partial pressure in mmHg with mercury monometers for centuries and most workers in the field today are most familiar with these units, at least in the USA, so these will be used rather than units derived from the cgs system. Cubic centimeters (cc) and milliliter is the volume of 1 g water at 41C and is equal to 1.0000 27 cc, a discrepancy immeasurable by physiological or clinical techniques. Cubic centimeters will be used in this article as the measure of volume primarily to balance the units in several equations.) The rate of diffusion of a molecular species is proportional to the mean linear velocity

16

DIFFUSION OF GASES

(v in cm s
1) of its molecules. The kinetic energy of the gas, (1/2) mv2, where m is the molecular weight in g mol
1, approximately equals its thermal energy, RT. Therefore RT ¼ ð1=2Þ mv2

½2

where R ¼ perfect gas constant in ergs mol
1 1C
1 and T ¼absolute temperature in kelvin. Therefore, the rate of diffusion of a molecular species is proportional to (T/m)1/2. A lighter gas diffuses more rapidly and all diffuse faster at higher temperatures. Diffusion in a gas phase, as in alveoli, is about a million times faster than in watery solution; it is difficult to separate from convection and will not be discussed here. Gas diffusion in the airways can be calculated from changes in volume of the dead space. In the body, gases exist in gas phases and liquid phases. At chemical equilibrium the concentration of a gas in the liquid equals its partial pressure, p (in mmHg), in the gas to which it is exposed, times its solubility in the liquid, a (in mol cm
3 mmHg
1), a relation which is known as Henry’s law: c ¼ ap

½3

A corollary to this is that a dissolved gas in a liquid exerts a measurable partial pressure or escaping tendency, even though there is no gas phase present. Substituting eqn [3] in eqn [1] we obtain dq=dt ¼ Aad@p=@x

½4

Much of gas transport in the body takes place between a gas phase and a liquid phase or between two liquid phases with different solubilities. At the boundary there is a discontinuity in gas concentration but not in p, in which case eqn [4] should be used.

Inert Gases

volume (lefthand side or LHS) equals the change in concentration of the gas in that volume. Solutions for this differential equation for various geometries are available in the literature and show that inert gases equilibrate between alveolar gas and capillary blood early in its transit. This conclusion is supported by the finding that calculations of pulmonary blood by inert gas uptake in the lungs, which assume such equilibria, agree with measurements by other techniques. Similarly, the halftime computed for inert gas to equilibrate between blood in a 8 mm diameter capillary and a surrounding peripheral tissue cylinder of 36 mm diameter is less than 0.1 s. With a capillary transit time of 1 s the gas would be completely equilibrated in the tissue cylinder. This means that inert gas exchange in body tissues is not rate limited by diffusion but by capillary blood/tissue volume and relative gas solubility. This is pertinent to exchange of anesthetic gases and decompression disease (bends).

Diffusion and Simultaneous Chemical Reaction: Steady State The rate of diffusion of gases that react with the cytoplasm is determined as much or more by the rate of the chemical reaction as by diffusion itself. Some gases are consumed (O2), bound (CO, NO), or produced (CO2) as they diffuse through a tissue. Under steady-state conditions this is described as follows, using O2 as a model: d@ 2 ½O2 =@½x2  ¼ m

The term on the left-hand side is the difference between the rate of diffusion of O2 into and out of a differential volume, and m is the rate of consumption of O2 in that volume, measured in mol s
1. The solution of this equation for a spherical cell with p as the driving force is PO2 ;o
PO2 ¼ ðm=6adÞðr2o
r3 Þ

Gases that do not react chemically with cell cytoplasm are called inert gases; these include nitrogen, helium, and anesthetic gases. They diffuse so rapidly that their rate of equilibration with tissues cannot be measured directly by experiment. By default, reliance has been on theoretical calculations using solutions of the following equation (Fick’s second law of diffusion) for nonsteady, that is, transient conditions: d@ 2 c=@x2 ¼
dc=dt

½5

This equation states that the difference between the diffusion of a gas into and out of a differential

½6

½7

where PO2 ;o ¼ oxygen partial pressure in mmHg at the surface of the cell, PO2 ¼ oxygen partial pressure in mmHg at the radial distance r (cm) from the surface of the cell, d ¼ diffusion coefficient of O2 in 7.6  10
6 cm2 s
1, and m is the rate of oxygen metabolism in mol cm
3 s
1 and is considered uniform with r and with PO2 above 2 mmHg. PO2 ;o
PO2 is the partial pressure drop at a distance r from the center of the cell. The greater the metabolic rate, m, and the lower the diffusion coefficient, d, the greater the drop. The maximum size of the cell is reached when PO2 in the center (r ¼ 0) becomes less

DIFFUSION OF GASES 17

PO2 ¼ PO2 ;o þ M=ð2aÞ½ðr2
r2c Þ=2
r2t ln r=rc 

½8

where PO2 ¼ the oxygen partial pressure at any radial point in the tissue cylinder, PO2 ;o is the oxygen partial pressure at the junction of the surface of the capillary and the tissue, r is the radial distance, rc is the radius of the capillary, the inner cylinder, and rt is the radius of the tissue cylinder of the muscle fiber. This is known as the Krogh–Erlang equation because it was developed for August Krogh in his original explanation of how tissue capillaries delivered oxygen to cells. The decrease in PO2 with radial distance from the capillary surface as calculated using eqn [8] is plotted in Figure 1 for resting skeletal muscle cells and cardiac cells. The important point is that these graphs demonstrate the maximum distance a muscle fiber of a given O2 consumption can be from a capillary and still be supplied with O2. Resting striated muscle can survive at a greater distance from a capillary than cardiac muscle because of its lower metabolic rate. During exercise additional capillaries open up, effectively reducing the diffusion distance to the perimeter of the model cylinder. In the early part of the twentieth century it was known that blood entered the lung with HbO2 about 75%, corresponding to 60 mmHg PO2 , and left the lung nearly 100% saturated with O2 corresponding to 100 mmHg. The mechanism by which O2 was transported from alveolar gas to the capillary blood hemoglobin was in dispute. Thus, it became important to determine the ability of the lung to equilibrate mixed venous blood with alveolar gas. In the lung, blood capillaries with the internal diameter of a red blood cell (8 mm) are separated from gas in the alveoli only by the alveolar membrane, consisting of single layers of capillary endothelium

scle l mu leta ske

10

Res ting

Hear t mus cle

60

PO 2 difference (mmHg)

than approximately 2 mmHg. For resting skeletal muscle and cardiac muscle, with m equal to 0.00196 and 0.08 mM s
1, respectively, the maximal cell radii are 354 and 50 mm, respectively. In order to get around this limitation on size, multicellular organisms have developed circulatory systems to convect a fluid that binds oxygen between an exchanger that equilibrates with the ambient medium (lungs, gills) and one that exchanges with peripheral cells (tissue capillaries). Peripheral capillaries deliver O2 to only one surface of a cell, but do not surround a cell as is implied in eqn [7]. A more realistic model of mammalian skeletal muscle is a capillary as a tube surrounded by a concentric cylinder of muscle cells for which the decrease in PO2 with radial distance is described by this solution of eqn [6]:

1 5

10

100

200

Tissue cylinder radius (µm) Figure 1 The log10 of the PO2 difference between the surface of a skeletal muscle capillary and the perimeter of a surrounding concentric cylinder of cells is plotted against the radius of the cylinder for heart muscle (open circles) and resting skeletal muscle (solid circles) with O2 consumption of 0.08 and 0.002 mM s
1, respectively. Data points were computed using eqn [8]. After Landis and Pappenheimer.

and alveolar epithelium, a diffusion distance of only several micrometers. O2 was considered to diffuse from alveolar gas into the capillary blood where it bound reversibly and instantaneously with hemoglobin in the erythrocytes. Any O2 gradients in capillary blood were neglected in comparison to the diffusion resistance of the alveolar membrane. O2 uptake in this model could be calculated by eqn [1] as follows, with units changed to those commonly using clinically: dVO2 =dt ¼ DL ðPA;O2
average PC;O2 Þ

½9

where VO2 is the amount of oxygen diffusing across the capillary wall (alveolar membrane) in cc, t ¼ time in min, DL was originally called the ‘diffusing capacity’ of the lung, a phenomenological transfer factor in cc mmHg
1 min
1, PA;O2 ¼ alveolar PO2 in mmHg, and PC;O2 ¼ capillary blood PO2 in mmHg. VO2 can be measured as the volume of inspired minus expired O2, or the rate of change of alveolar O2 concentration  alveolar volume. Alveolar PO2 varies with time during the respiratory cycle and amongst alveoli, depending on the ventilation/capillary blood flow. While a reasonable average value

18

DIFFUSION OF GASES

can be obtained for the alveolar PO2, this is not true for the capillary blood PO2 . As an increment of O2 is taken up by the blood and combines with hemoglobin, the blood PO2 rises by an amount determined by the ‘S’ shaped oxygen–hemoglobin equilibrium curve, for which there is no useful analytical expression. Equation [9] can be solved by a numerical integration (Bohr integration), but the difference between alveolar and end capillary blood PO2 must be known and is critical. First, a sample of this blood cannot be obtained; only arterial blood is available, and this is a mixture from all the alveoli and is contaminated by small amounts of blood that bypass the alveoli. Second, although it was possible to measure blood O2 content and HbO2 saturation, prior to the 1950s it was not possible to measure PO2 accurately. Thus, it was not certain whether end capillary blood PO2 equilibrated with alveolar gas or not. J S Haldane in fact argued for a long time that the lung could secrete O2 actively when necessary, for example, at high altitude or during heavy exercise, raising arterial PO2 higher than alveolar gas. A great advance in studies on gas diffusion in the lung was made by Christian Bohr and August and Marie Krogh who used CO exchange instead of O2. CO, like O2, binds reversibly with hemoglobin but with 225 times the affinity. The Pco for 50% saturation of HbCO is about 0.1 mmHg, while that of O2 is 28 mmHg. Therefore, with a low and safe, for a short period, alveolar Pco, the increase in blood HbCO during capillary transit is negligible. Thus, DL,CO can be calculated from eqn [9] substituting dVCO/dt and PA,CO for those terms in O2 and neglecting blood HbCO. DL;O2 can then be calculated from DL,CO on the assumption that diffusion through a watery alveolar membrane between alveolar gas and capillary blood is rate limiting. In this case the rate of diffusion is proportional to the solubility of the gas in water and inversely proportional to the square root of its molecular weight (Graham’s law): DL;O2 ¼ DL;CO ðmCO =mO2 Þ1=2 aO2 =aCO

½10

giving a value for the ratio of 1.20. A normal value for DL,CO is 31 cm3 mmHg
1 min
1 (single breath technique) and doubles with heavy exercise, increasing the uptake of O2 for the same mean O2 gradient from alveolar gas to capillary blood. This explains how arterial blood PO2 is maintained during exercise, obviating the need to propose O2 secretion. From the O2 consumption and DL;O2 , using the Bohr integration, one can show that end capillary PO2 is almost completely equilibrated with alveolar gas.

It was next found that DL,CO decreases when alveolar PO2 increases, which is explained by the competition of O2 and CO for hemoglobin. The rate of formation of HbCO in hemoglobin solution is given by the bimolecular reaction: d½HbCO=dt ¼ k1 ½CO½Hb
k
1 ½HbCO

½11

where k1 is the reaction velocity constant for the binding of the first CO on tetrameric hemoglobin in mM
1 s
1, k
1 is the reaction velocity constant for the dissociation of HbCO in s
1, and CO, Hb and HbCO are the concentrations of carbon monoxide, unliganded (‘reduced’) hemoglobin and carbonmonoxyhemoglobin in mM. k1 and k
1 can be measured in a solution of hemoglobin by several types of rapid reaction apparatus. When PO2 is present, as it in this case, [Hb] decreases slowing the rate of formation of HbCO by mass action.

Diffusion and Simultaneous Chemical Reaction: Transient The rate of uptake of CO by red blood cells can be measured best in a continuous-flow rapidmixing apparatus and at a normal alveolar PO2 of 100 mmHg is found to be 33% of that in solution. This reduction in CO uptake rate is caused by the interaction of diffusion and chemical reaction, an important phenomenon in reactive gas exchange. Theoretically this combined process is described by the following equation: @ 2 ½CO=@x2 ¼ @ ½CO=@ t þ k1 ½CO½Hb
k
1 ½HbCO

½12

which states that the difference between the rate of CO diffusing into and out of an infinitesimal volume equals the rate of change of CO less the net consumption of CO to form HbCO in that volume. It is not possible to obtain an analytical solution of eqn [12] because of the nonlinear term k
1 [CO][Hb]. However, representing the red blood cell as a semiinfinite layer of 30% hemoglobin 0.7 mm thick, it can be shown that upon suddenly exposing the surface to CO, a profile of [CO] in the layer will be established before a significant increase in Hb occurs at any point. The average of this profile equals the mean CO concentration in the layer, which multiplied by the initial concentration of Hb, obtained from the hemoglobin equilibrium curve, gives an initial value of d[HbCO]/dt from eqn [11], HbCO remaining constant. This corresponds to the initial slope of the experimentally determined rate of [HbCO] increase in a red blood cell suspension. It

DIFFUSION OF GASES 19

Measurement of Capillary Blood Volume In Vivo

1.00

Fraction of surface partial pressure

CO + 598 mmHg O2

The decrease in DL,CO as alveolar PO2 increases proves that the uptake of CO in the lungs is rate limited by the rate at which red blood cells in the alveolar capillaries can take up CO. Considering CO uptake in the alveoli as passing through two resistances in series, that of the alveolar membrane and that of the red blood cells in the capillaries, we can state that

0.80

0.60 CO + 100 mmHg O2 0.40

Total lung resistance ¼ membrane resistance þ red blood cell resistance

½13

0.20 NO + 100 mmHg O2 0.00 0.00

0.14

0.28

0.42

0.56

0.70

Distance from surface into hemoglobin layer (µm) Figure 2 ‘Initial’ partial pressure of a ligand gas in a 0.7 mm layer of 30% hemoglobin as a fraction of its value at the surface calculated from eqn [12] and plotted against distance into the layer. Unliganded Hb was considered constant, at pH 7.4 and 371C. PCO and PNO were 75 mmHg.

was found experimentally that the rate of uptake of a ligand gas by red blood cells did not increase indefinitely with the reaction rate in hemoglobin solution, but approached a limit. This is illustrated in Figure 2, which presents the initial profile of the partial pressure of ligand gas in the hemoglobin layer as a fraction of its value at the surface for three cases with increasing reaction velocity constants. These are CO in the presence of 585 mmHg PO2 and CO and NO in the presence of 100 mmHg with reaction velocity constants of 137 525, and 25 000 mM
1 s
1, respectively. The mean ligand gas partial pressure in the layer as a fraction of its value at the surface is 0.85, 0.52, and 0.02, respectively. Therefore, the velocity constants for the uptake of ligand in the layer would be 116, 273, and 500 mM
1 s
1 and experimental measurements on red blood cells are in agreement. The uptake of NO by red blood cells approximates the conditions for an infinite reaction velocity, the so-called ‘advancing front’ process, in which the ligand reacts instantaneously with the hemoglobin in the first differential volume, but requires time to diffuse through it to the next step, and so on. Red blood cells cannot take up a ligand any faster than this limit.

By analogy with Ohm’s law, resistance equals (partial pressure difference across the path)/(rate of gas flow). The total resistance is thus the reciprocal of the diffusion capacity. In succinct form: 1=DL;CO ¼ 1=Dm þ 1=DRBC

½14

where Dm ¼ diffusion capacity of the membrane alone in cc min
1 mmHg
1 and DRBC ¼ so-called CO diffusion capacity of the red blood cells in the capillary in cc min
1 mmHg
1. This can be broken down into YCO  VC, where YCO is the rate of uptake of CO by a cubic centimeter of blood in the alveolar capillaries in cc min
1 mm Hg
1 and VC is the volume of blood in the capillaries in cc: YCO ¼ k1;c aCO  blood CO capacity  60 s per min

½15

k1,c is a pseudo bimolecular reaction velocity constant calculated from eqn [11] with: (1) d[HbCO]/dt taken from the initial slope of a graph of [HbCO] versus time measured with a continuous-flow rapid reaction apparatus; (2) [CO] considered that at the surface of the cells; (3) [Hb] obtained from the PO2 and an HbO2 equilibrium curve; and (4) [HbCO] neglected. Blood CO capacity in cubic centimeters of gas per cubic centimeter of blood is that found in the capillary, which is considered the same as that in peripheral blood. Normally this is 0.2 cc cc
1 but is decreased in anemia, as is DL,CO. By measuring DL,CO at different alveolar PO2 greater than 100 mmHg, eqn [14] can be solved for VC and Dm. Representative values are about 100 cc for VC, and about 65 cc min
1 mmHg
1 for Dm.

20

DIFFUSION OF GASES

Facilitated Diffusion Facilitation of gas diffusion occurs when the gas combines rapidly and reversibly with another molecule, a carrier, which also diffuses, adding to the total transport. The carrier molecule is generally much larger than the gas molecule but is in greater concentration. Examples are O2 and CO reacting with hemoglobin in red blood cells and myoglobin in muscle, and CO2 forming H þ and HCO3
. The pair of equations describing steadystate-facilitated transport, using O2 and hemoglobin as an example, are as follows: dO2 @2½O2 =@x2 ¼ dHbO2 @½HbO2 =@x2 ¼
k
1 ½HbO2  þ k1 ½Hb½O2 

½16

Analogous sets of equations describe other facilitated pairs. These equations cannot be solved analytically because of the nonlinear (bimolecular) reaction term but have been approximated with numerical integration. The expression for facilitation of isotopically labeled 13CO2 transport at chemical equilibrium can be solved analytically because H þ in the nonlinear term in the equation is constant. Facilitation has been measured experimentally in artificial layers, but is more difficult to prove in vivo.

Diffusion through Cell Membranes Until recently it has been assumed that gases pass easily through cell membranes by dissolving in them. It is technically difficult to substantiate this experimentally because exchanges across the membrane are so rapid and the membranes so permeable that diffusion gradients outside the cells, stagnant layers, and diffusion within the cells cannot be differentiated from gradients across the membrane itself. However, the apical membranes of some epithelial cells of the gastrointestinal tract have recently been found experimentally to be relatively impermeable to CO2, permitting measurements. There is some evidence that gases such as CO2 and NH3 may pass through channels or membrane proteins.

Dilators

See also: Arterial Blood Gases. Carbon Dioxide. Carbon Monoxide. Diving. Extracorporeal Membrane-Gas Exchange. Hemoglobin. Myoglobin. Nitric Oxide and Nitrogen Oxides. Oxygen– Hemoglobin Dissociation Curve. Peripheral Gas Exchange. Pleural Effusions: Overview. Pulmonary Function Testing in Infants.

Further Reading Cotes JE (1979) Lung Function, 4th edn., chs. 8 and 9, pp. 203–250. Oxford: Blackwell. Crank J (1956) Mathematics of Diffusion. Oxford: Clarendon. Donaldson TL and Quinn JA (1974) Kinetic constants determined from membrane transport measurements: carbonic anhydrase activity at high concentrations. Proceedings of the National Academy of Sciences, USA 71: 4995–4999. Forster RE, II, Dubois AB, Briscoe WA, and Fischer AB (1986) The Lung, 3rd edn., ch. 8 and appendices, pp. 190–222. Chicago, IL: Year Book Medical Publishers. Forster RE (1987) Diffusion of gas across the alveolar membrane. In: Fishman AP, Farhi L, Tenney SM, and Geiger SR (eds.) The Respiratory System, Handbook of Physiology, vol. IV, ch. 5, sect. 3, pp. 71–88. Bethesda, MD: American Physiological Society. Forster RE (1987) Rate of gas uptake by red cells. In: Fenn WO and Rahn H (eds.) Respiration, Handbook of Physiology, vol. I, ch. 33, sect. 3, pp. 827–838. Bethesda, MD: American Physiological Society. Forster RE (1987) Diffusion of gases. In: Fenn WO and Rahn H (eds.) Respiration, Handbook of Physiology, vol. I, ch. 32, sect. 3, pp. 839–872. Bethesda, MD: American Physiological Society. Forster RE, Gros G, Lin L, Ono Y, and Wunder M (1998) The effect of 4,40 -isostilbene-2-20 -disulfonate on CO2 permeability of the red blood cell membrane. Proceedings of the National Academy of Sciences USA 95: 15815–15820. Kety SS (1951) The theory and application of the exchange of inert gas at the lungs and tissues. Pharmacological Reviews 3: 1–40. Landis EM and Pappenheimer JR (1963) Exchange of substances through capillary walls. In: Hamilton WF (ed.) Circulation, Handbook of Physiology, vol. II, ch. 29, sect. 2, pp. 961–1034. Bethesda, MD: American Physiological Society. Nicholson P and Roughton FJW (1951) A theoretical study of the influence of diffusion and chemical reaction velocity on the rate of exchange of carbon monoxide and oxygen between the red cell corpuscle and surrounding fluid. Proceedings of the Royal Society of London, Series B 138: 241–264. Roughton FJW (1987) Transport of oxygen and carbon dioxide. In: Fenn WO and Rahn H (eds.) Respiration, Handbook of Physiology, vol. I, ch. 32, sect. 3, pp. 767–826. Bethesda, MD: American Physiological Society.

see Bronchodilators: Anticholinergic Agents; Beta Agonists; Theophylline.

DIVING 21

DIVING R E Moon and J P Longphre, Duke University Medical Center, Durham, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Humans are poorly adapted to the underwater environment. Breath-hold diving is limited by limits on the capacity of the thorax to compress during descent, and ability to withstand hyperoxia and hypercapnia. Using compressed gas technology humans can breathe underwater, although with effects on pulmonary statics, dynamics, and gas exchange. Breathing compressed gas causes an increase in density, leading to impairment of both inspiratory and expiratory flow, and hence a reduction in maximum exercise ventilation. In addition, there is an increased Bohr dead space, which can further lower alveolar ventilation. Hypercapnia is common during diving. Immersion causes blood redistribution from the periphery into the lung, and a decrease in compliance. Gaseous compounds exert their physiological effects in proportion to their partial pressures. Thus, gases that are in concentrations that are safe to breathe at 1 ATA can become toxic during diving, including oxygen. Pulmonary O2 toxicity can occur with prolonged breathing of gas with a PO2 that exceeds 0.5–0.6 atmospheres (ATA). Other diving hazards include pulmonary barotrauma, and pulmonary edema due to venous gas embolism or the hemodynamics of immersed exercise. Mild long-term reductions in pulmonary function have been observed in professional divers and may be due to effects other than diving. Some lung conditions preclude safe diving.

Introduction Humans are poorly adapted to the underwater environment, and breath-hold dives are limited to only a few minutes duration. Early attempts to make use of compressed gas technology were impaired by physiological problems such as hypothermia and decompression sickness. The latter occurs during decompression, when the reduction in ambient pressure occurs at a rate such that the inert gas (e.g., N2) in tissues supersaturates and forms bubbles. The practical realization of commercial, military, and recreational diving has occurred through technological advances in compressed gas delivery systems, thermal protection, and an understanding of the physiology of inert gas elimination. Divers breathing compressed gas mixtures can stay underwater for prolonged periods, even up to weeks at a time. Nevertheless, both breath-hold and compressed gas diving induce physiological changes in respiratory function that can limit diver performance and cause morbidity.

Immersion Effects In the upright position on dry land gravity tends to cause blood to pool in the legs. During water immersion the hydrostatic pressure of the water counteracts the venous pressure, reducing venodilatation and causing blood in the legs to redistribute into the large veins and pulmonary vessels. The resulting increase in cardiac preload is equal to a transfusion of 500–800 ml of blood. The reduction in lung volumes (e.g., vital capacity) is augmented by cold water, in which active vasoconstriction increases the volume of redistributed blood, and a negative static lung load when the diver is in the head-up position (Figures 1 and 7). Lung engorgement also reduces lung compliance (Figure 2).

Breath-Hold Diving The world record for a breath-hold dive is currently 171 m (18 atm absolute, ATA, Figure 3). In order for a select group of humans to reach this depth and return alive, several physiological challenges must be met. During a breath-hold dive lung volume decreases with the increase in hydrostatic pressure according to Boyle’s law. During descent, lung volume will eventually reach residual volume (RV). Total lung capacity (TLC)/RV ratio is a predictor of maximum achievable. In order to allow for further lung volume reduction, continued descent will cause stretching of the diaphragm. Additional lung volume reductions can be accommodated by translocation of blood into pulmonary vessels to a limit determined by the allowable vascular transmural pressure. When this limit is exceeded, pulmonary capillary rupture can occur, causing intrapulmonary hemorrhage (referred to as ‘lung squeeze’). Adaptations that facilitate deep breath-hold diving include compliant chest wall, high TLC/RV ratio, and high pulmonary vascular compliance. During the descent, alveolar gas tensions rise in direct proportion to the ambient pressure. For instance, if alveolar PO2 is 100 mmHg at the surface, then an instantaneous compression to 10 m (2 ATA) would cause the alveolar PO2 to rise to 200 mmHg. At depth, O2 is continuously taken up by the pulmonary blood and CO2 is released. The breath-hold break point is determined mainly by the rising PCO2 , thus the diver begins ascending while the PO2 is still high. However, during ascent the alveolar gas partial pressure falls due to the reduced ambient pressure.

22

DIVING

RV

Standing dry

Immersed in water

VT

ERV

25 mmHg

FRC

RV

70 mmHg

140 mmHg

Figure 1 During immersion there is a central redistribution of blood due to the hydrostatic effect of the water column. In addition, depending upon the diver’s position, there is a static lung load, which in the upright position amounts to approximately 25 mmHg. Redrawn from Moon RE and Stolp BW (1997) The lung during anesthesia and in adverse environments. In: Schwinn D (ed.) Scientific Principles of Anesthesia, Atlas of Anesthesia (Miller RD, series ed.) vol. 2, pp. 6.2–6.15. Philadelphia, PA: Current Medicine, with permission.

As the diver nears the surface he may become hypoxic and lose consciousness (see Figure 4). Breath-hold time can be increased by at least two mechanisms. The first is
a low respiratory exchange ratio ¼V’ CO2 =V’ O2 : This can occur if carbohydrates have not been consumed for several hours, when basal metabolism is fueled to a greater extent by lipid. This slows the rise in PCO2 during a breath-hold, and thus postpones the break point. The second mechanism by which breath-hold time can be prolonged is prior hyperventilation. This reduces the PCO2 at the start, but increases O2 tension minimally. Both of these factors increase the likelihood of hypoxia. Other

mechanisms are probably in play for maintaining brain PO2 . Arterial gas embolism has been observed in individuals who repetitively breath-hold dive. The mechanism may involve air entering pulmonary blood vessels due to pulmonary stretch injury or vascular rupture as described above.

Compressed Gas Diving Anyone attempting to breathe from a snorkel at a depth exceeding a few centimeters will realize the impossibility of inhaling against the high hydrostatic

DIVING 23

pressure (see Figure 5). Thus, underwater breathing can only occur if the breathing gas is delivered at an ambient pressure close to that of the diver. Each 10 m (33 feet) of depth is equivalent to 1 atm of pressure. Thus, the ambient pressure at 30 m depth is 4 ATA (1 ATA at the surface plus an additional 3 ATA). Air is the most commonly used breathing gas. The maximum working depth breathing air is limited by

Lung volume (ml)

Submersion to neck

Submersion to xiphoid

1000

750

500

250

0

0

20

5 10 15 Pressure (cmH2O)

Figure 2 Pressure–volume relationships in the lung during immersion. Redistribution of blood into the thorax during immersion to the neck reduces lung compliance. Reproduced from Hong SK, Cerretelli P, Cruz JC, and Rahn H (1969) Mechanics of respiration during submersion in water. Journal of Applied Physiology 27: 535–538, used with permission from The American Physiological Society.

the narcotic properties of high pressure nitrogen (‘nitrogen narcosis’), which significantly affects psychomotor function at depths exceeding around 50 m. Helium is not narcotic, and thus can be used for much deeper dives. However, another clinical entity impairs human performance at depths: high pressure nervous syndrome. Its manifestations, including tremor, ataxia, nausea and vomiting, can be ameliorated to a large extent either by slow compression or addition of a small amount of narcotic gas to a helium–oxygen mixture. The record for human exposure during an experimental dive in a hyperbaric chamber is 701 m equivalent depth, using a mixture of helium, hydrogen, and oxygen. Typically for a self-contained underwater breathing apparatus (scuba) the gas is delivered to the diver via a regulator close to the mouthpiece (Figure 6). Depending upon the diver’s angle in the water, this may induce a small negative or positive static lung load (see Figure 7). Commercial divers do not use scuba, but instead gas is delivered from the surface via an umbilical hose (Figure 6), which runs in parallel with communication cables and sometimes a hot water source in order to maintain thermal balance. ‘Saturation diving’ refers to prolonged exposure to increased depth for a period of time sufficient to allow tissues to be saturated with inert gas (i.e., tissue and ambient inert gas partial pressures are equal). In saturation diving the divers are usually compressed to bottom depth in a hyperbaric chamber. Entry into

Year 1940 0

1950

1960

20 40

Depth (m)

60 80 100 120 140 160

Women Men

180 Figure 3 Breath-hold diving records (from various sources).

1970

1980

1990

2000

24

DIVING 340 320

Surface Dive

300 280

O2 CO2

260 240

Partial pressure (mmHg)

220 200 180 160 140 120 100

80 60 55 50 45 40 35 30

Descent

0 0

5

10

15

20

20 m 25

30 35 40 Time (s)

Ascent 45

50

55

60

65

70

75

Figure 4 Partial pressures of O2 and CO2 in the lungs during submersed breath-holds at the surface and during simulated dives to 20 m in a hyperbaric chamber, both under resting conditions. During descent, both PO2 and PCO2 increase. During the time at maximum depth PO2 drops faster than it does at the surface because of the increased blood content of O2 due to its higher partial pressure and a higher cardiac output. At the end of the dive, alveolar PO2 is lower after a dive than after a breath-hold at the surface of similar duration. During exercise, PO2 would drop even faster at depth, thus increasing the likelihood of hypoxemia and loss of consciousness when the diver approaches the surface. Redrawn from Liner MH, Ferrigno M, and Lundgren CE (1993) Alveolar gas exchange during simulated breath-hold diving to 20 m. Undersea Hyperbaric Medicine 20: 27–38, with permission.

1 ATA

1 ATA 1 ATA + water pressure

Figure 5 Breathing underwater via a snorkel longer than a few inches is impossible because the inspiratory muscles cannot overcome a pressure greater than around 100 mmHg. Adapted from Lundgren CEG and Miller JN (eds.) (1999) The Lung at Depth. New York: Dekker.

DIVING 25

Figure 6 (a) Scuba diver. The diver is wearing a wetsuit for thermal protection. The gas delivery hose is seen winding around the right side of her head. The red ‘bib-like’ device is a buoyancy compensator. Photographer Reese. (b) Tethered diver. Breathing gas is supplied from the surface. Behind the diver’s head can be seen the tip of a tank containing an emergency gas supply (‘bailout bottle’). Photos courtesy of National Oceanographic and Atmospheric Administration OAR/National Undersea Research Program (NURP).

SLL = −20 cmH2O

SLL = +25 cmH2O

Figure 7 Static lung load is the difference in pressure between the breathing regulator at the mouth and the lung centroid pressure. In the head-up position the inspiratory muscles experience an additional static load; in the head-down position the diver experiences positive pressure (CPAP). Reproduced from Lundgren CE (1999) Immersion effects. In: Lundgren CE and Miller JN (eds.) The Lung at Depth, pp. 91–128. New York: Dekker, with permission.

the water can be accomplished via a closed diving bell lowered into the water. When the water pressure is equal to the internal bell pressure, the hatch can be opened and the divers can exit, connected to the bell and receiving breathing gas by an umbilical. At the end of a diving shift the divers re-enter the bell, which is then closed, lifted to the surface, and mated with a living chamber where the divers can relax, eat, and sleep. Provided appropriate environmental conditions are maintained, divers can remain at pressure indefinitely. The advantage for commercial diving operations is the ability to perform multiple days of work with the requirement for only a single decompression, which may take several days. The increased ambient pressure requires that the physical characteristics of the gas will change. Air at 1 ATA has a density of around 1.1 g l
1 at 371C. At 50 m depth, compressed air has a density of 6.6 g l
1. In direct proportion to the ambient pressure, the density and partial pressures of the component gases increase. Gas viscosity is unchanged by the increased pressure. The increase in gas density has effects on both flow resistances and pulmonary gas exchange. Although vital capacity is not affected by gas density, all flow-related spirometric indices (e.g., forced expiratory volume in 1 s (FEV1), FEF25–75) are reduced. From studies in humans during dry experimental dives in hyperbaric chambers while breathing gas up to 25 times as dense as air at 1 ATA, it can be deduced that airways resistance increases approximately in proportion to the square root of gas density. Expiratory flow limitation occurs at higher lung volume and lower flow rates (Figures 8 and 9).

DIVING

MEF (l s–1)

26

10

10

8

8

6

6

4

4

2

2

0 100

80 60 40 20 Volume (% VC)

0

0

2

4 6 8 Pressure (atm)

10

Figure 8 Expiratory flow-volume relationships breathing air at increased ambient pressure in a dry hyperbaric chamber. VC, vital capacity. Reproduced from Miller JN, Lanphier EH, and Wangensteen OD (1971) Ventilatory limitations on exertion at depth. In: Lambertsen CJ (ed.) Underwater Physiology IV. Proceedings of the Fourth Symposium on Underwater Physiology, pp. 317–323. New York: Academic Press.

8

75% VC

7

1 ATA

6

V (l s–1)

5

4

4 ATA

3 10 ATA

2

1

0 20

10

−0+

10

20

30

40

50

Ppl (cmH2O) Figure 9 Isovolume pressure–flow curves at 75% vital capacity at increased ambient pressure breathing air. Reproduced from Wood LD and Bryan AC (1978) Exercise ventilatory mechanics at increased ambient pressure. Journal of Applied Physiology 44: 231–237, used with permission from The American Physiological Society.

Inspiratory flow limitation in the upper airway has also been observed. The functional result is a lower maximum breathing capacity (maximal voluntary ventilation, MVV) (see Figure 10). MVV at any gas density r can be estimated from the baseline MVV measurement (MVV0 obtained at r0) according to the following equation:  
0:4 r MVV ¼ MVV0 ½1 r0

MVV while immersed is 5–10% lower than in the dry. External breathing resistance in the breathing apparatus can induce a further reduction in MVV. At 1 ATA maximum exercise ventilation is generally no higher than 70% of MVV. However, during a dive, the exercise ventilation required for normocarbia may exceed MVV, leading to CO2 retention. In addition to the pulmonary mechanical changes induced by breathing dense gas, there is impaired alveolar–capillary gas exchange. Oxygen exchange

DIVING 27

130 110

Dry

90

Immersed

1

70 0

200

400

600

800

1000

Depth (fsw) Figure 10 Effect of depth and immersion on maximum voluntary ventilation (MMV) in divers breathing helium–oxygen. fsw, feet of sea water (33 fsw is equivalent to 1 atmosphere pressure (760 mmHg)). Redrawn from Wright WB and Crothers JR (1973) Effects of immersion and high pressures on pulmonary mechanical functions in man. Aerospace Medical Association Annual Scientific Meeting, p. 39.

VD (l, BTPS)

MVV (l min–1)

150

Depth

0.5 Surface

0

1

2

3

VT (l, BTPS)

does not appear to be affected. Additionally, the PO2 in the breathing mix is usually increased above normal. For instance, if the breathing gas is air, the ambient PO2 at 1 ATA is 159 mmHg, while at 10 m (2 ATA) it is 318 mmHg. However, CO2 exchange, as assessed by the physiological (Bohr) dead space, is impaired. Studies in experimental dives (ranging from 18 to 650 m equivalent depth, breathing gas densities 3.1–17.1 g l
1) during rest and exercise have revealed that physiological dead space is approximately twice normal (Figure 11). The most likely reasons for this are: (1) a higher anatomic dead space due to increased lung volume (to overcome the added breathing resistance induced by a greater gas density); and (2) nonuniformity of ventilation induced by altered gas density, thus creating gas exchange units with high ventilation/perfusion ratios. The human lung appears to have evolved to work most efficiently when the breathing gas density is around 1 g l
1, not higher as is more common in diving. The net effect of this dead space increase is to reduce alveolar ventilation, which in divers is not compensated by higher total ventilation, thus PCO2 increases. In the depth range of most scuba diving, during mild exertion, significant hypercapnia is unlikely, although respiratory compensation for exercise-induced metabolic acidosis is impaired. During heavy exercise in deep diving, arterial PCO2 above 60 mmHg has been observed.

Health Consequences Clinical problems associated with diving, pertinent to the lung, are listed in Table 1.

Figure 11 Dead space as a function of tidal volume during experimental deep dives (equivalent depths 18–650 m, breathing gas densities 7.9–17.1 g l
1). The data points correspond to exercise levels of rest: 360, 720, 960, and 1080 kp m min
1. Reproduced from Salzano JV, Camporesi EM, Stolp BW, and Moon RE (1984) Physiological responses to exercise at 47 and 66 ATA. Journal of Applied Physiology 57: 1055–1068, used with permission from The American Physiological Society.

Table 1 Clinical problems of diving related to the lung Pulmonary oxygen toxicity Pulmonary barotrauma Pneumomediastinum Pneumothorax Arterial gas embolism Effects of venous gas embolism Cardiorespiratory decompression sickness (‘chokes’) Chronic effects of saturation diving Immersion pulmonary edema

Oxygen Toxicity

Gaseous compounds exert their physiological effects in proportion to their partial pressures. Thus, gases that are in concentrations that are safe to breathe at 1 ATA can become toxic during diving. This includes oxygen, for which pulmonary toxicity can occur when the breathing gas PO2 exceeds 0.5–0.6 ATA (380–450 mmHg) for a prolonged time. This most commonly occurs during treatment for decompression sickness or arterial gas embolism, when the diver is administered 100% O2 at 2.8 ATA (breathing gas PO2 ¼ 2143 mmHg). However, pulmonary O2 toxicity can also occur when lower O2 percentages are breathed, especially during saturation diving. For example, breathing air for a prolonged time would produce pulmonary O2 toxicity at ambient pressures

28

DIVING

exceeding 3 ATA (20 m). Therefore, for long, deep dives the O2 percentage in the breathing gas must be adjusted to achieve an appropriate PO2 (for instance, during an experimental deep dive to 686 m, the oxygen concentration was 0.6%). Pulmonary Barotrauma

During ascent intrapulmonary gas expands and must be exhaled. If the diver holds his breath or regional airway obstruction causes gas trapping, alveolar rupture can occur (pulmonary barotrauma, PBT), causing pneumomediastinum, pneumothorax, or arterial gas embolism (AGE). Pneumomediastinum often presents as altered voice or subcutaneous emphysema. Pneumothorax is the least common manifestation of PBT. AGE typically presents with impaired consciousness and cortical signs such as hemiparesis, within minutes of surfacing. Risk factors for PBT include rapid ascent, as well as obstructive diseases, bullae, blebs, and interstitial restrictive lung diseases. Immediate treatment for AGE includes O2 administration; the definitive treatment is hyperbaric oxygen treatment in a recompression chamber.

capillary network, and generally no symptoms occur. However, high-grade VGE can cause cough, dyspnea, and pulmonary edema (cardiorespiratory decompression sickness or ‘chokes’), for which the initial treatment is high concentration O2, appropriate support of cardiovascular parameters. The definitive treatment is hyperbaric O2 in a recompression chamber. Immersion Pulmonary Edema

Immersion pulmonary edema is a condition in which the diver develops a cough with pink frothy sputum, usually shortly after entering the water. Chest radiography reveals pulmonary edema indistinguishable from other forms (Figure 12). The condition is believed to be more common in individuals who subsequently develop hypertension or after diving in cold water, although it has occurred in tropical water. Immersion pulmonary edema has been fatal. The pathophysiology of immersion pulmonary edema is believed to be high pulmonary capillary pressure due to the normal pulmonary vascular response to immersed exercise, augmented by transient cardiac failure due to the afterload induced by inspiring against high intrapulmonary and extrapulmonary resistance.

Venous Gas Embolism

Venous gas embolism (VGE) occurs during and after ascent as a result of inert gas supersaturation. VGE usually consists of low levels of bubbles, which can only be detected by ultrasound. During a week of diving 80% of recreational divers will have detectable VGE. VGE also commonly occurs during decompression from saturation dives. In the absence of an intracardiac or intrapulmonary shunt, VGE are cleared from the circulation by the pulmonary

Long-Term Effects of Diving on Pulmonary Function

Following deep saturation dives increases have been observed in dead space and reductions in FEV1, CO transfer factor, and exercise capacity. These have been attributed variously to deconditioning, persistent low levels of otherwise asymptomatic VGE during decompression, high oxygen partial pressures, and breathing gas contaminants.

Figure 12 Immersion pulmonary edema. (a) A 55-year-old male who dived to 20 m for 24 min in 171C water. He experienced respiratory distress at depth and surfaced with dyspnea, chest tightness, and cough productive of pink frothy sputum. Chest CT shows patchy densities in both lungs. (b) A male of the same age with a history of hypertension, who also experienced respiratory distress after diving to 24 m for 7 min.He surfaced with dyspnea, cough, and hemoptysis. Plain chest radiograph was normal. CT shows densities in the right middle lobe. From Cochard G, Arvieux J, Lacour JM, et al. (2005) Pulmonary edema in scuba divers: recurrence and fatal outcome. Undersea Hyperbaric Medicine 32: 39–44, with permission.

DIVING 29

Fitness to Dive Evaluation Pulmonary evaluation for fitness to dive with compressed gas consists of two aspects: pulmonary function and susceptibility to pulmonary barotrauma. Pulmonary Function

One way of considering this issue is as follows. According to eqn [1], a normal diver’s predicted MVV is at 70% of his or her surface value when he or she reaches a depth of 10 m. An exercise ventilation of 70% of MVV may be required to maintain a normal arterial PCO2 . Therefore, if the diver has a low MVV due to respiratory disease, he or she will not be able to achieve adequate exercise ventilation even at this shallow depth. Thus, abnormal 1 ATA spirometry would be disqualifying. Susceptibility to Pulmonary Barotrauma

Diseases in which air trapping is likely are disqualifying for diving. This includes a history of spontaneous pneumothorax, blebs, bullae or other lung cysts, or interstitial diseases. Asthmatics may also be at increased risk. However, the risk appears to be similar to the general population if there are no active symptoms of asthma (e.g., dyspnea, cough, or wheezing), spirometry is normal, and a provocative test, such as exercise while breathing dry air, does not produce a decrement in spirometry (see American Thoracic Society’s Guidelines for Methacholine and Exercise Challenge Testing, 1999). See also: Arterial Blood Gases. Diffusion of Gases. Exercise Physiology. Hyperbaric Oxygen Therapy. Mediastinitis, Acute and Chronic. Oxygen Toxicity. Pneumothorax. Space, Respiratory System. Hypoxia and Hypoxemia.

Further Reading American Thoracic Society (2000) Guidelines for Methacholine and Exercise Challenge Testing, 1999: official statement of the American Thoracic Society. American Journal of Respiratory and Critical Care Medicine 161: 309–329. Bert P (1978) Barometric Pressure (La Pression Barome´trique) (Translated by Hitchcock MA and Hitchcock FA). Bethesda, MD: Undersea Medical Society. Bove AA (ed.) (2004) Diving Medicine. Philadelphia, PA: Saunders. Brubakk AO and Neuman TS (2003) The Physiology and Medicine of Diving. New York: Elsevier.

Camporesi EM and Bosco G (2003) Ventilation, gas exchange and exercise under pressure. In: Brubakk AO and Neuman TS (eds.) The Physiology and Medicine of Diving, pp. 77–114. New York: Elsevier. Cochard G, Arvieux J, Lacour JM, et al. (2005) Pulmonary edema in scuba divers: recurrence and fatal outcome. Undersea Hyperbaric Medicine 32: 39–44. Flook V and Fraser IM (1989) Inspiratory flow limitation in divers. Undersea Biomedical Research 16: 305–311. Hong SK, Cerretelli P, Cruz JC, and Rahn H (1969) Mechanics of respiration during submersion in water. Journal of Applied Physiology 27: 535–538. Kohshi K, Wong RM, Abe H, et al. (2005) Neurological manifestations in Japanese Ama divers. Undersea Hyperbaric Medicine 32: 11–20. Liner MH, Ferrigno M, and Lundgren CE (1993) Alveolar gas exchange during simulated breath-hold diving to 20m. Undersea Hyperbaric Medicine 20: 27–38. Lundgren CEG and Miller JN (eds.) (1999) The Lung at Depth. New York: Dekker. Miller JN, Lanphier EH, and Wangensteen OD (1971) Ventilatory limitations on exertion at depth. In: Lambertsen CJ (ed.) Underwater Physiology IV. Proceedings of the Fourth Symposium on Underwater Physiology, pp. 317–323. New York: Academic Press. Moon RE and Stolp BW (1997) The lung during anesthesia and in adverse environments. In: Schwinn D (ed.) Scientific Principles of Anesthesia, Atlas of Anesthesia (Miller RD, series ed.), vol. 2, pp. 6.2–6.15. Philadelphia, PA: Current Medicine. Mummery HJ, Stolp BW, Dear GdL, et al. (2003) Effects of age and exercise on physiological dead space during simulated dives at 2.8 ATA. Journal of Applied Physiology 94: 507–517. Saltzman HA, Salzano JV, Blenkarn GD, and Kylstra JA (1971) Effects of pressure on ventilation and gas exchange in man. Journal of Applied Physiology 30: 443–449. Salzano J, Rausch DC, and Saltzman HA (1970) Cardiorespiratory responses to exercise at a simulated seawater depth of 1,000 feet. Journal of Applied Physiology 28: 34–41. Salzano JV, Camporesi EM, Stolp BW, and Moon RE (1984) Physiological responses to exercise at 47 and 66 ATA. Journal of Applied Physiology 57: 1055–1068. Skogstad M, Thorsen E, Haldorsen T, and Kjuus H (2002) Lung function over six years among professional divers. Occupational and Environmental Medicine 59: 629–633. Thorsen E, Hjelle J, Segadal K, and Gulsvik A (1990) Exercise tolerance and pulmonary gas exchange after deep saturation dives. Journal of Applied Physiology 68: 1809–1814. Thorsen E, Risberg J, Segadal K, and Hope A (1995) Effects of venous gas microemboli on pulmonary gas transfer function. Undersea Hyperbaric Medicine 22: 347–353. Wood LD and Bryan AC (1969) Effect of increased ambient pressure on flow–volume curve of the lung. Journal of Applied Physiology 27: 4–8. Wood LD and Bryan AC (1978) Exercise ventilatory mechanics at increased ambient pressure. Journal of Applied Physiology 44: 231–237. Wright WB and Croltiers JR (1973) Effects of immersion and high pressures on pulmonary mechanical functions in man. Aerospace Medical Association Annual Scientific Meeting, p. 39.

30

DNA / Repair

DNA Contents

Repair Structure and Function

Repair D M Wilson III, H-K Wong, D R McNeill and J Fan, National Institutes of Health, Baltimore, MD, USA Published by Elsevier Ltd.

Abstract Our genetic material, DNA, can be damaged by environmental (including sunlight and ionizing radiation and manmade products) and intracellular (e.g., reactive oxygen species) chemical and physical agents. These agents create lesions that if unrepaired can lead to genetic change (mutations) and accompanying cellular dysfunction. Thus, to avert or limit cell death and the mutagenic consequences of DNA damage, organisms are equipped with an array of DNA repair systems. Such systems function to recognize DNA damage, remove it, and restore genome integrity. Significantly, defects in these repair responses are associated with human disease, most notably cancer and tissue (namely neuronal) degeneration. In this article, we discuss the general mechanisms of the major DNA repair systems and highlight their relationship to specific human disorders, and we also present what little is known about their connection to respiratory disease.

DNA encodes the blueprint of the organism. In brief, genes (located within the DNA) are expressed in a complex, orchestrated manner to generate proteins that comprise the structural architecture and carry out the functional operations of the cell. The gene expression pattern, which is unique for each cell type, produces specific and distinctive cellular characteristics.

Base modification: spontaneous deamination, alkylation, oxidation

If this highly regulated network is upset or disrupted, either by dysregulation of gene expression or by the production of mutant (dysfunctional) proteins, cells can become ‘atypical’, creating problems for the organism as a whole. DNA is subject to constant molecular change (Figure 1) resulting from reactions with DNA-damaging agents that exist both within the environment and as natural byproducts within the cell. In particular, extracellular physical and chemical agents, such as ultraviolet light (sunlight), ionizing radiation, aromatic and heterocyclic amines, asbestos, vinyl chloride, and certain heavy metals can generate, directly or indirectly, harmful DNA damage. Additionally, aerobic organisms (e.g., humans) metabolize oxygen to generate energy, and during this process they form chemicals known as free radicals (or reactive oxygen species) that can react with DNA to produce dangerous intermediates. Unrepaired DNA damage can disrupt gene expression or promote permanent genetic mutations and, if excessive, can drive programmed cell death (apoptosis) or irreversible proliferation arrest (senescence). Dysregulation of or mutations in genes that control cell growth and function can promote disease development. To avert the deleterious effects of DNA damage and to maintain the fidelity of the genome, organisms have evolved a complex array of systems, known collectively as DNA repair, that recognize specific forms of DNA damage, remove the damage from DNA, and ultimately restore genome integrity.

Thymine dimer

Mismatched nucleotides

Abasic site

G T

Double-strand break Single-strand break Figure 1 Several major forms of DNA damage. See Table 1 for associated repair pathways. Note that the alkylation damage referred to here is distinct from the active methylation used to regulate promoter activity (i.e., enzyme-directed methylation of cytosine).

DNA / Repair 31 Table 1 Summary of the major human DNA repair pathways Major damage substrates

DNA repair pathway

Relationship to disease a

Nucleotide mismatches (e.g., G:T) and short nucleotide loop segments Spontaneous and oxidative forms of DNA damage (e.g., base modifications, abasic sites, and single-strand breaks) Helix-distorting bulky DNA lesions (e.g., T-T photodimers)

Mismatch repair

Colon and uterus cancer predominantly, but several sporadic tumors as well, including lung Colon cancer and potentially other forms of cancer (e.g., lung and B cell); indirect connection to spinocerebellar ataxias Skin cancer predominates; some instances of neurological degeneration; clinical features vary depending on gene mutated Likely role in preventing alkylating agent-induced carcinogenesis; role in influencing the therapeutic effectiveness of alkylating agents Leukemia, lymphoma, brain, skin, breast, ovary, and bone tumors

Base excision repair

Nucleotide excision repair

Alkylation base damage (e.g., O6alkylguanine)

Direct reversal

Double-strand breaks and unresolved, complex DNA intermediates

Recombinational repair

a

This is a broad representation. The precise clinical features will be determined by the specific gene mutated.

We briefly discuss aspects of the five major mammalian DNA repair pathways (summarized in Table 1) and report known associations to human disease.

Mismatch Repair The field of DNA repair remained relatively obscure until the discovery that mutations in components of mismatch repair (MMR) are genetically linked to forms of hereditary nonpolyposis colorectal cancer (HNPCC). This repair process is largely responsible for correcting replication errors, most notably mismatched nucleotides or small insertion/deletion loops, which arise during copying of the doublestranded genome. Defects in MMR lead to genomewide instability, particularly in simple repetitive sequences (known as microsatellite instability). The current understanding of the molecular details of this pathway is illustrated in Figure 2, yet is limited, particularly following the recognition step. Since the association of MMR with HNPCC was established, it has been found that defects in MMR are linked to some types of sporadic tumors as well. Many of these fall into the ‘HNPCC spectrum’ – that is, colon, rectum, uterine endometrium, stomach, and ovarian cancer. Recently, it has been realized that nonHNPCC-related sporadic cancers may also result from defects in MMR, including breast, prostate, and lung cancer. However, the mutational profiles of these cancers differ from those found in the HNPCC-related tumors, implying different molecular mechanisms. In addition to its role in processing DNA replication intermediates (Figure 2), the MMR system contributes to genomic stability by mediating programmed cell death in response to certain DNA-damaging agents. In this process, MMR operates to recognize specific DNA lesions and initiate an apoptotic response to eliminate severely damaged cells

from the organism, thereby preventing tumorigenesis. In fact, in the absence of MMR, cells display increased resistance to certain therapeutic drugs and a concomitant propensity for mutagenesis. These observations have prompted investigators to further examine the role of MMR in determining responsiveness to cell killing by certain anticancer agents, namely alkylating and DNA cross-linking compounds.

Base Excision Repair Base excision repair (BER) is the major pathway for dealing with spontaneous, nonbulky forms of DNA damage, including simple oxidative and alkylation damage. These include base modifications (e.g., the common mutagenic 8-oxoguanine lesion), sites of base loss, and single-strand breaks in DNA. The fundamental molecular details of this ‘workhorse’ pathway are well understood and are detailed in Figure 3. Note that although multiple subpathways exist in BER, the general biochemical steps remain the same. Until recently, no direct link of BER with human disease had been demonstrated. However, mutations in the human MutY homolog (MYH) DNA glyco sylase, which recognizes and initiates repair of certain base abnormalities, were linked to cases of HNPCC. Genetic analysis has also revealed that B30% of human tumors express a DNA polymerase b (POLb) variant, with one of these mutant proteins (Lys289Met) exhibiting reduced polymerase fidelity. Moreover, several epidemiological association studies have suggested correlations of specific BER gene variants with certain cancer types, although direct links have not been established. Last, it is worth noting that a central component of BER, X-ray cross-complementing 1 (XRCC1) (Figure 3), has been connected to two proteins – tyrosyl DNA phosphodiesterase 1 (TDP1)

32

DNA / Repair

G T Large insertion/deletion loops DNA damage recognition

MSH2

MSH2

MSH6

MSH3

PCNA

G MSH6

MSH2 T Incision, excision

+ ATP

MLH1

EXO1 RFC PCNA

PMS2 RPA

MLH3

MLH1 MLH1 MLH1 PMS1

A G MSH2 T PCNA

MSH6

RFC Gap-filling

T LIG1 Ligation

Figure 2 Mismatch repair (MMR). MMR is responsible for correcting mismatched nucleotides or insertion/deletion loops that inadvertently arise during copying of the genome. MSH2–MSH6 is primarily involved in targeting base–base (e.g., G:T) and small insertion/ deletion loop mismatches, whereas MSH2–MSH3 acts predominantly on insertion/deletion loops up to 12 nucleotides in length (details not shown). The MSH proteins have been argued to interact with the replication fork through associations with the replication processivity factor, proliferating cell nuclear antigen (PCNA). This interaction is thought to be important for determining which strand is the newly replicated DNA (i.e., the target strand) during the so-called strand discrimination step of MMR. ATP binding and hydrolysis are likely critical for the early steps of MMR, including MSH-mediated mismatch recognition and subsequent MSH–MLH interactions. The MLH1–PMS2 complex (or one of the putative backup complexes shown to the right) is hypothesized to signal mismatch recognition to downstream effectors, which are largely unknown. MLH1 has been shown to physically interact with EXO1, a 50 to 30 double-stranded DNA exonuclease implicated in both 50 and 30 nick-mediated MMR. EXO1 operates, likely in cooperation with PCNA and replication factor C (RFC), to remove the mismatched nucleotide or loop region, with the generated single-stranded portion (hundreds of nucleotides; not drawn to scale here) being stabilized by replication protein A (RPA). The replicative DNA polymerases e and d and DNA ligase 1 (LIG1) complete the process by filling the gap and sealing the final nick, respectively.

and aprataxin (APTX) – in which defects have been associated with spinocerebellar ataxias.

Nucleotide Excision Repair The connection of DNA repair to cancer susceptibility was first recognized upon the discovery that the rare human disorder xeroderma pigmentosum (XP), which is characterized by severe sensitivity to ultraviolet light and a 1000-fold increase in skin cancer susceptibility, is defective in components of the nucleotide excision repair (NER) response. In general,

NER copes with helix-distorting base lesions, which arise from sunlight or bulky chemicals such as benzopyrene. In Figure 4, the well-understood molecular mechanics of NER are shown. It is important to emphasize that NER has two functionally distinct branches – one that operates to repair lesions in the bulk ‘inactive’ genome and one that works in cooperation with the transcription machinery to remove damage from actively transcribed regions. Defects in NER are connected to at least three human genetic disorders: XP, Cockayne’s syndrome (CS), and trichothiodystrophy. Seven complementation

DNA / Repair 33

PP, P, or PG

IR OH

NEIL 1 Base excision

PARP1

UNG

XRCC1 PNK AP site incision

OH

dRP

APE1

End processing

APE1

Strand displacement synthesis Gap-filling

dRP excision

PCNA,RFC,RPA, and POL/ POL


POL


POL
FEN1 Flap excision

SP-BER

LP-BER Nick ligation

XRCC1 LIG3 LIG1

Nick ligation

Figure 3 Base excision repair (BER). BER is typically initiated by recognition and excision of a base damage (indicated as a black residue) by a DNA glycosylase. Such enzymes include uracil DNA glycosylase (UNG) and endonuclease VIII-like 1 protein (NEIL1). Monofunctional DNA glycosylases (e.g., UNG) generate an abasic (AP) site product, whereas bifunctional DNA glycosylases (e.g., NEIL1) create an AP site and then nick the DNA backbone. In the former case (pathway to the left), the AP site is cleaved by AP endonuclease 1 (APE1), creating a strand break with a normal 30 -hydroxyl (OH) group and a nonconventional 50 -terminus harboring a deoxyribose phosphate moiety (dRP). The one nucleotide gap and 50 -dRP residue are filled and excised, respectively, by DNA POLb. The remaining nick is sealed by a protein complex containing XRCC1 and DNA ligase 3 (LIG3), which together complete the process of short-patch BER (SP-BER). In the case of a multifunctional DNA glycosylase, or situations in which a strand break arises from the direct action of ionizing radiation (IR) or free radical attack (pathway to the right), the DNA ends are processed by a series of proteins, including poly(ADP-ribose) polymerase 1 (PARP1), XRCC1, polynucleotide kinase/phosphatase (PNKP), and APE1. Once the termini have been converted to normal 30 -OH ends by the appropriate phosphodiesterase and 50 -phosphate (P) groups by the PNKP kinase, the gap is filled by a DNA polymerase. In certain situations (not fully understood), the replicative polymerases (namely, d and e) operate to perform repair synthesis in cooperation with their accessory factors PCNA, RFC, and RPA. At times (again, not fully understood), gap-filling involves strand displacement synthesis, which generates a 50 -flap structure that requires subsequent removal by the flap endonuclease 1 (FEN1). In cases of long-patch BER (LP-BER), DNA ligase 1 (LIG1) functions to seal the final nick. 4-Hydroxypentenal phosphate (PP) is the 30 -terminal strand break product of some bifunctional DNA glycosylases. Phosphate (P) and phosphoglycolate (PP) are products of IR and reactive oxygen species attack. NEIL1 also generates 30 -P termini.

groups have been associated with XP (A–G), whereas two groups have been linked to CS (A and B), with each group representing a different repair gene. Although clinical manifestations differ considerably, varying from high cancer predisposition to aging symptoms, the common feature of the three syndromes is photosensitivity, manifested at different levels. Evidence has also pointed toward a role for NER in the repair of oxidative DNA damage that may be related to the neurodegenerative and premature

aging phenotypes associated with certain NERdefective individuals.

Direct Reversal Unlike the other repair processes that entail multiprotein complexes, direct reversal involves a single protein that transfers the modified portion of DNA to a residue within the protein’s active site. This action restores DNA back to its natural state but leads

34

DNA / Repair

DNA damage recognition

HR23B XPC

CSA CSB

GGR

RNA Pol

TCR

DNA unwinding XPA

TFIIH

RPA XPD

XPB

TFB5

Incision of damaged DNA XPF

ERCC1

XPG

XPA

TFIIH

RPA XPD

XPB

TFB5

Fragment release, repair synthesis, and DNA ligation

RFC

LIG1 DNA Pol PCNA RPA

Repaired double stranded DNA Figure 4 Nucleotide excision repair (NER). NER is subdivided into two major, distinct pathways. In global genome repair (GGR), the protein complex XPC/HR23B initially recognizes and binds to the helix-distorting damage site (shown as a thymine dimer photoproduct). In contrast, transcription-coupled repair (TCR) begins when an RNA polymerase (RNA Pol) stalls at the site of the lesion during transcription, followed by recruitment of CSA and CSB. Either of these events causes the main repair apparatus to be recruited in a sequential manner to the scene of the damage. The site of the lesion is first opened by the action of the transcription factor II H (TFIIH) complex (which contains two bidirectional helicase subunits, XPD and XPB (which unwind DNA in an ATP-dependent manner), and the recruitment subunit TFB5) and stabilized by XPA and RPA. Next, the ERCC1/XPF complex is directed to cut the damaged region on the 50 side of the lesion, while XPG is positioned to cleave 30 to the damage (resulting in an approximately 20-nucleotide incised fragment). Excision of the damage occurs due to the interaction between RPA, RFC, DNA polymerases (DNA Pol, likely d and e), and PCNA. At the end of the synthesis stage, the resulting nick is sealed by the action of DNA LIG1, producing a repaired double-stranded DNA molecule.

to irreversible inactivation of the covalently modified protein. Such a repair response has been termed ‘suicidal’ because the protein is no longer functional. In mammals, the repair protein O6-methylguanineDNA-methyltransferase (MGMT) transfers alkylation damage at the O6 position of guanine, and to a lesser extent the O4 position of thymine, to an active site cysteine residue. This repair step leads to the production of normal guanine or cytosine while leading to protein degradation. If unrepaired, O6alkylguanine lesions can promote mutagenesis upon replication of the strand containing damage.

MGMT is of particular interest because of its role in both cancer prevention and treatment. First, MGMT repair of O6-alkylguanine, a damage that promotes G to A transition, suppresses the mutagenic effects of exogenous carcinogens. For instance, transgenic mouse models indicate that MGMT expression levels are inversely related to the frequency of alkylating agent-induced tumorigenesis. Second, many anticancer agents, such as the alkylating compounds temozolomide, streptozotocin, procarbazine, dacarbazine, and the nitrosoureas, kill actively dividing cells by introducing cytotoxic DNA intermediates.

DNA / Repair 35

Thus, strategic manipulation of MGMT activity can have a profound impact on the efficacy of certain anticancer treatments for cancers. In particular, normal cells can be protected from the cytotoxicity of alkylating agents by increasing MGMT activity via gene therapy techniques, whereas cancerous cells (e.g., gliomas, brain tumors, lymphomas, and Hodgkin’s disease) can be killed more effectively by selectively inhibiting MGMT function. Current work on MGMT (as well as other DNA repair responses) focuses largely on maximizing its potential as a target for improved anticancer treatment strategies.

Recombinational Repair DNA double-strand breaks (DSBs) are direct products of ionizing radiation but are also formed during DNA replication when the progressing polymerase is disrupted by a DNA damage (e.g., a single-strand break) and the fork collapses. DNA DSBs are potent cytotoxic lesions and are capable of promoting gross chromosomal rearrangements (e.g., translocations). To repair DSBs, organisms are equipped with two corrective processes: homologous recombination (HR) and nonhomologous end-joining (NHEJ). HR occurs in S and G2 phase cells and relies on the extensive nucleotide sequence identity between the damaged chromatid and the intact homologous chromatid as the basis for strand exchange and repair. Since it involves an undamaged complementary template for repair, this process is error-free and restores the genome to its natural state; repair by HR is therefore conservative. That is, in the repaired chromosome, both strands are restored to the original template. NHEJ requires little or no sequence homology and is mediated by direct end-to-end ligation. However, depending on the terminal processing that occurs, NHEJ can result in the loss or gain of unwanted DNA nucleotides. The current understanding of these complex processes is depicted in Figure 5. Besides operating as key pathways for repair of DSBs, it is important to keep in mind that HR and NHEJ also function in essential cellular processes, such as meiotic recombination, which creates genetic diversity among the reproductive cell population, and V(D)J recombination, which generates diversity in the antigen receptor genes during lymphocyte development. Defects in HR or NHEJ are associated with chromosome instability, DNA-damaging agent hypersensitivity, cancer susceptibility (particularly hematological cancers), and immunodeficiency. This fact is perhaps best exemplified by the human diseases ataxia-like disorder and Nijmegen breakage syndrome (NBS), which are defective in MRE11 and

NBS1, respectively. Evidence also indicates that BRCA1 and BRCA2, originally identified as the ‘breast cancer genes’, and components of the autosomal recessive disorder, Fanconi anemia, contribute to the HR process. WRN (Werner’s syndrome) and BLM (Bloom’s syndrome), proteins associated with premature aging and cancer diseases, have been shown to help resolve or process complex replication/recombination DNA intermediates. Significantly, a network linking the various DSB repair participants to cell-cycle checkpoint responses has emerged. Defects in components of cell-cycle regulation (most notably, ATM, p53, and CHEK2), which operate to arrest cell-cycle progression to allow chromatin remodeling, repair of DNA damage, and avoidance of error-prone replication across a damaged template, are commonly associated with cancer or specific cancer-prone syndromes (i.e., ataxia telangiectasia and Li–Fraumeni). In cases of extensive damage, these processes also function to activate cell death and remove such precarious cells from the organism.

Associations with Lung Cancer and Respiratory Disease As outlined previously, evidence clearly links defects in DNA repair to cancer predisposition and neurological abnormalities. Not surprisingly, both epidemiological and molecular biology studies have indicated an inverse correlation between DNA repair capacity and lung cancer risk. Although associations have been reported, studies examining the direct relationship of DNA repair defects and lung cancer susceptibility are limited and still in the early stages. A hypothetical model outlining the potential relationship of DNA repair with respiratory disease is presented in Figure 6. The major risk factor for lung cancer – the leading cause of cancer-related death in the world – is exposure to tobacco smoke. In vivo experiments in rodents have shown that exposure to tobacco smoke causes bulky aromatic DNA adducts, oxidative damage (including single-strand breaks), and chromosome aberrations. Human biomarker studies conducted on smokers and nonsmokers have uncovered similar results, corroborating that tobacco smoke (and its constituents) is a potent genotoxin/mutagen. As alluded to previously, initial studies suggest that defects in components of BER, NER, and the cellcycle response are most prominently associated with increased risk of lung cancer. For instance, experiments indicate that deficiencies or genetic polymorphisms in the oxidative DNA damage repair

36

DNA / Repair

Recognition and end binding DNA−PKcs

Ku70/80 3′

5′ Recruitment of proteins for end processing

End Recognition, processing,singlestrand binding

Artemis

RAD51-Mediated homology search, strand invasion Recruitment of proteins for ligation

5′

3′ RAD51 3′ 5′

5′ 3′

XRCC4/LIG4

MRN

RPA RAD52

Polymerization, branch migration 5′ 3′ End ligation

3′ 5′

HJ resolution, end ligation 5′ 3′

(a)

5′ End trimming

3′

5′ Polymerization, ligation 5′

3′

3′

3′ 5′

(b)

Figure 5 Double-strand break (DSB) repair pathways. (a) NHEJ. A DSB is recognized and bound by the Ku70/80 heterodimer, which recruits and activates the catalytic subunit of DNA-dependent kinase (DNA-PKcs). Artemis, which possesses both exo- and endonuclease activities, is subsequently recruited by DNA-PKcs to clean up the DSB ends for ligation. The two DSB ends are ligated by LIG4 (present in an XRCC4/LIG4 complex), which is recruited by the Ku heterodimer. The Mre11/Rad51/Nbs1 (MRN) nuclease complex, and other proteins such as PNK, may also play a role in cleaning up the DNA ends. In situations in which there is resection at the break, polymerase X family members can fill the end prior to ligation (not shown). (b) HR. The MRN complex presumably resects the ends of the DSB, creating long 30 single-strand overhangs. Rad52 and RPA then bind to the end and single-strand portion, respectively, of the 30 overhang. Two subpathways could subsequently follow. In conservative HR (left), Rad51 replaces RPA and mediates the homology search between two sister chromatids, a process also facilitated by RAD54, followed by stand invasion. After polymerization and branch migration, the formed Holliday junctions (HJs) are resolved (it is currently unclear how), and a ligase then seals the ends to complete repair. BRCA1/2 may play a role in HR that is not completely understood. Repair by this subpathway is error-free. In nonconservative HR (right) (i.e., single-strand annealing (SSA)), the ends are brought together by homologous sequences (i.e., repeats) located on the opposite strands of the two DNA fragments (solid rectangles). After trimming of the ends (perhaps by the ERCC1/XPF endonuclease), ligation of the ends is performed by a currently unidentified ligase. Since this subpathway features deletion of a portion of DNA between the homologous segments, repair is inaccurate.

Stress Exogenous

Endogenous

DNA Lesion

• Mutations

DNA Repair

Unrepaired

• Cell death or senescence • Disease (e.g., lung cancer) and aging

Figure 6 DNA repair capacity model. As detailed in the text, DNA is exposed to various exogenous and endogenous stresses (i.e., DNA-damaging agents). Resulting DNA damage is typically repaired by one of the specialized DNA repair responses. However, in instances in which either the DNA repair capacity is insufficient or the DNA damage is excessive, unrepaired lesions can promote cytotoxic or mutagenic outcomes. Such end points can drive human disease (namely cancer) and age-related disorders.

glycosylase, OGG1, may be related to lung tumorigenesis. In addition, mutations or sequence variants in genes of the NER pathway (e.g., XPC and ERCC1; Figure 4) appear to have susceptibility as well as lung cancer pharmacogenetic and survival significance. Polymorphisms in the tumor suppressor protein p53

may also be linked to increased risk of lung cancer. Interestingly, accumulating data suggest that the chances for development of lung cancer are higher for women than men, and that this increased susceptibility may be due to higher levels of DNA adducts, decreased DNA repair capacity, increased

DNA / Structure and Function 37

frequency of mutations in tumor suppressor genes, as well as hormonal differences. Note that many inhaled occupational and environmental particles (e.g., airborne asbestos) have the capacity to be genotoxic/mutagenic. Many of these particles are harmful as a consequence of their ability to generate oxidants. Laboratory results indicate that there is increased inflammation, oxidative stress, and lipid peroxidation in lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis, and asthma. However, very little has been done to determine the effect of environmental and occupational airborne particles on DNA damage levels or the relationship of DNA repair to the prevention of such respiratory diseases. In conclusion, future work on elucidating the association of environmental exposures (including tobacco and other particles) to lung disease is paramount, as are devising strategies to suppress the harmful effects of identified compounds and clarifying the role of DNA damage and repair capacity in common respiratory ailments. See also: Apoptosis. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Cystic Fibrosis: Overview. DNA: Structure and Function.

Further Reading Frosina G (2004) DNA base excision repair defects in human pathologies. Free Radical Research 38: 1037–1054. Husgafvel-Pursiainen K (2004) Genotoxicity of environmental tobacco smoke: a review. Mutation Research 567: 427–445. Karagiannis TC and El-Osta A (2004) Double-strand breaks: signaling pathways and repair mechanisms. Cellular and Molecular Life Sciences 61: 2137–2147. Kastan MB and Bartek J (2004) Cell-cycle checkpoints and cancer. Nature 432: 316–323. Krokan HE, Kavli B, and Slupphaug G (2004) Novel aspects of macromolecular repair and relationship to human disease. Journal of Molecular Medicine 82: 280–297. Neumann AS, Sturgis EM, and Wei Q (2005) Nucleotide excision repair as a marker for susceptibility to tobacco-related cancers: a review of molecular epidemiological studies. Molecular Carcinogenesis 42: 65–92. Opresko PL, Cheng WH, and Bohr VA (2004) Junction of RecQ helicase biochemistry and human disease. Journal of Biological Chemistry 279: 18099–18102. Patel JD, Back PB, and Kris MG (2004) Lung cancer in US women: a contemporary epidemic. Journal of the American Medical Association 291: 1763–1768. Rosell R, Taron M, Ariza A, et al. (2004) Molecular predictors of response to chemotherapy in lung cancer. Seminars in Oncology 31(supplement 1): 20–27. Spitz MR, Wei Q, Dong Q, Amos CI, and Wu X (2003) Genetic susceptibility to lung cancer: the role of DNA damage and repair. Cancer, Epidemiology, Biomarkers and Prevention 12: 689–698. Stanton GL (2004) MGMT: its role in cancer aetiology and cancer therapeutics. Nature Reviews: Cancer 4: 296–307.

Taroni F and DiDonato S (2004) Pathways to motor incoordination: the inherited ataxias. Nature Reviews: Neuroscience 5: 641–655. Venkitaraman AR (2004) Tracing the network connecting BRCA and Fanconi anemia proteins. Nature Reviews: Cancer 4: 266– 276. Wu M (2005) DNA repair proteins as molecular therapeutics for oxidative and alkylating lung injury. Current Gene Therapy 5: 225–236. Yokota J and Kohno T (2004) Molecular footprints of human lung cancer progression. Cancer Science 95: 197–204.

Structure and Function A Ehrenhofer-Murray, Universita¨t Duisburg – Essen, Essen, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract DNA is the carrier of genetic information in all living organisms. DNA is a double helix whose strands are held together by complementary pairing between the bases adenine and thymine and between guanine and cytosine. Transcription generates an RNA template of the DNA sequence that is then translated into a protein, and the sequence of the bases determines the amino acid sequence of the protein. In eukaryotes, DNA is packaged into chromatin in the nucleus. Chromatin is a dynamic protein– DNA structure that modulates access of regulatory activities (transcription, replication, and DNA repair) to the genetic material. Histone modifying complexes (e.g., for histone acetylation and methylation) and chromatin remodeling complexes cooperate to regulate chromatin access during nuclear processes on the DNA template. Among others, alterations in DNA sequence can cause respiratory diseases, such as in the hereditary disorder cystic fibrosis. Genetic polymorphisms also increase susceptibility to lung disease and lung cancer. Numerous changes in DNA sequence, including changes in DNA methylation, as well as structural and numerical chromosome aberrations are found in (lung) cancerous tissues. Chromatin dysfunction also affects lung disease, and transcriptional mechanisms play an important role in the pathogenesis of acute lung inflammation.

Introduction The nucleic acid DNA serves as the genetic material in all living organisms. Seminal experiments carried out in the first half of the twentieth century led to the conclusion that DNA, and not other molecules such as proteins, carbohydrates, or lipids, is the genetic material and that it carries the hereditary factors (‘genes’) described by Gregor Mendel in the 1860s. In 1928, Frederick Griffith established the principle of transformation – the transfer of genetic material between bacteria. Subsequent experiments by Oswald Avery (1944) as well as Alfred Hershey and Martha Chase (1952) demonstrated that DNA is the

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transforming agent. Based on work from Erwin Chargaff on the base composition of DNA and X-ray diffraction pictures of DNA from Rosalind Franklin and Maurice Wilkins, James Watson and Francis Crick proposed a model for the double-helical structure for DNA in 1953.

The ribose moieties are linked by phosphodiester bonds and form the backbone of the DNA. The backbone strands are held together by complementary base pairing between the bases of the opposite strands that are attached to the ribose. The pairings depend on the shape of the bases and on their ability to form hydrogen bonds. A pairs with T, forming two hydrogen bonds, and G forms three hydrogen bonds with C (Figure 2). The base pairs are planar structures and stack on top of each other at the center of the double helix, which further stabilizes the helix by excluding water molecules. DNA is a right-handed helix with 10.4 base pairs (bp) per helix turn and has two distinct sizes of grooves running in a spiral, the major groove and the minor groove (Figure 2). The

Structure DNA is composed of three basic units: deoxyribose (a five-carbon sugar), phosphate, and four nitrogenous bases – the purines adenine (A) and guanine (G) and the pyrimidines thymine (T) and cytosine (C) (Figure 1). These units form a long helical structure of two antiparallel strands termed the double helix.

Adenine

Guanine

NH2 N N

O O

P

N

O

N

Cytosine NH2

O

N

H N

N

N N

O CH3

H

N

Thymine

NH2

O

N

N

O

Nitrogenous base

CH2

O

OH Phosphate

Deoxyribose

Figure 1 Chemical structure of the building blocks of DNA.

..T A ....

...

G ... C

...

C...G

..T A ....

P A

T

..T A ....

...

C ... G

...

.. C G ....

P P C

T ... A

G

P P

...T A...

G

..A T....

... C ...G ...T A...

C

P P T

A

..T A....

P

... G...C Double helix Major groove Minor groove

Backbone

Complementary base pairing

Figure 2 The structure of DNA. (Left) Simplified model of the DNA double helix. The sticks represent base pairs, and the ribbons represent the deoxyribose phosphate backbones of the antiparallel strands. (Right) Schematic of complementary base pairing in DNA. Dotted lines represent hydrogen bonds between bases. A, adenine; T, thymine; C, cytosine; G, guanine; P, phosphate.

DNA / Structure and Function 39

helix is a flexible structure in that, depending on its sequence, it can be bent along the helix axis, which influences the ability of proteins to bind DNA. The sequence of bases along the DNA strands constitutes the genetic code and determines the structure of the protein specified by a given genetic unit, a gene. Complementary base pairing is also employed to copy DNA during DNA replication and to transcribe DNA into an RNA template that is used for protein synthesis by the ribosome. The human genome consists of approximately 3 billion base pairs of DNA that are organized on 44 autosomes (22 pairs of homologous chromosomes) and two sex chromosomes, the X and the Y chromosomes. The first draft of the human genome sequence was completed in 2001 by an international consortium, and the high-accuracy sequence was reported in 2004. The human genome carries 20 000– 25 000 protein-coding genes. The coding regions comprise less than 3% of the genomic sequence and are often interrupted by noncoding regions (introns). Approximately 50% of the human genome consists of repeat sequences. They are categorized into five classes: (1) transposon-derived repeats, also referred to as interspersed repeats, include the long interspersed repetitive elements (LINE elements), the short interspersed repetitive elements (SINE elements), long terminal repeat (LTR) retrotransposons, and DNA transposons; (2) inactive copies of cellular genes (so-called pseudogenes); (3) simple sequence repeats, which consist of direct repeats of short sequences such as (A)n, (CA)n, or (CGG)n; (4) segmental duplications consisting of blocks of 10–300 kb that have been copied from one region of the genome to another; and (5) blocks of tandemly repeated sequences, for instance, at centromeres and telomeres. Centromeres are the basis for building kinetochores, the attachment sites of the microtubules during mitosis. Telomeres serve to protect the ends of the chromosomes from sequence loss and recombination. Otherwise, little is known about the function of repetitive sequences in the human genome. Simple sequence repeats (SSRs) are perfect or slightly imperfect tandem repeats of a particular short sequence. SSRs with a short repeat unit (1– 13 bp) are often termed microsatellites, and those with longer repeat units (40–500 bp) are termed minisatellites. The human genome consists of approximately 3% SSRs, which amounts to approximately one SSR per 2 kb of sequence. SSRs are very important in human genetic studies because they show a high degree of length polymorphism in the human population. Thus, SSRs can be used as genetic markers by measuring the number of tandem repeats of a simple sequence at a given genomic location. A

comprehensive map of SSRs is very useful in human genetic studies, for instance, to map disease mutations, and single SSRs can be used in forensics or paternity tests to identify individuals.

Regulation of DNA Function: Chromatin DNA in the eukaryotic nucleus is packaged into a macromolecular structure termed chromatin (Figure 3). The basic repeat unit of chromatin is the nucleosome, which consists of two each of the core histones H2A, H2B, H3, and H4, around which the DNA is wrapped in two turns totaling 146 bp of DNA. Nucleosomes are positioned along the DNA strand to form a filament 10 nm in diameter. The next level of chromatin organization is the 30 nm fiber, which is stabilized by the linker histone H1. The structure of this fiber is alternatively proposed to be a solenoid or a two-stop helix. The 30 nm fiber is further compacted into chromatin loops that are attached to a protein matrix and build individual chromosomes. In addition to the histones, there exist several types of nonhistone chromatin proteins. One major group is the high mobility group (HMG) of proteins. The HMG1- and HMG2-type proteins enhance transcription, HMG14 and HMG17 bind nucleosomes via interactions with the N-terminal tails of histones, and HMGI(Y) and HMG I-C bind to the minor groove of the DNA and are architectural components of chromosomes. Also, several transcription factors contain HMG-like domains.

Histone H1

30 nm fiber

Nucleosome Histones H3/H4 H2A/H2B

DNA

Figure 3 Model of the packing of DNA into chromatin. Blue disks represent nucleosomes.

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DNA / Structure and Function

more repressive to extraneous actions than naked DNA. Two main enzymatic activities can be distinguished that regulate chromatin access in the nucleus: histone/chromatin modifying complexes and chromatin remodeling complexes.

Eukaryotic genomes are organized into functionally distinct domains that differ in their chromatin composition. So-called euchromatin mostly contains actively transcribed genes and has an open chromatin configuration. Conversely, heterochromatin is gene poor, and the genes in heterochromatic regions generally are not expressed – a phenomenon termed ‘gene silencing’. Repression is achieved by the binding of specialized nonhistone proteins to the chromatin, which prevents transcription, and some heterochromatic regions also require structural RNA molecules for repression. For instance, the inactive X chromosome in female cells contains as a structural component the XIST RNA. Furthermore, pericentric heterochromatin is repressed by the action of small inhibitory RNAs. Euchromatic and heterochromatic regions can be distinguished by a cytological staining procedure that was developed by Emil Heitz in 1928. One type of change in DNA that plays a role in gene regulation involves the presence or absence of methyl groups on the bases of DNA. After replication, approximately 5% of cytosine residues within a eukaryotic genome are methylated by DNA methyltransferases, often in clusters of CpG sequence – so-called CpG islands. DNA methylation alters the ability of transcription factors to bind DNA. Furthermore, methyl-cytosine binding proteins bind methylated DNA and influence chromatin function. All DNA metabolic processes, such as DNA replication, transcription, and DNA repair in eukaryotic cells, operate on chromatin as a template, which is

P

Ac

Ac

The term ‘histone modification’ describes posttranslational modifications on the core histones. Potential modifications include lysine acetylation, arginine or lysine methylation, serine or threonine phosphorylation, and lysine ubiquitylation (Figure 4). The individual modifications are attached to the side chains of the amino acids. Most known modifications are found in the amino-terminal tails of the histones that protrude from the core nucleosome, but modifications also exist in the core region of the nucleosome. The position of individual modifications is highly conserved between different organisms (Figure 4). Posttranslational modification of histones is carried out by enzyme complexes. For instance, histone acetyltransferases (HATs) perform acetylation, and histone methyltransferases (HMTs) carry out methylation reactions. Some modifications can be enzymatically reversed. For example, histone deacetylases (HDACs) remove acetyl groups. Histone modifications may affect chromatin structure directly by altering DNA–histone contacts within and between nucleosomes, thus changing higher order chromatin structure. Also, protein domains are known that bind certain modifications. For

Me Ac Ac

Ub

H2A SGRGKQGGKARAKAKTRS 5 1 13 Me Ac

Histone Modifications

P Ac Ac

H2A

Ac

LPKKTESH 119

Me Ac

H2B PEPAKSAPAPKKGSKKAVTKAQKKDG 5 20 24 12 15 P Me Me

P P Me Me Ac Ac

Ub H2B

Me

H3 ARTKQTARKSTGGKAPRKQLATKAARKSA...GVKK 4 14 18 23 27 36 9

P

Me Ac

Ac

Me Ac

Ac

Me

DFKTD

P Me Me Me Ac Ac

Me Ac

TKYTSAK 120

79 H3

Me Ac

H4 SGRGKGGKGLGKGGAKRHRKVLRDN 5 12 16 20 8

H4

Figure 4 Posttranslational modifications on the four human core histones. Ac, acetylation; Me, methylation; P, phosphorylation; Ub, ubiquitylation.

DNA / Structure and Function 41

instance, a module called a bromodomain binds acetylated lysine residues in histones, and individual bromodomains in different proteins differ with regard to the precise residue they are able to bind. Similarly, some types of chromodomains bind methylated lysine side chains. Combinations of histone modifications thus constitute an interaction surface for nonhistone chromatin proteins that interpret this so-called histone code and translate it into a gene expression pattern. Moreover, histone modifications can also influence DNA methylation and vice versa. HATs use acetyl-coenzyme A (acetyl-CoA) as a cofactor in the acetylation reaction in which an acetyl group is added to the e-N position of a given lysine side chain. HATs are grouped into five families based on sequence comparison of their acetyl-CoA binding site. First is the Gcn5 family, which includes human GCN5 and PCAF. The HATs of the Gcn5 family carry a bromodomain. Second is the MYST family, named after the founding members MOZ, Ybf3/Sas3, Sas2, and Tip60. Translocations that create fusions between MOZ and other HATs are associated with certain types of leukemia. Third is the p300 family of HATs, which includes p300 and CREB binding protein (CBP), two highly related HATs. Mutations in p300 are found in epithelial cancers, and mutations in CBP are the cause of Rubinstein–Taybi syndrome. Fourth, several general transcription factors, such as TAF250 or TFIIIC, contain a HAT domain as well as a bromodomain. Finally, some nuclear receptor cofactors have intrinsic HAT activity that is thought to aid in transcriptional activation. HDAC families include the HDAC I family, which resembles yeast Rpd3, and the HDAC II family, which is similar to yeast Hda1. Four class I and five class II HDACs exist in humans. A third group of HDACs, the Sirtuin family, whose founding member is the yeast heterochromatin protein Sir2, is structurally unrelated to the other two families and, unlike the other families, requires the cofactor NAD þ in the deacetylation reaction. Seven human sirtuins are recognized. Mammalian sirtuins fulfill diverse functions in development, heterochromatin formation, and apoptosis and are suggested to link the metabolic rate of an organism to aging. Posttranslational acetylation is not restricted to histones. Some HATs also have nonhistone targets, an activity that is termed factor acetylation. Notably, some HMG proteins are also acetylated by HATs, and the acetylation is postulated to regulate their function within chromatin. Another prominent example is acetylation of the tumor suppressor p53. p53 is a transcription factor that responds to DNA damage. Acetylation of p53 by p300/CBP and PCAF

increases its DNA binding capacity and thus its activity in transcription. p53 deacetylation is carried out by sirtuins. In contrast to acetylation, lysine methylation can result in lysine residues that are mono-, di-, or trimethylated. The HMTs use S-adenosyl-methionine as a cofactor. One class of HMTs is the SET family of proteins, named after the chromatin regulators Su(var)3–9, Enhancer of Zeste, and Trithorax from Drosophila. They perform methylation of lysine residues in the amino-terminal tails of the histones. Twenty-seven human homologs are found, and many of them are confirmed or potential tumor suppressors or oncogenes. In contrast to the SET HMTs, the Dot1 family of HMTs methylates the residue K79, which lies in the core region of the histones. There is no sequence similarity between the two classes of HMTs. Chromatin Remodeling

In contrast to chromatin modification, chromatin remodeling leaves the biochemical composition of chromatin unaffected but alters histone–DNA contacts. Four basic types of remodeling are recognized: (1) nucleosome sliding, which changes the location of a nucleosome along a DNA sequence; (2) the creation of a remodeled state of the nucleosome that is characterized by altered histone–DNA contacts; (3) the removal of histones (histone eviction); and (4) histone exchange. Chromatin remodeling is catalyzed by protein complexes termed remodelers. Depending on the remodeler and the target nucleosome, the DNA sequence can be more or less amenable to accessory factors after chromatin remodeling. Chromatin remodelers use the energy of ATP hydrolysis to loosen DNA–histone contacts in order to facilitate the movement of nucleosomes. Thus, all chromatin remodeling complexes have an ATPase subunit, the motor of the complex. Two remodelers of the SWI/SNF family exist in human cells, hBRM (brahma) and BRG1 (brahma-related gene), that are highly related to each other and to yeast Snf2. Whereas BRM is dispensable for mouse development, the absence of BRG1 in mice causes death early in development. BRG1 is a central cell-cycle regulator and a tumor suppressor. A second class of remodelers contains an ATPase of the ISWI (imitation switch) protein family (termed SNF2L in mammals). In human cells, at least four complexes are observed with hSNF2h as a subunit. They are distinct in protein composition and play roles in chromatin replication and sister chromatid cohesion. A third category of remodeling ATPases is the CHD type of remodelers, which are characterized by the presence of two chromodomains. The human homolog Mi-2 is observed in several protein

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complexes that also contain HDACs and methyl-cytosine binding proteins, thus providing a functional link between chromatin remodeling, histone deacetylation, and DNA methylation.

Biological Function Transcription

DNA is the repository of genetic information in the cell. Genes are transcribed into messenger RNA (mRNA), which is translated into proteins. The genetic code consists of a linear series of triplet nucleotides present in the mRNA that reflects the sequence in the DNA and specifies the insertion of a specific amino acid as the mRNA is translated into a growing protein chain. During transcription, eukaryotic gene promoters are first recognized by general transcription factors (GTFs). The TATA box, a sequence approximately 30 bp upstream of the start site, is bound by TATA binding protein (TBP). TBP is part of the GTF complex TFIID that, in cooperation with other GTFs, attracts the core RNA polymerase II to start RNA synthesis. Transcription works on DNA packaged into chromatin as the native template. In this process, chromatin modifiers and remodelers cooperate with sequence-specific DNA binding factors to help the transcriptional apparatus gain access to the DNA. For transcription initiation, a transcriptional activator binds to its target sequence in the promoter region of a gene. The activator then recruits HAT complexes that acetylate the histones in the chromatin at the promoter. This facilitates the recruitment of a chromatin remodeler, which then mobilizes nucleosomes in the proximity of the promoter. In the elongation phase of transcription, the RNA polymerase proceeds through chromatin along the body of the gene. It is aided in its progression by a HAT complex that is tightly associated with the elongating polymerase. Elongation is also accompanied by histone methylation, which serves to recruit chromatin remodelers that increase chromatin fluidity and thus promote progression of the polymerase. Also, factors exist that remove H2A and H2B from the front of the transcribing polymerase and reassemble nucleosomes after its passage. Together, all these factors and activities contribute to successful gene expression. Replication

As cells divide and give rise to a whole organism, the genome must be accurately duplicated and passed on to the daughter cells in order to ensure that the genetic information remains constant over many cell

divisions. The primary DNA sequence is duplicated by semiconservative DNA replication. Replication starts at origins of replication that are bound by the replication initiator, the origin recognition complex (ORC). Replication initiation is tightly coordinated with the cell cycle. The protein Cdc6 is only present in the early G1 phase of the cell cycle. Its binding to ORC allows subsequent binding of so-called licensing factors that limit initiation to once per cell cycle. Initiation is then triggered through a cyclin-dependent kinase; the DNA duplex is locally unwound, and DNA polymerases are recruited that synthesize a complementary DNA strand. Regarding transcription, DNA replication operates on the chromatin template. Accordingly, histone modifications and chromatin remodeling are observed to modulate origin activity and to influence the progression of DNA replication. Also, chromatin has to be restored on the newly replicated DNA. This process is termed chromatin assembly and is catalyzed by chromatin assembly factors that recognize replicated DNA by the presence of PCNA and deposit histones onto the DNA. DNA Repair

The precise DNA sequence needs to be maintained during cell division and reproduction because changes in DNA sequence can have detrimental effects. Somatic mutations can cause disease of an individual, for instance, by causing genomic instability and resulting in cancer, and mutations in the germline can cause genetic diseases in the offspring. Thus, cells have developed efficient repair systems that recognize and eliminate DNA mismatches or lesions. Regarding transcription and replication, circumstantial evidence indicates a role for chromatin modifiers and remodelers in regulating access of repair machineries to the DNA in chromatin.

DNA in Respiratory Diseases Several categories with regard to how DNA is involved in respiratory diseases can be distinguished. They are briefly described here and are discussed in more detail in other chapters of this encyclopedia. In one scenario, the DNA sequence of a molecule important for lung function is mutated and causes disease, as is found in hereditary disorders. A prominent example is cystic fibrosis (CF), an autosomal recessive disorder in which the CFTR transmembrane ion transporter is mutated, which causes the lung to produce abnormally viscous mucus. Since CF is caused by a single gene mutation, treatment by gene therapy is being pursued.

DNA / Structure and Function 43

Heritable mutations can also modulate the susceptibility of an individual to lung disease. Certain alleles of the gene encoding a1-antitrypsin (AAT deficiency) increase the likelihood of developing chronic obstructive pulmonary disease, but there are probably many genetic modifiers as well as environmental factors (e.g., smoking) that modulate the disease risk and that remain to be characterized. Certain genetic polymorphisms have also been associated with a predisposition to lung cancer. Subtle differences in gene expression or activity of cellular components can influence an individual’s susceptibility to tobacco smoke and other environmental factors. Candidates for susceptibility genes are alleles of enzymes that metabolize exogenous and endogenous compounds (e.g., cytochrome P450 enzymes or microsomal epoxide hydroxylase), DNA repair genes (e.g., ERCC and XRCC), and alleles of the tumor suppressor gene p53. It is likely that there are complex interactions between many susceptible genes, ethnic background, and environmental exposure that modulate lung cancer risk in the population. As in all cancer tissues, major chromosomal aberrations are also a hallmark of lung cancer cells, a phenotype that has been termed ‘chromosomal instability’. These abnormalities can be of structural or numerical nature. Cytological investigations of lung cancerous tissues have highlighted some regions in the genome as being more frequently altered (chromosomal gains or losses) in lung cancer than in other types of cancer. Research is aimed at determining which genes are affected and how they affect disease progression. Interestingly, chromosomal rearrangements involving HMGI-C and HMG-I(Y) have been identified in pulmonary chondroid hamartomas, suggesting that they cause alterations in gene expression and thus cause disease. There are many causes for genomic instability leading to cancer, including mutations in cell-cycle control and checkpoint proteins or in the repair of mutations or double-strand breaks. For instance, as in many other cancer types, lung cancer cells are frequently mutated in p53, which may cause genomic instability by abrogating p53-dependent DNA damage checkpoint control. Also, oncogenes such as the ERBB and myc gene families are occasionally amplified in lung cancer, which disrupts cell-cycle control. One route of repression of cell regulators is through DNA methylation in their promoter region, which results in gene repression mediated by methylcytosine binding proteins and subsequent recruitment of HDAC complexes. Hence, gene-promoter hypermethylation is being exploited as an early diagnostic indicator of lung cancer. Specifically, aberrant promoter methylation of the CDKN2A gene,

which encodes the cyclin-dependent kinase inhibitor INK4A, is being evaluated in sputum samples for its predictive value. Similarly, methylation in the promoter region of the MGMT gene encoding the DNA repair enzyme O6-methylguanine DNA methyltransferase is being investigated. In addition to DNA sequence mutations, alterations in chromatin function also affect (lung) disease. Major genomic rearrangements as seen in cancer cells necessarily change cellular gene expression. Thus, compounds are being developed as general anticancer drugs that affect chromatin function. For instance, HDAC inhibitors are in clinical trials, although their application is not restricted to lung cancer. Transcriptional mechanisms also play a critical role in the pathogenesis of acute lung inflammation. In particular, the transcription factor NF-kB, along with other proinflammatory transcription factors, is activated by signaling cascades from several external stimuli, such as proinflammatory cytokines, bacterial and viral products, and reactive oxygen species. NFkB interacts with HAT coactivators such as CBP and p300/PCAF and activates transcription of many cytokines and growth factors, adhesion molecules, immunoreceptors, and acute phase proteins, all of which contribute to pathogenesis. In addition, acetylation of NF-kB by CBP increases its transcriptional activity. NF-kB-mediated gene expression is further enhanced by binding of HMG-I, which thus constitutes a mediator of lung inflammation. The effectiveness of corticosteroids in therapy of inflammatory lung disease is attributed to their ability to suppress the action of NF-kB and other proinflammatory transcription factors. They bind to nuclear hormone receptors (glucocorticoid receptor), which then directly activate anti-inflammatory genes, inhibit coactivators of proinflammatory transcription factors, or recruit HDACs to repress transcription. See also: Cell Cycle and Cell-Cycle Checkpoints. Cystic Fibrosis: Overview. DNA: Repair. Gene Regulation. Genetics: Overview. Transcription Factors: Overview. Tumors, Malignant: Overview.

Further Reading Barnes PJ, Adcock IM, and Ito K (2005) Histone acetylation and deacetylation: importance in inflammatory lung disease. European Respiratory Journal 25: 552–563. Becker PB and Ho¨rz W (2002) ATP-dependent nucleosome remodeling. Annual Review of Biochemistry 71: 247–273. Bell SP and Dutta A (2002) DNA replication in eukaryotic cells. Annual Review of Biochemistry 71: 333–374. de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, and van Kuilenburg AB (2003) Histone deacetylases (HDACs):

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characterization of the classical HDAC family. Biochemical Journal 370: 737–749. Ehrenhofer-Murray A (2004) Chromatin dynamics at DNA replication, transcription and repair. European Journal of Biochemistry 271(12): 2335–2349. International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature 431(7011): 931–945. Luger K, Mader AW, Richmond RK, Sargent DF, and Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389(6648): 251–260.

Masuda A and Takahashi T (2002) Chromosome instability in human lung cancers: possible underlying mechanisms and potential consequences in the pathogenesis. Oncogene 21: 6884– 6897. Roth S, Denu JM, and Allis CD (2001) Histone acetyltransferases. Annual Review of Biochemistry 70: 81–120. Sims RJ III, Nishioka K, and Reinberg D (2003) Histone lysine methylation: a signature for chromatin function. Trends in Genetics 19(11): 629–639. Strahl BD and Allis CD (2000) The language of covalent histone modifications. Nature 403(6765): 41–45.

DRUG-INDUCED PULMONARY DISEASE R A Dweik, The Cleveland Clinic Foundation, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Drug-induced lung disease is a common iatrogenic illness. Different drugs can cause similar pulmonary syndromes/presentations. The most common presentation is an abnormality on the chest X-ray in an asymptomatic patient or a symptom-sign complex. Early diagnosis is very important and requires maintaining a high index of suspicion in the appropriate clinical settings. The final diagnosis is usually one of exclusion. Cessation of the drug is usually enough for therapy for most drug toxicities; corticosteroid administration may be needed.

Introduction The lung is being increasingly recognized as a target organ for drug toxicity and currently more than 150 pharmacologic agents have been reported to cause adverse pulmonary reactions. In drug-induced pulmonary disease, an abnormality on the chest X-ray film or a symptom-sign complex is the most common form of presentation. Since the clinical presentation and radiologic and histologic findings are mostly nonspecific, a high index of suspicion is needed to diagnose drug-induced pulmonary disease in the appropriate clinical setting. In this article, we provide a brief overview of drug-induced pulmonary disease. Table 1 summarizes the more common clinical presentations of pulmonary toxicity induced by noncytotoxic medications, including illicit drugs, while Table 2 does the same for pulmonary toxicity induced by cytotoxic agents, including radiation. Some unusual manifestations of drug-induced pulmonary toxicity are listed in Table 3. Not all the medications listed in the tables are discussed in the text. In some instances it is useful to discuss the pulmonary complications associated with a specific agent; however, we will also discuss the clinical syndromes induced by a number of different agents.

Amiodarone Amiodarone, an antiarrhythmic drug, can cause pulmonary toxicity in about 6% of patients receiving the drug. The diagnosis of amiodarone pneumonitis is really one of exclusion. Pulmonary embolism, congestive heart failure, and pneumonia are the main differential diagnoses in patients suspected of having amiodarone pulmonary toxicity, and should be excluded first. Etiology

In most cases of amiodarone pneumonitis the patients have received daily doses exceeding 400 mg for two months or more. Pulmonary disease can occur with maintenance daily doses less than 400 mg but the incidence is lower (1.6%) as reported from combined placebo controlled trials involving 3439 patients. Pre-existing lung disease does not seem to increase the risk of development of toxicity (as evidenced by a lack of accelerated decrease in diffusing capacity in patients with chronic obstructive pulmonary disease (COPD) receiving amiodarone versus those receiving placebo in a randomized trial). Clinical Features

The most common clinical presentation is a chronic interstitial pneumonitis characterized by insidious onset of dyspnea, dry cough, weakness, weight loss, and occasionally fever. Organizing pneumonia, presenting more acutely than the chronic interstitial pneumonitis, is seen in 25% of cases. A picture suggestive of adult respiratory distress syndrome (ARDS) has been described in patients undergoing general anesthesia for surgical procedures or after pulmonary angiography. Furthermore, in a retrospective study of 67 patients undergoing map-guided surgical procedures for ventricular tachycardia, the incidence of postoperative acute respiratory failure was 17% in patients previously receiving amiodarone and zero in

DRUG-INDUCED PULMONARY DISEASE 45 Table 1 Clinical presentation of pulmonary toxicity induced by noncytotoxic drugs PIE

Noncardiogenic IP-F pulmonary edema

Acute Chronic PVD pleural pleural effusion effusion

Anorexigens Dexfenfluramine Fenfluramine Anti-inflammatory Acetylsalicylic acid Colchicine Gold Interleukin-2 Methotrexate NSAIDs Penicillamine Antimicrobial Amphotericin B Ethambutol Isoniazid Nitrofurantoin Para-aminosalicylic acid Penicillins Pentamidine Sulfasalazine Tetracycline Cardiovascular Alpha-methyldopa Amiodarone Anticoagulants Beta-blockers Dipyridamole Flecainide Hydralazine Hydrochlorothiazide Lidocaine Procainamide Propafenone Tocainide Illicit drugs Cocaine Heroin Methadone Methylphenidate Psychotropic/ antiepileptic Carbamazepine Diphenylhydantoin Phenothiazine Trazodone Tricyclics Miscellaneous Bromocriptine Contrast media Esophageal variceal sclerotherapy agents Estrogen Timolol (ophthalmic) Tocolytic agents

Parenchymal hemorrhage

DI-SLE

BOOP Asthma

þ þ þ þ þ þ þ

þ þ þ

þ

þ

þ þ

þ

þ

þ

þ þ þ þ þ

þ

þ

þ

þ þ þ

þ

þ

þ

þ þ

þ þ

þ þ

þ þ þ

þ þ þ þ þ þ þ þ

þ

þ

þ þ þ

þ þ þ þ þ þ þ þ þ þ þ

BOOP, bronchiolitis obliterans organizing pneumonia; IP-F, interstitial pneumonitis or fibrosis; PIE, pulmonary infiltrate with eosinophilia; PVD, pulmonary vascular disease; DI-SLE, drug-induced systemic lupus erythematosus. From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: Lippincott-Raven Publishers.

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DRUG-INDUCED PULMONARY DISEASE

Table 2 Clinical presentation of pulmonary toxicity induced by antineoplastic drugs Hypersensitivity PIE Noncardiogenic IP-F Acute Chronic PVD Parenchymal BOOP Asthma infiltrate pulmonary pleural pleural hemorrhage edema effusion effusion Azathioprine Bleomycin Busulfan Chlorambucil Cyclophosphamide Cytosine arabinoside Fludarabine Gemcitabine Melphalan Methotrexate Mitomycin C Nitrosoureas Paclitaxel Procarbazine Retinoic acid

þ

þ þ

þ þ þ þ þ

þ

þ

þ

þ

þ þ þ þ þ

þ þ þ þ þ

þ

þ þ

þ

þ

þ þ

þ þ

BOOP, bronchiolitis obliterans organizing pneumonia; IP-F, interstitial pneumonitis or fibrosis; PIE, pulmonary infiltrate with eosinophilia; PVD, pulmonary vascular disease. From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: Lippincott-Raven Publishers.

Table 3 Unusual manifestations of drug-induced pulmonary toxicity Presentation

Causative agent(s)

Alveolar proteinosis Bronchial necrosis

Busulfan Radiation therapy, brachytherapy b2-Adrenergic agonists Intravenous hydrocortisone Antacids, calcium, phosphorous, vitamin D ACE inhibitors Penicillamine Amiodarone, bleomycin Corticosteroids Diphenylhydantoin, methotrexate Esophageal variceal sclerotherapy Methylphenidate Nitrosoureas, bleomycin Chronic salicylate intoxication

Bronchospasm (paradoxical) Calcification (parenchymal) Cough Goodpasture’s syndrome Lung mass(es)7cavitation Mediastinal lipomatosis Mediastinal widening/ lymphadenopathy Mediastinitis Panlobular emphysema Pneumothorax Pseudosepsis syndrome

From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: Lippincott-Raven Publishers.

patients not exposed to the drug ðp ¼ 0:03Þ. Finally, in an unusual presentation, amiodarone pulmonary toxicity may take the form of parenchymal mass lesion(s), which may show cavitation (Table 3). Chest radiographs usually reveal a diffuse reticular pattern or bilateral airspace disease and, less commonly, nodular opacities or masses, pleural thickening, and pleural effusions. Computed tomography (CT) of the lung without the administration of

contrast (Figure 1(a)) may be helpful in making the diagnosis. Opacities caused by amiodarone toxicity have a high attenuation value (since the drug is an iodinated compound, Figure 1(b)) and appear more dense than usual opacities of other etiologies. A positive gallium scan can help differentiate amiodarone pneumonitis from pulmonary embolism or congestive heart failure (in which the gallium scan is negative) as long as infection has been excluded as a cause for the positive scan. One important limitation of gallium scan was evidenced by the lack of resolution of the abnormal uptake in three out of six patients with resolving amiodarone toxicity. Pulmonary function studies show restrictive defect and decreased carbon monoxide diffusing capacity (DLCO) and abnormal results correlate with exposure to the drug. As an example, in a trial of 519 patients, use of amiodarone for 1 year resulted in a significant decrease in DLCO in comparison to placebo ðp ¼ 0:02Þ. Although an isolated decrease in DLCO is a sensitive indicator of toxicity, it lacks specificity. In a prospective study evaluating the diagnostic utility of spirometry and DLCO in 91 patients on amiodarone, none of the 15 patients who had an asymptomatic decrease in diffusing capacity developed pulmonary toxicity in spite of continuing to take the drug in the 11 months of follow-up. Recently, measurement of serum concentrations of KL-6, a glycoprotein secreted by proliferating type II pneumocytes, has been proposed as a noninvasive marker of amiodarone toxicity. In a small study of 14 men receiving amiodarone, KL-6 levels above the

DRUG-INDUCED PULMONARY DISEASE 47

Figure 1 Radiographic appearance of amiodarone toxicity. (a) A computed tomographic scan without contrast in a patient receiving amiodarone reveals infiltrates in both lung bases, due to the iodine content in amiodarone. This enhancement, however, is not pathognomonic for amiodarone toxicity. (b) The chemical structure of amiodarone showing the iodine content. From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: Lippincott-Raven Publishers.

upper limit of 520 U ml
1 were seen in the two patients with pulmonary toxicity as opposed to normal levels in five patients with other pulmonary abnormalities (including pneumonia, congestive heart failure, and bronchogenic carcinoma) and seven asymptomatic individuals. Pathology

Histologically, a chronic interstitial pneumonitis associated with accumulation of foamy alveolar macrophages (Figure 2(a)) in the alveolar spaces, hyperplasia of type II pneumocytes, and widening of alveolar septa are the most common findings; diffuse alveolar damage and bronchiolitis obliterans organizing pneumonia (BOOP) are much less common. Bronchoalveolar lavage demonstrates the same foamy alveolar macrophages (Figure 2(a)) both in

Figure 2 Bronchoalveolar lavage findings in amiodarone exposure/toxicity. Bronchoalveolar lavage cytologic preparation showing: (a) foamy appearance of alveolar macrophages seen by light microscopy; and (b) lamellar bodies seen by electron microscopy. Both of these findings are characteristic of amiodarone exposure but not necessarily toxicity. From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: LippincottRaven Publishers.

patients with or without pulmonary toxicity, but the absence of this finding makes the diagnosis of toxicity unlikely.

Management and Current Therapy Cessation of the drug is the most important aspect of therapy. Corticosteroids have been used in uncontrolled studies and seem to be of greatest value in patients with severe disease and in those in whom the drug cannot be discontinued. Relapse of toxicity has been demonstrated after withdrawal of steroid therapy. Owing to the long elimination half-life of amiodarone, toxicity can progress for a while in spite of discontinuation of the drug. Overall, the prognosis is favorable with improvement or stabilization in threequarters of patients after discontinuation of the drug with or without steroid treatment. Unfortunately, in

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patients developing ARDS, mortality can be as high as 50%.

Drug-Induced Bronchospasm or Cough Drug-induced bronchospasm is caused by a variety of agents (Tables 1 and 2). In most instances, the drug exacerbates bronchospasm in a patient with underlying airway hyperreactivity. Acetylsalicylic Acid

Acetylsalicylic acid (ASA) produces bronchospasm in 0.3% of normal adults and in from 4% to 20% of asthmatic patients. The ASA triad is a syndrome characterized by asthma, nasal polyposis, and drug sensitivity. Furthermore, other nonsteroidal anti-inflammatory agents (NSAIDs) can produce a similar reaction and should be avoided in aspirin-sensitive patients. The first manifestation of the syndrome is vasomotor rhinitis with a watery discharge. Typically, this develops in the second or third decade in a person who is not atopic and who has previously taken ASA. The reaction is at first intermittent, later perennial. It is followed by the appearance of nasal polyps, and by midlife most patients demonstrate an asthmatic response. The syndrome is not a hypersensitivity response, despite the asthma and angioedema that may be seen following absorption of the drugs. It seems to be mediated by the proinflammatory and

bronchoconstrictive properties of active metabolites of arachidonic acid derived through the action of 5-lipoxygenase (Figure 3). Treatment starts with avoidance of the offending drug. In addition, leukotriene modifiers have been shown to improve pulmonary function and decrease beta-agonist use in patients already receiving corticosteroids. Aspirin desensitization can improve refractory upper and lower airway symptoms but is usually reserved for severe cases or when ASA use is absolutely necessary. Nasal polypectomy is used for symptoms of nasal obstruction only; it does not alter the response to ASA, and the polyps usually recur. Beta-Blockers

Beta-blockers can exacerbate chronic obstructive lung disease and precipitate bronchospasm. Some examples, in decreasing order of likelihood to cause bronchoconstriction, are propranolol, timolol, nadolol, atenolol, and labetolol. They should be used with extreme caution in patients with a potential for bronchospasm. Other Agents

Dipyridamole increases the concentration of the bronchoconstrictor adenosine, which can cause significant bronchospasm in some patients with underlying obstructive lung disease. Theophylline is the drug of choice for treatment and/or prophylaxis in these patients. Vinblastine appears to act synergistically

Phospholipids Phospholipase —Phospholipase inhibitors —Corticosteroids Arachidonic acid Cyclooxygenase

Lipoxygenase —Lipoxygenase inhibitors (e.g., zileuton)

—Aspirin —Nonsteroidal anti-inflammatory drugs

Leukotrienes Thromboxane

Prostaglandins Prostacyclin

—Receptor-level antagonists (e.g., zafirlukast, montelukast)

LTB4

Inflammation

Inflammation

LTC4, LTD4, LTE4

Bronchospasm Congestion Mucus plugging

Figure 3 How aspirin and nonsteroidal anti-inflammatory drugs may worsen asthma. Aspirin and nonsteroidal anti-inflammatory drugs inhibit cyclooxygenase activity, diverting arachidonic acid to the alternate lipoxygenase pathway, which converts arachidonic acid to potent bronchoconstrictor mediators: leukotrienes, LTC4, LTD4, and LTE4. Lipoxygenase inhibitors and receptor-level antagonists may counteract the effect. From Ozkan M and Dweik RA (2001) Drug-induced lung disease: an update. Cleveland Clinic Journal of Medicine 68: 782–795.

DRUG-INDUCED PULMONARY DISEASE 49

with mitomycin C to produce bronchospasm. Administration of nebulized or intravenous pentamidine or propellants can further irritate already hyperreactive airways. Paradoxical bronchospasm (Table 3) has been reported with the use of nebulized beta agonists as well as intravenous hydrocortisone (not reported with other steroid preparations). There is anecdotal evidence that patients developing paradoxical bronchospasm with beta agonists may benefit from the newly available levo-albuterol preparation. Estrogen therapy may worsen bronchospasm in women with chronic obstructive lung disease or asthma, and may even be associated with the onset of asthma. Angiotensin-Converting Enzyme Inhibitors

Angiotensin-converting enzyme (ACE) inhibitors can produce an irritating cough in about 5–25% of patients receiving this class of drugs. The cough occurs with all ACE inhibitors and does not respond to substitution within the same class. The pathogenesis of ACE inhibitor-induced cough probably involves accumulation of kinins and substance P (due to ACE inhibition) leading to bronchial irritation and cough (Figure 4). Another postulated mechanism is activation of the arachidonic acid pathway with consequent elevated levels of thromboxane potentiating the symptoms. In support of this hypothesis, a study using picotamide (an inhibitor of thromboxane synthase and antagonist of the thromboxane receptor) found that eight of nine patients with cough induced by enalapril had complete resolution of their symptoms. Cough usually completely resolves within 10 days of cessation of therapy. Angiotensin II receptor antagonists, which do not interfere with the synthesis

Angiotensinogen Renin

pathway, are not associated with this side effect and can be used in patients with ACE inhibitor-induced cough.

Pulmonary Manifestations of Anti-Inflammatory Agents Methotrexate

Methotrexate is currently used in low doses as an anti-inflammatory drug for many conditions, especially rheumatoid arthritis and Wegener’s granulomatosis. Typically, the patient is given less than 20 mg weekly. The prevalence of pulmonary toxicity in these patients is 3–5% and most cases occur within 2 years of initiation of therapy. In a casecontrol study, increasing age (odds ratio (OR) 5.1), presence of diabetes (OR 35.6), hypoalbuminemia (OR 19.5), use of disease modifying antirheumatic drugs (OR 5.6), and rheumatoid pleuropulmonary involvement (OR 7.1) were independent predictors for the development of pulmonary toxicity. The onset is insidious and associated with cough, dyspnea, and low-grade fever. Radiographically, the disease most commonly reveals basal or diffuse interstitial opacities that can progress to diffuse airspace disease. Pulmonary function studies classically demonstrate a restrictive pattern with a decrease in DLCO. However, there are some reports suggesting development of airflow obstruction with methotrexate therapy. Unfortunately, in a prospective evaluation of 124 patients receiving low-dose methotrexate, surveillance with pulmonary function tests (spirometry, lung volumes, and DLCO) did not allow for detection of toxicity prior to the onset of symptoms.

Kinins Desired effect

Adverse effect

—Remikiren, enalkiren Angiotensin I Angiotensin-converting enzyme (ACE) —ACE inhibitors

ACE —ACE inhibitors

Angiotensin II —Angiotensin-receptor blockers Binding to angiotensin receptors Vasoconstriction

Inactive metabolites

Increased levels of kinins, substance P Bronchial irritation, cough

Figure 4 A possible explanation for why angiotensin-converting enzyme (ACE) inhibitors cause cough. ACE inhibitors have the favorable effect of blocking conversion of angiotensin I to angiotensin II (left), but also lead to accumulation of kinins and substance P (right), which may explain the side effects of bronchial irritation and cough associated with this class of medication. Newer agents (angiotensin II receptor blockers, remikiren, enalkiren) in theory would not be associated with cough. From Ozkan M and Dweik RA (2001) Drug-induced lung disease: an update. Cleveland Clinic Journal of Medicine 68: 782–795.

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Bronchoalveolar lavage fluid demonstrates an increase in CD4 lymphocytes, a non-specific finding seen in a variety of inflammatory lung diseases. Histologically, changes resembling hypersensitivity pneumonitis (loosely formed granulomas) overlapping with a chronic interstitial pneumonitis are seen in most patients; BOOP and diffuse alveolar damage were also demonstrated in a few cases. The majority of patients improve with discontinuation of the drug. Corticosteroids have been recommended based on a favorable clinical response in case series and the opinion of experts in the field, but no controlled studies have examined its efficacy. Leukotriene Inhibitors

The leukotriene inhibitors zafirlukast, montelukast, and pranlukast have been associated with the development of Churg–Strauss syndrome in asthmatics. It is not clear whether the syndrome develops due to the use of the leukotriene inhibitors or it is unmasked due to the dose reduction or discontinuation of systemic steroid use achieved by the introduction of the leukotriene antagonists. The latter hypothesis is supported by the occurrence of Churg–Strauss syndrome in patients switched from systemic to inhaled corticosteroids. However, in one case report the syndrome was reported in one patient given montelukast in the absence of systemic steroid use. Other Agents

Pulmonary edema, which is probably due to a combination of increased capillary permeability and fluid overload, has been reported in association with treatment with interleukin-2. An asymptomatic, isolated decrease in diffusing capacity was demonstrated in patients receiving recombinant tumor necrosis factor for the treatment of metastatic malignancies.

Pulmonary Manifestations of Antimicrobial Agents Nitrofurantoin

Pulmonary toxicity is the most severe adverse effect of nitrofurantoin therapy. It seems to be an infrequent complication given the extensive use of the drug. In fact, during a 16-year period, 237 cases of adverse pleuropulmonary reactions were reported in an estimated 44 million courses of the drug. Reactions to nitrofurantoin are classified into two types: acute, developing hours to days after the institution of treatment; and chronic, occurring after weeks to years of continuous therapy. The former is more common, being reported in approximately 90% of cases of toxicity in two series. In the acute form,

fever is seen in approximately 80% of cases in conjunction with cough, dyspnea, rash (20%), and arthralgias (10–15%). In the patients with the chronic form, fever and rash are not seen and the respiratory symptoms develop more insidiously. Peripheral blood eosinophilia occurs in 83% of individuals with the acute form. Radiographic abnormalities are seen in the vast majority of patients and most commonly include diffuse alveolar or interstitial opacities. Pleural effusions may be present in conjunction with the parenchymal abnormalities or as an isolated finding. In the acute form, histologic findings seem to represent a type I hypersensitivity response, including signs of pulmonary vasculitis and interstitial and alveolar monocytes or polymorphonuclear inflammation with variable presence of eosinophilia. Other uncommonly reported findings include diffuse alveolar damage, desquamative interstitial pneumonitis (DIP)-like reaction, hypersensitivity pneumonitis-like reaction, BOOP, and diffuse alveolar hemorrhage. A chronic interstitial pneumonia resembling usual interstitial pneumonitis (UIP) is the commonest pathologic finding in patients with chronic nitrofurantoin pulmonary toxicity. Discontinuation of the drug is the mainstay of therapy in both forms of nitrofurantoin pulmonary toxicity. Eighty-eight percent of patients with the acute reactions will have resolution of symptoms within 3 days of discontinuation of the drug. Clinical improvement is slower (weeks to months) in the chronic form. Beneficial effects of steroid use have only been demonstrated in case reports and in a series of 447 patients its use did not accelerate recovery. In that same cohort, six cases were fatal, four of which occurred in patients with the chronic form of toxicity. Sulfasalazine has been reported to cause several pulmonary reactions including pulmonary opacities with eosinophilia, hypersensitivity pneumonitis, BOOP, DIP, pulmonary fibrosis, and bronchospasm. Pulmonary function studies can demonstrate both a restrictive or obstructive pattern. One patient with bronchospasm and another with bronchiolitis had resolution of their symptoms with steroid use.

Other Agents

A number of antimicrobials have been associated with an eosinophilic or hypersensitivity lung reactions including tetracycline, minocycline, sulfonamides penicillins, para-aminosalicylic acid, and ethambutol. Fatal pulmonary hemorrhage was demonstrated in 14 of 22 patients receiving amphotericin B in association with granulocyte transfusions. Although the

DRUG-INDUCED PULMONARY DISEASE 51

effect was not demonstrated in another study, a real association cannot be discounted.

Heroin and Other Illicit Drugs The use of illicit drugs has reached epidemic proportions, resulting in an increased incidence of pulmonary toxicity. The pulmonary manifestations relate not only to the substance used, but also to the route of administration. Narcotic addiction remains a major health problem, with noncardiogenic pulmonary edema occurring as a complication of heroin, methadone, and cocaine abuse. Opiates and related drugs are well-known causes of pulmonary edema occurring in 50–70% of overdoses. It has also been demonstrated with naloxone use, an opiate antagonist. Pulmonary edema develops within a few hours of use, and the patient presents with constricted pupils and depressed respiration. The chest radiograph shows alveolar opacities, usually in ‘bat wing’ distribution. A high protein concentration in the edema fluid suggests increase in capillary permeability as the etiologic mechanism. The therapy is supportive, with administration of oxygen, narcotic antagonists, and mechanical ventilation if needed. Cocaine is a highly addictive substance. Pulmonary complications are related to all forms of administration. Cough productive of black sputum, dyspnea, chest pain, and hemoptysis occur within minutes of crack inhalation. Pulmonary edema can occur with cocaine regardless of the route of administration and it has been reported with freebase inhalation, intravenous freebase, and even ‘body packing’ (smugglers swallowing packets of cocaine, which leak into the gastrointestinal tract). Increased membrane permeability and a possible component of myocardial dysfunction are the mechanisms responsible for the development of pulmonary edema. An acute pulmonary syndrome temporally related to inhalation of crack cocaine has also been described and is characterized by fever, hemoptysis, alveolar opacities on chest radiographs, and respiratory failure. Diffuse alveolar damage is the most common pathologic manifestation and it can be associated with alveolar hemorrhage and eosinophilic infiltration. The patients tend to respond to systemic corticosteroids. Other reported complications of smoking freebase cocaine include barotrauma, interstitial pneumonitis, thermal airway injury, bronchospasm, and BOOP. Pulmonary edema has been reported with amphetamine abuse. Early panlobular emphysema was described in intravenous abusers of methylphenidate and in individuals who inject talc-containing drugs intended for oral use.

Pulmonary Manifestations of Anti-Neoplastic Drugs The delayed adverse effect of cancer treatment is an increasing health problem as therapeutic regimens for neoplastic diseases evolve and an increased number of long-term survivors occur. For instance, nearly 30% of long-term survivors of Hodgkin’s disease complain of dyspnea and have pulmonary function abnormalities. Furthermore, in a series of 77 patients who underwent high-dose therapy and autologous bone marrow transplantation, the incidence of drug- or radiotherapy-induced lung toxicity was 16%. The adverse pulmonary responses to various antineoplastic drugs are usually similar and most often include chronic interstitial pneumonitis/fibrosis, acute hypersensitivity-type reactions and noncardiogenic pulmonary edema (Table 2). Typical symptoms of the interstitial pneumonitis/fibrosis develop over weeks to months and include progressive exertional dyspnea, dry cough, and malaise associated with crackles on physical exam. Dyspnea, nonproductive cough, fever and occasional peripheral blood eosinophilia are the most common features of hypersensitivity-type reactions. Symptoms generally progress rapidly over the course of days. Occasionally, factors such as total cumulative dosage, patient age, or prior use of agents that may act synergistically (e.g., radiation) can be used to anticipate an untoward response. Interstitial opacities resembling pulmonary fibrosis are the most common radiographic findings. Alveolar opacities can also be seen in patients presenting with hypersensitivity lung reactions. Pleural effusions are uncommon. Pulmonary function testing is usually consistent with a restrictive ventilatory defect (low vital capacity or total lung capacity) and a reduced DLCO; the flow rates are usually preserved. The list of causes of pulmonary opacities in the immunocompromised host always includes infection, which should be excluded as an etiology of opacities suspected to be due to drugs. Treatment is discontinuation of the agent. The role of steroids is not well established since no randomized trials have been conducted but they should probably be tried especially in severely symptomatic patients. Bleomycin

Bleomycin is deposited in the skin and lungs due to decreased drug inactivation in these tissues. Thus, the lungs and the skin are the two organs displaying the most serious side effects. Approximately 10% of patients receiving bleomycin will develop pulmonary toxicity. Interstitial fibrosis is the most commonly mentioned adverse pulmonary response, followed by hypersensitivity pneumonitis and BOOP with

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parenchymal nodular densities. Symptoms usually develop within the first 6 months of therapy and include dry cough and dyspnea. Physical examination reveals tachypnea, basilar crackles, and concomitant hyperpigmentation of the skin. Acute chest pain can also occur in 2.8% of patients during bleomycin infusion, being severe enough to require narcotics and warrant evaluations for myocardial infarction or pulmonary embolism. Factors that appear to increase the toxic potential include age, total cumulative dose, oxygen therapy, renal function, and prior radiation to the thorax. In one review, the incidence of toxicity was significantly higher in patients older than 70 years (6% in patients 20–70 years versus 15% in those above 70 years). Although toxicity can develop with doses as low as 49 units, toxicity and fatality sharply increases with doses greater than 450 units. Supplemental oxygen administration (FiO2 0.35 to 0.42) was associated with fatal pulmonary toxicity in five patients undergoing surgical procedures, while 12 patients maintained at an FiO2 of less than 0.25 during surgery showed no toxicity. In a series of 20 patients with metastatic testicular cancer receiving bleomycin, presence of renal failure was the most significant factor associated with the development of abnormal pulmonary function tests. Evidence for the enhancement of toxicity by concomitant use of other antineoplastic agents is inconclusive. In a series of 100 patients, radiographic abnormalities were detected by chest radiograph in 15% of cases and in 38% by chest CT. They normally include a non-specific interstitial pattern with the most severe changes appearing in the lung bases and subpleural areas. Nodular abnormalities, which can be confused with metastatic disease, can be encountered. Vital capacity, total lung capacity, and diffusing capacity have been reported to be sensitive markers for detection of early signs of toxicity but serial measurements were unable to specifically predict development of toxicity. The histologic changes seen in patients with fibrosis are most commonly those of diffuse alveolar damage (Figure 5). Eosinophilic pneumonia is seen in patients with hypersensitivity pneumonitis. Treatment is discontinuation of the drug. Resolution of interstitial disease has been reported with steroids in case reports as well as in cases of hypersensitivity pneumonitis and BOOP. If the patient’s condition demonstrates reversibility, the radiographic findings and pulmonary function test results usually improve. In a series of 180 patients treated for germ-cell tumors, the mortality related to bleomycin pulmonary toxicity was 2.8%.

Figure 5 Bleomycin toxicity. This is the typical appearance of interstitial lung disease due to drug toxicity of which bleomycin is a typical example. Characteristic features include hyperplastic alveolar epithelium, septal thickening, and lymphocytic infiltration. From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: Lippincott-Raven Publishers.

Cyclophosphamide

Pulmonary toxicity is a rare complication of cyclophosphamide use either in malignant or nonmalignant disorders. It appears to occur in two distinct forms: an early-onset interstitial pneumonitis and a late-onset interstitial pneumonitis with fibrosis, which is seen more frequently. In the early-onset pneumonitis, patients present within 1–6 months of onset of therapy with dyspnea, cough, fatigue, and frequently fever. Insidious and progressive dyspnea and cough occurring after months to years of therapy are characteristic findings of the late-onset form. There are no clearly defined risk factors for the development of toxicity. Interstitial reticular or nodular opacities are seen on the chest roentgenogram and in some cases in association with bilateral pleural thickening. Ground glass attenuation on high-resolution CT scans has also been reported. The pulmonary function tests show restrictive defects and a decreased DLCO. Histologic studies typically show organizing diffuse alveolar damage (DAD) and less frequently chronic interstitial pneumonitis and BOOP. Early-onset pneumonitis usually responds to discontinuation of the drug and, in the majority of series, steroids, whereas the course of late onset pneumonitis is most often progressive and irreversible. Bischloroethylnitrosourea

When used as a single agent, bischloroethylnitrosourea (BCNU) has been reported to cause pulmonary toxicity in up 20% of patients. Incidence can be as high as 60% when the drug is used as part of

DRUG-INDUCED PULMONARY DISEASE 53

high-dose combination protocols for bone marrow transplantation. The incidence of toxicity increases with dose increments. There is also evidence of an increased incidence of toxicity in patients treated before the age of 5 years. Pulmonary fibrosis can develop as an early (within 36 months) or late (up to 17 years) complication of therapy. The course of the disease is usually insidious with progressive cough, dyspnea, and fatigue, although rapidly progressive and fatal presentations have been described. Radiographic findings include bibasilar reticular opacities and ground glass attenuation in the lower lung zones. Less commonly, upper lobe disease, patchy consolidation, and pneumothorax can be seen. The radiograph may also be normal even in the presence of histologically proven fibrosis. Histologic findings typically include interstitial pneumonitis and fibrosis with atypia of type II cells or diffuse alveolar damage. In patients with early lung toxicity, corticosteroids seem to be helpful. Prednisolone used at a dose of 1 mg kg
1 for 10 days followed by a slow taper resulted in complete recovery in 15 of 17 patients developing pulmonary toxicity after carmustine-based preparative regimens for bone marrow transplant. Some authors have also recommended discontinuation of therapy and institution of steroids in patients with significant (410%) decrease in diffusing capacity. Steroids are not thought to be beneficial in patients with late-onset fibrosis. The prognosis seems to be more favorable in cases of early-onset fibrosis. A mortality rate of 36% has been reported in 26 patients who developed pulmonary toxicity within 1 year of therapy. In a study of 17 survivors of childhood brain tumors who were treated with BCNU 12% died of lung fibrosis within 3 years of treatment and 47% after 16–20 years of follow-up. Furthermore, all of the long-term survivors had evidence of lung fibrosis. Busulfan

Busulfan is considered the prototypic drug for cytotoxic drug-induced pulmonary damage. It is estimated to occur in less than 5% of patients taking the drug. The usual case is one of long-term toxic damage to the lungs, with an insidious onset of symptoms after the patient has taken the drug for 3–4 years. Symptoms include cough, dyspnea, and fever and occasionally hyperpigmentation. Diffuse interstitial and alveolar opacities are typically seen on the chest X-ray, but nodular densities, pleural effusions, or a normal picture may be seen as well. The pulmonary function tests show restrictive defects, and a decrease in diffusing capacity. However, as demonstrated in

two studies, pulmonary function studies could not predict development of pulmonary toxicity. Histologic evidence of toxicity may be seen in up to 46% of all patients treated and typically include the presence of large, cytologically atypical type II pneumocytes. Alveolar proteinosis has been reported in a number of patients receiving busulfan (Table 3). Response to steroids has been reported in case series. The prognosis is generally poor with a postdiagnosis mean survival of 5%. Mitomycin C

The incidence of pulmonary toxicity with mitomycin C ranges from 3% to 6%. Toxicity has been reported with mitomycin C alone or in combined treatments, particularly including vinca alkaloids. Pulmonary manifestations are somewhat unique and can include bronchospasm, acute and chronic pneumonitis, pleural abnormalities, and hemolytic uremic syndrome. Acute bronchospasm occurs in about 5% of patients and is associated with concomitant administration of a vinca alkaloid. Acute pneumonitis with concomitant vinca alkaloid administration, noncardiogenic pulmonary edema, and acute respiratory failure with diffuse alveolar damage comprise the other forms of acute presentation, occurring on average within 3 weeks of the onset of therapy. Chronic interstitial pneumonitis seems to be a less frequent complication, usually occurring in patients receiving more than 30 mg m
2 of the drug. Pleural disease presenting as exudates or pleural fibrosis seems to occur at a greater frequency with mitomycin C than other antineoplastic agents. Finally, a hemolytic-uremic-like syndrome associated with mitomycin C has been described. Fifty percent of patients presenting with the syndrome develop ARDS. Radiographic manifestations consist of bilateral reticular opacities predominantly in the lower lung zones and are seen in both the acute and chronic forms. Findings consistent with ARDS are also less frequently seen. The DLCO declines by 420% in approximately a quarter of patients after they have received three cycles of chemotherapy. Unfortunately, the use of serial DLCO measurements in patients receiving mitomycin C cannot predict pulmonary toxicity. Histologic findings are typically similar to those found with other cytotoxic agents and less frequently include BOOP. Lung tissue from patients dying from the hemolytic uremic syndrome shows capillary angiomatoid changes. In cases of acute and chronic pneumonitis, prednisone therapy can result in dramatic resolution of both symptoms and radiographic abnormalities. However, mortality exceeds 70% in cases of hemolytic uremic syndrome.

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See also: Acute Respiratory Distress Syndrome. Bronchoalveolar Lavage. Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations; Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General; Smoking Cessation. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia. Pneumonia: Overview and Epidemiology. Pulmonary Thromboembolism: Pulmonary Emboli and Pulmonary Infarcts.

Further Reading Carson CW, Cannon GW, Egger MJ, Ward JR, and Clegg DO (1987) Pulmonary disease during the treatment of rheumatoid arthritis with low-dose pulse methotrexate. Seminars in Arthritis and Rheumatism 16: 186–195. Cooper JA Jr, White DA, and Mathay RA (1986) Drug-induced pulmonary disease. Part 1: cytotoxic drugs. American Review of Respiratory Diseases 133: 321–340.

Cooper JAD, White DA, and Mathay RA (1986) Drug-induced pulmonary disease. Part 2: noncytotoxic drugs. American Review of Respiratory Diseases 133: 488–505. Cottin V, Tebib J, Massonnet B, Souquet PJ, and Bernard JP (1996) Pulmonary function in patients receiving long-term low-dose methotrexate. Chest 109: 933–938. Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Druginduced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: LippincotRaven Publishers. Meyers JL (1990) Pathology of drug-induced pulmonary disease. In: Katzenstein AL and Askin F (eds.) Surgical Pathology of Non-Neoplastic Lung Disease, 2nd edn., pp. 97–127. Philadelphia: Saunders. Ozkan M and Dweik RA (2001) Drug-induced lung disease: an update. Cleveland Clinic Journal of Medicine 68: 782–795. Rosenow EC (1994) Drug-induced pulmonary disease. Disease a Month 40: 255–310. Zitnick RJ and Cooper JA Jr (1990) Pulmonary disease due to antirheumatic agents. Clinical Chest Medicine 11: 139–150.

DUST MITE A Custovic and A Simpson, University of Manchester, Manchester, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The discovery in 1964 that allergen from dust mite Dermatophagoides pteronyssinus was the main house dust allergen marked the beginning of extensive research into mite biology, the relationship between mites and allergic diseases, and methods to reduce exposure. In order to digest food, mites produce a number of enzymes, which accumulate in their feces and in the dust reservoirs in the house, and may become inhaled and cause immune response in predisposed individuals. To date, 19 different allergens from dust mite have been characterized and found to elicit varying degrees of IgE reactivity and T-cell responses. Several mite allergens are biochemically active, including hydrolytic and nonhydrolytic enzymes. Proteolytic activity of mite allergens increases the permeability of epithelium in vitro. Opening up of tight junctions by environmental proteases and their delivery to antigen-presenting cells may be an important step in the development of sensitization and may contribute to ongoing symptoms of allergic airways disease. However, extrapolation of studies conducted in cell culture to in vivo scenarios must be made with caution. Whilst enzymatic activity may play a role in modulating allergenicity, current evidence suggests that it is not a prerequisite for allergy. There is a clear dose–response relationship between mite allergens exposure and development of sensitization, and between mite sensitization and allergic disease. However, there is no relationship between mite exposure and the development of symptomatic disease. In contrast, in established asthma and rhinitis, mite allergen exposure in mite-allergic individuals contributes to the severity of symptoms. However, although mite avoidance should lead to an improvement of symptoms, there is little evidence to support the use of physical or chemical methods to reduce exposure.

Introduction The term ‘house dust mite’ usually refers to the mites of the family Pyroglyphidae, while ‘domestic mites’ includes all mite taxa which can be found in homes (e.g., Pyroglyphidae and storage mites). In 1964 Voorhorst et al. suggested that allergen from dust mite Dermatophagoides pteronyssinus was the main house dust allergen, opposing the widely accepted hypothesis of the time that the dust antigen was produced by an abiotic chemical reaction. This marked the beginning of extensive research into mite biology and ecology, the relationship between mites and allergic diseases, and methods to reduce mite allergen exposure.

Taxonomy, Biology, and Ecology House dust mites belong to the family Pyroglyphidae, in the suborder Acaridida. Dust mites are not insects and belong to the same class as spiders, scorpions, and ticks. D. pteronyssinus mites are approximately 0.3 mm in length and are therefore not visible to the naked eye (Figure 1). They have eight legs and no distinct head. Pyroglyphid mites reproduce sexually; under optimal laboratory conditions, adults live for about 6 weeks, during which time a female may produce 40–80 eggs. They feed on human skin scales, fungi, bacteria, and various organic detritus. Certain fungi present in house dust may partially digest human skin scales making them more palatable for dust mites. Furthermore, fungi are a food source for

DUST MITE 55

furniture in most homes in areas where mites are prevalent. In most European countries D. pteronyssinus has been found to be the most abundant species, whereas D. farinae is predominant in North America. The geographic distribution and occurrence of these two species within homes is largely dependent on the level of humidity in the mites’ microhabitat; D. farinae appears to survive better in drier climates than D. pteronyssinus. Blomia tropicalis from the family Glycophagidae is another mite species often found in houses in tropical and subtropical areas and is an important cause of sensitization and asthma in these areas. Figure 1 House dust mites. Reproduced with permission of Dr B Hart, Royal Agricultural College, Cirencester, Gloucs, UK.

10 µm 12.0 kV 1.05E3 3403/00 Zoology Figure 2 House dust mite faecal pellets. Reproduced with permission of Dr B Hart, Royal Agricultural College, Cirencester, Gloucs, UK.

mites, and enhanced mite population growth after fungi are added to skin scales is not only due to fungal predigestion of skin scales. They have a well-developed digestive system, which produces spherical fecal pellets surrounded by a peritrophic membrane (Figure 2). It has been reported from laboratory studies that adult mites deposit on average 20 fecal pellets per day, but this may not reflect accurately what is happening in wild populations. These fecal pellets are usually between 20 and 40 mm in diameter and contain a large number of allergens. The respiration is cutaneous, with skin serving as a barrier for the exchange of gas and water vapor. Dust mites live in an environment with no liquid water present. Active life stages require relatively high indoor humidity for their survival, and the level of air humidity largely governs the occurrence of mites in homes. Analysis of dust samples collected from homes in different parts of the world shows that many mite species occur in household dust. In spite of this diversity, pyroglyphid mites constitute more than 90% of the mite population in mattresses, and 70–95% of the mite population in carpets and upholstered

Mite Occurrence in Buildings

Mites are free-living (i.e., not parasitic) and are usually found in the high-use areas of home (e.g., beds, carpets, and upholstered furniture). The conditions rendering an indoor microhabitat ideal for mite population growth are not as yet clear. The habitation of domestic dwellings results in the creation of house dust, including the accumulation of human skin scales. Suitable relative humidity and temperature in the microhabitat are essential for survival of mites. However, even in the regions where climate is conducive for mite population growth there may be huge differences in the mite density between different homes. The reasons for this variation probably include the age of the home and soft furnishings (older having higher levels), type of glazing (lower if double glazed), and presence of damp in the home. No apparent differences in the level of humidity nor the presence of adequate food are usually found between homes with high and low mite densities. The mite population of any one home is geographically isolated from that of others, but dispersal does occur on clothing. Some physical factors in the home may determine mite population density (e.g., long or loose-pile carpets contain more mites than tile or wood floors; short, tight-pile carpets harbor fewer mites than long or loose-pile carpets). Mite abundance does not appear to be affected by cleaning, and clean, well-maintained homes may still contain a large number of dust mites. The Effect of Humidity on House Dust Mites

Whilst the abundance of dust mites in homes is related to a number of environmental factors, only the effects of temperature and humidity have been studied in detail. Mites are able to extract water vapor from unsaturated air by passive and active absorption and amount of the passive gain is directly proportional to the humidity of the environment. The optimum relative humidity for mite growth is 75–95%,

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DUST MITE

at temperatures of 15–301C. The lowest relative humidity at which the passive and active uptake combined compensate for water loss under fasting conditions is known as the critical equilibrium humidity; at this point water gain is equal to water loss. Humidity below this point can dehydrate and kill mites, but will threaten the mite population only if sustained for long enough periods. At room temperature, relative humidity levels below 50% may be associated with the desiccation of mites. Studies have shown that within a room, the relative humidity of the carpet tends to be higher than the room air. Furthermore reducing the relative humidity of the room does not necessarily have an effect in mite microhabitats (carpet, mattress, and upholstery).

House Dust Mite Allergens The exact number of dust mite allergens that are clinically significant has not yet been determined. To date, 19 different allergens from dust mite have been characterized (Table 1) and found to exhibit varying degrees of IgE reactivity and T-cell proliferative responses. Sequence polymorphisms have been identified for several mite allergens. In order to digest food, mites produce a number of enzymes, which accumulate in their feces. Mites are probably coprophagic and this accumulation of highly immunogenic enzymes is consequent to the manner in which they digest the food. These enzymes may accumulate in the dust reservoirs within the house, and as a result of any activity that causes settled dust to become airborne (e.g., vacuuming, bed making, etc.) the fecal

pellets may become inhaled and cause immune response in predisposed individuals (see Allergy: Allergic Reactions). Proteolytic Enzyme Activity

Several clinically important dust mite allergens are biochemically active, and their biological function amongst others include hydrolytic enzymes (proteases; see Cysteine Proteases, Cathepsins) and nonhydrolytic enzymes (glutathione-S-transferase and amylase). This led to the hypothesis that in addition to their intrinsic properties (e.g., protein integrity, resistance to degradation, binding capacity, and the degree of sequence similarity to host proteins), the biologic function of mite allergens may contribute to the enhanced allergenicity. Within this context, the protease activity may facilitate the allergen access to immunocompetent cells by compromising airway barrier function (e.g., by reversibly disrupting intercellular epithelial adhesion). Having facilitated the passage through epithelial barrier, the proteolytic activity of allergens may enable them to modulate immunological signaling (either by directly promoting IgE synthesis through cleavage of the low affinity IgE receptor on B cells, or indirectly through cleavage of CD25 and CD40). A typical example for such function is group 1 allergens, comprising 25 kDa acidic to neutral allergenic proteins which function as digestive enzymes. In addition, allergen groups 3, 6, and 9 have also been identified as digestive enzymes exhibiting serine protease activity. Proteolytic activity of mite allergens increases the permeability of epithelium in vitro. Der p 1 and mite

Table 1 House dust mite allergens Mite species

Allergen

Molecular weight (kDa)

Function

Dermatophagoides spp. (D. pteronyssinus and D. farinae)

Group 1 Group 2 Group 3 Der p 4 Der p 5 Der p 6 Group 7 Der p 8 Der p 9 Group 10 Group 11 Group 14 Der f 15 Der f 16 Der f 17 Der f 18w Der p 20

25 14 25 60 14 25 22–31 26 24 36 B100 Unknown 98 53 53 60 40

Cysteine protease MD-2 related lipid recognition proteins Trypsin Amylase Unknown Chymotrypsin Unknown Glutathione transferase Collagenolytic serine protease Tropomyosin Paramyosin Apolipoprotein-like Chitinase Gelosolin/villin Ca-binding EF protein Chitinase Arginine kinase

Euroglyphus maynei

Eur m 2 Eur m 14

14 177

MD-2 related lipid recognition proteins Apolipoprotein-like

DUST MITE 57

spent growth medium extract detach cultured Madin–Darby canine kidney (MDCK) cells and canine tracheal epithelial cells grown on artificial and natural biopolymer substrata, causing visible disruption of the epithelial architecture and lead to an increase in albumin flux across isolated sheets of bovine bronchial mucosa. Extract of D. pteronyssinus causes cell detachment of human alveolar type II epithelial-like cells and primary nasal epithelial cultures, but this is inhibited by the presence of protease inhibitors. Both cysteine and serine protease activities of mite fecal pellets lead to a concentration-dependent increase in mannitol permeability across cultured epithelial cell monolayers, but again these effects are abolished in the presence of class-specific protease inhibitors. However, it has to be emphasized that the concentrations of purified allergen and extracts used in these experiments were in the range of several hundred micrograms per milliliter. In contrast, personal inhaled exposure to Der p 1 has been estimated to be only 2 mg year
1. Proteolytically active Der p 1 may alter epithelial permeability by increasing paracellular conductance as opposed to transcellular conductance. Paracellular channels are normally sealed by tight junctions (TJ), which form the most apical element of the junctional complex between polarized epithelial cells. Both cysteine and serine proteases from D. pteronyssinus can disrupt barrier function through the degradation of TJ proteins. Immunopurified Der p 1 and serine proteases from mite fecal pellets evoke a time-dependent decrease in TJ staining which can be detected within 30 min of exposure. Increase in the mannitol permeability is related to the total number of breaks visualized by immunostaining. In addition, it was confirmed by immunoblotting that reduction in fluorescence intensity was a result of proteolysis of the transmembrane proteins occludin and claudin and of the cytoskeletal protein zona occludens 1 (ZO-1). The TJ degradation and the resulting increase in transepithelial permeability are dependent upon the protease activity of the allergens and can be inhibited by the presence of protease inhibitors. Thus, it is hypothesized that the opening up of TJ by environmental proteases and their delivery to dendritic antigen-presenting cells may be an important early step in the development of sensitization and may contribute to ongoing symptoms of allergic airways disease. However, interpretation of such studies conducted in cell culture and extrapolation to in vivo scenarios with intact airway mucosa must be made with caution, as it is difficult to imply clinical relevance from such experiments given the actual human exposure levels. In in vitro studies varying concentrations of allergen with different levels of proteolytic

activity were applied to monolayers of cells. Comparable levels of activity in experiments yielded disparate levels of junctional breakdown. In order to evaluate the role of proteolytically active allergens in allergic disease, it is essential for the studies examining tight junctions in MDCK cells and bronchial epithelial cells to be replicated in primary nasal epithelial cell cultures or in ex vivo biopsy samples. Future studies also need to take into account the possible cytoprotective effect of antiproteases (e.g., a1-antitrypsin and secretory leukocyte protease inhibitor, which inhibit the protease activity of Der p 1). Thus, although enzymatic activity may play a role in modulating allergenicity, current evidence would suggest that it is not a prerequisite for allergy and this is supported by the finding that many allergens implicated in allergic rhinitis including cat, birch pollen and Der p 2 are not enzymatically active.

Dust Mites, Allergic Sensitization and Respiratory Diseases Mite Allergen Exposure and the Development of Sensitization and Asthma

Several studies have confirmed a simple dose–response relationship between dust mite allergen exposure and development of specific sensitization in infants and children, and early infancy has been suggested as a critical period for primary sensitization (see Allergy: Overview). The threshold concentration of 2 mg Der p 1 per gram of dust for developing mite sensitization in children already sensitized to other allergens has been suggested. In addition, sensitization to mites is a risk factor for the development of asthma in both children and adults (see Asthma: Overview). However, although a clear relationship has been demonstrated between sensitization and exposure to mite allergens, and between mite sensitization and allergic disease (especially asthma), the relationship between exposure to mite allergens and the development of manifest clinical disease is much less clear. Several longitudinal studies have examined the importance of mite allergen exposure in early life and subsequent development of asthma and mostly reported no significant relationship between exposure and symptomatic disease. For example, data from the large prospective birth cohort (German Multicentre Allergy Study) showed: (1) a strong association between sensitization to mite and wheezing and (2) mite allergen exposure strongly related to specific sensitization. However, there was no dose– response relation for mite allergen exposure and doctor-diagnosed asthma, wheezing within the last 12 months, or wheezing ever. In contrast to these

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DUST MITE

European data, several recent studies from the developing countries in Africa (Ghana and Ethiopia) have suggested that asthmatic children are exposed to significantly higher levels of dust mite allergen compared to controls, and that the risk for asthma in urban areas increases significantly with increasing dust mite allergen exposure. Mite Allergen Exposure in Established Disease

Mite allergen exposure in mite-allergic individuals is one of the numerous factors that has been related to the severity of asthma and rhinitis (see Asthma: Extrinsic/Intrinsic). If mite exposure contributes to the severity of symptoms, then reducing exposure should improve the control of disease. However, the evidence is equivocal as to whether measures to change the indoor environment are effective. Unequivocal findings about mite exposure and allergic respiratory diseases A controlled low-dose mite allergen challenge which mimics ‘real life’ increases non-specific airway reactivity in sensitized asthmatics. Peak expiratory flow rate (PEFR) variability, and pulmonary function in mite-sensitive subjects correlate with dust mite allergen levels in beds. Patients with severe asthma are significantly more often both sensitized and exposed to high levels of mite allergen compared with patients with mild disease, and the combination of mite sensitization and high exposure, and respiratory viral infection substantially increases the risk of hospital admission. However, demonstrating the link between exposure and disease severity does not equate to demonstrating the benefits of allergen avoidance. The equivocal findings about mite exposure and allergic respiratory diseases: clinical trials of mite allergen avoidance Although complete absence of allergen exposure is usually associated with improvement in symptoms amongst sensitized patients (e.g., in patients with hay fever or seasonal asthma, the absence of exposure outside the season is associated with a dramatic improvement in symptoms), it remains unclear whether a major ‘real-life’ reduction in personal exposure to mite allergens can be achieved. A Cochrane meta-analysis concluded in 1998 that current chemical and physical methods aimed at reducing exposure to mite allergens are ineffective and cannot be recommended for mite-sensitive asthmatics. The update of this meta-analysis in 2004 included 49 studies (search until June 2004), with no change in conclusions or recommendations. The largest double-blind, placebo-controlled trial on the effectiveness of mite allergen-impermeable mattress,

pillow, and duvet encasings, as a single intervention was carried out in 1122 adult patients with physician-diagnosed asthma taking regular inhaled corticosteroids and using at least 100 mg day
1 of salbutamol. There was no difference in either the primary outcomes (mean morning PEFR over a 4-week period during the run-in and at 6 months; the proportion of patients discontinuing inhaled corticosteroids in a phased reduction during months 7–12) or in the secondary outcomes (e.g., mean proportionate inhaled steroid reduction). Several small studies aimed at reducing mite allergen exposure have been conducted in children, and results of these have been more positive. Recently a large study adopting a wide-ranging intervention (including smoking cessation and education of carers) in children living in poor quality housing in the US reported improvements in asthma control amongst mitesensitized children. A systematic review dust mite avoidance measures for perennial allergic rhinitis has been published in 2003, suggesting no beneficial effect of physical or chemical intervention. Subsequent to this metaanalysis, a study in 279 patients investigated the effectiveness of mite-allergen-impermeable encasings in mite-sensitized subjects with perennial rhinitis. Both groups reported a decrease in symptom scores during the 12-month follow-up period, with no difference in any of the outcomes between the groups. Most of the studies in adults demonstrate that in the absence of other mite control measures, allergenimpermeable covers are clinically ineffective for routine management of adult asthma or rhinitis. It remains possible that a much more extensive intervention in carefully selected subgroup of patients could have some effect, but that has not as yet been adequately addressed. Although the general consensus is that mite allergen avoidance should lead to an improvement of symptoms in mite-sensitive patients, there is little evidence to support the use of physical or chemical methods. The use of single intervention for dust mite allergy (e.g., mattress encasings) in adults with asthma or rhinitis cannot be advocated. For children, environmental control measures may be of some benefit. See also: Allergy: Overview; Allergic Reactions. Asthma: Overview; Extrinsic/Intrinsic. Cysteine Proteases, Cathepsins.

Further Reading Aalberse RC (2000) Structural biology of allergens. Journal of Allergy and Clinical Immunology 106: 228–238.

DYNAMICS OF GAS FLOW AND PRESSURE–FLOW RELATIONSHIPS 59 Colloff MJ (1991) Practical and theoretical aspects of the ecology of house dust mites (Acari: pyroglyphidae) in relation to the study of mite-mediated allergy. Review of Medical and Veterinary Entomology 79: 611–629. Custovic A and Murray CS (2002) The effect of allergen exposure in early childhood on the development of atopy. Current Allergy and Asthma Reports 2: 417–423. Custovic A, Murray CS, Gore RB, and Woodcock A (2002) Environmental allergen control. Annals of Allergy, Asthma and Immunology 88(5): 432–441. Custovic A and Simpson A (2004) Environmental allergen exposure, sensitisation and asthma: from whole population to individuals at risk. Thorax 59: 825–827. Custovic A and Woodcock A (2001) On allergens and asthma (again): does exposure to allergens in homes exacerbate asthma. Clinical and Experimental Allergy 31: 670–673. Go/tzsche PC, Johansen HK, Schmidt LM, and Burr ML (2004). House dust mite control measures for asthma. Cochrane Database of Systematic Reviews, Issue 4, Art. No.: CD001187.pub2.DOI: 10.1002/14651858. Platts-Mills TAE and de Weck AL (1989) Dust mite allergens and asthma: a worldwide problem. Journal of Allergy and Clinical Immunology 83: 416–427. Platts-Mills TAE, Vervloet D, Thomas WR, Aalberse RC, and Chapman MD (1997) Indoor allergens and asthma: report of the

third international workshop. Journal of Allergy and Clinical Immunology 100(6): S1–S21. Pomes A (2002) Intrinsic properties of allergens and environmental exposure as determinants of allergenicity. Allergy 57: 673–679. Simpson A and Custovic A (2004) Allergen avoidance in the primary prevention of asthma. Current Opinion in Allergy and Clinical Immunology 4: 45–51. Simpson A, Woodcock A, and Custovic A (2001) Housing characteristics and mite allergen levels: to humidity and beyond. Clinical and Experimental Allergy 31: 803–805. Smith AM, Pomes A, and Chapman MD (2000) Molecular biology of indoor allergens. Clinical Reviews in Allergy and Immunology 18: 265–283. Stewart GA and Robinson C (2003) The immunobiology of allergenic peptidases. Clinical and Experimental Allergy 33: 3–6. Thomas WR, Smith WA, Hales BJ, et al. (2002) Characterization and immunobiology of house dust mite allergens. International Archives of Allergy and Immunology 129: 1–18.

Relevant Website http://www.allergen.org – This website provides allergen nomenclature maintained by IUIS Allergen Nomenclature Sub-Committee.

DYNAMICS OF GAS FLOW AND PRESSURE–FLOW RELATIONSHIPS J B Grotberg, University of Michigan, Ann Arbor, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Airflow in the lungs results from an imposed pressure difference between the mouth and the alveoli, both for inspiration and expiration. The airways conducting the flow are a branching network of tubes which makes the relationship between pressure and flow complicated. This article covers the basic information known about these flows and how they relate to engineering principles of fluid mechanics. These features include the effects of Poiseuille flow, boundary layer flow, converging/ diverging flow, flow in curved tubes and turbulent flow. Different aspects of these flows tend to dominate depending on the flow speed or Reynolds number. The concepts are integrated into a discussion of the overall flow versus pressure relationship over a wide range of Reynolds numbers. In addition, during forced expiration the outflow can become limited as a result of partial collapse of the flexible airways. The basis for this phenomenon is also presented.

Introduction The lung airways are a branching network of tubes and each level of branching is called a generation (see Figure 1). There are n ¼ 23 generations of branches, each essentially a bifurcation of one parent airway

into two daughter airways. So at any generation there are approximately 2n tubes. The trachea is the first, and largest tube, and it is the n ¼ 0 generation. The tubes decrease in length and diameter as n increases (see Table 1). Because of this structure it is useful to review first some basic aspects of gas flow in a single tube. The mechanics of flow pertains to any fluid, which includes gases and liquids. So this subject falls within the interdisciplinary field of biofluid mechanics which joins together the physics/engineering discipline of fluid mechanics with biosciences. Early contributions to biofluid mechanics in many ways stemmed from this interest in defining the pressure–flow relationship in the lung as a means of detecting disease, monitoring gas exchange, designing respiratory devices, delivering aerosol medications, predicting toxin deposition, and advancing respiratory therapy.

Pressure–Flow Relationships in a Single Tube Poiseuille Flow

Consider steady fluid flow in a tube of diameter d. This particular velocity profile does not change with axial position, x, so is said to be fully developed. It

60

DYNAMICS OF GAS FLOW AND PRESSURE–FLOW RELATIONSHIPS

Trachea n=0

n=1

n=2 n=3

Figure 1 Lung airways as a branching network of tubes. Reproduced with permission from SOMSO Modelle (www.somso.de). & Copyright by SOMSO Modelle. Table 1 Weibel airway geometry data Generation n

Number of airways

Diameter (cm)

Length (cm)

Total X–S (cm2)

Re @ 500 cc s
1

LE =LA

0 1 2 3 4 5 6 7 8 9 10 15 20

1 2 4 8 16 32 64 128 256 512 1 024 32 768 1 048 576

1.800 1.220 0.830 0.560 0.450 0.350 0.280 0.230 0.186 0.154 0.130 0.066 0.045

12.000 4.760 1.900 1.760 1.270 1.070 0.900 0.760 0.640 0.540 0.460 0.200 0.083

2.54 2.33 2.13 2.00 2.48 3.11 3.96 5.10 6.95 9.56 13.40 113.00 1600.00

2362.20 1745.35 1298.90 933.33 604.84 375.13 235.69 150.33 89.21 53.70 32.34 1.95 0.09

10.629 13.420 17.022 8.909 6.429 3.681 2.199 1.364 0.777 0.459 NA NA NA

has a parabolic shape and is known as Poiseuille flow. The relationship between the pressure drop, ’ is the P1
P2 ¼ DP, and the volumetric flow rate, V, well-known Poiseuille law
 128mL ’ DPP ¼ V ½1 pd4 where m is the fluid viscosity, L is the distance in x over which the pressure drop pertains, and d is the tube diameter. Since the tube has a constant diameter for all x, there is no local acceleration (if the tube narrows) or deceleration (if the tube widens) and DP is due entirely to viscous dissipation or resistance. Resistance, R, for any type of tube flow is defined generally as the ratio of the pressure drop from vis’ cous dissipation, DPV , to the flow rate ðR ¼ DPV =VÞ. The resistance for Poiseuille flow, RP , is then RP ¼

DPP 128mL ¼ pd4 V’

½2

Often pressure–flow relationships are given in dimensionless form by dividing the viscous pressure drop by (1/2)rU2, where U is the cross-sectional

92 13 49 091 388 221 776 809 788 399

average velocity. At any value of n, the cross-sectional ’ n , where An is the crossaverage velocity is Un ¼ V=A sectional area listed in Table 1. This dimensionless coefficient of friction, CF , for Poiseuille flow is C FP ¼

DPV L 1 ¼ 64 2 d Re ð1=2ÞrU

½3

where Re is the Reynolds number ðRe ¼ rUd=mÞ. Some authors prefer to make their pressure drop measurements or calculations dimensionless by dividing them by DPP. Then they are assured of results whose values must be greater than or equal to unity. Entrance Flow

For inspiratory air flow in the lung, it is more likely that Poiseuille flow is not present in the larger airways where most of the resistance occurs. Instead, from the flow division at each branch, we expect entrance flow where the velocity profile is x-dependent. The flow enters a long tube and has a flat velocity profile at x ¼ 0. Further down the tube, a boundary layer dðxÞ grows along the wall as the viscous retarding forces

DYNAMICS OF GAS FLOW AND PRESSURE–FLOW RELATIONSHIPS 61

slow the fluid in this region. If the tube is long enough, eventually the velocity profile becomes parabolic (Poiseuille) at a distance LE from the entrance. LE depends on the Reynolds number, a reasonable approximation to this dependence is LE =d ¼ 0:03Re which holds for laminar flow rates where 50o Reo2300. The important issue for flows in the lung is that the entrance length, LE, is usually longer than the airway length, LA, in the larger airways where the major resistance to flow exists. This can be seen in Table 1 where the Reynolds number for V’ ¼ 500 cm3 s
1 is calculated at each n. Multiplying each Re value by 0.03d yields LE and the last column is the ratio LE =LA . Only at generation 8 does there start to be a ratio less than unity, and the calculation is not applicable (NA) when Reo50. The coefficient of friction for entrance flow is given by
1=2 DPE L 1 CFE ¼ ¼ b ½4 d ð1=2ÞrU2 Re1=2

circumstances, the Re
1/2 term in eqn [5] is much larger than the Re
1 term when Rec100, which is fairly common in the large airways. So in this regime, both entrance flow and curved tube flow have an Re
1/2 dependence on CF .

where the constant bB6 for a uniform tube. In dimensional terms, this relationship shows that DPE BV’ 3=2 , which is a stronger dependence on flow rate than the linear relationship for Poiseuille flow.

where the term 0.04 for the rough tube is an estimate when the height of the roughness is B1/100 the tube diameter. These relationships yield DPTsmooth BV’ 7=4 and DPTrough BV’ 2 , which are stronger dependences ’ or enon flow rate compared to either Poiseuille (V) trance (V’ 3=2 ) flows.

Flow in a Curved Tube

Airway bifurcations have axial curvature along the flow path with radius of curvature Rax. Centrifugal forces preferentially throw the faster moving fluid of the midline toward the outer wall of the curve. This skews the axial velocity profile toward the outer wall compared to an equivalent, symmetric Poiseuille flow. The same mechanism creates a pair of vortices in the cross section. Individual fluid particles, then, move in a helical pattern down the tube. The viscous pressure drop for fully developed flow in curved tubes depends on the Dean number, De ¼ ððd=2Þ=Rax Þ1=2 Re=2, which involves the ratio of the tube radius to the axial radius of curvature, d/2Rax and the Reynolds number. A studied relationship for the ratio of the curved tube pressure drop, DPC , to the equivalent Poiseuille pressure drop is DPC =DPP ¼ 0:509 þ 0:0918De1=2 . Rewritten in terms of the coefficient of friction results in CFC

DPC ¼ ð1=2ÞrU2

!
 L d=2 1=4
1=2
1 0:509Re þ 0:0918 ¼ 64 Re d Rax ½5 Typical values of d=2Rax for airways can be B1/6, depending on the generation. Under these

Turbulent Flow

When the Reynolds numbers are large enough, the flow becomes turbulent. The critical value of Re that separates laminar from turbulent flow in a long, smooth tube is Re ¼ 2300. However, it can be a lower value in the airways where the tubes are aerodynamically short and the geometry is varied. For turbulent flow in a straight tube, the pressure drop depends on whether the tube walls are smooth or have roughness. The respective frictional coefficients are given by DPTsmooth ¼ 0:32ðL=dÞRe
1=4 ð1=2ÞrU2 DPTrough ¼ ¼ 0:04ðL=dÞ ð1=2ÞrU2

ðCFT Þsmooth ¼ ðCFT Þrough

½6

Convergent and Divergent Flow

Because the total cross-sectional area changes with generation n, the flow can be experiencing a convergence (decreasing area) or divergence (increasing area). There is a local minimum in cross-sectional area at n ¼ 3 (see Table 1). In divergent flow the profile becomes more pointed as Re increases. For Re high enough there can be backward flow near the walls. For convergent flow, the profile becomes more blunted as Re increases, and the velocity gradient near the wall is steeper. For large Re, it becomes a boundary layer whose thickness matches that of entrance flow. By similar means the coefficient of friction has an Re
1/2 dependence, as it does in eqn [4]. It also depends on the change in cross-sectional area or, equivalently, the convergence angle, a: CFconv ¼

DPconv B ðaReÞ
1=2 ð1=2ÞrU2

½7

Inspiratory Gas Flow at an Airway Bifurcation ’ results from a The total ventilation gas flow rate, V, combination of the pressures imposed at the alveolar and tracheal ends of the respiratory system and its

62

DYNAMICS OF GAS FLOW AND PRESSURE–FLOW RELATIONSHIPS

resistance to the flow. At a typical bifurcation, the flow in the parent tube splits at the carina and a complicated flow in the daughter tubes results. The velocity profile in the daughters is a developing flow, and because of the axial curvature there are secondary swirling flows. Detailed studies have used the structure of entrance-flow viscous-pressure drop, as given in eqn [4], for inspiratory flowpffiffiffiinto a model airway bifurcation and found b ¼ 8 2C, where C was experimentally determined to be C ¼ 1:85. So for the range of Re explored, bB21, a much stronger dependence than simple entrance flow due to high shear rates in the boundary layer. The total viscous resistance at any generation can be derived by adding up all of the individual airway resistances as parallel components. Using the equation for adding parallel resistances, we find 1 1 1 1 2n R - Rn ¼ n ¼ þ þ?þ ¼ Rn R R R R 2

½8

When this is done for the lung, it is readily seen that the major component of viscous pressure drop occurs within the first 8–10 generations, and that Poiseuille law significantly underestimates the losses. The smaller airways where n410 do not contribute much to the overall pressure drop. They form the ‘silent zone’ of the lung, so called because diseases which affect them in early stages are not readily diagnosed by measuring overall lung resistance. The total viscous resistance of the airway tree is calculated by adding the generational resistances assumed to be in series.

Expiratory Gas Flow at an Airway Bifurcation Expiratory flow at a bifurcation results in two vortex pairs, one pair from each daughter, due to the axial curvature of the daughters. So there are four vortices in the parent tube. The flow is also converging, so there will be a higher level of viscous dissipation in this flow compared to the Poiseuille equivalent due to the effects of convergence and swirling. Both effects have frictional coefficients that depend on Re
1/2.

Total Pressure Drop The total pressure drop is due to kinetic energy changes, DPK, and due to frictional, viscous, effects, DPV, so that DPtotal ¼ DPK þ DPV

½9

It is the viscous pressure drop DPV that we have discussed in all of the flows above. DPK is the kinetic

energy change due to convective acceleration of the fluid because of changes in the cross-sectional area of the conduit. It would tell us that between generation n and n þ 1, 2 Þ ðDPK Þexpiration ¼ ð1=2ÞrðUn2
Unþ1

¼
ðDPK Þinspiration

½10

Other than the region of n ¼ 3, where there is a local minimum in cross-sectional area, generally Anþ1 4An, and from mass conservation we know that Un An ¼ Unþ1 Anþ1 , so Unþ1 oUn for most airways. This implies that DPK o0 for inspiration, whereas DPK 40 for expiration. The magnitude of DPK can be up to B10% of DPV under various conditions.

Coefficient of Friction for an Airway Model The pressure–flow relationships from experiments of inspiratory and expiratory flow in a five-generation, rigid cast of the central airways are shown in Figure 2. Three different gases were used at various flow rates to achieve a wide range of Reynolds numbers. Now the forms of CF discussed above for single tubes become important guides in terms of their dependence on Re. Note that in Figure 2 the region of Reo200 has a slope of
1 in the log–log plot. This is the Re
1 dependence from Poiseuille flow. Then in the range 500oReo1500, the slope is
1/2, as expected when entrance flow dominates for inspiratory flow. For Re44000, the slopes of both inspiratory and expiratory curves are essentially zero, as is found in rough-walled turbulence. We note that the pressure drop at any Reynolds number is larger during expiration than during inspiration. This is due to the converging flow.

Expiratory Flow Limitation During expiratory flow, the flexibility of the airway walls can become a very significant issue in terms of restricting the flow rate. Figure 3(a) shows a flexible tube arrangement for an airway, where one could consider the external pressure, Pext, to be the pleural pressure, and the flow is driven by the difference between the upstream pressure, Pu, and the downstream pressure, Pd. In forced expiration, Pext increases with Pu, so an equivalent system in a tube is to decrease Pd while keeping Pu fixed. As this happens the flow increases until the internal pressure of the tube drops somewhat below the external pressure, Pext. Then the tube partially collapses, as shown in the dashed wall shape of Figure 3(a), decreasing in

DYNAMICS OF GAS FLOW AND PRESSURE–FLOW RELATIONSHIPS 63 30

HeO2

20

SF6O2

op e –1

10 9 8 7 6

Sl

∆P/(1/2
)(V/A)2

Air

Steady expiratory flow

5 4 3

Slo

pe

–1/

2

2

Steady inspiratory flow

7 8 9 102

2

3

5 6 7 8 9 103

4

2

3

4

5

6 7 8 9 104

2

3

Re = (
D/) (V/A) Figure 2 Moody diagram of dimensionless pressure drop vs. Re for inspiration and expiration using three gases in a rigid model of the central airways. Reproduced from Isabey D and Chang HK (1981) Steady and unsteady pressure–flow relationships in central airways. Journal of Applied Physiology 51: 1338–1348, with permission from American Physiological Society.

Pd

Pu

Pext

P, A

V

(a) A/A0 1

P − Pext

0

(b)

V

Rigid tube

(c)

cross-sectional area according to its pressure–area or ‘tube law’ relationship, A(P) shown in Figure 3(b). As Pd is further decreased, the tube reduces its crosssectional area while the average velocity of the flow increases. However, their product, the volumetric ’ does not increase as shown in Figure flow rate, V, ’ 3(c). The fact that VðPÞ in Figure 3(c) has a maximum, where the slope of the curve is zero, can be used to deduce a criterion for flow limitation called the ‘wave speed’ theory. The outcome of the theory is that flow limitation will occur when the local flow speed, U, equals the local wave speed, C, of long fluid-elastic waves that can propagate along the airway: sffiffiffiffi E U¼C¼ ½11 r

Flexible tubes

Pu − Pd

Figure 3 Flow through a flexible tube showing: (a) lateral view of collapsed segment, (b) tube law, and (c) flow limitation.

where E is the specific elastance of the tube, E ¼ AðdP=dAÞ, from tube law shown in Figure 3(b). When U ¼ C, pressure information can no longer propagate upstream since waves carrying the new pressure information are all swept downstream. The implication for lungs is that flow–volume curves at different levels of effort share a common curve toward the end of expiration when flow limitation is achieved and the flow rates are effort independent. Diseases which lower E, and hence C, will lower U at flow limitation, and expiration can be pathologically slow, as in asthma and emphysema. To counteract this, patients often breathe at a much higher functional

64

DYNAMICS OF GAS FLOW AND PRESSURE–FLOW RELATIONSHIPS

residual capacity. This helps to pull outward radially on the airways to keep them from collapsing. Since airway properties and E vary along the network, the most susceptible place for the flow limiting segment seems to be the proximal airways. For example, we expect gas speeds prior to flow limitation to be largest at generation n ¼ 3 where the total cross-sectional area of the network is minimal. So flow limitation is likely near that generation. An interesting feature that can occur during flow limitation in airways is the production of flutter oscillations which are heard as wheezing breath sounds. Wheezing is prevalent in asthma and emphysema patients.

Dyspnea

See also: Alveolar Surface Mechanics. Alveolar Wall Micromechanics. Ventilation: Control; Uneven.

Further Reading Grotberg JB (1994) Pulmonary flow and transport phenomena. Annual Review of Fluid Mechanics 26: 529–571. Grotberg JB (2001) Respiratory fluid mechanics and transport processes. Annual Review of Biomedical Engineering 3: 421– 457. Isabey D and Chang HK (1981) Steady and unsteady pressure-flow relationships in central airways. Journal of Applied Physiology 51: 1338–1348. Pedley TJ (1977) Pulmonary fluid dynamics. Annual Review of Fluid Mechanics 9: 229–274.

see Symptoms of Respiratory Disease: Dyspnea.

E Eicosanoids

Elastin

see Lipid Mediators: Overview.

see Extracellular Matrix: Elastin and Microfibrils.

ENDOMETRIOSIS S A Sahn and J T Huggins, Medical University of South Carolina, Charleston, SC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Thoracic endometriosis is a clinical syndrome whereby ectopic endometrial tissue is deposited in thoracic structures. Growth of endometriotic implants is dependent on the presence of ovarian steroids. The four major presentations of thoracic endometriosis include catamenial pneumothorax, catamenial hemothorax, catamenial hemoptysis, and asymptomatic pulmonary nodules. Catamenial pneumothorax accounts for 73% of the cases of thoracic endometriosis described. Symptoms occur within 24–48 h after the onset of menses. Current theories on the pathogenesis of thoracic endometriosis involve microembolization of endometrial tissue from pelvic veins and the movement of endometrial tissue into the pleural space via congenital diaphragmatic defects. The latter mechanism explains the right-side predilection of catamenial pneumothorax. The diagnosis is typically established clinically, as pathologic documentation is generally not required. The diagnosis rarely can be confirmed by pleural fluid cytology, cytology from bronchoscopic aspiration, and needle aspiration of lung nodules. Elevated levels of serum CA-125 and measurements of endometrial antibody have been reported in patients with thoracic endometriosis. Treatment consists of suppression of the ectopic endometrium by interfering with ovarian estrogen secretion. Despite estrogen suppression, recurrence rates remain high, and pleurodesis should be considered in those who develop recurrent pneumothorax or hemothorax.

Introduction Endometriosis is defined as the presence of endometrial glands and stroma outside of the uterus. This clinical entity is most often diagnosed in women between the ages of 25 and 35 years. The prevalence of endometriosis in the general population is not known. The incidence of pelvic endometriosis

is estimated at 1% for women undergoing surgery for all gynecologic indications but may be as high as 50% if the indication for surgery is to evaluate the cause for pelvic pain or infertility. Growth and maintenance of endometriotic implants are dependent upon the presence of ovarian steroids. Endometriosis most commonly involves structures within the pelvis resulting in dysmenorrhea, dyspareunia, and infertility. Endometrial tissue can occur outside the pelvis and has been described in the abdomen, central nervous system, skin, aorta, and thoracic cavity. In 1958, Maurer and colleagues were the first to report thoracic endometriosis in a patient with recurrent spontaneous pneumothorax associated with menstruation. At thoracostomy, their patient was found to have a normal-appearing lung; however, the right hemidiaphragm had a notable defect, which was studded with endometrial tissue. They postulated that air entered the peritoneal cavity by way of the genital tract during menses. Air within the peritoneal cavity could transverse into the pleural space via the diaphragmatic defect to induce a pneumothorax. Since this initial description, thoracic endometriosis remains a rare disease with varying clinical presentations. To date, there are slightly more than 100 cases reported in the medical literature. The most common presentation of thoracic endometriosis is right-side pneumothorax. Hemothorax, hemoptysis, chest pain, and asymptomatic pulmonary nodule occur less frequently. The symptoms of thoracic endometriosis are catamenial. ‘Catamenial’ is a Greek word meaning monthly or periodic. Symptoms occur with 24–48 h after the onset of menses. Chest pain is the most common symptom occurring in 90% of patients with thoracic endometriosis.

66

ENDOMETRIOSIS

Successful treatment requires both eradication and suppression of existing thoracic endometrial tissue and prevention of reseeding from the pelvis. Suppressing ovulation using oral contraceptives, progestins, danazol, or gonadotropin-releasing hormone (GNRH) analogs are currently the most accepted medical strategies. Hysterectomy with bilateral salpingo-oophorectomy should be considered only in the patient who is also suffering from disabling endometriosis–pelvic pain. Despite estrogen suppression, pneumothorax recurrence approaches 50%. Pleurodesis should be considered in those who develop a recurrent pneumothorax or hemothorax.

Etiology The causative and genetic factors for endometriosis are not well understood. Growth and maintenance of endometrial tissue is dependent upon ovarian steroids. Previous epidemiologic studies have suggested that delayed pregnancy is a risk factor for the development of endometriosis. Therefore, Caucasian women are reported to have a higher occurrence of endometriosis compared to other races. This trend, however, was a result of this group having greater access to medical care. Most believe there is no difference between the incidence of endometriosis in different ethnic groups. Genetic factors probably impact a woman’s susceptibility to endometriosis. A familial tendency for endometriosis has been recognized, and concordance in twins has been observed. Multiple genes interacting with environmental triggers are probably operative in conferring the disease. However, future studies are needed to identify the genes and environmental triggers involved in pathogenesis.

Pathology Thoracic endometriosis can be classified according to its clinical presentation. These clinical presentations include: (1) catamenial pneumothorax; (2) catamenial hemothorax; (3) catamenial hemoptysis;

(4) asymptomatic pulmonary nodules; and (5) aortic dissection. Histopathological diagnosis of thoracic endometriosis involves identifying endometrial glands and stroma within the thoracic cavity. Usually, histopathologic documentation of thoracic endometrial tissue is not required and signs are catamenial.

Clinical Features Of the 111 cases of thoracic endometriosis reported in the English language since 1958, catamenial pneumothorax is by far the most common presentation of thoracic endometriosis occurring in 80 (73%). Catamenial hemothorax occurred in 15 (14%), catamenial hemoptysis in 8 (7%), lung nodules in 7 (5%), and aortic dissection in 1 (o1%) (Figure 1). Thoracic endometriosis most commonly presents in premenopausal women with a mean age of 35 years and a reported age range of 19 to 54 years. Postmenopausal women on hormonal replacement therapy can also develop thoracic endometriosis. The vast majority of women with thoracic endometriosis will have evidence of pelvic endometriosis. In 15% of cases, pelvic endometriosis will be lacking in those with thoracic involvement. In catamenial pneumothorax, endometriotic implants are seen in less than 15% of patients examined by thoracoscopy or thoracostomy. The symptoms of thoracic endometriosis are catamenial, presenting in 24–48 h after the onset of menses. Chest pain is the most common symptom, occurring in 90% of patients with catamenial pneumothorax. In more than 90% of cases of catamenial pneumothorax, the pneumothorax is seen on the right side. Two cases of bilateral pneumothorax and one of isolated left-side occurrence has been reported. There should be a high index of suspicion for thoracic endometriosis in women of childbearing age who present with recurrent chest pain, pneumothorax, or hemoptysis. The diagnosis is established clinically; however, it is often delayed until multiple episodes have occurred as the patient and clinician fail to recognize the association between symptoms and the onset of menses.

<1% 14%

7%

5%

n = 111 Pneumothorax Hemothorax

73%

Hemoptysis Lung nodules Aortic dissection

Figure 1 Clinical syndromes of thoracic endometriosis. Major manifestation of thoracic endometriosis is a right-sided pneumothorax.

ENDOMETRIOSIS 67

Although a histopathologic documentation of thoracic endometriosis is not required, there have been reports of the diagnosis being established by cytology of pleural fluid, bronchoscopic aspirates, and needle aspiration of lung nodules. Elevated serum CA-125 (elevated CA-125 serum levels are typically seen in ovarian cancer and occasionally in benign disorders such as endometriosis) and endometrial antibody have been reported in patients with thoracic endometriosis. Pleural and pulmonary endometriotic implants have been described on computed tomography and magnetic resonance imaging scans. These scans may be negative unless obtained during menses. Bronchial arteriography has been used to establish the diagnosis in a few women. Regardless of the radiographic modality used, the appearance of thoracic endometriosis is not specific, and the diagnosis remains indirect. Thoracoscopic findings may consist of diaphragmatic perforations and pleural implants which vary in size depending on the time of menstruation. The diaphragmatic perforations vary in size from 1 mm to 2 cm and are found predominately on the right diaphragm. Macroscopically, the endometriotic implants appear as brown–yellow and sometimes red nodules surrounded by neovascularization. Although these ‘gunshot’ lesions are diagnostic of endometrial foci, pleural biopsy should be performed to confirm the diagnosis.

cells may lead to the proliferation of endometriotic implants. Endometriosis in locations outside the pelvis is explained by dissemination of endometrial cells through pelvic lymphatics and veins. This microembolization phenomenon explains why endometrial tissue can be found in locations like the skin, central nervous system, vertebrae, and aorta. However, in catamenial pneumothorax, more than 90% of cases occur on the right side, suggesting another plausible mechanism for this form of thoracic endometriosis. Congenital diaphragmatic holes are more commonly found on the right. These holes allow for a direct peritoneal–pleural communication, thereby allowing for endometrial tissue to preferentially migrate into the right pleural space. However, fewer than 25% of patients have visible thoracic implants during thoracoscopy. Others have theorized that air originates from abdominal endometriosis which can then move preferentially through these diaphragmatic defects into the pleural space. Less-accepted theories involve high circulating levels of prostaglandin F2 during menstruation causing bronchoconstriction and leading to alveolar rupture and pneumothorax. Another speculates that swelling of intrapulmonary endometriotic implants at menses causes airway obstruction resulting in alveolar wall rupture and pneumothorax. Neither of theses theories, however, explains the right-side predilection for catamenial pneumothorax.

Pathogenesis Several theories have been proposed to explain the pathogenesis of endometriosis. The coelomic metaplasia theory proposes that undifferentiated cells within the peritoneal cavity are capable of dedifferentiating into endometrial tissue. The implantation theory states that endometrial tissue from the uterus shed during menstruation is transported by the fallopian tubes and implants on pelvic structures. Another theory involves retrograde menstruation. This theory has been supported by the observation that females with genital tract obstruction have an increased likelihood of tubal reflux; however, the incidence of retrograde menstruation is similar among those with and without endometriosis. Therefore, the development of endometriosis may be dependent upon the quantity of endometrial tissue reaching the peritoneal cavity as well as the capacity of the immune system to recognize and destroy the displaced endometrial cells. Some authors have speculated that altered immunity may play a role in the development of endometriosis. An inability of cellular immunity to recognize the presence of endometrial tissue outside the uterus may be pivotal. Various cytokines and proinflammatory

Animal Models Animal models have shown that intravenous injected endometrial tissue remain viable, invade lung parenchyma, and undergo cyclic changes during the menstrual cycle.

Management and Current Therapy Successful treatment requires both eradication and suppression of existing thoracic endometrial tissue and prevention of reseeding from the pelvis. Medical treatment, the primary modality, consists of suppression of the ectopic endometrium by interfering with ovarian estrogen secretion. This can be accomplished by suppressing ovulation using oral contraceptives, progestins, danazol, or GNRH analogs. We do not routinely recommend hysterectomy with bilateral salpingo-oophorectomy unless the patient is also suffering from disabling endometriosis–pelvic pain. Progestins cause decidualization of endometrial tissue. Adverse side effects are problematic and include nausea, fluid retention, abnormal uterine bleeding, and depression.

68

ENDOMETRIOSIS

Danazol, an isoxazol derivative of 17a-ethinyltesterone, induces anovulation by attenuating the mid-cycle surge of luteinizing hormone secretion, increasing free testosterone concentration, and inhibiting multiple enzymes in the steroid pathway. The recommended dose of danazol in the treatment of endometriosis is 600–800 mg daily. At these doses, danazol has significant androgenic side effects, and irreversible liver toxicity has been reported. GNRH agonists decrease the secretion of follicle stimulating hormone and luteinizing hormone, resulting in hypogonadotropic hypogonadism. A persistent hypoestrogenic state ensues resulting in endometrial tissue atrophy and amenorrhea. Adverse effects associated with GNRH agonists include vaginal bleeding, hot flashes, decreased libido, depression, osteoporosis, and sleep disturbance. All medical regimens appear to be effective in the treatment of endometriosis. Several case reports have shown that GNRH agonists were effective in the treatment of thoracic endometriosis when oral contraceptives had failed. Nevertheless, recurrence

rates are as high as 50% with hormonal therapy. This suggests that endometrial implant regression is not complete and/or that recurrent embolization continues. The management of catamenial pneumothorax is similar to the acute management of other forms of spontaneous pneumothorax. Observation is appropriate for a small, asymptomatic pneumothorax. Catheter aspiration or closed tube thoracostomy should be considered for patients with symptoms or larger pneumothoraces. With these conservative approaches, pneumothorax recurrence will occur in all who do not receive appropriate estrogen suppression therapy. Moreover, despite estrogen suppression, pneumothorax recurrence approaches 50% over the next 18 months. Therefore, pneumothorax prevention strategies should be considered in those who develop a recurrent pneumothorax. Chemical pleurodesis (via tube thoracostomy, thoracoscopic talc poudrage, or surgical pleurodesis using pleural abrasion and partial parietal

Anovulatory medications Examples: Progestins, oral contraceptives, danazol, GNRH agonists Effective Continue anovulatory medications

Yes

Recurrent catamenial pneumothorax or hemothorax (50% recurrence in first 18 months) Chemical or surgical pleurodesis

Catamenial chest pain

Anovulatory medications Examples : Progestins, oral contraceptives, danazol, or GNRH agonists Figure 2 Management algorithm for thoracic endometriosis.

Yes

Continue anovulatory medications

Recurrent hemoptysis

Surgical or laser resection of endobronchial implants

ENDOTHELIAL CELLS AND ENDOTHELIUM 69

pleurectomy with or without talc poudrage) is highly effective in preventing catamenial pneumothorax and hemothorax. However, patients may continue to experience catamenial chest pain despite these therapeutic interventions. Catamenial chest pain is presumed to be due to cyclic proliferation of pleuropulmonary endometriotic implants in response to ovarian steroids. These symptoms can be relieved with hormonal suppression but recur if estrogen replacement therapy is initiated. It is well recognized that women suffering from thoracic endometriosis are in their reproductive years and may wish to conceive children. The use of hormonal ablation strategies would inhibit conception. On the other hand, pregnancy would be a protective state in preventing the occurrence of a pneumothorax. In women with thoracic endometriosis who are considering pregnancy, a pleurodesis procedure can be performed prior to discontinuation of estrogen suppression. Surgical resection of intraparenchymal implants and neodymium–yttrium aluminum garnet (Nd– YAG) laser resection of endobronchial implants have been successful in a limited number of reports. These strategies should be entertained in those with limited disease who have recurrent hemoptysis despite appropriate estrogen suppression (Figure 2).

See also: Pleural Effusions: Hemothorax. Pneumothorax. Symptoms of Respiratory Disease: Chest Pain.

Further Reading Elliot DL, Barker AF, and Dixon LM (1985) Catamenial hemoptysis: new methods of diagnosis and therapy. Chest 87: 687–688. Fonseca P (1998) Catamenial pneumothorax: a multifactorial etiology. Journal of Thoracic and Cardiovascular Surgery 116: 872–873. Hibbard LT, Schumann WR, and Goldstein GE (1981) Thoracic endometriosis: a review and report of two cases. American Journal of Obstetrics Gynecology 140: 227–232. Joseph J and Sahn SA (1996) Thoracic endometriosis syndrome: new observations from an analysis of 110 cases. American Journal of Medicine 100: 164–170. Lillington GA, Mitchell SP, and Wood GA (1972) Catamenial pneumothorax. Journal of the American Medical Association 219: 1328–1332. Olive DL and Pritts EA (2001) Treatment of endometriosis. New England Journal of Medicine 345: 266–275. Olive DL and Schwartz LB (1993) Endometriosis. New England Journal of Medicine 328: 1759–1769. Sahn SA (2004) Thoracic endometriosis. Up to Date American Thoracic Society (electronic media). Wilkins SB, Bell-Thomson J, and Tyras DH (1985) Hemothorax associated with endometriosis. Journal of Thoracic and Cardiovascular Surgery 89: 636–638. Wood DJ, Krishnan K, Stocks P, et al. (1993) Catamenial haemoptysis: a rare cause. Thorax 48: 1048–1049. Yamazaki S, Ogawa J, Koide S, et al. (1980) Catamenial pneumothorax associated with endometriosis of the diaphragm. Chest 77: 107–109.

ENDOTHELIAL CELLS AND ENDOTHELIUM A Hislop, Institute of Child Health, London, UK B Wojciak-Stothard, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Endothelial dysfunction has been implicated in several pulmonary disorders including pulmonary arterial hypertension, lung edema, acute lung injury, and acute respiratory distress syndrome (ARDS). Current research aims to identify signaling pathways leading to endothelial malfunction in search for new effective therapeutic strategies.

Abstract The pulmonary endothelium is a single-cell layer forming the inner lining of a vast network of pulmonary arteries, veins, and capillaries. All circulating blood passes through the lungs before it enters the systemic circulation, thus the endothelium can be regarded as an endocrine organ for the whole body. In addition to allowing gas exchange the endothelium has a number of other functions. It acts as a semipermeable barrier between air and blood, regulating fluid balance between the capillaries and alveolar space. The endothelial cells synthesize and transport to the smooth muscle cells relaxant and contractile mediators maintaining tone in the pulmonary circulation. It is involved in extravasation of white blood cells to areas of inflammation and regulates the coagulation process important to prevent vessel injury. The agents produced by the endothelial cells have multiple tasks and there is a carefully orchestrated interaction.

Introduction The pulmonary endothelium made up of a continuous sheet of endothelial cells is a large organ: its surface area is approximately 90 m2 (the size of a tennis court) and it lines all the blood vessels in the lung. It acts as a semipermeable barrier between the blood and the surrounding tissues regulating bidirectional exchange of fluids, gases, nutrients, and other metabolic mediators. Endothelial cells produce growth factors and substances that control vascular tone and are involved in the inflammatory and coagulant processes. Since the entire circulating blood passes

70

ENDOTHELIAL CELLS AND ENDOTHELIUM

through the lungs before it enters the systemic circulation, the endothelium can be regarded as an endocrine organ for the whole body. Endothelial cells are first in line to detect and respond to changes in blood composition and flow.

Structural Considerations The pulmonary circulation consists of three vascular segments that are arranged in series: the arteries, the microvessels or capillaries, and the veins. A second circulation in the lung, the bronchial circulation which is part of the systemic system, supplies the walls of airways and large pulmonary vessels with oxygenated blood. The pulmonary endothelial cell population is heterogeneous, and structural and functional differences exist between cells from arteries and veins, between cells from large and small vessels, and between cells within the same vascular region. Generally, the endothelial cell is flattened and is only 1–2 mm thick and about 10–20 mm in diameter. They are barely visible with the light microscope but a nucleus can be identified centrally at its thickest point. Ultrastructural studies are needed to describe the cell. It sits on a basement membrane and each cell is attached to its neighbor by tight junctions (Figure 1). These junctions are connected to the actin-based myofilament system in the cytoplasm which in the resting cell is located around the cell periphery. The surface is covered by many pinocytotic vesicles first described by Palade as plasmalemmal vesicles and now called caveolae. They are involved in transporting substances such as albumin from the circulating blood into the interstitium of the alveolar wall. The greatest density of caveolae is found in the alveolar capillaries where 20 000 caveolae per cell effectively double the surface area of the cell. Weibel–Palade bodies, originally called rod shaped bodies, are found sparsely through the cytoplasm and more are found in arterial than capillary endothelium. They store P-selectin involved in leukocyte adhesion and von Willebrand factor (vWF) involved in platelet aggregation. Endothelial cells are formed by vasculogenesis from mesothelial cells surrounding the epithelial lung bud which become either hematopoietic cells or endothelial cells which join first to form capillary tubes and then coalesce to form arteries and veins. This differentiation is the result of vascular endothelial growth factor (VEGF) produced by the epithelial cells. The airways impose organization on the randomly forming endothelial tubes and thus the preacinar pulmonary arteries and veins and airways develop together during early fetal life. Endothelial cells facilitate vessel differentiation and maturation

by secreting factors promoting smooth muscle cell proliferation and migration such as angiopoietins, endothelin, transforming growth factor beta (TGFb), and platelet-derived growth factor (PDGF). Later, in fetal life and during childhood, peripheral vessels form by angiogenesis, by sprouting from the existing blood vessels as the capillary bed forms within the alveolar walls, mediated by VEGF. During fetal life, in the peripheral vessels, the endothelial cells are not flattened and obstruct the lumen increasing resistance to flow. After birth, flattening their shape helps to increase blood flow to the lung. At the same time, fluid is removed from the lung via the epithelial and endothelial cells.

Functions of the Endothelium in the Normal Lung In the alveolar region, endothelial cells are primarily part of the blood gas barrier for diffusion of gases between blood and air. However, other functions may be equally as important. These include maintenance of vascular tone, barrier function, coagulation, leukocyte trafficking, production of growth factors, and cell signals with autocrine and paracrine effects (Figure 2). Regulation of Pulmonary Vascular Resistance

The lung is a low-pressure system but there is a requirement to maintain tone in the arteries and veins. The pulmonary endothelium secretes a range of vasoconstrictors and vasorelaxants that by interacting regulate pulmonary vascular resistance (PVR) or tone. In the adult lung, the major sites of resistance appear to be the small arteries and capillaries; however, larger arteries and veins may also play a role. When oxygen tension is low, the small vessels respond by vasoconstriction a reaction different to that seen in systemic vasculature. The vasoconstriction allows blood to be diverted away from areas of low oxygen to areas of higher oxygen for the gas exchange to take place. Factors that stimulate release of vasoconstrictors also cause release of vasodilators and thus the overall response is a complex interaction. The main mediators of tone are described. Nitric oxide Nitric oxide (NO) or endotheliumderived relaxing factor (EDRF) is a powerful vasorelaxant of vascular smooth muscle cells. It is a free-radical gas produced by the vascular endothelium during conversion of L-arginine to L-citrulline by the enzyme NO synthase (NOS). Both constitutive endothelial NOS (eNOS) and inducible NOS (iNOS) are produced by the endothelial cells. eNOS is

ENDOTHELIAL CELLS AND ENDOTHELIUM 71

ctm

m

I

Nucleus

rbc

j I

bm

0.75 µm

Alveolar space (a)

c

m c

v

bm I 0.25 µm (b) 5 µm

m wp

wp

j j j el

coll smc (c) Figure 1 (a) Capillary in the alveolar wall of a normal human lung. The flattened endothelial cell lines the vessel giving a thin blood gas barrier. bm, basement membrane; ctm, connective tissue matrix; j, tight cell junction; m, mitochondrion; I, epithelial type I pneumonocyte; rbc, red blood cell. (b) High-power micrograph of the endothelial cell showing numerous caveolae (c) on both surfaces and vesicles (v) within the cytoplasm. bm, basement membrane; m, mitochondrion; I, epithelial type I pneumonocyte. (c) Endothelial cell from a porcine small muscular artery. The vessel is not distended. el, elastic lamina between endothelial cells and the muscle wall; coll, collagen fibrils in the matrix; smc, smooth muscle cell; j, tight junction; wp, Weibel–Palade body; m-mitochondrion. All electron micrographs courtesy of Ann Dewar, NHLI.

located in the caveolae. NO production in endothelial cells is influenced by a variety of factors, including shear stress, oxygen tension, humoral factors acting via receptors, and growth factors. At a cellular level, NO production is regulated by transcription, posttranscriptional modification, substrate availability, intracellular localization, superoxide production, and cofactor availability. NO diffuses into the

smooth muscle cell where it stimulates soluble guanylate cyclase to form cGMP leading to relaxation. Hypoxia-induced pulmonary vasoconstriction and pulmonary hypertension are associated with decreasing production of NO by endothelial cells. Apart from acting as a potent vasodilator, low concentrations of NO inhibit smooth muscle cell proliferation, and prevent inflammatory responses, platelet

72

ENDOTHELIAL CELLS AND ENDOTHELIUM

Humoral factors

Physical factors

Inflammatory mediators

Acetylcholine Bradykinin

Shear stress, stretch, hypoxia

Histamine, thrombin, TNF


Paracellular permeability

Adhesion Antithrombotic

Platelets

Sepsis NO

Caveolin

Endothelium

Transcytosis NO

Fluid

PGI2

PGI2

ET-1 TxA

Myofilaments

Selectins

Neutrophils

ICAM, VCAM

PAF vWF

PECAM

Intercellular space

Matrix Dilatation

SMC

Constriction

SMC

Barrier function

SMC

Vascular tone

Host defense

Figure 2 Diagram illustrating some of the main functions of the pulmonary endothelial cells. Under normal conditions, the endothelial cells are influenced by humoral factors in the blood and the flow of blood. With an increase inflow and thus shear stress, the presence of hypoxemia, or inflammatory mediators, there is a change in balance of production of vasoactive mediators, an increase in permeability, and upregulation of adhesion molecules with platelet and neutrophil activation leading to cell migration. ET-1, endothelin-1; ICAM, intercellular adhesion molecule; NO, nitric oxide; PAF, platelet-activating factor; PECAM, platelet endothelial cell intercellular adhesion protein; PGI2, prostaglandin I2; SMC, smooth muscle cell; TNF-a, tumor necrosis factor alpha, TxA, thromboxane; VCAM, vascular cell adhesion molecule; vWF, von Willebrand factor.

and leukocyte adhesion, and increased endothelial leakage associated with pulmonary hypertension (see below). Prostacyclin Prostacyclin (Prostaglandin I2, PGI2) is a powerful vasodilator of both systemic and pulmonary vascular beds produced constitutively by the endothelium but increased in response to receptor stimulation or shear stress. It is derived from arachidonic acid, by the action of cyclooxygenases COX-1 and COX-2 and diffuses to the smooth muscle cells. The PGI2 receptor belongs to the family of G-protein-coupled receptors and its effects are mediated by cAMP. The levels of PGI2 during early fetal life are low and increase with the onset of breathing air. PGI2 also inhibits smooth muscle cell proliferation and is anti adhesive. Blood-borne bradykinin which acts through receptors on the endothelial cells causes release of NO and PGI2 and thus is a potent vasodilator in vivo; several other mediators such as potassium channels and S-nitrosothiols have also been proposed.

Thromboxane Thromboxane is another arachidonic acid metabolite produced by cyclooxygenases in endothelial cells and platelets. It is a potent vasoconstrictor, a smooth muscle mitogen, and an inducer of platelet aggregation. An increase in the production of thromboxane A2 metabolites is seen in pulmonary hypertension.

Endothelin-1 Endothelin-1 (ET-1) produced by pulmonary endothelial cells is a potent vasoconstrictor which also stimulates the proliferation of pulmonary smooth muscle cells. Release of ET-1 is stimulated by hypoxia or lung inflammation. There are two types of endothelin receptors in the pulmonary vasculature. ETA and ETB receptors are found on smooth muscle cells where they mediate vasoconstriction. Endothelial cells have only ETB receptors where stimulation induces vasodilation via the release of NO, PGI2, or via ATP-gated K þ channels. The plasma endothelin levels are high in utero and fall rapidly during the first week of life. They are

ENDOTHELIAL CELLS AND ENDOTHELIUM 73

increased in pulmonary arterial hypertension. ET-1 also has pro-inflammatory properties in the lung. Angiotensin Angiotensin II (Ang II), the key effector of the renin–angiotensin system, plays a central role in the regulation of blood pressure and electrolyte homeostasis. Angiotensin-converting enzyme, produced by endothelial cells, hydrolyzes angiotensin I to angiotensin II which acts as a powerful vasoconstrictor of pulmonary vascular smooth muscle cells. The vasoconstricting effects of this peptide are largely due to angiotensin-mediated increases in the production of ET-1. It also enhances proliferation and hypertrophy of smooth muscle cells. Paracellular Permeability and Transcytosis

The pulmonary microcirculation is in close proximity to the gas-exchanging alveolar epithelium. The integrity of both the alveolar endothelial and epithelial barriers is essential for lung fluid balance. After birth, pulmonary endothelium takes part in the clearance of the lung fluid and then endothelial cell junctions tighten to prevent leakage of plasma proteins into the interstitium and airspaces. In the normal lung, the endothelium delivers circulating substances such as albumin through transcellular trafficking of vesicles. Albumin carries a variety of peptides and amino acids from the blood through the endothelial cell via transcytosis into the interstitium. The protein binds to the caveolae on the luminal surface; these internalize, then move through the cell to the interstitium where they are released. Caveolin-1 is a scaffolding protein required for caveolae formation and caveolar transcytosis. In caveolin-1-null mice there is failure of transcytosis. In response to sepsis and intravascular inflammatory mediators such as thrombin, tumor necrosis factor alpha (TNF-a), and histamine, there is increased paracellular permeability through gaps in intercellular junctions resulting in increased protein fluid in the interstitial space. The intercellular gap formation is partly a result of actinomyosin-dependent contraction of endothelial cells. Pulmonary barrier function can also be affected by hypoxia and shear stress, resulting in changes in the levels of circulating VEGF, angiotensin II, and NO. VEGF, originally identified as vascular permeability factor (VPF), is a potent inducer of plasma extravasation. Angiotensin II may affect endothelial permeability via the release of prostaglandins and VEGF. By contrast, an increase in NO has been shown to prevent endothelial leakage in the lung. Changes in the levels and distribution of platelet endothelial cell intercellular adhesion protein PEC AM-1 (CD31) have also been shown to contribute to

the changes in endothelial barrier function. PECAM1 is a scaffolding protein in the intercellular region that also plays a critical role in transendothelial migration of inflammatory cells. Inflammatory Responses

Endothelial cells are normally nonadherent but can help recruit inflammatory cells to sites of tissue injury or infection. On stimulation by damage or infection they produce cytokines, growth factors, and adhesion molecules which allow leukocytes to leave the flowing blood and to roll along the vessel wall, both the leukocytes and the endothelial cells expressing adhesion molecules E-selectin and P-selectin. The leukocytes adhere to the endothelial cells depending upon upregulation of B2 integrins and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1), and finally migrate through the cell junctions via action of PECAM-1 which in the normal cell is concentrated at intercellular junctions. The cells move through the junction while the integrity of the endothelium is maintained. Coagulation

The pulmonary endothelium plays a key role in maintenance of the normal coagulation. The anticoagulant/antithrombotic substances include NO, PGI2, and thrombomodulin. After damage the procoagulant/prothrombotic factors increase these include platelet-activating factor which promotes platelet adhesion and vWF which is made by the endothelial cells and stored for rapid release in the Weibel–Palade bodies.

Endothelium in Respiratory Disease Pulmonary Hypertension

Pulmonary arterial hypertension is defined as a sustained elevation of pulmonary arterial pressure to more than 25 mmHg at rest or to more than 30 mmHg with exercise. It may be idiopathic pulmonary arterial hypertension (formerly, primary pulmonary hypertension) or secondary to a variety of lung, heart, or connective tissue disorders. There may also be a lesser increase in pulmonary artery pressure associated with respiratory problems such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis when the periphery of the lung is hypoxic leading to vasoconstriction. The main vascular changes in pulmonary arterial hypertension are an increase in arterial wall thickness due to smooth muscle cell proliferation or hypertrophy, increase in matrix deposition, and pulmonary endothelial cell dysfunction or injury. Pulmonary

74

ENDOTHELIAL CELLS AND ENDOTHELIUM

hypertension is associated with a decrease in prostacyclin production and eNOS and an increase of thromboxane. The plasma levels of ET-1 are increased. These changes will lead to vasoconstriction and a promitogenic system. Endothelial and smooth muscle cell dysfunction has been attributed to the increase in the activity of Rho GTPase RhoA, a protein regulating actin dynamics and actomyosin contractility. Endothelial dysfunction may also contribute to the thrombotic process, a feature of most forms of pulmonary hypertension. Acute Lung Injury/Acute Respiratory Distress Syndrome

Acute lung injury may be caused by a variety of insults including sepsis, trauma, pneumonia, and drug toxicity. There is severe oxygenation impairment, alveolar edema, and eventually pulmonary hypertension. The most extreme form of acute lung injury is acute respiratory distress syndrome (ARDS). The initial cause is an inflammatory process injuring the functional processes and structure of the endothelial cells resulting in the breakdown of gas exchange and an increase in barrier permeability of the endothelium and changes in the levels of vasoactive mediators towards a balance in favor of vasoconstriction. Therapeutic Considerations

Ultimately, our aim will be to control excessive reactivity and structural remodeling and to develop therapeutic agents which exploit the interactions between the signaling pathways controlling these events. Similar treatment of both pulmonary hypertension and ARDS focuses on increasing pulmonary arterial vasodilatation by giving either inhaled or intravenous prostacyclin or inhalation of NO or its enhancement by the use of a phosphodiesterase type 5 inhibitor. More recently, ET-1 receptor antagonists have been used to treat pulmonary hypertension in adults and children. This may effect a decrease in vasoconstriction and some prevention of the muscle cell hyperplasia. Statins might be helpful in treatment of ARDS because they have antileakage and anti-inflammatory effects and lead to an increase in NO production by endothelial cells, as well as increases in heme oxygenase-1 whose enzymic products, carbon monoxide and bilirubin, have potent anti-inflammatory and antioxidant properties. Gene transfer of iNOS is so far only experimental.

See also: Acute Respiratory Distress Syndrome. Angiogenesis, Angiogenic Growth Factors and Development Factors. Anticoagulants. Arteries and Veins. Bronchial Circulation. Caveolins. Chronic Obstructive Pulmonary Disease: Overview. Coagulation Cascade: Antithrombin III. Diffusion of Gases. Endothelins. Epithelial Cells: Type I Cells; Type II Cells. Fluid Balance in the Lung. Hypoxia and Hypoxemia. Kinins and Neuropeptides: Bradykinin. Leukocytes: Neutrophils. Lung Development: Overview. Nitric Oxide and Nitrogen Oxides. Peripheral Gas Exchange. Pulmonary Circulation. Pulmonary Vascular Remodeling. Vascular Disease. Vascular Endothelial Growth Factor.

Further Reading Corrin B (ed.) (2000) Normal lung structure. Pathology of the Lungs, 2nd edn., pp. 1–34. London: Churchill Livingstone. Dudek SM and Garcia JG (2001) Cytoskeletal regulation of pulmonary vascular permeability. Journal of Applied Physiology 91: 1487–1500. Fernandez N, Jancar S, and Sanchez CM (2004) Blood and endothelium in immune complex-mediated tissue injury. Trends in Pharmacological Science 25: 512–517. Galley HF and Webster NR (2004) Physiology of the endothelium. British Journal of Anaesthesia 93: 105–113. Hislop AA (2002) Airway and blood vessel interaction during lung development. Journal of Anatomy 201: 325–334. Jeffery TK and Wanstall JC (2001) Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension. Pharmacological Therapeutics 92: 1–20. Kirchner K, Dobyns EL, and Stenmark KR (1999) Lung cell biology. In: Taussig LM and Landau LI (eds.) Pediatric Respiratory Medicine, pp. 37–56. St Louis: Mosby. Lee TS, Chang CC, Zhu Y, and Shyy JY (2004) Simvastatin induces heme oxygenase-1: a novel mechanism of vessel protection. Ciculation 110: 1296–1302. Mehta D, Bhattacharya J, Matthay MA, and Malik AB (2004) Integrated control of lung fluid balance. American Journal of Physiology 287: L1081–L1090. Michiels C (2003) Endothelial cell functions. Journal of Cell Physiology 196: 430–443. Moloney ED and Evans TW (2003) Pathophysiology and pharmacological treatment of pulmonary hypertension in acute respiratory distress syndrome. European Respiratory Journal 21: 720–727. Orfanos SE, Mavrommati I, Korovesi I, and Roussos C (2004) Pulmonary endothelium in acute lung injury: from basic science to the critically ill. Intensive Care Medicine 30: 1702–1714. Piantadosi CA and Schwartz DA (2004) The acute respiratory distress syndrome. Annals of Internal Medicine 141: 460–470. Predescu D, Vogel SM, and Malik AB (2004) Functional and morphological studies of protein transcytosis in continuous endothelia. American Journal of Physiology 287: L895–L901. Rosenzweig EB, Widlitz AC, and Barst RJ (2004) Pulmonary arterial hypertension in children. Pediatric Pulmonology 38: 2–22. Stevens T, Rosenberg R, Aird W, et al. (2001) NHLBI workshop report: endothelial cell phenotypes in heart, lung, and blood diseases. American Journal of Physiology 281: C1422–C1433.

ENDOTHELINS 75

ENDOTHELINS ranging from diseases of the airways to pulmonary vascular disorders. This greater understanding has allowed for therapeutic manipulation of the endothelin system in vivo. An example of this is the use of endothelin antagonist therapy, which is now licensed for use in pulmonary arterial hypertension.

N Morrell, University of Cambridge School of Clinical Medicine, Cambridge, UK J Suntharalingam, Papworth Hospital, Cambridge, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Structure

Since their initial discovery in 1988, the importance of endothelins (ET) in both health and disease has been increasingly recognized. This family of peptides can be found throughout the body, but are found in particularly high concentrations within the pulmonary system. Here, they are synthesized on demand and act predominantly as paracrine and autocrine agents. Their actions are mediated by two endothelin receptors (ETA and ETB) that are themselves members of the G-protein-coupled receptor family. Although endothelins were initially recognized for their vasoconstrictor effects, they are now known to have mitogenic and immunoregulatory actions as well. The relative contribution of these effects in health is still unclear, but has become better understood in disease. ET-1 has been implicated in the pathogenesis of many pulmonary disorders, including pulmonary hypertension, pulmonary fibrosis, asthma, acute respiratory distress syndrome, lung transplant rejection, and lung cancer. This greater understanding has aided in development of drugs that can target the endothelin system; endothelin receptor antagonist therapy, for example, is now licensed for the treatment of pulmonary arterial hypertension.

The endothelin family consists of three isoforms ET1, ET-2, and ET-3, of which ET-1 is both the most prevalent and the best characterized. Although all three are 21 amino acid peptides and are structurally related, they are coded by genes from different chromosomes (chromosomes 6, 1, and 20 respectively). As Figure 1 shows, ET-1 takes the form of a hairpin loop linked by two intrachain disulfide bridges with a free carboxy-terminus at one end and an amino-terminus at the other. ET-2 and ET-3 have similar forms, but differ from ET-1 by two and six amino acid residues, respectively. The functions of ET-2 and ET-3 in humans are less well characterized, so the rest of this article will concentrate on ET-1.

Regulation of Production and Activity Although ET-1 synthesis occurs within several organs, the pulmonary system is one of the main sites of its production and clearance. Normally, ET-1 is produced predominantly by endothelial cells, with some contribution from airway epithelial and vascular smooth muscle cells. In disease, the relative contribution from these cells may vary and there may be additional production by other cells such as inflammatory cells within the pulmonary system. ET-1 is initially synthesized as the inactive molecule preproET-1, a 212 amino acid peptide which undergoes posttranslational modification to its active

Introduction In 1988, Masashi Yanagisawa and co-workers described an endothelial-derived peptide that caused potent vasospasm. The peptide was cloned, sequenced, and named endothelin (ET), and within a short time, the receptors were also characterized. Since then, work on understanding the role of endothelin has continued at a rapid rate. As a result, it is now accepted that endothelin has a wide range of actions in addition to its effects on vascular tone. It has also been implicated in many pulmonary disorders, Leu

Ser Ser

Met

Cys Ser

Asp

Cys

N

Lys Disulfide bridges

Glu Cys Val

Figure 1 Structure of ET-1.

Tyr

Phe

Cys

His

Leu

Asp

Ile

IIe

Try

C

76

ENDOTHELINS Promoters

Inhibitors

+

−

PreproET-1 Gene

PreproET-1 mRNA

PreproET-1 Peptide (212 AAs)

Endothelial cell

Endopeptidases Nucleus

Big ET-1 (38 AAs) Endothelinconverting enzyme ET-1 (21AA)

ET-1 Figure 2 Synthesis of ET-1.

Table 1 Factors regulating release of ET-1

Biological Function

Increased synthesis of ET-1

Inhibition of ET-1 synthesis

Mechanism of Action

Growth factors (TGF-b, PDGF, EGF) Cytokines (TNF-a, IL-1b, IFN-b) Thrombin Angiotensin II Vasopressin Catecholamines Lipopolysaccharides Hypoxia Shear stress Stretch

Prostacyclin

Only a small proportion of ET-1 is released apically across the luminal surface into vessels. As such, its systemic action as a hormone is limited. Instead, the majority of ET-1 is released basolaterally and acts on neighboring cells in a paracrine manner. Under certain circumstances, it also exerts autocrine effects. ET-1 exerts its actions by binding to the endothelin receptors ETA and ETB. The expression and function of these receptors and their structure are discussed in more detail below.

Adrenomedullin Heparin Nitric oxide Atrial natriuretic peptide

form. At first, preproET-1 is proteolytically cleaved by endopeptidases to produce big ET-1, a 38 amino acid peptide, following which it is further cleaved by soluble ET-converting enzyme (ECE) to produce ET-1 itself (Figure 2). ET-1 is not stored but is synthesized in response to demand. As such, regulation of its activity occurs at the transcriptional level. A wide variety of stimuli have been shown to either promote or inhibit the production of ET-1 (Table 1) and thus influence its biological function. Further levels of regulatory control exist at the level of endothelin receptor expression and subsequent intracellular signal transduction. ET-1 is cleared within both the pulmonary and renal circulations. Clearance is mediated by ETB receptors which bind ET-1 and are subsequently internalized and degraded. In health, there is a balance between pulmonary production of ET-1 and its clearance in such a way that no gradient results across the pulmonary circulation.

Action on Vascular Tone

ET-1 is a potent vasoconstrictor and takes on a prominent role in the process of maintaining pulmonary vascular tone. Much of its contractile action in health is mediated through ETA receptors which are situated on vascular smooth muscle cells found largely within the proximal circulation. However, the smooth muscle cells located within the distal pulmonary circulation also host ETB receptors, which may be increased in numbers in patients with pulmonary hypertension. Binding of ET-1 to these receptors also leads to vasoconstriction directly, and is possibly more relevant during disease. Intriguingly, ETB receptors can also be found on endothelial cells where ligand binding leads to vasodilatation indirectly through the release of factors such as nitric oxide and prostacyclin. Although the net effect of all these actions still allows ET-1 to act predominantly as a vasoconstrictor, the precise interplay between these contractile and relaxant effects is dependent on

ENDOTHELINS 77

the differential expression of ET receptors under different circumstances. Within airways, ET-1 is similarly involved in bronchomotor tone. It has been shown to produce bronchoconstriction in vitro through a direct effect on airway smooth muscle cells. Both ETA and ETB receptors have been implicated, although the latter are thought to be the more dominant receptors in peripheral human airways.

Other Actions

The effects of ET-1 are far-reaching and not just limited to actions on smooth muscle cells and epithelial cells. ET-1, acting as a cytokine, has been shown to have a chemotactic effect on many other cell types and subsequently alter their function. Animal models of eosinophilic inflammation, for example, have shown that ET-1 mRNA expression increases just prior to the influx of inflammatory cells, and that the degree of inflammation seen can be attenuated by the use of endothelin antagonists. Fibroblast migration and activation is also seen in response to ET-1, possibly leading to collagen deposition and altered matrix composition. These, and other actions that have been described, generally tend to show ET-1 to be a promoter of inflammation within the pulmonary system. Some of these effects are due to direct interaction between ET-1 and ET receptors on the cells concerned, whilst others are mediated via the production of other cytokines such as tumor necrosis factor alpha and interleukin-1b. The extent to which these effects play a role in maintaining normal function in the lungs as opposed to playing a pathophysiological role in disease is as yet unclear. Figure 3 summarizes some of the biological functions of ET-1 within the respiratory system.

Mitogenic Actions

ET-1 stimulates DNA synthesis and encourages proliferation of human vascular smooth muscle cells in culture. This process appears to involve both ETA and ETB receptors. When ET-1 synthesis is inhibited, by adding the ECE inhibitor phosphoramidon, this proliferative action is attenuated. This implies that not only can ET-1 be synthesized by smooth muscle cells, but also that it can then act as an autocrine mediator. Evidence from animal studies suggests that other growth factors, such as platelet-derived growth factor and epidermal growth factor, may be necessary cofactors for ET-1 to work effectively in this role. Both airway smooth muscle cells and airway epithelial cells in culture have been shown to proliferate in response to ET-1. Again, both ETA and ETB receptors are thought to be involved, although studies of human cell lines suggest that ETA may be the more relevant receptor. Furthermore, ET-1 is found to have a synergistic action when combined with other growth factors, suggesting again that it may be acting more as a co-mitogen rather than alone as a mitogen. Monocytes Macrophages Leukocytes

Receptors As discussed, two endothelin receptors, ETA and ETB, appear to mediate most of the actions of endothelins in human tissue. ETA binds ET-1 and ET-2 Chemotaxis cytokine release

Epithelial cells

ET-1 Pulmonary artery

ET-1

ET-1

Endothelial cells ET-1

ET-A ET-B

ET-B

Airway

Mucus gland

Mucus production

ET-1

Clearance No/PGI2 production Vasoconstriction proliferation ET-1

Bronchial smooth muscle Fibroblasts Collagen deposition

Vascular smooth muscle cells

Bronchoconstriction proliferation

Postcapillary venules Vasodilatation Figure 3 Biological functions of ET-1.

Permeability

78

ENDOTHELINS ET1 Ca2+ H2N

ET receptors Phospholipase C

Receptoroperated channel

G protein


q GDP



COOH

p p

q

DAG

GTP

GTP

GDP

IP3

Activation of protein kinase C

Activation of MAP kinase cascade

↑ intracellular calcium Proliferation Contraction Figure 4 Intracellular signaling mechanisms of ET-1.

with greater affinity, whereas ETB binds ET-1, ET-2, and ET-3 equally. Both receptors are members of the rhodopsin-like G-protein-coupled receptor family and preferentially interact with the Gq subfamily of guanine nucleotide binding (G) proteins (Figure 4). The binding of endothelin to ETA or ETB leads to the receptor undergoing a conformational change resulting in dissociation of the Ga subunit and activation of phospholipase C. This in turn leads to the hydrolysis of phosphoinositol 4,5-bisphosphate to the intracellular messengers 1,2 diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 increases intracellular calcium levels by inducing the release of intracellular stores, and also by opening receptoroperated calcium channels, causing an influx of extracellular calcium. This rise in intracellular calcium promotes ET-1’s vasoconstrictor actions. DAG, on the other hand, activates protein kinase C which initiates several other signaling pathways further downstream including the phosphorylation of intermediates of the mitogen-activated protein-kinase cascade system. These pathways are involved in promoting cell proliferation and growth.

Endothelins in Respiratory Diseases Pulmonary Arterial Hypertension

Pulmonary arterial hypertension is characterized by pulmonary vascular remodeling and increased

resting pulmonary vascular tone that ultimately leads to death through decompensation of the right ventricle. Given the biological functions described above, it is clear that ET-1 could be a key factor in the development of this disease. Animal studies have demonstrated that ET-1 expression is increased in models of pulmonary hypertension. Furthermore, ET antagonists prevent development of pulmonary hypertension in these models whilst addition of such antagonists to well established pulmonary hypertension appears to partially reverse the remodeling process. As such, it appears that remodeling is a dynamic process and not a fixed one, and that altering the effects of ET-1 can potentially reverse this. In humans, clinical trials of endothelin antagonists have had promising results; bosentan, a dual ETA and ETB antagonist, is now used for the treatment of idiopathic pulmonary arterial hypertension and scleroderma-associated pulmonary hypertension. Bosentan has been shown to improve functional status, reduce clinical worsening, and improve survival in patients. Studies in other conditions associated with pulmonary hypertension (e.g., congenital heart disease, HIV, and chronic thromboembolic disease) have also suggested a role for dual-receptor endothelin antagonists. However, the relative contribution of ETB receptor-mediated vasodilatation and ET-1 clearance as opposed to ETB receptor-mediated vasoconstriction in pulmonary hypertension is still under debate. In view of this, selective ETA

ENDOTHELINS 79

antagonists (e.g., sitaxsentan and ambrisentan) have also been developed and are now undergoing Phase III clinical trials. Pulmonary Fibrosis

Pulmonary fibrosis represents an abnormal woundhealing response to injury, either as a result of environmental insult or local inflammation. As ET-1 is known to have proinflammatory effects and is known to promote fibroblast migration, proliferation, and activation, it could act as either the trigger or the mediator of fibrotic change. Transgenic mice that overexpress ET-1 have been shown to develop pulmonary fibrosis, whilst increased levels of ET-1 have been found in the plasma, bronchoalveolar lavage (BAL) samples, and biopsies of patients with fibrosis. Some beneficial effects of endothelin antagonism have been described in animal models, and so randomized placebo-controlled trials are currently underway to study the effects of bosentan on patients with idiopathic pulmonary fibrosis and fibrosing alveolitis associated with systemic sclerosis.

Lung Transplantation

Through its vasoactive properties, endothelin may play a role in the ischemia-reperfusion injury that underlies early postoperative graft failure in lung transplantation. Its proliferative actions, on the other hand, may be more relevant in the pathogenesis of bronchiolitis obliterans, a condition that often results from chronic rejection. Lung Cancer

Several studies have demonstrated expression of ET1, big ET-1, ECE, and ETA and ETB receptors within pulmonary neoplasms. Furthermore, elevated plasma levels of big ET-1 have been correlated with a worse prognosis in non-small cell lung cancer patients. ET-1 expression by tumor cells may encourage tumor growth either through a proliferative effect on the cells themselves or through angiogenesis, allowing the tumor to extend beyond its margins. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. G-Protein-Coupled Receptors. Pulmonary Fibrosis. Pulmonary Vascular Remodeling. Surgery: Transplantation. Tumors, Malignant: Overview. Vascular Disease.

Asthma

Given its vasoactive, mitogenic, and immunomodulatory actions, it is not surprising that ET-1 has been suggested as a possible mediator in the development of asthma. Certainly, bronchial epithelial cell cultures from patients with asthma do show increased expression of both preproET-1 and ET-1. In addition, both serum and BAL levels of ET-1 are increased in these patients, with levels which inversely correlate with measures of airflow obstruction. Furthermore, successful treatment of asthma exacerbations is associated with a fall in these levels. Animal studies have demonstrated that endothelin antagonists modify many of the individual actions of ET-1, but whether or not endothelin antagonist therapy will be helpful in the management of asthma in the future is unknown. Acute Respiratory Distress Syndrome

Both elevated serum ET-1 levels and increased pulmonary immunostaining for ET-1 in patients with ARDS have implicated this peptide as a mediating factor. Endothelins may well contribute to the pathophysiology of ARDS through their effects on vascular tone, vascular permeability, and their proinflammatory actions. Alternatively, they may be innocent bystanders, released as a result of endothelial dysfunction and disruption.

Further Reading Barst RJ, Langleben D, Frost A, et al. (2004) Sitaxsentan therapy for pulmonary arterial hypertension. American Journal of Respiratory and Critical Care Medicine 169(4): 441–447. Billington CK and Penn RB (2003) Signalling and regulation of G protein-coupled receptors in airway smooth muscle. Respiratory Research 4: 2. Boscoe MJ, Goodwin AT, Amrani M, and Yacoub MH (2000) Endothelins and the lung. The International Journal of Biochemistry & Cell Biology 32: 41–62. Fagan KA, McMurtry IF, and Rodman DM (2001) Role of endothelin-1 in lung disease. Respiratory Research 2: 90–101. Galie N, Manes A, and Branzei A (2004) The endothelin system in pulmonary arterial hypertension. Cardiovascular Research 61: 227–237. Henry PJ (1999) Endothelin receptor distribution and function in the airways. Clinical and Experimental Pharmacology and Physiology 26(2): 162–167. Jeffrey TK and Morrell NW (2002) Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension. Progress in Cardiovascular Diseases 45(3): 173–202. Michael JR and Markewitz BA (1996) Endothelins and the lung. American Journal of Respiratory and Critical Care Medicine 154(1): 555–581. Rubin LJ, Badesch DB, Barst RJ, et al. (2002) Bosentan therapy for pulmonary arterial hypertension. The New England Journal of Medicine 346(12): 896–903. Teder P and Noble PW (2000) A cytokine reborn? Endothelin in pulmonary inflammation and fibrosis. American Journal of Respiratory Cell and Molecular Biology 23: 7–10. Yanagisawa M, Kurihara H, Kimura S, et al. (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411–415.

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ENDOTOXINS

ENDOTOXINS P J Bertics, M L Gavala, and L C Denlinger, University of Wisconsin Medical School, Madison, WI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Endotoxin is a major component of the outer leaflet of the outer membrane of Gram-negative bacteria and is composed of carbohydrates, fatty acids, phosphates, and associated metal ions. Endotoxin is also known as lipopolysaccharide (LPS) and varies in its carbohydrate and lipid composition between bacterial strains and species. However, several structural features are common to most LPS molecules, including a negatively charged hydrophilic heteropolysaccharide (O-antigen), a core oligosaccharide region, and a lipid A portion that usually contains two glucosamines coupled to six fatty acids. Lipid A is the toxic component of LPS and its bioactivity depends on its disaccharide, phosphate, and fatty acid content. Endotoxin is widely present in the environment, including dust, animal waste, foods, and other materials generated from, or exposed to, Gram-negative bacterial products. Occupational exposure to endotoxin (e.g., as a bioaerosol) is associated with airway disease, and endotoxin presentation to the tissues or blood initiates a profound immunological response. In the lung, the alveolar macrophage is the key in the recognition and host response to inhaled endotoxin. Endotoxin detection by the macrophage via cell surface receptors such as Toll-like receptor-4 results in the activation of multiple signaling cascades that trigger the production of both pro- and anti-inflammatory mediators. Host responses following endotoxin challenge are dependent on the individual genetic background and the anatomical site and associated cell types. If the responses are too robust, adverse consequences, such as pulmonary edema and bronchospasm, can occur. Also, lobar pneumonia or endotoxemia from infections of other organ systems can cause diffuse acute lung injury, septic shock, and death. The management and treatment of individuals exposed to LPS encompass the use of antibiotics, bronchodilators, inhaled corticosteroids, and activated protein C. In cases of an overwhelming bacterial infection, supportive measures include supplemental oxygen, intravascular fluid administration, vasopressors, stress-dose corticosteroids, and mechanical ventilation.

Structure and Physicochemical Properties Endotoxin Discovery and General Properties

The pathobiological activity of bacterial endotoxin, also known as lipopolysaccharide (LPS), was first reported more than a century ago. Because LPS was considered an integral part of the organism, it was named endotoxin as opposed to toxins that were freely released, which were termed exotoxins. When filtrates of dead Gram-negative bacteria were infused into animals, they produced shock and death, thus fulfilling Koch’s fifth postulate, wherein

a toxin harvested from a bacterial filtrate will reproduce the disease. Gram-negative bacteria differ from other bacteria and mammalian cells by having both an inner and outer membrane that consist of lipid bilayers and transmembrane proteins. The outer leaflet of the outer membrane of Gram-negative bacteria is largely composed of an amphipathic molecule defined as LPS (Figure 1). Functionally, LPS provides a barrier to heavy metals, lipid-disrupting agents, and larger molecules (e.g., lytic enzymes and DNA). The lipid portion of LPS allows it to pack tightly to form a planar membrane, and one bacterial cell contains approximately 3.5 million LPS molecules. Using the bacterium Escherichia coli as an example, nearly 75% of the outer leaflet is composed of LPS, whereas the other 25% is largely transmembrane proteins. The orientation of the LPS in intact bacteria is believed to obscure the lipid domains beneath the polysaccharide components of the molecule. However, upon bacterial death and cell wall lysis, LPS can be released and can interact with other hydrophobic molecules, including mammalian membrane lipids and the hydrophobic domains of various serum and cell surface proteins. Released LPS is thought to play a major role in the development of the septic shock that accompanies Gram-negative bacterial infectious diseases. Endotoxin Structure and Variation

Although the exact structural composition of LPS varies between different Gram-negative bacterial species and strains, there are several structural features that are common to most LPS molecules. These structural features include a negatively charged hydrophilic heteropolysaccharide (O-antigen), a core oligosaccharide region, and a lipid A portion that generally contains two glucosamines that are coupled at the 2, 3, 20 , and 30 positions with fatty acids (Figure 2). The O-antigen is composed of repeating groups of three to five sugars. The sugars in the O-antigen vary between bacterial strains, and they contribute to the antigenic differences observed within a species. Furthermore, bacteria with bulky O-antigen polysaccharides are often more resistant to complementmediated lysis and/or phagocytosis and exhibit an array of antigenic variants. In contrast to the Oantigen, the polysaccharides of the core region are less variable and can be divided into groups known as the outer and inner core. The outer core varies somewhat between genera and within species but is

ENDOTOXINS 81 Endotoxin (lipopolysaccharide O-antigen or LPS) n

n

n

n

Porin n proteins

n

Outer core Inner core Lipid A

Cell wall

Phospholipids Peptidoglycan

Plasma membrane

Integral proteins Figure 1 Schematic representation of endotoxin (lipopolysaccharide (LPS)) structure and localization in Gram-negative bacteria. The diagram provides an overview of the cell wall of the bacterium E. coli, which serves as a model of comparison for the localization/ structure of endotoxin molecules from other genera. The outer membrane contains fewer proteins (e.g., the porins) as compared to the larger integral protein content of the inner membrane. Between the outer membrane and the inner membrane, there exists a peptidoglycan layer. In general, endotoxin is the major component of the outer leaflet of the outer membrane, whereas the other three leaflets of the outer and inner (plasma) membranes are composed of phospholipids resembling those of mammalian cells. The lipid A portion directly constitutes the outer leaflet of the lipid bilayer and is responsible for the toxicity associated with LPS. The inner core (pink ovals) is composed of two or three KDOs and three heptoses, which contain phosphoethanolamine and phosphate groups that bind calcium and magnesium ions and help maintain the structural integrity of the outer membrane. The outer core (gold ovals) is usually composed of hexoses (five are shown), whereas the O-antigen (blue ovals) contains hexoses that are arranged as repeating units of trito hexasaccharides; the number of repeats (n) may be as large as 20–30 units and can even vary within the same culture.

usually composed of four to six hexoses, primarily glucose, galactose, and N-acetyl-glucosamine. The inner core contains three heptoses that are coupled to phosphates and phosphoenthanolamines as well as to two or three unique sugars that form the bridge between lipid A and the polysaccharide regions of LPS; this unique sugar is known as 3-deoxy-D-mannooctulosonic acid (KDO). The inner core is highly conserved across diverse genera. The lipid A present in common enteric and some oral–pharyngeal organisms usually contains six fatty acids, distributed such that four fatty acids are localized on one glucosamine and two are present on the other glucosamine (Figure 2). In contrast, the lipid A molecules synthesized by Neisseria meningitidis, Neisseria gonorrheae, and Pseudomonas aeruginosa possess three fatty acids on each glucosamine. An exception to the six fatty acid pattern is seen in Salmonella LPS, which contains seven fatty acids. Most of the fatty acids in enteric organisms contain 12–16 carbons, whereas the LPS from oral–pharyngeal pathogens generally have 10–14 carbon fatty acids. The LPS molecules from Endobacteriaceae and P. aeruginosa possess a large O-antigen, a complete outer and inner core, two or three KDOs, and a lipid A with four hydroxymyristic acids and two acyloxyacyl fatty acids linked to the fatty acids on the 20 and 30 positions of the disaccharide. Conversely, the

LPS of nasopharyngeal pathogens, such as Neisseria species, Haemophilus influenzae, and Bordetella pertussis, lack the O-antigen typical of the lower gastrointestinal tract organisms, and chlamydial LPS is essentially devoid of core polysaccharides except for the presence of a KDO trisaccharide. Instead of an O-antigen, other groups that confer serum resistance, such as the N-acetyl neuraminic acid found in Neisseria species, are found. The outer core hexoses, characteristic of enteric bacteria, are also missing in these nasopharyngeal organisms, but an inner core that is similar to that of E. coli and Salmonella species is present. Heptoses in the inner core from both enteric and nasopharyngeal species often contain negatively charged groups (phosphates and aminoethyl-phosphates) that tightly bind Ca2 þ and Mg2 þ . Structure–Activity Relationships

The lipid A portion is the toxic component of LPS, and every toxic lipid A is composed of a disaccharide containing either D-glucosamines or 2,3-diamino2,3-deoxy-D-glucose. Monosaccharide substructures of lipid A are B107-fold less toxic than LPS. In addition, phosphates at the 1 and 40 positions of lipid A are needed for its activity (e.g., monophosphoryl lipid A is approximately 1000-fold less toxic than LPS). Also, the number and configuration of the fatty

82

ENDOTOXINS HO

O −

O

O

HO

P

O

−O O

−

O O O

NH

O O

O

O

O O

HO

O

HO

O−

NH O

O HO

H3N +

−O

O O

NH O

O O

O P

O−

O−

S. minnesota

O P

O

O

O

E. coli

O

HO

HO

O

O

O−

O

O O

O P

O O

O

P −O

NH O

O

HO

O

O O

P −O

HO

O O

O O

NH

O HO

O

O

O

HO

O

HO

O

O

O

O O

NH O

O P O−

O O

P

+

O

NH3

O−

N. meningitidis Figure 2 Examples of structural variations in the lipid A portions of endotoxin molecules isolated from the Gram-negative bacteria E. coli, S. minnesota, and N. meningitidis. In each case, the remaining polysaccharides that constitute the LPS molecule, namely the inner core, outer core, and O-antigen, are attached to the free hydroxyl at the 60 position of the lipid A disaccharide (the HO group on the left-hand sugar residue in the figure).

acids attached to the disaccharide affect lipid A bioactivity. For example, lipid A substructures containing only four hydroxymyristic acids coupled to the glucosamine disaccharides are as much as 107-fold less toxic than the parent hexa-acyl synthetic lipid A. Moreover, the removal or addition of one fatty acid (i.e., penta-acyl or hepta-acyl lipid A) leads to a less toxic form of lipid A. Lastly, there is some indication that the chain length of the fatty acids is the key; that is, when the fatty acids are p12 carbons in length, the toxic activity of the LPS is reduced.

Source and Exposure Endotoxin is found in Gram-negative bacteria and bacterial products or debris. Thus, endotoxin is widely present in the environment, including dust, animal waste, foods, and other materials generated from, or exposed to, Gram-negative bacterial products. Accordingly, individuals involved in occupations

associated with intensive livestock and/or agricultural operations are frequently exposed to elevated levels of endotoxin. Given its prevalence in the environment, endotoxin is also generally present endogenously in sites such as the airway and the gastrointestinal tract. In fact, endotoxin presentation as a bioaerosol is an important route of occupational exposure associated with the development and progression of airway disease. In addition, mechanical- or pathogen-induced (e.g., virus) injury may allow for secondary bacterial introduction and thus lead to endotoxin presentation to the tissues or blood, whereupon it initiates a robust immunological response.

Clinical Consequences Endotoxin-Induced Cellular Responses

The innate immune system is part of an organism’s first line of defense against bacterial invasion. The

ENDOTOXINS 83

and monocytes, and this LBP/LPS/CD14 complex can lead to the activation of cell surface receptors including Toll-like receptor 4 (TLR4) (Figure 3). The LBP/ LPS/CD14 complex allows monocytes/macrophages to detect LPS at concentrations as low as 10 pg ml
1. During the past several decades, LPS has been reported to activate numerous intracellular signaling events, and many of these appear to be mediated by TLR4. For example, with the aid of MD-2, which is a small secreted accessory glycoprotein, LPS recognition by TLR4 activates signaling cascades such as those associated with mitogen-activated protein (MAP) kinases, phosphatidylinositol (PI) 3-kinase, and sphingolipid metabolites, and these pathways converge to regulate LPS-induced cellular responses (Figure 3). LPS-induced activation of multiple transcription factors, including nuclear factor kappa B (NF-kB), activator protein-1 (AP-1), and the cAMP response element-binding protein (CREB), along with

principal components of the innate immune system include antimicrobial substances produced by epithelial cells, polymorphonuclear leukocytes, and macrophages. These cells, along with B lymphocytes, are the primary targets for endotoxin-induced cellular responses. In the lung, epithelial cells are often involved in mediating the physical defenses to bacterial infection, whereas the alveolar macrophage is the key in the early recognition and host response to inhaled endotoxin. This early detection of endotoxin by the macrophage results in the activation of multiple signaling cascades that trigger the production of both pro- and anti-inflammatory mediators involved in the initial immune response. When LPS is released into the blood or tissues, it is generally bound to a lipid transfer protein referred to as LPS-binding protein (LBP). LBP facilitates the binding of endotoxin to soluble or membrane-bound CD14 found on both polymorphonuclear leukocytes

LBP

Other receptor systems

LPS

CD14

MD2

T

T

L

L

R

R

4

4

Extracellular MD2

Plasma membrane

Intracellular

PI3K

NF-
B

Sphingolipids

MAP kinases

Gene transcription mRNA stability Protein processing Cytoskeletal reorganization Figure 3 General view of endotoxin-mediated signaling in target cells such as monocytes/macrophages. Endotoxin (LPS) interaction with LPS-binding protein (LBP) and CD14, which can be either soluble or membrane bound via a glycosylphosphatidylinositol moiety (shown as a black line), facilitates LPS recognition by receptor systems such as TLR4/MD2. Stimulation of these receptor systems by endotoxin results in the activation of multiple signaling cascades, including those involving PI3-kinase, NF-kB, sphingosine metabolites, and MAP kinase cascades (e.g., those associated with the MAP kinases p38 and extracellular signal-regulated kinases ERK1 and ERK2). These events can ultimately alter cellular transcriptional activity, mRNA stability, protein processing, and cytoarchitecture, leading to cellular responses involved in cytokine and chemokine production/release, phagocytosis, and reactive oxygen species generation. Other receptor systems, such as b-integrins, TLR2, scavenger receptors, and nucleotide receptors, have also been proposed to modulate/amplify certain LPS-induced signaling events and biological activities. Pl3K, phosphatodylinositol 3-kinase; MAP, mitogen-activation protein; TLR4, Toll-like receptor 4.

84

ENDOTOXINS

LPS-initiated effects on mRNA stability and protein processing, leads to the coordinate induction and/or release of many inflammatory mediators. In addition, other membrane receptors, such as b-integrins, TLR2, scavenger receptors, and nucleotide receptors, have been proposed to act as systems that can serve to modulate or amplify the signaling induced by certain LPS molecules (Figure 3). Physiological Responses and Respiratory Diseases

The magnitude and severity of the host response following endotoxin challenge are dependent on the individual genetic background and on the anatomical site and associated cell types. For example, Gramnegative bacteria are resident organisms of the oral mucosa and upper airway that cause minimal toxic effects if contained outside of the bloodstream, in part due to a lack of MD-2 expression by respiratory epithelial cells. In contrast, infection of lung parenchyma is quickly recognized by alveolar macrophages that produce a cascade of mediators that can be both beneficial and harmful. Early host cellular responses are amplified by the release of prostaglandins, platelet-activating factor, chemokines, adenine nucleotides, and a variety of preformed cytokines (e.g., tumor necrosis factor alpha and interleukin-1b) that can promote autocrine effects on macrophage function. Several of these agents can have direct microbicidal activities, whereas others can initiate a variety of physiological responses, such as the induction of fever (which can attenuate bacterial growth) and microcoagulation. The resulting vascular congestion, cellular inflammation, and secondary mediator production further limit bacterial growth. However, if these responses are too robust, adverse consequences, such as pulmonary edema and bronchospasm,

can occur manifesting as cough, dyspnea, wheeze, tachypnea, and hypoxemia in severe cases (Table 1). Clinical presentations resulting from LPS exposure are diverse, and again depend on the location involved. Inhaled LPS from various environmental or industrial sources by itself is sufficient to cause asthma exacerbations or hypersensitivity pneumonitis. With respect to bacterial infection, if the physical defense system of the upper airway or bronchial tree is impaired due to cilial damage (e.g., in response to aspiration or tobacco exposure) or defective mucus clearance (e.g., cystic fibrosis), then bronchitis ensues with the hallmark production of purulent sputum in the absence of radiographic findings. Chronic inflammation may result if there is ineffective bacterial removal, thereby leading to irreversible airway damage known as bronchiectasis. Conversely, overwhelming infection of the distal airways and alveoli causes pneumonia, which presents with high fever, sputum production, and characteristic dense infiltrates on a plain chest film. Finally, lobar pneumonia or endotoxemia from infections of other organ systems (e.g., peritonitis associated with a ruptured appendix) can cause diffuse acute lung injury, septic shock, and death (100 000 deaths annually in the United States) due to lung inflammation incited by filtration of platelet and/or bacterial debris in the bloodstream as well as from mediator-induced vascular leak.

Management and Treatment The therapies for LPS exposure can be classified as those associated with either airway or parenchymal disorders. Pure airway disorders are often only minimally benefited by administration of antibiotics. Wheeze and cough due to bronchospasm

Table 1 Selected physiological consequences of LPS exposure and associated strategies for therapeutic intervention Symptom or sign

Associated causes or mediators

Biological effects or outcomes

Therapeutic interventions

Fever

IL-1, IL-18

Bacterial stasis

Inflammation

Prostaglandins, chemokines

Phagocyte recruitment, microbial killing

Tachypnea

Hypoxia

Hypotension

TNF-a, NO, vascular leak

Compensation for increased metabolic demands Decreased organ perfusion, lactic acidosis

Disseminated intravascular coagulation

PAF, platelet degranulation, thrombin

Antibiotics if coupled with high suspicion of a bacterial infection Source control such as device removal or drainage if present in areas not effectively cleared (e.g., pleural empyema) Supplemental oxygen 7 mechanical ventilation Goal-directed fluid resuscitation, vasopressors, stress-dose steroids Activated protein C

Disruption of microcirculation, cellular anoxia

TNF-a, tumor necrosis factor alpha; NO, nitric oxide; PAF, platelet-activating factor.

ENDOTOXINS 85

can be abated with the use of bronchodilators such as albuterol. Inhaled corticosteroids may be required to attenuate inflammation and other immune responses if the source of the LPS exposure cannot be eliminated. Conditions with chronic sputum production are also improved by the use of physical treatments (e.g., an expiratory flutter valve or chest physiotherapy) to improve secretion management. Treatment options for parenchymal disorders, such as pneumonia and septic shock, are reviewed in Table 1. In the clinical context of an overwhelming bacterial infection, antibiotics are the mainstay of treatment. A cautionary note is that some of the most effective bactericidal antibiotics, particularly those with cell wall-directed activity such as b-lactams, also facilitate the release of LPS into the local environment or systemic circulation. If the infection breeches an anatomic space not well protected by the immune system (e.g., pleural empyema), drainage and/or a surgical procedure may be required. Supportive measures for conditions of increasing severity include supplemental oxygen, intravascular fluid administration, vasopressors, stress-dose corticosteroids (improves vasopressor efficacy), and mechanical ventilation. Activated protein C, which is used for the treatment of septic shock associated with disseminated intravascular coagulation, is a unique agent in that it has both anticoagulant and antiinflammatory actions. Finally, although mediator-directed neutralization therapies have been ineffective in the past, future application of genomic and proteomic data may direct patient-customized immunomodulatory treatments. See also: Acute Respiratory Distress Syndrome. Adenosine and Adenine Nucleotides. Adhesion, Cell–Cell: Epithelial. Aerosols. Asthma: Occupational Asthma (Including Byssinosis); Exercise-Induced. Bronchiectasis. CD11/18. CD14. Chemokines. Chronic Obstructive Pulmonary Disease: Acute Exacerbations. Coagulation Cascade: Overview. Complement. Defense Systems. Dendritic Cells. Endothelial Cells and Endothelium. Environmental Pollutants: Overview. Histamine. Immunoglobulins. Interleukins: IL-1 and IL-18; IL-10; IL-6. Kinins and Neuropeptides: Bradykinin. Leukocytes: Mast Cells and Basophils; Neutrophils; Monocytes; Pulmonary Macrophages. Lipid Mediators: Overview; Leukotrienes; Prostanoids; Lipoxins. Lymphatic System. Macrophage Inflammatory Protein. Nitric Oxide and

Nitrogen Oxides. Occupational Diseases: Overview. Panbronchiolitis. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Viral. Signal Transduction. Toll-Like Receptors. Transcription Factors: AP-1; NF-kB and Ikb; Overview. Tumor Necrosis Factor Alpha (TNF-a). Tumors, Malignant: Chemotherapeutic Agents. Upper Respiratory Tract Infection. Vaccinations: Bacterial, for Pneumonia.

Further Reading Arbour NC, Lorenz E, Schutte BC, et al. (2000) TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nature Genetics 25: 187–191. Beutler B and Rietschel ET (2003) Innate immune sensing and its roots: the story of endotoxin. Nature Reviews: Immunology 3: 169–176. Denlinger LC, Fisette PL, Sommer JA, et al. (2001) The nucleotide receptor P2X7 contains multiple protein- and lipid-interaction motifs including a potential binding site for bacterial lipopolysaccharide. Journal of Immunology 167: 1871–1876. Falkow S (1988) Molecular Koch’s postulates applied to microbial pathogenecity. Review of Infectious Diseases 10: S274– S276. Freudenberg MA, Keppler D, and Galanos C (1986) Requirement for lipopolysaccharide-responsive macrophages in galactosamine-induced sensitization to endotoxin. Infection and Immunity 51: 891–895. Hotchkiss RS and Karl IE (2003) The pathophysiology and treatment of sepsis. New England Journal of Medicine 348: 138– 150. Jia HP, Kline JN, Penisten A, et al. (2004) Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2. American Journal of Physiology 287: L428–L437. Lorenz E, Jones M, Wohlford-Lenane C, et al. (2001) Genes other than TLR4 are involved in the response to inhaled LPS. American Journal of Physiology 281: L1106–L1114. Miller SI, Ernst RK, and Bader MW (2005) LPS, TLR4 and infectious disease diversity. Nature Reviews: Microbiology 3: 36–46. Monick MM and Hunninghake GW (2002) Activation of second messenger pathways in alveolar macrophages by endotoxin. European Respiratory Journal 20: 210–222. Proctor RA, Denlinger LC, and Bertics PJ (1995) Lipopolysaccharide and bacterial virulence. In: Roth JA, Bolin CA, Brogden KA, Minion FC, and Wannemuehler MJ (eds.) Virulence Mechanisms of Bacterial Pathogens, pp. 173–194. Washington, DC: ASM Press. Raetz CRH and Whitfield C (2002) Lipopolysaccharide endotoxins. Annual Review of Biochemistry 71: 635–700. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, and Mathison JC (1990) CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249: 1431–1433. Zhao L, Ohtaki Y, Yamaguchi K, et al. (2002) LPS-induced platelet response and rapid shock in mice: Contribution of O-antigen region of LPS and involvement of the lectin pathway of the complement system. Blood 100: 3233–3239.

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ENERGY METABOLISM

ENERGY METABOLISM E F M Wouters, University Hospital, Maastricht, The Netherlands E M Baarends, Zuyd University, Zuyd, The Netherlands & 2006 Elsevier Ltd. All rights reserved.

Abstract Energy metabolism is central to life and the main function of the respiratory system is to maintain aerobic metabolic processes in the body. Despite this important role, energy metabolism is poorly integrated in the diagnostic workup of chronic respiratory diseases. Increased attention during the last decade has focused on the contribution of energy imbalance in the pathogenesis of weight loss, particularly in chronic obstructive pulmonary disease (COPD) patients. Several factors contribute to the amount of energy spent by an individual: resting energy expenditure, physical activity, and to a lesser extent diet-induced thermogenesis. The article deals particularly with resting energy expenditure as well as total daily energy expenditure data, measured in COPD patients. Factors responsible for increase in resting energy expenditure as well as total energy expenditure are discussed in detail. Food intake as well as food utilization are essential components in the maintenance of energy balance. Recent progress in the field of energy homeostasis reveals the complexity of the regulatory neuroendocrine network. The role of the anorexigenic and adipostatic hormone leptin is discussed in detail.

Introduction Although energy metabolism is central to life and the main function of the respiratory system is to exchange oxygen (O2) and carbon dioxide (CO2) in order to maintain aerobic metabolic processes in the body, energy metabolism is poorly integrated in the diagnosis and management of patients with chronic respiratory conditions. While cell physiologists approach energy expenditure as the sum of at least membrane transport, protein turnover, and metabolic cycles, the clinical physiologist considers energy expenditure in terms of basal metabolic rate, thermic effect of food, and physical activities. This article deals with the clinical physiological aspects of energy expenditure and will discuss new insights in energy intake regulation. The growing interest in metabolic aspects of chronic respiratory disease conditions such as chronic obstructive pulmonary disease (COPD) as well as the rapid growth in measurement devices for indirect calorimetry, have created new interests in calorimetry. Insights in energy metabolism significantly contribute to a better understanding of pathogenetic processes in patients suffering from respiratory pathology.

Energy Balance Energy Expenditure

Total daily energy expenditure (TDEE) can be divided into three components: (1) resting energy expenditure (REE) which comprises sleeping metabolic rate and the energy expenditure for arousal, (2) dietinduced thermogenesis, and (3) physical activity-induced thermogenesis. Resting energy expenditure Indirect calorimetry is the method by which measurements of respiratory gas exchange (oxygen consumption, VO2 and carbon dioxide production, VCO2 ) are used to estimate the type and amount of substrate oxidized and the amount of energy produced by biological oxidation. Indirect calorimetry considers the whole organism in an idealized system as one big compartment containing all living cells, bathed by a homogeneous extracellular fluid and renewed by a constant blood flow. Input–output analysis in such a system allows measurement of V_ O2 and V_ CO2 . Since the REE accounts for the major component of TDEE in sedentary subjects and can be relatively easy measured (Figure 1), several studies have measured REE particularly in COPD, based on the growing interest in weight loss and muscle wasting in this disease condition. Data obtained by indirect calorimetry in these studies are generally related to predicted REE values. The most common approach to predict REE for an individual in clinical practice is to apply the Harris and Benedict (HB) equations, which are based on sex, age, height, and body mass. Different studies

Figure 1 Picture of a resting energy expenditure measurement.

ENERGY METABOLISM 87 40 ∆REE (%)

have investigated hypermetabolism in stable patients with COPD on base of the HB equations, most studies revealing an increased REE compared to the HB equations or to a control group. Few studies are known about the prevalence of hypermetabolism in COPD. Hypermetabolism at rest is reported in about 25% of stable COPD patients. Besides fat free mass (FFM) other factors like work of breathing, medication, and systemic inflammation are reported in order to explain intersubject variability in REE in patients with COPD. The increase in oxygen cost of breathing (OCB) is assumed to be one of the main explanations for the elevated REE seen in patients suffering from COPD. Several studies have investigated the OCB in patients with COPD and all found a higher OCB in patients compared to healthy controls. This OCB is generally measured by augmenting the ventilation or ventilatory effort: it is well accepted that increasing the ventilation poses an extra elastic load on the respiratory system due to progressive dynamic hyperinflation. Another possible explanation for the elevated REE seen in patients with COPD may be a thermogenetic effect of pharmacological maintenance treatment as part of the symptomatic therapy in patients with chronic respiratory conditions as COPD. Bronchodilating agents such as theophyllines and b2sympathicomimetics are widely prescribed to these patients to reduce breathlessness and improve exercise tolerance. Both drugs show an elevating effect on REE in younger healthy subjects, while the increase in REE in patients with COPD is very limited (Figure 2). Finally, it is shown that COPD patients demonstrate a blunted b-adrenoreceptor-mediated thermogenic response compared to control subjects matched for age and body composition. Together, these slight effects of b-adrenergic stimulation on REE cannot fully explain the elevated REE in these patients. Another contributing factor to hypermetabolism may be related to systemic inflammation. The polypeptide cytokine tumor necrosis factor alpha (TNF-a) is a proinflammatory mediator produced by different cell types. TNF-a inhibits lipoprotein lipase activity and is pyrogenic. It also triggers the release of other cytokines, which themselves mediate an increase in energy expenditure, as well as mobilization of amino acids and muscle protein catabolism. Using different markers, several studies provide clear evidence for involvement of TNF-a-related systemic inflammation in the pathogenesis of tissue depletion. It is demonstrated that hypermetabolism at rest is related to plasma TNF-a concentrations as well as to the level of acute phase response in COPD patients.

30 20 10 0

0

5

10

15

20 25 30 Time (min)

35

40

45

Figure 2 Change in resting energy expenditure (y-axis) during 45 min (x-axis) after nebulization of 5 mg of a b2-sympathicomimetic drug in three groups. The upper line are young healthy subjects, the lower lines are the patients with COPD and elderly healthy subjects.

Oxygen cost of breathing

Systemic inflammation

Bronchodilating agents

Increased resting energy expenditure 26% Prevalence Figure 3 Summary of the possible factors contributing to an increased resting energy expenditure in COPD.

A summary of possible factors contributing to an increased REE in the prevalence of hypermetabolism is shown in Figure 3. Total daily energy expenditure Measurements of energy expenditure over periods of days or at least over 24 h periods were advised to better determine energy requirements in humans. Respiratory chambers allow assessment of energy metabolism in humans in sedentary conditions or during exercise testing. Using a respiratory chamber, the different components of daily sedentary physical activity can be assessed: the sleeping metabolic rate, the energy costs of arousal (basal metabolic rate minus sleeping metabolic rate), the thermic effects of the meals, and the energy cost of spontaneous physical activity. However, one of the major components of the daily energy expenditure, and certainly the most variable, is the energy expenditure associated with motion and physical activity. Day-to-day variability in energy expenditure is most likely to be related to these changes in physical activity. The doubly labeled water method allows measurement of energy expenditure in totally free-living conditions.

88

ENERGY METABOLISM Energy Intake

2000

TDE−REE (kcal day–1)

p < 0.01 1500

1000

500

0 Controls

COPD

Figure 4 Nonresting component of total daily energy expenditure in patients with COPD and healthy subjects matched for age, sex, and body composition.

Despite the fact that measuring TDEE is methodologically difficult and expensive, recent studies have focused attention on the activity-related energy expenditure in patients with COPD. Using the doubly labeled water ð2 H2 18 OÞ technique to measure TDEE it is demonstrated that patients with COPD have a significantly higher TDEE than healthy subjects. Remarkably, the nonresting component of total daily energy expenditure is significantly higher in the patients with COPD than in the healthy subjects (Figure 4), resulting in a ratio between TDEE and REE of 1.7 in patients with COPD and 1.4 in healthy volunteers, matched for age, sex, and body weight. This increased activity related energy expenditure suggests a mechanical inefficiency during activities. Otherwise, when TDEE is measured in a respiration chamber, no differences in TDEE are found between patients with COPD and healthy controls, possibly as a consequence of the limited activity in the respiration chamber. A high variability of TDEE in patients with COPD is a constant finding in different studies. This variability in TDEE has to be considered in maintaining energy balance in COPD patients, particularly when exercise is advised as part of an integrated management program. Part of the increased oxygen consumption during exercise can be related to an inefficiency in muscles. Several studies indeed show a severely impaired oxidative phosphorylation during exercise in COPD. Further studies are indicated in order to investigate the possible relationship between an inefficient or relatively enhanced energy expenditure during activities and changes in substrate metabolism. In any case, there is increasing evidence suggesting that, in order to estimate energy requirements of patients with COPD, it is necessary to measure physical activity as well as metabolic efficiency during exercise.

Increments in energy expenditure caused by physical and metabolic tasks are related to utilization of the chemical energy stores. Fuel intake as well as fuel utilization are essential components in the maintenance of the energy balance. Therefore, hypermetabolism can explain why some COPD patients lose weight despite an apparent normal or even high dietary intake. Nevertheless, it has been shown that dietary intake in weight-losing patients is lower than in weight-stable patients, both in absolute terms as well as in relation to measured REE. This is quite remarkable because the normal adaptation to an increase in energy requirements in healthy men is an increase in dietary intake. The reasons for a relatively low dietary intake in COPD are not completely understood. In the past, impaired dietary intake in patients with chronic respiratory conditions was related to local organ impairment such as respiratory mechanics or gas exchange abnormalities. It has been suggested that patients with COPD eat suboptimally because chewing and swallowing change breathing pattern and decrease arterial oxygen saturation. Particularly in hypoxemic patients a rapid decrease in SaO2 during a meal with slow recovery after completion of the meal is reported; the decrease in SaO2 is associated with an increased dyspnea sensation. Gastric emptying time of a meal may also affect dietary intake since gastric filling in these patients may impair diaphragmatic functioning, contributing to an increase in dyspnea. Besides these effects on the pulmonary system, recent progress in the field of energy homeostasis has revealed a complex regulatory neuroendocrine network. The discovery of the anorexigenic and adipostatic hormone leptin and the neural circuits responsible for mediating these leptin actions has indicated the important role of the hypothalamus as the center for the integration of feeding and associated autonomic, neuroendocrine, and gastrointestinal activities. The adipocyte-derived hormone leptin represents the afferent hormonal signal to the brain in a feedback mechanism regulating fat mass. Leptin regulates adaptive feeding responses by interaction with the hypothalamic signals such as neuropeptide Y and agouti-related peptide (AGRP) resulting in a reduction in food intake as well as in an increase in energy expenditure. In addition, leptin has a regulating role in lipid metabolism and glucose homeostasis and increases thermogenesis. In Figure 5, the role of leptin in regulating energy intake and metabolism is summarized. Few data have been reported on leptin metabolism in COPD. Circulating leptin correlates well with

ENERGY METABOLISM 89

Hypothalamus Neuropeptide

Food intake Glucose metabolism

Leptin White adipose tissue

Fat Energy expenditure

Neuroendocrine function

Figure 5 The mechanism of leptin in regulating energy intake and metabolism.

body mass index (BMI) and fat percentage, as expected. In stable disease, plasma leptin corrected for fat mass was inversely correlated with dietary intake as well as with the degree of weight change after 8 weeks of nutritional supplementation in depleted patients with COPD. During an exacerbation of COPD, systemic leptin concentrations are increased and the normal feedback regulation of leptin by fat mass (FM) seems to be disrupted. In patients with emphysema as well as in patients suffering from acute exacerbations, leptin is reported to be related to systemic inflammation, independently of the amount of FM. Ghrelin has recently been discovered as the missing link between enteric nutrition and central regulation of energy balance and growth. Ghrelin functions as an orexigenic or appetite-stimulating signal from the stomach. Ghrelin regulates, in an antagonistic manner to leptin, synthesis and secretion of several neuropeptides in the hypothalamus involved in the regulation of feeding and associated hypothalamic functions. Further studies are needed to unravel the pathogenesis of disturbed energy regulation in relation to systemic inflammation in patients suffering from chronic respiratory diseases.

Conclusions The growing interest in energy metabolism in respiratory medicine during the last two decades has significantly contributed to a better understanding of pathogenetic processes underlying these disease conditions. The important role of activity-related energy

expenditure is stressed and new insights in the field of energy homeostasis open new fascinating perspectives of research and therapeutic potential in the management of these processes in the clinical course of a variety of disorders. Future research has to integrate metabolic cellular processes in the pathogenesis of energy disorders in respiratory diseases. See also: Primary Myelofibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Vascular Disease.

Further Reading Baarends EM, Schols AMWJ, Pannemans DL, Westerterp KR, and Wouters EFM (1997) Total free living energy expenditure in patients with severe chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 155: 549–554. Cherniack RM (1958) The oxygen consumption and efficiency of the respiratory muscles in health and emphysema. Journal of Clinical Investigation 38: 494–499. Coward WA, Prentice AM, Murgatroyd PR, et al. (1984) Measurement of CO2 and water production rates in man using 2H, 18 O-labeled H2O: comparisons between calorimeter and isotope values. In: van Es AJH (ed.) Human Energy Metabolism: Physical Activity and Energy Expenditure Measurements in Epidemiological Research Based upon Direct and Indirect Calorimetry, pp. 126–128. Wageningen: Stichting Nederlands Instituut voor de Voeding. Creutzberg EC, Schols AMWJ, Bothmer-Quaedvlieg FCM, Wesseling G, and Wouters EFM (1998) Acute effects of nebulized salbutamol on resting energy expenditure in patients with chronic obstructive pulmonary disease and in healthy subjects. Respiration 65(5): 375–380. Creutzberg EC, Schols AMWJ, Bothmer-Quaedvlieg FCM, and Wouters EFM (1998) Prevalence of an elevated resting energy

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expenditure in patients with chronic obstructive pulmonary disease in relation to body composition and lung function. European Journal of Clinical Nutrition 52: 1–6. Creutzberg EC, Wouters EF, Vanderhoven-Augustin IM, Dentener MA, and Schols AM (2000) Disturbances in leptin metabolism are related to energy imbalance during acute exacerbations of chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 162: 1239–1245. Cunningham JJ (1991) Body composition as a determinant of energy expenditure: a synthetic review and a proposed general prediction equation. American Journal of Clinical Nutrition 54: 963–969. Durnin JVGA, Edholm OG, Miller DS, and Waterlow J (1973) How much food does man require? Nature 242: 418. Friedman JM and Halaas JL (1998) Leptin and the regulation of body weight in mammals. Nature 395: 763–770. Harris JA and Benedict EG (1919) A Biometric Study of Basal Metabolism. Washington: Carnegie Institute of Washington. Inui A (2001) Ghrelin: an orexigenic and somatotrophic signal from stomach. Nature Reviews: Neuroscience 2: 551–560. Klein PD, James WP, Wong WW, et al. (1984) Calorimetric validation of the doubly-labelled water method for determination of

energy expenditure in man. Human Nutrition: Clinical Nutrition 38(2): 95–106. Kojima M, Hosoda H, Date Y, et al. (1999) Ghrelin is a growthhormone-releasing acylated peptide from stomach. Nature 402: 656–660. McGregor M and Becklake MR (1961) The relationship of oxygen cost of breathing to respiratory mechanical work and respiratory force. Journal of Clinical Investigation 40: 971–980. Nguyen LT, Bedu M, Caillaud D, et al. (1999) Increased resting energy expenditure is related to plasma TNF-alpha concentration in stable COPD patients. Clinical Nutrition 18: 269–274. Schoeller DA and van Santen E (1982) Measurement of energy expenditure in humans by double-labeled water method. Journal of Applied Physiology 53: 955–959. Schols AMWJ, Creutzberg EC, Buurman WA, et al. (1999) Plasma leptin is related to proinflammatory status and dietary intake in patients with chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 160: 1220– 1226. Schols AMWJ, Fredrix EW, Soeters PB, Westerterp KR, and Wouters EFM (1991) Resting energy expenditure in patients with chronic obstructive pulmonary disease. American Journal of Clinical Nutrition 54: 983–987.

ENVIRONMENTAL POLLUTANTS Contents

Overview Diesel Exhaust Particles Oxidant Gases Particulate Matter, Ultrafine Particles Particulate and Dust Pollution, Inorganic and Organic Compounds Radon

Overview

Introduction

V Stone, Napier University, Edinburgh, UK K Donaldson, University of Edinburgh, Edinburgh, UK

History

& 2006 Elsevier Ltd. All rights reserved.

Abstract The adverse health effects of air pollutants include respiratory and cardiovascular morbidity and mortality. The major pollutants of concern in the early twenty-first century include particles (PM10) and gases such as ozone, oxides of nitrogen, sulfur dioxide, and carbon monoxide. Such pollutants are monitored widely and a number of authorities have generated exposure limit values or guidelines aimed to minimize the impact of these pollutants on the health of populations. Susceptibility to the health effects of pollutants varies widely according to age, health status, and socioeconomic standing. Inflammation and oxidative stress are common mechanisms in the induction of adverse health effects by a number of pollutants, suggesting that these pollutants could interact to enhance the biological effect.

The term ‘air pollution’ relates to any substance, gaseous, liquid or particulate, that contaminates the air. Air pollution is derived from a variety of sources, many of which are anthropogenic. In the early twentieth century most air pollutant sources were derived from industry and from the domestic burning of fossil fuels. High smoke stacks were introduced at this time to increase pollution dissipation with the belief that by the time it precipitated back down to ground level it would be sufficiently dispersed to be insignificant with respect to its health effects. A number of major pollution episodes occurred and were well-documented during this time. For example, a pollution episode in the Meuse Valley of Belgium in December 1930 resulted in the deaths of both humans and cattle. Autopsy results of those who

ENVIRONMENTAL POLLUTANTS / Overview 91

Figure 1 Smog in Zebo city, China, 2004. Photograph courtesy of J Philp and C Cunningham.

died revealed the airways to be irritated and congested with mucus. Perhaps, the most famous pollution episode occurred in London in December 1952 when bad smog developed due to the entrapment of pollutants at ground level by still, cold weather and a temperature inversion. The density of particles, measured as black smoke, reached 4000 mg cm 3 (compare with current levels observed in London of 25–40 mg m 3). The number of extra deaths caused by this pollution episode is debatable due to the subsequent emergence of an influenza episode, however, estimates range from 4000 to 12 000 people. In the mid-twentieth century many governments, including that of the UK, introduced a Clean Air Act, largely in response to episodes such as those described above. This Act limited the burning of fossil fuels in towns and cities for both industrial and domestic use. This has had a dramatic effect on air pollution, leading to an overall improvement in air quality, especially for sulfur dioxide and particulates. However, the profile of airborne pollutants encountered in towns and cities in the developed world has altered over the years due to changes in the predominant sources of pollutants, namely the large and progressive increase in traffic-derived pollution. Static industrial sources remain major contributors despite the introduction of a number of systems, such as filters, to minimize emissions to the atmosphere. Traffic pollution is composed of combustion-derived products including ultrafine or nanoparticles associated with metals and organic matter. In addition, traffic pollution includes gases such as carbon monoxide and

oxides of nitrogen. Pollution levels in some countries such as China however remain elevated generating visible smogs on a regular basis (Figure 1). Pollution Monitoring

Pollution levels for a spectrum of pollutants are monitored daily in many cities in many countries. For example, the European Union (EU) has published guidelines that state air pollution ‘limit values’ in an attempt to improve air quality. An EU First Daughter Directive (99/30/EC) gives the limit for each air pollutant considered, including hourly and annual means, together with a date by which these limit values must be achieved (Table 1). Each country within the EU now has an obligation to meet these objectives. In the US, air pollution is regulated by federal law of the 1990 Clean Air Act, although individual states can choose to have more stringent pollution controls. Each state has responsibility for its pollution control, since this area requires specialist knowledge of factors such as local industries, housing, and geography that effect pollution production and dissipation, and must generate a state implementation plan (SIP) to explain how the local pollution will be controlled, with each SIP being approved by the EPA.

Current Day Pollutants Pollutant Gases

The predominant pollutant gases found in many cities include ozone (O3), oxides of nitrogen (NOx), and

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ENVIRONMENTAL POLLUTANTS / Overview

Table 1 EU limit values and EPA air quality guidelines for the major pollutants encountered in the developed world Pollutant

Symbol

EU Air Quality Daughter Directive limit values

EPA National Ambient Air Quality Standards

Carbon monoxide

CO

10 mg m 3 8 h mean Target date 2005

40 mg m 10 mg m

3 3

1 h mean 8 h mean

Sulfur dioxide

SO2

350 mg m 3 1 h mean not to be exceeded 424 times per year 125 mg m 3 24 h mean not to be exceeded 43 times per year Target date 2005

316 mg m 3 24 h mean not to be exceeded 41 time per year 68 mg m 3 annual mean

Nitrogen dioxide

NO2

200 mg m 3 1 h mean 40 mg m 3 annual mean Target date 2010

100 mg m

3

Ozone

O3

120 mg m 3 8 h mean not to be exceeded 420 times per year Target date 2010

235 mg m 157 mg m

3

50 mg m 3 24 h mean not to be exceeded 435 times per year 40 mg m 3 annual mean Target date 2005

150 mg m 3 24 h mean not to be exceeded 41 time per year 50 mg m 3 annual mean 65 mg m 3 24 h mean 15 mg m 3 annual mean

Particles

PM10

PM2.5

3

WHO Air Quality Guidelines 3

11.6 mg m

8 h mean

266 mg m

3

15 min mean

annual mean

287 mg m

3

1 h mean

1 h mean 8 h mean

100 mg m

3

8 h mean

50 mg m

3

24 h mean

Limit values may be exceeded during specific adverse conditions, e.g., bad weather.

sulfur dioxide (SO2). These pollutants are described in more detail elsewhere in this encyclopedia. Briefly, nitrogen oxides (NOx) are a group of pollutant gases that include nitrogen dioxide (NO2) and nitric oxide (NO) formed by the high-temperature burning of fossil fuels; hence, in ambient air they are derived principally from traffic and industrial sources. Both O3 and NOx are oxidant gases, with O3 being produced by the action of ultraviolet sunlight on NOx and volatile organic compounds (VOCs) that are found in the air and are derived from combustion sources such as traffic pollution. The requirement for UV light to generate ozone means that this pollutant is most commonly associated with sunny climates; hence, busy cities in hot climates often experience high ozone levels. The development and fitting of three-way catalysts to petrol cars in order to meet legislative standards is responsible for a decline in NOx pollution in many countries. In the lungs, both O3 and NOx can generate free radicals that induce oxidative damage to the lung lining fluid and inflammation. In experiments on human volunteers NO2 at high concentration was found to decrease lung function and increase airway responsiveness, respiratory symptoms, and the response of asthmatics to allergens. However, the concentrations required to elicit such responses are greater than those typically found in the developed

world, and are slightly higher than the values recorded during high pollution episodes. Most studies have suggested a small short-term effect of NO2 on health, although the possibility that these effects are caused by another component of traffic pollution cannot be ruled out. Elevated levels of ozone can also induce a pulmonary inflammatory response and decrease lung function in humans. Symptoms observed on exposure to high levels of ozone include cough, chest pain, and breathing problems, as well as headaches and eye irritation. However, data from laboratory and epidemiological studies suggest that not all people have the same susceptibility to ozone with the effects being more pronounced in children than in adults. Those who are most susceptible are not exclusively asthmatic, although specific genotypes related to the production of antioxidants have been linked to ozone susceptibility. Sulfur dioxide differs from O3 and NOx in that it is not an oxidant gas. It is generated by the combustion of sulfur-containing fossil fuels including coal and gasoline petrol. During the London smog pollution episode, SO2 was a major pollutant, contributing to both the smog and many of the adverse health effects including respiratory irritation and toxicity. Legislation controlling the burning of fossil fuels in towns and cities, as well as the sulfur content of gasoline, has led to a significant

ENVIRONMENTAL POLLUTANTS / Overview 93

decline in SO2 levels over the last 50 years. As a consequence, far less emphasis is now placed on SO2 as an important gaseous pollutant in many towns and cities. Particulate Matter (PM10)

Prior to the 1990s, particulate air pollution was monitored via a variety of different procedures including total suspended particulate (TSP) and black smoke. In the 1990s a worldwide initiative introduced sampling by the PM10 convention, that is, the mass of particulate matter collected with 50% efficiency for particles of 10 mm aerodynamic diameter. This means that particles within PM10 in general possess a diameter of 10 mm or less; this is the point at which particles are small enough to be inhaled and deposited throughout the respiratory tract. The coarse fraction of PM10 (2.5–10 mm) tends to deposit in the upper airways, while the fine fraction of PM10 (less than 2.5 mm) can deposit throughout the whole respiratory system. A number of countries, including the US, now also regulate the level of PM2.5. The adverse health effects induced by inhalation of increased levels of PM10 are numerous and are described in more detail elsewhere in this encyclopedia. Epidemiological studies clearly indicate that acute effects include increased risk of hospital admission or death due to a respiratory or cardiovascular cause. Long-term adverse health effects induced by PM10 are also becoming more apparent and include lung cancer as well as the potential for increased risk of asthma. The acute effects tend to be most prominent in susceptible groups such as the elderly, and patients with pre-existing lung or cardiovascular disease. Acute effects on healthy individuals include a decrease in lung function, but occur only at much higher levels of PM10 than are usually measured in most towns and cities. As is the case with other pollutants such as ozone, the adverse effects of PM10 are believed to be the result of oxidative stress-mediated inflammation. PM10 (and PM2.5) is a cocktail of components including ultrafine or nanoparticles, usually derived from combustion processes, consisting of a carbon core that is often associated with a variety of metals, organic molecules, and biological molecules (e.g., bacterial endotoxin). PM10 also includes secondary particles (sulfates and nitrates) that are derived from photochemical reactions in the atmosphere, as well as biological particles (e.g., pollen and spores) and wind-blown dust. The composition of PM10 is variable over time and location, with primary sources varying between rural and urban

locations, among different towns and cities, and among countries. For example, highly industrialized cities or countries emit PM10 rich in combustion-derived nanoparticles and metals, although the type of fuel employed will tend to be low in sulfur. In contrast, developing countries may have fewer sources of pollution, but may use lower grade fuel and hence generate pollutant particles with a different chemical and size profile. Rural locations are often associated with coarse particles generated by wind-blown dust. The current conundrum for particle toxicologists is to determine which components within PM10 are responsible for inducing oxidative stress and for driving inflammation. However, this is extremely difficult since the composition is highly variable and interaction between components is likely to modulate their relative toxicity. There is substantial evidence that the primary combustion-derived nanoparticles are strongly involved in driving particle-induced inflammation. Other studies suggest that bacterial components such as endotoxin drive the PM10-induced inflammation. These differences may be accounted for by differences in sampling location, with rural locations being more dominated than cities by endotoxin-contaminated wind-blown dust. One of the major problems in this field is to obtain a particle sample that is representative of the particles inhaled by humans, since the sampling procedure often requires the capture of particles onto a filter. Recovery of particulate matter from the filter is rarely 100% efficient and may result in the preferential release or retainment of some components. Furthermore, the stable sampling and storage of volatile components is almost impossible, leading to their loss from the sample. The World Health Organization (WHO) has recommended that PM2.5 be routinely monitored, because epidemiological and toxicological studies suggest PM2.5 to be more closely associated with health effects than PM10. However, the WHO recognized the substantial body of evidence that some toxicological activity resides within the coarse fraction of PM10 and hence PM10 measurements should continue. Protocols to standardize PM2.5 sampling across Europe are currently under development and an EU standard for this pollutant has yet to be set. In the US, the EPA has set an air quality standard for PM2.5 of 65 mg m 3, 24 h mean. Other Pollutants of Importance

The list of pollutants discussed above is not exhaustive, but these are the main pollutants of concern at the present time. Other important pollutants include benzene, carbon monoxide, and sulfur dioxide.

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ENVIRONMENTAL POLLUTANTS / Overview

Benzene is a volatile organic compound that is classified as a genotoxic carcinogen. In ambient air, benzene is derived from the combustion and distribution of petrol. Benzene is a genotoxic carcinogen and so a safe level of exposure cannot be recommended. Substantial effort has gone into ensuring that emissions of benzene are minimal and the EU Air Quality Directive limit value target of 5 mg m 3 must be met by 2010. Current annual mean values for benzene in countries such as the UK are 16.5 mg m 3. Carbon monoxide (CO) is a gas that is generated from the incomplete combustion of fossil fuels. Historically, CO has been mostly associated with indoor sources of pollution such as ill-maintained gas appliances. In the indoor environment such appliances can rapidly generate sufficient CO to cause death through asphyxiation. CO irreversibly binds to hemoglobin preventing oxygen binding and transport around the body, resulting in ill health or death. The main outdoor sources of CO are again traffic exhaust fumes, particularly from petrol engines. Both the EU Air Quality Directive limit values and the USA National Ambient Air Quality Standards (NAAQS) for CO are set at 10 mg m 3 (9 ppm) as an 8 h mean. Values in countries such as the UK are currently in the order of 10 mg m 3 (8 h mean).

The Adverse Health Effects of Air Pollutants Historically, humans have always been exposed to respirable particles in the form of biological particles (e.g., bacteria), fire smoke, and wind-blown dust, and have developed effective defense mechanisms to clear these substances from the lung and body. Lung lining fluid and lung cells contain a number of antioxidants that provide protection against the oxidative effects of many pollutants such as PM10 and NO2. In addition, acidic pollutants can generally be neutralized by proteins and buffers within the lung lining fluid. Mucus and ciliated cells protect the upper airways of the respiratory tract from particulate air pollutants. Particles and pollutants landing in the mucus are trapped and cleared from the airways by the beating motion of the cilia, allowing the particles to move via this mucociliary escalator and to be swallowed or blown from the nose. Within the respiratory regions of the lung, particles are cleared by immune cells such as macrophages or neutrophils. Biological particles are easily degraded in the phagolysosome of such cells, however, inorganic particles must be physically carried from the lung surface, either into

the lymphatic system or via the mucociliary escalator described above. Gaseous pollutants will dissolve within the mucus or lung lining fluid of the lung and either react quickly with biological molecules or diffuse away. The adverse health effects of air pollutants are numerous but are frequently reported to include, especially for particles and ozone, the exacerbation of asthma symptoms in both children and adults. There are also data to suggest that some air pollutants, such as particles, actually increase the incidence of asthma. For example, children living near to a busy road, such as a motorway or freeway, are reported to have an increased risk of developing asthma. However, the body of data suggesting that pollution causes asthma is less convincing than that indicating it enhances asthma symptoms. However, the potential ability of air pollution to cause development of asthma cannot be ruled out. In relation to other causes of asthma, air pollution is not likely to be a major factor, but could contribute to other causes to increase the risk of developing this disease. Effects of pollutants on lung function in healthy people are not usually related to asthma. Such effects are relatively less important since they only truly occur at very high exposure levels, greater than those typically achieved in the ambient air environment. Chronic obstructive pulmonary disease (COPD), an inflammatory airway disease most commonly observed in smokers, is made worse by exposure to pollutants such as particles. People suffering from cardiovascular disease are also more vulnerable to air pollution, resulting in an increased incidence of heart attacks and strokes. Both respiratory and cardiovascular effects can result in hospitalization or even death. A number of cohort studies suggest that particulate air pollution shortens life expectancy by 1–2 years, although these effects are not uniformly distributed throughout the population and depend upon a number of susceptibility factors.

Susceptibility to Air Pollutants Not all individuals have the same susceptibility to air pollution; the very old and the very young, as well as those suffering from a pre-existing disease appear to be the most vulnerable. In 2004, the WHO published a monograph on the vulnerability and susceptibility of children to air pollution. The WHO concluded that children are more susceptible than adults due to the ‘‘ongoing process of lung growth and development, incomplete metabolic systems, immature host defenses, and high rates of infection

ENVIRONMENTAL POLLUTANTS / Overview 95

by respiratory pathogens.’’ Pre- and postnatal lung development, as well as the efficiency of detoxification systems, was reported to exhibit time-dependent development, which might partly account for their enhanced susceptibility. The report also suggested that children are exposed to a higher level of air pollution than adults due to their activity patterns. The WHO report concluded that the adverse effects of pollution in children include longer term effects such as a lesser maximal functional capacity on reaching adulthood. However, not all children are the same, and the WHO recognized that the most susceptible individuals are those with ‘‘underlying chronic lung disease, particularly asthma.’’ The unborn child and infant are also at risk from air pollution, with evidence suggesting a causal link between particulate air pollution and respiratory deaths in the postneonatal period. The adverse health effects of indoor air pollution are not within the scope of this article, however, exposure to high levels of indoor air pollutants, such as environmental tobacco smoke or occupational dusts, may increase susceptibility of adults and children to outdoor pollutant exposure. Socioeconomic factors such as education and diet might also influence susceptibility to air pollution, and might partly explain why life expectancy is shorter in people from disadvantaged population groups.

Common Mechanisms of Toxicity Of the pollutants discussed above, particles and ozone pose the greatest health threat and so have received the greatest scientific interest with respect to adverse health effects. These pollutants demonstrate significant health effects in both epidemiological and toxicological studies. As described above ozone and particles induce oxidative stress that stimulates an inflammatory response, and the combination of the oxidative stress and inflammation is thought to produce the disease and ill health. Ozone is a highly reactive oxidizing gas that quickly oxidizes biological molecules that it comes into contact with, leading to oxidative stress. Many studies also provide evidence that PM10 and ultrafine particles can generate free radicals and reactive oxygen species (ROSs) in vitro and in vivo. The ultrafine or nanoparticles have a huge surface area per unit mass over which they can interact with biological molecules, causing oxidation. However, PM10 also contains other components capable of generating ROSs, including transition metals and quinone molecules. Transition metals, in the presence of an oxidant such as hydrogen peroxide, will redox

cycle by Fenton chemistry to generate hydroxyl radicals, one of the most potent forms of ROS found in the body. There is also evidence that metals can interact with ultrafine particles to potentiate the production of ROSs in vitro and inflammation in vivo. Some types of quinone molecules found within PM10 also have the capacity to redox cycle in cells, generating superoxide anion radicals and other ROSs. In addition, some quinones can arylate protein thiol groups. Since protein thiol groups play a critical role in antioxidant defense mechanisms, such as in the antioxidant glutathione, arylation of these groups by quinines may increase the susceptibility of the cells to the effects of ROSs and hence oxidative stress. Oxidative stress involves a perturbation of the antioxidant defense mechanism of the lung, allowing ROSs and free radicals to persist and cause damage to proteins, lipids, and DNA. Damage to proteins and lipids results in altered cell function that can range, according to the scale of the oxidative stress, from activation of the cell to elicit a proinflammatory response to cell death. The proinflammatory response entails the production of mediators, usually cytokines, which stimulate the recruitment and activation of immune cells such as macrophages and neutrophils. Inflammation is a normal healthy process that is required to clear foreign objects, including bacteria and pollution particles, from the body. If the inflammation is acute, then the inflammation is resolved and normal tissue is regenerated. However, if the inflammation is disproportionately large, prolonged, or is superimposed on top of a pre-existing inflammation then the result is ill health. Oxidative stress induced by pollutants can stimulate inflammation via redox-sensitive signaling pathways that culminate in activation of transcription factors such as nuclear factor kappa B (NF-kB). In an unstimulated form NF-kB resides in the cell cytoplasm associated with an inhibitor protein IkB. Via an unknown mechanism, oxidative stress stimulates the activation of NF-kB that involves the release of IkB allowing NF-kB to migrate into the nucleus where it binds to the promoter regions of proinflammatory genes in order to initiate their transcription. A number of other signaling pathways that control cytokine gene expression are also sensitive to oxidative stress; such pathways include cytosolic calcium. Cytosolic calcium has been shown to be important in the upregulation of cytokine production by macrophages treated with particles. For example, in macrophages calcium is involved in stimulating the production of tumor necrosis factor alpha by ultrafine particles, and interleukin 1 by PM10. The relationship between calcium and oxidative stress is

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ENVIRONMENTAL POLLUTANTS / Diesel Exhaust Particles

complicated. Many studies suggest that ROSs can stimulate calcium signaling; they also suggest that calcium signaling can stimulate cells to produce endogenous ROSs. Since both ozone and particles induce oxidative stress and inflammation, it is possible that these pollutants may interact, either enhancing or altering the subsequent health effects. Interactions among other pollutants are also possible, with the toxic effects of one pollutant making the lung more vulnerable to the effects of the other. Investigation of such interactions is difficult. A number of epidemiological studies have struggled to attribute adverse health effects to a single pollutant, since increases in a number of pollutants often occur concurrently. For example, traffic pollution enhances both NO2 and PM10 levels, while hot sunny weather enhances ozone and PM10 levels. Toxicological studies are required to investigate the plausibility of such interactions.

Conclusion Much of the information provided here pertains to the ambient environment of the developed world and clearly illustrates a link between air pollution and disease. The developing world frequently encounters higher pollution levels with a different composition. It is possible that the lessons learned by the developed world could be used to minimize the health impact of pollutants during financial and industrial development of other countries. However, since often finances are limited and ambitions great, other issues are likely to be considered to be of greater short-term importance (Figure 1). See also: Carbon Monoxide. Chronic Obstructive Pulmonary Disease: Overview. Environmental Pollutants: Diesel Exhaust Particles; Oxidant Gases; Particulate Matter, Ultrafine Particles; Particulate and Dust Pollution, Inorganic and Organic Compounds; Radon.

C, and Whyte G (eds.) ABC of Sports Medicine, 3rd edn., pp. 67–70. London: BMJ Books. Holgate S, Samet JM, Koren HS, and Maynard RL (eds.) (1999) Air Pollution and Health: Academic Press. Kelly FJ, Dunster C, and Mudway I (2003) Air pollution and the elderly: oxidant/antioxidant issues worth consideration. European Respiratory Journal 40: 70s–75s. Mehta S (2003) Air pollution and health in rapidly developing countries. Bulletin of the World Health Organization 81(10): 771. Stone V, Wilson M, Lightbody J, and Donaldson K (2003) Investigating the potential for interaction between the components of PM10. Environmental Health Preventative Medicine 7: 246–253. Wilson R (1996) Introduction. In: Wilson R and Spengler J (eds.) Particles in our Air. Concentrations and Health Effects, pp. 1–14. Harvard University Press.

Relevant Websites http://www.defra.gov.uk – This web link provides a full description of the air quality strategy for England, Scotland, Wales, and Northern Ireland as written by representatives of the UK government. It also provides access to figures relating to the actual current and historical pollution values. http://www.dh.gov.uk – This website provides access to UK Policy guides, research reports and other DH-published documents about air pollution. Details regarding UK funded air pollution research projects are also listed. A link to the committee on the Medical Effects of Air Pollution (COMEAP) is also provided. http://www.epa.gov – This website provides details of the US Environmental Protection Agency Air Quality Standards and includes links to the Clean Air Act. http://www.euro.who.int – Air pollution information collected and reported by the World Health Organization. http://www.scielosp.org – SciELO Public Health is an electronic library online covering health science articles published by scientific journals.

Diesel Exhaust Particles H-E Wichmann, GSF – Institute of Epidemiology, Neuherberg, Germany & 2006 Elsevier Ltd. All rights reserved.

Further Reading

Abstract

Air Quality Expert Group (AQEG) (2004) Nitrogen Dioxide in the United Kingdom. London: Department of Environment, Food and Rural Affairs Publications. Bell ML, Davis DL, and Fletcher T (2004) A retrospective assessment of mortality from the London smog episode of 1952: the role of influenza and pollution. Environmental Health Perspectives 112: 6–8. Donaldson K, Stone V, Borm PJ, et al. (2003) Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM(10)). Free Radical Biology and Medicine 34: 1369– 1382. Florida-James GD, Donaldson K, and Stone V (2005) Sports performance in a polluted environment. In: Harries M, Williams

Diesel motor emission is a complex mixture of hundreds of constituents in either gas or particle form. Diesel particulate matter (DPM) is composed of a center core of elemental carbon and adsorbed organic compounds including PAHs and nitroPAHs, and small amounts of sulfate, nitrate, metals, and other trace elements. DPM consists of fine particles including a high number of ultrafine particles. These particles are highly respirable and have a large surface area where organics can adsorb easily. Exposure to DPM can cause acute irritation, neurophysiological, respiratory and asthma-like symptoms and can exacerbate allergenic responses to known allergens. Consistently lung cancer risk is elevated among workers in occupations where diesel engines have been used. However, quantification of

ENVIRONMENTAL POLLUTANTS / Diesel Exhaust Particles 97 the cancer risk with respect to DPM concentrations is not possible. Furthermore, ambient fine and ultrafine particles, of which DPM is an important component, contribute to cardiopulmonal morbidity and mortality, and lung cancer. In conclusion, diesel exhaust poses a cancer risk greater than that of any other air pollutant, as well as causing other short- and long-term health problems. One effective way to effectively reduce emission of DPM are particle traps.

Solid carbonaceous/ ash particle with adsorbed hydrocarbon/ sulfate layer

Introduction Sulfuric acid particles

Diesel motor emission (DME) and especially diesel particulate matter (DPM) play an important role in the discussions regarding the risk to health posed by pollutants from ambient air or in the workplace. Several international and national bodies have dealt with the evaluation of composition, exposure, health effects, and risk assessment, as well as taking steps to prevent possible health risks by setting environmental standards for both emission and ambient concentrations.

Hydrocarbon/ sulfate particles

0.2 µm

Composition and Physicochemical Properties DME is a complex mixture of hundreds of constituents in either gas or particulate form. Gaseous components of DME include carbon monoxide, nitrogen compounds, sulfur compounds, and numerous lowmolecular-weight hydrocarbons such as aldehydes, benzene, polycyclic aromatic hydrocarbons (PAHs), and nitro-PAHs. The particles present in DME are composed of a central core of elemental carbon and adsorbed organic compounds, as well as small amounts of sulfate, nitrate, metals, and other trace elements. DPM consists of fine particles (particulate matter (PM2.5) including a high number of ultrafine particles (o0.1 mm diameter) (Figure 1)). Collectively, these particles have a large surface area, which makes them an excellent medium for adsorbing organics. Also, their small size makes them highly respirable and they have the potential to reach the deep lung and even the blood circulation. The organics, in general, range from about 20% to 40% of the particle mass. Many of the organic compounds present on the particle and in the gases are individually known to have mutagenic and carcinogenic properties. The typical chemical composition of DPM from new heavy duty diesel vehicle exhaust is shown in Table 1: 75% (33–90%) elemental carbon, 19% (7–49%) organic carbon, 2% (1–5%) metals and elements, 1% (1–4%) sulfate and nitrate, and 3% (1–10%) other.

Sources and Exposure The most important sources of DME relevant for the general population are on-road vehicles such as

Figure 1 Schematic diagram of diesel engine exhaust particles. Reproduced from US-EPA (2002) Health effects assessment document for diesel engine exhaust. EPA/600/8-90/057F, May 2002.

Table 1 Typical chemical composition of fine particulate matter in the US

Elemental carbon Organic carbon Sulfate, nitrate, ammonium Minerals Unknown

Eastern US

Western US

Diesel PM2.5

4% 21% 48%

15% 39% 35%

75% 19% 1%

4% 23%

15%

2% 3%

Reproduced from US-EPA (2002) Health effects assessment document for diesel engine exhaust. EPA/600/8-90/057F, May 2002.

passenger cars and heavy-duty and light-duty vehicles. However, nonroad diesel engines in locomotives, marine vessels, heavy-duty equipment, etc. also play a role, especially in exposure at the workplace. DME varies substantially in chemical composition and particle sizes among different engine types, engine operating conditions (idle, acceleration, deceleration), and fuel formulations (high/low sulfur fuel). The mass of particles emitted and the organic components on the particles have been reduced over recent years. The toxicologically relevant organic compounds of DME (e.g., PAHs and nitro-PAHs) emitted from older vehicle engines are still present in emissions from newer engines, though relative amounts have decreased.

98

ENVIRONMENTAL POLLUTANTS / Diesel Exhaust Particles

DPM mass (expressed as micrograms of DPM per cubic meter) has historically been used as a surrogate measure of exposure for whole DME. Although uncertainty exists as to whether DPM is the most appropriate parameter to correlate with human health effects, it is considered a reasonable choice until more definitive information about the mechanisms of toxicity or mode(s) of action of DME becomes available. A large percentage of the population is exposed to ambient PM2.5 and ultrafine particles, of which DPM is an important constituent. Exposure estimates for the early to mid-1990s in the US suggest that national annual average DME exposure from on-road engines alone was in the range of about 0.5–0.8 mg DPM m 3 of inhaled air in many rural and urban areas, respectively. Exposures could be higher if there was a nonroad DME source that added to the exposure from on-road vehicles. For example, it has been estimated that in the US, on a national average basis, nonroad DME emissions are mighter than on-road emissions. For localized urban areas where people spend a large portion of their time outdoors, the exposures are higher and, for example, may range up to 4.0 mg DPM m 3 of inhaled air. In some urban situations the annual average fraction of PM2.5 attributable to DPM (on a mass basis) is about 35% at the high end, although for the US the proportion appears to be more typically in the range of 10%. The situation in Europe is different since diesel passenger cars are used more frequently. For Germany it has been estimated that on-road traffic generates 3.6 mg DPM(EC) m 3 in urban areas and 1.8 mg DPM(EC) m 3 in rural areas. On average, in Germany on-road cars are estimated to contribute about 3 mg PM2.5 m 3, which corresponds to about 20% of the mean PM2.5 concentration.

There is also evidence for an immunologic effect – the exacerbation of allergenic responses to known allergens and asthma-like symptoms. A large number of epidemiological studies have been performed to investigate the health effects of ambient fine (PM2.5) and ultrafine particles. They show impact on respiratory and cardiovascular endpoints such as daily mortality, hospital admissions, increase of respiratory symptoms, increased numbers of myocardial infarction, and changes of inflammatory and ECG parameters. However, the contribution of DPM has not been investigated specifically in these studies. Studies in which volunteers have been exposed to concentrated ambient air have demonstrated airway effects and changes in the heart and blood vessel system that link to the adverse health effects found in the epidemiological studies. They also provide mechanistic and other toxicological information that helps explain the adverse health effects of air pollutants. The toxicological database on PM-related health effects analyzed in animal experiments is not yet able to provide definite answers regarding causal factors, whether relating to physical parameters, chemical components, or biological aspects. Nevertheless, clear differences have been identified as to induction of airway inflammatory response, heart/blood vessel and allergen adjuvant effects, and between ambient PM samples from different locations. Cellular and mechanistic studies provide important information about how various particles and components related to air pollution interact with cells and cellular systems. The studies describe inflammatory responses and provide support for the hypothesis that very small particles (ultrafine particles) are an especially harmful component, but suggest that the more coarse and intermediate-sized PM also is important.

Health Effects Available evidence indicates that there are human health hazards associated with exposure to DME. The hazards include acute exposure-related symptoms, chronic exposure-related noncarcinogenic effects, and lung cancer. Acute (Short-Term Exposure) Effects

Information is limited for characterizing the potential health effects associated with acute exposure to DME. However, on the basis of available human and animal evidence, it is concluded that acute or shortterm (e.g., episodic) exposure to DME can cause acute irritation (e.g., eye, throat, bronchial), neurophysiological symptoms (e.g., lightheadedness, nausea), and respiratory symptoms (cough, phlegm).

Chronic (Long-Term Exposure) Noncarcinogenic Effects

Information from the available human studies is inadequate for a definitive evaluation of possible noncancer health effects from chronic exposure to DME. However, on the basis of extensive animal evidence, DME poses a chronic respiratory hazard to humans. Chronic-exposure, animal inhalation studies show a spectrum of dose-dependent inflammation and histopathological changes in the lung in several animal species including rats, mice, hamsters, and monkeys. In epidemiological studies it has been shown that long-term exposure to ambient PM2.5 affects morbidity and mortality. In children, the lung function was impaired as well as the age-dependent lung

ENVIRONMENTAL POLLUTANTS / Diesel Exhaust Particles 99

growth; the prevalence of bronchitis, sinusitis, and common cold was increased and the effects were reduced with reduced exposure. Some evidence is described for increased infant mortality in areas of high exposure. In adults, effects on chronic bronchitis and chronic obstructive pulmonary disease (COPD) were found. In addition, there is strong evidence that long-term exposure to PM2.5 significantly increases the risk for total mortality and cardiopulmonary mortality. Furthermore, in a European study significantly increased mortality was observed close to busy roads suggesting an adverse influence of PM emitted from vehicles, 90% of which are DPM. Chronic (Long-Term Exposure) Carcinogenic Effects

The US-EPA, like several other boards, concludes that DME is ‘‘likely to be carcinogenic to humans by

0.5

inhalation.’’ This conclusion is based on the totality of evidence from human, animal, and other supporting studies. There is considerable evidence demonstrating an association between DME exposure and increased lung cancer risk among workers in varied occupations where diesel engines historically have been used (Figure 2). Most of the individual studies and all meta-analyses showed an increased risk of lung cancer in occupations with exposure to DME. However, in most studies only data on job title and duration of stay at a workplace with exposure to DME were available. Quantification of the exposure to DPM concentration was not possible in these studies, and this is the main reason why DME has not yet been classified as carcinogenic to humans. At ambient PM2.5 concentrations also a significantly increased risk of lung cancer mortality was observed for people living in cities with exposure to high versus low concentrations of fine particulates. In

RR estimates & 85% Cl 1 1.5

2

All studies

Case–control studies

Colhort studies Internal comparison population External comparison population Smoking adjusted

Smoking not adjusted Sub-analysis by occupation Railroad workers

Equipment operators

Truck drivers

Bus workers Figure 2 Pooled relative risk (RR) estimates and heterogeneity-adjusted 95% confidence intervals (CI) for all studies and subgroups of studies included. Reproduced from Bhatia R, Lopipero P, and Smith AH (1998) Diesel exhaust exposure and lung cancer. Epidemiology 9: 84–91, with permission from Lippincott Williams & Wilkins.

100 ENVIRONMENTAL POLLUTANTS / Diesel Exhaust Particles

addition to the human evidence, there is supporting evidence of DPM’s carcinogenicity and associated DPM organic compound extracts in rats and mice by noninhalation routes of exposure. Other supporting evidence includes the demonstrated mutagenic and chromosomal effects of DME and its organic constituents, and evidence that suggests bioavailability of the DPM organics in humans and animals. Although chronic high-exposure rat inhalation studies showed a significant lung cancer response, according to the US-EPA this is not thought to be predictive of a human hazard at lower environmental exposures. The response in the rat experiments mainly resulted from an overload of particles in the lung due to high exposure, and such an overload is not expected to occur in humans exposed to environmental levels.

Possible Regulatory Steps In 2003 a report by the American Lung Association and Environmental Defense concluded that ‘‘Diesel exhaust contains a host of harmful contaminants that together pose a cancer risk greater than that of any other air pollutant, as well as causing other short and long term health problems.’’ A easy and quick way to reduce DPM might be the obligatory fitting of particle traps to all diesel engines. Based on an epidemiologically derived exposure–response relationship for PM2.5 and mortality, additional data on ambient particle concentrations, and estimates on the contribution of DPM, the positive health effects of the use of particle traps have been estimated for Germany. As a result, approximately 1–2% of the annual deaths have been attributed to DPM from on-road vehicles. Using particle traps would prevent most of these premature deaths and would lead to an expected mean increase in life expectancy of 1–3 months. In Europe diesel cars are very popular. For example, in Germany the percentage of newly registered diesel passenger cars increased from 5% in 1980 to 38% in 2003, and in 2020, diesel vehicles are expected to account for 36% of total vehicle kilometers. On the one hand, total emission of DPM has decreased by 40% in the last 10 years and will decline further due to measures taken for heavy-duty vehicles; on the other hand, DPM and nitric oxide emissions from diesel passenger cars would stagnate if limit values remained unchanged. Even with the introduction of better standards for the particulate mass, the number of ultrafine particles could not be controlled, and these extremely small particles pose a potential health risk that is not yet clearly understood. The carcinogenic potency of DME from current car models without particulate traps is 10 times

higher than that of petrol engine exhaust. Particle traps have the potential to solve all the problems outlined above as they are very effective at retaining coarse, fine, and ultrafine particulates. See also: Environmental Pollutants: Overview; Particulate and Dust Pollution, Inorganic and Organic Compounds. Particle Deposition in the Lung.

Further Reading American Lung Association, Environmental Defense (2003) Closing the Diesel Divide – Protecting Public Health from Diesel Air Pollution. Bhatia R, Lopipero P, and Smith AH (1998) Diesel exhaust exposure and lung cancer. Epidemiology 9: 84–91. Bru¨ske-Hohlfeld I, Mo¨hner M, Ahrens W, et al. (1999) Lung cancer risk in male workers occupationally exposed to diesel motor emissions in Germany. American Journal of Industrial Medicine 36: 405–414. Cohen AJ (2000) Outdoor air pollution and lung cancer. Environmental Health Perspectives 108: 743–750. Garshick E, Laden F, Hart JE, et al. (2004) Lung cancer in railroad workers exposed to diesel exhaust. Environmental Health Perspectives 112(15): 1539–1543. Health Effects Institute (HEI) (1999) Diesel Emissions and Lung cancer: Epidemiology and Quantitative Risk Assessment. A Special Report of the Institute’s Diesel Epidemiology Expert Panel. Heinrich J and Wichmann HE (2004) Traffic related pollutants in Europe and their effects on allergic disease. Current Opinion in Allergy and Clinical Immunology 4: 341–348. Lipsett M and Campleman S (1999) Occupational exposure to diesel exhaust and lung cancer: a meta-analysis. American Journal of Public Health 89: 1009–1017. Pope CA, Burnett RT, Thun MJ, et al. (2002) Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution. Journal of the American Medical Association 287: 1132–1141. Riedl M and Diaz-Sanchez D (2005) Biology of diesel exhaust effects on respiratory function. Journal of Allergy and Clinical Immunology 115(2): 221–228 (quiz 229). UBA: Umweltbundesamt – Federal Environmental Agency Germany (2003) Future Diesel. US-EPA (2002) Health effects assessment document for diesel engine exhaust. EPA/600/8-90/057F, May 2002. US Environmental Protection Agency (2004) Air quality criteria for particulate matter. Washington EPA/600/P-99/D02bF. WHO (2003) Health aspects of air pollution with particulate matter, ozone and nitrogen dioxide. Report from WHO Working Group meeting, 13–15 January, Bonn. Copenhagen: WHO Regional Office for Europe. Wichmann HE (2004) Positive health effects by the use of particle traps in Diesel cars – risk assessment for mortality in Germany (in German). UmweltmedForschPrax 9: 85–99.

Relevant Websites www.environmentaldefense.org – This website is maintained by Environmental Defense Fund (EDF). It provides information on environmental rights of all people, including future generations. www.unweltdaten.de – This website provides information on environmental planning and sustainability strategies, health-related environmental protection, chemical and biological safety, etc.

ENVIRONMENTAL POLLUTANTS / Oxidant Gases 101

Oxidant Gases M Nickmilder and A Bernard, Catholic University of Louvain, Brussels, Belgium & 2006 Elsevier Ltd. All rights reserved.

Abstract Ozone, nitrogen dioxide, chlorine, and trichloramine are the principal oxidant gases to which man can be exposed in the work or general environment. Ozone is the main oxidant produced in the troposphere through UV-driven reactions involving nitrogen oxides and volatile organic compounds. Nitrogen dioxide is an indoor and outdoor pollutant generated by all combustion processes when at high temperatures oxygen combines with nitrogen. Chlorine is a corrosive gas mainly used in industry but is also released by some cleaning and disinfecting products. Trichloramine is a volatile by-product of water chlorination that contaminates the air of indoor swimming pools and various other indoor environments. All these oxidant gases are potential irritants to the eyes, the upper respiratory tract, and the lungs. Depending on the inhaled dose and the individual susceptibility, they can produce a wide spectrum of short-term effects including irritation symptoms, lung function impairment, airways inflammation, asthma exacerbation, and in extreme cases pulmonary edema and even death. The long-term effects of these gases are much less well documented although there is little doubt that repeated or chronic exposures to high levels of these oxidants are detrimental to the lung tissue and may lead to lung disorders, especially asthma or asthma-like syndromes (e.g., reactive airway dysfunction syndrome caused by chlorine). No specific antidote exists for these pulmonary irritants. Treatment is basically supportive according to the clinical manifestations.

Introduction

considered as a transboundary problem. Current background levels are in the range of 40–70 mg m
3 (0.02–0.035 ppm) but, as a result of its photochemical origin, it displays strong seasonal and diurnal patterns. In some areas, O3 can reach very high concentrations during the summer months (e.g., up to 41000 mg m
3 in Mexico city). NO2 is a common pollutant of ambient and indoor air and also an occupational hazard for some professions (e.g., welders). Its main indoor source is unvented combustion appliances. NO2 in ambient air largely derives from the oxidation of nitrogen monoxide, the major sources of which are combustion emissions, mainly from vehicles. Current levels of NO2 are in the order of 10–50 mg m
3 but may reach several hundreds mg m
3 in areas affected by high pollution episodes. It is involved in a number of physicochemical reactions in the atmosphere, some of them leading to the formation of harmful secondary air pollutants such as O3 and nitric acid. Cl2 is an important raw material for the chemical industry. The general population can be exposed to chlorine gas in cases of accidental leakage from industrial facilities and also when using household chlorine bleach, which can release some chlorine gas. Cl2 reacts with water to form mainly hypochlorous acid (HClO) depending on the pH. NCl3 is a volatile by-product of water chlorination produced when hypochlorous acid released from

OH•

The principal oxidant gases to which man can be exposed in the environment are ozone (O3), nitrogen dioxide (NO2), chlorine (Cl2), and nitrogen trichloride or trichloramine (NCl3). Their physicochemical properties are shown in Table 1. O3 is formed by photochemical reactions in the presence of precursor pollutants such as nitrogen oxides and volatile organic compounds (Figure 1). Because of its high reactivity with other oxidizable pollutants (e.g., NO) or materials, O3 is usually present at lower levels in indoor compared to outdoor air and in busy urban centers compared to suburban and adjacent rural areas. O3 is also subject to long-range atmospheric transport and is therefore

RCH3

O2 •

RCH2

OH•

NO RCOO•

RCO•

RCHO

NO2

O + NO

O2

NO2 + O2

O3

Figure 1 Ozone formation in photochemical smog. RCOO, radical peroxy; OH, radical hydroxy; NO, nitrogen oxide; NO2, nitrogen dioxide; O3, ozone.

Table 1 Physicochemical properties of oxidant gases

Ozone Nitrogen dioxide Chlorine Nitrogen trichloride

Formula

MW

MP ( 1C)

BP ( 1C)

SW (g/100 g)

Color

Odor

O3 NO2 Cl2 NCl3

48 46 71 120


193
11
101
40


112 21
34.5 71

3  10
5 (Very low solubility) Low solubility 7.3  10
1 (Slightly soluble) Virtually insoluble

Colorless to blue Reddish to brown Greenish yellow Brownish yellow

Pungent Pungent Suffocating Swimming pool

MW, molecular weight; MP, melting point; BP, boiling point; SW, solubility in water.

102 ENVIRONMENTAL POLLUTANTS / Oxidant Gases

chlorine-based disinfectants reacts with nitrogenous compounds. NCl3 is particularly concentrated in the air of poorly ventilated indoor pools where it can reach concentrations of more than 1000 mg m
3. This gas frequently contaminates many other indoor areas where chlorine bleach is used as a cleaning or disinfecting agent.

Clinical Consequences O3 toxicity depends on its concentration, exposure duration, and respiratory rate of an individual. Because of its low solubility in water, it is carried over into the deep lung. The greatest deposition and the most severe damage are located mainly in terminal and respiratory bronchioles where O3 causes oxidative stress injury to surfactant components and epithelial cells. Short-term effects of O3 include upper respiratory tract irritation symptoms, pulmonary function changes, increased airway responsiveness, and airway inflammation, characterized by both an influx of inflammatory cells and an increased permeability of the air–blood barrier associated with an increased flux of proteins across it (i.e., CC16 and albumin) (Figure 2). The mechanism by which O3 increases the epithelial permeability appears to involve mainly the tight junctions between cells lining the respiratory epithelium, which are disrupted primarily as a consequence of oxidative damage and secondarily by proinflammatory mediators.

In children and adults, a decrease in pulmonary function can occur after only short-term exposure to O3 concentrations in the range 120–240 mg m
3; for lung inflammation, a threshold of around 135 mg m
3 has recently been estimated. Asthmatic subjects are generally not more sensitive to O3 than healthy subjects with regard to airway narrowing and hyperreactivity but they develop a more pronounced airway inflammation. However, the pulmonary response to O3 shows a great interindividual variability, which can be explained by differences in the lung protective mechanisms but also by other factors such as age, obesity, diet, or exposure to other lung toxicants (e.g., cigarette smoke). In studies where exposure to O3 was repeated, some health effects such as lung function decline and inflammatory reaction showed a progressive attenuation. However, this adaptation should not be interpreted as an intrinsic positive response, as it may impair or prevent the normal physiological responses and the ability of the lung to face new challenges such as infectious or allergic agents. In addition, there is some evidence to suggest that exposure to O3 can enhance the inflammatory response to allergens, viruses, and particles. The long-term effects of O3 are much less well documented. At levels currently observed in ambient air, the evidence linking O3 exposure to asthma incidence and prevalence in children or adults is not consistent. There is some epidemiological evidence

Lymphatic drainage

Lymphatic drainage Airways

Ep

In

En

Ep Blood

Airways

In

En

Blood

O3 Albumin

Albumin

CC16

Albumin

CC16

CC16

O3

(a)

Lymphatic drainage

(b)

Lymphatic drainage

Figure 2 Passage of albumin and the lung-specific Clara cell protein (CC16) across the different barriers separating the airways from the blood at the level of a terminal bronchiole under normal conditions (a) and after acute exposure to O3 (b). O3 causes an increased permeability of the epithelial barriers to proteins, resulting in an increased leakage of plasma albumin into the airways and, in the opposite direction, of CC16 into the interstitium from which it is cleared by lymphatic drainage or directly into the blood across the endothelium. The thickness of the arrows is not proportional to the concentration of protein but is used to illustrate the relative permeability of the different barriers and the increased fluxes casued by O3 exposure. En, endothelium; Ep, epithelium; In, interstitium.

ENVIRONMENTAL POLLUTANTS / Oxidant Gases 103

suggesting that long-term O3 exposure could reduce lung growth in children. The suggestion that prolonged O3 exposure causes chronic damage to human lung is supported by animal studies. At different levels of O3 exposure, animals show various biological responses including inflammation and increased airways permeability, morphological alterations in the lung, increased susceptibility to bacterial infections, increased production of certain lung proteins active in oxidant defenses, and increase in collagen content. Furthermore, the susceptibility of lung to O3 is remarkably species and strain dependent. The effects of NO2 on health are more dependent on the level of exposure than on the duration. It is a highly reactive, poorly water-soluble gas. Biologically, its effects result primarily from a reaction with components in the cell lining fluid at various sites in the respiratory tract, such as the region reaching the distal airways and air sack spaces, resulting in the formation of reactive oxygen that can damage cells or stress them to release proinflammatory substances. Short-term acute effects of NO2 include lung function impairment and increase in bronchial hyperresponsiveness. Subjects with asthma are much more sensitive to NO2 exposure than healthy subjects. They show an increase of bronchial hyperresponsiveness after 180 mg m
3 of NO2, whereas healthy subjects require a 10-fold higher level to elicit the same response. In allergic asthmatics, NO2 can also enhance the response to allergens but only at concentrations that can be attained in some indoor environments (e.g., houses with unvented gas stoves). In subjects with chronic obstructive diseases, a decline of lung function has been demonstrated at a NO2 exposure of 540 mg m
3 but no threshold has really been established for this population. In nonallergic subjects, exposure to NO2 can cause a shift towards an allergic immune cell response in the cells of the airway wall, but this occurs only at very high levels ranging from a thousand to several thousand mg m
3 over several hours. In animals, long-term exposures to NO2 concentrations higher than those encountered in ambient air produce changes in lung structure and increase susceptibility to microbial lung infections. In humans, long-term exposure to NO2 may decrease lung function and increase the risk of respiratory symptoms. NO2 also exacerbates the symptoms of asthma, and this effect appears to be greater in children than in adults. However, ambient NO2 often has complex interrelations with other pollutants, such as particulate matter and O3, making it difficult to disentangle its effect per se. Furthermore, because homes with gas appliances have peak NO2 levels

causing clinical effects, the observed effects cannot always be clearly attributed to either the repeated short-term high-level exposures or long-term exposures (or possibly both). Cl2 is a potential irritant gas to the eyes, upper respiratory tract, and the lungs. Because of its solubility in water, Cl2 can damage both the airways and the alveolar–capillary structures. At physiological pH, Cl2 combines with tissue water to form mainly HClO, which diffuses into cells and forms chloramines by reaction with amino acids or proteins. These derivatives are believed to be largely responsible for the tissue damage and inflammation caused by chlorine gas. Acute exposure to low concentrations (1–10 ppm) may cause sore throat, coughing, and eye and skin irritation. At higher levels, Cl2 causes burning of the eyes and skin, chest pain, vomiting, dypsnea, wheezing, cough, pneumonitis, and pulmonary edema. Exposure to even higher levels can produce severe injuries leading to death. Some people may develop a type of asthma due to irritating effects of Cl2, called reactive airways dysfunction syndrome (RADS). Chronic exposure of animals to Cl2 produces decreased body weight gain, eye and nose irritation, non-neoplasic lesions, and respiratory epithelial hyperplasia. Workers chronically exposed to Cl2 complain of eye irritation and respiratory effects such as throat irritation and airflow obstruction. Except in cases of industrial accident, the general population is unlikely to be exposed to high levels of Cl2 but it is frequently in contact with NCl3, the main volatile by-product of water chlorination. NCl3 is a strong oxidant that is virtually insoluble in water and exerts its toxic effects more deeply in the lung than Cl2. This gas is capable of disrupting acutely or chronically the epithelial barrier in the deep lung, thereby increasing the transepithelial leakage of proteins. NCl3 is an eye and upper respiratory tract irritant, which can also produce acute lung injury in accidental exposures particularly when chlorine bleach is mixed with ammonia. In rodents, this gas has the same irritating potency as chlorine or formaldehyde and causes fatal lung edema at high doses. Information concerning the potential risks of chronic exposure to NCl3 are very scarce and are limited to studies on children attending indoor chlorinated swimming pools and on lifeguards and swimming teachers. In children, the regular attendance at chlorinated pools has recently been found to be associated with an exposure-dependent increase in lung epithelium permeability and in the risk of developing asthma. NCl3 can be a cause of occupational asthma in lifeguards.

104 ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrafine Particles Table 2 Air Quality Guidelines by WHO, odor threshold, TLV, EGRP, and IDLH for oxidant gases

O3 NO2 Cl2 NCl3

Air Quality Guidelines

Odor threshold

TLV

ERPG

IDLH

0.06 (8 h) 0.11 (1 h)

0.05 0.12 0.31

0.1 3 0.5

0.5 1 1

5 20 10

0.06 (2 h)a

a Air standard for the Brussels capital region. All values are given in ppm except air guidelines. O3: 1 ppm ¼ 1.96 mg m
3; NO2: 1 ppm ¼ 1.88 mg m
3; Cl2: 1 ppm ¼ 2.90 mg m
3; NCl3: 1 ppm ¼ 4.90 mg m
3. TLV, threshold limit values; ERPG, emergency responses planning guidelines; IDLH, immediately dangerous for life or health concentrations.

Management and Treatment O3, NO2, Cl2, and NCl3 exert their toxicity in a dose-dependent way. No specific antidote exists for these pulmonary irritants. Treatment is basically supportive according to the clinical manifestations. It includes support of vital functions with focus on free airways, supplemental oxygen, and bronchodilators. Owing to the role of oxidative stress in pathogenesis, treatment with oxygen should only continue as long as required to keep sufficient oxygenation. Prevention of the adverse effects of these gases is mainly based on air quality guidelines from the World Health Organization (WHO) and from other national or international organizations and the establishment of odor thresholds, threshold limit values (TLV), emergency responses planning guidelines (ERPG), and immediately dangerous for life or health concentrations (IDLH) (Table 2). See also: Asthma: Overview. Environmental Pollutants: Overview. Lung Anatomy (Including the Aging Lung).

EU Directive (1999) EU Directive 1999/30/EC of the European Council of 29 September 1999 relating to limit values for sulphur dioxide, nitrogen dioxide and oxides of nitrogen, particulate matter and lead in ambient air. Official Journal of the European Communities L163: 41–60. EU Directive (2002) EU Directive 2002/3/EC of the European Parliament and the Council of 12 February 2002 relating to ozone in ambient air. Official Journal of the European Communities L67: 14–30. Holgate ST, Samet JM, Koren HS, and Maynard RL (eds.) (1999) Air Pollution and Health. London: Academic Press. Lippmann M (ed.) (2000) Environmental Toxicants, 2nd edn. New York, NY: Wiley-Interscience. Massin N, Bohadana AB, Wild P, et al. (1998) Respiratory symptoms and bronchial responsiveness in lifeguards exposed to nitrogen trichloride in indoor swimming pools. Occupational and Environmental Medicine 55: 258–263. Tickett KM, McCoach JS, Gerber JM, Sadhra S, and Burge PS (2002) Occupational asthma caused by chloramines in indoor swimming-pool air. European Respiratory Journal 19: 827–832. US EPA (1997) National Ambient Air Quality Standards for Ozone. Federal Registration 62: 38855–38896. WHO (2000) Classical pollutants. In: Air Quality Guidelines for Europe, European Series, no. 91, 2nd edn., pp. 173–198. Copenhagen: WHO Regional Publications.

Further Reading Bernard A, Carbonnelle S, Michel O, et al. (2003) Lung hyperpermeability and asthma prevalence in schoolchildren: unexpected associations with the attendance at indoor chlorinated swimming pools. Occupational and Environmental Medicine 60: 385–394. Broeckaert F, Arsalane K, Hermans C, et al. (2000) Serum Clara cell protein: a sensitive biomarker of increased lung epithelium permeability caused by ambient ozone. Environmental Health Perspective 108: 533–537. Bylin G, Cotgraeve I, Gustafsson L, et al. (1996) Health risk evaluation of ozone. Scandinavian Journal of Work and Environmental Health 22: 5–104. Carbonnelle S, Francaux M, Doyle I, et al. (2002) Changes in serum pneumoproteins caused by short-term exposures to nitrogen trichloride in indoor chlorinated swimming pools. Biomarkers 7: 464–478. Committee of the Environmental and Occupational Health Assembly of the American Thoracic Society (1996) Health effects of outdoor air pollution. American Journal of Respiratory and Critical Care Medicine 153: 3–50.

Particulate Matter, Ultrafine Particles K Donaldson and W MacNee, University of Edinburgh, Edinburgh, UK V Stone, Napier University, Edinburgh, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Particulate matter (PM), the particulate fraction, is the most harmful component of the air pollution cocktail. It is measured using the PM10 and PM2.5 conventions that approximately collect the tracheobronchial and respirable fractions, respectively. Daily increases in PM correspond to increases in deaths and hospitalizations for airway disease and cardiovascular disease 1 or 2 days later. Living in a particle-polluted area also increases the incidence of chronic lung and cardiovascular disease and

ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrafine Particles 105

0.6 Extra-thoracic 0.4 Respirable 0.2

Tracheobronchial

0.01

0.1

1 MMAD (µm)

10

100

Figure 1 Deposition curves. The relationship between particle size and the efficiency of deposition in various anatomical compartments of the lungs. MMAD, mass median aerodynamic diameter.

100

80 In

la

ble

M 10)

prox. P

cic (ap

) x. PM 2.5 appro

( irable

able espir

risk r

40

ha

Thora

60

Resp

High-

Air pollution is a cocktail of different components, both gaseous and particulate. The particulate component of environmental air pollution is measured according to a sampling convention, either as PM10 or PM2.5, which are described later. Particles deposit in different regions of the respiratory tract depending on their aerodynamic diameter (dae). Aerodynamic diameter is defined as the diameter of a sphere with the same density as water (‘unit density’, 1 gm cm
3) and the same gravitational settling speed as the particle of interest. This settling speed is determined by the equilibrium between gravitational force (proportional to density multiplied by volume) and the aerodynamic resistance (proportional to particle diameter multiplied by velocity). The various regions of the lungs in which particles of different aerodynamic size deposit are shown in the deposition curves in Figure 1; the tracheobronchial region is the region beyond the larynx, and the respirable region is the area beyond the ciliated airways, also known as the centriacinar region. Occupational hygienists, who have long had an interest in particles and the lungs, have developed sampling conventions based on the relationship between particle aerodynamic diameter and the fraction of particles penetrating to the different regions of the respiratory tract; these International Standard Organization (ISO) conventions are illustrated in Figure 2. In air pollution measurement, the conventions of PM10 (approximately thoracic) and PM2.5 (approximately respirable) are often used. It is important to note that sampling conventions differ from deposition curves. Deposition curves show the likelihood of a particle of any size being deposited in different parts of the lung, whereas sampling conventions show the ability of samplers of a specified convention to capture particles of a certain size. There is an important relationship between the two, however, that allows sampling of particles of sizes that are relevant to health effects. These sampling conventions are the global environmental standards for measuring the mass of airborne particles, and measurements are expressed as mass per unit volume sampled – conventionally mg m
3. In the UK, particulate matter (PM) and the gaseous

0.8 Deposition fraction

Measurement

Inhalable

1.0

% of total particles in convention

lung cancer. The underlying causes of these adverse effects are becoming better understood. There is heterogeneity in the potency of the components of PM, with some parts that contribute to the PM mass being essentially nontoxic (e.g., sea salt). The primary combustion-derived nanoparticulate component seems to be especially important in causing oxidative stress and inflammation, which may explain all the observed adverse health effects.

20

0 1

10

100

Aerodynamic diameter (µm) Figure 2 ISO health-based sampling conventions for particles.

pollutants are measured at sites in cities by Automated Urban Network (AUN) monitors run by the Department of Environment, Food and Rural Affairs (DEFRA).

Nomenclature There are some problems of nomenclature for particles because of the continued use of older sampling conventions. For the purposes of risk assessment, the health-based conventions are preferable, but the

106 ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrafine Particles

following are measurement conventions of PM that are also used: Total suspended particles (TSP). A TSP monitor captures atmospheric particulate smaller than approximately 40 mm in diameter. This means that TSP is not representative of the large particles that do not enter the respiratory tract and the lungs. Black smoke. This system was used in the UK and other countries until the end of the 1980s. Air was drawn through a size-selective orifice onto a white paper and the blackness of the ‘smudge’ was measured by reflectance. This method is obviously biased toward black (i.e., carbon-based) particles. There is a variable relationship between particles as measured by black smoke and other measures of PM. PM10. This is the commonly used size-selective sampling convention that captures particles of 10 mm with 50% efficiency; it roughly corresponds to the thoracic fraction of particles as defined by ISO. PM10 is routinely measured in the UK by DEFRA in their AUN. PM2.5. This is a size-selective sampling convention that captures particles of 2.5 mm with 50% efficiency; it roughly corresponds to the respirable fraction of particles as defined by ISO.

Levels of PM AUN measurements show that PM10 levels can fluctuate over a day, with higher levels usually occurring during rush hours on weekdays. A typical UK city in the twenty-first century has an average PM10 level of approximately 20 mg m
3, with short-term peaks of up to 100 mg m
3. In heavily polluted cities such as Athens or Mexico City, the levels of PM10 can be much higher, commonly reaching 50–75 mg m
3 on average with very high peaks. The fixed monitors of the AUN, or their equivalents worldwide, form the basis for virtually all epidemiological studies of the human health effects of PM. However, personal sampling studies show higher concentrations of particles kerbside in busy streets or tunnels and also in homes where there is cigarette smoking or certain forms of cooking. In some countries, indoor burning of biomass fuels in poorly ventilated dwellings produces very high levels of exposure, but the effects of these are relatively unstudied. In the past in the UK, there was high air pollution and very severe episodes. As a comparison with the approximately 20 mg m
3 found in the air of most UK cities today, the famous great London ‘pea-soup smogs’ of the 1950s had particle levels estimated

at over 1500 mg m
3. Levels of PM, along with other pollutants, have decreased markedly during the past 50 years, but the traffic-derived component, which is hypothesized to be the most harmful component, has increased, in line with increases in road traffic.

Adverse Effects of PM in Epidemiological Studies The levels of PM in any city vary both temporally, as a level fluctuating hourly around a mean, and spatially, dependent on average levels or traffic in an area or due to local industrial sources. The temporal and spatial variation in air pollution underlie the two main approaches to detecting and quantifying the adverse effects of PM (and indeed any air pollutant) in human populations. Time series studies utilize the temporal dimension and seek to relate the moving average of PM level to the moving average of a defined end point (e.g., mortality). Various lag times are used to detect the relation between the level of PM and the end point (e.g., 1 day). Environmental epidemiological studies seek to compare an end point such as the death rate in cities with high air pollution with the death rate in a city with low air pollution. Panel studies form a third category, in which small, well-characterized populations are followed for a defined end point that can be related to PM levels measured by personal or fixed point samplers. When these different approaches are taken together and examined over several hundred such studies, there is good coherence between the acute effects seen in time series and panel studies and the chronic effects seen in environmental studies. More important, many of these studies show no evidence of a threshold; that is, these adverse effects are occurring at the levels of PM that pertain in our cities today. These adverse effects are summarized in Table 1; the quantitative extent of the mortality effects of PM in European studies is shown in Figure 3. Within a short lag time of 1 or 2 days following an increase in PM, there are increases in all-cause mortality, significant increases in attacks of asthma and

Table 1 Summary of the human adverse health end points that are increased following increases in PM, as detected in epidemiological studies Mortality from cardiovascular and respiratory causes Admission to hospital for cardiovascular causes Exacerbation of asthma in pre-existing asthmatics Symptoms and use of asthma medication in asthmatics Exacerbation of chronic obstructive pulmonary disease Lung function decrease Lung cancer

ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrafine Particles 107 The Central Role of Inflammation

1.025

1.020

1.010

All causes

Relative risk

1.015

Cardiovascular

Respiratory

1.030

1.005

1.000

0.995

0.990 Figure 3 Relative risk for all cause, respiratory, and cardiovascular mortality following a 10 mg m
3 increase in PM10: results from a meta-analysis of European studies performed for a World Health Organization task group.

increased usage of asthma medication, increased deaths in chronic obstructive pulmonary disease (COPD) patients, exacerbations of COPD, and increased deaths and hospitalizations for cardiovascular disease. The adverse cardiovascular effects associated with increases in PM are well documented. Panel studies have documented associations between elevated levels of particles and onset of myocardial infarction, increased heart rate, and decreased heart rate variability. Chamber studies with concentrated airborne particles (CAPs) have also shown increased lung inflammation and altered brachial artery diameter in relation to increased exposure.

PM in Toxicological Studies: Mechanism of the Inflammatory Effects of PM Toxicological studies have helped to understand the mechanism of the adverse effects on the lungs and cardiovascular system found in the epidemiology studies described previously. Toxicological studies have used both PM and surrogate particles. Surrogate particles are used because of the variability of PM and the difficulty of obtaining bulk material for testing and for testing specific hypotheses related to the relative potency of the different components of PM.

Inflammation plays a role in the effects of all harmful particles, and toxicology studies have emphasized the central role of inflammation in the pulmonary and cardiovascular effects of PM. PM has shown a variety of proinflammatory effects in CAPs exposure studies in humans and rats and in animals exposed to PM by instillation. The mechanism of the proinflammatory effects of PM has been investigated extensively, and the ability of PM to cause proinflammatory effects in toxicological models of the lung has been amply demonstrated. Thus, PM or surrogate particles have been shown to activate nuclear factor kappa B and other proinflammatory signaling pathways in lungs and cells in culture and to increase release of proinflammatory mediators such as the chemokine interleukin (IL)-8 by cells in culture. These proinflammatory effects help to explain the increased exacerbations of asthma and COPD seen when pollution increases. Inflammation and Cardiovascular Effects

Inflammation can also be seen as a driver of the cardiovascular effects. There is evidence of systemic inflammation following increases in PM, as shown by elevated C-reactive protein, blood leukocytes, platelets, fibrinogen, and increased plasma viscosity. Atherosclerosis is the underlying cause of acute coronary syndrome, the main cause of cardiovascular morbidity and mortality. Atherogenesis is an inflammatory process initiated via endothelial injury and producing systemic markers of inflammation that are risk factors for myocardial and cerebral infarction. Repeated exposure to PM10 may, by increasing systemic inflammation, exacerbate the vascular inflammation of atherosclerosis and promote plaque development or rupture. Thus, inflammation in the lungs may explain both the local and the systemic cardiovascular effects of PM (Figure 4). The inflammatory response to particles or the particles themselves may also affect the neural regulation of the heart, leading to death from fatal dysrhythmia. In support of this hypothesis, studies of humans and animals have shown changes in the heart rate and heart rate variability in response to particle exposures.

Relative Potency of the Components of PM in Causing Inflammation and the Central Role of Oxidative Stress PM is a complex and variable mixture of particle types (Table 2). Many of the common components of PM have low potency in causing inflammation in the

108 ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrafine Particles Asthma/COPD

Cardiovascular disease

Background inflammation in the lungs

Susceptible lungs? Lung inflammation

PM Worsening of lung inflammation

Effects on clotting cascade Exacerbations and deaths

Effects on atherogenic plaques

Thrombosis

Deaths and hospitalizations for CV Figure 4 Hypothetical pathways from inflammation to local pulmonary and cardiovascular effects.

Table 2 Typical composition of the particles in PM PM10 component

Comment

Primary combustion-derived nanoparticles

Large surface area per unit mass and contain transition metals and organic volatiles; derived from combustion (e.g., vehicle exhaust particles) Derived from sea spray Predominantly ammonium sulfate Predominantly ammonium nitrate Derived from the earth’s crust (e.g., clay) Reentrained dust from wear on road surfaces, tyre wear, etc. For example, endotoxin

Sodium and magnesium compounds Sulfate Nitrate Compounds of calcium and potassium and insoluble minerals Road dust Biologically derived materials

lungs. Among those components that contribute to the mass of PM but are unlikely to cause inflammation are sea salt, ammonium nitrates and sulfates, road dust, and crustal dust. For example, in one epidemiological study, days when the origin of the particle cloud was predominantly from wind-blown dust were removed from the analysis. This greatly strengthened the relationship with adverse health effects and emphasized the importance of the combustion-derived particles. In contrast, the primary combustion-derived nanoparticles (PCDNs), derived predominantly from automobiles, especially diesel, are known to cause pulmonary inflammation in humans and animals. Nanoparticles are extremely small particles with a diameter less than 100 nm, and those found in the general environment are principally derived from traffic. They have enhanced ability to generate highly reactive ‘free radical’ molecules that damage and activate lung cells to produce proinflammatory mediators. This is a consequence of the fact that PCDNs possess three important components: high surface

area, organic contamination, and metals. All of these components are able to generate oxidative stress, which acts on key redox-sensitive, proinflammatory signaling pathways in lung cells. This leads to inflammatory gene transcription, which causes the adverse health effects (Figure 5). Oxidative Activity of PCDNs

PCDNs contain components able to generate oxidative stress when the particles deposit on the lung surface. Transition metals Formation of hydroxyl radicals (dOH), generated by ionic iron and other transition metals via the Fenton reaction, has been implicated in the oxidative stressing and inflammatory effects of PM. Other transition metals, such as chromium, vanadium, and copper, are also found to occur in PM and generate dOH. Electron paramagnetic resonance spectroscopy using a spintrap has been used to directly demonstrate the generation of dOH by

ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrafine Particles 109 PM10 Primary combustion-derived nanoparticles (transition metals, particle surface, organics)

Salts, crustal dust, road dust etc.

No effect Oxidative stress

Depletion of anti-oxidants

Lipid peroxidation

Activation of NF-
B/AP-1 and other redox-sensitive signaling pathways Gene expression Inflammatory mediators Inflammation

Exacerbations of airways disease/ effects on the cardiovascular system Figure 5 Molecular pathways for the proinflammatory effects of the PCDN component of PM.

PM in the presence of H2O2. In these studies, dOH formation is prevented by the transition metal chelator desferoxamine. Nanoparticle surfaces Surface area of particles is known to be a major factor in driving inflammation. A large surface area lung burden of even low-toxicity particles is known to cause inflammation. Nanoparticles of low-toxicity, low-solubility materials have a very high surface area per unit mass. They are also highly inflammogenic by virtue of this high surface area, and oxidative stress, generated through mechanisms that are not well understood, appears to play a role. PM can contain very high numbers of PCDNs as singlet particles but also as aggregates; these aggregates can be aerodynamically larger than the classical definition of a nanoparticle (i.e., they can be larger than 100 nm). However, nanoparticles in aggregates still express toxicity consistent with their geometric surface area and not in relation to their surface area as deduced from their aerodynamic diameter as an aggregate. Organics PCDNs contain organic molecules that have the ability to generate free radicals and cause oxidative stress. These organics include polycyclic

aromatic hydrocarbons (PAHs), n-alkanes, nitroPAHs, and quinones formed by combustion or derived from unburned fuel. Interaction of these organics with enzyme systems can result in oxidative stress via the production of oxidants such as superoxide anion. These also have the ability to activate oxidative stress-responsive signaling pathways in cells, leading to the release of proinflammatory chemokines such as IL-8. Translocation of Nanoparticles

Nanoparticles also seem to be able to traverse the epithelial barrier and gain access to the pulmonary interstitium. Particles in the interstitium are unlikely to be cleared and so the dose builds up. In the interstitium, they can activate cells, leading to more harmful interstitial inflammation. In the interstitium, the particles are very close to the blood, and experimentally nanoparticles have been shown to gain access to the blood. If this is confirmed for environmental nanoparticles, then we may postulate that bloodborne nanoparticles may interact with endothelial cells and clotting proteins, thus enhancing thrombogenesis. Additionally, they may interact directly with the endothelium overlying atherogenic plaques and may even gain access to plaques. This could cause activation of cells in the plaques and hasten plaque worsening or rupture, thus making a direct contribution to acute coronary syndrome. Endotoxins

Endotoxins are biological components, present in some particle samples, that are released from the outer membrane of Gram-negative bacteria. Chemically, they are defined as lipopolysaccharides and found in variable amounts in PM from various locations. Studies indicate that the PM endotoxin can be a major component in stimulating cells, especially macrophages, to produce inflammatory mediators. Endotoxins are commonly present in the coarse fraction (PM10 and PM2.5–10).

Cancer and PM Lung cancer is among the major chronic effects of living in an area with high PM10. In the famous six-cities study, more than 8000 deaths were studied in relation to the levels of air pollution in six US cities. Adjusted mortality rate ratio, corrected for smoking, for the most polluted cities compared to the least polluted showed a 25% increase in deaths from lung cancer. The risk of death from lung cancer due to living in an area with high levels of air pollution particles was further confirmed in a study using

110 ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution

data from the American Cancer Society. In this study, living in an area with a 10 mg m
3 elevation in fine particulate air pollution was associated with an 8% increase in the risk of lung cancer mortality. This has been supported by toxicological studies on the genotoxic effects of PM, which have demonstrated that PM has the ability to cause specific DNA adducts that form through oxidative stress pathways. The pathway from DNA adducts to accumulations of mutations, culminating in transformation to a cancer cell, is well documented. Coarse and fine PM samples have been shown to generate hydroxyl radical, the most harmful free radical formed in tissue under oxidative stress conditions, and this combines with guanine in DNA to form a specific adduct, 8-hydroxy-20 -deoxyguanosine (8-OHdG). PM samples are able to generate hydroxyl radical via transition metals, and these can chemically react with lung molecules to produce the hydroxyl radical. Organic molecules such as PAHs and quinones, both present on combustion-derived PM10, have been implicated in redox cycling and oxidative stress. When the PM is mixed with naked DNA, the 8-OHdG adduct is formed, and it is also seen in lung epithelial cells incubated with particles, a more realistic exposure than direct incubation of PM with DNA.

Interactions with Other Components of Air Pollution Because other components of air pollution, such as ozone, also cause oxidative stress and inflammation, there is potential for additive and possibly synergistic interactions between PM and the gaseous components in causing adverse effects. See also: Environmental Pollutants: Overview; Particulate and Dust Pollution, Inorganic and Organic Compounds. Particle Deposition in the Lung.

Further Reading Brook RD, Brook JR, Urch B, et al. (2002) Inhalation of fine particulate air pollution and ozone causes acute arterial vasoconstriction in healthy adults. Circulation 105: 1534–1536. Brook RD, Franklin B, Cascio W, et al. (2004) Air pollution and cardiovascular disease: a statement for healthcare professionals from the expert panel on population and prevention science of the American Heart Association. Circulation 109: 2655–2671. Brunekreef B and Holgate ST (2002) Air pollution and health. Lancet 360: 1233–1242. Donaldson K, Jimenez LA, Rahman I, et al. (2004) Respiratory health effects of ambient air pollution particles: role of reactive species. In: Vallyathan V, Shi X, and Castranova V (eds.) Oxygen/Nitrogen Radicals: Lung Injury and Disease, Lung Biology in Health and Disease (Lenfant C, series ed.), vol. 187. New York: Dekker.

Donaldson K, Stone V, Borm PJ, et al. (2003) Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM10). Free Radical Biology and Medicine 34: 1369– 1382. Donaldson K, Stone V, Seaton A, and MacNee W (2001) Ambient particle inhalation and the cardiovascular system: potential mechanisms. Environmental Health Perspectives 109(supplement 4): 523–527. Donaldson K and Tran CL (2002) Inflammation caused by particles and fibres. Inhalation Toxicology 14: 5–27. Donaldson K, Tran CL, and MacNee W (2002) Deposition and effects of fine and ultrafine particles in the respiratory tract. European Respiratory Monograph 7: 77–92. Dreher K (2000) Paticulate matter physicochemistry and toxicology: in search of causality – a critical perspective. Inhalation Toxicology 12(supplement 3): 45–57. Ghio AJ, Kim C, and Devlin RB (2000) Concentrated ambient air particles induce mild pulmonary inflammation in healthy human volunteers. American Journal of Respiratory and Critical Care Medicine 162: 981–988. Gilmour PS, Rahman I, Donaldson K, and MacNee W (2003) Histone acetylation regulates epithelial IL-8 release mediated by oxidative stress from environmental particles. American Journal of Physiology: Lung Cellular and Molecular Physiology 284: L533–L540. Jimenez LA, Thompson J, Brown DA, et al. (2000) Activation of NF-kappaB by PM(10) occurs via an iron-mediated mechanism in the absence of IkappaB degradation. Toxicology and Applied Pharmacology 166: 101–110. Peters A, Dockery DW, Muller JE, and Mittleman MA (2001) Increased particulate air pollution and the triggering of myocardial infarction. Circulation 103: 2810–2815. Pope CA III, Burnett RT, Thun MJ, et al. (2002) Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution. Journal of the American Medical Association 287: 1132–1141. Routledge HC, Ayres JG, and Townend JN (2003) Why cardiologists should be interested in air pollution. Heart 89: 1383–1388. Stone R (2002) Air pollution. Counting the cost of London’s killer smog. Science 298: 2106–2107. World Health Organization (2004) Health Aspects of Air Pollution. Results from the WHO Project ‘Systematic Review of Health Aspects of Air Pollution in Europe’. Copenhagen: World Health Organization.

Particulate and Dust Pollution, Inorganic and Organic Compounds W Birmili, Leibniz Institute for Tropospheric Research, Leipzig, Germany T Hoffmann, Johannes Gutenberg – Universita¨t Mainz, Mainz, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract This article reviews the basic properties and behavior of airborne environmental particles (particulate matter, PM). The basic relevance of environmental PM to health and air quality is

ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution 111 outlined, and current knowledge on the sources and generation mechanisms of natural and anthropogenic PM is summarized. It is shown that the interaction between the sources, atmospheric transformation processes, and sinks of PM determines the shape of the physical particle size distribution. The general aspects of PM measurement as well as the abundance and behavior of inorganic PM compounds, notably metals, are discussed. The article concludes with an overview of carbonaceous PM and its contribution to particulate pollution, and includes the definitions and properties of the frequently discussed subfractions – elemental carbon, black carbon, soot, and organic carbon.

Introduction Airborne particles in our environment are an integral part of what we breathe in. Besides the uptake of environmental pollutants through nutrition, humans are exposed to airborne particles through the skin and, importantly, through the respiratory tract, that is, nose, thorax, the upper airways, and the lung. The biological effects of harmful pollutants strongly depend on where these pollutants are deposited in the body. In the case of airborne particulates, physical size is the main determinant of where in the airways a particle will actually be deposited. While coarse particles are deposited in the nose and the throat, fine (o1 mm grain size) and ultrafine (o0.1 mm grain size) particulates may enter deep into the bronchial and even alveolar regions, where gas exchange takes place with the blood circulation. Depending on their chemical composition and morphological structure, inhaled particles may cause harmful effects such as oxidative stress and inflammation, which over time may affect the body’s ability to regenerate and lead to respiratory or cardiovascular disease. Small insoluble particles have been shown to translocate to almost every organ within the human body thereby allowing for complex possibilities of disease. In this article, we introduce the natural and anthropogenic sources, the atmospheric life cycle of airborne particulates, and their properties relevant for health. Current knowledge regarding the abundance and behavior of particulate inorganic and organic species is summarized. A number of conditions found in the atmosphere, ranging from an unpolluted baseline atmosphere to polluted, near-source conditions such as those found in urban areas, are included.

Atmospheric Aerosols and Air Pollution Definitions

The definitions relating to particulate air pollution include ‘particulate matter’ (PM), ‘suspended particulate matter’, ‘dust’ (typically referring to large visible particles), or ‘atmospheric aerosol’. The definition of ‘aerosol’ dates back to the beginning of the

twentieth century; the term was intended to be analogous to that of ‘hydrosol’, and was soon adopted for particulates suspended in the atmosphere. The physical size of a particle dictates its behavior in the atmosphere, the length of its life, its climaterelevant effects (such as light scattering or absorption, cloud formation), and its zone of deposition in the respiratory tract. Several conventions originating from public health needs have been established to quantify the entity of particles that are respirable down to a certain depth in the human airways (PM10, PM2.5). The convention ‘PMx’ includes the ensemble of all particles below a certain diameter in micrometers. Often, the term PMx is used as a synonym for total particle mass concentration below this diameter. Atmosphere scientists, in contrast, tend to distinguish among particles using their mechanism of generation, that is, coarse particles (41 mm), which are mainly mechanically generated, and fine particles (o1 mm), which arise from condensation, agglomeration, and liquid phase processes. The term ‘ultrafine’ typically refers to particles smaller than 0.1 mm. Scientists working in the field of atmospheric electricity would call ultrafine particles ‘intermediate ions’. The relatively new term ‘nano-particles’ (typically up to about 50 nm in diameter), which is used in the innovative fields of material science and molecular medicine, refers to particles and processes where atomic and molecular effects are felt. Air Quality

Since making use of the convenience of domestic fire humankind has been exposed to particulate pollution in the form of smoke. The first reports on urban air pollution date back to Roman times. However, frequently recurring episodes of air pollution over large areas have only been experienced since the industrialization of manufacturing processes and the agglomeration of populations in modern cities, both of which are associated with an increased generation of energy from fossil fuel. The problems caused by air pollution are manifold: odor, deterioration in visibility (dry or wet fog), damage to vegetation and buildings, adverse effects on health, and, on a larger scale, acid rain, overfertilization, and modification of the earth’s radiative balance. Air pollution events are usually associated with a complex and locally dependent mixture of gaseous and particulate pollutants. Two rather specific types of air pollution mixtures have become known during the twentieth century: London smog and Los Angeles smog. The term London smog (smog ¼ smoke þ fog) refers to a mixture of pollutants notably including sulfur dioxide (SO2) and coarse PM (e.g., fly ash)

112 ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution

Figure 1 Good and poor visibility due to pollution aerosols in Beijing. Photographs courtesy of B Wehner, IfT Leipzig.

that is representative for a period of industrialization where the unregulated combustion of coal played the dominant role in heat and energy generation. Particle and dust sources include a plethora of industrial and domestic coal fires for the generation of heat, steam, motion, and electricity. In London, the absence of systematic emission regulations until well into the 1950s meant that the city was regularly subjected to episodes of pollution, which tended to occur under cool weather conditions associated with fog and atmospheric inversion. It was only after the infamous smog event of December 1952, which caused approximately 4000 premature deaths, that legislation enforced a swift technological transition toward the cleaning of exhaust gases and the substitution of coal by natural gas as a heating fuel. While London smog is usually a phenomenon of cool weather, the Los Angeles smog, in contrast, is associated with high levels of ozone (O3) and organic PM at warm temperatures. First noted in California in the 1950s, there was initially considerable confusion about the origin of this air pollution covering the wider area of Los Angeles. By 1960, motor vehicles had been identified as the main sources of pollution, primarily by their emissions of nitrogen oxides and gaseous and particulate hydrocarbons. Nitrogen oxides react photochemically under the impact of sunlight with anthropogenic and biogenic hydrocarbons, thereby producing an excess of ground-level ozone as well as significant amounts of organic PM, giving the atmospheric boundary layer a brownish color. Owing to the complex nature of chemical reactions that lead to Los Angeles smog, abatement measures have proved difficult to establish. Indeed, reductions in nitrogen oxide emissions may under certain circumstances lead to an increase in ozone concentrations rather than to a decrease. Today, variations on air pollution abatement measures are found in many large conurbations, especially in regions where the population, energy consumption,

and volumes of motorized traffic are rapidly increasing, such as parts of China (see Figure 1), South east Asia, and India. Air pollution problems may be aggravated by local topography in cities like Mexico City, Santiago de Chile, Sa˜o Paulo or Kathmandu, where surrounding mountains or hills frequently block the dispersion of pollutants. Despite the spread of improved technologies for cleaning of exhaust emissions and efficient energy use, it is anticipated that air quality problems will increase on a global level along with economic growth during the decades to come. Natural Sources of Atmospheric Aerosols

Even if the emission of all anthropogenic sources of PM could be eliminated, environmental concentrations of aerosol particles would not go down to zero because of a number of natural sources that continuously maintain a significant atmospheric aerosol population even in the remotest regions of the globe. It is a generally accepted practice to distinguish particles in terms of their class of generation mechanism. ‘Primary particles’ or primary PM are those that are emitted directly from source, such as sea spray particles or desert dust. ‘Secondary particles’ or secondary PM are those that are generated in the atmosphere by nucleation, condensation, or liquid phase processes from gaseous precursor species, such as particulate sulfate or secondary organic aerosol (SOA). The ocean, being the largest part of the earth’s surface, emits particles in several ways: Sea spray particles emerge from bursting bubbles just above the water surface, a process overwhelmingly controlled by the intensity of breaking waves and thus mainly influenced by the wind speed. The major fraction of these particles is coarse (41 mm) and composed of the ions contained in the seawater, mainly sodium and chloride. It is noteworthy that sea spray aerosol

ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution 113

can be a relevant contributor to particle mass (e.g., PM10) in coastal areas. A further important oceanic source is the formation of secondary aerosol through the pathway of gaseous dimethylsulfide (DMS) emissions by plankton, followed by atmospheric oxidation to methane sulfonic acid (MSA) and subsequent nucleation or condensation to atmospheric particulates. This process is part of the atmospheric sulfur cycle, and generates particles that are nano-sized at the start and grow to bigger sizes (0.1–1 mm) within a matter of days or weeks. Likewise, volatile iodine compounds released from marine biota, especially from macroalgae, have been observed to be efficient precursors for particle formation in coastal areas. Hypotheses about the eventual relevance of oceanic organic material for the formation of primary aerosol are currently under investigation. Continental sources of atmospheric particles are even greater. In arid regions, the wind-driven resuspension of soil material, manifested by the formation of dust storms, can be the most important source of particulate mass. Transported to the free troposphere by deep convection, Saharan desert dust, for example, regularly reaches Europe and South America where it causes overcast skies even in the absence of clouds. Enrichments of mineral dust can usually be detected by particle shape (irregular; crystal layers) and analytically by the presence of high levels of elements from the earth’s crust such as silicon, titanium, and the rare earth elements. Natural sources of carbonaceous and sulfate aerosols include continuous volcanic emissions or the more dramatic events of volcanic eruptions that send up large amounts of fly ash into the stratospheric regions from where aerosol layers can spread across the entire globe. Finally, natural forest fires, which emit mostly elemental and organic particulate carbon, are another source of natural primary aerosols. Adverse effects on human health through inhalation of smoke, however, are only expected in the immediate vicinity of the fire, since atmospheric dilution reduces concentrations relatively fast downwind of a source. Important secondary sources over the continents include the formation of nitrogen-containing inorganic aerosols (especially ammonium and nitrate), and secondary aerosols from biogenic precursors, such as isoprene and terpenes, whose formation rates are especially relevant in the warm season of the year and in forested areas. In terms of their health effects, surprisingly little evidence is available for natural (especially secondary) aerosol particles. Ionic material, as well as the soluble fractions of organic carbon (OC), is readily water soluble and is therefore rapidly excreted in biological organisms. Unless associated with high

acidity, the health effects corresponding to these compounds are therefore assumed to be of minor concern. Anthropogenic Sources of Atmospheric Aerosols

Man’s impact on the environment has already heavily changed the composition of the atmosphere. The most noticeable effects are concerned with general air quality and visibility on a local to regional scale, whereas their effects upon human health, the oxidation capacity of the atmosphere, and the globe’s radiative balance are more complex and less evident. In contrast to the natural sources, it may be costeffective for a society to put anthropogenic PM sources under regulation mechanisms if there is evidence for a sustained disadvantage to society. Particulate pollution may originate from the wide range of industrial activities, particularly those involving the combustion of fossil fuel (steel smelters, coke generation, electric power generation), and also from waste incinerators, domestic heating, and vehicular and air traffic. Additional exposure results from domestic combustion sources (stoves). In rural valleys, wood burning may be the most relevant source for PM exposure. An important man-made source is the practice of forest clearance by burning. It is important to note that in indoor vehicular, and public transport microenvironments, emissions from vehicular sources can accumulate to the most extreme levels due to poor ventilation. Combustion aerosols are overwhelmingly carbonaceous by mass, including elemental carbon (EC) and OC, some of the latter being present in the form of toxins (polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyles (PCBs)). Combustion particles are largely insoluble and thus represent a challenge for the defense mechanisms in the human lung. The anthropogenic combustion aerosols range in size from smoke and fly ash particles (41 mm) through die sel soot particulates (20–200 nm) to sulfuric and organic combustion nano-particles (o50 nm). Notable sources of coarse particle emissions through anthropogenic land use are wind-blown dust, agricultural aerosols, and opencast mines, which may be of considerable importance for local exposure. Anthropogenic secondary particle emissions include nitrate aerosol resulting from agricultural fertilization and the growth of livestock, sulfate aerosol stemming from the gradual oxidation of sulfur dioxide, and SOAs. Most anthropogenic effects are socially driven in that diurnal and weekly profiles are observed in pollutant concentrations. In urban areas total particle mass, particle number, size distribution, and soot concentration develop a clear diurnal cycle linked to

114 ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution

working and traffic patterns. These idealized anthropogenic cycles are modified by meteorological effects, notably atmospheric stability, which account for varying dispersion conditions. Persistent pollution episodes are more likely to develop during the winter in cities of the Northern hemisphere. This is because in the cold season, anthropogenic emissions rise through the need for heating and atmospheric inversion situations are more frequent. Further meteorologically driven effects include the long-range transport of pollutants in tropospheric air masses, or even in the stratosphere so that particulate pollution can intrude into areas that are remote from their original source.

particles). An effective pathway to build up secondary ionic particulate mass is through liquid phase processes in activated cloud droplets; this is, for example, the major pathway for particulate sulfate production. The combination of emission and transformation processes leads to most atmospheric particles being chemically mixed. Figure 2 shows two main types of particles in urban air. The main removal processes for airborne particles are dry deposition and wet deposition. Dry deposition (mainly sedimentation) to the surface (ocean, land surface, vegetation) is most relevant for coarse particles. Wet deposition includes in-cloud scavenging (i.e., activation of particles to fog or cloud droplets under supersaturation of water vapor, and subsequent growth to rain droplets) and below-cloud scavenging (i.e., removal by falling rain droplets). Wet deposition is the main removal process for particles between 0.1 and 1 mm in diameter. Depending on the humidity available in the atmosphere wet deposition limits the lifetime of accumulation mode particles to between 3 and 14 days. Coalescence is an important process to decrease the number of small particles (diameter o50 nm). The probability that a particle will coagulate with another particle increases strongly with decreasing particle size. Coalescence is also very efficient in forming agglomerations of particles in or near particle sources, such as in vehicle exhaust. At a given time, an atmospheric particle size distribution is shaped by past and present aerosol

Lifetime of Atmospheric Aerosols and Particle Modes

The lifetime of atmospheric particles is determined by the balance between their source and removal rates. Since aerosol dynamic processes are, in general, a function of particle size, all lifetime relevant effects will depend on the particle size distribution. The basic processes that generate aerosol particles are mechanical generation (abrasion, resuspension), nucleation (formation of new individual particles exclusively from supersaturated gas phase precursors), condensation (transfer of supersaturated gases onto the surfaces of existing particles), and coagulation (build-up of larger agglomerates from small

a 100 nm b b a

b

b

a 1 µm (a)

a

b

(b)

Figure 2 Scanning electron micrographs of particles in urban air. (a) Soot agglomerates made of primary particles B70 nm in diameter. (b) Internally mixed sulfate/soot particle ‘a’ indicates areas of soot, ‘b’ indicates areas of ammonium sulfate). Courtesy of M Ebert, TU Darmstadt, Germany.

ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution 115

Homogeneous nucleation

Wet deposition

Combustion sources

Condensational growth

Cloud + fog activation

Abrasion + resuspension Defragmentation

Sedimentation

1E-3 Diameter (µm) Fresh

0.01

0.1 Aged

Nucleation modes

Accumulation mode

1 Droplet mode

10 Coarse mode

100

Super coarse mode

Figure 3 The size spectrum of atmospheric aerosol particles featuring idealized particle modes and some of the shape-giving processes. Increased spatial and temporal variability is indicated by dot-dash and dashed line.

generation and removal processes, and liquid phase transformations such as fog and cloud processing. Figure 3 shows a multimodal paradigm of the atmospheric particle size distribution, including the various modes corresponding to distinct source and transformation processes.

Atmospheric Aerosols: General Abundance and Inorganic Compounds Measurement and Chemical Analysis: General Techniques

A number of robust experimental techniques can be applied to determine integral as well as specific properties of PM in near real-time. These methods include: gravimetry (tempered oscillating microbalance; TEOM) to determine total particle mass concentrations; epiphaniometry to determine total particle surface area; aethalometry to determine total light attenuation; nephelometry to determine total optical scattering; optical particle sizers; ion spectrometry (to determine size distributions of charged particles); differential and scanning mobility particle sizers; aerodynamic particle sizers; and condensation nucleus counters. A relatively new but strongly emerging field is the development and application of real-time mass spectrometric techniques for particle characterization. Two competing instrumental approaches are currently under development: individual particle analysis based on laser detection/laser ionization mass spectrometry, which is especially

suited for the characterization of inorganic aerosol constituents; and thermal desorption/electron ionization mass spectrometry, which appears to be the more promising technique for analysis of the organic aerosol fraction. Several off-line methods allow a more specific morphological analysis, but first require the capture of particles on a filter or an impaction substrate. These methods include gravimetry (filter weighing), optical microscopy, scanning and transmission electron microscopy, and atomic force microscopy. Importantly, particle samples collected on filters and substrates can be subject to detailed elemental speciation, for example, through proton-induced Xray emission, neutron activation analysis, or atomic absorption spectroscopy. After dissolution of PM in a solvent, or evaporation, further speciation methods can be applied. Hybrid techniques usually involve a species-selective stage, such as ion chromatography, gas chromatography, liquid chromatography, capillary electrophoresis, and an actual detection stage for atoms, ions, or molecules. Sensitive multielement techniques for higher weight atoms include, for example, inductively coupled plasma atomic emission and mass spectrometry. Comprehensive details of the current aerosol measurement and chemical analytical techniques can be found in the ‘Further reading’ list. Ambient PM: General Characteristics and Inorganic Ions

Table 1 summarizes observations of integral parameters of ambient PM across Europe. Notably, mass

116 ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution Table 1 Typical ambient levels of PM mass concentration and particle number concentration (D 410 nm) in Europe

Remote Rural Urban influenced Urban background Kerbside

PM10 (mg m3)

PM2.5 (mg m3)

8 15 25 30 40

5 10 20 22 26

(3–35) (8–32) (22–50) (19–72) (28–76)

Number (103 cm
3)

(2–22) (5–29) (12–32) (11–62) (17–50)

1 (0.2–2) 2.5 (2–5) 6 (5–12) 14 (7–23) 50 (40–60)

The values in brackets indicate the range of levels at different observation sites. From Pataud JP, van Dingenen R, Baltensperger U, et al. (2003) A European Aerosol Phenomenology, Joint Research Centre.

Rural PM10−2.5

Urban

Elemental carbon Organic matter NO3− NH4+ SO42− Sea salt Mineral dust Unspecified

Kerbside

Rural PM2.5

Urban Kerbside 0

20

40 60 Mass fraction (%)

80

100

Figure 4 Average chemical composition of ambient PM in Europe. Data from Pataud JP, van Dingenen R, Baltensperger U, et al. (2003) A European Aerosol Phenomenology, Joint Research Centre.

and number concentrations increase with increasing proximity to anthropogenic sources. Evidence is available for an overwhelming contribution of vehicular traffic to particle number concentration. Figure 4 shows the bulk chemical composition of PM10–2.5 (coarse PM) and PM2.5 (fine PM). Coarse PM includes large portions of mineral dust and sea salt, both the result of resuspension processes of material from the earth’s crust and seawater. A class of unspecified material includes strongly bound metals such as silicates, which are not accessible by standard analytical techniques. Fine PM, in contrast, is made up mainly of carbonaceous compounds and inorganic ions. In a kerbside environment (i.e., immediately next to a road), half of the fine PM may consist of carbonaceous compounds. The strong enrichments of inorganic compounds, such as sulfate, nitrate, and ammonium ions, reflect the formation of secondary aerosol over continental areas. Sulfate (SO24
) is known to be formed during liquid phase processes (cloud and fog processes) by oxidation of S(IV) to S(VI) by ozone (O3) and hydrogen peroxide (H2O2); an alternative pathway is direct gas-to-particle conversion of sulfuric acid (H2SO4), which is of relevance in anthropogenic plumes of smoke under photochemically active conditions. Nitrate (NO3
) and ammonium (NH4þ ) ions are mainly formed through oxidation of emitted nitrogen oxides (NO, NO2)

and ammonia (NH3), respectively. Particulate nitrate concentrations may be especially high downwind of agricultural areas, and may contribute to up to half of total PM10 mass concentration in rural areas. Owing to their semivolatile character, nitrates tend to partition into either the particulate or the gas phase depending on ambient temperature. Therefore, the concentration of nitrates is considerably higher in the winter than in the summer. The volatility of nitrates requires special care during experimental procedures, notably sampling and sample storage, in order to prevent sampling artifacts. Characteristics of Metals in Ambient PM

Much of the work on the speciation of inorganic PM constituents has focused on compounds containing oxygen, carbon, nitrogen, sulfur, and halogens. This is due to their role in global warming, acid rain, declining air quality, and tropospheric ozone increase. The speciation of metals has received comparably little attention because of the relatively small concentrations in the atmosphere and the great difficulties involved in sampling and analysis. However, it is now known that metallic species exert toxic effects in animals and humans, and metals have recently become the focus of health-related PM research. A recent hypothesis is that transition metals

ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution 117 Na Mn

Ca Sn

Fe Ti

K Hg

Mg Ni

Al Se

Zn Cd

Cu Co

Pb Ag

Ba Pt

10−1

Mass fraction

10−2

10−3

10−4

10−5

10−6

Total PM (<7.2 µm)

Coarse PM (3−7.2 µm)

Fine PM (<0.5 µm)

Figure 5 Mass fractions of individual metals in different particle size fractions in the urban PM of Birmingham (UK). Data from Birmili W, Allen AG, Bary F, and Harrison RM. Trace metal concentrations and water solubility in size-fractionated atmospheric particles and influence of road traffic. Environmental Science and Technology (in press).

such as iron, zinc, and copper may release free radicals in lung fluid via the Fenton reaction and cause cellular inflammation. The important properties of atmospheric metals are not only their ambient concentrations but also their chemical form, that is, oxidation state, solubility, and ability to form organic complexes. Since particle-bound metals need to dissolve and become free ions in the lung fluid, particle solubility in lung fluid is a major criterion for the bioavailability and, therefore, toxicity of environmental PM. Alternatively, metals have been exploited as tracers in order to identify individual sources of PM because it has been recognized that the search of reliable tracers may be crucial for developing cost-effective air pollution control strategies. Some elemental combinations have been identified to represent individual anthropogenic sources: oil combustion (vanadium, sulfur, arsenic), coal combustion (aluminum, scandium, sulfur), steel industry (manganese, iron, chromium), cement (calcium), waste incineration (zinc, potassium, lead, antimony), abrasive vehicle emissions (iron, barium, copper, zinc), and soil dust (aluminum, silicon, titanium, scandium, manganese). It is interesting to note that since the almost universal removal of lead from gasoline, there is no longer a significant marker available to identify vehicular exhaust PM through an inorganic multielement analysis. Figure 5 illustrates the relative abundance (mass fraction) of some metal species in urban PM. Besides the more abundant earth alkali metals, most elements occur in small traces only, usually in PM fractions below 1 per 1000. In addition, specific

differences are evident between coarse and fine PM. Metals that occur predominantly in the coarse mode are: manganese and sodium (sea water); and calcium, barium, iron, titanium, and aluminum (typical elements of the earth’s crust). Metals that occur predominantly in the fine mode are mercury, selenium, cadmium, tin, nickel, lead, and platinum, all of which are related to high-temperature combustion sources. It can be seen that metals reflect the fundamentally different generation mechanisms of the different PM size fractions.

Carbonaceous Compounds in Atmospheric Aerosols The concentration of inorganic carbon, essentially as carbonate, is on average negligible, at least when considering PM2.5. If inorganic carbon occurs in atmospheric PM, it is limited to the form of carbonatecontaining mineral dust, which is part of the coarse mode of PM. In contrast to their naming, EC and OC are not well-defined groups of chemical compounds. In fact, the definitions of EC and OC are strongly related to the commonly used analytical technique. The differentiation between EC and OC is based on thermal treatment of the particle-loaded filter media and measurement of the released carbon compounds as a function of temperature. The amount of carbon released below a certain temperature (typically 340– 3501C) is considered to be organic (CxHyOz). The amount released above this arbitrarily chosen default temperature is considered to be EC. The original idea

118 ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution

of this classification was to obtain a measure for the ‘pure’ element carbon itself, since one characteristic feature of EC is the absorption of visible light, and it is exactly this feature that is vital to understanding direct radiative forcing of atmospheric aerosol particles. Nevertheless, the terms black carbon (BC), graphitic carbon, and soot are nowadays used synonymously with EC. Other measurement techniques for the determination of the EC fraction exist and are based on the light absorption characteristics, notably the light reflectance and transmittance through particles collected on a filter, and the in situ heating of particles in an air stream with photoacoustic detection. Unfortunately, none of these techniques can provide reliable information on the EC content; for example, certain higher molecular weight organic compounds can undergo charring to form pyrolytically generated EC during the thermal treatment of the aerosol samples. Not only are the names of the two classes misleading in terms of the chemical composition, but also the different carbon analysis methods do not consistently provide comparable EC or OC concentrations, as demonstrated in many comparisons between methods used and among laboratories. The degree of equivalence depends on the nature of the sample as well as on the analytical method and protocol. Unfortunately, different protocols can be based on the same method, but potentially important differences that might affect the results have not been adequately documented. Therefore, whenever EC, BC, or OC concentrations are taken from the literature, these implicit uncertainties and inaccuracies have to be taken into account. Elemental Carbon, Black Carbon, and Soot

As mentioned above, EC (also called graphitic carbon, black carbon, or soot), is ubiquitous in combustion emissions. It is predominantly found in diesel exhaust, but it is emitted from all combustion sources such as biomass, coal, and oil burning. EC is neither pure nor highly structured carbon because real EC, such as diamond and graphite, rarely occurs as pure particles unmixed with other atmospheric constituents. The combustion-generated fraction of primary carbonaceous aerosol, commonly termed ‘soot’, can be regarded as a form of impure EC consisting of plains of hexagonal arrays of carbon atoms, similar to graphite’s crystalline structure. Unlike graphite these plains can be curved forming spherical bodies. If the number of graphitic layers is very small, carbon might be called amorphous; if the number is high, it might be called graphitic carbon. The size of soot particles depends on the history of

their formation (substance, temperature, oxygen content in flame, etc.). Primary soot particles (from diesel engines or oil burning) have diameters between 10 and 50 nm and a spherical onion-layered structure, but usually they agglomerate rapidly during the combustion process forming chain aggregates measuring from 100 nm to about 1 mm in length (Figure 2). Black carbon and soot also contain various amounts of hydrogen and other atoms as well as some extractable organic matter like PAHs or alkanes. The organic matter may be adsorbed onto the soot surface or even enclosed inside the primary soot particles. In fact, since the carbon:hydrogen ratio approaches infinity within the homologous series of PAHs, the transition between organic and EC is smooth. Organic Carbon

Although organic compounds typically account for 10–70% of the total dry fine particle mass in the atmosphere, their concentrations, composition, and the processes that control their formation and transformation in the atmosphere are not well understood, particularly in relation to the other major fine particle constituents, such as sulfate and nitrate compounds. While the classification between EC and OC was an operational one, the further subdivision of OC is based on the different mechanisms of formation. Primary organic aerosol components include all compounds, which are released directly as PM into the atmosphere. They comprise particle phase OC from anthropogenic combustion sources (biomass burning, use of fossil fuels, and mechanically formed aerosol particles from road dusts or tire debris). In very densely populated environments even the direct input of organic particulates from domestic activities like cooking or cigarette smoking can be significant. In contrast, the natural contributions to the primary organic aerosol are less well understood. Field measurements of the content of macromolecular organic compounds in airborne particles indicate that biogenic sources play a significant role. For example, cellulose and humic-like substances have been found in atmospheric aerosol particles, pointing to a direct input of OC from vegetation, probably originating from plant surfaces. Furthermore, pollen, airborne bacteria, viruses, and spores have to be considered as contributors to the organic aerosol fraction, although some of these primary biological aerosol particles only contribute to the coarse and super-coarse particle mode. However, a detailed quantification of the different natural contributions to the atmospheric particle phase is still missing, especially due to the complex chemical composition of biological matrices.

ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution 119

that anthropogenic SOA formation in an urban airshed can be modeled on the basis of the aromatic content of the complex hydrocarbon mixture. However, based on the current knowledge about precursor emissions and the aerosol formation potential of the individual VOCs, SOA formation from natural precursors dominates the production of SOAs in the troposphere on a global scale. Interestingly, a significant anthropogenic impact on natural SOA formation is expected, resulting from the influence of the oxidation pathway of the biogenic VOCs (reaction with O3, OH, or NO3) on the aerosol formation potential of the biogenics. Besides the direct transfer of low volatile organics into the tropospheric particle phase by gas-to-particle conversion, atmospheric processes involving heterogeneous processes are suspected to contribute to SOAs. Partially oxidized organic gases such as alcohols, aldehydes, or peroxides, which are too volatile to condense directly onto existing particles, might be absorbed into water droplets in clouds and fogs and, after undergoing chemical reactions, result in the addition of organic matter when the droplets evaporate. Another recently discovered pathway for SOA component formation involves polymerization reactions of small-chain precursor molecules, such as C2 or C3 aldehydes, in the organic particle phase; these were formerly believed not to be involved in aerosol formation. In fact, this build up of higher molecular weight compounds might also explain part

In addition, organic aerosol material is formed in the troposphere by the mass transfer of low vapor pressure substances to the aerosol phase. The condensable species are formed in the gas phase by the reaction of volatile organic compounds (VOCs) with the principal atmospheric oxidizing agents
O3, OH, and NO3 radicals. To distinguish this fraction of tropospheric aerosols from the direct input of particulate organics into the atmosphere as described above, it is specified as SOA. VOCs are emitted into the troposphere from anthropogenic and biogenic sources. Anthropogenic sources comprise organics such as alkanes, alkenes, aromatics, and carbonyls, while biogenic sources include organics such as isoprene, monoterpenes, and sesquiterpenes, as well as a series of oxygen-containing compounds. Since the volatility of the oxidation products is one of the most important parameters that determines the significance of precursor gases in terms of their aerosolforming potential, hydrocarbons with a higher number of carbon atoms in particular will contribute to SOAs under atmospheric conditions. Thus, natural SOA formation is believed to result mainly from the oxidation of C10- and C15-terpenoids, although recent investigations showed that isoprene, a biogenic C5 hydrocarbon that is released in huge amounts into the atmosphere, can also form condensable products. Among the anthropogenic precursor gases, aromatics have been shown to dominate the process of SOA formation, suggesting

Primary OC >57 Secondary OC 11−46

Gas/particle conversion

?

240 210

22 35

18

12

Vegetation Biomass burning

Fossil fuels

Figure 6 Estimation of global primary and secondary organic particle contributions.

?

120 ENVIRONMENTAL POLLUTANTS / Radon

of what is still defined as ‘primary’ OC (i.e., humiclike substances). What is the significance of carbonaceous compounds from the point of view of health? The few epidemiological studies that have addressed this important question specifically suggest that combustion sources are particularly important for health. Toxicological studies have also indicated that primary combustion-derived particles have a higher toxic potential. These particles are often rich in organic compounds, which are thought to be involved in the adverse health effects of PM2.5. In addition, many organic compounds are electrophilic and reactive molecules, which can cause oxidative damage to proteins and DNA and induce cytokines and inflammation. However, at present it is not possible to quantify the effect of organic compounds from the different sources on health caused by exposure to ambient PM. Based on the currently available figures, Figure 6 attempts to summarize the estimated global contributions to the organic fraction of tropospheric aerosols. It should be noted that the uncertainties regarding these figures are still very great and that some potential formation pathways, such as chemical processing in the liquid phase or polymerization of gas phase precursors, are not included. Furthermore, the specific natural sources for organic aerosol particles, such as primary organic contributions from terrestrial or marine sources, are still poorly understood.

Putaud JP, van Dingenen R, Baltensperger U, et al. (2003) A European Aerosol Phenomenology. Joint Research Centre. Seinfeld JH and Pandis SN (1997) Atmospheric Chemistry and Physics. New York: Wiley Interscience. Spokes LJ and Jickells TD (2002) Speciation of metals in the atmosphere. In: Ure AM and Davidson CM (eds.) Chemical Speciation in the Environment, 2nd edn., pp. 161–187. Oxford: Blackwell. Spurny KR (ed.) (1999) Analytical Chemistry of Aerosols. Boca Raton, FL: CRC Press. Spurny KR (ed.) (2000) Aerosol Chemical Processes in the Environment. Boca Raton, FL: CRC Press. The Mayor of London (2002) 50 Years On: The Struggle for Air Quality in London since the Great Smog of December 1952. Greater London Authority.

See also: Environmental Pollutants: Overview; Diesel Exhaust Particles; Particulate Matter, Ultrafine Particles.

Radon gas is the most important source of natural radioactivity. It is a radioactive noble gas that emanates from the ground. Radon concentrations in outdoor air are low, but in underground mines or inside houses radon concentrations can reach high levels. The biologic effects of radon are mostly due to its decay products. These are solid heavy metal isotopes that are themselves radioactive. They can be deposited on the bronchial epithelium, where they emit ionizing radiation in the form of aparticles. Alpha radiation can effectively cause permanent damage to the DNA and can eventually lead to cancer. Epidemiological studies have given conclusive evidence that radon is carcinogenic to humans and can cause lung cancer. The first studies were performed in underground miners exposed to high levels of radon. Later studies performed in the general population showed that radon can cause lung cancer even at the lower concentrations found in houses. This indoor radon exposure is currently estimated to contribute to 5–10% of lung cancer deaths.

Further Reading Birmili W, Allen AG, Bary F, and Harrison RM. Trace metal concentrations and water solubility in size-fractionated atmospheric particles and influence of road traffic. Environmental Science and Technology (in press). Brasseur GP, Prinn RG, and Pszenny AP (eds.) (2003) Atmospheric Chemistry in a Changing World. Berlin: Springer. Brimblecombe P and Maynard RL (eds.) (2000) Urban Atmosphere and its Effect. London: Imperial College Press. Brown LM, Collings N, Harrison RM, Maynard AD, and Maynard RL (eds.) (2003) Ultrafine Particles in the Atmosphere. London: Imperial College Press. Davidson CI (2000) Human exposure to toxic metals. In: Rubin ES (ed.) Introduction to Engineering and the Environment, pp. 402–433. New York: McGraw Hill. Friedlander SK (2000) Smoke, Dust, and Haze. Fundamentals of Aerosol Dynamics, 2nd edn. Oxford: Oxford University Press. Gelencser A (2004) Carbonaceous Aerosol. Dordrecht: Springer. Hinds WC (1999) Aerosol Technology, 2nd edn. New York: John Wiley & Sons Inc. IPCC (2001) Intergovernmental Panel of Climate Change, Third Assessment Report. Cambridge: Cambridge University Press. Kulmala M, et al. (2004) Formation and growth rates of ultrafine atmospheric particles: a review of observations. Journal of Aerosol Science 35: 143–176.

Relevant Website http://ies.jrc.cec.eu.int – European Commission DirectorateGeneral Joint Research Center which monitors air pollution from a cruise ship.

Radon A S Rosario and H-E Wichmann, GSF – Institute of Epidemiology, Neuherberg, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract

Structure and Physicochemical Properties Radon is a naturally occurring, radioactive noble gas. It is chemically inert, colorless, odorless, and soluble in water. There are three naturally occurring radon isotopes: radon-222 (radon), radon-220 (thoron), and radon-219 (actinon), of which radon-222 is the most important. It is the only gaseous member

ENVIRONMENTAL POLLUTANTS / Radon 121 Uranium-238 4500 million years




..

Radon-222 3.82 days


.




Atomic mass

Radium-226 1600 years



Half-life Type of radioactive decay


Radon-222 3.82 days
Short-lived radon progeny

Polonium-218 3.05 min
Lead-214 26.8 min



Bismuth-214 19.9 min



Polonium-214 164 µs
Lead-210 22.3 years



Bismuth-210 5.0 days



Polonium-210 138.4 days
Lead-206 (stable)

Figure 1 Natural decay chain of uranium-238.

of the radioactive decay chain of uranium-238 (Figure 1). Uranium is ubiquitously present in the earth’s crust, albeit in variable concentrations. Radon decays with a half-life of 3.8 days, emitting ionizing radiation in the form of a-particles (helium nuclei). The biologic effects of radon are mainly due to its radioactive decay products (also called radon progeny or radon daughters), in particular the shortlived decay products with a half-life of up to 30 min, such as the a-emitters polonium-218 and polonium214. These are solid heavy metal isotopes, which mostly attach to dust particles or form clusters with water molecules. Radon gas concentrations are usually measured in becquerels (Bq) per cubic meter (1 Bq m
3 corresponds to one radioactive decay per second and cubic meter of air). Another unit in current use is picoCurie (pCi) per liter (1 pCi l
1 ¼ 37 Bq m
3).

Source and Exposure Radon gas is the most important source of natural radioactivity, accounting for about half of the effective radiation dose received from natural sources in the

worldwide average. It is formed in rocks and soils through the radioactive decay of radium-226, which itself is a decay product of uranium-238. As a gas, it can ascend and diffuse from the soil into the air. Alternatively, it can dissolve in ground water. The amount of radon released from the soil depends on its radium content, but also on factors affecting radon transport such as underground air and water flow and the porosity and permeability of the soil. In outdoor air, radon concentrations are usually low because of its dilution with atmospheric air. In underground mines and caves or inside buildings, however, radon concentrations can build up. Occupational Radon Exposure: Underground Miners and Others

Underground miners, especially uranium miners, can be subject to very high radon concentrations. Other potentially exposed workplaces are radon spas or underground caves and mines (e.g., for tourist guides). Above ground, employees in water supply facilities can be exposed to high radon concentrations. Radon concentrations in typical workplaces (factories,

122 ENVIRONMENTAL POLLUTANTS / Radon

offices, schools, etc.) tend to be lower than in the home, although the data are sparse and there can be exceptions to the rule. Indoor Radon Exposure

For the general population, radon gas concentrations in houses pose a potential health hazard, although the concentrations are usually considerably lower than those encountered in occupationally exposed groups. Apart from the geological conditions described above, the influx of radon into houses is influenced by meteorological factors such as wind speed and indoor–outdoor differences in barometric pressure and temperature. Indoor radon concentrations are also determined by structural characteristics of the house and by ventilation habits (Table 1). Radon levels are generally highest in the basement and lower in the higher floors. Concentrations tend to be higher in single-family homes and lower in apartment blocks. Urban areas are likely to have lower radon levels than rural areas. The radon concentration inside a house is subject to diurnal and seasonal variation (usually higher at night and in the winter months). Indoor radon concentrations within a country can vary by several orders of magnitude. Variation within a country is considerably larger than variation among countries. The worldwide average indoor radon concentration is estimated to be 40–50 Bq m
3, but up to several thousands or even tens of thousands of Bq m
3 have been reported in many countries. Table 1 Factors contributing to increased indoor radon concentrationsa Geological factors

High uranium/radium content of the soil, e.g., in granite rocks High porosity and/or permeability of the soil Tendency for higher concentrations in mountainous regions

House characteristics

Presence of cracks and fissures in the basement floor or walls Insufficiently sealed entrance of tubes or cables into the basement Houses without basement or without separation of the living area from the basement Living area located in a floor near the basement Single-family home Insulation windows and similar sealing efforts to enhance energy efficiency

Behavior

Low ventilation rate

a

This list can only serve as a rough guide.

Radon concentrations typically show a skewed distribution (Figure 2). Most individuals in a population are exposed to low radon concentrations, while a small number of people are exposed to high or very high concentrations. Soil is generally the main source of indoor radon exposure. Other less important sources are building materials, radon dissolved in tap water (especially from private wells), or radon contained in coal or natural gas used for heating and cooking.

Clinical Consequences Radon is of public health concern as it has been found to increase the risk of lung cancer. An excess of lung diseases among miners in the German Ore Mountains was observed as early as the sixteenth century by Paracelsus, and in 1913 a possible etiologic link between radon exposure and lung cancer was postulated for the first time. Radon has been classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC) in 1988 and 2001. Mechanisms of Radon-Induced Carcinogenesis

Most of the radon gas itself is exhaled again after inhalation, but its solid decay products can be deposited in the bronchi and the lung. These decay products undergo further radioactive decay on the surface of the bronchial epithelium before they can be removed from the airways by clearance mechanisms. They emit a-radiation, which is known to be more effective than other types of radiation in terms of biological damage. Whereas high doses of ionizing radiation can lead to acute effects, low doses lead to stochastic effects, that is, they increase the probability of mutations, either through direct DNA damage or through the formation of free radicals and other oxidative agents. Radiation typically causes large deletions and rearrangements, rather than simple base-pair exchanges. Alpha radiation in particular tends to cause more complex, clustered DNA damage that is more difficult for the cell to repair. If not repaired, it can be one of the critical events in the multistep process eventually leading to cancer. Radiation can affect neighboring cells (probably via cell–cell communication) or descendant cells that have not themselves been irradiated. On the other hand, small doses of radiation might render the cell more resistant to subsequent carcinogenic stimuli by activating repair enzymes. There is no clear evidence that radiation causes mutations at specific sites of the genome.

ENVIRONMENTAL POLLUTANTS / Radon 123

0

100

200

300

Radon level (Bq

400

500

m–3)

Figure 2 Distribution of indoor radon concentrations in West Germany. Ten values between 500 and 1111 Bq m
3 not shown. Data from Schmier H and Wicke A (1985) Results from a survey of indoor exposure in the Federal Republic of Germany. Science of the Total Environment 45: 307–310.

In various mammalian cell systems, radon and its progeny were found to cause malignant transformation of cells. In cytogenetic studies both in vivo and in vitro, radon and its progeny have led to chromosome aberrations and increased cell proliferation in a dose-dependent fashion. An increased number of chromosome aberrations have also been found in humans exposed to high indoor radon concentrations. Experiments in rats have shown that radon inhalation can lead to the formation of tumors in the respiratory tract. Epidemiological Studies in Underground Miners

The epidemiological evidence regarding the carcinogenicity of radon in humans was first assembled in studies of large cohorts of underground miners. In a combined analysis of 11 studies performed worldwide, nearly 68 000 miners were evaluated. A statistically significant relationship between their cumulative radon progeny exposure and the subsequent lung cancer risk was found. The relationship was modeled assuming that the relative risk (the ratio of the lung cancer risk of an exposed individual to the risk of an unexposed or low-exposed individual) for lung cancer increases linearly with increasing radon exposure. This assumption was strengthened by the experimental observation that the traversal of a single a-particle through a cell has the potential to cause permanent changes to the DNA, although the existence of a threshold could not be ruled out completely.

Epidemiological Studies on Indoor Radon and Lung Cancer

The evidence found in the studies carried out in miners led to concern about the potential risk of elevated radon concentrations in houses. Consequently, a series of population-based (mostly case– control) studies were carried out. Not all of these studies showed an association between indoor radon concentrations and lung cancer. Methodological differences among the studies partly explain the different results. The main problem, however, lies in the insufficient sample size of the single studies. Although radon is an important risk factor for lung cancer, this risk is quite small as only a small proportion of the population experiences high radon concentrations; hence, sample sizes in studies of radon exposure need to be large. For a precise quantitative risk assessment, the data from the single studies need to be combined. Such efforts have been undertaken both in North America and Europe. The results of the European analysis show a linear dose–response relationship with no evidence of a threshold. Relative lung cancer risk increased by 8% per 100 Bq m
3 increase in indoor radon concentrations (95% confidence interval: 3–16%). In the North American analysis, relative risk increased by 11% per 100 Bq m
3 (95% confidence interval 0–28%). It is crucial that smoking behavior is taken into account in such studies, since smoking is a very strong risk factor for lung cancer and there is an association

124 ENVIRONMENTAL POLLUTANTS / Radon

between radon and smoking; in rural areas, smoking tends to be less frequent, while radon exposures tend to be higher. Therefore, adjustment for smoking increases the risk estimates for radon in most studies. A major problem for the case–control studies is the fact that cancer develops over a long time period; consequently, the level of radon exposure in houses inhabited during the previous decades has to be estimated based on measurements taken in the present. This uncertainty generally leads to risk estimates that are biased downwards. In the collaborative European analysis, accounting for these uncertainties doubled the risk estimate, that is, the linear increase in relative risk was estimated to be 16% per 100 Bq m
3 (95% confidence interval: 5–31%). Radon seems to be more likely to cause small-cell carcinomas than other histological subtypes of lung cancer. Similarly, the risk estimate in the North American analysis increased when restrictions regarding exposure assessment were taken into account. Lung Cancer Cases Attributable to Radon; Influence of Smoking

Radon is considered the second most important single agent that can cause lung cancer, after smoking. Whereas smoking causes about 90% of all lung cancer cases in male populations, the proportion of cases attributable to radon is considerably lower, in the order of 5–10%, depending, among others, on the distribution of indoor radon concentrations in the country of interest. Only a small part of these cases occur at high radon concentrations, since the number of people exposed to very high concentrations is usually quite low. A substantial number of cases arise in houses with moderate radon concentrations. Current evidence suggests that the combined effect of smoking and radon on lung cancer risk is multiplicative or slightly less than multiplicative. This means that smoking greatly increases the radon-related lung cancer risk. Because smoking is such a strong risk factor for lung cancer and because it is still a widespread habit in the population, the absolute number of radon-induced lung cancer cases is considerably higher among smokers than among nonsmokers.

Management and Treatment Increased indoor radon concentrations can and should be reduced. Official action levels vary from country to country. The European Commission recommends that 400 Bq m
3 should not be exceeded in existing houses and 200 Bq m
3 in newly built houses. The US Environmental Protection Agency (EPA) sets the action level at 148 Bq m
3.

Under normal conditions, frequent and thorough ventilation helps to reduce radon levels. In houses with high radon concentrations, simple and effective steps can be taken to reduce radon exposure (e.g., improved sealing of the basement or installation of ventilation systems). Radon concentrations can vary substantially even among nearby houses, and so a definite conclusion about the radon level in a given house can only be derived from direct measurement. Measurements should preferably be taken over a whole year to cancel out seasonal effects. Information on regions that are likely to suffer from higher radon concentrations, laboratories that offer radon detectors, and how to reduce radon concentrations in buildings is usually available from national radiation protection institutions. Reducing the number of smokers in the population and enabling smokers to quit will considerably diminish the number of radon-related lung cancers. As far as treatment and prognosis are concerned, radiation-induced lung cancers do not differ from other lung cancers. See also: Environmental Pollutants: Tumors, Malignant: Overview.

Overview.

Further Reading Burkart W (1989) Radiation biology of the lung. Science of the Total Environment 89: 1–230. Darby S, Hill D, Auvinen A, et al. (2005) Radon in homes and risk of lung cancer: collaborative analysis of individual data from 13 European case–control studies. British Medical Journal 330: 223–227. IARC International Agency for Research on Cancer (2001) Ionizing Radiation, Part 2: Some Internally Deposited Radionuclides. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 78. Lyon: IARC Press. ICRP International Commission for Radiation Protection (1987) Lung Cancer Risk from Indoor Exposure to Radon Daughters. ICRP Publication No. 60. New York: Pergamon. Jostes RF (1996) Genetic, cytogenetic, and carcinogenic effects of radon: a review. Mutation Research 340: 125–139. Krewski D, Lubin JH, Zielinski JM, et al. (2005) A combined analysis of North American case–control studies of residential radon and lung cancer. Epidemiology 16: 137–145. Little JB (2000) Radiation carcinogenesis. Carcinogenesis 21: 397–404. Lubin JH, Boice JD, Edling C, et al. (1995) Lung cancer in radonexposed miners and the estimation of risk from indoor exposure. Journal of the National Cancer Institute 87: 817–827. Lubin JH, Toma´sˇek L, Edling C, et al. (1997) Estimating lung cancer mortality from residential radon using data for low exposures of miners. Radiation Research 147: 126–134. Lubin JH, Wang ZY, Boice JD, et al. (2004) Risk of lung cancer and residential radon in China: pooled results of two studies. International Journal of Cancer 109: 132–137. NAS National Academy of Sciences (1999) Health Effects of Exposure to Radon (BEIR VI Report). Washington, DC: National Academy Press. Samet JM and Eradze GR (2000) Radon and lung cancer risk: taking stock at the millenium. Environmental Health Perspectives 108(supplement 4): 635–641.

EOTAXINS 125 Schmier H and Wicke A (1985) Results from a survey of indoor exposure in the Federal Republic of Germany. Science of the Total Environment 45: 307–310. UNSCEAR (United Nations Scientific Committee on the Effects of Atomic Radiation) (2000) Sources and Effects of Ionizing Radiation. UNSCEAR 2000 Report to the General Assembly. vol. I: Sources; vol. II: Effects. Wichmann HE, Rosario AS, Heid IM, et al. (2005) Increased lung cancer risk due to residential radon in a pooled and extended analysis of studies in Germany. Health Physics 88: 71–79.

Relevant Websites http://www.epa.gov – US Environmental Protection Agency: y to protect human health and the environment. http://www.unscear.org – This website includes an overview of average indoor radon concentrations in several countries. http://www.nap.edu – The National Academy Press publishes on agriculture, behavioral science, biology, chemistry, computer science, etc.

EOTAXINS I Sabroe, University of Sheffield, Sheffield, UK T J Williams and J E Pease, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The Eotaxins are small proteins secreted during allergic reactions that have the ability to attract eosinophils and certain other cell types from the blood. Three of these CC-chemokines have been described, Eotaxin-1, -2, and -3. They induce leukocyte recruitment by acting on a receptor, CCR3, which is highly expressed on the surface of eosinophils and also on basophils, mast cells, and a subpopulation of the Th2 lymphocytes. Neutralization of Eotaxin-1 by monoclonal antibodies has been shown to suppress eosinophil accumulation during allergic reactions in man and animal models. Small molecule antagonists are under development with the aim of blocking eosinophil recruitment and, as a consequence, inhibiting the lung dysfunction and tissue remodeling associated with eosinophil activation.

Introduction The eotaxins comprise a family of three molecules related by their biological activity. Eotaxin was first identified in guinea pigs, where the biological activity responsible for pulmonary eosinophil recruitment in response to allergen challenge was shown to be a protein, and was then revealed by microsequencing to be a CC chemokine. This molecule induced a relatively selective recruitment of eosinophils (the first chemokine found to do so) and was thus named according to its biological function. The human homolog of the guinea pig molecule was identified and shown also to mediate eosinophil chemotaxis. This was followed by the identification of the receptor for eotaxin, which was named CC chemokine receptor-3 (CCR3). Two further CC chemokines were subsequently identified in man that also stimulate chemotaxis of eosinophils via CCR3. Though they are quite dissimilar to eotaxin (eotaxin-1) in

terms of amino acid sequence, they were named eotaxin-2 and eotaxin-3 on the basis of their functional homology. A recently adopted systematic nomenclature has resulted in these molecules being renamed CCL11, CCL24, and CCL26. Subsequent work has implicated the eotaxin/CCR3 axis in many aspects of leukocyte trafficking in allergic inflammation, and has provided powerful arguments for targeting this pathway in the treatment of diseases such as asthma and allergic rhinitis.

Structure The fidelity of the three eotaxins for their receptor CCR3 is curious, as at the primary sequence level, the mature proteins share only 30–37% identity. Furthermore, the eotaxins are not all encoded on the same chromosome: the gene for CCL11 is on chromosome 17 whereas the genes for CCL24 and CCL26 are on chromosome 11. Like most chemokines described to date, the three eotaxins share a common protein fold, the ‘Greek-key’ motif, consisting of three antiparallel b-pleated sheets, overlaid by a C-terminal a-helix (Figure 1(a)). Truncation studies of the eotaxins imply that the N-terminus of each chemokine is important for the activation of CCR3 and, notably, these domains contain considerable sequence divergence between the molecules. This may explain their distinct rank orders of potency/efficacy at CCR3 as measured by assays of eosinophil chemotaxis or shape change, with CCL11 being the more potent and efficacious ligand. Local pH and ionic strength have been reported to strongly influence the ability of CCL11 to bind to CCR3, suggesting that interactions between these molecules involve charged amino acid residues, and posttranslational modifications such as glycosylation may affect availability and receptor binding of the eotaxins. The interaction of CCL11 with CCR3 at the molecular level has been

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Figure 1 (a) A cartoon representation of eotaxin-1, illustrating the Greek-key structural motif typical of chemokines. (b) A surface model of eotaxin-1, with the basic residues lining the extended groove and postulated to bind the acidic CCR3 N-terminus more darkly shaded. Both models were produced by the package PyMOL using the solution structure of Crump et al. as template (file 1EOT available from the protein data bank).

examined by the use of chimeric receptor constructs and conforms to the two-site model of receptor activation in which a high-affinity binding site within the receptor N-terminus tethers the chemokine, facilitating delivery of the ligand to a second, lower affinity site composed of the remaining extracellular loops. This latter interaction is necessary for signal transduction in response to CCL11 and correlates with recent data from studies employing a soluble protein mimic that have shown that the juxtaposition of both the N-terminus and third extracellular loop of CCR3 are required for the optimal binding of this chemokine. Residues within the N-loop of CCL11 have been shown to play a role in binding to CCR3, and binding studies utilizing a CCR3 N-terminal peptide reported that it bound within an extended groove in the CCL11 surface (Figure 1(b)).

Regulation of Production In general, allergic inflammation is associated with an induction of CCL11, and probably CCL24 and CCL26. These proteins may be directly released from early response cells (e.g., mast cells), and then synthesis upregulated in a wide range of tissue cells (e.g., epithelia, endothelial cells, airway smooth muscle) and leukocytes (including eosinophils and macrophages) as a result of an inflammatory cascade. Th2 cells are able to regulate the infiltration of eosinophils in allergic reactions by secreting interleukin-4 (IL-4) and IL-13, which switch on the genes for the eotaxins in others cells in the tissue. In airway smooth muscle, IL-9 can also induce CCL11 generation. Synergistic induction of CCL11 generation by

Th2 cytokines and other proinflammatory molecules such as IL-1b has also been demonstrated in airway smooth muscle cells. There is evidence for separate regulation of the production of eotaxins, since intradermal allergen challenge in man results in phased induction of CCL11 and CCL24. Additionally, CCL24 is induced from monocytes by lipopolysaccharide (LPS) stimulation, whilst CCL11 is not, and since allergen exposure often occurs together with contaminants such as LPS, this provides a mechanism by which innate immunity may amplify allergic inflammation. (Further complicating this picture is a change in phenotype as monocytes mature, such that IL-4, but not LPS, induces CCL24 generation from macrophages.) The opportunities for phased regulation of CCL11, CCL24, and CCL26 generation provides a potential mechanism to sequentially regulate trafficking of cells expressing CCR3.

Biological Function Eotaxins are chemokines (chemoattractant cytokines) and, as such, their principal actions are to facilitate the recruitment of leukocytes to inflammatory sites. The eotaxin family were described through their apparently selective recruitment of eosinophils. In humans and animals, intradermal injection of CCL11 and CCL24 has been shown to recruit eosinophils and, likewise, nasal administration also recruits eosinophils into nasal lavage fluid. It remains unclear whether these molecules truly serve individual nonredundant functions or whether they have subtly different functions, as eotaxins can show

EOTAXINS 127

distinct patterns of cellular expression and can be differentially regulated by different cytokines. Studies of allergic inflammation in human skin induced by intradermal injection of allergen have suggested that CCL11 generation correlates with early eosinophil recruitment, whilst CCL24 generation may be more associated with later phases of tissue eosinophilia. CCL11 also plays roles in constitutive trafficking of eosinophils to noninflamed mucosal sites (including the gastrointestinal tract). In addition to mediating the trafficking of eosinophils to tissues, the eotaxins are also able to mobilize eosinophils from the bone marrow (facilitating local recruitment at sites of inflammation by increasing systemic supply). Immature cells can also be mobilized: CCR3 has been demonstrated on CD34 þ stem cells and committed eosinophil progenitors. Eotaxins can also activate eosinophils, inducing cytokine generation and modulating their life span. In addition to substantial roles in the regulation of eosinophil recruitment and activation, it has become apparent that the eotaxin receptor, CCR3, is expressed on a wider range of cells (Figure 2). Regulated expression on human mast cells, basophils, and subsets of macrophages and Th2-type T cells suggests that eotaxins may play important roles in the

trafficking of these cells as well. The ability of eotaxins to mediate recruitment of so many cell types involved in allergic inflammation, from Th2-type T cells to basophils, mast cells, and eosinophils, with amplification of generation of eotaxins by Th2-type cells, affirms that eotaxins are likely to play major roles in respiratory diseases.

Receptors The eotaxins exert their common actions through their ability to bind to CCR3. In common with other chemokine receptors, CCR3 couples to G proteins (principally of the Gai subtype), to mediate activation of signaling pathways including [Ca2 þ ]i flux, MAP kinases, PI-3 kinases, and protein kinase C. CCR3 is very promiscuous and binds several other chemokines in addition to the three eotaxins, including members of the monocyte chemoattractant protein (MCP) family. There is some evidence that CCR3 ligands show varying abilities to activate CCR3, and there may be temporal roles for different CCR3 ligands in eosinophilic inflammation. Therefore, although therapeutic antibodies against CCL11 have been developed, targeting the eotaxin/CCR3 axis in disease may be more effectively achieved using CCR3

AM

EC

ASM

MC

Eotaxins Eotaxin 3

Inhibition of CCR1, 2, and 5 may alter T cell recruitment Eotaxin

MC Eos Th2-type T cells

Partial agonist of CCR2: may recruit monocytes

CCR3-mediated actions Figure 2 Eotaxins can be generated by a range of cell types within the lung, including macrophages, epithelia, mast cells, and airway smooth muscle. Their principal action is probably to cause the recruitment of eosinophils, but they also act on other CCR3-expressing cells including mast cells and Th2-type T cells. Partial agonist and antagonist actions at other receptors may also result in alterations of patterns of leukocyte recruitment. AM, alveolar macrophage; EC, epithelial cells; MC, mast cell; ASM, airway smooth muscle; Eos, eosinophil.

128 EOTAXINS

small molecule antagonists, which are in clinical development. Intriguingly, the eotaxins show varying abilities to antagonize or partially stimulate other chemokine receptors: CCL11 is a weak agonist at CCR2, whilst CCL26 can antagonize CCR1, CCR2, and CCR5 (possibly influencing in vivo recruitment of monocytes and T cell subsets). Meanwhile, ligands of the Th1-associated chemokine receptor, CXCR3, can antagonize signaling at CCR3. The summated actions of eotaxins may therefore be a subtle blend of actions on more than one chemokine receptor, and Th1-associated chemokines may exert opposing actions on the Th2-favored CCR3 axis.

Eotaxins in Respiratory Diseases Eotaxins, and their major receptor CCR3, are expressed wherever allergic inflammation is present in the human respiratory tract, and levels of expression show correlations with degree of severity. Upregulation of eotaxins in allergic inflammation in the nose and lung are consistent with an important role in lung disease, and these molecules may play additional roles in the regulation of sensitization. The roles of eotaxins in lung diseases have been both fairly and unfairly caught up in a current, wideranging debate on the role of eosinophils in asthma. In animal models, eotaxin has incontrovertibly important roles in eosinophil, and to some extent Th2type T cell, recruitment in allergic inflammation. Depending upon the strain and exact model, removal of eotaxins by neutralizing antibodies or knockout strategies can reduce eosinophil recruitment and airway hyperreactivity (AHR), but a causal link between eosinophilia and AHR is not a feature of all models. The CCR3(–/–) mouse provides a further layer of complexity where routes of sensitization exert an effect on the phenotype. Intraperitoneal allergen sensitization results in increased lung mast cell numbers versus the wild-type mice and no abolition of AHR in response to allergen challenge. Where mice are sensitized epicutaneously, there is no increase in lung mast cell numbers and allergen challenge of knockouts is associated with severely impaired eosinophil recruitment and prevention of AHR. In man, the role of eosinophils in allergic airway disease remains controversial. Anti-IL-5 has been used to dramatically reduce eosinophil numbers from the circulation, but is less effective at depleting tissue eosinophils (perhaps because eosinophil progenitors, potentially originally recruited by eotaxins, are resident in lung tissues). Anti-IL-5 treatment has been associated with reductions in AHR in certain animal models, but not in small clinical studies to

date. The failure to deplete lung tissue cells means that these studies have been inconclusive with respect to the involvement of eosinophils in bronchial hyperreactivity in man. More recent studies of the results of anti-IL-5 treatment in man indicate that the eosinophil may be more important in tissue remodeling than in hyperreactivity. Eotaxin expression is prominent in allergic diseases and the eotaxin receptor CCR3 is expressed by key cell types involved in the process. Clinical trials of small molecule CCR3 antagonists, currently under development, will provide valuable information on the importance of the eotaxins in the disease process and hold out the promise of future selective therapy for allergy and asthma. See also: Adhesion, Cell–Matrix: Focal Contacts and Signaling; Integrins. Asthma: Overview. Chemokines, CC: C10 (CCL6); MCP-1 (CCL2)–MCP-5 (CCL12); RANTES (CCL5); TARC (CCL17); TECK (CCL25). G-Protein-Coupled Receptors. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; Monocytes.

Further Reading Dent G, Hadjicharalambous C, Yoshikawa T, et al. (2004) Contribution of eotaxin-1 to eosinophil chemotactic activity of moderate and severe asthmatic sputum. American Journal of Respiratory and Critical Care Medicine 169: 1110–1117. Flood-Page PT, Menzies-Gow AN, Kay AB, and Robinson DS (2003) Eosinophil’s role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. American Journal of Respiratory and Critical Care Medicine 167: 199–204. Forsythe P and Befus AD (2003) CCR3: A key to mast cell phenotypic and functional diversity? American Journal of Respiratory Cell and Molecular Biology 28: 405–409. Gutierrez-Ramos JC, Lloyd C, Kapsenberg ML, Gonzalo JA, and Coyle AJ (2000) Non-redundant functional groups of chemokines operate in a coordinate manner during the inflammatory response in the lung. Immunological Reviews 177: 31–42. Humbles AA, Conroy DM, Marleau S, et al. (1997) Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo. Journal of Experimental Medicine 186: 601–612. Humbles AA, Lu B, Friend DS, et al. (2002) The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inflammation and hyperresponsiveness. Proceeding of the National Academy of Sciences, USA 99: 1479–1484. Jose PJ, Griffiths-Johnson DA, Collins PD, et al. (1994) Eotaxin: a potent chemoattractant cytokine detected in a guinea-pig model of allergic airways inflammation. Journal of Experimental Medicine 179: 881–887. Lamkhioued B, Abdelilah SG, Hamid Q, et al. (2003) The CCR3 receptor is involved in eosinophil differentiation and is up-regulated by Th2 cytokines in CD34 þ progenitor cells. Journal of Immunology 170: 537–547. Ma W, Bryce PJ, Humbles AA, et al. (2002) CCR3 is essential for skin eosinophilia and airway hyperresponsiveness in a murine

EPIDERMAL GROWTH FACTORS 129 model of allergic skin inflammation. Journal of Clinical Investigation 109: 621–628. Menzies-Gow A, Ying S, Sabroe I, et al. (2002) Eotaxin (CCL11) and eotaxin-2 (CCL24) induce recruitment of eosinophils, basophils, neutrophils, and macrophages as well as features of early- and late-phase allergic reactions following cutaneous injection in human atopic and nonatopic volunteers. Journal of Immunology 169: 2712–2718. Palframan RT, Collins PD, Williams TJ, and Rankin SM (1998) Eotaxin induces a rapid release of eosinophils and their progenitors from the bone marrow. Blood 91: 2240–2248. Ponath PD, Qin S, Post TW, et al. (1996) Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. Journal of Experimental Medicine 183: 2437–2448. Rothenberg ME (1999) Eotaxin. An essential mediator of eosinophil trafficking into mucosal tissues. American Journal of Respiratory Cell and Molecular Biology 21: 291–295. Rothenberg ME, Ownbey R, Mehlhop PD, et al. (1996) Eotaxin triggers eosinophil-selective chemotaxis and calcium flux via a

distinct receptor and induces pulmonary eosinophilia in the presence of interleukin 5 in mice. Molecular Medicine 2: 334–348. Ying S, Robinson DS, Meng Q, et al. (1999) C-C chemokines in allergen-induced late-phase cutaneous responses in atopic subjects: association of eotaxin with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C-C chemokines (monocyte chemoattractant protein3 and RANTES). Journal of Immunology 163: 3976–3984.

Relevant Websites http://www.pymol.org – A user-sponsored molecular visualization system on an open-source foundation providing the package PyMol. http://www.rcsb.org – A protein data bank: worldwide reporitory for the processing and distribution of three-dimensional biological macromolecular structure data.

EPIDERMAL GROWTH FACTORS J C Pache, University Hospital of Geneva, Geneva, Switzerland & 2006 Elsevier Ltd. All rights reserved.

Abstract Epidermal growth factor (EGF) was the first growth factor to be discovered. EGF precursor is a transmembranous glycoprotein from which the active EGF is cleaved by an endopepsidase. With the other members of the epidermal growth factors family, it binds to the epidermal growth factor receptor (EGFR). After ligand binding, EGFR (ErbB-1) dimerizes with itself or with its homologs ErbB-2, ErbB-3, or ErbB-4 and consequently increases its intracellular tyrosine kinase activity. This activates signaling cascades that have many effects: cell proliferation, reduced apoptosis, and angiogenesis. The alveolar septation that occurs in embryonic life relies on the presence of EGF. The bronchial wall in chronic bronchitis overexpresses EGF and EGFR. Bronchial epithelial cells, in asthma, demonstrate an increased expression of EGFR. EGFR activation occurs in non-small cell lung carcinoma by overexpression of ligand and/ or receptors, or by a partial deletion of the receptor that results in a ligand-independent activation. Many EGFR-targeted agents are in development and include antibodies directed against the receptor extracellular domain, or small molecules that interfere with the intracellular kinase domain, and various clinical trials are currently testing the relevance of these inhibitors.

Introduction Cohen discovered epidermal growth factor (EGF) in 1962 in submaxillary glands as a factor inducing premature eyelid opening, incisor tooth eruption, and hair growth inhibition when injected in the

newborn mice. Subsequent studies with skin explants revealed a direct effect on epidermal growth. Ligands of the EGFR include EGF, transforming growth factor alpha (TGF-a), heparin-binding EGFlike growth factor (HB-EGF), amphiregulin (AR), betacellulin (BTC), epiregulin (EPR), and epigen. They have a high-affinity binding to the EGFR. Ligand-independent activation of EGFR may also be achieved by oxidative stress produced by cigarette smoke or activated neutrophils.

Structure Chromosome 4 (q25–q27) contains the gene for EGF, which measures approximately 120 kb. Exon 24 encodes the precursor EGF, while exons 20 and 21 encode the mature EGF. The EGF precursor molecule is a glycoprotein composed of 1207 amino acids, which consist of an intracytoplasmic (149 amino acids) and an extracellular (1032 amino acids) domain, respectively. The transmembranous part of the molecule anchores EGFR to the cytoplasmic membrane. The active EGF molecule (53 residues) is cleaved from the precursor by an endopepsidase (Figure 1). Both membrane-anchored EGF and soluble EGF bind and activate the EGFR.

Regulation of Productivity and Activity Activation of EGFR can, in turn, activate Ras signaling, but in reverse, mutant Ras upregulates EGFR

130 EPIDERMAL GROWTH FACTORS

Extracellular

Cytoplasmic

Mature form of EGF with three intramolecular disulfide bridges

EGF precursor transmembranous (prepro-EGF) Figure 1 EGF precursor and mature molecules.

ligands. Various stimuli that include tumor necrosis factor alpha (TNF-a), cigarette smoke, and inhalation of toxins like bleomycin increase the expression of EGFR in conducting airways epithelial cells. In rodents, submaxillary glands mostly expressed EGF. It is also located in the apical membrane of the ascending limb of the kidney, in exocrine glands of the gastrointestinal tract, and in serous acini of the nasal cavity. In Madin–Darby canine kidney (MDCK) cells transfected with prepro-EGF, molecule immunoreactivity appears predominantly at the apical membrane, but the metalloproteinase-dependent cleavage happens mostly at the basolateral protein. Simultaneously, EGFR locates at the basolateral cell surface. The metalloproteinase involved in prepro-EGF cleavage is unknown. TNF-a converting enzyme and metalloproteinase 17 (TACE/ADAM17) cleaves the TGF-a precursor molecule. EGFR located basolaterally binds avidly to the active ligands.

Biological Function The EGFR gene is on chromosome 7 (p12.3–p12.1), contains 26 exons, and covers approximately 110 kb. The 1186 residues molecule inserts across the cell membrane. After ligand binds the receptor, EGFR dimerizes with itself or with its three known homologs – ErbB-2 (also known as neu or HER2), ErbB-3 (HER3), and ErbB-4 (HER4) – to form heterodimers. This increases the quantity of dimerized receptor and the enzymatic activity of their intracellular kinase (Figure 2). The intracellular effects of EGF activation take place predominantly via tyrosine kinase activity. The

intracytoplasmic molecule acts as a docking site for many signaling proteins. After activation of the EGFR kinase and autophosphorylation, the molecule Grb2 binds to EGFR. This will result in the activation of the proto-oncogene Ras which will further activate different kinases, including extracellular signal-related kinases (ERKs) 1 and 2, leading ultimately to activation of intranuclear transcription factors. Members of the Src family of cytosolic tyrosine kinases are also involved in EGFR signaling, whereas overexpression of Src proteins strongly enhances EGF-mediated proliferation and transformation in epithelial and fibroblastic cells. STAT proteins are associated with the EGFR as inactive transcription factors. After activation by EGFR kinase activity, they form homo- or heterodimers, become activated, and can move to the nucleus. EGF stimulation of a cell produces strong effects on the phospholipid metabolism: EGFR directly activates phosphatidylinositol-3 kinase (PI3K), phospholipase C-g (PLC-g), and phospholipase D (PLD).

Role in Development

EGF and EGFR play predominant roles in development. The Drosophila epidermal growth factor receptor, called DER, plays multiple roles in embryonic or postembryonic stages, such as proliferation and guidance of migration. DER activates tracheal invagination and branching, as well as the specification of muscle precursor. EGFR activation can lead to cell migration by activation of many different pathways that include the activation of PLC-g.

EPIDERMAL GROWTH FACTORS 131 EGF

NH2 EGFR homodimer Cell membrane Ras P Kinase domain

Raf

P Src

P

MEK 1/2

P

Erk 1/2

P

P P Grb2

Nucleus

P P

Cell proliferation

P STAT

P

STAT

P




C-

PI3K

PL

COOH

Cell mobility Figure 2 EGFR homodimer after EGF binding with consecutive activation of intracellular signaling molecules.

EGF stimulates DNA synthesis in cultured embryonic chick tracheal and bronchial epithelium. EGF intravenous infusion into fetal lambs induces tracheal, bronchial, and type II pneumocytes hyperplasia. Immunohistochemical studies in mouse embryo localize EGF and EGFR in epithelial and smooth muscle cells of the bronchi and bronchioles. In the human fetal and postnatal lung, EGF and EGFR colocalize within the large airways. Mature epithelial cells also express EGF which regulates proliferation of type II pneumocytes. In the fetal mouse lung, EGF stimulates branching morphogenesis, and production of surfactant proteins A and C. Abrogation of the EGFR signaling results in decreased branching morphogenesis, and a pulmonary lethal phenotype. EGF injection into neonatal mice induces precocious eyelid opening and tooth eruption, subtle effects on neurobehavioral development, and a reduction in growth rates. Genetic Experiments

Transgenic mice have been produced in which TGF-a is expressed under the control of the surfactant

protein C (SP-C) promoter. The lungs show enlarged alveoli and subpleural and peribronchial fibrosis. This implicates a paracrine signal between epithelial and mesenchymal cells, which results in fibroblast proliferation and collagen deposition. Results of gain-of-function gene experiments focus on the role of EGF/EGFR on proliferation whose consequences will be discussed later. Loss-of-function data, in contrast, produce information on the role of EGF in development and physiology. Sialadenectomy was first used to study the effects of reduced EGF in the body. The most affected organs were the mammary gland and the epidermis. Both organs showed a reduction in size and thickness that could be reversed by EGF administration. Genetic manipulations have produced animals null for EGF, amphiregulin, and TGF-a. The phenotypes of TGF-anull mice are mild with a waxy fur and eyes that at birth are open and opaque. EGF-null and amphiregulin-null animals have no obvious phenotype. EGFR knockout mice, dependent on their genetic background, may have a severely affected phenotype that varies from mid-gestational death to severe abnormalities of the lung. They include impaired branching, and deficient alveolization and septation; type II pneumocytes have a decreased number of lamellar bodies. EGFR is primarily expressed at the cell surface, constantly internalized and recycled to the surface. Ligand activation similarly produces a rapid internalization of EGFR and eventually degradation in lysosomes, or recycling to the cell surface.

Respiratory Diseases In children with bronchopulmonary dysplasia, bronchial cells present EGF. This suggests the implication of EGF in pulmonary remodeling processes. TGF-a and EGFR overexpress in idiopathic pulmonary fibrosis and in cystic fibrosis. In vitro, hyperoxia induces release of TGF-a by lung fibroblasts. In acute lung injury, TGF-a increases during hyperoxia and the consequent fibrosis. Fluid from bronchioloalveolar lavage of patients with acute lung injury and idiopathic fibrosis contains an increased level of TGF-a. In mucosal biopsies of patients with chronic bronchitis, in comparison to controls, EGF expression significantly increases in the epithelium and submucosal cells. Furthermore, in comparison to controls, smokers have an increased expression of EGFR in bronchial mucosal epithelial cells. In asthma, bronchial epithelial cells show an increased expression of EGFR which does not cause a proliferative activity. After in vitro stimulation of airway epithelial cells by various stimuli, there is an expression of

132 EPIDERMAL GROWTH FACTORS

MUC5AC and MUC2 mucin gene that depends on activation of EGFR. In chronic obstructive pulmonary disease or asthma, the activation of EGFR by increased levels of EGF therefore leads to mucin hypersecretion and airways obstruction. After epithelial cell injury in vitro or in vivo, EGFR activation promotes repair by stimulating cell proliferation and migration. After in vitro asbestos exposure, EGFR message and protein are upregulated. In addition, asbestos will cause phosphorylation of EGFR and causally activate ERK 1 and 2 as well as expression of the proto-oncogene c-fos. In asbestos or naphthalene exposed animals, the region of the bronchioles and the alveolar ducts demonstrates an increased expression of TGF-a and EGF. In mice that inhale asbestos fibers, immunostain for p-ERK increases in foci of epithelial cells and is associated with proliferation and local fibrosis. A mouse line (9527) has been developed that expresses a mutant EGFR under the control of the lung-specific SP-C promoter. This mutant receptor (dnEGFR) lacks a portion of the intracytoplasmic phosphorylation domain, which inhibits signal transduction by the endogenous EGFR. When exposed in vivo to asbestos, the terminal bronchioles of dnEGFR mice

showed significantly less PCNA-positive cells than those of controls. After binding to a wall receptor, EGF stimulates the growth of extracellular Mycobacterium avium and M. tuberculosis. Furthermore, granulomas contain large amounts of EGF. Evidence has accumulated that overexpression of ligand and/or receptor as well as ligand-independent receptor activation occurs in many lung cancers. EGFR is overexpressed in up to 80% of squamous cell carcinomas of the lung. Deletion of the receptor extracellular portion results in a dimerization and production of an inappropriate signal, even in the absence of ligand binding. In practice, there are no universal methods to evaluate EGFR expression. Furthermore, the relation between EGFR expression and prognosis in non-small cell lung cancer is not clear; most studies do not identify EGFR as a prognostic factor. Studies on EGFR DNA may produce information on gene amplification, deletion, or mutation. Because of possible posttranslational modifications that could affect the quantity of protein produced, RNA studies may not be appropriate. EGFR protein can easily be detected by immunohistochemistry (Figure 3). In addition, activation of the receptor and of the signaling

Figure 3 Squamous cell carcinoma of the lung: immunostain for EGFR (Dakocytomation) shows a membranous localization (arrows) Original magnification
400.

EPITHELIAL CELLS / Type I Cells 133

network can be studied by use of antibodies directed against phosphorylated EGFR or against p-ERK and/ or by markers of cell proliferation or maturation. Many EGFR-targeted agents are in development and/ or use in clinical trials. They include antibodies directed against the receptor extracellular domain, and small molecules that interfere with the intracellular kinase domain. Recently, studies have described mutations of the EGFR gene, located around the region encoding the ATP-binding pocket of the receptor tyrosine kinase. In a particular group of patients suffering mostly from lung adenocarcinoma, these mutations may predict the response to gefitinib, a tyrosine kinase inhibitor. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Bronchiolitis. Cystic Fibrosis: Overview. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Lung Development: Overview. Mucins.

Further Reading Burgel PR and Nadel JA (2004) Roles of epidermal growth factor receptor activation in epithelial cell repair and mucin production in airway epithelium. Thorax 59: 992–996. Ciardiello F and Tortora G (2003) Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. European Journal of Cancer 39: 1348–1354. Guillermo Paez J, Ja¨nne PA, Lee JC, et al. (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304: 1497–1500.

Jorissen RN, Walker F, Pouliot N, et al. (2004) Epidermal growth factor receptor: mechanisms of activation and signalling. In: Carpenter G (ed.) The EGF Receptor Family. Biologic Mechanisms and Role in Cancer, pp. 33–55. Amsterdam: Elsevier. Knight DA and Holgate ST (2003) The airway epithelium: structural and functional properties in health and disease. Respirology 8: 432–446. Kumar VH and Ryan RM (2004) Growth factors in the fetal and neonatal lung. Frontiers in Bioscience 9: 464–480. Liu JY, Morris GF, Lei WH, Corti M, and Brody AR (1996) Upregulated expression of transforming growth factor-alpha in the bronchiolar-alveolar duct regions of asbestos-exposed rats. American Journal of Pathology 149: 205–217. Lynch TJ, Bell DW, Sordella R, et al. (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. New England Journal of Medicine 350: 2129–2139. O’Donnell RA, Richter A, Ward J, et al. (2004) Expression of ErbB receptors and mucins in the airways of long term current smokers. Thorax 59: 1032–1040. Pache JC, Janssen YM, Walsh ES, et al. (1998) Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos fibers. American Journal of Pathology 152: 333–340. Vignola AM, Chanez P, Chiappara G, et al. (1997) Transforming growth factor-b expression in mucosal biopsies in asthma and chronic bronchitis. American Journal of Respiratory and Critical Care Medicine 156: 591–599. Whitsett JA and Zhou L (1996) Use of transgenic mice to study autocrine–paracrine signaling in lung morphogenesis and differentiation. Clinics in Perinatology 23: 753–769. Zanella CL, Posada J, Tritton TR, and Mossman BT (1996) Asbestos causes stimulation of the extracellular signal-regulated kinase 1 mitogen-activated protein kinase cascade after phosphorylation of the epidermal growth factor receptor. Cancer Research 56: 5334–5338.

EPITHELIAL CELLS Contents

Type I Cells Type II Cells

Type I Cells Z Borok and E D Crandall, University of Southern California, Los Angeles, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Alveolar epithelial type I (AT1) cells are large, amorphous cells with elongated thin cytoplasmic processes that cover the majority of the gas exchange surface of the lung. AT1 cells are believed to be derived from AT2 cells during development and following injury. Recent studies demonstrating differentiation of AT1 cells

from bone marrow-derived stem cells suggest other paradigms. Until recently, the only known function of AT1 cells was a presumptive passive role in gas exchange. Improvements in methods for isolation of AT1 cells and recent identification of genes and proteins in AT1 cells suggest additional functions in alveolar homeostasis for this previously elusive cell type, including water flux, active ion transport, cytoprotection, procoagulant and proinflammatory properties, development, remodeling, and cell proliferation. AT1 cells have extremely high water permeability due to the presence of the water channel aquaporin-5. Identification of Na channel and Na pump subunits, and amiloridesensitive Na uptake and oubain-inhibitable Rb þ uptake, confirm a role in active Na transport. AT1 cells are highly susceptible to injury, and release of AT1 cell proteins into bronchoalveolar

134 EPITHELIAL CELLS / Type I Cells lavage fluid and serum may serve as an index of injury. The contributions of AT1 cell injury and repair mechanisms to pathogenesis of lung diseases are being investigated.

open onto the alveolar surface or interstitial space, although these are not specific to AT1 cells, being present also in endothelial cells (Figure 2). Investigation of the biological and functional properties of AT1 cells has been hampered by a paucity of specific nonmorphologic phenotypic markers for cell identification as well as lack, until recently, of reproducible methods for isolation and culture of AT1 cells.

Introduction Alveolar epithelial type I (AT1) cells were first identified in rat lung by electron microsocopy in 1952. Together with type II (AT2) cells, they form a continuous surface epithelial layer lining the peripheral lung. AT1 cells cover more than 95% of the alveolar surface of the distal lung and, together with closely apposed capillary endothelial cells and thin interstitium, form the air–blood barrier across which gas exchange occurs (Figure 1). Within the alveolar space, AT1 cells make contact with other AT1 cells and with AT2 cells via tight junctions located at the cell periphery. Together with AT2 cells, AT1 cells form a barrier to the passive leak of solutes and fluid into the alveolar space, an important prerequisite for normal gas exchange. AT1 cells are large cells with a mean cellular volume of 4900 mm3 in the rat and 1800 mm3 in humans, and covering a surface of 4500 and 5100 mm2 in rats and humans, respectively. AT1 cells have attenuated cytoplasmic extensions that can help form the surface of more than one alveolus. Nuclei are surrounded by few mitochondria, endoplasmic reticulum with ribosomes, interspersed microfilaments, and an inconspicuous Golgi apparatus. Unlike AT2 cells which contain lamellar bodies, AT1 cells do not possess any other identifying morphologic hallmarks. Throughout the cell are several flask-shaped vesicles, or caveolae, some of which

Ontogeny

AT1 cells have been thought to arise from AT2 cells during development and following injury, although during development there is evidence that they may also arise from an undifferentiated pool of precursor cells expressing markers of both AT2 and AT1 cells. The notion of the AT2 cell as the progenitor cell in the adult is derived primarily from serial morphologic analyses of the lungs of animals exposed to sublethal concentrations of oxidant gases (e.g., NO2 and O2) and from autoradiographic analyses of regenerating lung pulse-labeled with 3H-thymidine immediately following injury, in which initial incorporation of label into AT2 cells is followed by the appearance of label in AT1 cells. Reflecting this in vivo paradigm following injury, AT2 cells in culture lose their characteristic hallmarks and acquire many of the morphologic and phenotypic features of AT1 cells in situ, consistent with transdifferentiation toward an AT1 cell-like phenotype in vitro. This process is significantly influenced by substratum, soluble factors (including homologous serum), and changes in cell shape. AT1 cells are believed to be terminally differentiated cells incapable I

I A

E Nu

COL R

C

EI I I A

1 µm

Figure 1 Alveolar wall from human lung. The air–blood barrier (short double arrow) consists of type I epithelial cells with long cytoplasmic extensions (I), interstitium (*) between basal laminae of epithelial and endothelial cells, and capillary endothelium (E). A, alveolus; C, plasma in the alveolar capillary; R, cytoplasm of the red cell; El, elastin; COL, collagen; Nu, nucleus of capillary endothelial cell. Reproduced from Albertine KH, Williams MC, and Hyde DM (2000) in Murray JF, Nadel JA, Mason RJ, and Boushey HA (eds.) Textbook of Respiratory Medicine, 3rd edn., pp. 3–33. Philadelphia: Saunders, with permission.

EPITHELIAL CELLS / Type I Cells 135

S

C

Figure 2 Caveolae in rat lung. Transmission electron micrograph shows flask-shaped plasmalemmal invaginations and cytoplasmic vesicles in both capillary endothelium (right-hand side) and alveolar epithelial type I cells (left-hand side). C, capillary lumen; S, surfactant in airspace. Reproduced with permission from Newman GR, Campbell L, von Ruhland C, Jasani B, and Gumbleton M (1999) Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type 1 cell function. Cell and Tissue Research 295: 116.

of proliferation or self-renewal, although recent in vitro studies suggest that, at least in culture, AT1 cells can revert to a AT2 cell phenotype. The paradigm in which AT2 cells serve as sole progenitors of AT1 cells in the adult has been challenged by recent studies indicating that transplanted bone-marrowderived stem cells may engraft as differentiated AT1 cells in animal models following bleomycin injury. The regulation of AT1 cell differentiation at the molecular level is poorly understood. Animals in which the zinc finger transcription factor GATA-6 is repressed have an absence of AT1 cells with relatively normal AT2 cells, suggesting an important role for GATA-6 in alveolar epithelial cytodifferentiation. Several other key regulatory pathways have been implicated in AT1 cell development and differentiation (e.g., Hox genes, Wnt ligands), but their precise roles remain to be determined. Phenotypic Markers

Among the earliest methods used to distinguish between AT2 and AT1 cells was differential binding of lectins to glycoproteins on their apical surfaces, although their specificity for each cell type has been questioned. Lectins that have been described predominantly on AT1 compared with AT2 cells include

Ricinus communis agglutinin I (RCA1), Lycopersicon esculentum agglutinin (LEA), and Bauhinia purpurea agglutinin (BPA), although BPA is also present on macrophages. More specific phenotypic markers for AT1 cells have become available through a combination of monoclonal antibody (mAb) and molecular approaches. mAbs VIIIB2 and SF-1/RTI40 bind selectively to the apical surface of rat AT1 cells but not to AT2 cells or endothelial cells, and both increase during transdifferentiation from AT2 toward AT1 cell phenotype in vitro. VIIIB2 recognizes an as yet unidentified epitope in the apical membrane of AT1 cells in the rat and is not reactive with other lung and nonlung cell types. SF-1/RTI40 was raised using partially purified populations of AT1 cells. Screening of a rat lung library with SF-1/RTI40 led to cloning of the gene T1a that encodes a 38–40 kDa glycoprotein. Within lung, T1a is selectively expressed in AT1 cells, although it is also expressed at other sites including choroid plexus, ciliary epithelium, salivary gland, and lymphatic endothelial cells. A mAb, HTI56, has been identified that binds to the apical plasma membrane of human AT1 cells. Aquaporin-5 (AQP5), a member of the aquaporin family of water channels, is a transmembrane protein of B28 kDa selectively expressed by AT1 cells in distal lung, although it is also expressed in other tissues, including salivary and lacrimal glands. Other AT1 cell markers include carboxypeptidase M, intercelluar adhesion molecule 1 (ICAM-l), receptor for advanced glycation end-products (RAGE), and caveolin-1 (an integral membrane protein associated with caveolae), none of which is unique to lung.

Function in Normal Lung Until recently, no major functions had been attributed to AT1 cells other than a presumptive passive role in gas exchange. Early insights into AT1 cell function were gained from studies using isolated AT2 cells grown as monolayers on inflexible substrata. Consistent with observations in vivo, under these conditions AT2 cells transdifferentiate toward an AT1 cell-like phenotype (AT1-like cells) and express phenotypic markers characteristic of AT1 cells in situ (Figure 3). Using this in vitro model, several putative functions were ascribed to AT1 cells, including the ability to vectorially transport Na in both an amiloride-sensitive and -insensitive manner. Consistent with a role in ion transport, subunits of the rat epithelial Na channel (ENaC) and Na pump were identified in AT1-like cells, findings which have now been confirmed in isolated AT1 cells. Given the need for additional models for characterization of AT1 cells, both mouse (E10/C10) and rat (R3/1) cell lines

136 EPITHELIAL CELLS / Type I Cells

Figure 3 Type I-like alveolar epithelial cell in culture (day 5) on polycarbonate filter. Transmission electron micrograph shows a cell with prominent nucleus and long cytoplasmic extensions resembling type I cells in vivo. Reproduced from Cheek JM, Evans MJ, and Crandall ED (1989) Type 1 cell-like morphology in tight alveolar epithelial monolayers. Experimental Cell Research 184: 380, with permission from Elsevier.

Figure 4 Expression of Na pump a1-subunit in type I (AT1) alveolar epithelial cells in situ. (a) Linear staining (red) using a mouse monoclonal antibody (Ab) localizes the Na pump a1-subunit to the basolateral surface of AT1 cells that are labeled with a polyclonal antiaquaporin-5 (anti-AQP5) Ab (green) on the apical surface. (b) Substitution of mouse monoclonal IgG for anti-a1 Ab reveals no reactivity with AT1 cells labeled with anti-AQP5 on the apical surface. (c) AT1 cells labeled on the apical surface with anti-AQP5 Ab (green) and on the basolateral surface with anti-a1 Ab (red). The AT2 cell is not reactive with anti-AQP5 Ab but expresses the a1-subunit on the basolateral surface. Reproduced with permission from Borok Z, Liebler JM, Lubman RL, et al. (2002) Sodium transport proteins are expressed by rat alveolar epithelial type I cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 282: L606.

have been developed that have some features of AT1 cells in situ. Expression of the a-subunit of the Na pump has recently been identified in AT1 cells in situ (Figure 4). Advances in methods for isolation of AT1 cells have confirmed expression of Na channel and Na pump subunits in these cells using double-label immunofluorescence in partially purified cells and reverse transcriptase polymerase chain reaction (RT-PCR) in highly purified populations. Further evidence for a

role of AT1 cells in ion transport was the observation of amiloride-sensitive Na uptake and ouabain-inhibitable Rb þ uptake, consistent with a role in ion transport. AT1 cells have also been shown to express b2-adrenergic receptors (b2-AR) and G-protein-coupled receptor kinase 2 which plays a role in desensitization of b2-AR. Further analyses of the specific ion channels expressed by AT1 cells will benefit from the recent development of a lung slice model, in which cells that are positively identified using an AT1

EPITHELIAL CELLS / Type I Cells 137

cell-specific mAb are amenable to patch clamp analyses in situ. Recent identification of genes and proteins in AT1 cells is beginning to provide insights into additional functions of this previously elusive cell type. In studies using partially purified populations, AT1 cells were shown to have extremely high water permeability. Subsequent deletion of AQP5 in knockout mice demonstrated a dramatic decrease in airspace–capillary osmotic water permeability, confirming a role for AQP5 in osmotically driven water clearance. However, clearance of hydrostatic lung edema and active alveolar fluid absorption were not affected, indicating that AQP5 is not required for active near-isosmolar fluid clearance. Since AQP5 is regulated by osmotic stimuli, a role for this water channel has been suggested in regulation of cell volume or airway surface liquid. T1a is required for normal lung development. T1a knockout mice die at birth of respiratory failure and have a defect in AT1 cell differentiation, as demonstrated by fewer AT1 cells and reduced levels of AQP5. Increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells in these mice at a time when cell division has normally ceased suggest a role for T1a in regulating cell proliferation in the distal lung. Caveolae are known to function in internalization of small molecules or ions and in intracellular trafficking of macromolecules, and also play a role in integrating signaling pathways. Abundant numbers of caveolae in AT1 cells have suggested a role for AT1 cells in macromolecule transport. Studies comparing patterns of gene expression in AT1-like cells and freshly isolated AT1 cells with AT2 cells have identified several new genes associated with AT1 cells that suggest additional functions for these cells, some of which still require functional verification. Although some of these findings await immunohistochemical confirmation and definitive functional analyses, these studies suggest roles for AT1 cells in addition to active ion transport in alveolar homeostasis, including cytoprotective functions, proinfiammatory and procoagulant properties, development and remodeling, tumor/growth suppression, and surfactant metabolism.

Function in Respiratory Diseases AT1 cells are highly susceptible to injury from diverse causes (e.g., bleomycin, radiation, inhaled NO2). Regardless of the cause, the response to injury is fairly stereotypic, with AT1 cells becoming detached and leaving behind denuded basement membrane. This is followed by proliferation of AT2 cells with subsequent differentiation of postproliferative AT2 cells into AT1 cells via an intermediate cell phenotype.

Little is known of the molecular regulation of these events. Several lung-enriched and ubiquitous transcription factors activate the T1a promoter, and binding sites for several of these are also identified in the promoter region of AQP5. However, these transcription factors also activate AT2 cell-specific genes, indicating that none of these elements alone is responsible for distinguishing between gene expression in AT2 and AT1 cells. Following lung injury, there may be loss of cell-specific proteins from the apical surface of AT1 cells, and SF-1/RTI40 has been found to be upregulated in bronchoalveolar lavage (BAL) fluid in rodent models of lung injury (e.g., bleomycin, Pseudomonas pneumonia). Similarly, the human AT1 cell marker HTI56 is increased in pulmonary edema fluid and serum in human patients with acute respiratory distress syndrome (ARDS), suggesting that release of a biochemical AT1 cell marker may be useful as an index of acute lung injury. There is an increasing appreciation for a central role of ongoing alveolar epithelial injury in lung diseases such as usual interstitial pneumonitis, with the concept being that progressive fibrosis results from failure of normal reepithelialization. It has been speculated that inhibition of the transition from AT2 to AT1 cell phenotype may play a role in the pathogenesis of this disorder due to upregulation of b-catenin and Wnt signaling in AT2 cells, which would tend to maintain a proliferative rather than terminally differentiated phenotype. The precise mediators and molecular events that are involved in failure of normal epithelial repair mechanisms are the focus of ongoing investigation. See also: Acute Respiratory Distress Syndrome. Adrenergic Receptors. Aquaporins. Epithelial Cells: Type II Cells. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Ion Transport: ENaC (Epithelial Sodium Channel). Lung Development: Overview. Panbronchiolitis. Stem Cells.

Further Reading Albertine KH, Williams MC, and Hyde DM (2000) Anatomy of the lungs. In: Murray JF, Nadel JA, Mason RJ, and Boushey HA (eds.) Textbook of Respiratory Medicine, 3rd edn., pp. 3–33. Philadelphia: Saunders. Borok Z, Liebler JM, Lubman RL, et al. (2002) Sodium transport proteins are expressed by rat alveolar epithelial type I cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 282: L599–L608. Borok Z and Verkman AS (2002) Role of aquaporin water channels in fluid transport in lung and airways. Journal of Applied Physiology 93: 2199–2206. Brody JS and Williams MC (1992) Pulmonary alveolar epithelial cell differentiation. Annual Review of Physiology 54: 351–371.

138 EPITHELIAL CELLS / Type II Cells Cheek JM, Evans MJ, and Crandall ED (1989) Type 1 cell-like morphology in tight alveolar epithelial monolayers. Experimental Cell Research 184: 375–387. Crandall ED and Matthay MA (2001) Alveolar epithelial transport: basic science to clinical medicine. American Journal of Respiratory and Critical Care Medicine 163: 1021–1029. Dobbs LG, Gonzalez R, Matthay MA, et al. (1998) Highly water-permeable type I alveolar epithelial cells confer high water permeability between the airspace and vasculature in rat lung. Proceedings of the National Academy of Sciences, USA 95: 2991–2996. Gumbleton M (2001) Caveolae as potential macromolecule trafficking compartments within alveolar epithelium. Advanced Drug Delivery Reviews 49: 281–300. Kasper M and Singh G (1995) Epithelial lung cell marker: current tools for cell typing. Histology and Histopathology 10: 155–169. Kemp PJ and Kim KJ (2004) Spectrum of ion channels in alveolar epithelial cells: implications for alveolar fluid balance. American Journal of Physiology 287: L460–L464. Kotton DN, Summer R, and Fine A (2004) Lung stem cells: new paradigms. Experimental Hematology 32: 340–343. Newman GR, Campbell L, von Ruhland C, Jasani B, and Gumbleton M (1999) Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type 1 cell function. Cell and Tissue Research 295: 111–120. Schneeberger EE (1997) Alveolar type 1 cells. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 535–542. Philadelphia: LippincottRaven. Williams MC (2003) Alveolar type I cells: molecular phenotype and development. Annual Review of Physiology 65: 669–695.

Type II Cells R J Mason, National Jewish Medical and Research Center, Denver, CO, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The alveolar epithelium is composed of two types of epithelial cells, named alveolar type I and type II cells. Type I cells are large flat cells that comprise about 95% of the alveolar surface. Type II cells are small cuboidal cells with characteristic lamellar inclusions and apical microvilli and cover about 5% of the alveolar surface. Type II cells make and secrete pulmonary surfactant. They are the progenitor cells for the alveolar epithelium in the adult lung. Type II cells also transport sodium from the apical to the basolateral surface to keep the alveolus relatively free of fluid and participate in the innate immune system. Type I cells are much more susceptible to injury than type II cells. In the injured lung, type II cells spread to reform the alveolar epithelium after a loss of type I cells and then proliferate to restore the differentiated epithelium. Many interstitial lung diseases are characterized by hyperplastic type II cells which are envisioned as part of normal epithelial wound healing. However, it is also possible that type II cells contribute to the fibroproliferative reaction by secreting growth factors and proinflammatory molecules. Finally, type II cells can give rise to alveolar cell carcinomas.

Introduction The alveolar type II cell is the defender of the alveolus (Figure 1). The alveolus is the anatomic site for gas exchange, the critical function of the lung. For gas exchange to take place, the diffusion distance between inhaled gas and the red cell has to be minimized. This is accomplished by keeping the air passages open (the low surface tension at the air–liquid interface provided by pulmonary surfactant), the alveolar fluid volume low (transepithelial sodium transport), and inflammation and edema absent (the innate immune system). Type II cells are also considered to be the progenitor cells for the alveolar epithelium. The alveolar type II cell provides many components required for the special mircoenvironment in the alveolus. Some of these components are pulmonary surfactant, the collectins SP-A and SP-D, and a variety of anti-inflammatory and antimicrobial substances such as lysozyme, b-defensin 2, secretory leukocyte proteinase inhibitor (SLPI), and lipocalin 2. Finally, type II cells also provide part of the initial antioxidant defense by secreting glutathione, which is present in high concentration in alveolar fluid. The domain that the type II cell defends is a special microenvironment. The alveolar unit as stated above has evolved to facilitate gas exchange, the uptake of oxygen, and the removal of carbon dioxide. The alveolar epithelium is composed of two main cell types. The type I cells are large flat thin cells that cover about 95% of the surface area. They are a major part of the epithelial barrier and express genes that indicate they have significant metabolic functions. However, because of their low density of mitochondria per surface area they may lack the energetics. They represent about 11% of the cells in the alveolar wall and each type I cell in the human lung has a surface area of 7000 mm2. Type II cells are smaller cuboidal cells with the anatomic features of an active metabolic epithelial cells with a high density of mitochondria and special apical microvilli. The structural characteristics of this cell are the lamellar inclusions, which are the intracellular storage form of surfactant. Type II cells comprise 17% of the cells of the alveolar wall and cover about 5% of the alveolar surface. Each type II cell in the human lung has a surface area of 250 mm2. In the rat there is only about one alveolar macrophage per alveolus, six type II cells, and four type I cells. Hence, the initial defense to inhaled particles and gases is likely the epithelium. The alveolar fluid also has important characteristics. The volume is very low, about 35 ml per adult human lung. This is about 4 ml cm 2, and the pH is acidic, about pH 6.9.

EPITHELIAL CELLS / Type II Cells 139

Type I cell

Type II cell

Figure 1 Type II cells are defenders of the alveoli by secreting surfactant, keeping the alveolar space relatively free from fluid, serving as progenitor cells to repopulate the epithelium after injury, and providing important components of the innate immune system.

Type II Cell Function in the Normal Lung

Precursor of Alveolar Epithelial Cells

Production of Surfactant

Another important function for type II cells is to serve as progenitor cells for all alveolar epithelial cells. Type II cells are thought to be an asynchronous transient proliferating cell population. It is thought that in vivo type II cells divide and some of the daughter cells remain as type II cells, whereas others transdifferentiate to become type I cells. In vitro there is tremendous plasticity of the epithelial phenotype, so that phenotypic type II cells and type I cells can cycle back and forth depending on the culture substratum and presence or absence of growth factors (Figure 2). However, in the fetal lung it appears that primitive lung epithelial cells can express type I cell markers without having to display type II cell markers. Hence, it is possible that not all type I cells come from differentiated type II cells. It is also not known whether or not there are bona fide omnipotent stem cells in the adult lung that could give rise to alveolar epithelial cells. However, it has recently been reported that there are omnipotent epithelial stem cells in the terminal bronchioles. There is also evidence that circulating stem cells from the bone marrow can lodge in the lung and differentiate into type I and type II cells. However, this is a rare event. The relative importance of circulating stem cells in

The production of surfactant is the best-known function of type II cells. The surface tension in the alveoli at the end of expiration is very low (o10 mN m 1), and this provides for alveolar stability, effortless breathing, and prevents pulmonary edema and atelectasis. The low surface tension is provided by phospholipid, predominantly dipalmitoylphosphatidylcholine, and is assisted by the hydrophobic surfactant proteins SP-B and SP-C. The type II cells synthesize the phospholipids of surfactant, SP-B, and SP-C and package them together in lamellar bodies. These lamellar bodies are then secreted by exocytosis. Exocytosis is regulated by a variety of factors including stretch, intracellular calcium, extracellular ATP, b-adrenergic agonists, and agents that activate protein kinase C. Once the lamellar bodies are secreted, the contents unfold to form tubular myelin and what is termed large aggregate surfactant. This material is adsorbed into the air– liquid interface at the end of inspiration. Some of the surfactant is squeezed out of the monolayer at the end of expiration, and part of this material is taken up by type II cells for reprocessing and reutilization and part is ingested by macrophages for degradation.

140 EPITHELIAL CELLS / Type II Cells In vivo

In vitro

Type II cell

In vivo

In vitro SP-C

Type I cell T1


In vivo Figure 2 The phenotype of alveolar type II cells is reversible in primary culture. The type II cell phenotype is close but probably not the same as the type II cell in the normal lung, and the type I-like cells in vitro are significantly different from type I cells in vivo. The phenotypes are reversible in vitro depending on the matrix on which the cells are grown and the presence of growth factors such as KGF. The type II cells in vitro are probably like those intermediate cells observed during lung repair.

normal cell turnover and in response to injury is not known but is probably very, very limited. Transepithelial Sodium Transport

A third function of type II cells is to keep the alveolus relatively dry by transporting sodium from the apical surface into the interstitium. Type II cells have apical sodium channels, abundant Na/K ATPase, and a rich supply of mitochondria to generate ATP, and they have been shown to transport sodium in vitro. Type I cells also have these transport components, but it does not appear that they have the energy supply as indicated by mitochondrial density to be effective sodium transporters. Beta-adrenergic agonists can increase transepithelial fluid transport and alveolar fluid resorption. This process requires an intact epithelium as would be present in congestive heart failure but not in adult respiratory distress syndrome or severe pneumonia where the epithelium is severely damaged. Regulation of Innate Immunity

The final important function of type II cells is to be an integral part of the innate immune system and to recognize and deal with inhaled microbes, toxic gases, and particulates. We inhale about 10 000 l or 16 kg of air each day. The air we exhale is cleaner in some ways than the air we inhale. The alveolus must deal with low levels of inhaled organisms without inducing much of an inflammatory response but then must switch and produce a significant immune response once an overt infection is established. Type II cells participate in the innate immune function of the lung in several ways. They secrete the collectins SP-A and SP-D, which are polyvalent calcium-dependent lectins. These proteins bind and aggregate a variety of microorganisms. They also can suppress the inflammatory response of alveolar macrophages under some circumstances as in the normal lung and

yet stimulate macrophages under other circumstances as in acute infection. It should be recognized that the lung should not develop an acute inflammatory state or stimulate a T-cell response with every inhaled antigen or microorganism. Both the innate and the adaptive immune systems should be dampened. Inflammation impairs gas exchange. For example, alveolar macrophages are relatively poor antigen-presenting cells. In addition, type II cells can secrete a variety of antimicrobial peptides such as b-defensins 2, lipocalin 2, and lysozyme. Moreover, they provide the reducing substances in the alveolar fluid to neutralize oxidant gases such as the phospholipids of surfactant, reduced glutathione, ascorbate, and urate. Finally, type II cells can also signal to other more active immunoeffector cells such as macrophages and neutrophils by secreting chemokines and cytokines. Type II cells secrete a variety of cytokines and chemokines, including the interleukins IL-1b, IL-1a, IL-6, IL-8 (CXCL8), epithelial neutrophil activating peptide-78 (ENA-78; CXCL5), growth related oncogene alpha (GRO-a; CXCL1), macrophage inflammatory protein-2 (MIP2; CXCL2), monocyte chemoattractant protein 1 (MCP-1; CCL2), exotaxin (CCL11), ‘regulated upon activation normally T cell expressed and secreted’ (RANTES; CCL5), and undoubtedly many others. Type I cells can also secrete cytokine and chemokines.

Type II Cell Function in Respiratory Diseases The type I cells comprise 95% of the alveolar surface and are very susceptible to injury. After damage to type I cells, the type II cells spread, proliferate, and then differentiate to reform the epithelium (Figure 3). If type II cell hyperplasia is induced by a single instillation of KGF, some of the proliferating type II cells

EPITHELIAL CELLS / Type II Cells 141

Normal

Dedifferentiation and migration

Proliferation

Differentiation

Transdifferentiation

Figure 3 Epithelial cells respond to injury or wound healing in a characteristic way, and this occurs in the lung, although it is difficult to visualize. After injury to type I cells, type II cells spread, dedifferentiate, and migrate to cover the wound. The type II cells then proliferate to form a hyperplastic epithelium and then differentiate as type II cells. Finally, some of the epithelial cells undergo apoptosis and some transdifferentiate into type I cells.

undergo apoptosis and others transdifferentiate to form type I cells with the result of normal appearing lung 1 week after instillation of KGF. However, in numerous interstitial lung diseases this process is not complete. The hyperplastic type II cells persist. The reason for this is not known but there is clearly continued proliferation as indicated by PCNA and Ki67 immunostaining as well as an apparent failure to transdifferentiate into type I cells. The role of hyperplastic type II cells in interstitial lung disease is not known. From the perspective of cutaneous wound healing they are reforming the epithelium and can limit granulation tissue and fibroblast proliferation. For example, in vitro type II cells release IL-1a and other substances that increase prostaglandin E2 (PGE2) production in fibroblasts that in turn inhibit fibroblast proliferation. However, type II cells can also secrete growth factors for fibroblasts and transforming growth factor beta (TGF-b) and may in some circumstances stimulate fibroblast proliferation and matrix production. It is interesting to note that the epithelial lining over fibroblastic foci in usual interstitial pneumonia is commonly incomplete and suggests that the incomplete epithelium may promote growth of fibroblastic foci and matrix production. Surfactant production is decreased in a variety of lung injuries and contributes the alveolar collapse, pulmonary edema, and increased work of breathing. Changes in phosphatidylglycerol are the earliest changes in the composition of surfactant phospholipid with lung injury, but the physiologic consequences of this change are not apparent. Similarly, transepithelial sodium transport is impaired in acute lung injury. It should be noted that the resolution of alveolar

exudation requires an intact alveolar epithelium. Until the epithelium is reformed, effective transepithelial transport cannot take place. The alveolar epithelium can also facilitate clearance of the alveolar exudate by producing tissue factor. Finally, alveolar type II cells can give rise to alveolar adenomas and adenocarcinomas. In mice nearly all of the lung adenomas express SP-C and are therefore derived from type II cells. Hence, type II cells are truly the defender of the alveolus by providing normal gas exchange and effortless breathing, dealing with low levels of toxic substances that we inadvertently inhale, and initiating an inflammation response if needed to deal with a more virulent pathogen. See also: Adhesion, Cell–Cell: Epithelial. Epithelial Cells: Type I Cells. Extracellular Matrix: Basement Membranes. Fibroblast Growth Factors. Fluid Balance in the Lung. Keratinocyte Growth Factor. Permeability of the Blood–Gas Barrier. Stem Cells. Surfactant: Overview. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Vascular Endothelial Growth Factor.

Further Reading Fehrenbach H (2001) Alveolar epithelial type II cell: defender of the alveolus revisited. Respiratory Research 2: 33–46. Gonzalez R, Yang YH, Griffin C, et al. (2005) Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes. American Journal of Physiology: Lung Cellular and Molecular Physiology 288: L179–L189. Mason RJ, Lewis MC, Edeen KE, et al. (2002) Maintenance of surfactant protein A and D secretion by rat alveolar type II cells

142 ERYTHROCYTES in vitro. American Journal of Physiology: Lung Cellular and Molecular Physiology 282: L249–L258. Mason RJ, Pan T, Edeen KE, et al. (2003) Keratinocyte growth factor and the transcription factors C/EBPa, C/EBPd, and SREBP-1c regulate fatty acid synthesis in alveolar type II cells. Journal of Clinical Investigation 112: 244–255. Mason RJ, Williams MC, Widdicombe JH, et al. (1982) Transepithelial transport by pulmonary alveolar type II cells in primary culture. Proceedings of the National Academy of Sciences, USA 79: 6033–6037. Matthay MA, Folkesson HG, and Clerici C (2002) Lung epithelial fluid transport and the resolution of pulmonary edema. Physiological Reviews 82: 569–600. Shannon JM (1994) Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme. Developmental Biology 166: 600–614.

Shannon JM, Pan T, Nielsen LD, Edeen KE, and Mason RJ (2001) Lung fibroblasts improve differentiation of rat type II cells in primary culture. American Journal of Respiratory Cell and Molecular Biology 24: 235–244. Veldhuizen R and Possmayer F (2004) Phospholipid metabolism in lung surfactant. Sub-Cellular Biochemistry 37: 359–388. Warburton D and Bellusci S (2004) The molecular genetics of lung morphogenesis and injury repair. Pediatric Respiratory Reviews 5(Supplement A): S283–S287. Williams MC (2003) Alveolar type I cells: molecular phenotype and development. Annual Review of Physiology 65: 669–695. Zemans RL and Matthay MA (2004) Bench-to-bedside review: the role of the alveolar epithelium in the resolution of pulmonary edema in acute lung injury. Critical Care 8: 469–477.

ERYTHROCYTES E Ingley and S P Klinken, Western Australian Institute for Medical Research and Centre for Medical Research, Perth, WA, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Erythrocytes are biconcave enucleate red blood cells responsible for transport of O2/CO2 between the body’s tissues and lungs; their oxygen-carrying capacity is due to their high hemoglobin content. Red blood cells are derived from hemopoietic stem cells in a process controlled by the hormone erythropoietin. Oxygenation of the erythrocyte in the lung capillaries facilitates unloading of CO2, causing shrinkage and ATP release. ATP activates endothelial cells to synthesize nitric oxide (NO), some of which becomes complexed with hemoglobin. Deoxygenation in peripheral tissues initiates NO release and CO2 loading, resulting in swelling of the red cell. Red cell defects can affect the lung. Anemias can manifest as respiratory abnormalities, although polycythemias rarely display pulmonary problems. Hemoglobinopathies, such as sickle cell disease, often show pulmonary complications causing significant morbidity and mortality. When erythrocytes contact lung fibroblasts, due to lung injury, they stimulate release of interleukin-8 (IL-8), enhancing inflammatory responses. Cystic fibrosis (CF) patients develop numerous lung pathologies due to mutations in the ATP-transporting cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF erythrocytes are unable to release ATP on mechanical deformation in the lung, preventing ATPstimulated synthesis of NO from the endothelial cells, resulting in pulmonary hypertension. Malaria-infected erythrocytes display multiple abnormalities, which result in alveolar and endothelial damage.

Introduction Erythrocytes, or red blood cells, with their wellknown biconcave shape, are critical for exchange

and transport of O2/CO2 between the lungs and the body’s tissues and organs. This is achieved through red cells containing high levels of the oxygen carrier hemoglobin (Hb). While the eminent Egyptian physician Ibn al-Nafis was the first to describe pulmonary circulation (in the thirteenth century), Jan Swammerdam, a 21-year-old Dutch microscopist, is thought to be the first person to observe and describe red blood cells (in 1658). These cells are found within the capillaries surrounding the alveoli of the lung (Figure 1(a)). Enucleated red cells have an average diameter of 8.4 mm and a mean volume of 90 fL; due to their highly flexible membrane, erythrocytes can easily expand up to 150 fL, or be ‘squeezed’ to traverse capillaries that are less than 8 mm in diameter. They are produced primarily in the bone marrow from pluripotent hemopoietic stem cells. This development of red blood cells from stem cells occurs via a process called erythropoiesis; a variety of cytokines and hormones initiate a series of proliferation and maturation steps, which result in release of reticulocytes into the bloodstream. Reticulocytes then mature into erythrocytes within 1–2 days (Figures 1(b) and 1(c)). The earliest committed progenitor of the erythroid lineage is the burst-forming unit-erythroid (BFU-E). This stage is not defined morphologically, rather functionally, as these cells produce large burst colonies of erythroid cells in semisolid culture media; BFU-E are highly responsive to stem cell factor (SCF) stimulation. The next recognizable stage is that of the colony forming unit-erythroid (CFU-E), which again is defined functionally via the generation of compact colonies. Erythropoietin (Epo), the hormone

ERYTHROCYTES 143

HSC

Tc

E

RBC

BFU-E

Po

CFU-E

E E

R

Or

B

Pr

En

Or

Mac

Py

Py

R

(b) Alvll R

E

R

E E

E

Or

Or Po

Tc (a)

E

R Or Po

Or

Pr

B Po

Po

R

R

(c)

Figure 1 (a) Transmission electron micrograph of airway cells showing erythrocytes (E), endothelial cells (En), type II alveolar cells with distinctive multilamellar bodies (AlvII), and the thin-walled capillaries (Tc). Scale bar ¼ 2 mm. Photograph courtesy of the School of Anatomy and Human Biology, UWA. (b) Ontogeny of erythrocytes. The development of a red blood cell (RBC) from a hemopoietic stem cell (HSC) is illustrated. Stages from the colony forming unit-erythroid (CFU-E) through to the enucleated reticulocyte (R) are found in erythroblastic islands surrounding a central macrophage (Mac). Other stages shown are burst-forming unit-erythroid (BFU-E), proerythroblast (Pr), basophilic erythroblast (B), polychromatic erythroblast (Po), orthochromatic erythroblast (Or), and the pyknotic nucleus (Py) of the enucleating erythroblast, which is engulfed by the macrophage. (c) Neutral benzidine and Wright’s stain of erythroblasts at different stages of development isolated from an erythroblastic island. Hb is stained yellow/orange. Cells present are proerythroblast (Pr), basophilic erythroblast (B), polychromatic erythroblast (Po), orthochromatic erythroblast (Or), and reticulocyte (R).

principally responsible for the development of the latter stages of erythropoiesis, is produced in the kidneys in response to a low oxygen tension. Responsiveness to Epo begins at the BFU-E stage and is maximal in CFU-E through to proerythroblasts, the next stage of erythroid development. This latter phase is the first morphologically recognizable stage and is characterized by large cells with a high nuclear to cytoplasmic ratio. The subsequent stages of development (viz. basophilic erythroblast, polychromatic erythroblast, and orthochromatic erythroblast) are typified by nuclear condensation, a decrease in cell size, and increasing Hb levels. Up to the orthochromatic erythroblast stage, cells are able to proliferate. Thereafter, nuclei become excentric, Hb synthesis increases, and eventually the nuclei are extruded, releasing reticulocytes, which in turn develop into erythrocytes. Mature red blood cells, which lack a nucleus and organelles, remain in circulation for approximately 120 days before displaying phosphatidylserine on their outer membrane, identifying them as ‘old’ red blood cells that need to be removed by the reticuloendothelial system (macrophages). Nearly all of the constituents of aged erythrocytes are recycled, except for the heme component of Hb, which

is eventually excreted through the bile duct as bilirubin.

Erythrocyte Function in the Normal Lung The specialized function of the red cell is to mediate transport of O2 from the lungs to peripheral tissues, and CO2 in the reverse direction. Erythrocytes influence O2 transport in two main ways viz. the total concentration of Hb in the blood and its affinity for O2. Ventilation of the lungs not only mediates the O2 loading of red cells but also CO2 release, and maintenance of the blood’s acid–base balance, which also influence O2 loading (Figure 2(a)). Cardiac output and blood shunting can also affect the amount of O2 loading of erythrocytes. An essential feature of O2 transport via the red blood cell is the need to bind O2 firmly in the pulmonary capillaries under high O2 tension, and subsequently release it in the periphery under lower O2 tension. Oxygenation of the red cell is extremely rapid; almost complete oxygenation occurs within the first third of the 780 ms transit time within the lung capillaries. To achieve maximal O2 saturation, the Hb tetramer binds O2 in a cooperative manner. The first O2 molecule to interact with deoxygenated Hb binds

144 ERYTHROCYTES

H2CO3 HbH+

CO2

CO2

HbO2

HCO3−

O2

ATP NO SNOHb

O2

O2

O2

Alveoli (a)

Lung capillary

CO2

H2CO3 HbH+

CO2

CO2 −

HCO3 NO

HbO2 SNOHb

(b)

Tissue capillary

CO2

O2

Tissue cells

Figure 2 Normal function of erythrocytes in the lung and peripheral tissues. (a) Erythrocytes in pulmonary capillaries release CO2 and take up O2 via their gradients. Oxygenation of Hb initiates unloading of CO2, which induces erythrocyte shrinkage. Mechanical deformation initiates release of ATP, stimulating endothelial cells to synthesize NO, which reacts with Hb to form S-nitrosohemoglobin (SNOHb). (b) Erythrocytes in the peripheral tissues release O2 and take up CO2 due to their gradients. Deoxygenation of the Hb stimulates release of NO. Loading of CO2 stimulates swelling of the erythrocyte.

relatively weakly as the Hb is in a ‘relaxed’ or Rform. However, once the first O2 molecule has bound, the other O2-binding sites of the Hb are transformed into high-affinity sites. This allosteric state of Hb is known as the ‘tense’ or T-form. The extensive packing of Hb within the red cell provides numerous benefits, including juxtaposition to enzymes required to regulate its O2 affinity, for example, 2,3-diphosphoglycerate, which is present at approximately equimolar concentrations with Hb in red cells. The bolus flow of red cells, as opposed to a laminar flow, means there are no stagnant flow layers along the capillary wall. The rolling action of red cells within the circulation further enhances the rate of gas exchange. Interestingly, oxygenation of Hb in the lungs promotes the binding of nitric oxide (NO) to cystineb93

of the globin moiety, forming S-nitrosohemoglobin (SNOHb) (Figure 2(a)). Deoxygenation in the periphery, accompanied by the allosteric transition of the SNOHb from the R to the T form, releases the NO group, which is known to help mediate O2 uptake by the peripheral tissues through relaxation of vessels and increasing blood flow (Figure 2(b)). Perfusion of the lung results in a relaxation of the pulmonary smooth muscle vasculature to approximately 10-fold less than that of the systemic circulation, due to induced NO synthesis in the endothelium. Red blood cells are necessary for this vasodilation as they are mechanically deformed during their traversal of the pulmonary capillary. This initiates release of ATP, which then stimulates the endothelium to synthesize NO via their P2y purinergic receptors (Figure 2(a)). Erythrocytes are the main vessel for CO2 transport to the lungs in the form of H2CO3, a reaction facilitated by the rapid hydration of CO2 by carbonic anhydrase. Red cells acquire CO2 in the periphery through a CO2 gradient; while approximately 25% of the CO2 reacts with the deoxygenated Hb to form carbamino-Hb, most is converted to H2CO3 by carbonic anhydrase, which in turn dissociates to form HCO3
and H þ . Deoxygenated Hb accepts the proton and buffers the pH of the cell. The HCO3
then diffuses out of the cell into the plasma and Cl
moves in to restore the electrostatic balance; however, this upsets the osmotic equilibrium so H2O also enters, swelling the cell. Thus, gaseous exchange in red cells also results in a volume change (Figure 2(b)). The oxygenation of Hb results in the dissociation of a proton from the globin chain, known as the Bohr effect, initiating simultaneous release of CO2. The proton combines with the HCO3
to form H2CO3 and the carbonic anhydrase now converts this to CO2 and H2O, allowing release of the CO2 along its pressure gradient (Figure 2(a)). Thus, erythrocytes do not expend any energy in transporting O2 and CO2, due to the gradients of these gases and the unique properties of Hb.

Erythrocytes in Respiratory Diseases Anemias and Polycythemias

Anemias, depending on their severity, can manifest as respiratory abnormalities, most notably dyspnea. The general decrease in O2-carrying capacity caused by anemia often results in compensatory increases in cardiac output and blood shunting, particularly in chronic anemias. Treatment of anemias caused by kidney disease with recombinant Epo results in the normalization of pulmonary functions. Anemias caused by deficiencies of iron, vitamin B12, or

ERYTHROCYTES 145

folic acid, which can be corrected by appropriate supplementation, also result in the disappearance of pulmonary dysfunction. More often a primary respiratory disease will be the cause of a general hypoxic state within the erythroid compartment. Polycythemia can, on rare occasions, present with respiratory problems due to the high blood viscosity impairing blood flow. Interestingly, chronic polycythemia associated with high altitude is usually accompanied by decreased resistance in the periphery, preventing an increase in blood pressure and the appearance of respiratory problems. Hypoxemia due to pulmonary disorders that produce impaired O2 diffusion through the alveolar walls can result in a secondary polycythemia through the excessive induction of Epo. The increased hematocrit and blood viscosity results in an increase in circumference of pulmonary and tissue capillaries so the cardiac output is not significantly increased. The use of regular phlebotomy to treat polycythemia corrects the pulmonary and circulatory changes. Hemoglobinopathies

Hemoglobinopathies, including sickle cell disease and thalassemias, can result in lung pathologies/respiratory problems. Indeed, in sickle cell disease, pulmonary complications of acute chest syndrome and chronic sickle cell lung disease are significant causes of morbidity and mortality. Erythrocytes are high in antioxidants, which can prevent oxidation of iron in the heme moiety, and prevent oxidative stress damage in the lung. Interestingly, sickle cell erythrocytes have reduced antioxidant enzymes and are less effective against reactive oxygen species during oxidative injury. Sickle cells retain adhesion factors that are normally lost as reticulocytes are released into the circulation, resulting in increased adherence to the endothelial cell surface, which leads to endothelial injury in the lung. Owing to a variety of factors, sickle cells impair host defenses and, consequently, patients have an increased incidence and severity of pulmonary infections. There are no specific treatments for lung diseases associated with sickle cells; generally, treatments are aimed at reducing the proportion of sickled erythrocytes. Hydroxyurea treatment induces fetal Hb and, thus, decreases the relative concentration of the sickle (S) Hb. Epo treatment can also be employed. Transfusions and bone marrow transplantation have been successfully undertaken with patients with severe complications. Inflammatory Lung Injury

Recent studies have indicated that erythrocytes have the potential to participate in inflammation

and repair following lung injury. Resident fibroblasts of the lung can respond to inflammatory mediators and release other molecules that regulate inflammation, including interleukin-8 (IL-8), which acts as a chemoattractant for neutrophils. Erythrocytes, or factors released by them, can stimulate fibroblasts to release IL-8. Thus, an injury that leads to red cell contact with the extracellular matrix/fibroblasts has the potential to initiate an inflammatory response by stimulating IL-8 secretion and neutrophil infiltration. Cystic Fibrosis

Cystic fibrosis (CF) patients develop numerous lung pathologies, including severe airway disease, alveolar hypoxia, and pulmonary hypertension. CF is caused by a diminution, or loss, of the cystic fibrosis transmembrane conductance regulator (CFTR), caused by specific genetic mutations. This potential ion channel is one of a family of molecules responsible for regulating the movement of ATP out of a cell. Red blood cells are known to release ATP upon mechanical deformation and specific inhibitors of CFTR negate this activity. Erythrocytes from CF patients are unable to display this ATP efflux after physical stimulation due to their lack of functional CFTR. Furthermore, ATP released into the pulmonary circulation enhances NO synthesis in the lung. Consequently, the pulmonary hypertension seen in CF could well be due to the lack of ATP-stimulated NO synthesis in the pulmonary vasculature, normally initiated by red blood cell deformation. Additionally, in primary pulmonary hypertension, which has no known etiology, there is again a hindrance of this pathway and a lack of ATP release from red cells. Malaria

Malaria is one of the world’s major public health concerns and significant pulmonary problems are reported; up to 5% of patients infected with Plasmodium falciparum are known to develop respiratory failure. This is caused by sequestration of parasitized red blood cells in the microvasculature of the lung; infected erythrocytes express parasite-derived proteins, which induce adhesion to endothelial cells. The development of the inflammatory response to parasite antigens also leads to release of inflammatory mediators, which results in alveolar and endothelial damage and capillary leakage. Many factors contribute to hypoxemia in severe malaria, including sequestration of parasitized red cells in the pulmonary vasculature, aspiration pneumonia, fluid overload, and secondary Gram-negative bacteremia. Ventilation, replacement transfusion, and fluid management

146 EXERCISE PHYSIOLOGY

are important for rectifying pulmonary functions during treatment with antimalarial drugs. See also: Arterial Blood Gases. Colony Stimulating Factors. Hemoglobin. High Altitude, Physiology and Diseases. Peripheral Gas Exchange. Systemic Disease: Sickle Cell Disease.

Further Reading Fredriksson K, Lundahl J, Palmberg L, et al. (2003) Red blood cells stimulate human lung fibroblasts to secrete interleukin-8. Inflammation 27: 71–78. Harris JW and Kellermeyer RW (eds.) (1970) The Red Cell – Production, Metabolism, Destruction: Normal and Abnormal. Cambridge: Harvard University Press. Hillman RS and Finch CA (eds.) (1974) Red Cell Manual, 4th edn. Philadelphia: F A Davis Company. Ingley E, Tilbrook PA, and Klinken SP (2004) New insights into the regulation of erythroid cells. IUBMB Life 56: 177–184.

James MF (1985) Pulmonary damage associated with falciparum malaria: a report of ten cases. Annals of Tropical Medicine and Parasitology 79: 123–138. Klinken SP (2002) Red blood cells. International Journal of Biochemistry and Cell Biology 34: 1513–1518. Knight J, Murphy TM, and Browning I (1999) The lung in sickle cell disease. Pediatric Pulmonology 28: 205–216. Lane DJ (ed.) (1976) Respiratory Disease. London: William Heinemann Medical Books Ltd. Pawloski JR, Hess DT, and Stamler JS (2001) Export by red blood cells of nitric oxide bioactivity. Nature 409: 622–626. Pouvelle B, Buffet PA, Lepolard C, Scherf A, and Gysin J (2000) Cytoadhesion of plasmodium falciparum ring-stage-infected erythrocytes. Nature Medicine 6: 1264–1268. Sprague RS, Ellsworth ML, Stephenson AH, Kleinhenz ME, and Lonigro AJ (1998) Deformation-induced ATP release from red blood cells requires cystic fibrosis transmembrane conductance regulator activity. American Journal of Physiology 275: H1726– H1732. Sprague RS, Stephenson AH, Ellsworth ML, Keller C, and Lonigro AJ (2001) Impaired release of ATP from red blood cells of humans with primary pulmonary hypertension. Experimental Biology and Medicine 226: 434–439.

EXERCISE PHYSIOLOGY B J Whipp, University of Leeds, Leeds, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Exercise intolerance is a consequence of the inability to meet the energy requirements of the chosen, or imposed, task. The goal of clinical exercise testing is to stress the organ systems contributing to the intolerance to a level at which the abnormality becomes discernible from the magnitude or profile of appropriately selected response variables. Assessing the normalcy, or otherwise, of these responses to exercise requires the investigator to select and appropriately display the cluster of response variables that are themselves reflective of the particular system(s) behavior. Interpretation is then based on two interrelated perspectives: discriminating a magnitude or pattern of deviation from the normal response (age, gender, and activity matched standard subject); and matching the magnitude or pattern of abnormality with that characteristic of particular impairments of physiological system function. Exercise is not a mere variant of rest: it is the essence of the machine – Joseph Barcroft

Introduction Purposeful increases in muscular activity are essential components of human cultural expression. However, the limits to plausible physical aspiration are set by the adequacy of functioning of the physiological

systems that link and support oxygen transfer from the atmosphere to the energy transduction sites within contracting muscles. When the ability to meet the energy requirements of the chosen or imposed tasks becomes limited, exercise intolerance ensues. Physiological, as other, systems tend to fail under stress; an optimized stress profile can therefore be utilized to allow abnormalities in the magnitude or contour of change of selected variables of interest to be discerned – providing clues to the source of the intolerance. This naturally requires a context of normal response.

Determinants of Normal Responses Metabolic Considerations

Exercise is fueled by the chemical energy of ingested food, which is transformed into the mechanical energy of muscular force generation. Skeletal muscle, however, is composed of different fiber types with different mechanical and metabolic characteristics. For tasks requiring relatively low force generation, the more efficient and more aerobic Type I fibers are predominantly recruited; with higher demands for force generation (or for rapid contractions), the less efficient and more glycolytic (‘anaerobic’) Type II fibers are also recruited. The contraction profile of

EXERCISE PHYSIOLOGY 147 Table 1 Sources of ATP resynthesis during exercise O2 transport O2 stores

Aerobic

 PCr stores O2 defðmuscÞ
þ Lactate and H production

Anaerobic

O2 def (lung)

the muscles must be effectively orchestrated for skillful performance. While adenosine triphosphate (ATP) is the obligatory energy resource for muscle contraction, its low concentration (B5 mM kg
1) is maintained by increased aerobic and anaerobic production (Table 1). The immediate reaction for ATP resynthesis is splitting of locally stored phosphocreatine (PCr) to creatine (Cr) and inorganic phosphate (Pi):

WR for moderate intensity cycle ergometry at constant pedaling frequency; not varying appreciably with ‘fitness’, training, gender, or age. The typical slope (or ‘gain’) of this V’ O2 –WR relationship (i.e., DV’ O2 =DWR) averages an easy-to-remember B10 ml min
1 W. For a 100-W WR, the V’ O2 ss of the ‘standard’ 70-kg slim adult cycling at 60–70 rpm is composed of:

PCr-Cr þ Pi þ DG

1. resting V’ O2 (B250 ml min
1); 2. the ‘unmeasured’ additional V’ O2 to move legs with no load on the flywheel (‘0’ W) (B250 ml min
1); and 3. an increase ðDV’ O2 ssÞ of B1000 ml min
1, reflecting the 100 W applied WR, to an absolute V’ O2 ss of B1500 ml min
1.

½1

where DG is the free energy of ATP hydrolysis. PCr consequently decreases in proportion to work rate (WR), the magnitude of decrease depending on the rate at which O2 utilization increases towards its steady-state value. Consequently, less ‘fit’ subjects have a greater PCr decrease for a given WR than fitter subjects. The major route of ATP resynthesis, however, is oxidative phosphorylation, deriving from atmospheric O2 transported to the muscle during the exercise and O2 already stored in the body. The pulmonary O2 uptake ðV’ O2 Þ therefore conflates the im’ mediate influence of the increased cardiac output ðQÞ and the delayed influence of the increased muscle arteriovenous O2 content difference. In the steady state, all the energy transformations derive from aerobic exchange from the transported O2 with no further contribution from O2 stores or anaerobic mechanisms. V’ O2 subsequently remains steady, as long as WR remains constant. The steadystate level of CO2 output ðV’ CO2 Þ will, in addition, depend on the substrate mixture being metabolized. Prior to the steady state, energy must be provided from other sources. The O2-equivalent of these reactions is termed the O2 deficit (O2def). While it takes B3 min for V’ O2 to attain steady state ðV’ O2 ssÞ in healthy young subjects, it takes considerably longer in older and/or sedentary subjects, whose O2def will consequently be greater. As a result of the greater tissue capacitance for CO2, the V’ CO2 time course will be appreciably longer; consequently, the respiratory exchange ratio ðR ¼ V’ CO2 =V’ O2 Þ undershoots during the transient. Below the lactate threshold (yL), the V’ O2 ss increment is a relatively constant function of increasing

Although obesity increases V’ O2 ss at ‘0’ W, the DV’ O2 ss associated with a measured WR increment is not. And while the task performance is ‘inefficient’, in the sense that the total energy and O2 costs are high, the efficiency of transducing substrate energy into effective muscular work is not (B25–30% in both cases). The treadmill is less suitable for interpreting the V’ O2 response to the apparent WR. That is, inefficient gait patterns, the subject being partially supported by the handrails or by an investigator (e.g., during a blood-sampling procedure) modify metabolic cost of the task. The substrate mixture being oxidized influences the O2 cost of the task. Carbohydrate is the more ‘efficient’ fuel (by B6%) in terms of O2 utilization (thereby minimizing the cardiovascular demands for O2 delivery). However, fats produce appreciably less CO2 (by B40%) per unit ATP yield (thereby reducing the ventilatory demands for acid– base regulation). The rate at which V’ O2 increases towards its steady state (faster in ‘trained’ subjects) is a major determinant of the O2def. A large O2def for a given WR requires greater utilization of stored energy resources and predisposes to early-onset lactic acidosis (low yL ). When O2 is not utilized at appropriate rates (either inadequate delivery or impediment to utilization), ATP can only be formed through the low-yield

148 EXERCISE PHYSIOLOGY

anaerobic–glycolytic pathway: C6 H12 O6 ðglucoseÞ-2ðlactate
þ Hþ Þ þ 2ATP

½2

This increase in cellular proton load generates greater stress, influencing muscle contractile properties and ventilatory control mechanisms. The benefit, however, is that the ATP supply can be maintained: 1 ATP per lactate molecule formed from glucose, or 1.5 ATP from glycogen. The bicarbonate (HCO3
) system is the most important nonrespiratory contributor to acid–base regulation during exercise, being functionally ‘open’ with respect to the atmosphere:


CH3  CHOH  COO H lactate þ proton

þ

þ NaHCO3 sodium bicarbonate

-CH3  CHOH  COONa sodium lactate

þ H2 CO3 carbonic acid

½3

and

V’ E ¼ 863V’ CO2 =PaCO2 ð1
VD =VT Þ

While each proton that combines with an HCO
3 ion produces one additional CO2 molecule to be vented ðVCO2 Þ, the rate at which this CO2 is produced ðV’ CO2 Þ depends on the rate at which HCO
3 falls. Ventilatory Considerations

The major ventilatory demands of exercise are arterial blood-gas and acid–base regulation, while optimizing the ‘cost’ in terms of the respiratory muscle work. Alveolar (A), and hence arterial (a), PCO2 and PO2 can only be maintained constant during exercise if V’ A changes in precise proportion to V’ CO2 and V’ O2 , respectively: ½4

(The small effect of the difference in inspiratory and expiratory ventilation that occurs when R does not equal 1 is neglected.) As V’ A is common to both relationships, it cannot meet the regulatory demands of both O2 and CO2 exchange when they differ during exercise. V’ E changes in close proportion to V’ CO2 with PaCO2 being the more closely regulated variable, although the control mechanisms remain poorly understood. Any consequent changes in PaO2 normally only traverse the relatively flat region of the

½5

where VD/VT is the physiologic dead space fraction of the breath. The relationship between V’ E and V’ CO2 during exercise is highly linear (but with a small positive intercept on the V’ E axis, normally of B3–6 l min
1) up to the onset of compensatory hyperventilation. Consequently, the magnitude and profile of the ventilatory equivalent for CO2 ðV’ E =V’ CO2 Þ is closely linked to that of VD/VT: V’ E =V’ CO2 ¼ 863=PaCO2 ð1
VD =VT Þ

H2 CO3 -H2 O þ CO2

863  V’ CO2 863  V’ O2 ¼ V’ A ¼ PA CO2 ðPI O2
PA O2 Þ

oxyhemoglobin dissociation curve, with little effect on O2 saturation. As there is no sustained lactic acidosis during moderate exercise, arterial pH (pHa) will be regulated at its set-point level only if PaCO2 is maintained at the appropriate level. This depends on matching V’ A to the CO2 exchange rate at the lung – not its production rate in muscle. As a consequence of the physiologic dead space (VD), a proportion of the total ventilation ðV’ E Þ does not contribute to V’ A :

½6

High V’ E =V’ CO2 reflects a low PaCO2, high VD/VT, or both. Consequently, V’ E =V’ CO2 will be increased under conditions which impair pulmonary gas exchange (high VD/VT) or induce ‘excessive’ V’ E with respect to CO2 exchange (low PaCO2) caused, for example, by hypoxemia, metabolic acidosis, or anxiety. High VD/VT, however, does not necessarily reflect abnormal pulmonary function, as rapid, shallow breathing yields a high VD/VT even in normal subjects. The alveolar-to-arterial partial pressure difference (A
a) for the gas of interest provides an index of its exchange efficiency. Thus, while alveolar hypoventilation leads to arterial hypoxemia and hypercapnia, it does not widen (A
a); diffusion impairment, right-to-left shunt, and maldistribution ’ ðV’ A =QÞ ’ all do, particularly of V’ A with respect to Q for O2. PaO2 and PaCO2 are maintained at, or close to, resting levels during moderate exercise in normal subjects at sea level. Arterial hypocapnia, however, is a common feature of supra-yL exercise. Why, therefore, does PaO2 not systematically increase? It tends to be maintained close to resting levels at all WRs despite both V’ E =V’ O2 and end-tidal PO2 (PETO2) increasing progressively 4yL. That is (A
a) PO2 either begins to widen at yL , or begins to widen more rapidly, normally without significant hypoxemia (although this is seen in some highly fit normal subjects). In lung disease patients, pulmonary gas exchange inefficiencies leads to often-marked differences

EXERCISE PHYSIOLOGY 149

between alveolar (either ‘ideal’ or ‘real’) and arterial partial pressures. And while this increases the V’ E demand during the exercise, the ability to respond appropriately is constrained and, in the extreme, limited by the impaired pulmonary mechanics.

Normal Response Profiles Virgil’s dictum hoc opus hic labor est (‘there is the task and there is the toil’) recognizes the distinction between work rate, an absolute physical construct, which we measure in units of power, and work intensity, a relative construct reflecting its impact on the degree and perception of the consequent stress. Moderate Exercise

Sub-yL WRs are considered to be of moderate intensity as they are (1) generally not stressful; (2) sustainable for prolonged periods of time; and (3) typified by steady states of cardiopulmonary and metabolic response (e.g., Figure 1(b)). The response time courses are different, however; V’ O2 normally increases with a time constant (t) of B30–40 s while tV’ CO2 is B50– 60 s, reflecting the influence of the fraction of metabolically produced CO2 taken up into muscle stores during the on-transient. V’ E changes marginally more slowly (tV’ E is B55–65 s). Consequently, as tV’ E is appreciably longer than tV’ O2 , PAO2 and PaO2 fall transiently. But, owing to the relatively small kinetic dissociation between V’ E and V’ CO2 , the transient PaCO2 increase is hardly discernible. The values of PaCO2 and PaO2 are typically averages of blood sampled over several respiratory cycles, providing a mean of the intrabreath oscillations. For example, end-inspiratory PACO2 is reduced because of the greater dilution of alveolar gas, while end-expiratory, or end-tidal, PCO2 (PETCO2) is increased because of increased CO2 flux across the alveolar–capillary membrane during exhalation (the increased mixed-venous PCO2 ðPv% CO2 Þ causing the slope of the ‘alveolar phase’ to increase). The intrabreath PaO2 profile will, naturally, closely mirror that of PaCO2. In normal exercising subjects therefore, PETCO2 is typically higher than PaCO2 (by as much as 6 mmHg), depending on Pv% CO2 and the expiratory duration. PETCO2 should not therefore be used to represent PaCO2 in computing VD/VT. Algorithms for estimating PaCO2 from PETCO2 are poor in normal subjects and do not work in subjects with lung disease. Heavy and Very Heavy Exercise

For the more stressful range of WRs above yL , at which the increase in arterial lactate (L
a) and H þ a can stabilize (or even decrease) with time (heavy

intensity), V’ O2 can also attain a delayed steady state – but with a higher-than-expected V’ O2 that is a result of a slow component of the V’ O2 kinetics which supplements the early response. This additional increment can result in DV’ O2 ss=DWR values as high as 14 ml min
1 W (rather than the sub-yL value of B10 ml min
1 W). At even higher WRs, beyond what is termed ‘critical power’ (CP), V’ O2 steady states are not attainable (very heavy intensity). All very heavy WRs are associated with inexorable increases in V’ O2 , V’ E , and H þ a to the limit of tolerance (Figure 1(a)). In contrast, the V’ CO2 profile appears more exponential-like (Figure 1(a)). This can be easily misinterpreted, however; its profile conflates the influence of the aerobic component with that from HCO3
-buffering (which peaks early in the transient and then declines as the rate of HCO3
decrease progressively slows) and that from hyperventilation (which develops slowly). Above CP, V’ O2 increases towards its maximal value ðV’ O2 max Þ at a rate that is greater the higher the WR, progressively reducing tolerance time. On average yL occurs at B50% V’ O2 max and CP at B75% (Figure 2) in groups of normal subjects. But both yL and CP occur at highly variable fractions of V’ O2 max in individual subjects. Consequently, two subjects exercising at the same %V’ O2 max , as in Figure 1, could manifest markedly different patterns of response. The use of the %V’ O2 max as an index of work intensity among subjects is therefore not justifiable; a common temporal profile of V’ E , pulmonary gas exchange, and arterial acid–base response should be characterized as a common intensity. When the subject’s limit of tolerance is set by the ability to increase V’ E , as in chronic obstructive pulmonary disease (COPD) patients, for example, CP (and yL ) occurs at a higher %V’ O2 max than normal (e.g., Figure 2). This provides the potential for using a higher fraction of the V’ O2 max in a training regimen. Above CP, each progressive increase in WR results in the same V’ O2 max . In contrast to normal subjects, however, this is also associated with effectively the same (limiting?) V’ E (Figure 2). Endurance training increases CP in normal subjects (Figure 3) and COPD patients. And even if the training only leads to a relatively small increase in the maximum WR (WRmax) achieved on an incremental exercise test, small changes in CP can translate into considerable improvements in the tolerable duration of some high fraction (e.g., 80%) of that maximum. Unfortunately, while CP is an important parameter characterizing the upper limit of sustainable power generation for this kind of exercise, it can at present only be determined as the power asymptote of a series of exhausting WRs.

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Figure 1 Oxygen uptake ðV’ O2 Þ, CO2 output ðV’ CO2 Þ, and ventilatory ðV’ E Þ responses to constant-load exercise on a cycle ergometer in two subjects (a, b) exercising at the same % V’ O2 max ; this work rate was below the lactate threshold for subject b, but above for subject a. Note the marked V’ O2 slow component in subject a, and its absence in subject b. Reproduced from Whipp BJ, Ward SA, and Rossiter HB (2005) Pulmonary O2 uptake during exercise: conflating muscular and cardiovascular responses. Medicine and Science in Sports and Exercise 37: 1574–1585, with permission from Lippincott Williams & Wilkins.

Response Limitations When a subject has ostensibly exercised to the limit of tolerance, it is important to determine whether

particular systems that contribute to the energy exchange have reached their limit. For example, if the maximum voluntary ventilation (MVV) determined at rest is considered the maximum attainable V’ E ,

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Figure 2 The responses of V’ O2 and V’ E to five progressively greater work rates performed on a cycle ergometer, to the limit of tolerance or for 20 min (whichever came first) in a healthy control (left panels) and a COPD patient (right panels), matched for age. Note that, both in the control and the patient, the lowest WR (closed circles) corresponded to critical power, with consequent stability of V’ O2 ; at the higher four WRs, V’ O2 at end exercise attained V’ O2 max . In the control subject, V’ E at end exercise was progressively higher with increasing work rate, but in no instance was the maximum voluntary ventilation (MVV) approached; in the COPD patient, however, all WRs above critical power resulted in V’ E attaining MVV (i.e., with zero breathing reserve). Reproduced from Neder JA, Jones PW, Nery LE, and Whipp BJ (2000) Determinants of the exercise endurance capacity in patients with chronic obstructive pulmonary disease: the power–duration relationship. American Journal of Respiratory and Critical Care Medicine 162: 497–504, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

then the difference between this and the value actually attained at the end of exercise (Figure 4) can be considered to represent the subject’s breathing reserve (BR). This can be zero (or even negative in a subject who bronchodilates during exercise) either as a result of MVV being low (lung disease patients) or achievable WRs and V’ E being high (highly fit normal subjects). Also, if the maximum effort expiratory airflow is considered to reflect the greatest possible flow at a particular lung volume (not necessarily the case in COPD patients), then achieving these flows during exercise indicates an absence of flow reserve (Figure 5). Similarly, a tidal volume (VT) that encroaches upon the inspiratory capacity (IC) is reflective of lack of volume reserve. Significant heart rate (HR) reserve at maximum exercise is usually judged relative to the subject’s age-predicted maximum; unfortunately, this has high variability. These expected maxima therefore provide useful frames of reference for discriminating among potential causes of the exercise limitation.

Unlike normal (especially young) subjects, the tolerable WR range in patients with lung disease is constrained by a combination of pulmonary factors, chief of which are: 1. impaired pulmonary-mechanical and gas-exchange functions, which increase the V’ E demands; 2. limitations of airflow generation or lung distention; 3. increased physiological costs of meeting the V’ E demands, in terms of respiratory muscle work, perfusion, and O2 consumption; 4. predisposition to shortness-of-breath or dyspnea, consequent to the high fraction of the achievable flow or lung distention demanded by the WR, commonly exacerbated by arterial hypoxemia and early-onset metabolic acidosis; along with 5. other factors, such as coexistent heart disease, pulmonary hypertension, nutritional deficiencies, and detraining as a result of low-activity patterns of daily living – the latter helping explain the high

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Figure 5 Spontaneous flow-volume curves from a normal subject (left) and a COPD patient at rest (inner black loop), at submaximal exercise (blue loop), and at maximal exercise (outer black loop) (right); inspiration downwards; expiration upwards. Note that for the COPD patient, the maximal exercise curve impacts on the volitionally generated maximal flow-volume curve, with dynamic hyperinflation; this is not the case for the normal subject. Modified from Leaver DJ and Pride NB (1971) Flow– Volume curves and expiratory pressures during exercise in patients with chronic airway obstruction. Scandinavian Journal of Respiratory Diseases Supplement 77: 23–27, with permission.

incidence of ‘fatigue’ rather than dyspnea as the dominant reported cause of exercise limitation. MVV BR BR BR VE

VO2 Figure 4 Schematic of the V’ E response to incremental exercise, as a function of V’ O2 . In healthy subjects (black), note that V’ E at the lactate threshold (filled symbols) and at V’ O2 max (open symbols) becomes progressively greater as fitness increases: sedentary (circles), normal fitness (squares), high fitness (diamonds). As maximum voluntary ventilation (MVV) is unaffected by training status, breathing reserve (BR) therefore decreases. In a COPD patient (blue), V’ E at any particular V’ O2 is increased (reflecting the characteristically increased ventilatory requirement) and MVV (dashed-dotted line) is reduced. Modified from Whipp BJ, and Pardy R (1986) Breathing during exercise. In: Macklem P and Mead J (eds.) Handbook of Physiology, Respiration (Pulmonary Mechanics), pp. 605–629. Washington, DC: American Physiological Society, used with permission from the American Physiological Society.

The increased airways resistance and/or decreased pulmonary recoil pressure reduces maximum expiratory airflow in COPD patients. This reduces the effective operating range of the subject’s V’ E during exercise. The V’ E demands, however, are commonly greater than normal, reflecting the high VD/VT (a component of which is also seen, as a result of loss of lung recoil, in the otherwise healthy elderly). This leads to high V’ E =V’ O2 and V’ E =V’ CO2 – which may be further increased from arterial hypoxemia. However, some COPD patients can have higher-than-normal PaCO2, especially those with poor peripheral chemosensitivity. The combination of increased V’ E demands and decreased maximum-attainable V’ E leads to little or no BR at maximum exercise (Figures 2–5). COPD patients, especially, also generate spontaneous expiratory airflows during exercise which equal, or even exceed, those achieved at a given lung volume during a maximal flow-volume maneuver (Figure 5). The seeming paradox of expiratory airflow during exercise exceeding that generated during a maximal expiratory effort at rest can be explained by: 1. bronchodilatation from exercise-induced increases in circulating catecholamines;

EXERCISE PHYSIOLOGY 153

2. the forced-expiratory maneuver from total lung capacity allows lung units with fast mechanical t’s (the product of airways resistance and thoracic compliance) to empty at high lung volumes, leaving the longer t units to empty at lower volumes. When these less-than-maximum lung volumes are attained during spontaneous breathing, the fast t units are now recruited at these lower lung volumes, resulting in greater airflow – at that lung volume; and 3. maximum expiratory airflow not being achieved with maximum expiratory effort (especially at low lung volumes) because of dynamic airway compression, and even closure in some cases, during the forced maneuver. The increase in end-expiratory lung volume during exercise (dynamic hyperinflation) in COPD patients (Figure 5) is a result of the mechanical t values being long with respect to the time available for expiration. This results in the VT encroaching upon the compliance limits, adding a ‘restriction-like’ component to the obstruction. Factors that reduce breathing frequency ( fB), such as hyperoxic inspirates and/or physical training, prove effective at reducing the hyperinflation and improving exercise tolerance. When the upper limit for exercise tolerance is set by pulmonary determinants, other components of the body’s energy supply systems may not be stressed to their limits. Consequently, maximum HR, O2 pulse, and L
a are often markedly less than predicted in COPD patients. Although the pattern of blood-gas response to exercise is highly variable in such patients, in many cases the resting levels of PaO2 can be maintained without further hypoxemia; diffusion limitation across the alveolar–capillary bed does not seem to be significantly contributory and the in’ dispersion does not appear to worsen. creased V’ A =Q Also, those patients who are capable of generating a metabolic acidosis during exercise usually evidence little or no respiratory compensation owing to the obstructive constraint. In patients with ‘restrictive’ lung diseases, such as diffuse interstitial fibrosis, reduced airflow generation (at a particular lung volume) is not of concern. Rather, the increased pulmonary elastance demands greater inspiratory muscle force and increased work of breathing. This predisposes to their typical tachypnea (fB450 min
1 being common at maximum exercise), with VT often reaching their (reduced) IC. Unlike COPD, however, the hypoxemia in these patients typically worsens as WR increases – not, it ’ matching seems, because of further impaired V’ A =Q but because of increasing diffusion limitation, often

leading to (A
a)O2 in excess of 60 mmHg at the limit of tolerance. While exercise does not usually worsen the degree of airway obstruction in the emphysematous and/or bronchitic forms of COPD, it typically does so in patients with asthma. Although the exercise-induced bronchoconstriction – which can also occur in subjects with no history (or even a recognition) of airway hyperreactivity – is sometimes manifest during exercise, it is most typically a postexercise phenomenon, with high-intensity exercise the more potent trigger. A prior moderate-intensity warm-up can ameliorate the degree of bronchoconstriction during a subsequent high-intensity exercise bout, as does – for a short period – a prior exercise-induced bronchospastic episode itself.

Patterns of Response Indicative of Disease While the constant-load exercise test provides a closer simulation of the demands for sustained occupational or daily living tasks, the appropriateness of the integrated system responses to exercise is best studied (at least, for the initial exercise evaluation) by means of an incremental test which spans the entire tolerance range. This allows both cause(s) and severity of exercise intolerance to be identified from considerations of the normalcy of response of variables of interest, compared with those for an appropriate reference population. It can also establish both the limits and effective operating range of system function, allowing ‘disability’ and ‘impairment’ to be assessed and training protocols to be optimized, and providing a frame of reference for discriminating the need for, or benefits of, therapeutic interventions (including those related to major surgery). A test duration of 20 min or less is usually sufficient, comprising: 1. a resting phase, 2. a control phase of at least 3 min of ‘unloaded’ exercise (or another suitably low WR) to ensure that the responses have stabilized, 3. a linearly incremental exercise phase to the limit of tolerance, and 4. a recovery phase. The results are not appreciably different when WR is increased continuously (ramp test) or by uniformly small amounts over short intervals (e.g., 1-min incremental test). An incremental phase duration of B10 min provides optimum discrimination, with 10–20 W min
1 being an appropriate incremental rate for healthy nonathletic subjects but as low as

154 EXERCISE PHYSIOLOGY

5 W min
1 in a patient. A subsequent constant-load test (at a now-known intensity) may, on occasion, be needed to gain additional information about particular system response kinetics. Useful Noninvasive Relationships

The response profiles and maximum values of certain noninvasive variables, in addition to the standardly assessed electrocardiogram (ECG) and arterial blood pressure, have proven especially informative with respect to inferring system pathophysiology, in the context of the available normal predicted values. V’ O2 –WR relationship In response to a constant WR, V’ O2 attains a constant steady-state value (over the WR range for which steady states are attainable). For a ramp test (e.g., Figure 6), a constant rate of change of WR yields a constant rate of change of V’ O2 , after a small lag-phase (reflecting the system response kinetics). The gain of this response ðDV’ O2 =DWRÞ has been shown not to differ from that of the steady-state response (normally 9– 11 ml min
1 W). The incremental gain is therefore often used as an index of the work (in)efficiency. But while the rate of change, in the linear region of the ramp, is normally the same as that for the steadystate test, the actual value of V’ O2 at any WR is lower. However, in many patients with cardiopulmonary diseases, this incremental gain can be very low (e.g., 8 ml min
1 W
1 or less), suggestive of impaired aerobic exchange. The highest value achieved with good subject effort is termed the peak V’ O2 ðV’ O2 peak Þ or V’ O2 max when V’ O2 does not continue to increase with further increase in WR. While the good effort V’ O2 peak is not different with different ramp slopes, the WRmax is progressively greater the faster the ramp; this must be accounted for when choosing a %WRmax for a subsequent constant-WR strategy. Abnormalities of the V’ O2 response predominantly reflect inadequacies of cardiovascular function. Consequently, poorly fit (but otherwise normal) subjects typically evidence low V’ O2 peak and yL but with a normal DV’ O2 =DWR. Patients with lung disease, other than those with significant pulmonary vascular dysfunction, present a V’ O2 response pattern similar to the sedentary normal subject but with a V’ O2 peak that is even more significantly reduced, that is, when impaired lung mechanics limit the tolerable WR range. Patients with cardiac, peripheral, and/or pulmonary vascular disorders, on the other hand, often also manifest abnormally low V’ O2 –WR slope throughout the incremental test. Those with coronary heart disease commonly present with a relatively normal response

slope over the lower reaches of their WR range, which then becomes reduced in concert with ECG evidence of developing ischemia. V’ CO2 –V’ O2 relationship The profile of V’ CO2 as a function of V’ O2 (V slope) is one in which the V’ CO2 response initially lags behind that of V’ O2 early in the transient (as some of the metabolically produced CO2 is diverted into the body stores) and then increases often as a linear function of V’ O2 (Figure 6). The slope of the relationship ðDV’ CO2 =DV’ O2 Þ in this region has been termed S1, with a value typically close to unity in subjects on a typical Western diet. When the aerobically produced CO2 is supplemented by additional CO2 released from HCO3
buffering, the slope increases (Figure 6) – termed S2 in this higher WR region. As the amount of CO2 released in the proton-buffering process is a function not of the magnitude of HCO3
decrease but its rate of decrease, S2 is highly dependent on the WR incrementation rate. At slow incrementation rates, additional CO2 from compensatory hyperventilation supplements V’ CO2 in the S2 range. The presence of an isocapnic buffering region with more rapid WR incrementation rates obviates the concern for hyperventilation being the cause of the slope increase. Having ruled out both aerobic metabolism and hyperventilation as the source of the increased S2, one is left with the remaining source – HCO3
decrease reflecting yL . Why V’ E retains the same relationship to V’ CO2 in this supra-yL region as below yL, that is, V’ E increases as a function of V’ O2 but not V’ CO2 (Figure 6) is not fully understood. But when the V-slope plot may not be partitioned into two defensibly linear segments, the unit tangent to the curve may be used as a second-order estimate of yL . At a higher V’ CO2, however, the beginning of an increase in V’ E =V’ CO2 and decrease in PETCO2 provides evidence of compensatory hyperventilation. This is termed the respiratory compensation point (RCP). V’ E –V’ CO2 relationship and ventilatory equivalents The V’ E –V’ CO2 relationship is linear up to RCP (Figure 6), with a slope of B25 in healthy young subjects but increasing with age. V’ E =V’ CO2 however declines hyperbolically with respect to V’ CO2 (toward the value of the V’ E –V’ CO2 slope itself), reflecting the influence of the small positive V’ E intercept. As discussed previously, V’ E =V’ CO2 is closely linked to VD/VT in the regulation of PaCO2 and pHa (eqn [6]). Thus, high values of V’ E =V’ CO2 reflect either a low PaCO2, high VD/VT, or both. But, without arterial blood sampling, how can a low PaCO2 be ruled out as the cause of the high V’ E =V’ CO2 ? It can’t – at least not conclusively! This is especially so when

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Figure 6 Left and middle panels show a multipanel display of the system responses of a series of physiological variables to an incremental cycleergometer exercise test performed to the limit of tolerance. The selected clusters of variables are those that have been shown to be particularly useful both for establishing parameters of system function (e.g., the estimated lactate threshold (yL), the gain (DV’ O2 =DWR) and peak of the V’ O2 response, and the ventilatory ‘efficiency’ (DV’ E =DV’ CO2 ) and for assessing the normalcy, or otherwise, of the magnitude and profile of system responses. The values commonly derived from such a display are designated A, B, C, D, E, F and G in the data display; their physiological equivalents are given in the right panel. Left panel, top to bottom: the steady-state level of CO2 output V’ CO2 , ventilatory equivalents for O2 and CO2 (V’ E =V’ O2 , V’ E =V’ CO2 ), end-tidal PCO2 and PO2 (PETCO2, PETO2), and the respiratory exchange ratio (RER) as a function of V’ O2 . Middle panel, top to bottom: V’ O2 vs. WR; V’ E vs. V’ CO2 ; heart rate (HR) and O2-pulse (V’ O2 / HR) vs. WR; and tidal volume (VT) vs. V’ E .

PETCO2 is low simultaneously, less so if it is ‘normal’, but it is an unlikely cause if PETCO2 is high. Patients with lung disease consequently manifest high values for both the V’ E 2V’ CO2 slope and

V’ E =V’ CO2 measured at its minimum or at yL . Patients with heart disease also evidence high values of these variables during exercise, predominantly (it appears) to an increased VD/VT resulting from uneven

156 EXERCISE PHYSIOLOGY

distribution of pulmonary perfusion. The magnitude of the increase is correlative of poor prognosis, especially when yL and V’ O2 peak are also low. The increase in V’ E =V’ O2 following its initial VD/VT -linked decline is used as an index of the additional V’ E drive above yL . End-tidal gas tensions These are easy to measure and extremely difficult to interpret. As described above, PETCO2 normally increases progressively with increasing WR up to yL (Figure 6); it then stabilizes with increasing WR in the region of isocapnic buffering (as the effect of the increasing Pv% CO2 is offset by the decreasing TE), and subsequently decreases as frank compensatory hyperventilation is manifest. PETO2, in contrast, decreases progressively up to yL, at which point it begins to increase systematically as the rate of V’ E increase exceeds that of V’ O2 (Figure 6), that is, there is no ‘iso-oxic’ equivalent of the isocapnic period. Mean PaCO2, however, differs from mean PACO2 ’ inhomogeneities and/or right-toas a result of V’ A =Q left shunt, leading to PETCO2 being commonly less than PaCO2 in patients with COPD or pulmonary vascular disease, for example. Consequently, a PETCO2 which is less than mean PaCO2 during exercise (or even equal to it) is reflective of abnormal gas exchange. Breathing pattern While some individuals entrain fB to some unit multiple of their cycling or stride frequency, V’ E during exercise normally increases predominantly through changes in VT over the relatively linear thoracic compliance range, thereby minimizing airflow-related work of breathing. At higher WRs, further increases in V’ E are usually mediated largely through increases in fB, obviating large increases in elastic work of breathing over the ‘shallow’ thoracic compliance region. The VT–V’ E is commonly used to discern both the breathing pattern response and the extent to which V’ E and/or VT reach limiting values. Patients with COPD have, in addition to an augmented V’ E response, a disproportionately large fB increase during the exercise and little or no BR at the limit of tolerance. This is also true of the patients with ‘restrictive’ disorders, but in this case VT increases to the level of the IC early in exercise with fB often in excess of 50 min
1. V’ O2 –HR relationship and O2 pulse HR during exercise is normally a linear function of V’ O2 to an agedependent maximum (averaging B220–age, but with a large standard deviation of B10 min
1) and a slope that is an inverse function of a physical fitness. As this linear relationship has a positive intercept on the HR axis, the O2 pulse ðO2 -P ¼ V’ O2 =HRÞ increases

hyperbolically as WR increases. But the O2-P is of further interest, as it is numerically equivalent to the product of stroke volume and the arteriovenous O2 content difference. And so, if O2-P fails to increase with increasing WR, then either both variables are constant or one is increasing while the other decreases – either way a reflection of poor cardiovascular functioning. The rate at which O2-P increases is not only abnormally low but actually becomes flat in patients with impaired cardiovascular function, including heart failure, hypertrophic cardiomyopathy, and coronary artery disease, and also patients with significant pulmonary circulatory pathology.

Conclusion A clinical exercise tests is a device for educing flaws or abnormalities of physiological system function that contribute to exercise intolerance. The results of an appropriately designed test can also provide a frame of reference for the need for, and any benefits of, interventions designed to improve performance. Appropriately interpreting deviations from an expected response profile (i.e., characteristic of a reference population) needs a clear justification of their underlying physiological determinants. Commercially available ‘carts’ now readily provide a wealth of relevant evidence. But this is information; appropriate interpretation remains the investigator’s challenge. See also: Asthma: Exercise-Induced. Carbon Dioxide. Energy Metabolism. Hypoxia and Hypoxemia. Neurophysiology: Neuroanatomy. Space, Respiratory System. Ventilation: Overview; Control.

Further Reading American College of Sports Medicine (1995) Guidelines for Exercise Testing and Prescription. Baltimore: Williams & Wilkins. American Thoracic Society/American College of Chest Physicians (2003) Statement on cardiopulmonary exercise testing. American Journal of Respiratory and Critical Care Medicine 167: 211–277. Casaburi R and Petty TL (eds.) (1993) Principles and Practice of Pulmonary Rehabilitation. Philadelphia: Saunders. Gallagher C (1990) Exercise and chronic obstructive pulmonary disease. Medical Clinics of North America 74: 619–641. Johnson BD, Badr MS, and Dempsey JA (1994) Impact of the aging pulmonary system on the response to exercise. Clinics in Chest Medicine 15: 229–246. La¨nnergren J and Westerblad H (2003) Limits to human performance caused by muscle fatigue. Physiology News 53: 7–10. Leaver DJ and Pride NB (1971) Flow–volume curves and expiratory pressures during exercise in patients with chronic airway obstruction. Scandinavian Journal of Respiratory Diseases Supplement 77: 23–27. Neder JA, Jones PW, Nery LE, and Whipp BJ (2000) Determinants of the exercise endurance capacity in patients with chronic obstructive pulmonary disease: the power–duration relationship.

EXTRACELLULAR MATRIX / Basement Membranes 157 American Journal of Respiratory and Critical and Care Medicine 162: 497–504. Roca J and Whipp BJ (eds.) (1997) Clinical Exercise Testing, European Respiratory Monograph, vol. 2, no. 6. Sheffield: European Respiratory Journals. Rowell LB (1993) Human Cardiovascular Control. New York: Oxford University Press. Wagner PD (1996) Determinants of maximal oxygen transport and utilization. Annual Review of Physiology 58: 21–50. Wasserman K, Hansen JE, Sue DY, Stringer WW, and Whipp BJ (2004) Principles of Exercise Testing and Interpretation. Philadelphia: Lippincott Williams & Wilkins.

Weisman IM and Zeballos RJ (eds.) (1994) Clinical exercise testing. Clinics in Chest Medicine 15. Whipp BJ and Pardy R (1986) Breathing during exercise. In: Macklem P and Mead J (eds.) Handbook of Physiology, Respiration (Pulmonary Mechanics), pp. 605–629. Washington, DC: American Physiological Society. Whipp BJ and Sargeant AJ (eds.) (1999) Physiological Determinants of Exercise Tolerance in Humans. London: Portland Press. Whipp BJ, Ward SA, and Rossiter HB (2005) Pulmonary O2 uptake during exercise: conflating muscular and cardiovascular responses. Medicine and Science in Sports and Exercise 37: 1574–1585.

EXTRACELLULAR MATRIX Contents

Basement Membranes Elastin and Microfibrils Collagens Matricellular Proteins Matrix Proteoglycans Surface Proteoglycans Degradation by Proteases

Basement Membranes

Introduction

J H Miner and N M Nguyen, Washington University School of Medicine, St Louis, MO, USA

Basement membranes (BMs) are thin sheets of specialized extracellular matrix that were first identified by transmission electron microscopy as continuous ribbon-like structures adjacent to a subset of cells. They are evolutionarily ancient structures, being present even in primitive organisms such as sponges and Hydra. In mammals, BMs underlie endothelial and epithelial cells and surround all muscle cells, fat cells, and peripheral nerves. They play important roles in filtration, in compartmentalization within tissues, and in maintenance of epithelial integrity, and they influence cell proliferation, differentiation, migration, and survival. In the lung, BMs are associated with bronchial and vascular smooth muscle cells, bronchial epithelium, nerve, and pleura, and they are part of the air–blood barrier between microvascular endothelial cells and alveolar epithelial cells (Figure 1). The biochemical characterization of BM proteins was facilitated by the Engelbreth–Holm–Swarm (EHS) tumor, a transplantable tumor of mouse origin that produces large amounts of BM components that can be easily isolated. Studies of EHS tumor matrix proteins led to the identification of the four

& 2006 Elsevier Ltd. All rights reserved.

Abstract Basement membranes (BMs) are thin sheets of specialized extracellular matrix that underlie endothelial and epithelial cells and surround all muscle cells, fat cells, and peripheral nerves. They play important roles in filtration, in compartmentalization within tissues, and in maintenance of epithelial integrity, and they influence cell proliferation, differentiation, migration, and survival. In the lung, BMs are associated with bronchial and vascular smooth muscle cells, bronchial epithelium, nerve, and pleura, and they are part of the air–blood barrier between microvascular endothelial cells and alveolar epithelial cells. Collagen IV and laminin are the major components of all BMs which also contain entactin/nidogen and various heparan sulfate proteoglycans, including perlecan. Two collagen IV heterotrimeric protomers, (a1)2a2 and a3a4a5, are found in alveolar basement membranes, but the latter does not appear to have an important function in the lung. However, the a3 chain contains the epitope that is attacked in Goodpasture’s syndrome, an autoimmune disease characterized by pulmonary hemorrhage and glomerulonephritis. Laminins containing the a5 chain are widespread in both developing and adult lung. Preventing expression of laminin a5 via targeted mutagenesis interferes with lung lobe septation and maturation of the lung parenchyma.

158 EXTRACELLULAR MATRIX / Basement Membranes

BM ALV

BM IM

RBC

AT 1

FIB

AT 1

EC ALV BM IM

Figure 1 Schematic diagram of the air–blood interface. A basement membrane (BM) surrounds the capillary and separates the endothelial cell (EC) from the thin-walled alveolar epithelial cell. ALV, alveolus; AT 1, alveolar type I cell; RBC, red blood cell; FIB, fibroblast; IM, interstitial matrix.

major classes of proteins present in all BMs: type IV collagen, laminin, entactin/nidogen, and sulfated proteoglycans. Though all BMs contain these four components, in vertebrates there are different isoforms of each that are differentially distributed in BMs, leading to significant molecular heterogeneity among BMs. This presumably translates into functional heterogeneity. In this article, we focus on the isoforms of type IV collagen and laminin that are expressed in the lung and how they relate to lung function and disease.

Type IV collagen
-chain

7S

NC1

Trimerization

(a)

Protomers
2
1
1
4
3
5

Structure Type IV Collagen

Type IV collagen describes a family of six genetically distinct a-chains, a1–a6, which heterotrimerize with each other to form protomers (Figure 2). Protomers represent the building blocks of the collagen IV network. Mature collagen IV a-chains are B180 kDa and contain three major domains: a short N-terminal 7S domain, a long central collagenous domain, and a C-terminal noncollagenous 1 (NC1) domain of B230 amino acids. The collagenous domain is defined by the presence of stretches of Gly X–Y triplet repeats, which allows formation of stable, stiff triple helices. X and Y can be any amino acid but are most commonly proline. Collagen IV is unique compared to other collagens (there are 26 other types in humans) because its chains all contain multiple interruptions of the Gly X–Y repeats within the

Collagenous

(b)


6
5
5

Figure 2 Structure of type IV collagen. (a) Each of the six individual a-chains contains an N-terminal 7S domain, a central collagenous domain with multiple interruptions, and a C-terminal noncollagenous domain (NC1). (b) Three a-chains assemble in a defined fashion to form the three types of protomers. All three are found in the lung, though the (a5)2a6 protomer is restricted to bronchial smooth muscle BMs.

collagenous domain. These interruptions are presumed to impart flexibility to the trimer and thus to the BM of which it is a part. Laminin

Laminins constitute the major noncollagenous component of BMs. Laminins are heterotrimeric

EXTRACELLULAR MATRIX / Basement Membranes 159
1,2,3B,5 LN LE

L4 L4

LN

L4

L4

1,3

1,2


3A


3A,4

LN

1,3

1,2

2

3

Coiled-coil

LG1

LG5

LG1

LG5

LG2

LG2 LG3 LG4

LG3

LG4

LG1 LG2

LG3

LG Lm-1,2,3,4,6B,7B,10,11,12,15

Lm-6A,7A,8,9,14

Lm-5A

Figure 3 General structure of laminins. Laminin (Lm) heterotrimers (with Arabic numbers) exist in three shapes: cruciform, Y- or Tshaped, and rod-shaped. The globular domains of the short arms, laminin N-terminal (LN) and laminin four (L4), are separated by rodlike laminin EGF-like repeat (LE) domains. The a–b–g chains trimerize via the coiled-coil domains to form the long arm. The C-terminal LG domain is unique to a chains.

Table 1 Laminin heterotrimers and chain composition Laminin-1 Laminin-2 Laminin-3 Laminin-4 Laminin-5A Laminin-5B Laminin-6A/B Laminin-7A/B Laminin-8 Laminin-9 Laminin-10 Laminin-11 Laminin-12 Laminin-14 Laminin-15

a1b1g1 a2b2g2 a1b2g1 a2b2g1 a3Ab3g2 a3Bb3g a3A/Bb1g1 a3A/Bb2g1 a4b1g1 a4b2g1 a5b1g1 a5b2g1 a2b1g3 a1b2g3 a1b5g3

glycoproteins comprised of one a, one b, and one gchain (Figure 3). Fifteen laminin isoforms have been identified from combinations of five a-, four b-, and three g-chains (Table 1). The a-, b-, and g-chains share structural homologies, including the laminin N-terminal domains (LN), laminin epidermal growth factor (EGF)-like repeats domains (LE), laminin 4 domains (L4), and the a-helical coiled-coil long arms. Unique to a-chains are the large C-terminal laminin globular (LG) domains that consist of five smaller units (LG1–5). These a-chain LG domains are sometimes cleaved by proteases to make them smaller. For a subset of laminin chains (especially

laminin a3b3g2, or laminin-5), the N-terminal short arms are truncated and/or cleaved by proteases.

Regulation of Production and Assembly Type IV Collagen

The six collagen IV a-chain genes are arranged in head-to-head pairs on three different chromosomes, and each pair is thought to be regulated by a bidirectional regulatory element. Col4a1 and Col4a2 are the most widely expressed pair. The a1- and a2(IV)-chains are produced by the EHS tumor and are found in virtually all BMs throughout the body. Col4a3 and Col4a4 have a more restricted expression pattern, but the a3- and a4(IV)-chains are especially prominent in kidney glomerular and lung alveolar BMs. Col4a5 and Col4a6 have different but overlapping expression patterns. The a6(IV)-chain is found in smooth muscle and in some kidney BMs, always localizing with the a5(IV)-chain, but a5(IV) is present at additional sites wherever a3 and a4(IV) are present, including kidney glomerular and lung alveolar BMs. Biochemical studies have revealed the molecular basis for these findings. The six a-chains can trimerize in only three specific combinations to make three different protomers: (a1)2a2, a3a4a5, and (a5)2a6 (Figure 2). Specificity for trimerization is encoded by residues in the NC1 domains. The protomers

160 EXTRACELLULAR MATRIX / Basement Membranes

assemble in the endoplasmic reticulum and are then secreted into the extracellular matrix. A collagen IV network forms from these protomers via both NC1 domain interactions in which two protomers participate, and N-terminal 7S domain interactions in which four protomers participate. Covalent cross-linking of 7S domains and of lateral protomer associations provides increased stability to the network.

alv aw bv

alv

Laminin

Laminins form by intracellular self-trimerization of individual a-, b-, and g-chains (100–400 kDa) into a cruciform structure (400–800 kDa) with a common triple helical coiled-coil in the long arm that terminates in the large LG domain contributed by the a chain (Figure 3). Secreted laminins self-assemble into a fine network through interactions at the N-terminal globular domains, and this network then connects with the collagen IV network via entactin/nidogen and proteoglycans to stabilize the BM. Numerous cell types including epithelial, endothelial, and mesenchymal cells are capable of synthesizing laminins. In the developing lung, laminin a1 is expressed by both epithelial and mesenchymal cells and is deposited in the BM of developing airways. By the second trimester of gestation, laminin a1 can be weakly detected by antibody staining but not by in situ hybridization. Laminin a1 is absent from late embryonic and postnatal lung. Laminins a2–a5 are present in both embryonic and adult lung but differ in source and distribution. Laminin a2 is expressed by mesenchymal cells and deposited in the airway BM. Laminins a3A and a3B are expressed by epithelial cells and deposited in the epithelial BM of the airways and alveoli. Laminin a4 is expressed by mesenchymal cells and endothelium is detected in vascular BMs and in BMs surrounding smooth muscle cells of the large airways. Laminin a5 is expressed by epithelial and endothelial cells and is present in epithelial BM of the airways, alveoli, blood vessels, and pleura (Figure 4). It is of note that while epithelial BMs often contain multiple laminin a chains, and thus multiple laminin heterotrimers, the only laminin a-chain present in the visceral pleura is laminin a5. Laminins b3 and g2 are exclusively found in laminin-5 with laminin a3 and parallel laminin a3 in expression and distribution. Laminins b1, b2, and g1 exhibit a wide pattern of expression and distribution. Laminin chain expression and production are regulated by multiple factors, such as growth factors, transcription factors, cytokines, and hormones. In injury states, transforming growth factor beta

alv

Figure 4 Laminin a5 is widely deposited in lung BMs. A frozen section of adult mouse lung was stained with a rabbit antimouse laminin a5 antibody. Alveolar, bronchial epithelial, smooth muscle, and endothelial BMs contain laminin a5. alv, alveolus; bv, blood vessel; aw, airway. Original magnification
200.

(TGF-b) is a potent stimulator of extracellular matrix production, but its effect on laminin synthesis varies according to cell type and laminin chain examined. In general, epithelial and endothelial cells increase laminin production in response to TGF-b, while no response or a decrease in laminin production is seen in fibroblasts. In cell culture or in differentiating or diseased animal tissues, interleukin-1b, glucose, interferon a and g, and glucocorticoids have all been shown to increase laminin transcription.

Biological Function Collagen IV

For many years the collagen IV network was thought to serve as a required scaffold for BM assembly. However, targeted mutation of the linked mouse Col4a1 and Col4a2 genes shows that BMs can form in the total absence of collagen IV, though they exhibit impaired integrity, and homozygous mutant embryos die just past mid-gestation. This suggests that the main function of a1 and a2(IV) is to maintain the stability of BMs. This may also be true for the a3-, a4-, and a5(IV)-chains. In the absence of any one of them due to mutation of the encoding gene, the a3a4a5 protomer cannot form, so all three chains are missing from sites where they normally colocalize. The physiological result of this is a disease called Alport syndrome, defined as hereditary glomerulonephritis associated with sensorineural hearing loss and lens abnormalities. The major histopathological finding is thickening and splitting of the glomerular BM, suggesting that its integrity is

EXTRACELLULAR MATRIX / Basement Membranes 161

impaired. However, despite the abundance of the a3a4a5 protomer in the normal lung, there is no obvious lung phenotype associated with Alport syndrome in humans or in animal models for the disease. Laminin

Numerous studies have shown that laminins are important for maintenance of alveolar epithelial cell (AEC) phenotype, promotion of adult AEC proliferation, and protection of AECs from apoptosis. Laminins affect lung development at multiple stages and in multiple compartments. With regard to the lung, laminin-1 (a1b1g1) is the most studied laminin, but it is present only during early stages of lung development. Nonetheless, in vitro studies with lung bud cultures show that addition of laminin-1 antibodies or proteolytic fragments disturbs lung bud branching morphogenesis. On the other hand, laminin a2 is important for bronchial smooth muscle cell differentiation in vitro, and ablation of the entactin/nidogen binding site in laminin g1 causes impaired sacculation. Targeted mutagenesis in mice has been used extensively to study the biological functions of laminins. Lama1  /  , Lamb1  /  , and Lamc1  /  embryos die very early in development long before lung formation. Mutation of the Lama2, Lama4, or Lamb2 genes in mice does not cause embryonic or perinatal death and does not result in any described lung phenotype. Lama3  /  , Lamb3  /  , and Lamg2  /  mice die 1–3 days after birth from nonrespiratory causes, but their lungs have not been closely examined. Most Lama5  /  mice die between embryonic day 14 and 17, after completion of the pseudoglandular stage of lung development but before saccular and alveolar stages. Examination of Lama5  /  mice through E16.5 revealed normal branching morphogenesis but incomplete to absent lobar septation, lack of visceral pleura BM formation, and ectopic deposition of laminin a4 in airway BMs. Production of a lung-epithelial cell-specific laminin a5 knockout mouse shows a role for laminin a5 in the latter stages of lung development. These mice exhibit abnormal sacculation/alveolization with enlarged, emphysematous-appearing distal airspaces, and they died within hours after birth from respiratory compromise. Distal epithelial cell differentiation and maturation were severely impaired, with markedly reduced alveolar type II cells and a near absence of alveolar type I cells. In addition, cell proliferation was decreased, and apoptosis was increased. The density of the capillary network was diminished, and this was associated with decreased production of vascular endothelial growth factor by lung epithelial

cells. Thus, laminin a5 has a crucial role in maturation of the lung parenchyma.

Receptors The primary receptors that bind collagen IV and laminin are the integrins. Integrins are a family of ab heterodimeric transmembrane glycoproteins that serve as adhesion receptors, signaling receptors, components of mechanotransducers, and modulators of cell proliferation and apoptosis. Pulmonary epithelial cells express many integrins, but those that interact with laminin are integrins a3b1 and a6b4. a1b1, a2b1, and a3b1 have been reported to interact with type IV collagen. The importance of integrins to lung development is demonstrated in mice lacking integrin a3, which exhibit reduced airway branching and proximalization of distal epithelium. Additional, nonintegrin receptors for laminin include dystroglycan, cell surface heparan sulfate proteoglycans (syndecans), and the Lutheran blood group glycoprotein.

Collagen IV in Respiratory Disease There are no apparent lung abnormalities typically associated with Alport syndrome, despite the absence of the a3a4a5 protomer. This is probably due to compensation by the a1/a2(IV) network, which is normally present throughout the lung. However, there is a rare X-linked form of Alport syndrome that is associated with diffuse leiomyomatosis (DL). This usually affects esophageal smooth muscle, but benign tumors of bronchial smooth muscle have also been reported. Alport syndrome with DL is always accompanied by deletions that encompass the 50 end of COL4A5 and extend into the COL4A6 gene no farther than the second intron. Interestingly, deletions that extend farther into COL4A6 do not result in DL, suggesting it is not merely the absence of a6 in smooth muscle that causes the tumors. The autoimmune disorder Goodpasture syndrome severely affects both the kidney glomerulus and the lung parenchyma. In Goodpasture syndrome, antibodies to a cryptic epitope in the NC1 domain of collagen a3(IV) bind both to the kidney glomerular BM and to the lung alveolar BM and cause glomerulonephritis and pulmonary hemorrhage, respectively. The mechanism of exposure of the epitope is unknown, but there has been speculation that tobacco smoke, hydrocarbons, or endogenous oxidants may be involved.

Laminin in Respiratory Disease While laminins are both numerous and prominent in the lung, as of yet, no clinical human pulmonary

162 EXTRACELLULAR MATRIX / Elastin and Microfibrils

disease has been definitively associated with laminins. Lung injuries, whether from infection, acute respiratory distress syndrome, toxins, or non-specific causes, can manifest abnormalities and discontinuities of the BM, as well as elevated laminin fragments and laminin concentration in bronchoalveolar lavage fluid. However, no causative relationship has yet been discovered. See also: Adhesion, Cell–Matrix: Focal Contacts and Signaling; Integrins. Angiogenesis, Angiogenic Growth Factors and Development Factors. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Lung Development: Overview. Matrix Metalloproteinases. Mesothelial Cells. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Transgenic Models. Tumors, Benign. Vascular Endothelial Growth Factor.

Further Reading Aberdam D, Virolle T, and Simon-Assmann P (2000) Transcriptional regulation of laminin gene expression. Microscopy Research and Technique 51: 228–237. Colognato H and Yurchenco PD (2000) Form and function: the laminin family of heterotrimers. Developmental Dynamics 218: 213–234. Crouch EC, Martin GR, Brody JH, and Laurie GW (1997) Basement membranes. In: Crystal RG and West JB (eds.) The Lung: Scientific Foundations, pp. 769–791. Philadelphia: LippincottRaven. Hudson BG (2004) The molecular basis of Goodpasture and Alport syndromes: beacons for the discovery of the collagen IV family. Journal of the American Society of Nephrology 15: 2514–2527. Hudson BG, Tryggvason K, Sundaramoorthy M, and Neilson EG (2003) Alport’s syndrome, Goodpasture’s syndrome, and type IV collagen. New England Journal of Medicine 348: 2543– 2556. Miner JH and Yurchenco PD (2004) Laminin functions in tissue morphogenesis. Annual Review of Cell and Developmental Biology 20: 255–284. Nguyen NM, Kelley DG, Schlueter JA, et al. (2005) Epithelial laminin a5 is necessary for distal epithelial cell maturation VEGF production and alveolization in the developing murine lung. Developmental Biology 282: 111–125. Nguyen NM, Miner JH, Pierce RA, and Senior RM (2002) Laminin a5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung. Developmental Biology 246: 231–244. Sheppard D (2002) Functions of pulmonary epithelial integrins: from development to disease. Physiological Reviews 83: 673–686. Yurchenco PD, Amenta PS, and Patton BL (2004) Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biology 22: 521–538.

Elastin and Microfibrils E C Davis and B Quiney, McGill University, Montreal, QC, Canada & 2006 Elsevier Ltd. All rights reserved.

Abstract Elastin is an extracellular matrix protein that is critical for lung development and function. Elastin comprises B2.5% of the dry weight of the lung and is distributed as elastic fibers throughout virtually all pulmonary structures. Closer investigation of elastic fibers reveals that they actually contain two components, a core of insoluble elastin and a peripheral mantle of 10-nm microfibrils. The microfibrils are composed of a number of proteins; however, most of these proteins are only associated proteins and not true constituents. The main constituent protein of microfibrils is fibrillin. As the name implies, elastin provides the lung with the property of elastic stretch and recoil. Since this property is clearly essential for lung function, abnormal assembly or destruction of lung elastic fibers can lead to devastating pulmonary disease.

As early as 1891, the distribution and organization of elastic fibers in various tissues was demonstrated by light microscopy using characteristic staining reactions. Several of these histological stains, such as Weigert’s resorcin-fuchsin (from 1898) and Verhoeff’s iron hematoxylin (from 1908), are still routinely used today. By the 1960s, the morphological appearance of elastin in different tissues was being documented in detail: interwoven rope-like fibers in elastic ligaments, three-dimensional networks in the lung, concentric sheets or lamellae in the great vessels, and an anastomosing, honeycomb pattern in elastic cartilage. At approximately the same time, the presence of small filaments at the surface of developing elastic fibers was also noted by electron microscopists. These fine extracellular filaments became so routinely observed in a variety of tissues that they were assigned a specific name, collectively termed ‘microfibrils’ (Figure 1). The microfibrils were established as distinct components of elastic fibers based on characteristic staining properties that were different from those of elastin and on their separation from elastic fibers and partial characterization. Even at this early date, it was evident that the microfibrils appeared first in the extracellular matrix followed by the deposition of elastin, suggesting that they played some role in elastogenesis. In 1986, the major core constituent of microfibrils was identified and named ‘fibrillin’. Since then, considerable research has been conducted on the molecular assembly of fibrillin into microfibrils, the role of microfibrils in elastic fiber assembly, additional functions of elastin and microfibrils apart from their structural role in elastogenic

EXTRACELLULAR MATRIX / Elastin and Microfibrils 163

Ep

Ep

EI

Coll

F (b)

SMC

mf

Ep

mf EI

SMC F

(a)

(c)

Coll

Figure 1 Electron micrographs of elastic fibers in the lung. (a) Elastic fibers (black spots) form an extensive network interposed between the airway epithelium (Ep) and the subjacent smooth muscle cell layer (SMC). Fibroblasts (F) in this region are responsible for much of the elastic synthesis. Higher magnification of the elastic fibers in longitudinal (b) and cross-section (c) shows the close association of elastin (El) with the filamentous microfibrils (mf). An abundance of collagen (Coll) is also seen in this area.

tissues, and genetic diseases linked to the elastin and fibrillin genes.

Structure Tropoelastin is the monomeric form of elastin. The human tropoelastin gene (ELN) is a single copy on chromosome 7 (band q11.2). The gene contains 34 exons interspersed between large introns, giving a primary transcript of approximately 40 kb (Figure 2). The primary transcript undergoes considerable alternative splicing, generating multiple heterogeneous tropoelastin isoforms. The production of these different isoforms is spatially and temporally regulated, suggesting that the ratio of tropoelastin isoforms may be important for the structural function of the mature elastin polymer. In humans, the most frequently observed isoform lacks exon 26A, which has been reported to be expressed only in certain disease states. The tropoelastin protein ranges from 62 to 75 kDa, depending on the isoform, and is characterized by a distinct, periodic organization of hydrophobic and cross-linking domains. The protein is particularly rich in glycine, valine, alanine, and proline, with more than 75% of the amino acids being nonpolar or

uncharged. The only two cysteine residues are located in the C terminal, where they form an intramolecular disulfide bond. Once outside the cell, individual tropoelastin monomers are cross-linked to form an extremely durable, insoluble polymer. Three fibrillin genes exist in humans with distinct but overlapping expression patterns. Of these, fibrillin-1 is the major structural component of microfibrils. The human fibrillin-1 gene (FBN1), consisting of 65 exons that extend over 200 kb, is located on chromosome 15 and encodes a protein of B350 kDa. The protein is composed of multiple domains, the most abundant being tandem repeats of calcium-binding epidermal growth factor (EGF)-like modules (Figure 2). Within each of these modules are six cysteine residues that form three intradomain disulfide bonds, giving the protein a rigid, rod-shaped structure. At the cell surface, fibrillin monomers are thought to align in a head-to-tail fashion, with a lateral packing of eight monomers to form the microfibril.

Regulation of Production Elastin is synthesized and secreted by several different cell types, depending on anatomic location within

164 EXTRACELLULAR MATRIX / Elastin and Microfibrils Tropoelastin 2 4

6

Exon 36 8

10 12

15

18

20

22

24

26

∗ ∗ ∗

28 30 32 36 ∗

∗ ∗∗

G

K +

L

S 0H

C

Signal peptide

Hydrophobic exons

exon 26A

A

∗ Alternatively spliced exons

Cross-linking exons

exon 36

G

S-S

C G

+ R

+ K

+ R

+ K

G F

P

(b)

(a)

Fibrillin-1

RGD

N-terminus

8-cysteine/TGF-
binding domain

EGF-like domain

C-terminus

Hybrid domain

Calcium-binding EGF-like domain

Proline rich region

Transglutaminase cross-link

N-linked sugar

(c) Figure 2 Schematic representation of the exon organization of tropoelastin, the amino acid sequence of exon 36 of tropoelastin, and the domain structure of fibrillin-1. (a) Tropoelastin contains alternating hydrophobic and cross-linking domains. Multiple exons are subjected to alternative splicing, with exon 26A being most commonly spliced out. (b) The C-terminal exon of tropoelastin contains the only two cysteine residues. These form an intramolecular disulfide bond creating a hairpin loop and a positively charged pocket. This region is thought to be important for elastic fiber assembly. (c) Fibrillin-1 has 47 calcium-binding EGF-like domains, interspersed with TGF-b binding modules. The protein has many N-glycosylation sites and an integrin-binding RGD motif in the center of the molecule.

the lung. These cells include chondroblasts, myofibroblasts, interstitial fibroblasts, and mesothelial, endothelial, and smooth muscle cells. Apart from cleavage of the signal sequence as the completed polypeptide enters the endoplasmic reticulum, the tropoelastin monomer remains relatively unchanged as it traverses the secretory pathway en route to the cell surface, with no glycosylation or proteolytic processing. Once secreted, individual monomers are cross-linked between lysine residues on adjacent monomers, forming desmosine cross-links that are unique to elastin. The enzyme responsible for crosslink formation is lysyl oxidase. In contrast to tropoelastin, the fibrillin monomer is N-glycosylated and undergoes C-terminal proteolytic processing by a furin/PACE-type protease. Mutations in FBN1 that alter these posttranslational modifications can result in intracellular retention of the fibrillin monomer or defective microfibril assembly. Once secreted, pericellular proteoglycans such as heparan sulfate proteoglycan and chondroitin sulfate proteoglycan may

aid in the assembly of the fibrillin monomers into microfibrils. Based on biomechanical approaches, it has been suggested that transglutaminase cross-links play a major role in strengthening the microfibrillar network and, in terms of microfibril assembly, may facilitate correct alignment of the fibrillin monomers at fixed positions during initial formation. As in other elastogenic tissues, elastin in the lung is primarily produced during late fetal, neonatal, and postnatal development. Elastin is first expressed in smooth muscle cells of the developing intralobar pulmonary arteries near airway branch points during the pseudoglandular stage. Expression increases during the canalicular and saccular stages of development as elastic fibers are assembled between the smooth muscle cells and epithelium of developing airways. Elastin expression reaches a peak during alveolarization in the neonate, with high levels seen in the alveolar walls, particularly at bends, and in the alveolar septal tips. At the end of development, prominent elastic fibers can be seen at these sites as

EXTRACELLULAR MATRIX / Elastin and Microfibrils 165

Airway Alveolar tips

(a)

Vessels

Pleura

Alveolar walls

(b)

(c)

Figure 3 Light microscope images showing the appearance of elastic fibers in various regions of the lung using Hart’s stain for elastin. (a) Elastic fibers form a linear, filamentous network underlying the airway epithelium. Concentric layers of elastin are also seen surrounding the pulmonary vessels. (b) A continuous sheet of elastin is found subjacent to the mesothelial cells of the visceral pleura. (c) Elastic fibers are present in the alveolar walls and septal tips. At the septal tips, the elastin forms a ring around the mouth of each alveolus.

well as in longitudinal bands subjacent to the airway epithelium, as concentric lamellae in the arteries and arterioles, and as a continuous sheet under the mesothelium of the visceral pleura (Figure 3). At different stages of lung development, therefore, different cells are responsible for the production and assembly of elastin into the distinct architectural forms necessitated by function. During development, elastin expression is controlled by pretranslational regulation of the amount of the elastin mRNA transcript available. One mechanism for upregulating elastin expression as the lung develops is mechanical stress. Fetal and neonatal breathing movements are important for lung development and coincide with the onset of elastin synthesis. Furthermore, in vitro studies have shown that intermittent stretching of organotypic cultures of mixed fetal rat lung cells can increase elastin gene expression. Specific soluble factors can also influence the level of elastin expression. Stimulating factors include insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-b1, glucocorticoids, and retinoids, and repressing factors include EGF, basic fibroblast growth factor, phorbol esters, tumor necrosis factor alpha, interleukin-1b, and vitamin D. Following development, elastin expression ceases.

Since the protein is incredibly long-lived, with a halflife that exceeds the life span of the organism, little or no elastin synthesis is needed in the healthy adult. Elastin expression can be reactivated following lung injury, such as in fibrosis or pulmonary hypertension. The elastin synthesized during repair, however, is often disorganized with tortuous, clumped, and frayed fibers. The cessation of lung elastin production after development is controlled strictly by a posttranscriptional mechanism. Although tropoelastin premessenger RNA is transcribed at the same rate in neonatal and adult cells, the fully processed transcript does not accumulate in adult lung. In the cytosol of adult cells, a trans-acting protein has been reported to bind to exon 30 and destabilize the elastin transcript. TGF-b1, which is known to stabilize tropoelastin mRNA, acts by interfering with the exon 30–binding protein interaction. Although cross-linking experiments have suggested that the binding protein is approximately 52 kDa, the identity of this protein remains to be determined.

Biological Function Elastin is the key structural protein of the lung that allows for repeated deformability: stretch during the

166 EXTRACELLULAR MATRIX / Elastin and Microfibrils

active process of inspiration and elastic recoil during the passive process of quiet expiration. The property of distensibility is important for the function of not only the terminal airspaces but also the conducting airways, pleura, and pulmonary vasculature. The function of the microfibrils appears to be as a scaffold for the deposition, orientation, and assembly of the tropoelastin monomers. This notion is supported by observations that elastin is always seen in the presence of microfibrils, microfibrils appear prior to elastin in the extracellular matrix during development, and tropoelastin can bind to fibrillin-1 with high affinity. The appearance of elastin in the airspaces coincident with the formation of the septal walls led early investigators to hypothesize not only that elastin may play a mechanical role in the lung but also that elastin could be the driving force behind alveolar development. Support for this hypothesis came from the development of transgenic mice in which elastic fiber assembly was disrupted. In mice deficient in platelet-derived growth factor-A, myofibroblasts fail to form in the alveolar tips and elastin is not produced. Until postnatal day 4, lung development appears normal. As development continues, however, the distal airspaces become distended and thin walled, and alveolar sacs fail to form. Progressive disruption of normal development continues and the mice die by 6 weeks of age. Not only is the physical presence of elastin needed for alveolar development but it must be properly assembled too. Evidence to support this concept has come from studies of fibulin-5. Fibulin-5 is an elastic

(a)

fiber-associated protein essential for normal elastic fiber assembly. In fibulin-5 null mice, expanded distal airspaces are observed by 2 weeks of age. Elastic fibers are present in the alveolar walls and tips; however, they are abnormal in distribution and appearance. These results indicate that elastin must be both produced and correctly assembled by the myofibroblasts in the alveolae for the normal development of these structures. Ultimately, the earliest role of elastin in lung development was revealed by investigating lungs of mice deficient in elastin. In the elastin-null mice, distal airways failed to branch, possibly through a signaling mechanism from the elastin to the mesenchymal cells surrounding the developing airways (Figure 4). Elastin-null mice die by postnatal day 3 due to overproliferation of smooth muscle cells in the aortic wall, precluding any further studies of postnatal lung development in these animals. Similar to elastin, studies of transgenic mice have revealed an additional function for fibrillin apart from merely aiding in elastic fiber assembly. Mice homozygous for a centrally deleted FBN1 allele die by postnatal day 10 due to aortic rupture. Immediately after birth, however, these mice showed a significant increase in distal airspace caliber, with only the rare formation of a secondary alveolar septum. This phenotype was independent of elastic fiber assembly since smooth muscle cell differentiation and distribution appeared normal in the mutant lung and elastin was localized to the alveolar walls and septal tips. Additional research showed that fibrillin can bind to and regulate the activity of TGF-b1. In

(b)

Figure 4 Lung development is severely disrupted in the absence of elastin. Comparison of lungs from postnatal day 1.5 mouse pups from wild-type (a) and elastin-null (b) mice shows a dramatic difference in lung structure. In the absence of elastin, terminal airway branching is greatly impaired. The defect is even evident in the intact lung (insets show whole heart/lung blocks). Note the expanded airspaces in the peripheral lung tissue of the elastin-null mouse compared to the wild-type animal (arrows).

EXTRACELLULAR MATRIX / Elastin and Microfibrils 167

mice harboring the mutant fibrillin-1 gene, therefore, dysregulation of TGF-b1 activation and signaling leading to an increase in cell death was found to be the cause of the lung phenotype. A similar lung defect was found in the tight-skin mouse, a model for emphysema, in which a genomic duplication in FBN1 generates a larger than normal fibrillin protein, leading to abnormal microfibrils with altered molecular organization. It is thought that in the neonatal tight-skin mouse lung, the inability of normal growth factor signaling, together with defective elastic fiber assembly, contributes to the cessation of alveolarization seen in these mice.

Receptors Both tropoelastin and fibrillin have been reported to interact with cell surface proteins. Until recently, the binding of tropoelastin to cells has been solely attributed to the elastin/laminin receptor complex. This complex includes the 67 kDa elastin-binding protein (EBP), an alternatively spliced enzymatically inactive form of b-galactosidase. EBP recognizes a hexapeptide repeat (VGVAPG) in the central region of tropoelastin and has been implicated in protecting tropoelastin from degradation and aiding in the secretion and assembly of the monomer into the elastic fiber. Purified EBP has yet to be examined in vitro for its ability to bind tropoelastin. Interestingly, mice lacking the b-galactosidase gene have no reported vascular defects. A direct interaction of tropoelastin via the cell surface avb3 integrin was reported despite the fact that tropoelastin does not contain an RGD–integrin binding sequence. A single specific binding site was localized to the C-terminal 16 residues of tropoelastin, a highly charged region encompassed by exon 36 (Figure 2). In contrast to tropoelastin, fibrillin-1 contains an RGD sequence located in the fourth eight-cysteine motif. This RGD sequence is responsible for the binding of fibrillin-1 to both the avb3 and a5b1 integrins and has been suggested to provide an important link between elastic fibers and the cell surface. Such a function is supported by the appearance of abnormal cell–elastic fiber connections in mice with mutations in the fibrillin-1 gene. Incomplete inhibition of binding by competing RGD peptides or antibodies against the integrin proteins suggests that a second non-RGD-mediated receptor for the fibrillin molecule exists in certain cell types.

Elastin and Microfibrils in Respiratory Disease Elastic fiber abnormalities leading to respiratory disease can result from either destruction or

overproduction of elastin in the adult or by aberrant initial assembly. Destruction of elastin in the lung is a key factor in the pathogenesis of emphysema. For the most part, this occurs due to an imbalance between elastases and anti-elastases. Enhanced elastase activity can occur by the recruitment of elastase-secreting inflammatory cells into the lung, often as a result of air pollution or cigarette smoking, or by deficiency in the elastase inhibitor, a1-antitrypsin. a1-Antitrypsin deficiency is a rare hereditary disorder, but it is likely widely under- and misdiagnosed. An overproduction of elastin can also adversely affect lung function. Inhalation of mineral dust, which often occurs as an occupational hazard, can reinitiate tropoelastin gene expression leading to an aberrant accumulation of mature elastin and, ultimately, granulomatous lung diseases or pulmonary fibrosis. Abnormal assembly of elastic fiber during development can also occur in premature infants subject to mechanical ventilation and oxygen supplementation due to respiratory complications at birth. In this respiratory disorder, termed bronchopulmonary dysplasia, patients have decreased alveolar number, impaired lung development, and irregular elastic fiber structure. Altered lung structure and function can also occur as a consequence of mutations in the elastin or fibrillin genes. In humans, heterozygous null mutations of ELN cause supravalvular aortic stenosis (SVAS), a generalized segmental stenotic arterial disease characterized by overproliferation of vascular smooth muscle cells. No lung phenotype has been reported in these patients. In contrast, a distinct class of ELN mutations cause autosomal dominant cutis laxa, with the main manifestation being lax, redundant, and inelastic skin. In these patients, a mutant tropoelastin protein is synthesized that appears to act through a dominant negative mechanism to disrupt normal elastic fiber assembly and function. Unlike SVAS, some cutis laxa patients present with pulmonary emphysema, suggesting that the quality of the elastic fiber is more important for lung development than the quantity. Mutations in the fibrillin-1 gene cause Marfan syndrome, which is an autosomal dominant disorder characterized by tall stature and other skeletal defects, aortic dilatation, mitral valve prolapse, and ectopic lentis. Some individuals with Marfan syndrome present with pneumothorax secondary to rupture of emphysematous bullae in the lung. Approximately 10–15% of patients have emphysema, although this is likely underdiagnosed. Similar to that described previously for mouse models with defective fibrillin-1 production, the lung phenotype in Marfan patients likely results from reduced targeting and sequestration of latent TGF-b1

168 EXTRACELLULAR MATRIX / Collagens

in the lung tissue, leading to increased TGF-b1 activation, inhibition of alveolar development, and eventual emphysema.

Collagens

See also: Acute Respiratory Distress Syndrome. Alveolar Wall Micromechanics. Breathing: Breathing in the Newborn. Bronchopulmonary Dysplasia. Chest Wall Abnormalities. Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General. Environmental Pollutants: Particulate Matter, Ultrafine Particles; Particulate and Dust Pollution, Inorganic and Organic Compounds. Extracellular Matrix: Degradation by Proteases. Fibroblasts. Genetics: Overview. Lung Anatomy (Including the Aging Lung). Lung Development: Overview. Myofibroblasts. Occupational Diseases: Overview; Asbestos-Related Lung Disease; Silicosis. Pectus Excavatum. Platelet-Derived Growth Factor. Pulmonary Vascular Remodeling. Smooth Muscle Cells: Airway; Vascular. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Transgenic Models.

& 2006 Elsevier Ltd. All rights reserved.

S E Dunsmore, Brigham and Women’s Hospital at Havard Medical School, Boston, MA, USA

Abstract Collagens, the most abundant proteins in the animal kingdom, are best known for providing mechanical strength to skin, bone, and other tissues. In the lung, collagens limit expansion of the parenchyma and prevent airway collapse. Structurally, collagens are characterized by a triple helical region containing a Gly-x-y motif. Collagens are found in the extracellular matrix where they are assembled into fibrils and other supramolecular structures. Normal growth and development are dependent on the proper assembly of collagens. Collagens interact with three types of receptors: integrins, discoidins, and glycoprotein VI. Respiratory disease can result from inappropriate synthesis, assembly, or deposition of collagen. Current research indicates that collagen fragments may be important endogenous inhibitors of tumorigenesis and mediators of lung transplant rejection.

Introduction Further Reading American Thoracic Society Ad Hoc Statement Committee (2004) Mechanisms and limits of induced postnatal lung growth. American Journal of Respiratory and Critical Care Medicine 170: 319–343. Dietz HC and Mecham RP (2000) Mouse models of genetic diseases resulting from mutations in elastic fiber proteins. Matrix Biology 19: 481–488. Foster JA and Curtiss SW (1990) The regulation of lung elastin synthesis. American Journal of Physiology 259: L13–L23. Gardi C, Martorana PA, de Santi MM, van Even P, and Lungarella G (1989) A biochemical and morphological investigation of the early development of genetic emphysema in tight-skin mice. Experimental and Molecular Pathology 50: 398–410. Kielty CM, Sherratt MJ, Marson A, and Baldock C (2005) Fibrillin microfibrils. Advances in Protein Chemistry 70: 405–436. Kielty CM, Sherratt MJ, and Shuttleworth CA (2002) Elastic fibres. Journal of Cell Science 115: 2817–2828. Lindahl P, Karlsson L, Hellstrom M, et al. (1997) Alveogenesis failure in PDGF-A-deficient mice is coupled to lack of distal spreading of alveolar smooth muscle cell progenitors during lung development. Development 124: 3943–3953. Mariani TJ, Sandefur S, and Pierce RA (1997) Elastin in lung development. Experimental Lung Research 23: 131–145. Milewicz DM, Urban Z, and Boyd C (2000) Genetic disorders of the elastic fiber system. Matrix Biology 19: 471–480. Mithieux SM and Weiss AS (2005) Elastin. Advances in Protein Chemistry 70: 437–461. Neptune ER, Frischmeyer PA, Arking DE, et al. (2003) Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome. Nature Genetics 33: 407–411. Starcher BC (2000) Lung elastin and matrix. Chest 117: 229S–234S. Wendel DP, Taylor DG, Albertine KH, Keating MT, and Li DY (2000) Impaired distal airway development in mice lacking elastin. American Journal of Respiratory Cell and Molecular Biology 23: 320–326.

Collagen came into use as a word in the English language around 1865. It is derived from the French term collage`ne, which stems from the Greek word for glue, Kolla. Early definitions of collagen centered on its role as a constituent of connective tissue which yielded glue or gelatin upon boiling and indeed, chemical means of solubilizing collagen were examined in some of the first reports of experimentation with the molecule. By the early 1940s, the techniques of electron microscopy and X-ray diffraction were being used to define the structure of collagen fibrils. The triple-chain helical nature of the collagen molecule became evident in 1955, but it was not until 1969 that the existence of more than one type of collagen was recognized. Knowledge of the existence and structure of different collagen types was advanced by the application of recombinant DNA technology which was first used in 1978 to isolate the gene for the proa2(I) chain of chicken type I procollagen. Analysis of the human genome indicates that there may be more than 40 collagen genes encoding the subunits of at least 27 different collagen types. Collagens are traditionally divided into two classes, fibrillar (Table 1) and nonfibrillar. Fibrillar collagens (types I, II, III, V, XI, XXIV, and XXVII) are the major structural components of connective tissues, cartilage, and bone. Types I and III collagens are the most abundant proteins in the lung accounting for about 15–20% of the dry weight of the tissue. Nonfibrillar collagens may be further subdivided into basement membrane (types IV and VII) (Table 2), short-chain (types VI, VIII, and X) (Table 3), FACIT

EXTRACELLULAR MATRIX / Collagens 169 Table 1 Fibrillar collagens Collagen type

Gene symbol

Chain composition

Supramolecular structure

Tissue localization

Lung distribution

I

COL1A1 COL1A2

[a1(I)]2a2(I) [a1(I)]3

67 nm banded fibrils

Bone, tendon, dermis, cornea

Interstitium, vasculature, pleura, trachea, bronchi

II

COL2A1

[a1(II)]3

67 nm banded fibrils

Cartilage, vitreous body of eye

Bronchial and tracheal cartilage

III

COL3A1

[a1(III)]3

67 nm banded fibrils

Dermis, aorta, uterus, intestine

Interstitium, vasculature, pleura, trachea, bronchi

V

COL5A1 COL5A2 COL5A3

[a1(V)]2a2(V) [a1(V)a2(V)a3(V)] [a1(V)]3

9 nm banded fibrils

Placenta, bone, dermis, cornea

Interstitium, vasculature, basement membranes

XI

COL11A1 COL11A2 COL2A1

[a1(XI)a2(XI)a3(XI)]

Fine fibrils (similar to type V collagen fibrils)

Cartilage, intervertebral disk

Bronchial and tracheal cartilage

XXIV

COL24A1

[a1(XXIV)]3

Presumed to form homotrimeric fibrils

Cornea, bone

May not be present

XXVII

COL27A1

[a1(XXVII)]3

Presumed to form homotrimeric fibrils

Cartilage, eye, ear, lung, colon

Expressed by chondrocytes and epithelial cells

Table 2 Basement membrane collagens Collagen type

Gene symbol

Chain composition

Supramolecular structure

Tissue localization

Lung distribution

IV

COL4A1 COL4A2 COL4A3 COL4A4 COL4A5 COL4A6

[a1(IV)]2a2(IV) [a1(IV)]2a3(IV) [a1(IV)]2a4(IV) [a1(IV)]2a5(IV) [a1(IV)]2a6(IV)

Nonfibrillar meshwork

Basement membranes

Underneath airway and alveolar epithelium

VII

COL7A1

[a1(VII)]3

Anchoring fibrils

Skin, amniotic membrane, mucosal epithelium

Underneath airway epithelial cells

Table 3 Short-chain collagens Collagen type

Gene symbol

Chain composition

Supramolecular structure

Tissue localization

Lung distribution

VI

COL6A1 COL6A2 COL6A3

[a1(VI)a2(VI)a3(VI)]

5–10 nm beaded microfibrils

Uterus, dermis, cornea, cartilage

Interstitium, vasculature

VIIIa

COL8A1 COL8A2

[a1(VIII)]2a2(VIII)

Nonfibrillar hexagonal lattice

Endothelial cells, Descemet’s membrane

Vasculature

Xa

COL10A1

[a1(X)]3

Nonfibrillar hexagonal lattice

Calcifying cartilage

Not present in adult lung

a

Many molecules such as collectins, complement related factors, metabolic regulators (ACRP30, Hib27, acetylcholinesterase), and elastic fiber-associated molecules (emilins) are structurally homologous to collagens VIII and X possessing similar triple helical and C-terminal noncollagenous (NC1) domains.

170 EXTRACELLULAR MATRIX / Collagens Table 4 FACIT (Fibril-associated Collagens with Interrupted Triple helices) collagens Collagen type

Gene symbol

Chain composition

Supramolecular structure

Tissue localization

Lung distribution

IX

COL9A1 COL9A2 COL9A3

[a1(IX)a2(IX)a3(IX)]

Associates with type II collagen fibrils

Cartilage, vitreous body of eye

Tracheal and bronchial cartilage

XII

COL12A1

[a1(XII)]3

Associates with type I collagen fibrils

Dermis, tendon, cartilage

Interstitium of fibrotic lungs

XIV

COL14A1

[a1(XIV)]3

Associates with type I and II collagen fibrils

Dermis, tendon, cartilage

Interstitium of fibrotic lungs

XVI

COL16A1

a1(XVI)

Associates with fibrillin-1 rich microfibrils and Dbanded type II collagen fibrils

Heart, kidney, smooth muscle

Minimal expression

XIX

COL19A1

a1(XIX) [a1(XIX)]2 [a1(XIX)]3 [a1(XIX)]4 [a1(XIX)]5 [a1(XIX)]6

N-terminal-interacting oligomers

Basement membrane of differentiating muscle cells

Minimal expression in adult lung

XX

COL20A1

a1(XX)

Presumed to bind to collagen fibrils

Corneal epithelium

May not be present

XXI

COL21A1

a1(XXI)

Unknown

Placenta, vasculature of heart, stomach, kidney, skeletal muscle

Minimal expression

XXII

COL22A1

a1(XXII)

Presumed to bind to basement membrane components

Myotendinous junction, articular cartilage–synovial fluid junction, hair follicles

Not expressed

(types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII) (Table 4), transmembrane (types XIII, XVII, XXIII, and XXV) (Table 5), and multiplexin (types XV and XVIII) collagens (Table 6). Type IV collagen is the most abundant nonfibrillar collagen in the lung constituting approximately 5% of parenchymal collagen. Type IV collagen is responsible for the tensile strength of the blood–gas barrier and plays a large part in preventing stress failure of the pulmonary capillaries under normal conditions.

proteins so that this amino acid is used as a specific index of collagen synthesis and concentration. The three polypeptide chains associate via the Gly-x-y sequences to form triple helices. All collagens contain at least one triple helical region. In this conformation, peptide bonds linking adjacent amino acids are buried within the interior of the molecule which renders the triple helical region highly resistant to proteolysis (Figure 1).

Regulation of Production and Activity Structure Collagens consist of three polypeptide chains containing the amino acid sequence (Gly-x-y). Although x and y may be any amino acid, proline and hydroxyproline predominate in these positions such that approximately every third x is proline and every third y is hydroxyproline. Hydroxyproline is not unique to collagen; however, concentrations present in collagen are much higher than for other

The pattern of collagen synthesis is tissue specific and is regulated at both the transcriptional and translational levels. Collagens are also subject to numerous posttranslational modifications. In most cases, biological activity is dependent upon the assembly of supramolecular structures (Figure 2). Fibril formation by types I, II, and III collagen is well characterized. Likewise, the macromolecular assembly of the open network structure of type IV collagen has been

EXTRACELLULAR MATRIX / Collagens 171 Table 5 Transmembrane collagensa Collagen type

Gene symbol

Chain composition

Supramolecular structure

Tissue localization

Lung distribution

XIII

COL13A1

[a1(XIII)]3

Homotrimeric transmembrane

Adhesive junctions in cartilage, bone, skeletal muscle, lung, intestine, skin

Sites of branching morphogenesis

XVII

COL17A1

[a1(XVII)]3

Homotrimeric transmembrane

Hemidesmosomes

Not expressed

XXIII

COL23A1

[a1(XXIII)]3

Homotrimeric transmembrane

Placenta, kidney, skin, neural cells

Not expressed

XXV

COL25A1

[a1(XXV)]3

Homotrimeric transmembrane

Alzheimer amyloid plaques, exclusively produced by neuronal cells

Not expressed

a

Several molecules with small triple helical domains: ectodysplasin A, the class A macrophage scavenger receptors, the MARCO receptor, and the colmedins (gliomedin, UNC-122, COF-2, CG6867) are classified as collagenous transmembrane proteins.

Table 6 Multiplexin collagens Collagen type

Gene symbol

Chain composition

Supramolecular structure

Tissue localization

Lung distribution

XV

COL15A1

a1(XV)

Forms bridge between basement membrane and collagen fibrils

Vasculature with the exception of alveolar capillaries

XVIII

COL18A1

a1(XVIII)

Unknown

Vascular, neuronal, mesenchymal basement membranes, most epithelial basement membranes Endothelial and epithelial basement membranes

extensively studied. Details of the molecular assembly of type VI collagen into beaded microfibrils, type VII collagen into anchoring fibrils, and types VIII and X collagen into nonfibrillar hexagonal lattices have been reported. Transmembrane collagens form homotrimers with the collagenous domain extracellularly oriented. FACIT collagens associate with interstitial collagen fibers. In the case of type IX collagen, this association occurs through covalent cross-linking of lysine/hydroxylysine residues in the N-terminal of the second collagenous domain of type IX collagen to sequences in the C-telopeptide region of the type II collagen fibril. Collagenases (matrix metalloproteinase (MMP)-1, MMP-8, and MMP-13) are the only proteases capable of degrading fibrillar collagen. These MMPs cleave at a site within the triple helical region of fibrillar collagen producing fragments that are threefourth and one-fourth the size of the original molecule. This single cleavage event destroys the triple helical structure of the collagen fibril and renders the molecule biologically inactive. In contrast, biologically active fragments may be produced from proteolytic degradation of the noncollagenous domains

Alveolar capillaries

(NC1) of types IV, XV, and XVIII collagen. Although the proteolytic events that generate these fragments are not entirely clear, the potent effects of endostatin on tumor growth and angiogenesis have been of great interest.

Biological Function Collagens are essential for normal growth and development. A variety of clinical disorders primarily manifested in the skeleton, skin, and joints are attributed to mutations in the various collagen genes. Collagens also play important roles in wound repair. Fibrous collagen activates platelets to initiate formation of a hemostatic plug. Cell migration following injury is directed by collagens which are important constituents of granulation tissue. Collagens can function as survival factors and generally prevent apoptosis of adherent cells. Recently, it has become evident that collagen fragments may have biological functions. Endostatin, a proteolytic fragment of collagen type XVIII, is a potent inhibitor of angiogenesis.

172 EXTRACELLULAR MATRIX / Collagens Glycine O

Proline

Hydroxyproline

O

O

H2N OH

OH

OH NH2

NH2 OH

(a)

(Gly–Pro–Hyp)2 O

O

H2N NH

OH

OH

O HN O

N H

O

OH

HN HN O (b) Figure 1 Constituents of the collagen triple helix. (a) The three most common amino acids found in the collagen triple helix: glycine (Gly), proline, and hydroxyproline. (b) Collagen chains associate via Gly-x-y sequences to form triple helices. In the triple helices, side chains of proline and hydroxyproline (shown in pink) are on the outside of the molecule, and peptide bonds between the amino acids (shown in blue) are on the inside of the helix. Hydrogen bonds (denoted by green dashed line) stabilize the triple helix and occur between the HN-group of glycine and the OQC group of the amino acid in position x.

Angiogenesis is also inhibited by restin, a collagen XV fragment, and by arrestin, canstatin, and tumstatin, type IV collagen fragments. These fragments have been identified in serum and in the extracellular matrix and may be important endogenous inhibitors of tumorigenesis. A type XXV collagen fragment, CLAC-P, has been linked to neuronal degeneration in Alzheimer disease. CLAC-P binds to and stabilizes aggregates of amyloid-b peptides.

Receptors Four integrin receptors, a1b1, a2b1, a10b1, and a11b1, interact with collagens. These integrins contain an I domain which recognizes the hexapeptide motif Gly–Phe–Hyp–Gly–Glu–Arg found in the triple helical region of collagens. Integrins a1b1 and a2b1 are widely expressed and differ in binding affinities for fibrillar and basement membrane collagens. Fibrillar collagens preferentially associate with integrin a2b1 while integrin a1b1 has a higher affinity for type IV collagen. Tissue distribution of integrins a10b1 and a11b1 is limited. Chondrocytes express a10b1, and its primary ligand is type II collagen. Integrin

a11b1 is predominantly found in muscle tissue where it preferentially interacts with type I collagen. Two kinases, focal adhesion kinase and integrin-linked kinase, interact with the cytoplasmic tail of the b1 subunit to modulate signaling pathways that regulate cell proliferation, migration, and survival. Collagens also bind to two discoidin domain receptors (DDRl and DDR2) which are expressed in a variety of tissues including the lung. Binding of collagen types I, II, III, IV, V, VI, and VIII to DDR1 has been documented. DDR2 appears to have a higher affinity for fibrillar collagens and in particular for collagen types I and III. DDRs are receptor tyrosine kinases, but their intracellular signaling partners are incompletely characterized. Both DDR1 and DDR2 null mice are smaller than wild-type littermates, however, suggesting that the interaction of collagen with DDRs is functionally important. Fibrous collagen binds to glycoprotein VI to induce platelet aggregation. Glycoprotein VI is found exclusively on platelets and signals through a well-known cascade involving Src kinases, phosphatidylinositol 3-kinase, phospholipase Cg2, and intracellular calcium to effect platelet activation.

EXTRACELLULAR MATRIX / Collagens 173

Fibril Types I, II, III, V, and XI collagen

FACIT Types IX, XII, and XIV collagen

Beaded microfibrils Type VI collagen

Open network Type IV collagen

Transmembrane Types XIII, XVII, XXIII, and XXV collagen

Anchoring fibrils Segment long spacing-like structures Type VII collagen

Hexagonal lattice Types VIII and X collagen

Figure 2 Collagen supramolecular structures. Lysyl oxidase-induced cross-links are shown in orange. FACIT cross-links are depicted in green.

The biologically active collagen fragments (endostatin, restin, arrestin, canstatin, tumstatin) have been shown to bind to a variety of receptors including integrins (a1b1, a2b1, a3b1, a5b1, avb5, avb1, and a6b1), glypican-3, and vascular endothelial growth factor receptor 2. It is not clear, however, which of these receptors mediates the in vivo effects of these collagen fragments.

Collagens in Respiratory Disease Pulmonary fibrosis, the end result of a variety of clinical entities, is the respiratory disease most often associated with collagens. The primary pathological

defect in this disease is the inappropriate synthesis and deposition of interstitial collagens in the lung parenchyma. Blood oxygenation is adversely affected by the loss of capillary beds and disruption of the blood–gas diffusion barrier. In the airways, increased subepithelial deposition of collagen is observed in patients with asthma, chronic obstructive pulmonary disease (COPD), or obliterative bronchiolitis. Although the functional significance of increased collagen deposition in these diseases remains unclear, airway fibrosis often correlates with disease severity. Collagens are associated with a group of immune disorders termed collagen vascular diseases. Systemic lupus erythematosus, rheumatoid arthritis, Sjo¨gren’s

174 EXTRACELLULAR MATRIX / Collagens

syndrome, systemic sclerosis, polymyositis, and dermatomyositis are examples of collagen vascular diseases with pulmonary complications. Pleuritis and pulmonary infections are common in systemic lupus erythematosus patients. Rheumatoid arthritis patients may also suffer from pleuritis, but interstitial lung disease or fibrosis is the predominant pulmonary manifestation. Interstitial lung disease is common in patients with Sjo¨gren’s syndrome or systemic sclerosis and may occur in patients with polymyositis and/or dermatomyositis. Although grouped together because of the common connective tissue or collagen manifestations, many of these diseases are also characterized by the presence of autoantibodies to nuclear proteins. Pulmonary hypertension can result from a variety of causes including genetic defects, hypoxia, and chronic thrombotic disease. In all cases, type I collagen is increased in the vasculature. In primary pulmonary hypertension, type I collagen is increased in the neointima of the vessels. When vascular remodeling is induced by hypoxia, however, increased collagen is observed in the medial and adventitial layers of the vessels. Osteogenesis imperfecta and Ehlers–Danlos syndrome are the most common manifestations of collagen gene mutations. Approximately 60% of osteogenesis imperfecta patients who have altered or deficient type I collagen present with severe chest wall deformities. Pulmonary compromise is the leading cause of death in these patients. Pulmonary complications are rare in Ehler–Danlos syndrome although cavitary lesions, pneumothorax, and pulmonary hemorrhage are occasionally observed in patients with the type IV subtype in which the gene encoding type III collagen is mutated. Pulmonary hemorrhage also occurs in patients with Goodpasture syndrome which is characterized by the production of autoantibodies to type IV collagen. Recent evidence suggests that type V collagen may be a major antigen involved in lung allograft rejection. In animal models, type V collagen-reactive T cells perpetuate the graft rejection response. Oral administration of type V collagen can induce tolerance to lung allografts and prevent the development of obliterative bronchiolitis. Although preliminary, these results may be very important in the future therapy of chronic respiratory disease as the efficacy of lung transplant as a treatment modality is presently limited by the high rate of graft rejection. See also: Acute Respiratory Distress Syndrome. ADAMs and ADAMTSs. Adhesion, Cell–Cell; Vascular; Epithelial. Adhesion, Cell–Matrix: Focal Contacts and Signaling; Integrins. Alveolar Hemorrhage.

Alveolar Wall Micromechanics. Apoptosis. Arteries and Veins. Autoantibodies. Bronchopulmonary Dysplasia. Chronic Obstructive Pulmonary Disease: Overview. Coagulation Cascade: Overview. Complement. Connective Tissue Growth Factor. Cytoskeletal Proteins. Drug-Induced Pulmonary Disease. Extracellular Matrix: Basement Membranes; Matrix Proteoglycans; Degradation by Proteases. Fibroblasts. Granulomatosis: Lymphomatoid Granulomatosis; Wegener’s Disease. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Matrix Metalloproteinases. Myofibroblasts. Neurofibromatosis. Occupational Diseases: Overview; Coal Workers’ Pneumoconiosis; Asbestos-Related Lung Disease; Silicosis. Permeability of the Blood–Gas Barrier. Platelets. Polymyositis and Dermatomyositis. Progressive Systemic Sclerosis. Pulmonary Alveolar Microlithiasis. Pulmonary Effects of Systemic Disease. Pulmonary Fibrosis. Pulmonary Vascular Remodeling. Radiation-Induced Pulmonary Disease. Signal Transduction. Systemic Disease: Sarcoidosis; Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Aouacheria A, Cluzel C, Lethias C, et al. (2004) Invertebrate data predict an early emergence of vertebrate fibrillar collagen clades and an anti-incest model. Journal of Biological Chemistry 279: 47711–47719. Boot-Handford RP and Tuckwell DS (2003) Fibrillar collagen: the key to vertebrate evolution? A tale of molecular incest. BioEssays 25: 142–151. Chu M-L and Prockop DJ (2002) Gene structure. In: Royce PM and Steinmann B (eds.) Connective Tissue and Its Heritable Disorders, pp. 223–248. New York: Wiley-Liss. Eble JA (2005) Collagen-binding integrins as pharmaceutical targets. Current Pharmaceutical Design 11: 867–880. Exposito JY, Cluzel C, Garrone R, and Lethias C (2002) Evolution of collagens. Anatomical Record 268: 302–316. Franzke CW, Bruckner P, and Bruckner-Tuderman L (2005) Collagenous transmembrane proteins: recent insights into biology and pathology. Journal of Biological Chemistry 280: 4005–4008. Gelse K, Poschl E, and Aigner T (2003) Collagens: structure, function, and biosynthesis. Advanced Drug Delivery Reviews 55: 1531–1546. Kielty CM and Grant ME (2002) The collagen family: structure, assembly, and organization in the extracellular matrix. In: Royce PM and Steinmann B (eds.) Connective Tissue and Its Heritable Disorders, pp. 159–221. New York: Wiley-Liss. Knupp C and Squire JM (2005) Molecular packing in networkforming collagens. Advances in Protein Chemistry 70: 375–403. Myllyharju J and Kivirikko KI (2004) Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends in Genetics 20: 33–43. Ortega N and Werb Z (2002) New functional roles for non-collagenous domains of basement membrane collagens. Journal of Cell Science 115: 4201–4214. Suki B, Ito S, Stamenovic D, Lutchen KR, and Ingenito EP (2005) Biomechanics of the lung parenchyma: critical roles of collagen and mechanical forces. Journal of Applied Physiology 98: 1892–1899.

EXTRACELLULAR MATRIX / Matricellular Proteins 175 Verrecchia F and Mauviel A (2004) TGF-beta and TNF-alpha: antagonistic cytokines controlling type I collagen gene expression. Cellular Signalling 16: 873–880. Vogel WF (2001) Collagen-receptor signaling in health and disease. European Journal of Dermatology 11: 506–514. White DJ, Puranen S, Johnson MS, and Heino J (2004) The collagen receptor subfamily of the integrins. International Journal of Biochemistry and Cell Biology 36: 1405–1410.

Matricellular Proteins P Bornstein, University of Washington, Seattle, WA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The term ‘matricellular’ was coined to identify a group of proteins that are secreted and function in the extracellular matrix, but do not play a structural role as an integral component of a physical entity such as a fiber or basement membrane. Instead, matricellular proteins primarily serve to modulate cell function. These functions are achieved in part by interaction with a variety of cell-surface receptors, with the resulting engagement of signal transduction pathways. Matricellular proteins are also capable of interacting with structural matrix proteins, such as collagens and fibronectin, and with a number of bioactive proteins, that is, cytokines, growth factors, and proteases. As a consequence, these bioactive molecules can either be sequestered in the matrix, presented to their appropriate receptors, or in the case of proteases, inhibited by direct binding or cleared from the pericellular environment by formation of protein– enzyme complexes that are recognized by a scavenger receptor. It should be noted, however, that the distinction between matricellular and structural matrix proteins is not sharp, since structural proteins such as collagens, laminins, and fibronectin also influence cell function by interaction with cell-surface receptors, and they also bind bioactive proteins. The classification of proteins as matricellular is based on similarity of mode of action rather than on homology in amino acid sequence and three-dimensional structure. Thus, the thrombospondins, SPARC family members, tenascins, CCN proteins, and osteopontin, the core group of matricellular proteins considered in this chapter, are structurally unrelated. The partial dependence of the function of matricellular proteins on the proteins with which they interact gives rise to the characterization of their function as contextual. The relevance of these proteins to the biochemistry and physiology of the lung will therefore depend to a large extent on the co-expression of interacting proteins. Matricellular proteins are frequently expressed in pulmonary fibrosis and in neoplasms of the lung, and early evidence supports the ability of these proteins to influence the progress of these disorders.

cellular behavior, had its origin in studies of thrombospondin (TSP)-1, SPARC (secreted protein, acidic, and rich in cysteine), and tenascin-C. In contrast to typical adhesive matrix proteins such as fibronectin and type I collagen, these proteins were found to be anti-adhesive in that a variety of cells attached poorly to the proteins in vitro, and attachment of cells to collagen or fibronectin was reduced when these matricellular proteins were added. It is also known that these proteins are expressed predominantly, but transiently, in tissues that are undergoing rapid change, for example, during embryonic development, and in wound healing, angiogenesis, and bone remodeling. Therefore, it was surmised that matricellular proteins serve to modulate cell attachment and detachment, and thus regulate cell migration during morphogenesis and tissue repair. Subsequent studies indicated that the properties of osteopontin and the six members of the CCN family (an acronym derived from the initial names of the first three members in this family: Cyr61, connective tissue growth factor, and Nov) justified their inclusion in the group of matricellular proteins. It is likely that additional proteins and/or proteoglycans will be found to qualify for inclusion in this group. An additional reason to consider inclusion of matricellular proteins in a separate category of extracellular proteins was provided by studies of the functions of TSP1 and TSP2, first in vitro and subsequently deduced from the phenotypes of knockout mice. The diversity of the functions that were attributed to TSP1, based on studies in vitro, was puzzling and could not be readily explained by the existence of a close homolog, TSP2, or by modification of the protein, either by alternative splicing or by limited proteolysis. This problem was compounded when the full panoply of effects from a lack of TSP2 was revealed in the phenotype of the TSP2 knockout mouse. Similarly, the phenotype of the SPARC-null mouse, which is still under active investigation, has turned out to be far more complex than initially anticipated. This wide range of functions can be more readily understood when one postulates that the complexity of the pericellular environment can provide an explanation for the diversity of function displayed by these and other matricellular proteins.

The Structure of Matricellular Proteins Introduction

Thrombospondins 1 and 2

The realization that proteins exist in the extracellular milieu, distinct from cytokines, growth factors, and proteases, whose primary function is to modulate

Matricellular proteins typically display a multidomain structure, resulting from the process of exon shuffling during evolution, and differences among

176 EXTRACELLULAR MATRIX / Matricellular Proteins Thrombospondin 1 and 2 S NH2

PC

I

I

I

II

II

II

III III III III III III III

COOH

S

S II

NH2

II

II

II

III III III III III III III

COOH

S Thrombospondin 3, 4, and 5 Figure 1 A schematic representation of the structures of individual chains in the thrombospondins. TSP1 and TSP2 are trimers and TSPs 3–5 are pentamers. The structure of the NH2-terminal domains is not known and the amino acid sequences in this domain vary considerably among the TSPs. The oligomerization domain, containing interchain disulfide bonds, is followed by a procollagen homology domain (PC), also known as a von Willebrand type C repeat, and in the case of TSP1 and TSP2, by three TSR (thrombospondin structural repeats) or properdin-like repeats. All TSPs have type II or EGF-like repeats, type III or calcium-binding repeats, and a COOHterminal domain.

homologous members of a subfamily among different species (orthologs) can be attributed to selection for the properties unique to the individual paralogs (related proteins within a single species). The TSP family (Figure 1) consists of five members. TSP1 and TSP2 are disulfide-bonded trimers with a chain molecular weight (MW) of 145 000, while TSP3, TSP4, and TSP5 (also known as COMP, cartilage oligomeric protein) are pentamers of about 105–110 000 molecular weight. The differences in the MWs of the pentameric TSPs result from a variation in their NH2terminal domains. Currently, only TSP1 and TSP2 can be considered to be matricellular proteins. While functional information for the pentameric TSPs is limited, at least TSP3 and TSP5 appear more likely to play a structural role in the matrix. The conservation in amino acid sequence between mouse TSP1 and TSP2 increases monotonically from 32% identity in the NH2-terminal domain to 82% identity in the COOH-terminal domain. As shown in Figure 1, a TSP1 or TSP2 chain is composed of an NH2-terminal domain, a disulfide knot or oligomerization region that links the three chains in the protein, a PC (procollagen/von Willebrand type C) domain, the types I, II, and III repeats, and a COOHterminal domain. The type I repeats, also known as the properdin or thrombospondin structural repeats (TSR) repeats, are broadly distributed in nature and are found in other vertebrate proteins (properdin, ADAMTS proteases, F-spondin), Drosophila (F-spondin, papilin), Caenorhabditis elegans (Fspondin), and the malarial parasite, Plasmodium falciparum (circumsporozoite protein). The type II

or EGF-like repeats are also found in many other proteins including fibrillins, tenascins, and in the extracellular domain of many transmembrane proteins. On the other hand, sequences in the type III repeats and the COOH domain are unique to members of the TSP family. Rotary shadowing electron microscopy of TSP1 molecules revealed a bola-like structure in which the three NH2-termini are coalesced into a single globe that is connected by protein strands to three separate COOH-terminal globes. The latter were thought to represent only the COOH-terminal domains, but recent biophysical and crystallographic studies indicate that the COOH-terminal half of each chain is folded into a compact structure that is stabilized by the binding of calcium ions. Thus, the strand visible by rotary shadowing may be limited to the procollagen domain and the TSR repeats in the calcium-replete structure. The crystal structure of the second and third TSR repeats of human TSP1 has revealed a novel, antiparallel, three-stranded fold with alternating stacked layers of tryptophan and arginine residues. SPARC and Family Members

SPARC is a 32 kDa glycoprotein that is widely expressed during development and growth of vertebrates, but expression in the adult is more restricted to tissues such as bone and the lens of the eye. Reexpression occurs following injury and in tumors. SPARC orthologs also exist in Drosophila and C. elegans. The structure of SPARC and its paralogs are shown schematically in Figure 2. The crystal

EXTRACELLULAR MATRIX / Matricellular Proteins 177 SPARC (285)

−C

N−

Hevin (634) QR1 (660) Testican1 (421) tsc 36 (288) SMOC-1 (434) Figure 2 A schematic representation of the structures of six members of the SPARC family. There are two additional testicans and two additional tsc proteins. The individual domains in the proteins are represented by different colors and are not drawn to scale. The number of amino acids in each protein is indicated in parentheses. Yellow: the acidic domain, which is highly variable in length; blue: follistatin domain; orange: the highly conserved E-C domain, containing two EF hand calcium-binding structures (black bars); white: thyroglobulinlike domain. The green, hatched, and cross-hatched domains are specific for the indicated proteins. Figure kindly provided by Dr E Helene Sage.

structure of two of the three domains of human SPARC, the best-studied member of this ten-protein family (Figure 2) has been solved. While the acidic NH2-terminal domain was poorly structured and could not be resolved by X-ray analysis, the structure of the middle, follistatin domain was shown to resemble that of serine protease inhibitors of the Kazal family. The COOH-terminal domain consists of two EF-hand calcium-binding sites and a number of ahelices. These two domains interact through a small interface. The highly variable, acidic NH2-terminal domains of different SPARC family members are thought to determine the properties that are unique to each member of the family. Tenascins

The tenascins (TNs) are a family of four or possibly five structurally related extracellular proteins that exist as trimers due to the presence of an NH2-terminal TN assembly (TA) domain (Figure 3). Chain association initially occurs by interaction of heptad repeats and further stabilization is achieved by formation of interchain disulfide bonds. Despite the presence of additional domains in human TN-X, this TN and avian TN-Y may be orthologs, based on similarities in sequence and expression patterns. In the case of tenascins-C and -W, two trimers associate to form hexamers (see rotary shadowing image in Figure 3(a)), which is the basis for the term hexabrachion, applied initially to tenascin-C; this protein has also been termed cytotactin. The structures of the tenascins are described in detail in the legend to Figure 3. Whereas the formation of heterotypic multimers is theoretically possible, the tenascins are thought to be homo-trimeric or -hexameric in the sense that all subunit chains are the products of the same gene. However, the chains in any given

molecule can vary considerably in molecular weight (from 190 to 300 kDa in tenascin-C) as a result of extensive alternative splicing. Osteopontin

Osteopontin (OPN) is sometimes classified as a member of an integrin-binding family of five N-linked glycoproteins that includes bone sialoprotein, dentin matrix protein I, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein. The genes encoding these proteins are closely linked on human chromosome 4. However, since the other members of the family appear to perform structural roles in the matrix and do not share the wide range of nonstructural functions displayed by OPN, only the latter will be considered here. The name OPN was coined to suggest a bridging function for the protein between hydroxyapatite and cells in bone. In addition to calcium-containing minerals, OPN binds to collagen, fibronectin, and osteocalcin. OPN is a highly acidic protein that is rich in aspartic and glutamic acids and serine (Figure 4). Additional negative charges are introduced by phosphorylation of serine and threonine residues. Judging by its highly acidic character and amino acid sequence, OPN is likely to be highly flexible, with very little a-helical or b-sheet structure. The molecular weight of the protein varies as a result of alternative splicing, as well as variable N- and O-glycosylation and phosphorylation. In addition, OPN is subject to cleavage by thrombin between Arg168 and Ser169 (in the human protein). Thrombin cleavage modulates its interactions with cell-surface receptors, and consequently influences cell adhesion and migration. Recently, OPN has also been shown to be a substrate for matrix metalloproteinases (MMPs), although the function of MMP-derived fragments is uncertain. In

178 EXTRACELLULAR MATRIX / Matricellular Proteins

Region of oligomer assembly

Conserved FN-III domains

Spliced FN-III domains

EGF L repeats

Fibrinogen globe

(b)

(a) TA domain

EGFL repeats

TN-C:

FG globe

FN-III domains 1

2

3

4

5

A1 A2 A3

A4

1

2

3

4

5

R

8

B AD2 AD1 C

D

6

7

8

Heptad TN-R:

TN-W:

7

WA WB WC WC WD

TN-X:

XA XA

XR1 XR2 XR3 XR4 XR5 XR6 XR7 XR8 XR9

TN-Y:

6

YA

SPX

SPX

1 X1 X2 X3 2

X4 X5

X6 X7 X8 X9 X10 X11 X12

XR X13 X14 X15 X16 X17 X18 X19 X20 X21 10

1 YB1 YC1 YD1 YE1 YE2 YE3 YD2 YE4 YE2 YD1 YE5 8 2

(c) Figure 3 The structures of the members of the tenascin family. (a) Rotary shadowing EM image of two mouse TN-C hexamers. (b) A model of a TN-C hexamer. The domains are color-coded as in (c). FN III, fibronectin type III repeats; EGF-like, epidermal growth factorlike. (c) Models of the structures of the four or five members of the tenascin family. Despite their apparently different structures, mammalian TN-X and avian TN-Y may be orthologs (see text). The TN assembly (TA) domain (green) is comprised of four heptad repeats and a number of cysteines that form interchain disulfide bonds. In the cases of TNs X and Y, the white ovals indicate that the cysteines required to link trimers in higher order multimers are missing. The EGF-like repeats that are common to all vertebrates (salmon ovals) vary from 18.5 in TN-C to one and two halves in TN-Y. In the case of TN-C, an additional repeat specific to mammals (navy blue oval) is present. The FN-III repeats (blue, red, or purple rectangles) fall into several different categories that reflect their composition or origin during evolution. A serine- and proline-rich domain (purple diamond) is found in TNs X and Y. A fibrinogen globe (yellow circle) is common to all TNs. Reproduced from Developmental Dynamics; Jones FS and Jones PL; copy right & 2000, Wiley-Liss. Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

comparisons of the OPN sequences among vertebrate species, a polyaspartic acid sequence, the thrombinand MMP-cleavage sites, and a GRGDS integrinbinding sequence are highly conserved.

CCN Proteins

The CCN proteins are a family of six homologous, secreted proteins that share a very similar structure.

EXTRACELLULAR MATRIX / Matricellular Proteins 179

Osteopontin

Thrombin cleavage site (
v3,
v5,
v1,
81)

(CD44)

RGD

PPP P P

NH2

P P

1

P P

SVVYGLR

P PP COOH 300

169 Ca2+-binding domain

P

Ca2+-binding domain

(
91,
41,
47) MMP cleavage site Figure 4 A schematic structure of human OPN. The boxes from N- to C-terminus indicate domains that have some confirmed function as follows: (1) the signal sequence; (2) conserved casein–kinase-2 phosphorylation sites; (3) calcium-binding domain containing a polyaspartic acid sequence; (4) RGD site; (5) MMP cleavage site; (6) calcium-binding domain; and (7) heparin-binding domain. The positions of phosphorylated serines (P) are indicated. The site of thrombin cleavage occurs between amino acids 168 and 169 in the human protein. The approximate sites of interaction with a number of integrins and CD44 are shown. Kindly provided by Dr Cecilia Giachelli.

Cyr61, CTGF, and Nov are now termed CCN1, CCN2, and CCN3; CCN 4–6 were previously known as WISP (Wnt-1-induced secreted protein) 4–6. With the exception of CCN5, which lacks a C-terminal module, each of the proteins contains a signal peptide, an insulin-like growth factor-binding protein domain, a von Willebrand factor C repeat, a TSR or TSP1 repeat, and a C-terminal domain (Figure 5). Each of these modules is, in turn, encoded by a separate exon. This conservation of structure extends to the presence of 38 homologously placed cysteines in all CCN proteins except CCN6, which contains only six cysteines in its von Willebrand factor C repeat. However, the complexity of the family is increased considerably by the additional presence of variants that are either truncated N- or C-terminally by limited proteolysis, or that originate by alternative splicing. CCN variants or isoforms can perform functions that differ considerably from those of the parent protein.

Biological Functions of Matricellular Proteins Thrombospondins 1 and 2

TSP1 was first discovered as a major component of a-granules in platelets and was thought to be important in the formation of a platelet plug in response to injury. However, TSP1-null mice show no defects in clot formation and do not have a bleeding diathesis.

Instead, it seems likely that a major function of platelet TSP1 is to serve as a chemotactic factor that attracts inflammatory cells (monocytes/macrophages), which in turn initiate wound repair. The phenotypes of TSP1- and TSP2-null mice have played an important role in our understanding of the function these proteins. This is true because it is difficult to determine the biologically relevant functions of these proteins by experiments in vitro. Thus, such experiments cannot replicate the interactions with multiple cell-surface receptors (Table 1) and other extracellular proteins displayed by TSP1 and TSP2, nor do they take into account the temporal and spatial specificity of their expression. A characteristic of matricellular proteins such as TSP1 and TSP2 is that the consequences of their absence is often not evident in uninjured adult animals. TSP1-null mice appear normal, but display a mild spinal lordosis, and have a higher than normal fetal mortality. A major manifestation is increased susceptibility to pneumonias, apparently caused by the normal flora of the upper respiratory tract. While the problem has not been studied carefully, a lack of the chemotactic activity of TSP1 for inflammatory cells may be a factor. In addition, TSP1 functions to bind and activate latent transforming growth factor (TGF)-b1, and there is evidence for a deficiency of active TGF-b1 in some tissues, particularly the lung, of TSP1-null mice, which could contribute to an increased predisposition to infection. The best-studied function of TSP1 is its ability

180 EXTRACELLULAR MATRIX / Matricellular Proteins CCN2 CCN3 CCN5 CCN1 CCN4 CCN6

(a)

Exon 1

E2

E3

E4

E5

Promoter

3′-UTR

Signal peptide NH2 No. of cysteine residues

IGFBP

VWC

Cysteine knot

TSP1

COOH 12

10(6)

Variable

6

10

(b) Figure 5 The CCN proteins. (a) Dendogram showing the evolutionary relationships of the six CCN proteins. (b) Schematic representations of a typical CCN gene and protein. Each CCN gene consists of five exons, each of which encodes a module in the protein. IGFBP, insulin-like growth factor binding protein; VWC, von Willebrand factor type C repeat; TSP1, thrombospondin-1 or TSR repeat. All CCN proteins contain the same number of cysteines in homologous modules, except CCN6, which contains 6 instead of 10 cysteines in the VWC module. Reproduced from Perbal B and Takigawa M (eds.) (2004) CCN Proteins: A New Family of Cell Growth and Differentiation Regulators. London: Imperial College Press, with permission from Imperial College Press.

Table 1 Interactions of thrombospondins with cell-surface receptorsa NH2-terminal domain Syndecans Sulfatides Calreticulin LRP1 a3b1c a4b1 a6b1

Type I repeats CD36 HIVgp120b ab1d ab1d

Type II repeats d

ab1 ab1d

Type III repeats

C-terminal domain

avb3 a5b1

CD47

a

Unless otherwise specified, both TSP1 and TSP2 interact with these receptors. No receptors have yet been found to interact with the procollagen domain. b 120 kDa glycoprotein on the surface of human immunodeficiency virus. c Applies only to TSP1. d The sequences in the type I and II repeats are pan-b1 specific, i.e., they serve as ligands for several ab1 integrins.

to inhibit angiogenesis. As a consequence, tumors grow more rapidly and metastases are increased in the absence of TSP1. A number of studies have implicated the TSR repeats of TSP1 in its antiangiogenic properties. These repeats interact with the scavenger receptor CD36 on endothelial cells, and this interaction culminates in activation of caspases and apoptosis. TSP2-null mice also appear overtly normal and are normally fertile. However, their skin is unusually fragile, and tendons and ligaments are lax to an extent that the end of the tail can be tied into a knot. These findings suggest a defect in collagen

fibrillogenesis; indeed, collagen fibrils in skin and tendon are generally wider in diameter and are irregular in contour by electron microscopy. The basis for these changes in collagen structure is not known, but an increase in levels of matrix metalloproteinase (MMP)-2 levels may be partially responsible. Both TSP2-null fibroblasts in culture and injured tissues in TSP2-null mice show increased levels of MMP2. The cause of this increase can be attributed to the fact that TSP2 serves as a clearance factor for MMP2. The TSP2–MMP2 complex is recognized by the LRP1 scavenger receptor, endocytosed, and degraded in lysosomes. MMP2 and MMP9 levels are strikingly

EXTRACELLULAR MATRIX / Matricellular Proteins 181

increased in the myocardium of angiotensin II-induced hypertensive TSP2-null mice, and a high proportion of these mice die of cardiac rupture. TSP2, like TSP1, is an inhibitor of angiogenesis. While there is a modest increase in vascularity in uninjured skin, vessel density is markedly increased in healing excisional skin wounds and in the foreign body capsule surrounding implanted biomaterials in TSP2-null mice. As a consequence, these mice heal their wounds more quickly, and studies are under way to determine whether inhibition of TSP2 levels by local gene therapy can prolong the functional lifetime of implanted biosensors and delivery devices. SPARC and Family Members

SPARC was first identified, more or less simultaneously, as a secreted protein in cultures of endothelial cells, as a major noncollagenous protein in bone (termed osteonectin), and as a putative constituent of basement membranes (BM-40). However, its presence as an integral protein in basement membranes has not been confirmed. Purified SPARC protein inhibits cell attachment by interfering with focal adhesions and causes cells to arrest in the G1 phase of the cell cycle. SPARC also interacts with at least two growth factors (vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF)) and this binding probably accounts for its anti-angiogenic activity in vitro. As with targeted disruption of several other matricellular protein genes (gene knockouts), the study of SPARC-null mice has been highly informative and has revealed functions of SPARC that were unsuspected. The complex phenotype of the SPARC-null mouse is characterized by formation of cataracts at an early age, laxity, reduced tensile strength and collagen content of skin, accelerated closure of excisional dermal wounds, a marked increase in adipose tissue, severe premature osteopenia, and a propensity for increased growth and metastatic spread of a variety of ectopically transplanted tumors. Studies are under way to identify cell-surface receptors for SPARC and to elucidate the biochemical mechanisms responsible for the unusual findings in the SPARC-null mouse. Hevin, also known as SC1, a close structural paralog of SPARC, is widely expressed in brain and in high endothelial venules, from which it receives its name. Hevin shares some functions with SPARC, including its ability to inhibit focal adhesions and cell attachment and proliferation. Hevin-null mice also heal excisional skin wounds more rapidly than normal. Hevin is thought to be involved in regulation of cell migration during embryonic development, although hevin-null mice appear normal at birth

and grow normally. However, the major, striking manifestations of the SPARC-null mouse, such as cataracts, increased adiposity, and premature osteopenia, are lacking in the hevin-null mouse. Very little is known about the functions of other SPARC family members. Tenascins

The phylogenetic distribution of TNs, which contain a sequential arrangement of EGF-like and fibronectin-III repeats and a fibrinogen domain, appears to be limited to vertebrates. In keeping with their classification as matricellular proteins, TNs are expressed primarily during development, in response to injury or mechanical stress, and in tumors. Indeed, TN-C was first discovered as a glioma-associated extracellular protein. Despite a number of studies to determine whether the level of expression of TN-C can serve as a prognostic indicator, no consistent association has been found between the levels of TN-C and the invasiveness of tumors. Like TSPs and SPARC, TN-C shows counter-adhesive properties. There is evidence that TN-C interferes with the synergy between the major fibronectin receptor, a5b1, and its co-receptor, syndecan-4, leading to reduced activity of Rho and disassembly of actin stress fibers. However, like the TSPs, the effects of TN-C, as well as that of other TNs, can be cell specific. The TN-C-null mouse was initially described as normal, but subsequent analyses have revealed subtle defects. Thus, TN-C-null mice are more susceptible to snake venom-induced glomerulonephritis and to chemically induced dermatitis, findings that are consistent with an anti-inflammatory function of the protein. The lungs in TN-C-deficient mice also show a defect in vascular branching. TN-C, -R, and -Y are all expressed in the central nervous system in patterns that are distinct, both with respect to anatomical location and appearance during development. It is thought that the alternatively spliced forms of these TNs may have subtly different properties. TN-C-null mice display abnormalities in motor coordination and exploratory behavior, and TN-R-null mice have reduced velocities in axonal conduction. TN-X is widely expressed in connective tissues, primarily in skin, tendon, heart, and smooth and skeletal muscle. The TN-X gene is part of a multigene cluster that includes the steroid 21-hydroxylase gene (CYP21A2), and a defect in TN-X was first identified in a patient who also had adrenal hyperplasia due to CYP21A2 deficiency. In this patient a single deletion removed the entire CYP21A2 gene and truncated the contiguous TN-X gene. Patients with TN-X deficiency have a form of the

182 EXTRACELLULAR MATRIX / Matricellular Proteins

Ehlers–Danlos syndrome (benign joint hypermobility syndrome) that is characterized by hypermobile joints, hyperelastic skin, and easy bruisability. There is evidence that heterozygosity for the TN-X-null allele results in haploinsufficiency. TN-X mice also show a constellation of findings that mimic the hypermobility-type Ehlers–Danlos syndrome. These mice have hyperextensible skin and a reduced collagen content in dermis and tendon. Although the synthesis of collagen by cultured TN-X fibroblasts was normal, these fibroblasts failed to incorporate the collagen into the matrix. Tissues and fibroblasts from TN-X-null mice have increased levels of both proand active MMP2 and MMP9, and these proteases may be partly responsible for the adhesive defect seen in TN-X-null fibroblasts and the increased tumor spread and metastases in these animals. Osteopontin

In keeping with the expression patterns of other matricellular proteins, the distribution of OPN is limited in healthy adult animals and humans, but expression is markedly increased in disease and in response to injury. In normal bone, OPN is synthesized by both osteoblasts and osteoclasts and is thought to regulate the mineralization of bone matrix and bone remodeling, both by direct interaction with bioapatite and by binding to integrin cell-surface receptors. OPN similarly inhibits and promotes regression of ectopic calcification in vascular tissues by physical interaction with hydroxyapatite crystals and by induction of carbonic anhydrase II, thus promoting acidification of the extracellular milieu. In the kidney, OPN regulates renal inducible nitric oxide synthase (iNOS) and the deposition of urinary calcium oxalate. OPN is found normally in urine, saliva, milk, and bile, where the protein is also thought to inhibit the formation of concretions. Studies of OPN-null mice have provided support for these functions. Thus, mice that are null for matrix Gla protein, which develop precocious vascular calcification that is associated with increased OPN deposition, are even more prone to ectopic calcification when crossed with OPN-null mice. In acute and chronic injury both pro- and antiinflammatory modes of action of OPN have been documented. As a pro-inflammatory protein, OPN functions as a chemoattractant for macrophages and T cells and enhances the expression of Th1 cytokine by interaction with a number of cell-surface receptors, including CD44 and several integrins. As an anti-inflammatory agent, OPN inhibits the induction of iNOS mRNA and thus blocks formation of NO and the production of reactive oxygen species

in macrophages. OPN also plays an important role in the immune system by stimulating T cell-mediated immunity, as well as B cell activation and antibody production. CCN Proteins

The constellation of domains present in CCN proteins is limited to vertebrates, although Drosophila appears to have a truncated homolog and individual domains are more widely distributed among metazoan species. CCN proteins are normally expressed during embryonic development, and in adult animals in the nervous, vascular, and musculoskeletal systems, and in the adrenal gland. However, in keeping with their matricellular properties, the expression of these proteins is enhanced during tissue repair, and in fibrosis, inflammation, and tumor growth. CCN proteins are involved in regulation of cell-cycle progression, cell adhesion and migration, and control of both production and degradation of ECM proteins. As a result, these proteins participate in diverse processes such as angiogenesis, chondro- and osteogenesis, and tissue remodeling. CCN1 influences the function of fibroblasts, endothelial and smooth muscle cells, platelets, and monocytes by interaction with at least five different integrins. In some cases, heparan sulfate proteoglycans participate as co-receptors. In the case of actively motile cells, such as fibroblasts and smooth muscle cells, CCN1 stimulates formation of filopodia and lamellipodia that enable migration. Signal transduction leads to increased expression of MMP1 and 3. Adherence to CCN1 also protects endothelial cells from apoptosis upon serum withdrawal. CCN1 is rapidly activated transcriptionally by basic fibroblast growth factor, PDGF, and TGF-b1. In keeping with its important role as a proangiogenic factor, CCN1null mice die in early to midgestation as a result of defective vascular bifurcation and placental development, as well as compromised vascular and cardiac integrity. The histopathological findings resemble those in av-null mice and thus identify CCN1 as a major ligand for av integrins. CCN2 was initially termed connective tissue growth factor, but it is generally recognized that its function in stimulating cell proliferation is indirect. CCN2 is also proangiogenic and stimulates endothelial adhesion, migration, and proliferation. Stimulation of migration is associated with dissolution of focal adhesions and disassembly of the actin cytoskeleton. The protein is expressed in the skeletal system and promotes synthesis of type II collagen and proteoglycans. CCN2-null mice die soon after birth from respiratory failure caused by extensive

EXTRACELLULAR MATRIX / Matricellular Proteins 183

skeletal defects in the thorax and elsewhere. Matrix remodeling in the hypertrophic zones of growth plates in long bones is defective, and this can be attributed to decreased expression of VEGF and reduced numbers of osteoblasts and osteoclasts that produce MMPs. Less is known about the functions of CCN3 (previously termed Nov) and CCN4-6 (WISP 1-3). CCN3 interacts with several integrins as well as with extracellular proteins, and appears to be involved in regulation of cell growth and differentiation. The protein is proangiogenic and induces neovascularization in corneal implants. Recently, CCN3 was shown to interact with the gap junction protein, connexin 43. This interaction may play a role in the growth control exerted by gap junction proteins. The expression of CCN4 is restricted to osteoblasts and to osteoblast progenitor cells of the perichondrial mesenchyme. CCN4 binds decorin and biglycan, two small, leucinerich proteoglycans, and promotes proliferation of mesenchymal cells and differentiation of osteoblasts, while repressing differentiation of chondrocytes. The protein functions similarly during fracture repair. The expression and function of CCN5 are also restricted largely to the regulation of osteoblast function. Mutations in the gene encoding CCN6 in humans cause recessive progressive pseudorheumatoid dysplasia, a disorder characterized by postnatal degeneration of articular cartilage.

The Role of Matricellular Proteins in Respiratory Diseases There is a small but growing literature that documents the association of increased expression of tenascin C, SPARC, TSP1, osteopontin, and CCN 1 and 4 with a variety of disorders, including neonatal respiratory distress syndrome, pulmonary tumors, and fibrosing diseases of the lung. In the majority of published studies the relation of these proteins to the disease states has been limited to the documentation of a spatial association. Since matricellular proteins are typically expressed in response to a variety of injuries, this association is to be expected. Nevertheless, in some cases the expression of the matricellular protein appears to have predictive value for patient outcome. Furthermore, preliminary evidence suggests that these proteins can exert regulatory functions that can either promote or inhibit the progression of the disease. Such opposing effects very likely reflect the complex roles that matricellular proteins play in inflammation, and in the control of cell adhesion, migration, division, and viability.

Acknowledgments I thank Drs Helene Sage, Cecilia Giachelli, Fred Jones, and Lester Lau for their review of parts of the article. See also: Adhesion, Cell-Matrix: Integrins. Angiogenesis, Angiogenic Growth Factors and Development Factors. Extracellular Matrix: Collagens; Matrix Proteoglycans; Surface Proteoglycans.

Further Reading Adams JC (2001) Thrombospondins: multifunctional regulators of cell interactions. Annual Review of Cell and Developmental Biology 17: 25–51. Bornstein P (2001) Thrombospondins as matricellular modulators of cell function. Journal of Clinical Investigation 107: 929–934. Bornstein P, Armstrong LC, Hankenson K, Kyriakides TR, and Yang Z (2000) Thrombospondin 2, a matricellular protein with diverse functions. Matrix Biology 19: 557–568. Bornstein P and Sage EH (2002) Matricellular proteins: extracellular modulators of cell function. Current Opinion in Cell Biology 14: 608–616. Brekken RA and Sage EH (2001) SPARC, a matricellular protein: at the crossroads of cell–matrix communication. Matrix Biology 19: 815–827. Chiquet-Ehrismann R and Chiquet M (2003) Tenascins: regulation and putative functions during pathological stress. Journal of Pathology 200: 488–499. Denhardt DT, Noda M, O’Regan AW, Pavlin D, and Berman JS (2001) Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. Journal of Clinical Investigation 107: 1055–1061. Giachelli CM and Steitz S (2000) Osteopontin: a versatile regulator of inflammation and biomineralization. Matrix Biology 19: 615–622. Ivkovic S, Yoon BS, Popoff SN, et al. (2003) Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130: 2779–2791. Jones FS and Jones PL (2000) The tenascin family of ECM glycoproteins: structure, function, and regulation during embryonic development and tissue remodeling. Developmental Dynamics 218: 235–259. Lau LF and Lam SCT (1999) The CCN family of angiogenic regulators: the integrin connection. Experimental Cell Research 248: 44–57. Mo F-E, Muntean AG, Chen CC, et al. (2002) CYR61 (CCN1) is essential for placental development and vascular integrity. Molecular and Cellular Biology 22: 8709–8720. O’Regan A (2003) The role of osteopontin in lung disease. Cytokine and Growth Factor Reviews 14: 479–488. Perbal B and Takigawa M (eds.) (2004) CCN Proteins: A New Family of Cell Growth and Differentiation Regulators. London: Imperial College Press. Sullivan MM and Sage EH (2004) Hevin/Sc1, a matricellular glycoprotein and potential tumor-suppressor of the SPARC/BM40/osteonectin family. International Journal of Biochemistry and Cell Biology 36: 991–996. Zweers MC, Hakim AJ, Grahame R, and Schalkwijk J (2004) Joint hypermobility syndromes: the pathophysiologic role of tenascin-X gene defects. Arthritis and Rheumatism 50: 2742– 2749.

184 EXTRACELLULAR MATRIX / Matrix Proteoglycans

Matrix Proteoglycans C W Frevert, VA Puget Sound Medical Center, Seattle, WA, USA T N Wight, The Hope Heart Institute, Seattle, WA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Matrix proteoglycans, complex molecules composed of a core protein and glycosaminoglycan side chains, impart biomechanical properties to lung tissue and are important biological modifiers which regulate processes such as lung development, homeostasis, inflammation, and wound healing. The diverse roles of matrix proteoglycans suggest that these molecules play a critical role in normal and diseased lungs.

are four classes of glycosaminoglycans: (1) hyaluronan, (2) chondroitin sulfate (CS)/dermatan sulfate (DS), (3) heparan sulfate (HS)/heparin, and (4) keratan sulfate (KS). All four classes of glycosaminoglycan are found in normal lungs and all except hyaluronan are bound to core proteins. The predominant glycosaminoglycan in normal lungs is HS (40–60%) followed by CS/DS (31%), hyaluronan (14%), and heparin (5%). Sulfation of glycosaminoglycans, a structural modification that occurs in the Golgi during chain elongation, has important biological consequences as specific sulfation patterns on glycosaminoglycans form the binding sites for a number of proteins including morphogens, growth factors, cytokines, and chemokines.

Introduction

Glycosaminoglycan Structure

Proteoglycans are a family of charged molecules containing a core protein and one or more covalently attached glycosaminoglycan side chains. The complexity and diversity of proteoglycans is derived from approximately 30 different core proteins of varying size and structure (10–500 kDa) and the considerable variability in the number (1 to 4100) and types of glycosaminoglycan side chains attached. Proteoglycans are found in the extracellular matrix, plasma membrane of cells, and intracellular structures. Matrix proteoglycans such as perlecan, collagen XVIII, and agrin are found in the basal laminal of cells, and decorin, biglycan, and versican are found in the interstitial spaces of the lungs. The glycosaminoglycan side chains and core proteins of matrix proteoglycans regulate a number of biomechanical and cellular processes in normal and diseased lungs.

Hyaluronan is a repeating disaccharide structure composed of glucuronic acid and N-acetyl glucosamine that is nonsulfated and is not attached to a core protein (Table 1). CS has a disaccharide repeat pattern similar to hyaluronan, but it contains galactosamine instead of glucosamine. The galactosamine can have a sulfate ester attached at the 4- or 6-position (e.g., chondroitin 4-sulfate) and the glucuronic acid can have a sulfate ester placed at the 2-position. The degree of sulfation of CS is variable and can differ within a single preparation and from one tissue to another. Dermatan sulfate is a copolymer composed of two types of disaccharide repeats, D-glucuronate-N-acetyl galactosamine and L-iduronate-N-acetyl galactosamine. The formation of iduronic acid residues occurs by the conversion of glucuronic acid already incorporated into the growing polymer by an epimerization reaction that is tightly coupled to the sulfation process. KS has a repeating disaccharide residue containing galactose and N-acetyl glucosamine. The linkage of KS to the core protein differs from other glycosaminoglycans because it is not formed on the typical xylose–serine linkage. KS expression in lungs is

Glycosaminoglycan Side Chains Glycosaminoglycans are linear polymers of repeating disaccharides with a high negative charge imparted by sulfate and/or carboxyl groups in their structure. There

Table 1 Structure of glycosaminoglycans Glycosaminoglycan

Monosaccharide (1)

Monosaccharide (2)

Sulfation patterns

Hyaluronan Chondroitin sulfate

D-glucuronic acid D-glucuronic acid

N-acetyl glucosamine N-acetyl galactosamine

Dermatan sulfate

D-glucuronic acid or L-iduronic acid D-galactose D-glucuronic acid or L-iduronic acid D-glucuronic acid or L-iduronic acid

N-acetyl galactosamine

No sulfation 2-O-sulfation, 4-O-sulfation, 6-O-sulfation 2-O-sulfation, 4-O-sulfation, 6-O-sulfation 6-O-sulfation 2-O-sulfation, 3-O-sulfation, 6-O-sulfation, N-sulfation 2-O-sulfation, 3-O-sulfation, 6-O-sulfation, N-sulfation

Keratan sulfate Heparan sulfate Heparin

N-acetyl glucosamine N-acetyl glucosamine N-acetyl glucosamine

EXTRACELLULAR MATRIX / Matrix Proteoglycans

limited but this glycosaminoglycan is present in the cartilage of the trachea. Heparin and HS are glycosaminoglycans of closely related structure and contain repeating disaccharide units composed of glucosamine and either D-glucuronic acid or L-iduronic acid. Glucuronic acid is the predominant uronic acid component in HS, whereas iduronic acid is the largest component in heparin. Heparin and HS contain a number of glucosamine residues that contain N-sulfate groups instead of N-acetyl groups. HS contains a lower degree of O-sulfation than heparin and therefore has fewer sulfates per disaccharide than heparin. In mature HS about 40–50% of the amino sugars are converted from the N-acetylated form to the N-sulfated form. The N-sulfated residues occur predominantly in contiguous sequences or S-domains, which are separated by 15 disaccharides that are made in large part by N-acetyl-rich domains. This results in HS being a more complex molecule than heparin.

Core Proteins of Matrix Proteoglycans A number of HS proteoglycans (HSPGs), CS proteoglycans (CSPGs), and DS proteoglycans (DSPGs) are found in the matrix of lungs (Table 2). Perlecan is a large proteoglycan consisting of five domains, which contain low-density lipid (LDL) receptor class A modules, Ig-like repeats, laminin-like repeats, and EGF-like repeats. Electron micrographs of perlecan showed a protein with the appearance of beads on a string, which resulted in the name perlecan. Whereas HS is the predominant glycosaminoglycan on perlecan, CS chains are found on domain V of recombinant perlecan. Collagen XVIII is an HSPG with features typical of collagen including sensitivity of the core protein to collagenase. This HSPG is of particular interest because of the presence of endostatin, a 22 kDa antiangiogenic peptide located in the C-terminal domain of collagen XVIII. Agrin is an HSPG that contains nine follistatin-like domains which share similarity to Kazal-type protease inhibitors, including pancreatic trypsin inhibitor, follistatin, thrombin inhibitor, and elastase inhibitor. The N-terminal laminin-binding domain of chick agrin has a high

Table 2 Matrix proteoglycans Proteoglycan

Core protein (kDa)

Location

GAG type

Perlecan Collagen XVIII Agrin Decorin Biglycan Versican

450 180 220 40 40 350

Basal lamina Basal lamina Basal lamina Interstitium Interstitium Interstitium

HS/CS HS HS/CS CS/DS CS/DS CS

185

structural similarity to the protease inhibition domain in tissue inhibitor of metalloproteinase-1. Decorin and biglycan are small leucine-rich CSPGs with core proteins of similar size and structure (Table 2). The core protein of decorin has one CS/DS chain, whereas biglycan has two CS/DS chains. Versican is a large CSPG with a core protein consisting of an N-terminal globular domain (G1) which binds hyaluronan and a C-terminal binding domain (G3) which contains lectin-like, EGF-like, and CRP-like subdomains. The two globular domains, G1 and G3, are separated by two domains that contain CS chains. Alternative splicing of versican mRNA leads to four isoforms, V0, V1, V2, and V3, which carry varying amounts of CS side chains, and mRNA for all isoforms except V2 have been found in normal lungs.

Distribution of Matrix Proteoglycans Basement membrane proteoglycans. The basement membrane or basal lamina of the cell contains proteoglycans that interact directly with cells (Figure 1). Proteoglycans found in the basement membrane include the HSPGs, perlecan, agrin, and collagen XVIII. Collagen IV is the only collagen which is more abundant in the basement membrane than collagen XVIII. Interstitial proteoglycans. The large CS proteoglycan, versican, is located in the interstitial spaces in the lungs where it interacts with hyaluronan (HA), which fixes this proteoglycan in tissue as a very high-molecular-weight aggregate (Figure 1). Decorin and biglycan are also located in the interstitial spaces of the lungs where decorin binds collagen fibers with a regular periodicity. Basal surface of cells. Syndecans are a small family of transmembrane HSPGs which interact with a number of matrix proteins (Figure 1). Whereas syndecans are not considered matrix proteoglycans (and are therefore not discussed in detail in this article), their interaction with matrix transmits signals plays a role in focal adhesion and cell death pathways. Syndecans are described in greater detail in Extracellular Matrix: Surface Proteoglycans.

Biomechanical Properties of Proteoglycans Extracellular matrix proteoglycans confer important biomechanical properties to lung tissue. Glycosaminoglycans are strongly hydrophilic and their high negative charge attracts cations such as Naþ , which pulls water into the matrix through osmotic activity. The large amount of water pulled into the

186 EXTRACELLULAR MATRIX / Matrix Proteoglycans

Alveolar space

Type II cell

Alveolar macrophage

Epithelium

Interstitial space

Perlecan Syndecan-1

Collagen

Versican-HA Interstitial cell

Decorin Syndecans

Endothelium

Vascular space Figure 1 Matrix proteoglycans in normal lungs. Perlecan, a HS proteoglycan, is found in the basal lamina of epithelial and endothelial cells. The CS proteoglycans, versican and decorin, are found in the interstitial space of the lungs. Versican binds to the glycosaminoglycan, hyaluronan, to form high-molecular-weight complexes. Decorin binds to collagen and helps stabilize the collagen–elastin network. Syndecans are membrane proteoglycans that interact with matrix proteins. This figure is not meant to represent the concentrations of the different proteoglycans in normal lungs and is meant to only show their location in lung tissue.

matrix by glycosaminoglycans results in swelling of the matrix, which allows the matrix to withstand compressive forces. Proteoglycans interact with other matrix molecules such as collagen, laminin, and elastin to form a structured matrix. Work performed in decorin knockout mice suggests that the stabilization of the collagen–elastin network by this proteoglycan contributes to lung elasticity and alveolar stability. Proteoglycans also provide a selective charge filtration barrier for the exchange of oxygen and plasma constituents and studies suggest that disruption of this charge barrier could lead to marked alterations in vascular permeability.

Proteoglycans as Determinants of Cellular Phenotype Proteoglycans are important determinants of cellular phenotype, playing a critical role in development, cellular proliferation, cell death, inflammation, and wound healing. A critical function of proteoglycans

in matrix is mediated by glycosaminoglycan side chains which bind to growth factors, cytokines, and chemokines. The binding of these proteins to glycosaminoglycans was initially believed to be a non-specific ionic interaction between positively charged amino acids on proteins and the negatively charged sulfates on glycosaminoglycans. It is now known that considerable specificity exists in the interaction between proteins and glycosaminoglycans with unique binding sites being identified on glycosaminoglycans for proteins such as thrombin and growth factors. The degree of specificity between proteins and glycosaminoglycans is still not known but there is speculation that there may be a specific sulfation pattern (e.g., binding site) for each protein that binds to a glycosaminoglycan. Current evidence suggests that the specificity observed in protein–glycosaminoglycan interactions plays an important role in the function of proteins in lung tissue. Listed below are five potential functions of protein– glycosaminoglycan interactions.

EXTRACELLULAR MATRIX / Matrix Proteoglycans

1. Determination of protein binding sites. Because of the specificity observed in protein–glycosaminoglycan interactions, matrix proteoglycans position proteins such as chemokines and fibroblast growth factors to distinct anatomical locations (e.g., basement membrane or intersitial spaces). 2. Storage/sequestration. Glycosaminoglycans provide a site for proteins to bind, which sequesters a protein and prolongs its retention in lung tissue. The binding of chemokines to glycosaminoglycans provides a site for dimerization of chemokines which is a mechanism shown to increase the amount of chemokine binding in the lungs. Undersulfation of the basement membrane matrix of alveolar type II cells compared with that of neighboring type I cells is believed to account for some of the known morphological and functional differences between these alveolar epithelial cells. Increased sulfation of glycosaminoglycans under type I cells sequesters fibroblast growth factors, limiting their effect on this cell. In contrast, decreased sulfation of the matrix under type II cells allows fibroblast growth factors to activate signaling pathways in this cell. 3. Formation and stabilization of morphogen and chemokine gradients. The binding of morphogens and chemokines to glycosaminoglycans has been proposed as a mechanism whereby gradients develop in tissue. The binding affinity of chemokines to glycosaminoglycans, which is in the low micromolar range, provides a mechanism whereby the chemokine is able to diffuse and form chemotactic gradients in tissue. 4. Cell signaling. The binding of growth factors and cytokines to glycosaminoglycans has been shown to play a role in cellular activation. For proper presentation to fibroblast growth factor receptors, members of the fibroblast growth factor family interact with HSPG. 5. Protection from proteolysis. The binding of a protein to a glycosaminoglycan may sequester proteolytic cleavage sites, which protects proteins from proteolysis. Glycosaminoglycans have been shown to protect chemokines and growth factors from proteolytic cleavage.

Proteoglycans and Disease Changes in the composition of glycosaminoglycans and proteoglycans in the lungs have been reported in animal models and human lung disease. A consistent finding in animal models such as exposure to lipopolysaccharide, bleomycin, and silica is an increase in the synthesis of CS and DS. This change occurs within the first 72 h following exposure and results in

187

a shift from the normal lung with HS predominating to the diseased lung where the predominant GAG is CS and DS. An increased deposition of the CSPG, versican, is a feature common to a number of interstitial lung diseases, including acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis, bronchiolitis obliterans organizing pneumonia, and lymphangioleiomyomatosis. Often associated with the increased expression of versican in the lungs is an increased deposition of hyaluronan and the CSPGs, decorin and biglycan. Patients with severe asthma have increased expression of versican and biglycan in their airways, which is correlated with airway responsiveness. Because of the potential for matrix PG and hyaluronan to affect mechanical properties and cellular function in the lungs, changes in their expression will most likely affect the clinical course of lung diseases. However, further work is required to determine the role of proteoglycans in the pathogenesis of lung disease. See also: Alveolar Wall Micromechanics. Extracellular Matrix: Matricellular Proteins; Surface Proteoglycans. Fibroblast Growth Factors. Smooth Muscle Cells: Airway.

Further Reading Allen BL, Filla MS, and Rapraeger AC (2001) Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition. Journal of Cell Biology 155(5): 845–858. Bensadoun ES, Burke AK, Hogg JC, and Roberts CR (1996) Proteoglycan deposition in pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine 154(6 Pt 1): 1819–1828. Cavalcante FS, Ito S, Brewer K, et al. (2005) Mechanical interactions between collagen and proteoglycans: implications for the stability of lung tissue. Journal of Applied Physiology 98(2): 672–679. Frevert CW, Kinsella MG, Vathanaprida C, et al. (2003) Binding of interleukin-8 to heparan sulfate and chondroitin sulfate in lung tissue. American Journal Respiratory Cell and Molecular Biology 28(4): 464–472. Fust A, LeBellego F, Iozzo RV, Roughley PJ, and Ludwig MS (2005) Alterations in lung mechanics in decorin-deficient mice. American Journal of Physiology: Lung Cellular and Molecular Physiology 288(1): L159–L166. Giri SN, Hyde DM, Braun RK, et al. (1997) Antifibrotic effect of decorin in a bleomycin hamster model of lung fibrosis. Biochemical Pharmacology 54(11): 1205–1216. Halfter W, Dong S, Schurer B, and Cole GJ (1998) Collagen XVIII is a basement membrane heparan sulfate proteoglycan. Journal of Biological Chemistry 273(39): 25404–25412. Karlinsky JB, Bucay PJ, Ciccolella DE, and Crowley MP (1991) Effects of intratracheal endotoxin administration on hamster lung glycosaminoglycans. American Journal of Physiology 261(2 Pt 1): L148–L155. Noonan DM, Fulle A, Valente P, et al. (1991) The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule. Journal of Biological Chemistry 266(34): 22939–22947.

188 EXTRACELLULAR MATRIX / Surface Proteoglycans Sannes PL, Khosla J, Li CM, and Pagan I (1998) Sulfation of extracellar matrices modifies growth factor effects on type II cells on laminin substrata. American Journal of Physiology 275(4 Pt 1): L701–L708. Sasaki T, Fukai N, Mann K, et al. (1998) Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO Journal 17(15): 4249–4256. Smits NC, Robbesom AA, Versteeg EM, et al. (2004) Heterogeneity of heparan sulfates in human lung. American Journal of respiratory Cell and Molecular Biology 30(2): 166–173. Spillmann D, Witt D, and Lindahl U (1998) Defining the interleukin-8-binding domain of heparan sulfate. Journal of Biological Chemistry 273(25): 15487–15493. Tapanadechopone P, Hassell JR, Rigatti B, and Couchman JR (1999) Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan. Biochemical and Biophysical Research Communications 265(3): 680–690. Westergren-Thorsson G, Chakir J, Lafreniere-Allard MJ, Boulet LP, and Tremblay GM (2002) Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects. International Journal of Biochemistry & Cell Biology 34(10): 1256–1267.

Surface Proteoglycans P W Park, Y Chen, and K Hayashida, Baylor College of Medicine, Houston, TX, USA

defines proteoglycans as heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), or keratan sulfate proteoglycans (KSPGs). Some proteoglycan core proteins carry both heparan sulfate (HS) and chondroitin sulfate (CS) chains. Surface proteoglycans include the syndecan and glypican families with four and six members in mammals respectively, NG2 (AN2 in mice), betaglycan, thrombomodulin, a5b1 integrin, and CD44. The first studies on GAGs and proteoglycans date back to 1916 when heparin, a highly sulfated version of HS, was unexpectedly identified as a potent anticoagulant in liver extracts (hence the name heparin) by a medical student who was trying to isolate a procoagulant molecule. HS was first thought to be a contaminant in the heparin preparation, but was later distinguished from heparin in 1948 by the difference in the extent of sulfation and greater structural variability. For a long time, biological functions of proteoglycans were largely speculative and, in fact, most proteoglycans were thought to be specific to cartilage, functioning as cushions in joints for variable, compressive loads. Recent studies have revealed that surface proteoglycans also function as key modulators of many molecular interactions, including those relevant to lung biology.

& 2006 Elsevier Ltd. All rights reserved.

Structure Abstract Cell surface proteoglycans, such as syndecans, glypicans, and CD44, regulate a wide variety of molecular interactions that mediate cell adhesion, migration, proliferation, and differentiation. Through these activities, surface proteoglycans modulate many pathophysiological processes, including development, inflammation, tissue repair, cancer metastasis, and infection. Proteoglycans are composites of glycosaminoglycans (GAGs) attached covalently to core proteins. The majority of the ligandbinding activities of proteoglycans are mediated through GAGs. Recent studies have revealed that surface proteoglycans play key roles in lung biology. In humans, expression of several surface proteoglycans is reduced in lung cancers, suggesting that these proteoglycans are potential prognostic markers. Mice lacking certain GAG biosynthetic enzymes die soon after birth from conditions resembling acute respiratory distress syndrome (ARDS), indicating that proteoglycans are essential for lung development and function. Furthermore, inflammatory responses in mice made null for syndecan-1 and CD44 are drastically altered in various models of lung inflammation. These data highlight the physiological significance of surface proteoglycans in lung development and in the pathogenesis of respiratory diseases.

Introduction Nearly all mammalian cells express proteoglycans on their cell surface. The chemical nature of the glucosaminoglycans (GAGs) attached to core proteins

A proteoglycan consists of a core protein and one or several covalently attached GAG chains. GAGs are attached to and polymerized on certain Ser residues of a Ser-Gly dipeptide sequence, often repeated two or more times. GAGs are linear polysaccharides consisting of repeating disaccharide units that are defined by the composition and chemical linkage of the amino sugar and uronic acid monosaccharides in the disaccharide unit. The signature disaccharide repeat of an HS/heparin polysaccharide is GlcUAb1-4GlcNAca1–4, CS (including dermatan sulfate) is GlcUAb1–3GalNAcb1–4, keratan sulfate (KS) is Galb1-4GalNAcb1–3, and hyaluronan (HA) is GlcUAb1–3GlcNAcb1–4. Except for HA, these primary structures are modified in the Golgi apparatus by several sulfation and epimerization reactions that are catalyzed by distinct enzymes. Because the polymerization and modification reactions do not go to completion, the biosynthetic process generates an exceptionally diverse array of GAG structures, both in length and extent of modification. For example, HS, the most structurally heterogeneous GAG, varies in length from 50 to 150 disaccharides with cell type and core protein. However, a mere HS decasaccharide can potentially assume over 106 distinct sequences, which is already in vast excess of the estimated gene products

EXTRACELLULAR MATRIX / Surface Proteoglycans

that the whole human genome can generate. This enormous structural diversity largely explains why and how HS binds to so many proteins. Except for HA, HS, CS, and KS are all found covalently linked to specific core proteins in vivo as proteoglycans. Surface proteoglycans harbor HS, CS, or both. Core proteins of syndecans, NG2, CD44, betaglycan, and thrombomodulin are type I transmembrane proteins, whereas core proteins of glypicans are attached to the surface through a glycosylphosphatidylinositol (GPI) anchor. As shown in Figure 1, these core proteins have distinct structural designs and GAG-attachment sites. For example, syndecan core proteins contain an extracellular domain extended in conformation due to a high Pro content, whereas glypican extracellular domains are globular because they contain 14 highly conserved Cys residues that form intramolecular disulfide

Syndecans (core proteins: 22– 46 kDa) HS

189

bonds. HS chains are attached distal to the plasma membrane on all syndecans and CS chains are also attached proximal to the cell surface on some syndecans (e.g., syndecan-1). GAG-attachment sites in NG2 (CS) and CD44 (HS/CS) are located in the middle portion of the core protein, whereas those of glypicans (HS), thrombomodulin (CS), and betaglycan (HS/CS) are proximal to the cell surface. The short cytoplasmic domain of syndecans contains several signaling and scaffolding motifs, such as three highly conserved Tyr and one highly conserved Ser for potential phosphorylation, and a C-terminal PDZ-binding domain. The NG2 cytoplasmic domain also contains a PDZ-binding domain and several Thr residues that may be phosphorylated. The CD44 cytoplasmic domain contains an ezrin, radixin, moesin (ERM) motif that may link surface CD44 to the actin cytoskeleton. No obvious signaling or

Glypicans (core proteins: 62– 66 kDa)

CD44 (core protein: varies with alt. splicing)

HS S-S HS or CS CS

CS

CS HS

Betaglycan (core protein: 94 kDa)

HS GPI

Thrombomodulin (core protein: 62 kDa)

NG2 (core protein: 200 kDa)

CS HS or CS

HS or CS

CS

Figure 1 Core proteins of syndecan and glypicans: structural designs and GAG-attachment sites. HS, heparan sulfate; CS, chondroitin sulfate.

190 EXTRACELLULAR MATRIX / Surface Proteoglycans

scaffolding motifs have so far been found in the cytoplasmic domains of thrombomodulin and betaglycan.

Regulation of Production and Activity The temporal and spatial expression patterns of surface proteoglycans are highly regulated. For example, syndecan-1 is first detected at the four-cell stage in mouse embryos, suggesting that its expression is zygotically activated. In adult lung, syndecan-1 is the predominant HSPG expressed on the basolateral surface of airway and alveolar epithelia. Lung epithelia also express syndecan-4, although its expression is much lower than that of syndecan-1. Several inflammatory mediators (e.g., transforming growth factor beta (TGF-b), antimicrobial peptides) and pathological conditions (e.g., cancer, myocardial infarction) regulate the expression of surface proteoglycans at the transcriptional and posttranscriptional level. Furthermore, surface expression of syndecans and CD44 can be regulated by a proteolytic cleavage mechanism called ectodomain shedding. Ectodomain shedding is of paramount importance to proteoglycan biology because it both rapidly changes the surface phenotype of affected cells by reducing the amount of surface GAGs and generates soluble proteoglycan ectodomains replete with all their GAGs that can function as autocrine or paracrine effectors. Glypicans are also released from the cell surface by cleavage of their GPI anchor by phopholipases in vitro, suggesting that glypicans also function as soluble molecules in vivo. Other surface proteoglycans are also found in soluble form, but how they are released from the cell surface is not known. Because GAG structures dictate ligand-binding activities of proteoglycans, regulation of GAG biosynthetic enzymes is also thought to modulate proteoglycan function.

Biological Functions Surface proteoglycans, especially those harboring the highly structurally diverse HS chains, bind and regulate a plethora of biological molecules through their GAG chains. The list includes growth factors, cytokines, chemokines, proteinases and inhibitors, antimicrobial peptides, coagulation factors, surfactant proteins, extracellular matrix components, and many more. Surface proteoglycans can serve as primary receptors for some ligands (e.g., lipoprotein lipase). However, in most cases, surface proteoglycans function as co-receptors that capture ligands and catalyze the encounter between ligands and their respective signaling receptors. Surface proteoglycans can also regulate receptor–ligand interactions by

affecting the stability, conformation, and oligomerization state of either ligand or signaling receptor. Furthermore, binding of some chemokines to surface proteoglycans generates a chemotactic gradient that directs migration of inflammatory cells to specific sites of tissue injury. Surface proteoglycans also function as soluble molecules because they can be released from the surface by ectodomain shedding and other mechanisms. Once solubilized, surface proteoglycans show functions similar to or distinct from their immobilized counterparts. The biological significance of surface proteoglycan functions is best exemplified by phenotypes of mice made null for the various biosynthetic enzyme and core protein genes. For example, mice lacking N-deacetylase/N-sulfotransferase-1 or C5 epimerase, enzymes required for the synthesis of sulfated HS, die shortly after birth due to respiratory failure. CD44 null mice show defects in the tissue distribution of myeloid progenitors. Glypican-3 null mice show phenotypes resembling Simpson–Golabi–Behmel syndrome, a human overgrowth disorder, and they also show abnormal lung development. No apparent congenic abnormalities have so far been found in syndecan-1, -3, and -4, and glypican-2 and -4 null mice. However, syndecan-1 and -4 null mice show abnormal phenotypes when subjected to experimental models of inflammatory diseases, and syndecan-1 null mice, in particular, exhibit various lung phenotypes as described below.

Surface Proteoglycans in Respiratory Diseases Several lines of evidence indicate that surface proteoglycans are central players in the pathogenesis of major respiratory diseases (Figure 2). In lung cancer, surface expression of syndecan-1 and glypican-3 is significantly reduced in lung carcinomas. In general, low expression of surface proteoglycans is associated with a poor prognosis in lung and other cancers. High levels of soluble syndecan-1 ectodomains are also associated with a poor outcome in lung carcinomas, suggesting that aberrant activation of syndecan-1 shedding may promote lung cancer progression. The biological basis for the correlation between surface proteoglycan expression and lung cancer has yet to be clearly defined, but it is speculated that normal expression of surface proteoglycans is required to regulate cellular activities central to cancer progression, such as adhesion, migration, and proliferation. Lung inflammation is exacerbated in CD44 null mice instilled intratracheally with bleomycin and in syndecan-1 null mice instilled intranasally with

EXTRACELLULAR MATRIX / Surface Proteoglycans

191

Asthma: inhibit CC chemokine-mediated Th2 cell homing, attenuate allergic lung inflammation Ectodomain

Shedding

Lung infection: inhibit host defense, promote infection Lung cancer: promote cancer growth, metastasis

Acute lung injury: generate CXC chemokine gradient that guides neutrophil transepithelial migration

Lung infection: promote microbial adhesion and invasion Lung cancer: regulate cell proliferation and migration Lumen

Airway epithelia

Figure 2 Functions of surface proteoglycans in lung diseases.

allergens, suggesting that these surface proteoglycans function as anti-inflammatory molecules in idiopathic interstitial pneumonia and asthma. In the mouse model of interstitial pneumonia, CD44 is required to clear apoptotic neutrophils and accumulated HA fragments at sites of tissue injury, and to assure the correct activation of TGF-b1. In the mouse model of asthma, syndecan-1 attenuates allergic lung inflammation by inhibiting the recruitment of Th2 cells to the lung, a central process in asthma pathogenesis. Here, syndecan-1 shedding by airway epithelial cells is activated by allergen challenge and shed ectodomains attenuate lung inflammation by binding and inhibiting the CC chemokines CCL7, CCL11, and CCL17 through their HS chains. Syndecan-1 shedding has also been found to modulate lung inflammation in bleomycin-induced acute lung injury. Upon injury, newly synthesized KC (a CXC chemokine, mouse functional homolog of human interleukin-8 (IL-8)) binds to syndecan-1 HS on alveolar epithelial cells and the syndecan-1/KC complex is shed by MMP-7. This process generates a KC gradient that guides the transepithelial migration of neutrophils into the alveolar compartment. Interestingly, syndecan-1 null mice are more susceptible to bleomycin-induced tissue damage and lethality, indicating that syndecan-1 shedding protects the host in acute lung injury by regulating and confining inflammation to specific sites of tissue injury. In fact, these and other available data suggest that activation of epithelial syndecan-1 shedding is an innate host

response that assures the adequate and correct functioning of host inflammatory responses. In lung infection, accumulating evidence indicates that many bacterial and viral pathogens exploit surface proteoglycans to promote their pathogenesis. First, respiratory pathogens, such as Mycobacterium spp., Bordetella pertussis, Haemophilus influenzae, parainfluenza virus, and respiratory syncytial virus, bind to the HS moiety of surface HSPGs to promote their adhesion and also their invasion of host cells if they are intracellular pathogens. In fact, microbial exploitation of surface HSPGs for host cell adhesion and invasion is a mechanism common to many intracellular pathogens, including nonrespiratory pathogens such as Neisseria gonorrhoeae, Leishmania spp., and herpes simplex virus. Second, several respiratory pathogens, such as Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae, also take advantage of airway syndecan-1 by inducing ectodomain shedding to promote their lung pathogenesis. These pathogens activate syndecan-1 shedding through secreted virulence factors, and the shed ectodomains inhibit various host defense factors (e.g., antimicrobial peptides, collectins) through their HS chains. These processes modify the normally sterile pulmonary environment to favor infection over eradication. Consistent with this mechanism, syndecan-1 null mice resist intranasal P. aeruginosa, S. aureus, and S. pneumoniae lung infections, and wild-type mice are protected from infection by intranasal administration of ectodomain

192 EXTRACELLULAR MATRIX / Degradation by Proteases

shedding and HS antagonists. Together, these studies underscore the importance of surface proteoglycan functions in the pathogenesis of major respiratory diseases. Current efforts aimed at further dissecting the molecular mechanisms of how surface proteoglycans function in lung biology should yield relevant information needed to design new therapeutic approaches to attenuate, halt, or reverse various lung disorders. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Chemokines. Chemokines, CXC: IL-8. Extracellular Matrix: Matricellular Proteins. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Degradation by Proteases P J Stone and S M Morris, Boston University School of Medicine, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Matrix degrading enzymes are proteinases that are capable of processing substrates that are bound to surfaces or lie beneath the surface of tissue. The in vitro method of assessing matrix proteinases using purified substrates is relatively straightforward. However, extrapolation of the result to the case of the complex substrate or in vivo situation is often difficult and may lead to inaccurate predictions. An example of this is two proteinases that have similar elastolytic potency with purified elastin substrates but substantially different emphysema-inducing potencies in animal models.

Further Reading Bernfield M, Go¨tte M, Park PW, et al. (1999) Functions of cell surface heparan sulfate proteoglycans. Annual Review of Biochemistry 68: 729–777. Bernfield M, Kokenyesi R, Kato M, et al. (1992) Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annual Review of Cell Biology 8: 365–393. Conrad HE (1998) Heparin-Binding Proteins, pp. 1–527. San Diego, CA: Academic Press. Esko JD and Selleck SB (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annual Review of Biochemistry 71: 435–471. Filmus J and Selleck SB (2001) Glypicans: proteoglycans with a surprise. Journal of Clinical Investigation 108: 497–501. Forsberg E and Kjelle´n L (2001) Heparan sulfate: lessons from knockout mice. Journal of Clinical Investigation 108: 175–180. Gallagher JT (2001) Heparan sulfate: growth control with a restricted sequence menu. Journal of Clinical Investigation 108: 357–361. Li Q, Park PW, Wilson CL, and Parks WC (2002) Matrilysinmediated shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell 111: 635–646. Park PW, Pier GB, Hinkes MT, and Bernfield M (2001) Exploitation of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Nature 411: 98–102. Park PW, Reizes O, and Bernfield M (2000) Cell surface heparan sulfate proteoglycans: selective regulators of ligand– receptor encounters. Journal of Biological Chemistry 275: 29923–29926. Reizes O, Park PW, and Bernfield M (2000) Cell surface heparan sulfate proteoglycans. In: Beckerle M (ed.) Cell Adhesion: Frontiers in Molecular Biology, pp. 155–188. Oxford: Oxford University Press. Rostand KS and Esko JD (1997) Microbial adherence to and invasion through proteoglycans. Infection and Immunity 65: 1–8. Sanderson RD (2004) Heparan sulfate proteoglycans and heparanase: partners in osteolytic tumor growth and metastasis. Matrix Biology 23: 341–352. Teder P, Vandivier RW, Jiang D, et al. (2002) Resolution of lung inflammation by CD44. Science 296: 155–158. Tyrrell DJ, Horne AP, Holme KR, Preuss JM, and Page CP (1999) Heparin in inflammation: potential therapeutic applications beyond anticoagulation. Advances in Pharmacology 46: 151–208.

Definition of Matrix Degrading Enzymes Proteolytic enzymes (proteinases) catalyze the thermodynamically favored (but, normally, extremely slow) hydrolysis of peptide bonds in their selected substrates. The catalytic hydrolysis results in the addition of one water molecule across a peptide bond, usually in a number of steps, resulting in a new N-terminal amino group and a new C-terminal carboxyl group. Proteolytic processing often regulates processes involving those substrates and their products. Antiproteases regulate proteinase activity by slowing or inactivating the catalytic site or interfering with proteinase–substrate interactions. Matrix degrading enzymes are proteinases that are capable of processing substrates that are bound to surfaces or lie beneath the surface of tissue. Thus, matrix processing enzymes are able to catalytically hydrolyze peptide bonds within a polymeric extracellular matrix protein, such as elastin. The core protein chain of proteoglycans can be degraded by a number of proteinases, including human neutrophil elastase. In addition, the glycosaminoglycan chains attached to the core protein can be degraded by a number of glycosaminoglycan degrading enzymes. Chronic excess elastin degradation is thought to play a central role in the pathogenesis of pulmonary emphysema.

Classification of Matrix Degrading Enzymes Proteinases are usually categorized by their active site components that catalyze peptide bond hydrolysis: serine proteinases, (matrix) metalloproteinases, cysteine proteinases, and aspartyl proteinases. Serine

EXTRACELLULAR MATRIX / Degradation by Proteases 193

proteinases, metalloproteinases, and cysteine proteinases are discussed in detail elsewhere. Excess proteolysis by these enzymes has been linked to a number of pathologic processes. More than 500 genes in the human genome encode proteinases or proteinase-like sequences.

Identification of Matrix Degrading Enzymes and Their Sources Sources of matrix degrading enzymes in the lung include blood-derived inflammatory cells, microorganisms, and endogenous lung cells. Fibroblasts and smooth muscle cells can be induced to remodel surrounding extracellular matrix by expressing proteinases, including serine and metalloproteinases. Studies using transgenic animal models in which genes for individual proteinases are knocked out have been used to help identify suspect host proteinases in lung diseases such as pulmonary emphysema. Studies in which the substrate preferences of proteinases are altered by site-directed mutagenesis of individual amino acids in the proteinase may further pinpoint the role of these proteinases.

Assessment of Matrix Degradation There is a wide range of assays to assess matrix degradation. These include in vitro methods that use a purified substrate, or a complex substrate derived from a living system that contains the substrate of interest. There are also in vivo methods in which degraded substrate is measured in biological fluids or tissues. While the in vitro method using purified substrate is relatively straightforward, extrapolation of the result to the case of the complex substrate or in vivo situation is often difficult and may lead to inaccurate predictions. In Vivo Assays

Elevated expression of a matrix degrading proteinase in vivo may not necessarily result in a proportionate increase in degradation product. Factors such as compensating increases in proteinase inhibitor levels or lack of access of proteinase to the substrate may intervene. Degradation may also be decreased by the distance between the source of the proteinase and its substrate. On the other hand, the presence of a protected microenvironment could favor degradation, such as one created by an adherent neutrophil releasing proteinase in close juxtaposition to the substrate. Measurement of substrate degradation in vivo is often complicated by the presence of many interfering

materials in the sampled biologic fluid that are similar to substrate degradation products. For example, the measurement in the urine of desmosine, a crosslink amino acid characteristic of elastin, is rendered difficult by the presence of chemically similar nitrogen metabolites. In principle, the amount of desmosine in the urine should reflect the degradation of elastin in the organism, since desmosine does not appear to be metabolized by the liver. However, the kidneys may slow by sequestration the excretion of increased levels of desmosine and release the excess desmosine over several days following in vivo treatment with elastase. On the other hand, measurement of desmosine in lung fluids such as sputum or bronchoalveolar lavage fluid should be relatively simple, but the correlation with lung elastin degradation is challenging, due to sampling and dilution effects. Two elastase enzymes exhibit different emphysemainducing potency An example of the discrepant results between in vivo and in vitro assays is provided by studies of two elastolytic enzymes, porcine pancreatic elastase and human neutrophil elastase, that have been used to produce experimental emphysema in rodent models by introduction into the lungs. It was observed that hamsters receiving nearly equal molar amounts of porcine pancreatic elastase or human neutrophil elastase exhibited a 59% increase in airspace size with the former but only a 19% increase with the latter. There was also a positive correlation between the increase in airspace size and the urine desmosine levels for the first 3 days after treatment with elastase. Elevated urine desmosine levels were evidence for increased elastin destruction. Urine desmosine levels for animals receiving saline only, human neutrophil elastase, or porcine pancreatic elastase were 74, 212, and 816 ng per animal per day for the first 3 days. However, both porcine pancreatic elastase and human neutrophil elastase exhibited similar enzymatic half-lives in the lung as measured in lavage fluid, 51 and 45 min, respectively. Assays Using Purified Substrates: The Two Elastases Exhibit Similar Activity

The elastin solubilizing activities of these two elastases were compared using purified elastin substrates. With elastin purified from calf neck ligament using a relatively gentle autoclaving method, human neutrophil elastase and porcine pancreatic elastase exhibited comparable elastolytic activity at 371C, that is, 367743 and 381746 mg elastin solubilized per nmole of elastase h
1 (mean7SD), respectively.

194 EXTRACELLULAR MATRIX / Degradation by Proteases Assays Using Reconstituted Systems Elucidate Differences in Potency of the Two Elastases

Cell culture models When the same amount of porcine pancreatic elastase or human neutrophil elastase was placed in a tissue culture flask containing a 5-week-old cell culture of primary smooth muscle cells with the serum washed away, the porcine pancreatic elastase solubilized 117728 mg elastin per nmol elastase and the human neutrophil elastase solubilized only 1173 mg elastin per nmol elastase or 971% of the 117 mg elastin solubilized by porcine pancreatic elastase. Yet, with elastin purified from the cultures porcine pancreatic elastase and human neutrophil elastase exhibited comparable levels of elastin solubilizing activity, 297738 and 2707133 mg elastin solublized per nmol of elastase h
1, respectively. Thus, human neutrophil elastase exhibited a 6.7- to 25-fold reduction in its elastin solubilizing activity with intact cell cultures as compared with the purified elastin substrate, whereas porcine pancreatic elastase exhibited only a 1.5- to 2.5-fold reduction. Desmosine cross-links were used as the marker for elastin in the protein mixture. This effect could not satisfactorily be explained as preferential inhibition of human neutrophil elastase activity in the culture system, because the amount of protein solubilized by human neutrophil elastase was 59% that of porcine pancreatic elastase. The mean elastin content of porcine pancreatic elastase-solubilized protein was 110% that of the elastin content of the corresponding cell culture; the value for human neutrophil elastase-solubilized protein was only 16%. The amount of elastin per microgram of solubilized protein for human neutrophil elastase was 15% that for porcine pancreatic elastase. Additional insights into the mechanism behind this greatly decreased activity of human neutrophil elastase in solubilizing elastin in complex substrates were obtained by immunocytochemical study of these cultures employing antibodies to human neutrophil elastase and porcine pancreatic elastase. This study revealed that porcine pancreatic elastase penetrates the smooth muscle cell cultures more readily than does human neutrophil elastase (Figure 1). Because the amount of elastin in these cultures increases with increasing distance from the free surface, the lesser amounts of elastin solubilized by human neutrophil elastase may be partly due to poor penetration of human neutrophil elastase into the living extracellular matrix, resulting in limited access to elastin substrate. Ultrastructural and immunocytochemical studies indicated, however, that even when human neutrophil elastase does have access to elastin substrate, it is less efficient than porcine pancreatic elastase at penetrating and degrading individual elastic fibers (not shown).

(a)

(b) Figure 1 Smooth muscle cell cultures treated with (a) porcine pancreatic elastase and (b) human neutrophil elastase. Elastase was localized with a primary antibody specific for each enzyme followed by a secondary antibody conjugated to colloidal gold followed by silver enhancement to produce a black precipitate at the site of the elastase. Equivalent amounts of enzyme and equal exposure times resulted in complete penetration of the culture by porcine pancreatic elastase, while human neutrophil elastase remained near the top of the culture. Scale bar ¼ 10 mm.

Human lung tissue A similar approach was used in comparing the activity of human neutrophil elastase and porcine pancreatic elastase in solubilizing elastin in human lung tissue. Lung tissue was exposed in vitro to human neutrophil elastase or porcine pancreatic elastase. Although both enzymes solubilized protein at similar rates, porcine pancreatic elastase solubilized elastin five times faster than did human neutrophil elastase, based on measurement of soluble desmosine cross-links characteristic of elastin. Only 1% of the protein solubilized by human neutrophil elastase was elastin, whereas elastin accounted for 8% of the protein solubilized by porcine pancreatic elastase. Ultrastructurally, human neutrophil elastase-exposed tissue exhibited fewer damaged elastic fibers as well as some fibers that were damaged at the edges, whereas the interior of the fiber appeared intact. Elastic fibers in tissue exposed to porcine pancreatic elastase always showed extensive damage in both the exterior and interior of the fiber. Immunocytochemical studies in which antibodies to human neutrophil elastase and porcine pancreatic elastase were applied to thin sections of Lowicryl-embedded tissue indicated that both of these elastases could be detected in association with elastic fibers, but only in areas of the fiber that showed morphological evidence of elastase injury (Figure 2). Factors that might explain the relatively low elastolytic activity of human neutrophil elastase in vivo or in reconstituted systems include the lower ability

EXTRACORPOREAL MEMBRANE-GAS EXCHANGE 195

(a)

(b)

Figure 2 Elastic fibers in human lung tissue exposed to (a) porcine pancreatic elastase or (b) human neutrophil elastase. Primary antibody specific for each elastase was followed by a secondary antibody conjugated to colloidal gold. Elastolytic digestion is evident throughout the elastic fiber in tissue treated with pancreatic elastase and colloidal gold particles are seen over the entire fiber. The elastic fiber exposed to neutrophil elastase shows damage and colloidal gold particles only at the periphery. (a) Scale bar ¼ 0.5 mm; (b) scale bar ¼ 0.25 mm.

of human neutrophil elastase to move through tissue as well as penetrating individual elastic fibers. Purification of elastin involves the homogenization of tissue and the formation of finely divided elastin that is more susceptible to degradation by human neutrophil elastase. Other possibilities include the presence of sulfated proteoglycans in the extracellular matrix, such as proteoglycans containing chondroitin sulfate and heparan sulfate that are inhibitors of human neutrophil elastase and might exert a protective effect. See also: Chronic Obstructive Pulmonary Disease: Emphysema, General. Extracellular Matrix: Elastin and Microfibrils; Matrix Proteoglycans; Surface Proteoglycans. Matrix Metalloproteinases. Serine Proteinases.

Further Reading Lopez-Otin C and Overall CM (2002) Protease degradomics: a new challenge for proteomics. Nature Reviews 3: 509–519. Morris SM and Stone PJ (1995) Immunocytochemical study of the degradation of elastic fibers in a living extracellular matrix. Journal of Histochemistry and Cytochemistry 43: 1145–1153.

Morris SM, Stone PJ, and Snider GL (1993) Electron microscopic study of human lung tissue after in vitro exposure to elastase. Journal of Histochemistry and Cytochemistry 41: 851–866. Owen CA, Campbell MA, Sannes PL, Boukedes SS, and Campbell EJ (1995) Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. Journal of Cell Biology 131: 775–789. Shapiro SD, Goldstein NM, Houghton AM, et al. (2003) Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. American Journal of Pathology 163: 2329–2335. Starcher B and Peterson B (1999) The kinetics of elastolysis: elastin catabolism during experimentally induced fibrosis. Experimental Lung Research 25: 407–424. Stone PJ, Bryan-Rhadfi J, Lucey EC, et al. (1991) Measurement of urinary desmosine by isotope dilution and high performance liquid chromatography: correlation between elastase-induced air-space enlargement in the hamster and elevation of urinary desmosine. American Review of Respiratory Disease 144: 284–290. Stone PJ, McMahon MP, Morris SM, Calore JD, and Franzblau C (1987) Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase: an in vitro model of elastolytic injury. In Vitro Cellular & Developmental Biology 23: 663–676.

EXTRACORPOREAL MEMBRANE-GAS EXCHANGE K Lewandowski and M Lewandowski, Elisabeth Krankenhaus, Essen, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract For patients with severe acute respiratory failure whose pulmonary gas exchange cannot be improved sufficiently by means of

advanced intensive care, extracorporeal membrane oxygenation may be established as an additional therapeutic option during the acute phase. Extracorporeal membrane oxygenation is able to partly take over oxygenation and carbon dioxide removal and thereby allow respirator settings to be adjusted to the aims of lung protective mechanical ventilation. The key features of an extracorporeal circuit are catheters for vascular access, tubing for blood drainage and return, pump, membrane lung, heat exchanger, and monitoring unit. Venovenous circuits are established

196 EXTRACORPOREAL MEMBRANE-GAS EXCHANGE if the major goal is respiratory support while venoarterial techniques are considered if combined cardiac and respiratory failure is an issue. Bleeding remains the most prevailing complication in adult patients with acute respiratory distress syndrome (ARDS) undergoing extracorporeal gas exchange, and seems partly related to anticoagulation. Surface-heparinized extracorporeal circuits and membranes markedly reduce the daily blood loss and survival rates are higher than in patients treated with nonheparinized systems. Survival rates of adult ARDS patients treated with extracorporeal gas exchange are 33–50%. In neonates with severe respiratory failure, survival rates of 68% and higher are reported.

Introduction Extracorporeal gas exchange has an established role as salvage therapy for severe respiratory or cardiac failure in adults, children, and neonates unresponsive to optimal ventilator and intensive care management. In patients with acute respiratory distress syndrome (ARDS), the technique may be helpful in establishing lung protective mechanical ventilation and thereby avoiding ventilator-associated lung injury. Further indications of extracorporeal gas exchange include treatment of acute exacerbations of chronic lung diseases and ‘bridging’ therapy for lung transplant recipients until an organ becomes available. It is the purpose of this article to review the history, rationale, technique, and outcome of this demanding procedure.

History Gibbon began developing the heart-lung machine in 1937 to permit open-heart surgery. He designed a system in which anticoagulated blood was directly exposed to oxygen (‘film’ or ‘bubble oxygenators’). However, due to the direct contact between blood and the gaseous phase severe hemolysis, thrombocytopenia, hemorrhage, and organ failure complicated the treatment and limited the use of this device to a few hours. In 1956, Clowes and his associates developed an artificial lung that separated the gaseous from the liquid phase by a membrane. This ‘membrane oxygenator’, with subsequent improvements in materials, provided faster and more efficient blood oxygenation with fewer complications than the ‘film’ or ‘bubble oxygenators’ and became practical for cardiopulmonary bypass taking more than a few hours. In 1972, Hill and colleagues reported the survival of a 24-year-old polytraumatized patient with ARDS who had been treated with extracorporeal membrane oxygenation (ECMO) during the acute phase of the disease. Four years later, Bartlett and colleagues described the first newborn to be treated with ECMO, who survived. These enthusiastic publications nourished the hope that an effective new symptomatic therapy for

severe hypoxia in ARDS was now available. A large randomized multicenter trial was launched in 1974 to test venoarterial ECMO versus conventional therapy in adult ARDS patients. The study revealed that mortality rates in the ECMO therapy group were as high as 90% and not significantly different from those in the conventionally treated group. In the following years, Kolobow and Gattinoni presented an advanced technique where oxygen uptake and carbon dioxide (CO2) removal were dissociated. Oxygenation was primarily accomplished through the nearly motionless natural lung via apneic oxygenation or low-frequency positive-pressure ventilation (LFPPV), while CO2 was cleared through the artificial lung (extracorporeal CO2-removal, ECCO2R). ECCO2-R was performed at low extracorporeal blood flows (20–30% of cardiac output) so that a venovenous bypass technique instead of a venoarterial one sufficed; this turned out to be less harmful to blood cells, coagulation, and internal organs. With this technique, survival rates of up to 49% were reported. In 1994, a second randomized controlled trial tested the LFPPV-ECCO2-R technique against advanced standard treatment. Again, survival rates in the ECCO2-R group were not significantly different from those in the conventionally treated group (42% conventional vs. 33% ECCO2-R). In 1983, Swedish researchers further improved the ECMO technique by binding the heparin molecule covalently to all synthetic surfaces of the circuit. It was thereby possible to avoid full systemic anticoagulation.

Uses in Respiratory Medicine Terminology

Several acronyms have been used to describe the variety of techniques that have been invented to extracorporeally oxygenate blood and remove CO2. When the acronym ECMO was coined in the 1970s, it stood for a high-flow venoarterial bypass system that was aimed primarily at blood oxygenation. Subsequently, this method was refined and other forms of extracorporeal bypass techniques were introduced (see Table 1). The acronym ECMO survived the changing technologies and today is a general byword for the wide range of methods that are currently in use for extracorporeal blood oxygenation and CO2 removal. However, the question of which acronym to use is of less importance than characterizing the applied extracorporeal circulation and pulmonary support system. At least three criteria need to be reported: the vascular access used (venovenous, venoarterial, or arteriovenous), the proportion of

EXTRACORPOREAL MEMBRANE-GAS EXCHANGE 197 Table 1 Common acronyms for various types of extracorporeal gas exchange Acronym

Meaning

System

ECMO

Extracorporeal membrane oxygenation Extracorporeal CO2 removal

Traditional high-flow venoarterial bypass system aiming primarily at blood oxygenation Low-flow venovenous bypass technique for extracorporeal gas exchange and CO2 removal Low-flow venovenous bypass to eliminate a part of the body’s CO2 load in patients with chronic lung diseases Venovenous low-flow bypass system in patients who were not endotracheally intubated and mechanically ventilated Prolonged but temporary (1–30 days) support of heart or lung function using mechanical devices Arteriovenous pumpless low-flow bypass system for patients with respiratory failure

ECCO2-R PECOR ECLA

Partial extracorporeal CO2 removal Extracorporeal lung assist

ECLS

Extracorporeal life support

AVCO2, av-ECLA, pECLA

Pumpless extracorporeal lung assist

cardiac output pumped, and the ventilatory regimen of the natural lung. Rationale of ECMO

Within the last decades it has become evident from laboratory and clinical research that in patients with severe acute respiratory failure, mechanical ventilation contributes to the progression of the disease. The term ‘ventilator-induced lung injury’ (VILI) was coined to describe the non-specific radiographic, physiologic, and pathologic manifestations of acute lung injury and its complications. VILI is viewed as a systemic disease that closely resembles the symptoms and the macroand microscopical features of experimental acute lung injury and is not markedly different from the diffuse alveolar damage present in human ARDS. It may be associated with pulmonary and systemic infections, multisystem organ dysfunction, volutrauma, barotrauma, and increased mortality. Establishing a ‘lung-protective-ventilation strategy’ in severe acute respiratory failure may be incompatible with the goal of maintaining sufficient gas exchange. ECMO is able to take over part of oxygenation and CO2 removal and thereby may allow respirator settings to be adjusted to the mechanical and gas exchange properties of the diseased lung so that the aims of lung-protective mechanical ventilation, even in severe acute respiratory failure, can be achieved. ECMO Technique

Important components of an extracorporeal circuit for membrane oxygenation include catheters for vascular access, tubing for blood drainage and return, pump, membrane lung, heat exchanger, and monitoring unit. The circuit design is essentially the same in neonatal, pediatric, and adult ECMO. Nowadays a wide array of vascular access cannulas is available

for surgical introduction or percutaneous cannulation of the femoral, iliac, and jugular veins or the femoral, carotid, axillar, or iliac arteries; they are usually made of polyvinylchloride. Extracorporeal blood flow may be routed in three possible ways: venoarterial, venovenous, and arteriovenous. Venoarterial access is the traditional method and is required when there is combined cardiac and respiratory failure. Venoarterial ECMO is the most common mode of extracorporeal support in children and has so far accounted for two-thirds of treatments in this group. Venovenous access is the method of choice for respiratory support in all age groups as long as adequate cardiac function permits. In the majority of patients, especially in adults with primary respiratory disease, venovenous ECMO has proved to be satisfactory. Arteriovenous access is applied when a pumpless ECMO technique is used. This system works with the pressure gradient between artery and vein. To move the blood through the circuit, either nonocclusive or occlusive roller pumps or centrifugal pumps are employed. However, most centers prefer to use the roller pump because it generates only mild pressure fluctuations during blood inflow and outflow, and produces little hemolysis. The membrane oxygenator with a surface area of up to 4.5 m2 is the heart of the extracorporeal circuit. Most available devices work in a similar fashion. Blood flows through the tubing into the membrane lung, which is either assembled like an envelope or consists of thousands of microporous fibers of a hollow fiber device. Oxygen flows through the membrane lung in the opposite direction to flow of the bloodstream, diffuses into the blood, and CO2 is eliminated blood then enters the tubing and is perfused via the heat exchanger and via the return cannula into the patient. The initial contact of blood with foreign surfaces induces profound activation of the coagulation

198 EXTRACORPOREAL MEMBRANE-GAS EXCHANGE

system. To avoid clotting, either systemic heparin should be administered or the entire circuit needs to be coated with heparin. Finally, to make the extracorporeal system safe, it is crucial that a sophisticated level of monitoring is employed. This should include online circuit pressure monitors and flow monitors, bubble detectors, and premembrane and postmembrane oxygen saturation assessment. Figure 1 shows an example of a typical venovenous bypass. Complications of ECMO

Known hazards of the ECMO technique can be classified into mechanical and patient-related medical complications. Mechanical complications include oxygenator failure, tubing/circuit disruption, pump or heat exchanger malfunction, and problems associated with cannula placement or removal. Patient-related medical problems are bleeding, neurologic complications, additional organ failure (e.g., renal, cardiovascular, liver), barotrauma, infection, and metabolic disorders. Available literature suggests that bleeding remains the most important complication in adult patients with ARDS undergoing extracorporeal gas exchange and seems partly related to constant

anticoagulation. It has been shown that the use of surface-heparinized ECMO circuits and membranes reduces the daily blood loss, the amount of substituted red cells, and the necessary i.v. heparin dose. Survival rates are higher than in patients treated with nonheparinized systems. Outcome from ECMO

Over the last decade, survival rates of adult ARDS patients treated with extracorporeal gas exchange in prospective observational studies have stabilized at a level of 50–70%. This is in sharp contrast to the extremely low survival rates of 10% reported in the first US ECMO study of 1974 and also markedly better than those of the second randomized controlled trial of 1994 (33%). In 1996, the results from a UK ECMO randomized controlled trial in 185 neonates with severe respiratory failure showed a survival rate of 68% in the ECMO group. Survival was significantly worse in the conventionally treated control group (41%). Since long, extensive information regarding survival in ECMO-treated adults, neonates, and children with severe acute respiratory failure has been provided by the Extracorporeal Life Support Organization

Respirator

Membrane oxygenator

Roller pump

Figure 1 Schematic drawing of a low-flow venovenous ECMO circuit. In the typical low-flow venovenous bypass often used today, venous blood is drained through two heparin-coated catheters percutaneously inserted from both groins into the inferior vena cava and then connected by a Y-piece. The oxygenated blood is returned into the superior vena cava through a catheter, which is advanced percutaneously via the right internal jugular vein. A femoral-jugular venovenous bypass is established using a near-occlusive roller pump and a parallel configuration of two hollow fiber oxygenators. The gas flow rate is set to the required CO2 levels. Oxygenation is accomplished through the mechanically ventilated natural lung as well as by arterializing the circulating blood via the membrane oxygenator. Reproduced from Lewandowski K (2000) Extracorporeal membrane oxygenation for severe acute respiratory failure. Critical Care 4: 156–168, with permission from BioMed Central Ltd, Current Science Group.

EXTRACORPOREAL MEMBRANE-GAS EXCHANGE 199

(ELSO) Registry. Yearly survival rates have remained constant at 80% for neonatal cases regardless of diagnosis. In children, a survival rate of slightly more than 50% has been documented since 1989, which is a great improvement on the 30% survival rate recorded previously. Other Gas Exchange Devices

Strictly speaking, the intravenous oxygenator (IVOX) and the Hattler catheter, an intravenous membrane oxygenator, are not extracorporeal membrane-gas exchangers. Nevertheless, they are interesting in that they represent options to reduce the technical complexity, the pump-associated complications, and costs and manpower usually associated with ECMO. The IVOX device is a membrane oxygenator system (surface area 0.21–0.52 m2) configured as a complex of thin hollow fibers mounted on a catheter. It is positioned in the vena cava and venous blood circulates through the pores of the oxygenator while pure oxygen is supplied to the hollow fibers by an external pump. Compared with ECMO the gas exchange capacity appears to be significantly inferior. The average O2 transfer in preliminary human studies was 50–100 ml min
1 and the average CO2 elimination was 48–74 ml min
1. Clinicians rarely used this device; however, it has served as a prototype for future models, such as the Hattler Catheter. The features of the Hattler Catheter (see Figure 2) are somewhat similar to those of the IVOX. However, the key component of the Hattler catheter is a central pulsating balloon, helically surrounded by 1000 hollow fiber membranes. Through three exteriorized gas pathways, O2 is introduced into the body and CO2 is removed from the blood. The Hattler catheter is inserted simply through a vein in the leg, which can be done using percutaneous insertion, and placed in the vena cava with its tip located in the vena cava superior. The balloon pulsates with a frequency of 150 per min to move the fibers and mix the blood in that region. Oxygen, which is suctioned through the fibers, diffuses from the membranes into the blood. CO2 diffuses into the membranes and is removed by a vacuum tube. The device is not able to replace fully the function of the lungs, but is only able to supplement oxygenation of the body. Human lungs have a gas exchange area of 100 m2 while the Hattler catheter works with just 0.21–0.23 m2 of exchange area. The gas exchange capacity was determined to be 115–305 ml min
1 m
2 membrane surface for O2 and 128–340 ml min
1 m
2 for CO2. Finally, the pumpless arteriovenous extracorporeal lung assist (av-ECLA), also termed pumpless

Superior vena cava

Hollow fiber membranes

Pulsating central balloon

Right atrium Inferior vena cava

Insertion through peripheral vein site Exteriorized gas pathways

Figure 2 The Hattler Catheter in situ. Modified from http:// www.alung.com/cath.html, with permission from Professor B G Hattler.

extracorporeal lung assist (pECLA), is another true extracorporeal device for gas exchange (see Figure 3). The system is driven by the pressure gradient between the femoral artery and the respective vein. This is technically feasible because the drop in pressure induced by the oxygenator is approximately only 15 mmHg. Extracorporeal blood flow ranges between 1 and 2.6 l and allows for sufficient CO2 elimination (minimally 38 ml l
1 membrane oxygenator blood flow) and a remarkable increase in oxygenation (minimally 45 ml l
1 membrane oxygenator blood flow). However, these levels will only be reached if a relatively high blood flow through the oxygenator and a sufficient mean arterial blood pressure of around 70 mmHg can be established. Preliminary results in a limited

200 EXTRACORPOREAL MEMBRANE-GAS EXCHANGE

Flow sensor

Femoral artery

Oxygen inflow

Femoral vein

Figure 3 A pumpless extracorporeal lung assist placed between the patient’s legs. The device is an arteriovenous shunt with an interposed membrane oxygenator. Reproduced from Reng CM, Philipp A, Kaiser M, et al. (2000) Pumpless extracorporeal lung assist and adult respiratory distress syndrome. Lancet 356: 219–220, with permission from Elsevier.

number of patients with ARDS have revealed encouraging results. See also: Acute Respiratory Distress Syndrome. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation; Ventilator-Associated Pneumonia.

Further Reading Bartlett RH, Roloff DW, Custer JR, et al. (2000) Extracorporeal life support. The University of Michigan experience. Journal of American Medical Association 283: 904–908. Clowes GH, Hopkins AL, and Neville WE (1956) An artificial lung dependent upon diffusion of oxygen and carbon dioxide through plastic membranes. Journal of Thoracic and Cardiovascular Surgery 32: 630–637. Dreyfuss D and Saumon G (1998) Ventilator-induced lung injury: lessons from experimental studies. American Journal of Respiratory and Critical Care Medicine 157: 294–323. Gattinoni L, Pesenti A, Mascheroni D, et al. (1986) Low-frequency positive pressure ventilation with extracorporeal CO2-removal in severe acute respiratory failure. Journal of American Medical Association 256: 881–886. Gibbon JH (1937) Artificial maintenance of circulation during experimental occlusion of pulmonary artery. Archives of Surgery 34: 1105–1131. Hemmila MR, Rowe SA, Boules TN, et al. (2004) Extracorporeal life support for severe acute respiratory distress syndrome in adults. Annals of Surgery 240: 595–607. Hill JD, O’Brien TG, Murray JJ, et al. (1972) Prolonged extracorporeal oxygenation for acute post-traumatic respiratory failure (shock-lung syndrome). New England Journal of Medicine 286: 629–634. Kolobow T and Bowman RL (1963) Construction and evaluation of an alveolar membrane artificial heart-lung. Transactions of the American Society of Artificial Internal Organs 9: 238–243.

Kolobow T, Gattinoni L, Tomlinson T, and Pierce JE (1977) Control of artificial breathing using an extracorporeal membrane lung. Anesthesiology 46: 138–141. Lewandowski K (2000) Extracorporeal membrane oxygenation for severe acute respiratory failure. Critical Care 4: 156–168. Lewandowski K, Rossaint R, Pappert D, et al. (1997) High survival rate in 122 ARDS patients managed according to a clinical algorithm including extracorporeal membrane oxygenation. Intensive Care Medicine 23: 819–835. Liebold A, Reng CM, Philipp A, et al. (2000) Pumpless extracorporeal lung assist – experience with the first 20 cases. European Journal of Cardio-Thoracic Surgery 17: 608–613. Morris AH, Wallace CJ, Menlove RL, et al. (1994) Randomized clinical trial of pressure-controlled inverse ratio ventilation and extracorporeal CO2 removal for adult respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 149: 295–305. Pesenti A, Bombino M, and Gattinoni L (1998) Extracorporeal support of gas exchange. In: Marini JJ and Slutsky AS (eds.) Physiological Basis of Ventilatory Support, pp. 997–1020. New York: Dekker. Reng CM, Philipp A, Kaiser M, et al. (2000) Pumpless extracorporeal lung assist and adult respiratory distress syndrome. Lancet 356: 219–220. UK Neonatal ECMO Trial Group (1996) UK collaborative randomized trial of neonatal extracorporeal membrane oxygenation. Lancet 348: 75–82. Zapol WM, Snider MT, Hill JD, et al. (1979) Extracorporeal membrane oxygenation in severe acute respiratory failure. A randomized prospective study. Journal of American Medical Association 242: 2193–2196. Zwischenberger JB and Alpard SK (2002) Artificial lungs: a new inspiration. Perfusion 17: 253–268.

Relevant Website http://www.med.umich.edu – Extracorporeal Life Support Organization (ELSO) Registry website with information regarding survival in ECMO-treated adults, neonates, and children with severe acute respiratory failure.

F FIBRINOLYSIS Contents

Overview Plasminogen Activator and Plasmin

Overview A A-R Higazi, Hadassah–Hebrew University Medical Center, Jerusalem, Israel & 2006 Elsevier Ltd. All rights reserved.

Abstract The fibrinolytic system consists of serine proteases, their substrates, receptors, and inhibitors. Activation of the fibrinolytic cascade leads to the generation of the protease, plasmin, which cleaves the insoluble polymeric network composed primarily of fibrin. Imbalances in the system that result in the inhibition of plasmin formation lead to the deposition of fibrin in tissue as well as in vessels, common features of diverse pulmonary diseases. In addition to fibrin cleavage, each component in the plasminogen activator (PA) system is involved in a wide range of (patho)physiological processes. This involvement may result from: (1) the catalytic activity of the proteases, for example, the activity of plasmin in tissue remodeling by degrading the extracellular matrix; (2) interaction with cellular integrins and integrin ligands on cells or in matrix to regulate cell adhesion and migration and hereby immunity, tumor invasiveness, and metastasis; (3) direct or indirect induction of signal transduction by the proteases and/or their inhibitors, which can be mitogenic or regulate vascular reactivity. The multifunctionality of the components of the PA cascade has led to extensive research by groups from diverse fields. Notwithstanding the dramatic increase in recent knowledge, our understanding is far from complete and additional research is needed to clarify several aspects of the system, especially novel ways to regulate its activity in vivo.

coagulation and the fibrinolytic systems under pathologic conditions. This includes both increases in the expression of tissue factor (TF), the primary initiator of clotting, and suppression of fibrinolytic activity due, in large part, to increased synthesis and release of plasminogen activator inhibitor type 1 (PAI-1). Increased concentrations of the urokinase plasminogen activator (uPA) have also been reported in lung diseases characterized by fibrin deposition, such as pneumonia, but the presence of PAI-1 and TF seems to overcome its fibrinolytic effect. Similarly, pleural injury is characterized by early and persistent fibrin deposition and disordered fibrin turnover plays a significant role in the pathogenesis of pleural inflammation, repair, and scarring.

Components of the Fibrinolytic System The fibrinolytic system consists of serine proteases and their inhibitors. The enzymes of the fibrinolytic PA PAI-1 Plasminogen

(–) Fibrin clot Plasmin (–)

Introduction The fibrinolytic system is designed to cleave the insoluble polymeric network of fibrin into soluble fragments (Figure 1). In addition to providing the structure of blood clots, fibrin deposition is a common feature of many lung diseases, including acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pneumonia, and pleural injury. Fibrin deposition in the lung results from both systemic and local (alveolar, interstitial) imbalance between the

SFDPs


2-AP Figure 1 The fibrinolytic cascade. The plasminogen activators (PAs), tPA, uPA, scuPA (or pro-uPA), or scuPA/uPAR complex (among others), activate the proenzyme plasminogen to the powerful proteolytic enzyme plasmin. Plasmin cleaves the insoluble network of fibrin, converting it into soluble fibrin degradation products (SFDPs). Plasmin can cleave other proteins and activate other proteases, such as metalloproteinases. Plasmin can compromise the vascular integrity at sites of injury and thereby accelerate dissolution of the vascular basal lamina. Plasminogen activator inhibitor type 1 (PAI-1) inhibits tPA and uPA. a2-antiplasmin (a2-AP) inhibits the activity of plasmin.

202 FIBRINOLYSIS / Overview

cascade are expressed, in part, as proenzymes that are activated under certain physiological and pathological conditions. Activation of the system ends with the conversion of the proenzyme plasminogen into the active serine protease plasmin. The classic function of the system is summarized in Figure 1. It is designed to generate the proteolytic enzyme plasmin, which cleaves fibrin into soluble fragments. However, each component in the system has other activities and is involved in a wide range of pathophysiological processes. Plasminogen and Plasmin

Plasminogen is a zymogen or proenzyme. Plasminogen is activated to plasmin by cleavage of the Arg561– Val562 peptide bond by plasminogen activators (PAs). The resultant plasmin is a serine protease that is considered to be the major extracellular protease in plasma. In addition to fibrinolysis, plasmin can directly degrade the extracellular matrix (ECM) through its activity on fibronectin, laminin, and type IV collagen, as well as indirectly by activating prometalloproteases (MMPs), such as stromelysin and procollagenase, thereby facilitating cell migration and tissue remodeling. Plasmin also activates latent cell-associated transforming growth factor beta 1, which regulates the inflammatory response, among other effects. Plasmin can also activate the classic and alternate complement pathways and generate bradykinin from kininogen. Plasmin(ogen) in thrombotic diseases The wide specificity of plasmin poses advantages and disadvantages to the host. Clearance of intravascular fibrin averts tissues ischemia and lysis of extravascular fibrin limits ingress of inflammatory cells that use it as a matrix and thereby promotes tissue healing. On the other hand, the pleiotropic effects of plasmin are problematic when PAs are used in therapeutic concentrations to treat myocardial infarction or ischemic stroke. For example, tissue-type plasminogen activator (tPA) increases the risk of bleeding by cleaving fibrinogen (among other plasma proteins), activating MMP-9 and extravasating into extravascular tissue where it encounters additional substrates, a situation of relevance to the lung in settings in which the endothelial and epithelial barrier is impaired. Plasmin(ogen) in infectious diseases There is considerable evidence that surface-bound plasmin may facilitate bacterial invasion of pericellular matrix. Pathogenic bacteria, such as group A, C, and G streptococci and Staphylococcus aureus, or mycoplasma express plasminogen-binding sites and some

organisms, such as Mycobacterium tuberculosis, have been shown to both bind and activate plasminogen. Plasminogen and plasmin bind to cells and to bacteria through lysine binding sites in their kringle domains. Several bacterial plasminogen receptors have been reported, including a-enolase. In addition, a ‘pseudo-lysine arrangement’ has been implicated in the high-affinity binding of plasminogen to the M-like protein of group A streptococci. Whether plasmin(ogen) is directly involved in the immune response during infectious diseases is under study. Binding of plasmin(ogen) to macrophages and other leukocytes increases cell surface proteolytic activity, thereby enhancing migration and ECM degradation. Furthermore, peritoneal recruitment of monocytes and lymphocytes in response to thioglycollate inflammation is impaired in plasminogen knockout (plasminogen / ) mice. On the other hand, no changes were found in the number and distribution of lung macrophages between WT and plasminogen / mice after infection by Mycobacterium avium. Differences related to tissue (lung vs. peritoneum) and the inflammatory agent (thioglycollate vs. M. avium) may be responsible for the discrepancy. Certain fragments of plasmin(ogen) devoid of any catalytic activity have been shown to have a signal transduction effect that inhibits angiogenesis. These fragments (including kringles 1–3) known as angiostatin have also been found to inhibit the PA activity of the scuPA/uPAR complex and to regulate contraction of isolated arterial rings (Higazi, unpublished data). Urokinase Plasminogen Activator (uPA) and Its Receptor (uPAR)

uPA uPA is synthesized and released as a 411 amino acid proenzyme composed of an N-terminal fragment (ATF, amino acids 1–135) that contains the growth factor domain (GFD, amino acids 1–43) required for binding to uPAR, a kringle (amino acids 44–135), and a C-terminal proteolytic domain (lowmolecular-weight uPA; amino acids 136–411). uPA is synthesized as a single-chain proenzyme (scuPA) with little or no intrinsic enzymatic activity. scuPA is activated when it is cleaved by plasmin at Lys158–Ile159 to generate a two-chain molecule (tcuPA) or by binding, as a single-chain molecule, to uPAR. The scuPA/uPAR complex and tcuPA have similar catalytic rate constants, but differ markedly in kinetic parameters and reversibility of activation and inactivation by the PAI-1 where the scuPA/suPAR complex is more resistant to PAI-1 inhibition than tcuPA. Furthermore, the scuPA/uPAR complex is more

FIBRINOLYSIS / Overview 203

active than tcuPA in models of pulmonary microembolism and pleural effusion.

tcuPA also dramatically increases its susceptibility to irreversible inhibition by PAI-1. Inactive PAI-1–uPA– uPAR complexes are rapidly internalized by LRP. Thus, PAI-1 initiates an LRP-dependent decrease in cell surface tcuPA and uPAR. Interestingly, uPAR is elevated on the surface of LRP-deficient cells and this increase correlates with increased cell mobility. The binding of uPA to uPAR stimulates intracellular signaling and also induces conformational changes in uPAR, which increase its affinity for vitronectin (VN) and promotes its interaction with a variety of integrins. uPAR occupancy can also activate certain integrins and growth factor receptors. Another pathway to initiate intracellular signaling by uPA is by direct interaction with LRP (without the intervention of uPAR). The interaction of uPA with LRP regulates vascular contractility, including that of pulmonary arteries (Higazi, unpublished data). This pathway is inhibited by PAI-1 and by a PAI-1-derived peptide that does not affect the catalytic activity of uPA (Figure 2).

uPAR uPAR is a glycolipid-anchored (GPI) 281 amino acid protein composed of three domains (DI– III) that are partial repeats, each containing two to three paired disulfide bonds linked by flexible linker sequences. Binding of uPA–uPAR to various transmembrane adapters, such as integrin ligands/integrins and the low-density lipoprotein receptor-related protein (LRP), has been implicated in cell adhesion and intracellular signaling, although a direct productive association of GPI-uPAR with intracellular signaling proteins in lipid rafts has not been excluded. uPA/uPAR in lung disease uPA and uPAR have been implicated in diverse physiological and pathological processes involving cell migration through fibrin and subcellular matrices, that are of direct relevance to lung injury and repair, for example, angiogenesis, wound repair, inflammation, immunity, and tumor metastasis. uPA / mice have defects in fibrinolysis, wound healing, neointima formation, immunity, and dissemination of certain tumors. In uPAR / mice leukocyte mobilization to sites of infection is impaired. uPA / mice have increased susceptibility to lethal pulmonary infection by certain organisms. Furthermore, deficiency of uPA has been associated with more severe pulmonary hypertension and impaired vascular remodeling during chronic hypoxia in mice.

Tissue-Type Plasminogen Activator

Tissue-type plasminogen activator (tPA) is the second principal plasminogen activator in mammals. tPA / mice accumulate fibrin in several organs and their capacity to lyze pulmonary microemboli is impaired. tPA also modulates vascular (including pulmonary arterial) contractility through LRP (Higazi, unpublished data) and exerts proteolytic and nonproteolytic toxic effects on extravascular tissues, best characterized in the central nervous system. PAI-1 and a PAI-1-derived peptide (Figure 2) inhibit tPA-mediated vasoactivity without affecting its fibrinolytic activity. As in the case of uPA / mice, bleomycin-induced pulmonary fibrosis in tPA / mice is enhanced.

Nonproteolytic effects of uPA and uPAR As mentioned above, uPA is active as a proteolytically cleaved two-chain molecule or as a single-chain molecule bound to uPAR. Proteolytic conversion to

EEIIMD

Docking site

Catalytic site

PAI-1 tPA or uPA

PAI-1 tPA or uPA

(a)

(b)

PA-EEIIMD (c)

Figure 2 PAI-1 interactions with PAs. (a) PAI-1 binds to tPA or uPA through two independent sites: the catalytic site and a docking site. The docking site, comprising amino acids 296–299 in tPA and 179–184 in uPA, lies outside the catalytic site. PAI-1 binds to the docking site through amino acids 350–355. (b) The PAI-1-derived peptide EEIIMD, which corresponds to amino acids 350–355, interacts with the docking site in uPA and tPA and competes for the binding of PAI-1. (c) Binding of EEIIMD to uPA or tPA may induce conformational changes that inhibit their effect on vasoactivity and other signal transduction pathways.

204 FIBRINOLYSIS / Overview Plasminogen Activator Inhibitor Type 1

PAI-1 is a member of the serine protease inhibitor (SERPIN) family and is the primary physiological inhibitor of both tPA and uPA. It is a trace protein in plasma where it circulates in a complex with the adhesive glycoprotein vitronectin. Binding of PAI-1 to VN stabilizes the inhibitor in its active conformation, and this binding is reversed when the inhibitor forms complexes with tPA or uPA. tPA and uPA bind PAI-1 through two independent sites (Figure 2), the catalytic site and a docking site. The docking site is located outside the catalytic site and is composed of amino acids 296–299 in tPA and 179–184 in uPA. PAI-1 binds to the docking site of tPA or uPA through the amino acids 350–355 (Figure 2). The peptide EEIIMD, which corresponds to amino acids 350–355 in PAI-1, interacts with the docking site in uPA and tPA and competes for the binding of PAI-1. EEIIMD inhibits tPA- and uPA-mediated vasocontractility without affecting their catalytic activities. We hypothesize that binding of EEIIMD to the PAI-1 docking site on tPA or uPA produces conformational changes in the plasminogen activators that neutralize their signal transduction capacity (Figure 2). Fibrinogen

Fibrinogen is a 340 000 kDa protein consisting of three pairs of disulfide-bonded nonidentical polypeptide chains. Fibrinogen is proteolyzed by thrombin during coagulation, releasing fibrin monomer. Fibrin monomers polymerize into an insoluble network that gives tensile strength to the blood clot and is the substrate of the fibrinolytic system. Fibrin(ogen) binds to integrin and nonintegrin receptors on many cell types. Fibrin(ogen) enhances fibroblast migration and proliferation and is involved in the early stages of the immune inflammatory responses by stimulating leukocyte–endothelial cell adhesion and migration. Furthermore, fibrin colocalizes with collagen-rich fibrotic lung lesions, suggesting the involvement of fibrin(ogen) in the progression of inflammatory diseases in general and in the fibroproliferative response after lung injury specifically. Involvement of fibrin(ogen) in lung fibrosis was initially supported by studies showing fibrin within fibrotic lesions and other studies showing that bleomycin-induced pulmonary fibrosis is exacerbated in tPA / and uPA / mice. Fibrosis is also greater in PAI-1 overexpressing mice and attenuated in PAI-1deficient animals. However, genetic ablation of fibrinogen does not alter either the early inflammatory response to bleomycin injury or the development of collagen-rich pulmonary fibrosis. Therefore, plasminogen and plasminogen activators may regulate

pulmonary fibrosis through other mechanisms, including the regulation of collagen turnover by activation of collagenases and metalloproteinases. An alternative explanation for the increased pulmonary fibrosis reported in PA knockout mice may be their involvement in signal transduction, specifically in regulation of vasoactivity; since PAI-1 abolishes tPA- and uPA-induced signal transduction and vasoactivity, this hypothesis could also explain the phenotype seen in PAI-1 deficient and overexpressing mice. See also: Acute Respiratory Distress Syndrome. Coagulation Cascade: Overview; Fibrinogen and Fibrin. Fibrinolysis: Plasminogen Activator and Plasmin. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia. Proteinase Inhibitors: Alpha-2 Antiplasmin. Serine Proteinases. Thrombolytic Therapy.

Further Reading Bdeir K, Murciano J-C, Tomaszewski J, et al. (2000) Urokinase contributes to fibrinolysis in the pulmonary microcirculation. Blood 96: 1820–1826. Bertozzi P, Astedt B, Zenzius L, et al. (1990) Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. New England Journal of Medicine 322: 890–897. Gunther A, Mosavi P, Heinemann S, et al. (2000) Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 161: 454–462. Gyetko MR, Chen GH, McDonald RA, et al. (1996) Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans. A murine transgenic model. Journal of Clinical Investigation 97: 1818–1826. Higazi AA-R, Cohen RL, Henkin J, et al. (1995) Enhancement of the enzymatic activity of single-chain urokinase plasminogen activator by soluble urokinase receptor. Journal of Biological Chemistry 270: 17375–17380. Idell S (1994) Extravascular coagulation and fibrin deposition in acute lung injury. New Horizons 2: 566–574. Khalil N, Corne S, Whitman C, and Yacyshyn H (1996) Plasmin regulates the activation of cell-associated latent TGF-beta(1) secreted by rat alveolar macrophages after in vivo bleomycin injury. American Journal of Respiratory Cell and Molecular Biology 15: 252–259. Lahteenmaki K, Kuusela P, and Korhonen TK (2001) Bacterial plasminogen activators and receptors. FEMS Microbiology Reviews 25: 531–552. Lijnen HR (2001) Plasmin and matrix metalloproteinases in vascular remodeling. Thrombosis and Haemostasis 86: 324–333. Lijnen HR and Collen D (2005) Molecular and cellular basis of fibrinolysis. In: Hoffman Hematology: Basic Principles and Practice, 4th edn., pp. 1955–1959. MacKay AR, Corbitt RH, Hartzler JL, and Thorgeirsson UP (1990) Basement membrane type IV collagen degradation: evidence for the involvement of a proteolytic cascade independent of metalloproteinases. Cancer Research 50: 5997–6001. Miles LA, Dahlberg CM, Plescia J, et al. (1991) Role of cell-surface lysines in plasminogen binding to cells: identification of a-enolase as a candidate plasminogen receptor. Biochemistry 30: 1682–1691.

FIBRINOLYSIS / Plasminogen Activator and Plasmin 205 Nassar T, Akkawi S, Shina A, et al. (2004) The in vitro and in vivo effect of tPA and PAI-1 on blood vessel tone. Blood 103: 897–902. Ploplis VA, French EL, Carmeliet P, Collen D, and Plow EF (1998) Plasminogen deficiency differentially affects recruitment of inflammatory cell populations in mice. Blood 91: 2005–2009. Plow EF, Herren T, Redlitz A, Miles LA, and Hoover-Plow JL (1995) The cell biology of the plasminogen system. EMBO Journal 9: 939–945. Wilberding J, Ploplis V, McLennan L, et al. (2001) Development of pulmonary fibrosis in fibrinogen-deficient mice. Annals of the New York Academy of Sciences 936: 542–548. Wistedt AC, Kotarsky H, Marti D, et al. (1998) Kringle 2 mediates high affinity binding of plasminogen to an internal sequence in streptococcal surface protein PAM. Journal of Biological Chemistry 273: 24420–24424.

Plasminogen Activator and Plasmin S Shetty and S Idell, University of Texas Health Center, Tyler, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The plasminogen activation system involves several components, including two plasminogen activators, urokinase-type (uPA) and tissue-type (tPA), a plasma membrane-associated receptor (uPAR), and two specific inhibitors, plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2). The plasminogen activator/plasmin system plays an important role in both physiological and pathophysiological processes involving degradation of intravascular thrombi and extracellular matrices that may contribute to wound healing in the setting of tissue inflammation and repair and desmoplasia in the setting of neoplasia. Recent studies have revealed that the plasminogen activation system, through interaction with integrins and other components of the extracellular matrix, is also involved in the regulation of cellular signaling, proliferation, adhesion, and migration in a manner that is independent of plasminogen activator-mediated proteolytic activity. Overexpression of uPA, uPAR, and PAI-1 is commonly associated with acute or chronic lung injury and lung carcinomas. The expression of components of the plasminogen activation system is regulated both at the transcriptional and posttranscriptional level by a variety of proinflammatory cytokines or other proinflammatory mediators through interactions of specific transcription factors or mRNA binding proteins with plasminogen activation system component DNAs or mRNAs, respectively. Most recently, it has been found that uPA itself can stimulate its own expression as well as that of uPAR and PAI-1. The induction of components of the plasminogen activation system by uPA could likewise contribute to the alteration in fibrin turnover that may occur in the injured lung and in lung neoplasia.

Introduction The zymogen plasminogen is converted to the active serine protease plasmin by proteolytic cleavage

effected by either urokinase-type plasminogen activator (uPA) or tissue-type plasminogen activator (tPA). Plasmin is, in turn, capable of degrading protein constituents of the extracellular matrix and basement membranes. Extracellular proteolytic enzymes such as serine proteinases including uPA and tPA and matrix metalloproteinases play an important role in tissue remodeling. uPA and tPA are produced by a wide range of normal and transformed cells. tPA appears to play a major role in intravascular thrombolysis whereas uPA is mainly involved in tissue remodeling during evolving inflammation and repair or in neoplasia. The proteolytic activities of these proteases are tightly regulated by serine protease inhibitors (serpins), including plasminogen activator inhibitors (PAI)-1 and PAI-2. A shift towards proteolysis is observed during tissue remodeling and angiogenesis. A shift towards proteolysis is likewise observed in malignant tissues and neoplastic cells enabling tumors to invade adjacent normal tissues, to induce tumor-associated angiogenesis, and to promote metastatic spread. The balance of proteolysis and inhibition of proteolysis is therefore contingent on variable expression of PAs and PAIs and may therefore influence the virulence of tumors in terms of both local extension and distant spread.

Biochemical Properties of uPA The uPA is secreted as a 53 kDa prouPA otherwise known as single-chain uPA, which binds to its cell surface receptor uPAR and is later converted by plasmin to the relatively more active two-chain form of uPA, which in turn converts membrane-bound plasminogen to plasmin. The two polypeptide chains of uPA are linked by several disulfide bonds (Figure 1). The uPA molecule contains three functional domains. It contains an N-terminal receptor binding growth factor (EGF)-like domain and a C-terminal serine protease domain, which are interconnected by the kringle domain. In addition to effecting the activation of plasminogen, uPA also activates prohepatocyte growth factor/scatter factor (HGF/SF) and transforming growth factor beta (TGF-b).

Biochemical Properties of tPA tPA is a 70 kDa protein secreted in a proenzyme form that is also converted by plasmin to the active twochain form. The tPA molecule has four functionally distinct domains. These include a fibronectin-like or finger domain, an EGF-like domain, two kringle domains, and a serine protease domain. The presence of receptors for tPA has been reported in several cell

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Growth factor domain

G G

T

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T V Y

180 R V S G G R H

C F I D Y P H T F A K P P S L C S A E W V I 200 M E C E N S P Y S N K R T L N 300 N G G D D T E F Y P K Q L G Q S G Y T F R E M P K I I G E 260 E K T S C Q T M R L G L L L Q L T S C V K A 360 L I K V V T M G P D I L G Y Y V N I 320 H G S W G P H S S G S R H D Q H E G R G Q E C Q A V 380 C C T L A T T S L D P K D K T K W Q P D A A C L M K 340 Q

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Serine protease part Figure 1 Primary structure of uPA. The diagrammatic model shows the primary structure of uPA and illustrates the three major domains of the molecule. Reproduced from Collen D and Lijnen HR (1994) American Journal of Physiology: Lung Cellular and Molecular Physiology 288: L319–L328, used with permission from The American Physiological Society.

FIBRINOLYSIS / Plasminogen Activator and Plasmin 207

types. tPA has high affinity for fibrin, which promotes its activation. tPA is mainly associated with intravascular fibrinolysis, but is also represented at sites of extravascular inflammation, including pleural and parenchymal lung injuries. tPA is induced by thrombosis such as may occur in tissue ischemia and inflammation.

Plasminogen Activator Inhibitors Plasminogen activator inhibitors belong to the serpin protease inhibitor (SERPIN) superfamily. The inhibitors include PAI-1, PAI-2, and protease nexin I. These inhibitors exhibit a peptide bond for targeting proteases and stably coupling them with a 1:1 stoichiometry. These are very fast-acting inactivators of the plasminogen activators and are present in most body fluids and tissues.

Plasminogen Activator Inhibitor 1 PAI-1 is a 50 kDa glycoprotein serpin secreted by several cell types. PAI-1 can bind to free and receptorbound uPA, thereby inhibiting uPA-mediated extracellular matrix degradation. The receptor-bound PAI-1 also mediates internalization of trimeric cell surface uPA–PAI-1–uPAR complexes and the subsequent recycling of uPAR to the cell surface. PAI-1 is also involved in the regulation of cell adhesion and migration. PAI-1 is rapidly induced by TGF-b, representing a potentially critical mechanism that promotes an antifibrinolytic balance of the plasminogen activator system and favors maintenance of transitional fibrin and fibrotic repair.

Plasminogen Activator Inhibitor 2 PAI-2 is a 47 kDa protein and is less potent in inhibiting receptor-bound uPA than PAI-1. PAI-2 is rapidly induced by proinflammatory mediators including tumor necrosis factor alpha (TNF-a) and LPS. PAI-2 protects cells from TNF-a-mediated apoptosis. PAI-2 exists both in intracellular and secreted form and the latter participates in the control of tissue remodeling and fibrinolysis. PAI-2 is generally undetectable in the circulation, except in pregnancy and in association with selected neoplasms. PAI-2 also occurs in extravascular fluids, including bronchoalveolar lavage from patients with acute respiratory distress syndrome (ARDS) or chronic lung injury or in pleural effusions from patients with exudative pleuritis.

The uPA Receptor The receptor for uPA, uPAR, is a plasma membrane protein that consists of three cysteine-rich domains.

The N-terminal domain has uPA binding activity, while the other two domains bind to vitronectin, a key constituent of the extracellular matrix. uPAR has an approximate molecular weight of 50 kDa and is highly glycosylated. uPAR is associated with the plasma membrane through a glycosylphosphotidyl inositol (GPI) anchor. Several cytokines and growth factors including TNF-a, TGF-b, HGF, vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) induce uPAR expression in a variety of cell types.

Pathophysiological Role of the Plasminogen Activator System The plasminogen activator system has been shown to influence a broad array of cellular responses that extend well beyond traditional fibrinolysis. Alteration of the plasminogen activator system assumes particular importance in promoting fibrin deposition and remodeling in diverse forms of tissue injury. Similarly, it has been shown that expression of fibrinolytic components is altered in the setting of lung cancer as well as other solid neoplasms.

Specific Derangements of the Plasminogen Activator System Associated with Acute and Chronic Lung Injury It is now well known that uPA is the major plasminogen activator expressed in alveolar lining fluids in the normal lung. In the absence of disease, fibrinolytic activity is readily detectable in bronchoalveolar lavage specimens obtained from normal healthy subjects. Conversely, fibrinolytic activity is depressed in the alveolar compartment in ARDS. This defect in local fibrinolysis in part accounts for the florid extravascular fibrin that is consistently observed in the alveolar and interstitial neomatrix of the injured lung. The fibrinolytic defect in the alveolar space in ARDS is largely attributable to increased local expression of PAI-1. PAI-2 and antiplasmins also contribute to the decrease in fibrinolytic activity in ARDS, so that the plasminogen activator system is downregulated in series at the levels of plasminogen activators and plasmin (Figure 2). Similar abnormalities were found in the bronchoalveolar lavage specimens of patients with idiopathic pulmonary fibrosis, sarcoidosis, and other forms of interstitial lung diseases. In ARDS and interstitial lung diseases, the local defect in uPA-dependent fibrinolytic activity is largely attributable to local overexpression of PAI-1.

208 FIBRINOLYSIS / Plasminogen Activator and Plasmin Fibrinolysis

PAI Antiplasmins Coagulation

ARDS Pneumonias Interstitial lung disease Figure 2 Alterations of local fibrinolysis and coagulation in acute and chronic lung injuries.

Fibrin turnover is similarly altered in the setting of pleural injury, which is likewise characterized by intrapleural fibrin deposition. In pleural effusions of patients with exudative pleuritis due to pneumonia, metastatic cancer, malignant mesothelioma, or emphysema, fibrinolytic activity is generally undetectable. The decrease in fibrinolytic activity under these circumstances is also due to overexpression of PAI-1, which is increased several-fold versus the usual plasma level. Both uPA- and tPA-related fibrinolytic activities are also downregulated by PAI-2 and antiplasmins within the pleural compartment. These abnormalities favor the maintenance of intrapleural fibrin in evolving pleural loculation. Reversal of these abnormalities by the intrapleural administration of fibrinolysins has been part of the therapeutic armamentarium for over 50 years, although recent multicenter clinical trials are now being conducted to evaluate their efficacy for patients with organizing pleural loculation and fibrosis. In both parenchymal and pleural injuries, a variety of resident cell types express components of the plasminogen activator system. Alveolar macrophages, lung epithelial cells, lung fibroblasts, and pleural mesothelial cells are sources of uPA, tPA, and fibrinolytic inhibitors. These cells also express the urokinase receptor. In these cells, inflammatory mediators released during lung injury influence the regulation of uPA, uPAR, and PAI-1 and PAI-2. For example, TNF-a and TGF-b increase uPA, uPAR, and PAI-1 expression by lung epithelial cells and lung fibroblasts.

Disordered Fibrinolysis in Lung and Pleural Neoplasia There are consistent abnormalities in fibrin turnover in inflammation, evolving fibrosis, and cancer. Extravascular fibrin deposition in solid neoplasms promotes the desmoplastic response as well as the

growth and spread of solid tumors. Abnormalities of the plasminogen activator system have likewise been implicated in the pathogenesis of tumor invasiveness and metastatic spread. Mechanisms by which the uPA–uPAR system contributes to the pathogenesis of tumor invasiveness include proteolytic degradation of basement membrane and extracellular matrix constituents and intracellular signaling that initiates mitogenic or altered adhesion responses. Several reports link the expression of uPA, uPAR, PAI-1, and PAI-2 to both tumor cell invasiveness and to differential prognosis in cancer patients. For instance, high levels of uPA have been correlated with poor outcome or shorter survival in a variety of tumors, including those of the breast and lung as well as a variety of other solid neoplasms. Expression of PAI-1 has likewise been linked to aggressive tumor behavior and uPAR expression also appears to be a prognostic marker in some forms of malignancy, including breast or colorectal cancer. Increased expression of uPA, uPAR, and PAI-1 has been demonstrated in different histologic forms of lung carcinoma and linked to poor prognosis, suggesting that clinical outcome extends beyond predictable alterations in fibrinolytic capacity and may, rather, be broadly related to pathophysiologic responses initiated by these proteins through cellular signaling.

Autocrine Regulation of the Plasminogen Activator System Recent reports confirm that uPA itself can stimulate its own expression or that of uPAR or PAI-1 in lung epithelial cells in culture (Figure 3). While clear evidence of the role of these pathways in lung or pleural injury has yet to be determined, they may likewise contribute to the derangements of fibrin turnover that have been reported to occur in the injured lung and in lung neoplasia. Tumor cell invasion is facilitated by saturation of uPAR with either exogenous or overexpressed endogenous uPA. Tumor cells generally exhibit several characteristics such as anchorage-independent growth, high migratory capacity, the ability to produce relatively large quantities of tumor proteases, and enhanced proliferation. In several human tumors, the balance between proteases encompasses alterations of the uPA–uPAR–PAI-1 system. The efficiency of plasminogen activation by uPA is enhanced several-fold when uPA is bound to uPAR. The uPA/uPAR complex also functions as a vitronectin receptor, which could thereby influence tumor cell motility and invasiveness. It now appears that uPA, uPAR, and PAI-1 are integrally involved in the pathogenesis of lung carcinomas, raising the

FIBRINOLYSIS / Plasminogen Activator and Plasmin 209

The uPAR promoter contains AP-1, SP-1, AP-2, and SP-1/-3 binding sites.

Fibrinolysis

Remodeling of transitional (alveolar & interstitial) fibrin

Autoregulation of uPA Regulation of uPAR and PAI-1

Signaling of proinflammatory and profibrogenic cytokines Figure 3 Role of fibrinolytic pathways in lung injury and repair. Urokinase-type plasminogen activator (uPA), its receptor (uPAR), and its major inhibitor plasminogen activator inhibitor-1 (PAI-1).

important possibility that overexpression of uPA, uPAR, and PAI-1 by these cells and clinical prognosis may rely upon autocrine or paracrine induction by uPA.

Transcriptional Regulation of the Plasminogen Activator System Expression of uPA, uPAR, PAI-1, and PAI-2 genes is regulated at both transcriptional and posttranscriptional levels. uPA gene expression is induced by growth factor, hormones, UV light, changes in cell morphology and contact, and, as noted above, by uPA itself. The best-characterized regulatory regions of the uPA promoter are the Ets/AP-1a composite site, the AP-1b site, and the connecting 74 bp cooperation mediator region. Ets1 and Ets2 have been shown to activate the uPA promoter. Similarly, tPA expression is enhanced by a wide range of stimuli, including retinoids and steroids. The transcription factors SP1, CREB1, NF1, and ATF2 are responsible for induction of tPA mRNA expression. PAI-1 gene expression is likewise regulated by many growth factors and cytokines, including TGF-b, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), FGF, interleukin-1 (IL-1), and TNF-a and hormones. SP-1, AP-2 proteins as well as a 72 kDa unknown component have been found to interact with the PAI-1 promoter. Transcription factor nuclear factor I and the ubiquitous factor (USF) are responsive to TGF-b induction in the natural promoter. PAI-2 gene expression is regulated by multiple growth factors, hormones, cytokines, and vasoactive peptides. Site-directed mutagenesis studies revealed the involvement of AP-1a and CRE-like sites for both basal and PMA-induced PAI-2 transcription. Finally, the uPAR gene is induced by various signals including PMA, TGF-b, TNF-a, cAMP, and HIV infection.

Posttranscriptional Regulation of the Plasminogen Activator System uPA, uPAR, PAI-1, and PAI-2 mRNAs are all regulated by posttranscriptional mechanisms that operate at the level of mRNA stability. The uPA mRNA 30 untranslated region (30 UTR) contains multiple instability determinants and specific mRNA-binding protein recognition sequences. Interaction of two specific mRNA-binding proteins has been reported for uPA mRNA. Levels of tPA protein are determined by activation of tPA mRNA, which is controlled by poly(A) tail length. PAI-1 mRNA stability is altered via TGFb, cAMP, and insulin-like growth factor. At least two stability determinants have been identified within the PAI-1 30 UTR. Multiple PAI-1 mRNA-binding proteins interacting with the PAI mRNA 30 UTR have been reported. PAI-2 mRNA is also induced by PMA and TNF-a at the posttranscriptional level. Posttranscriptional regulation of PAI-2 mRNA is regulated by determinants present in both the coding region and 30 UTR. uPAR mRNA is also induced by several agents including PMA, LPS, or TGF-b at the posttranscriptional level. Stability of uPAR mRNA is regulated by determinants present both in the coding and 30 UTR. A 50 kDa protein interacts with the uPAR mRNAcoding region and a 40 kDa protein with 30 UTR determinants to regulate uPAR mRNA stability. See also: Acute Respiratory Distress Syndrome. Epidermal Growth Factors. Fibrinolysis: Overview. Fibroblast Growth Factors. Gene Regulation. Proteinase Inhibitors: Antichymotrypsin. Pulmonary Fibrosis. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumors, Malignant: Overview. Vascular Endothelial Growth Factor.

Further Reading Andreasen PA, Kjoller L, Christensen L, and Duffy M (1997) The urokinase-type plasminogen activator system in cancer metastasis: a review. International Journal of Cancer 72: 1–22. Besser D, Verde P, Nagamine Y, and Blasi F (1996) Signal transduction and the uPA/uPAR system. Fibrinolysis 10: 215–237. Collen D and Lijnen HR (1994) American Journal of Physiology: Lung Cellular and Molecular Physiology 288: L319–L328. Collen D and Lijnen HR (1994) Fibrinolysis and the control of hemostasis. In: Stamatoyannopoulos GS, Nienhuis AW, Majerus PW, and Vermus HV (eds.) The Molecular Basis of Blood Diseases, pp. 725–752. Philadelphia: Saunders. Dickenson JL, Bates EJ, Ferrante A, and Antalis TM (1995) Plasminogen activator inhibitor type 2 inhibits tumor necrosis factor alpha-induced apoptosis: evidence for an alternate biological function. Journal of Biological Chemistry 270: 27894–27904. Garcia R, Schweigerer L, and Medina MA (1998) Modulation of the proteolytic balance plasminogen activator/plasminogen activator

210 FIBROBLAST GROWTH FACTORS inhibitor by enhanced N-myc oncogene expression or application of genistein. European Journal of Cancer 34: 1736–1740. Idell S (1995) Coagulation, fibrinolysis and fibrin deposition in lung injury and repair. In: Phan SH and Thrall RS (eds.) Pulmonary Fibrosis: Lung Biology in Health and Disease. vol. 80, pp. 743–776. New York: Dekker. Irigoyen JP, Munoz-Canoves P, Montero L, Koiczak M, and Nagamine Y (1999) The plasminogen activator system: biology and regulation. Cell and Molecular Life Sciences 56: 104–132. Mazar AP, Henkin J, and Goldfarb RH (1999) The urokinase plasminogen activator system in cancer: implications for tumor angiogenesis and metastasis. Angiogenesis 3: 15–32. Nykjaer A, Conese M, Christensen EI, et al. (1997) Recycling of the urokinase receptor upon internalization of the uPA: serpin complexes. EMBO Journal 16: 2610–2620. Pedersen H, Brunner N, Francis D, et al. (1994) Prognostic impact of urokinase, urokinase receptor, and type 1 plasminogen activator inhibitor in squamous and large cell lung cancer tissue. Cancer Research 54: 4671–4675.

Planus E, Barlovatz-Meimon G, Rogers RA, et al. (1997) Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading. Journal of Cell Science 110: 1091– 1098. Scheneiderman J, Adar R, and Savion N (1991) Changes in plasmatic tissue-type plasminogen activator and plasminogen activator inhibitor activity during acute arterial occlusion associated with severe ischemia. Thrombosis Research 62: 401–408. Shetty S and Idell S (2000) Posttranscriptional regulation of the fibrinolytic system in lung and pleural disease. Research in Advanced Pathology 1: 1–16. Shetty S and Idell S (2002) New perspectives on the regulation of fibrinolysis in the lung. Recent Research Development in Biological Chemistry 1: 223–230. Xing RH, Mazar A, Henkin J, and Rabbani SA (1997) Prevention of breast cancer growth, invasion, and metastasis by anti-estrogen tamoxifen alone or in combination with urokinase inhibitor B-428. Cancer Research 57: 3585–3593.

FIBROBLAST GROWTH FACTORS D Ornitz, Washington University School of Medicine, St Louis, MO, USA P Sannes, North Carolina State University, Raleigh, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Fibroblast growth factors (FGFs) are a large group of small polypeptide growth factors, some of which play key roles in pulmonary biology. Their molecular sizes vary from 17 to 34 kDa and they share a wide range of amino acid homology with highly conserved gene structures and amino acid sequences. They also share the common property of avidity for heparin, which enables them to bind to components of extracellular matrices and cell surfaces. FGFs form a trimolecular complex with heparin-like molecules (e.g., heparan sulfate) and one of four polypeptide tyrosine kinase receptors. The chemical heterogeneity of heparan sulfate and alternative splicing of FGF receptors both determine FGF binding specificity and receptor dimerization and activation. Functions for FGFs are, in many cases, narrowly defined to specific stages of development or repair, while others are broadly applied to physiological maintenance and responses to injury. FGF signaling has been shown to affect cell proliferation, cell survival, chemotaxis, migration, and cell adhesion. FGFs are known to play specific roles in tumorigenesis and pulmonary fibrosis, and have unique capacities to protect against DNA damage induced by oxidants and some environmental toxicants.

Introduction The fibroblast growth factor (FGF) family members have wide-ranging and sometimes overlapping functions. FGFs have been identified in vertebrates and invertebrates, and even in some viral genomes.

Fgf-like sequences have been identified in the unicellular organisms Drosophila and Caenorhabditis elegans. It is believed that the Fgf gene expansion coincided with a phase of global gene duplication that occurred during the period leading to the emergence of vertebrates. Early work on FGFs was focused, not surprisingly, on fibroblasts, although it soon became apparent that the FGFs impacted a wide variety of cell types and functional modalities. In the lung, FGFs were first noted in bleomycin-induced fibrosis where they were found to stimulate DNA synthesis in type II alveolar epithelial cells. FGFs are essential for lung development and in the adult are expressed in a variety of respiratory diseases, with some having distinctly protective qualities.

Structure FGFs are encoded by 22 distinct genes. The FGFs have a central core region of increased similarity, containing 28 highly conserved amino acid residues and six identical residues. Ten of the conserved residues interact with one or more of the various FGF receptors and are separate from regions that bind heparin/heparan sulfate. FGF1 and FGF2 have been studied in the greatest detail. X-ray crystallography has identified a b-trefoil 12-core structure containing 12 antiparallel and fourstranded b sheets in a triangular array. Two of the 12 strands in FGF2 contain basic amino acids that are the primary binding sites for heparin. Fgf genes typically contain three coding exons, with exon 1 containing the initiation methionine. However, several have an additional 50 transcribed

FIBROBLAST GROWTH FACTORS 211 1

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Encodes the C-terminal domain divergent between Fgf s

(a)

N-terminus

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SP (b) Figure 1 The prototypical Fgf gene (a) contains 3 coding exons, including a conserved core (2) and 50 and 30 exons encoding N-terminal (1) and C-terminal (3) domains, respectively, that are divergent between Fgfs. Open boxes represent untranslated regions; solid boxes represent coding sequence. The 50 transcribed sequence encodes an N-terminal signal peptide (SP) in most of the FGFs (b).

sequence that initiates from upstream codons. The genes vary in size from under 5 to over 100 kb (Figure 1). Fgf genes are scattered throughout the genome, suggesting that the Fgf gene family was generated by gene and chromosomal duplications and translocations during evolution.

Regulation of Production and Activity Most of the FGFs (FGFs 3–8, 10, 15, 17–19, and 21– 23) have N-terminal signal peptides and are readily secreted from cells. Several FGFs lack these peptides and are released from cells by nonclassical mechanisms (FGFs 1 and 2), several FGFs have internal signal sequences (FGFs 9, 16, and 20), and several FGFs are retained intracellularly (FGFs 11–14). Some (FGFs 2 and 3) even possess nuclear-localization signals and can be found in the nucleus, although the functional consequences of this remain unclear. FGF2 is found prominently in the basal laminae of epithelium and is bound to the structural heparan sulfate proteoglycan (Perlecan).

Biological Function FGFs play critical roles in the morphogenesis and maturation of the developing lung, the specificity and impact of which are determined by spatiotemporal patterns of expression. Initial bud formation and epithelial proliferation are stimulated by FGF10, while FGF7 promotes epithelial differentiation and, along with FGF2, expression of surfactant proteins-A (SPA), SP-B, and SP-C. FGF7 and FGF10 share an unusual ability to signal through epithelial splice forms

Pleura Mesenchyme FGF9 FGFR2b

FGF10

Airway epithelium

FGF9 Figure 2 FGF regulatory pathways in lung development. The expression of Fgf10 in lung mesenchyme is regulated by FGF9 produced by both pleura and airway epithelium. FGF10 in turn influences epithelial proliferation and bud formation through the FGFR2b receptor expressed only by epithelium.

of FGF receptors (FGFR2b). The expression of FGF7 and FGF10 is regulated in part by FGF9, which signals from lung epithelium and mesothelium to mesenchyme (Figure 2). This stimulatory regulation is opposed by sonic hedgehog (Shh), bone morphogenetic protein-4 (BMP-4), and transforming growth factor beta 1 (TGF-b1). FGF10 is essential for lung bud initiation and its expression is selectively induced by retinoic acid. Furthermore, FGF10 acts as a strong chemoattractant to distal but not proximal epithelium. These effects are greatly influenced by the presence of extracellular matrices, such as heparan sulfate proteoglycans (HSPGs) and Netrin-1 and -4. Genetic manipulation of FGF expression during development has served to emphasize further the

212 FIBROBLAST GROWTH FACTORS

importance of spatiotemporal factors. Inducible transgenic expression of FGF7 results in increased lung size and cystadenomatoid malformations prenatally, while postnatally, FGF7 induces marked epithelial cell proliferation, adenomatous hyperplasia, and mononuclear infiltration. Fgf9-null mice exhibit hypoplastic lungs and early postnatal death. Interestingly, their lungs have reduced mesenchyme and decreased airway-branching but show significant distal airspace formation and pneumocyte differentiation. Prenatal disruption of Fgf10 causes adenomatous malformations, perturbed branching morphogenesis, and respiratory failure at birth. Postnatal transgene interference results in differentiated papillary and lepidic pulmonary adenomas. Notably, the epithelial cells lining the tumors have high expression of thyroid transcription factor (TTF)-1 and SP-C, but not Clara cell secretory protein, suggesting that FGF10 promotes differentiation of peripheral alveolar type II cells. Fgf18-null mice have lungs that are smaller than normal without impairment of proximal or distal airways. They appear to have reduced alveolar space, thicker interstitial compartments, and many embedded capillaries, suggesting an important role for FGF18 during later stage embryonic lung development. In the adult lung, FGF1, FGF2, and FGF7 may be important in maintaining normal physiological homeostasis. These FGFs, when overexpressed, stimulate proliferation of type II alveolar cells, and FGF7 increases mRNA levels and secretion of SP-A and SP-D too. FGF7 can also increase the expression of Naþ -Kþ -ATPase and aquaporin 5 and regulate ion and fluid balance. FGF2 has been shown to decrease elastin production in fibroblasts; induced expression of FGF3 at low levels results in increased free alveolar macrophage infiltration and at high levels in diffuse alveolar type II cell hyperplasia.

Receptors FGFs bind to tyrosine kinase receptors (FGFRs). Binding specificity of FGFRs 1–3 is determined by alternative splicing of immunoglobulin-like domain III (Figure 3). FGFR4 has only one known spliced form in the alternatively spliced region of immunoglobulin-like domain III. The ligands specific for each splice form tend to be expressed in adjacent tissues with resultant directional signaling. The exon encoding immunoglobulin-like domain IIIb is expressed by epithelium while the exon encoding immunoglobulin-like domain IIIc is expressed in mesenchymally derived cells. Receptor isoforms expressed by epithelium bind mesenchymal sourced ligands. Interestingly, the alternative splice form of the same receptor expressed in neighboring mesenchyme will

SP

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Figure 3 FGFRs contain two or three extracellular immunoglobulin-like domains (I, II, or III) defined by disulfide bonds (S-S), a transmembrane domain (TM), and kinase (KI) domain. Alternative mRNA splicing specifies the sequence of the C-terminal half of the immunoglobulin III resulting in isoforms IIIb or IIIc.

not react with the same mesenchymal ligand. Instead, the mesenchymally expressed variant has specificity for a different FGF that is sourced from epitheliallike tissue. FGFRs typically contain two or three immunoglobulin-like domains and a heparin-binding sequence. The heparin-binding character of both FGFs and FGFRs is a critical determinant of binding specificity and receptor activation. Heparin and related heparan sulfate proteoglycans on cell surfaces and/or in extracellular matrices also serve as binding or storage sites for FGFs and protect them from thermal denaturation and proteolysis.

Relevance to Respiratory Diseases Expression of FGF2 has been linked to progression of lung tumors and ultimately outcomes in patient. Increased serum levels of FGF2 found in small cell lung carcinoma correlated inversely with responsiveness to chemotherapy. On the other hand, expression of FGF2 in tumor-associated stromal cells and vessels correlated inversely with progression of non-small cell lung cancer (NSCLC). Expression of FGFR1 strongly correlated with NSCLC responsiveness to the anticancer drug, doxorubicin. The angiogenic properties of FGF2 have been shown to be of particular importance in pulmonary adenocarcinomas, where they have a strong correlation with metastasis and patient survival. Similarly, in mesothelioma pleural effusions, elevated FGF2 levels correlated with poor patient survival. Interestingly, elevated levels of extracellular FGF1 and FGF2 appear to correlate with resistance to anticancer therapy. Pulmonary fibrosis seems to be connected with FGFs as well. FGF2 has been shown to be increased in macrophages in alveolar fibrosis and in mast cells in idiopathic pulmonary fibrosis, chronic beryllium disease, and silicosis. Experimentally, fibrogenic exposures to hyperoxia or paraquat are associated with rises in expression of FGF2 and its FGFR1 receptor or FGF1, respectively. FGF7 has been shown to prevent bleomycin-induced lung injury and fibrosis,

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in part by reducing pulmonary edema, transforming growth factor beta and platelet-derived growth factor-BB expression, and alveolar type II cell loss. FGF7 has been shown to have rather unique properties of protection against certain cellular injuries. It facilitates repair of radiation- or oxidant-induced DNA damage in pulmonary epithelial cells by mechanisms involving tyrosine kinases, protein kinase C, and DNA polymerases. This repair also involves antiapoptic effects of FGF7 by virtue of its receptor interaction with a p21-activated protein kinase 4 and inhibition of caspase-3. Alpha-naphthylthiourea-induced lung leakage is attenuated by FGF7 by inducing alveolar type II cell hyperplasia and attendant Naþ Kþ -ATPase activity. FGF7 also protects against Pseudomonas aeruginosa-induced lung injury and reduces lung damage due to acid instillation. FGF7 enhances postpneumonectomy lung growth by alveolar proliferation and prevents ventilator-induced lung injury. Perhaps similarly to the latter, FGF10 prevents stretchinduced alveolar epithelial cell DNA damage through mitogen-activated protein kinase (MAPK) activation. FGF10 has also been shown to substantially rescue pulmonary hypoplasia induced by nitrofen.

Acknowledgments This work was supported in part by a grant from the March of Dimes (DMO) and PHS grant HL-44497 (PLS). See also: Epithelial Cells: Type II Cells. Extracellular Matrix: Basement Membranes; Matrix Proteoglycans; Surface Proteoglycans. Keratinocyte Growth Factor. Lung Development: Overview; Congenital Vascular Disorders.

Further Reading Bellusci S, Grindley J, Emoto H, Itoh N, and Hogan BL (1997) Fibroblast growth factor 10 (FGF10) and branching

morphogenesis in the embryonic mouse lung. Development 124: 4867–4878. Cardoso WV (2001) Molecular regulation of lung development. Annual Review of Physiology 63: 471–494. Colvin JS, White A, Pratt SJ, and Ornitz DM (2001) Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme. Development 128: 2095–2106. Deterding RR, Havill AM, Yano T, et al. (1997) Prevention of bleomycin-induced lung injury in rats by keratinocyte growth factor. Proceeding of the Association of American Physicians 109: 254–268. Itoh N and Ornitz DM (2004) Evolution of the Fgf and Fgfr gene families. Trends in Genetics 20: 563–569. Liu Y, Stein E, Oliver T, et al. (2004) Novel role for Netrins in regulating epithelial behavior during lung branching morphogenesis. Current Biology 14: 897–905. Lu Y, Pan ZZ, Devaux Y, and Ray P (2003) p21-activated protein kinase 4 (PAK4) interacts with the keratinocyte growth factor receptor and participates in keratinocyte growth factor-mediated inhibition of oxidant-induced cell death. Journal of Biological Chemistry 278: 10374–10380. Metzger RJ and Krasnow MA (1999) Genetic control of branching morphogenesis. Science 284(5420): 1635–1639. Ornitz DM and Itoh N (2001) Fibroblast growth factors. Genome Biology 2: 1–12. Ornitz DM, Xu J, Colvin JS, et al. (1996) Receptor specificity of the fibroblast growth factor family. Journal of Biological Chemistry 271: 15292–15297. Powers CJ, McLeskey SW, and Wellstein A (2000) Fibroblast growth factors, their receptors and signaling. Endocrine Related Cancer 7: 165–197. Song S, Wientges MG, Gan Y, and Au JL (2000) Fibroblast growth factors: an epigenetic mechanism of broad spectrum resistance to anticancer drugs. Proceedings of the National Academy of Sciences, USA 97: 8658–8663. Takeoka M, Ward WF, Pollack N, Kamp DW, and Panos RJ (1997) KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells. American Journal of Physiology 272: L1174–L1180. Weaver M, Batts L, and Hogan BL (2003) Tissue interactions pattern the mesenchyme of the embryonic mouse lung. Developmental Biology 258: 169–184. Wu KI, Pollack N, Panos RJ, Sporn PH, and Kamp DW (1998) Keratinocyte growth factor promotes alveolar epithelial cell DNA repair after H202 exposure. American Journal of Physiology 275: L780–L787.

FIBROBLASTS F Chua and G J Laurent, The Royal Free Hospital, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Fibroblasts are found in abundance in the airway and distal lung. They orchestrate the continual production and turnover of the pulmonary extracellular matrix, the correct organization of which is vital for efficient gas exchange. The lung matrix also

provides crucial structural support for various cells and exerts profound influences on their function. These interactions regulate normal airway and alveolar morphogenesis during fetal development, and regain prominence during repair responses following injury to these tissues. In addition to matrix formation, immune functions have also been ascribed to fibroblasts, including antigen presentation and the secretion of bioactive molecules. These roles highlight the versatility and indispensability of fibroblasts to the maintenance of normal lung physiology. Consequently, fibroblast dysfunction has been implicated in the pathogenesis of pulmonary disorders in which there is

214 FIBROBLASTS disproportionate extracellular matrix deposition (e.g., fibrotic lung diseases) or destruction (e.g., emphysematous and cystic lung disorders). In these settings, uncontrolled fibroblast activation and proliferation or, conversely, the loss of fibroblast number or function, contribute to the initiation and progression of abnormal lung repair. Substantial research efforts are being directed at identifying molecular targets and novel strategies to treat disorders characterized by pathological matrix remodeling.

LR Ep Eo

Introduction Fibroblasts are derived from mesenchymal progenitors and are relatively easy to isolate from the lung and other tissues. In fact, the first electron micrograph of an intact cell was that of a fibroblast cultured from a chick embryo, reported by Porter and colleagues in 1945. They become most prominent in late fetal life, coinciding temporally with periods of heightened matrix production. In the adult lung, fibroblasts represent approximately one-third of all lung cells. With an elongated, spindle-shaped appearance and an estimated volume of approximately 1600 mm3, the fibroblast is also one of the largest cells in the lung. Parenchymal lung fibroblasts are located mainly within the interstitial space, while airway fibroblasts have a predominantly subepithelial distribution (Figures 1 and 2). Wherever their location, these cells are inherently associated with connective tissue fibers and possess specialized cytoplasmic protrusions and invaginations that facilitate interactions with other cells, vascular and neutral structures, as well as the surrounding extracellular matrix (ECM).

Fibroblast Functions in the Normal Lung Lung fibroblasts undergo activation prior to discharging many of their most important cellular functions. Relatively passive tissue fibroblasts transdifferentiate into contractile myofibroblasts, with the acquisition of enhanced synthetic and migratory potential, as well as the expression of alpha-smooth muscle actin (a-SMA). Activation of fibroblasts can occur through several different mechanisms: stimulation by growth factors in an autocrine or paracrine manner (e.g., transforming growth factor beta or TGF-b), signaling interactions with various ECM proteins (e.g., fibronectin), dynamic cell–cell communications (e.g., alveolar epithelial cells), or changes in physicochemical gradients in their immediate environment (e.g., mechanical stress or hypoxia).

Synthesis of Extracellular Matrix Proteins The mammalian pulmonary ECM constitutes roughly 25% of the dry weight of the lung and is the result of a fine balance between the synthesis and degradation of a variety of proteins. Activated

Fib 10 µm (a)

Ep

Ery Endo

Col Fib

El

BM 2 µm

(b) Figure 1 Transmission electron micrograph of (a) human airway and (b) alveolar wall. (a) A biopsy specimen of an asthmatic airway with thickened lamina reticularis (LR), epithelial cells (Ep), a fibroblast/myofibroblast (Fib), and an eosinophil (Eo). Courtesy of Professor P K Jeffrey. (b) An interstitial fibroblast (Fib) closely associated with elastin fibers (El), collagen fibrils (Col), an endothelial cell (Endo), and the basement membrane (BM). The attenuated cytoplasm of an epithelial cell (Ep) is also seen, as well as an intracapillary erythrocyte (Ery). Courtesy of Ann Dewar and Professor P K Jeffrey.

fibroblasts are the archetypal producers of this ECM and synthesize, amongst others, a family of collagens (predominantly fibrillar types I and III), elastin, proteoglycans, laminin, fibronectin, and tenascin. Collagens constitute the commonest ECM proteins, and line conducting airways, interstitium, and vascular walls. They contain a characteristic right-handed triple helix that is formed by three tightly binding polypeptide a-chains. The helical regions of these chains are composed of repeating Gly-X-Y triplets, with approximately every third X being proline and every third Y, hydroxyproline. Although five types of fibrillar collagens are found in the human lung (types I, II, III, V, and XI), their relative abundance may vary quite significantly. For example, types I and III form almost 90% of all collagens within the pulmonary interstitial compartment and present in a fairly consistent ratio of 2:1 under homeostatic conditions.

FIBROBLASTS 215

Injury

Soluble signals (e.g., TGF-
, PDGF, GM-CSF, IL-4)

Physicochemical stimuli (cell−ECM interactions, stretch, hypoxia)

Fibroblast

Proliferation and migration

Apoptosis

Transdifferentiation and synthesis

Immune functions

Extracellular matrix remodeling

Excess matrix deposition (interstitial lung diseases, airway remodeling)

Inadequate matrix repair (pulmonary emphysema, cystic fibrosis)

Figure 2 Fibroblasts play a key role in the pathogenesis of a number of pulmonary diseases. In response to diverse stimuli, they undergo functional alterations crucial for pathological matrix remodeling characteristic of these disorders.

The most intense period of collagen fiber synthesis occurs during the perinatal period. However, the synthetic capacity of fibroblasts persists well into adulthood. Consequently, daily rates of collagen turnover reaching as high as 10% have been reported in the lungs of adult rodents and rabbits. Even though the rate of collagen synthesis may decrease with increasing age, the degradation rate of newly synthesized procollagen may remain high. It has been estimated that normal adult lung fibroblasts can degrade up to 1
106 molecules of procollagen per hour. In fibrotic states, enhanced collagen synthesis and its decreased degradation are thought to underlie the excessive accumulation of ECM that characterizes such disorders. While collagen fibers are the main determinants of the expansile properties of the lung, elastic fibers provide the resiliency for restoring shape and volume known as pulmonary elastic recoil. Elastin accounts for 20–30% of the dry weight of the lung ECM and contains regions of extreme hydrophobicity essential for its designated biological roles. It is synthesized as a 70 kDa soluble precursor (tropoelastin) that is rapidly cross-linked and polymerized into a random network of elastin molecules. Proteoglycans are a distinct group of complex macromolecules containing a core protein linked to variable numbers of

glycosaminoglycan polysaccharide side-chains. Aggregating proteoglycans regulate ECM hydration and contribute to its compressive properties. In the alveolar wall, the major proteoglycans are heparin sulfate, chondroitin sulfate, and dermatan sulfate. On the other hand, heparin sulfate and laminin are the central proteoglycans in the alveolar basement membrane.

Secretion of Inflammatory Mediators and Growth Factors Fibroblasts also produce a variety of inflammatory mediators and growth factors. In turn, they are subject to regulation by these and other signals produced by neighboring cells or released from the ECM. TGFb is one prime example. Many fibroblast-dependent processes are governed by the activation of TGF-bmediated autocrine and paracrine pathways. Indeed, TGF-b appears to be the most effective inducer of extracellular matrix production yet identified. During fibroblast differentiation, TGF-b upregulates the expression of myofibroblast contractile proteins essential for phenotypic switching and also inhibits fibroblast/ myofibroblast apoptosis. Insulin-like growth factor II (IGF-II) and interleukin-4 (IL-4) are other important

216 FIBROBLASTS

mediators of fibroblast activation. On the other hand, platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF-a), and connective tissue growth factor (CTGF), a member of the PDGF family, appear primarily to promote fibroblast proliferation. Fibroblast-derived soluble factors such as chemokines and lymphokines can also set up chemical gradients to attract and stimulate inflammatory cells migrating into the matrix. Secretory products of these cells may then act directly on fibroblasts, or indirectly to mobilize stored growth factors with matrix-modifying properties. Fibroblast–matrix interactions are mediated by specific cell surface receptors such as a1b1 and a2b1 integrins that facilitate fibroblast interactions with fibrillar collagens (types I and III, respectively), and a5b1 and aVb3 integrins that promote fibroblast–fibronectin interactions. These processes are temporally and spatially relevant to the overall ability of the lung ECM to respond to constitutive and adaptive stimuli. Tissue inhibitors of metalloproteinases (TIMPs) produced by fibroblasts oppose the degradation of ECM by matrix metalloproteinases (MMPs), a family of secreted and membrane-localized Zn2 þ -dependent enzymes. A correct balance in relative MMP–TIMP activity is critical for the physiological turnover of pulmonary ECM. In addition to these proteins, matrix synthesis and breakdown are also controlled by the activity of morphogens and other mediators such as retinoids, patched (PTCH), epidermal growth factor (EGF), and hepatocyte growth factor. Overall, a high degree of molecular regulation ensures that airway branching and alveologenesis occur normally and that repair responses following tissue injury are not subverted by unwanted fibroproliferation.

Fibroblasts in Respiratory Diseases Fibroblasts in Interstitial Lung Diseases

Fibroblasts are central to the pathogenesis of chronic interstitial lung diseases (ILDs) such as idiopathic pulmonary fibrosis (IPF), pulmonary sarcoidosis, and asbestosis. One current hypothesis suggests that repeated or sequential episodes of lung injury provide the pathological signals to drive uncontrolled fibroproliferation, a process that ultimately results in ECM overproduction. Recent studies have indicated that aberrant alveolar–fibroblast (epithelial– mesenchymal) interactions in the parenchymal environment may also contribute to such fibrogenic responses. Indeed, myofibroblasts in the interstitial space have been shown to produce soluble factors that, amongst other things, induce alveolar epithelial cell apoptosis, a pathologic determinant of pulmonary fibrosis.

Numerous cytokines and growth factors are known to regulate matrix accumulation at the molecular level. The most widely described group of profibrotic mediators belongs to the TGF-b family of proteins. These polypeptides promote fibroblast activation, growth, and collagen synthesis and counterregulate the function of MMPs and TIMPs. Increased TGF-b expression has been identified in lungs from patients with a variety of fibrotic disorders. In rats, the overexpression of constitutively active TGF-b following direct gene delivery to the distal lung leads to the development of severe and protracted pulmonary fibrosis. Conversely, neutralization of TGF-b activity or signaling inhibits lung collagen accumulation as well as histologic parameters of pulmonary fibrosis. New insights have also been gained into signal transduction pathways downstream of the TGF-b receptor complex. Early findings in the bleomycin model of pulmonary fibrosis have shown that excessive ECM accumulation is associated with decreased expression of Smad3 (a TGF-b receptor-associated signaling molecule) whereas inhibition of pulmonary fibrosis correlates with increased expression of Smad7, a protein that inhibits signaling of the same pathway. At least 20 other cytokines, including IGF-1, endothelin-1 and TNF-a, have been shown to exert profibrotic effects in vitro and have been implicated in disease pathogenesis in vivo. In addition, abnormalities of the coagulation and fibrinolytic systems also appear to play a role in promoting fibrotic lung repair. More specifically, thrombin has been shown to stimulate fibroblast mitogenesis via the production of autocrine mediators (e.g., PDGF-AA) and promote connective tissue synthesis by the activation of cell surface protease-activated receptors (PARs). Decreased protein C activation leading to impaired fibrinolysis and enhanced procoagulability has also been described in the lungs of patients with ILDs. In the quest to further delineate fibroblast activation pathways in pulmonary fibrosis, emerging evidence from animal studies suggests that bone marrowderived fibroblast progenitors termed fibrocytes may populate the repairing lung during the fibrotic response. These cells express, amongst others, CD45 and collagen I as well as the chemokine receptors CCR7 and CXCR4. In mice treated with bleomycin, increased sequestration of such cells occurs in areas of enhanced fibrotic matrix deposition. Although the mechanisms by which these cells traffic to the lung are still unclear, a role for the CXCL12 chemokine (a ligand for CXCR4) has been implicated. Antifibrotic molecules also appear to act by suppressing fibroblast function. In a preliminary study, the administration of interferon gamma (IFN-g), a cytokine that inhibits fibroblast differentiation,

FIBROBLASTS 217

proliferation, and collagen production in vitro, was associated with improved lung function in a small group of patients with corticosteroid-resistant IPF. The prostaglandin PGE2, a potent inhibitor of fibroblast proliferation and matrix production in vitro, may also exert similar actions in the lung. Fibroblasts obtained from patients with IPF have a diminished capacity to synthesize PGE2 under basal conditions and when stimulated with TGF-b. Antifibrotic properties have also been ascribed to IL-7, a molecule that is able to inhibit TGF-b signaling in lung fibroblasts and attenuate bleomycin-induced pulmonary fibrosis in vivo. The role of the fibroblast in acute fibroproliferative lung injury, in particular the acute respiratory distress syndrome (ARDS), is also increasingly appreciated. Abnormal lung matrix turnover in this devastating disorder is now believed to occur much earlier than previously thought. In ARDS, the formation of hyaline membranes, a type of provisional matrix, within damaged alveoli is a key feature of the accelerated fibroproliferative response. However, the molecular controls that distinguish this form of acute fibrosis from the more chronic pattern in ILDs have not been elucidated.

proinflammatory mediators, including chemoattractants such as eotaxin and monocyte chemotactic protein-1. In the chronically inflamed airway, the constant exchange of information between fibroblasts and infiltrating leukocytes sets up a fertile ground upon which airway remodeling is augmented and propagated. Although these observations provide strong circumstantial evidence linking pathological airway remodeling to bronchial hyperresponsiveness and airflow obstruction, the mechanisms underlying these processes are far from established. Studies in individuals with chronic asthma have shown that circulating fibrocyte-like cells expressing CD34, collagen type I, and a-SMA accumulate in areas of collagen deposition within the bronchial subepithelial environment following allergen challenge. When stimulated in vitro by fibrogenic mediators such as endothelin-1 and TGF-b, such cells acquire a myofibroblast phenotype and secrete fibronectin and collagen type III. In a corresponding murine allergen model, the use of in vivo cell-labeling techniques has enabled the trafficking of similar fibrocytes to be monitored as they move from the circulation to the bronchial submucosa where they subsequently differentiate into tissue-embedded myofibroblasts.

Fibroblasts in Asthma

Fibroblasts in Chronic Obstructive Pulmonary Disease (Chronic Bronchitis and Emphysema)

Dramatic changes in the anatomy of the asthmatic airway are well described, but interest has historically focused on smooth muscle cell and glandular hyperplasia. However, there has been a recent surge in the appreciation of bronchial myofibroblast accumulation and subepithelial fibrosis in the airways of chronically asthmatic patients. In these individuals, the lamina reticularis, the layer between the epithelial basement membrane and the underlying smooth muscle layer, may be increased by two- to three fold in thickness. This anomaly is the direct result of quantitative increases in a variety of extracellular matrix proteins, including collagen types I, III, and V, elastin, and fibronectin. Most airway fibroblasts are also found within this layer. In fact, the concept of the epithelial–mesenchymal trophic unit in the airway preceded an analogous description in the pulmonary interstitium. These sub-basal lamina fibroblasts lie in close proximity to bronchial epithelial cells, and are thought to be the primary elements of the fibroproliferative machinery responsible for matrix overproduction. In addition, thickening of the subepithelial basement membrane itself is well documented. T-helper type 2 cytokines, particularly IL-4 and IL-13, can promote fibroblast activation, extracellular matrix accumulation, and the secretion of

In chronic obstructive pulmonary disease (COPD), most of the changes in the extracellular matrix occur in the peripheral airways and surrounding parenchyma. Although airway remodeling is less well understood than in asthma, increased thickness of the inner (lamina reticularis) and outer (adventitial) walls of the airways has been documented. However, less is known about the composition of individual matrix proteins or the specific changes in fibroblast number or phenotype associated with this process. In addition to changes in the airway wall, emphysematous lesions of the lung parenchyma are also associated with destruction of alveolar connections to the outer bronchial wall, a phenomenon implicated as a contributor to overall airway obstruction. However, a causative link between airway ECM alterations and clinical airflow limitation in COPD remains unproven. The predominant hypothesis of emphysema suggests that a persistent protease–antiprotease imbalance in the distal lung favors net lung matrix degradation, particularly of elastin and its associated proteins. This view emphasizes the notion that both uncontrolled matrix breakdown and inadequate tissue repair are required to produce the abnormal enlargement of alveoli and loss of alveolar septal tissue that characterize this condition. Cigarette smoke constituents and

218 FIBROBLASTS

cadmium are among the agents known to be deleterious to the lung matrix. Their ability to inhibit various fibroblast functions including proliferation, matrix production, and chemotaxis in vitro has broader implications for the capacity of the lung matrix to regenerate following degradative injury. Paradoxically, discrete areas of increased interstitial ECM may be evident in lungs affected by the emphysematous process. It is possible that while the overall extent of matrix loss may overwhelm the tissue restorative potential in such lungs, focal ECM renewal may occur where reparative efforts are particularly successful. The increased expression of TGF-b and epidermal growth factor (EGF) in epithelial and submucosal cells of patients with COPD has been reported. These mediators may contribute to the peribronchiolar fibrosis present on histological analysis although the nature of the localized fibrogenic response is inadequately understood.

Therapeutic Considerations

There is as yet no specific therapy to prevent or reduce the pathological accumulation of airway or parenchymal lung matrix. Corticosteroids remain the cornerstone of treatment of symptomatic asthma and COPD, but their ability to alter the pathobiology of fibrotic lung diseases is unproven. The propensity of these and other immunosuppressive agents to cause severe adverse effects significantly limits their clinical use. Attention has recently shifted to agents that may interfere directly with the fibrogenic process in the lung. Although distinct subpopulations of fibroblasts are suspected to initiate or intensify airway or parenchymal lung fibrosis, the mechanisms by which they operate are still unclear. In vitro and in vivo studies have highlighted the potential value of angiotensin-converting enzyme inhibitors/angiotensin II receptor antagonists, PGE2, colchicine, N-acetylcysteine, and lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in this context. The few agents to have produced any measurable benefit in very small numbers of patients with pulmonary fibrosis include pirfenidone, relaxin, and IFN-g. However, their true clinical value remains unknown. The use of increasingly sensitive molecular techniques and refined disease models is yielding important information regarding the role of the fibroblast in major pulmonary disorders. These insights may help to define aspects of fibroblast biology that are amenable to therapeutic manipulation. Such efforts are necessary in order to improve the efficacy and specificity of future strategies aimed at interfering with the fibrotic process in the lung.

See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fibroblast Growth Factors. Interferons. Matrix Metalloproteinases. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Chambers RC and Laurent GJ (1997) Collagens. In: Crystal RG, West J, Weibel E, and Barnes P (eds.) The Lung: Scientific Foundations, 2nd edn., vol. 49, pp. 709–727. Philadelphia: Lippincott-Raven. Dunsmore SE, Chambers RC, and Laurent GJ (2003) Matrix proteins. In: Gibson GJ, Geddes DM, Costabel U, Ster PJ, and Corrin B (eds.) Respiratory Medicine, 3rd edn., pp. 82–92. London: Elsevier. Hashimoto N, Jin H, Liu T, Chensue SW, and Phan SH (2003) Bone-marrow-derived progenitor cells in pulmonary fibrosis. Journal of Clinical Investigation 113: 243–252. Hogaboam CM, Smith RE, and Kunkel SL (1998) Dynamic interactions between lung fibroblasts and leukocytes: implications for fibrotic lung disease. Proceedings of the Association of American Physicians 110: 313–320. Huang M, Sharma S, Zhu LX, et al. (2002) IL-7 inhibits fibroblast TGF-beta production and signaling in pulmonary fibrosis. Journal of Clinical Investigation 109: 931–937. Keerthisingam CB, Jenkins RG, Harrison NK, et al. (2001) Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-b in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice. American Journal of Pathology 158: 1411–1422. McAnulty RJ and Laurent GJ (2002) Fibroblasts. In: Barnes PJ, Drazen J, Rennard S, and Thomson N (eds.) Asthma and COPD: Basic Mechanisms and Clinical Management, pp. 139– 144. London: Academic Press. McGowan SE and Torday JS (1997) The pulmonary lipofibroblast (lipid interstitial cell) and its contributions to alveolar development. Annual Review of Physiology 59: 43–62. Nakamura Y, Romberger DJ, Tate L, et al. (1995) Cigarette smoke inhibits lung fibroblast proliferation and chemotaxis. American Journal of Respiratory and Critical Care Medicine 151: 1497– 1503. Phillips RJ, Burdick MD, Hong K, et al. (2004) Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. Journal of Clinical Investigation 114: 438–446. Powell DW, Mifflin RC, Valentich JD, et al. (1999) Myofibroblasts. I. Paracrine cells important in health and disease. American Journal of Physiology 277(1 pt. 1): C1–C19. Schmidt M, Sun G, Stacey MA, Mori L, and Matoli S (2003) Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. Journal of Immunology 171: 380–389. Sempowski GD, Beckmann MP, Derdak S, and Phipps RP (1994) Subsets of murine lung fibroblasts express membrane-bound soluble IL-4 receptors. Role of IL-4 in enhancing fibroblast proliferation and collagen synthesis. Journal of Immunology 152: 3606–3614. Sime PJ, Xing Z, Graham FL, Csaky KG, and Gauldie J (1997) Adenovector-mediated gene transfer of active transforming growth factor-b1 induces prolonged severe fibrosis in rat lung. Journal of Clinical Investigation 100: 768–776.

FLUID BALANCE IN THE LUNG 219 Stenmark KR and Mecham RP (1997) Cellular and molecular mechanisms of pulmonary vascular remodeling. Annual Review of Physiology 59: 89–144. Swiderski RE, Bencoff JE, Floerchinger CS, Shapiro SD, and Hunninghake GW (1998) Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary fibrosis. American Journal of Pathology 152: 821–828.

Uhal BD, Joshi I, Hughes WF, et al. (1998) Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung. American Journal of Physiology 275: L1192– L1199. Vlahovic G, Russell ML, Mercer RR, and Crapo JD (1999) Cellular and connective tissue changes in alveolar septal walls in emphysema. American Journal of Respiratory and Critical Care Medicine 160: 2086–2092.

FLUID BALANCE IN THE LUNG A E Taylor, University of South Alabama, Mobile, AL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Airway edema is a very common occurrence in patients with heart failure and also in different forms of lung inflammation (sepsis). The lung’s circulation, airways, and alveoli have tremendous surface areas and can fully oxygenate the venous blood returning from the body into the right atrium and also rid the body of the high amounts of CO2 contained in the venous blood. However, the large gas-exchanging areas in the lungs can also create a problem when left-sided heart failure is present. During pathology, the pulmonary capillary hydrostatic pressure (PPC ) increases in the lung’s circulation and fluid leaves the circulation (JV ) to enter the lung’s interstitium. This is opposed by the protein osmotic pressure in the plasma (pP ). The lymph flow increases (JL ) and removes a large amount of this fluid. The interstitial protein osmotic pressure (pT ) will also decrease as proteins are removed by lymph flow and diluted by protein-free fluid entering the lung’s interstitium. Interstitial fluid pressure (PT ) also increases, which opposes the increased microvascular pressure. Increases in PT and JL coupled with the decreased interstitial protein osmotic pressure (pT ) have been defined as edema safety factors because they provide a substantial protective effect against alveolar edema formation. When PPC increases, the sum of these forces can provide a safety factor of approximately 23 mmHg before the lung tissues fill with edema fluid.

Physiologists derived this equation for muscle fluid exchange, and sd is a recent addition to the fluid flux equation. Since KFC and PPC are the primary components in eqn [1] that produces JV , early studies developed ingenious ways to measure them. First, an organ was isolated and perfused, and the arterial and venous pressures and flow were measured. Then, arterial and venous pressures were adjusted until the organ did not gain or lose weight. The blood flow and arterial pressure (PA ) were decreased and venous outflow pressure (PV ) increased to maintain a noweight-change condition in the organ – an isogravimetric state. This procedure was done several times, and the intercept of PV versus flow curve on the pressure axis was PPC since the resistance in the arterial and venous systems were known. Normal flow

Tissue

PT (−5) (28)
P (+1) Net pressure

The following equation describes the microvascular filtration rate (JV ) occurring across the lung’s microcirculation and is shown schematically in Figure 1: JV ¼ KFC ½ðPPC
PT Þ
sd ðpP
pT Þ

½1

where KFC is the filtration coefficient of the capillary wall; PPC is the hydrostatic pressure in the lung’s microvessels; PT is the interstitial pressure of the lung; pP and pT are protein osmotic pressures of the plasma and tissues, respectively; and sd is the reflection coefficient for the plasma protein. If sd is 1 for the proteins, the membranes are impermeable to the protein, and if sd is 0, the protein is freely permeable.

(−5)

(7) PPC

(−5) (Surface tension) Alveolus


T (17) (−6) PLYM

Capillary

Lymphatic filling pressure (PT –PLYM) Figure 1 Schematic representation of the Starling forces operating between pulmonary capillaries and the interstitium. Note that the alveolar surface tension is exactly opposed by the subatmospheric interstitial fluid pressure (
5), and the lymphatic filling pressure (PT
PLYM ) [
5–(
6)] is positive, which promotes the filling of lymphatic vessels. Reproduced from Taylor AE, Rehder K, Hyatt RE, and Parker JC (1989) Clinical Respiratory Physiology, Ch. 10, p. 178. Philadelphia, PA: Saunders, with permission from Elsevier.

220 FLUID BALANCE IN THE LUNG

was again instituted, and knowing PA , both PV and PPC could be calculated. These techniques were used to evaluate the forces (PPC and PT ), flows ( JV and JL ), and plasma (pP ) and lymph (pL ) protein osmotic pressure. In addition, we developed a protein osmometer to measure pT and pP . Thus, for the first time, techniques were in place to apply similar techniques in lung preparations to measure all components of eqn [1]. The first quantitative study of edema formation conducted in dog lungs was done by Guyton and Lindsey in 1959. Left atrial pressures were increased to different levels in in situ dog lungs, and then the lungs were removed and weighed, both before and after they were dried. Surprisingly, the amount of fluid in the lung’s interstitium as determined by the lung’s wet/dry weight ratios did not increase until left atrial pressures (LAPs) exceeded 25 mmHg. However, once LAPs exceeded this value, a large increase in pulmonary edema formation occurred. This study produced two major conclusions. First, no substantial amount of edema accumulated in lung tissues until LAPs exceeded 25 mmHg. Second, edema did not occur at lower LAPs, but the reason for this was not clear. We now know that interstitial fluid pressure (PT ) increases as fluid enters the lung’s interstitium, lung lymph flow ( JL ) removes some of the microvascular filtration, and tissue plasma proteins are also removed by the lymph flow, which decreases the tissue osmotic pressure (pT ). The average value of these ‘edema safety factors’, which act to oppose edema formation when microvascular pressures increase, is 50% of the edema safety factor in lungs as a result of the decreased tissue protein osmotic pressure (pT ). The increased lymph flow ( JL ) and the increased tissue hydrostatic pressures (PT ) each provide approximately 22% of the safety factors. The sum of these forces provides an edema safety factor of approximately 23 mmHg. This means that lungs are normally protected against pulmonary edema formation by these safety factors; however, when the microvascular pressure increase exceeds approximately 25 mmHg, intra-alveolar edema forms very rapidly even in undamaged lungs. When the endothelial barrier is damaged by sepsis and other inflammatory responses, severe lung edema occurs even at very low PPC because the microvascular walls are extremely permeable to all plasma proteins. KFC will also increase when the endothelial barrier is damaged, which increases the amount of fluid entering the tissues as PPC increases. Also, the coefficient that determines the activity of osmotic pressure gradient acting across the microvascular wall (sd ) is decreased, causing pP
pT to become very small. In this condition, even though the lymph

flow ( JL ) increases to very high levels, the flow is insufficient to remove the excessive amount of fluid that enters the lung’s tissue. Severe intra-alveolar edema that contains high protein concentrations will result and cause death if the process causing the edema cannot be corrected. The major conclusion of this first quantitative study of pulmonary edema formation was that future pulmonary edema studies had to be performed to determine the values of KFC , lymph flow, PT , PPC , pP , pL , and sd . The techniques needed to measure these requirements were first studied in isolated and perfused animal lungs that were continuously weighed in order to accurately measure the rate of edema formation. This was accomplished by simply placing the pump-perfused lungs on a sensitive weight device. Second, ways to measure lymph flow ( JL ) and its plasma protein osmotic pressure (pT ) were also developed. The existing microvascular pressure (PPC ) was measured using gravimetric procedures and the interstitial pressure (PT ) was measured using previously implanted devices; lung weight changes were also continuously measured. The parameters shown in eqn [1] also provide some small amount of edema protection across the pulmonary circulation following elevation of capillary pressures when lungs are severely damaged by pathology but it is more difficult to reverse than hydrostatic edema. Finally, a biophysical factor, the osmotic reflection coefficient (sd ), which is multiplied by (pP
pL ), is close to 1 for normal capillaries but has been shown to be reduced to less than 0.3 when the pulmonary vascular walls are severely damaged, that is, if sd ¼ 0:9, then the proteins in plasma and tissues would exert 90% of the measured protein osmotic pressure. This important factor can be measured using the following equation, which requires that the concentration of a protein in both lymph (CL ) and plasma (CP ) be measured when lymph flow is high and CL =CP approaches a constant but before edema results: CL =CP ¼ ð1
sÞ=ð1
se
x Þ

½2

where x ¼ ð1
sÞJL =PS, and PS is the permeability surface area product of the lung’s microcirculation. Importantly, when JL is very high, eqn [2] reduces to CL =CP ¼ 1
s and sd can be easily determined by using the following equation and analyzing the lymph and plasma concentrations of the proteins under study as they attain a constant value: s ¼ 1
ðCL =CP Þ

½3

Obviously, all components responsible for producing volume flow occurring across the capillary wall

FLUID BALANCE IN THE LUNG 221

were finally defined, and now each protein in plasma can be measured and studied in both normal and damaged lungs. Importantly, safety factors allow PPC to vary from moment to moment in normal lungs; that is, PT increases, JL increases, and the reduction in tissue proteins (pT ) all change in a direction to oppose further filtration into the lung’s tissue. As long as PPC is not greatly elevated, the lungs will not become edematous but will continue to properly exchange gases with the circulation and atmosphere unless the edema becomes severe. It is important to understand that when the endothelial barrier in the lung’s circulation is damaged, edema is likely to develop, even if the PPC remains low. The following calculations can be used to illustrate this point. Using eqn [1], we can calculate JV , assuming the following forces are normal transcapillary filtration values: PT ¼
5 mmHg, pP ¼ 28 mmHg, pT ¼ 17 mmHg, sd ¼ 1, KFC ¼ 0:2 ml min
1, and PPC ¼ 7 mmHg. Thus, JV ¼ 0:2 ml min
1. This is the normal amount of fluid entering the lung’s tissues each minute, and it can easily be removed by lymph flow. However, when capillary walls are damaged, a very different effect occurs. Assume the following forces (in mmHg): PPC ¼ 7, PT ¼
5, sd ¼ 0:5, pP ¼ 28, pT ¼ 19, and KFC is doubled. Then, JV ¼ 3 ml min
1, as compared to 0.2 ml min
1 in normal lungs. Note that when the endothelial barrier is damaged and KFC is doubled, tissue protein osmotic pressure increases, and sd is reduced to 0.5 with damaged capillaries. This change in capillary permeability causes a 15-fold difference in transcapillary filtration. Edema will certainly develop in the lung because lymph flow is unable to remove this amount of excess fluid and protein entering the tissues, and the edema fluid continues to enter the tissues and alveoli. The magnitude of safety factors increases when left atrial pressures rise during chronic heart failure and in intact lungs of in vivo animals such as sheep, but acute studies are usually conducted in experiments using isolated perfused lungs. However, it is well-known that patients with left atrial pressure of 40 mmHg should have extensive pulmonary edema, but edema does not occur! Previous animal studies have shown that chronic elevations of left atrial pressure cause 25 times as much lymph flow as does acute elevation of left atrial pressure. A larger lymph flow safety factor is present during chronic elevation of left atrial pressure which produces a 25-fold increase in conductivity of pulmonary lymphatics. Therefore, the safety factors that oppose edema formation must become far greater than the 20– 25 mmHg occurring in acute studies. However,

chronically elevated left atrial pressure is due to the extremely large lymph flows. The safety factor might well be 40–45 mmHg, that is, double the 23 mmHg total safety factor seen in acute pulmonary edema formation because of the huge lymph flow. However, there are vertical gradients in PPC , PT , pT , and JL in upright intact lungs that are very different from the average values used in the examples given previously, as shown in Figure 2. For example, the capillary pressure increases from 2 to 12 mmHg from the apex to the base of a lung, which is 10 cm high. These different capillary pressures result in a 10 mmHg difference in force acting at the bottom of the lung compared to the apex, and they will produce a greater transcapillary filtration in the more dependent regions of the lungs. Since a greater filtration occurs in the bottom of the lung relative to the top, pT would be less in the base tissues (17 mmHg) than at the apex (19 mmHg) of the lung. Lymph flow would also be higher at the base of the lung compared to the apex, and the lymphatic system can remove a larger portion of the filtration. PT decreases from
8 to 0 mmHg from the top to the bottom of the lung, which opposes filtration at the bottom of the lung. The combination of greater transcapillary filtration and lymph flow in the basal regions of the lung results in decreased pT and increased PT . The differences between these capillary, tissue forces, and lymph flows at the apex and base of the

Top

10 cm

PT


T

PC

∆P

−8

19

2

1

−4

18

7

1

0

17

12

1

(
C = 28)

Heart level

Bottom Figure 2 Schematic representation of tissue fluid pressure (PT ), plasma protein osmotic pressure of the tissue fluids (pT ), and capillary pressure (PC ) at the bottom, midpoint, and top of an upright lung. The imbalance in tissue forces (DP) is the same at the top and bottom of the lung. pC is the plasma protein osmotic pressure. However, the filtration coefficient will be higher at the bottom of the lung than the top and more transcapillary filtration will occur. Lymph flow will increase at the bottom of the lung and remove some of the capillary filtration. Reproduced from Taylor AE, Khimenko PL, Moore TM and Adkins WK (1997) Fluid balance. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung Scientific Foundations, 2nd edn., p. 1557. New York: Raven Press, with permission from Lippincott–Raven Publishers.

222 FLUID BALANCE IN THE LUNG

lungs (i.e., PPC , PT , pP , pT , and even KFC , JV , and JL ) are usually studied in animal lungs laid flat on a scale. However, it is necessary to know how these forces change in the upright lung in order to accurately evaluate the fluid exchange in the upright human lung. The differences given here are sufficient for a general analysis of transcapillary filtration, but for more precise measures, lungs must be studied in an upright position since the KPC will be much smaller at the top of the lung as compared to the lower portion. Although isolated and perfused lungs have provided investigators with new insight into the factors responsible for both producing and opposing pulmonary edema, a more clinical-type animal model has also been developed to study edema formation in awake and standing sheep. The LAPs were measured and the sheep’s lung lymph was also collected. LAPs were usually elevated while the sheep were being studied, lymph collected, and its plasma proteins measured. Then, sheep were subjected to sepsis or some form of inflammation, such as a period of ischemia in the lungs, followed by reperfusion, and the JL and its protein concentrations were measured before and after treatment with different interventions to determine whether specific interventions could prevent and/or reverse the damage to the sheep’s microvascular membrane and lessen pulmonary edema. Obviously, when the pulmonary capillaries are normal, CL =CP will decrease when LAPs are increased; however, when the capillaries are damaged, lymph flow and the plasma protein concentrations will also increase even when LAP increases. This animal model was, and still is, a most useful experimental procedure because the correction of sepsis and other forms of lung pathology can be sufficiently defined to perhaps provide clinically useful data; certain interventions have been found that prevent and even reverse capillary wall damage in the awake-sheep model. Finally, what is known about the reabsorption of edema fluid once it fills large portions of the lungs? Obviously, reducing capillary pressure will decrease filtration, and although it is controversial, it is now believed that the pulmonary epithelial cells can actively move ions from the intra-alveolar fluid into the interstitial tissues of the lung which could be removed from the lung by the lymphatics, or the transported fluid can also enter the blood that flows through the lung, if the forces are consistent with capillary reabsorption. It appears that amiloride-sensitive Na channels are located in the epithelial cells of the alveoli and when airways are activated by Naþ / Kþ ATPase, alveolar fluid removal is accelerated and can be removed by capillary absorption or lymph

flow. Ouabain, which blocks these channels, and amiloride, an Na þ channel blocker, increase lung wet/dry weight ratios exposed to ischemia/reperfusion injury from 10 (damage with only ischemia/ reperfusion) to 18, when both Na channels and the Naþ /Kþ ATPase are blocked. However, if the capillary endothelial barrier is damaged, it cannot absorb the transported fluid, which complicates data interpretation. Future studies must be performed to gain a much better understanding of the transport systems in the lung’s epithelial barrier to clarify its role in edema clearance. It is not clear how much fluid can be absorbed by active transport from the airways and whether the transport is upregulated in edema. See also: Acute Respiratory Distress Syndrome. Carbon Dioxide. Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Infant Respiratory Distress Syndrome. Ion Transport: Calcium Channels; ENaC (Epithelial Sodium Channel). Lymphatic System. Nitric Oxide and Nitrogen Oxides. Oxidants and Antioxidants: Antioxidants, Enzymatic. Permeability of the Blood–Gas Barrier. Pleural Effusions: Pleural Fibrosis. Pulmonary Circulation. Vascular Disease.

Further Reading Borok Z and Verkman AS (2002) Lung edema clearance: 20 years of progress. Invited review: role of aquaporin water channels in fluid transport in lung and airways. Journal of Applied Physiology 93: 2199–2206. Guyton AC and Lindsey AE (1959) Effect of elevated left atrial pressure and decreased plasma protein concentration on the development of pulmonary edema. Circulation Research 7: 649–657. Khimenko PL and Taylor AE (1999) Physiology of body fluids. In: Webb AR, Shapiro MJ, Singer M, and Suter PM (eds.) Oxford Textbook of Critical Care, pp. 24–27. Oxford: Oxford University Press. Moore TM, Shirah WB, Khimenko PL, et al. (2002) Involvement of CD40–CD40L signaling in postischemic lung injury. American Journal of Physiology 283: L1255–L1262. Pappenheimer JR, Renkin EM, and Borrero LM (1951) Filtration, diffusion and molecular sieving through peripheral capillary membranes. A contribution to the pore theory of capillary permeability. American Journal of Physiology 167: 13–46. Parker JC and Townsley MI (2004) Evaluation of lung injury in rats and mice. American Journal of Physiology 286: L231– L246. Patlak CS, Goldstein DA, and Hoffman JF (1963) The flow of solute and solvents across a two-membrane system. Journal of Theoretical Biology 5: 426–442. Rippe B and Haraldsson B (1992) Transport of macromolecules across microvascular walls: the two-pore theory. Physiology Review 74(1): 163–219. Rippe B and Taylor A (2001) NEM and filipin increase albumin transport in lung microvessels. American Journal of Physiology 280: H34–H41.

FLUID BALANCE IN THE LUNG 223 Staub NC (1978) Lung water and solute exchange. In: Lefant C (ed.) Lung Biology in Health and Disease, vol. 7. New York: Dekker. Taylor AE (1997) Microvascular fluid and solute exchange. In: Encyclopedia of Human Biology, 2nd edn., pp. 2581–2592. San Diego: Academic Press. Taylor AE and Ballard ST (1992) Microvascular function. American Review of Respiratory Disease 146: S24–S27. Taylor AE, Khimenko PL, Moore TM, and Adkins WK (1997) Fluid balance. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung Scientific Foundations, 2nd edn., p. 1557. New York: Raven Press.

Taylor AE and Parker JC (1985) Pulmonary interstitial spaces and lymphatics. In: Fishman AP and Fisher AB (eds.) Handbook of Physiology, Respiratory System I, pp. 167–230. Baltimore: Williams & Wilkins. Taylor AE, Rehder K, Hyatt RE, Parker JC (1989) Clinical Respiratory Physiology, Ch. 10, p. 178. Philadelphia, PA: Saunders. Uhley HN, Leeds SE, Sampson JJ, and Friedman M (1961) Some observations on the role of the lymphatics in experimental acute pulmonary edema. Circulation Research 9: 688–693.

G GASTROESOPHAGEAL REFLUX P O Katz and N Tajong, Albert Einstein Medical Center, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract This article reviews the presentation, epidemiology, diagnosis, and therapy of gastroesophageal reflux and its relationship to lung disease. Focus is on cough, asthma, and idiopathic pulmonary fibrosis. The approach to treatment can be applied to any patient with a pulmonary disease or symptom believed to be associated with gastroesophageal reflux disease.

Gastroesophageal Reflux Disease Gastroesophageal reflux disease (GERD) is a very common yet complex disease with widely variable presentations and complications that extend beyond the esophagus. The most common esophageal presentation is heartburn, which is caused by reflux of acid and gastric contents into the esophagus. Approximately 50% of Americans experience heartburn once a month with 25% using over-the-counter antacids three or more times a month with 4–7% of the patient population experiencing daily symptoms. Recent reviews have suggested the annual cost of treating GERD with both over the counter (OTC) and prescription drugs to be over 8 billion US dollars. Normal physiology involves transient relaxation of the lower esophageal sphincter (LES), especially after meals. This allows gastric contents (acid) to enter the esophageal lumen, which can lead to mucosal damage in susceptible patients. The main determinants of injury to esophagus are the duration of contact as well as epithelial resistance to injury, and factors other than acid. The duration of contact depends on the number of reflux episodes and the efficiency of esophageal peristalsis, which normally clears the refluxed gastric contents. Patients with GERD can have a wide range of clinical presentations ranging from esophageal symptoms with or without mucosal damage to evidence of mucosal damage without symptoms, or few esophageal symptoms with extensive extraesophageal manifestations. Typical symptoms of GERD include heartburn, which is described as a retrosternal

burning sensation with radiation to throat. Often there is regurgitation of acidic gastric contents into the mouth with complaints of sour taste. Symptoms are worse after high-fat meals, in the supine position, after heavy meals, spicy foods, alcohol consumption, and citrus products. Of all GERD symptoms a history of heartburn and regurgitation has the highest specificity for diagnosing GERD. Since there is no accepted gold standard the diagnosis of GERD can be made using endoscopy, pH monitoring, radiologic testing, provocative testing, and therapeutic trial of acid suppression. The first line approach to the patient with typical GERD symptoms is a therapeutic trial of acid suppression usually with a proton pump inhibitor (PPI) once or twice daily. Some advocate twice daily dosing for 1–2 weeks, while others recommend once a day for 4–8 weeks. Unfortunately, there are no definitive studies suggesting the optimal drug, dose, or duration of trial; however, multiple studies have supported this approach to patients with typical symptoms of GERD. Patients who fail to experience symptom relief despite a therapeutic trial or those with unusual or alarm symptoms (weight loss, dysphagia) are referred for upper endoscopy to rule out other etiologies and to grade the severity of esophageal mucosal damage. Upper endoscopy is also indicated for patients with longstanding disease to screen for Barrett’s esophagus. The main drawbacks of endoscopy are that it lacks sensitivity for GERD as many studies reveal that only 30–40% of patients with heartburn will have erosive esophagitis. The measurement of esophageal pH by an intraluminal monitor is another method for diagnosing GERD. A pH probe is placed 5 cm above the manometrically located lower esophageal sphincter with continuous recordings of pH in the distal esophagus over a defined period. Recent advances have allowed this to be accomplished with a tubeless recording device that is taped to the esophagus. Monitoring is for 48 h. Patients’ symptoms are recorded so that correlation of symptoms with acid reflux can be made. Limitations of pH monitoring include patient acceptance and reproducibility, which may be as low

226 GASTROESOPHAGEAL REFLUX Table 1 Atypical symptoms or signs of gastroesophageal reflux disease EENT a

Pulmonary

Pharyngitis Hoarseness/voice changes Otitis Sinusitus Vocal cord granulomas Subglottic stenosis Laryngitis Laryngeal cancer Cough

Asthma Cough Idiopathic pulmonary fibrosis Chronic bronchitis Pneumonia Unclassified Unexplained chest pain Sleep apnea Dental erosions

a

EENT, eyes, ears, nose, and throat.

as 85% in patients with respiratory symptoms, and that the test only measures acid reflux into the esophagus. Combined impedance and pH monitoring will clarify the role of nonacid reflux. Barium studies can diagnose anatomical abnormalities that may contribute to GERD, but are of limited use in the evaluation of GERD without alarm symptoms as they lack sensitivity for mucosal disease. Provocative testing involves the infusion of 0.1 N hydrochloric acid into the esophagus (Bernstein test) with a positive test defined as reproduction of patients’ typical symptoms with the infusion. This test is highly specific for acid reflux, but not sensitive and so reserved for situations when a pH monitor is not available. Respiratory Complications

The respiratory manifestations of acid reflux disease include bronchial asthma, cough, pulmonary fibrosis, and pneumonia. A more extensive list of extraesophageal manifestations of GERD is seen in Table 1.

Chronic Cough Chronic cough is a problem that plagues 3–40% of the population. The three most common etiologies include asthma, esophageal reflux, and rhinitis. Approximately 20% of nonsmoking patients with chronic cough and normal chest X-ray have esophageal reflux, either as sole cause or in combination with the other causes of chronic cough. The pathophysiology of GERD-induced cough is not well understood, but involves microaspiration with exposure of esophageal contents into the bronchial tree as well as vagally mediated tracheoesophageal reflex. Exposure of esophageal contents to the airway directly irritates cough receptors in small and larger airways through vagal mediation. The tracheobronchial reflex has been theorized to cause GERD-induced cough in several ways.

Stimulation of cough sensory nerve ends in the esophagus may cause transmission to and from the cough center via the vagus nerve. This nervous impulse may also be reflexively transmitted via vagal efferents from the brain, which stimulate secretion of neurotransmitters in the lower respiratory tract causing cough. Lastly, stimulation of nerve endings in the esophagus may send impulses directly to the trachea, causing cough, independent of the central nervous system (Figure 1). Esophageal dysmotility is also seen in patients with chronic cough and may be an underestimated mechanism for GERD-induced cough and present new possibilities for treatment of these difficult patients. Clinical Presentation

The likelihood of GERD as etiology of cough is high when typical symptoms such as heartburn, sour taste, and regurgitation are present. It is not unusual to have cough as the initial presentation of GERD, even in the absence of heartburn. Cough related to reflux is traditionally thought to be nocturnal, but this association is hard to prove. Patients with microaspiration-triggered cough may present with more reflux symptoms, usually occurring prior to cough. It is important to note that cough may induce reflux, further complicating the presentation. Diagnosis

There is no accepted gold standard for diagnosing cough induced by GERD. Excluding causes of chronic cough such as asthma and postnasal drip is crucial. The most reliable test to determine if cough is due to GERD is resolution of cough with treatment of reflux. Many authors including us support a therapeutic trial of antisecretory therapy after other nonreflux causes have been excluded, in light of the fact that GERD is so prevalent in this population. One study found that of 17 patients with a positive pH test, six (35%) had striking resolution in their symptoms within 2 weeks of omeprazole therapy (40 mg b.i.d.), which was sustained over 1 year. This approach if successful avoids the cost of diagnostic procedures and limits them to those who do not respond to an empiric trial. Twenty-four hour esophageal pH measurement is the most sensitive and specific test for diagnosing GERD-induced cough with positive and negative predictive values of 89% and 100%, respectively. Abnormal findings on pH study include increased frequency of reflux episodes, decreased acid clearance times, and increased acid exposure times. However, an abnormal pH study does not reliably predict which patients with cough and abnormal pH results

GASTROESOPHAGEAL REFLUX Esophagus

CNS

Reflux

Tracheobronchial tree Microaspiration

227

Airway

Mediator release Inflammation

Airway vagal afferents Esophageal vagal afferents

CNS

Edema Mucus

Airway vagal efferents

Smooth muscle

Heightened bronchial reactivity Figure 1 Gastroesophageal reflux disease may lead to microaspiration with direct effects on the epithelium. Refluxate can stimulate vagal afferents in the esophageal or tracheobronchial tree. Axon reflexes may cause direct pulmonary neuroinflammatory changes or can lead to central nervous system (CNS) stimulation. The addition of nerve growth factor can lead to switching of airway ‘A’ fibers to tachykinin-expressing fibers, which result in greater CNS stimulation and peripheral neuroinflammatory effects. Reproduced from Stein M (2003) Symposium: possible mechanisms of influence of esophageal acid on airway hyperresponsiveness. American Journal of Medicine 115(3A): 55S–59S, with permission from Elsevier.

will respond to treatment. In a recent study 11 of 17 patients with abnormal pH did not respond to treatment with omeprazole. Some with a normal pH study (thus physiologic reflux) may still have GERD as cause of cough. In this group of patients there is a temporal relationship between cough during or within 5 min of periods when esophageal pH is less than 4 despite normal reflux time. Endoscopy may reveal the mucosal damage caused by GERD but has little role in the diagnostic evaluation of GERD-induced cough. Esophageal manometry has a limited role, but may be important as an adjunct to pH monitoring in selected groups of patients.

Asthma Airway hyperresponsiveness is now the accepted underlying pathophysiology in asthma. The association between asthma and GERD is derived from multiple studies showing a high prevalence of acid reflux in asthmatics, as well as the observed improvement in some asthma patients with treatment of acid reflux. As many as 80% of adult asthmatics have abnormal acid reflux. Compared to normal controls asthma patients have decreased lower esophageal sphincter pressures, more frequent reflux episodes, and take longer to clear the esophagus of acid. A large study of veterans found a higher likelihood of asthma in patients with esophagitis and stricture, and a more recent study found asthmatics to have significantly more frequent and more severe GERD symptoms

than nonasthmatics. More evidence for the relationship between asthma and GERD was noted in a large prospective study of asthmatics, which not only found heartburn to be common in asthmatics, but also found that heartburn was independently associated with future hospitalizations in asthmatics. A population-based study in three European countries found that subjects with nocturnal GERD were more likely to be diagnosed with asthma and have evidence of airway reactivity as measured by peak expiratory flow rate. The mechanism by which GERD causes respiratory symptoms is unknown, but esophageal acidinduced airway hyperresponsiveness is the most accepted theory. This is thought to occur via microaspiration of refluxate, esophageal triggered vagal reflexes, and esophageal triggered necroinflammation. The interplay between these mechanisms is thought to result in airway hyperreactivity (Figure 2). Microaspiration may lead to airway inflammation through damage of epithelial cells, which release inflammatory cytokines, and this leads to increased vagal tone. Vagally mediated airway reflexes due to acid reflux have been demonstrated in many studies including one in which 20 patients with asthma and GERD were compared to nonasthmatics with GERD using acid infusion. The peak expiratory flow decreased in the asthma group with acid challenge compared to the nonasthmatic group and it was found that administration of vagolytic doses of atropine ablated the response to acid infusion. Increased airway hyperresponsiveness, as measured

228 GASTROESOPHAGEAL REFLUX Esophagus

CNS

Tracheobronchial tree

Airways

Neurokinins

Axon reflex

Reflux

Axon reflex

Inflammation

Microaspiration

Edema

NGF

Mucus Vagal afferents

CNS

Vagal afferents Vagal efferents

Mediators

Bronchial hyperreactivity

Smooth muscle

NGF

Figure 2 Both esophageal afferents and airway afferents lead to central nervous system (CNS) stimulation with resultant increases in vagal efferent impulses. This leads to increased hyperresponsiveness. NGF, nerve growth factor. Reproduced from Harding S (1997) Ambulatory Esophageal pH Monitoring: Practical Approach and Clinical Applications, pp. 149–164. Baltimore: Williams & Wilkins, with permission.

by maximal expiratory flow at 50% vital capacity, is seen with infusion of 0.1 N HCl in the esophagus. This response can be blocked by pretreatment with atropine, further implicating the vagal link. Medications used to treat asthma may also exacerbate reflux and produce further symptoms. Inhaled beta2 agonists were shown to cause a dose-dependent decrease in LES pressures as well as a dose-dependent decrease in esophageal body amplitudes. A study on the effects of oral steroids on reflux in asthma subjects showed significant increase in esophageal acid contact time at the proximal and distal pH probes. There was no significant increase in asthma or GERD symptoms, or basal stimulated gastric acid secretion. These results were limited by the small sample size. Diagnosis of GERD-Related Asthma

The diagnosis of GERD-related asthma relies mainly on the clinical presentation. As in chronic cough asthmatics can have GERD in the absence of typical symptoms. This so-called clinically ‘silent GERD’ was demonstrated in a study in which 62% of asthmatics had acid reflux on pH study without clinical symptoms. Many patients come to attention after usual therapies for asthma have failed. The workup

is similar to chronic cough in that most experts advocate a trial of PPI and pursue further diagnostic testing only if this fails.

Idiopathic Pulmonary Fibrosis Idiopathic pulmonary fibrosis (IPF) is a severe form of interstitial lung disease of unknown etiology that has been associated with GERD. The evidence for this association comes from observational and epidemiologic studies as well as from animal models. The histology of this disease is characterized by interstitial fibrosis, inflammation, and honeycomb changes interspersed by normal lung epithelium. Various animal studies dating back to the 1950s demonstrated similar changes in the lung epithelium when exposed to 0.1 N hydrochloric acid as well as gastric acid. A case-control study using patients with esophagitis and strictures were compared to controls for the occurrence of laryngeal, pharyngeal, sinus, and pulmonary diseases. Patients with erosive esophagitis were more likely to have various pulmonary diseases, including pulmonary fibrosis with odds ratio 1.36 for IPF. Further association of pulmonary fibrosis with GERD was demonstrated in a study that showed that the incidence of GERD was significantly higher in patients with confirmed

GASTROESOPHAGEAL REFLUX

pulmonary fibrosis than in controls. Sixteen of 17 IPF subjects had abnormal acid exposure compared to 4 of 8 controls. Twenty-five per cent of the patients with abnormal pH results had no symptoms of acid reflux. Thus far, the data linking GERD to IPF is compelling, but more studies are needed. Large-scale randomized trials looking specifically at GERD and IPF as well as prospective therapeutic trials would shed more light on this fatal disease.

Treatment There have been few clinical trials of treatment involving patients with extraesophageal manifestations of GERD, specifically asthma, cough, and voice changes. The majority are uncontrolled and do not address long-term maintenance. Treatment is based on the principles advocated for treating patients with heartburn and erosive esophagitis, observations from available clinical trials, and clinical experience. It appears in general that patients with extraesophageal manifestations of GERD must be treated with higher doses of pharmacologic therapy, principally with PPIs (twice daily), and with longer periods of treatment (up to 3–4 months) to achieve complete relief of symptoms as compared to patients with heartburn and erosive esophagitis (usually 8 weeks). Important insights into treatment of patients with extraesophageal GERD can be gleaned from one well-designed study in which 30 patients with documented asthma and GERD proven by prolonged pH monitoring were treated with increasing doses of omeprazole. Starting at 20 mg daily, the medication was increased by 20 mg after each 4-week treatment period for 3 months or until esophageal acid exposure was reduced to ‘normal’. Normalization of esophageal acid exposure resulted in improvement in pulmonary symptoms in 70% of patients. Several observations emerge from this trial: 8/30 (28%) of patients needed greater than 20 mg of omeprazole a day to normalize esophageal acid exposure; and many patients required the entire 3-month period of treatment to achieve optimal symptom relief with improvement progressing continuously over the 3-month period, confirming the observations of others. A favorable response to omeprazole was seen in patients who presented with frequent regurgitation (greater than once a week) and those with abnormal proximal acid exposure demonstrated by ambulatory pH monitoring. This study emphasizes the importance of adequate esophageal acid control to achieve improvement in patients with extraesophageal symptoms. Further studies are needed to determine the optimal reduction in esophageal exposure required

229

for successful treatment, as simple ‘normalization’ of acid control may not be sufficient. Complete elimination of esophageal acid exposure may be needed in patients with extraesophageal disease for adequate symptom control. Suggested Approach to Treatment of Pulmonary Symptoms due to GERD

It is our current practice to begin therapy with PPIs twice daily, before breakfast and dinner for 2–3 months (Figure 3). Clinical experience suggests patients with GERD will respond to this length of therapy, though many will require longer treatment periods to achieve optimal results. Patients who have a good initial but incomplete response to this therapeutic trial are to be continued at the same dose for an additional 4–8-week period to assess continued improvement. If satisfactory symptom relief is not achieved at this point, we evaluate the patient with prolonged reflux pH monitoring studies while on continued therapy with PPI to assess the adequacy of intragastric acid suppression and elimination of distal and proximal esophageal acid exposure. If acid suppression is incomplete, additional medical therapy is indicated prior to assuming that the patient is a medical failure. If the patient has a poor response to the initial therapeutic trial, prolonged ambulatory pH monitoring should be performed while on therapy to evaluate drug efficacy.

Possible GERD symptoms

Trial of PPI Rx

Success (confirm dx)

Persistent symptoms

Ambulatory MII-pH monitoring on Rx

Acid GERD with symptoms

No GERD

Nonacid GERD with symptoms Figure 3 GERD diagnostic algorithm. PPI Rx, proton pump inhibitor reflux.

230 GASTROESOPHAGEAL REFLUX

Recent studies from our laboratory have shown that 70% of patients with GERD treated with twicedaily PPI therapy will continue to have gastric acid breakthrough (pH o 4) for at least 1 h in the overnight period. Reflux will occur in 30–50% of these patients during this breakthrough period. Another study from our laboratory found that 30% of patients with extraesophageal reflux (asthma, cough, LPR) continue to have abnormal acid exposure (GER) despite therapy twice daily with a PPI. These patients require more aggressive medical therapy, as even small amounts of acid exposure may be injurious to the mucosa of the larynx and airways. Doubling the dose of PPI (e.g., omeprazole 40 mg b.i.d.) may be sufficient to control esophageal acid exposure effectively. However, some patients will have continued nocturnal gastric acid breakthrough and esophageal reflux despite high-dose PPI and may require the addition of an H2-receptor antagonist to achieve optimal acid suppression. Referral for ambulatory pH monitoring to document the effectiveness of therapy and assessment of the presence of continued acid exposure or nonacid reflux is indicated if patients are refractory to PPIs. We do not routinely recommend adding a prokinetic agent to PPIs. We reserve prokinetic agents for patients in who adequate acid exposure cannot be accomplished with acid suppressive agents, and for the patient with documented delayed gastric emptying. Clinical experience suggests that most patients with extraesophageal GERD will have chronic GERD and will require long-term medical therapy and/or consideration of antireflux surgery for longterm control. The dose of PPI and other medications, and the decision to perform antireflux surgery should be individualized to maintain symptom relief and mucosal healing. Current evidence suggests that long-term medical therapy is safe and tolerance or tachyphylaxis extremely rare. Patients who choose long-term medical therapy can be confident of excellent long-term control of acid-induced mucosal injury without the worry of serious complications. See also: Asthma: Overview; Extrinsic/Intrinsic. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Laryngitis and Pharyngitis. Pulmonary Fibrosis. Upper Respiratory Tract Infection.

Further Reading Avidan B, Sonnenberg A, Schnell TG, and Sontag SJ (2001) Temporal associations between coughing or wheezing and reflux in asthmatics. Gut 49(6): 767–772.

Canning BJ and Mazzone SB (2003) Reflex mechanisms in gastroesophageal reflux disease and asthma. American Journal of Medicine 115(3A): 45S–48S. Diette GB, Krishnan JA, et al. (2002) Asthma in older adults, factors associated with hospitalization. Archives of Internal Medicine 162: 1123–1132. El-Serag HB and Sonnenberg A (1997) Comorbid occurrence of laryngeal or pulmonary disease with esophagitis in the United States Military Veterans. Gastroenterology 113: 755–760. Field and Stephen K (1999) A critical review of the studies of the effects of stimulated or real gastroesophageal reflux on pulmonary function in asthmatic adults. Chest 115: 848–856. Fontana GA and Pistolesi (2003) Cough 3: chronic cough and gastroesophageal reflux. Thorax 58(12): 1092–1095. Gislason T, Jansen C, Vermeire P, et al. (2002) Respiratory symptoms and nocturnal gastroesophageal reflux: a population-based study of young adults in three European countries. Chest 121: 158–163. Harding S (1997) Ambulatory Esophageal pH monitoring: Practical Approach and Clinical Applications, pp. 149–164. Baltimore: Williams & Wilkins. Harding SM, et al. (1995) GERD induced bronchospasm. American Journal of Respiratory and Critical Care Medicine 151: A589. Harding SM, Richter JE, et al. (1996) Asthma and gastroesophageal reflux: acid suppression therapy improves asthma outcome. American Journal of Medicine 100: 395–405. Harding SM and Richter JE (1997) The role of gastroesophageal reflux in chronic cough and asthma. Chest 111: 1389–1402. Harding and Susan M (2003) Recent clinical investigations examining the association of asthma and gastroesophageal reflux. American Journal of Medicine 115(3A): 39S–44S. Irwin RS, Azwacki JK, Curley FJ, French CL, and Hoffman PJ (1989) Chronic cough as the sole presenting manifestation of gastroesophageal reflux. American Review of Respiratory Diseases 140: 1294–1300. Irwin RS, Curley FJ, and French CL (1990) Chronic cough: the spectrum and frequency of causes, key components of the diagnostic evaluation, and outcome of specific therapy. American Review of Respiratory Diseases 141: 640–647. Irwin RS and Richter JE (2000) Gastroesophageal reflux and chronic cough. American Journal of Gastroenterology 95(8): S9–S14. Kastelik JA, Redington AE, Aziz I, et al. (2003) Abnormal esophageal motility in patients with chronic cough. Thorax 58(8): 699–702. Katz PO, Anderson C, Khoury R, and Castell DO (1998) Gastrooesophageal reflux associated with nocturnal gastric acid breakthrough on proton pump inhibitors. Alimentary Pharmacology and Therapeutics 12: 1231–1234. Khoury R, Mohiuddin M, Katz P, et al. (1998) Influence of body position on nighttime recumbent reflux in patients with GERD. Gastroenterology 114(4): A174. Kiljander TO (2003) The role of proton pump inhibitors in the management of gastroesophageal reflux disease-related chronic cough. American Journal of Medicine 115(3A): 65S–71S. Morice AH and Kastelik JA (2003) Cough: chronic cough in adults. Thorax 58(10): 901–907. Peghini PL, Katz PO, Bracy NA, and Castell DO (1998) Nocturnal recovery of gastric acid secretion with twice-daily dosing of proton pump inhibitors. American Journal of Gastroenterology 93: 763–767. Schnatz PF, Castell JA, and Castell DO (1996) Pulmonary symptoms associated with gastroesophageal reflux: use of ambulatory pH monitoring to diagnose and to direct therapy. American Journal of Gastroenterology 91(9): 1715–1718.

GENE REGULATION 231 Stein M (2003) Symposium: possible mechanisms of influence of esophageal acid on airway hyperresponsiveness. American Journal of Medicine 115(3A): 55S–59S.

Zaid Y and Johnson DA (1999) Diagnostic evaluation in gastroesophageal reflux disease. Gastroenterology Clinics of North America 28(4): 809–827.

GENE REGULATION M A Perrella, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

expression of genes can be regulated, with emphasis at the level of gene transcription and mRNA processing. Figure 1 provides an overview of the gene regulation process.

Abstract The genome of an organism contains the complete set of genetic information passed on from generation to generation. The gene is a subunit of this genetic information, consisting of a sequence of nucleic acids. These nucleic acids provide a code, which in many circumstances lead to the production of functional proteins. The inheritable information contained in our genes determines the development of a person or organism, and their eventual appearance, functional status, and even their susceptibility to contract diseases. Variations in gene content or alterations in gene expression may increase a person’s risk for disease development. Through the study of gene sequences as well as their regulation, medical science has the potential to make major breakthroughs in the management (detection, prevention, and therapy) of various disease processes. The present article provides an overview of how gene expression can be regulated: from gene transcription in the nucleus, to various mRNA processing events, to export and decay of mRNA in the cytoplasm.

Introduction Genes are the genetic material that helps to determine how a person or organism develops, their appearance and functional status, and what diseases they may eventually contract. The complete set of genes within the body of a person is known as the genome. A project was initiated in 1990 to sequence the entire human genome and provide a roadmap of the genetic material that makes up our bodies. The information provided by the genome project is helping to give scientists and physicians a better understanding of how diseases occur, and is being used to develop medications that specifically target the cause of a disease, not just the symptoms. Diseases may be caused by variations in gene content or by alterations in gene expression, which increases a person’s risk for disease development. Through the study of gene sequences and their regulation, medical science has the potential to make major breakthroughs in the pathophysiology, early detection, treatment, and prevention of distinct disease processes. The present article focuses on the mechanisms by which the

Transcriptional Regulation A gene is a subunit of genetic information consisting of a sequence of nucleic acids. These nucleic acids provide a code, which in many circumstances leads to the production of functional proteins. If the protein binds in a sequence-specific manner to DNA, it is referred to as a trans-acting factor. The segment of genomic DNA, typically 6–12 bp in length, that acts as a binding site for trans-acting factors is called a cis-acting element. The interaction of trans-acting factors and their cognate cis-acting elements contribute to gene regulation, and is discussed in detail below. Sequence-specific trans-acting factors are often composed of functional modules. For example, a DNA-binding domain may be linked to one or more activation or repression modules, as well as modules for dimerization or regulation. These factors are frequently classified according to the type of DNAbinding domain, usually a short motif in this domain responsible for DNA binding. Examples of these DNA-binding domains include zinc finger, steroid receptors, helix–turn–helix, helix–loop–helix, and leucine zippers. For further details regarding transcription factors and their cognate DNA-binding sites, please refer to Transcription Factors: Overview or the articles on specific transcription factors of interest. Gene regulation by transcription involves the synthesis of RNA, which is catalyzed by RNA polymerase (type II in higher eukaryotes). Genes that contain RNA polymerase II promoters have a short conserved sequence, termed the initiator (Inr) at the start point of transcription. Most of these RNA polymerase II promoters contain a sequence approximately 25 bp upstream of the transcription start point called the TATA box. The TATA box sequence consists entirely of A . T nucleotides, and it tends to be surrounded by G . C rich sequences. The TATA box (one of the first cis-acting elements to be identified) is

232 GENE REGULATION Enhancer Transcription factors (trans-acting factors)

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AAAA 3′ poly(A) tail

Transport into cytoplasm Translation Figure 1 Overview of gene regulation. In conjunction with the general transcription apparatus, trans-acting factors bind to their cognate regulatory sequences (cis-acting elements) in the 50 -flanking sequence of a gene to initiate and drive transcription. The general transcription apparatus contains RNA polymerase II (Pol II) and TATA-binding protein (TBP) and associated factors that form a transcriptional complex at the TATA box (TATAA), and subsequently initiates transcription (bent arrow). Trans-acting factors bind to cisacting elements contained in promoter and enhancer regions of the gene. These two regions of the 50 -flanking sequence of a gene can be separated by several kilobases. The mRNA that is transcribed undergoes a variety of processing events, including the splicing out of introns and joining of exons to generate a smaller mRNA that contains the intact coding sequence, the addition of a 50 methylated cap (MeGppp) and a poly(A) tail (AAAA), and the export of the mature mRNA into the cytoplasm for translation of the functional protein.

considered to be involved in the positioning of eukaryotic RNA polymerase II (a sequence-specific DNA binding transcription factor or trans-acting factor) for correct initiation. RNA polymerase II cannot initiate transcription by itself, but is dependent on auxiliary transcription factors. For RNA polymerase II, binding of TFIID to a region that extends upstream of the TATA box is the first step in initiation. TFIID consists of the TATA-binding protein (TBP) and TAFs (TBP-associated factors). Binding of TBP to the TATA box is the first step in initiation, followed by TAFs binding to the initiation complex in a defined order. When RNA polymerase II binds to this complex, transcription is initiated. The polymerase plus the auxiliary factors constitutes the general or basal transcription apparatus. Promoters that do not contain a TATA box are called TATA-less promoters. In TATA-less promoters, the same general apparatus, including TFIID, are needed. However, in these promoters TFIID binds to the Inr via one or more of the TAFs (and not TBP) directly recognizing the Inr element. In promoters that contain a TATA box, transcriptional initiation typically occurs at a defined start point. The TATA box aligns the RNA polymerase by its interaction

with TFIID allowing initiation to occur at the proper site. However, in the case of TATA-less promoters, they often lack a unique start point, with transcriptional initiation occurring at one of a number of potential start points. It is also of interest that in TATA-less promoters, binding sites for a subset of sequence-specific activators, for instance Sp1, are commonly located in the proximal promoter region (within the first 250 bp upstream from the transcription start site). These activators, in conjunction with their cognate DNA binding sites, interact with the basal transcriptional machinery to efficiently regulate gene transcription in TATA-less promoters. The largest subunit of RNA polymerase II contains a C-terminal domain (CTD) that consists of multiple repeats of a 7 amino acid sequence. The CTD is unique to RNA polymerase II, and CTD can be highly phosphorylated on serine and threonine residues. This phosphorylation process is involved with the transcriptional initiation process. Once initiation has taken place, RNA polymerase II moves along the DNA template, synthesizing RNA until it reaches a termination sequence. The production of eukaryotic mRNA involves additional processing after transcription is complete, allowing production of mature

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mRNA. Further details regarding these procedures will be described in a later section on mRNA processing. In conjunction with RNA polymerase II, expression of eukaryotic genes is tightly regulated by additional trans-acting factors interacting with their cognate cis-acting elements. These elements are contained in promoter or enhancer regions of the gene. The promoters are typically located immediately upstream of the transcription initiation site, while enhancers may act at long distances, either upstream or downstream, of the RNA start site. The enhancer region(s) may be separated by several kilobases from the promoter. While enhancers are made up of the same type of cis-acting elements, the density of these elements is greater in the enhancer than in the promoter. This gives an enhancer the ability to form a regulatory complex. When a group of trans-acting factors assembles on an enhancer region to regulate gene transcription, this complex is referred to as an enhanceosome. Together, sequence-specific transcription factors and their associated DNA binding sites play an important role in determining the regulation of gene expression.

Chromatin Beyond the interaction of trans-acting factors with cis-acting elements, the chromosomal environment also plays an essential role in the transcriptional regulation of gene expression. In the cell, DNA is packaged into chromatin by histone and nonhistone proteins. Moreover, whether a gene is expressed depends on the structure of chromatin. Thus, the control of chromatin structure has a great influence on the regulation of gene expression. Changes in chromatin structure can occur in a very localized way (affecting a promoter of a specific gene), or these changes may affect large regions of a chromosome. Changes that affect large regions of a gene control the potential of that gene to be expressed. For instance, by affecting a large region of a gene the expression of the gene may be silenced, by preventing transcription factors and RNA polymerase from binding to DNA and activating gene transcription. On the other hand, changes in chromatin structure can also occur at an individual promoter, controlling whether the expression of a particular gene is activated or repressed.

Histone Proteins Events leading to alterations in chromatin structure depend on interactions with histones. For example, recently it has been determined that modifications of

histones have an effect on chromatin structure and the initiation of gene transcription. One such modification, acetylation, has been shown to be associated with gene activation. On the other hand, some repressors of transcription function by deacetylating histones. Thus, the acetylation process is reversible, with each modification being catalyzed by a specific enzyme. Enzymes that support the acetylation of histones are referred to as histone acetyltransferases (HATs), while enzymes that subsequently remove the acetyl groups are histone deacetylases (HDACs). Another modification of DNA that is associated with altering gene expression is methylation. Methylation of both histones and DNA, by methyltransferase enzymes, is a feature of inactive chromatin. If methylation of DNA occurs in the vicinity of a promoter, then transcription cannot proceed. Most of the methylation sites in DNA are CpG islands. The methylation of these CpG islands can regulate transcription by either preventing a transcription factor from binding to DNA, or by causing specific repressors to bind DNA. Thus, by regulating methyltransferase enzyme activity, the methylation status of DNA can be altered and gene expression regulated. Phosphorylation of histone H1 occurs during mitosis of the cell cycle, and it has been shown that histone H1 is a good substrate for Cdc2 kinase, which is a critical regulator of the cell cycle. Another process that has been shown to be associated with histone phosphorylation (H3) is chromatin remodeling. Loss of a kinase that phosphorylates histone H3 has devastating effects on chromatin structure. Taken together, modifications of histones and DNA including acetylation, methylation, and phosphorylation have a dramatic effect on gene regulation.

Nonhistone Proteins or Architectural Transcription Factors As mentioned previously, a group of trans-acting factors that assembles on an enhancer region of a gene to regulate gene transcription is referred to as an enhanceosome. The combinatorial assembly, or disassembly, of an enhanceosome is critical to allowing tight control over the regulation of gene expression. The effective formation of an enhanceosome depends on appropriate DNA conformation to allow the binding of transcription factors, and thus the assembly of a functional complex. Architectural transcription factors are a group of proteins that lack the intrinsic ability to directly transactivate a gene, yet they have been shown to be important in the regulation of specific genes, in part, by controlling DNA conformation. These architectural transcription factors, which

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are nonhistone proteins, contribute to the regulation of chromatin structure and thus the regulation of gene expression. An example of a group of architectural transcription factors is the high-mobility group (HMG) proteins. HMG proteins can be separated into three different superfamily members: HMG-I/Y (HMGA), HMG1/2 (HMGB), and HMG-14/-17 (HMGN), each of which is characterized by a functional sequence motif. For example, HMG-I/Y proteins bind to AT-rich regions in the minor groove of DNA. Each HMG-I/Y protein has three separate DNA-binding motifs, which are referred to as AT-hooks, separated by flexible linker peptides. A single AT-hook motif preferentially binds to a stretch of DNA containing 4–6 bp of AT-rich sequence. Binding of HMG-I/Y proteins to DNA can promote DNA structural changes including bending, straightening, unwinding, and loop formation. Depending on the target gene, these DNA conformational changes introduced by HMG-I/Y can either facilitate or prevent the assembly of enhanceosomes, and thus affect gene transcription. Beyond modifying the structure of DNA, architectural transcription factors such as HMG-I/Y are also known to recruit transcription factors into an enhanceosome complex. By binding in the minor groove of DNA, HMG-I/Y is able to recruit transcription factors to the major groove. Taken together, studies have demonstrated that HMG-I/Y is able to orchestrate the regulation of gene expression by forming an enhanceosome complex to efficiently drive transcription by altering DNA conformation and recruiting transcription factors to DNA. Figure 1 depicts a simplified two-dimensional view of gene transcription, with one or a few transcription factors binding to regulatory elements of a gene, subsequently leading to the up- or downregulation of that gene. However, a much more complex process occurs when taking into account chromatin and the role of architectural transcription factors. In this model, a group of transcription factors assembles into a complex that regulates gene expression through protein–DNA and protein–protein interactions. This complex of factors assembles on an enhancer region of a gene (enhanceosome), and then through DNA binding interacts with the promoter region to regulate gene transcription. Taking into account our studies and the work of others, we have devised a model of nitric oxide synthase (NOS)2 enhanceosome formation (Figure 2) that may form during lipopolysaccharide (LPS) and inflammatory cytokine stimulation. NOS2 is a highly inducible gene that is regulated at the level of gene transcription. NOS2 plays an important role in vivo in disease processes such as endotoxemia and sepsis, where an

HMG-I/Y IRF RNA Pol II HMG-I/Y

p50/p65

HMG-I/Y TBP TA

TA

A

DNA Figure 2 Assembly of an enhanceosome complex by HMG-I/Y. A model of NOS2 enhanceosome formation by an inflammatory stimulus is depicted. In this model, HMG-I/Y proteins bind DNA, recruit transcription factors to DNA (such as the p50 and p65 subunits of nuclear factor kappa B (NF-kB) and IRF family proteins), and facilitate interactions between these transcription factors and the transcriptional initiation complex that includes the TATA-binding protein (TBP) and associated factors and RNA polymerase II (RNA Pol II). Reproduced from Carvajal IM, Baron RM, and Perrella MA (2002) High mobility group-I/Y proteins: potential role in the pathophysiology of critical illness. Critical Care Medicine 30(supplement 1): S36–S42, with permission from Lippincott Williams & Wilkins.

overproduction of nitric oxide contributes to disease pathophysiology. This model is meant to provide a theoretical image of how architectural factors (such as HMG-I/Y) can interact with transcription factors and DNA to orchestrate the assembly of an enhanceosome complex (through DNA–protein and protein–protein interactions) that allows the initiation of transcription along with the general transcriptional machinery and RNA polymerase II. The above example demonstrates how HMG-I/Y is able to enhance the transcription of NOS2. However, HMG-I/Y is also able to suppress transcription in other genes. For example, HMG-I/Y is able to prevent induction of the interleukin-4 promoter by competing with the nuclear factor of activated T cells (NF-AT) proteins for DNA binding. Thus, nonhistone architectural transcription factors, such as HMG-I/Y, are able to play a significant role (either positive or negative) in the transcriptional regulation of gene expression.

mRNA Processing For most genes, transcription is the major control point and the most common level of gene regulation. However, control points exist beyond transcriptional initiation and synthesis, including mRNA processing,

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export of mRNA from the nucleus to the cytoplasm, and mRNA degradation in the cytoplasm. This section focuses on mRNA processing and degradation. As mentioned above, the largest subunit of RNA polymerase II contains a CTD that is unique to RNA polymerase II. This CTD can be highly phosphorylated on serine and threonine residues. CTD is critical for transcriptional initiation; however, it may also be involved in processing RNA after its synthesis by RNA polymerase II is complete. For instance, guanylyl transferase binds to phosphorylated CTD and adds a G residue (in the reverse orientation to all other nucleotides) to the 50 end of the newly synthesized RNA. This structure is termed a ‘cap’. The 50 cap is a substrate for methylation events (1–3 methyl groups are added to the new terminal G). An additional set of proteins binds to CTD, and then binds with splicing factors, allowing a coordination of transcription and splicing. RNAs that are derived from interrupted genes require splicing to remove the introns and thus generating a smaller mRNA that contains the intact coding sequence. Thus, CTD appears to help coordinate transcription with additional RNA processing. Finally, following the synthesis of RNA, a stretch of approximately 200 bases of adenylic acid, termed poly(A), is added to the 30 end of mRNA. The 30 poly(A) tail, along with the 50 cap, stabilizes the mRNA against degradation (Figure 3). Once processing is complete, the mature RNA must then be exported from the nucleus to the cytoplasm, where translation of the mRNA into a protein takes place. Besides stability, the 30 poly(A) tail may also play a role in nuclear mRNA processing,

5′-UTR

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3′ 5′ 5′ 3′ Degrade Exonuclease Exonuclease

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Figure 3 mRNA degradation. The 30 poly(A) tail (AAAA) bound by poly(A)-binding protein (PABP), along with the 50 cap (MeGppp), stabilizes the mRNA and protects against exonuclease activity. However, binding of a trans-acting factor (e.g., AUF-1) to the A þ U-rich elements triggers destabilization by deadenylation and loss of PABP, and removal of the 50 cap. This allows both 30 -50 and 50 -30 exonuclease activity to degrade the mRNA.

export of the mRNA into the cytoplasm, and translation events. While the regulation of gene expression may involve any or all of these stages of RNA processing, changes do not uncommonly occur in splicing (i.e., removal of introns and joining of exons in RNA). Most genes are transcribed into RNA that gives rise to a single type of mRNA, with no variation in exons and introns. However, in some genes, alternative splicing of RNA occurs giving rise to more than one isoform of mRNA. These alternative spliced messages may lead to the expression of different protein products. In some cases, multiple products of gene transcription are made in a cell; however, in other circumstances the splicing process is regulated so that certain transcripts are generated in specific cell types or under specific cellular conditions.

mRNA Stability In many genes, the regulation of gene expression and subsequent protein production is achieved by controlling the mRNA levels. Thus, the level of cytoplasmic mRNA is a balance between nuclear events (rate of nuclear mRNA synthesis, modification, and export) and the rate of cytoplasmic mRNA degradation. Thus, mRNAs of genes with a short half-life respond more acutely to changes in transcription compared with genes having stable mRNAs with a long half-life. Different decay pathways have been identified that regulate mRNA degradation. Nevertheless, the process of regulated mRNA decay involves various structural components of the message including the 50 cap, the 50 -untranslated region (UTR), the coding region, the 30 -UTR, and the 30 poly(A) tail. Most mammalian mRNAs are polyadenylated, and the poly(A) tails of these genes are typically bound to a poly(A)binding protein (PABP). This complex of the poly(A) tail and PABP provides stability to the mRNA and prevents rapid degradation by exoribonucleases in a 30 -50 direction (Figure 3). Deadenylation of the poly(A) tail is the first step in the degradation of many mammalian mRNAs. In deadenylation-dependent pathways, poly(A) shortening is often followed by removal of the 50 cap, thus also allowing 50 -30 degradation and a bidirectional decay of the message (Figure 3). mRNA decay may also occur in a deadenylation-independent manner. In this pathway, endonucleolytic cleavage of the message occurs, followed by exoribonuclease decay. The deadenylationdependent pathway appears to be the more prevalent pathway of mRNA degradation. The interaction of specific cis-acting structural elements and their associated trans-acting factors

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allow stabilizing or destabilizing influences on mRNA. The biological activity of these interactions at the mRNA level relies on a combination of primary and secondary structural elements, assembled in a complex, and recognized by RNA-binding proteins. Protein binding to the 30 -UTR may thus be sequence specific (as in DNA-mediated regulatory signals) or promoted by structural stem-loops formed in the mRNA. Thus, 30 -UTR plays a major role in the control of mRNA decay. Well recognized are the A þ U-rich elements (ARE), containing AUUUA motifs (Figure 3). The ARE are potent destabilizing elements that play a critical role in many genes with short half-lives. As stated, these elements are located in the 30 -UTR, and several ARE-binding proteins have been described that either support mRNA decay (e.g., AUF-1 in several interleukins [IL-1,2,3,6], c-myc, and b1-adrenergic receptor; and tristetrapolin (TTP) in granulocyte-macrophage colony-simulating factor (GM-CSF), growth-related oncogene alpha (GRO-a), and tumor necrosis factor alpha (TNF-a) mRNAs) or mRNA stability (e.g., HuR in VEGF and ELAV proteins in interferon-a). Regulatory elements may also exist in the coding region (CRD-1 and CRD-2 in c-fos mRNA) and the 50 -UTR (JNK-response elements, which bind stability determinants nucleolin and YB-1 in IL-2 mRNA). While it is clear that the binding of trans-acting factors to cis-acting elements in mRNAs plays an important role in mRNA decay or stability, the details regarding the signals that this interaction provides to the degradation machinery is still being elucidated. Moreover, questions remain regarding the mechanisms of mRNA degradation in a gene-specific (individual gene) or function-specific (class of genes) manner.

RNA-Mediated Gene Silencing Recently, a new avenue of gene regulation has developed, using regulatory RNAs to induce gene silencing. Gene silencing by nucleotide sequence-specific interactions mediated by RNA can function in at least three ways. First, RNAs can recognize DNA and produce alterations in the genome, such as DNA methylation and histone modifications, to target specific regions of the gene and modify chromatin structure altering gene expression at the transcriptional level. Second, micro (mi)RNAs bind to the complementary 30 -UTRs and act as translational repressors; miRNAs are generally 21–25 bp noncoding RNAs that are cleaved from precursors (by RNase III activity termed Dicer) and form stem–loop structures. Third, the most recognized form of RNA gene silencing is RNA interference (RNAi). RNAi is a sequence-specific

gene silencing mechanism that is induced by processing of double-stranded RNA (dsRNA) by Dicer into short dsRNAs of approximately 21–25 bp in length. These small dsRNAs are referred to as small interfering RNAs (siRNAs). Following unwinding of the siRNAs, one strand of the siRNA duplex is incorporated into an RNA-induced silencing complex (RISC). The RISC targets the cognate mRNA for degradation. Taken together, these small RNAs are capable of altering gene expression from the level of gene transcription, to mRNA stability, to translational events.

Future Directions At all levels of investigation within an organism, scientists and physician scientists hope to have an impact on understanding the development and function of an organism, and to help determine how and why a disease process may occur. Through the study of gene sequences as well as their regulation, medical science has the potential to make major breakthroughs in the pathophysiology, early detection, treatment, and prevention of distinct disease processes. This overview aims to provide a basic understanding of the potential importance of studying gene regulation, and its potential impact on the biomedical sciences and discovery opportunities in the future. See also: Transcription Factors: Overview; AP-1; ATF; Fox; NF-kB and Ikb; POU; PU.1.

Further Reading Bevilacqua A, Ceriani MC, Capaccioli S, and Nicolin A (2003) Post-transcriptional regulation of gene expression by degradation of messenger RNAs. Journal of Cellular Physiology 195: 356–372. Carvajal IM, Baron RM, and Perrella MA (2002) High mobility group-I/Y proteins: potential role in the pathophysiology of critical illnesses. Critical Care Medicine 30(supplement 1): S36–S42. Guhaniyogi J and Brewer G (2001) Regulation of mRNA stability in mammalian cells. Gene 265: 11–23. He L and Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nature Reviews: Genetics 5: 522–531. Kadonaga JT (2004) Regulation of RNA polymerase II transcription by sequence-specific DNA binding factors. Cell 116: 247–257. Lemon B and Tjian R (2000) Orchestrated response: a symphony of transcription factors for gene control. Genes and Development 14: 2551–2569. Lewin B (2004) Transcription. In: Challice J (ed.) Genes VIII, pp. 241–278. Upper Saddle River, NJ: Pearson Prentice Hall. Matzke M, Aufsatz W, Kanno T, et al. (2004) Genetic analysis of RNA-mediated transcriptional gene silencing. Biochimica et Biophysica Acta 1677: 129–141.

GENETICS / Overview 237 Reeves R and Beckerbauer L (2001) HMGI/Y proteins: flexible regulators of transcription and chromatin structure. Biochimica et Biophysica Acta 1519: 13–29.

Smale ST (1997) Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes. Biochimica et Biophysica Acta 1351: 73–88.

GENETICS Contents

Overview Gene Association Studies

Overview E K Silverman, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The major techniques used in respiratory genetic epidemiology include familial aggregation studies, linkage analysis, and association studies. These approaches are being used to localize genetic susceptibility determinants for many respiratory diseases.

Introduction Genetic determinants likely have some degree of influence on every major respiratory disease. In some cases, such as cystic fibrosis, the importance of genetic factors is obvious; however, even diseases with a major environmental basis, such as respiratory infections, likely are influenced by genetic susceptibility as well. Traditionally, genetic disorders have been classified as monogenic or complex. Monogenic disorders are strongly influenced by a single major gene. These typically rare conditions include cystic fibrosis and a1-antitrypsin deficiency. Complex disorders are likely influenced by multiple genetic and environmental influences, as well as gene-by-gene and geneby-environment interactions. However, diseases which are classified as complex may be influenced by major genes in monogenic subtypes, such as Netherton disease and atopy, or cutis laxa and emphysema. In addition, monogenic disorders may have variable expression that is determined by environmental factors and genetic modifiers. Thus, even classical monogenic disorders may have features of a complex disorder, and complex disorders may have monogenic subtypes. The identification of genetic determinants typically involves demonstration of a phenotype/genotype correlation. A phenotype is an observable trait in an

individual, which may be the presence/absence of disease or a quantitative disease-related characteristic. Quantitative disease-related phenotypes are often known as intermediate phenotypes, because they can be viewed as capturing intermediate pathways between a genetic determinant and a disease. Intermediate phenotypes are likely often more powerful for susceptibility gene identification than the assessment of the presence or absence of a disease, which is subject to diagnostic bias. For example, serum IgE levels and airway responsiveness are intermediate phenotypes that have been widely studied in asthma genetics. Although intermediate phenotypes can be quite useful, the genetic determinants of an intermediate phenotype may not overlap completely with the genetic determinants of the disease of interest. Thus, demonstrating a relationship of purported susceptibility genes identified from studies of intermediate phenotypes to disease status is still important. The two general approaches to susceptibility-gene identification are candidate-gene approaches and positional cloning. Candidate-gene approaches assess the relationship between variants in a gene hypothesized to be important in the disease of interest, based on the current concepts of disease pathophysiology (or based on other evidence, such as geneexpression differences). Positional cloning involves a more systematic search through the genome without a preconceived opinion about the location of a novel susceptibility gene; such genome-wide approaches using linkage analysis and association studies will be discussed below.

Familial Aggregation Several genetic epidemiological methods, which are generally referred to as familial aggregation studies, linkage analysis, and association studies, are used to determine whether genetic factors influence a phenotype. Familial aggregation studies determine

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whether a phenotype tends to be more similar in relatives compared to unrelated individuals. Familial aggregation can be caused by genetic or environmental factors. For instance, nuclear family members typically share a household environment, and diseases that would be influenced by that household environment (e.g., lung cancer and cigarette smoke exposure) will aggregate in families even without genetic influences. On the other hand, genetic factors are unlikely to be involved if familial aggregation is not present. Familial aggregation can be demonstrated in several ways. Comparison of disease prevalence in relatives of affected individuals to control subjects can be used to assess the statistical significance and magnitude of the risk to relatives. Family studies can be used to assess heritability, the estimated fraction of phenotypic variation that is due to genetic causes. Twin studies, which compare concordance between monozygotic (identical) and dizygotic (fraternal) twins, can also be used to assess the heritability of a phenotype. Higher concordance in monozygotic than dizygotic twins suggests genetic influences, but the potentially more similar environmental exposures in monozygotic than dizygotic twins should also be considered.

Assessment of Genetic Variation The human genome includes approximately 3 billion nucleotide bases on 22 pairs of autosomal chromosomes and two sex chromosomes. There are approximately 30 000 genes in the human genome; much of the human genome sequence is intergenic and of uncertain function and significance. There are approximately 10 million locations in the 3 billion-letter ‘genetic alphabet’ at which the genetic sequence commonly varies between people. When there are at least two alternate genetic variants (referred to as alleles) at a location in the genome that each occur with allele frequencies above 1%, that locus is said to be a polymorphic locus, or polymorphism. If there is a genetic variant with allele frequency below 1%, it is often referred to as a mutation, although this 1% threshold is somewhat arbitrary. Genetic variation determines important inherited differences between people, but also includes nonfunctional, evolutionarily neutral changes. Most of the approximately 10 million single nucleotide polymorphisms (SNPs) in the genome probably have no functional effects. Functional genetic variants include nonsynonymous SNPs that change the amino acid sequence of the corresponding protein, but regulatory variation is possibly even more critical for complex diseases. Although more difficult to prove a functional effect,

regulatory variation can affect the magnitude, timing, and location of gene expression. The mechanisms of regulatory variation are still being determined, but important regulatory variants could be present in multiple locations, including the promoter region, the introns, the 30 untranslated region, or in enhancers that are located far upstream or downstream from a gene. Identification of evolutionarily conserved sequences across multiple species appears to be a promising approach to identify sites of potential regulatory genetic variation. There are several common types of genetic variation, including SNPs, short tandem repeat polymorphisms (STRs), and insertion-deletion variants (indels). SNPs occur approximately once every 300 nucleotide bases, but the occurrence of SNPs varies widely throughout the genome; some genes have many hundreds of SNPs while others have very few. Most genetic determinants of respiratory diseases (coding or regulatory) are likely to be SNPs, which represent the most common type of genetic variation. The optimal genotyping approach for SNPs has not been determined; many different methods are available, and most of them rely on the polymerase chain reaction (PCR). Historically, differences in recognition patterns for cleavage of DNA by microbial restriction enzymes were used to assess for the presence of an SNP; this approach is laborious and requires identification of a restriction enzyme recognition sequence at a specific locus of interest. Commonly used approaches for SNP genotyping today include hybridization (e.g., TaqMan), minisequencing (e.g., Sequenom), and oligonucleotide ligation reactions (combined with minisequencing in genotyping platforms such as Illumina). STR polymorphisms are less frequent than SNPs in the genome; they include 2, 3, or 4 bp repeat sequences that have a high mutation rate. The number of repeats at an STR polymorphism often varies between individuals, so they are often informative in linkage studies that attempt to track specific transmitted chromosomal regions through families. Genotyping of STR markers is reasonably standardized; fluorescent labels are attached to PCR primers that anneal to nonrepetitive unique DNA sequences surrounding the repeat sequence, and the length of the repeat is assessed after PCR. Although previously genotyped using polyacrylamide gel-based approaches, capillary electrophoresis has increased the throughput and reliability of STR genotyping. Indels are also reasonably common; they typically include short insertions or deletions of 1 to 4 nucleotide bases in length. Genotyping of indels can be performed by assessing the length of the sequence, analogous to STR genotyping.

GENETICS / Overview 239

Genetic variants can be analyzed individually, or as a set of adjacent variants on a particular chromosome known as a haplotype. Haplotypes can be determined from extended pedigree information or by intensive molecular characterization; quite often, statistical methods are used to impute the likely haplotypes for genetic association analysis.

Linkage Analysis Linkage analysis is a commonly used genetic epidemiological approach to identify the general location of a genetic determinant of a phenotype of interest. Genetic variants (traditionally short tandem repeat markers, but more recently SNPs as well) are genotyped within families, and linkage to a hypothetical disease gene locus is assessed. If two genetic loci are located on different chromosomes (or far apart on one chromosome), the transmission of the variants at those loci will be independent and will follow Mendelian segregation. On the other hand, if those two loci are located close enough together such that recombination is reduced below the Mendelian expectation of 0.5 (50%), then they are linked. The null hypothesis that is being tested in linkage analysis is that the recombination fraction between two genetic loci is equal to 0.5; in effect, there is random genetic assortment of these loci. The statistical certainty of rejecting this null hypothesis of no linkage can be assessed by a p value or by a Lod score. A Lod score is the logarithm of the odds for linkage. Lod scores above 3 generally correspond to significant linkage, although more stringent thresholds have been suggested, varying with the type of families that are included. Genetic loci that are separate by relatively large physical distances can still show evidence for linkage; this is both a strength and a weakness of this approach (Table 1). The strength lies in the fact that

only a modest number of genetic variants need to be genotyped to perform a genome scan for linkage, typically on the order of 400 STR markers. It is a weakness in that the physical location of the susceptibility locus could reside within a large number of nucleotide base pairs. With a classical monogenic disorder, specific recombination events can be identified and additional families can be included to narrow the physical region of interest. In complex traits, where recombination events are not determined with certainty due to potential complicating factors such as environmental phenocopies (subjects with the disorder of interest due to purely environmental rather than genetic causes) and genetic heterogeneity (multiple genetic mechanisms for a disorder), localization only to broad genomic regions (e.g., 20 million base pairs) is typical. Linkage analysis can be performed with modelbased approaches, which require assumptions regarding a variety of disease allele parameters, such as allele frequency and penetrance. Although these model-based or parametric approaches have been extremely successful for monogenic disorders, they have been less successful in complex disorders, at least in part, because the model-based parameters for complex disorders are often completely unknown. Model-free or nonparametric approaches have been more widely used in complex disorders; these methods typically assess allele-sharing between affected relatives. In addition to presence/absence of phenotypes, linkage to quantitative phenotypes can also be performed, for example, using variance component approaches.

Genetic Association Analysis Genetic association studies assess whether a specific genetic variant (an allele) tracks with a disease, that is, whether there is significant evidence for linkage

Table 1 Comparison of linkage and association analysis Issue

Linkage analysis

Association analysis

Subjects included

Families (nuclear families or extended pedigrees) 400 STRs or 1500–10 000 SNPs

Cases/controls or families (often parent/child trios) 100 000 to 1 000 000 SNPs

Meiotic recombination in families

Linkage disequilibrium in populations

Genetic markers for a genome scan Driving property of statistical test Resolution for localization of a susceptibility gene for a complex disorder Utility in monogenic disorders

Approx. 20 000 000 bases

Approx. 200 000 bases

Very high

Utility in complex disorders

Useful, but replication has been difficult

Potentially useful; typically performed after linkage studies Useful, but replication has been difficult

240 GENETICS / Gene Association Studies

disequilibrium. As discussed in the article Genetics: Gene Association Studies, both case-control and family-based approaches can be used to assess for the presence of linkage disequilibrium between a measured genetic variant and a hypothetical disease locus. Linkage disequilibrium measures the tendency of alleles at nearby genetic loci to track together. As opposed to linkage analysis, which determines whether a particular location in the genome is shared more often than expected by chance among relatives with a particular phenotype, association analysis determines whether a specific allele tracks with disease more often than expected by chance in a population of individuals. Linkage disequilibrium is variable across the genome, but it is uncommon for significant linkage disequilibrium to extend beyond 200 kilobases. Thus, the resolution of genetic association studies is typically finer than genetic linkage studies. Although it is difficult to select the right genetic variant to test for association, if a valid association is uncovered, the functional variant is likely nearby if such an association can be found. Although still quite expensive, genome-wide association studies are becoming feasible. These studies will require genotyping hundreds of thousands of SNPs to provide adequate genomic coverage (fewer in Caucasian and Asian populations, and more in African populations due to the differing extents of average linkage disequilibrium in those populations). Such largescale association studies have the potential to identify many genetic determinants of complex respiratory diseases. See also: Cystic Fibrosis: Overview. DNA: Structure and Function. Gene Regulation. Genetics: Gene Association Studies. Neurofibromatosis. Proteinase Inhibitors: Alpha-2 Antiplasmin. Pulmonary Function Testing in Infants. Systemic Disease: Sarcoidosis; Langerhans’ Cell Histiocytosis (Histiocytosis X); Sickle Cell Disease. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Hartl DL and Clark AG (1997) Principles of Population Genetics, 3rd edn. Sunderland, MA: Sinauer Associates. Hirschhorn JN and Daly MJ (2005) Genome-wide association studies for common diseases and complex traits. Nature Reviews: Genetics 6: 95–108. Khoury MJ, Little J, and Burke W (2004) Human Genome Epidemiology. Oxford: Oxford University Press. Rao DC and Province MA (eds.) (2001) Genetic Dissection of Complex Traits. San Diego, CA: Academic Press. Silverman EK, Shapiro SD, Lomas DA, and Weiss ST (2005) Respiratory Genetics. London: Hodder-Arnold. Thomas DC (2004) Statistical Methods in Genetic Epidemiology. Oxford: Oxford University Press.

Gene Association Studies A A Litonjua and J C Celedo´n, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract With the completion of the Human Genome Project and the rapid progress in genotyping technology, the number of genetic association studies in respiratory diseases has increased exponentially in recent years. Most of these association studies have employed a population-based case-control design, and very few have provided results that have been replicated by others. While disagreement in results between studies may be due to true differences in genetic variation and environmental exposures among populations, attention must be paid to potential problems that lead to lack of replication. These potential problems include differences in phenotype definitions, lack of attention to Hardy–Weinberg equilibrium in controls, small sample sizes, multiple testing, and potential population stratification. While methods have now been developed to test and control for population stratification, family-based methods have also been developed in response to this problem. This article provides an overview of the design and conduct of genetic association studies for complex respiratory diseases.

Introduction Genetic association studies can be defined as investigations that assess the relationships between genetic variants and either aspects of a disease or the disease itself. In its simplest form, association studies determine whether a particular form of a genetic marker occurs more frequently in subjects with a particular trait or disease than in those without the trait or disease of interest. Association studies play an important role in identifying genetic determinants of complex human diseases. The rapid progress of the Human Genome Project has propelled the use of genetic association studies as a tool to better understand complex respiratory disorders. The most popular design that has been employed is the population-based case-control study. Because this design is susceptible to potential bias by population stratification (see below), family-based designs have been developed. Each of these designs have their advantages and disadvantages, and the investigators’ choice of the most efficient design will depend on factors such as the age of onset of the disease and the availability of subjects and specimens. In general, when a significant association is found between a genetic variant (e.g., a polymorphism) and a disease, there are three possible explanations: (1) the finding is a false positive due to a spurious association; (2) the association is due to close proximity to a functional variant in the same locus or in an

GENETICS / Gene Association Studies 241

adjacent locus (linkage disequilibrium (LD); see below); or (3) the locus is functional and directly affects phenotype expression. Spurious associations may arise due to chance or bias. In genetic association studies, the main type of bias that must be considered is that which results from population stratification, which may be thought of as a form of confounding by ethnicity. Population stratification results when cases and controls are not drawn from the same underlying population, and two necessary conditions exist: (1) the prevalence of the disease of interest is different in the two populations, and (2) the frequency of alleles is different in the two populations. Population-based case-control designs are susceptible to this form of bias, whereas family-based designs are generally immune to the effects of population stratification. Chance alone may explain false positive results in genetic association studies, and the importance of replication of initial findings in independent populations cannot be overemphasized. When potential sources of bias have been adequately controlled, the most likely explanation for a positive association between a genetic marker(s) and a disease of interest is LD between the marker(s) studied and a nearby functional locus. LD is the association between particular alleles at two or more loci because of their proximity (tight linkage) on the same chromosome. When alleles at two distinct loci occur more frequently together than expected based on their frequencies, these alleles are said to be in LD. Finally, a positive genetic association may represent the finding of a true disease susceptibility locus. However, this needs to be evaluated by functional studies in humans and/or animal models of the disease of interest.

Genetic Contribution to Disease Before embarking on a gene association study, it is first necessary to determine whether the disease of interest has a significant heritable component. Several methods exist to determine the genetic contribution to a disease, and in many instances, these studies have already been performed and results have been published. One method to determine whether a disease has a significant genetic component is to perform familial aggregation studies. For continuous traits, these studies determine the correlations of the trait between relatives of different types (e.g., between siblings, between parents and children) and compare these correlations. For example, if the correlation of the trait among siblings is stronger than that found between spouses (who are not biologically related),

then the trait may be heritable. For dichotomous traits, studies typically examine the proportion of relatives of the probands who also have the trait and compare this with the proportion of relatives of control subjects who have the trait. Studies of monozyotic and dizygotic twins are helpful in providing clues to the heritability (the proportional contribution of genetic factors) of a trait. A higher concordance of disease in monozyotic twins than in dizygotic twins suggests that the disease has a significant genetic component. Finally, segregation analysis can be used to evaluate the most likely mode of inheritance of a disease of interest; this can be particularly helpful if a monogenic variant of a complex disease is suspected. In this method, a variety of models (both genetic and nongenetic) are fit to family data and the results are assessed to determine which model best fits the data. Because segregation analysis entails the use of family data, it is generally performed as part of gene mapping studies, before genetic marker information is available.

Phenotypes for Gene Association Studies The phenotype of an individual refers to the observable trait or characteristic of that individual, for example hair color or height. In the case of association studies, phenotypes that are studied are usually the presence or absence of a disease. More recently, studies have also begun to investigate genetic modifiers of a disease (e.g., modifiers of disease presentation of a-1-antitrypsin deficiency or genes associated with the severity of asthma). The choice of phenotype is critical to the success of gene association studies. It may appear to be straightforward and appealing to use the disease diagnosis as the phenotype, since most studies are investigating the risk factors for the condition of interest. However, diagnostic misclassification is problematic and can seriously affect the power of the study. Disease diagnosis is a qualitative (all or none) rather than a quantitative (continuous) trait, and generally quantitative phenotypes are more powerful in identifying disease susceptibility genes. Furthermore, complex diseases, such as asthma and chronic obstructive pulmonary disease (COPD), are heterogeneous in their presentation. For example, one must decide whether they are studying atopic vs. nonatopic asthma or COPD that primarily presents as emphysema or as chronic bronchitis. In narrowing the phenotype definition, it is necessary to collect information on disease-associated traits, or intermediate phenotypes. Intermediate phenotypes are appealing because they are objective, are measured

242 GENETICS / Gene Association Studies Table 1 Intermediate phenotypes for respiratory gene association studies Phenotype

Tool or measurement

Symptoms

Self-administered questionnaires; interviewer-administered questionnaires; video questionnaires Questionnaires on medication use, healthcare utilization including urgent care and emergency room visits Spirometry: FEV1, FVC, FEV1/FVC, FEV25–75/FVC, PEF Challenge testing using non-specific (e.g., methacholine, histamine) or specific (e.g., platelet activating factor) agents FEV1 change after administration of bronchodilator (albuterol) Total and specific IgE; skin test reactivity; eosinophil count

Disease severity or control Lung function Bronchoconstrictor response Bronchodilator response Atopy Biomarkers of inflammation Lung specific Generalized Imaging

Markers measured in sputum, exhaled gases and exhaled breath condensates, BAL fluid Markers measured in blood and urine CT analysis of parenchyma and airways

with more precision and reliability than physicians’ diagnoses and most are continuous measures (quantitative traits), thus potentially increasing the power for the analyses. Table 1 summarizes some intermediate phenotypes that may be used in respiratory disease genetics. Questionnaires for symptoms and disease control, spirometry, bronchoconstrictor and bronchodilator responses, and atopy measures have been extensively used in association analyses, while imaging modalities and biomarker measurement hold promise for better phenotyping in the future. Information on symptoms is collected with the use of questionnaires. Several standardized, and in some instances validated, questionnaires are available. These include the questionnaires from the American Thoracic Society and Division of Lung Diseases (ATS-DLD), the International Study of Asthma and Allergy in Childhood (ISAAC), the Medical Research Council (MRC) of Great Britain, and the International Union against Tuberculosis and Lung Disease (IUATLD) to name a few. These have been in use for many years in the field of respiratory epidemiology. For asthma in particular, the ISAAC questionnaire has incorporated a video questionnaire, designed to overcome language and cultural differences in the interpretation of the symptom of wheeze, making standard definitions for multinational studies a possibility. In addition to collecting information on baseline symptoms, information on disease severity or control, and medication use may also be collected. These will be important for studies investigating genetic modifiers of disease or determinants of frequency of exacerbation of a whole range of respiratory diseases. Spirometry is widely used in gene association studies in respiratory diseases. Field studies using spirometry have been conducted for many decades, making it applicable for measurement of lung function in families and populations. Measures of forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), forced

expiratory flow 25–75% (FEF25–75), which is the flow between 25% and 75% of the vital capacity, and the peak expiratory flow (PEF) can all be determined from the spirometric maneuver. Similary, bronchoconstrictor response testing and bronchodilator response testing are invaluable in defining phenotypes for respiratory disease. While some studies have incorporated one or the other measure in the phenotype definition, it is likely that these traits are defined by different sets of genes. Because asthma and atopy are closely associated, measurements of total and specific immunoglobulin E (IgE) in serum and skin test reactivity to a range of allergens have been used to define the phenotype in many asthma genetic association studies. Additionally, studying the genetic determinants of these traits may also uncover mechanisms involved in the development of asthma. Inflammation is a major process in many respiratory disorders. While the exact cell type and the location of the inflammatory infiltrate vary in each disorder, biomarkers have been used in studies of the presence of disease, disease severity, or response to treatment, and have been useful in elucidating the pathophysiologic processes occurring in each one. A wide range of cytokines, products of oxidation, and other mediators have been studied. These can be roughly categorized as biomarkers of generalized or lung-specific inflammation. Early studies used biomarkers measured in blood components and urine, because obtaining these samples was reasonably simple. However, these biomarkers may not faithfully reflect the inflammatory process occurring in the lung, and their levels are confounded by comorbid conditions. While specimens obtained using the fiberoptic bronchoscope, including bronchoalveolar lavage (BAL), are specific, the procedure is too invasive to be of routine use in human genetic association studies, where large numbers of cases and controls, or family

GENETICS / Gene Association Studies 243

members need to be phenotyped. Sputum induction is a reasonably noninvasive method of obtaining samples, and many biomarkers that are measured in blood have been shown to be measurable in these sputum samples. There is more research interest, however, in the newer noninvasive methods of sample collection. Collection and examination of both exhaled gases and exhaled breath condensates are promising methods of obtaining respiratory samples. However, standardization of equipment and techniques is needed before these methods can be advocated as means of phenotyping subjects. The advent of high-resolution computed tomographic (HRCT) scanning has allowed better anatomical understanding of the changes occurring in various lung diseases. Assessment of small airways dimensions has aided in the diagnoses of various lung disorders, such as constrictive bronchiolitis, hypersensitivity pneumonitis, sarcoidosis, and several occupational lung disorders. HRCT has also been used to assess the extent and distribution of parenchymal abnormalities in pulmonary emphysema. Other imaging modalities (e.g., hyperpolarized helium-3 gas magnetic resonance imaging) of the lung are rapidly being developed, and these hold great promise in noninvasively assessing the structural characteristics of the lung to aid in phenotyping subjects for respiratory genetics studies. The absence of so-called ‘gold standards’ for the diagnosis of respiratory disorders has led to the use of variable definitions in association studies. This variability in defining phenotypes is surely one reason for the lack of replication in association studies, and collecting information on objective intermediate phenotypes helps to standardize phenotype definition across studies. A strategy for defining phenotypes has been to combine a physician’s diagnosis with symptoms and an objective intermediate phenotype (e.g., spirometry in the case of COPD, bronchoconstrictor response or bronchodilator response in the case of asthma).

Selection of Candidate Genes and Polymorphisms Genetic association studies can examine alleles in candidate genes or in a genomic region(s) of interest (fine-mapping association studies). Because fine-mapping association studies are more expensive and complex, candidate gene association studies are frequently conducted. A candidate gene is defined as a gene that is identified either by its protein product that suggests that it could be the determinant of the disease in question (a biological candidate), or by its position in a chromosomal region that has been

linked with the disease (positional candidate). Examples of biological candidates are the b2 -adrenergic receptor gene (ADRB2) for asthma because of the central role of b agonists in asthma therapy; oxidant– antioxidant genes such as glutathione-S-transferase gene for emphysema based on the pathobiology of the disease; and the tumor necrosis factor alpha (TNF-a) gene in pulmonary fibrosis because of the role of cytokines and inflammation in the development of fibrosis. Positional information derived from linkage studies is another way to identify candidate genes. There are fewer examples of this, since most of the linkage analyses have been performed in the two most common complex respiratory disorders: asthma and COPD. In asthma, several genome scans have been conducted, and some chromosomal regions that have shown consistent linkage to asthma-related phenotypes in several populations (e.g., 5q, 6p, 12q, and 13q). Thus, genes located in these regions are all potential candidate genes for association studies of asthma. To date, there have only been three genome scans for COPD-related phenotypes, with regions in chromosome 2q in one study and chromosome 4 in another having log10 of the odds of linkage (LOD) scores above 3.3. Once candidate genes have been selected for study, the next step is to find variants in the gene. Variation in DNA sequence in a gene that has a frequency of greater than 1% is called a polymorphism. There are different types of polymorphisms in the genome: single nucleotide polymorphisms (SNPs), repeat polymorphisms, and insertions or deletions. By far, the most common type of variation is the SNP, which is a single base variation in DNA sequence. SNPs occur commonly, at about 1 out of every 300 bases in the human genome, and it is estimated that there are about 10 million SNPs across the human genome. Sources of information on SNPs include the National Center for Biotechnology Information (NCBI) SNP database and the International Haplotype Map (HapMap) Project. SNPs are ubiquitous and are found in both coding and non-coding regions of a gene. When an SNP occurs in a coding region of a gene, it may have one of three consequences. The first is a nonsense SNP, which is a single nucleotide variation that results in a STOP codon, thus giving rise to a nonfunctional protein. The second is a missense (also called nonsynonymous) SNP, in which the single nucleotide variation results in the coding of a different amino acid, thus giving rise to a protein with altered structure or function. Thirdly, SNPs in coding regions may also be synonymous SNPs, in which the single nucleotide variation does not change the amino acid sequence of the protein. Most SNPs

244 GENETICS / Gene Association Studies

are located in the noncoding regions of the genome, but may still be important because they may lie in gene promoter or regulatory regions that may affect gene expression. For practical reasons, genotyping all known SNPs in a gene for an association study is often not feasible. Thus, choosing a subset of SNPs for genotyping is an important step for a successful association study, and investigators should have an idea of how they want to prioritize available SNPs. Priority should be given to SNPs that have known function, nonsynonymous SNPs in coding regions, and SNPs known to alter gene splicing or expression. SNPs that have previously been associated with the disease of interest should also be chosen, as this would allow comparison of results across studies. Preference should also be given to SNPs that are common (i.e., SNPs with minor allele frequency of at least 5%), so that statistical power of the study will not suffer. Additionally, consideration should be given to SNPs that can be reliably genotyped (e.g., HapMap SNPs). Finally, since SNPs in a gene may be closely correlated with other SNPs in the same gene because of LD, it is possible to choose a subset of SNPs that captures most of the information that one would get by typing the whole set. In the extreme case (complete LD), an allele found at one locus can predict completely which allele will be found at another locus, making one of the loci redundant for the purpose of genotyping. Empirical studies have reported blocklike segments in the genome with high LD. These studies have also shown that areas of the genome have a much smaller number of haplotypes (the linear, ordered arrangement of alleles in a segment of a chromosome) than would be predicted by the number of markers alone. Thus, these studies have motivated a marker selection strategy for association studies, whereby a subset of SNPs is chosen that can stand as a proxy for many other SNPs. These ‘tag’ SNPs would then capture most of the common variation within the region of interest. There are various algorithms that choose tag SNPs based on LD or based on haplotypes. However, these assume that there is prior knowledge of the LD patterns of the region of interest. This data may arise from resequencing a subset of subjects in a study or from large genotyping studies in reference samples such as the HapMap project.

Study Designs for Gene Association Studies There are two basic study designs for gene association studies: population-based designs and family-based

designs. Population-based designs entail collection of unrelated individuals, some who have the disease of interest (cases) and others who do not (controls), while family-based designs entail collection of information from families of individuals who have the disease (probands). Each of these designs has its advantages and disadvantages. Ultimately, the choice of which study design to use will depend on a host of factors such as the expense of collecting phenotype information and samples, genotyping costs, and characteristics of the disease of interest (e.g., for late-onset diseases, obtaining parental genotype data may not be feasible).

Population-Based Designs Probably the simplest design for gene association studies is the case-control design, where a comparison is made in the frequency of SNP alleles in two well-defined groups of individuals: cases who have the disease or the trait of interest, and controls who are known to be unaffected. This is no different from the standard case-control design used in epidemiologic studies, and the finding of an increased frequency of an SNP allele or genotype in cases compared with controls indicates that the presence of the SNP may increase the risk of disease (Figure 1). Because of the ease of the concept and the familiarity with this design, it is no surprise that this is the most popular design that researchers have adopted. However, many of the limitations of this design in the epidemiologic setting also apply in association studies, and basic principles of performing good casecontrol association studies are often overlooked. In selecting cases and controls, the source population should be well defined. Cases should be a representative sample of all the cases in this precisely defined and identified population. Hence, the source population is determined by the eligibility criteria for cases to enter the study. For example, if in a casecontrol association study for asthma, cases were made up of admissions to a city hospital, the source population would be defined as all the patients admitted to that hospital who would have been given a diagnosis of asthma, if indeed they had asthma. Controls should be sampled from this source population. Controls should also be representative of the source population with respect to exposure. In the particular case of gene association studies, this means that allele frequencies found in controls should reflect those in the source population. Obviously, it is impossible to determine whether this is indeed the case, but random sampling of the source population when selecting controls assures a greater likelihood of the representativeness of the control group.

GENETICS / Gene Association Studies 245

A

B

Case Control Allele counts
2 =

Σ

(Observed − Expected) 2

cells AA

AB

Expected

BB

Case Control Genotype counts Figure 1 Population-based case-control association studies. Associations may be analyzed as either allele counts or genotype counts. In either case, the analysis consists of comparing the observed counts to the expected counts in a w2 analysis with either one (allele counts) or two (genotype counts) degrees of freedom. When nongenetic covariates, such as age, are collected in a study, these may be accounted for in the analysis by fitting multiple logistic regression models with the case status as the dependent variable and the genetic and nongenetic variables as the independent variables.

A second type of population-based design is the cohort study. A cohort study consists of assembling a group of subjects, who are then followed in time to determine the frequency with which disease develops (e.g., identifying infants at birth and following them for the development of asthma; or assembling a group of smokers and seeing who will develop clinically significant emphysema). Cohort studies are more expensive and labor intensive, but are less prone to ascertainment bias than case-control studies. It is also possible to select cases and controls from existing cohorts (nested case-control studies). Numerous population-based association studies have been published, particularly for asthma and COPD, yet very few of these studies have been replicated, and in some instances, contradictory results have been found. There are many reasons for the lack of replication in association studies, and some of these include: (1) no assessment and control for population stratification, (2) small sample sizes, (3) multiple testing, (4) differences in phenotype definitions between studies, and (5) failure to consider linkage disequilibrium with adjacent loci. As explained earlier, population stratification is a potential problem in population-based case-control association studies, and is one reason for false positive findings. In the design phase of a study, this potential problem may be minimized by careful matching of cases and controls on ethnicity. However, subtle differences in the ethnic make-up of the population may be easily missed and formal assessment and control (if it is present) of population stratification is needed for a well-designed casecontrol association study. Determining whether Hardy–Weinberg equilibrium (HWE) is present

among controls is important (Figure 2). True genetic associations can cause deviations from HWE among cases, so it should be tested in control subjects. If genotypes are not in HWE, this is a clue of potential genotyping error (most commonly) or population stratification. Finally, a set of markers that are not known to be linked to or associated with the disease of interest can be genotyped, and an analysis of association between these markers and the trait of interest can be used as a formal test of population stratification. It is currently unclear how many unlinked markers need to be genotyped to exclude significant population stratification. If indeed stratification is found, then methods such as genomic control, exist to control this in the analysis. While there are numerous association studies in respiratory disease in the literature, many of these have been composed of small numbers of cases and controls. Ideally, case-control association studies should have adequate numbers of subjects. The numbers should be guided by several factors such as the frequency of the polymorphisms and the effect estimates related to these polymorphisms, but investigators should aim to collect as many cases and controls as is feasible. Having large numbers of cases and controls improves the chances for replication of a positive association. Multiple testing is another problem in case-control association studies, and arises from testing multiple phenotypes, multiple SNPs in genes, and/or multiple genes. While it is clear that the classical Bonferroni correction is too conservative, the best method for addressing the multiple testing issue for association studies remains unresolved. Probably the best strategy so far is replication of results in an independent population.

246 GENETICS / Gene Association Studies • Fundamental principle in population genetics • Simple explanation for how genetic diversity for Mendelian traits can be maintained in a population • Named after the English mathematician G. H. Hardy (1877−1947) and the German physiologist W. Weinberg (1882−1937) who independently formulated the model and deduced its theoretical predictions of genotype frequency • A locus with two alleles, A and a, where: p = frequency of A q = frequency of a • Equilibrium distribution of genotypes is defined as: Genotype Frequency

AA p2

Aa 2pq

aa q2

• Absence of Hardy−Weinberg equilibrium among control subjects in a population-based case-control study is likely due to either genotyping error or population stratification, while its absence in parents of offspring in a family-based study of association may be due to genotyping error. Figure 2 The Hardy–Weinberg principle.

As indicated in the earlier section on phenotypes, there is a lack of standard definitions for respiratory phenotypes used in association studies. Thus, differences in phenotype definition can lead to the contradictory findings from study to study. Obtaining information on intermediate phenotypes that are collected in a standardized manner may help in replication studies. Despite attention to these details of the case-control association study, findings may still not be replicated in other populations. It is possible that one locus may be associated with a trait in one population, but another locus is associated with the same trait in another population (locus heterogeneity). It is also likely that different environmental stimuli in separate populations interact with the same locus to produce different associations. In this case, the associations seen in each population may be real, but differing environmental exposures may produce dissimilar, sometimes even conflicting, results. Finally, different patterns of LD may exist in different populations, and if the causative SNP (or SNPs) is not the one being tested, these different patterns of LD will also produce dissimilar results. To summarize, various issues may affect the validity of gene association studies. Careful attention to these study design particulars is needed to have a well designed study. Finally, even if a study is well designed, the need for replication of findings in independent populations cannot be overemphasized. Family-Based Association

The problem of potential population stratification in case-control designs was the main impetus to the

development of methods that incorporated family designs in association studies. One of the earliest approaches to the problem of population stratification is the transmission disequilibrium test (TDT), reported by Spielman and colleagues in 1993. The rationale behind the TDT is that in the absence of both linkage and association between the marker locus being studied and the true disease locus, marker alleles will be transmitted randomly from parents to offspring. In the original TDT design, a pedigree with both parents and a single affected offspring was required, and it compared the transmission of marker alleles to affected offspring against the nontransmission of alleles by means of a simple w2 statistic (Figure 3). Many extensions and variations to the TDT have been developed since the original report, which now allow the incorporation of multiallelic markers, multiple markers, missing parental information, extended pedigrees, quantitative traits, and non-genetic covariates. Compared with case-control association studies, family-based association studies have three main advantages: (1) control for potential population stratification, (2) increased ability to detect genotyping errors because of availability of parental data, and (3) reduced costs of phenotyping, particularly for the TDT design (only affected offspring is phenotyped). It should be remembered, however, that parental data may not be available for family-based association studies because of multiple factors, including late-onset of the disease of interest and divorce. In addition, more individuals have to be genotyped for a familybased study than in a case-control study, although this is offset somewhat by the need to genotype a set of unlinked markers in the population-based design.

GENETICS / Gene Association Studies 247 (1)

(2)

A B

A

A B

A

B

A

A

A

A

A

Untransmitted allele

Transmitted allele

A

B

A

a

b

B

c

d

Test statistic T=

(b − c)2 ∼ χ12 b+c

Figure 3 Transmission disequilibrium test. Family-based association studies may be analyzed by the transmission disequilibrium test (TDT) or the various extensions of the TDT. In the simplest case of a trio (i.e., genotypes from both parents and from the affected offspring), the TDT compares the transmission of an allele to the affected offspring against the nontransmission of the allele. In the illustration above for family (1), the A allele is transmitted to the offspring by each parent; thus, in the table, b ¼ 2, and a, c, and d are all equal to 0. For family (2), the A allele is transmitted by the father to the offspring and the B allele is untransmitted, while one A allele is transmitted by the mother and the other A allele is untransmitted. Thus, for this trio, the table should be filled out with a ¼ 1, b ¼ 1, and c and d are equal to 0. These counts are performed for all the trios in the study and these are summed over all the tables and analyzed as a w2 test with one degree of freedom. It is clear from this illustration that homozygous parents do not contribute to the test statistic.

Ultimately, investigators have to carefully weigh the advantages and disadvantages of a particular study design for a disease or trait of interest. See also: Asthma: Overview. CD1. Gene Regulation. Genetics: Overview.

Further Reading Abecasis GR, Cardon LR, and Cookson WOC (2000) A general test of association for quantitative traits in nuclear families. American Journal of Human Genetics 66: 279–292. Cardon LR and Bell JI (2001) Association study designs for complex diseases. Nature Reviews: Genetics 2(2): 91–99. Cardon LR and Palmer LJ (2003) Population stratification and spurious allelic association. Lancet 361(9357): 598–604. Clark AG (2004) The role of haplotypes in candidate gene studies. Genetic Epidemiology 27: 321–333. Davies JC (2004) Modifier genes in cystic fibrosis. Pediatric Pulmonology (Supplement) 26: 86–87. Garcia CK and Raghu G (2004) Inherited interstitial lung disease. Clinics in Chest Medicine 25(3): 421–433. (Erratum in: Clinics in Chest Medicine 25(4): xi.) Haines JL and Pericak-Vance MA (eds.) (1988) Approaches to Gene Mapping in Complex Human Diseases. New York: Wiley-Liss.

Hartl DL, Clark AG (1997) The Hardy–Weinberg principle. In: Hartl DL and Clark AG (eds.) Principles of Population Genetics, 3rd edn. Sunderland: Sinauer Associates. Hoffjan S, Nicolae D, and Ober C (2003) Association studies for asthma and atopic diseases: a comprehensive review of the literature. Respiratory Research 4(1): 14. Horvath S and Laird NM (1998) A discordant-sibship test for disequilibrium and linkage: no need for parental data. American Journal of Human Genetics 63: 1886–1897. Horvath S, Xu X, and Laird NM (2001) The family based association test method: strategies for studying general genotype– phenotype associations. European Journal of Human Genetics 9: 301–306. Khoury MJ, Beaty TH, and Cohen BH (1993) Fundamentals of Genetic Epidemiology. New York: Oxford University Press. Laird NM, Horvath S, and Xu X (2000) Implementing a unified approach to family based tests of association. Genetic Epidemiology (Supplement) 19: 36–42. Lake SL, Blacker D, and Laird NM (2000) Family based tests of association in the presence of linkage. American Journal of Human Genetics 67: 1515–1525. Lange C, DeMeo D, Silverman EK, Weiss ST, and Laird NM (2004) PBAT: tools for family based association studies. American Journal of Human Genetics 74(2): 367–369. Meyers DA, Larj MJ, and Lange L (2004) Genetics of asthma and COPD: similar results for different phenotypes. Chest 126(2 supplement): 105S–110S. Molfino NA (2004) Genetics of COPD. Chest 125(5): 1929–1940. Monks SA and Kaplan NL (2000) Removing the sampling restrictions from family-based tests of association for a quantitativetrait locus. Amercian Journal of Human Genetics 66: 576–592. Newton-Cheh C and Hirschhorn JN (2005) Genetic association studies of complex traits: design and analysis issues. Mutation Research 573: 54–69. Risch NJ (2000) Searching for genetic determinants in the new millennium. Nature 405(6788): 847–856. Rothman KJ and Greenland S (1998) Case-control studies. In: Rothman KJ and Greenland S (eds.) Modern Epidemiology, 2nd edn., pp. 93–114. Philadelphia: Lippincott-Raven. Rothman KJ and Greenland S (1998) Cohort studies. In: Rothman KJ and Greenland S (eds.) Modern Epidemiology, 2nd edn., pp. 79–91. Philadelphia: Lippincott-Raven. Schulze TG and McMahon FJ (2002) Genetic association mapping at the crossroads: which test and why? Overview and practical guidelines. American Journal of Medical Genetics 114: 1–11. Silverman EK (2002) Genetic epidemiology of COPD. Chest 121(3 supplement): 1S–6S. Steen KV and Lange C (2005) PBAT: a comprehensive software package for genome-wide association analysis of complex family-based studies. Human Genomics 2(1): 67–69. Tabor HK, Risch NJ, and Myers RM (2002) Opinion: candidategene approaches for studying complex genetic traits: practical considerations. Nature Reviews: Genetics 3(5): 391–397. Villar J, Flores C, and Mendez-Alvarez S (2003) Genetic susceptibility to acute lung injury. Critical Care Medicine 31(4 supplement): S272–S275. Weiss KM and Terwilliger JD (2000) How many diseases does it take to map a gene with SNPs? Nature Genetics 26(2): 151–157.

Relevant Websites http://www.hapmap.org – International Haplotype Map Project. http://www.ncbi.nlm.nih.gov – National Center for Biotechnology Information.

248 G-PROTEIN-COUPLED RECEPTORS

G-PROTEIN-COUPLED RECEPTORS S B Liggett, University of Maryland, Baltimore, MD, USA D W McGraw, University of Cincinnati, Cincinnati, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract G-protein-coupled receptors (GPCRs) are a large and diverse superfamily of cell surface receptors that transduce their signals via membrane-associated heterotrimeric guanine nucleotidebinding proteins (G-proteins). It is estimated that there are over 1000 GPCRs that may make up to 5–10% of the human genome. Members of the G-protein superfamily share a common structural motif of seven transmembrane spanning domains that includes an extracellular N-terminus and an intracellular C-terminus, interspersed by seven domains that span the cell membrane in tandem, connected by three extracellular loops and three intracellular loops. Although the general scheme of signal transduction is similar for all members of the GPCR superfamily, there is considerable structural diversity in the receptor proteins that provides for plasticity at nearly every step of regulation including receptor synthesis, ligand binding, G-protein coupling, and intracellular trafficking (desensitization, resensitization, and downregulation). Through activation or inhibition of their downstream messengers, GPCRs regulate a wide array of physiological and compensatory functions in the lung. GPCRs are currently important targets for drugs used to treat lung diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary hypertension, and are a major focus of pharmaceutical development. Because so many aspects of GPCR function are governed by receptor structure, it is not surprising that an increasing amount of data indicate that naturally occurring genetic variation in GPCRs may encode receptors that differ in signal transduction, and thereby contribute to differences in physiology or therapeutic responses.

Introduction G-protein-coupled receptors (GPCRs) represent a large and diverse superfamily of integral membrane receptor proteins that transduce extracellular stimuli to an intracellular signal via a comparatively smaller group of membrane-associated heterotrimeric guanine nucleotide-binding proteins (G-proteins). Since the cloning of bovine rhodopsin and the b2-adrenergic receptor (b2-AR), several hundred GPCRs have been isolated by molecular cloning techniques, with estimates that the human genome may have over 1500 members of the superfamily based on bioinformatics techniques of the genome sequence. GPCRs are thus the largest superfamily of plasma membrane receptors, comprising up to 5–10% of the human genome. GPCRs respond to a wide array of external stimuli including neurotransmitters, hormones, chemokines,

nucleotides, odorants, light, and undoubtedly many others. That so many different signaling events are accomplished via the same basic mechanism suggests that the process is highly efficient. While the general scheme of signal transduction is similar for all GPCRs, significant structural diversity has evolved among members of the GPCR family which provides for diversity at virtually every step of the process.

Structure All GPCRs share a common structural topology that has been derived from their homology to bovine rhodopsin, the only mammalian GPCR for which high-resolution three-dimensional crystal structure has been resolved, and by studies utilizing proteolytic digestion, peptide mappings, and site-directed mutagenesis. This unique structure consists of seven hydrophobic regions that adopt a-helical conformations which serve as transmembrane spanning domains. This structure orients the receptor in the cell membrane such that the N-terminal end is extracellular, the C-terminal end is intracellular, and the seven transmembrane (7-TM) spanning domains are linked by three extracellular loops and either three or four intracellular loops depending upon the presence of a palmitoylated cysteine residue in the proximal C-terminal tail (Figure 1). The presence of specific residues within a GPCR may additionally permit the receptor to undergo a variety of regulatory posttranslational modifications including N-linked glycosylation, palmitoylation, formation of disulfide bonds, and phosphorylation. GPCR families have been categorized into five or six different groups based upon similarities in their structural features or sequence, as well as their function. Over 90% of known GPCRs are in family A, which includes rhodopsin, olfactory, biogenic amine, nucleic acid, and bioactive lipid and peptide receptors. The biogenic amine group includes the well-characterized adrenergic, muscarinic, prostanoid, histamine, and adenosine receptors. Family A receptors have a short N-terminal extracellular domain and several conserved amino acids within the transmembrane spanning domains. In most cases, the transmembrane spanning domains of family A members serve as the ligand-binding site, while the intracellular connecting loops serve as coupling and regulatory domains. Family B receptors include the glycoprotein hormone receptors (e.g., luteinizing hormone, thyroid stimulating hormone, and follicle stimulating

G-PROTEIN-COUPLED RECEPTORS 249 NH2

Extracellular

Intracellular

HOOC Figure 1 Schematic representation of the putative membrane topology GPCRs. Seven hydrophobic transmembrane spanning are connected in tandem by three extracellular and three intracellular loops. The N-terminal tail is extracellular and the C-terminal tail is intracellular.

hormone receptors), secretin, calcitonin, and parathyroid hormone receptors. Family B receptors are characterized by a long extracellular N-terminal domain (4400 amino acids) that interacts with portions of the extracellular connecting loops to form the ligand-binding domain. Family C receptors contain an even longer extracellular N-terminus (4600 amino acids) where the ligand-binding domain is exclusively located. This group includes the metabotropic glutamate (mGlu) and g-aminobutyric acid (GABA) neurotransmitter receptors, the extracellular calcium sensing receptor, and members of the taste receptor group. Additional families of GPCRs include those related to fungal pheromone P- and a-factor receptors (family D), fungal pheromone A- and M-factor receptors (family D), and those related to slime mold cAMP receptors (family E). A growing number of new GPCR families continue to be reported, including those of the frizzled and smoothened families. It is thus likely that any classification of GPCRs will continue to expand and change, especially since many GPCRs identified by cloning or bioinformatics are presently orphan receptors with no function yet ascribed to them.

Signal Transduction The classic feature of GPCRs is their utilization of G-proteins for intracellular signal transduction. Signal transduction is initiated when the receptor adopts an ‘activated’ confirmation that can then bind to one or more members of the heterotrimeric G-protein family. In an early model of receptor signaling, the

activated conformation was thought to be achieved upon receptor binding to agonist. However, numerous GPCRs have been observed in vitro to couple to G-proteins and transduce signals in the absence of agonists. Such behavior is better explained by a multistate model wherein there is equilibrium between active and inactive receptor conformation states that is influenced by the presence or absence of agonists. In this scenario, ligands that promote stabilization of the active conformation are agonists, those that do not affect the active/inactive equilibrium are neutral antagonists, and those that favor the inactive conformation are inverse agonists. Once a GPCR adopts an active conformation, it can associate with one or more of the B20 members of the G-protein family to form the ‘ternary complex’ (Figure 2). Each G-protein heterotrimer is composed of a Ga subunit that can bind guanine nucleotides (GTP and GDP) in combination with nondissociable Gb and Gg subunits. Binding of the activated GPCR is accompanied by Ga subunit-mediated exchange of GDP for GTP and subsequent separation of the G-protein into Ga and nondissociable Gbg subunits. Depending upon the G-protein family members involved, these Ga and Gbg subunits act individually or synergistically to activate or inactivate various downstream effectors such as adenylyl cyclase, phospholipase C, and ion channels. Subsequent hydrolysis of GTP to GDP, which enables the Ga subunit to reassociate with Gbg subunits, completes the receptoractivated portion of the cycle. A family of proteins termed regulators of G-protein signaling (RGS protein) serve a major role in this GTP hydrolysis.

250 G-PROTEIN-COUPLED RECEPTORS GTP

GDP

G
(GDP)

G
(GTP)



Signal

Signal

 RGS G
(GDP)

Figure 2 Signal transduction by GPCRs. Receptors may exist in an activated or inactivated state. Agonist binding stabilizes or induces the activated state and enables the receptor to associate with its G-protein. Receptor activation causes the Ga subunit to exchange GDP for bound GTP, and to dissociate from the Gbg subunits. Either of subunit may then act to stimulate or inhibit downstream effectors such as adenylyl cyclase or phospholipase C.

The downstream second messengers and effectors activated by a particular GPCR are determined in large extent by the G-protein to which the receptor preferentially couples. Well-recognized examples of G-protein signal pathways include GS stimulation of adenylyl cyclase, Gi inhibition of adenylyl cyclase, and Gq/11 stimulation of phospholipase C. Other G-protein families include GT, GO, GH, and GZ. Studies using mutagenesis and chimeric receptors have demonstrated that specificity of GPCR coupling to G-proteins appears to be dictated in large part by the structure and conformation of the second and third intracellular loops and the portion of the cytoplasmic tail near the membrane. While it was thought early on that a given GPCR activated a single predominant G-protein and its associated signaling pathway, it has since been recognized that many GPCRs can couple to multiple G-protein families to simultaneously activate or inhibit additional signaling cascades. It is interesting to note that for some receptors that can couple to multiple Gproteins, synthetic agonists can bind in such a way to favor conformational states that provide preferential coupling to one G-protein over the other. Moreover, within these major G-protein families, it is now known that multiple isoforms of the a, b, and g subunits exist. For example, at least three different isoforms of Gai exist. Evidence suggests these subunit isoforms may differ in their signal transduction capacity and specificity, thereby providing for additional levels of complexity and plasticity in GPCR signal transduction. Finally, it is now recognized that ‘signaling’, in its most liberal interpretation, does not necessarily require receptor–G-protein interaction. For example, the b2-AR couples to the sodium hydrogen exchanger regulatory factor via direct interaction between the factor and a small region in the distal portion of the C-terminal cytoplasmic tail of the receptor. Another method of signaling without the need for efficient G-protein

interaction is via the agonist-promoted binding of the receptor and scaffolding proteins such as b-arrestin. Taken together, the term GPCR may not be most appropriate, but will undoubtedly be retained for historical reasons. Rather, ‘7-TM’ receptors best describe the superfamily.

Regulation of Production and Activity Following activation of signal transduction, many, but not all, GPCRs display a characteristic reduction in signaling intensity. This phenomenon, termed desensitization, is a negative feedback mechanism to prevent or limit the consequences receptor overstimulation. Overall GPCR activity thus reflects the balance between activating events, such as coupling of the receptor to its G-protein, and the events responsible for its desensitization. Although a combination of events may be responsible for the latter, three predominant mechanisms appear to be common among GPCRs: (1) uncoupling of the receptor from its G-protein by receptor phosphorylation, (2) internalization of the receptor from the cell surface to internal compartments, and (3) downregulation of the total number of receptors due to decreased production or increased degradation. These events are temporally distributed with phosphorylation occurring most rapidly, followed by internalization (within minutes) and downregulation (hours). These events are dictated not only by factors such as the nature of the agonist or length of exposure, but also by the presence or absence of requisite structural features (e.g., appropriate serine/threonine residues to act as substrates for phosphorylation) within the receptor itself. For susceptible GPCRs, phosphorylation is the most rapid desensitization event. Phosphorylation may be mediated by second-messenger-dependent kinases such as PKA or PKC, or by a group of secondmessenger-independent kinases termed G-proteincoupled receptors kinases (GRKs) of which seven family members have been identified. The critical substrates for phosphorylation appear to be serine and threonine residues within the intracellular loops and C-terminal tail regions, although the specific residues differ for the two groups of kinases. GRK-dependent phosphorylation promotes binding of cytosolic proteins from the arrestin family, which serves to uncouple the protein from its G-protein. Unlike second messenger-dependent phosphorylation, activation of downstream effectors is not requisite for GRK-mediated phosphorylation but an activated, or agonist-occupied, receptor is. Because of signal amplification, second-messenger-dependent phosphorylation is thought to be the primary desensitization event at low levels of agonist, whereas higher

G-PROTEIN-COUPLED RECEPTORS 251

levels of agonist additionally activate the more receptor-specific GRK-dependent kinases. Evidence is mounting that other kinases (e.g., tyrosine kinases and casein kinase 1a) may phosphorylate GPCRs as well, although the effect of these events on GPCR signaling is less well known. Internalization is a mechanism of desensitization whereby a GPCR is translocated from the plasma membrane to an internal cellular compartment where it is recycled or degraded. It is also where dephosphorylation occurs, and is often considered a mechanism of resensitization, if the receptor is rapidly recycled to the cell surface rather than degraded. The process is governed by the interaction of multiple determinants that include receptor structure, the nature of the activating ligand, the cell type expressing the receptor, and the surrounding cellular environment. No consensus structural motif for internalization has been identified, and the relevant structural determinants appear to vary among GPCR family members. For some GPCRs, internalization requires arrestin interaction; thus receptor phosphorylation and internalization can be intimately linked. Downregulation occurs following prolonged receptor activation and differs from internalization in that the total receptor pool (not just the cell surface receptors) is reduced. Downregulation may also be evoked by second messengers (i.e., cAMP/PKA activation by other GS-coupled receptors downregulates b2-AR). Molecular mechanisms responsible for mediating downregulation include decreased receptor synthesis rates due to transcriptional, translational, or posttranslational changes, and increased rates of receptor degradation. The genetic or structural determinants that affect downregulation may therefore reside outside the coding region of the gene, such as in the promoter or 30 untranslated region. However, as with other aspects of GPCR regulation, structural features of the receptor protein are also important. This is exemplified in the human b2-adrenergic receptor where a single N-linked glycosylation site is important for downregulation, and naturally occurring polymorphisms in the coding region differ in their resistance or sensitivity to downregulation.

Biological Function GPCRs regulate a wide variety of physiological and compensatory functions in the lung including maintenance of bronchomotor and vasomotor tone, secretory and absorptive function of the bronchial and alveolar epithelium, cell growth and regeneration, ciliary function, and mediator release by both resident and circulating inflammatory cells. It is thus not surprising that GPCRs have been and continue to be

important targets for therapeutic agents, with estimates that up to 50% of modern drugs target GPCRs. Important examples for pulmonary disease include the use of b2-AR agonists, muscarinic antagonists, and leukotriene antagonists for the treatment of asthma and chronic obstructive pulmonary disease, and the use of endothelin receptor antagonists and prostacyclin receptor agonists for treatment of pulmonary hypertension. The intimate relationship between GPCR structure and its function further suggests that differences within a GPCR due to genetic variation (mutations or polymorphisms) could potentially impact physiology, pathophysiology, and therapeutic responses. For example, polymorphisms in the b2-AR appear to have affects on risk, disease phenotypes, and response to therapy in the treatment of asthma. See also: Acetylcholine. Adrenergic Receptors. Asthma: Overview. Chemokines. Chronic Obstructive Pulmonary Disease: Overview. Cyclic Nucleotide Phosphodiesterases. Fluid Balance in the Lung. Histamine. Ion Transport: Overview. Lipid Mediators: Lipoxins. Neurophysiology: Neural Control of Airway Smooth Muscle. Signal Transduction. Smooth Muscle Cells: Airway; Vascular.

Further Reading Ferguson SS (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Phamacology Reviews 53: 1–24. Filipek S, Stenkamp RE, Teller DC, and Palczewski K (2003) G protein-coupled receptor rhodopsin: a prospectus. Annual Review of Physiology 65: 851–879. Filipek S, Teller DC, Palczewski K, and Stenkamp R (2003) The crystallographic model of rhodopsin and its use in studies of other G protein-coupled receptors. Annual Review of Biophysics and Biomolecular Structure 32: 375–397. Foord SM, Bonner TI, Neubig RR, et al. (2005) International Union of Pharmacology. XLVI. G protein-coupled receptor list. Phamacology Reviews 57: 279–288. Kenakin T (2002) Drug efficacy at G protein-coupled receptors. Annual Review of Pharmacology and Toxicology 42: 349–379. Lefkowitz RJ (2004) Historical review: a brief history and personal retrospective of seven-transmembrane receptors. Trends in Pharmacological Sciences 25: 413–422. Liggett SB (2000) Pharmacogenetics of beta-1- and beta-2adrenergic receptors. Pharmacology 61: 167–173. Marchese A, Chen C, Kim YM, and Benovic JL (2003) The ins and outs of G protein-coupled receptor trafficking. Trends in Biochemical Sciences 28: 369–376. Palczewski K, Kumasaka T, Hori T, et al. (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289: 739–745. Pitcher JA, Freedman NJ, and Lefkowitz RJ (1998) G proteincoupled receptor kinases. Annual Review of Biochemistry 67: 653–692. Small KM, McGraw DW, and Liggett SB (2003) Pharmacology and physiology of human adrenergic receptor polymorphisms. Annual Review of Pharmacology and Toxicology 43: 381–411.

252 GRANULOMATOSIS / Lymphomatoid Granulomatosis

GRANULOMATOSIS Contents

Lymphomatoid Granulomatosis Wegener’s Disease

Lymphomatoid Granulomatosis M I Schwarz and G P Cosgrove, University of Colorado Health Sciences Center, Denver, CO, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Lymphatoid granulomatosis (LG) is a rare lymphoproliferative disorder of unknown etiology affecting multiple sites, most notably within the lungs, skin, and central nervous system. The clinical presentation is similar to a systemic vasculitis, thereby necessitating a definitive evaluation to differentiate the two entities, as LG is now believed to be a malignant lymphoma. Characteristic of LG is a triad of histology including: (1) polymorphic lymphocytic infiltrates with small lymphocytes, plasma cells, and atypical mononuclear cells termed immunoblasts, (2) angioinvasion with transmural infiltration of arteries and veins by T lymphocytes, previously termed ‘angiitis’, and (3) focal necrosis within the vessel walls, termed ‘granulomatosis’. In association with the majority of cases is the concurrent or recent infection with Epstein–Barr virus (EBV). Optimal therapy has yet to be determined but chemotherapy, usually corticosteroids with cyclophosphamide, has been successful in some patients, especially in the more aggressive variants. In light of the association of EBV and clonal proliferation of B lymphocytes, novel antiviral therapy and B-cell-specific therapies have been used with success.

Introduction

malignant lymphoma, with B- and T-cell proliferation. More specifically, it is thought to be a T-cellrich B-cell lymphoreticular disorder. The majority of cases are caused by neoplastic transformation of B lymphocytes by Epstein–Barr virus (EBV) infection, with reactive T-lymphocyte proliferation. Isolated cases of T-lymphocyte predominant proliferation have been reported in the absence of evidence to suggest recent EBV infection. In these cases, the disorder may represent an isolated peripheral T-cell lymphoma.

Pathology A triad of histology characterizes LG: (1) polymorphic lymphocytic infiltrates with small lymphocytes, plasma cells, and atypical mononuclear cells termed immunoblasts, (2) angioinvasion with transmural infiltration of arteries and veins by T lymphocytes, previously termed ‘angiitis’, and (3) focal necrosis within the vessel walls, termed ‘granulomatosis’ (see Figure 1). The T lymphocytes appear to be reactive and typically are polyclonal without significant atypia. The large, atypical mononuclear cells, or immunoblasts, frequently have significant cellular atypia and represent transformed B lymphocytes

Lymphomatoid granulomatosis (LG) is an uncommon, multisystem, immunoproliferative disorder of uncertain etiology. It was first described as a distinct clinicopathologic entity by Liebow and colleagues in 1972. LG is characterized by angiocentric lymphoproliferation of T and B lymphocytes, with the lung being the most common organ of involvement. Divergent pathogenic mechanisms such as autoimmunity, postviral lymphoproliferation, and malignant conversion of B and T lymphocytes have been suggested. As a result, the terms angiocentric immunoproliferative lesion and angiocentric lymphoma have also been used to describe this disorder. The preferred term remains LG.

Etiology The pathogenesis of LG remains unclear but the current body of evidence suggests that it is a form of a

Figure 1 High-resolution computed tomogram in a 58-year-old female with LG. Multiple subcentimeter nodules adjacent to the bronchovascular bundle (black arrows) as well as within the interlobular septa were identified (white arrows). A confident diagnosis was obtained following a surgical lung biopsy.

GRANULOMATOSIS / Lymphomatoid Granulomatosis

253

secondary to EBV infection, based on immunophenotyping and EBER1/2 RNA in situ hybridization. Histopathologic grading systems based on the presence of EBV-positive cells, cellular atypia, and the degree of necrosis have been described, but their clinical usefulness is limited due to inconsistency and poor correlations with outcome.

Table 1 Common presenting symptoms and signs in patients with lymphomatoid granulomatosis

Clinical Features LG is a rare disease affecting multiple organs during its course. Based on the original report by Liebow and colleagues, and then a continuation by Katzenstein and colleagues, patients of all ages may be affected. The usual age of onset is between 40 and 60 years with a mean of 48 years. Rarely, it may also affect children. Males are affected twice as often as females. LG occurs in individuals who are otherwise healthy, with the exception of sporadic cases in which it is associated with primary or secondary immunodeficiency disorders such as Wiskott–Aldrich syndrome or postchemotherapy immunodeficiency or organ transplantation, HIV, and human T-cell leukemia virus infections. This association has led some to suggest that patients who develop LG have a selective immunodeficiency for controlling EBV. Pulmonary manifestations occur in all patients at some point during the course of their disease. The most common extrapulmonary sites of involvement are within the skin and nervous system. Manifestations within the spleen, liver, lymph nodes, kidney, adrenal gland, and heart were also noted at the time of autopsy by Katzenstein and colleagues but did not result in clinically significant symptoms or signs. It is in this manner that it contrasts to Wegener’s granulomatosis. Pulmonary involvement manifests as cough, dyspnea, and chest pain (see Table 1). Systemic symptoms include weight loss, fever, and night sweats. Skin lesions are occasionally the only presenting manifestation, but they are often found in conjunction with the pulmonary disease in 20–40% of cases. These are both dermal and subcutaneous nodules, which may be generalized but are more commonly localized to the lower extremities. Although typically nontender, ulcerative, tender, and erythematosus lesions resembling erythema nodosum may occur. Neurologic involvement occurs in up to 30% of cases and can result in peripheral neuropathy, cranial nerve palsies, or direct involvement of the brain with seizures. Destructive nasopharyngeal and paranasal sinus involvement may also occur and, in the setting of nodular pulmonary disease, cannot be distinguished from Wegener’s granulomatosis in the absence of a surgical lung biopsy. The presence of clinical manifestations in organ systems other than

Symptom/sign

152 cases (%)

Cough Fever Rash/nodules Malaise Weight loss

56 58 39 35 35

Neurologic abnormalities Central nervous system symptoms Cranial nerve abnormalities Peripheral nerve abnormalities

30 19 11 7

Dyspnea Splenomegaly Chest pain

30 18 13

Hepatomegaly Lymphadenopathy

12 8

Reproduced from Cancer; Katzenstein AL, Carrington CB, and Liebow AA; Copyright & 1979, Wiley. Reprinted with permission of John Wiley & Sons, Inc.

the lungs is associated with more malignant lymphomatous disease and a poorer prognosis. There are no characteristic laboratory abnormalities associated with LG. Non-specific immunodeficiencies, such as anergy and impairment in in vitro antigen stimulation assays, were reported by Sordillo and colleagues. Hypergammaglobulinemia (IgG and IgM) was noted in 47% of patients reported by Katzenstein and colleagues. Serologies for systemic autoimmune diseases and systemic vasculitides are typically unremarkable. EBV is detected in the majority of patients. Chest radiography shows primarily middle- and lower-zone involvement with multiple nodules, which have a tendency to coalesce and cavitate (see Figure 2). Alveolar infiltrates and diffuse reticular opacities may also occur. Hilar and mediastinal adenopathy does not occur in LG, in contrast to other pulmonary lymphomas, primary or secondary. High-resolution computed tomography demonstrates diffuse, poorly defined nodular infiltrates in a peribronchovascular distribution or within the interlobular septa. Thin-walled cysts in association with nodular infiltrates may also be seen. An accurate diagnosis of LG is dependent upon histologic inspection of specimen from an involved site, most commonly being the lung. In light of similar clinical and radiographic abnormalities, it must be differentiated from Wegener’s granulomatosis, lymphocytic interstitial pneumonia, malignant primary or secondary pulmonary lymphoma, or extranodal NK/T-cell lymphoma, nasal type, which is also associated with EBV infection but rarely involves the

254 GRANULOMATOSIS / Lymphomatoid Granulomatosis

Figure 2 Histologic appearance of lymphomatoid granulomatosis. Photomicrograph demonstrating prominent perivascular lymphocytic inflammation (a). The cellular composition was determined using immunohistochemistry. CD3 ( þ ) T lymphocytes (b) were more prominent than CD20 ( þ ) B lymphocytes in this case (c). Few atypical cells, the absence of necrosis, and a negative EBER1/2 RNA in situ hybridization resulted in a classification of low-grade LG. Original magnifications: (a) 200
; (b and c) 100
.

lung. Immunohistochemistry studies serve to define the repertoire of B and T lymphocytes and in situ hybridization for EBV provide corroborative evidence to support a diagnosis of LG.

Animal Models Animal or other biologic systems modeling LG have yet to be developed. The disease has been reported in felines, canines, and horses, but these were spontaneous cases similar to the human disease.

Prognosis and Current Therapy The clinical course of LG varies from benign, selflimited disease to malignant lymphoma. The majority of cases are progressive and requires therapy. Survival ranges from 14 to 72 months from the time of diagnosis. In those with malignant variants, mortality approaches 60% at 1 year. Poor prognostic features include central nervous system involvement, highgrade atypia, younger age, and hepatosplenomegaly. In those without extensive systemic involvement, prolonged survival may occur without treatment. Optimal therapy has yet to be determined for those with progressive disease. Aggressive therapy consisting of a combination of corticosteroids and cyclophosphamide resulted in durable remission in approximately 50% of patients. Antiviral therapy, specifically interferon-a2b, has been used in light of the association of LG and EBV. In a small series by Wilson and colleagues, 3/4 patients achieved durable remission at 40 months following therapy. More recently, therapy directed against transformed B lymphocytes, using the anti-CD20 antibody, rituximab, has shown promise with minimal toxicity. Future studies will need to address the efficacy of such novel therapies as interferon-a2b and rituximab in a larger cohort of patients.

Acknowledgment This work is supported by the NIH (NHLBI SCOR HL67671). See also: Granulomatosis: Wegener’s Disease. Leukocytes: T cells. Tumors, Malignant: Lymphoma. Vasculitis: Overview.

Further Reading Cosgrove GP, Fessler MB, and Schwarz MI (2003) Lymphoplasmocytic infiltrations of the lung. In: Schwarz MI and King TE (eds.) Interstitial Lung Disease, 4th edn., pp. 825–847. Malden, MA: Blackwell Science. Fauci AS, Haynes BF, Costa J, Katz P, and Wolff SM (1982) Lymphomatoid granulomatosis. Prospective clinical and therapeutic experience over 10 years. The New England Journal of Medicine 306(2): 68–74. Isaacson PG (1990) Lymphomatoid granulomatosis. British Medical Journal 300(6724): 612. Jaffe ES and Wilson WH (1997) Lymphomatoid granulomatosis: pathogenesis, pathology and clinical implications. Cancer Surveys 30: 233–248. Jaffe ES and Wilson WH (2001) Lymphomatoid granulomatosis. In: Jaffe ES, Harris NL, Stein H, et al. (eds.) World Health Organization Classification of Tumors. Pathology and Genetics of Tumors of Haematopeotic and Lymphoid Tissues, 1st edn., p. 185. Lyon, France: IARC Press. Katzenstein AL, Carrington CB, and Liebow AA (1979) Lymphomatoid granulomatosis: a clinicopathologic study of 152 cases. Cancer 43(1): 360–373. Lee JS, Tuder R, and Lynch DA (2000) Lymphomatoid granulomatosis: radiologic features and pathologic correlations. American Journal of Roentgenology 175(5): 1335–1339. Liebow AA, Carrington CR, and Friedman PJ (1972) Lymphomatoid granulomatosis. Human Pathology 3(4): 457–558. Myers JL, Kurtin PJ, Katzenstein AL, et al. (1995) Lymphomatoid granulomatosis. Evidence of immunophenotypic diversity and relationship to Epstein–Barr virus infection. The American Journal of Surgical Pathology 19(11): 1300–1312. Sordillo PP, Epremian B, Koziner B, Lacher M, and Lieberman P (1982) Lymphomatoid granulomatosis: an analysis of clinical and immunologic characteristics. Cancer 49(10): 2070–2076. Wilson WH, Kingma DW, Raffeld M, Wittes RE, and Jaffe ES (1996) Association of lymphomatoid granulomatosis with

GRANULOMATOSIS / Wegener’s Disease 255 Epstein–Barr viral infection of B lymphocytes and response to interferon-alpha 2b. Blood 87(11): 4531–4537. Zaidi A, Kampalath B, Peltier WL, and Vesole DH (2004) Successful treatment of systemic and central nervous system lymphomatoid granulomatosis with rituximab. Leukemia & Lymphoma 45(4): 777–780.

Wegener’s Disease W L Gross, P M Aries, and P Lamprecht, University Hospital Schleswig – Holstein, Luebeck, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract Wegener’s granulomatosis (WG) is a potentially life-threatening chronic inflammatory disease of as yet unknown etiology, characterized by necrotizing granulomatous lesions and an autoimmune vasculitis mediated by antineutrophil cytoplasmatic autoantibody (ANCA). The histopathologic hallmark is the triad of granulomatous inflammation, necrosis and vasculitis. WG usually takes a biphasic course starting in the upper and/or lower respiratory tract (so-called localized WG) with subsequent generalization with prominent clinical features of systemic vasculitis. Approximately 95% of patients with active generalized WG are ANCA positive, which is usually directed against ‘Wegener’s autoantigen’ proteinase 3 (PR3). In vitro and animal models suggest that the interaction of ANCA with cytokine-primed neutrophils results in premature activation, respiratory burst, and degranulation of neutrophil granulocytes with subsequent endothelial cell damage and necrotizing vasculitis. Altered T-cell responses with predominant Th1-type cytokine release might facilitate autoantigen recognition in WG. WG is a multifocal inflammatory disease that affects most often the upper and lower tract and the kidneys, but involvement of any other organ such as eye, skin, heart, gastrointestinal tract, central and peripheral nervous system may occur and give rise to serious or life-threatening complications. In few cases involvement of the upper and/or lower respiratory tract may be the only manifestation for years (localized WG). Cyclophosphamide plus steroids (NIH-scheme) is the standard therapy for the induction of remission in severe organ- or life threatening disease followed by azathioprine or other drugs for the maintenance of remission. In localized and early systemic WG trimetoprim-sulfamethoxazole and/or less aggressive immunosuppressions (e.g., MTX) can induce remission.

Introduction Wegener’s disease (WG) is a potentially organ- and life-threatening autoimmune disease of as yet unknown etiology characterized by granulomatous lesions and a necrotizing vasculitis predominantly affecting small vessels, that is, arterioles, capillaries, and venules. A prevalence of 23.7 per million adults has been reported in Europe. The peak incidence is in the fourth and fifth decade of life. The overall incidence ranges between 2.9 and 12/106/year depending on the geographic region. In recent years, early diagnosis has led to the recognition of less aggressive

disease courses and even long-term variants restricted to the upper respiratory tract in the absence of kidney involvement. Fifty years ago, Carrington and Liebow introduced the term ‘limited’ WG in order to define disease stages based on clinical and pathological findings. Today, the European Vasculitis Study Group (EUVAS) distinguishes ‘localized’ (i.e., WG restricted to the respiratory tract) and ‘early systemic’ WG (i.e., nonimminent WG without renal organ involvement) from ‘generalized’ WG. In most patients the disease progresses to generalized WG, but in a smaller fraction of patients the disease remains ‘localized’ or ‘early systemic’ for years. In generalized WG the upper and/or lower respiratory tract and the kidneys are involved in 80% of the patients. Rapidly progressive glomerulonephritis and pulmonary hemorrhage (pulmonary-renal syndrome) are typical symptoms of life-threatening full-blown WG (Table 1). Highly specific antineutrophil cytoplasmatic autoantibodies targeting ‘Wegener’s autoantigen’ PR3 (PR3-ANCA) are detected in about 95% of patients with generalized WG. A large number of in vitro studies and two recently published in vivo studies provide evidence that systemic vasculitis is induced by antineutrophil cytoplasmatic autoantibody (ANCA).

Etiology The etiology of WG is still subject to intensive research. The detection of ANCA directed to proteinase 3 (PR3-ANCA) is highly specific for WG, but incomplete knowledge is achieved about how granulomatous lesions, autoimmune vasculitis, and PR3ANCA evolve. Infections and other environmental influences may play a role in triggering or maintaining disease activity on the basis of a genetic predisposition. Recently, a strong association of HLADPB1*0401 has been reported. Epidemiological studies have revealed an association of noninfectious agents such as silica and exposure to farming with WG. Chronic nasal carriage of Staphylococcus aureus is an independent risk factor for a relapse. Animal models and anecdotal reports suggest that further infectious agents might have a role in triggering disease activity but definitive proof has been lacking so far. The risk of relapses in the respiratory tract can significantly be reduced by the use of trimethoprimsulfamethoxazole.

Pathology WG is characterized by the classic pathological triad of granulomatous inflammation predominantly of the

256 GRANULOMATOSIS / Wegener’s Disease Table 1 Differential diagnosis of the pulmonary syndrome: distinction is dependent on pathomechanism Diagnostic feature

Immunohistochemistry

Antibasalmembrane-antibodies

Linear immunodeposits

Goodpasture syndrome Idiopathic alveolar hemorrhagic syndrome

Immunocomplexes

Granular immunodeposits

Systemic lupus erythematodes Essential mixed cryoglobulinemic vasculitis Henoch–Scho¨nlein purpura IgA-nephritis Idiopathic alveolar hemorrhagic syndrome

ANCA (PR3-ANCA/ MPO-ANCA)

Negative or minimal immunodeposits (pauci-immune)

Wegener’s granulomatosis Microscopic polyangiitis Churg–Strauss syndrome

respiratory tract, necrotizing glomerulonephritis, and systemic vasculitis. The classical triad is most often found in specimens from open lung biopsies. However, not all elements of the triad may be seen and only one or two features may be detected in the biopsies. Renal granulomas are rare and found in o10% of kidney biopsies. Granulomatous lesions are built up by CD4 þ and CD8 þ T cells, CD28
T cells, histiocytes, CD20 þ B lymphocytes, neutrophil granulocytes, macrophages, and multinucleated giant cells surrounding an area of central necrosis. The term ‘geographic’ necrosis is used for a confluent or serpiginous pattern of the central necrosis. ‘Geographic’ necrosis and histiocytes lined up in a pallisading fashion are characteristic morphological features most often found in open lung biopsies. Scattered eosinophils are seen and sometimes may cause difficulties in distinguishing WG from Churg–Strauss syndrome. The necrotizing vasculitis affects predominantly small vessels and mediumsize arteries, that is, capillaries, venules, arterioles, and arteries. Endothelial cells are the target of the initial injury. The earliest changes affect the vascular endothelium with swelling, necrosis, and platelet deposition. In the lung capillaries, venules and arterioles are infiltrated by polymorphonuclear leukocytes. Pulmonary microvascular necrotizing vasculitis (capillaritis) is the cause of pulmonary hemorrhage. In the kidney, rupture of the basement membrane subsequent to neutrophil degranulation gives rise to glomerular capillary thrombosis followed by segmental necrosis of the tuft. Necrotic material, blood, and fibrin spill into the Bowman’s space. As a consequence, accumulation and proliferation of monocytes and parietal (Bowman’s) epithelial cells with the formation of crescents is seen. The growing crescent compresses the capillary tuft, leading to segmental and global loss of circulation through the glomerulus. In WG, the histological picture of vasculitis is polymorphic. Apart from necrotizing vasculitic lesions, leukocytoclastic vasculitis of the skin may be seen as cutaneous vasculitis

Disease

manifestation. Necrotizing granulomatous arteritis involving medium-sized vessels is commonly found close to large necrotizing granulomatous foci in the lung. Nongranulomatous fibrinoid necrosis may affect bronchial arteries. Finally, vasculitis morphologically identical to that seen in polyarteritis, both in size of the vessels involved and the presence of fibrinoid necrosis, may also be seen in WG in some cases. No or only a few immunodeposits are detected in glomerular lesions, hence the name ‘pauci-immune’ vasculitis.

Clinical Features Localized Wegener’s Granulomatosis

Localized WG has been defined by the EUVAS as WG restricted to the upper and/or lower respiratory tract (Table 2). Retrospective studies revealed that 50% of WG patients initially present with solitary ear, nose, and throat (ENT) involvement. The duration of the localized disease stage may last from months to several years. Typically symptoms are nasal obstruction by mucosal swelling, serosanguinous discharge, and epistaxis. Nasal crusting, mucosal ulcerations, hoarseness, sinusitis, or mastoiditis are further typical symptoms of upper respiratory tract involvement. Persistent disease activity causes saddle-nose deformity, septum perforation, and subglottic tracheal stenosis (Figure 1). Proptosis may be a sign of retrobulbary granuloma, often due to per continuum infiltration from the sinus. Granulomatous inflammation of the lower respiratory tract may lead to bronchus stenosis, atelectasis, and obstructive pneumonia. Localized WG usually progresses to early systemic or generalized WG in over 95% of cases within weeks or months. Generalized Wegener’s Granulomatosis

‘Early systemic’ WG has been defined by the EUVAS as WG with any organ involvement except renal or

GRANULOMATOSIS / Wegener’s Disease 257 Table 2 Clinical subgroups of Wegener’s granulomatosis according to the definitions of the European Vasculitis Study Group (EUVAS) Subgroup

Organ involvement

Constitutional symptoms

Presence of ANCA

Localized WG Early systemic WG Generalized WG

Upper and/or lower respiratory tract Any except renal involvement or imminent organ failure Renal with serum creatinine p500 mmol l
1 and/or other imminent organ failure Renal with serum creatinine 4500 mmol l
1 Progressive disease despite therapy with GC ad Cyc

No Yes Yes

Yes/No Usually yes Yes

Yes Yes

Yes Yes/No

Severe renal WG Refractory WG

WG, Wegener’s granulomatosis; GC, glycocorticoids; Cyc, cyclophosphamide.

Figure 1 Wegener’s granulomatosis with saddle-nose deformity.

imminent vital organ failure. ‘Generalized WG’ has been defined as WG including renal involvement and/ or imminent organ failure. Classical WG usually takes a biphasic course, starting in the respiratory tract (localized WG), followed by subsequent generalization of the disease. Generalization is often heralded by constitutional symptoms (weight loss, fever, and night-sweat) and rheumatic complaints such as migratory-arthralgia and myalgia. Otological manifestations include sensorineural hearing loss, vestibular impairment, and facial nerve palsy due to otitis media, mastoiditis, or neuropathy. Relapsing polychondritis may occasionally be encountered. Retroorbital masses, mostly developing by granulomatous inflammation per continuum of the sinus, may cause proptosis, compression of the optic nerve, and progressive visual loss. Dacryocystoadenitis and nasolacrimal duct inflammation are peculiar organ manifestations of WG and are usually not found in other vasculitides. Episcleritis presenting as ‘red eye’, uveitis, retinal exudates, optic nerve vasculitis, and retinal artery occlusion reflect occular vasculitic lesions (Figure 2). Pulmonary involvement is one of the cardinal features in WG and occurs in over 80% of cases

Figure 2 Wegener’s granulomatosis with scleritis.

during the course of the disease. Even if pulmonary symptoms are clinically absent, 30% may have radiographically demonstrable pulmonary lesions. Abnormalities of pulmonary function such as reduced CO diffusion may be early signs of pulmonary involvement. In case of subglottic stenosis an inspiratory stridor or severe dyspnea may be present. Frequently, early complaints related to the lower respiratory tract are cough, dyspnea, pleuritic pain, or hemoptysis. Bland hemoptysis or alveolar hemorrhage with respiratory insufficiency and anemia reflects pulmonary capillaritis and has a high mortality rate (Figure 3). Multiple pulmonary nodules may be asymptomatic; however, solitary granulomatous masses can cause severe bronchial stenosis. Spontaneous pneumothorax may be induced by rupture of a subpleural cavity nodule. Pleural effusions and mediastinal or hilar adenopathy are less common. Cardiac manifestations are pericarditis with pericardial effusion, myocarditis, and coronaritis with or without myocardial infarction and rarely subsequent cardiomyopathy. Renal involvement ranges from asymptomatic hematuria to rapidly progressive glomerulonephritis.

258 GRANULOMATOSIS / Wegener’s Disease

Cytokine-primed neutrophils release PR3 from azurophilic granula, and express PR3 on the cell surface. Binding of PR3-ANCA to the membrane-located PR3 induces further premature activation and degranulation of the cells with subsequent endothelial damage and induction of necrotizing vasculitis (ANCA-cytokine-sequence theory) (Figure 4). Recently, murine transfer models showing the in vivo induction of a necrotizing systemic vasculitis and glomerulonephritis by myeloperoxidase-specific (MPO)-ANCA and exacerbation of inflammatory panniculitis by PR3-ANCA have further underscored the pathophysiological role of ANCA in inducing vasculitis. Figure 3 Wegener’s granulomatosis: clinical presentation with the recent development of hemoptysis. CT scan shows classical signs of diffuse alveolar hemorrhage.

A nephritic urine sediment (microscopic hematuria with red cell casts and glomerular selective or unselective proteinuria) and reduction of creatinine clearance demonstrate renal involvement. In over 50% of cases neurological involvement is seen as a consequence of vasculitis or compression by mass-like lesions or frank vasculitis. In most patients, sensory peripheral neuropathy or motor mononeuritis multiplex occurs within the first 2 years. An effect on the CNS is less frequent (about 10%). Further manifestations, including involvement of the gall bladder, ovaries, parotis, and breast among others, add to the broad spectrum of symptoms.

Pathogenesis American pathologist Fienberg suggested that WG starts as granulomatous disease in the upper and/or lower respiratory tract with subsequent development of vasculitis. A predominance of T-helper-1 (Th1)type cytokines is seen in early granulomatous lesions in localized WG whereas this is less evident in generalized WG. Disease progression is associated with a ‘switch’ or further complexity of the collective T-cell response with the appearance of a subset of Th2-type cells. This ‘switch’ or increasing complexity of the cytokine profile might be a consequence of further Bcell expansion and T-cell-dependent PR3-ANCA production during disease progression. Infections such as S. aureus, other environmental influences, or ‘Wegener’s autoantigen’ PR3 itself are thought to play a role in triggering and/or maintaining disease activity in Wegener’s granulomatosis on the basis of a genetic predisposition to an exaggerated Th1-type response early in the disease process. Neutrophils become activated by tumor necrosis factor alpha (TNF-a).

Diagnosis Diagnosis of WG is based on clinical manifestations, serological parameters, and histomorphological findings. The classification criteria of the American College of Rheumatology (ACR), defining clinical criteria for distinguishing patients with WG from those with other vasculitides, were proposed in 1990 before the implementation of ANCA as a routine diagnostic procedure and are not intended to be used as diagnostic criteria (Table 3). Non-specific serological parameters indicative of inflammation such as elevated erythrocyte sedimentation rate and C-reactive protein are usually found. With regard to ANCA highest specificity (99%) and sensitivity (73%) can be reached when the results of the immunofluorescence (IFT) and enzyme-linked immunosorbent assay (ELISA) technique are combined. In IFT a cytoplasmic pattern C-ANCA is found in 50% of patients with localized WG and in 95% with generalized WG. The target autoantigen detected by ELISA is PR3 in X95% of WG patients. Few patients (p5%) present with an MPO-ANCA. Tissue biopsies should be obtained from easily accessible symptomatic organs such as the upper or lower respiratory tract. Transbronchial biopsies are seldom representative, whereas nasal biopsies produce a much higher yield of representative biopsies. However, most often specimens from open lung biopsies exhibit the classic pathological triad of granulomatous inflammation, necrosis, and vasculitis. Kidney biopsies typically provide evidence of segmental necrotizing glomerulonephritis with little or no immunoglobulin deposition (pauciimmune) which is characteristic of ANCA-associated vasculitis. Disease activity, extent, and damage can be scored with validated instruments such as the Birmingham vasculitis activity score (BVAS), the disease extent index (DEI), and the vasculitis damage index (VDI) (Table 4). ANCA titers can be used for the follow-up of patients: a fourfold or greater rise in the titer may indicate an imminent relapse and should be

3 Expression of adhesion molecules

2 PR3 expression on the cell surface

1 Resting stage

sPR3 PR3 TNF-


TNF-


IL-8, IL-1

PR3

IL-8, IL-1

PR3

siCAM-1 LFA-1

LFA-1

ANCA PR3 CAM-1

PR3

E-selectin

VCAM-1

Activated

Endothelial cells: resting 4 Ligation and activation

5 Destruction and granuloma formation

PR3 PR3 PR3
, AT

ANCA

F
RII LTB4


.1AT sPR3

PR3-ANCA-IC

· O2

HLE

PR3

Figure 4 ANCA-cytokine sequence theory. 1, Resting polymorphonuclear granulocytes (PMNs) with proteinase-3 (PR3) stored in vesicles. 2/3, Activation of PMNs and endothelial cells by proinflammatory cytokines resulting in expression of adhesion molecules and translocation of the intracytoplasmic PR3 on the cell surface. 3/4, Adhesion of PMN on endothelial cells. 4/5, Binding of ANCA to membrane-located PR3 leads to further activation and is followed by degranulation of PMNs. Thus, toxic radicals and lytic proteins are released near the endothelial cells, out of reach for proteinase inhibitors like alpha-1 antitrypsin. Subsequently, endothelial damage and necrotizing vasculitis takes place.

260 GRANULOMATOSIS / Wegener’s Disease

followed by close monitoring of the patient for clinical signs of WG activity.

Therapy In severe organ- and life-threatening WG induction of remission with the NIH-standard or so-called

Table 3 Definitions and criteria for Wegener’s granulomatosis The American College of Rheumatology proposed clinical classification criteria (vasculitis must be present) for Wegener’s granulomatosis (1990): * Nasal or oral inflammation (painful or painless oral ulcers or purulent or bloody nasal discharge) * Abnormal chest radiograph showing nodules, fixed infiltrates, or cavities * Abnormal urinary sediment (microscopic hematuria with or without red cell casts) * Granulomatous inflammation on biopsy of an artery or perivascular area Definition and classification of Wegener’s granulomatosis according to the Chapel Hill Consensus (1994): * Granulomatous inflammation involving the respiratory tract, and necrotizing vasculitis affecting small to medium-sized vessels (i.e., capillaries, venules, arterioles, and arteries) * Necrotizing glomerulonephritis is common * Cytoplasmic pattern ANCA (C-ANCA) with antigen specificity for proteinase 3 (PR3) is a very sensitive marker for Wegener’s granulomatosis

‘Fauci’s scheme’ remains the gold standard, with oral cyclophosphamide (2 mg kg
1 day
1 po for 3 days) and steroids (initially 1–2 mg kg
1 day
1 prednisolone iv or orally, with subsequent tapering of dose) given for 3–6 months. Data from a recent EUVAS trial (CYCAZAREM) provided evidence that azathioprine is equally effective for the maintenance of remission. Therefore, azathioprine should be regarded as the treatment of choice for maintenance therapy. The use of mesna is recommended for prophylaxis of the cyclophosphamide urotoxicity. Several trials analyzed the potential benefit of plasmapheresis in patients with severe renal involvement. Preliminary data from the latest EUVAS trial (MEPEX) analyzing initial plasmapheresis versus intravenous high-dose methyl-prednisolone (1 g) in addition to standard treatment with cyclophosphamide and steroids showed higher efficacy of plasmapheresis in patients with a creatinine of 4500 mmol l
1. Disease and treatment-related mortality was shown to be high in the subgroup of patients in this trial. In non-life-threatening, nonrenal ‘early systemic’ WG, MTX can be used. The efficacy of other agents (leflunomide, mycophenolate mofetil, and cyclosporin A) has been reported in case reports or smaller case series. The optimal duration of maintenance therapy is the subject of another presently ongoing EUVAS study (REMAIN) comparing 2 years versus 5 years of maintenance remission with azathioprine.

Table 4 Organ manifestations of Wegener’s granulomatosis and common disease activity, extent and damage scores Organ manifestation

ELK classification

Disease extent index (DEI)

Birmingham vasculitis activity score (BVAS)

Vasculitis damage index (VDI)

ENT Lung Kidney Mucous membrane Skin Eye Joint/muscles Central nervous system Peripheral nervous system Cardiovascular system/ peripheral vascular disease Intestine Constitutional symptoms Side effect of therapy

E L L na S Ey A C P

2 2 2 na 2 2 2 2 2

6 6 12 na 6 6 2 9 9

6 7 3 1 2 7 5 6 1

H

2

6

15

Gi B na

2 1 na

9 3 na

4 na 6

Maximal score

na

21

63

64

na, not applicable. ELK: reflecting the disease extent without weighting scoring. DEI: every organ involved that is attributable to active WG is added up. BVAS: disease features are scored when they are due to active vasculitis. Scores have been weighted according to the severity. Each section has a different maximum score. VDI: this is for recording organ damage that has occurred in patients since the onset of vasculitis.

GRANULOMATOSIS / Wegener’s Disease 261

For prophylaxis, treatment with trimetoprim-sulfamethoxazole reduces S. areus colonization in the respiratory tract and thereby reduces the risk of respiratory relapses. Trimetoprim-sulfamethoxazole also induces remission in localized WG. The primary goal of all therapies is to achieve remission. Using the BVAS, remission is defined as the absence of signs of new or worse disease activity and persistent disease activity for no more then one item on the BVAS evaluation form. Some disease manifestations regress completely, but others do not such as nasal septal perforation or persistent ‘defect’ proteinuria after glomerulonephritis. Despite the efficacy of the above-mentioned regimen in the majority of patients, up to 10% achieve no complete remission and/or early relapses occur in up to 50% of patients. Thus, the aim of new treatment options is to combine improved efficacy with less toxicity. TNF-a inhibitors infiximab and etanercept, anti-CD20 antibody rituximab, azathioprine pulse therapy, highdose cyclophosphamide, anti-thymocyte globin, intravenous immunoglobulins, 15-deoxyspergualin, and humanized monoclonal anti-CD52 or anti-CD4 antibodies have been reported to be beneficial in refractory WG in smaller case series. One trial found no significant differences between the addition of etanercept to standard treatment and standard treatment alone in the rates of sustained remission in nonrefractory WG (WGET study by eight American centers). However, it has been shown that TNF-a production is elevated in active and refractory WG. TNF-a levels are normalized with successful standard treatment. Therefore, as suggested by these in vitro data, patients responding to standard treatment will have no further benefit from the addition of a TNF-a blocking agent, whereas patients refractory to standard treatment will benefit from the addition of a TNF-a inhibitor. In line with this consideration are data from an open-label, multicenter, prospective clinical trial by Booth, et al. who successfully induced remission with infliximab in addition to standard or existing treatment regimens both in active and refractory patients (n ¼ 32). Further, higher rates of nonsustained remission in the WGET trial might have been caused by early tapering of steroids in that study. See also: Alveolar Hemorrhage. Corticosteroids: Therapy. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome. Vasculitis: Microscopic Polyangiitis.

Further Reading Jagiello P, Gencik M, Arning L, et al. (2004) New genomic region for Wegener’s granulomatosis as revealed by an extended association screen with 202 apoptosis-related genes. Human Genetics 114: 468–477. Jayne D for the European Vasculitis Study Group (EUVAS) (2001) Update on the European Vasculitis Study Group trials. Current Opinion in Rheumatology 13: 48–55. Jayne D, Rasmussen N, Andrassy K, et al., for the European Vasculitis Study Group (2003) A randomized trial of maintenance therapy for vasculitis associated with antineutrophil autoantibodies. New England Journal of Medicine 349: 36–44. Jennette JC and Falk RJ (1997) Small-vessel vasculitis. New England Journal of Medicine 337: 1512–1523. Jennette JC, Falk RJ, Andrassy K, et al. (1994) Nomenclature of systemic vasculitides. Proposal of an International Consensus Conference. Arthritis and Rheumatism 37: 187–192. Komocsi A, Lamprecht P, Csernok E, et al. (2002) Peripheral blood and granuloma CD4 þ CD28
T-cells are a major source of IFN-g and TNF-a in Wegener’s granulomatosis. American Journal of Pathology 160: 1717–1724. Lamprecht P, Erdmann A, Muller A, et al. (2003) Heterogeneity of CD4 þ and CD8 þ memory T cells in localized and generalized Wegener’s granulomatosis. Arthritis Research and Therapy 5: R25–R31. Leavitt RY, Fauci AS, Bloch DA, et al. (1990) The American College of Rheumatology 1990 criteria for the classification of Wegener’s granulomatosis. Arthritis and Rheumatism 33: 1101–1107. Pfister H, Ollert M, Frohlich LF, et al. (2004) Antineutrophil cytoplasmic autoantibodies against the murine homolog of proteinase 3 (Wegener autoantigen) are pathogenic in vivo. Blood 104: 1411–1418. Popa ER, Stegeman CA, Kallenberg CGM, and Cohen Tervaert JW (2002) Staphylococcus aureus and Wegener’s granulomatosis. Arthritis Research 4: 77–79. Reinhold-Keller E, Beuge N, Latza U, et al. (2000) An interdisciplinary approach to the care of patients with Wegener’s granulomatosis. Long-term outcome in 155 patients. Arthritis and Rheumatism 43: 1021–1032. Stone JH, Hoffman GS, Merkel PA, et al. for the International Network for the Study of the Systemic Vasculitides (INSSYS) (2001) A disease-specific activity index for Wegener’s granulomatosis: modification of the Birmingham Vasculitis Activity Score. Arthritis and Rheumatism 44: 912–920. Xiao H, Heeringa P, Hu P, et al. (2002) Jennette: antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. Journal of Clinical Investigation 110: 955–963.

Relevant Website http://www.vasculitis.org – Homepage of the ‘The European Vasculitis Study Group (EUVAS)’ with information about trial protocols, trial progress, meetings, nomenclature, disease scoring and ANCA-testing.

H HEMOGLOBIN R Winslow, University of California, San Diego, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Hemoglobin is the red oxygen-carrying pigment of the blood, contained within red blood cells (erythrocytes). It is composed of four subunits, two a and two b polypeptide chains, each of which contains a heme group with iron (Fe) which is the site of oxygen binding. The detailed structure of each of the chains has been defined and the reversible binding of oxygen is explained by two distinct structures of hemoglobin. The synthesis of the a and b chains of hemoglobin is controlled by specific genes that are activated at different times of fetal development. A large number of mutations of the chains have been detected. Most of them are of no clinical consequence, but some, like the sickle cell anemia mutation, can cause severe disease. Mutations that affect the relative quantities of the chains can also cause disease (the thalassemias).

a and two b) of about 140 amino acids each. Each chain carries a tetrapyrrole iron-containing prosthetic group, heme, which can bind one molecule of oxygen. All vertebrate and most nonvertebrate animals use hemoglobin to transport oxygen from the atmosphere to tissues. In humans several hemoglobins occur normally, but all contain four subunits: the differences are limited to the primary structure (amino acid sequence) of globin. Each polypeptide chain is the product of a specific gene. For the composition of hemoglobin A, the major component of A adult human red blood cells, is aA 2 b2 . During human fetal development at least three different non-a globin chains are produced, each a product of a separate gene (Figure 1). The z and e chains are found in very early fetuses and g chains later and in newborns. By about 6 months after birth, the newborn exhibits only the adult pattern of hemoglobin (Table 1).

Structure Hemoglobin is a globular protein whose molecular mass is about 64 kDa. The protein portion (globin) is composed of four subunit polypeptide chains (two

Primary Structure

Hemoglobin was one of the first proteins whose primary structure (amino acid sequence) was determined

100

Polypeptide chains present (%)





80  60



40

20


3 Gestation in months

 3

6 Birth

6

Age in months

Figure 1 Ontogeny of the human globin chains. The earliest detectable globins are the e chains of hemoglobins Gower 1 and 2. The z chain of hemoglobins Portland 1 and 2 appear only transiently. By 3 months of gestation, hemoglobin F predominates; by 6 months after birth, hemoglobin A constitutes about 97% of the total. The d chains are synthesized at approximately the same time as the b chain. See Table 1 for the chain compositions of hemoglobin A.

264 HEMOGLOBIN

(Figure 2). The high-resolution X-ray crystallographic analysis by Perutz and co-workers allowed an extensive integration of sequence and functional data. This integration leads to an appreciation of structure– function relationships so delicate that substitution of Table 1 The hemoglobins of human red blood cells A0 F A2 Portland 1 Portland 2 Gower 1 Gower 2

a2b2 a2g2 a2d2 g2z2 z4 e4 a2z2

Main component of human red blood cells Fetal hemoglobin Minor component of human red blood cells Embryonic hemoglobin Embryonic hemoglobin Embryonic hemoglobin Embryonic hemoglobin

one amino acid in 280 can lead to serious clinical consequences, as in sickle cell anemia. Secondary Structure

About two-thirds of the residues of each chain are arranged in seven and eight helices of the a and b chains, respectively (see Figure 3). The helices are designated from A to H, and vary from 7 to 20 residues in length. There are 3.6 residues per turn of helix, which are stabilized by hydrogen bonds formed longitudinally along their axes. The remaining onethird of the residues are distributed over corners or nonhelical regions of the chain.

 

Val Val

His

Leu Leu

Ser Thr

A Pro Pro

Ala Glu

Asp Glu

Lys Lys

Thr Ser

Asn Ala

Val Val

Lys Thr

 

Ala Ala

Ala Leu

Try Try

Gly Gly

Lys Lys

Val Val

A Gly Asn

Ala Val

B His -

Ala -

Gly Asp

Glu Glu

 

Tyr Val

Gly Gly

Ala Gly

Glu Glu

Ala Ala

Leu Leu

Glu Gly

Ar g Ar g

Met Leu

Phe Leu

Leu Val

Ser Val

 

C Phe Tyr

Pro Pro

Thr Try

Thr Thr

Lys Gln

Thr Arg

Phe Phe

Pro Glu

His Ser

Phe Phe

Gly

Asp Asp

 

Leu Leu

Ser Ser

D His Thr

Gly Pro

Ser Asp

Ala Ala

Val

Met

Gly

Asn

Pro

Gln Lys

 

Val Val

Lys Lys

Gly Ala

His His

Gly Gly

Lys Lys

Lys Lys

Val Val

Ala Leu

Asp Gly

Ala Ala

Leu Phe

 

Thr Ser

Asn Asp

Ala Gly

Val Leu

Ala Ala

Val Leu

Asp Asp

Asp Asn

Met Leu

Pro Lys

Asn Gly

 

Ala Thr

F Leu Phe

Ser Ala

Ala Thr

Leu Leu

Ser Ser

Asp Glu

Leu Leu

His His

Ala Cys

His Asp

Lys Lys

 

Leu Leu

Arg His

Val Val

G Asp Asp

Pro Pro

Val Glu

Asn Asn

Phe Phe

Lys Arg

Leu Leu

Leu Leu

Ser Gly

 

His Asn

Cys Val

Leu Leu

Leu Val

Val Cys

Thr Val

Leu Leu

Ala Ala

Ala His

His His

Leu Phe

Pro Gly

 

Ala Lys

Glu Glu

Phe Phe

H Thr Thr

Pro Pro

Ala Pro

Val Val

His Gin

Ala Ala

Ser Ala

Leu Tyr

Asp Gln

 

Lys Lys

Phe Val

Leu Val

Ala Ala

Ser Gly

Val Val

Ser Ala

Thr Asn

Val Ala

Leu Leu

Thr Ala

Ser His

 

Lys Lys

Tyr Tyr

Arg His

E

His His

Figure 2 The amino acid sequences of the normal adult globin chains. Helical regions are designated by letters A to H. The numbers above each row refer to residues of the a chain, those below the rows to the non-a chains. Residues that are common to more than one chain are enclosed in boxes.

HEMOGLOBIN 265 C

C D

H

F

E

B

G

A N

Figure 3 Schematic diagram of a globin chain. Any of the globin chains (or myoglobin) could fit the diagram with only minor alteration. The a-helical regions are lettered, and heme is shown as a disk. The only covalent link between heme and globin is between the iron atom and histidine F8, the ‘proximal’ histidine.

Myoglobin and all the various hemoglobin chains resemble each other to a remarkable degree. To emphasize this resemblance and to facilitate comparison among chains, residues are given designations according to their position in or between helical regions. For example, residue 10 of the a chain is valine A7(10)a because it occupies position 7 from the beginning of the A helix. Although the corresponding residue in the b chain is valine A7(11)b, it occupies the eleventh position from the N-terminus of that chain. Similarly, the residues in the interhelical regions are given designations using letters of adjacent helices: the third residue in the C–D region of the a chain, for example, is histidine CD3(45)a, and in the b chain, serine CD3(44)b. Tertiary Structure

The three-dimensional folding of a globin chain is also shown in Figure 3. The similarity among the various chains is sufficient so that the figure could represent any of them with only minor alterations. Heme is represented as a flat ring between the E and F helices. Proline does not fit into a helix because of its pyrrolidine ring; it cannot form hydrogen bonds with the next coil in the helix. Therefore, it is likely

to occur at corners and interhelical regions. Other corners are stabilized by a variety of stereochemical mechanisms, some of which involve portions of chains that lie nearby. Interactions can also occur between helical segments that lie in proximity to one another. For example, the B and E helices are stabilized in each helix by glycine residues, which afford a compact fit at their crossing. A very important force stabilizing the globin chain structure is the presence of heme itself. Free globin is much less soluble than the molecule containing heme. It is embedded firmly into a ‘pocket’ in the chain as shown in Figure 3. It has polar and nonpolar ends, one formed by propionic acid groups and the other by vinyl and methyl groups, respectively. The amino acid side groups lining the heme pocket are largely nonpolar and allow the heme to enter, vinyl end first. It is then anchored there by a large number of interactions between the pyrrole ring and amino acids of the heme pocket. In addition, it is linked covalently to histidine F8, which is called the proximal histidine. The distal histidine E7 is not linked covalently to the heme group, but its presence in the heme pocket is important in maintaining spatial relationships. The hydrophobic nature of the heme pocket is extremely important in the maintenance of

266 HEMOGLOBIN

the heme iron in its reduced (Fe2 þ ) state, which is required for oxygenation. A number of mutations in the environment of the heme pocket can lead to the entry of water, oxidation of the iron, and as a result, methemoglobin formation. The distal histidine (E7) acts as a ‘gate’ to allow ligand to enter the heme pocket only when it swings out. In the a chain (or myoglobin), a hydrogen bond forms between Ne of histidine E7 and the oxygen molecule. Quaternary Structure

The association of globin chains into a tetrameric structure leads to cooperativity, the Bohr effect, the 2,3-diphosphoglycerate (2,3-DPG) effect, and suitable oxygen affinity so that tissue oxygen requirements can be met (see Oxygen–Hemoglobin Dissociation Curve). The molecule has a twofold axis of symmetry, each half containing one a and one b chain, which are held tightly together by a large number of atomic interactions. There are four areas of contact between the subunits: a1b1, a2b2, a2b1, and a1b2. The a1b2 and a2b1 regions are somewhat weaker than the a1b1 and a2b2 contacts and allow movement during oxygenation. Isolated hemoglobin chains are less stable than the fully associated tetramers. Thus, the regions of contact between them are important in maintaining normal solubility. Several unstable mutants involve the a1b1 and, to a lesser degree, the a1b2 contacts. The central cavity of hemoglobin is filled with water, allowing entrance of charged molecules that affect molecular function, such as 2,3-DPG and salts. Most of the charged amino acid side groups are directed toward the outside of the molecule and enter into electrostatic relations with solvents, allowing very high solubility within the red blood cells. Buried in the midst of the globin chains are the four heme groups containing ferrous iron, protected against oxidation by a hydrophobic environment. Two quaternary conformations The function of hemoglobin in oxygen binding can be most easily understood on the basis of at least two separate quaternary conformations, corresponding to the oxy and deoxy states. Perutz and his associates described these two structures using X-ray crystallography and related them to the R (for relaxed) and T (for tense) conformations. They found that the combination of deoxyhemoglobin with oxygen produced shifts of the helical regions of 2 to 3 A˚, and a change in the tilt of the heme group relative to the globin chain. This is accompanied by shifts of the iron atom within the porphyrin and of the heme-linked histidine. In the deoxy subunit the e-methyl group of valine E11 (67) blocks the heme pocket. In the oxy form (R)

this pocket widens, making room for the binding of an oxygen molecule. There is little difference at the a1b1 (and a2b2) contact, but a significant change at the a1b2 (and a2b1) contact occurs. The region is dovetailed so that the CDa region of one chain fits into the FG region of the other. During transition from R to T, the dovetailing between CDa and FG changes so that the hydrogen bond linking aspartate G1(94)a to asparagine G4(102) is replaced by another between tyrosine C7(42)a and aspartate G1(99). Finally, large differences occur in the areas of the C-terminal residues. In deoxyhemoglobin the tyrosines (HC2) occupy pockets between helices F and H, and their phenolic groups are hydrogen-bonded to valines FG5. The Cterminal residues are constrained by participation in salt bridges. In oxyhemoglobin, the phenolic groups of tyrosine HC2a are ejected from their pockets, and the salt bridges are broken. Perutz and co-workers proposed a model that relates these molecular changes to oxygen binding. The first, or trigger, step results from the combination of iron with oxygen. When this occurs, the atomic radius of iron decreases (high-to-low spin transition), allowing it to move closer to the plane of the porphyrin ring. This movement is less than 1 A˚, but it is sufficient to change the distance between the heme-linked histidine and the plane of the porphyrin ring, initiating the sequence of changes leading to the T-to-R transition. This description applies to the a chain; but in the b chain, the hydrophobic side group of valine E11 is in the heme pocket, providing an additional block to oxygen binding. Movement of the porphyrin ring relative to the heme-linked histidine results in a narrowing of the pocket between helices F and H into which the phenolic group of tyrosine HC2 fits, which leads to its ejection. Rotation of this group distorts the C-terminal amino acids so that the salt bridges are no longer possible, and they rupture. Each subunit undergoes change in the tertiary structure with successive oxygenation, as described above; after two or three of them have done so, there is a change in the quaternary structure. This change involves a rotation of the two halves of the molecules (a subunits) relative to one another, and a ‘sliding’ occurs at the a1b2 and a2b1 interfaces. The equilibrium between the R and T conformations can be influenced by a number of small molecules that bind at specific sites. These heterotropic or allosteric effectors are mainly hydrogen ion (H þ ), chloride (Cl
), 2,3-DPG (within the red cell), and carbon dioxide (CO2), but many other compounds bind to hemoglobin as well (see Oxygen–Hemoglobin Dissociation Curve).

HEMOGLOBIN 267

Hemoglobin Variants: The Hemoglobinopathies

Globin – the protein part of hemoglobin (without the heme prosthetic group)

More than 500 structural variants of human hemoglobin have been identified. Most of them have been discovered because of altered electrophoretic mobility. The great majority of these mutations have no clinical consequences, but some affect critical properties such as solubility, oxygen affinity, or oxidation rate. Fewer a chain than b chain variants have been found, most likely because a chain mutations would affect fetal hemoglobin as well as adult hemoglobin (see Figure 1). While most of these variants are the result of point mutation, other genetic mechanisms, such as deletions, insertions, additions, and unequal crossing-over have occurred as well. In addition to these structural mutations, the regulation of the quantities of the various gene products is subject to genetic control as well. Altered regulation of a or b chain expression occurs in the thalassemias. The hemoglobin variant with the greatest clinical impact S is hemoglobin S (aA 2 b2). In this mutation, the normal glutamic acid at b6 is replaced by valine, giving that region of the molecule a slightly more hydrophobic environment such that polymers with adjacent molecules can form when the molecules are in the deoxygenated conformation. The polymers become extensive, eventually deforming the red blood cell, causing them to occlude small vessels. This leads to lack of oxygenation of the tissue and, if not reversed, death of the tissue. The clinical entity caused by this process is called sickle cell anemia, and is characterized by marked anemia and intermittent episodes of severe pain. The discovery of the hemoglobin S mutation was the first instance of a genetic disease caused by a point mutation.

Heme – an iron-containing tetrapyrrole prosthetic group that binds one molecule of oxygen

See also: Oxygen–Hemoglobin Dissociation Curve. Systemic Disease: Sickle Cell Disease.

Further Reading Bolton W and Perutz M (1970) Three dimensional Fourier synthesis of horse deoxyhemoglobin at 2 Angstrom units resolution. Nature 228: 551–552. Bunn HF and Forget BG 2005 In: Dyson J (ed.) Hemoglobin: Molecular, Genetic and Clinical Aspects. Philadelphia: Saunders. Ingram VM (1957) Gene mutations in human haemoglobin: the chemical difference between normal and sickle cell haemoglobin. Nature 180: 326–328. Perutz M (1989) Myoglobin and haemoglobin: role of distal residues in reactions with haem ligands. Trends in Biochemical Sciences 14: 42–44.

Hemoglobinopathy – a clinical disease caused by any mutation either of a structural hemoglobin peptide subunit or of an imbalance in their synthesis Point mutation – substitution of one amino acid for another Polypeptide chain – a protein subunit consisting of a series of amino acids Primary structure – amino acid sequence Prosthetic group – a nonprotein group, contained within a protein; heme is a prosthetic group Quaternary structure – arrangement of protein subunits into a fully functional molecule R state – the high oxygen affinity hemoglobin conformation Secondary structure – arrangement of amino acids into helical segments Sickle cell anemia – a genetic disease caused by mutation of glutamic acid to valine at position 6 of the b chain Tertiary structure – arrangement of helical segments into a three-dimensional protein structure Thalassemia – a genetic disease in which the synthesis of polypeptide chains of hemoglobin is unbalanced, leading to hemolysis and anemia T state – the low oxygen affinity hemoglobin conformation a chain – one of the peptide subunits of adult human hemoglobin (A) a helix – a helical sequence of amino acids in a peptide chain b chain – one of the peptide subunits of adult human hemoglobin (A) g chain – one of the peptide subunits of the human fetal hemoglobin (F) d chain – one of the peptide subunits of the human adult minor hemoglobin (A2)

Glossary

e chain – one of the peptide subunits of the human fetal hemoglobin (Gower 1 and 2)

Cooperativity – the increase of O2 affinity with successive ligation of O2 molecules

z chain – one of the peptide subunits of the human fetal hemoglobin (Portland 1 and 2)

268 HEPATOCYTE GROWTH (SCATTER) FACTOR

Hemorrhage

see Alveolar Hemorrhage.

Hemothorax

see Pleural Effusions: Hemothorax.

HEPATOCYTE GROWTH (SCATTER) FACTOR S E Mutsaers, The University of Western Australia, Nedlands, WA, Australia S E Herrick, University of Manchester, Manchester, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Hepatocyte growth factor (HGF), also known as scatter factor and tumor cytotoxic factor, is a multifunctional polypeptide secreted as an inactive single-chain propeptide which is cleaved extracellularly by serine proteases to give an active heterodimer consisting of an a- and b-chain. The a-chain contains an N-terminal hairpin loop and four kringle domains, whereas the b-chain resembles the catalytic domain of serine proteases but does not have enzymatic activity. Cells of mesenchymal origin are the primary producers of HGF, whereas epithelial and endothelial cells are the main responding cell types. HGF acts through the cell-surface receptor, c-met, to cause proliferation and migration, induce tubulogenesis, inhibit apoptosis, and promote invasiveness. During development, HGF plays a major role in epithelial– mesenchymal interactions essential to organogenesis especially of the lung, heart, liver, and kidney. In the adult, HGF maintains normal tissue function and injury leads to increased systemic HGF levels and tissue mRNA expression with associated tissue regeneration. Studies in experimental models suggest that administration of HGF may provide therapeutic benefit for conditions including pulmonary fibrosis, emphysema, asthma, and pulmonary hypertension. However, excessive HGF activity or cmet expression is associated with tumor growth and metastasis and c-met mutations have been detected in a variety of human cancers including mesothelioma and non-small cell lung carcinomas, and may contribute to the invasive growth of lung cancer.

Introduction Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional polypeptide that has been implicated in embryo development, tissue repair, and cancer growth. HGF and SF were independently isolated based on two different biological activities. In the mid-1980s, HGF was initially isolated from rat platelets as a factor that stimulated DNA synthesis in primary cultures of rat hepatocytes, in human plasma from patients with fulminant hepatic failure and in rabbit serum. Around the same time, humanembryo-fibroblast conditioned medium was found to contain a factor which induced scattering of tightly packed colonies of epithelial cells and so was named

SF. Subsequently, molecular cloning and physiological activity demonstrated that HGF and SF were identical. Another factor found to be identical to human placental HGF is tumor cytotoxic factor, initially purified from the conditioned medium of human embryonic lung fibroblasts, IMR-90 cells. HGF/ SF forms part of a family of plasminogen-related growth factors along with HGF-like/macrophage stimulating protein (an effector of macrophage chemotaxis and phagocytosis), plasminogen (a proenzyme of the serine protease family), and apolipoprotein (a family of glycoproteins associated with lipid transport), as they all are thought to have evolved from the same ancestral gene. HGF is produced mainly by mesenchymal cells with activity mediated through the c-met receptor found principally on epithelial and endothelial cells.

Structure The gene-encoding HGF maps on chromosome 7q21.1 in humans and is composed of 18 exons interrupted by 17 introns spanning approximately 70 kb. In the mouse, the HGF gene maps on chromosome 5; however, the primary structure of HGF is 490% homologous in humans and rodents. The signal peptide involved with intracellular processing and secretion and the 50 -untranslated region are contained in the first exon, whereas the next five pairs of exons correspond to the amino-terminal and four successive kringle domains, respectively. The 12th exon encodes the region that links the heavy and light chain, and the remaining six exons specify the serineprotease domain. The promoter sequence of the HGF gene contains a number of putative regulatory elements which include a transforming growth factor beta (TGF-b) inhibitory element, interleukin (IL)-6, cAMP, oestrogen and vitamin D responsive elements, and potential binding sites for tumor necrosis factor alpha, IL-6, IL-1, and liver-specific, B-cell-specific and macrophage-specific transcription factors. HGF is derived from a biologically inactive singlechain precursor (pro-HGF) of 728 amino acids (Figure 1). Amino acid sequence is highly homogeneous with plasminogen. The mature active form is

HEPATOCYTE GROWTH (SCATTER) FACTOR 269 Kringle 2 Pro-HGF

Kringle 1

Kringle 3 -Chain Kringle 4

Processing Arg-Val

Hairpin loop
-Chain

Processing site

Mature HGF

Figure 1 Structure of the mature active HGF molecule following processing from its biologically inactive single-chain precursor, proHGF. The mature HGF consists of a heavy a-chain and a light b-chain held together by a disulfide bond. The a-chain contains an N-terminal hairpin loop and four kringle domains, whereas the b-chain resembles the catalystic domain of serine proteases but does not have enzymatic activity. Adapted from Funakoshi H and Nakamura T (2003) Hepatocyte growth factor: from diagnosis to clinical applications. Clinica Chimica Acta 327(1–2): 1–23, with permission from Elsevier.

a heparin-binding, heterodimeric glycoprotein consisting of a heavy 69 kDa a-chain and a light 34 kDa b-chain held together by a disulfide bond. The heavy a-chain contains an N-terminal hairpin loop, homologous to the plasminogen activation peptide and four kringle domains involved in protein–protein interactions, whereas the b-chain resembles the catalytic domain of serine proteases but does not have enzymatic activity. The first and second kringle domains are particularly necessary for the correct biological function of the molecule. Naturally occurring variants of HGF have been identified resulting from alternative RNA splicing. Natural variants, NK1 and NK2, which contain the N-terminal hairpin loop and either the first or the first two kringle domains, respectively, retain the ability to interact with the c-met receptor. However, although NK1 demonstrates the full spectrum of HGF responses when expressed as a transgene in vivo, NK2 antagonizes the pathological consequences of HGF overexpression. A third natural variant, an isoform of 723 amino acids (dHGF) with a deletion of five residues located in the first kringle, retains all the biological activity of fulllength HGF.

Regulation of Production and Activity A variety of cells including platelets, hepatocytes, monocytes, fibroblasts, and certain tumor cells produce pro-HGF following stimulation by inflammatory mediators such as IL-1b and prostaglandin E2 but also branched amino acids, heparin, basic fibroblast growth factor (bFGF), epidermal growth factor, and platelet-derived growth factor. Following secretion of pro-HGF from the cell, it becomes bound to the cell surface or to extracellular matrix (ECM) components, in particular heparan sulfate and dermatan sulfate containing proteoglycans. The active disulfide-linked heterodimer is formed by proteolytic cleavage of inactive single-chain pro-HGF between arginine and valine residues, at position 494 and

495, respectively. HGF activation is induced by a number of factors including urokinase plasminogen activator (uPA), tissue plasminogen activator, HGFactivator (HGF-A), matriptase, kallikrein, and blood coagulation factor XIIa. HGF induces the expression of uPA and so participates in a positive feedback loop. Activation of HGF is a finely regulated process, given that some of these molecules require activation from inactive zymogens and are also under inhibitory control. For example, HGF-A, the most powerful activator, is also secreted as a proenzyme, which requires activation by serine proteases such as thrombin. Furthermore, the identification of endogenous HGF-A inhibitors (HAIs) has provided further information about the regulation of HGF-A activity. Currently, two types of HAIs, namely HAI-1 and HAI-2, have been found. Active HGF mainly exerts its actions on epithelial cells through the c-met receptor; however, non-epithelial cells such as hematopoietic, endothelial, lymphoid, neural, muscle, and mesothelial cells may also respond.

Biological Function HGF regulates a wide range of cellular processes such as cell survival, proliferation, migration, and differentiation (Figure 2). It acts as an endocrine, paracrine, and autocrine factor and can be detected in sera, bronchoalveolar lavage, cerebrospinal and synovial fluids, although levels change with age and during pregnancy. Mice genetically deficient in HGF are embryonic lethal with severe placental insufficiency, reduction of sensory nerves, and developmental defects in the liver and muscle. Transgenic mice that overexpress HGF develop diverse tumors, polycystic kidney disease, and inflammatory bowel disease. In development, HGF is involved in the formation of multiple tissue/organ systems such as the lung, placenta, tooth, kidney, liver, heart, pancreas, prostate, mammary gland, ovary, testes, muscle, and

270 HEPATOCYTE GROWTH (SCATTER) FACTOR

Kringle 2 Kringle 1 Hairpin loop

Kringle 3 -chain Kringle 4

HGF HGF


-chain

c-Met

HGF

Plasma membrane

PI3-K Ras SOS

P

S

S

P

P

Y

Y

P

P

Y

Y

P

p85 P

Y

Y

P

P

Y

Y

P

PH P

PLC-

Ga

b1

P

Grb2

Grb2

P

Bag-1

PI3-K p85

STAT

P

Rap1

Crk

Shp2 MAPK

Antiapoptosis

Mitogenesis Angiogenesis

Morphogenesis

Motogenesis

Neurite extension

Figure 2 Biological activities elicited by HGF mediated through intracellular signaling of the c-met receptor. Adapted from Funakoshi H and Nakamura T (2003) Hepatocyte growth factor: from diagnosis to clinical applications. Clinica Chimica Acta 327(1–2): 1–23, with permission from Elsevier.

neuronal tissue. During organogenesis, HGF plays a key role in epithelial–mesenchymal transition. In situ hybridization analysis has demonstrated HGF and c-met gene expression in mesenchyme and epithelium, respectively, of the developing lung. Furthermore, HGF has been shown to be a mesenchyme-derived morphogen-regulating lung morphogenesis by mediating epithelial branching in collaboration with FGF family members. HGF is also involved in regulating bone remodeling where HGF is secreted by osteoclasts and activates its receptor on both osteoclasts and osteoblasts. In the brain, HGF is a new member of the family of neurotrophic factors. In lung and breast cancer, HGF concentration correlates with disease relapse and reduced overall survival suggesting that HGF may promote tumor progression. In the lung, squamous cell carcinoma cells over expressing c-met show increased tumorigenesis and metastases in response to HGF in vivo. HGF’s ability to upregulate uPA and its receptor uPAR may increase the invasive potential of tumor cells. However, HGF has also been shown to have cytotoxic activity on certain tumor cells implying HGF inhibits some cancer growth. It is likely that the level of c-met signaling is key to the control of carcinogenesis.

HGF plays a key role in the proper maintainance and repair of adult tissue including the lung. It is a potent mitogen for epithelial and hematopoietic cells and chrondrocytes and is involved in tubule formation of many epithelial cell lines including those of the lung, mammary gland, kidney, pancreas, and liver. Following cutaneous wounding, overexpression of HGF induces keratinocyte proliferation and enhanced expression of matrix metalloproteinases leading to increased re-epithelialization, and through upregulating vascular endothelial growth factor expression, increased blood vessel formation, and granulation tissue expansion. In contrast, neutralization of HGF leads to retarded wound healing associated with reduced vascularization, granulation tissue formation, and re-epithelialization. Administration of antibodies to HGF in normal mice leads to the onset and progression of tissue fibrosis, and renal dysfunction again confirming one of the key activities of HGF is the preservation of normal tissue structure and function. Prevention of fibrosis and normal regeneration of lung, liver, heart, and kidney has been associated with the antiapoptotic activity of HGF and the protection of epithelial cells against DNA damaging agents. In addition, HGF prevents the initiation and progression of chronic fibrosis

HEPATOCYTE GROWTH (SCATTER) FACTOR 271

by directly antagonizing the profibrotic actions of TGF-b1 possibly through inhibiting myofibroblast activation.

HGF Receptor The HGF receptor is the Met protein product of the c-met proto-oncogene (p190 met). c-Met consists of a transmembrane 145 kDa b-chain and an extracellular 50 kDa a-chain to form a dimeric 190 kDa protein with structural features of a tyrosine kinase receptor (Figure 2). The a-chain is heavily glycosylated and is disulfide-linked to the b-chain. The latter contains the kinase domain, the tyrosine autophosphorylation sites, and the multifunctional docking sites that comprise a specific stretch of amino acids located in the carboxy-terminal part of the protein. HGF also binds to cell-surface heparan sulfate proteoglycans that serve as low-affinity HGF receptors and modulate the interaction between HGF and c-met receptor. The various biological functions of HGF on target cells must be attributable to different intracellular signal transduction pathways as only one receptor has so far been identified. HGF binding, in the presence of ATP and Mg2 þ , triggers receptor dimerization and autophosphorylation at a conserved two-tyrosine motif within the receptor docking site resulting in the recruitment of intracellular signaling molecules containing the src homology (SH) domain. c-Met signals in the large part via the Ras signaling pathway after binding the docking protein Grb2. Data have shown that activation of the Ras pathway leads to cell proliferation, whereas the PI3 kinase pathway is needed to induce motogenesis. Both pathways are essential for invasive growth. Dysregulation of c-met signaling is observed in carcinogenesis. Point mutations in c-met detected in the catalytic domain of the receptor have been associated with carcinomas, and overexpression of c-met gene in transgenic mice leads to carcinomas of the thyroid gland, breast, liver, pancreas, and ovary. Increased expression of c-met is associated with a poor prognosis.

Hepatocyte Growth (Scatter) Factor in Respiratory Diseases HGF acts as a lung-regenerating factor after injury as it stimulates proliferation of alveolar type II epithelial cells, thus contributing to regeneration of alveolar structures. Hence, HGF levels are often elevated in disease conditions such as in bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis, sarcoidosis, and the interstitial lung disease associated with rheumatoid arthritis. However,

excessive levels of activated HGF are reported in mesotheliomas and non-small cell lung carcinomas and may contribute to the invasive growth of lung cancer. It was reported that HGF mRNA and HGF activity increased in whole lung 3–6 h after intratracheal administration of hydrochloric acid, and an increase in whole lung HGF expression has been reported in a rat model of ischemia-reperfusion. HGF is also upregulated following bleomycin administration in mice. However, if levels are further increased through administering HGF systemically or intratracheally, the fibrosis is attenuated. In contrast, administration of neutralizing antibodies to HGF markedly increased collagen accumulation in the lungs of bleomycin-injured mice. Administration of HGF to the airways of mice also reduced airway inflammation, hyperresponsiveness, and remodeling following sensitization and challenge with ovalbumin. Recent studies have demonstrated a potential therapeutic application for HGF in emphysema. Human fibroblasts from patients with emphysema produce less HGF than from normal patients. When overexpressed in an animal model of emphysema, HGF improved pulmonary function by inhibiting alveolar cell apoptosis, enhancing alveolar regeneration, and promoting angiogenesis. HGF also promoted compensatory growth in remnant lung tissue of emphysamateous rats following lung volume reduction surgery. The major sources of HGF are thought to be macrophages and interstitial fibroblasts and the cellular target, alveolar epithelial cells. Evidence suggests that the increase in HGF following lung injury is controlled by the plasminogen activation system, primarily through plasmin-mediated breakdown of the ECM releasing proteoglycanbound HGF into the alveolar space. Interestingly, the lung may also be a source of HGF after injury to other organs. For instance, after partial hepatectomy, unilateral nephrectomy, or induction of hepatitis in rats, HGF mRNA in the intact lung increased at 6 h. These findings suggest that the lung may contribute to organ repair and regeneration in an endocrine fashion through production of circulating HGF. The factors that stimulate upregulation of HGF expression following lung or other organ injury have not been fully elucidated. There is a vast amount of evidence that shows raised HGF levels in lung disease but no studies exploring the therapeutic value of HGF in human clinical conditions. In vivo gene transfection with HGF gene attenuated the medial hypertrophy of pulmonary arteries and enhanced the ameliorating effect of prostacyclin for pulmonary hypertension in monocrotoline-treated rats. Thus, gene therapy with HGF and prostaglandin I2 (prostacyclin) synthase may be a

272 HIGH ALTITUDE, PHYSIOLOGY AND DISEASES

promising strategy for severe pulmonary hypertension. In addition, administration of HGF might exhibit a potent function in vivo for protection and improvement of acute and chronic lung injuries induced by inflammation and/or oxidative stress. Overall, there is tremendous potential for HGF to be used as a novel therapeutic agent in combating various inflammatory and fibrotic disorders including those of the lung.

Conclusion HGF has trophic, mitogenic, regenerative, and therapeutic effects on lung, kidney, pancreas, spleen, liver, heart, and spinal cord. While HGF was originally identified as a potent mitogen for mature hepatocytes, the biological functions of this factor reach far beyond its original identification. Due to its wealth of regeneration potential, use of HGF for purposes of therapeutics is being given increasing attention. See also: Adhesion, Cell–Cell: Vascular. Angiogenesis, Angiogenic Growth Factors and Development Factors. Apoptosis. Chemokines. Chronic Obstructive Pulmonary Disease: Emphysema, General. Coagulation Cascade: iuPA, tPA, uPAR. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Fibrinolysis: Plasminogen Activator and Plasmin. Fibroblasts. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Pulmonary Macrophages. Lung Development: Overview. Mesothelial Cells. Mesothelioma, Malignant. Oncogenes and Proto-Oncogenes: Overview. Platelet-Derived Growth Factor. Platelets. Pleural Effusions: Overview. Pulmonary Fibrosis. Serine Proteinases. Signal Transduction. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Transgenic Models. Tumors, Malignant: Overview; Metastases from Lung Cancer. Vascular Disease.

Further Reading Dohi M, Hasegawa T, Yamamoto K, and Marshall BC (2000) Hepatocyte growth factor attenuates collagen accumulation in a murine model of pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine 162(6): 2302–2307.

Funakoshi H and Nakamura T (2003) Hepatocyte growth factor: from diagnosis to clinical applications. Clinica Chimica Acta 327(1–2): 1–23. Hattori N, Mizuno S, Yoshida Y, et al. (2004) The plasminogen activation system reduces fibrosis in the lung by a hepatocyte growth factor-dependent mechanism. American Journal of Pathology 164(3): 1091–1098. Higashio K, Shima N, Goto M, et al. (1990) Identity of a tumor cytotoxic factor from human fibroblasts and hepatocyte growth factor. Biochemical and Biophysical Research Communications 170(1): 397–404. Jiang W, Hiscox S, Matsumoto K, and Nakamura T (1999) Hepatocyte growth factor/scatter factor, its molecular, cellular and clinical implications in cancer. Critical Reviews in Oncology/ Hematology 29(3): 209–248. Ma PC, Maulik G, Christensen J, and Salgia R (2003) c-Met: structure, functions and potential for therapeutic inhibition. Cancer and Metastasis Reviews 22(4): 309–325. Matsumoto K and Nakamura T (1997) Hepatocyte growth factor (HGF) as a tissue organizer for organogenesis and regeneration. Biochemical and Biophysical Research Communications 239(3): 639–644. Ono M, Sawa Y, Mizuno S, et al. (2004) Hepatocyte growth factor suppresses vascular medial hyperplasia and matrix accumulation in advanced pulmonary hypertension of rats. Circulation 110(18): 2896–2902. Rubin JS, Bottaro DP, and Aaronson SA (1993) Hepatocyte growth factor/scatter factor and its receptor, the c-met protooncogene product. Biochimica et Biophysica Acta 1155(3): 357–371. Stella MC and Comoglio PM (1999) HGF: a multifunctional growth factor controlling cell scattering. The International Journal of Biochemistry & Cell Biology 31(12): 1357–1362. Stoker M and Perryman M (1985) An epithelial scatter factor released by embryo fibroblasts. Journal of Cell Science 77: 209–223. Stuart KA, Riordan SM, Lidder S, et al. (2000) Hepatocyte growth factor/scatter factor-induced intracellular signalling. International Journal of Experimental Pathology 81(1): 17–30. Ware LB and Matthay MA (2002) Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair. American Journal of Physiology. Lung Cell Molecular Physiology 282(5): L924–L940. Yanagita K, Matsumoto K, Sekiguchi K, et al. (1993) Hepatocyte growth factor may act as a pulmotrophic factor on lung regeneration after acute lung injury. Journal of Biological Chemistry 268(28): 21212–21217. Yanagita K, Nagaike M, Ishibashi H, et al. (1992) Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs. Biochemical and Biophysical Research Communications 182(2): 802–809.

HIGH ALTITUDE, PHYSIOLOGY AND DISEASES J B West, University of California – San Diego, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Exposure to high altitude causes generalized hypoxia throughout the body because the reduction in barometric pressure

reduces the inspired partial pressure of oxygen. Physiological consequences include impairment of physical performance, mental performance, and sleep. The deleterious effects of high altitude are reduced by the process of acclimatization, the most important feature of which is an increase in ventilation. Other features include polycythemia, changes in oxidative enzymes in cells, and reduced intercapillary distances in some tissues. However acclimatization does not return the body to its sea level status. Three principal high-altitude diseases affect people who

HIGH ALTITUDE, PHYSIOLOGY AND DISEASES 273

Physiology of High Altitude What is Meant by High Altitude?

In this context, there is no precise definition of high altitude but, in general, it refers to altitudes that affect the human body. At altitudes of 1500–2500 m, physiological changes such as increased ventilation are seen. From 2500 to 3500 m, high-altitude illness is common if ascent is rapid. At higher altitudes of 3500–5500 m, altitude illnesses are frequently seen but long periods of altitude exposure are possible. Above that, progressive deterioration is inevitable. A surprising number of people live, work, or play at high altitude. For example, 140 million people reside at altitudes over 2500 m mainly in the Americas, Asia, and eastern Africa. Some people work at very high altitudes, e.g., in mines at over 4500 m and in observatories at over 5000 m. Mountaineers ascend to altitudes of over 8000 m. All of these groups are prone to high-altitude diseases with occasional fatal outcomes. Barometric Pressures at High Altitude

As the altitude increases, the barometric pressure falls because of the smaller column of air above. Figure 1 shows the relationship between altitude and barometric pressure in those regions of the world where people normally go to high altitude. At an altitude of 3000 m, which commonly occurs in ski resorts, the barometric pressure and inspired PO2 are only about 70% of the sea level value. The highest altitude at which humans permanently reside is about 5000 m, where the inspired PO2 is only onehalf of the sea level value. The highest point in the world, Mt Everest (altitude 8848 m), has a barometric pressure only one-third of the sea level value. The barometric pressures shown in Figure 1 are higher than those of the standard atmosphere, which is used by the aeronautical industry and included in some medical textbooks. The reason is that regions of the world with high mountains such as the Andes

800

Altitude (ft) 20 000 10 000 Sea level Denver

150

600 Commercial aircraft cabin

100

Pikes peak

400 Highest human habitation 200

50 Mt Everest

Inspired PO2 (mmHg)

0 Barometric pressure (mmHg)

normally live near sea level. Acute mountain sickness causes headache, fatigue, insomnia, and anorexia but is generally a selflimiting disease that resolves after 2–3 days. High-altitude pulmonary edema is a much more serious condition characterized by severe dyspnea, which may progress to coughing up frothy fluid. The pulmonary hypertension that occurs at high altitude because of the alveolar hypoxia damages some pulmonary capillaries, a condition known as stress failure. High-altitude cerebral edema is uncommon but potentially fatal and causes confusion, ataxia, and eventually coma. Permanent residents of high altitude sometimes develop chronic mountain sickness characterized by severe polycythemia and a constellation of neurological symptoms.

0 0

0

2000

4000 6000 Altitude (m)

8000

Figure 1 Relationship between altitude and barometric pressure. Note that at 1520 m (5000 ft) in Denver, CO, the PO2 of moist inspired gas is about 130 mmHg, while on the summit of Mt Everest it is only 43 mmHg. Reproduced from West JB, Respiratory Physiology – The Essentials, 7th edn., Lippincott Williams & Wilkins, 2005, with permission from Lippincott Williams & Wilkins.

and Himalayas are at low latitudes where the barometric pressure for a given altitude tends to be relatively high. Physiological Effects of High Altitude

The hypoxia of high altitude affects almost all the organ systems in the body. However, in the present context the most important changes are in physical performance, mental performance, and sleep. Physical performance as measured by maximal oxygen consumption falls as the inspired PO2 is lowered. At an altitude of 3000 m, the maximal oxygen consumption is reduced to about 85% of the sea level value and at an altitude of 5000 m, it is down to about 60%. On the summit of Mt Everest, the maximal oxygen consumption is only about 20% of the sea level value. These substantial reductions in physical performance are accompanied by a greatly increased sense of fatigue. The impaired physical performance is presumably mainly caused by the reduced availability of oxygen to mitochondria, which interferes with the normal function of the electron transport chain. However, some investigators believe that central inhibition from the brain may play a part in limiting exercise capacity. Mental performance is impaired at high altitude. For example, visual perception is altered and night vision is reduced at altitudes as low as 2000 m. People working at an altitude of 4000 m have greater mental fatigue, reduced attention span, and make an unusually large number of arithmetical errors. Mountaineers at altitudes over 6000 m show impairment of short-term memory and manipulative skills, and it is

274 HIGH ALTITUDE, PHYSIOLOGY AND DISEASES

interesting that some residual impairment of these functions can be demonstrated after return to sea level when compared with the pre-exposure values. Sleep is poor at high altitude and a common complaint is that people do not feel refreshed in the morning. Periodic breathing is almost universal above an altitude of 4000 m and the periods of apnea may result in arousals. Acclimatization to High Altitude

When lowlanders (that is, people who normally live near sea level) go to high altitude, a series of adaptive changes occur, collectively known as acclimatization. The most important of these is an increase in pulmonary ventilation, but other changes also occur including polycythemia, increase in the density of capillaries in peripheral tissues, and changes in oxidative enzymes inside cells. Hyperventilation is the most important feature of acclimatization to high altitude. The mechanism is stimulation of the peripheral chemoreceptors by the low PO2 in the arterial blood. There is a prompt increase in ventilation on ascent to high altitude but the ventilation continues to rise over several days apparently because of increased sensitivity of the peripheral chemoreceptors to the low PO2. The extent of the hyperventilation at extreme altitude is remarkable. For example, on the summit of Mt Everest the alveolar ventilation is increased about fivefold, which drives the alveolar PCO2 down from its normal value of about 40 mmHg to below 10 mmHg. Polycythemia is often thought to be an important feature of acclimatization but for visits of a week or so to high altitude it plays almost no role because of the delay in the production of new red cells. However, the hematocrit often increases within a day or so of going to high altitude because of a reduced plasma volume. This is partly caused by dehydration and partly by hormonal changes regulating plasma volume. Longer-term stays at high altitude are accompanied by polycythemia which increases the oxygen carrying capacity of the blood. However, residence at very high altitudes may cause such high hematocrits (over 70%) that the accompanying viscosity of the blood actually reduces exercise performance. Some misconceptions about acclimatization have developed, particularly among nonmedical people. Although acclimatization greatly reduces the incidence of high-altitude diseases, it does not return the body to its sea level status. In other words, even in the well-acclimatized person at high altitude, the hypoxia impairs physical and mental performance, and sleep. As an example, astronomers working on

the summit of Mauna Kea, Hawaii (altitude 4200 m) have such a low alveolar and arterial PO2 even when fully acclimatized that if this were caused by chronic obstructive pulmonary disease at sea level, they would be entitled to continuous oxygen therapy under Medicare. This emphasizes the great hypoxic stress of these altitudes. A recent technical advance has improved the quality of life and efficiency of people who are required to work at high altitude. If the oxygen concentration of a room is raised from its normal value of 21% to 25% or higher, the deleterious effects of high-altitude hypoxia can be mitigated. A radiotelescope operated by the California Institute of Technology at an altitude of 5050 m in north Chile has rooms where the oxygen concentration is maintained at 27%. In effect, this reduces the equivalent altitude (based on the level of the inspired PO2 ) to about 3200 m, which is easily tolerated. The oxygen is produced from air by electrically powered concentrators that inject the oxygen into the ventilation of the room. The result has been a tremendous increase in productivity and quality of life for people who must work at these great altitudes. The same technique has been used in a mine in north Chile where the dormitories are at an altitude of 3800 m. Miners who have difficulties with sleeping have much improved nights if they sleep in an oxygen-enriched room. Physiological Changes at Extreme Altitudes

Mountaineers who ascend to extremely high altitudes such as those near the summit of Mt Everest undergo extraordinary physiological changes. There is an enormous increase in alveolar ventilation, which, on the Everest summit, drives the alveolar PCO2 down to about 7–8 mmHg. This very high ventilation maintains the alveolar PO2 at about 35 mmHg, which is just sufficient to maintain life. The very low PCO2 is accompanied by an extreme respiratory alkalosis with an arterial pH (based on the measured alveolar PCO2 and base excess of the blood) of over 7.7. Even so, the maximal oxygen consumption is only about 1 l min 1, a miserably low value but just sufficient to allow a climber to reach the summit. It is a remarkable coincidence that a climber at the highest point on Earth appears to be so close to the limit of tolerance to oxygen deprivation.

Diseases of High Altitude There are three major diseases affecting lowlanders who go to high altitude: acute mountain sickness, high-altitude pulmonary edema, and high-altitude cerebral edema. In addition, there are several other

HIGH ALTITUDE, PHYSIOLOGY AND DISEASES 275

pathological conditions such as high-altitude retinal hemorrhage. In special situations, lowlanders may develop a condition known as subacute mountain sickness. Permanent residents of high altitude, especially in the Andes, sometimes develop chronic mountain sickness. Acute Mountain Sickness

This condition is common in people who ascend from near sea level to altitudes of 3000 m and above. However, it occasionally occurs at altitudes as low as 2000 m. The most important features are headache, lightheadedness, breathlessness, fatigue, insomnia, anorexia, and occasionally nausea. Commonly, the symptoms begin 2–3 h after ascent and often the first night at high altitude is particularly unpleasant. However, the condition is usually self-limiting and most of the symptoms disappear after 2–3 days, although the insomnia may persist. Acute mountain sickness responds well to descent. The best way to reduce the incidence of acute mountain sickness is by gradual ascent, which allows time for acclimatization. Handbooks for trekkers often advise that above an altitude of 3000 m the ascent rate should be no more than 300 m per day with a rest day every 2–3 days. However, this is rather conservative and many people are able to ascend more rapidly. An interesting feature of acute mountain sickness is that even a brief recent exposure to high altitude reduces the incidence of the disease. The cause of acute mountain sickness is poorly understood. Of course hypoxia is a major factor because this is the only unavoidable physiological stress at high altitude. Other factors such as cold, high winds, and intense solar radiation can be removed by appropriate protection. Some people think that the respiratory alkalosis that occurs as a result of the hyperventilation may be partly responsible. This would fit with the time course because the alkalosis also becomes less evident after 2–3 days as a result of renal compensation. There is some evidence that mild cerebral edema plays a role. In fact, there is a continuum between severe acute mountain sickness and high-altitude cerebral edema, which is discussed below. The cause of the cerebral edema is not fully understood but contributing factors may be an increased cerebral blood flow or increased permeability of the cerebral capillaries. There is some evidence of slight brain swelling and since the cranium is essentially rigid, even a small increase in brain tissue volume may cause a large rise in pressure. Note that at high altitude there are two opposing influences on cerebral blood flow. The arterial hypoxemia causes

cerebral vasodilatation whereas the low PCO2 and alkalosis result in cerebral vasoconstriction. The incidence of acute mountain sickness can be reduced by taking the carbonic anhydrase inhibitor acetazolamide. This is not necessary if one ascends slowly but, for example, a rapid ascent to high altitude as in flying from Lima to Cuzco in Peru, or into La Paz, Bolivia inevitably means an abrupt exposure to high altitude. The dose of acetazolamide is 250 mg once or twice a day. Some people recommend 125 mg at night to reduce the incidence of periodic breathing and thus improve sleep. The mechanism of action of acetazolamide is probably the metabolic acidosis, which stimulates ventilation. Side effects to the drug are common including paresthesia of fingers and toes, diuresis, and an unpleasant taste to carbonated drinks. In addition, acetazolamide is a sulfonamide drug and therefore some people have a hypersensitivity to it. Dexamethasone has also been used to reduce the incidence of acute mountain sickness, the recommended dosage being 2 mg every 6–8 h. Some studies have shown that gingko biloba reduces the incidence of acute mountain sickness but additional work is necessary to establish its effectiveness. At the present time, it does not seem to be as effective as acetazolamide. Acute mountain sickness usually does not need treatment although the headache may respond to aspirin, acetaminophen, or ibuprofen. If the condition is severe, oxygen can be administered. Acetazolamide 250 mg three times a day or dexamethasone 4 mg four times a day are also useful for severe mountain sickness. The best treatment is descent, which generally causes a rapid improvement. High-Altitude Pulmonary Edema

Whereas acute mountain sickness is usually self-limiting and a minor nuisance, high-altitude pulmonary edema is a serious condition that is occasionally fatal. The incidence depends very much on the ascent rate. Commonly, the incidence is about 1–2% but in reports of people ascending rapidly to 4500 m, the incidence was as high as 10%. The condition typically occurs about 1–2 days after reaching high altitude and an interesting feature is that if it does not occur within a week it is unlikely to occur at all unless the subject ascends even higher. One form of high-altitude pulmonary edema is seen in high-altitude residents who travel to low altitude for a few days and then return to high altitude. This is called reascent high-altitude pulmonary edema. It is not easy to predict who is at risk from highaltitude pulmonary edema but people who have developed it on one occasion are more likely to do so

276 HIGH ALTITUDE, PHYSIOLOGY AND DISEASES

on a subsequent ascent. There is some evidence that an upper respiratory tract infection may increase the likelihood, particularly in children. High-altitude pulmonary edema may be preceded by acute mountain sickness but this is certainly not always the case and failure to realize this has resulted in some missed diagnoses. A major symptom of high-altitude pulmonary edema is dyspnea with orthopnea being common. Exercise tolerance is reduced. Frequently, there is a dry cough initially but this may progress to one that produces frothy pink sputum because of blood staining. On examination tachypnea and tachycardia frequently occur, there may be a mild pyrexia, and crackles can be heard over the lung fields by auscultation. Occasionally, the patient is aware of fluid in his or her lungs because of gurgling sounds. The pathogenesis of high-altitude pulmonary edema has been puzzling in the past and has stimulated much recent research. Left ventricular failure can be ruled out because cardiac catheterization has shown normal wedge pressures. However, there is strong evidence of an association with pulmonary hypertension. This is brought about through vasoconstriction of small pulmonary arteries as a result of the alveolar hypoxia. The evidence for the importance of pulmonary hypertension in the pathogenesis can be summarized as follows. Susceptible individuals tend to have an unusually vigorous hypoxic pulmonary vasoconstrictor response, and also unusually high pulmonary artery pressures have been measured prior to the onset of edema. Cardiac catherization studies in patients with high-altitude pulmonary edema have shown pulmonary artery systolic pressures as high as 144 mmHg with a usual range of 60–80 mmHg. The normal value is in the 20s. Exercise at high altitude is recognized as a provocative factor because this increases pulmonary artery pressure. Patients with a restricted pulmonary vascular bed, for example unilateral absence of a pulmonary artery, are especially at risk. Finally, pulmonary vasodilator drugs such as nifedipine are useful both in the prevention and treatment of the disease. An important finding from bronchoalveolar lavage studies was that the alveolar fluid has a large concentration of high molecular weight proteins and cells, and therefore is of the high-permeability type. After a day or so the edema fluid may contain markers of an inflammatory response but, importantly, this is not seen in the early stages. There are also changes in blood coagulation and platelet activation later in the disease. The absence of an inflammatory response early on implicates a mechanical origin. There is now convincing evidence that the pathogenic mechanism is uneven hypoxic pulmonary vasoconstriction, which results in the high pressure in the

pulmonary arteries being transmitted to some of the capillaries. Direct evidence of uneven hypoxic pulmonary vasoconstriction has recently been reported. It is not particularly surprising that this occurs because it is known that in the adult lung, the distribution of vascular smooth muscle is uneven with some small pulmonary arteries having very little. As a result vasoconstriction cannot occur, or is weak, in these regions. The resulting high pressure in some of the capillaries means that the stresses in the capillary wall become extremely high, and the vessels therefore develop ultrastructural damage. It is not surprising that the stresses are high because the blood–gas barrier in some places is extraordinarily thin, being only 0.2–0.4 mm in thickness. Since the wall stress is inversely proportional to the wall thickness, other factors being equal, it is easy to see how the circumferential or hoop stresses in the capillaries become enormously high. Ultrastructural changes in pulmonary capillaries in animal preparations have been demonstrated when the capillary pressure is raised to high levels, though not as high as apparently can occur in high-altitude pulmonary edema. The ultrastructural changes include disruption of the capillary endothelial layer, alveolar epithelial layer, and, in some cases, all layers of the wall. The result of this ultrastructural damage is that fluid containing high molecular weight proteins and cells leaks out of the capillaries. In addition, exposure of the endothelial basement membranes causes platelet adhesion, which may be responsible for the blood coagulation and inflammatory response that develops later in the disease. The damage to the capillaries is termed stress failure because it results from the very high stresses in their walls. The sequence of events in the pathogenesis of high-altitude pulmonary edema is outlined in Figure 2. As stated above, high-altitude pulmonary edema normally comes on about 2–4 days after ascent, and if it does not occur within the first week it is very unlikely to occur at all. The explanation for this may be remodeling in the arteries in response to the high pulmonary artery pressure. This is well known to occur in animals in experimental conditions where there is alveolar hypoxia. The remodeling causes an increase in vascular smooth muscle in the small pulmonary arteries and this presumably protects the capillaries that would otherwise be at risk. A related explanation may be responsible for the reascent highaltitude pulmonary edema mentioned above. In this case, the abundant smooth muscle in the small pulmonary arteries of high-altitude residents presumably undergoes involution when they go to a lower altitude and this makes them susceptible again to high-altitude pulmonary edema.

HIGH ALTITUDE, PHYSIOLOGY AND DISEASES 277 Alveolar hypoxia Hyperresponsive pulmonary vasculature

Exercise

Descent, oxygen, nifedipine

Hypoxic pulmonary vasoconstriction (uneven)

Restricted vascular bed (unilateral PA)

Increased capillary pressure (some capillaries)

Damage to capillary wall (stress failure)

High permeability edema (patchy distribution)

Exposed basement membranes

Neutrophil activation

Platelet activation

Release of inflammatory markers

Fibrin thrombi

Figure 2 The sequence of events in the pathogenesis of high-altitude pulmonary edema. Reproduced from West JB and MathieuCostello O (1992) High altitude pulmonary edema is caused by stress failure of pulmonary capillaries. International Journal of Sports Medicine 13(supplement 1): S54–S58, with permission.

The pathogenic mechanism outlined above is consistent with the prevention and treatment of the disease. Subjects should ascend slowly to allow some remodeling of the pulmonary circulation to occur. Strenuous exercise, particularly immediately after ascent, is a risk factor because it raises the pulmonary artery pressure and therefore it should be avoided. Pulmonary vasodilator drugs such as the calcium channel blocker nifedipine given prophylactically reduce the incidence of high-altitude pulmonary edema. The best treatment is to remove the patient to a lower altitude as quickly as possible. Recovery is usually rapid if this is done. Of course oxygen should be administered if it is available to relieve the alveolar hypoxia. Nifedipine has been shown to relieve symptoms, a suggested dose being 20 mg of the slowrelease preparation orally every 6–8 h. Other vasodilators such as nitric oxide may be effective but are generally difficult to use in the field. Some recent research suggests that salmeterol and sildenafil may also be helpful. High-Altitude Cerebral Edema

This is an uncommon but potentially fatal condition. It is usually associated with acute mountain sickness and indeed many people think that it is the far end of the spectrum of this condition. The incidence is uncertain but is perhaps as high as 1–2% in people who ascend above 4500 m. Headache is common as part of the acute mountain sickness. However, the onset of high-altitude cerebral edema is indicated by ataxia, mental confusion, and

mood changes. Ultimately coma may supervene, followed by death. Hallucination has been described in some patients. On examination ataxia can be demonstrated, there is often papilledema and occasionally there are focal neurological signs affecting the cranial nerves. Hemiparesis has been described. The pathogenesis of high-altitude cerebral edema is the subject of intense study but is still not clear. Certainly, there is evidence of cerebral edema. Autopsies have shown edema of the brain with swollen flattened gyri, and in a few patients magnetic resonance imaging has revealed intense T2 signals in white matter, particularly in the splenium and corpus callosum. This is consistent with cerebral edema. Why the edema occurs is unclear. Possibly an increased cerebral blood flow plays a role. As indicated earlier, arterial hypoxemia causes cerebral vasodilatation and increased retinal blood flows have been demonstrated at high altitude. It is also possible that there is an increased permeability of the blood–brain barrier. By far the most important treatment is to remove the patient to a lower altitude as quickly as possible. This often results in rapid improvement. Oxygen should be administered if this is available. Dexamethasone is also valuable, the suggested dose being 8 mg initially followed by 4 mg every 6 h. Sometimes, as on an expedition, rapid removal to a lower altitude is not practicable. In this case, portable hyperbaric bags such as the Gamow bag have a place. The patient is placed inside the bag and the pressure is raised with a foot pump. This simulates removal to a lower altitude. The Gamow bag can

278 HIGH ALTITUDE, PHYSIOLOGY AND DISEASES

also be used for treatment of high-altitude pulmonary edema. Chronic Mountain Sickness

This is seen in permanent residents of high altitude, often after many years of residence. The condition is characterized by severe polycythemia with very high hematocrits that may exceed 70%. Values above 80% have even been recorded. The polycythemia is accompanied by cyanosis and a constellation of neurological symptoms including headache, fatigue, somnolence, and mood changes. Treatment is difficult. Patients usually improve if they go down to a lower altitude but often this is not practicable for economic reasons. Therapeutic phlebotomy provides temporary relief but the polycythemia returns. Some success has been obtained with respiratory stimulants such as medroxyprogesterone acetate, the rationale for using these being that some patients develop hypoventilation. An interesting feature of this condition is that while it is relatively common in the Andes at high altitude, it is much rarer in Tibet. This is of great interest to anthropologists some of whom believe that the residents of the Tibetan plateau have progressed further in genetic adaptation to high altitude than the highlanders of the Andes. It is argued that the Andean residents have only been at high altitude for some 10 000 years or so whereas Tibetans have been high-altitude residents for a very much longer period. Subacute Mountain Sickness

This term is confusing and there are currently efforts to replace it with better descriptors. It refers to two separate conditions. One affects young adults who spend long periods of time at extremely high altitudes. The best example has been soldiers in the Indian army who have been posted to altitudes of approximately 6000 m for several months. They develop dyspnea and dependent edema, and this may be associated with a cough and even effort angina. On investigation, there are signs of right heart hypertrophy and the condition is thought to be caused by right heart failure as a result of the severe pulmonary hypertension over a long period. Interestingly, cattle that are grazed at high altitude develop a similar condition known as Brisket disease, so-called because the edema is prominent in the soft tissue of the neck or brisket. The other group of patients to whom the term subacute mountain sickness is given are infants at high altitude who develop severe respiratory distress,

cyanosis, and congestive heart failure. This is particularly evident in Han Chinese infants in Tibet. Interestingly, Tibetan infants rarely develop the condition. Again the pathogenesis is apparently right heart failure as a result of the severe pulmonary hypertension. Retinal Hemorrhage

This condition is common in members of climbing expeditions who go above 5000 m. The flame-shaped hemorrhages can be seen by ophthalmoscopy but usually cause no visual impairment. The ophthalmic appearances become normal when the climbers return to sea level. See also: Acid–Base Balance. Arterial Blood Gases. Diffusion of Gases. Exercise Physiology. Fluid Balance in the Lung. Oxygen–Hemoglobin Dissociation Curve. Peripheral Gas Exchange. Permeability of the Blood–Gas Barrier. Pulmonary Circulation. Vascular Disease. Ventilation: Control.

Further Reading Anand IS, Malhotra RM, Chandrashekhar Y, et al. (1990) Adult subacute mountain sickness – a syndrome of congestive heart failure in man at very high altitude. Lancet 335: 561–565. Hackett PH and Roach RC (2001) Current concepts: high-altitude illness. New England Journal of Medicine 345: 107–114. Hackett PH and Roach RC (2004) High altitude cerebral edema. High Altitude Medicine and Biology 5: 136–146. Hornbein TF and Schoene RB (2001) High Altitude: An Exploration of Human Adaptation. New York: Dekker. Moore LG, Niermeyer S, and Zamudio S (1998) Human adaptation to high altitude: regional and life cycle perspectives. American Journal of Physical Anthropology Yearbook 41: 25–64. Pollard AJ and Murdoch DR (2003) The High Altitude Medicine Handbook, 3rd edn. Oxford, New York: Radcliffe Medical Press. Reeves JT and Leo´n-Velarde F (2004) Chronic mountain sickness: recent studies of the relationship between hemoglobin concentration and oxygen transport. High Altitude Medicine and Biology 5: 147–155. Schoene RB (2004) Unraveling the mechanism of high altitude pulmonary edema. High Altitude Medicine and Biology 5: 125–135. Ward MP, Milledge JS, and West JB (2000) High Altitude Medicine and Physiology, 3rd edn. London: Arnold. West JB (1984) Human physiology at extreme altitudes on Mount Everest. Science 223: 784–788. West JB (2005) Respiratory Physiology – The Essentials, 7th edn. Baltimore: Lippincott Williams & Wilkins. West JB and Mathieu-Costello O (1992) High altitude pulmonary edema is caused by stress failure of pulmonary capillaries. International Journal of Sports Medicine 13(supplement 1): S54–S58.

HISTAMINE 279

HISTAMINE W W Busse and J E Knuffman, University of Wisconsin Medical School, Madison, WI, USA

The Molecule

& 2006 Elsevier Ltd. All rights reserved.

Abstract Histamine is a molecule that has long been recognized as crucial to the allergic response. In this article, the authors discuss the metabolism of histamine, its interaction with cells of the immune system and, briefly, how development of antagonists of the histamine receptor can modulate the molecule’s effector functions. Specific focus is given to the various types of histamine receptors and histamine’s release from the mast cell. Histamine’s role in T-cell function is also addressed. In the upper airway, histamine is a primary mediator of symptoms comprising allergic rhinitis such as nasal and ocular pruritus, sneeze, and nasal congestion. Regarding allergen-induced lower airway symptoms, histamine contributes to bronchoconstriction leading to wheeze and cough.

Introduction Histamine is one of the principle molecules associated with the allergic-response mechanism, and was one of the first products identified as originating from mast cells. First described in the early 1900s by Drs Dale, Laidlaw, and Barger, histamine has been extensively studied, not only for its own effects on the body, but also for the effects of its antagonists on preventing certain responses. As early as the late 1940s, there were 20 formulations available to modulate histamine’s actions. In this chapter, the highlights of histamine that will be covered include a discussion of the molecule and its receptors, release from and interaction with cells of the immune system, as well as a brief discussion of clinically useful antagonists of the histamine receptor.

Histamine (b-imidazolethylamine) comes from the Greek ‘histos’ which means ‘tissue’, derived undoubtedly from the molecule’s presence in a variety of tissue types. Negatively charged side chains of proteoglycans within the cytoplasmic matrix associate with histamine, including heparin and chondroitin 4-sulfate. Histamine is derived from decarboxylation of the amino acid histidine by the enzyme L-histidine decarboxylase (HDC). This decarboxylation occurs primarily in the Golgi apparatus of mast cells and basophils. Histamine is stored in its active form within granules in the cytoplasm of these cells. In humans, the basophil contains most of the histamine content of the body. In addition to mast cells and basophils, dendritic cells and T cells also express HDC, although histamine is not stored in the latter. Histamine is also synthesized in platelets, endothelial cells, enterochromaffin-like cells, and neuronal cells (Figure 1). Histamine reaches peak serum concentrations in about 5 min and returns to baseline values in less than 30 min, giving it a half-life of about 20 s. Mast cells contribute local histamine release in asthma pathophysiology, but account for only about 2% of the histamine found in the circulation, the basophil producing most of the remainder. The metabolism of the histamine molecule is divided into two separate pathways. One path accounts for 70% of the molecule’s metabolism and involves a methylation step utilizing histamine N-methyltransferase. This methylation can be followed by an oxidation step before the final product, N-methylimidazole acetic acid, is excreted via the renal system. The other route of histamine metabolism involves direct oxidation of histamine followed by successive phosphate transfers to arrive at the final metabolite, riboside-N-3imidazole. Less than 5% of histamine is excreted

Histamine

Bronchoconstriction

Wheeze

Chest tightness

Figure 1 Putative effects of histamine.

Increased mucus production

Cough

Mucus plugging

Vasodilatation

Nasal congestion

Itch

Nasal/ocular

Urticaria

280 HISTAMINE

unchanged in the urine, but elevations are more prolonged than that of serum, making measurement of urinary histamine a more useful clinical tool.

Table 1 Histamine receptors Receptor

Chromosome

G protein

Location Nasal mucosa, endothelium, lung, lymphocytes, monocytes, neurons, hepatocytes, gastric mucosa, heart, CNS Gastric mucosa, lung, uterus, heart, CNS Central and peripheral nervous system, perivascular autonomic nerve terminals, GI tract Hematopoietic cells, colon, heart, lung, thymus, spleen, small bowel

HR1

3

Gq/11

HR2

5

Gas

HR3

20

Gi/o

HR4

18

Gi/o

The Histamine Receptors Histamine receptors are 7-transmembrane receptors, which mediate a variety of physiologic responses and are encoded for by up to 1000 genes. At this point in time, there are four histamine receptors (HR1–4) that have been identified, with a fifth less sufficiently characterized, molecularly. All histamine receptors are associated with a G-protein-coupled receptor, a very common target for modern pharmacotherapy. Histamine interacts with peptide stretches located on the third and the fifth transmembrane regions of the receptor. The H1 receptor gene is encoded on chromosome 3 and is involved in several physiologic and pathophysiologic roles, the most notable being that of allergic disease. The basis of this receptor’s function is to mobilize intracellular calcium levels. This calcium release is coupled to phospholipase C (PLC) catalyzing breakdown of membrane phospholipids. Phosphatidyl 4,5-biphosphate (PIP2) is broken down into 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 mediates calcium release which leads to activation of numerous potential second messengers (cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), just to name two) which further promote downstream cellular processes. In the lung, the H1 receptor mediates the contraction of airway smooth muscle and has been implicated in heightened airway hyperresponsiveness. Other effects of the H1 receptor include urticaria and increased vascular permeability which have clinical applications in the treatment of chronic urticaria and allergic rhinitis. The H2 receptor is encoded for on chromosome 5 and relies not only the phosphoinositide metabolic pathway but also the adenylate cyclase system that uses cAMP as a mediator. H2-receptor stimulation leads to gastric acid secretion, inotropic and chronotropic cardiac stimulation, and downregulation of the immune system. Other effects mediated by H1 and H2 receptors include vasodilatation and increased mucus production. The third histamine receptor to be identified, H3, is encoded for on chromosome 20. It has been implicated in the modulation of histamine storage and release specifically in the central and peripheral nervous systems. It is believed that the H3 receptor functions as a negative feedback mechanism on histamine synthesis. The H4 receptor gene is found on chromosome 18 and is significantly homologous to the H3 receptor, creating

overlap in the experimental use of H3 and H4 agonists and antagonists, presently. There is a fifth histamine receptor labeled H(ic), which is not well defined currently (Table 1).

Cellular Histamine Release To understand the effects of the histamine molecule, it is necessary to review the mechanisms which mediate its release from pertinent cells, namely mast cells and basophils. As mentioned above, the mast cell accounts for much of the local histamine release pertinent to the asthmatic and rhinitis processes. The initial step leading to histamine release begins with a cross-linking, by antigen, of adjacent IgE molecules on the mast cell surface. The Fc portions of these IgE molecules bind to a high-affinity Fc receptor which is specific for these heavy chain regions. The relative strength of this bond is high, meaning that although serum IgE levels are among the lowest of all immunoglobulin subtypes, the effects of IgE are quite powerful. This Fc receptor, FceRI, is constitutively expressed on the surfaces of mast cells and basophils and is central in mediating the effector functions of these cells (Figure 2). The FceRI is composed of three transmembrane components: a, b, and g. The a subunit mediates ligand binding through two Ig-like domains. The b subunit is a 4-pass transmembrane protein containing an immunoreceptor tyrosine-based activation motif (ITAM) near its cytoplasmic carboxyterminus and is involved in the subsequent signaling cascade. There are two identical, linked g chains involved in

HISTAMINE 281 IgE molecule binding site

N S S N

N

S S S–S

C

N

C

C

Syk

ITAMs

Lyn




C







Figure 2 High-affinity IgE receptor, FceRI. IgE, produced from B cells, binds in the region of the Ig-like domains, created by disulfide linkages in the a subunit. The tyrosine kinase, Lyn, phosphorylates ITAMs on the cytoplasmic portions of the b and g chains. Another tyrosine kinase, Syk, is activated when it associates with the phosphorylated ITAMs on the g chains. Syk, then, is able to continue the intracellular signaling process.

intracellular signaling which contain one ITAM apiece on their cytoplasmic aspects. There are at least three main pathways leading to the effector function of the mast cell. The first two involve the formation and release of cytokines such as tumor necrosis factor (TNF) and lipid-derived products, such as prostaglandin D2, leukotriene C4 (LTC4), LTD4, and LTE4, among others. The third pathway involves the release of granule contents, namely histamine and others. Going back to the FceRI complex described above, there exists a tyrosine kinase on the cytoplasmic aspect of the b chain called Lyn. Soon after antigen cross-linking of IgE has taken place, Lyn phosphorylates the ITAMs located on the other cytoplasmic b chain and the g chain. At that juncture, another tyrosine kinase called Syk phosphorylates an isoform of PLC, introduced previously during the discussion of the H1 receptor. PLC allows for an overall increase in intracellular calcium levels which contributes to the activation of protein kinase C (PKC). PKC is important because it is this molecule that dissolves the actin–myosin complexes near the cellular membrane, allowing the granules containing histamine, and other preformed mediators, to fuse with the plasma membrane. After the fusion is complete, these mediators are released into the surrounding tissues to exert their effects. Besides histamine, other preformed mediators from mast cells and basophils include heparin, chondroitin sulfate, tryptase, chymase, acid hydrolases, cathepsin G, carboxypeptidase, and lysophospholipase, among others (Figure 3).

The Interaction of Histamine with Cells of the Immune System In the asthmatic process, histamine release from mast cells is associated with airway obstruction, mucosal edema, and mucus hypersecretion, undeniably related to its inherent properties of promoting smooth muscle contraction in the airway as well as increasing vascular permeability. These effects are largely mediated through HR1, although HR2 plays a significant role in mucus secretion. The cellular components involved in promoting asthma and rhinitis symptoms is a topic of intense research. A number of immune cell types express H1 and H2 receptors, such as regulatory T cells, B cells, helper T cells, and monocytes. An important concept to introduce is the Th1/Th2 dichotomy. CD4 þ T cells can differentiate into either the Th1 or Th2 subsets, based on the profile of cytokines secreted. Th1-like cytokines include IFN-g and TNF-b which are linked to cell-mediated immune responses, while Th2-like cytokines include IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, linked to a humoral response pattern including the allergic phenotype. Dendritic cells play a sizable role in influencing which subset a certain CD4 þ T-cell clone becomes, potentially based on early antigen presentation in the periphery. When immature dendritic cells are exposed to IFN-g, the IL-12 production is increased, which is important for cultivating a Th1 response. Similarly, the effects of increased levels of IL-10 on mature dendritic cells can result in suppressed levels of IL-12 and a resultant Th2 response.

282 HISTAMINE PIP2

FcRI complex

Mast cell plasma membrane

PLC Activated Syk

IP3

DAG

Ca2+

PKC

MAP kinase cascade

Lipid mediator synthesis and secretion

Cytokine expression and secretion

Histaminecontaining granule

Actin−myosin complexes dissolved

Histamine released after granule fuses with mast cell membrane Figure 3 Intracellular signaling in the mast cell. After the binding of antigen cross-linked IgE to the FceRI and subsequent activation of the Syk tyrosine kinase, several distinct intracellular signaling pathways result. Syk may activate a mitogen-activated protein (MAP) kinase cascade leading to the production of lipid mediators and the production of cytokine gene expression. Syk also activates an isoform of phospholipase C (PLC) which breaks down phosphatidylinositol biphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). DAG, along with increased intracellular calcium stimulated by IP3, both activate protein kinase C (PKC), which breaks down actin–myosin complexes near the mast cell membrane allowing fusion of granules with it. These granules contain histamine and a number of other preformed mediators. After fusion with the cell membrane and subsequent exocytosis of the granule contents occur, histamine is released to the extracellular tissue.

With the Th1/Th2 paradigm in mind, studies evaluating histamine’s effects can be placed into perspective. Some antigen-presenting cells (APCs) such as immature dendritic cells can express all four histamine receptors. It has been shown that in these cells, the H1 and H3 receptors can promote antigen-presenting properties as well as Th1 phenotype while H2-receptor stimulation contributes to an APC more capable of secreting IL-10 consistent with a Th2 cell type. In addition, the administration of histamine has been shown to inhibit IL-12 and other pro-inflammatory cytokines in mature dendritic cells by way of the H2 receptor. With regard to T cells, it has been shown that the Th2 subtype has a significantly higher expression of HR2 and that these cells express more Th2 cytokines, such as IL-4 and IL-13. The Th1 cells show higher expression of HR1 and are capable of producing more IFN-g. The differences between HR1 and HR2 potentially explain part of the concept of tolerance invoked by HR2 stimulation and autoimmunity fueled by HR1 stimulation.

Histamine Antagonists’ Use in the Respiratory, Gastrointestinal, and Integumentary Systems A number of pharmacologic agents that antagonize the histamine receptors, namely HR1 and HR2, are available for clinical use. Many of these uses apply to rhinitis, allergic conjunctivitis, urticaria, and gastroesophageal reflux disease. Some in vitro data with antihistamines in asthma have been intriguing, but studies looking at antihistamines for treating asthma in humans to date have been less encouraging. With the advent of the low or nonsedating, second-generation antihistamines aimed at HR1, treatment of rhinitis has become far more tolerable for the average patient. Two of these second-generation HR1 antagonists, astemizole and terfenadine, were removed from the market due to concerns over prolongation of the QT interval, especially when combined with azole antifungal agents or macrolide antimicrobials (Table 2). In general, the HR1 antagonists have proven extremely useful in treating the symptoms of allergic

HISTAMINE 283 Table 2 Histamine receptor antagonists HR1 antagonists First generation Pheniramine Brompheniramine Chlorpheniramine Triprolidine Dimetindene Hydroxyzine Meclizine Buclizine Diphenylpyraline Azatadine Cyproheptadine Second generation Acrivastine Oxatomide Cetirizine Fexofenadine Ebastine Astemizole Loratadine

HR2 antagonists Diphenhydramine Clemastine Dimenhydrinate Doxylamine Phenyltoloxamine Carbinoxamine Pyrilamine Tripelennamine Antazoline Promethazine Methdilazine

Cimetidine Ranitidine Famotidine Nizatidine

Desloratadine Mizolastine Terfenadine Ketotifen Azelastine Levocarbastine

rhinitis, specifically addressing sneezing, pruritus, and rhinorrhea. They have been shown to improve quality of life. They are not used solely for the purpose of relieving nasal congestion like nasal corticosteroids. Several prescription antihistamines are currently marketed in combination with pseudoephedrine, as the latter medication can address nasal congestion. In addition to their clinical efficacy in treating allergic rhinitis, HR1 antagonists are widely used in the treatment of chronic urticaria and also given in addition to intramuscular epinephrine for cases of anaphylaxis. Side effects from the firstgeneration HR1 antagonists may include somnolence, impaired mental efficacy, irritability, and blurred vision among a host of other central nervous system (CNS) adverse effects. Although the HR2 antagonists have an adjunctive role in the treatment of chronic urticaria, these medications are widely used in treating conditions of excessive acid production from the gastrointestinal tract.

Conclusion The properties of histamine, its receptors, and a brief introduction to its role in the immune response have been addressed in this article. Current research

is focused on evaluating histamine’s modulation of the immune system especially during its formative period. Knowledge of the mechanisms of pharmacotherapy currently available for the treatment of upper airway symptoms will undeniably lead to a further understanding of histamine’s role in the lower airway as well. See also: Allergy: Overview; Allergic Rhinitis. Asthma: Overview; Acute Exacerbations; Exercise-Induced. Chymase and Tryptase. Dendritic Cells. Gastroesophageal Reflux. G-Protein-Coupled Receptors. Interleukins: IL-4; IL-6; IL-9; IL-10; IL-12; IL-13. Leukocytes: Mast Cells and Basophils; T cells. Lipid Mediators: Leukotrienes. Neurophysiology: Neural Control of Airway Smooth Muscle. Reactive Airways Dysfunction Syndrome. Signal Transduction.

Further Reading Abbas AK and Lichtman AH (eds.) (2003) Immediate hypersensitivity. In: Cellular and Molecular Immunology, 5th edn., pp. 432–452. Philadelphia, PA: Saunders. Akdis CA and Blaser K (2003) Histamine in the immune regulation of allergic inflammation. Journal of Allergy and Clinical Immunology 112: 15–22. Hart PH (2001) Regulation of the inflammatory response in asthma by mast cell products. Immunology and Cell Biology 79: 149–153. Idzko M, La Sala A, Ferrari D, et al. (2002) Expression and function of histamine receptors in human monocyte-derived dendritic cells. Journal of Allergy and Clinical Immunology 109: 839–846. Leurs R, Smit MJ, Timmerman H, et al. (1996) Histamine receptors and histamine. In: Simons FER (ed.) Histamine and H1Receptor Antagonists in Allergic Disease, pp. 1–90. New York: Dekker. Lieberman P (2001) Prevention and therapy of Immunologic diseases: antiinflammatory medications. In: Rich RR (ed.) Clinical Immunology, 2nd edn., pp. 109.1–109.11. New York: Mosby. MacGlashan D (2003) Histamine: a mediator of inflammation. Journal of Allergy and Clinical Immunology 112: S53–S59. Mazzoni A, Young HA, Spitzer JH, Visintin A, and Segal DM (2001) Histamine regulates cytokine production in maturing dendritic cells, resulting in altered T cell polarization. Journal of Clinical Investigation 108: 1865–1873. Simons FER (2003) Antihistamines. In: Adkinson NF, Yunginger JW, Busse WW, et al. (eds.) Middleton’s Allergy: Principles and Practice, 6th edn., pp. 834–863. New York: Mosby. Simons FER (2004) Drug therapy: advances in H1-antihistamines. New England Journal of Medicine 351: 2203–2217. Togias A (2003) H1 receptors: localization and role in airway physiology and in immune functions. Journal of Allergy and Clinical Immunology 112: S60–S68.

284 HUMAN IMMUNODEFICIENCY VIRUS

HUMAN IMMUNODEFICIENCY VIRUS M Lipman, The Royal Free Hospital, London, UK

Table 1 Adult AIDS indicator diseases (1993)

& 2006 Elsevier Ltd. All rights reserved.

Candidiasis of the esophagus, trachea, bronchi, or lungs Cervical cancer, invasive Coccidioidomycosis, disseminated or extrapulmonary Cryptococcosis, extrapulmonary Cryptosporidiosis with diarrhea for over 1 month Cytomegalovirus disease outside the liver, spleen, or lymph nodes Herpes simplex skin ulcers for over 1 month or involving the lungs or esophagus HIV-related encephalopathy Isosporiasis with diarrhea for over 1 month Kaposi’s sarcoma Lymphoma: Burkitt’s or immunoblastic or primary (i.e., not involving other parts of the body) brain Disseminated mycobacteriosis (including tuberculosis) Pneumocystis jirovecii pneumonia (PCP) Recurrent bacterial pneumonia within a 12-month period Progressive multifocal leukoencephalopathy (PML) Recurrent Salmonella septicemia Toxoplasmosis of the brain HIV wasting syndrome

Abstract Human immunodeficiency virus (HIV) is a human retrovirus belonging to the lentivirus family. It is responsible for the progressive immune dysfunction that leads to acquired immunodeficiency syndrome; and has resulted in over 20 million deaths. Many of these are due to respiratory disease with pathogens such as bacteria (pneumococcus and Gram-negative organisms), the fungus Pneumocystis jirovecii, and Mycobacterium tuberculosis. The immune dysregulation induced by HIV also leads to a variety of noninfectious pulmonary complications. Highly active antiretroviral therapy has transformed HIV care: the rates of severe opportunist disease have plummeted in countries where the drugs are widely available.

Introduction Human immunodeficiency virus (HIV) is a human retrovirus belonging to the lentivirus family. It is responsible for the progressive immune dysfunction that leads to acquired immunodeficiency syndrome (AIDS); and has resulted in over 20 million deaths. Two subtypes (HIV-1 and HIV-2) have been identified. HIV-1 is responsible for the vast majority of infections. HIV-2 is mainly prevalent in West Africa, and has a similar, though slower, clinical course. This article focuses on HIV-1 infection in adults. Life-threatening opportunist illnesses associated with HIV infection were first reported in 1981. These included the fungal pneumonia due to Pneumocystis jirovecii (formerly known as Pneumocystis carinii) and the herpes virus-driven tumor Kaposi’s sarcoma (KS). The term AIDS was created early within the epidemic as an epidemiologic surveillance definition to capture the diseases which resulted from profound immune dysregulation. This has been modified over time to incorporate the expanding spectrum of recognized opportunist illness (Table 1). By the end of 2004 it was estimated that approximately 39 million children and adults were HIV infected. This number has increased year on year, as for every 3 million deaths there are 5 million new infections worldwide. Of these, 800 000 are children or neonates. Most HIV-positive individuals live in either middle or lower income countries, in particular in sub-Saharan Africa, with limited healthcare resources. HIV/AIDS is the fourth biggest global killer, and the leading cause of death in Africa. HIV infection is mainly spread through sexual contact. Worldwide over 70% of infections are

Adapted from Centers for Disease Control, Atlanta, USA.

acquired heterosexually. In higher income countries, homosexual/bisexual males account for the largest number of infected individuals, although in many Western nations the principal route of new transmission is now heterosexual sex. Injecting drug use is responsible for about 10% of all HIV infections; while fewer than 5% of cases result from infected blood and blood products. Vertical mother-to-child HIV transmission is an important route of spread, especially in lower income countries.

Etiology and Pathology Infectious HIV can be recovered from most body fluids including blood, semen, and cervical or vaginal secretions. At initial infection, HIV spreads to the local lymph nodes, circulating immune cells and thymus. The acute viremia may be partly cleared by a host immune response. However, persistent trapping of HIV by dendritic cells exposes vulnerable host cells to infection, leading to ongoing viral replication and dissemination. The virus preferentially infects activated HIV-specific CD4 þ T lymphocytes, though it can also directly attack other T and B cells as well as monocytes, tissue macrophages, and cells expressing surface CD4. Viral entry requires binding of the HIV external envelope glycoprotein gp120 to the CD4 receptor as well as protein co-receptors such as chemokine receptor 5 (CCR5), or other co-receptors including

HUMAN IMMUNODEFICIENCY VIRUS 285

CXCR4, dependent on host cell type. Once HIV is inside the cell it can, using viral reverse transcriptase, integrate its genetic material into the host genome. The virus (in the form of proviral DNA) remains latent in many cells until the cell itself becomes activated. This constitutes a long-term reservoir of potentially infectious virus. Cytokine or antigen stimulation (e.g., with HIV or other viruses) will switch on viral transcription into new RNA which ultimately produces more infectious virus particles. These can leave the cell and infect other CD4-bearing cells. Following an acute seroconversion illness (often clinically evident as a flu-like syndrome in up to 50% of cases), an HIV-infected individual will remain well despite there being detectable circulating HIV. As the virus targets the very cells responsible for immune regulation, persistent infection of CD4 þ cells leads to increasing degeneration of the immune system. This is mirrored by CD4 þ cell depletion and the onset of clinical symptoms. The immune dysregulation ultimately results in opportunistic infections and tumors. There is much debate over the precise mechanisms involved. Possibilities include enhanced cell apoptosis, syncytia formation with cell death, chronic immune activation, and HIV glycoprotein binding to uninfected CD4 þ cells causing widespread activation of cytotoxic T lymphocytes (CTL). The end result is that in the majority of infected people the blood CD4 þ count falls over time. The situation in the lung is similar in that profound changes occur in the resident alveolar and interstitial cell populations. Several different pulmonary cell populations can be infected by HIV, and studies show that there are higher viral loads in lung cells compared to blood during episodes of respiratory disease. In many patients, the presence of HIV in the lung leads to an asymptomatic macrophage alveolitis and CD8 þ lymphocytosis. The CTL are directed against fibroblasts, macrophages, and lymphocytes. With progressive failure of CD4 þ regulation, and consequent changes in the cytokine milieu, the residual cells are less able to maintain the normal homeostasis which usually can mount a strong local immune response against pathogens yet also exhibit tolerance toward selected antigen. The consequence of progressive HIV disease is therefore an increased incidence of disseminated pulmonary infection, the development of specific respiratory illness through defects in the normal immune surveillance mechanisms, and failure of the local, controlled immune response. Although blood contains approximately only 2% of total body lymphocytes, the decline in blood absolute CD4 þ and CD4 þ percentage of total lymphocytes are useful indicators of the risk of developing a severe illness. Individuals can be stratified as having

relatively intact (blood CD4 þ 4 500 cells ml
1), moderate (200–500 cells ml
1), or severely impaired immunity (o200 cells ml
1). This is useful as a pragmatic guide when planning treatments and assessing disease episodes. For example the risk of individuals developing Pneumocystis pneumonia rises precipitously as the blood CD4 þ count falls below 200 cells ml
1. Hence specific primary prophylaxis has been recommended at this level in all adults. High plasma HIV loads are also associated with more rapid disease progression and risk of death. HIV-related clinical symptoms can provide important prognostic information independent of CD4 þ count, oral thrush and constitutional illness being the strongest clinical predictors of progression to AIDS. The US Centers for Disease Control and Prevention (CDC) clinical classification of disease describes the various levels of HIV infection, though does not predict individual outcome. It is based on evidence of HIV infection with clinical indicators of impairment in cell-mediated immunity (Table 2). The 1993 CDC classification includes an immunologic criteria for AIDS (CD4 þ counto200 cells ml
1 or CD4 þ percentageo14%) irrespective of clinical symptoms (Table 3). Antiretroviral therapy directly targets HIV at different stages within its life cycle. Currently drugs Table 2 Centers for Disease Control (CDC) classification of HIV infections (1985)a Group I Group II Group III Group IV Subgroup A

Subgroup B

Subgroup C

Subgroup D Subgroup E a

Acute primary infection Asymptomatic infection Persistent generalized lymphadenopathy (PGL) Other disease Constitutional disease, e.g., weight loss 410% body weight or 44.5 kg fevers 438.5, diarrhea lasting 41 month Neurological disease: myelopathy, peripheral neuropathy, HIV encephalopathy Secondary infectious diseases: C1: AIDS defining secondary infectious disease, e.g., Pneumocystis jirovecii pneumonia, cerebral toxoplasmosis, cytomegalovirus retinitis C2: other specified secondary infectious diseases, e.g., oral candidiasis, multidermatomal varicella zoster Secondary cancers, e.g., Kaposi’s sarcoma, non-Hodgkin’s lymphoma Other conditions, e.g., lymphoid interstitial pneumonitis

The CDC definition of the stages of HIV disease was updated in 1993 (see Table 3). However, the older classification remains in use for some research studies and is therefore reproduced here. Adapted from Centers for Disease Control, Atlanta, USA.

286 HUMAN IMMUNODEFICIENCY VIRUS Table 3 1993 Revised CDC classification system for HIV infection 1. Classification categoriesa Category A: one or more of the conditions listed below in an adolescent or adult (13 years or older) with documented HIV infection. Conditions listed in categories B and C must not have occurred. Asymptomatic HIV infection Persistent generalized lymphadenopathy (PGL) Acute (primary) HIV infection with accompanying illness (sometimes known as seroconversion illness) or history of acute HIV infection Category B: symptomatic conditions in an HIV-infected adolescent or adult that are not included among conditions listed in category C and that meet one of the following criteria: (1) the conditions are attributed to HIV infection or are indicative of a defect in cell-mediated immunity, or (2) the conditions are considered by physicians to have a clinical course or to require management that is complicated by HIV infection. This category includes all such symptomatic conditions, with the exception of those placed in category C. Examples of conditions in this category include, but are not limited to: Bacillary angiomatosis Candidiasis (thrush) in the mouth and/or upper throat Candidiasis of the vagina and/or vulva which is persistent, frequent, or responds poorly to treatment Cervical abnormalities of moderate or severe extent or cervical cancer Constitutional symptoms such as fever (38.5 1C) or diarrhea lasting longer than 1 month Herpes zoster (shingles) involving at least two distinct episodes or more than one dermatone (skin area) Idiopathic thrombocytopenia purpura Listeriosis Oral hairy leukoplakia Pelvic inflammatory disease, particularly if complicated by tuboovarian abscess Peripheral neuropathy Category C: includes the following conditions listed in the AIDS (see Table 1) surveillance case definition. For classification purposes, once a category C condition has occurred, the person will remain in category C. 2. Clinical categoriesb CD4 þ T cell categories X500 cells ml
1 200–499 cells ml
1 o200 cells ml
1

(A) Acute (primary) HIV, asymptomatic or PGL A1 A2 A3

(B) Symptomatic (not A or C – see explanation) B1 B2 B3

(C) AIDS indicator conditions C1 C2 C3

a

For classification purposes, category B clinical conditions take precedence over those in category A. For example, someone previously treated for oral or persistent vaginal candidiasis (and who has not developed a category C disease) but who is now asymptomatic should be classified in clinical category B. b This classification stratifies patients clinically (A to C) and immunologically (1 to 3). Groups A3 and B3 satisfy the immunologic but not the clinical criteria for AIDS. Category B consists of symptomatic conditions that are not included in category C but can be either attributed to, or are complicated by, HIV infection. Examples include thrush (oral or persistent vulvovaginal); moderate or severe cervical dysplasia; thrombocytopenia; and peripheral neuropathy. Adapted from Centers for Disease Control, Atlanta, USA.

block entry to host cells, reverse transcription, and viral production and virion release. In 1996 effective drug combinations were introduced. This highly active antiretroviral therapy (HAART) has had a profound impact on those who can access medication. Major opportunistic infections as well as mortality have been reduced by over 80%. Diseases associated with severe immunosuppression such as cytomegalovirus (CMV) retinitis and Mycobacterium avium intracellulare complex (MAC) are much less common. However there has been an increase in AIDS-defining malignant disease, for example, non-Hodgkin’s lymphoma (NHL), as well as non-AIDS deaths due to liver and cardiovascular disease. The drugs are also not without significant side effects. Despite the efforts of the World Health Organization (WHO), HAART remains largely confined to the wealthier nations – and for the majority of infected individuals, the natural history of HIV is the same as

that seen prior to HAART. It is likely that almost all will progress, with a median time from infection to AIDS of approximately 10 years. However the estimated range is very wide, at 18 months to 25 years. Without HAART, 94% of AIDS patients will die within 5 years. Respiratory disease occurs at all stages of HIV infection. A wide variety of infectious and noninfectious diseases can occur (Table 4). Prior to HAART, Pneumocystis jirovecii pneumonia was the commonest life-threatening disease, accounting for up to 40% of all AIDS-defining illnesses. Tuberculosis (TB) is now the leading cause of HIV-related death in Africa. Even with HAART, it is the most frequent global presentation of severe opportunistic infection. TB can present at any stage of HIV disease, and acts synergistically to suppress CD4 þ counts and elevate viral loads. Although TB can affect immunocompetent individuals, the impairment of cell-mediated

HUMAN IMMUNODEFICIENCY VIRUS 287 Table 4 Common etiologies of HIV-related pulmonary disease Bacteria

Fungi

Parasites

Viruses

Noninfectious causes

Streptococcus pneumoniae Hemophilus infuenzae Staphylococcus aureus Pseudomonas aruginosa Escherichia coli Nocardia asteroides Mycobacterium tuberculosis Mycobacterium avium intracellulare complex Mycobacterium kansasii

Pneumocystis jirovecii Cryptococcus neoformans Histoplasma capsulatum Candida albicans Aspergilus spp. Penicillium marneffei

Toxoplasma gondii Cryptosporidium spp. Strongyloides stercoralis

Cytomegalovirus Adenovirus

Lymphoid interstitial pneumonitis Non-specific interstitial pneumonitis Pulmonary hypertension Emphysema Lung cancer Kaposi’s sarcoma Lymphoma, Hodgkin’s and non-Hodgkin’s Immune reconstitution inflammatory syndrome

Adapted from Centers for Disease Control, Atlanta, USA.

immunity in HIV greatly increases individual susceptibility to the development of active TB. It is estimated that HIV has increased worldwide rates of TB by 10%. In Africa up to one-third of cases of TB occur in HIV-positive individuals. Within 1 year of HIV infection, subjects living in TB-endemic areas have a doubling of risk of active disease; whilst subsequent risk per year is similar to that found in HIVnegatives over a lifetime (5–10%). Pneumocystis pneumonia (PCP) is historically important, though the use of HAART and targeted specific primary prophylaxis has greatly reduced its incidence. Patients who present with PCP in higher income countries are usually those who are unable or unwilling to access medical care. Pulmonary CD4 þ cell depletion as well as probable CD8 þ dysfunction contributes to its pathogenesis. Acute bronchitis is seen in up to 30% of individuals with early HIV infection. Bacterial pneumonia has an attack rate approximately six times that found in the HIV uninfected population. The risk of specific conditions is determined to some extent by individuals as well as geographic factors. Injecting drug users have a high incidence of wasting syndrome, recurrent bacterial pneumonia, and TB, whilst subjects from populations with high background rates of TB are at greatly increased risk of this disease. Thus it is the combination of locally impaired pulmonary immunity together with the presence of specific environmental organisms that appears to determine the spectrum of opportunist respiratory disease present within a community. Respiratory disease in children is also common. PCP is often the presenting illness in undiagnosed HIV-infected babies and neonates. Recurrent, severe bacterial infections occur frequently; and, together with TB and cytomegalovirus (CMV) infection, are typical post-mortem findings. Lymphoid interstitial pneumonitis is almost exclusively seen in children.

Clinical Features of Respiratory Disease The clinical presentation of both infectious and noninfectious respiratory illness can be very similar. Typically an individual may complain of cough and breathlessness. A short history (a few days), or one associated with fever and purulent sputum, suggests a bacterial cause. A longer duration with minimal respiratory signs and evidence of significant immunosuppression elsewhere, for example, oral thrush, implicates PCP. Marked weight loss and constitutional symptoms may point to an underlying mycobacterial etiology. Gradual breathlessness over several months is present in conditions such as lymphoid interstitial pneumonitis (LIP) and HIVrelated pulmonary hypertension. However, clinical features are rarely cause-specific: for example, several pathogens including viruses, bacteria, protozoa, and fungi can mimic PCP. Early investigation, with the aim of targeting treatment, is thus a critical part of respiratory disease management. In practical terms, useful information can be derived from knowledge of an individual’s previous medical history. For example, PCP is much more likely if a patient has a low CD4 þ count (o200 cells ml
1) and either no history of adequate specific Pneumocystis prophylaxis or poor adherence to a prescribed regimen. TB is especially likely (at any CD4 þ count) in subjects who have lived in endemic areas or have a personal history of mycobacterial exposure. It should be remembered that two or more pathogens are recovered from up to 20% of patients during a respiratory illness. Bacterial Pneumonia

This may be the first clinical manifestation of HIV infection. Its presentation is no different to that seen in an immunocompetent population. Typical organisms are listed in Table 4. At higher blood

288 HUMAN IMMUNODEFICIENCY VIRUS

CD4 þ counts Streptococcus pneumoniae is the commonest cause. More unusual bacteria occur with increasing frequency at later stages of HIV infection. Such individuals are also at greater risk of recurrent pneumonia, septicemia, and the development of rapid-onset bronchiectasis. Up to a quarter of HIV infected children will have episodes of bacterial septicemia, usually secondary to pulmonary disease. Gram-negative septicemia is a well-recognized cause of HIV-related death. The commonest radiological feature is focal consolidation, found in 50% of cases. However, bacterial pneumonia may present as solitary or multiple pulmonary nodules, interstitial opacities, or with a pleural effusion. Diagnosis rests on clinical suspicion, and culture of sputum, lung fluid, and blood. Rapid antigen testing for pneumococcus can be helpful. However a large number of bacterial pneumonias are treated empirically with no specific organism isolated. Mycobacterial Disease

HIV infection has transformed the clinical presentation and management of mycobacterial disease. The changes produced in local pulmonary immunity lead to a failure of granuloma formation and thus a high incidence of extrapulmonary and disseminated disease. In such cases, the radiological features of pulmonary TB are often atypical: instead of ‘post primary’ features of upper zone infiltrates with cavitation, there is non-specific mid and lower zone shadowing, pleural effusions, and thoracic lymphadenopathy. This pattern is much more common as the blood CD4 þ count falls. The cornerstone of TB diagnosis is sputum smear microscopy and culture. Three early morning (or overnight) samples should be obtained if the patient is productive. If not, then either induced sputum or bronchoalveolar lavage (BAL) will be helpful. These should be carried out in negative pressure isolation to reduce the risk of TB transmission. However, it is common for TB to be recovered from more than one body site; and blood, urine, and, if clinically indicated, spinal fluid cultures also should be collected. New methods of rapid diagnosis include nucleic acid amplification (NAA) tests as well as serological and immunological techniques. The NAA tests are increasingly useful, as they have a greater sensitivity than microscopy, can distinguish TB from other mycobacteria, and allow early detection of drug resistant strains. Immune-based tests (often using T cell responses to mycobacteria-specific antigens) appear to be less sensitive in HIV-infected individuals than the immunocompetent. This is not surprising given

the reduced T-cell number and function seen in patients with advancing HIV. Such tests, however, may have a role in predicting the future risk of developing mycobacterial disease, which is especially important in this population. Other mycobacterial opportunist pathogens such as MAC and Mycobacterium kansasii usually occur when the blood CD4 þ count is less than 100 cells ml
1. Their clinical presentation may be similar to TB (with cough, sputum, weight loss, and fever) though can be very non-specific. MAC in particular is seen in a disseminated form and can be diagnosed in such cases on the basis of culture from any normally sterile site such as blood or bone marrow. Pneumocystis Pneumonia

PCP typically presents with a history lasting several days to weeks of dry cough, breathlessness, fever, and malaise. There are often surprisingly few pulmonary findings, though the chest radiograph typically reveals bilateral mid and lower zone interstitial and alveolar shadowing. In up to 10% of cases this may be very subtle and the radiograph will appear normal. Computed tomography (CT) scanning can be helpful in such cases, demonstrating the ‘ground glass’ appearance consistent with alveolar pathology. It should be noted that this is not specific for PCP and that provided the patient is fit enough, lung sampling is necessary to make a definitive diagnosis. Pneumocystis is difficult to culture, and PCP diagnosis relies on direct visualization of the cysts. Immunofluorescence techniques increase the sensitivity of microscopy; and NAA tests are generally not required for diagnosis. Sampling can be performed using induced sputum, BAL, or tissue biopsy. The procedure of choice may be determined by local expertise. Induced sputum has a yield of 50–80% but a low negative predictive value, and is easiest to perform. BAL is more sensitive (over 90%) though requires greater technical expertise, facilities, and expense. Transbronchial and open lung biopsies are bigger procedures, with greater patient risk. They tend to be reserved for cases that remain diagnostically challenging despite imaging and BAL. The distribution of Pneumocystis and its inflammatory response within the interstitium and alveolar space mean that exercise testing can be helpful in those patients with a compatible history and a normal chest radiograph. Here, after exercise (such as ‘step-ups’ or cycling on a fixed frame for 5–10 min to increase heart rate to 80% of maximum predicted), exercise desaturation (o90%) is more likely to be due to PCP than bacterial or other etiologies.

HUMAN IMMUNODEFICIENCY VIRUS 289 Other Infections

The following pulmonary infections occur in individuals with markedly impaired pulmonary immunity. This is usually reflected in blood CD4 þ counts o100 cells ml
1. CMV is often recovered from the lung of patients with respiratory disease. The significance of this is unclear. When associated with PCP, the overall outcome appears to be worse, although there is little evidence to show that specific CMV treatment is beneficial. The presence of CMV may therefore reflect the extent of profound local immunosuppression rather than active disease per se. Toxoplasma gondii pulmonary disease presents with cough and fever. The lung may be the only site of disease or, just as with the fungus Cryptococcus neoformans, can be associated with severe, disseminated infection. Radiographic appearances are nonspecific and the diagnosis can be made using cytology or NAA techniques. Pulmonary cryptococcosis is typically confirmed using antigen tests (on lung fluid as well as blood) and culture. Noninfectious Conditions

LIP is reported in up to 40% of children aged over 1 year with perinatally acquired HIV infection. It presents with slowly progressive breathlessness associated with cough, wheeze, and digital clubbing. It is much less common in adults, though can be mistaken for PCP and other infections. The diagnosis may require confirmation by tissue biopsy, where massive inflammatory cell infiltration will be evident throughout the alveolar and interstitial spaces. A less aggressive form of disease is seen in adults presenting with widespread CD8 þ T cell infiltrates involving the parotids, muscle, and bone as well as lung. This diffuse infiltrative syndrome can occur in subjects with either relatively well-preserved immunity or who have had a good response to antiretroviral treatment. A further and less well-characterized condition is that of non-specific interstitial pneumonitis (NSIP). This has been used to describe individuals who have typically mild symptoms of dry cough, breathlessness, and occasionally fever but where no infectious cause has been determined. The findings are often associated with pulmonary function abnormalities such as a reduced transfer factor. Chest radiology may show infiltrates or nodularity. It is thought that NSIP is due to the increased non-specific proinflammatory state present within the HIV-infected lung. Pulmonary hypertension of unknown etiology appears to be more common in HIV-infected individuals. It presents as increasing breathlessness, with ultimately right-sided cardiac failure. The plexiform arteriopathy found on histology is thought to arise

from HIV protein- and cytokine-mediated vasoconstriction and endothelial proliferation. This needs to be distinguished from other pulmonary vascular abnormalities such as those associated with injected foreign material (e.g., talc in injecting drug users), chronic thromboembolic disease, and hepatopulmonary syndromes in cirrhotics. HIV-infected cigarette smokers appear, in some studies, to have an accelerated loss of lung volume and early onset bullous emphysema. This may be due to increased local production of matrix metalloproteases, which in turn results from dysregulation of HIV-infected macrophages. KS is the tumor most strongly associated with HIV infection. The relative risk compared to HIV-negative populations is measured in the hundreds. Its incidence has declined in countries where HAART has been introduced, though it remains the commonest HIVrelated cancer. Pulmonary KS will often present as progressive breathlessness, cough, and hemoptysis in patients with KS elsewhere. Typical other sites include the skin, mouth (especially hard palate), and abdominal viscera. Chest radiology often shows reticulonodular shadowing in a bronchocentric distribution, intrathoracic adenopathy, or large (blood-stained) pleural effusions. Diagnosis can usually be made on the basis of compatible symptoms with radiology or direct visualization of lesions at bronchoscopy. Infection with human herpes virus 8 (HHV-8), a sexually transmitted virus, is necessary for an individual to develop KS. This to some extent explains the tumor’s high prevalence in HIV-infected populations; though it is likely that HIV proteins are also responsible for accelerating tumorigenesis. Other HHV-8 associated diseases include primary effusion lymphoma and angiofollicular lymphoid hyperplasia (Castleman’s syndrome), both of which are more common in HIV-positive populations. NHL is the second most common HIV-associated tumor. It is typically of B cell origin; and in many cases is associated with Epstein–Barr virus infection. Approximately 10% of NHL involves the lung. Its presentation is often non-specific and the radiological features are equally varied. These include infiltrates, mass lesions, and effusions. Diagnosis is often based on tissue biopsy. Lung cancer appears to be about four times more common in HIV-infected smokers. Unlike viral-associated tumors, there does not seem to be an obvious relationship with the degree of immunosuppression. The tumors are predominantly peripheral non-small cell lung cancers; and patients tend to be younger than HIV-negative subjects. The diagnosis is usually established by sputum or pleural fluid cytology, histologic and cytologic evaluation of samples obtained

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via bronchoscopy, or by percutaneous fine-needle aspiration. The immune restoration inflammatory syndrome (IRIS) is a clinical phenomenon associated with the use of HAART. This may take a number of different forms; and similar presentations have been seen in posttransplant populations as well as HIV-negative individuals on treatment for TB. In general terms, patients experience either a recurrence of previously controlled symptoms of an opportunistic infection shortly after starting HAART, or the antiretrovirals unmask subclinical disease. IRIS has been reported with a great number of infectious agents, typically those due to intracellular pathogens. These include MAC, TB, CMV, herpes, and hepatitis viruses. In fact, one problem with IRIS is that it is a diagnosis of exclusion (of other intercurrent events, and relapse of the original disease process). As such it may be overdiagnosed clinically. Patients with mycobacterial disease seem especially prone to IRIS. Up to 15% of HIV-negative individuals experience a transient deterioration in symptoms after starting antimycobacterial treatment (the paradoxical reaction). This more than doubles when HAART is coadministered, and the reaction is often much more severe. There are no good predictors of who is at risk of IRIS; though given its association with changes in local immunity it would seem reasonable that a rapid reduction in HIV load and possibly increases in blood CD4 þ count (as a reflection of cell trafficking to the site of activity) may be relevant. Symptoms can last anything from a few days to more than a year. They are most often at the original site of disease. In cases where HAART activates latent infection, the location may be very atypical, for example, focal bone disease due to MAC.

3.

4.

5.

6.

7.

Management and Current Therapy The detailed discussion of specific treatments is covered in other sections of this encyclopedia. Some general principles which apply to HIV management are as follows: 1. Clinical suspicion can guide initial therapy and some treatments should not be deferred for outstanding investigations if the patient is sick (e.g., treatment of PCP in an individual with compatible symptoms and a low CD4 þ count). 2. Rapid investigation is vital – there may be a short period of time during which pulmonary samples or tissue biopsies can be obtained. At a later stage, the procedure may be deemed too risky (e.g., bronchoscopy in a hypoxic patient) or

8.

the prescribed treatments may reduce the sensitivity of the laboratory tests. Get to know what resources are available within the local pathology services. Diagnostic tests for relatively rare conditions are dependent on the expertise of those performing and interpreting them. Thus, there may be a high rate of false negative results in a center with little experience of a particular condition. Several different pathologies may coexist at the same time; hence nonresponse should prompt early reassessment and investigation. Drug–drug interactions and adverse effects are common in HIV-infected individuals. This is especially the case for HAART and anti-TB agents; whilst anti-PCP treatments can cause adverse effects in up to 50% of individuals. Specialist input from a team experienced in HIV disease is crucial. In practice this may require contributions from multiple disciplines which then need to be coordinated as one seamless, management plan. Prevention is better than cure. Prophylactic regimens should be considered in all individuals at risk of significant opportunist infection. HAART has transformed the spectrum of respiratory illness and its management. It undoubtedly prevents disease by improving local and specific immune responses. Individuals who have a sustained increase in blood CD4 þ count, an undetectable viral load, and good drug concordance can stop specific infection prophylaxis. For example, PCP prophylaxis can be discontinued at CD4 þ counts above 200 cells ml
1 (i.e., similar levels to that at which it is usually instituted). Immunization schedules with noninfectious vaccines may be useful. Pneumococcal immunization, despite its rather limited efficacy, has been shown to reduce rates of bacterial infection and decrease mortality. At present its uptake in HIV-infected populations is low. Scrupulous infection control is critical. Although many infections are not clearly transmissible between subjects within a healthcare setting, there is an increased incidence of bacterial nosocomial pneumonia, and TB is a constant concern in HIVinfected subjects. Respiratory precautions should be adopted when managing patients with pulmonary symptoms. These include the use of isolation facilities, with negative pressure ventilation if they are admitted to a ward with other immunocompromised individuals. The use of particulate dust filter masks by staff is important if the patient is undergoing investigation, for example, bronchoscopy. Procedures that involve the production of an aerosol (such as sputum induction or bronchoscopy)

HUMAN IMMUNODEFICIENCY VIRUS 291

should be performed in negative pressure facilities with air vented directly to the outside. Avoidance of unnecessary procedures is also relevant; and the importance of careful, repeat sputum examinations for acid-fast bacilli cannot be overstated. Tuberculosis

HIV-related TB usually responds to standard short course (6 month) four drug rifamycin-based therapy. There are some concerns regarding risk of relapse with this duration of therapy. Indication for treatment prolongation are persistence of culture positive samples (usually sputum) after 2 months of treatment with what should be effective therapy; low CD4 þ counts (o100 cells ml
1) with no option for HAART, in areas where TB is endemic; extrapulmonary disease with cerebral involvement; and mycobacterial drug resistance. In general, there is a good response to standard therapy, though short-term mortality is increased in patients with advanced immunosuppression. A key issue concerns the optimum time at which to start HAART. Ensuring adequate TB treatment is most important, though if HIV-infected individuals with CD4 þ counts o100 cells ml
1 defer antiretrovirals for too long, there is a high risk of further opportunist infections. The early use of HAART needs to be balanced against a possible increase in adverse effects, drug–drug interactions, reduced adherence through large pill burden, and a potential for significant IRIS during this period. There is no standard approach and most clinicians tend to assess such cases on an individual basis, using the small amount of evidence that currently exists. Drug-resistant TB is increasingly common. It is almost always due to inferior treatment either from inadequate prescribed therapy or poor patient concordance. It is associated with HIV infection, though this may result from the social clustering of infectious cases in both conditions. Multidrug-resistant TB, where the organism is resistant to at least isoniazid and rifampicin, is difficult to treat, has a high mortality, and has been implicated in several outbreaks of nosocomial TB. HIV infection is a risk factor for acquired rifampicin monoresistance. This may be due to underdosing or impaired absorption of medication. Treatment adherence is important for all TB patients. Directly observed therapy as part of a package of care may be helpful when there is possible noncompliance. TB control requires organized contact tracing, chemoprophylaxis for those at risk (i.e., HIV-infected individuals with a history of significant TB exposure and positive skin/immune-based tests),

and effective local measures to reduce spread. The issue of chemoprophylaxis in high-income countries is thorny. Some authorities recommend that it should be offered to HIV-positive patients with declining CD4 þ counts who have a positive purified protein derivative skin test. Historically, in resource-poor settings, treatment and prevention of established TB was an effective strategy. However, HIV infection had made this approach much less beneficial, and notwithstanding the problems associated with widescale implementation of HAART, HIV itself must also be treated. Pneumocystis Pneumonia

In practical terms, if PCP seems the most likely diagnosis, treatment should be started early and a diagnostic procedure planned for the next few days, as Pneumocystis can be recovered during the first week of treatment. Respiratory support has altered the management of severe PCP. With all currently available drug therapies (including cotrimoxazole, pentamidine, and clindamycin plus primaquine) there is a period of several days before clinical improvement occurs. The use of adjuvant steroids (to reduce interstitial leak) and support techniques such as continuous positive airways pressure (CPAP) circuits can help to maintain respiratory health during this time, often avoiding the need for full mechanical ventilation in those with severe PCP. Patients with first-episode PCP and/or a good quality of life are usually those considered for formal ventilation. Pneumocystis prophylaxis can be given in either a primary form (when an individual is deemed to be at high risk of future disease) or as secondary prophylaxis, following an episode of PCP. This is a sensible policy as the risk of relapse/reinfection is estimated at almost 50% over a 12-month period if HAART and prophylaxis are not available. The increase in use of agents such as co-trimoxazole (a combination of trimethoprim and sulfamethoxazole) in this context and also in low-income countries to reduce risk of infection and long-term mortality, has led to concerns regarding the development of drug-resistant Pneumocystis. Mutations have been observed in the organism’s dihydropteroate synthase gene; and these do appear to be associated with use of such sulfacontaining drugs. There is no good evidence that this leads to worse outcomes or that these effective drugs should not be used to treat active disease; though this seems a future possibility. Noninfectious Conditions

In higher-income countries up to 50% of HIV-infected individuals are cigarette smokers. This has

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been associated with an increased incidence of upper and lower respiratory tract infection, recurrent PCP, and overall accelerated mortality. Given the compelling evidence for high rates of lung cancer and chronic obstructive pulmonary disease, it would seem that smoking cessation strategies should be prioritized as part of a healthy living program. This is especially important in the context of HAART prolonging survival at the expense of an increase in blood lipids and possibly cardiovascular events. HAART has undoubtedly improved the outcome for HIV-related tumors. This is reflected in the reported reduction in KS and NHL. Lung cancer, however, is different. Despite HAART and cancer chemotherapy, the outlook tends to be poor. The good scientific rationale for treating HIV-associated pulmonary hypertension with HAART and endothelin-1 inhibitors has been proven in practice. Survival is no longer measured in months; and the quality of life for individuals tolerating therapy is greatly improved. See also: Bronchoalveolar Lavage. Interstitial Lung Disease: Hypersensitivity Pneumonitis. Laryngitis and Pharyngitis. Pneumonia: Overview and Epidemiology; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Mycobacterial; The Immunocompromised Host. Vaccinations: Viral. Viruses of the Lung.

Further Reading Agostini C, Zambello R, Trentin L, and Semenzato G (1996) HIV and pulmonary immune responses. Immunology Today 17: 359–364. Badri M, Wilson D, and Wood R (2002) Effect of highly active antiretroviral therapy on incidence of tuberculosis in South Africa: a cohort study. Lancet 359: 2059–2064. Bonnet F, Lewden C, May T, et al. (2004) Malignancy-related causes of death in human immunodeficiency virus-infected patients in the era of highly active antiretroviral therapy. Cancer 101: 317–324.

Castro M (1998) Treatment and prophylaxis of pneumocystis carinii pneumonia. Seminars in Respiratory Infections 13: 296–303. Huang L, Crothers K, Atzori C, et al. (2004) Dihydropteroate synthase gene mutations in pneumocystis and sulfa resistance. Emerging Infectious Diseases 10: 1721–1728. Interdepartmental Working Group on Tuberculosis (1998) UK Guidance on the Prevention and Control of Transmission of (1) HIV-Related Tuberculosis and (2) Drug-Resistant, including Multiple Drug-Resistant Tuberculosis: The Prevention and Control of Tuberculosis in the United Kingdom. London: Department of Health. Lawn SD, Bekker LG, and Miller RF (2005) Immune reconstitution disease associated with mycobacterial infections in HIVinfected individuals receiving antiretrovirals. Lancet Infectious Diseases 5: 361–373. Masur H, Kaplan JE, and Holmes K (2002) Guidelines for preventing opportunistic infections among HIV-infected persons – 2002: recommendations of the US Public Health Service and the Infectious Diseases Society of America. Annals of Internal Medicine 137: 435–478. Mayaud C, Parrot A, and Cadranel J (2002) Pyogenic bacterial lower respiratory tract infections in human immunodeficiency virus-infected patients. European Respiratory Journal 20(supplement 36): 28s–39s. Pozniak AL, Miller RF, Lipman MC, et al. on behalf of the BHIVA Guidelines Writing Committee (2005) BHIVA treatment guidelines for tuberculosis (TB)/HIV infection 2005. HIV Medicine 6 (supplement 2): 62–83. Smith CJ, Levy I, Sabin CA, et al. (2004) Cardiovascular disease risk factors and antiretroviral therapy in an HIV-positive UK population. HIV Medicine 5: 88–92. Stebbing J, Gazzard B, and Douek DC (2004) Where does HIV live? New England Journal of Medicine 350: 1872–1880. Subcommittee of the Joint Tuberculosis Committee of the British Thoracic Society (2000) Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 55: 210–218. UNAIDS (2004) Report on Global AIDS Epidemic. Geneva: UNAIDS. White NC, Agostini C, lsrael-Biet D, Semenzato G, and Clarke JR (1999) The growth and the control of human immunodeficiency virus in the lung: implications for highly active antiretroviral therapy. European Journal of Clinical Investigation 29: 964– 972. Zumla A, Johnson MA, and Miller R (eds.) (1997) AIDS and Respiratory Medicine. London: Chapman & Hall.

HYPERBARIC OXYGEN THERAPY I Grover and T Neuman, University of California, San Diego, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Hyperbaric oxygen therapy is the use of oxygen at increased atmospheric pressure. The history of hyperbaric oxygen therapy can be traced back to the late seventeenth century, when Henshaw treated patients in a pressurized chamber. Initially, patients

were treated with pressurized air. In 1955, the modern era of hyperbaric oxygen therapy was born. Today, patients are treated in either monoplace chambers or multiplace chambers. The pressurized oxygen exerts its effects by several different mechanisms, including creating a diffusion gradient for inert gases, oxygenating ischemic tissues, limiting reperfusion injuries, inactivating certain toxins, and supporting angiogenesis and leukocyte function. There are 13 indications for which scientific evidence supports the use of hyperbaric oxygen therapy: arterial gas embolism; decompression sickness; carbon monoxide poisoning; clostridial myonecrosis; crush injuries, compartment

HYPERBARIC OXYGEN THERAPY 293 syndrome, and other acute ischemias; enhancement of healing in selected problem wounds; exceptional blood loss anemia; intracranial abscess; necrotizing soft tissue infections; refractory osteomyelitis; delayed radiation injury; preservation of skin grafts and flaps; and thermal burns.

Using hyperbaric therapy for treatment of multiple disorders dates back to 1662, when Reverend Henshaw used a pressurized chamber for therapeutic purposes. In 1889, a British engineer, Ernest Moir, became the first person to treat decompression sickness (DCS) with recompression. When he took over as the superintendent of the Hudson River tunnel project, workers had a 25% mortality rate from DCS. After he installed a recompression chamber at the work site, the mortality rate from DCS declined to 1.7%. In 1955, Ite Boerema, from the University of Amsterdam, performed an experiment using hyperbaric oxygen to keep exsanguinated pigs alive by the oxygen dissolved in plasma alone. At the same time, W H Brummelkamp at the University of Amsterdam discovered that hyperbaric oxygen could be used to inhibit anaerobic infections. In Scotland, studies suggested that hyperbaric oxygen was beneficial in the treatment of carbon monoxide poisoning in humans. There are 13 indications for the use of hyperbaric oxygen therapy (HBOT) which are backed by scientific evidence. HBOT consists of treating a patient with 100% oxygen at pressures above 1.4 atm absolute (4 m of seawater) in a hyperbaric chamber. The oxygen is delivered to the patient by inhalation. There are two types of chambers in which patients are treated: monoplace and multiplace. They (Figure 1) are used to treat a single patient. They are pressurized with oxygen or air, and attendants do not have to go into the chamber with the patient. Monoplace chambers are less expensive than multiplace chambers, and they require less operating space. Multiplace chambers (Figure 2) are larger and used to treat multiple patients simultaneously. These chambers are pressurized with air, and oxygen is delivered to the patients via plastic hoods or face masks. Multiplace chambers have the advantage of treating multiple patients at a single time. Also, critically ill patients can be treated with a nurse and other support personnel in the chamber. Hyperbaric oxygenation has two primary mechanisms of action that are the basis of its use: the mechanical effect and the effect of an increased partial pressure of oxygen. The mechanical effects of pressure shrink gas bubbles and gas-filled spaces in the body following Boyle’s law (volume is inversely proportional to absolute pressure). There are multiple other physiologic effects that supernormal partial pressures of oxygen exert on the body. These benefits

Figure 1 Monoplace chamber. Photo courtesy of OxyHeal Health Group, Inc.

Figure 2 Multiplace hyperbaric chamber. Photo courtesy of OxyHeal Health Group, Inc.

include angiogenesis, fibroblast growth and collagen production, improved osteoclast function, enhanced removal of carbon monoxide (CO) from hemoglobin, inhibition of a-toxin production in clostridial myonecrosis, improved leukocyte killing, decreased neutrophil adherence to capillary walls, increased production of superoxide dismutase, and vasoconstriction in normal vessels. These effects are the physiologic rationale for the use of HBOT in the conditions discussed next.

Indications for Hyperbaric Oxygen Therapy Gas Embolism

A gas embolism occurs from any number of mechanisms, including mechanical ventilation, penetrating

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chest trauma, chest tube placement, bronchoscopy, and pulmonary barotrauma from scuba diving. Gas bubbles enter the arteries or veins and cause a number of different symptoms, including loss of consciousness, altered mental status, focal neurological deficits, hypotension, pulmonary edema, or cardiac arrhythmias including cardiac arrest. A patient who exhibits neurologic symptoms or cardiac symptoms from a gas embolism should be treated with HBOT. First, HBOT exerts a mechanical effect on bubble size with recompression. As the pressure in the chamber increases, the bubble size will decrease. Second, the increased amount of oxygen in the blood creates a diffusion gradient to help further reduce the size of the bubbles. Third, HBOT inhibits neutrophil adherence to capillary walls, reducing the inflammatory response caused by damage to the endothelium from the presence of a gas bubble. Finally, the increased oxygen carried by the plasma may help oxygenate partially ischemic tissues. Decompression Sickness

DCS encompasses a myriad of syndromes caused by bubbles of inert gas that are generated from rapid decompression during ascent from diving, flying, or in a hyperbaric or hypobaric chamber. When these bubbles occur in large enough numbers to cause pain or impede normal organ function, symptoms of DCS arise. As with gas embolism, the treatment for DCS is HBOT. The mechanism of action of HBOT is the same as for gas embolism. Carbon Monoxide Poisoning

Carbon monoxide is a colorless, odorless, and tasteless gas that is a leading cause of injury and death from poisoning worldwide. Patients who suffer from CO poisoning can present with tachycardia, tachypnea, chest pain, headache, altered mental status, seizures, amnesia, or peripheral neuropathy. Laboratory abnormalities include elevated carboxyhemoglobin levels and a metabolic acidosis. There are five randomized clinical trials in the literature using HBOT in patients with CO poisoning. Although there have been contradictory results from these studies, the best was done by Weaver and coworkers and was published in 2002. This study showed a statistically significant reduction in neuropsychological sequelae in patients treated with HBOT. Patients with evidence of metabolic acidosis, ischemic chest pain and/or electrocardiographic evidence of ischemia, abnormal psychometric testing, history of unconsciousness, carboxyhemoglobin level greater than 15% in a pregnant patient (because of increased binding of CO to fetal hemoglobin), or a

carboxyhemoglobin level greater than 40% should be treated with HBOT. HBOT benefits the CO-poisoned patient by decreasing the half-life of carboxyhemoglobin from more than 300 minutes breathing room air to 23 minutes breathing 100% oxygen at 3 atm absolute (ATA). The half-life of carboxyhemoglobin remains 90 minutes if breathing 100% oxygen at sea level. Also, CO has been shown to inhibit cellular oxidative metabolism by binding to cytochrome c oxidase. HBOT at 3 ATA has been shown to expedite the dissociation of CO from cytochrome c oxidase, thereby allowing the process of oxidative phosphorylation to return to normal. Other benefits of HBOT in CO poisoning are the prevention of lipid peroxidation of the cell membrane and prevention of the adherence of neutrophils to the vascular endothelium by inhibiting the b2 integrin system. Clostridial Myonecrosis

Clostridial myonecrosis is a severe, life-threatening infection caused by anaerobic, spore-forming, grampositive, encapsulated bacilli of the genus Clostridium. These infections are rapidly progressive and treatment involves the combined use of surgery, antibiotics, and HBOT. HBOT has been shown to be bacteriostatic to clostridia in vivo and in vitro by the formation of oxygen free radicals. The clostridia organisms have no free radical-degrading enzymes, such as superoxide dismutases, catalases, and peroxidases, thereby making them susceptible to the oxygen free radicals produced during HBOT. Also, it has been shown that oxygen tensions of 250 mmHg, which can be achieved on 100% oxygen at 3 ATA, inhibit the production of a-toxin, which is one of the main hemolytic and tissue-necrotizing toxins produced by the clostridial organisms. Necrotizing Soft Tissue Infections

Necrotizing soft tissue infections are an increasing problem in current medical practice. These infections are caused by aerobic or anaerobic bacteria, but most commonly they are caused by mixed bacterial flora. Often, hosts are immunocompromised, contributing to the rapid spread of the infection. Necrotizing infections are hypoxic and an occlusive endarteritis caused by the infection contributes to the wound hypoxia. There are a limited number of neutrophils at the wound site due to intravascular sequestration of the polymorphonuclear leukocytes (PMNs). The PMNs that are present function poorly due to hypoxia and a decreased oxidation–reduction potential (Eh) from the accumulation of metabolic products from the aerobic organisms. HBOT is recommended

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as an adjunct to surgical debridement and antibiotics. With HBOT, there is increased tissue oxygenation, which inhibits anaerobic bacteria growth by direct toxic mechanisms and by improving the Eh as well. Also, with improved tissue oxygenation, PMN function improves. Patients are treated twice a day until the infection is controlled and then daily until no further debridement is required. Crush Injury and Other Acute Traumatic Peripheral Ischemias

Traumatic injury causes a wound with damaged blood vessels. Tissue ischemia and a hypoxic wound environment can then ensue. This hypoxic wound environment causes increasing edema, which contributes to a vicious cycle of wound ischemia and edema. Several surgical and orthopedic conditions have this pathophysiology: crush injuries, compartment syndromes, threatened grafts and flaps, threatened replantations, burns, and frostbitten extremities. As such, HBOT is used as an adjunct for the treatment of these conditions. Initially, HBOT will provide oxygen to hypoxic tissues. PMN function is improved, fibroblast migration and proliferation improve, collagen is laid down, and neovascularization proceeds because tissue oxygen tensions are increased due to HBOT. Another benefit of HBOT is edema reduction. Hyperoxygenation during HBOT causes vasoconstriction, which has been shown to reduce blood flow by 20%. Venous outflow continues unabated, so there is an overall edema reduction. Oxygen delivery to tissues is unaffected by vasoconstriction because of the hyperoxygenation from HBOT. Finally, HBOT limits the reperfusion injury by preventing lipid peroxidation of the cell membrane; antagonizing the b2 integrin system, thereby inhibiting the sequestration of neutrophils to the vascular endothelium; and providing additional oxygen for reperfused tissues to generate scavengers such as superoxide dismutase, catalase, peroxidase, and glutathione. Enhancement of Healing in Selected Problem Wounds

Problem wounds can result in significant morbidity and mortality, and they represent an increasing burden on the healthcare system as the population ages. Diabetic ulcers are an example of problem wounds that are difficult to heal by conventional methods. These wounds are hypoxic due to the small vessel disease caused by diabetes. Since wound healing has been shown to be oxygen-dependent, these hypoxic wounds heal, if at all, at a much slower rate. The processes that contribute to wound healing, such as fibroblast replication, collagen placement,

angiogenesis, intracellular leukocyte bacterial destruction, and infection resistance, are all reliant upon oxygen. This is why hypoxic wounds may not heal. HBOT increases tissue oxygenation, thereby reversing the effects of the hypoxic wound environment. Exceptional Anemia

Occasionally, a profoundly anemic patient will be unable to be transfused with packed red blood cells either because of religious objections or because the patient cannot be cross-matched. If the patient’s hemoglobin is so low that oxygen delivery is insufficient to offset the basic metabolic demands of the body, then oxygen debt will accumulate. As oxygen debt increases, signs of end-organ failure develop. HBOT is a method that repays the accumulated oxygen debt. The patient can be treated with frequent HBO treatments until blood becomes available or the patient produces sufficient hemoglobin. Intracranial Abscesses

Intracranial abscesses encompass several disorders: cerebral abscess, subdural empyema, and epidural empyema. HBOT is recommended as an adjunct to antibiotics and surgical debridement. Benefits of HBOT in these disorders include high concentrations of oxygen in brain tissue that inhibit the mainly anaerobic organisms that cause intracranial abscesses, decreased edema around the abscess site by mechanisms mentioned previously, and improved leukocyte function. Refractory Osteomyelitis

Patients suffering from refractory osteomyelitis (i.e., osteomyelitis that does not respond or reoccurs after appropriate antibiotics and surgical debridement) usually have systemic problems or local wound factors that inhibit their ability to heal these infections. HBOT is an adjunct to systemic antibiotics and surgical debridement. The benefits of HBOT include enhancement of osteogenesis by improving the oxygen-dependent osteoclast function of removing necrotic bone. Additionally, PMNs destroy bacteria by improved oxidative killing when oxygen tensions in the infected bone are raised to normal or supernormal levels. Studies have also shown that aminoglycoside transport across the bacteria cell wall is oxygendependent. HBOT improves aminoglycoside efficacy in hypoxic wound environments by improving delivery of the antibiotic across the bacterial cell wall. Finally, HBOT reduces tissue edema, limits the inflammatory response, and promotes neovascularization and wound healing, as described previously.

296 HYPERBARIC OXYGEN THERAPY Delayed Radiation Injuries

Delayed radiation injury leads to progressive endarteritis that results in tissue hypoxia and secondary fibrosis. Once the oxygen demand of the tissues outstrips oxygen supply, tissue breakdown occurs. The most common delayed radiation injuries that benefit from HBOT are prevention of osteoradionecrosis of the mandible in patients requiring dental work following radiation to the oropharynx, treatment of radiation cystitis, and treatment of radiation proctitis. HBOT promotes angiogenesis, which leads to increased cellularity in irradiated tissues. Studies have shown that HBOT can return to radiated tissue 80% of the capillary density of nonradiated tissue.

Contraindications and Side Effects HBOT is very safe and there are few contraindications to therapy. The major absolute contraindication to HBOT is an untreated pneumothorax because it could progress to a tension pneumothorax during the ascent phase of a treatment when gas volume will expand due to the decrease in barometric pressure. Relative contraindications include pregnancy because of unknown risks to the fetus, chronic sinusitis because of the risk of sinus barotrauma, emphysema with CO2 retention because these patients may lose their stimulus to breath, high fever because this can predispose the patient to seizures from oxygen toxicity, a history of spontaneous pneumothorax because of the risk of recurrent pneumothorax, a seizure disorder because of the risk of oxygen-induced seizures, or a history of middle ear surgery because otic barotrauma could damage the repaired structures. Several medications are contraindicated in HBOT, including doxorubicin due to cardiac toxicity, bleomycin due to pulmonary toxicity, cis-platinum due to problems with wound healing, disulfiram, and sulfamylon. The most common side effect of HBOT is barotrauma of the middle ear. This occurs in 2% of patients and is a result of eustachian tube dysfunction or an inability to properly perform the Valsalva maneuver. Disruption of the round window is a rare consequence of HBOT and results from a forceful Valsalva maneuver. Sinus squeeze is another form of barotrauma that results from the expansion of trapped gases in the sinuses on ascent. Another side effect of treatment is a temporary refractive change. These changes typically occur after numerous treatments and consist of progressive myopia. It is theorized that the changes are due to oxygen uptake by

the lens. Claustrophobia may result from the patient being treated in a relatively small enclosed space. These symptoms can be treated with anxiolytics. Oxygen toxicity causes effects in the central nervous system (CNS) and pulmonary systems. The manifestation of CNS oxygen toxicity is seizures. Oxygen toxicity seizures have an incidence of 1 in 10 000 treatments at 2.4 ATA. Pulmonary oxygen toxicity is another possible side effect. The symptoms are substernal chest pain, dry cough, and a decrease in vital capacity. Finally, there is a theoretical risk of pneumothorax and arterial gas embolism if the patient were to have trapping of gas in his or her lungs or if the patient held his or her breath during ascent. See also: Carbon Monoxide. Diving. Lung Abscess.

Further Reading Boerema I, Meijne NG, Brummelkamp WH, et al. (1960) Life without blood. A study of the influence of high atmospheric pressure and hypothermia on dilution of the blood. Journal of Cardiovascular Surgery 1: 133–146. Brummelkamp WH (1965) Considerations on hyperbaric oxygen therapy at three atmospheres absolute for clostridial infections type welchii. Annals of the New York Academy of Sciences 117: 688–699. Feldmeier JJ (ed.) (2003) Hyperbaric Oxygen 2003: Indications and Results. The Hyperbaric Oxygen Therapy Committee Report. Kensington MD: Undersea and Hyperbaric Medical Society. Kindwall EP and Whelan HT (eds.) (2002) Hyperbaric Medicine Practice, 2nd rev. edn. Flagstaff AZ: Best. Marx RE, Ehler WJ, Tayapongsak P, and Pierce LW (1990) Relationship of oxygen dose to angiogenesis induction in irradiated tissue. American Journal of Surgery 160: 519–524. Marx RE, Johnson RP, and Kline SN (1985) Prevention of osteoradionecrosis: a randomized prospective clinical trial of hyperbaric oxygen versus penicillin. Journal of the American Dental Association 111: 49–54. Moon RE (1997) Treatment of decompression sickness and arterial gas embolism. In: Bove AA (ed.) Diving Medicine, 3rd edn., pp. 184–204. Philadelphia: Saunders. Thom SR (1993) Functional inhibition of leukocyte beta-2 integrins by hyperbaric oxygen in carbon monoxide mediated brain injury in rats. Toxicology and Applied Pharmacology 123: 248–256. Weaver LK, Hopkins RO, Chan KJ, et al. (2002) Hyperbaric oxygen for acute carbon monoxide poisoning. New England Journal of Medicine 347: 1057–1067. Zamboni WA, Roth AC, Russell RC, et al. (1989) The effect of acute hyperbaric oxygen therapy on axial pattern skin flap survival when administered during and after total ischemia. Journal of Reconstructive Microsurgery 5: 343–347. Zamboni WA, Wong H, Stephenson L, et al. (1997) Evaluation of hyperbaric oxygen for diabetic wounds: prospective study. Undersea Hyperbaric Medicine 24: 175–179.

HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME 297

HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME J D Brewer and M D P Davis, Mayo Clinic, Rochester, MN, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Hypocomplementemic urticarial vasculitis syndrome (HUVS) is a form of urticarial vasculitis (UV) characterized as a clinicopathologic entity comprised of chronic urticaria clinically and vasculitis pathologically. When patients with UV have an associated hypocomplementemia, they are more likely to manifest systemic complications of the disease and are characterized as having HUVS. HUVS is affected by both genetic and environmental factors; however, the exact cause remains unknown. HUVS is a rare disease with approximately 100 cases described, is more common in women, usually affects people in their 40s, and is rare in children but associated with a more complicated course when found in the pediatric population. Patients with HUVS have chronic urticaria for more than 6 months, hypocomplementemia, and a various array of systemic manifestations including musculoskeletal, respiratory, renal, gastrointestinal, cardiac, ophthalmologic, central nervous system, and rheumatologic features. Biopsies of active urticarial lesions show leukocytoclastic vasculitis with extravasation of white blood cells and endothelial cell damage. Patients almost invariably have low levels of the C1q component of complement as well as the presence of an IgG antibody directed against a collagen-like region of C1q, also refered to as C1q precipitans. Biopsies of certain organs, especially the kidney, may show deposits of these immune complexes within the basement membrane of inflamed tissue. The pathogenesis of HUVS is largely unknown, although hypotheses exist about exacerbating factors of the disease. HUVS is believed to be an autoimmune disease, much like systemic lupus erythematosus, with the formation of autoantibodies that cause immune complexes, deposition in certain parts of the body, and activation of the immune system causing inflammation and destruction. For example, immune complexes are thought to deposit in the lung, causing aggregation and activation of neutrophils, which actively secrete elastase, causing destruction of the lung architecture and the development of chronic obstructive pulmonary disease (COPD). There is no specific therapy for HUVS; however, many therapeutic agents have been found to be beneficial. Pruritus is treated with antihistamines and cutaneous manifestations have responded to dapsone, colchicine, antimalarials, nonsteroidal anti-inflammatory drugs (NSAIDs), interferon alpha, and corticosteroids. Severe systemic aspects of HUVS have been treated with chemotherapeutic/immunosuppressive agents such as azathioprine, cyclophosphamide, mycophenolate mofetil, cyclosporine, and methotrexate, as well as combinations of previously mentioned agents. The prognosis of HUVS is relatively good; however, morbidity and mortality do occur, and are more likely to be seen in pediatric patients. Death from disease is rare, and is most commonly due to laryngeal edema or COPD.

Introduction Urticarial vasculitis (UV) is a clinicopathologic entity requiring both chronic urticarial wheals clinically

and the evidence of cutaneous vasculitis or leukocytoclastic vasculitis (LCV) histopathologically. Biopsy of the urticarial lesions reveals this vasculitis as having leukocytoclasis and vessel wall destruction, which may or may not be accompanied by fibrinoid deposits. UV is sometimes associated with hypocomplementemia, and hence many authors divide UV into normocomplementemic urticarial vasculitis (NUV) and hypocomplementemic urticarial vasculitis (HUV). Patients with HUV (UV with associated complement activation) are much more likely to have systemic manifestations compared to patients with NUV, and many of these systemic features resemble those of systemic lupus erythematosus (SLE). These systemic manifestations include angioedema, glomerulonephritis, abdominal pain, and obstructive lung disease. It has been speculated that a continuum exists between UV, HUV, and hypocomplementemic urticarial vasculitis syndrome (HUVS), with urticaria and minimal vasculitis at one end of the spectrum and urticaria with marked systemic vasculitis and hypocomplementemia at the other end. One study, however, followed patients for over 20 years, and found that patients did not move or progress down this continuum with time, suggesting that if a continuum does exist between these disease entities, progression from one disease subtype to another is rare. HUVS was first described in 1971, when a patient with hypocomplementemia and hypergammaglobulinemia was labeled as having an unusual SLE-related syndrome. Later a similar presentation in patients was described as hypocomplementemia with cutaneous vasculitis, and UV with associated hypocomplementemia was noted to be a more serious and systemic form of UV. In 1980, patients with urticarial vasculitis and hypocomplementemia were categorized as having a hypocomplementemic urticarial vasculitis syndrome (HUVS), which has also been called McDuffie syndrome. Since then, more than 100 cases have been described, and this syndrome is now widely recognized as HUVS. When first discovered, much of its nature, causes, and associated disease were unknown. The term syndrome was used to characterize patients with HUV who had variable organ system involvement. The term syndrome, however, may be inappropriate for HUVS based on the way syndrome has been defined in medical terminology, and hypocomplementemic urticarial vasculitis disease may be a more appropriate name for HUVS.

298 HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME

HUVS is a relatively rare disease, more common in women (60–80% of cases), with a peak incidence in the fifth decade (average age at diagnosis being 43). HUVS is less common in the pediatric population, but has been described as more severe in these patients. Although rare, HUVS should be considered in children with glomerulonephritis, urticarial rash, arthralgias or arthritis, and pulmonary disease.

Etiology HUVS is an autoimmune disease of unclear etiology. Although more common in females, HUVS has not been associated with other genetic factors. A description of HUVS in identical twins has showed variation in disease severity and time of onset, suggesting HUVS as a disease entity requiring and influenced by both genetic and environmental factors. HUVS is characterized by anti-C1q antibodies and low complement levels as well as manifestations of systemic inflammatory vasculitis. Whether anti-C1q antibodies contribute to the pathogenesis of HUVS or SLE is not fully understood. It is hypothesized that anti-C1q antibodies contribute to organ dysfunction (especially renal) by forming complexes with C1q, dsDNA, and anti-dsDNA antibodies. These autoimmune complexes could then become less soluble and deposit in the membranes of vascular beds, giving rise to an inflammatory autoimmune disease with unknown environmental exacerbating factors. These antibodies may also represent an epiphenomenon of HUVS as something commonly seen during the course of the disease, but not necessarily associated with the disease.

HUVS has been associated with viral infections, Henoch–Scho¨nlein purpura, SLE, Sjo¨gren’s syndrome, IgM paraproteinemia (Schnitzler’s syndrome), and malignancies. There have been various case reports describing the association of HUVS and malignancy and recently, the association with a diffuse large B cell lymphoma was noted. This recent finding is particularly interesting in that complement deficiencies are known to precede the development of B cell lymphomas. It is proposed that HUVS may cause an underlying stimulation of B cells, much like that seen in mixed cryoglobulinemia, eventually leading to B cell overgrowth/expansion and lymphoma. If this proposed association is true, close follow-up of patients with HUVS would be needed due to the possible development of non-Hodgkin’s lymphoma. As previously mentioned, HUVS also shares many features characteristic of SLE, and can have renal involvement virtually indistinguishable from SLE nephritis, even histologically. Because of the many similarities between these two disease entities, some authors have suggested HUVS as being an SLErelated syndrome, and that a spectrum of diseases may occur ranging from SLE to HUVS (Table 1).

Pathology Obtaining a biopsy of an active lesion remains the gold standard for diagnosing UV. Leukocytoclastic vasculitis (LCV) is what is typically seen histopathologically. The vasculitis involved in UV is most commonly described as a venulitis, affecting the postcapillary venules, and is considered to be a type III

Table 1 Causes and associations of UV and HUVS Category 1. 2. 3. 4. 5. 6.

No cause Infections Hematologic diseases Connective tissue diseases Immune complex diseases Therapeutics and supplements

7. Complement involvement 8. Immunoglobulin abnormalities 9. Physical conditions 10. Malignancy 11. Other

Examples Idiopathic (most common) Hepatitis B, hepatitis C, infectious mononucleosis, Lyme disease Leukemia, lymphoma, polycythemia rubra vera, idiopathic thrombocytopenia purpura SLE, Sjo¨gren’s syndrome Serum sickness Diltiazem, potassium iodide, cimetidine, procarbazine, fluoxetine, butylhydroxytoluene (preservative in chewing gum), methotrexate, etanercept, cocaine, Bacille–Gue´rin vaccine, procainamide, dexfenfluramine (and other centrally acting appetite suppressants), butylated hydroxyanisole, and certain herbs C3 deficiencies, C4 deficiencies, C3 nephritic factor activity IgM monoclonal gammopathy (Schnitzler’s syndrome), IgG gammopathy (paraproteinemia), IgD gammopathy, IgA gammopathy, cryoglobulinemia Cold exposure (cold urticaria), sun exposure (solar urticaria), exercise-induced urticaria Hodgkin’s disease, malignant teratoma, paraneoplastic B cell non-Hodgkin’s lymphoma, IgA myeloma, metastatic adenocarcinoma of the colon Inflammatory bowel disease, striae distensae of pregnancy, Cogan’s syndrome, amyloidosis (Muckle–Wells syndrome)

HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME 299

hypersensitivity reaction. The minimal criteria used to characterize an urticarial syndrome as UV include leukocytoclasis or fibrin deposits within the lesion, whether or not blood cell extravasation exists. The exact definition of LCV remains ambiguous. LCV has many features including injury and swelling of endothelial cells (usually of the venules), extravasation of red blood cells, fragmentation of leukocytes with nuclear debris (leukocytoclasis), fibrin deposits in and around the venules, and a perivascular infiltrate composed mostly of neutrophils. A predominance of neutrophils in the infiltrate is characteristic of HUV. Leukocytoclasis and fibrinoid deposits have been deemed as the most important aspects of LCV because they represent direct signs of vessel damage. Inflammation is seen within and around capillaries and postcapillary venules, with an infiltrate composed mostly of neutrophils and occasional lymphocytes and eosinophils. Leukocytoclasis, defined as the fragmentation of neutrophils resulting in scattered nuclear fragments or nuclear dust, is frequently seen in the infiltrate. Erythrocytes may extravasate, and can be seen perivascularly as well as in the interstitium. Swelling of the endothelial cells also occurs, and may be so extensive as to occlude the lumen of affected vessels. Finally, fibrinoid degeneration of the endothelial cells occurs, characterized by a blunting of the vascular outline due to edema and fibrin deposits.

Clinical Features The most common presentation of HUVS clinically is the urticarial wheal. These cutaneous manifestations are usually persistent, painful, and tender, and sometimes have a burning sensation. As noted in the major criteria for the diagnosis of HUVS, these urticarial lesions must be persistent and present for at least 6 months. When the urticarial wheals go away, they characteristically leave purpura or hyperpigmentation as residual effects. Other cutaneous

manifestations include livedo reticularis, an erythema multiform-like eruption, angioedema, and rarely bullae or Raynaud’s phenomenon. When approaching patients with the possibility of HUVS, clinicians should first establish the diagnosis of UV, and then look for systemic involvement and hypocomplementemia characteristic of HUVS. The first step in the investigation involves a skin biopsy of a lesion for histologic examination. Histologic examination includes a hematoxylin and eosin stain of the biopsy specimen, to see if the criteria for vasculitis are fulfilled. Direct immunofluorescence usually demonstrates immune complexes, complement, and fibrin deposits in the basement membrane. Hypocomplementemia can then be quantified, in terms of reduced levels of C1, C2, C3, and C4. These components represent factors from the classical complement pathway, and can result in reduced levels of CH50/100. With respect to the C1 component of complement, the C1q levels are almost exclusively reduced more significantly than levels of C1r and C1s (the other components of C1). The diagnosis of HUVS involves major, minor, and exclusion criteria. For the diagnosis of HUVS, the patient must have both major criteria, at least two minor criteria, and none of the exclusion criteria (Table 2). Once the diagnosis of HUVS has been established the astute clinician should be aware of and evaluate for systemic involvement (Table 3). Many systemic associations have been linked to HUVS, some of which are capable of causing a great deal of morbidity and mortality. A chest X-ray and pulmonary function tests may be appropriate for suspected pulmonary involvement, and an echocardiogram may give light to cardiac abnormalities. Infections, certain drugs, immunoglobulin abnormalities, and hematologic diseases have been linked to the development of HUVS, and should be considered as possible coexisting entities (see Table 1). Angioedema occurs in as many as 50% of patients with HUVS, frequently involving the lips, tongue,

Table 2 Diagnostic criteria for HUVS Level of criteria

Specifics

Needed for the diagnosis

1. Major criteria

Urticaria for more than 6 months and hypocomplementemia

2. Minor criteria

Venulitis of the dermis (established via biopsy), arthralgia or arthritis, mild glomerulonephritis, uveitis or episcleritis, recurrent abdominal pain, and a positive C1q precipitin test by immunodiffusion with an associated suppressed C1q level Significant cryoglobulinemia (cryocrit 41%), an elevated anti-DNA antibody level, a high titer of antinuclear antibodies, hepatitis B antigenemia, depressed C1 esterase inhibitor level, or an inherited complement deficiency

The patient must have both major criteria The patient must have at least two of the minor criteria

3. Exclusion criteria

The patient cannot have any of the exclusion criteria

300 HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME Table 3 Approach to diagnosis of HUVS Step of investigation

Laboratory data or study modality employed

1. Confirm the diagnosis of UV 2. Confirm the diagnosis of HUVS

Biopsy of early lesions, histopathologic evaluation for vasculitis Confirm hypocomplementemia (serum complement levels: C1q, C3, C4, and CH50) and apply the diagnostic criteria Complete blood count, erythrocyte sedimentation rate, urea, urinalysis, chest X-ray, liver function tests, circulating immune complexes, electrolytes (including creatinine to evaluate for renal involvement), immunoglobulins, cryoglobulin Echocardiogram, bronchial lavage, pulmonary function tests, joint and skeletal X-rays

3. Evaluate for systemic disease

4. Special tests that may be ordered according to the history 5. Other tests for suspected associations

Antinuclear antibodies, anti-DNA antibody, anti-SSA(Ro)/SSB(La) antibodies, Borrelia antibody titer, antiphospholipid antibodies, cryoglobulins, C1q precipitin, C3 nephritic factor, hepatitis B and C serology, monospot test for Epstein–Barr virus infection

periorbital tissues, and hands, but rarely life-threatening. Reversible tracheal stenosis has also been seen. Musculoskeletal complaints include arthritis and arthralgias, seen in nearly all patients (incidence as high as 75%). Jaccoud’s arthropathy, a condition paralleling rheumatoid arthritis (RA) clinically, has been seen in a number of patients with HUVS, as well as patients with SLE, scleroderma, Sjo¨gren’s syndrome, mixed connective tissue disease, acquired immuno-deficiency syndrome (AIDS), mycosis fungoides, and angioimmunoblastic lymphadenopathy. In patients with HUVS, Jaccoud’s arthropathy has been associated with severe acute noninfectious endocarditis with superficial fibrinoid necrosis, and fibrin deposition in the cardiac valves, causing a cardiac valvulopathy, which in many instances requires cardiac valve replacement. Pulmonary involvement is seen up to 50% of the time in patients with HUVS, with pleuritis and chronic obstructive pulmonary disease (COPD) being common presentations. Lung biopsies in these patients demonstrate a leukocytoclasitic vasculitis characteristic of the disease. Lung involvement in HUVS patients is a frequent cause of morbidity and mortality, associated with respiratory failure and death. The mechanism of development of COPD in these patients is unclear; however, it is proposed that there is a local release of elastase by the neutrophils present, inducing a proteolytic lung destructive process. In addition, cigarette smoking may increase the number of neutrophils present as well as decrease the antiproteolytic activity of a1-antitrypsin, exacerbating the lung destructive process. Like C1q, surfactant apoproteins contain a collagen-like region thought to be a potential site of anti-C1q antibody cross-reactivity, proposed to be a potential cause of surfactant dysfunction and lung disease in these patients. Most patients with lung involvement are concomitant smokers. Although smoking is considered to be a contributing factor in these patients with lung disease, many have disease out of proportion to the number of years

smoked, and lung disease has been seen in patients in their 30s and 40s with no history of tobacco use. Cigarette smoking appears to be a risk factor for fatal lung disease, and is thought to potentiate the proposed neutrophil-mediated elastase release in areas of vasculitis within the lung, leading to breakdown of lung architecture and emphysema. Renal involvement is seen in 50% of patients, with proteinuria and hematuria occurring 20–30% of the time. Renal involvement is generally mild; however, there have been a few instances of end-stage renal failure requiring dialysis and even kidney transplant in patients with HUVS. In most cases of renal involvement, deposits of immunoglobulins and complement have been recognized suggesting an immune-mediated component, or immune-complex type nephritis. Anti-C1q antibodies have been associated with renal involvement in both SLE and HUVS, making it difficult to distinguish between these two entities. In fact, it is virtually impossible to differentiate histologically between SLE- and HUVSrelated nephritis; however, the renal prognosis is better in patients with HUVS, and steroid therapy seems to be very effective in these patients. It has recently been suggested that monitoring the C1q levels could be an important tool in predicting renal flares. Gastrointestinal symptoms including abdominal pain, nausea, vomiting, and diarrhea are seen in approximately 17–30% of patients. Ocular involvement is rare, and usually involves the uveal tract. Other ocular manifestations include conjunctivitis and episcleritis. More rare systemic manifestations of HUVS include pericarditis and cardiac tamponade, constitutional symptoms (fever, fatigue, malaise), myositis, dermal ulcerations, Raynaud’s phenomenon, digital infarction, dermatographism, livedo reticularis, serpiginous choroidopathy with vision loss, hypothyroidism, pseudotumor cerebri, peripheral neuropathy, lower cranial nerve palsies, and transverse myelitis.

HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME 301

Pathogenesis By definition, HUVS is associated with low complement levels. Autoantibodies to C1q with a subsequent decreased C1q level is seen almost exclusively in patients with HUVS, and such levels may be predictive of severity. These autoantibodies are referred to as C1q precipitins, identified as IgG antibodies directed toward the collagen-like, Fc-dependent portion of C1q. C1q antibodies and low levels of C1q may also be seen in SLE, RA, Felty’s or Sjo¨gren’s syndrome, glomerulonephritis, and cryoglobulinemia. Occasionally, depressed C3, C4, C5, and hemolytic complement levels (CH50) are also seen. Female sex, interstitial neutrophilia, and the presence of a lupus band on direct immunofluorescence studies of skin biopsy specimens have been shown to predict hypocomplementemia. In addition, the erythrocyte sedimentation rate (ESR) is usually elevated, and 50% of the time patients have positive antinuclear antibody (ANA) titers. Patients do not usually have anti-dsDNA or anti-Smith (anti-Sm) antibodies.

Animal Models There are currently no known available animal models of HUVS.

Management and Current Therapy There is no definite therapy for HUVS that works all the time. Multiple treatments have been tried with attempts to manage the underlying causes, if one is identified (Table 4). Some therapies include nonsteroidal anti-inflammatory drugs (NSAIDs) (indomethacin) and antihistamines for joint pain and pruritus. Moderate to high doses of glucocorticoids have proven effective; however, due to the multiple complications associated with long-term therapy, other regimens have been used including antimalarial and immunosuppressive therapy with dapsone,

colchicine, hydroxychloroquine, azathioprine, methotrexate and cyclophosphamide, mycophenolate mofetil (MMF) and intravenous immunoglobulin (IVIG). MMF is a relatively new immunosuppressant agent, recently shown to be efficacious as maintenance therapy in a couple of patients with HUV. In a recent report, another patient resistant to MMF and IVIG had a rapid response to rituximab. Hydroxychloroquine is an antimalarial sometimes used as therapy in RA and HUVS. Although the mechanism of action is unclear, dramatic responses to therapy have been reported in the literature in patients resistant to other therapeutic modalities. Dapsone is a relatively nontoxic drug compared to long-term corticosteroid or cytotoxic therapy, and sustained remissions in HUVS patients treated with dapsone have been described. Dapsone suppresses neutrophil-mediated inflammation and has been proposed as a first-line agent in HUVS therapy due to its high therapeutic index, ease of administration, low cost, and persistent beneficial effects. Recently, the addition of pentoxyphylline to dapsone therapy was found to be helpful in inducing remission in HUV. Additionally, the use of dapsone followed by colchicine caused clearing of disease in one patient. Due to the proposed neutrophil involvement in HUVS-associated lung disease, dapsone may be an ideal drug in the early stages in efforts to arrest lung damage. Cyclosporin A (CyA) primarily suppresses T cell activation and has been used to treat a number of glomerular diseases associated with nephrotic syndrome. Recently CyA was found to be useful in a patient with HUVS-associated nephrotic syndrome. Although the great majority of patients with HUVS have multi-organ involvement, the prognosis still remains good, with few patients ever dying as a direct result of the disease. Some systemic complications, however, especially pulmonary (COPD) and laryngeal edema can be the source of great morbidity and at times mortality.

Table 4 Approaches to therapy for HUVS Symptom treated

Therapeutic agent usually effective

1. Pruritus 2. Cutaneous manifestations (urticaria) 3. Systemic manifestations 4. Severe systemic disease or refractory to other therapies 5. Proposed combination therapy, also employed in refractory disease

Antihistamines Dapsone, hydroxychloroquine, colchicine, indomethacine, and corticosteroids Dapsone, corticosteroids, and interferon alpha Azathioprine, methotrexate, mycophenolate mofetil, to cyclophosphamide, cyclosporin, and plasma exchange Cyclophosphamide and dexamethasome pulse therapy, interferon alpha and ribavirin (and occasionally doxepin), prednisone and azathioprine, dapsone and pentoxifylline, dapsone and corticosteroids, and prednisone and low-dose gold therapy

302 HYPOXIA AND HYPOXEMIA See also: Asthma: Overview. Bronchiectasis. Bronchiolitis. Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations; Emphysema, Alpha-1Antitrypsin Deficiency; Emphysema, General; Smoking Cessation. Histamine. Panbronchiolitis. Signs of Respiratory Disease: Breathing Patterns. Symptoms of Respiratory Disease: Dyspnea. Vasculitis: Overview.

Further Reading Berg RE, Kantor GR, and Bergfeld WF (1988) Urticarial vasculitis. International Journal of Dermatology 27: 468–472. Berg RE, Kantor GR, and Bergfeld WF (1988) Urticarial vasculitis in adults. International Journal of Dermatology 27: 504–505. Black AK (1999) Urticarial vasculitis. Clinics in Dermatology 17: 565–569. Chen HJ and Bloch KJ (2001) Hypocomplementemic urticarial vasculitis, Jaccoud’s arthropathy, valvular heart disease, and reversible tracheal sienosis: a surfeit of syndromes. Journal of Rheumatology 28: 383–386. Davis MDP and Brewer JD (2004) Urticarial vasculitis and hypocomplementemic urticarial vasculitis syndrome. Immunology and Allergy Clinics of North America 24: 183–213. Davis MDP, Daoud MS, Kirby B, Gibson LE, and Rogers RS (1998) Clinicopathologic correlation of hypocomplementemic and normocomplementemic urticarial vasculitis. Journal of the American Academy of Dermatology 38: 899–905.

Falk DK (1984) Pulmonary disease in idiopathic urticarial vasculitis. Journal of the American Academy of Dermatology 1: 346–352. Gammon WR and Wheeler CE (1979) Urticarial vasculitis: report of a case and review of the literature. Archives of Dermatology 115: 76–80. Ghamra Z and Stoller JK (2003) Basilar hyperlucency in a patient with emphysema due to hypocomplementemic urticarial vasculitis syndrome. Respiratory Care 48: 697–699. Jones MD, Tsou E, Lack E, and Cupps TR (1990) Pulmonary disease in systemic urticarial vasculitis: the role of bronchoalveolar lavage. American Journal of medicine 88: 431–434. Mehregan DR, Hall MJ, and Gibson LE (1992) Urticarial vasculitis: a histopathologic and clinical review of 76 cases. Journal of the American Academy of Dermatology 26: 441–448. Monroe EW (1999) Urticarial vasculitis: an updated review. Journal of the American Academy of Dermatology 5: 88–95. O’Donnell B and Black AK (1995) Urticarial vasculitis. International Angiology 14: 166–174. Schwartz HR, McDuffie FC, Black LF, Schroeter AL, and Conn DL (1982) Hypocomplementemic urticarial vasculitis: association with chronic obstructive pulmonary disease. Mayo Clinic Proceedings 57: 231–238. Venzor J, Lee WL, and Huston DP (2002) Urticarial vasculitis. Clinical Reviews in Allergy and Immunology 23: 201–216. Wisnieski JJ (2000) Urticarial vasculitis. Current Opinion in Rheumatology l2: 24–31. Wisnieski JJ, Baer An, Christensen J, et al. (1995) Hypocomplementemic urticarial vasculitis syndrome: clinical and serologic findings in 18 patients. Medicine (Baltimore) 74: 24–41.

HYPOXIA AND HYPOXEMIA G Gutierrez, George Washington University, Washington, DC, USA

may lead to hypoxia, a condition of low tissue O2 concentration.

& 2006 Elsevier Ltd. All rights reserved.

Normal Physiological Processes Abstract Hypoxemia and hypoxia refer to conditions of decreased oxygen concentration in arterial blood and in the peripheral tissues, respectively, that may be experienced by individuals exposed to extreme environmental conditions (high altitude), accidental conditions (diving accidents, near drowning, and carbon monoxide poisoning), or with diseases that affect the transport of oxygen to the tissues. If untreated, hypoxemia and hypoxia may lead to organ dysfunction and eventually to death. Anaerobic sources of energy help supplement faltering aerobic energy production during hypoxia. Treatment of hypoxemia and hypoxia should be directed at the resolution of the precipitating cause.

Description Hypoxemia, a condition that literally means low blood O2 concentration, results from conditions or diseases that impede the transfer of O2 from the lungs into arterial blood (Table 1). Hypoxemia

Prokaryotic cells derive their energy from glycolysis, a process that oxidizes and splits the six-carbon glucose molecule into two three-carbon pyruvate molecules (Figure 1). The energy released during glycolysis is conserved by the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) and by the reduction of nicotinamide adenine dinucleotide (NAD þ ) to NADH. Glycolysis would stop if it lacks supply of NAD þ . Higher organisms regenerate NAD þ by reducing pyruvate to lactate, allowing glycolysis to continue indefinitely: Glucose þ 2 ADP þ 2 Pi-2 lactate þ 2 ATP where Pi denotes inorganic phosphate. The hydrolysis of ATP provides the energy required to power vital cellular processes, such as protein synthesis, locomotion, and membrane-associated

HYPOXIA AND HYPOXEMIA 303 Table 1 Causes of hypoxemia, hypoxia, and dysoxia Hypoxemia High altitude Enclosed spaces with low PO2 Near drowning Airway obstruction Hypoventilation Central hypoventilation Neuromuscular disorders Hypoventilation–obesity syndrome Ventilation–perfusion mismatch Adult respiratory distress syndrome Alveolar hemorrhage Asthma/obstructive pulmonary disease Interstitial lung disease Lung contusion Pneumonia Pneumothorax Pulmonary hypertension Pulmonary and intracardiac vascular shunts Pulmonary emboli – thrombus and fat Heart failure Anemia, including sickle cell anemia Carbon monoxide intoxication Hemoglobinopathies Methemoglobinemia Hypoxia Peripheral embolic phenomena Diffuse intravascular coagulation Shock – cardiogenic, hypovolemic, and septic Dysoxia Pyruvate dehydrogenase inhibition Mitochondrial enzymes and cytochrome chain (a,a3) inhibition NAD þ depletion by poly-ADP ribose polymerase (PARP-1) activation Cyanide with uncoupling of oxidative phosphorylation

ionic pumps:

until transferred to oxygen, the final electron acceptor. The energy liberated by the mitochondrial electron cascade is coupled to the partly understood mechanism of oxidative phosphorylation, producing 36 ATP molecules per glucose molecule. The abundant cellular energy provided by aerobic metabolism allowed eukaryotic cells to form complex multicellular organisms. Growth, however, was limited by the diffusion of O2 from the environment. Species adapted to prevailing ecological conditions by developing organs to carry O2 from the surrounding media to all tissue cells. Animal O2 transport systems consist of heme pigments to reversibly bind O2 (hemoglobin), specialized cells to enfold but not consume the potentially toxic O2 (erythrocytes), organs to promote O2 diffusion from the environment into blood (lungs or gills), and the hydraulic pumps and circulatory conduits needed to convey O2-containing cells to and from the tissues (the heart and blood vessels). Air reaches the alveolar spaces following a negative pressure gradient during inspiration. Alveolar O2 partial pressure (PAO2) may be estimated from the alveolar air equation PA O2 ¼ FI O2 ðPatm
Pwater Þ
Pa CO2 ½FI O2 þ ð1
FI O2 Þ=RQ mmHg where FIO2 is the inspired O2 fraction (0.21 for air), Patm is the atmospheric pressure, Pwater is the water vapor pressure, PaCO2 is the arterial blood CO2 partial pressure, and RQ is the respiratory quotient. When breathing air at sea level, this equation approximates to

2 ATP-2 ADP þ 2 Pi þ 2 Hþ þ energy

PA O2 ¼ 150
Pa CO2 =0:8 mmHg

Combining the previous equations yields the general expression for glycolysis:

Oxygen diffuses from the alveoli into pulmonary capillary blood, where it dissolves in plasma or binds to hemoglobin. The alveolar–arterial gradient is the difference between alveolar and arterial PO2 ðA
a ¼ PA O2
Pa O2 Þ. Ventilation perfusion inequality, shunt, or (rarely) diffusion limitation of oxygen can widen the A–a gradient across the blood– gas barrier. The sigmoid-shaped oxyhemoglobin dissociation curve (ODC) defines the relationship between plasma PO2 and fractional hemoglobin O2 saturation (SO2). The concentration or O2 content of blood is the sum of O2 bound to hemoglobin and O2 dissolved in plasma:

Glucose-2 lactate þ 2 Hþ þ energy The emergence of mitochondria, remarkable organelles capable of handling oxygen safely, provided the energy required for the evolution of eukaryotic cells. Mitochondria are thought to be descendants from symbiotic bacteria since they contain their own DNA and replicate independently. Mitochondria generate vast quantities of energy from O2, generating CO2 and water as by-products. In the mitochondria, pyruvate is oxidized to acetylCoA, which enters the tricarboxylic acid cycle, producing CO2 and electrons. These electrons move down a cascade of progressively lower energy states

O2 content ¼ 13:9  SO2  ½Hb þ 0:031  PO2

ml O2 per liter of blood

304 HYPOXIA AND HYPOXEMIA Glycolysis Fatty acids

Glucose

Amino acids

ADP + Pi

NADH

NAD+

ATP

Lactate

Pyruvate Cytosol e−

Mitochondrion

e−

CO2 Acetyl-CoA Citrate Oxaloacetate e− e−

Tricarboxylic acid cycle CO2

e−

e− CO2

2H− + 1/2 O2

H2O

Transfer of electrons coupled to oxidative phosphorylation ADP + Pi

ATP

Figure 1 Glycolysis and oxidative phosphorylation.

where the term 13.9 is the O2 carrying capacity of hemoglobin, [Hb] is the concentration of hemoglobin in blood (g l
1), and the term 0.031 is the solubility of O2 in plasma for PO2 in mmHg. Arterial blood delivers oxygen and metabolic substrate to the tissues in accordance to local energy requirements. Higher level control of the circulation is aimed primarily at counteracting gravitational effects by stimulation of arteriolar a and b receptors. The microvasculature – the vast array of terminal arterioles, capillaries, and venules – regulates intraorgan blood flow through vasomotion, rhythmic oscillations in vascular tone. Accurate measurements of regional O2 delivery are difficult to obtain. A first approximation to the adequacy of cellular O2 transport may be obtained by calculating the systemic rates of O2 delivery (DO2) and O2 uptake (VO2), DO2 ¼ cardiac output  O2 content

ml O2 per minute

Systemic O2 uptake (VO2) may be measured directly from the expired gases as VO2 ¼ ½FI O2 ð1
FE O2
FE CO2 Þ=ð1
FI O2 Þ
FE O2   VE ml O2 per minute where FIO2 and FEO2 denote the inspired and expired fractional O2 concentration, respectively; FECO2 is the fractional concentration of expired CO2; and VE is the expired minute volume. A practical problem with this expression is that small inaccuracies in measuring FIO2 at values 40.60 may produce large errors in VO2. Systemic VO2 may also be calculated with the aid of Fick’s principle as VO2 ¼ cardiac output  ðarterial O2 content
mixed venous O2 contentÞ ml O2 per minute This calculation requires the insertion of a pulmonary artery catheter to gain access to pulmonary

HYPOXIA AND HYPOXEMIA 305

artery blood. Fick’s principle underestimates total VO2 since it does not account for pulmonary VO2. The latter can be substantial in severe pneumonia or in adult respiratory distress syndrome. The ratio of tissue CO2 production (VCO2) to VO2 is the respiratory quotient, RQ ¼ VCO2 =VO2 The type of substrate consumed determines the value of RQ, being 0.7 for free fatty acids and 1.0 for glucose. The O2 extraction ratio (ERO2), the fraction of DO2 taken up by the tissues, is another useful parameter: ERO2 ¼ VO2 =DO2

Systemic oxygen uptake (VO2)

ERO2 is approximately 20–25% at rest, increasing to 50% or greater with exercise or during cardiac failure at rest. ERO2 values o15% are suggestive of functional peripheral shunting, as might occur during sepsis. The DO2–VO2 relationship (Figure 2) is the subject of great clinical interest. Experimental preparations subjected to decreases in DO2 initially show little variation in VO2 resulting from microvascular responses that increase ERO2. Decreases in VO2 occur below a critical DO2 because cellular O2 supply cannot keep up with the rate of aerobic metabolism. Anaerobic processes now must be recruited to complement the faltering mitochondrial supply of ATP. This condition, if protracted, may lead to the death of the animal. Measurements of DO2 and VO2 from critically ill individuals uniformly show a straight line, one lacking a critical DO2 defining the threshold for anaerobic metabolism. Failure to account for the passage

Critical DO2 Anaerobic region

Aerobic region

Systemic oxygen delivery (DO2) Figure 2 Schematic DO2–VO2 relationship derived from experimental studies.

of time in the DO2–VO2 relationship may explain the difference in findings between clinical and experimental data. Animal experiments usually take a few hours, during which time DO2 is relentlessly lowered. Clinical measures are gathered over a longer period, typically several days, and no efforts are made to decrease DO2. During this time, the patient’s metabolic rate is bound to change, leading to variations in DO2 in response to changes in VO2, not the reverse. By considering time as an additional dimension, the two-dimensional DO2–VO2 curve becomes a surface whose contour varies in concert with the patient’s clinical condition. Figure 3 illustrates the temporal evolution of a hypothetical DO2–VO2 relationship. In this example, the person’s condition worsens with time as increases in DO2 become less effective in their ability to increase VO2. The figure also shows that samples of DO2 and VO2 taken at different times (red circles) may project as a straight line if viewed from a two-dimensional perspective.

Physiological Processes in Respiratory Diseases Diseases that affect the lungs will impair O2 diffusion, widen the A–a gradient, and produce hypoxemia, defined by a Pa O2 o60 mmHg or Sa O2 o90%. An acute response to hypoxemia is hyperventilation, resulting in decreased PaCO2 and, according to the alveolar air equation, a modest increase in PAO2. A more protracted response to hypoxemia is polycythemia produced by the release of erythropoietin by the kidneys. The arterial blood O2 content is a more precise index of hypoxemia than either PaO2 or SaO2. Decreases in [Hb] or conditions that interfere with hemoglobin O2 binding will diminish arterial blood O2 concentration but may not affect PaO2 or SaO2. Shown in Figure 4 are the O2 content curves for blood with ½Hb ¼ 150 g l
1 , a normal value, and ½Hb ¼ 100 g l
1 , a moderate degree of anemia. The O2 content at Pa O2 ¼ 100 mmHg for the anemic case corresponds to a Pa O2 ¼ 35 mmHg in the nonanemic case. Decreases in cardiac output, [Hb], and SaO2 are quantitatively similar in lowering DO2, but they elicit different systemic responses. Perhaps as an evolutionary response to blood loss, humans appear to tolerate decreases in [Hb] better than equivalent reductions in cardiac output or SO2. As long as circulating blood volume is maintained, humans tolerate acute decreases in [Hb] to 50% of normal values (i.e., 70 g l
1 ) with remarkable ease. On the other hand, decreases in SaO2 or cardiac output up to 50% of normal values are likely to prove fatal.

O2 consumption

306 HYPOXIA AND HYPOXEMIA

O

2

de liv er y

e

Tim

Figure 3 Three-dimensional representation of the DO2–VO2 relationship as a function of time. Darker regions represent conditions likely to produce hypoxemia and hypoxia. In this hypothetical example, the ability of the individual to increase VO2 in response to increases in DO2 decreases with time. This is perhaps the result of regional microcirculatory alterations or of cellular apoptosis. The red circles represent samples obtained at different times during the patient’s illness. When projected to a two-dimensional perspective, the DO2–VO2 relationship traced by these samples will appear as a straight line.

O2 content (ml O2 per liter of blood)

200 [Hb] = 150 g l–1 150 [Hb] = 100 g l–1

100

50

0 0

20

40

60

80

100

PO2 (mmHg) Figure 4 Blood O2 content curves for hemoglobin concentrations of 150 g l
1 (normal) and 100 g l
1 (anemia). The O2 content of fully saturated anemic blood corresponds to the O2 content attained at PO2 ¼ 35 mmHg for normal blood.

Considerable debate exists on the benefits of therapeutic increases in DO2 during critical illness. One strategy used to augment DO2 in critically ill patients is to increase arterial blood O2 content by raising

either [Hb] or SaO2. This approach is problematic due to the slow process of de novo hemoglobin generation. Moreover, blood transfusions can be detrimental to some critically ill patients. Similarly, significant increases in arterial O2 content by increasing FIO2 are nearly impossible to attain, given the flat portion of the ODC (unless the patient is taken to a hyperbaric chamber). A more common strategy used when increasing DO2 is to increase cardiac output produced by the infusion of inotropic agents, such as dobutamine. Randomized trials of protocol-driven increases in DO2 have not demonstrated improved survival. However, studies have shown decreases in mortality when resuscitation is initiated early in the course of the patient’s illness. Hypoxia refers to decreases in tissue oxygenation capable of limiting aerobic metabolism. Measures of venous SO2 and lactate concentration can detect individual organ hypoxia. On the other hand, systemic measures of mixed venous SO2 and arterial lactate concentration are unreliable indices of regional tissue hypoxia. Other, less practical techniques to measure intracellular oxygen concentration include surface

HYPOXIA AND HYPOXEMIA 307 Table 2 Anaerobic sources of ATP Glycolysis Glucose þ 2 ADP þ 2 Pi - 2 lactate þ 2 ATP Creatine kinase reaction PCr þ ADP þ H þ - Cr þ ATP Adenylate kinase reaction (myokinase) ADP þ ADP - AMP þ ATP

polarographic electrodes, oxygen microelectrodes, myoglobin cryospectrophotometry, fluorescence techniques, and electron paramagnetic resonance oximetry. These methods provide little information on intramitochondrial PO2, a difficult parameter to measure with a magnitude o0.1 mmHg. Moreover, there may be instances in which mitochondrial oxygen may be plentiful but the cellular bioenergetic machinery is impaired and cannot use it – a condition termed cytopathic hypoxia. Dysoxia is defined as an imbalance between the rates of aerobic energy production and cellular energy utilization. Dysoxia can be inferred by measuring metabolites involved in the anaerobic generation of ATP. There are three metabolic sources of anaerobic ATP: glycolysis, the creatine kinase reaction, and the adenylate kinase reaction (Table 2). As mentioned previously, glycolysis can be monitored by measures of blood lactate concentration, although clinical interpretation of hyperlactatemia can sometimes be challenging. The creatine kinase reaction occurs in selected organs (skeletal muscle, heart, and brain) and may be monitored by magnetic resonance spectroscopy. The adenylate kinase reaction occurs in most cells, and the products of AMP degradation, xanthine and hypoxanthine, may be monitored using high-pressure liquid chromatography. A more practical method to estimate regional dysoxia is to measure tissue CO2 concentration. Tissue acidosis occurs in hypoxia since the major anaerobic sources of ATP production, glycolysis and the adenylate kinase reaction, do not recycle H þ produced by the hydrolysis of ATP. Buffering of H þ by bicarbonate liberates CO2, a gas that can be measured with relative ease using gastric or sublingual tonometry. See also: Acute Respiratory Distress Syndrome. Adenosine and Adenine Nucleotides. Alveolar

Hemorrhage. Arterial Blood Gases. Asthma: Acute Exacerbations. Carbon Monoxide. Diffusion of Gases. Diving. Endotoxins. Erythrocytes. Exercise Physiology. Hemoglobin. High Altitude, Physiology and Diseases. Hyperbaric Oxygen Therapy. Interstitial Lung Disease: Overview. Oxygen Therapy. Oxygen–Hemoglobin Dissociation Curve. Peripheral Gas Exchange. Pulmonary Circulation. Pulmonary Edema. Pulmonary Thromboembolism: Pulmonary Emboli and Pulmonary Infarcts. Sleep Disorders: Hypoventilation. Vascular Disease. Ventilation, Perfusion Matching. Ventilation: Overview.

Further Reading Connett RJ, Honig CR, Gayeski TEJ, and Brooks GA (1990) Defining hypoxia: a systems view of VO2, glycolysis, energetics, and intracellular PO2. Journal of Applied Physiology 68: 842–883. Fink M (2002) Cytopathic hypoxia. Critical Care 6: 491–499. Gattinoni L, Brazzi L, Pelosi P, et al. (1995) A trial of goal-oriented hemodynamic therapy in critically ill patients. SvO2 Collaborative Group. New England Journal of Medicine 333: 1025–1032. Gladden LB (2004) Lactate metabolism: a new paradigm for the third millennium. Journal of Physiology 558: 5–30. Gutierrez G (2004) A mathematical model of tissue–blood carbon dioxide exchange during hypoxia. American Journal of Respiratory and Critical Care Medicine 169: 525–533. Gutierrez G, Wulf-Gutierrez ME, and Reines HD (2004) Monitoring oxygen transport and tissue oxygenation. Current Opinion in Anesthesiology 17: 107–117. Lopez-Barneo J, del Toro R, Levitsky KL, Chiara MD, and OrtegaSaenz P (2004) Regulation of oxygen sensing by ion channels. Journal of Applied Physiology 96: 1187–1195. Masson N and Ratcliffe PJ (2003) HIF prolyl and asparaginyl hydroxylases in the biological response to intracellular O2 levels. Journal of Cell Science 116: 3041–3049. Nelson DL and Cox MM (eds.) (2004) Lehninger Principles of Biochemistry, 4th edn. New York: Worth. Nilsson H and Aalkjær C (2003) Vasomotion: mechanisms and physiological importance. Molecular Interventions 3: 79–89. Rivers E, Nguyen B, Havstad S, et al. (2001) Early Goal-Directed Therapy Collaborative Group. Early goal-directed therapy in the treatment of severe sepsis and septic shock. New England Journal of Medicine 345: 1368–1377. Robin ED (1977) Special report: dysoxia. Abnormal tissue oxygen utilization. Archives of Internal Medicine 137: 905–910. Vincent JL and De Backer D (2004) Oxygen transport – the oxygen delivery controversy. Intensive Care Medicine 30: 1990–1996. Walsh TS (2003) Recent advances in gas exchange measurement in intensive care patients. British Journal of Anaesthesiology 91: 120–131. West JB (2004) A century of pulmonary gas exchange. American Journal of Respiratory and Critical Care Medicine 169: 897–902.

I IDIOPATHIC PULMONARY HEMOSIDEROSIS O C Ioachimescu, Cleveland Clinic Foundation, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Idiopathic pulmonary hemosiderosis (IPH) is a rare disease of unknown etiology, characterized by recurrent episodes of diffuse alveolar hemorrhage and sideropenic anemia. IPH occurs most commonly in children. During an acute episode, a constellation of cough, dyspnea, and hemoptysis with alveolar infiltrates and worsening anemia should raise the suspicion for intrapulmonary bleeding. Sputum, bronchoalveolar lavage (BAL), and eventually lung tissue examination show numerous hemosiderin-laden macrophages in the alveoli, without evidence of capillaritis/vasculitis, granulomatous inflammation, deposition of immunoglobulins, or immune complexes. Glucocorticoids and other immunosuppressive drugs seem to be effective during IPH exacerbations and, possibly, also during the remission phase. The significant improvement in morbidity and mortality of IPH during the past several decades is probably due to the long-term use of immunosuppressive therapy.

had large amounts of hemosiderin in the lungs. The clinical triad of hemoptysis, pulmonary opacities, and anemia with no other identified cause was considered diagnostic if the lung biopsy excluded vasculitic processes, granulomatous diseases, or immune depositions. In the past, many IPH cases may have been misdiagnosed, especially without a lung biopsy and before the advent of newer diagnostic tools and disease markers (e.g., antinuclear antibodies (ANA), antineutrophil cytoplasmic antibodies (ANCA), antiglomerular basement membrane (anti-GBM) antibodies, immunofluorescence studies). Approximately 80% of cases have the onset during childhood, most of them during the first decade of life. In adults there are two categories of IPH: primary-pediatric cases followed into adulthood and primary adult onset. Gender distribution appears to be balanced in childhood-onset IPH, and is slightly skewed towards a male predominance in adult onset. The incidence is estimated to be between 0.24 and 1.23 cases per million children per year.

Introduction Pulmonary hemosiderosis represents a disease characterized by iron accumulation in the local macrophages, as a consequence of bleeding and subsequent erythrocyte phagocytosis (Table 1). Hemorrhage into the lower respiratory tract can be a severe, lifethreatening condition. Conditions associated with diffuse alveolar hemorrhage (DAH) are very diverse and the diagnosis of idiopathic pulmonary hemosiderosis (IPH) is rendered only after meticulous exclusion of other conditions (see Alveolar Hemorrhage). DAH is characterized by hemoptysis, dyspnea, alveolar opacities on chest radiograph, and various degrees of anemia. Following a hemorrhagic episode, the alveolar macrophages convert the hemoglobin’s iron into hemosiderin within 72 h. Since the hemosiderin-laden macrophages reside for up to 4–8 weeks in the lungs, the term pulmonary hemosiderosis should be reserved for persistent or recurrent intra-alveolar bleeding beyond an 8-week interval. Virchow initially described the entity of IPH in 1864 as ‘‘brown lung induration.’’ In 1931, Ceelen published the autopsy findings in two children who

Diagnosis IPH is a diagnosis of exclusion. The clinical presentation varies significantly from acute, fulminant hemoptysis, to chronic cough and dyspnea, repetitive hemoptysis, fatigue, or only asymptomatic anemia. In adults the respiratory symptoms tend to be more pronounced, while in children failure to thrive and anemia (and less often hemoptysis) can be the presenting findings. The clinical course waxes and wanes between two phases, alternating in an unpredictable fashion: 1. Acute phase, corresponding to active DAH episodes, manifested by cough, dyspnea, hemoptysis, and, sometimes, respiratory failure. Almost 100% of adults experience hemoptysis during the disease course. The physical examination ranges from normal to that consistent with chronic anemia. 2. Chronic phase, which is characterized by a slow resolution of previous symptoms, with or without treatment. Although the physical exam may be normal, there are usually signs of chronic

310 IDIOPATHIC PULMONARY HEMOSIDEROSIS Table 1 Dictionary of terms Term

Etymology

Definition

Iron

Lat. Ferrum

Element, atomic weight 55.847, atomic number 26, specific gravity 7.874 (201C), valence 2, 3, 4, or 6; by weight, the fourth most abundant element on earth, making up the crust. It is found in nature as hematite (Fe2O3), taconite or common ore. It is a vital component of plants and animal organisms, and appears in hemoglobin

Hemosiderin

Gr. Hemo, blood þ Gr. Sideros, iron

Intracellular storage form of iron, containing a complex of ferric hydroxides, polysaccharides, proteins and iron 433% by weight, which stains blue with Prussian stain (Perls’ method)

Hemosiderosis

As above

Focal or general increase in tissue iron stores, especially in local macrophages

Pulmonary B

As above

Deposition of abnormal amounts of hemosiderin in the lungs, especially in the alveolar macrophages and interstitium, which may be due to various conditions (congestive heart failure, valvulopathies, vasculitides, etc.)

Idiopatic pulmonary B

As above

Repeated, sudden attacks of dyspnea, hemoptysis, diffuse alveolar bleeding, and anemia; seen mostly in children, but also in adults (also called Heiner– Ceelen disease) of unknown cause

Hemochromatosis

Gr. Hemo, blood þ Gr. Chromatosis, staining

Disorder due to deposition of hemosiderin in the parenchymal cells, causing tissue damage of the liver, pancreas, heart, and pituitary

Siderosis

Gr. Sideros, iron þ Gr. -osis, condition

Pneumoconiosis siderotica, a form of pneumoconiosis due to the presence of iron dust, usually serious when combined with silica exposure (silicosiderosis) Hemosiderosis Hyperferremia

Siderophage

Gr. Sideros þ Gr. Phagos, ingestion

Macrophage laden with phagocytosed iron-containing particles

Siderophilin

Gr. Sideros, iron þ Gr. Phyllos, loving

Non-heme iron-binding protein, also called transferrin (in vertebrate blood), which serves as the major circulatory transporter of iron

B, hemoriderosis. Modified from Ioachimescu O, Kotch A, and Stoller JK (2005) Idiopathic pulmonary hemosiderosis in adults. Clinical Pulmonary Medicine 12(1): 16–25, with permission from Lippincott Williams & Wilkins.

illness: pallor, emaciation, failure to thrive, and hepatosplenomegaly. In those with advanced fibrosis, cyanosis, bilateral crackles and clubbing, and signs of cor pulmonale may be present. Pulmonary Function Tests

Pulmonary function tests show variable degrees of ventilatory restrictive defects; no obstructive defects should be seen. The diffusing lung capacity for CO (DLCO) is low or low-normal during the chronic phase and elevated in the acute phase, as a manifestation of high CO uptake during active DAH. Arterial blood gas analysis may reveal hypoxemia or, rarely, hypercapneic respiratory failure. Laboratory Features

Other causes of DAH must be ruled out (see Alveolar Hemorrhage). The laboratory investigation reveals

variable degrees of anemia, without any quantitative or qualitative platelet defects, liver or kidney disease, coagulopathies, hemorrhagic diatheses, or any inflammatory syndromes. The sideropenic (transferrin saturation less than 40%), microcytic (mean corpuscular volume (MCV) o 80 fl) anemia is the direct consequence of recurrent DAH. Plasma ferritin concentration is normal or elevated due to alveolar macrophage synthesis and releases into the circulation. Bone marrow biopsy typically shows low iron stores. Although not a sensitive test, sputum examination may demonstrate intra-alveolar bleeding (erythrocytes and hemosiderin-laden macrophages) by hematoxylin-eosin and Prussian blue (Perls’) stains. Bronchoalveolar lavage (BAL) from involved areas has conceivably a higher diagnostic yield. The predominant cellular types are the alveolar macrophages, siderophages, and occasionally neutrophils and eosinophils.

IDIOPATHIC PULMONARY HEMOSIDEROSIS 311 Imaging Studies

The radiographic features of IPH vary in relation to the clinical phase. During the acute phase, the radiographs show diffuse alveolar opacities, predominantly in the dependant lung fields, with corresponding ground-glass attenuation or frank consolidations on the high-resolution computed axial tomography (CAT) scan (Figure 1). During the remission phase, the

alveolar opacities tend to resorb and an interstitial reticular or micronodular pattern of opacities may occur in the same areas, with variable degrees of traction bronchiectasis and, rarely, honeycombing. Technetium 99m (99mTc) or chromium 51 (51Cr) perfusion scans may demonstrate intra-alveolar bleeding, as opposed to a pneumonic process or alveolar edema, but their clinical utility has not been validated.

Lung Biopsy

Figure 1 CAT scan of the chest of a patient with IPH, showing fine bilateral nodules, fine subpleural reticulations, and discrete areas of ground-glass attenuation, the latter probably due to recent alveolar hemorrhage.

Transbronchial lung biopsy from involved areas is the initial diagnostic procedure of choice. Larger and multiple lobe samples can be obtained by videoassisted thoracoscopic surgery (VATS) or open lung biopsy. The lung biopsy can exclude specifically any granulomas, as seen in fungal, mycobacterial or other infections, in sarcoidosis, berylliosis, foreign-body reactions, Wegener’s granulomatosis, Churg–Strauss syndrome, microscopic polyangiitis, or isolated pauciimmune pulmonary capillaritis. In IPH, in addition to light microscopy (Figure 2), immunofluorescence or immunohistochemistry stains are needed to evaluate for immunoglobulin or immune complex depositions. Iron stains, such as Prussian blue (Perls’ stain), typically show numerous siderophages in alveolar and distal airways (Figure 3), as in other diseases characterized by DAH. The hemosiderotic lungs have a macroscopic appearance, called by Virchow as ‘‘brown induration.’’ On light microscopy, the alveolar walls are thickened, type 2 pneumocytes are hyperplastic, and the interstitium contains mild to moderate amounts

Figure 2 Hematoxylin-eosin stain of a hemosiderotic lung specimen, showing non-specific interstitial infiltration, scattered areas of collagen deposition, and significant alveolar hemorrhage. Magnification
100. Courtesy of Dr C Farver.

312 IDIOPATHIC PULMONARY HEMOSIDEROSIS

Figure 3 Perls’ (Prussian blue) stain of the same lung biopsy specimen as in Figure 2, showing multiple intra-alveolar and interstitial siderophages. Magnification
200. Courtesy of Dr C Farver.

of collagen. In addition, three other features are important: *

*

*

presence of intact or minimally fragmented erythrocytes in the distal airways and alveoli, as a reflection of recent or active DAH; multiple hemosiderin-laden macrophages (siderophages) in the alveolar spaces and interstitium, as an expression of subacute/chronic or recurrent intrapulmonary bleeding; and absence of any of the following: focal or diffuse smooth muscle cell proliferations, vascular malformations, malignancy, pulmonary infarcts, capillaritis/vasculitis, granulomatous inflammation, or infectious agents.

*

Useful ancillary tests are immunohistochemistry and immunofluorescence to exclude any intrapulmonary immunoglobulin or immune complex depositions. Electron microscopy (not mandatory for diagnosis) may reveal swelling of the alveolar cells, with minimal thickenings and localized disruptions of the basement membranes, but no electron-dense deposits in the alveolar basement membrane.

Pathogenesis The etiology of IPH remains largely unknown. There are several hypotheses that try to explain the occurrence of the structural lesions of the alveolar-endothelial membrane: *

Genetic theory. Familial clustering of IPH has been described, suggesting either a heritable trait

*

or a genetic predisposition to the influence of as yet unidentified environmental agents. So far, no specific genetic linkages or polymorphisms have been reported. Environmental theory. A series of outbreaks of alveolar hemorrhage among infants living in water-damaged houses in Cleveland and Chicago led to an extensive investigation of possible infectious or mycotoxigenic pathogenesis. Environmental mold/fungi, especially Stachybotrys atra, were thought to be associated with the development of the process. However, both the association with the exposure to molds, that is, S. atra, and the diagnosis of IPH have been questioned by a Centers for Disease Control and Prevention (CDC) review. Other molds, such as Alternaria, Aspergillus, Penicillium, and Trichoderma spp., have also been implicated, but a clear relationship with IPH has not been established. Trichotecens, fungal toxins with potent protein synthesis inhibitory capacity, have been shown to impede the angiogenesis underneath the rapidly forming alveolar membranes of infants and create in the acinar region an area susceptible to bleeding. Other authors proposed an association between IPH and exposure to insecticides, but this theory has not been verified. Autoimmune theory. An autoimmune pathogenesis has been suggested by the demonstration of circulating immune complexes. Immunohistochemical examinations of lung tissue in IPH generally have not supported an immune pathogenesis. Interestingly, approximately 25% of the patients who

IDIOPATHIC PULMONARY HEMOSIDEROSIS 313

*

*

survive more than 10 years with this disease subsequently develop some form of autoimmune disease. Furthermore, the treatment of choice is immunosuppressive, which is another observation in support of an immunopathogenetic mechanism for this disease. Allergic theory. Several children with IPH had detectable plasma antibodies (precipitins and IgE) against cows’ milk, which has led to the hypothesis of a systemic allergy/hypersensitivity reaction to milk components, although these findings have not been reproduced. Another interesting association is between IPH and celiac sprue. To date, there are more than 10 case reports of celiac disease (celiac sprue) with IPH, in whom the lung disease went into complete remission after institution of a gluten-free diet. Metabolic theory. In IPH, iron metabolism appears abnormal. In DAH, the alveolar macrophages accumulate acutely large amounts of iron, which quickly saturate intracytoplasmic ferritin, and then the free iron launches the sequence of cellular oxidative injury. Later, the iron derived from the metabolism of heme is found as trapped iron in hemosiderin (at 50–2000 times normal values) and, to a lesser extent, bound to other proteins (ferritin, etc.). Furthermore, in contrast to other macrophages, alveolar macrophages are less able to process and release iron. Within the macrophages, iron is removed from hemoglobin in an essential step catalyzed by heme-oxygenase (HO), which is subject to regulation. Heme-oxygenase 1 (HO-1) is induced by a variety of cell stressors, including various cytokines. The limited capacity of alveolar macrophages to metabolize hemoglobin may be related to its lack of HO, an enzyme that can be induced following the phagocytosis of alveolar erythrocytes. The exact relationship between HO and iron metabolism deserves further study.

The total amount of iron in the alveolar macrophages can be quantified using cytochemical (Perls’ stain), colorimetric (ferrozine stain), or particleinduced X-ray emission (PIXE) techniques. It is as yet unclear if the metabolic ‘arrest’ within the alveolar macrophages occurs at the level of HO involved in the degradation of hemoglobin in the endoplasmic reticulum, at the level of ferroportin 1, ceruloplasmin, or hephaestin (or other proteins involved in the iron ion egress from the macrophages), or at a different site.

Animal Model In animals, stachybotryomycotoxicosis leads to alveolar hemorrhage associated with significant

inflammation. Different animal models have been used (rabbits, hamsters, etc.) to study the effect of neutrophil elastase administration; the significance of DAH, diffuse alveolar damage (DAD) and emphysema caused by the elastase, and the interrelation with neutrophils’ recruitment process is still unclear.

Current Therapy There are no controlled studies and no IPH registries, so the management of this disease is the result of observations on a relatively small number of patients, which are frequently lost in follow-up over the years. A number of measures have been tried, as follows: *

*

*

Splenectomy, without significant results (there is no evidence of hypersplenism in hemosiderosis, as opposed to hemochromatosis). Inhaled corticosteroids, with insufficient experience to date. Systemic glucocorticoids, commonly started during episodes of DAH, with apparent good control and apparent impact on mortality, but with unclear effect on the chronic phase. We recommend a starting dose of 1 mg kg  1 day  1 prednisolone or equivalent for a couple of months, at which time the alveolar infiltrates tend to resorb, and then a slow taper over the next months, if symptoms do not recur. The majority of patients seem to respond favorably to long-term treatment with oral corticosteroids, with decreased number of IPH exacerbations and, possibly, a decline in fibrogenesis. Any attempt to stop the treatment portends a high risk of recurrence. However, in children and adolescents, the long-term treatment can be problematic because of side effects. Other immunosuppressive agents that have been tried with variable results include: azathioprine, hydroxychloroquine, cyclophosphamide, and methotrexate. Among them, azathioprine or hydroxychloroquine in combination with steroids are the most popular regimens. Singlelung transplantation has been reported in two cases. Unfortunately, both cases had a clinical course complicated by IPH recurrence in the transplanted lungs.

The response to therapy can be assessed during the episodes of DAH by improvement in symptoms, anemia, pulmonary opacities, and the return of DLCO to baseline, and on a long-term basis by reduction in the number and severity of hemorrhagic episodes and the progression of interstitial lung disease (as assessed by the decline of DLCO).

314 IMMUNOGLOBULINS

Prognosis The lack of prospective studies or large clinical registries makes the evaluation of short/long-term prognosis and the benefits of therapy very difficult. Overall, it appears that children and adolescents have a more severe course and prognosis, while adults have a more protracted course, with milder symptoms and more favorable prognosis. The most frequent cause of death is acute respiratory failure accompanying massive DAH, or chronic respiratory failure and cor pulmonale due to severe pulmonary fibrosis. The mean survival after the onset of symptoms is approximately 2.5–3 years (range 3 months to 10.5 years). In a small series of pediatric patients the 5-year survival rate was 86%, while in another retrospective analysis of 15 children with IPH followed for a mean of 17 years, 80% of the patients had a benign course. The better outcome might have been related to the more prolonged and sustained immunosuppressive therapy in these series. With improved immunosuppressive and corticosteroid therapy, most IPH patients tend to survive to childbearing age, so that exacerbations of pre-existing disease during pregnancy are more likely to occur. During pregnancy, both corticosteroids and azathioprine may be used in severe IPH flares, which tend to occur during the last trimester. There seems to be a general agreement that untreated disease is more deleterious than the side effects of the immunosuppressive agents, for both fetal and maternal health (at least with corticosteroids). See also: Alveolar Hemorrhage. Bronchoalveolar Lavage. Corticosteroids: Therapy. Granulomatosis: Wegener’s Disease. Lung Imaging. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome. Vasculitis: Overview.

Further Reading Centers for Disease Control and Prevention (1994) Acute pulmonary hemorrhage/hemosiderosis among infants – Cleveland,

January 1993–November 1994. MMWR. Morbidity and Mortality Weekly Report 43: 881–883. Centers for Disease Control and Prevention (2004) Investigation of acute idiopathic pulmonary hemorrhage among infants – Massachusetts, December 2002–June 2003. MMWR. Morbidity and Mortality Weekly Report 53: 817–820. Calabrese F, Giacometti C, Rea F, et al. (2002) Recurrence of idiopathic pulmonary hemosiderosis in a young adult patient after bilateral single-lung transplantation. Transplantation 74: 1643. Chryssanthopoulos C, Cassimos C, and Panagiotidou C (1983) Prognostic criteria in idiopathic pulmonary hemosiderosis in children. European Journal of Pediatrics 140: 123–125. Helman DL, Sullivan A, Kariya ST, et al. (2003) Management of idiopathic pulmonary haemosiderosis in pregnancy: report of two cases. Respirology 8: 398. Ioachimescu OC, Kotch A, and Stoller JK (2005) Idiopathic pulmonary hemosiderosis in adults. Clinical Pulmonary Medicine 12(1): 16–25. Ioachimescu OC, Sieber S, and Kotch A (2004) Idiopathic pulmonary haemosiderosis revisited. European Respiratory Journal 24: 162–170. Jarvis BB (2003) Stachybotrys chartarum: a fungus for our time. Phytochemistry 64: 53–60. Kiper N, Gocmen A, Ozcelik U, Dilber E, and Anadol D (1999) Long-term clinical course of patients with idiopathic pulmonary hemosiderosis (1979–1994): prolonged survival with low-dose corticosteroid therapy. Pediatric Pulmonology 27: 180–184. Kjellman B, Elinder G, Garwicz S, and Svan H (1984) Idiopathic pulmonary haemosiderosis in Swedish children. Acta Paediatrica Scandinavica 73: 584–588. Le Clainche L, Le Bourgeois M, Fauroux B, et al. (2000) Longterm outcome of idiopathic pulmonary hemosiderosis in children. Medicine (Baltimore) 79: 318–326. Milman N and Pedersen FM (1998) Idiopathic pulmonary haemosiderosis. Epidemiology, pathogenic aspects and diagnosis. Respiratory Medicine 92: 902–907. Ohga S, Takahashi K, Miyazaki S, Kato H, and Ueda K (1995) Idiopathic pulmonary haemosiderosis in Japan: 39 possible cases from a survey questionnaire. European Journal of Pediatrics 154: 994–995. Saeed MM, Woo MS, MacLaughlin EF, Margetis MF, and Keens TG (1999) Prognosis in pediatric idiopathic pulmonary hemosiderosis. Chest 116: 721–725. Soergel K and Sommers SC (1962) Idiopathic pulmonary hemosiderosis and related syndromes. American Journal of Medicine 32: 499–511.

IMMUNOGLOBULINS A August, The Pennsylvania State University, University Park, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Immunoglobulins or antibodies are proteins secreted by B cells, specialized cells in the immune system, and interact with antigens

or foreign material. These proteins are responsible for humoral immunity (or immunity found in serum) against pathogens, toxins, or in some pathological conditions, autoimmune diseases. Immunoglobulins are made by rearrangement of multiple gene segments in two separate genes, and are generally premade prior to exposure to pathogens. There are five main types or classes of immunoglobulins, all of which have two functions, antigen binding and effector function, via their Fc regions. These classes include m, d, g, e, and a. The effector functions of immunoglobulins

IMMUNOGLOBULINS include the ability to activate the complement pathway as well as mast cells, macrophages, neutrophils, and natural killer cells via specific Fc receptors on these cells. Specific classes of immunoglobulins have been demonstrated to have roles in the development of different respiratory diseases, including transfusionrelated acute lung injury, pulmonary complications arising from systemic lupus erythematosus, and asthma, acting via various effector functions to induce pathology.

Introduction Immunoglobulins (or antibodies) were first discovered in serum responsible for humoral immunity (i.e., binding to foreign material or antigens). Subsequent analysis indicated that these proteins had two main functions, antigen binding and effector function (in this case, fixation of complement). Immunoglobulins were later found to be part of the gamma globulins in serum, hence the name immune globulins or immunoglobulins. In serum, antibodies are a heterogeneous mixture with different recognition specificities, and so attempts to characterize them at the molecular level were initially not successful. A major breakthrough came with that realization that multiple myeloma patients carried tumors derived

315

from B cells that secrete antibodies, thus providing a source of antibodies with a single specificity. Purification and sequencing studies demonstrated that antibodies are homodimers with up to five domains that fold in a particular manner, now referred to as immunoglobulin domains or folds (Figure 1). Subsequent cloning of the immunoglobulin genes illustrated that these proteins are actually assembled from two different gene products, a light chain and a heavy chain (Figures 1 and 2). Furthermore, it was shown that these genes undergo rearrangements prior to being assembled into functional proteins.

Structure Immunoglobulins are homodimers of two light chains and two heavy chains. Each light chain has a single variable domain that is unique to each immunoglobulin and a constant domain that is one of two isotypes, k or l. Each heavy chain also has a single V or variable domain that is unique and a constant region that is made up of three or four constant domains. The constant region identifies the immunoglobulin isotype or class as m (IgM), d (IgD),

Ag binding

Hinge region Fc lgD

lgG

lgE

J-chain J-chain

lgA

lgM

Figure 1 Structure of immunoglobulin isotypes. Immunoglobulins are symmetrical dimers with two light chains and two heavy chains. Darker ovals represent variable domains of light and heavy chains. These form the antigen-binding domains (one variable domain from each chain) and are also referred to as Fab regions. Lighter ovals represent the constant domains of the light and heavy chains. IgG, IgD, and IgA have hinge regions and three constant domains. IgE and IgM lack hinge regions and have an extra constant domain. IgA can exist as a monomer (left) or a dimer (right) held together by the J-chain or secretory component. IgM is secreted as a pentamer held together by the J-chain. The antigen-binding regions and Fc portions of the antibody are indicated.

316 IMMUNOGLOBULINS JLn

VLn

CL DNA

Rearranged DNA (AAAA)n

(a)

DHn

VHn

JHn

mRNA






3

1

1

2

4



2






3

1

1

2

4



2



2

Alternate splicing
(b)

(AAAA)n &



(AAAA)n Isotype switch

Figure 2 Structure of immunoglobulin loci. (a) Generic structure of the light-chain locus. l and k light-chain loci are similar. The darker boxes represent variable (V) segments and the lighter box represents joining (J) segments. The constant domain is shown as the lightest box. Broken lines represent large distances between segments. Light-chain DNA is rearranged in developing B cells to place a V segment next to a J segment. The intervening DNA is deleted. The resulting DNA is then transcribed into RNA, which is spliced to make mRNA encoding the light-chain proteins. (b) Structure of the heavy-chain locus. Heavy-chain DNA is rearranged in developing B cells to place V, D, and J segments next to each other. This DNA is transcribed and alternatively spliced to make mRNA for IgM and IgD carrying the same VDJ segments. In mature B cells, isotype switching can occur – in this example, to make IgE – by placing the rearranged VDJ segments upstream of the e constant domains. The intervening DNA is deleted.

g (IgG), e (IgE), or a (IgA) (Figure 1). In many cases, subtypes of the different isotypes exist, such that there are four subclasses of IgG and two types of IgA in humans, and these are unique genes. There are also allotypes of immunoglobulin isotypes, which usually represent amino acid differences in the constant domains between individuals. An immunoglobulin molecule is thus a symmetrical dimer of two light chains and two heavy chains, with the light- and heavy-chain V domains folding to form the antigenbinding site (Figure 1). Immunoglobulins are assembled from the products of two separate genes – light chains encoded on chromosomes 22 and 2 (l and k light chains) and heavy chains encoded on chromosome 14 in the human. These loci have multiple segments of DNA, which are brought together during B cell development to form a functional immunoglobulin. The light chains have multiple V segments, Vk or Vl, that are organized next to each other in the locus (Figure 2). These segments are followed by J or joining segments, which are followed by a segment that encodes for the constant domain of the k or l. These V and J segments are brought together during B cell development to make a functional light chain, as described

later. The heavy-chain locus is similar, except that it has more V segments, diversity or D segments that are not found in the light chains, and J segments upstream of the constant segments. The constant segments encode those of the m, d, g, e, and a isotypes. These V, D, J, and constant segments are brought together during B cell development to make a functional heavy chain via a process that is carried out by the RAG proteins. The variable domain forms part of the antigen-binding site of the immunoglobulin molecule, and the D and J segments form the most variable region of this domain – the antigen contact site. This junction between the VD and J segments is a spot for insertion of extra nucleotides by the enzyme terminal deoxynucleotide transferase (TdT), which increases the diversity of immunoglobulins. Two other regions found in all variable domains are also hypervariable. Together, these three regions are called hypervariable regions 1, 2, and 3 or complementary determining regions (CDRs), and they form the antigen contact sites, with the hypervariable or CDR3 region being the most variable. The light-chain genes are rearranged in a similar way but are usually less variable because they lack the D segments and do not have extra added nucleotides.

IMMUNOGLOBULINS

The light-chain variable domains also have three CDR regions, with the third formed by the junction of the V segment and the J segment. Upon the formation of a functional light-chain protein, the light and heavy chains fold together to form the functional immunoglobulin molecule. Structure of Immunoglobulin Isotypes

IgD, IgG, and IgE isotypes have 2 antigen-binding sites; however, IgM exists as a monomer on the surface of the B cell and as a pentamer with 10 antigen binding sites when secreted. IgA can exist either as a monomer (with 2 antigen-binding sites) or as a dimer (with 4 antigen-binding sites). IgM and IgA multimers are held together by a J chain, which is not encoded within the immunoglobulin locus (Figure 1 and Table 1). The constant domains or Fc regions of immunoglobulins control the effector functions of the molecule. This differs based on the isotype of the molecule, as discussed later. IgD, IgA, and IgG have a proline and disulfide bond-rich hinge region in their constant domain that provides flexibility within the molecule for access to antigens. These isotypes have three constant domains (CH1–3). In contrast, IgM and IgE do not have a hinge region but have an extra constant domain that may provide a similar function as these molecules (CH1–4). Although these domains are highly homologous and are all members of the immunoglobulin superfamily, they differ, are encoded by unique genes, and are responsible for the different effector functions of these molecules. Table 1 Cytokines that induce Ig isotype switch Ig isotypea

Structure

Cytokines inducing isotype switching b

IgM

Part of BCR, secreted form is a pentamer and interacts with J chain Part of BCR, secreted form is a monomer Monomer Monomer Monomer or dimer, the latter form interacts with J chain Monomer Monomer Monomer Monomer or dimer, the latter form interacts with J chain

N/A

IgD IgG3 IgG1 IgA1

IgG2 IgG4 IgE IgA2

a

317

IgM and IgD, both of which carry the same variable domains and associate with the same light chains, are also expressed as part of the B cell receptor (BCR). Both of these events (expression of a surface form of immunoglobulin and expression of the two isotypes with the same variable domains) are accomplished by alternative splicing to generate m and d heavy chains, both of which contain a hydrophobic membrane-spanning region that allows anchoring in the plasma membrane. Thus, these two isotypes expressed on the surface of B cells both carry the same antigen-binding specificity. The five different isotypes of immunoglobulins are generated by a process called isotype- or class-switching, which rearranges the DNA of the VDJ segment of the heavy chain, placing it upstream of the desired isotype. For example, B cells undergoing class-switch to generate IgE will rearrange the VDJ segment DNAs of their immunoglobulins, deleting the genes for m, d, g, and a1, and place this VDJ region upstream of the e heavy-chain segments (Figure 2). These cells will then make IgE that has the same antigenic specificity of the original immunoglobulin. Immunoglobulins Undergo Somatic Hypermutation

Antibody responses increase in affinity during the course of an immune response, a process referred to as affinity maturation. This occurs when the variable regions of the rearranged heavy chain undergo somatic mutations. These somatic mutations are selected by antigen such that hot spots within the CDR1, -2, and -3 of the V region exhibit the most changes. This process does not change the overall structure of the antibody but results in select changes in the antigen-binding pockets, increasing the affinity of the antibody for the antigen.

Regulation of Production and Activity N/A

Regulation of Immunoglobulin Secretion

IFN-g IFN-g TGF-b

Immunoglobulins are made and secreted only by B cells. B cells are activated upon binding of specific antigen to their BCRs, and in the presence of Thelper (CD4 þ ) cell help – in the form of cytokines and cell–cell contact, generally via CD40–CD40L interactions – complete differentiation to plasma cells (T-dependent antigens). Plasma cells switch most of the immunoglobulin heavy-chain molecules (by alternative splicing of the primary RNA transcript) to molecules that lack the membrane-spanning domain, allowing them to be secreted. Plasma cells are antibody factories and the majority of their cellular physiology and processes are geared to synthesizing and secreting antibody, which they can

IL-2, IFN-g, TGF-b IL-4 IL-4 TGF-b

Ig Isotypes are listed in the order they are found in the human Ig heavy-chain locus. b Cytokines also require CD40 signals via CD40L-mediated help from T cells. BCR, B-cell receptor; N/A, not applicable.

318 IMMUNOGLOBULINS

do at the rate of approximately 120 000 molecules
1 min
1 cell. The nature of the antibodies and the level of the different isotypes generally depend on the nature of the immune response as well as the anatomical location being analyzed. Plasma cells secreting IgA and some IgE are generally found in lymph nodes draining the respiratory system or in the lungs. Plasma cells have a relatively short half-life, so antibody secretion is not thought to be further regulated. Plasma cells can live for 4 or 5 days and have to be replenished by newly differentiating B cells. However, the different isotypes may have much longer half-lives in serum. In some cases, B cells can be activated by antigens that are T-cell independent; in these cases, T cell help is not required (T-independent antigen). Under these conditions, B cells can differentiate into plasma cells and secrete antibodies. Regulation of Immunoglobulin Isotype Switching

Immunoglobulin isotype- or class-switching is regulated by the nature of the immune response and thus the nature of cytokines being made by other cells responding, particularly cytokines and cell–cell contact from CD4 þ T cells. Activated B cells can undergo class-switch to change the isotype of the antibody in the presence of T cell help. The specific isotype that is switched depends on the cytokine milieu (Table 1). For example, in the presence of

CD40L-mediated T cell help and interleukin (IL)-4, a T-helper-2 (Th2) type cytokine, B cells will undergo class-switch to make IgE or IgG4. Thus, in diseases in which there is a predominant Th2 response, such as allergic asthma, high levels of circulating IgE are usually observed. Transforming growth factor beta (TGF-b), on the other hand, induces class-switch to IgA, usually found in mucosal surfaces, including the respiratory system and gut. Signals that induce classswitch initially induce the opening of the locus being switched, resulting in the rearrangement of the VDJ segment being used by this antibody to the desired isotype segments by a mechanism that is not clear but that involves the DNA repair machinery of the cell as well as the enzyme activation-induced cytosine deaminase (AID), the latter being inducible by IL-4 and CD40 signals (Figure 2). Due to the requirement for T cell help, T-dependent antigens are usually the only antigens that can induce class-switching. Regulation of Immunoglobulin Somatic Hypermutation

Immunoglobulins also undergo somatic hypermutation during the immune response, which usually results in an overall increase in the affinity of the Ag binding

Table 2 Serum concentration and function of Ig isotypes Ig isotype

Serum concentration (mg/dl); half-life (days)

Function

IgM

0.075–0.3; 5

IgD IgG3

0.003–0.01; 3 0.1; 8

Part of the BCR; activates complement Part of the BCR Opsonization; activates complement; ADCC by NK cells Opsonization; activates complement; ADCC by NK cells Opsonization; activates complement; ADCC by NK cells Crosses mucosal surfaces and found in the respiratory system Opsonization; weakly activates complement; ADCC by NK cells Mast cells and basophil activation Crosses mucosal surfaces and found in the respiratory system

IgG1

0.9; 23

IgG3

0.1; 8

IgA1

0.3; 6

IgG2

0.3; 23

IgE

0.000 03; 2.5

IgA2

0.05; 6

Fc

BCR, B-cell receptor; ADCC, antibody-dependent cytotoxicity; NK, natural killer cells.

Complement activation (lgM > lgG3 > lgG1 > lgG2) Anaphylatoxins MAC

Fc receptors on neutrophils, macrophages mast cells, basophils NK cells

ADCC, degranulation phagocytosis

Fc receptors on epithelial cells

Transport across mucous membranes Figure 3 Functions of immunoglobulins. Immunoglobulins bind to antigen via their N-terminal variable domains, leading to neutralization or clearance of antigen. The Fc is referred to as the effector domain, and it interacts with various Fc receptors on different cell types, resulting in cell activation, degranulation, or induction of antibody-dependent cytotoxicity (ADCC), depending on the cell type. Alternatively, depending on the isotype, the Fc region can activate the classical complement pathway, leading to the production of anaphylatoxins as well as assembly of the membrane attack complex (MAC) on cells, bacteria, or viruses. Certain isotypes also can interact with Fc receptors on epithelial cells, resulting in transport across mucous membranes, including the respiratory system, and into milk.

IMMUNOGLOBULINS

antibody response. This process results in genetic changes in the variable regions of the heavy chain of immunoglobulins. Like isotype switching, this process is dependent on T cell help, cytokines, as well as cell–cell contact, and although the contributions of specific cytokines are not clear, CD40–CD40L signals contribute to this process. Again, similar to isotype switching, this process is dependent on DNA repair machinery of the cell as well as AID.

Biological Function Immunoglobulins have two functional domains – the N-terminal regions with variable domains for antigen binding and the C-terminal isotype-specific domains for effector functions. These effector functions differ based on the isotype of the antibody but fall into two main types – complement activation and Fc receptor binding. In general, the different immunoglobulin isotopes have different activities in activating the classical pathway of complement, such that IgM4IgG34IgG14IgG2. Table 2 summarizes the main functions of the different isotypes of

319

immunoglobulins with regard to complement activation and Fc receptor binding (Figure 3).

Receptors Immunoglobulins serve as receptors for antigen, either as soluble secreted proteins or as part of the BCR (IgM and IgD). However, a number of cells carry receptors for different immunoglobulin isotypes that allow these cells to respond in an antigenspecific manner via these Fc receptors (FcRs). Other cells carry these receptors in order to transport specific isotypes across membranes (e.g., PIgR transports IgM and IgA across mucous membranes). Table 3 summarizes the various FcRs for the different immunoglobulin isotypes, where they are expressed, and some of their functions.

Immunoglobulins in Respiratory Diseases Deficiencies in IgG2, IgG3, and IgG4 have been associated with increased respiratory infections. There is also a high correlation between increased levels of IgE

Table 3 Ig receptors, expression pattern, and function Ig isotype

Receptor

Expression

Function

IgM

PIgR

IgD IgG3

None identified FcRg I (CD64, high affinity) FcRg II (CD32, intermediate affinity) FcRg III (CD16, low Affinity) FcRn FcRg I (CD64, high affinity) FcRg II (CD32, intermediate affinity) FcRg III (CD16, low affinity) FcRn

Epithelial cells lining mucous membranes N/A Mono, Macs Mono, Macs, Neuts, B

Transport IgM, IgA across mucous membranes N/A ADCC, antigen uptake and clearance ADCC, antigen uptake and clearance

Macs, Neuts, NK

ADCC, antigen uptake and clearance Transport into milk to neonates ADCC, antigen uptake and clearance ADCC, antigen uptake and clearance

IgG1

IgA1

IgG2

IgE IgG2

IgA2

FcaR (serum IgA, CD89) PolyIgR (mucosal IgA, PIgR) FcRg II (CD32, intermediate affinity) FcRg III (CD16, low affinity) FcRn FceRI (high affinity) FceRII (CD23, low affinity) FcRg II (CD32, intermediate affinity) FcRg III (CD16, low affinity) FcRn FcaR (serum IgA, CD89) PolyIgR (mucosal IgA, PIgR)

Mono, Macs Mono, Macs, Neuts, B Macs, Neuts, NK Placental tissue and gut epithelia of neonates Neuts, Eos, T Epithelial cells lining mucous membranes Mono, Macs, Neuts, B

ADCC, antigen uptake and clearance Transport into milk to neonates

Macs, Neuts, NK

ADCC, antigen uptake and clearance Transport into milk to neonates Activation of mast and basophils Unknown ADCC, antigen uptake and clearance

Mast, Baso Neuts, Eos, T Mono, Macs, Neuts, B Macs, Neuts, NK Neuts, Eos, T Epithelial cells lining mucous membranes

Antigen uptake and clearance Transport across mucous membranes ADCC, antigen uptake and clearance

ADCC, antigen uptake and clearance Transport into milk to neonates Antigen uptake and clearance Transport IgM, IgA across mucous membranes

PIgR, poly Ig receptor; Mono, monocytes; Macs, macrophages; Neuts, neutrophils; Eos, eosinophils; NK, natural killer cells; T, T cells; B, B cells.

320 INFANT RESPIRATORY DISTRESS SYNDROME

and allergic asthma. High levels of allergen-specific IgE can result in binding of allergen to IgE, and interaction of this complex with the FceRI on mast cells and basophils leads to degranulation and release of pharmacological agents, including histamine, serotonin, proteoglycans, prostaglandins, leukotrienes, mast cell tryptase, as well as cytokines and chemokines. These agents have effects on the epithelial and smooth muscle cells in the lung and contribute to the development of goblet cell hyperplasia, mucous production, tissue remodeling, and airway hyperresponsiveness. Therapies based on blocking the interaction between IgE and the FceRI are currently being used for patients with allergies or asthma. rhumAb-E25 (omalizumab) is a humanized monoclonal antibody against the Fc region of IgE and is indicated for patients who have moderate to severe allergic asthma and those with seasonal allergic disease accompanying asthma and allergic rhinitis. A prominent role for immunoglobulin has also been demonstrated for transfusion-related acute lung injury (TRALI). The recognition of antigens expressed by neutrophils and granulocytes by immunoglobulins in transfused plasma can result in large-scale activation of these cells. These cells release products that lead to damage to the lung, resulting in bilateral pulmonary edema seen in TRALI. This lung damage has also been observed in rare cases following allogeneic bone marrow transplantation. Similar roles for immunoglobulins have been suggested for the presence of pulmonary injury in some patients with complications arising from systemic lupus erythematosus. See also: Allergy: Overview. Asthma: Overview. Chymase and Tryptase. Complement. Histamine.

Interleukins: IL-4. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; Pulmonary Macrophages. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading D’Amato G, Liccardi G, Noschese P, et al. (2004) Anti-IgE monoclonal antibody (omalizumab) in the treatment of atopic asthma and allergic respiratory diseases. Current Drug Targets – Inflammation and Allergy 3: 227–229. Davies DR and Metzger H (1983) Structural basis of antibody function. Annual Review of Immunology 1: 87–115. Finkelman FD, Holmes J, Katona IM, et al. (1990) Lymphokine control of in vivo immunoglobulin isotype selection. Annual Review of Immunology 8: 303–333. Ghetie V and Ward ES (2000) Multiple roles for the major histocompatibility complex class I-related receptor FcRn. Annual Review of Immunology 18: 739–766. Goldsby RA, Kindt TJ, Osborne BA, and Kuby J (2003) Immunology, 5th edn. New York: WH Freeman. Gould HJ, Sutton BJ, Beavil AJ, et al. (2003) The biology of IgE and the basis of allergic disease. Annual Review of Immunology 21: 579–628. Heyman B (2000) Regulation of antibody responses via antibodies, complement, and Fc receptors. Annual Review of Immunology 18: 709–737. Honjo T (1983) Immunoglobulin genes. Annual Review of Immunology 1: 499–528. Honjo T, Kinoshita K, and Muramatsu M (2002) Molecular mechanism of class switch recombination: linkage with somatic hypermutation. Annual Review of Immunology 20: 165–196. Kinet J-P (1999) The high-affinity IgE receptor (FceRI). Annual Review of Immunology 17: 931–972. Looney MR, Gropper MA, and Matthay MA (2004) Transfusionrelated acute lung injury: a review. Chest 126: 249–258. Mostov KE (1994) Transepithelial transport of immunoglobulins. Annual Review of Immunology 12: 63–84. Ravetch JV and Bolland S (2001) IgG Fc receptors. Annual Review of Immunology 19: 275–290.

INFANT RESPIRATORY DISTRESS SYNDROME P A Dargaville, Royal Hobart Hospital, Hobart, TAS, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Infant respiratory distress syndrome (IRDS) is a disease of the premature infant caused by a deficiency of pulmonary surfactant. Its incidence is inversely proportional to gestational age, with a heightened risk following Cesarean delivery without labor, and after perinatal asphyxia. Infants with IRDS have an early onset of a symptom complex including tachypnea, grunt, and retractions, associated with a requirement for supplemental oxygen. Chest radiograph shows loss of lung volume and diffuse

airspace opacification. Histologically, the lack of surfactant results in collapse of primordial airspaces, epithelial disruption, and proteinaceous exudation with the formation of hyaline membranes. The functional consequence of surfactant deficiency is poor lung volume at end expiration, with unfavorable pulmonary mechanics during inspiration. The pathogenesis of IRDS involves a deficiency of all elements of pulmonary surfactant, in particular dipalmitoyl phosphatidylcholine, along with surfactant inhibition and a global immaturity of lung architecture commensurate with the degree of prematurity. The disease is in part preventable by the administration of antenatal glucocorticoids; the use of these agents is associated with a 50% reduction in the risk of IRDS, and a decreased mortality. Other than general intensive care measures and respiratory support, specific treatment for IRDS is available in the form of

INFANT RESPIRATORY DISTRESS SYNDROME 321 exogenous surfactant administered directly into the trachea. Both natural and synthetic surfactant preparations are used, and have been seen to result in a 2–40% reduction in mortality, and a decrease in pneumothorax.

Introduction Infant respiratory distress syndrome (IRDS) (synonyms: hyaline membrane disease, idiopathic respiratory distress syndrome) is a life-threatening neonatal lung disease occurring in premature infants. It is, in essence, the clinical and morphological expression of an immature lung forced to adapt to extrauterine life. IRDS is the single most common indication for applying assisted ventilation in the newborn period, and is a significant contributor to the relatively high mortality noted amongst preterm infants. The burden associated with IRDS in neonatal units worldwide is largely a consequence of the high prevalence of preterm birth; in the developed world around 6% of infants are liveborn before 37 weeks gestation, and somewhat more than 1% before 32 weeks gestation. These proportions have been rising in many countries in the past two decades, and when combined with the increasing proportion of preterm infants to whom life-sustaining intensive care therapy is being offered, the result is a larger than ever population of preterm infants that require treatment for IRDS and other complications of prematurity. Epidemiology of IRDS

IRDS occurs worldwide, sparing no population or ethnic groups. The disease is inextricably linked to

premature birth, with both the incidence of developing IRDS, and the risk of dying of the condition, being inversely related to length of gestation (Figure 1). Many other factors combine to modulate the risk and severity of IRDS at a given gestation (Table 1). Antenatal factors that most commonly lead to an increase in IRDS risk are birth asphyxia and Cesarean delivery without antecedent labor. Male gender is associated not only with an increased incidence, but also with an increased mortality risk. Several antenatal factors, including fetal growth restriction, prolonged rupture of membranes, and illicit drug exposure, are seen as relative protective factors. Additionally, negroid ethnicity lends some protection against IRDS. Postnatally, the adequacy of resuscitative measures, in particular positive pressure ventilation and avoidance of hypothermia, have a bearing on the subsequent risk and severity of IRDS.

Table 1 Factors modulating risk of IRDS at a given gestation Factors increasing IRDS risk

Factors decreasing IRDS risk

Perinatal asphyxia Cesarean delivery without labor Male gender Maternal diabetes mellitusa Second of twins Familial risk Inadequate resuscitation Hypothermia

Prolonged rupture of membranesa Fetal growth restrictiona Maternal illicit drug use Negroid ethnicity

a

Not all available data support the assertion.

100

Incidence (%)

80

60 IRDS 40

20

26

27

28

29

30 31 32 33 34 Gestational age (weeks)

35

36

39−40

25

37−38

0

Figure 1 Relationship between gestational age and IRDS. Plot of the incidence of IRDS at each gestation. Reproduced from Robertson PA, Sniderman SH, Laros RK Jr, et al. (1992) Neonatal morbidity according to gestational age and birth weight from five tertiary care centers in the United States, 1983 through 1986. American Journal of Obstetrics and Gynecology 166: 1629, with permission from Elsevier.

322 INFANT RESPIRATORY DISTRESS SYNDROME Historical Perspective

IRDS has been recognized as a distinct clinical entity occurring in the preterm infant only since the early 1950s. Prior to this, the recorded observations on neonatal lung disease were dominated by pathologic descriptions of hyaline membranes occurring in a host of neonatal lung diseases. Even with the recognition of IRDS as a distinct entity, the preoccupation with eosinophilic hyaline membranes persisted, and gave the disease its original name (hyaline membrane disease) and also a spurious causation (influx of eosinophilic material via aspiration of amniotic fluid). Only with the understanding that hyaline membranes were derived from plasma protein came the recognition that these eosinophilic ‘red herrings’ were not the cause of IRDS, but merely a reflection of disruption of the alveolocapillary barrier. The seminal paper of Avery and Mead in 1959, in which lung effluent from preterm infants dying of IRDS was demonstrated to have poor surface activity, established the primacy of surfactant deficiency in the pathogenesis of IRDS. A wealth of research followed in the decades thereafter, with the result that the biology and ontogeny of surfactant were elucidated, the precise nature of the surfactant abnormalities in IRDS was established, and exogenous surfactant replacement evolved from a distant hope to a therapeutic reality.

Etiology The primary cause of IRDS is a lack of pulmonary surfactant in the airspaces of the preterm infant. There is both deficiency and dysfunction of surfactant in IRDS, and, in addition, global immaturity of the preterm respiratory system. The complex interplay of these factors is further discussed in section ‘Pathogenesis’ below.

Pathology The gross findings at autopsy in IRDS are of large areas of atelectasis and congestion, particularly in the dependent areas, giving the lung a liver-like appearance. The distensibility of the lung is markedly reduced. Histologically, the appearances vary with age, with the earliest features being distal airspace collapse accompanied by epithelial necrosis and sloughing within the pathologically distended terminal bronchioles. These changes, present within 1–2 h of birth, are followed beyond 3 h by the appearance of hyaline membranes, generally within the terminal and respiratory bronchioles and extending into the primordial alveolar ducts (Figure 2). The hyaline membranes are eosinophilic linear aggregations of

Figure 2 Pathology of IRDS. Lung section from a preterm infant dying of IRDS. Strongly eosinophilic hyaline membranes are seen at the airspace margins, with lightly stained proteinaceous exudate filling the remainder of several airspaces. Note the primitive lung architecture, and the highly cellular stroma.

plasma protein in which may be encased nuclear debris from sloughed pneumocytes. They occur as a direct result of disruption of the alveolocapillary barrier, and are not specific to IRDS, being found in other diseases of the newborn lung (e.g., meconium aspiration syndrome), and in lung injury beyond the newborn period (e.g., acute respiratory distress syndrome). The putative mechanism by which epithelial damage occurs is uneven distribution of transpulmonary pressure, generated by the infant’s own respiratory efforts, by positive pressure ventilation, or both. The resultant shear forces damage the lining epithelium and associated endothelium, leading to an influx of proteinaceous material some of which coalesces into hyaline membranes. By 24 h, inflammatory cells (mostly macrophages with some neutrophils) appear in the airway lumen and interstitium, and debris from hyaline membranes is prominent within intraluminal macrophages by 48–72 h. The epithelium begins to regenerate after 48 h, with the initial appearance of thick epithelial squames. Around this time, surfactant synthesis and secretion begins to occur. By 7 days, infants recovering from IRDS show resorption of hyaline membranes and further epithelial regeneration. Those still ventilated will show a mixture of ongoing damage and regeneration, ultimately with the development of fibrosis and scarring characteristic of neonatal chronic lung disease, also known as bronchopulmonary dysplasia. In the extremely preterm infant (o28 weeks gestation), these findings will be accompanied by evidence of a failure of ex utero maturation, with arrest of alveolarization and secondary crest formation.

Clinical Features Hallmarks of the clinical diagnosis of IRDS are the early onset (before 4 h) of a symptom complex

INFANT RESPIRATORY DISTRESS SYNDROME 323 Table 2 Clinical features of IRDS Pulmonary

Extrapulmonary

Tachypnea Retractions Grunting Cyanosis without supplemental oxygen Apnea

Hypotension Oliguria Hypotonia Ileus Exaggerated neonatal jaundice

characterized by tachypnea, retractions, grunting, and cyanosis without supplemental oxygen (Table 2). Tachypnea has historically been defined as a respiratory rate above 60 breaths/min, although it is clear that extremely preterm infants may respire at rates of 70–80 min
1 in the absence of parenchymal lung disease. Retractions indicate poor lung compliance, and refer to the suprasternal, intercostal, and subcostal skin recession noted during inspiration, and also to the recession of the highly compliant sternum and anterior chest wall in the preterm infant. Grunting is the expiratory noise generated by forced exhalation through a partially closed glottis, and is a reflex mechanism to preserve the volume of the diseased lung in expiration. The clinical features of neonatal respiratory distress are by no means specific for IRDS, and the context in which they occur may be of more help in securing a clinical diagnosis, with the likelihood of IRDS being greatest at lower gestations (Figure 1), and in the presence of other risk factors (Table 1). Table 3 shows a differential diagnosis of respiratory distress in the newborn infant. Several extrapulmonary features are regularly encountered in IRDS, including hypotension, hypotonia, ileus, and oliguria with impaired capacity to excrete a water load (Table 2). These abnormalities are seen to resolve in parallel with restoration of lung function; a diuresis may in fact herald the onset of respiratory improvement. The natural course of IRDS, without assisted ventilation, is characterized by progressive deterioration in the first 24–48 h, highest mortality in the first 3 days, and a recovery period in survivors beginning at approximately 72 h. The introduction of prenatal glucocorticoid therapy, assisted ventilation, and exogenous surfactant therapy has considerably changed the natural history and outcome of IRDS (see section ‘Management and current therapy’ below). Radiology

Chest radiograph is an indispensable tool in the diagnosis and management of IRDS, and should be performed in any infant with respiratory distress, regardless of gestation. The image thus obtained will

distinguish IRDS from a legion of other causes of neonatal respiratory compromise (Table 3), and give an impression of disease severity. The response to treatment and the development of complications can be ascertained on sequential radiographs. At their most prominent, the radiographic features of IRDS are striking and distinct (Figure 3). The lung fields show diffuse reticulogranular opacification, which may on occasions be more prominent on the right side, and in the lower zones. Anatomically, the radiolucent ‘granules’ in the opacified lung represent distended terminal bronchioles surrounded by microatelectasis. The volume of the lung fields may be considerably reduced. With dense parenchymal opacification, there is loss of normal contours, including the cardiac silhouette and the diaphragms, and the development of air bronchograms. The radiographic appearances in IRDS take some hours to evolve, and are significantly moderated by the application of positive airway pressure, and by the administration of exogenous surfactant. IRDS in its natural form shows progressive parenchymal opacification over the first 48–72 h, followed by a gradual clearing of the chest radiograph, coincident with the resolution of surfactant deficiency. Respiratory complications of IRDS may be apparent radiographically from early in the course of the disease, including pulmonary interstitial emphysema (Figure 4), pneumothorax (Figure 5), and other air leaks. Lung Function Testing

Lung volumes and pulmonary mechanics The lack of surfactant and resultant high surface tension within fluid-lined airspaces leads to low lung volume in expiration, and unfavorable pulmonary mechanics on inspiration (Table 4). Functional residual capacity is much reduced, and with the higher proportion of atelectatic lung units comes a higher alveolar–arterial oxygen difference. Lung compliance is poor, and improves gradually to normal values as the disease resolves. As a result, work of breathing is increased, but breathing efficiency is decreased given the highly compliant chest wall and suboptimal diaphragmatic configuration of the premature infant. Arterial blood gas analysis Hypoxemia of some degree is universal in IRDS, and is usually accompanied by respiratory acidosis with elevated PaCO2. Where supplemental oxygen is being administered the disturbance of oxygenation is best quantified using measures such as the alveolar–arterial oxygen difference (see Table 4). These correlate well with radiographic severity and other indices of pulmonary mechanics.

Table 3 Differential diagnosis of respiratory distress in the newborna Condition

Causea

Affects term or preterm infants

Usual time of symptom onset

Distinguishing features, comments

IRDS

Primary surfactant deficiency and lung immaturity

Preterm

o6 h

Pulmonary insufficiency of prematurity

Global immaturity of the respiratory system

Extremely preterm

o6 h

Transient tachypnea of the newborn

Retained fetal lung fluid

Both (especially following elective Cesarean section)

o6 h

Meconium aspiration syndrome

Perinatal inhalation of meconium causing airway obstruction and pneumonitis

Term

o6 h

Neonatal pneumonia

Transplacental, transtracheal, or hematogenous acquisition of pathogenic organisms

Both

Transplacental: o6 h; others: variable

Spontaneous pneumothorax

Gas in the pleural space

Both

o6 h if isolated; at any time if associated with positive pressure ventilation

Pulmonary hypoplasia

(a) oligohydramnios (b) neuromuscular or skeletal anomalies (c) developmental lung anomalies or cysts

Both

o6 h

Tachypnea, retractions, and grunting, with a significant requirement for supplemental oxygen, associated with respiratory acidosis; diffuse reticulogranular opacification radiographically Minimal oxygen requirement but persistent need for respiratory support, often for many weeks Transient relatively mild respiratory distress, rarely needing assisted ventilation; interstitial edema radiographically Meconium-stained infant with moderate or severe respiratory distress often with pulmonary hypertension; patchy opacities and regional hyperinflation radiographically Usually focal changes radiographically, but group B streptococcal pneumonia can mimic IRDS; nosocomial pneumonia may complicate the course of other neonatal lung diseases, especially if prolonged intubation is necessary May cause acute cardiorespiratory deterioration; small pneumothoraces can be detected on up to 1% of chest radiographs after delivery Bell-shaped chest and/or high diaphragms radiographically; risk of early pneumothorax

Pulmonary hemorrhage

Hemorrhagic pulmonary edema

Both

Usually o24 h

Congenital diaphragmatic hernia

Posterolateral diaphragmatic defect (usually left-sided) resulting in abdominal contents occupying hemithorax in fetal life; associated pulmonary hypoplasia

Term

o6 h

Pulmonary hypertension

Increased pulmonary vascular resistance

Term or occasionally preterm

o6 h

Cyanotic congenital heart disease

Obligatory right-to-left shunt or mixing lesion

Term

Acyanotic congenital heart disease

Obligatory left-to-right shunt or left ventricular outflow obstruction

Term

Variable depending on ductal closure and/or degree of mixing 424 h

Airway obstruction

Anatomical þ /
functional obstruction at any level

Term

Variable

Lactic acidosis

Tissue ischemia related to perinatal asphyxia

Both

o6 h

a

Interstitial or patchy changes radiographically; can complicate the respiratory disease of an extremely preterm infant (especially after surfactant therapy), or a term infant with meconium aspiration syndrome and/or asphyxia Scaphoid abdomen and bowel sounds in hemithorax, often associated with prominent evidence of pulmonary hypertension; chest radiograph shows malpositioned abdominal organs associated with hypoplastic ipsilateral and contralateral lung Marked hypoxemia with pre/postductal difference in oxygenation, prominent right ventricular impulse and triscuspid incompetence; pulmonary hypertension often accompanies other parenchymal lung diseases, especially in the full-term infant Hypoxemia not overcome by oxygen therapy; often effortless tachypnea with normal or low PaCO2 Left ventricular outflow obstruction may remain clinically silent until closure of the ductus arteriosus; left-to-right shunt lesions become symptomatic once pulmonary vascular resistance falls in the first weeks or months Usually audible stridor or other airway noise associated with retractions; hypoxemia is a late and ominous sign Tachypnea often with minimal retraction but prominent pallor

Most common causes are listed; see ‘Further reading’ section for more information on these and other rarer causes of neonatal respiratory distress.

326 INFANT RESPIRATORY DISTRESS SYNDROME

Pathogenesis Biochemical Immaturity in IRDS

Figure 3 Radiographic appearance of IRDS. Chest radiograph of a preterm infant at 28 weeks gestation on day 1 of life showing moderately severe IRDS. Note the relatively uniform reticulogranular opacity in the lung fields, with loss of normal contours related to microatelectasis. Air bronchograms are noted bilaterally.

Pulmonary surfactant Deficiency of pulmonary surfactant is the fundamental cause of IRDS. Surfactant is a mixture of phospholipid and protein vital for the normal function of the lungs, and instrumental in smoothing the passage from intrauterine to extrauterine life. The major phospholipid component, dipalmitoyl phosphatidylcholine (DPPC), forms a monolayer on the alveolar surface which lowers surface tension, thereby maintaining alveolar volume in expiration, and conferring favorable pulmonary mechanics during inspiration from low lung volume. Several other components of surfactant assist in the formation of the DPPC monolayer, including other phospholipids (e.g., phosphatidylglycerol), and surfactant-associated proteins A, B, and C (SP-A, SP-B, SP-C). All elements of surfactant are synthesized, stored, secreted, and recycled by the alveolar type II cell.

Figure 4 Radiographic appearance of pulmonary interstitial emphysema. Chest radiographs of separate preterm infants with pulmonary interstitial emphysema: (a) taken at 12 h of life shows rounded lucencies within the opacified lung, representing pathologically distended airways proximal to collapsed primordial alveoli; (b) is from an infant at 1 month with marked cystic emphysematous change in the right lung, with midline shift and volume loss on the left side.

Figure 5 Radiographic appearance of pneumothorax. Chest radiographs in a 29-week gestation infant. At 18 h of age (a) there is a left pneumothorax under some tension, with marked loss of ipsilateral lung volume. Chest radiograph 1 h later (b) shows that the extrapleural air in the left hemithorax has been removed by an intercostal drain tube (arrow). The infant also has an endotracheal tube. A pneumothorax is now present on the right side.

INFANT RESPIRATORY DISTRESS SYNDROME 327 Table 4 Lung function in IRDS Parameter

IRDS

Preterm infant with normal lungs

Tidal volume Functional residual capacity Compliance of the lung Alveolar–arterial oxygen differencea PaCO2

4–6 ml kg
1 10–20 ml kg
1 body weight 0.2–0.5 ml cmH2O
1 kg
1 Elevated (up to 650 mmHg) Elevated

Similar 30 ml kg
1 1–2 ml cmH2O
1 kg
1 40–60 mmHg 35–45 mmHg

a

Alveolar–arterial oxygen difference ¼ ðFiO2  Pbarometric Þ
PH2 O
ðPaCO2 =RÞ
PaO2 .

Surfactant ontogeny: the basis for biochemical immaturity IRDS Nature’s parsimony has dictated that during fetal life, the elements of surfactant only appear in the lung when there is a reasonable chance of survival ex utero. There is a natural delay in the development of the surfactant system, with phospholipid-containing lamellar bodies first recognizable within type II cells at around 24 weeks gestation, and surfactant phospholipids only appearing in trace amounts in amniotic fluid before 30 weeks of gestation. Beyond 30 weeks, there is a progressive rise in the ratio of PC (lecithin) to sphingomyelin (i.e., the L : S ratio) in amniotic fluid, reflecting gradual increase in secretion of surfactant from the type II cell and efflux from the lung in fetal lung fluid. A ‘mature’ L : S ratio of 2 or above (an excellent predictor of surfactant sufficiency and absence of IRDS in postnatal life) is only noted close to full term in a nonstressed pregnancy. In fetal life, the regulation of the surfactant system is under the control of many competing influences, but particularly the levels of circulating hormones. The concentration of endogenous glucocorticoids is a fundamental determinant of the state of the surfactant system in the fetus, impacting upon the synthesis of many elements of surfactant, as well as many other facets of lung maturation. Upregulation of several key synthetic enzymes, most particularly choline phosphate cytidylyl transferase, and fatty acid synthetase, leads to a marked increase in surfactant phospholipid production. These glucocorticoid effects have been successfully exploited in the form of antenatal steroid therapy in the setting of threatened premature delivery (see section ‘Treatment’ below). Levels of other hormones also impact upon fetal surfactant maturation. Circulating catecholamines promote surfactant synthesis in a similar way to glucocorticoids. On the other hand, both androgens and insulin depress cytidylyl transferase activity, contributing to the higher incidence of IRDS in males, and infants of diabetic mothers, respectively. In the days prior to birth, there are considerable changes in the fetal lung in preparation for extrauterine life. The normal secretion of lung fluid slows, and synthesis of surfactant elements increases. Additional maturation occurs during labor, as catecholamine and

glucocorticoid concentrations surge. Postnatally, as the air–liquid interface is formed, there is a rapid increase in the size of the surfactant pool, with establishment of an intra-alveolar surfactant compartment which evolves rapidly in the first 24 h. The importance of this final maturation process is demonstrated by the increased risk of IRDS in the infant delivered by Cesarean section without labor. On the basis of the above, it might be expected that many more infants less than 36 weeks would suffer from the effects of surfactant deficiency. The fact that many premature infants (even some much less than 30 weeks) do not have IRDS is an indication of the effective escape route that nature can apply to the lungs of the preterm infant when delivery is imminent – the rapid inducibility of surfactant synthesis in response to stress, mediated by endogenous catecholamines and glucocorticoids. Surfactant deficiency in IRDS All elements of the surfactant system have been found to be deficient in IRDS. Based on studies of phosphatidylcholine (PC) content, the size of the surfactant pool is markedly reduced in preterm animals with IRDS (e.g., for sheep, 20 mmol PC kg
1 in RDS vs. 100 mmol PC kg
1 in the normal newborn). In human preterm infants with IRDS surfactant pool size has been estimated at 10% of that at full term. Estimated pool size correlates well with measures of lung dysfunction, for example, lung compliance. In addition to an overall reduction in phospholipid content, the ratio of DPPC to total PC in lung fluid is also considerably reduced in IRDS. At the outset, the DPPC : PC ratio may be 0.3 or less (compared to a normal value of 40.5), and has been noted to normalize during recovery. Estimates of alveolar PC and DPPC concentration from lung lavage samples are lower in IRDS than normal controls, and correlate well with other measures of lung dysfunction, in particular oxygenation. Concentrations of other surfactant-associated phospholipids are also reduced in IRDS. Phosphatidylglycerol is regularly absent in the first days of life in lavage fluid from preterm infants with respiratory distress, and is seen to appear during the recovery phase. Delay in the appearance of phosphatidylglycerol in the lung

328 INFANT RESPIRATORY DISTRESS SYNDROME

has been particularly noted in infants born to diabetic mothers, and may contribute to the higher risk of IRDS in this group. Reduced concentration of surfactant proteins has also been noted in lung fluid from infants with IRDS. Content of SP-A in lavage fluid is low at the height of disease, and increases in parallel with recovery in the first week of life. SP-A concentration shows a lesser or no increase in infants who ultimately do not survive, or go on to develop chronic lung disease. Similarly, concentrations of the surfactant proteolipids SP-B and SP-C are measurably low in lavage fluid from infants with IRDS. Inhibition of surfactant function In addition to surfactant deficiency, there is evidence that considerable inhibition of surfactant function occurs in IRDS. At equimolar DPPC concentration, lung fluid from afflicted infants shows impaired surface-active properties. Putative mechanisms by which surfactant function is impaired include sequestration of surfactant in hyaline membranes, and inhibition both by plasma proteins and edema fluid. Proteinaceous inhibitors of surfactant function are found at high levels in the airspaces in IRDS.

biochemical immaturity in around 2% of infants born at 38 weeks gestation by Cesarean section without labor, and on the other as prolonged respiratory insufficiency and susceptibility to ventilatorinduced damage in the structurally immature lung of an infant born at 25 weeks gestation needing no supplemental oxygen from the outset. The biochemical and structural immaturity of the preterm lung not only contributes to the pathogenesis of IRDS, but also renders the lung susceptible to structural damage as both the infant and the clinician attempt to overcome the respiratory insufficiency. A critical event here is the pathological distension of primitive airspaces related to abnormally high transpulmonary pressure, generated by the infant’s extreme respiratory efforts and/or the application of positive pressure to the airways by the clinician. Rupture of these distended airspaces heralds several important complications of IRDS, pulmonary interstitial emphysema and pneumothorax. More globally, the initiation of a process of epithelial damage and repair is a key step in the pathogenesis of chronic lung disease, a relatively frequent complication in the preterm infant with IRDS (see below).

Animal Models Structural and Cellular Immaturity in IRDS

Other factors apart from surfactant deficiency contribute to the risk and severity of IRDS in the premature infant. Whilst survival in the extrauterine environment is possible around 22–24 weeks gestation, the lung at this stage is approaching the end of the canalicular phase of lung development, with a cellular interstitium and primitive air spaces with a thick blood–air barrier. The pulmonary epithelium is undifferentiated, relatively cuboidal, and susceptible to shear forces, rendering the lung unable to cope with superadded surfactant deficiency, and uniquely vulnerable to injury from positive pressure ventilation. A number of extraparenchymal factors, such as the compliance of the chest wall, the function of the diaphragm, and the maturity of the neural respiratory control centers, profoundly influence the capacity of the premature infant to sustain an air-breathing existence. These factors all contribute to the severity of respiratory dysfunction in IRDS. There may be a considerable disparity between the structural and biochemical maturity of the lung in a newborn preterm infant, related not only to inherent differences in ontogeny of these elements, but also to a difference in the capacity of the lung to rapidly mature in response to environmental and therapeutic agents. This disparity manifests on the one hand as severe but transient respiratory distress related to

Animal models of IRDS aim to replicate all elements of the biochemical immaturity seen in the preterm human lung, along with a concomitant degree of structural and cellular immaturity. Once delivered, the animal should manifest the characteristic clinical and physiological disturbances of IRDS, and yet be salvageable with standard neonatal intensive care so as to allow prolonged study and evaluation of specific postnatal therapies. Over more than four decades, a number of mammalian models of IRDS have been extensively studied (Table 5), and have yielded much information about the biology of surfactant, the pathophysiology of IRDS, and the efficacy of exogenous surfactant preparations. Each model has limitations, chief amongst which is that the unique combination of circumstances that result in the birth of a preterm infant are difficult to reproduce experimentally. Additionally, the balance of structural, cellular, and biochemical immaturity noted at any given gestation in the human infant is somewhat different in each of the animal species.

Management and Current Therapy Prevention of IRDS

Given the relationship between prematurity and IRDS, avoidance of preterm delivery is a logical way to reduce the incidence of IRDS. This said, the

INFANT RESPIRATORY DISTRESS SYNDROME 329 Table 5 Animal models of IRDSa Species

Term gestation (days)

Preterm gestation studied (days)

Preterm weight

Comment

Rabbit

31

27

30 g

Lamb

147

120–130

2–3 kg

Rhesus monkey

160

127–131

250–350 g

Baboon

179

134–147

500–700 g

Mouse lacking SP-B

18–21

Studied at or near full gestation

1–2 g (at full gestation)

Small size precludes intricate postnatal testing of lung function, or prolonged survival Morphological development in advance of surfactant ontogeny; prolonged survival and creation of a model of chronic lung disease is possible Survival for several days postnatally is possible with respiratory support Prolonged survival and creation of a model of chronic lung disease is possible Targeted disruption of SP-B gene causes fatal respiratory failure in the newborn period, with global atelectasisb

a Animals at any age may also be rendered surfactant deficient by repeated saline lavage. This model is not included in the above table as the resultant lung injury has more features in common with acute respiratory distress syndrome than with IRDS, and is not accompanied by structural and cellular immaturity. b A human homolog exists, in which lack of functional SP-B leads to respiratory failure at birth in infants at or near full term.

rates of preterm birth are in fact rising in the developed world, fueled by a combination of factors including advanced maternal age, multiple pregnancy, and uptake of assisted conception. Further, there is an increased preparedness in obstetric practice to deliver extremely preterm infants if the health of mother or fetus appears likely to be compromised by continuing the pregnancy. Ablation of uterine contractions using tocolytic drugs does have a role in spontaneous preterm labor, if only to allow sufficient time for glucocorticoids administered to the mother to have an effect on the fetus. Accepting that preterm birth is often inevitable, the incidence and severity of subsequent IRDS can be reduced by careful obstetric management, including acceleration of fetal maturation with glucocorticoids, avoidance of traumatic delivery and/or intrapartum asphyxia, and application of appropriate resuscitation to the infant immediately after birth. Antenatal glucocorticoid therapy The impact of antenatal glucocorticoids on human fetal lung maturation was first documented in 1972, and this therapy is now widely applied in pregnancies threatening to end prematurely. Pooled data from randomized controlled trials of antenatal glucocorticoids show a 50% reduction in risk of IRDS, a lesser severity of IRDS, and a lower mortality. The risks of intracranial hemorrhage and periventricular leukomalacia are also reduced. The benefits of glucocorticoids are apparent at all gestations below 32 weeks, with a maximal effect if delivery occurred between 24 h and 7 days after administration. Antenatal glucocorticoid therapy is not associated with an increase in maternal, fetal, or neonatal infection, and appears to

be safe even in the context of premature membrane rupture. The agents used are betamethasone (12 mg intramuscularly, two doses 24 h apart) and dexamethasone (6 mg intramuscularly, four doses 12 h apart), with the former resulting in the best neurological outcome for the premature infant. Other agents that have been administered antenatally for induction of fetal lung maturation include thyrotropin-releasing hormone and ambroxol, with no conclusive evidence of benefit. Postnatal Management

The management of the preterm infant with IRDS includes supportive care to overcome respiratory failure and other organ dysfunction, and specific therapy with exogenous surfactant to counter the primary surfactant deficiency (Table 6). The importance of general supportive measures such as adequate delivery room resuscitation, temperature control, and circulatory stabilization cannot be overestimated, and improvements in these aspects of care led to a marked increase in survival for the preterm infant well before the advent of exogenous surfactant. Specific Therapy for IRDS

Respiratory support Oxygen therapy, continuous positive airway pressure (CPAP), and positive pressure ventilation are the mainstays of conventional therapy to support the respiratory system in a preterm infant with IRDS. Current evidence favors the following approach in the early management of preterm infants with IRDS: 1. Judicious use of oxygen therapy, targeting arterial oxygen saturation values no higher than 95%

330 INFANT RESPIRATORY DISTRESS SYNDROME Table 6 Management of IRDS Antenatal preventative therapy

Tocolysis for spontaneous preterm labor Antenatal glucocorticoid administration Avoidance of birth trauma and intrapartum asphyxia

a

Postnatal therapy Supportive measures

Drug therapy

Resuscitation Temperature control Intravenous fluid therapy, avoiding fluid overload Supplemental oxygen Nasal or nasopharyngeal continuous positive airway pressure Intubation and positive pressure ventilationa Circulatory support with volume and/or inotropes

Exogenous surfactant therapy Diuretics Indomethacin (or ibuprofen) for prevention or treatment of patent ductus arteriosus Antibiotics if perinatal infection suspected

Multiple ventilatory modes are used in preterm infants with IRDS (see Goldsmith and Karotkin).

(corresponding to a PaO2 value of around 60 mmHg) so as to lower the risk of retinopathy of prematurity. The lower limits of the safe range of oxygenation for the preterm neonate remain to be determined; an oxygen saturation of at least 88% (PaO2 around 45 mmHg) is currently deemed acceptable in most neonatal intensive care units. 2. Intubation and positive pressure ventilation for preterm infants with: (a) significant respiratory failure (PaCO24 60 mmHg) and arterial pHo7.2) unresponsive to lesser measures, (b) marked hypoxemia (failure to maintain oxygenation in the acceptable range despite an FiO2 of 0.6 or more), and (c) a single apneic episode requiring positive pressure ventilation, or multiple lesser episodes causing physiological disturbance. 3. Early administration of exogenous surfactant (see below) for any intubated preterm infant with an FiO240.25, unless there is clinical suspicion of a diagnosis other than IRDS. 4. Nasal CPAP via short binasal prongs as primary therapy for infants with lesser respiratory dysfunction, and after extubation of those initially managed with an endotracheal tube. There remains uncertainty (and controversy) regarding: 1. The question of routine intubation of preterm infants o29 weeks gestation in the delivery room, given that many such infants can be effectively supported with nasal CPAP unless apneic. 2. The value of intubation solely to administer surfactant in infants where nasal CPAP is to be used as primary therapy.

3. The indications for intubation of a preterm infant on nasal continuous positive airway pressure. 4. The most appropriate mode of mechanical ventilation to use in preterm infants with IRDS (including the place of high-frequency oscillatory ventilation). 5. The value of synchronized ventilation. 6. The safe limits of oxygenation in the preterm infant. Exogenous surfactant therapy Exogenous surfactant preparations available for clinical use may be categorized as natural (derived from mammalian lung tissue) or synthetic (Table 7). The animal products are lipid extracts of either lung mince or alveolar lavage fluid. In lung mince surfactants, the phospholipid profile of the lipid extract is modified, either by purification or fortification, to more closely replicate that of natural surfactant. All natural surfactants contain the surfactant proteolipids SP-B and SP-C (which coisolate with lipids in chloroform–methanol extraction), but there is significantly less SP-B in surfactant preparations derived from lung mince. Second-generation synthetic surfactants contain no surfactant proteins, and are simply phospholipid preparations to which other components are added to facilitate surface adsorption. These have now been superseded by thirdgeneration surfactants having surfactant proteolipid activity, the efficacy of which appears similar to natural surfactants in comparative trials. The dose of surfactant phospholipid used for treatment of IRDS (50–200 mg kg
1) has been chosen to replenish the deficient lung with an amount of phospholipid equivalent to that present in the normal newborn lung (100 mg kg
1). This dose is far in excess of the estimated amount of phospholipid required to form a surfactant film over the entire respiratory epithelium (around 3 mg kg
1), but must

Table 7 Commercially available exogenous surfactant preparations Trade name (generic name)

Product derivation

Natural surfactants: lung mince extracts Survanta Cow lung mince extract (beractant) fortified with DPPC, palmitic acid, and tripalmitin Curosurf (poractant)

Surfacten (surfactenTA)

Pig lung mince extract, chromatographically purified by removal of neutral lipids Cow lung mince extract fortified with DPPC, palmitic acid, and tripalmitin

Natural surfactants: lung lavage extracts Alveofact Cow lung lavage extract (bovactant) bLES (bovine lung extract surfactant) Infasurf (calfactant)

SP-C content per mg phospholipid (%wt./wt.)

Phospholipid concentration in fullstrength preparation (mg ml
1)

Usual phospholipid dose in IRDS (mg kg
1)

84%

PC 83%a DPPC 59% PG 3.2%

0.1%a

2.1%a

25

100

99%

PC 73%a DPPC 41%a PG 1.2%

0.3%a

1.3%

80

100–200

84%

PC 83%a DPPC 59% PG 3.2%

0.1%a

2.1%

25b

100

90%

PC 84% DPPC 33% PG 9% PC 82% DPPC 46% PG 12% PC 74% DPPC 46% PG 3.6%

1.2%a

1.7%

42

50–100

27

135

1.2%a

1.3%

35

105

84%

DPPC 100%

Nil

Nil

13.5b

67.5

100%

PC 75% DPPC 75% PG 25%

Nil

Nil

12.5b

100

82%

PC 75% DPPC 75% PG 25%

KL4 3%

Nil

30

175

92%

PC69% DPPC 69% PG 31%

Nil

rSP-C 2%

46b

50

98%

Calf lung lavage extract

91%

Synthetic surfactantsc: 3rd generation Surfaxin Synthetic phospholipid (lucinactant) mixture supplemented with KL4 peptide (sinapultide)

a

Phospholipid SP-B content per mg composition (% phospholipid total phospholipid) (%wt./wt.)

Cow lung lavage extract, acetone precipitated

Synthetic surfactantsc: 2nd generation Exosurf Synthetic phospholipid mixture (colfoscleril supplemented with tyloxapol palmitate) and hexadecanol ALEC Synthetic phospholipid (pumactant) mixture

Venticute (rSP-C surfactant)

Phospholipid content (% dry weight)

Synthetic phospholipid mixture supplemented with recombinant SP-C (lusupultide)

2% Combined

Value is an average of several estimates. After reconstitution of powder with aqueous vehicle. c First-generation synthetic surfactants were suspensions of pure DPPC, without any agents added to promote spreading. These were found to be ineffective in treating IRDS in trials conducted in the 1960s. Reprinted with modification from Dargaville PA and Mills JF (2005) Surfactant therapy for meconium aspiration syndrome: current status. Drugs 65 (in press), with permission. b

332 INFANT RESPIRATORY DISTRESS SYNDROME

compensate for uneven distribution and protein inhibition, as well as primary phospholipid deficiency. A number of different methods of surfactant instillation have been devised, including delivery via a side-port connector attached to the endotracheal tube, or via a separate catheter inserted through the endotracheal tube. The latter method appears to have been more widely adopted in clinical practice. Surfactant has also been given as a bolus into the pharynx, and as an aerosol, and has even been instilled into the amniotic fluid near the face of the fetus, gaining access to the lung during fetal respiration. These alternative methods of delivery remain in the province of research. Since the first report of successful use of exogenous surfactant in human infants in 1980, more than 30 randomized controlled trials of surfactant treatment have been conducted. Pooled data from these trials show that mortality from IRDS is considerably reduced (odds ratio 0.59–0.73) after treatment with either natural or synthetic surfactant preparations (Figure 6). The effect is consistent, whether the drug is given prophylactically, or as a rescue therapy when

the features of IRDS have become established. Risk of air leak is also consistently lower in surfactanttreated infants, with the odds ratio for pneumothorax being around 0.35 for natural surfactant, and 0.6 for synthetic surfactants. Where tested, a more favorable outcome has been noted in infants given two doses of surfactant, rather than one. There appears to be minimal gain from a third and fourth surfactant dose. The timing of surfactant therapy is of considerable importance, with most trials showing benefit in early treatment, given either in the delivery room or shortly after arrival and stabilization in the neonatal intensive care unit. Administration of surfactant beyond 24 h of life is associated with a higher risk of treatment failure, presumably related to the progression of lung injury and accumulation of proteinaceous exudate in the alveolar space. A number of comparative trials of different surfactant preparations have been conducted. Those comparing natural with second generation synthetic surfactants have highlighted the shortcomings of the latter, in particular delayed clinical response and

Natural surfactant prophylaxis

Natural surfactant treatment

Mortality

Mortality

Pneumothorax

Pneumothorax

PDA

PDA

CLD

CLD

IVH

IVH 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80

(a)

Odds ratio

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 (b)

Odds ratio

Synthetic surfactant prophylaxis

Synthetic surfactant treatment

Mortality

Mortality

Pneumothorax

Pneumothorax

PDA

PDA

CLD

CLD

IVH

IVH 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80

(c)

Odds ratio

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 (d)

Odds ratio

Figure 6 Meta-analysis of clinical trials of IRDS. Meta-analysis of exogenous surfactant treatment in IRDS, showing odds ratios and 95% confidence intervals for relevant outcomes in (a) eight trials (involving 988 infants) of natural surfactant administered in the delivery room; (b) 12 trials (1600 infants) of natural surfactant for treatment of established IRDS; (c) seven trials (1560 infants) of synthetic surfactant administered in the delivery room; and (d) five trials (2352 infants) of synthetic surfactant for treatment of IRDS. Mortality is defined as death prior to day 29. PDA, patent ductus arteriosus; CLD, chronic lung disease, defined as the need for oxygen therapy at 28 days; IVH, all grades of intraventricular hemorrhage. Data from the Cochrane library.

INFANT RESPIRATORY DISTRESS SYNDROME 333

greater risk of pneumothorax. More recent comparative trials have generally demonstrated equivalence between natural and third-generation synthetic surfactants. In the developed world, exogenous surfactant is widely used in the treatment of IRDS, with the relatively high cost of the drug being offset by its known benefits. Surfactant usage is inversely related to gestation, being administered to 70–90% of infants at 24–27 weeks gestation, and 30–60% of those born at 28–31 weeks. There are considerable regional differences in surfactant administration, governed largely by delivery room intubation practices, and the readiness to use nasal CPAP as a primary therapy for IRDS. Diuretic therapy Infants with IRDS are oliguric and susceptible to water overload, and in addition have a significant pulmonary edema. Their response to diuretic therapy is, however, modest at best, and the small gain in pulmonary function appears more than offset by an increased risk of patent ductus arteriosus. Complications of IRDS

Complications of IRDS (Table 8) occur in relation to (1) the surfactant-deficient lung, (2) the general insult visited upon the preterm organism, and (3) the treatment applied. Some are consequences of all three. The most notable acute respiratory complication is air leak, in particular pulmonary interstitial emphysema and pneumothorax. Pulmonary interstitial emphysema is a common accompaniment of severe IRDS treated with positive pressure ventilation (Figure 4). It is a consequence of bronchiolar rupture and tracking of gas into the interstitium. At its most aggressive, multiple gas-filled cysts form within the interstitium, leading to local gas trapping and atelectasis in lesser affected regions, markedly compromising gas exchange. Management includes downward positioning of affected areas, minimization of positive pressure excursions (including the use of high-frequency ventilation), and selective intubation of the lesser-affected lung. Pneumothorax occurs when interstitial gas enters the pleural space via a breach in the visceral pleura (Figure 5). If under

tension, there may be dramatic cardiorespiratory compromise, with hypotension, bradycardia, and hypoxemia. Pneumothorax occurs in around 5% of nonventilated infants with IRDS, 10–15% of those receiving nasal CPAP, and between 10% and 30% of cases requiring intubation and ventilation. Its successful treatment relies on early clinical and radiographic recognition, and, if necessary, expeditious drainage of intrapleural gas via needling and intercostal drain insertion. Presence of pneumothorax increases mortality in IRDS, and also increases risk of intracranial hemorrhage. Patency of the ductus arteriosus is a regular feature of IRDS, particularly in the extremely preterm infant. At the height of parenchymal lung disease, whilst pulmonary vascular resistance remains elevated, there may be bidirectional shunt with little net flow through the ductus. As lung disease resolves, however, the resultant fall in vascular resistance and pulmonary arterial pressure can lead to a left-to-right ductal shunt of significant degree. The combination of increased pulmonary blood flow and pulmonary edema that results from this shunt causes a delay in recovery of respiratory function, and a prolonged need for assisted ventilation. Treatment with indomethacin (or ibuprofen) affects closure of the ductus in most instances; recalcitrant or recurrent cases may require surgical ligation. Chronic lung disease (CLD), also known as bronchopulmonary dysplasia, is a common complication of IRDS, occurring in 10–30% of all infants less than 32 weeks, and 30–60% of those less than 28 weeks. The pathogenesis of CLD is multifaceted, complex and continually evolving; the key features are outlined in Table 9. With the recognition of the contribution of barotrauma and volutrauma to CLD has come refinements in the application of assisted ventilation to preterm infants, such that gross fibrous dysplasia and squamous metaplasia is now less common, whereas lung dysmaturity and arrest of alveolarization has become more common, particularly in infants at gestations of less than 28 weeks. With the increased survival at low gestation, care of infants with CLD now places a considerable burden on hospital resources. Treatment for such infants

Table 8 Complications of IRDS Respiratory

Cardiovascular

Neurological

Other

Pulmonary interstitial emphysema Pneumothorax Pneumomediastinum Nosocomial pneumonia Chronic lung disease/ bronchopulmonary dysplasia

Patent ductus arteriosus Poor left ventricular function Pulmonary hypertension

Intraventricular hemorrhage Periventricular hemorrhage Periventricular leukomalacia

Necrotizing enterocolitis Systemic infection Retinopathy of prematurity

334 INFANT RESPIRATORY DISTRESS SYNDROME Table 9 Features of neonatal chronic lung disease Definitions

Risk factors

Pathology

Need for any form of respiratory support (including oxygen) at 36 weeks postconceptional age in a preterm infant born at less than 32 weeks gestation Oxygen dependency (7 abnormal chest radiograph) at 36 weeks postconceptional age in a preterm infant born at less than 32 weeks gestation

Maternal chorioamnionitis Lower gestation and birth weight Increasing severity of IRDSa

Airways Loss of bronchial and bronchiolar epithelium with fibroproliferative response and squamous metaplasia; obliterative bronchiolitis in severe cases Interstitium/respiratory unit Interstitial fibrosis, smooth muscle proliferation and type II cell metaplasia In extremely preterm infants: large, poorly septated primitive airspaces with minimal inflammation or fibrosis Vasculature Smooth muscle hypertrophy and adventitial fibrosis In extremely preterm infants: arrested vascular development

Oxygen dependency at 28 days with an abnormal chest X-ray

a

Associated pulmonary hypoplasia Oxygen therapy Male gender

Ventilation with high positive pressure and high tidal volume Patent ductus arteriosus Fluid overload Infection

Chronic lung disease can occur in preterm infants with minimal early IRDS, including those receiving no initial ventilatory support.

includes supplemental oxygen therapy, nasal CPAP, inhaled bronchodilators and/or glucocorticoids, and diuretic therapy. Systemic glucocorticoid therapy has been linked to poor neurodevelopmental outcome, in particular cerebral palsy, and is now infrequently used in CLD. Preterm infants with IRDS are at risk of serious neurological complications, in particular intracranial hemorrhage and injury to the periventricular white matter. There is a clear association between severity of IRDS and the incidence and severity of intra- and periventricular hemorrhage and periventricular leukomalacia. Hemorrhage within the ventricular system in the preterm neonate begins in the highly vascular germinal matrix, and may remain in the subependymal region, or rupture into the ventricular system itself. Venous infarction in the adjacent brain parenchyma may then occur. The developing white matter tracts in the internal capsule may also be injured; in this case, systemic hypotension and a pressure-passive cerebral circulation are causally implicated. See also: Alveolar Surface Mechanics. Breathing: Breathing in the Newborn. Bronchiolitis. Bronchopulmonary Dysplasia. Fluid Balance in the Lung. Pediatric Aspiration Syndromes. Pediatric Pulmonary Diseases. Pulmonary Function Testing in Infants.

Further Reading Askin FB (1988) Pulmonary disorders in the neonate infant and child. In: Thurlbeck WM (ed.) Pathology of the Lung, pp. 123–146. New York: Thieme.

Comroe JH (1977) Premature science and immature lungs. Parts I– III. American Review of Respiratory Disease 116: 127–135, 311–323, 497–518. Dargaville PA and Mills JF (2005) Surfactant therapy for meconium aspiration syndrome: current status. Drugs 65 (in press). Farrell PM and Avery ME (1975) Hyaline membrane disease. American Review of Respiratory Disease 111: 657–688. Goerke J and Clements JA (1986) Alveolar surface tension and lung surfactant. In: Macklem PT and Mead J (eds.) Handbook of Physiology, Section 3, The Respiratory System, pp. 247–261. Bethesda: American Physiological Society. Goldsmith JP and Karotkin EH (eds.) (2003) Assisted Ventilation of the Neonate. Philadelphia: WB Saunders. Greenough A and Milner AD (eds.) (2003) Neonatal Respiratory Disorders, 2nd edn. London: Hodder Arnold. Jobe AH (1998) Pathophysiology of respiratory distress syndrome and surfactant metabolism. In: Polin RA and Fox WW (eds.) Fetal and Neonatal Physiology, pp. 1299–1314. Philadelphia: WB Saunders. Jobe AH (1999) The new BPD: an arrest of lung development. Pediatric Research 46: 641–643. Martinez AM, Dargaville PA, and Taeusch HW (2000) Epidemiology of bronchopulmonary dysplasia: clinical risk factors and associated clinical conditions. In: Bland RD and Coalson JJ (eds.) Chronic Lung Disease of Early Infancy, pp. 20–40. New York: Dekker. Robertson B and Halliday HL (1998) Principles of surfactant replacement. Biochimica et Biophysica Acta 1408: 346–361. Robertson PA, Sniderman SH, Laros RK Jr, et al. (1992) Neonatal morbidity according to gestational age and birth weight from tertiary care centers in the United States, 1983 through 1986. American Journal of Obstetrics and Gynecology 166: 1629. Slattery MM and Morrison JJ (2002) Preterm delivery. Lancet 360: 1489–1497. Tooley WH (1977) Hyaline membrane disease: telling it like it was. American Review of Respiratory Disease 115: 19–28.

INHIBITION OF DIFFERENTIATION (ID) PROTEINS 335

INHIBITION OF DIFFERENTIATION (ID) PROTEINS bHLH heterodimer

D Morse, University of Pittsburgh, Pittsburgh, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Inhibition of differentiation (Id) proteins were originally described as negative regulators of basic helix-loop-helix transcription factors that play key roles in cell differentiation and proliferation. As more is learned about the regulation of Id proteins, their interactions, and downstream effects, it is becoming evident that Id proteins have more complex activities than originally suspected. Id proteins have now been shown to both induce apoptosis and sustain cell survival, to promote angiogenesis and tumorigenesis, to regulate lymphocyte development and perhaps to regulate matrix deposition. Here, we summarize and discuss the functions of Id proteins as they relate to lung biology.

Introduction Among the many factors known to regulate transcription and thereby fundamentally control lung development, function, and pathology, the inhibition of differentiation (Id) proteins are a relatively new addition. Originally described in 1990 as dominant negative antagonists of the basic helix-loop-helix (bHLH) transcription factors, a growing number of biological functions are now known to be under the influence of Id proteins. Definition of specific roles of Id proteins in the lung is, for the most part, lacking. Nevertheless, the functions that they play in cell growth and differentiation, cancer development, immune function, and angiogenesis ensure that these newcomers will establish themselves in the canon of learning required for a sound understanding of lung biology.

Structure The family of helix–loop–helix (HLH) transcription factors contains over 200 members that function broadly as regulators of developmental processes. Common to all members of the family is a highly conserved HLH region containing two amphipathic a helices separated by a shorter intervening loop. The HLH domain allows these proteins to homo- or heterodimerize, a function that is necessary for their activity. Adjacent to this sequence is a basic region capable of binding to DNA elements containing the canonical E-box sequence, CANNTG. Members of the class of HLH transcription factors known as Id proteins

CANNTG (a)

Gene transcription

E-box

Id protein

CANNTG (b)

No gene transcription

E-box

Figure 1 Effect of Id proteins on gene transcription by bHLH transcription factors. (a) In the absence of Id proteins, bHLH proteins form dimers and bind DNA with their basic regions to activate transcription. (b) In the presence of Id proteins, dimers form between Id and bHLH proteins. Because Id proteins lack the basic region needed for DNA binding, the resulting dimers cannot initiate transcription and gene expression is inhibited.

possess the defining HLH sequence without the adjacent basic region necessary for DNA binding (Figure 1). These proteins are thus capable of binding to other HLH transcription factors and preventing their participation in gene transcription. The term ‘Id’ refers to the ability of these proteins to both inhibit DNA binding and, given the control of HLH transcription factors over cell fate, cell differentiation.

Regulation of Activity The expression of Id proteins, like other early response genes, is rapidly induced by stimulation with growth factors. Regulation of expression at the promoter level is incompletely understood, but Smad proteins, Egr1, Sp1, and ATF3, have been shown to affect promoter activity. Intracellular levels are also regulated through the ubiquitin-proteasome degradation pathway, resulting in half-lives for the Id proteins in the range of 20–60 min. Heterodimerization with other bHLH proteins modulates the rate of degradation of Id proteins and thus can significantly extend their half-lives.

Biological Functions Well-established functions of the HLH family of proteins in multicellular organisms include regulation of cell cycle, lineage commitment, and cell differentiation. The HLH transcriptional regulators

336 INHIBITION OF DIFFERENTIATION (ID) PROTEINS

and their inhibitors are expressed throughout phylogeny from yeast to humans. Four vertebrate Id proteins have been identified and are designated Id1 to Id4. Despite shared homology in their HLH regions and similar binding affinities for bHLH transcription factors, the four Id family members differ in their expression patterns during embryogenesis, and their known biological functions overlap only partially. The sections that follow introduce the reader to those functions beyond embryonic development that are currently being explored for these four proteins. Cell Cycle Control

Cellular differentiation is in many cases accompanied by cell-cycle arrest, and by virtue of their role in cell differentiation, it is reasonable to postulate that the Id proteins may be involved in cell-cycle regulation. Indeed, the expression level of Id genes is high in proliferating cells, and the expression of Id1, Id2, and Id3 transcripts increases rapidly following mitogenic stimulation of serum-starved human fibroblasts. After stimulation with serum or growth factors, there are two peaks of expression of Id1 and Id2, in early and late G1. Inhibition of Id protein synthesis by antisense oligonucleotides prevents the progression of G0-arrested fibroblasts into the cell cycle, providing further evidence that the Id proteins exert control over cell proliferation. The mechanism of this effect is incompletely understood, but a number of recent studies

have elucidated interactions between Id proteins and other cell-cycle regulatory proteins. Id2 and Id3 are now known to be phosphorylated in late G1 by cyclin-dependent kinase 2 (cdk2), although the consequence of this phosphorylation is not clear. Id2 has been shown to antagonize retinoblastoma (Rb) protein and other Rb family proteins through direct protein protein interaction via its HLH domain (Figure 2). Negative regulation of growth-inhibitory proteins such as p21 and p16 by Id1 has been reported by a number of investigators; this presumably occurs via suppression of bHLH transcriptional activity, and leads to cell-cycle progression into the S-phase. Taken together, current evidence suggests that each Id family member is responsible for unique effects on growth regulation, and that the effect of each protein depends upon its specific binding partner and probably upon its state of phosphorylation. Ongoing work in this area of research should lead to a better understanding of the mechanism of action of each Id family protein in cell-cycle control. Tumorigenesis

Given the evidence that Id proteins function as proliferative factors for a wide variety of cell types and regulate cell differentiation, it is natural to hypothesize that Id might also play a role in the development of cancer. There is a wealth of evidence, mainly gleaned from tissue immunostaining, that Id protein

Mitogen

Id Id Proliferation

E2F

1 E2F

Id Rb

Rb

cdk

2 bHLH

P Id

Id

CANNTG

p16 p21

3

Cell cycle arrest

E-box Figure 2 Tentative scheme for the role of Id1 in cell-cycle regulation. 1. Id2 binds and antagonizes unphosphorylated forms of the Rb family proteins, which causes the release of E2F. Once E2F has dissociated, it activates genes involved in cell-cycle progression. 2. Id proteins antagonize basic helix–loop–helix (bHLH) proteins that regulate the expression of target genes, including p21 and p16. Expression of p21 and p16 is thus decreased, and their inhibitory effect on cell-cycle progression is lost. 3. During late G1 phase, cyclindependent kinases (cdk) phosphorylate Id2 and Id3, which may affect the avidity or specificity of their binding to bHLH proteins. Rb protein, retinoblastoma protein.

INHIBITION OF DIFFERENTIATION (ID) PROTEINS 337

levels are elevated in multiple types of human tumors. Ablation of Id protein expression in carcinoma cells leads to reduced proliferation and sometimes to inhibition of their malignant phenotype, indicating that these cells may depend upon Id expression for unrestricted proliferation. Ectopic expression of Id proteins in a variety of cell lines enhances cell proliferation and decreases sensitivity to growth factor depletion; in some cases it confers features of transformation, such as the ability for a fibroblast cell line to grow on soft agar. Despite abundant evidence implicating Id protein expression in tumorigenesis, there have been no mutations of Id genes reported in human cancers. It appears that Id proteins are downstream targets of altered oncogenes that affect Id expression via a wide variety of cellsignaling pathways. Angiogenesis

In order to examine the role of Id proteins in differentiation, a number of groups of investigators have generated mice with targeted disruption of Id1, Id2, or Id3. Mice lacking either Id2 or Id3 exhibit hematopoietic abnormalities, but these mice otherwise appear to develop normally. On the assumption that the Id proteins have overlapping functions, Benezra and colleagues examined the effects of inactivating two members of the family, Id1 and Id3 simultaneously. The deletion of all alleles for Id1 and Id3 resulted in embryonic lethality, but the preservation of one single allele for Id1 allowed for normal development of adult mice. These mice were unique in their response to inoculation with primary tumor cells: they were resistant to tumor growth and metastasis. This elucidated a previously unsuspected function of the Id protein family as key mediators of tumor neovascularization. The mechanism by which the Id proteins promote angiogenesis in this setting is not known, but loss of Id function has been shown to lead to a decrease in vascular endothelial growth factor (VEGF) expression, and the expression of matrix metalloproteinases has been shown to vary with Id protein expression. Since this discovery, thrombospondin-1 has been identified as the transcriptional target of Id1, and a probable effector of its role in angiogenesis. One of the intriguing aspects of the newly discovered role of Id proteins in angiogenesis is that the blood vessel formation associated with tumor growth and metastasis is more sensitive to a decrease in Id protein levels than is the angiogenesis required to support fetal development. This may provide further insight into mechanisms of angiogenesis and make the Id proteins a potential target for anti-angiogenic therapy.

Lymphopoiesis

The bHLH class of transcription factors is crucial to lymphocyte development and function, and Id proteins play important roles in this process. Id2deficient mice are defective in the production of natural killer (NK) cells, and lack Peyer’s patches and peripheral lymph nodes. Given that in the absence of activity of the bHLH transcription factor E2A, NK cell development is enhanced, it seems likely that the differential dose of the bHLH transcription factor E2A and Id2 in early thymocyte differentiation decides lineage commitment. Id3-null mutant mice exhibit defects in positive selection of both MHC class I- and MHC class II-restricted thymocytes, while Id1-null mutant mice do not appear to have defects in lymphocyte development or function. It is evident from the observations made in these mutant mice that the Id proteins act upon a number of stages of lymphocyte development, but the signaling mechanisms involved have yet to be fully defined. There is some evidence implicating both the mitogenactivated protein-kinase cascade and the transforming growth factor beta (TGF-b)/SMAD signaling cascade in Id3 activation, and current studies should further clarify these relationships. Apoptosis

A number of proteins share a dual ability to stimulate proliferation of cells and induce cell death, and the Id proteins are such Janus-faced molecules. Id2 has been shown to augment apoptosis in two cell lines by a mechanism that is completely separate from its ability to dimerize with bHLH transcription factors. The N-terminal region of Id2 alone is sufficient to bring about cell death via increased expression of a known modulator of programmed cell death, Bax. Id3 has also been shown to induce apoptosis of lymphocyte progenitors following TGF-b1 stimulation, although in this case the effect was assumed to be due to modulation of bHLH transcription factor activity. As with other functions of Id, it is perplexing that these proteins can share similar functions and yet employ disparate mechanisms of action; understanding these mechanistic differences will be crucial to clarifying the role of Id proteins in cell death. Fibrosis

Recent evidence suggests that Id1 may play a role in regulating the elaboration of extracellular matrix by fibroblasts (Figure 3). As in lymphocytes, fibroblast Id proteins levels are increased by stimulation with TGF-b1, a key mediator of fibrosis. Chambers and colleagues have shown that when human fetal and adult lung fibroblasts are stimulated with TGF-b1,

338 INHIBITION OF DIFFERENTIATION (ID) PROTEINS TGF-
1

Id1 Metalloproteinases Matrix production Myofibroblast differentiation

Figure 3 Hypothetical scheme for the role of Id1 in TGF-b1-mediated fibroblast behavior. Increased TGF-b1 levels lead to the transition of fibroblasts towards a myofibroblast phenotype with increased a-smooth muscle actin expression and matrix formation. TGFb1 also increases intracellular levels of Id1, which may act to suppress matrix formation and a-smooth muscle actin expression and increase matrix turnover.

Id1 expression increases in a time course typical for immediate-early response genes. The functional implication of this increased Id1 expression is not well understood, but differences in matrix expression between wild-type fibroblasts and fibroblasts deficient in the gene for Id1 have been reported in two different experimental settings. Additionally, Id1 immunostaining is increased in the lung fibrotic lesions within the parenchyma of bleomycin-treated rats, suggesting a relationship between Id1 expression in vivo and lung fibrosis. Interestingly, the effect of TGF-b on Id1 expression in fibroblasts is opposite to that in epithelial cells, where Id1, Id2, and Id3 are all rapidly repressed by treatment with TGF-b1. As with other functions of Id proteins, their role in fibrosis will require clarification by further investigations.

Id Proteins in Respiratory Diseases The adaptive responses of the lung to various injuries and pathologies depend upon a coordinated balanced

cell proliferation, differentiation, and death. The Id proteins play a key role in these cellular behaviors, but to date there is little known about their influence in specific lung diseases. There is mounting evidence that Id1 is a downstream effector of TGF-b1 effects in lung fibrosis, and that Id protein expression can alter the behavior of lung tumors. Nevertheless, as Chairman Mao famously replied when asked his thoughts on the impact of the French revolution, ‘‘It is too early to tell’’ to what extent improved understanding of Id protein function will expand our grasp of lung biology. The discovery of Id proteins is relatively recent, and there is much yet to be learned about how they are regulated and how they integrate into the multiple systems that they are known to influence. The currently known functions of Id proteins are relevant to the study of lung development, lung tumors, diseases involving the adaptive and innate immune responses, and interstitial lung diseases, and so a conservative guess would predict that we will be hearing more about Id proteins in the future.

INSULIN-LIKE GROWTH FACTORS 339 See also: Apoptosis. Cell Cycle and Cell-Cycle Checkpoints. Colony Stimulating Factors. Fibroblasts. Pulmonary Fibrosis. Tumors, Malignant: Overview.

Further Reading Atchley W and Fitch W (1997) A natural classification of the basic helix–loop–helix class of transcription factors. Proceedings of the National Academy of Sciences, USA 94: 5172–5176. Benezra R, Davis R, Lockshon D, Turner D, and Weintraub H (1990) The protein ID a negative regulator of helix– loop–helix DNA binding proteins. Cell 61: 49–59. Chambers RC, Leoni P, Kaminski N, Laurent GJ, and Heller RA (2003) Global expression profiling of fibroblast responses to transforming growth factor-beta1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching. American Journal of Pathology 162(2): 533–546. Florio M, Hernandez MC, Yang H, et al. (1998) Id2 promotes apoptosis by a novel mechanism independent of dimerization to basic helix–loop–helix factors. Molecular and Cellular Biology 18(9): 5435–5444. Heemskert MH, Blom B, Nolan G, et al. (1997) Inhibition of T cell and promotion natural killer cell development by the

dominant negative helix loop helix factor Id3. Journal of Experimental Medicine 186: 1597–1602. Jen Y, Manova K, and Benezra R (1997) Each member of the Id gene family exhibits a unique expression pattern in mouse gastrulation and neurogenesis. Developmental Dynamics 208: 92–106. Langlands K, Yin X, Anand G, and Prochownik EV (1997) Differential interaction of Id proteins with basic helix–loop–helix transcription factors. Journal of Biological Chemistry 272: 19785–19793. Lasorella A, Uo T, and Iavarone A (2001) Id proteins at the cross-road of development and cancer. Oncogene 20: 8326– 8333. Lyden D, Young AZ, Zagzag D, et al. (1999) Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature 401: 670–677. Norton JD (2000) ID helix–loop–helix proteins in cell growth, differentiation and tumorigenesis. Journal of Cell Science 113: 3897–3905. Pan L, Sato S, Frederick JP, Sun XH, and Zhuang Y (1999) Impaired immune responses and B-cell proliferation in mice lacking the Id3 gene. Molecular and Cellular Biology 19(9): 5969– 5980. Rivera R and Murre C (2001) The regulation and function of the Id proteins in lymphocyte development. Oncogene 20(58): 8308–8316.

INSULIN-LIKE GROWTH FACTORS B W Winston, A Ni, and R C Arora, University of Calgary, Calgary, AB, Canada & 2006 Elsevier Ltd. All rights reserved.

Abstract Insulin-like growth factors, IGF-I and IGF-II, are single-chain polypeptides with significant homology to proinsulin. Mature IGF-I peptide is derived from two classes of prepro-IGF-I peptides after posttranslational processing. The majority of circulating IGF-I molecules bind to IGF binding protein-3 and an acid-labile subunit to form a ternary complex. The majority of biological functions of IGFs are mediated by the IGF-I receptor, a tyrosine kinase membrane receptor. Both IGF-I and IGF-II play important roles in development and growth by promoting mitogenesis, cell differentiation, and prevention of cellular apoptosis. IGF-I is the most active peptide at the postnatal stage, while IGF-II is principally expressed and exerts the majority of its actions prenatally. Owing to their strong mitogenic and anti apoptotic effects, IGFs, when present at excessive levels, have the potential to contribute to carcinogenesis. An increasing body of evidence suggests that IGFs are involved in a number of respiratory diseases. The elevated circulating IGF-I level, for example, has been positively related to the risk of lung cancer. Local IGF-I level is elevated in the lungs of patients with idiopathic pulmonary fibrosis, fibroproliferative acute respiratory distress syndrome, sarcoidosis, and other fibrotic lung disorders.

Introduction Insulin-like growth factors (IGFs) (formerly known as somatomedans) are serum peptides with insulinlike activities. The two principal ligands are IGF-I and IGF-II. Unlike other peptide hormones, which are by and large stored within the intracellular space, IGFs circulate throughout the body at concentrations approximately 1000 times higher than those of most peptide hormones. Tissue levels of IGFs, in general, are at lower levels than those present in serum and the IGF levels are still in excess of the level required for maximal cellular stimulation. IGF-I, produced primarily by hepatocytes, is the predominant form of circulating IGF. The existence of these hormones was first proposed by Salmon and Daughaday in 1957, due to the observation that impaired cartilage growth in growth hormone (GH)-deficient rats could be rapidly repaired by the administration of GH in vivo, but the cartilage growth activity in culture media could not be restored by its addition in vitro. It was therefore hypothesized that there were other factors in the serum that mediated GH activity and acted directly

340 INSULIN-LIKE GROWTH FACTORS

on target tissues. They collectively called those unknown factors somatomedins. In 1978, Rinderknecht and Humbel isolated two peptides from Cohn plasma–protein fractions. Their amino acid sequences shared about 70% identity. Since their tertiary structures are highly homologous to each other and to proinsulin, and their biological activities similar to those of insulin, they were named insulinlike growth factor-I and -II. It was later revealed that IGF-I and -II were identical to somatomedin C and somatomedin A, respectively. Both IGFs are crucial for the development and growth of bone, muscle, and other organs in mammals. They also exert insulin-like effects such as decreasing blood sugar levels and stimulating metabolism. A growing body of evidence indicates that IGF-I and IGF-II are also involved in the development of lung cancer and a number of other respiratory diseases such as IPF, sarcoidosis, and fibroproliferative acute respiratory distress syndrome (ARDS).

IGF Gene and Protein Structure Gene Structure

The human IGF-I gene is a single copy, six-exon gene spanning approximately 70 kb on chromosome 12 (Figure 1). Both exon 1 and 2 contain promoter regions with multiple transcription start sites. The mechanism of IGF-I gene regulation is complex (Figure 1), Class 1

with resultant multiple mRNA and peptide species. Only one of exon 1 or 2 can be used for each transcript. The exon 1- and exon 2-derived mRNAs are collectively denoted as class I and class II mRNA, respectively. Class I mRNA is in virtually every tissue except liver. On the other hand, class II mRNA is relatively high in liver. In humans, due to alternative splicing, there are a further two subtypes within class I and II transcripts. Therefore, there are in total four pre-pro-IGF-I peptides, all of which are processed posttranslationally, giving rise to a common mature (70 amino acids, 7.6 kDa) IGF-I peptide. Only the mature form of IGF-I exerts biological activity. The IGF-II gene consists of 10 exons with the mature peptide encoded principally by exons 7–9. The mature peptide is most commonly a 67 amino acid single-chain residue, although there are several human variants that are 67–70 amino acids in length. The IGF-II gene also has complex gene regulation. Alternative splicing, multiple polyadenylation sites, and site-specific endonucleolytic cleavage of IGF-II RNAs can produce proteins of different molecular weights, which increase the complexity of gene transcription (Figure 2). Protein Structure

Both human mature IGF-I and IGF-II proteins are single-chain nonglycosylated peptides that are highly

Class 2

5′ Gene

3′ 1

2

3

4

5

6

Alternative transcription initiation and splicing Class 1 or 2 mRNA 1 or 2

Pre-pro-IGF-IA peptide

3

4

E-region

Signaling sequence

6

1 or 2

3

Pre-pro-IGF-IB peptide

4

5

E-region

Signaling sequence Posttranslational processing

Mature IGF-I ( 70 amino acids, 7.6 kDa ) Figure 1 IGF-I gene, mRNA structure, and posttranslational processing. Multiple transcription initiation sites exist in both exon 1 and 2. Transcription initiated from any site in exon 1 gives rise to class 1 mRNA, while transcription initiated from any site in exon 2 gives rise to class 2 mRNA. The usage of exon 1 and exon 2 is mutually exclusive. Within each class, two species of mRNA exist due to alternative splicing. IGF-IA mRNA lacks exon 5 and IGF-IB mRNA lacks exon 6. The four species of IGF-I mRNA resulting from the combination of alternative transcription initiation and splicing produce four pre-pro-IGF-I peptides with different N-terminal signaling sequences and Cterminal E-regions. During posttranslational processing, both signaling sequence and E-region are cleaved by proteases, resulting in a common mature IGF-I peptide.

INSULIN-LIKE GROWTH FACTORS 341 Gene P1

P2

P3

P4

3′

5′ 1

2

4

3

5

6

8

7

10

9

P1 mRNAs

1

2

3

8

9

10

8

9

10

P2 4 Alternative transcription initiation and splicing

P2 4

5

8

9

10

P3 8

6

10

9

P4 7

8

9

10

Posttranslational processing Pre-pro-IGF-II peptide

E-region

Signaling sequence

Mature IGF-II (67–70 amino acids)

Figure 2 IGF-II gene, mRNA structure, and posttranslational processing. The IGF-II gene consists of 10 exons with alternate transcription initiated by four known promoters. The mature peptide is encoded principally by exons 7–9. Posttranslational processing results in a mature IGF-II peptide of varying size based on the promoter region used to initiate the transcription process.

conserved among both mammalian and nonmammalian species. They both consist of four contiguous domains (Figure 3): an N-terminal B-domain, a central C- and A-domain, and a C-terminal D-domain. This structure is similar to that of proinsulin, which consists of B-, C-, and A-domains. There is substantial amino acid sequence homology between IGFs and proinsulin in their B- and A-domains, but their C-domains contain little sequence identity. Unlike proinsulin, the C-domain in IGFs is not cleaved off during posttranslational processing.

Regulation of Production and Activity Regulation of Production

Although liver is the primary organ producing circulating IGFs, most organs can also produce IGFs locally. The regulation of IGF-I and IGF-II production are quite different. IGF-I The production of IGF-I is regulated by GH, developmental stage, and nutrition status.

GH stimulates IGF-I production at a gene transcriptional level and increases the abundance of all species of IGF-I mRNA. The robust correlation between serum IGF-I level and GH level has led to the use of serum IGF-I measurement as a diagnostic method for detecting GH deficiency. Although the main stimulatory effect of GH is on circulating IGFI, GH can also stimulate local IGF-I expression, such as in ovary and kidney. The regulatory effect of GH on IGF-I is seen only during postnatal life. Both circulating and local IGF-I levels in the fetus are unresponsive to GH. IGF-I levels vary widely during different developmental stages. In humans, prenatal IGF-I is expressed at relatively low level in multiple organs. After birth, serum IGF-I levels rise progressively and reach the level of normal adults before puberty. During puberty, there is a two- to threefold increase in serum IGF-I level, which correlates well with sex hormone levels. After puberty, serum IGF-I levels gradually fall to normal adult levels that are reached in the early thirties, and remains constant until approximately the age of 60, after which serum IGF-I levels begin to decline.

342 INSULIN-LIKE GROWTH FACTORS

C-terminal N-terminal D-domain

B-domain

S S

S S

S S A-domain

C-domain Figure 3 Protein structure of IGFs. The protein structures of IGF-I and IGF-II are very similar. They both consist of four domains (B-, C-, A-, D-domains from N-terminal to C-terminal). The amino acid sequences of B- and A-domains are highly homologous with proinsulin, while C-domain contains little homology with proinsulin. D-domain is unique to IGFs and not found in proinsulin. There are two disulfide bonds linking B- and A-domains, and a third disulfide bond within A domain.

The nutrition status is another important factor that influences IGF-I production. Malnutrition decreases serum IGF-I levels by reducing hepatic IGF-I production. The fact that GH administration to protein-deprived rats restores hepatic IGF-I mRNA to normal levels without changing hepatic and serum IGF-I protein levels, suggests that at least part of the regulation acts at a posttranscriptional stage. IGF-II In humans, IGF-II mRNA and protein levels are highest in fetal tissue, particularly in the liver. After birth, IGF-II levels decrease but do persist in neuronal and liver tissue. There are four known promoters (P1–P4) of gene transcription (Figure 2). During fetal development, promoters P2–P4 direct most of IGF-II transcription. P3, which produces a 6.0 kb mRNA transcript, is the most active. After birth, P1 functions as an ‘adult-stage’ promoter that maintains IGF-II production in the liver. The most active form is a 7.5 kDa peptide. Promoters P2, P3, and P4 continue to initiate IGF-II gene expression in other tissues in the adult, but at much lower levels than P1-initiated expression in the liver. Regulation of Activity

The in vivo activity of IGFs is regulated by a family of proteins named IGF-binding proteins (IGFBP). Six IGF-binding proteins (denoted IGFBP1–6) have been identified to date, which act as a sophisticated regulatory system for IGF activity. About 80% of circulating mature IGF-I and -II bind to IGFBP3 and an acid-labile subunit (ALS) to form 150 kDa ternary

complexes, which stabilize mature IGFs and extend their half-life from about 10 min to several hours. The other five IGFBPs can also bind to IGFs with high affinity to form other complexes. It has been shown that the insulin-like activities of IGFs, such as the hypoglycemic effect, are blocked by the formation of ternary complexes. Also, because this large complex is unable to get through the capillary barrier, it prevents bound IGFs from leaving the circulation, and therefore restricts the availability and activity of IGFs in tissues and organs. In vitro studies show that all six IGFBPs can inhibit IGF activities, primarily by attenuating the binding of IGFs to their receptors. IGFBP3 is the most efficient inhibitor of IGF activities, and is capable of reducing almost every effect of IGFs on cells. However, evidence suggests that IGFBPs also exhibit a stimulatory effect on IGF functions in some situations. The IGF-I-IGFBP3 binary complex has been shown to be able to enhance the effects of IGF-I in wound healing and bone growth in the rat and pig. Preincubation of bovine fibroblasts with IGFBP3 enhances the response of fibroblasts to subsequent IGF-I exposure. Similar enhancement has been seen in dephosphorylated-IGFBP1-treated fibroblasts. In the in vivo environment, multiple IGFBPs can interact with IGFs at the same time. Thus, the final regulatory effect of IGFBPs on IGF activities results from the combined inhibitory and stimulating effects. Furthermore, the interactions between IGFs and IGFBPs are mutual, resulting in reciprocal regulation of both peptides. Taken together, IGFs and IGFBPs form an extremely complex regulatory network, much of which is still not elucidated.

Biological Function IGF-I

IGF-I is a pleiotropic peptide with a wide variety of physiological and cellular functions, one of which is controlling glucose metabolism. Free IGF-I peptide infusion can efficiently decrease serum glucose level by stimulating peripheral glucose uptake as well as decreasing hepatic glucose production, resulting in hypoglycemia. In addition, unlike insulin, very high concentrations of IGF-I can also reduce circulating free fatty acid levels. A more important physiological function of IGF-I is mediating many of the effects of GH. GH controls IGF-I levels and its subsequent ability to enhance somatic growth. Studies on double transgenic mice with GH deficiency and IGF-I overexpression show that IGF-I expression can completely correct the effects of GH deficiency on body weight. Human GH insensitivity syndrome is a disease caused by GH

INSULIN-LIKE GROWTH FACTORS 343

receptor dysfunction. Whereas increasing serum GH level does not alter the clinical symptoms, administration of IGF-I to these patients restores the effects of GH resulting in an increase in nitrogen and phosphate retention and height growth velocity. In addition to its critical role in somatic growth, IGF-I also participates in the development and growth of multiple organs and tissues. IGF-I-deficient mice suffer impaired fetal growth and development, with high perinatal death rates due to muscle hypoplasia and lung immaturity. IGF-I exerts these functions by stimulating cell proliferation, promoting cells to progress through G1 phase and continue through the cell cycle. A wide variety of cells demonstrate mitogenic and differentiation response to IGF-I. Complementary to its mitogenic activity, IGF-I is also capable of inhibiting apoptosis of certain cells. This antiapoptotic action has been well characterized in hematopoietic cells such as pre-B cells and bone marrow-derived mast cells. IGF-I can also promote differentiation of various cells. IGF-I increases the rate and extent of terminal differentiation of myoblasts in culture. IGF-I produced by bone marrow stromal cells in the hematopoietic microenvironment induces differentiation of pro-B cells into pre-B cells. IGF-I also promotes vitamin D3-induced promyeloid cell differentiation into a macrophage cell type. The strong mitogenic and antiapoptotic activities of IGF-I have the potential to contribute to carcinogenesis. A large amount of epidemiological data has provided evidence for a significant positive relationship between circulating IGF-I level and risk of several common cancers, such as breast, prostate, colon, and lung cancers. This relationship, however, is only an association and does not prove causation. Fifty per cent of IGF-I transgenic mice that specifically express IGF-I in the basal prostatic epithelium eventually develop small cell carcinomas or prostatic adenocarcinomas. In vitro studies have shown that IGF-I can stimulate growth of a variety of primary cancer cells and cancer cell lines. IGF-II

IGF-II appears to be essential for normal embryonic development and, as such, IGF-II is thought to be a fetal growth factor. IGF-II is highly expressed in embryonic and neonatal tissues and promotes proliferation of many cell types primarily of fetal origin. In IGF-II knockout mice, fetal growth is significantly retarded. Postnatally, however, growth rates in IGF-II deficient mice parallel the rates in normal mice, but attain only 60% of the normal size as ‘catch-up’ growth does not occur in these animals. However, it should be noted that the levels of IGF-II are

substantially lower in the postnatal mouse as compared to the human. There has not been a syndrome of IGF-II deficiency as such described in humans. IGF-II is the principal IGF peptide expressed in skeletal tissue. It contributes to the proliferation of myocytes during fetal development, and once a sufficient density of cells has been formed, an autocrine source of IGFs is produced to maintain survival and the differentiated state of the myocytes. In vitro, IGF-II has been shown to stimulate type I collagen synthesis and osteoblast proliferation; in vivo, it has been shown to increase bone formation. IGF-II, like IGF-I, is mitogenic and antiapoptotic for a number of cell lines. IGF-II has been shown to be upregulated during normal wound healing, specifically, during the final part of the proliferative phase in granulation tissue formation. Similar to IGF-I, overexpression of the IGF-II gene has been frequently observed in both human and animal tumors and has been implicated in tumorigenesis. Abnormal expression of fetal IGF-II has been implicated in pathologic tissue development. For example, a switch in the regulation of IGF-II promoter usage has been shown to be involved in hepato cellular carcinoma. Similar observations have been made in a number of other tumorous tissues. There is also evidence that IGF-II may play a role in angiogenesis and it has been associated with the development of hemangiomas.

IGF Receptors The biological activities of IGFs are mediated by two IGF cell-surface receptors, IGF-I receptor (IGF-IR) and IGF-II receptor (IGF-IIR), and by the insulin receptor (IR). The molecular structures of IGF-IR and IR are strikingly similar. They are both tetramers with two a-chains and two b-chains linked by disulfide bonds (Figure 4). Both IGF-IR and IR belong to the classic type 2 group of the tyrosine kinase membrane receptor family. Both IGF-I and IGF-II peptides can bind to IGF-IR, and most of their physiological and cellular effects are mediated by IGF-IR. The binding of IGFs to IGF-IR induces a conformational change leading to autophosphorylation of three tyrosine residues in the cytoplasmic domain of the b-chain, which greatly enhances the tyrosine kinase activity of IGF-IR. Activated IGF-IR then phosphorylates a wide range of cytoplasmic adaptor proteins such as Src homology and collagen domain protein (SHC) and insulin receptor substrate (IRS), triggering downstream signaling pathways resulting in various altered cellular behaviors and functions such as proliferation, transformation, and inhibition of apoptosis (Figure 4).

344 INSULIN-LIKE GROWTH FACTORS IGF-I

IR

IGF-II

IGF-IR

 




IGF-IIR (M6PR)


Tyrosine kinase domain

P

SHC

IRS-1/2

SOS/Grb-2

Ras-Raf-MAPK pathway

P110

P

P85

PI3 kinase pathway

Cell responses Figure 4 Interaction between IGFs and IGF-IR, IGF-IIR, IR, and IGF-IR signaling pathway. Both IGF-I and IGF-II can interact with IGF-IR with high affinity and initiate intracellular signal transduction. IGF-I and IGF-II can also interact with IR in some circumstances, with much lower affinity. Only IGF-II can bind to IGF-IIR, which is identical to mannose-6-phosphate receptor. This binding does not induce any known intracellular signal transduction. The thickness of the arrows between IGFs and the receptors indicates the relative affinity of their interactions. The interaction between IGF-IR and its IGF ligands activates the b-chain tyrosine kinase domain by tyrosine residue phosphorylation. Activation of this tyrosine kinase domain phosphorylates adaptor proteins IRS-1/2 and SHC, both of which can interact with either son of sevenless (SOS) growth factor receptor-binding protein (Grb-2) complex or P85 and P110. SOS/Grb-2 complex then associates with Ras, resulting in membrane association and activation of Raf, followed by induction of mitogen-activated protein-kinase (MAPK) pathway. P85 and P110 activate phosphoinositide 3 kinase (PI3-K), which induces PI3 kinase pathway.

IGF-IIR is identical to the mannose-6-phosphate receptor. It is quite different from IGF-IR and IR (Figure 4). IGF-II, but not IGF-I, interacts with the IGF-IIR. IGF-IIR acts as a clearance receptor for IGFII, regulating the extracellular IGF-II levels, but has not been reported to mediate any IGF-II biological function. Several investigators have provided evidence that the IGF-IIR has a tumor suppression function. Genetic mutations of the gene allele transcripts for the receptor have been reported in a number of malignancies, such as hepatocarcinoma, melanoma, renal cell carcinoma, non-Hodgkin’s lymphoma, adrenocortical tumors, and aggressive breast cancer. Compared to the IGF-IR and the IGF-IIR, the IR exhibits much lower affinities to IGFs. The affinity of IGF-I-IR binding is about two orders of magnitude lower than that of IGF-I–IGF-IR binding. Thus, only at pharmacological doses can IGF-I interact with the IR. IGF-II has been shown to bind to the IR at a low affinity during the early stages of development when IGF-IR expression is absent.

IGFs in Respiratory Diseases The critical roles of IGFs in the prenatal and postnatal development of the respiratory system have been described above. Recent data has shown, however, that dysregulation of the IGF and IGFBP have been implicated in the pathogenesis of many respiratory diseases. As stated above, IGFs stimulate proliferation of a number of cell types, including fibroblasts. This interaction may have synergistic effects with other fibrogenic growth factors with the potential of inducing collagen synthesis. Local expression of IGF-I by alveolar macrophages has been well recognized since 1983, explaining why there is an alternative name for IGF-I: alveolar macrophage-derived growth factor (AMDGF). In a number of fibrotic lung disorders including idiopathic pulmonary fibrosis (IPF), fibroproliferative ARDS, and bleomycin-induced pulmonary fibrosis increased local IGF-I production by alveolar macrophages, epithelial cells, and interstitial

INSULIN-LIKE GROWTH FACTORS 345

macrophages has been suggested. In fact, an increased bronchoalveolar lavage (BAL) IGF-I level has been positively associated with disease severity. It has been found that the cytokine production in the lung shifts towards a T-helper-2 (Th2) profile in fibrotic lung disorders, and the Th2 cytokines interleukin (IL)-4 and IL-13 can stimulate the expression of IGFI by alveolar macrophages. IGF-IR expression in alveolar epithelial cells is also upregulated in response to enhanced levels of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) in patients with fibrotic lung disorders. These observations suggest a paracrine or autocrine effect of IGF-I may be operating in these disorders. While the involvement of IGF-I in fibrotic lung diseases is likely, its exact role in these diseases is largely unknown. It has been proposed that IGF-I can stimulate proliferation and differentiation of lung fibroblast, as well as extracellular matrix synthesis, and therefore may augment the severity of pulmonary fibroproliferation. Conversely, it has been suggested that apoptosis of alveolar epithelial cells, which is a key event to induce subsequent fibrogenesis, can be inhibited by IGF-I due to its strong antiapoptotic effect. Further studies are needed to elucidate the role of IGF-I in fibrotic lung diseases. IGFBPs have regulatory functions. IGFBPs exert both stimulatory and inhibitory effects on IGF-I-mediated actions and also exert IGF-independent effects themselves. Importantly, recent data has shown that both IGFBP-2 and -3 levels are increased in BAL fluid taken from patients with idiopathic pulmonary fibrosis and in type II pneumocytes exposed to oxidant injury. It has also been demonstrated that IGFBP-3 may be induced by other inflammatory mediators, such as transforming growth factor beta 1 (TGF-b1). In a recent experimental analysis of normal human adult lung tissue, both IGFBP-3 and -5 were shown to induce production and deposition of collagen and fibronectin in primary adult lung fibroblasts. Thus, several IGFBPs may exert IGF-independent functions in the development of pulmonary fibrogenesis. Owing to its carcinogenic effects, IGF-I may also play a role in lung cancer. Population studies have shown a positive relationship between circulating IGF-I level and risk of lung cancer. Local expression of IGF-I has been found in various primary lung cancer cells in culture. Paradoxically, serum IGF-I levels are not raised in patients with advanced lung cancer. In some cases, IGF-I levels may even decrease due to malnutrition. Functional IGF-I-binding sites are also expressed in these primary cancer cells as well as lung cancer cell lines. In vitro studies on lung cancer cells possessing IGF-I-binding sites have shown that these cells exhibit enhanced mitogenesis,

invasive potential, and resistance to apoptosis in response to exogenous IGF-I. As the IGF-IR has been known to mediate the effects of IGF-I on tumor cells, anti-IGF-IR strategy is being evaluated as a potential therapy for cancer. A number of anti-IGFIR strategies, including dominant negative transgene, RNA interference, and antibodies have been shown to be very effective in preventing carcinogenesis as well as causing tumor regression in rodent models. While the IGF-IR has been deemed as a promising therapeutic target in cancers, the difficulties in specific targeting and delivery need to be resolved before this strategy can be properly studied and used therapeutically.

Acknowledgment BWW is supported by a scholarship from the Alberta Heritage Foundation for Medical Research. See also: Acute Respiratory Distress Syndrome. Epidermal Growth Factors. Fibroblast Growth Factors. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Interleukins: IL-4; IL-13 Platelet-Derived Growth Factor. Systemic Disease: Sarcoidosis. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Allen JT and Spiteri MA (2002) Growth factors in idiopathic pulmonary fibrosis: relative roles. Respiration Research 3(1): 1–13. Baserga R (1996) Controlling IGF-receptor function: a possible strategy for tumor therapy. Trends in Biotechnology 14(5): 150– 152. Daughaday WH (2000) Growth hormone axis overview somatomedin hypothesis. Pediatric Nephrology 14(7): 537–540. Daughaday WH and Rotwein P (1989) Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocrine Reviews 10(1): 68–91. Firth SM and Baxter RC (2002) Cellular actions of the insulinlike growth factor binding proteins. Endocrine Reviews 23(6): 824–854. Jones JI and Clemmons DR (1995) Insulin-like growth factors and their binding proteins: biological actions. Endocrine Reviews 16(1): 3–34. Khandwala HM, McCutcheon IE, Flyvbjerg A, et al. (2000) The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocrine Reviews 21(3): 215–244. Pavelic´ K, Bukovic D, and Pavelic´ J (2002) The role of insulin-like growth factor 2 and its receptors in human tumors. Molecular Medicine 8(12): 771–780. Phillips LS, Pao CI, and Villafuerte BC (1998) Molecular regulation of insulin-like growth factor-I and its principal binding protein, IGFBP-3. Progress in Nucleic Acid Research and Molecular Biology 60: 195–265. Pilewski JM, Liv L, Henry AC, et al. (2005) Insulin-like growth factor binding proteins 3 and 5 are overexpressed in

346 INTERFERONS idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition. American Journal of Pathology 166: 399–407. Rosenfeld RG and Roberts CT (1999) The IGF System: Molecular Biology, Physiology, and Clinical Applications. Totowa, NJ: Humana Press.

Integrins

Stewart CE and Rotwein P (1996) Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiological Reviews 76(4): 1005–1026. Van den Brande JL (1999) A personal view on the early history of the insulin-like growth factors. Hormone Research 51(supplement 3): 149–175.

see Adhesion, Cell–Matrix: Integrins.

INTERFERONS J N Kline and K Kitagaki, University of Iowa, Iowa City, IA, USA & 2006 Elsevier Ltd. All rights reserved.

identified as antiviral substances, they have subsequently been found to engender a variety of significant effects on tumor growth, immune function, and reproductive responses.

Abstract Interferons (IFNs) are a family of cytokines that were first identified almost half a century ago through their antiviral properties. IFNs not only have important antiviral effects but also have a role in antitumor and immunomodulatory responses. There are two major classes of IFNs: type I (IFN-a subtypes, IFN-b, etc.) and type II (IFN-g). Additional IFNs (IFN-like cytokines; IFN-l subtype) have recently been discovered, but they are not as well characterized. Type I and II IFNs use distinct but similar receptor systems. Research on these receptor systems has helped to provide a fundamental base to understand the function and signal pathways of other cytokines and their receptors. The biological effects of IFNs result primarily from the IFN-inducible proteins in responsive cells. Type I IFNs were the first to be produced by recombinant DNA technology and used therapeutically for viral infections, cancers, and autoimmune diseases. IFN-g plays important roles in controlling diseases caused by intracellular bacteria, parasites, and fungi by induction of reactive oxidant species. It may also be important in modulating adaptive immune responses in the lung and participates in the pathogenesis of pulmonary diseases such as pulmonary fibrosis and asthma.

Introduction In 1957, Isaacs and Lindermann discovered a substance that was capable of protecting cells from viral infections. This was one of the first cytokine bioresponses to be indentified, and Isaacs and Lindermann named the substance interferon (IFN). IFNs belong to the class II family of a-helical cytokines and constitute a multimember cytokine family (type I IFNs: IFN-a subtypes, -b, -e, -k, -o, -d, and -t; type II IFNs: IFN-g, IFN-like cytokines, IL-28A (IFN-l2), IL-28B (IFN-l3), IL-29 (IFN-l1), and limitin). Some of these members (IFN-d, IFN-t, and limitin) have not been isolated in humans. Although IFNs were initially

Structure Type I IFNs are a closely related family of 165–172 amino acid proteins that are produced by leukocytes (a subtypes), fibroblasts (b subtypes), lymphocytes (o subtypes), and ruminant embryos (t subtypes). Transcripts of type I IFNs lack introns. The IFN-a gene family resides on human chromosome 9. Excluding pseudogenes, 13 genes encode 12 IFN-a species and many allelic variants, which are single polypeptide chains of 165–166 amino acids, with a molecular mass of approximately 20 kDa. There is only one form of IFN-b. It is encoded by a distinct gene located adjacent to the IFN-a locus in human chromosome 9 and contains a single polypeptide chain of 166 amino acids with a molecular mass of 20 kDa. The IFN-g gene is localized to human chromosome 12. It is 6 kb in size and contains 4 exons and 3 introns. Activation of the gene generates a 1.2 kb mRNA that encodes a 166 amino acid polypeptide. After proteolytic removal of the terminal 23 amino residues, a mature IFN-g consists of a 143 amino acid polypeptide with a predicted molecular mass of 17 kDa. IFN-g functions as a noncovalent homodimer.

Regulation of Production and Activity Production of IFNs is regulated by several transcription factors. One important class of these factors contains a DNA-binding motif and members are termed IFN regulatory factors (IRFs). There are at

INTERFERONS 347

least nine members: IRF-1 to IRF-9. IRF-4, -5, and -7 are primarily expressed in lymphoid cells, whereas others are ubiquitously expressed. Although a variety of cells can produce IFN-a/b in response to viral and bacterial stimuli, plasmacytoid dendritic cells are the most potent producers of type I IFNs. Recent studies demonstrate that the family of Toll-like receptors (TLRs) plays an important role in inducing IFN responses. Pathogen-associated molecular patterns (PAMPs) on a broad range of microbial products bind to TLRs; for example, dsRNA binds to TLR3, ssRNA to TLR7 (mouse) or TLR8 (human), lipopolysaccharide to TLR4, and prokaryotic unmethylated CpG-DNA to TLR9. These interactions lead to the activation of IRF-3, initiating transcription of type I IFNs. IFN-g is produced mainly by T cells (CD4 Th1 cells and CD8 cytotoxic lymphocytes) and natural killer cells in response to mitogenic and antigenic stimuli. Interleukin (IL)-12 and IL-18 induce IFN-g. Production of IFN-g is inhibited by transforming growth factor beta (TGF-b) and IL-10, which are important products of regulatory T cells and other regulatory cells.

JAK-STAT Pathway of IFN Signal Transduction

IFN-a/b signaling The monomer of IFN-a/b binds to the IFN-a receptor (IFNAR) complex, comprised of IFNAR1 and IFNAR2c. The receptor-associated JAKs, tyrosine kinase 2 (TYK2) and JAK1, are crossphosphorylated. STAT2 then binds to IFNAR2c and recruits STAT1. Both STAT1 and STAT2 are phosphorylated and detached, forming a heterodimer. The STAT1–STAT2 heterodimer and IRF9 (p48) migrate to the nucleus, forming interferonstimulated gene factor 3 (ISGF3), and stimulate interferon-stimulated regulatory element (ISRE)-dependent transcription. IFN-l also utilizes STAT1 and STAT2, and stimulates ISRE-dependent transcription as well (see Figure 1). IFN-c signaling The dimeric IFN-g binds to two IFN-g receptor (IFNGR)-1 chains, after which two IFNGR2 are recruited into the ligand–receptor complex. This aggregation of receptor chains phosphorylates JAKs associated with IFNGR. Phosphorylation of JAK1 provides IFNGR1 with a docking site for STAT1. Once STAT1 is phosphorylated by JAK2, the STAT1 detaches from IFNGR1, forming a homodimer. The dimeric STAT1 migrates to the nucleus and stimulates gamma-activated sequence (GAS)-dependent transcription (see Figure 1).

Biological Function The biological responses to all IFNs are mediated by the numerous and pleiotropic proteins generated through induction of Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways.

Termination of IFN Signal Transduction

Following the initiation of ISRE- and GAS-dependent transcription, mRNA for suppression of cytokine

IFN-
/

IFN- IFN- IFNGR1

IFNAR1

IFNGR2

IFNAR2

TYK2

JAK1

JAK2 JAK1

Phosphorylation

STAT2 STAT1

Heterodimer

Nuclear translocation

IRF9

Phosphorylation

STAT1

Homodimer Nuclear translocation

Nuclear ISGF3 ISRE

Gene transcription

Figure 1 The IFN-stimulated JAK-STAT signaling pathways.

GAS

Gene transcription

348 INTERFERONS

signaling 1 (SOCS-1) is synthesized over much the same time course as the mRNAs responsible for the IFN effects. SOCS-1 mRNA is translated into protein, which binds to JAK1, JAK2 and TYK2, and suppresses their tyrosine kinase activity, resulting in inhibition of JAK-STAT signal transduction. IFNinducible protein inhibitors of activated STAT1 (PIAS1) bind tyrosine-phosphorylated STAT1 and inhibit its transcription response. Antiviral Effect of Type I IFN

Both type I and type II IFNs are essential aspects of the innate immune response, providing an early line of defense against viral infections. Type I IFNs induce antiviral and antiproliferative effects on target cells and also modulate immune responses by enhanced expression of major histocompatibility complex (MHC) molecules and cytokine receptors. Although IFNs are reported to inhibit a variety of steps of virus replication, including entry, transcription, translation, maturation, and release, the bestcharacterized IFN-induced antiviral pathways are the dsRNA-dependent protein kinase (PKR), the 2-5A system, and Mx protein pathways (see Figure 2). The PKR pathway IFN-inducible PKR is normally inactive but it autophosphorylates after binding to dsRNA in the presence of ATP. The phosphorylated (activated) PKR inactivates the initiation factor, eIF2, by phosphorylation of the alpha subunit (eIF2a), resulting in rapid inhibition of dsRNA translation. The 2-5A system IFN-inducible 2-5A synthetase is activated by dsRNA and then produces

The PKR pathway

20 ,50 -oligoadenylates (2-5A). 2-5A activates the 2-5A-dependent RNAase L (ribonuclease), resulting in extensive cleavage of mRNA. The Mx pathway IFN-inducible Mx protein interferes with viral replication, impairing the growth of influenza and other negative-strand RNA viruses at the level of viral transcription and at other steps. It is believed to interfere with the trafficking or activity of viral polymerases.

Receptors Although the exact composition of the active IFNAR is still controversial, the active IFNAR consists of at least two polypeptides, IFNAR1 and IFNAR2c. IFNAR1 is a 100 kDa ligand-binding subunit encoded by genes on human chromosome 21 and including a TYK2 binding site. IFNAR2c, a 125 kDa polypeptide encoded by a gene on human chromosome 21, also plays an important role in ligand binding and signal transduction, which is mediated by JAK1. There are three forms of the IFNAR2 chain. IFNAR2a is a soluble form of the extracellular domain of the IFNAR2 subunit. IFNAR2b is an alternatively spliced variant with a short cytoplasmic domain, which has dominant negative activity. The active IFNAR requires IFNAR2c. Active IFNGR consists of two subunits. IFNGR1 is a 90 kDa polypeptide encoded by a gene on human chromosome 6. It binds the ligand and JAK1, which plays an important role in signal transduction. IFNGR2 is a 62 kDa polypeptide encoded by a gene on human chromosome 21. Although it plays only a

The 2-5A system

The Mx pathway

Induction by IFNs

PKR (inactive)

2-5A synthetase (inactive)

Mx protein

Viral dsRNA PKR (active)

2-5A synthetase (active) 2-5A

eIF-2
(active)

eIF-2
(inactive)

RNase L (inactive)

Inhibition of protein synthesis Figure 2 Antiviral mechanisms of IFN action.

RNase L (active)

RNA cleavage

Transcription inhibition

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minor role in ligand binding, it has a JAK2 binding site that is important for signaling. The IFN-l family utilizes IL-28R1 and IL-10R2 chains encoded by genes on human chromosome 1 and 21, respectively. Like IFNAR, they have JAK1 and TYK2 binding sites.

IFN-c in Asthma and Other Respiratory Diseases Airway hyperresponsiveness and reversible airflow obstruction are characteristic features of asthma. Currently, asthma is recognized as chronic eosinophilic airway inflammation that is mediated and modulated by CD4 þ T-helper (Th) cells. These T lymphocytes are divided into two populations, Th1 and Th2, based on their contrasting cytokine expression profiles and distinct functional properties. Th2 cells secrete cytokines implicated in the pathogenesis of allergic disorders, particularly asthma. For example, IL-4 and IL-13 play a major role in promoting Th2 differentiation and IgE production. IL-5 is a critical factor for eosinophil maturation, chemotaxis, activation, and survival. On the other hand, Th1 cells produce IFN-g that promotes Th1 differentiation, while inhibiting Th2 cells and their cytokine production. Therefore, enhancement of Th1 cells and their cytokine production has been considered a potentially useful response to promote in the treatment of allergic disorders including asthma. This concept is supported by a large number of experimental studies. Hanifin and colleagues reported in 1993 that subcutaneous injection of recombinant IFN-g (rIFN-g) results in clinical improvement along with a decrease in circulating eosinophil numbers in patients with atopic dermatitis compared with placebo-treated control patients in a double-blind trial. However, subcutaneously injected and nebulized rIFN-g was not significantly effective in patients with asthma, possibly due to the difficulty in obtaining a sufficiently high concentration locally in the airways. The persistent imbalance toward Th2 is also one of the major mechanisms responsible for the progression of pulmonary fibrosis. IFN-g regulates collagen deposition, probably through reduction of profibrotic cytokines such as TGF-b. A recent clinical trail demonstrated that rIFN-g was effective in improving lung function in patients with idiopathic pulmonary fibrosis. A phase III clinical trail is ongoing in the US. Mycobacterial infections, especially multidrugresistant tuberculosis in the HIV-positive population, have recently been resurgent, becoming a serious

public health problem. Evaluation of the safety and efficacy of inhaled rIFN-g with antimycobacterial drugs is currently ongoing in a randomized, doubleblind, placebo-controlled phase II study.

Other Clinical Applications of Type I IFNs IFN-a has been approved for the treatment of chronic hepatitis B and C as well as condyloma acuminata and labial and genital herpes. Recombinant human IFN-a2a and -a2b were first approved for clinical use in the treatment of hairy cell leukemia and Kaposi’s sarcoma in many countries in the mid 1980s. Since then, they have been approved for the treatment of chronic myelogenous leukemia and metastatic malignant melanoma. Clinical trails continue for their use in other cancers such as renal cell cancer. IFN-b has been approved for the treatment of relapsing-remitting multiple sclerosis, although the mechanism of action still remains unclear. See also: Allergy: Overview; Allergic Reactions. Asthma: Overview. CD14. Defense Systems. Dendritic Cells. Interstitial Lung Disease: Overview. Leukocytes: T cells. Toll-Like Receptors. Interleukins: IL-1 and IL-18; IL-12. Antiviral Agents.

Further Reading Antoniou KM, Ferdoutsis E, and Bouros D (2003) Interferons and their application in the diseases of the lung. Chest 123: 209–216. Bach EA, Aguet M, and Schreiber RD (1997) The IFN gamma receptor: a paradigm for cytokine receptor signaling. Annual Review of Immunology 15: 563–591. Barnes PJ (2004) New drugs for asthma. Nature Reviews: Drug Discovery 3: 831–844. Boehm U, Klamp T, Groot M, and Howard JC (1997) Cellular responses to interferon-gamma. Annual Review of Immunology 15: 749–795. Farrar MA and Schreiber RD (1993) The molecular cell biology of interferon-gamma and its receptor. Annual Review of Immunology 11: 571–611. Hertzog PJ, O’Neill LA, and Hamilton JA (2003) The interferon in TLR signalling: more than just antiviral. Trends in Immunology 24: 534–539. Kalvakolanu DV (2003) Alternate interferon signaling pathways. Pharmacolgy and Therapeutics 100: 1–29. Katze MG, He Y, and Gale M Jr (2002) Viruses and interferon: a fight for supremacy. Nature Reviews: Immunology 2: 675–687. Pestka S, Krause CD, Sarkar D, et al. (2004) Interleukin-10 and related cytokines and receptors. Annual Review of Immunology 22: 929–979. Pestka S, Krause CD, and Walter MR (2004) Interferons, interferon-like cytokines, and their receptors. Immunological Review 202: 8–32.

350 INTERLEUKINS / IL-1 and IL-18 Ramana CV, Gil MP, Schreiber RD, and Stark GR (2002) Stat1-dependent and -independent pathways in IFN-gammadependent signaling. Trends in Immunology 23: 96–101. Sen GC (2001) Viruses and interferons. Annual Review of Microbiology 55: 255–281. Shtrichman R and Samuel CE (2001) The role of gamma interferon in antimicrobial immunity. Current Opinion in Microbiology 4: 251–259.

Stark GR, Kerr IM, Williams BR, Silverman RH, and Schreiber RD (1998) How cells respond to interferons. Annual Review of Biochemistry 67: 227–264. Trinchieri G (2003) Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nature Reviews: Immunology 3: 133–146.

INTERLEUKINS Contents

IL-1 and IL-18 IL-4 IL-5 IL-6 IL-7 IL-9 IL-10 IL-12 IL-13 IL-15 IL-16 IL-17 IL-23 and IL-27

IL-1 and IL-18 E Stylianou, University of Nottingham Medical School, Nottingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Although identified almost half a century apart, the cytokines interleukin-1 (IL-1) and interleukin-18 (IL-18) have marked structural and functional similarities. Research into these two molecules has significantly advanced knowledge of the molecular basis of innate immunity and inflammatory disease. Both cytokines have similar intron–exon arrangements and secondary structure, and are synthesized as precursor proteins that require cleavage by caspase 1. IL-1 and IL-18 bind different receptors but their receptor complexes and signaling pathways share similarities. This is reflected in their distinct and overlapping functions in regulating inflammatory and immune responses. Both cytokines have attracted huge interest as potential therapeutic targets but further research is required to determine whether blocking their effects would be efficacious in respiratory disease.

Introduction Interleukin-1 (IL-1) was first described in the 1940s as a heat-labile protein called endogenous pyrogen

that produced fever. Since then it has been purified as lymphocyte activating factor, thymocyte proliferation factor, and catabolin. The unifying term interleukin-1 was assigned to it in 1979. Considerable interest remains in this ‘master’ cytokine as a key coordinator of immune and inflammatory responses in almost every tissue. The term IL-1 refers to two distinct molecules IL-1a and IL-1b. The expression of these is regulated independently, but they bind to the same receptor and exert virtually identical biological effects. IL-1 markedly resembles its naturally occurring competitive inhibitor, the IL-1 receptor antagonist (IL-lra), IL-18 and six other members of the IL-1 family called IL-F5-10. Interleukin-18 (IL-18) is a 24 kDa nonglycosylated polypeptide that was first reported as interferon gamma-inducing factor (IGIF) in 1995. It was found circulating during endotoxemia in Propionibacterium acnes-infected mice and induced interferon gamma (IFN-g) production by T cells and natural killer cells. In 1999 it was renamed interleukin-18. It has structural and functional similarities to IL-1 but uses a distinct receptor.

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Structure The genomic structure and intron–exon arrangement of the IL-1b, IL-1a, and IL-1ra loci suggests that they are derived from duplication of a common ancestral gene 350 Ma. All three of these genes map to human chromosome 2q. IL-18 and other IL-1 gene family members may also be descended from the same ancestral gene since they share a similar gene structure (Figure 1). Of the members of the IL-1 family, only IL-18 lies on a separate chromosome, 11q22.2–22.3. IL-18 and IL-1a/b consist of b sheets that are packed against each other to form a distinctive structure called a b-trefoil fold. IL-1b and IL-18 exhibit 28.6% amino acid similarity and 17% sequence identity. IL-1b and IL-1a are 23% identical.

Regulation of Production and Activity Monocytes/macrophages are the best-studied sources of IL-18 and IL-1b and both can be synthesized by cells of hemopoietic and nonhemopoietic lineages. For example, IL-18 is synthesized by keratinocytes, osteoblasts, and adrenal cortex cells and IL-1a/b by T cells, endothelial cells, epithelial cells, and fibroblasts, amongst others. Human IL-1a and IL-1b are synthesized as 31– 33 kDa, glycosylated precursor proteins. The structures of the 50 flanking regions of the IL-18, IL-1a, IL-1b, and IL-1ra genes differ. The IL-1b and IL-1ra (but not the IL-1a) promoters have a typical TATA box but all three promoters contain regulatory elements for the nuclear factor kappa B (NF-kB) transcription factor family. IL1b and IL-1ra have binding sites for the transcription factors AP-1 and CREB. The promoters of IL-1a and IL-1b contain sites for the NF-IL6 family of transcription factors. Unusually, IL18 has two promoter regions but neither has a TATA box. The promoter upstream of exon 2 acts constitutively and the one upstream of exon 1 is inducible.

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Both promoters have an NF-kB site which is important in the inducibility of IL-18 gene expression. IL-1b in human monocytes is synthesized extremely rapidly (within 15 min) and can induce its own synthesis, as can other cytokines, for example, tumor necrosis factor (TNF) that can enhance the stability of IL-1b mRNA. The 30 untranslated region contains elements (AUUUA and AU) that render the IL-1b mRNA relatively unstable whereas IL-18 mRNA is unusually stable due to the lack of similar motifs. IL-18 can be synthesized in response to stress-inducing stimuli (neurogenic and bacterial) but is constitutively expressed in human monocytes. Macrophages and keratinocytes store pro-IL-18 at high levels under normal conditions in contrast to little pro-IL-1b. IL-1a/b and IL-18 are enzymatically processed to generate active, mature cytokines. Only a small amount of precursor IL-1b (pro-IL-1b) is secreted. Most of pro-IL-1b is cytosolic; and some of it is retained in lysosomes associated with the microtubules. Most IL-18 also remains cytosolic until it is enzymatically cleaved and transported out of the cell. Neither IL-1b nor IL-18 contains a signal peptide and their precursors are cleaved by the interleukin-1b converting enzyme (ICE), now known as caspase 1. Other caspases can also cleave pro-IL-1b and pro-IL-18. In contrast to pro-IL-1b, pro-IL1a is fully active and it remains in the cytosol. It can be cleaved by calpain and is found in the membranes of monocytes and B cells. Unlike IL-1a/b or IL-18, pro-IL-1ra has a leader sequence, and is synthesized, processed, and secreted.

Receptors The IL-1b and IL-18 receptors are members of the IL1 receptor (IL-1R) family. The type I IL-1 receptor (80 kDa) and the type II receptor (60–68 kDa) are transmembrane glycoproteins. IL-1 receptors are found on all cells except red blood cells. IL-1a and

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Figure 1 Structure of the human IL-18 and IL-1b genes. Exons are represented by white boxes, 50 UTR (untranslated region) and 30 UTR are shown as shaded boxes and the size of these is given within the boxes. Single lines connecting boxes are introns and length of these is given in italics. Adapted from Huising et al. (2004) Developmental & Comparative Immunology 28: 395–413, with permission.

352 INTERLEUKINS / IL-1 and IL-18

have a natural antagonist or soluble form, there is an IL-18 binding protein (IL-18bp) that binds IL-18 with similar affinity to the IL-18R complex. This modulates the activity of IL-18 during inflammation. In addition to the two subunits in each of the IL-1R and IL-18R there are six other IL-1 receptor homologs and members of the IL-1R family which includes IL-1R type II. These are presumed capable of mediating biological responses but limited information is available regarding their ligands and function.

IL-1b each bind at subnanomolar concentrations to the type I IL-1 receptor. The latter has an extracellular, ligand-binding region that comprises three immunoglobulin-like domains. Its cytoplasmic domain is 213 amino acids in length and is homologous to the same regions in the other members of the IL-1R and Toll families. Binding of IL-1 leads to recruitment of the IL-1 accessory protein (IL-1RAP) which initiates signal transduction to activate target cells. The crystal structure of each of IL-1b and IL-1ra bound to the IL-1 receptor has been solved. IL-1ra can antagonize the IL-1 signaling pathway as it binds with slightly higher affinity to the type I receptor. It occupies the ligand-binding site, prevents IL-1a/b binding, and so does not recruit IL-1RAP. Binding of the type II receptor by IL-1b prevents it binding the type I receptor. Hence the type II receptor has been termed a decoy receptor. The type II receptor has a short cytoplasmic tail 29 amino acids in length which cannot transmit an intracellular signal. By internalizing it, it regulates IL-b levels for which it has a higher affinity than for IL-1a or IL-1ra. It can be cleaved to generate a soluble receptor which can also bind IL-1. The IL-18R complex is remarkably similar to the IL-IR complex. The IL-18R consists of two distinct but structurally related immunoglobulin-like domains that are members of the IL-1 receptor family: IL-18Ra and IL-18Rb. Secreted, mature IL-18 interacts with IL-18Ra. This complex heterodimerizes with the signal-transducing IL-18Rb accessory protein that facilitates a conformational change in the receptor to allow high-affinity binding of ligand. As with the IL-1R complex, both chains are required for signal transduction. Although the IL-18R does not

Prostanoids, nitric oxide

Biological Function IL-1 exerts its effects via signaling events many of which are similar to those of IL-18. Both appear to use the adapter molecules myeloid differentiation primary response gene 88 (MyD88), interleukin 1 receptor-associated kinase (IRAK), and TNF receptor-associated factor 6 (TRAF 6), though it is unclear how many of the downstream responses are common to both cytokines. There is evidence for IL-1 and IL18 activation of JNK and p38 mitogen-activated protein kinase (MAPK) but there are conflicting data as to whether IL-18 is a major activator of NF-kB. A principal function of IL-1 is to cause leukocyte accumulation by inducing adhesion receptors on vascular endothelium and to stimulate the production of a variety of chemokines, for example, IL-8 and monocyte chemoattractant protein-1 (Figure 2). IL-1 is a potent stimulator of hematopoiesis and the adaptive immune system and prepares the organism to deal with infection or injury. IL-1 is also required for the efficient synthesis of IFN-g, a key activator of macrophages. It also causes the production of other

Cytokine and chemokine synthesis

Extracellular matrix turnover

Neuroimmune responses, e.g., fever Cartilage and bone formation and remodeling

IL-1

+IL-12

Adhesion molecule expression

+/− IL-12 IFN- production

Allergic reactions

IL-18

B and T lymphocyte activation

Acute phase protein synthesis

FasL expression and Fas-mediated apoptosis

Figure 2 Biological functions of IL-1b and IL-18. The main functions of each cytokine are shown to demonstrate that these have distinct as well as overlapping effects. Fas, an apoptosis-inducing receptor protein; FasL, ligand of the Fas receptor.

INTERLEUKINS / IL-1 and IL-18 353

cytokines, for example, TNF-a, IL-1 itself, along with prostanoids and nitric oxide. Another major role is to stimulate hepatic acute phase protein synthesis. IL-1 can also act as an accessory signal for lymphocyte activation. It can cause cartilage and bone resorption, bone formation, and insulin secretion, and regulates extracellular matrix turnover. There are diverse effects of this cytokine in the brain where IL-1 is the major endogenous pyrogen. A role for IL-1 in mediating hypophagia, slow-wave sleep, and neuroendocrine changes has been documented as is a role in neurodegeneration and neuronal cell death. IL-18 has a widespread though more restricted role than IL-1 in innate and adaptive immunity (Figure 2). It appears to act as an early-warning signal to the immune system of the presence of potentially dangerous pathogens. It acts synergistically with IL-12 to induce IFN-g production. IL-18 upregulates the cytolytic activity of lymphocytes and natural killer cells and promotes the development and differentiation of Th1 and Th2 cells. Like IL-1b, IL-18 is a key mediator in the pathogenesis of a variety of chronic inflammatory disorders and can also induce the production of chemokines, for example, IL-8 and cytokines, and IL-1 and TNF-a. It can activate endothelial cells, upregulate adhesion molecule expression, and stimulate neutrophils, T cells, and dendritic cells, all pivotal events in immune and inflammatory responses.

IL-1 and IL-18 in Respiratory Diseases IL-1b is one of the major proinflammatory cytokines along with IL-1ra that has been identified in bronchial alveolar lavage fluids from patients with acute respiratory distress syndrome (ARDS). In patients the levels of IL-1b relative to its antagonist are elevated 10-fold. Both soluble type II IL-1R and shed type IL1R that can bind antagonists have also been detected in these patients indicating that the balance of production is tilted in favour of proinflammatory activity in these conditions. Also, IL-1b has an important role in the injury of resident epithelial cells and endothelial cells and in the pathogenesis of both acute lung injury and acute respiratory distress syndrome. It is part of the complex network of cytokines that mediate, amplify, and perpetuate lung injury. Biopsy studies have demonstrated that there is increased expression of IL-1b and other proinflammatory and Th2 type cytokines in this disease. IL-1b is one of the cytokines that regulate the inflammatory response of the lower airways that cause the remodeling and structural alterations of the airway in asthma. IL-18 exacerbates a number of severe inflammatory diseases. Its aberrant expression with IL-12 is linked to Th1-related inflammatory diseases, for

example, rheumatoid arthritis. Data from transgenic mice studies indicate that IL-18 can influence Th2related responses in asthma and allergic rhinitis in which elevated levels of the cytokine have been correlated with disease severity. Keratinocyte-specific caspase 1 transgenic mice have a phenotype linked with Th2 type responses. Increased levels of IL-18 in these mice cause the appearance of IL-4 and IL-13 in the airways and skin in an immunoglobulin E (IgE)dependent manner. IL-18 in the presence of antigen transforms Th1 cells into cells that can produce cytokines and chemokines specific for Th1 and Th2 responses, for example, in bronchial asthma. In this way IL-18 directly induces allergic inflammatory responses relevant to allergic rhinitis and asthma. Interestingly, common single nucleotide polymorphisms (SNPs) associated with allergic rhinitis have been identified in the human il-18 gene. One SNP in promoter is strongly associated with total IgE and specific sentisitization to common antigens, for example, pollen and dust mites, but the effect of this SNP on IL-18 gene expression is unknown. Anti-inflammatory agents that target IL-1 and IL18 are required for therapeutic benefit and are in clinical development. Anti-IL-1 strategies have been efficacious in ameliorating a variety of inflammatory diseases, for example, rheumatoid arthritis, in preclinical studies. Neutralization of IL-1b through employment of a recombinant form of the IL-1ra called anakinra has decreased disease activity. However, the efficacy of anti-IL-1 and anti-1L-18 strategies in respiratory diseases is unknown. In animal models, blockade of IL-1 (and TNF) has been successful in limiting lung injury associated with sepsis and respiratory distress syndrome. Recent evidence indicates that in patients with severe disease, anti-Th1 cytokines (e.g., TNF-a) may be highly effective. However, the complexity of the signaling events involved makes the selection of appropriate targets difficult and suggests that a combination of anticytokine therapies might be required. Further research must address the problem of non-specific side effects associated with such strategies and increase understanding of inflammatory signaling pathways to provide new therapeutic targets for respiratory diseases. See also: Acute Respiratory Distress Syndrome. Allergy: Allergic Rhinitis. Asthma: Overview. Bronchoalveolar Lavage. Matrix Metalloproteinases.

Further Reading Dinarello CA (1997) Interleukin-1. Cytokine and Growth Factor Reviews 8: 253–265. Dinarello CA (1999) Interleukin-18. Methods 19: 121–132.

354 INTERLEUKINS / IL-4 Dinarello CA (2004) Therapeutic strategies to reduce IL-1 activity in treating local and systemic inflammation. Current Opinion in Pharmacology 4: 378–385. Fantuzzi G and Dinarello CA (1999) Interleukin-18 and Interleukin-1b: two cytokine substrates for ICE (caspase-1). Journal of Clinical Immunology 19: 1–11. Huising, et al. (2004) Developmental & Comparative Immunology 28: 395–413. Kato Z, Jee JG, Shikano H, et al. (2003) The structure and binding mode of interleukin-18. Nature Structural Biology 10: 966–971. Lee J-K, Kim S-H, Lewis EC, et al. (2004) Differences in signaling pathways by IL-1b and IL-18. Proceedings of the National Academy of Sciences USA 101: 8815–8820. Sims JE (2002) IL-1 and IL-18 receptors, and their extended family. Current Opinions in Immunology 14: 117–122. Stylianou E and Saklatvala J (1998) Interleukin-1. International Journal of Biochemistry and Cell Biology 30: 1075–1079. Tone M, Thompson SAJ, Tone Y, et al. (1997) Regulation of IL-18 (IFN-g-inducing factor) gene expression. Journal of Immunology 159: 6156–6163. Towne JE, Garka KE, Renshaw BR, et al. (2004) Interleukin (IL)1F6, IL-1F8, and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP to activate the pathway leading to NF-kB and MAPKs. Journal of Biological Chemistry 279: 13677–13688. Tsutui H, Yoshimoto T, Hayashi N, et al. (2004) Induction of allergic inflammation by interleukin-18 in experimental animal models. Immunological Reviews 202: 115–138. Ushio S, Namba M, Okura T, et al. (1996) Cloning of the cDNA for human IFN-g-inducing factor, expression in the Escherichia coli, and studies on the biologic activities of the protein. Journal of Immunology 156: 4274–4279. Vigers GPA, Anderson LJ, Caffes P, and Brandhuber BJ (1997) Crystal structure of the type-1 interleukin-1 receptor complexed with interleukin-1b. Nature 386: 190–194. Vigers GPA, Caffes P, Evans RJ, et al. (1994) X-ray structure of ˚ resolution. Journal of interleukin-1 receptor antagonist at 2.0-A Biological Chemistry 269: 12874–12879. Yoshimoto T, Mizutani H, Tstsui H, et al. (2000) IL-18 induction of IgE: dependence on CD4 þ T cells, IL-1 and STAT 6. Nature Immunology 1: 132–137.

tissue repair. All these same actions, however, could also be detrimental to the host in some circumstances, by antagonizing interferon gamma (IFN-g)-driven TH1 responses or by synergizing with IL-13 to foster tissue fibrosis. IL-4 is a ligand for the IL4Ra chain, which can form two distinct signaling complexes, by pairing either with the gamma common chain shared with specific receptors for other four a-helix bundle family cytokines, or with IL-13Ra1, which also binds IL-13. Polymorphisms of IL-4 or of the IL-4Ra chain have been associated with increased severity of asthma.

Introduction As the central immunoregulatory cytokine responsible for initiating type 2 immune responses, interleukin 4 (IL-4) is of great importance to atopy, allergic responses including asthma, and pulmonary fibrosis. IL-4 is a glycoprotein produced by a limited spectrum of hematopoietic cell types, including CD4 þ TH2 lymphocytes, mast cells, and basophils. IL-4 binds to specific cell-surface receptor complexes expressed on a wide variety of immune and parenchymal cell types. The pleiotropic actions of IL-4 include crucial roles in IgE production and polarization of T cells to the TH2 cytokine phenotype, as well as driving production of mucus and extracellular matrix proteins, while counteracting inflammation driven by type 1 responses. IL-4 was first identified in 1982 based on its ability to induce activated murine B cells to proliferate and secrete IgG1. This bioassay explains its original historical name, B-cell stimulatory factor-1 (BSF-1). The cDNA encoding murine IL-4 was cloned virtually simultaneously by groups in the US and Japan in 1996. Human IL-4 and the IL-4Ra chain were both cloned soon thereafter.

Structure

IL-4

IL-4 Protein

J L Curtis, University of Michigan, Ann Arbor, MI, USA

The cDNA for human IL-4 encodes a single open reading frame of 153 amino acids which is processed to a mature protein of 129 amino acids. Full-length human IL-4 contains three disulfide bridges and two predicted N-linked glycosylation sites. Consistent with this prediction, expression of IL-4 in mammalian cells produces proteins of 15, 18, and 19 kDa, corresponding to un-, mono-, and di-glycosylated isoforms. There is no cross-reactivity between murine IL-4 and human IL-4. The three-dimensional structure of IL-4 has been solved using both nuclear magnetic resonance (NMR) and X-ray crystallography. The expressed IL-4 protein has a compact globular configuration similar to that of IL-3, macrophage colony stimulating factor (M-CSF), and granulocyte-macrophage

& 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin 4 (IL-4) is the prototypic immunoregulatory cytokine responsible for initiating allergic responses. IL-4 is a small (15–19 kDa) single-chain glycoprotein that is a member of the four a-helix bundle family of cytokines. It is produced chiefly by activated TH2 lymphocytes, mast cells, and basophils. IL-4 is uniquely required to polarize uncommitted T cells to a type 2 phenotype, characterized by secretion of IL-4 itself, plus IL-5, IL-9, IL-10, and IL-13. IL-4 also induces B cells to switch their immunoglobulin production to IgE secretion. Through a broad range of actions on many other hematopoietic and parenchymal cell types, IL-4 drives immune responses towards antibody production, reduced proinflammatory cytokine elaboration, and

INTERLEUKINS / IL-4

colony-stimulating factor (GM-CSF). Like those cytokines, IL-4’s principal structural feature is a four a-helix bundle, consisting of helices arranged in a left-handed antiparallel bundle that are connected by peptide loops. IL-4 Gene

The human IL-4 gene (Gene ID 3565) is located on the long arm of chromosome 5 (5q31.1), at the centromeric end of a cluster of cytokine genes that also contains the genes for IL-3, IL-5, IL-9, IL-13, and GM-CSF. The IL-4 gene is approximately 10 kb in size, and consists of four exons (encoding 45, 16, 59, and 33 amino acids, respectively) and three introns (Figure 1). A variant IL-4 mRNA transcript, called IL-4d2, occurs naturally and is found in greater abundance in bronchoalveolar lavage cells and thymocytes. The IL-4d2 transcript lacks 48 base pairs (bp) due to alternative splicing of exon 2. This change deletes one disulfide bridge and part of a peptide loop. IL-4d2 has no T-cell proliferative capacity and in vitro antagonizes T-cell proliferation induced by full-size IL-4. A biological role for IL-4d2 is best established in the response to Mycobacterium tuberculosis, in which increased production of IL-4d2 is seen in healthy contacts (relative to unexposed control subjects) and following successful chemotherapy. Relatively increased production of IL-4d2 has also been postulated to reduce asthma susceptibility. What regulates the balance between full-length IL-4 and this apparent natural antagonist, an important subject for vaccine research, is unknown.

Regulation of Production and Activity IL-4 is not secreted constitutively, but is induced on cellular activation via transcriptional regulation. Principal sources are CD4 þ TH2 lymphocytes, mast cells, and basophils. Early in immune responses, NKT cells are also a key source of IL-4. NKT cells are an innate cell type that shares expression of some 5′

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surface receptors with classical natural killer (NK) cells; however, NKT cells display a restricted T-cell antigen repertoire and depend on the major histocompatibility complex (MHC) class I-like molecule CD1d for development and activation. Secretion of IL-4 has also been reported by g/d T cells, eosinophils, and alveolar macrophages.

Biological Function IL-4 acts on many cell types to polarize immune responses toward antibody production and away from cell-mediated cytotoxicity (Figure 2). The next eight sections outline direct effects of IL-4 on individual cell types, followed by a summary of the integrated effects of IL-4 on immune responses and of conclusions derived from transgenic mice that either overexpress or lack IL-4 or the IL-4Ra chain. T-Cell Polarization

Probably the most important action of IL-4 is to induce differentiation of uncommitted T cells to the type 2 phenotype. T cells of this phenotype (called TH2 cells if CD4 þ and TC2 cells if CD8 þ ) produces IL-4 itself, plus IL-5, IL-9, IL-10, and IL-13. This differentiative effect of IL-4 is mediated by induction of the fate-determining transcription factor GATA-3. IL-4 further promotes the survival and proliferation of TH2 cells, by acting as an autocrine growth factor. Additionally, IL-4 antagonizes development of the TH1 subset, by blocking interferon gamma (IFN-g) production that would lead to TH1 commitment, and by inhibiting IL-2-induced proliferation needed to sustain TH1 and TC1 cells. Unlike many other actions of IL-4, these inductive effects to TH2 polarization cannot be replaced by IL-13. B-Cell Differentiation

IL-4 enhances the proliferation of activated B cells and potently induces them to class-switch their immunoglobulin production to IgE and IgG4 (IgE and IgG1 in mice). IL-4 also increases expression of several receptors on B cells, including surface IgM, class II MHC antigens, CD40, and CD23, the lowaffinity receptor for IgE (FceRII). By downregulating FcgRII (CD32), IL-4 counteracts the inhibitory effect of immune complexes on B-cell activation. IL-4 also induces B cells to secrete IL-6 and tumor necrosis factor alpha (TNF-a).

29

Murine IL-4 Figure 1 Diagram of IL-4 gene structure in humans and mice. For exons, translated sequences are indicated in white, and untranslated sequence in blue. Adapted from data in the National Center for Biotechnology Information.

Mononuclear Phagocytes

IL-4 inhibits macrophage production of proinflammatory cytokines, including TNF-a, IL-1, and IL-6, in part via transcriptional repression. IL-4 also enhances

356 INTERLEUKINS / IL-4 APC

Naive T cell

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Basophil IL-4 IL-4

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Endothelial cells Mast cell VCAM

E-selectin Fibroblasts Epithelial cells Chemotaxis, proliferation Mucus production

ICAM-1, collagen, fibronectin expression

Figure 2 IL-4 exerts pleiotropic actions on hematopoietic and parenchymal cell types. The antigen-presenting cell (APC) delivers appropriate signals (including antigen in the context of MHC and costimulation), activating the naive T cell (yellow starburst). After activation, the naive T cell becomes a TH2 cell via exposure to IL-4. This exposure could come either from an NKT cell, an innate immune cell type that rapidly makes IL-4 on stimulation, or from the T cell itself, via a stochastic process following T-cell activation. Once polarized, the TH2 cell produces IL-4 (and the additional type 2 cytokines IL-13, IL-5, IL-6, and IL-9). Cell types are shown in black text and IL-4 effects in red text; Mø, macrophage. This diagram highlights the known actions of IL-4, but during in vivo immune responses many of these actions synergize with those of other type 2 cytokines, especially IL-13.

production of molecules that counteract these cytokines, such as IL-1R antagonist and the decoy receptor IL-1RII. In monocytes, IL-4 decreases expression of surface and soluble p75 TNF receptors. Additionally, IL-4 upregulates CD13, a cell-surface aminopeptidase which inactivates inflammatory peptides. IL-4 upregulates macrophage expression of MHC class II antigens LFA-1 and CD23, while decreasing CD14 and all three FcgRs. IL-4 also induces formation of multinucleated giant cells, and suppresses macrophage apoptosis. Macrophage production of superoxide and prostaglandin E2 (PGE2), and induction of cyclooxygenase activity are all profoundly inhibited by IL-4. Furthermore, IL-4 switches eiscosanoid production in both macrophages and dendritic cells by downregulating 5-lipoxygenase and upregulating 15-lipoxygenase. Granulocytes

Acting early in development of bone marrow progenitors, IL-4 favors granulopoiesis over production of monocyte–macrophages. Synergizing with IL-3, IL-4 drives basophil production. IL-4 is a chemoattractant for eosinophils and neutrophils, and induces eosinophils to produce TH2 cytokines.

Mast Cells

IL-4 induces mast cells to proliferate and increase surface expression of FceRI, the receptor that triggers release of granular products such as histamine and tryptase on binding of IgE. IL-4 also regulates expression by mast cells of leukotriene C4 synthase. Epithelial Cells

IL-4 induces goblet cell metaplasia in murine models and mucin gene activation in mice and cultured human airway epithelial cells. Vascular Endothelial Cells

Acting together with TNF-a, IL-4 alters adhesion molecule expression on vascular endothelial cells, by inducing vascular cell adhesion molecule 1 (VCAM1) and suppressing E-selectin. IL-4 also counteracts the procoagulant effect of lipopolysaccharides (LPS), IL-6, and TNF-a. Fibroblasts

IL-4 exerts several actions on fibroblasts that have the net effect of increasing fibrosis. IL-4 induces fibroblast chemotaxis and proliferation. It increases

INTERLEUKINS / IL-4

intercellular adhesion molecule 1 (ICAM-1) expression and synthesis of such extracellular matrix proteins as fibronectin and types I and II collagen. Integrated Effects of IL-4

Collectively, IL-4-induced changes enhance the capacity of B cells to survive and present antigen to T cells. IL-4 reduces many of the inflammatory effects of TH1 responses, by suppressing production of IFN-g, IL-1, and TNF-a, and by reducing the responsiveness of macrophages to those cytokines. Induction of VCAM and suppression of E-selectin on endothelial cells by IL-4 favors recruitment of Th2 cells, basophils, and eosinophils over neutrophils. Inhibiting IL-4 activity via neutralizing antibody against the cytokine or IL-4Ra chain, or by gene targeting either of these molecules, diminishes TH2 responses. This finding substantiates the central role of IL-4 in T-cell differentiation, but also does not exclude the possibility that the biological effects seen in these experimental models may derive from effects of other TH2 cytokines. In mice, such treatments ablate the protective effect of previous infection with two nematode parasites, and has a striking effect on murine models of allergic airways disease. Overexpression of IL-4 in the lungs of transgenic mice impairs the elimination of Histoplasma

Type I receptor complex

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capsulatum that is normally mediated by development of a type 1 immune response. However, IL-4 does not cause a universally defective host response, as these transgenic mice show accelerated clearance of Pseudomonas aeruginosa.

Receptors The reason for the overlapping effects of IL-4 and IL13 can best be appreciated by understanding the cell surface receptors that bind these two related cytokines (Figure 3). IL-4 binds to the IL-4Ra chain with high affinity (KdB20–300 pM). The IL-4Ra chain can then dimerize with either of two chains: (1) the common gamma subunit (gc), which is shared by receptors for multiple members of the four a-helix bundle family of cytokines, to form the type I receptor complex; or (2) with low affinity to the IL-13Ra1 chain, to form the type II receptor complex. By contrast, IL13 first binds with high affinity to the IL-13Ra1 chain, which then dimerizes with the IL-4Ra chain to form a complex identical to the type II receptor. IL-13 is unable to bind the IL-4Ra chain directly. Both type I and type II receptor complexes transduce signals; how the cell distinguishes between IL-4 and IL-13 bound to type II receptor complexes is currently unclear. Type I receptor complexes predominate on hematopoietic cells, while type II

Type II receptor complex IL-4

IL-13 OR

IL-4R chain


c Chain

Jak3

Jak1

IL-13R1 chain

Jak1

IL-13R2 chain

Tyk2

STAT3 STAT6

(a)

(b)

(c)

Figure 3 IL-4 and IL-13 receptors. The IL-4Ra chain can pair with either of two transmembrane receptors. (a) Pairing of the IL-4Ra chain with the gc chain in response to IL-4 forms the type I receptor complex. This complex is associated with Jak3 and Jak1 (members of the Janns tyrosine kinase family), and leads to activation of signal transducer and activator of transcription 6 (STAT6). (b) Pairing of the IL-4Ra chain with the IL-13Ra1 chain in response to IL-4 or IL-13 forms the type II receptor complex. This complex is associated with Jak3 and Tyk2, and leads to activation of STAT3. Thus, IL-4 can bind to either the type I receptor complex (a) or the type II receptor complex (b), and in either case, can activate receptor-associated kinases to induce STAT activation. By contrast, IL-13 cannot bind to the type I receptor complex, but does signal via the type II receptor complex. (c) IL-3 can also bind to the monomeric IL-13Ra2 chain, a decoy receptor.

358 INTERLEUKINS / IL-4

receptor complexes are expressed on both hematopoietic cells and nonhematopoietic cells, including airway epithelium. Expression of IL-4Ra chain on lymphocytes is upregulated by activation (e.g., by LPS or anti-IgM stimulation of B cells or by antigen–stimulation of T cells). IL-4 itself also upregulates expression of the IL-4Ra chain, setting the stage for a positive feedback loop. In addition to the cDNA encoding the full-length IL-4Ra chain, cDNAs for two shorter forms have been isolated. One encodes a membrane-bound form lacking a cytoplasmic domain, predicted to be a decoy receptor. The other encodes a soluble form of the receptor, which has been found to be secreted and to block the ability of IL-4 to induce T-cell proliferation in vitro. Neither IL-4Ra nor gc has enzymatic activity. Instead, binding of IL-4 recruits and activates a pair of Janus family tyrosine kinases (Jak-1 to IL-4Ra, Jak-3 to gc, Tyk2 to IL-13Ra) (Figure 3). The Jak kinases first transphorphorylate and activate each other. Next, they phosphorylate tyrosines in the cytoplasmic domain of IL-4Ra (or IL-13Ra), providing docking sites for several signaling molecules. Among these are the latent transcription factors STAT6 (which binds to phosphorylated IL-4Ra), and STAT3 (which binds to phosphorylated IL-13Ra1). Phosphorylation induces STAT6 monomers to dimerize, migrate to the nucleus, and bind to specific sites in the promoters of many genes to mediate most effects of IL-4.

IL-4 in Respiratory Diseases Polymorphisms of the IL-4 gene have been associated with severity of atopic asthma in several ethnic groups. Two naturally occurring polymorphisms of the IL-4Ra chain have been implicated in atopy. One of these, substitution of Val50 by isoleucine, enhances signal transduction resulting in increased IgE production. The exact importance of IL-4 in respiratory diseases remains to be determined. The unique role of IL-4 polarization of uncommitted T cells to the TH2 phenotype argues that it should play a key role in allergies and host defense against pathogens. However, virtually all other actions of IL-4 can be replaced by other TH2 cytokines, especially IL-13, which appears to be more crucial as a mediator of immune responses in tissues outside organized lymph nodes. Mast cells, basophils, and eosinophils have recently been shown to be capable of producing IL-13 transcripts in an IL-4Ra- and STAT6-independent fashion.

Supporting this generalization, soluble IL-4 receptor that inhibited allergen-induced IgE production in preclinical models has been administered by inhalation in two small trials in asthmatic patients. The modest clinical efficacy seen in these short trials awaits confirmation in larger, longer studies. See also: Adhesion, Cell–Matrix: Focal Contacts and Signaling. Allergy: Allergic Reactions. Apoptosis. Asthma: Extrinsic/Intrinsic. Chymase and Tryptase. Colony Stimulating Factors. Dendritic Cells. Dust Mite. Extracellular Matrix: Collagens. Fibroblasts. Histamine. Immunoglobulins. Interferons. Interleukins: IL-1 and IL-18; IL-5; IL-6; IL-9; IL-13. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Mast Cells and Basophils; Neutrophils; Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Leukotrienes. Mucins. Mucus. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens); Parasitic; Viral. Pulmonary Fibrosis. Signal Transduction. Smooth Muscle Cells: Airway. Transcription Factors: Overview. Transgenic Models.

Further Reading Barnes PJ (2002) Cytokine modulators as novel therapies for asthma. Annual Review of Pharmacology and Toxicology 42: 81–98. Beghe B, Barton S, Rorke S, et al. (2003) Polymorphisms in the interleukin-4 and interleukin-4 receptor alpha chain genes confer susceptibility to asthma and atopy in a Caucasian population. Clinical and Experimental Allergy 33: 1111–1117. Chatila TA (2004) Interleukin-4 receptor signaling pathways in asthma pathogenesis. Trends in Molecular Medicine 10: 493– 499. Chiu BC, Freeman CM, Stolberg VR, et al. (2003) Cytokine– chemokine networks in experimental mycobacterial and schistosomal pulmonary granuloma formation. American Journal of Respiratory Cell and Molecular Biology 29: 106–116. Finkelman FD and Urban JF Jr (2001) The other side of the coin: the protective role of the TH2 cytokines. Journal of Allergy and Clinical Immunology 107: 772–780. Gordon S (2003) Alternative activation of macrophages. Nature Reviews: Immunology 3: 23–35. Jakubzick C, Kunkel SL, Puri RK, and Hogaboam CM (2004) Therapeutic targeting of IL-4- and IL-13-responsive cells in pulmonary fibrosis. Immunologic Research 30: 339–349. Kronenberg M and Gapin L (2002) The unconventional lifestyle of NKT cells. Nature Reviews: Immunology 2: 557–568. Martin JG, Suzuki M, Ramos-Barbon D, and Isogai S (2004) T cell cytokines: animal models. Paediatric Respiratory Reviews 5(supplement A): S47–S51. Mueller TD, Zhang JL, Sebald W, and Duschl A (2002) Structure, binding, and antagonists in the IL4-/IL-13 receptor system. Biochimica et Biophysica Acta 1592: 237–250. Nelms K, Keegan AD, Zamorano J, Ryan JJ, and Paul WE (1999) The IL-4 receptor: signaling mechanisms and biologic functions. Annual Review of Immunology 17: 701–738. O’Byrne PM, Inman MD, and Adelroth E (2004) Reassessing the Th2 cytokine basis of asthma. Trends in Pharmacological Sciences 25: 244–248.

INTERLEUKINS / IL-5 Steinke JW (2004) Anti-interleukin-4 therapy. Immunology and Allergy Clinics of North America 24: 599–614. Wills-Karp M (1999) Immunologic basis of antigen-induced airway hyperresponsiveness. Annual Review of Immunology 17: 255–281. Zhu J, Guo L, Watson CJ, Hu-Li J, and Paul WE (2001) Stat6 is necessary and sufficient for IL-4’s role in Th2 differentiation and cell expansion. Journal of Immunology 166: 7276–7281.

Relevant Website www.ncbi.nim.nih.gov – National Center for Biotechnology Information.

IL-5 M L Moore and R S Peebles Jr, Vanderbilt University School of Medicine, Nashville, TN, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin-5 (IL-5) is a cytokine secreted principally by activated helper T cells of the T-helper-2 (Th2) subset. IL-5 is a short chain helical bundle cytokine structurally similar to IL-3, IL-4, and granulocyte-macrophage colony-stimulating factor. The IL-5 gene is tightly linked with the IL-4 and IL-13 genes in a Th2 cytokine gene cluster on chromosome 5q, and the expression of these genes is coordinately regulated. The bioogy of IL-5 has been best studied in the context of asthma in humans and allergic airway inflammation in mouse models. IL-5 directly mediates eosinophilic inflammation by stimulating the differentiation, proliferation, recruitment, survival, and activity of eosinophils. Eosinophils release granule proteins and leukotrienes that are hypothesized to contribute to airway hyperreactivity and epithelial damage in allergic asthma. However, recent studies indicate that the immunopathogenic role of IL-5 and eosinophils in asthma is not clear.

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eosinophil differentiation activity, and it was renamed IL-5. DNA sequencing revealed that IL-5 was identical to B-cell growth factor II (BCGF-II). By 1987, complementary DNA (cDNA) clones for mouse and human IgA-enhancing factor (IgA-EF), eosinophil colony-stimulating factor (Eo-CSF), and human eosinophil differentiation factor (EDF) were all found to encode for the same molecule, IL-5. In 1988, it was demonstrated that recombinant hIL-5 stimulates the differentiation, proliferation, recruitment, survival, and activity of mouse eosinophils. Since then, a body of evidence has firmly established the requirement for IL-5 in airway eosinophilia. Mortality and morbidity associated with asthma has increased over the last two decades, prompting research aimed at elucidating the role of IL-5 and eosinophils in this disease.

Structure IL-5 is a member of the helical bundle family of cytokines, along with IL-3, IL-4, and granulocytemacrophage colony-stimulating factor (GM-CSF). hIL-5 and mIL-5 contain 114 and 113 amino acids, respectively, and they are 70% identical. IL-5 is a disulfide-linked, homodimeric protein with an apparent molecular weight of 45–60 kDa; it is heavily glycosylated. Each monomer of IL-5 consists of four a-helical domains with a small antiparallel b-sheet between each of the helices. The two monomers are interdigitating, such that the C-terminal end and helix of each polypeptide interacts with the N-terminal three helices of the other polypeptide (Figure 1). The formation of an interdigitating homodimer is necessary for the function of IL-5. Among the short chain helical bundle cytokines, dimerization is unique to IL-5.

Introduction Interleukin-5 (IL-5) is a cytokine synthesized and secreted mainly by activated CD4 þ helper T cells of the T-helper-2 (Th2) subset, and also by mast cells and eosinophils. The effector functions of human IL5 (hIL-5) are principally associated with a single cell type, the eosinophil. In mice, IL-5 stimulates B cells in addition to eosinophils. One hundred years after eosinophils were first associated with symptomatic asthma, molecular cloning enabled the identification of IL-5 as a cytokine that stimulates the functions of eosinophils. In the 1970s and 1980s, soluble factors produced by T cells cultured in vitro were purified biochemically and named according to their biological activities. Mouse IL-5 (mIL-5) was originally identified as T-cellreplacing factor (TRF). TRF was shown to have

N

N′ Figure 1 View of the homodimeric IL-5 protein along the dimer twofold axis. IL-5 consists of two interdigitating polypeptides and contains two helical bundle domains. Each helix is represented by a cylinder. There are four helices per polypeptide (shaded or unshaded) with a small antiparallel b-sheet (represented by a solid or dashed line) connecting each of the helices. The C-terminal end and helix of each polypeptide interacts with the N-terminal three helices of the other polypeptide. Dimerization is required for function.

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Regulation of IL-5 Synthesis hIL-5 is located on chromosome 5 and mIL-5 is located on chromosome 11. In both species, the IL-5 gene is tightly linked to the genes for IL-4 and IL-13, and these three genes are contained in a Th2 cytokine gene cluster that spans 140 kb. Coding and noncoding regions within the Th2 cytokine gene cluster are conserved between mouse and man. The IL-5 gene is also linked to the IL-3 and GM-CSF genes, which are approximately 500 kb from IL-5 in both mouse and man. In humans, the region containing the Th2 cytokine cluster and the genes for IL-3, GM-CSF, IL-9, and fibroblast growth factor 1 is called the chromosome 5q cytokine cluster. The genes for IL-4, IL-5, and IL-13 are coordinately expressed and regulated. Evidence suggests that chromatin remodeling mechanisms, such as histone modification and DNA methylation, are an integral part of transcriptional regulation of the Th2 cytokine cluster. In differentiating Th2 cells, ligation of the T-cell receptor results in activation of the transcription factors signal transducer and activator of transcription 6 (STAT6) and GATA-3, which direct chromatin remodeling and specifically stimulate transcription from the Th2 cytokine cluster. Mast cells, another source of IL-5, do not express STAT6 or GATA-3. However, mast cells and Th2 cells contain the same DNase I hypersensitivity (HS) sites at this locus. HS sites mark accessible chromatin and reflect noncoding regions of DNA that act as docking sites for chromatin remodeling and transcriptional protein complexes. Thus, Th2 cells and mast cells may regulate IL-5 transcription via similar chromatin remodeling pathways but distinct signaling pathways. Elements regulating IL-5 synthesis are not fully defined. The proximal 50 IL-5 promoter contains conserved sequence motifs also found in the promoters of the IL-3, IL-4, and GM-CSF genes. However, like the IL-3, IL-4, and GM-CSF promoters, the IL-5 promoter alone cannot reproduce physiological expression patterns. A number of distal DNA elements that regulate IL-5 transcription have been defined, including a 6.2 kb upstream enhancer with potential GATA-3 binding sites. Cytokines that stimulate the differentiation and activation of Th2 cells can promote IL-5 production, including IL-2, IL-4, and IL-13. IL-2 is produced by T cells and is mainly an autocrine T-cell growth factor. The IL-4 and IL-13 receptors have a common subunit (IL-4Ra) that activates STAT6, resulting in transcription of many genes, including IL-4, IL-5, and IL-13. Prostaglandin E2 (PGE2) can increase or decrease IL-5 expression in T cells in vitro, depending on the culture conditions. Cytokines that can

suppress IL-5 production via inhibition of Th2 cells include interferon (IFN)-a, IFN-g, and IL-12. The cytokine transforming growth factor beta (TGF-b) can directly block the antiapoptotic effect of IL-5 on eosinophils and inhibit eosinophil differentiation.

Biological Function IL-5 promotes the differentiation of eosinophil precursor cells in the bone marrow, the recruitment of eosinophils from the bone marrow to tissues, the activation of tissue eosinophils, and eosinophil survival (Figure 2). Eosinophils are bone marrow-derived granulocytes that are important for defense against helminthic parasites (flukes, tapeworms, and roundworms) but contribute to pathogenesis in allergic diseases. IL-5 is the predominant hematopoietic growth factor for eosinophils, stimulating colony formation by CD34 þ myeloid progenitor cells in the bone marrow and their development into mature eosinophils. The transcription factors CCAAT/enhancer binding protein (C/EBP) is required for eosinophil and neutrophil maturation, whereas GATA-1 specifically promotes the terminal differentiation of eosinophils. Bone marrow

CD34+

IL-5

CCR3

EO

PAF-R

Blood CD11b IL-5 Lung parenchyma Leukotrienes, ECP, EPO, MBP, EDN, PAF, cytokines

EO

IL-5 Apoptosis

Figure 2 IL-5 promotes the maturation, recruitment, activation, and survival of eosinophils. IL-5 is the predominant growth factor for eosinophils, stimulating the differentiation of CD34 þ myeloid progenitor cells in the bone marrow into mature eosinophils (EO). IL-5 mediates eosinophil recruitment by upregulating the cell surface expression of the adhesion molecule CD11b and by upregulating the expression of PAF-R and CCR3, the receptors for PAF and eotaxin, respectively. IL-5 promotes eosinophil activation by stimulating the production of leukotrienes, granular proteins (ECP, EPO, MBP, and EDN), PAF, and cytokines; IL-5 induces eosinophil degranulation. IL-5 promotes the survival of eosinophils by inhibiting apoptosis.

INTERLEUKINS / IL-5

IL-5 mediates eosinophil recruitment by a variety of mechanisms. The direct chemotactic potential of IL-5 for eosinophils is modest. However, IL-5 upregulates the expression of the adhesion molecule CD11b on the surface of eosinophils, thereby facilitating recruitment. IL-5 also increases the expression of the platelet-activating factor (PAF) receptor on eosinophils. PAF is a known chemotactic factor for eosinophils. A potent inducer of eosinophil recruitment is the chemokine eotaxin, which functions cooperatively with IL-5 to mediate eosinophil extravasation from the blood to the lung parenchyma. IL-5 also primes eosinophils for chemotaxis mediated by the chemokines IL-8 and regulated upon activation normally T cell expressed and secreted (RANTES). Cells of the promyelocytic leukemia cell line HL-60 can be induced to differentiate into eosinophils by treatment with butyric acid. In this model of eosinophil development, addition of IL-5 to the culture media increases expression of CCR3, the receptor for eotaxin. IL-5 augments eosinophil activation and cytotoxicity. Eosinophils release a number of effector molecules, including the eosinophil granule proteins eosinophilic cationic protein (ECP), major basic protein (MBP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), as well as leukotrienes, PGE2, a number of cytokines (IL-2, IL-3, IL-4, IL-5, GM-CSF, IL-6, INF-g, IL-12, and TGF-b), and reactive oxygen intermediates, such as hydrogen peroxide and hydroxy radicals. The granule proteins released by activated eosinophils are highly cytotoxic to cells, including helminthic cells, in the case of parasitic infections, and mammalian airway epithelial cells, in the case of asthma. mRNAs for the eosinophil granule proteins are among the most abundant transcripts in IL-5-differentiated umbilical cord precursor cells, with MBP being the most abundant mRNA. IL-5 stimulates eosinophil production of leukotrienes, superoxide, and cytokines. In addition, IL-5 promotes eosinophil degranulation. PAF may play a role in IL-5-mediated eosinophil activation since antagonists of PAF inhibit IL-5-induced eosinophil superoxide production and degranulation. In addition to IL-5, IL-3, and GM-CSF can stimulate eosinophil effector functions. Mature eosinophils express approximately 1000 receptors per cell for IL-3, IL-5, and GM-CSF. IL-5 and GM-CSF promote eosinophil survival by inhibiting apoptosis. Mature eosinophils undergo apoptosis in vitro and in vivo if these cytokines are absent from the milieu. Mitochondria play an important role in regulating apoptosis, in addition to facilitating cellular respiration. Interestingly, human peripheral blood eosinophils contain few

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mitochondria, and eosinophil mitochondria are deficient in cellular respiration but maintain the ability to induce apoptosis via release of cytochrome c. Recent studies suggest that IL-5 inhibits eosinophil apoptosis by signaling through the Janus kinase (JAK)/STAT pathway to prevent the translocation of the pro-apoptotic protein Bax from the cytosol to the mitochondrial membrane, thereby inhibiting Bax-induced cytochrome c release.

IL-5 Receptor Receptors for the cytokines IL-5, IL-3, and GM-CSF consist of a cytokine-specific a receptor subunit and a common bc subunit. The a subunit of the IL-5 receptor (IL-5Ra) binds to its ligand with low affinity. Association of the bc subunit with the a subunit results in a high-affinity receptor. Both a and bc subunits contain intracellular domains, but the bc subunit plays a more significant role in signal transduction. When IL-5 binds to the IL-5Ra/bc receptor, receptor-mediated signal transduction is initiated. The juxtamembranous protein kinases JAK-2 and Lyn are physically associated with the bc subunit of the IL-5 receptor at basal conditions. Receptor dimerization leads to a cascade of tyrosine phosphorylation and activation of downstream targets, such as STAT1a and STAT5 transcription factors in the JAK/STAT pathway, G proteins of the mitogen-activated protein-kinase (MAPK) pathway, phosphatidylinositol-3 kinase (PI3 kinase), and the tyrosine kinases Syk and Fyn. Recent studies have begun to determine which signaling molecules and pathways are involved in specific IL-5-mediated functions. Lyn and Syk are important for IL-5-mediated inhibition of apoptosis, whereas Raf-1 kinase of the MAPK pathway is important for eosinophil degranulation.

IL-5 in Respiratory Diseases IL-5 is important for respiratory diseases that involve eosinophilic inflammation, such as nasal polyps, eosinophilic pneumonias, hypereosinophilic syndrome, and, especially, asthma. Asthma is an inflammatory disease of the lung characterized by reversible airway obstruction, airway hyperresponsiveness (AHR), pulmonary eosinophilia, and excessive airway mucus production. Allergic asthma is a Th2 cell-dependent disease. Th2 cells mediate asthmatic inflammation via secretion of the cytokines IL-4, IL-5, and IL-13. Allergen inhalation leads to antigen uptake by antigen-presenting cells, particularly dendritic cells, which reside in the airway epithelium and migrate to draining

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lymph nodes of the lung to present antigen to CD4 þ T cells. In asthmatic inflammation, a Th2 immune response ensues that can initiate two phases of pathogenesis. The immediate brochospastic phase occurs within minutes and is characterized by IgEmediated mast cell activation and degranulation. Mast cells, the principal effector cells of the immediate phase, release histamine and leukotrienes that contribute to acute bronchoconstriction. The late phase occurs 3–6 h after inhalation and can persist for several days without therapy. The eosinophil is hypothesized to be the principal effector cell of the allergen-induced pulmonary late-phase reaction. Th2 cells and mast cells produce IL-5, resulting in eosinophil maturation, recruitment to the lung, and activation (Figure 2). Activated eosinophils release granular proteins (discussed above) and leukotrienes, which are thought to contribute to AHR and airway epithelial damage in asthma. The relationship between IL-5 and eosinophilia is clear. In anti-IL-5 antibody-treated, IL-5 gene knockout, and IL-5 transgenic mice it has been demonstrated that IL-5 is necessary and sufficient for the development of eosinophilia in allergic airway inflammation. In asthma patients, the causal relationship between IL-5 production in the airways and the development of eosinophilia is also well established. However, the relationship between IL-5 and AHR is not clear. In mice, there is a body of evidence indicating that IL-5 contributes to AHR. IL-5 gene knockout mice and mice treated with anti-IL-5 antibodies exhibit reduced AHR in allergen challenge models. IL-5 transgenic mice that constitutively express IL-5 in the lung epithelium spontaneously develop AHR. Mice that express the diphtheria toxin A chain under control of the EPO promoter, resulting in the complete absence of eosinophils, do not develop AHR upon allergen challenge. However, some groups have reported that depletion of eosinophils by anti-IL-5 antibody treatment had no effect on AHR. Also, GATA-1-deficient mice, which lack eosinophils, exhibit AHR upon allergen challenge. Treatment of mice with anti-IL-13 antibody reduces AHR without affecting the number of eosinophils in the lung. Therefore, the role of IL-5 in mouse models of allergic lung inflammation remains controversial. In humans, the role of IL-5 in asthma pathogenesis is also controversial. Eosinophil levels correlate with asthma disease kinetics and severity. Effective corticosteroid therapy is associated with reduced eosinophilia. Independent investigators have found that susceptibility to asthma maps to the human chromosome 5q cytokine cluster. Depletion of IL-5 in humans has provided important clues for the role of this cytokine and eosinophils in asthma pathogenesis. In asthmatic

patients, anti-IL-5 did not alter blood or bone marrow CD34 þ myeloid progenitor cells, but reduced the number of eosinophils in the bone marrow by 70%, blood eosinophils by 85%, and ECP concentrations by two-thirds, without affecting the composition of T-cell subsets or T-cell cytokine production. In another study of allergic asthmatics, anti-IL-5 decreased airway eosinophils by 55% without having an appreciable effect on bronchial mucosal staining of MBP. In this report, anti-IL-5 had no effect on airway responsiveness, forced expiratory volume in 1 s, or peak flow measurements. Additionally, anti-IL-5 had no effect on the allergen-induced late-phase response in a different group of allergic asthmatics. However, anti-IL-5 has reduced the bronchial expression of proteins that are associated with airway wall remodeling in patients with allergic asthma. Therefore, these data would suggest that the current route, dose, and duration of anti-IL-5 given in human studies does not affect pulmonary function, but may have an effect on airway remodeling. In conclusion, IL-5 is a critical eosinophil differentiation, recruitment, and activation factor. However, the role of this cytokine in human diseases such as asthma remains to be clearly defined. See also: Allergy: Overview. Apoptosis. Asthma: Overview. CD11/18. Chemokines. Chemokines, CC: RANTES (CCL5). Chemokines, CXC: IL-8. Dendritic Cells. Gene Regulation. Genetics: Gene Association Studies. Histamine. Interferons. Interleukins: IL-9; IL-13. Leukocytes: Mast Cells and Basophils; Eosinophils; T cells. Lipid Mediators: Overview; Leukotrienes. Mucus. Platelet-Derived Growth Factor. Signal Transduction. Systemic Disease: Eosinophilic Lung Diseases. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Transgenic Models. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Abbas AK, Lichtman AH, and Pober JS (2003) Cellular and Molecular Immunology, 5th edn., pp. 243–274. Philadelphia: Saunders. Adachi T and Alam R (1998) The mechanism of IL-5 signal transduction. American Journal of Physiology 275: C623–C633. Ansel KM, Lee DU, and Rao A (2003) An epigenetic view of helper T cell differentiation. Nature Immunology 4: 616–623. Borish L and Rosenwasser LJ (2003) Cytokines in allergic inflammation. In: Adkinson NF Jr, Yunginger JW, Busse WW, et al. (eds.) Middleton’s Allergy Principles & Practice, 6th edn., pp. 135–157. Philadelphia: Mosby. Foster PS, Mould AW, Yang M, et al. (2001) Elemental signals regulating eosinophil accumulation in the lung. Immunological Reviews 179: 173–181. Gleich GJ (2000) Mechanisms of eosinophil-associated inflammation. Journal of Allergy and Clinical Immunology 105: 651–663. Gleich GJ, Adolphson CR, and Leiferman KM (1993) The biology of the eosinophilic leukocyte. Annual Review of Medicine 44: 85–101.

INTERLEUKINS / IL-6 Hamelmann E and Gelfand EW (2001) IL-5-induced airway eosinophilia – the key to asthma? Immunological Reviews 179: 182–191. Lalani T, Simmons RK, and Ahmed AR (1999) The biology of IL-5 in health and disease. Annals of Allergy and Asthma Immunology 82: 317–333. Renauld JC (2001) New insights into the role of cytokines in asthma. Journal of Clinical Pathology 54: 577–589. Viola JPB and Rao A (1999) Molecular regulation of cytokine gene expression during the immune response. Journal of Clinical Immunology 19: 98–108. Wills-Karp M (1999) Immunological basis of antigen-induced airway hyperresponsiveness. Annual Review of Immunology 17: 255–281. Wills-Karp M and Karp CL (2004) Eosinophils in asthma: remodeling a tangled tale. Science 305: 1726–1728.

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IL-6 was originally discovered as a cytokine that stimulated B lymphocytes to become plasma cells and was first called B-cell stimulatory factor in the early 1980s. It has subsequently been found to be one of the most broad-ranging cytokines there are in terms of its effector profile. In addition to stimulating lymphocytes it affects osteoclasts, neural cells, keratinocytes, renal cancer cells, hepatocytes, and many other cell types. Its clinical effects range from both inflammatory to anti-inflammatory, making it one of the most complex cytokines currently known.

Structure

IL-6 N Ward, Brown University, Providence, RI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin-6 (IL-6) is a pleiotropic cytokine that is generally produced at sites of inflammation. It is a member of a family of cytokines classified as IL-6-type cytokines which also includes IL-11, leukemia inhibitory factor, cardiotrophin-1, oncostatin M, and ciliary neurotrophic factor based on the overlapping effector profiles of these cytokines and their shared use of gp130 as the b-subunit in their multimeric receptor complexes. IL-6 induces fever, activates B and T lymphocytes, and stimulates hepatocytes to produce acute phase proteins. IL-6 is produced early in most forms of inflammation by many different cell types and so it plays a role in almost all forms of lung disease that involve infection or inflammation. Its levels in serum and lung fluid have been shown to correlate with some clinical outcomes in the acute respiratory distress syndrome but other than that, little is known about its role in specific diseases. It is not currently thought to play a significant role in asthma or allergic lung diseases. Despite often being classified as a proinflammatory cytokine, recent studies have demonstrated that IL-6 also may have potent antiinflammatory and protective properties. These include the ability to inhibit the production of tumor necrosis factor (TNF), IL-1, and MIP-2; decrease neutrophil sequestration; increase levels of IL-1 receptor antagonist (IL-1ra) and TNF-soluble receptor (TNFsr); stimulate the production of metalloproteinase inhibitors; reduce intracellular superoxide production; reduce tissue matrix degradation; and inhibit cellular apoptosis.

Introduction Interleukin-6 (IL-6) is a pleiotropic cytokine that is generally produced at sites of tissue inflammation. It is part of a family of cytokines classified as IL-6type cytokines. IL-6 type cytokines have in common overlapping effector profiles and their shared use of gp130 as the b-subunit in their multimeric receptor complexes. This family of cytokines also includes IL-11, leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF).

IL-6 is produced by a single gene but exists in many different forms based on posttranslational modification. The IL-6 gene has been localized to the p21 region of chromosome 7. It is extremely similar to the gene coding G-CSF and is thought to perhaps have arisen from a common ancestor. The primary gene product is 212 amino acids long but is cleaved to 184 amino acids within the cell. This peptide is then further modified to a final protein that can range from 17 to 84 kDa in size. The different subtypes of IL-6 produced tend to be tissue specific with monocytes characteristically producing a 24 kDa species and endothelial cells producing a 45 kDa form.

Regulation of Production and Activity IL-6 is produced by wide variety of cell types including monocytes, lymphocytes, fibroblasts, endothelial cells, mesangial cells, and keratinocytes. Its transcription is stimulated by a variety of mediators including tumor necrosis factor alpha (TNF-a), IL-1, and transforming growth factor beta. Viruses and endotoxin are also known to stimulate the production of IL-6. Studies have shown that different cell types are more or less sensitive to different stimuli. For example, in alveolar macrophages endotoxin is a potent stimulator of IL-6 but produces little in response to IL-1. Fibroblasts produce little IL-6 to endotoxin but are heavily stimulated by IL-1. Glucocorticoids are known to inhibit the production of IL-6 by binding to the IL-6 promoter site on DNA. There is evidence that glucocorticoids also inhibit IL6 production by decreasing mRNA stability.

Receptors IL-6 binds to an 80 kDa receptor protein, called IL6Ra, which has both extracellular and intracellular domains. The IL-6/IL-6Ra complex then interacts with and binds to the 130 kDa signal transducing molecule, gp130, which together with IL-6Ra forms the functional IL-6 receptor. The gpl30 subunit is

364 INTERLEUKINS / IL-6

also used by a variety of related cytokines with some similar functions such as IL-11, LIF, CT-1, OSM, and CNTF. IL-6 receptors are found on a diverse array of cells including hepatocytes, lymphocytes and other myeloid cells, keratinocytes, various nervous system cells, hematopoietic stem cells, and many other tissue epithelial and endothelial cells.

Interestingly, megakaryocytes both produce IL-6 and have IL-6 receptors on their surface. Other functions of IL-6 include effects on the stimulation of adrenal axis through stimulation of ACTH production and effects on skin keratinocytes which both express IL-6 receptors as well as produce IL-6.

IL-6 in Respiratory Disease Biological Functions Stimulator of Acute Phase Proteins

One of the most important functions of IL-6 appears to be its role as a stimulator of the acute phase proteins, a heterogeneous collection of proteins generally produced by the liver during inflammation and major stresses which act to limit the proinflammatory response. This is one of the reasons IL-6 is now classified as having both pro-and anti-inflammatory properties in vivo. When administered exogenously IL-6 stimulates fever as well as production of the acute phase proteins. Some of these proteins are produced in response to IL-6 alone and others need costimulation with IL-1. Glucocorticoids may positively interact with this system as well. Stimulator of B Cells and T Cells

The ability of IL-6 to stimulate B lymphocytes has been known since its discovery and was thought initially to be its main function. While IL-4 and IL-5 are more important cytokines for the proliferation, activation, and maturation of B cells, IL-6 appears to be essential for the later phase of activation leading to production and secretion of antibodies. IL-6 stimulates B lymphocytes to produce immunoglobulins IgG, IgM, and IgA. IL-6 has important roles in the activation of T lymphocytes as well yet acts in the early stages of Tcell activation, in contrast to its role in B-cell activation. IL-6 induces T-cell proliferation and increases the sensitivity of T cells to the stimulatory factor IL-2. Interestingly, IL-6 is also produced by some activated T cells. Finally, IL-6 is a stimulatory factor for cytotoxic T cells and lymphokine-activated killer cells. Other Functions

IL-6 has a broad array of other functions that have been discovered over the last several decades. Most notably, IL-6 plays a role in hematopoiesis. It can stimulate the growth of stem cells and has several noted effects on the maturation and survival of myeloid cells. IL-6 also has potent effects on thrombopoiesis. It is a stimulator of platelet production and mice will actually increase their serum IL-6 levels in response to artificially induced thrombocytopenia.

As a mediator of the inflammatory response, IL-6 plays a prominent role in many systemic diseases which can affect the lung including (but not limited to) transplant rejection, human immunodeficiency virus, rheumatoid arthritis, systemic lupus erythematosus, and other vasculitic and chronic inflammatory diseases. It is also an important mediator of disease in several hematologic malignancies such as leukemia, lymphoma, and multiple myeloma. Its most active role in the lung, however, is in states of acute inflammation such as sepsis, bacterial pneumonia, and trauma. IL-6 is produced by almost all different types of lung cells including endothelial cells, airway epithelial cells, fibroblasts, and, most notably, alveolar macrophages. IL-6 is thought to play a role in all types of acute lung inflammation through its stimulatory effects on lymphocytes and the acute phase response. IL-6 is also a modulator of both the proinflammatory cytokines IL-1 and TNF. IL-6 has been used extensively as a marker of inflammation in systemic infectious diseases such as sepsis and in lung-specific conditions such as acute respiratory distress syndrome (ARDS). IL-6 levels in both brochoalveolar lavage fluid and serum have been shown to rise early in the development of ARDS and accurately track with the clinical course of the disease. Several studies have linked early and/or late levels of IL-6 with clinical outcomes in ARDS such as mortality. It is important to note, however, that while IL-6 is often used as a marker of the proinflammatory state it may not be acting as a proinflammatory mediator. As previously mentioned, IL-6 can have many antiinflammatory effects such as the stimulation of acute phase proteins. In one study of ARDS in mice, the lack of the IL-6 gene dramatically increased the inflammatory response and prolonged tissue leukocytosis in response to lung injury. Giving IL-6 to these mice reversed the effect. Other studies have shown IL-6 to inhibit the production of TNF, IL-1, and MIP-2, increase levels of IL-1 receptor antagonist (IL-1ra) and TNF-soluble receptor (TNFsr), stimulate the production of metalloproteinase inhibitors, reduce intracellular superoxide production, reduce tissue matrix degradation, and inhibit cellular apoptosis.

INTERLEUKINS / IL-7 See also: Acute Respiratory Distress Syndrome. Interleukins: IL-1 and IL-18. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Barnes PJ (2001) Cytokine modulators as novel therapies for airway disease. European Respiratory Journal 34(supplement): 67s–77s. Chung KF (2001) Cytokines in chronic obstructive pulmonary disease. European Respiratory Journal 34(supplement): 50s–59s. Dreyfuss D, Ricard JD, and Saumon G (2003) On the physiologic and clinical relevance of lung-borne cytokines during ventilatorinduced lung injury. American Journal of Respiratory and Critical Care Medicine 167(11): 1467–1471. Hirano T (1992) Interleukin-6 and its relation to inflammation and disease. Clinical Immunology and Immunopathology 62(1 Pt 2): S60–S65. Hirano T (1992) The biology of interleukin-6. Chemical Immunology 51: 153–180. Martin TR (1999) Lung cytokines and ARDS: Roger S. Mitchell Lecture. Chest 116(1 supplement): 2S–8S. Parsons PE, Eisner MD, Thompson BT, et al. (2005) Lower tidal volume ventilation and plasma cytokine markers of inflammation in patients with acute lung injury. Critical Care Medicine 33(1): 1–6. discussion 230–232. Ranieri VM, Suter PM, Tortorella C, et al. (1999) Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized controlled trial. Journal of the American Medical Association 282(1): 54–61. Strieter RM and Kunkel SL (1994) Acute lung injury: the role of cytokines in the elicitation of neutrophils. Journal of Investigative Medicine 42(4): 640–651. Toews GB (2001) Cytokines and the lung. European Respiratory Journal 34(supplement): 3s–17s. Ward NS, Waxman AB, Homer RJ, et al. (2000) Interleukin-6-induced protection in hyperoxic acute lung injury. American Journal of Respiratory Cell and Molecular Biology 22(5): 535–542. Xing Z, Gauldie J, Cox G, et al. (1998) IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. The Journal of Clinical Investigation 101(2): 311–320. Zitnik RJ, Elias JA (1993) Interleukin-6 and the Lung. In: Kelley J (ed.) Cytokines of the Lung, vol. 1, pp. New York: Dekker.

IL-7 W J Leonard and H-H Xue, National Heart, Lung, and Blood Institute, Bethesda, MD, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin-7 (IL-7) is a stroma-derived cytokine that is indispensable for thymopoiesis in mice and humans. IL-7 also plays a pivotal role in the generation of memory CD4 þ and CD8 þ cells, and in controlling the numbers of naı¨ve and memory T cells. Its combined effects of increasing thymic output and enhancing homeostatic expansion of T cells make IL-7 a potentially ideal agent to restore immune competence in patients with lymphopenia.

365

Introduction IL-7 was originally identified as a stromal factor that could support the growth of murine B-cell progenitors in long-term bone marrow cultures, and subsequently was shown to play critical roles in the development of T and B cells in mice and T cells in humans. IL-7 exerts its effects, both as a growth and survival factor. Although mouse IL-7 cannot stimulate human pre-B cells, human IL-7 has activity on murine cells.

Structure IL-7 is a type I cytokine that has a typical four-a helical bundle structure with an up-up-down-down topology. A three-dimensional model of human IL-7 was constructed by using the X-ray crystal structure of IL-4 as a template and superimposing the IL-7 helices onto those of IL-4. The model was completed by incorporating the disulfide bond assignments as determined by MALDI spectroscopy and site-directed mutagenesis. The IL-7 gene is located on human chromosome 8q12–13 and murine chromosome 3, and the human and mouse genes share about 80% homology within their protein-coding regions. Alternatively spliced isoforms of IL-7 have been described, the predominant active isoform being 25 kDa. The increased apparent molecular weight over the size of the predicted amino acid sequence (17.4 kDa) is due to glycosylation.

Regulation of Production and Activity Regulation of the IL-7 gene is not well understood, with no apparent upstream TATA or CAAT sequences typical of inducible genes. Unlike other cytokines whose receptors utilize the common cytokine receptor g chain, gc, the production of IL-7 is constitutive rather than inducible, and there is an ambient, low level in the serum. Its production in bone marrow is downregulated by transforming growth factor beta (TGF-b), and may be positively regulated by the transcription factor C/EBPb, given that C/EBPb-deficient bone marrow cells have decreased production of IL-7. IL-7 is produced by fetal liver cells and stromal tissues in bone marrow, thymic epithelial cells, and intestinal epithelial cells. Its production is tightly regulated during ontogeny, with peak production in fetal development, accompanied by massive thymic expansion, and a substantial decrease in the aging thymus.

Receptors The IL-7 receptor (IL-7R) complex consists of the IL7Ra chain and gc. IL-7Ra is also utilized by another

366 INTERLEUKINS / IL-7

cytokine, thymic stromal lymphopoietin (TSLP) (Figure 1), whereas gc is shared by other cytokines including IL-2, IL-4, IL-9, IL-15, and IL-21. The binding of IL-7 to its receptor complex activates multiple signal cascades, including the Janus family tyrosine kinase (Jak)-signal transducer and activator of transcription (STAT) pathway, with Jak1, Jak3, Stat5a, and Stat5b being primarily activated, and the phosphatidyl inositol 3-kinase (PI3-K) pathway. The intracellular domain of IL-7Ra is 195 amino acids long and lacks intrinsic kinase activity but instead provides docking sites for signaling molecules. An 8 amino acid motif termed Box1 in the membrane proximal region is conserved among the type I cytokine receptor family members and in IL-7Ra, this motif mediates the recruitment of Jak1. Tyrosine 449 in the cytoplasmic domain is part of a YXXM motif that mediates the docking of both Stat5 proteins and the p85-binding subunit of PI3-K. IL-7Ra is dynamically regulated during thymic development and the T-cell immune response. The most immature thymocytes are double negative cells that lack expression of CD4 and CD8 (CD4
CD8
) but express IL-7Ra. These cells then downregulate expression of IL-7Ra as they mature to CD4 þ CD8 þ double-positive thymocytes. IL-7Ra is highly expressed in mature CD4 þ or CD8 þ single positive thymocytes and remains so in peripheral resting T cells. Upon exposure to antigens, naı¨ve T cells become activated, undergo clonal expansion, and differentiate into effector T cells that express much lower IL-7Ra. IL-2 is a cytokine produced by activated T cells; it decreases expression of IL-7Ra via a PI3-K/Akt-dependent mechanism. The low IL-7Ra expression on effector T cells is thus maintained by IL-2. When the antigen is

TSLP

IL-7

cleared and the immune response subsides, memory T cells to the antigen are formed and IL-7Ra expression is regained on these cells. During B-cell development, IL-7Ra is expressed on early B-cell precursors and pro-B cells, with its expression progressively downregulating as B cells mature.

Biological Functions The phenotypes manifested in human genetic deficiencies and gene-targeted mouse strains have demonstrated an absolute requirement of IL-7 signaling for lymphopoiesis. Ablation of IL-7 or IL-7Ra gene expression in mice results in a severe decrease in thymocytes and peripheral ab T cells. In the most severe cases, T-cell development is blocked at the double negative (CD4
CD8
) stage. B-cell development in these mice is also impaired, showing a block at CD43 þ pro-B cells or earlier. Interestingly, genetic deficiencies in the IL-7Ra gene in humans result in severe combined immunodeficiency (SCID) with a T
B þ NK þ phenotype, indicating that in humans the IL-7/IL-7R system is essential for T-cell development, but not for B-cell maturation (see Table 1 for the major actions of IL-7). So far, more than 20 SCID patients manifesting a T
B þ NK þ phenotype have been found to have mutations in the IL-7Ra gene. These mutations are clustered in the first five exons, resulting in diminished expression of IL-7Ra, impaired IL-7 binding to the receptor, or in presumably perturbed receptor structure and/or function. In addition to its role in ab T-cell development, IL-7 has a nonredundant role in the development of gd T cells, as indicated by their complete absence in IL-7 or IL-7Ra-deficient mice. The important role of IL-7 in ab T-, gd T-, and B-cell development is reflected in its contribution to variable diversity joining (V(D)J) rearrangements in T-cell receptor b chain (TCRb), TCRg, and immunoglobulin heavy chain (IgH) loci. IL-7 contributes to the regulation of accessibility of these loci to the V(D)J recombinase. For the TCRg locus, IL-7 increases histone acetylation, resulting in an open chromatin structure. Besides its effects on developing lymphocytes, IL-7 provides survival signals to naive T cells in the periphery and to memory T cells. For naı¨ve T cells,

TSLPR Table 1 Major biological action of IL-7


c IL-7R Figure 1 IL-7Ra is a shared receptor component for IL-7 and TSLP. The functional IL-7 receptor is IL-7Ra þ gc whereas the functional TSLP receptor is IL-7Ra þ TSLPR. TSLPR is the TSLP-binding protein.

T-cell development B-cell development T-cell homeostasis Memory T-cell generation and maintenance

Human

Mouse

Yes No Yes Unkown

Yes Yes Yes Yes

INTERLEUKINS / IL-7

survival also requires contact with self-peptide major histocompatibility complexes (MHCs). The survival effect of IL-7 has been attributed at least in part to its upregulation of antiapoptotic protein Bcl-2, and the down-modulation of pro-apoptotic proteins Bad and Bax, given that a Bcl-2 transgene rescued and Bax deficiency partially corrected T lymphopoiesis in IL-7Radeficient mice. Thus, IL-7 plays key roles in the homeostatic maintenance of naı¨ve T cells, and because of its limited availability, IL-7 also contributes to control the size of T-lymphocyte pool in the periphery. In athymic mice and patients with T-cell depletion (e.g., postchemotherapy), changes in the immune system milieu activate peripheral mechanisms to restore T-cell numbers in the host, a process known as peripheral ‘homeostatic’ expansion. When transferred to an IL-7-deficient lymphopenic host, normal T cells undergo little if any homeostatic proliferation, indicating a key role of IL-7 in this process. IL-7 contributes to both the generation and efficient maintenance of antigen-specific memory T cells, which are essential for long-lasting immunological protection. IL-7 promotes the transition of CD4 þ effector T cells to persistent memory cells. In the absence of IL-7, the generation of memory CD4 þ T cells is greatly impaired in both lymphoid and nonlymphoid compartments, in spite of normal CD4 þ T-cell responses upon antigen challenge. The selective expression of IL-7Ra on the CD8 þ effector T cells (especially CD8aa cells) is a prerequisite for the generation of memory CD8 þ T cells; IL-7 signals are required for the survival of CD8 þ memory T-cell precursors also. Memory T cells maintain their numbers for long periods after antigen exposure through homeostatic proliferation. Along with IL-15, IL-7 contributes to the maintenance of memory CD8 þ T cells; in lymphopenic hosts especially, IL-7 can compensate for the absence of IL-15. Whereas memory CD8 þ T cells persist in the absence of MHC-I ligand, homeostasis of memory CD4 þ T cells requires both IL-7 and MHC-II-derived TCR signals. IL-7 is also a potent modulator of immune responses. First, IL-7 acts as a potent co-stimulatory signal for T-cell activation by enhancing proliferation and cytokine production. It not only can substantially increase the number of antigen-specific T cells that respond to stimuli with high affinity for the TCR, but it can also convert an otherwise nonreactive stimulus to a reactive one when TCR/peptide interactions are weak. Second, in human peripheral blood mononuclear cells, IL-7 can enhance the generation and function of cytolytic T lymphocytes (CTLs), which substantially contribute to cell-mediated immune responses. In addition, IL-7 induces lymphokineactivated killer activity and the cytotoxicity of

367

CD56 þ NK cells. Moreover, IL-7 can synergistically act with IL-21 to expand CD8 þ T cells in the absence of TCR stimulation, accompanied by increased interferon g expression. Third, IL-7 is a co-factor for the development of lymphoid dendritic cells in the thymus and for the differentiation of monocytic dendritic cells from myeloid precursors. Several lines of IL-7 transgenic mice have been described. When placed under the control of an MHC-II promoter, IL-7 overexpression resulted in a 30-fold increase in peripheral T and B cells without affecting thymic cellularity or CD4/CD8 ratios. Similarly, expression of IL-7 under control of an immunoglobulin k light chain promoter resulted in an increase in immature and mature B cells, and all mature T-cell subsets, but did not lead to an increase in CD4
CD8
double-negative T cells. On the other hand, when driven by the IgH promoter and enhancer, IL-7 overproduction caused a severe perturbation in thymic development with a profound decrease in DP thymocytes, a marked increase in mature T cells, and a lymphoproliferative disorder in the periphery characterized by T- and B-cell lymphomas. However, in transgenic mice in which IL-7Ra expression is driven by the human CD2 promoter, T cells developed normally but total thymocyte numbers decreased as a function of age, consistent with decreased IL-7 production in aging thymus. The biological actions of IL-7 are coordinately regulated by the concentration of available IL-7 and its expression levels, and cell-type specific expression of IL-7Ra. The expression pattern of IL-7Ra during T-cell development and after antigen stimulation corresponds to the actions of IL-7. As IL-7 is a limited resource during thymopoiesis, the downregulation of IL-7Ra on CD4 þ CD8 þ double-positive thymocytes minimizes competition with CD4
CD8
double-negative thymocytes for local IL-7, and allows the elimination of autoreactive T cells by the process of negative selection. IL-7Ra is expressed at a high level on naı¨ve T cells, allowing their receipt of survival signals from IL-7, thus maintaining T-cell homeostasis. The diminished expression of IL-7Ra on effector T cells may make the cells more susceptible to apoptosis during the contraction phase of immune response, whereas the selective expression of IL-7Ra on effector T cells can facilitate the transition to CD4 þ or CD8 þ memory T cells. The expression of IL-7Ra, at a level higher than that on naı¨ve T cells, contributes to the maintenance of these CD4 þ and CD8 þ memory T-cell pools by supporting both survival and homeostatic division. The expression of IL-7Ra in multipotential hematopoietic progenitors, early B-cell precursors, and pro-B cells is modulated by an Ets factor, PU.1,

368 INTERLEUKINS / IL-9

through an Ets-binding motif in the promoter region of IL-7Ra gene. PU.1 is also expressed in immature thymocytes, but is downregulated as T cells mature. The regulation of IL-7Ra in T cells is regulated through the same Ets-binding motif, but by a different Ets factor, GA-binding protein.

IL-7 and Clinical Implications IL-7 acts as a potent modulator of T-cell immune reconstitution, and is able to restore immune competence through its combined effects on increased thymic output and enhanced homeostatic expansion of T cells. The biological actions of IL-7 and preclinical data provide a strong rationale for its potential clinical efficacy. Chemotherapy of cancer patients, lympho-depleting therapy of autoimmune diseases such as rheumatoid arthritis, and transplantation of allogeneic bone marrow grafts are all accompanied by therapy-induced lymphopenia. IL-7 could be an effective agent in augmenting immune recovery and helping to optimize immune therapies. IL-7 may also enhance de novo T-cell generation after highly active antiretroviral treatment in human immunodeficiency virus patients. Pulmonary fibrosis is a devastating disease that is characterized by a perturbed balance between the synthesis and breakdown of the extracellular matrix. TGF-b contributes to the pathogenesis of pulmonary fibrosis by enhancing synthesis and deposition of extracellular matrix components including collagen. Fibroblasts from patients with pulmonary fibrosis express IL-7Ra, and treatment of these cells with IL7 significantly decreases the production of TGF-b. Additionally, IL-7 can potently inhibit the actions of TGF-b by (1) the upregulation of Smad7, which inhibits TGF-b-mediated phosphorylation of Smad2/3, (2) the inhibition of TGF-b-induced activation and protein kinase C-d, and (3) the suppression of TGFb-induced collagen synthesis. These effects indicate that IL-7 is a potential antifibrotic agent. In mouse models, when IL-7 is administered systemically or its production is induced along with co-stimulatory molecules within a tumor microenvironment through gene therapy, it can enhance antitumor response. Such effects are mediated by multiple mechanisms including the expansion and maintenance of T cells expressing TCRs with high affinity for tumor antigens and the recruitment of low-affinity T cells to the site of tumor. Translation of these findings to clinical use awaits further preclinical tests. See also: Apoptosis. Dendritic Cells. Gene Regulation. Human Immunodeficiency Virus. Leukocytes: T cells. Pulmonary Fibrosis. Signal Transduction.

Transcription Factors: Overview; PU.1. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Alpdogan O and van den Brink MRM (2005) IL-7 and IL-15: therapeutic cytokines for immunodeficiency. Trends in Immunology 26: 56–64. Beq S, Delfraissy JF, and Theze J (2004) Interleukin-7 (IL-7): immune function, involvement in the pathogenesis of HIV infection and therapeutic potential. European Cytokine Network 15: 279–289. Bradley LM, Haynes L, and Swain SL (2005) IL-7 maintaining T-cell memory and achieving homeostasis. Trends in Immunology 26: 172–176. Cosenza L, Rosenbach A, White JV, Murphy JR, and Smith T (2000) Comparative model building of interleukin-7 using interleukin-4 as a template: a structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation. Protein Science 9: 916–926. DeKoter RP, Lee HJ, and Singh H (2002) PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors. Immunity 16: 297–309. Fry TJ and Mackall C (2002) Interleukin-7: from bench to clinic. Blood 99: 3892–3905. Hofmeister R, Khaled AR, Benbernou N, et al. (1999) Interleukin7: physiological roles and mechanisms of action. Cytokine Growth Factor Review 10: 41–60. Kroemer RT, Kroncke R, Gerdes J, and Richards WG (1998) Comparison of the 3D models of four different human IL-7 isoforms with human and murine IL-7. Protein Engineering 11: 31–40. Leonard WJ (2003) Type I cytokines and interferons and their receptors. In: Paul WE (ed.) Fundamental Immunology, 5th edn., pp. 701–747. Philadelphia: Lippincott Williams & Wilkins. Leonard WJ (2005) Interleukin-7 deficiency in rheumatoid arthritis. Arthritis Research & Therapy 7: 42–43. Namen AE, Lupton S, Hjerrild K, et al. (1988) Stimulation of B-cell progenitors by cloned murine interleukin-7. Nature 333: 571–573. Park LS, Friend DJ, Schmierer AE, Dower SK, and Namen AE (1990) Murine interleukin 7 (IL-7) receptor. Characterization on an IL-7-dependent cell line. Journal of Experimental Medicine 171: 1073–1089. Xue HH, Bollenbacher J, Rovella V, et al. (2004) GA binding protein regulates interleukin 7 receptor alpha-chain gene expression in T cells. Nature Immunology 5: 1036–1044. Zeng R, Spolski R, Finkelstein SE, et al. (2005) Synergy of IL-21 and IL-15 in regulating CD8 þ T cell expansion and function. Journal of Experimental Medicine 201: 139–148.

IL-9 A Dasvarma and A N J McKenzie, Laboratory of Molecular Biology, Cambridge, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin-9 (IL-9) was originally cloned as a growth factor for certain mouse T helper cell clones. It is a 30–40 kDa glycoprotein produced by activated T cells, with a variety of roles

INTERLEUKINS / IL-9 including T-cell stimulation, mast cell proliferation and differentiation, erythroid differentiation, Ig production, protection against parasite infection, and neuronal differentiation. Its deregulated expression has been implicated in a variety of disorders, including asthma and lymphomagenesis.

The distinctive activity of p40 from other immune growth factors was also confirmed by its inability to stimulate cell lines dependent on IL-3, IL-5, or IL-6. In addition to T cells, p40 was also found to be able to stimulate bone-marrow-derived mast cell lines (BMMC): recombinant p40 supported the growth of BMMC lines sensitive to mast cell-growth enhancing activity, induced IL-6 secretion, and enhanced the survival of primary mast cell cultures. p40 was renamed IL-9 in light of the fact that it was able to support the growth of several different immune cell types.

Introduction Uyttenhove and colleagues identified interleukin-9 (IL-9), originally called p40, in 1988, as a growth factor for T cell clones. A collection of T helper cell lines was established from lymph nodes of antigenprimed mice. Cells were cultured in the presence of antigen and maintained via regular feeding with antigen and irradiated splenic antigen-presenting cells. Two clones derived from these cells proliferated in response to their own conditioned medium without the aid of antigen and feeder cells. The supernatant was obtained after concanalavin A (Con A) stimulation, indicating that the growth factor was associated with stimulated, not resting T cells. The growth factor eluted as a symmetrical peak in the region of 30–40 kDa, leading to its designation as p40. Murine p40 was cloned and characterized via screening of a cDNA library, from a T helper cell clone that produced large amounts of p40 postConA stimulation. The cDNA encoding the human homolog of mouse p40 was identified by expression cloning of hematopoietic growth factors stimulating the proliferation of a megakaryoblastic leukemic cell line.

1

2 3

114

36 33

1

2 3

369

Structure Analysis of genomic clones of IL-9 revealed that both human and mouse p40 genes consist of five exons and four introns spread over approximately 4 kb of DNA (Figure 1(a)). Both the mouse and human genes are highly similar with significant identity in the 50 untranslated region (50 UTR) and coding regions. The human gene is 3.58 kb in length, whereas the mouse gene is 2.97 kb. The human gene is located on chromosome 5, at cytoband 5q31.1, whereas the mouse gene is located at cytoband 13B2. In humans the gene is located in a region containing genes encoding various cytokines and immune-related molecules, including IL-3, IL-4, IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor (Figure 1(b)). This region is commonly deleted in patients with acquired 5q abnormalities, leading to

4

5

132 4

120

120

5

(a)

IL-3 GM-CSF IL-5

IL-13

IL-4

IL-9

5q31.1 131.4 Mb

(b)

135.3

Human chromosome 5

Figure 1 (a) Genomic organization of the human and mouse IL-9 genes. Exons are represented as numbered boxes, with the empty region on exon 1 representing the 50 UTR. Exon sizes in base pairs are represented by numbers in italics. (b) Location of the IL-9 gene on human chromosome 5 in relation to other cytokine genes.

370 INTERLEUKINS / IL-9

hematologic disorders, although this does not necessarily implicate any of the individual cytokines in the pathologies. The IL-9 protein is a cationic cysteine-rich polypeptide that exists as a secreted monomer, with four N-linked glycosylation sites. Both the mouse and human proteins are 144 amino acids in length, with a signal peptide of 18 residues and a molecular weight of approximately 16 kDa.

Cellular Synthesis IL-9 is synthesized by activated T cells, which participate in an adaptive immune response. T cells develop in the thymus, after which they enter the bloodstream and circulate to peripheral lymphoid organs. Following exposure to specific antigens, the T cells develop into armed effector T cells which are induced to proliferate and differentiate into cells that contribute to the elimination of pathogens. T cells fall into two classes, CD4 or CD8 T cells. CD4 T cells undergo differentiation into either T-helpertype-1 (Th1) or T-helper-type-2 (Th2) cells. Th2 cells produce IL-9 in response to pathogen exposure. Following synthesis in the endoplasmic reticulum and posttranslational modifications, IL-9 molecules are secreted and act in an autocrine manner to further

activate the T cell to proliferate and produce more IL-9, along with other roles.

Receptors IL-9 binds to the IL-9 receptor (IL-9R), which is a 54 kDa polypeptide associated with 10 kDa N-linked sugars, spanning the cell membrane. The receptor peptide is 468 and 521 amino acids in length, in humans and mice respectively, and belongs to the hematopoeitin receptor superfamily. In mice, a signal peptide exists between residues 15 and 40; there is a transmembrane domain between residues 271 and 291 and an extracellular domain, which comprises 233 amino acids with two potential N-linked glycosylation sites (Figure 2). The receptor is constitutively bound to Janus kinase 1 (JAK1), which allows induction of several different signaling pathways, leading to transcription of further genes. The region required for JAK binding in the IL-9 receptor is the Box1 motif, a proline-rich region close to the transmembrane domain. The IL-9 specific chain can bind IL-9 with high affinity, but cannot mediate any signal on its own. The functional IL-9 receptor consists of the IL-9R described above and the common cytokine receptor g

Extracellular domain
c Chain Hematopoietic receptor consensus sequence

Cell membrane

Cytoplasmic domain

Box1 motif

Transmembrane domain

Figure 2 Structure of the IL-9R chain of the IL-9 receptor, which comprises the IL-9R chain and the gc chain.

INTERLEUKINS / IL-9

chain, gc, which is a shared component of receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. While the IL9R binds JAK1, the gc chain binds JAK3.

Table 1 Roles of IL-9 in various cell types and processes Biological process

Cells/systems affected

Effect

Hematopoiesis

Erythroid and myeloid precursors

Clonogenic differentiation

Immunity

Mast cells

Proliferation and differentiation Ig production Proliferation and differentiation Increased expression

Biological Function and Mode of Action To exert its effects, IL-9 first binds to its receptor (IL-9R). As described previously, the receptor comprises two separate chains, the IL-9 specific receptor and the gc chain. Upon receptor ligation, a change in receptor conformation leads to auto and/or transphosphorylation of JAK kinases, phosphorylation of the receptor, and activation of one of three different pathways involved in mediation of IL-9 intracellular signaling: the signal transducer and activator of transcription (STAT) pathway, the PI3/ IRS-1/2 pathway, and the mitogen-activated protein(MAP) kinase pathway, all of which are involved in cell differentiation, proliferation, and cell death and development. The STAT pathway involves signal transduction and transcription factors 1, 3, and 5. Upon ligand binding to the IL-9 receptor, the residue tyrosine 407 in the IL-9R chain is phosphorylated and serves as a docking site for the SH2 domain of the STAT transcription factors. The STAT factors are then phosphorylated, inducing their dimerization, migration to the nucleus, and binding to specific DNA sites in the promoter of cytokine-regulated genes. The PI3 pathway is mediated by phosphatidylinositol 3-kinase (PI3K), belonging to a family of lipid and serine/threonine kinases. Various cytokines including IL-9 recruit PI3K to the plasma membrane where it phosphorylates phosphoinositides, which function as important second messengers for intracellular signaling in several processes. The IL-9R cannot recruit PI3K directly owing to a lack of PI3K binding sites; however, the Box1 domain and a downstream region are required for insulin receptor substrate 2 (IRS-2) activation, which leads to PI3K activation via its regulatory subunit, p85. IL-9 induces IRS-2 phosphorylation and association with the PI3K p85 subunit, and cell proliferation is induced. The IL9-induced MAP kinase pathway is not as prominent in IL-9 signaling as the other two pathways, and is restricted to only certain cell types. The signaling cascade is linked to IL-9R through the adaptor protein, Shc. Shc phosphorylation is thought to lead to activation of rat sarcoma (RAS) GTPases, and the mitogenic cascade is mediated further through the phosphorylation and activation of Raf-1 and Map/Erk kinase 1/2 (MEK1/2) extracellular signal-regualted kinases 1 and 2 (ERK1– 2) leads to activation of the MAP kinase pathway. IL-9 affects a variety of cell types (Table 1). It is produced by Th2 cells and acts in an autocrine manner to stimulate the proliferation of T cells as

371

B cells T cells Th2 and other cytokines Tumorigenesis

Pulmonary response/ asthma

Other

T lymphoma Hodgkin’s lymphoma

Survival and proliferation Survival and proliferation

Mast cells

Proliferation

Goblet cells Th2 cytokines

Proliferation Increased expression

Neurones

Neuronal precursor differentiation

part of the humoral immune response. The existence of specific high-affinity receptors for IL-9 has been demonstrated in T cells, mast cells, macrophages, and tumor T cells. IL-9 is also involved in T-cell stimulation, erythroid differentiation, antibody production, and neuronal differentiation. Blockade of the IL-9R a-chain resulted in a severe reduction in the number of human thymocytes, with a major decrease in the number of cells maturing from thymic precursor cells, illustrating that IL-9R signaling is essential in early T-lymphoid development. Injection of mice with T cells transfected with IL-9 led to widespread T-cell proliferation in vivo, illustrating IL-9’s role as a T cell growth factor. In IL-9 lung-specific transgenic mice, the Th2 cytokines IL-4, IL-5, and IL-13 (Figure 3) were expressed in response to IL-9, demonstrating its role in potentiating a Th2 response. IL-9 induces mast cell proliferation and differentiation and stimulates expression of mast cell proteases. In IL-9-deficient mice, a decrease in mastocytosis was observed, demonstrating a specific role for IL-9 in mast cell proliferation. It is also required for an efficient mast cell-mediated immune response against intestinal parasites such as Trichuris muris. IL-9 has also been implicated in IL-4-induced IgG1 and IgE production in B cells. IL-9 has also been demonstrated to have an antiapoptotic role in that it was shown to protect a murine thymic lymphoma cell line from apoptosis

372 INTERLEUKINS / IL-9

Mast cell hyperplasia

Airway eosinophilia

Increased Th2 cytokine expression, including IL-4, IL-5, IL-13 Increased IL-9

Goblet cell hyperplasia Mucus hyperproduction

Figure 3 IL-9 overexpression, both systemically and lung-specifically, has been shown to lead to various asthmatic symptoms, including, (a) increased mast cell hyperplasia, proliferation, and differentiation; (b) increased eosinophil and lymphocyte infiltration; (c) increased cytokine expression; and (d) goblet cell hyperplasia and mucus hyperproduction.

induced by dexamethasone and maintained cellular proliferation in its presence.

Regulation of IL-9 Activity IL-9 signal attenuation (and hence modulation of its activity) occurs mainly through the activity of the suppressors of cytokine signaling (SOCS) or cytokine-inducible SH2-containing proteins (CIS), which comprise a family of signaling inhibitors. Expression of these inhibitors is induced by several cytokines and negatively regulates signal transduction. In a T-cell lymphoma line, IL-9 induced the rapid expression of CIS, SOCS-2, and SOCS-3. STAT activation was shown to be required for CIS/SOCS induction using IL-9R mutants. Each of these inhibitor proteins was used to transiently transfect a cell line with demonstrated IL-9 and STAT activity, with only SOCS3 being able to inhibit IL-9-induced signal transduction as well as downstream effects such as STAT activation, gene induction, and antiapoptotic activity.

IL-9 in Respiratory and Other Diseases The first association of IL-9 with disease pathology was made through the observation of elevated IL-9 expression in human malignant lymphomas, with lymph nodes from patients with Hodgkin’s disease (HD) and large cell anaplastic lymphoma (LCAL) constitutively producing IL-9. Using patient-derived cell lines, elevated IL-9 expression was demonstrated in two of six cases of LCAL and 6 of 13 cases of HD.

Increased levels of IL-9 were detected in the sera from 18 out of 44 HD patients but not in the sera from healthy controls. IL-9 mRNA was also detected in 42% of HD cases via Northern blot analysis. In vitro, IL-9 induces the proliferation of murine T lymphoma and myeloid leukemia cells: in one of the studies, IL-9 significantly stimulated the proliferation of 26 of 45 mouse thymic lymphoma cell lines, induced by mutagen or X-ray treatment. IL-9 transgenic mice were found to develop thymic lymphomas spontaneously 3–9 months after birth, implicating IL-9 as a potential tumorigenic factor. In another study, more than 85% of IL-9 transgenic mice were found to have a severe lymphoproliferative disorder resulting in highly enlarged lymphoid tissues and lymphocyte infiltration into other tissues. Various studies have demonstrated a link between asthma and IL-9. It was originally designated as a risk factor for asthma since its expression was markedly reduced in bronchial hyporesponsive mice. IL-9 has been shown to stimulate goblet cell proliferation in differentiating primary cultures of human airway epithelia, and has also been implicated in repair of injured epithelia, both characteristics of asthma. A significant difference in IL-9 mRNA expression was detected in human peripheral blood eosinophils freshly isolated from asthmatic subjects compared with those isolated from normal control subjects. The proportion of IL-9 immunoreactive eosinophils from asthmatic patients was increased compared with that found in normal control subjects. Mice overexpressing IL-9, either systemically

INTERLEUKINS / IL-10 373

or lung-specifically, developed more severe asthmatic symptoms than wild-type littermates. IL-9 expression in the lungs of such mice was shown to cause various asthmatic symptoms, including airway inflammation, mast cell hyperplasia, mucus hyperproduction, eosinophil and lymphocyte infiltration into lung tissues, and bronchial hyperresponsiveness (Figure 3). Mice deficient in IL-9 have decreased goblet cell proliferation and mast cell proliferation in response to a pulmonary challenge, illustrating IL-9’s essential role in the rapid onset of these two phenomena in a pulmonary Th2 response. See also: Histamine. Interleukins: IL-4; IL-5; IL-13; IL16. Leukocytes: T cells.

Further Reading Benczik M and Gaffen SL (2004) The interleukin (IL)-2 family cytokines: survival and proliferation signalling pathways in t lymphocytes. Immunological Investigations 33(2): 109–142. Demoulin JB and Renauld JC (1998) Interleukin 9 and its receptor: an overview of structure and function. International Reviews of Immunology 16: 345–364. Knoops L and Renauld JC (2004) IL-9 and its receptor: from signal transduction to tumorigenesis. Growth Factors 22(4): 207–215. Skinnider BF and Mak TW (2002) The role of cytokines in classical Hodgkin lymphoma. Blood 99(2): 4283–4297. Uyttenhove C, Simpson RJ, and Van Snick J (1988) Functional and structural characterization of p40, a mouse glycoprotein with t-cell growth factor activity. Proceedings of National Academy of Sciences 85: 6934–6938.

IL-10 T J Standiford and J C Deng, University of Michigan, Ann Arbor, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin-10 (IL-10) is an immunomodulatory cytokine that regulates important aspects of innate and cell-mediated immunity. It is synthesized by hematopoietic and nonhematopoietic cells, including macrophages and epithelial cells within the lung. The biologically active form of IL-10 is a homodimer that binds to the IL-10 receptor, which is expressed on leukocytes as well as structural cells. The primary function of this cytokine is to downregulate inflammation via inhibition of cytokine production and other inflammatory mediators. IL-10 is secreted in response to acute inflammation and under conditions that promote tolerance or anergy. The absence of IL-10 has been implicated in the pathogenesis of uncontrolled inflammation during the development of acute respiratory distress syndrome, chronic Pseudomonas colonization in cystic fibrosis, and allergic airways disease. However, IL-10 also possesses immunostimulatory properties for cytotoxic T cells and natural killer cells. Furthermore, IL-10 has been implicated as a mediator of critical

illness-induced immunosuppression and matrix deposition/ remodeling in interstitial lung disease. Thus, IL-10 is clearly a complex molecule with potential therapeutic uses in modulating inflammation, but its salutary anti-inflammatory effects must be weighed against its potentially stimulatory effects on cytotoxic activity and adverse impacts on host defense and fibrosis.

Introduction Interleukin-10 (IL-10) is an immunomodulatory cytokine with pleiotrophic activities. When initially discovered in 1989, IL-10 was named cytokine synthesis inhibitory factor because of its ability to inhibit the production of proinflammatory cytokines, including interferon gamma (IFN-g), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-a) by various cell types. Subsequently, a viral homolog of IL-10 was identified in Epstein–Barr virus (EBV), which was believed to facilitate immune evasion, thereby enabling EBV to survive in the host and promote the development of B cell lymphoma. While the predominant role of IL-10 is to suppress inflammation, this cytokine also possesses immunostimulatory properties for certain cell types, including cytotoxic T cells and natural killer (NK) cells. Thus, it is evident that this molecule displays a complex array of biological activities – effects that are dependent upon the timing of its release, cellular targets, and the immune context within which this cytokine is acting.

Structure The IL-10 gene locus spans approximately 2 kb and resides on chromosome 1 in humans and mice. The IL10 gene is composed of five exons, with sequences that are involved in transcriptional regulation (e.g., binding sites for transcriptional factors nuclear factor kappa B (NF-kB) and AP-1; see Figure 1). No splice variants are known to exist. The IL-10 protein is synthesized as a monomer composed of six a-helices, with a molecular weight of 18.5 kDa, but mainly exists in its biologically active homodimeric form. Murine and human IL-10 share approximately 72% sequence homology, with limited crossreactivity as described below. At low protein concentrations, the IL-10 dimer is relatively unstable. Its activity may therefore be curtailed by this instability, in that IL-10 may only exert its effects locally where the concentrations of secreted protein are high. The IL-10 interdomain angle is approximately 901, which reduces the number of hydrophobic interdomain interactions, rendering this cytokine relatively hydrophilic.

Regulation of Production and Activity IL-10 is produced by a variety of cell types, including CD8 þ and CD4 þ T cells (e.g., T-helper-2 (Th2)

374 INTERLEUKINS / IL-10 TATA I 2+4 200 AP-1 binding

10+4 1084 NF-
B like reg

1531

1535 1563

II 1795 2680

III 2739 3045

IV 3197 4205

V 4270 5892

6686

Figure 1 Schematic depicting human IL-10 gene structure and putative regulatory elements.

cells, regulatory T cells), gd-T cells, NK cells, NK T cells, B cells, dendritic cells, eosinophils, mast cells, and monocytic cells (e.g., microglia, monocytes, macrophages). Macrophages are the major source of IL-10, although regulatory T cells are now recognized as an important subpopulation of T cells that release IL-10. Nonleukocyte cell populations, including epithelial cells, keratinocytes, and melanoma cells, also express IL-10. In the lung, human alveolar macrophages (AMs), bronchial epithelial cells, and alveolar epithelial cells have been shown to express IL-10 mRNA and protein. In contrast, murine resident AMs secrete minimal amounts of IL-10, although these cells do express IL-10 receptors. Macrophages produce IL-10 in response to inflammatory or infectious stimuli, including lipopolysaccharide, TNF-a, and catecholamines. High levels of IL-10 are also produced under conditions that induce anergy and tolerance, such as repeated antigen stimulation. In these contexts, IL-10 likely provides negative feedback to dampen the magnitude of immune responses. Interestingly, several viruses, such as EBV, produce IL-10 or IL-10-like molecules (vIL-10). The genomes of other viruses, including members of the herpesvirus family, poxvirus, and primate cytomegaloviruses (CMV), also contain homologs of the IL-10 gene. Many vIL-10 proteins share extensive sequence homology with human IL-10. Viral IL-10 appears to engage the same IL-10 receptor in the host and is capable of producing some of the same biological effects as host-derived IL-10. Given the anti-inflammatory properties of IL-10, vIL-10 likely confers a survival advantage for these viruses by suppressing antiviral host immune responses.

Biological Function IL-10 is a critical regulator of innate and cellmediated immune responses. The main cellular targets of IL-10 are lymphocytes and antigen-presenting cells. In early studies, IL-10 was found to inhibit cytokine synthesis by T cells, which was attributed to its ability to inhibit antigen presentation by macrophages and dendritic cells. Subsequent studies have demonstrated that IL-10 can suppress cytokine production directly by inhibiting inhibitor kappa B (IkB) kinase activation and blocking NF-kB DNA

binding, thereby inhibiting NF-kB activity. IL-10 also inhibits inflammatory cytokine signaling by activating suppressors of cytokine signaling (SOCS)-1 and SOCS-3, which suppress Janus kinase (JAK)/signal transducers and activators of transcription (STAT) cytokine signaling pathways. Importantly, IL-10 appears to be a potent inhibitor of type-1 immune responses. Mice deficient in IL-10 (IL-10
/
) have augmented Th1 responses, as evidenced by the enhanced ability of these animals to clear both bacterial and fungal pathogens. IL-10 also suppresses the production of IL-12 and interferon-inducible protein-10 (IP-10) and counteracts the effects of IFN-g, all of which are important type 1-associated cytokines. In activated monocytes and macrophages, IL-10 inhibits the synthesis of many inflammatory mediators, including cytokines and reactive nitrogen species instrumental to the generation of protective innate and cell-mediated immune responses in the lung. Conversely, IL-10 directly stimulates the expression of IL-1 receptor antagonist and soluble TNF receptors, molecules that antagonize the effects of selected proinflammatory cytokines. IL-10 has similar anti-inflammatory effects on neutrophils. In addition, IL-10 impairs the ability of monocytes and macrophages to present antigen and activate T cells by decreasing expression of MHC class II molecules, CD52 (ICAM-1), and the costimulatory molecules CD80 and CD86. Furthermore, IL-10 inhibits monocyte differentiation into dendritic cells, further suppressing the antigen-presenting capabilities of the innate immune system. Finally, IL-10 enhances the phagocytic ability of monocytes and macrophages, but suppresses microbicidal activity of these cells. In addition to prominent inhibitory effects on monocyte/macrophage effector cell functions, IL-10 also exerts suppressive effects on selected T-cell activities. For instance, IL-10 inhibits mitogen-induced proliferation and cytokine production (e.g., IL-2, IFN-g) by CD4 þ T cells. IL-10 also promotes the development of regulatory CD4 þ T cells, cells that are instrumental in the development of immune tolerance. Inhibitory effects of IL-10 on CD8 þ T cells are less pronounced and, under certain conditions, IL-10 can even activate CD8 þ T-cell populations, including cytotoxic T cells. Likewise, IL-10 can stimulate NK cells to produce IFN-g, TNF-a, and granulocytemacrophage colony-stimulating factor (GM-CSF).

INTERLEUKINS / IL-10 375

Acquired immunity

Innate immunity Differentiation

Dendritic cell

Phagocytosis Microbicidal activity

Inflammatory cytokine production Mitogen-induced proliferation Th0 Th2 phenotype

Inflammatory cytokine secretion

Th cell

Monocyte

Treg phenotype development

Inflammatory cytokine secretion Antigen Macrophage presentation

Treg cell IL-10 Cytotoxic activity Proliferation

Inflammatory cytokine secretion Neutrophil

Tc cell Y

Cytokine production Cytoxicity

YY

NK cell

YY

Proliferation Immunoglobulin secretion

Structural cells Cytokine production Collagen production MMP production

B cell

Epithelial cell fibroblast

Figure 2 Overview depicting the biological effects of IL-10. Treg, regulatory T cell; Tc, cytotoxic T cell.

IL-10 also stimulates mast cell growth, B-lymphocyte proliferation, and immunoglobulin secretion. IL-10 exerts diverse effects on functions of structural cells. Similar to effects in macrophages, IL-10 inhibits the expression of inflammatory cytokines from various parenchymal and stromal cell populations, including alveolar and airway epithelial cells and fibroblasts. This cytokine also appears to modulate matrix production and remodeling. In isolated fibroblast populations, IL-10 generally inhibits collagen and fibronectin expression. Effects on the release of matrix metalloproteinase proteins (MMPs) involved in matrix remodeling are varied, as IL-10 stimulates MMP-1 and stomelysin release, whereas MMP-9 expression is diminished in the presence of IL-10. Therefore, given the complexity of IL-10 effects on immune and structural cell responses, it is more accurate to consider IL-10 as an immunomodulatory cytokine, rather than simply an immunosuppressive molecule. A schematic depicting the biological effects of IL-10 is shown in Figure 2.

Receptors The IL-10 receptor complex is composed of four transmembrane chains, two of which comprise IL10R1 and two of which form IL-10R2. Expressed on

most hematopoietic cells and a few nonhematopoietic cell types (e.g., fibroblasts, epithelial cells), IL10R1 binds ligand and has a predicted molecular weight of 90–120 kDa. IL-10R1 binds both cellular and viral IL-10 proteins, but its affinity for vIL-10 is approximately 1000-fold less than cell-derived IL10. The IL-10R1 receptor exhibits some species specificity. For example, murine IL-10R1 binds both human and murine IL-10, whereas the human receptor binds only human IL-10. IL-10R2 initiates signal transduction upon IL-10 binding via the JAK/ STAT pathway, using the Jak1 and Tyk3 kinases and STAT3 transcription factor. Expressed on most cell types, IL-10R2 also complexes with the IL-22, IL-28A, IL-28B, and IL-29 receptors. Although no splice variants have been found for either IL-10R1 or IL-10R2 mRNA, it is plausible that these variants exist. In particular, the removal of exon 6 or 7 could result in the formation of a soluble IL-10 receptor. An engineered soluble IL-10R has displayed antagonist activity.

IL-10 in Respiratory Disease States Acute Lung Injury

IL-10 is produced by many cells of the lung in respiratory disease states, although the exact role of this

376 INTERLEUKINS / IL-10

cytokine in disease pathogenesis is unclear. In general, IL-10 appears to play a pivotal role in controlling the magnitude of inflammatory responses during either systemic or pulmonary inflammation. In the gut, for example, mice lacking the IL-10 gene (IL-10
/
) develop spontaneous enterocolitis that mimics inflammatory bowel disease due to the unchecked immune responses to endogenous bowel flora. Induction of sepsis (either due to endotoxin administration or cecal ligation and puncture) results in the systemic production of IL-10, and blockade of IL-10 during the septic response dramatically enhances sepsis-induced inflammatory cytokine release, tissue injury, and mortality. Similarly, IL-10
/
mice have prolonged inflammation following acute intrapulmonary challenge with cytotoxic strains of Pseudomonas aeruginosa. Conversely, endotoxin-induced tissue injury in animal models of sepsis can be substantially reduced by the administration of recombinant IL-10. IL-10 is expressed in the airspace of patients with acute respiratory distress syndrome (ARDS), and lower levels of IL-10 in bronchoalveolar lavage fluid have been associated with increased mortality in these patients. These data suggest that insufficient IL-10 responses may contribute to enhanced and/or persistent lung inflammation in ARDS, which in turn may lead to adverse clinical outcomes. Lung Host Defense

While the endogenous production of IL-10 is required in order to appropriately compartmentalize inflammatory responses, the IL-10-mediated suppression of cytokines involved in innate and acquired immunity can substantially impair antimicrobial host defense mechanisms. For instance, virulent bacterial pathogens such as Klebsiella pneumoniae and Streptococcus pneumoniae require a robust innate immune response for effective clearance from the lung, and the endogenous production of IL-10 significantly impairs host antibacterial immunity against these respiratory pathogens. IL-10 also suppresses cellmediated responses against intracellular pathogens of the lung, including fungi and Mycobacterium spp. Moreover, IL-10 appears to be a major mediator of leukocyte deactivation that occurs in states of critical illness, most notably sepsis. In particular, the release of IL-10 during the septic response causes alveolar macrophage dysfunction and resultant impairment of pulmonary host defenses. Collectively, these observations illustrate the need for a balanced IL-10 response in order to limit inflammation while preserving sufficient host immune function against microbial challenge.

Airways Disease

Mounting evidence suggests that IL-10 plays a fundamental role in shaping immune responses within the airway. Studies in animal models demonstrate that IL-10 promotes the development of tolerance to inhaled allergen challenge, resulting in decreased eosinophilic inflammation and airway hyperreactivity. The mechanism of protection is due to suppression of type 2 cytokine release (e.g., IL-4, IL-5, and IL-13) and resultant attenuation of allergic airways inflammation. In allergic diseases, including asthma, the severity of disease appears to be inversely correlated with local IL-10 levels. The expression of IL-10 is elevated in some patients with asthma, with further upregulation by allergen challenge, perhaps released in order to counterbalance the actions of type 2 cytokines. This cytokine also participates in control of airway inflammation in response to infectious challenge of the respiratory tract. For example, prolonged and more severe airway inflammation is observed in IL-10-deficient mice during chronic endobronchial Pseudomonas infection, as compared to wild-type animals, and insufficient IL-10 responses may account for the persistent airway inflammation seen in patients with cystic fibrosis. Indeed, patients with cystic fibrosis have lower levels of airway epithelial cell-derived IL-10, as compared to healthy subjects. Similarly, in patients with chronic obstructive pulmonary disease (COPD), IL-10 levels in sputum are diminished compared to healthy controls. As IL-10 has been shown to inhibit MMP-9 release, reductions in IL-10 observed in COPD patients may promote a lung microenvironment characterized by accelerated parenchymal destruction. Taking into account the dominant anti-inflammatory properties of IL-10, exogenous administration of this cytokine has been tested and found to be beneficial in several immune-mediated diseases, including inflammatory bowel disease, rheumatoid arthritis, and psoriasis. Given the putative roles that IL-10 plays in sepsis and sepsis-induced lung injury, allergic airways disease, and COPD, it is possible that IL-10 may be a therapeutic target in the development of immunomodulatory therapies for these diseases. The potential beneficial effects of IL-10 immunotherapy must be reconciled with the less desirable effects on immune host defenses and selective T and NK cell activation. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Chemokines. Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-12. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: T cells; Pulmonary Macrophages.

INTERLEUKINS / IL-12 377

Further Reading Asadullah K, Sterry W, and Volk HD (2003) Interleukin-10 therapy – review of a new approach. Pharmacological Reviews 55(2): 241–269. Barbarin V, Arras M, Misson P, et al. (2004) Characterization of the effect of interleukin-10 on silica-induced lung fibrosis in mice. American Journal of Respiratory Cell and Molecular Biology 31: 78–85. Barnes PJ (2003) Cytokine-directed therapies for the treatment of chronic airway diseases. Cytokine and Growth Factor Reviews 14: 511–522. Bergeron A, Soler P, Kambouchner M, et al. (2003) Cytokine profiles in idiopathic pulmonary fibrosis suggest an important role for TGF-b and IL-10. European Respiratory Journal 22: 69–76. Chmiel JF, Konstan MW, Saadane A, et al. (2002) Prolonged inflammatory response to acute Pseudomonas challenge in interleukin-10 knockout mice. American Journal of Respiratory and Critical Care Medicine 165: 1176–1181. Conti P, Kempuraj D, Kandere K, et al. (2003) IL-10, an inflammatory/inhibitory cytokine, but not always. Immunology Letters 86: 123–129. Goodman RB, Pugin J, Lee JS, and Matthay MA (2003) Cytokinemediated inflammation in acute lung injury. Cytokine and Growth Factor Reviews 14: 523–535. Hawrylowicz CM and O’Garra A (2005) Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma. Nature Reviews: Immunology 5(4): 271–283. Kay AB (2003) Immunomodulation in asthma: mechanisms and possible pitfalls. Current Opinion in Pharmacology 3: 220–226. Kim JM, Brannan CL, Copeland NG, et al. (1992) Structure of the mouse IL-10 gene and chromosomal localization of the mouse and human genes. Journal of Immunology 148: 3618–3623. Lynch EL, Little FF, Wilson KC, Center DM, and Cruikshank WW (2003) Immunomodulatory cytokines in asthmatic inflammation. Cytokine and Growth Factor Reviews 14: 489–502. Mocellin S, Panelli MC, Wang E, Nagorsen D, and Marincola FM (2003) The dual role of IL-10. Trends in Immunology 24(1): 36–43. Moore KW, Malefyt RD, Coffman RL, and O’Garra A (2001) Interleukin-10 and the interleukin-10 receptor. Annual Review of Immunology 19: 683–765. Oberholzer A, Oberholzer C, and Moldawer LL (2002) Interleukin-10: a complex role in the pathogenesis of sepsis syndromes and its potential as an anti-inflammatory drug. Critical Care Medicine 30(supplement 1): S58–S63. Oltmanns U, Schmidt B, Hoernig S, Witt C, and John M (2003) Increased spontaneous interleukin-10 release from alveolar macrophages in active pulmonary sarcoidosis. Experimental Lung Research 29: 315–328. Pestka S, Krause CD, Sarkar D, et al. (2004) Interleukin-10 and related cytokines and receptors. Annual Review of Immunology 22: 929–979.

IL-12 M J Walter, Washington University School of Medicine, St Louis, MO, USA

proteins, termed the IL-12 related cytokines. The IL-12 related cytokines are generated from five independently regulated genes that homodimerize and heterodimerize to form seven secreted family members. These proteins have overlapping as well as distinct biologic properties and this article focuses specifically on IL12, p80, and p40. IL-12, a disulfide-linked heterodimer composed of a p40 and p35 subunit, has been identified as an immune cell stimulator that promotes differentiation and proliferation of T cells and enhances the production of interferon gamma. Human studies and murine experimental models have implicated IL-12 in the regulation of multiple immunologic processes including regulation of CD4 þ and CD8 þ T-cell differentiation, host defense, autoimmunity, tumor cell immune recognition, alloimmunity, and host response to vaccine administration. p80 is a disulfide-linked p40 homodimer that can function as a competitive antagonist of IL-12 and a macrophage chemoattractant. Specific topics discussed in this article include the structure, regulation, function, receptor utilization, and role of IL-12 related cytokines in respiratory disease.

Introduction IL-12 is a heterodimeric cytokine with broad immunoregulatory properties that belongs to a larger family of related proteins called the IL-12 related cytokines. IL-12 has been identified as an immune cell stimulator that promotes differentiation and proliferation of T cells and enhances the production of interferon gamma (IFN-g). Since its discovery, IL-12 has been implicated in the regulation of multiple immunologic processes that include regulation of CD4 þ and CD8 þ T-cell differentiation, host defense, autoimmunity, tumor cell immune recognition, alloimmunity, and host response to vaccine administration. Molecular cloning revealed that IL-12 was a heterodimeric cytokine composed of two disulfide-linked glycoproteins. These individual subunits were named p40 and p35 according to their electrophoretic molecular weight. A p40 homolog named Epstein–Barr virus-induced gene 3 (EBI3), and two p35 homologs named p19 and p28 have been identified. These five independently regulated genes encode the proteins that comprise the IL-12 related cytokines. Due to monomer secretion as well as homodimeric and heterodimeric partnering, the IL-12 family currently consists of seven secreted proteins: IL-12 (a p40/p35 heterodimer), p80 (a p40/p40 homodimer), p40 (a p40 monomer), EBI3/p35 (an EBI3/p35 heterodimer), EBI3 (an EBI3 monomer), IL-23 (a p40/p19 heterodimer), and IL-27 (EBI3/p28 heterodimer) (Table 1). These family members have overlapping as well as distinct biologic properties. This article focuses specifically on the IL-12, p80, and p40 family members.

& 2006 Elsevier Ltd. All rights reserved.

Structure of IL-12 Abstract Interleukin-12 (IL-12) is a heterodimeric cytokine with broad immunoregulatory properties that belongs to a larger family of

The human p40 gene was originally termed IL-12B and is located within a cytokine gene cluster on chromosome 5q31. The gene encodes a 328 amino

378 INTERLEUKINS / IL-12 Table 1 Characteristics of IL-12 related cytokines IL-12 member

Protein components

Receptors

Receptor affinity (humana/mouseb)

Biologic functions

IL-12

p35 and p40

IL-12Rb1 and IL-12Rb2

4-7/2-3

p80

p40 and p40

IL-12Rb1

20-70/4

p40 IL-23

p40 p19 and p40

IL-12Rb1 IL-12Rb1 and IL-23R

400-700/25-100 ND

EBI3 EBI3/p35 IL-27

EBI3 EBI3 and p35 EBI3 and p28

ND ND TCCR and gp130

ND ND ND

Proliferation and differentiation of T cells IFN-g secretion from T/NK cells IL-12 antagonist Macrophage chemotaxis ND Proliferation of memory T cells IFN-g, IL-17A, IL-17F secretion from T cell ND ND Proliferation of naive T cells IFN-g secretion from T cells

Inhibitory concentration (IC) 50% (ng ml
1) in KIT225/K6 cells. IC 50% (ng ml
1) in mouse concavalin A blasts. ND, not determined. EBI3, Epstein–Barr virus-induced gene 3; IFN-g, interferon gamma; IL, interleukin; IL-12Rb1, IL-12 receptor b1; NK, natural killer; TCCR, T-cell cytokine receptor; gp130, glycoprotein 130. a b

acid polypeptide which contains a 22 amino acid signal peptide, four potential N-glycosylation sites, four intramolecular disulfide bonds, one free cysteine, and one cysteine that forms a disulfide bond with p35. The p40 protein shares homology with the extracellular domain of the hematopoietic cytokine receptor family and is most closely related to the IL-6 receptor a-subunit. Although related to this receptor family, the p40 protein does not contain a transmembrane domain and membrane-bound p40 has not been identified. Murine p40 is located on chromosome 11 in a cytokine gene cluster that is equivalent to the human locus and is 70% homologous to human p40. The human p35 gene was originally named IL-12A and is located on chromosome 3. The gene encodes a 253 ammo acid with two separate initiation codons. Initiation at the second methionine results in a 22 amino acid hydrophobic region that is characteristic of a signal peptide, whereas initiation at the first methionine results in a longer but less hydrophobic region that may serve to anchor p35 to the cell membrane. The p35 protein is an a-helix-rich structure that contains three potential N-glycosylation sites, three intramolecular disulfide bonds, and one cysteine that forms a disulfide bond with the p40 protein. p35 does not have any sequence homology with p40 but does share high homology with IL-6 and granulocyte colony stimulating factor (G-CSF). Murine p35 is located on chromosome 3 and is 67% homologous to human p35.

Regulation of IL-12 Production Numerous cell types can produce IL-12 and many stimuli are involved in the regulation of its production.

IL-12 production has been found in dendritic cells, monocytes, macrophages, neutrophils, keratinocytes, and airway epithelial cells. These cell types are integral members of the innate immune response, suggesting a critical role for IL-12 in this arm of the immune system. Secretion depends on simultaneous expression of both p40 and p35 subunits, and cell-specific differences in the expression of p40, p35, and IL-12 have been noted. Cytokines, microbiologic products, and costimulatory protein ligation induce p40 gene transcription and the secretion of IL-12 correlates closely with p40 induction. p35 expression is constitutive and regulated through transcriptional and translational mechanisms; however, p35 monomer is not secreted. Although initial reports indicated p35 expression was not enhanced by inflammatory cytokines, recent reports demonstrate IFN-g priming can result in lipopolysaccharide (LPS)-dependent interferon regulator factor (IRF)-1-driven p35 gene transcription. While a large number of mediators are involved in activating IL-12 expression, an equally large number of inflammatory cytokines, chemoattractants, and G-protein-coupled receptors have been shown to inhibit its production (Table 2). A p40 monomer can dimerize with p35 to form IL-12, but it can also be secreted as a p40 monomer, homodimerize with itself to form p80, or heterodimerize with p19 to form IL-23. Many stimuli that induce p40 result in the secretion of large molar excesses of p40 monomer and p80 compared to IL-12. The cellular mechanisms that control p40 monomer secretion and the disulfide linking of p40 with either p35, p40, or p19 are not known. The cellspecific responses to the wide variety of activating and inhibitory mediators and the ability of p40 to couple with different partners likely provides the

INTERLEUKINS / IL-12 379 Table 2 Regulation of IL-12 secretion IL-12 activators

IL-12 inhibitors

IFN-g IL-4 (o24 h) IL-13 (o24 h) Bacterial DNA CpG oligonucleotides Lipopolysaccharide Brucella Viruses Double-stranded RNA Fungi Intracellular parasites Toxoplasma CD40 ligation

IFN-a IFN-b IL-4 (424 h) IL-13 (424 h) IL-10 Transforming growth factor beta Monocyte chemoattractant protein 1-4 C5a FMLP Prostaglandin E2

fMLP, formylmethionylleucylphenylaline.

immune system with numerous regulatory checkpoints to finely control the temporal and spatial concentrations of the IL-12 related cytokines.

Biologic Function of IL-12, p80, and p40 IL-12 evokes potent immunoregulatory properties by promoting CD4 þ T-helper-1 (Th1) cell differentiation, inhibiting CD4 þ Th2 cell differentiation and enhancing immune cell production of IFN-g. IL-12 is required for the development of an appropriate Th1 response to some, but not all, microbial pathogens. For example, IL-12 is required for the development of a Th1 response to Leishmania major, Mycobacterium tuberculosis, Histoplasma capsulatum, Salmonella, and Klebsiella pneumoniae. By contrast, IL-12 is not required for the Th1 response to lymphocytic choriomeningitis virus, vesicular stomatitis virus, mouse hepatitis virus, and mouse parainfluenza virus. In the context of a murine Th1 response to pathogens, exogenously administered recombinant IL-12 can provide protection or cause enhanced inflammation depending on the microbial challenge and administration regimens. Additional studies have demonstrated the influence of IL-12 during a Th2 immunologic response. For example, in mice recombinant IL-12 administration concurrent with allergen sensitization or challenge prevented airway hyperreactivity, eosinophil recruitment, and IL-4 and IL-5 production while it increased IFN-g levels. In addition, IL-12 blockade with neutralizing antibody at the time of allergen challenge allowed for the development of a Th2 response in an otherwise resistant mouse strain and increased the Th2 response in a sensitive strain. Ovalbumin sensitized and challenged p40 deficient mice develop an enhanced Th2 response with increased airway, peripheral blood and bone marrow eosinophils and

increased IL-4 and IgE concentrations, although this effect is not found in all studies. p80 has an affinity for IL-12Rb1 that is comparable to IL-12. Originally, p80 was found to be a competitive antagonist for IL-12 binding, but recent experiments indicate p80 has distinct functions that are independent from the other IL-12 related cytokines. As an IL-12 antagonist, p80 can effectively block in vitro IL-12-dependent IFN-g production, proliferation of lymphocytes, and natural killer (NK) cell activation. Similarly, exogenously administered p80 can inhibit murine endotoxin-induced IFN-g production and decrease mortality. Transgenic mice with p40 and p80 overexpression in the liver exhibit a blunted Th1 immune response to Plasmodium berghei, and selective overexpression of p40 and p80 in the basal epithelial cells of the skin generates an inflammatory skin lesion resembling eczema. Recombinant p80, but not IL-12 or p40, signals through IL-12Rb1 to cause macrophage chemotaxis and can induce ‘inducible nitric-oxide synthetase’ expression and activate nuclear factor kappa B (NF-kB) in isolated mouse peritoneal macrophages and microglial cells. Although p40 monomer is secreted, its affinity for IL-12Rb1 is at least 100-fold less than IL-12 and a biologic role for this protein has not been identified.

IL-12 Receptors The high-affinity receptor for human IL-12 is composed of two independently regulated type I transmembrane glycoproteins referred to as IL-12 receptor b1 (IL-12Rb1) and IL-12Rb2. Both of these receptors belong to the hematopoietin receptor superfamily and share homology with gpl30, G-CSF, and leukemia-inhibitory factor receptors. The human IL-12Rb1 gene is located on chromosome 19pl3 and encodes a 662 amino acid polypeptide with a 24 amino acid signal peptide, a 516 amino acid extracellular domain, a 31 amino acid transmembrane domain, and a 91 amino acid cytoplasmic tail. IL12Rb1 expression is confined to immune cells and is found on T cells, NK cells, B cells, dendritic cells, and macrophages. The extracellular cytokine-binding domain interacts with the p40 subunit of IL-12 and the cytoplasmic portion interacts with the Janus kinase family member tyrosine kinase 2 (Tyk2). The mouse IL-12Rb1 gene encodes a 738 amino acid polypeptide with 54% homology to the human at the amino acid level and has identical protein interactions with p40 and Tyk2. The human IL-12Rb2 receptor is located on chromosome 1p31 and encodes a 862 amino acid polypeptide with a 27 amino acid signal peptide, a 595 amino acid extracellular domain, a 24 amino

380 INTERLEUKINS / IL-12 p80

IL-12 p40

p35

IL-12R
2

IL-12R
1 Tyk2

p40

p40

IL-12R
1

Jak2

p40

p40

Tyk2

IL-12R
1 Tyk2

Unknown Stat4

Stat4-P

NF-B activation chemotaxis

Figure 1 Signal transduction pathways for IL-12 related cytokines. IL-12 binds to IL-12Rb1 and IL-12Rb2 to activate the transcription factor Stat4. p80 binds to IL-12Rb1 to activate NF-kB and chemotaxis. p40 binds to IL-12b1 with very low affinity and activation of intracellular signaling has not been described. IL-12, interleukin-12; IL-12Rb1, IL-12 receptor b1; IL-12Rb2, IL-12 receptor b2; Jak2, Janus kinase 2; NFkB, nuclear factor kappa B; Stat4, signal transducer and activator of transcription-4; Stat4-P, phosphorylated Stat4; Tyk2, tyrosine kinase 2.

acid transmembrane domain, and a 216 amino acid cytoplasmic tail. The extracellular region binds to the p35 subunit of IL-12 and the cytoplasmic tail associates with Janus kinase 2 (Jak2) and contains three conserved tyrosines that are required for intracellular signaling. The binding of IL-12 to the receptor chains initiates subsequent tyrosine phosphorylation of the receptor associated tyrosine kinases (Tyk2 and Jak2, respectively), IL-12Rb2, and the transcription factors signal transducer and activator of transcription-4 (Stat4), Stat3, Stat1, and Stat5. Although IL-12 has been reported to activate multiple members of the Stat family as well as mitogen-activated protein kinase (MAPK), NF-kB, and Lck, the predominant signaling molecule appears to be Stat4 (Figure 1). IL-12Rb2 is absent on resting immune cells, and the induction of IL-12Rb2 expression confers the ability to respond to IL-12. The mouse IL-12Rb2 gene encodes a 875 amino acid polypeptide with 68% homology to the human at the amino acid level.

IL-12-Related Cytokines in Respiratory Disease Identification of individuals with susceptibility to mycobacterial disease has led to a description of a human syndrome known as Mendelian susceptibility to mycobacterial disease. Molecular characterization of these individuals has identified 73 patients from 21 countries that are completely deficient in p40 (n ¼ l9) or IL-12Rb1 (n ¼ 54). Since p40 and IL-12Rb1 are required for the function of IL-12, IL-23, p80, and p40 monomers, these individuals are deficient in all of these IL-12 related cytokines. Collectively, these individuals

display an increased susceptibility to primary infection with environmental mycobacteria, live Bacille Calmette-Guerin (BCG), and Salmonella. Follow-up of these individuals noted essentially no recurrence of mycobacterial disease, whereas secondary infection with Salmonella did occur. Penetrance for this infectious phenotype is low because 18% of probands failed to develop BCG infection after vaccination, and affected siblings often do not develop these spontaneous infections. This group of individuals demonstrates no reported increased susceptibility to viral, Grampositive, Gram-negative, protozoa, or fungal infections (one case of Nocardia asteroides and Paracoccidioides braziliensis) suggesting a redundant role for these IL-12-related cytokines in these infections. IL-12-related cytokines have also been implicated in asthma. In one report, bronchoalveolar lavage fluid from human subjects with asthma had low protein levels of IL-12 but increased amounts of p80. The increased p80 expression was independent of concurrent inhaled glucocorticoids and associated with increased accumulation of macrophages. In contrast, others have found decreased p40 mRNA in mild asthmatics at baseline, and in the steroidsensitive subjects these levels increased to normal after oral glucocorticoid administration. In this study the protein levels of IL-12 family members were not determined so the relative contribution of IL-12 and p80 was not defined. Lastly, in an attempt to modulate asthma, four escalating doses of weekly subcutaneous IL-12 were administered to mild asthmatic subjects. This treatment decreased peripheral blood and sputum eosinophilia but failed to alter airway reactivity to histamine or inhaled antigen.

INTERLEUKINS / IL-12 381 Table 3 Associations between respiratory disease and polymorphisms in the IL-12 gene family Disease

Gene

Authors (year)

Population

Polymorphisma

Association

Asthma

p40

Morahan et al. (2002)

Australia

IL-12B promoter

Khoo et al. (2004) Noguchi et al. (2001)

Australia Japan

Randolf et al. (2004)

USA

Birkisson et al. (2004)

Iceland

p35

Birkisson et al. (2004)

Iceland

IL-12B promoter Ser226Asn 1188A/C IL-12B promoter IL-12B 1025 flanking IL-12B 2508 intron IL-12B 4237 intron IL-12B 4496 intron IL-12B 6402 intron IL-12B 13478 30 UTR None detected in promoter (1883 bp) None detected in promoter (1579 bp)

Heterozygosity with sever asthma None None None None None None Positive None Positive Positive None

p40

Ma et al. (2003) Tso et al. (2004)

USA China

IL-12Rb1

Akahoshi et al. (2003)

Japan

Remus et al. (2004)

Latsi et al. (2003)

Tuberculosis

Idiopathic pulmonary fibrosis

p40

None None Positive Positive Positive None Positive Positive

Morocco

IL-12B 30 UTR Promoter Intron 2 Intron 4 Exon 5 30 UTR Q214R, M365T, G378R haplotype
111A-T
2C-T 378G-C 409 þ 21C-A 467G-A 641A-G 1094 T-C 1791 þ 46 T-C 1989 þ 34C-T

UK

1188 (A/C) 30 UTR

None

Positive Positive None None None None None None None

a Nomenclature as reported in manuscript. 30 UTR, 30 untranslated region of gene.

The p40 gene is located within a cytokine gene cluster on chromosome 5q31-33. This region of chromosome 5 has been associated with numerous nonrespiratory and respiratory human diseases in population-based linkage analysis studies. Specific polymorphisms in the p40 gene have been associated with type I diabetes, atopic dermatitis, psoriasis, multiple sclerosis, malaria, and hepatitis C viral infection in some but not all studies. No association with p40 polymorphisms and celiac disease, aphthous stomatitis, Crohn’s disease, or Behc¸et’s disease has been reported. Additional studies have been performed to identify associations between polymorphisms in the IL-12-related cytokines and respiratory diseases such as asthma, tuberculosis, and idiopathic pulmonary fibrosis (Table 3). See also: Allergy: Overview. Asthma: Overview. Genetics: Gene Association Studies. Interferons.

Interleukins: IL-23 and IL-27. Leukocytes: Monocytes; T cells; Pulmonary Macrophages.

Further Reading Abdi K (2002) IL-12: the role of p40 versus p75. Scandinavian Journal of Immunology 56: 1–11. Devergne O, Hummel M, Koeppen H, et al. (1996) A novel interleukin-12 p40 related protein induced by latent Epstein–Barr virus infection in B lymphocytes. Journal of Virology 70: 1143–1153. Fieschi C and Casanova JL (2003) The role of interleukin-12 in human infectious diseases: only a faint signature. European Journal of Immunology 33: 1461–1464. Gately MK, Renzetti LM, Magram J, et al. (1998) The interleukin12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annual Review of Immunology 16: 495–521. Ha SJ, Lee CH, Lee SB, et al. (1999) A novel function of IL-12 p40 as a chemotactic molecule for macrophages. Journal of Immunology 163: 2902–2908. Kobayashi M, Fitz L, Ryan M, et al. (1989) Identification and purification of natural killer cell stimulatory factor (NKSF), a

382 INTERLEUKINS / IL-13 cytokine with multiple biologic effects on human lymphocytes. Journal of Experimental Medicine 170: 827–845. Oppmann B, Lesley R, Blom B, et al. (2000) Novel p19 protein engages IL-12 p40 to form a cytokine, IL-23, with biologic activities similar as well as distinct from IL-12. Immunity 13: 715– 725. Pflanz S, Timans JC, Cheung J, et al. (2002) IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4( þ ) T cells. Immunity 16: 779–790. Russell TD, Yan Q, Fan G, et al. (2003) IL-12 p40 homodimerdependent macrophage chemotaxis and respiratory viral inflammation are mediated through IL-12 receptor beta. Journal of Immunology 171: 6866–6874. Trinchieri G (2003) Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nature Reviews: Immunology 3: 133–146. Trinchieri G, Pflanz S, and Kastelein RA (2003) The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses. Immunity 19: 641–644. van de Vosse E, Lichtenauer-Kaligis EG, van Dissel JT, and Ottenhoff TH (2003) Genetic variations in the interleukin-12/interleukin-23 receptor (beta1) chain, and implications for IL-12 and IL-23 receptor structure and function. Immunogenetics 54: 817–829. Walter MJ, Kajiwara N, Karanja P, Castro M, and Holtzman MJ (2001) IL-12 p40 production by barrier epithelial cells during airway inflammation. Journal of Experimental Medicine 193: 339–351. Watford WT, Moriguchi M, Morinobu A, and O’Shea JJ (2003) The biology of IL-12: coordinating innate and adaptive immune responses. Cytokine & Growth Factor Reviews 14: 361–368. Wolf SF, Temple PA, Kobayashi M, et al. (1991) Cloning of cDNA for natural killer cell stimulatory factor, a heterodimer cytokine with multiple biologic effects on T and natural killer cells. Journal of Immunology 146: 3074–3081.

IL-13 D B Corry and F Kheradmand, Baylor College of Medicine, Houston, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin-13 (IL-13) is a cytokine of T-helper-2 cells that is an important mediator of allergic inflammation and disease. In addition to effects on immune cells that are similar to those of the closely related cytokine IL-4, IL-13 is more importantly implicated as a central mediator of the physiologic changes induced by allergic inflammation in many tissues. IL-13 induces its effects through a multisubunit receptor that includes the a-chain of the IL-4 receptor (IL-4Ra), which is also part of the IL-4 receptor, and at least one of two known IL-13-specific binding chains. Most of the biological effects of IL-13, like those of IL-4, are linked to a single transcription factor
signal transducer and activator of transcription 6. Depending on the organ involved, IL-13 plays either beneficial or deleterious roles in immunity. IL13 is required for the expulsion of a variety of parasites from the gut. Conversely, IL-13 mediates granulomatous disease, which causes dysfunction of many organs in the setting of allergic inflammation, and may suppress beneficial immune functions of macrophages. In the lung, IL-13 coordinates the recruitment of

allergic inflammatory cells, induces airway obstruction through direct effects on target airway cells, and is associated with airway fibrosis. In addition to its proinflammatory role, IL-13 also induces matrix metalloproteinases of the airway that promote resolution of allergic inflammation.

Introduction Originally described as a secreted polypeptide, p600, and a product of the newly described T-helper-2 (Th2) cells in 1989, interleukin-13 (IL-13) was subsequently shown to be homologous to IL-4, a cytokine described several years earlier. Indeed, the marked structural and functional similarities between IL-4 and IL-13 led to the belief among many investigators that IL-13 was functionally redundant. Like IL-4, IL-13 was shown to inhibit activation of monocytes and stimulate immunoglobulin E (IgE) secretion from CD40-activated human B cells. IL-13 also induces expression of the low-affinity IgE receptor, CD23, and major histocompatibility class II molecules on hematopoietic cells, as does IL-4. Almost 10 years after its discovery, the more important role of IL-13 as a potent effector cytokine during allergic inflammatory reactions finally came to light. Today, IL-13 is recognized as a key mediator of allergic effector responses and disease rather than as a growth and differentiation factor for hematopoietic cells, a role that is best defined by IL-4. The biology of IL-4 and IL-13 overlaps further with respect to the molecules that they share as part of their receptor complexes. Despite such insight, a complete molecular explanation for the distinct functions of IL-4 and IL-13 remains an important and elusive goal.

Structure Both human and mouse IL-13 are located in a cluster of immunologically important genes in the distal arms of chromosomes 5q 23-31 and 11q, respectively, and encode a polypeptide of approximately 12 400 Da. The IL-4 gene is located 12 kb from the IL-13 gene and both are transcribed in an opposite direction to all other genes in the region. Human IL-13 complementary DNA (cDNA) and polypeptides are 66% and 58% homologous with the mouse cytokine, respectively, and human IL-13 is between 20% and 25% identical with human IL-4. These findings suggest that IL-4 and IL-13 have descended from the same ancestral gene. IL-13 exists in two alternatively spliced forms, differing only by the addition or not of a single glycine residue.

Production and Activity Like most hematopoietin cytokines (the ‘interleukins’), production of IL-13 is usually regulated by

INTERLEUKINS / IL-13 383

transcription, the major exception being the preformed IL-13 that is released as part of the granule contents of activated eosinophils and mast cells. Other cells that are reported to make IL-13 include monocytes/macrophages, basophils, and natural killer cells. However, the principal source of IL-13 during allergic inflammation is probably the T cell, especially Th2 cells. Posttranslational modifications to IL-13 such as glycosylation, although documented, do not contribute to activity; other modifications such as phosphorylation, sulfation, and proteolytic cleavage have not been described. IL-13 is secreted during activation of allergic effector cells in response to a wide variety of stimuli. Perhaps best described in this regard are Th2 cells, which release IL-13 simultaneously with other Th2 cytokines following antigen stimulation. The close proximity of the IL-4 and IL-13 genes suggested a shared regulatory pathway and, indeed, the promoters for IL-13 and IL-4, although not identical, are activated by many of the same transcription factors (activator protein 1 (AP-1), nuclear factor kappa B (NF-kB), signal transducer and activator of transcription 6 (STAT6), GATA3). Additional regulatory elements are critical to the production of these cytokines, including a 400 bp noncoding genomic element that coordinately regulates IL-4, IL-5, and IL-13 production and which lies in the intergenic region between the IL-4 and IL-13 loci, termed central nervous system (CNS)-1. How organisms such as parasitic worms of the gut and common allergens such as pollens, dust mites, and fungi elicit IL-4 and IL-13 responses is not fully understood. However, these allergenic agents all have strong proteinase activity, which at least for some fungi is required to elicit strong Th2 responses and production of IL-4 and IL-13 in mice.

Biological Function The functions of IL-13 overlap considerably with those of IL-4, especially with regard to changes induced on hematopoietic cells, but these effects are probably less important given the more potent role of IL-4. Thus, although IL-13 can induce IgE secretion from activated human B cells, deletion of IL-13 from mice does not markedly affect either Th2 cell development or antigen-specific IgE responses induced by potent allergens. In comparison, deletion of IL-4 abrogates these responses. Instead of a lymphoid cytokine, IL-13 acts as an important molecular bridge linking allergic inflammatory cells to the nonimmune cells in contact with them and drastically altering their function. IL-13 specifically induces physiologic changes in parasitized organs that are

required to expel the offending organisms or their products. For example, expulsion from the gut of a variety of murine helminths requires IL-13 secreted by Th2 cells. IL-13 induces several changes in the gut that create a hostile environment for the parasite, including enhanced contractions and hypersecretion of glycoprotein from gut epithelial cells, which ultimately lead to detachment of the organism from the gut wall and their removal. The eggs of the parasite Schistosoma may lodge in a variety of organs including the gut wall, liver, lung, and even the CNS, inducing the formation of granulomas under the control of IL-13. In this case, the eventual result is organ damage and often profound or even fatal disease, not resolution of the infection. An emerging concept is that IL-13 may antagonize Th1 responses that are required to resolve intracellular infections. In this immune dysregulated context, marked by the recruitment of aberrantly large numbers of Th2 cells, IL-13 inhibits the ability of macrophages to destroy intracellular pathogens such as Leishmania major and probably other organisms as well.

Receptors The IL-13 receptor remains the least understood of all the interleukins. The a-chain of the IL-4 receptor (IL-4Ra) is the principal signaling chain for both the IL-4 and IL-13 receptors. However, like all hematopoietin cytokine receptors, the IL-4 and IL-13 receptors are comprised of several molecules (Figure 1). In addition to IL-4Ra, the IL-4 receptor includes the g-chain of the IL-2 receptor (IL-2Rg), forming the type I IL-4 receptor. The type1 IL-4 receptor is, however, probably incapable of signaling with IL-13 as the ligand neither binds IL-4Ra nor IL-2Rg to IL-13. Therefore, the IL-13 receptor must include at least one of two known IL-13 binding chains, IL13Ra1 or IL-13Ra2; the requirement of IL-2Rg for IL-13 signaling remains controversial. However, current evidence based on IL-13Ra2-deficient mice indicates that IL-13Ra2, which actually binds to IL13 with much greater affinity compared to IL-13Ra1, is a decoy receptor that serves to downregulate IL-13 responses. Thus, current evidence indicates that the principal IL-13 receptor consists of IL-13Ra1 together with IL-4Ra, with or without IL-2Rg. IL-4 can also signal through this receptor, which is referred to as the type II IL-4 receptor. Activation of the IL-13 receptor induces recruitment of two of the four known cytoplasmic tyrosine kinases of the Janus kinase family (Jaks) to the receptor complex. While the usage of Jaks is typically highly predictable among cytokine receptors, IL-13 appears to be capable of recruiting different combinations of Jaks

384 INTERLEUKINS / IL-13

IL-4 receptor

IL-13 receptor IL-4R
IL-13R
1

IL-4R


IL-13R
2

IL-2R STAT6 Allergic disease

Figure 1 The IL-4 and IL-13 receptors. The IL-4 and IL-13 receptors share the IL-4Ra subunit, but also have their unique components. The IL-2Rg subunit is only found as part of the IL-4 receptor, whereas IL-13Ra1 is thought to be the principal unique subunit of the IL-13 receptor. IL-13Ra2 may function as a decoy receptor that inhibits IL-13 signaling (dashed line). IL-4 may also signal through the combination of IL-4Ra and IL-13Ra1. Both IL-4 and IL-13 activate the transcription factor STAT6, which in turn regulates most aspects of allergic inflammation and disease.

Goblet cell metaplasia Macrophage intracytoplasmic killing

IL-13

Airway hyperresponsiveness

{

Subepithelial fibrosis

Enhanced secretion of: Proallergic chemokines Matrix metalloproteinases Glycoproteins Th2 cell

Stimulatory role

Inhibitory role

Figure 2 Pulmonary effects of IL-13. Although the functions of IL-13 overlap with those of IL-4, these are less important compared to IL-13-mediated physiological effects during allergic inflammation. These include prominent changes of the airway epithelium and smooth muscle resulting in airway hyperresponsiveness, goblet cell metaplasia, and subepithelial fibrosis. Th2 cell-derived IL-13 further enhances secretion of airway glycoproteins, matrix metalloproteinases, and proallergic chemokines. Under some conditions, IL-13 may compromise the ability of macrophages to destroy intracellular pathogens.

depending on the exact tissue expressing the IL-13 receptor. Regardless of which Jaks are recruited, they phosphorylate STAT6. There is now widespread agreement that STAT6 is the principal transcription factor activated by both the IL-4 and IL-13 receptors,

although the IL-4 receptor (through IL-2Rg) may also activate additional factors of the insulin response substrate (IRS) family. STAT6 in turn activates most if not all genes induced by IL-13 and is therefore responsible for most biological effects of IL-13.

INTERLEUKINS / IL-15 385

IL-13 in Respiratory Diseases IL-13 induces many features of allergic lung disease, including airway hyperresponsiveness, goblet cell metaplasia, and glycoprotein hypersecretion, which all contribute to airway obstruction (Figure 2). IL-4 contributes to these physiologic changes, but is less important than IL-13 as established by IL-4- and IL-13-deficient mice, mice deficient in both of these cytokines, and wild-type mice in which either IL-4 or IL-13 was inhibited using specific neutralizing agents. IL-13 also induces secretion of chemokines that are required for recruitment of allergic effector cells to the lung. Studies of STAT6 transgenic mice suggest the interesting possibility that IL-13 signaling occurring only through the airway epithelium is required for most of these effects. The chronic presence of IL-13 in the airway is further associated with subepithelial collagen deposition, widespread deposits of Charcot-Leyden-like crystals, and early mortality, indicating that IL-13 is an important mediator of airway disease during both acute and chronic allergen challenge. While no studies have as yet directly implicated IL-13 in the control of human diseases, many polymorphisms in the IL-13 gene have been shown to confer an enhanced risk of atopic respiratory diseases such as asthma. Although IL-13 is associated primarily with the induction of airway disease it also has anti-inflammatory properties. Airway matrix metalloproteinases (MMPs), such as MMP2 and MMP9, are required to establish chemokine gradients across the airway epithelium that serve to induce egression of effete parenchymal inflammatory cells into the airway lumen where they are then cleared. Among other factors, IL-13 induces these MMPs as part of a mechanism that protects against excessive allergic inflammation that predisposes to asphyxiation. See also: Allergy: Overview; Allergic Reactions; Allergic Rhinitis. Asthma: Overview. Chemokines, CC: TARC (CCL17). Chymase and Tryptase. Dust Mite. Eotaxins. Epithelial Cells: Type II Cells. Interleukins: IL-4; IL-5; IL-9. Lipid Mediators: Overview. Matrix Metalloproteinases. Mucus. Transcription Factors: Overview. Transgenic Models.

Further Reading Corry DB (1999) IL-13 in allergy: home at last. Current Opinion in Immunology 11: 610–614. Corry DB and Kheradmand F (2002) Biology and therapeutic potential of the interleukin-4/interleukin-13 signaling pathway in asthma. American Journal of Respiratory Medicine 1: 185–193. Finkelman FD, Shea-Donohue T, Morris SC, et al. (2004) Interleukin-4- and interleukin-13-mediated host protection against

intestinal nematode parasites. Immunology Reviews 201: 139–155. Jiang H, Harris MB, and Rothman P (2000) IL-4/IL-13 signaling beyond JAK/STAT. Journal of Allergy and Clinical Immunology 105: 1063–1070. Murata T, Taguchi J, Puri RK, and Mohri H (1999) Sharing of receptor subunits and signal transduction pathway between the IL-4 and IL-13 receptor system. International Journal of Hematology 69: 13–20. Murphy KM, Ouyang W, Farrar JD, et al. (2000) Signaling and transcription in T helper development. Annual Review of Immunology 18: 451–494. Townley RG and Horiba M (2003) Airway hyperresponsiveness: a story of mice and men and cytokines. Clinical Review of Allergy and Immunology 24: 85–110. Wills-Karp M (1999) Immunologic basis of antigen-induced airway hyperresponsiveness. Annual Review of Immunology 17: 255–281. Wynn TA (2003) IL-13 effector functions. Annual Review of Immunology 21: 425–456.

IL-15 Q Hamid and I Ito, McGill University, Montreal, QC, Canada S Muro, Kyoto University, Kyoto, Japan & 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin-15 (IL-15) is a 14–15 kDa protein that shares many biological activities with IL-2. IL-15 binds and signals via a trimetric receptor consisting of a common gamma chain, IL2Rb and IL-15Ra. Both IL-15 and IL-15Ra, which are essential for the high-affinity binding, have a much broader tissue distribution than IL-2/IL-2Ra. Multiple complex posttranscriptional regulatory mechanisms tightly control IL-15 expression. These findings suggest that IL-15 has pleiotropic functions in immune and nonimmune systems. It is possible that IL-15 has a role in respiratory diseases such as sarcoidosis and tuberculosis through its role in Th1-driven-inflammation.

Introduction Interleukin-15 (IL-15) was first isolated from cell culture supernatants of a simian kidney epithelial cell line in 1994 by Grabstein and co-workers as a novel T-cell growth factor whose biological activity is similar to IL-2. At the same time, Burton and co-workers showed that interleukin-T (IL-T), a lymphokine secreted by a human T-cell lymphotrophic virus-1 (HTLV-1)-associated leukemia cell line, stimulates T cells and lymphokine-activated cells. This was subsequently proven to be identical to IL-15. IL-15 is a 14–15 kDa protein of 114 amino acids (AAs), and belongs to the four a-helix bundle cytokine family, which includes IL-2, IL-3, IL-6, IL-7, IL-21, granulocyte-colony stimulating

386 INTERLEUKINS / IL-15

factor (G-CSF) and granulocyte-macrophage colonystimulating factor (GM-CSF). IL-15 mRNA is widely expressed in monocytes, macrophages, bone marrow stroma, and thymic epithelium, as well as by epithelial cells in the kidney, skin, and intestines. IL-15 binds to a receptor complex that consists of its own IL-15Ra, IL-2Rb and the gc chain. IL-2 and IL-15 share the same beta and gamma subunits, both of which are common subunits for signal transduction. IL-15 mediates functions similar to IL-2 in vitro. However, the IL-15Ra shows broader tissue distribution compared to IL-2Ra. Studies using mice with targeted disruptions of the IL-15 and IL-15Ra genes has demonstrated that IL-15 and IL-2 play different functions in vivo.

Structure The IL-15 gene is located on human chromosome 4q31 and the central region of mouse chromosome 8. The genomic structures of human and murine IL-15 are similar, with a length of 34 kb or more, and contain nine exons (7 coding exons). IL-15 is highly conserved between the two species (97% identity comparing human and simian, 73% identity comparing human with murine). The mature IL-15

protein consists of 114 AAs with the molecular weight of 14–15 kDa, and is encoded by exons 5–8 (Figure 1). IL-15 protein contains two disulfide bonds at positions C42–C88 that are identical to IL-2 at C35– C85. There are two N-linked glycosylation sites at the C-terminus of the IL-15 protein, at N79 and N112. The nucleotide and protein sequences of IL-15 show little homology with IL-2. However, the intron/ exon structure (4 exons and 3 introns) is similar to that of the IL-2 gene and that of other four a-helix bundle cytokines. Therefore, the mature gene and protein structure of all members of the four a-helix bundle cytokine family appear similar. The originally identified human IL-15, long-signaling peptide (LSP) IL-15, contains a 50 untranslated region (UTR) of at least 316 bp, and a 486 bp open reading frame; and a 30 UTR of at least 400 bp, encoding a precursor IL-15 with an unusually long 48 AA leader peptide and a 114 AA mature protein. Recently, an additional sequence, exon 4A, has been found between exons 4 and 5 that encodes an alternative, shorter leader peptide (Figure 1). SSP-IL-15 lacks exons 1 and 2 of the original cDNA, and instead contains 119 nucleotides (nt) within intron 4 resulting in a smaller spliced mRNA product (Figure 1). This

Human IL-15 gene Exon (bp) 91 125 Intron (kp) ? 1

2

99/11 >10

0.59

3

45

98 119 35/51 0.49

4

0.75

4A

5.8

LSP-IL-15 classical 5′ UTR LSP IL-15

5′ UTR

3′ UTR

8

7

SSP-IL-15 alternative

IL-15 mRNA isoforms

1 2 3 4 5 6 7

2.8

6

5

112/397

137 1.9

SSP IL-15

3′ UTR

In2 3 4 4A 5 6 7

8

8

IL-15 Precursor proteins LSP (48AA) N

N

C 3,4,5,

SSP (21AA) C N 4A,5

IL-15 (114AA) 3,4

5,6,7,8

IL-15 (114AA) C 5,6,7,8

IL-15 Mature protein C

N 5,6,7,8

Figure 1 Human IL-15 gene, mRNA, and protein structure. The human IL-15 locus consists of nine exons and eight introns and is located on chromosome 4q31. Two IL-15 mRNA isoforms have been described, the classical LSP and alternative SSP, with both encoding an identical IL-15 mature protein of 114 AAs (see text for details). Translational start sites (3) and stopcodons (F) are indicated; In2 indicates intron 2. Reproduced from Fehniger TA and Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood 2001; 97: 14–32. Copyright American Society of Hematology, used with permission.

INTERLEUKINS / IL-15 387

exonic sequence, intron 4A, encodes three premature stopcodons and a novel downstream ATG translation start site. As a consequence, the translated product contains a 21 AA short-signal peptide. Thus, there are two isoforms of IL-15: LSP form (48 AAs) that is secreted from the cell, and a shortsignaling peptide (SSP) form (21 AAs) that remains intracellular and localized to nonendoplasmic regions in both cytoplasmic and nuclear compartments (see below). Although the signal sequences are different, both IL-15 isoforms encode an identical mature IL-15 protein in human and mouse.

Regulation of Production and Activity The two IL-15 mRNA isoforms contain significant variance in their leader peptides (Figure 1). These differences in leader peptides are thought to determine the intracellular localization and potential secretion of the associated mature protein. LSP IL-15 is targeted to the secretory pathway (endoplasmic reticulum (ER)/ Golgi apparatus), whereas SSP IL-15 appears to be restricted to the cytoplasm and nucleus. The distribution of IL-15 mRNA is different between LSP IL-15 and SSP IL-15. LSP IL-15 is shown to be expressed in the heart, thymus, lung, liver, kidney, skeletal muscle, and placenta. On the other hand, SSP IL-15 mRNA is detected in the heart, thymus, appendix, and testis. The biological significance of this differential expression is still unclear.

Regulation of IL-15 Gene Expression IL-15 transcription, translation, and secretion are shown to be regulated through multiple complex mechanisms and the most important stage of IL-15 regulation appears to be posttranscriptional.

identified at
348 to
336 relative to the cap site of the murine IL-15 promoter. The observation that IRF-1
/
mice lack inducible IL-15 expression and natural killer (NK) cells suggests that IRF-induced IL-15 expression is essential for the NK cell development in the bone marrow. Translation, Intracellular Trafficking, and Secretion

Multiple startcodons (AUGs) in the 50 UTR, the unusual LSP and SSP, and a negative regulator near the C-terminus of the precursor proteins, are shown to regulate IL-15 mRNA translation into the IL-15 precursor protein. LSP IL-15 was shown to have a lower translational efficiency than SSP IL-15. The LSP–IL-15 50 UTR is relatively long (more than 316 nt in humans) and contains multiple (12 in humans) AUGs upstream of the actual translation start site. These multiple AUGs have been shown to reduce translational efficiency. The expression of SSP IL-15 was shown to be localized to the cytoplasm and nucleus, whereas LSP IL-15 was within the ER/Golgi apparatus. Thus, SSP IL-15 seems to be translated efficiently but remains intracellular, whereas the translation of LSP IL-15 is less efficient but the protein is transported to the ER/ Golgi pathway and is secreted from the cell at low levels. It is suggested that a signal in the carboxyl portion of the mature IL-15 protein may act as a retention signal, resulting in low secretion. Transgenic mice that overexpress IL-15, as a result of elimination of these posttranscriptional regulations, developed early expansions in NK and CD8 þ T cells, followed by a fatal lymphocytic leukemia, suggesting that the tight regulation of IL-15 is critical for the homeostasis of immune system.

Control of Transcription

Biological Function

Mouse and human IL-15 promoter regions contain transcription factor-binding motifs including a–INF-2, NF–IL-6, g-IRE, myb, Gc factor (GCF), and NF-kB. The NF-kB site, located at
75 to
65 relative to the transcription start site of the human IL-15 promoter, was shown to be important for HTLV-1 tax protein-induced IL-15 mRNA upregulation and for lipopolysaccharide (LPS)-induced IL-15 gene expression in murine macrophages. The 50 deletion of region
201 and
141 in the human IL-15 promoter region was shown to result in a dramatic increase in IL-15 promoter activity, suggesting that this region contains an unidentified site responsible for negative regulation of IL-15 expression. An essential interferon regulatory factor (IRF)-E consensus-binding site was

IL-15 shares a number of biological activities with IL-2, including stimulation of the proliferation of activated CD4 þ , CD8 þ , as well as the gd subset of T cells. However, in general, IL-15 has antiapoptotic actions to activated T cells and inhibits IL-2-mediated activation-induced cell death. IL-15 stimulates proliferation and persistence of memory CD8 T cells whereas IL-2 inhibits these functions. IL-15 also stimulates the proliferation of NK cells and acts as a co-stimulator with IL-12 to facilitate the production of interferon gamma (IFN-g) and tumor necrosis factor-a by these cells. The coexistence of IL-15 and IL-12 is also shown to increase the IFN-g production from T cells. However, IL-15 cannot substitute for IL-2 in the differentiation of

388 INTERLEUKINS / IL-15

CD4 þ T cells into a Th2 type phenotype in the presence of IL-4, suggesting that IL-15 has the potential to skew the immunological response in favor of Th1 rather than a Th2 response. On the other hand, IL-15 promotes the production of IL-5 in human T helper cells, and stimulates and induces proliferation of mast cells through a distinct, unidentified receptor. IL-15 has been shown to induce the production of IL-8 in human neutrophils and to stimulate the induction of cytolytic effector cells such as cytotoxic T cells and lymphokineactivated killer cells. IL-15 also acts as a potent T-cell chemoattractant. Thus, IL-15 seems to play an important role in innate and acquired immunity. Human monocyte/macrophage cell lines and primary human monocytes have been shown to express constitutively bioactive IL-15 protein on their cell-surface membrane. IL-15 protein is also detected in human peripheral blood mononuclear cells by immunohistochemistry, Western analysis, and flow cytometry. These IL-15 membrane expressions are shown to be upregulated by stimulation with IFN-g, suggesting that IL-15 could exert a biological effect while being undetectable in culture supernatant. Further study should elucidate the functional significance of constitutive cell-surface expression of IL-15. Furthermore, IL-15 induces the proliferation and synthesis of immunoglobulin by human tonsillar B cells activated by CD40 ligand or an immobilized antibody to IgM. IL-15 has been shown to take effect on nonimmune cells. For example, skeletal muscle cells express IL-15 mRNA and its receptor, and IL-15 induces skeletal muscle fiber hypertrophy. Other nonimmunological systems such as the kidney, lung, liver, epithelial cell lines, and activated endothelial cells are also shown to express mRNAs for IL-15 and its receptor.

Receptor and Signal Transduction Like the IL-2 receptor, the IL-15 receptor is also composed of three subunits: a, b, and g (Figure 2). The b and g subunits of the IL-15 receptor are shared with IL-2. In contrast to the restricted expression of IL-2Ra to subsets of lymphocytes, IL-15Ra expression is far more diverse. The expression of IL-15Ra has been reported in lymphoid and myeloid cells as well as nonhematopoietic cells: CD8C T cells (naı¨ve and activated), NK, NKT and g/d T cells, monocytes, macrophages, dendritic cells, neutrophils, endothelial cells, kidney epithelial cells, colonic epithelial cells, brain microglial cells, and skeletal muscle cells. In addition, both receptors are inducible and a recent study indicates that both IL-2 and IL-15 increase

IL-2Ra mRNA and protein expression, while IL-15 selectively causes IL-2Ra expression on the cell surface. Thus, different regulation of cell-surface expression of IL-2Ra and IL-15Ra may contribute to the distinct functions of IL-2Ra and IL-15Ra in vivo. Although isolated IL-15Ra has high affinity for IL15 (KdB10 pM), IL-15Ra alone has been reported to exert no signal transduction. However, recent evidence suggests that IL-15 may indeed mediate certain intracellular signals. IL-15Ra has been shown to interact with tumor necrosis factor receptorassociated factor 2 (TRAF2) and further investigation is necessary to clarify the biological significance of IL-15Ra-mediated signals. So far, only IL-2 and IL-15 have been shown to utilize the IL-2/15Rb. The IL-2Rg, also referred to as the common gamma chain (gc), is shared by receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. IL-15Ra, IL-2/15Rb, and gc constitute a complete heterotrimeric high-affinity complex which exerts signals through IL-2/15Rb and gc, identical to the IL2R after the ligand binding. In the absence of IL-15Ra, the heterodimeric IL-2/15Rbgc can bind to IL-15 with intermediate affinity (KdB1 nM) with signal transduction. It has been shown that mast cells respond to IL-15 with unique high-affinity binding receptor (60– 65 kDa, IL-15RX) that does not include the identified IL-2/15Rb, gc, or IL-15Ra, and transduces different intracellular signals compared to IL-15Rabgc. As IL-2 and IL-15 share common signaling components (IL-2/15Rbgc), the interaction of IL-2 or IL-15 with their respective receptors leads to similar signaling events such as the activation of the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathway. IL-2/15Rb is associated with Jak1 and the gc is associated with Jak3, resulting in STAT3 and STAT5 phosphorylation respectively, following ligation with IL-15 (Figure 2). The stimulation of IL-2/15R complexes are also shown to activate the src-related tyrosine kinases, induce Bcl-2, and stimulate the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway that ultimately results in fos/jun activation. The IL-2/15Rb also activates the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway.

IL-15 in Respiratory Diseases It has been shown that alveolar macrophages isolated from bronchoalveolar lavage (BAL) of patients with active sarcoidosis and tuberculosis produce increased levels of IL-15 compared to normal subjects. BAL cells from individuals with granulomatous sarcoid lesions show a marked increase in the numbers of CD4 þ lymphocytes, which have been shown to

INTERLEUKINS / IL-15 389

IL-15

Kd 10–11 M

IL-2

(a)

Kd 10–11 M

Kd 10–8 M

Intermediate affinity IL-2R or IL-15R

S A

(b)

Kd 10–9 M

Kd 10–11 M

Jak1

?

No signal

Jak3

P (Syk) (Src) Shc STAT3 Grb2/SOS STAT5a/b Ras Raf PI-3K MAPK Akt

Figure 2 (a) Schematic figure of the IL-15 and IL-2 receptor complexes. The high-affinity, heterotrimeric IL-2 receptor is composed of the IL-2a, IL-2/15Rb and gc, while the intermediate-affinity receptor is made up of the heterodimer of IL-2/15Rb and gc. The isolated IL-2Ra binds IL-2 with low-affinity. The high-affinity heterotrimeric IL-15 receptor is comprised of IL-15Ra, IL-2/15Rb and gc, and similar to IL-2, IL-15 binds to the heterodimeric IL-2/15Rbgc with intermediate-affinity. In contrast to IL-2Ra, the isolated IL-15Ra alone has high-affinity for IL-15. Blue ovals represent conserved cysteine bonds, while the WXSWS motif shared between hematopoietin receptor members is depicted as red ovals. (b) Signaling cascade from the IL-15 and IL-2 receptor complexes. Signals from the IL-2/15Rb and gc are shared between the IL-15 and IL-2 receptor complexes through the signaling pathways shown. Recent studies have shown that the IL-15Ra cytoplasmic tail binds to TRAF2, preventing its interaction with the pro-apoptotic cascade members and may increase Bcl2. IL-2Ra does not mediate any known signals. The serine-rich (S), acidic (A), and proline-rich (P) portions of the IL-2/15Rb cytoplasmic domain are indicated, as well as tyrosine residues (Y). Reproduced from Fehniger TA, Cooper MA, and Caligiuri MA (2002) Interleukin-2 and interleukin-15: immunotherapy for cancer. Cytokine & Growth Factor Reviews 13: 169–183, with permission from Elsevier.

390 INTERLEUKINS / IL-16

Mph/DC

IL-12

Memory CD8+ T cell

IL-15

NKT cell

Persistence

IFN-


Th1 response ?

NK cell

Sarcoidosis tuberculosis

Figure 3 Possible role in the pathogenesis of respiratory diseases. IL-15 stimulates proliferation of NK cells, NKT cells, and memoryphenotype CD8 þ T cells in concert with IL-12. Memory-phenotype CD8 þ T cells are maintained by IL-15. These cells produce IFN-g, which leads to Th1 response seen in respiratory diseases such as sarcoidosis and tuberculosis.

proliferate in response to IL-15. Therefore, IL-15 may contribute to the development of T-cell alveolitis in this disease. Its expression is also increased in bronchial biopsies from patients with sarcoidosis and tuberculosis patients and, to a lesser extent in patients with chronic bronchitis, compared to asthmatics and normal control subjects. Thus, it is suggested that IL-15 is involved in Th1-dominant inflammation in respiratory diseases. (Figure 3). To date, administration of IL-15 has not been used in clinical trials for disease therapy. In mice, IL-15 administration has been shown to enhance T- and NK-cell function resulting in increased antitumor activity in mice, and IL-15 was less toxic than IL-2. Thus IL-15 is one of the candidates for antitumor immunotherapy. IL-15 is shown to be involved in the chronic inflammation in rheumatoid arthritis and clinical intervention studies on IL-15 blockade in inflammatory synovitis are ongoing. See also: Interferons. Leukocytes: T cells. Signal Transduction. Systemic Disease: Sarcoidosis.

Further Reading Agostini C, Trentin L, Facco M, et al. (1996) Role of IL-15, IL-2, and their receptors in the development of T cell alveolitis in pulmonary sarcoidosis. Journal of Immunology 157: 910–918. Alpdogan O and van den Brink MR (2005) IL-7 and IL-15: therapeutic cytokines for immunodeficiency. Trends in Immunology 26: 56–64. Fehniger TA and Caligiuri MA (2001) Interleukin 15: biology and relevance to human disease. Blood 97: 14–32. Fehniger TA, Cooper MA, and Caligiuri MA (2002) Interleukin-2 and interleukin-15: immunotherapy for cancer. Cytokine & Growth Factor Reviews 13: 169–183. Giri JG, Kumaki S, Ahdieh M, et al. (1995) Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor. EMBO Journal 114: 3654–3663. Muro S, Taha R, Tsicopoulos A, et al. (2001) Expression of IL-15 in inflammatory pulmonary diseases. Journal of Allergy and Clinical Immunology 108: 970–975.

Musso T, Calosso L, Zucca M, et al. (1999) Human monocytes constitutively express membranebound, biologically active, and interferon-gamma-upregulated interleukin-15. Blood 93: 3531–3539. Waldmann TA, Dubois S, and Tagaya Y (2001) Contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for immunotherapy. Immunity 14: 105–110. Waldmann TA and Tagaya Y (1999) The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens. Annual Review of Immunology 17: 19–49.

IL-16 F F Little, W W Cruikshank, and D M Center, Pulmonary Center, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin-16 (IL-16) is a pleotropic cytokine that is produced by immune cells and epithelial cells. The secreted C-terminal product of caspase-3 cleavage is a PDZ-domain-containing protein whose terminal 16 amino acids bind the D4 domain of CD4, its major receptor. IL-16 elicits monocyte, eosinophil, and T-cell chemoattraction in vitro that is preferential for TH1 cells, yet also anergizes T cells resulting in downregulation of antigen recognition and T-cell activation. In addition, IL-16 has direct effects on the generation of tolerogenic dentritic cells and is a cofactor in mast cell development. The dominant T-cell immunomodulatory effect in vivo appears to be downregulation of TH2-mediated allergic inflammation.

Introduction Interleukin-16 (IL-16) was originally described as a T-cell chemoattractant (lymphocyte chemoattractant factor (LCF)) found in the supernatants of mitogen-stimulated peripheral blood mononuclear cells (PBMCs). IL-16 is synthesized by varied immune and

INTERLEUKINS / IL-16 391

parenchymal cells, including CD4 and CD8 T cells, eosinophils, macrophage/dendritic cells, mast cells, bronchial epithelium, and fibroblasts. IL-16 has two major in vitro effects on CD4 þ T cells: chemoattraction, preferentially of TH1 cells, and inhibition of CD3/T-cell receptor mediated activation, preferentially of TH2 cells. The binding site of IL-16 on CD4 maps to the sequence in the D4 domain of CD4 that is responsible for autodimerization, supporting a molecular explanation for the interference of IL-16 on CD4 þ T-cell activation seen in vitro. As the most studied and primary receptor for IL-16 is CD4, its therapeutic potential in HIV infection and allergic lung disease has been investigated in detail.

IL-16 Gene Structure, Expression, and Protein Product The IL-16 gene is a single copy gene located on human chromosome 15.26.1–3, and on chromosome 7 D2–D3 in the mouse. The entire gene stretches over 20 kb and encodes for two forms of the precursor molecule, synthesized by alternative splicing. Neuronal 1322 and 1332 amino acid precursor forms (in mouse and human, respectively) are expressed exclusively in the brain and have an unknown number of exons. The non-neuronal (‘leukocyte’) form of the human IL-16 gene contains seven exons, of which the first is noncoding, and results in a 2.6 kb mRNA species. The promoter is TATA-less, yet it contains two CAAT box-like motifs and three GA-binding protein transcription factor binding site consensus sequences which are functional in T cells, one of which lies in the 50 untranslated region (UTR). Leukocyte IL-16 is a 631 (human) and 624 (mouse) amino acid protein whose major biologic function relevant to pulmonary inflammation resides in the 121/118 amino acid C-terminal, secreted end of the molecule. The N-terminal product of cleavage (by caspase-3, see next section) is a PSD-95, dlg, Z01 homology (PDZ) domain-containing protein which localizes to the nucleus (Figure 1). In addition to two PDZ domains, this B60 kDa N-terminal product of caspase-3 cleavage has a dual phosphorylationregulated nuclear localization sequence permitting nuclear translocation, where it plays a role in regulating the levels of p27KIP1 and G0/G1 arrest of T cells. The structure of the secreted C-terminal inteleukin, IL-16, is unusual as it is a functional extracellular protein that contains a PDZ domain. There is low homology between the originally described PDZ proteins and IL-16; NMR spectroscopy has revealed that the core GLGF region in IL-16 is partially occluded by a tryptophan residue. Yeast-2

D/S

CK2 cdc2 NLS PDZ

PDZ

N-terminal ‘Pro-IL-16’

PDZ RRKS... C-terminal: ‘secreted’

Figure 1 Protein structure of IL-16. Leukocyte IL-16 is synthesized as a ca. 80 kd precursor which is cleaved at Asp510-Ser511 by caspase-3, releasing the C-terminal peptide whose mechanism of secretion is unknown. This protein has a PDZ domain in the proximal end and the RRKS (RRTS in the mouse) CD4-binding site in the distal 16 amino acids. Also depicted is the dual phosphorylation-regulated nuclear localization sequence (CcN motif) of pro-IL-16, which includes a casein-kinase 2 substrate site, cdc2 kinase substrate site, and bipartite nuclear localization sequence (NLS).

hybrid experiments using lymphocyte and splenocyte cDNA libraries have not identified an intracellular binding partner for this PDZ domain. While its mechanism is unclear, autoaggregation of IL-16 monomers is required for bioactivity and occurs intracellularly before secretion. Contained within the distal 16 amino acids is a core 4 amino acid sequence (arg-arg-lys-ser (RRKS) in humans, arg-arg-thr-ser (RRTS) in the mouse) which is required for CD4binding ability and consequent T-cell chemotactic and immunomodulatory properties. There is a high degree of functional and sequence homology of IL-16 from all species (e.g., human, simian, and murine). Both murine and simian IL-16 are chemotactic to human T cells, and monoclonal antibodies generated to secreted human IL-16 can be used to neutralize the bioactivity as well as to affinity purify murine IL-16. The conservation across species of protein structure and biologic actions of IL-16 implies a conserved evolutionary function and bears on the application of animal models to understanding IL-16’s biologic effects in humans.

Regulation and Cellular Synthesis of IL-16 While the transcriptional regulation of IL-16 has not been fully characterized, the nature of IL-16 message production in different cell types has been examined in detail. CD4 þ and CD8 þ T cells, eosinophils, mast cells, and dendritic cells constitutively generate IL-16 mRNA. By contrast, nonimmune cells such as airway epithelial cells and fibroblasts must be induced to transcribe IL-16 message. Full-length leukocyte IL-16 is synthesized as a 631 amino acid precursor that is cleaved into two fragments by caspase-3. This occurs readily without other associated apoptotic events. With the notable exception of CD8 þ T cells, caspase-3 activation is a major regulator of IL-16 release in all cells that constitutively express mRNA and protein. CD8 þ T cells express constitutively active caspase-3, and store cleaved

392 INTERLEUKINS / IL-16

bioactive IL-16 that is released within hours following stimulation by histamine or serotonin via H2 or S2 receptors. While only one functional caspase-3 cleavage site has been definitively identified in ProIL-16 from T cells, further proteolytic processing has been suggested in dendritic cells. It is likely that the stimuli responsible for IL-16 release in non-T-cells, such as fibroblasts, result in new transcription, translation, and posttranslational cleavage by activated caspase-3. The mechanism of secretion has not been identified.

IL-16 Receptor: Relationship between IL-16 and CD4 The ability of IL-16 to function as a chemotactic stimulus for CD4 þ T cells led to an investigation of the interaction between IL-16 and cell surface CD4. All cells with a well-described effector response to IL16 express surface CD4, including T lymphocytes, eosinophils, dendritic cells, and neuronal cells. IL-16 has a distinct binding site on CD4 from other CD4 ligands, including HIV gp120 and major histocompatibility class II antigens. T-cell migration to IL-16 can be inhibited by coincubation with monomeric Fab fragments directed against CD4, and murine Tcell hybridomas transfected with CD4 gain migratory function to IL-16 which is eliminated by various mutations of CD4. Similarly, CD4 expression on CD8 þ T cells following anti-CD3/anti-CD28 costimulation confers IL-16 responsiveness, which is blocked by antibodies to CD4. While it appears that alternate receptors for IL-16 exist on monocytes and dendritic cells, the evidence for a predominant IL-16-CD4 ligand–receptor relationship in T cells is compelling. Following ligation of CD4 by IL-16, activation of the CD4-associated src-related tyrosine kinase p56lck occurs followed by rises in intracellular Ca2 þ , IP3, and activation of PI-3 kinase. This first response is associated with autocatalytic activity of p56lck with tyrosine phosphorylation and translocation of protein kinase-C to the membrane. While the catalytic activity of p56lck is not necessary to mediate T-cell chemoattraction, an adaptor function of p56lck coupling to PI-3 Kinase via the SH3 domain of p56lck is required. Point mutations in key intracytoplasmic regions of CD4 necessary for CD4
p56lck association abrogate the T-cell chemotactic response to IL16. Cells expressing these mutations retain migratory responses to other chemoattractants. The chemotactic response also appears independent of the rise in Ca2 þ and IP3 turnover. It is likely that these two latter signals and the enzymatic activity of p56lck are required for IL-16-induced expression of CD25; however, this has not been proved. These findings

imply that there are multiple CD4-associated signals induced by IL-16 that lead separately to lymphocyte migration and selective gene expression. The precise binding sites on CD4 for IL-16 have been mapped by peptide inhibition and CD4 mutagenesis studies, and are comprised of two six amino acid stretches that form a groove in the tertiary structure of the D4 region. This sequence of the D4 domain of CD4, closest to the cell membrane, is responsible for autoaggregation that facilitates activation of the TCR/CD3 antigen recognition complex. The presence of CCR5, expressed primarily on TH1 cells, appears to contribute to IL-16 binding and likely accounts for the preferential migratory response of these cells. The mechanism by which CCR5 increases IL-16 binding to CD4 has not been identified as yet.

Biologic Effects of IL-16 This section briefly discusses the in vitro and in vivo effects of IL-16 (Figure 2). Chemotactic Activity of IL-16

CD4-mediated chemoattraction A variety of target cells for IL-16 stimulation have been identified. IL-16 was initially described as a chemoattractant specifically for CD4 þ T cells. Recent evidence has shown that IL-16 is preferentially chemoattractant to TH1 cells, suggesting a contributory role in the downregulation of TH2-cell-mediated lung disease such as allergic asthma. IL-16 is also a potent chemoattractant for all peripheral immune cells expressing CD4, including CD4 þ monocytes, eosinophils, and dendritic cells. In vitro studies have indicated that for lymphocytes and eosinophils, the ED50 (half maximal effective dose) for recombinant IL-16 is 10–11 M, which is consistent with other reported chemoattractants such as regulated on activation, normal T-cell expressed and secreted (RANTES); (CCL5) and monocyte chemoattractant protein-1 (MCP-1); (CCL2). Indirect effects of IL-16 on chemoattraction via chemokine receptors Indications that the CD4/C-C chemokine receptor 5 (CCR5) receptor complex is more than a docking site for human immunodeficiency-1 (HIV-1) binding and internalization were suggested by the observation that HIV-1 gp120 activates intracellular signaling of both CD4 and CCR5. This concept was furthered by the observation that ligation of either CD4 or CCR5 by their natural ligands results in reciprocal receptor cross-desensitization. IL-16 treatment of CD4 þ T cells results in transient inhibition of macrophage

INTERLEUKINS / IL-16 393 Lung

Anergy TH1 cell influx ? Treg activity Chemokine receptor desensitization

Epithelium ?Chemoattraction CD4+ and CD8+ T cells

IL-16 DC tolerization

Eosinophil

Expansion

Mast cell

Bone marrow Expansion of pre-B cells ?

IL-16 Progenitor cell

Expansion

Figure 2 Major cellular sources and biologic effects of IL-16 in the lung and bone marrow (refer to text for details). In the lung, there are varied sources of IL-16, some of which require stimulation to synthesize and secrete IL-16 (epithelial cells), while others constitutively synthesize IL-16 (T lymphocytes, eosinophils, mast cells). There are consequent direct effects on T cells and eosinophils as outlined. The effects on dendritic cells and mast cells have yet to be explicitly demonstrated. The source of IL-16 in the bone marrow is unknown, but stimulates CD4 þ progenitor cells to expand in number and percentage of pre-B cells and markedly increase the production of chymase and tryptase in mast cells.

inflammatory protein 1b (CCL4) induced chemoattraction, indicating that an IL-16–CD4 association desensitizes CCR5 to stimulation by a natural CCR5 ligand. Reciprocal receptor cross-desensitization is also observed: CCL4 treatment of the same cells prevents IL-16-induced migration. This autoregulatory effect is not due to steric inhibition nor changes in surface receptor expression; CCR5 desensitization by IL-16 requires intact signal transduction by CD4. Both of these effects are likely to be relevant to the finding that rIL-16 inhibits HIV-1 replication, but not infectivity, in peripheral blood mononuclear cells. In addition, ligation of CCR5 can induce phosphorylation of CD4-associated p56lck. Similarly, the presence of CCR5 contributes not only to IL-16/CD4 binding, but to CD4 signaling as well. T cells from CCR5
/
mice have a markedly diminished migratory response to IL-16. CCR5 signaling likely contributes to this response as pertussis toxin treatment of CCR5 þ cells reduces the migratory response to IL-16 to levels noted in CCR5
/
cells. The previously noted preferential migration of TH1 cells to IL-16 noted in these experiments has direct implications on the role of IL-16 in inflammatory lung disease; the enhanced migration of TH1 cells over TH2 cells is not due to differences in surface CD4 expression, but due to the co-receptor function of

CCR5. The receptor cross-desensitization phenomenon is not unique to CCR5. IL-16/CD4 signaling also desensitizes C-X-C chemokine receptor 4 (CXCR4) response to stimulation with its ligand stromal derived factor 1a (CXCL12). IL-16/CD4 signaling has no effect on migration induced by CCL2/CCR2b, eotaxin (CCL11)/CCR3, or macrophage-derived chemokine (CCL22)/CCR4. Modulation of the Primary Immune Response

Downregulation of antigen-mediated T-cell activation The location of IL-16’s binding site in the D4 region of CD4 bears directly on its ability to inhibit aspects of CD3-T-cell receptor (TCR)-mediated Tcell activation by antigen or that induced by a mixed lymphocyte reaction (MLR) (Figure 3). In both humans and mice, 1 h rIL-16 pretreatment of responder cells inhibits the MLR by 50%; this inhibition is reversible by cotreatment with either anti-IL-16 antibody or recombinant soluble CD4. These findings are consistent with the report that activation of T-cells by antigen or anti-CD3 is inhibited by IL-16 pretreatment. Furthermore, treatment of antigen-specific murine T cells with IL-16 during antigen stimulation reduces production of the TH2 cytokines IL-4 and IL-5 without affecting release of interferon gamma (IFN-g) or IL-10. It is noteworthy that the T-cell

394 INTERLEUKINS / IL-16

D1

D1

DC

DC

D2 D3

D3





IL-16 D4

Ag-MHC II

Ag-MHC II

D2 CD4

D4

  p56lck

p56lck phosphorylation CD25 induction TH1-cell chemoattraction

CD4 (autodimerized)


 

  

TCR/CD3


 

TCR/CD3

Antigen-driven activation TH2 cytokine production Augmented allergic response

CD4 (undimerized)

IL-16

p56lck

Alteration of CD4-TCR/CD3 interaction Anergy Downregulation of allergic response

Figure 3 Putative mechanism of IL-16-mediated immunomodulation in CD4 þ T-lymphocyte activation. In the absence of antigen, IL-16 oligomers bind CD4 in the D4 region causing a complex steric relationship that leads to coupling and autophosphorylation of p56lck (see text (‘IL-16 receptor’) for details), with subsequent induction of CD25 and preferential TH1-cell chemotaxis (left). In the presence of antigen/MHC II (center), TCR/CD3-mediated cell activation is facilitated by dimerized CD4 of a different steric conformation than that bound by IL-16, leading to antigen-driven proliferation and cytokine production that augments the allergic response. In the presence of IL-16 (right), CD4 dimerization and facilitation of TCR/CD3 activation by antigen/MHC II is altered, causing anergy and modulation of antigen-driven responses.

chemoattractant and immunomodulatory effects of IL-16 occur at equimolar concentrations. Thus, as a consequence of being bound by IL-16, CD4 can function to transduce signals sufficient to induce Tcell chemotaxis and expression of IL-2Ra and b while CD4’s facilitation of antigen recognition function is inhibited. Altered phenotype of T cells and dendritic cells IL16 treatment of T cells increases surface expression of human leukocyte antigen-DR (HLA-DR), suggesting that IL-16 contributes to facilitation of antigen recognition by T cells. Four-week culture of T cells in the presence of IL-2 demonstrates increased surface expression of surface CD25, CD122, and cell proliferation, providing a competence growth function for IL-16. While often a marker of activated T cells, the increased surface expression of CD25 attributable to IL-16 could reflect induction of a regulatory phenotype, though this hypothesis has not been directly tested. This concept is supported by the finding that a tolerogenic dendritic cell (DC) phenotype is induced by culturing CD34 þ hematopoetic progenitor cells with IL-16 in addition to standard DC generating factors. These DCs tolerize cocultured T cells in a manner that persists upon restimulation with cognate immunogenic DCs. This may have a human in vivo correlate in that monocyte-derived dendritic cells from normal individuals store and secrete IL-16, while similarly derived cells

from individuals with atopic dermatitis have impaired synthesis and secretion of IL-16. Cofactor in Hematopoetic Cell Development into Pre-B and Mast Cells

The reversal of declining levels of recombinationactivating gene (RAG) mRNA hematopoetic cells in old and nude mice by supernatants of activated T cells is attributable to the presence of IL-16 as the major factor. IL-16 not only increased RAG mRNA but also increased the numbers and percentages of bone marrow pre-B cells. Related developmental effects have been observed in umbilical cord and peripheral blood cell expansion into mast cells. After 3 weeks of culture in the presence of c-kit ligand, IL-16 induced a 20-fold increase in tryptase and chymase in CD4 þ hematopoetic progenitor-derived mast cells from normal individuals. Along with its role as a competence growth factor for T cells noted earlier, these experiments demonstrate that the IL-16/CD4 interaction is relevant in hematopoetic progenitor cells of varied lineages as it is in mature T cells. Role of IL-16 in Lung Diseases

The role of IL-16 in lung disease is incompletely defined. Its role as a putative modulator of the allergic immune response in the lung is supported by its attenuation of airway reactivity and eosinophilia in a mouse model of allergy airway inflammation. In humans and mice, IL-16 is readily detected in allergic

INTERLEUKINS / IL-17 395

bronchial epithelium and in the bronchoalveolar lavage after allergen or histamine challenge. In concert with well described in vitro immunomodulation of Tcell activation by antigen, it is postulated that IL-16 acts as a natural regulator of T-cell-mediated inflammation by its direct interaction with CD4. See also: Allergy: Overview. Asthma: Overview. Chemokines. Chemokines, CC: RANTES (CCL5). Chymase and Tryptase. Dendritic Cells. Interleukins: IL-4; IL-5; IL-9. Leukocytes: T cells. Macrophage Inflammatory Protein. Stem Cells.

Further Reading Center DM, Kornfeld H, Ryan TC, and Cruikshank WW (2000) Interleukin 16: implications for CD4 functions and HIV-1 progression. Immunology Today 21(6): 273–280. Cruikshank WW, Center DM, Nisar N, et al. (1994) Molecular and functional analysis of a lymphocyte chemoattractant factor: association of biologic function with CD4 expression. Proceedings of the National Academy of Sciences, USA 91(11): 5109– 5113. Cruikshank WW, Lim K, Theodore AC, et al. (1996) IL-16 inhibition of CD3-dependent lymphocyte activation and proliferation. Journal of Immunology 157(12): 5240–5248. de Bie JJ, Jonker EH, Henricks PA, et al. (2002) Exogenous interleukin-16 inhibits antigen-induced airway hyper-reactivity, eosinophilia and Th2-type cytokine production in mice. Clinical and Experimental Allergy 32(11): 1651–1658. Kornfeld H and Cruikshank WW (2001) Prospects for IL-16 in the treatment of AIDS. Expert Opinion on Biological Therapy 1(3): 425–432. Kurschner C and Yuzaki M (1999) Neuronal interleukin-16 (NIL16): a dual function PDZ domain protein. Journal of Neuroscience 19(18): 7770–7780. Little FF and Cruikshank WW (2004) Interleukin-16 and peptide derivatives as immunomodulatory therapy in allergic lung disease. Expert Opinion on Biological Therapy 4(6): 837–846. Liu Y, Cruikshank WW, O’Loughlin T, et al. (1999) Identification of a CD4 domain required for interleukin-16 binding and lymphocyte activation. Journal of Biological Chemistry 274(33): 23387–23395. Lynch EA, Heijens CA, Horst NF, Center DM, and Cruikshank WW (2003) Cutting edge: IL-16/CD4 preferentially induces Th1 cell migration: requirement of CCR5. Journal of Immunology 171(10): 4965–4968. Mashikian MV, Ryan TC, Seman A, et al. (1999) Reciprocal desensitization of CCR5 and CD4 is mediated by IL-16 and macrophage-inflammatory protein-1 beta, respectively. Journal of Immunology 163(6): 3123–3130. Muhlhahn P, Zweckstetter M, Georgescu J, et al. (1998) Structure of interleukin 16 resembles a PDZ domain with an occluded peptide binding site. Nature Structural Biology 5(8): 682–686. Nicoll J, Cruikshank WW, Brazer W, et al. (1999) Identification of domains in IL-16 critical for biological activity. Journal of Immunology 163(4): 1827–1832. Rand TH, Cruikshank WW, Center DM, and Weller PF (1991) CD4-mediated stimulation of human eosinophils: lymphocyte chemoattractant factor and other CD4-binding ligands elicit eosinophil migration. Journal of Experimental Medicine 173(6): 1521–1528.

IL-17 Q Hamid, S Lajoie-Kadoch, S Le´tuve´, and Z Mu¨ller, McGill University, Montreal, QC, Canada & 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin-17 (IL-17) has become the focus of many recent studies, specifically with respect to its roles in host defense and the increasing evidence supporting its involvement in lung pathology. This cytokine has a unique structure containing an atypical cystine knot and is the founding member of the IL-17 family of cytokines that shares a high degree of structural homology. IL-17’s biological effects relate to its ability to induce the release of various inflammatory mediators following binding to its receptor IL-17R. Since it is produced by activated T lymphocytes, this cytokine is thought to play a critical role at the interface between innate and adaptive immunity. The most notable property of IL-17 is its ability to promote neutrophilic inflammation via the release of specific chemoattractants and activators. Neutrophil activation by IL-17 leads to the production of cytotoxic mediators such as free oxygen radicals and elastase with resultant tissue injury. Thus, elevated concentrations of IL-17, as recorded in several inflammatory conditions of the airways, implicate this cytokine in the onset, maintenance, and progression of many respiratory diseases.

Introduction Interleukin-17 (IL-17), also referred to as IL-17A, is the prototype for a relatively novel, six-membered family of cytokines, including IL-17B, IL-17C, IL17D, IL-17E (IL-25), and IL-17F. These additional cytokines have been identified based on their sequence homology with IL-17, and appear to share proinflammatory properties among the family to an extent related to their degree of sequence similarity, as assessed by phylogenic analysis (Figure 1). Indeed, the most homologous members IL-17A and IL-17F have very similar biological effects. IL-17E is the most divergent member sequence-wise and displays a distinct, Th2, activity in vivo. Secreted by activated T lymphocytes, IL-17 is a pleiotropic proinflammatory cytokine playing a major part in the neutrophilic airway inflammation common to many respiratory disorders. In particular, IL-17 biology is strongly associated with the recruitment of neutrophils via stimulation of the release of specific cytokines and chemokines acting on these granulocytes. In line with this, IL-17 administration into the airways of mice promotes the local release of neutrophil mediators. IL-17 was cloned in 1993 using a library of murineactivated T-cell hybridoma cDNA. The resulting protein was first named cytotoxic T-lymphocyteassociated antigen (CTLA) 8 and was found to have strong sequence homology with the open reading frame 13 of the T-lymphotrophic virus Herpesvirus

396 INTERLEUKINS / IL-17 IL-17A IL-17F IL-17B IL-17D IL-17C IL-17E Figure 1 Dendogram representing the degree of sequence similarly and evolutionary relationship among members of the IL-17 family of cytokines. Analysis was performed using the Clustal W method.

1 5′

5′ 5′- UTR

3 2

3′ 4.25 kb IL-17 gene

3′

1.86 kb IL-17 mRNA

3′- UTR

468 bp ORF

155 aa IL-17 protein 155

19 1

Amino acid (aa) #

2 1

Legend Gene Exon sequences Intron sequences

Protein region

mRNA: 3′-UTR element

Signal peptide

Shaw–Kamen sequence (AUUUA)

Figure 2 IL-17 gene, mRNA, and protein structure.

saimiri. Messenger RNA encoding IL-17 was then identified in human peripheral blood T cells and its expression was seen to increase significantly upon Tcell activation. Mapped to chromosome 6p12, human IL-17 shares a 60–70% sequence homology with its rodent counterparts and has a highly unique structure when compared to all other identified interleukins.

Structure IL-17 is synthesized as a monomer of 155 amino acids, with a molecular weight ranging from 15 to 22 kDa (Figure 2). Secreted as a disulfide-linked homodimeric glycoprotein, IL-17 has a unique structure and is highly conserved throughout vertebrate evolution. The structural homology reported between rodent and human species is found most notably at sites of glycosylation and suggests a critical role for this cytokine in the mammalian immune system.

The IL-17 family of cytokines is characterized by the presence of a cystine knot conformation. This structural motif resembles the canonical cystine knot-fold formed by six cysteine residues which is typical of the growth factor superfamily. In the case of the IL-17 group of related cytokines, only four cysteines are involved in this three-dimensional (3D) structure and they have been shown to form disulfide bridges within IL-17 chains and to be involved in the ultimate formation of the dimeric cytokine.

Regulation of Production and Activity Human IL-17 mRNA is predictably unstable, as the 30 untranslated region contains many Shaw–Kamen sequences characterized by the presence of AU enriched motifs (50 -AUUUA-30 ) Figure 2. These motifs, common to several other cytokines, growth factors, and oncogenes, are characterized by the instability which they confer to mRNA. Both CD4 þ and CD8 þ

INTERLEUKINS / IL-17 397 Chemotaxis (through IL-8) NO, elastase IL-8 IL-6 GM-CSF CXCL1 CXCL6 Neutrophils

Epithelium

IL-1
TNF- IL-6 IL-10 IL-12 IL-1R COX-2 PGE2

Monocytes/ macrophages

Sources of IL-17 Eosinophil

CD4+ T cell

PMA Ionomycin CD8+ T cell

Neutrophil

IL-15 IL-23

Endothelium G-CSF IL-8 IL-6 NO

Fibroblasts IL-6 G-CSF PGE2

Figure 3 Cellular sources and targets for IL-17.

T cells, mainly of the memory phenotype, have been identified as major sources of IL-17. This cytokine is not easily characterized as either a Th1 or Th2 cell type and its cellular distribution resembles that reported for the proinflammatory cytokines tumor necrosis factor alpha (TNF-a) or IL-1b. However, it is not a T-cell-specific cytokine since it is also synthesized by neutrophils and eosinophils. Little is known so far about the regulation of IL-17 synthesis. High levels of this cytokine can be induced in vitro in human T cells and peripheral blood mononuclear cells, following their stimulation with a combination of the mitogen, phorbol myristate acetate, and the calcium ionophore, ionomycin. It is also induced by IL-15 in human CD4 þ T lymphocytes and this finding is complemented by data obtained from murine studies which point to IL-15 as a key

cytokine for the induction of IL-17 in lymphocytes and neutrophils. IL-23 is yet another important cytokine implicated in the activation of IL-17. Produced by dendritic cells, IL-23 is upregulated during infection and promotes the release of IL-17 by CD4 þ and CD8 þ T cells. Importantly, IL-23 drives the differentiation of a specialized subset of Th cells, namely ThIL-17, that coexpresses IL-17 and TNF-a (Figure 3).

Biological Function Cytokine and Chemokine Induction

When exogenously expressed in mouse airways, IL-17 promotes local production of chemokines belonging to the CXC group and alveolar neutrophilia. However, it does not directly elicit neutrophil

398 INTERLEUKINS / IL-17

chemotaxis, but rather induces neutrophil chemotactic activity in a number of structural cells. IL-17 upregulates CXC chemokine production in many types of airway structural cells in vitro, such as epithelial and smooth muscle cells as well as fibroblasts. This arises primarily through up-regulation of the chemokine IL-8/CXCL8, a strong neutrophil chemoattractant. IL-17 is also known to increase the production of the chemokines CXCL1/GROa; CXCL6/GCP-2 is known to act on neutrophils in vitro (Figure 3). Interaction with Inflammatory Mediators

The proinflammatory effects of IL-17 most likely relate to its ability to induce TNF-a and IL-1b production by human monocytes. Importantly, IL-17 also synergizes with these mediators to induce high levels of chemokines such as IL-8 and CXCL1 that act on neutrophils. This synergism is also responsible for increased levels of growth factors such as granulocytecolony stimulating factor (G-CSF) and granulocyte macrophage-CSF (GM-CSF) in human bronchial epithelial cells, venous endothelial cells, and fibroblasts. IL-17 also acts concomitantly with the Th1 cytokine IFN-g, and with the Th2 cytokines IL-4 and IL-13, to upregulate IL-8 production (Figure 3). Other inflammatory mediators upregulated by IL17 include IL-6, IL-10, IL-12, IL-1 receptor antagonist, cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). IL-17 also likely contributes to the in situ production of oxygen radicals through inducible nitric oxide synthase (iNOS) and the resultant increase in nitric oxide (NO) release as shown in stromal cells. These observations underline IL-17’s role as a potent inflammatory mediator of the inflammatory process in both Th1 and Th2 settings. IL-17 may also favor neutrophil migration to the sites of inflammation since it amplifies IFN-g-mediated increases in intercellular adhesion molecule-1 (ICAM-1) in fibroblasts and human bronchial epithelial cells. In addition, IL-17 increases neutrophil survival, a process involved in cell accumulation in the tissues. The production of G-CSF and GM-CSF, two growth factors that are known to inhibit neutrophil death by apoptosis, is upregulated in human airway epithelial cells. It is through this increase in growth factors as well as in stem cell factor, that IL-17 enhances the capacity of fibroblasts to sustain the proliferation of CD34 þ hematopoietic progenitors and promotes their subsequent differentiation into neutrophils. In line with these properties, transgenic mice that systemically overexpress IL-17 are characterized by enhanced granulopoiesis and massive peripheral neutrophilia.

Immune Response

IL-17 is produced early during airway allergen challenge in antigen-sensitized mice and inhibition of endogenous IL-17 is seen to decrease neutrophil numbers at the expense of an exacerbation of eosinophilic inflammation linked to higher expression of IL-5. These observations suggest a role for IL17 in the balanced production and accumulation of granulocytes in tissues. However, the exact function of IL-17 in airway antigen challenge requires further definition, as it has also been shown to contribute to allergic inflammation and the development of non-specific airway hyperresponsiveness in mice. As described above, IL-17 can activate on cells specialized in the recognition of pathogens and antigen presentation (innate immunity) leading to subsequent generation of specific (adaptive) immune responses. Although IL-17 has not been shown to interact directly with the biology of the pathogen receptor Toll-like receptor (TLR) 4, several reports have proposed a link between this cytokine and the activation of innate immune systems. IL-17 is induced in mice upon infection with endotoxin and the Gram-negative bacteria Klebsiella pneumoniae. IL-17 knockout mice are indeed documented to have impaired clearance of this microorganism in the lung. This phenomenon is associated with a lack of G-CSF production and the absence of neutrophils in the bronchoalveolar lavage. IL-17-mediated host defense may also involve the production of the antimicrobial peptide b-defensin-2, as well as macrophage inflammatory protein-3 (MIP-3). The importance of IL-17 in antigen response may also involve its capacity to promote dendritic cell differentiation, as well as its ability to activate T cells in allergic models of hapten-specific hypersensitivity responses in mice. Tissue Remodeling

IL-17 plays a critical role in tissue remodeling in bone homeostasis and inflammatory joint disease by enhancing the expression of enzymes involved in matrix degradation, such as matrix metalloproteinases (MMPs)-1,-3, -9, and -13 in osteoblasts, synoviocytes, chondrocytes, and macrophages. It may also promote bone degradation via the induction of the receptor activator of NF-kB ligand (RANKL) in osteoblasts. IL-17 has been implicated in tissue fibrosis through induction of IL-11 and IL-6 production by cultured airway fibroblasts. Additionally, this cytokine promotes angiogenesis by acting on endothelial cell growth and favoring neovascularization and production of angiogenic factors such as vascular endothelial growth factor, PGE1 and PGE2 and NO in lung fibroblasts and endothelial cells.

INTERLEUKINS / IL-17 399

Receptor

IL-17 in Respiratory Diseases

Structure

IL-17 is present at low levels in the airways under physiological conditions. Apart from T cells, eosinophils within airways represent an important source of this cytokine during allergic inflammation, especially in asthma where these granulocytes show increased intracellular levels of IL-17. In line with this observation, IL-17 levels are increased in the bronchoalveolar lavage and the bronchial submucosa. IL-17 is similarly upregulated in induced sputum of individuals with asthma, as compared to controls and subjects with chronic obstructive pulmonary disease. Furthermore, these elevated cytokine levels were found to be associated with the extent of non-specific reactivity of the airways, and correlated with PC20 values in response to metacholine challenge. Higher expression of IL-17 was detected in bronchial biopsies from subjects with moderate-to-severe asthma, as compared to mild asthmatics and control donors. This suggests that IL-17 is involved in the more severe forms of asthma which are characterized by neutrophilic inflammation. In this study, importantly, IL-17 levels observed in moderate-to-severe asthma were decreased by a 2-week course of oral corticosteroids. Little data are available to date in relation to the presence of IL-17 in other respiratory diseases. IL-17 expression is increased in both nasal polyps of subjects with chronic hyperplastic sinusitis and induced sputum of subjects with chronic bronchitis. In healthy human volunteers challenged by exposure to inhalation of swine dust, IL-17 levels were noted to be elevated within the bronchoalveolar lavage coinciding with the development of severe neutrophilic lung inflammation. This effect is likely to be related to IL-17’s property to amplify cellular responses to proinflammatory mediators, such as TNF-a and IL-1b. In this context, IL-17 may thus represent a tissue-specific target for pharmacotherapies aimed at resolving airway inflammation. It is therefore expected that pharmacotherapy that directly targets IL-17 will ensure more specificity and produce fewer side effects than current anti-inflammatory drugs or anti-IL-1b/TNF-a antibody treatments. Neutralizing antibodies against IL-17 have been tested in mice and can effectively reduce the influx of neutrophils into the lung. However, such treatments are also seen to aggravate symptoms in mice suffering from colitis. Therefore, given the critical role of IL-17 and neutrophils in host defense, anti-IL-17 therapy would most likely require selective, tissue-restricted use.

IL-17 receptor (IL-17R) is expressed by a wide range of human cell types. The array of cells includes epithelial, smooth muscle and vascular endothelial cells, fibroblasts, marrow mesenchymal stem cells, as well as cells from the immune system, that is, B and T lymphocytes and myelomonocytic cells. IL-17R is transcribed from multiple exons, giving rise to a protein that displays a molecular weight of 130 kDa. IL-17R is a type I receptor, that is, a single-pass transmembrane protein containing an extracellular amino terminus. Experiments involving blockage of IL-17R with antibodies were seen to inhibit the release of IL-6 upon IL-17 induction, indicating that IL-17R is integral to the IL-17-mediated response. Notable however is IL-17’s weak affinity for its receptor, with a Ka value that ranges from only 2
107 to 2
108 M  1. In view of the low concentrations of IL-17 needed to induce biological reactions in vivo, it is hypothesized that IL-17 binds to additional receptors. Signal Transduction

The signal transduction involved in IL-17-induced cytokine and chemokine production involves activation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated (ERK) and c-Jun terminal kinase (JNK) pathways. MAPK activation is indeed the central pathway mediating IL-17-induced IL-6 and IL-8 production by human bronchial epithelial cells. The synergy observed between IL-17 and other proinflammatory cytokines is likely to occur at the mRNA level, in a process involving mRNA stabilization and/or increased transcription. The transcription factor NF-kB appears to have a widespread role in the mediation of cellular responses to IL-17. Activation of MAPKs and NF-kB is required for the induction of the anti-microbial peptide b-defensin-2 as well as iNOS and COX-2 by IL-17 in airway epithelial cells. The adaptor molecule TNF-associated factor (TRAF) 6, rather than TRAF2, is required for IL-17R-mediated activation of NF-kB and JNK and subsequent production of IL-6 and ICAM-1 in fibroblasts. IL-17-mediated production of inflammatory mediators in fibroblasts can display corticosteroid sensitivity similar to that reported for IL-8, CXCL1, and CXCL6. However, the intracellular pathways involved in cytokine production by IL-17 are probably cell type specific since IL-8 production by IL-17 is not inhibited by corticosteroids in bronchial epithelial and smooth muscle cells.

See also: Chemokines, CXC: IL-8. Interleukins: IL-15; IL-23 and IL-27. Leukocytes: Neutrophils; T cells.

400 INTERLEUKINS / IL-23 and IL-27

Further Reading Aggarwal S and Gurney AL (2002) IL-17: prototype member of an emerging cytokine family. Journal of Leukocyte Biology 71(1): 1–8. Awane M, Andres PG, Li DJ, and Reinecker HC (1999) NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells. Journal of Immunology 162(9): 5337–5344. Barczyk A, Pierzchala W, and Sozanska E (2003) Interleukin-17 in sputum correlates with airway hyperresponsiveness to metacholine. Respiratory Medicine 97(6): 726–733. Chakir J, Shannon J, Molet S, et al. (2003) Airway remodelingassociated mediators in moderate to severe asthma: effect of steroids on TGF-beta, IL-11, IL-17, and type I and type III collagen expression. Journal of Allergy and Clinical Immunology 111(6): 1293–1298. Hellings PW, Kasran A, Liu Z, et al. (2003) Interleukin-17 orchestrates the granulocyte influx into airways after allergen inhalation in a mouse model of allergic asthma. American Journal of Respiratory Cell and Molecular Biology 28(1): 42–50. Kolls JK and Linden A (2004) Interleukin-17 family members and inflammation. Immunity 21(4): 467–476. Laan M, Cui ZH, Hoshino H, et al. (1999) Neutrophil recruitment by human IL-17 via C-X-C chemokine release in the airways. Journal of Immunology 162(4): 2347–2352. Linden A and Adachi M (2002) Neutrophilic airway inflammation and IL-17. Allergy 57(9): 769–775. Miljkovic D and Trajkovic V (2004) Inducible nitric oxide synthase activation by interleukin-17. Cytokine & Growth Factor Reviews 15: 21–32. Molet S, Hamid Q, Davoine F, et al. (2001) IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines. Journal of Allergy and Clinical Immunology 108(3): 430–438. Moseley TA, Haudenschild DR, Rose L, and Reddi AH (2003) Interleukin-17 family and IL-17 receptors. Cytokine & Growth Factor Reviews 14(2): 155–174. Prause O, Laan M, Lotvall J, and Linden A (2003) Pharmacological modulation of interleukin-17-induced GCP-2-, GRO-alphaand interleukin-8 release in human bronchial epithelial cells. European Journal of Pharmacology 462(1–3): 193–198. Yao Z, Spriggs MK, Derry JM, et al. (1997) Molecular characterization of the human interleukin (IL)-17 receptor. Cytokine 9(11): 794–800. Ye P, Garvey PB, Zhang P, et al. (2001) Interleukin-17 and lung host defense against Klebsiella pneumoniae infection. American Journal of Respiratory Cell and Molecular Biology 25(3): 335–340.

Relevant Website http://www.ncbi.nlm.nih.gov – IL-17 in NCBI database.

IL-23 and IL-27 J K Kolls and P J Dubin, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Interleukin-23 (IL-23) and IL-27 are recently identified heterodimer cytokines with components related to the IL-6 family of

cytokines. Structurally, both are closely related to IL-12; however, they are functionally distinct. In response to Gramnegative infection in the airway, the most prominent result of IL-23 activation is the induction of IL-17 as part of a proinflammatory axis critical for host defense. Lipopolysaccharide ligation of TLR4 induces IL-23 production by antigen-presenting cells, most prominently myeloid dendritic cells and macrophages. IL-23 stimulates memory CD4 þ T cells to produce IL-17 resulting in neutrophilic infiltration. IL-23 has also been implicated in autoimmune responses, specifically in arthritis and experimental autoimmune encephalitis. The antitumorigenic and antimetastatic properties of IL-23 have also been characterized and, in contrast to those of IL-12, rely on CD8 þ T cells to mediate the effects; IL-23 activation also induces a memory response to tumors. While IL-27 also primarily induces a T-helper-1 response to infection and is produced by antigen-presenting cells in response to infectious triggers, it acts primarily on naive CD4 þ T cells, augmenting the actions of IL-12 by inducing high levels of IFN-g. IL-27 also exhibits antitumorigenic properties that are similar in magnitude to those of IL-23 and IL-12. In contrast to IL-23, IL-27 suppresses IL-17 and thus may downregulate autoimmunity.

Introduction In 2000, Oppmann et al. described a novel factor, which they called p19 or IL-23p19. This member of the IL-6 family of cytokines was noted to have sequence homology with the p35 subunit of IL-12 and was also biologically inactive unless associated with the p40 subunit of IL-12, a soluble cytokine receptor. The p19/p40 heterodimer was named IL-23 and was shown to be produced by activated dendritic cells. Acting primarily on memory CD4 þ T cells, as well as on CD8 þ T cells, IL-23 triggers the production of IL-17, a proinflammatory cytokine that is critical for neutrophil recruitment in infection by Gram-negative organisms. The identification of IL-23 also helped to explain inconsistencies in the IL-12 literature resulting from differences in the phenotype of p35
/
and p40
/
mice in experimental models of infection and autoimmunity. Concurrent with investigations defining the role of IL-23 in linking the innate and adaptive immune response to infection, studies demonstrated that IL-23 plays a significant role in the autoimmune response, specifically in experimental autoimmune encephalomyelitis and collagen-induced arthritis. Other groups have also demonstrated that IL-23 has significant activity in the inhibition of growth and metastasis of tumors. In 2002, IL-27 was described. Again, the structural and functional similarities to IL-12 are notable. IL-27 is a heterodimer that consists of p28, an IL12p35-related polypeptide, and EBI3, an IL-12p40related protein. IL-27 is also produced by activated antigen-presenting cells and stimulates naive T-cell subsets, exhibiting synergy with IL-12 to induce

INTERLEUKINS / IL-23 and IL-27 401

interferon gamma (IFN-g) production. Though structurally IL-12, IL-23, and IL-27 are closely related, there are distinct differences in their functions; in some cases the cytokines act synergistically, in others they act antagonistically.

Table 1 Cytokine components of IL-12, IL-23, and IL-27: IL-12p35, IL-23p19, and IL-27p28 are unique type I cytokine, IL-6 subfamily subunits; IL-12p40 and EBI13 are soluble type I receptors Cytokine

Structure IL-23 is comprised of covalently linked p19 and IL-12p40 subunits. The p19 subunit is unique to IL23 and has been classified as a member of the type I cytokines, specifically the IL-6 family of cytokines, based on its sequence homology and a-helical structure. Encoded on chromosome 10 in mouse and chromosome 12 in humans, it appears to be most closely related structurally to the p35 subunit of IL-12, though neither the human nor the mouse p19 exhibit N-glycosylation sites. Both proteins also contain five cysteines and the p19/p40 complex is linked by disulfide bonds. Mouse and human p19 are 196 and 189 amino acids in length, respectively, and there is 70% homology between the two. The IL-12p40 subunit is a type I receptor, more specifically a member of the group 3 soluble receptors. Though it complexes with the p35 subunit of IL-12 and IL-23p19, p40 can also be secreted as a monomer or homodimer. In mice, p40 or (p40)2 has been noted to inhibit IL-12 function; this effect is less prominent when studying human IL-12 and IL-12p40. Of note, this effect was studied using antiIL-12p40 neutralizing antibodies and the effects of the IL-23p19/p40 heterodimer were not controlled for in these experiments. There are a number of parallels between the structures of IL-27 and IL-23. IL-27 is also a heterodimeric cytokine, which consists of a unique p28 subunit that complexes with EBI3, a group 3 soluble cytokine receptor. The p28 subunit, also a member of the IL-6 family of cytokines, is coded on human chromosome 16 and mouse chromosome 7, ultimately forming mature proteins of 24.5 and 23.6 kDa, respectively. Though neither mouse nor human p28 are heavily glycosylated, both exhibit differences in N- and O-glycosylation. Both also exhibit a highly negatively charged loop region, which has not been described in any of the other IL-6 subfamily cytokines. The EBI3, or Epstein–Barr virus-induced gene3, is a group 3 soluble receptor that has roughly 30% homology to IL-12p40. It was initially identified in EBV-infected B lymphocytes, but is also expressed in monocytes and macrophages. EBI3 does not exclusively dimerize with p28; dimerization with IL-12p35 has been documented but is of unclear significance (Table 1).

IL-12 IL-23 IL-27

Heterodimer constituents Type I cytokine Unique IL-6 subfamily subunit

Type I receptors Soluble receptors

p35 p19 p28

IL-12p40 IL-12p40 EBI3

Regulation of Production and Activity Though IL-23p19 contains a signal peptide, it is not secreted from mouse or human dendritic cells in appreciable amounts and does not exhibit activity as a single molecule. As a result, coexpression with a variety of nonsignaling group 3 soluble receptors was studied (including EBI3); and only the coexpression of IL-23p19 and IL-12p40 led to detection of enhanced p19 secretion and co-immunoprecipitation of the p19 subunit as a heterodimer. It is also worth noting that because IL-12p40 is found as part of the IL-12 heterodimer, as a monomer (p40) and as a homodimer (p40)2, there is usually detectable IL-12p40 in the absence of IL-23 induction. As a result, IL-23 activity is dependent on IL-23p19 induction, in the presence of IL-12p40 production. Also, though p40 monomer and homodimers inhibit IL-12 activity, it is not clear if the same holds true for IL-23. Because IL-12 and IL-23 are structurally similar and there is a history of apparent inconsistencies in the IL-12 literature pertaining to phenotypic differences of mice deficient in IL-12p40 versus IL-12p35, controls for the effect of both of these cytokines are requisite in current studies of IL-23 and IL-12. The initiating trigger for IL-23 production in infection is activation of antigen-presenting cells (dendritic cells or macrophages); lipopolysaccharide (LPS) ligation of TLR4 is the primary trigger for this, though other bacterial components such as other lipopeptides (TLR2) and unmethylated CpG DNA (TLR9) augment this response. The most prominent target of IL-23 is the memory CD4 þ T cell creating an IL-23/IL-17 proinflammatory axis; however, IL-23 receptors are also expressed on subpopulations of antigen-presenting cells suggesting that this is an additional route of IL-23 regulation. Assessment of IL-27 regulation again demonstrates striking parallels between IL-23 and IL-27. That IL27 activity requires coexpression of p28 and EBI3 was determined by coexpressing p28 with other soluble receptors including IL-12p40, CLF-1, and soluble IL-11 receptor. Biological activity occurs only

402 INTERLEUKINS / IL-23 and IL-27

when the heterodimeric form is present. IL-27 production is induced by LPS activation of antigenpresenting cells within 3–6 h of stimulation in vitro. This time frame is shorter than that required for IL12 production and similar to that observed for IL-23.

STAT3, and STAT4. Though the structure does not clearly predict this, there is also an observed association with Janus kinase (Jak) 2. The differences between the IL-12 b2 chain and IL-23R chain correlated with differences in STAT activation: most notably, IL-12 ligation preferentially activates STAT4 and IL-23 STAT3, and STAT3/ STAT4 dimers; and IL-23’s weaker activation of STAT4 correlates with significantly lower IFN-g induction. Though the mechanism is unknown, IL23 ligation on T cells, specifically memory T cells, results in the production of IL-17 A and F. The IL-27 receptor consists of WSX-1 or alternately, the T-cell cytokine receptor (TCCR) and gp130. Though both components are members of the group 2, type I receptors or IL-6 subfamily cytokine receptors, WSX-1/TCCR had previously been identified and its ligand was unknown; gp130 is a promiscuous receptor that binds IL-6 and IL-11 among others (Figure 1 and Table 2). Both WSX-1/TCCR and gp130 are coexpressed on T and B cells, natural killer (NK) cells, monocytes, dendritic cells, mast cells, and endothelial cells. In T cells, Jak1, STAT1, STAT3, STAT4, and STAT5 are phosphorylated, while in monocytes and mast cells STAT3 is phosphorylated.

IL-23 and IL-27 Receptors Just as there are many parallels between IL-12, IL23, and IL-27 there are striking similarities among their receptors. While the IL-12 receptor is a heterodimer consisting of IL-12Rb1 and IL-12Rb2 chains, the IL-23 receptor consists of the 12Rb1 chain and an IL-23-specific chain, IL-23R. The IL-23R chain is encoded on human chromosome 1, upstream of the IL-12Rb2. In addition, the extracellular domains of the IL-23R chain are all related to corresponding domains of IL-12Rb2, containing signal sequence, an N-terminal Ig-like domain, and two cytokine receptor domains. The major difference is that IL-23R does not contain three transmembrane-proximal fibronectin type III domains found in the IL-12Rb2 chain. There are also potential N-linked glycosylation sites in IL-23R, which are not found in the IL12Rb2 chain. Whether a cell responds to IL-12 or IL-23 depends on the expression of an IL-12Rb1/IL12Rb2 or an IL-23R/IL-12Rb1 receptor complex. The IL-23R chain does not complex with IL-12Rb2 and functionality of the intact IL-23R complex was demonstrated with binding studies as well as cell proliferation studies. The transmembrane domain of IL-23R contains a WQPWS sequence that, in mice, is repeated in a 20 amino acid duplication not found in human IL-23R. This WQPWS is similar to the WSXWS motif observed in other cytokine receptors. The cytoplasmic domain of the human IL-23R chain contains seven tyrosines, six of which are conserved in the mouse IL23R. These tyrosines define potential binding sites for major components of the intracellular signaling cascades including Src homology 2 domain, signal transducer and activator of transcription (STAT) 1,

Biological Function Studies of the biological function of IL-23 and IL27 preceded or were conducted concurrently with Table 2 Cytokine receptor components of IL-12, IL-23, and IL27 receptors: all subunits are members of the type I cytokine receptor family; IL-12Rb2, IL-23R, and WSX-1/TCCR are unique components for the IL-12, IL-23, and IL-27 receptors, respectively; IL-12Rb1 and gp130 subunits are promiscuous Cytokine receptor

EBI3 p28 gp130

p40 p35 IL-12R
2

p40 p19

IL-12R
1

IL-27

II-23R

IL-12

IL-12R
1

IL-23

Type I receptors

Type I receptors Promiscuous/common

IL-12Rb2 IL-23R WSX-1/TCCR

IL-12Rb1 IL-12Rb1 gp130

WSX-1

IL-12R IL-23R complex IL-27R

Heterodimer constituents

Figure 1 IL-12, IL-23, and IL-27 cytokine and receptor complexes. The IL-23 cytokine consists of a heterodimer of IL-12p40 and IL-23p19, which is similar in structure to the IL-12p35 subunit. IL-12 is comprised of the IL-12p40 subunit and a p35 subunit. IL-27 consists of EBI3 and p28. IL-12p40 and EBI3 are group 3 soluble cytokine receptors. IL-23p19, p35, and p28 are members of the IL-6 subfamily of type I cytokines. The IL-23 receptor consists of the IL-12Rb1 and IL-23R subunits. The IL-12 receptor consists of the IL-12Rb1 and IL-12Rb2 subunits. The IL-12Rb2 and IL-23R subunits are structurally similar. The IL-27 receptor consists of a gp130 and WSX-1 subunit.

INTERLEUKINS / IL-23 and IL-27 403

studies of IL-23 and IL-27 receptors. The earliest studies by Weikowski et al. compared the effects of ubiquitous, liver-specific, and bone-marrow-specific constitutive expression of IL-23p19 in vivo. With constitutive, ubiquitous expression, the affected mice exhibited runting, shortened life span, infertility, and systemic inflammatory changes. Specifically, levels of IL-1, tumor necrosis factor alpha (TNF-a), and the number of circulating neutrophils were increased (IL-17 levels were not reported as this interaction was not known at the time). Similar problems were observed with bone-marrow-specific, but not liverspecific overexpression; this difference was attributed to the lack of p40 expression in the liver and suggested that the main mediator of IL-23 production was hematopoietic cells. Other studies confirmed that stimulated antigen-presenting cells were the primary source of IL-23. After the identification of the IL-23 receptor, the pattern of IL-23 receptor expression was also utilized to study IL-23 function; IL-23 receptor mRNA was present primarily in T-helper-1 (Th1) and Th2 cells and NK cells. p40
/
and p35
/
mice initially developed to study IL-12 function were also utilized early on and it was not until the development of p19
/
mice that the direct analysis of IL-23 function could be performed. Some of these studies demonstrated that IL-23 played an important role in

regulating the production of CXC chemokines (a subfamily of the SCY chemokines), particularly CXCL1 (KC and MIP-2), to a variety of infectious agents, the most prominent pulmonary pathogens being extracellular Gram-negative bacteria such as Klebsiella pneumoniae. These effects were mediated by IL-23 induction of IL-17, a key regulator of the CXC chemokines KC, MIP-2, and CXCL1 and CXCL5 (LIX), as well as G-CSF (Figure 2). IL-23 has also been shown to play a key role in autoimmune encephalitis and arthritis, though a role for IL-23 in autoimmune pulmonary manifestations has not yet been demonstrated. In the model of autoimmune encephalitis, IL-23 generates a unique population of CD4 þ T cells termed ThIL-17 cells, which coexpress IL-17A, IL-17F, IL-6, and TNF and upon adoptive transfer are the encephalopathogenic T-cell populations. In contrast, classic Th1 cells grown in the presence of IL-12, which produced IFN-g upon antigen restimulation, were largely devoid of IL-17 production and failed to confer encephalitis upon adoptive transfer. More recently, IL-23 has been shown to have antitumor properties. Studies of IL-23 antitumorigenic and antimetastatic properties demonstrated that IL23 induced long-term regression of cancers similar to that of IL-12 transduced tumors; in addition, IL-23 induced a memory response to tumor rechallenge,

TLR4/TLR2/TLR9 Pathogen

IL-23 + CD8+ T cell

CD4+ T cell

+ IL-17

Vascular endothelium

Fibroblasts

Airway epithelium

ICAM-1, CXCL8, GM-CSF

CXCL1, 5, 8

IL-6, CXCL1, CXCL5, CXCL8, G-CSF, GM-CSF

Figure 2 IL-23
its proximate targets and downstream effects. On TLR stimulation by a pathogenic stimulus, the antigen-presenting cell is stimulated to produce IL-23. IL-23 activation of CD4 þ and CD8 þ T cells results in IL-17 production. IL-17 stimulates the vascular endothelium, fibroblasts, and the airway epithelium to produce CXC chemokines and G-CSF, GM-CSF, and IL-6.

404 INTERLEUKINS / IL-23 and IL-27

Pathogen

IL-27 +

− Mast

CD8+ T cell

CD4+ T cell

IFN-

NK

NK

IL-4/IL-5

CD4+ T cell

IFN-

CD8+ T cell

Monocyte

Degranulation

IL-6/TNF-


Figure 3 IL-27
its targets and downstream effects. The antigen-presenting cell produces IL-27 in response to stimulation by a pathogenic antigen. IL-27 has both pro- and anti-inflammatory effects on CD8 þ , CD4 þ , and natural killer (NK) cells as well as antiinflammatory effects on mast cells and monocytes. IL-27 is involved most prominently in IFN-g, IL-4, and IL-5 regulation. It blocks degranulation of mast cells and downregulates IL-6 and TNF-a production by monocytes.

which was mediated through CD8 þ T cells. Other studies also have shown that CD40 ligand expression on lung tumors in mice activates the immune response, upregulating p19 and p40 at a transcriptional level, with resulting regression of the tumors. Currently, the main function of IL-27 appears to be stimulation of naive CD4 þ T cells and production of IFN-g (Figure 3). Though IL-27 does promote an early Th1 response, it is not absolutely required, working synergistically with IL-12 for the development of a Th1 response. IL-27 promotes IFN-g by STAT1-dependent and STAT1-independent induction of T-bet. T-bet is a transcription factor that also induces expression of IL-12Rb2 on effector cells, allowing increased responsiveness to IL-12. The IL27R also activates STAT4, which acts to polarize Th1 effector cells. As with many other IL-6 family cytokines, IL-27 exhibits both proinflammatory and anti-inflammatory properties. In toxoplasmosis, WSX-1
/
mice develop immune-mediated liver necrosis, implying that the presence of IL-27 signaling plays an inhibitory role in the immune response. The mechanisms for this are currently unknown and similar anti-inflammatory roles have not been specifically described for IL-27 in the lung; of note,

however, WSX-1-deficient mice do develop more severe lung pathology and succumb to infection, in spite of a lower organism burden.

IL-23 and IL-27 in Respiratory Diseases To date, IL-23 has been studied in murine models of Gram-negative (Klebsiella) and mycobacterial pulmonary infection as well as lung tumors. Though studies have not defined the role of IL-23 in human lung diseases, the key differences between IL-12 and IL-23 are the ability of IL-23 to induce IL-17 production through activation of memory CD4 þ and, possibly, CD8 þ T cells, as well as the induction of a memory response to IL-23-sensitive tumors. It appears that optimal IL-23 is required for optimal IL17 production, which subsequently regulates CXC chemokines and G-CSF production. Though IL-27 has been even less well studied, experimental models demonstrate that it plays a significant role in Listeria monocytogenes and mycobacterial infection by inducing Th1 immune mechanisms. See also: Acute Respiratory Distress Syndrome. Chemokines. Defense Systems. Dendritic Cells. Interferons. Interleukins: IL-12. Leukocytes: T cells; Pulmonary Macrophages. Pneumonia: Community

INTERSTITIAL LUNG DISEASE / Overview 405 Acquired Pneumonia, Bacterial and Other Common Pathogens. Toll-Like Receptors. Transgenic Models.

Further Reading Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, and Gurney AL (2003) Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. Journal of Biological Chemistry 278(3): 1910–1914. Brombacher F, Kastelein R, and Gottfried A (2003) Novel IL-12 family members shed light on the orchestration of Th1 responses. Trends in Immunology 24(4): 207–212. Cooper AM, Kipnis A, Turner J, et al. (2002) Mice lacking bioactive IL-12 can generate protective, antigen-specific cellular responses to mycobacterial infection only if the IL-12 p40 subunit is present. Journal of Immunology 168(3): 1322–1327. Ghilardi N, Kljavin N, Chen Q, et al. (2004) Compromised humoral and delayed-type hypersensitivity responses in IL-23deficient mice. Journal of Immunology 172(5): 2827–2833. Happel KI, Zheng M, Young E, et al. (2003) Cutting edge: roles of Toll-like receptor 4 and IL-23 in IL-17 expression in response to Klebsiella pneumoniae infection. Journal of Immunology 170(9): 4432–4436. Harrington LE, Hatton RD, Mangan PR, et al. (2005) Interleukin17-producing CD4 þ effector cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nature Immunology 6(11): 1123–1132. Hunter CA (2005) New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions. Nature Reviews: Immunology 5(7): 521–531. Kolls JK and Linden A (2004) Interleukin-17 family members and inflammation. Immunity 21(4): 467–476.

Langrish CL, Mckenzie BS, Wilson NJ, et al. (2004) IL-12 and IL-23: master regulators of innate and adaptive immunity. Immunology Reviews 2002: 96–105. Lankford CS and Frucht DM (2003) A unique role for IL-23 in promoting cellular immunity. Journal of Leukocyte Biology 73(1): 49–56. Oppmann B, Lesley R, Blom B, et al. (2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar to as well as distinct from IL-12. Immunity 13(5): 715–725. Parham C, Chirica M, Timans J, et al. (2002) A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R. Journal of Immunology 168(11): 5699–5708. Park H, Li Z, Yang XO, et al. (2005) A distinct lineage of CD4 T cells regulates inflammation by producing IL-17. Nature Immunology 6(11): 1133–1141. Pflanz S, Hibbert L, Mattson J, et al. (2004) WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27. Journal of Immunology 172(4): 2225–2231. Pflanz S, Timans JC, Cheung J, et al. (2002) IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4( þ ) T cells. Immunity 16(6): 779–790. Villarino A, Hibbert L, Lieberman L, et al. (2003) The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection. Immunity 19(5): 645–655. Villarino A, Huang E, and Hunter CA (2004) Understanding the pro- and anti-inflammatory properties of IL-27. Journal of Immunology 173(2): 715–720. Wiekowski MT, Leach MW, Evans EW, et al. (2001) Ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inflammation, runting, infertility, and premature death. Journal of Immunology 166(12): 7563–7570.

INTERSTITIAL LUNG DISEASE Contents

Overview Alveolar Proteinosis Amyloidosis Cryptogenic Organizing Pneumonia Hypersensitivity Pneumonitis Idiopathic Pulmonary Fibrosis Lymphangioleiomyomatosis

Overview K K Brown and G P Cosgrove, University of Colorado Health Sciences Center, Denver, CO, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Comprised of over 150 named disorders, interstitial lung disease (ILD) is a complex area of pulmonary medicine for generalists and experts alike. While many of the disorders share common

clinical features, the mechanism of disease, outcome, type, or even availability of therapy for each specific disease often differs dramatically. An accurate diagnosis is therefore essential for the determination of prognosis and appropriate management. This article provides a framework for the classification and evaluation of patients with ILD.

Introduction The interstitial lung disease (ILD) is a heterogeneous group of disorders that adversely affects the

406 INTERSTITIAL LUNG DISEASE / Overview

pulmonary parenchyma. They are classified together due to similar clinical characteristics (Table 1). A more appropriate term for this group of disorders would be the diffuse parenchymal lung diseases since the entire lung, not just the interstitium, may be involved. For the purposes of this article, these terms can be considered synonymous. Pulmonary infections can mimic ILD, and while they may share Table 1 Clinical classification of interstitial lung disease (with examples) Systemic diseases Collagen vascular diseases Scleroderma Rheumatoid arthritis Sjo¨gren’s syndrome Polymyositis/dermatomyositis Immunodeficiency Common variable immunodeficiency Exposures Avocational Hypersensitivity pneumonitis Hot tub lung Bird breeder’s lung Compost lung Environmental Hypersensitivity pneumonitis Farmer’s lung Environmental molds Occupational Asbestosis Berylliosis Silicosis Popcorn worker’s lung Drug-induced lung disease Amiodarone Methotrexate Nitrofurantoin Genetic Familial interstitial pneumonia Hermansky–Pudlack syndrome Surfactant protein-C mutation Idiopathic Idiopathic interstitial pneumonias Idiopathic pulmonary fibrosis Non-specific interstitial pneumonia Cryptogenic organizing pneumonia Desquamative interstitial pneumonia/respiratory bronchiolitis interstitial pneumonia Lymphoid interstitial pneumonia Acute interstitial pneumonia Primary disorders Sarcoidosis Pulmonary Langerhans’ cell histiocytosis Pulmonary alveolar proteinosis Small vessel vasculitides of the lung Wegener’s granulomatosis Churg–Strauss syndrome Microscopic polyarteritis

clinical and radiographic features, they will not be considered a form of ILD herein. The focus of this article is to provide a framework for the physician to think about the evaluation and classification of the patient with ILD.

Etiology The sheer number and variety of clinical entities that fall under the heading of ILDs (greater than 150 have been described) make any generalizations regarding etiology only marginally helpful. While many of the defined disorders share clinical, radiographic, and in some cases even pathologic features, the mechanism of disease, prognosis, type of therapy, and expected response for each specific disease often differs drastically, making identification of the specific etiology essential. Categorization on the basis of an etiologic scheme leads to the following groupings: those disorders associated with systemic disease; those associated with inhaled or systemic exposures (medication, drug, avocations, occupations, environmental, and accidental); those associated with heritable genetic traits (familial interstitial pneumonia); and those that are currently idiopathic, offers a potentially useful approach to initial evaluation. There are approximately 100 000 hospital admissions that result from acute or chronic ILDs. The prevalence has been estimated to be 80.9 per 100 000 in males and 67.2 per 100 000 in females with the respective incidence of 31.5 per 100 000 and 26.1 per 100 000. Of those patients, idiopathic pulmonary fibrosis (IPF) accounted for 45% of the cases, making it the most common ILD (see Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis). The basic mechanisms of disease for the majority of these disorders have yet to be determined. A generalized hypothesis suggests an initial injury to the epithelium (and less frequently to the endothelium), with a subsequent pathologic repair response, with varying degrees of cellular inflammation, extracellular matrix deposition (fibrosis), and architectural distortion. In progressive fibrotic disease, it has been suggested that a process of disordered and incomplete wound healing in response to recurrent epithelial injury occurs. Known causes of lung injury leading to cellular infiltration or lung fibrosis include: inhalation of environmental inorganic particles (such as silica), or organic antigens (such as avian antigens as occurs in hypersensitivity pneumonitis); vascular dissemination of an injurious agent to the lung (as occurs in drug-induced or some collagen vascular disease-associated ILDs). In several primary diseases such as lymphangioleiomyomatosis, amyloidosis, and lymphangitic carcinomatosis, rather than excessive

INTERSTITIAL LUNG DISEASE / Overview 407

deposition of extracellular matrix or infiltration with inflammatory cells, accumulations of smooth muscle cells, amyloid fibrils, and malignant cells occur, respectively.

Clinical Features ILDs, although diverse in etiology, generally present with similar symptoms and findings. The value of a thorough medical history and examination cannot be overemphasized and will often identify both the underlying diagnosis and its etiology. Dyspnea on exertion, a nonproductive cough, or an abnormal chest radiograph are the most common presenting features. The duration of symptoms and the rate of their progression differentiate the acute (days to weeks) and subacute (weeks to months) from the much more common chronic (months to years) ILDs (see Table 2). Patients should be specifically questioned about the presence of wheezing, hemoptysis, or chest pain, as the presence of these more unusual symptoms helps pare the differential diagnostic possibilities and may suggest an airwaycentered process (e.g., bronchiolitis), diffuse alveolar hemorrhage, or those diseases that often present with pleural involvement (e.g., rheumatoid arthritis) or pneumothoraces (e.g., lymphangioleiomyomatosis). A detailed past medical history and review of systems should be obtained to identify prior or evolving systemic diseases that may be associated with ILDs. For example, a subset of patients can develop parenchymal lung disease prior to the onset of the characteristic systemic manifestations of collagen vascular disease; adult onset Raynaud’s phenomenon, sicca symptoms, or dysphagia in the setting of an ILD would raise this concern. Inflammatory bowel diseases may manifest with ILD. Therefore, chronic diarrhea or abdominal complaints should be queried. Neurologic symptoms, such as polydypsia and polyuria, may be extrapulmonary manifestations of sarcoidosis or pulmonary Langerhans’ cell histiocytosis. Vasculitides, such as Wegener’s granulomatosis or Churg–Strauss syndrome, often present with systemic manifestations such as hematuria, mononeuritis multiplex, and ILD. Table 2 Acute or subacute interstitial lung diseases Acute interstitial pneumonia Idiopathic Collagen vascular disease-associated ILD Acute eosinophilic pneumonia Cryptogenic organizing pneumonia Drug-induced ILD Diffuse alveolar hemorrhage Hypersensitivity pneumonitis

Current and previously used medications, including over-the-counter and naturopathic medications as well as nutritional supplements, should be reviewed to identify any potential agents predisposing patients to ILD. Specific examples are prior exposure to amiodarone in patients with dysrhythmias, nitrofurantoin prophylaxis in those with recurrent urinary tract infections, or chemotherapy such as bleomycin in those with remote malignancies such as lymphomas. All medications should be considered in the differential diagnoses of ILD, especially when there is a clear temporal association with the onset of symptoms. An exhaustive investigation into clinically significant environmental, occupational, avocational, or accidental exposures should be performed in all patients with ILD. In the case of hypersensitivity pneumonitis, the immunologic response and lung injury is driven by continued exposure (see Interstitial Lung Disease: Hypersensitivity Pneumonitis). To avoid persistent and/or progressive lung disease, antigen avoidance with abatement of the exposure is almost always needed. Patients should be questioned about prior or ongoing exposure to birds (an avocation highly associated with hypersensitivity pneumonitis), hot tubs, or other damp environments (potential exposure to molds and fungi). While it is important to document a patient’s previous and current occupations, it is just as important to identify the specific duties performed, as general job titles often fail to alert one to clinically pertinent exposures. Heritable interstitial lung disease is more common than previously appreciated, and a detailed family medical history serves to identify potential disorders such as tuberous sclerosis, familial interstitial pneumonia, sarcoidosis, or Hermansky–Pudlak syndrome. Siblings or a first-degree relative with documented ILD are important historical keys. As with most pulmonary diseases, quantifying the extent of current or former tobacco use is important. There are several smoking-related ILDs that should be considered in a current smoker or individual with significant passive exposure: respiratory bronchiolitis ILD (RB-ILD), desquamative interstitial pneumonitis (DIP), pulmonary Langerhans’ cell histiocytosis, and Goodpasture’s syndrome with diffuse alveolar hemorrhage. In all of these diseases, active tobacco use is associated with the development of the disease and/ or increased disease activity. On physical examination, resting tachypnea may exist depending on the severity of disease. Clubbing, while most commonly found in patients with IPF, may be seen in other chronic fibrotic ILDs such as asbestosis, silicosis, or chronic or fibrotic

408 INTERSTITIAL LUNG DISEASE / Overview

hypersensitivity pneumonitis. Bibasilar, mid-late-inspiratory crackles are a hallmark of most fibrosing ILDs. A mid-inspiratory wheeze or squeak may be heard in airway-centered ILD such as the bronchiolitides. Alternatively, patients with granulomatous lung disease such as sarcoidosis or berylliosis may have few if any demonstrable findings on auscultation, despite significant physiologic or radiographic abnormalities. An increased second heart sound, a holosystolic murmur in the tricuspid area, or overt cor pulmonale with ascites and lower extremity edema suggests pulmonary hypertension. Extrathoracic signs of collagen vascular diseases, such as xerostomia, xero-ophthalmia, synovitis with deforming arthritis, sclerodactyly, telangectasias, or proximal muscle weakness should be specifically sought.

Physiology Pulmonary physiologic abnormalities are present in the majority of patients at the time of presentation and may be restrictive, obstructive, or mixed. An isolated abnormality in gas exchange, as indicated by an isolated decrease in carbon monoxide diffusing capacity (DLCO) or exercise-induced desaturation as measured by pulse oximetry, may exist particularly early in the course or with milder ILD. Pulmonary physiology serves both as a guide to the diagnosis and as a way to grade the severity of impairment, which may be important in determining prognosis and response to therapy. Occasionally, pulmonary function testing may lack the sensitivity to detect clinically significant disease, especially in early disease. In some diseases, physiological findings may under-represent the extent of disease, as is the case in some patients with hypersensitivity pneumonitis or mixed disease with emphysema and ILD. In those patients in which ILD is clinically a concern but the pulmonary physiology is normal, formal cardiopulmonary exercise testing should be considered to more accurately quantify ventilatory, cardiovascular, and gas exchange abnormalities.

In general, plain chest radiographic abnormalities can be classified as alveolar-filling, interstitial, or mixed disorders. Alveolar-filling disorders often result in poorly defined coalescent opacities and consolidation with air bronchograms, as occurs in alveolar proteinosis or cryptogenic organizing pneumonia (COP). Interstitial opacities denote either reticular (linear) or nodular opacities, typically the result of expansion of the interstitial compartment with extracellular matrix, inflammatory or malignant cells, or smooth muscle proliferation. In most disorders, a combination of reticular and nodular opacities occurs. High-resolution computed tomography (HRCT) is the most sensitive and accurate radiographic study in the evaluation of ILD and should be considered a standard piece of the evaluation. Specific abnormal radiographic features (such as ground-glass opacities or honeycomb cysts) can be combined into an overall radiographic pattern, and this pattern of disease almost always gives a strong indication of the underlying histopathology, serving to suggest a small group of clinical disorders or even a specific disease (see Table 3 and Figure 1). Broadly speaking, abnormal HRCT features in ILD can be classified as parenchymal opacification (ground-glass attenuation and consolidation), lines (reticular lines and septal thickening), cysts (isolated Table 3 High-resolution computed tomographic features in ILD Abnormal feature Parenchymal opacification Ground-glass opacities Consolidation

Lines Reticular Septal thickening

Cysts Isolated

Radiology An abnormal plain chest radiograph is a common initial clue to the presence of ILD. While certain characteristic patterns may suggest specific disorders (e.g., chronic eosinophilic pneumonia), the plain radiograph is almost never diagnostic by itself, and up to 10% of patients with clinically significant disease may have a normal plain radiograph at presentation, as is the case with some patients with desquamative interstitial pneumonitis, sarcoidosis, or hypersensitivity pneumonitis.

Honeycombing

Nodules Ground-glass Soft tissue Calcific Traction bronchiectasis Mosaic pattern Air trapping

Diseases (e.g.) NSIP DIP COP Eosinophilic pneumonia

IPF Asbestosis Sarcoid Lymphangitic cancer

Lymphangioleiomyomatosis PLCH IPF Other fibrosing lung disease

Hypersensitivity pneumonitis Sarcoidosis/berylliosis Remote granulomatous infection IPF Other fibrosing lung disease Bronchiolitis Bronchiolitis

INTERSTITIAL LUNG DISEASE / Overview 409

Figure 1 High-resolution computed tomograms demonstrating the radiographic features commonly present in ILD. (a) Dense alveolar consolidations with air bronchograms in a patient with pulmonary lymphoma. (b) Reticular opacities with traction bronchiectasis in a patient with idiopathic non-specific interstitial pneumonitis. (c) Subpleural honeycombing, traction bronchiectasis, and reticular opacities in a patient with IPF. (d) Ground-glass opacities with associated pulmonary cysts in a patient with desquamative interstitial pneumonia.

Table 4 Diagnostic utility of bronchoalveolar lavage in the correct clinical context

Table 5 Probability of definitive histologic diagnosis on transbronchial biopsy

Often diagnostic Alveolar proteinosis Alveolar hemorrhage Eosinophilic pneumonia Bronchoalveolar cell carcinoma Certain infections

Often diagnostic Sarcoidosis/beryliosis Hypersensitivity pneumonitis Eosinophilic pneumonia Lymphangitic malignancy Alveolar proteinosis Certain infections

Often useful Hypersensitivity pneumonitis IPF Often useful when combined with transbronchial biopsy Sarcoidosis/beryliosis COP PLCH

and honeycomb), nodules (soft tissue, ground-glass and calcific density), traction bronchiectasis, mosaic pattern, and air trapping. By combining these features with their distribution, a differential diagnosis can be

Occasionally diagnostic Vasculitis Amyloidosis PLCH Lymphangioleiomyomatosis Never diagnostic Usual interstitial pneumonitis Organizing pneumonia Non-specific interstitial pneumonia DIP/RBILD LIP Bronchiolitis

410 INTERSTITIAL LUNG DISEASE / Overview

Figure 2 Photomicrographs of histologic patterns that may occur with different interstitial lung diseases. (a, b) Dense alveolar consolidation with areas of intraluminal fibrin plugs termed Mason bodies (mb) in a patient with cryptogenic organizing pneumonia. (c, d) Homogeneous septal expansion with lymphoplasmocytic infiltration in a patient with idiopathic non-specific interstitial pneumonia. (e, f) Heterogeneous fibrosing process with septal expansion, honeycombing, and fibroblastic foci (ff) directly adjacent to relatively normal alveoli in a patient with IPF/UIP. (g, h) Peribronchiolar inflammation and septal thickening are accompanied by multinucleated gaint cells (arrow) and septal noncaseasting granulomas in a patient with hypersensitivity pneumonitis. Original magnifications: (a, c, e, g)
40; (b, d, f, h)
200. Photomicrographs courtesy of C D Cool.

generated. For example, the combination of peripheral predominant, basal predominant reticular lines, honeycombing, and traction bronchiectasis with minimal ground-glass opacity and in the absence of other abnormal features can provide a highly confident

diagnosis of pathologic usual interstitial pneumonia (UIP). Abnormal CT findings outside of the abnormal lung parenchyma such as pleural thickening with calcification, esophageal dilation, and mediastinal lymphadenopathy may add to the discriminative

INTERSTITIAL LUNG DISEASE / Overview 411

ability of the scan and suggest disorders such as asbestos-related lung disease, collagen vascular disorders such as scleroderma, and sarcoidosis. However, while a particular radiographic pattern may suggest a small group of disorders, or even a specific disorder, it must be viewed within the entire clinical context using the comprehensive history, examination, physiology, and pathologic data to allow for an accurate clinical diagnosis.

Pathology In those in whom a confident diagnosis cannot be obtained by clinical, physiologic, radiographic, and laboratory testing, additional invasive or semi-invasive testing is often necessary. Bronchoscopy can be used to visually inspect the airway, perform bronchoalveolar lavage (BAL), and perform endobronchial and transbronchial lung biopsies. BAL is rarely diagnostic (Table 4), must be interpreted within the clinical context, and generally serves to rule out or lower the likelihood of competing diagnoses. Lung biopsy is often required to provide a definitive pathologic diagnosis and can be performed via a bronchoscope (transbronchial biopsy) and/or via a surgical route (video-assisted thoracoscopic (VATS) or open procedure). Transbronchial and endobronchial biopsies are small, focal, and limited in their ability to provide a pathologic diagnosis. However, they can be performed on an outpatient basis and can be diagnostic in the correct clinical settings (see Table 5). Surgical lung biopsies are performed in those patients in whom a confident clinico-radiographic diagnosis cannot be obtained and transbronchial biopsies are thought to be of limited usefulness or demonstrate non-specific findings. The histopathologic features identified on surgical lung biopsy samples lead to an accurate diagnosis in greater than 90% of patients (see Figure 2). The morbidity and mortality of surgical lung biopsies has significantly decreased with the advent of the VATS approach. In light of the dramatic differences in prognosis between several ILDs that may mimic one another, a surgical lung biopsy is recommended when a confident diagnosis is unattainable in its absence, or a definitive diagnosis is required.

Animal Models One of the major limitations to understanding the pathobiology of ILD is the lack of an appropriate model to mimic the overwhelming majority of ILD. Bleomycin- or paraquat-induced pulmonary fibrosis serves as a model of fibrosing lung disease but is likely more representative of acute toxic or drug-induced

lung injury. It remains the most common model used today to investigate the mechanisms underlying interstitial lung disease. A newer model has been developed in which transforming growth factor beta1 is overexpressed within the lung and appears to result in abnormalities more representative of the progressive fibrotic response noted in the human diseases.

Management and Current Therapy The management of patients with ILD is critically dependent upon the underlying etiology. Diseasespecific treatment is addressed in each individual disease article. In all patients, regardless of available therapy, supplemental oxygen should be utilized in those with hypoxemia at rest, with activity, or nocturnally to avoid the secondary development of right heart disease. Pulmonary rehabilitation should be considered in all patients to maximize their functional status, and the use of vaccination to decrease the frequency of respiratory infections is strongly encouraged. In those with chronic, progressive disorders, psychosocial support in the form of local or national support groups is often very helpful.

Acknowledgment This work is supported by the NIH (NHLBI SCOR HL67671). Dr Cosgrove is a Parker B Francis fellow in Pulmonary Research. See also: Interstitial Lung Disease: Hypersensitivity Pneumonitis; Idiopathic Pulmonary Fibrosis.

Further Reading American Thoracic Society Committee of the Scientific Assembly on Environmental and Occupational Health. (1997) Adverse effects of crystalline silica exposure. American Journal of Respiratory and Critical Care Medicine 155(2): 761–768. American Thoracic Society (2000) Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS), and the European Respiratory Society (ERS). American Journal of Respiratory and Critical Care Medicine 161(2 Pt 1): 646–664. American Thoracic Society/European Respiratory Society (2002) American Thoracic Society/European Respiratory Society international multidisciplinary consensus classification of the idiopathic interstitial pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. American Journal of Respiratory and Critical Care Medicine 165(2): 277–304. American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and Other Granulomatous Disorders (1999) Statement on sarcoidosis. Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other

412 INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee, February 1999. American Journal of Respiratory and Critical Care Medicine 160(2): 736–755. Akira M, Hara H, and Sakatani M (1999) Interstitial lung disease in association with polymyositis–dermatomyositis: long-term follow-up CT evaluation in seven patients. Radiology 210(2): 333–338. Bodolay E, Szekanecz Z, De´ve´nyi K, et al. (2005) Evaluation of interstitial lung disease in mixed connective tissue disease (MCTD). Rheumatology (Oxford) 44(5): 656–661. Coultas DB, Zumwalt RE, Black WC, and Sobonya RE (1994) The epidemiology of interstitial lung diseases. American Journal of Respiratory and Critical Care Medicine 150(4): 967–972. Fan LL, Kozinetz CA, Deterding RR, and Brugman SM (1998) Evaluation of a diagnostic approach to pediatric interstitial lung disease. Pediatrics 101(1 Pt 1): 82–85. Guidotti TL, Miller A, Christiani D, et al. (2004) Diagnosis and initial management of nonmalignant diseases related to asbestos. American Journal of Respiratory and Critical Care Medicine 170(6): 691–715. Lacasse Y, Selman M, Costabel U, et al. (2003) HP Study Group. Clinical diagnosis of hypersensitivity pneumonitis. American Journal of Respiratory and Critical Care Medicine 168(8): 952– 958. Raghu G and Brown KK (2004) Interstitial lung disease: clinical evaluation and keys to an accurate diagnosis. Clinics in Chest Medicine 25(3): 409–419, v. Research and Scholarship (1999) Respiratory health hazards in agriculture. American Journal of Respiratory and Critical Care Medicine 158(5 Pt 2): S1–S76. Schwarz MI, King TE, and Raghu G (2003) Approach to the evaluation and diagnosis of interstitial lung disease. In: Schwarz MI and King TE (eds.) Interstitial Lung Disease, pp. 1–30. ON: Blackwell. Selman M (2004) Hypersensitivity pneumonitis: a multifaceted deceiving disorder. Clinics in Chest Medicine 25(3): 531–547, vi. Strimlan CV (1997) Interstitial lung disease and primary Sjogren’s syndrome: clinical-pathological evaluation and response to treatment. American Journal of Respiratory and Critical Care Medicine 155(4): 1489.

Alveolar Proteinosis J J Presneill, The Royal Melbourne Hospital, Melbourne, VIC, Australia K Nakata, Niigata University Medical and Dental Hospital, Niigata, Japan J F Seymour, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Pulmonary alveolar proteinosis (PAP) is a rare clinical syndrome with three currently recognized forms: congenital, secondary, and acquired. More than 90% of cases are an acquired autoimmune disorder. In PAP excess accumulation of surfactant leads to bilateral airspace opacities on chest radiograph and extensive alveolar densities with thickened interlobular septa on computed tomography lung scan. PAP typically afflicts

previously healthy individuals, who present with several months of dyspnea and impaired pulmonary gas transfer of variable severity. ‘Milky’ appearing, lipid-rich fluid recovered at bronchoalveolar lavage is typical. Lung biopsy, when performed, shows minimally inflamed alveoli uniformly filled with amorphous eosinophilic material that on electron microscopy contains characteristic lamellar bodies. Current concepts of pathogenesis of acquired PAP are that the excess surfactant results from impaired clearance by alveolar macrophages, as a result of inhibition by specific polyclonal autoantibodies of trophic physiological signals from the endogenous cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). Current therapy consists of whole-lung lavage under general anesthesia, repeated as guided by clinical status. Some adult patients also respond to the repeated administration of recombinant human GM-CSF.

Introduction Pulmonary alveolar proteinosis (PAP), first recognized in 1958, is a rare but distinctive condition characterized by the widespread intra-alveolar accumulation of excess surfactant with associated lung dysfunction of variable severity. The most common form (490%) is that acquired by previously healthy adults without any familial predisposition, but congenital and secondary forms also exist. Cases of acquired PAP have been described from all populated continents, without any clear racial or geographic predisposition. The median age of adult patients is B40 years, with more than 70% being smokers prior to diagnosis. Men are affected twice as commonly as women, but this may be confounded by a globally greater proportion of men who smoke, as the genders are equally affected among nonsmoking patients. PAP in infants is a pathogenetically separate process to that in adults with a distinct histopathology, resulting from one of several identified genetic disorders. Even the most common adult acquired form of PAP is rare, with an estimated annual incidence of 0.36 cases per million, and a prevalence of 3.70 cases per million.

Etiology More than 90% of cases are acquired by apparently previously healthy adults. This disease was previously termed idiopathic PAP and is now regarded as an acquired autoimmune disease based upon the recognition of high-affinity polyclonal autoantibodies that specifically eliminate physiologically important granulocyte-macrophage colony-stimulating factor (GM-CSF) activity in the lungs of essentially all cases so far examined. In contrast, congenital PAP (2% of PAP cases) results from various genetic disorders of surfactant proteins B and C, or the common signal transducing b-chain of the GM-CSF

INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis 413

Figure 1 Lung biopsy appearance of acquired PAP, showing alveoli filled with eosinophilic lipoproteinaceous material with otherwise relatively preserved lung architecture (hematoxylin–eosin,  40). Reproduced from Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the first 44 years. American Journal of Respiratory and Critical Care Medicine 166(2): 215–235, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

receptor shared by interleukin-3 (IL-3) and IL-5 (bc). This congenital form of PAP has a very poor prognosis and is optimally managed with lung transplantation (in contrast to acquired PAP, which has recurred in transplanted lungs). PAP occurs occasionally as a secondary process associated with a range of underlying disorders affecting the number and function of alveolar macrophages, most commonly myeloid hematopoietic malignancies, but also other hematopoietic and immunodeficiency diseases, acute silicosis and other inhalation syndromes, and the rare genetic disorder lysinuric protein intolerance. At least in the case of myeloid malignancies, secondary PAP may resolve following therapies that restore normal hematopoiesis.

Pathology Lung biopsies in affected adults show the characteristic appearance of minimally inflamed alveoli with preserved normal architecture filled with foamy macrophages amongst granular acellular eosinophilic, periodic acid-Schiff positive lipoproteinaceous material (Figure 1). In some cases the diagnosis may be established less invasively by bronchoalveolar lavage that typically returns ‘milky’ appearing fluid with a microscopic appearance identical to that described above (Figure 2). Electron microscopy can confirm the diagnosis in difficult cases by demonstrating the presence of characteristic concentrically laminated phospholipid lamellar bodies (Figure 3).

Figure 2 Cytological preparation of BAL showing alveolar macrophages containing abundant PAS-positive material in a background of PAS-positive granular lipoproteinaceous material (PAS,  400). Reproduced from Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the first 44 years. American Journal of Respiratory and Critical Care Medicine 166(2): 215–235, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

500 nm Figure 3 Ultrastructural appearance of BAL fluid showing cellular debris and characteristic concentrically laminated phospholipid lamellar bodies. Reproduced from Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the first 44 years. American Journal of Respiratory and Critical Care Medicine 166(2): 215–235, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

Clinical Features In adults, the usual presentation of PAP is slowly progressive dyspnea of several months’ duration, sometimes with minimally productive cough or fatigue and occasional weight loss. A minority of patients is minimally symptomatic. Fever and hemoptysis are uncommon, and suggest complicating secondary infection, most commonly with Nocardia spp. Physical signs on examination are often inconspicuous, but crackles are audible in up to 50% of patients. Bilateral patchy, nodular, or confluent

414 INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis

clinically distinctive, PAP may be mistaken initially for any of the numerous infective, inflammatory, infiltrative, or neoplastic causes of bilateral pulmonary infiltrates.

Pathogenesis and Animal Models Surfactant Catabolism in Human PAP: Defect in Surfactant Clearance

Figure 4 Thin-section, high-resolution computed tomography scan of the thorax showing features typical of acquired PAP. Extensive opacity involves most of the lungs. The areas of ground-glass opacity demonstrate thickening of the interlobular septa leading to the characteristic ‘crazy paving’ appearance typical of PAP. Reproduced from Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the first 44 years. American Journal of Respiratory and Critical Care Medicine 166(2): 215–235, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

airspace opacities on chest radiology are revealed by computed tomographic scans to be due to widespread airspace consolidation with thickened interlobular and intralobular septa (‘crazy paving’ appearance) (Figure 4). The mean (SD) arterial oxygen tension at the time of diagnosis in a large group of reported cases was 58.6 (15.8) mmHg. Pulmonary function tests typically show a relatively mild restrictive ventilatory defect that contrasts with a moderate or severe impairment in pulmonary gas transfer. The most common biochemical abnormality is a slight to moderate elevation in serum levels of lactate dehydrogenase, serial measurement of which can be used as a useful index of disease activity. Although

The initial report of Rosen et al. recognized that the eosinophilic material accumulating within alveoli in the lung of patients with PAP was rich in lipids, proteins, and some carbohydrate, with similar biochemical composition and electron microscopic appearance to pulmonary surfactant. Surfactant, composed of approximately 90% lipids, 10% proteins, and a small amount of carbohydrates, plays an important role in reducing surface tension at the alveolar wall air–liquid–tissue interface thus preventing alveolar collapse and transudation of capillary fluid. Surfactant proteins comprising SP-A, SP-B, SP-C, and SP-D contribute to the surface-active properties and support the host defense functions of alveolar macrophages. Surfactant lipids and proteins are synthesized and partially recycled by alveolar type II cells, with catabolism in both alveolar type II cells and alveolar macrophages. Both production and degradation of surfactant materials have been extensively evaluated in PAP, with impaired clearance being the predominant cause of the increased alveolar pool size. Markedly elevated levels of these surfactant proteins are found in the blood of all patients with PAP, but these findings are not specific for PAP and can occur in other interstitial lung diseases, although usually at lower levels.

Figure 5 Surfactant homeostasis and impaired surfactant catabolism in pulmonary alveolar proteinosis. GM-CSF has a critical role in surfactant homeostasis in the normal lung (a). Interruption of GM-CSF signaling in the lung results in pulmonary alveolar proteinosis (b). Surfactant lipids and surfactant proteins A, B, C, and D are produced by alveolar type II epithelial cells (solid black arrows). Surfactant protein precursors are processed in the Golgi network. Some (surfactant proteins B and C) are then assembled, along with surfactant phospholipids, in lamellar bodies; surfactant proteins A and D are secreted by other secretory vesicles. After exocytosis into the alveolar surface liquid, the lamellar bodies assemble into surfactant structures known as tubular myelin as well as into large and small aggregates. Phospholipids from extracellular surfactant structures form continuous monolayers and multilayers of phospholipids that line the alveolar spaces and airways, with their polar heads oriented toward the liquid and their acyl chains toward the air. The large aggregates, extracellular lamellar bodies, and tubular myelin all have surface-active properties. Normally (a) surfactant is inactivated by mechanical and biologic processes and converted into small, surface-inactive aggregates. Approximately 70–80% of the small aggregates are taken up by alveolar type II cells, transported to phagolysosomes, and reused (green arrows) or catabolized (red arrows). Alveolar macrophages internalize and catabolize the remaining surfactant pool, a process critically dependent on GM-CSF. Although GM-CSF stimulates lung growth and causes alveolar type II epithelial-cell hyperplasia, a potential role for GM-CSF in surfactant recycling by these cells has not been defined (dashed black arrows). In pulmonary alveolar proteinosis (b), interruption of GM-CSF signaling (small red bar) in the alveolar macrophage for example, by targeted ablation of the gene encoding GM-CSF or its receptor in mice or, presumably, by neutralizing anti-GM-CSF autoantibodies in humans impairs the catabolism of surfactant by alveolar macrophages without impairing its uptake. This results in the intracellular buildup of membrane-bound, concentrically laminated surfactant aggregates. Progressive expansion of the extracellular surfactant pool and accumulation of cellular debris due to the impaired catabolism eventually cause filling of the alveoli, thus reducing the size of the available gas-exchange surface and eventually leading to the clinical syndrome. Reproduced from Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349(26): 2527–2539, with permission. Copyright & 2003 Massachusetts Medical Society. All rights reserved.

INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis 415 GM-CSF Plays a Critical Role in Surfactant Homeostasis in the Lung

Gene-targeted mice lacking either GM-CSF or bc develop PAP, indicating that lack of GM-CSF bioactivity leads to reduced surfactant clearance. Local expression of GM-CSF in airway epithelial cells or

Lamellar body

Surfactant

inhalation of GM-CSF in GM-CSF-knockout mice corrected PAP. Surfactant metabolism studies in GMCSF-knockout mice have demonstrated normal levels of surfactant protein mRNA and normal secretion rates, but grossly impaired clearance of surfactant phospholipids and SP-A by alveolar macrophages.

Phospholipid monolayer

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416 INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis Dysfunction of Alveolar Macrophages in Patients with PAP and Gene-Targeted Mice

Impaired Differentiation of Alveolar Macrophages in PAP and GM-CSF-Knockout Mice: Role of PU.1

The abnormal accumulation of surfactant materials in both patients and murine PAP models establishes that a defect in surfactant catabolism by alveolar macrophages is causative (Figure 5). Functional defects in the macrophages from patients with PAP include impaired chemotaxis, adhesion, phagocytosis, microbicidal activity, and phagolysosome fusion. In GM-CSF-knockout mice, alveolar macrophages have similar functional abnormalities in cell adhesion, cell-surface pathogen recognition receptor expression, phagocytosis, endocytosis, superoxide production, microbial killing, cytokine secretion, and catabolism of both surfactant phospholipids and proteins. It is likely that alveolar macrophages from patients with PAP will share this spectrum of functional defects.

The functional abnormalities in alveolar macro phages suggest a defect in maturation and, indeed, alveolar macrophages from patients with PAP are ultrastructurally immature. This is caused by an absence of PU.1, an ets-family transcription factor that normally leads to the terminal differentiation of alveolar macrophages. In the physiological state, bioactive GM-CSF in the lung leads to upregulation of PU.1 in alveolar macrophages. Thus, it appears that PU.1 mediates a number of the biological effects of GM-CSF in alveolar macrophages (Figure 6). Acquired PAP is a manifestation of functional GM-CSF-deficiency caused by an autoantibody against GM-CSF. It had long been recognized that a factor in the bronchoalveolar lavage fluid and sera from patients

Innate immunity

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B cell IL-12 Natural killer cell Increased PU.1 Phagolysosome

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Figure 6 GM-CSF in modulating the function of alveolar macrophages in mice. In vivo, pulmonary GM-CSF stimulates an increase in the level of PU.1, a transcription factor, in alveolar macrophages in the lung. In vitro, alveolar macrophages from knockout mice without the GM-CSF gene have a number of functional defects, including defects in cellular adhesion, catabolism of surfactant proteins and surfactant lipids, expression of pathogen-associated molecular pattern receptors (e.g., Toll-like receptors and the mannose receptor), Toll-like-receptor signaling, phagocytosis of pathogens (bacteria, fungi, and viruses), intracellular killing of bacteria (independent of uptake), pathogen-stimulated secretion of cytokines (tumor necrosis factor, IL-12, and IL-18), and Fc receptor-mediated phagocytosis. Cytoskeletal organization is abnormal and may in part account for defects in phagocytosis. The inability of alveolar macrophages to release IL-12 and IL-18 severely impairs the interferon response to pulmonary infection, thus impairing an important molecular connection between innate and adaptive immunity in the lung. Retroviral vector-mediated, constitutive expression of PU.1 in alveolar macrophages from GM–/– mice corrects all these defects, suggesting that GM-CSF stimulates terminal differentiation of the macrophages through the global transcription factor PU.1. The purple arrows represent the functions of PU.1 that are affected by the absence of GM-CSF. Reproduced from Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349(26): 2527–2539, with permission.

INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis 417

Serum anti-GM-CSF autoantibody level (µg ml–1)

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50

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Pulmonary alveolar proteinosis Figure 7 Presence of autoantibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF) within the bronchoalveolar lavage fluid and serum of patients with acquired pulmonary alveolar proteinosis. The autoantibodies are not present in persons with congenital or secondary pulmonary alveolar proteinosis or other lung diseases or in normal persons. Each point represents an individual patient. Some data are taken from Kitamura T, Uchida K, Tanaka N, et al. (2000) Serological diagnosis of idiopathic pulmonary alveolar proteinosis. American Journal of Respiratory and Critical Care Medicine 162: 658–662. Reproduced from Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349(26): 2527–2539, with permission.

with PAP inhibited the mitogen-stimulated proliferation of normal monocytes. In 1999, very high levels of a high-affinity neutralizing IgG autoantibody against GM-CSF were identified in the bronchoalveolar lavage fluid and sera of patients with acquired PAP from many geographic regions. This autoantibody specifically inhibited GM-CSF-induced cell growth in vitro. Various assays for this autoantibody have identified patients specifically with acquired PAP (as opposed to congenital or secondary forms) with high sensitivity and specificity, making this a very efficient screening and diagnostic test (Figure 7). It is likely that the autoantibody against GM-CSF obliterates the bioactivity of GM-CSF in vivo, resulting in impaired alveolar macrophage maturation, functional impairment, and surfactant accumulation. This pathogenetic model is supported by the observation that patients with acquired PAP do not develop the expected peripheral blood neutrophilia after systemic administration of exogenous GM-CSF, despite the absence of mutations in either the GMCSF or GM-CSF receptor genes, and normal antigenic levels of GM-CSF in bronchoalveolar lavage fluid.

Management and Current Therapy After a diagnosis of PAP is established, therapy directed at improving gas exchange may not immediately be

required. Commonly used and broadly accepted criteria for the institution of therapy are: at least moderately severe hypoxia (arterial PO2 p65 mmHg), persistent and functionally significant respiratory symptoms attributable to PAP, and rarely progressive systemic deterioration (weight loss, repeated respiratory infections, etc.). However, in all patients, it is mandatory that the supervising physician be vigilant in surveillance for opportunistic infections, both respiratory and systemic. While the prognosis for adult patients with acquired PAP is very good and has improved in recent years (predicted 5-year disease specific survival of B90%), approximately 20% of deaths in patients with PAP are due to infection. Although there are anecdotal cases claiming improvements in respiratory function with corticosteroids in the older literature, there is currently no role for the use of these agents in patients with uncomplicated PAP. Approximately 30% of patients with PAP will have minimal or mild symptoms and have stable or minimally fluctuating disease severity and never require therapy, and B10% of patients will have some degree of ‘spontaneous improvement’ in their PAP with diminished symptoms and functional improvement. These patients have an extremely good prognosis; however, it is not known if the underlying disease process has truly resolved. For the remaining patients who require therapy, the standard approach to treatment for the last

418 INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis

40 years has been whole lung lavage. This treatment is based on the revolutionary studies of Ramı´rezRivera in the 1960s at the Veterans’ Administration Hospital in Baltimore. He used repeated ‘segmental flooding’ through a percutaneous transtracheal endobronchial catheter in awake patients. Over the decades, the original procedure has been sequentially refined through the routine use of general anesthesia, increased lavage volumes, the recognition that saline is safer and as efficacious as other lavage fluids, the additional benefit of concomitant chest percussion, and the successful completion of bilateral sequential whole lung lavage in the same treatment session. Whole lung lavage remains the current standard of care for patients with PAP, and although the procedure is complex to perform and the associated physiologic changes are quite profound, it is usually well tolerated when performed by an experienced team. However, it does require a prolonged general anesthetic and has associated morbidity and very rarely mortality. Unfortunately, there are no established response criteria for therapeutic lavage. However, significant clinical, physiological, and radiological improvements

are usually seen after the initial therapeutic lavage in 80–90% of patients. The median duration of symptomatic benefit for patients is B15 months, with 20–30% of patients remaining free of recurrent symptoms beyond 3 years. Recent single-center reports have suggested meticulous attention to lavage technique in highly experienced centers may achieve more durable benefits. A review of published cases described a mean improvement in arterial PO2 of 20 mmHg, a 31 mmHg improvement in (A–a)DO2 and a 0.5 l improvement in vital capacity, and a 4.4 ml mmHg 1 min 1 improvement in DLCO (corresponding to B30% improvement in percent predicted). Some degree of physiological improvement is usually seen within a few days postlavage, making this therapy the preferred approach for severely hypoxic patients. The other treatment option is the therapeutic use of GM-CSF. This has mostly been used subcutaneously starting at a dose of 5 mg kg 1 day 1, with some patients requiring dose escalation to achieve responses, and less commonly as a nebulized inhalation. Unlike whole lung lavage, responses to GM-CSF are generally slow to be manifest, with

40

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Time on treatment (days) Figure 8 Kinetics of improvement in (A a)DO2 for patients responding to their first effective course of GM-CSF therapy (5 mg kg 1 day 1 for five patients and 20 mg kg 1 day 1 for one patient). Each individual responding patient is represented by a separate color, with the patient responding only at the higher dose of GM-CSF represented by a broken line with open circles. The patient represented by a line with solid circles had only one baseline ABG evaluation. For other patients, pretreatment values are shown as mean 7 SEM for all ABG analyses performed within 28 days of commencing therapy (in two cases SEM are contained within symbols). Reproduced from Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the first 44 years. American Journal of Respiratory and Critical Care Medicine 166(2): 215–235, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

INTERSTITIAL LUNG DISEASE / Amyloidosis 419

improvements in (A–a)DO2 not evident until 4–6 weeks, with maximal improvements not achieved until 6–10 weeks of therapy (Figure 8). Subcutaneous GM-CSF treatment is easily delivered but expensive, with few patients ceasing treatment due to adverse effects, usually rash or local reaction at the injection site. Indeed, patients with PAP also appear to have a lower incidence of adverse effects with GM-CSF than anticipated. The presence of GM-CSF-neutralizing antibodies likely results in a reduced effective biologic dose. The likelihood of response to GM-CSF, predominantly among patients no longer deriving benefit from whole lung lavage, is 40–50%, with the magnitude of physiological improvement among responders similar to that seen with lavage. Given the detection of GM-CSF antibodies in all of the treated patients, it is not apparent why therapeutic responses are variable. Although limited data are available, the effective antibody titer, reflecting total GM-CSF-neutralizing capacity, may be lower in responders. The sequential application of increasing doses seeking evidence of biologic activity of GM-CSF, such as peripheral blood eosinophilia, may identify appropriate dose ranges for further evaluation. While the subcutaneous route was used in most patients, based on its proven safety and efficacy in other settings, recent animal work suggests that inhaled GM-CSF may have greater pulmonary effects, but conversely may not ameliorate any extrapulmonary aspects of the disease. Although aerosolized GM-CSF is well tolerated in humans, and this route of GM-CSF delivery has been successfully used in a small number of patients with acquired PAP, the issues of pharmacokinetics and reliable drug delivery remain unresolved. See also: Autoantibodies. Leukocytes: Pulmonary Macrophages. Surfactant: Overview.

factor in patients with acquired pulmonary alveolar proteinosis. Blood 92: 2657–2667. Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the first 44 years. American Journal of Respiratory and Critical Care Medicine 166: 215–235. Seymour JF and Presneill JJ (2004) Pulmonary alveolar proteinosis. What is the role of GM-CSF in disease pathogenesis and treatment? Treatments in Respiratory Medicine 3: 229–234. Seymour JF, Presneill JJ, Schoch OD, et al. (2001) Therapeutic efficacy of granulocyte-macrophage colony-stimulating factor in patients with idiopathic acquired alveolar proteinosis. American Journal of Respiratory and Critical Care Medicine 163: 524– 531. Shah PL, Hansell D, Lawson PR, Reid KB, and Morgan C (2000) Pulmonary alveolar proteinosis: clinical aspects and current concepts on pathogenesis. Thorax 55: 67–77. Shibata Y, Berclaz P-Y, Chroneos Z, et al. (2001) GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1. Immunity 15: 557–567. Trapnell BC and Whitsett JA (2002) GM-CSF regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense. Annual Review of Physiology 64: 775–802. Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349: 2527– 2539. Uchida K, Nakata K, Trapnell BC, et al. (2004) High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis. Blood 103: 1089– 1098. Yoshida M, Ikegami M, Reed JA, Chroneos ZC, and Whitsett JA (2001) GM-CSF regulates surfactant protein-A and lipid catabolism by alveolar macrophages. American Journal of Physiology. Lung Cellular and Molecular Physiology 280: L379–L386.

Amyloidosis J P Lynch III, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA H D Tazelaar, Mayo Clinic Arizona, Scottsdale, AZ, USA & 2006 Elsevier Ltd. All rights reserved.

Further Reading Beccaria M, Luisetti M, Rodi G, et al. (2004) Long-term durable benefit after whole lung lavage in pulmonary alveolar proteinosis. European Respiratory Journal 23: 526–532. Dranoff G, Crawford AD, Sadelain M, et al. (1994) Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 264: 713–716. Goldstein LS, Kavuru MS, Curtis-McCarthy P, et al. (1998) Pulmonary alveolar proteinosis: clinical features and outcomes. Chest 114: 1357–1362. Kitamura T, Uchida K, Tanaka N, et al. (2000) Serological diagnosis of idiopathic pulmonary alveolar proteinosis. American Journal of Respiratory and Critical Care Medicine 162: 658– 662. Presneill JJ, Nakata K, Inoue Y, and Seymour JF (2004) Pulmonary alveolar proteinosis. Clinics in Chest Medicine 25: 593–613. Seymour JF, Begley CG, Dirksen U, et al. (1998) Attenuated hematopoietic response to granulocyte-macrophage colony-stimulating

Abstract Amyloidosis is a heterogeneous group of diseases characterized by deposition of amyloid protein, a fibrillar insoluble crystalline material, in the extracellular matrix of involved tissues. Amyloidosis can be localized or systemic; both inherited and acquired forms exist. Four major categories of systemic amyloidosis include primary or immunoglobulin light-chain (AL) disease, secondary or amyloid protein A (AA) disease, familial or mutant transthyretin (ATTR) disease, and dialysis-associated or b-microglobulin (b2-M) disease. Primary amyloidosis is associated with deposition of monoclonal k or l immunoglobulin light chains (amyloid AL) and can be idiopathic or associated with plasma cell dyscrasias. Secondary amyloidosis results from excessive production of serum amyloid A (SAA), an acute phase protein released in response to inflammation. Amyloid AA may complicate diverse chronic inflammatory conditions. Hereditary or familial amyloidosis comprises 4% of cases of amyloidosis.

420 INTERSTITIAL LUNG DISEASE / Amyloidosis Most cases are due to mutations in the transthyretin gene. In this article, we discuss the salient clinical features of the various forms of amyloidosis, and emphasize specific complications of respiratory tract involvement. Specific types of intrathoracic involvement in AL amyloidosis include tracheobronchial amyloidosis, diffuse interstitial or alveolar–septal disease, nodular disease (amyloidomas), intrathoracic adenopathy, and pleural disease.

Introduction Amyloidosis is a heterogeneous group of diseases characterized by deposition of amyloid protein, a fibrillar insoluble crystalline material, in the extracellular matrix of involved tissues. Amyloidosis can be localized or systemic; both inherited and acquired forms exist. Classification of amyloidosis is based on different subunit proteins. Nomenclature includes an ‘A’ for amyloid and a letter indicating the respective proteins involved. The four major categories of systemic amyloidosis include (1) primary or immunoglobulin light-chain (AL) disease, (2) secondary or amyloid protein A (AA) disease, (3) familial or mutant transthyretin (ATTR) disease, and (4) dialysis-associated or b-microglobulin (b2M) disease. Additional diseases associated with amyloid deposition include Alzheimer’s disease (b-amyloid precursor protein), type II diabetes (islet amyloid polypeptide), spongiform encephalopathies, and medullary carcinoma of the thyroid and other malignancies. These latter entities are beyond the scope of this article. Pathogenesis

Amyloid is an insoluble b-pleated fibrillar glycoprotein that forms rigid fibrils that are resistant to proteolysis. Deposits of amyloidosis in extracellular spaces disrupt tissue architecture and function and can impair organ function by space-occupying effects. Amyloid fibrils may be cytotoxic and promote apoptosis. Amyloid deposits are unusually stable, but can regress when the supply of fibril precursors is reduced. Certain glycosaminoglycans (GAGs) are invariably associated with the fibrils. Although the pathogenesis of amyloidosis has not been elucidated, serum amyloid protein (SAP) and GAGs may be important. In SAP knockout mice, experimentally-induced AA amyloid is attenuated. Little is known regarding individual susceptibility to amyloidosis or why specific amyloid proteins preferentially affect specific organs. Histopathology

Amyloid is a homogeneous amorphous eosinophilic material that binds the dye Congo red, appearing orange-red by conventional light microscopy (Figure 1). Under polarized light microscopy, amyloid exhibits apple-green birefringence. AA amyloid loses its

Figure 1 Nodular amyloidosis stained with Congo-red and examined under polarized light showing apple-green birefringence.

Figure 2 Transmission electron microscopic imaging comparing collagen (upper left) with a diameter of 20–100 nm to amyloid ˚. fibrils (lower right) with a mean diameter of 100 A

Congo red-associated birefringence after oxidation in permanganate solution, whereas AL amyloid retains this feature. Amyloid also stains with sulfated alcian blue. Electron microscopic examination of amyloid reveals a dominant (95%) fibrillar component (Figure 2), with distinct periodicity. A glycoprotein component, derived from soluble amyloid protein (SAP), is also present. More than 15 types of amyloid, which share common morphological features, are recognized. Immunohistochemical techniques to detect k and l light chains, amyloid-associated protein, or transthyretin (TTR) can be done to classify the fibril type. Negative stains for serum amyloid A (SAA) protein and TTR exclude AA-type and TTRtype amyloidosis, but stains for k and l chains are positive in only 60% of cases of AL amyloidosis.

Primary (AL) Amyloidosis Primary (AL) amyloidosis is associated with deposition of monoclonal k and l immunoglobulin light

INTERSTITIAL LUNG DISEASE / Amyloidosis 421

chains (amyloid AL) and can be idiopathic or associated with plasma cell dyscrasias (e.g., multiple myeloma). Both, systemic and localized, forms of AL exist. AL amyloidosis is the most common type of amyloidosis, but only 1275–3200 new cases are diagnosed annually in the United States. Clinical Features

Clinical manifestations of AL amyloidosis are protean. Amyloidosis can affect any organ, but predominant sites include kidney, heart, peripheral nervous system, gastrointestinal (GI) tract, joints, the tongue, spleen, liver, and upper respiratory tract. Initial symptoms are often fatigue and weight loss. Specific symptoms related to amyloidosis develop, affecting specific target organs. Predominant features include kidney failure, congestive heart failure (CHF), and peripheral neuropathy. Renal amyloid may result in nephrotic syndrome and varying degrees of renal insufficiency. Amyloid deposits in the myocardium or pericardium may lead to conduction disturbances, restrictive cardiomyopathy, and CHF. Autonomonic and sensory neuropathies and carpal tunnel syndrome are relatively common but the central nervous system is not involved in AL amyloidosis. Amyloidosis may affect any site in the GI tract; hemorrhage, ulcerations, obstruction, diarrhea, or malabsorption caused by bowel involvement may occur. Rectal biopsy may reveal amyloid deposits in systemic AL amyloidosis. Hepatomegaly is common in AL amyloidosis but splenomegaly occurs in o5% of cases. However, splenic dysfunction (i.e., the presence of Howell–Jolly bodies in peripheral blood smear) occurs in 24% of cases. Macroglossia, a classical feature of amyloidosis, occurs in 15–20% of patients. Infiltration of thyroid or adrenal glands may result in hypothyroidism or hypoadrenalism, respectively. Skin findings include waxy thickening and easy bruising. Amyloidosis may also be associated with a bleeding diathesis, due to factor X deficiency as well as amyloid involving blood vessels. Clinically significant pulmonary involvement is rare in other forms of amyloidosis, but occurs in 5–20% of patients with AL amyloidosis. Localized AL amyloidosis most often affects the upper respiratory, urogenital, or GI tracts, skin, or orbit. In such cases, the amyloidogenic light chains are produced by clones of lymphocytes or plasma cells localized in proximity to the amyloid deposits. Diagnostic evaluation of AL amyloidosis requires an assessment of the extent and nature of organ involvement. Biopsies of affected site(s) may establish the diagnosis. Biopsies of subcutaneous abdominal fat or bone marrow are positive in 485% of patients

with systemic AL amyloidosis. Examination of bone marrow aspirates and biopsies is critical to determine whether plasma cell dyscrasias or myeloma is present. Serum and urine light chains should be measured to assess for monoclonality. Twenty fourhour urine should be obtained to rule out nephrotic syndrome or renal involvement. Radionuclide scintiscans using 123I-labeled SAP component provide a macroscopic whole body survey that may assess anatomical distribution. SAP scans are expensive and not available in most centers. Electrocardiograms and echocardiograms should be performed to assess cardiac involvement, which is the major determinant of prognosis in AL amyloidosis. 2D echocardiographic features of cardiac involvement include septal thickening, concentrically thickened left ventricle, normal-to-small ventricular cavity, reduced ejection fraction (EF), high left-sided pressures (a restrictive filling pattern), and a ‘speckled’ pattern. A clinical clue to cardiac amyloidosis is marked worsening of heart failure after treatment with calcium channel blockers. Serial studies of urine and serum light chains may be invaluable to assess the course of the disease. Pathogenesis

The fibrils in systemic AL amyloidosis are derived from circulating monoclonal light chains produced by clonal plasma cell dyscrasias. In localized forms of AL amyloidosis, the light chains are produced by a clone of lymphoplasmacytes localized in proximity to the amyloid deposits. Clinical expression of the disease is determined by the extent and anatomic distribution of amyloid deposits. Management and Current Therapy

Prognosis of AL amyloidosis is determined by extrapulmonary organ involvement. Mean survival in untreated AL amyloidosis is 1–2 years. Most deaths are due to cardiac, renal, or neurological causes. Multisystem involvement portends a dismal prognosis. Optimal therapy for AL amyloidosis is not clear. Alkylating agents (particularly melphelan) plus prednisone are appropriate for amyloidosis associated with plasma cell dyscrasias (e.g., multiple myeloma). For idiopathic AL amyloidosis, 25% of patients respond to melphelan/prednisone but only modest gains in survival (8–10 month improvement) are achieved. Colchicine and a-interferon are ineffective. High-dose chemotherapy followed by peripheral blood stem cell transplantation (SCT) achieves remissions in 65% of patients, and is the most promising therapy. However, late relapses may occur and data are limited regarding long-term survival.

422 INTERSTITIAL LUNG DISEASE / Amyloidosis

Anecdotal responses were reported with thalidomide; dimethyl sulfoxide (DMSO); vincristin, adriamycin, and dexamethasone (VAD); and 40 -iodo40 -deoxy-doxorubicin (a new anthracycline), but data are sparse. Novel agents that stabilize amyloid precursor proteins, inhibit fibril formation, or enhance fibril degradation are currently under development. Candidate drugs include GAG mimetics and SAP-binding inhibitors. In addition, treatment needs to be directed towards the organ involved. Treatment of specific complications is important.

Secondary Amyloidosis (Amyloid AA) Secondary amyloidosis (amyloid AA) results from excessive production and organ-deposition of amyloid formed from SAA, an acute phase protein released in response to inflammation. There are several SAA proteins in existence. In humans, AA amyloid deposits consist of fragments of at least five different molecular forms. Etiology

Amyloid AA occurs in 1–5% of patients with diverse chronic inflammatory conditions, for example, osteomyelitis or chronic infections (i.e., tuberculosis, bronchiectasis, leprosy, malaria); familial Mediterranean fever; connective tissue disease; and inflammatory bowel disease. Secondary amyloidosis may complicate renal cell carcinoma or other malignancies. Most cases (but not all) are AA amyloid. Given the marked reduction of chronic infectious diseases in developed countries, AA amyloidosis is exceptionally rare. Clinical Features

The onset of AA amyloidosis ranges from several months to many decades after acquiring the underlying disorder. Principal target organs include kidneys (91%), GI tract (22%), liver (5%), nerves (3%), and lymph nodes (2%). Lung involvement is rare (o1%) in AA amyloidosis. Survival in AA amyloidosis is influenced primarily by renal involvement. Renal failure is associated with a mean survival of 11 months, compared to 57 months among patients with normal renal function. In contrast to AL amyloidosis, cardiac involvement is rare in AA amyloidosis. As in ATTR amyloidosis, macroglossia is not a feature of AA amyloidosis.

Hereditary Amyloidosis Hereditary or familial amyloidosis comprises 4% of cases of amyloidosis.

Etiology

Most cases of hereditary amyloidosis are due to mutations in the TTR gene found on chromosome 18 (autosomal dominant with high penetrance). More than 50 mutations of TTR have been identified in familial amyloidosis; however, mutations of apolipoprotein A-1, gelsolin, fibrinogen A a, and lysozyme also lead to amyloidosis. Clinical Features

The onset of familial amyloidosis is usually after age 50. Most common features include peripheral neuropathy (480%), autonomic neuropathy (470%), cardiomyopathy (450%), carpal tunnel syndrome (440%), and nephropathy (o20%). Macroglossia does not occur. Lung involvement is rare (o4%). Biopsies or aspirates of subcutaneous fat or bone marrow are the standard tests to diagnose amyloidosis. Immunohistochemical stains to detect TTRderived amyloid confirm the diagnosis. Molecular genetic studies of blood can determine the specific mutant TTR. Management and Current Therapy

Survival among patients with ATTR amyloidosis varies with the specific mutation, but median survival rates approximate 4–7 years after diagnosis. The most common causes of death in ATTR amyloidosis are progressive CHF or sudden cardiac death and inanition due to chronic autonomic neuropathy. In one study, median duration of survival was 3.4 years among ATTR patients with cardiomyopathy and 7 years without cardiomyopathy. For patients with familial amyloidosis, treatment of specific complications (e.g., orthostatic hypotension, endocrine insufficiency, and cardiac arrhythmias) is critical. Since TTR is synthesized in the liver, liver transplantation is the optimal therapy for ATTR amyloidosis. Liver transplantation may prevent further deposition of amyloid, and may effect regression of amyloid deposits and symptoms. Combined heart and liver transplantation has been successful in a few patients.

Other Rare Causes of Amyloidosis Deposition of wild-type TTR amyloid protein may be found with advanced age (senile amyloidosis), usually as an incidental finding. Autopsy studies detected senile pulmonary amyloidosis in approximately 10% of patients older than 80 years of age and in 50% of patients older than 90 years of age. These deposits are usually incidental findings and are not associated with specific symptoms. Other rare

INTERSTITIAL LUNG DISEASE / Amyloidosis 423

sources of amyloidosis include b2-microglobulin in chronic renal failure patients on dialysis, b-protein amyloid in the cerebral senile plaques of Alzheimer’s disease, procalcitonin in medullary carcinoma of the thyroid, and islet cell amyloid polypeptide in the pancreas in patients with type II diabetes.

Pulmonary Involvement in Amyloidosis Clinically significant pulmonary involvement occurs in o10–20% of patients with AL amyloidosis, but at necropsy, amyloid deposits in pulmonary vessels are present in up to 90% of patients. Lung involvement is rare in AA and ATTR amyloidosis, and, when present, is usually an incidental finding (e.g., diffuse alveolar septal opacities without clinical symptoms). In the following sections, the specific forms of pulmonary amyloidosis (principally due to AL amyloid) will be discussed. Five types of extravascular lung involvement are seen in AL amyloidosis (systemic or localized). These include (1) diffuse interstitial or alveolar–septal disease, (2) nodular disease, (3) intrathoracic adenopathy, (4) pleural disease, and (5) diaphragmatic deposition and miscellaneous sites. As was previously noted, pulmonary involvement is rarely lethal in AL amyloidosis. Diffuse Interstitial (Alveolar–Septal) Amyloidosis

Diffuse parenchymal (alveolar–septal) amyloidosis is an uncommon complication of systemic or localized AL amyloidosis (Figure 3). Deposits of AL amyloid in the alveolar septae and interstitium may be associated with reticulonodular or micronodular lesions, with or without pleural effusions, on chest radiographs. Chest computed tomographic (CT) scans typically demonstrate reticular opacities, interlobular septal thickening, small, 2–4 mm nodules in the subpleural regions, honeycomb change, and groundglass opacities. Physiological aberrations include reduced lung volumes, decreased diffusing capacity for carbon monoxide (DLCO), and widened alveolar– arterial O2 gradient. Pathologically, pulmonary vascular involvement is relatively common in this form of amyloid (Figure 4), but clinically significant pulmonary hypertension is rare. Pulmonary involvement is rarely severe, and most patients with alveolar–septal amyloidosis do not have specific pulmonary symptoms. The prognosis is dictated by the cardiac status. In one study of AL patients with cardiac failure (i.e., left ventricular EF o40%), median survival was only 12 weeks, compared to 16 months with alveolar–septal infiltrates and normal cardiac function.

Figure 3 (a)Alveolar–septal amyloid stained with sulfated alcian blue in which the amyloid appears bright green. (b) Without the special stain, it is very difficult to appreciate the amyloid even at a higher microscopic power.

Nodular Amyloid Lesions (Amyloidomas)

Localized pulmonary amyloid nodules (amyloidomas) most commonly reflect a localized abnormal immune response of bronchus-associated lymphoid tissue (BALT), but rarely complicate systemic AL amyloidosis, orthotopic liver transplantation, IgA nephropathy, or systemic light-chain deposition disease. Histopathological features of nodular amyloidosis Grossly, nodules exhibit gray-yellow translucence on a cut section (Figure 5). Histologically, irregular, amorphous, and predominantly acellular masses of amyloid replace the lung parenchyma (Figure 6) and are frequently surrounded by a lymphoplasmacytic infiltrate (Figure 7) that is usually light-chain restricted. AL amyloid may be found in vessels and interstitium adjacent to the nodules. Fibrosis, calcification, ossification, giant cells, and mononuclear inflammatory cells may be present.

424 INTERSTITIAL LUNG DISEASE / Amyloidosis

Figure 4 Vascular amyloid deposition highlighted by sulfated alcian blue stain.

Figure 6 Low-power photomicrograph from an amyloidoma. Note irregularly shaped deposits of amorphous eosinophilic amyloid.

Figure 7 High-power photomicrograph showing an amyloidoma associated with a marked lymphoplasmacytic infiltrate. Figure 5 Surgically excised nodule of amyloid showing yellow homogeneous cut section.

Clinical features Chest radiographs or CT scans demonstrate single or multiple nodular densities, ranging in size from 0.4 to 15 cm, with a proclivity for the lower lobes. Calcification is present in 25–50% of lesions. Metaplastic bone or cartilage formation may be present. Cavitation is rare. The nodules usually grow slowly over the years. Most patients are asymptomatic but cough or hemoptysis may occur. Pulmonary function is usually normal. Management and current therapy Prognosis of nodular amyloid is generally excellent. Since systemic disease is lacking, chemotherapy is not necessary. Surgical resection is advised for solitary symptomatic lesions. Intrathoracic Lymphadenopathy

Hilar and mediastinal adenopathy due to amyloid deposition occurs in 8% of patients with AL amyloidosis and may occur in AA amyloidosis or

tracheobronchial amyloidosis (TBA) (discussed later). Amyloid-infiltrated nodes may calcify. Pleural Effusions Complicating AL Amyloidosis

Pleural effusions in primary AL amyloidosis may reflect CHF, nephrotic syndrome, or amyloid deposits along the pleural surface. Transudative effusions suggest CHF or nephrotic syndrome; hemorrhagic, exudative effusions suggest direct deposits along the pleural surface. Rarely, chylous pleural effusions occur in patients with extensive amyloid burden. Rare Respiratory Tract Complications of AL Amyloidosis

Rare manifestations of AL amyloidosis include respiratory muscle weakness (due to diaphragmatic amyloid), obstructive sleep apnea (secondary to involvement of the tongue), recurrent laryngeal nerve palsy, hoarseness or stridor due to amyloid deposits in the supraglottic larynx, and pulmonary marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) type.

INTERSTITIAL LUNG DISEASE / Amyloidosis 425 Primary Pulmonary Amyloidosis

‘Primary’ pulmonary amyloidosis (PPA) refers to amyloidosis that is confined to the lungs and associated structures (e.g., tracheobronchial tree, pleura, hilar, or mediastinal lymph nodes). Investigators from the Mayo Clinic reported 55 patients with PPA from 1980 to 1993. Forms of amyloidosis included AL amyloidosis (n ¼ 35), AA amyloid (n ¼ 2), localized pulmonary amyloid (n ¼ 11), senile amyloid (n ¼ 6), and familial (n ¼ 1). A review of 126 cases of localized AL amyloid identified three major forms of pulmonary disease namely (1) tracheobronchial disease (53%), (2) nodular opacities (44%), and (3) diffuse opacities (3%). Multinodular parenchymal amyloidosis associated with thin-walled pulmonary cysts has been described in patients with Sjogren’s syndrome (Table 1). Tracheobronchial Amyloidosis

TBA may be the only site of involvement, with submucosal amyloid deposits in the airways ranging from the larynx to the distal airways. It is rare; a review of the literature in 2002 found fewer than 140 published cases of TBA. Even at large referral centers, less than 1–2 cases per year of TBA are diagnosed. Etiology Most cases of TBA are due to excessive deposition of light chains, produced from localized collections of plasma cells or BALT. Clinical features Narrowing or involvement of the nasopharynx, sinuses, larynx, trachea, or bronchi may give rise to symptoms such as hoarseness, wheezing, dyspnea, hemoptysis, cough, atelectasis,

Table 1 Pulmonary and TBA Radiographic features Single or multiple nodular or mass lesions (0.4–15 cm) Tracheobronchial amyloidosis: airway wall thickening, irregularly narrowed airway lumens, heterotopic tracheal wall calcifications Diffuse interstitial, reticular, or micronodular lesions Hilar or mediastinal lymphadenopathy Pleural effusions

recurrent pneumonia, or chronic infections. In TBA, the lung parenchyma is spared. Bronchoscopic features of TBA include single or multifocal submucosal plaques or nodules (Figure 8) and/or varying degrees of stenosis of the trachea or bronchi. Some cases are associated with tracheobronchopathia osteoplastica, a rare disorder characterized by calcified or cartilaginous submucosal nodules within the tracheobronchial tree. Amyloidosis involving the trachea may cause severe (even fatal) airflow obstruction. Pulmonary function tests (PFTs) in patients with proximal tracheal stenosis reveal truncation of peak inspiratory and expiratory flow rates (consistent with fixed upper airway obstruction (UAO)). Involvement of distal trachea or bronchi may manifest in air trapping; flow rates may be normal or obstructed. The configuration of flow-volume loops can usually distinguish UAO from distal stenoses. Spirometry is invaluable in detecting progressive airflow obstruction. Spiral CT scans are useful to quantitatively assess the degree and sites of airway narrowing, and follow the course of the disease. CT scans demonstrate tracheal- or bronchial-wall thickening, nodules, or plaques, irregularly narrowed airway lumens, and heterotopic tracheal-wall calcifications in patients with long-standing TBA. Postobstructive features comprise atelectasis, bronchiectasis, and consolidation, or hyperinflation. Management and current therapy The course of TBA is usually slowly progressive; one-third of patients with proximal or severe mid-airway disease die from progressive respiratory insufficiency within 7 to 12 years after diagnosis. Symptomatic foci involving trachea, larynx, or bronchi require intermittent bronchoscopic dilatation or debridement, surgical resection, laser therapy, tracheostomy, or tracheal resection. Recurrence is frequent, and stenosis of airways may ensue. Unfortunately, repetitive

Histologic features Plaques, nodules, interstitial or vascular deposits amyloid protein Congo red ( þ ); green birefringence under polarized light microscopy Submucosal nodules of amyloid with or without calcification (tracheobronchial tree) Therapy Localized pulmonary amyloidosis: surgical resection or no treatment

Figure 8 Tracheal amyloid. The submucosa is filled with deposits of amyloid.

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bronchoscopy or laser treatments denude airways and promote collagen deposition. In rare cases, tracheostomy is required. External beam radiation therapy has been used, with anecdotal successes, for severe airflow obstruction. Chemotherapeutic agents, colchicine, or corticosteroids are of doubtful efficacy. Because TBA is localized, systemic agents should generally be avoided. See also: Stem Cells. Surfactant: Surfactant Proteins B and C (SP-B and SP-C).

in patients with amyloidosis in the respiratory tract. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 19: 134–142. Thompson PJ and Citron KM (1983) Amyloidosis and the lower respiratory tract. Thorax 38: 84–87. Utz JP, Swensen SJ, and Gertz MA (1996) Pulmonary amyloidosis. The Mayo Clinic Experience from 1980–1993. Annals of Internal Medicine 124: 407–413.

Cryptogenic Organizing Pneumonia J-F Cordier, Claude Bernard University, Lyon, France

Further Reading Bergethon PR, Sabin TD, Lewis D, et al. (1996) Improvement in the polyneuropathy associated with familial amyloid polyneuropathy after liver transplantation. Neurology 47: 951–955. Berk JL, O’Regan A, and Skinner M (2002) Pulmonary and tracheobronchial amyloidosis. Seminars in Respiratory Critical Care Medicine 23: 155–166. Capizzi SA, Betancourt E, and Prakash UB (2000) Tracheobronchial amyloidosis. Mayo Clinic Proceedings 75: 1148–1152. Comenzo FL, Vosburgh E, Falk RH, et al. (1998) Dose–intensive melphalan with blood stem-cell support for the treatment of AL (amyloid light-chain) amyloidosis: survival and responses in 25 patients. Blood 191: 3662–3670. Cordier JF, Loire R, and Brune J (1986) Amyloidosis of the lower respiratory tract. Clinical and pathologic features in a series of 21 patients. Chest 90: 827–831. Falk RH, Comenzo RL, and Skinner M (1997) The systemic amyloidoses: current approaches to diagnosis and treatment. The New England Journal of Medicine 337: 898–909. Gertz MA, Kyle RA, and Thibodeau SN (1992) Familial amyloidosis: a study of 52 North American-born patients examined during a 30-year period. Mayo Clinic Proceedings 67: 428–440. Gillmore JD and Hawkins PN (1999) Amyloidosis and the respiratory tract. Thorax 54: 444–451. Hawkins PN, Lavender JP, and Pepys MB (1990) Evaluation of systemic amyloidosis by scintigraphy with 123I-labelled serum amyloid P component. The New England Journal of Medicine 323: 508–513. Hui HN, Koss MN, Hochholzer L, et al. (1986) Amyloidosis presenting in the lower respiratory tract: clinicopathologic, radiologic, immunohistochemical, and histochemical studies on 48 cases. Archives of Pathology & Laboratory Medicine 110: 212– 218. Khoor A, Myers JL, Tazelaar HD, and Kurtini PJ (2004) Amyloidlike pulmonary nodules, including localized light-chain deposition: clinicopathologic analysis of three cases. American Journal of Clinical Pathology 121: 200–204. Kim HY, Im JG, Song KS, et al. (1999) Localized amyloidosis of the respiratory system: CT features. Journal of Computer Assisted Tomography 23: 627–631. Kyle RA, Gertz MA, Greipp PR, et al. (1997) A trial of three regimens for primary amyloidosis: colchicine alone, melphalan and prednisone, and melphalan, prednisone and colchicine. The New England Journal of Medicine 336: 1202–1207. O’Regan A, Fenlon HM, Beamis JF Jr, et al. (2000) Tracheobronchial amyloidosis: the Boston University experience from 1984 to 1999. Medicine (Baltimore) 79: 69–79. Shah PL, Gillmore JD, Copley SJ, et al. (2002) The importance of complete screening for amyloid fibril type and systemic disease

& 2006 Elsevier Ltd. All rights reserved.

Abstract Cryptogenic organizing pneumonia (formerly called idiopathic bronchiolitis obliterans organizing pneumonia) is an infiltrative lung disease of unknown cause defined by a pathologic pattern of organizing pneumonia consisting of buds of granulation tissue in the distal airspaces. Symptoms include mild dyspnea, fever, and weight loss. Sparse crackles are heard at auscultation over involved areas. Imaging features typically consist of multiple patchy subpleural alveolar opacities, often containing an air bronchogram, and sometimes migratory. Although many causes of organizing pneumonia have been reported (especially infection, and drug-induced reaction) or its occurrence in specific contexts (especially in the connective tissue diseases), it is more often cryptogenic (idiopathic). Improvement is obtained within a few days with corticosteroid treatment, but relapses are common after reducing or stopping treatment.

Introduction Cryptogenic organizing pneumonia (COP) is a rare distinct idiopathic interstitial pneumonia included in the American Thoracic Society (ATS)/European Respiratory Society (ERS) international multidisciplinary consensus classification of the idiopathic interstitial pneumonias. Organizing pneumonia was historically described as a process of nonresolving infectious pneumonia. The four successive macroscopic stages of acute lobar pneumonia were described by Laennec at the beginning of the nineteenth century. In the first stage the affected lobe is congested and heavy. The following stage of red hepatization is characterized by a red and dense lobe (as the normal liver). Then gray hepatization takes place with the lobe becoming gray and airless. Finally, in favorable cases, the pneumonia resolves with normalization of the lobe. Failure of the latter process characterizes nonresolving pneumonia. By the end of the nineteenth and the beginning of the twentieth century the usual cause of acute lobar pneumonia was identified as being an infection due to Streptococcus pneumoniae, and the four

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macroscopic stages described by Laennec were further studied histopathologically. The sequential stages of pneumonia were characterized by the following lesions: intra-alveolar edema with many pneumococci, polymorphonuclear leukocytes, and erythrocytes; fibrin intra-alveolar exudates; liquefaction of fibrin exudates; and eventual resolution of pneumonia without significant sequelae. Nevertheless, before antibiotics were available a number of patients died from pneumococcal pneumonia, including nonresolving pneumonia. Pathologists observed that nonresolving pneumonia was characterized by a process of intra-alveolar organization of the inflammatory fibrinous exudates with a final characteristic lesion consisting of granulation tissue (as seen in wound healing) formed by intra-alveolar buds of fibroblasts embedded in connective matrix. Pathologists later considered for a long time that the histopathological finding of the organizing pneumonia pattern was just a witness of a previous nonresolving infection with little clinical significance. However, this pathologic pattern and the concept of organizing pneumonia were revisited two decades ago when it was matched with characteristic clinical imaging features, thus allowing the identification of a clinical syndrome of unknown origin called COP.

pneumophila. Other bacteria as well as viruses, fungi, and parasites have been reported to induce organizing pneumonia (Table 1). Drug-induced organizing pneumonia (Table 2) represents one of the histopathological pulmonary patterns secondary to drug reactions which also comprise diffuse alveolar damage, eosinophilic pneumonia, alveolar hemorrhage, etc. Drug-induced organizing pneumonia is often difficult to diagnose especially when the disorder for which the drug has been prescribed may by itself be associated with organizing pneumonia (e.g., connective tissue disease or immunopathological processes associated with transplantation). The eventual diagnosis of druginduced organizing pneumonia often remains elusive because corticosteroid treatment is generally given to hasten improvement after drug withdrawal. Iatrogenic organizing pneumonia after radiation therapy for breast cancer is distinct from radiation lung injury which is confined to the radiation field. It differs from the latter by the involvement of the nonirradiated parts of the lungs and complete improvement under corticosteroid treatment without significant sequelae. It develops within 3–6 months after radiotherapy. Migration of the pulmonary opacities on chest imaging occurs in a majority of patients, before treatment or at relapse.

Etiology Table 2 Drugs causing drug-induced organizing pneumonia

By definition, the cause of COP is unknown. The final diagnosis is thus made only once the known causes of organizing pneumonia have been carefully excluded. Organizing pneumonia may also be diagnosed in specific contexts or conditions of unknown etiology, such as the connective tissue diseases. Furthermore several causes or specific associated conditions may contribute to the development of organizing pneumonia. As mentioned above, bacterial infection was the first established cause of organizing pneumonia. The main bacteria causing organizing pneumonia are probably Streptococcus pneumoniae and Legionella

Acebutolol 5-Aminosalicylic acid Amiodarone Amphotericin B Bleomycin Busulphan Carbamazepine Chlorambucil Doxorubicin Fluvastatin Gold salts Interferon alpha Interferon beta 1a

Mesalazine Methotrexate Minocycline Nilutamide Nitrofurantoin Phenytoin Sirolimus Sotalol Sulfasalazine Tacrolimus Ticlopidine Transtuzumab L-Tryptophan

Table 1 Infectious agents causing organizing pneumonia Bacteria

Viruses

Parasites

Fungi

Streptococcus pneumoniae Legionella pneumophila Others Burkholderia cepacia Chlamydia pneumoniae Coxiella burnetii Mycoplasma pneumoniae Nocardia asteroides Pseudomonas aeruginosa Serratia marcescens Staphylococcus aureus

Adenovirus Cytomegalovirus Herpesvirus Human immunodeficiency virus Influenza virus Parainfluenza virus Human herpesvirus-7 Respiratory syncytial virus

Plasmodium vivax Dirofilaria immitis

Cryptococcus neoformans Penicillium janthinellum Pneumocystis jiroveci

428 INTERSTITIAL LUNG DISEASE / Cryptogenic Organizing Pneumonia

The organizing pneumonia histopathological pattern has been described in association with the connective tissue diseases as well as those of nonspecific interstitial pneumonia (NSIP) and of usual interstitial pneumonia. It may develop in the course of a previously diagnosed connective tissue disease, or it may precede the clinical systemic manifestations, or be contemporaneous with these. Organizing pneumonia is especially common in dermatomyositis/polymyositis. The muscular and/or cutaneous involvement may manifest only when corticosteroid treatment given for organizing pneumonia is decreased or stopped. Organizing pneumonia has also been reported in rheumatoid arthritis, but is much more rarely seen in the other connective tissue diseases (systemic lupus erythematosus, scleroderma, Sjo¨gren syndrome). Organizing pneumonia has increasingly been described in patients after lung transplantation or allogenie bone marrow graft, as a process distinct from the constrictive bronchiolitis with airflow obstruction common in these conditions. Organizing pneumonia is often associated with acute rejection after lung transplant, and it is correlated with chronic bronchiolitis obliterans. After allogenic bone marrow graft organizing pneumonia may be associated with graft-versus-host disease. Although organizing pneumonia is likely a process of rejection, or graft-versus-host disease in the respective context of lung transplantation and bone marrow graft, it may also result from infection, aspiration pneumonitis, drug-induced reaction, or from several associated processes. Case reports or short series have described organizing pneumonia occurring in various settings susceptible to induce an inflammatory pulmonary process (Table 3).

Table 3 Organizing pneumonia of undetermined cause in specific contexts Connective tissue disorders

Bone marrow graft and transplantation

Miscellaneous

Polymyositis/ dermatomyositis Rheumatoid arthritis

Bone marrow graft Lung transplantation Liver transplantation

Myelodysplasia Leukemia Myeloproliferative disorders Lung cancer Sarcoidosis Ulcerative colitis Crohn’s disease Sweet’s syndrome Polymyalgia rheumatica Common variable immunodeficiency Wegener’s granulomatosis Polyarteritis nodosa Thyroid disease Cystic fibrosis Coronary artery bypass graft surgery Spice processing

Pathology The histopathologic pattern of organizing pneumonia is defined by the presence of buds of granulation tissue (consisting of fibroblasts and myofibroblasts embedded in a loose connective matrix) within the lumen of the distal airspaces (i.e., the alveoli, the alveolar ducts, and possibly the bronchioles). This pattern has also been called bronchiolitis obliterans organizing pneumonia (BOOP). However, since organizing pneumonia is the characteristic and predominant pathologic pattern with bronchiolitis being only an accessory finding, the present international terminology is organizing pneumonia which further avoids confusion with the different types of bronchiolitis obliterans (especially bronchiolitis obliterans with airflow obstruction).

Figure 1 Histopathological pattern of organizing pneumonia with buds of granulation tissue within the alveoli.

The histopathologic diagnosis of the pattern of organizing pneumonia first necessitates the finding of the characteristic buds of granulation tissue in the distal airspaces especially the alveoli (Figure 1). These may extend from one alveolus to the adjacent one through the pores of Kohn thus forming butterfly-shaped figures. The process is patchy, and usually the lesions are all the same age. The different stages of organization (from fibrinoid inflammatory cell clusters to mature fibrotic buds) may be found on the

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same biopsy specimen but the lesions are usually temporally uniform. The lesions are bronchiolocentric and distal to the bronchioles. Cellular inflammatory interstitial infiltration is usually present but it is mild or moderate. Foamy macrophages are commonly present in the airspaces. In order to make a diagnosis of COP the pattern of organizing pneumonia must clearly be the major type of lesion and not just an accessory finding of another interstitial pneumonia as NSIP, especially cellular or cellular and fibrotic, where intra-alveolar buds are commonly found. Negative findings of the organizing pneumonia pattern include the lack of prominent interstitial fibrosis, the lack of fibroblastic foci, granulomas, hyaline membranes, vasculitis, and eosinophilia. Other processes where organizing pneumonia may be present and even prominent must be excluded, as the BOOP variant of Wegener’s granulomatosis. Although lung specimens obtained by transbronchial lung biopsy may show the characteristic intraalveolar and occasionally intrabronchiolar buds of granulation tissue, it does not allow the exclusion of other possible specific lesions or etiologic agents (such as uncommon infections, neoplasia, vasculitis) which may be demonstrated on larger lung specimens (including with the help of special stains or microbiological analysis of lung tissue). Videothoracoscopic lung biopsy is currently the preferred technique both to establish the histopathologic diagnosis of organizing pneumonia and to exclude associated lesions specific for other disorders.

Physical examination is non-specific. Some fine crackles are often heard over affected pulmonary areas but there are no wheezes and no diffuse velcro rales. Finger clubbing is not present. Imaging

The chest X-ray and especially high-resolution computed tomography (HRCT) findings have a major role in the diagnosis and further define the main characteristic types of COP. The most typical imaging features of COP (classical COP) comprise patchy alveolar opacities on chest X-ray, usually bilateral (Figure 2), asymmetric, and predominating in the lower lung zones. These may migrate in the lungs within a few days (with the ensuing necessity of making a chest X-ray within the 2 days preceding a lung biopsy to ensure it is done in an affected area). On HRCT, the density of the opacities varies from ‘ground glass’ to consolidation (Figure 3). Their size ranges from a few

Clinical Features COP affects men and women equally (a female predominance is found in some series). It usually occurs at the age of 50–60 years, and more commonly in nonsmokers or ex-smokers.

Figure 2 Typical COP with bilateral alveolar opacities on chest X-ray.

Clinical Manifestations

Often preceded by a flu-like syndrome, symptoms generally develop subacutely over a few weeks with mild and persistent fever, anorexia, weight loss, sweats, nonproductive cough, and mild dyspnea. Chest pain, hemoptysis, and bronchorrhea are uncommon. The initial diagnosis being usually that of an infectious pulmonary disease, most patients receive antibiotics which are not efficient, thus leading to consideration of another diagnosis. Diagnosis of COP is usually obtained only after about 6–12 weeks. Occasionally symptoms are more severe and some patients present with features of acute respiratory distress syndrome (ARDS).

Figure 3 Typical COP with patchy alveolar opacities on HRCT (with an air bronchogram on the left).

430 INTERSTITIAL LUNG DISEASE / Cryptogenic Organizing Pneumonia

centimeters to an entire lobe. They are generally peripheral, but they may predominate in a peribronchovascular location (bronchocentric pattern). Sometimes these opacities may present as large nodules or masses often containing an air bronchogram. The clinical syndrome together with the above typical imaging features must evoke the possible diagnosis of COP to the clinician. Based on the finding of the characteristic features of patchy subpleural and/ or peribronchovascular airspace consolidation often associated with areas of ground glass attenuation, COP is diagnosed correctly in about 80% of cases in patients with idiopathic interstitial pneumonia. The second common clinical imaging presentation of COP consists of solitary focal COP presenting as a solitary nodule or mass, often located in the upper lobes. Some patients are asymptomatic, with both diagnosis and cure obtained by surgical excision of the lesion suspected to correspond to lung cancer (intense tracer uptake may occur on 18F fluorodeoxyglucose positron emission tomography in organizing pneumonia). Other patients present with persistent fever and inflammatory biologic abnormalities despite antibiotics. Hemoptysis and cavitation of the mass may be present. The third distinct and more confusing clinical imaging presentation of COP is that of diffuse infiltrative lung disease with interstitial opacities on HRCT often associated with superimposed small alveolar opacities (Figure 4). Although there is no honeycombing initially, some reticular subpleural opacities and eventually honeycombing may later develop. Histopathologically this type of COP is usually associated with more interstitial mononuclear inflammation and overlaps with NSIP especially cellular with foci of intra-alveolar buds of granulation tissue.

Many other less characteristic imaging features of COP have been occasionally reported, including small nodular opacities, irregular linear opacities, bronchial wall thickening, dilatation pneumatocele, and multiple cavitary lung nodules. The micronodular pattern centered on the bronchioles may correspond to bronchiolitis with peribronchiolar organizing. Band-like areas of consolidation in the subpleural area (paralleling the chest wall) may be observed especially during healing on corticosteroid treatment (a finding also present in idiopathic chronic eosinophilic pneumonia). Central ground glass opacity and surrounding airspace consolidation of crescent or ring shape (reversed halo sign) may be found in some patients, usually in association with other types of opacity. Laboratory Findings

There is no laboratory finding really helpful in the diagnosis of COP. An increased level of blood leukocytes with an increase in the proportion of neutrophils is usual. The erythrocyte sedimentation rate and C-reactive protein level are increased. Occasionally antinuclear antibodies and rheumatoid factor are present (usually at a low titer) in the absence of patent connective tissue disease. Gammaglutamyl transferase and alkaline phosphatase are mildly increased. Bronchoalveolar Lavage

Bronchoalveolar lavage (BAL) differential cell count is helpful especially when showing a ‘mixed pattern’ with an increase in lymphocytes (about 25–45% with a decreased CD4 þ /CD8 þ ratio in the majority of cases), neutrophils (about 10%), and eosinophils (about 5%). Foamy macrophages are often present, and interestingly some mast cells and plasma cells may be found in some patients. In addition to providing arguments for the diagnosis of COP the BAL helps in excluding causes of organizing pneumonia as infectious processes. It also helps in the differential diagnosis (eosinophilia pneumonia, lymphoma, bronchioloalveolar carcinoma). Lung Function Tests

Figure 4 COP with an infiltrative pattern on HRCT.

Lung function tests in patients with COP usually show a mild (or moderate) restrictive pattern. Airflow obstruction is not typically present, although it may be found in smoker patients with underlying chronic obstructive pulmonary disease. The CO transfer factor is reduced, but the CO transfer coefficient is usually normal. Mild hypoxemia is usually present. More severe hypoxemia may occur in patients with severe, rapidly

INTERSTITIAL LUNG DISEASE / Cryptogenic Organizing Pneumonia 431

progressive diffuse COP; in patients with an underlying respiratory disease (e.g., emphysema); or in patients with a right-to-left shunt at the alveolar level demonstrated by the presence of an increased alveolar–arterial oxygen gradient while breathing 100% oxygen together with negative contrast echocardiography.

Pathogenesis COP is a quite original model of pulmonary inflammatory and fibrosing disease because of the usual complete disappareance of the intra-alveolar buds of connective matrix with corticosteroid treatment. This is in marked contrast with the other fibrosing interstitial lung diseases, especially idiopathic pulmonary fibrosis, where dense and mutilating fibrosis relentlessly progresses despite all therapeutic attempts. The dynamic histopathological features offer some insight on the mechanisms of organization, with the individualization of a sequence of distinct processes. Alveolar epithelial cells are the first target of injury. Pneumocytes undergo necrosis thus leaving the epithelial basal laminae denuded. Whereas cell necrosis is intense the basal laminae remain well preserved although some gaps are present. The striking difference with the diffuse alveolar damage seen in ARDS (or acute interstitial pneumonia) is the lack of hyaline membranes. Inflammatory cells are present in the interstitium. The endothelial cells are not markedly damaged. Fibrinoid inflammatory cell clusters appearing within the alveolar lumen represent the first stage of the process of organization. Inflammatory cells (especially mononuclear cells) are associated with bands of fibrin. Then fibrin is fragmented and inflammatory cells are less numerous as fibroblasts colonize the fibrin deposits after they have entered the alveolar lumen through gaps in the basal laminae. The proliferation of fibroblasts is illustrated by the presence of mitotic figures. The fibroblasts further undergo phenotypic modulation whereby they acquire intracellular filaments characteristic of myofibroblasts. The parallel re-epithelialization of the basal laminae is probably a key to avoid the interstitial incorporation of the intra-alveolar connective matrix into the alveolar interstitium. The final stage of organization is that of the typical mature fibrotic buds. At this stage usually no more fibrin is present and only few inflammatory cells may persist within the buds. Alternating circular layers of fibroblast/ solidus myofibroblasts and connective matrix deposits compose the mature buds. Whereas in usual interstitial pneumonia (the pathologic pattern of idiopathic pulmonary fibrosis) the connective matrix

consists predominantly of dense bundles of collagen I, the loose connective matrix of the buds in COP comprises collagen III and fibronectin in addition to collagen I, together with glycoproteins and other collagen types. Intra-alveolar bud capillarization is prominent (with vascular endothelial growth factor and fibroblast growth factor widely expressed in the buds). This organization of the intra-alveolar buds shares many features with the granulation tissue developing during cutaneous wound healing. Interestingly both conditions represent young reversible fibrosis capable of complete healing without significant fibrotic scarring. The biopathological interpretation of the above morphological processes is largely inferred from human or experimental studies of early diffuse alveolar damage. The formation of fibrin with the alveolar lumen results from the leakage of plasma coagulation proteins as a consequence of epithelial–endothelial alveolar damage, with clotting resulting from an intra-alveolar inbalance between the coagulation and fibrinolytic cascades. The coagulation and fibrinolysis factors and inhibitors including plasminogen activator inhibitor 1 have a complex role in the process of fibrogenesis. Tissue remodeling further necessitates the cleaving of the connective matrix components by matrix metalloproteinases which may play a major role in the disappearance of the buds. The main factors for the parallel disappearance of the fibroblasts and myofibroblasts remain a mystery. This contrasts with the fibrosing and mutilating role attributed to the fibroblast foci (which morphologically resemble the buds) in usual interstitial pneumonia.

Animal Models Only few experimental animal studies have been conducted in organizing pneumonia. The early stage of alveolar damage may be studied in monkeys receiving paraquat or in rats after intratracheal instillation of bleomycin, but the most interesting animal model of organizing pneumonia used intranasal inoculation of reovirus at various titers in CBA/J mice. This model especially demonstrated the possible role of genetic factors as the lesions develop in CBA/J strains of mice but not in other strains. Furthermore, whereas moderate titer of reovirus induces organizing pneumonia, higher titers determine the development of diffuse alveolar damage. Interestingly only intraluminal alveolar lesions produced by moderate doses of reovirus are improved by corticosteroids. Thus this animal model suggests that both genetic factors and the degree of severity of the initial alveolar injury determine the evolution toward

432 INTERSTITIAL LUNG DISEASE / Cryptogenic Organizing Pneumonia

either nonreversible diffuse alveolar damage or organizing pneumonia capable of improvement with corticosteroids.

Management and Current Therapy The main initial prerequisite for management is the confidence of diagnosis, both positive and etiologic. The differential diagnosis of typical COP is listed in Table 4. The current history and investigations usually exclude these alternative diagnoses rather easily. The diagnosis of COP on transbronchial biopsy findings is reliable only in patients with the typical clinicoradiological profile of COP and a careful follow-up so that any unusual evolution may be detected soon and a surgical lung biopsy done (allowing for example a diagnosis of pulmonary lymphoma having initially responded to corticosteroid treatment). The diagnosis of COP without any histopathologic confirmation is hazardous and may be accepted only in rare cases by an experienced clinician. Videothoracoscopic lung biopsy is the reference diagnostic procedure. It is a safe procedure which allows a definite diagnosis of COP and allows the recognition of other disorders requiring specific treatment (as vasculitis). It is also useful in convincing patients reluctant to prolonged corticosteroid treatment to accept it. The etiologic diagnosis of organizing pneumonia always need to be very carefully examined to disclose any definite cause (especially drugs) or underlying disorder (as paucisymptomatic connective tissue disease). When the etiologic diagnosis is negative, the diagnosis of cryptogenic (idiopathic) organizing pneumonia may be definitely accepted. Although occasional improvement of typical COP has been reported spontaneously or after treatment with antibiotics, corticosteroids remain the current reference treatment. The response to corticosteroids is very rapid. The clinical manifestations Table 4 Differential diagnosis of typical COP presenting with multiple patchy alveolar opacities Infectious pneumonia Aspiration pneumonia and aspiration pneumonitis Pulmonary thromboembolism Bronchioloalveolar carcinoma Primary pulmonary lymphoma Chronic eosinophilic pneumonia Exogeneous lipid pneumonia Sarcoidosis Alveolar lipoproteinosis Alveolar hemorrhage Wegener’s granulomatosis

improve within 48 h, and complete resolution of the pulmonary opacities on imaging is obtained within a few days or weeks without significant functional or imaging sequelae. Although the efficiency of corticosteroids is well established, the best dose and duration of treatment have not been determined. High doses (1 mg kg
1 day
1) of prednisone for 1–3 months, then 40 mg day
1 for 3 months, then 10 mg daily (or 20 mg every other day) for 1 year, have been proposed. We use lower doses and shorter duration of treatment, with 0.75 mg kg
1 day
1 of prednisone for 4 weeks, then 0.5 mg kg
1 day
1 for 6 weeks, then 20 mg day
1 for 6 weeks, then 5 mg day
1 for 6 weeks. Although the response to corticosteroids is excellent, a proportion of patients (about 15–60%) experience a relapse of COP, and several relapses for some of them. About two-thirds of patients still receive corticosteroids (at a mean dose of about 10 mg day
1 prednisone) when they experience relapse. Relapse on more than 20 mg day
1 prednisone is rare and must lead to reconsider the diagnosis of COP (especially in patients with disease diagnosed without surgical biopsy). The treatment of relapses may use moderate doses of prednisone (about 20 mg day
1). The predictors of multiple relapses occurrence comprise delayed treatment and mild cholestasis (elevated gamma-glutamyl transferase and alkaline phosphatase). Relapses are not associated with increased mortality nor increased long-term functional morbidity. Treatment aims at obtaining an optimal balance between efficiency and the adverse effects of corticosteroids. The prognosis of COP treated with corticosteroids is excellent in the vast majority of cases. Patients with severe progressive disease not responding to corticosteroids often have overlapping conditions with pathologic features of organizing diffuse alveolar damage or acute fibrinous and organizing pneumonia. See also: Acute Respiratory Distress Syndrome. Bronchoalveolar Lavage. Coagulation Cascade: Overview. Corticosteroids: Glucocorticoid Receptors; Therapy. Drug-Induced Pulmonary Disease. Extracellular Matrix: Matricellular Proteins; Degradation by Proteases. Fibrinolysis: Overview. Fibroblasts. Interstitial Lung Disease: Overview. Lung Imaging. Matrix Metalloproteinase Inhibitors. Matrix Metalloproteinases. Myofibroblasts. Permeability of the Blood–Gas Barrier. Pneumonia: Overview and Epidemiology. Polymyositis and Dermatomyositis. Pulmonary Effects of Systemic Disease. Pulmonary Fibrosis. Radiation-Induced Pulmonary Disease. Surgery: Transplantation.

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Further Reading American Thoracic Society/European Respiratory Society (2002) Classification of the idiopathic interstitial pneumonias: international multidisciplinary consensus. American Journal of Respiratory and Critical Care Medicine 165: 277–304. Beasley MB, Franks TJ, Galvin JR, Gochuico B, and Travis WD (2002) Acute fibrinous and organizing pneumonia: a histological pattern of lung injury and possible variant of diffuse alveolar damage. Archives of Pathology & Laboratory Medicine 126: 1064–1070. Colby TV (1992) Pathologic aspects of bronchiolitis obliterans organizing pneumonia. Chest 102: 38S–43S. Cordier JF, Loire R, and Brune J (1989) Idiopathic bronchiolitis obliterans organizing pneumonia: definition of characteristic clinical profiles in a series of 16 patients. Chest 96: 999–1004. Cordier JF, Peyrol S, and Loire R (1994) Bronchiolitis obliterans organizing pneumonia as a model of inflammatory lung disease. In: Epler GR (ed.) Diseases of the Bronchioles, pp. 313–345. New York: Raven Press. Crestani B, Valeyre D, Roden S, et al. (1998) Bronchiolitis obliterans organizing pneumonia syndrome primed by radiation therapy to the breast. American Journal of Respiratory and Critical Care Medicine 158: 1929–1935. Davison AG, Heard BE, McAllister WAC, and Turner-Warwick ME (1983) Cryptogenic organizing pneumonitis. Quarterly Journal of Medicine 52: 382–394. Epler GR, Colby TV, McLoud TC, Carrington CB, and Gaensler EA (1985) Bronchiolitis obliterans organizing pneumonia. New England Journal of Medicine 312: 152–158. Johkoh T, Muller NL, Cartier Y, et al. (1999) Idiopathic interstitial pneumonias: diagnostic accuracy of thin-section CT in 129 patients. Radiology 211: 555–560. Lazor R, Vandevenne A, Pelletier A, et al. (2000) Cryptogenic organizing pneumonia: characteristics of relapses in a series of 48 patients. American Journal of Respiratory and Critical Care Medicine 162: 571–577. Lohr RH, Boland BJ, Douglas WW, et al. (1997) Organizing pneumonia: features and prognosis of cryptogenic, secondary, and focal variants. Archives of Internal Medicine 157: 1323–1329. Lynch DA, Travis WD, Mu¨ller NL, et al. (2005) Idiopathic interstitial pneumonias: CT features. Radiology 236: 10–21. Oikonomou A and Hansell DM (2002) Organizing pneumonia: the many morphological faces. European Radiology 12: 1486–1496. Raghu G (ed.) (2004) Interstitial lung disease: idiopathic interstitial pneumonia. Clinics in Chest Medicine 25: 621–782. Travis WD, Colby TV, Koss MN, et al. (2002) Atlas of Nontumor pathology: Non-Neoplastic Disorders of the Lower Respiratory Tract. Washington, DC: American Registry of Pathology and Armed Forces Institute of Pathology.

Hypersensitivity Pneumonitis V Poletti, Ospedale GB Morgagni, Forli, Italy M Zompatori, University of Parma, Parma, Italy P Vailati, Ospedale GB Morgagni, Forli, Italy M Chilosi, University of Verona, Verona, Italy & 2006 Elsevier Ltd. All rights reserved.

Abstract Hypersensitivity pneumonitis (HP), also called extrinsic allergic alveolitis, is a group of immunologically mediated lung diseases

in which the repeated inhalation of certain finely dispersed antigens, mainly organic particles or low-molecular-weight chemicals, provokes a hypersensitivity reaction with granulomatous inflammation in the distal bronchioles and alveoli of susceptible subjects. The disease has three kinds of clinical presentation – acute, subacute, or chronic – depending on the frequency and intensity of exposure to the antigen and the duration and clinical course of the disease. The diagnosis of HP is often made on the basis of clinical history of exposure with resulting respiratory symptoms, but the definitive diagnosis requires a constellation of clinical, radiologic, laboratory, and pathologic findings. The complete avoidance of exposure to the inciting antigen may result in a spontaneous resolution of the symptoms, especially for the acute form; the chronic progressive form may improve, stabilize, or, rarely, evolve to fibrosis and respiratory insufficiency even though the patient completely avoids re-exposure to the inciting antigen. Corticosteroid therapy can significantly and quickly reduce acute symptoms; however, the long-term beneficial effects on arresting disease progression in subacute and chronic HP remain to be definitively established.

Introduction Hypersensitivity pneumonitis (HP), also called extrinsic allergic alveolitis, is a group of immunologically mediated lung diseases in which the repeated inhalation of certain finely dispersed antigens, mainly organic particles or low-molecular-weight chemicals, provokes a hypersensitivity reaction with granulomatous inflammation in the distal bronchioles and alveoli of susceptible subjects. In 1713, B. Ramazzini from Carpi, one of the fathers of occupational medicine, described a grain harvester disease in De Morbis Artificum Diatriba. In 1932, J. Campbell described the clinical features of an acute illness in a farmer following work with hay and coined the term ‘Farmer’s lung’. Subsequently, a large number of etiologic agents have been recognized and new causes continue to be recognized. The disease has three general forms of clinical presentation: acute, subacute, or chronic. These presentations depend mainly on the intensity of exposure to the antigen and frequency and the duration and clinical course of the disease. The diagnosis of HP is often considered on the basis of history of exposure to an antigen known to cause the illness. However, definitive diagnosis requires a constellation of clinical, radiologic, laboratory, and pathologic findings.

Etiology Agents implicated as causes of HP are numerous and can be found in different occupations, hobbies, and environments (Table 1). Causative antigens include microbes, animal and plant proteins, and organic and inorganic low-molecular-weight particles.

434 INTERSTITIAL LUNG DISEASE / Hypersensitivity Pneumonitis Table 1 Causative agents of hypersensitivity pneumonitisa Agent

Source

Disease

Fungal and bacterial Thermophilic actinomycetes Saccharopolyspora rectivirgula Thermoactinomyces vulgaris

Moldy plant materials Moldy hay Moldy hay, compost

Farmer’s lung

Sugarcane residue Detergent enzymes Moldy grains Animal bedding Tobacco mold Esparto dust Cheese mold Moldy cork Moldy wood dust Moldy maple bark Moldy sequoia dust Contaminated water Wood or wood pulp Rotten wood Grape mold Mold in Japanese homes Sewage Paprika Saxophone mouthpiece Contaminated water Aerosolized metalworking fluids

Farmer’s lung, mushroom worker’s lung, composter’s lung Bagassosis Detergent worker’s lung Malt worker’s lung Doghouse disease Tobacco worker’s lung Stipatosis Cheese washer’s lung Suberosis Woodworker’s lung Maple bark stripper’s lung Sequoiosis Sauna taker’s disease Woodworker’s lung Dry rot lung Wine grower’s lung or Spa¨etlase lung Summer-type HP Sewage worker’s lung Paprika splitter’s lung Sax lung Hot tub lung Machine operator’s lung

Amoebae Naegleria gruberi, Acanthamoeba

Contaminated water

Humidifier lung

Animals Avian proteins Rat proteins Gerbil proteins Animal fur protein Ox and pork protein Mollusk shell protein Fish Wheat weevil Silkworm larvae proteins

Bird excreta, blood, or feather Rat urine or serum Gerbil Animal fur Pituitary snuff Mollusk shell dust Fishmeal dust Flour Silkworm larvae

Bird breeder’s lung, bird fancier’s lung Rodent handler’s lung Gerbil keeper’s lung Furrier’s lung Pituitary snuff taker’s lung Oyster shell lung Fishmeal worker’s lung Miller’s lung Sericulturist’s lung

Plants Soybean Coffee Lycoperdon species

Soybean hulls Coffee bean dust Puffballs

Soybean worker’s lung Coffee worker’s lung Lycoperdonosis

Chemicals Isocyanates Anhydrides

Paints, plastics Plastics

Paint refinisher’s lung Chemical worker’s lung, plastic worker’s lung, epoxy worker’s lung Pauli’s reagent lung Vineyard sprayer’s lung Insecticide lung Epoxy resin lung

Thermoactinomyces sacchari Bacillus subtilis enzimes Aspergillus clavatus Aspergillus versicolor Aspergillus species Aspergillus fumigatus Penicillium casei, A. Clavatus Chrysonilia sitophila Penicillium chrysogenum Cryptostroma corticale Aureobasidium pullulans Aureobasidium species Alternaria species Merulius lacrymans Botrytis cinerea Trichosporon cutaneum Cephalosporium Mucor stolonifer Candida albicans Mycobacterium avium-intracellulare Pseudomonas fluorescens

Sodium diazobenzene sulfate Copper sulfate Pyrethrum Phtalyc anhydride

Laboratory reagent Bordeaux mixture Insecticides Heated epoxy resin

Metals Cobalt Beryllium Unknown

Hard metal lung disease Berylliosis Moldy typesetting water Cloth wrappings of mummies Cereal grain

Bible printer’s lung Coptic lung (mummy handler’s lung) Grain measurer’s lung

INTERSTITIAL LUNG DISEASE / Hypersensitivity Pneumonitis 435 Table 1 (Continued ) Agent

Source

Disease

Coffee bean dust Contaminated tap water Tea plants Sea snails shell Aerosolized endotoxin from pool water sprays and fountains

Coffee worker’s lung Tap water lung Tea grower’s lung Mollusk shell HP Swimming pool worker’s lung

a

The more frequent causative agents are listed in bold type. Adapted from Patel AM, Ryu JH, and Reed CE (2001) Hypersensitivity pneumonitis: current concepts and future questions. Journal of Allergy and Clinical Immunology 108: 661–670.

However, only a few of these are responsible for the majority of cases of HP. The diagnosis of HP and consequently the avoidance of exposure to HP caused by rare or unknown inciting antigens can be very difficult to confirm. The more common clinical entities include farmer’s lung, bird breeder’s lung, mushroom worker’s disease, and air-conditioner lung, but the prevalence of the different forms of HP diagnosed varies greatly within and between countries as well as with seasonal changes; in Japan, summer-type hypersensitivity pneumonitis due to Trichosporon cutaneum mold seems to account for the majority of cases of HP (74.4%).

Pathology Although the earliest pathologic changes in HP are not well defined, lung changes seem to be characterized by a centrilobular interstitial and alveolar inflammation and edema, epithelial alveolar necrosis, and acute bronchiolitis. Subacute HP shows typical histopathologic features that may affect the air–blood barrier and peripheral airways configuring a bronchiolocentric interstitial granulomatous pneumonitis. The characteristic triad of (1) patchy interstitial infiltrate consisting of lymphocytes, mainly CD8 þ , plasma cells, and monocytes with peribronchiolar accentuation, (2) small, loosely formed, and poorly circumscribed non-necrotizing granulomas, and (3) intra-alveolar buds of granulation tissue is considered diagnostic (Figures 1 and 2). Ancillary findings are intra-alveolar accumulation of foamy macrophages and peribronchiolar lymphoid follicle hyperplasia. Rarely, histology may be less specific: patchy interstitial cellular pneumonitis, dense lymphoid interstitial infiltrate, and organizing pneumonia have been reported as the only or predominant feature. The chronic phase (Figure 3) may maintain some histopathologic features suggesting the diagnosis of HP, such as a moderate lymphocytic infiltrate or poorly formed granulomas, but mostly non-specific extensive interstitial fibrosis and honeycombing in the upper lobes may be found. Typically,

Figure 1 HP, subacute form. Open lung biopsy: centrilobular patchy interstitial pneumonitis and lymphoid follicular hyperplasia are evident (H&E, low power).

Figure 2 HP, subacute form. Loose non-necrotizing granuloma (H&E, high power).

436 INTERSTITIAL LUNG DISEASE / Hypersensitivity Pneumonitis

exposure and the symptoms experienced during every episode of exacerbation tend to become progressively more severe and long-lasting. Chronic HP

Figure 3 HP, chronic form. Interstitial fibrosis with exuberant bronchiolar epitelization in the centrilobular zone (H&E, midpower).

bronchiolar epithelium extending from the centrilobular site to the surrounding alveoli are evident (so-called ‘lambertosis’).

The chronic form of HP may develop from unrecognized and untreated subacute HP or begin with very mild progressive symptoms mimicking chronic bronchitis. Typically, patients present with cough, mucus production, exertional dyspnea, progressive weight loss and anorexia, malaise, and, sometimes, low-grade fever. Finger clubbing and chest crackles are uncommon. The symptoms are not clearly related to each antigen exposure. Chronic HP may be progressive and irreversible, with or without continued exposure to the inciting antigen. The illness may lead to chronic respiratory failure. The development of pulmonary fibrosis is associated with older age, greater restrictive lung physiology, and diminished survival. Strict definitions of acute, subacute, and chronic stages of HP have not been generally agreed on. It has been proposed that HP be described as recently diagnosed, recurrent or progressive, or residual disease.

Diagnostic Approach Clinical Features Acute HP

Acute HP is generally subsequent to heavy exposure to the inciting antigen and is probably the easiest form to recognize. The classical symptoms of presentation are fever, chills, malaise, nausea, scarcely productive cough, dyspnea, and chest tightness arising 4–6 h after exposure and reaching a peak intensity between 12 and 24 h after exposure. Spontaneous resolution generally occurs within 48 h after cessation of exposure. Physical examination may identify diffuse fine inspiratory rales associated with tachypnea.

The diagnosis of HP has most often relied on a high index of suspicion and on an array of non-specific clinical symptoms and signs developed in an appropriate setting, with demonstration of interstitial markings on chest radiographs, serum precipitating antibodies against offending antigens, characteristic bronchoalveolar lavage (BAL) findings, and a granulomatous reaction on lung biopsies (Table 2). One study used a stepwise linear regression model to evaluate the importance of different clinical, laboratory, and radiologic variables in the diagnosis of HP. This study identified six significant predictors of HP: * *

Subacute HP

The distinction between acute and subacute HP is often difficult. The subacute form generally has the same symptoms (dyspnea on effort, cough, and lowgrade fever) and signs as the acute form but with a more gradual period of development. As in the acute form, during exposure-free periods patients are asymptomatic. Individuals who have recurrent episodes of subacute HP over a period of months to years are at high risk of developing chronic HP; in this case, the subsequent episodes are elicited by a lower dose

* * * *

exposure to a known offending antigen; positive precipitating antibodies; recurrent episodes of symptoms; inspiratory crackles on physical examination; symptoms occurring 4–8 h after exposure; and weight loss.

When all six predictors were evident, the probability of HP was 98%. Chronic HP may mimic idiopathic pulmonary fibrosis or the advanced fibrotic stage of any infiltrative diffuse lung disease. In these cases, environmental or laboratory-controlled challenge test with the suspected antigen and surgical lung biopsy are usually necessary to confirm the diagnosis.

INTERSTITIAL LUNG DISEASE / Hypersensitivity Pneumonitis 437 Table 2 Diagnostic criteria for hypersensitivity pneumonitisa Major criteria 1. History of symptoms compatible with hypersensitivity pneumonitis that appear or worsen within hours after antigen exposure 2. Confirmation of exposure to the offending agent by history, investigation of the environment, serum precipitin test, and/or bronchoalveolar lavage fluid antibody 3. Compatible changes on chest radiography or high-resolution computed tomography of the chest 4. Bronchoalveolar lavage fluid lymphocytosis, if bronchoalveolar lavage performed 5. Compatible histologic changes, if lung biopsy performed 6. Positive ‘natural challenge’ (reproduction of symptoms and laboratory abnormalities after exposure to the suspected environment) or by controlled inhalational challenge Minor criteria 1. Basilar crackles 2. Decreased diffusion capacity 3. Arterial hypoxemia, either at rest or with exercise a The diagnosis is considered confirmed if the patient fulfills four of the major criteria and at least two of the minor criteria and if all other diseases with similar symptoms and signs have been excluded. Although these criteria provide useful guidelines with regard to the diagnosis of the disease, strict clinical application of the criteria appears unwarranted. From Patel AM, Ryu JH, and Reed CE (2001) Hypersensitivity pneumonitis: current concepts and future questions. Journal of Allergy and Clinical Immunology 108: 661–670.

Routine Laboratory Tests

Especially during acute exacerbation, moderately elevated leukocyte count, lymphopenia, and increased erythrocyte sedimentation rate and C-reactive protein, immunoglobulins, and immune complexes are found. Peripheral eosinophil count and serum IgE concentration are generally normal. Serum precipitins The presence of serum precipitins is useful for identifying exposure to a potential causative antigen. The level of serum precipitins against many antigens is rarely helpful in the diagnosis of HP. Serum precipitins are classically assessed by radial diffusion (Ouchterlony) analysis, although enzyme-linked immunosorbent assay, which is more sensitive, is increasingly being used. The analysis of serum precipitating IgG antibodies is not diagnostic but requires a more complex analysis of clinical, radiologic, and immunologic data. In fact, as many as 40–50% of asymptomatic exposed people may have precipitins in their serum. Another problem is that the absence of serum precipitins does not rule out HP; negative results are quite frequent both for the wide variety of causative antigen and for the possibility to test precipitins with different laboratory methods with different sensitivity and specificity for any single causative antigen. Skin tests are not helpful in the diagnosis of HP and are not routinely used.

Inhalation challenge If antibodies cannot be detected, the patient may undergo an inhalation challenge with two modalities – through exposure to the environment of the supposed antigen or through inhalation of the purified antigen through a hand nebulizer for a short time (approximately 10 min) in a clinical laboratory setting. Many parameters are monitored during the 24 h after challenge, such as clinical symptoms including cough, dyspnea, chills, fever, and malaise; leukocyte counts; C-reactive protein; arterial blood gas analyses; pulmonary function tests; chest radiographs; and high-resolution computed tomography (HRCT). Postchallenge monitoring is time-consuming but may be helpful in determining a potential diagnosis, and, fortunately, the reactions induced are usually well tolerated. Some authors do not recommend the performance of inhalation challenge because of the risk that it will induce progressive disease and because of the absence of standardized antigen preparations. Pulmonary function tests Pulmonary function tests in all forms of HP may show a restrictive or mixed ventilatory deficit, impaired diffusion capacity, decreased compliance, and exercise-induced hypoxemia. A resting hypoxemia and hypocapnia may also be found. Bronchospasm and bronchial hyperreactivity are sometimes found in acute cases. BAL The typical BAL profile is characterized by a three- to fivefold increase in cell count, with high lymphocytes rates ranging between 20% and 70% and often higher than 50%, with a predominance of CD3 þ CD8 þ cells and a consequent CD4 þ /CD8 þ ratio usually o1 (in Western countries the CD4/CD8 ratio is 43 in only approximately 3% of cases). Lymphocytes show the morphology of ‘activation’ with irregular nuclei, evident cytoplasm, and, in a minority, small azurophilic intracytoplasmatic granules (large granular lymphocytes). Immunophenotypical analysis shows that there is also an increase in CD16 þ CD3
(NK cells) and CD3 þ cells expressing the IL-2 receptor (CD25 þ cells) and HLA-DR antigens. Plasma cells and mast cells (better seen using Alcian staining) are also present. This BAL profile is not diagnostic by itself because it may also be observed in subjects exposed to antigens but without clinical evidence of the disease; however, this BAL profile may be helpful to exclude other interstitial disorders, such as idiopathic pulmonary fibrosis (IPF) and sarcoidosis. A different pattern with high neutrophilic rates can be found if the procedure is performed within 48 h after acute exposure. Neutrophil also seems to play a major role in lung damage and fibrosis; a positive

438 INTERSTITIAL LUNG DISEASE / Hypersensitivity Pneumonitis

correlation between the percentage of lung neutrophils and the percentage of lung fibrosis has been demonstrated, and in advanced disease a neutrophilic rate 45% is common, often accompanied by mild eosinophilia and an increase in the CD4 þ / CD8 þ ratio. Transbronchial biopsy The role of transbronchial biopsy in HP diagnosis seems to be limited. The characteristic histopathologic triad may also be discernable in tiny specimens; however, it should probably be performed in patients with intermediate pretest probability of HP. Imaging Findings

The radiological features of HP are similar, regardless of the responsible agent, and can be divided into three stages: acute, subacute, and chronic. Different features may coexist in the same patient. Chest X-ray can be normal, especially between acute episodes, and can demonstrate diffuse opacities during relapses, but plain radiography is not sensitive or specific (abnormalities detected in less than 50% of proven cases). On the other hand, HRCT is abnormal in nearly 100% of patients with HP and can suggest the correct diagnosis in the appropriate clinical setting, particularly when the patient is exposed to recognized organic antigens. The acute phase of HP, poorly documented in the literature, is characterized by diffuse, bilateral areas of consolidation or ground glass density, predominating in the mid- to lower zones, with sparing of the costophrenic angles (Figure 4). In the subacute phase (Figure 5), HRCT shows diffuse ground glass opacities in a panlobular distribution (52–90% of cases) and/or small, poorly defined centrilobular nodules less than 5 mm in diameter (40–76%). These indistinct, peribronchiolar nodules should suggest the diagnosis of HP but are

Figure 4 Acute HP; HRCT features (see text for details).

not pathognomonic. They can also be found in other entities (e.g., respiratory bronchiolitis). In the axial plane, the distribution may be diffuse or patchy. The HRCT findings are generally most evident in the parahilar and basal regions. Small, thinwalled cystic spaces containing air have been reported in a small percentage of patients with subacute HP. Lobular areas of decreased attenuation and expiratory air trapping are common (50–75% and up to 92% in patients with dynamic inspiratory–expiratory HRCT). Air trapping is particularly common within the areas of ground glass density and represents small airways obstruction. Its detection is regularly enhanced and sometimes only demonstrated by performing expiratory scans. In the chronic phase (Figure 6), HRCT shows signs of fibrosis, characterized by inter- and intralobular septal thickening, honeycombing, traction

Figure 5 Subacute HP; HRCT features (see text for details).

Figure 6 Chronic HP; HRCT features (see text for details).

INTERSTITIAL LUNG DISEASE / Hypersensitivity Pneumonitis 439

bronchiectasis, and volume loss. The presentation can overlap that of sarcoidosis or usual interstitial pneumonitis (UIP)/IPF, but in case of HP, lesions are more patchy and predominantly distributed to midlung regions, with relative sparing of apices and bases, and need not be subpleural. In some cases of chronic HP, however, lower lobe predominance can be found.

Pathogenesis The pathogenesis of HP involves both type III and type IV hypersensitivity reactions. A genetic predisposition to the development of HP has also been identified. It has been shown that pigeon fanciers with HP have an increased frequency of HLA-DRB1*1305 and HLA-DQB1*0501 alleles, a decreased frequency of the HLA-RB1*0802 allele, and an increased frequency of the TNF-2(-308) polymorphism of the tumor necrosis factor alpha (TNF-a) promoter gene. Type III hypersensitivity seems to characterize the acute phase of HP alveolitis, which is mainly neutrophilic. The immunologic process is probably triggered, in a susceptible host, by the formation of immune complexes that activate the complement cascade and macrophages that secrete chemokines and cytokines (MCP-1, MIP-1a, IL-1a, IL-6, and CXCL8/IL-8) responsible for the neutrophilic recruitment and migration, release of toxic oxygen metabolites and proteinases, the differentiation of B cells into plasma cells, and maturation of CD8 þ cells into cytotoxic cells. TNF-a may play a role in the induction of the adhesion molecules L-selectin and Eselectin and alveolar damage. Respiratory epithelial cells and mast cells are other actors in this phase. If antigenic stimulation persists, this early phase is followed by an influx of mononuclear cells in the lung, with type IV cell-mediated delayed hypersensitivity assuming a pivotal role in the immunopathogenetic events. Particularly alveolar recruitment and/ local proliferation of suppressor/cytotoxic CD8 þ lymphocytes and macrophages, associated with increased production of IL-1, TNF-a, IL-12, IL-15, and IL-18 and with a reduction in IL-10 production, increased production of interferon gamma (IFN-g), and elaboration by dendritic cells of CC chemokine-1 interact to promote granulomatous inflammation. Oligoclonal expansion of T cells that express homologous or identical complementary-determining region 3 has been documented in blood and lung cells, indicating that the immune reaction that occurs at the lung level gives rise to a systemic reaction. Although several chemokines contribute to the development of the characteristic HP cellular infiltrate

and granulomas, the specific biologic steps leading to transformation of lymphocytes and macrophages in organized granulomas are unknown. A particularly intriguing question is why only a small percentage of individuals exposed to the different antigens develop the disease. Cofactors are probably needed to render patients hypersensitive to environmental antigens: viral infection, bacterial endotoxin, or fungal glucans have been proposed as possible factors enhancing host susceptibility to the antigen. Using polymerase chain reaction techniques, common respiratory viruses have also been demonstrated in the lower airways of patients with an acute episode of HP, and both respiratory syncytial and Sendai virus increase the expression of HP in mice. Convincing evidence indicates that active cigarette smoking protects against the development of HP. The pathogenetic events leading to abnormal extracellular remodeling and progression to fibrosis seem to involve neutrophils, endothelin-1 upregulation, and transforming growth factor-b overproduction.

Animal Models Many animal models of HP have been performed using a wide variety of animal species (rabbits, guinea pigs, mice, primate, and calves); the most common route of administration is via intratracheal instillation of various antigens (Saccharopolydpora rectivirgula, Penicillium notatum, bat feces, tubercle bacilli antigens, Corynebacterium parvum, and many others). These studies were useful to understand the different roles of macrophages, T cells, mast cells, cytokines and chemokines (mainly IFN-g, IL-12, IL-10, and IL-4), and vascular adhesion molecules (E-selectin, P-selectin, and vascular cell adhesion molecule-1) in HP. Obviously, these animal models have important limitations, such as the administration of large amounts of antigenic material as an intratracheal bolus, the difficulties in producing a granulomatous reaction and ongoing fibrosis, and the numerous obvious differences between experimental animals and humans. The development of the inflammatory reactions and the formation of granuloma and fibrosis scars derive from a complicated sequence of tissue and cellular events that still needs better characterization.

Treatment The treatment of HP is not well standardized and the most common recommendations are based on studies of farmer’s lung and pigeon breeder’s lung. Avoidance of antigen exposure and corticosteroid therapy are the key elements in the treatment of HP.

440 INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis Avoidance of Exposure

The complete avoidance of exposure to the inciting antigen may result in spontaneous resolution of the symptoms, especially for the acute form. The chronic progressive form may improve, stabilize, or, rarely, evolve to fibrosis and respiratory insufficiency even though the patient completely avoids re-exposure to the inciting antigen. Patients with progressive disease should strongly be recommended to avoid the causative environment. After an acute first episode the risk of recurrence is unclear and drastic measures to avoid inciting antigens may not always be indicated. In many cases, the source of exposure (birds and domestic yeast) can easily be removed, but if occupational exposure is involved, an initial attempt to avoid the antigen’s heaviest areas of exposure and the use of preventative measures should be tried. Dust and pollen masks with filters, airstream helmets, and ventilated helmets with a supply of fresh air are increasingly efficient means of purifying inhaled air and, in association with mechanization of the feeding process on farms, industrial hygiene, and alterations in forced-air ventilatory systems may permit adequate control of the disease. Corticosteroid Therapy and Other Immunomodulating Drugs

Although there are no controlled clinical trials regarding the treatment of chronic HP, it is recommended that patients with severe acute or progressive chronic HP be treated with a trial of corticosteroids. The recommended therapy is usually a starting dose of 0.5– 1 mg kg
1day
1 of prednisone (maximum, 60 mg) for 2–4 weeks, followed by a slow tapering of the dosage until suspension or the minimal maintenance dose, if required, is reached. Corticosteroid therapy can significantly and quickly reduce acute symptoms; however, the long-term beneficial effects on arresting disease progression in subacute and chronic HP remain to be definitively established. There is also concern that steroid-treated patients are more likely to relapse than those who are untreated. Little experience has been reported on the use of systemic immune modulators (azathioprine and cyclophosphamide) in the treatment of severe refractory HP. See also: Bronchiectasis. Bronchoalveolar Lavage. Bronchoscopy, General and Interventional. Chemokines. Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12). Chemokines, CXC: IL-8. Corticosteroids: Glucocorticoid Receptors; Therapy. Dendritic Cells. Endothelins. Endotoxins. Environmental Pollutants: Overview; Particulate and Dust Pollution, Inorganic and Organic Compounds. Genetics: Overview. Infant Respiratory Distress Syndrome. Interferons.

Interleukins: IL-1 and IL-18; IL-6; IL-10; IL-12; IL-15. Interstitial Lung Disease: Overview; Alveolar Proteinosis; Cryptogenic Organizing Pneumonia; Idiopathic Pulmonary Fibrosis. Leukocytes: Mast Cells and Basophils; Neutrophils. Pulmonary Fibrosis. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Agostini C, Trentin L, et al. (2004) New aspects of hypersensitivity pneumonitis. Current Opinion in Pulmonary Medicine 10: 378–382. Bourke SJ, Dalphin JC, et al. (2001) Hypersensitivity pneumonitis: current concepts. European Respiratory Journal 32: 81S–92S. King TE Jr (2005) Classification and clinical manifestations of hypersensitivity pneumonitis (extrinsic allergic alveolitis). King TE Jr (2005) Diagnosis of hypersensitivity pneumonitis (extrinsic allergic alveolitis). Up To Date. King TE Jr (2005) Treatment and prognosis of hypersensitivity pneumonitis (extrinsic allergic alveolitis). Lacasse Y, Selman M, et al. (2003) Clinical diagnosis of hypersensitivity pneumonitis. American Journal of Respiratory and Critical Care Medicine 168: 952–958. Mohr LC (2004) Hypersensitivity pneumonitis. Current Opinion in Pulmonary Medicine 10: 401–411. Patel AM, Ryu JH, and Reed CE (2001) Hypersensitivity pneumonitis: current concepts and future questions. Journal of Allergy and Clinical Immunology 108: 661–670. Schuyler M (2004) Hypersensitivity pneumonitis. In: Crapo JD, Glassroth J, Karlinsky J, and King TE Jr (eds.) Baum’s Textbook of Pulmonary Diseases, pp. 481–498. Philadelphia: Lippincott Williams & Wilkins. Selman M (2004) Hypersensitivity pneumonitis: a multifaceted deceiving disorder. Clinics in Chest Medicine 25: 531–547. Travis WD, Colby TV, et al. (2001) Hypersensitivity pneumonitis. In: Non-Neoplastic Disorders of the Lower Respiratory Tract, pp. 115–122. Washington, DC: Armed Forces Institute of Pathology/American Registry of Pathology.

Relevant Websites http://germop.univ-lyon1.fr – Group of Studies and Research on the ‘Orphan’ Diseases Pulmonary. www.uptodate.com – UpToDate is specifically designed to answer the clinical questions that arise in daily practice and to do so quickly and easily so that it can be used right at the print of case.

Idiopathic Pulmonary Fibrosis F J Martinez, C M Hogaboam, E S Choi, G B Toews, K R Flaherty, and V J Thannickal, University of Michigan, Ann Arbor, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Idiopathic pulmonary fibrosis (IPF) is a clinical syndrome of unknown cause affecting primarily middle aged or elderly patients. IPF follows an inexorably progressive and, usually, fatal

INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis 441 course. It is associated with the histopathological pattern of usual interstitial pneumonia on surgical lung biopsy (SLB), showing heterogeneous distribution of fibroblastic foci interspersed with normal-appearing lung and minimal inflammation. Clinical features include progressive dyspnea with/without dry cough, end-expiratory ‘crackles’ on lung auscultation, and digital clubbing in up to half of all patients. Interstitial (fibrotic) lung diseases associated with environmental or drug exposures and connective tissue diseases must be excluded. Pulmonary function testing typically shows restrictive physiology with gas exchange abnormalities. High-resolution computed tomography (HRCT) classically demonstrates subpleural and lower lobe-predominant interstitial/septal thickening in a reticular pattern and ‘honeycomb’ change. SLB is recommended when the clinical and/or HRCT findings are atypical. Current concepts on pathogenesis of IPF suggest an aberrant host response to alveolar epithelial injury, although the etiology of the ‘injury’ remains unknown. Dysregulated repair involving altered communication between the injured epithelium and fibroblasts/myofibroblasts (in fibroblastic foci) plays a dominant role in disease pathogenesis. Traditional therapies with steroids and potent immunosuppressive agents have been disappointing. Newer ‘antifibrotic’ agents are currently under investigation.

inconclusive. Cigarette smoking has been reported to be an independent risk factor for the development of IPF; however, prognosis in established cases of IPF appears to be ‘paradoxically’ improved in active smokers. Familial IPF accounts for less than 3% of IPF patients. Recent evidence suggests that some families harbor mutations in the prosurfactant protein-C (proSP-C) gene expressed in alveolar epithelial cells; this alters protein processing and results in epithelial injury. Further studies are required to determine if germline/somatic mutations of this or other proteins may account for ‘injury’ of alveolar epithelial cells. Gene polymorphisms involving transforming growth factor beta-1 (TGF-b1), tumor necrosis factor alpha, interleukin-1-receptor antagonist, interleukin-6, and major histocompatibility complex loci among others have been associated with increased risk or progression of IPF.

Pathology Introduction Idiopathic pulmonary fibrosis (IPF) is a clinical– radiological–pathological syndrome that belongs to a group of enigmatic diffuse parenchymal lung disorders known as the idiopathic interstitial pneumonias (IIPs). In 1975, Liebow first proposed a classification of the IIPs into five distinct histopathological categories that were subsequently ‘lumped’ into a single IPF disease category in the 1980s up until the late 1990s. More recently, these disorders have been reclassified in a new classification scheme that was adopted, by consensus, jointly by the American Thoracic Society and the European Respiratory Society in 2002. Although overlapping histopathological patterns of IIPs are seen in the individual patient with IPF, classification of patients into as distinctive a group as is currently possible will allow for more meaningful studies of prognoses and responses to emerging drug therapies for these disorders.

Etiology The etiology of IPF is not known. Based on the observations that the disease is limited to the lungs and that alveolar epithelial cells (AECs) viewed under electron microcopy appear to be ‘injured’, potential causative agents that directly target these cells have been postulated. Respirable environmental agents and occupational exposures to inorganic/organic dusts have been proposed, but none have been clearly linked to IPF. Studies of latent viral infections, particularly Epstein–Barr virus (EBV) and cytomegalovirus (CMV), as risk factors in IPF have been

The key histologic features of the usual interstitial pneumonia (UIP) pattern have been well defined and include architectural remodeling, fibrosis often with honeycombing, scattered fibroblastic foci, patchy distribution, and involvement of the periphery of the acinus (Figure 1). A cardinal feature is the bilateral but heterogeneous (patchy) involvement, with a predilection for the lower lobes and peripheral (subpleural) regions. A key feature of this temporal heterogeneity is the presence of dense acellular collagen and ‘fibroblastic foci’ (aggregates of proliferating fibroblasts and myofibroblasts). Fibroblastic foci develop in areas of lung injury and are associated with active collagen synthesis, suggesting that fibrosis is active. Although fibroblastic foci are not pathognomonic, they are necessary for the histologic diagnosis of UIP. The extent of interstitial inflammation is usually mild to moderate, patchy, and characterized by a septal infiltrate of lymphocytes, plasma cells, and histiocytes associated with hyperplastic type II pneumocytes. Additional features include smooth muscle hypertrophy, reactive metaplasia and hyperplasia of type II pneumocytes, mucostasis, secondary pulmonary hypertensive changes, traction bronchiectasis and bronchiolectasis, reduced airspace volume, and destroyed and distorted alveolar architecture. Subpleural fat and metaplastic bone are noted in severe disease. Without areas of relatively normal lung in the surgical specimen, the presence of other interstitial disorders may be difficult for the pathologist to exclude and the UIP histological pattern may be difficult to recognize. Recently, several groups have confirmed that in some patients a UIP pattern may be seen in one specimen from a surgical

442 INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis

Figure 1 (a) Lower power image illustrating heterogeneous (patchy) involvement with a predilection for the peripheral (subpleural) regions. (b) Higher power image illustrating a key histological feature of UIP, the presence of dense acellular collagen and ‘fibroblastic foci’ (aggregates of proliferating fibroblasts and myofibroblasts).

lung biopsy (SLB), while specimens from a second or third lobe of lung exhibit other histologic patterns. Importantly, these patients exhibit the survival characteristics of idiopathic UIP or IPF. The histologic approach to differential diagnosis includes a careful search for histologic clues to potential causes such as asbestos bodies, infectious agents, or other exogenous agents. The differential diagnosis of the UIP histologic pattern includes other IIPs including fibrosing non-specific interstitial pneumonia (NSIP), desquamative interstitial pneumonia (DIP), organizing pneumonia (OP), and diffuse alveolar damage (DAD). Lesions that can be histologically similar (but not identical) to UIP include asbestosis, collagen vascular disease, fibrosing hypersensitivity pneumonitis, radiation pneumonitis, and Hermansky–Pudlak syndrome. Temporal heterogeneity is a central histologic feature that may aid in distinguishing UIP from other IIPs, as non-UIP IIPs are generally temporally uniform. In addition, fibroblastic foci, which are prominent in UIP, are rarely numerous in DIP, respiratory bronchiolitis interstitial lung disease (RBILD), or NSIP. Although fibroblastic foci are present in acute interstitial pneumonia, this disorder is diffuse and temporally uniform, in contrast to UIP. Furthermore, inflammatory cells are generally more conspicuous in other IIPs. Intraalveolar macrophages may be seen in UIP, but are not a dominant histologic feature, while dense intraalveolar or peribronchiolar alveolar macrophages suggest DIP or RBILD. Nevertheless, recent work by a group of expert pathologists suggests that the differential diagnosis of UIP remains difficult, particularly in separating UIP from cases of fibrosing NSIP.

Clinical Features The typical clinical features of IPF include dry cough, exertional dyspnea, and end-inspiratory ‘velcro-like’

rales on physical examination. Exertional dyspnea is one of the most common symptoms and progresses inexorably over months to years. A dry, hacking cough may be the initial symptom, and is often paroxysmal. Bibasilar end-inspiratory velcro rales are present in the majority of patients with IPF, while clubbing is present in a smaller proportion of patients. Cyanosis and cor pulmonale are features noted in advanced clinical disease. The physical examination is likely most useful in identifying extrapulmonary signs; extrapulmonary involvement does not occur in IPF and, if present, suggests an alternative diagnosis. Laboratory abnormalities are non-specific and may include an elevated erythrocyte sedimentation rate, rheumatoid factor, or circulating antinuclear antibodies (ANA). In general, serologic studies do not correlate with the extent or activity of the disease and have limited prognostic value. However, some investigators have suggested that serum levels of surfactant protein (SP)-A and SP-D correlate with the extent of alveolitis, and may be predictive of longterm survival. The physiologic abnormalities in IPF patients characteristically include reduced lung volumes (e.g., vital capacity (VC), and total lung capacity (TLC)), normal or increased expiratory flow rates, increased forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC) ratio, and a reduced diffusing capacity for carbon monoxide (DLCO). Hypoxemia or increased alveolar-arterial oxygen difference (PðA-aO2 Þ) may be present, which is generally accentuated by exercise. Cardiopulmonary exercise testing (CPET) generally documents hypoxemia or widened PðA-aO2 Þ. Additional findings may include decreased oxygen consumption ðVO2 Þ, increased physiologic dead space, increased minute ventilation for the level of VO2 , high respiratory rate/low tidal volume respiratory pattern, and a decreased O2 pulse. The

INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis 443

impairment in gas exchange (DLCO) and oxygenation are likely the earliest abnormalities. Although a restrictive defect with reduced FVC and TLC is characteristic of IPF, lung volumes may be normal if emphysema coexists. In this setting, DLCO and oxygenation are disproportionately reduced. In general, physiological parameters provide only a rough estimate of disease severity, with the DLCO correlating better with the extent of disease on high-resolution computed tomography (HRCT) scans. Chest radiographs generally exhibit diffuse, bilateral reticular opacities, with a predilection for basilar and peripheral (subpleural) regions (Figure 2). As disease progresses, lung volumes decrease. HRCT scans, using 1–2 mm thin sections, are superior to conventional radiographs in depicting the characteristics, extent, and distribution of parenchymal infiltration. The three major HRCT patterns include ground glass opacity (GGO), reticular pattern, and honeycomb cysts. The characteristic HRCT features of IPF include a basilar, subpleural predominance of abnormality, patchy involvement with large areas of spared lung parenchyma, coarse reticular or linear opacities (intralobular and interlobular septal lines), honeycomb cysts, and traction bronchiectasis or bronchiolectasis (Figure 3). More than focal areas of GGO (i.e., ill-defined, hazy zones of increased alveolar attenuation) are unusual and should suggest an alternative diagnosis (e.g., DIP, NSIP, or hypersensitivity pneumonia (HP)). Honeycomb cysts are the key feature in ensuring a radiographic diagnosis of IPF. Zones of emphysema (usually upper lobe predominant) may be present in smokers; these cysts are generally distinguished from honeycombing by the lack of well-defined walls but may make radiographic diagnosis difficult.

Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) has been quite valuable in the evaluation of some ILDs, although its value in the diagnosis of IPF appears to be limited. Increases in polymorphonuclear leukocytes (PMNs), mast cells, alveolar macrophages, eosinophils, and various cytokines have been noted in BAL fluid from IPF patients, although such measurements are rarely diagnostic or clinically valuable. Radionuclide scans have been used to evaluate numerous ILDs. Increased intrapulmonary uptake of gallium 67 citrate had been touted to be a marker of alveolitis, although its diagnostic and prognostic value in IPF has been disappointing. Increased clearance of technetium 99m-diethylenetriamine penta-acetate aerosol, a marker of increased lung permeability, is accelerated in IPF, which some investigators have argued may have prognostic value. Unfortunately, this technique has not become widely utilized. The onset of IPF is indolent, but progresses inexorably over months to years. Spontaneous remissions do not occur, although some patients stabilize for long periods of time; this can sometimes occur after an initial decline. Most patients die of respiratory failure within 3–8 years of onset of symptoms, with a mean survival of several years. Other causes of death include cardiac failure, pulmonary embolism (due to immobility or cor pulmonale), pulmonary infections, and cerebrovascular accidents. Lung cancer occurs in 6–13% of patients with IPF. Acute exacerbation of IPF, also known as the accelerated form of IPF, is characterized by acute progression of dyspnea over 1 month or less, in association with new, diffuse opacities on chest radiography, worsening hypoxemia, and rapid development of respiratory failure in the absence of infection or alternative diagnoses.

Figure 2 (a) Chest radiograph of a patient with mild to moderate IPF. Note the basilar predominance of reticular findings. (b) Chest radiograph of a patient with more advanced IPF. Note the reduction in lung volumes and diffuse, bilateral interstitial or reticulonodular pattern of infiltration.

444 INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis

Figure 3 (a, b) HRCT images of a patient with moderately severe UIP proven using SLB. Upper and lower lung zone reticular infiltrates are very subtle; basilar predominant, subpleural honeycomb cysts are noted. (c, d) HRCT images of a patient with advanced IPF. Note the predominately peripheral honeycomb change.

Pathologic examination typically shows acute alveolar injury with or without hyaline membrane formation. While the exact incidence of this acute syndrome is unknown, one recent series noted that approximately half of patients with mild to moderate IPF who died of an IPF-related cause appeared to follow such a pattern of accelerated disease. As such, rapid respiratory decompensation in patients with mild to moderate IPF is likely more common than previously suspected.

Pathogenesis The pathogenesis of IPF is complex. It is, perhaps, best understood as an abnormal host tissue response to injury. The inciting event appears to be injury to the alveolar epithelial cells (AECs), although the precise nature of the ‘injury’ is not known. Host responses to tissue injury, in general, typically involve both inflammation and repair (Figure 4). UIP represents a dysregulated repair response in which there is

persistent activation of fibroblasts/myofibroblasts in fibroblastic foci, a defining lesion in IPF. Dysregulated communication between AECs and mesenchymal cells are likely central to the ongoing, unresolving repair process that culminates in fibrosis. AECs appear to be in a state of dysrepair with an inability of these cells to reconstitute the alveolar wall. Exaggerated proliferation of AECs is observed in bronchoalveolar junctions; whereas epithelial cells directly overlying fibroblastic foci appear to be undergoing cell death. It is not known if a defect exists in the ability of replicating type II cells to differentiate into type I AECs or if there is impairment in the migratory capacity of these cells. Fibroblast/myofibroblast-secreted factors, such as angiotensin peptides and oxidants, may contribute to AEC injury by paracrine mechanisms. Fas ligand and mitochondria-mediated apoptosis of AECs in the lungs of IPF patients have also been reported. In contrast to AECs, fibroblasts/myofibroblasts in fibroblastic foci are highly activated and resistant to

INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis 445 Lung injury

apoptosis. Myofibroblasts are an intriguing group of cells that play a central role in connective tissue remodeling in the lung as well as other tissues. They are cells with contractile and synthetic properties that may account, in large part, for the restrictive physiology and gas-exchange abnormalities in IPF. The profibrotic cytokine TGF-b1 plays a central role in the differentiation, activation, and survival of myofibroblasts. Other pathogenetic mechanisms that play significant roles in different phases of the disease include: imbalances in the chemokine/cytokine milieu, angiogenesis, impaired fibrinolysis, reduced matrix degradative capacity, and oxidative stress (Figure 5) (see Pulmonary Fibrosis for details). There has been recent interest in bone-marrowderived cells in both the regeneration of injured alveolar epithelium (‘stem cells’) as well as a source of fibroblasts and myofibroblasts in fibrotic lesions. Most of these studies have been performed in animal models and whether similar mechanisms are important in human disease awaits further study. The recruitment of ‘repair’ cells to the lung in response to injury is likely to involve small molecular weight peptides known as chemokines. Several groups have examined the expression of chemokines in SLB and other samples from IIP and non-IIP patient populations. The published data have provided a ‘snapshot’

Age Genetic factors Environmental factors Nature of injury - etiologic agent - recurrent vs. single - endothelial vs. epithelial

Histopathologic pattern DIP

RB-ILD

LIP

COP

Inflammation

NSIP

AIP

UIP

Fibrosis

Figure 4 The histopathological spectrum of IIPs represents varied tissue reaction with varying degrees of inflammation and fibrosis. This reaction pattern probably depends on multiple factors, including age, genetic susceptibility, environmental factors, and perhaps the nature of the injurious agent. DIP, desquamative interstitial pneumonia; RB-ILD, respiratory bronchiolitis-associated interstitial lung disease; LIP, lymphocytic interstitial pneumonia; COP, cryptogenic organizing pneumonia; NSIP, non-specific interstitial pneumonia; AIP, acute interstitial pneumonia; UIP, usual interstitial pneumonia. Reproduced from Thannickal VJ, Toews GB, White ES, Lynch JP III, and Martinez FJ (2004) Mechanisms of pulmonary fibrosis. Annual Review of Medicine 55: 395–417.

• Age • Genetic factors • Extrinsic factors

Repetitive alveolar epithelial injury

Oxidative stress

Profibrotic cytokines

Altered alveolar microenvironment

TIMPs–MMPs imbalance Fibrosis

Impaired fibrinolysis

Chemokines

Eicosanoid imbalance

Dysregulated repair Loss of epithelial cells Accumulation of mesenchymal cells Figure 5 Progressive fibrosis results from dynamic alterations in the alveolar microenvironment eventually promoting the loss of alveolar epithelial cells and accumulation of activated fibroblasts/myofibroblasts. Such alterations include: the presence or activation of profibrotic cytokines, growth factors, and chemokines; eicosanoid imbalance with increased production of profibrotic leukotrienes and deficiency in prostaglandin E2; impaired fibrinolysis; overproduction of tissue inhibitors of matrix metalloproteinases (TIMPs) relative to matrix metalloproteinases (MMPs); and a state of elevated oxidative stress. Reproduced from Thannickal VJ, Toews GB, White ES, Lynch JP III, and Martinez FJ (2004) Mechanisms of pulmonary fibrosis. Annual Review of Medicine 55: 395–417.

446 INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis

of the various chemokines that may contribute to the pathology of IIP, although the understanding of the sequence of events that lead to changes in chemokine synthesis is limited.

Animal Models An animal model of pulmonary fibrosis that recapitulates the cardinal pathologic and physiologic features of human IPF has been difficult to establish. The most commonly utilized model of pulmonary fibrosis relies on a single injurious insult with intratracheal bleomycin administered to rodents (mice or rats). Bleomycin injury in the lungs of these animals is typically followed by an acute inflammatory response and fibrosis that is typically centered around peribronchiolar structures. The fibrotic response to bleomycin is self-limited and does not lead to the classical restrictive physiology of human IPF. A large number of studies using this model, however, has provided significant insights into basic mechanisms of host inflammatory and repair responses. Newer animal models that are better at reproducing the progressive nature of the fibrotic response have been studied. A number of groups have utilized an active (mutant) form of TGF-b1 expressed either by an inducible promoter in Clara cells of transgenic mice, by adenovirus-mediated gene transfer to rat lung, or by epithelial cell-specific expression of the mutant gene in mice lung explants. Nonrodent mammalian species may offer better models of human IPF; a recent report suggests the development of UIP-like histopathological lesions in spontaneous feline pulmonary fibrosis.

Management and Current Therapy The general approach to the patient with a suspected IIP is to identify the most likely diagnosis in an expeditious fashion; this is particularly important with regard to a diagnosis of IPF, given its particularly ominous prognosis. The American Thoracic Society (ATS) and European Respiratory Society (ERS) have recently recommended an integrated clinical, radiologic, and pathologic approach to the diagnosis of diffuse parenchymal lung disease. This integrated approach includes a comprehensive assessment of clinical features, physical examination findings, selected laboratory studies, imaging studies, and, in selected patients, transbronchial biopsy or SLB. We have confirmed that such an iterative approach maximizes interobserver agreement among expert clinicians, radiologists and pathologists. A careful history is required in the initial evaluation with attention to identifying diagnostic and prognostic clinical

features. These include gender, age, the presence of comorbid diseases, medication exposures, and identification of potential exposures at home or work. For example, it is particularly important to identify the presence of a collagen vascular illness, as collagen vascular diseases associated with histologic UIP exhibit an improved prognosis. A multicenter, case-control study identified several occupational exposures that were associated with an increased likelihood of IPF, including farming, working with livestock, hairdressing, metal dust work, raising birds, stone cutting/polishing, and vegetable dust/animal dust exposure. Significant exposure to asbestos is crucial to identify, as asbestosis may present with a clinical picture remarkably similar to IPF but is associated with a longer, more benign clinical course. Exposure to potential antigens can result in HP; advanced cases may clinically mimic IPF. Cigarette smoking has been implicated in the development of IPF and may be associated with improved survival. Familial types of pulmonary fibrosis are well described, making a careful family history crucial in the evaluation of patients with suspected IPF. Routine laboratory tests and specific serologic studies may be useful in the exclusion of alternative diagnoses, particularly HP and an associated collagen vascular illness. Imaging studies have assumed a pre-eminent role in the evaluation of suspected IPF. HRCT is particularly likely to be diagnostic in patients with IPF. As such, numerous investigators have confirmed that typical HRCT findings of IPF are associated with a high positive predictive value for a histological diagnosis of UIP in the appropriate clinical setting. In addition, recent work confirms that patients with these typical HRCT features of IPF (all of whom exhibited typical histological features of UIP) exhibited the worst survival. The importance of SLB in the diagnosis of IPF has been confirmed by numerous investigative groups. In the setting of a nonstereotypical HRCT, identification of histological UIP at SLB is associated with a significantly worse prognosis. Importantly, three groups have documented that histologic findings of NSIP and UIP are frequently noted in multiple lobes (and even in the same lobe) of patients undergoing SLB; two of these groups have confirmed that the presence of histologic UIP in any sample is associated with a much worse survival. The totality of these data has led to the diagnostic algorithm depicted in Figure 6. Current therapy for IPF is limited. Traditional pharmacotherapy has involved prolonged courses of high-dose corticosteroids with or without the addition of cytotoxic agents such as azathioprine or cyclophosphamide. Although some patients appear to improve or stabilize with this approach, the overall

INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis 447

Patient with suspected ILD

Hx, PE, CXR, PFT, Labs

Yes

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Yes

Is bronch diagnostic?

No

No

Stop

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Figure 6 A potential algorithm for the evaluation of a patient with a diffuse parenchymal lung disease. Hx, history; PE, physical examination; CXR, chest radiograph; PFT, pulmonary function test; bronch, bronchoscopy; LIP, lymphocytic interstitial pneumonitis. Reproduced from Martinez FJ and Flaherty (2004) Diagnosis of interstitial lung disease. Pulmonary and Critical Care Update 18: Lesson 3.

effectiveness of such therapies is disappointing. The assessment of anti-inflammatory therapy for IPF is further complicated by the fact that previous trials were underpowered, often unblinded, lacked placebo/ controls, and involved variable dosage regimens and endpoints. Moreover, these studies were performed prior to the recent ATS/ERS reclassification of the IIPs. It is suspected that many of the patients that responded to immunosuppressive therapy in early clinical trials of presumed IPF may have had other forms of IIP such as NSIP. Newer therapeutic approaches that are designed to target fibrogenic responses per se have received greater attention. Recently, a large phase III clinical trial has been completed using interferon gamma, an immunomodulatory T-helper-1 (Th1) cell cytokine. In this double-blind study of 330 IPF patients randomized to receive subcutaneous interferon gamma (200 mg three times weekly) or placebo, no significant

difference was noted in the primary end point of progression-free survival, defined as the time to disease progression or death. Moreover, no differences were observed in any of the pulmonary physiologic parameters, dyspnea, or quality-of-life measures. Overall, survival in interferon-gamma-treated and placebo groups was not statistically different (10% and 17%, respectively, p ¼ 0:08). In post hoc explorative subgroup analyses, patients with less severe disease (baseline FVC above the median, 62% of predicted) and a ‘treatment adherent’ cohort (patients receiving at least 80% of scheduled doses) appeared to have a survival advantage in the interferon-gamma-treated group. Another prospective, randomized trial (International Study of Survival Outcomes in Idiopathic Pulmonary Fibrosis with Interferon-gamma 1b, ‘INSPIRE’ trial, sponsored by Intermune Pharmaceuticals) is now underway to test the validity of these findings in patients

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with mild-moderate disease (baseline FVCX55% of predicted). Other novel ‘antifibrotic’ agents in various stages of clinical development include a small molecule inhibitor of fibroblast activation (Pirfenidones), endothelin-1 antagonists (Bosentans), a soluble tumor necrosis factor-receptor fusion protein (Etanercept), leukotriene inhibitors (Zileutons), a copper-chelating anti-angiogenic compound (Tetrathiomolybdate), monoclonal antibodies against TGF-b1 and connective tissue growth factor, a protein kinase inhibitor (Gleevacs), and the glutathione-antioxidant precursor N-acetylcysteine (NAC). It is hoped that one or more of these approaches will be more effective than conventional therapy solely with immunosuppressive agents. It is unlikely that a single approach will be more effective than combination therapy given the heterogeneity of the pathologic lesions found in individual patients with IPF. Thus, accurate clinical, and perhaps biological, phenotyping will be necessary to ‘individualize’ therapy for maximal benefit. See also: Interstitial Lung Disease: Overview, Leukocytes: Pulmonary Macrophages, Matrix Metalloproteinase Inhibitors, Matrix Metalloproteinases, Pulmonary Fibrosis.

Further Reading Ambrosini V, Cancellieri A, Chilosi M, et al. (2003) Acute exacerbations of idiopathic pulmonary fibrosis: report of a series. European Respiratory Journal 22: 821–826. American Thoracic Society (2000) Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Journal of Respiratory and Critical Care Medicine 161: 646–664. American Thoracic Society/European Respiratory Society (2002) American Thoracic Society/European Respiratory Society international multidisciplinary consensus classification of the idiopathic interstitial pneumonias. American Journal of Respiratory and Critical Care Medicine 165: 277–304. Azuma A, Nukiwa T, Tsuboi E, et al. (2005) Double blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine 171: 1040–1047. Charbeneau RP and Peters-Golden M (2005) Eicosanoids: mediators and therapeutic targets in fibrotic lung disease. Clinical Science (London) 108: 479–491. Collard HR and King TE Jr (2003) Demystifying idiopathic interstitial pneumonia. Archives of Internal Medicine 163: 17–19. Egan JJ, Martinez FJ, Wells AU, and Williams T (2005) Lung function estimates in idiopathic pulmonary fibrosis: the potential for a simple classification. Thorax 60: 270–273. Flaherty KR, King TE Jr, Raghu G, et al. (2004) Idiopathic interstitial pneumonia: what is the effect of a multi-disciplinary approach to diagnosis? American Journal of Respiratory and Critical Care Medicine 170: 904–910. Flaherty KR, Thwaite EL, Kazerooni EA, et al. (2003) Radiological versus histological diagnosis in UIP and NSIP: survival implications. Thorax 58: 143–148.

Hunninghake GW, Lynch DA, Galvin JR, et al. (2003) Radiologic findings are strongly associated with a pathologic diagnosis of usual interstitial pneumonia. Chest 124: 1215–1223. Hunninghake GW, Zimmerman MB, Schwartz DA, et al. (2001) Utility of a lung biopsy for the diagnosis of idiopathic pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine 164: 193–196. Katzenstein ALA and Myers JL (1998) Idiopathic pulmonary fibrosis. Clinical relevance of pathologic classification. American Journal of Respiratory and Critical Care Medicine 157: 1301–1315. Lynch JP III, Wurfel M, Flaherty K, et al. (2001) Usual interstitial pneumonia. Seminars in Respiratory and Critical Care Medicine 22: 357–385. Martinez FJ and Flaherty KR (2004) Diagnosis of interstitial lung disease. Pulmonary and Critical Care Update 18: Lesson 3. Martinez FJ, Safrin S, Weycker D, et al. (2005) The IPF Study Group. Clinical course of patients with idiopathic pulmonary fibrosis. Annals of Internal Medicine 142: 963–967. Nicholson AG, Addis BJ, Bharucha H, et al. (2004) Inter-observer variation between pathologists in diffuse parenchymal lung disease. Thorax 59: 500–505. Raghu G, Brown KK, Bradford WZ, et al., for the Idiopathic Pulmonary Fibrosis Study Group (2004) A placebo-controlled trial of interferon gamma-1b in patients with idiopathic pulmonary fibrosis. New England Journal of Medicine 350: 125–133. Selman M, King TE Jr, and Pardo A (2001) Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Annals of Internal Medicine 134: 136–151. Selman M, Thannickal VJ, Pardo A, et al. (2004) Idiopathic pulmonary fibrosis: pathogenesis and therapeutic approaches. Drugs 64(4): 405–430. Thannickal VJ, Flaherty KR, Martinez FJ, and Lynch JP III (2004) Idiopathic pulmonary fibrosis: emerging concepts on pharmacotherapy. Expert Opinion in Pharmacotherapy 5(8): 1671–1686. Thannickal VJ, Toews GB, White ES, Lynch JP III, and Martinez FJ (2004) Mechanisms of pulmonary fibrosis. Annual Review of Medicine 55: 395–417. Thomas AQ, Lane K, Phillips J III, et al. (2002) Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred. American Journal of Respiratory and Critical Care Medicine 165: 1322–1328.

Lymphangioleiomyomatosis J Moss, W K Steagall, and A Taveira-DaSilva, National Institutes of Health, Bethesda, MD, USA Published by Elsevier Ltd.

Abstract Lymphangioleiomyomatosis (LAM), a rare multisystem disease affecting women, is characterized by cystic lung disease and abdominal tumors (e.g., angiomyolipomas (AMLs) and lymphangioleiomyomas). Proliferation of abnormal smooth musclelike cells (LAM cells) is observed throughout the lung, along axial lymphatics in the thorax and abdomen, and in AMLs. Patients can present with progressive dyspnea, recurrent pneumothoraces, hemoptysis, chylous pleural effusions, and/ or ascites. Diagnosis may be made by lung biopsy or by clinical and roentgenographic data. LAM occurs sporadically or in

INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis association with tuberous sclerosis complex (TSC), an autosomal dominant disorder with variable penetrance caused by mutations in the tumor suppressor genes TSC1 and/or TSC2. LAM cells from sporadic LAM patients contain TSC mutations. TSC1 encodes hamartin, a protein with a role in cell adhesion; TSC2 encodes tuberin, a protein involved in cell-cycle progression and control of cell growth and proliferation, in part, through its regulation of mammalian target of rapamycin (mTOR). The natural course of LAM is highly variable. Therapy includes hormonal manipulations, bronchodilators, supplemental oxygen, and lung transplantation. Clinical trials are under way to study the effects of rapamycin, an inhibitor of mTOR.

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one-third of female TSC patients have some degree of cystic involvement in their lungs. TSC is caused by mutations in either the TSC1 or TSC2 genes and may be characterized by hamartomatous lesions, seizures, and mental retardation. There are more than 500 known patients with sporadic LAM in North America. Tuberous sclerosis-associated LAM was first described in 1918 by Lutembacher. Sporadic LAM was first described in 1937 by von Stossel.

Etiology Introduction Lymphangioleiomyomatosis (LAM), a rare multisystem disorder affecting primarily middle-aged women, is characterized by cystic lung lesions, involvement of the axial lymphatics, and abdominal tumors (e.g., angiomyolipomas (AMLs)). A hallmark of the disease is the proliferation of abnormal smooth muscle-like cells (LAM cells), which leads to the formation of thin-walled cysts in the lungs and fluid-filled cystic structures in the axial lymphatics (i.e., lymphangioleiomyomas). AMLs are composed of LAM cells and adipocytes, intermixed with incompletely developed vascular structures. LAM occurs sporadically or in association with tuberous sclerosis complex (TSC), an autosomal dominant disorder with variable penetrance. Approximately

LAM cells from patients with sporadic LAM contain TSC2 mutations. Identical mutations in TSC2 have been detected in lung lesions and AMLs from the same patient with sporadic LAM, and in the donor lung transplanted in a LAM patient. These data suggest that LAM cells can metastasize. Because 50% of sporadic LAM cases do not have demonstrable AMLs, another ‘primary’ source of LAM cells may exist, perhaps in the lymphatic system. There is no evidence for involvement of infectious, carcinogenic, or inflammatory agents in LAM pathogenesis.

Pathology Lung lesions are characterized by nodules of LAM cells in the interstitium and walls of cysts (Figure 1).

Figure 1 Lung pathology in LAM. Thin-walled cysts (a) and nodules of various sizes (b) are seen in involved lung (H&E, original magnification
50). (c) Bronchiolitis: deposition of mucus in the bronchial lumen, focal goblet cell hyperplasia, and moderate infiltration of inflammatory cells, including eosinophils, in the wall of a bronchiole. Note the infiltrates of smooth muscle cells around the bronchiole (H&E, original magnification
160).

450 INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis

Figure 2 Immunohistochemical reactivity of LAM cells. Immunoperoxidase method and counterstaining with hematoxylin. (A and B) aSmooth muscle actin. Most LAM cells show strong reactivity (a). Vascular smooth muscle cells are also strongly positive (arrow). Most of the LAM cells in the thin walls of the cysts are also strongly reactive (b) (original magnification
250 for each). (c) HMB-45. Positive cells are mainly distributed in the periphery of LAM nodules (original magnification
250). (d) HMB-45. Higher magnification view of tissue shown in (c). A strong granular reaction is present in large epithelioid LAM cells adjacent to epithelial cells covering LAM nodules (original magnification
1000).

Bronchiolitis and lung hemorrhage may also be present. The LAM nodules, covered with hyperplastic type II pneumocytes, contain spindle-shaped cells in the center and epithelioid-like cells at the periphery. Both types of cells react with antibodies against smooth muscle cell-specific antigens. The epithelioid cells are more likely to react with HMB-45, which is a monoclonal antibody that recognizes gp100, a premelanosomal protein (Figure 2); the spindle-shaped cells are more likely than epithelioid cells to react with anti-PCNA antibodies, consistent with a more proliferative state. LAM lesions contain damaged elastic fibers and collagen fibrils. Matrix metalloproteinases (MMPs), including MMP-2 (gelatinase A), MMP-1, MMP-9 (gelatinase B), and MMP-14, are associated with the lesions. Decreased expression of tissue inhibitor of metalloproteinase (TIMP)-3, an inhibitor of some MMPs, has been reported, leading to the hypothesis that the destructive lesions are due to an imbalance between proteases and their inhibitors.

Clinical Features Clinical Manifestations

LAM occurs in both pre- and postmenopausal women; it has been observed in patients in the eighth

decade of life. In a large cohort of LAM patients, the average age of patients at the time of diagnosis was 41 years; it was estimated that these patients may have had the disease for approximately 5 years prior to the diagnosis. Patients with LAM present with progressive dyspnea, recurrent pneumothoraces, chylous pleural effusions, hemoptysis, and ascites. Less frequently, the first manifestation of LAM is an acute hemorrhage into an AML requiring surgical intervention. In 80% of cases, spontaneous pneumothorax and pleural effusions are the events leading to the diagnosis. Roentgenographic Findings

Chest X-rays may be normal or show pleural effusions or pneumothorax. Chest computed tomography (CT) shows uniquely circular, thin-walled cysts distributed throughout the lungs (Figure 3). Other abnormalities seen on chest CT include adenopathy, pleural effusions, or pericardial effusions. Abdominal CT scans may show renal masses (i.e., AMLs), lymphangioleiomyomas, and ascites (Figure 3). Pulmonary Function Abnormalities

Reduction of single-breath diffusing capacity (DLCO) and airflow obstruction, causing a decrease in forced expiratory volume in 1 s (FEV1), are the main

INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis

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Figure 3 CT scans of four patients with LAM. (a) The CT scan of a patient with severe disease (CT scan grade 3) with a predominance of large cysts. (b) Another case of severe LAM in which there are numerous small cysts distributed throughout the lungs. (c) Multiple angiomyolipomas of the left kidney in a patient who had already undergone a right nephrectomy for treatment of a bleeding angiomyolipoma. (d) A large lymphangioleiomyoma located posteriorly, between the tip of the spleen and the kidney.

abnormalities. Both are seen in more than half of patients. Some patients have decreased DLCO with preservation of FEV1, whereas others have predominantly airflow obstruction. Restriction is uncommon except in patients who have undergone extensive pleurodesis. Normal spirometry is seen in early stages of LAM or in patients who have mild lung disease, such as those patients with TSC who are screened for LAM. Diagnosis

The diagnosis of LAM can be made by lung biopsy or by clinical and roentgenographic data. The latter criteria consist of a history of recurrent pneumothoraces and/or chylothorax, chylous ascites, abdominal lymphangioleiomyomas and/or AMLs, and a chest CT scan showing multiple thin-walled cysts throughout the lungs. In patients with TSC, the presence of lung cysts is usually sufficient to establish a diagnosis. In patients without TSC, the presence of lung cysts in the absence of extrapulmonary disease is not diagnostic of LAM. In those cases, tissue biopsy may be necessary to confirm the diagnosis. LAM cells can

also be isolated from blood and other body fluids for genetic analysis. Natural Course: Measures of Disease Severity

The natural course of LAM is highly variable. Severity of disease may be assessed by lung biopsy (LAM histology score), pulmonary function testing, high-resolution chest CT scans (CT grade), and cardiopulmonary exercise testing. The LAM histologic score (LHS), based on the extent of replacement of lung tissue by cystic lesions and infiltration by LAM cells (LHS-1, less than 25%; LHS-2, 25–50%; LHS-3, more than 50%), correlates with time to transplantation or death. The severity of disease can also be graded on CT scans by estimating the amount of the lung involved with LAM (grade 0, no involvement; grade 1, less than 30% involved; grade 2, 30–60% involved; grade 3, more than 60% involved). DLCO and, to a lesser extent, FEV1 correlate with LHS scores and CT grade of severity. Cardiopulmonary exercise testing with measurement of maximal oxygen uptake (VO2 max) is another method of assessing disease severity in LAM. VO2

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membrane proteins through the amino termini, thus bridging the plasma membrane and cortical actin filaments. Hamartin also binds neurofilament L in cortical neurons, perhaps influencing neuronal migration. Hamartin regulates Rho activity; the absence of hamartin leads to loss of focal adhesions, detachment from substrate, and cell rounding.

Percent predicted

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Figure 4 VO2 max, DLCO, and FEV1, according to CT scan grade of disease severity. Severity grade 1, white bars; severity grade 2, solid bars; severity grade 3, cross-hatched bars. *Significantly different by ANOVA (po0:001) from severity score 1 patients. **Significantly different (po0:001) from severity scores 1 and 2.

max correlates with DLCO and FEV1, CT scans, use of oxygen, and LHS scores (Figure 4). DLCO is the single best predictor of VO2 max and exerciseinduced hypoxemia. Exercise-induced hypoxemia, however, may occur in patients with near normal DLCO and FEV1.

Pathogenesis TSC1 and TSC2 are tumor suppressor genes, and, consistent with Knudson’s ‘two-hit’ hypothesis of tumor development, loss of heterozygosity of TSC2 has been detected in LAM lesions from lung and kidney. In vivo, 130 kDa hamartin, the protein product of the TSC1 gene, and 198 kDa tuberin, that of the TSC2 gene, form a 450 kDa complex that is primarily localized in the cytosol, with a small fraction associated with the cytoskeleton or membranes. Complex formation is important for the stability of both proteins: tuberin is a cytosolic chaperone of hamartin, and hamartin stabilizes tuberin by preventing its proteasomal degradation. TSC1

The TSC1 gene maps to chromosome 9 (9q34) and contains 23 exons. Hamartin is generated from exons 3–23. It has limited similarity with other known proteins and is postulated to play a role in cell adhesion by binding activated ezrin–radixin–moesin (ERM) proteins. These proteins bind filamentous actin through their carboxy termini and integral

The TSC2 gene, containing 41 exons, is located on chromosome 16 (16p13.3) and is postulated to have roles in pathways that control cell growth and proliferation. Cell cycle Tuberin is a negative regulator of cellcycle progression because loss of tuberin function shortens the G1 phase of the cell cycle (Figure 5). Tuberin stabilizes levels of p27KIP1, a cyclindependent kinase inhibitor, and thus inhibits cellcycle progression. With mutation of tuberin, p27 is unstable and mislocated to the cytoplasm, allowing the cell cycle to progress. cyclin-dependent kinase-1 (CDK1) and cyclins A and B1 have been shown to interact with the hamartin–tuberin complex. Cell growth and proliferation Tuberin is sensitive to at least two types of signals that are important for growth: mitogens and energy status. Mitogenic stimuli activate phosphatidylinositide-3-OH kinase, which activates Akt, leading to phosphorylation and inhibition of tuberin, thus relieving the negative regulation of Ras homolog enriched in brain (Rheb), a GTP-binding protein, and mammalian target of rapamycin (mTOR), resulting in cell growth and proliferation (Figure 5). Tuberin is also a substrate of AMP-activated kinase, which is activated by high levels of AMP, indicative of a low-energy state. This phosphorylation activates tuberin and enforces the inhibition of cell growth. Transcriptional activation Tuberin can interact with members of the retinoid X receptor family, and it has different effects on transcription depending on which family member is bound. Tuberin facilitates transcription of genes controlled by the vitamin D receptor and peroxisome proliferator-activated receptor, but it negatively regulates transcription of genes controlled by the glucocorticoid receptor. Rab5 and endocytosis Tuberin interacts with rabaptin-5, a protein that also associates with Rab5. Tuberin acts as a Rab5-GAP (GTPase-activating protein), influencing a critical step in docking and

INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis Mitogens AMP

PI3K Akt

AMPK

TSC1/2

Rheb

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hemangiosarcomas of the spleen, leiomyomas/ leiomyosarcomas of the uterus, pituitary adenoma, and brain tumors. TSC1 or TSC2 knockout mice are embryonically lethal, whereas the heterozygous mice develop kidney cystadenomas and liver hemangiomas. As seen in the human disease, the TSC1 heterozygous phenotype seems somewhat milder than that of the TSC2 heterozygote.

Management and Therapy

p27

Hormonal Therapy Cell cycle

mTOR

4E-BP1

S6K1

eIF-4e

S6

Because estrogens are implicated in the pathogenesis of LAM, antiestrogen therapy, especially progesterone, had been widely used as treatment. However, a retrospective study of a large group of patients found no significant difference in rates of decline in FEV1 and DLCO between patients treated with intramuscular or oral progesterone and untreated patients. Bronchodilators

Cell growth, proliferation Figure 5 Model of TSC1/2 pathways. The TSC1/2 complex controls cell-cycle progression and cell growth and proliferation. Tuberin stabilizes levels of p27KIP1, a cyclin-dependent kinase inhibitor, and thus inhibits cell-cycle progression. Tuberin also has Rheb GAP (GTPase-activating protein) activity, which leads to inactivation of Rheb by conversion of Rheb-GTP to RhebGDP. Rheb controls mTOR, which regulates the phosphorylation of at least two substrates: 4E-BP1, relieving the inhibition of eIF4E and allowing cap-dependent translation, and S6K1, resulting in the phosphorylation of S6 and 50 TOP (terminal oligopyrimidine tract) translation. TSC1/2 complex is sensitive to at least two types of signals: mitogens and energy status. Mitogenic stimulation activates phosphatidylinositide-3-OH kinase (PI3K), which activates Akt serine/threonine kinase, leading to phosphorylation and inhibition of tuberin and thus resulting in increased translation and cell growth and proliferation. A state of low cellular energy is signaled by increased AMP, which activates AMP-activated kinase (AMPK). AMPK phosphorylates and, in this case, activates tuberin, slowing translation and cell growth and proliferation.

fusion during endocytosis. Loss of tuberin function may increase Rab5-GTP, leading to an increased rate of fluid-phase endocytosis, possibly resulting in improper targeting of proteins.

Animal Models There is no animal model for pulmonary LAM. However, there are models of other effects of TSC1 or TSC2 gene loss. The Eker rat has a germline retrotransposon insertion in TSC2. The homozygous mutant dies at approximately 13 days of fetal life. Renal carcinomas develop in heterozygous rats, with the potential for development of hemangiomas/

Approximately 25% of LAM patients respond to bronchodilators with an increase in airflow. Treatment with these drugs is recommended for those who show a positive response. Pneumothorax

Uncomplicated pneumothorax should be treated by closed chest tube drainage. If the lung does not expand or if the pneumothorax recurs, pleurodesis is recommended. Talc pleurodesis is most effective but is also more likely to result in compromise of lung volumes. Chylothorax

Repeated thoracentesis may lead to progressive malnutrition and infectious complications. Talc pleurodesis by video-assisted thorascopic surgery may be effective if the rate of chyle generation is reduced. To minimize the volume of chyle generated, the patient should be placed on a fat-free total parenteral nutrition regime prior to, during, and after surgery. Additional treatments to be tried are medroxyprogesterone injections and octreotide. No treatment has proven consistently effective. Pleuroperitoneal shunts have also been tried. Ascites and Edema

Ascites, peripheral edema, and compression of the bladder and other viscera by large lymphangioleiomyomata may cause severe symptomatology, including pain, obstipation, and urinary frequency. A low-fat diet, diuretics, and medroxyprogesterone have

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not proven effective. A peritoneal venous shunt may be considered for most severe cases when the ascites is disabling and causing mechanical/nutritional problems. Oxygen Therapy

Since exercise-induced hypoxemia occurs in patients with near normal DLCO and FEV1, exercise oximetry should be considered to determine the need for supplemental oxygen. Transplantation

Patients with severe LAM in whom DLCO and/or FEV1 have decreased to levels under 40% of predicted should be referred to a transplantation center for evaluation. The survival rate of LAM patients undergoing lung transplantation has been reported to be slightly better than that seen in other lung transplant patients (69 versus 53%). However, this report referred to a small number of patients, and the difference was not statistically significant. Operative morbidity, because of prior pleurodesis, tends to be higher. In addition, recurrence of LAM in the allograft has been reported. However, the clinical implications of LAM cells seeding in the allograft lung appear to be minor, and this is not a contraindication to lung transplantation. Rapamycin

Rapamycin is a microbial macrolide that inhibits mTOR. It is a Food and Drug Administrationapproved drug that is used for immunosuppression after organ transplantation. Clinical trials to study the effect of rapamycin on growth of AMLs are under way. Studies in Eker rats showed a decrease in the size of kidney tumors with rapamycin. However, active disease was still detected after 2 months of therapy. It was not clear if this was residual disease or new tumors. Osteoporosis

Osteopenia and osteoporosis occur in 70% of patients with LAM. It is recommended that LAM patients undergo periodic evaluation of bone density and those with osteoporosis be treated with calcium, supplementary vitamin D, and bisphosphonates. In view of the rapid deterioration of bone density after lung transplantation, early initiation of therapy in LAM patients with severe lung disease and osteopenia is recommended. See also: Lung Imaging. Matrix Metalloproteinase Inhibitors. Matrix Metalloproteinases. Oxygen Therapy. Pleural Effusions: Chylothorax, Psuedochylothorax, LAM,

and Yellow Nail Syndrome. Pneumothorax. Smooth Muscle Cells: Airway. Tuberous Sclerosis.

Further Reading Bittman I, Rolf B, Amann G, et al. (2003) Recurrence of lymphangioleiomyomatosis after single lung transplantation: new insights into pathogenesis. Human Pathology 34: 95–98. Chu SC, Horiba K, Usuki J, et al. (1999) Comprehensive evaluation of 35 patients with lymphangioleiomyomatosis. Chest 115: 1041–1052. Costello LC, Hartman TE, and Ryu JH (2000) High frequency of pulmonary lymphangioleiomyomatosis in women with tuberous sclerosis complex. Mayo Clinic Proceedings 75: 591–594. Franz DN, Brody A, Meyer C, et al. (2001) Mutational and radiographic analysis of pulmonary disease consistent with lymphangioleiomyomatosis and micronodular pneumocyte hyperplasia in women with tuberous sclerosis. American Journal of Respiratory and Critical Care Medicine 164: 661–668. Henske EP (2003) Metastasis of benign tumor cells in tuberous sclerosis complex. Genes, Chromosomes and Cancer 38: 376– 381. Karbowniczek M, Astrinidis A, Balsara BR, et al. (2003) Recurrent lymphangioleiomyomatosis after transplantation. American Journal of Respiratory and Critical Care Medicine 167: 976–982. Kitaichi M, Nishimura K, Harumi I, et al. (1995) Pulmonary lymphangioleiomyomatosis: a report of 46 patients including a clinicopathologic study of prognostic factors. American Journal of Respiratory and Critical Care Medicine 151: 527–533. Krymskaya V (2003) Tumour suppressors hamartin and tuberin: intracellular signalling. Cellular Signalling 15: 729–739. Li Y, Corradetti MN, Inoki K, and Guan K (2004) TSC2: filling the GAP in the mTOR signaling pathway. Trends in Biochemical Sciences 29: 32–38. Matsui K, Takeda K, Yu Z, et al. (2001) Prognostic significance of pulmonary lymphangioleiomyomatosis histologic score. American Journal of Surgical Pathology 25: 479–484. Moss J, Avila NA, Barnes PM, et al. (2001) Prevalence and clinical characteristics of lymphangioleiomyomatosis (LAM) in patients with tuberous sclerosis complex. American Journal of Respiratory and Critical Care Medicine 163: 669–671. Pechet TT, Meyers BF, Guthrie TJ, et al. (2004) Lung transplantation for lymphangioleiomyomatosis. Journal of Heart and Lung Transplantation 23: 301–308. Sullivan EJ (1998) Lymphangioleiomyomatosis. A review. Chest 114: 1689–1703. Taveira-DaSilva AM, Hedin CJ, Stylianou MP, et al. (2001) Reversible airflow obstruction, proliferation of abnormal smooth muscle cells and impairment of gas exchange as predictors of outcome in lymphangioleiomyomatosis. American Journal of Respiratory and Critical Care Medicine 164: 1072–1076. Taveira-DaSilva AM, Stylianou MP, Hedin CJ, et al. (2003) Maximal oxygen uptake and severity of disease in lymphangioleiomyomatosis. American Journal of Respiratory and Critical Care Medicine 168: 1427–1431. Taveira-DaSilva AM, Stylianou MP, Hedin CJ, et al. (2004) Decline in lung function in patients with lymphangioleiomyomatosis treated with or without progesterone. Chest 126: 1867–1874. Urban TJ, Lazor R, Lacronique J, et al. (1999) Pulmonary lymphangioleiomyomatosis. A study of 69 patients. Medicine 78: 321– 337. Yeung RS (2003) Multiple roles of the tuberous sclerosis complex genes. Genes, Chromosomes and Cancer 38: 368–375.

ION TRANSPORT / Overview 455

ION TRANSPORT Contents

Overview Calcium Channels Chloride Channels ENaC (Epithelial Sodium Channel) Potassium Channels

Overview D C Eaton and L Jain, Emory University School of Medicine, Atlanta, GA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Efficient gas exchange in the lungs depends on precise regulation of the amount of fluid in the thin liquid layer lining the alveolar epithelium. Studies in vivo and in isolated lungs have shown that fluid can be transported from the alveolar into the interstitial spaces, and that this process can be inhibited by the addition of amiloride, an Naþ channel inhibitor, to the alveolar space. These results suggested that alveolar epithelial cells are the major sites of Naþ transport and fluid absorption in the adult lung. The alveolar epithelium, which covers more than 99% of the large internal surface area of the lung (100–150 m2 in humans), is composed of two distinct cell types, alveolar type I (T1) and type II (T2) cells. T2 cells, which cover only 2–5% of the internal surface area of the lung, are cuboidal cells that secrete pulmonary surfactant. T2 cells contain ion channels, including several forms of the amiloride-sensitive epithelial sodium channel (ENaC), and a chloride channel, the cystic fibrosis transmembrane regulator (CFTR). T1 cells are large squamous cells whose thin cytoplasmic extensions cover 495% of the internal surface area of the lung. T1 cells contain functional ENaC, pimozide-sensitive, cyclic nucleotide-gated, nonselective cation channels, and K þ channels, and appear to contain some CFTR. Besides these ion channels, the apical membrane of most airway epithelia contains several different other Cl
channels. The Ca2 þ -activated Cl-channel (CLCA) is another anion channel at the apical membrane of airway epithelia that responds acutely to increases in intracellular calcium and is involved in transepithelial fluid transport. In some airway cells, there are also volume-regulated Cl-channels (ClC family of channels) that respond to changes in cell volume to alter chloride transport.

Introduction Normal lung function is critically dependent upon maintaining a thin layer of fluid on the cell surfaces of the airway, but this fluid layer is especially critical in the alveoli where abnormalities in lung fluid balance can lead to substantial pathology including pulmonary edema (see Fluid Balance in the Lung), a propensity to develop airway infections (see Cystic Fibrosis: Cystic

Fibrosis Transmembrane Conductance Regulator (CFTR) Gene), and acute respiratory distress syndrome (see Acute Respiratory Distress Syndrome).

The Role of Airway Epithelium in Fluid Clearance Epithelial cells line the compartments of the body and separate the interior compartments (cells, blood, and interstitial fluid) from compartments that are contiguous with the outside world (GI tract, urinary space, and, particularly important for this text, airways). Epithelial tissues have specifically evolved to move substances from one of these compartments to the other using ATP energy in the process. It is the active movement or transport of ions across the airway epithelia that drives airway fluid clearance. Fluid appears in the airways because of the passive movement of fluid driven by hydrostatic pressure out of the pulmonary capillaries across the airway epithelium. The amount secreted is relatively constant (because the pulmonary capillary pressure and permeability of the epithelium are relatively constant) except under pathological conditions when either the pulmonary blood pressure may be abnormally elevated (see Pulmonary Circulation) or the permeability of the epithelium may be increased by inflammation or disease (see Acute Respiratory Distress Syndrome). The amount of fluid on the airway surface, therefore, represents a balance between the rate at which fluid is passively secreted and the rate at which it is actively reabsorbed. Since fluid absorption depends upon the rate of ion transport, regulation of the ion transport provides the mechanism by which the amount of airway fluid is regulated.

A General Mechanism for Airway Epithelial Ion Transport: The Transcellular Route Epithelial transport requires that epithelial cells are polarized, that is, the proteins present in the apical

456 ION TRANSPORT / Overview

Sodium permeability pathways

Interstitium Tight junctions

Lateral space

Pulmonary capillary

Airway lumen

Step 1 Na+

Na+

ATP

Na+

K+

Step 4

Step 2

K+

Anions

Anions

Anions

Water

Water

Water

Aquaporins Apical membrane

Step 3 Basolateral membrane

Figure 1 A schematic of generalized airway epithelium salt and water transport. Transport can be viewed as a four step process. Step 1 is the active extrusion of sodium via (Na,K)-ATPase from the cell to the interstitium. This creates a low concentration of sodium within the cell so that sodium moves downhill from the lumen to cell interior via a variety of sodium-permeable ion channels. The consequence of this transcellular sodium movement is separation of charge (excess Naþ on the interstitial side) that promotes step 2, the movement of anions to balance the positive charge. The accumulation of sodium and anions in the interstitial space produces an osmotic gradient from lumen to interstitium that promotes water movement (step 3). Finally, the accumulation of salt and water in the interstitium promotes the bulk flow of solute and water into the pulmonary capillaries (step 4). The low intracellular sodium generated by the (Na,K)-ATPase also plays a role in establishing the energy gradient necessary for the transport of other ions.

(airway facing) and the basolateral (blood facing) membrane are not the same. This polarization can promote net flux of sodium from lumen to inte rstitium. Figure 1 shows the morphology of a generalized airway epithelium in which salt and water transport can be viewed as a four-step process. Step 1 is the active extrusion of sodium via (Na,K)-ATPase from the cell to the interstitium. This creates a low concentration of sodium within the cell so that sodium moves downhill from the lumen to cell interior via a variety of sodium-permeable ion channels (see Ion Transport: ENaC (Epithelial Sodium Channel)). The consequence of this transcellular sodium movement is separation of charge (excess Naþ on the interstitial side) that promotes step 2, the movement of anions to balance the positive charge (see Ion Transport: Chloride Channels). The accumulation of sodium and anions in the interstitial space produces an osmotic gradient from lumen to interstitium that promotes water movement (step 3). Finally, the accumulation of salt and water in the interstitium promotes the bulk flow of solute and water into the pulmonary capillaries (step 4). The low intracellular sodium generated by the (Na,K)-ATPase also plays a role in establishing the energy gradient necessary for the transport of other ions (see Ion Transport: Calcium Channels; Potassium Channels). The general point is that the transcellular

route involves crossing two membranes – apical and basolateral – and the transporters in the two membranes are different. Despite the initial requirement for the energy-dependent removal from airway epithelial cells of sodium by the (Na,K)-ATPase at the basolateral membrane, the ion channels lining the airway lumen constitute the rate-limiting step for both secretion and absorption (see Ion Transport: Calcium Channels; Chloride Channels; ENaC (Epithelial Sodium Channel); Potassium Channels).

The Paracellular Route In the absence of any other process, as water follows sodium and its anions across the epithelium, the fluid volume remaining in the airway lumen would be reduced. Therefore, any solute that has not been specifically transported via the transcellular route would become more concentrated. As the luminal concentration rises, this would generate a concentration gradient across the tight junctions between lumen and interstitium. In addition, if the tight junctions are permeable to the substance in question, then the substance will diffuse from lumen to interstitium. This does happen for some ions such as sodium, potassium, chloride, calcium, and magnesium. The amounts reabsorbed depend on the permeability of

ION TRANSPORT / Overview 457

the tight junctions. Since ions are driven not only by concentration gradients, but also by voltage gradients, the transepithelial voltage also plays a role. The lumen of airway epithelium is negative relative to the interstitium and enhances paracellular anion absorption and reduces cation absorption. Because of the paracellular transport pathway, ion transport across airway epithelium can only establish a small concentration gradient: any significant lowering of luminal concentration relative to the interstitium results in a leak back into the lumen as fast as the substance is transported out. The epithelium has a finite passive permeability to the substance, usually through the tight junctions, such that a large concentration gradient between the interstitium and lumen results in a large passive flux. Consider the case of sodium. In the alveolar epithelium, the tight junctions are quite permeable to sodium. As sodium is transported into the interstitium, the interstitial concentration begins to rise. This rise creates a gradient that drives a flux of sodium out of the interstitium, both into the pulmonary capillaries, and back through the tight junctions into the lumen. Most of the sodium moves into the blood, accomplishing the goal of absorption, but some does leak back into the lumen. When the concentration of sodium reaches a high enough value, the concentration gradient between the interstitium and the lumen drives sodium back across the tight junctions as fast as it can be transported through the transcellular pathway from lumen to interstitium. At this point transport through paracellular and transcellular pathways is large, but net transport is zero: the system has established the largest gradient possible, its gradient limit. The leakier the epithelium, the lower is the gradient limit which is presumably why, when epithelial integrity is compromised, the epithelium has difficulty in absorbing sufficient salt and fluid to compensate for the large passive flux through the paracellular pathway. Of course, the same is true for other ions, in particular chloride. The transport of sodium generates a potential driving force to move chloride. However, there can be no substantial concentration gradient since the chloride will begin to leak back across the tight junctions as fast as it moves into the interstitium. As pointed out above, the gradient limit on the movement of ions establishes a balance between the volume of alveolar fluid and salt and the interstitial volume of fluid and salt. If salt and water absorption increases, the accumulation of isotonic fluid in the interstitium will force salt and water across the tight junctions to almost exactly balance the increase in absorption. Thus, large changes in transport often produce only small changes in alveolar fluid amounts. By the same token, increases in permeability of the tight junctions

with concomitant increase in secretion of salt and water can be compensated by increasing the rate of absorption (until the upper limit of the transport capacity of the apical ion channels and the basolateral (Na,K)-ATPase is reached after which alveolar flooding will occur) (see Acute Respiratory Distress Syndrome).

Ion Channels in Airway Epithelium From the discussion above, it is clear that absorption and secretion of ions across the airway epithelium is responsible for maintaining the correct composition and volume of the airway surface liquid. Each of the major ion channels present in airway epithelial cells are dealt with in more depth in Ion Transport: Calcium Channels; Chloride Channels; ENaC (Epithelial Sodium Channel); Potassium Channels.

Cation Channels The amiloride-sensitive, epithelial Naþ channel (ENaC) is expressed at the apical membrane of all airway epithelial cells and is rate-limiting step in controlling the rate of transepithelial Naþ and fluid absorption (see Ion Transport: ENaC (Epithelial Sodium Channel)). Besides ENaC, cyclic-nucleotidegated cation channels (CNGC) are widely expressed in airway (alveoli, trachea, bronchi, and bronchioles). This channel is stimulated by increases in intracellular cGMP. The channel is equally permeable to Naþ and K þ but apparently does still contribute to transepithelial sodium transport in airway epithelial cells. Blockers of cyclic- nucleotide-gated cation channels (l-cis-diltiazem and dichlorobenzamil) inhibit an amiloride-insensitive component of sodium and fluid absorption in airways. Also, lung epithelial cells have several other types of cation channels whose molecular identity has been less well defined. The most common types are several species of nonselective cation channels that are stimulated by relatively high concentrations of intracellular calcium and are particularly prevalent in fetal lung epithelial cells. Potassium channels in airway epithelial cells mostly reside in the basolateral membrane where they form a pathway to allow exit of the potassium transported into the cell by the (Na,K)-ATPase. Many different types of potassium channels have been described (for a complete description, see Ion Transport: Potassium Channels). More surprising than the basolateral K þ channels are potassium channels on the apical surface of airway epithelial cells. There appear to be several members of the voltage-gated potassium channel (Kv) family present in T1 cells. Such channels offer the interesting prospect of Naþ

458 ION TRANSPORT / Overview

for K þ exchange rather than sodium chloride uptake. This would make these cells analogous to principal cells of the cortical collecting duct in the mammalian kidney and would offer yet another way to adjust the volume and ionic composition of the alveolar fluid.

Anion Channels There are also several different Cl
channels in the apical membrane of most airway epithelia. The cystic fibrosis transport regulator (CFTR) is a member of the ATP-binding cassette (ABC) family of membrane transporters. CFTR is a Cl
channel that is stimulated by increases in intracellular cAMP and is the channel that is responsible for basal Cl
transport (either absorption or secretion depending upon the location and circumstances) in airway epithelia text (see Cystic Fibrosis: Overview). The Ca2 þ -activated Cl-channel (CLCA) is another anion channel at the apical membrane of airway epithelia that responds acutely to increases in intracellular calcium and is involved in transepithelial fluid transport (see Ion Transport: Chloride Channels). There are at least four members of this family with widespread tissue distribution, but only CLCA2 is present in tracheal epithelial cells. In some airway cells, there are also volume-regulated Cl-channels (ClC family of channels) that respond to changes in cell volume to alter chloride transport (see Ion Transport: Chloride

Channels). ClC-2 is ubiquitously distributed including lung epithelial cells; ClC-3 is a volume-activated chloride channel that appears to be present in all cells including lung epithelial cells. ClC-6 and ClC-7 are also present in the airways but are not functionally expressed as membrane Cl
channels, but rather as intracellular organelle channels contributing to acidification of endosomal compartments. Recently, a so-called maxi-Cl channel has been cloned and is present ubiquitously in airway epithelial cells. The physiological role of this channel is unclear since the conductance is so high that the activity of even one channel would flood epithelial cells with chloride. Some have suggested that its normal function is in the mitochondrial membrane, but that during apoptosis, the channel is translocated to the plasma membrane where it contributes to programmed cell death. Besides ion channels, airway cells also have water channels (members of the aquaporin family of proteins), with different members of the family expressed in different regions of the mammalian lung. These water channels mediate the osmotically induced fluid movement in response to ion transport (see Aquaporins).

Summary Ion channels are critical for normal fluid balance in the lung. The large diversity of ion channels present + Cl− ENaC Na Na+ ,K+ HSC

Alveolus

Paracellular Na+,K+,Cl−

Paracellular Na+,K+,Cl− CNGC Na+,K+

CNGC H2O Na+,K+ AQP5

K els nn ha

C

K+ K+ K Channels

T1 cell

ENaC ENaC HSC NSC Na+ Na+,K+ H2O AQP5 Na+

K+ Na,K-ATPase

Cl− CFTR

CLC

Paracellular

ENaC NSC

Na+,K+,Cl− T2 cell

Cl−

Na+

ENaC HSC Na+ K+

K K+ Channels CLC (Na,K)-ATPase

Interstitium Pulmonary capillary Figure 2 Schematic depiction of alveolar epithelial ion transport. Naþ is absorbed from the apical surface of both T1 and T2 cells via ENaC (HSC and NSC channels). Electroneutrality is conserved with Cl– movement through CFTR or CLC channels in T2 cells, and/or paracellularly through tight junctions. Naþ is transported from the basal surface of both cell types into the interstitial space by (Naþ ,K þ )ATPase. K þ may be transported from alveolar epithelial cells via K þ channels located on the apical surface of T1 cells or through potential basolateral K þ channels in T2I cells. Cyclic-nucleotide-gated channels (CNGC) are alternative pathway for the movement of Naþ that would be amiloride insensitive. CNG channels could also be a pathway for calcium entry into cells. If net ion transport is from the apical surface to the interstitium, an osmotic gradient would be created, which would in turn direct water transport in the same direction, either through aquaporins or by diffusion.

ION TRANSPORT / Calcium Channels 459

in airway epithelium means that there are multiple, redundant pathways for the control of fluid balance. Abnormalities in ion channel function can strongly exacerbate the tendency for edema formation in sepsis or acute lung injury. A model for ion transport in airway is shown in Figure 2.

Acknowledgments This study was supported by R01 HL063306 to LJ; and R01 HL071621 and P01 DK061521 to DCE. See also: Acute Respiratory Distress Syndrome. Apoptosis. Aquaporins. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Epithelial Cells: Type I Cells; Type II Cells. Fluid Balance in the Lung. Ion Transport: Calcium Channels; Chloride Channels; ENaC (Epithelial Sodium Channel); Potassium Channels. Pulmonary Circulation.

Further Reading Chinet TC, Gabriel SE, Penland CM, et al. (1997) CFTR-like chloride channels in non-ciliated bronchiolar epithelial (Clara) cells. Biochemical and Biophysical Research Communications 230: 470–475. Frank J, Roux J, Kawakatsu H, et al. (2003) TGF-beta 1 decreases expression of the epithelial sodium channel alpha ENaC and alveolar epithelial vectorial sodium and fluid transport via an ERK 1/2-dependent mechanism. Journal of Biological Chemistry 278(45): 43939–43950. Giebisch G, Hebert SC, and Wang WH (2003) New aspects of renal potassium transport. Pflugers Archives 446: 289–297. Jentsch TJ, Friedrich T, Schriever A, and Yamada H (1999) The CLC chloride channel family. Pflugers Archives 437: 783–795. Knowles MR, Olivier K, Noone P, and Boucher RC (1995) Pharmacologic modulation of salt and water in the airway epithelium in cystic fibrosis. American Journal of Respiratory and Critical Care Medicine 151: 65–69. Leroy C, Dagenais A, Berthiaume Y, and Brochiero E (2004) Molecular identity and function in transepithelial transport of K(ATP) channels in alveolar epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 286: L1027–L1037. Matalon S, Lazrak A, Jain L, and Eaton DC (2002) Invited review: biophysical properties of sodium channels in lung alveolar epithelial cells. Journal of Applied Physiology 93: 1852–1859. Matthay MA, Folkesson HG, and Clerici C (2002) Lung epithelial fluid transport and the resolution of pulmonary edema. Physiological Reviews 82: 569–600. Matulef K and Zagotta WN (2003) Cyclic nucleotide-gated ion channels. Annual Review of Cell and Developmental Biology 19: 23–44. O’Grady SM and Lee SY (2003) Chloride and potassium channel function in alveolar epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 284: 689–700. Schwiebert EM, Cid-Soto LP, Stafford D, et al. (1998) Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic fibrosis airway cells. Proceedings of the National Academy of Sciences, USA 95: 3879–3884.

Schwiebert EM, Egan ME, Hwang TH, et al. (1995) CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell 81: 1063–1073. Strange K, Emma F, and Jackson PS (1996) Cellular and molecular physiology of volume-sensitive anion channels. American Journal of Physiology 270: C711–C730.

Calcium Channels Y Marunaka and N Niisato, Kyoto Prefectural University of Medicine, Kyoto, Japan & 2006 Elsevier Ltd. All rights reserved.

Abstract The Ca2 þ channels are classified into: (1) voltage-gated Ca2 þ channel, (2) inositol 1,4,5-triphosphate (IP3)-activated Ca2 þ channel, (3) ryanodine-receptor (RyRs) Ca2 þ -release channel, (4) store-operated Ca2 þ (Ca2 þ -release-activated Ca2 þ (CRAC)) channel, and (5) transient receptor potential (TRP)-related Ca2 þ channel. The cytosolic Ca2 þ has function as a regulator of various enzymes and plays an important role as an intracellular second messenger of various hormones in tissues such as the lung. Ca2 þ enters into the cytosolic space via several types of Ca2 þ channels such as voltage-gated Ca2 þ channels across the plasma membrane and is also released into the cytosolic space from intracellular organelles such as endoplasmic reticulum via Ca2 þ -release channels associated with IP3 and RyRs receptors. Ca2 þ also enters into the cytosolic space through CRAC channels triggered by emptying of intracellular Ca2 þ stores so-called capacitative Ca2 þ entry. The molecular basis of the CRAC channel would be the TRP-related Ca2 þ channel. These channels cooperatively regulate the cytosolic Ca2 þ concentration ([Ca2 þ ]c) in the lung. Regulation of the [Ca2 þ ]c maintains cell function in the lung such as contraction of smooth muscles, cytokine secretion, ion channel activity etc. On the other hand, disorders of these channels might cause pathogenesis of lung function such as lung edema.

Introduction Cytosolic Ca2 þ plays essential roles in respiratory tissues including airway epithelium, alveolar epithelium, endothelium of blood vessels, and smooth muscles. In the airway epithelium, the cytosolic Ca2 þ stimulates fluid secretion via activation of Cl
efflux to prevent the airway epithelium from drying out. In the alveolar epithelium, the cytosolic Ca2 þ plays an important role in the regulation of the thin fluid layer covering the epithelium via regulation of Na þ absorption. An increase in the cytosolic Ca2 þ concentration elevates the endothelial permeability to protein and other solutes, leading to lung edema. Further, smooth muscles contract as a result of an increase in the cytosolic Ca2 þ concentration. This leads to a decrease in the diameter of airways and blood vessels, resulting in an increase of resistance.

460 ION TRANSPORT / Calcium Channels

Thus, an increase in the cytosolic Ca2 þ concentration has various important roles in the function of respiratory tissues. This increase in the cytosolic Ca2 þ concentration is mediated by Ca2 þ influx from the extracellular space via Ca2 þ channels and also is caused by Ca2 þ release from intracellular organelles such as endoplasmic reticulum via Ca2 þ -release channels associated with the inositol 1,4,5-triphosphate (IP3) receptors and ryanodine receptors (RyRs). Table 1 summarizes the classification of Ca2 þ channels: (1) voltage-gated Ca2 þ channel; (2) inositol 1,4,5-triphosphate (IP3)-activated Ca2 þ channel; (3) ryanodine-receptor (RyRs) Ca2 þ -release channel; (4) store-operated Ca2 þ (Ca2 þ -releaseactivated Ca2 þ (CRAC)) channel; and (5) transient receptor potential (TRP)-related Ca2 þ channel.

Voltage-Gated Ca2+ Channels Ca2 þ channels at the plasma membrane are characterized, in general, as voltage-gated Ca2 þ channels (Table 2 and Figure 1).This means that the voltagegated Ca2 þ channel is activated by membrane depolarization, that is, open probability of the Ca2 þ channel is increased by depolarization of the plasma membrane. The voltage-gated Ca2 þ channels are classified as L, T, N, P/Q, and R types based on the current profiles through the channels. These types of channels are, in general, located in skeletal muscles, heart muscles, and neurons. The voltage-gated Ca2 þ channel belongs to the superfamily of voltagegated cation channels including Na þ , K þ , and Ca2 þ channels. The voltage-gated cation channels have the

Table 1 Classification of Ca2 þ channels Classification of channels Voltage-gated Ca

2þ

Function

channels

IP3-activated Ca2 þ channel Ryanodine-receptor Ca2 þ -release channel

Store-operated Ca2 þ channel Transient receptor potential (TRP)-related Ca2 þ channel

Ca2 þ influx into the cytoplasmic space from the extracellular space linking membrane depolarization Ca2 þ release to the cytoplasmic space from the ER at IP3 binding to this ligandgated Ca2 þ channel Ca2 þ release to the cytoplasmic space from the SR blocked by ryanodine binding to its receptor in the channel facing the L-type voltage-gated Ca2 þ channel located on the T tubules in skeletal muscles and activated by activation of L-type voltage-gated Ca2 þ channel Ca2 þ entry through store-operated Ca2 þ channels triggered by emptying of intracellular Ca2 þ stores; capacitative Ca2 þ entry TRP-related Ca2 þ channel reported as a candidate for the store-operated Ca2 þ channel (or Ca2 þ -release-activated Ca2 þ (CRAC) channel)

ER, endoplasmic reticulum; SR, sarcoplasmic reticulum. Table 2 Properties and classification of voltage-gated Ca2 þ channels Channel type of property

Kinetics

Voltage activation

Pharmacology

Location

Function

Genes

L

Long duration

High threshold (4
30 mV)

Blocked by DHPs

Transient

Low threshold (o
30 mV)

Blocked by DHPs to a less extent

Link membrane depolarization to cytosolic Ca2 þ signaling Repetitive firing of action potentials

aıS, aıC, aıD, aıF

T

N

Intermediate– long duration

High threshold (4
30 mV)

Insensitive to DHPs, blocked by o-conotoxin GVIA

Exocytotic neurotransmitter release

aıB

P/Q

Intermediate– long duration

High threshold (4
30 mV)

Insensitive to DHPs, blocked by o-agatoxin IVA

Exocytotic neurotransmitter release

aıA

R

Intermediate

High threshold (4
30 mV)

Insensitive to DHPs, blocked by o-agatoxin IIIA

Heart, skeletal, muscle, vascular smooth muscle, neuroendocrine Sinoatrial node of heart, brain neurons Presynaptic terminals, dendrites, cell bodies of neurons Cerebellar Purkinje cells, granule cells, and cell bodies of central neurons Cerebellar granule cells, neurons

Exocytotic neurotransmitter release

aıE

DHP, 1,4-dihydropyridine.

aıG, aıH, aıI

ION TRANSPORT / Calcium Channels 461 2


1 N Domain I

Domain II

4 p 1 2 3 4 + 5 6 1 2 3 + 5 p6 N

Domain III 1 2 3 4 + 5p 6

s s ss

Domain IV 1 2 3 4 + 5 p6

C

C

N

C  N

C 

Figure 1 A membrane-folding model of voltage-gated Ca2 þ channels. A voltage-gated cation channel has the sequence of the S4 segments generating pore for ion permeation across the membrane, which is called an a1 subunit. A voltage-gated Ca2 þ channel is composed of a pseudo-oligomeric a1 subunit, as well as an extracellular a2 subunit, a cytoplasmic b subunit, and membrane-spanning g and d subunits. The a1 subunit consists of four internally homologous repeats (domains I, II, III, and IV) and this subunit forms an ionpermeable pore. Each domain contains an S1 through S6 transmembrane motif. Thus, the a1 subunit has 24 transmembrane domains in total. Data from Isom LL, De jongh KS, and Catterall WA (1994) Auxiliary subunits of voltage-gated ion channels. Neuron 12: 1183–1194.

sequence of the S4 segments generating pore for ion permeation across the membrane, the a1 subunit. The voltage-gated Ca2 þ channel is composed of a pseudo-oligomeric a1 subunit, as well as an extracellular a2 subunit, a cytoplasmic b subunit, and membrane-spanning g and d subunits. The a1 subunit consists of four internally homologous repeats: domains I, II, III, and IV. Each domain contains an S1 through S6 transmembrane motif. Thus, the a1 subunit has 24 transmembrane domains in total. Mammalian a1 subunits of the L-, T-, N-, P/Q-, and R- type Ca2 þ channels are encoded by distinct genes: a1S, a1C, a1D, and a1F genes for the L-type Ca2 þ channel; a1G, a1H, and a1I genes for the T-type Ca2 þ channel; a1B gene for the N-type Ca2 þ channel; a1A gene for the P/Q-type Ca2 þ channel; and a1E gene for the R-type Ca2 þ channel. In respiratory tissues, smooth muscles have an L-type Ca2 þ channel. L-Type Ca2+ Channel

The L-type Ca2 þ channel is activated when the plasma membrane depolarizes (4
30 mV, high threshold). Furthermore, once the L-type Ca2 þ channel is activated, it maintains its activity over a long period of time. The L-type Ca2 þ channel is blocked by 1,4-dihydropyridine (1,4-DHP) such as nitrendipine via binding of 1,4-DHP to the channel, while Bay K 8644, an analog of 1,4-DHP, activates the channel by locking it into an open state. The L-type Ca2 þ channel is located in heart muscle, skeletal

muscle, smooth muscle, neurons, uterus, and neuroendocrine cells. The L-type Ca2 þ channel is activated by protein kinase A via an increase of open probability, and plays a role in excitation-contraction coupling. The L-type Ca2 þ channels are classified as CaV1.1, CaV1.2, CaV1.3, and CaV1.4. T-Type Ca2+ Channel

The T-type Ca2 þ channel is activated when the plasma membrane depolarizes (o
30 mV; low threshold), and is inactivated over a more negative voltage range. When the T-type Ca2 þ channel is activated, it maintains its activity during a short time period (transient duration). These characteristics of the T-type Ca2 þ channel indicate that it functions at the initial time of action potential and produces repetitive firing. The T-type Ca2 þ channel is located in the sinoatrial (SA) node of heart and brain neurons. The T-type Ca2 þ channel is blocked by 1,4-dihydropyridines to a lesser extent than the L-type Ca2 þ channel. T-type Ca2 þ channels are classified as CaV3.1, CaV3.2, and CaV3.3 based on the amino acid sequence. N-Type Ca2+ Channel

Similar to L-type Ca2 þ channels, N-type Ca2 þ channels are activated when the plasma membrane depolarizes (4
30 mV; high threshold). When the N-type Ca2 þ channel is activated, it maintains its activity over a relatively long time period (intermediate

462 ION TRANSPORT / Calcium Channels

to long duration). The N-type Ca2 þ channel is insensitive to 1,4-dihydropyridines, and is blocked by o-conotoxin GVIA. This type of Ca2 þ channel is located in presynaptic terminals, dendrites, and neuronal bodies, and is involved in exocytotic neurotransmitter release. The N-type Ca2 þ channel is classified as CaV2.2. P/Q-Type Ca2+ Channel

The P/Q-type Ca2 þ channel is activated when the plasma membrane depolarizes (4
30 mV; high threshold). When the P/Q-type Ca2 þ channel is activated, the channel maintains its activity during a relatively long time period (intermediate to long duration). The P/Q-type Ca2 þ channel is insensitive to 1,4-dihydropyridine, and is blocked by o-agatoxin IVA. This type of Ca2 þ channel is located in cerebellar Purkinje cells, granule cells, and cell bodies of central neurons. This channel plays a role in exocytotic neurotransmitter release. The P/Q-type Ca2 þ channel is classified as CaV2.1 based on the amino acid sequence. R-Type Ca2+ Channel

The R-type Ca2 þ channel is activated when the plasma membrane depolarizes (4
30 mV, high threshold). Once this channel is activated, it maintains its activity for a relatively long time period (intermediate duration), but less time than the T-, N-, and P/Q-type Ca2 þ channels. The N-type Ca2 þ channel is insensitive to 1,4-dihydropyridines, and is blocked by o-agatoxin IIIA. This type of Ca2 þ channel is located in cerebellar granule cells and neurons, and plays a role in exocytotic neurotransmitter release like the N-type Ca2 þ channel. The R-type Ca2 þ channel is classified as CaV2.3 based on the amino acid sequence.

through the IP3-activated Ca2 þ channel is terminated by dephosphorylation of IP3.

Ryanodine-Receptor Ca2+-Release Channel The Ca2 þ -release channel is located in the sarcoplasmic reticulum of muscles. This type of Ca2 þ channel is called a ryanodine-receptor Ca2 þ -release channel, since the channel is blocked by ryanodine. The ryanodine-receptor Ca2 þ -release channel is a tetramer and each subunit has four transmembrane domains. This channel faces the L-type Ca2 þ channel located on the T tubules in skeletal muscles.

Store-Operated Ca2+ Channels Store-operated Ca2 þ channels play a role in regulating intracellular Ca2 þ . Emptying of intracellular Ca2 þ stores triggers the opening of store-operated Ca2 þ channels, and the Ca2 þ entry through these channels is known as capacitative Ca2 þ entry; hence, these channels are also called capacitative Ca2 þ entry (CCE) channels. Furthermore, the terms of Ca2 þ -release-activated Ca2 þ current (ICRAC)/ Ca2 þ -release-activated Ca2 þ (CRAC) channel are used to distinguish this Ca2 þ entry through storeoperated Ca2 þ channels from other Ca2 þ entry channels. Two models for activation of store-operated Ca2 þ channels (or CCE channels) are proposed. One is a direct conformational coupling between store-operated Ca2 þ channels and IP3-activated Ca2 þ channels. The other involves Ca2 þ -influx factor (CIF), that is, a diffusible messenger based on the fact that CIF is released when the intracellular Ca2 þ store is empty.

IP3-Activated Ca2+ Channels

Transient Receptor Potential-Related Ca2+ Channel

Phospholipase Cb stimulated by G-protein generates IP3 and diacylglycerol from phosphatidylinositol 4,5-biphosphate (PIP2). IP3 interacts with a receptor located in the membrane of endoplasmic reticulum (ER). The receptor is a ligand-gated Ca2 þ channel. When IP3 binds to the ligand-gated Ca2 þ channel, Ca2 þ is released to the cytoplasmic space from the ER. Therefore, this type of Ca2 þ channel is called an IP3-activated Ca2 þ channel. The IP3 receptor is a tetramer (approximately 260 kDa), and is phosphorylated by PKA, PKC, and Ca2 þ /calmodulin (CaM)-dependent protein kinase. Each subunit has six transmembrane domains. The Ca2 þ release

This is a large class of channel subunits based on a modestly similar primary structure and permeability to monovalent cations and Ca2 þ . The TRP-related Ca2 þ channel is a candidate for the store-operated Ca2 þ channel (i.e., CRAC channel). This family is subclassified into three major subfamilies (TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid)), and four more distantly related subfamilies (TRPP, TRPML, TRPN, and TRPA). All TRP channels have six transmembrane regions with cytoplasmic N- and C-termini. In this section, only three major TRPC, TRPM, and TRPV are described. TRPC has seven members (TRPC1 through TRPC7), TRPM

ION TRANSPORT / Calcium Channels 463

has eight members (TRPM1 though TRPM8), and TRPV has six members (TRPV1 through TRPV6): *

*

*

TRPC1: TRPC1 shows a nonselective cation current including Ca2 þ . The TRPC1 is activated by IP3 or thapsigargin to deplete intracellular Ca2 þ stores, and is blocked by Gd3 þ . The TRPC1 is widely expressed throughout the body. The TRPC1 is the intracellular Ca2 þ release channel (store-operated channel). TRPC2: TRPC2 is activated by diacylglycerol, but it is not confirmed whether the TRPC2 is activated by depletion of the intracellular Ca2 þ store. TRPC3, 6, and 7: TRPC3, TRPC6, and TRPC7 form nonselective cation channels. TRPC3, 6, and 7 are activated by diacylglycerol with no requirement for either IP3 or the IP3 receptor. TRPC3 is blocked by SKF96365, verapamil, La3 þ , Gd3 þ , and Ni2 þ . The differences among TRPC3, TRPC6, and TRPC7 are their ion selectivities. TRPC6 is Ca2 þ selective. TRPC6 is highly

*

*

*

*

expressed in the lung tissues including pulmonary arteries. TRPC3 and TRPC7 have relatively low selectivity for Ca2 þ compared with TRPC6. TRPC4 and 5: TRPC4 and TRPC5 show Ca2 þ selective currents that are activated by either IP3 or thapsigargin-induced depletion of intracellular Ca2 þ stores. TRPC4 is activated by G-protein-coupled receptors. TRPC5 is reported as a receptor-operated cation channel. TRPM1: TRPM1 might elevate the intracellular Ca2 þ concentration by an unknown mechanism. TRPM2: TRPM2 is expressed in the brain, bone marrow, spleen, heart, leukocytes, liver, and lung. TRPM2 is activated by ADP-ribose, NAD, and H2O2. TRPM2 permeates not only monovalent cations such as Na þ , K þ , and Cs þ , but also Ca2 þ . TRPM3: TRPM3 elevates the basal level of intracellular Ca2 þ concentration. TRPM3 permeates not only monovalent cations such as Na þ and Cs þ , but also Ca2 þ and Mg2 þ . This channel is activated by extracellular hypotonicity.

Voltage-gated Ca2+ channel

In skeletal muscles

Ca2+

L-type voltage-gated Ca2+ channel

SR

Ca2+

Ca2+-activated K+ channel K+

RyRs Ca2+-release channel (+)

(+)

[Ca2+]c Cl –

Ca2+

Contraction of muscles

(+)

IP3-activated Ca2+ channel

Ca2+-activated Cl– channel Ca2+ Empty of Ca2+ (+)

Ca2+ ADP

ATP

Ca2+

Na+/Ca2+ exchanger

Ca2+ -ATPase Na+

Ca2+ Store-operated Ca2+ channel (TRP-related Ca2+ channel)

Figure 2 Function of Ca2 þ channels in cells: regulation of cytosolic Ca2 þ concentration ([Ca2 þ ]c) by Ca2 þ channels and Ca2 þ transporters in smooth and skeletal muscles (see area indicated by ‘skeletal muscle’). IP3-activated Ca2 þ channel, inositol 1,4,5triphosphate (IP3)-activated Ca2 þ channel; RyRs Ca2 þ -release channel, ryanodine-receptor (RyRs) Ca2 þ -release channel; TRP-related Ca2 þ channel, transient receptor potential (TRP)-related Ca2 þ channel. ( þ ), stimulation or activation.

464 ION TRANSPORT / Calcium Channels *

*

*

*

*

TRPM4: TRPM4 was initially reported to be a plasma protein of 1040 amino acid residues with Ca2 þ permeability: TRPM4a. Subsequently, a full-length TRPM4 (TRPM4b) was revealed that is activated by Ca2 þ but is Ca2 þ -impermeable. TRPM5: TRPM5 is reported to be a Ca2 þ -permeable, store depletion-activated ion channel. This channel is also regulated by a receptor-mediated mechanism that is coupled to phospholipase C activation. TRPM6: TRPM6 produces currents that are activated by a reduction in free Mg2 þ and Mg-ATP, and have identical permeability to Ca2 þ and Mg2 þ . TRPM6 mainly contributes to Mg2 þ balance in the body. TRPM7: TRPM7 forms a Ca2 þ -permeable, nonselective cation channel. The TRPM7-induced channel is activated by increasing intracellular ATP concentration, but is inhibited by high intracellular Mg2 þ and Mg-ATP. The ion selectivity of TRPM7-induced channels is Zn2 þ 4Ni2 þ 4Ba2 þ 4Co2 þ 4Mg2 þ 4Mn2 þ 4Sr2 þ 4Cd2 þ 4Ca2 þ . TRPM8: TRPM8 is responsible for cold-sensing mechanisms. It is activated by cooling cells such as dorsal root ganglia neurons p241C and by cooling agents such as menthol and icilin. TRPM8 is permeable to Ca2 þ .

*

*

TRPV1, 2, 3, and 4: TRPV1, TRPV2, TRPV3, and TRPV4 play a role in sensing of body temperature. The heat thresholds are 4431C in the TRPV1 channel, 4531C in the TRPV2 channel, 431–391C in the TRPV3 channel, and 424–331C in the TRPV4 channel. These channels are expressed widely in dorsal root ganglia neurons, hypothalamus, mechanosensory cochlear hair cells, and skin epidermal keratinocytes. TRPV5 and 6: TRPV5 is known as ECaC1, and TRPV6 as ECaC2. TRPV5 and TRPV6 have different characteristics from TRPV1, TRPV2, TRPV3, and TRPV4. They are highly Ca2 þ selective compared with other TRPVs. These channels are expressed in epithelia. The term ECaC (epithelial Ca2 þ channel) originates from the expression of the channel in epithelia. These channels are constitutively active.

Regulation of the Cytosolic Ca2+ Concentration The cytosolic Ca2 þ concentration is regulated by the various types of Ca2 þ channels mentioned above and other ion transporters such as Na þ /Ca2 þ exchanger and Ca2 þ -ATPase (pump) in different ways depending on the type of cell or tissue in various organs. Figure 2 gives a summary of how the

Chronic hypoxia

Acute hypoxia

(+)

(Vascular smooth muscle)

Voltage-gated Ca2+ channel

(+)

Store-operated Ca2+ channel (TRP-related Ca2+ channel)

(+)

(+)

Expression of TRP-related Ca2+ channel in endothelial cell

Expression of TRP-related Ca2+ channel in vascular smooth muscle

[Ca2+]c

[Ca2+]c in endothelial cell

[Ca2+]c in vascular smooth muscle

Contraction of muscle

ET-1 in endothelial cell

Contraction of muscle

IP3 in vascular smooth muscle

Pulmonary vascular resistance

Pulmonary vascular resistance

(+) Pulmonary artery hypertension

IP3-activated Ca2+ channel in vascular smooth muscle

Pulmonary artery hypertension

Figure 3 Pathogenesis in abnormal regulation of Ca2 þ channels in respiratory system. A model scheme of pulmonary artery hypertension induced by acute and chronic hypoxia. ET-1, endothelin 1; IP3-activated Ca2 þ channel, inositol 1,4,5-triphosphate (IP3)-activated Ca2 þ channel; TRP-related Ca2 þ channel, transient receptor potential (TRP)-related Ca2 þ channel. ( þ ), stimulation or activation.

ION TRANSPORT / Chloride Channels 465

Ca2 þ channels described above and other Ca2 þ transporters regulate the cytosolic Ca2 þ concentration in smooth and skeletal muscles.

Role of Ca2+ Channels in Respiratory Diseases Regulation of the cytosolic Ca2 þ concentration maintains cell function in the lung such as contraction of smooth and skeletal muscles, secretion of cytokine, and activity of ion channel (e.g., Ca2 þ -activated K þ and Cl
channels), etc. On the other hand, disorders of these channels might cause defects in lung function. Genetic defects of Ca2 þ channels are known. Lambert–Eaton syndrome, an autoimmune disorder that is most often seen in patients with small cell lung cancer, is known to be due to an impairment of presynaptic Ca2 þ channels at motor nerve terminals. Antibodies against presynaptic Ca2 þ channels reducing the channel activity (number) cause dysfunction of neurotransmitter release from the presynaptic terminals leading to pathogenesis of neuronal activity in patients. Abnormal environments also cause some diseases. For example, hypoxia causes pulmonary artery hypertension via elevation of cytosolic Ca2 þ concentration. Figure 3 shows a scheme of how acute and chronic hypoxia cause pulmonary artery hypertension. Acute hypoxia directly elevates the cytosolic Ca2 þ concentration in smooth muscle of the pulmonary artery by activating voltage-gated Ca2 þ channels and store-operated Ca2 þ channels (possibly TRP-related Ca2 þ channels), resulting in pulmonary artery hypertension (Figure 3). Chronic hypoxia also acts on endothelial cells of the pulmonary artery by elevating the cytosolic Ca2 þ concentration, which stimulates secretion of endothelin. Endothelin acts on its receptor in smooth muscle of the pulmonary artery, elevating the cytosolic Ca2 þ concentration of the smooth muscle, which causes pulmonary artery hypertension via an increase in the resistance of pulmonary artery resulting from Ca2 þ induced contraction of smooth muscle (Figure 3). The diseases mentioned above are just a few of a number of disorders of Ca2 þ channels; in other words, the Ca2 þ channel plays a key role in the maintenance of normal function of respiratory organs. See also: Fluid Balance in the Lung. Ion Transport: Overview; Chloride Channels. Smooth Muscle Cells: Airway; Vascular.

Further Reading Benham CD, Gunthorpe MJ, and Davis JB (2003) TRPV channels as temperature sensors. Cell Calcium 33: 479–487.

Catterall WA, Striessnic J, Snutch TP, and Perez-Reyes E (2003) International Union of Pharmacology. XL. Compendium of voltage-gated ion channels: calcium channels. Pharmacological Reviews 55: 579–581. Cortright DN and Szallasi A (2004) Biochemical pharmacology of the vanilloid receptor TRPV1. European Journal of Biochemistry 271: 1814–1819. Fleig A and Penner R (2004) The TRPM ion channel subfamily: molecular, biophysical and functional features. Trends in Pharmacological Sciences 25: 633–639. Hoenderop JGJ, Nilius B, and Bindels RJM (2005) Calcium absorption across epithelia. Physiological Reviews 85: 373–422. Isom LL, De jongh KS, and Catterall WA (1994) Auxiliary subunits of voltage-gated ion channels. Neuron 12: 1183–1194. McGeown JG (2004) Interactions between inositol 1,4,5-triphosphate receptors and ryanodine receptors in smooth muscle: one store or two? Cell Calcium 35: 613–619. Mehta D, Bhattacharya J, Matthay MA, and Malik AB (2004) Integrated control of lung fluid balance. American Journal of Physiology. Lung Cellular and Molecular Physiology 287: L1081–L1090. Moczydlowski EG (2003) Electrophysiology of the cell membrane. In: Boron WF and Boulpaep EL (eds.) Medical Physiology, pp. 145–171. Philadelphia: Saunders/Elsevier Science (USA). Moczydlowski EG (2003) Electrical excitability and action potentials. In: Boron WF and Boulpaep EL (eds.) Medical Physiology, pp. 172–203. Philadelphia: Saunders/Elsevier Science (USA). O’Neil RG and Brown RC (2003) The vanilloid receptor family of calcium-permeable channels: molecular integrators of microenvironmental stimuli. News in Physiological Sciences 18: 226–231. Revens U, Wettwer E, and Hala O (2004) Pharmacological modulation of ion channels and transporters. Cell Calcium 35: 575–582. Striessnig J, Hoda J-C, Koschak A, et al. (2004) L-type Ca2 þ channels in Ca2 þ channelopathies. Biochemical and Biophysical Research Communications 322: 1341–1346. Strock J and Diverse-Pierluissi MA (2004) Ca2 þ channels as integrators of G protein-mediated signaling in neurons. Molecular Pharmacology 66: 1071–1076. Thevenod F (2002) Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins. American Journal of Physiology. Cell Physiology 283: C651–C672.

Chloride Channels H R de Jonge and B C Tilly, Erasmus University Medical Center, Rotterdam, The Netherlands & 2006 Elsevier Ltd. All rights reserved.

Abstract Chloride channels in the airways are especially abundant in the surface epithelium and gland compartments where they play an essential role in the transepithelial movement of electrolytes and water. The cystic fibrosis transmembrane conductance regulator, a member of the superfamily of ATP-binding cassette transport proteins, functions as a cyclic AMP- and cyclic GMP-activated chloride channel and is a major determinant of airway fluid homeostasis by triple action: it inhibits isotonic NaCl and fluid absorption by the ciliated cells through its interaction with epithelial sodium channels, it promotes fluid secretion by serous cells in the submucosal glands, and it mediates b-adrenergic

466 ION TRANSPORT / Chloride Channels stimulation of NaCl and fluid absorption in the distal airways and alveolar compartment. Consequently, CF airways show a depletion of airway surface liquid resulting in impaired mucociliary clearance, and a propensity to pulmonary edema. Calcium-activated chloride channels are abundantly expressed in tracheal and bronchial epithelium. Upon activation by Ca2 þ mobilizing, purinergic, or cholinergic receptors, they may serve as compensatory chloride channels in CF. Their molecular nature is not yet elucidated. Putative molecular candidates include the CLCAs (hCLCA1–4). hCLCA1 (gob-5) is associated with mucin granule membranes and plays a key role in mucus overproduction and the pathogenesis of bronchial asthma. In addition, numerous other chloride channel species (e.g., CLCs, bestrophins, CLICs, and ORCCs) have been identified in the respiratory tract and are briefly discussed in this review.

other gene families are less well understood (e.g., calcium-activated chloride channels (CLCAs), bestrophins, chloride intracellular channels (CLICs)) or not yet identified at the molecular level (e.g., volume-regulated anion channel (VRAC), outwardly rectifying chloride channel (ORCC)). This review focuses on the role of Cl
channels in the vectorial transport of salt and water across proximal airways and distal pulmonary epithelium and their involvement in the pathophysiology of cystic fibrosis and pulmonary edema. It also delineates a putative role in other airway functions, including the secretion of mucus and antimicrobial molecules, and in the pathogenesis of asthma.

Introduction Chloride channels form proteinaceous pores in plasma lemma and intracellular membranes that allow the passive diffusion of negatively charged ions along their electrochemical gradient. Their functions range from ion homeostasis to regulation of electrical excitability, cell volume, cell cycle and apoptosis, pH of intracellular organelles, and transepithelial ion transport. Whereas three molecularly distinct Cl
channel families (cystic fibrosis transmembrane conductance regulator (CFTR), CLCs, ligand-gated g-aminobutyric acid (GABA) and glycine receptors) are structurally and functionally well characterized,

Structure CFTR emerged from the search for the cystic fibrosis locus and was the first anion channel identified by expression cloning and reconstitution in lipid bilayers. It belongs to the superfamily of ATP-binding cassette (ABC) transporters for organic compounds but displays Cl
conducting properties. Its unique regulatory domain (R) serves as a target for cAMPand cGMP-dependent protein kinases (Figure 1). The molecular nature of calcium-activated chloride channels (CaCCs) is not yet resolved. Putative

CFTR

CLCA Cleavage site N

C N

ATP NBD1 ATP

NBD2 C

R Pn

?

CBS1

N C

N

N C

CBS2 C

CLC

Barttin

Bestrophin

Figure 1 Topology models of the major classes of chloride channels expressed in the respiratory tract. NBD1,2: nucleotide-binding domains; binding of ATP to NBD2 promotes NBD1–NBD2 cassette heterodimerization and allows channel opening following multisite phosphorylation of the regulatory (R) domain by cAMP- and cGMP-dependent protein kinase. CBS1,2: conserved structural domain of unknown function (named after cystathione-b synthase). Barttin: needed as a b-subunit in ClC-K channel isoforms.

ION TRANSPORT / Chloride Channels 467

candidates belong to a new family of cloned membrane proteins, the CLCAs, including multiple human and murine homologs (hCLCA1–4; mCLCA1–6). Proteins of this family are characterized by sizes of 902–943 amino acids, four or five transmembrane regions, and a highly conserved pattern of five cysteine residues in the extracellular amino-terminus. These proteins are processed by proteolytic cleavage into B90 and B35 kDa fragments (Figure 1). Although transfection studies and reconstitution studies in lipid bilayers suggest that some of the CLCA isoforms may represent genuine Ca2 þ -activated chloride channels, major discrepancies were found between functionally expressed CLCAs and native CaCC currents. At least in one example, this appeared due to the absence of an essential regulatory subunit. In other cases, it remains possible that CLCA proteins activate endogenous Cl
channels rather than being channels themselves. In mammals, at least nine CLC genes have been discovered, encoding for ClCs1–7 and ClCs Ka/b. A topology model of monomeric CLC is depicted in Figure 1. From early electrophysiological studies, it has been proposed that these CLC channels have a so-called ‘double barrel’ structure, for example, each functional unit has two chloride selective pores that are gated independently. High-resolution structures of two bacterial homologs of CLCs (EcClC from Escherichia coli and StClC from Salmonella typhimurium) have confirmed this model. Bestrophins were originally identified in Caenorhabditis elegans by their sequence homology as a distinct group of membrane proteins. Recent models of bestrophin (hBest1–4) suggest that the protein consists of four to six transmembrane domains (Figure 1) and that it is functional as a heterooligomer (tetra- or pentamer). The protein has a large cytoplasmic C-terminus containing consensus sequences for phosphorylation by cyclic AMP- and cyclic GMP-dependent protein kinases and for protein kinase C, as well as a binding site for the b-catalytic subunit of protein phosphatase 2A. The first member of the CLIC family, p64, was isolated from bovine kidney microsomes and subsequently cloned from a kidney cDNA library. To date, six additional homologs genes have been identified, encoding for CLIC1–5 and parchorin. These ubiquitous proteins exist in both soluble and membrane forms, with a single putative transmembrane domain. Recently, the three-dimensional structure of CLIC1 has been elucidated. Surprisingly, the N-terminal domain resembles a so-called ‘thioredoxin fold’ as is present in members of the superfamily of glutathione transferases.

In addition, several other proteins have been reported to function as putative anion selective channels, including ICln, a ubiquitously expressed soluble phosphoprotein that translocates to the membrane upon cell swelling; an outwardly rectifying chloride channel (ORCC), of unknown molecular identity; Mid-1-related chloride channel (MClC), an intracellular chloride channel containing four putative transmembrane domains and expressed moderately in the lung; and Tweety, a novel class of large conductance chloride channels (hTTY1–3), showing sequence homology with the BK-type of calcium-activated potassium channels and activated by cell swelling or a rise in intracellular Ca2 þ . However, current information about the expression and function of these channels in the respiratory tract is rudimentary or absent.

Expression and Regulation The CFTR protein is localized in the apical membrane of epithelial cells including the ciliated cells in human nasal and bronchial epithelium, and the airway serous glandular cells (but not the goblet cells) (Figure 2). Low but functionally important expression is found in the Clara cells of the distal airways and in alveolar epithelia. CFTR activation is triggered primarily by cAMP signals evoked in response to b-adrenergic, VIPergic, and purinergic agonists. In addition, air-side activation of CFTR in Clara cells mediated through cGMP signals is triggered autocrinically by guanylin, a bioactive peptide stored in secretory granules underneath the apical membrane. CaCCs have been functionally identified in the apical membrane of many epithelial tissues, including tracheal and bronchial cells. The putative anion channel protein mCLCA3 (gob-5) is associated exclusively with mucin granule membranes of gastrointestinal and respiratory goblet cells (Figure 2). The mode of activation of CaCC, involving direct voltage-dependent binding of Ca2 þ to the channel or phosphorylation by the Ca2 þ /calmodulin-dependent protein kinase II, differs from CFTR channel gating. Activation of CaCC in epithelial cells requires agonist stimulation of G-protein-coupled receptors (e.g., purinergic and cholinergic) in the apical or basolateral membrane followed by intracellular Ca2 þ mobilization and/or Ca2 þ influx through transient receptor potential (TRP) or cyclic nucleotide-gated (CNG) cation channels. Whereas the expression of some CLCs is tissue specific, for example, ClC-1 and ClC-Ka/b are predominantly found in skeletal muscle and kidneys/inner ear, respectively, the other members of the family

468 ION TRANSPORT / Chloride Channels

Cilium

Apical membrane

ORCC

CFTR

CaCC

ENaC Mucin

NO/cGMP PKG

cAMP PKA

Bestrophin

ORCC

CIC-2

Ca2+

Gob-5 (mCLCA3)

Tight junction ER/golgi

CIC-3/6

CLIC-1/3

CIC-2

Basolateral membrane

Figure 2 Subcellular localization of anion channels and sodium channels (ENaC) in airway epithelial cells. The direction of chloride and sodium ion movements is indicated by black or blue arrows, respectively. Signaling molecules and their interaction with the molecular targets are indicated in red.

are more ubiquitously expressed. In mammalian lung, the presence of at least six different CLC channels (ClC-2–7) has been reported, some of them being expressed only at specific stages during lung development. ClC-1, -2, -Ka, and -Kb are targeted towards the plasma membrane, whereas the other members are localized predominantly in intracellular compartments. With the exception of ClC-Ka and -Kb, which require barttin for activation (Figure 1), all CLCs channels are fully functional by themselves and do not need the presence of accessory proteins. The CLC chloride channels are opened and closed in a voltage-dependent manner. Changes in the extracellular Cl
concentration as well as alterations of the pH modulate the activation kinetics. The presence of ClC-2 has been established in nasal polyps, at the luminal side of the trachea, at the apex of bronchial ciliated cells, in bronchioles, as well as type II alveolar cells. However, chloride current measurements in colonic epithelium suggest a predominantly basolateral localization of ClC-2 in polarized epithelia (Figure 2). ClC-2 mRNA is highest in fetal lung and drops rapidly after birth. In addition to transcriptional and translational regulation, ClC-2 is acutely activated in response to a low extracellular pH. Like ClC-2, the highly conserved ClC-3 is widely expressed in mammals. In the airways, ClC-3 expression has been found in tracheal, bronchial, and bronchiolar epithelia, as well as in the submucosal

glands of nasal tissue. The subcellular localization of ClC-3 is primarily intracellular, although upon overexpression some of the channels may traffic to the plasma membrane. Like ClC-2, expression of ClC-3 is developmentally regulated, although species differences may exist. In rat lung, the expression of ClC3 increases robustly after birth. In humans, however, ClC-3 expression is most prominent mid-gestation. At least three other members of the CLC family are expressed in the airways, ClC-4, -5, and -6, all of them primarily localized intracellularly. ClC-4 and -5 share a high-sequence homology and have similar functional properties, including their ability to function as Cl
/H þ antiporters rather than as ion channels. The highest expression of ClC-5 was found in the kidneys and the intestine, but lower amounts were observed in tracheal epithelium and alveoli. In rat, ClC-5 expression in the lungs is most abundant during embryonic development, but significant levels are also present in neonatal and adult animals. Both ClC-4 and -6 are more ubiquitously expressed. Bestrophins are widely expressed throughout the animal kingdom. Four members of this family have been found in humans (hBest1–4). Bestrophin is localized in the basolateral membranes of airway epithelia and in the Calu-3 human airway serous cell line. Using hBest1- directed siRNA to achieve gene silencing, a prominent contribution of bestrophin to the basolateral anion conductance was demonstrated in this cell type. The conductance could be activated

ION TRANSPORT / Chloride Channels 469

by cyclic AMP- and NO/cyclic GMP-dependent signaling pathways. Moreover, several isoforms have been found to be activated by intracellular Ca2 þ . CLIC channels have a broad tissue distribution and are mainly expressed intracellularly. Despite the fact that these relatively small proteins contain only a single transmembrane domain and the observation that at least one of them, parchorin, exists predominantly in a soluble form, heterologous expression of several members resulted in an increase in the chloride conductance of intracellular vesicles. This, together with the relatively high expression of these proteins in tissues involved in water transport as well as the similarity of their C-termini with pore-forming bacterial proteins, led to the notion that they might function as (regulators of) intracellular chloride channels. Parchorin, a highly hydrophilic phosphoprotein named after its high expression in parietal cells and the choroid plexus, has been studied in lung. Expression of parchorin was found in the mucous epithelial cells of the trachea, as well as in the bronchioles and type II alveolar cells. Although parchorin is largely localized intracellularly, a translocation to the apical plasma membrane has been observed in response to cAMP, supporting a putative role in chloride and water transport. The ORCC is found in many different types of epithelial cells, including airway epithelial cells. Although its biophysical properties are well established, the molecular identity of the channel has not yet been resolved. ORCC has been found both in the apical and basolateral membranes of airway cells, but is regulated differently (Figure 2). In the apical membrane, the channel is activated by cAMP and CFTR, either directly or by a CFTR-dependent release of ATP. In the basolateral membrane, ORCC is stimulated by adenosine receptor activation. Exposure of lung epithelial cells to reactive oxygen species was found to inhibit ORCC activation.

Biological Function and Involvement in Respiratory Disease The biological function and involvement in respiratory disease are discussed as follows (see Table 1). Cystic Fibrosis Transmembrane Conductance Regulator

The apparent function of CFTR in airway ion and fluid transport, and the consequences of its dysfunctioning in CF disease, differ in each airway compartment. In superficial epithelium, by concomitantly inhibiting Naþ absorption through the epithelial Naþ channels (ENaC) and promoting isotonic NaCl and fluid secretion, CFTR regulates the thickness of the thin film (B30 mm) of airway surface liquid (ASL). This ASL consists of a periciliary sol and a mucus gel that are propelled toward the mouth by coordinated ciliary beating. Consequently, CF airways show increased Naþ absorption driving increased absorption of Cl
across non-CFTR shunt pathways and promoting fluid absorption. This results in a depletion of ASL volume, dehydration of mucus, and a reduced mucociliary clearance of pathogens. Symptoms of CF disease could be mimicked in transgenic mice by overexpression of the b-subunit of ENaC channels in the bronchial compartment. The mechanism by which ENaC is upregulated in CF is not yet fully resolved. In submucosal glands, CFTR is essential as a mediator of VIPergic/cyclic AMP-mediated as well as cholinergic/calcium-mediated NaCl/NaHCO3 and fluid secretion by the serous cells. It also promotes the secretion of other serous cell products, that is, antimicrobial, anti-inflammatory, and antioxidant molecules involved in microbial growth inhibition, inhibition of serine protease-activation of ENaC channels, and protection against oxidative damage,

Table 1 Summary of key functions of chloride channels in the respiratory tract, and associated respiratory diseases Chloride channel

Key function(s) in the respiratory tract

Respiratory disease

CFTR

Regulator of ASL volume (ciliated cells) Regulates secretion of serous cell products (salt, water, antimicrobial, anti-inflammatory, antioxidant molecules) Promotes salt and fluid absorption in alveoli Regulator of ASL volume (upregulated in CF) Synthesis, condensation, or secretion of mucins Fetal fluid secretion Endosomal acidification, endocytosis Endosomal acidfication, endocytosis Cell volume regulation? Cellular detoxification? cAMP-triggered salt and water secretion? Coactivated with CFTR

Cystic fibrosis

CaCC hCLCA1/gob-5 CIC-2 CIC-3 CIC5 Bestrophin CLIC Parchorin ORCC

Pulmonary edema Cystic fibrosis Bronchial asthma, CF (?) Lung cyst formation Not known Not known Not known Not known Not known Cystic fibrosis

470 ION TRANSPORT / Chloride Channels

respectively. Loss of these functions in CF may contribute to a reduced ASL, mucus plugging, mucosal obstruction, and lung infection. In the distal airways and alveolar compartment, the lack of pathologic lung development in newborns with CF suggests that active chloride secretion and the production of lung fluid in fetal pulmonary epithelium occurs through CFTR-independent pathways. Likewise, alveolar clearance of perinatal fluid at the time of birth involves Naþ uptake through ENaC and Cl
uptake through CFTR-independent chloride shunt pathways; this explains why the risk of acute respiratory failure at birth does not increase in CF patients and CF mice. However, in vivo studies in CF mice and ex vivo studies in human CF lungs have shown that CFTR is necessary for b-adrenergic, cyclic AMP-stimulated salt, and fluid absorption that is important in minimizing pulmonary edema in septic and hypovolemic shock. Expression of human CFTR increased Naþ absorption and fluid clearance in alveolar epithelium of normal rats and mice, and administration of isoproterenol or overexpression of a human b2-adrenergic receptor enhanced alveolar fluid clearance in normal, but not in CF, mice. Whereas the cross-talk between the b2 receptor and the CFTR channel is mediated through physical interactions involving PDZ (postsynaptic density protein 95/disc large/Zonula occludens protein 1) domain-scaffholding proteins, the coupling between CFTR and ENaC is most plausibly electrogenic, that is, CFTR activation results in hyperpolarization and increases the driving force for Naþ uptake through the sodium channels. Calcium-Activated Chloride Channels

The apparent downregulation of CaCCs by CFTR, presumably through interaction with the CFTR-R domain, and their upregulation under conditions of CFTR dysfunction, suggests a putative role of the CaCCs as compensatory chloride channels in CF and may at least in part explain the lack of airway disease in most CF mouse models. Apart from chloride transport, additional putative roles include the promotion of apoptosis (mCLCA1 and 2), cell adhesion of lung metastatic cells through b4 integrin-binding motifs (hCLCA2 and mCLCA1 and 5), and the synthesis, condensation, or secretion of mucins (mCLCA3 and its human ortholog, hCLCA1, gob-5). Gob-5 expression was induced specifically in murine lung tissue associated with airway hyperresponsiveness (AHR), and in patients with bronchial asthma. Moreover, intratracheal adenovirus expression of antisense gob-5 RNA into AHR mice suppressed the asthma phenotype,

including AHR and mucus overproduction, whereas adenovirus overexpression of gob-5 exacerbated the asthma phenotype. These observations indicate that mCLCA3/hCLCA1 plays a key role in mucus overproduction and the pathogenesis of murine and human asthma. Consequently, CLCA1 gene polymorphisms may be useful for predicting the susceptibility of chronic obstructive pulmonary disease, and may also contribute to the phenotypic variability of CF lung disease. CLCs

Numerous physiological roles have been proposed including stabilization of the membrane potential (ClC-1), transepithelial ion transport (ClC-2, -Ka, -Kb), cell volume regulation (ClC-2, -3), and regulation of the intracellular pH and acidification of endosomes (ClC-2, -3, -5, -7). The pathophysiological importance of ClC members is exemplified by the findings that myotonia congenita, Dent’s disease, osteopetrosis, and Bartter’s syndrome, all human genetic disorders, are due to mutations in CLC genes (ClC-1, ClC-5, ClC-7, and barttin, respectively). ClC-2

It is now well established that ClC-2 is activated in the presence of a reduced extracellular pH. Because the alveolar pH of fetal lambs is acidic, it was suggested that ClC-2 had a role to play during fetal fluid secretion. Indeed, using primary cultures of rat fetal lung cells, an increased chloride secretion was observed in the presence of an acidic luminal fluid. In addition, inhibition of ClC-2 was found to disrupt the formation of lung cysts in fetal explants. Although these results suggest that ClC-2 is essential during lung morphogenesis, the lungs of ClC-2 knockout mice were found to develop normally. ClC-3

Although a role for ClC-3 in the regulation of cell volume has been reported by several groups, its intracellular localization as well as the observation that ClC-3 channels are constitutively active, argues against the concept that ClC-3 may represent the volume-regulated anion channel. Accordingly, cellvolume regulation is not impaired in ClC-3 KO mice. In contrast, present evidence indicates that ClC-3 has an important function during endosomal acidification, acting as a shunt conductance for the vesicular proton ATPases, similar to ClC-5. ClC-4–6

From these three isoforms, ClC-5 has been studied most intensively, due to the fact that mutations in

ION TRANSPORT / ENaC (Epithelial Sodium Channel) 471

ClC-5 results in Dent’s disease, an X-linked hereditary kidney disease characterized by low-molecular-weight proteinuria, hypercalciuria, and hyperphosphaturia. ClC-5 is localized intracellularly at the luminal surface, associated with early or recycling endosomes, and, like ClC-3, is involved in vesicular acidification and receptor-mediated endocytosis. A possible role of ClC-4 and -6 in lung development and function remains to be established. Bestrophins

Mutations in human bestrophin1 are responsible for Best disease, a dominant autosomal hereditary disorder, which leads to macula degeneration and a subsequent loss of vision. The bestrophins do not share any sequence homology with other groups of ion channels; however, early observations that the electroretinogram is altered in Best patients suggested that bestrophins could function as anion channels. Further studies, which included heterologs expression of cloned isoforms as well as the generation of specific mutations within the protein that alter the biophysical properties of the conductance, demonstrated that these proteins could function as bona fide (parts of) anion channels. The function of bestrophin in the basolateral membrane of airway cells has not been not clarified yet, but, in analogy to retinal pigmented epithelium, may be pertinent to cell-volume regulation.

vesicular function. Journal of Clinical Investigation 115: 2039–2046. Mall M, Grubb BR, Harkema JR, O’Neal WK, and Boucher RC (2004) Increased airway epithelial Na þ absorption produces cystic fibrosis-like lung disease in mice. Nature Medicine 10: 487–493. Mutlu GM, Adir Y, Jameel M, et al. (2005) Interdependency of b-adrenergic receptors and CFTR in regulation of alveolar active transport. Circulation Research 96: 999–1005. Nakanishi A, Morita S, Iwashita H, et al. (2001) Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma. Proceedings of the National Academy of Sciences. USA 98: 5175–5180. Nilius B and Droogmans G (2003) Amazing chloride channels: an overview. Acta Physiologia Scandinavica 177: 119–147. Rowe SM, Miller S, and Sorscher EJ (2005) Cystic fibrosis. New England Journal of Medicine 352: 1992–2001. Schwiebert EM, Cid-Soto LP, Stafford D, et al. (1998) Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic fibrosis airway cells. Proceedings of the National Academy of Sciences USA 95: 3879–3884. Suzuki M and Mizuno A (2004) A novel human Cl
channel family related to Drosophila flightless locus. Journal of Biological Chemistry 279: 22461–22468.

ENaC (Epithelial Sodium Channel) D C Eaton and L Jain, Emory University School of Medicine, Atlanta, GA, USA & 2006 Elsevier Ltd. All rights reserved.

Chloride Intracellular Channels

The finding of a thioredoxin fold in the CLIC structure suggests that CLIC proteins, in addition to their putative function as an anion channel, may serve a role in cellular detoxification. See also: Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Ion Transport: Overview; Calcium Channels.

Further Reading Ashley RH (2003) Challenging accepted ion channel biology: p64 and the CLIC family of putative intracellular anion channel proteins. Molecular Membrane Biology 20: 1–11. Duta V, Szkotak AJ, Nahirney D, and Duszyk M (2004) The role of bestrophin in airway epithelial ion transport. FEBS Letters 577: 551–554. Fang X, Fukuda N, Barbry P, Sartori C, Verkman AS, and Matthay MA (2002) Novel role for CFTR in fluid absorption from the distal airspaces of the lung. Journal of General Physiology 119: 199–207. Fuller CM and Benos DJ (2000) Ca2 þ -activated Cl
channels: a newly emerging anion transport family. News in Physiological Sciences 15: 165–171. Jentsch TJ, Maritzen T, and Zdebik AA (2005) Chloride channel diseases resulting from impaired transepithelial transport or

Abstract Amiloride-sensitive sodium channels in the lung play an important role in lung fluid balance. Particularly in the alveoli, sodium transport is closely regulated to maintain an appropriate fluid layer on the surface of the alveoli. Both alveolar type 1 and 2 cells (T1 and T2) have several different amiloride-sensitive, sodiumpermeable channels in their apical membranes. This diversity appears to play a role in both normal lung physiology and pathological states. In many epithelial tissues, amiloride-sensitive epithelial sodium channels (ENaC) are formed from three subunit proteins designated a-, b-, and g-ENaC. At least part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. This leads to epithelial tissue in the lung that has enormous flexibility to alter the magnitude and regulation of salt and water transport. In lung, ENaC is regulated by many transmitter and hormonal agents. The mechanism of regulation depends upon which type of sodium channel is present, but involves controlling either the number of channels present in the apical membrane or the activity of individual channels.

Introduction Efficient gas exchange in the lungs depends on precise regulation of the amount of fluid in the thin

472 ION TRANSPORT / ENaC (Epithelial Sodium Channel)

liquid layer lining the alveolar epithelium (see Fluid Balance in the Lung. Peripheral Gas Exchange). In airway epithelium, fluid is transported from the airway lumen into the interstitial spaces, and this process can be substantially inhibited by the addition of amiloride, an epithelial Naþ channel (ENaC) inhibitor, to the alveolar space. While all parts of the airway have ENaC and absorb Naþ , the alveolar epithelium covers more than 99% of the large internal surface area of the lung (100–150 m2 in humans) suggesting that alveolar epithelial cells are the major sites of Naþ transport and fluid absorption in the adult lung. The alveolar epithelium is composed of two distinct cell types, alveolar type 1 (T1) and type 2 (T2) cells (see Epithelial Cells: Type I Cells; Type II Cells). T2 cells, which cover only 2–5% of the internal surface area of the lung, are cuboidal cells that secrete pulmonary surfactant (see Surfactant: Overview). T2 cells contain ion channels, including the amiloridesensitive ENaC and the cystic fibrosis transmembrane regulator (CFTR). T1 cells are large squamous cells whose thin cytoplasmic extensions cover 495% of the internal surface area of the lung; and, although less is known about these cells, they also appear to have a full complement of ion channels including ENaC and contribute to alveolar Naþ absorption.

Single-Channel Measurements from Alveolar Cells: A Family of AmilorideSensitive Channels Because of the diversity of ENaC channels in airway epithelial cells, they are best examined using patch clamp methods to examine the properties of single ENaC channels. Single-channel studies have identified at least four different amiloride-blockable

channels with either high selectivity, moderate selectivity, or no selectivity for Naþ over Kþ in the apical membranes of alveolar epithelial cells (Table 1). These channels differ not only in their ion selectivity, but also in their single-channel conductance and other characteristics; nevertheless, all appear to play some role in alveolar Naþ absorption. The channels can be grouped based on their different biophysical characteristics into four main categories: (1) highly Naþ selective cation (HSC) channels with a singlechannel conductance of 4–5 pS and Naþ /Kþ selectivity of 440; (2) moderately selective cation (MSC) channels with a single-channel conductance of 8–9 pS and Naþ /K þ selectivity of 5–8; (3) a different moderately selective channel with similar Naþ / Kþ selectivity but a much greater single-channel conductance of 56 pS; and (4) nonselective cation (NSC) channels with a single-channel conductance of 19–24 pS and Naþ /Kþ selectivity of about 1.5. The cloning of an epithelial Naþ channel consisting of three homologous subunits – a, b, and g from rat colon provided a molecular definition of one class of Naþ channel. Expression studies of various combinations of the different ENaC subunits in heterologous expression systems showed that HSC channels were composed of a-, b-, and g-ENaC subunits (see Figure 1), and that NSC channels were composed of a subunits alone. The moderately selective Naþ channels were composed of the a ENaC subunit in combination with either b- or g ENaC (see Figure 2). Both T1 and T2 cells contain all types of ENaC channels: HSC, MSC, and NSC, although HSC and NSC are by far the most prevalent. The density of all forms of ENaC is similar in both T1 and T2 cells, but T1 cells have slightly more NSC than HSC channels per cell while the reverse is true for T2 cells.

Table 1 Comparison of different amiloride-blockable channels in epithelial apical membranes (including lung epithelial cells) Channel property

Highly selective (HSC)

Moderately selective (type I; MSC)

Moderately selective (type II)

Nonselective (NSC)

Naþ /K þ selectivity

440

5–8

7

1.5

Unit conductance (pS)

4–5

8–9

56

19–24

Amiloride sensitivity (K0.5 nM)

450

700

44000

2000

Increased cAMP

Increases N

Increases Po

None

Increases Po

?

Decreases Po

Increased cGMP þ

None þ

None þ

þ

In the table, Na /K selectivity is the ratio of Na permeability (PNa) to the K permeability (PK) determined from the reversal potentials of single channel currents. N is the channel density per unit area of membrane which is equivalent to the average number of channels per patch and Po is the open probability of a single channel defined as the ratio of amount of time the channel is open to the total recording time.

ION TRANSPORT / ENaC (Epithelial Sodium Channel) 473 Extracellular loop

Pore?

M2



M1





N-terminus

C-terminus

 Figure 1 The structure of an ENaC channel consisting of three homologous subunits – a, b, and g. The subunits consist of protein with short amino- and carboxy-terminal tails, two membrane spanning domains, M1 and M2, a putative pore region near the second membrane spanning domain, and a very large extracellular loop containing about 400 amino acids. Assembly of the channel appears to require four subunits, at least two of which must be the a-subunit (although the exact stoichiometry is controversial).








Unassembled ENaC subunits in membrane

Hypoxia, steroid depletion, or impermeable matrix

Steroid repletion and impermeable matrix




Normoxia, steroid repletion, and permeable matrix  


















NSC channel (20 pS):
-ENaC only

 MSC channel (10 pS):
-, -, or
-, -ENaC

HSC channel (6 pS):
-, -, -ENaC

Figure 2 A schematic model for assembly and regulation of amiloride-sensitive sodium channels in the lung. Alveolar environment, particularly oxygen tension, steroid exposure, and alveolar distension, are likely to influence assembly of ENaC subunits. Signal transduction pathways mediated by several protein kinases including A, G, and C regulate each of these channel types in different ways (see Figure 3). T1 and T2 cells with different channel types (and, therefore, different regulation) will have very different levels of sodium transport that will respond quite differently to hormonal and transmitter agents.

Regulation of ENaC in the Lung Excessive accumulation of fluid in the alveolar spaces frequently accompanies acute lung injury, and the failure of the lungs to rapidly clear this edema fluid has been related to higher morbidity and mortality. Since alveolar fluid clearance is driven by active transport of sodium and water across the epithelial

lining of air spaces (see Fluid Balance in the Lung), significant effort has focused on strategies to enhance this process particularly under pathological conditions. This has often times involved trying to enhance the activity of ENaC at the apical membrane of alveolar epithelial cells. ENaC in the airways is regulated by an enormous variety of agents (see Signal Transduction). These include transmitters

474 ION TRANSPORT / ENaC (Epithelial Sodium Channel) ENaC Airway lumen

Na+ D1R

P2YR

Gi

Gs

Gq

Inhibitory pathway

PIP2

cAMP

PIP3

PKC

PI-3-K

Stimulatory pathway

GR PKA

GC

cAMP Na+ Gs

Gs

Interstitium d2AR

K+ (Na,K)-ATPase

D2R

Figure 3 Major signaling pathways that regulate ENaC. For clarity, not all elements of the pathways are shown. A typical airway cell is shown with, starting from the top left, purinergic signaling mediated by P2Y receptors (P2YR). Purinergic signaling reduces ENaC activity by reducing the amount of membrane phosphatidylinositol-4,5-bis-phosphate (PIP2) by phospholipase C hydrolysis and by activating protein kinase C (PKC) which also inhibits ENaC. At the top right is the type 1 dopamine receptor (D1R) that promotes apical production of cyclic adenosine monophosphate (cAMP) and then, through a complicated signaling pathway that involves the small G protein, Rap1, stimulates phosphatidylinositol-3-kinase to form phosphatidylinositol-3,4,5-tris-phosphate (PIP3) that strongly stimulates ENaC. At the basolateral membrane is the type 2 dopamine receptor (D2R) that activates ENaC in a manner similar to the type receptor, but also activates the basolateral (Na,K)-ATPase. The basolateral membrane also contains b2-adrenergic receptors (b2-AR) that stimulate production of an alternative pool of cAMP that activates PKA to promote ENaC insertion in the apical membrane. Glucocorticoids (GC) bind to their receptor (GR) which when bound activates a complicated signaling pathway that requires gene expression but finally leads to activation of phosphatidylinositol-3-kinase to form PIP3 that strongly stimulates ENaC. Gs, Gi and Gq are G proteins that stimulate or inhibit adenylyl cyclase or stimulate phospholipase, respectively.

interacting with G-protein-coupled receptors (e.g., purinergic, adrenergic, and dopaminergic) (see GProtein-Coupled Receptors), circulating hormones (e.g., glucocorticoids, angiotensin, and eicosanoids), and chemokines (e.g., tumor necrosis factor alpha (TNF-a), transforming growth factor beta (TGF-b), and interleukins (ILs)) (see Chemokines) among others. The large number of regulatory agents attests to the fact that regulation of lung Naþ absorption is so critical for normal lung fluid balance and function (see Fluid Balance in the Lung). Several agents have a particularly profound effect on Naþ reabsorption (summarized in Figure 3).

across T2 cells, can be upregulated by b-agonists suggests that these agents might be useful in limiting alveolar edema and decreasing morbidity and mortality in patients with acute lung injury. Activation of b2 receptors (see G-Protein-Coupled Receptors) on T2 cells stimulates adenylyl cyclase that, in turn, increases intracellular cyclic adenosine monophosphate (cAMP) levels and increases the number of HSC channels in the apical membrane and increases the activity of NSC channels already in the apical membrane. The effects of increased cAMP were totally blocked by the b-antagonist, propranolol, and by the protein kinase A (PKA) blocker, H89.

Regulation of ENaC by b-Adrenergic Agents

Regulation of ENaC by Purinergic Agonists

The demonstration that Naþ transport across the alveolar epithelium in vivo and ex vivo, as well as

The luminal surface of airway epithelial cells contains several different types of purinergic receptors.

ION TRANSPORT / ENaC (Epithelial Sodium Channel) 475

These include adenosine type 1 and 2a receptors (see G-Protein-Coupled Receptors). Adenosine at low concentrations (o100 nM) stimulates ENaC by increasing the open probability of single channels presumably by activating A1 receptors. At higher concentrations (41 mM), adenosine inhibits amiloridesensitive Naþ transport through A2a receptors. Besides adenosine receptors, there are several P2 purinergic receptors sensitive to ATP and UTP. The receptors are of both the P2Y metabotropic and P2X ligand-gated ion channel type. P2Y2 receptors (see G-Protein-Coupled Receptors) are sensitive to ATP and strongly inhibit ENaC through a protein kinase C (PKC)-dependent mechanism. The chloride channel, CFTR (see Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene), is often associated with release of ATP and some have suggested that ATP, acting through the P2Y2 receptor, allows for coordinated control of sodium and chloride absorption across the apical membrane of T2 cells. P2X receptors (probably P2X5) are, when activated by purines, ion channels themselves; however, they can also alter intracellular calcium and, therefore, would be expected to activate NSC channels and increase Na þ transport in alveolar epithelial cells.

Regulation of ENaC by Dopamine Dopamine increases lung liquid clearance under basal conditions and in situations where edema accompanies lung injury. Alveolar epithelial cells appear to contain both type 1 and 2 dopamine receptors (D1 and D2) (see G-Protein-Coupled Receptors). Stimulation of D1 or D2 receptors on the basolateral surface of T2 cells activates (Na,K)-ATPase, but some of the increase in lung fluid clearance is mediated by D1 activation of apical ENaC. Apical D1 receptors stimulate the production of cAMP which, through a complicated signaling pathway that involves the small G protein, Rp-1, but not PKA, finally increases the activity of individual ENaC proteins. The effect of dopamine on both ENaC and (Na,K)-ATPase suggests a coordinated response of both transporters to promote maximal increases in transport and alveolar fluid clearance. The action of dopamine also demonstrates that signaling within alveolar cells is compartmentalized. Basolateral production of cAMP by b-adrenergic receptors produces a PKA-dependent increase in the number of ENaC in the apical membrane with little or no change in the activity of individual channels. On the other hand, the apical production of cAMP by D1 receptors produces a PKA-independent increase in the activity of individual channels with little or no change in the number of channels.

Regulation of ENaC by Steroids Lung epithelial cells contain both mineralocorticoid and glucocorticoid receptors (see Corticosteroids: Glucocorticoid Receptors), but since circulating levels of aldosterone are usually low compared to glucocorticoids, it is likely that the major steroids regulating lung ENaC are glucocorticoids. Glucocorticoids regulate sodium absorption by short- and long-term processes: an initial phase which increases transport four- to sixfold in the first 2–6 h and a late phase which requires 12–48 h and increases transport another three- to fourfold. A glucocorticoid, like other steroid hormones, enters target cells and binds to cytosolic, glucocorticoid receptor complexes. After some rearrangement, the glucocorticoid-bound receptor moves to the nucleus and acts as a DNAbinding protein which targets steroid response elements on genetic DNA. Binding to the response elements alters gene expression. Increases in Naþ transport can be measured within 1 h of exposure to glucocorticoids, and this increase is dependent on gene transcription and translation with the gene products generically referred to as steroid-induced proteins (SIPs). In the short term, glucocorticoids regulate sodium transport by inducing expression of the small G protein, K-Ras2A, and by subsequent K-Ras2A-induced activation of phosphatidylinositol phosphate-5-kinase (PIP-5-K) and phosphatidylinositol-3-kinase (PI-3-K) to produce phosphotidylinositol-3,4,5-phosphate (see Signal Transduction) that ultimately increases the activity of individual ENaC channels. In the long term, glucocorticoids regulate sodium transport by inducing SIPs that alter trafficking, assembly, and degradation of ENaC, and thereby change the number of ENaC in the surface membrane. Glucocorticoids also apparently increase the number of HSC channels at the expense of NSC channels ensuring that the epithelium becomes highly selective for Naþ so that Naþ is preferentially absorbed by the lung epithelium by a mechanism that involves altering rates of channel insertion and degradation (see Figure 4).

Regulation of ENaC by Inflammatory Chemokines Many inflammatory cytokines reduce ENaC activity and, thus, exacerbate the pulmonary edema associated with sepsis. TNF-a, IL-1b, and TGF-b all reduce ENaC activity via mitogen-activated protein kinase signaling pathways (see Chemokines). Besides direct inhibition, IL-1b and TGF-b also interfere with glucocorticoid gene activation. Therefore, they not only reduce ENaC activity, but prevent glucocorticoid-mediated

476 ION TRANSPORT / ENaC (Epithelial Sodium Channel) 3

HSC channel c.d.i channel NSC channel c only channel

Insertion

Ubiquitination 4 Retrieval

Recycling

2

6 5

Trafficking

Resorting d

Processing 7 Degradation

ER

Assembly

Proteasome

1

i

c Figure 4 A schematic diagram of the assembly, trafficking, and degradation of ENaC with major components numbered. At (1) is the initial translation of separate ENaC subunits and accumulation in the ER pool. Only if the subunits are assembled into heteromeric complexes are they efficiently transported out of the ER. (2) Trafficking through the Golgi prior to insertion into the membrane. There may be a substantial pool of assembled HSC channels in a subapical vesicular pool which can be stimulated to insert into the membrane in response to increases in cAMP (b
adrenergic stimulation). (3) Aging of the channels which appears to involve Nedd4-mediated ubiquitination that leads to retrieval of channels (4) and resorting and separation of subunits (5) where the b and g subunits are destined for rapid proteasomal degradation (7), but the a subunits are resistant to degradation and may be recycled to the surface to form a-only NSC channels (6).

increases in ENaC activity and hamper the effectiveness of exogenously administered steroids.

ENaC’s Role in Lung Disease The effect of inflammatory cytokines on ENaC implies that under certain pathological conditions, lung fluid clearance could be compromised by reduced ENaC activity. Obviously, several pathological conditions can lead to an increase in TGF-b and, in these conditions, TGF-b will have many effects, of which two will be to increase alveolar epithelial permeability and inhibit ENaC. Thus, at the same time that excess fluid enters the alveoli from the pulmonary capillaries because of the loss of epithelial barrier function, the ability of the epithelium to reabsorb fluid is severely compromised due to reduction in ENaC activity (see Fluid Balance in the Lung). ENaC dysregulation has also been implicated in other lung pathologies. Recently, several investigators have shown that respiratory viruses can reduce ENaC activity when the viruses bind to the surfaces of lung epithelial cells. The consequence of this inhibition is to reduce alveolar fluid clearance, thereby, promoting movement of excess fluid containing viral

particles up the airways and out of the lungs which allows subsequent aerosol formation and transmission of the viruses. There are also genetic defects that lead to abnormalities of lung fluid clearance some of which are due to ENaC dysfunction. As mentioned above, a-ENaC knockout mice die shortly after birth due to an inability to clear fetal fluid from their lungs. In humans, a loss-of-function mutation in a-ENaC (pseudohypoaldosteronism type I) is not lethal, but does cause excessive lung fluid production that can be cleared successfully, and does put individuals with the defect at risk for pulmonary infections. Cystic fibrosis (CF) is a defect in the gene for a chloride channel, but surprisingly is characterized by increased ENaC activity. This leads to reduced alveolar and airway fluid depth that, in the upper airways, causes significant problems with the mucus accumulation characteristic of the disease. The defect in ENaC regulation associated with a CF suggests a tight regulation between sodium and chloride reabsorption in normal lung which is probably controlled by the CF gene product, CFTR. The genetic defect in CF disrupts this normal regulation and allows unregulated, high, levels of ENaC activity that disturbs fluid balance (Cystic Fibrosis: Cystic Fibrosis

ION TRANSPORT / Potassium Channels 477

Transmembrane Conductance Regulator (CFTR) Gene). That there is extremely tight control of ENaC in the lung is underscored by another genetic defect, Liddle’s syndrome which is an ENaC gain-of-function mutation that leads to such excessive reabsorption in the kidney that individuals with the disorder have life-threatening hypertension. However, they have no lung symptoms suggesting that ENaC regulatory mechanisms in the lung are sufficient to maintain proper fluid clearance.

Glucocorticoid Receptors. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Epithelial Cells: Type I Cells; Type II Cells. Fluid Balance in the Lung. G-Protein-Coupled Receptors. Ion Transport: Overview; Calcium Channels; Chloride Channels; Potassium Channels. Lipid Mediators: Overview. Peripheral Gas Exchange. Proteasomes and Ubiquitin. Signal Transduction. Surfactant: Overview. Tumor Necrosis Factor Alpha (TNF-a). Vesicular Trafficking.

Further Reading Summary It seems clear that ion channels that are members of the ENaC family of proteins play an important role in lung fluid balance. Because the ENaC channel density/ patch is similar in T1 and T2 cells and because T1 cells cover 495% of the alveolar surface area, it seems likely that T1 cells may be responsible for the bulk of Naþ transport in the lung. In alveolar T1 and T2 cells, there are a variety of different amiloridesensitive, sodium-permeable channels. This significant diversity appears to play a role in both normal lung physiology and pathological states. At least part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of a-, b-, and g-ENaC subunits to form channels with different biophysical properties and different mechanisms for regulation. In particular, when only a-ENaC subunits are assembled together to form channels, the result is a 21–28 pS nonselective cation channel which is sensitive to intracellular calcium and whose open probability is increased by cAMP. In marked contrast, when a-, b-, and g-ENaC subunits are assembled together, the result is a 4–6 pS channel that is highly selective for Naþ over K þ , is insensitive to intracellular calcium, and cAMP increases channel number with no effect on open probability. The diversity of channel expression and functional properties leads to epithelial tissue in the lung that has enormous flexibility to alter the magnitude and regulation of salt and water transport. Better understanding of these pathways will help delineate the mechanisms that regulate alveolar fluid balance and may provide the basis for developing strategies to prevent or treat the respiratory compromise associated with alveolar flooding.

Chen XJ, Eaton DC, and Jain L (2002) Beta-adrenergic regulation of amiloride-sensitive lung sodium channels. American Journal of Physiology: Lung Cellular and Molecular Physiology 282: 609–620. Eaton DC, Malik B, Saxena NC, Al Khalili OK, and Yue G (2001) Mechanisms of Aldosterone’s action on epithelial Na þ transport. Journal of Membrane Biology 184: 313–319. Farman N, Talbot CR, Boucher RC, et al. (1997) Noncoordinated expression of a-, b-, and g-subunit mRNAs of epithelial Na þ channel along rat respiratory tract. American Journal of Physiology 272: C131–C141. Firsov D, Gautschi I, Merillat AM, Rossier BC, and Schild L (1998) The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO Journal 17: 344–352. Fukuda N, Jayr C, Lazrak A, et al. (2001) Mechanisms of TNF-a stimulation of amiloride-sensitive sodium transport across alveolar epithelium. American Journal of Physiology: Lung Cellular and Molecular Physiology 280: L1258–L1265. Jain L, Chen XJ, Ramosevac S, Brown LA, and Eaton DC (2001) Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions. American Journal of Physiology: Lung Cellular and Molecular Physiology 280: L646–L658. Matalon S and O’Brodovich HM (1999) Sodium channels in alveolar epithelial cells: molecular characterization, biophysical properties, and physiological significance. Annual Review of Physiology 61: 627–661. Matalon S, Lazrak A, Jain L, and Eaton DC (2002) Invited review: biophysical properties of sodium channels in lung alveolar epithelial cells. Journal of Applied Physiology 93: 1852–1859. Rossier BC, Pradervand S, Schild L, and Hummler E (2002) Epithelial sodium channel and the control of sodium balance: interaction between genetic and environmental factors. Annual Review of Physiology 64: 877–897. Yue G, Malik B, Yue G, and Eaton DC (2002) Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates epithelial sodium channel activity in A6 cells. Journal of Biological Chemistry 277: 11965–11969.

Potassium Channels

Acknowledgments

S M O’Grady, University of Minnesota, St Paul, MN, USA

This study was supported by R01 HL063306 to LJ; and by R01 HL071621 and P01 DK061521 to DCE.

& 2006 Elsevier Ltd. All rights reserved.

See also: Acute Respiratory Distress Syndrome. Adenosine and Adenine Nucleotides. Adrenergic Receptors. Aquaporins. Chemokines. Corticosteroids:

Abstract Airway and alveolar epithelial cells express a variety of K þ channels that are specifically localized to either the apical or

478 ION TRANSPORT / Potassium Channels basolateral membrane depending upon their physiological function. These channels play important roles in maintenance of membrane potential, control of cell volume, regulation of cell proliferation, and support of transepithelial electrolyte and nutrient transport. K þ channels present in the apical membrane of alveolar epithelial cells, for example, facilitate K þ secretion, thus contributing to the elevated K þ concentration observed in alveolar fluid. K þ channels localized to the basolateral membrane of airway epithelial cells contribute to transepithelial electrolyte and fluid transport by sustaining the electrical driving force necessary for anion efflux or cation influx and limit fluctuations in intracellular K þ concentration associated with changes in Na þ –K þ ATPase activity. Signaling molecules that control the absorptive and secretory functions of lung epithelia have been shown to regulate specific K þ channel subtypes through the mobilization of intracellular second messengers such as Ca2 þ and cAMP. In this review, the diversity of K þ channel expression and the functional role of these channels in transepithelial electrolyte and fluid transport in lung epithelia are addressed.

K+ Channel Expression in Lung Epithelia K þ channels are multimeric structures composed of pore-forming a-subunits and specific accessory proteins that serve to regulate the expression and

gating properties of the channel. Approximately 80 K þ channel-related genes have been identified in the human genome. Airway epithelial cells express several distinct families of K þ channels, many of which have been previously identified in excitable cells. Table 1 presents a summary of K þ channel families and specific subfamilies known to be expressed in lung epithelia and their membrane localization. Multiple subtypes of voltage-gated (Kv) K þ channels (the Shaker-related channels, Kv1
4), the ether-a-go-go-related K þ channel, Kv7 (KCNQ channels), inwardly rectifying K þ channels (Kir), ATP-regulated K þ channels (KATP) and calcium-activated K þ channels (SK4/IK1) have been identified. In addition, several accessory K þ channel proteins are also expressed. Voltage-gated K þ channels possess six transmembrane domains and one pore region (6TM/1P) where both the N-terminal and C-terminal domains are located within the cytoplasm (Figure 1). The S4 segment contains several regularly spaced, positively charged amino acids that play a role in voltage sensing by the channel. The pore loop located between S5

Table 1 K þ channel-related genes identified in lung epithelial cells Gene

Channel name

Auxilary subunits

Location

Membrane

Activators

Blockers

KCNQ1

Kv7.1;KvLQT1

KCNE2; KCNE3

Airways

Basolateral

Chromanol 293B Clofilium

KCNN4

KCa3.1;SK4; K1

Calmodulin

Airways

Basolateral

KCNA1

Kv1.1

Kvb1; Kvb2

Alveolus

Apical

cAMP L364373 DIDS Niflumic acid Ca2 þ 1-EBIO Chlorzoxazone Voltage

KCNA3

Kv1.3

Kvb

Alveolus

Apical

Voltage

KCNA4

Kv1.4

Kvb

Alveolus

Apical

Voltage

KCND1

Kv4.1

KChIP1

Alveolus

?

Voltage

KCND2

Kv4.2

KChIP2

Alveolus

Apical

Voltage

KCND3

Kv4.3

KChIP2

Alveolus

Apical

Voltage

KCNS3 KCNMA1

Kv9.3 Slo; BK

Kv2 a-subunits BKb

Alveolus A549 cells

? ?

None Ca2 þ voltage

KCNJ2

Kir2.1

Kir2.2 a-subunits

Alveolus trachea

?

None

KCNJ8

Kir6.1

SUR2B

Alveolus

(r) basolateral (h) apical

Pinacidil YM934 Diazoxide nucleotide diphosphates

Clotrimazole Charybdotoxin TEA 4-AP Dendrotoxin Charybdotoxin 4-AP TEA 4-AP TEA Riluzole 4-AP TEA Arachidonic acid 4-AP Heteropodatoxins Bupivacane Nicotin 4-AP Hypoxia Iberiotoxin Charybdotoxin TEA Mg2 þ Polyamines CS þ Glibenclamide

ION TRANSPORT / Potassium Channels 479

6TM/1P

NH2

β

S1

S2

COOH

+ + + S3 S4 + + +

G Y G S5

P-X-P S6

T1

(a)

NH2

COOH

2TM/1P G Y/F G

Kvα

M2 M1

T1

COOH NH2

(b)

(c)

þ

Figure 1 Topology of the two classes of K channel a-subunits present in lung epithelial cells. (a) Schematic diagram of a voltagegated K þ channel with six transmembrane domains and one pore domain (6TM/1P). The single transmembrane b-subunit (KCNE) is also represented. (b) Schematic diagram of an inwardly rectifying K channel a-subunit. (c) Tetramer organization of a voltage-gated K þ channel showing the orientation of the pore domains.

and S6 contains the selectivity filter sequence motif (YTxGYG). The pore of the channel is formed when four identical a-subunits (homotetramer) or four different a-subunits from the same subfamily (heterotetramer) interact to create the walls of the pore. In the Kv1 subfamily, the T1 region, located in the Nterminus preceding the first transmembrane domain (S1), appears to be involved in tetramerization. In KCNQ1, however, sequence motifs (K535 to L650) within the C-terminus appear to be necessary for heterotetramer formation. The intermediate conductance, Ca2 þ -activated K þ channel KCa3.1 also belongs to the group of K þ channels that possess six transmembrane domains and one pore region. However, these channels differ from Kv channels in that they exhibit voltage-independent gating and bind calmodulin at the C-terminus. Calmodulin association with the a-subunits permits Ca2 þ binding and subsequent activation of the channel. In contrast to voltage-gated and KCa K þ channels, inwardly rectifying and ATP-regulated K þ channels

have a-subunits composed of two transmembrane domains and one pore region (2TM/1P). These channels lack the voltage-sensing S4 domain and thus do not exhibit strong voltage-dependent activation. The degree of inward rectification varies among subfamilies and depends upon the binding affinity of the channel for intracellular Mg2 þ or polyamines. Kir channels possess a selectivity filter sequence motif, G(Y/F)G, that is similar to Kv channels, making them significantly more selective for K þ over Na þ . As with Kv channels, functional Kir channels are formed by the interaction of four homologous or heterologous a-subunits within the same subfamily. The N-terminus also contains the T1 sequence motif (N96 to D184) found in certain Kv channel subtypes and appears to be involved in tetramer assembly. Auxiliary subunits are typically required for reconstitution of the physiological properties of K þ channels expressed in native tissues. In lung epithelial cells, several Kv channel b-subunits (Kvb) and at least

480 ION TRANSPORT / Potassium Channels

two K channel-interacting protein (KChIP) subfamilies are thought to associate with Shaker-related Kv channels in alveolar epithelial cells. The minimum K þ channel (minK; KCNE1) subunit has been shown to coassemble with Kv7.1 (KCNQ1) in airway epithelial cells and the sulfonourea receptor (SUR2B) coassembles with Kir6.1 to confer ATP regulation to this inwardly rectifying K þ channel in alveolar epithelial cells.

K+ Channel Function in Airway Epithelial Cells K þ channel activation occurs in parallel with increases in anion secretion. Signaling molecules that increase intracellular Ca2 þ or cAMP increase anion channel activity in the apical membrane and activate specific K þ channel subtypes located in the basolateral membrane. A conceptual model illustrating the pathways involved in cAMP-dependent anion secretion is shown in Figure 2. The principal cAMPregulated anion channel is the cystic fibrosis transmembrane conductance regulator (CFTR). Cyclic AMP produces activation of protein kinase A and subsequent stimulation of apical CFTR. Associated with the increase in CFTR activity is activation of basolateral KCNQ1 K þ channels. The auxiliary subunit KCNE3, identified in certain airway epithelial cells, presumably coassembles with KCNQ1 to modify its functional activity. Studies in colonic epithelia have indicated that KCNE3 alters the gating properties of KCNQ1, rendering the channel constitutively active and voltage independent. Activation of KCNQ1/KCNE3 facilitates electrogenic Cl
transport by increasing the electrical driving force CI− −

Na+ CFTR

P PKA

CI−

cAMP V

ATP

AC

KCNQ1

2K+

K+

Na+

ATP +

3Na+

for Cl
exit across the apical membrane. In addition, the recycling of K þ across the basolateral membrane prevents K þ accumulation within the cells and ensures that K þ depletion within the extracellular unstirred layer adjacent to the basolateral membrane does not occur so that Na þ –K þ ATPase activity is not rate limited by K þ availability. The importance of KCNQ1 activation is highlighted by the fact that inhibition of these channels by chromanol compounds such as 293B, significantly inhibits cAMPstimulated anion secretion. A similar role for basolateral K þ channels holds for Ca2 þ -dependent anion secretion. Stimulation of apical P2Y receptors in bronchial epithelial cells or trachea, for example, leads to a rapid increase in intracellular [Ca2 þ ] that activates Ca2 þ -dependent Cl
channels in the apical membrane and intermediate conductance, Ca2 þ -activated K þ channels (KCNN4) in the basolateral membrane. KCNN4, as mentioned earlier, forms a tight association with calmodulin at its C-terminus, which allows for Ca2 þ binding and subsequent regulation of channel function. Activation of KCNN4 hyperpolarizes the basolateral and apical membranes of the cell (the degree of apical membrane hyperpolarization depending on the ionic permeability of the paracellular junctions), thus offsetting the depolarization of the apical membrane produced by anion efflux and sustaining the electrical driving force required for net anion secretion. It is worth noting that basolateral K þ channel activation can also increase electrogenic Naþ transport across airway epithelia. In tracheal epithelial cells (CFT1 cells) derived from cystic fibrosis (CF) patients, for example, K þ channel opening compounds such as chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) increase transepithelial Naþ absorption. Airway epithelia from most cystic fibrosis patients do not effectively traffic CFTR to the apical membrane and Naþ uptake by epithelial Naþ channels (ENaC) is elevated compared to non-CF individuals. Under these conditions chlorzoxazone or 1-EBIO stimulates Ca2 þ -activated K þ channels (presumably KCNN4), which can be blocked by the antifungal agent clotrimazole. The hyperpolarization produced by K þ exit from the cell compensates for the depolarization produced by Naþ uptake across the apical membrane, thus enabling the continued absorption of Naþ across the epithelium.

NKCC KCNE3

K+

Na+ 2CI−

Figure 2 Model of cAMP-stimulated Cl
secretion across the airway epithelium. AC, adenylyl cyclase; PKA, protein kinase A; NKCC, Na-K-2Cl cotransporter; CFTR, cystic fibrosis transmembrane conductance regulator.

K+ Channel Function in Alveolar Epithelial cells The K þ concentration in alveolar lining fluid is greater than that of plasma, suggesting that the alveolar epithelium is capable of K þ secretion. Some

ION TRANSPORT / Potassium Channels 481

K þ secretion presumably occurs through nonselective cation channels located in the apical membrane; however, immunocytochemical studies have shown that several Kv channel subfamilies are expressed in the apical membrane of adult alveolar epithelial cells. Given that Kv channel subfamilies gating is dependent on the membrane potential, their possible contribution to K þ secretion will depend on the apical membrane voltage. Analysis of the voltage dependence of activation and inactivation of alveolar cell Kv channels indicates that a measurable open probability exists between
40 and þ 10 mV, suggesting that these channels may contribute to K þ secretion if the apical membrane voltage lies within this range. Some Kv channel subtypes identified in alveolar epithelial cells have been shown to play a role in oxygen sensing in vascular smooth muscle and when expressed in HEK cells. For example, the electrically silent Kv9.3 a-subunit has been shown to associate with Kv2.1 to produce functional K þ channels that are oxygen sensitive. Moreover, Kv b-subunits also confer oxygen sensitivity to Kv4.2 channels expressed in HEK293 cells. In chronic hyperoxia, expression of Kv1.1, Kv4.3, and Kv9.3 is reduced in pulmonary artery smooth muscle cells. These observations suggest that certain Kv channels may function as part of an oxygen-sensing mechanism within the alveolar epithelium to detect changes in alveolar ventilation and oxygen diffusion. Rat and guinea pig adult alveolar epithelial cells are known to express Kir2.1, but the membrane localization of this inwardly rectifying K þ channel is not certain. Kir6.1 and its auxiliary subunit SUR2B have also been detected in adult rat alveolar epithelial cells. This ATP-regulated K þ channel is expressed in the basolateral membrane and is inhibited by glibenclamide. Treatment with the KATP channel opener 2-(3,4-dihydro-2,2-dimethyl-6nitro-2K-1, 4-benzoxazin-4-yl) pyridine N-oxide (YM934) increases alveolar liquid clearance in human lung. In cultured rat alveolar epithelial cells, the KATP channel opener penacidil increased Naþ and Cl
transport by approximately 35%, consistent with the observation in human lung that activation of this KATP results in an increase in alveolar fluid clearance. Two types of Ca2 þ -activated K þ channels have been identified in A549 cells, an epithelial cell line derived from human lung. The large conductance BK channel with a single channel conductance of 242 pS was activated by both increases in [Ca2 þ ] and membrane depolarization. BK channel blockers including Ba2 þ , TEA, and quinidine were all found to inhibit the large conductance channel in A549 cells. The intermediate conductance KCNN4 channel was also identified and shown to be activated by adenosine

and by nucleoside transport inhibitors and inhibited by clotrimazole. Attempts to detect mRNA for slo1, the ion-conducting subunit of BK and KCNN4, which encodes for the intermediate conductance channel in rat alveolar epithelial cells, were unsuccessful, thus the expression of these channels may vary among species.

Clinical Significance Compounds that open basolateral K þ channels in airway epithelial cells may have therapeutic potential for the treatment of CF. At this time two lead compounds (1-EBIO and chlorzoxasone) have been identified and their effects on airway ion transport function characterized. As previously stated, these agents have been shown to increase electrogenic Cl
secretion in CFTR-expressing epithelial cells by activating intermediate conductance Ca2 þ -dependent K þ channels. These K þ channels are activated following stimulation with Ca2 þ mobilizing agonists such as UTP. In airway epithelia from CF patients, apical P2Y receptor stimulation using stable analogs of UTP produces activation of Ca2 þ -dependent anion channels that provide an alternative pathway to that of CFTR for increasing net anion and fluid secretion. Compounds related to 1-EBIO or chlorzoxasone could potentially augment P2Y receptor-mediated anion and fluid secretion by enhancing the driving force for anion efflux. However, as previously mentioned, airway epithelia from CF patients exhibit increased rates of ENaC-dependent Naþ absorption and increasing the basolateral membrane K þ conductance with 1-EBIO or chlorzoxasone was shown to stimulate Naþ absorption. Thus, the therapeutic benefit of K þ channelactivating compounds in CF may probably depend on strategies that limit their effect on Naþ absorption. The identification of KATP channels in alveolar epithelia suggests that KATP channel openers related to YM934 or penacidil could have therapeutic potential in the treatment of conditions that cause lung edema. In human lung, YM934 was shown to increase K þ influx into the alveolar space. In contrast to the results in rat alveolar epithelial cells, this result suggested that the channels were localized to the apical membrane. Treatment with the KATP channel blocker, glibenclamide, inhibited the YM934-stimulated increase in fluid clearance, indicating that these channels play a role in transalveolar electrolyte and fluid absorption. However, it is worth noting that glibenclamide is an inhibitor of CFTR, which has been identified in alveolar and airway epithelial cells. Thus, it is possible that at least some of the effects of glibenclamide on fluid clearance may have been due to CFTR inhibition.

482 ION TRANSPORT / Potassium Channels

Conclusions Lung epithelial cells express a remarkable diversity of K þ channel genes and this list continues to expand. Many of these channels are present in excitable cells, but some have distinct physiological and pharmacologic properties in epithelial cells. Such differences appear to be the result of associations with auxiliary subunits that modify gating, cell surface expression, and pharmacological characteristics of the ion-conducting a subunits. Studies of ion transport function in airway and alveolar epithelial cells have definitively demonstrated that specific K þ channel subtypes are activated by signaling molecules that regulate anion secretion and that K þ efflux through these channels is required to sustain the electrical driving force necessary for anion exit across the apical membrane. However, there are several K þ channel genes expressed in lung epithelia where a functional role is less certain. Additional studies will be necessary to elucidate the physiologic functions of these channels and any newly discovered K þ channel genes in the future. See also: Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Epithelial Cells: Type I Cells; Type II Cells. Ion Transport: Overview; Chloride Channels; ENaC (Epithelial Sodium Channel).

Further Reading Armstrong CM (2003) Voltage-gated K channels. Science STKE 10: 1–7.

Bleich M and Warth R (2000) The very small-conductance K þ channel KvLQT1 and epithelial function. Pflugers Archives 440: 202–206. Cowley EA and Linsdell P (2002) Characterization of basolateral K þ channels underlying anion secretion in the human airway cell line Calu-3. Journal of Physiology 538: 747–757. Gutman GA and Chandy GK (2002) Potassium channels. In: Catterall WA, Chandy GK, and Gutman GA (eds.) The IUPHAR Compendium of Voltage-Gated Ion Channels, pp. 57–190. Leeds: IUPHAR Media. Leroy C, Dagenais A, Berthiaume Y, and Brochiero E (2004) Molecular identity and function in transepithelial transport of KATP channels in alveolar epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 286: L1027–L1037. Mall M, Wissner A, Schreiber R, et al. (2000) Role of KvLQT1 in cyclic adenosine monophosphate-mediated Cl
secretion in human airway epithelia. American Journal of Respiratory Cell and Molecular Biology 23: 283–289. MacKinnon R (2003) Potassium channels. FEBS Letters 555: 62–65. Mannhold R (2004) KATP channel openers: structure–activity relationships and therapeutic potential. Medical Research Reviews 24: 213–266. O’Grady SM and Lee SY (2003) Chloride and potassium channel function in alveolar epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 284: L689– L700. Schultz SG and Dubinski WP (2001) Sodium absorption, volume control and potassium channels: in tribute to a great biologist. Journal of Membrane Biology 184: 255–261. Warth R (2003) Potassium channels in epithelial transport. Pflugers Archives 446(5): 505–513. Wu JV, Krouse ME, Rustagi A, Joo NS, and Wine JJ (2004) An inwardly rectifying potassium channel in apical membrane of Calu-3 cells. Journal of Biological Chemistry 279(45): 46558– 46565.

K KALLIKREIN–KININ CASCADE K D Bhoola, The University of Western Australia, Perth, WA, Australia E Fink, Ludwig-Maximilians-Universita¨t Mu¨nchen, Munich, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract Recent evidence increasingly supports the view that kinins exercise an important regulatory control in inflammation and in the growth and proliferation of cancer cells. Kinins are formed by the serine proteases plasma and tissue kallikreins. In their functional capacity as proteases, the two enzymes release the vasoactive kinin peptides (bradykinin and lys-bradykinin (kallidin)) from multifunctional protein molecules called kininogens, of which two species have so far been described in humans. Plasma kallikrein is expressed in many epithelial and endocrine cells apart from hepatocytes. Tissue kallikrein is found in the endothelial and endocrine cells, and neutrophils. The kinins released by the kallikreins are considered to play a primary role as inflammatory mediators (local hormones) by causing contraction of smooth muscles, dilation of arterioles, increasing permeability of the capillary membrane, and interacting with sensory nerve terminal transmitters to evoke pain. The various cellular actions of kinins are mediated by the activation of two specific receptors, B1 and B2. Both these seven-transmembrane receptors of the rhodopsin-like subfamily have been cloned, and are linked to specific Gaq=11 , Gas, Gbg, and Ga12,13 proteincoupled second messenger signaling systems. The development of kallikrein inhibitors as well as kinin receptor antagonists for use in immune-modulated disorders (e.g., asthma) and in tumors (namely lung cancer) may provide a new generation of drugs of therapeutic value in inflammation and in carcinogenesis.

Historical Discovery of Kallikrein and Kinins The first observation on the kallikrein–kinin cascade was made at the turn of the twentieth century, and in 1928 by the German surgeon E K Frey in Munich, who demonstrated the presence of a hypotensive molecule in the human urine and later from a pancreatic cyst. The observed hypotension was considered at that time to be a hormone (Kreislauf hormone) secreted by the pancreas into the circulation. It was called ‘kallikrein’ after the Greek synonym for pancreas. In further studies kallikrein was identified in the blood, pancreas, and salivary glands. Subsequent pharmacological experiments of Eugene

Werle and colleagues identified a new substance ‘kallidin’, which was shown to be the enzymatic product of kallikrein. Kallidin (now known as lysbradykinin) contracted isolated smooth muscle preparations and showed marked hypotensive activity. Only a few years later, bradykinin was discovered by William Beraldo working in Rocha e Silva’s laboratory in Brazil. He showed that when trypsin or snake venom proteases were incubated with serum, a hypotensive substance was formed. Bradykinin was so called because it caused a slow sustained contraction of the isolated guinea pig ileum (brady ¼ slow and kinin ¼ movement). The kinin peptides, lys-bradykinin (decapeptide) and bradykinin (nanopeptide), show a similar structure–activity profile but differ in the potency of their cellular actions.

Kallikreins Plasma Kallikrein

Sites of synthesis and properties Plasma prekallikrein (PPK), the zymogen of the serine protease plasma kallikrein (PK, EC 3.4.21.34), is a singlechain glycoprotein which is the product of the gene KLKB1 located on chromosome 4q34–q35. It had been accepted for several decades that PPK is synthesized only in the liver, but recently, expression of the mRNA of PPK has been proven in multiple tissues; hence, it is evident that the protein is also produced extrahepatically. Furthermore, in immunocytochemical studies PK/PPK has been specifically visualized in several human tissues such as pancreas, kidney, testis, stomach, and lung (unpublished data). Immunolocalization of PPK in the epithelial lining cells of the human bronchus is illustrated in Figure 1 and on the external surface membrane of the human neutrophil in Figure 2. The PPK synthesized in hepatocytes is secreted into the blood where it circulates at a concentration of 35–50 mg l
1 mainly as a complex with high-molecular-weight kininogen (HMWK; H-kininogen). Two forms of PPK with different carbohydrate contents (Mr ¼ 85 and 88 kDa) exist in blood plasma. PPK is a single-chain protein with 619 amino acids. Conversion to active PK is achieved by cleavage of the

484 KALLIKREIN–KININ CASCADE

Bronchial epithelium

Figure 1 Visualization of the immunolabeled PPK on the epithelial cells of the human bronchus. Arrows indicate labeled epithelial cells; Positive label shown by the dark brown immunoprecipitated chromogen complex (DAB, diaminobenzidine); PPK, plasma prekallikrein. Magnification  20.

Figure 2 Confocal images of the immunolabeled plasma prekallikrein on the cell membrane of the human neutrophil. Intensity of the signal expressed in pixels (250–0) is shown in the color chart. Reproduced from Henderson LM, Figueroa CD, Mu¨llerEsterl W, and Bhoola KD (1994) Assembly of contact phase factors on the surface of the human neutrophil membrane. Blood 84(2): 474–482, with permission.

peptide bond Arg371–Ile372 on negatively charged surfaces by activated factor XII (FXII, Hageman factor) with HMWK as cofactor. Alternatively, when PPK is linked to surface-bound HMWK on endothelial cells, it can be activated by prolylcarboxypeptidase. The two chains of active PK are held together by a disulfide bond. The N-terminal heavy chain contains four tandemly arranged apple domains of 90 or 91 amino acids which are important for the interaction with proteins. The light chain contains the catalytic domain and is homologous to the members of the chymotrypsin family of peptidases. Biological activities Biological activities attributed to the PPK circulating in the blood are involved in the activation of neutrophils and C3 convertase of the alternative pathway, induction of the fibrinolytic cascade by converting the prourokinase plasminogen activator to an active molecule, and the release from

HMWK of bradykinin, which through specific G-protein-coupled receptor is linked to second messengers that regulate cellular events. The PPK in the blood originates essentially or totally from the liver; therefore, it is reasonable to conclude that the extrahepatically synthesized PPK has special functions at or near the site of its synthesis. Interestingly, novel biological roles of PK which fit into the concept of such special local functions have been reported recently. PK and other proteases activate bradykinin B2 receptor directly, independent of kinin release. PK is required for adipogenesis; it mediates activation of a plasminogen cascade, which in turn promotes adipocyte differentiation. PK can activate prohepatocyte growth factor and thus may also regulate processes that involve the hepatocyte growth factor/c-Met signaling pathway. Impaired liver regeneration by circulating endotoxin after partial hepatectomy seems to be caused by a PK-mediated activation of latent transforming growth factor beta (TGF-b). Involvement in pathologic disorders The two protease zymogens, PPK and FXII, together with the nonenzymatic cofactor HMWK represent the components of the contact activation system. The contact system becomes activated when blood is exposed to negatively charged artificial surfaces or, physiologically, when PPK, complexed with HMWK, binds to endothelial cells and is activated by prolylcarboxypeptidase or FXIIa bound to the cell surface (details of these processes are still unclear). Upon contact system activation, the two zymogens FXII and PPK are converted into the active enzymes. Subsequently, FXIIa activates both PPK and FXII, and PPK activates FXII. Thus, the result is an accumulation of PK and FXIIa. Spatial and temporal

KALLIKREIN–KININ CASCADE 485

control of their activities is provided mainly by the C1 inhibitor (C1INH). The active PK releases the nanopeptide bradykinin from HK which is the activator of kinin B2 receptor. By a carboxypeptidase, bradykinin can be transformed to des-Arg9-bradykinin, which is a kinin B1 receptor agonist. Bradykinin and des-Arg9-bradykinin are involved in various biological activities (cf. kinins). Control of the bradykinin plasma concentration is achieved, on the one hand, by the regulation of PK activity and, on the other hand, by the degradation of bradykinin to inactive peptides by peptidases (cf. kininases). Elevated plasma levels of bradykinin are thought to be co-responsible for anaphylactoid reactions with symptoms ranging from flushing, cough, edema, and dizziness to life-threatening reactions with severe bronchospasm, hypotension, and cardiorespiratory arrest. Such increased bradykinin plasma concentrations may be due to the following: 1. excess activation of PPK resulting from contact activation on artificial surfaces (e.g., hemodialysis membranes, low density lipoprotein-apheresis with immobilized dextran sulfate, filtration of platelet concentrates with white cell-removal filters, colloid volume substitutes, and radiocontrast media); 2. normal PPK activation, but reduced bradykinin degradation because of angiotensin converting enzyme (ACE)-inhibitor treatment or decreased level(s) of kinin degrading enzymes, especially in combination with (1); and 3. insufficient regulation of PK activity due to reduced inhibitory activity of C1 inhibitor in hereditary or acquired angioedema.

As a whole, due to its bradykinin-releasing activity PK is directly involved in such pathologic disorders which result from increased bradykinin levels in the circulation. On the other hand, partial or complete PPK deficiency is not connected to any adverse clinical phenotype. Tissue Kallikrein Family

Genomics and molecular structure Tissue kallikreins (TKs) belong to a closely related multigene family of 15 members, designated as KLK1–KLK15, which enact different patterns of tissue-specific gene expression. Of the 15 gene members, KLK1 (hK1, kininogenase), KLK2 (hK2), and KLK3 (hK3, prostate-specific antigen (PSA)) show about 60–70% homology, whereas KLK4–KLK15 show about 30– 40% homology. All 15 are clustered within a 320 kb region. They have several features in common. They

are located in a distinct gene region on the human chromosome 19q13.3–q13.4. TK genes (KLK) comprise five exons and four introns, and are 4–10 kb in size. The differences in size are attributed to the introns, whereas the exons are highly conserved. The alignment of the genes commences with KLK1– KLK15–KLK3–KLK2 and then continues downstream with the gene spacing of 25 kb. Gene products The KLK genes encode for the pre– pro form of serine proteases, which are synthesized bound to a 17-amino-acid-signal peptide. All of these gene products (except KLK1, which shows kininforming activity (kininogenase)) have extensive sequence similarity at DNA and protein levels. Phylogenetic analysis of the 15 gene products showed that hK1 (tissue kininogenase), hK2, and hK3 (PSA) reside on the same branch but have evolved away from a branch that links with trypsin and chymotrypsin B. The KLK1 gene product (hK1, tissue kininogenase, EC.3.4.21.35) is a single-chain acid glycoprotein of 238 amino acids with Ile at the amino-terminal and Ser at the carboxy-terminal, and has a molecular weight of 25–45 kDa. By its catalytic triad of His41, Asp96, and Ser189, hK1 hydrolyzes one methionyl and one arginyl bond of domain 4 of its physiological substrates, L- and H-kininogens to release the decapeptide, kallidin. Cellular expression Expression of the TK genes is wide spread and tissue specific. The KLK1 gene is expressed predominantly in the salivary glands (Figure 3), pancreas, kidney (Figure 4), and lung. In the normal human lung, high mRNA expression for KLK1–KLK12 genes has been reported. The expression of hK1 has also been demonstrated in many tissues (colon, endometrium and myometrium of the uterus, placenta and aminiotic fluid, cardiac tissue extracts), bronchial lavage from asthmatics, and nasal secretions from patients with allergic rhinitis. In addition, tissue kininogenase has been identified in synovial fluid from arthritic joints, and in the circulating and synovial fluid neutrophils. KLK2 and KLK3 are expressed almost exclusively in the prostate. Similarly, KLK11 trypsin-like serine protease is expressed in many tissues, including the lung. Essentially, some of the genes show prostate-restricted expression (KLK2–KLK4) or pancreas-restricted expression (KLK6–KLK13). Lower expression is noted in kidney, ovary, testis, and salivary gland. KLK14 showed high expression in brain and bone marrow. Expression of TKs in cancer Recent reports implicate TK (KLK1) in the carcinogenic process arising

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TK 200 TK

150 100 50 0

SD 10 µm Figure 3 Confocal image of the human salivary gland showing intensely labeled tissue kallikrein in the duct cells of the gland. Arrows pointing at immunolabeled TK in the DT; intensity of the signal expressed in pixels (250–0) is shown in the color chart.

TK TK DT

Figure 4 Visualization of tissue kallikrein in granules within the connecting tubule cells of the human kidney. Positive label is shown by the dark brown DAB chromogen; arrows pointing at immunolabeled TK in the CT. Magnification 40.

from the induction of one or more genes. Expression of hK1 has been localized in a number of human carcinomas, namely esophageal, gastric, and renal carcinomas and astrocytomas. Such studies predict that the TK gene (KLK1) and its protein product (hK1) are expressed in lung tumors. Of the two kallikrein genes, KLK2 and KLK3 (PSA) present in the prostate, KLK2 is overexpressed in prostate cancer, with increasing expression, as the benign epithelium is transformed into high-grade intraepithelial prostatic neoplasia. Tissue analysis also shows high expression of the kallikreins in prostate (hK2, hK3, hK4, hK10, hK11, and hK15), breast (hK5 and hK10), testicular (hK10, hK11, and hK15), ovarian (hK10), and renal (hK1) cancers. Elevated circulating levels of tissue-specific kallikreins may prove to be of value as markers for lung carcinomas in which there may be induction of one or more kallikrein genes. Most kallikrein genes, for example, KLK2–KLK7, KLK10–KLK13, and KLK15, are upregulated by estrogens, whereas

KLK4 is downregulated in breast cancer and KLK11 (NES1) in breast and possibly other cancers. As cells become pleomorphic and the tumor invades surrounding parenchyma, the continuing upregulation of kallikrein genes increasingly assumes importance as prognostic indicator of advancing carcinogenesis and disease progression. Circulating levels of hKs may be of value in monitoring progression as well as therapeutic efficacy. Gene induction of TK and the subsequently formed kinins enhances the proliferation of tumor cells. As the carcinoma invades the surrounding parenchymal tissue, endothelial cells migrate into the tumor to form new blood vessels. The endothelial cells are attracted into the carcinoma by tumorkines and a number of promoters of angiogenesis which include vascular endothelial growth factor (VEGF), transforming growth factors, angiopoietins, and the proteins of the kallikrein–kinin cascade. The role of TK (hK1) is illustrated in Figure 5 which shows the sequential

KALLIKREIN–KININ CASCADE 487

TK

TK (a)

(b)

CP

(c)

(d)

Figure 5 Images of microvascular endothelial cells. The sequential events in the formation of new capillary blood vessels (a, b, c) are regulated by proteins of the kallikrein–kinin cascade. Confocal images show immunolabeled tissue kallikrein (TK, white label – see color chart) in the body and pseudopodia of the angiogenic endothelial cells as they progress toward forming a new capillary blood vessel. c1, is a phase contrast image; intensity of the signal expressed in pixels (250–0) is shown in the color chart. (a) Reproduced from Plendl J, Snyman C, Naidoo S, Sawant S, Mahabeer R, and Bhoola KD (2000) Expression of tissue kallikrein and kinin receptors in angiogenic microvascular endothelial cells. Biological Chemistry 381:1103–1115, with permission from Walter de Gruyter GmbH & Co. K G. (b) Reproduced with kind permission of Springer Science and Business Media.

orientation of endothelial cells that are involved in the formation of new capillary blood vessel. Although there are no data on the functional consequences of kallikrein (hK1) expression in tumors, it can be postulated that this enzyme may have growth regulatory, extracellular matrix remodeling, and angiogenic effects in lung cancers and their metastasis. Functions – biological role TKs are considered to process in vitro a variety of promolecules that may include matrix metalloproteinases, procollagenase, and progelatinase, and the processing of growth and peptide hormones. The potential activation of these proteinases by TKs suggests that it may play a role in endothelial and tumor cell migration, neutrophil diapedesis, leukocyte aggregation, angiogenesis, and lung tissue remodeling. The primary function of human tissue kininogenase (hK1) is the cleavage of L- or H-kininogen to release the decapeptide lysbradykinin (kallidin).

Kallikrein Inhibitors 1. The specific endogenous inhibitor of hK1 TK known as kallistatin has been purified and cloned.

Kallistatin also binds to elastase and chymotrypsin, but not to PK, urokinase, or collagenase. At the gene and protein level, kallistatins are members of the serpin (serine protease inhibitor) superfamily. The enzyme and inhibitor form a complex of 92 kDa, which is endocytosed by hepatocytes and cleared from the circulation. Human kallistatin and TK are colocalized in a variety of tissues. 2. A low-molecular-weight inhibitor of TK (CH2856) reduced eosinophilia in an animal model of allergic inflammation, and further such peptide inhibitors of TK have also been designed. 3. Aprotinin (trasylol) is an in vitro inhibitor of both kallikreins, and the substitution of an amino acid has resulted in the synthesis of [Val 15] aprotinin which selectively binds PK and neutrophil elastase. 4. In addition, whereas the actions of hK1 are inhibited by a1 antitrypsin, PK is inhibited by a2 macroglobulin and C1 esterase inhibitor; the genetic reduction or absence of the latter is associated with angioneurotic edema. DX88, a novel recombinant-specific inhibitor of PPK, inhibits hereditary angioedema in mice, and is currently

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undergoing clinical trials in patients with angioneurotic edema and also those undergoing cardiopulmonary bypass.

Kininogens Genomics and Molecular Structure

In humans there are only two species of kininogens, H-kininogen (HMWK, HK) and L-kininogen (lowmolecular-weight kininogen (LMWK), LK). H-kininogen and L-kininogen are encoded by a gene located on chromosome 3q26-qter consisting of 11 exons. Distinct mRNAs for H-kininogen and L-kininogen result from alternative splicing. H-kininogen and L-kininogen share the coding regions of the first nine exons, a part of exon 10 which transcribes the kinin sequence, and the first 12 amino acids that follow the carboxy-terminal of the sequence. Exon 11 codes for the 4 kDa light chain of L-kininogen. The complete exon 10 possesses the full coding sequence for the 56 kDa light chain of Hkininogen. The kininogen molecule consists of an amino-acid-terminal heavy chain and a carboxy-terminal light chain, with the kinin moiety interleaved between the two domains. L-kininogen is a 66 kDa bglobulin with a plasma concentration of 60 mg ml
1 (2.4 mM) and an isoelectric point of 4.7, whereas H-kininogen is a 120 kDa a-globulin with a plasma concentration of 80 mg ml
1 (0.67 mM). The heavy chain is common to both H-kininogen and L-kininogen, but the light chain is unique to either H-kininogen or L-kininogen. The molecular structure is illustrated in Figure 6. Cellular Expression

The kininogens are present in extracellular fluids and have been localized on human neutrophils, platelets, and endothelial cells, the collecting ducts of human kidney and sweat glands. The close relationship

D2 D1

D3

D4

D6

between the cells containing TK and its substrate, L-kininogen, suggests that kinins could be generated in the lumen of tubular structure of glandular organs, namely kidney, salivary gland, and bronchi. Specific, reversible, and saturable binding sites for kininogens have been reported on human neutrophils, platelets, and endothelial cells. On the surface membrane of the human neutrophil, H-kininogen is bound to PPK, the substrate and enzyme forming a unique complex. Functions and Biological Role

Although each domain of the complex molecule has one or more specific functions, the protein as a whole participates in several biological processes. The heavy chain consists of three domains (D1–D3): domain 1 has a low-affinity Ca2 þ binding site, the function of which is unknown; domain 2 is the calpain inhibitory region; and domains 2 and 3 with the sequence Glu–Val–Val–Ala–Gly are potent cysteine protease inhibitors. Domain 4 contains the kinin moiety which is released from the kininogen molecule by the enzymic action of kininogenases. In addition, domain 4 has the ability to inhibit athrombin-induced platelet aggregation, and also serves as a cell-binding site for the parent molecule. The carboxy-terminal portion of the kinin moiety and the amino-terminal portion of the light chain participate as low-affinity binding sites to endothelial cells. More importantly, the domain 4 cell-binding region holds the kininogens in the appropriate conformation for optimal cell binding. The light chain of L-kininogen is 4 kDa and consists of one domain (D5) of unknown function. The light chain of H-kininogen is 56 kDa and consists of domains 5 (D5) and 6 (D6). Domain 5 contains two histidineand glycine-rich regions, one at its carboxy-terminal and the other on its amino-terminal. It serves as a cell-binding site on platelets, neutrophils, and endothelial cells. In addition, the histidine- and glycinerich regions have the ability to bind to anionic surfaces such as Zn2 þ and heparin. Domain 5 of Hkininogen (kininostatin) inhibits critical steps in the angiogenic process, namely endothelial cell proliferation and apoptosis. This finding emphasizes the pertinent role that is likely to be played by domain 5 in tumor angiogenesis. Domain 6 of H-kininogen has a PPK and FXI binding site.

Kinin domain

Kinins D5

H-kininogen Figure 6 Molecular architecture of the kininogen molecule.

Cellular Actions of Kinins

Kinins are most important primary mediators of inflammation. Kinins express their functional effects by activating specific kinin receptors situated on the

KALLIKREIN–KININ CASCADE 489

surface membranes of many cell types. The kinin peptides are potent contractors of smooth muscle, cause arteriolar dilatation, and increase vascular permeability. They are powerful pain-producing substances, and they cause pain through two mechanisms: (1) by the direct stimulation of nociceptor fibers (C and Ad), and (2) by the sensitization of sensory fibers to physical and chemical stimuli. This algesic effect of BK is potentiated by thromboxanes and prostaglandins, and 50 -HT. It is postulated that the release of the kinin moiety from the kininogen molecule on the surface of the neutrophil by the enzymic action of kiningenases results in opening the junctions between the endothelial cells, and thus promotes the local diapedesis of neutrophils and cancer cells, as well as the extravasation of plasma. In addition, kinins stimulate the release of the proinflammatory cytokines, interleukin (IL-1) and tumor necrosis factor (TNF), osteoclastic bone resorption, and stimulate the release of several second-generation mediators, for example, platelet activating factor, leucotrienes, prostaglandins, substance P, acetlycholine, and noradrenaline. Other biological activities of kinins include the ability to lower systemic blood pressure (the plasma concentration has been reported to be about 10 pg ml
1). Kinins also stimulate the secretion of renin from the kidney and the release of vasopressin from the neurohypophysis. In the nervous system BK is involved in the central regulation of blood pressure and increasing neuronal excitability. Mitogenic Actions of Kinins

Although kinins are considered to play a primary role in inflammation, they are also mitogenic and regulate the proliferation of cancer cells. Several studies support the view that the kinin peptides stimulate DNA synthesis and thereby promote the process of tumorigenesis through their cell-differentiating and growth-promoting actions. Transformation of cells in vitro by the ras oncogene, which is mutated in a large number of human tumors, is associated with increased expression of kinin receptors. Kinins activate nuclear factor kappa B (NF-kB) through coupling with Gaq/11, which requires the induction of protein kinase C, and is independent of the TNF-a pathway. These findings together with the known release of kinin imply that kinin peptides formed in the tumor parenchyma could enhance the process of tumorigenesis. The ability of kinins to induce cell division and proliferation of tumor cells enhances the spread of cancerous cells, and by increasing vascular permeability they initiate the metastatic migration of cancer cells. These carcinogenic effects are mediated

by kinin receptors, which have been identified in a number of human carcinomas. Kinin B2 receptors have been immunolocalized in squamous cell and papillary adenocarcinomas of the lung. Direct ligand binding studies have demonstrated kinin B2 receptors in squamous cell and adenocarcinomas and in normal lung membranes. The observation of increased kinin receptor expression due to oncogenic transformation gives further credence to a mitogenic role for kinins in tumor tissue. Sequential analysis of in vitro cultures of endothelial cells immunolabeled with TK (hK1) clearly unfolds the role that the kallikrein– kinin protein components play in the formation of new blood vessels in the angiogenic processes linked to tumor invasiveness.

Kininases The turnover of kinins depends on both the rate of formation and the rate of destruction. After kinins are formed, they are destroyed at variable rates in the circulation, extracellular fluid space and in cells by the enzymatic action of peptidases. This family of enzymes is generally called kininases. There are two families of kininases: kininase I carboxypeptidases (KI) and kininase II peptidylpeptidases (KII). The KI family comprises kininase I-carboxypeptidase N (KICPN) and kininase I-carboxypeptidase M (KI-CPM) and the KII family includes kininase II-angiotensin I-converting enzyme (KII-ACE) and kininase II-neutral endopeptidase (KII-NEP). The enzymes which mainly cleave kinins are ACE (KII), CPN (KI), and NEP (KII).

Kinin Receptors Genomics and Molecular Structure

The kinins B1 and B2 are members of the superfamily of G-protein-coupled rhodopsin-like receptors characterized by seven-transmembrane regions connected by three extracellular and three intracellular loops, linked to second messenger signaling systems. The kinin binding site is located at the amino-terminal part of the third extracellular loop. Homology between the family members of the kinin receptors is most pronounced in the transmembrane regions, whereas the loop regions are more divergent in their sequences. The receptors have been mapped to chromosome 14q32, comprising more than 28 kb, and organized in three exons and two introns. The cDNA clone encoding a human B1 kinin receptor was isolated from a human embryonic lung fibroblast cDNA library by expression cloning. The mRNA encoding the B1 receptor is approximately 2 kb shorter than

490 KALLIKREIN–KININ CASCADE

that of the B2 receptor. The kinin B1 receptor has a predicted sequence of 353 amino acids, whereas the B2 receptor protein comprises 364 amino acids, is highly glycosylated, and exists in multiple isoforms at 69 kDa with an isoelectric point of pH 6.8–7.1. The two receptors show 36% protein sequence and 54% nucleotide homology.

receptor. In vitro, the B1 receptor is rapidly upregulated in experimental inflammation, by noxious stimuli, bacterial products (endotoxins, lipopolysaccharide), and inflammatory mediators, including proinflammatory cytokines (IL-1b, IL-8, TNF-a). Several growth factors such as epidermal growth factor (EGF) and endothelial growth factor also induce a kinin B1 receptor response. There is an increase in the number of B1 binding sites in inflamed or septic tissue. Normally, the B1 receptor is latent but appears to be rapidly upregulated in infections, carcinoma, and immune-modulated disorders, including rheumatoid arthritis, transplant rejection, and glomerulonephritis (Figure 8), and has been identified also in human fibrotic lung tissue. They also participate in mechanisms of hyperalgesia. The upregulation of the B1 receptors may be a mechanism of host defense.

Signal Transduction

The signal coupling of kinin receptors results in the activation of protein kinase C and tyrosine kinase pathways, coordinated with the activation of mitogen activated protein kinase (MAPK) and NF-kB. Kinin receptors also stimulate phosphatidylinositol hydrolysis in smooth muscle leading to mobilization of intracellular Ca2 þ , phospholipase C or phospholipase A, and appear to stimulate biosynthesis and release of prostaglandins. Stimulation of the kinin receptors on macrophages has been reported to release IL-1 and TNF. These bioactive peptides on signaling through the Gaq-linked receptor initiate the release of nitric oxide (EDRF) in endothelial cells. One attractive hypothesis is that on conversion of BK or lys-BK to the B1 agonist, the released carboxyterminal arginine may act as a substrate for eNOS (Figure 7).

Kinin B2 Receptors

These receptors are ubiquitous and are expressed in most tissues. They are constitutively expressed in all mammalian tissues, and are activated specifically by bradykinin and lys-bradykinin. The B2 receptor accounts for the majority of the physiological actions of the kinins. Thus, the acute nociceptive and inflammatory responses as well as the vasoactive properties elicited by the parent kinin molecules appear to be mediated through B2 receptors. In vivo, the B2 receptors have been implicated in hypotension, acute bronchoconstriction, and edema formation. Upregulation of the B2 receptors has been reported following

Kinin B1 Receptors

The pharmacological activity of the B1 receptor is regulated by the carboxypeptidase metabolites; des-Arg9-BK and des-Arg10-kallidin, in contrast to bradykinin, which is essentially inactive at the B1

BK P M

P K

G

F

S

P

F

R BK1 BK2

AA

Gp

Gp GC

R

CPM GC

cGMP

NO synthase

Arginase

Arginine

NO

Orn

Figure 7 Diagramatic illustration of the conversion of bradykinin to des-Arg9-bradykinin by a membrane carboxypeptidase M on the surface of cells that expresses both kinin receptors which are linked to cyclic GMP second messenger signaling pathway. MK–BK, methly-lys-bradykinin; K–BK, lys-bradykinin; R–R, bradykinin; BK2, bradykinin B2 receptor; BK1, bradykinin B1 receptor; NO, nitric oxide; Gp, G protein; GC, guanyl cyclase.

KALLIKREIN–KININ CASCADE 491

100.00

µm

DT DT

Kinin B1 receptors

Figure 8 Visualization of immunolabeled kinin B1 receptors on the renal tubule cells of the human kidney. Positive label is shown by the dark brown DAB chromogen; DT, distal tubules. Magnification 20.

myocardial infarction and in response to cytokines. TNF-a and IL-1b both induced a rapid and transient increase in B1 and B2 receptor mRNA expression in human embryonic lung fibroblasts, and IL-1b has been shown to increase the expression of B2 receptors in human deciduas derived cells. In addition, immunolabeling for B2 receptors has been observed on the astrocytic cells in patients with astrocytomas, and in esophageal, gastric, and renal carcinomas. A recent interesting observation with regard to bradykinin receptors is that PK and other serine proteases such as trypsin and cathepsin G are able to activate the B2 receptor. Thus, by a mechanism analogous to that whereby thrombin and trypsin activate the protease-activated receptors (PAR-1 and PAR-2, respectively), the B2 receptor may function as a novel serine-protease-activated receptor. However, kallikrein was able to activate a mutated B2 receptor missing the 19 N-terminal amino acids, suggesting a different mechanism of activation to that of the thrombin-activated PAR-1 receptor. The B1 receptor has not been investigated in this regard.

Kinin Receptor Antagonists The understanding of the critical role the kinin receptor plays in health and disease has been greatly enhanced by the development of several antagonists. By definition, antagonists are compounds that bind to the receptors without invoking intrinsic activity. Over several decades kinin receptor antagonists have been developed comprising five generations of synthetic molecules. The first selective antagonist of the B1 receptor was des-Arg9 [leu8] bradykinin. Other antagonists such as des-Arg10 [leu9] lys-bradykinin (kallidin), with higher affinity and a longer duration of action, have been reported. The second-generation kinin B2 receptor antagonist, DArg–[Hyp3, Thi5,

Dtic7, Oic8]–bradykinin (Hoe 140 or icatibant), proved to be more potent than the first-generation molecules with a longer in vitro lifetime and has been efficacious in clinical trials for allergic asthma and rhinitis. The recently synthesized kinin receptor antagonists are anticancer agents. Their mode of action is unique in that they inhibit the growth of cancer cells by a ‘novel-agonist-based’ mechanism that involves silencing Gaq11 and stimulating Ga12,13. The most potent growth inhibitor of small-cell lung carcinoma, in this series of antagonists, is the N-terminal cross-linked molecule dimerized with suberimide [Darg-arg-pro-hyp (trans-4-hydroxy-L-proline)gly-Igl (alpha-2-indanylglycine)-ser-DIgl-Oic(octahydroindole-2-carboxylic acid)-arg]. Small mimetic (BKM-570, FSC-OC2Y-Atmp) antagonists are also potent inhibitors of the growth of lung cancer cell lines. Domain 5 (kininostatin) of H-kininogen, the endogenous substrate of PK, inhibits critical steps in the angiogenic process, namely endothelial cell proliferation and apoptosis. This finding further emphasizes the pertinent role that is being played by the kallikrein–kinin proteins in new capillary blood vessel formation and in carcinogenesis. Nonpeptide Kinin Antagonists

Several orally available nonpeptide kinin receptor antagonists, namely FR 173657, 165649, and 167344, have been developed. These have been found to be potent against B2 binding, with no effect on B1 binding. In vivo, they blocked human B2-receptor-mediated phosphatidylinositol hydrolysis, bradykinin-induced bronchoconstriction in guinea pigs, and carrageenin-induced paw edema. In addition, a nonpeptide antagonist for the B1 receptor (PS020990) has been developed recently.

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Kallikrein–Kinin Cascade in Lung Diseases Cellular Expression and Mediator Release

The kinin peptides play an important role in the regulation of airway inflammation. All of the components of the cascade – kallikrein, kininogen, and free kinins – have been detected in bronchial lavage fluid of asthmatics but not in control subjects. Allergen challenge of asthmatics resulted in an increase of TK and kinin in the airways. Neutrophils, monocyte/ macrophages, and submucous glands are likely to be the sources of TK in the airways, whereas the kininogen substrates are probably derived from neutrophils and plasma transudate. When the bronchial epithelium is inflamed, as is the case in asthma, kinins are capable of producing a marked and persistent contraction of bronchial smooth muscle; bradykinin is a potent bronchoconstrictor in asthmatics, but not in normal subjects. The release of prostanoids, thromboxane, substance P, and C-fiber evoked axon reflexes, and direct stimulation of kinin receptors has been implicated in the bronchoconstrictor actions of bradykinin. In addition to stimulating contraction of airway smooth muscle, bradykinin induces IL-8, VEGF, COX-2, and PGE2 production, and extracellular signal-regulated kinase (ERK)1/2 and p38 MAPK dependent IL-6 expression by cultured airway smooth muscle cells. Airway hyperresponsiveness to bradykinin has recently been identified as a sensitive indicator of corticosteroid responsiveness in asthma. Bradykinin increases IL-8 generation in the airway epithelial cells by a mechanism dependent on COX2-derived prostanoids. Furthermore, bradykinin increases the expression of the IL-8 chemokine receptors, CXCR1 and CXCR2, in nasal tissue and epithelium of patients with allergic rhinitis. Since IL-8 is a potent neutrophil chemoattractant, bradykinin may contribute to neutrophilic inflammation in asthma and chronic obstructive pulmonary disease by stimulating IL-8 production from airway epithelial cells. Other effects of bradykinin that may be important in the context of airway inflammation are its ability to increase capillary permeability producing mucosal edema, and to enhance signal traffic in pulmonary nerves by stimulating the secretion of acetylcholine as well as neuropeptides. Bradykinin stimulates alveolar macrophages to release eosinophil chemotactic factors (predominantly leukotriene B4). The kinin-related link between neutrophils and eosinophils appears to be bidirectional in that eosinophil–granule-derived major basic protein increases airway hyperreactivity. Evidence of an eosinophil–kinin link in airway inflammation comes

from a study showing that in subjects with mild asthma, the magnitude of eosinophilic inflammation of the airways was more strongly associated with responsiveness to inhaled bradykinin compared with methacholine. In asthmatics, pulmonary function gradually improved during 4 weeks of treatment with icatibant (Hoe 140, specific B2 receptor antagonist), suggesting that B2 receptor blockade may have anti-inflammatory effects.

Signal Transduction

Most of the biological effects of bradykinin in human airways appear to be mediated by the constitutively expressed B2 receptor, which signals through p38, ERK-1/2, MAP kinases, protein kinase C, and NFkB. Furthermore, nasal challenge with the B1 receptor agonist lys-[des-Arg10]-bradykinin activated ERK in subjects with allergic rhinitis, but not in normal subjects, and additionally lys-[des-Arg10]-bradykinin activated the transcription factor AP-1 in human airway epithelial cells. However, the precise mechanisms by which kinins may influence the pathogenesis of inflammatory diseases such as asthma and chronic pulmonary obstructive disease are unknown. See also: Kinins and Neuropeptides: Bradykinin; Neuropeptides and Neurotransmission; Tachykinins; Vasoactive Intestinal Peptide.

Further Reading Barnes PJ (1992) Bradykinin and asthma. Thorax 47: 979–983. Bhoola KD, Figueroa CD, and Worthy K (1992) Bioregulation of kinins: kallikreins, kiniogens and kininases. Pharmacological Reviews 44: 1–80. Blaukat A (2003) Structure and signaling pathways of kinin receptors. Andrologia 35: 17–23. Borgono CA and Diamandis EP (2004) The emerging roles of human tissue kallikreins in cancer. Nature Reviews: Cancer 4: 876–890. Chan DC, Gera L, Stewart JM, et al. (2002) Bradykinin antagonist dimer, CU201, inhibits the growth of human lung cancer cell lines by a ‘biased agonist’ mechanism. Proceedings of the National Academy of Sciences, USA 99: 4608–4613. Chan DC, Gera L, Stewart JM, et al. (2002) Bradykinin antagonist dimer, CU201, inhibits the growth of human cancer cell lines in vitro and in vivo and produces synergistic growth inhibition in combination with other antitumour agents. Clinical Cancer Research 8: 1280–1287. Clements J, Hooper J, Dong Y, and Harvey T (2001) The expanded human kallikrein (KLK) gene family: genomic organization, tissue-specific expression and potential functions. Biological Chemistry 382: 5–14. Clements JA, Willemsen NM, Myers SA, and Dong Y (2004) The tissue kallikrein family of serine proteases: functional roles in human disease and potential as clinical biomarkers. Critical Reviews in Clinical Laboratory Sciences 41: 265–312.

KERATIN 493 Colman RW and Schmaier AH (1997) Contact system: a vascular biology modulator with anticoagulant, profibrinolytic, antiadhesive, and proinflammatory attributes. Blood 90: 3819–3843. Cyr M, Eastlund T, Blais C, Rouleau JL, and Adam A (2001) Bradykinin metabolism and hypotensive transfusion reactions. Transfusion 41: 136–150. Davis AE (2005) The pathophysiology of hereditary angioedema. Clinical Immunology 114: 3–9. Figueroa CD, Henderson LM, Kaufmann J, et al. (1992) Immunovisualization of high (HK) and low (LK) molecular weight kininogens on isolated human neutrophils. Blood 79: 754–759. Figueroa CD, MacIver AG, and Bhoola KD (1989) Identification of a tissue kallikrein in human polymorphonuclear leucocytes. British Journal of Haematology 72: 321–328. Guo Y-L, Wang S, and Colman RL (2001) Kininostatin an angiogenic inhibitor, inhibits proliferation and induces apoptosis of human endothelial cells. Arteriosclerosis, Thrombosis, and Vascular Biology 21: 1427–1433. Haasemann M, Figueroa CD, Henderson L, et al. (1994) Distribution of bradykinin B2 receptors in target cells of kinin action. Visualization of the receptor protein in A431 cells, neutrophils and kidney sections. Brazilian Journal of Medical and Biological Research 27: 1739–1756. Hecquet C, Becker RP, Tan FL, and Erdo¨s EG (2002) Kallikreins when activating bradykinin B-2 receptor induce its redistribution on plasma membrane. International Immunopharmacology 2: 1795–1806. Henderson LM, Figueroa CD, Muller-Esterl W, and Bhoola KD (1994) Assembly of contact-phase factors on the surface of the human neutrophil membrane. Blood 84: 474–482. Hermann A, Arnhold M, Kresse H, Neth P, and Fink E (1999) Expression of plasma prekallikrein mRNA in human nonhepatic tissues and cell lineages suggests special local functions of the enzyme. Biological Chemistry 380: 1097–1102.

Mahabeer R and Bhoola KD (2000) Kallikrein and kinin receptor genes. Pharmacology & Therapeutics 88: 77–89. Marceau F, Hess FJ, and Bachvarov DR (1998) The B1 receptor for kinins. Pharmacological Reviews 50: 357–386. Marceau F and Regoli D (2004) Bradykinin receptor ligands. Nature Reviews 3: 845–847. Neth P, Arnhold M, Nitschko H, and Fink E (2001) The mRNAs of prekallikrein, factors XI and XII, and kininogen, components of the contact phase cascade are differentially expressed in multiple non-hepatic human tissues. Thrombosis and Haemostasis 85: 1043–1047. Peek M, Moran P, Mendoza N, Wickramasinghe D, and Kirchhofer D (2002) Unusual proteolytic activation of prohepatocyte growth factor by plasma kallikrein and coagulation factor XIa. Journal of Biological Chemistry 277: 47804–47809. Plendl J, Snyman C, Naidoo S, Sawant S, Mahabeer R, and Bhoola KD (2000) Expression of tissue kallikrein and kinin receptors in angiogenic microvascular endothelial cells. Biological Chemistry 381: 1103–1115. Poblete MT, Garces G, Figueroa CD, and Bhoola KD (1993) Localization of immunoreactive tissue kallikrein in the seromucous glands of the human and guinea-pig respiratory tree. Histochemical Journal 25: 834–839. Renne T, Dedio J, Meijers CM, Chung D, and Mu¨ller-Esterl W (1999) Mapping of the discontinuous H-kininogen binding site of plasma prekallikrein – evidence for a critical role of apple domain-2. Journal of Biological Chemistry 274: 25777–25784. Stuardo M, Gonzalez CB, Nualart F, et al. (2004) Stimulated human neutrophils form biologically active kinin peptides from high low molecular weight kininogens. Journal of Leukocyte Biology 75: 631–640. Vidal MA, Astroza A, Matus CE, et al. (2005) Kinin B2 receptorcoupled signal transduction in human cultured keratinocytes. Journal of Investigative Dermatology 124: 178–186.

KERATIN S H Randell, University of North Carolina, Chapel Hill, NC, USA J L V Broers, Eindhoven University of Technology, Eindhoven, The Netherlands F C S Ramaekers, University of Maastricht, Maastricht, The Netherlands & 2006 Elsevier Ltd. All rights reserved.

role to maintain epithelial integrity. Emerging evidence indicates that keratin intermediate filaments modulate additional cellular functions such as receptor-mediated signaling and cellular-protein targeting. Keratins are a defining feature of epithelial cells and the predictable expression pattern in each lung epithelial cell type underlies their use for cell identification. Since keratin fragments are released from tumors and in other pathological conditions affecting epithelial cells, they may also serve as disease markers. Importantly, immunostaining for keratin may assist the differential diagnosis of lung neoplasms.

Abstract Keratins, represented by more than 50 genes and 20 known polypeptides, are amongst the most abundant proteins of lung epithelial cells. They comprise keratin intermediate filaments which, together with microfilaments and microtubules, form the cytoskeleton that dynamically maintains epithelial cell structure. The diverse keratin intermediate filament family evolved from a common ancestral gene and shares a common structural plan. Type I and II subtypes of keratin form polymers in an epithelial cell-type-specific manner, which is also influenced by developmental stage, cellular differentiation, and pathophysiology. Mutations in keratins, while not resulting in a primary pulmonary phenotype, cause genetic diseases, establishing their

Introduction Early electron micrographs demonstrated fibrous, detergent-resistant elements extending throughout the cytoplasm of metazoan cells. This network was subsequently termed the cytoskeleton. It is now known to consist of three polymeric structures microfilaments (6 nm diameter), microtubules (23 nm diameter), and intermediate filaments (10 nm diameter) that serve as an internal scaffold, maintaining cellular shape,

494 KERATIN Table 1 Intermediate filament classes, genes, and expression patterns (a) Intermediate filaments Class

Molecule

Cell type

Type I

K9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23 KRTHA1-8 CHaAKRT K1, 2, 3, 4, 5, 6, 7, 8 Hb1-6, ChHb A, B C, D Vimentin Desmin GFAP Peripherin NF-L, M, H a-internexin Lamin A/C, B1, B2 Phakinin, filensin

Epithelial

Type I hair Type II Type II hair Type III

Type IV Type V Other

Hair and nail Epithelial Epithelial Hair and nail Epithelial Fibro, endo, leuko Muscle Glia Peripheral neurons CNS neurons CNS neurons Nucleus, widespread Lens

(b) Keratin intermediate filaments in normal adult lung Pair

Distribution

K4, K13 K5, K14 K8, K18

Occasional columnar cells Basal cells in pseudostratified epithelium Columnar cells in pseudostratified epithelium, cuboidal bronchiolar cells, and alveolar cells

Unpaired

Distribution

K7

Columnar cells in pseudostratified epithelium, cuboidal bronchiolar cells, and alveolar cells Basal cells in pseudostratified epithelium Subset of columnar and basal cells in pseudostratified epithelium, cuboidal bronchiolar cells, and alveolar cells

K15 K19

Data from Broers JLV, de Leij L, Klein Rot M, et al. (1989) Expression of intermediate filament proteins in fetal and adult human lung tissues. Differentiation 40: 119–128; Coulombe PA and Wong P (2004) Cytoplasmic intermediate filaments revealed as dynamic and multipurpose scaffolds. Nature Cell Biology 6: 699–706; Hesse M, Magin TM, and Weber K (2001) Genes for intermediate filament proteins and the draft sequences of the human genome: novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18. Journal of Cell Science 114: 2569–2575; Oshima RG (2002) Apoptosis and keratin intermediate filaments. Cell Death and Differentiation 9: 486–492; and Porter RM and Lane EB (2003) Phenotypes, genotypes and their contribution to understanding keratin function. Trends in Genetics 19: 278–285.

imparting structural integrity, and regulating motility. Microfilaments and microtubules are evolutionarily conserved, similar in many different cell types within an organism, and undergo distinct and rapid processes of regulated, polar assembly and disassembly utilizing nucleotide hydrolysis. In contrast, intermediate filaments are highly diverse, and distinct groups of intermediate filaments are expressed in different cell types (Table 1). Keratin intermediate filaments are a defining feature of epithelial cells and are visible as tonofilament bundles in basal cells of the airway epithelium. Keratin proteins are subdivided into two groups and systematically numbered 1–23 (except 11 and 22) based on molecular weight and isoelectric point. Type I keratins (K9, 10, 12–20, and 23) are large and acidic, while type II keratins (K1–8), are relatively smaller and basic. In addition to the epithelial (soft) keratins given

above, type I and II keratins are further subdivided into hair and nail (hard) type keratins. Unlike microfilaments and microtubules, assembly of intermediate filaments does not require nucleotide hydrolysis. Turnover is highly variable and is regulated by posttranslational modifications including phosphorylation, ubiquitinylation, glycosylation, transglutamination, and protease cleavage. Furthermore, diversity in keratin stability is imparted by variable degrees of disulfide bridging, progressively increasing in keratins of stratified epithelia of internal organs, epidermal keratinocytes, and hair and nail keratins, respectively.

Structure The intermediate filament gene family ranks amongst the 100 largest in the human genome. All functional

KERATIN 495 Rod

Head DNA

1A

1B L1

Tail 2B

2A L12

L2

Type I/type II protein dimer

Staggered, antiparallel tetramer

Keratin fiber

Figure 1 Keratin structure and filament assembly. The tripartite keratin monomer consists of head, central rod, and tail domains. The a helical segments within the rod (1A, 1B, 2A, 2B) are connected by linker segments (L1, L12, L2). The cross-hatched areas represent the highly conserved domains that drive formation of coiled-coil heterodimers. Two heterodimers in a staggered, reverse orientation create tetramers that, in turn, form linear polymers. Keratin intermediate filament fibers consist of four aligned octameric protofilaments.

keratin genes are contained within clusters on chromosomes 12q13 and 17q21. There are numerous keratin pseudogenes and gene fragments, many related to keratins 8 and 18, which are spread amongst most human chromosomes. Alignment of the multiple DNA sequences of intermediate filament genes reveals a common tripartite structure. The keratin monomer consists of a conserved central a-helical section, the rod domain, flanked by nonhelical head and tail domains of variable length and sequence (Figure 1). The highly conserved ends of the rod domain drive filament assembly via the formation of coiled-coil heterodimers. In turn, two dimers form tetramers and these associate in a staggered, reverse orientation to form linear polymers called protofilaments. When a purified type I keratin and a purified type II keratin are mixed in vitro, they spontaneously form filaments (Figure 2(a)). Although there may be variability among different intermediate filament types, mature fibers apparently consist of four aligned octameric protofilaments. Keratin filaments in human airway epithelial cells in culture are illustrated in Figure 2(b).

Regulation of Keratin Expression Keratin intermediate filament expression is inextricably linked to the formation of an epithelial sheet and to its type and differentiation state. A simple epithelium, made up of a single layer of polarized cells expressing keratins 8 and 18, first appears during early embryogenesis. These keratins continue to be expressed in all simple epithelia, which may then acquire additional keratins characteristic of the particular epithelium. For example, the simple cuboidal bronchiolar epithelium and type II epithelial cells express keratins 7, 8, 18, and 19. Complex epithelia, such as the pseudostratified tracheobronchial epithelium, express characteristic keratin pairs and these pairs systematically vary according to cell type. Typically, keratins 5, 14, and 15 are present in the basal cells and gland myoepithelial cells, while keratins 7, 8, 18, and 19 predominate in columnar cells and gland acinar cells. Images of keratins in lung cells are shown in Figure 3. Detailed expression of keratins in normal and pathological lung has not been comprehensively documented as yet.

496 KERATIN

Figure 2 Keratin intermediate filaments in vitro and in cells. (a) Negative stain electron micrograph illustrating spontaneous reconstitution of filaments upon mixing keratin 5 and keratin 14 proteins in vitro. Reproduced from Coulombe PA and Omary MB (2002) ‘Hard’ and ‘soft’ principles defining the structure, function and regulation of keratin and intermediate filaments. Current Opinion in Cell Biology 14: 110–122, with permission from Elsevier. (b) Confocal light microscope image of human airway epithelial cells in culture. Keratin intermediate filaments were detected with an anti-pan cytokeratin antibody (green), actin was detected with phalloidin (red), and the nucleus was stained with DAPI (blue).

Figure 3 Keratin expression in human lungs. Immunoperoxidase staining with different antikeratin antibodies as indicated. Magnification 400.

Transcriptional control of keratin gene expression in developing and mature epithelia has been extensively studied, using both in vitro cell models and transgenic mice. However, the complex mechanisms regulating tissue- and cell-type-specific expression of keratins are not fully understood. Furthermore, keratin expression is modulated by developmental stage

and physiologic or pathologic conditions. A dramatic example is development of squamous metaplasia in the airway epithelium as a function of chronic injury, carcinogen exposure, or vitamin A deficiency. In this case, the keratin 4 and 13 pair, normally found in stratified oral, genital, and esophageal epithelia, is dramatically induced in the pathological airway.

KERATIN 497

Biological Function

Interacting Molecules

Early investigators suggested that the cytoskeleton, including intermediate filaments, functioned to maintain cellular shape and integrity, a theory subsequently proven by the phenotype of humans and mice with genetic abnormalities of keratin. Disruption of keratin 5 and 14 filaments, found in basal cells, causes extreme mechanical fragility of the epidermis, resulting in blistering skin disorders collectively known as epidermolysis bullosa simplex. Nearly 20 human genetic disorders of keratin have been discovered. In addition, a large and growing panel of keratin mutants, knockouts, and double knockouts has been created in mice. In humans and mice, the pathologic consequences depend upon both, the expression pattern of the mutated keratin and the specific defect. Disruptive mutations may result in a more severe phenotype than complete absence, presumably due to functional redundancy in which an alternative keratin replaces a missing heteromeric partner. The spectrum of genetic alterations and their resulting phenotypes has provided key insights regarding keratin structure and function, firmly establishing the role of keratin intermediate filaments in maintaining epithelial integrity. The diverse phenotypes of mice and humans with genetic alterations in keratins have suggested additional functions for keratin intermediate filaments. Keratin 8 is important for placental function. Keratin 8 gene-deleted mice surviving after birth develop colorectal abnormalities including loose stools and colonocyte hyperplasia, as well as subtle liver pathology that becomes more evident upon liver stress and regeneration. Cells from keratin 8- and 18-deficient animals are strikingly more sensitive to the pro-apoptotic effects of tumor necrosis factor (TNF). Keratin 8 and 18 intermediate filaments bind to the TNF receptor 2, indicating a likely role in the modulation of TNF signaling. Physical association of filaments with both receptors and signaling intermediates is likely a general mechanism for modulating diverse signal transduction pathways. The intestinal symptoms of keratin 8/18 deficiency are linked to ion transport abnormalities and reflect the importance of keratin intermediate filaments to facilitate protein-targeting to the correct plasma membrane domain. Additionally, keratins are targets for, and participate in, the compartmentalization of caspases during apoptosis. Keratins may sequester growth-modulating proteins during physiologic regulation of the cell cycle, thereby participating in homeostatic cell turnover and regulation of proliferation during wound healing or in transformed cells. Although correlates of these functions have not been explicitly studied in lung cells, analogous roles are likely.

There is a long and growing list of proteins that interact with keratins, falling into broad functional categories related to whether they are involved in intermediate filament synthesis and assembly (e.g., HSPs), cross-linking (e.g., filaggrin), attachment to the plasma membrane, nucleus or other cell structures (e.g., desmoplakin), or in modulation of signaling pathways (e.g., TNFR2). The human type I keratin gene cluster is interrupted by a domain of multiple, structurally related genes encoding keratinassociated proteins, but these are mostly involved in disulfide bridging of hair keratins.

Keratin in Respiratory Diseases Genetic diseases due to abnormalities in keratin 5 and 14 (epidermolysis bullosa simplex) profoundly affect the skin, whereas alterations in keratin 8 and 18 primarily predispose to liver (cryptogenic cirrhosis) and bowel disease. In epidermolysis bullosa, upper airway pathology is not uncommon and manifests as hoarseness and inspiratory stridor, sometimes progressing to laryngotracheal stenosis and obstruction, which can be life threatening. To our knowledge, there are no reports of pulmonary manifestations of genetic diseases of keratins 8 and 18. Other known diseasecausing mutations affect keratins that are not highly expressed in lung, and thus are not expected to result in a pulmonary phenotype. In the gut, pathogenic salmonella strains may utilize keratins as scaffolds for cell-type-specific intracellular invasion and, in the lung, keratin 13 has been reported as a receptor for the cable pilus of Burkholderia cenocepacia, an epidemic pathogen of cystic fibrosis patients. There are reports that the presence of keratin fragments in bronchopulmonary lavage fluid may be used to monitor disease intensity in chronic bronchitis, bronchiectasis, panbronchiolitis, idiopathic pulmonary fibrosis, alveolar proteinosis, and lung allograft rejection. Chronic airway and lung injury results in progressive epithelial changes, including modulation of keratin expression. As noted previously, a prominent example is induction of keratin 4 and 13 accompanying the development of squamous metaplasia. As metaplasia progresses to atypia and dysplasia during multistage carcinogenesis, changes in keratin expression are prominent; keratins have become important in the differential diagnosis and monitoring of thoracic neoplasms. Briefly, all lung adenocarcinomas express keratins found in normal simple, cuboidal epithelial cells, namely keratins 7, 8, 18, and 19, and some express keratin 4. Squamous carcinomas show greater complexity in their keratin-expression pattern and may additionally

498 KERATINOCYTE GROWTH FACTOR

express keratins 5, 6, 10, 13, 14, 15, and 17. Small cell carcinomas of the lung and carcinoid tumors typically express low levels of keratin 8, 18, and 19, while cytokeratin 7 is strikingly absent in these tumors. Mesothelioma expresses keratins 5, 7, 8, 18, and 19, and some express keratins 4, 6, 14, and 17. Examination of keratin expression has proven useful to solve difficult diagnostic problems in lung cancer. Keratin 5/6 is amongst a panel of immunochemical markers discriminating between mesothelioma and adenocarcinoma. The combined use of keratin 7 (present) and 20 (absent) may help distinguish primary lung adenocarcinomas versus metastases from other sites, and in combination with other markers, may help to distinguish even highly related tumors such as pulmonary versus metastatic GI carcinoid or endocrine tumors. In addition, detection of keratin fragments in the serum of lung cancer patients may be of prognostic importance. Tumor growth is accompanied by increased apoptosis, and during apoptosis, keratins are cleaved in a highly specific manner, generating stable protein fragments detectable in serum. The CYFRA21.1 test detects keratin 19 fragments and appears to have value as a covariable for monitoring response to treatment and predicting outcome in non-small cell lung cancer. See also: Mesothelioma, Malignant. Tumors, Malignant: Bronchogenic Carcinoma; Bronchial Gland and Carcinoid Tumor; Carcinoma, Lymph Node Involvement.

Further Reading Broers JLV, de Leij L, Klein Rot M, et al. (1989) Expression of intermediate filament proteins in fetal and adult human lung tissues. Differentiation 40: 119–128.

Broers JLV, Ramaekers FCS, Klein Rot M, et al. (1988) Cytokeratins in different types of human lung cancer as monitored by chain-specific monoclonal antibodies. Cancer Research 48: 3221–3229. Coulombe PA and Omary MB (2002) ‘Hard’ and ‘soft’ principles defining the structure, function and regulation of keratin and intermediate filaments. Current Opinion in Cell Biology 14: 110–122. Coulombe PA and Wong P (2004) Cytoplasmic intermediate filaments revealed as dynamic and multipurpose scaffolds. Nature Cell Biology 6: 699–706. Hesse M, Magin TM, and Weber K (2001) Genes for intermediate filament proteins and the draft sequences of the human genome: novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18. Journal of Cell Science 114: 2569–2575. Ordonez NG (2003) The immunohistochemical diagnosis of mesothelioma: a comparative study of epithelioid mesothelioma and lung adenocarcinoma. American Journal of Surgery and Pathology 27: 1031–1051. Oshima RG (2002) Apoptosis and keratin intermediate filaments. Cell Death and Differentiation 9: 486–492. Owens DW and Lane EB (2003) The quest for the function of simple epithelial keratins. Bioessays 25: 748–758. Porter RM and Lane EB (2003) Phenotypes, genotypes and their contribution to understanding keratin function. Trends in Genetics 19: 278–285. Pujol JL, Molinier O, Ebert W, et al. (2004) CYFRA 21-1 is a prognostic determinant in non-small-cell lung cancer: results of a meta-analysis in 2063 patients. British Journal of Cancer 90: 2097–2105. Schaafsma HE and Ramaekers FCS (1994) Cytokeratin subtyping in normal and neoplastic epithelium: basic principles and diagnostic applications. Pathology Annual 29: 21–62. Schutte B, Henfling M, Koelgen W, et al. (2004) Keratin 8/18 breakdown and reorganization during apoptosis. Experimental Cell Research 297: 11–26. Strelkov SV, Herrmann H, and Aebi U (2003) Molecular architecture of intermediate filaments. Bioessays 25: 243–251. Tot T (2002) Cytokeratins 20 and 7 as biomarkers: usefulness in discriminating primary from metastatic adenocarcinoma. European Journal of Cancer 38: 758–763.

KERATINOCYTE GROWTH FACTOR R J Mason and Y Chang, National Jewish Medical and Research Center, Denver, CO, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Keratinocyte growth factor (KGF); also designated fibroblast growth factor-7 (FGF-7) and similar to FGF-10, is a mitogen produced primarily by fibroblasts and is a potent growth factor for alveolar and bronchial epithelial cells. The special significance of KGF is that it can prevent lung injury due to a variety of insults, if given before the injury. There have been no definitive reports of KGF effects given solely after lung injury. KGF is an approved growth factor for the treatment of mucositis due to

toxic chemotherapeutic agents used to prevent rejection in allogeneic bone marrow transplantation. In vitro KGF stimulates proliferation and differentiation of alveolar type II cells. KGF signals through a tyrosine kinase receptor (KGFR, FGFR2-IIIb) and activates several signal cascades, including ERK, Akt, and c-jun N-terminal kinase (JNK) as well as PAK4. The KGFR dominant negative mice have essentially no lungs, whereas the lungs of KGF knockout mice are relatively normal. These observations have been interpreted as indicating that FGF-10, not FGF-7, is required for fetal lung development and that FGF-10 can replace FGF-7 in the FGF-7 null animal. KGF has the potential as an important treatment for acute lung injury, and studies on the mechanism of how KGF increases differentiation in vitro may provide new therapeutic targets for the treatment of many lung diseases.

KERATINOCYTE GROWTH FACTOR 499

Introduction Keratinocyte growth factor (KGF); also designated fibroblast growth factor-7 (FGF-7) is a potent relatively specific growth factor for epithelial cells produced primarily by fibroblasts. FGF-7 is very similar in its action to FGF-10, which is also referred to as KGF-2. Both use the same tyrosine kinase receptor (FGFR2-IIIb). Human recombinant KGF is available for the treatment of mucositis produced by toxic chemotherapeutic agents and is marked under the name of palifermin. The importance of KGF for lung biology is that it is a potent mitogen for normal alveolar type II cells as well as for bronchial epithelial cells and microvascular endothelial cells and has been used to prevent lung injury, if given before a variety of insults. It is not known if KGF can be developed to use effectively in the treatment of acute lung injury after the injury has occurred. KGF was cloned from fetal lung fibroblasts in the search for growth factors for epithelial cells. KGF was first shown to be a potent mitogen for rat alveolar type II cells in vitro. These initial studies were performed to determine the mitogenic factors for type II cells in fibroblast-conditioned medium. The two growth factors identified were KGF and hepatocyte growth factor (HGF). Soon thereafter, recombinant human KGF was shown to induce proliferation of rat type II cells when it was instilled into the normal lung in vivo. KGF instillation causes a marked increase in BdrU labeling 2 and 3 days after instillation and increases the expression of sufactant protein A (SP-A), SP-B, SP-C, and SP-D. In contrast, KGF decreases the expression of the bronchiolar epithelial protein CC-10. KGF also increases the expression of the Na/K-ATPase, which is the pump for the transepithelial sodium transport and is important for transporting fluid from the alveolar into the interstitial compartment, where it can be cleared by lymphatics. In other studies, KGF was also shown to stimulate the proliferation of bronchial epithelial cells and microvascular endothelial cells. Because KGF can cause the proliferative effect of both the epithelial and endothelial components of the alveolar wall, it is a potentially important therapeutic agent for the treatment of acute lung injury.

Structure and Regulation of Production of KGF KGF is a small glycosylated heparin-binding 26– 28 kDa protein. It is produced by fibroblasts, and secretion by fibroblasts can be stimulated by IL-1b and PDGF and inhibited by dexamethasone. KGF is also produced by gdT cells in the skin and intestine.

However, it is not known if gdT cells in the lung produce KGF. KGF (FGF-7) has significant homology to FGF-10 (KGF-2) (see Figure 1).

Biologic Function of KGF Effects of KGF on the Lung In vivo

KGF can stimulate epithelial proliferation if administered intratracheally, intravenously, or subcutaneously, although the largest effect is seen after intratracheal administration. The effect is not species restricted, in that rats and mice also respond to human recombinant KGF. The response in the rat appears to be more marked than the effect in mice or with human type II cells in vitro. Marked type II cell hyperplasia occurs with an adenovirus overexpressing KGF or with conditional expression of KGF in transgenic mice (see Figure 2). KGF is also a major mitogen for keratinocytes, hair follicles, the epithelium lining the GI tract, and presumably any other epithelial tissue. The epithelial cells express KGFR2IIIb (the high-affinity receptor for KGF), but do not express KGF. The effect on the epithelium is a paracrine effect, whereby local mesenchymal cells produce KGF and regulate the local epithelial population. This occurs in the lung, the skin, the gastrointestinal track, and the prostate. Numerous studies have shown KGF to offer a preventative treatment for acute lung injury. Unfortunately, KGF administration has only been shown to be effective to protect against a given injurious agent if given before the insult, and postinjury treatment regimens have been ineffective. However, it is very effective as a pretreatment. KGF prevents lung injury due to oxygen, bleomycin, acid instillation, radiation, and a variety of cytotoxic agents. For example, instillation of bleomycin into rats reduces the total capacity by 25%; this effect is entirely blocked by pretreatment with KGF for 48 or 72 h but not by saline pretreatment. Bleomycin markedly reduces SP-C expression 1 or 7 days after instillation; this effect is completely inhibited by pretreatment with KGF for 48 h but not by saline pretreatment. KGF also prevents pulmonary complications of allogenic bone marrow transplantation. For injuries that are slow (oxygen toxicity), KGF can be given at the onset of injury; for injuries that take 1–2 days to develop (bleomycin injury), KGF needs to be given 48 h prior to the insult; and for injuries that cause immediate damage (HCl instillation), KGF needs to be given 3 days prior to the insult. It seems that the alveolar epithelium is maximally protected about 3 days after intratracheal instillation. Maximum proliferation also occurs 2–3 days after instillation. Time will tell

500 KERATINOCYTE GROWTH FACTOR KGF receptor Heparan sulfate

KGF KGF

PIP3

p

PIP2

RAS S p O grb2 Raf S

SHP2

p

FRS2

p

PLC
PDK-1

grb2 MLK3

MEK1/2

PIP3

P85 P110

MEKK1/4

DAG P13K MKK3/6

pH

MKK4/7

PKB/ AKT

PKC

P70S6K ERK 1/2

p

38

p

JNK

GSK-3

FAS ?

SCD-1

SREBP1 ?

? ?

E-FABP

C/EBPα ?

Figure 1

Normal alveolar epithelium

KGF

Hyperplastic alveolar epithelium Figure 2 KGF stimulates type II cell hyperplasia. The normal alveolar surface is covered primarily by type I cells, and there is a low rate of cell proliferation by the progenitor type II cells. However, after KGF treatment type II cells undergo rapid cell division and form an alveolar epithelium composed primarily of hyperplastic type II cells. This is readily seen in the rat lung, and there may be a less dramatic response in other species. After a week without additional stimulation by KGF the hyperplastic type II cells undergo apoptosis, and the lung appears normal.

if a treatment regimen can be developed for clinical human acute lung injury. Since the injury is usually ongoing, there appears to be a window of opportunity if the drug is given early. In addition, in bleomycin lung injury, KGF and HGF protein increase in lavage. In our studies, we were not able

to demonstrate a convincing increase in KGF mRNA after a variety of lung injuries. Importantly, KGF can also enhance postpneumonectomy lung growth. As contrasted with epidermal growth factor (EGF) and retinoic acid, KGF can induce formation of new alveoli. It should be noted that KGF also has the

KERATINOCYTE GROWTH FACTOR 501

capability of inducing autocrine growth factor signals in epithelial cells. In rat alveolar type II cells and keratinocytes, KGF increases the expression of EGF ligands such as transforming growth factor alpha (TGF-a), heparin-binding epidermal growth factor (HB-EGF), and amphiregulin. Effects of KGF on Lung Cells In Vitro

KGF has marked effects on type II cells in vitro. It causes more type II cell proliferation than any other growth factor tested to date. The mitogenic effect of KGF can be blocked by TGF-b. KGF also induces type II cell differentiation in vitro. This is likely related to proliferation and an increase in the cuboidal cytoarchitecture of type II cells, which appears to be critical for type II cell differentiation, but the effect is also seen on matrigel where the cells are already cuboidal. KGF increases type II cell differentiation, as measured by secretion of SP-A and SP-D much more effectively than other growth factors such as HGF, FGF-10, and HB-EGF. KGF signals through Akt and c-jun N-terminal kinase (JNK) to increase a variety of transcription factors. Our laboratory has focused on the ability of KGF to stimulate lipogenesis in type II cells. KGF increases SREBP-1c and C/EBP a and d, which in turn increases the expression of lipogenic enzymes specifically acetyl CoA carboxylase, ATP-citrate lyase, fatty acid synthase, stearoyl CoA desaturase, and glycerol3-P acyltransferase. KGF also increases SP-A, SP-B, SP-C, and SP-D expression through other pathways.

Receptors KGF binds to the KGFR in association with heparan sulfate proteoglycans, such as syndecan-1. The KGFR contains an extracellular domain composed of three immunoglobulin-like loops, a transmembrane segment, and an intracellular domain with tyrosine kinase activity. Its third IgG loop is encoded by two exons, IIIa and IIIb, producing a receptor with affinity for KGF, FGF-10, FGF-1, and FGF-3. The KGFR signals through several pathways, including ERK, JNK, Akt, and PAK 4. However, the precise signaling pathways for the divergent functions of KGF are not defined. Since the biologic effects of KGF take 24–28 h, numerous signaling pathways and a host of transcription factors and coactivators are likely involved. SU 5402 is an FGF receptor kinase inhibitor that can block the effect of KGF.

Properties of FGF-10 FGF-10 (KGF-2) is similar to KGF (FGF-7) but differs from it in some important respects. It also signals

through the KGFR (FGFR2-IIIb). For rat type II cells, the concentration of FGF-10 required for maximal thymidine incorporation is about 500 ng ml 1, whereas the concentration for maximal activity of KGF is 5–10 ng ml 1. FGF-10 binds heparin and presumably heparin sulfate proteoglycans more avidly than KGF. Heparin can stimulate mitogenic activity of FGF-10, whereas it can inhibit the mitogenic activity of KGF. FGF-10 binds to cells and extracellular matrix avidly and is not released easily into culture medium. FGF-10 likely has a very local effect, whereas some KGF may circulate. In the fetal lung, FGF-10 also causes more migration but less proliferation than KGF. FGF-10 is also being investigated as a therapeutic agent under the name repifermin.

Role of KGF in Respiratory Disease KGF has the potential to be very important in the treatment of acute lung injury. However, its potential role has not been validated in studies on humans. KGF protects rodent lungs from a variety of acute lung injuries, if it is administered before the injury. However, postinjury treatment regimens have not been reported. The apparent cytoprotection probably requires a time of 2–3 days and the stimulation of type II cell proliferation. KGF increases rapidly and dramatically in the dermis after cutaneous wound healing. However, the increase in KGF mRNA or protein levels in the rodent lung after injury has been difficult to demonstrate. We know relatively little about KGF levels in human lung diseases. It is also not clear if KGF-deficient animals are more susceptible to lung injury. There is much more to learn about the role of KGF and FGF-10 in human lung diseases. See also: Acute Respiratory Distress Syndrome. Angiogenesis, Angiogenic Growth Factors and Development Factors. Fibroblast Growth Factors. Stem Cells.

Further Reading (2004) Palifermin: AMJ 9701, KGF-Amgen, recombinant human keratinocyte growth factor, rHu-KGF. Drugs in R&D 5: 351– 354. auf demKeller U, Krampert M, Kumin A, Braun S, and Werner S (2004) Keratinocyte growth factor: effects on keratinocytes and mechanisms of action. European Journal of Cell Biology 83: 607–612. Bao S, Wang Y, Sweeney P, et al. (2005) Keratinocyte growth factor induces akt kinase activity and inhibits fas-mediated apoptosis in A549 lung epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 288: L36–L42. Danilenko DM (1999) Preclinical and early clinical development of keratinocyte growth factor, an epithelial-specific tissue growth factor. Toxicologic Pathology 27: 64–71.

502 KININS AND NEUROPEPTIDES / Bradykinin Deterding RR, Havill AM, Yano T, et al. (1997) Keratinocyte growth factor prevents bleomycin lung injury in rats. Proceedings of the Association of American Physician 109: 254–268. Finch PW and Rubin JS (2004) Keratinocyte growth factor/fibroblast growth factor 7, a homeostatic factor with therapeutic potential for epithelial protection and repair. Advances in Cancer Research 91: 69–136. Kaza AK, Kron IL, Leuwerke SM, Tribble CG, and Laubach VE (2002) Keratinocyte growth factor enhances post-pneumonectomy lung growth by alveolar proliferation. Circulation 106: I120–I124. Mason RJ, Pan T, Edeen KE, et al. (2003) Keratinocyte growth factor and the transcription factors C/EBP{alpha}, C/EBP{delta}, and SREBP-1c regulate fatty acid synthesis in alveolar type II cells. Journal of Clinical Investigation 112: 244–255. Morikawa O, Walker TA, Nielsen LD, et al. (2000) Effect of adenovector-mediated gene transfer of keratinocyte growth factor on the proliferation of alveolar type II cells in vitro and in vivo. American Journal of Respiratory Cell and Molecular Biology 23: 626–635. Panos RJ, Bak PM, Simonet WS, Rubin JS, and Smith LJ (1995) Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats. Journal of Clinical Investigation 96: 2026–2033. Panos RJ, Rubin JS, Aaronson SA, and Mason RJ (1993) Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II

cells in fibroblast-conditioned medium. Journal of Clinical Investigation 92: 969–977. Ulich TR, Yi ES, Longmuir K, et al. (1994) Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. Journal of Clinical Investigation 93: 1298–1306. Ware LB and Matthay MA (2002) Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair. American Journal of Physiology: Lung Cellular and Molecular Physiology 282: L924–L940. Werner S (1998) Keratinocyte growth factor: a unique player in epithelial repair processes. Cytokine Growth Factor Reviews 9: 153–165. Yano T, Deterding RR, Simonet WS, Shannon JM, and Mason RJ (1996) Keratinocyte growth factor reduces lung damage due to acid instillation in rats. American Journal of Respiratory Cell and Molecular Biology 15: 433–442. Yano T, Mason RJ, Pan T, et al. (2000) KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in the adult rat lung. American Journal of Physiology: Lung Cellular and Molecular Physiology 279: L1146–L1158. Yi ES, Williams ST, Lee H, et al. (1996) Keratinocyte growth factor ameliorates radiation- and bleomycin-induced lung injury and mortality. American Journal of Pathology 149: 1963–1970. Zhang F, Nielsen LD, Lucas JJ, and Mason RJ (2004) Transforming growth factor-beta antagonizes alveolar type II cell proliferation induced by keratinocyte growth factor. American Journal of Respiratory Cell and Molecular Biology 31: 679–686.

KININS AND NEUROPEPTIDES Contents

Bradykinin Neuropeptides and Neurotransmission Tachykinins Vasoactive Intestinal Peptide Other Important Neuropeptides

Bradykinin J Eddleston, S C Christiansen, and B L Zuraw, University of California – San Diego, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Bradykinin (BK) and Lys-BK are vasoactive peptides generated via the cleavage of kininogen by the action of kallikrein proteases. Both have a half-life of only seconds within the circulation, being rapidly hydrolyzed to biologically active des-Arg kinin derivatives. BK and Lys-BK cause the classical signs of inflammation – redness, local heat, swelling, and pain – whereas their des-Arg derivatives induce increased collagen synthesis, fibroblast proliferation, and cytokine release from macrophages. The effects of these peptides are mediated by two distinct G-coupled receptors. The kinin system has been implicated in

the pathophysiology of human airway inflammation. Inhalation of BK by asthmatics results in chest tightness, cough, and bronchoconstriction. Furthermore, increased levels of BK and Lys-BK, their derivatives, and tissue kallikrein are observed within the human airway following allergen provocation, with levels of BK correlating with symptom severity. Respiratory viral infections are also associated with increase levels of BK and Lys-BK within the airway. Research has demonstrated that BK is intricately involved in the pathogenesis of allergic airway inflammation and respiratory viral infections; as such, therapeutic methods for intervening in the activation of the kinin system may alleviate airway inflammation and symptoms of respiratory infections.

Introduction A substance in urine that was capable of causing prolonged hypotension when injected into animals

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was identified in the early 1900s and named kallidin (Lys-bradykinin). The enzyme required for its generation was called kallikrein (named after the Greek word for pancreas). In 1940, Rocha e Silva et al. discovered an enzyme in snake venom that generated a smooth muscle contracting factor in plasma that they labeled bradykinin (BK). The primary structure of BK, however, was not unraveled until the 1960s. A series of elegant pharmacologic studies defined two distinct kinin receptors whose agonists were BK and its desArg product (des-Arg-BK), respectively. Molecular analysis of the kallikrein–kinin system has confirmed the general organization described previously, whereas the diverse biological effects of kinins have been clarified by functional and genetic studies. Kinins have a range of beneficial actions, including effects on insulin sensitivity, skeletal muscle function, fluid balance, vascular tone, and cardioprotection. As is the case for other potent bioactive substances, however, kinins can mediate adverse effects on the host when present either inappropriately or in excessive concentrations. The control of kinin generation, degradation, and receptor interactions is therefore central to understanding the role of kinins in respiratory disease.

Structure, Production, and Regulation Kinins are positively charged vasoactive peptides generated by proteolytic cleavage of the b2-globulin kininogen. Two human kininogens, high-molecularweight kininogen (HMWK) and low-molecular-weight kininogen (LMWK), are synthesized via alternative splicing from a single gene and circulate as single-chain glycoproteins. The primary sequences of HMWK (115 kDa) and LMWK (68 kDa) are identical from the N terminus through the BK moiety (heavy chain) Exon 5′

1

but differ at the C terminus (light chain). The common heavy chain mediates cysteine protease inhibitory activity, whereas the light chain of HMWK (but not LMWK) mediates procoagulant activity. Kinin is generated following cleavage of kininogen by one of several serine proteases, termed kallikreins (Figure 1). Plasma kallikrein circulates in plasma as the zymogen prekallikrein primarily bound to the light chain of HMWK. During contact system activation, prekallikrein is cleaved into active plasma kallikrein, which then cleaves HMWK at two sites releasing BK. Plasma kallikrein is efficiently inhibited by C1 inhibitor and, to a lesser extent, by a2-macroglobulin. A separate and distinct family of tissue kallikreins is widely distributed in mammalian tissues. At least one of these tissue kallikreins (hK1) has kininogenase activity and releases Lys-BK (kallidin) from LMWK or HMWK. hK1 is synthesized as a preprokallikrein protein that is sequentially cleaved to generate active tissue kallikrein. Once activated, tissue kallikrein is relatively resistant to inhibition. BK and Lys-BK are rapidly hydrolyzed by peptidases called kininases. In blood, the half-life of kinin is estimated to be only a few seconds, and BK is essentially completely cleaved during one passage through the pulmonary circulation. As such, BK is active only close to the site of formation in a paracrine manner, acting as a local hormone (autocoid). The major kininases in the lung are carboxypeptidase N (kininase I), angiotensin-converting enzyme (kininase II), neutral endopeptidase (enkephalinase), and aminopeptidases. Of particular importance is the fact that carboxypeptidase processes BK and Lys-BK via the removal of the N-terminal arginine to produce the biologically active kinins des-Arg9-BK and Lysdes-Arg9-BK, respectively.

2 3 4 5 6 7 8 9

10

11 3′

Kininogen COOH

NH2 Kallikrein

380

389 Lysyl-Bradykinin (tissue kallikrein + LMWK )

Kinins

Bradykinin (plasma kallikrein + HMWK ) 1

2

3

4

5

6

7

8

9

Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

Kininases

Aminopeptidase M

Carboxypeptidase N Neutral endopeptidase (kininase I ) (enkephalinase) Angiotensin I-converting enzyme (kininase II )

Figure 1 Generation and metabolism of kinins. The kininogen gene is alternatively spliced to yield HMWK (exons 1–10) or LMWK (exons 1–9, 50 end of exon 10, and exon 11). Kininogen is cleaved by kallikrein yielding kinins, which are then metabolized by kininases.

504 KININS AND NEUROPEPTIDES / Bradykinin

Biological Function In the 1960s, Elloit and colleagues demonstrated that synthetic BK injected into human or animal tissue reproduced the four classical signs of inflammation: redness, local heat, swelling, and pain. The redness and local heat can be attributed to BK-induced local endothelium-dependent vasodilation, whereas the swelling is due to increased vascular permeability. BK also causes pain and triggers the autonomic nervous system as a result of activation of afferent neurons. In addition, BK causes the contraction of several types of smooth muscle, including human small bronchi and strips of human colon and urinary bladder. Furthermore, stimulation with BK can result in increased intracellular levels of arachidonic acid, eicosanoids, diacylglycerol (DAG), inositol-1,4,5triphosphate (IP3), cAMP, and Ca2 þ . BK has been shown to activate multiple phospholipases (PLs), including PLA2 (leading to arachidonic acid release and eicosanoid generation), PLC (leading to generation of DAG and IP3), and PLD. DAG can subsequently activate protein kinase C (PKC), whereas IP3 can release intracellular Ca2 þ stores. BK stimulation is associated with activation and membrane translocation of multiple PKC isoforms, including the Ca2 þ -independent PKCe and the atypical PKCz isoforms. It has also become clear that BK can contribute to inflammation by activating transcription factors such as nuclear factor kappa B and activation protein 1. BK and Lys-BK in general share the same spectrum of biologic activity. The des-Arg-derivatives of BK and Lys-BK, however, do not induce signs of acute inflammation or smooth muscle contraction. They do activate many of the same intracellular signaling pathways as BK, share some of the vascular effects of BK, and have also been found to mediate increased collagen synthesis, fibroblast proliferation, and cytokine release from macrophages (Figure 2).

Bradykinin Receptors Kinins exert their biological effects by stimulating seven-transmembrane domain G-protein-coupled receptors (GPCRs). Two distinct kinin receptors, termed B1 and B2 receptors (B1R and B2R, respectively), were characterized by Regoli and co-workers in the 1970s. Tissues that responded to des-Arg9-BK or Lys-des-Arg9-BK better than BK or Lys-BK were considered to have B1R, whereas tissues that responding to BK or Lys-BK better than the des-Arg derivatives were considered to have B2R. These predictions were subsequently confirmed by cloning of two distinct genes for the B1R and the B2R that map

SMC contraction, mucus secretion, neuronal activation, vascular effects, inflammation

B2 BK receptor

Plasma Kallikrein

BK

Lys-BK kininase I

des-Arg-BK

Tissue Kallikrein

Lys-des-Arg-BK

B1 BK receptor

Vascular effects, inflammation, cell proliferation Figure 2 Biological effect of kinins. BK and Lys-des-Arg-BK act through the B2R and transduce the classic kinin effects. Their des-Arg metabolites act through the B1R and transduce vascular effects as well as effects on inflammation and cell growth.

in close proximity to each other on human chromosome 14 but are only distantly related, with their amino acid sequence sharing 36% identity. The B1R gene has a single exon, whereas the B2R gene is composed of three exons separated by two introns, with the third exon encoding the receptor. In the noncoding region, several SNPs have been found that affect the transcriptional rate of the receptors. Coupling of B2R and B1R to hererotrimeric G proteins and the downstream signaling pathways that are activated appear to be tissue and cell specific. BK has also been shown to bind to the orphan GPCR, GPR100, although the significance of this is uncertain. B2R appear to be constitutively expressed and mediate most of the classic inflammatory action of BK, whereas B1R are generally absent from normal tissue but are rapidly induced following certain types of injury. In vitro, B1R numbers are induced in many tissues by a number of factors, the most potent of which is interleukin (IL)-1b. Upregulation of B1R expression has also been shown to occur in vivo in inflamed tissues isolated from human subjects. Following ligand stimulation, B2R show specific and rapid internalization and desensitization. B1R, however, display high basal activity even in the absence of ligand and do not show appreciable internalization or desensitization. Thus, the function of B1R during inflammatory insult may be to amplify the tissue effects of kinins.

Bradykinin in Respiratory Disease The pluripotent activities of kinin make it an attractive candidate for participating in the pathogenesis of respiratory tract disease. BK is a potent

KININS AND NEUROPEPTIDES / Bradykinin 505

bronchoconstrictor ex vivo, causing a biphasic response consisting of a brief contraction followed by sustained relaxation. Indeed, symptomatic and physiologic changes following BK challenge have suggested a key role for the kinin system in asthma and allergic rhinitis. Nasal insufflation of BK causes acute symptoms of nasal congestion, drainage, and throat irritation, and inhalation of BK in asthmatic but not normal subjects results in cough, chest tightness, and measurable decreases in flow rates indicative of bronchoconstriction. These effects appear to be mediated in large part by neural mechanisms with involvement of both vagal cholinergic and sensory C fibers. Other cardinal features of asthma induced by BK include mucus hypersection and airway edema. In subjects with perennial asthma, airway responsiveness to BK was found to correlate with the intensity of eosinophilia in bronchial biopsies, an accepted marker of asthma severity. The significance of these effects is supported by direct evidence of kinin generation in the airway during allergic inflammation and other respiratory diseases. Proud and colleagues first reported that BK and Lys-BK were generated in nasal secretions following allergen provocation, and they showed that the kinin levels correlated with reported symptoms and release of mast cell mediators. Elevated levels of both tissue kallikrein activity and kinins were found by Christiansen et al. in the bronchoalveolar lavage fluid of patients with active asthma or chronic bronchitis. Furthermore, local segmental challenge with relevant allergen in allergic asthmatics increased tissue kallikrein and kinin levels in conjunction with histamine release at both immediate and late-phase time points. Kinins are also generated during naturally occurring rhinitis. Autoradiographic and immunohistochemical studies have confirmed the expression of B2R on a variety of airway structural cells, including epithelial cells, fibroblasts, and smooth muscle cells. Although airway challenge with B1R agonists results in a conspicuous lack of acute effects, B1R have been shown to acquire an increasingly primary role during chronic inflammation in a variety of animal models. Indeed, allergic rhinitis subjects, but not normal subjects, were found to express B1R in nasal tissue, and allergen challenge further increased nasal expression of B1R mRNA. Nasal challenge with the B1R agonist Lys-des-Arg9-BK in subjects with mild allergic rhinitis activated the ERK MAPK in allergic rhinitis but not in normal subjects, suggesting that the B1R expressed in nasal tissue is functional and may participate in human airway inflammation. Subsequent animal studies have supported these human results. Murine tracheal explants display enhanced

contractile responses to des-Arg9-BK and BK following long-term culture with IL-4, a signature cytokine for allergic inflammation. This effect was mediated by upregulation of B1R. Furthermore, allergen-induced bronchial hyperresponsiveness was shown to parallel B1R upregulation in Brown Norway rats and was partially attenuated by the B1R antagonist des-Arg10-Hoe-140. Several lines of evidence support a role for kinins in the propagation of airway inflammation beyond their direct effects on permeability, mucus production, and smooth muscle contraction. Kinins activate inflammatory cells, thereby amplifying the release or production of other mediators of asthma, notably histamine, lipid-derived mediators, and neuropeptides. Treatment of moderate asthmatics for 4 weeks with an inhaled B2R antagonist (HOE-140) resulted in a gradual dose-dependent increase in FEV1, suggesting an anti-inflammatory effect resulting from the BR blockade. HOE-140 has also been shown to abrogate the allergen-induced increases in nasal histamine hyperresponsiveness and the appearance of eosinophils, ECP, kinin, and IL-8 in lavage fluid in allergic rhinitis patients. Conversely, nasal BK challenge selectively upregulated in vivo expression of the chemokine receptors CXCR1 and CXCR2 in allergic rhinitis subjects. Kinins may also exert effects on cell growth, and treatment with B2R antagonists has been shown to result in the killing of small cell lung carcinoma cells in vitro. The kinin system may also have an important role in infectious diseases affecting the respiratory tract. Objective signs of respiratory disease in ferrets inoculated with influenza virus corresponded with the appearance of kinins in the nasal lavages, and airway hyperresponsiveness following parainfluenza3 infection in guinea pig correlated with generation of BK. Furthermore, experimental rhinovirus infections in human subjects resulted in the generation of both Lys-BK and BK that directly correlated with symptom severity. Other studies have suggested a relationship between kinin generation and activation of the innate immune response. Exposure of mouse trachea to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide or the TLR-3 agonist poly I:C increased B1R expression and enhanced sensitivity of the tissue to both B1R and B2R ligands, suggesting that bacterial and viral infections of the airway could lead to increased sensitization of the airways to kinins. Molecular definition of the kinin system has led to the development of animals with targeted deletions or overexpression of kinin system components as well as improved tools to assess the role of the kinin system in human respiratory diseases. The availability of potent and specific BK receptor antagonists

506 KININS AND NEUROPEPTIDES / Neuropeptides and Neurotransmission

provides an opportunity to translate these advances into potential therapeutic interventions. See also: Capsaicin. Kinins and Neuropeptides: Neuropeptides and Neurotransmission; Tachykinins; Vasoactive Intestinal Peptide; Other Important Neuropeptides. Proteinase Inhibitors: Cystatins.

Further Reading Bryborn M, Adner M, and Cardell LO (2004) Interleukin-4 increases murine airway response to kinins, via up-regulation of bradykinin B1-receptors and altered signalling along mitogenactivated protein kinase pathways. Clinical and Experimental Allergy 34: 1291–1298. Casalino-Matsuda SM, Monzon ME, Conner GE, Salathe M, and Forteza RM (2004) Role of hyaluronan and reactive oxygen species in tissue kallikrein-mediated epidermal growth factor receptor activation in human airways. Journal of Biological Chemistry 279: 21606–21616. Christiansen SC, Eddleston J, Woessner KM, et al. (2002) Upregulation of functional kinin B1 receptors in allergic airway inflammation. Journal of Immunology 169: 2054–2060. Christiansen SC, Proud D, and Cochrane CG (1987) Detection of tissue kallikrein in the bronchoalveolar lavage fluid of asthmatic subjects. Journal of Clinical Investigation 79: 188–197. Eddleston J, Christiansen SC, Jenkins GR, Koziol JA, and Zuraw BL (2003) Bradykinin increases the in vivo expression of the CXC chemokine receptors CXCR1 and CXCR2 in patients with allergic rhinitis. Journal of Allergy and Clinical Immunology 111: 106–112. Fuller RW, Dixon CM, Cuss FM, and Barnes PJ (1987) Bradykinin-induced bronchoconstriction in humans. Mode of action. American Review of Respiratory Disease 135: 176–180. Huang TJ, Haddad EB, Fox AJ, et al. (1999) Contribution of bradykinin B(1) and B(2) receptors in allergen-induced bronchial hyperresponsiveness. American Journal of Respiratory and Critical Care Medicine 160: 1717–1723. Leeb-Lundberg LM (2004) Bradykinin specificity and signaling at GPR100 and B2 kinin receptors. British Journal of Pharmacology 143: 931–932. Leeb-Lundberg LM, Marceau F, Mu¨ller-Esterl W, Pettibone DJ, and Zuraw BL (2005) Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacologic Reviews 57: 27–77. Marceau F and Regoli D (2004) Bradykinin receptor ligands: therapeutic perspectives. Nature Reviews: Drug Discovery 10: 845–852. Naclerio RM, Proud D, Lichtenstein LM, et al. (1988) Kinins are generated during experimental rhinovirus colds. Journal of Infectious Disease 157: 133–142. Pan ZK, Zuraw BL, Lung CC, et al. (1996) Bradykinin stimulates NF-kappaB activation and interleukin 1beta gene expression in cultured human fibroblasts. Journal of Clinical Investigation 98: 2042–2049. Proud D, Togias A, Naclerio RM, et al. (1983) Kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen. Journal of Clinical Investigation 72: 1678–1685. Roisman GL, Lacronique JG, Desmazes-Dufeu N, et al. (1996) Airway responsiveness to bradykinin is related to eosinophilic inflammation in asthma. American Journal of Respiratory and Critical Care Medicine 153: 381–390.

Turner P, Dear J, Scadding G, and Foreman JC (2001) Role of kinins in seasonal allergic rhinitis: icatibant, a bradykinin B2 receptor antagonist, abolishes the hyperresponsiveness and nasal eosinophilia induced by antigen. Journal of Allergy and Clinical Immunology 107: 105–113.

Neuropeptides and Neurotransmission N R Prabhakar and G K Kumar, Case Western Reserve University, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Neurotransmitters influence breathing by acting either on central neurons that regulate breathing or on the peripheral nerves that innervate lungs, airways, and O2-sensing chemoreceptors. In addition to amino acids and biogenic amines, neuropeptides emerged as another class of molecules that profoundly influence neuronal activities. Substance P (SP) is one such neuropeptide that profoundly impacts breathing via its actions on central and peripheral nervous systems. SP belongs to a group of structurally related peptides called tachykinins. It participates in the generation of respiratory rhythm by acting on pre-Botzinger neurons in the medulla oblongata and phrenic motoneurons in cervical cord that innervate the diaphragm. Selective destruction of neurons expressing SP-receptors leads to apneas, irregular breathing, and hypoxemia. SP also contributes to the integration of sensory inputs from lungs, airways, and peripheral chemoreceptors by acting on the neurons in the nucleus of the tractus solitarious. In the peripheral nervous system, SP acting as a sensory transmitter participates in the hypoxic sensing at the carotid body. In the lungs and airways, SP stimulates ‘irritant’ receptors innervated by the vagus nerves and participates in the generation of cough reflex. Also, SP is linked to asthma and hyperreactivity. In response to allergens, SP-like peptides are released from the nerve fibers innervating airways, and SP is a potent constrictor of central and peripheral airways. It also stimulates airway secretions. SP-receptor antagonists are emerging as a new class of therapeutic interventions to alleviate asthma and chronic cough.

Introduction Breathing is a rhythmic motor act that requires generation of respiratory rhythm by brainstem neurons, which is continuously under the control of information from lungs, airways, and chemoreceptors that senses changes in arterial blood gases via their respective sensory receptors. Neurotransmitters profoundly influence breathing by acting either centrally or peripherally on sensory receptors and/or airways. For instance, amino acid transmitters (e.g., glutamate and g-amino butyric acid) modulate breathing by acting on central neurons. Biogenic amines such as acetylcholine (ACh), on the other hand, exert their influence on breathing by acting peripherally on the

KININS AND NEUROPEPTIDES / Neuropeptides and Neurotransmission 507

Tachykinins SP

Effectors

Pre-Botzinger neurons

Nucleus of the tractus solitarious

Effects

Central

Generation of respiratory rhythm

Integration of afferent inputs (lungs, airways, chemoreceptors)

Peripheral

Carotid body

Hypoxic sensing

Irritant receptors

Airways

Cough

Airway hyperreactivity

Figure 1 Schematic representation of the effects of substance P (SP), a tachykinin peptide, on breathing. The figure depicts the central and peripheral targets of SP and effect on breathing.

airways more so than their actions on the central nervous system. In recent years, neuropeptides have emerged as another class of molecules that profoundly influence neuronal activities. Substance P (SP) is one such neuropeptide that modulates breathing by acting centrally as well as peripherally (Figure 1). SP belongs to a group of structurally related peptides called tachykinins. Other members of the tachykinin family include neurokinin A, neuropeptide K , neuropeptide g, and neurokinin B. Amongst the different tachykinins, SP is the most extensively studied with regard to its action on breathing.

Central Effects of SP SP and Generation of Respiratory Rhythm

Neural circuitry that is critical for generating the respiratory rhythm (at least in neonates) has been localized to the pre-Botzinger complex, a region in the medulla oblongata located ventrolateral to the nucleus ambiguous. Microinjections of SP into the pre-Botzinger area stimulate breathing primarily by increasing respiratory frequency in newborn rats. Biological actions of tachykinins are mediated by G-protein-coupled receptors that are classified as neurokinin-1, -2, and -3 receptors (NK-1, -2, and -3). Many of the neurons in this region express NK-1 receptors. More importantly, selective destruction of these neurons leads to irregular breathing, frequent occurrence of apneas, and hypoxemia in awake rats. However, in adult animals, generation of respiratory rhythm is more complex and involves other areas of the brain that make extensive synaptic contacts with

the pre-Botzinger neurons. In adult animals, in addition to its effects on pre-Botzinger neurons, SP modulates respiratory rhythm by directly stimulating phrenic motoneurons that innervate the diaphragm. SP and Integration of Respiratory Afferent Information at Brainstem Neurons

Respiratory responses to activation of pulmonary afferents innervated by vagal sensory fibers and peripheral chemoreceptors require integration of afferent information by brainstem neurons, especially those localized in the nucleus of the tractus solitarius (nTS). Neurons in the nTS translate afferent information into appropriate respiratory motor behavior. Information from lungs (lung volume, intrapulmonary pressure) during each inspiration is conveyed to nTS neurons via sensory stretch receptors innervated by vagus nerves (pulmonary stretch receptors, PSR). After reaching a certain threshold, pulmonary stretch receptors turn off inspiration reflexly involving brainstem neurons and expiration ensues passively. This phenomenon, known as ‘inspiratory off-switch’ is critical for determining the magnitude of the tidal volume and duration of inspiration and, as a consequence, respiratory rate. SP alters the threshold of the ‘inspiratory off-switch’ reflex primarily by affecting nTS neurons. Local application of SP produces longlasting activation of nTS neurons, and stimulates breathing by increasing both tidal volume and respiratory rate. SP antagonists prevent these responses. In addition to its effects on inspiratory off-switch threshold, SP is also involved in integration of sensory information from peripheral chemoreceptors

508 KININS AND NEUROPEPTIDES / Neuropeptides and Neurotransmission

that sense hypoxia in arterial blood. Hypoxia facilitates SP release in the nTS region, and denervation of peripheral chemoreceptors prevents this effect.

Peripheral Effects of SP SP and Peripheral Chemoreceptors: Implications for Hypoxic Ventilatory Response

Compensatory hyperventilation that ensues during hypoxemia is reflex in nature and mediated entirely by activation of peripheral chemoreceptors, especially the carotid bodies. Carotid bodies are composed of two cell types: type I and type II. Type I cells (also called glomus cells) are of neural crest origin and express a variety of neurotransmitters. Type II cells resemble glial cells and are supporting cells. Type I cells are critical for sensing hypoxia. During hypoxia type I cells release neurotransmitter(s), which by acting on the nearby afferent nerve-ending, produces an increase in sensory activity that is eventually conveyed to brainstem neurons to evoke stimulation of breathing. SP satisfies several of the criteria for its role as a transmitter in hypoxia-induced sensory activation of the carotid body. SP occurs at femtomole levels in the carotid body, and is localized to type I cells of the carotid body in several species including humans, cats, and rabbits. Carboxypeptidase E, an enzyme essential for SP biosynthesis, is expressed in the carotid body. Hypoxia releases SP from the carotid bodies and requires activation of voltage-dependent calcium channels. SP augments carotid body sensory activity in several species both in vivo and ex vivo. Studies on ex vivo preparations suggest that the stimulatory effects of SP are due to its direct action on the carotid body rather than secondary to its systemic effects on the cardiovascular system. The stimulatory effects of SP on carotid body sensory activity requires the C-terminus region of the peptide to be intact, whereas the N-terminal fragment of the peptide was without any effect. Other tachykinin peptides including NKA, which has a similar C-terminal sequence to SP, also augment the sensory response of the carotid body. Autoradiographic analysis revealed SP-binding sites in the cat carotid body, and blockade of NK-1 receptors prevented SP-induced carotid body stimulation. In addition to its receptor-mediated actions, SP may directly influence mitochondrial metabolism by acting as a protonophore as shown in mitochondrial preparations in vitro. Systemic administration of phosphoramidon, a selective and potent inhibitor of neutral endopeptidase, an enzyme that

terminates the actions of SP, potentiates sensory response of the carotid body to hypoxia. Blockade of NK-1 receptors prevents this effect. There is considerable evidence that SP profoundly influences hypoxic ventilatory responses in humans and experimental animals. In conscious human subjects, systemic administration of SP potentiates hypoxic but not hypercapnic ventilatory response. In experimental animals (rats), NK-1 receptor antagonist attenuates hypoxic ventilatory response. In rats, depletion of SP, by neonatal exposure to capsaicin, markedly reduces the magnitude of the hypoxic ventilatory response, by attenuating the respiratory frequency. Furthermore, hypoxic but not the hypercapnic ventilatory responses are selectively blunted in genetically engineered mice with targeted deletion of the genes encoding pre-pro-tachykinin-1, the precursor for SP.

SP and Pulmonary and Airway Sensory Receptors: Implications in Generation of Cough

Cough is critical for maintaining the defense of airways and in clearing mucus and other foreign particles. It is a complex reflex and involves (1) sensory receptors, (2) the afferent pathway, (3) the cough center, (4) the efferent pathway, and (5) the effectors. ‘Irritant’ receptors (also called rapidly-adapting type of mechanosensory receptors) in airways innervated by vagus nerves constitute the sensory afferents and the afferent pathway. The cough center is located in the brainstem, and efferent signals are transmitted to glottis, diaphragm, intercostals, and abdominal muscles via cranial and spinal motor pathways. It has been proposed that chemical stimuli that induce cough do so by directly stimulating ‘irritant’ receptors, and also secondarily by stimulating ‘C’ fibers (slowly-conducting mechanosensory receptors of pulmonary origin), which release tachykinins. Once released, tachykinins act on irritant receptors to promote cough further (positive feedback). SP is present in vagal sensory fibers innervating the bronchopulmonary system. SP stimulates ‘irritant’ receptors in rabbits and cats and SP-antagonists abolish these effects. Further support for the involvement of tachykinins in generation of cough has come from a number of studies demonstrating that neurokinin receptor antagonists prevent cough in several species including humans. Furthermore, cough can be elicited by exogenous administration of SP or by increasing the endogenous level of SP by preventing its degradation by neutral endopeptidase (NEP). Thus, interventions with selective neurokinin receptors might be of therapeutic value in alleviating cough.

KININS AND NEUROPEPTIDES / Tachykinins 509 SP and Airway Hyperreactivity: Implications in Asthma

Asthma is a chronic airway inflammatory disorder and is characterized by increased airway responsiveness to various physiologic and environmental stimuli. Airway hyperreactivity has been attributed in part to the transmitters released from the nerve fibers innervating airways. There is also considerable evidence for the role of tachykinins, especially SP in airway hyperreactivity. SP-like peptides are present in the nerve fibers innervating airways. Antigens, ozone, and inflammatory mediators release SP from airway sensory nerves. Challenging guinea pigs with antigen markedly potentiates action-potential-driven tachykinin release from afferent fibers in the airways. Anatomical and electrophysiological studies indicate that tachykinin-containing sensory fibers directly innervate the local parasympathetic ganglion neurons in the airways. SP is a potent bronchoconstrictor and affects both the central and peripheral airway tone, via NK-1 receptors. Atropine and hexamethonium reduce SP-induced bronchoconstriction. Allergic reaction increases vagal sensory and parasympathetic activity that involves cross-talk between tachykinins and the cholinergic system. Thus, tachykinins control airway reactivity by directly affecting airway smooth muscle via NK-1 receptors as well as via their interaction with cholinergic neurons innervating airways. Eosinophils, macrophages, lymphocytes, and dendritic cells (inflammatory cells) that are resident to airways also produce SP. Immune stimuli boost the production and secretion of SP. Apart from its action on bronchomotor tone, SP also promotes airway secretions and bronchial circulation (vasodilatation and microvascular leakage). Bronchomotor actions of SP are terminated by the enzymes NEP and angiotensin-converting enzyme (ACE). Lung contains large amounts of ACE. Therapeutic interventions with ACE are commonly associated with increased incidence of cough, secondary to increased airway sensitivity. The side effects of ACE inhibitors on breathing may in part be due to elevated levels of endogenous SP in lungs and airways. In view of their potent effects on the airways, tachykinins have been put forward as possible mediators of asthma, and neurokinin receptor antagonists are emerging as a potential class of anti-asthmatic medications.

Symptoms of Respiratory Disease: Cough and Other Symptoms.

Further Reading Barnes PJ (1987) Neuropeptides in human airways: function and clinical implications. American Review of Respiratory Diseases 136: S77–S83. Fox AJ (1996) Modulation of cough and airway sensory fibers. Pulmonary Pharmacology 9: 335–342. Hokfelt T, Pernow B, and Wahren J (2001) Substance P: a pioneer amongst neuropeptides. Journal of Internal Medicine 249: 27–40. Joos GF, Germonpre PR, and Paulwels RA (2000) Role of tachykinins in asthma. Allergy 55: 321–337. Kumar GK and Prabhakar NR (2003) Tachykinins in the control of breathing by hypoxia. Pre- and Post-genomic era. Respiration Physiology and Neurobiology 135: 145–154. Prabhakar NR and Chearniack NS (1989) Importance of tachykinin peptides in hypoxic ventilatory drive. In: Lahiri S, Forster RE, Davies RO, and Pack AI (eds.) Chemoreceptors and Reflexes in Breathing: Cellular and Molecular Aspects, pp. 99–112. New York: Oxford University Press. Widdicombe JG (1995) Neurophysiology of the cough reflex. European Respiratory Journal 8: 1193–1202.

Tachykinins G F Joos and K De Swert, Ghent University Hospital, Ghent, Belgium & 2006 Elsevier Ltd. All rights reserved.

Abstract The four major mammalian tachykinins are substance P, neurokinin A, neurokinin B, and hemokinin 1. They are derived from three preprotachykinin genes, termed TAC1, TAC3, and TAC4. Tachykinins are present in human airways, both in sensory nerves and in non-neuronal cells, including airway epithelium, airway smooth muscle, and immune cells such as eosinophils, monocytes, macrophages, lymphocytes, and dendritic cells. Tachykinins can be recovered from the airways after inhalation of ozone, cigarette smoke, or allergen. They interact in the airways with tachykinin NK1, NK2, and NK3 receptors to cause bronchoconstriction, plasma protein extravasation, and mucus secretion and to attract and activate immune cells. In preclinical studies substance P and neurokinin A have been implicated in the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD), including airway inflammation and bronchial hyperresponsiveness induced by allergen or cigarette smoke. Tachykinin receptor antagonists are potential new drugs for the treatment of asthma and/or COPD.

Acknowledgment

Introduction

This work was supported by grants from National Institutes of Health, HL-25830.

The tachykinin peptide family represents one of the largest peptide families described in the animal organism. They have been isolated from invertebrate, protochordate, and vertebrate tissues. The tachykinins substance P and neurokinins A and B have

See also: Asthma: Overview. Hypoxia and Hypoxemia. Kinins and Neuropeptides: Tachykinins.

510 KININS AND NEUROPEPTIDES / Tachykinins

previously been considered as a group of neuropeptides because of their widespread distribution in the central and the peripheral nervous system. Tachykinins are released from sensory nerves by a variety of stimuli such as capsaicin, electrical nerve stimulation, low pH, ether, formalin, toluene diisocyanate, histamine, bradykinin, methacholine, prostaglandins, leukotrienes, and cigarette smoke. In addition, tachykinins are present in a variety of non-neuronal cells, including immune cells.

Structure Tachykinins, defined as peptides having the characteristic C-terminal pentapeptide F-X-G-L-M-NH2, are identified as ‘aromatic’ when X is an aromatic amino acid residue (F or Y) or ‘aliphatic’ when X is an aliphatic amino acid residue (V or I). All tachykinins are amidated at their C-terminus and this is crucial for their biological activity. The four major mammalian tachykinins are substance P, neurokinin A, neurokinin B, and hemokinin 1. Neurokinin A has also two N-terminally extended forms, neuropeptide g and neuropeptide k. Mammalian tachykinin peptide sequences are species invariant except for hemokinin 1. Human hemokinin 1 has also two elongated forms, endokinin A (47 amino acids) and endokinin B (41 amino acids) (Table 1).

Synthesis, Localization, and Degradation Synthesis

Mammalian tachykinins are derived from three preprotachykinin genes which according to the Human Genome Organization (HUGO) Gene Nomenclature Committee are termed TAC1, TAC3, and TAC4 (Figure 1). The TAC1 gene (previously known as the

Table 1 Amino acid sequences of mammalian tachykinins Peptide

Amino acid sequence

Substance P Neurokinin A Neuropeptide g

R-P-K-P-Q-Q-F-F-G-L-M-NH2 H-K-T-D-S-F-V-G-L-M-NH2 D-A-G-H-G-Q-I-S-H-L-R-L-DS-F-V-G-L-M-NH2 D-A-D-S-S-I-E-L-Q-V-A-L-L-KA-L-Y-G-H-G-Q-I-S-H-K-RH-G-Q-I-S-H-L-R-L-D-S-FV-G-L-M-NH2 D-M-H-D-F-F-V-G-L-M-NH2 (R)-S-R-T-R-Q-F-Y-G-L-MNH2 T-G-K-A-S-Q-F-F-G-L-M-NH2 R-H-R-T-P-M-F-Y-G-L-M-NH2

Neuropeptide k

Neurokinin B Hemokinin 1 m (mouse) Hemokinin 1 h (human) Chromosome 14 tachykininlike peptide 1 (C14TKL-1)

PPT-I or PPT-A gene) is coding for substance P, neurokinin A, and its extended forms. The TAC3 gene (also known as PPT-II or PPT-B) encodes the sequence for neurokinin B. Hemokinin 1 is coded by the TAC4 gene (or PPT-C). The precursor RNA of the TAC1 gene is alternatively spliced to yield four different mRNAs, a-, b-, g-, and d-TAC1 mRNA. All four mRNAs can generate substance P. Posttranslational processing of the b-TAC1 mRNA product can yield neurokinin A or neuropeptide k, while the g-form can generate neurokinin A and neuropeptide g. Usage of two differential promoters and alternative splicing of the TAC3 DNA transcript yields two mRNAs coding for neurokinin B (a- and b-TAC3). In contrast to the mouse and rat TAC4 gene which predicts one homologous transcript, the human ortholog encodes a different undecapeptide (Table 1) and has also the potential to generate a truncated form of the hemokinin 1 (hemokinin 1(4-11)). It should be noted that endokinin A and B are elongated forms of the human hemokinin 1. Translation of the mature mRNA yields a large polypeptide. The preprotachykinin consists of a signal peptide, one or several copies of a neuropeptide, and one or more spacer parts. The signal peptide is cleaved off after polypeptide synthesis leading to the propeptide. Tachykinins are liberated from their propeptides through proteolytic cleavage at specific sites of single basic residues (R, L) or at two adjacent basic residues, carried out by six groups of enzymes called convertases. The C-terminal methionine residues of the preprotachykinin precursors are all followed by glycine that donates its amine group thereby generating the C-terminal amidation. Localization

Tachykinins (substance P, neurokinin A, and neurokinin B) have previously been considered as a group of neuropeptides because of their widespread distribution in the central and the peripheral nervous system (capsaicin-sensitive primary afferent neurons and capsaicin-insensitive intrinsic neurons). This terminology is no longer held since their presence in a variety of non-neuronal structures has been proved repeatedly. Furthermore, mRNA expression studies suggest that hemokinin 1 has a unique distribution outside neuronal tissues. To date, this interpretation is altered to a more abundant expression outside neuronal tissue as low levels are detected in brain tissue. The alternative splicing of the TAC1 gene (Figure 1) affects the regional distribution of substance P and neurokinin A. Substance P can be expressed alone (translation of the a- and the d-form) while neurokinin A expression is always

TAC1 gene 5′

1

2

SP

4

TAC4 gene

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NKA

7

5′

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1′′

1′

1

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4

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6

7

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1

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Transcription and RNA splicing

1

2 SP 4

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7 AAA

1

2 SP

1

2 SP 4 NKA

4

5

NKA

1

7 AAA

2

7 AAA


-TAC1 mRNA

1

2 SP 5

3

4

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6

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1

-TAC3 mRNA

-TAC1 mRNA

-TAC1 mRNA

7 AAA

1′′ 1′

-TAC1 mRNA

1

2

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7 AAA

-TAC3 mRNA

2

3

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5 AAA

-TAC4 mRNA

1

2

3

5 AAA


-TAC4 mRNA

1

4

2

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-TAC4 mRNA

1

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-TAC4 mRNA

Translation and posttranslational processing

Substance P

Substance P Neurokinin A Neuropeptide 

Substance P Neurokinin A Neuropeptide


Substance P

Neurokinin B

Neurokinin B

hHemokinin 1 Endokinin A (Endokinin C)

hHemokinin 1 Endokinin B

hHemokinin 1 Endokinin B

hHemokinin 1 Endokinin B (Endokinin D)

Figure 1 Tachykinin peptides, mRNAs, and genes. Mammalian tachykinins are encoded by three different genes (TAC1, TAC3, and TAC4). TAC1 yields four different mRNA splice variants, which generate substance P, neurokinin A, and the elongated forms of neurokinin A. The TAC3 gene encodes for neurokinin B. The TAC4 gene is not species invariant. In humans, the gene yields also four splice variants which are finally translated into endokinin A, B, C, and D.

512 KININS AND NEUROPEPTIDES / Tachykinins Table 2 Tissue distribution of mammalian tachykinins Peptide

MRNA

Main sources

Substance P

a-, b-, g-, d-TAC1

Neurokinin A

b-, g-TAC1

Neurokinin B

TAC3

Hemokinin 1 m

mTAC4

CNS, primary afferent neurons, enteric neurons, immunocytes, inflammatory cells, endothelium, epithelium, Leydig cells, enterochromaffin cells, fibroblasts, smooth muscle, female reproductive tracta CNS, primary afferent neurons, enteric neurons, immunocytes, inflammatory cells, female reproductive tracta CNS, spinal chord, syncytiotrophoblasts of human placenta, lunga Lymphoid B and myeloid lineage cells of bone marrow,a T cell precursors of the thymus, uterus, blastocyst-stage embryos, ovary, testis, spleen, stomach, skin, lactating breast, heart, kidney, liver, lung, adrenal tissue, brain Heart,a skeletal muscle, skin, thyroid, kidney, liver, lung, stomach, testis, placenta, prostate Adrenal gland,a fetal liver, spleen Adrenal gland,a placenta, heart, liver, bone marrow, prostate, testis Liver,a kidney, prostate, bronchus, CNS

Hemokinin 1 h

hTAC4

Endokinin A

a-hTAC4

Endokinin B

b-, g-, d-hTAC4

C14TKL-1

?

a Only mRNA expression evaluated. CNS, central nervous system.

accompanied by substance P expression (translation of the b- and the g-form). The most abundant mRNAs are the b- and the g-form. This means that in many occasions, substance P and neurokinin A are co-synthesized and co-released. The tissue distribution of mammalian tachykinins is reported in Table 2. In the airways, a distinct subpopulation of primary afferent nerves that are characterized by their sensitivity to capsaicin are considered as the principal source of substance P and neurokinin A. Although they can also be expressed in capsaicin-resistant neurons, capsaicin pretreatment of experimental animals causes an almost total depletion of substance P and neurokinin A immunoreactivity. Radioimmunoassay and immunohistochemistry demonstrated substance P and neurokinin A positive nerves beneath and within the epithelium, around blood vessels and submucosal glands, and within the bronchial smooth

muscle layer. In guinea pigs, airway sensory nerves containing tachykinins are easily demonstrated, but in human airways, tachykinergic innervation is sparse. In the bronchial tree, tachykinergic sensory innervation originates from the (right) vagus nerve, but in the lung, the fibers are both from vagal parasympathetic and thoracic spinal origin. Non-neuronal sources of tachykinins in the airways are: airway epithelium, airway smooth muscle cells and immune cells such as eosinophils, monocytes and macrophages, lymphocytes, and dendritic cells. Immunoreactivity for neurokinin B has not been found in the airways yet. Transcripts of the mouse and the human TAC4 gene in lung tissue have been reported and these may result in the formation of the hemokinin 1. Degradation

The physiological actions of neuropeptides are normally terminated by extracellular metabolism. As far as the metabolism of substance P and neurokinin A is concerned, much attention has been paid to the role of the neutral endopeptidase 24.11 (also called NEP, enkephalinase, CALLA, CD10, or E.C.3.4.24.11) and to the role of peptidyl dipeptidase-15.1, better known as angiotensin-converting enzyme (ACE, kininase II, or E.C.3.4.15.1). A series of potent and relative selective inhibitors of NEP are available, such as the thiol inhibitors (e.g., thiorphan), and phosphorus-containing inhibitors (e.g., phosphoramidon). In guinea pigs, both NEP and ACE participate in the metabolism of substance P when administered intravascularly, while aerosolized tachykinins are degraded by NEP only. NEP mRNA and NEP-immunoreactive material has been identified in human airways. NEP is present in the basal cells of the epithelium, alveolar cells type II, submucosal glands, neutrophils, airway smooth muscle, postcapillary venules, and nerves.

Biological Functions Tachykinins elicit a broad spectrum of biological actions in the cardiovascular, gastrointestinal, urogenital, immune, and nervous system. These may vary in different species and even in the various strains of single species. This suggests the concept of a general, important functional significance of these peptides. However, mice with a disrupted preprotachykinin I gene (TAC1 gene, encoding for substance P, neurokinin A, and its extended forms) or a disrupted tachykinin NK1 or tachykinin NK3 receptor gene (the TACR1 and TACR3 gene) are in good health conditions and fertile, which demonstrates that the

KININS AND NEUROPEPTIDES / Tachykinins 513

tachykinins substance P and neurokinin A and the tachykinin NK1 and NK3 receptor are not essential for life and health, at least in mice. The adequate stimuli for tachykinin release from the sensory nerves in the airways are of chemical nature, especially those chemicals that are produced during inflammation and tissue damage. Airway Smooth Muscle Tone and Airway Responsiveness

The bronchoconstrictor effect of exogenous substance P was first reported in 1977, in guinea pigs and cats. Since then, substance P was shown to cause a dosedependent bronchoconstriction in various animal species, in vitro as well in vivo. Exogenously administered tachykinins also contract human airways, in vitro and in vivo. In vitro, substance P contracts human bronchi and bronchioli, but is less potent than histamine or acetylcholine. Neurokinin A however is a more potent constrictor, being, on a molar base, two to three orders of magnitude more potent than histamine or acetylcholine. Neurokinin B does not exert a contractile activity on human airways. The role of tachykinins in the regulation of airway function has been under even more intense investigation, since the demonstration that the atropine-resistant contraction (about 60% of the contraction induced by electrical field stimulation) in guinea pig airways was ascribable to tachykinins released from sensory nerves. Such a noncholinergic bronchoconstriction has, however, not been consistently demonstrated in human airways. Cough

An afferent (sensory) and an efferent arm that synapses in the brainstem areas, including the nucleus tractus solitarius, make up the reflex arch that mediates the cough reflex. Mediators and mechanisms of the sensory arm of the reflex pathway are not completely understood. Pharmacological evidence exists for an involvement of substance P. The role of tachykinins in the regulation of the cough response appears to be complex since antagonists for the three tachykinin receptors have antitussive activity in preclinical models. In guinea pigs, substance P locally applied into the airways has been shown to produce or potentiate cough produced by other stimuli. These findings support a role for substance P at a peripheral site of action. Central sites of action have also been reported. Secretion of Mucus, Water, and Electrolytes

Relatively strong evidence exists that tachykinins released from sensory nerve fibers are physiological

(and pathological) regulators of secretion in the upper and lower airways. A local release of tachykinins in the nasal mucosa plays a role in the defensive response to irritants. Intranasally administered capsaicin induces sneezing, pain, and nasal secretion in both experimental animals and humans. Similarly, local tachykinin administration evokes secretion. In the lower tracheobronchial tree, seromucous glands and goblet cells produce mucus. Both sources are under neural control. Depending upon species and airway level, innervation comprises parasympathetic, sympathetic and ‘sensory-efferent’ pathways. The transmitters of the latter pathway are substance P and neurokinin A, which act via tachykinin NK1 receptors. An involvement of tachykinin NK3 receptors has been suggested. Mucus secretion is closely coupled to liquid secretion to ensure optimal ciliary transport and presence of adequate concentrations of antibacterial substances on the airway surface. Substance P has also been shown to be involved in this process. Alveolar Epithelial and Microvascular Permeability

The involvement of sensory nerves in producing increased vascular permeability and plasma extravasation was first described in skin and conjunctiva and termed neurogenic inflammation. Neurogenic inflammation has also been described in the airways. Substance P, released from the sensory nerves, mediates the neurogenic inflammation in the airways. Plasma extravasation, induced by cold air, hypertonic saline, or isocapnic hyperpnea, depends on stimulation of sensory fibers and is inhibited by tachykinin receptor antagonists. Once plasma extravasation occurs, leukocytes initiate a process that results in slowing down their velocity, rolling on and adhering to the venular endothelium. The involvement of the tachykinin NK1 receptor in all these phenomena has been demonstrated by the use of specific antagonists. Tachykinin NK2 receptors also mediate part of the neurogenic plasma extravasation in the secondary bronchi and intraparenchymal airways of the guinea pig. Direct evidence for neurogenic plasma extravasation in human airways could not be demonstrated for long time due to difficulties in the assessment. However, microvascular leakage can now be measured in human airways with a ‘dual induction’ model. First, substance P is inhaled and then sputum is induced by inhalation of hypertonic saline solution. With this method, it was demonstrated that inhalation of substance P induced significant increases in the levels of a2-macroglobulin, ceruloplasmin, and albumin in induced sputum.

514 KININS AND NEUROPEPTIDES / Tachykinins

tachykinin NK1, NK2, and NK3 receptors. These receptors belong to the rhodopsin-like G-proteincoupled receptor family. This group of proteins share the same structural motif: a bundle of seven hydrophobic transmembrane domains with three extracellular loops, three intracellular loops, an extracellular N-terminus, and a cytoplasmic C-terminus. The human tachykinin NK1 and NK2 receptors are proteins of 407 and 398 amino acids respectively. The human tachykinin NK3 receptor is N-terminally extended and has 465 residues. The genes encoding the three mammalian tachykinin receptors have a similar structural organization. They all contain five exons, with introns interrupting the protein-coding sequence in identical positions (Figure 2). Within the family of the G-protein-coupled receptors, the tachykinin receptor family is one of the few that retains introns. The splice sites of the exons occur at the border of the sequences encoding the putative transmembrane domains. The tachykinin receptor displaying the highest affinity for substance P was termed the tachykinin NK1 receptor. The receptor showing the highest affinity for neurokinin A was termed the tachykinin NK2 receptor and the receptor with the highest affinity for neurokinin B was called the tachykinin NK3 receptor. Hemokinin 1 and its elongated forms act as tachykinin NK1 receptor preferring agonists. It should be emphasized that all tachykinins can act as full agonists on the three different receptors but with lower affinities than on the preferred receptor. The extracellular loops of the receptors are involved in the selection of a specific ligand, whereas the interaction of the ligand with the transmembrane domains is responsible for receptor activation. Upon ligand binding, receptor activation and subsequent interaction with a G-protein occurs. The

Cell Growth and Proliferation of Mesenchymal Cells

Tachykinins stimulate the proliferation of a number of cell types, including fibroblasts, smooth muscle cells, epithelial cells, and endothelial cells. The substance P-induced proliferation of human fibroblasts is mediated by interaction with tachykinin NK1 receptors. The proliferation of rabbit airway smooth muscle involves also tachykinin NK1 receptors. The proliferation of smooth muscle cells in response to tachykinins is coupled to inositoltriphosphate formation and DNA synthesis. Immunomodulation

Tachykinins are known to have a wide variety of modulatory effects on inflammatory and immune cells. Substance P degranulates mast cells, leading to the release of histamine and 5-hydroxytryptamine (5HT, serotonin), and the production of tumor necrosis factor alpha (TNF-a). Tachykinins cause adherence and chemotaxis of human neutrophils. Substance P induces chemotaxis of human eosinophils. Several in vitro and in vivo studies have shown that substance P is able to modulate the chemotaxis, proliferation, and activation of lymphocytes. Tachykinins also have a number of stimulatory effects on monocytes and macrophages: substance P has been shown to evoke superoxide anion production, enhance phagocytosis, and cause production of interleukin-1 and interleukin-12 by macrophages. Tachykinins are also able to activate mesenchymal cells in the airways.

Receptors The biological activity of tachykinins depends on their interaction with three specific receptors: the ATG

130

195

245

311

TAG (407) TACR1

1

ATG

2

131

3

196

5

4

247

313

TGA (398) TACR2

1

3

2

183

ATG

4

246

5

296

362

TAA (465) TACR3

1

2

3

4

5

Figure 2 Schematic structure of the genes encoding for the tachykinin NK1 receptor (TACR1), the NK2 receptor (TACR2), and the NK3 receptor (TACR3). The coding regions of the genes are interrupted by four introns. The dark blue boxes indicate the transmembrane segments. Amino acid positions at splicing sites are indicated.

KININS AND NEUROPEPTIDES / Tachykinins 515

main second messenger system coupled to the receptors is the stimulation of phospholipase C (PLC), leading to phosphoinositide breakdown, inositol triphosphate (IP3) formation which leads to elevation of intracellular calcium and formation of diacylglycerol (DAG) which activates protein kinase C (PKC). Receptor activation leads also to activation of adenylyl cyclase (AC) and subsequent increases in cAMP levels but this requires about one order of magnitude higher concentrations of the tachykinin peptides (Figure 3). In Chinese hamster ovary (CHO) cells, transfected with the human tachykinin NK1 receptor, activation of the receptor results not only in IP3 and DAG accumulation and increase of cAMP levels but also in the mobilization of arachidonic

acid. In these cells, receptor activation also leads to stimulation of the phospholipase D (PLD), with formation of phosphatidic acid (PA) and sustained activation of protein kinase C. Also Rho family GTPases are involved in NK1 receptor signaling. Native tachykinin NK1 receptors, expressed in CaCo-2 (colon adenocarcinoma cell line), tracheal smooth muscle, U-373 MG (astrocytoma), and peritoneal mast cells, are coupled downstream to the activation of mitogen-activated kinases (MAPKs). Downstream involvement of NF-kB has been demonstrated in murine macrophages and dendritic cells, U-373 MG cells, and lymphocytes. An overwhelming amount of functional data demonstrates indirectly the broad expression of

HK SP

NKA

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PLD

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GTP

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Prostaglandins Leukotrienes Lipoxins




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[DAG]

NO

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PKA

[Ca2+]

MAPK NF-B IB

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Transcription

CREB

Figure 3 Intracellular signaling pathways. The tachykinin NK1 receptor is known to interact with several G proteins (Gq, Gs, Gi), which in their turn interact with effector molecules as adenylyl cyclase (AC) and phospholipase C (PLC). Protein kinases are depicted in red, GTPases in blue, and nuclear factors in gray. CREB, cAMP response element-binding protein; DAG, diacylglycerol; eNOS, endothelial nitric oxide synthase; HK, hemokinin; lkB, inhibitor kappa B; NKA, neurokinin A; NO, nitric oxide; P13K, phosphatidylinositol-3-kinase; PKA, protein kinase A; RhoA, a small GTP-binding protein; SP, substance P.

516 KININS AND NEUROPEPTIDES / Tachykinins

Epithelium Secretion Submucosal gland

Inflammatory cell Stimulation

Sensory nerve

Facilitation

Vasodilatation Blood vessel

Contraction

Smooth muscle

Plasma leakage Figure 4 Nerve fibers containing substance P and neurokinin A are present from the pharynx down to the bronchioli, occasionally extending into the alveoli. They are located beneath and within the respiratory epithelium, among bundles of smooth muscle, around blood vessels, submucosal glands, and local tracheobronchial ganglion cells; and in close association with lymphoid aggregates and 5-hydroxytryptamine (5-HT)-positive cells. The sensory nerves can be activated by mechanical and chemical stimuli, generating antidromic impulses and a local axon reflex, which lead to noncholinergic bronchoconstriction and neurogenic inflammation.

tachykinin NK1 and NK2 receptors in the airways. Also functional evidence for the presence of tachykinin NK3 receptors exists. TACR1 and TACR3 mRNA are found in human pulmonary veins, arteries, and bronchi. TACR2 mRNA is abundantly expressed in human bronchi. A low expression of this receptor is found in human pulmonary veins and arteries. On surgical specimens, using specific antibodies, tachykinin NK1 and NK2 receptor protein can be detected in human bronchial glands, bronchial vessels, and bronchial smooth muscle. Tachykinin NK1 receptors are occasionally found in nerves and tachykinin NK2 receptors in inflammatory cells, such as T lymphocytes, macrophages, and mast cells. On endobronchial biopsies, immunoreactivity for the tachykinin NK1 receptor is found in the epithelium and the submucosa. Goblet cells appear to be the cells with the strongest staining. In the submucosa, staining is localized on the endothelial cells of the blood vessels, the surfaces of inflammatory cells, and some smooth muscle cells. Immunohistochemistry on human pulmonary vessels from tissue obtained at lobectomy reveals positive staining for tachykinin NK1 receptors on the endothelium of both veins and arteries. Inflammatory cells such as macrophages, lymphocytes, neutrophils, dendritic cells, and mast cells, whose presence becomes important in inflammatory states, also express functional tachykinin NK1 receptors. In guinea pig airways, tachykinin NK2 receptors and to a lesser extent tachykinin NK1 receptors are involved in the bronchoconstrictor response to exogenous tachykinins, capsaicin, and electrical field

stimulation. In humans, it has long been thought that only tachykinin NK2 receptors are involved in the direct contraction of isolated bronchi. However, in small- and medium-sized bronchi, tachykinins also cause contraction via tachykinin NK1 receptor stimulation.

Role of Tachykinins in Respiratory Diseases The tachykinins substance P and neurokinin A are present in human airways, sensory nerves, and immune cells. Tachykinins can be recovered from the airways after inhalation of ozone, cigarette smoke, or allergen. They interact in the airways with tachykinin NK1, NK2, and NK3 receptors to cause bronchoconstriction, plasma protein extravasation, and mucus secretion and to attract and activate immune cells (Figure 4). In preclinical studies they have been implicated in the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD), including airway inflammation and bronchial hyperresponsiveness induced by allergens and cigarette smoke. Clinical studies with potent tachykinin receptor antagonists (such as dual NK1/NK2 or triple NK1/NK2/NK3 tachykinin receptor antagonists) can bring a definite judgment on their importance in obstructive airway diseases. See also: Capsaicin. Kinins and Neuropeptides: Bradykinin; Neuropeptides and Neurotransmission; Other Important Neuropeptides. Symptoms of Respiratory Disease: Cough and Other Symptoms.

KININS AND NEUROPEPTIDES / Vasoactive Intestinal Peptide

Further Reading Advenier C, Joos G, Molimard M, Lagente V, and Pauwels R (1999) Role of tachykinins as contractile agonists of human airways in asthma. Clinical and Experimental Allergy 29: 579–584. Advenier C, Lagente V, and Boichot E (1997) The role of tachykinin receptor antagonists in the prevention of bronchial hyperresponsiveness, airway inflammation and cough. European Respiratory Journal 10: 1892–1906. Barnes PJ (2001) Neurogenic inflammation in the airways. Respiratory Physiology 125: 145–154. Barnes PJ, Baraniuk JN, and Belvisi MG (1991) Neuropeptides in the respiratory tract. Part I. American Review of Respiratory Disease 144: 1187–1198. Canning BJ and Fischer A (2001) Neural regulation of airway smooth muscle tone. Respiratory Physiology 125: 113–127. Carr MJ and Undem BJ (2003) Pharmacology of vagal afferent nerve activity in guinea pig airways. Pulmonary Pharmacology & Therapeutics 16: 45–52. Groneberg DA, Quarcoo D, Frossard N, and Fischer A (2004) Neurogenic mechanisms in bronchial inflammatory diseases. Allergy 59: 1139–1152. Joos GF, De Swert KO, and Pauwels RA (2001) Airway inflammation and tachykinins: prospects for the development of tachykinin receptor antagonists. European Journal of Pharmacology 429: 239–250. Joos GF, Germonpre´ PR, Kips JC, Peleman RA, and Pauwels RA (1994) Sensory neuropeptides and the human lower airways: present state and future directions. European Respiratory Journal 7: 1161–1171. Joos GF, Germonpre´ PR, and Pauwels RA (2000) Role of tachykinins in asthma. Allergy 55: 321–337. Maggi CA (1997) The effects of tachykinins on inflammatory and immune cells. Regulatory Peptides 70: 75–90. Pennefather JN, Lecci A, Candenas ML, et al. (2004) Tachykinins and tachykinin receptors: a growing family. Life Sciences 74: 1445–1463. Rogers DF (2001) Motor control of airway goblet cells and glands. Respiratory Physiology 125: 129–144. Tai CF and Baraniuk JN (2002) Upper airway neurogenic mechanisms. Current Opinion in Allergy and Clinical Immunology 2: 11–19.

Relevant Website http://www.gene.ucl.ac.uk – The Centre for Human Genetics.

Vasoactive Intestinal Peptide S I Said, State University of New York, Stony Brook, NY, USA & 2006 Elsevier Ltd. All rights reserved.

airway and vascular smooth muscle, inhibition of the proliferation of both smooth muscle types, modulation of inflammatory and immune responses, and protection of the lung against acute injury and apoptotic cell death. These actions suggest potential therapeutic potential in bronchial asthma, pulmonary hypertension and acute lung injury.

Introduction Although vasoactive intestinal polypeptide (VIP) was first isolated from porcine small intestine, its principal biological activity, as a vasodilator, had previously been discovered in lung extracts. VIP was later rediscovered as a neuropeptide with neurotransmitter/neuromodulator properties, and is now known to occur widely in the central and peripheral nervous systems and to have a wide spectrum of biological actions.

Structure VIP is a 28 amino acid residue peptide (Figure 1). It belongs to a family of structurally related peptides that include a peptide with N-terminal histidine and C-terminal isoleucine amide (PHI) and its counterpart in human tissues, a peptide with N-terminal histidine and C-terminal methionine amide (PHM), secretin, glucagon, gastric inhibitory peptide or glucose-dependent insulinotropic peptide (GIP), corticotropinreleasing hormone (CRH), growth hormone-releasing hormone (GHRH), sauvagine, urotensin I, helodermin, and pituitary adenylate cyclase-activating peptide (PACAP).

Distribution and Localization The distribution of VIP in the central and peripheral nervous systems has been defined by immunofluorescence, radioimmunoassay, and, more recently, by measurement of VIP mRNA. In the brain the peptide is found in highest concentrations in the cerebral cortex, suprachiasmatic nucleus, amygdala, and striatum, but it is also present in the midbrain periaqueductal gray and the sacral spinal cord. In the peripheral nervous system, VIP is localized in the sympathetic ganglia, the vagus nerve, predominantly HOOC

His

Ser Asp Ala

Val Phe Thr Asp Asn Tyr

Abstract Vasoactive intestinal peptide (VIP) is a neuropeptide that is widely distributed in the central and peripheral nervous systems. Both VIP and its receptors are expressed in various pulmonary structures, including airway epithelial cells, airway and pulmonary vascular smooth muscle, secretory glands, and immune and inflammatory cells. Pulmonary effects include relaxation of

517

Ala

Met Gln

Lys

Tyr

Lys

Arg Leu Arg

Thr

Val Lys Leu Asn Ser

lle

Leu Asn

NH2

Figure 1 Amino acid sequence of human VIP. The sequence is well conserved in mammalian and other species.

518 KININS AND NEUROPEPTIDES / Vasoactive Intestinal Peptide

motor nerves such as the sciatic nerve, and nerves supplying exocrine glands, blood vessels, and nonvascular smooth muscle. In the lungs, VIP-containing nerve fibers and nerve terminals are principally localized in the smooth muscle layer of the airways from the trachea through the small bronchioles, around submucosal mucous and serous glands, and in the walls of both pulmonary and bronchial arteries. Immunoreactive VIP is also present in neuronal cell bodies forming microganglia that provide a source of intrinsic innervation of pulmonary structures. In addition to its neuronal localization, VIP is present in abundance in the immune system, including inflammatory cells such as mast cells, eosinophils, and B and T lymphocytes. Colocalization with Other Peptides and Neurotransmitters

Strong evidence suggests that VIP coexists with acetylcholine in many postganglionic cholinergic neurons supplying exocrine glands and blood vessels. VIP may also be colocalized with other peptides: It coexists with PHI (or PHM, its human counterpart), both peptides being synthesized from the same precursor molecule, and may be colocalized with substance P. Other neurotransmitters that coexist with VIP in some of the neurons of bronchial ganglia include nitric oxide (nitric oxide synthase) and glutamate.

Actions on Lung Structures Airways

VIP has potent effects on all major structural components of the airways.

exocrine glands brings together the vasodilator effect of VIP and the secretory effect of acetylcholine. Bronchial and pulmonary vessels VIP dilates the vessels supplying the nose, upper airways, trachea, and bronchi, as well as pulmonary vessels. As a pulmonary vasodilator, VIP is 50 times as potent as prostacyclin, and its action is independent of endothelium. Immunologic release of mediators VIP has a moderate inhibitory effect on antigen-induced release of histamine from guinea pig lung. Since the peptide is normally present in mast cells, it may thus act as a natural modulator of mast cell function. Other immunomodulator activities of VIP are outlined below.

Biological Actions Pertinent to the Lung Multiple biological actions of VIP have been identified. Those relevant to lung function and disease are summarized in Table 1.

Receptors Specific, high-affinity receptors for VIP have been identified in membrane preparations of normal lungs and human lung tumor cells. These receptors, localized immunocytochemically and by the increased cAMP levels resulting from activation by VIP, exist ;in a variety of pulmonary cells. Molecular and pharmacologic studies have led to the identification, characterization, and cloning of at least three receptors Table 1 Actions of VIP in the lung

Airway smooth muscle VIP relaxes airway smooth muscle both in vitro and in vivo, as demonstrated in airways of guinea pigs, rabbits, dogs, and humans. VIP also prevents or attenuates airway smooth muscle contraction by a variety of agents, including histamine, prostaglandin F2a, kallikrein, leukotriene D4, neurokinin A, serotonin, and endothelin. VIP-induced airway relaxation is long lasting and is unaffected by blockade of adrenergic or cholinergic receptors or of cyclooxygenase activity. However, in human asthmatics VIP has been found to be generally less effective in relieving bronchoconstriction, probably because of its rapid inactivation by proteases in airway secretions. Tracheobronchial secretion VIP stimulates water and ion transport in canine tracheal epithelium and may also promote the secretion of sulfated macromolecules. The coexistence of VIP and acetylcholine in cholinergic neurons supplying bronchial and other

Site

Action

Airways

Relaxation of smooth muscle, inhibition of smooth muscle proliferation, suppression of airway inflammation and airway hyperreactivity Relaxation of smooth muscle, inhibition of smooth muscle proliferation Modulation of multiple aspects of the immune and inflammatory responses: (a) inhibition of proinflammatory cytokines (TNF-a, IL-6, IL-12, IL-18), chemokines (RANTES, MCP-1, 1a, 1b, 2), i NOS, proteases (MMP-2 gelatinase), (b) upregulation of antiinflammatory cytokine IL-10 Inhibition of apoptotic cell death, promotion of cell survival, suppression of certain neoplastic cell proliferation, e.g., small cell cancer

Pulmonary vessels Immune and inflammatory responses

Cell death, cell survival, or proliferation

IL, interleukin; i NOS, inducible NOS; MMP, matrix metalloprotease; MCP, monocyte chemotactic protein.

KININS AND NEUROPEPTIDES / Vasoactive Intestinal Peptide

519

for VIP and the related peptide PACAP. Now called VPAC receptors, these receptors belong to a distinct family of seven transmembrane-domain receptors coupled to G proteins. The three VPAC receptors are VPAC1 and 2, which respond to VIP and PACAP with comparable affinity, and PAC1, which binds PACAP with high affinity and VIP with considerably lower affinity.

together to bring about the relaxation and to inhibit mitogenesis and smooth muscle proliferation.

Signal Transduction Pathways

1. mediation of neurogenic (noncholinergic, nonadrenergic) airway smooth muscle relaxation; 2. mediation of pulmonary vascular relaxation; 3. modulation of inflammation (see Table 1); 4. modulation of airway and pulmonary smooth muscle proliferation; 5. modulation of the pulmonary immune response; and 6. protection against apoptotic cell death and promotion of cell survival (Figure 2).

The actions of VIP are mediated principally by adenylyl cyclase stimulation and cAMP production. VIP biosynthesis itself is promoted by higher cAMP levels, and thus the peptide is potentially capable of stimulating its own generation. In some VIP actions, additional second messengers and signal transduction pathways may be involved, for example, intracellular mobilization of Ca2 þ in the case of the PACAP-preferring PAC1 receptor. The actions of VIP on some tissues and organs, including airway and vascular smooth muscle, may be mediated in part by the release of nitric oxide (NO) from neurons or smooth muscle cells. Because of its activation of a constitutive NO synthase (NOS) in neural elements and smooth muscle, and the subsequent activation of cytosolic guanylyl cyclase, VIP may stimulate intracellular cyclic GMP formation. The role of neuronal NOS in mediating the effects of VIP in the airways was recently assessed in neuronal NOS-knockout mice, and estimated to account for a significant proportion of VIP-induced tracheal relaxation. It is now clear that NO, along with carbon monoxide (CO), mediates VIP-induced relaxation of airway, gastrointestinal, and other smooth muscle. The cyclic AMP and cyclic GMP pathways work

Physiological Roles Evidence, largely collected from experimental studies, suggests that VIP probably plays a number of important physiological roles in relationship to the lung, including:

Antiapoptotic Actions of VIP

VIP exerts its antiapoptotic activity by three complementary mechanisms: inhibition of the activation of caspases, key effectors of apoptosis; upregulation of antiapoptotic bcl2; and suppression of cytoplasmic translocation of cytochrome c, a critical step in mitochondria-mediated apoptosis.

VIP in Pulmonary Disease Bronchial Asthma

The airways of several severely asthmatic subjects who had died in accidents were found to be lacking

VIP

Mediation of neurogenic airway smooth muscle relaxation

Mediation of pulmonary vascular relaxation

Modulation of airway & pulmonary vascular smooth muscle proliferation

Modulation of inflammation

Modulation of immune responses

Protection against apoptotic cell death, promotion of cell survival

Figure 2 Physiological roles of VIP relevant to the lung.

520 KININS AND NEUROPEPTIDES / Vasoactive Intestinal Peptide

VIP

Airways

• Relaxation of smooth muscle • Inhibition of smooth muscle proliferation • Suppression of inflammation & hyperreactivity

Potentially beneficial in bronchial asthma (VIP-immunoreactive nerves may be lacking in asthmatic airways)

Pulmonary circulation

• Relaxation of smooth muscle • Inhibition of smooth muscle proliferation

Potentially beneficial in primary pulmonary hypertension

Parenchymal lung cells

• Inhibition of apoptotic cell death; promotion of cell survival • Modulation of inflammatory/immune damage

Protection against acute lung injury

Small cell lung cancer cells

• Suppression of proliferation in vitro & in vivo

Potentially beneficial in small cell lung cancer

(VIP-containing nerves are lacking in pulmonary arteries in PPH)

Figure 3 Key effects of VIP on various structural components of the lungs, and their potential therapeutic implications. PPH, primary pulmonary hypertension.

in VIP-immunoreactive nerves, raising the possibility of a causative link between VIP deficiency and the pathogenesis of asthma. This observation still awaits confirmation, but further evidence suggesting such a link comes from knockout mice. Mice with targeted deletion of the VIP gene exhibited airway hyperreactivity and, upon sensitization and challenge with ovalbumin, showed pathologic features of airway inflammation (see Figure 3). Cystic Fibrosis

VIP-containing nerves were absent around the sweat glands of a group of children with cystic fibrosis (see Figure 3). How this deficiency may relate to the pathophysiology of cystic fibrosis has not been established.

Therapeutic Potential Strong preclinical data and early clinical trials support a therapeutic role for VIP, or more appropriate agonists, in a variety of lung diseases (Table 2). Bronchial Asthma

The airway-relaxant, anti-inflammatory, and antiproliferative effects of VIP make it an attractive candidate as an antiasthma medication. As mentioned above, early clinical trials of VIP as an aerosol in asthmatics showed modest benefit, probably because of its degradation by airway proteases. On the other hand, VIP-like peptides that are more resistant to degradation, such as heloderma and PACAP, have not been adequately tested.

Primary Pulmonary Hypertension (PPH)

Acute Lung Injury/Acute Respiratory Distress Syndrome

In PPH patients, serum VIP levels are low, and VIPcontaining nerves are lacking in pulmonary arterial walls. Conversely, VIP receptors are upregulated in pulmonary arterial smooth muscle cells, and VIP inhibits the proliferation of these smooth muscle cells in vitro. These findings suggest that a deficiency of VIP may be a factor in the genesis of PPH (see Figure 3).

VIP prevents or delays acute lung injury (ALI) in 10 experimental models, in isolated lungs and in vivo. This protection is attributable to a combination of anti-inflammatory, antioxidant, and antiapoptotic properties. Recently, male VIP knockout mice were reported to be more susceptible than wild-type mice to endotoxemia. Clinical trials of VIP in acute respiratory distress syndrome (ARDS) are in progress,

KININS AND NEUROPEPTIDES / Other Important Neuropeptides Table 2 Possible therapeutic applications of VIP Disorder Airways Bronchial asthma

Pulmonary circulation Pulmonary hypertension Other ALI/ARDS Small cell lung cancer

Potential benefit from VIP Suppresses bronchoconstriction, airway hyperreactivity, airway inflammation, airway remodeling

Reduces pulmonary hypertension

Attenuates or prevents ALI Inhibits growth and proliferation of cancer cells

ALI, acute lung injury; ARDS, acute respiratory distress syndrome.

with the peptide being delivered by IV infusion or as an aerosol.

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Maruno K, Absood A, and Said SI (1998) Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo. Proceedings of the National Academy of Sciences, USA 95: 14373–14378. Petkov V, Mosgoeller W, Ziesche R, et al. (2003) Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension. Journal of Clinical Investigation 111: 1339–1346. Said SI (1988) Vasoactive intestinal peptide in the lung. Annals of the New York Academy of Sciences 527: 450–464. Said SI (1989) Vasoactive intestinal polypeptide and asthma (Editorial). New England Journal of Medicine 320: 1271–1273. Said SI (1996) Molecules that protect: the defense of neurons and other cells (editorial). Journal of Clinical Investigation 97: 2163–2164. Said SI (2004) Vasoactive intestinal polypeptide. In: Adelman G and Smith BH (eds.) Encyclopedia of Neurosciences, 3rd edn. (CD-ROM). Amsterdam: Elsevier. Said SI and Rattan S (2004) The multiple mediators of neurogenic smooth muscle relaxation. Trends in Endocrinology and Metabolism 15: 189–191.

Pulmonary Hypertension

VIP, given as an aerosol over 12–24 weeks, markedly improved hemodynamics and exercise tolerance in patients with PPH. Lung Cancer

Because VIP suppresses the growth of small cell lung cancer cells in culture and in athymic nude mice in vivo, it may offer a less toxic alternative to conventional chemotherapy in the management of this highly malignant form of cancer. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Cystic Fibrosis: Overview. Vascular Disease.

Further Reading Delgado M (2003) VIP: A ‘‘very important peptide’’ in T helper differentiation. Trends in Immunology 24: 221–224. Delgado M, Abad C, Martinez C, Leceta J, and Gomariz RP (2001) Vasoactive intestinal peptide prevents experimental arthritis by down-regulating both autoimmune and inflammatory components of the disease. Nature Medicine 7: 563–568. Fahrenkrug J (1989) Transmitter role of vasoactive intestinal peptide. Pharmacology and Toxicology 72: 354–363. Goetzl EJ, Voice JK, Shen S, et al. (2001) Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC2 receptor for vasoactive intestinal peptide. Proceedings of the National Academy of Sciences, USA 98: 13854–13859. Gozes I and Brenneman DE (1989) VIP: Molecular biology and neurobiological function. Molecular Biology 3: 201–236. Harmar AJ, Arimura A, Gozes I, et al. (1998) International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacological Reviews 50: 265–270. Laburthe M, Couvineau A, and Marie JC (2002) VPAC receptors for VIP and PACAP. Receptors and Channels 8: 137–153.

Other Important Neuropeptides M G Belvisi and D J Hele, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Neuropeptides have been implicated in the physiology and pathophysiology of chronic inflammatory diseases of the airways such as asthma, allergic rhinitis, and chronic obstructive pulmonary disease. Detailed distribution studies have provided data on the putative role of neuropeptides in the control of normal respiratory functions, and studies point to the involvement of regulatory neuropeptides in diseases of the lung. Neuropeptides such as bradykinin, the tachykinins, and vasoactive intestinal peptide have been described as having a role in inflammatory diseases of the airways but a number of other neuropeptides, which include calcitonin gene-related peptide, secretoneurin, bombesin, galanin, somatostatin, cholecystokinin, neuropeptide-Y, enkephalins, neurotensin, and pituitary adenylate cyclase-activating polypeptide, have been implicated in the etiology or pathophysiology of inflammatory diseases of the airways.

Introduction Neuropeptides have been implicated in the pathophysiology of chronic inflammatory diseases of the airways such as asthma, allergic rhinitis, and chronic obstructive pulmonary disease (COPD). Many factors stimulate primary sensory neurons including cigarette smoke, airborne pollutants, and allergens, all of which may participate in the generation of airway inflammation. Pathological activation of sensory neurons and the resulting inflammatory events are known as neurogenic inflammation and appear to be

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important in many organ systems, including the lung. Neuropeptides form part of that inflammatory response and neurogenic inflammation may participate in the development and progression of chronic airway inflammatory diseases such as asthma or COPD. The molecular mechanisms underlying neurogenic inflammation are orchestrated by a large number of neuropeptides. Neuropeptides in the lung originate either from local neurons (e.g., vasoactive intestinal peptide (VIP) and galanin), extrinsic neurons localized in sensory ganglia (e.g., substance P and calcitonin generelated peptide (CGRP)), or the sympathetic chain (e.g., neuropeptide Y (NPY)). Detailed distribution studies have provided data on the putative role of the peptides in the control of normal respiratory functions and further studies point to the involvement of regulatory peptides in diseases of the lung. Their widespread distribution and physiological effects suggest they may have a role to play and in fact plasma levels of neuropeptides such as CGRP, substance P, and NPY have been shown to be raised during exacerbations in asthma patients. Neuropeptides such as bradykinin, the tachykinins, and VIP have been described elsewhere (see Kinins and Neuropeptides: Bradykinin; Tachykinins; Vasoactive Intestinal Peptide). This article will discuss the neuropeptides CGRP, secretoneurin, bombesin, galanin, somatostatin, cholecystokinin, NPY, enkephalins, neurotensin, and pituitary adenylate cyclase-activating polypeptide (PACAP), all of which have been implicated in the etiology or pathophysiology of airway inflammatory disease.

Calcitonin Gene-Related Peptide CGRP is a 37 amino acid peptide that is constitutively expressed in normal lungs where it localizes to a specialized subset of epithelial cells (neuroendocrine cells) and sensory C fibers distributed in the pulmonary airways, blood vessels, and lymphoid tissue. Two CGRP isoforms generated from different genes (a- and b-CGRP) have been identified in rats and humans. Comparison of the peptide sequences of a- and b-CGRP reveals a striking degree of sequence homology (human a-CGRP differs from b-CGRP in 3 of 37 amino acids, and rat a-CGRP differs from b-CGRP in 1 of 37 amino acids), and the two isoforms have been considered to be almost equivalent in function. Two CGRP receptors have been identified pharmacologically on the basis of their differential affinities for the competitive peptide antagonist CGRP8–37. A candidate receptor was initially cloned from the sequence of the calcitonin receptor by polymerase chain reaction (PCR) in rat lung and was named calcitonin receptor-like receptor (CRLR).

Human CRLR has subsequently been cloned and proposed as the CGRP1 receptor. It has recently been determined that when coexpressed with receptor activity-modifying proteins (RAMPs), in particular RAMP1, CRLR functions as a CGRP1 receptor in a variety of cells. In addition, an intracellular accessory protein termed CGRP-receptor component protein (RCP) has recently been identified and shown to be required for signal transduction through CGRP receptors. A recent study identified a 66 kDa protein as a new receptor for CGRP, which is distinct from CRLR and RAMP1, and proposed a new classification for CGRP1 and CGRP2 receptors as CGRP-A and CGRP-B, respectively. Binding of CGRP to its receptor is known to activate adenylyl cyclase and increase cAMP production, a pathway that involves Gas protein. CGRP has long been suggested to participate in airway physiology and pathophysiology and may play a key role in the pathogenesis of a number of respiratory diseases such as asthma, COPD, and chronic cough (Figure 1). A study has shown CGRP to be a constrictor of human bronchus airway smooth muscle in vitro, which is surprising since it stimulates intracellular cAMP generation; an effect normally associated with bronchodilation. There are two possible mechanisms that may account for the constrictor effect: either CGRP activates phospholipase C-b1 and leads via the inositol 1,4,5-trisphosphate-related pathways to constriction or CGRP does not have a direct effect on the airway smooth muscle itself but liberates other constrictor mediators. In this regard, a few binding sites for CGRP were found in the airway smooth muscle layer of human lung, suggesting an indirect mode of CGRPmediated bronchoconstriction in human airways. In the guinea pig respiratory tract, the effects of CGRP on airway smooth muscle are controversial and no consistent bronchoconstrictor or bronchodilator effect has been demonstrated. In addition, CGRP also causes other pulmonary effects including the induction of eosinophil migration and the stimulation of T cell adhesion to fibronectin at sites of inflammation. Furthermore, an increase in CGRP content in the lungs of rats and guinea pigs by allergic inflammation has been reported. Interestingly, a recent study evaluated the role of CGRP in the development of airway hyperresponsiveness (AHR) in a-CGRP knockout mice and described increases in CGRP expression in the lungs of ovalbumin-sensitized and challenged wild-type mice, and an attenuated AHR in sensitized and challenged a-CGRP knockout mice. It was concluded that CGRP is a mediator of AHR, but not eosinophilia, in this model.

KININS AND NEUROPEPTIDES / Other Important Neuropeptides Inflammatory cells

523

Structural cells

Eosinophil

Vessels

Migration

T lymphocyte Vasodilation Adhesion

CGRP Airway smooth muscle

Macrophage

Secretion T-cell activation

Bronchospasm?

Figure 1 Cellular effects of CGRP.

In contrast to the effects in allergic animal models described above, another study has shown that CGRP was significantly depleted in pulmonary neuroepithelial bodies (NEBs) and submucosal nerve fibers following airway allergen challenge of sensitized mice and that these mice developed a robust allergic airway inflammation and AHR. In addition, the CGRP1 receptor antagonist CGRP8–37 did not inhibit the development of AHR or airway inflammation in these animals but exogenously administered CGRP (systemically or by inhalation) did inhibit the development of AHR. Similar observations of CGRP depletion have been reported in the guinea pig following sensitization and airway challenge with toluene-diisocyanate. Furthermore, CGRP has been also shown to inhibit macrophage secretion and the capacity of macrophages to activate T cells, indicating a potential anti-inflammatory effect. A role for CGRP in tissue repair is also suggested based on its capacity to induce migration and proliferation of fibroblasts. The ability of CGRP to exert multiple effects such as vasoregulation, anti-inflammatory actions and tissue repair suggests a role in airway homeostasis. However, in order to clearly define the functional effects of CGRP it is necessary to develop potent and highly selective pharmacological agents, which can be used as tools to dissect the role of this peptide in airway physiology and pathophysiology.

Secretoneurin Chromogranins belong to an evolutionarily conserved family of proteins that serve as neuropeptide proproteins and the secretogranin-II-derived peptide secretoneurin is a 33 amino acid polypeptide that exerts its

effects by binding to specific receptors. Secretoneurin is derived by proteolytic processing from its precursor secretogranin II (chromogranin C). Expression data suggests the presence of secretoneurin in rat tracheal tissue and in human bronchial neuroendocrine cells. Studies in rats have shown that afferent nerve fibers may contain secretoneurin. Secretoneurin appears to have a similar distribution to the sensory neuropeptides substance P and CGRP in the nervous system of the rat. Furthermore, studies in rats have demonstrated that levels of secretoneurin are reduced in peripheral organs, including the trachea, after neonatal capsaicin treatment, although the reduction may be less pronounced than that of substance P and CGRP. Interestingly, it has been shown that this neuropeptide may have effects on various immune cells. For example, secretoneurin has been demonstrated to attract immature, and arrest mature, blood-derived dendritic cells. Moreover, secretoneurin may trigger migration of human monocytes in vitro and in vivo, and combinations of secretoneurin and sensory neuropeptides (substance P, somatostatin) synergistically stimulate this migration. Intriguingly, secretoneurin may also act as a potent eosinophil chemoattractant. Taken together, these previous studies indicate that secretoneurin may play a role in inflammatory conditions. In fact, recent data have demonstrated that secretoneurin is widely distributed in the nasal mucosal nerves of patients with seasonal allergic rhinitis and ongoing allergen exposure is characterized by increased levels of secretoneurin in nasal lavage fluid. Therefore, secretoneurin may have a role in the local traffic of immune cells in human airway mucosa and may play an important role in the early stages of allergic inflammation.

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Bombesin Bombesin is a 14 amino acid bioactive peptide that was first isolated from frog skin; bombesin-like peptides (BLPs) are mammalian homologs of bombesin. BLPs comprise a family of related peptides, several of which could account for the BLP immunoreactivity localized to pulmonary neuroendocrine cells. One major pulmonary BLP has been identified as gastrinreleasing peptide (GRP). GRP and bombesin are highly similar both structurally and functionally, and neuromedin B (NMB) is also closely related. BLPs are growth factors for fibroblasts and normal airway epithelial cells. They have neuroregulatory functions in the brain and gut; and are potent bronchoconstrictors. GRP and bombesin are potent stimulators of airway mucus secretion in animals and humans. Multiple receptor subtypes mediate the effects of bombesin-related peptides. The bombesin receptor family is currently comprised of four receptor subtypes. These subtypes are classified as the NMB subtype (BB1), the GRP subtype (BB2), the orphan receptor subtype (BB3), and the bombesin (BBN) receptor subtype (BB4). BLPs promote fetal lung development, including lung-branching morphogenesis, epithelial and mesenchymal cell proliferation, and type II cell differentiation. A possible role has been suggested for BLPs in promoting cellular responses that may contribute to inflammatory processes, including macrophage activation and phagocytosis, chemotaxis of macrophages and fibroblasts, smooth muscle constriction, and proliferation of fibroblasts, lymphocytes, and endothelial cells. BLPs have also been shown to be elevated in newborns who later develop bronchopulmonary dysplasia. Bombesin stimulates superoxide anion production and interleukin (IL)-8 release by peripheral monocytes. Increased lung levels of BLPs have been described in smokers and patients with COPD. Bombesin significantly enhanced the phagocytosis of monocytes and alveolar macrophages from normal and COPD patients and restored deficient phagocytosis in a group of COPD patients. Increased numbers of BLP-positive neuroendocrine cells have been associated with other chronic inflammatory lung diseases, including cystic fibrosis, lung cancer, eosinophilic granuloma, and pulmonary hypertension. Evidence suggests that BLPs are involved in immunoregulatory activities in the lung.

Galanin Galanin is a 29 (30 in human) amino acid neuropeptide found in central and peripheral nervous systems. It appears to have excitatory and inhibitory

actions via three subtypes of G-protein-coupled receptor (denoted GALR1, GALR2, and GALR3), and has modulatory effects on sensory nerve fibers. Galanin potently modulates the mechanosensitivity of gastroesophageal vagal afferents with either facilitatory or inhibitory actions on individual afferent fibers. Both intrinsic and extrinsic vagal neurons contain galanin and are therefore potential sources of endogenous galanin. Galanin immunoreactive fibers have been detected in the nerve cell bodies of the airways and in the trachea, bronchus, and major intrapulmonary airways, predominantly in smooth muscle, around seromucous glands, and in the adventitia of blood vessels but rarely in lung parenchyma. Galanin-containing nerve fibers in the airways originate from nerve cell bodies of intrinsic airway ganglia, and galanin and VIP are frequently colocalized in these neurons. The distribution of galanin suggests that it may have some influence on airway, vascular, and secretory functions in the mammalian respiratory tract although its role appears to be in nociception rather than as an inflammatory mediator. There is evidence to suggest that secretion of mucus into airways is regulated by a complex network of peptidergic stimulators and inhibitors, which includes galanin, and it has been demonstrated that galanin inhibits secretion of mucus.

Somatostatin The neuropeptide somatostatin (or somatotroph release inhibiting factor (SRIF)-14) is a cyclic decatetrapeptide initially isolated from hypothalamus extracts but has been found widely distributed in the body. Somatostatin is the predominant isoform of the SRIF family (the other being SRIF-28). The actions of these peptides are mediated by a family of seven transmembrane (TM) domain G-protein-coupled receptors containing five distinct subtypes (termed SSTR1–5) that are encoded by separate genes segregated on different chromosomes. The five receptor subtypes bind the natural SST peptides, SST-14 and SST-28, with low nanomolar affinity. The five receptors share common signaling pathways such as the inhibition of adenylyl cyclase, activation of phosphotyrosine phosphatase (PTP), and modulation of mitogen-activated proteinkinase (MAPK) through G-protein-dependent mechanisms. Some of the subtypes are also coupled to inward rectifying K þ channels (SSTR2, 3, 4, 5), to voltage-dependent Ca2 þ channels (SSTR1, 2), a Naþ / H þ exchanger (SSTR1), AMPA/kainate glutamate channels (SSTR1, 2), phospholipase C (SSTR2, 5), and phospholipase A(2) (SSTR4). Five different G-protein-coupled receptors for somatostatin have been cloned and at least three

KININS AND NEUROPEPTIDES / Other Important Neuropeptides

subtypes have been found in the lung. One of these receptors, sst4, is elevated in disease states in the lung. Furthermore, the expression of sst2A and sst3 receptor mRNAs has been reported in cells of the immune system such as activated macrophages and T and B lymphocytes. Significant quantities of somatostatin have been found in the lung and there is evidence to suggest that it may have anti-inflammatory properties and may play a regulatory role in secretion of mucus. Somatostatin has been shown to have potent immunomodulatory actions on secretion by immune cells such as immunoglobulin (Ig) production by activated B cells and cytokine production by activated T cells and macrophages. It has also been shown to be involved in the regulation of the Th1/Th2 pattern of cytokine secretion. Somatostatin also inhibits IgE-stimulated histamine from basophils and tumor necrosis factor alpha (TNF-a), IL-1b, and IL-6 from monocytes. Somatostatin has been shown to be involved in the inhibition of neutrophil chemotaxis, natural killer cell activity, and the phagocytic activity of monocytes/macrophages. The proliferative response to antigens and mitogens is modulated by somatostatin; this can be inhibitory or stimulatory depending on the concentration of somatostatin present. Somatostatin has been shown to inhibit secretion of mucus and somatostatin analogs have been shown to inhibit the bronchoconstriction that occurs in patients with carcinoid syndrome. The evidence suggests that somatostatin may be of importance in the pathophysiology of a variety of lung diseases and may have a beneficial anti-inflammatory role in inflammatory diseases of the airways.

Cholecystokinin The neuropeptide cholecystokinin octapeptide (CCK-8) has been found in significant quantities in the lungs of various mammalian species but the portion colocalized with nerve fibers is found to be very low. CCK-8 has been shown to protect the lung against injury by endotoxin, reducing the inflammatory response and protecting against the development of pulmonary hypertension. Conversely, CCK-8 potently constricts airways in guinea pigs and humans and this effect is potentiated by prior sensitization to allergen. This direct constrictor effect on airway smooth muscle can be reversed by pretreatment with a specific CCK antagonist. There are two known receptors, CCK-A and CCK-B, and the constrictor effect is thought to be mediated via the CCK-A receptor, which is more widely distributed in the peripheral tissues. The CCK-A and CCK-B receptor genes have been found in the lung in vascular endothelial cells, macrophages, bronchial epithelial cells,

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and alveolar epithelial cells where they mediate the regulatory actions of CCK-8 on these cells. Although CCK-8 is not closely associated with nerves in the airways, its constrictor effect on airway smooth muscle and its presence in many cell types may suggest a role in airway disease.

Neuropeptide Y NPY is a 36 amino acid neuropeptide that participates via at least five G-protein-coupled receptors in the regulation of a large number of physiological and pathophysiological processes in the respiratory system, immune system, nervous system, and endocrine system. Subtypes Y1, Y2, Y4, and Y5 are expressed in humans. Their physiologic ligands are the neurotransmitter NPY and the two hormones peptide YY (PYY) and pancreatic polypeptide (PP). NPY is distributed in the region of sympathetic nerves found in the nasal mucosa, bronchial vessels, and seromucous glands and is often colocalized with catecholamines. NPY has long been proposed to play a role in the pathogenesis of inflammatory diseases and serum levels of NPY are increased during asthma exacerbations. The number of NPY-immunoreactive nerves in the airways may remain constant in patients with airway inflammatory disease although studies have shown NPY-immunoreactive nerves to be significantly decreased in the smooth muscle of patients with asthma and chronic bronchitis. NPY exerts a major influence on humoral and cellular immune functions and is known to modulate immune cell distribution, T-helper cell differentiation, mediator release, and natural killer cell activation. In addition to these direct effects, NPY also acts as an immunomodulator by influencing the effects of a variety of other neurotransmitters. NPY has no direct effect on airway smooth muscle or on mucus secretion but may cause bronchoconstriction via the release of prostaglandins and has been shown to enhance cholinergic and adrenergic stimulation of mucus secretion. Conversely, local treatment of the nasal mucosa with NPY reduced nasal obstruction and mucus secretion evoked by allergen challenge in humans. This evidence suggests a possible role for NPY in the development of pulmonary inflammatory disorders and studies with selective antagonists and agonists may help to elucidate that role.

Opioids Endogenous opioids are formed by proteolysis of larger precursor molecules. There are three such precursor molecules: proenkephalin A, which is processed to form the family of enkephalins; prodynorphin,

526 KININS AND NEUROPEPTIDES / Other Important Neuropeptides

which yields dynorphin A and B; and a- and b-neoendomorphins and propriomelanocortin, which give rise to b-endorphin. Endogenous opioids endomorphin-1 and -2 have also been described that act at the MOP (m ¼ mu for morphine) receptor in a naloxonesensitive fashion. Relatively recently another endogenous opioid-like peptide, nociceptin/orphanin FQ, was isolated which has some homology with dynorphin but lacks the N-terminal tyrosine residue that defines the ‘classical’ opioids and is required for receptor activation. Both the endomorphins and nociceptin differ structurally from one another and from the classical opioids and are thought to derive from different precursor molecules. Endogenous opioids are synthesized by nerves innervating the airways. Immunoreactivity for [Met]enkephalin-Arg6-Gly7Leu8 and Met and Leu-enkephalin have been demonstrated in nerve fibers innervating the trachea and major bronchi in the rat and guinea pig. These fibers were present in smooth muscle bundles, around airway glands, in the lamina propria, and in the walls of pulmonary vessels. Pulmonary neuroepithelial endocrine cells have been shown to contain a number of regulatory neuropeptides, including enkephalins. Enkephalins have been found in normal lung tissues and possess both immunostimulant and immunosuppressive properties. The cells of the immune system such as T and B cells can synthesize and secrete significant amounts of enkephalins. Leu-enkephalin has been localized to neuroendocrine cells in mammalian airways and met-enkephalin immunoreactive nerves have also been found. To date, four opioid receptors have been cloned: the MOP, the KOP (k ¼ kappa for ketocyclazocine), the DOP (d ¼ delta for deferens because it was first identified in mouse vas deferens), and the NOP-R (initially called LC132, ORL-1, or nociceptin/orphanin FQ receptor). Met-enkephalin and enkephalin-containing intermediary peptides of the prohormone pro-enkephalin A are produced and secreted by human peripheral blood T cells and monocytes and play an important regulatory role in the immune response. If the production of met-enkephalin and enkephalin-containing intermediary peptides is interrupted, it results in an enhancement of the proliferative T cell response and an inhibition of monocyte IL-6 secretion. The immunomodulatory effects of met-enkephalin are blocked by the opioid antagonist naloxone, suggesting these effects are mediated by opioid receptors on the cells involved. Opioids have also been shown to inhibit neurogenic mucus secretion and vagal nerve mediated plasma leakage in the airways by presynaptic inhibition of the release of neuropeptides from sensory nerve endings. Furthermore, an inhibitory action was

also revealed on plasma exudation induced by cigarette smoke in guinea pig bronchi. Similarly, opioids have also been reported to inhibit nonadrenergic, noncholinergic, sensory nerve-mediated bronchoconstriction in guinea pig airways and cholinergic neurotransmission in both guinea pig and human airways in vitro. The opioid receptor mediating these inhibitory actions on airway nerves appears to be the MOP subtype. The mechanism of the inhibition is unknown but may be via the opening of large conductance calcium-activated potassium channels. Opioids such as codeine, morphine, and dihydrocodone have also been demonstrated to be antitussives due to their action at central and peripheral MOP opioid receptors located on sensory nerve endings. Recently, both DOP receptor antagonists and agonists have been found to be antitussive in animal models. Additionally, antitussive KOP receptor agonists have been reported. Therefore the evidence suggests that endogenous opioids may act as regulators of the neural and inflammatory response and may have a role to play in inflammation in the airways and in the suppression of reflex events such as cough.

Nociceptin Nociceptin/orphanin FQ (N/OFQ) is an opioid-like peptide and is the endogenous ligand for the NOP receptor. NOP receptors are distributed widely in the CNS and on airway nerves. Nociceptin has been found to inhibit the release of sensory neuropeptides following depolarization of C fibers and to inhibit bronchospasm in the guinea pig. Recently, it has been demonstrated that N/OFQ inhibited cough induced by mechanical stimuli or capsaicin in guinea pigs and cats. This suggests that NOP receptors are involved in the modulation of the cough reflex and that selective NOP receptor agonists might therefore have potential as novel peripherally acting antitussives, although to date there have been no studies with such drugs in humans.

Neurotensin Neurotensin (NT) is a 13 amino acid tridecapeptide localized to epithelial cells and nerves that fulfils a dual function: as a neurotransmitter/neuromodulator in the nervous system, and a paracrine and circulating hormone in the periphery. Three NT receptors, NTS1, NTS2, and NTS3, have been cloned to date. NTS1, and NTS2 belong to the family of seven transmembrane G-protein-coupled receptors, whereas NTS3 is a single transmembrane domain protein (also known as sortilin) that belongs to a recently identified family of sorting receptors. Most of the known peripheral

KININS AND NEUROPEPTIDES / Other Important Neuropeptides

and central effects of NT are mediated through NTS1. NT is a potent bronchoconstrictor, an effect that is modulated by the enzyme neutral endopeptidase. Neutral endopeptidase levels are reduced in airway inflammatory disease states and this leads to enhanced neurogenic inflammation, which may involve NT. Further evidence for the proinflammatory nature of NT is that it promotes the adhesion of neutrophils to bronchial epithelial cells and causes the degranulation of certain types of mast cell. There are no reported studies as yet on the effects of NT in human airways.

Pituitary Adenylate Cyclase-Activating Polypeptide PACAP is a 38 amino acid neuropeptide widely distributed in the central and peripheral nervous systems. PACAP-containing nerve fibers have been found in association with bronchial and vascular smooth muscle and appear to be more abundant than VIP-containing nerves in human bronchi. PACAP receptors have been found at all levels in the respiratory system. There are three distinct receptor subtypes: the PACAP-specific PAC1 receptor, which is coupled to several transduction systems; and the two PACAP/VIP-indifferent VPAC1 and VPAC2 receptors, which are primarily coupled to adenylyl cyclase. PAC1 receptors are particularly abundant in the brain and pituitary and adrenal glands whereas VPAC receptors are expressed mainly in the lung, liver, and testis. PACAP exists in two endogenous forms, PACAP-27 and PACAP-38, and these display several activities that may be relevant to the understanding of airway diseases such as asthma and COPD. They modulate inflammatory cell activity, mediating effects on lymphocyte mobility. PACAP-38 is present in human lung and is a more potent dilator of human bronchi than VIP, suggesting a role for PACAP-38 in the endogenous regulation of airway tone. This effect on smooth muscle tone is more sustained than that seen with PACAP-27. PACAP-38 has also been shown to enhance phagocytosis in macrophages. PACAP-38 inhibits chemotaxis and the release of the proinflammatory cytokines TNFa, IL-2, IL-6, and IL-12, and promotes the release of the anti-inflammatory cytokine IL-10, suggesting an overall anti-inflammatory action. PACAP-38 has also been shown to be a potent stimulator of airway mucus secretion. Owing to the bronchodilator properties of PACAP, synthetic analogs are currently under

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investigation to examine their potential as putative treatments for asthma. See also: Asthma: Overview. Capsaicin. Chronic Obstructive Pulmonary Disease: Overview. Kinins and Neuropeptides: Bradykinin; Neuropeptides and Neurotransmission; Tachykinins; Vasoactive Intestinal Peptide. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; T cells; Pulmonary Macrophages. Mucus. Neurophysiology: Neuroendocrine Cells. Smooth Muscle Cells: Airway.

Further Reading Barnes PJ, Baraniuk JN, and Belvisi MG (1991) State of the art: neuropeptides in the respiratory tract. Part 1 (review). American Review of Respiratory Diseases 144(5): 1187–1198. Barnes PJ, Baraniuk JN, and Belvisi MG (1991) State of the art: neuropeptides in the respiratory tract. Part 2 (review). American Review of Respiratory Diseases 144(6): 1391–1399. Bartfai T, Hokfelt T, and Langel U (1993) Galanin – a neuroendocrine peptide (review). Critical Review of Neurobiology 7(3–4): 229–274. Crawley JN and Corwin RL (1994) Biological actions of cholecystokinin (review). Peptides 15(4): 731–755. Cutz E (1982) Neuroendocrine cells of the lung. An overview of morphologic characteristics and development (review). Experimental Lung Research 3(3–4): 185–208. Groneberg DA, Folkerts G, Peiser C, Chung KF, and Fischer A (2004) Neuropeptide Y (NPY) (review). Pulmonary Pharmacology & Therapeutics. 17(4): 173–180. Groneberg DA, Quarcoo D, Frossard N, and Fischer A (2004) Neurogenic mechanisms in bronchial inflammatory diseases. Allergy 59: 1139–1152. Krantic S (2000) Peptides as regulators of the immune system: emphasis on somatostatin (review). Peptides 21(12): 1941– 1964. Scheuermann DW, Adriansen D, Timmermanns JP, and De Groodt-Lasseel MH (1992) Comparative histological overview of the chemical coding of the pulmonary neuroepithelial endocrine system in health and disease (review). European Journal of Morphology 30(2): 101–112. Springer J, Geppetti P, Fischer A, and Groneberg DA (2003) Calcitonin gene-related peptide as inflammatory mediator (review). Pulmonary Pharmacology and Therapeutics 16(3): 121–130. Sunday ME (1997) Neuropeptides and lung development. In: McDonald JA (ed.) Lung Growth and Development, pp. 401– 494. New York: Dekker. Uddman R, Hakanson R, Luts A, and Sundler F (1997) Distribution of neuropeptides in airways. In: Barnes PJ (ed.) Autonomic Innervation of the Respiratory Tract, pp. 21–37. London: Harwood Academic. Vaudry D, Gonzalez BJ, Basille M, Fournier A, and Vaudry H (2000) Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions (review). Pharmacological Reviews 52(2): 269–324. Wiedermann CJ (2000) Secretoneurin: a functional neuropeptide in health and disease (review). Peptides 21(8): 1289–1298.

L LARYNGITIS AND PHARYNGITIS H Coates, The University of Western Australia, Perth, WA, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Pharyngitis and laryngitis are inflammatory conditions affecting the upper airway. The majority of these inflammatory conditions are viral in origin and self-limiting. In adults, cigarette smoking and gastroesophageal reflux are major causes of chronic laryngitis, whilst in infants and children gastroesophageal reflux and viral and bacterial infections play the major role in etiology. Symptoms that occur secondarily to the pathologic features of mucosal inflammation and submucosal edema include hoarseness, sore throat, cough, pain, or difficulty in swallowing or, occasionally, airway obstruction. Diagnostic tests, depending on the presumptive diagnosis, may include bacterial culture and serology, histopathologic biopsy, barium radiography, or pH studies. Management may include avoidance or treatment of the underlying cause with viral conditions managed symptomatically, some bacterial infections treated with antibiotics and for more serious conditions such as epiglottitis (supraglottitis) and diphtheria occasionally requiring endotracheal intubation or tracheotomy.

Introduction Laryngitis is an inflammation of the larynx, which frequently results in hoarseness or loss of the voice. Laryngitis may be acute or chronic and the most common form associated with a viral upper respiratory tract infection is self-limiting although bacterial infection may also cause laryngitis in association with bronchitis or pneumonia. Chronic laryngitis is frequently seen in smokers, and also in adults with gastroesophageal reflux disease (GERD). In children acute laryngotracheobronchitis (croup) and epiglottitis (supraglottitis) can lead to severe or even fatal respiratory obstruction. Pharyngitis is an inflammatory condition of the pharynx accompanied by a sore throat and occasionally difficulty in swallowing. It is usually viral but may be caused by bacterial or fungal infection. GERD or particularly extraesophageal reflux (EER) can also cause an acid pharyngitis in adults and children. Serious complications of pharyngitis may include peritonsillar abscess or retropharyngeal abscess, airway obstruction with infectious mononucleosis

or rheumatic fever, glomerulonephritis, or bacteremia from group A beta hemolytic streptococcus (GABHS) infection. Historically, laryngitis and pharyngitis have been known since the earliest medical writings but it is only in the last two centuries that visualization of the larynx has been achieved. The incidence of epiglottitis (supraglottitis) and diphtheria with severe airway obstruction and the need for urgent tracheotomy has declined precipitously since the introduction of vaccinations for both of these conditions.

Epidemiology Pharyngitis and laryngitis are relatively common conditions, especially those spread by viruses, and are more common in late winter and spring as is laryngotracheobronchitis (croup). Three to five per cent of children have at least one episode of croup and it reoccurs in about 5% of these cases. The majority of episodes occur between the ages of 6 months and 4 years, and boys are affected twice as commonly as girls. It has been estimated that prior to the introduction of the Haemophilus influenzae (Hib) vaccine, there were between 3.2 and 8.6 cases of epiglottitis (supraglottitis) per 10 000 pediatric hospital admissions.

Etiology The major etiologic causes of laryngitis are shown in Table 1 and include infective, allergic, and traumatic causes. The great majority of cases of laryngitis, both acute and chronic, are caused by infective agents and GERD in children and adults, as well as cigarette smoking in adults. Vocal cord abuse in both age groups is another common cause of laryngitis. Viral croup covers a group of infections caused by viruses, including laryngotracheitis, laryngotracheobronchitis, and spasmodic croup. The organisms involved include adenoviruses, influenza viruses, parainfluenza virus, rhinovirus, and respiratory syncytial viruses. In addition, there are other factors that predispose a child to croup, including cold temperatures, low humidity, pollution, and passive smoking, as well as

530 LARYNGITIS AND PHARYNGITIS Table 1 Etiology of laryngitis

Table 2 Etiology of pharyngitis

Infective Viral Common cold virus Adenovirus Influenza virus Parainfluenza virus Rhinovirus Respiratory syncytial virus Herpes simplex viruses Bacteria Haemophilus influenzae Diphtheria Syphilis Tuberculosis Fungal Candida albicans Histoplasmosis

Infective Viral Common cold virus Adenovirus Influenza virus Infective mononucleosis (Epstein–Barr virus) Human immunodeficiency virus Bacteria Group A streptococcus Corynebacterium diphtheriae Neisseria gonorrhoea Haemophilus influenzae Borrelia vincentii Ludwigs cellulitis (Anaerobic fusobacteria and spirochetes) Chlamydia Syphilis Tuberculosis Fungal Candida albicans

Allergic Angioedema Trauma GERD/EER Cigarettes Alcohol Burns Corrosives Vocal cord abuse

Trauma GERD/EER Cigarette abuse Alcohol abuse

EER. In pediatric patients, epiglottitis (supraglottitis) is almost always caused by H. influenzae infection although Streptococcus pneumoniae, Streptococcus group A, B, and C, Staphylococcus aureus, Pseudomonas aeruginosa, Candida, and Herpes simplex viruses have been implicated in less than 1% of cases in this age group and usually in immunocompromised patients. Diphtheria remains endemic in many developing countries although it has virtually disappeared from most developed countries with the introduction of diphtheria toxoid and immunization programs. The causative organism is Corynebacterium diphtheriae. GERD and, in particular, EER affect the larynx giving a high incidence of hoarseness, excessive throat clearing, chronic cough, and other otolaryngologic complaints with little in the way of esophagitis. The larynx and pharynx do not have protective mechanisms to prevent mucosal injury, while the esophagus demonstrates a mucosal barrier, bicarbonate production, and peristaltic clearance. Pharyngitis includes inflammation of the structures of the nasopharynx, oropharynx, and laryngopharynx (see Table 2). Nasopharyngitis is common during winter months with adenovirus, influenza virus, parainfluenza viruses, and enterovirus being the most common etiologic factors. Ninety per cent of pharyngotonsillitis is related to GABHS, adenoviruses,

influenza viruses, parainfluenza viruses, enteroviruses, Epstein–Barr virus, and mycoplasma infection. GABHS is the most common and serious bacterium causing pediatric pharyngitis. There is a small risk of rheumatic fever (0.3%) and acute glomerulonephritis (10–15% of those infected with nephritogenic strains). Infectious mononucleosis is caused by Epstein–Barr virus, a member of the herpes virus family, and is a major cause of pharyngitis in adolescents and young adults. EER may cause an acid pharyngitis and has been implicated in sinus and middle ear disease.

Other Kawasaki disease

Pathology Viral croup occurs when the causative virus spreads from the nose and pharyngeal airway to the larynx and trachea. Local cellular defenses are impaired and the ciliary function is inhibited resulting in edema and infiltration of inflammatory cells producing narrowing of the subglottis. Secondary bacterial infection may occur causing purulent secretions, which may compromise the airway. Epiglottitis (supraglottitis) is caused by H. influenzae B bacterium, which is a mesomorphic Gramnegative organism capable of both anaerobic and aerobic growth. The type B strain is unique in that it possesses an antigenic capsule and is capable of invading mucosal tissue. The organism may be spread

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hematogenously and blood cultures may be positive. Invasion of the supraglottic structures results in an intense inflammatory and infective reaction and occasionally microabscess formation in the region that causes the epiglottis to swell and compromise the airway. Diphtheria caused by C. diphtheriae Gram-positive bacillus affects the respiratory passages with an exotoxin released after 2–4 days of incubation causing tissue necrosis and exudate. This exudate eventually can develop into an adherent gray membrane of fibrinous nature, which may cause edema and airway obstruction. EER causes laryngeal and pharyngeal erythema with edema of the vocal cords and false vocal cords, larynx, and pharynx. Because of the lack of protective factors mentioned earlier, less acid or pepsin exposure is needed to cause mucosal damage to the pharynx and larynx. Tonsillitis may be viral or bacterial, particularly with GABHS. Chronic tonsillitis where there is cryptic debris secondary to bacterial interaction with undigested food may also show bacterial biofilm formation within the crypts. Peritonsillar and retroesophageal abscesses occur when bacterial organisms proliferate in between the tonsillar capsule and tonsillar bed and the retropharyngeal tissues and the prevertebral fascia, respectively. Chronic laryngitis in adults is often related to cigarette abuse and may be aggravated by alcohol consumption and EER. Pathologically, there is edema of the submucosa and epithelial changes that may undergo malignant transformation.

Clinical Features Pharyngitis

General features Major symptoms are a sore throat, usually of sudden onset, accompanied by pain and difficulty in swallowing, and fever. In cases of viral pharyngitis there will be rhinitis, conjunctivitis, diarrhea, or cough, which will be absent in ‘strep throat’, caused by GABHS. Fever, headache, and swollen cervical lymph nodes may accompany ‘strep throat’. Gastroesophageal reflux in infants may be normal and in small quantities but persistent reflux may be accompanied by excessive vomiting during the first few weeks of life, forceful vomiting, chronic cough, wheezing, apnea or brief breath holding spells, and excessive crying. In more severe cases, there may be weight loss or slow weight gain. In adults, GERD symptoms may include heartburn, particularly at night, belching, nausea and vomiting, and regurgitation of food with some hoarseness, sore

throat, difficulty in swallowing, and occasionally cough or wheezing. Signs Signs of pharyngitis include erythematous pharynx including the tonsils and soft palate on occasion with a coating or membrane. There may be accompanying cervical lymph node enlargement and tenderness. Classically, streptococcal pharyngitis shows a red pharynx with exudate on the tonsils and petechiae on the soft palate. It may be accompanied by scarlet fever with a red rash on the trunk primarily with a strawberry tongue and pallor around the mouth. The main sign of gastroesophageal reflux in infants and children is diffuse erythema of the oropharynx, unaccompanied by exudate or fever. Investigations If a diagnosis of strep pharyngitis is suspected then throat culture or an antigen detection test should be performed. The throat culture is 90–95% sensitive while the rapid strep test is less sensitive but as specific as the throat culture. Culture must be taken prior to starting antibiotic treatment. There is a 10% false-positive rate in patients who are carriers of GABHS without clinical symptoms. Testing for infectious mononucleosis is indicated if there is generalized lymphadenopathy together with a membrane over the pharynx. If there is suspicion of other infections such as gonorrhea or HIV, then these should be tested for using the appropriate swab or serologic testing. Gastroesophageal reflux may be tested for using a barium swallow examination or esophageal pH monitoring. Intraluminal impedance testing is a new modality for assessing GERD. An endoscopy may be necessary to demonstrate ulceration or inflammation of the esophageal walls or obtain biopsies of the mucosa. In adults, esophageal manometry is used to demonstrate abnormal sphincter pressure. Tonsillitis

General features Acute tonsillitis presents with fever, sore throat, and odynophagia and usually in the absence of acute coryzal symptoms. Chronic tonsillitis is less well defined but may present with chronic sore throat, halitosis, malaise, and coughing up of cryptic debris. Signs The tonsils are usually enlarged, erythematous, and with an exudate on the medial surface, accompanied by tender cervical lymph nodes. The chronically infected tonsil may show dilated surface vessels and it may be possible to express cryptic debris with compression of the tonsil.

532 LARYNGITIS AND PHARYNGITIS

Investigations Throat cultures may show GABHS on testing surface swabs while full blood counts, monospot, or Epstein–Barr virus serology will differentiate infectious mononucleosis from other conditions. Laryngitis

General features Laryngitis may be accompanied by an upper respiratory tract infection or have a history of a recent infection. There is often hoarseness. In the acute form, there will be fever and a possible cervical lymphadenopathy. Chronic laryngitis will have a history of months to years of hoarseness, possible cough or accompanying heartburn and there may be a history of cigarette or alcohol abuse. Laryngotracheobronchitis (croup) often is associated with a mild upper respiratory tract infection with a ‘seal bark’ type cough. This may progress to inspiratory stridor and labored breathing. There may be signs of chest retraction and on auscultation of the chest there may be decreased breath sounds, wheezing, or prolonged inspiration or expiration. Epiglottitis (supraglottitis) presents with a sore throat, difficulty in swallowing, drooling, and difficulty in breathing with a crowing inspiratory stridor. There may be hoarseness, fever, chills, and occasionally cyanosis (Figures 1 and 2). Signs Apart from an accompanying upper respiratory tract infection, the larynx may show a diffusely erythematous larynx with swelling of the vocal cord. It is critical in adults with a long smoking history for a careful mirror or fiber-optic examination of the larynx to rule out carcinoma of the larynx. With epiglottitis (supraglottitis), it is contraindicated to examine the larynx with a tongue blade as this may cause an acute laryngospasm and obstruction.

Figure 1 Transoral view of classical epiglottitis.

Investigations In adult smokers with persistent hoarseness, a microlaryngoscopy examination under anesthesia may be necessary for diagnosis and biopsy purposes to rule out carcinoma of the larynx. Radiography of the neck may reveal a classical ‘rat-tail’ appearance of the subglottis in croup. Epiglottitis (supraglottitis) with airway obstruction is an emergency situation and requires careful assessment and investigation under controlled conditions. Neck X-rays, which might show an enlarged epiglottis, may well be academic and pose a danger of obstruction while the patient is in the radiology suite (Figure 3). A blood or throat culture may show H. influenzae or other bacteria and a full blood count may indicate elevated white blood cell levels.

Figure 2 Classical appearance of child with epiglottitis, sitting up, mouth open, head forward, drooling.

Figure 3 Radiograph showing lateral view with swollen epiglottis.

LARYNGITIS AND PHARYNGITIS 533

Pathogenesis Pediatric esophageal reflux and EER is increasingly being diagnosed with a range of symptoms from postprandial vomiting through to failure to thrive and airway manifestations. GERD in children is generally defined using four categories: 1. Physiologic reflux. This is usually largely asymptomatic with infrequent emesis in children. 2. Functional reflux. This is silent or asymptomatic and can be confirmed by pH monitoring. 3. Pathologic gastroesophageal reflux. This can interfere with growth and cause respiratory complications. 4. Secondary GERD. This is related to a secondary disorder such as neurologic or anatomic abnormality of the esophagus. Respiratory complications of reflux include recurrent bronchitis, pneumonia, croup, and chronic asthma and it may complicate or possibly cause laryngeal contact granulomas and ulcers.

Management and Current Therapy Gastroesophageal Reflux in Children and Adults

There are three phases for treatment of EER in children, which include lifestyle modifications, pharmacologic treatment, and antireflux surgery; the severity of the reflux determines the level of treatment. Conservative measures including elevating the head when in bed, frequent small feeds, thickening of milk, and fasting before bedtime are useful. Adjuncts for conservative management of EER may include H2 receptor blockers, prokinetic agents, or protein pump inhibitors. Conservative measures, antacids, and H2 antagonists are the treatment of choice for mild reflux and in the more severe cases prokinetic agents or protein pump inhibitors are suggested. Surgical intervention includes Nissen fundoplication, which has a 90% success rate in symptom control and a low mortality rate. Management of adults with gastroesophageal reflux is similar to that in children but particular attention should be paid to weight reduction. Laryngitis

The majority of cases of viral laryngitis correspond to conservative management with voice rest, humidification, and anesthetic lozenges. Croup of mild degree can be managed at home with a cool air nebulizer and symptomatic therapy. Occasionally, oral or inhaled steroids are necessary. Serious illness requires hospitalization with aerosolized racemic epinephrine, oxygen, humidification, and consideration of antibiotic therapy for bacterial infections.

Infrequently, intubation is necessary. Epiglottitis (supraglottitis) requires hospitalization, usually in the intensive care unit, and endotracheal intubation may be required and, occasionally, emergency tracheotomy. Intravenous fluids, systemic steroids, and antibiotics are often required. Family members should be treated in view of the contagious nature of the infections. GABHS Pharyngitis

Viral pharyngitis is treated symptomatically but GABHS pharyngitis is managed with penicillin V for 10 days. There are concerns about the length of the regimen and bacteriologic treatment failures occur in up to 35% of young patients. Cephalosporins are an alternative treatment to penicillin therapy. The new generation macrolides such as azithromycin and chlorithromycin are effective in penicillin-resistant patients although there appears to be increasing GABHS resistance to this group of antibiotics. Clindamycin may be used in cases of GABHS carrier states or failure with other antibiotics. Bacterial biofilm in tonsillar crypts may mitigate against effective antibiotic therapy for the chronic tonsillitis states. Bacterial replacement therapy utilizing probiotic bacteria, particularly Streptococcus salivarius, may be applied to prevention of streptococcal pharyngitis. Tonsillitis

Tonsillectomy is indicated for severe recurrent tonsillitis where there are three or more tonsillar infections per year despite adequate medical therapy that are accompanied by fever, dysphagia, cervical adenopathy, or positive GABHS culture and tonsillar exudates. Other indications for tonsillectomy include quinsy (peritonsillar abscess) as well as hypertrophy causing obstructive sleep disorder. If chronic tonsillitis does not respond to a 3–6 week therapeutic trial of clindamycin or amoxicillin clavulanate, then tonsillectomy may need to be considered.

Facets of Treatment Although vaccination for diphtheria and Hib has significantly decreased the presentation of these diseases, a high index of suspicion should still be exercised. There may be compliance problems with a 10 day penicillin treatment for GABHS pharyngitis but a 5-day course of azithromycin or macrolides, in view of the high rate of failure with these agents, must be regarded with caution. Recurrent croup may require further investigation to rule out coexisting anatomical abnormalities such as subglottic stenosis. Adults with chronic laryngitis who persist in smoking require regular follow-up to detect malignant change in the larynx.

534 LEUKOCYTES / Mast Cells and Basophils See also: Aerosols. Allergy: Overview; Allergic Reactions. Asthma: Extrinsic/Intrinsic. Bronchoscopy, General and Interventional. Chronic Obstructive Pulmonary Disease: Smoking Cessation. Corticosteroids: Glucocorticoid Receptors. Croup. Endotoxins. Environmental Pollutants: Overview; Particulate Matter, Ultrafine Particles; Particulate and Dust Pollution, Inorganic and Organic Compounds. Gastroesophageal Reflux. Human Immunodeficiency Virus. Mucus. Occupational Diseases: Inhalation Injury, Chemical. Pediatric Aspiration Syndromes. Pediatric Pulmonary Diseases. Tracheostomy. Tumors, Malignant: Overview. Upper Airway Obstruction. Upper Respiratory Tract Infection. Vaccinations: Bacterial, for Pneumonia; Viral.

Further Reading Bisno AL (2001) Acute pharyngitis. New England Journal of Medicine 344(3): 205–211.

De Souza C, Stankiewicz J, and Pellitteri PK (1999) Textbook of Pediatric Otorhinolaryngology: Head and Neck Surgery, vol. 11. San Diego: Singular Publishing Group. Del Mar CB, Glasziou PP, and Spinks AB (2000) Antibiotics for sore throat. Cochrane Database of Systematic Reviews 4: CD000023. Discolo CM, Darrow DH, and Koltai PJ (2003) Infectious indications for tonsillectomy. Pediatric Clinics of North America 50: 445–458. Jacobs RF (2000) Judicious use of antibiotics for common pediatric respiratory infections. Pediatric Infectious Diseases Journal 19(9): 938–943. McGuirt WF (2003) Gastroesophageal reflux and the upper airway. Pediatric Clinics of North America 50: 487–502. Pichichero ME (2000) Short course antibiotic therapy for respiratory infections: a review of the evidence. Pediatric Infectious Diseases Journal 19(9): 929–937. Wetmore RF, Muntz HR, McGill TJ, et al. (2000) Pediatric Otolaryngology: Principles and Practice Pathways. New York: Thieme. Woodson GE (2001) Ear, Nose and Throat Disorders in Primary Care. Philadelphia: Saunders.

LEUKOCYTES Contents

Mast Cells and Basophils Eosinophils Neutrophils Monocytes T cells Pulmonary Macrophages

Mast Cells and Basophils F Hollins, P Bradding, and C E Brightling, University of Leicester, Leicester, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Mast cells and basophils develop from pluripotential stem cells in the bone marrow. Mast cells migrate into tissue, where they mature into resident tissue mast cells. In contrast, basophils are resident in the circulation and migrate into tissue as part of an allergic response. Following activation, both cell types can degranulate, releasing preformed mediators from their granules and synthesizing lipid mediators and cytokines. Mast cell and basophil degranulation is classically mediated by cross-linking of IgE bound to the high-affinity IgE receptor, but there is increasing recognition that several mechanisms can result in degranulation of these cells. Mast cells have an important role in the immune response to infections, and both mast cells and basophils play a pivotal role in the allergic response and are implicated in the pathogenesis of a number of respiratory diseases, particularly asthma and allergic rhinitis.

In the late 1870s, Paul Ehrlich first identified the mast cell and basophil. For many years, mast cells and basophils were thought to be the same cell or one a progenitor of the other. It has only been in recent years that the emergence of a differing developmental pathway from a common precursor has come to light and is gradually becoming accepted. Mast cells develop from pluripotential hematopoietic stem cells within the bone marrow. They enter the circulation as nongranular immature mast cell precursors and express CD34 þ , CD13 þ , and CD117 þ on their surface. They migrate to tissue, and under the influence of local mediators they differentiate into mature tissue mast cells. Mature human mast cells are classified based on their serine protease content into cells that contain chymase and tryptase (MCTC) or cells that contain tryptase alone (MCT). Basophils are the rarest blood leukocyte, accounting for less than 1% of the total cell population. Like mast cells, basophils develop from pluripotential

LEUKOCYTES / Mast Cells and Basophils 535 Bone marrow

Circulation

Tissue

Fc
RI+ CD34+ Basophil precursor

Mature basophil

bsp-1

CD117low

CD117high

Pluripotent hematopoietic stem cell

Fc
RI+

Mast cell precursor

+ Immature CD13 mast cell

+ Mature CD13 mast cell

Figure 1 Comparison of the ontogeny of the mast cell and basophil. Although they both develop from a common pluripotent hematopoietic stem cell precursor, they have separate development and maturation pathways. Much of the maturation process is yet to be investigated.

respiratory diseases. This article summarizes mast cell and basophil biology and describes their role in health and disease.

Mast Cell and Basophil Biology Cell Trafficking to the Lung

Figure 2 Electron micrograph of a mast cell within the lung tissue showing the rounded nucleus and granules of varying density. Note the dissolution of some of the granules.

hematopoietic stem cells within the bone marrow, where the cell differentiates into a basophil precursor. The basophil matures upon entering the circulation, expressing basophil-specific antibody-1 (Bsp-1). The ontogeny of mast cells and basophils is illustrated in Figure 1, whereas the structure of the mature mast cell is shown in Figure 2. Mast cells have an important role in both the innate and the adaptive immune response to infections. Mast cells and basophils play a central role in the allergic inflammatory response, and both cells are implicated in the pathogenesis of a number of

Immature mast cell precursors are drawn into the circulation from the bone marrow by chemotactic factors and chemokines. Chemokines are a superfamily of low-molecular-weight heparin-binding molecules that serve as potent chemoattractants for cells of the immune system. The chemokine family is divided into four groups, designated CXC, CC, C, and CX3C, depending on the spacing of conserved cysteines (X represents an amino acid). The CC and CXC chemokines are the major chemokine families, consisting of more than 50 members. The C and CX3C chemokines are much smaller families, with each consisting of only 1 or a few members. Chemokines exert their effects through binding to heterotrimeric, seven-transmembrane-spanning, G-protein-coupled receptors. CCR1, -3, -4, -5, and -7 and CXCR1, -2, -3, -4, and -6 receptors have been identified on mast cells, which can bind a multitude of ligands. Some chemokines found to be important in the recruitment of mast cells include interleukin (IL)-8 (CXCL8 binding to CXCR1), SDF-1a (CXCL12 binding to CXCR4), IP-10 (CXCL10 binding to CXCR3), RANTES (CCL5 binding to CCR1, -3, and -5), and eotaxin (CCL11 binding to CCR3).

536 LEUKOCYTES / Mast Cells and Basophils Bone marrow

Circulation

Bronchus

Epithelium

Airway lumen

Smooth muscle

SCF TGF- CXCL10 CCR3 CCR1 CCR4

CXCR3

CD117 CXCR4 CXCR1

Rolling adhesion and migration

CD117 CXCR4 CCR3

Mast cell survival and differentiation

CXCL8 CXCL10 CXCL12 CCL11 TGF- NGF

Precursor recruitment SCF CCL11 CXCL12 Submucosal gland

Figure 3 Mast cell trafficking from the bone marrow into the circulation and then into the tissue in asthma. The important receptors expressed on the mast cell at each point and the cytokines and chemokines that are critical in controlling the mast cell movement are shown.

These chemokines, in conjunction with other chemotaxins such as stem cell factor (SCF), transforming growth factor beta (TGF-b), and nerve growth factor (NGF), are involved in the migration of mast cell precursors into the circulation and the movement of mast cells into tissue. Like mast cells, basophils express several chemokine receptors, including CCR1, -2, -3, and -5 and CXCR1, -2, and -4. The chemokine CCL11, acting via CCR3, induces the most potent basophil migration. In addition, basophils express the prostaglandin-D2 receptor DP2/CRTh2, and ligand activation of this receptor results in basophil migration. Mature mast cells localize to specific structures in the lung, namely nerves, blood vessels, lymphatics, airway epithelium, glands, and airway smooth muscle. This microlocalization of mast cells is likely to be important in the pathogenesis of many lung diseases. The mechanisms involved in mast cell trafficking are summarized in Figure 3. Mast Cell Differentiation and Survival

Once within the tissues, mast cells can survive for a relatively long period of time, possibly days to weeks, in comparison to neutrophils, which survive only for hours. Mast cell survival is thought to occur through the action of cytokines, particularly SCF, which also plays an important role throughout the development and migration of mast cells. Several other growth factors and cytokines are also known to affect the growth and differentiation of mast cells, including

IL-3, IL-4, IL-9, IL-10, and NGF. Basophil growth and survival are mediated by granulocyte macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-5. Mechanisms Mediating Degranulation

The extracellular release of both preformed and newly synthesized mediators by mast cells and basophils, a process termed degranulation, can be mediated by both IgE-dependent and IgE-independent mechanisms. IgE molecules produced by B lymphocytes bind to high-affinity IgE receptors (FceRI) on the surface of mast cells and basophils. IgE can also bind to FcgRII, FcgRIII, and galectin-3, which are also found on the surface of mast cells. However, IgE elicits its functions only through binding to FceRI. FceRI is a heterotetrameric receptor composed of an IgE-binding a subunit, a four-transmembranespanning b subunit, and two disulfide-bonded g subunits (abg2). The g subunits are signal-transducing molecules, producing a signal that can then be amplified by the b subunits. Cross-linking of the FceRI with IgE and antigen induces activation of protein tyrosine kinases, including those of the Src, Syk, and Tec families. Pathways that involve the activation of these protein tyrosine kinases ultimately lead to degranulation. In addition, several IgE-independent mechanisms can cause degranulation: (1) physical factors (i.e., high temperature, ionizing irradiation, and mechanical trauma); (2) chemical substances (i.e., proteases,

LEUKOCYTES / Mast Cells and Basophils 537

toxins, and venoms); (3) endogenous mediators (i.e., tissue proteases and cationic proteins derived from eosinophils and neutrophils); (4) osmotic effects; (5) adenosine, via activation of the adenosine A2B receptor; and (6) anaphylatoxins C3a, C4a, and C5a, formed during activation of complement can trigger degranulation through activation of the C5a receptors on the surface of mast cells and basophils. However, it is important to note that human lung mast cells are devoid of the C5a receptor; hence, it is unlikely that this mechanism of mast cell degranulation is important in the lung. Following degranulation of mast cells and basophils, there is an immediate release of granule products. Subsequently, there is synthesis and release of lipid mediators over several minutes and the production and secretion of cytokines, chemokines, and growth factors over several hours. Preformed Mediators

Histamine Two major sources of histamine within the body are mast cells and basophils, containing approximately 3 and 1 pg per cell, respectively. Histamine plays a pivotal role in allergic inflammation. Following mast cell degranulation, there is a local and systemic increase in histamine concentration. Basophils are then attracted into the tissue by cytokines and chemokines, at which point they too degranulate, releasing histamine, and contribute to the delayed late-phase reaction. The histamine receptor family is a group of seventransmembrane-spanning units that couple ligandbinding with intracellular reactions through guanosine triphosphate (GTP)-binding heterotrimeric proteins. The histamine receptors are subclassified into four groups: H1–H4. The different receptor subtypes also have differing intracellular secondary messengers; H1 and H4 receptors mobilize intracellular calcium, whereas H2 receptor utilizes cyclic adenosine monophosphate and the H3 receptor can signal through both of these pathways. There is evidence of H1, H2, and H4 expression on both human mast cells and basophils. These receptors act to induce or modulate cytokine synthesis in allergic inflammation, having varying effects on different cell types. Proteases The mast cell granules contain neutral proteases. Four such proteases have been identified: tryptase, chymase, carboxypeptidase, and cathepsin G. Tryptase Mast cell tryptase is a tetrameric neutral serine protease with a molecular weight of 134 kDa, and it composes 20% of the total cellular mast cell protein. Tryptase is found in negligible quantities in

other cells, making it a good mast cell marker for identification of both the cells and their causative properties. The enzyme is made up of four noncovalently bound subunits, with each subunit having one enzymatically active site. There are two main types of mast cell tryptase, a-tryptase and b-tryptase, with approximately 90% sequence homology between them. a-Tryptases are classified into aI- and aII-tryptases, and b-tryptases are classified into bI-, bII-, and bIII-tryptases. bII-Tryptase is stored in the secretory granules of mast cells. In contrast, a-protryptase is secreted constitutively from mast cells as an inactive proenzyme. The activation of b-II-pro-tryptase involves two proteolytic steps. The first is an autocatalytic intermolecular cleavage, which occurs optimally at an acidic pH and in the presence of heparin or dextran sulfate. The resulting product is a monomer, which is approximately 50 times less active than the final tetramer. The second step involves the removal of the remaining precursor dipeptide by dipeptidyl peptidase I, thus allowing the mature peptide to spontaneously form the active tetramer. This process also requires the presence of heparin or dextran sulfate. Tryptase can activate a number of peptides by cleaving certain extracellular substrates, such as vasoactive intestinal peptide, calcitonin gene-related peptide, fibronectin, kininogens, and the protease activated receptor (PAR)-2. Airway epithelial and smooth muscle cells, endothelial and vascular smooth muscle cells, terminal bronchial epithelium, type II pneumocytes, and mast cells within the respiratory tract express PARs. Tryptase is a potent mitogen for epithelial cells, airway smooth muscle cells, and fibroblasts. During inflammation, tryptase can stimulate the release of granulocyte chemoattractant IL-8 and upregulate expression of ICAM-1 on epithelial cells. It induces the expression of IL-1b mRNA, which may be important for the recruitment of inflammatory cells to the sites of mast cell activation. The release of tryptase also promotes the degranulation of neighboring mast cells via activation of PAR-2, thereby initiating a positive feedback mechanism. Chymase As outlined previously, there are two types of mast cells: MCT and MCTC. MCT mast cells are the predominant mast cell subtype in the lung, comprising 90–95% of the lung mast cell population. The MCTC mast cells are found in increased numbers in the airway smooth muscle, epithelium, and glands, and their numbers increase in disease. Mast cell chymase is a glycosylated chymotryptic serine protease with a molecular weight of 30 kDa. It is stored in the same secretory granules as those that contain tryptase, but it is released from mast cells in

538 LEUKOCYTES / Mast Cells and Basophils

a macromolecular complex with carboxypeptidase and proteoglycans, which is distinct from tryptase. Like tryptase, chymase is present in the granules as a catalytically active form, but due to the acidic condition within the granules, this activity is suppressed. Chymase has a net charge of þ 13, allowing it to bind strongly with negatively charged granule proteoglycans and to cells and basement membranes. Angiotensin I is readily hydrolyzed by chymase to form angiotensin II, a reaction that is catalyzed more efficiently than with the angiotensin enzyme. Circulating a-antichymotrypsin and a1-antitrypsin and the respiratory tract inhibitors bronchial secretion inhibitor and antileukoprotease inhibit chymase. Chymase can modulate inflammatory processes. It can convert cytokines such as IL-1b from their inactive to active form and it can degrade others, such as IL-4. In addition, chymase has a role in tissue destruction and matrix remodeling because chymase can activate stromelysin and interstitial collagenase, and it can convert procollagen I to collagen-sized fragments and degrade type IV collagen. Interestingly, chymase, like tryptase, can activate cells via PARs. Chymase acts on PAR-1, which is expressed on several inflammatory and mesenchymal cells and can lead to cell activation and proliferation. Carboxypeptidase Human mast cell carboxypeptidase is a zinc metalloexopeptidase, 34.5-kDa monomer only found in mast cells of the tryptase/ chymase phenotype. It cleaves the carboxy terminal of the His-Leu bond of angiotensin I, generating desLeu10 angiotensin I, an inhibitor of angiotensinconverting enzyme. Cathepsin G Various physiological effects are attributed to cathepsin G, including antimicrobial activity, degradation of extracellular matrix, vasoregulation, activation of neutrophil elastase, processing of IL-8, and mast cell degranulation. Synthesized Mediators

Lipid-Derived Mediators Arachidonic acid is released from the phospholipid membrane following activation, the metabolites of which are cyclooxygenase and lipooxygenase. The cyclooxygenase pathway results in the synthesis of prostaglandin D2 (PGD2), whereas the lipooxygenase pathway liberates leukotrienes, particularly leukotriene C4 (LTC4). PGD2, which is synthesized by mast cells but not basophils, can bind to receptors on smooth muscle and can act as a vasodilator and a bronchoconstrictor. It can also cause histamine release from basophils, and it causes chemotaxis and accumulation

of inflammatory cells at the site of inflammation, aided by its effect on increasing the microvascular permeability. There are two types of receptor for PGD2: DP1 and DP2 (CRTH2). Both of these receptors are G-protein-coupled rhodopsin-type receptors with seven transmembrane domains. DP1 receptors are prostanoid receptors found on various types of hematological and nonhematological cells, including platelets, neutrophils, and smooth muscle cells. DP2 (CRTH2) receptors are a member of the chemokine receptor family and are found exclusively on the hematological cells: basophils, eosinophils, and Th2 cells. Leukotrienes are produced upon the activation of mucosal mast cells and basophils. They bind to receptors on smooth muscle and cause prolonged bronchoconstriction (10–1000 times more potent than histamine), induce prolonged wheal-and-flare response, enhance venular permeability, promote mucus secretion, and act as chemotactic agents for neutrophils and eosinophils. The other type of lipid mediator produced by mast cells and basophils is platelet-activating factor (PAF). PAF has a diverse range of physiological actions, varying from aggregation and degranulation of platelets and neutrophils to a variety of cellular effects, including stimulation of chemotaxis and chemokinesis. It can also result in bronchoconstriction and vasodilatation, and it may cause retraction of endothelial cells. Cytokines/Chemokines Mast cells and basophils produce many different cytokines and chemokines, at differing amounts, that may contribute toward an inflammatory response. The cytokines and chemokines produced are shown in Table 1. Mast cell and basophil activation results in the de novo synthesis of these cytokines; however, TNF and IL-4 may already be stored preformed in granules and rapidly released upon FceRI cross-linking.

Mast Cell and Basophil Functions in the Normal Lung Mast cells are found within tissues that interface with the local environment, such as the skin, airways, and intestine, and are therefore well placed to initiate and enhance early responses to a variety of challenges. In response to pathogens, mast cells produce cytokines, proteases, and lipid-associated mediators. This leads to immune vascular permeability and recruitment of other inflammatory cells, such as neutrophils and eosinophils. The acquired immune response is enhanced through influencing the function and maturation of dendritic cells and increasing recruitment

LEUKOCYTES / Mast Cells and Basophils 539 Table 1 Mast cell basophil contents Mast cell

Basophil

Function

Resting

Activated

Activated

Resting

Histamine

þ

þ

þ

þ

Vascular permeability

Proteases Tryptase Chymase Carboxypeptidase Cathepsin G

þ MCTC only MCTC only MCTC only

þ MCTC only MCTC only MCTC only











Tissue remodeling

Lipid-derived mediators Prostaglandin D2 Leukotrienes





þ þ






þ




þ




þ





þ

þ þ þ þ þ





þ þ


þ


þ

IL-3 IL-4 IL-5 IL-13


þ



þ þ þ þ








þ
þ

IL-12 IFN-g





þ þ







IL-10




þ







TGF-b




þ

Chemokines CCL1 (TCA3/I309) CCL2 (MCP-1) CCL3 (MIP-1a) CCL4 (MIP-1b) CCL5 (RANTES) CCL20 (LARC)









þ þ þ þ þ þ

Recruitment of effector cells

CXCL8 (IL-8) CXCL10 (IP-10)





þ þ

Recruitment of effector cells and immune response regulation

Platelet-activating factor

Cytokines IL-1a and -b IL-6 IFN-a and -b GM-CSF TNF-a




Recruitment of effector cells, immune response regulation, and angiogenesis, bronchoconstriction, and edema promotion Effector cell activation and chemoattractant

Inflammation induction

Th2 cytokines

Th1 cytokines

Inflammation and angiogenesis induction

þ , presence;
, absence; blank, unknown.

of T cells in the lymph nodes through the actions of TNF-a, which optimizes the environment for effective antigen presentation. Both mast cells and basophils are able to detect and respond to pathogens through the use of direct or indirect receptors. Direct cell activation is essential for initiation of an early innate immune response as well as the generation of appropriate acquired immunity. For this direct interaction to occur, these

cells have cell surface receptors that can interact directly with pathogens. Toll-like receptors (TLRs) are an example of such a receptor type. TLRs recognize products from pathogens such as gram-positive and gram-negative bacteria. Binding of these pathogen products to TLRs can result in their activation, which could ultimately lead to a proinflammatory response. In mammals, 11 TLRs have been identified, each having a distinct function with regard to its

540 LEUKOCYTES / Mast Cells and Basophils

immune recognition properties. Mast cells have been found to express TLR1, -2, -3, -4, -6, and -9, each capable of activating mast cells upon binding of the appropriate ligand. Indirect cell activation is important in the event of secondary or subsequent infections and can occur through several mechanisms, including Fc receptormediated activation. Wound Repair

Mast cells are resident in tissues, particularly in association with endothelial and epithelial cell basement membranes, and are increased at sites of inflammation, injury, and fibrosis. Although mast cells are known to both release and generate proinflammatory mediators in response to inflammatory stimuli, the cells express large amounts of mRNA for collagen IV, laminin, and heparin sulfate proteoglycan, suggesting that these cells may contribute to normal tissue repair.

Mast Cells and Basophils in Respiratory Disease Asthma

Asthma is an important cause of morbidity and mortality worldwide, and its prevalence is increasing. More than five million people in the United Kingdom are currently taking treatment for asthma. It is characterized by the presence of variable airflow obstruction and airway hyperresponsiveness (AHR), which is the exaggerated bronchoconstrictor response of the airways to a wide range of specific and non-specific stimuli. In asthma, mast cells are present in an ‘activated’ secretory state. Mast cell mediators have the capacity to contribute to the development and maintenance of the airway inflammation and associated disordered airway physiology, which characterizes asthma. Secretion of the autacoid mediators histamine, PGD2, and LTC4 induces bronchoconstriction, mucus secretion, and mucosal edema and thus contributes to the symptoms of asthma. Basophils are increased in number in the bronchial submucosa of asthmatics and play an important role in the late asthmatic reaction in response to allergens. The mast cell has been implicated as playing an important role in the development of AHR. Strong correlations have been observed between the severity of AHR and mast cell numbers, histamine concentrations, and spontaneous histamine release in bronchoalveolar lavage (BAL). In biopsy and postmortem studies, mast cell numbers were found to be increased in the airway epithelium, glands, and airway smooth

muscle. The strongest evidence supporting the role of the mast cell in the development of the asthmatic phenotype is derived from comparisons of the immunopathology of asthma and nonasthmatic eosinophilic bronchitis. Eosinophilic bronchitis accounts for 15% of the cases of cough referred to respiratory specialists. It is characterized by corticosteroid responsive cough and the presence of a sputum eosinophilia occurring in the absence of variable airflow obstruction or AHR. Thus, any differences in the immunopathology of asthma compared to eosinophilic bronchitis may yield specific clues as to the ‘essential’ components of asthma. Both conditions share many immunopathological features, including eosinophilic airway inflammation, Th2 cytokine expression, and subbasement membrane collagen deposition. The only immunopathological feature that is different is that there are significantly increased numbers of mast cells within the airway smooth muscle bundles in bronchial biopsies in patients with asthma but virtually none in patients with eosinophilic bronchitis or in normal subjects. The majority of these mast cells are of the MCTC phenotype containing both tryptase and chymase, with increased IL-4 and IL-13, but not IL-5, expression. In contrast, there are almost no T cells or eosinophils in the smooth muscle of any of these groups. The number of airway smooth muscle mast cells in asthma is negatively correlated with the degree of AHR. Taken together, this body of evidence suggests that the microlocalization of mast cells is a critical event in the development of the asthmatic phenotype.

Allergic Rhinitis

Allergic rhinitis is a very common condition, affecting 10–25% of the world’s population. Allergic rhinitis is an inflammatory disorder of the nasal mucosa typified by the symptoms of nasal itch, sneeze, anterior nasal secretions, and nasal blockage. These symptoms arise from the interaction between mediators and neural, vascular, and glandular structures within the nose. Nasal itch, sneezes, and rhinorrhea are predominantly neural in origin, whereas nasal obstruction is predominantly vascular. Nasal biopsy studies show accumulation of eosinophils, mast cells, and basophils in an activated state within the lamina propria and epithelium in both seasonal and perennial allergic rhinitis. The role of the mast cell and basophil in allergic rhinitis is supported by the observation that histamine and leukotriene nasal insufflation induces the symptoms of rhinitis, and that these symptoms are improved by nasal corticosteroids and H1 receptor antagonists.

LEUKOCYTES / Mast Cells and Basophils 541 Anaphylaxis

Systemic anaphylaxis arises when mast cells, possibly along with other cell types, are provoked to secrete mediators that evoke a systemic response. Increased tryptase levels in the circulation provide a precise indicator of mast cell involvement. Although it is recognized that the mast cell is the predominant cell mediating anaphylaxis, in some circumstances there are elevations of histamine but not tryptase, suggesting that basophil-dependent anaphylaxis may occur. Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a common condition predominantly caused by smoking. COPD is the major cause of respiratory failure and a common cause of chronic disability. In contrast to asthma, COPD is characterized by irreversible airflow obstruction. The physiological abnormalities observed in COPD are due to a combination of emphysema and obliteration of the small airways. In some, but not all, reports, histamine and tryptase sputum concentrations are increased in COPD and chronic bronchitis. However, mast cell numbers are not increased in the airway wall. Thus, the role of mast cells and basophils in COPD is unclear. Lung Fibrosis/Interstitial Lung Disease

Lung fibrosis is characterized by inappropriate tissue remodeling and enhanced vascular remodeling, fibroproliferation, and deposition of extracellular matrix. Without resolution, continued fibrosis leads to loss of lung function and ultimately death. In chronic fibrotic lesions, a correlation exists between the number of mast cells and the severity of fibrosis. Mast cells and fibroblasts are usually found in close proximity to one another within the alveolar septae in both the normal and the fibrotic lung. These mast cells often appear to contain fewer numbers of granules, indicating that there is partial ongoing degranulation. In support of this, elevated histamine levels have also been described in the BAL of patients with lung fibrosis. Basic fibroblast growth factor (bFGF) is also produced by mast cells and is a potent fibrogenic cytokine. Mast cells are thought to be the predominant cells within the lung interstitium expressing bFGF. Indeed, the distribution of bFGF þ mast cells correlates with the deposition of extracellular matrix and, accordingly, the extent of fibrosis. This therefore suggests that mast cells do contribute to the fibrotic process. Fibroblasts have been held to be the predominant cells responsible for repair and remodeling within the

lung, but evidence suggests that blood-derived fibroblast precursors, fibrocytes, may play a critical role in the deposition of matrix proteins in the development of lung fibrosis. How mast cells and fibrocytes interact in terms of fibrocyte activation, differentiation, and survival is unknown. Lung Cancer

Mast cell numbers are increased in several malignant tumors, including non-small cell carcinoma (NSCLC). The functional significance of the accumulation of mast cells within lung tumors is a subject of controversy because of contradictory experimental and clinical data. Mast cells are a rich source of TNF-a, cytotoxic for some tumors, and mast cell-derived cytokine, IL-4, inhibits growth and induces apoptosis in human carcinoma cells. In patients with NSCLC, especially lung adenocarcinoma, the high mast cell count group has a significantly worse prognosis than the low mast cell count group. In vitro, mast cell conditioned medium stimulates capillary endothelial cell migration. Mast cell granules localize within endothelial cells and stimulate their proliferation. Mast cell products also degrade connective tissue matrix to provide space for neovascular sprouts to form. Mast cell histamine induces angiogenesis and heparin promotes new vessel formation in vivo. Tryptase derived from mast cells stimulates vascular tube formation and functions as a mitogen for microvascular endothelial cells. Chymase promotes angiogenesis through the local chymase-dependent angiotensin II-generating system in pathophysiological angiogenesis. Mast cells produce angiogenic factors vascular endothelial growth factor (VEGF) and bFGF; VEGF expression is documented in intratumoral stromal mast cells in NSCLC. Mast cells synthesize and store large amounts of matrix metalloproteinases-2 and -9, which can degrade collagen types IV, V, VII, and X and fibronectin, the major components of the interstitial stroma and subendothelial basement membrane. This may contribute to the progression from in situ to invasive and metastatic solid tumors.

Summary and Therapeutic Considerations Mast cells are present in the respiratory mucosal surface and are localized with epithelial and mesenchymal cells. They have an important role in the innate immune response and wound repair, and they play a role in the development of lung fibrosis and angiogenesis in lung cancer. Both mast cells and basophils have an established role in allergic

542 LEUKOCYTES / Eosinophils

disease. Mast cells are the key in the development and maintenance of chronic airway inflammation in asthma, and their microlocalization within the airway smooth muscle bundles is critical in the development of disordered airway physiology in the disease. Furthering our understanding of mast cell and basophil migration to the lung and their survival, maturation, and activation in tissue may provide novel targets for the effective treatment of lung disease. In particular, treatments directed at modifying interactions between mast cells and airway smooth muscle may provide a novel approach to the effective treatment of asthma. See also: Adenosine and Adenine Nucleotides. Adhesion, Cell–Cell: Epithelial. Allergy: Overview; Allergic Reactions; Allergic Rhinitis. Angiogenesis, Angiogenic Growth Factors and Development Factors. Apoptosis. Asthma: Overview; Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; Exercise-Induced; Extrinsic/Intrinsic. Bronchiolitis. Bronchodilators: Beta Agonists. Chemokines. Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations; Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General. Chymase and Tryptase. Complement. Defense Systems. Eotaxins. Fibroblast Growth Factors. Fibroblasts. Histamine. Interstitial Lung Disease: Overview; Cryptogenic Organizing Pneumonia; Hypersensitivity Pneumonitis; Idiopathic Pulmonary Fibrosis. Lipid Mediators: Overview; Leukotrienes; Platelet-Activating Factors; Prostanoids. Pulmonary Fibrosis.

Further Reading Bradding P and Holgate ST (1999) Immunopathology and human mast cell cytokines. Critical Reviews in Oncology/Hematology 31: 119–133. Brightling CE and Bradding P (2005) The re-emergence of the mast cell as a pivotal cell in asthma pathogenesis. Current Allergy and Asthma Report 5(2): 130–135. Galli SJ, Nakae S, and Tsai M (2005) Mast cells in the development of adaptive immune responses. Nature Immunology 6(2): 135–142. Gurish MF and Austen KF (2001) The diverse roles of mast cells. Journal of Experimental Medicine 194(1): F1–F5. Kawakami T and Galli SJ (2002) Regulation of mast cell and basophil function and survival by IgE. Nature Reviews: Immunology 2: 773–786. Marone G, Triggiani M, and de Paulis A (2005) Mast cells and basophils: friends as well as foes in bronchial asthma? Trends in Immunology 26(1): 25–31. Marshall JS (2004) Mast cell responses to pathogens. Nature Reviews: Immunology 4: 787–799. Nagata K and Hirai H (2003) The second PGD2 receptor CRTH2: structure, properties and functions in leukocytes. Prostaglandins, Leukotrienes and Essential Fatty Acids 69: 169–177.

Eosinophils D S Robinson and H H Kariyawasam, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Eosinophils are predominantly tissue resident cells with a bilobed nucleus and granular cytoplasm and are derived from bone marrow CD34 þ hemopoietic progenitor cells. Lineage selection and maturation is critically dependent on interleukin-5 (IL-5), and release from marrow occurs in response to further IL5 and eotaxin. In disease states, selective accumulation into tissue occurs in response to a series of interactions between eosinophils and endothelial cells with migration along specific chemokine gradients. Lung eosinophilia is implicated in a variety of allergic and nonallergic diseases. Mature eosinophils are an important source of basic proteins, lipid-derived mediators, growth factors, and proinflammatory cytokines that can result in a spectrum of lung diseases that range from allergic airway inflammation to parenchymal and vasculitic diseases. As a result, there is a substantial effort to target therapy at critical events involved in the eosinophil life cycle and functional pathways.

Originally described by Paul Ehrlich in 1879, the eosinophil was named with regard to the cell’s avidity for the negatively charged fluorescein compound eosin that confers on the cell a characteristic pink color. In health, eosinophils comprise only 1–3% of the circulating leukocyte population and are essentially tissue resident cells with confinement predominantly to the gastrointestinal (GI) tract. In disease states, however, the cells can be rapidly mobilized from the bone marrow into the peripheral circulation and selectively accumulate at sites of tissue inflammation with an estimated half-life of 13 days. Eosinophils are approximately 8 mm in diameter (this can vary with the state of activation) and are morphologically distinct with a bilobed nucleus and ovoid granules that confer the characteristic granular cytoplasm to the cell. Electron micrographs reveal four distinct populations of cytoplasmic granules that house specific cellular contents. Primary granules are the main site of Charcot–Leyden crystals that display intrinsic lysophospholipase activity. Secondary granules are composed of a prominent crystalloid core containing major basic protein (MBP) and a noncrystalloid matrix that houses other highly cationic proteins, such as eosinophilic-cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase (EPO), together with a number of cytokines and proteins with enzymatic activity. Lipid bodies are the site of major lipidderived mediator synthesis. Finally, there are small granules seen in mature tissue eosinophils that store proteins such as arylsulfatase B and acid phosphatase (Figure 1). The role of the small-granule proteins in eosinophil biology is not known.

LEUKOCYTES / Eosinophils 543

E P L E

E

Figure 1 Electron micrograph of a bronchial biopsy from a mild stable asthmatic showing three eosinophils (E) (one degranulating) in close association with a lymphocyte (L) and plasma cell (P). Scale ¼ 2 mm. Kindly provided by P K Jeffery and Dr A Rogers, National Heart & Lung Institute, Imperial College London.

Life Cycle Eosinophils are derived from CD34 þ bone marrow (BM) hemopoietic progenitor cells that differentiate into precursors with both basophil and eosinophil properties in response to T helper type 2 (Th2)secreted cytokines interleukin (IL)-3 and granulocyte macrophage colony-stimulating factor (GM-CSF). Ensuing cell surface expression of the IL-5Ra chain receptor confers IL-5 responsiveness. Cells can then undergo early lineage selection, proliferation, and terminal differentiation in direct response to IL-5. Although the marrow is the predominant site of eosinophilopoiesis, progenitors (CD34 þ IL-5Ra) are detectable in both the bronchial tissue and the sputum of atopic asthmatics, confirming that progenitors can traffic to tissue and thus eosinophilopoiesis is not marrow restricted. There is thus a pool of immature and mature eosinophils that can be rapidly mobilized into the circulation during inflammatory responses. This mobilization is critically dependent on IL-5 and the eosinophil-specific chemokine eotaxin. Chemokines are chemoattractant cytokines selectively produced at sites of inflammation and are subdivided into families based on the position of cysteine residues, such as CC, CXC, and CX3C, where X is any amino acid. Eotaxin (a CC chemokine) and the homologs eotaxin-2 and -3 display very high selectivity for eosinophils exclusively through the chemokine receptor CCR3. Recently differentiated eosinophils constitutively express CCR3, but in response to priming signals generated by the local tissue cytokine milieu, eosinophils can upregulate the expression of several other chemokine receptors, including CCR1. The chemokines RANTES (regulated

upon activation, normal T cell expressed and secreted) and MCP-3 and -4 (monocyte chemoattractant protein) can regulate eosinophils through CCR3, and MIP-1a (macrophage inflammatory protein) can regulate them through CCR1. However, such chemokines are not eosinophil specific, with effects on both T cells and basophils. The selective trafficking of eosinophils from the circulation into tissue sites is a highly specific and regulated stepwise process between the eosinophil and endothelium that requires cell–endothelial adhesion and then extravasation. Cell rolling is facilitated by enodothelial expression of P-selectin that specifically binds eosinophil-expressed P-selectin glycoprotein-1 (PSGL-1). As eosinophil activation proceeds in response to the local inflammatory and chemokine microenvironment, such as platelet-activating factor (PAF) and particularly eotaxin, conformational change of the b2 integrins Mac-1 and lymphocyte function-associated antigen (LFA)-1 on the eosinophil surface allows binding to endothelial intercellular adhesion molecule (ICAM)-1, thus allowing the cell to halt. Expression of very late activation antigen (VLA)-4 integrin (a4b1) is specific to eosinophils. VLA-4 attaches to the vascular cell adhesion molecule (VCAM)-1 on endothelium, allowing selective eosinophil transmigration across the vessel wall into tissues. Both IL-4 and IL-13, key inflammatory cytokines, markedly upregulate the expression of VCAM-1. Also, eosinophil a4b7 can bind the mucosal addressin cell adhesion molecule 1 (MadCam-1), enabling further adhesion. This interaction is particularly important for eosinophil recruitment into the GI tract. High chemokine concentrations induce chemokine receptor internalization, thus preventing eosinophils from egressing from the tissue. IL-5 plays a key role in priming eosinophils for selective chemotaxis into tissues and local IL-5, and GM-CSF prolongs eosinophil survival by delaying apoptosis (Figure 2).

Eosinophil Functions in the Normal Lung Granule Proteins

Eosinophils are ideally equipped to provide the primary defense against parasitic infection. The immune response to such invading organisms is characterized by IgE production, marked blood eosinophilia, and eosinophil accumulation in infected tissues and tissueresident mast cells. Thus, allergic responses may be considered as an aberration of this defense mechanism whereby environmental agents elicit a Th2 polarized adaptive immune response with production of the key cytokines IL-4 and IL-5. Eosinophil contents exert direct toxicity against parasites. MBP, which

544 LEUKOCYTES / Eosinophils Eotaxin CCR3

Eosinophil

Marrow

Structural cells Tissue infiltration

Vessel

Chemotaxis Eotaxin MCP - 4, RANTES

Lineage selection Maturation Release

CCR3

IL- 5

VLA- 4

Migration Adhesion VCAM - 1 Endothelial cell

IL- 4 IL- 13

IL - 3 GM - CSF

Th2

PAF

Activation mediator release

CCR3

Survival IL - 3, IL - 5, GM - CSF

Figure 2 The key stages involved in the recruitment of eosinophils from the bone marrow to the tissue are illustrated. Eosinophilopoiesis is dependent on IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and particularly IL-5. On entering the circulation in response to IL-5 and eotaxin, the cells are recruited specifically to sites of tissue inflammation through interaction of various adhesion molecules, including VLA-4/VCAM-1. Several cytokines can upregulate such adhesion molecules. Eosinophils undergo conformational change to transmigrate into tissues along chemokine gradients before interacting with the surrounding inflammatory microenvironment to undergo activation. VLA, very late antigen; VCAM, vascular cell adhesion molecule; PAF, platelet-activating factor; MCP-4, monocyte chemoattractant protein; RANTES, regulated upon activation normal T cell expressed and secreted.

comprises 50% of the granule proteins, and ECP are both helminthotoxic and cytotoxic. Their highly basic property is conferred by the high arginine content and allows surface charge interactions with target cells that disrupt membrane permeability with consequent cell damage. In addition, both possess bactericidal activity. MBP can degranulate basophils and ECP mast cells. Both ECP and EDN have ribonuclease activity, but ECP is 100 times less potent than EDN in this respect. EDN displays markedly reduced toxicity compared to ECP due to lack of basicity. In addition, eosinophils can directly generate nitrite compounds, superoxide, and hydroxyl radicals through the action of EPO and NADPH oxidase in response to cell activation and priming. Lipid Mediators and Cytokines

Primed eosinophils have the capacity to rapidly produce an array of lipid mediators and cytokines. The eicosanoids are a family of bioactive lipids derived from the cell membrane phospholipid arachidonic acid (AA). AA is cleaved by activated phospholipase A2 (PLA2), with the cleavage product entering the principal pathways of eicosanoid production in

eosinophilia. The cyclooxygenase (COX) pathway is responsible for the generation of prostanoids that include prostaglandins, prostacyclin (PGI2), and thromboxane A2 (TxA2). The 5- and 15-lipooxygenase (LO) pathway generates leukotrienes (LTs). Eosinophils are a predominant source of cysteinyl leukotrienes, particularly LTC4, which can induce bronchoconstriction, mucous hypersecretion, airway hyperresponsiveness (AHR), and increased microvascular permeability. Eosinophils have the capacity to produce and store cytokines that include IL-1–6, -10, -16, and GM-CSF. They are an important source of transforming growth factor (TGF)-b1, a potent stimulus for fibroblast activation and extracellular matrix (ECM) production. TGF acts as a chemotactic factor for mast cell recruitment, and mast cell-derived tumor necrosis factor (TNF) can induce further TGF release from eosinophils. ECP has been shown to inhibit the degradation of proteoglycans, and eosinophils store and release matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and -2. This suggests an important novel role for eosinophils in the process of lung repair and remodeling. Several fibrotic diseases are associated with eosinophilic infiltration.

LEUKOCYTES / Eosinophils 545

Chemokines Eotaxin RANTES MCP - 3,4 MIP - 1


Cytotoxic compounds Hydrogen peroxide Superoxides Halide acids Cytokines / Growth factors IL-1,IL-2,IL - 3,IL - 4,IL - 5,IL - 6,IL - 8,IL - 12,IL - 16, IL- 18 GM - CSF,TNF -
,IFN -  TGF -
/ ,VEGF SCF

Mucous hypersecretion

Lipid mediators LTC4 LTD4 LTE4 PAF Prostanoids

Smooth muscle constriction

Inflammation

Granule proteins MBP ECP EPO EDN

Cellular toxicity AHR

Vascular leakage / airway edema Remodeling

Figure 3 Important eosinophil secretory products and the resultant effector functions of the cell. AHR, airway hyperresponsiveness; IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; TNF, tumor necrosis factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; SCF, stem cell factor; PAF, platelet-activating factor; MCP-4, monocyte chemoattractant protein; RANTES, regulated upon activation normal T cell expressed and secreted; MIP, macrophage inflammatory protein; LT, leukotriene; MBP, major basic protein; ECP, eosinophil cationic protein; EPO, eosinophil peroxidase; EDN, eosinophilderived neurotoxin.

Eosinophilia of tissues is often associated with mast cell recruitment. Eosinophils secrete mast cell stem cell factor (SCF), which is essential for mast cell growth, activation, chemotaxis, and degranulation. Mast cells in turn regulate eosinophils both directly and indirectly, thus creating a regulatory dependence between cells. Eosinophil maturation and migration is in turn effected by mast cell secreted cytokines (including IL-3–5 and GM-CSF) and chemokines, including eotaxin, RANTES, and MCP-1. Mast cell-derived TNF-a is a potent stimulus for EPO release. The possibility that eosinophils may act as antigen-presenting cells to T cells has been raised by the in vitro demonstration of the expression of molecules involved in antigen presentation on the eosinophil surface. The emerging theme is that eosinophils and other key effector inflammatory cells crossregulate each other (Figure 3).

Eosinophil Function in Respiratory Diseases Asthma

Airway eosinophilia is a hallmark of asthma. The degree of tissue eosinophilia and the level of eosinophil activation correlate with the severity of asthma. The

characteristic late phase response following allergen exposure in asthma is associated with airway eosinophilic infiltration. Many of the products released by eosinophils, such as LTC4 and MBP, can promote the pathophysiological hallmarks of asthma, such as airway hyperresponsiveness. MBP can also enhance vagally mediated bronchoconstriction by acting as an antagonist at the neuronal M2 muscarinic receptor. Collectively, these findings suggest that eosinophils are the major effector cells in the pathogenesis of asthma (i.e., the eosinophil hypothesis in asthma). However, this view has been challenged by the results of human studies in asthma that targeted tissue eosinophil depletion with an anti-IL-5 monoclonal antibody. Eosinophil depletion failed to show much effect on the asthma phenotype in man, such as airway hyperresponsiveness. This may be a reflection of the fact that only a 55% reduction in bronchial wall eosinophil numbers was achieved, and further studies are needed before definite conclusions can be made. Importantly, however, eosinophil reduction is associated with decreased airway remodeling, confirming that eosinophil-derived growth factors such as TGF-b1 are pivotal to this process. This has important therapeutic implications given that structural changes in asthmatic airways are correlated with the severity of disease and progressive decline in lung function.

546 LEUKOCYTES / Eosinophils Eosinophilic Bronchitis

The degree of airway eosinophilia and other inflammatory cell infiltrates in EB is comparable to that of asthma, and both EB and asthma display the characteristic basement membrane thickening. However, EB syndrome is characterized by cough, normal lung function, and, importantly, a complete absence of AHR. Glucocorticoid eosinophil suppression leads to rapid improvement. The significance of eosinophilic infiltration in this condition is unknown. Eosinophilic Parenchymal Diseases

Pulmonary eosinophilia describes the association of a raised blood eosinophil count with areas of pulmonary consolidation, sometimes ‘flitting’ in nature. Pulmonary eosinophilia can occur either as an association with another underlying disease process or as an idiopathic disease entity. The common causes are listed in Table 1. Allergic Bronchopulmonary Aspergillosis

This is a syndrome of pulmonary eosinophilia and proximal bronchiectasis that can sometimes complicate asthma and cystic fibrosis. Airway inflammation and subsequent damage is driven by eosinophil recruitment to the airway in response to fungal allergen-driven Th2-secreted IL-4 and IL-5. Drug Reactions

A number of drugs associated with pulmonary eosinophilia have been documented, and drug reactions are probably the most common cause of eosinophilia in the United Kingdom. The symptoms are generally mild, sometimes with cough and systemic symptoms, and usually improve on stopping the medication. In more severe or persistent cases, treatment with corticosteroids is required. Parasitic Infections

A number of parasitic infections can be associated with pulmonary eosinophilia. Symptoms are

predominantly confined to the GI tract, with respiratory symptoms comprising mild cough and wheeze. Loeffler’s syndrome is considered a response to the migration of parasitic larvae, usually Ascaris, through the lung. A more severe clinical presentation is seen in tropical pulmonary eosinophilia caused by the pulmonary presence of the filarial worms Wuchereria bancrofti and Brugia malayi. The corresponding eosinophil recruitment and activation in the lung is a host defense response. Pulmonary Vasculitis

Churg–Strauss syndrome is characterized by systemic vasculitis in association with hypereosinophilia and asthma. A background of rhinosinusitis with atopy is common, and asthma may precede the syndrome by up to 30 years. Eosinophilic Pneumonia

This is a disorder of unknown etiology that mimics pneumonia-like illness, with both respiratory and systemic symptoms. There is an association with atopy and recent-onset asthma. Therapeutic Manipulation of Eosinophils in Respiratory Disease

Corticosteroids are considered the most effective treatment for eosinophilic disorders by virtue of their ability to inhibit eosinophil recruitment and activation by suppression of a variety of inflammatory genes, including IL-5. Corticosteroids induce eosinophil apoptosis by the release of proapoptogenic factors and also by stimulating intracellular apoptotic events. The non-specific effects of corticosteroids and a failure of some patients with asthma to respond to steroids have driven the search to find more specific therapies targeted at eosinophils alone, directed at different stages of the eosinophil life cycle. Anti-IL-5 therapy failed to affect lung function and symptoms in asthma in clinical trials. Only a 55% reduction in the degree of tissue eosinophilia was achieved. Such therapy may be ineffective because the pathways that

Table 1 Causes of pulmonary eosinophilia Drugs

Parasites

Systemic disease

Idiopathic

Sulfonamides Nitrofurantoin Minocycline Penicillins Tetracycline Clarithromicin Leukotriene inhibitors Sulfasalazine Aspirin

Ascaris species Toxocara species Ancyclostoma species Wuchereria bancrofti Brugia malayi Schistosoma species Strongyloides species

Hodgkin’s lymphoma Bronchogenic carcinoma Ulcerative colitis Mycobacterial infection Vasculitis

Acute eosinophilic pneumonia Chronic eosinophilic pneumonia

LEUKOCYTES / Neutrophils 547

recruit and activate eosinophils remain active. Eotaxin and CCR3 receptor blockade offer obvious therapeutic potential. Antagonists against these molecules are expected to enter clinical trials in the near future. See also: Allergy: Overview. Asthma: Overview. Chemokines. Eotaxins. Interleukins: IL-5. Lipid Mediators: Overview; Leukotrienes; Prostanoids.

Further Reading Alberts WM (2004) Eosinophilic interstitial lung disease. Current Opinion in Pulmonary Medicine 10(5): 419–424. Bochner BS (2004) Verdict in the case of therapies versus eosinophils: the jury is still out. Journal of Allergy and Clinical Immunology 113(1): 3–10. Flood-Page PT, Menzies-Gow AN, Kay AB, and Robinson DS (2003) Eosinophil’s role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. American Journal of Respiratory and Critical Care Medicine 167(2): 199–204. Gleich GJ (2000) Mechanisms of eosinophil-associated inflammation. Journal of Allergy and Clinical Immunology 105(4): 651–663. Kay AB, Phipps S, and Robinson DS (2004) A role for eosinophils in airway remodelling in asthma. Trends in Immunology 25(9): 477–482. Klion AD and Nutman TB (2004) The role of eosinophils in host defense against helminth parasites. Journal of Allergy and Clinical Immunology 113(1): 30–37. Menzies-Gow A and Robinson DS (2001) Eosinophil chemokines and chemokine receptors: their role in eosinophil accumulation and activation in asthma and potential as therapeutic targets. Journal of Asthma 38(8): 605–613. Robinson DS, Kay AB, and Wardlaw AJ (2002) Eosinophils. Clinical Allergy & Immunology 16: 43–75. Rothenberg ME (2004) Eosinophilic gastrointestinal disorders (EGID). Journal of Allergy and Clinical Immunology 113(1): 11–29.

Neutrophils I Sabroe, University of Sheffield, Sheffield, UK E R Chilvers, Addenbrooke’s Hospital, Cambridge, UK M K B Whyte, University of Sheffield, Sheffield, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The neutrophil is a phagocytic leukocyte, the most abundant in the human body and the most short-lived of all myeloid cells. This polymorphonuclear cell is packed with granules containing potent antimicrobial compounds, and is absolutely essential for day-to-day successful defense against bacterial and fungal infections. Its recruitment and activation is carefully regulated, and control of its life span coupled with mechanisms allowing for a noninflammatory death pathway (apoptosis) reduce risks of tissue damage and disease arising from unwanted neutrophil activation. Given their destructive potential, and despite intensive

regulation of their function, it is unsurprising that neutrophilic inflammatory diseases are prevalent; these include asthma, chronic obstructive pulmonary disease, the acute respiratory distress syndrome, and several vasculitides.

Introduction The existence of white blood corpuscles was described by Hewson in the eighteenth century. In the mid-nineteenth century, Ehrlich was able to demonstrate the existence of several subtypes of these cells using a triacid stain; he identified acidophils (eosinophils), neutrophils, and basophils. The ability of white blood cells to migrate through blood vessel walls and comprise the chief cellular constituent of pus was established at around this time by Addison and Cornheim, but it was Metchnikov who, in the late nineteenth century, realized that these cells ingested and destroyed microbes, coining the term ‘phagocyte’ from the Greek ‘phagein’ (‘to eat’). This crucial view of the benefits of inflammation was in contradiction to beliefs held since the time of the early Greek physicians in and before the first century, in which inflammation was viewed as an entirely negative and destructive process. Furthermore, it defined the principal role of neutrophils: to mobilize to sites of infection by emigration from the circulation and to destroy invading microbes. Since that date, the goals of neutrophil research have changed little: to understand how recruitment and antimicrobial responses are regulated. The neutrophil is our most numerous phagocyte (numbering 2.5–7.5
109 cells l  1 blood), whose function is primarily to defend against bacterial infections, but it may also play a role in antiviral responses and particle clearance. A striking feature of this cell type is its short life span, enhancement of which is one of the most important ways of regulating its antibacterial capabilities. Neutrophils only survive for about 6–8 h in the circulation. To maintain their numbers in spite of a short life span, the bone marrow manufactures 1
1011 neutrophils per day in health, with the capacity to markedly upregulate this rate of production when required. Development from CD34 þ stem cells to mature neutrophils typically takes about 14 days, and is dependent upon cytokines such as granulocytemacrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), the latter cytokine being a useful therapeutic agent to enhance neutrophil production in the neutropenic patient (e.g., during chemotherapy). The bone marrow contains a large pool of mature or near-mature neutrophils available for rapid mobilization, and specific pathways regulate the release of mature cells,

548 LEUKOCYTES / Neutrophils

and the return of senescent cells, from and to the marrow (see below). Physiological removal of circulating cells also takes place in the reticuloendothelial systems of the liver and spleen. Within the circulation, a substantial proportion of neutrophils marginate into microvascular capillary beds such as the lung. Their large size (10–12 mm diameter, i.e., larger than red cells) delays their transit through these beds, and it is possible that in these environments, neutrophils play important roles filtering out bacteria and other particles from the circulation. Certainly, sudden systemic exposure to bacteria or chemoattractants such as activated complement results in neutrophil activation in the circulation and a dramatic increase in margination, with a consequent transient neutropenia. Neutrophils contain large numbers of primary and secondary granules. Primary (azurophil) granules contain hydrolases, matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and cationic proteins; secondary granules contain lactoferrin, histaminase, and collagenase. Delivery of these proteins to phagosomes containing ingested microorganisms results in their destruction (though some bacteria have evolved strategies to protect themselves from these microbicidal systems). These act in concert with the NADPH oxidase system, which generates reactive oxygen species, and influences phagosome alkalinization and the activation of proteases within the phagosome. Release of granule contents and superoxides into the extracellular milieu can be extremely destructive (see below), and excessive or inappropriate neutrophil activation is a major potential cause of tissue damage. (The oft-repeated and over-used phrase describes this as the ‘double-edged sword’ of inflammation, but we should bear in mind that the daily survival of every individual depends on neutrophil function, and whilst diseases involving unwanted neutrophil activation are prevalent in the developed world, the relative number of individuals killed by their own neutrophils remains small.) The histotoxic potential of neutrophils dictates the need for efficient physiological mechanisms to safeguard against inadvertent activation. The key mechanism for this is neutrophil priming, whereby degranulation and activation of the NADPH oxidase occurs only in cells pre-exposed to agents such as lipopolysaccharides (LPS) or GM-CSF. As well as controlling secretory responses, priming also affects neutrophil shape (creating a cell with a highly irregular polarized cell contour), deformability, integrin expression and longevity, and, as a consequence, has a profound impact on the rheological, adhesive, and survival properties of these cells and their injurious potential.

Neutrophil Function in the Normal Lung The distal airways and acini are generally held to be sterile, yet small numbers of neutrophils may often be found on cellular analysis of normal bronchoalveolar lavage fluid, or in normal endobronchial biopsies. Constitutive trafficking of granulocytes to normal mucosa has been seen in other organs (e.g., eosinophil trafficking to the gut); thus, whether neutrophil recruitment to the lung is the result of chronic low-grade inflammatory insults (infectious organisms, inhaled endotoxin, pollution), or a surveillance and defense pathway that would act in the absence of any measurable stimulus, is unknown (and, since our only sterile environment is antenatal, broadly irrelevant). Large numbers of neutrophils reside temporarily in the pulmonary microvascular bed, where they may help filter the blood of pathogens and particles. In either setting, the principal role of the cell remains host defense.

The Neutrophil in Respiratory Diseases The neutrophil is principally active against bacterial infections such as those that cause community- and hospital-acquired pneumonias, though there is also some evidence that neutrophil activation may contribute to the early response to tuberculosis (TB) and successful granuloma formation. Neutrophils also provide important responses to fungal infections and contribute to antiviral responses (e.g., to influenza A). Trafficking and Recruitment

Recruitment to inflamed sites is regulated at multiple levels. Neutrophils are actively released from the marrow in processes regulated by adhesive interactions (blockade of CD11b/CD18 enhances release, whilst blockade of CD49d (VLA-4) inhibits release) and chemokine signaling. Neutrophils express the chemokine receptors CXCR1 and CXCR2, whose ligands, in particular IL-8/CXCL8, mobilize neutrophils from the marrow. Surprisingly, recent work has also shown that immature neutrophils express CXCR4, and SDF-1a/CXCL12 signals to retain neutrophils within the marrow. Furthermore, expression of CXCR4 increases with age and provides a mechanism that causes senescent neutrophils to traffic back to the marrow, presumably for removal by bone marrow macrophages. Recruitment into tissues from the microcirculation is driven by a series of adhesive interactions between the cell and the vessel wall, dependent chiefly on L-selectin and CD11b/ CD18, and directed by a large panel of neutrophil chemoattractants. Again, the chemokines have received considerable attention, as ligands of CXCR1

LEUKOCYTES / Neutrophils 549

and CXCR2 provide a degree of selectivity for neutrophils (though these receptors may be expressed by monocytic cells and lymphocytes to some degree). Activated neutrophils are themselves a source of CXCL8, potentially mobilizing more phagocytes to sites of inflammation. However, many other molecules, such as activated complement fragments (C5a) and bacterially derived peptides, are potent chemoattractants; in contrast to chemokines these are also excellent stimulators of neutrophil degranulation and respiratory burst, and are important regulators of neutrophil function. In pneumonia, there is evidence from animal models that neutrophil recruitment, activation, and apoptosis proceeds in relatively discrete waves over a period of days, rather than continuous neutrophil recruitment occurring whilst inflammation persists. Detection of Pathogens

Neutrophils detect pathogens through a broad range of pathogen-recognition receptors, including integrins, Toll-like receptors (TLRs; see Toll-Like Receptors), and recently identified molecules such as dectin-1. Their signaling is typically primed by cytokines such as GM-CSF and lipid mediators such as platelet-activating factor (PAF), through signaling pathways including activation of MAPKs and PI-3K. The same pathways are involved in antimicrobial defenses activated at sites of infection, including generation of reactive oxygen species (ROS), cationic proteins (defensins), and release of granule contents into phagolysosomes or the extracellular milieu. Neutrophilic responses are regulated at every level by other lung cells and leukocytes. Neutrophil-recruiting chemokines are manufactured by many cell types, including epithelia, airway smooth muscle, and endothelia. Monocytic cells serve important roles in enhancing responses of lung tissue cells to infection, synergistically increasing inflammatory responses. Furthermore, monocytic and other cells are also responsible in large measure for the increase in neutrophil survival at sites of infection, generating factors that activate nuclear factor kappa B (NF-kB), probably one of the most important signaling pathways in granulocyte survival. Disruption of Function and Disease

When normal neutrophil functions are disrupted by disease, severe infection can result. Chronic granulomatous disease is an X-linked recessive condition in which neutrophils are unable to make hydrogen peroxide: in these patients, severe infections with organisms such as Staphylococcus aureus affect multiple organs. In the Che´diak–Higashi syndrome,

failure of granule fusion with the phagosomes also results in vulnerability to staphylococcal infection. Patients with deficiencies in CD11b/CD18 also show multiple defects in aspects of neutrophil recruitment and activation, and are subject to multiple bacterial and fungal infections. The latter abnormality may affect other leukocytes; similarly, defects in signaling of interleukin-1 (IL-1) and TLRs (particularly TLR4) resulting from abnormalities in the intracellular protein IRAK-4 affect both neutrophils and monocytes, and dramatically increase susceptibility to bacterial, though not viral, infections. Mutations significantly affecting neutrophil function are extremely rare, presumably as the odds of the affected individuals making it to reproductive age are dramatically reduced. In the war between host and pathogen, the neutrophil retains the upper hand, though some pathogens have also developed strategies to evade neutrophil function, for example, by expression of capsules that inhibit phagocytosis (some streptococci) or production of toxins that cause neutrophil death (Pseudomonas aeruginosa). Resolution of Inflammation

The most extraordinary feature of neutrophilic inflammation is its ability to resolve. Lobar consolidation dissipates to leave normal lung architecture, an amazing feat given the delicacy of alveoli and lung soft tissue. Neutrophils typically die by apoptosis, a programmed cell death that retains granules intact inside the dying cell, and prevents unwanted local release of cellular contents. These apoptotic neutrophils are taken up locally by macrophages, without activating the macrophage inflammatory responses that typically follow phagocytosis. Where cell death proceeds more rapidly than macrophages can handle, progression to necrosis results in neutrophil lysis, release of granule contents, and tissue damage. Neutrophilic Inflammation and Disease

Unwanted neutrophil inflammation has substantial potential to cause disease. Classical neutrophilic inflammatory diseases include the acute respiratory distress syndrome (ARDS), where a predominantly neutrophilic infiltrate causes massive lung tissue damage and is associated with a high mortality. Animal models using ischemic reperfusion injury (in the lung and other organs) recapitulate acute insults, and demonstrate the dependence of tissue injury on neutrophil recruitment and activation. In the human, levels of CXCL8 in bronchoalveolar lavage of patients at risk of ARDS are predictive of subsequent disease development. The inability of corticosteroids to suppress neutrophil activation or induce neutrophil

550 LEUKOCYTES / Neutrophils

(PR3)), as opposed to the perinuclear binding pattern (pANCAs) associated with antibodies to myeloperoxide. Increasing evidence shows these antibodies to be pathological. Priming of the neutrophil by cytokines such as tumor necrosis factor alpha (TNF-a) moves PR3 to the cell surface; engagement by cANCAs delivers an activating signal that is reinforced by interaction of the Fc portion of the cANCA with IgG receptors on the same neutrophil. Generation of ROS

apoptosis may, in part, explain their lack of effectiveness in this disease. Activation of the neutrophil in the vasculature in diseases such as Wegener’s granulomatosis (WG) is associated with a severe multisystem vasculitis that has its greatest impact in the lung and kidney. In WG, patients typically have autoantibodies that target neutrophils, eliciting a cytoplasmic binding pattern (cytoplasmic antineutrophil cytoplasmic antibodies (cANCAs) that bind proteinase 3

Normal function

rece ptor s

CXCRs 1, 2, 4 C5aR

tant

Pathogen clearance

moa

ttrac

fMLPR

Che

PAFR External regulation (e.g., by monocytes)

Asthma

=

Bronchiectasis/CF

Cytokine production

Vasculitis

COPD External regulation (e.g., by monocytes)

tors

ARDS

n re

cep

Nonrespiratory diseases CD11b/ CD18

Path

oge

n re

cog

nitio

Resolution by apoptosis

CD14 TLR2 In disease TLR4

Figure 1 Neutrophil function in health and disease. Neutrophils function to maintain health by removal of pathogens. Their recruitment into tissues is regulated by many chemoattractants, principal amongst which are chemokines acting on CXCR1 and CXCR2 (such as CXCL8), and the activated complement fragment, C5a. Bacterial peptides may also activate the fMLP receptor, though this receptor may also respond to as yet unidentified endogenous molecules. Recruitment into tissues and to the site of infection is followed by neutrophil activation, as the cells detect pathogens through a range of pathogen-recognition receptors, which probably act in concert to switch on bacterial killing systems such as ROS generation and the release of toxic proteases. Simultaneously, neutrophils generate more CXCL8, facilitating further cell recruitment. It is likely that other cell types, e.g., monocytes and macrophages, oversee the neutrophil response, providing survival cytokines that control the duration and amplitude of the inflammatory response. After removal of pathogens, neutrophils die by apoptosis and are engulfed by monocytic cells, which remove them from the local environment, preventing tissue damage. However, where inappropriate or excessive neutrophil activation occurs, there is considerable potential for tissue damage. Neutrophil-mediated tissue damage has been linked with the pathology of a range of respiratory and nonrespiratory diseases, and to date remains extremely difficult to target therapeutically.

LEUKOCYTES / Neutrophils 551

results, and this neutrophil activation appears central to disease pathogenesis. CXCL8 generation and some measure of neutrophil recruitment is a common feature of many other respiratory diseases, but it can be surprisingly hard to discern the unique contribution of neutrophils to the pathology. Such contributions have occasionally been obscured further when roles for neutrophils ran counter to dogma, even when pointers to their importance were apparent. There is now clear evidence that subgroups of asthmatics show marked airway neutrophilia (alone or with eosinophilia), which may be amplified by treatment with corticosteroids, since they induce eosinophil apoptosis but prolong neutrophil survival. Acute severe asthma is relatively hard to study, but neutrophils often dominate the airway inflammation in these settings. Treatments for neutrophilic asthma variants may offer new ways of intervening in asthma that has responded poorly to conventional management strategies. Likewise in COPD, airway neutrophilia correlates with severity of airway obstruction, and overexpression of IL-1b in the lung results in airway inflammation with neutrophil and monocyte recruitment, airway remodeling, and emphysema. In COPD, neutrophil proteases and ROS may contribute to tissue damage and remodeling, though the exact contribution of the neutrophil versus the monocyte/macrophage is difficult to determine in such a chronic setting. Likewise, arguments over the role of the neutrophil in some variants of fibrotic lung disease persist, and they may be involved in tissue damage where chronic infection is present, for example, bronchiectasis and cystic fibrosis.

Summary In summary, the neutrophil is central to host defense, particularly against bacterial and fungal infections. Although diseases with a basis in neutrophilic inflammation are prevalent, given the extraordinary efficacy of these cells in causing microbial and tissue destruction, it is impressive how well controlled neutrophil function is on a day-to-day basis in the majority of the population. In the future, targeting of neutrophils may provide effective treatments for inflammatory diseases where currently treatments are absent or rely on heavy immunosuppression (Figure 1). See also: Adhesion, Cell–Cell: Vascular; Epithelial. Adhesion, Cell–Matrix: Focal Contacts and Signaling; Integrins. Asthma: Overview; Acute Exacerbations; Extrinsic/Intrinsic. Autoantibodies. Bronchiectasis. Bronchiolitis. Bronchoalveolar Lavage. CD11/18. CD14. Chemokines. Chemokines, CXC: CXCL12 (SDF-1); IL-8; CXCL1 (GRO1)–CXCL3 (GRO3). Chronic

Obstructive Pulmonary Disease: Overview; Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General. Complement. Defense Systems. Defensins. Endotoxins. Granulomatosis: Wegener’s Disease. Interstitial Lung Disease: Overview. Lipid Mediators: Platelet-Activating Factors. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens). Toll-Like Receptors. Transcription Factors: NF-kB and Ikb. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Burdon PC, Martin C, and Rankin SM (2005) The CXC chemokine MIP-2 stimulates neutrophil mobilization from the rat bone marrow in a CD49d-dependent manner. Blood 105: 2543–2548. Cowburn AS, Cadwallader KA, Reed BJ, Farahi N, and Chilvers ER (2002) Role of PI3-kinase-dependent bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival. Blood 100: 2607–2616. Donnelly SC and Haslett C (1992) Cellular mechanisms of acute lung injury: implications for future treatment in the adult respiratory distress syndrome. Thorax 47: 260–263. Haslett C, Savill JS, Whyte MK, Stern M, Dransfield I, and Meagher LC (1994) Granulocyte apoptosis and the control of inflammation. Philosophical Transactions of the Royal Society of London 345: 327–333. Hopken UE, Lu B, Gerard NP, and Gerard C (1996) The C5a chemoattractant receptor mediates mucosal defence to infection. Nature 383: 86–89. Lamblin C, Gosset P, Tillie-Leblond I, et al. (1998) Bronchial neutrophilia in patients with noninfectious status asthmaticus. American Journal of Respiratory and Critical Care Medicine 157: 394–402. Martin C, Burdon PC, Bridger G, et al. (2003) Chemokines acting via CXCR2 and CXCR4 control the release of neutrophils from the bone marrow and their return following senescence. Immunity 19: 583–593. Medvedev AE, Lentschat A, Kuhns DB, et al. (2003) Distinct mutations in IRAK-4 confer hyporesponsiveness to lipopolysaccharide and interleukin-1 in a patient with recurrent bacterial infections. Journal of Experimental Medicine 198: 521–531. Parker LC, Whyte MK, Dower SK, and Sabroe I. The expression and roles of Toll-like receptors in the biology of the human neutrophil. Journal of Leukocyte Biology (in press). Peters AM (1998) Just how big is the pulmonary granulocyte pool? Clinical Science (London) 94: 7–19. Segal AW (2005) How neutrophils kill microbes? Annual Review of Immunology 23: 197–223. Tonnel AB, Gosset P, and Tillie-Leblond I (2001) Characteristics of the inflammatory response in bronchial lavage fluids from patients with status asthmaticus. International Archives of Allergy and Immunology 124: 267–271. Ward C, Chilvers ER, Lawson MF, et al. (1999) NF-kappa B activation is a critical regulator of human granulocyte apoptosis in vitro. Journal of Biological Chemistry 274: 4309–4318. Wenzel SE, Schwartz LB, Langmack EL, et al. (1999) Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics. American Journal of Respiratory and Critical Care Medicine 160: 1001–1008. Williams JM, Kamesh L, and Savage CO (2005) Translating basic science into patient therapy for anca-associated small vessel vasculitis. Clinical Science (London) 108: 101–112.

552 LEUKOCYTES / Monocytes

Monocytes P A Stumbles and C von Garnier, The University of Western Australia, Perth, WA, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Monocytes are bone marrow-derived phagocytic cells that circulate through the blood and into tissues, playing a major role in respiratory defense. Through their phagocytic and lysosomal activity, monocytes play a major primary role in innate immunity and pathogen clearance. Furthermore, through their capacity for cytokine-driven differentiation into macrophages (Mf) and dendritic cells, monocytes play an important role in respiratory homeostasis by acting as a resident precursor pool for these two major classes of immune cells. Dendritic cells (DCs) are the major antigen-presenting cell type of the mucosal surfaces and interstitial tissues of the respiratory tract and play an important role in the regulation of adaptive T-cell immunity to inhaled allergens and pathogens. Alveolar, interstitial, and mucosal Mf are the major phagocytes within the respiratory tract that, in addition to their important innate immune activity, also play a crucial role in regulating the antigen-presenting activity of DCs and the prevention of unchecked lymphoproliferation. In addition to their homeostatic activity, products of monocytes, such as chemokines, and the recruitment and deregulated differentiation of these cells are relevant in a wide variety of important pulmonary diseases such as tuberculosis and allergic asthma. Strategies aimed at modifying the recruitment and/or differentiation of these cells, as well as regulating their production of proinflammatory mediators, may provide new therapeutic directions for the treatment of a wide variety of respiratory inflammatory disorders.

Introduction Monocytes are phagocytic cells that contribute to innate immunity through their phagolysosomal activity and adaptive immunity through their ability to differentiate into antigen-presenting cells. Cells of the mononuclear phagocyte system originate from stem

Bone marrow Stem cell

Common myeloid precursor

Blood Circulating monocyte precursor

cells in the bone marrow (BM) that exit into the blood as circulating monocyte precursors prior to migration into tissues and differentiation into macrophages (Mf) and dendritic cells (DCs) (Figure 1). Pulmonary alveolar Mf can be obtained by lung lavage and are responsible for the clearance of inhaled particles and lung surfactant. They contribute to innate immunity through their capacity to phagocytose and are able to modulate the adaptive immune response of the dendritic cell–T cell axis. In contrast, respiratory tract DCs (RTDCs) are pivotal in the initiation of T and B cell-mediated immunity due to their potent antigen uptake and presenting capacity. Ultrastructurally, monocytes are large (10– 18 mm) cells with a characteristic lobulated nucleus and prominent intracytoplasmic lysosomes (Figure 2(a)). Based on surface expression of CD11c, major histocompatibility complex (MHC) class II, and other markers, cells that phenotypically and ultrastructurally resemble tissue monocyte precursor cells can be isolated from peripheral lung tissue of mice that, under appropriate stimulatory conditions, can differentiate into Mf and RTDCs (Figure 2(a)). In situ, these can be identified as Mf located at the alveolar interseptal junction in parenchymal lung tissue in association with type I alveolar epithelial cells. Monocytic precursor cells can also give rise to RTDCs, which lie within the underlying interstitium in close proximity to Mf (Figure 2(b)). Mf populations have also been identified in the pleural lining and peribronchial and perivascular connective tissue of peripheral lung. Within the mucosal tissue of the conducting airways, RTDCs are found both within the epithelium and in the proximal (luminal) regions of the underlying lamina propria, overlaying the cartilage rings. In contrast, airway mucosal Mf are located distally in a band overlaying smooth muscle and underlaying the cartilage rings, and are also

Respiratory tract Resident monocyte tissue precursor

Macrophage

DC Sca1+ cKit+ Thy1low CD34low/+

F4/80+ CD11b+ Gr-1+

Sca1+ cKit− F4/80+ CD11clow CD11b+ Gr-1+

F4/80+ CD11c+ CD11b− Gr-1−

F4/80− CD11c+ CD11blow Gr-1−

Figure 1 Monocyte origins and ontogeny. Monocytes develop from the bone marrow stem cells and enter the blood as circulating monocyte precursors, which enter tissues as resident monocyte precursors. Local factors produced within the respiratory tract will then promote differentiation into either Mf or dendritic cells.

LEUKOCYTES / Monocytes 553

MHC class II

RTDC

M
Mono

CD11c (a)

Alveolar interseptal junction

M
Cap

M


Cap Type I alveolar epithelial cell

Type I epithelial cell membrane

RTDC (b) Ep DC

Airway lumen

Ep bm

Lp DC C C

C

M
sm

(c) Figure 2 Morphology and distribution of monocytes, Mf, and RTDCs. (a) Transmission electron micrograph (TEM) of a monocytic precursor cell purified from mouse lung tissue (left) and a schematic representation of the differentiation pathway of these cells into Mf or RTDCs following in vitro differentiation as defined by expression of CD11c and MHC class II. (b) Schematic representation of a crosssection of lung tissue showing the location of Mf at the alveolar interseptal junction and in close proximity to RTDCs and capillaries (Cap). (c) Schematic representation of a longitudinal section of conducting airway showing the distribution of RTDCs within the epithelium (Ep DC), underlying the basement membrane (bm) and within the lamina propria (Lp DC), and the location of Mf below the cartilage rings (c) and in juxtaposition with smooth muscle cells (sm).

554 LEUKOCYTES / Monocytes

present in mucous layers covering the epithelial cells of small and large airways (Figure 2(c)).

Monocyte Function in the Normal Lung The primary function of cells of the monocyte/macrophage lineage in peripheral lung is phagocytosis and killing of inhaled microorganisms. However, these cells also play an equally important role in the maintenance of tissue-specific homeostatic processes. Influxing blood monocytes are thought to be the principal cell source for renewal of resident Mf and DCs in lung and airway tissue, although the processes governing these differentiation pathways in vivo are poorly understood. In vitro, macrophage colonystimulating factor (M-CSF) has been identified as a potent Mf differentiation factor in humans and rodents. Expression of the M-CSF receptor (CD115) on monocytes is regulated by a number of cytokines including IL-6, IL-10, and interferon gamma (IFN-g). Exposure of monocytes to these factors shifts monocyte differentiation towards Mf. In vitro studies of human peripheral blood monocytes also identified the capacity of these cells to differentiate into DCs following exposure to granulocyte-macrophage colonystimulating factor (GM-CSF) together with IL-4. Similar differentiation pathways exist in vitro for mouse monocytes and have also been demonstrated to occur in vivo in this species. GM-CSF receptor expression is also regulated by cytokines, with GMCSF itself acting dominantly to suppress M-CSF-driven Mf differentiation. While monocyte differentiation in the respiratory tract is poorly understood, this process is likely to be controlled by products of epithelial cells and other stromal cells in the steady state acting to replenish populations of local tissue RTDCs and Mf. Given that the half-life of RTDC at mucosal surfaces is estimated to be in the order of 12–24 h, and that rapid recruitment of these cells occurs following inflammatory challenge within a similar time frame, the differentiation of monocytes into RTDCs is likely to represent a dominant role for monocytes in the maintenance of respiratory tract homeostasis. Signaling through surface receptors for bacterial lipopolysaccharides (LPS) such as CD14 and Toll-like receptor 4 (TLR4) and through TLRs specific for other conserved microbial sequences will also accelerate the differentiation of respiratory monocytes into Mf and RTDCs during infection-induced inflammation. Chemokines

Chemokines are produced by, and act on, virtually all cells of the immune system to direct homeostatic

Table 1 Monocyte-derived or -acting chemokines in respiratory disease Systematic name

Common name

Receptor

Respiratory disease

CCL2

MCP-1

CCR2

CCL7

MCP-3

CCR1,2,3

CCL13 CCL22 CXCL1 CXCL7

MCP-4 MDC GROa NAP-2

CCR2,3 CCR4 CXCR2 CXCR2

Allergic asthma Bacterial pneumonia Mycoplasmainduced BPD Interstitial pneumonia Allergic asthma Allergic asthma COPD COPD

COPD, chronic obstructive pulmonary disease.

cell migration (chemotaxis), and their abnormal production has been implicated in a large range of disease states. While monocytes produce and respond to a wide range of chemokines, only two families of chemokines have been described to act in a relatively monocyte-specific manner: activity of the monocyte chemoattractant protein (MCP) chemokines MCP-1 (CCL2), MCP-3 (CCL7), and MCP-4 (CCL13) is restricted to recruitment of monocytes and basophils and has been associated with monocyte recruitment in a variety of respiratory diseases; it is most often associated with allergic airways disease (Table 1). In contrast monocyte-derived chemokine (MDC, CCL22) is a potent attractor and activator of Th2type T cells and has been implicated in the pathogenesis of allergic asthma (Table 1). Enhanced chemotactic responses of monocytes towards other chemokines such as growth-related oncogene alpha and neutrophil-activating peptide (GROa/NAP-2, CXCL1, CXCL7) have also been identified in diseases such as cigarette smoke-induced chronic obstructive pulmonary disease. Immunoregulation

An important homeostatic process within lung tissue is the regulation of local immune reactivity in order to avoid local immunopathology and damage of the blood-to-air interface. In this respect, a major function of cells of the monocyte/macrophage lineage in normal lung tissue is their capacity to downmodulate local T cell responsiveness through cytostatic signals delivered to both T cells and antigen-presenting DCs. Initial studies in rodents showed that in vivo depletion of alveolar Mf enhanced immune priming to aerosolized antigens. Subsequent studies showed that this effect could operate on both RTDCs through inhibition of the maturation of these cells into competent antigen presenters, and also on the T cell

LEUKOCYTES / Monocytes 555 CD4+ T cell

CD4+ T cell

NO • GM-CSF • TNF- • IL-4 • TGF-

NO M


M
RTDC

M-CSF

(a)

RTDC GM-CSF IL-4

GM-CSF IL-4

Monocyte precursor

(b)

Monocyte precursor

Figure 3 Immunoregulatory functions of monocytes and Mf. (a) In normal tissue, monocytes differentiate into RTDCs and Mf, the latter acting to inhibit CD4 þ T cell activation by RTDCs through production of NO. (b) Under inflammatory conditions, proinflammatory cytokines (GM-CSF, TNF-a, IL-4, TGF-b) act to overcome the immunosuppressive activity of Mf while at the same time acting to promote the differentiation of monocytes into immunostimulatory RTDCs.

through inhibition of lymphoproliferative activity. The principal mediator of the lymphostatic effect was shown to be Mf-derived nitric oxide (NO), which inhibits IL-2 receptor signaling in T cells through disruption of the Jak3/STAT5 intracellular signaling pathway. Similarly, NO was also demonstrated to be the principal mediator of alveolar Mf-mediated inhibition of RTDC maturation. The inhibitory effects of alveolar Mf can be abrogated by exposure to the cytokine GM-CSF and to a lesser extent transforming growth factor beta (TGF-b), IL-4, and tumor necrosis factor alpha (TNF-a), the latter acting in a synergistic fashion with GM-CSF to inhibit NO production by Mf and also promote maturation of RTDCs. Thus, monocyte-derived alveolar Mf and RTDCs participate in a tricellular interaction with T cells that in normal lung tissue inhibits T cell activation and priming to nonpathogenic inhaled antigens through a suppressive mechanism mediated by Mf-derived NO (Figure 3(a)). Under inflammatory conditions, production of cytokines such as GM-CSF together with other proinflammatory cytokines such as TNF-a and IL-4 will act to promote RTDC–T cell interactions and T cell priming through inhibition of Mf-mediated T cell suppression as well as upregulating the antigenpresenting capacity of Mf and the differentiation of monocytes towards mature antigen-presenting RTDCs (Figure 3(b)).

Monocytes in Respiratory Diseases Pulmonary Alveolar Proteinosis

Accumulation of surfactant protein leads to a rare disorder termed pulmonary alveolar proteinosis. The

clinical course of this disease can range from severe respiratory failure to spontaneous resolution, but an important feature is susceptibility to pulmonary infections with opportunistic infections. Pulmonary alveolar proteinosis may present in three clinically different forms: congenital, secondary, and acquired. The congenital form is caused by mutations in the genes encoding surfactant protein B or C, or the bc chain of the GM-CSF receptor. Certain infections, inhalation of toxic fumes or inorganic dust, hematological malignancies, and pharmacological immunosuppression cause the secondary form, which results in functionally impaired or reduced numbers of alveolar Mf. In acquired (or idiopathic) pulmonary alveolar proteinosis, a neutralizing autoantibody against GM-CSF causes a functional defect in alveolar Mf, including impaired surfactant lipoprotein catabolism and homeostasis. Though mechanisms for cytokine changes are unknown, both MCP-1 and M-CSF are raised in acquired disease. Treatment with GM-CSF improves acquired pulmonary alveolar proteinosis in some patients, though the mechanism of this effect remains to be elucidated. Tuberculosis

In primary pulmonary tuberculosis (TB), Mycobacterium tuberculosis organisms reaching the alveolar space are phagocytosed by alveolar Mf and are either killed or survive. In the latter instance, bacteria multiply and kill their host Mf; this is followed by the release of mycobacteria and subsequent infection of other host cells. Chemotactic factors attract circulating monocytes, lymphocytes, and neutrophils, none of which kills bacteria efficiently, leading to the formation of granuloma. These granulomas display a

556 LEUKOCYTES / Monocytes

high rate of monocyte and lymphocyte turnover, indicative of the toxicity of M. tuberculosis bacilli and requiring continuous replacement of host cells. For protective immunity, a strong nonantigen-specific granulomatous reaction within days is essential, followed by an antigen-specific T cell response within weeks, which is embodied by a delayed type hypersensitivity (DTH) reaction. The DTH response leads to the killing of infected Mf as the periphery of the granuloma becomes fibrotic and caseated. Necrotic areas are surrounded by a layer of epithelioid histiocytes and multinucleated giant cells, which in turn are enclosed by fibroblasts, lymphocytes, and bloodderived monocytes. If a sufficient cell-mediated immunity is generated, infection is permanently terminated at this stage. If this response is insufficient, Mf containing viable M. tuberculosis escape from the granuloma, or tubercle, rapidly leading to lymphatic dissemination of organisms to regional lymph nodes. In an attempt to control infection, the host DTH response leads to further pulmonary parenchymal destruction and eventually causes liquefaction of the caseous granuloma center, providing a rich and oxygenated environment for enhanced mycobacterial proliferation. A repeat infectious episode, or postprimary TB can be caused either by reactivation of a dormant lesion, or by inhalation of additional mycobacteria, leading to an infection that proceeds in spite of existing immunity. Tissue necrosis and caseation then occur due to a DTH response. Several cell surface molecules are involved in the binding and internalization of M. tuberculosis: complement receptors, macrophage mannose receptors (MMR), and CD14. Following binding and phagocytosis of mycobacteria, intracellular organisms are able to proliferate only if they avoid eradication by lysosomal enzymes, reactive oxygen intermediates (H2O2 and O2
), and reactive nitrogen intermediates (NO and NO2
). One of the mechanisms by which intracellular M. tuberculosis might evade destruction is through decreased fusion of mycobacteriumcontaining phagosomes with lysosomes. The presence of live mycobacteria may lead to a reduction in antigen processing, loading, and transport of MHCII to the cell surface. Following infection by M. tuberculosis, activated Mf may efficiently kill mycobacteria, while immature monocytes recruited from the periphery following infection are less effective and serve as the preferred host. Leukocyte-derived local production of IFN-g and TNF is central to activation and differentiation of recruited monocytes. Activated Mf present mycobacterial antigen to T cells in association with MHC class I, MHC class II, or nonpolymorphic MHC-like proteins such as CD1. Moreover, Mf have been shown to transfer

mycobacterial antigen to dendritic cells, thereby enhancing activation of naive T cells. Allergic Asthma

Alveolar Mf must display the capacity to both enhance and suppress an inflammatory response. Suppressive actions of Mf are mediated by soluble molecules and mechanisms requiring cell-to-cell interaction. NO has been suggested to promote Th2 responses, but may also suppress other inflammatory responses including leukotriene synthesis, cytokine production, and smooth muscle contraction. Regulatory cytokines TGF-b and IL-10 elaborated by Mf have generalized suppressive effects on inflammatory and immune responses. The gaseous molecule CO possesses generalized anti-inflammatory effects. Its administration following allergen sensitization/challenge reduced eosinophilia and levels of inflammatory cytokines in bronchoalveolar lavage. The enzymatic source of CO, heme oxygenase I, is increased in alveolar Mf from individuals with uncontrolled asthma. Prostaglandin E2 (PGE2) has been reported to possess contradictory effects on eosinophilic inflammation. PGE2 causes bronchodilatation, inhibits proinflammatory leukocyte function, and suppresses functions of fibroblasts as well as smooth muscle cells. While PGE2 promotes lymphocyte-dependent Th2 cytokine secretion in vitro, it suppresses Th2 cytokine secretion and allergic asthmatic responses. The PGD2 degradation product, 15-deoxy-PGJ2, exerts an in vitro anti-asthmatic effect by inhibition of T cell-dependent IL-5 generation and epithelial cell-derived IL-8 production. Both 15-deoxy-PGJ2 and the arachidonate 15-lipoxygenase product 15 hydroxyeicosatetraenoic acid (15-HETE) bind to the nuclear hormone receptor, peroxisome proliferatoractivated receptor-g (PPAR-g), which inhibits activation of the transcription factor nuclear factor-kB. Lipoxin A4, a dual 5- and 15-lipoxygenation product from arachidonic acid, exerts in vivo and in vitro antiinflammatory actions. Apoptotic leukocytes generated during any inflammatory process are phagocytosed by alveolar Mf, a process that triggers production of anti-inflammatory molecules such as PGE2 and TGF-b. In contrast, phagocytosis of pathogens via opsonin receptors such as the Fcg receptor for IgG leads to proinflammatory responses. Alveolar Mf from human asthmatics have been shown to have impaired Fcg receptor-mediated phagocytosis due to decreased surface expression of Fcg receptor I. See also: Asthma: Overview. Chemokines. Chemokines, CC: C10 (CCL6); MCP-1 (CCL2)–MCP-5 (CCL12); RANTES (CCL5); TARC (CCL17); TECK (CCL25).

LEUKOCYTES / T Cells 557 Chemokines, CXC: CXCL12 (SDF-1); IL-8; CXCL9 (MIG); CXCL10 (IP-10); CXCL1 (GRO1)–CXCL3 (GRO3). Dendritic Cells. Interstitial Lung Disease: Alveolar Proteinosis. Leukocytes: Pulmonary Macrophages.

role in innate, nonantigen-dependent immunity. Substantial research efforts are being directed at identifying mechanisms by which T or NK cells may contribute to disease pathogenesis or as targets for preventative strategies.

Further Reading

Introduction

Bilyk N and Holt PG (1993) Inhibition of the immunosuppressive activity of resident pulmonary alveolar macrophages by granulocyte/macrophage colony-stimulating factor. Journal of Experimental Medicine 177: 1773–1777. Bingisser RM, Tilbrook PA, Holt PG, et al. (1998) Macrophagederived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway. Journal of Immunology 160: 5729–5734. Blusse van oud Alblas AB and van Furth R (1979) Origin, kinetics, and characteristics of pulmonary macrophages in the normal steady state. Journal of Experimental Medicine 149: 1504–1518. Holt PG, Oliver J, Bilyk N, et al. (1993) Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. Journal of Experimental Medicine 177: 397–407. Imhof BA and Aurand-Lions M (2004) Adhesion mechanisms regulating the migration of monocytes. Nature Reviews: Immunology 4: 433–444. Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349: 2527–2539. Zlotnik A and Yoshie O (2000) Chemokines: a new classification system and their role in immunity. Immunity 12: 121–127.

Leukocytes, erythrocytes, and platelets are all derived from common progenitor cells, known as hematopoietic stem cells, which are located in the bone marrow. The myeloid progenitor is the precursor of granulocytes, macrophages, dendritic cells, and mast cells, whereas the lymphoid progenitor is the precursor of lymphocytes, which are the main topic of this article. The interaction of T cells with mononuclear cells including antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B lymphocytes is crucial for the onset of specific immune responses. T cells are derived from hematopoietic stem cells in the bone marrow or fetal liver (Figure 1). Following stimulation by stem cell factor (SCF), they differentiate into lymphoid stem cells, a common precursor for both T (thymic-derived) and B (bonemarrow derived) cells as well as for natural killer (NK) and some dendritic cells (DCs). Like SCF, interleukins such as IL-7 contribute to induce maturation of lymphoid stem cells into T- or B-cell precursor cells. Mature lymphocytes emerging from the thymus or bone marrow are in a quiescent or ‘resting’ state. In a healthy state, T cells in the blood are present at a concentration of approximately 2500 cells mm
3. Most naive lymphocytes, unless activated, are programmed to die within a few days after leaving the bone marrow or thymus. Once dispersed into the bloodstream, the lymphocytes migrate efficiently to various secondary (peripheral) lymphoid organs, such as the white pulp of the spleen, lymph nodes, tonsils, or mucosal regions of the respiratory or digestive tract, to carry out the different immune functions described below. The mucosal immune system of the lung includes antigen-dependent organized lymphoid tissues consisting of mucosal follicles of the respiratory tract, i.e., bronchus-associated lymphoid tissue (BALT) and diffuse lymphoid tissue consisting of widely distributed cells in the mucosal lamina propria.

T Cells P W Finn, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA B Schaub, University Children’s Hospital, Munich, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract Leukocytes emanate from hematopoietic stem cells in the bone marrow. T cells, derived from lymphoid progenitor cells, and natural killer (NK) cells mature and emerge from the bone marrow and thymus. Following antigen-presentation and costimulation, T cells proliferate and elicit their effector functions, including cytokine secretion. Communication between T and antigen-presenting cells is crucial in normal immune function, and key in immune dysfunction. T cells, whose main function is host defense, are classically thought to be important in allergic pulmonary diseases, including asthma. Recently, it has been suggested that T cells also play a prominent role in other pulmonary diseases including tuberculosis, sarcoidosis, Wegener’s granulomatosis, and cystic fibrosis. The main function of NK cells is to kill lymphoid tumor cells by producing effector proteins that penetrate the cell membrane and induce programmed cell death. NK cells are recognized as being important in asthma and tuberculosis, among other pulmonary disorders. In addition to the role of T cells and NK cells in adaptive, antigen-dependent immunity, there is also great interest in understanding their

Activation of T Cells and NK Cells This article will focus on antigen-dependent adaptive immune responses, which involve T and B cells, and are characterized by recognition of antigen via receptors such as the T-cell receptor (TCR) or B-cell receptor (BCR). In contrast, innate immune responses are antigen-independent, involving multiple cell

558 LEUKOCYTES / T Cells Bone marrow

Thymus

SCF Hematopoietic stem cell

Lymphoid stem cell

IL - 7 SCF

IL - 7 SCF

T- and B-cell precursors Naive T and B cells

Secondary Iymphoid organ

NK cells Figure 1 Lymphopoiesis: schematic overview of lymphocyte development. SCF, stem cell factor; IL, interleukin; NK cells, natural killer cells.

Antigen presentation co-stimulation

Proliferation amplification

Differentiation cytokine production

Th 2 cell Activated APC

CD86 / CD80 CD28 / CTLA4

MHC II TCR

T reg cell

IL - 10 IL - 13 IL - 4 IL - 5 IL - 6

TGF -
IL - 10

Activated T cell Th 1 cell

IFN -  TGF -  IL - 15

Figure 2 Antigen presentation and T-cell activation. Induction of different T-cell populations and cytokine secretion. APC, antigenpresenting cell; CTLA4, cytotoxic T lymphocyte-associated antigen 4; MHC, major histocompatibility complex; TCR, T-cell receptor; T reg cell, T regulatory cell.

types and receptors that recognize pathogen-associated molecular patterns (PAMPs) such as the Toll-like receptors (TLRs), which in turn recognize endotoxins and bacteria, among other ligands. The interaction of adaptive and innate immunity in pulmonary diseases such as asthma is a topic of current interest. The first step in adaptive immunity is the activation of naive T cells, requiring the presentation of specific antigen via major histocompatibility complex (MHC) II on APC to the TCR (Figure 2). This recognition of antigen occurs in the lymphoid organs and tissues, such as the lung, through which T cells are constantly crossing. TCRs recognize peptide fragments of intracellular pathogens transported to the cell surface by glycoproteins of the MHC. Two classes of MHC molecule (MHC I, II) transport peptides from different intracellular compartments to present them to distinct types of effector T cells: cytotoxic T cells (CD8 þ ), which kill cells infected with viruses; and

T-helper (Th) cells (CD4 þ ), which differentiate into Th1 and Th2 cells producing Th1 (e.g., interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), IL-15), Th2 cytokines (e.g., IL-4, IL-5, IL-6, IL-10, IL-13), and chemokines, activating other cells such as B cells and macrophages. The balance of Th1/Th2 cytokines, named the Th1/Th2 paradigm, has been a key immunological concept, and is a model that will be repeatedly referred to in this article. In addition, we will also consider the recent discovery of new Tcell subsets that may be as important or more important than the Th1/Th2 balance in the control of immune responses. T cells are crucial for both humoral and cellmediated responses of adaptive immunity. In addition to APC recognition and presentation of antigen to TCRs, additional co-stimulatory pathways mediate full T-cell activation (Figure 2). The best-characterized co-stimulatory molecules on T cells and APCs

LEUKOCYTES / T Cells 559

are the CD80 (B7.1) and CD86 (B7.2) molecules on APC, which interact with CD28 on T cells. CD28 is a glycoprotein homologous with cytotoxic Tlymphocyte-associated antigen 4 (CTLA4). Both CD28 and CTLA4 bind CD80 and CD86, but are positive and negative signals, respectively, for T-cell activation. In addition to the negative signal CTLA4, other negative signals suppress T-cell activation including programmed death-1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) molecules. PD-1 and BTLA also interact with members of the B7 family, providing negative regulation of immune function. Proliferation and differentiation of T cells is driven by interleukin-2 (IL-2), which is itself produced by activated T cells. In the case of specific antigen encounter, there is induction of IL-2 synthesis together with the a-chain of the IL-2 receptor (CD25). The b- and g-chains of the IL-2 receptor are expressed in resting T cells. Following activation, the association of the a-chain with the b- and g-chains induces a high affinity for IL-2, permitting the T cells to respond to very low IL-2 concentrations. These activated T cells are characteristically CD25 þ , a marker expressed in the maturation of thymocytes following expression of CD44 and c-kit. After rearrangement of the b-chain locus, expression of T a: b on the cell surface is associated with small amounts of CD3, followed by loss of CD25. This triggers progression through the cell cycle from the initial G1 phase. For complete T cell activation, signal 1 (antigen-presentation) and signal 2 (co-stimulation) are required. In the absence of co-stimulation, anergy occurs, characterized by the inability to produce IL-2, resulting in a lack of proliferation and differentiation even when antigen is presented. Anergy ensures the tolerance of T cells to self-tissue antigens. Besides macrophages and B cells, DCs are the most potent APCs, eliciting distinct functions; these are discussed in a separate article (see Dendritic Cells). NK cells develop in the bone marrow from the common lymphoid progenitor cells and circulate in the blood. Larger than T cells, they have distinct granules in the cytoplasm. NK cells, which lack expression of the TCR/CD3 complex, kill lymphoid tumor cell lines, releasing granules onto the surface of the bound target cell. The effector proteins penetrate the cell membrane, inducing programmed cell death. Activation of NK cells is induced by interferons or macrophage-derived cytokines. Unlike NK cells, NK T cells display rearranged TCR chains in association with the CD3 complex and express constitutive markers and receptors of both NK cells and T cells, supporting a potential relationship between NK T cells and NK cells.

T-Cell Function in the Normal Lung The main functions of T cells in the lung are host defense at mucosal surfaces and prevention of the entry of mucosal antigen. T cells protect the systemic immune system from inappropriate antigenic exposure, and the induction of helper or cytotoxic activities of T cells in the diffuse lamina propria. Follicular T cells are scattered throughout the dome area, including the germinal centers (densest in the interfollicular area), expressing activation markers such as CD25. CD4 þ T cells are widely distributed throughout the mucosal follicle, and CD8 þ T cells are found exclusively in the interfollicular area. T cells produce a variety of cytokines characterized as Th1 or Th2, but overlapping expression of these cytokines is increasingly being recognized. Rates of antigen exposure and use of adjuvants (substances that boost immunity through undefined mechanisms, likely involving innate immunity) are important factors in determining immune responses. For example, in murine models, Th1 responses follow oral protein antigen administration (without adjuvant). Th2 responses predominate following administration with certain adjuvants such as aluminum hydroxide (alum) and cholera toxins. In the diffuse mucosal lymphoid tissue, lymphocytes cluster in the intraepithelial lymphocyte (IEL) compartment (predominantly CD8 þ T cells) and the lamina propria lymphocyte (LPL) compartment (predominantly CD4 þ T cells), expressing the ab TCR and the human memory cell marker CD45 RO (495%). Specific to the lung, alveolar T cells are a primed memory cell phenotype that does not usually proliferate, nor does it normally undergo apoptosis. The percent of B and NK cells found in bronchoalveolar lavage (BAL) is very low; B cells are localized in the interstitial space around the pulmonary alveoli, while NK cells do not seem to play an important role in the local pulmonary defense. The evaluation of the CD4 þ /CD8 þ ratio in BAL fluid can be used as an auxiliary tool in the diagnosis of lung diseases. For example, the BAL CD4 þ /CD8 þ amounts in interstitial lung disease (ILD) may be decreased, reflecting a local imbalance between T helper primed memory cells and cytotoxic T cells.

T Cells in Respiratory Disease In the following sections, we detail pulmonary diseases involving immune cells, primarily T cells, and NK cells, as relevant. The mucosal surface of the lungs poses tremendous problems for an immune system charged with maintaining a sterile pulmonary environment. Despite challenges posed by different

560 LEUKOCYTES / T Cells

diseases, the immune system is quite effective in controlling most pulmonary dangers. To date, most data regarding pulmonary immunity derive from animal models or BAL in humans. The results of BAL-cell differentials indicating lymphocytic, neutrophilic, eosinophilic, or a mixed cellular pattern have been associated with diseases such as sarcoidosis, occupational exposure, or eosinophilic pneumonia, respectively. In some cases, BAL may aid in the diagnosis of immune-mediated pulmonary disease, when accompanied by disease history and clinical and radiological findings. Evaluation of the lymphocyte surface antigen phenotype may suggest sarcoidosis (increased proportion of CD4 þ lymphocytes) or extrinsic allergic alveolitis (increased proportion of CD8 þ lymphocytes). The BAL characterization of idiopathic pulmonary fibrosis (IPF) has been associated primarily with Th2 cytokines, while sarcoidosis or hypersensitivity pneumonitis are associated with a Th1 profile. High neutrophil counts in the BAL are characteristic of fibrosing processes or of occupational diseases caused by inhalation of inorganic dust.

T Cells in Asthma T cells play a critical role in the pathogenesis of atopic asthma, a disease characterized by mucus hypersecretion, bronchoconstriction, edema, and a Th2-prominent immune response. CD4 þ T-helper cells respond to inhaled antigens from a variety of nonmicrobial sources. The cytokines secreted by CD4 T þ cells recruit a variety of other cell populations, either directly via cytokine receptors on the secondary effector cells, or indirectly, via IgE antibody. The variety of recruited cells includes B cells, neutrophils, mast cells, macrophages, platelets, and eosinophils. Recently, the neonatal period has been recognized as playing an important role in the determination of immune responses and the later onset of diseases like asthma. Focusing on the major population of T cells, those that express ab-chains of the TCR, neonatal populations typically contain CD4 þ /CD8 þ -positive cells. In neonatal immunity, 90% of CD4 þ T cells express CD45RA, representing the ‘naive’ precursors of CD45RO cells. This proportion of CD45RO cells, loosely termed ‘memory’, steadily increases during childhood with exposure to environmental antigens. Some data suggest a predominance of specific cytokine patterns according to age. At birth, a Th2 cytokine pattern appears to dominate. During healthy childhood, Th1 cytokines increase. Since 1986, the Th1/Th2 paradigm, originally described in murine cells as an increase in Th2

(e.g., IL-4, IL-5, IL-13) and a decrease in Th1 (e.g., IFN-g) cytokine secretion, has dominated our understanding of asthma and allergic diseases. In asthmatic patients, this Th2 predominance may be modulated by factors not yet identified. An opposing paradigm, termed the hygiene hypothesis, suggests that exposure to a less hygienic environment is associated with a lower prevalence of allergic diseases. Exposure to different microbial substances, family size, older siblings, environment, and infections may account for differences in the development of allergen disease or severity. To date, the immunological mechanisms that protect against allergic responses are not well understood, but should take into account cell types and distinct pathways in addition to the Th1/Th2 paradigm. Thus, a spectrum of T cells including Th1 cells, CD4 þ T cells, different subsets of T regulatory cells (T regs) (e.g., Th3, Tr1 cells), and NK T cells may play a critical role in regulating allergy and asthma. CD4 þ CD25 þ cells (T regs), a minor fraction of CD4 þ T cells (10%), express the IL-2 Ra chain and are produced by the thymus as a functionally mature T-cell subpopulation. The CD4 þ CD25 þ T regs are characterized by markers such as the transcription factor Foxp3, glucocorticoid-induced TNF-receptor (GITR), lymphocyte activation gene (LAG)-3, and constitutive levels of CTLA4. T regs in the circulation, and possibly at the site of injury, may play an important role in the determination of disease or a healthy state by providing a balance between Th1 and Th2 cells, inducing tolerance, or other as yet undescribed mechanisms. Th3 cells produce transforming growth factor beta (TGF-b) as well as IL-4 and IL-10, and suppress the development of experimental autoimmune encephalomyelitis. Th3 suppressive effects in autoimmune models suggest that they may inhibit the effects of pathogenic autoantigenspecific T cells. Tr1 cells, induced with IL-10, can regulate antigen-specific immune responses, inhibiting the function of pathological autoantigen-specific T cells in vitro. NK T cells recognize self-glycolipid antigens in the respiratory tract after exogenous allergens enter the lung, followed by production of large quantities of cytokines. NK T cells produce IL-4 and IL-13, cytokines presumed to regulate the development of airway hyperresponsiveness (AHR) in allergic asthma. Whether NK T cells potentiate the development of asthma via cytokine production of IL-4 and IL-13 has yet to be determined. Therapies enhancing the development of regulatory T cells and inhibiting the function of NK T cells in patients with allergic disease offer exciting opportunities in the prevention or treatment of these disorders.

LEUKOCYTES / T Cells 561

T Cells in Chronic Obstructive Pulmonary Disease Chronic obstructive pulmonary disease (COPD) is defined as chronic irreversible airflow limitation, characterized by slowly progressive and irreversible deterioration in lung function, often induced by heavy smoking. T cells are increased in patients with COPD, showing a correlation between the number of T cells mm
3 of lung and the extent of emphysema. While both CD4 þ and CD8 þ T cells are increased in the airways and lung parenchyma, CD8 þ T cells predominate in studies to date. In addition to macrophages, CD8 þ T cells are the prevalent cell population in COPD. Increased CD8 þ T cells are also seen in healthy smokers. In contrast, there is a predominance of CD4 þ T cells in asthma. In exacerbations of COPD, bronchial biopsies demonstrated increased T-cell infiltration; in addition, increased infiltration of T cells is noted in the alveolar walls, small airways, and by the presence of lymphoid follicles that contain a core of B lymphocytes surrounded by T cells. The CD4 þ and CD8 þ T cells in COPD seem to be fully activated, showing a predominant Th2/cytotoxic T1 (Tc) pattern with increased IFN-g and Th1 chemokines. These data suggest that specific T-cell populations focused toward T-cell activation in COPD may be of importance.

T Cells in Tuberculosis, Sarcoidosis, and Wegener’s Granulomatosis In tuberculosis, the mycobacterium enters mainly by the respiratory route, and the alveolar macrophage is the important cell type combating the pathogen. However, in tuberculosis, within 1 week of infection, the number of activated CD4 þ and CD8 þ T cells in the lung-draining lymph nodes increases, followed by migration to the lung and to site of infection. In murine experiments, lack of CD4 þ cells results in delayed distribution of activated CD8 þ T cells from draining lymph nodes to distant organs and, consequently, delayed acquisition of immune protection. In humans, the tuberculosis granuloma contains both CD4 þ and CD8 þ T cells. Both CD4 þ and CD8 þ cells sustain infection within the granuloma and prevent reactivation. CD4 þ T cells are most important in the protective response to control the infection, with the primary effector function being IFN-g production and, possibly, other cytokines such as TNF-a, which are sufficient to activate macrophages. IFN-g is presumed to activate macrophages to combat intracellular infection via acidification of the phagosomal compartment housing the infection and production of nitric

oxide (NO), which is thought to have bactericidal effects. Also, apoptosis or lysis of infected cells by CD4 þ T cells may play a role in controlling infection. Whether Th1 or Th2 cytokines predominate in tuberculosis is not yet fully understood, but appears to depend on the site of location (peripheral blood or lung) and different stage or severity of disease. Primarily Th1 responses have been observed at the site of infection, and the strength of the Th1 type response is known to relate directly to the clinical manifestation of the disease. While the mechanism by which mycobacterial proteins gain access to the MHC I molecules is not fully understood, CD8 þ T cells may play a role in the balance of pulmonary Th1/Th2 cells. NK cells, as effector cells of innate immunity, may directly lyze the pathogens or infected monocytes. Decreased NK cell activity during tuberculosis infection may be the effect, rather than the cause, of the disease. In addition, apoptosis is a likely mechanism of NK cytotoxicity. In sarcoidosis, the salient pathological feature is the formation of noncaseating granulomas in more than one tissue system. Cytokines and chemokines produced by CD4 þ T cells, and mononuclear phagocytes guide the development and function of granuloma formation in the lung. A Th1 polarized immune response is demonstrated by increase in IFN-g and IL-2, as well as IL-12 and IL-18 (monocyte/macrophage-derived), providing a positive feedback loop, consistent with enhanced Th1 responses. IL-15, secreted by mononuclear cells among others, is involved in T-cell activation; TNFa and chemokine expression contribute to trafficking of immune cells to the inflammatory site. Sarcoidosis patients with persistent inflammation can develop pulmonary fibrosis or fibrosis can develop de novo, for example, idiopathic pulmonary fibrosis (IPF). As IPF is rare, human data are limited regarding cytokine profiles to assess the contribution of Th1/Th2 distribution. From experimental models, Th2 cytokines are profibrotic, enhancing fibroblast production of collagen. In contrast, the Th1 cytokine, IFN-g acts against fibrosis by downregulating fibroblast production of collagen and TGF-b expression, but can also be proinflammatory. Indeed, a phase III trial administering IFN-g to IPF patients is ongoing in the US. Alternatively, IFN-g may play a profibrotic role by enhancing tissue injury and subsequent repair processes, depending on timing of expression, and the presence of other proinflammatory cytokines. Whether patients who develop pulmonary fibrosis convert to a more profibrotic Th2 phenotype later in the disease or initially present with a Th2 dominant response is unknown. Another possibility

562 LEUKOCYTES / T Cells

is that fibrotic changes occur in a Th1 environment, or that other profibrotic mediators such as TGF-b, insulin-like growth factor-1, and platelet-derived growth factor may contribute to a fibrotic outcome regardless of Th1/Th2 cytokine milieu. In Wegener’s granulomatosis, an autoimmune disease of unknown etiology, a clinical triad consists of necrotizing granulomatous inflammation of the upper/ lower respiratory tract, necrotizing glomerulonephritis, and an autoimmune systemic vasculitis affecting predominantly small vessels. Recent data suggest that TNF-a-producing Th1 type CD4 þ CD28
T-cell effector memory T cells may be involved in the pathogenesis of Wegener’s granulomatosis. Their presence as a Th1-type cytokine population within granulomas provided the rationale for using TNF-a blocking agents in Wegener’s disease refractory to standard induction therapy with cyclophosphamide and corticosteroids. The discovery of complex immunoregulatory networks to downregulate inflammation, for example, CD4 þ CD25 þ T regs, suggests novel potential immune pathways that may be useful in the modulation of granulomatous inflammation.

T Cells in Hypersensitivity Pneumonitis, Cystic Fibrosis, and Allergic Bronchopulmonary Aspergillosis In other diseases with pulmonary fibrosis such as hypersensitivity pneumonitis, pneumoconiosis, or chronic beryllium disease, Th1 cytokines may also play a role. In hypersensitivity pneumonitis, both type III and type IV hypersensitivity reactions are mediated by immune complexes and Th1-type cells. Proinflammatory cytokines and chemokines activate alveolar macrophages, which secrete IL-12, promoting the differentiation of CD4 Th0 cells into T cells of the Th1 phenotype. IFN-g production by Th1 cells further stimulates alveolar macrophages to produce greater amounts of IL-1 and TNF-a, resulting in a positive feedback loop with additional IFN-g production. Also, alveolar macrophages induce an influx of CD8 þ and, to a lesser degree, CD4 þ T cells into the lung, facilitating granuloma formation, and promoting the development of pulmonary fibrosis. In cystic fibrosis (CF), chronic pulmonary infection and destructive inflammation are still the major causes of morbidity and mortality. A dysregulation of immune responses in CF includes antibody production by B cells, and perpetuation of the inflammatory response by neutrophils. Overall, T-cell function may be of crucial importance for inflammatory control. For example, IL-10, a major regulatory cytokine, produced not only by macrophages and NK cells but

also by Th1, Th2, and T regs is increased in CF. Furthermore, an imbalance of cytokines, for example, high IL-2 (proliferation) and low IL-8 production (activation of granulocytes, chemotaxis), may contribute to the immune dysregulation in CF. Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease mediated by an allergic late-phase inflammatory response to Aspergillus fumigatus antigens that affects approximately 10% of patients with CF. ABPA is characterized by markedly elevated Aspergillus-specific and total IgE levels and eosinophilia, and manifested by wheezing, pulmonary infiltrates, bronchiectasis, and fibrosis, which afflict asthmatic and CF patients. The pathogenesis of ABPA seems to be a prolonged asthmatic late-phase reaction orchestrated by CD4 þ Th2-like cells in response to persistent pulmonary A. fumigatus allergen exposure. Thus, polyclonal and A. fumigatus-specific IgE antibodies (and IgA and IgG) and blood pulmonary eosinophilia are stimulated by Th2-derived cytokines such as IL-4 and IL-5. In addition, IL-4 would also promote pulmonary transendothelial migration of eosinophils, basophils, and lymphocytes via induction of cell adhesion molecules and their ligands. In addition, A. fumigatus proteases play a role in facilitation of antigen transport across the epithelial cell layer by damaging the epithelial integrity and by interacting directly with epithelial cell-surface receptors, producing proinflammatory cytokines and corresponding inflammatory responses.

Consideration of Present and Proposed Therapeutic Options Corticosteroids, by general dampening of the immune function directed at T cells as well as other immune cells such as eosinophils, monocytes, and neutrophils, have been used as the mainstay of therapy for a number of lung diseases, including asthma, sarcoidosis, IPF, hypersensitivity pneumonitis, and even pleural tuberculosis. Corticosteroids exert their effects through binding to cytoplasmic glucocorticoid receptors, and modulate the transcription rate of inflammatory factors such as transcription factors. They act as inhibitors of proinflammatory cytokine production, also suppressing fibroblast proliferation and collagen deposition. However, the side effects of generalized immunosuppressive therapy can result in devastating side effects in addition to those of the disease itself, for example, osteoporosis, increased infection, diabetes, and cataracts. Strategies of directed therapy in asthma have included efforts at inhibiting events downstream of antigen presentation to T cells. Administration of anti-IL-5 to block

LEUKOCYTES / Pulmonary Macrophages 563

eosinophils has yielded only marginal effects, while anti-IgE treatment has been shown to be efficacious in some severe allergic asthmatics. Recently, suppression of TNF-a in severe asthma was shown to be efficacious in a small open study, but further examination is necessary. Use of a leukotriene receptor antagonist, which blocks the leukotriene pathway and has weak anti-inflammatory activity, has had variable success in decreasing asthma symptoms or reducing the need for corticosteroids. These data and others suggest that the success of therapeutic interventions may depend not only on the selection of one specific target molecule, but potentially on a combination of several molecules and importantly also on the genetic background of the patient as well as environmental exposures, supporting the expanding field of pharmacogenetics and epigenetics. Potential new therapeutic approaches include target cell populations such as T regs or NK cells. T regs may perform essential functions in modulating immune responses, including regulation of Th1/Th2 cytokine secretion. In murine models of autoimmunity, the adoptive transfer of T regs cures ongoing disease. Manipulating T regs or NK cells may be important not only in common diseases of unknown etiology such as asthma, but also for less common pulmonary entities such as sarcoidosis or granulomatosis. Prior to any strategies regarding therapeutic intervention, it is crucial to evaluate the multifaceted functions of both cell types, and, importantly, as they interact with each other and other cells, including APCs. Substantial research efforts are being directed at identifying mechanisms by which T cells and/or NK cells may contribute to disease pathogenesis or may become targets for preventive strategies.

Further Reading Akbari O, Stock P, DeKruyff A, and Umetsu DT (2003) Role of regulatory T cells in allergy and asthma. Current Opinion in Immunology 15: 627–633. Barnes PJ and Cosino M (2004) Characterization of T lymphocytes in chronic obstructive pulmonary disease. Public Library of Science Medicine 1: 1. Costabel U and Guzman J (2001) Bronchoalveolar lavage in interstitial lung disease. Current Opinion in Pulmonary Medicine 7: 255–261. Di Stefano A (2004) Cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview. Clinical and Experimental Allergy 34: 1156–1167. Finkelstein R, Fraser RS, Ghezzo H, and Cosio MG (1995) Alveolar inflammation and its relation to emphysema in smokers. American Journal of Respiratory and Critical Care Medicine 152(5 Pt 1): 1666–1672. Korsgren M (2002) NK cells and asthma. Current Pharmaceutical Design 8: 1871–1876. Lamprecht P and Gross WL (2004) Wegener’s granulomatosis. Herz 29: 1. Mohr LC (2004) Hypersensitivity pneumonitis. Current Opinion in Pulmonary Medicine 10: 401–411. Moller DR (2002) Pulmonary fibrosis of sarcoidosis. New approaches, old ideas. American Journal of Respiratory Cell and Molecular Biology 29: S37–S40. Mosmann TR and Coffman RL (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annual Review of Immunology 7: 145–173. Raja A (2004) Immunology of tuberculosis. Indian Journal of Medical Research 120: 213–232. Sharma OP (2002) Sarcoidosis and other autoimmune disorders. Current Opinion in Pulmonary Medicine 8: 452–456. Umetsu DT, et al. (2003) Regulatory T cells control the development of allergic disease and asthma. Journal of Allergy and Clinical Immunology 112(3): 480–487. Woodland D (2003) Cell-mediated immunity to respiratory virus infections. Current Opinion in Immunology 15: 430–435.

Pulmonary Macrophages Conclusion Recent discoveries in molecular immunology have advanced our understanding of the role of immune cells in the pathogenesis of pulmonary diseases. Immune cells such as T cells, and T cell subsets such as T regs and NK cells, play important roles in the activation of regulatory pathways in several lung diseases and, importantly, in host defense. T cells, as crucial regulators of the interplay between innate and adaptive immune responses, could provide promising targets for future therapeutic options in pulmonary diseases. See also: Asthma: Overview. Dendritic Cells. Interleukins: IL-7. Leukocytes: Monocytes; Pulmonary Macrophages. Toll-Like Receptors.

G S Davis and M E Poynter, University of Vermont, Burlington, VT, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Macrophages serve as the first cellular line of defense against inhaled particulates and infectious agents. They are key participants in both innate non-antigen-specific immune responses and adaptive antigen-specific immune responses in the lung. They may play central roles in the transition from lung injury and inflammation to fibrosis in both chronic airway and diffuse interstitial lung diseases. The macrophages of the lung arise from hematopoietic stem cells in the bone marrow. Several populations of macrophages reside in the normal healthy lung: alveolar, interstitial, airway and intravascular macrophages, dendritic cells, and monocytes. The pulmonary macrophage plays a central role in the homeostasis of the lung. Under normal

564 LEUKOCYTES / Pulmonary Macrophages resting conditions it protects against the daily burden of inhaled particulates, microbes, and cellular debris without triggering an inflammatory or immune response that could injure the lung. When challenged by virulent infectious agents, the macrophage is a first line of defense, a sentinel that calls forth additional immune inflammatory resources, and when activated it is an effective participant in microbial killing. Its potent degradative enzymes, inflammatory cytokines, and mediators that signal fibroblast growth and collagen production appear to be key elements in a wide variety of environmental and idiopathic lung diseases.

Introduction Macrophages serve as the first cellular line of defense against inhaled particulates and infectious agents. They are key participants in both innate non-antigen-specific immune responses and adaptive antigen-specific immune responses in the lung. They may play central roles in the transition from lung injury and inflammation to fibrosis in both chronic airway and diffuse interstitial lung diseases.

Macrophage Subpopulations in the Lung Several populations of macrophages reside in the normal healthy lung (Table 1). The macrophages of the lung arise from hematopoietic stem cells in the bone marrow. Resident alveolar macrophages are large mobile phagocytes, loosely adherent to the epithelial surface. They appear to move about in the epithelial lining fluid in order to encounter, engulf, sequester, and dispose off the particulates, microbes, and debris that are inhaled as part of normal life. It is postulated that the primary job of these cells is sanitation: cleansing the alveolus promptly to prevent injury to structural surfaces and limit extracellular replication of microbial agents without triggering an immune or inflammatory response. In

many instances the phagocytosed material can be killed or degraded within the alveolar macrophage. Even if the material cannot be eliminated effectively, it can be isolated and sequestered while the cell and its contents are removed from the lung by the mucociliary escalator or while immune inflammatory responses are mobilized. Macrophages also exist embedded in the airway wall and parenchymal tissue of normal subjects, albeit at lower numbers than in the alveolar spaces. These interstitial macrophages are thought to represent a self-renewing population of monocytes, some of which may go on to populate the alveolar compartment as alveolar macrophages, as well as to serve functions as innate sentinels. Within the lungs of large animals (possibly including humans) but not of rodents, a population of macrophages, termed pulmonary intravascular macrophages (PIMs), exist adhered to the endothelial cells comprising the pulmonary capillaries. The PIMs are phagocytic, capable of ingesting bacteria and producing inflammatory mediators. Dendritic cells (DCs) share a number of qualities with monocytes and macrophages, and are interspersed throughout the lung tissues. DCs, in their immature form, are especially proficient at engulfing antigenic materials and processing antigenic proteins into smaller peptide fragments. Ingestion of bacterial products and/or exogenous cytokines stimulates activation, mobilization, and maturation of DC. Activated DC upregulates CCR7, a chemotactic receptor that induces the DC to travel to lymphoid aggregates or regional lymph nodes where large numbers of T cells and B cells are located. Upon maturation, the DC may present the antigenic peptide on the cell surface in the context of major histocompatibility complex (MHC) class II molecules. DCs are considered to be professional antigen-presenting cells

Table 1 Populations of macrophages in the lung Name

Location in the lung

Features

Antimicrobial activity

Antigen presenting function

Inflammatory recruiting function

Alveolar macrophage Interstitial macrophage Vascular macrophage Airway macrophage Dendritic cell

Alveolar surface

Large complex cell, many inclusions Small cell, some inclusions Small cell, some inclusions Small cell, some inclusions Large cell, long processes Small cell, few inclusions

Excellent

Very poor

Good (better when activated) Good (better when activated) Good (better when activated) Poor

Probably poor Probably poor

Excellent recruitment Activation of fibroblasts Probably good

Probably poor

Probably good

Excellent, primary role Poor

Poor

Monocyte

Alveolar interstitium Capillaries and venules Airway wall, submucosa Interstitium, lymphoid tissues Recruited from blood stream

Good (better when activated)

Good

LEUKOCYTES / Pulmonary Macrophages 565

(APCs). With activation, the DC also upregulates cell-surface molecules that act as co-receptors in Tcell activation and greatly enhance the ability to activate T cells during antigen presentation. DCs are named for their characteristic appearance in tissues, wherein they extend cellular processes away from the main cell body perhaps to sample antigenic material at more remote sites. Several subclasses of DC have been described, including plasmacytoid and myeloid dendritic cells. In general, the plasmacytoid DCs (PDCs) elicit suppressive effects, while the myeloid DC (MDC) subset elicits immunostimulatory effects. The MDC subset shares many characteristics with the monocyte/macrophage population, and is likely to be derived from them. Monocytes are large mononuclear phagocytes of the peripheral blood, ranging in size from 10 to 30 mm in diameter with band- or kidney-shaped nuclei. Blood monocytes possess limited capabilities as phagocytes or as secretors of enzymes and cytokines. Instead, their main function may be to act as precursors for tissue macrophages. Blood monocytes form an important component of the lung macrophage population, both as precursors to mature macrophages and as recruited inflammatory leukocytes. Monocytes from the blood enter tissues and increase in size, phagocytic activity, and lysosomal enzyme content and become macrophages. In addition to circulating blood monocytes, there appear to be local replicating populations of mononuclear cells within the parenchyma and lymphoid aggregates of the lung that may serve as precursors for the resident populations of pulmonary macrophages. Blood monocytes are recruited into the lung rapidly and abundantly by a variety of inflammatory or defense-related signals. The number of macrophage/monocyte cells is greatly increased above resident levels in virtually all infectious or inflammatory lung diseases, as evidenced by recovery from enzyme digestates or bronchoalveolar lavage (BAL) samples. Thus, lung inflammatory cell populations contain a mixture of mature, activated macrophages and monocytes in transition.

Structure and Function The most detailed information about macrophages in the lung pertains to the alveolar macrophage because this cell can be obtained in large numbers and relatively high purity by BAL from normal human subjects, from patients with lung diseases, and from laboratory animals. The alveolar macrophage is a large pleomorphic cell, spherical in suspension but spread into a ‘fried egg’ shape on the alveolar surface or in cell culture (Figure 1). A highly ruffled cell membrane with numerous folds, pseudopods, and

Figure 1 Human alveolar macrophage cell surface features. Scanning electron micrograph of a human alveolar macrophage recovered by bronchoalveloar lavage and placed in cell culture. The cell is adherent and spreading on the plastic surface by extending thin cytoplasmic psuedopods. The complex folded and ruffled surface of the cell is prominent. Courtesy of A R Brody, PhD, Department of Pathology, Tulane University, New Orleans, LA, USA.

projections offers a very large surface area for the cell volume, and provides maximum contact with materials in its environment. The alveolar macrophage is mobile and can move across surfaces and through the pores of experimental membranes, although it moves more slowly and less readily than a blood monocyte. The shape of the folded surface is conferred by the extent of polymerization of cytoplasmic actin filaments, and changes substantially with activation. The intracellular anatomy of the alveolar macrophage is also complex (Figure 2). The nucleus is typically crescent- or kidney-shaped and is placed eccentrically within the cell. Nuclear chromatin is clumped at its periphery. There is an abundant Golgi apparatus and plentiful mitochondria and endoplasmic reticulum, perhaps reflecting the active role of the alveolar macrophage in producing enzymes, cytokines, and oxidants. There are numerous lysosomal granules that contain cathepsins, proteolytic enzymes, collagenases, and other enzymes. These chemicals may be excreted into the environment near the cell by exocytosis, or utilized for intracellular action by the merger of lysosomes with phagosomes containing engulfed materials. The alveolar macrophage cytoplasm usually features large and small phagolysosomes that represent engulfed debris in various stages of digestion or detoxification. Living or dead bacteria, inhaled particles and fibers, and

566 LEUKOCYTES / Pulmonary Macrophages

M

N

L

PL

Figure 2 Human alveolar macrophage intracellular structure. Transmission electron micrograph of a human alveolar macrophage recovered by bronchoalveolar lavage shows a complex ultrastructure. The eccentrically placed kidney-shaped nucleus (N) with peripheral chromatin, numerous cell surface folds, and abundant intracellular inclusions are typical of this cell type. Lysosomes (L), mitochondria (M), and phago-lysosomes (PL) containing ingested debris can be identified. Courtesy of A R Brody, PhD, Department of Pathology, Tulane University, New Orleans, LA, USA.

debris from nearby dead cells can be identified. Most alveolar macrophages also contain distinctive whorled inclusions (lamellar bodies) that represent phagocytosed surfactant lipids and proteins. The alveolar macrophage is believed to be an important mechanism for disposing of (or possibly for recycling) pulmonary surfactant. The inclusion of the alveolar macrophage is a marker of the history of the alveolus. Human subjects or experimental animals exposed to environmental dusts and fibers carry these materials within the alveolar macrophage for months or years after exposure. Bacteria within alveolar macrophages in sputum samples are taken as proof of pneumonia, an active infection in the alveolar spaces. Hemorrhage is reflected as erythrocytes in alveolar macrophages within minutes after alveolar bleeding, and hemosiderin pigment from hemoglobin digestion appears in alveolar macrophages about 48 h after bleeding and may last for several weeks. Retired granite workers show numerous silica particles in their alveolar macrophages recovered by BAL more than 20 years after leaving the work place. Metabolic Functions

The metabolic functions and products of the macrophage reflect its many roles in lung homeostasis and disease. Most of these materials have many different activities, targets, and interactions with other mediators depending on the exact location and circumstances. Table 2 provides an overview, not a comprehensive summary.

Enzymes Macrophages produce many enzymes that appear designed primarily to act within the phagolysosome to create an acidic environment, inhibit microbial growth, kill susceptible microorganisms, and degrade organic debris. Most of these hydrolytic enzymes are upregulated by the act of phagocytosis. If they leak into the extracellular milieu they may be irritating and destructive. The matrix metalloproteinases can degrade structural proteins such as elastin and collagen. While normal tissue remodeling depends on their action, lung disease can result from excess degradation (e.g., emphysema) or inhibited activity (e.g., pulmonary fibrosis). Reactive oxygen species (ROS) such as superoxide, hydroxyl radical, and hydrogen peroxide appear to be essential agents for killing microbes within the phagolysosome. Accordingly, an oxidative burst of ROS generation is a central feature of macrophage activation following ingestion of microbes. Humans and experimental animals that lack the enzymes responsible for generating these oxidant materials have impaired pulmonary host defenses and develop pyogenic bacterial infections. If these products are released extracellularly they may have the potential to kill target cells or damage proteins. Lipid mediators Macrophages produce lipid mediators derived from cell membrane arachidonic acid. The lipid mediator linked most securely to pulmonary macrophages is leukotriene B4, an important mechanism for attracting and activating neutrophils. The cysteinyl leukotriene C4 (and its metabolites D4 and E4) is an important mediator in asthma and bronchoconstriction, acting through the CysLT1 receptor. Mast cells and eosinophils appear to be the major source, but pulmonary macrophages may contribute as well. New attention focuses on profibrotic activities mediated through the CysLT2 receptor, and could be another role for macrophages in chronic interstitial lung diseases. Cytokines Pulmonary macrophages are a very important source for the cytokines that drive acute and chronic lung diseases. The synthesis and secretion of cytokines by macrophages probably does not occur substantially under basal conditions, although BAL alveolar macrophages from humans and animals do produce small quantities of many cytokines when placed in resting or unstimulated cell culture. Activation of macrophages by mediators or direct cell contact triggers an intense, selective, and specific upregulation of families of cytokines with defined functions. Although gene expression of many of these proteins is mediated through nuclear factor kappa B (NF-kB), the panoply of surface receptors carried by

LEUKOCYTES / Pulmonary Macrophages 567 Table 2 Macrophage products and their functions Macrophage product

Function

Enzymes Cathepsin B Lysozyme Acid hydrolases Angiotensin convertase Matrix metalloproteinases (MMPs) Collagenase Elastase Peroxidase Superoxide dismutase Tissue inhibitors of metalloproteinases (TIMPs)

Antimicrobial (intracellular) Antimicrobial (intracellular) Degradation of debris (intracellular) Activate angiotensin Degrade connective matrix proteins (extracellular) Degrade collagen (extracellular) Degrade elastin (extracellular) Generate hydrogen peroxide Convert superoxide to H2O2 and H2O Inhibit matrix metalloproteinases

Oxidant products Superoxide (Od
2 ) Hydrogen peroxide (H2O2) Nitric oxide (NO) Hydroxyl radical (dOH) Peroxynitrite (ONOO
)

Antimicrobial Antimicrobial Antimicrobial Antimicrobial Antimicrobial

Lipid mediators Leukotriene B4 (LTB4) Leukotriene C4 (LTB4) Platelet activating factor (PAF) Prostaglandin E2 (PGE2)

Attract/activate neutrophils Bronchoconstriction Vascular tone, bronchoconstriction Anti-inflammatory

Cytokines Tumor necrosis factor alpha (TNF-a) Interleukin-1-beta (IL-1b) Interleukin-6 (IL-6) Interleukin-12 (IL-12) Interleukin-15 (IL-15) Interleukin-18 Fibroblast growth factor Transforming growth factor beta (TGF-b1)

Lymphocyte activation, cachexia, death Lymphocyte activation Proinflammatory reactant TH1 T-cell activation TH1 T-cell and natural killer (NK) cell activation TH1 T-cell activation Promote fibroblast growth Fibroblast collagen synthesis, inhibitor of macrophages

Chemokines Interleukin-8 (IL-8; CXCL8) Macrophage chemotactic protein 1 (MCP-1; CCL2) Interferon gamma inducible protein 10 (IP-10; CXCL10) RANTES (CCL5, MCP-2) Macrophage inflammatory protein 1 (MIP-1a; CCL3)

Attract/activate neutrophils Attract/activate macrophages, TH1 T cells, NK cells Attract/activate TH1 T cells, NK cells Attract monocytes Attract monocytes, macrophages

the macrophage (see below) allows focused responses to many distinct ligands. Acute reactants such as tumor necrosis factor alpha (TNF-a), interleukin-1beta (IL-1b), and interleukin-6 (IL-6) are generated by many stimuli including bacteria (endotoxin), silica, or asbestos. The elaboration of IL-12 and IL-15 by macrophages appears to be essential for effective activation of T cells and natural killer (NK) cells in the innate immune response, and an essential step in converting to an antigen-specific adaptive immune response. Mice that lack either of these cytokines are highly susceptible to tuberculosis and other intracellular pathogens. Macrophages are believed to be a primary source for the cytokines that promote fibroblast proliferation and increased collagen production in pulmonary fibrosis. Unlike macrophages in other

(intracellular; (intracellular; (intracellular; (intracellular; (intracellular;

? ? ? ? ?

extracellular extracellular extracellular extracellular extracellular

injury?) injury?) injury?) injury?) injury?)

tissues, alveolar macrophages play an important role in the modulation of inflammatory and immune responses through the constitutive secretion of antiinflammatory cytokines, especially IL-10 and transforming growth factor beta (TGF-b). These cytokines function to retain the lung in a relatively silent state under normal conditions, which allows the resident phagocytes to deal inconspicuously with foreign inhaled material without the need to elicit inflammatory or adaptive immune responses. Chemokines When needed, attracting and activating other leukocytes for pulmonary inflammation and immune responses is a vital function performed by macrophages through the production of chemokines. These chemoattractant proteins are structurally

568 LEUKOCYTES / Pulmonary Macrophages

related and signal through a family of G-protein-coupled membrane receptors. The most common chemokines are divided into CC and CXC families based on amino acid structure. There is substantial redundancy in the secretion, activities, and receptor expression related to these polypeptides, but chemokines and their cognate receptors do exhibit substantial specificity that allows selective recruitment of neutrophils, eosinophils, TH-1 or TH-2 T cells, or NK cells. Cell Surface Receptors and Ligands

The cell surface molecules displayed by the pulmonary macrophage are its interface with the particles, microbes, signal peptides, and other cell types that surround it. Some of the more important surface molecules are listed in Table 3. Macrophages recognize material destined for phagocytosis through a myriad of cell surface receptors. The scavenger receptors are not specific for individual ligands, but can bind dust particles, fibers, and debris. Mannosyl

receptors bind glycoconjugates common on fungal and bacterial cell walls. Toll-like receptors (TLRs) facilitate interactions with microbes, including Gram-positive and Gram-negative bacteria, fungi, parasites, and viruses. Signaling through TLRs (particularly TLR4) is believed to be an important route for macrophage activation. Receptors for the Fc portion of immunoglobulins mediate phagocytosis of microbes (or other particulates) that have been coated with antibody, a more rapid and efficient ingestion than through other receptors. An important macrophage phagocytic function is clearance of apoptotic cells from the lungs. This occurs under normal conditions of homeostasis, as well as during resolution of inflammation. The ingestion of apoptotic bodies, necrotic cells, and their debris does not elicit an inflammatory response on the part of the macrophages. Receptors for cytokines and chemokines allow the macrophage to respond to endocrine, paracrine, and autocrine cell signals, to become activated, to migrate to sites of inflammation, and to participate in

Table 3 Cell surface molecules on pulmonary macrophages Cell surface receptors and ligands

Function

Phagocytic receptors Scavenger receptors Toll-like receptors Mannosyl receptors Immunoglobulin Fc receptors (IgG, IgM, IgA) Complement receptors (C3, C5)

Bind Bind Bind Bind Bind

Cytokine receptors Tumor necrosis factor R1 and R2 Interferon gamma R1 and R2 (CDw119) Interleukin-1 receptor (IL-IR CD121b) Interleukin-4 receptor (IL-4R)

Bind TNF-a/TNF-b IFNGR1 binds IFN-g, activates macrophage Low levels, autocrine effects Binds IL-4, may downregulate macrophage function

Chemokine receptors CXCR1 (CD128a) CXCR2 (CD128b) CXCR3 (CD183) CXCR4 (CD184) CCR5 (CD195)

Binds Binds Binds Binds Binds

Adhesion molecules Intercellular adhesion molecule-1, 2, 3 (ICAM-1, 2, 3 CD54, 102, 50) Sialoadhesin receptor (CD169) Integrin aM (Mac-1a CD11b) PECAM-1 (CD31) L-Selectin (CD62L)

Intercellular adhesion Migration, neutrophil interactions Intercellular adhesion Intercellular adhesion Intercellular adhesion

Immune regulatory molecules MHC Class II MHC Class I CD4

Present antigen peptides to CD4 þ T cells, B cells Confer cell type recognition, present antigen to CD8 þ cells Antigen presentation costimulatory molecule

Other epitopes Lipopolysaccharide receptor (endotoxin; CD14) Macrosialin (CD68) Fas (CD95; APO-1)

Signaling results in broad activation Binds oxidized lipoproteins (surfactants) Signaling may trigger apoptosis

particles, fibers, debris bacteria, organic materials fungal cell wall elements antibody-coated particulates activated complement components

IL-8 (CXCL8), chemoattractant signal IL-8 (CXCL8), chemoattractant signal IP-10 (CXCL10), ITAC CXCL4, chemoattractant signal MIP1a (CCL3), MIP-1b, RANTES (CCL5)

LEUKOCYTES / Pulmonary Macrophages 569

immune responses. The surface repertoire of the monocyte evolves to that of a mature and/or activated tissue macrophage after recruitment into the lung. Adhesion molecules, sialoadhesin receptors, and integrins attach the macrophage to lung surfaces and facilitate close contact with other cells for signaling. A group of immune regulatory molecules allow macrophages, particularly DCs, to present processed antigen peptides to T lymphocytes and B cells in an appropriate context so as to trigger clonal expansion or antibody production. Endotoxin, a cell wall lipopolysaccharide (LPS) from Gram-negative bacteria, is one of the most potent and well-studied signals that trigger a stepchange in macrophage function. LPS in serum (or possibly in alveolar epithelial lining fluid) is joined to a specific binding protein. This LPS-binding protein complex is recognized by the macrophage surface receptor CD14. Signal transduction via TLR4 results in prompt, strong cell activation and the new or increased production of TNF-a, IL-1b, and other cytokines. Macrophages carry abundant Fas (CD95) molecules, making them susceptible to death signaling for apoptosis upon encountering cells bearing Fas ligand.

experiments utilizing colored latex particles show that alveolar macrophages from all parts of the lung migrate to mediastinal lymph nodes. Inhaled silica particles follow a similar path in both humans and experimental animals. This appears to be an important route for antigen processing and adaptive immunity, as well as a mechanism for sequestering durable particulates. The kinetics of macrophage turnover in the lung probably varies greatly between normal health and disease. Alveolar macrophages appear to be longlived cells under normal circumstances. Early observations used BAL to sample airspace cells from patients who had received bone marrow transplantation from a donor of the opposite sex, and to track the appearance or disappearance of the Y chromosome in their alveolar macrophages. At least 6 months was required for near-complete conversion from host to donor phenotype. With active infection, an intense immune response, or brisk inflammation there may be large numbers of monocytes recruited to the lung and many macrophages that die or leave. Thus, some macrophages may serve a brief lifespan of several days, while others may live for months or perhaps longer.

Life History of the Pulmonary Macrophage

Macrophages in the lung arise ultimately from hematopoietic stem cell precursors in the bone marrow. Monoblasts become promonocytes and then monocytes, enter the blood stream, circulate for up to 40 h, and then adhere to pulmonary vascular endothelium, migrate into the interstitium, and finally take up residence in the alveolus or at other sites as listed above. During this process of differentiation and maturation their metabolic functions and surface molecule repertoire evolve from monocyte to tissue macrophage. It is likely that there are also local replicating populations of precursors to macrophages and DCs within the lung. The alveolar macrophage serves its life primarily on the epithelial surface. The end of life may occur by death or by exit from the alveolus. Death may be by necrosis from virulent microbial replication or ingestion of toxic particles, or by apoptosis from Fas signaling or other means. Macrophages may leave the alveolus via the airway mucociliary escalator, carrying away potentially injurious materials before they can harm more delicate tissues, and viable macrophages are found in expectorated sputum. Macrophages may also leave the alveolus by traversing the epithelium, entering the lymphatics, and migrating to bronchial-associated lymphoid tissue (BALT) aggregates and to regional lymph nodes. Animal

The Role of the Macrophage in Pulmonary Infection The respiratory system is protected from inhaled infection by a complex network of physical barriers such as filtration of air by inertial impaction and removal of particulates by the mucociliary stream. It is also protected by molecular mechanisms such as surfactant, defensins, complement proteins, and immunoglobulins in the epithelial lining fluid. When microbes evade these mechanisms, the resident alveolar macrophages are the next line of defense. When these resident defenses are insufficient and microbes multiply then other non-specific innate immune defenses are called forth. If these are still not sufficient, and the host survives, the most powerful specific adaptive immune responses appear in the lung. Airborne and oropharyngeal bacteria are inhaled frequently. The alveolar macrophages can effectively recognize and adhere to bacteria utilizing scavenger, Toll-like, and mannose receptors, or immunoglobulin Fc receptors if the bacterium has been coated with antibody. Binding of these receptors usually triggers prompt phagocytosis. The alveolar macrophages are capable of killing most inhaled bacteria, including virulent strains of staphylococci and Gram-negative organisms. When the bacterial load is high or macrophage function is impaired, bacteria can grow

570 LEUKOCYTES / Pulmonary Macrophages

extracellularly in the epithelial lining fluid and may replicate faster than they can be removed. While small numbers of bacteria or nonvirulent bacteria may be eliminated without stimulating an inflammatory response, larger numbers of organisms, bacteria that produce endotoxin, or virulent multiplying bacteria will trigger inflammation. Resident alveolar macrophages are stimulated to secrete chemokines that attract and activate other leukocytes, particularly neutrophils (see Table 2). Cytokines, lipid mediators, and ROS cause increased permeability, alveolar edema, and functional changes in epithelial cells. Disruption of macrophages by growing bacteria releases lysosomal enzymes that can further damage the alveolar wall. Pneumonia is the result of this expanding alveolar infection, injury, edema, and inflammation. Some pathogens, such as Legionella species, corrupt the activities of alveolar macrophages, creating within the cells a niche for bacterial replication. These types of organisms are considered obligatory or facultative intracellular pathogens. Having entered the alveolar macrophage by coiling phagocytosis mediated through the complement receptor, Legionella pneumophila induces a blockade of phagosome fusion with the lysosome. The bacteria replicate within the phagosome and then the cytoplasm, reaching a burden of up to 10 000 bacteria per cell within 48 h. Ultimately, death and lysis of alveolar macrophages release these bacteria to infect other nearby cells. When the adaptive cellular immune responses come into play, interferon gamma (IFN-g) produced by TH1-like lymphocytes activates the alveolar macrophages and decreases the rate of intracellular bacterial replication. Humoral immune responses provide IgM and then IgG to assist phagocytosis, activate complement, and assist with killing. Mycobacterium tuberculosis (TB) enters the alveolar macrophages via mannose and complement receptors. It survives in the phagocytes by interfering with fusion of the phagosome with the acidic lysosome. Infected macrophages selectively undergo apoptosis (which engenders less inflammation than necrosis) and the infected apoptotic bodies are subsequently ingested and by other macrophages. The effective clearance of TB requires the generation of a potent cell-mediated adaptive immune response in order to activate macrophages adequately to kill these bacteria. Most healthy human hosts are able to mount a successful initial immune response and kill or at least sequester TB bacteria without growth. Small numbers of these hardy organisms may survive for decades within macrophages in apparently healed lesions, only to break out again and cause active infection when the defenses of the host become impaired. Similar processes, albeit without long-term

sequestration, also occur during infection with most fungi and protozoan parasites.

Tobacco Smoke and the Macrophage Tobacco smokers have a dramatic increase in the number of alveolar macrophages in their lungs, and show accompanying changes in the morphology and function of these cells. A 240 ml BAL sample from a normal nonsmoker yields approximately 20 million alveolar macrophages, while 80–100 million are recovered from normal current smokers. Smokers have a significantly increased number of alveolar macrophages per unit lung volume and per unit surface area, though the relative increase is less than from BAL. The BAL fluid from smokers contains more chemotactic activity for monocytes, apparently in keeping with enhanced recruitment to the airspaces. The smokers’ alveolar macrophages are generally larger, show a golden-brown color, and contain more cytoplasmic inclusions, some of which are small kaolinite mineral particles derived from field dust or from clay added to tobacco in processing. Smokers’ alveolar macrophages exhibit a slightly higher basal oxidant production, but less capacity to increase superoxide production in response to endotoxin LPS. The smokers’ cells may also have a reduced ability to phagocytose particles and kill bacteria in vitro, implying an explanation for increased susceptibility to respiratory infection. Smokers’ macrophages stimulated with LPS secrete less IL-1 and other cytokines than nonsmokers’ cells. Smokers’ alveolar macrophages produce more elastin-degrading capacity and more matrix metalloproteinase activity than nonsmokers’ alveolar macrophages. When combined with the greatly increased cell number, the enhanced production of matrix-degrading enzymes on a per cell basis implies a very large increase in an elastolytic burden in the lungs of smokers. One hallmark of chronic obstructive pulmonary disease (COPD) is the accumulation of macrophages and neutrophils around small airways (respiratory bronchiolitis) and the subsequent development of centrilobular emphysema at these sites. Thus the pulmonary macrophage, recruited and modified by the effects of tobacco smoke, appears to play a central role in the pathogenesis of COPD and emphysema.

The Macrophage in Fibrotic and Inflammatory Lung Diseases Many chronic lung diseases feature accumulations of inflammatory cells, proliferating fibroblasts, epithelial

LEUKOCYTES / Pulmonary Macrophages 571

cells, and connective tissue matrix in distinctive pathological patterns. Macrophages are part of this mixture in most conditions, and in many are seen as essential elements orchestrating inflammation and fibrosis. Macrophage function has been studied extensively in sarcoidosis, idiopathic pulmonary fibrosis, asbestosis, silicosis, and other diseases. Sarcoidosis will serve as an example. Sarcoidosis

The cardinal feature of sarcoidosis in all organs is the noncaseating granuloma, a spherical collection of lymphocytes, monocytes, and macrophages surrounding occasional large multinucleated giant cells. These lesions appear in lymph nodes with tremendous enlargement, and in the lung and other organs with resulting dysfunction. Although the inciting agent or antigen is not known, sarcoidosis appears to be a TH1-like chronic cell-mediated immune response. CD4 þ T cells predominate in sarcoid tissues and are greatly increased in BAL samples, both in number and proportion. Local and systemic levels of TNF-a and IFN-g are high, and are believed to be pathogenic in driving the inflammatory response. BAL macrophages from patients with sarcoidosis have served as a source for many studies, and upregulation of a variety of functions has been observed. In most instances the changes were compartmentalized to the lung, and peripheral blood monocytes did not show similarly altered activities. Cytokines and other mediators are produced by macrophages in sarcoidosis. Alveolar macrophages from sarcoidosis patients show greatly enhanced mRNA expression, basal secretion, and stimulated production of TNF-a, believed to be a primary cytokine driving the inflammatory process and granuloma formation in this disease. Levels of potential antagonists and soluble TNF receptors 1 and 2 are also increased. The production of other cytokines such as IL-1b, IL-6, platelet-derived growth factor beta (PDGF-b), and granulocyte-macrophage colonystimulating factor (GM-CSF) is also increased. ROS (superoxide, hydrogen peroxide) are generated in elevated amounts by alveolar macrophages from patients with active sarcoidosis compared to normal subjects or patients with inactive disease. Sarcoidosis macrophages attract lymphocytes and monocytes to evolving granulomas. Elevated levels of macrophage inflammatory protein 1a (MlP-1a), a CD4 þ lymphocyte chemoattractant, and IFN-inducible protein 10 (IP-10), a CXC chemokine that stimulates the directional migration of activated T cells and NK cells, appear in the BAL fluid of patients with pulmonary sarcoidosis, and production of these

mediators is localized to macrophages. Stimulated alveolar macrophages from patients with active sarcoidosis release higher levels of the chemoattractant leukotriene B4 than those from normal subjects. Sarcoidosis macrophages appear to activate T cells. Alveolar macrophages from patients with active sarcoidosis produce IL-15 (while those from healthy subjects and patients with inactive sarcoidosis do not), and tissue staining localizes IL-15 mRNA and protein to alveolar macrophages. Thus alveolar macrophages may trigger the growth of sarcoid T cells through IL-2 receptor signaling, and could deliver proliferative signals for the development of the T-cell alveolitis. Alveolar macrophages that produce IL-18, an accessory cytokine with IL-12 and IL-15 that drives T cells toward a TH1-like phenotype, can be found within the boundary zone of sarcoid granulomas. Although normal alveolar macrophages do not bear costimulatory molecules, those from sarcoid patients carry CD80 and CD86 (members of the B7 family) and the CD5 coligand CD72, and thus may have enhanced lymphocyte activation and/or enhanced antigen presentation abilities. Sarcoidosis macrophages also exhibit increased abundance of cell adhesion molecules. The percentages of macrophages expressing the aM integrin CD11b (Mac1, CR3) and the adhesion molecule CD54 (ICAM-1) are significantly increased in patients with active sarcoidosis compared with patients with inactive disease or control subjects. These changes may, in part, reflect the influx of less mature and more monocyte-like cells. Pulmonary macrophages in active sarcoidosis demonstrate an activated inflammatory phenotype that accompanies, and may drive, a TH1-like activated T-cell response.

Conclusions The pulmonary macrophage plays a central role in the homeostasis of the lung. Under normal resting conditions it protects against the daily burden of inhaled particulates, microbes, and cellular debris without triggering an inflammatory or immune response that could injure the lung. When challenged by virulent infectious agents, the macrophage is a first line of defense, a sentinel that calls forth additional immune-inflammatory resources, and when activated an effective participant in microbial killing. Its potent degradative enzymes, inflammatory cytokines, and mediators that signal fibroblast growth and collagen production appear to be key elements in a wide variety of environmental and idiopathic lung diseases. A better understanding of this key cell, and more selective ways to modify its functions, will be important directions for lung research.

572 LIPID MEDIATORS / Overview See also: Bronchoalveolar Lavage. Dendritic Cells. Interleukins: IL-6; IL-10; IL-12; IL-15. Leukocytes: Monocytes. Systemic Disease: Sarcoidosis. Toll-Like Receptors. Transcription Factors: NF-kB and Ikb.

Further Reading Barnes PJ (2003) New concepts in chronic obstructive pulmonary disease. Annual Review of Medicine 4: 113–129. Delclaux C and Azoulay E (2003) Inflammatory response to infectious pulmonary injury. European Respiratory Journal 42(supplement): 10s–14s. Gal AA and Koss MN (2002) The pathology of scarcoidosis. Current Opinion in Pulmonary Medicine 8(5): 445–451. Gordon SB and Hughes D (1997) Macrophages and their origins: heterogeneity in relation to tissue microenvironment. In: Lipscomb MF and Russell SW (eds.) Lung Macrophages and Dendritic Cells in Health and Disease, 1st edn., pp. 3–31. New York: Dekker. Gordon SB and Read RC (2002) Macrophage defences against respiratory tract infections. British Medical Bulletin 61: 45–61.

Lohmann-Matthes ML, Steinmuller C, and Franke-Ullmann G (1994) Pulmonary macrophages. European Respiratory Journal 7: 1678–1689. Reynolds HY (2005) Lung inflammation and fibrosis: an alveolar macrophage-centered perspective from the 1970s to 1980s. American Journal of Respiratory and Critical Care Medicine 171(2): 98–102. Schneeberger EE and Suda T (1997) Ontogeny and heterogeneity of lung dendritic cells. In: Lipscom MF and Russell SW (eds.) Lung Macrophages and Dendritic Cells in Health and Disease, 1st edn., pp. 403–469. New York: Dekker. Warner AE (1996) Pulmonary intravascular macrophages: role in acute lung injury. Clinics in Chest Medicine 17: 125–135. Wesselius LJ, Murphy WJ, Morrison DC, and Russell SW (1997) Macrophage activation: concepts and principles applicable to the lung. In: Lipscom MF and Russel SW (eds.) Lung Macrophages and Dendritic Cells in Health and Disease, 1st edn., pp. 33–86. New York: Dekker. Zhang P, Summmer WR, Bagby GJ, and Nelson S (2000) Innate immunity and pulmonary host defense. Immunological Reviews 173: 39–51.

LIPID MEDIATORS Contents

Overview Leukotrienes Prostanoids Platelet-Activating Factors Lipoxins

Overview M Peters-Golden, University of Michigan, Ann Arbor, MI, USA

overproduction of pathogenic lipids as well as the underproduction of protective lipids. A variety of pharmacologic agents that block either the synthesis of, or the receptors for, lipid mediators are currently employed in the clinic, and additional applications for such therapy are on the horizon.

& 2006 Elsevier Ltd. All rights reserved.

Abstract

Introduction

Lipid mediators comprise a family of enzymatically generated phospholipid hydrolysis products that act via receptors to elicit a wide spectrum of biological responses. The best known of these are platelet-activating factor and the eicosanoids, oxygenated metabolites that include prostanoids, leukotrienes, and lipoxins. These molecules are synthesized and secreted within minutes of agonist stimulation by virtually all cell types of bone marrow and lung origin, and exert autocrine and paracrine effects that influence almost every conceivable category of biological response. Lipid mediator synthesis is modulated by, and mediates many of the actions of, polypeptides such as hormones and cytokines. By virtue of their diverse actions, lipid mediators are implicated in a variety of lung diseases, including asthma, interstitial lung disease, pulmonary vascular disease, and acute lung injury. They are also increasingly recognized to contribute to homeostatic or protective responses, such as innate immunity. Precedent exists for disease states to reflect both the relative

The term ‘lipid mediators’ generally refers to enzymatically generated derivatives of membrane phospholipids that are secreted from the source cell and act at nanomolar concentrations on target cells, most classically via G-protein-coupled receptors (GPCRs). These substances include platelet-activating factor (PAF) as well as oxygenated metabolites of the 20-carbon fatty acid arachidonic acid (AA), which are collectively known as ‘eicosanoids’ (eicosa ¼ Greek for twenty). The best-studied eicosanoids include prostanoids (prostaglandins (PGs) and thromboxane), leukotrienes (LTs), and lipoxins. Although originally recognized for their capacity to elicit biological responses such as smooth muscle

LIPID MEDIATORS / Overview 573

contraction, edema, and platelet aggregation, lipid mediators are now known to influence processes ranging from inflammation and immune responses to chronic tissue remodeling and cancer. Indeed, there is scarcely an area of biology or medicine in which these molecules have not been implicated. Not surprisingly, the lung is an important source of and target for lipid mediators. A variety of pharmacotherapeutic strategies based on lipid mediators are in current use and others are on the horizon. The generation and lung biology of PGs, LTs, lipoxins, and PAF are specifically addressed elsewhere in this encyclopedia. This article provides an historical perspective on lipid mediators and an overview of their synthesis, receptors, signaling, actions, and roles in respiratory diseases. As lipid mediators are likely to be less familiar to many readers than peptide mediators such as cytokines, it is particularly instructive to compare and contrast these two classes of mediators. Finally, the interplay between lipid and peptide mediators is discussed.

Brief History of Lipid Mediators Although not recognized as such at the time, research into what we now term lipid mediators dates back to two disparate lines of investigation that commenced in the 1930s. First, Von Euler described a smooth muscle contractile bioactivity found in seminal vesicle and prostate, which he termed ‘prostaglandin’. A few years later, Feldberg and Kellaway described a bioactivity released by cobra venom-challenged guinea pig lung that caused a very potent and protracted contraction of the smooth muscle, and which was termed ‘slow reacting substance’ (SRS). Over the next 40 years, SRS was identified as a mixture of LTs, and both PGs and LTs were recognized as families of enzymatically generated oxygenation products of AA. Synthesis of PGs was initiated by the cyclooxygenase (COX) enzyme, and that of LTs by the 5-lipoxygenase (5-LO) enzyme. In 1982, the Nobel Prize in Physiology or Medicine was awarded to three investigators – Sune Bergstro¨m, John Vane, and Bengt Samuelsson – who pioneered studies of the chemical structures, biosynthetic pathways, pharmacologic modulation, and biologic actions of eicosanoids during this era. Subsequently, many other families of eicosanoids were identified, including products of other LO enzymes, products formed from the interaction of two different LO enzymes (lipoxins), and cytochrome P-450 metabolites of AA. Free AA, the substrate for eicosanoid synthesis, is the product of phospholipase A2 (PLA2)-mediated hydrolysis of membrane phospholipids. Lysophospholipid is the other product of PLA2 action, and is the precursor for

PAF. During the last 20 years, the tools of modern cell and molecular biology, biochemistry, genetics, transgenic animal modeling, and clinical medicine have provided new insights into the generation and actions of these lipid mediators.

Generation of Lipid Mediators Biosynthetic Pathways

Synthesis of both PAF and eicosanoids (Figure 1) is initiated by a single enzymatic step, cleavage of AA (or other unsaturated fatty acids) at the sn-2 position of membrane phospholipids by the enzyme PLA2. This process is stimulated by cellular agonists, acting via the generation of second messengers. More than 15 different mammalian PLA2 enzymes have been identified, which differ in their intracellular localization and biochemical characteristics. The enzyme that appears to be the most universally important in mediating the stimulated synthesis of lipid mediators is called cytosolic PLA2 (cPLA2). This is an AA-preferring enzyme whose activation involves its calcium-dependent translocation from the cytosol to intracellular membranes. Its catalytic activity is enhanced following phosphorylation by mitogen-activated protein kinases. Besides PLA2 action, another factor that determines the availability of AA for conversion to eicosanoids is the relative proportion of AA among the unsaturated fatty acids esterified at the sn-2 position of phospholipids. This can vary among different cell types. It is also influenced by dietary intake, as increased consumption of foods rich in n-3 fatty acids (e.g., fish oils), such as eicosapentaenoic and docosahexaenoic acids, results in decreased AA content within phospholipids and a corresponding decrease in synthesis of bioactive eicosanoids. PAF formation requires that the fatty acid at the sn-1 position of the parent phospholipid molecule be linked to the glycerol backbone by an ether rather than an acyl bond. Since ether-linked phospholipids tend to be enriched in AA, PAF synthesis and AA release are thereby linked. The sn-1 ether-linked lysophospholipid (lyso-PAF) generated by PLA2 is then acetylated by the enzyme acetyltransferase to generate PAF. Free AA is potentially available for bioconversion to eicosanoids via a variety of metabolic pathways, each generally referred to by the name of its initial oxygenase enzyme. The two pathways for which the most information exists are the PGH synthase or COX pathway and the 5-LO pathway. In both of these, the oxygenase catalyzes the synthesis of an unstable intermediate, which is further metabolized by downstream terminal enzymes.

574 LIPID MEDIATORS / Overview Phospholipid

AT lyso-PAF

PAF

Agonist

PLA2 2 AA

5-LO 12-LO

COX-1 COX-2

12-HPETE

15-LO

P-450 Epoxides HETEs

FLAP

15-HPETE

5-HPETE

PGH2 12-HETE

15-HETE

5-HETE

LTA4

Lx PGD2

PGF2
PGE2

PGI2

TxA2

LTB4

LTC4

LTD4

LTE4

15-d-PGJ2 Figure 1 Pathways of lipid mediator formation. Enzymes are indicated by enclosure in rectangles and terminal lipid mediators are indicated in italics. AA, arachidonic acid; PAF, platelet-activating factor; PG, prostaglandin; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; LT, leukotriene; Lx, lipoxins; 15-d-PGJ2, 15-deoxy-D12,14-PGJ2; PLA2, phospholipase A2; AT, acetyltransferase; COX, cyclooxygenase; LO, lipoxygenase; FLAP, 5-LO activating protein; P-450, cytochrome P-450.

COX is an integral membrane enzyme that acts on AA to form the unstable endoperoxide PGH2, which can then be converted by a variety of synthases to the individual bioactive prostanoids: PGE2, PGF2a, PGD2, thromboxane (Tx) A2, and PGI2 (prostacyclin). COX exists in two isoforms, COX-1 and COX-2, which differ in their tissue distribution and regulation of expression. Although the conventional notion holds that COX-1 is constitutive and COX-2 is inducible, many exceptions to this dogma are now recognized. COX is the target for the actions of nonselective nonsteroidal anti-inflammatory drugs, such as indomethacin and aspirin, and for selective COX-2 inhibitors (coxibs). 5-LO is a calcium-dependent and ATP-activated enzyme that acts on AA to form the unstable epoxide LTA4. A helper protein for 5-LO, known as 5-LO activating protein (FLAP), serves to bind AA liberated by PLA2 and ‘present’ it to 5-LO, thereby facilitating its enzymatic oxygenation. Agonist activation causes a calcium-dependent translocation of soluble 5-LO to intracellular membranes, where FLAP is located. LTA4 is either hydrolyzed by LTA4 hydrolase to LTB4, or is conjugated with a glutathione molecule by LTC4 synthase to form LTC4. Sequential removal of the amino acids of glutathione can be mediated extracellularly by: (1) g-glutamyl transpeptidases, including g-glutamyl leukotrienase, to yield LTD4; and then (2) a dipeptidase to yield LTE4. LTs C4, D4, and E4 are collectively known as the cysteinyl LTs

(cysLTs), and these account for the bioactivity originally described as SRS. Regulation of Lipid Mediator Generation

Consistent with the biological importance of lipid mediators, their synthesis is carefully regulated (Table 1). The most obvious mechanism for regulation of lipid mediator synthesis is via changes in expression of their biosynthetic enzymes. Expression of virtually all of the proteins involved in lipid mediator synthesis is subject to regulation, either up or down. Changes in expression most often involve regulation at the transcriptional level. Factors modulating expression of eicosanoid-forming proteins include cytokines, hormones, vitamins, and microbial products. In addition to such exogenous modifiers, genetic factors can also influence the expression of many of these proteins, as polymorphisms in the promoter or coding elements of their corresponding genes have been identified. The catalytic efficiency of eicosanoid-forming enzymes is also subject to regulation. This can occur through a variety of mechanisms. First, levels of essential cofactors for these enzymes can vary. For example, ATP is required for optimal 5-LO action, and reduced glutathione is required for optimal action of PGE synthase and as a cosubstrate for synthesis of LTC4; depletion of ATP or glutathione, as might occur during metabolic or oxidative stress, can thereby

LIPID MEDIATORS / Overview 575 Table 1 Mechanisms for the regulation of lipid mediator biosynthesis and secretion

Table 2 Representative ‘complete’ agonists that activate lipid mediator biosynthesis

Alterations in expression levels of biosynthetic proteins Genetic Acquired (modulation by cytokines, hormones, lipopolysaccharide, etc.) Alterations in function of biosynthetic proteins Levels of essential cofactors (e.g., calcium, ATP, glutathione) for enzymes Posttranslational modifications that influence catalytic activity of proteins (e.g., phosphorylation, nitration) Intracellular localization of proteins Alterations in expression or function of transport proteins required for export

Soluble ‘model’ agonists Calcium ionophores Phorbol esters Receptor-mediated agonists Thrombin Bradykinin Interleukin-8 Endothelin Immune complexes Leukotrienes PAF Microorganisms Viruses Bacteria Fungi Protozoa Silica Phagocytic particles Zymosan Asbestos Silica Oxidants Hyperoxia Ozone Nitrogen dioxide

influence the profile of eicosanoids generated. Second, posttranslational modification of enzymes can influence their catalytic efficiency; examples include phosphorylation by protein kinases and nitration by nitric oxide. In this way, mitogen-activated protein kinase can enhance the actions of cPLA2, protein kinase A can reduce the actions of 5-LO, and nitric oxide can directly inhibit 5-LO. Third, intracellular compartmentalization of eicosanoid-forming proteins has emerged as an important means of regulating their functions. As lipid mediators are predominantly secreted into the extracellular space, it was long assumed that their synthesis depended on phospholipid hydrolysis and metabolism at the plasma membrane. Unexpectedly, many of the enzymes involved in their generation have instead been localized to the nuclear membrane and perinuclear endoplasmic reticulum. It may be inferred that topographic colocalization of a group of enzymes that must function in series serves to optimize their coupling and the efficiency for lipid mediator generation. As colocalization of enzymes may depend on specific molecular motifs or phosphorylation events, it too may be subject to genetic or acquired variation. Finally, the fact that lipid mediators are synthesized within the cellular interior mandates that mechanisms for their export must exist. Indeed, evidence suggests the presence of distinct cellular transporters for prostanoids, LTB4, and LTC4. Although much remains to be clarified about these export mechanisms, they represent an additional target for variation and regulation. Agonists for Lipid Mediator Synthesis

A myriad of substances can stimulate or potentiate the synthesis of lipid mediators (see Table 2). Three types of stimuli are recognized. (1) ‘Complete agonists’ are capable of rapidly triggering AA release and its subsequent metabolism to an array of eicosanoids,

often because they increase intracellular calcium either directly or via receptor-dependent signal transduction. Examples include the model agonist calcium ionophore as well as physiologically relevant substances such as hormones, microbes, antigens, and oxidants. (2) Certain agonists are only weak triggers by themselves, but can potentiate the subsequent response to a more complete agonist in a process referred to as ‘priming’. A priming effect may reflect activation of protein kinases that amplify the catalytic activity or translocation of eicosanoid-forming enzymes. Lipopolysaccharide and various cytokines act in this manner. (3) Finally, a number of substances can potentiate lipid mediator synthesis by upregulating expression of eicosanoid-forming enzymes, as noted above; cytokines exemplify such actions. The effects of a given agonist on lipid mediator generation may vary depending on the cell type. Cellular Participation in Lipid Mediator Synthesis

Although virtually all cell types participate in lipid mediator synthesis, the specific pathways expressed and therefore the spectrum of products synthesized depend on the cell type. As a general rule, structural or parenchymal cell types (such as epithelial cells, endothelial cells, fibroblasts, and smooth muscle cells) metabolize AA primarily via the COX pathway, often with some modest capacity for 12- or 15-LO metabolism. By contrast, only bone marrow-derived cells

576 LIPID MEDIATORS / Overview Table 3 Lipid mediator synthetic profiles of various cell types Cell type

TxA2

Platelet Neutrophil Alveolar macrophage Mast cell Endothelial cell Epithelial cell Smooth muscle cell Fibroblast

þþþ 7 þ

PGI2

PGD2

7 þþþ þþþ þ 7 7

tend to synthesize large amounts of 5-LO products, and some also generate substantial amounts of COX, 12-LO, or 15-LO metabolites. PAF is synthesized by endothelial cells, platelets, and various leukocyte populations. The specific profile of eicosanoids synthesized by a given cell type is dictated by its complement of distal enzymes (see Table 3). However, it is instructive to note that both tissue and species specificity in eicosanoid synthetic profiles can also be manifest. For example, alveolar macrophages tend to synthesize substantially more LTs and less prostanoids than do macrophages from other sites. Furthermore, mouse alveolar macrophages differ from human or rat cells in that LTC4, rather than LTB4, is their predominant 5-LO product. In view of the differences in biological actions between these two LTs (see below), this species discrepancy must be kept in mind when seeking to interpret the human significance of studies employing transgenic mouse models in which macrophages are a key source of these mediators. In vivo, lipid mediator synthesis reflects interactions among neighboring cells. In a process known as transcellular eicosanoid synthesis, one cell type releases an intermediate in the AA metabolic pathway, which is subsequently taken up and processed to an eicosanoid by a second cell type. Thus, studies in a variety of co-culture systems have documented transcellular metabolism of intermediates including AA itself, PGH2, and LTA4 to classical eicosanoids such as prostanoids and LTs. Furthermore, transcellular exchange of LO intermediates can result in dual oxygenation of AA by both 5-LO and either 12- or 15LO to yield lipoxygenase interaction products, or lipoxins. Such transcellular interactions can involve virtually any combination of bone marrow-derived and structural cell types, and are clearly important determinants of tissue output of lipid mediators. Degradation of Lipid Mediators

One further determinant of the levels of bioactive lipid mediators in tissues is the capacity for their

PGE2 7 þ

cysLT

LTB4

þ þþþ

þþþ þþþ þ

þ þþþ þþþ þþþ

PAF þþ þþ þ þþþ

degradation or subsequent metabolism. Such conversion can proceed either spontaneously or enzymatically. Importantly, it can result in either inactivation or activation of bioactivity. Given their inherent reactivity, some eicosanoids, but not all, undergo almost instantaneous spontaneous conversion in an aqueous milieu; examples include the conversion of PGI2 to 6-keto-PGF1a and of TxA2 to TxB2 that dictate that these bioactive eicosanoids be measured as their more stable but biologically inactive metabolites. Similarly, PGD2 can spontaneously break down to PGJ2 and then to 15-deoxy-D12,14-PGJ2, which has been implicated as a possible ligand for intranuclear receptors (see below). Likewise, certain eicosanoids are susceptible to oxidative attack and subsequent bioinactivation, and this has been described for LTs. Finally, lipid mediators can be enzymatically metabolized with divergent consequences for biological activity. The conversion of LTC4 to LTD4 by g-glutamyl transpeptidases generally results in enhanced biological activity (see below). By contrast, PGs can be inactivated in the lung by the enzyme 15-hydroxy PG dehydrogenase, PAF can be inactivated by the enzyme PAF acetylhydrolase, and LTs can be metabolized and inactivated by cytochrome P-450 enzyme pathways.

Measuring Lipid Mediators and Their Synthesis Lipid mediators or their stable metabolites or breakdown products can often be successfully measured in biological samples. This can be accomplished using immunoassays; greater specificity may be obtained by an initial lipid extraction step or by utilizing chromatographic separation techniques. These approaches have been successfully applied to specimens including cell culture supernatants, plasma, urine, lung lavage fluid, lung tissue homogenates, induced sputum, and even exhaled breath condensate. Knowledge of the pitfalls, sensitivity, and specificity of each of these approaches is essential to their successful implementation.

LIPID MEDIATORS / Overview 577

and cellular proliferation. It is attractive to speculate that interaction with nuclear receptors may mediate some of the intracellular (‘intracrine’) effects of lipids formed locally at the nucleus, without the need for their prior export. It is now recognized that, as is true for certain synthetic enzymes, receptors for a given eicosanoid can be encoded by multiple genes. This is best exemplified by the receptors for PGE2 (termed the E prostanoid, or EP, receptors), as four distinct EP receptors are known. Multiple receptors also bind PGD2, LTB4, and cysLTs. As these distinct receptors differ in their tissue distributions, ligand specificities or affinities, and downstream signaling pathways, it is likely that such a complex receptor variety provides a further means for functional and cellular specificity in the actions of lipid mediators. Studies employing selective receptor agonists and antagonists as well as transgenic mouse lines are beginning to clarify the role of specific receptors in the biological actions of lipid mediators. Although relatively little is currently known about the regulation of receptor expression, it is apparent that genetic and acquired variation in such expression will also be of pathophysiologic importance.

A variety of indirect or surrogate approaches can also provide information about lipid mediator synthesis. The enzymatic activity or the mRNA or protein expression of specific eicosanoid-forming enzymes can be assayed in cell or tissue lysates. Such expression can also be assessed in situ using microscopic techniques. The activation of enzymes such as cPLA2 or 5-LO that translocate to membranes during cell stimulation can also be inferred by microscopic detection of changes in their intracellular localization. Combining direct mediator measurements with assessments of specific enzymes provides the most comprehensive information about the processes underlying lipid mediator generation.

Lipid Mediator Receptors and Signaling Lipid mediators can exert biological actions on the source cell (autocrine actions) or on neighboring cells (paracrine actions). In either case, their actions are generally presumed to be mediated by their binding to specific receptors on target cells. Ligation of these receptors results in the generation of intracellular signals that, in turn, transduce the biological actions. Receptors for most of the major lipid mediators have been characterized using classic pharmacologic techniques, and many have been cloned. These are GPCRs, distributed mainly on the plasma membrane, that are coupled to signal transduction events including alterations in intracellular calcium and cyclic AMP levels. These, in turn, influence the activation of diverse protein kinases such as protein kinases A and C and mitogen-activated protein kinases. Certain lipid mediators and free fatty acids can also bind to an alternative type of receptor known as the peroxisome proliferator-activated receptor (PPAR). These are intranuclear receptors that also act as transcription factors, regulating the expression of proteins involved in lipid metabolism, inflammation,

Actions of Lipid Mediators in the Lung As noted in the introductory comments, lipid mediators are now recognized to influence the entire spectrum of biological responses, ranging from ‘macro’-level processes such as smooth muscle contraction to more subtle intracellular processes such as transcription and growth regulation. Although they will be considered in greater detail elsewhere, some of their key actions most relevant to lung biology are summarized in Table 4. Certain central paradigms about lipid mediator actions are apparent. First, many of these mediators

Table 4 Major biological actions of lipid mediators Lipid Platelet Airway or Bronchial Vascular Leukocyte Release of Fibroblast Immune mediator aggregation vascular responsiveness permeability recruitment mediators proliferation function tone and collagen synthesis PGE2 PGI2 PGF2a TxA2 PGD2 LTB4 5-HETE LTC4/D4/E4 PAF Lx

k m

m

k k m m m m m k

k k

m m

k k

k k

k k

k k

m m

m

m m k

m m

m m m m m k

m m m m m k

m m m m

m m m m k

578 LIPID MEDIATORS / Overview

demonstrate redundancy in their actions. Thus, PGD2, TxA2, cysLTs, and PAF all cause bronchoconstriction, and PGD2, PAF, LTB4, and cysLTs all promote leukocyte recruitment. Second, the actions of particular lipid mediators demonstrate context dependency. For example, PGE2 can either promote or inhibit the generation of proinflammatory cytokines as well as the egress of leukocytes from the vasculature; this may reflect, at least in part, the roles of distinct EP receptors in different cells or circumstances. Third, certain of these molecules oppose or antagonize the actions of others. Such a yin-yang relationship is exemplified by LTs versus PGE2. For example, LTs generally promote leukocyte activation while PGE2 generally inhibits this process; cysLTs are bronchoconstrictive whereas PGE2 is bronchodilatory; and LTs promote while PGE2 opposes fibrogenesis.

Lipid Mediators in Respiratory Disease and Homeostasis The determination that a given mediator participates in the pathogenesis of a disease process is based on the fulfillment of three criteria: (1) it can be identified in appropriate body fluids or tissues (or it can be synthesized by relevant cells); (2) its administration to experimental subjects (humans, animals, organs, or cells) reproduces the pathophysiologic features of the disease process; and (3) its antagonism or inhibition by pharmacologic, immunologic, or genetic strategies ameliorates the manifestations of the disease. By these criteria, lipid mediators have been implicated in a variety of respiratory disease states; a partial list of such conditions is presented in Table 5. The link between LTs and asthma is the most firmly established, but a growing body of evidence also suggests roles for LTs in chronic obstructive pulmonary disease (COPD), acute lung injury, and interstitial lung diseases. Likewise, potential roles have

Table 5 Lung diseases in which abnormalities of lipid mediator production are implicated Airway diseases Asthma Aspirin-sensitive asthma Chronic bronchitis Cystic fibrosis Acute lung injury ARDS Oxygen toxicity

Vascular disorders Pulmonary hypertension Pulmonary thromboembolism Immune complex vasculitis

Lung cancer

Interstitial lung diseases Impaired antimicrobial defense Idiopathic pulmonary fibrosis HIV infection Asbestosis Malnutrition Scleroderma Smoking

emerged for PGD2 in asthma and for both LTs and PGE2 in lung cancer. More recently, the homeostatic role of these molecules has been recognized. Examples of homeostatic functions include the protective role of PGI2 in the pulmonary vasculature, of PGE2 and lipoxins in asthma, of PGE2 in fibrotic lung diseases, and of LTs in antimicrobial defense. Two important paradigms are noteworthy in this context. The first is that disease states can be characterized not only by overproduction of injurious/ contractile/proinflammatory lipid mediators (e.g., LTs, PGD2, and PAF), but also by underproduction of protective/relaxant/anti-inflammatory mediators (e.g., PGE2, PGI2, and lipoxins). Although states of relative deficiency of homeostatic lipid mediators are much less well recognized, relevant examples include deficient synthesis of PGI2 in primary pulmonary hypertension, of lipoxins in asthma, and of PGE2 in idiopathic pulmonary fibrosis. The second is that value judgments regarding the actions of lipid mediators are of necessity context dependent. Thus, the proinflammatory actions of LTs can be considered protective, rather than deleterious, in the context of antimicrobial defense; moreover, increased susceptibility to infection may result when capacity for LT biosynthesis is relatively impaired, as occurs in HIV infection and in malnutrition. Likewise, the antiinflammatory effects of PGE2 may be deleterious in the context of antimicrobial defense, and increased susceptibility to infection may also result from states of PGE2 overproduction, as occurs in cancer and advanced age.

Therapeutic Strategies to Alter the Synthesis or Actions of Lipid Mediators Given the pathophysiologic importance of lipid mediators, therapeutic opportunities exist for modulating their tissue concentrations or tissue responsiveness to them. The generic strategies that can be considered are: (1) administration of a potentially beneficial substance (or overexpression of the gene(s) encoding for its synthesis); (2) inhibition of the synthesis of a potentially deleterious substance; and (3) inhibition of the actions of a potentially deleterious substance. Each of these strategies has been successfully employed. Examples include: (1) systemic or inhalational administration of PGI2, PGE2, or analogs thereof for the treatment of pulmonary vascular disease; (2) inhibition of COX for the treatment of pain, fever, and cancer, and of 5-LO for the treatment of asthma; and (3) cysLT receptor blockade for the treatment of asthma. It is easy to envision many other circumstances in which these basic approaches

LIPID MEDIATORS / Overview 579

Lipid mediator receptor

Lipid mediator

Cytokine

Cytokine receptor

Figure 2 Bidirectional interactions between lipid mediators and cytokines. Lipid mediators can modulate both the generation of other mediators such as cytokines as well as the expression level of their receptors. In this way, cytokines mediate some of the actions of lipid mediators. In an analogous manner, cytokines can also modulate both the production of lipid mediators as well as the expression of lipid mediator receptors. Thereby, lipid mediators mediate some of the actions of cytokines.

can be applied to further exploit the actions of lipid mediators in lung disease or homeostasis, and these are considered elsewhere. Apart from issues unique to a particular candidate therapeutic agent, the relative merit of each of the above therapeutic approaches ultimately depends on its specificity and the clinical context in which it is applied. In general terms, specificity of actions increases as one moves down the biosynthetic pathways and to specific receptors. For example, an inhibitor of 5-LO will have far greater specificity than will an inhibitor of PLA2, but far less specificity than will an antagonist of the cysLT1 receptor. Achievement of the maximal level of specificity, however, depends on the disease being treated and the relative roles of different mediators. If asthma is the disease target and the only mediators of relevance are cysLTs acting through cysLT1, a cysLT1 antagonist should prove optimal. By contrast, if cysLT2 also mediates actions of cysLTs relevant to asthma, a 5-LO inhibitor would be expected to yield a more desirable therapeutic outcome. With further advances in our knowledge of the actions of lipid mediators and the receptors through which they act, the versatility of our repertoire for therapeutic targeting of lipid mediators will continue to grow.

Lipid Mediators and Peptide Mediators: Similarities, Differences, and Interactions It is interesting to note that the actions of lipid mediators bear striking similarities to those of other mediator classes, and to cytokines in particular. Like cytokines, lipid mediators can elicit intracellular signal transduction events, which ultimately influence every aspect of cellular biology. Like cytokines, different lipid mediators exhibit functional redundancy. They also resemble cytokines in manifesting context dependence in their actions. Finally, pairs of lipid mediators exhibit functional opposition, or yin-yang relationships, in their actions. However, important differences from cytokines also exist. Thus, while lipid mediators can match virtually all of the actions of cytokines, they can

exert actions that cytokines generally cannot. These include the ability to modulate such processes as smooth muscle contraction, vascular permeability, and platelet aggregation. Another noteworthy difference relates to the temporal aspects of lipid mediator synthesis. Since cytokine generation usually requires transcriptional and translational events, it tends to require a lag period of at least several hours. By contrast, since the enzymes necessary for biosynthesis of lipid mediators are constitutively expressed, these can generally be elaborated within 1–15 min following agonist exposure. For this reason, lipids are far more important than cytokines in mediating immediate and early responses to various perturbations. Moreover, because enzymes involved in their biosynthesis can be further upregulated by induction or phosphorylation in response to stimuli or other mediators, subsequent waves of lipid mediator generation can also occur, permitting these substances to also be elaborated in the later phases of a biological response. Finally, a comprehensive understanding of the biological roles of both lipid mediators and peptide mediators, such as cytokines, growth factors, proteases, and histamine, mandates an appreciation of the interactions between them (Figure 2). In fact, lipid mediators can modulate the expression of peptide mediators, as well as of their receptors. Likewise, peptide mediators modulate the expression of eicosanoid-forming enzymes and of eicosanoid receptors. By virtue of such interactive networks, cytokines actually mediate many of the actions of lipid mediators and vice versa. Recognizing these interactions has important pathophysiologic as well as therapeutic implications. See also: Asthma: Overview. G-Protein-Coupled Receptors. Lipid Mediators: Leukotrienes; Prostanoids; Platelet-Activating Factors. Peroxisome ProliferatorActivated Receptors.

Further Reading Calder PC (2003) N-3 polyunsaturated fatty acids and inflammation: from molecular biology to the clinic. Lipids 38: 343–352.

580 LIPID MEDIATORS / Leukotrienes Charbeneau RP and Peters-Golden M (2005) Eicosanoids: mediators and therapeutic targets in fibrotic lung disease. Clinical Science (London) 108: 479–491. Christie PE and Henderson WR Jr (2002) Lipid inflammatory mediators: leukotrienes, prostaglandins, platelet-activating factor. Clinical Allergy and Immunology 16: 233–254. Diaz BL and Arm JP (2003) Phospholipase A2. Prostaglandins Leukotrienes, and Essential Fatty Acids 69: 87–97. Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294: 1871–1875. Laskin JJ and Sandler AB (2003) The importance of the eicosanoid pathway in lung cancer. Lung Cancer 41(supplement 1): S73–S79. Leslie CC (2004) Regulation of arachidonic acid availability for eicosanoid production. Biochemistry and Cell Biology 82: 1–17. McMahon B and Godson C (2004) Lipoxins: endogenous regulators of inflammation. American Journal of Physiology. Renal Physiology 286: F189–F201. Peters-Golden M (2003) Eicosanoids as mediators and therapeutic targets in airway inflammation. In: Eissa NT and Huston DP (eds.) Airway Inflammation, pp. 627–656. New York: Dekker. Peters-Golden M, Canetti C, Mancuso P, and Coffey MJ (2005) Leukotrienes: underappreciated mediators of innate immune responses. Journal of Immunology 174: 589–594. Poff CD and Balazy M (2004) Drugs that target lipoxygenases and leukotrienes as emerging therapies for asthma and cancer. Current Drug Targets in Inflammation and Allergy 3: 19–33. Prescott SM, Zimmerman GA, Stafforini DM, and McIntyre TM (2000) Platelet-activating factor and related lipid mediators. Annual Review of Biochemistry 69: 419–445.

Leukotrienes D C Zeldin and J W Card, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Published by Elsevier Ltd.

Abstract Leukotrienes (LTs) are potent bioactive lipid mediators whose synthesis is increased in response to inflammatory stimuli. LTB4 and the cysteinyl LTs (LTC4, LTD4, and LTE4) are products of the 5-lipoxygenase pathway of arachidonic acid metabolism and exert their biological effects through specific receptors located on the surface of target cells. In addition to promoting the recruitment and retention of inflammatory cells in the lung, LTs increase mucus secretion, enhance vascular permeability, and cause airway constriction. Genetic and pharmacologic manipulation of LT biosynthetic and signaling pathways in experimental animals has uncovered a variety of functions of LTs in models of respiratory disease including allergen-induced airway inflammation and hyperresponsiveness, pulmonary fibrosis, and cancer. Clinical data, including increased bronchoalveolar lavage fluid levels of LTs in numerous disease states, also indicate a prominent role for LTs in respiratory disease and dysfunction. While the effects of LT modifiers in many lung diseases have not been thoroughly investigated, the use of LT biosynthesis inhibitors and cysteinyl LT receptor antagonists has proven valuable for the treatment of asthma. Continued investigation into the roles of LTs in the lung will undoubtedly yield significant advancements in the treatment of a variety of respiratory diseases.

Introduction Leukotrienes (LTs) are endogenously produced inflammatory lipid molecules that affect lung function and disease pathogenesis via receptor-mediated effects on a variety of cell types. The slow-reacting substance of anaphylaxis, first described in the 1930s, was later shown to be comprised of the cysteinyl LTs (cysLTs). Since then, interest in the biochemical and physiological effects of LTs has stemmed primarily from their deduced and predicted roles in asthma pathogenesis. Efforts to diminish the proinflammatory and bronchoconstrictive effects of LTs via inhibition of their production and/or blockade of their receptors have resulted in improved therapeutic modalities for this disease. Additionally, clinical data and experimental studies have identified LTs as having potentially significant roles in other lung diseases including chronic obstructive pulmonary disease, fibrosis, and cancer.

Structure LTs are so called because they are generated by white blood cells (hence the term ‘leuko’) and contain a conjugated ‘triene’ system of double bonds. They are metabolites of arachidonic acid that are comprised of a 20-carbon backbone, and differ from one another in subtle but important structural ways as a result of progressive enzymatic processing (Figure 1). Oxygenation of arachidonic acid leads to formation of LTA4; subsequent enzymatic hydroxylation of LTA4 at the 12 position or conjugation with reduced glutathione at the 6 position generates LTB4 or LTC4, respectively. LTD4 and LTE4 are formed by the enzymatic removal of L-glutamate and L-glycine, respectively, from the glutathione moiety of LTC4.

Regulation of Production and Activity The production of LTs is mediated by the coordinated activity of several enzymes (Figure 1) and is dependent on the availability of free arachidonic acid within the cell. Importantly, it is influenced by a variety of endogenous (e.g., cytokines, chemokines, growth factors) and exogenous (e.g., dietary factors, toxins) factors. The primary cells that generate LTs are eosinophils, mast cells, polymorphonuclear leukocytes (neutrophils), and monocytes/macrophages, whereas structural cells (e.g., epithelial and endothelial cells) exhibit low LT synthetic capacity. LTs are not stored in cells, but are generated de novo following cellular activation by bacterial peptides, immune complexes, cytokines, growth factors, and other stimuli. Such stimuli lead to translocation of

LIPID MEDIATORS / Leukotrienes 581 Cell activation

cPLA2 5-LO Arachidonic acid

5-L

FLAP

O

COOH

FLAP

Nucleus

COOH

LTA4

LTC4S

5-LO

O

LTA4H OH

OH COOH

OH

LTB4

COOH

LTC4

S-Cys-Gly
Glu

S-Cys-Gly
Glu

MRP1

LTB4 transporter

Receptor

Cell/tissue expression

Effect

BLT1 (high affinity)

Leukocytes Lung, spleen, thymus

Chemotaxis, adhesion, activation Uncharacterized

BLT2 (low affinity)

Most tissues

Uncharacterized

cysLT1 (high affinity)

Leukocytes Airway smooth muscle Endothelium

Activation, cytokine production Contraction Increased vascular permeability

cysLT2 (low affinity)

Mast cells, macrophages Endothelium

Activation, cytokine production Activation

OH COOH

LTC4

S-Cys-Gly
Glu OH COOH S-Cys-Gly OH

LTD4

COOH S-Cys

LTE4

Figure 1 Synthesis and actions of LTs. Cell activation leads to liberation of arachidonic acid from cellular membranes by cPLA2. 5-LO, in association with FLAP on the nuclear membrane, then converts free arachidonic acid to LTA4. Formation of LTB4 occurs via the action of LTA4 hydrolase on LTA4, whereas LTC4 is formed via the action of LTC4 synthase on LTA4. Both LTB4 and LTC4 are actively transported out of the cell, LTB4 by an uncloned transporter and LTC4 by multidrug resistance protein-1 (MRP1). Extracellular metabolism of LTC4 leads to generation of LTD4 and LTE4; these three molecules are collectively referred to as the cysLTs. LTB4 and the cysLTs act on specific receptors on target cells to elicit a variety of biological effects.

cytosolic phospholipase A2 (cPLA2) to intracellular membranes, which results in the liberation of free arachidonic acid. 5-lipoxygenase (5-LO), which translocates from the nucleus and/or the cytoplasm to the nuclear envelope, converts free arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5HPETE) and then to LTA4. The presence of the cofactor 5-LO activating protein (FLAP) is required for 5-LO activity and hence for LTA4 generation. Indeed, cells lacking FLAP or that are treated with a FLAP inhibitor, and mice genetically deficient in FLAP, are unable to generate LTs. LTA4 is the precursor for the intracellular generation of LTB4, formed via LTA4 hydrolase activity, and of LTC4, formed via LTC4 synthase activity. Following their generation, LTB4 and LTC4 are actively transported out of the cell, although LTB4 may also act intracellularly to regulate gene expression via nuclear receptor interaction. Extracellular metabolism of the glutathione moiety of LTC4 results in the formation of LTD4 and LTE4; collectively, these three molecules are referred to as the cysLTs or sulfidopeptide LTs. The cell-specific expression of LT biosynthetic enzymes contributes significantly to the regulation of

LT effects in the lung. Neutrophils and dendritic cells primarily synthesize LTB4, mast cells and eosinophils primarily synthesize cysLTs, and macrophages produce a balance of both. As the cells that generate LTs are primarily inflammatory in nature, they are usually only present in the lung in substantial numbers following recruitment by inflammatory mediators secreted by injured or stressed pulmonary epithelial and/or endothelial cells and resident macrophages. In addition to recruiting inflammatory cells that generate LTs, inflammatory mediators can upregulate the expression of LT biosynthetic enzymes and receptors within inflammatory and other cell types, including airway smooth muscle and epithelial cells. The net result is an environment in which LT synthesis contributes to decrements in lung function and to the recruitment and retention of more inflammatory cells via direct chemotactic effects and through the continued generation of proinflammatory cytokines and chemokines. Inflammatory mediators that amplify LT biosynthesis include interleukin-4, interleukin-5, transforming growth factor beta, and others. It is of interest that the hormone leptin, which is secreted by adipocytes in correlation with total body fat mass,

582 LIPID MEDIATORS / Leukotrienes

has also been shown to augment LT production, suggesting a possible mechanistic link between obesity and asthma. Regulation of LT production also appears to exist at the genetic level, as polymorphisms in the promoter regions of the 5-LO and LTC4 synthase genes have been identified and may contribute to respiratory diseases and influence therapeutic responses to anti-LT agents. The biological actions of LTs are mediated by the regulated expression, distribution, and activation of their receptors. Specific cell surface receptors for LTB4 and for the cysLTs are found on a variety of lung and inflammatory cells. Expression levels of these receptors can be altered by an assortment of factors including cytokines, chemokines, LTs, and other mediators whose levels themselves vary with disease state and severity.

Biological Functions LTs exert several important effects in the lung including promotion of inflammatory cell recruitment and adhesion, mucus secretion, airway smooth muscle contractility, and vascular permeability. The primary role of LTB4 is as a potent chemoattractant and activator of myeloid leukocytes including neutrophils, monocytes, and eosinophils. As an example, direct intratracheal administration of LTB4 to humans induces neutrophil recruitment into the airways without altering protein permeability. LTB4 is a recognized autocrine stimulator of neutrophils, and exerts anti-apoptotic effects to prolong the survival of neutrophils and eosinophils. It is also involved in the recruitment and trafficking of T cells in asthma, acting in cooperation with various cytokines and chemokines to direct T cell migration to sites of inflammation. cysLTs are potent mediators of bronchoconstriction resulting from exposure to allergic and other stimuli, and are more potent than histamines in eliciting a decrease in airflow in humans. Blockade of the cysLT1 receptor attenuates the bronchoconstrictive response to various stimuli including allergen, cold air, exercise, and adenosine inhalation, confirming the central role of cysLTs in airflow obstruction. In addition to their bronchoconstrictive effects, cysLTs contribute significantly to the recruitment and retention of inflammatory cells in the lung, promote the induction of cytokine generation by eosinophils and mast cells, and increase vascular permeability. cysLTs also exert numerous effects on dendritic cells including influencing their development, trafficking, and immunomodulatory functions. Given the importance of dendritic cells as antigen-presenting cells, the

effects of cysLTs indicate an additional significant role in asthma pathogenesis.

Receptors The biological actions of LTs result from their interaction with the specific 7 transmembrane domain, G-protein-coupled receptors on the surface of target cells (Figure 1). Two receptors have been identified for LTB4, namely BLT1 and BLT2, which are highand low-affinity LTB4 receptors, respectively. BLT1 is not strongly expressed in lung tissue, but is present on a variety of leukocytes including neutrophils, eosinophils, and effector CD4 þ and CD8 þ T cells. BLT2 is widely expressed in most human tissues and is also found on leukocytes. Two receptors have also been identified for the cysLTs, namely cysLT1 and cysLT2, which are high- and low-affinity cysLT receptors, respectively. Both cysLT1 and cysLT2 are expressed in a variety of tissues and cell types. The expression of cysLT1 on airway smooth muscles is primarily responsible for asthmatic bronchoconstriction resulting from antigen-induced generation of the cysLTs, and targeted antagonism of this receptor has provided an effective adjuvant in the treatment of this disease. Expression of LTB4 and cysLT receptors can be modified by cytokines and other factors present in the microenvironment of injured or diseased lung tissue. For example, interferon gamma stimulates BLT1 expression in macrophages, whereas interleukin-4 or interleukin-13, proinflammatory cytokines believed to play crucial roles in asthma pathogenesis, upregulate cysLT1 expression. As such, the effects of LTs in respiratory diseases are probably mediated by a complex interplay between LTB4 and cysLT receptors and the regulated/dysregulated production of LTB4 and the cysLTs. As LTB4 and LTC4 are synthesized deep within cells, the existence of novel intracellular receptors that mediate autocrine effects of LTB4 and the cysLTs has long been suspected. The nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR-a) was the first molecule identified as a receptor for LTB4 and may be involved in transcriptional regulation of genes involved in LTB4 inactivation and clearance. An intracellular cysLT receptor has been suggested based on the ability of cysLTs to liberate interleukin-4 from membrane-permeabilized but not from nonpermeabilized eosinophils treated with cysLT1/2 inhibitors.

Leukotrienes in Respiratory Diseases A wealth of clinical and experimental evidence supports the involvement of LTs and their receptors in several respiratory diseases (Table 1), the most

LIPID MEDIATORS / Leukotrienes 583 Table 1 Murine models of respiratory disease in which genetic and pharmacological approaches have identified detrimental roles for LTs Disease model

Approach

Allergen-induced inflammation

5-LO gene deletion cysLT1 gene deletion cysLT1 antagonism

Bleomycin-induced fibrosis

cPLA2 gene deletion 5-LO gene deletion LTC4 synthase gene deletion cysLT2 gene deletion

Chemical-induced carcinogenesis

cPLA2 gene deletion 5-LO inhibition FLAP inhibition cysLT1 antagonism

thoroughly studied of which is asthma. Bronchoalveolar lavage (BAL) fluid LT levels are increased in asthmatics compared with normal subjects, and animal models of allergen-induced airway inflammation reveal similar findings. Inhibition of LT biosynthesis or abrogation of cysLT signaling via cysLT1 blockade reduces allergen-induced airway inflammation and hyperresponsiveness in mice, and clinically efficacious cysLT1 antagonists and inhibitors of cysLT synthesis have been developed and employed in the treatment of asthma. Recent recognition that LTs influence dendritic cell maturation, trafficking, and immunomodulatory function may lead to the development of further beneficial therapies for this disease. In addition to asthma, elevated levels of LTs have been reported in BAL fluid or exhaled breath condensate from patients with pneumonia, cystic fibrosis, lung cancer, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis. While little data is available regarding LTB4, cysLTs appear to be intimately involved in the pathogenesis of pulmonary fibrosis. Constitutive activation of 5-LO is observed in lung tissue of humans with idiopathic pulmonary fibrosis. In addition, bleomycin-induced pulmonary fibrosis in mice is characterized by increased lung cysLT production and is attenuated by disruption of the 5-LO gene. Among other mechanisms, cysLTs appear to promote a profibrotic phenotype via inflammatory cell recruitment and induction of fibroblast proliferation and collagen production. The phenomenon of transcellular metabolism may contribute to the increased LT biosynthesis observed in respiratory diseases such as asthma and fibrosis. Under normal conditions, pulmonary epithelial cells metabolize arachidonic acid primarily via

the cyclooxygenase pathway, leading to generation of the anti-inflammatory, antifibrotic, and bronchodilatory prostaglandin E2 (PGE2). In conditions of disease, epithelial cell injury or dysfunction results in impaired cyclooxygenase activity and decreased PGE2 production. Consequently, there is an increased availability of free arachidonic acid that can be metabolized by leukocytes located in close proximity to the epithelial cells. This transcellular metabolism of arachidonic acid results in increased LT biosynthesis by leukocytes and may contribute to the inflammatory and bronchoconstrictive phenotype of various respiratory diseases. Interestingly, LTs have been shown to exert antimicrobial activity and to be involved in protecting the lung from injury in a mouse model of pneumonia, indicating that not all actions of these mediators in the lung are deleterious. While the observed involvement of LTs in an array of respiratory diseases underscores their central importance in the diseased lung, recent advances indicate that much remains to be understood with regard to the varied roles of these lipid mediators. The evolving recognition and elucidation of the diverse roles of LTs in the lung suggests a favorable future for the development of therapeutic approaches for the treatment of respiratory diseases based on LT biology. See also: Asthma: Overview. Lipid Overview; Lipoxins. Pulmonary Fibrosis.

Mediators:

Further Reading Coffey M and Peters-Golden M (2003) Extending the understanding of leukotrienes in asthma. Current Opinions in Allergy and Clinical Immunology 3: 57–63. Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294: 1871–1875. Holgate ST, Peters-Golden M, Panettieri RA, and Henderson WR (2003) Roles of cysteinyl leukotrienes in airway inflammation, smooth muscle function, and remodeling. Journal of Allergy and Clinical Immunology 111: S18–S36. Kanaoka Y and Boyce JA (2004) Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses. Journal of Immunology 173: 1503–1510. Luster AD and Tager AM (2004) T-cell trafficking in asthma: lipid mediators grease the way. Nature Reviews: Immunology 4: 711–724. McMillan RM (2001) Leukotrienes in respiratory disease. Pediatric Respiratory Reviews 2: 238–244. Peters-Golden M and Brock TG (2001) Intracellular compartmentalization of leukotriene synthesis: unexpected nuclear secrets. FEBS Letters 487: 323–326. Peters-Golden M, Canetti C, Mancuso P, and Coffey MJ (2004) Leukotrienes: underappreciated mediators of innate immune responses. Journal of Immunology 173: 589–594. Sampson AP, Pizzichini E, and Bisgaard H (2003) Effects of cysteinyl leukotrienes and leukotriene receptor antagonists on markers of inflammation. Journal of Allergy and Clinical Immunology 111: S49–S61.

584 LIPID MEDIATORS / Prostanoids Soberman RJ and Christmas P (2003) The organization and consequences of eicosanoid signaling. Journal of Clinical Investigation 111: 1107–1113. Tager AM and Luster AD (2003) BLT1 and BLT2: the leukotriene B4 receptors. Prostaglandins, Leukotrienes and Essential Fatty Acids 69: 123–134. Yokomizo T, Izumi T, and Shimizu T (2001) Leukotriene B4: metabolism and signal transduction. Archives of Biochemistry and Biophysics 385: 231–241.

roles of prostanoids in pathologic and homeostatic processes. This article reviews the current state of knowledge of the synthesis, actions, and pathophysiologic roles of prostanoids in the respiratory tract, as well as the therapeutic potential of their modulation.

Pulmonary Prostanoid Synthesis and Metabolism

Prostanoids M Peters-Golden, University of Michigan, Ann Arbor, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Prostanoids are a family of lipid mediators that include prostaglandins, prostacyclin, and thromboxane. Their synthesis from arachidonic acid is initiated by the cyclooxygenase enzyme, which is the molecular target of the widely employed class of nonsteroidal anti-inflammatory drugs. Terminal synthase enzymes, expressed in cell-specific fashion, complete the synthesis of bioactive prostanoids. Via ligation of G-protein-coupled receptors, they exert autocrine and paracrine effects including modulation of smooth muscle tone, microvascular permeability, tissue repair and remodeling, inflammation, and immune responses. Either overproduction or underproduction of prostanoids has been implicated in a variety of lung diseases, including asthma, interstitial lung disease, pulmonary vascular disease, cancer, and infections. Current therapies employ both blockade and administration of prostanoids, and additional applications of increasing selectivity can be envisioned in the future.

Introduction Prostanoids comprise a family of lipid mediators formed from arachidonic acid (AA) via the prostaglandin (PG) H synthase or cyclooxygenase (COX) pathway. They include PGE2, PGD2, and PGF2a, as well as prostacyclin (PGI2) and thromboxane A2 (TxA2). Prostanoids have long been known to modulate such biological processes as smooth muscle tone, vascular permeability, pain, fever, and platelet aggregation. More recently, roles for these molecules in cellular proliferation and survival, immune responses, and even sleep have been identified. The clinical importance of prostanoids is emphasized by the fact that their biosynthesis is the target for nonsteroidal anti-inflammatory drugs (NSAIDs), one of the most widely used classes of pharmacotherapeutic agents. The last decade has witnessed remarkable advances in our understanding of both prostanoid synthetic mechanisms as well as the receptors that mediate prostanoid action. In addition, transgenic animal models have shed a great deal of light on the

Arachidonic acid liberated from membrane phospholipids by the actions of phospholipase A2 (PLA2) is transformed to the unstable endoperoxide PGH2 by the COX enzyme (see Figure 1). Since the early 1990s it has been recognized that COX exists in two isoforms, generally designated COX-1 and COX-2. Most cell types are capable of expressing both COX isoforms, and both can initiate prostanoid synthesis from AA. The initial paradigm held that COX-1 was constitutively expressed in most tissues and was responsible for basal synthesis of prostanoids that mediated ‘housekeeping’ functions, while COX-2 expression required induction (by cytokines, hormones, growth factors, lipopolysaccharide), which was glucocorticoid inhibitable, and was responsible for augmented synthesis of prostanoids that mediated pathobiologic effects. From this came the notion that inhibition of COX-2 accounted for the desirable therapeutic effects of nonselective NSAIDs (e.g., aspirin, indomethacin, and ibuprofen) that inhibited both isoforms, while inhibition of COX-1 accounted for their unwanted side effects (e.g., gastrointestinal irritation and impaired renal blood flow). This paradigm promptly ascended to the level of dogma, and helped drive the swift development of highly selective COX-2 inhibitors (coxibs). In fact, only some of the predictions of the original dogma have been proven to be accurate. The prediction that synthesis of pathologic prostanoids most often derives from COX-2 has generally been borne out. Not anticipated, however, was the fact that COX-2 was expressed constitutively in certain tissues (e.g., the vascular endothelium and the respiratory epithelium) and was consequently responsible for elaboration of prostanoids serving certain homeostatic functions. The COX-derived intermediate PGH2 is converted to bioactive prostanoids by terminal synthase enzymes, whose expression profiles exhibit cell type specificity. Although macrophages synthesize a broad range of prostanoids, most cell types elaborate a single predominant COX product, as follows: platelets, TxA2; mast cells, PGD2; endothelial cells, PGI2; and epithelial cells, fibroblasts, and smooth muscle cells, PGE2. PGF2a, PGI2, and TxA2 synthases appear to

LIPID MEDIATORS / Prostanoids 585 COOH

Arachidonic acid

NSAIDs O

PGH2

+

COX-2

COX-1

LPS TNF-


GCs

Coxibs COOH

O OH

Prostaglandin synthases PGDS

PGIS

PGES

OH COOH

−

COO Na

PGD2

HO

OH

TxA2

HO OH

HO OH

PGI2 PGJ2

OH COOH

COOH

O

COOH

O

OH

COOH

TxS OH

+

O

O

PGFS

OH

PGF2


PGE2

COOH O

15-deoxy-∆12,14 –PGJ2 Figure 1 Pathway of prostanoid production. Free AA is oxygenated by either COX-1 or COX-2 to form the unstable endoperoxide intermediate prostaglandin (PG) H2. Traditional NSAIDs such as aspirin, indomethacin, and ibuprofen nonselectively inhibit COX-1 and -2, while coxibs selectively inhibit COX-2. The COX-2 enzyme can be induced by inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-a), a process that can be prevented by glucocorticoids (GCs). PGH2 can be converted by terminal PG synthase enzymes to the stable prostanoids, PGD2, prostacyclin (PGI2), PGE2, PGF2a, and TxA2, each of which ligates specific G-protein-coupled prostanoid receptors. PGD2 can be nonenzymatically dehydrated to PGJ2 and then to 15-deoxy-D12,14-PGJ2; the latter compound is a putative ligand for the intranuclear receptor peroxisome proliferator-activated receptor-g (PPAR-g).

represent single enzyme isoforms. Lipocalin and hematopoietic PGD synthases represent two distinct gene products present in brain and mast cells/macrophages, respectively. At least three PGE synthases exist, all of which require reduced glutathione as a cofactor. Cytosolic PGE synthase and membrane-associated PGE synthase-2 are both primarily constitutively expressed enzymes. Membrane-associated PGE synthase-1, by contrast, resembles COX-2 in its induction by inflammatory stimuli, which can be inhibited by glucocorticoids. Incremental prostanoid synthesis requires either an increase in free AA or increased expression of COX or terminal synthase enzymes capable of metabolizing the relatively small amount of AA liberated during constitutive phospholipid remodeling. Substances such as bradykinin, thrombin, antigen, leukotrienes, oxidants, silica, and microbes can activate PLA2 to trigger AA release, while lipopolysaccharide, growth factors, and cytokines can enhance expression of downstream enzymes. Prostanoids produced in the lung can be measured directly in lung tissue, bronchoalveolar lavage fluid, induced sputum, exhaled breath

condensate, or plasma, or indirectly as urinary metabolites. In addition to a substantial capacity to elaborate COX products, the lung plays an important role in their catabolism. During a single pass through the pulmonary vascular bed, the majority of prostanoids are inactivated. This occurs via transporter-mediated uptake into endothelial cells followed by metabolism via the enzyme 15-hydroxy PG dehydrogenase. Mice, in which this enzyme has been deleted, demonstrate enhanced levels of PGE2 and biological responses mediated by PGE2.

Prostanoid Receptors The autocrine or paracrine actions of prostanoids are mediated via ligation of receptors. The best characterized receptors for prostanoids are high-affinity (EC50 values in the nanomolar range) G-proteincoupled receptors (GPCRs). Binding to these receptors results in the generation of second messengers and subsequent activation of signal transduction pathways. Figure 2 depicts the GPCRs for various

586 LIPID MEDIATORS / Prostanoids PGI2 PGD2

Table 1 Actions of prostanoids other than PGE2 in the respiratory tract

IP DP1

EP4

PGD2 * sleep * airflow obstruction * Th2 immune responses

EP3

TxA2 * bronchoconstriction * vasoconstriction * platelet aggregation

EP2

Gs

↑cAMP

PGE2

TxA2

TP

Gi

↓cAMP

DP2

EP1 PGF2


FP

Gq

↑Ca2 +

Figure 2 GPCRs for prostanoids. Prostanoids and the GPCRs, major G proteins, and second messengers through which they act are depicted. The dashed line represents the capacity of PGD2 to ligate the TP receptor with a relatively low affinity.

prostanoids, and the G proteins and second messengers through which they generally signal. Based on their expected effects on smooth muscle tone, those receptors that signal through Gs-mediated increases in intracellular cyclic AMP can be considered ‘relaxant’ receptors, while those that signal through Gqmediated increases in intracellular calcium can be considered ‘contractile’ in nature. The profile of prostanoid receptors varies with the cell type. In addition, the expression of prostanoid receptors is under genetic control and can also be modulated by exogenous factors, such as lipopolysaccharide, cytokines, and tissue injury. Certain prostanoids can bind to and act via multiple GPCRs that are encoded by distinct genes. PGE2 represents the most striking example of this phenomenon, as it acts via four distinct E prostanoid (EP) receptors; such receptor diversity probably contributes to the pleiotropic actions of this most ubiquitous of prostanoids. Certain prostanoids can bind to another class of receptor, the peroxisome proliferator-activated receptors (PPARs). PPARs comprise a family of intranuclear receptors and transcription factors. PPAR-g regulates the expression of genes involved in lipid metabolism, inflammation, and cellular proliferation. While it is the target for the glitazone class of antidiabetic drugs, the nature of the endogenous ligand(s) for this receptor has remained elusive. Substantial attention in recent years has surrounded the possibility that 15-deoxy-D12,14-PGJ2, a nonenzymatic dehydration product of PGD2, may fulfill this role and thereby serve as a break on inflammatory and proliferative events. However, the EC50 of PPAR-g for

PGI2 * vasodilation * inhibition of platelet aggregation * inhibition of vascular remodeling PGF2a * bronchoconstriction * vasoconstriction

exogenous 15-deoxy-D12,14-PGJ2 is in the 10 5 M range, and sophisticated mass spectrometric analyses have cast serious doubts on whether such levels of 15deoxy-D12,14-PGJ2 are generated endogenously. For this and other reasons, the role of this prostanoid as an endogenous activator of PPAR-g remains to be established.

Prostanoids in Respiratory Disease and Homeostasis Prostanoids have been implicated in virtually every type of pathologic or homeostatic process conceivable. The best-known actions of prostanoids pertinent to the respiratory tract are shown in Table 1 and Figure 3, and their key roles in clinically relevant pathophysiologic states are summarized below. It is essential to point out that respiratory disease states can be linked to both the overproduction as well as the underproduction of various prostanoids. Asthma and Aspirin-Sensitive Asthma

Prostanoids have the potential to influence every aspect of the pathobiology of asthma. The recognition of both contractile and relaxant groups of prostanoids has been previously noted, and the effects of individual prostanoids on bronchial tone are depicted in Table 1. The greatest interest currently surrounds the possible proasthmatic role of PGD2 and the antiasthmatic role of PGE2. PGD2 can reproduce many of the airway abnormalities observed in asthma and its levels are increased in asthmatics. The fact that DP1 null mice are protected in a model of antigen-induced ‘asthma’ is somewhat surprising, since DP1 signals as a ‘relaxant’ receptor. Therefore, the proasthmatic actions of

LIPID MEDIATORS / Prostanoids 587

Vascular responses

Airway responses

• Vasodilation • ↑ Microvascular permeability

• Bronchodilation • Protection against bronchoconstriction • Bronchial hyperresponsiveness

Epithelial cells

Neoplasia

• ↑ Wound closure by airway epithelium

• ↑ Tumor cell proliferation • ↓ Tumor cell apoptosis

• ↑ Surfactant secretion by alveolar type ΙΙ cells • ↓ Apoptosis

PGE2

• ↑ Tumor cell adhesion & migration • ↑ Angiogenesis

Inflammatory responses

Mesenchymal cell (fibroblast & smooth muscle cell) functions

• ↓ Myelopoiesis

• ↓ Migration

• ↓ Leukocyte chemotaxis

• ↓ Proliferation

• ↓ Adhesion molecule expression • ↓ Generation of inflammatory mediators

• ↓ Collagen synthesis and ↑ collagen degradation • ↓ Fibroblast transition → myofibroblasts • ↑ Fibroblast production of hepatocyte growth factor, a survival factor for epithelial cells

• ↑ Generation of proinflammatory IL- 6

Immune responses

• ↑ Generation of anti-inflammatory IL-10

• ↑ Maturation of immature dendritic cells • ↓ Antigen presentation by mature dendritic cells • ↓ T and B lymphocyte proliferation and functions • Context-dependent effects on Th1 vs. Th2 polarization • ↓ Phagocyte antimicrobial functions

Figure 3 Pleiotropic actions of PGE2 in the respiratory tract.

PGD2 may instead involve DP1-mediated increases in airway edema (or other unknown actions), contractile effects mediated by its ability to also ligate the TP receptor, and chemoattractant and proinflammatory effects mediated via a chemokine family receptor previously termed ‘chemoattractant receptor expressed on Th2 cells’ or CRTH2 and now designated as DP2. Antagonists for both DP1 and DP2 are currently under development. Inhalation of PGE2 or its analogs in humans causes bronchodilation, protects against bronchoprovocation, and attenuates induced airway inflammation. Such actions reflect not only its well-known relaxant properties, but also its diverse anti-inflammatory and immunosuppressive actions (Figure 3). Most of these salutary effects are mediated by EP2- or EP4-mediated increases in cyclic AMP. Recent studies have shown a worsening in models of experimental asthma when COX inhibitors or COX null mice are employed. As PGE2 is the predominant prostanoid found in lung lavage fluid, these data are most consistent with the conclusion that endogenously produced PGE2 serves a bronchoprotective role in vivo. Limited data from humans and animal models suggest that, in some instances, asthma may be associated with a relative deficiency of endogenous PGE2. In most asthmatics, administration of NSAIDs has negligible effect, probably reflecting the global inhibition of prostanoids that exert opposing actions on the

airways. In B5–10% of asthmatics, however, nonselective COX inhibitors dramatically worsen their airway function; this is termed aspirin-sensitive or aspirin-intolerant asthma. Interestingly, these patients can safely tolerate COX-2-selective inhibitors. These observations imply that in this subset of patients, COX-1-dependent synthesis of a prostanoid (most likely PGE2) serves a critical bronchoprotective role. Inhibitors of COX-1 remove this ‘brake’, and also allow the accumulation of unmetabolized AA, which can be metabolized to proasthmatic leukotrienes. A relative deficiency of COX-2-derived PGE2 in aspirinsensitive patients may help to explain both their ability to tolerate COX-2-selective inhibitors as well as the overall severity of their asthma at baseline even when they avoid NSAIDs. As for other types of bronchoprovocation, inhalation of PGE2 can also protect against aspirin-induced bronchospasm. However, such treatment frequently provokes cough. If the EP receptors that mediate cough are distinct from those that exert bronchoprotective actions, inhalation of EP-selective PGE2 agonists may represent an attractive therapeutic approach in both aspirin-sensitive and aspirin-tolerant asthma. Fibrotic Lung Disease

By virtue of its capacity to inhibit inflammation as well as fibroblast migration, proliferation, differentiation

588 LIPID MEDIATORS / Prostanoids

into myofibroblasts, and collagen synthesis, PGE2 attenuates fibrotic responses to lung injury (Figure 3). These actions are largely mediated via increases in cyclic AMP. In animal models of pulmonary fibrosis, pharmacologic or genetic inhibition of COX enzymes exaggerates the degree of fibrosis. Interestingly, patients with idiopathic pulmonary fibrosis have been shown to have a relative deficiency of PGE2 in their lung lavage fluid, and impaired PGE2 synthetic capacity has been documented in both alveolar macrophages and lung fibroblasts from such patients. PGE2 deficiency would tend to promote an activated phenotype in fibroblasts. Activation of fibroblasts and smooth muscle cells also contributes to airway remodeling in asthma, and defective PGE2 synthesis has also been described in these cell types from asthmatics. Taken together, these data strongly suggest that a relative deficiency of PGE2 synthesis characterizes and may contribute to remodeling of both parenchyma and airways in chronic lung disease. Again, administration of PGE2 or of PGE2 analogs that selectively activate cyclic AMP-coupled EP receptors is an appealing therapeutic strategy in these conditions. Pulmonary Hypertension

It is well known that PGI2 is the major prostanoid product of vascular endothelial cells and that it inhibits platelet aggregation as well as contraction and proliferation of vascular smooth muscle cells. TxA2 is produced by platelets, and has opposing effects. Patients with primary pulmonary hypertension have been identified as having both increased TxA2 production and impaired PGI2 generation, with the former probably reflecting platelet aggregation and the latter reflecting a deficiency of PGI2 synthase. In view of their deficiency of this important endogenous vasodilator, it is not surprising that exogenous administration of PGI2, PGE2, or analogs thereof by the intravenous or inhaled route has become established as standard therapy for pulmonary hypertensive disorders. These prostanoids have proven not only to cause vasodilation, but also to ameliorate vascular wall remodeling. Lung Cancer

Many cancers, including lung cancers, overexpress COX-2 and elaborate high levels of PGE2. Pharmacologic and genetic interruption of PGE2 synthesis or EP receptors reduces tumor growth in vitro and in vivo. Such strategies target the ability of PGE2 to promote tumor cell migration, survival, and proliferation, to stimulate tumor angiogenesis, and to reduce immune surveillance against malignant cells

(Figure 3). Trials of COX inhibitors in humans with lung cancer are currently underway. Immune Function

PGE2 modulates immune responses in a complex and sometimes conflicting manner (Figure 3). For example, it can either stimulate or inhibit antigen-presenting dendritic cells, depending on their degree of maturation. Its effects on Th1 versus Th2 T lymphocyte phenotypes have also been controversial. In general, however, PGE2 suppresses most aspects of lymphocyte, macrophage, and neutrophil functions. Therefore, it might be expected to interfere with immune responses to vaccines, immune surveillance of cancer cells, and antimicrobial defense against infections. It is interesting to speculate that overproduction of PGE2 – as has been described in cancer, bone marrow transplantation, the elderly, and in infections themselves – might increase susceptibility to infections and compromise innate immune responses to them. In such instances, inhibition of PGE2 synthesis or blockade of relevant EP receptors might have a therapeutically desirable immunostimulatory effect. See also: Asthma: Overview; Aspirin-Intolerant. Corticosteroids: Glucocorticoid Receptors; Therapy. Epithelial Cells: Type I Cells; Type II Cells. Fibroblasts. G-ProteinCoupled Receptors. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Mast Cells and Basophils; Pulmonary Macrophages. Myofibroblasts. Peroxisome Proliferator-Activated Receptors. Platelets. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Signal Transduction. Smooth Muscle Cells: Airway. Transgenic Models. Tumors, Malignant: Bronchogenic Carcinoma. Vascular Disease.

Further Reading Badesch DB, McLaughlin VV, Delcroix M, et al. (2004) Prostanoid therapy for pulmonary arterial hypertension. Journal of American College of Cardiology 43(12 supplement S): 56S–61S. Breyer RM, Bagdassarian CK, Myers SK, and Breyer MD (2001) Prostanoid receptors: subtypes and signaling. Annual Review of Pharmacology and Toxicology 41: 661–690. Carey MA, Germolec DR, Langenbach R, and Zeldin DC (2003) Cyclooxygenase enzymes in allergic inflammation and asthma. Prostaglandins Leukotrienes and Essential Fatty Acids 69: 157–162. FitzGerald GA and Patrono C (2001) The coxibs, selective inhibitors of cyclooxygenase-2. New England Journal of Medicine 345: 433–442. Harris SG, Padilla J, Koumas L, Ray D, and Phipps RP (2002) Prostaglandins as modulators of immunity. Trends in Immunology 23: 144–150. Hayaishi O and Urade Y (2002) Prostaglandin D2 in sleep-wake regulation: recent progress and perspectives. Neuroscientist 2002. 8: 12–15.

LIPID MEDIATORS / Platelet-Activating Factors Helliwell RJ, Adams LF, and Mitchell MD (2004) Prostaglandin synthases: recent developments and a novel hypothesis. Prostaglandins, Leukotrienes and Essential Fatty Acids 70: 101–113. Ide T, Egan K, Bell-Parikh LC, and FitzGerald GA (2003) Activation of nuclear receptors by prostaglandins. Thrombosis Research 110: 311–315. Narumiya S, Sugimoto Y, and Ushikubi F (1999) Prostanoid receptors: structures, properties, and functions. Physiological Reviews 79: 1193–1226. Sandler AB and Dubinett SM (2004) COX-2 inhibition and lung cancer. Seminars in Oncology 31(2 supplement 7): 45–52. Szczeklik A, Sanak M, Nizankowska-Mogilnicka E, and Kielbasa B (2004) Aspirin intolerance and the cyclooxygenaseleukotriene pathway. Current Opinion in Pulmonary Medicine 10: 51–56. Vancheri C, Mastruzzo C, Sortino MA, and Crimi N (2004) The lung as a privileged site for the beneficial actions of PGE2. Trends in Immunology 25: 40–46. Warner TD and Mitchell JA (2004) Cyclooxygenases: new forms, new inhibitors, and lessons from the clinic. FASEB Journal 18: 790–804. Zha S, Yegnasubramanian V, Nelson WG, Isaacs WB, and De Marzo AM (2004) Cyclooxygenases in cancer: progress and perspective. Cancer Letters 215: 1–20.

Platelet-Activating Factors K F Chung, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Platelet-activating factor (PAF) is a biologically active phospholipid mediator belonging to a family of alkylphosphoglycerides with potent actions as a mediator of inflammation. It acts through a G-protein-coupled receptor linked to phospholipase A2 and C. PAF is metabolized by PAF acetylhydrolases which reduce its activity. Low plasma PAF acetylhydrolase activity has been associated with inflammatory disorders such as asthma and rheumatoid arthritis. PAF is produced by endothelial cells, macrophages, neutrophils, eosinophils, monocytes, and mast cells, and activates many inflammatory cells by intercellular and intracellular mechanisms. PAF may be an important inflammatory mediator in various lung diseases such as asthma, acute respiratory distress syndrome, and pulmonary vascular diseases. Although there is some support for a role of PAF in animal models, the effects of inhibiting the actions of PAF in human asthma in particular have been negligible. PAF may play an important role in normal physiological homeostasis and in the primary response to inflammation and injury. This role remains to be defined.

Introduction Platelet-activating factor (PAF) or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine was first discovered as an active moiety released from rabbit basophils after stimulation with immunoglobulin E (IgE). PAF has turned out since to be a pleiotropic lipid mediator of physiologic and pathophysiologic events occurring during inflammation.

589

Metabolism of PAF

PAF is synthesized in many inflammatory cells such as monocytes, macrophages, neutrophils, eosinophils, mast cells, and endothelial cells by the remodeling pathway (Figure 1), which involves the activation of phospholipase A2 to hydrolyze a longchain acyl group from 1-alkyl-2-acyl-sn-glycero-3 phosphocholine in membrane phospholipids to form 1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lysoPAF), followed by acetylation by an acetyltransferase enzyme. Acetyltransferase activity can be induced by the calcium ionophore A23187, thrombin, bradykinin, elastase, cathepsin G, leukotrienes C4 and D4, interleukin (IL)-8, and tumor necrosis factor (TNF) and IL-1a. Activation of the remodeling pathway results in the biosynthesis of excessive amounts of PAF. By contrast, the de novo pathway involves the direct generation of PAF from etherlinked phospholipids, such as alkylacetylglycerols, under the action of a highly specific cholinephosphotransferase present in several tissues including rat lung and human neutrophils. This production of PAF relates to basal physiological levels. PAF is rapidly inactivated by the removal of the acetate moiety to form lyso-PAF, which is biologically inactive, by acetylhydrolases, of which the most important one is PAF-specific acetylhydrolases (PAFAH) which is present in cells and in plasma. Cellular acetylhydrolase activity may be released into the supernatant after stimulation of platelets with various agonists, including PAF. Low levels of plasma PAF-AH have been associated with several inflammatory diseases such as asthma, rheumatoid arthritis, and Crohn’s disease. PAF-AH deficiency was found to be more frequent in Japanese children with severe asthma, and was accounted for by a single point mutation at position 279 (V279F) on the PAFAH. The significance of plasma PAF-AH deficiency is unclear since those deficient in plasma PAF-AH bronchoconstrict equally to inhaled PAF as those with normal PAF-AH levels.

PAFs Although most studies have concentrated on two molecular species of PAF (i.e., C16-PAF and C18-PAF), stimulated inflammatory cells also synthesize and release a variety of PAF homologs and analogs, including saturated and unsaturated alkyl-chain homologs and 1-O-acyl-PAF analogs and acetylated glycerylphosphoethanolamine. Some human cell types (such as neutrophils, eosinophils, and macrophages) produce almost exclusively PAF on stimulation. Other cell types (mast cells, basophils, and endothelial cells)

590 LIPID MEDIATORS / Platelet-Activating Factors Remodeling

De novo

Membrane phospholipid l-Alkyl-2-acyl-sn-GPC

Ether-linked phospholipid l-Alkyl-2-acetylsn-glycerol

Acyltransferase

Phospholipase A2

Arachidonic acid

Cholinephosphotransferase

l-Alkyl-2-lyso-sn-GPC (lyso-PAF)

Acetylhydrolase

Acetyltransferase

l-Alkyl-2-acetyl-sn-GPC (PAF)

Eicosanoids

CH2

O PGs

LTs CH3

C

O

O

(CH2)n O

CH CH2

O

P

O

CH3 n = 15,17 Choline

O Figure 1 Synthesis and catabolism of PAF. GPC, glycerol-3-phosphocholine; PGs, prostaglandins; LTs, leukotrienes. Reproduced with permission from Chung KF (1992) Platelet-activating factors in inflammation and pulmonary disorders. Clinical Science 83: 127–138, & the Biochemical Society and the Medical Research Society.

produce predominantly 1-alkyl-2-acyl-sn-glycero-3phosphocholine. This profile of PAF generation also depends on the stimulus; for example, human basophils synthesize predominantly 1-alkyl-2-acyl-snglycero-3-phosphocholine in response to the calcium ionophore A23187. Lung tissue can also be stimulated via cell-surface IgE to produce a large proportion of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine relative to PAF. The biological activities of these PAF analogs and homologs can differ. Alkyl-chain PAF homologs are also active in stimulating human neutrophil in vitro when compared with C16-PAF. There are differences in rank orders of potency of alkyl and acyl PAF molecules in vitro on human neutrophils and in vivo thrombocytopenic and leukopenic effects when injected into rabbits. 1-alkyl-2-acyl-sn-glycero-3phosphocholine is a relatively weak stimulus of human neutrophils and platelets, and may actually inhibit some of the stimulating effects of PAF on the human neutrophil.

Cellular Sources A wide range of cell types produce PAF in vitro in response to several stimuli. Within a few minutes of

activation of neutrophils by opsonized zymosan or the calcium ionophore A23187, PAF is synthesized, although only 3–4% is released. Leukotriene B4 also stimulates PAF biosynthesis and 5-hydroxyeicosatetraenoic acid synergistically increases PAF-stimulated PAF synthesis, and PAF itself could stimulate PAF acetyltransferase in neutrophils. The cytokine granulocyte–macrophage colony stimulating factor (GM-CSF) can augment the production of PAF and increase the activity of acetyltransferase in human neutrophils. Eosinophils from patients with eosinophilia show high activity of acetyltransferase and release PAF after stimulation with various chemotactic factors. Human eosinophils contain the most abundant PAF precursor, 1-alkyl-2-acyl-sn-glycero-3-phosphocholine. Purified human hypodense eosinophils release PAF after IgE (but not IgG) mediated activation. Human alveolar macrophages obtained by bronchoalveolar lavage of allergic asthmatic patients also release small amount of PAF after stimulation in vitro. Human alveolar macrophages also produce and release a large amount of lyso-PAF after stimulation with the calcium ionophore A23187 or after IgEmediated mechanisms. Cultured human endothelial cells release a small percentage of synthesized PAF

LIPID MEDIATORS / Platelet-Activating Factors

after activation by a calcium ionophore, bradykinin, thrombin, and IL-1. Purified human lung mast cells also generate PAF when stimulated with anti-IgE, but most of the PAF is also retained intracellularly. Human lymphocytes appear to lack acetyltransferase activity. PAF biosynthesis can be stimulated in human monocytes in response to the cytokines IL-1b, TNF, and interferon alpha, in a biphasic process. During the first 2 h of stimulation, most of the PAF is retained, whereas during the later phase of activation, PAF is largely secreted.

Role for Intracellular PAF Many cell types such as neutrophils, endothelial cells, and macrophages can produce large amounts of PAF but retain it within the cell. This has led to the possibility that PAF may have an intracellular role as a second messenger. The ability of endotoxin to prime neutrophils with an enhanced secretion of superoxide anions may be related to the primed increase in intracellular PAF. Intracellular PAF in endothelial cells stimulated by agents such as thrombin or leukotrienes may mediate neutrophil adherence. Thus, PAF generated intracellularly in endothelial cells can be translocated by mechanisms that are not yet clear to the outer part of the surface membrane, where it can bind to the PAF receptor on target neutrophils (or eosinophils).

591

This type of action of PAF may lead to the priming and activation of leukocytes at surfaces of endothelial cells that have been activated. One advantage of this mechanism to PAF is that there is less chance of PAF being rapidly degraded by acetylhydrolases and ensures that only low concentrations of PAF are needed. Thus, PAF may be considered as a juxtacrine signaling molecule.

PAF Receptors PAF activates specific membrane receptors (Figure 2). Intracellular endosomal receptors have also been described but their function is unknown. The PAF receptor is a serpentine G-protein-coupled receptor containing seven a-helical membrane-spanning domains and is linked to phosphoinositide metabolism by a G-protein coupled to phospholipase C and A2. Stimulation of PAF receptors causes transient production of diacyglycerol, which activates protein kinase C, and of inositol triphosphate which leads to the release of internal calcium stores. PAF stimulates tyrosine phosphorylation of many proteins in neutrophils, macrophages, and platelets, induces the stimulation of nuclear factor kappa B (NF-kB) and transcription of c-fos and c-jun in inflammatory cells. PAF can activate a mitogen-activated protein kinase (MAPK) kinase-3, a known activator of p38 MAPK,

PAF Acetyl Choline

P

O Extracellular space

NH2

n2

PAF receptor

n1

n1 G-protein

P

G-protein

PLA2

PLC

PtdInaP2 O P

Base Arachidonic acid

n2

GTP

DAG OH

DAG PKC

Ca2+

GTP O COOH

Inositol-P2

Eicosanoids

Ca2+

n1 n2

Endoplasmic reticulum

InsP3

Protein phosphorylation

InsP3 Cytosol

Figure 2 Schematic representation of the PAF receptor and PAF signaling pathways. PLA2, phospholipase A2; PLC, phospholipase C; PtdinsP2, phosphatidyl 4,5-bisphosphate; Inositol-P2, inositol 4,5-bisphosphate; InsP3, inositol 1,4,5-triphosphate; DAG, diacylglycerol; PKC, protein kinase C. Reproduced from Montuccio G, Alloatti G, and Camussi G (2000) Role of platelet-activating factor in cardiovascular pathophysiology. Physiological Reviews 80: 1669–1699, used with permission from The American Physiological Society.

592 LIPID MEDIATORS / Platelet-Activating Factors

and also the Jak/STAT pathway. Following ligand activation, the PAF receptor is degraded via both the proteasome and lysosomal pathways. Potent selective PAF antagonists have been developed from natural products (e.g., ginkgolides and kadsurenone) or by synthesis (e.g., phospholipid analogs, tetrahydrofuran derivatives, and triazolobenzodiazepine derivatives). Another approach to inhibiting the effects of PAF has been to use recombinant PAF-AH.

Effects of PAF on Cell Activation and Lipid Metabolism PAF, at picomolar concentrations, causes aggregation of washed rabbit and human platelets, with release of 5-hyroxytryptamine (serotonin). PAF causes the release of lysosomal enzymes and superoxide anions, the generation of LTB4, and chemotaxis from neutrophils. PAF causes the release of the granule-associated enzyme eosinophil peroxidase from human and guinea pig eosinophils. Eosinophils activated by PAF induce tracheal epithelial shedding in vitro, associated with a slowing of ciliary beat frequency. It induces a dose-dependent enhancement of eosinophil cytotoxicity. PAF is a potent chemotactic and chemokinetic mediator for eosinophils in vitro and promotes the adhesion of neutrophils and eosinophils to vascular endothelial cell. PAF inhibits human lymphocyte proliferation when stimulated with mitogens such as phytohemagglutinin or concanavalin A and also suppresses IL-2 production. At higher concentrations of PAF, CD4 þ T-cell proliferation is inhibited. PAF induce suppressor cells, accompanied by an increase in CD8 þ T cells and a small decrease in CD4 þ T cells. PAF interacts with cytokines and arachidonic acid products. Cleavage of the preformed precursor of PAF liberates two important precursor molecules, lyso-PAF and arachidonic acid, which are then metabolized to PAF and lipoxygenase and/or cyclooxygenase products. LTB4 and PAF production from neutrophils stimulated by the ionophore A23187 are tightly coupled while PAF itself can stimulate LTB4 synthesis in the neutrophil and the eosinophil. IL-1b, TNF, and interferon alpha can induce the synthesis of PAF from human monocytes and GM-CSF can augment the production of PAF from human neutrophils. PAF can also increase the release of cytokines such as TNF and IL-1 from alveolar macrophages and monocytes, respectively. Preincubation of blood neutrophils with GM-CSF enhances the ability of PAF to stimulate leukotriene synthesis by increasing both arachidonic acid availability and

5-lipoxygenase activity. GM-CSF also enhances PAF-induced eosinophil accumulation in the lungs of guinea pigs.

Actions of PAF in the Lung Airways

PAF induces contraction of human bronchial smooth muscle in vitro. When inhaled by humans, PAF is a potent bronchoconstrictor but there is a rapid development of tachyphylaxis with repeated doses; the bronchoconstrictor effect is inhibited by Cyst-LT1 receptor antagonists. PAF induces a rapid increase in microvascular leakage throughout the respiratory tract, an effect independent of circulating platelets and of eicosanoid generation. Inhalation of PAF in normal subjects caused an increase in alveolar–arterial oxygen partial pressure gradient and a fall in the arterial oxygen partial pressure, together with ventilation/perfusion mismatching. PAF stimulates the secretion of mucus from tracheal explants, secondary to lipoxygenase activation. PAF increases the protein content of tracheal fluid by inducing plasma extravasation. PAF also induces small changes in ion transport and epithelial conductance. Inhaled PAF impairs tracheobronchial mucociliary clearance in normal subjects. PAF causes an increase in bronchial responsiveness to metacholine in normal subjects and in a variety of animal species, including guinea pig, dog, and sheep; however, asthmatic patients do not demonstrate an increase in bronchial responsiveness after PAF.

Circulating and Bronchovascular Cells

PAF causes a profound neutropenia immediately after inhalation, followed by a rebound neutrophilia. Circulating neutrophils become hypodense within 15 min of inhalation as a result of an increase in cell volume. The falls in circulating blood cell levels could be due to a combination of hemodynamic factors, such as a slowing of pulmonary blood flow and increased adhesiveness of cells to the pulmonary vascular endothelium. Radiolabeled neutrophils transiently accumulate within the pulmonary vasculature after inhalation of PAF. PAF inhalation causes neutrophil influx into the airways of normal volunteers probably through increasing the expression of the b2-integrins, Mac-1 (CD11a/CD18) and LFA-1 (CD11b/CD18) on neutrophils. Intradermal injection of PAF in allergic subjects causes the selective accumulation of eosinophils. PAF induces eosinophilic infiltration of the airways of guinea pigs.

LIPID MEDIATORS / Platelet-Activating Factors Pulmonary Vasculature and Microvascular Permeability

Infusion of PAF in sheep induces an acute transient rise in pulmonary arterial pressure, together with an increase in the lung lymph flow and the lymph-toplasma protein concentration ratio, indicating an increase in pulmonary microvascular permeability. PAF infusion causes pulmonary edema in many lung preparations, an effect that may be dependent on leukotrienes. PAF may mediate pulmonary edema through acid sphingomyelinase-dependent production of ceramide, and activation of the cyclooxygenase pathway particularly through the production of prostaglandin E2. In the rat, low doses of PAF reduced pulmonary hypertension induced by hypoxia, prostaglandin F2a, or norepinephrine. On the other hand, chronic infusion of PAF into rabbits over a 4-week period resulted in pulmonary hypertension, an enlargement of the right ventricle, and internal thickening and a decreased number of small pulmonary arteries.

PAF in Pulmonary Diseases Because of its wide range of effects on cellular function and on the priming of the inflammatory response, PAF has been considered as a mediator of inflammation in many pulmonary diseases, including asthma and allergy, acute respiratory distress syndrome (ARDS), and pulmonary hypertension. Asthma and Allergic Diseases

Because PAF can mimic many of the features of asthma such as bronchoconstriction, bronchial hyperresponsiveness, airway edema, and eosinophil chemotaxis and activation, it has been implicated in this disease. PAF has been measured in plasma and bronchoalveolar lavage fluid from asthmatic patients. An increase in PAF activity derived from acetylation of lyso-PAF and detected using a platelet-aggregating assay has been observed in the plasma of asthmatic patients during the late asthmatic response, but not in those with a single immediate response after antigen challenge. Plasma PAF levels increased immediately after allergen challenge of mild seasonal asthmatic subjects, but not after methacholine challenge. High levels of lyso-PAF have been detected in bronchoalveolar lavage fluid from allergic subjects after allergen challenge, but no significant increase in PAF levels was seen. Studies in mice support a role for PAF in allergic inflammation. Transgenic mice overexpressing PAF receptors demonstrate bronchial hyperresponsiveness to inhaled methacholine. In addition, mice

593

lacking PAF receptor do not develop bronchial hyperresponsiveness following chronic allergen exposure, but have persistent lung inflammation. Recombinant PAF-acetylhydrolase (r-PAF-AH), which converts PAF into inactive lyso-PAF, inhibited allergen-induced bronchial hyperresponsiveness, mucus hypersecretion, and airway eosinophilia in a sensitized murine model. However, no such effects were found in asthmatic subjects undergoing allergen challenge when pretreated with r-PAF-AH. PAF receptor antagonists have been used to probe the role of PAF. They inhibit eosinophil infiltration in the airways and bronchial hyperresponsiveness induced by ovalbumin in sensitized guinea pigs, and to reduce allergen-induced late phase response in allergic sheep and rabbits. However, in allergic asthmatic patients, the PAFWEB 2086 and MK-287 had no significant effects on the late phase response. Overall, while PAF may have a role in animal models of allergic inflammation, it appears to contribute insignificantly in human asthma. This is consistent with a lack of clinical effect of a PAF antagonist in the treatment of mild-to-moderate asthma or of acute exacerbations of asthma. Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a complex clinical syndrome characterized by refractory hypoxemia and high-permeability pulmonary edema in the presence of a normal pulmonary wedge pressure. PAF can cause alveolar-capillary damage, high-permeability pulmonary edema, pulmonary vasoconstriction, and bronchoconstriction. Lungs taken from PAF-treated animals reveal frank damage of endothelial cells. A PAF antagonist inhibited hypoxemia, pulmonary edema, and the increased permeability of the alveolar capillary membrane after endotoxin-induced lung injury in pigs. In overexpressing PAF receptor mice, acid-induced lung injury, lung edema, and hypoxemia were increased, while in mice with PAF receptor gene disruption, these effects were reduced. In a trial of patients with severe sepsis but without established ARDS, r-PAF-AH showed a trend towards prevention of ARDS and death. Pulmonary Vascular Disease

The role of PAF in the regulation of the normal pulmonary circulation remains unclear. Lung homogenates obtained from normal rats contain measurable levels of PAF and PAF is released into bronchoalveolar lavage fluids in rats exposed to hypoxia. High plasma levels of PAF have been detected in persistent pulmonary hypertension in the newborn. In some experiments, hypoxic pulmonary vasoconstriction

594 LIPID MEDIATORS / Lipoxins

can be reversed by exogenous PAF while it can be reduced by PAF antagonist WEB 2086. These conflicting observations are difficult to reconcile.

Conclusion The biosynthesis, metabolism, cellular response, and control of PAF is complex. Its role in normal homeostasis and inflammation remains to be defined. By acting both as an intercellular and an intracellular messenger, it may participate as a cell-to-cell communicator and in so doing allow cooperation of cells in recruitment and activation of leukocytes to inflammatory sites both under physiological or pathophysiological conditions. This would also be a mechanism for priming of inflammation. The fact that no specific beneficial effects in several pulmonary diseases has been observed in humans with inhibition of PAF’s effects does not exclude these important roles for PAF as a mediator of inflammation. A more localized targeted approach on PAF may be required. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Endothelial Cells and Endothelium. Leukocytes: Eosinophils; Neutrophils; Pulmonary Macrophages. Vascular Disease.

Further Reading Benveniste J, Henson PM, and Cochrane CG (1972) Leukocytedependent histamine release from rabbit platelets: the role of IgE, basophils, and a platelet-activating factor. Journal of Experimental Medicine 136: 1356–1377. Chung KF (1992) Platelet-activating factor in inflammation and pulmonary disorders. Clinical Science 83: 127–138. Goggel R, Winoto-Morbach S, Vielhaber G, et al. (2004) PAFmediated pulmonary edema: a new role for acid sphingomyelinase and ceramide. Nature Medicine 10: 155–160. Gomez FP, Marrades RM, Iglesia R, et al. (1999) Gas exchange response to a PAF receptor antagonist, SR 27417A, in acute asthma: a pilot study. European Respiratory Journal 14: 622–626. Henig NR, Aitken ML, Liu MC, Yu AS, and Henderson WR Jr. (2000) Effect of recombinant human platelet-activating factoracetylhydrolase on allergen-induced asthmatic responses. American Journal of Respiratory and Critical Care Medicine 162: 523–527. Ishii S, Nagase T, and Shimizu T (2002) Platelet-activating factor receptor. Prostaglandins and Other Lipid Mediators 68–69: 599–609. Ishii S, Nagase T, Shindou H, et al. (2004) Platelet-activating factor receptor develops airway hyperresponsiveness independently of airway inflammation in a murine asthma model. Journal of Immunology 172: 7095–7102. Kuitert LM, Hui KP, Uthayarkumar S, et al. (1993) Effect of a platelet activating factor (PAF) antagonist UK-74, 505 on allergen-induced early and late response. American Review of Respiratory Disease 147: 82–86.

Montuccio G, Alloatti G, and Camussi G (2000) Role of Plateletactivating factor in cardiovascular pathophysiology. Physiological Reviews 80: 1669–1699. Nagase T, Ishii S, Shindou H, Ouchi Y, and Shimizu T (2002) Airway hyperresponsiveness in transgenic mice overexpressing platelet activating factor receptor is mediated by an atropinesensitive pathway. American Journal of Respiratory and Critical Care Medicine 165: 200–205. Prescott SM, Zimmerman GA, Stafforini DM, and McIntyre TM (2000) Platelet-activating factor and related lipid mediators. Annual Review of Biochemistry 69: 419–445. Rodriguez-Rosin R, Felez MA, Chung KF, et al. (1994) Plateletactivating factor causes ventilation-perfusion mismatch in humans. Journal of Clinical Investigation 93: 188–194. Tjoelker LW and Stafforini DM (2000) Platelet-activating factor acetylhydrolases in health and disease. Biochimica et Biophysica Acta 1488: 102–123. Tokuyama K, Lo¨tvall JO, Barnes PJ, and Chung KF (1991) Mechanism of airway narrowing caused by inhaled platelet-activating factor. Role of airway microvascular leakage. American Reiview of Respiratory Disease 143: 1345–1349.

Lipoxins C N Serhan and B D Levy, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Lipoxins (LXs) are lipoxygenase interaction products of arachidonic acid metabolism that are distinct from leukotrienes and prostaglandins in structure and function. This interesting class of eicosanoids is generated via bidirectional transcellular biosynthesis during multicellular host responses to inflammation, infection, or injury. Similar to other autacoids, LXs are rapidly formed, act locally, and then are rapidly inactivated by specific metabolic enzymes. Unlike the proinflammatory prostaglandins and leukotrienes, LXs display counterregulatory actions. A broad (and growing) array of cell-type specific biological actions has been uncovered for LXs that together serve to promote resolution of acute inflammatory responses. When administered to human cells in vitro or murine systems in vivo, at least two classes of receptors, CysLT1 receptors and LXA4 receptors (designated ALX), can interact with LXs to mediate their actions. LXs are generated during several thoracic inflammatory conditions, including airway and parenchymal lung diseases and exudative pleural effusions. Of interest, respiratory diseases of chronic inflammation, such as severe asthma and cystic fibrosis, display defective generation of these protective lipid signals. Together, these findings indicate a pivotal role for LXs in mediating respiratory homeostasis.

Introduction Enzymatic oxygenation of arachidonic acid occurs in a wide range of human cell types in the lung and results in the formation of several classes of biologically active products, termed eicosanoids because they contain 20 carbons. These compounds are involved in the regulation of inflammatory reactions,

LIPID MEDIATORS / Lipoxins 595

smooth muscle tone, hemodynamic events, allergy, and asthma, as well as many other important physiological and pathophysiological processes. Lipoxygenase (LO)-derived eicosanoids display diverse and potent actions on leukocytes, endothelia, and epithelia. While studying the transformation of [1-14C]arachidonic acid in suspensions of mixed human leukocytes, Charles Serhan and colleagues in 1984 reported that labeled arachidonate was transformed into polar compounds with physical properties distinct from prostaglandins (PGs), thromboxanes, and leukotrienes. Of particular interest was the initial 15-lipoxygenation of arachidonate by activated leukocytes because this enzyme is abundant in the lung and active during airway inflammation. 15-lipoxygenase (15-LO)-derived intermediates were determined to be substrates for further conversion by leukocyte-derived 5-LO to novel derivatives, which were named lipoxins (lipoxygenase interaction products). It is now appreciated that lipoxin (LX) formation occurs in vivo and is conserved across species, from fish to humans. Further investigation has uncovered additional biosynthetic pathways, intriguing biological actions, and routes for metabolic inactivation.

Structure LXs are trihydroxytetraene-containing eicosanoids. 5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid is also called lipoxin A4 (LXA4) and its positional isomer, 5S,14R,15S-trihydroxy-6,10,12trans-8-cis-eicosatetraenoic acid, is denoted lipoxin B4 (LXB4) (Figure 1). The structural features of LXs are ideally suited for spectroscopic methods in their detection. The conjugated tetraene imparts a UV absorption spectrum on both LXA4 and LXB4, with three intense absorbance bands at lMeOH 278, 300, max and 315 nm. A fourth, weaker absorbance band at 270 nm is also present. The most intense absorbance band at 300 nm, with a molar extinction coefficient of 50 000 M
1 cm
1, is typically used for UV quantification. In addition, LXs’ tetraene structure provides fluorescent properties for quantitation with an emission maximum at 410 nm with an excitation maximum at 320 nm at room temperature. Several different extraction and chromatographic methods have been employed for LX isolation from complex sample matrices. The charge state of the LX carboxylic acid can be easily changed to alter the affinity of the compound for chromatography

COOH Airway

Arachidonic acid EPI ALOX15 WBC ALOX5

COOH

O

OH

COOH

LTA4

15(S) - HETE

HO

OH

OH COOH

OH LXA4

COOH

OH HO LXB4

Figure 1 LX generation in airway inflammation. As circulating leukocytes (WBC) traffic into the interstitium and airspaces of the lung, their 5-lipoxygenase activity (WBC ALOX5) can collaborate with airway epithelial 15-lipoxygenase activity (EPI ALOX15) to generate LXA4 and LXB4 during cell–cell interactions. Biosynthetic intermediates, such as 15(S)-hydroxy-eicosatetraenoic acid (15(S)-HETE) from ALOX15 and LTA4 from ALOX5, can serve as substrate for conversion to LXs by the complementary enzyme during transcellular biosynthesis.

596 LIPID MEDIATORS / Lipoxins

stationary phases. For isolation by solid phase extraction, samples are acidified to protonate LXs prior to loading onto a C18 (i.e., octadecyl) cartridge. Following loading, the cartridge is first washed with water to remove more polar moieties, such as salts, and then with hexane to remove more nonpolar materials. LXs are then eluted with a solvent of intermediate polarity, such as methyl formate. Because the tetraene geometry and conformation are fragile, careful handling of LXs is required to prevent cis- to trans-double bond isomerization. In this regard, LXs are thermally labile and sensitive to light. Therefore, they should be kept cold in an organic solvent, such as methanol, and in an environment enriched with nitrogen to prevent non-specific oxygenation. By taking advantage of LXA4’s unique overall three-dimensional conformation, enzyme-linked immunosorbent assays (ELISAs) have been developed for rapid detection of LXA4 as well as stereoisomerspecific detection of 15-epi-LXA4 (vide infra). These ELISAs are commercially available, show no crossreactivity for 5S-HETE, 12S-HETE, 15S-HETE, LTB4, LTC4, LTD4 or arachidonic acid, and have detection limits of approximately 100 fmol ml
1.

Regulation of Production At sites of injury or inflammation, LXs are generated via biosynthetic routes engaged during cell–cell interactions. 15-LO is present in airway epithelial cells, eosinophils, and other leukocytes, and is capable of both initiating LX biosynthesis as well as converting the leukocyte 5-LO-derived intermediate LTA4 to LX. In the lung, mobilization of an LX biosynthetic circuit occurs, for example, when infiltrating polymorphonuclear leukocytes (PMN) (WBC ALOX5, Genbank accession #NP 000689 interact with airway epithelia (EPI ALOX15, Genbank accession #NP 001131) in inflamed target organs (Figure 1). In addition to this 15-LO pathway, LX formation also occurs in the vasculature during platelet–leukocyte interactions by platelet 12-LO-catalyzed conversion of leukocyte LTA4. While human platelets are unable to generate LXs on their own, they can be a major source of LXA4 and LXB4 when adherent to activated neutrophils (PMNs) at sites of vascular injury or inflammation. At both anti-inflammatory and low-dose, aspirin therapy can promote LX biosynthesis. Aspirin-acetylated cyclooxygenase-2 (COX-2) no longer produces PGs, but remains catalytically active in cells to generate the endogenous LX mimetics, 15-epi-lipoxins (15-epi-LXs). These pathways illustrate anti-inflammatory mechanisms that can account for some of aspirin’s beneficial actions. Because LXs and the aspirin-triggered 15-epi-LXs

display leukocyte-selective actions, they are the main candidates for functional roles as endogenous mediators of resolution for acute inflammation. Like other autacoids, LXs are rapidly formed to act locally and rapidly inactivated by metabolic enzymes via pathways shared with other eicosanoids. LXs are primarily inactivated via dehydrogenation converting LXA4 to 15-oxo-LXA4, and LXB4 to 5-oxo-LXB4, followed by specific reduction of the double bond adjacent to the ketone. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of LXA4 and LXB4 to their oxo-metabolites. This compound is biologically inactive and, in the case of 15-oxo-LXA4, further converted to 13,14-dihydro15-oxo-LXA4 by the action of eicosanoid oxidoreductase (EOR) (also known as LXA4/PGE 13,14-reductase/LTB4 12-hydroxydehydrogenase). Reduction of the 15-oxo-group by 15-PGDH yields 13,14-dihydro-LXA4, which reveals an additional catalytic activity for this enzyme. LX dehydrogenation is highly stereospecific, as compared with native LXA4, 15-epi-LXA4 is converted less effectively to the 15-oxo-metabolite. When 15-epi-LXs are generated, their biologic half-life is increased by about twofold greater than LXA4, thereby enhancing their ability to evoke bioactions. LXB4 shares a common route of inactivation with LXA4. LX structural analogs have been constructed with specific modifications of the native structures of LXA4 and LXB4 to block metabolic inactivation by 15-PGDH. Modifications have included synthesis of an LXA4 analog that has a phenoxy group bonded to carbon-16 to replace the o-end of the molecule to protect from dehydrogenation in vivo and addition of halide to the para-position of the phenoxy ring to hinder degradation further. A 15-epi, 16-para-fluorophenoxy-lipoxin A4 analog is active and, like LXA4 and 15-epi-LXA4, signals via competition for endogenous lipoxin A4 receptors (Figure 2). Thus, these modifications to native LXA4 serve to prolong in vivo half-life in the circulation and enhance both the pharmacokinetic and biological properties of the compound. In response to inflammatory stimuli, the in vivo generation of eicosanoid classes is temporally staged with increases in LXA4 levels in experimental exudates that occur later than maximal LTB4 and PGE2, coincident with decreases in exudate PMN during resolution of acute inflammation. The expression and activity of the LX biosynthetic enzymes 5-LO and 15-LO are regulated by inflammatory mediators, including select cytokines and early response lipids (e.g., PGE2) to increase LX biosynthetic capacity at sites of inflammation. During an inflammatory program, early administration of a metabolically stable

LIPID MEDIATORS / Lipoxins 597 HO

OH

O HO

OH

OH

LXA4

COOH X

OH

LXA4 stable analog HO

OH

COOH

COOH

OH 15-epi-LXA4

ALX

G
LSP-1

Cell-type specific responses: Neutrophils − stops adhesion, chemotaxis, transmigration, degranulation Eosinophils − stops chemotaxis, allergen-induced trafficking T-lymphocytes − stops cytokine release, NK cell cytotoxicity Monocytes − stimulates adhesion, chemotaxis, phagocytosis of apoptotic PMNs Epithelial cells − stops cytokine release and promotes bacterial killing Figure 2 ALX signals for cell-type specific responses. LXA4, 15-epi-LXA4, and bioactive LXA4 stable analogs can interact with ALX to regulate leukocyte and nonmyeloid cell functional responses in a cell-type specific manner. ALX signals LX’s anti-inflammatory effects in PMN, in part, by blocking phosphorylation of leukocyte-specific protein-1 (LSP-1) and activation of the p38-MAPK cascade.

LXA4 analog inhibits PMN recruitment, providing evidence for a functional link between LXA4 and cessation of PMN infiltration. Increased LX metabolism by local instillation of recombinant EOR (pivotal to LX endogenous inactivation) dramatically exacerbates PMN infiltration at sites of inflammation. Together, these findings indicate that the onset of LX formation is delayed from PGE2 and LTB4 in acute inflammation and that LXA4 is a regulator of PMN trafficking during the progression of acute inflammatory responses.

Biological Function LXA4, 15-epi-LXA4 and bioactive structural analogs display leukocyte selective actions (Figure 2). This is exemplified by LXA4, which, on the one hand, is a potent stop signal for neutrophil chemotaxis, adhesion, transmigration, azurophilic granule degranulation, and superoxide anion generation, and on the other hand, stimulates monocyte locomotion, adherence, and monocyte-derived macrophage phagocytosis of apoptotic PMNs. Like PMNs, eosinophil chemotaxis and tissue accumulation, dendritic cell mobilization and IL-12 release, T-lymphocyte cytokine release, and natural killer (NK) cell cytotoxicity

are all inhibited by LXA4. In sum, LXs regulate both innate and acquired immune effector cells to promote resolution of acute inflammation. Many nonmyeloid cell functional responses are also regulated by LXs. LXA4 is a potent regulator of proinflammatory cytokine and chemokine release and gene expression via nuclear factor kappa B (NFkB) in fibroblasts and epithelial cells (Figure 2). In addition, LXA4 blocks endothelial P-selectin expression, renal mesangial cell and adenocarcinoma-transformed airway epithelial cell proliferation, and hepatocyte PPARa (peroxisome proliferator-activated receptor a) signaling. Distinct from an immunosuppressive compound, LX signaling in epithelial cells promotes expression of bacterial permeability-inducing protein to enhance mucosal bacterial killing. Thus, in addition to anti-inflammation, LXA4 signals for host protection. These cellular effects have now been investigated in a wide range of experimental animal models through the use of LXA4 stable analogs and generation of transgenic animals that either overexpress 15-LO or utilize a component of the CD11b promoter to target myeloid expression of the human lipoxin A4 receptor (ALX). Outside the lung, LXA4 signaling increases glomerular filtration rate, renal

598 LIPID MEDIATORS / Lipoxins

plasma flow rate, and protects renal tissues from ischemic acute renal failure and glomerulonephritis. In the gastrointestinal tract, LXA4 decreases leukocyte rolling and can promote detachment to lessen mucosal injury from gastric and colonic toxins. LXA4 signaling blocks the development of allergy in a murine model of asthma and promotes resolution of allergic pleural edema. Dermal, ocular, peritoneal, and periodontal inflammation is also potently regulated by LXA4. Increased 15-LO activity also increases LX formation in vivo and in 15-LO transgenic rabbits protects against the development of atherosclerosis and periodontitis. Human ALX transgenic mice display markedly decreased inflammatory responses to zymosan-induced peritonitis and experimental asthma. LX signaling also potently regulates pulmonary responses, including relaxation of precontracted pulmonary arteries and bronchi, prevention of PMN-mediated second organ injury in the lung from hind limb ischemia, and inhibition of both airway hyperresponsiveness and allergic inflammation in experimental asthma (vide infra). These and other findings with LX generation and impact in vivo in the lung are reviewed in Table 1. Table 1 Action of LXs and 15-epi-LXs in the lung – animal models and human diseases Action/impact Animal model Asthma

Microbial infection Pseudomonas aeruginosa infection Angiostrongylus costaricensis infection Human lung diseases

Inhibit airway hyperresponsiveness and allergic inflammation

A novel secreted microbial lipoxygenase

Shorten the duration of pleural exudation LX and 15-epi-LX stimulate host antimicrobial activity LXA4 detected in bronchoalveolar lavage fluids from patients who have pulmonary disease and asthma LXA4 detected in human exudative pleural fluids Production of LX by nasal polyps and bronchial tissue LXA4 inhalation shifts and reduces LTC4-induced contraction in patients who have asthma ASA-intolerant asthmatics display a lower biosynthetic capacity than ASA-tolerant patients LXA4 inhibits IL-8 release by monocytes in patients who have asthma LX generation decreased in severe asthma LX generation defective in cystic fibrosis

LXs have been identified in human subjects with a wide range of illnesses involving leukocyte activation. Of interest, a lower biosynthetic capacity of these potentially protective signals (i.e., LX or 15epi-LX) is linked to more severe or chronic forms of disease, including chronic myelogenous leukemia, chronic liver disease, aspirin-intolerant and severe asthma, and cystic fibrosis. Because of the rapid inactivation of LXs, there is only limited information on their actions in humans in vivo. In one clinical trial, LXA4 was administered to individuals with asthma, leading to protection from LTC4-induced bronchoconstriction. The recent development of new LX stable analogs (Figure 2) that are topically and orally active should enable further investigation on LX regulation of human illness. Together, findings on the biological functions of LXs indicate that altered LX or 15-epi-LX production can regulate both inflammation and organ-specific functions and is mechanistically related to the pathogenesis of human disease.

Receptors The actions of LXs can be attributed to interactions at one or more specific receptors with three potential mechanisms: (1) acting at their own specific receptor; (2) interacting with a subclass of LTD4 receptors (i.e., CysLT1); or (3) acting at intracellular recognition sites after transport and uptake (or within its cell of origin). Specific LXA4 binding sites were first identified on human PMNs. ALX (also known as formyl peptide receptor like-1 and formyl peptide receptor-2) is also expressed in several other mammalian cells and tissues, including human monocytes, enterocytes, synovial fibroblasts, murine spleen and lung, and rat leukocytes. ALX expression is dramatically induced in vitro by cytokines in airway epithelial cells and enterocytes, most notably by interleukin-13 and interferon-g. This cytokine regulation is also evident in vivo during disease states, as ALX is upregulated in airway epithelial and inflammatory cells during allergic airway inflammation in a murine model of asthma that has high levels of IL-13 in the respiratory tract. These functional receptors are 7-transmembrane spanning, G-protein-coupled proteins that bind LXA4 with high affinity (KD ¼ 1.7 nM) (Figure 2). Human, mouse, and rat ALX amino acid sequences share 74% or greater homology with the highest homology found in their second intracellular loop (100% identical) and sixth transmembrane segments (93% identical). As a class, ALX belongs to the cluster of chemoattractant peptide receptors. Of interest for asthma therapy, annexin 1 is induced by corticosteroids and enzymatically cleaved to peptides that,

LIPID MEDIATORS / Lipoxins 599

similar to the lipids LXA4 and 15-epi-LXA4, can interact with ALX to initiate anti-inflammatory circuits. In PMNs, ALX signals for the anti-inflammatory effects of LXs, in part, by polyisoprenyl phosphate remodeling and by inhibition of leukocytespecific protein-1 (LSP-1) phosphorylation, a downstream regulator of the p38-mitogen-activated protein-kinase (MAPK) cascade. ALX was the first receptor to be identified that binds both lipid and peptide ligands. In addition to low-affinity interactions with annexin-1 peptide and fMLP (KDB5 mM), ALX binds several other peptides in the micromolar range, including naturally produced, cleaved forms of the urokinase-type plasminogen activator. The functional consequences of lipid and peptide ligands for ALX differ, probably secondary to distinct intracellular signaling that depends on the cell type and system.

LXs in Respiratory Diseases Both LXs and 15-epi-LXs are generated during respiratory inflammation. LXA4 (0.4–2.8 ng ml
1) has been detected in bronchoalveolar lavage and pleural fluid from individuals with inflammatory lung disease (Table 1). In an experimental model of asthma, LX formation at peak airway inflammation is similar to LTB4, yet one to two log orders less than cysteinyl LTs and PGE2. An LX stable analog, designed using the 15-epi-LXA4 structure as a template, markedly inhibits (with as little as 1 mg; B0.05 mg kg
1) murine allergen-driven airway hyperresponsiveness and inflammation. Targeted expression of human ALX to murine leukocytes in transgenic mice also dramatically inhibits allergen sensitization, eosinophil trafficking, and airway inflammation. With an ED50 of less than 0.05 mg kg
1 in this murine model of asthma, the LX analog compares favorably with both the CysLT1 receptor antagonist montelukast (0.03 mg kg
1 in rats) and the synthetic glucocorticoid dexamethasone (0.5–3 mg kg
1 in mice). Thus, ALX activation by endogenous ligands or an LX stable analog evokes potent stop signals for airway responses. Diminished formation of these counterregulatory mediators has been identified in severe forms of human respiratory illness, including aspirinintolerant asthma, severe steroid-dependent asthma, and cystic fibrosis. Decreased production of these counterregulatory substances may predispose the host to more severe inflammatory responses in the lung. Elucidation of endogenous regulators of LX pathways will provide further insight into the role of LX in inflammatory lung disease and its potential as a new therapeutic strategy that emphasizes natural counterregulatory pathways.

See also: Allergy: Allergic Reactions. Asthma: Overview; Aspirin-Intolerant. CD11/18. Cystic Fibrosis: Overview. Dendritic Cells. Leukocytes: Eosinophils; Neutrophils; Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Overview; Leukotrienes; Prostanoids; Platelet-Activating Factors. NADPH and NADPH Oxidase. Pleural Effusions. Pleural Fluid, Transudate and Exudate.

Further Reading Badr KF, DeBoer DK, Schwartzberg M, and Serhan CN (1989) Lipoxin A4 antagonizes cellular and in vivo actions of leukotriene D4 in rat glomerular mesangial cells: evidence for competition at a common receptor. Proceedings of the National Academy of Sciences, USA 86: 3438–3442. Fiorucci S, Wallace JL, Mencarelli A, et al. (2004) A beta-oxidation-resistant lipoxin A4 analog treats hapten-induced colitis by attenuating inflammation and immune dysfunction. Proceedings of the National Academy of Sciences, USA 101: 15736–15741. Gilroy DW, Lawrence T, Perretti M, and Rossi AG (2004) Inflammatory resolution: new opportunities for drug discovery. Nature Reviews: Drug Discovery 3: 401–416. Godson C, Mitchell S, Harvey K, et al. (2000) Cutting edge: lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. Journal of Immunology 164: 1663–1667. Karp CL, Flick LM, Park KW, et al. (2004) Defective lipoxinmediated anti-inflammatory activity in the cystic fibrosis airway. Nature Immunology 5: 388–392. Levy BD, Bertram S, Tai HH, et al. (1993) Agonist-induced lipoxin A4 generation: detection by a novel lipoxin A4-ELISA. Lipids 28: 1047–1053. Levy BD, Clish CB, Schmidt B, Gronert K, and Serhan CN (2001) Lipid mediator class switching during acute inflammation: signals in resolution. Nature Immunology 2: 612–619. Levy BD, De Sanctis GT, Devchand PR, et al. (2002) Multipronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A(4). Nature Medicine 8: 1018–1023. Ohira T, Bannenberg G, Arita M, et al. (2004) A stable aspirintriggered lipoxin A4 analog blocks phosphorylation of leukocyte-specific protein 1 in human neutrophils. Journal of Immunology 173: 2091–2098. Perretti M, Chiang N, La M, et al. (2002) Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor. Nature Medicine 8: 1296–1302. Samuelsson B, Dahlen SE, Lindgren JA, Rouzer CA, and Serhan CN (1987) Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science 237: 1171–1176. Serhan CN and Chiang N (2004) Novel endogenous small molecules as the checkpoint controllers in inflammation and resolution: entree for resoleomics. Rheumatic Diseases Clinics of North America 30: 69–95. Serhan CN, Fiore S, Brezinski DA, and Lynch S (1993) Lipoxin A4 metabolism by differentiated HL-60 cells and human monocytes: conversion to novel 15-oxo and dihydro products. Biochemistry 32: 6313–6319. Serhan CN, Jain A, Marleau S, et al. (2003) Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15lipoxygenase and endogenous anti-inflammatory lipid mediators. Journal of Immunology 171: 6856–6865. Serhan CN, Maddox JF, Petasis NA, et al. (1995) Design of lipoxin A4 stable analogs that block transmigration and adhesion of human neutrophils. Biochemistry 34: 14609–14615.

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LUNG ABSCESS H Koziel, Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Lung abscess is a clinical condition characterized by focal suppurative necrosis of lung parenchyma. The incidence of lung abscess is unknown. Current concepts suggest that most lung abscesses occur as a complication of aspiration pneumonia and are often attributed to anaerobic organisms from the gingival crevices of the oral cavity. Patients typically present with indolent onset of fever, malaise, night sweats, weight loss, cough, pleurisy, and purulent sputum production, of several weeks duration and often with an antecedent history of loss of consciousness or aspiration. The chest radiograph typically demonstrates parenchymal radiolucency with surrounding thick wall, or a radiolucency with an air–fluid level, and may require chest CT to confirm the diagnosis. Anaerobic bacteria frequently account for the majority of community-acquired lung abscess cases (often polymicrobial), whereas anaerobes may account for most cases of hospital-acquired lung abscess (often a single pathogen). Evaluation of sputum is controversial and blood cultures are rarely positive, so isolating the etiological agent often requires invasive techniques. Treatment includes prolonged antimicrobial therapy, and in general, the prognosis is favorable. Surgical resection is reserved for patients with poor clinical responses. Techniques of percutaneous or endobronchial drainage may be effective alternatives for select patients with poor clinical responses who are considered high surgical risk.

Introduction Lung abscess is a suppurative process often considered in the spectrum of anaerobic pleuropulmonary diseases that include aspiration pneumonia, necrotizing pneumonia, lung abscess and empyema. As abscesses can be of varied sizes, the distinction between necrotizing pneumonia (or pulmonary gangrene) and lung abscess is somewhat arbitrary as these entities represent a progression along a continuum of lung infections. Generally, lung abscess is often defined as X2 cm in diameter whereas abscesses o2 cm are considered necrotizing pneumonia. Lung abscess has been recognized since the ancient Greek times of Hippocrates. Anaerobic bacteria were first identified in empyema fluid in 1899, and subsequently determined in 1927 to be a critical component of lung abscess formation based on experimental animal data. The role of aspiration was appreciated in 1934 with the association of high rate of lung abscess complicating head and neck surgery performed in the upright sitting position. The benefit of drainage of lung abscesses was realized in 1945,

and the introduction of penicillin was associated with an apparent reduction in the incidence (especially in persons o50 years of age) and mortality. The major role of anaerobes in lung abscess formation was confirmed during the 1970s through investigations using newly developed laboratory culturing techniques that allowed for improved isolation and culturing of anaerobic bacteria. The incidence of lung abscess is not known, although lung abscesses likely develop relatively infrequently and the incidence appears to have decreased since the introduction of antibiotics (especially penicillin). From 1943 to 1956, the Massachusetts General Hospital reported 10–11 cases of lung abscess per 10 000 hospital admissions in the pre-antibiotic era compared to 1–2 cases per 10 000 admissions in the post-antibiotic era. The Beth Israel Deaconess Medical Center’s experience during 1984–96 showed that lung abscesses represented approximately 0.2% of all cases of pneumonia requiring hospitalization. The decrease in cases of lung abscess is primarily attributed to early and broad use of effective antimicrobials, improved management of hospitalized unconscious patients, and improved management of patients undergoing anesthesia. Patients with lung abscess typically present with indolent onset of fever, malaise, night sweats, weight loss, cough, pleurisy, and purulent sputum production, of several weeks duration and often with an antecedent history of loss of consciousness or aspiration. The chest radiograph typically demonstrates parenchymal radiolucency with surrounding thick wall, or a radiolucency with an air–fluid level, and may require chest CT to confirm the diagnosis (Figure 1). Mortality for untreated lung abscess may approach 40%.

Etiology Anaerobic bacteria are considered the most common pathogens in community-acquired lung abscess, reflecting both pathogenic potential and representing the predominant component of normal flora of the upper airways (although any pathogen can create a lung abscess under the appropriate circumstances). Prior studies using transtracheal aspirates or transthoracic needle aspirates reported recovery rates of anaerobic bacteria from 85% to 93% of lung abscess cases. Anaerobes were the only isolates in up to 46% of cases, whereas 43% of cases had a mixture of aerobes and anaerobes (Table 1). Although anaerobic abscesses generally contain multiple anaerobic

LUNG ABSCESS 601 Table 1 Reported microbiology of lung abscess Anaerobic organisms (common)a Fusobacterium nucleatum Peptopstreptococcus species Prevotella melaninogenica Bacteroides species Clostridium species Eubacterium species Lactobacillus Propionibacteria Aeorobic organisms (common) Staphylococcus aureus Streptococcus pyogenes Klebsiella pneumoniae Pseudomonas aeruginosa Aeorobic organisms (uncommon) Escherichia coli Haemophilus influenzae type B Other pathogens (rare) Nocardia asteroids Paragonimus westermani Legionella species Burkholdaria pseudomallei Burkholdaria mallei (glanders) Mycobacterium tuberculosis Mucoraceae species Aspergillus species Entameoba histolytica

Figure 1 Radiological demonstration of lung abscess development in a patient with necrotizing Staphylococcus aureus pneumonia. A 47-year-old HIV þ male admitted to the medical intensive care unit following an acute drug overdose and loss of consciousness. (a) Admission anteroposterior chest radiograph demonstrated a dense RUL infiltrate, and bronchoscopy with quantitative BAL revealed Staphylococcus aureus; (b) chest CT scan on day 14 of hospitalization confirmed the interval development of a cavitary lesion in the posterior segment of the RUL despite appropriate antimicrobials.

isolates, occasionally lung abscesses may result from a single anaerobic organism recognized as highly virulent, such as Fusobacterium nucleatum or Peptopstreptococcus species. Anaerobes may also be in combination with aerobic or facultative anaerobic species such as microaerophilic streptococci. In contrast to community-acquired lung abscess, aerobic bacteria may be a more important etiological agent in hospital-acquired lung abscess. Nosocomial aspiration is often associated with Gram-negative bacteria and Staphylococcus aureus, including organisms with hospital-acquired antibiotic resistance patterns. Furthermore, the spectrum of microbiological agents responsible for lung abscess in

Organisms associated with lung abscess in immunocompromised hosts (including HIV) Pseudomonas aeruginosa Streptococcus pneumonia Pneumocystis jiroveci Klebsiella pneumoniae Staphylococcus aureus Haemophilus influenzae Stenotrophomonoas maltophilia Legionella species (nonpneumophilia) Enterobacter species Streptococcus milleri Proteus mirabilis Cryptococcus neoformans Aspergillus species Mycobacteria (nontuberculous) Nocardia Rhodococcus Zygomycetes a

Mouth flora.

immunocompromised hosts may be quite different and distinct from that of immunocompetent patients. This is evident in the study of the Beth Israel Deaconess Medical Center’s experience during 1984–96 in 34 cases of lung abscesses in adults. Aerobic bacteria were the most common isolates in both immunocompromised and non-immunocompromised patients. However, immunocompromised patients more often had multiple pathogens, and certain

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pathogens were exclusively isolated from the lung abscess of immunocompromised patients, including Pseudomonas aeruginosa, and Hemophilus spp. (non-influenzae), Stenotrophomonas maltophilia, Haemophilus influenza, Enterobacter spp., Klebsiella oxytoca, nonpneumophilia Legionella spp., Mycobacterium avium complex and Candida spp. Anaerobes were isolated in only 20% of the cases of non-immunocompromised patients, and not isolated in any specimens from immunocompromised patients. Other reports of lung abscess in HIV þ patients demonstrate bacteria in 65% of isolates, followed by fungi (9%), and mixed infections (16%), although despite effective treatment, high relapse (36%) and mortality (19%) rates were experienced. More recent data suggest that the microbiology of lung abscess may be evolving, perhaps in part in response to external pressures induced by the indiscriminate use of broad-spectrum antimicrobials, development of bacterial resistance, presence of multiple or complex medical comorbidities in patients, and the expanding population of immuosuppressed individuals. As the microbiology of communityacquired pneumonia changes (e.g., the expanding role of methicillin-resistant Staphylococcus aureus), the agents responsible for community-acquired lung abscess are likely to change. Although certain genetic diseases that result in chronic lung disease (e.g., cystic fibrosis, Kartangener’s syndrome) or chronic infection (e.g., Mycobacterium tuberculosis) may predispose to lung abscess formation, there are no known specific genetic markers that identify persons at risk for the development of lung abscess.

Pathology Lung abscess is defined as a pus-containing necrotic lesion of the lung parenchyma. The cardinal histologic change in all abscesses is suppurative destruction of the lung parenchyma within the central area of cavitation. Lung abscess can be of any size, from a few millimeters to several centimeters, often manifest as a single lesion (e.g., as a complication of aspiration pneumonia), but can develop multiple foci (e.g., as a complication of right-sided endocarditis). Lung abscesses are often infected and generally develop after inflammation and produce tissue necrosis with cavitation. In the absence of communication with an airway, the abscess cavity may be filled with suppurative debris. However, when the abscess cavity communicates with an airway, the contents may be partially drained to create an air-containing cavity. In cases of pre-existing cavitary lung disease (such as that associated with emphysema or prior

Table 2 Predisposing conditions or risk factors to development of lung abscessa Primary lung abscessb Alcoholism Seizure disorder General anesthesia Drug abuse Esophogeal lesions Neurological deficits (e.g., CVA, bulbar disease) Insulin-treated diabetes mellitus Secondary lung abscessc Pulmonary embolism with infarction Septic emboli with pulmonary infarction Endobronchial obstruction by neoplasm (benign or malignant) Obstruction by foreign body Bronchiectasis Cryptogenic lung abscess Absence of identifiable risk factor a

10–15% of patients with lung abscess may not have obvious risk factor. b Symptoms often indolent over weeks. c Symptoms often abrupt over days.

Mycobacterium tuberculosis infection), infection of the cavity may proceed without overt necrosis. The abscess cavity may become lined with regenerated lung epithelium, and in cases of chronic lung abscess, fibroblastic proliferation may result in a fibrinous wall. Early autopsy observations that bacteria in the walls of the lung abscess at autopsy were similar to the bacteria found in the oral gingival crevice (between the tooth and gum tissue) prompted investigators to hypothesize that aspiration was the major mechanism in disease pathogenesis. Acute lung abscess is arbitrarily defined as o4 weeks duration, whereas chronic lung abscess is 44 weeks duration, although the clinical implications of this distinction are not clearly established. Abscess formation as a consequence of aspiration and pneumonia in a relatively normal host are defined as primary lung abscess (Table 2). In contrast, abscess formation as a consequence of central airway obstruction, bronchiectasis, immunocompromised state, or due to hematogenous spread from a distant site is defined as secondary lung abscess. Cases with no recognized or identifiable risk factors are termed cryptogenic lung abscesses. Approximately 80% of cases of lung abscesses are primary lung abscess.

Clinical Features Lung abscess is suspected from the history and physical exam, but requires radiographic confirmation. Clinical suspicion for lung abscess should be heightened in persons with a recognized pre-existing

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condition that increases risk for development of lung abscess (Table 2). In primary lung abscess, symptoms of lung abscess are similar to other anaerobic lung infections, and include indolent (1–3 weeks) development of fever, cough, pleurisy, dyspnea, sweats, weight loss, and sputum production (often foulsmelling). Sputum production may be absent in the absence of a communication of the abscess cavity and the airways. Most patients present with fever (median T ¼ 39.11C) and leukocytosis (median peripheral WBC ¼ 15 000 cells mm
3). The production of putrid sputum occurs in approximately 49% of the cases, and is generally considered diagnostic of anaerobic infection (as aerobic bacteria are generally not capable of producing this characteristic odor). In patients with chronic sputum production (such as bronchiectasis or chronic bronchitis), a change in the character, quality, or quantity of sputum should raise suspicion of a new lung infection or development of lung abscess. In secondary lung abscess, the development of symptoms may be more rapid, evolving over days, and may include localized symptoms that provide a clue to the underlying condition predisposing to rapid lung abscess formation (such as endobronchial tumor or endocarditis with multiple septic emboli). Radiographic appearances include a radiolucency surrounded by a visible irregular thick wall, or an air–fluid level within an area of pneumonia. Chest radiographs generally reveal cavity formation distributed in dependent lung segments. In recumbent patients, these include the superior segments of the lower lobes and posterior segments of the upper lobes. In patients who aspirate in the upright position, the basilar segments of the lower lobes are favored. For all patients, the right lung is more often involved in aspiration events due to the more direct vertical takeoff of the right mainstem bronchus compared to the angulated takeoff of the left mainstem bronchus. A solitary lung cavity is generally associated with primary lung abscess, whereas multiple small cavities suggest secondary lung abscess associated with hematogenous spread of infection. Peripheral parenchymal lung abscess near the chest wall may require chest CT to distinguish an abscess from a loculated empyema with bronchopleural fistula. Chest CT scan is more sensitive than a routine chest radiograph, and may detect small cavities, demonstrate obstructing endobronchial lesions, or distinguish lung abscess from air–fluid levels in the pleural space. Cavitary lung lesions may represent lung abscess, but also represent noninfectious causes of cavitary lung lesions including pulmonary infarction, neoplasm, septic emboli, vasculitis, bullae or cystic

bronchiectasis, pulmonary sequestration, or sarcoidosis. These conditions represent conditions that may become secondarily infected with the subsequent development of a lung abscess. Complications of lung abscess include extension into the pleural space, hemorrhage, and septic emboli (brain abscess or meningitis). In an aspiration-prone patient with gingival disease presenting with a subacute illness, foul-smelling sputum, and a cavitary lesion on chest radiograph, a putative diagnosis of anaerobic lung abscess can be made without further microbiological studies. In other cases, the major challenges to establishing the bacterial diagnosis of lung abscess requires sampling the cavity without contamination by other oropharyngeal microbes (to distinguish pathogenic from colonizing bacteria) and sampling prior to the administration of antimicrobials (which may reduce the ability to cultivate bacteria from lung specimens). Generally, most patients receive antecedent antimicrobials as treatment for a pneumonitis, prior to the development of lung abscess. Techniques used to obtain relatively uncontaminated specimens include transtracheal aspirates (rarely used in current times) or quantitative cultures following bronchoalveolar lavage or protected brush specimens using fiberoptic bronchoscopy. Quantitative culture of lower respiratory specimens using bronchoscopy may improve the diagnostic yield for aerobes, whereas the detection of anaerobic bacteria by these methods is less well studied. In addition to sampling techniques, proper specimen storage, rapid transportation, and appropriate processing by the clinical laboratory are essential to optimizing isolation of etiological agents, especially anaerobes. As laboratory isolation of pathogens and in vitro sensitivity testing may require several days, empirical antimicrobial selection is necessary for initial treatment in patients with lung abscess. Gram-stain of expectorated sputum may provide initial guidance to the selection of antimicrobials, based on characteristic findings of polymicrobial flora or the unique morphological features of certain Gram-negative anaerobic organisms and may provide clues as to the presence of anaerobic infection. Furthermore, selection of empirical antimicrobials should also consider the environmental exposures and immune status of the host, and whether the abscess is primary or secondary.

Pathogenesis Aspiration of oropharyngeal contents is considered the main mechanism for the development of lung abscess (Figure 2). The association of peridontal

604 LUNG ABSCESS Pathogenesis of lung abscess Aspiration of oral flora (especially anaerobes)* Large inoculum Altered host defense Aspiration pneumonia Pathogen virulence factors Anatomical abnormalities Necrotizing pneumonia

Lung abscess * Increased risk in persons with peridontal disease and predisposing factor for aspiration Figure 2 Predominant mechanism for the development of lung abscess.

disease with lung abscess formation underscores the importance of anaerobic pathogens and aspiration. The bacteria implicated in anaerobic lung infections represent the normal flora of the oral cavity, and concentrations of anaerobic bacteria in the gingival crevice may approach 1012 organisms per gram, especially in the context of gingivitis. Lung abscess is rare in edentulous persons. Although aspiration of oropharyngeal materials may occur in 45% of healthy individuals during sleep, these occult aspirations may be of relatively small volume and normal host defense and clearance mechanisms allow spontaneous resolution preventing the development of clinical disease. However, clinically significant aspiration of these organisms occurs in persons with compromised consciousness or dysphagia. Predisposing conditions require compromised host defense mechanisms that protect the lower airways (such as cough, glottic closure, or mucocilliary clearance), and an infectious inoculum sufficient to induce disease (such as a large inoculum that overwhelms defenses, induction of a host inflammatory response, or direct toxicity to the host) or a combination of these factors. The decisive factor for the development of lung complications following aspiration may depend on the frequency of aspiration events, volume of the aspirate, and character of the aspirated inoculum. Conditions that promote stasis or necrosis of tissue also predispose to anaerobic lung abscess including pulmonary infarction, obstruction due to endobronchial carcinoma, obstruction due to foreign body, and bronchiectasis. Aspiration of substances such as gastric acid pHo 2.5 (Mendelson’s syndrome), milk and mineral oil, and volatile hydrocarbons are directly toxic to the lower respiratory tract. The inflammatory process

induced by these substances (especially acid) may predispose to bacterial superinfections and result in abscess formation. Based on serial radiographic studies in patients following a defined period of aspiration, lung cavity formation was observed in 7–14 days. Anaerobic virulence factors promote abscess formation. The propensity for tissue necrosis by anaerobic bacteria may relate in part to a specific family of capsular polysaccharide of anaerobic Gram-negative bacilli. Specific repeating oligosaccharides containing sugars with positively charged free amino acid groups and negatively charged carboxyl or phosphate groups mediate abscess formation in experimental animal models. In addition, metabolic production of short-chain fatty acids by anaerobic bacteria (which may be responsible for the putrid odor of anaerobic infections) may inhibit phagocytosis and bacterial killing by host cell at low pH levels. The pathogenesis of lung abscess is in part dependent on the nature of the specific organism. For example, amoebic lung abscess formation generally involves the right lower lobe by direct extension of the liver abscess through the diaphragm.

Animal Models Development of a reliable animal model for lung abscess has been elusive. Validation of the hypothesis that aspiration of oral bacteria was the major mechanism for the development of lung abscess was demonstrated in the pre-antibiotic era by Smith in 1927. Inoculating the trachea of rabbits with human gingival crevice material resulted in pneumonitis followed by the development of lung abscess formation in 7–10 days. The bacteriologic studies of the gingival crevice inoculum revealed predominantly anaerobic bacteria, including Fusobacterium nuclatum, Prevotella melaninogenica, Peptostreptococcus, and an anaerobic spirochete. Interestingly, lung abscess formation required a combination of anaerobes, as lung abscess formation did not occur when animals were inoculated individually with these anaerobic bacteria. In 1929, Varney’s inoculation of human abscess material into experimental dogs produced lung abscess, although the microbiology of the inoculum was not fully defined. More recent experiments using rabbits inoculated with well-characterized laboratory anaerobic bacterial isolates suggested an important role for Bacteroides fragilis, although the nature of the stored bacteria may not be clinically relevant. Murine models of lung abscess using Porphyromonas gingivalis and Treponema denticola are associated with a high mortality and thus of limited value.

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Management and Current Therapy Antibiotics represent the main component of treatment for lung abscess (Table 3). However, most published trials of antimicrobials in the treament of lung abscess have been relatively small. Penicillin represented the main agent during 1950–75 as most patients responded to it positively, whereas patients who failed to respond were treated with tetracycline. More recent data indicate that approximately 15–25% of anaerobic bacteria responsible for lung infections are resistant to penicillin, generally due to penicillinase production. Penicillin resistance is of particular importance with infections by Fusobacteria, Prevotella, and Bacteroides species, where 40–60% of these organisms may produce b-lactamases. In small randomized trials, clindamycin is superior to penicillin in terms of clinical response rates, time to defervescence, radiological improvement, clearance of sputum, and relapse rates. Metronidazole monotherapy for the treatment of anaerobic lung infections is associated with poor response in 50% of patients (possibly due to the presence of aerobic or microaerophilic streptococci), but may be effective in combination with penicillin. Other regimens are limited to anecdotal experience or efficacy in treatment of anaerobic infections at other anatomical sites, including amoxicillin–clavulanate, a combination of b-lactam with a b-lactamase inhibitor, chloremphenicol, imipenem, cefoxitin, and cefotetan. The efficacy of macrolides in the treatment of anaerobic lung infections has not been established. Vancomycin has effect against Gram-positive anaerobes. Aminoglycosides, quinolones, aztreonam, and trimethoprim–sulfamethoxazole Table 3 Recommended antimicrobial treatment regimens for lung abscessa Recommended treatment for community-acquired lung abscess Clindamycin, or 300–600 mg every 8 h Penicillin G, plus 1 gm every 8 h Metronidazoleb 500 mg every 8 h Suggested alternative treatments Combination penicillin/b-lactamase inhibitors Combination b-lactam/b-lactamase inhibitors Carbapenems Quinilones (moxifloxacin, gatafloxacin) Recommended treatment for hospital-acquired lung abscess Agents directed at aerobes and facultative bacteria, with particular regards to major institutional pathogens (especially Staphylococcus aureus, Pseudomonas, and Enterobacteriaceae). a Recommended 3–6 weeks duration, or until constitutional symptoms are resolved and radiographic appearance of abscess is resolved or stable. b Possible disulfiram-like reaction in persons with chronic alcoholism.

have no significant activity against anaerobes and are generally not recommended. The need for drainage of abscess is in part dependent on the clinical response. The absence of an air–fluid level suggests the absence of communication with a bronchus, and may require drainage. Radiological-guided percutaneous catheter drainage of lung abscess may be associated with contamination of the pleural space and associated morbidity and mortality. However, a series of small reports suggests that percutaneous drainage may be used effectively in selected patients. Although bronchoscopy is useful in defining unanticipated endobronchial findings or the development of bacterial resistance in cases of poorly resolving lung abscess, the role of bronchoscopic drainage is controversial as clinical outcomes are not necessarily improved, and complications of spread of infection to other areas of the lung, development of ARDS, and death, limit the enthusiasm for this intervention. Recent data suggest that endobronchial drainage combined with catheter-instilled antimicrobials may represent a viable option for select patients with a nonresolving lung abscess. In general, most patients with lung abscess improve with medial management including antimicrobial therapy alone, with collapse of the abscess in approximately 90% of patients. Patients generally defervesce in 7–10 days. Radiographic improvement lags behind clinical improvement, and cavity closure can occur in a median of 4 weeks, but may take several months. The role of chest physiotherapy, postural drainage, and bronchodilators has not been established. Antibiotics reduce the overall mortality from lung abscess to o10% (primary or communityacquired lung abscess mortality of approximately 2%) and reduce the pulmonary complications such as recurrent infections, recurrent abscess, pleural empyema, chronic bronchitis, and bronchiectasis. The prognosis for special populations such as HIV þ patients with lung abscess remains worse despite effective treatment and is associated with high relapse rates (36%) and high mortality (19%). As lung abscesses generally drain spontaneously, surgical resection may only be necessary in 10–15% of cases, including cases of failed medical therapy, rapidly progressive infection, abscess due to an obstructing endobronchial lesion, large lung abscess (48 cm), abscess due to resistant organisms (e.g., Pseudomonas aeruginosa), infarcted lung, poorly functioning lung, or in cases of rapidly progressive lung abscess formation in a compromised host. Surgical intervention generally requires lung resection, lobectomy, and less often, pneumonotomy. Lung abscess can be complicated by empyema in approximately 30% of cases. Increased mortality

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from lung abscess is associated with advanced age, systemic disability, immunocompromised state, abscess due to aerobic pathogens, large abscess (45 cm), progressive lung necrosis, presence of obstructing lesion, and delays in seeking medical attention. Mortality may exceed 65% in cases of lung abscess associated with obstructing airway lesions, impaired host defenses, or nosocomial acquisition. Furthermore, approximately 10–15% of cases of lung abscess may be associated with malignancy. See also: Bronchiectasis. Pleural Effusions: Parapneumonic Effusion and Empyema. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens. Systemic Disease: Sarcoidosis. Upper Airway Obstruction. Upper Respiratory Tract Infection. Viruses of the Lung.

Further Reading Bartlett JG (1991) Antibiotics in lung abscess. Seminars in Respiratory Infections 6: 103–111. Bartlett JG (1993) Anaerobic bacterial infections of the lung and pleural space. Clinical Infectious Diseases 16: S248–S255. Bartlett JG, Gorbach SL, Tally FP, et al. (1974) Bacteriology and treatment of primary lung abscess. American Review of Respiratory Diseases 109: 510–518. Finegold SM and Fishman JA (1998) Empyema and lung abscess. In: Fishman AP (ed.) Fishman’s Pulmonary Diseases and Disorders, vol. 1, pp. 2021–2033. New York, NY: McGraw-Hill.

Fraser RG, Pare JAP, Pare PD, Fraser RS, and Genereux GP (1988) Diagnosis of Diseases of the Chest, 3rd edn., vol. 1, pp. 458– 684. Philadelphia, PA: Saunders. Furman AC, Jacobs J, and Sepkowitz KA (1996) Lung abscess in patients with AIDS. Clinical Infectious Diseases 22: 81–85. Griffiths JK and Snydman DR (1994) Anaerobic pleuropulmonary infections: aspiration pneumonia, abscess, and empyema. In: Niederman MS, Sarosi GA, and Glassroth J (eds.) Respiratory Infections: A Scientific Basis for Management, pp. 345–357. Philadelphia, PA: Saunders. Hammond JMJ, Potgieter PD, Hanslo D, et al. (1995) The etiology and antimicrobial susceptability patterns of microorganisms in acute community acquired lung abscess. Chest 108: 937–941. Herth F, Ernst A, and Becker HD (2005) Endoscopic drainage of lung abscesses: technique and outcome. Chest 127: 1378–1381. Hirshberg B, Sklair-Levi M, Nir-Paz R, et al. (1999) Factors predicting mortality of patients with lung abscess. Chest 115: 746–750. Husain AN and Kumar V (2005) The lung. In: Kumar V, Abbas AK, and Fausto N (eds.) Robbins and Cotran Pathologic Basis of Disease, pp. 711–772. Philadelphia, PA: Elsevier Saunders. Johnson CC and Finegold SM (1988) Pyogenic bacterial pneumonia, lung abscess and empyema. In: Murray JF and Nadel JA (eds.) Textbook of Respiratory Medicine, pp. 803–855. Philadelphia, PA: Saunders. Lorger B (2005) Lung abscess. In: Mandell GL, Bennett JE, and Dolin R (eds.) Principles and Practice of Infectious Diseases, pp. 853–857. New York, NY: Churchill Livingstone. Mansharamani NG, Balachandran D, Delaney D, et al. (2002) Lung abscess in adults: clinical comparison of immunocompromised to non-immunocompromised patients. Respiratory Medicine 96: 178–185. Mansharamani NG and Koziel H (2003) Chronic lung sepsis: lung abscess, bronchiectasis and empyema. Current Opinion in Pulmonary Medicine 9: 181–185.

LUNG ANATOMY (INCLUDING THE AGING LUNG) R Harding, Monash University, Melbourne, VIC, Australia K E Pinkerton, University of California, Davis, CA, USA & 2006 Elsevier Ltd. All rights reserved.

and basement membrane deposition with focal increases in the interstitium also appear in the normal aging process, while the metabolic function of the lungs decreases with age. Environmental factors are likely to influence changes during the aging process that may be associated with asthma, emphysema, and chronic obstructive pulmonary disease.

Abstract

Introduction

The respiratory system undergoes a continual process of change with age. A number of similarities have been noted in the mouse, rat, dog, and human with distension of alveolar ducts immediately beyond terminal and respiratory bronchioles. This enlargement is typically observed in the form of ductasia with alveoli becoming stretched and shallow in affected regions. Although destruction of alveolar walls may occur with age in the normal lung, these changes reflect in large measure an increase in the size and frequency of alveolar pores connecting adjacent alveoli. An increase in the number of alveolar macrophages in the lungs is a common finding with aging. Increased collagen

This article briefly reviews basic lung anatomy and changes associated with normal aging. The focus will be the human lung, but data are also presented from the mouse, rat, and dog. Within these species there are many structural features in aging that are common. We will consider separately the gas-exchanging regions of the lung (alveoli) and the conductive structures (bronchial tree) as both contribute to lung function.

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General Lung Structure in Humans Over 40 different cell types form the epithelial, interstitial, and vascular compartments of the lungs. The epithelium is composed of secretory cells (such as mucous, serous, Clara, and type II) interspersed among a wide variety of other epithelial cells lining the airways (ciliated, basal, brush, neuroendocrine, and Kulchitsky) and airspaces (alveolar type I and type II), each with uniquely different functions. Interstitial cells (fibroblasts, smooth muscle, myofibroblasts, and chondrocytes) create the essential scaffolding to give form and shape to the airways and parenchyma, while also creating a conduit for nerves and lymphatics as well as trafficking of cells (monocytes, macrophages, lymphocytes, neutrophils, mast cells, eosinophils, and basophils) and substances to and from the blood and airspaces. The vascular compartment of the lung allows for the flow of blood from the heart, while also creating a portion of the thin tissue partition (endothelial cells and pericytes) in the alveoli that allows efficient gas exchange. The lung parenchyma is composed of alveoli, alveolar ducts, and alveolar sacs; this gas-exchanging portion of the lungs occupies 80–90% of the total lung tissue volume. Alveoli (B300 mm in diameter) are the smallest anatomical units involved in gas exchange and are composed of the airspace bounded by the alveolar wall and its opening into the alveolar duct. The alveolar duct is formed by the airspace shared in common with alveoli opening along a common channel created by tissue ridges forming the mouths of individual alveoli. The branching of alveolar ducts to form discrete alveolar duct generations begins at the bronchiole–alveolar duct junction and ends three to five generations away as a blind alveolar sac formed by several alveoli. The highly vascular alveolar gas–tissue interface creates a surface area (B70 m2 in an adult male) which is B25-fold greater than that of the body. This tissue barrier separates the alveolar gas from blood by less than 1 mm (Figure 1). A highly ordered branching of about 6–23 generations of airways (depending on which airway path is followed), beginning with the trachea, gives rise to millions of alveoli which line several generations of alveolar ducts and sacs to bring about this enormous increase in surface area. The typical male human lung contains B300 million alveoli. In humans, alveoli start being formed before birth but 70–80% are formed after birth. Alveolarization is thought to be complete by 8–10 years of age, after which lung growth occurs by enlargement of existing structures. During adolescence, the lungs continue to expand as the thoracic cavity enlarges; by age 18, lung growth is considered to be complete.

II C

I

Figure 1 The alveolar septum is composed of epithelial type I and II cells, interstitial components, endothelium, and a capillary (c) bed to form a highly efficient gas-exchange tissue barrier. Scale bar is 3 mm. Reproduced with permission from Pinkerton KE, Barry BE, O’Neil JJ, et al. (1982) Morphologic changes in the lung during the life span of Fischer 344 rats. American Journal of Anatomy 164: 155–174.

With further aging the number of alveoli may be reduced through a destructive process, with loss of alveolar septal wall surface area, and/or the rearrangement of essential extracellular matrix components such as collagen and elastin. These processes cause the alveoli to become stretched and shallow leading to distention of alveolar ducts referred to as alveolar ductasia.

Functional Changes in the Human Respiratory System with Age In humans, maximal lung function is reached at B20 years in females and 25 years in males. After this, lung function progressively declines even in the absence of disease, but remains able to meet respiratory requirements. The major functional changes are an increase in lung compliance (0.1–0.2 cmH2O/year), a decrease in chest wall compliance (increased stiffness), and reduction in respiratory muscle strength. Lung Function

Due to increased lung compliance and decreased chest wall compliance with age, residual volume (RV) increases by B50% between 20 and 70 years; functional residual capacity (FRC) also increases, but total lung capacity (TLC) does not typically change. The changes in RV and FRC are partly attributable to gas trapping, and result in the elderly breathing at a higher lung volume and hence chest wall expansion, increasing the work of breathing. Dynamic airway compression increases with age, which may be due in part to a progressive loss of tethering of small airways by alveoli; hence the airway closing volume increases with age. Closing volume may reach FRC, resulting in many peripheral airways not being ventilated and hence not contributing to gas exchange.

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The consequent reduction in the regional ventilation–perfusion ratio leads to a fall in arterial PO2, an increase in AaDO2, and a reduction in pulmonary diffusing capacity. Forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) increase with age in both males and females up to early adulthood, after which they progressively decline. In females and males, the peak values are reached at B20 and B27 years respectively, after which the rate of decline in FEV1 is greater in males than females, and greater in those with greater airway reactivity. Age-related changes in the expiratory flow–volume relationship provide evidence of an increase in small airway obstruction with age; similarly, peak expiratory and inspiratory flow velocities decline with age. In relation to lung volume, airway resistance does not decline with age; this may be due to the fact that the resistance of small airways is not reflected in total airway resistance. Pulmonary gas exchange declines with age, apparently due to poor matching of regional ventilation (V) and perfusion (Q); in particular, dependent regions of the lung are relatively underventilated due to increased airway closing volume. This increased heterogeneity of V/Q contributes to a lowering of PaO2, AaDO2, and diffusing capacity with advancing age, such that PaO2 values of 80–85 mmHg may be normal in subjects older than 65 years. Other factors contributing to declining diffusing capacity with age are a reducing alveolar surface area and a decreasing density of alveolar capillaries. Chest Wall Function

The age-related increase in chest wall stiffness is likely a result of structural alterations, including calcification, of the thoracic cage and its articulations. In addition, the shape of the thoracic cage alters with age due to increasing spinal curvature, leading to ‘barrel chest’. These changes may also alter the curvature of the diaphragm and hence its ability to generate force. Respiratory Muscle Function

The diaphragm is formed by skeletal muscle fibers that radiate from a central tendon and attach along the costal margin; both motor and sensory innervation is provided by the phrenic nerves. The posterior part of the diaphragm is formed by two muscular pillars, the left and right crura, which attach to the lumbar vertebrae. Contraction of the muscle causes downward displacement of the dome of the diaphragm during inspiration. The force-generating capacity of the diaphragm, as with other skeletal muscles, declines with age; it is assumed that the

intercostal and accessory respiratory muscles show similar changes. The muscle strength of the diaphragm is assessed as the maximal transdiaphragmatic pressure (Pdi) that can be generated voluntarily or by nerve stimulation. The decline in muscle strength is likely due to reduced muscle mass, fewer muscle fibers, and fewer peripheral motoneurons. In addition, individual muscle fibers may produce less force or contract more slowly due to impaired intracellular calcium handling, a reduction in the synthesis of myosin and a decrease in mitochondrial function.

Structural Changes in Lung Parenchyma with Age The age-related increase in lung compliance is not thought to be due to a change in the pulmonary surfactant system but rather to structural changes in the lung parenchyma. Although the total lung content of elastin and collagen does not change with age, it is apparent that elastic recoil declines due to changes in the elastic fibers of the lungs; collagen, in contrast, becomes more stable with age. There is evidence that the spatial arrangement and/or crosslinking of mature elastin is altered, or that pseudoelastin becomes more prominent. Most data on the changes in lung structure with aging are derived from animal studies, in particular those using mice, rats, and dogs. However, especially with short-lived rodent species, it is important to bear in mind the differences in lung development in relation to birth, the different postnatal growth patterns, and the shorter life span between these species and humans. Owing to differences between species, data from each will be discussed separately. Data from Mice

The life span of the laboratory mouse is typically 2–3 years. Total lung volume (measured at 25 cmH2O) increases with age to 25–28 months. The volume of air within alveolar ducts and sacs increases in a consistent and significant degree with increasing age; a threefold increase occurs from 1 to 28 months of age. An increase in total alveolar surface area in combination with a continual increase in the total air volumes of the alveoli, ducts, and sacs suggests that aging in the mouse results in a hyperinflated lung with significant airspace distention. In older animals (19 months), alveolar wall thickness is greater than in younger animals. Interalveolar pores form communication between adjacent alveoli. At 1 month, these pores are sparse but increase in number and frequency with age; the number per alveolus more than doubles by 24 months. The total area of interalveolar pores in the BALB/cNNia

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mouse increases more than fourfold between 1 and 28 months. The increase in the total area of interalveolar pores as well as the number of pores per alveolus corresponds to the increase in total alveolar surface area during the life span of the mouse. Rapid expansion of the lungs postnatally may account in part for the formation of some pores; however, degenerative processes during aging may also increase the number of pores per alveolus and their total surface area. Pore enlargement with age may occur as a result of the rupture of tissue strands between adjacent pores, especially in senescent animals. The proportion of alveolar wall formed by pores is B3.5% at 1 month, 5.9% at 2 months, and 8.5–9% after 9 months. As in humans, lung compliance increases with age (i.e., pulmonary elasticity declines). Measurements of pulmonary elastin show that there is no change in total elastic fiber length, despite increasing pulmonary volume. However, if elastic fiber length is normalized to pulmonary volume, an age-related decrease in elastic fibers is seen in BALB/c mice. Biochemical analysis of elastic content demonstrated a loss of elastic fibers in aging lungs. Unless pseudoelastin, a form of elastin in human lungs that increases with age, is separated from elastin during biochemical analysis, the actual amount of elastin will be overestimated. Pseudoelastin has not been found in mice and this absence may account for the decreased elastin content in aging BALB/c mouse lungs. Measurements of changes in lung elastic fibers in senescence-accelerated mice (SAM) have not shown evidence of destruction of the alveolar wall or elastic fibers during aging. Therefore, age-related increases in lung distension in SAM occur in a manner similar to that noted for other strains of mice. In contrast, for mice of the SAM-P/I strain, lung hyperdistension occurs in an accelerated manner compared with the resistant strain (SAM-R/1) or other mouse strains. Therefore, SAM-P/I strain mice could prove useful in the study of senile-related lung hyperinflation. Data from the Rat

The total alveolar air volume of rat lungs progressively increases over a 2-year period of growth. During the first months after birth, alveolar tissue volume increases dramatically, with little change occurring from 5 to 26 months. The peak rate of formation for new alveoli is B1000 min 1. Examination of terminal bronchioles, alveoli, and alveolar septa demonstrated no differences from 5 to 26 months of age. Animals aged 26 months appeared to have slightly enlarged alveolar ducts compared with younger animals but were otherwise indistinguishable from them.

From 1.5 to 5 months of age alveolar surface area almost doubled, but then remained unchanged from 5 to 26 months. Since airspace volume increased by 50% from 5 to 26 months of age without a significant change in alveolar surface area, alveolar duct enlargement with age could explain, in part, how airspace volume increased without a loss of surface area within the lung parenchyma. Enlargement of alveolar ducts was not quantitatively confirmed, but emphysematous changes (alveolar wall destruction) appeared to be absent in the aging Fischer 344 rat. The volume of the capillary bed also increases dramatically during the first 5 months of life, but remains relatively unchanged from 5 to 26 months. Changes in capillary surface area with age are proportional to changes in alveolar surface area. The capillaries within the alveolar septa of the neonatal rat undergo a fascinating transformation from a double capillary system to a single capillary system associated with the growth of secondary alveolar septa to form new alveoli. This reorganization of the pulmonary vasculature is complete by 3 weeks of age. From 5 to 26 months, the total surface area of the capillary bed remained unchanged in both male and female rat lungs. The composition of the alveolar epithelium in the rat changes dramatically during postnatal development. The total volume of type 1 alveolar epithelial cells (AECs) increased more than sixfold from 1 to 6 weeks of age, while the volume of type II AECs displayed a threefold increase. The total surface area of type I and type II AECs also increased more than fivefold from 1 to 6 weeks of age. The ratio of type II to type I AECs at 6 weeks of age was 1.8, but it decreased to 1.0 by 26 months of age. The reduction in type II to type I cell ratio may contribute to altered surfactant secretion in type II cells of old rats since a greater surface area must be served per type II cell to form the surfactant lining layer of the lungs compared with that in younger animals. Type II cellular composition between birth and adulthood differs mainly by a doubling of the volume density of the lamellar bodies and a 50% increase in mitochondrial volume density. Neither the appearance nor the size distribution of lamellar bodies changes during postnatal life; in contrast, dramatic changes occur in mitochondria which shift from an isolated spheroid form to a highly branched interconnected web in the adult cell. The meaning of such a shift is unknown but similar changes occur in other phyla (e.g., insect flight muscle) and may accompany cell division. Although the notion is untested, it is easy to see how division of the mitochondria would be simplified by the fetal disconnected form. Adult mitochondria have only a small surface area–volume

610 LUNG ANATOMY (INCLUDING THE AGING LUNG)

ratio advantage over the spheroid form and it seems unlikely to be the entire advantage of that shape. The extended shape would allow an entire mitochondrion to respond to very focal cellular changes in high-energy phosphates. Over the life span of the Fischer 344 rat, the absolute volume of the cellular interstitium of the lung parenchyma does not change. Interstitial cell number and cell size were similar at 1 week and 26 months of age, although the types and ratios of interstitial cell types forming the parenchyma cell pool were markedly different in neonatal pups compared with adult rats. In contrast, the noncellular components of the interstitium demonstrated dramatic volume changes from 1 week to 5 months of age, increasing approximately tenfold in total volume. From 5 to 26 months, interstitial volume continued to change at a lower rate. Compared to 5 month old rats, the interstitial matrix volume increased 18% by 14 months of age and 39% by 26 months of age. These changes could result from either pulmonary edema or an increase in the noncellular matrix components of the lung (e.g., collagen or elastin). Morphometric analysis of alveoli arising immediately beyond the terminal bronchiole shows that the increase in interstitial matrix volume in the parenchyma with age could be due almost entirely to a thickening of basement membranes and collagen deposition. Basement membrane thickness increased from 40–45 nm at 4–6 months to 75–80 nm at 20–24 months. Collagen fiber volume, normalized to alveolar surface area, increased by more than 100% during this period. Basement membrane volume and collagen fiber volume were similar in the proximal alveolar regions and accounted for 50% of the noncellular matrix at 4–6 months and 80% at 20–24 months of age. By comparison, no change occurred in the relative volume of elastin and remaining acellular space. With age, the volume ratio of collagen to elastin shifted from 3–5 at 4–6 months to 10, at 20–24 months. It appears that the volume of the pulmonary ground substance, relative to epithelial surface, is not significantly modified in aging Fischer 344 rats. Therefore, it is unlikely that edema contributes significantly to the increase in interstitial matrix volume with age. The absence of detectable changes in the volume of elastin is consistent with its low rate of synthesis and slow turnover in adults. A progressive thickening of basement membranes may be explained by epithelial and endothelial cell turnover if the cells lay down basement membrane material during each cell cycle, and possibly also during interphase. The increase in the volume of collagen fibers observed histologically in adult rats is consistent with biochemical studies of collagen content.

Morphologic and biochemical evidence supports the notion that there is a continuous deposition of mature cross-linked extracellular collagen in the lung parenchyma of aging rats which can be interpreted as an age-related excess of collagen (or fibrosis). However, there is no visible difference between the overall gross architecture of the matrix in older animals compared to that of younger rats. The dominant stimulus for collagen fiber deposition, through life, may be the mechanical stress exerted on fibers and then translated to fibroblasts. Following this concept, fibroblasts may add fibrils to the existing network while maintaining the general spatial relationship between collagen fibers and other tissue components and result in a net enrichment in collagen. It is not clear how this increase in collagen mass and the shift in the ratio of collagen to elastin with age impact on the micromechanical behavior of the lungs. A logical assumption would be stiffer lungs, but physiologic measurements suggest otherwise. Three-dimensional reconstructions and physiologic measurements could address this issue in the senescent rat lung. An interesting feature of pulmonary aging in the Lewis rat is an abrupt fourfold decrease in the rate of collagen synthesis between 15 and 24 months. More than 80% of newly synthesized collagen is apparently degraded intracellularly in 15-month-old animals and 60% degraded intracellularly at 24 months. The result is only a small fraction of product being deposited as extracellular cross-linked collagen. A high rate of collagen synthesis may allow fibroblasts to rapidly redirect procollagen from intracellular degradation pathways towards secretory pathways in response to injury. In Fischer 344 rats there is no decline in fibroblast numbers with age. A fall in the rate of collagen synthesis may be an intrinsic characteristic of the senescent lung fibroblast in vivo resulting in a compromised ability to repair collagen fibers. The endothelium forms the third major tissue compartment of the alveolar septum. The endothelial volume of Fischer 344 rat lungs increases more than threefold from 1 to 6 weeks of age. After 6 weeks, total endothelial cell volume does not increase significantly to 26 months. The total surface area of the capillary endothelium, like the epithelial surface, increases almost 10-fold from 1 to 6 weeks; from 6 weeks to 5 months of age it increases by a further 20%, and from 5 to 26 months, it does not change significantly. Data from the Dog

Changes in alveolar size, enlargement of respiratory bronchioles and alveolar ducts, and the accumulation of anthracotic pigment are all common features

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of the aging dog lung. In beagle dogs the volume density of alveolar air and tissue decreases with age, while the volume density of alveolar ducts and sacs increases with age. The number of alveoli per unit volume also decreases with age. No significant correlation was observed between the numerical density of alveolar ducts and alveolar sacs with age. In aging dogs, alveolar duct profile area increases, with minimal evidence of emphysema and fibrosis. The increase in alveolar ducts is associated with decreases in the volumetric density of alveoli and parenchymal tissue, as well as decreases in the numerical density of alveoli, and the surface density of parenchymal tissues. These observations strongly correlate with pulmonary hyperdistention or ductasia in aging dogs; similar findings have been made in the human lung, without a significant decrease in the number of alveoli. In ductasia, alveoli adjacent to enlarged alveolar ducts in respiratory bronchioles decrease in depth, maintaining a constant number of alveoli in the lungs. The observed decrease in pulmonary surface density with age can be explained by an increase in lung volume, a loss of interalveolar septa, a rearrangement of geometry due to alveolar flattening and duct enlargement, or a combination of any of these changes. Similar changes have been observed in aged human lungs with a geometric rearrangement of alveoli in alveolar ducts, resulting in an increase in the average distance between interalveolar septa. The total alveolar surface area decreased as a result of an increase in the average interalveolar septal distance rather than an increase in lung volume. The reduction in volume density of alveolar parenchymal tissue could be interpreted as a loss of interalveolar septa, as observed in human lungs. Alveolar pores appear to be absent at birth in dogs, but become prominent within the first year of life; similar observations have been made in sheep. The average number and size of these pores remain constant through the first 10 years of life. Prolonged exposure to environmental pollutants increases the number and size of these pores, resulting in extensive fenestration of alveolar walls.

the mouse as Clara cells are typically found only in more distal bronchioles in most species. At 2–3 months, the tracheal epithelium consists of 10% basal cells, 39% ciliated cells, 49% Clara cells, and 2% unknown cells; in the mainstem bronchi, basal cells constitute 4% of the total population, ciliated cells 47%, Clara cells 46%, and unknown cells 3%. With aging, the proportions of epithelial cell types change, with basal cells accounting for 1%, ciliated cells 36%, Clara cells 61%, and unknown cells 2%. The bronchiolar epithelium consists of a simple cuboidal to columnar epithelial layer formed by ciliated cells and Clara cells. The proportion of Clara cells in the bronchiole is B80%, with ciliated cells constituting the remainder. Clara cells within the terminal bronchiole of the mouse have dome-shaped apical surfaces protruding into the lumen of the airway and numerous secretory granules and mitochondria within the apical cytoplasm. Data from the Rat

The tracheobronchial epithelium of adult rats varies in terms of the types of cells present and the relative proportions of specific cell types. In the trachea, there are four major epithelial cell types: basal cells, serous cells, ciliated cells, and mucous goblet cells. In contrast, the epithelium of bronchi and bronchioles consists of ciliated cells and nonciliated bronchiolar epithelial cells. At birth, the rat trachea contains some mature ciliated cells, obvious secretory cells, and the beginning of basal cell differentiation. Ciliogenesis begins at about 80% gestation in the rat. Nonciliated secretory cells appear in the tracheal epithelium at about 90–95% gestation. The general characteristics of the airway epithelium have been examined in Fischer 344 rats at 5 and 22 months of age. In the bronchi, no significant differences in the abundance of ciliated, nonciliated, and basal cells were seen from 5 to 22 months; total epithelial cell volume also remained constant over this period. These findings suggest that few changes occur in the proportions of epithelial cell types in the tracheobronchial tree during aging. Data from the Dog

Changes in Airway Structure with Age Data from Mice

In the trachea and proximal bronchi of mice, the pseudostratified epithelium contains three primary cell types: basal cells, ciliated cells, and secretory cells. In the trachea, secretory cells form the nonciliated bronchiolar epithelial cells, or Clara cells; the location of these cells within the trachea is unique to

Little information is available on the effects of aging in the trachea or conducting airways in the dog lung. Age-related calcification of cartilaginous plates occurs within the bronchi as well as hypertrophic changes of the submucosal glands. No changes have been described for the nonrespiratory bronchioles of dogs with aging. The most peripheral airways in the lungs of dogs are formed by respiratory bronchioles and alveolar ducts.

612 LUNG ANATOMY (INCLUDING THE AGING LUNG) Human (19−28 yr) 12

Velocity (mm min–1)

10 Human (56−70 yr)

8 Do

g

6 4 2 0

0

4

8 Age (years)

12

16

Figure 2 Tracheal mucous velocity for beagle dogs with increasing age. Values are expressed as mean7S.E. Available data for humans are also plotted after transforming for age. Reproduced with permission from Pinkerton KE, Murphy KM, Hyde DM (2001) Morphology and morphometry of the lung. In: Capen CC, Benjamin SA, Hahn FF, et al. (eds.) Pathobiology of the Aging Dog, vol. 1, pp. 43–56. Ames: Iowa State University Press.

Mucous velocity in the trachea has been studied in dogs between 1 and 15 years of age; the velocity increased during the first few years of life, followed by a gradual age-related reduction in flow rates beginning around 4 years. With extrapolation of equivalent dog years to human years, a close correlation has been found between dogs and humans with similar patterns of decline in tracheal mucous velocity rates with increasing years during adult life (Figure 2). The branching pattern of the tracheobronchial tree in dogs changes with postnatal age. Resin casts were made from the bronchial tree of four Labrador dogs weighing 0.5 (1 week old), 3.4, 7.5, and 30.0 kg, and the branching pattern was ordered by number, mean diameter, and mean length of branches in each generation. The diameter and length of similar airway orders ran in a parallel fashion for dogs of varying age. If these measurements were normalized to body weight, they were found to be identical. This correction factor was equal to the cube root of body weight. These observations indicate that airway branching morphogenesis of the bronchial tree of the dog is complete at birth and postnatal growth reflects a simple expansion of the length and diameter of each airway generation in the absence of any further airway branching. See also: Alveolar Surface Mechanics. Arteries and Veins. Clara Cells. Transcription Factors: AP-1.

Further Reading Burri PH, Dbaly J, and Weibel ER (1974) The postnatal growth of the rat lung: I. Morphometry. Anatomical Record 178: 711–730.

Coleman GL, Barthold SW, Osbaldistan GW, Foster SJ, and Jonas AM (1977) Pathological changes during aging in barrier-reared Fischer 344 rats. Journal of Gerontology 32: 258–278. Crapo RO (1993) The aging lung. In: Mahler DA (ed.) Pulmonary Diseases in the Elderly Patient, vol. 63, pp. 1–21. New York: Dekker. Horsefield K and Cumming G (1976) Morphology of the bronchial tree in the dog. Respiratory Physiology 26: 173–182. Hyde D, Orthoefer J, Dungworth D, et al. (1978) Morphometric and morphologic evaluation of pulmonary lesions in beagle dogs chronically exposed to high ambient levels of air pollutants. Laboratory Investigation; A Journal of Technical Method & Pathology 4: 455–469. Janssens JP, Pache JC, and Nicod LP (1999) Physiological changes in respiratory function associated with aging. European Respiratory Journal 13: 197–205. Jeffery PK and Reid LM (1977) Ultrastructure of airway epithelium and submucosal glands during development. In: Hodson E (ed.) Development of the Lung, pp. 87–134. New York: Dekker. Kawakami M, Paul JL, Thurlbeck WM (1984) The effect of age on lung structure in male BALB/cNNia inbred mice. American Journal of Anatomy 170–171. Kuhn C (1997) Morphology of the aging lung. In: Crystal RG, West JB, Barnes PJ, and Weibel ER (eds.) The Lung: Scientific Foundations, pp. 2187–2191. Philadelphia: Lippincott-Raven. Kurozumi M, Matsushita T, Hosokawa M, and Takeda T (1994) Age-related changes in lung structure and function in the senescence-accelerated mouse (SAM): SAM-P/I as a new murine model of senile hyperinflation of lung. American Journal of Respiratory & Critical Care Medicine 149: 776–782. Liebow AA (1964) Summary: biochemical and structural changes in the aging lung. In: Cander L and Moyer JH (eds.) Aging of the Lung, pp. 97–104. New York: Grune and Stratton. Martin H (1963) The effect of aging on the alveolar pores of Kohn in the dog. American Review of Respiratory Disease 88: 773–778. Massaro GD and Massaro D (1986) Development of bronchiolar epithelium in rats. American Journal of Physiology 250: R783–R788. Mauderly JL and Hahn FF (1982) The effects of age on lung function and structure of adult animals. Advances in Veterinary Science & Comparative Medicine 26: 35–77.

LUNG DEVELOPMENT / Overview 613 Mays PK, McAnulty RJ, and Laurent GJ (1989) Age-related changes in lung collagen metabolism: a role for degradation in regulating lung collagen production. American Review of Respiratory Disease 140: 410–416. Pinkerton KE, Barry BE, O’Neil JJ, et al. (1982) Morphologic changes in the lung during the life span of Fischer 344 rats. American Journal of Anatomy 164: 155–174. Pinkerton KE, Murphy KM, and Hyde DM (2001) Morphology and morphometry of the lung. In: Capen CC, Benjamin SA, Hahn FF, et al. (eds.) Pathobiology of the Aging Dog, vol. 1, pp. 43–56. Ames: Iowa State University Press.

Pinkerton KE and Green FHY (2004) Normal aging of the lung. In: Harding R, Pinkerton KE, and Plopper CG (eds.) The Lung: Development, Aging and the Environment, pp. 213–233. London: Elsevier Academic Press. Pump K (1976) Emphysema and its relation to age. American Review of Respiratory Disease 114: 5–13. Ranga V and Kleinerman J (1980) Interalveolar pores in mouse lungs. American Review of Respiratory Disease 122: 477–481. Ranga V, Kleinerman J, and Sorensen J (1979) Age-related changes in elastic fibers and elastin of lung. American Review of Respiratory Disease 119: 369–376.

LUNG DEVELOPMENT Contents

Overview Congenital Parenchymal Disorders Congenital Vascular Disorders

Overview R Harding, S B Hooper, and M J Wallace, Monash University, Melbourne, VIC, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Lung development begins within 2–3 weeks of conception and is completed when thoracic growth ceases in late adolescence. Human lung morphogenesis largely occurs before birth and during infancy under the control of a genetic program that is modulated by endocrine and physical factors. Lung development occurs by branching morphogenesis which is closely regulated by intrinsic growth factors. After the bronchial tree has been laid down, lung growth occurs by expansion of existing lung tissue rather than by the establishment of new structural units. In the fetus, the development of major lung components including the vascular bed, alveoli, and innervation occurs in parallel such that by the time of term birth, the lung is capable of effective gas exchange. During fetal life, the lung is expanded by liquid, which is critical for tissue growth and remodeling; after birth, growth of the thoracic cage is likely to drive lung growth. The genetic program of lung development can be altered by prenatal and early postnatal challenges leading to lasting effects on lung structure and function. Fetal exposure to adverse intrauterine conditions such as reduced amniotic fluid, excess glucocorticoids, nutritional and oxygen restriction, or maternal tobacco smoking can interfere with the genetic program of development.

placenta the vital role of gas exchange. Before birth the airways contain a unique fluid (lung liquid) that is actively secreted by the lung epithelium. That is, the fetal lungs do not develop in a collapsed state but are maintained in an expanded state by this liquid, which is essential for normal lung development. At birth, the lungs are cleared of lung liquid and within moments of disconnection from the placenta the infant must initiate continuous breathing movements and the lungs must be capable of sustaining the gas exchange requirements of the newborn. Despite this degree of structural and functional maturity, lung development is not complete by the time of birth and much of it continues after birth whilst filled with air and potentially exposed to environmental pollutants and pathogens.

Stages of Lung Development Lung development can be broadly divided into an initial period of structural differentiation, which ends in infancy or early childhood, followed by a period of growth which continues until somatic growth ceases after puberty. The structural development of the lung has, by convention, been divided in a series of stages based on its morphology. Embryonic Stage

Introduction In humans and most other mammals, the lung reaches a state of functional maturity by the time of birth, such that it can rapidly take over from the

The lung first appears as an outgrowth of the ventral surface of the embryonic foregut; in humans this occurs at 22–26 days post conception. The endodermal tissue of the lung bud divides to form the right and

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left lung anlages; each ‘bronchus’ divides repeatedly to form branched, tubelike structures that invade the surrounding mesoderm. In humans, the major bronchopulmonary segments have formed by 34 days. Left–right asymmetry is apparent from an early stage, such that the right lung bud initially forms three major divisions while on the left side only two are formed; these correspond to the lobes of the adult lungs. The primitive ‘airways’ are lined with tall columnar cells rich in glycogen granules. The splanchnic mesoderm which invests the ‘airways’ gives rise to all nonepithelial structures of the lung including blood vessels, fibroblasts, lymphatics, cartilage, and smooth muscle. The primitive endodermal and mesenchymal cells of the embryo eventually give rise to more than 40 different cell types in the mature lung. Pseudoglandular Stage

At 5–17 weeks of human gestation, the lung resembles a typical secretory gland; the future airways are liquid-filled and are lined with tall columnar epithelial cells. During this time, the major airways (bronchi) form and the respiratory units of the lung (acini) develop due to growth and repeated branching of the epithelial tubes into the surrounding mesenchyme. This branching morphogenesis is dependent upon signaling between the growing epithelial tubes and the surrounding mesenchyme. The modest vascular bed of the lung and the absence of a dense capillary network preclude the pseudoglandular lung functioning as an organ of gas exchange. During this stage of development, the epithelial cells lining the ‘airway’ tubes begin to differentiate, beginning at the hilum and extending towards the periphery. These cells give rise to goblet cells and mucous glands, as well as flatter epithelial cells with cilia; this occurs by 11–13 weeks in human gestation. Canalicular Stage

During this stage (16–26 weeks in humans), the airways widen and lengthen, resulting in a large increase in the potential air space of the lung. This stage of ‘canalization’ is characterized by the formation of respiratory units (lobules), each being a cluster of terminal bronchioles, several respiratory bronchioles, and a peripheral cluster of closely branched buds which subsequently form terminal saccules. With the growth of the airways, the mesenchyme surrounding the distal respiratory units thins and the mesenchymal capillaries investing the tubules come into close contact with the epithelium. Distally, the distance between the tubule lumen and capillaries (i.e., the gas diffusion distance) becomes smaller due to a reduction in mesenchymal tissue

volume as well as growth and branching of pulmonary blood vessels and thinning of the epithelium. Differentiated epithelial cells first appear in the terminal airsacs during this stage of development. Those cells destined to become type I epithelial cells become flattened, forming thin cytoplasmic extensions that ultimately provide a large surface area for gas exchange and a thin barrier to diffusion; this marks the first stages of the formation of a functional air–blood barrier. Epithelial cells destined to become type II cells retain their spherical shape and develop cytoplasmic inclusions (lamellar bodies) that contain surfactant. Respiratory gas exchange becomes possible during this period, with some pre-term infants born at 22–23 weeks of gestation being viable with suitable ventilatory support. Saccular and Alveolar Stages

These stages are characterized by the formation of the respiratory zone, such that by term (37–40 weeks in humans) the lung can function independently as an effective organ of gas exchange. During the saccular stage, before definitive alveoli are formed, the distal airspaces enlarge to form sacs or transient alveolar ducts from which alveoli develop. The capillary network around the terminal sacs proliferates and the separation between the epithelium and blood continues to diminish due to a reduction in perialveolar tissue. Starting before birth and continuing into the postnatal period in humans, the terminal saccules progressively subdivide into definitive alveoli by the outgrowth of secondary septal crests from the saccules’ walls. This process of septation begins with the formation of a ridge that projects into the saccule lumen. These ridges increase in height, forming walls (secondary septa) that subdivide the saccules into separate alveoli. The secretion and deposition of elastin at the tips of these septal crests is critical for alveolar formation; mice with an ablated tropoelastin gene are unable to form definitive alveoli. After alveolarization is complete, a ring of elastic fibers surrounds the mouth of each alveolus. The formation of secondary septa greatly increases the surface area of the distal airspaces and hence the area for gas exchange; the luminal surface of the human lung increases from 1–2 m2 in the fetus to 50–100 m2 in the adult, largely due to alveolarization. In humans, although 20–50 million alveoli develop before birth, the majority form during infancy to reach the adult number of B300 million. The formation of alveoli is intimately linked with the development of an extensive capillary network surrounding each alveolus. With further attenuation of perialveolar tissue, the distance between the alveolar epithelium and capillary blood is further

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reduced, such that by term birth the air–blood barrier is little thicker than in the adult.

Regulation of Lung Morphogenesis Most of the information on the molecular and cellular events controlling lung development has been obtained from mice. Although birth occurs at a much earlier stage of lung development in this species, relative to humans, studies in mice have provided critical insights into the regulation of tissue remodeling and differentiation processes that give rise to a mature lung from a primordial bud. In general, endodermal development is determined by transcription factors that are locally expressed, as well as by soluble factors that diffuse from adjacent cells. These agents mediate paracrine interactions and regulatory feedback loops. Primary lung bud formation appears to require fibroblast growth factor (FGF) signaling within foregut endodermal cells, as mice lacking FGF10 or its receptor (FGFR2-IIIb) develop a ‘trachea’ but not lung buds. Similarly, studies in knockout mice and vitamin A-deficient rats suggest that initial lung bud formation may be dependent upon the Gli family of transcription factors and retinoic acid signaling. Branching morphogenesis is critical to lung development as it leads to the formation of the highly branched bronchial tree from the primary bud. This process involves bud outgrowth, bud elongation, and subdivision of the terminal units. The epithelial bud (or tube) grows into the surrounding mesenchyme and epithelial–mesenchymal interactions regulate branching of the tube; this process is mediated by diffusible factors which activate or suppress specific transcription factors to induce branching of buds or to allow tubule elongation. Numerous studies have indicated that FGF10, produced by mesenchymal cells, and its receptor FGFR2 located in epithelial cells, play an essential role in the pattern of airway branching. FGF10 is expressed dynamically in mesenchyme at sites of eventual distal epithelial bud formation, while FGFR2 is expressed evenly along the respiratory tract epithelium. FGF10 appears to act as a chemoattractant inducing FGFR2 expressing epithelial cells to grow towards the source of FGF10, thus inducing new branch formation. Locally expressed signaling molecules modulate the responses to FGF10 at the branching point of tubules, to regulate spatial and temporal branch formation (Figure 1). Sonic hedgehog (Shh) and bone morphogenetic protein 4 (BMP4, a member of the transforming growth factor beta (TGF-b) superfamily) are highly expressed in the epithelial cells at the tips of growing branches and can be induced by FGF10. Both Shh and BMP4

Lumen Epithelium

Initiation of lung budding and outgrowth +

Shh BMP4 +

FGF10 secreting cells

Mesenchyme (a)

Lumen Shh –

TGF-
1 BMP4 –

(b)

New site of FGF10 secretion

Figure 1 Branching morphogenesis in the developing lung. Mesenchymal cells express and secrete FGF10 producing a chemoattracting gradient for lung bud outgrowth. FGF10 stimulates sonic hedgehog (Shh) and bone morphogenetic protein 4 (BMP4) production in epithelial cells lining the tubule, which in turn inhibits FGF10 expression and abolishes the stimulus for bud outgrowth. Alternate sites of FGF10 expression appear and new buds are initiated. Transforming growth factor beta 1 (TGF-b1) is deposited in the clefts between buds where it induces the expression of extracellular matrix components and prevents FGF10 expression, thereby preventing bud formation and stabilizing the cleft.

can inhibit FGF10 expression and are thought to abolish FGF10 production and its chemoattracting properties when the growing branch tip reaches the FGF10 secreting cells, thus forming a negative feedback loop. FGF10 expression reappears at alternate sites inducing new sites of branch formation. Mice lacking Shh exhibit a virtual failure of branching morphogenesis. Sprouty (Spry2) is also expressed in the branch epithelium and is believed to act in a similar manner to that proposed for Shh and BMP4. TGF-b1 protein accumulates in the clefts between

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branches and its ability to inhibit epithelial cell proliferation and FGF10 expression and to induce synthesis of extracellular matrix is thought to stabilize the cleft and inhibit local branch formation.

Development of Alveoli Alveolarization is a late event that occurs after the conducting airway system has been laid down by branching morphogenesis. Relative to our understanding of early lung morphogenesis, much less is known about the molecular control of alveolarization in which saccules become subdivided, by a process of septation, into mature alveoli. However, some processes are common to both. Alveolar formation can be divided into three phases: (1) secondary septation in which new alveoli are formed by septation of the terminal sacs; (2) remodeling of secondary septa and maturation of the single capillary network surrounding the alveoli; and (3) occurrence of a phase of growth after alveolar formation ends. Septation starts by the protrusion of secondary crests from the primary septa (i.e., the walls of the terminal sacs). The septa contain a central core of fibroblasts and other connective tissue components, which is flanked on each side by capillaries and epithelium. Fibroblast proliferation is an important part of septation as it increases the height of the septa; the tips of the septa contain myofibroblasts which form a ring around the mouth of the alveoli and produce elastic fibers giving the septa, and ultimately the alveolar mouth, strength and elasticity. Airway wall remodeling occurs after septation, resulting in the lengthening and thinning of the secondary septa. This involves loss of interstitial mesenchymal cells and capillary remodeling in which the dual capillary system becomes a single capillary system. In this single system, capillaries become interconnected with each other, leading to a dense network of capillaries with a large cross-sectional area. Following the end of alveolar formation, which in humans occurs at 2–3 years after birth, the lung enters a growth phase in which the surface area of alveoli and volume of airspace in the lung greatly increase while the septal tissue mass decreases. This growth phase ends prior to the growth of long bones in humans, but in rodents may continue throughout life. An important aspect of alveolar formation is maturation of the alveolar epithelium. This maturation typically occurs in synchrony with secondary septation and the increase in pulmonary surface area. The mature lung contains two major types of alveolar epithelial cells (AECs), both of which arise from the endoderm-derived epithelial cells lining the distal duct system in the developing lung. The differentiation of

the epithelial progenitor cells into type I and type II AECs begins prior to the formation of true alveoli, during the saccular phase of lung development. Type I AECs constitute 45–50% of the epithelial cells in mature lung and cover B90% of the surface of alveoli; hence they are the major cells through which gas exchange takes place. These cells contain few organelles and have an extremely flattened cytoplasm that can spread across the surface of more than one alveolus. Type I AECs contain water channels (aquaporins) and caveolae suggesting they may transport water and other materials across the cell. Although type I cells were thought to be terminally differentiated, recent evidence indicates that they may be capable of transdifferentiation to type I cells. Type II AECs constitute 50–55% of the epithelial cells in the mature lung but occupy only around 7% of the alveolar surface area. They are characterized by their spherical/cuboidal shape, apical surface microvilli, and surfactant-containing lamellar bodies. Type II AECs are thought to be the epithelial progenitor cells, giving rise to type I cells by transdifferentiation and daughter type II cells by division. The most important function of type II AECs is the production of pulmonary surfactant and surfactant proteins, without which the lungs are unable to be expanded with air and collapse during expiration; surfactant proteins are also important in host defense. Alveolarization is regulated by a number of factors, several of which are related to elastin deposition. Platelet-derived growth factor A (PDGF-A) is critical for alveolarization as PDGF-A knockout mice fail to develop alveoli, likely due to reduced myofibroblast proliferation, migration, and elastin production. Increased fetal production of glucocorticoids prior to birth has a major role in remodeling the lung extracellular matrix, reducing interalveolar tissue volume, stimulating surfactant production, and enhancing the mechanical properties of the lung. Therefore, glucocorticoids are routinely used to induce fetal lung maturation in women at risk of preterm labor. However, it is now clear that exogenous glucocorticoids can lead to a permanent inhibition of alveolarization, raising concerns about the doses and duration of treatment. An impairment of alveolarization and reduction in capillary formation are hallmarks of bronchopulmonary dysplasia in pre-term infants and are likely due to a combination of insults to the pre-term lung. Studies in knockout mice and vitamin A-deficient pre-term babies suggest that retinoic acid and its receptors are also important for alveolarization. In the rat, retinoic acid administration can increase alveolarization and can reverse the alveolarization defect induced by glucocorticoids. Vascular endothelial growth factor (VEGF) is also

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important for alveolarization, likely due to its critical role in capillary formation.

Pulmonary Vascular Development The pulmonary vasculature develops in close coordination with the bronchial tree and distal airspaces and recent evidence indicates a close interdependence between capillary development and alveolar formation. For effective gas exchange to occur at birth it is essential that the pulmonary vascular bed has developed to such an extent that alveoli are richly endowed with a dense capillary network and that the vascular bed can accept the entire cardiac output once the ductus arteriosus closes shortly after birth. Vascular development in the lung occurs by growth and remodeling of existing structures, processes that continue up to the time at which thoracic growth ceases. In the embryo, the lung buds are vascularized by ingrowth of a vascular plexus from the heart. Then a branching network develops by angiogenesis from the central arterial and venous trunks; these increase in diameter and length and develop offshoots by a process of dichotomous branching. In humans, the adult pattern of airway and vascular structures, with lobar and segmental branches, is in place by 6 weeks of gestation. Distal networks of vessels and capillaries form by vasculogenesis and then fuse with the proximal networks. Pulmonary vascular development is paralleled by development of the bronchial vascular bed and their associated venous systems. Vascular development occurs by both vasculogenesis and by angiogenesis. Vasculogenesis is the creation of new vessels from mesenchymal progenitor cells; these cells migrate and differentiate into angioblasts, which eventually differentiate into endothelial cells, or hemangioblasts, which are blood cell precursors. ‘Blood islands’ of cells and spaces form and develop into capillary-like structures as the angioblasts form simple vascular networks. Angiogenesis involves sprouting of existing capillaries and small vessels due to endothelial cell migration through sites of degradation in the vessel walls. The sprouts then elongate and branch, and may fuse with adjacent sprouts, and eventually form a lumen allowing the passage of blood. Intussusceptive microvascular growth is initiated by contact between endothelial cells on opposite sides of the vessel, forming interendothelial bridges. The area of contact is sealed and organized into interendothelial junctions that become thinner and are then invaded by connective tissue, forming a post or division within the capillary channel. Numerous growth factors have been implicated in the development of the pulmonary vascular bed. These include VEGF isoforms and their

receptors, which are critical for angiogenesis and endothelial cell survival. PDGF isoforms are important for both pulmonary vascular and alveolar development. Fibroblast growth factors, endothelial nitric oxide synthase, and angiopoietins have also been implicated in pulmonary vascular development. The preacinar pattern of pulmonary arteries and veins is completed by 36 weeks of gestation in humans. With the onset of alveolarization, there is an increase in length and diameter of existing units and new intra-acinar units are formed. After birth, with continuing alveolarization, the number of arteries and density of capillary networks continue to increase. In the fetus, the pulmonary vascular bed has a high resistance and consequently it receives only a small proportion (5–8%) of cardiac output, with most blood bypassing the lung via the ductus arteriosus. At birth, the abrupt fall in pulmonary vascular resistance results in an 8–10-fold increase in pulmonary blood flow and a decrease in pulmonary arterial pressure. This fall in resistance allows the lung, for the first time, to accept the entire cardiac output and to function as an organ of gas exchange. If pulmonary vascular resistance fails to decrease at birth, persistent pulmonary hypertension of the newborn (PPHN) develops, leading to hypoxemia; PPHN is a common cause of neonatal morbidity and death, especially in pre-term infants. The ability of the pulmonary vascular bed to respond to birth and air-breathing by a reduction in vascular resistance increases with fetal development. A number of factors contribute to the maintenance of a high vascular resistance in the fetus: low oxygen tension, low production of vasodilators such as prostaglandin I2 and nitric oxide, high levels of production of vasoconstrictors such as endothelin-1 and leukotrienes, and increased smooth muscle reactivity. Alterations in these factors at birth serve to increase the dilation of resistance vessels. Another important factor is the capillary transmural pressure that exists in the fetus, due to the presence of lung liquid which maintains a constant distending pressure within the terminal airspaces. At birth, the lung is cleared of luminal liquid and the capillary transmural pressure gradient increases because the lung recoils with the creation of an air–liquid interface, thereby reducing interstitial pressure and compression of blood vessels and allowing increased blood flow.

Development of Pulmonary Innervation The sensory and motor innervation of the pulmonary airways is important for normal pulmonary function after birth. Neural tissue is prominent in the developing lung, as revealed by recent studies using

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confocal microscopy and immunofluorescent staining. Such studies have shown that neural tissue and airway smooth muscle are integral components of the lung from early in development and throughout fetal life and postnatal development. By the pseudoglandular stage, chains of ganglia connected by nerve trunks to the vagus can be observed. Nerve trunks run along the airways to their tips, lying above the airway smooth muscle, and terminate in fine branches over the muscle cells. During the canalicular stage, large nerve trunks run along the length of the airways and terminate together with a network of ganglia. Most neurotransmitters and the beginnings of a neural network that can serve both efferent and afferent functions of the vagus nerve are present. In the saccular stage (i.e., most of the third trimester), the bronchial mucosa and glands are richly innervated and afferent innervation is prominent. Further development occurs after birth, including myelination of vagal fibers and increasing density of both efferent and afferent innervation. A striking feature of the developing airways is the presence of neuroendocrine (NE) cells in the epithelium. They are derived from undifferentiated epithelial cells of endodermal origin and are present as isolated cells or as clusters in conjunction with Clara cells (neuroepithelial bodies, NEB) from the nasal mucosa to the terminal airways. NE cells and NEB contain abundant bioactive amines and peptides and are present in the human fetal airways from as early as 10 weeks of gestation. Their function is largely unknown although it has been suggested that they may sense hypoxia or play a paracrine role in epithelial cell growth and regeneration.

Control of Lung Growth Before and After Birth Before Birth

In the fetus the pulmonary airways are filled with lung liquid, which differs in composition from plasma and amniotic fluid. It is actively secreted by the lung epithelium (see Breathing: Fetal Lung Liquid), and provides mechanical support to the developing lung tissue. There is some evidence that the airway smooth muscle of the fetus is rhythmically active, which may redistribute the fluid regionally within the lung. Expansion of the lung tissue by this luminal fluid is essential for normal lung growth and maturation (see Breathing: Fetal Breathing; Fetal Lung Liquid). Fetal breathing movements occur episodically in the healthy human fetus from around 10–12 weeks of gestation and play an important role in stimulating lung growth by retarding efflux of lung

liquid, thereby helping to maintain lung expansion (see Breathing: Fetal Breathing). The prolonged absence of fetal breathing movements retards lung growth by reducing the degree of lung expansion. Similarly, a lack of amniotic fluid (oligohydramnios) reduces lung expansion by increasing the degree of fetal trunk flexion, and if prolonged can thereby cause pulmonary hypoplasia. The critical factor is apparently the maintenance of a small pressure gradient across the lungs which results in an appropriate level of expansion. After Birth

Once lung liquid is replaced by air at birth, the lungs recoil away from the thoracic wall due to creation of an air–liquid interface within the alveoli; a luminal distending pressure is no longer present. Postnatal lung growth most likely occurs due to growth and expansion of the thoracic cage; lung growth is believed to stop when somatic growth stops. Thoracic expansion due to somatic growth would be expected to cause a transpulmonary pressure gradient; augmentation of this gradient by continuous positive airway pressure provides a further stimulus to lung growth. Although the cause of the mechanical strain on lung tissue is different before and after birth, the molecular basis for postnatal lung growth may be similar to that in the fetus. After the cessation of alveolar formation at 2–3 years after birth, lung growth occurs by enlargement of existing alveoli and an associated increase in lung parenchyma. See also: Breathing: Fetal Breathing; Fetal Lung Liquid. Bronchopulmonary Dysplasia. Lung Development: Congenital Parenchymal Disorders; Congenital Vascular Disorders.

Further Reading Abman SH (2004) Developmental physiology of the pulmonary circulation. In: Harding R, Pinkerton KE, and Plopper CG (eds.) The Lung: Development, Aging and the Environment, pp. 105–117. London: Elsevier. Cardoso WV (2004) Lung morphogenesis, role of growth factors and transcription factors. In: Harding R, Pinkerton KE, and Plopper CG (eds.) The Lung: Development, Aging and the Environment, pp. 3–11. London: Elsevier. Groenman F, Unger S, and Post M (2005) The molecular basis for abnormal human lung development. Biol Neonate 87: 164–177. Hooper SB and Harding R (2001) Respiratory system. In: Harding R and Bocking AD (eds.) Fetal Growth and Development, pp. 114–136. Cambridge: Cambridge University Press. Hooper SB and Wallace MJ (2004) Physical, endocrine and growth factors in lung development. In: Harding R, Pinkerton KE, and Plopper CG (eds.) The Lung: Development, Aging and the Environment, pp. 131–148. London: Elsevier. Hsia CCW, Berberich B, Laubach VE, et al. (2004) Mechanisms and limits of induced postnatal lung growth. American Journal of Respiratory & Critical Care Medicine 170: 319–343.

LUNG DEVELOPMENT / Congenital Parenchymal Disorders 619 Jones R and Reid LM (2004) Development of the pulmonary vasculature. In: Harding R, Pinkerton KE, and Plopper CG (eds.) The Lung: Development, Aging and the Environment, pp. 81–103. London: Elsevier. McGowan SE and Snyder JM (2004) Development of alveoli. In: Harding R, Pinkerton KE, and Plopper CG (eds.) The Lung: Development, Aging and the Environment, pp. 55–73. London: Elsevier. Miller LA (2004) Development of the pulmonary immune system. In: Harding R, Pinkerton KE, and Plopper CG (eds.) The Lung: Development, Aging and the Environment, pp. 169–176. London: Elsevier. Sparrow MP and Lamb JP (2003) Ontogeny of airway smooth muscle: structure, innervation, myogenesis and function in the fetal lung. Respiratory Physiology & Neurobiology 137: 361–372. Stenmark KR and Abman SH (2005) Lung vascular development: implications for the development of bronchopulmonary dysplasia. Annual Review of Physiology 67. 27.1–27.39. Warburton D and Bellusci S (2004) The molecular genetics of lung morphogenesis and injury repair. Paediatric Respiratory Reviews 5(supplement A): S283–S287.

Congenital Parenchymal Disorders G B Mychaliska and R Seetharamaiah, University of Michigan, Ann Arbor, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The major congenital parenchymal disorders of the lung include pulmonary agenesis, pulmonary hypoplasia, congenital diaphragmatic hernia, bronchogenic cyst, congenital lobar emphysema, congenital cystic adenomatoid malformation, and bronchopulmonary malformation. The majority of abnormalities will manifest clinically shortly after birth or within the first year of life. A few anomalies may remain dormant until young adulthood. The advent of prenatal ultrasonography and fetal magnetic resonance imaging has led to earlier detection of these lesions and shed light on their natural history and pathogenesis. Postnatal diagnosis is based on history, physical examination, and radiographic evaluation. Current therapy involves complex medical and surgical management. Select lesions have become amenable to fetal treatment.

Pulmonary Agenesis Pulmonary agenesis is a congenital disorder in which there is complete absence or severe hypoplasia of one or both lungs. The true incidence is unknown, although autopsy studies reveal a prenatal incidence of 1 in 15 000. Unilateral agenesis is 25 times more frequent than bilateral agenesis, and the right and left lungs are equally affected. Isolated lobar agenesis is rare. There is no gender predominance. Unilateral pulmonary agenesis is associated with a variety of

other congenital anomalies. Associated anomalies include cardiac (patent ductus arteriosis, atrial and ventricular septal defects), anomalous pulmonary venous drainage, tracheoesophageal anomalies, gastrointestinal anomalies, renal anomalies, and spinal anomalies (specifically hemivertebrae). Occasionally, there are ipsilateral facial or jaw abnormalities, and ipsilateral limb abnormalities including the ulna, radius, and particularly the thumb. The pathogenesis remains unknown. Folic acid and vitamin A deficiencies may be involved. Additional theories include vascular disruption, teratogenic insult, or a possible genetic cause. The association with other malformations suggests embryological abnormalities occurring during the fourth week of life. Three classes of agenesis have been described based on the degree of underdevelopment of the lung. Class I refers to agenesis of the lung or total absence of the bronchus and lung. Class II refers to aplasia with rudimentary bronchus without lung tissue. Class III refers to severe pulmonary and bronchial hypoplasia. Symptoms depend on the degree of lung agenesis and associated malformations. Bilateral agenesis is incompatible with life. Symptoms are usually related to respiratory insufficiency and central airway stenosis or tracheobronchomalacia. Symptoms of central airway compression are seen more frequently in patients with unilateral absence of the right lung, because the aorta can compress the trachea. Because of the higher incidence of associated cardiac abnormalities, right lung agenesis has a higher morbidity and mortality compared to left lung agenesis. Approximately 50% of patients with agenesis are stillborn or die in the first few years. Prenatally, the diagnosis is made with sonographic evidence of mediastinal shift toward the affected side, ipsilateral diaphragmatic elevation, and enlarged echogenic lung herniated into the contralateral chest. Given the associated anomalies, a thorough examination with ultrasound, fetal magnetic resonance imaging (MRI), and fetal echocardiogram is required. Postnatally, the diagnosis may be made on chest radiograph in which there is shift of the mediastinum to the affected side with opacification of the ipsilateral hemithorax and hyperinflation of the contralateral side. Definitive diagnosis is made with bronchoscopy, multidetector computed tomography (CT), MRI, and angiography. Treatment consists of respiratory support and management of associated anomalies. Although gas exchange may be improved with compensatory lung growth, persistent mediastinal shift can lead to airway compression and scoliosis. Prognosis of patients with unilateral agenesis depends largely on the presence

620 LUNG DEVELOPMENT / Congenital Parenchymal Disorders

of associated congenital malformations. Survivors remain at risk for recurrent pulmonary infections. In the absence of the associated anomalies, compensatory hypertrophy of the solitary lung may lead to longterm survival.

Pulmonary Hypoplasia Pulmonary hypoplasia is the underdevelopment of the lung which may occur as a result of interference with development of the alveolar system. The majority of patients with pulmonary hypoplasia have lesions that compete for space in the pleural cavity or have physiologic impairment of lung fluid dynamics. Methods of determining hypoplasia are shown in Table 1. Hypoplasia may result from: (1) lung compression from an intrathoracic source (congenital cystic adenomatoid malformation (CCAM), congenital Table 1 Methods of determining pulmonary hypoplasia Lung weight Lung weight/body weight ratio Radial alveolar count Alveolar count per unit volume Lung volume measurement and correlation with crown–rump length Airway branching count using latex injection DNA content Reproduced from Travis WD, Colby TV, Koss MN, et al. (2002) Congenital anomalies and pediatric disorders. In: Travis WD and Colby TV (eds.) Atlas of Nontumor Pathology: Non-Neoplastic Disorders of the Lower Respiratory Tract, 1st Series, Fascicle 2, pp. 473–538. Washington, DC: American Registry of Pathology and Armed Forces Institute of Pathology, with permission from American Registry of Pathology.

diaphragmatic hernia (CDH), pulmonary sequestration or a large pleural effusion), or an extrathoracic source (malformed thorax), (2) amniotic and lung fluid volume disturbances, (3) abnormal or absent fetal breathing movements (phrenic nerve abnormalities), (4) environmental agents, (5) decreased blood flow to the lungs, (6) primary mesodermal defects, and (7) unknown or idiopathic causes. Table 2 delineates multiple anomalies associated with pulmonary hypoplasia. The presence of bilateral lung constraint, as occurs in oligohydramnios or thoracic dystrophy, can produce bilateral hypoplasia. However, a large unilateral lesion, such as CDH or a large CCAM, may displace the mediastinum and produce a contralateral hypoplasia which is usually not as severe as the ipsilateral side. Fetal lamb models of CDH and CCAM demonstrate the resulting pulmonary hypoplasia resulting from a space occupying lesion. Fetal lamb models of tracheal drainage and renal obstruction demonstrate the effects of altered lung fluid mechanics and oligohydramnios, respectively. Pulmonary hypoplasia is recognized in the newborn period because of severe respiratory distress and hypoxia despite mechanical ventilation and often is associated with persistent pulmonary hypertension. The degree of pulmonary hypoplasia depends on the timing and duration of the underlying cause. Prenatal history and imaging is helpful in establishing a diagnosis of pulmonary hypoplasia. Mechanical ventilation is required to support gas exchange. Extracorporeal membrane oxygenation (ECMO) may provide gas exchange for a critical period of time and may permit survival in cases that are not amenable to less invasive treatment. Severe

Table 2 Anomalies associated with pulmonary hypoplasia Common

Unusual

Rare

Diaphragmatic hernia Renal agenesis (bilateral) Renal dysgenesis (bilateral) Obstructive uropathy Polycystic renal disease (Potter, type I) Large abdominal wall defects

Large congenital cystic adenomatoid malformation, bronchopulmonary sequestration Diaphragmatic hypoplasia or eventration Hemolytic disease Musculoskeletal abnormalities (such as thoracic dystrophy) Pleural effusion, as with nonimmune fetal hydrops Oligohydramnios caused by prolonged amniotic fluid leakage Chromosomal anomalies, including trisomy 13,18, and 21 Anencephaly Scimitar syndrome

Ascites secondary to congenital infection Abdominal pregnancy Thoracic neuroblastoma Phrenic nerve agenesis Cloacal dysgenesis Congenital hydropericardium Gastroschisis Right-sided cardiovascular malformation as with hypoplastic right ventricle Glutaric academia, type II Laryngotracheoesophageal cleft Upper cervical spinal cord injury

Adapted from Travis WD, Colby TV, Koss MN, et al. (2002) Congenital anomalies and pediatric disorders. In: Travis WD and Colby TV (eds.) Atlas of Nontumor Pathology: Non-Neoplastic Disorders of the Lower Respiratory Tract, 1st Series, Fascicle 2, pp. 473–538. Washington, DC: American Registry of Pathology and Armed Forces Institute of Pathology and Stocker JT (1994) Congenital and developmental diseases. In: Dial DH and Hammar SP (eds.) Pulmonary Pathology, 2nd edn., pp. 155–190. New York: Springer.

LUNG DEVELOPMENT / Congenital Parenchymal Disorders 621

cases of pulmonary hypoplasia are incompatible with life.

Congenital Diaphragmatic Hernia CDH is a defect in the diaphragm that results in pulmonary hypoplasia primarily from compression of the developing lung by herniated viscera. The incidence of CDH is 1 in 3000 live births. However, this incidence does not account for the ‘hidden mortality’ of CDH (fetal death, stillbirths, and immediate neonatal death). Therefore, the true incidence may be as high as 1 in 2000 births. The morbidity and mortality of CDH is related to pulmonary hypoplasia and pulmonary hypertension. Associated anomalies are found in 40–50% of all cases of CDH. These include congenital heart defects, hydronephrosis, renal agenesis, intestinal atresia, extralobar sequestration, and neurological defects. Chromosomal abnormalities, particularly trisomies 21, 18, and 13, are seen in 10–20% of prenatally diagnosed cases. There is a female preponderance with female to male ratio of 3 : 2. The cause of CDH is unknown. There are anecdotal reports in association with maternal ingestion of thalidomide, quinine, and antiepileptic drugs but no definitive causal relation is known. Most cases are sporadic, but some familial cases of CDH are inherited in a autosomal dominant manner. The association of CDH with other genetic abnormalities suggests an underlying genetic etiology. The pathogenesis of CDH is complex and includes intrinsic lung abnormalities and hypoplasia caused by mechanical factors. The posterolateral defect in the diaphragm occurs from abnormal organogenesis within the pleuroperitoneal fold. Knockout mice for c-met receptors show formation of an amuscular diaphragm. Lung development is impaired from herniated viscera resulting in a smaller amount of bronchial divisions and alveoli. Murine models have demonstrated altered patterns of gene expression responsible for lung development. The decreased pulmonary vascular cross-sectional area coupled with muscularized arterioles contributes to the degree of pulmonary hypertension. Although the surgically created fetal lamb CDH model does not recapitulate the early embryological aspects of human CDH, it has served to delineate the mechanical factors responsible for lung hypoplasia and pulmonary vascular abnormalities. Studies in fetal lamb tracheal drainage and tracheal occlusion have demonstrated the importance of fetal lung fluid dynamics in lung development. These studies stimulated research in fetal tracheal occlusion which has been shown experimentally and clinically to accelerate lung growth in CDH.

The Nitrofen rodent model is the most widely used small animal model of CDH. A multitude of studies have contributed to our understanding of the pathogenesis of CDH in the areas of altered gene expression, retinoids, extracellular matrix, and mechanical factors. Prenatal treatments with tracheal occlusion, glucocorticoids, and retinoids have been tested with this model with moderate success. The left-sided posterolateral defect (Bochdalek’s hernia), accounts for 80% of cases. Right-sided hernias account for 15–20% of CDHs. Analysis of lung tissue from CDH shows evidence of lung hypoplasia such as small air spaces, thicker septa, immature alveolar type II cells, and abnormal vasculature. Some infants may have parietal pleural and peritoneal membrane acting as hernia sac in addition to the diaphragmatic defect. The pathophysiology of CDH is complex. Prenatally, the diagnosis is made by ultrasound demonstrating herniated abdominal viscera in the thoracic cavity. The fluid-filled stomach and small bowel contrast with the more echogenic lungs. Diagnosis of right-sided CDH is often challenging because only the liver may be herniated and the echo densities of the liver and the lung are similar. Therefore, fetal MRI has been advocated for patients suspected of fetal CDH to accurately define the lesion, exclude other thoracic abnormalities, and determine liver position. The most accurate sonographic predictors of survival in left-sided CDH are liver position and the lung to head ratio (LHR). The most severe cases have a LHR o0.9 and liver herniation. Postnatally, the majority of infants present with respiratory distress at birth or within the first few hours of life. Physical examination reveals a scaphoid abdomen and decreased breath sounds on the affected side. The diagnosis is confirmed by a chest radiograph (Figure 1). The radiographic findings of the left-sided CDH include presence of bowel loops within the left hemithorax with mediastinal shift to the right. Right-sided lesions are often harder to appreciate and may appear as lobar consolidation, pleural effusion, or diaphragmatic eventration. Although postnatal survival has improved to over 80% in select centers, overall survival remains 65% in large series. Despite improved survival, there is growing recognition of the significant long-term morbidity in survivors. The majority of patients with CDH are best treated by planned delivery in a tertiary center with ECMO support. Over the past decade, significant advances have been made in the postnatal management of CDH. Key aspects include avoidance of barotrauma using a ‘gentle ventilation’ strategy, meticulous management of pulmonary hypertension with NO, judicious use of ECMO, and

622 LUNG DEVELOPMENT / Congenital Parenchymal Disorders

delayed surgery until the infant is stabilized. For select patients, thoracoscopic repair had been reported. Over the past 25 years, there has been a tremendous amount of experimental and clinical work directed toward fetal surgery for CDH, primarily at University of California at San Francisco (UCSF) and Children’s Hospital of Philadelphia (CHOP). Fetal surgical techniques and strategies have evolved considerably, from open fetal surgery to percutaneous

placement of a detachable balloon for tracheal occlusion (Figure 2). Although a recent randomized clinical trial demonstrated equivalent survival in patients treated with fetal tracheal occlusion and conventional postnatal management, inclusion criteria did not select for the most severe cases. Therefore, novel treatment strategies are still being explored for the subset of severe patients to increase survival and also reduce the devastating long-term morbidity among survivors. To avoid neonatal hypoxemia and barotrauma, a novel unpublished strategy is the ex utero intrapartum treatment (EXIT) to ECMO technique. Another strategy to avoid barotrauma and promote lung growth is the use of partial or total liquid ventilation. One small trial with partial liquid ventilation while on ECMO support suggested a benefit with perfluorocarbon induced lung growth (PILG). Total liquid ventilation, while promising, remains experimental.

Bronchogenic Cysts

Figure 1 Anteroposterior chest radiograph in a neonate with congenital diaphragmatic hernia (CDH) demonstrating bowel in the left chest. The heart and mediastinum are shifted to the right. Note intrathoracic position of nasogastric tube.

Bronchogenic cysts arise from abnormal budding of the ventral diverticulum of the foregut that leads to a focal cystic duplication of the tracheobronchial tree. There is a slight male predominance. The true incidence is unknown. Embryologically, they are thought to be supernumerary foregut buds that become isolated from the tracheobronchial tree and progressively enlarge to form a discrete cystic mass of nonfunctioning pulmonary tissue. Hilar and paratracheal cysts are considered to have been derived from the tracheal diverticula.

Perfusion scope

Ultrasound

Balloon inflated Balloon detached

Figure 2 Technique of ultrasound guided fetoscopic tracheal occlusion with a detachable balloon.

LUNG DEVELOPMENT / Congenital Parenchymal Disorders 623

Central cysts are of early embryonic origin and present as a solitary cyst. Peripheral cysts result from developmental abnormalities during the 6th to 16th week of gestation and may be multiple and extensive. Approximately two-thirds of congenital bronchogenic cysts are located centrally near the mediastinum or hilum, and one-third are intraparenchymal. In the mediastinum carinal location is most frequent, but they may also be found in the posterior or anterior mediastinum. Unusual locations include the neck, pericardium, and abdominal cavity. The cysts are uni- or multilocular. Bronchogenic cysts have cuboidal to columnar ciliated (respiratory) epithelial lining surrounded by a fibromuscular wall which may contain cartilage and nests of glands resembling bronchial submucosal glands. Bronchogenic cysts are usually identified in children or young adults. Younger patients present with respiratory symptoms due to mass effect on the tracheobronchial tree, whereas older patients typically develop symptoms secondary to infection of the cyst. Infections occur in 20% of patients with intraparenchymal cysts, usually secondary to communication with the tracheobronchial tree. However, most bronchogenic cysts in children are found incidentally on imaging. Radiographically, these cysts are spherical and may be air-filled if they communicate with the airway or solid if there is no communication. Air–fluid levels suggest infection. Bronchial communication is more common with peripheral cysts. If the communication exists, a flap-valve type of obstruction produces rapid enlargement of a tension cyst which may produce sudden respiratory distress and may even resemble pneumothorax. The CT attenuation varies with the composition of the cyst fluid (Figure 3). Approximately half of them have

water attenuation and the rest may appear solid or hyperattenuated secondary to high protein content, hemorrhage, or calcium content. MRI demonstrates relatively high signal intensity on T1 and a high homogeneous signal on T2-weighted images. Treatment consists of surgical excision. Surgical excision is justified even in asymptomatic patients because of the risk of infection and enlargement leading to respiratory distress. Many of these lesions are amenable to minimally invasive surgical excision using thoracoscopic or robotic techniques (Figure 4). Intraparenchymal cysts require segmental resection or lobectomy.

Figure 3 A chest CT scan demonstrating a well-defined hyperattenuated mass (bronchogenic cyst) in the mediastinum.

Figure 4 Intraoperative photograph of minimally invasive robotic excision of a mediastinal bronchogenic cyst.

Congenital Lobar Emphysema Congenital lobar emphysema (CLE) is an uncommon cause of respiratory distress in infancy which presents with hyperinflation of one or more lobes and compression of normal lung. The left upper lobe is most frequently affected, followed by right middle lobe and right upper lobe. Associated cardiac defects are seen in 14–20% of patients. There is a male predominance. The most frequently identified cause of CLE is obstruction of the developing airway and creation of a ball-valve mechanism in the bronchus causing air trapping. Intrinsic obstruction may be caused by bronchomalacia, mucosal plugging, granulomas, or mucosal infolding. Extrinsic obstruction may be caused by vascular compression or intrathoracic masses. There have been case reports suggesting a possible heritable basis for this condition. Although the total alveolar number is significantly increased, the airways and arteries are normal. This alveolar proliferation is postulated to be secondary

624 LUNG DEVELOPMENT / Congenital Parenchymal Disorders

Figure 5 Anteroposterior chest radiograph demonstrating massive overdistension of the left upper lobe with shifting of the mediastinum to the right, and compression of the ipsilateral and contralateral lobes.

to chronic obstruction. In most cases, a hypoplastic bronchial cartilage with bronchial collapse creates a ball-valve mechanism leading to hyperinflation. This bronchial defect probably occurs during the fourth to sixth week of embryonic development. Normal lobes are collapsed and the mediastinum is shifted to the contralateral side. CLE is a rare cause of respiratory distress in the neonatal period. Infants present with tachypnea and hypoxemia usually within the first few days of life. On examination, breath sounds are reduced on the affected side. Chest radiograph demonstrates hyperinflation of the affected lobe with compression of the unaffected ipsilateral lobes and mediastinal shift (Figure 5). CT scan and ventilation/perfusion (V/P) scan may be helpful in confirming the diagnosis. Bronchoscopy may reveal bronchomalacia of the affected lobe. Prenatal diagnosis of CLE has been reported and may optimize postnatal therapy. CLE is treated with surgical resection of the abnormal lobe. During induction of anesthesia, positive pressure ventilation may rapidly expand the already overinflated lung, and immediate thoracotomy may be required. Outcome after surgical treatment for patients with CLE diagnosed early is generally good.

Congenital Cystic Adenomatoid Malformation CCAM is a congenital abnormality of the lung characterized by a cystic mass of disorganized pulmonary

parenchyma with proliferation of terminal respiratory bronchioles and lack of normal alveoli. There is a slight male predominance, with equal occurrence in right and left lung. The pathogenesis of CCAM is unknown. Evidence suggests cell proliferation and decreased apoptosis. Pathologically, it has been attributed to an overgrowth of the bronchioles, with almost complete suppression of alveolar development between the seventh and tenth weeks of embryonic life. Recent studies of the cellular components of CCAM found that discordant development of the epithelial and mesenchymal parts of developing lung leads to an abnormal proliferation of these components. Characteristics of rapidly enlarging fetal CCAMs include elevated platelet-derived growth factor B. A successful model to understand pathophysiology of fetal hydrops and CCAM was created by inflating an intrathoracic tissue expander in fetal sheep. Based on this model, hydrops associated with CCAM results from obstruction of cardiac venous return and central venous hypertension. This was reversed by expander deflation, which simulates in utero CCAM resection. The multicystic mass of pulmonary tissue in CCAMs resemble terminal bronchioles lined by cuboidal or columnar epithelium with increased underlying stromal elastica and smooth muscle. This malformation has been classically subdivided into three types, based on combination of gross and pathologic features and presence or absence of other anomalies. In a subsequent classification two additional types (type 0 and type 4) were proposed based upon the site of origin (Table 3). Gross findings vary from almost entirely solid to cystic lesions. CCAM type 0 corresponds to acinar dysplasia or dysgenesis with lung showing only bronchial-like structures, cartilage plates, and loose vascularized mesenchyme. The smaller cysts in type 1 lesions and cysts in type 2 resemble dilated terminal bronchioles. Mucinous cells are found in all of type 0 and a one-third of type 1 lesions. Type 3 CCAMs are bulky masses with histological resemblance to early canalicular stage of lung development. Type 4 lesions are thin-walled cysts in distal lung tissue. There is a high incidence of associate anomalies in type 2 CCAM such as renal agenesis or dysgenesis, cardiac including truncus arteriosis and tetralogy of Fallot, jejunal atresia, diaphragmatic hernia, hydrocephalus, and skeletal anomalies. CCAMs are equally distributed between right and left lungs, but lesions are usually limited to one lobe. Presence of hybrid lesions in CCAMs with bronchopulmonary sequestration (BPS) has been well described. It has been shown that 50% of extralobar sequestration may be associated with a coexisting

LUNG DEVELOPMENT / Congenital Parenchymal Disorders 625 Table 3 Pathologic features of congenital cystic adenomatoid malformation Features

Type 0

Type 1

Type 2

Type 3

Type 4

Approximate frequency Cyst size (maximum cm) Epithelial lining (cysts)

1–3

465

20–25

8

2–4

0.5

10

2.5

1.5

7.0

Ciliated; pseudostratified; tall columnar with goblet cell 100–500

Ciliated; pseudostratified; tall columnar

Ciliated; cuboidal or columnar

Ciliated; cuboidal

Flattened; alveolar lining cells

100–300

50–100

0–50

25–100

Present in 33% of cases Present in 5–10% of cases Absent

Absent

Absent

Absent

Absent

Absent

Absent

Present in 5% of cases

Absent

Absent

Muscular wall thickness of cysts (in mm) Mucous cells

Present in all cases

Cartilage

Present in all cases

Skeletal muscle

Absent

Reproduced from Pulmonary Pathology, 2nd edn., 1994, pp. 155–190, Congential and developmental diseases, Stocker JT, table 7-12, with kind permission of Springer Science and Business Media.

CCAM. Typically these lesions show histological features of CCAM with an anomalous systemic feeding vessel. Patients with small CCAMs may be asymptomatic. Patients with large lesions present with respiratory distress caused by overdistension of the involved lobe and compression of normal lung. Chest radiograph usually demonstrates a unilateral, multicystic, airfilled lung lesion. They may become larger because of air and fluid trapping, resulting in mass effect on the adjacent lung and mediastinum. CT imaging of these lesions demonstrates the cystic nature with the intervening septa and delineates the extent and distribution of this disease (Figure 6). Prenatal ultrasonography can detect CCAMs showing a cystic or multicystic mass in the fetal thorax with associated mass effect. Sequential prenatal ultrasonography has demonstrated regression in approximately 15% of cases while hydrops develop in up to 10%. Large CCAMs may cause cardiac and caval compression leading to cardiac failure and hydrops which is lethal without treatment in utero. The development of fetal hydrops mandates fetal intervention because of certain fetal demise without treatment. Lesions with a dominant cyst may be decompressed with a thoracoamniotic shunt. Large solid lesions require open fetal surgery with lobectomy to reverse hydrops and allow compensatory lung growth of the unaffected lung. In large fetal CCAMs thought to cause severe cardiopulmonary compromise at birth, resection on placental support during the EXIT procedure with ECMO standby is advocated. Postnatally, the treatment is pulmonary lobectomy of the entire lobe containing the CCAM. Although

Figure 6 A chest CT scan demonstrates a multicystic mass with intervening septa in the right lower lobe consistent with a CCAM.

somewhat controversial, current recommendations include resection for asymptomatic CCAMs due to the risks of infection and malignant transformation, particularly mucinous bronchioloalveolar carcinoma and mesenchymal rhabdomyosarcoma. Despite successful resection of large lesions, severe respiratory failure and shunting may develop from pulmonary hypoplasia and pulmonary hypertension which can be treated with ECMO. The overall prognosis depends on the size and pathophysiology of the lesion.

Bronchopulmonary Sequestration Bronchopulmonary sequestration (BPS) is defined as nonfunctioning lung tissue that lacks an obvious

626 LUNG DEVELOPMENT / Congenital Parenchymal Disorders Table 4 Intralobar versus extralobar sequestration Feature

Intralobar sequestration

Extralobar sequestration

Age at diagnosis Sex ðM : FÞ Side affected Arterial supply Venous drainage Associated anomalies Pathogenesis

50% over age 20 years (15% asymptomatic) 1:5 : 1 Left, 55% Systemic Pulmonary (rarely systemic) Uncommon (6–12%) Congenital anomaly

60% less than 6 months (10% asymptomatic) 3:1 Left, 65% Systemic (rarely pulmonary) Systemic or pulmonary Over 60% Congenital anomaly

Reproduced from Travis WD, Colby TV, Koss MN, et al. (2002) Congenital anomalies and pediatric disorders. In: Travis WD and Colby TV (eds.) Atlas of Nontumor Pathology: Non-Neoplastic Disorders of the Lower Respiratory Tract, 1st Series, Fascicle 2, pp. 473–538. Washington, DC: American Registry of Pathology and Armed Forces Institute of Pathology, with permission from American Registry of Pathology.

communication with the tracheobronchial tree and receives its arterial supply from the systemic circulation. There is a spectrum of malformations, and many combinations of systemic and pulmonary arterial blood supply, venous drainage, and pulmonary tissue have been reported. Sequestrations are classified as either intralobar or extralobar (Table 4). BPS occurs as a sporadic anomaly with no genetic predisposition. Intralobar Sequestration

In intralobar sequestration the abnormal tissue is contained within the normal pleural investment of the lung. They are predominantly found in the lower lobes, most commonly in the posterior basilar segment of the left lobe. They lack communication with the tracheobronchial tree and are supplied by a systemic artery arising from the aorta below the diaphragm. More than one vessel may supply the sequestration. The venous drainage is usually via pulmonary veins, but occasionally they drain to the azygous system. Intralobar sequestration is the more common malformation seen in infants and children. There is a slight male predominance, with male to female ratio of 1:5 : 1. Other associated anomalies are unusual. Intralobar sequestrations may develop following obstruction of the tracheobronchial tree and secondary development of a systemic arterial supply from hypertrophied pulmonary ligament arteries. This theory explains the propensity of these lesions to occur in the medial lower lobe. Grossly, intralobar sequestration demonstrates the effect of chronic inflammation with thickened and adhered pleura resulting from recurrent or prolonged infection and/or inflammation. Histologically, parenchyma shows effects of inflammation and fibrosis by dilated bronchi filled with mucus or purulent material and alveoli filled with alveolar macrophages. Thick-walled vessels are noted reflecting the systemic vascular supply. The development of malignancy has been described in intralobar sequestration.

Figure 7 A chest CT scan demonstrating a heterogeneous, multicystic mass in the left lower lung consistent with an infected intralobar pulmonary sequestration.

Patients usually present with symptoms of pulmonary infection in childhood or early adulthood. As a result of anomalous vascular supply, arteriovenous fistula with high output failure or massive hemoptysis or hemothorax may occur. Radiographic findings range from opacification from persistent parenchymal consolidation with irregular borders to a cystic parenchymal lesion with an air–fluid level. Homogeneous or heterogeneous mass or consolidation with heterogeneous contrast enhancement is seen on the CT imaging (Figure 7). These lesions may have irregular borders. Multiple thin-walled cystic spaces with or without air–fluid levels may be seen within the lesion. To delineate the blood supply to these lesions, cross-sectional imaging is particularly helpful. Less invasive studies have supplanted angiography to delineate the anomalous arterial supply. Ultrasound with Doppler interrogation, multidetector CT with three-dimensional reconstruction, and magnetic resonance angiography (MRA) clearly delineate the pulmonary and vascular anatomy. Treatment is surgical resection of the involved lobe. Inflammatory changes from previous infections

LUNG DEVELOPMENT / Congenital Parenchymal Disorders 627

can produce dense adhesions, obliterate intersegmental planes, and make intraoperative identification of the systemic artery difficult. Prognosis is excellent following resection. In select cases, thoracoscopic resection may be possible. Extralobar Sequestration

Extralobar sequestration (also known as accessory lung or Rakitansky lobe) is a congenital anomaly in which the sequestration is separate from the normal pulmonary lobe and has its own pleural investment. Extralobar sequestration is more common than intralobar sequestration in the fetus and neonates. It may occur within the thorax or in a subdiaphragmatic location. Extralobar sequestrations are associated with multiple other anomalies such as chest wall deformity, pericardial defects, enteric duplication, congenital heart defects, and CDH. A widely accepted theory of embryogenesis postulates that a supernumerary lung bud arises caudal to the normal lung bud and migrates caudally with the esophagus. If this development occurs subsequent to pleural formation, the bud will grow separately and becomes invested with its own pleura, forming extralobar sequestration. However, if the development occurs prior to pleural formation, the bud is invested with adjacent pleura and may become an intralobar sequestration. Extralobar sequestrations are generally single and pyramidal or ovoid in shape. Remnants of bronchial cartilage may be found in half of these cases. Microscopically, dilated airways lined by bronchiolar type epithelium and dilated airspaces lined with type I and II pneumocytes are found. In infected cases, there is non-specific acute and chronic inflammation, fibrosis, and purulent exudate. Extralobar sequestration has a male predominance, with male to female ratio of 3 : 1 and almost always involves the left lung. Most patients present before the age of 6 months, but some have been described in adults. These lesions have been associated with other abnormalities, especially CDH. Many patients are asymptomatic and the lesion is detected as a part of evaluation for an associated anomaly. Although there is no connection with the tracheobronchial tree, infection can occur by hematogenous spread of bacteria. Subdiaphragmatic extrapulmonary sequestrations may present as an abdominal mass on prenatal ultrasonograms. These lesions typically appear more homogeneous and hyperechoeic relative to liver and spleen and an arterial-feeding vessel is characteristically seen. Color Doppler has been used to demonstrate the systemic vessels supplying sequestration on prenatal ultrasonograms. There have been reports of

Figure 8 Coronal reconstruction from a multidetector CT scan demonstrating a left extralobar sequestration with two systemic arteries originating from the abdominal aorta.

development of fetal hydrops in association with sequestration. Fetal pulmonary sequestrations may spontaneously regress by serial prenatal ultrasonography. The mechanism by which these lesions shrink is unknown. Differential diagnosis in these cases should include neuroblastoma, germ cell tumor, CDH, collapsed lung, and foregut duplication. Radiographically, they appear as a well-marginated, homogeneous, triangular mass in the thorax. These lesions usually appear to be homogeneous on CT, ultrasonogram, or MRI (Figure 8). Extralobar sequestration is treated by surgical resection. Thoracoscopic resection is easily performed for this lesion. Operative results are excellent, but overall prognosis depends on associated anomalies. See also: Chronic Obstructive Pulmonary Disease: Emphysema, General. Lung Development: Overview.

Further Reading Baptista MJ, Melo-Rocha G, Pedrosa C, et al. (2005) Antenatal vitamin A administration attenuates lung hypoplasia by interfering with early instead of late determinants of lung underdevelopment in congenital diaphragmatic hernia. Journal of Pediatric Surgery 40: 658–665. Bouchard S, Johnson MP, Flake AW, et al. (2002) The EXIT procedure: experience and outcome in 31 cases. Journal of Pediatric Surgery 37: 418–426. Cass DL, Quinn TM, Yang EY, et al. (1998) Increased cell proliferation and decreased apoptosis characterizes congenital cystic adenomatoid malformation of the lung. Journal of Pediatric Surgery 33(7): 1043–1047. Chowdhury M, Samuel M, Ramsay A, et al. (2004) Spontaneous postnatal involution of intraabdominal pulmonary sequestration. Journal of Pediatric Surgery 39(8): 1273–1275. Corbett HJ and Humphrey GM (2004) Pulmonary sequestration. Paediatric Respiratory Reviews 5(1): 59–68. Deprest J, Gratacos E, Nocolaides KH, FETO Task Group (2004) Fetoscopic tracheal occlusion (FETO) for severe congenital

628 LUNG DEVELOPMENT / Congenital Vascular Disorders diaphragmatic hernia: evolution of a technique and preliminary results. Ultrasound in Obstetrics & Gynecology 24(2): 121– 126. Evans MG (1996) Hydrops fetalis and pulmonary sequestration. Journal of Pediatric Surgery 31(6): 761–764. Gabarre JA, Izquierdo AG, Ponferrada MR, et al. (2005) Isolated unilateral pulmonary agenesis: early prenatal diagnosis and long term follow-up. Journal of Ultrasound in Medicine 24: 865–868. Guilbert TW, Gebb SA, and Shannon JM (2000) Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development. American Journal of Physiology: Lung Cellular and Molecular Physiology 279: L1159–L1171. Liechty KW, Crombleholme TM, Quinn TM, et al. (1999) Elevated platelet-derived growth factor-B in congenital cystic adenomatoid malformations requiring fetal resection. Journal of Pediatric Surgery 34(5): 805–810. McLean SE, Pfeifer JD, Siegel MJ, et al. (2004) Congenital cystic adenomatoid malformation connected to an extralobar pulmonary sequestration in the contralateral chest: common origin? Journal of Pediatric Surgery 39(8): 13–17. Olutoye OO, Coleman BG, Hubbard AM, et al. (2000) Prenatal diagnosis and management of congenital lobar emphysema. Journal of Pediatric Surgery 35(5): 792–795. Ozcan C, Celik A, Ural Z, et al. (2001) Primary pulmonary rhabdomyosarcoma arising within cystic adenoid malformation: a case report and review of the literature. Journal of Pediatric Surgery 36(7): 1062–1065. Paterson A (2005) Imaging evaluation of congenital lung abnormalities in infants and children. Radiologic Clinics of North America 43: 303–323. Shanmugam G, MacArthur K, and Pollock JC (2005) Congenital lung malformations: antenatal and postnatal evaluation and management. European Journal of Cardiothoracic Surgery 27: 45–52. Stocker JT (1994) Congenital and developmental diseases. In: Dial DH and Hammar SP (eds.) Pulmonary Pathology, 2nd edn., pp. 155–190. New York: Springer. Travis WD, Colby TV, Koss MN, et al. (2002) Congenital anomalies and pediatric disorders. In: Travis WD and Colby TV (eds.) Atlas of Nontumor Pathology: Non-Neoplastic Disorders of the Lower Respiratory Tract, 1st Series, Fascicle 2, pp. 473–538. Washington, DC: American Registry of Pathology and Armed Forces Institute of Pathology. Wilson RD, Baxter JK, Johnson MP, et al. (2004) Thoracoamniotic shunts: fetal treatment of pleural effusion and congenital cystic adenomatoid malformations. Fetal Diagnosis and Therapy 19: 413–420.

abnormalities in early organogenesis are postulated. Pulmonary vascular abnormalities are usually associated with other congenital abnormalities. These anomalies are rarely diagnosed prenatally. Symptoms begin in infancy or childhood. Diagnosis is made by history, physical examination, radiologic examination, and angiography. Current therapy consists of complex medical and surgical management.

Vascular Sling Anomaly

& 2006 Elsevier Ltd. All rights reserved.

A vascular sling anomaly refers to an anomalous or aberrant left pulmonary artery that arises from the right pulmonary artery, and causes airway obstruction as it courses between the esophagus and trachea to the left lung. More than half of all these cases have associated cardiac defects, such as patent ductus arteriosus (PDA), ventricular septal defect (VSD), atrial septal defect, atrioventricular canal, single ventricle, or aortic arch anomalies. The diagnosis is usually made during infancy. Both respiratory symptoms and feeding problems may occur. Common symptoms include coughing, wheezing, stridor, choking, cyanosis, and apnea. A history of atelectasis, emphysema, or pneumonia of the right lung is found with vascular sling anomalies. A variety of radiographic tests are used for diagnosis. Barium esophagogram demonstrates a characteristic anterior indentation of esophagus seen in the lateral view at the level of carina. A right-sided indentation is usually seen on the posteroanterior view. The right lung is either hyperlucent or atelectatic with pneumonic infiltrations (Figure 1). Angiography is still the current standard, although multidetector computed tomography (CT) and magnetic resonance imaging (MRI)/magnetic resonance angiography (MRA) are novel, less invasive imaging modalities that demonstrate the aberrant course of the left pulmonary artery and allow for assessment of the tracheal lumen (Figure 2). Surgical correction requires left thoracotomy, with division of the left pulmonary artery and reimplantation to the main pulmonary artery. Associated tracheal lesions often require tracheoplasty. Simultaneous correction of associated cardiac defects may be performed in select patients. In infants who have had surgery for severe symptoms, airway obstruction may persist for weeks or months. Pre-existing tracheomalacia requires careful respiratory management in the postoperative period.

Abstract

Pulmonary Arteriovenous Malformations

Congenital vascular and lymphatic disorders of the lung include vascular sling anomaly, pulmonary arteriovenous malformations, scimitar syndrome, diffuse pulmonary lymphangiectasia, and diffuse pulmonary lymphangiomatosis. The pathogenesis of most pulmonary vascular anomalies is not known, but developmental

Pulmonary arteriovenous malformations (PAVMs) were first described at autopsy in 1897 and consist of abnormal communication between pulmonary artery and veins; they are usually congenital in nature. They

Congenital Vascular Disorders G B Mychaliska and R Seetharamaiah, University of Michigan, Ann Arbor, MI, USA

LUNG DEVELOPMENT / Congenital Vascular Disorders 629

Figure 1 Barium esophagogram in a child with a vascular sling anomaly. Anteroposterior view showing significant indentation of the esophagus at the level of the carina.

Figure 2 A chest CT scan showing the aberrant course of the left pulmonary artery as it arises from the proximal right pulmonary artery and courses between the narrowed trachea and esophagus.

may exist as single or multiple pulmonary artery to pulmonary vein connections that bypass the pulmonary–capillary bed resulting in a right-to-left shunt. About 70% of these patients have hereditary

hemorrhagic telangiectasia (HHT or Osler–Weber– Rendu syndrome), an autosomal dominant vascular dysplasia. Conversely, 30% of patients with HHT have PAVMs. PAVMs are classified as simple or complex. A simple PAVM consists of a single or multiple feeding arteries originating from a single segmental artery. In complex PAVMs, feeding arteries originate from two or more segmental arteries. Approximately 50% are small (o1 cm) and tend to be multiple; the rest are greater than 1 cm and tend to be subpleural. Individual PAVMs are typically 1–5 cm in size. Fifty to seventy per cent of them are found in the lower lobes and 75% are unilateral. PAVMs occur twice as often in women as in men, but there is a male predominance in newborns. Grossly, arteriovenous malformations (AVMs) show abnormal dilated vessels of varying sizes. These malformations may appear as a large, single sac or as a plexiform mass of dilated vascular channels or a dilated and tortuous direct communication between artery and vein. Histologically, there is a tangle of abnormal vessels of varying sizes that may contain secondary thrombosis. The pathogenesis of PAVMs is not known. However, there are several proposed theories of their pathogenesis: a defect in terminal arterial loops may allow dilation of thin-walled capillary sacs; during fetal development there may be incomplete resorption of the vascular septae that separate the arterial and venous plexuses; and, lastly, failure of capillary development during fetal development may be followed by progressive dilation of favored limbs of smaller plexuses and formation of large saccular PAVMs. There is preliminary evidence that PAVMs associated with HHT have abnormalities on chromosome 5. The classic triad of dyspnea on exertion, cyanosis, and clubbing of fingers are seen in AVMs. However, at least 30% right-to-left shunting is required. The presence of symptoms correlates best with the lesion size. Small arteriovenous malformations (shunting o25% of pulmonary blood flow) have no true cyanosis and half of these patients are asymptomatic. The incidence of symptoms is greater in patients with multiple rather than single PAVMs. The presence of a PAVM should be suspected in patients with one or more of the features presented in Table 1. The onset of symptoms is usually in adolescence or adulthood. On examination, a soft systolic or continuous murmur or a bruit may be present over the lesion. Patients with PAVMs are at risk for rupture, bleeding, or paradoxical emboli resulting in stroke or cerebral abscess. A more extensive list of complications is listed in Table 2. Neurologic complications occur in approximately 40% of adults with untreated PAVMs

630 LUNG DEVELOPMENT / Congenital Vascular Disorders Table 1 Features suspicious for the presence of PAVMs One or more pulmonary nodules associated with suspicious X-ray findings Mucocutaneous telangiectases Hemoptysis Symptoms of right-to-left shunt, such as dyspnea, hypoxemia, polycythemia, clubbing, cyanosis, cerebral embolism, or brain abscess A relative with hereditary hemorrhagic telangiectasia (HHT)

Table 2 Complications of PAVMs Seizure Transient ischemic attack Cerebrovascular accident Migraine headache Cerebral thrombosis Brain abscess Hypoxemia Hemothorax Hemoptysis Pulmonary hypertension Polycythemia Anemia Congestive heart failure Infectious endocarditis

and hemorrhagic complications occur in approximately 15%. In contrast, the largest series in children noted neurologic complications in 19% and hemorrhagic complications in 7% before treatment. On chest radiograph, these malformations appear as round or oval, somewhat lobulated pulmonary nodules or masses of uniform density in various sizes. Less frequently, a tangle of dilated blood vessels presents as parenchymal opacity that mimics consolidation. Although pulmonary angiography remains the gold standard for diagnosis, several other less invasive imaging modalities have been developed. Cross-sectional imaging with CT or MRI demonstrates excellent sensitivity for the detection of PAVMs. Contrast echocardiography is an excellent tool for evaluation of cardiac and intrapulmonary shunts, and is able to identify small shunts. The finding of an intrapulmonary shunt warrants additional evaluation for PAVMs with pulmonary angiography. Transcatheter embolotherapy has supplanted surgical resection as the preferred treatment of PAVMs in children and adults. Symptomatic patients with PAVMs should be treated, and also asymptomatic patients with PAVMs 42 cm in diameter, or with feeding vessels X3mm in diameter. Embolization using coils or detachable balloons is safe, well tolerated, and results in excellent symptomatic improvement. Long-term follow-up with radiographic and physiologic evaluation should be performed to document involution of embolized PAVMs and

Figure 3 Intraoperative thoracoscopic photograph of a PAVM localized to one lobe.

detect enlargement of small previously undetected PAVMs. Reperfusion and recurrence of treated PAVMs may occur due to recanalization of embolized arteries, recruitment of normal branches, or development of a systemic arterial supply. Antimicrobial prophylaxis (for dental procedures, etc.) is advocated. Surgical resection of arteriovenous malformation should be considered if transcatheter embolotherapy is unsuccessful (Figure 3). Extensive disease isolated to one lobe may respond better to treatment with surgical lobectomy. Surgical resection of PAVMs is associated with negligible mortality and minimal morbidity associated with thoracotomy.

Scimitar Syndrome Scimitar syndrome is also known as hypogenetic lung syndrome and congenital venolobar syndrome. The main features of this rare congenital malformation include anomalous pulmonary venous return (usually draining to the inferior vena cava (IVC), portal or hepatic vein, or the right atrium), right lung hypoplasia, absent or small pulmonary artery perfusing the abnormal lung, systemic arterial supply from the thoracic or abdominal aorta, anomalies of the right hemidiaphragm, and dextroposition of the heart. Approximately 30% of patients have associated cardiac anomalies, especially atrial septal defects. There is a slight female predominance. The moniker scimitar syndrome stems from the anomalous pulmonary vein, which may be visible on chest radiographs in the shape of a Turkish sword or scimitar (Figure 4). Three types of the syndrome have been described: 1. The adult form is characterized by a small shunt from right pulmonary vein to IVC and intracardiac defect. This is usually well tolerated and has

LUNG DEVELOPMENT / Congenital Vascular Disorders 631

Figure 5 An oblique coronal (maximum intensity projection) CT image of scimitar syndrome. Note the right lung hypoplasia and the anomalous origin of the systemic blood supply to the right lower lung from the abdominal aorta.

Figure 4 Chest radiograph in a child with scimitar syndrome. Note the sickle-shaped shadow in the medial aspect of the right lower lung.

a good prognosis. It is detected incidentally or when a child is evaluated for dyspnea or recurrent pulmonary infection. 2. The infantile form is characterized by a large shunt from systemic arterial supply to the lower part of the right lung (Figure 5). This is symptomatic in early infancy with respiratory distress and heart failure, and has a poor prognosis. 3. A third form is characterized by predominant multiple cardiac and extracardiac malformations. The age of presentation and clinical features depend on the size of the right-to-left shunt. Patients with this disorder may be asymptomatic or may present with symptoms related to congenital heart disease, pulmonary hypertension, or repeated pulmonary infections. About a third of these patients present during infancy and childhood. Radiographically, the anomalous vein classically appears as a sickle-shaped shadow in the medial aspect of the right lower lung which resembles a Turkish sword or scimitar. However, the typical

appearance is seen in only a third of the cases. Definitive diagnosis is made with cardiac catheterization and angiography. Velocity-encoded cine MR may be used as a noninvasive method to evaluate associated congenital heart disease and quantification of shunting. Surgical treatment consists of repair of the anomalous venous return and ligation of collaterals. Treatment of scimitar syndrome is controversial. An indication for surgery is the presence of severe cardiac abnormality. A two-stage surgical approach has been suggested for the infantile form. This involves initially ligating the systemic arteries with or without lobectomy, and then rerouting the abnormal pulmonary venous return of the right lung, and if present, correcting the cardiac defects later. Coil embolization has been reported as an alternative to surgical ligation in symptomatic infants before cardiac surgery. Anomalous venous drainage may be redirected to the left atrium in the adult form if the left-to-right shunt is significant. Also, pneumonectomy or lobectomy may be required in selected cases. The prognosis depends on the severity of the shunt, associated cardiac abnormalities, and the severity of pulmonary hypoplasia.

Abnormalities of the Pulmonary Lymphatics Abnormalities of pulmonary lymphatics produce many clinicopathological syndromes such as lymphangioma, lymphangiomatosis, lymphangiectasia, lymphatic dysplastic syndrome, and lymphangioleiomyomatosis. Most of these are rare, although

632 LUNG DEVELOPMENT / Congenital Vascular Disorders

lymphangiectasia and lymphangiomatosis deserve mention. Diffuse Pulmonary Lymphangiectasia

This is a rare but fatal condition with very few patients surviving past infancy. It is a poorly documented congenital anomaly first described by Virchow in 1856. It is characterized by diffusely dilated lymphatic ducts throughout the lung and is associated with several syndromes including Noonan’s, Ulrich–Turner, and Down’s syndromes. There is a male preponderance. The cause of the primary pulmonary lymphangiectasia is not known. However, it appears that pulmonary venous obstruction produces elevated transvascular pressure and engorges the pulmonary lymphatics. Three groups are identified, and referred to as Noonan’s classification: (1) primary pulmonary involvement; (2) secondary pulmonary lymphangiectasia acquired after pulmonary venous obstruction owing to venous or cardiovascular anomalies; and (3) generalized lymphangiectasia. Patients with generalized lymphangiectasia usually have mild pulmonary involvement and are known to have a better prognosis. Microscopically, the disease is characterized by abnormal lymphatics in the subpleural and interlobar connective tissue such as dilation of the septal, perivascular, and subpleural lymphatic vessels. Patients present with dyspnea and cyanosis in the newborn period. Respiratory compromise occurs because of the space-occupying nature of the lesion and decreased compliance with increased work of breathing. Chest radiographs reveal dilated lymphatics as diffuse, bilateral, dense, reticular interstitial opacities. Thickened interlobar septa (Kerley-B lines) and chylous effusions may be noted. The spared areas appear hyperlucent if the lung is not completely involved. Right heart catheterization, CT scan, and open lung biopsy help in the diagnosis. Treatment is mainly supportive, and includes supplemental oxygen, mechanical ventilation, and careful fluid management with diuretics. In rare circumstances of localized disease, surgical resection can be curative. Most patients die shortly after birth, but occasional long-term survival has been reported. Diffuse Pulmonary Lymphangiomatosis

This is a rare, progressive congenital anomaly in which the lymphatics of the lung including the pleura, septa, and bronchovascular bundles are expanded by an increased number of complex anastomosing lymphatic spaces. Males and females are equally affected.

Gross and histological findings reflect the proliferation of the lymphatics. Histologically, this disease is characterized by the distribution of the anastomosing spaces along pulmonary lymphatic pathways, the absence of blood elements in the spaces, single layered lining cells with associated chylous effusion, and spindle cells with elongated, cigar-shaped nuclei. The distinction between the lymphangiectasia and diffuse lymphangiomatosis should be made on the basis of the size and number of participating lymphatic spaces. In lymphangiomatosis the spaces tend to be smaller and more numerous, and the smooth muscle component is more pronounced. Most patients present with respiratory distress or dyspnea in childhood or early adulthood. Hemoptysis is frequent owing to the involvement of airway submucosa by lymphangiomatosis. Chest radiographs reveal increased interstitial marking and chylous effusion. Involvement of the mediastinum or generalized lymphangiomatosis may be evident on radiographs or during surgery. CT scan is helpful in evaluation of the disease. The rate of progression varies among patients. Treatment is mainly supportive. Palliative treatment to alleviate pleural effusion may be helpful. Recent data suggests that interferon therapy may be beneficial. See also: Chest Wall Abnormalities. Genetics: Overview. Lung Development: Overview; Congenital Parenchymal Disorders. Pulmonary Function Testing in Infants.

Further Reading Barker PM, Esther CR Jr, Fordham LA, et al. (2004) Primary pulmonary lymphangiectasia in infancy and childhood. European Respiratory Journal 24: 413–419. Bouchard S, Di Lorenzo M, Youssef S, et al. (2000) Pulmonary lymphangiectasia revisited. Journal of Pediatric Surgery 35(5): 796–800. Gossage JR and Kanj G (1998) Pulmonary arteriovenous malformations: a state of the art review. American Journal of Respiratory and Critical Care Medicine 158: 643–661. Guttmacher AE, Marchuck DA, and White RI Jr (1995) Hereditary hemorrhagic telangiectasia. New England Journal of Medicine 333(14): 918–924. Holt PD, Berdon WE, Marans Z, et al. (2004) Scimitar vein draining to the left atrium and a historical review of the scimitar syndrome. Pediatric Radiology 34: 409–413. Sahin S, Celebi A, Yalcin Y, et al. (2005) Embolization of the systemic arterial supply via a detachable silicone balloon in a child with scimitar syndrome. Cardiovascular and Interventional Radiology 28: 249–253. Scott FE, Cheatham JP, and Bolam DL (2000) Primary transcatheter treatment of congenital pulmonary arteriovenous malformation causing cyanosis of the newborn. Catheter and Cardiovascular Interventions 50: 48–51. Tazelaar HD, Kerr D, Yousem SA, et al. (1993) Diffuse pulmonary lymphangiomatosis. Human Pathology 24(12): 1313–1322.

LUNG IMAGING 633

LUNG IMAGING K Marten, Technical University, Munich, Germany D M Hansell, Royal Brompton Hospital, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract During the past two decades, lung imaging has undergone a marked transformation. In many clinical settings, the chest radiograph remains the first step in radiologic diagnosis. However, the increased availability of multidetector computed tomography (CT) has greatly improved the assessment of the mediastinum, the large airways, and the pulmonary vascular tree, and refined high-resolution CT techniques allow insights into the patterns and distribution of parenchymal and small airways diseases. Current techniques for lung imaging and the most important imaging features encountered in patients with focal and diffuse lung diseases and diseases of the airways are reviewed in this article.

During the past two decades, lung imaging has undergone a marked transformation. In many clinical settings, the chest radiograph remains the first step in radiologic diagnosis. However, the increased availability of multidetector computed tomography (CT) has greatly improved the assessment of the mediastinum, the large airways, and the pulmonary vascular tree, and refined high-resolution CT techniques allow insights into the patterns and distribution of parenchymal and small airways diseases. These CT techniques are, to a limited extent, complemented by magnetic resonance imaging, which can be used for the assessment of the mediastinum and the chest wall. Current techniques for lung imaging and the most important imaging features encountered in patients with focal and diffuse lung diseases and diseases of the airways are reviewed in this article.

Scientific Basis Conventional Chest Radiography

The routine radiographic views of the chest are the posteroanterior (PA) and lateral projections with the patient in upright position. The patient must be positioned such that the X-ray beam is centered properly – the scapulae are rotated anteriorly to be projected laterally away from the lungs. Radiographs are taken at suspended respiration, ideally at full inspiration. Exposure time is as short as possible, with the mean radiation dose at skin entrance not exceeding 0.3 mGy per exposure for a PA chest radiograph. The effective radiation dose for a PA chest X-ray is

approximately 0.02 mSv, corresponding to approximately 30–40% of the observed annual background radiation dose rate, which ranges from 0.5 to 0.9 mSv. The focus-film distance (i.e., the distance between the focal spot of the X-ray tube and the radiograph) should be at least 180 cm to minimize magnification. A high-kilovoltage technique, between 115 and 150 kVp, is generally recommended. When using a grid in order to reduce scatter, a minimum of 10:1 aluminum interspace with 103 lines per inch is recommended. Digital radiography systems provide many advantages over conventional screen-film systems, such as wider exposure latitude, which allows use under a broad range of exposure conditions. Digital systems automatically determine the range of appropriate gray levels and generate image data within that range, rendering the final output image virtually independent of absolute X-ray exposure levels. Image processing yields digital images that are adapted to conventional image characteristics with a simultaneously increased transparency of high-absorption areas such as the mediastinum. Digital detector systems currently available are storage phosphor plates, selenium detectors, or flat-panel direct detectors. Improved dose efficiency of these detector systems and improved image processing lead to excellent image quality that is equivalent or superior to that of conventional radiography film. In addition, the new direct detector systems have the potential for significant dose reduction. Computed Tomography

Three major CT techniques are employed in clinical investigations of the chest. Spiral and multidetector row CTs are standard technologies for the majority of clinical indications of chest CT, whereas high-resolution CT is used for the assessment of diffuse lung diseases as well as airways diseases. Spiral ( ¼ helical) CT requires a scanner with a continuously rotating X-ray tube. The patient travels through the scan plane at a continuous table speed (table feed) during raw data acquisition. A CT image can then be generated from any segment within the scanned volume. The section collimation (SC) determines the spatial resolution that is achieved along the z-axis (i.e., the direction of patient travel) and can be incrementally varied; for example, SC ¼ 0.5, 1, 2, 3, 5, 7, or 10 mm. The table feed (TF) can be selected independently of the SC. The ratio of the TF per

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gantry rotation to the SC is referred to as pitch (P). The pitch is inversely proportional to the patient radiation exposure and directly proportional to the z-axis scan coverage. In image reconstruction, the spacing between the reconstructed sections is called the reconstruction increment (RI), defining the degree of overlap between the axial sections. In contrast to spiral CT systems using a single detector arc or ring, multidetector row CT systems employ two or more parallel detector arrays. Systems with 8, 10, or 16 detector arrays are commonly available. Simultaneous acquisition of multiple sections and reduced gantry rotation times to 0.42 s have greatly increased the performance of these systems, resulting in a shorter scan duration, longer scan ranges, and thinner sections compared to single-slice spiral CT. Shorter scan duration allows for the reduction of motion artifacts and is beneficial in children or critically ill patients. Moreover, it improves CT angiography (CTA) of the pulmonary arteries (e.g., in acute pulmonary embolism) by allowing for scans with a more well-defined contrast enhancement phase and a reduction of the amount of contrast material needed. Longer scan ranges allow for thin section coverage of the entire lungs within a single breath-hold. Thin section isotropic (‘volumetric’) imaging can yield identical submillimeter spatial resolution in all directions, allowing for isotropic multiplanar imaging at arbitrary planes. Multidetector row CT uses various detector types, with matrix detectors, adaptive array detectors, and hybrid detectors being most common. Matrix detectors consist of multiple detector rows of identical width – for example, 16 parallel detector arrays with a width of 1.25 mm each. With four-slice scanners, exposure of only the innermost four detector rows leads to a collimation of 4
1.25 mm. A wider collimation (e.g., 4
2.5, 4
3.75, or 4
5 mm) is achieved by grouping the signal of two or more adjacent detector rows. Accordingly, 16-slice scanners allow for 16
0.75 or 16
1.25 collimations. Hybrid detectors are similar to matrix detectors but have narrower innermost detector rows. Adaptive array detectors consist of detector rows with increasing width from the center of the section toward the periphery, allowing different collimations to be chosen. As in spiral CT, the most important acquisition parameters in multidetector row CT are SC, TF, and P. SC is determined by the available detector configuration, as noted previously. Two definitions of pitch factor are currently used, depending on whether a single SC or the total collimation of the detector array (N
SC) is chosen as the reference: P ¼ TF/ (N
SC), or P* ¼ TF/SC (volume pitch). Whereas P is

independent of the number of detector rows, P* increases with an increasing number of detector rows. The most important reconstruction parameters are the RI, as in spiral CT, and the effective section width (SW) of the reconstructed images. SW must be larger than or equal to the SC, and it may otherwise be chosen independently from SC for most multidetector row scanners. For routine chest CT applications, RI should be chosen to allow a moderate overlap of approximately 20% of SW. A 50% or greater overlap is recommended for three-dimensional (3D) reconstructions. Thin section volumetric multidetector row CT acquisitions of the entire lungs allow for the assessment of focal or diffuse, as well as proximal to distal, airway disease. In this context, postprocessing tools such as multiplanar reformations, minimum intensity projections, or 3D rendering with surface shaded or volume rendered displays play a major role. Moreover, virtual bronchography and virtual bronchoscopy have been developed as dedicated volume-rendering techniques for display of the airways. Virtual bronchography is based on a volume-rendering technique applied at the level of the central airways, allowing reconstruction of 3D images of the airways visualized in semitransparent mode, similar to conventional bronchograms. Virtual bronchoscopy provides an internal rendering of the tracheobronchial walls and lumen, using a perspective-rendering algorithm that simulates an endoscopist’s view of the internal airway surface. This technique is applicable to the central airways, but because of technical limitations, it cannot be used to examine subsegmental bronchi. High-resolution CT (HRCT) may be performed with either single-slice or multidetector row CT scanners. Its principle is based on 1 or 2 mm slice thickness and discontinuous scan acquisition with a 10–20 mm gap between the sections. Thin collimation is mandatory for reduction of partial volume averaging, which occurs with thicker sections and blurs fine detail. The application of a high-resolution filter kernel for image reconstruction increases spatial resolution at the expense of increased conspicuity of image noise. Most HRCTs are performed with the patient in the supine position, and inspiratory images suffice if the lung abnormality is diffusely distributed or severe. Additional HRCTs with the patient in the prone position are useful when the abnormality is confined to the dependent portions of the lungs, allowing for differentiation of normal hypostatic changes from subpleural fibrosis. HRCTs in the prone position should be confined to those areas that are suspicious on the supine scan. Expiratory HRCT scans may be obtained to detect air trapping indicating peripheral airway obstruction. Usually,

LUNG IMAGING 635 Table 1 Standard chest CT scan protocol

Table 3 CT pulmonary angiography scan protocol

Patient position Scan range

Patient position Scan range

Respiratory phase Contrast injection

Scan parameters

Supine with arms elevated Standard: thoracic inlet – posterior pleural recess Suspected lung cancer: thoracic inlet – below renal veins, to include liver and adrenal glands End-inspiratory Single-slice CT: 80 ml CM, 2 ml s  1, 20 s delay 4-slice CT: 80 ml CM þ 50 ml saline flush, 2 ml s  1, 20 s scan delay 16-slice CT: 70 ml CM þ 50 ml saline flush, 2 ml s  1, 30 s scan delay Single-slice CT: SC/TF/RI ¼ 5/10/5 mm 4-slice CT: SC ¼ 1.25–3.75, P ¼ 1.5, SW/ RI ¼ 5/4 16-slice CT: SC ¼ 1–1.5, P ¼ 1.3–1.5, SW/ RI ¼ 5/4

SC, slice collimation (mm); TF, table feed (mm/rotation); RI, reconstruction increment (mm); P, pitch; SW, effective section width (mm); CM, contrast material, contrast concentration ¼ 200 mg ml  1 iodine.

Respiratory phase Contrast injection

Scan parameters

Supine with arms elevated Single-slice CT: top of aortic arch – top of diaphragm Multidetector row CT: entire lungs End-inspiratory Single-slice CT: 150 ml CM þ 50 ml saline flush, 4–5 ml s  1, bolus trigger right ventricle 4-slice CT: 120 ml CM þ 50 ml saline flush, 4–5 ml s  1, bolus trigger pulmonary artery 16-slice CT: 80–100 ml CM þ 50 ml saline flush, 4–5 ml s  1, bolus trigger right ventricle Single-slice CT: SC/TF/RI ¼ 1–2/3–4/ 0.7–1 4-slice CT: SC ¼ 1–1.25, P ¼ 1.5, SW/ RI ¼ 1–2/0.75–1 16-slice CT: SC ¼ 0.63–1, P ¼ 1.5, SW/ RI ¼ 1/0.7

SC, slice collimation (mm); TF, table feed (mm/rotation); RI, reconstruction increment (mm); P, pitch; SW, effective section width (mm); CM, contrast material, contrast concentration ¼ 200 mg ml  1 iodine.

Table 2 High-resolution CT scan protocol Patient position

Scan range Respiratory phase

Contrast injection Scan parameters

Supine with elevated arms Prone for differentiation of normal hypostatic change from subpleural fibrosis Thoracic inlet – posterior pleural recess Standard: end-inspiratory To demonstrate air trapping: endexpiratory None Discontinuous scanning with 10–20 mm increment between sections Single-slice CT: SC ¼ 1–1.5 4-slice CT: SC ¼ 1–1.25, SW ¼ 1–2 16-slice CT: SC ¼ 0.5–1, SW ¼ 1–2

SC, slice collimation (mm); SW, effective section width (mm).

fewer expiratory HRCT scans are obtained, at 2 cm or more spacing. Typical scan protocols for standard chest CT, HRCT, and pulmonary CTA examinations are given in Tables 1–3. Magnetic Resonance Imaging

In magnetic resonance imaging (MRI), the patient is exposed to a static external magnetic field (B0), and a short selective excitatory radiofrequency (RF) pulse using the resonance frequency of B0 is applied through an RF coil mimicking a constant B1 field, which flips the macroscopic magnetization rotating around B0 toward the RF pulse direction. With the RF switched off, the excitation will decay and shift the macroscopic magnetization back toward the direction

of B0 (free induction decay). The time required to realign to the main field is termed T1 relaxation time. The specific time constant that characterizes the pace of dephasing due to spin–spin interactions is termed T2 relaxation time. During these processes, while the macroscopic magnetization continues to rotate around B0 an ‘echo’ signal is induced in a receiver coil. Creating a magnetic field gradient along the direction of B0 and applying RF pulses to a specific resonance frequency allows for slice-selective excitation. Simultaneous spatial encoding in the x–y plane along a frequency-encoding perpendicular field gradient and a short phase shift gradient allows for the signals to be spatially resolved, and an image of the distribution of the emitting nuclei can be formed. In MRI of the chest, the spin echo sequence that is composed around a 901 excitation pulse and a 1801 refocusing pulse is most commonly used. The time between the centers of the excitation pulse and the received spin echo is termed echo time (TE). The time interval required to separate two successive 901 pulses on spin echo MRI is known as the repetition time (TR). Short repetition times allow distinction between tissues with different T1 relaxation times and are called T1-weighted sequences. To distinguish tissues with different T2 characteristics, a long TE is required. To maximize T2 differences and to minimize the effect of T1 relaxation, T2-weighted images require long TR intervals. Proton density (PD) sequences minimize the T1 effect (long TR) and the T2 effect (short TE). The signal on PD-weighted images

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is influenced primarily by the PD or relative water content. MRI assessment of the lung parenchyma is hampered by low signal-to-noise ratio due to low proton density, by signal loss from susceptibility and magnetic field inhomogeneity, and by physiologic motion that may partly be compensated by the use of cardiac and respiratory gating. Acquisition times of SE sequences can be reduced by fast spin echo (echotrain including multiple phase-encoding sets of 1801 refocusing pulses and free induction decays with spin echoes) or breath-hold gradient echo imaging (the 1081 refocusing pulse is replaced by gradient inversion). An emerging application is hyperpolarized 3He MRI of the lungs, a technique that is capable of producing high spatial resolution images of the airspaces following inhalation of laser-polarized helium gas. Radionuclide Imaging

Ventilation–perfusion scanning For perfusion lung scanning, either technetium-99m labeled human albumin microspheres or macroaggregated albumin are used. Ventilation imaging is commonly performed using 133xenon. Although sensitive, perfusion imaging is non-specific for diagnosing pulmonary disease because numerous parenchymal diseases result in decreased pulmonary arterial perfusion within the affected lung region. Thromboemboli characteristically cause abnormal blood flow with preserved ventilation (mismatched defects), whereas parenchymal lung disease most commonly causes ventilation and perfusion abnormalities within the same lung zone (matched defects). Positron emission tomography In positron emission tomography (PET) imaging, tomographic images are obtained after administration of positron-emitting radiopharmaceuticals. Emitted positrons collide with electrons, thereby destroying each other upon contact, producing two gamma ray photons of 511 keV that are emitted in opposite directions. These photons are simultaneously detected at opposite sides of a ring-type gantry. The most important agent used is fluorodeoxyglucose (FDG), which is incorporated and phosphorylated by metabolically active, particularly malignant cells, in the same way as glucose. FDG is not metabolized further, remaining within the cell. Therefore, the amount of FDG-6-phosphate imaged by PET is proportional to glucose uptake and metabolism. Ultrasonography

The application of ultrasonography in lung imaging is limited by the physical composition of the

intrathoracic structures because neither aerated lung nor bone transmit sound. Instead, these tissues absorb and reflect incoming sonic energy, respectively, thereby preventing the collection of information about acoustic interfaces behind ribs or within aerated lung tissue. Nevertheless, ultrasonography is extremely helpful in identifying fluid in the pleural space and especially for guiding needle aspiration and drainage.

Indications Chest radiography constitutes the first step in radiologic diagnosis in most clinical settings. According to the American College of Radiology, indications include investigation of signs and symptoms related to the respiratory and cardiovascular systems, followup of previously diagnosed thoracic disease, staging of intrathoracic and extrathoracic tumors, preoperative assessment of patients scheduled for intrathoracic surgery or patients with increased perioperative morbidity or mortality, as well as monitoring of patients using life-support devices and patients who have undergone cardiac or thoracic surgery. Contrast-enhanced spiral CT is performed in patients with suspected mediastinal abnormalities on chest radiography and for the detection and staging of neoplastic disease; diagnosis of (occult) infectious processes and their complications; diagnosis of acute and chronic pulmonary thromboembolism; assessment of the pleura and chest wall; and guidance for percutaneous biopsy of mediastinal, pleural, or pulmonary nodules or masses. The choice of the pulmonary CT examination technique depends on whether the whole lung needs to be evaluated (e.g., with expected focal or multiple pathologies) or merely sampled, in which case discontinuous HRCT can be employed (e.g., for the evaluation of diffuse lung disease). Indications for HRCT include the assessment of large and small airways disease and the evaluation and differential diagnosis of diffuse lung diseases, including the detection of subtle parenchymal changes, morphologic characterization, quantification of parenchymal abnormalities, and localization for open lung biopsy or bronchoalveolar lavage. Virtual bronchography is rarely indicated for improving understanding of complex tracheobronchial abnormalities. Virtual bronchoscopy is occasionally helpful in patients with obstructive airway lesions, in whom it allows visualization of the bronchial tree beyond the obstruction. MRI is a secondary imaging modality for the evaluation of the mediastinum and hila in patients in

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whom CT findings are equivocal. MRI may be superior to spiral CT in the assessment of mediastinal and vascular invasion. MRI is an excellent technique for assessment of the chest wall, including primary chest wall tumors and chest wall extension by lymphoma or pulmonary carcinoma, particularly if the lesions are located in the superior sulcus region. Ventilation/perfusion (V/Q) scanning has been widely replaced by CT as the first-line imaging technique in patients with suspected acute pulmonary embolism because approximately 60% of V/Q scans in these patients are not definitively diagnostic. It serves as a secondary imaging modality in patients unable to undergo CT in cases in which the exact localization of the pulmonary embolus is not required. Current

indications for FDG PET include differentiation of benign and malignant nodules in patients with suspected pulmonary carcinoma, mediastinal lymph node staging in non-small cell lung cancer, and differentiation between parenchymal scarring and recurrent malignancy in patients who have had previous therapy for lung cancer. Ultrasonography is most frequently used for the diagnosis of pleural, diaphragmatic, and infradiaphragmatic abnormalities. Its role is limited to assessment of pulmonary masses or consolidation abutting the chest wall, diaphragm, or mediastinum and investigating the presence and nature of pleural fluid. In addition, it serves as an alternative guide to needle biopsy and catheter placement for pleural sclerotherapy or drainage of empyema.

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Figure 1 CT scans of solitary pulmonary nodules (arrows) showing different margin (a and b) and density characteristics (c and d). (a) Pulmonary nodule in the anterobasal segment of the left lower lobe showing a well-defined lobulated margin, commonly seen in benign lesions. However, histopathologic examination revealed an adenocarcinoma. (b) Pulmonary nodule in the apical segment of the left upper lobe showing a spiculated margin typical of a malignant nodule. Histopathology revealed a non-small cell lung cancer. (c) Calcified pulmonary nodule in the posterior segment of the left upper lobe, typical of a granuloma. (d) Pulmonary nodule in the apical segment of the right lower lobe continuous with a subsegmental pulmonary artery showing punctuate eccentric calcification. This was proved to be metastasis from chondrosarcoma.

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Common CT and HRCT Patterns

Diffuse Lung Disease

Focal Lung Disease

Nodular opacities Nodules as small as 1 mm can be detected on HRCT. According to their anatomic distribution, they can be categorized as random, centrilobular, or perilymphatic. Random nodules are diffusely and uniformly distributed throughout the lung and are usually well defined (Figure 2(a)). This pattern is seen in hematogenous metastasis and miliary tuberculosis, but it is also encountered in diffuse sarcoidosis. Centrilobular nodules commonly reflect bronchiolar or peribronchiolar abnormalities in a centrilobular location. If they are the predominant finding, diseases involving the small airways are most likely (Figure 2(b)). Centrilobular nodules are centered 5–10 mm away from the pleural surface and are often evenly spaced. Centrilobular nodules may be well defined or ill defined and are seen in endobronchial spread of tuberculosis or tumor, hypersensitivity pneumonitis, silicosis, asbestosis, respiratory bronchiolitis-associated interstitial lung disease, and Langerhans cell histiocytosis. Perilymphatic nodules occur in relation to lymphatic vessels within the lung and involve specific lung regions (Figure 2(c)). They are commonly well defined and are typically found in sarcoidosis, showing a peribronchovascular and subpleural distribution, or in lymphangitis carcinomatosa, where they occur in close relation to the peribronchovascular interstitium and the interlobular septa. Less common diseases exhibiting perilymphatic nodules include coal workers pneumoconiosis, silicosis, and, rarely, lymphocytic interstitial pneumonia.

Solitary pulmonary nodule CT has a major role in the noninvasive assessment of nodule morphology, growth, and perfusion patterns, and it may allow estimation of a nodule’s benignity in a few cases; however, a clear-cut diagnosis is possible only rarely. Size criterion has been shown to be of limited value in nodule assessment because small nodules may be malignant and large nodules may be benign. With regard to margin characteristics, most of the malignant nodules display an irregular contour as well as spiculated margins, whereas benign nodules have a smooth margin and a sharply defined edge (Figure 1). However, up to 38% of lung cancers present with sharply defined margins, and benign lesions may exhibit a spiculated margin due to a fibrotic response. Benign as well as malignant nodules may demonstrate homogeneous attenuation. The wall thickness of cavitary nodules can be a useful criterion for differentiation of malignant and benign pulmonary nodules, with the majority of malignant nodules displaying thick, irregular walls and benign nodules exhibiting rather thin walls. Furthermore, the presence of intranodular calcification in solid nodules is strongly predictive of benignity, particularly if the calcification pattern is either laminated or concentric or if a dense central nidus or popcorn-like pattern is found (Figure 1). A punctuate pattern, however, may also be seen in malignancy (Figure 1). Unfortunately, less than one-third of benign nodules contain calcification.

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Figure 2 Different nodular patterns on CT. (a) Randomly distributed nodules in a patient with hematogenous pulmonary metastases from a pancreatic cancer. (b) Micronodules in a centrilobular distribution in a patient with diffuse panbronchiolitis. Nodules as well as small branching centrilobular opacities represent dilated, fluid-filled bronchioles (tree-in-bud pattern). (c) Nodules in a perilymphatic, and thus bronchocentric, distribution in a patient with sarcoidosis.

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Reticular opacities Reticular opacities reflect thickening of the interstitial fibrous network of the lung. Interlobular septal thickening and honeycombing are the most frequently encountered types of reticulation in practice. Interlobular septal thickening has a characteristic appearance because interlobular septa form the secondary pulmonary lobular margins. Thickening may be smooth, nodular, or irregular, and these characteristics may suggest the underlying pathology. Common causes of smooth septal thickening include pulmonary edema and lymphangitic tumor spread (Figure 3). Honeycombing is characterized by the appearance of thickwalled, air-filled, wall-sharing cysts and generally reflects end-stage pulmonary fibrosis (Figure 4). The lung structure is distorted, and associated findings include traction bronchiectasis and bronchiolectasis. Honeycombing is most frequently found in usual interstitial pneumonia (less commonly in non-specific interstitial pneumonia) but also in fibrotic drug reactions, asbestosis, sarcoidosis, and chronic hypersensitivity pneumonitis. Airspace consolidation The pattern of airspace consolidation is seen secondary to replacement of alveolar air with fluid or cells. It is characterized by an increase in lung attenuation with obscuration of underlying vessels. A pattern of patent bronchi within the consolidated lung is referred to as an air bronchogram. Infective pneumonia is the most common cause of consolidation (Figure 5(a)). In patients with chronic diffuse infiltrative lung diseases, the most frequent causes are cryptogenic organizing pneumonia

(a)

(Figure 5(b)), eosinophilic pneumonia, and bronchoalveolar carcinoma. Ground-glass opacification Ground-glass opacification refers to a hazy increase in lung attenuation that does not obscure the underlying pulmonary structures. The finding may reflect many different pathologic processes, including filling of the alveoli with fluid or cells or minimal interstitial fibrosis, and it is therefore non-specific. Acute causes of ground-glass opacification

Figure 4 CT pattern of honeycombing. In this patient with usual interstitial pneumonia, multiple small thick-walled cystic airspaces are present, located in the subpleural, anterolateral, and right paravertebral parts of the lungs (arrowheads). This pattern is referred to as honeycombing, indicating severe fibrosis. Note the traction bronchiectasis in the right middle lobe caused by the retractile fibrosis.

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Figure 3 CT patterns of interlobular septal thickening. (a) Interlobular septal thickening in a patient with pulmonary edema. Note the smoothly thickened interlobular septa in the lung apex, outlining the secondary pulmonary lobules. (b) Interlobular septal thickening in a patient with lymphangitic spread secondary to breast cancer, located predominantly in the posterior segment of the right upper lobe, showing a slightly more irregular appearance than in Figure 3(a).

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Figure 5 CT pattern of consolidation in acute and chronic lung diseases. (a) Homogeneous airspace opacification obscuring the underlying pulmonary vessels in the right middle lobe secondary to infectious pneumonia. Note the patent bronchi within the consolidated lung (‘air bronchogram’). (b) Airspace consolidation in the right middle lobe in a patient with Wegener’s granulomatosis. Additional foci of consolidation and ground-glass opacification are present in the right middle and right lower lobes.

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Figure 6 CT pattern of ground-glass attenuation. (a) Patchy areas of ground-glass attenuation are noted in a patient with pulmonary hemorrhage secondary to Wegener’s granulomatosis-associated pulmonary vasculitis. Note that the underlying pulmonary vessels can still be identified, a finding that differentiates this pattern from frank consolidation. (b) More widespread areas of ground-glass opacification in a patient with Pneumocystis carinii pneumonia.

include pulmonary edema, pulmonary hemorrhage, viral pneumonia, diffuse alveolar damage, or hypersensitivity pneumonitis (Figure 6). Subacute or chronically progressive disorders include non-specific and desquamative interstitial pneumonias, sarcoidosis, alveolar proteinosis, or bronchoalveolar carcinoma. The coexistence of ground-glass opacification and septal thickening is called ‘crazy paving’, a non-specific pattern encountered in alveolar proteinosis, subacute pulmonary hemorrhage, viral pneumonia, or pulmonary edema. If ground-glass opacification is seen in

conjunction with traction bronchiectasis or other findings of fibrosis, it is likely to reflect fine interstitial fibrosis. Cysts and emphysema Lung cysts are well-defined, thin-walled, air-containing lesions that are commonly 1 cm or larger in size. Multiple lung cysts are found in Langerhans cell histiocytosis (LCH) and lymphangioleiomyomatosis (LAM). In LCH, cysts evolve from cavitating lung nodules. They display thick or thin walls and have a tendency to coalesce, displaying

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bizarre shapes (Figure 7(a)). They show an apical preponderance and usually spare the costophrenic angles. The interspersed lung parenchyma is frequently normal. In LAM, cysts are evenly distributed throughout the lung parenchyma, showing a rounded and more uniform appearance than in LCH (Figure 7(b)). Cysts may also be a feature of Pneumocystis carinii pneumonia or in lymphocytic interstitial pneumonia. Typical findings in emphysema are focal areas of low attenuation within normal lung parenchyma

(a)

(Figure 7(c)). It can easily be differentiated from honeycombing because most areas of centrilobular emphysematous destruction lack visible walls. Airways Disease

Large airways disease: bronchiectasis Bronchiectasis is the prototype pathology in large airways disease. On HRCT, dilatation of the bronchi with or without bronchial wall thickening is seen (Figure 8). In cylindrical bronchiectasis, a bronchial diameter exceeding

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Figure 7 CT patterns of cysts and emphysema. (a) Cysts in Langerhans cell histiocytosis. Thin-walled cysts are present in an advanced case, with some of them displaying unusual shapes. Note that the interspersed lung parenchyma appears to be relatively normal. (b) Cysts in lymphangioleiomyomatosis. Thin-walled, relatively uniform-size cysts are seen throughout the pulmonary parenchyma. Pneumothorax. (c) Advanced centrilobular emphysema showing widespread areas of low attenuation throughout the right upper lobe.

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Figure 8 CT patterns of bronchiectasis. (a and b) Extensive bronchiectasis in a patient with cystic fibrosis. Cylindrical (black arrows), varicose (white arrows), and cystic forms of bronchiectasis (arrowheads) are present. Additional abnormalities include bronchial wall thickening, mucus plugging, and mosaic of attenuation, reflecting small airways obliteration (a).

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Figure 9 CT pattern of mosaic attenuation in a patient with obliterative bronchiolitis secondary to mycoplasma infection. Note the patchy attenuation differences resulting from underventilated, and consequently underperfused, areas of the lung, with the relatively higher attenuation regions representing relatively increased perfusion of the normally ventilated lung (asterisks). Within the areas of decreased attenuation, the caliber of pulmonary vessels is reduced. Bronchial dilatation (arrowheads) and mild bronchial wall thickening are additional, but inconsistent, findings. Image courtesy of Dr C Engelke, Technical University, Munich, Germany.

that of the accompanying pulmonary artery, a lack of tapering of the bronchial lumen, and visualization of a bronchus within 1 cm of the costal pleura are the major findings. In varicose bronchiectasis, a beaded appearance of dilated bronchi is characteristic, whereas in cystic bronchiectasis, cystic spaces with or without fluid levels are found. Secretion accumulation within bronchiectasis yields a characteristic glove-finger, V- or Y-shaped appearance. Small airways disease Mosaic attenuation The mosaic attenuation pattern is a key HRCT finding in small airways disease (Figure 9). It refers to the finding of patchy attenuation differences resulting from underventilated, and consequently underperfused, areas of lung, with the relatively higher attenuation regions representing relatively increased perfusion of the normally ventilated lung. This inhomogeneity is accentuated on expiratory scans. Although normal lung should increase in attenuation at end-expiration, air trapping will reduce or annihilate this expiratory attenuation increase, which is characteristic of small airway obliteration. Associated findings in patients with obliterative bronchiolitis include a reduction in caliber of pulmonary vessels within areas of air trapping, abnormal macroscopic airways (e.g., bronchial dilatation and wall thickening), and a lack of change of cross-sectional area of affected lung parts on expiratory scans. In patients with widespread small airways disease and severe air trapping, expiratory CT scans may appear identical to inspiratory CT sections. Tree-in-bud pattern The tree-in-bud pattern is indicative of exudative rather than obliterative small airways disease. HRCT shows centrilobular nodular and branching linear opacities resulting from dilated and fluid-filled bronchioles, detectable in a centrilobular distribution (Figure 10). These otherwise

Figure 10 Tree-in-bud pattern on CT. In this patient with endobronchial spread of tuberculosis, multiple round and branching small opacities are seen (arrowheads), resembling a tree in bud. This pattern reflects exudates in and around otherwise invisible bronchioles.

invisible bronchioles become opacified by florid plugging and thickening of the small airways, with exudate in the immediately surrounding alveoli. The tree-in-bud pattern is most characteristic of diffuse panbronchiolitis, but it may also be seen in bacterial or fungal infection, tuberculosis, cystic fibrosis, and other forms of bronchiectasis. See also: Bronchiectasis. Bronchiolitis. Chronic Obstructive Pulmonary Disease: Overview. Cystic

LYMPHATIC SYSTEM 643 Fibrosis: Overview. Drug-Induced Pulmonary Disease. Granulomatosis: Lymphomatoid Granulomatosis. Interstitial Lung Disease: Overview. Mediastinal Masses. Mediastinitis, Acute and Chronic. Occupational Diseases: Overview. Panbronchiolitis. Pleural Effusions: Overview. Pneumonia: Overview and Epidemiology. Pulmonary Circulation. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome. Tumors, Benign. Tumors, Malignant: Overview. Vasculitis: Overview.

Further Reading Cutillo AG (1996) Application of Magnetic Resonance to the Study of the Lung. New York: Futura.

Lung Volume Reduction

Grenier PA, Beigelman-Aubry C, Fetita C, et al. (2002) New frontiers in CT imaging of airways disease. European Radiology 12: 1022–1044. Hansell DM (2001) Small airways diseases: detection and insights with computed tomography. European Respiratory Journal 17: 1294–1313. Hansell DM, Armstrong P, Lynch DA, and McAdams HP (2005) Imaging of Diseases of the Chest. New York: Elsevier. Mu¨ller NL, Fraser RS, Colman NC and Pare´ PD (eds.) (2001) Methods of radiologic investigation. In: Radiologic Diagnosis of Diseases of the Chest. Philadelphia: Saunders. Prokop M (2003) Principles of CT, spiral CT, and multislice CT. In: Prokop M and Galanski M (eds.) Computed Tomography of the Body. New York: Thieme. Remy J and Remy-Jardin M (1998) Spiral CT of the Chest. Berlin: Springer. Webb WR, Mu¨ller NL, and Naidich DP (2001) High-Resolution CT of the Lung. Philadelphia: Lippincott Williams & Wilkins.

see Surgery: Lung Volume Reduction Surgery.

Lymphangioleiomyomatosis

see Interstitial Lung Disease: Lymphangioleiomyomatosis.

LYMPHATIC SYSTEM C J Carati and B J Gannon, Flinders University, Adelaide, SA, Australia & 2006 Elsevier Ltd. All rights reserved.

external forces; obstruction of the lymphatics by tumor; or increased central venous pressure. Pleural fluid drainage is via intercellular gaps called stomata in the pleural mesothelial lining, which directly connect the pleural cavities to lymphatics that drain the dependent regions of the parietal pleura.

Abstract Lymphatic networks in the lung are found in close association with blood vessels and bronchi, and in the pleura. These networks anastomose at the lung surface and interlobular septa, and drain via the hilar region into the mediastinal and tracheobronchial lymphatic system, and thence to the right lymphatic duct or thoracic duct. Interstitial fluid from the alveolar walls drains into the parenchyma of the alveolar ducts, where it enters blindended lymphatic capillaries consisting of simple discontinuous but overlapping endothelial cells. These in turn drain into collecting lymphatic vessels that contain smooth muscle and oneway valves to help pump the lymph centrally along the network, significantly aided by respiratory and vascular movement. This drainage maintains normal tissue hydration, but is overcome in cardiovascular and lung disease resulting in fluid accumulation at the alveolar level. The lymphatics also provide a route of removal of inflammatory and pathological material, including tumor cells, which often end up in lymph nodes. Factors that can compromise lymphatic drainage from the lungs include reduced lymphatic pumping by compromised respiratory, vascular, or body movement; inhibition of lymphatic pumping by inflammatory cytokines or cells; constriction of lymphatic vessels by

Anatomy, Histology, and Structure Lung lymphatics are arranged in two main beds, one in the pleura and the other running in the peribronchovascular (PBV) connective tissue sheath accompanying the bronchi and pulmonary arteries (Figures 1 and 2). They merge at the hilum, and at the interlobular septa, where the pleural lymphatics meet lymphatic branches associated with the pulmonary veins. Thus, there are two potential pathways for drainage from the lung parenchyma, although they are widely anastomotic. Drainage from the lung itself is via the tracheobronchial and mediastinal lymph nodes, and thence to the right lymphatic duct or thoracic duct, or both. A third lymphatic bed, that of the parietal pleura, also affects respiratory system function. Pleural fluid (0.3 ml kg 1, about 20 ml in adults) is largely derived from capillaries of the upper

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Bronchial lymphatics Hilar lymphatics

Pleural plexus

Figure 1 Scan of injected lymphatic system of the lung, showing continuity between the pleural, peribronchovascular (PBV), and hilar lymphatics. Adapted from Sorokin SP, (1983) The respiratory system. In: Weiss L (ed.) Histology: Cell and Tissue Biology, 5th edn., pp. 853–855. New York: Elsevier Science, with permission.

thoracic wall, and probably exclusively drained by lymphatics of the more dependent parietal peritoneum (lower intercostal, mediastinal, and diaphragm). Major drainage of diaphragmatic pleural lymphatics is via trunks running in the pulmonary ligaments, connecting to mediastinal and hilar tracheobronchial lymph node chains. As in other organs, lung lymphatics originate as initial lymphatic capillaries, blind channels with discontinuous basal lamina and flaplike overlapping endothelial cells that act as primary valves (Figure 3). One end of the endothelial cell is anchored to the interstitial matrix by connective tissue filaments, while the other end merges into the lymphatic capillary. These drain into the collecting lymphatics which have thin walls, consisting of one to three layers of smooth muscle cells, a simple basement lamina, and endothelium. They are arranged as lymphangions, a single propulsive unit up to 1 mm in length, between two centripetally oriented valves. These collecting lymphatics pump lymph serially into larger lymph vessels, which then drain via the lymphatic systems associated with pulmonary vessels and bronchi or the pleura. Lymphatics are more variable and complex in their organization than the blood vascular system, but are usually found in close association with pulmonary arteries and veins.

Alveoli Peribronchiolar lymphatics

Alveolar duct region Alveoli

Respiratory bronchiole

Peribronchiolar pulmonary arteriole

Anastomotic lymphatics and veins Visceral pleura and lymphatics

Conduit lymphatics Pulmonary venule

Figure 2 The lymphatic drainage of the alveolar region, illustrating the close association between lymphatic and blood vessels, especially in the PBV region. Adapted from Sorokin SP, (1983) The respiratory system. In: Weiss L (ed.) Histology: Cell and Tissue Biology, 5th edn., pp. 853–855. New York: Elsevier Science, with permission.

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Arteriole

Alveoli

Alveoli

Anchoring filaments open lymphatic endothelial gaps

Para-alveolar initial lymphatic drains perivascular interstitium

Direction of fluid flux

Venule

Figure 3 The alveolar duct region illustrating the absence of lymphatics in the alveolar wall, which ‘pushes’ fluid into the alveolar duct region where it enters the initial lymphatics. Note the anchoring filaments that assist in opening the lymphatics during variation in interstitial pressure. Adapted from Sorokin SP, (1983) The respiratory system. In: Weiss L (ed.) Histology: Cell and Tissue Biology, 5th edn., pp. 853–855. New York: Elsevier Science, with permission.

Larger bronchi contain a deep and superficial lymphatic plexus that communicate freely. The para-alveolar lymphatic capillaries arise in the area around pulmonary vessels, pulmonary septum, and bronchioles in the region of the alveolar duct, but they are absent from the interalveolar walls, as is any significant interstitium. Drainage from the interalveolar region is into the looser connective tissue of the alveolar ducts, and thence into the lymphatic capillaries that originate there. These capillaries then drain into the larger lymphatic system. There is variable lymphoid tissue (single nodules or nodes) along the PBV tree in adults, being most dense at the bifurcation of the bronchi. These appear to develop in response to antigenic stimulation, since they are not seen at birth or in germ-free animals, and provide a likely focus for antibody and cellmediated immune responses. Lymphatics subjacent to the parietal pleura have discontinuous dilated initial segments connecting to gaps between overlying mesothelial cells, forming stomata which drain pleural fluid directly into these lymphatics. Stomata are concentrated in the more dependent regions of parietal pleura; their size (1–30 mm) allows free intrapleural cells and particulate debris as well as fluid and protein to enter the lymphatic system (as occurs from the peritoneal cavity).

Lymphatics in Normal Lung Function Lung lymphatics perform two key functions. First, interstitial fluid volume is maintained by a balance between fluid filtration from the microvasculature and fluid drainage via the lymphatics. The conventional view is that the net balance of hydrostatic and oncotic pressures of plasma and interstitial fluid (the Starling Principle) results in a low protein transudate that leaks from the arterial end of capillaries, most of which is then resorbed at the venous end of the same capillary bed due to the lower hydrostatic pressure there. Lymph is the difference between these arterial and venous fluid fluxes. A more recent view is that venous resorption is unlikely under normal conditions, and that fluid and protein transit the capillary wall by different paths. The endothelial glycocalyx, very narrow inter-endothelial cell clefts, and selective transport of plasma proteins by transendothelial vesicles play critical roles in controlling normal fluid fluxes across the blood capillary lining. In inflamed or injured tissues, major disturbances to the blood endothelial barrier occur, allowing much greater transcapillary fluid efflux into the interstitium. There is limited functional capacity for lung tissue to accommodate excess interstitial fluid; normal functioning of the pleura relies on a thin layer of intrapleural fluid at pressures below atmospheric,

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and lung parenchyma needs to be thin to allow for gas exchange at the alveoli. Hydration of the lung parenchyma also affects composition of the alveolar mucous/surfactant layer. Homeostatic maintenance of the lung parenchyma involves the transport of fluids from the vascular system, through the interstitium into the peribronchovascular space and into the lymphatics. Second, the lymphatics are responsible for removing immunologically relevant and/or inflammatory material and presenting it to the immune system at lymph nodes. The airways and alveolar surface of the lung are constantly exposed to inhaled and particulate matter, much of which is removed by the ‘mucociliary escalator’. Material that infiltrates the lung parenchyma is dealt with by the cell-mediated immune and macrophage systems and then cleared by the lymphatics. This material is also exposed to the systemic immune system at lymph nodes. Lymphatic endothelium has a molecular profile, protein expression pattern, and ultrastructure consistent with key active roles in transcellular transport and cell trafficking. There are abundant vesicles, clefts, and caveolae in lymphatic endothelial cells, suggestive of active regulation of transport between the interstitial and intralymphatic environments. There are also abundant aquaporin-1 (water) transmembrane channels to aid water flux. Lymphatic endothelium secretes a number of cytokines both basally and apically that may play a role in attracting certain cells, such as macrophages, under normal physiological conditions and tumor or inflammatory cells under pathological conditions (see below).

Control of Lymphatic Propulsion Lymph flows centripetally through the lung lymphatic systems, being propelled by variations in interstitial volume and pressure, respiratory movement, and Transmural pressure = interstitial pressure minus intraluminal pressure

pulsations of the associated blood vessels, and by cyclic intrinsic lymphatic propulsion through lymphangions (Figure 4). Interstitial pressure provides the driving force for interstitial fluids (called lymph once in the lymphatic vessels) to enter the initial lymphatics (Figure 3). These have filaments that anchor the initial endothelial cells to the surrounding interstitium. As interstitial volume and pressure vary, these filaments open the lymphatic endothelium and permit entry of material into the initial lymphatics down a pressure gradient. This gradient forces lymph into the collecting lymphatics, where both extrinsic and intrinsic forces move the lymph up the lymphatic tree from one lymphangion to the next, in the direction of the lymphatic valves (Figure 4). Both extrinsic and intrinsic factors influence the contractile rate and stroke volume of the lymphangion units, which combine to determine lymph flow, in a manner analogous to cardiac contractility. Extrinsic Mechanisms

Lymphatic propulsion occurs at low pressures, and involves changes in transmural pressure across the lymphatic wall that assist in lymph propulsion. Such movements include respiratory movements, vascular pulsations, and body movements. Respiratory movements Respiratory movements provide a major component of absorptive and propulsive forces. Changes in transmural pressure across the lymphatic wall range from negative (e.g., 5.370.5 mmHg (mean7SEM) at end expiration in pleural lymphatics of spontaneously breathing rats) to slightly positive (0.570.3 mmHg at end inspiration, ditto) pressures during the respiratory cycle. Negative pressures imply absorption of lymph from the interstitium, while positive pressures imply

Lymphatic smooth muscle and endothelium

Autonomic innervation

Lymph flow Respiratory pulsation

Blood vessel Vascular pulsation Figure 4 Many intrinsic and extrinsic factors affect conduit lymphatic pumping, which in turn determines lymph flow (see also Table 1).

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centripetal propulsion, with backflow prevented by the one-way valves. Any particular lymphatic vessel may play both an absorptive and/or a propulsive role, depending on the complex tissue stresses acting on its wall during a respiratory cycle. Note that lymphatics with negative transmural pressures do not collapse due to the tensile forces applied by tissue fibers that tether the outer surface of the vessel to surrounding tissues. Lung lymph flow increases proportional to tidal volume and (less so) frequency, through direct mechanical effects on the interstitium and lymphatic vessels. Vascular pulsations Vascular pulsations are transmitted to adjacent lymphatics embedded in the PBV tissue. Lung lymphatics are often wrapped around arteries and veins in the PBV tissue (called sacculotubular lymphatics), and are thus ‘massaged’ with every heartbeat. This effect is more variable than for respiratory movements, but still changes intraluminal lymphatic pressure by 5.572 mmHg in rat pleural lymphatics. The presence of frequent one-way valves ensure that lymph moves centrally. Body movements Movements of the torso or body assist generalized lymphatic clearance through the thoracic cavity.

Table 1 Factors affecting lymphatic contractility, based on studies of the behaviour of lymphatics in vitro from a number of different organs, not including the lung Increases lymphatic contractility Interstitial factors Increased interstitial pressure Bradykinin Histamine Serotonin Vasopressin

Endothelium-derived factors Arachidonic acid metabolites (thromboxanes, PGF2a) Neural factors Norepinephrine (a) Substance P Neuropeptide Y Adenosine triphosphate

Decreases lymphatic contractility Nitric oxide Prostaglandins Atrial naturetic peptide Hydrogen peroxide Free radicals Endothelin Endotoxin N-formyl-methionylleucyl-phenylalanine Heme (derived from hemoglobin) Inflammatory cytokines Nitric oxide Prostaglandins (PGE) Norepinephrine (b) Calcitonin gene-related peptide Vasoactive intestinal polypeptide Acetylcholine Serotonin

Intrinsic Mechanisms

Intrinsic mechanisms are less likely to be critical in lung lymphatics, which are subjected to more significant rhythmic tissue stretching (similar to skeletal and cardiac muscle), than in other well-studied organs such as the mesentery, with its much looser connective tissue. However, lymphatics have spontaneous contractions of the lymphatic smooth muscle that constrict the lymphatic lumen to propel lymph. These arise from spontaneous transient depolarizations caused by brief rhythmic release of calcium from intracellular stores that generate calcium-activated chloride currents. If of sufficient amplitude, these depolarizations generate an action potential in that cell which spreads to cause contraction of the smooth muscle cells of the whole lymphangion. These depolarizations can be modified by humoral (derived from the interstitium), intrinsic (derived from the smooth muscle or endothelium), and/or neural (due to a sparse autonomic innervation) influences that act to increase or decrease the rate of spontaneous contractions and their amplitude (Figure 4, Table 1). The contractile status of the collecting lymphatic may also be directly modified by factors in the lymph, such as cytokines or other by-products of tissue metabolism, pathology, and repair, or by the local interstitial environment.

Lymphatics in Respiratory Diseases Deficient or hypoplastic pulmonary lymphatics result in impaired lymphatic drainage, or lymphedema, and there is increased susceptibility to pulmonary infections. There are rare instances of primary or acquired lymphangiectasia, a diffuse dilation of discontinuous pulmonary lymphatics that leads to respiratory distress syndrome and is often fatal. Role of Lymphatics in Clearing Lung Edema

Lymphatics have a certain reserve capacity to clear excess interstitial fluid (interstitial edema), but once this is exceeded, elevated interstitial pressure drives fluid into the alveoli (alveolar edema). There are many conditions which give rise to excess fluid in the lungs. Interstitial edema is characterized by engorgement of the PBV space (so-called interstitial ‘cuffing’) and lymphatics. There may be widening of the thicker side of the alveolar wall, and of the interlobular septum. Lymph flow increases up to 15-fold to clear excess interstitial fluid, with little overt effect on pulmonary function. Once this drainage capacity is exceeded, fluid enters the alveoli and causes them to

648 LYMPHATIC SYSTEM

collapse through increased surface tension, with consequent adverse sequelae. Ventilation is compromised and fluid moves from one alveolus to another and into the larger airways. If this is associated with alveolar epithelial barrier breakdown, protein may leak into the alveoli, drawing more water by osmosis. Blood capillary permeability may also increase, resulting in increased interstitial protein and edema and further adding to the lymphatic load. Any concomitant compromise in lymphatic drainage will exacerbate this problem, such as: *

*

*

*

*

*

reduced lymphatic pumping by compromised respiratory, vascular, or body movement; inhibition of lymphatic pumping by inflammatory cytokines or cells; impaired flow due to increased lymph viscosity caused by excess protein or cells; constriction of lymphatic vessels by excessive interstitial pressure or interstitial fibrosis; obstruction of the lymphatics by infection or tumor; or increased central venous pressure, which opposes return of lymph to the venous system via the thoracic and right lymphatic ducts.

Obstructive Diseases: Involvement of Lymphatics in Spread of Tumors

Most obstructive diseases involving the lymphatic system arise because they show a predilection for lymphatic drainage routes in the PBV space. These include sarcoidosis (noncaseating granulomas that may progress to fibrosis), silicosis (caused by inhalation of micro-particulate dusts that become embedded in the lung tissue), lymphoproliferative disorders (involving abnormal proliferation of lymphoid cells), and pulmonary lymphangitic carcinomatosis (tumor growth in the lymphatics). Tumor growth in the lymphatic system of the lungs is associated with direct lymphatic spread of tumor. Such tumors grow primarily in the axial PBV space and in the peripheral interstitial compartment, resulting in thickening of the PBV space, interlobular septa, and subpleural interstitium. In general, the greater the lymphatic involvement in tumor growth, the poorer the prognosis. Some tumors induce new lymphatic growth or lymphangiogenesis within or around the tumor. Lymphangiogenesis is also seen in asthma and other lung inflammatory diseases where there is increased fluid load and edema. Lymphangiogenesis is due to localized secretion of vascular endothelial growth factor (VEGF)-C or VEGF-D to the cell surface receptor VEGFR3 on lymphatic endothelial cells, under the influence of a range of both physiological and

pathophysiological stimuli. Whether or not there is growth of new lymphatic vessels within tumors is controversial, but lymphatic endothelium is found in certain tumors, due either to formation of new lymphatics or existing lymphatics being trapped by the expanding tumor. There is better evidence for peritumor lymphatic growth, where enlarged hyperplastic lymphatics are observed, often containing tumor cells or clusters. These lymphatics provide an enlarged endothelial surface for tumor cells to attach, and potentially to subsequently migrate into the lymphatic system, especially to the lymph nodes where they may develop as metastases. Lymphatic endothelium expresses at least 34 receptors, adhesion molecules, and chemokines that may assist in this process; many of these interactions are modulated by local factors, such as inflammatory cytokines. This is an area of active current research. Tumor growth is associated with swollen lymphatics and localized interstitial edema due to increased vascular permeability and fluid fluxes associated with tumor growth. There may also be lymphatic blockade (lymphedema) consequent upon either growth within the lymphatic system itself, or compression of a lymphatic trunk. This fluid load is likely to end up in the alveoli, if it cannot be adequately drained by the lymphatic system. Pleural effusion occurs in a variety of pulmonary and cardiovascular conditions, and indicates that the maximal capacity of the parietal pleural lymphatics (up to B700 ml/day) is overwhelmed. A pleural effusion is due to either transudate (excess pleural fluid formation by parietal thoracic wall capillaries, such as occurs when central venous pressure is raised) or exudate (fluid leakage across the pleura due to lung or pleural infection, tumor, pulmonary embolus, or other lung injury, which release vasoactive cytokines that disturb the affected vascular bed). See also: Acute Respiratory Distress Syndrome. Alveolar Surface Mechanics. Alveolar Wall Micromechanics. Angiogenesis, Angiogenic Growth Factors and Development Factors. Aquaporins. Bronchial Circulation. Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations; Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General; Smoking Cessation. Chymase and Tryptase. Defense Systems. Dendritic Cells. Endothelial Cells and Endothelium. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fluid Balance in the Lung. Interstitial Lung Disease: Overview; Alveolar Proteinosis; Amyloidosis; Cryptogenic Organizing Pneumonia; Hypersensitivity Pneumonitis; Idiopathic Pulmonary Fibrosis; Lymphangioleiomyomatosis. Leukocytes: Pulmonary

LYSOZYME 649 Macrophages. Lung Anatomy (Including the Aging Lung). Lymphatic System. Macrophage Inflammatory Protein. Macrophage Migration Inhibitor Factor. Matrix Metalloproteinase Inhibitors. Matrix Metalloproteinases. Mediastinal Masses. Pleural Effusions: Overview; Pleural Fluid, Transudate and Exudate; Parapneumonic Effusion and Empyema; Malignant Pleural Effusions; Postsurgical Effusions; Pleural Fibrosis; Chylothorax, Psuedochylothorax, LAM, and Yellow Nail Syndrome; Hemothorax. Pleural Space. Pneumonia: Overview and Epidemiology. Pulmonary Circulation. Pulmonary Edema. Smooth Muscle Cells: Vascular. Tumors, Malignant: Overview; Lymphoma; Metastases from Lung Cancer. Vascular Endothelial Growth Factor. Vasculitis: Overview. Ventilation: Overview; Collateral; Control. Vesicular Trafficking. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation; VentilatorAssociated Pneumonia.

Further Reading Baluk P, Tammela T, Ator E, et al. (2005) Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway in flammation. Journal of Clinical Investigation 115(2): 247–257. Castaner E, Gallardo X, Pallardo Y, et al. (2005) Diseases affecting the peribronchovascular interstitium: CT findings and pathologic correlation. Current Problem in Diagnostic Radiology 34(2): 63–75. Farnsworth RH, Achen MG, and Stacker SA (2005) Lymphatic endothelium: an important interactive surface for malignant cells. Pulmonary Pharmacology and Therapeutics doi 10.1016/ j.pupt.2005.02.003 Flick MR (1994) Pulmonary edema and acute lung injury. In: Murray JF and Nagel JA (eds.) Textbook of Respiratory Medicine, 2nd edn., pp. 1725–1777. Philadelphia: WB Saunders.

Lymphoma

Ham H and Light RW (1997) The pleura: the outer space of pulmonary medicine. European Respiratory Journal 10(1): 2–3. Hansen-Flaschen J (1995) Cardiogenic and non-cardiogenic pulmonary edema. In: Grippi M (ed.) Pulmonary Pathophysiology, pp. 195–210. Philadelphia: J Lippincott. Levick JR (2004) Revision of the starling principle: new views of tissue fluid balance. Journal of physiology 557(3): 704. Miserocci G (1997) Physiology and pathophysiology of pleural fluid turnover. European Respiratory Journal 10(1): 219–225. Moriondo A, Mukenge S, and Negrini D (2005) Transmural pressure in rat initial subpleural lymphatics during spontaneously or mechanical ventilation. American Journal of Physiology. Heart and Circulatory Physiology 289(1): H263–H269. Negrini D, Moriondo A, and Mukenge S (2004) Transmural pressure during cardiogenic oscillations in rodent diaphragmatic lymphatic vessels. Lymphatic Research and Biology 2(2): 69–81. Ohhashi T, Mizuno R, Ikomi F, and Kawai Y (2004) Current topics of physiology and pharmacology in the lymphatic system. Pharmacology and Therapeutics 105(2): 165–188. Pearse DB, Searcy RM, Mitzner W, Permutt S, and Sylvester JT (2005) Effects of tidal volume and respiratory frequency on lung lymph flow. Journal of Applied Physiology 99(2): 556–563. Schraufnagel DE, Agaram NP, Faruqui A, et al. (2003) Pulmonary lymphatics and edema accumulation after brief lung injury. American Journal of Physiology: Lung Cellular and Molecular Physiology 284(5): 891–897. Sorokin SP (1983) The respiratory system. In: Weiss L (ed.) Histology: Cell and Tissue Biology, 5th edn., pp. 853–855. New York: Elsevier Science. Stack BHR and Thomas J (1990) Lymphatic and lymphoproliferative disorders. In: Brewis R, Gibson G, and Geddes D (eds.) Respiratory Medicine, pp. 1198–1205. London: Baillie`re Tindal. Staub NC and Albertine KH (1994) Anatomy of the lung. In: Murray JF and Nagel JA (eds.) Textbook of Respiratory Medicine, 2nd edn., pp. 3–35. Philadelphia: Saunders. von der Weid P-Y (2001) Review article: lymphatic vessel pumping and inflammation – the role of spontaneous constrictions and underlying electrical pacemaker potentials. Alimentary Pharmacology & Therapeutics 15: 1115–1129.

see Tumors, Malignant: Lymphoma.

LYSOZYME T Ganz, University of California, Los Angeles, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Lysozyme, a 14 kDa cationic protein, is one of the principal components of airway fluid. In the human respiratory tract, its main function is in the host defense of the airways. Lysozyme, through its dual activities as a lytic enzyme and a small cationic protein, damages or kills bacteria by lysing their cell wall peptidoglycan, by disrupting bacterial membranes, and by activating

autolytic enzymes in the bacterial cell wall. Lysozyme is secreted by submucosal glands, neutrophils, and macrophages. Against most bacteria, lysozyme acts synergistically with other antimicrobial polypeptides. Local lysozyme deficiency may contribute to the pathogenesis of recurrent sinusitis, hyaline membrane disease, and early-stage cystic fibrosis.

Definition Lysozyme is a widely distributed highly cationic 14 kDa enzyme that cleaves the beta-glycosidic bond between the C1 of N-acetyl muramate (NAM) and

650 LYSOZYME

C4 of N-acetyl glucosamine (NAG). The principal target of lysozyme is peptidoglycan, a glycosidic polymer and structural component of the bacterial cell wall that gives bacteria shape and osmotic resistance. Peptidoglycan (sometimes called murein) consists of chains of alternating NAM and NAG molecules cross-linked by short peptides between parallel chains. Functionally and structurally related enzymes, chitinases, degrade chitin, a polymer of NAG that forms the exoskeleton of fungi, worms, insects, crustaceans, and other invertebrates.

Distribution Sir Alexander Fleming discovered and named lysozyme in 1922 after noticing that droplets inadvertently sneezed onto an agar plate containing bacteria caused the bacterial colonies to dissolve. He partially purified lysozyme from various biological fluids and called it ‘a remarkable bacteriolytic element’. Enzymes with lysozyme activity are found in many animals in host defense settings as well as in digestive organs. High concentrations of lysozyme are found in avian egg white, and in mammalian tears, milk, respiratory secretions, granulocytes, and macrophages. In human granulocytes, lysozyme is a highly abundant protein in both phagocytic and secretory granules, and is the principal secreted protein of macrophages. Locally, at sites of infection, the concentrations of lysozyme in respiratory secretions are prominently modulated by inflammation, in part by the release of chemoattractants that recruit neutrophils, monocytes, and macrophages, which contain large amounts of lysozyme.

Lysozyme in the Respiratory Tract Analysis of mixed samples of respiratory fluid obtained from the nose by mechanical or chemical stimulation or as sputum from patients with chronic bronchitis indicates that lysozyme is one of the most abundant antimicrobial proteins of human airway secretions, and is present at about 0.1–1 mg ml 1. These estimates agree with bronchoalveolar lavage sampling assuming that the lavage fluid represents about a 100-fold dilution of respiratory fluid, an approximate dilution factor based on measurements of urea concentrations in bronchoalveolar lavage fluid (BALF). Lysozyme is about 10-fold more abundant in the initial ‘airway’ aliquot than in subsequent aliquots of bronchoalveolar lavage, and its concentration correlates poorly with neutrophil concentrations suggesting that, on the average, airway epithelium and its glands are the major sources of lysozyme in airway secretions. Immunohistochemical staining of

human lungs shows that both alveolar macrophages and submucosal glands are major sources of lysozyme. In contrast, in mouse and rat lungs, type II alveolar cells and alveolar macrophages are the predominant lysozyme producers.

Lysozyme Genes The human enzyme is encoded by a single lysozyme gene on chromosome 12 but in the mouse there are two: one gene (lysozyme M) is expressed in macrophages, neutrophils, and most epithelia, and the other (lysozyme P) in Paneth cells of the small intestine. In other animals, the number of lysozyme genes and their tissue-specific patterns of expression are highly variable. The abundance and wide distribution of lysozyme would suggest that it is more important than hitherto appreciated and that not all of its functions are known.

The Catalytic Mechanism of Lysozyme Both the molecular structure and catalytic mechanism of lysozyme have been described in such great detail that the lysozyme molecule serves as one of the best-understood paradigms of protein structure and function. The catalytic site of lysozyme holds six saccharide units of the peptidoglycan chain. Two amino acids contribute side chains that play a critical role in the catalytic site: glutamic acid in position 35 in the lysozyme polypeptide and aspartic acid in position 52 (Figure 1). The cleavage takes place between sugars 4 and 5 in the cleft. In the first step, glutamic acid 35 donates a proton that breaks the bond between sugars 4 and 5. A covalent intermediate involving the aspartic acid 52 and the sugars 1 through 4 forms and the peptidoglycan fragment containing sugars 5 and 6 is released. The aspartate intermediate reacts with water to restore the enzyme and free the other peptidoglycan fragment containing sugars 1 through 4.

Mechanism of Antimicrobial Action Lysozyme damages microbes through at least three distinct mechanisms (Figure 2). The disruption of the peptidoglycan layer by lysozyme is sufficient to kill some bacteria directly. Most bacteria are not killed but some become more susceptible to other antimicrobial substances as well as to osmotic stress. Organisms that are susceptible to low concentrations of lysozyme include certain nonpathogenic Grampositive bacteria (e.g., Micrococcus luteus, also called M. lysodeicticus, and Bacillus species). Other Gram-positive bacteria are variably defended against

LYSOZYME 651

Glu35

Asp52

Figure 1 The structure of human lysozyme. The molecule is oriented to show its catalytic cleft through which the polysaccharide chain of peptidoglycan passes during its cleavage. The polypeptide backbone is shown as a thick tube with side chains as thin wires. The arrows point to the two catalytic residues marked in yellow. The cationic and anionic amino acid side chains are shown in blue and red, respectively, and the disulfide bonds in dark gold. The predominance of cationic residues is readily seen.

Lysozyme

As an enzyme, digests bacterial peptidoglycan

As a cationic and amphipathic protein, disrupts bacterial membranes

As a small cationic protein, releases autolytic enzymes from bacteria

Figure 2 Lysozyme, a triple threat antimicrobial protein.

the effects of lysozyme because of covalent modifications (O-acetylation of NAM and de-N-acetylation of NAG) that render the peptidoglycan molecule resistant to the enzymatic action of lysozyme. In general, Gram-negative bacteria are more resistant due to their outer membranes that hinder the access of lysozyme to peptidoglycan. At high but still realistic concentrations of lysozyme, nonenzymatic antimicrobial activity, dependent on the highly cationic nature of this molecule, is also observed. This is best illustrated by the preservation of antimicrobial activity after site-directed mutagenesis in the catalytic site of lysozyme. In this nonenzymatic role, lysozyme may act by disrupting membrane integrity or by releasing autolytic enzymes from bacteria where they

652 LYSOZYME

are normally used for cell wall remodeling during bacterial growth and division.

Biological Roles By itself, the bactericidal activity of lysozyme against many pathogenic species of bacteria is weak, especially against Gram-negative bacteria, but it is greatly potentiated by other host defense substances (lactoferrin, antibody-complement, or hydrogen peroxide–ascorbic acid). These cofactors presumably disrupt the outer membrane of Gram-negative bacteria and allow lysozyme access to the sensitive peptidoglycan layer. The antimicrobial activity of human nasal fluid is largely dependent on lysozyme, which acts synergistically with other antimicrobial components of nasal secretions. Mice overexpressing rat lysozyme under the control of the human surfactant protein C promoter have increased resistance to lung infection with both gp B Streptococcus and P. aeruginosa. Mice lacking lysozyme M compensate by newly expressing Paneth cell lysozyme (lysozyme P) in inflammatory macrophages. One defect in these mice is greatly increased inflammatory response to subcutaneous injection of M. luteus, due to the inability of these mice to rapidly degrade peptidoglycan. Although there is a significant delay before M. luteus are killed in the lysozyme M-deficient mice, both normal and deficient mice sterilize the subcutaneous abscess by day 5 after infection. However, lysozyme M-deficient mice infected intranasally with Klebsiella pneumoniae show diminished clearance of the bacteria and are more susceptible to death from the infection. These studies suggest that lysozyme is important both for the killing of certain microbes and for the breakdown of their proinflammatory cell wall components.

Role of Lysozyme in Human Diseases To date, no human cases of genetic lysozyme deficiency have been reported. Some mutations in lysozyme cause the protein to aggregate abnormally, leading to familial amyloidosis but no associated host defense defects have been reported, presumably because these lysozymes are fully functional. Decreased lysozyme concentrations in nasal secretions have been reported in patients with perennial allergic rhinitis and recurrent sinusitis as compared to those with perennial allergic rhinitis alone or to healthy controls. This abnormality could be related to decreased mucosal secretory response previously noted in patients with recurrent sinusitis. In newborns with hyaline membrane disease, low lysozyme concentrations in respiratory secretions correlated with more severe disease and the development of bronchopulmonary

dysplasia. In this setting, low lysozyme production may reflect the immaturity of submucosal glands, which could predispose to infectious complications. Based on limited studies in model systems, local deficiency of lysozyme and of other antimicrobial factors produced by submucosal glands could also exist in cystic fibrosis. In this disease, lysozyme secretion could be decreased because functional cystic fibrosis transmembrane regulator (CFTR) is required for efficient secretion and because the lack of CFTR function leads to increased viscosity of secretions and obstruction of submucosal glands. Diminished lysozyme secretion during the earliest stages of cystic fibrosis could predispose to colonization. The functional deficiency would eventually resolve as neutrophils are recruited and deliver more lysozyme, but neutrophils also contribute to tissue injury. Studies of lysozyme secretion in the airways of uninfected patients with cystic fibrosis will be required to assess whether the deficiency of lysozyme and other antimicrobial polypeptides in the airways could contribute to the pathogenesis of this disease. See also: Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Fluid Balance in the Lung. Pleural Effusions: Pleural Fluid, Transudate and Exudate.

Further Reading Akinbi HT, Epaud R, Bhatt H, and Weaver TE (2000) Bacterial killing is enhanced by expression of lysozyme in the lungs of transgenic mice. Journal of Immunology 165: 5760–5766. Cole AM, Liao HI, Stuchlik O, et al. (2002) Cationic polypeptides are required for antibacterial activity of human airway fluid. Journal of Immunology 169: 6985–6991. Ellison RT and Giehl TJ (1991) Killing of Gram-negative bacteria by lactoferrin and lysozyme. Journal of Clinical Investigation 88: 1080–1091. Fleming A (1922) On a remarkable bacteriolytic element found in tissues and secretions. Proceedings of the Royal Society of London. Series B: Biological Sciences 93: 306–317. Ganz T, Gabayan V, Liao HI, et al. (2003) Increased inflammation in lysozyme M-deficient mice in response to Micrococcus luteus and its peptidoglycan. Blood 101: 2388. Laible NJ and Germaine GR (1985) Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides. Infection and Immunity 48: 720–728. Markart P, Korfhagen TR, Weaver TE, and Akinbi HT (2004) Mouse lysozyme M is important in pulmonary host defense against Klebsiella pneumoniae infection. American Journal of Respiratory and Critical Care Medicine 169: 454–458. Masschalck B and Michiels CW (2003) Antimicrobial properties of lysozyme in relation to foodborne vegetative bacteria. Critical Review of Microbiology 29: 191–214. Serpell LC, Sunde M, and Blake CC (1997) The molecular basis of amyloidosis. Cellular and Molecular Life Sciences 53: 871–887. Singh PK, Tack BF, McCray PB Jr, and Welsh MJ (2000) Synergistic and additive killing by antimicrobial factors found in human

LYSOZYME 653 airway surface liquid. American Journal of Physiology: Lung Cellular and Molecular Physiology 279: L799–L805. Strynadka NC and James MN (1996) Lysozyme: a model enzyme in protein crystallography. EXS 75: 185–222. Thompson AB, Bohling T, Payvandi F, and Rennard SI (1990) Lower respiratory tract lactoferrin and lysozyme arise primarily

in the airways and are elevated in association with chronic bronchitis. Journal of Laboratory and Clinical Medicine 115: 148–158. Wecke J, Lahav M, Ginsburg I, and Giesbrecht P (1982) Cell wall degradation of Staphylococcus aureus by lysozyme. Archives of Microbiology 131: 116–123.

M MACROPHAGE INFLAMMATORY PROTEIN M P Keane, R M Strieter, and J A Belperio, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract There are several macrophage inflammatory proteins (MIP), MIP-1a/CCL3, MIP-1b/CCL4, MIP-1g/CCL9/10, MIP-1d/ CCL15, MIP-4/CCL18, MIP-3a/CCL19, MIP-3b/CCL20, and MIP-2/CXCL2. With the exception of MIP-2/CXCL2 they are all CC chemokines. In contrast MIP-2/CXCL2 is a CXC chemokine that is a murine functional homolog of CXCL8/IL-8. With the exception of MIP-2/CXCL2 and in common with the other CC chemokines they are all chemotactic for mononuclear leukocytes with varying specificity for particular cell types. In contrast the sole member of the CXC chemokine family, MIP-2/ CXCL2, is a potent neutrophil chemoattractant and promotes angiogenesis in vivo. Various members of the MIP family have been implicated in pulmonary host defense and the pathogenesis of pulmonary fibrosis.

from 7 to 10 kDa and are characteristically basic heparin-binding proteins. The chemokines display four highly conserved cysteine amino acid residues. The CXC chemokine family has the first two N-terminal cysteines separated by one nonconserved amino acid residue, the CXC cysteine motif. The CC chemokine family has the first two N-terminal cysteines in juxtaposition, designated the CC cysteine motif. CCL15 and CCL9/10 have six instead of four conserved cysteines. Figure 1 shows a schematic representation of a ‘ribbon model of MIP-1a’.

Regulation of Production and Activity All of the MIPs are upregulated by the early response cytokines IL-1 and tumor necrosis factor (TNF). The

Introduction

CYS35

There are several macrophage inflammatory proteins (MIPs): MIP-1a/CCL3, MIP-1b/CCL4, MIP-1g/ CCL9/10, MIP-1d/CCL15, MIP-4/CCL18, MIP-3b/ CCL19, MIP-3a/CCL20, and MIP-2/CXCL2. With the exception of MIP-2/CXCL2 they are all CC chemokines. In contrast MIP-2/CXCL2 is a CXC chemokine that is a murine functional homolog of CXCL8/interleukin-8 (IL-8).

CYS111

CYS51

CYS12

Structure The structural properties of MIPs are outlined in Table 1. Chemokines in their monomeric form range

Figure 1 Schematic representation of the structural 3-D model (ribbon) of MIP-1a as analyzed by SwissModel version 3.5.

Table 1 Name, chromosomal location of the gene, peptide size, and receptors for the macrophage inflammatory proteins Name

Human chromosome

Peptide size (precursor/mature)

Receptor

MIP-1a/CCL3 MIP-1b/CCL4 MIP-1g/CCL9/10 MIP-1d/CCL15 MIP-4/CCL18 MIP-3b/CCL19 MIP-3a/CCL20 MIP-2/CXCL2

17q11.2 17q11.2 Unknown 17q11.2 17q11.2 9p13 2q33-q37 4q12-q21

92 aa/69–70 aa 92 aa/69–70 aa 122 aa/101 aa 113 aa/92 aa 89 aa/69 aa 98 aa/77 aa 96 aa/70 aa 100 aa/73 aa

CCR1, CCR5 CCR5 CCR1 CCR1, CCR3 Unknown CCR7 CCR6 CXCR2

2 MACROPHAGE INFLAMMATORY PROTEIN

genes are inducible in most hematopoietic cells. MIP-4/CCL18 is induced by IL-4 and IL-10 and is inhibited by interferon gamma (IFN-g). Interestingly IL-4, IL-10, and dexamethasone downregulate MIP-1/ CCL3/4 expression. MIP-1g/CCL9/10 in contrast to other CC chemokines is constitutively expressed in macrophages, dendritic cells, and myeloid cell lines. Both MIP-3b/CCL19 and MIP-3a/CCL20 are downregulated by IL-10. There is limited information on the degradation and half-life of the MIP proteins. MIP-1 activity may be limited by proteases although proteolytic cleavage has also been reported to increase activity of MIP-1a/CCL3, MIP-1b/CCL4, and MIP-1d/ CCL15. Binding to proteoglycans such as heparin enhances MIP activity.

Biological Function MIP-1a/CCL3 in common with the other CC chemokines is a potent chemoattractant for monocytes/ macrophages. In addition, MIP-1a/CCL3 is chemotactic for T cells, B cells, basophils, eosinophils, mast cells, and natural killer (NK) cells. MIP-1b/CCL4 is chemotactic for monocytes, T cells, and NK cells. Interestingly, MIP-1a/CCL3 suppresses colony formation of immature myeloid progenitor cells and this effect is antagonized by MIP-1b/CCL4. MIP-1a/CCL3 and MIP-1b/CCL4 preferentially attract CD8 and CD4 T cells, respectively. In mice and rabbits both MIP-1a/CCL3 and MIP-1b/CCL4 have been shown to induce neutrophil infiltration. Both MIP-1a/CCL3 and MIP-1b/CCL4 induce fever that is nonprostaglandin mediated. MIP-1d/CCL15 is a potent chemoattractant for monocytes and T cells and is angiogenic in vitro. MIP-1g/CCL9/10 induces chemotaxis in CD4 and CD8 cells and in both activated and unactivated T cells (Table 2). MIP-3a/CCL20 is chemotactic for memory T cells, naive B cells, and immature dendritic cells; at high concentrations it may chemoattract neutrophils but has no effect on monocytes. MIP-3b/CCL19 attracts memory T cells, naive B cells, NK cells, immature dendritic cells, and macrophage progenitor cells. MIP-3b/CCL19 is not chemotactic for monocytes.

MIP-2/CXCL2 is a CXC chemokine that is a potent chemoattractant for neutrophils. CXC chemokines can also chemoattract basophils and are weakly chemotactic for eosinophils. In common with other members of the CXC chemokine family MIP-2/ CXCL2 is chemotactic for endothelial cells and induces angiogenesis in vivo and in vitro. MIP-4/CCL18 is chemotactic for naive lymphocytes but interestingly has been shown to antagonize CCR3 and thereby may limit infiltration of eosinophils. Similar to MIP-3a/CCL20 and MIP-3b/ CCL19 it has no activity on monocytes (Table 2).

Receptors MIP-1a/CCL3 binds to CCR1 and CCR5, MIP-1b/ CCL4 binds to CCR5, and MIP-2/CXCL2 binds to CXCR2. MIP-1g/CCL9/10 binds to CCR1 and MIP1d/CCL15 binds to CCR1 and CCR3. MIP-3a/ CCL20 binds to CCR6 and MIP-3b/CCL19 binds to CCR7. The receptor for MIP-4/CCL18 is unknown (Table 1).

MIPs in Respiratory Disease MIP-la/CCL3 protein and mRNA expression in lung tissue homogenates has been found to be elevated post-bleomycin challenge with detectable levels of MIP-la/CCL3 protein peaking at 2 and 16 days. The kinetics of expression of MIP-la/CCL3 during the first week post-bleomycin challenge temporally correlates with accumulation of lung mononuclear cells. In the second week post-bleomycin challenge, increased MIP-la expression coincided with increases in macrophages. The predominant cellular source of MIP-1a/CCL3 in this model is the alveolar macrophage. In addition, eosinophils, epithelial cells, and interstitial macrophages are also significant cellular sources of MIP-1a/CCL3. Passive immunization of mice with neutralizing antibodies to murine MIP-1a/CCL3 resulted in a reduction of infiltrating cells into the lungs of bleomycin-treated animals by 35%. Depletion of MIP-1a/CCL3 reduced B-lymphocyte, macrophage, and neutrophil infiltration.

Table 2 Biological function of the macrophage inflammatory protein family of chemokines Chemokine

Chemotactic function

MIP-1a/CCL3 MIP-1b/CCL4 MIP-1g/CCL9/10 MIP-1d/CCL15 MIP-4/CCL18 MIP-3b/CCL19 MIP-3a/CCL20 MIP-2/CXCL2

Monocytes/macrophages, T cells (CD8), B cells, basophils, eosinophils, mast cells, and NK cells Monocytes, T cells (CD4) and NK cells CD4 and CD8 T cells Monocytes and T cells, promotes angiogenesis Naive lymphocytes but antagonizes CCR3 limiting infiltration of eosinophils Memory T cells, naive B cells, NK cells, immature dendritic cells, macrophage precursors Memory T cells, naive B cells, NK cells, immature dendritic cells Neutrophils, endothelial cells, promotes angiogenesis

MACROPHAGE INFLAMMATORY PROTEIN 3

Bleomycin-challenged mice that were passively immunized with neutralizing anti-MIP-la/CCL3 antibodies demonstrated a 49% decrease in total lung collagen, as measured by lung hydroxyproline content. Depletion of MIP-la/CCL3 did not completely abrogate either the inflammatory or fibrotic response to bleomycin. This suggests the existence of other mediators with similar or overlapping activities. Certainly, other CC chemokines have been shown to have activities similar to MIP-1a/CCL3 and may be concomitantly expressed and induce the observed leukocyte recruitment that supports a profibrotic environment. CCR1, which binds MIP-1a/CCL3, has been shown to play an important role in the pathogenesis of bleomycin-induced pulmonary fibrosis. Following the administration of bleomycin the expression of CCR1 mRNA peaked at 7 days. This paralleled the expression of RANTES/CCL5 and MIP-1a/CCL3, the major ligands for CCR1. Treatment with antibodies to CCR1 leads to a reduction in both inflammatory cell infiltrates and the development of fibrosis. MIP-2/CXCL2 was found to be directly correlated with total lung hydroxyproline levels, a measure of lung collagen deposition, during bleomycin-induced pulmonary fibrosis. When MIP-2/CXCL2 was depleted by passive immunization with neutralizing antibodies, there was a marked attenuation of pulmonary fibrosis that was entirely attributable to a reduction in angiogenesis in the lung. These findings support the notion that angiogenesis is a critical biological event that supports fibroplasia and deposition of extracellular matrix (ECM) in the lung during pulmonary fibrosis, and that angiogenic and angiostatic factors, such as CXC chemokines play an important role in the pathogenesis of this process. MIP-1a/CCL3 has been found in the bronchoalveolar lavage fluid (BALF) of interstitial lung disease patients. MIP-1a/CCL3 was found in equivalent levels in the BALF of 22 of 23 patients with sarcoidosis and 9 of 9 patients with idiopathic pulmonary fibrosis (IPF), whereas detectable MIP-1a/CCL3 was found in only 1 of 7 healthy subjects. In addition, these levels correlated with increased monocyte chemotactic activity in the BALF obtained from patients with sarcoidosis and IPF, respectively, as compared to healthy subjects. The monocyte chemotactic activity was reduced by approximately 22% when BALF from sarcoidosis and IPF patients were preincubated with rabbit antihuman MIP-1a/CCL3 antibodies. The predominant cellular sources of MIP-1a/CCL3 within the lung of these patients, by immunolocalization, were both alveolar and interstitial macrophages and pulmonary fibroblasts. Minimal to

no detectable MIP-1a/CCL3 was expressed in normal subjects. Furthermore, pulmonary fibroblasts isolated from patients with IPF produced greater amounts of MIP-1a/CCL3 after challenge with IL-1b than did similarly treated pulmonary fibroblasts recovered from patients without fibrotic lung disease. Similar to the findings for MIP-1a/CCL3, MIP-3b/CCL19 has been found to be elevated in sarcoid BALF and to correlate with BALF lymphocytes and T cell subsets. The predominant cellular source of MIP-3b/CCL19 was the alveolar macrophage. Using a mouse adapted strain of influenza A infected in CCR5
/
(CCR5 is the receptor for RANTES (CCL5), MIP-1a (CCL3), and MIP-1b(CCL4)) and CCR2
/
(CCR2 is the receptor for MCP-1 (CCL2)) mice, as compared to control þ / þ mice, Dawson and colleagues demonstrated that CCR5
/
mice displayed increased mortality related to severe pneumonitis, whereas CCR2
/
mice were protected from the severe pneumonitis due to defective macrophage recruitment. The delay in macrophage accumulation in CCR2
/
mice was correlated with high pulmonary viral titers. These studies support the potential of different roles that CC chemokine ligand/receptor biology plays in influenza infection. In addition, this study also demonstrates that the importance of macrophage recruitment during the innate response is critical to the development of adaptive immunity to this microbe. Paramyxovirus pneumonia virus infection in mice is associated with predominant neutrophil and eosinophil infiltration into the lung that is accompanied by expression of CCR1 ligands. However, in CCR1
/
mice infected with Paramyxovirus, the inflammatory response was found to be minimal, the clearance of virus from lung tissue was reduced, and mortality was markedly increased. These results indicate that CC chemokine-dependent innate responses limited the rate of virus replication in vivo and played an important role in reducing mortality. The effect of MIP-1a/CCL3 in mediating the recruitment of mononuclear cells during the innate host defense is not limited to viral infections. MIP-1a/CCL3 and the recruitment of mononuclear cells plays an important role in the eradication of invasive pulmonary aspergillosis. In both immunocompetent and neutropenic mice, MIP-1a/CCL3 is induced in the lungs in response to intratracheal inoculation of Aspergillus fumigatus. Depletion of endogenous MIP-1a/CCL3 by passive immunization with neutralizing antibodies to MIP-1a/CCL3 resulted in increased mortality in neutropenic mice, which was associated with a reduced mononuclear cell infiltration and markedly decreased clearance of lung fungal burden. Furthermore, CCR1
/
mice

4 MACROPHAGE INFLAMMATORY PROTEIN

exposed to A. fumigatus had markedly increased mortality compared to wild-type CCR1 þ / þ control mice. These studies indicate that MIP-1a/CCL3 and elicitation of mononuclear cells are crucial in mediating host defense against A. fumigatus in the setting of neutropenia, and this understanding may be important in devising future therapeutic strategies against invasive pulmonary aspergillosis. Cryptococcus neoformans is acquired via the respiratory tract and is a significant cause of fatal mycosis in immunocompromised patients. Both the innate and adaptive immune response are necessary to clear the microbe from the lung and prevent dissemination to the meninges. MIP-1a/CCL3 plays an important role in the eradication of C. neoformans from the lung and prevention of cryptococcal meningitis. In mice exposed to intratracheal C. neoformans, MIP-1a/CCL3 expression directly correlates with the magnitude of infiltrating leukocytes. Depletion of MIP-1a/CCL3 resulted in a significant reduction in total leukocytes and an increase in the burden of C. neoformans in the lungs of these animals. Interestingly, depletion of MIP-1a/CCL3 did not decrease the levels of MCP-1/CCL2; however, depletion of MCP-1/CCL2 significantly reduced MIP-1a/CCL3 levels, demonstrating that induction of MIP-1a/CCL3 was largely dependent on MCP-1/ CCL2 production. Neutralization of MIP-1a/CCL3 also blocked the cellular recruitment phase of a recall response to cryptococcal antigen in the lungs of immunized mice. Thus, in both the context of active cryptococcal infection or rechallenge with cryptococcal antigen, MIP-1a/CCL3 was required for maximal leukocyte recruitment into the lungs, most notably the recruitment of phagocytic effector cells (neutrophils and macrophages). These studies support the notion that CC chemokine ligand/receptor biology plays a critical role in innate host defense and development of pulmonary inflammation that is important in eradication of microorganisms. Recently, the importance of receptor polymorphisms in various disease states has been demonstrated. CCR5 is a major receptor for MIP-1a/CCL3, MIP-1b/CCL4, and RANTES/CCL5. Homozygosity for the CCR5D32 mutation has been shown to predict prolonged renal allograft survival (90% at 20 years), reduced risk of asthma, and decreased severity of rheumatoid arthritis. In contrast, there was an increased frequency of the CCR5D32 allele in patients with sarcoidosis that was associated with more apparent disease and an increased need for corticosteroids. This suggests that CCR5D32 is associated with altered susceptibility to immunologically mediated diseases and that the balance between chemokines and their appropriately expressed receptor is

necessary for the full manifestation of various diseases. Similarly, polymorphisms in the CXCR2 gene have been described in patients with systemic sclerosis both with and without evidence of interstitial lung disease, suggesting that CXCR2 may have a role in the fibrotic process. See also: Chemokines. Chemokines, CC: C10 (CCL6); MCP-1 (CCL2)–MCP-5 (CCL12). Chemokines, CXC: IL-8; CXCL1 (GRO1)–CXCL3 (GRO3). Chymase and Tryptase. Interleukins: IL-6; IL-10. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Kinins and Neuropeptides: Neuropeptides and Neurotransmission. Leukocytes: Eosinophils; Neutrophils; T cells; Pulmonary Macrophages. Matrix Metalloproteinases. Pulmonary Fibrosis. Transgenic Models.

Further Reading Dawson TC, Beck MA, Kuziel WA, Henderson F, and Maeda N (2000) Contrasting effects of CCR5 and CCR2 deficiency in the pulmonary inflammatory response to influenza A virus. American Journal of Pathology 156: 1951–1959. Domachowske JB, Bonville CA, Gao JL, et al. (2000) The chemokine macrophage-inflammatory protein-1 alpha and its receptor CCR1 control pulmonary inflammation and antiviral host defense in paramyxovirus infection. Journal of Immunology 165: 2677–2682. Fischereder M, Luckow B, Hocher B, et al. (2001) CC chemokine receptor 5 and renal-transplant survival. Lancet 357: 1758– 1761. Gao JL, Wynn TA, Chang Y, et al. (1997) Impaired host defense, hematopoiesis, granulomatous inflammation and type 1–type 2 cytokine balance in mice lacking CC chemokine receptor 1. Journal of Experimental Medicine 185: 1959–1968. Gibejova A, Mrazek F, Subrtova D, et al. (2003) Expression of macrophage inflammatory protein-3{beta}/CCL19 in pulmonary sarcoidosis. American Journal of Respiratory and Critical Care Medicine 167: 1695–1703. Huffnagle GB, Strieter RM, McNeil LK, et al. (1997) Macrophage inflammatory protein-1alpha (MIP-1alpha) is required for the efferent phase of pulmonary cell-mediated immunity to a Cryptococcus neoformans infection. Journal of Immunology 159: 318–327. Keane MP, Belperio JA, Moore TA, et al. (1999) Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis [In Process Citation]. Journal of Immunology 162: 5511–5518. Maurer M and von Stebut E (2004) Macrophage inflammatory protein-1. International Journal of Biochemistry & Cell Biology 36: 1882–1886. Mehrad B, Moore TA, and Standiford TJ (2000) Macrophage inflammatory protein-1 alpha is a critical mediator of host defense against invasive pulmonary aspergillosis in neutropenic hosts. Journal of Immunology 165: 962–968. Nibbs RJ, Salcedo TW, Campbell JD, et al. (2000) C–C chemokine receptor 3 antagonism by the beta-chemokine macrophage inflammatory protein 4, a property strongly enhanced by an amino-terminal alanine–methionine swap. Journal of Immunology 164: 1488–1497. Petrek M, Drabek J, Kolek V, et al. (2000) CC chemokine receptor gene polymorphisms in Czech patients with pulmonary

MACROPHAGE MIGRATION INHIBITOR FACTOR 5 sarcoidosis. American Journal of Respiratory and Critical Care Medicine 162: 1000–1003. Smith RE, Strieter RM, Phan SH, and Kunkel SL (1996) CC chemokines: novel mediators of the profibrotic inflammatory response to bleomycin challenge. American Journal of Respiratory Cell and Molecular Biology 15: 693–702.

Standiford TJ, Rolfe MW, Kunkel SL, et al. (1993) Macrophage inflammatory protein-1a expression in interstitial lung disease. Journal of Immunology 151: 2852–2863. Tokuda A, Itakura M, Onai N, et al. (2000) Pivotal role of CCR1positive leukocytes in bleomycin-induced lung fibrosis in mice. Journal of Immunology 164: 2745–2751.

MACROPHAGE MIGRATION INHIBITOR FACTOR S C Donnelly and J A Baugh, University College Dublin, Dublin, Republic of Ireland & 2006 Elsevier Ltd. All rights reserved.

Abstract Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator and integrator of the immunoneuroendocrine interface. An increasing body of data indicates that MIF’s position within the cytokine cascade is to act in concert with endogenous glucocorticoids to control the ‘set point’ and magnitude of the immune and inflammatory response. It is now well established that apart from over-riding the immunosuppressive effects of glucocorticoids, MIF also has a direct proinflammatory role in respiratory diseases such as ARDS, asthma, and fibrosis as well as pro-oncogenic and pro-angiogenic effects in cancer. The functions of MIF within the immune system are both unique and diverse, and whilst a unified molecular mechanism of action remains to be elucidated, there have been significant advances in understanding how MIF affects cellular processes. This article will discuss the pathogenic role of MIF in respiratory disease and highlight the novel structural, functional, and mechanistic properties of MIF.

Introduction Macrophage migration inhibitory factor (MIF) has been well described as a potent proinflammatory mediator, but is now emerging as a key integrator of the immuno-neuroendocrine interface. MIF was originally described in 1966 as a T-lymphocyte-derived ‘activity’ that inhibited the random migration of macrophages. Over the subsequent 20 years, MIF expression was found to correlate with general macrophage activation functions including adherence, spreading, phagocytosis, and enhanced tumoricidal activity. In this article, we will highlight the key role for MIF in driving an exaggerated inflammatory response particularly as it pertains to the lung and development of acute lung injury. We will also provide an overview of more recent data which translates important bench- based scientific observations in relation to MIF function in chronic inflammatory lung diseases particularly asthma, cystic fibrosis, and potentially, lung fibrosis. Although originally described as a T-lymphocyte product, studies in the early 1990s aimed at

identifying novel mediators that could counterregulate the effects of glucocorticoids on inflammation and immunity, led to the discovery that MIF is released by cells of the anterior pituitary gland. It was shown that MIF accounts for approximately 0.05% of the total pituitary content as compared to adrenocorticotropichormone (ACTH) (0.2%) and prolactin (0.08%). Lipopolysaccharide (LPS)- induced secretion of MIF from the pituitary gland and from circulating/tissue resident monocyte/macrophages is thought to play an important role in the pathogenesis of endotoxemia and sepsis. Treatment of mice with recombinant MIF has been shown to potentiate lethal endotoxemia and MIF’s therapeutic potential, in models of severe sepsis, has been highlighted by its ability to neutralize antiMIF antibodies to protect animals. Of clinical importance, this protection has been observed when given before, and up to 8 h after, the initiating septic insult. With the development of MIF knockout mice, it has become apparent that these animals are relatively resistant to LPS. Investigators have recently shown that this is due to downregulation of Toll-like receptor 4 (TLR-4), the signal-transducing molecule of the LPS receptor complex, and is associated with decreased activity of the transcription factor PU.1, which is required for optimal expression of the Tlr4 gene in myeloid cells (Table 1). In addition to being secreted from the pituitary, MIF is also found to be released from immune/inflammatory cells as a consequence of glucocorticoid stimulation. Recombinant MIF ‘overrides’ or counter-regulates the immunosuppressive effects of glucocorticoids on immune/inflammatory cell activation and proinflammatory cytokine release. An emerging body of data presently indicates that MIF’s position within the cytokine cascade is to act in concert with glucocorticoids to control the ‘set point’ of the immune and inflammatory response.

Structure The Mif gene (GeneBank Accession number: L19686) is located on chromosome 22q11.2. The gene is 2167

6 MACROPHAGE MIGRATION INHIBITOR FACTOR

bp and has three exons separated by two introns of 189 bp and 95 bp. The cDNA for MIF encodes a 115 amino acid protein with an apparent molecular weight of 12.5 kDa that has no significant sequence homology to any other protein. The unique structure of both rat and human MIF has been defined using Table 1 Tissue/cellular distribution of MIF protein Tissue

Stimuli

Anterior pituitary Corticotrophic cells

CRF, LPS

Immune system Monocytes/macrophages

T cells (Th2 4 Th1), mast cells Eosinophils HL-60, myelomonocytic Lung Bronchial epithelium Alveolar macrophages

LPS, TNF-a, IFN-g, glucocorticoids TSST-1, exotoxin A aCD3, PMA/ionomycin, PHA PMA, C5a, IL-5 LPS

LPS

Liver Hepatocytes, Kupffer cells

LPS

Vasculature Endothelial cells

LPS

X-ray crystallography. The amino acid sequences of human and murine MIF share 90% identity and this is reflected in almost identical three-dimensional crystal structures. Crystal structure studies show that MIF protein exists as a homo-trimer with each monomer consisting of two antiparallel a-helices and six b-strands. Thus three b-sheet domains are surrounded by six a-helices to form a central barrel with open ends (Figure 1). The barrel structure contains a solvent accessible channel that varies from 4 to 15 A˚ in diameter and is predominantly lined with hydrophilic atoms. Electrostatic potential mapping indicates that the core of the barrel structure is positively charged and could possibly interact with negatively charged moieties.

Regulation of Production and Activity MIF is ubiquitously expressed in response to various hormones, growth factors, and inflammatory stimuli such as LPS. Furthermore, MIF is expressed at high levels in many resting inflammatory cells and is stored preformed, ready for immediate secretion. Specific release of MIF is seen from macrophages in response to LPS, T cells in response to anti-CD3/CD28 stimulation, and fibroblasts in response to adhesion, integrin ligation, and growth factor stimulation.

Figure 1 Three-dimensional structure of MIF in trimeric form with each 12.5 kDa monomer respresented by a different color. The barrel-like structure is being viewed from above indicating the solvent accessible channel that is formed by the three molecules when they interact.

MACROPHAGE MIGRATION INHIBITOR FACTOR 7

The MIF promoter is responsive to several signaling pathways and many of the stimuli that induce MIF secretion also induce gene expression. In vitro promoter studies have indicated the importance of cyclic adenosine monophosphate (cAMP)-responsive elements in transcriptional responses to corticotrophinreleasing factor in the pituitary and have revealed the functional effects of polymorphic variations in the MIF gene-promoter. Indeed, high expression of MIF in many inflammatory diseases may be contributed to by promoter polymorphisms. A tetranucleotide CATTrepeat is present at position 794 with individuals being homozygous or heterozygous for 5-, 6-, 7- or 8-CATT repeats. Of these four genotypes, the 5-CATT allele has lowest transcriptional activity and is associated with less aggressive rheumatoid disease. Similarly, in cystic fibrosis, patients expressing a 5-CATT allele have less end-organ injury and a trend toward a higher body mass index (BMI) and improved pulmonary function. Genetic susceptibility to enhanced MIF expression may be a critical determinant in the pursuit of therapeutic strategies for inflammatory lung diseases and strongly influence the efficacy of glucocorticoid treatment.

Biological Functions Upon release, MIF is directly proinflammatory by activating or promoting the expression of several cytokines, chemokines, and inflammatory molecules such as TNF-a, IL-1b, IL-2, IL-6, IL-8, IFN-g, nitric oxide, matrix metalloproteinase, and cyclooxygenase-2 (Cox-2). MIF has also been shown to counteract glucocorticoid-induced inhibition of inflammatory cytokine secretion. Furthermore, MIF inhibits the ability of glucocorticoids to induce IkB synthesis in LPS- stimulated human peripheral blood mononuclear cells. Thus, by blocking glucocorticoid induced IkB synthesis, MIF promotes the translocation of NF-kB into the nucleus where it activates proinflammatory cytokine and adhesion molecule expression (Table 2). Recombinant MIF as well as endogenously released MIF, which is secreted as a consequence of serum stimulation and integrin ligation, induces the proliferation of quiescent fibroblasts. This response is associated with a sustained activation of the p44/p42 extracellular signal-regulated protein kinase (ERK) subfamily of mitogen-activated protein (MAP) kinases and is dependent upon the activity of protein kinase A. The ERK MAP kinase signaling cascade results in the phosphorylation and activation of a number of cytosolic proteins such as P90rsk, c-myc, and cytoplasmic phospholipase A2 (cPLA2). cPLA2 is a critical mediator of inflammatory responses and its

Table 2 Summary of MIF functions in respiratory pathologies Proinflammatory

Induces expression of TNF-a, IL-1b, IL-2, IL-6, IL-8, IFN-g, nitric oxide, matrix metalloproteinase, and cyclooxygenase-2

Glucocorticoid inhibition

Overrides anti-inflammatory effects of glucocorticoids, e.g., blocks inhibition of TNF-a and IL-8 expression in alveolar macrophages

Antiapoptotic

Blocks p53 function Prevents neutrophil apoptosis Attenuates activation induced apoptosis of macrophages

Proangiogenic

Promotes VEGF expression Promotes vascularization in angiogenesis models Elevated expression associated with areas of vascularization in various tumors

Pro-proliferative

Promotes proliferation of many tumor cells and fibroblasts, including primary human lung fibroblasts May influence oncogenic transformation

product, arachidonic acid, is the precursor for the synthesis of prostaglandins and leukotrienes. Further studies show that MIF induces the phosphorylation and activation of cPLA2 and, ultimately, the release of arachidonic acid by an ERK- but not p38-MAP kinase-dependent mechanism. Furthermore, it has been shown that adhesioninduced fibroblast MIF secretion promotes integrindependent activation of MAP kinase, cyclin D1 expression, and DNA synthesis in a Rho GTPase-, Rho kinase-, and myosin light-chain kinase-dependent manner. Cyclin D1 is an important upstream modulator of the tumor suppressor protein Retinoblastoma (Rb), phosphorylation of which in turn releases the transcription factor E2F and subsequently induces expression of critical S phase enzymes. MIF may play a significant role in integrin-mediated cell-cycle progression via the induction of Rb phosphorylation and E2F activity. In addition, embryonic fibroblasts from MIF knockout mice are refractory to malignant cell transformation induced by oncogenic H-Rasv12. The cell growth-promoting property of MIF may indeed be an important mechanism by which MIF contributes to the pathology of various inflammatory lung diseases. During the response to infection, the innate immune response is kept in check by specialized counter-regulatory mechanisms such as apoptosis or programmed cell death. Defective apoptosis of activated macrophages may lead to a prolonged inflammatory response. Recent data have linked the actions of MIF to the inhibition of the tumor

8 MACROPHAGE MIGRATION INHIBITOR FACTOR

suppressor p53. These data were extended to show that MIF sustains macrophage survival and proinflammatory function by suppressing activation induced, p53-dependent apoptosis. A likely scenario can now be envisaged whereby MIF’s prime role in the immune/inflammatory response is to regulate the infiltration, induce the activation, and promote the survival of inflammatory cells. Inappropriate expression of MIF could counterregulate the effects of endogenous glucocorticoids and tip the neuroendocrine balance in favor of cell proliferation/survival, an exaggerated inflammatory response, and the development of chronic disease.

Receptors MIF regulates cellular functions via interactions with both surface-expressed and intracellular proteins. The dominant cell-surface interaction of MIF is with CD74. Sustained ERK activation, fibroblast proliferation, and PGE2 release are all mediated via interaction of MIF with CD74.

MIF in Respiratory Diseases Although MIF is most well known as a key contributor to acute lung injury, it is also apparent that MIF plays a role in the development of allergic inflammatory disease. Human eosinophils, derived from atopic donors, are potent sources of MIF. Both C5a and IL-5, physiologically relevant stimuli in allergic inflammatory disease, cause significant immediate release of MIF from these ex vivo circulating cells. Eosinophils have been implicated as a key cell type in the pathogenesis of allergic inflammatory diseases such as bronchial asthma, allergic rhinitis, and atopic dermatitis. Analysis of bronchoalveolar lavage (BAL) samples from a cohort of stable asthmatics revealed an elevated level of MIF protein expression and it is suggested that the elevated numbers of eosinophils present in the alveolar airspaces are responsible for its production. More recently, in animal models of sepsis, C5a has also been shown to induce MIF expression and release from neutrophils. The identification of eosinophils and notably, the type II alveolar epithelial cells as additional cellular sources of MIF, and the correlation of MIF expression with the development of a Th2 phenotype highlights the potential importance of MIF in allergic pulmonary inflammatory disease. MIF has been demonstrated to play a key role in acute lung injury, highlighted by its ability to accentuate proinflammatory cytokine production and override glucocorticoid suppression of TNF-a and IL-8 expression in ex vivo alveolar cells derived from

patients with acute respiratory distress syndrome (ARDS). ARDS is a life-threatening, hyper acute inflammatory response that occurs in the lungs following trauma or sepsis. Inflammatory cell activation, and in particular neutrophil activation, has been implicated in the early stages of ARDS pathogenesis. Recent clinical studies have supported a role for MIF in ARDS by showing that increased MIF expression correlates with the development of ARDS in septic patients. Elevated MIF levels are seen in the serum and peripheral blood mononuclear cells of patients with ARDS and intense staining for MIF protein can be seen in the bronchial epithelium, alveolar epithelium, vascular smooth muscle, and alveolar macrophages. In a rat model of lung injury, anti-MIF treatment was shown to reduce the accumulation of activated neutrophils in the lungs. BAL samples from these animals were found to contain significantly reduced levels of the neutrophil chemoattractant macrophage inflammatory protein-2 (MIP-2). As MIF itself is not a chemoattractant for neutrophils, these data suggest that it may play a role in ARDS via upregulation of the neutrophil chemoattractant MIP-2. Further studies have also shown that recombinant MIF prolongs the life span of activated neutrophils by delaying mitochondria-dependent apoptosis. These data suggest that overproduction of MIF in the lung during acute trauma could lead to neutrophilia that is refractory to suppression by both endogenous and therapeutic glucocorticoids. Clinical treatment with anti-MIF strategies may help reduce early neutrophil accumulation in ARDS and increase the effectiveness of glucocorticoid treatment. A role for MIF in the development of acute lung injury has also been demonstrated in a mouse model of lung injury and fibrosis. Intratracheal administration of bleomycin induced a rapid elevation in MIF expression in both lung tissues and BAL fluids. Treatment of mice with a neutralizing anti-MIF antibody resulted in significantly reduced accumulation of inflammatory cells and reduced TNF-a expression in alveolar airspaces on day 7, decreased histopathological lung injury score on day 10, and decreased mortality by day 14. Anti-MIF treatment, however, did not appear to influence markers of lung fibrosis on day 21 in this model. These data highlight a role for MIF in the initiation of acute lung injury. Despite the inefficacy of anti-MIF treatment on the development of fibrosis in the murine bleomycin-induced lung injury model, a role for MIF in aberrant remodeling in the human lung is supported by the detection of elevated MIF levels in BAL fluids from patients with idiopathic pulmonary fibrosis. Furthermore, the CD74-dependent pro-proliferative

MATRIX METALLOPROTEINASE INHIBITORS 9

effects of MIF on primary human lung fibroblasts and the ability of integrin ligation to promote MIF release and cell-cycle progression highlight the potential role of MIF in the pathogenesis of fibrosis. The ability of MIF to promote cell-cycle progression and inhibit apoptosis may also play an important role in the pathogenesis of lung cancer. Increased MIF expression is seen in non-small cell lung cancer (NSCLC), squamous cell carcinoma and adenocarcinoma, and elevated serum levels of MIF in NSCLC have prompted the investigation of MIF as a potential biomarker. In NSCLC, elevated MIF expression is also associated with elevated risk or recurrence after tumor resection. In several tumors, elevated MIF levels correlate with increased vascular endothelial growth factor expression and in both in vitro and in vivo angiogenesis models, MIF promotes vascularization. These data are supported by the clinical observations that increased MIF expression correlates with elevated angiogenic CXC chemokine expression and vessel density in some NSCLC, and that increased MIF expression is seen in areas of cell proliferation and increased vascularization in atypical adenomatous hyperplasia. The fact that murine embryonic fibroblasts from MIF knockout mice resist oncogenic transformation further supports a role for MIF in the development of cancer. MIF is a key inflammatory cytokine and integrator of the immuno-neuroendocrine interface which is clearly implicated in the pathogenesis of inflammatory lung pathologies. At present, significant evidence exists to provide a rationale for focused anti-MIF clinical trials in the treatment of inflammatory lung disease. Furthermore, emerging evidence highlighting genetic susceptibility to enhanced

MIF expression may prove invaluable in the definition of patient populations in which a focused antiMIF treatment strategy would be efficacious. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Cell Cycle and Cell-Cycle Checkpoints. Corticosteroids: Therapy. Cystic Fibrosis: Overview. Fibroblasts. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Tumors, Malignant: Overview.

Further Reading Baugh JA and Donnelly SC (2003) Macrophage migration inhibitory factor: a neuroendocrine modulator of chronic inflammation. Journal of Endocrinology 179(1): 15–23. Donnelly SC and Bucala R (1997) Macrophage migration inhibitory factor: a regulator of glucocorticoid activity with a critical role in inflammatory disease. Molecular Medicine Today 3(11): 502–507. Donnelly SC, Bucala R, Metz CN, et al. (1999) Macrophage migration inhibitory factor and acute lung injury. Chest 116 (1 supplement): 111S. Donnelly SC, Haslett C, Reid PT, et al. (1997) Regulatory role for macrophage migration inhibitory factor in acute respiratory distress syndrome. Nature Medicine 3(3): 320–323. Leng L, Metz CN, Fang Y, et al. (2003) MIF signal transduction initiated by binding to CD74. Journal of Experimental Medicine 197(11): 1467–1476. Liao H, Bucala R, and Mitchell RA (2003) Adhesion-dependent signaling by macrophage migration inhibitory factor (MIF). Journal of Biological Chemistry 278(1): 76–81. Mitchell RA (2004) Mechanisms and effectors of MIF-dependent promotion of tumourigenesis. Cell Signal 16(1): 13–19. Rossi AG, Haslett C, Hirani N, et al. (1998) Human circulating eosinophils secrete macrophage migration inhibitory factor (MIF). Potential role in asthma. Journal of Clinical Investigation 101(12): 2869–2874. Swope MD and Lolis E (1999) Macrophage migration inhibitory factor: cytokine, hormone, or enzyme? Reviews of Physiology, Biochemistry and Pharmacology 139: 1–32.

MATRIX METALLOPROTEINASE INHIBITORS K J Leco, E L Martin, L C Barcroft, and S E Gill, University of Western Ontario, London, ON, Canada & 2006 Elsevier Ltd. All rights reserved.

Abstract The tissue inhibitors of metalloproteinases (TIMPs) are the primary endogenous inhibitors of matrix metalloproteinases (MMPs). The TIMPs are expressed in lung tissue during the initial stages of lung development and throughout the life of the organism. Through regulation of MMP activity, TIMPs indirectly regulate remodeling of the extracellular matrix (ECM) as well as cell signaling via ECM molecules. An imbalance between active MMPs and TIMPs in favor of enhanced MMP activity can lead to inappropriate ECM loss, or conversely, an

imbalance favoring the TIMPs can abrogate MMP activity leading to excess ECM deposition. Homeostasis between MMPs and TIMPs is also required to maintain correct growth factor signaling. MMPs are known to contribute to the release of growth factors from the ECM, and affect the activation or degradation of growth factors and/or growth factor receptors. The presence or absence of TIMPs can influence this MMP-dependent growth factor regulation. Maintenance of the MMP–TIMP balance is integral to normal lung development and function and a disruption in this balance has been implicated in numerous diseases of the lung. Overabundance of MMPs versus TIMPs can lead to emphysema while enhanced inhibition may contribute to fibrotic pulmonary diseases. Current research is focused on the function of these inhibitors within the lung and exploring the potential of MMP inhibition as a therapeutic target for multiple lung diseases.

10

MATRIX METALLOPROTEINASE INHIBITORS

Introduction The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which collectively, are capable of cleaving all of the protein components of the extracellular matrix (ECM). There are 25 mammalian MMPs which can be classified into subfamilies based on their major matrix substrates. These enzymes are inhibited by a separate endogenous family of four proteins known as the tissue inhibitors of metalloproteinases (TIMPs). As a group, TIMPs-1, -2, -3, and -4 are able to inhibit all MMPs; however, they each preferentially inhibit a subset of MMPs. Normally, a balance between the active MMPs and TIMPs contributes to a state of ECM homeostasis while a disruption of this balance in favor of the active MMPs leads to ECM degradation. Conversely, shifting the axis to favor the TIMPs promotes ECM deposition, which in a severe form is known as fibrosis. Coordinate regulation of MMP and TIMP expression is required for many physiological processes including ovulation, embryo implantation, embryo development, endometrial cycling, and wound healing. Inappropriate disruption of the MMP–TIMP balance is associated with many pathological conditions including multiple diseases of the lung, such as pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, acute lung injury (ALI), and cancer invasion and metastasis. The four TIMPs are highly homologous proteins ranging from 21 to 28 kDa in size, with a core protein molecular mass of approximately 21 kDa, and variable glycosylation contributing the remainder of the mass. In general, TIMPs-1, -2, and -4 are soluble or cell surface associated, while TIMP-3 is bound to the ECM. The TIMP proteins are composed of two domains, a three-loop N-terminal domain responsible for the MMP inhibitory activity, and a three-loop C-terminal domain, which can contribute to the specificity of interactions between TIMPs and MMPs. Each of the four TIMP proteins contains 12 conserved cysteine residues, which form six disulfide bonds required for correct folding and function of the protein (Figure 1). Inhibition of MMPs by TIMPs occurs through 1 : 1 enzyme-inhibitor complex mediated primarily by the first cysteine residue in the TIMP protein, which coordinates with the zinc atom in the active site of the enzyme. The remainder of the N-terminal domain of the TIMP molecule sits as a wedge within the active site of the MMP, with various amino acid residues providing selectivity of binding. The C-terminal domain of TIMP-1 mediates a specific interaction with pro-MMP-9, while the C-terminal domain of TIMP-2 can bind to a proMMP-2 molecule. The C-terminal domain of TIMP-3

L6

L5

9 8

L3

L2

L4

10 11

7 12 4 5 1 2 NH2

6 3

COOH L1

Figure 1 Structure of the TIMP-1 protein. The TIMP-1 molecule contains six peptide loops (L1 through L6) held together by six disulfide bonds formed between the 12 cysteine residues (numbered circles). All 12 cysteine residues are absolutely conserved among all four TIMP proteins as is the looped structure. The first four amino acids (Cys-x-Cys-x) of TIMP proteins bind to the active site of MMPs, similar to substrate binding; however, catalysis does not occur. Six distinct sections of the TIMP-1 molecule (surrounding the first two disulfide bonds at the N-terminus) make contact with MMP-3, with the cysteine residue at position one of TIMP-1 coordinating with the zinc atom of the active enzyme. Adapted with permission from Bodden MK, Harber GJ, BirkedalHansen B, et al. (1994) Functional domains of human TIMP-1 (tissue inhibitor of metalloproteinases). Journal of Biological Chemistry 269(29): 18943–18952, & American Society for Biochemistry and Molecular Biology. This work is protected by copyright and the making of this copy was with the permission of Access Copyright. Any alteration of it’s content or further copying in any form whatsoever is strictly prohibited.

can interact with both pro-MMP-2 and -9, and the C-terminal domain of TIMP-4 can mediate binding to pro-MMP-2. Kinetic studies between TIMP-1 and MMP-1 or TIMP-2 and MMP-2 have demonstrated that there is a rapid formation of a noninhibitory, reversible intermediate, followed by conversion to a stable inhibitory complex with an estimated half-life of one year. An inventory of structural and functional attributes of the TIMP proteins can be found in Table 1. TIMP expression can be regulated by various growth factors and/or cytokines, such as transforming growth factor beta (TGF-b), basic fibroblast growth

MATRIX METALLOPROTEINASE INHIBITORS 11 Table 1 Structural and functional attributes of the TIMP proteins TIMP-1

TIMP-2

TIMP-3

TIMP-4

21 0 Soluble/cell surface Pro-MMP-2 None

24/27 1 ECM Pro-MMP-2/-9 None

22 0 Soluble/cell surface Pro-MMP-2 None

ADAM inhibition

28 2 Soluble Pro-MMP-9 MT1-MMP MT2-MMP MT3-MMP MT4-MMP MT5-MMP MMP-19 ADAM 10

None

None

Cell proliferation

mErythroid precursors

mErythroid precursors

ADAM 12 ADAM 17 ADAM 19 (ADAM 10) ADAMTS-4, TS5 mSmooth muscle cells and cancer cells

mTumor cells

mTumor cells mFibroblasts mSmooth muscle cells kEndothelial cells mColorectal cancer cells kMelanoma

mSmooth muscle cells

mCardiac fibroblasts

Protein kDa N-glycosylation sites Protein localization Pro-MMP association MMPs poorly inhibited

Apoptosis

Tumor angiogenesis Tumorigenesis effects

kBurkitt’s lymphoma cells

mMammary kLiver Inhibits

kMelanoma kMammary Inhibits

mMammary tumor cells kWilm’s tumor cells

mTumor cells mRetinal pigmented epithelial cells kMelanoma Inhibits

Inhibits

Adapted from Baker AH, Edwards DR, and Murphy G (2002) Metalloproteinase inhibitors: biological actions and therapeutic opportunities. Journal of Cell Science 115: 3719–3727, with permission of The Company of Biologists Ltd.

factor, epidermal growth factor, and the interleukins. In particular, TGF-b has multiple effects on ECM metabolism; it can induce the synthesis of ECM molecules, potentiate growth factor induction of TIMP expression, and repress growth factor stimulation of MMP expression, thus promoting matrix deposition. The TIMPs are multifunctional proteins, in that they can act as growth factors or proapoptotic molecules in their own right. For instance, the Timp-1 cDNA was first identified due to the erythroid potentiating activity of the TIMP-1 protein. Recent evidence has indicated that TIMPs-2, -3, and -4 also possesses similar abilities. Further, TIMP proteins can either induce or be protective against apoptosis depending on the cell type examined (Table 1). In addition to altering cell growth and survival, TIMPs can affect cell adhesion and migration. MMPs can disrupt cell–cell adhesion by cleaving molecules such as b4 integrin and E-cadherin, MMP-2 cleavage of laminin-5 induces karatinocyte migration, and MT1-MMP cleavage of laminin-5 can induce migration of several cell types. Therefore, TIMP inhibition of MMPs can prevent such migration and promote cell–cell and cell–ECM adhesion. MMPs, and thus TIMPs, are also involved in the production (and/or

degradation) of various chemotactic gradients during growth, development and inflammation. This function may contribute to the TIMP protein’s antiangiogenic properties (Table 1). The TIMP-2 protein has a unique property when compared to the other TIMPs. It not only inhibits MMP-2, but is also required for pro-MMP-2 activation by MT1-MMP. This activation requires formation of a trimolecular complex between MT1-MMP, TIMP-2, and pro-MMP-2. More specifically, the Nterminal domain of TIMP-2 binds to and inhibits a cell surface MT1-MMP. The free TIMP-2 C-terminal domain then binds to the hemopexin site within the Cterminal domain of pro-MMP-2 and a second, nonTIMP-2 associated MT1-MMP creates an activation intermediate of the tethered pro-MMP-2. Full activation MMP-2 is then achieved by autocatalytic cleavage via adjacent, tethered, activated MMP-2 molecules. However, an excess of TIMP-2 can totally inhibit pro-MMP-2 activation by inhibition of all active MT1-MMP molecules. Although the physiological consequences of this interaction are not fully understood, it is believed this membrane-bound complex is able to localize MMP-2 activity to points of focal proteolysis such as lung bronchiole epithelial

12

MATRIX METALLOPROTEINASE INHIBITORS

invasion into distal mesenchyme during lung morphogenesis. Despite the fact that the C-terminal domain of TIMP-4 can bind the C-terminal domain of proMMP-2, and the N-terminal domain of TIMP-4 can inhibit MT-MMPs, formation of a similar trimolecular complex has not been observed for TIMP-4. The TIMP-3 protein is distinct from the other TIMP family members in several aspects. First, TIMP-3 is the only TIMP that binds tightly and specifically to the ECM. This occurs through interaction between the N-terminal domain of TIMP-3 and heparan sulfate and other sulfated glycosaminoglycans of the ECM. This characteristic positions TIMP-3 to protect discrete regions of the ECM, which may be especially important in pattern formation during lung morphogenesis. Second, TIMP-3 not only inhibits members of the MMP family, but it is also able to inhibit members of a related class of enzymes, the ADAMs (a disintegrin and metalloproteinase). ADAMs, commonly referred to as sheddases, are involved in numerous cellular processes such as cleavage of ECM molecules, disruption of cell–cell and cell–ECM adhesion and shedding of cell surface proteins. The proteolytic activity of ADAM-10 can be inhibited by both TIMP-1 and -3, but other members of the ADAM family are inhibited only by TIMP-3. For example, TIMP-3 is an efficient inhibitor of ADAM-TS4 and -TS5 which cleave aggrecan, a component of cartilage matrix. TIMP-3 can specifically inhibit tumor necrosis factor alpha (TNF-a) converting enzyme (ADAM-17), and therefore implicates TIMP-3 as a major player in the control of systemic inflammation. Finally, TIMP-3 is the only TIMP known to be directly involved in a human disease. Sorsby’s fundus dystrophy is an autosomal dominant disorder resulting in irreversible blindness between the ages of 30 and 50 due to macular degeneration. This disease is caused by mutation(s) of the Timp-3 gene, which introduce unpaired cysteine residue(s) in the C-terminal domain of TIMP-3, causing improper folding and accumulation of the defective protein in the ECM of the retina. Aberrant TIMP-3 protein complexes alter cell adhesion to the ECM, induce neovascularization of the macula, and, as a consequence, lead to blindness.

and the saccular and alveolar stages (alveologenesis). Coordinated interaction between epithelial and mesenchymal cells is required for the entire process of lung development and this interaction is regulated primarily by the MMPs and consequently, the TIMPs. This necessitates a finely orchestrated balance between the abundance of active MMPs and their inhibitors. The four TIMPs are found in the lung during each of the developmental stages. Although their presence in the developing lung is suggestive of a role for the TIMPs throughout lung development, genetic studies have implicated a function primarily for TIMP-3 during the pseudoglandular, saccular, and alveolar stages. Through regulation of MMP activity, TIMP-3 may indirectly regulate cell signaling mechanisms including growth factor signaling, cell-to-cell signaling, and ECMto-cell signaling during lung development. Bronchiole Branching Morphogenesis

All four Timp mRNA transcripts are expressed during the pseudoglandular phase of mouse lung development, suggesting a niche for TIMPs in the regulation of bronchiole branching. Bronchiole branching morphogenesis involves the repetitive generation of epithelial tube branch points (clefts) and invasion of the new epithelial buds into the distal mesenchyme. This recurring cleft and bud formation ultimately gives rise to the conducting airways within the lungs. TIMP-3 is required during bronchiole branching to regulate MMP activity, and thus regulates patterning of cleft and bud forming regions. Deletion of the Timp-3 gene (and consequently the TIMP-3 protein) impairs the formation of airways, with TIMP-3 null animals having a decreased number of terminal bronchioles (Figure 2). This is due to disruption in the MMP–TIMP balance in favor of the active MMPs, resulting in enhanced ECM degradation (specifically fibronectin) in the nascent cleft forming regions of embryonic TIMP-3-null lungs. Loss of fibronectin from these regions, in turn, alters ECM–cell signaling via focal adhesion kinase, leading to impaired bronchiole branching. In addition, enhanced MMP activity at the distal ends of epithelial buds may destroy ECM–cell signals, which are essential for proliferation of the epithelial cells and migration of those cells into the mesenchyme (Figure 3).

TIMPs in Lung Development Lung development is composed of five different stages based on the structures formed during each stage. These stages include the embryonic stage (lung bud formation), the pseudoglandular stage (bronchiole branching morphogenesis), the cannilicular stage (blood vessel and terminal air sac formation),

Alveologenesis

Alveologenesis in mammals begins in utero during the saccular stage of lung development, with the generation of terminal air sacs at the distal termini of bronchioles. Alveolar development continues well after birth (alveolar stage extends up to 4 weeks

MATRIX METALLOPROTEINASE INHIBITORS 13 TIMP-3 −/−

Ed13.5 (left lobe)

TIMP-3 +/+

(b)

(c)

(d)

~500 µm

PP1 (periphery of left lobe)

(a)

~200 µm

Figure 2 Bronchiole branching impairment in the TIMP-3-null murine lung. The cartoons depict the extent of bronchiole branching in (a,b) the whole left lung lobe at embryonic day 13.5 (Ed13.5) or (c,d) at the periphery of the left lung at postpartum day 1 (PP1) in wildtype (TIMP-3 þ / þ ) and TIMP-3 null (TIMP-3
/
) lungs. Bronchiole branching in the null lung is significantly reduced during embryogenesis, with a consequent reduction in lung volume. The diminished branching during embryogenesis is not due to a developmental delay in the TIMP-3 null lung, as branching of the terminal bronchioles is also impaired at postpartum day 1. Impaired branching is reflected in the reduced number of dilated terminal bronchioles in the postpartum null lung compared to the wild-type control.

Epithelial lung bud invading into the distal mesenchyme

Fibroblasts

(a)

Invading epithelial lung bud in the TIMP-3 wild-type lung

Invading epithelial lung bud in the absence of TIMP-3 Basement membrane

MMP molecules MMP inhibited by TIMP-3

(b)

(c)

ECM deposition in cleftforming region

Figure 3 A model for reduced branching in the TIMP-3 null lung. During the pseudoglandular stage of lung development, epithelial buds invade into the surrounding mesenchyme. The basement membrane surrounding the tip of the bud normally thins as MMP enzymes are produced. Accumulation of ECM proteins (elaborated by fibroblasts in the mesenchyme) presents an obstacle to the invading epithelial tube, which then forms a cleft region yielding two epithelial buds. The process is repeated forming the conducting airways of the lung. In the absence of TIMP-3, excess MMP activity prevents accumulation of basement membrane proteins forming a dilated epithelial tube. Excessive MMP activity also reduces the deposition of ECM proteins in cleft-forming regions of the mesenchyme, producing fewer barriers to epithelial cell invasion, and therefore fewer branch points.

14

MATRIX METALLOPROTEINASE INHIBITORS

postpartum in the mouse and 2 years in humans) culminating in the formation of alveoli, the functional units of gas exchange within the lung. This process requires budding of the epithelium in the terminal air sacs to form secondary septa, which serve to increase the surface area for gas exchange within the lung. This is followed by thinning of the mesenchyme within the alveoli to reduce the distance for gas exchange. During the saccular and alveolar stage of lung development, both Timp-2 and Timp-3 mRNAs are abundantly expressed in the mesenchyme of the developing alveoli, suggestive of a role in the regulation of alveologenesis. Loss of TIMP-3 leads to enlarged air spaces, demonstrable at birth in TIMP-3-null mice, which continue to enlarge through alveologenesis when compared to wild-type animals. Reduction in ECM molecule abundance (fibronectin and collagen) contributes to this observed phenotype, but conclusive evidence delineating the molecular mechanism of air space enlargement awaits further experimentation. Within the current published literature, there are no reports of mice deficient for any of the other three TIMPs having a developmental lung phenotype. However, it is apparent that expression of TIMP-3 is required during multiple stages of lung development. The presence of TIMP-3 is necessary for correct formation of both the conducting and respiratory zones of the lungs. Loss of TIMP-3 negatively impacts lung development through disruption of ECM–cell contacts resulting in aberrant epithelial intracellular signaling from the ECM, and also possibly through inappropriate growth factor processing via active MMPs.

TIMPs in Lung Diseases An imbalance between the activities of MMPs and the abundance of TIMPs is associated with many diseases including those of the lung, where disruption of the MMP–TIMP balance contributes to the pathogenesis of many lung diseases. Emphysema

Pulmonary emphysema is characterized by chronic inflammation and destruction of alveolar tissue, leading to airspace enlargement and loss of surface area for gas exchange within the lung. The major contributing factor in the pathogenesis of emphysema is an imbalance between protease and protease inhibitors, known as the protease/antiprotease hypothesis. The two main causes of emphysema are inhalation of toxins (cigarette smoke or air pollution) or a-1-antitrypsin deficiency. In the first instance, inhalation of

toxins induces an inflammatory reaction that leads to an imbalance between MMPs and TIMPs favoring excessive MMP activity. Other protease/antiprotease systems are also involved, but are beyond the scope of this article. In the second instance, a rare genetic deficiency for the endogenous trypsin inhibitor a-1-antitrypsin leads to excessive protease activity giving rise to early-onset emphysema. To date, emphysema has been correlated with increased levels of many of the MMPs, but MMP-1, -9, and -12 are thought to be the main contributors to the pathogenesis of emphysema and are increased in response to cigarette smoke. In addition, emphysema has been linked to high MMP-2 : TIMP-2 and MMP9 : TIMP-1 ratios, indicative of a protease/antiprotease imbalance. Further evidence in support of the protease/antiprotease hypothesis of emphysema comes from genetic manipulations in mice. Mice deficient for TIMP-3 develop spontaneous airspace enlargement similar to that of pulmonary emphysema. The airspace enlargement is observable from the onset of alveologenesis and continues throughout the lifespan of the animal, culminating in decreased efficiency for gas exchange and early mortality at 13–18 months of age (the normal lifespan of a laboratory mouse is 24–36 months). Disruption of the MMP–TIMP balance in the TIMP-3-null mice also results in significant collagen degradation, and resynthesis of disorganized collagen bundles in the lungs of adult mutant animals. This lack of ECM ultrastructure contributes to a significant increase in lung compliance (a measure of flexible recoil of the lung) in the TIMP-3-null animals when compared to control wild-type mice. This phenotype is enhanced further in mice null for Timp-3 and heterozygous for Timp-2, where mice succumb to respiratory distress starting at 6 months of age. In these animals, lung compliance is more than twice that of Timp-3-null mice. The work with TIMP knockout animals definitively demonstrates that an upset of the normal MMP–TIMP balance has devastating effects on lung structure and function (Figure 4). Based on the animal work described above, one might expect that humans who have Sorsby’s fundus dystrophy would also have early onset emphysema. However, recent studies have shown that recombinant TIMP-3 proteins encoding the Sorsby’s mutations maintain most or all of the MMP inhibitory capacity. Thus, while accumulation of misfolded protein in the eyes of these patients has dramatic effects on vision, loss of MMP inhibitory activity of the TIMP-3 protein is essential to generate an emphysematous phenotype. There are very few current studies examining the effects of synthetic MMP inhibitors on pulmonary

MATRIX METALLOPROTEINASE INHIBITORS 15 Alveolar macrophage

Alveolar airspace Type II cell

Alveolar airspace

Type I cell ECM Capillary Alveolar airspace

MMPs Alveolar airspace TIMPs

TIMP bound to MMP

(a)

Alveolar airspace

Alveolar airspace

ECM

Capillary containing macrophage and PMN

Alveolar airspace

Alveolar airspace

(b) Figure 4 The MMP–TIMP balance contributes to the normal and diseased lung structure. (a) Normal lung structure. The cartoon depicts the conjunction of four adjacent alveoli composed of type I and type II epithelial cells, which rest upon a basement membrane and a scaffold of extracellular matrix (ECM) composed mainly of collagen, elastin, fibronectin, and laminin. Held within the ECM are several cytokines, chemokines, and growth factors (pink dots). Within the airspace is a resident alveolar macrophage responsible for phagocytosis of foreign particles. Both alveolar epithelial cells and alveolar macrophages constitutively produce MMPs and TIMPs, which function to maintain normal ECM remodeling and ultrastructure homeostasis. (b) Diseased lung structure. An MMP–TIMP imbalance favoring the MMPs can be induced by excessive elaboration of MMPs in an immune response, or genetically by the deletion of TIMP-3 in experimental animals. In emphysema, inhalation of smoke particles induces an immune response whereby MMPs, released by macrophage cells over an extended period of time, destroy the ultrastructure of the lung leading to airspace enlargement. The TIMP-3-null animals demonstrate similar histology in the absence of macrophage infiltration, due to endogenous production of MMPs which, in the absence of TIMP-3, destroy the lung ECM. In acute lung injury, an initiating insult induces excessive production of MMPs by epithelial cells, alveolar macrophage cells, or invading polymorphonuclear cells (PMN). High MMP levels result in excessive ECM degradation, which can (1) release ECM bound cytokines, chemokines, and growth factors, creating chemotactic gradients for inflammatory cells which increase MMP abundance; (2) activate alveolar macrophages to release stored MMPs; and (3) cause ECMdependent apoptosis (anoikis) of epithelial cells as the ECM is compromised, resulting in destruction of overall lung structure.

emphysema development or progression, likely due to the inherent side effects of long-term broadspectrum MMP inhibitor use in humans. However, a recent study using a broad range MMP inhibitor (CP-471; 474) in guinea pigs found that it

attenuated smoke-induced emphysema and the associated inflammation. In humans with emphysema, treatment with retinoic acid concurrently reduces MMP-9 and increases TIMP-1, resulting in a marked reduction in the MMP-9 : TIMP-1 ratio. However,

16

MATRIX METALLOPROTEINASE INHIBITORS

the long-term benefit of this treatment has not been determined. In the near future, generation of selective pharmacologic MMP inhibitors may prove valuable for treatment of early stage pulmonary emphysema. That is, synthetic MMP inhibitors capable of inhibiting only MMP-9 or MMP-12, for example, may provide benefit to emphysema patients without the undesirable side effects of joint stiffening and skin thickening that are common to therapies involving broad-spectrum MMP inhibitors. Acute Lung Injury

ALI and its more severe form, acute respiratory distress syndrome (ARDS), are characterized by impaired pulmonary gas exchange, bilateral infiltrates, and noncardiogenic edema. A variety of insults including smoke inhalation, stomach acid aspiration, trauma, sepsis, and mechanical ventilation can induce either ALI or ARDS. Once acquired, the mortality rate for these illnesses is 30–50%. Within the airspace, alveolar macrophage cells release MMP-9 and MMP-12 in response to these insults, representing the initial step in disease progression. Recent studies have indicated that TIMP-3 is important in the defense against ALI, as TIMP-3-deficient mice are more susceptible to sepsis-induced ALI. This is also the case in ALI induced by endotoxin treatment, but not in ALI initiated by mechanical ventilation or hyperoxia. The TIMP-3 knockout animals show increased lung compliance following either the induction of sepsis or administration of endotoxin. Increased compliance is accompanied by reduced collagen and fibronectin abundance, as well as increased MMP-2 and -9 activity compared to controls. Thus, TIMP-3 is required to regulate MMP activity during a septic insult to prevent excessive lung injury. Its presence, however, is not imperative in response to direct lung insults such as hyperoxia or mechanical ventilation. ALI represents a clinical presentation where use of broad-spectrum MMP inhibitors shows promise for therapeutic intervention. For example, use of a synthetic MMP inhibitor, COL-3 (a chemically modified tetracycline), reduces severity of lung injury in a pig model of ALI induced by endotoxin infusion. Likewise, intratracheal instillation of human recombinant TIMP-2 or administration of synthetic inhibitors specific for ADAM-17 and MMPs improve outcome in rat models of endotoxin-induced lung injury. Clinical trials utilizing synthetic inhibitors of MMPs in humans have not yet been reported, but the course of treatment would be limited to several days to a few weeks at most. As such, this disease is a good candidate for pharmacological inhibition of MMPs as a management strategy.

Pulmonary Fibrosis

Idiopathic pulmonary fibrosis involves the development of fibrotic scar tissue throughout the lung with a resultant decrease in lung compliance and function. Although there is strong evidence to show that MMPs and TIMPs play an integral role in the development of pulmonary fibrosis, the mechanism through which they fulfill this role is largely unknown. Several studies have been performed on human samples from idiopathic pulmonary fibrosis patients with conflicting results regarding MMP expression/abundance dependent upon the type of sample used (sputum vs. lavage vs. biopsy). Conversely, all of the studies report an increase in the abundance of the TIMP proteins and/or mRNAs. Thus, the prevailing hypothesis is that an upset in the MMP–TIMP balance, this time in favor of the TIMPs, causes excessive MMP inhibition which, in turn, promotes ECM deposition and fibrotic scar formation. Bleomycin was originally characterized as an antibiotic and is still used clinically as a last-line chemotherapeutic agent. However, administration of bleomycin also induces inflammation, fibroblast proliferation, and fibrotic scarring of the lung. Bleomycin administration is used within the laboratory to induce pulmonary fibrosis in experimental animals in a dosedependent manner. In this model there is a consistent upregulation of MMP-2, -9, and -12 at the levels of mRNA and protein abundance and extent of proenzyme activation during pulmonary fibrosis. Increased MMP abundance and activity associated with excessive scar formation may seem counter-intuitive; however, upregulation of MMPs in pulmonary fibrosis is largely a byproduct of inflammation leading to dysregulated matrix remodeling. Further, both TIMP-1 and -2 are also upregulated during bleomycininduced pulmonary fibrosis, contributing to the extent of fibrosis. Conversely, complete inhibition of MMP activity with a synthetic MMP inhibitor, batimastat, prevents bleomycin-induced pulmonary fibrosis by dampening the inflammatory response. Batimastat acts to decrease macrophage and lymphocyte infiltration into the lung, and reduces the activity of MMP-2 and -9 and TIMP-1 abundance. Therefore, the potential for pharmacological inhibition of MMPs in human pulmonary fibrosis remains a viable therapeutic option, but one yet to be explored. Cancer

Numerous studies have associated enhanced MMP expression, abundance, and activation with all stages of cancer progression, including neoplasia, angiogenesis, primary tumor growth, tumor cell invasion, and metastasis in both humans and animal models.

MATRIX METALLOPROTEINASE INHIBITORS 17

Genetic upregulation of MMP expression in host animals and tumor cells has defined a causative role for MMPs in cancer initiation and progression. Moreover, an anti-oncogenic role for TIMPs is supported by the association of TIMPs with tumors that are less aggressive, as well as the correlation of TIMP expression with favorable cancer prognosis in humans. Furthermore, there are many reports demonstrating that exogenous expression of TIMPs, either in transgenic hosts or via transfection of tumor cells, reduces tumor burden, tumor size, and extent of angiogenesis. All of this evidence contributed to the generation of broad-spectrum synthetic MMP inhibitors by many pharmaceutical companies for the treatment of cancer in humans. Initial animal experiments were very supportive for the potential therapeutic application of these inhibitors in cancer chemotherapy. For example, the molecule AG3340 (Prinomastat) potently inhibits MMP-2, -3, -9, -13, and -14, and was effective at inhibiting tumor growth of orthotopic human lung cancer cells in nude mice by a cytostatic and/or antiangiogenic mechanism(s). Numerous other preclinical trials of synthetic MMP inhibitors in animal models of cancer demonstrated similar encouraging outcomes. Phase I clinical trials, however, uncovered painful musculoskeletal side effects including inflammation and joint stiffening as well as skin thickening. Despite the drawbacks of the synthetic inhibitors, several were used in phase III clinical trails (including Marimastat, Prinomastat, and Tanomastat) against a number of advanced cancers in humans including small-cell and non-small cell lung carcinomas. Invariably, these clinical trials were terminated due to the lack of a survival benefit. In hindsight, the differences in efficacy and survival between the preclinical animal experiments and the human trials can be explained by differences in experimental design. In the animal experiments, the inhibitors were administered either before or just after the inoculation with tumor cells. This is in contrast to the human studies where the inhibitors were given to patients with advanced disease. Thus, the human therapeutic approach was, in effect, too little too late. In order for the synthetic inhibitors to be effective, they must be administered during the early stages of the disease, preferably prior to invasion and/or metastasis. MMPs are critically important for local invasion in situ, and may be less important for tumor maintenance after metastasis has occurred. Therefore, in human lung cancer, advances in early detection are a necessary prerequisite to further rational use of MMP inhibitors in chemotherapy.

In the case of advanced small-cell lung cancer, the clinical trial with Tanomastat was terminated prematurely due to the fact that patients receiving the drugs demonstrated poorer survival than placebo controls. Tanomastat is an effective inhibitor of MMP-2, -3, and -9; however, MMP-3, -11, and -14 are the predominant MMPs expressed in human small-cell lung cancers. Thus, in this trial the drug used was not an appropriate inhibitor for the type of MMPs expressed by the cancer cells. Thorough evaluation of the expression profiles of MMPs from different cancers as prognostic indicators will also provide valuable information as to which MMPs should be targeted in specific diseases and should precede the use of targeted inhibitors. Additionally, since animal studies have shown that MMP therapy provides primarily cytostatic and antiangiogenic benefit to tumor burden, concomitant use of cytotoxic chemotherapeutic agents (possibly combined with radiotherapy) is warranted in future clinical trials.

Conclusions Until recently, the ECM was considered a static structure, responsible only for the maintenance of tissue architecture. We now know the ECM to be in a dynamic flux, constantly being remodeled to accommodate developmental and physiological processes. The composition of ECM, its three-dimensional structure, and proteolytic remodeling all contribute to the cellular microenvironment that controls cell growth, shape, motility, differentiation, and survival. Traditionally, the presumed role for MMPs was confined to ECM catabolism. In recent years it has been established that MMPs cleave a wide range of bioactive substrates including growth factors, growth factor binding proteins, and cytokines, either activating or inactivating them. Indeed this may be the predominant function of MMPs in vivo. Since the TIMP proteins are able to inhibit the activities of the MMPs, the TIMPs indirectly control the processes of matrix degradation and bioavailability of certain growth factors and cytokines. This unexpected, enhanced complexity of MMP– TIMP biology has likely contributed to the failure of synthetic MMP inhibitors in clinical trials. Compounds were rushed to phase III trials in patients with unprecedented momentum, before the implications of such therapies were known. Perhaps the synthetic inhibitor(s) actually protected or enhanced tumorassociated paracrine or autocrine growth factor signaling, leading to premature morbidity and mortality of patients. It is clear that there is now an evolving theme in MMP biology. Enhanced expression of MMPs (associated with many human pathological

18

MATRIX METALLOPROTEINASES

conditions) has pleiotrophic effects, which involve both matrix degradation and inappropriate activation/inactivation of other bioregulatory molecules. Identification of the natural substrates of MMPs, the mechanism of activation/inactivation of these substrates, and classification of MMP expression profiles are prerequisites to the further design of selective synthetic MMP inhibitors for clinical use in a variety of human lung disorders. Ultimately, treatment of acute conditions like ALI or myocardial infarction, where MMP inhibitors are administered for a short duration, are the most likely to provide maximal benefit to the patient with minimal side effects.

Acknowledgments Our laboratory is supported by grants from the Canadian Institutes of Health Research (37927) and the Canada Foundation for Innovation (2860). Salary support for SEG and ELM is provided by the Ontario Graduate Scholarship Program. KJL is a recipient of the Ontario Premier’s Research Excellence Award. See also: Acute Respiratory Distress Syndrome. ADAMs and ADAMTSs. Chronic Obstructive Pulmonary Disease: Emphysema, General. Epidermal Growth Factors. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans;

Degradation by Proteases. Fibroblast Growth Factors. Matrix Metalloproteinases. Pulmonary Fibrosis. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Baker AH, Edwards DR, and Murphy G (2002) Metalloproteinase inhibitors: biological actions and therapeutic opportunities. Journal of Cell Science 115: 3719–3727. Bodden MK, Harber GJ, Birkedal-Hansen B, et al. (1994) Functional domains of human TIMP-1 (tissue inhibitor of metalloproteinases). Journal of Biological Chemistry 269(29): 18943–18952. Brew K, Dinakarpandian D, and Nagase H (2000) Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochimica et Biophysica Acta 1477: 267–283. Coussens LM, Fingleton B, and Matrisian LM (2002) Matrix metalloproteinases inhibitors and cancer: trials and tribulations. Science 295: 2387–2392. Gill SE, Pape MC, Khokha R, Watson AJ, and Leco KJ (2002) A null mutation for tissue inhibitor of metalloproteinases-3 (TIMP-3) impairs murine bronchiole branching morphogenesis. Developmental Biology 261: 313–323. Leco KJ, Waterhouse P, Sanchez OH, et al. (2001) Spontaneous air space enlargement in the lungs of mice lacking tissue inhibitors metalloproteinases-3 (TIMP-3). Journal of Clinical Investigation 108: 817–829. Martin EL, Moyer BZ, Pape MC, et al. (2003) Negative impact of tissue inhibitor of metalloproteinase-3 null mutation on lung structure and function in response to sepsis. American Journal of Physiology: Lung Cellular and Molecular Physiology 285: L1222–L1232.

MATRIX METALLOPROTEINASES W C Parks, University of Washington School of Medicine, Seattle, WA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Matrix metalloproteinases (MMPs) are a family of extracellular proteinases defined by the presence of two conserved motifs. One motif is a cysteine-containing prodomain, which functions, in part, to restrain catalytic activity. The other conserved motif is a histine-rich catalytic domain responsible for the endopeptidase activity. Most MMPs possess other domains, which are thought to function in substrate recognition. In the lung, essentially all cell types, including epithelial, interstitial, vascular, and inflammatory cells, produce MMPs; however, both the pattern and levels of MMPs expressed vary among cell types and situations. Furthermore, the function of a given MMP produced by one cell type is likely distinct from the function of the same MMP produced by another cell. Although long thought to be responsible for the turnover and degradation of the extracellular matrix, matrix degradation per se is neither the sole nor the predominant function of these proteinases. Recent findings

indicate that MMPs act on a variety of extracellular proteins, such as cytokines, chemokines, antimicrobial peptides, and other proteins, that regulate varied aspects of inflammation and immunity.

Introduction As their name implies, matrix metalloproteinases (MMPs) are thought to be responsible for the turnover and degradation of connective tissue proteins. The first MMP was isolated in 1962 in the regressing tadpole tail by Jerold Gross and Charles Lapiere at Harvard University in their search for an endogenous collagenase. Following their discovery, essentially all MMPs since isolated have been shown to be capable of degrading various protein components of the extracellular matrix. Consequently, as a family, MMPs are often assigned the role of being the enzymes responsible for the turnover and destruction of matrix, an assumption that led to some unexpected results in

MATRIX METALLOPROTEINASES 19

clinical trials with MMP inhibitors and, eventually, a rethinking of MMP function.

Table 1 Mammalian matrix metalloproteinases Designationa

Common name

Other name(s)

Structure

MMP-1

Collagenase-1

Fibroblast collagenase, interstitial collagenase

MMP-2

Mcol-Ab Mcol-Bb Gelatinase-A

MMP-3 MMP-7 MMP-8 MMP-9

Stromelysin-1 Matrilysin Collagenase-2 Gelatinase-B

MMP-10 MMP-11 MMP-12

Stromelysin-2 Stromelysin-3

MMP-13 MMP-14 MMP-15 MMP-16 MMP-17 MMP-19 MMP-20 MMP-21 MMP-22 MMP-23 MMP-24 MMP-25 MMP-26 MMP-27 MMP-28

Collagenase-3 MT1-MMP MT2-MMP MT3-MMP MT4-MMP RASI-1 Enamelysin

MMPs comprise a family of 25 related but distinct vertebrate gene products, of which 24 are found in mammals (Table 1). MMPs are secreted or anchored to the cell surface, thereby confining their catalytic activity to membrane proteins and proteins within the secretory pathway or extracellular space. To be classified as an MMP, a protein must have at least the conserved pro- and catalytic domains (Figure 1). The prodomain of a typical MMP is approximately 80 amino acids and contains the consensus sequence PRCXXPD. The exception to this rule is MMP-23, in which the critical cysteine is found within a distinct run of amino acids. The catalytic domain contains an active site Zn2 þ (hence the prefix ‘metallo’) ligated to three conserved histidines in the sequence HEXXHXXGXXH. The glutamate residue within this catalytic motif provides the nucleophile that severs peptide bonds. The backbone structures of the MMP catalytic domain, including a characteristic Met-turn caused by a conserved methionine downstream of the zinc-binding site, are quite similar to those of the ADAM and ADAMTS metalloproteinase families. These three families are part of the much larger metzincins clan of the metalloendopeptidase superfamily. With the exceptions of MMP-7, -23, and -26, MMPs also have a flexible proline-rich hinge region and a hemopexin-like C-terminal domain (Figure 1), which is thought to function in substrate recognition. Other domains found in MMPs are restricted to subgroups of enzymes. For example, four membranetype MMPs (MMP-14, -15, -16, and -24) have transmembrane and cytosolic domains, and MMP-17 and -25 have C-terminal glycosylphosphatidylinositol (GPI) anchors. The two gelatinases (MMP-2 and MMP-9) have gelatin-binding domains that likely enhance their interaction with gelatin-like substrates. In addition to a remarkably common three-dimensional structure, MMPs share a similar gene arrangement suggesting that they arose by duplications of an ancestor gene. At least eight of the known human MMP genes (MMP-1, -3, -7, -8, -10, -12, -13, and -20) are clustered on chromosome 11 at 11q21–23. Other MMP genes are scattered along chromosomes 1, 8, 12, 14, 16, 20, and 22.

Regulation of Production and Activity As for all secreted proteinases, the catalytic activity of MMPs is regulated at four points – gene expression, compartmentalization (i.e., the pericellular

72 kDa gelatinase, 72 kDa type IV collagenase Transin-1 PUMP Neutrophil collagenase 92 kDa gelatinase, 92 kDa type IV collagenase Transin-2 Metalloelastase

CA-MMP MT5-MMP Leukolysin Endometase

Rat collagenase Membrane-type MMP

MT6-MMP Matrilysin-2

Epilysin

a

MMP-4, -5, and -6 turned out to either MMP-2 or MMP-3 and, hence, were not unique MMPs. MMP-18 (collagenase-4) has only been cloned from Xenopus. A mammalian homolog has not been found. b Mcol-A and -B are murine homologs of MMP-1. Based on its chromosome position and enzymatic activity, Mcol-A is a strong candidate to be the murine ortholog of human MMP-1.

accumulation of enzyme), proenzyme (or zymogen) activation, and enzyme inactivation – and is further controlled by substrate availability and affinity. Typically, MMPs are not expressed in normal, healthy tissues. In contrast, some level of MMP expression is seen in any repair or remodeling process, in any diseased or inflamed tissue, and in any cell type grown in culture. Although the qualitative patterns and quantitative levels of MMPs vary among cell types, tissues, and inflammatory conditions, activated cells, whether in tissue or in a culture dish, express MMPs. For the most part, the production of MMPs is regulated at the level of transcription by specific signals, such as cell–cell and cell–matrix interactions, cytokines, and infection. Pro-MMPs are kept in a catalytically inactive state by the interaction between the thiol of a prodomain cysteine residue and the zinc ion of the catalytic site. They are converted to active proteinases

20

MATRIX METALLOPROTEINASES

Zn

SH Typical

Pro

SP

Hinge

Catalytic

Hemopexin-like

MMP-1 (and MColA. MColB). -0. -8. -10. -12. -10. -18. -19. -20. -22. -27 Minimal

Pro

SP

Catalytic

MMP-7. -28 Furin activated

Pro

SP

Catalytic

Fr

Hemopexin-like

MMP-11. -28 Hemopexin-like Hemopexin-like Gelatin binding

SP

Pro

Catalytic

FN FN FN

Pro

SP

Hemopexin-like

CL

MMP-2. -9 (CL) Type I membrane

Hemopexin-like

or

Fr

Hemopexin-like

Catalytic

TM Cs

MMP-14. -15. -18. -24 Type II membrane

SA

Pro

Fr

Catalytic

Pro

Fr

Catalytic

Cys

IgG-like

MMP -20 GPI anchored

SP

Hemopexin-like

GPI

MMP -17. -25 SP

Signal peptide

TM

Transmembrane

FN Fibronectin repeat

Pro

Prodomain

Cs

Cytosolic

CL

Fr

GPI GPI anchor

Furin cleavage

(a)

SA N-terminal signal anchor Cys

Catalytic domain

Collagen-like

Cysteine array

Gelatin-binding domain (MMP-2, -9)

PRCXXPD

HEXXHXXGXXHS Propeptide

Hinge

Linker, transmembrane, or cytoplasmic domains (MT-MMPs) (b)

Hemopexin-like domain (absent Cys in MMP-7, -26)

Figure 1 Domain structure of MMPs. (a) Although MMPs are often subdivided into groups based on differences in domain composition, domain structure alone does not predict function. One division that can be made is between MMPs that are membrane associated (type I and II membrane and GPI anchored) and MMPs that are released (typical, minimal, furin activated, and gelatin binding). It is imprecise to consider the released MMPs as soluble enzymes. Data indicate that they anchor to membrane structures, which is a mechanism to confine proteolysis to defined pericellular compartments and to effectively increase enzyme concentration. Reproduced from Parks WC, Wilson CW, and Lopez-Boado YS (2004) Matrix metalloproteinases as modulators of inflammation and innate immunity. Nature Reviews: Immunology 4: 617–629, with permission from Nature Publishing Group. (b) More instructive information is revealed by the crystal structure of MMPs. Shown here is a composite structure of 17 different MMPs. The histidines in the conserved sequence HEXXHXXGXXHS ligate with the zinc atom (brown ball) in the catalytic domain (purple). The thiol of the cysteine residue in the conserved sequence PRCXXPD of the prodomain (green ribbon) ligates with the zinc ion to keep the pro-form (zymogen) in an inactive state. Once the zinc–thiol bond is broken, the enzyme is active, and peptide bonds of protein substrates are cleaved within the catalytic cleft (yellow dashed line). With the exception of MMP-7 and MMP-26, the catalytic domain is linked to a four-blade, b-sheet hemopexin domain (red and orange) by a short, flexible hinge (white). Models have predicted that the hemopexin swings toward the catalytic cleft to stabilize the interaction with substrate. Reproduced from Massova I, Kotra LP, Fridman R, and Mobashery S (1998) Matrix metalloproteinases: structures, evolution, and diversification. FASEB Journal 12: 1075–1095, with permission.

MATRIX METALLOPROTEINASES 21

by disruption of this interaction, the so-called cysteine switch mechanism, which can be achieved by proteolysis of the prodomain or by modification of the cysteine thiol group. Several MMPs contain an RXKR or RRKR sequence between the pro- and catalytic domains, which serves as a target sequence for pro-protein convertases or furins (Figure 1(a)). For the other MMPs, the mode of activation is more presumed than proved. Possibly the best described nonfurin activation mechanism of a pro-MMP is the cell surface activation of pro-MMP-2 by active MMP-14. Once the prodomain has been cleaved, the active MMPs can be inhibited by natural inhibitors such as TIMPs, internalization, and other mechanisms. For example, oxidants can both activate (via oxidation of the prodomain thiol) and subsequently inactivate MMPs (via modification of amino acids critical for catalysis), providing a mechanism to control bursts of proteolytic activity at sites of inflammation. In vitro studies have demonstrated significant overlap in the substrates that MMPs can cleave, particularly among the extracellular matrix (ECM) substrates. For example, fibronectin, laminins, elastin, and type IV collagen can be degraded by a variety of MMPs in vitro. In a setting such as inflammation, in which essentially all MMPs are present, the shared substrate potential among enzymes would seemingly permit biochemical redundancy. However, substrate selectivity can be honed by two processes: enzyme affinity and compartmentalization. Kinetic studies with model substrates have demonstrated that specific enzymes degrade some substrates more efficiently than others. For example, both MMP-2 and MMP-9 act on cleaved collagen better than other gelatinolytic MMPs, and MMP-7 is a more potent elastase than is MMP-9 or MMP-12. Thus, in a tissue environment, which contains many potential substrates, selectivity of MMP catalysis, in addition to being regulated by the concentration of active enzyme, may be in part directed by the concentration of a preferred substrate relative to that of other potential substrates within range of a secreted MMP. Compartmentalization (i.e., where and how in the pericellular environment an MMP is released and held) is equally important, if not more so, for regulating the specificity of proteolysis than is the affinity of the enzyme–substrate interaction. An important concept is that cells do not indiscriminately release proteases. Rather, proteinases such as MMPs are typically anchored to the cell membrane, thereby maintaining a locally high enzyme concentration and targeting their catalytic activity to specific substrates within the pericellular space. In addition to the membrane-bound MMPs, several examples of specific cell–MMP interactions have been reported, such as

the binding of MMP-2 to the avb3 integrin, MMP-1 to the a2b1 integrin, MMP-9 to CD44, and MMP-7 to surface proteoglycans. As suggested for CD44 and the a2b1 integrin, these membrane anchors may act as accessory factors that mediate both proenzyme activation and binding of both substrate and proteinase, thereby increasing the probability of proteolysis. Thus, as for most protein–protein interactions, MMP specificity may be driven by a third component. It is likely that other MMPs are also attached to cells or matrix via similar specific interactions, and determining these anchors will be a key advance toward identifying activation mechanisms and substrates.

Biological Function The role of MMPs as ECM-degrading enzymes has been based largely on findings within a defined system – typically a purified MMP incubated under optimal conditions with a purified ECM protein – showing what a proteinase can do, not what it does do in real tissues. Despite this caveat, scores of compelling biochemical and observational data, combined with recent genetic findings, indicate that some MMPs do act on ECM proteins in vivo. An important concept is that in a complex setting, such as an inflamed tissue or tumor environment, multiple, diverse cell types are present and perform a variety of processes, such as angiogenesis, remodeling, and phagocytosis. As stated previously, cells produce a spectrum of MMPs, and as a result of their distinct localization, a specific MMP made by one cell type, such as a macrophage, would likely perform a function distinct from the same enzyme produced by another cell type, such as an epithelial cell. Furthermore, a given MMP produced by one cell type likely does more than one thing. In other words, individual MMPs can serve multiple functions (i.e., act on multiple protein substrates) in a given tissue setting, and evidence that has emerged during the past several years indicates that these functions are not always related to ECM degradation. The key to understanding MMP function is to identify the physiologic substrates. Many proteinases, particularly MMPs, are woefully non-specific in vitro; thus, biologically relevant approaches were needed to identify enzyme function (i.e., to identify the natural, physiologic substrates). Because MMPs are secreted or anchored to the cell surface, their potential substrates include all membrane proteins or proteins within the secretory pathway or extracellular space, but it is likely that only a small proportion are actual substrates. The identification of substrates is not straightforward, but several approaches have been used, such as

22

MATRIX METALLOPROTEINASES

affinity-based approaches with substrate-binding motifs, proteomics, and deduction. Expression technology in cell systems and the development of genetically defined mice have proved to be extremely powerful investigative tools in modern biology. A lack of protein cleavage in a specific knockout mouse combined with the acquisition of proteolysis on ectopic expression of a specific MMP are useful tools for finding and verifying substrates. During the past few years, MMPs have been shown to act on as many, if not more, nonmatrix substrates as their more classical ECM substrates. Of the MMPs genetically targeted to date, all except MMP-14-deficient mice, which have severe bone deformations and markedly impaired alveolar morphogenesis, show no or only a minor phenotype in unchallenged mice. Although these negative observations could be interpreted to indicate that many MMPs do not serve a direct role in turnover of ECM proteins, this function is performed by some family members. For example, MMP-14 is a key physiologic collagenase, and gain-of-function studies indicate that MMP-7 expressed by human macrophages is the relevant elastase made by these cells. However, matrix degradation is neither the sole nor the predominant function of these enzymes. An emerging view of MMPs is that they act primarily on nonmatrix proteins, such as cytokines, chemokines, receptors, and antimicrobial peptides, often potentiating activity. Once challenged, such as by lung injury, inflammation, and infection, MMP knockout mice reveal a variety of phenotypes, indicating that these enzymes serve specific and, occasionally, essential roles in tissue repair, angiogenesis, host defense, tumor progression, and, in particular, inflammation (Table 2). Thus, it seems that MMPs, at least those that have been knocked out, have evolved to respond to the insults and pressures of extrauterine existence. If any generalization can be made about MMP function, it is that these proteinases function more in immunity than in matrix turnover. MMPs in Inflammation

Elevated levels of many MMPs are seen in any disease characterized by or associated with inflammation. Although MMP inhibitors have been suggested as a therapy to halt tissue destruction in inflammatory conditions such as chronic obstructive pulmonary disease, arthritis, and vascular disease, the exact role of most MMPs in inflammatory conditions has not been deciphered, even at the level of knowing if an individual proteinase functions to make matters better or worse. Indeed, using arthritis as an example, MMPs long suspected to contribute to joint

Table 2 Lung-related phenotypes of MMP-null mice Mouse

Phenotypes

MMP-2–/– MMP-3–/–

Decreased allergic inflammation Reduced neutrophil influx in immune-mediated lung injury Spatially constrained chemokine gradients Reduced neutrophil influx in injured lung Impaired repair (re-epithelialization) of airway wounds Reduced shedding of E-cadherin from injured airway Protection against endotoxin-mediated shock Reduced dendritic cell migration Reduced angiogenesis in ischemic tissues Impaired bronchiolization after acute lung injury Altered leukocyte influx in allergen-induced airway inflammation Altered chemokine gradients in allergen-induced airway inflammation Modulation of IL-13-induced lung inflammation Reduced macrophage influx in response to cigarette smoke Reduced emphysema in response to cigarette smoke Reduced release of active TNF from smokeexposed macrophages Reduced neutrophil influx and epithelial permeability in immune-mediated lung injury Modulation of IL-13-induced lung inflammation Disrupted alveolar development None examined nor reported

MMP-7–/–

MMP-9–/–

MMP-12–/–

MMP-13–/– MMP-8–/– MMP-11–/– MMP-19–/– MMP-20–/–

destruction, such as MMP-2 and -3, may actually be protective, as suggested by mouse studies. There are several examples that demonstrate that these proteinases are important effectors in a variety of processes controlling repair and leukocyte recruitment – processes central to inflammation. Epithelial Repair

Innate immunity comprises a variety of ready-to-go mechanisms that defend against invading microbes, contribute to tissue repair, and regulate the activity and influx of cells involved in acquired immunity. By maintaining a tight barrier against the external environment, secreting antibiotic peptides, and producing chemokines and homing receptors, epithelium is the first line of defense. Injury disrupts the barrier function of the epithelium, creating a portal for microbes and toxins. Fortunately, injured epithelial cells respond rapidly to close wounds, a process that involves cell proliferation, spreading, and migration. Injury induces the expression of several MMPs in lung epithelium. Among these, MMP-7 is expressed

MATRIX METALLOPROTEINASES 23

by wound edge epithelial cells and is required for re-epithelialization, presumably by shedding E-cadherin ectodomains, which would loosen cell–cell contacts and thereby facilitate cell migration. Although MMP-9 has been convincingly shown to be required for the migration of airway epithelial cells over a collagen matrix in cell culture, no defect in epithelial closure is seen in MMP-9 knockout mice. MMPs and Chemokines

Chemokines are a group of chemotactic molecules that specifically attract and recruit populations of immune effector cells, including neutrophils, monocytes, and eosinophils, to sites of injury or infection and shape the evolution and outcome of the inflammatory response. Several studies have demonstrated that specific MMPs control chemokine activity. This control is either direct by MMPs cleaving these molecules, resulting in enhanced, inactive, or antagonistic chemokine activities, or indirect by the proteinases acting on other substrates that bind, retain, or concentrate the chemoattractant molecules to particular locations. Direct Modulation of Chemokine Activity by MMPs

One potential action of MMPs is to convert chemokines from their true (or initial) nature as chemoattractants into antagonistic derivatives, thereby disrupting the further recruitment of cellular components and contributors to sites of inflammation. This repressive processing has been studied in detail by Overall and co-workers, who studied the monocyte chemoattractant protein (MCP) group of CC chemokines. Using the C-terminal hemopexin domain of MMP-2 as a bait in a yeast two-hybrid system, they discovered unexpectedly that MCP-3/CCL-7 is a substrate of MMP-2, which cleaves the N-terminal four amino acids of the chemokine. Truncated MCP3/CCL-7 still binds its CC chemokine receptor, but it no longer promotes chemotaxis; rather, it acts as a chemokine antagonist. Similarly, MMP-1, -3, -13, and -14 cleave the N terminus of MCP-1/CCL2, MCP-2/ CCL-8, and MCP-4/CCL-13, producing antagonist factors. Although long considered to augment inflammation and the associated tissue damage, the action on the MCPs indicates that MMPs can also dampen inflammatory processes. In addition to MCPs, the function of other CC and CXC chemokines is altered by direct MMP proteolysis. Stromal cell-derived factor-1 (SDF-1/ CXCL-11) is a substrate of multiple MMPs (1, 2, 3, 9, 13, and 14) both in vitro with purified proteins and in cell culture models, and SDF-1 processing by MMP-2 yields a product with potent neurotoxic

activity. Similarly, MMP-9 can process a variety of chemokines, such as GCP-2/CXCL-6, ENA-78/ CXCL-5, and mouse GCP-2/LIX (CXCL-5 or -6), leading to a loss of or, as for IL-8/CXCL-8, markedly increased chemoattractant activity. In vitro, MMP-8 can cleave GCP-2/LIX, generating peptides with enhanced neutrophil chemotactic activity, but in vivo high levels of processed GCP-2/LIX are recovered from inflamed lungs of MMP-8-null mice, suggesting that other proteinases function in regulating the activity of this CXC chemokine. In addition, MMPs may regulate the expression of chemokine receptors and signaling, providing an indirect mechanism by which these proteases can regulate leukocyte influx. Although the studies discussed in this section indicate that MMPs can act on chemokines and potently alter their activity, a complete deficiency in the proteolytic processing of a specific chemokine has not been demonstrated in any model of cellular recruitment in an MMP knockout mouse. MMPs Establish Chemokine Gradients

In addition to the biosynthesis, receptor expression, and modification of chemokines by proteinases, chemokine bioavailability is also regulated by immobilization to the ECM or to cell surfaces. In vivo, chemokines form chemoattractant gradients by binding to accessory macromolecules, typically the glycosaminoglycan side chains of proteoglycans, thereby providing directional cues to migrating leukocytes. By acting on these accessory molecules, MMPs can indirectly regulate chemokine activity and, in turn, the influx of inflammatory cells. A compelling demonstration of this mechanism is provided by the ability of alveolar epithelial-derived MMP-7 to shed the ectodomain of syndecan-1, a transmembrane heparan sulfate proteoglycan, as shown in a model of acute lung injury. In this scenario, three epithelial components – a secreted proteinase (MMP-7), a cell-bound proteoglycan (syndecan-1), and a chemokine (KC) – act coordinately to control and confine acute inflammation, specifically the transepithelial migration of neutrophils, to sites of injury. In response to lung injury, epithelial cells synthesize, secrete, and deposit KC (or IL-8 in humans) onto the heparan sulfate chains of pre-existing syndecan1 molecules. MMP-7, which is also induced by injury, cleaves the syndecan-1 core protein at a juxtamembrane site to release the ectodomain–KC complex. The shed complex, either actively or passively, creates a chemotactic gradient guiding neutrophils into the alveolar space. In bleomycininjured lungs of MMP-7-null mice, neutrophils extravasate from the vasculature without difficulty, but

24

MATRIX METALLOPROTEINASES

they are held at either the epithelial–interstitial interface or within markedly expanded perivascular spaces, and they do not enter the lumen of the lung. Interestingly, the interaction between MMP-7 and heparan-sulfated molecules increases its catalytic activity. Thus, the glycosaminoglycan chains on syndecan-1 may act as an accessory factor linking MMP-7 to its substrate – the core protein of the proteoglycan. The observation that MMP-7-null mice are protected against acute lung injury and the subsequent lethal effects of a proinflammatory insult does not mean that in all settings this MMP is a detrimental proteinase. Indeed, although in the experimental model discussed previously, massive neutrophil influx and oxidative burst cause indiscriminate, severe, and potentially mortal damage, neutrophils comprise an essential cellular component of innate host defense, and in alternative situations MMP-7 serves a beneficial function in neutrophil influx and activation, as well as in mucosal immunity and epithelial migration. Although MMP-7 may not directly process chemokines, it assumes a leading role in controlling the formation of a chemotactic gradient that controls neutrophil influx and activation. Other MMPs also regulate the formation of chemokine gradients. Allergen-induced airway inflammation is dampened in MMP-2-null mice, an observation associated with reduced levels of eotaxin/CCL-11 in lavage fluid. How MMP-2 controls the bioavailability of eotaxin/CCL-11 is not known, but it is interesting that this proinflammatory function is distinct from the anti-inflammatory action of MMP-2 on MCP chemokines. In the same allergen model, MMP-9 was also shown to be required for formation of transepithelial gradients of eotaxin/CCL-11, as well as MARC/CCL-7 and TARC/CCL-17, but again, the mechanism by which MMP-9 facilitates the movement of these chemokines from one tissue compartment to another is not known. MMP-9 also affects the ability of IL-8/ CXC-8 to stimulate the release of leukocytes from marrow, but as in the allergen model, the mechanism of this effect (i.e., the target substrate) has not been identified. MMP-3 (stromelysin-1) mediates the release of a macrophage chemotactic activity from chondrocytes, and MMP-12 is required for macrophage influx into smoke-exposed lung, but the nature of these activities, and indeed whether they involve chemokines, is not known. Collectively, these findings demonstrate that several MMPs can regulate an inflammatory response by controlling the activity and mobilization of chemokines. Identifying the key substrates in these processes will provide insight into fundamental mechanisms of inflammation.

MMPs and Cytokines

Several studies have suggested that MMPs can directly or indirectly affect the activity of a variety of cytokines that function in inflammation and repair processes, including interferon-b, VEGF, EGFs, FGFs, and transforming growth factor-b1 (TGFb1). As demonstrated in TGF-b1 knockout mice, this cytokine functions to restrain mononuclear inflammation. TGF-b1 is released by cells with its cleaved prodomain still latently associated, and a number of mechanisms, including MMP proteolysis, have been proposed to release the active cytokine from this complex. In both cells and tissue explant models, MMP-3, MMP-9, and MMP-14 have been shown or suggested to activate some amount of total TGF-b1. If these or other MMPs can activate TGF-b1 in vivo, then this would be another mechanism whereby MMPs restrain, rather than augment, inflammation. Similarly, interleukin-1b (IL-1b), a potent proinflammatory cytokine, requires proteolytic processing for activation, a process attributed to the IL-1b-converting enzyme (ICE; caspase-1). Although the function of ICE had been well established in vitro, studies of caspase-1-deficient mice have provided evidence for other mechanisms of IL-1b activation. At least three members of the MMP family, MMP-2, -3, and 9, can cleave and activate the IL-1b precursor. Furthermore, after activating it, MMP-3 degrades biologically active IL-1b, which can also be inactivated in vitro by MMP-1, -2, and -9. These data suggest a dual role for MMPs in biphasic modulation of inflammatory mediator activity, with both activation (MMP-3 and -9 and, more weakly, MMP-2) and inactivation (MMP-3). Another essential proinflammatory mediator regulated by metalloproteinase activity is tumor necrosis factor (TNF)-a, which is produced as a 26 kDa membrane-associated protein (pro-TNF-a) and cleaved by a TNF-a-converting enzyme (TACE) into a soluble 17.5 kDa cytokine. Because synthetic metalloproteinase inhibitors blocked this cleavage, it was suggested that TACE was an MMP. However, when the convertase activity was purified and cloned, it turned out to be ADAM-17, a member of the disintegrin family of metalloproteinases (ADAMs). The cleavage of pro-TNF-a by ADAM-17 is quite specific, and because the release of active TNF-a is reduced by 90% in cells derived from TACE-deficient mice, ADAM-17 does seem to be the principal physiological TNF-a-converting enzyme in vivo. Even if ADAM-17 is the primary modulator of the generation of TNF-a activity, several MMPs, including MMP-1, -2, -3, -9, and -17, can process pro-TNF-a to its active form in vitro, and as demonstrated with

MEDIASTINAL MASSES 25

cells from knockout mice, MMP-7 and MMP-12 also activate pro-TNF-a on macrophages (Table 1). Thus, whereas TACE is seemingly involved in the inducible, high-level release of TNF-a in response to bacteria and toxic shock, MMP-7 and MMP-12 may be responsible for the constitutive release of TNF-a from macrophages needed for day-to-day functions, such as tissue resorption and resolution in response to injury. See also: Acute Respiratory Distress Syndrome. ADAMs and ADAMTSs. Chemokines. Chemokines, CC: C10 (CCL6); MCP-1 (CCL2)–MCP-5 (CCL12); RANTES (CCL5); TARC (CCL17); TECK (CCL25). Chemokines, CXC: CXCL12 (SDF-1); IL-8; CXCL1 (GRO1)–CXCL3 (GRO3). Chronic Obstructive Pulmonary Disease: Emphysema, General. Defense Systems. Defensins. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; Monocytes; T cells. Matrix Metalloproteinase Inhibitors.

Further Reading Coussens LM, Fingleton B, and Matrisian LM (2002) Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 295: 2387–2392. Li CK, Pender SL, Pickard KM, et al. (2004) Impaired immunity to intestinal bacterial infection in stromelysin-1 (matrix metalloproteinase-3)-deficient mice. Journal of Immunology 173: 5171–5179. Massova I, Kotra LP, Fridman R, and Mobashery S (1998) Matrix metalloproteinases: structures, evolution, and diversification. FASEB Journal 12: 1075–1095. McCawley LJ and Matrisian LM (2001) Matrix metalloproteinases: they’re not just for matrix anymore! Current Opinion in Cell Biology 13: 534–540. Parks WC and Shapiro S (2001) Matrix metalloproteinases in lung biology. Respiratory Research 2: 10–19. Parks WC, Wilson CL, and Lopez-Boado YS (2004) Matrix metalloproteinases as modulators of inflammation and innate immunity. Nature Reviews: Immunology 4: 617–629. Sternlicht MD and Werb Z (2001) How matrix metalloproteinases regulate cell behavior. Annual Review of Cell and Developmental Biology 17: 463–516. Van Wart HE and Birkedal-Hansen H (1990) The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proceedings of the National Academy of Sciences, USA 87: 5578–5582.

MEDIASTINAL MASSES G Esposito, ‘Federico II’ University of Naples, Naples, Italy C Esposito, ‘Magna Graecia’ University of Catanzaro, Catanzaro, Italy M De Marco, ‘Federico II’ University of Naples, Naples, Italy A Centonze, ‘Magna Graecia’ University of Catanzaro, Catanzaro, Italy & 2006 Elsevier Ltd. All rights reserved.

Abstract Mediastinal masses are an important feature of mediastinal pathology. This article describes the surgical anatomy of the mediastinum and lists the various masses of the three mediastinal compartments. It also documents the different symptoms caused by the mediastinal masses (seen in the mediastinal syndrome), the diagnostic protocols currently employed in this area of respiratory medicine and oncology and, at last, the principles of therapy of the most important masses.

Introduction A short description of mediastinal topography and surgical anatomy is indispensable to understand the different mediastinal masses and decide any diagnostic and therapeutic program.

The mediastinum (from the Greek medium istemi) is an anatomic region localized at the center of the thorax, limited in front by the sternum, in the back by the spine, and laterally by the lungs with their pleural lining. There are several classifications for the mediastinum, although it is Shield who classically divided this region into three areas: anterior, visceral, and posterior, limited by two frontal planes, the first tangent to the anterior surface of the pericardium and the large vessels, the second tangent to the anterior surface of the vertebral bodies. As illustrated in Figure 1, thymus is found in the anterior mediastinum with the internal mammary vessels, the lymph nodes, connective tissue, fat-cell tissue, the lower pole of thyroid, and the ectopic parathyroids. In the visceral compartment there are the heart and pericardium; the great vessels: ascending and descending aorta, the aortic arch with the supraortic vessels, the pulmonary artery with the proximal segment of their branches, the distal segments of the pulmonary veins, the superior vena cava with the brachiocephalic trunks, the azygos vein, and Botallo’s ligament or Botallo’s pervious duct; the thoracic

26

MEDIASTINAL MASSES

Anterior compartment

Posterior compartment

• Thymus • Internal mammary vessels • Lymph nodes • Connective tissue • Fat-cell tissue • Lower pole of thyroid • Ectopic parathyroid

• Lateral surface of vertebral body • Proximal costal segments • Proximal segments of intercostal vessels • Proximal segments of intercostal nerves • Sympathetic chain with its ganglions

Visceral compartment • Heart and pericardium • Great vessels: ascending and descending aorta, aortic arch with supraortic vessels, pulmonary artery with the proximal segment of their branches, distal segment of pulmonary veins, superior vena cava with the brachiocephalic trunks, azygos vein • Ligamentun arteriosus or patent ductus arteriosus • Thoracic duct • Nerves: vagus, phrenic, recurrents • Lymph nodes chains • Anterior surface of vertebral body Figure 1 Anatomic structures of the three mediastinal compartments

duct; nerves: vagus, phrenic, recurrent laryngeal, lymph node chains, and the anterior surface of the vertebral bodies. In the posterior compartment there are the lateral surface of the vertebral bodies, the internal surface of the intercostal muscles, the proximal segment of the intercostal nerves, the sympathetic chain with its ganglions, and hemiazygos vein. The wide variety of mediastinal masses (Figure 2), different for embryological origin, anatomic constitution, location, and functional features are responsible for the various signs and symptoms, which are collectively called the mediastinal syndrome, and are due to compression, obstruction, or infiltration of the mass over near-structures. The mediastinal syndrome includes all the symptoms caused by the pathological environment of all mediastinal structures and systems, the cardiovascular, respiratory, digestive, and lastly, the peripheral and central nervous system. It can be total, affecting all three compartments, or partial, with anterior, median, or posterior mediastinal syndrome. In addition, it is important to mention systemic syndromes associated with tumoral masses (Table 1)

Table 1 Systemic syndromes associated with mediastinal masses Myastenia gravis Hypogammaglobulinemia Systemic lupus erythematosus Scleroderma Fever of unknown origin Claude Bernard–Horner Parfour-Depetit Opsoclonus-myoclonus Vertebral anomalies

Thymoma Thymoma Thymoma Thymoma Lymphoma Neuroblastoma Neuroblastoma Neuroblastoma Intestinal duplications

and syndromes of endocrinal hypersecretion caused by tumors (Table 2). The diagnosis (Figure 3) for different mediastinal masses is done step by step, from the evaluation of the clinical aspects, the epidemiological and laboratory data, to the diagnostic imaging (standard chest radiography, ultrasonography, echocardiography, CT scan, MRI, and scintigraphy), then endoscopic examinations (mediastinoscopy, thoracoscopy, esophagoscopy, and tracheobronchoscopy), and lastly, biopsy. After the evaluation of the symptoms and signs previously described, we will look at

MEDIASTINAL MASSES 27

Masses of anterior compartment

Mases of posterior compartment

• Masses of the thymus • Germ-cell tumors • Lymphomas • Lymphangiomas • Hemangiomas • Lipomas • Fibromas • Fibrosarcomas • Morgagni–Larrey hemia with hepatic content (rare) • Parathyroid adenoma (rare)

• Neuroblastomas • Ganglioneuroblastomas • Genglioneuromas • Schwannomas • Pheochromocytomas • Fibrosarcomas • Neuroenteric cysts • Lateral meningocele (rare) • Lymphomas • Aneurysmatic vertebral cysts

Masses of visceral compartment • Abnormalities of the primitive intestine (bronchogenic cysts, oesophageal duplications) • Lymphomas • Pericardial cysts • Vascular masses • Mesothelial cysts • Lymphoid hamartoma • Mediastinal granulomas • Paragangliomas • Pheochromocytomas • Thoracic duct cysts Figure 2 Masses commonly found in the three mediastinal compartments. Table 2 Endocrine hypersecretion syndromes associated with mediastinal masses S. Cushing Gynecomastia HCG increase Leyding’s cell stimulation Arterial hypertension Diarrhea

Hypercalcemia Hypoglycemia

Thymoma Germ-cell tumor Germ-cell tumor Germ-cell tumor Pheochromocytoma Ganglioneuroma Ganglioneuroblastoma Neuroblastoma Lymphoma Teratoma Fibrosarcoma Neurosarcoma

the epidemiological data as related to the distribution and number of all mediastinal masses. Apart from the routine laboratory tests related to the diagnosis of a tumor (red blood cells, white blood cells, sedimentation rate, serum proteins, etc.), other useful tests are the dosage of vanillylmandelic (VMA) and homovanillic acids (HVA) in sympathetic tumors, especially the neuroblastoma; b-HCG values, and a-fetoproteins in germinal cell tumors.

In relation to the instrumental diagnosis, given their safety and noninvasiveness, ultrasound methods are the first to be employed especially in pediatric populations. In fact, ultrasonography can define the cystic or solid nature of a suspected mass, and in the case of little skeletal ossification in children, it is able to show even mild central opacity not detected by X-rays. The plain antero-posterior/latero-lateral chest X-rays, remain the principal diagnostic study to evaluate a mass, if the previous ultrasonography does not provide useful information. In 90% of cases, it can define well-delimited lesions with clear borders (such as cystic masses or benign tumors), localize the position of a mass in one of the mediastinal compartments, and then reveal inner calcification (Figure 4). Other imaging procedures are CT scan, spiral CT, MRI, and PET, that may be done separately or sequentially and can confirm the site and extent of the masses and their characteristics (solid, cystic, with calcifications). CT scans are extremely useful in providing a clear delineation of the extent and nature of a mass and its

28

MEDIASTINAL MASSES Evolution of clinical features Symptoms specific:

compression of trachea compression of esophagus involvement of cardiovascular system involvement of nervous trunks

Symptoms non-specific: fever Syndromic symptoms

Evaluation of epidemiological data

Laboratory data

Instrumental diagnosis Standard X-ray; ultrasound; ecocardiography; CT scan; MRI; scintigraphy; esophagography; angiography; broncography Endoscopic investigations

Mediastinoscopy; thoracoscopy; esophagoscopy; tracheobroncoscopy

Bioptic techniques (surgical or by aspiration echo- or CT-guided) Figure 3 Diagnostic protocol of mediastinal masses.

Figure 4 Standard chest radiography in a case of mediastinal lymphangioma.

relation with the other mediastinal organs, because of their high sensitivity in differing connective tissue, fat-cell tissue, cystic mass, and vessels; they can also define a vessel’s pathology (venous or lymphatic dysplasia, aneurysm, external or inner obstruction). An intravenous contrast CT scan (angioCT) can detect the vascularization of the mass and its location with respect to vessel structures (Figure 5).

MRI is preferred in children because it avoids exposure to ion rays, but the lengthiness of the examination and the need for sedation in younger patients limits its use. MRI is nevertheless ideal to study vascular pathologies and any extension into the spinal canal is clearly defined (Figure 6). Other procedures are techniques performed with contrast medium such as barium swallow, aortography,

MEDIASTINAL MASSES 29

Figure 5 TC scan in a case of Morgagni–Larrey hernia showing the herniation of the liver into the anterior mediastinum.

Figure 7 Superior cavography showing trombotic obstruction of the left innominate vein and of the superior vena cava in a case of thymoma.

Figure 6 MRI in a case of mediastinal ganglioneuroma.

phlebography (Figure 7), lymphography, angiocardiography (Figure 8), and mediastinography, the last ones (angiocardiography and mediastinography) obsolete and very rarely performed. The contrast esophagogram and other means to show the trachea and bronchus are indicated to detect any deviation and/or compression caused by a mass. In some cases, scintigraphy is an important examination particularly for studying masses that have retained the isotopes. This examination is also useful during follow-up to find recurrences (Figure 9). The final step consists of endoscopic investigations: bronchoscopy, esophagoscopy, mediastinoscopy, and thoracoscopy, eventually integrated by an echo- or a CT-guided fine-needle biopsy.

Diseases of the Mediastinum As previously stated, a mediastinal mass presents a wide spectrum of symptoms, and can appear in every compartment in all areas at the same time. For this reason, a

Figure 8 Angiocardiography in a case of aortic aneurism complicating an aortic coartaction.

mass is generally not classified on the basis of the compartment of origin, but is divided into three groups: nontumoral masses, tumoral masses, and masses of endothorax organs with mediastinal extensions. The first group includes hypertrophy and cysts of the thymus, anomalies of primitive foregut, thyroid masses, echinococcus cysts, and tubercular and nontubercular adenopathy. The second group includes tumors of the thymus, lymphomas, germinal cell neoplasms, and neurogenous, mesenchymal, and metastatic tumors.

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MEDIASTINAL MASSES

Figure 9 Scintigraphy with MIBG in a case of mediastinal neuroblastoma.

The third group includes masses of the esophagus, the lungs, the pleura, and diaphragm. The most important ones of these masses are described here. Thymic Hyperplasia

The thymic hyperplasia occurs, especially in pediatric age, as a hypertrophy of the organ primitive or secondary to drugs. Histologically, the glands can appear normal or characterized by hypertrophic lymphoid follicles. The symptoms are due to a compression on near organs (stridor, dyspnea, cyanosis, and palpable suprasternal swelling); less frequently, it produces a myasthenia gravis (weakness of extraocular, bulbar, and skeletal muscles). On the chest X-ray, it appears as a triangular shadow, like a sail (sign of undersail) which exceeds the mediastinum superiorly and laterally. Conservative treatment is indicated in children, aiming at spontaneous reduction. In persistent forms or complicated by myasthenia gravis, surgery that may be done with a traditional or a mini-invasive approach, is the treatment of choice. Bronchogenic Cysts

They derive from an anomalous gemmation of the primitive foregut or tracheobronchial duct; they may occur at any point along the trachea and bronchi in the mediastinum or lungs (Figure 10). They are cystic masses that contain thick mucoid or purulent material, or even hemorrhagic material in case of complications. The cysts are lined with respiratory epithelium and can contain cartilage or muscle tissue. Respiratory symptoms may be aspecific at the beginning, with cough and then bronchopneumopathy and dyspnea.

Figure 10 Intraoperative view of a bronchogenic cyst.

If it adheres to the trachea and bronchi and complete excision is not possible, the mucosa layer of the wall adherent to the bronchial tree must nevertheless be removed. Esophageal Duplications

In the past, these conditions were called by different names (intestinal cyst, enterogen cyst, mediastinal cyst, etc.); these masses develop from an anomalous bud of the posterior wall of the primitive foregut after its division to form the laryngotracheal duct. As all digestive duplications, esophageal ones have three main characteristics: continuity or contiguity with the esophageal wall, duplication wall formed by longitudinal and circular muscle tissue, and lining with esophageal mucosa or ectopic mucosa. Duplications can be cystic (uni- or multilocular), rarely diverticular, or tubular, and may communicate exceptionally through the diaphragm and extend to an

MEDIASTINAL MASSES 31

Figure 11 Intraoperative view of a duplication of epicardial esophagus.

abdominal duplication (thoracoabdominal duplications). They may be very bulky and present during the first days of life with respiratory symptoms (cough, forced respirations, dyspnea, chirring) or with gastro-esophageal reflux episodes when localized in the epicardial region (Figure 11). X-rays show a roundish opacity in the visceral mediastinum behind the heart, frequently on the right side, appearing in the ultrasounds and CT scans as a cystic formation. The esophagogram may show a print on the esophagus or its dislocation. Treatment consists of complete excision by thoracotomy or thoracoscopy. Aneurysms of the Mediastinal Aorta

They represent one-third of all aortic aneurysms (37%). What is interesting is almost 50% of them are from the descending aorta (16%) and rarely from the ascending aorta (10%), and the arch (7%). Their percentage in relation to all mediastinal masses, is very low, ranging from 2.9% to 4.7%, according to various statistics. Having almost always an atherosclerotic origin, after the regression of syphilis, this disease is responsible for arterial aneurysms. Aneurysms may also have a congenital origin as in the case of the sinus of Valsalva or those that complicate the aortic coarctation (as in case treated by us means that we have treated an aortic aneurysm in a child with an aortic coarctation). The clinical symptomatology is beyond the 50% of light entity while the case initially asymptomatic is rare. The signs are due to the bulk and involvement of the adjacent organs, while the symptoms of exteriorisation (pulsatile parasternal mass) occur rarely. Among all symptoms, the thoracic pain frequently associated with obstinate cough is typical. Variously localized in relation to the site of the aneurysm, it is due to the involvement of nervous paraortic or pleural structures. Also typical, especially if they are associated with pain, are the signs due to compression of venous structures caused principally by the aneurysm at

anterosuperior extension (jugular vein turgescence, cephalalgia, epistatis, and mantelet-shaped or peripheral edema that are responsible for the superior and/or inferior cava syndrome (from 9% to 25%). (The mantelet-shaped edema is an edema localized at the superior part of the thorax and of the arms). Other symptoms are likely to be connected with the nervous system with intercostal pain, cervicobrachialgia, and Horner’s syndrome; the peripheral circulation with tracheal beats and pulsus anomalies; the respiratory system caused essentially by the aneurysm of the concavity of the arch with dyspnea, cough, dysphonia, syndrome of Delafoy, asmatiform raucousness or broncoconstrictive crisis, and hemoptysis; and the alimentary system with hiccup or dysphagia. They occupy an important place in the chapter of mediastinal masses and each roundish opacity of the middle mediastinal region is suspected to be an aortic aneurysm. The mediastinal involvement, caused by the aneurysm of the thoracic aorta, is conditioned by three factors: the bulk caused by the mass, the concomitant inflammation of the mediastinal tissue responsible for the irritation of the surrounding formations, and the eccentric development of the mass responsible for the compression on the adjacent organs. These may occur above the ascending aorta, above and on the left of the aneurysm of the arch, and behind and on the left of the descending aorta. Frequent complications are represented by the aortic dissection, the rupture of the aorta, and the formation of a fistula between the aorta and an adjacent organ. The rupture that may occur 2–3 years from the discovery of the aneurysm is almost always fatal, if not operated upon immediately. The diagnosis, at least in an initial phase, is not easy because the clinical features may be misleading. In any case, the standard radiogram of the thorax is useful because it highlights a mediastinal roundish opacity on the right and above the aneurysm of the aortic arch, and at left for the aneurysm of the descending aorta. A following echography and TC-scan offer more details than are available by other exams such as traditional angiography, digital angiography, angioTC, or angioMRI that allow to appreciate form, dimension, site of aneurysm, and eventual involvement of the adjacent formations. Surgery is the only option considering that the rupture of the aneurysm may be inoperable or immediately fatal. For modalities of the surgical treatment, that can vary according to the site and the extension of the aneurysm, we recommend consulting a volume of cardiovascular surgery. Lymphangioma

Also called cystic lymphangioma, it is a neoformation of lymphatic origin, arising from a mistake in

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the development of the jugular lymphatic sac. It may be localized only in the mediastinum or the neck. Generally, it occupies the visceral compartment and may even extend to compress the trachea and/or the esophagus, anteriorly reaching the thymic region. Rarely, it localizes in the postero-inferior mediastinum, in which case it rises from anomalies of the primitive thoracic ducts. The lymphangioma may be macrocystic and of mixed type and may be associated with chromosomal abnormalities such as Turner’s syndrome and trisomies. The mediastinal lymphangioma may be asymptomatic or, depending on its size, with symptoms of compression over the near organs (dysphagia, dyspnea, cough, or life-threatening conditions) or with a superior vena cava syndrome or with an atelectasis and chylothorax. The diagnosis is easy when there is a cervicomediastinal lymphangioma, whereas it may be difficult in the pure mediastinal form, the latter showing on X-rays a roundish opacity, generally a polylobular, homogeneous mass, localized in the anterosuperior mediastinum, that is mobile with breathing. More details are provided by ultrasound and CT scans, which confirm the cystic nature, and by angiographic studies to see the involvement of the superior vena cava and other vessels. MR imaging is the method of choice for assessing the extent of cervicomediastinal lymphangioma and its relationship to neurovascular structures. It can regress spontaneously; if not, the lymphangioma needs a complete surgical excision. An alternative treatment is the injection of sclerosal substances, but it does not always achieve recovery. Pericardial Cysts

Pericardial or pleuropericardial cysts are congenital cysts of celomatic origin. They are localized in the region included by the pericardium medially; the middle and inferior pulmonary lobes above and laterally: the sternocostal wall forward; and the diaphragm down. Generally single and monolocular, three out of five times, they are localized on the right side. They have a variable form (elongated or spherical) and variable dimensions (of the size of a mandarin to a grapefruit) with a weight of 100–200 g. The wall of the cyst, more or less tight, is smooth and translucid, white or yellowish, and poorly vascularized. Generally they are independent from the pericardium but may be connected, rarely, by a solid or hollow pedicle, in which case they can be differentiated by a diverticulum of pericardium. The vascularization is assured by a vascular pedicle of pericardial origin that comes from the internal mammalian artery. The

pericardial cysts contain clear fluid as in hydatid cyst and histologically are lined by flattened mesothelium. Pericardial cysts almost always asymptomatic may present with thoracic pain and respiratory or cardiovascular symptoms (such as cough, light dyspnea, expectoration, hemoptysis, athsmatic form of attacks, anginal pains, palpitations, and tachycardia). The physical examination may reveal a light right paracardiac right obtuseness, when the cyst is located at the right side or a light enlargement of the cardiac obtuseness, when the cyst is located on the right side. At the standard X-ray of the chest, an opaque image that is changeable depending on the incidence, is detected. With laterolateral incidence the image, hemispherical, ogival or quadrangular, is very characteristic because it occupies exactly the sternodiaphragmatic angle being superimposed to the cardiac shadow. With the anteroposterior incidence, the image presents as a superdiaphragmatic opacity that is round, ovoid, or polygonal, filling the right side of the phrenopericardic angle, while on the left side the opacity overflows the cardiac shadow or merges with the cardiac opacity. The diagnosis can be confirmed by ultrasonography that reveals the liquid nature of the contents of the cysts, by computerized tomography that reveals the densimetric values of the liquid of the cyst and its relation with the pericardium, and by magnetic resonance imaging that provides a middle-intense signal in the T1 image. The differential diagnosis can be made with all tumoral or pseudotumoral masses of the anterior mediastinum (lymphomas, cystic lymphangiomas, pulmonary sequestrations, and blocked pleurisies). The treatment is surgical and it consists in the excision of the cyst that may be achieved with a thoracoscopic approach as well. Some authors in the asymptomatic forms advise a conservative treatment. Aneurysmatic Vertebral Cysts

The aneurysmatic vertebral cysts have unknown etiology. They typically occur at a juvenile age, arising in three-quarters of cases before age 20, more frequently in females. They present as a mass at low increase, radiologically highlighted by an under periostal osteolysis, initially with soft edges delimited by a periostal reaction responsible of the classic ballooning aspect. Subsequently the radiological features are characterized by the opacification of the area of rarefaction that assumes a trabecular aspect. From anatomopathological point of view, it is a neoformation of sponge tissue containing osteoid trabecules, covered by a bluish capsule that may go toward a break producing

MEDIASTINAL MASSES 33

capillary hemorrage from anastomotic cavernous spaces with blood contained in the cysts. Histologically, the cyst constituted by fibroblastic tissue, presents cavernomatous spaces with septi having an endothelial lining, giant polynucleated cells, and osteoid tissue. Clinically, the aneurysmatic vertebral cysts may present with pain and nervous symptoms due to spinal cord compression that may mime the symptomatology of a tumor, especially an osteosarcoma. The treatment is surgical and consists of a complete evacuation of the contents of the cysts. In other cases, the radiotherapy may be useful. The Tubercular Adenitis

It is more frequent among young people, especially in women aged from 10 to 40 years of age. They may be an occasional radiological finding while investigating for other problems, or present with fever, poor general condition, signs of respiratory compression (dyspnea, hissing respiration, stridor, and cough), and emphysema or atelectasis. The chest radiogram can be negative in case of small adenopathy, may show a single paramediastinal roundish opacity or, in extensive forms, a pseudotumoral, polycyclic, and bilateral image. In contrast CT scans, we find many paratracheal and prevascular lymph nodes that are enlarged and hypodense, with a peripheral coat with high capture of contrast. The prophylaxis is based on vaccination and chemoprophylaxis, while treatment is based on terizidone and kanamicine. Thyroid Masses

Thyroid masses are endothorax goiters, generally originating from the migration of a cervical goiter and rarely from an ectopic thyroid gem in the mediastinum. They represent 8–9% of all mediastinal masses (range 1.7–8%, rarely 30%). They are localized in the lower part of the neck, the cervicothoracic region, the retrosternal, and ectopic region (paracardiac, supradiaphragmatic, retrotracheal). Thyroid masses may be diffuse or nodular, benign or rarely malignant. Asymptomatic at the beginning, they later manifest with signs of tracheal, venous, esophageal, and nervous compression. The first provokes dyspnea, stridor, forced respiration, and cough; venous compression produces vein turgidity and collateral circulation; and esophageal compression leads to dysphagia. At the clinical examination, we may find parasternal obtuseness and a decrease in breathing sound. The radiography shows a cuplike opacity in the superior mediastinum when the thyroid mass is in the cervicothoracic region, or an opacity in other places in case of ectopic goiter (thymic region,

paracardiac, or supradiaphragmatic). Thyroid scintigraphy is useful in uncertain cases. Surgery is the treatment of choice, and may be performed via the cervical or thoracic route, also in thoracoscopy or in video-assisted thoracic surgery (VATS). Thymic Tumors

Thymic tumors include the thymomas and thymolipomas. Thymomas They originate from epithelial cells of the thymus, and localize in the anterior mediastinum. Frequent in adulthood (10% of the anterior masses) and rare in childhood, they consist of a yellow or gray lump of variable size, sometimes with lobular aspects. They are made of epithelial cells mixed with lymphoid cells. According to their histological features they may be lymphocytic, epithelial, with fuse cells, malignant or undifferentiated. They present with three different clinical aspects: asymptomatic, with symptoms due to compression over near organs (cough, dyspnea, dysphagia, superior vena cava syndrome etc.), systemic symptoms (myasthenia gravis, lymphocytic aplasia, neutropenies, thrombocitopenia, hypogammaglobulinemia, polimyositits, systemic lupus erythematosus). Myasthenia or myasthenia gravis is a neuromuscular disorder characterized by a weakness, generally progressive, of the voluntary musculature. It is caused by a competition for acetylcholine receptors by autoantibodies produced by the thymus, with acetylcholine release from presynaptic nerves. The disease may present as an acquired form in infants, in children over 5 years of age, and in adults, or in infants as a congenital disease caused by a transplacental transmission of the antibodies by a mother affected by myasthenia. The myasthenia gravis may occur both in the hyperplasia of the thymus and in thymoma. Standard radiograms are able to reveal a mass that may contain irregular calcifications in the anterior mediastinum with well delimited borders, hiding the cardiac shadow extending through the two hemithoraces. It is rarely in the posterior mediastinum corresponding to a retropharingeal aberrant thymus. Scintigraphy, mediastinography, and cavography (used only if thrombosis is suspected) are obsolete, but CT scans can confirm the diagnosis. Malignant thymomas can give rise to metastases on lungs, bones, medulla, kidneys, thyroid, liver, and brain. Surgery is the treatment of choice, either with a cervical approach or by sternotomy also with a videoassisted approach. Radio- and chemotherapy is used for malignant forms.

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The treatment of myasthenia may be medical or surgical. The medical treatment includes anticholinesterase drugs (pyridostigmine bromide or neostigmine) and immunosuppressive drugs (corticosteroids, plasmapheresis). The surgical treatment consists of thymectomy, essentially to avoid a long-term medical treatment in case of a tumor. The probable beneficial effect of thymectomy is the reduction of autoantibodies production. The thymectomy may be effected with an open approach (transcervical, superior sternal splint, complete midline sternotomy) or with video-assisted surgery. Thymolipomas A thymolipoma is a rare benign tumor (200 cases described in literature), histologically formed by fat-cell tissue and parenchymal thymic tissue. It may be asymptomatic or present with cough, dyspnea, and chest pains. Diagnosis and treatment are the same as for thymomas. Lymphomas

Lymphomas are classified as either Hodgkin’s lymphoma (HL) or non-Hodgkin’s lymphoma (NHL); the first one rises from lymphoreticular lymph nodal tissue, the second from cells of the immunocompetent system localized in extralymph nodal regions. A lymphoma can be associated with syndromic diseases, as in the Wiskott–Aldrich syndrome. HL has a high incidence in the third and fourth decades, although there is a 20% chance before age 20; in children it rarely occurs below age 5 years, its frequency increasing from 7 to 14 years old. There are 4 histogenic types: nodular sclerosis (70%), mixed cellularity (16%), lymphocytic predominance (7%), and lymphocytic depletion (2%). Another group of not otherwise specified histogenic type accounts for 6% of cases. All are characterized by a particular cell, the Reed–Sternberg cell. HLs are classified in four stages, according to severity and evolution of the mass: First stage: only one lymph node or organ is involved. Second stage: only one lymph node or organ under and over the diaphragm is involved. Third stage: lymph nodes under and over the diaphragm, one organ, and spleen are affected. Fourth stage: organs under and over the diaphragm, with or without lymph nodes can be affected. The first sign is a lymph node swelling, frequently laterocervical or left supraclavear, that remains stable over time in the slow forms, then extending to other

lymph node stations: axillary, mediastinal (causing a mediastinal syndrome), inguinal, abdominal. General symptoms may be present (weakness, sweat, itch), often with intermittent fever. Lung infiltration causes purulent and hemorrhagic cough, dyspnea, pleural spilling. Patients may also have digestive (constipation and diarrhea) or neurological symptoms (Claude Bernard–Horner’s syndrome). Lymph-node biopsies generally lead to the diagnosis but CT, MRI, mediastinoscopy, thoracoscopy, and laparoscopy are also useful. The therapy is multidisciplinary (radio- and chemotherapy) and surgery may be useful for staging and then removing the spleen (seldom used in children). The prognosis is good, with a 90% survival rate at 5 years. Patients with lower-stage disease have a survival rate 491% whereas those with higher-stage disease have a 70% survival rate. NHL is a heterogeneous group of tumors derived from cells of the immunocompetent system, and for this reason may occur anywhere in the body. When the primitive localization is the abdomen, the anterior mediastinum is more frequently involved, followed by visceral sites. As to the histopathology, we have lymphoblastic lymphoma (50% of cases), undifferentiated Burkitt, or pleiomorphic non-Burkitt, and histocytic lymphoma. Clinically, it presents peripheral symptoms (laterocervical lymph node swelling), regional symptoms (respiratory, superior vena cava syndrome), and general symptoms (fever, weakness, loss of weight, night-sweats). Diagnosis is based on lymph node biopsy and, if necessary, on mediastinoscopy and/or thoracoscopy. Several types of staging have been described, although the one mostly used is Murphy’s four- stage classification: First stage: one lymph node or one extra lymphnodal tumor. Second stage: more lymph nodes or tumors on the same side of the diaphragm. Third stage: two or more lymph-nodal or extra lymph-nodal tumors on both sides of the diaphragm, a primitive thoracic tumor, extensive gastrointestinal and epidural forms. Fourth stage: all of the above, plus medullary and/ or central nervous system involvement. Chemotherapy is the only treatment, with a combination of drugs (up to 20), sometimes combined with radiotherapy, for a period of 18 months to 2 years. Surgery is indicated for the diagnosis and to relieve symptoms of compression over the trachea.

MEDIASTINAL MASSES 35 Germ Cell Tumors

Germ cell tumors are a group of neoplasms arising from a totipotential primitive germ cell, that can become a differentiated or undifferentiated cell; the first develops into seminoma or a disgerminoma, and second into embryonic cancer and, based on the differentiation, into extraembryonic structures, tumors of the endodermic sinus (yolk sac tumor), coriocarcinomas, and benign or malignant teratomas (Figure 12). Three types of tumors are described below: teratoma, malignant nonseminomatous germ cell tumors, and malignant seminomatous germ cell tumors (germinomas). Teratoma This includes a group of tumors formed by cells that differ from the tissue of the organ in which they develop. Its incidence is higher between 20 and 40 years of age, but can present also in pediatric ages, and may be benign or malignant. It consists of all three primitive layers (endoderm, mesoderm, and ectoderm). Most teratomas are cystic and localized in the anterior mediastinum, rarely in the visceral one. They are divided according to the cell’s maturity; mature forms are generally benign, and immature ones malignant. There are also teratomas with foci of malignant mixed germ cell tumor. The diagnosis of mediastinal teratomas may be incidental or subsequent to symptoms of compression of contiguous structures, especially the respiratory system, along with cough, chest pain, dyspnea, then respiratory distress, and venous compression. Teratomas located in the posterior mediastinum compress the

esophagus and the aorta; if present during pregnancy, there will be polyhydramnios. Radiologically, they appear as opacities within calcification, and are easily recognized by CT scan. Surgical removal is indicated. Malignant nonseminomatous germ cell tumors This group includes coriocarcinoma, yolk sac tumor, embryonal carcinoma, teratocarcinoma, and malignant teratoma. Most of them occur in the anterior mediastinum or in the posterior compartment; they are lobulated masses sometimes covered by a capsule, able to affect contiguous structures. These tumors produce biological markers such as alphafetoprotein (AFP) and chorionic gonadotropin (HCG), whose dosage is useful not only to diagnose but also to evaluate the prognosis. HCG is produced by the coriocarcinoma and AFP by the yolk sac tumor, and both by teratocarcinoma and embryonal carcinoma. Symptoms are cough, fever, loss of weight, dyspnea, chest pain, hemoptysis, and the superior vena cava syndrome. Standard chest X-rays and CT scans are utilized for the diagnosis; if yolk sac tumor is suspected, a CT scan of abdomen and an ultrasound of testis can be performed to exclude a tumor secondary to a seminoma of the testis. Treatment includes surgery, radio- and chemotherapy with bleomicyne, cisplatinum, etc. Yolk sac tumor is a malignant tumor which may lead to lymph node, lung, liver, and bone metastases. Histologically, it is a cystic mass with columnar or cuboidal epithelium containing glomerular structures called Schiller–Duval bodies.

Primordial germ cell (pluripotent)

Differentiation

Seminoma-dysgerminoma

Nondifferentiation

Embryonic carcinoma

Differentiation versus extraembryonic structures

Somatic differentiation ectoderm, mesoderm, endoderm

Yolk sac tumor, coriocarcinoma

Teratoma

Figure 12 Histogenetic relationship of the various tumors derived from the primitive germ cell.

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Malignant germ cell seminomatous tumors (germinomas) The name germinoma derives from the fact that they may occur also in females. Malignant seminomatous germ cell tumors occur more frequently between the ages of 20 and 40 years, and rarely in infancy. They are generally situated in the anterior mediastinum. Histologically, they are seminomatous masses, pure or mixed with nonseminomatous embryonal components or yolk sac tumors. The clinical outcome is frequently with thoracic pain associated with cough, fever, loss of weight, dyspnea, weakness, and superior vena cava syndrome. CT scans are useful to diagnose and detect paraaortic and retroperitoneal lymphonodes. Scintigraphy with gallium is also indicated because of the isotope’s affinity with seminomas. It is important to always exclude a primitive seminoma of the testis. Surgery is the treatment of choice, linked with radio- and chemotherapy.

Lipoblastomas and lipoblastomatosis are tumors arising from embryonal fat-cell tissue, the first in isolated form and the second diffuse (lipoblastomatosis). While the isolated form is asymptomatic, lipoblastomatosis presents severe respiratory distress. Surgery is the only treatment for malignant forms, along with radio- and chemotherapy. Tumors of Vessels

These rare tumors may be situated anywhere in the mediastinum; 90% of them are hemangiomas, divided into capillary, cavernous, venous, and mixed; and 10% are vascular amartomas, angiosarcomas, benign hemangioendotheliomas, benign angiopericitomas, juvenile hemangiomas, leiomyomas, and leiomyosarcomas. As for all vascular tumors, the treatment of choice is complete excision. Tumors of the Sympathetic Ganglia

Tumors of Fat-Cell Tissue

Belonging to the group of mesenchymal tumors, fat-cell tissue tumors include lipomas, liposarcomas, lipoblastomas, and lipoblastomatosis. Lipomas These are benign tumors generally located in the anterior mediastinum; they develop as a slowly growing masses, initially asymptomatic, then provoking compression over the contiguous organs. A CT (along with needle biopsy) easily detects the mass for the characteristics of fat-cell tissue. Liposarcomas They are malignant tumors, located anywhere in the mediastinum, causing atelectasis with dyspnea, tachypnea, stridor, and cough.

Table 3 Mediastinal neurogenic tumors Origin

Benign

Sympathetic ganglia Nerve sheath

Ganglioneuroma

Malignant

Ganglioneuroblastoma Neuroblastoma Schwannoma Malignant Neurofibroma schwannoma Malignant neurofibroma Spindel cell sarcoma Neurogenic fibrosarcoma Neuroectodermal Melanotic prognoma tumor Askin’s tumor Paraganglionic Phaeochromocytoma Malignant tumor Paraganglioma pheochromocytoma Malignant paraganglioma

These include benign, mixed, and malignant forms, and occur almost exclusively in infancy. All arise from differentiated nervous cells: sympathetic gangliar and chromaffine cells (Table 3). They are categorized as benign ganglioneuromas, ganglioneuroblastomas, and neuroblastomas (localized or disseminated). Ganglioneuroma This tumor arises from a mature gangliar cell and forms 30% of all sympathetic tumors. It generally affects children between the ages of 4 and 5 years. It is a well capsulated mass composed of typical gangliar cells in a fibrous stroma. It can be an incidental report or may develop compressive symptoms (dyspnea, dysphagia, Claude Bernard–Horner’s syndrome), or less frequently, diarrhea due to hypersecretion of the vasoactive intestinal hormone. The diagnosis is based on standard X-ray, CT, and MRI; the latter especially useful when there is a spinal cord involvement (dumbbell syndrome), where it presents with an intermediate signal on T1 and an high signal on T2. Surgical excision leads to recovery. The spinal cord involvement called ‘dumbbell syndrome’ presents as tumor extension through an intervertebral foramina causing spinal cord and/or spinal roots compression with paraparesis or paraplegia. It is present in about 10% of all tumors of the sympatic ganglia and requires a neurosurgical impact. Ganglioneuroblastoma It is the transient form between mature ganglioneuroma and immature neuroblastoma. It generally develops in adolescents as a

MEDIASTINAL MASSES 37

strong mass sited in the intervertebral grooves. It is composed of mature and immature gangliar cells. The patient suffers from regional symptoms to respiratory compression, paraplegia, Claude Bernard–Horner’s syndrome, and diarrhea due to hypersecretion of vasoactive intestinal hormones. The VMA and HVA tests are not always useful as with neuroblastoma. Standard X-ray, CT, and MRI lead to the diagnosis. Complete surgical excision gives a survival rate of 90% at 5 years. Neuroblastoma In 20% of patients, the neuroblastoma is located in the mediastinum, developing at 7–8 years of age in 90% and in the first two years in 50% of the cases. It is an immunogenic tumor, and therefore may undergo spontaneous recovery, as proven by the high incidence (from 1 in 23 to 1 in 200 cases) in infants aged o3 months, in autoptic studies in children who died of other causes, and the lower incidence in all ages with a frequency of 1:1000 and 1:3000 children. Recent studies concern the deletion or translocation of the short arm of chromosome 1, the presence of N-myc protoncogene on the short arm of chromosome 2, the nerve growth factor (NGF), and the DNA index. It is a polylobulated well-vascularized mass, with a pseudocapsule. It can present hemorrhagic or necrotic areas. Histologically, it is composed of little cells with a polygonal nucleus and chromatine disposed as ‘salt and pepper’. Often the cells look like a pseudo-rosette characterized by cells surrounding nerve fibers. Symptoms include respiratory ones (thoracic pains, cough, dyspnea), Claude Bernard–Horner’s syndrome, or digestive and general conditions such as fever and slow growth. It may have an intraspinal extension (dumbbell syndrome). To achieve the diagnosis, the dosage of urinary vanillylmandelic and homovanillic acids is very important, as are CT, MRI, and scintigraphy MIBG (iodine-123-labeled metaiodobenzylguanidine), especially when there is an invasion of spinal cord. Microcalcification within the opacity is characteristic. The INSS international classification (International Neuroblastoma Staging System), which has supplanted the old one by Evans, is useful for the prognosis and distinguishes five stages: Stage 1: tumors located in a totally removable organ, with negative lymph nodes. Stage 2A: tumors located on one side, not completely removable, with negative lymph nodes. Stage 2B: tumors located on one side, incompletely or totally removable with homolateral positive lymph nodes.

Stage 3: tumors that surpass the midline with or without positive omolateral lymphnodes, or with positive bilateral lymph nodes. Stage 4: dissemination of tumor to bone, marrow, liver, lungs, brain, and distant lymph nodes. Stage 4 s: tumors as in stages 1 or 2 with dissemination only to liver, skin, and/or bone marrow. Multitherapy includes primary surgery in stages 1 and 2, reductive or delayed surgery in stages 3 and 4 associated with polychemotherapy (cyclophosphamide, vincristine, cysplatinum, etoposide, doxorubicin, dacarbazine, etc.), and in selected cases, radiotherapy. The prognosis varies according to the stage, ranging from 95% (in stage 1) to 40% (in stages 3 and 4), if chemotherapy is associated with marrow transplant; without chemotherapy, survival rate after 4 years is o20%. The general prognosis of mediastinal neuroblastoma is better than for other localizations (78% vs. 59%). Tumors of Nerve Sheath

These are benign and malignant schwannomas and neurofibromas. More frequent in the second and third decade of life, they can also occur in infancy and represent the 15–16% of neurogenic tumors of mediastinum. The schwannomas arises from Schwann’s cell; it is a strong yellow tumor with a smooth thick capsule which originates from intercostal nerves. It develops from disorganised proliferation of the cells of the nerves including Schwann’s cells, myelinated, or unmyelinated fibers and fibroblasts. It can provoke a dumbbell syndrome. The neurofibroma (Figure 13) that can be distinguished from the fibromatosis consists of nervous and sheath elements and presents by itself or in patients with Recklinghausen syndrome. An MRI scan shows it as a low signal on T1 and a high signal on T2. Surgery is the treatment of choice combined with chemotherapy in malignancy.

Figure 13 Intraoperative view of a neurofibroma of mediastinum.

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Leydig-Cell Tumors

Leydig-cell tumors are generally testicular tumors but they may exist everywhere, for example, in the mediastinum. They secrete excessive testosterone resulting in pediatric age in peripheral precocious puberty, while in the adult they produce a gynecomastia. The symptomatology depends on the disturbance caused by the compression on the contiguous organs while the diagnosis is confirmed by an echo- or a CT-guided fineneedle biospy of the tumor. Askin’s Tumor

Askin’s tumor is an infrequent malignant tumor that affects mainly the parietal structures of the chest and, less frequently, the mediastinal ones. It has a marked local invasivity with little incidence of metastasis in other organs. Histologically, it is constituted of little round cells with primordial characteristics. Immunohistochemical, ultrastructural, and cytogenetic researches are necessary to distinguish this tumor from Ewing’s sarcoma and neuroblastoma. The diagnosis may also be confirmed by an echo- or a CT-guided fine-needle biospy of the tumor. See also: Bronchoscopy, General and Interventional. Lung Imaging. Lymphatic System. Mediastinitis, Acute and Chronic. Symptoms of Respiratory Disease: Chest Pain. Tumors, Malignant: Lymphoma.

Further Reading Adams GA, Schochat SJ, Smith IE, Shuster JJ, and Joshi VV (1993) Thoracic neuroblastoma: a pediatric oncology group study. Journal of Pediatric Surgery 3: 372–378. Ahmed A, Mirza S, and Rothera MP (2001) Mediastinal tuberculosis in a 10-month-old child. Journal of Laryngology and Otology 115: 161–163. Ahrendt MN and Wesselhoeft CW (1992) Chordoma presenting as a posterior mediastinal mass in a pediatric patient. Journal of Pediatric Surgery 12: 1515–1518. Akashi A, Hazama K, Miyoshi S, et al. (2001) An analysis of video-assisted thoracoscopic resection for mediastinal masses in 150 cases. An overview of the pansternal approach, histology, and complications. Surgical Endoscopy 15(10): 1167–1170. Androniku S, Joseph E, Lucas S, Brachmeyer S, and Du Toit G (2004) CT-scanning for the detection of tuberculous mediastinal and hylar lymphoadenophaty in children. Pediatric Radiology 34: 232–236. Azarow KS, Peal RH, Zurcher R, Edwards FH, and Cohen AJ (1993) Primary mediastinal masses. A comparison of adult and pediatric population. Journal of Thoracic and Cardiovascular Surgery 1: 67–72. Bangerter M, Behnisch W, and GriessHammer M (2000) Mediastinal masses diagnosed as thymus hyperplasia by fine needle aspiration cytology. Acta Cytologica 44(5): 743–747.

Bariety M and Coury Ch (1958) Le mediastin et sa pathologie. Paris: Masson. Castellote A, Vasquez E, Vera J, et al. (1999) Cervicothoracic lesions in infants and children. Radiographic 19: 583–600. Cigliano B, Esposito C, Vecchio P, and Esposito G (1989) Le masse mediastiniche nell’infanzia. Atti 911 Congresso della Societa` Italiana di Chirurgia, Genova, Settembre–ottobre. pp. 349–354. Cirino LM, Milanez de Campos JR, Fernandez A, et al. (2000) Diagnosis and treatment of mediastinal tumors by thoracoscopy. Chest 117(6): 1787–1792. Deeb ME, Brinster CJ, Kucharzuck J, Shrager JB, and Kaiser LR (2001) Expanded indications for transcervical thymectomy in the management of anterior mediastinal masses. Annals of Thoracic Surgery 72(1): 208–211. De Farias AP, Deheinzelin D, Younes RN, and Chojniak R (2003) Computed tomography-guided biopsy of mediastinal lesions: fine versus cutting needles. Revista do Hospital das Clinicas Faculdade de Medicina Sao Paulo 58(2): 69–74. De Ugarte DA, Shapiro NL, and Williams HL (2003) Tuberculous mediastinal mass presenting with stridor in a 3-month-old child. Journal of Pediatric Surgery 38: 624–625. Donaldson SS and Link MP (1991) Childhood lymphomas: Hodgkin’s disease and non-Hodgkin’s lymphoma. In: Moossa R, et al. Comprehensive Textbook of Oncology, vol. 2, pp. 1514–1532. Baltimore: Williams and Wilkins. Esposito C (2003) Patologia mediastinica di interesse chirugico in eta` pediatrica. Archivio ed Atti della Societa` Italiana di Chirurgia 2: 223–232. Esposito G (1999) Diagnosis of mediastinal masses and principles of surgical tactics and technique for their treatment. Seminars in Pediatric Surgery 8: 54–60. Esposito G, Cigliano B, De Luca V, and Giampaglia F (1982) Su tre casi di linfangioma cervico-mediastinico in eta` pediatrica. Atti 181 Congresso Nazionale di Chirurgia Toracica, S. Marino. Esposito G, Cigliano B, and Paludetto R (1983) Abdominothoracic gastric teratoma in a female newborn infant. Journal of Pediatric Surgery 18: 304–305. Esposito G, Cigliano B, Savanelli A, and Scala G (1982) Le duplicazioni intestinali a sede toracica: a proposito di tre osservazioni personali. Atti 181 Congresso Nazionale di Chirurgia Toracica, S. Marino. Esposito G, Cigliano B, Settimi A, and Vecchio P (1982) Descrizione di un caso di fibromatosi giovanile a localizzazione mediastinica. Atti 181 Congresso Nazionale di Chirurgia Toracica, S. Marino. Esposito G, Cigliano B, and Severino G (1985) Iperplasia e neoplasie timiche nell’infanzia. Atti Congresso Nazionale di Endocrinochirurgia, Ferrara. Esposito G, Settimi A, Tamburrini O, and Quattrin S (1983) Use of a dacron prosthesis for relief of superior vena cava obstruction in a child with thymoma. Italian Journal Pediatric Surgical Sciences 3: 239–243. Esposito G, Sodano A, Molea G, et al. (1980) Singolare evoluzione di coartazione aortica complicata da aneurisma sottostenotico. Rivista Italiana di Pediatria 6: 249–254. Evans AE, D’Angio GJ, and Randolph JG (1971) A proposed staging for children with neuroblastoma. Cancer 27: 374–378. Fadel E, Court B, Chapelier AR, Droz JP, and Dartevelle P (2000) One-stage approach for retroperitoneal and mediastinal metastatic tumor resection. Annals of Thoracic Surgery 69: 1717–1721. Glick RD and La Quaglia MP (1999) Lymphomas of the anterior mediastinum. Seminars in Pediatric Surgery 8: 69–77.

MEDIASTINAL MASSES 39 Grosfeld JL, Weinberg M, Kilman JW, and Weber TR (1971) Primary mediastinal neoplasm in infants and children. Annals of Thoracic Surgery 12: 179–190. Grosfeld JL, Weber TR, and Vane DW (1982) One stage resection of massive cervicomediastinal hygroma. Surgery 92: 693. Hattori Y, Sugimura S, Iriyama T, et al. (1999) Dumbbell type schwannoma of the posterior mediastinum: a report of two cases with different surgical approaches. Kyobu Geka 52: 728–732. Hoerbelt R, Keunecke L, Grimm H, Schwemmle K, and Padberg W (2003) The value of a noninvasive diagnostic approach to mediastinal masses. Annals of Thoracic Surgery 75(4): 1086–1090. Hujala KT, Sipila JI, and Grenman R (2001) Mediastinoscopy – its role and value today in the differential diagnosis of mediastinal pathology. Acta Oncology 40(1): 79–82. Hujala KT, Sipila JI, and Grenman R (2003) Mediastinoscopy in the diagnosis of mediastinal disease. An analysis of 181 explorations. Archivos de Bronconeumologia 39(1): 29–34. Kaji T, Takamatsu H, Noguchi H, Tahora H, and Yoshiyama K (2000) Cervico-mediastinal bronchogenic cyst occuring in the prenatal period: report of a case. Surgery Today 30: 1016–1018. Kern JA, Daniel TM, Tribble CG, Silem ML, and Rodgers BM (1993) Thoracoscopic diagnosis and treatment of mediastinal masses. Annals of Thoracic Surgery 56: 92–96. Kitano Y, Yokomori K, Ohkura M, et al. (1993) Giant thymolipoma in a child. Journal of Pediatric Surgery 12: 1622–1625. Knudson I and Greval H (2004) Thoracoscopic excision of paraesophageal bronchogenic cyst in a child. Journal of the Society of Laparoendoscopic Surgeons 8: 179–182. Koduri PR (2004) The diagnostic approach to mediastinal mass. Annals of Thoracic Surgery 78: 1888–1890. Lakhoo K, Boyle M, and Drake DP (1993) Mediastinal teratomas: review of 15 pediatric cases. Journal of Pediatric Surgery 9: 1161–1164. McAdams HP, Kirejczyk WM, Rosado-de Christenson ML, and Matsumoto S (2000) Bronchogenic cyst: imaging features with clinical and histopathologic correlation. Radiology 217(2): 441–446. Mekki M, Belghith M, Krichene I, et al. (2001) Esophageal duplication in children. Report of 7 cases. Archives de Pediatrie 8: 55–61. Merten DF (1992) Diagnostic imaging of mediastinal mass in children. American Journal of Roentgenology 158: 825–832. Metin M, Sayar A, Turna A, and Gurses A (2002) Extended cervical mediastinoscopy in the diagnosis of anterior mediastinal masses. Annals of Thoracic Surgery 73(1): 250–252. Noyes BE, Weber T, and Vogler C (2003) Pericardial cysts in children: surgical or conservative approach? Journal of Pediatric Surgery 38: 1263–1265. Ocal T, Turken A, Ciftci AO, et al. (2000) Thymic enlargement in childhood. Turkish Journal of Pediatrics 42(4): 298–303. Ogino S, Franks TJ, Deubner H, and Koss MN (2000) Thymohemangiolipoma, a rare histologic variant of thymolipoma: a case report and review of the literature. Annals of Diagnostic Pathology 4: 236–239. Partrick DA and Rothenberg SS (2001) Thoracoscopic resection of mediastinal masses in infants and children: an evaluation of technique and results. Journal of Pediatric Surgery 36(8): 1165–1167. Pedrero Campos C, Sanjuan Rodriguez S, Amaya Lozano JL, and Moran Penco JM (2001) Hydatic cyst in mediastinum. Cirugia Pediatrica 14: 127–128.

Pun YW, Moreno Balsalobre R, Prieto Vicente J, and Fernandez Fau L (2002) Multicenter experience of video-assisted thoracic surgery to treat mediastinal cysts and tumors. Archivos de Bronconeumologia 38(9): 410–414. Rescorla FJ, West KW, Gingalewski CA, et al. (2000) Efficacy of primary and secondary video-assisted thoracic surgery in children. Journal of Pediatric Surgery 35: 134–138. Ricketts RR (2001) Clinical management of anterior mediastinal tumors in children. Seminars in Pediatric Surgery 10(3): 161–168. Rodgers BM (1993) Thoracoscopic procedures in children. Seminars in Pediatric Surgery 3: 182–189. Saenz NC, Schinitez JJ, Eraklis AE, Hendren HW, and Grier HE (1993) Posterior mediastinal mass. Journal of Pediatric Surgery 2: 171–176. Shields TW (1991) Lesions masquerading as primary mediastinal tumors and cysts. In: Shields TW (ed.) Mediastinal Surgery, Cap 18, pp. 118–137. Philadelphia: Lea and Febiger. Shields TW (1991) Primary mediastinal tumors and cysts and their diagnostic investigation. In: Shields TW (ed.) Mediastinal Surgery, Cap 17, pp. 111–117. Philadelphia: Lea and Febiger. Shields TW (1991) The mediastinum and its compartments. In: Shields TW (ed.) Mediastinal Surgery, Cap 1, pp. 6–13. Philadelphia: Lea and Febiger. Shields TW and Robinson PG (1991) Mesenchymal tumors of the mediastinum. In: Shields TW (ed.) Mediastinal Surgery, Cap 31, pp. 272–288. Philadelphia: Lea and Febiger. Smaltino F and Porta E (1990) Mediastino. In: Idelson, Napoli (ed.) Radiologia oggi, Cap. VIII, pp. 479–502. Spector JA, Willis DN, and Ginsburg HB (2003) Paraganglioma (pheochromocytoma) of posterior mediastinum: a case report and review of literature. Journal of Pediatric Surgery 38: 1114– 1116. Suita S, Tajiri T, Sera Y, and Ikedu K (2000) The characteristics of mediastinal neuroblastoma. European Journal of Pediatric Surgery 10: 353–359. Superina RA, Ein SH, and Humphreys RP (1984) Cystic duplication of the esophagus and neuroenteric cysts. Journal of Pediatric Surgery 5: 527–530. Takeda S, Miyoshi S, Minami M, et al. (2003) Clinical spectrum of mediastinal cysts. Chest 124(1): 125–132. Tamburini O, Cigliano B, Cucchiara S, and Esposito G (1983) Epicardial esophageal duplication with hiatal hernia in a case of Turner’s syndrome. Pediatric Radiology 13: 342. Tan C, Alphonso N, Anderson D, and Austin C (2003) Mediastinal haemangiomas in children. European Journal of Cardio-Thoracic Surgery 23: 1065–1067. Todani T, Watanabe Y, Toki A, Umshihara N, and Sato Y (1990) Endodermal sinus tumor of the posteroinferior mediastinum resembling Dumbell Neuroblastoma in a child. Zeitschrift Fur Kinderchirurgie 45: 120–122. Wernecke K, Vassallo P, Potter R, Luckner HG, and Peters PE (1990) Mediastinal tumors: sensitivity of detection with sonography compared with CT and radiography. Radiology 175: 137–143. Williams HJ and Alton HM (2003) Imaging of pediatric mediastinal abnormalities. Pediatric Respiratory Reviews 4: 55–66. Zhang Y, Yang SR, Cheng DU, and Guan J (2003) Clinical and pathological features of congenital bronchial cyst. Zhonghua Jie He He Hu Xi Za Zhi. 26: 619–622.

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MEDIASTINITIS, ACUTE AND CHRONIC M E Balkan, Atatu¨rk Education and Training Hospital, Ankara, Turkey

mycobacteria), and to perform tests (e.g., fibrosing mediastinitis).

& 2006 Elsevier Ltd. All rights reserved.

Acute Mediastinitis Abstract

Etiology

Acute mediastinitis is an uncommon and potentially lethal clinical condition that is almost always a complication of other clinical problems. Chronic fibrosing mediastinitis is deposition of thick fibrous tissue encasing any of the various mediastinal structures outside of the mediastinal lymph nodes. A variety of etiologic factors contribute to acute and chronic infection of the mediastinum. Although many of these conditions can be diagnosed clinically, the surgical pathologist is called on to describe the type of presenting mediastinitis (e.g., acute, subacute, and chronic non-specific, granulomatous), to identify specific infectious agents (e.g., fungi and mycobacteria), and to perform tests (e.g., fibrosing mediastinitis).

Acute mediastinitis is an uncommon and potentially lethal clinical condition that is almost always a complication of other clinical problems (Table 1). The development of infection following esophageal perforation is potentiated by the dynamics of respiration because the fluctuations in negative intrathoracic pressures tend to suck esophageal contents, including air, saliva, digestive enzymes, bile, acid food, and bacteria, into the mediastinum.

Introduction A variety of etiologic factors contribute to acute and chronic infection of the mediastinum. Although many of these conditions can be diagnosed clinically, the surgical pathologist is called on to describe the type of presenting mediastinitis (e.g., acute, subacute, and chronic non-specific, granulomatous), to identify specific infectious agents (e.g., fungi and

Pathology

The pathology of acute mediastinitis is that of severe inflammation in the mediastinal soft tissues, often with abscess formation. Microbiology

The microbiology is related to cardiothoracic surgery, and that secondary to odontogenic or other head and neck infections is strikingly different, as summarized in Table 2.

Table 1 Etiologic factors in acute mediastinitis Esophageal perforation Iatrogenic Esophagogastroduodenoscopy, esophageal dilatation, esophageal variceal sclerotherapy, nasogastric tube, Sengstaken–Blakemore tube, endotracheal intubation, esophageal surgery, paraesophageal surgery, radiation-associated necrosis Swallowed foreign bodies Bone, coins, can pull-tabs, drug-filled condoms, swords Trauma Penetrating: gunshot wound, knife wound Blunt: steering wheel injury, seat belt injury, cardiopulmonary resuscitation, whiplash injury, barotrauma Spontaneous or other Emesis (Boerhaave’s syndrome, Mallory–Weiss syndrome), cricoid pressure during anesthesia induction, heavy lifting, defecation, parturition, erosion of the esophageal wall by malignant tumors Head and neck infections Odontogenic, Ludwig’s angina, pharyngitis, tonsillitis, parotitis, epiglottitis Infection originating at another site Pneumonia, lung abscess, pleural space infection or empyema, subphrenic abscess, pancreatis, cellulitis or soft tissue infection of the chest wall, osteomyelitis of sternum, clavicle, ribs, or vertebrae, hematogenous spread from distant foci Cardiothoracic surgery Coronary artery bypass grafting, cardiac valve replacement, repair of congenital heart defect, heart transplantation, heart–lung transplantation, cardiac-assist devices, other types of cardiothoracic surgery Adapted from Rupp ME (2000) Mediastinitis. In: Mandell GL, Bennett JE, and Dolin R (eds.) Principles and Practice of Infectious Diseases, 5th edn. London: Churchill Livingstone and Marchevsky AM and Kaneko M (1992) Surgical Pathology of the Mediastinum, 2nd edn. New York: Raven Press.

MEDIASTINITIS, ACUTE AND CHRONIC 41 Table 2 Microbiology of mediastinitis Organisms frequently recovered in mediastinitis secondary to infection of the head and neck or esophageal perforation Peptostreptococcus spp., Actinomyces, Eubacterium, Lactobacillus, Veillonella, Bacterioides spp., Fusobacterium spp., Prevotella spp., Porphyromonas spp., Streptococcus spp., Staphylococus spp., Corynebacterium, Branhamella, Enterobacteriaceae, Pseudomonas spp., Eikenella corrodens, Candida albicans Organisms recovered in mediastinitis secondary to cardiothoracic surgery S. auresus, S. epidermidis, Enterococcus spp., Streptococcus spp., E. coli, Enterobacter spp., Klebsiella spp., Proteus spp.; Pseudomnas spp.; Candida albicans; Others occasionally reported; Acinobacter, Legionella spp., B. fragilis, Corynebacterium spp., Mycoplasma hominis, Candida tropicalis, Nocardia spp., Kluyvera, M. fortuitum, M. chelonei, Rhodococcus bronchialsis Other unusual causes of mediastinitis Anthrax, Brucellosis, actinomycosis, paragonimiasis, S. pneumonia infection Adapted from Rupp ME (2000) Mediastinitis. In: Mandell GL, Bennett JE, and Dolin R (eds.) Principles and Practice of Infectious Diseases, 5th edn. London: Churchill Livingstone.

Figure 1 CT scan in a patient with a mediastinal fluid collection in the retrosternal space. The collection is circumscribed by a rim of contrast enhancement and contains a small gas bubble (arrow). This appearance corresponded to a poststernotomy mediastinal abscess.

Clinical Findings

Symptoms of acute mediastinitis generally appear suddenly and include chills, high fever, tachycardia, lethargy, respiratory distress, dysphagia, and evidence of extreme toxicity. Tachypnea and pain on swallowing are present. When the superior zone of the mediastinum is involved, the retrosternal pain radiating upward into the neck is accompanied by the sensation of earache. When involvement is primarily in the inferior–posterior zone, the pain often is between the shoulder blades and occasionally has a radicular component with radiation around the chest. The physical examination is usually not diagnostic, but there may be cervical tenderness in patients with cervical cellulitis and subcutaneous emphysema in cases of esophageal perforation. Chest roentgenograms may reveal mediastinal widening, but it may also be completely normal, and this finding must not preclude additional investigations. Air is present after rupture or perforation of the esophagus, trachea, or bronchi. Computed tomography (CT) scan often shows mediastinal abscess, air–fluid levels,

gas bubbles, and pleural effusion (Figure 1). Manifestations occur with compression of mediastinal structures. These include: (1) the superior vena cava syndrome or unilateral obstruction of brachiocephalic veins; (2) compression of brachiocephalic arteries resulting in differences in blood pressure between the two arms; (3) tracheal compression resulting in dyspnea, stridor, and obstructed breathing; and (4) chylothorax or chylous ascites from obstruction of the thoracic duct. Rarely does pressure on nerve structures result in functional disturbances such as hoarseness from recurrent laryngeal nerve involvement, diaphragmatic paralysis from phrenic nerve pressure, tachycardia from involvement of the vagus nerve, or Horner’s syndrome due to injury to a stellate ganglion. Treatment

The treatment of acute mediastinitis includes surgery for correction of the pathogenetic factors and drainage of abscesses, as well as appropriate antibiotic

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therapy and electrolyte, ventilatory, and nutritional support. Antibiotics to control both aerobic and anaerobic oropharyngeal infections should be given promptly. Surgical drainage is usually done through the supraclavicular notch unless the abscess is in the posterior mediastinum. If a mediastinal abscess is not drained, it often empties into the trachea, bronchi, esophagus, or the pleural space. In the latter case, a small rib resection immediately adjacent to the spine should be performed.

Acute Descending Necrotizing Mediastinitis Etiology

A less common but just as lethal form of acute mediastinitis may result as an extension of an oral infection into the neck; if unrecognized, these virulent cervical infections may extend into the visceral compartments of the mediastinum. Acute necrotizing mediastinitis usually develops anterior to the prevertebral fascia, or lymphatic spread of infections is present in structures such as the neck, pleura, lung, subphrenic spaces, and vertebrae adjacent to the mediastinum. Infection can also extend along the carotid sheaths (Figure 2). Pathology

Despite antibiotic therapy and, often, drainage of the deep cervical space by an anterior cervical mediastinotomy, the infection progresses to involve the mediastinum. Early diagnosis is often difficult because

of the vagueness of the early symptoms implicating involvement of the mediastinum. Microbiology

Symbiosis between one or more species of aerobic and anaerobic bacteria can result in a synergistic necrotizing cellulitis. Clinical Findings

Development of cervical infection manifests originally with signs and symptoms of sepsis, with stiffness, swelling, and pain in the neck. Dysphagia may or may not be present. The involvement of the mediastinum may occur as soon as 12 h to as late as 2 weeks, but most commonly it is seen within 48 h of the onset of the deep cervical infection. Pitting edema and crepitation in the neck and upper anterior chest wall may be present. Substernal pain, increased dysphagia, cough, and dyspnea may develop. Pleural or pericardial involvement (friction rub and effusion) may occur. Infection of the retroperitoneal space of the abdomen may even develop as the inflammatory process descends into the abdomen through the esophageal hiatus. On radiographic examination of the neck and chest, four features are usually present: widening of the retrocervical space with or without an air–fluid level, anterior displacement of the tracheal air column, mediastinal emphysema, and loss of the normal lordosis of the cervical spine (Figure 3). CT findings include abscess formation, soft tissue infiltration with loss of the normal fat planes, the absence of prominent lymphadenopathy, and the

Pretracheal fascia Thyroid gland Recurrent nerve Trachea

Carotid sheath (Lincoln highway)

Esophagus

Paraesophageal space

Common carotid artery

Internal jugular vein

Buccopharyngeal fascia VII

Cervical sympathetic chain

Vagus nerve Prevertebral fascia Retropharyngeal space Alar fascia Figure 2 Cross section of the neck at the level of the seventh cervical vertebra. Adapted from Shields TW (1994) Infections of the mediastinum. In: Shields TW (ed.) General Thoracic Surgery, 4th edn., vol. 2. Philadelphia: Williams & Wilkins.

MEDIASTINITIS, ACUTE AND CHRONIC 43

Figure 3 Posteroanterior and lateral CXRs show the right hydropneumothorax.

Figure 5 Gross pericardial effusion and bilateral parietal, and visceral pleural thickening on thoracic CT scan.

Figure 4 Nasopharyngeal CT scan demonstrates a large parapharyngeal abscess and gas collections in pharynx and deep cervical compartments.

presence of gas bubbles. The scan is helpful in determining the downward extent of the inflammatory process, especially below the fourth thoracic vertebra – the level of the carina. Pleural and pericardial effusion may also be demonstrated (Figures 4–6). CT examination is recommended for every patient with a deep cervical infection so that the presence of descending necrotizing mediastinitis can be identified.

Figure 6 CT scan shows anteromediastinitis with mediastinal abscess, hydropneumothorax, pleural thickening, and gas collection in the retrosternal space.

Treatment

Antibiotics to control both aerobic and anaerobic oropharyngeal infections should be given promptly. The surgical approach depends on the cause, and where the pretracheal fascia binds to the fourth vertebral body is anatomically important. Anterior cervical mediastinotomy is generally sufficient if the infection has not spread downward below the level of the fourth thoracic vertebra. Soft rubber tissue drains should be used; firm-walled drainage tubes

should be avoided because of the possibility of erosion of a major arterial vessel in the area. When the inflammatory process has extended lower than the fourth thoracic vertebra, adequate drainage must be accomplished by thoracotomy and sternotomy, wide incision, and drainage of mediastinum. When the infection is confined to the anterior mediastinal compartment, in addition to the transcervical drainage, subxyphoid drainage of the anterior space may

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be satisfactory to ensure adequate drainage. Tracheostomy is recommended by some authors for all patients with descending necrotizing mediastinitis to ensure access to the airway.

Subacute Mediastinitis Etiology

The definition of subacute mediastinitis is unclear, but this term should embrace those inflammatory processes involving the mediastinum. Pathology

Subacute infection is observed only infrequently in previously healthy people, but it is becoming more common in immunocompromised patients, particularly those with AIDS. Mediastinal or hilar adenopathy was reported to be one of the dominant findings in 59% of AIDS patients with Mycobacterium tuberculosis infections. Atypical mycobacterial infection of the sternum and mediastinum occasionally follows median sternotomy. Microbiology

Subacute infections attributed to histoplasmosis and to primary progressive Mycobacterium spp. infection are encountered most often. Actinomycotic infections are rare. Primary mediastinal infection due to Nocardia asteroides has been described. Clinical Findings

The inflammatory nature of the mediastinal mass may be suggested by the CT features of the lesions, but it is best confirmed by gallium scintigraphy, especially in AIDS patients. Indium leukocyte scintigraphy may also be useful in identifying these subacute infections. The final diagnosis is made by identification of the specific organism by tissue stains or culture. Adequate samples are usually obtained by needle aspiration, although occasionally a more invasive mediastinal exploration is necessary. Treatment

Treatment consists of the administration of the appropriate drug(s) for the specific organism identified. Occasionally, the organisms show in vitro resistance to the antituberculosis drugs, but this treatment is usually successful. Extensive debridement may be necessary.

Chronic Fibrosing Mediastinitis The mediastinal fibrosis and resulting clinical features are variously termed fibrosing mediastinitis,

Table 3 Etiologic factors of granulomatous mediastinitis with fibrosis Fungal infections Histoplasmosis Aspergillosis Mucormycosis Coccidioidosis Cryptococcosis Mycobacterial infections Tuberculosis Nontuberculous infections Bacterial infections Nocardiosis Actinomycosis Autoimmune disease Sarcoidosis Rheumatic fever Neoplasms Trauma Drugs Idiopathic Adapted from Marchevsky AM and Kaneko M (1992) Surgical Pathology of the Mediastinum, 2nd edn. New York: Raven Press.

fibrous mediastinitis, sclerosing mediastinitis, and granulomatous mediastinitis. Fibrosing mediastinitis is the deposition of thick fibrous tissue encasing any of the various mediastinal structures outside of the mediastinal lymph nodes. The process should involve and obstruct the major airways or the pulmonary arteries or veins or both. Etiology

Fibrosing mediastinitis may result from a number of factors (Table 3). In rare instances, the disease is familial and multifocal, and patients present with retroperitoneal and mediastinal fibrosis, sclerosing cholangitis, Riedel’s thyroiditis, and pseudotumor of the orbit. The actual mechanisms of the development of the process are unknown, but the process is hypothesized to occur as a result of an exaggerated delayed hypersensitivity reaction to antigens from infected lymph nodes. The presence of strongly positive skin and serum reactivity and hypergammaglobulinemia and hypercomplementemia support the theory. Pathology

Fibrosing mediastinitis is characterized grossly by a diffuse, fibrotic infiltration of the mediastinal structures by chronic inflammatory cells. The mass of tissue is described as woody hard; tissue planes are obscured, and the process may extend to involve the root of the lungs and the major pulmonary vessels.

MEDIASTINITIS, ACUTE AND CHRONIC 45 Clinical Features

Fibrosing mediastinitis may be self-limiting, but serious persistent complications can incapacitate the patient and even result in death. The condition is more common in young adult white women. Women are affected three times more often than men. The average age is between 19 and 25 years, although some patients are in the fourth or fifth decades of life. Approximately 40% of patients are asymptomatic, and the disease is discovered only as an incidental radiographic finding. In the other 60% of patients, the clinical features vary, with visceral mediastinal structures entrapped and compressed. Compression and occlusion of the superior vena cava results in the superior vena cava syndrome. Invasion of the pulmonary veins usually occurs around the left atrium and results in a clinical syndrome that closely mimics mitral valve stenosis with pulmonary hypertension. Severe stenosis of the trachea, carina, main stem bronchus, or esophagus was noted in 44% of patients in one study. Cough, dyspnea, chest pain, fever, wheezing, postobstructive pneumonia, dysphagia, and hemoptysis are common. Hoarseness occurs due to compression of the left recurrent nerve and resulting left vocal cord paralysis. It is usually accompanied by a secondary ectasia of veins and lymphatic vessels in the airway lamina propria, which in turn leads to hemoptysis, dyspnea, and markedly abnormal pulmonary function tests. A more frequent complication in patients with sclerosing mediastinitis is pulmonary interstitial fibrosis, which can follow a clinical and pathologic course similar to that in patients with usual interstitial pneumonitis. Rarely, a bronchoesophageal fistula may occur as a consequence of mediastinal granulomatous disease in the subcarinal area.

Radiological Findings

Patients who have major airway obstruction and in whom the disease is located primarily in the subcarinal region may also have normal radiographic findings. The mass, when present, is often asymmetric and commonly projects into the right hemithorax. CT examination demonstrates the degree of compression of the great arteries, trachea, and esophagus (Figures 7 and 8). Magnetic resonance imaging can be useful in demonstrating the extent of involvement of the great arteries, especially if the use of contrast material is contraindicated (Figure 9). Radionuclide venography with technetium 99m to demonstrate occlusion of the vena cava is a quick and safe method of investigation (Figure 10). Pulmonary arteriography may be helpful when involvement of the pulmonary vessels is suspected (Figure 11). Upper

Figure 7 Mediastinal granuloma due to fungal disease with chest pain. Posteroanterior CXR reveals a calcified right paratracheal mass (arrowhead).

Figure 8 CT scan (mediastinal window) reveals the focal paratracheal mass with a low-attenuation center and extensive calcification (arrowhead). Note the mass effect on the tracheal wall (T).

gastrointestinal contrast X-ray studies can demonstrate esophageal narrowing (Figure 12). Diagnostic Investigation

Bronchoscopy and mediastinoscopy are generally sufficient, but thoracotomy may be necessary to establish the benign nature of the fibrosis. Esophagoscopy is indicated when dysphagia is a major complaint. Skin testing for mycobacterial and fungal diseases is indicated. Complement fixation studies are established for fungal agents but are not diagnostic. Treatment

Although vascular reconstruction is not recommended, as a general rule, the use of spiral saphenous

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vein grafts or azygos vein transposition has been successful in vena caval obstruction. In such cases, bypass of the obstructed superior vena cava using a vascular or prosthetic graft from the left innominate vein to atrium is indicated. Whether or not a course of ketoconazol should be instituted before any surgical intervention should be discussed. In approximately 25% of patients with localized granulomas, complete excision of the lesion can be achieved. When a bronchoesophageal fistula is present, excision of the fistulous tract and closure of both the tracheal and esophageal openings are required. Unfortunately, surgical resection of the lung to eradicate obstruction or pulmonary complications is associated with Figure 9 MRI can be useful in demonstrating the extent of involvement of the great arteries. Arrows, heterogeneous right hilar and subcarinal mass; S, superior vena cava.

Figure 10 Radionuclide venography demonstrates occlusion of the vena cava. Figure 12 Upper gastrointestinal contrast X-ray studies demonstrate esophageal narrowing (arrows). D, superior esophageal diverticulum.

Figure 11 Pulmonary arteriography may be helpful when there is involvement of the pulmonary vessels. Arrow, long segment eccentric stenosis; arrowhead, narrowing of the right interlobar pulmonary artery; M, right main pulmonary artery; P, right pulmonary artery.

MESOTHELIAL CELLS 47

significant mortality. In one study, the interval between the onset of symptoms and death was slightly less than 6 years. Death resulted from cor pulmonale. See also: Lung Imaging. Mediastinal Masses. Pleural Effusions: Overview. Symptoms of Respiratory Disease: Cough and Other Symptoms; Dyspnea; Chest Pain.

Further Reading Akman C, Kantarci F, and Cetinkaya S (2004) Imaging in mediastinitis: a systematic review based on aetiology. Clinical Radiology 59(7): 573–585. Balkan ME, Oktar GL, and Oktar MA (2001) Descending necrotizing mediastinitis: a case report and review of the literature. International Surgery 86(1): 62–66. Brook I (2002) Anaerobic bacteria in upper respiratory tract and other head and neck infections. Annals of Otology, Rhinology & Laryngology 111(5 Pt 1): 430–440. Brook I and Frazier EH (1996) Microbiology of mediastinitis. Archives of Internal Medicine 156(3): 333–336. Brook I and Frazier EH (1996) Microbiology of mediastinitis: erratum. Archives of Internal Medicine 2(10): 1112. Friedman BC and Pickul DC (1990) Acute mediastinitis. What to do when the cause is nonsurgical. Postgraduate Medicine 87(4): 273–275, 278–280, 285.

Gardlund B, Bitkover CY, and Vaage J (2002) Postoperative mediastinitis in cardiac surgery – microbiology and pathogenesis. European Journal of Cardiothoracic Surgery 21(5): 825–830. Giamarellou H (2000) Anaerobic infection therapy. International Journal of Antimicrobial Agents 16(3): 341–346. Hasegawa T, Endo S, and Sohara Y (2000) Classification of descending necrotizing mediastinitis. Annals of Thoracic Surgery 69(4): 1296. Karnath B and Siddiqi A (2002) Acute mediastinal widening. Southern Medical Journal 95(10): 1222–1225. Marchevsky AM and Kaneko M (1992) Surgical Pathology of the Mediastinum, 2nd edn. New York: Raven Press. Murray PM and Finegold SM (1984) Anaerobic mediastinitis. Review of Infectious Diseases 6(supplement 1): S123–S127. Rodriguez E, Soler R, Pombo F, Requejo I, and Montero C (1998) Fibrosing mediastinitis: CT and MR findings. Clinical Radiology 53(12): 907–910. Rossi SE, McAdams HP, Rosado-de-Christenson ML, Franks TJ, and Galvin JR (2001) Fibrosing mediastinitis. Radiographics 21(3): 737–757. Rupp ME (2000) Mediastinitis. In: Mandell GL, Bennett JE, and Dolin R (eds.) Principles and Practice of Infectious Diseases, 5th edn. London: Churchill Livingstone. Shields TW (1994) Infections of the mediastinum. In: Shields TW (ed.) General Thoracic Surgery, 4th edn., vol. 2. Philadelphia: Williams & Wilkins.

MESOTHELIAL CELLS S E Mutsaers, The University of Western Australia, Nedlands, WA, Australia S E Herrick, University of Manchester, Manchester, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Mesothelial cells form a monolayer of cobblestone-like cells that line the pleural pericardial and peritoneal cavities and most internal organs. They are embryologically derived from the mesoderm but express many epithelial cell characteristics. Mesothelial cells are predominantly squamous-like cells approximately 25 mm in diameter, with a central nucleus and characteristic surface microvilli. However, cuboidal mesothelial cells can be found at various locations and following injury or stimulation of the serosal surface. The layer of mesothelial cells, termed the mesothelium, is a dynamic membrane that plays important roles in maintaining normal serosal membrane integrity and function. It provides a nonadhesive protective surface to allow movement of organs within the serosal cavities, provides a semipermeable membrane regulating transport of fluid and cells across the serosa, regulates the immune response by presenting antigen to T cells, secretes mediators that regulate serosal inflammation and tissue repair, controls fibrin deposition and breakdown, and possibly inhibits tumor cell adhesion and

dissemination. Injury to the mesothelium can result in excessive and disordered repair processes, which clinically manifest as fibrous adhesions/plaques and serosal thickening or, following asbestos exposure, cell transformation and the development of malignant mesothelioma. There is no effective treatment for either of these conditions, but developments in our understanding of mesothelial cell–matrix interactions and the growth factors that influence mesothelial cell functions are suggesting new therapeutic approaches.

Introduction Mesothelial cells line the pleural, peritoneal, and pericardial cavities with visceral and parietal surfaces covering the internal organs and body wall, respectively. They form a monolayer of specialized epithelial-like cells called the mesothelium, which rests on a thin basement membrane supported by subserosal connective tissue containing blood vessels, lymphatics, resident immune cells, and fibroblast-like cells. The mesothelium is bathed in serosal fluid that resembles an ultrafiltrate of plasma and contains blood proteins, resident inflammatory cells, sugars, and various enzymes.

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Embryological Origin The mesothelium was first described in 1827 by Bichart, who observed that serous cavities were lined by a layer of flattened cells similar to those of the lymphatics. Minot (1890) subsequently proposed the term ‘mesothelium’ following a detailed study of its embryological origin that showed this layer to be the ‘‘epithelial lining of mammalian mesodermic cavities.’’ During human development, the intraembryonic mesoderm on each side of the neural groove differentiates into paraxial, intermediate, and lateral mesoderm. The lateral mesoderm is continuous with the extraembryonic mesoderm covering the yolk sac and amnion. At the end of the third week, small spaces appear in the lateral mesoderm that fuse, dividing the mesoderm into two layers: the intraembryonic somatic or parietal layer and the intraembryonic splanchnic or visceral layer. The somatic

mesoderm and overlying embryonic ectoderm form the embryonic body wall, whereas the splanchnic mesoderm and embryonic endoderm form the embryonic gut wall. A continuous mesothelial membrane lines the margin of these two layers and so borders the intraembryonic coelom. Between 5 and 7 weeks, the coelom is subdivided, forming the early pericardial, pleural, and peritoneal cavities.

Morphology Mesothelial cells are morphologically similar at different serosal sites and between different mammalian species. They form a monolayer of predominantly squamous-like cells approximately 25 mm in diameter, with a central oval nucleus and characteristic surface microvilli and occasional cilia (Figure 1). The microvilli vary in shape, length, and density between

EL N

(a)

EL (b)

(c)

Figure 1 Morphology of respiratory mesothelium. (a) Transmission electron micrograph of a section of mouse lung showing the thin attenuated cytoplasm of a mesothelial cell (arrow) resting on an elastic lamina (EL) with underlying alveolar and endothelial cells. N, nucleus of underlying cell. Scale bar ¼ 1.3 mm. (b) High power of mesothelial cell showing characteristic surface microvilli (large arrow), tight-like junctions between cell membranes (small arrows), and pinocytic vesicles (arrowheads). Scale bar ¼ 0.5 mm. (c) Scanning electron micrograph of the luminal aspect of mesothelial cells covered with a carpet of microvilli. Some cells have a reduced number of microvilli over the central nuclear region (arrow). Scale bar ¼ 12 mm.

MESOTHELIAL CELLS 49

adjacent cells and different organs, which may reflect functional adaptation. Mesothelial cells express mesenchymal features, including the intermediate filaments vimentin and desmin and, upon stimulation, the microfilament a-smooth muscle actin (a-SMA). Mesothelial cells also display many epithelial characteristics, including a polygonal cell shape, cytokeratin (6, 8, 18, and 19) intermediate filaments, junctional complexes, apical/basal polarity, surface microvilli, and the ability to secrete a basement membrane. Mesothelial cells also have a well-developed system of vesicles and vacuoles. Most are micropinocytic, but multivesicular bodies and large vacuoles can be found. The boundaries between mesothelial cells are tortuous, with adjacent cells often overlapping. They have well-developed cell– cell junctional complexes, including tight junctions located toward their luminal aspect, adherens junctions, gap junctions, and desmosomes. Tight junctions in particular are crucial for the development of cell surface polarity and the establishment and maintenance of a semipermeable diffusion barrier. They also express E-, N-, and P-cadherins, but unlike true epithelia, N-cadherin predominates. Although mainly squamous in appearance, cuboidal mesothelial cells are found at various locations, including the septal folds of the mediastinal pleura, parenchymal organs (liver and spleen), the ‘milky spots’ of the omentum, and the peritoneal side of the diaphragm overlying the lymphatic lacunae. Mesothelial cells can also change to a cuboidal morphology after injury or stimulation of the serosal surface. The two forms of mesothelial cell, squamous-like and cuboidal, also show differences ultrastructurally. In particular, cuboidal cells have a larger nucleus with prominent nucleoli, abundant mitochondria and rough endoplasmic reticulum, a well-developed Golgi apparatus, microtubules, and a greater number of microfilaments compared with squamous cells, suggesting a more metabolically active state. Mesothelial cells can also change their phenotype, comparable to changes seen in epithelial-to-mesenchymal transition (EMT). This has implications in both normal repair and pathological processes. After several passages in culture, mesothelial cells adopt a fibroblast-like phenotype, consistent with ultrastructural observations in healing serosa. This is concomitant with increased expression of the transcription factor ‘snail’ and downregulation of E-cadherin and cytokeratin levels. Furthermore, incubation of cells with the wound repair and profibrotic mediator, transforming growth factor beta (TGF-b), induces EMT in mesothelial cells and upregulates a-SMA and type I collagen expression, consistent with a myofibroblast phenotype. This

implies that the mesothelium is a likely source of fibrogenic cells during serosal inflammation and wound healing and may play important roles in serosal fibrosis and adhesion formation.

Functions The mesothelium is a dynamic cellular membrane with many important functions (Figure 2). Protective Nonadhesive Barrier

Mesothelial cells form a protective barrier against invading pathogens, cells, and particulates. This protection is increased by the secretion of glycosaminoglycans (particularly hyaluronan), proteoglycans including syndecans and biglycan, and surfactant lubricants, which prevents attachment and penetration of cells by infective agents and possibly inhibits tumor dissemination. In addition, this provides a slippery nonadhesive surface to prevent serosal tissues from sticking together and forming adhesions. Mesothelial Transport

Despite a tight barrier preventing invasion of particulates across the serosal surface, mesothelial cells actively transport fluid, cells, and molecules through the serosa via pinocytic vesicles, intracellular junctions, and stomata. Stomata are cavities at the junction of two or more mesothelial cells and are generally found in regions where cuboidal mesothelial cells are present. These openings provide a direct access to the lymphatic system allowing rapid removal of fluid and cells from the serosal cavities, which is regulated by adhesion molecule/integrinmediated interactions. Inflammation

Mesothelial cells regulate serosal inflammation by secreting various pro-, anti-, and immunomodulatory mediators and by modulating microvilli density and adhesion molecule expression to regulate trafficking of leukocytes into the serosal cavities. Stimuli such as bacterial products, asbestos, instilled agents, and tissue injury induce the release of proinflammatory cytokines and chemokines, including interleukin (IL)-1, IL-8, monocyte chemoattractant protein-1, RANTES, growth-related oncogene alpha, interferongamma-inducible protein-10, stromal cell-derived factor-1, and eotaxin, which recruits inflammatory cells to the site of challenge. Further stimulation by hyaluronan secreted by mesothelial cells, and products released from activated macrophages such as IL-1b, tumor necrosis factor alpha (TNF-a), and interferon gamma, increases the production of chemokines

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Prevention of tumor cell adhesion

Coagulation and fibrinolysis

Antigen presentation

Release of surfactant, proteoglycans and GAGs Barrier against invading pathogens

HA

Secretion of inflammatory mediators

CD4+ T cell

Fluid transport

Secretion of cytokines, growth factors and ECM

CD44

Leukocyte migration through stomata

Figure 2 Mesothelial cell functions. The role of the mesothelium is to maintain serosal integrity and function. Mesothelial cells provide a protective barrier against abrasion and invading pathogens and secrete glycosaminoglycans, proteoglycans, and surfactant to provide a slippery, nonadhesive surface to allow intracoelomic movement. These molecules may also be important for preventing tumor growth and dissemination. Mesothelial cells facilitate transport of fluid and cells across the serosal cavities; present antigen to T cells; and through secretion of cytokines, growth factors, matrix, proteases, and other inflammatory mediators participate in the induction and resolution of inflammation and tissue repair. They are also the major source of plasminogen activators in serosal fluid involved in the breakdown of fibrin and prevention of adhesion formation. Reproduced from Mutsaers SE (2004) International Journal of Biochemistry and Cell Biology 36: 9–16, with permission from Elsevier.

by mesothelial cells, which potentiates the inflammatory response. Mesothelial cells also regulate the inflammatory response by secreting anti-inflammatory mediators, including prostaglandins, prostacyclin, and IL-6. In addition, mesothelial cells play an immunoregulatory role by presenting antigen to T cells. Tissue Repair

Mesothelial cells release growth factors, including TGF-b, platelet-derived growth factor (PDGF), fibroblast growth factor, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and members of the epidermal growth factor (EGF) family (EGF, heparin-binding EGF, and vascular EGF), that initiate cell proliferation, differentiation, and migration of mesothelial and submesothelial cells surrounding a lesion. Interestingly, KGF and HGF are considered epithelial growth factors; however, mesothelial cells also express receptors for KGF and HGF and respond to them, confirming their dual epithelial/mesenchymal properties. Mesothelial cells

also synthesize extracellular matrix (ECM) molecules, including collagen types I, III, and IV, elastin, fibronectin, and laminin, and regulate turnover through the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases. Deposition of ECM is important for the synthesis of a basement membrane and maintenance of submesothelial connective tissue. Coagulation and Fibrinolysis

Mesothelial cells regulate local fibrin turnover within serosal cavities through the secretion of both procoagulant and fibrinolytic enzymes. Procoagulant activity is due to tissue factor, the main cellular initiator of the extrinsic coagulation cascade. Fibrinolytic activity is mediated through secretion of tissue plasminogen activator (tPA) and urokinase PA (uPA) and their inhibitors, plasminogen activator inhibitor (PAI)-1 and PAI-2. Both pro- and antifibrinolytic mediators are regulated by inflammatory factors, including lipopolysaccharide, TNF-a, and IL-1, and

MESOTHELIAL CELLS 51

fibrogenic mediators such as TGF-b and thrombin. The PAs convert plasminogen into plasmin, which enzymatically breaks down fibrin. Their fibrinolytic activity is a key factor in the removal of fibrin deposits that form following mechanical injury, hemothoraces, and infection. If the fibrinolytic capacity is insufficient and fibrin accumulation is not resolved, fibrous adhesions/plaques form between opposing serosal surfaces. Tumor Growth and Dissemination

Mesothelial cells have been implicated in both the spread and the inhibition of tumor growth within serosal cavities. Traumatized mesothelial surfaces are privileged sites for tumor cell adhesion possibly due to upregulation of adhesion molecules on mesothelial cells in response to inflammatory mediators. However, binding via integrins to exposed submesothelial ECM is likely to the main mechanism of attachment. Tumor growth is then potentiated by growth factors released from activated mesothelial cells or from the tumors. Many studies have demonstrated that adhesion of tumor cells to hyaluronan on the mesothelial cell surface is important for the spread of ovarian and colorectal tumors. However, release of hyaluronan into the serosal fluid may inhibit tumor cell adhesion. Conditioned medium from cultured mesothelial cells containing large amounts of hyaluronan prevented tumor cell attachment to mesothelial cells, but this inhibition was overcome following hyaluronidase treatment. It is likely that hyaluronan in the conditioned medium bound to CD44 molecules on the tumor cells and prevented their interaction with hyaluronan on the mesothelial cell. In the lung, mesothelioma cells express high levels of CD44, and conditioned medium from mesothelioma cell lines stimulates hyaluronan synthesis in mesothelial cells. Hyaluronan induces mesothelioma cell proliferation and migration in vitro, but the role of CD44/hyaluronan interactions on malignant mesothelioma (MM) and mesothelial cell attachment has not been investigated.

Respiratory Diseases Mesothelial cells are important clinically because they are the likely progenitor cells of MM, whereas impaired mesothelial regeneration is a likely cause of pleural adhesion/plaque formation. Pleural Adhesions/Plaques and Serosal Thickening

Pleural diseases are common in clinical practice. Pleural mesothelial cells are often exposed to pathogens and particulates such as asbestos fibers or subjected to trauma. Injuries to the mesothelium are

often followed by excessive and disordered repair processes, which clinically can manifest as fibrous adhesions/plaques and serosal thickening or fibrosis. The mechanism for adhesion formation is unknown but involves persistence of fibrin between serosal surfaces, possibly due to impaired mesothelial regeneration and reduced PA levels. Clinically, fibrous adhesions produce loculations that prevent the complete drainage of pleural effusions, such as parapneumonic effusions or empyemas, interfering with the successful management of pleural infection. Pleural fibrosis and diffuse thickening is common in workers involved in mining asbestos and industries utilizing large quantities of asbestos. There is no effective treatment for pleural adhesions and fibrosis. Fibrinolytic therapy using streptokinase, pepsin, trypsin, papain, plasmin, actase, fibrinolysin, uPA and tPA, and corticosteroids has not shown any clear benefit to suggest that any of these approaches improve pleural drainage or avoid surgery/death. Materials, primarily barrier membranes and gels, have also been trialed to reduce adhesions by separating serosal surfaces. However, these materials are not widely used because of problems associated with their clinical application and their limited success. Malignant Mesothelioma

MM is a serosal tumor, predominantly of the pleura, strongly associated with previous asbestos exposure. It has a latency period of up to 40 years and is largely unresponsive to conventional chemotherapy or radiotherapy, with mean survival following diagnosis of less than 12 months. The mechanism regulating asbestos-induced mesothelial cell transformation is unclear, although fiber dimension, surface properties, physical durability, and cell and tissue responses are important factors that contribute to the carcinogenicity of asbestos fibers. Asbestos activates signaling pathways such as mitogen-activated protein kinase, which is associated with proliferation of mesothelial cells. Asbestos also induces chromosomal aberrations, sister chromatid exchanges, and multilocus deletions in cells; significantly, some deleted chromosomal fragments contain genes with known tumor suppressor functions. Studies have identified Simian virus 40 (SV40), a DNA virus recognized as a contaminant of early poliovirus vaccines, as a possible causative agent for MM. Mesothelial cells are particularly susceptible to transformation by SV40 in vitro and produce MM when injected into the visceral cavities of rodents. Its oncogenicity is associated with SV40 large and small t-antigen and its ability to inactivate p53 and

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retinoblastoma tumor suppressor proteins. SV40 DNA sequences have been detected in human pleural MM by some laboratories but not by others. Although SV40 DNA may be an incidental finding or a polymerase chain reaction contamination artifact, it is more likely that SV40 acts as a cocarcinogen and potentiates the effect of asbestos. Various approaches, including surgery, radiotherapy, chemotherapy, immunotherapy, and gene therapy, have been trialed to improve survival rates in patients with MM with no or limited success. Surgical management has made the greatest strides in palliating the major symptoms of the disease, with some improvement in survival seen in selected patients. Measurement of serum markers such as soluble mesothelin-related proteins together with thoracoscopy are leading to the earlier diagnosis of more patients with MM, making early diagnosed patients stronger candidates for more aggressive attempts at surgical and postsurgical cure. Promising results have been reported with extrapleural pneumonectomy in combination with chemotherapy and postoperative radiation, termed trimodality therapy, as well as combinational chemotherapy using combinations of agents including pemetrexed, raltitrexed, cisplatin, and gemcitabine. Other new directions in therapy include antifolates, antiangiogenesis agents, and drugs directed at EGF and PDGF receptors. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Cell: Vascular. Chemokines, CXC: IL-8. Chemokines. Chest Wall Abnormalities. Coagulation Cascade: Overview; Fibrinogen and Fibrin; iuPA, tPA, uPAR; Tissue Factor. Environmental Pollutants: Particulate and Dust Pollution, Inorganic and Organic Compounds. Extracellular Matrix: Basement Membranes; Collagens; Surface Proteoglycans. Fibrinolysis: Overview; Plasminogen Activator and Plasmin. Fibroblasts. Hepatocyte Growth (Scatter) Factor. Interleukins: IL-1 and IL-18; IL-6. Keratin. Keratinocyte Growth Factor. Leukocytes: Pulmonary

Macrophages. Lung Development: Overview. Matrix Metalloproteinases. Mesothelioma, Malignant. Occupational Diseases: Asbestos-Related Lung Disease. Platelet-Derived Growth Factor. Pleural Effusions: Overview; Parapneumonic Effusion and Empyema; Malignant Pleural Effusions; Pleural Fibrosis; Hemothorax. Stem Cells. Surfactant: Overview. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumor Necrosis Factor Alpha (TNF-a). Tumors, Malignant: Overview; Chemotherapeutic Agents. Vesicular Trafficking.

Further Reading Chegini N (2002) Peritoneal molecular environment, adhesion formation and clinical implication. Frontiers in Bioscience 7: e91–e115. Herrick SE and Mutsaers SE (2004) Mesothelial progenitor cells and their potential in tissue engineering. International Journal of Biochemistry & Cell Biology 36: 621–642. Holmdahl L (1997) The role of fibrinolysis in adhesion formation. European Journal of Surgery Supplement 577: 24–31. Kuwahara M and Kagan E (1995) The mesothelial cell and its role in asbestos-induced pleural injury. International Journal of Experimental Pathology 76: 163–170. Mutsaers SE (2002) Mesothelial cells: their structure, function and role in serosal repair. Respirology 7: 171–191. Mutsaers SE (2004) International Journal of Biochemistry and Cell Biology 36: 9–16. Mutsaers SE, Preˆle CM, Brody AR, and Idell S (2004) Pathogenesis of pleural fibrosis. Respirology 9: 428–440. Ohtani O, Ohtani Y, and Li RX (2001) Phylogeny and ontogeny of the lymphatic stomata connecting the pleural and peritoneal cavities with the lymphatic system – A review. Italian Journal of Anatomy and Embryology 106(2 supplement 1): 251–259. Pass HI, Bocchetta M, and Carbone M (2004) Evidence of an important role for SV40 in mesothelioma. Thoracic Surgery Clinics 14: 489–495. Robinson BWS and Chahinian AP (eds.) (2002) Mesothelioma. London: Dunitz. Sterman DH and Albelda SM (2005) Advances in the diagnosis, evaluation, and management of malignant pleural mesothelioma. Respirology (in press). Treutner K-H and Schumpelick V (eds.) (1996) Peritoneal Adhesions. New York: Springer.

MESOTHELIOMA, MALIGNANT B W S Robinson, Queen Elizabeth II Medical Centre, Nedlands, WA, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Malignant mesothelioma is an aggressive serosal tumor which is rarely cured and is increasing in frequency throughout the

world. Although the main risk factor is asbestos exposure, recent studies have suggested that the SV40 polyoma virus may have a role. There is no association with smoking. Mesothelioma has a molecular pathology characterized by loss of tumor-suppressor genes, particularly the P16INK4A, P14ARF, and NF2 genes, rather than the more common p53 and Rb tumorsuppressor genes. Cytopathology of mesothelioma effusions or fine needle aspirations can establish a diagnosis in many cases. Histopathology diagnoses the remainder of the cases accurately.

MESOTHELIOMA, MALIGNANT 53 Adenocarcinoma remains the most difficult differential diagnosis. Breathlessness and chest pain with pleural effusions is the most common presentation. Median survival is 12 months from diagnosis. Palliative chemotherapy regimes which have some benefit include pemetrexed or gemcitabine combined with platinum drugs. Aggressive surgery is used in some centers but its role remains controversial. Gene therapy and immunotherapy are used on an experimental basis only. Microarray patterns may be useful for diagnosis as well as prognostication.

Introduction Mesothelioma is a fatal and debilitating tumor of serosal surfaces such as pleura and peritoneum and is usually caused by asbestos. It has become more common in the past 30–40 years and is now approximately as common a cause of death as cancers of the cervix, ovary, liver, bone, and bladder. This incidence continues to increase.

Epidemiology Mesothelioma’s relationship with asbestos was first published in South Africa in 1960. The occurrence of mesothelioma can be predicted from the asbestos patterns of use. Erionite exposure (in Turkey), ionizing radiation, and possibly the SV40 virus are other likely causes. Median survival from the time of presentation is 12 months. There is a long incubation period between the onset of the disease and time since the first exposure, typically over 30 years. Mesothelioma is not related to cigarette smoking. In the developed world, increasing constraints on asbestos use and regulation of allowable exposures in industry over the past 30–40 years have meant that the peak in the number of cases of mesothelioma can be predicted to occur within the next 20 years as the number of exposed people decreases, even though the individual risks in the surviving exposed people continue to escalate. As numbers of mesothelioma cases decline in developed countries after another 10–20 years, they will rise in developing countries because of continued use of asbestos there, coupled with less stringent environmental controls.

2. Interference with mitosis – asbestos fibers alter the function of the mitotic spindle leading to aneuploidy and chromosome damage. 3. Toxic oxygen radicals – reactive oxygen species (ROS) induced by asbestos cause DNA damage. 4. Kinase-mediated signaling – phosphorylation of the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinases (ERKs) 1 and 2, induced expression of early response protooncogenes (fos/jun or activator protein 1 family members), plus other growth factors are induced and maintained by asbestos.

SV40 virus, a polyoma virus which blocks tumorsuppressor genes may have a role as SV40 DNA sequences are found in mesotheliomas but not in normal adjacent tissues or in lung cancers. SV40 contaminated the Salk polio vaccine in the 1950s and 1960s although evidence connecting vaccine-derived infection and the occurrence of mesothelioma is scant. Familial clustering of mesothelioma has been reported on several occasions. In each case, the conclusion offered has been that various genetic factors could be held to account for a predisposition to disease, although common exposure to asbestos is almost always present. Comparative genomic hybridization demonstrated in two cases, a loss at chromosome 9p as the only change. A genetic disposition to erionite-induced mesothelioma was postulated to play a role in the high rate of mesotheliomas in Cappadocia, Turkey. Various animal models of mesothelioma have been described but the most commonly used species are still the rat and the mouse, including inbred, knockout, and transgenic mice. Mice with a heterozygous mutation in the NF2 gene develop mesothelioma at a higher frequency than their wild-type counterparts after exposure to asbestos fibers and mice heterozygous for p53 also show an increased incidence and reduced latency to the induction of mesothelioma by asbestos.

Pathogenesis Asbestos causes mesothelioma by a number of processes. The main processes are given as follows. 1. Pleural irritation – the fiber shape and length-towidth ratio determines how deeply the fibers are respired and penetrate the lung to irritate the pleura. Long, thin fibers are the most notorious as they penetrate deeply, causing repeated damage and repair of pleural tissue.

Molecular Pathology Mesotheliomas have shown extensive aneuploidy and structural rearrangements, including loss of chromosome 22 and structural rearrangement of 1p, 3p, 9p, and 6q. Neurofibromatosis type 2 (NF2) is located at 22q and loss of heterozygosity (LOH) is found in 100% of mesothelioma cell lines. Loss of P16,INK4A, P14ARF, and NF2 implies that tumor-suppressor gene loss drives mesothelioma development.

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Clinical Presentation Mesothelioma patients usually present with breathlessness due to a pleural effusion, often with chest wall pain. Weight loss and fatigue generally appear later in the course of disease. Mesothelioma can be found on routine chest X-rays. Patients with peritoneal mesothelioma exhibit abdominal distension and pain. Pleural effusion or fixed hemithorax are common physical signs. Peritoneal mesothelioma presents with ascites and tenderness. Subcutaneous masses follow surgical intervention. Signs of local invasion are usually late features. Death from metastatic deposits of mesothelioma is uncommon.

Diagnosis Differentiation of mesothelioma from adenocarcinoma is the main challenge for pathologists. Cytology

Immunohistochemical and ultrastructural assessment of cells aspirated from serous effusions or masses is often sufficient in experienced hands. The immunocytochemical stains that distinguish mesothelioma from adenocarcinoma and other diseases are listed below. Histopathology

A suite of immunohistochemical stains is used to differentially diagnose mesothelioma on solid tissue obtained by closed biopsy, thoracoscopic biopsy, or video-assisted thoracoscopic biopsy (VATS). The most useful immunohistochemistry patterns are EMA, WT1, calretinin, HBME-1, TF-1, cytokeratin 5/6 or mesothelin positive, and CEA, CD15, BerEP4, B72.3, and MOC-3 1 negative. Electron microscopy can help confirm the mesothelial origin of the tissue (Figure 1).

Sarcomatoid mesothelioma has keratin-positive malignant spindle cells sometimes with extensive collagen deposition. If the latter is predominant, the term ‘desmoplastic’ is applied. Imaging

Chest X-rays usually show unilateral pleural effusions, localized chest wall masses and later, encasement by tumor rind or diffuse lobular masses. Pleural plaques signify asbestos exposure only and do not produce mesothelioma themselves. A ‘rounded atelectasis’ is a benign pleurally based mass lesion associated with asbestos but it can mimic mesothelioma. Computed tomography shows fluid, localized, or diffused pleural masses with thickening of interlobular fissures, chest wall invasion, and perhaps plaques. Peritoneal mesothelioma scans exhibit fluid and masses. Magnetic resonance imaging may be helpful in defining localized disease, for example, the tumor extent and chest wall invasion. Positron emission tomography is also useful for determining if a mass is likely to be a tumor and also defining its extent and invasion. Serum Mesothelin-Related Protein

Serum mesothelin-related protein (SMRP) is the circulating product of the mesothelial differentiation molecule mesothelin. In a recent study of 44 mesothelioma patients, 84% had elevated levels of SMRP versus o2% of patients with other pulmonary or pleural diseases, with a sensitivity of 84% and specificity of over 80% (Figure 2).

Staging Staging is mainly used for surgical management. The International Mesothelioma Interest Group (IMIG) staging system, based on the tumor-node-metastasis (TNM) system of lung cancer, parallels prognosis.

Prognostic Factors Poor prognosis is associated with poor-performance status, male gender, the sarcomatoid histological variant, and a high WBC count. cDNA microarrays may be able to predict outcome in mesothelioma.

Treatment Figure 1 Epithelial membrane antigen (EMA) in pleural biopsy. EMA immunostaining of mesothelioma cells in a pleural biopsy showing EMA positive mesothelioma cells. H&E and EMA immunostain. Courtesy of Dr A Segal.

Surgery

Diagnostic surgery is used to obtain tissue for diagnosis. Palliative surgery, for example, partial

MESOTHELIOMA, MALIGNANT 55 1.50

Absorbance (420 nm)

1.25

1.00

0.75

0.50

0.25

Healthy controls

Pleural effusions

Other

Other cancers

Other pleural diseases

TB

RA lung

Other ILD

SLE

Sarcoidosis

IPF

Asbestosis

Other cancer

Breast cancer

Lung cancer

Gross thickening

Plaques

Malignancy

Transudates

Inflammatory

Infection

Asb-exposed

Nonexposed

Mesothelioma

0.00

Inflammatory diseases

Other lung diseases

Figure 2 SMRP levels in mesothelioma. Levels of SMR in 44 patients with mesothelioma, 68 healthy adults (28 nonasbestos exposed, 40 asbestos exposed), and 92 patients with inflammatory diseases of the lung, plus results from 30 patients with cancers which were not mesothelioma and who had no pleural involvement, 20 patients with nonmesothelioma pleural effusions and 18 patients with other nonmalignant pleural diseases without effusions. Shaded area represents the ‘normal range’ healthy subjects. Reproduced from Robinson BWS, Creancy J, Lake R, et al. (2003) Mesothelin-family proteins and diagnosis of mesothelioma. Lancet 362: 1612–1616, with permission from Elsevier.

pleurectomy, controls symptoms. ‘Potentially curative’, for example, extrapleural pneumonectomy (EPP) followed by some form of adjuvant therapy has led to longer median survival times in specialized experienced centers. Chemotherapy

Pemetrexed plus Cisplatin demonstrated improved overall survival of nearly 3 months with the combination, compared to the single drug, with an objective response rate of 41%. Gemcitabine plus Cisplatin has also been shown to offer similar results, with objective response rates of 48% and 33% in 74 patients. Symptomatic improvement and quality-of-life benefits were also seen. The growth factor signaling blockers imatinib mesylate (Gleevec) and Gefitinib (Iressa) have shown no convincing evidence of efficacy.

Radiotherapy

The major limitation to radical radiotherapy is the anatomy of the lung – the tumor covers most of the pleural surface plus the interlobular fissures, so it is difficult to avoid radiation pneumonitis. Intensity modulated radiotherapy (IMRT) following EPP is promising. Immunotherapy and Gene Therapy

Local cytokine therapy using recombinant interleukin2 or gamma interferon into the tumor cavity or the tumor itself have induced some responses in patients with early disease but not in patients with advanced disease. Recombinant interferon alpha induces some responses and, curiously, some dramatic responses lasting 5–13 years. Gene therapy in mesothelioma has utilized engineered viruses, either ‘suicide gene’ or ‘immunogene’ vectors, with no dramatic responses in early studies but biologically relevant effects.

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Other Therapies

Photodynamic therapy, inducing toxic oxygen radicals in tumor cavities, is laborious and time consuming but an effective form of cytoreduction. Other novel agents, for instance, anti-angiogenic agents, copper lowering agents, histone deacetylase inhibitors, immunotoxins, and proteosome inhibitors are also being tested. Palliation

Recurrent effusions are typically controlled by talc pleurodesis or surgery. Neuropathic, somatic, and visceral types of pain are often severe and require opioids along with nonsteroidal anti-inflammatory drugs and nerve blockade at times. Psychosocial support is helpful. See also: Environmental Pollutants: Overview. Mesothelial Cells. Occupational Diseases: AsbestosRelated Lung Disease. Pleural Effusions: Overview; Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube; Malignant Pleural Effusions.

Further Reading Armstrong BK, Musk AW, Baker JE, et al. (1984) Epidemiology of mesothelioma in Western Australia. Medical Journal of Australia 141: 86–88.

Metastases

Gazdar AF and Carbone M (2003) Molecular pathogenesis of mesothelioma and its relationship to Simian Virus 40. Clinical Lung Cancer 5: 177–181. Kindler HL (2000) Malignant pleural mesothelioma. Current Treatment Options in Oncology 1(4): 313–326. Marom EM, Erasmus JJ, Pass HI, and Patz EF Jr (2002) The role of imaging in malignant pleural mesothelioma. Seminars in Oncology 29: 26–35. Nowak AK, Lake RA, Kindler HL, and Robinson BW (2002) New approaches for mesothelioma – biologics, vaccines, gene therapy, and other novel agents. Seminars in Oncology 29: 82–96. Robinson BWS (2004) Soluble mesothelin-related protein – a sensitive new marker for mesothelioma. American Journal of Oncology Review 3: 230–233. Robinson BWS, Creancy J, Lake R, et al. (2003) Mesothelinfamily proteins and diagnosis of mesothelioma. Lancet 362: 1612–1616. Rusch VW (1995) A proposed new international TNM staging system for malignant pleural mesothelioma. From the International Mesothelioma Interest Group. Chest 108: 1122–1128. Rusch VW (1999) Indications for pneumonectomy. Extrapleural pneumonectomy. Chest Surgery Clinics of North America 9: 327–338. Scott B, Mukherjee S, Lake R, and Robinson BWS (2000) Malignant mesothelioma. In: Hanson H (ed.) Textbook of Lung Cancer, pp. 273–293. London: Martin Dunitz. Segal A, Whitaker D, Henderson D, and Shilkin K (2002) Pathology of mesothelioma. In: Robinson BWS and Chahinian AP (eds.) Pathology and Mesothelioma. Mesothelioma, vol. 8, pp. 143–184. London: Martin Dunitz. Wagner JC, Sleggs CA, and Marchand P (1960) Diffuse pleural mesothelioma. British Journal of Industrial Medicine 17: 260–271. Whitaker D and Shilkin KB (1984) Diagnosis of pleural malignant mesothelioma in life – a practical approach. Pathology 143: 147–175.

see Pleural Effusions: Malignant Pleural Effusions. Tumors, Malignant: Metastases from

Lung Cancer.

Microfibrils

see Extracellular Matrix: Elastin and Microfibrils.

MUCINS J A Voynow and B M Fischer, Duke University Medical Center, Durham, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Mucins are the major macromolecular components of mucus. Each mucin has a unique and characteristic sequence of tandemly repeating amino acids rich in serine and/or threonine.

The tandem repeat domain is the primary region for O-linked glycosylation of the molecule. There are at least 20 different mucin genes (MUC) classified into two families: secreted and membrane-associated. At least 12 MUC genes are expressed in the respiratory tract and at least six MUC glycoproteins have been detected in lung tissue. A variety of stimuli including bacteria, cytokines, proteases, and pollutants regulate MUC gene expression; this regulation occurs at both the transcriptional and posttranscriptional level and is mediated by several intracellular signaling cascades. The functions of mucins in the

MUCINS 57 healthy lung are currently being elucidated. Secreted mucins act as a shield protecting the airway epithelia. Owing to their structure and charge, mucins may influence hydration at the cell surface. Their complex oligosaccharide structures may function as ligands for cellular lectins; mucin binding permits mucociliary clearance of pathogens and prevents access to the cell membrane. Membrane-associated mucins may interact via epidermal growth factor-like domains or cytoplasmic tails with receptor tyrosine kinases, activating intracellular signaling, and regulating gene expression. In chronic inflammatory airway diseases, overproduction and hypersecretion of mucin glycoproteins contribute to airway obstruction. In several cancers, aberrant expression and glycosylation of mucins contribute to tumor survival and proliferation. Thus, mucin structures play a critical role in determining their functions in health and disease.

components of mucus. They are large, heavily O-glycosylated proteins. Mucins are defined by a central protein domain of tandemly repeating sequences containing serine and/or threonine and proline and, typically, glycine and alanine. The tandem repeats are unique in sequence and size for each mucin (MUC) protein. The tandem repeat domain is the primary site for mucin O-linked glycosylation (Figure 1), which encompasses over 50% of the mass of the mucin glycoprotein. The following discussion will focus on mucin glycoproteins expressed in the respiratory tract, specifically on regulation of the MUC genes and expression of MUC mucin glycoproteins in health and disease.

Introduction The lumen of the upper airways is coated with mucus. Normal airway mucus is the first line of defense between the lung and the environment. It has many important functions required for protecting the lung: hydrating epithelial surfaces, binding and clearing bacteria, detoxifying and removing particulates, and protecting against oxidants and proteases. Bacteria and particulates are cleared by the mucociliary apparatus, a coordinated escalator of beating cilia on the apical surface of the epithelium that moves mucus upwards to the oropharynx where it can be swallowed. The reader is referred to Mucus for more information on this topic. Mucus is composed of water, ions, mucin glycoproteins, proteoglycans, proteins, including lysozyme, lactoferrin, defensins, serine leukoprotease inhibitor, glutathione, secretory component, serum proteins and immunoglobulins, and phospholipids. Mucin glycoproteins are the major macromolecular

Signal NH2 D domains

Mucin Genes and Gene Products There are currently 20 human MUC genes. Each MUC gene encodes a tandemly repeating amino acid sequence that is unique for each MUC protein. There are two main classes of mucins: secreted and membrane associated. The secreted mucins include large gel-forming mucins that oligomerize and are produced in the mucous cells of submucosal glands (MUC5B), or goblet cells (MUC2, MUC5AC), or serous cells of the submucosal glands (MUC7). In contrast, the membrane-associated mucins are associated with the cell surface and are expressed on superficial ciliated cells (MUC1, MUC4). The mucins in each class have similar domain structures. For example, the gel-forming mucins contain N- and C-terminal domains that have significant homology to Von Willebrand factor D domains (Figure 1); these function as sites for disulfide bonding and mucin oligomerization. Membrane-tethered mucins contain

Secreted COOH Tandem repeats

Membrane tethered

Cys-rich

Cytoplasmic tail

NH2

COOH Tandem repeats

EGF-like domains

TM

Figure 1 Major features of secreted and membrane-tethered mucins. Both families of mucins contain a tandem repeat domain(s) (green vertical stripes), which is the largest domain of the molecule and the primary region for O-linked glycosylation. Both families of mucins also have a signal sequence (dark blue) at the N-terminus. The secreted mucins have unique, nonrepeating amino acid domains in the N- and C-termini. The D domains contain cysteine (light blue) and have homology to von Willebrand factor D domains. There are other cysteine-rich domains in MUC2, MUC5AC, and MUC5B. In particular, one cysteine-rich domain, the cysteine knot (orange), is the site for mucin dimerization during posttranslational processing. The membrane-tethered mucins have a transmembrane domain (TM, gray) and a short cytoplasmic tail (cytoplasmic tail, black). Most membrane-tethered mucins (except MUC1) have two or three epidermal growth factor domains (EGF-like, pink) near the transmembrane domain.

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a transmembrane domain near the carboxyl end followed by a short cytoplasmic domain. All of the membrane-associated mucins except MUC1 also have epidermal growth factor (EGF)-like domains near their C-terminus, which may function to activate epidermal growth factor or other members of the ErbB family of receptor tyrosine kinases. In addition, MUC4 and MUC15 encode alternatively spliced transcripts that lack the transmembrane domain, yielding forms of the glycoproteins that are secreted. Several MUC genes are clustered on specific chromosomes suggesting they derive from a common ancestral gene; MUC2, MUC5AC, MUC5B, and MUC6 are clustered on chromosome 11p15, while MUC3, MUC11, MUC12, and MUC17 are clustered on chromosome 7q22. A summary of the different mucin genes, their major tissue localization, and their chromosomal location are listed in Table 1. At least 12 MUC genes (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7, MUC8, MUC11, MUC13, MUC15, MUC19, and MUC20) are expressed as mRNA in normal respiratory tract tissues. In addition, some of the other mucins, such as MUC6, may be expressed in lung cancers. The expression of these genes varies during fetal development (Table 2). MUC1, MUC2, and MUC4 are present during early branching morphogenesis; MUC5AC, MUC5B, and MUC7 appear at later stages during epithelial cytodifferentiation.

Mucin Gene Regulation A common pathologic feature of chronic inflammatory airway diseases is goblet cell hyperplasia and increased airway mucus production, especially during exacerbations of disease. There has been intense investigation to determine whether infectious or inflammatory mediators regulate expression of MUC genes and increase mucin glycoprotein production. From in vitro studies using cancer cell lines and cultured primary respiratory epithelial cells, a model of MUC gene regulation in response to bacterial products and cytokines has emerged (Figure 2). Bacterial exoproducts (e.g., lipopolysaccharide or leipoteichoic acid), cytokines (e.g., TNF-a, IL-1b, IL-6/IL-17), and reactive oxygen species activate specific cell surface receptors, which ultimately lead to activation of the downstream mitogen-activated protein-kinase (MAPK) signaling cascades. Ultimately, signaling pathways lead to increased transcription of MUC2 and MUC5AC via activation of nuclear transcription factors including nuclear factor kappa B (NF-kB) or Sp1. EGFR activation, by ligand-binding or by transactivation, also upregulates transcription of MUC5AC by activating downstream MAPK signaling. Interestingly, there are redundant intracellular signaling pathways that transcriptionally upregulate MUC5AC. For example, tobacco smoke activates both EGFR-dependent and

Table 1 Human mucin genes Gene

Major tissue localization

Respiratory tract

Chromosome

Secreted mucins MUC2 a MUC5AC a MUC5B a MUC6 a MUC7 a MUC8 MUC9 MUC19

Jejunum, ileum, colon, conjunctiva Airways, stomach, conjunctiva Airways, salivary, gall bladder Stomach, biliary, ileum Salivary, conjunctiva Airways, reproductive tract Oviduct Glandular tissues: sublingual, submaxillary, and tracheal submucosal glands

Yes Yes Yes 7 Yes Yes ? Yes

11p15.5 11p15.5 11p15.5 11p15.5 4 12q24.3 1p13 12

Yes 7 Yes Yes No Yes Yes ? No Yes

1q21–24 7q22 3q29 7q22 7q22 3q13.3 11p14.3 19q13.3 7q22 3q29

Membrane-tethered mucins MUC1 a Airways, pancreas, breast, conjunctiva MUC3A/3B Colon MUC4 a,b Airways, colon, female reproductive tract, mammary MUC11 a Colon MUC12 Colon MUC13 a Colon, trachea, kidney, duodenum, conjunctiva Colon, ovary, testis, breast, conjunctiva, lung MUC15 a,b MUC16 c Cancers of the ovary, breast, stomach, CNS; conjunctiva MUC17 Pancreas, colon, duodenum, stomach, conjunctiva MUC20 a Kidney, colon a

Complete cDNA sequences reported. May also have secreted forms of the protein. c Human MUC16 is also known as CA125, which is an ovarian cancer tumor serum antigen. Note: mouse MUC10 is the mouse homolog for human MUC7. There is no human homolog to the mouse MUC14. Human MUC18, also known as CD 146, is an adhesion molecule part of the immunoglobulin gene superfamily. b

MUCINS 59 Table 2 Airway MUC-mRNA expression during human lung developmenta MUC

Age (weeks)

Localization of expression

10

13

17

23

Adult

MUC1 MUC2 MUC4 MUC5AC MUC5B

þ þþ þ – –

þ þ þþ – þ

þ þþ þþþ þþ þ

þ þþ þþþ þ þþþ

þ þ þ þ þ

MUC7

–

–

–

þþþ

þþþ

þ þþ þþ þþ

Surface epithelium Surface epithelium; glands of adult but at lower level Surface epithelium Surface epithelium In goblet cells early in development and predominantly in the glands after 23 weeks Glands

a

Note: to date, there have been no reports on the expression of other mucin genes during development. MUC8 and MUC19 are expressed in the submucosal glands of adults. Bacteria/ endotoxin

Lymphocytes Neutrophil

Retinoic acid

IL-6/IL-17 TNF-


Elastase oxidants

PAF IL-1 IL-8 IL-4 IL-9 IL-13

Ozone, ROFA Acrolein EGFR activation

AEC

MUC5AC MUC2

MUC5AC MUC4

MUC5B

MUC5AC

Figure 2 Regulation of MUC expression in airway epithelial cells (AEC). Respiratory tract epithelial cells (AEC, ovals) are exposed to inflammatory cytokines, proteases, oxidants, and bacterial products. Several studies reveal that these mediators increase expression of MUC2 and MUC5AC. Recently, it has been recognized that MUC4 and MUC5B expression are also regulated by inflammatory mediators in the airway. Th2 cytokine (e.g., IL-4, IL-13, and IL-9)-induced increase in MUC5AC expression has primarily been reported in rodent models of airway disease.

EGFR-independent signaling to increase MUC5AC transcription via AP-1. These studies demonstrate that transcriptional regulation of MUC genes may be activated by several intracellular signaling pathways. Importantly, MUC gene regulation also occurs at the posttranscriptional level. Neutrophil elastase regulates MUC5AC and MUC4 gene expression by a posttranscriptional mechanism. MUC2 is posttranscriptionally regulated by phorbol ester and forskolin. MUC4 is also posttranslationally regulated by transforming growth factor beta. These studies illustrate the important concept that mucin regulation occurs via multiple signaling pathways and at several levels of gene and protein regulation.

Mucin Glycoproteins Our current understanding of the translation and posttranslational processing of secreted mucins stems from studies on MUC2 and porcine submaxillary mucin. These secreted gel-forming mucins are

synthesized as nascent peptides and translocated into the endoplasmic reticulum (ER). In the ER, mucins are N-glycosylated, and form disulfide-bonded dimers via the cysteine knot domains in the C-termini. The mucin dimers are then transported to the Golgi apparatus for processing of O-linked glycosylation. In the trans-Golgi, disulfide-bonded mucin oligomers form bonds via the Von Willebrand D domains. It is also here that terminal glycosylation and further packaging of the mucins occur (Figure 1). In contrast to the secreted, gel-forming mucins, membrane-associated mucins, MUC1 and MUC4, undergo proteolytic cleavage in the ER at the juxtamembrane portion of the molecule destined for the extracellular domain. The two molecular halves remain covalently linked as they are translocated to the Golgi for posttranslational processing and targeted to the cell surface. Most mucin carbohydrate chains are joined to the mucin protein core through N-acetylgalactosamine (GalNAc) in an O-glycosidic linkage to the hydroxyl oxygen of serine or threonine, which is then elongated or branched by specific glycosyltransferases to form one of four major core GalNAc-Gal/GlcNAc structures. The cores are elongated by glycosyltransferases to form type 1 or type 2 backbone structures. Extending still further out from the core and backbone oligosaccharides are peripheral carbohydrate structures comprised of fucose, galactose (Gal), N-acetylglucosamine (GlcNAc), or sialic acid. These oligosaccharides frequently have specific epitopes recognized by lectins or antibodies (e.g., type 1 or 2 disaccharide, blood group A or B, or Sialyl Lewisx). Numerous glycosyltransferases are expressed in the lung; each enzyme catalyzes a specific linkage for each monosaccharide during the posttranslational processing of the mature glycosylated mucins. In addition, the oligosaccharide structures may be terminated by the addition of sulfate groups catalyzed by sulfotransferases. The reader is referred to the article by Lamblin and co-workers for a more extensive description and illustration of the length of the mature glycosylated mucins and the diversity of mucin

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glycosylation patterns. The mucin glycosylation patterns appear to be tissue- and disease-specific. This great variability of oligosaccharide structure suggests that subtle changes in carbohydrate structure of the same mucin protein backbone may have profound effects on mucin function. Evaluation of MUC-specific proteins is required for investigating mucin regulation and function in health and diseases. However, the size and complexity of mucin oligosaccharide structures precluded the development of MUC-specific antibodies until recently. Many of the original antimucin antibodies detected mucin oligosaccharide structures and served as ‘pan-mucin’ antibodies, but were not useful for specific mucin identification. Recent advances in molecular technology have allowed specific MUC genes and deduced amino acid sequences to be identified, and have permitted the development of MUC-specific antibodies. Identification of mucin glycoproteins is still technically challenging because the huge size of these glycoproteins limits the usefulness of routine Western gel electophoresis. Investigators have developed different types of gels (agarose versus acrylamide) for electrophoresis prior to Western blot analysis, and other laboratories have developed nongel electrophoresis methods for MUC quantitation (dot/slot blot, ELISA). These methods are still fraught with the challenge of appropriate standards for quantitation, and the presence of large, charged oligosaccharides obscuring the mucin protein backbone from antibody detection.

Role of Mucins in Normal Lung Function The molecular functions of airway mucins are not yet well characterized for the secreted, gel-forming mucins. They constitute the major macromolecular components of mucus and therefore are important in mucociliary clearance of particulates and pathogens. The mucin oligosaccharides bind bacterial lectins, which in part explain their role in pathogen clearance. There is more information available for the functions of two membrane-associated mucins, MUC1 and MUC4 (Figure 3). For further details on membrane-associated mucins in cancer, please see the review by Hollingsworth and Swanson. The MUC1 protein backbone is a receptor for the bacterium Pseudomonas aeruginosa. Binding of the bacterial flagella to MUC1 stimulates tyrosine phosphorylation of the cytoplasmic domain of the mucin, leading to activation of the extracellular signal-regulated protein kinase (ERK) MAP kinase cascade. In breast carcinoma cells, MUC1 also activates EGFR and interacts directly with b-catenin. MUC1 indirectly has an antioxidant function as it regulates the

FOXOA3 transcription factor resulting in reduced intracellular oxidant stress and reduced apoptosis. MUC4 has EGF-like domains, which in cancer cell lines serve as a ligand(s) for ErbB2, a member of the ErbB receptor tyrosine kinase family. MUC4 may promote tumor growth, suppress tumor cell apoptosis, and shield tumor cells from immune detection. Both MUC1 and MUC4 have been reported to interfere with adenovirus-mediated gene therapy in the lung, suggesting that the glycosylated domains function to reduce cell–cell interactions.

Mucins in Disease Several chronic inflammatory airway diseases have mucus overproduction as a key component to their pathogenesis, including cystic fibrosis (CF), chronic bronchitis, and asthma. Goblet cell and submucosal gland hyperplasia are major pathologic features of these diseases, probably contributing to mucus overproduction and airway obstruction; the reader is referred to excellent reviews on in vivo models of secretory cell metaplasia relevant to these diseases. However, in addition to secretory cell metaplasia, there may be altered mucin expression and/or glycosylation in these diseases. Information is very limited on the identity, quantitation, and glycosylation of mucins in normal and diseased airways. In asthma, Th2 cytokines are key mediators regulating airway secretory cell remodeling. However, there is some controversy on the role of Th2 cytokines in upregulating MUC gene expression in human airway cells in vitro. A few studies demonstrate that in asthmatic airway secretions, MUC5AC and MUC5B are predominant secreted mucin glycoproteins and expression of MUC1, MUC2, and MUC4 mRNA are also increased. There is limited information concerning the identity and quantitation of mucin glycoproteins present in airway secretions of chronic bronchitis patients. However, both, in vitro studies using human airway epithelial cells and in vivo studies using rodent models, demonstrate that tobacco smoke, air pollutants, reactive oxygen species, and neutrophil elastase mediate secretory cell metaplasia and increase expression of MUC5AC. CF is characterized by mucus obstruction of bronchioles to bronchi associated with chronic bronchitis, neutrophil-dominant inflammation, and progressive bronchiectasis and parenchymal scarring leading to respiratory failure. Although CF is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the impact of abnormal CFTR protein on the pathogenesis of mucus obstruction is still highly controversial. Several

MUCINS 61 Secreted mucin oligomers

Membraneassociated mucin

Receptor tyrosine kinase

Intracellular signaling

Figure 3 Mucin functions in epithelial cells. Secreted mucins oligomerize and form a gel covering the cell surface. Mucin oligosaccharides (linear branched structures) are recognized by bacteria (green ovals); they bind to the bacteria and are removed from the airway by mucociliary clearance. Membrane-associated mucins (dark blue) interact with cell surface receptors (pink) and activate intracellular signaling, often via phosphorylated residues (yellow circles) in the cytoplasmic domain.

current theories of the primary pathogenesis include the following: (1) reduction in the sol layer causing failure of mucus clearance by the cilia; (2) altered glycosylation (increased sulfate and fucose, and decreased sialic acid) of CF mucins and CF epithelial cell-surface glycoproteins resulting in altered bacterial binding; (3) aberrant regulation of secretion causing impaired innate immunity and mucin obstruction of glands; and (4) increased mucus viscosity due to increased DNA, lipids, and proteoglycans, altering the normal relative amounts of mucin glycoprotein to other pathologic mucus constituents. In lung cancers, there is limited information on MUC expression. In general, MUC1 glycoprotein is expressed in most squamous cell carcinomas, adenocarcinomas, and small cell carcinomas of the lung, while MUC2, MUC4, MUC5AC, and MUC8 glycoproteins are not consistently expressed, with as few as 10% of the tumors tested expressing MUC5AC. Approximately 17% of the squamous cell carcinomas express MUC6, which is not normally expressed in the airways.

Summary In the last 15 years, there has been tremendous progress in our understanding of mucin molecular structure, regulation, and protein expression. With

the tools that have recently been developed to evaluate MUC gene and MUC protein expression, we are poised to carry out translational studies to determine the function of specific respiratory tract mucins in health and disease. See also: Asthma: Overview. Bronchiectasis. Bronchiolitis. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Mucus.

Further Reading Adler KB and Li Y (2001) Airway epithelium and mucus: intracellular signaling pathways for gene expression and secretion. American Journal of Respiratory Cell and Molecular Biology 25(4): 397–400. Asker N, Axelsson MA, Olofsson SO, and Hansson GC (1998) Dimerization of the human MUC2 mucin in the endoplasmic reticulum is followed by a N-glycosylation-dependent transfer of the mono- and dimers to the Golgi apparatus. Journal of Biological Chemistry 273: 18857–18863. Basbaum C, Lemjabbar H, Longphre M, et al. (1999) Control of mucin transcription by diverse injury-induced signaling pathways. American Journal of Respiratory and Critical Care Medicine 160: S44–S48. Beum PV and Cheng PW (2001) Biosynthesis and function of beta 1,6 branched mucin-type glycans. Advances in Experimental Medicine and Biology 491: 279–312. Buisine MP, Devisme L, Copin M-C, et al. (1999) Developmental mucin gene expression in the human respiratory tract. American Journal of Respiratory Cell and Molecular Biology 20: 209–218.

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Carraway KL, Perez A, Idris N, et al. (2002) Carraway. Muc4/ sialomucin complex, the intramembrane ErbB2 ligand, in cancer and epithelia: to protect and to survive. Progress in Nucleic Acid Research and Molecular Biology 71: 149–185. Chen Y, Thai P, Zhao Y-H, et al. (2003) Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop. Journal of Biological Chemistry 278: 17036–17043. Hollingsworth MA and Swanson BJ (2004) Mucins in cancer: protection and control of the cell surface. Nature Reviews: Cancer 4: 45–60. Kips JC, Anderson GP, Fredberg JJ, et al. (2003) Murine models of asthma. European Respiratory Journal 22(2): 374–382. Kirkham S, Sheehan JK, Knight D, Richardson PS, and Thornton DJ (2002) Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B. Biochemical Journal 361: 537–546. Lamblin G, Degroote S, Perini J-M, et al. (2001) Human airway mucin glycosylation: a combinatory of carbohydrate determinants which vary in cystic fibrosis. Glycoconjugate Journal 18: 661–684. Leikauf GD, Borchers MT, Prows DR, and Simpson LG (2002) Mucin apoprotein expression in COPD. Chest 121: 166S–182S. Lillehoj EP, Kim BT, and Kim KC (2002) Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin.

American Journal of Physiology. Lung Cellular and Molecular Physiology 282(4): L751–L756. Perez-Vilar J and Hill RL (1999) The structure and assembly of secreted mucins. Journal of Biological Chemistry 274(45): 31751–31754. Reid CJ, Gould S, and Harris A (1997) Developmental expression of mucin genes in the human respiratory tract. American Journal of Respiratory Cell and Molecular Biology 17(5): 592–598. Rose MC and Gendler SJ (1997) Airway mucin genes and gene products. In: Rogers DF and Lethem MI (eds.) Airway Mucus: Basic Mechanisms and Clinical Perspectives, pp. 41–66. Basel, Switzerland: Birkhauser Verlag. Rose MC, Nikola TJ, and Voynow JA (2001) Airway mucus obstruction: mucin glycoproteins, MUC gene regulation and goblet cell hyperplasia. American Journal of Respiratory Cell and Molecular Biology 25: 533–537. Rose MC and Voynow JA. Mucin glycoproteins in lung health and disease. Physiological Reviews (in press). Takeyama K, Dabbagh K, Lee H-M, et al. (1999) Epidermal growth factor system regulates mucin production in airways. Proceedings of the National Academy of Sciences, USA 96: 3081–3086. Voynow JA (2002) What does mucin have to do with lung disease? Paediatric Respiratory Reviews 3(2): 98–103.

MUCUS M C Rose, Children’s National Medical Center, Washington, DC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Mucus can be regarded as a multifunctional compartment that contributes to the innate defense system, thereby protecting the airways against pathogens and environmental toxins. The identification of specific mucosal components in each compartment and their functional role in the biophysical and biological properties of airway mucus are being investigated by numerous laboratories. Future studies on airway mucus will result in a better understanding of the molecular pathways and processes required for airway health and defense. This in turn should facilitate circumvention or amelioration of mucus hypersecretion and overproduction in specific airway diseases, thereby reducing the morbidity associated with these diseases, which are becoming more of a major health issue in industrialized countries.

Introduction The apical surface of the epithelium in the mammalian respiratory tract, like that of the gastrointestinal and reproductive tracts, is coated by mucus. This is a heterogeneous mixture of water, ions, and secreted molecules and macromolecules. These include mucin glycoproteins (characterized by O-glycans), glycoproteins (typically containing N-glycans), proteins,

proteoglycans (containing glycosaminoglycans), and lipids. Mucus coats the lumen of the upper (nose and sinuses) and the lower (trachea, bronchi, bronchioles) respiratory tract and hydrates the apical surfaces of the epithelium in the conducting airways. By its location and heterogeneous nature, mucus provides a protective barrier against pathogens and toxins, thereby serving as a first line of defense against the environment and contributing to the innate host defense system. Mucus protects the respiratory tract to a large extent by clearing pathogens and particulates via the mucociliary apparatus (see Cilia and Mucociliary Clearance). Additionally, mucosal components protect the epithelium by binding pathogens. The molecular details of such interactions are not yet well understood, but predictably, as the identity of specific components of mucus continue to be identified, these will be elucidated. Furthermore, mucosal components detoxify and remove particulates and protect against oxidants and proteases.

Airway Mucus Components In the older literature, the term ‘mucus’ is considered to encompass the respiratory tract secretions in the soluble (sol) and the viscoelastic mucosal gel layers.

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The current paradigm is that respiratory tract secretions, for example, airway surface liquid (ASL), comprise multifunctional compartments including a periciliary liquid layer and a mucosal gel layer (Figure 1). Surfactant components, which originate from the alveolar epithelium, can also contribute to the ASL (see Surfactant: Overview). The term airway mucus is used throughout this review to include all components in the ASL. Some of these components will likely prove to be in dynamic equilibrium between the compartments. These components are present in airway mucus of healthy individuals, as well as in the expectorated mucus produced by patients with chronic lung diseases like asthma, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD), including bronchitis. Expectorated mucus from patients with lung disease is typically referred to as sputum and contains mucus, as well as bacteria, cell debris, products released by inflammatory cells (including high-molecular-weight DNA, filamentous actin, and proteases), immunoglobulins, and serum protein transudates. The macromolecular components of airway mucus are typically secreted by secretory cells, for example, goblet cells in the surface epithelium and mucosal and serosal cells in the submucosal glands.

The components of the goblet/mucosal cells and of the serosal cells are briefly overviewed in the following paragraphs. Mucin glycoproteins (mucins) are major components of both the soluble and viscoelastic mucosal gel in airway mucus from healthy airways and sputum from patients with chronic airway diseases. For this reason alone, mucins have long been implicated in health and disease. Mucins are synthesized and stored in secretory cell granules until secretion is initiated by secretagogues. Specific mucins, identified by the cDNA sequences that encode MUC protein backbones, have now been identified. Of the 18 human MUC genes now recognized, at least 12 mucins are expressed at the mRNA level in lung epithelial cells or tissues. Many of these encode membrane-tethered mucins, which are localized to the apical surfaces of epithelial cells and function as receptors that protect epithelial cells by activating cellular signaling pathways. The predominant secretory mucins are MUC5AC and MUC5B, both of which may be overexpressed in asthmatic airway secretions and underexpressed in CF airway secretions. Secretory mucins may entrap particles in the viscoelastic mucus gel, acting perhaps in conjunction with tethered mucins at the apical surface of epithelial cells, or bind receptors on Mucosal gel layer

Airway surface liquid

Periciliary liquid layer

Ciliated cell

Goblet cell

Mucus cell Serosal cell Mucin glycoproteins Figure 1 Schematic of airway mucus. Airway mucus or airway surface liquid, has two compartments – the mucosal gel layer on top of the cilia and the sol or periciliary liquid layer. Goblet cells in the surface epithelium and serousal and mucosal cells in the submucosal glands biosynthesize, secrete, and store mucin glycoproteins and other mucosal components in their secretory granules. When secreted, the mucosal components integrate into specific compartments in the airway surface liquid, where perhaps some components are in dynamic equilibrium between the compartments. The components in airway mucus are poised to provide innate defense properties to the airways.

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inflammatory cells using motifs in specific mucin protein domains or in O-glycans (see Mucins on mucin glycoproteins for additional information and to listed references). Serous cells in the submucosal glands secrete lungspecific proteins in airway secretions/mucus, which have long been regarded as innate defense molecules that play a role in mucosal defense. These include lysozyme (Lysozyme), lactoferrin, lactoperoxidase, beta-defensins (Defensins), serine leukoprotease inhibitor, trefoil factors, and glutathione. Additionaly, hyaluron, a proteoglycan, is secreted by glandular cells and is thought to provide an extracellular reservoir at the airway epithelial apical surface through which enzymes (such as kallikrein or lactoperoxidase) are poised to respond as innate defense molecules.

Sample Procurement for Analysis of Mucosal Components Identification and quantitation of components in airway mucus have proven challenging because of the inherent physical properties of lung mucus and because healthy individuals produce much smaller amounts of ASL, which are not easily obtainable. Mucus samples can be obtained by endotracheal suctioning of patients without airway diseases who are undergoing surgical procedures. Samples from volunteers can be obtained following a standardized protocol of hypertonic saline exposure, which may increase the osmolarity of the ASL. Analyses have also been carried out using bronchoalveolar lavage (BAL) samples from control patients or patients with airway diseases. BAL samples are diluted by saline, enriched in alveolar and inflammatory cells washed out of the conducting airways, and low in mucosal components. Macromolecules in the mucosal gel may not be washed out by saline lavage. On the other hand, patients with chronic airway diseases expectorate copious amounts of sputum, which is why protocols for isolation and characterization of airway secretions have traditionally utilized this material. This inherent inequality of samples, (e.g., mucus and sputum) complicates comparative studies of components in airway secretions. Thus, reliable quantitation and comparison of components in mucus samples from healthy airways and sputum samples from diseased airways remains a challenging problem. This is encumbered by difficulties not only in procuring, but also in processing samples, as overviewed below. Mucus and sputum samples typically require the use of a dissociating solvent (sodium dodecyl sulfate, guanidinium hydrochloride, or urea) to solubilize

and a reducing agent (b-mercaptoethanol or dithiothreitol) to separate the protein components that associate/interact with mucins. Additionally, identification of mucins of known MUC protein backbones by MUC-specific antibodies can be blocked by O-glycosides. Nevertheless, determining the levels and identity of components in airway epithelial secretions is a crucially important step for investigating the functional roles of specific molecules in airway homeostasis, injury, and repair.

Biophysical/Biological Properties of Mucus Mucus is a viscoelastic gel that couples to the mucociliary escalator. Mucins are the macromolecular components primarily responsible for the biophysical, for example, viscoelastic and aggregate, properties of mucus. However, it is likely that mucosal components interact with specific mucins through inter- and intramolecular interactions. The molecular basis of mucin viscoelasticiy is thought to reside in the cysteine-rich domains of secretory mucins. Nevertheless, mucin carbohydrate components, especially the terminators of mucin O-glycosides (e.g., sialic acid and sulfate, which impart anionic properties, and fucose, which imparts hydrophobic properties), are also likely to contribute to the biophysical properties of airway mucins and thus airway mucus. Additionally, the O-glycoside structures of mucins are likely to be major determinants of the biological properties of mucins and impact on their binding to bacteria, viruses, and cells in the airway fluid. Future studies should reveal the molecular details wherein mucins of known MUC protein backbone and O-glycoside structures contribute to the biochemical and physical properties of mucus.

Mucus and Airway Diseases Mucus and mucins are overproduced in the airways of patients with chronic airway diseases, which greatly contribute to airway obstruction in patients with asthma, CF, or COPD. These diseases are characterized by inflammatory and immune response mediators, which are released into airway tissue and airway fluid/secretions. These mediators in turn sustain airway mucin overproduction, which ultimately contribute to airway obstruction and therefore to the high morbidity and mortality associated with these diseases. Investigations over the last decade have tested and validated the hypothesis that inflammatory and immune response mediators regulate mucin gene expression in vitro in airway epithelial cells,

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thereby directly linking infection and inflammation to the copious amounts of mucus produced in chronic airway diseases. An increased number of goblet cells, for example, goblet cell hyperplasia, occurs in patients with mild, moderate, or acute asthma and thus contributes to the overproduction of airway mucin glycoproteins and mucus in asthmatic patients. Patients who die in ‘status asthmaticus’ often have mucus plugs in their airways. MUC5B mucin has been identified in one mucus plug, but additional studies to define the mucin glycoproteins that are overproduced consequent to the inflammatory and immune response genes activated during asthmatic exacerbations are warranted. Other mucosal components may also be overproduced in asthma. For example, surfactant protein D has recently been localized to hyperplastic rat goblet cells following development of allergic asthma. Studies on the role of airway mucus in the recessive genetic disease CF are leading to a re-evaluation of the components and compartments of mucus. A current paradigm is based on demonstration of a saltinduced decrease in the volume of the periciliary layer in differentiated airway cells grown in culture, for example, human bronchial epithelial (HBE) cells, from patients with CF compared to volume levels in control HBE cells. The decreased volume appears to alter hydration of the mucosal gel and thus the rheological properties of CF airway mucus, which in turn is thought to increase the susceptibility of CF airways to bacterial infection. An emerging concept is that membrane-tethered mucins, which have been shown to be expressed at the mRNA level in ciliated and basal cells as well as in secretory cells, are expressed at the protein level on the apical surfaces of cells in the conducting airways. Thus, membrane-tethered mucins contribute to the functioning of the periciliary layer, thereby impacting on mucociliary mechanisms and ultimately on the innate defense system.

Clinical Approaches for Treating Airway Mucus Overproduction Mucolytics are pharmacological agents for thinning or dispersing sputum in the airways of patients with obstructive lung diseases. They include agents like N-acetyl cysteine, which contain free sulfhydryl groups that reduce disulfide bridges of mucins and/or mucosal components. More recently, other pharmacological agents like Pulmozyme (human recombinant DNase I), which cleaves DNA in sputum and reduces sputum viscosity and adhesitivity, and proteins like gelsolin or thymosin b4, which depolymerize F-actin filaments in sputum, have become available.

Summary Future studies to identify, quantify, and functionally characterize specific mucosal components will lead to a better understanding of the molecular pathways and processes that lead to mucus hypersecretion and overproduction in specific airway diseases. This, in turn, should facilitate circumvention or amelioration of these conditions, thereby reducing the morbidity associated with mucus-producing chronic airway diseases, which are an increasing major health issue in industrialized countries. See also: Bronchoalveolar Lavage. Cilia and Mucociliary Clearance. Defensins. Lysozyme. Mucins. Surfactant: Overview.

Further Reading Adler KB and Li Y (2001) Airway epithelium and mucus: intracellular signaling pathways for gene expression and secretion. American Journal of Respiratory Cell and Molecular Biology 25: 397–400. Barnes PJ (2002) Current and future therapies for airway mucus hypersecretion. Novartis Foundation Symposium 248: 237–249. Basbaum C and Finkbeiner W (1989) Mucus-producing cells of the airways. In: Massaro D (ed.) Lung Cell Biology, pp. 37–80. New York: Dekker. Basbaum C, Lemjabbar H, Longphre M, et al. (1999) Control of mucin transcription by diverse injury-induced signaling pathways. American Journal of Respiratory and Critical Care Medicine 160: 544–548. Boat TF, Cheng PW, Iyer RN, Carlsen DM, and Polony I (1976) Human respiratory tract secretions: mucus glycoproteins of nonpurulent tracheobronchial secretions, and sputum of patients with bronchitis and cystic fibrosis. Archives of Biochemistry and Biophysics 177: 95–104. Boucher RC (2004) New concepts of the pathogenesis of cystic fibrosis lung disease. European Respiratory Journal 23: 146–158. Casalino-Matsuda SM, Conner GE, Salathe M, and Forteza RM (2004) Role of hyaluronan and reactive oxygen species in tissue kallikrein-mediated epidermal growth factor receptor activation in human airways. Journal of Biological Chemistry 279: 21606–21616. Hollingsworth MA and Swanson BJ (2004) Mucins in cancer: protection and control of the cell surface. Nature Reviews Cancer 4: 45–60. Kirkham S, Sheehan JK, Knight D, Richardson PS, and Thornton DJ (2002) Heterogeneity of airway mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B. Biochemical Journal 361: 537–546. Knowles MR and Boucher RC (2002) Mucus clearance as a primary innate defense mechanism for mammalian airways. Journal of Clinical Investigation 109: 871–877. Rogers DF (2004) Airway mucus hypersecretion in asthma: an undervalued pathology? Current Opinion in Pharmacology 4: 241–250. Rose MC (1989) Characterization of human tracheobronchial mucin glycoproteins. In: Ginsburg V (ed.) Methods in Enzymology, vol. 179, pp. 3–17. New York: Academic Press.

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Rose MC, Lynn WS, and Kaufman B (1979) Resolution of the major components of human lung mucosal gel and their capabilities for reaggregation and gel formation. Biochemistry 18: 4030–4037. Rose MC and Voynow JA (2006) Respiratory tract mucin genes and mucin glycoproteins in health and disease. Physiological Reviews 86: 245–278. Rubin BK, Ramirez O, Zayas JG, Finegan B, and King M (1990) Collection and analysis of respiratory mucus from subjects without lung disease. American Review of Respiratory Disease 141: 1040–1043.

Muscle

Salathe M, Forteza R, and Conner GE (2002) Post-secretory fate of host defense components in mucus. Novartis Foundation Symposium 248: 20–26. Thomas PS, Yates DH, and Barnes PJ (1999) Sputum induction as a method of analyzing pulmonary cells: reproducibility and acceptability. Journal of Asthma 36: 335–341. Vestbo J, Prescott E, Lange P, and The Copenhagen City Heart Study Group (1996) Association of chronic mucus hypersecretion with FEV1 decline and chronic obstructive pulmonary disease morbidity. American Journal of Respiratory and Critical Care Medicine 153: 1530–1535.

see Neurophysiology: Neural Control of Airway Smooth Muscle. Respiratory Muscles, Chest Wall,

Diaphragm, and Other. Smooth Muscle Cells: Airway; Vascular.

Mycobacteria

see Pneumonia : Mycobacterial.

MYOFIBROBLASTS G Gabbiani, University of Geneva, Geneva, Switzerland A Orlandi, Tor Vergata University of Rome, Rome, Italy & 2006 Elsevier Ltd. All rights reserved.

Abstract The concept that fibroblastic cells can modulate into myofibroblasts, and thus participate in the evolution of wound healing and fibrocontractive diseases, has been developed over the last 30 years, starting from the ultrastructural observation that granulation tissue fibroblasts exhibit contractile features. It is now well accepted that the myofibroblast plays a role in the regulation of tension within the pulmonary alveolar tissue in normal conditions, and in the accumulation of extracellular matrix, as well as in the production of tissue deformation with decreased functional activity of lung parenchyma, during pathological situations, such as interstitial lung disease. Myofibroblasts originate predominatly from local fibroblasts, but they can also be produced from other local mesenchymal cells such as pericytes or smooth muscle cells and, as recently discovered, from transdifferentiation of adjacent epithelial cells or from bone marrow-derived circulating precursors. Force generation by myofibroblasts depends on the formation of intracellular stress fibers. These organelles may contain cytoplasmic b- and g-actin isoforms (protomyofibroblast) or, in addition, a-smooth muscle actin, the actin isoform typical of vascular smooth muscle (differentiated myofibroblast). In this second case, tension production is maximal. Transforming growth factor b1 (TGF-b1) is the most powerful cytokine that stimulates myofibroblast differentiation. In order to act, TGF-b1 needs the presence of the ED-A splice variant of cellular fibronectin in the

extracellular space. Myofibroblasts are responsible for the production of long lasting isometric tension that in turn induces a helpful tissue retraction, for example, during wound healing (wound contraction) or pathological tissue deformation, for example, during fibrocontractive diseases. These changes are stabilized by the deposition of extracellular matrix components such as collagen. They play a key role in connective tissue remodeling during pathological situations such as asthma and idiopathic pulmonary fibrosis. The possibility of interfering with force production by myofibroblasts could represent a new approach in order to control fibrocontractive diseases. Presently this approach seems to be efficient in few experimental situations. Further work along these lines may result in the development of clinically applicable tools.

Introduction The myofibroblast was first described in electron micrographs of contracting granulation tissue during experimental wound healing. Soon thereafter, its biochemical, pharmacological, and immunohistochemical features were delineated. Since then the myofibroblast has been identified in normal tissues, particularly where there is a necessity for tension development, and in pathological tissues, in relation to hypertrophic scarring, fibromatoses, and fibrocontractive diseases, as well as in cases of stroma reaction to epithelial tumors. The observation that most myofibroblasts express a-smooth muscle actin (a-SMA), the actin isoform typical of vascular smooth muscle (SM) cells, has

MYOFIBROBLASTS

furnished a reliable marker of myofibroblast differentiation. When present in the myofibroblast, a-SMA localizes in stress fibers, organelles that were at first considered to be characteristic of cultured cells, but are also present in vivo, particularly during myofibroblast accumulation. Myofibroblasts can also express other proteins characteristic of SM cells, such as SM-myosin heavy chains, according to the pathological situation; however, to date, they have not been shown to express late SM cell differentiation markers, such as smoothelin. This allows one to distinguish between the two phenotypes. It has been assumed for many years that myofibroblasts derive exclusively from local fibroblasts. Although it appears at present that this remains the most common mechanism of myofibroblast differentiation, it has become increasingly apparent that their origin is multiple. Locally, SM cells and pericytes as well as specialized mesenchymal cells, such as perisinusoidal cells in the liver and mesangial cells in the kidney, have been shown to modulate into myofibroblasts. Epithelial–mesenchymal transition plays an important role in myofibroblast accumulation that takes place in kidney interstitial fibrosis, the source of myofibroblasts being tubular epithelial cells, and in dialysis-induced peritoneal fibrosis, the source of myofibroblasts being mesothelial cells. In the last few years it has also been demonstrated that myofibroblasts can derive from circulating precursors such as mononuclear cells and a new category of circulating cells called fibrocytes. Peripheral blood fibrocytes rapidly enter the site of injury at the same time as circulating inflammatory cells. It has been suggested that circulating fibrocytes represent an important source of myofibroblasts during healing of extensive burn wounds where it may be difficult for local fibroblasts to migrate from the wound edge. Similarly, a variable proportion of myofibroblasts present in liver and pulmonary fibrosis is bone marrow-derived. Bone marrow-derived myofibroblasts contribute to the stroma reaction, at least in experimental situations. Another location in which fibrocytes contribute to myofibroblast population is the bronchial submucosa during asthma development (see below). Thus, it appears that several cells are capable of modulation into myofibroblasts according to the physiological situation and the pathological condition.

Fibroblast–Myofibroblast Transition and Force Generation by Myofibroblasts The mechanisms of fibroblast–myofibroblast transition have been the object of intensive investigation. Transforming growth factor beta 1 (TGF-b1) is now accepted as the crucial factor in this transition since

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it stimulates both the synthesis of collagen type I and of a-SMA by fibroblastic cells. Connective tissue growth factor has also been proposed to play a role in myofibroblast differentiation. Normal tissues can contain myofibroblasts with stress fibers expressing only cytoplasmic b- and g-actin isoforms, for example, alveolar septa or with stress fibers expressing in addition a-SMA. The first category has been named protomyofibroblast and the second differentiated myofibroblast. It is now accepted that during the healing of an open wound and possibly during the development of fibrotic changes, fibroblast–myofibroblast transition begins with the appearance of protomyofibroblasts. This first transition has not been well explored yet, but probably depends on mechanical tension development within the affected tissue and the permanent feedback between intracellular and extracellular tension. Several recent studies have demonstrated that mechanical stress is also a prerequisite for the second step of myofibroblast development, that is, the appearance of the differentiated myofibroblast, which is hallmarked by the neo expression of a-SMA in stress fibers. However, this step requires in addition the action of chemical mediators among which TGF-b1 is the most important. Recently, thrombin and endothelin-1 have been demonstrated to induce myofibroblast differentiation, the latter either directly or in conjunction with epithelial cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can also stimulate the development of abundant granulation tissue rich in a-SMA-positive myofibroblasts upon experimental intratracheal application; this action is however indirect and could be mediated by TGF-b. It should be noted that the action of TGF-b in stimulating both collagen type I and a-SMA synthesis strictly depends on the presence of cellular fibronectin and in particular its splice variant ED-A within the extracellular matrix. Thus, myofibroblast differentiation is a complex process regulated by at least one cytokine and an extracellular matrix component, as well as the presence of mechanical tension (Figure 1). As discussed above, a-SMA is the most widely used marker of the myofibroblastic phenotype. However, whether this protein is instrumental in force production by this cell has been debated for a long time. Recently, it has been shown that there is a good correlation between a-SMA expression and the efficiency in producing deformations in the silicone substrate on which fibroblastic cells are cultured; moreover transfection of 3T3 fibroblasts with a-SMA complementary DNA (cDNA) results in increased contractility, which is significantly higher compared to that of the same fibroblasts transfected with the cDNA of a-skeletal or g-cytoplasmic actins. This

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Focal adhesion site Cortical cytoplasmic actins Cytoplasmic actins
-Smooth muscle actin Fibronectin ED-A fibronectin

Nucleus ?

Fibroblast

Mechanical tension

Differentiated myofibroblast

TGF-1 ED-A fibronectin Mechanical tension Proto-myofibroblast Figure 1 The two-stage model of myofibroblast differentiation. In vivo, fibroblasts might contain actin in their cortex but they neither show stress fibers nor do they form adhesion complexes with the extracellular matrix. Under mechanical stress, fibroblasts will differentiate into proto-myofibroblasts, which form cytoplasmic actin-containing stress fibers that terminate in fibronexus adhesion complexes. Proto-myofibroblasts also express and organize cellular fibronectin, including the ED-A splice variant, at the cell’s surface. Functionally, these cells can generate contractile force. Transforming growth factor-b1 (TGF-b1) increases the expression of ED-A fibronectin. Both factors, in the presence of mechanical stress, promote the modulation of proto-myofibroblasts into myofibroblasts, which are characterized by the de novo expression of a-SMA in more extensively developed stress fibers, and by large fibronexus adhesion complexes (in vivo) or supermature focal adhesion (in vitro). Functionally, differentiated myofibroblasts generate greater contractile force than proto-myofibroblasts, which is reflected by a higher organization of extracellular fibronectin in fibrils. Reproduced with permission from Nature Reviews Molecular Cell Biology, Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, and Brown AR (2002) Myofibroblasts and mechano-regulation of connective tissue remodeling. Nature Reviews: Molecular Cell Biology 3: 349–363, copyright 2002 Macmillan Magazines Ltd.

increase in contractility takes place in the absence of any other change in protein expression, in particular SM myosin heavy chain expression. Similar to what happens in SM cells, stress fiber contraction may be regulated by elevated levels of intracellular Ca2 þ leading to activation of myosin light chain kinase and phosphorylation of myosin light chains. However, experimental and clinical observations support the assumption that granulation tissue retraction, in contrast to rapid and reversible contraction of SM cells, is the result of a continuous isometric force exerted on the surrounding connective tissue. This retraction is stabilized by newly synthesized matrix components, such as collagen, and thus becomes irreversible. Importantly, the isometric tension produced by stress fibers is regulated by Rho/ Rho-kinase, which in its active form leads to longlasting tensile activity by the inhibition of myosin

phosphatase. Phosphatase inhibitors stimulate myofibroblast contraction in vitro in the absence of any of the contraction agonists. Conversely, increasing intracellular Ca2 þ with ionophore does not induce contraction, indicating that activation of myosin light chain kinase alone is not sufficient to produce myofibroblast development. The force generated in stress fibers is transmitted to the extracellular tissue at sites of cell–matrix adhesions. In vivo, myofibroblasts form a specialized adhesion complex, the fibronexus, which is characterized by co-alignment of intracellular actin bundles with extracellular fibronectin fibrils; these in turn are connected to collagen fibrils. In vitro, differentiated myofibroblasts communicate with the extracellular matrix through specialized ‘supermature’ focal adhesions, which contain high levels of vinculin, paxillin, and tensin and transmembrane

MYOFIBROBLASTS

integrins avb3 and a5b1. Thus, the retraction of the whole tissue depends on the complex interplay of intracellular, cell surface, and extracellular factors. During the healing of an open wound, when epithelial reconstruction is achieved, an important wave of apoptosis affecting small vessel cells (endothelial cells, pericytes) and myofibroblasts takes place in the underlying granulation tissue, leading to the transition towards scar tissue. The lack of apoptosis has been suggested as one of the possible mechanisms involved in the development of hypertrophic scars and possibly of other fibrotic changes. However, this possibility has not been thoroughly explored. The loss of mechanical stress has been suggested as a major signal for myofibroblast disappearance from granulation tissue. Myofibroblast apoptosis has been induced by releasing stress applied to granulation tissue by means of a fixed frame inhibiting wound contraction (splinted wounds) or in vitro by releasing fibroblast populated collagen gels attached to the plastic substrate.

Myofibroblast Functions in the Normal Lung Perhaps the most widely accepted example of a functional myofibroblast in normal tissue is that present in the pulmonary interstitium, the spaces between the airspace epithelium (from the main bronchi to the alveoli), the vascular endothelium, and the pleural mesothelium. The role of the pulmonary interstitium is to connect these cell layers, to carry out specific functions, and to serve as a scaffold for the different parenchymal components of the lungs. Myofibroblasts were first identified in the alveolar interstitium by means of their ultrastructural features; their composition and function have now also been characterized. They are located within the thick portion of the air– blood barrier and are not surrounded by a proper basement membrane. At the alveolar aspect of the interalveolar septum they are delimited by the alveolar basement membrane and on the capillary edge by the capillary basement membrane. Their main feature is the presence of intracytoplasmic stress fibers that have been shown to contain cytoplasmic b- and g-actin isoforms, thus defining these cells as protomyofibroblasts. Given these features, it is not surprising that during pathological situations alveolar myofibroblasts rapidly modulate into differentiated myofibroblasts and contribute to the onset of fibrotic lesions. Morphometric studies have indicated that alveolar myofibroblasts make up around 42% of total interstitial volume of alveolar septa and represent approximately 18% of septal cell volume; hence, they are sufficiently

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numerous to imply a role in V/Q regulation as well as in the remodeling taking place during lung development. The cytoplasmic processes of alveolar myofibroblasts extend into the thick portion of the air–blood barrier adjacent and parallel to collagen fibers. They are disposed in a sinusoidal fashion and form a network along the capillary bed. Hence, when they contract, the length of the fiber lattice within the alveolar septum is reduced and the alveolus shrinks. The contractility of alveolar tissue has been demonstrated using lung strips that retract in vitro following exposition to different stimuli, such as hypoxia or epinephrine. This phenomenon is due largely to the contraction of myofibroblasts. It should be noted that myofibroblasts in different locations do not necessarily contract upon application of the same stimuli, thus supporting the notion of myofibroblast heterogeneity.

Myofibroblasts in Respiratory Diseases Myofibroblasts in Asthma

Asthma is characterized by functional and structural alterations of the bronchial epithelium, chronic airway inflammation, and remodeling of the bronchial structure. Recently, the work of several laboratories has indicated that during the evolution of this disease a prominent feature is the accumulation of a-SMA-positive myofibroblasts below the epithelial basement membrane, accompanying subepithelial fibrosis. It has been suggested that the bronchial remodeling process plays a major role in the irreversible airflow obstruction and permanently impaired pulmonary function observed in patients with chronic asthma. Since myofibroblast participation in bronchial remodeling during asthma evolution appears likely, altering the activities of this cell has been suggested as a new approach in the development of anti-asthma drugs. In allergic asthma, tissue alterations may be due to an increased susceptibility of the bronchial epithelium to allergen-induced injury or to ineffective repair mechanisms during chronic allergen exposure. The connective tissue response is essentially similar to that observed in pathological scarring: the accumulation of myofibroblasts following epithelial lesions is not followed by apoptosis when re-epithelialization is completed. This failure to resolve the inflammatory and structural changes leads to excessive extracellular matrix accumulation and tissue deformation. The origin of submucosal myofibroblasts in asthma has been the object of numerous investigations. It appears now well accepted that, in addition to a local origin, a proportion of such cells derive from

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MYOFIBROBLASTS

circulating fibrocytes. Thus, the control of factors regulating the extravasation of fibrocytes and the differentiation of myofibroblasts in the bronchial submucosa may represent a new way of controlling the evolution of asthmatic disease. Myofibroblasts in Interstitial Lung Disease

Lung fibrosis is the common final result of various diseases caused by different noxae, including toxic, autoimmune, drug-induced, infectious, and traumatic damage. Independently of the cause, resolution is more likely when inflammatory processes predominate. At the opposite end of the spectrum of pulmonary tissue responses to injury there is chronic interstitial lung disease, including idiopathic pulmonary fibrosis (IPF) in which myofibroblasts play a crucial role. IPF is a progressive and usually fatal disease characterized by fibroblast proliferation, extracellular matrix accumulation, and an important remodeling of lung tissue, which results in irreversible distortion of lung architecture. The primary sites of ongoing remodeling during IPF are areas of fibroblastic accumulation, the socalled fibroblastic foci, often localized at the ‘leading edge’ of the fibrotic lesion. They represent the histological hallmark of IPF and their frequency is one of the most reliable markers of poor prognosis with decreased survival. Several studies have demonstrated the presence of myofibroblasts in fibroblast foci of IPF (Figures 2 and 3). Ultrastructural and immunohistochemical investigations have revealed that myofibroblasts of such foci (as well as those present in bleomycin-induced pulmonary fibrosis) express a-SMA and/or desmin, the intermediate filament protein typical of SM cells. The source of myofibroblasts in fibroblastic foci is unclear: the most current evidence points to differentiation from fibroblasts of alveolar septa, which, as discussed above, display a protomyofibroblastic phenotype, under the local stimulation of growth factors and cytokines. Recent work additionally supports the contribution of circulating bone marrow-derived cells, such as fibrocytes or the transdifferentiation of epithelial and resident stem cells. Another hypothesis suggests that fibroblasts from patients with IPF are genetically susceptible to generating abnormal responses when subjected to the action of different inflammatory factors. Profibrotic molecules secreted by activated epithelial cells may stimulate genetically defective fibroblasts and thus facilitate the development of a chronic fibrotic process. Independently of their local or hematogenous origin, the apoptotic rate of fibroblastic cells cultured from IPF lesions appears to be lower than the apoptotic rate of similar cells

recovered from organizing pneumonia. It is conceivable that a defective apoptosis of myofibroblasts plays an important causal role in fibroblastic foci formation in areas of epithelial cell dropout. It has been shown that inhibition of fibroblast growth does not necessarily promote normal lung repair. Apoptotic alveolar epithelial cells are detected primarily in areas immediately adjacent to myofibroblast-rich fibroblastic foci. These findings emphasize the concept that IPF, rather than an inflammatory disorder, is an ‘epithelial–mesenchymal’ disease in which myofibroblast dysregulation actively participates in the development of the lesions. The high synthetic phenotype of myofibroblasts results in a de novo accumulation of high amounts of extracellular matrix proteins and integrins. This is accompanied by reduced extracellular matrix degradation, due to an imbalance in the production of metalloproteinases and their tissue inhibitors, in particular TIMP-2. Several studies have attempted to characterize in vitro the phenotype of myofibroblasts present in IPF lesions, with conflicting results. Such differences may relate to inherent fibroblast heterogeneity as well as to heterogeneity of the cellular microenvironment in the different situations. Fibroblasts cultured from fibrotic tissue of patients with IPF have been shown to possess both high and low proliferative capacities and anchorage-dependent growth in soft agar. Other features of IPF myofibroblasts include enhanced migratory activity and contractility, a defect in cyclooxygenase-2 expression and a low capacity to synthesize prostaglandin E2 (PGE2). It has also been reported that IPF myofibroblasts induce alveolar epithelial cell death in vitro. Moreover, these cells sustain their growth even in the absence of inflammatory cells and their products. Autocrine mechanisms and epithelial-derived factors may be sufficient to drive the fibrogenic process, similar to the ‘epithelial–mesenchymal trophic unit’ described in airway remodeling in asthma. This is supported by the high level of growth factor and of cell adhesion receptor expression by myofibroblasts allowing these cells to respond to microenvironmental mitogenic stimuli and extracellular matrix signals. Finally, it has been shown that myofibroblasts themselves produce a number of cytokines and chemokines. Thus, the myofibroblast appears to be the key cell involved in the pathogenesis of pulmonary fibrosis. Myofibroblasts in Asbestosis and Sarcoidosis

The respiratory diseases caused by asbestos inhalation are many and varied. Inhalation of asbestos fibers is well recognized to result in a characteristic lung fibrosis called asbestosis. Although progress has

MYOFIBROBLASTS

(a)

(b)

(c)

(d)

71

Figure 2 Myofibroblastic foci in IPF. (a,b) Ematoxylin-eosin stained sections; (c) Masson trichrome stain showing the collagen accumulation in blue; and (d) in the same area of lung parenchyma, immunochemical staining with anti-SMA monoclonal antibody confirms the myofibroblastic nature of interstitial cells, covered by hyperplastic epithelial cells.

(a)

(b)

Figure 3 Transmission electron micrographs showing myofibroblasts in the lung parenchyma of a patient with IPF. (a) Elongated cells with irregular nuclei are easily detected. (b) At higher magnification, peripheral bundles of filaments associated with dense bodies and well-developed rough endoplasmic reticulum are evident; cells are surrounded by a collagenous matrix. Scale bar ¼ 1 mm.

been made in the understanding of the mechanisms of asbestosis, many important questions remain unanswered. Once inhaled, asbestos fibers travel through the airways, and those not cleared by the

mucociliary system move into the alveoli, where they may be engulfed by macrophages and eliminated via the lymphatic system, or may produce fibrosing or carcinogenic effects. Asbestos fibers can also provoke

72

MYOFIBROBLASTS

chronic pleural inflammation through direct toxicity to mesothelial cells that determines the basis for mesothelioma progression. Experimental exposure of rats to chrysolite asbestos fibers results in an increase in the volume of the lung interstitium accompanied by a persistently high number of macrophages, accumulation of fibroblasts/myofibroblasts, and an increased volume of extracellular matrix. Successively, interstitial mesenchymal cells maintain a high accumulation of connective tissue matrix. In fact, macrophages activated by asbestos secrete proinflammatory and profibrotic cytokines, such as fibroblast growth factor, tumor necrosis factor alpha, GM-CSF, neutrophil chemotactic factor, fibronectin, and platelet-derived growth factor, as well as inflammatory mediators, such as leukotriene B4 and PGE2, which play an important role as mediators of asbestosis. Activation of ERK signaling within lung epithelium in vivo, following inhalation of asbestos fibers, also contributes to the advent of asbestos-associated apoptosis and proliferation of mesothelial and alveolar epithelial cells and is linked to the development of pulmonary fibrosis. Although myofibroblasts contribute to the development of asbestosis, the main difference of this type of pulmonary fibrosis with IPF is the prevalent interstitial matrix accumulation and the absence of a leading role of myofibroblasts in the distortion of lung architecture, characteristic of IPF. Sarcoidosis is a systemic disorder of unknown etiology in which an inflammatory lung process occurs, characterized by the presence of noncaseating granulomas. Although there is a spontaneous resolution of inflammatory phenomena in about 80% of cases, in the remaining cases a fibrotic process may develop. Nevertheless, unlike IPF, myofibroblasts are not detectable in lung specimens and in bronchoalveolar lavage of patients with sarcoidosis. Inflammatory and epithelial cells are mainly involved in the specific tissue reaction, which includes collagen and proteoglycan accumulation. It appears clear that different types of interstitial lung diseases are characterized by variable degrees of myofibroblast involvement, supporting different etiological and pathogenetic mechanisms.

Concluding Remarks The description and characterization of the myofibroblast has opened up new possibilities for the understanding of physiological and pathological phenomena in several organs including those of the respiratory system. Further understanding of

myofibroblast biology will be instrumental in the development of tools to control normal lung function and the evolution of several important diseases such as asthma and IPF. See also: Alveolar Wall Micromechanics. Apoptosis. Asthma: Overview. Coagulation Cascade: Overview. Colony Stimulating Factors. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Lipid Mediators: Prostanoids. Occupational Diseases: Asbestos-Related Lung Disease. Systemic Disease: Sarcoidosis. Transforming Growth Factor Beta (TGFb) Family of Molecules.

Further Reading Desmoulie`re A, Darby IA, and Gabbiani G (2003) Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Laboratory Investigation 83: 1689–1707. Gabbiani G, Hirschel BJ, Ryan GB, Statkov PR, and Majno G (1972) Granulation tissue as a contractile organ. A study of structure and function. Journal of Experimental Medicine 135: 719–734. Gharaee-Kermani M and Phan SH (2003) Role of fibroblasts and myofibroblasts in idiopathic pulmonary fibrosis. In: Lynch J (ed.) Idiopathic Pulmonary Fibrosis, pp. 501–563. New York: Dekker. Hinz B and Gabbiani G (2003) Mechanisms of force generation and transmission by myofibroblasts. Current Opinion in Biotechnology 14: 538–546. Kapanci Y, Assimacopoulos A, Irle C, Zwahlen A, and Gabbiani G (1974) ‘Contractile interstitial cells’ in pulmonary alveolar septa: a possible regulator of ventilation perfusion ratio? Ultrastructural, immunofluorescence, and in vitro studies. The Journal of Cell Biology 60: C375–C392. Kapanci Y and Gabbiani G (1997) Contractile cells in pulmonary alveolar tissue. In: Crystal RG, et al. (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 697–707. New York: LippincottRaven. Low RB (1999) Modulation of myofibroblast and smoothmuscle phenotypes in the lung. Current Topics in Pathology 93: 19–26. Selman M, King T, and Pardo A (2001) Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Annals of Internal Medicine 134: 136–151. Simpson J and Millar A (2004) The Year in Pulmonary Fibrosis Part I, Recent Advances, pp. 3–74. Clinical Publishing Oxford, CRC Press LLC, Boca Raton, London, New York, Washington, DC. Thannickal VJ, Toews GB, White ES, Lynch JP III, and Martinez FJ (2004) Mechanisms of pulmonary fibrosis. Annual Review of Medicine 55: 395–417. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, and Brown AR (2002) Myofibroblasts and mechano-regulation of connective tissue remodeling. Nature Reviews: Molecular Cell Biology 3: 349–363. White ES, Lazar MH, and Thannickal VJ (2003) Pathogenic mechanisms in usual interstitial pneumonia idiopathic pulmonary fibrosis. Journal of Pathology 201: 343–354.

MYOGLOBIN 73

MYOGLOBIN molecular mass of about 17 kDa and consists of a single polypeptide chain of around 153 amino acids with a secondary structure of eight a-helices (Figure 1). Between the fifth and sixth helices there is a hydrophobic region that acts as a pocket for a heme moiety (Fe-protoporphyrin-IX). The iron of the heme coordinates to four nitrogen atoms of the porphyrin ring and to the protein via the nitrogen from a histidine amino acid residue. The sixth coordination site of the heme iron is available to bind exogenous molecules such as oxygen, carbon monoxide (57), cyanide, water, depending on the valence state of the iron. In the physiologically active ferrous (FeII) oxidation state the iron can reversibly bind oxygen. Unlike its heterotetrameric ‘blood relation’ hemoglobin (Hb), monomeric Mb does not exhibit cooperative binding of oxygen, having a Hill coefficient of 1 compared to 2.8 for Hb (Figure 2). Mb has a higher affinity for oxygen than Hb and hence a low oxygen tension is required for oxygen release. Many textbooks refer to Mb as an oxygen-storage molecule or as an extra reserve of oxygen. While Mb is certainly capable of storing oxygen, the total oxygen-storage capacity of Mb is low compared to the oxygen demands of active muscles such as the heart. More recently it has been proposed that Mb facilitates oxygen diffusion down an oxygen gradient, enhancing oxygen transport to the mitochondria where oxygen is consumed by the respiratory enzyme cytochrome

M T Wilson and B J Reeder, University of Essex, Colchester, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The normal function of myoglobin is to bind oxygen reversibly and facilitate oxygen diffusion from blood capillaries to mitochondria in the muscle. It also appears that another main function of myoglobin is to scavenge nitric oxide, preventing inhibition of the mitochondrial enzyme cytochrome c oxidase. There is now a large body of research indicating that myoglobin can contribute to the pathogenesis of various disease states due to its ability to catalyze redox chemistry. These processes are particularly serious following ischemic/reperfusion injuries or following myolytic events. Under these conditions myoglobin can participate in oxidative reactions that include a cascade oxidation of lipid membranes that lead to the formation of powerful vasoactive compounds such as the isoprostanes.

Structure and Function Myoglobin (Mb) is a heme-containing globular protein that is found in abundance in myocyte cells of heart and skeletal muscle. Mb and Mb-like proteins are also found in many taxa, including bacteria, plants, fungi, and animals. In 1958 the structure of ˚ resolution, sperm whale Mb by X-ray diffraction (6 A ˚ refined to 2 A in 1960) was reported, the first three-dimensional structure obtained of any protein. Sperm whale Mb, like most mammalian Mbs, has a

Distal histidine N

C

C

N

CH HC NH

HC

CH NH O

Heme plane

N

Proximal histidine

(a)

N

Fe

Heme HC N

N

CH C

N

O Oxygen N

Fe HC N

N

CH C

(b)

Figure 1 (a) Three-dimensional ribbon model of sperm whale myoglobin showing the heme group and proximal histidine which links the iron of the heme group to the protein. (b) In the deoxy state the iron is pulled out of the plane of the heme by the proximal histidine. Oxygen binding pulls the iron back into the plane of the heme.

74

MYOGLOBIN 1 0.9 Myoglobin

Fraction saturation

0.8 0.7 0.6 0.5

Hemoglobin

0.4 0.3 0.2 0.1 0 0

2

4

6

8

10

12

pO (kPa) 2 Figure 2 Oxygen saturation curves for myoglobin and hemoglobin.

oxidase. Despite its apparent importance in oxygen transport, Mb knockout mice show no obvious phenotype. These ‘white muscle’ mice are able to function normally without Mb, but show several compensatory mechanisms, including increased cardiac capillary density, coronary flow, and Hb. These result in a reduction of the diffusion path length for O2 between capillary and the mitochondria.

Pathological Consequences of Myoglobin Acting as a Pro-Oxidant Redox Enzyme The physiological requirement for Mb to bind oxygen to a redox active metal creates a problem in that Mb, under certain conditions, can exhibit a peroxidase-like enzymatic activity that promotes reactions that facilitates tissue damage, a process termed oxidative stress. As O2 is an oxidizing agent, the binding of molecular oxygen to Mb can promote oxidation of the ferrous iron (FeII) to the ferric form (FeIII). This autoxidation reaction also results in the conversion of molecular oxygen to superoxide ðOd
a reactive oxygen species 2 Þ, ðFeII 2O2 -FeIII þ Od
2 Þ. The antioxidant defense system of the myocyte normally copes with the reactive oxygen species generated and returns the physiologically inactive ferric Mb to the physiologically active ferrous form. In the presence of peroxides, such as lipid hydroperoxides and hydrogen peroxide, Mb can react with these to generate the ferryl form of Mb (FeIV ¼ O2
), accompanied by the formation of a free radical (depending on the initial redox state of the Mb).

Unlike classical peroxidases, Mb does not hold the radical safely on the porphyrin ring as part of a catalytic cycle of peroxide consumption. Instead the radical rapidly escapes the heme to the protein with subsequent intra- and intermolecular electron migration. This radical leakage may result in the initiation of lipid oxidation reactions (catalyzing oxidation of either free or membrane-bound lipids/fatty acids). These reactions can also be initiated by the ferryl form of Mb (Figure 3). Such ‘rogue activities’ of Mb are cytotoxic and under severe conditions, such as ischemia reperfusion injuries, the antioxidant defense system can be overwhelmed. In pathological conditions the Mb may be separated from the antioxidant defenses, allowing Mb-induced oxidative reactions to thrive. Mb-induced oxidative damage to lipid membranes can result in the formation of a class of oxidized lipid molecules known as isoprostanes. These isoprostanes have powerful vasoactive properties. Following rhabdomyolysis (muscle trauma), Mb is released from the myocyte to eventually enter the kidney, leading to tissue damage and acute renal failure. High levels of isoprostanes are found in the kidney and urine following rhabdomyolysis, indicating that lipid oxidation reactions are involved in the mechanism of pathology. Isoprostane formation is widely considered as a reliable index of oxidative stress in vivo. Free iron as well as Mb can generate isoprostanes in vitro and there is debate over the relative importance of Mb-induced lipid oxidation versus free iron-induced lipid oxidation in vivo. It is interesting to note that heme oxygenase knockout mice given a myolytic shock are unable easily

MYOGLOBIN 75 Radicals Peroxides

−

O2 Autoxidation Myoglobin Fe2+–O2 or peroxides

Fe3+

Ferric/ferryl redox cycle

Oxidized lipids

(Heme to protein cross-linked myoglobin) Fe4+–OH−

Membrane lipids

Mb-X

Accelerates lipid oxidation

Acidosis

Peroxides

Isoprostanes

Vasoconstriction

Pathological complications Figure 3 Simplified schematic representation of some of the oxidation reactions that myoglobin catalyzes to lead to pathological complications.

to survive rhabdomyolysis, unlike their wild-type counterparts. Urinary levels of plasma creatinine, used as a marker of renal function, increased in both knockout and wild-type mice following induction of rhabdomyolysis. However, although the creatinine levels recovered over a period of a few days in wildtype mice, the knockout mice exhibited ever-increasing levels until death intervened. Consistent data are also available on subarachnoid hemorrhage models on heme oxygenase knockin animals. As heme oxygenase metabolizes heme, releasing free iron, these studies imply that heme proteins are active agents for lipid oxidation. The ability of Mb to catalyze these toxic reactions is influenced by the pH of its milieu. The pro-oxidant and pseudoperoxidase activities are enhanced at acidic pH, activities that have been linked to a protonation of the oxo-ferryl species. Acidosis often accompanies rhabdomyolysis, enhancing the catalytic activity of Mb which results in enhanced isoprostane formation, increasing acidosis further, producing a vicious cycle of tissue damage. Interestingly, it has been observed that alkalinization of the urine significantly attenuates renal function, which could be mechanistically linked to the pro-oxidant ability of Mb. The pro-oxidant ability of Mb can, under certain conditions, result in a side-reaction in which the Mb oxidizes itself, covalently binding the heme to the protein. This heme-to-protein cross-linked form is more cytotoxic than native protein, oxidizing lipids up to five times more rapidly. This type of altered Mb has been identified in the urine of patients following rhabdomyolysis, providing in vivo evidence that

Mb has been involved in redox cycling and thereby involved in oxidative reactions. The formation of this highly cytotoxic form of Mb is also pH dependent, being greatly enhanced at acid pH.

Nitric Oxide Scavenger: The Real Function of Myoglobin? Facilitation of oxygen diffusion may be compensated by other mechanisms in the absence of Mb. With the recent research indicating that Mb oxidative chemistry can be detrimental in certain disease states one may wonder why Mb is necessary. An answer may lie in the discovery that Mb, in addition to its oxygenbinding function, can act as a scavenger of nitric oxide (NO). NO is an important signaling molecule that is involved in a diverse range of physiological processes including maintaining blood pressure through its action on endothelial cells. Being a small gas NO can rapidly diffuse through cell membranes and reach the mitochondrion. The lifetime of NO in biological systems ranges from a few seconds to several minutes. In the mitochondrion, NO can inhibit cell respiration through its interaction with cytochrome c oxidase. Mb and Hb rapidly catalyze the conversion of NO to nitrate (MbII – O2 þ NOMbIII þ NO3
), decreasing the NO lifetime to a few milliseconds in the red muscle. As such Mb can protect cytochrome c oxidase from NO inhibition. Mb is one of the most studied and best-characterized proteins, in terms of its structure, function, and evolution. Yet there are still many questions about its true function and there is much to learn about its

76

MYOGLOBIN

redox and radical chemistry and its relationship to pathology in vivo. Hb is related to Mb, both in terms of its function and in its redox chemistry. Understanding the molecular processes of Mb in pathology relates to the development of cell-free hemoglobinbased blood substitutes as these oxygen carriers have cytotoxic side effects due to their redox chemistry. Recently two more globins have been discovered and added to the globin family: neuroglobin is expressed predominantly in the nervous system and cytoglobin (also called histoglobin) is found in almost all tissue types. The functions of these proteins are unknown, but have been proposed to be oxygen-consuming or oxygen-sensor proteins. The functions of these proteins may cast new light on the ancestral functions of vertebrate globins. See also: Endothelial Cells and Endothelium. Hemoglobin. Nitric Oxide and Nitrogen Oxides. Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants. Oxygen Toxicity. Oxygen–Hemoglobin Dissociation Curve.

Further Reading Better OS and Stein JH (1990) Early management of shock and prophylaxis of acute renal failure in traumatic rhabdomyolysis. New England Journal of Medicine 322: 825–829. Brunori M (2001) Nitric oxide, cytochrome-c oxidase and myoglobin. Trends in Biochemical Sciences 26: 21–23. Catalano CE, Choe YS, and Ortiz de Montellano PR (1989) Reactions of the protein radical in peroxide-treated myoglobin. Formation of a heme-protein cross-link. Journal of Biological Chemistry 264: 10534–10541. Cleeter MW, Cooper JM, Darley-Usmar VM, Moncada S, and Schapira AH (1994) Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Letters 345: 50–54. Doyle MP and Hoekstra JW (1981) Oxidation of nitrogen oxides by bound dioxygen in hemoproteins. Journal of Inorganic Biochemistry 14: 351–358. Eich RF, Li T, Lemon DD, et al. (1996) Mechanism of NO-induced oxidation of myoglobin and hemoglobin. Biochemistry 35: 6976–6983. Godecke A, Flogel U, Zanger K, et al. (1999) Disruption of myoglobin in mice induces multiple compensatory mechanisms.

Myopathies

Proceedings of the National Academy of Sciences of the United States of America 96: 10495–10500. Holt S, Reeder B, Wilson M, et al. (1999) Increased lipid peroxidation in patients with rhabdomyolysis. Lancet 353: 1241. Kendrew JC, Bodo G, Dintzis HM, et al. (1958) A threedimensional model of the myoglobin molecule obtained by X-ray analysis. Nature 181: 662–666. Moore KP, Holt SG, Patel RP, et al. (1998) A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure. Journal of Biological Chemistry 273: 31731– 31737. Morrow JD, Hill KE, Burk RF, et al. (1990) A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proceedings of the National Academy of Sciences of the United States of America 87: 9383–9387. Nath KA, Haggard JJ, Croatt AJ, et al. (2000) The indispensability of heme oxygenase-1 in protecting against acute heme proteininduced toxicity in vivo. American Journal of Pathology 156: 1527–1535. Osawa Y, Nakatsuka K, Williams MS, Kindt JT, and Nakatsuka M (1996) Reactions of reactive metabolites with hemoproteinstoxicological implications: covalent alteration of hemoproteins. Advances in Experimental Medicine and Biology 387: 37–45. Pesce A, Bolognesi M, Bocedi A, et al. (2002) Neuroglobin and cytoglobin. Fresh blood for the vertebrate globin family. EMBO Reports 3: 1146–1151. Reeder BJ and Wilson MT (2001) The effects of ph on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species. Free Radical Biology and Medicine 30: 1311–1318. Reeder BJ, Svistunenko DA, Cooper CE, and Wilson MT (2004) The radical and redox chemistry of myoglobin and hemoglobin: from in vitro studies to human pathology. Antioxidants and Redox Signalling 6: 954–966. Reeder BJ, Svistunenko DA, Sharpe MA, and Wilson MT (2002) Characteristics and mechanism of formation of peroxideinduced heme to protein cross-linking in myoglobin. Biochemistry 41: 367–375. Roberts LJ and Morrow JD (2000) Measurement of F(2)-isoprostanes as an index of oxidative stress in vivo. Free Radical Biology and Medicine 28: 505–513. Vuletich JL, Osawa Y, and Aviram M (2000) Enhanced lipid oxidation by oxidatively modified myoglobin: role of proteinbound heme. Biochemical and Biophysical Research Communications 269: 647–651. Zager RA (1989) Studies of mechanisms and protective maneuvers in myoglobinuric acute renal injury. Laboratory Investigation 60: 619–629.

see Neuromuscular Disease : Inherited Myopathies; Acquired Myopathies.

N NADPH AND NADPH OXIDASE A B Fisher and Q Zhang, University of Pennsylvania Medical Center, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract NADP in the cell exists in both reduced (NADPH) and oxidized (NADP þ ) forms. Reduction of NADP þ to NADPH occurs by the pentose phosphate pathway, which is an alternative route for the metabolism of glucose. NADPH provides reducing power for biosynthetic reactions, maintains the redox state of the cell, and is the electron donor for NADPH oxidase. This enzyme is present in polymorphonuclear neutrophil leukocytes (PMNs) and other phagocytic cells where it produces superoxide from oxygen and NADPH for cellular bactericidal function. In phagocytic cells, NADPH oxidase is composed of the flavocytochrome b558, an integral membrane heterodimer composed of gp91phox and p22phox subunits, and the cytosolic proteins, p40phox, p47phox, p67phox, and Rac. The subunits of NADPH oxidase are unassembled and the enzyme is inactive in the resting state but with the appropriate stimulus, activation occurs by translocation of cytosolic components to the cell membrane. NADPH oxidase also exists in nonphagocytic cells, such as endothelial cells and smooth muscle cells, although the gp91phox component may be replaced by homologous (Nox and Duox) proteins. The enzyme is largely responsible for basal and stimulated production of reactive oxygen species (ROS) that serves a cell-signaling function leading to changes in gene transcription and protein synthesis. Deficiency of NADPH oxidase results in chronic granulomatous disease, an inherited disease characterized by recurrent chronic infections due to abnormal bactericidal mechanisms. Overproduction of ROS by NADPH oxidase associated with PMN infiltration and activation results in oxidant stress that may exacerbate acute lung injury.

Introduction NADPH oxidase is a membrane-bound enzyme complex that generates superoxide anion to serve a variety of physiological functions. The system was initially described in polymorphonuclear neutrophil leukocytes (PMNs) but is now known to be widely distributed in many cell types. This enzyme plays an important role in PMN bactericidal mechanisms but the physiological roles in other cell types have only recently come under investigation and are not as well understood. Production of small amounts of superoxide anion by NADPH oxidase in some cells appears to serve the purpose of cellular signaling. This latter function is especially important in the vasculature, including vascular smooth muscle and endothelial cells. On the

other hand, production of superoxide and its subsequent reactions to form highly reactive chemical species may cause severe tissue damage and induce tissue inflammatory responses emphasizing the need for a tight regulation of this multiprotein enzyme complex.

Nicotinamide Adenine Dinucleotide Phosphate Overview

Nicotinamide adenine dinucleotide phosphate (NADP), like its homolog nicotinamide adenine dinucleotide (NAD), is a biological carrier of reducing equivalents, i.e., it can accept and deliver electrons. In that sense, it functions generally as a coenzyme. NADP differs from NAD by the presence of a phosphate group at the C-20 position of the adenosyl moiety. The molecule exists in cells in reduced (NADPH) and oxidized (NADP þ ) forms reflecting the redox state of the cell. NADPH is located predominantly in the cytosolic compartment while NADH is localized predominantly to mitochondria. NADPH Production

The primary source of reducing equivalents for reduction of NADP þ to NADPH in cells is the reactions of the pentose phosphate pathway of glucose metabolism. This pathway located in the cellular cytosol does not generate ATP directly but provides NADPH for reductive biosyntheses and redox reactions. The pentose phosphate pathway (also called the hexose monophosphate shunt pathway) is a multicyclic process in which three molecules of glucose 6-phosphate give rise to three molecules of CO2 and three residues containing five carbon atoms (pentoses). These residues are rearranged to regenerate two molecules of glucose 6-phosphate and one molecule of the glycolytic intermediate, glyceraldehyde 3phosphate, as shown in reaction [1]: 3 glucose 6-phosphate +

6NADP+

3 CO2 + 2 glucose 6-phosphate + glyceraldehyde 3-phosphate + 6 NADPH + 6H +

Glucose-6-phosphate dehydrogenase

½1


78

NADPH AND NADPH OXIDASE

Fructose-6-P, glyceraldehyde-3-P

Glucose-6-P dehydrogenase Ribulose-5-P

Glucose-6-P NADP +

NADP + + H +

NADPH

NADPH oxidases

GSH reductase

GSH

Ribose-5-P

O2− •

GSSG O2

SOD H2O

H2O2 GSH peroxidase

Figure 1 Generation of NADPH and its utilization by NADPH oxidase. The pentose phosphate pathway generates NADPH by reduction of NADP þ in a reaction catalyzed by glucose-6-P dehydrogenase (top). Depending on needs of a cell for ribose-5-phosphate, NADPH, and ATP, the pentose phosphate pathway can operate in various modes to maximize different products. To maximize formation of NADPH, glyceraldehyde-3-P and fructose-6-P are converted to G-6-P for reentry into the linear portion of the pentose phosphate pathway. Glutathione peroxidase catalyzes degradation of H2O2 by reduction, as two GSH molecules are oxidized to a disulfide (bottom). Regeneration of GSH by GSH reductase requires NADPH, which is oxidized to NADP þ (middle). NADPH þ in turn is regenerated by the GSH reductase reaction, completing the cycle. NADPH is also the substrate for NADPH oxidase that catalyzes the production of superoxide (O2 d), which dismutes to H2O2 in a reaction catalyzed by superoxide dismutase (SOD). These pathways are central to maintenance of cellular redox state.

Thus, glucose-6-phosphate dehydrogenase, through its linkage to NADP þ reduction in the pentose phosphate cycle, plays a key role in supplying cellular reducing equivalents (Figure 1). Cellular Functions of NADPH

An important cellular role for NADPH is to provide the reducing power for reductive biosynthesis of macromolecules including fatty acids and complex lipids, proteins, and nucleotides. NADPH also plays an important role in the control of cellular redox state. In this latter function, NADPH provides reducing equivalents for reduction of oxidized glutathione (GSSG) to the reduced form (GSH), a reaction catalyzed by glutathione reductase (Figure 1). GSH is an important intracellular reductant and plays an important role in cellular anti-oxidant defense. Finally, NADPH provides electrons for transfer to molecular O2 in the reaction catalyzed by NADPH oxidase. This reaction has several important physiological roles as described below. In addition, the NADPH oxidase reaction by generating excess superoxide can contribute to oxidant stress. Thus, by serving as a cofactor for both NADPH oxidase and GSH reductase, NADPH has a role in both promoting and protecting against oxidant stress.

NADPH Oxidase Overview

NADPH oxidase represents a family of enzymes that catalyze the production of superoxide (O2 d) from oxygen and NADPH as shown in reaction [2]: NADPH þ 2O2 -NADPþ þ Hþ þ 2Od 2

½2


NADPH oxidase is a multicomponent complex composed of integral membrane and cytosolic proteins. NADPH oxidase was originally described in PMNs and for many years was considered to be present only in these cells and other ‘professional’ phagocytes (eosinophils, macrophages). Recently, this enzyme system has been described in endothelial cells, smooth muscle cells, and a variety of other cells, and indeed may be present in most cell types. Based on its long history of study, the structure of the enzyme complex is best understood in PMNs, although information for vascular and other cells is rapidly accumulating. It appears that the components of the complex in most cells are similar to those in PMNs except for differences in the flavoprotein component.

NADPH AND NADPH OXIDASE 79

gp91phox

p22 phox

Extracellular

gp91phox

RacGTP RacGTP

p40phox

Intracellular

p67phox p47phox

F GE

RacGDP RhoGDI

p67phox P

P

p22phox

O2−

O2

P p47phox P

p40phox

P

e tid

leo uc -n nge e nin ha ua exc

NADP+ + H+

NADPH

G

Phosphorylation Resting

Activation

Figure 2 Structure and activation of the NADPH oxidase in PMN. In the resting state, the NADPH oxidase subunits p22phox and gp91phox (together called cytochrome b558) are located in the plasma membrane, while the other NADPH oxidase subunits (Rac, p40phox, p47phox, and p67phox) are cytosolic (left). Inactive cytosolic GDP-bound Rac exists in a complex with the Rho GDP dissociation inhibitor (RhoGDI). Activation (right) occurs as a result of phagocytosis of bacteria or associated with other signals. Dissociation of the Rac/Rho complex results in guanine-nucleotide exchange with formation of active GTP-bound Rac that translocates to the membrane to form the NADPH oxidase complex. Cytosolic protein kinases, including protein kinases C and B (Akt), catalyze phosphorylation of autoinhibitory regions of p47phox, which also translocates to the membrane and binds through its two SRC-homology 3 (SH3) domains to p22phox and to phosphorylated p67phox. Thus, p47phox serves as an organizer protein. The enzyme complex transfers an electron via the flavoprotein component of gp91phox across the cell membrane to the acceptor oxygen molecule thereby generating O2 d in the extracellular space. This reaction also generates NADP þ and a proton in the cytoplasm.

Components of NADPH Oxidase in PMNs and Other ‘Professional’ Phagocytes

The NADPH oxidase system of neutrophils and other phagocytes is composed of two integral membrane proteins (p22phox and gp91phox) and four cytosolic proteins (Rac, p40phox, p47phox, and p67phox). These components are named for the nominal molecular mass of the protein on gel electrophoresis (phox refers to phagocyte oxidase). gp91phox is the core catalytic component of the enzyme complex and, together with p22phox, constitutes the membraneassociated cytochrome (cyt b558), named because it shows maximal spectral absorption at 558 nm. The enzyme is inactive under resting conditions; upon stimulation, cytosolic proteins translocate to the plasma membrane where they associate with the membrane components to form an active enzyme. Activation of the NADPH oxidase is regulated by a complex set of reversible protein–protein interactions, although the precise molecular details of these events are not yet fully understood. Phosphorylation of the cytosolic components during the

activation process plays an important role by regulating protein–protein interactions and their association with the membrane-bound subunits (Figure 2). The two membrane components and several cytosolic proteins, p47phox and p67phox (and probably Rac), are essential for superoxide production, as evident from manifestations of chronic granulomatous disease where the absence of any one of these components results in abnormal PMN function (see below). gp91phox The gp91phox gene encodes an essential component of the oxidase and gene deletion or mutation results in chronic granulomatous disease. The primary translation product of the gp91phox gene is a 66 kDa protein that is extensively glycosylated to form the mature protein. gp91phox in cyt b558 represents the only catalytic component in NADPH oxidase and is responsible for electron transfer from NADPH to molecular O2. The gp91phox protein has two nonidentical hemes with midpoint redox potentials of  265 and  285 mV; the heme has six coordination sites in a low spin state, which is essential for O2 d generation in reconstituted NADPH oxidase.

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NADPH AND NADPH OXIDASE

The protein contains a flavin adenine dinucleotide (FAD) component that is the direct link to molecular oxygen for electron transfer. p22phox p22phox is the nonglycosylated 22 kDa a-subunit of the intrinsic cell membrane protein complex, cytochrome b558. The p22phox moiety serves as a docking site for p47phox, and accessorily for p67phox. This protein with its tail in the cytosol functions in a regulatory role and is essential for recruitment and binding of the cytosolic proteins to the complex leading to oxidase activation. p40phox p40phox is a cytosolic protein that trans locates to the cell membrane to form the active NADPH oxidase but lacks a clear function. It contains conserved ‘phox homology’ domains that preferentially bind phosphatidyl inositol 3,4-biphosphate and phosphatidyl inositol 3-phosphate. The protein might facilitate assembly of the NADPH oxidase complex under certain circumstances. However, it is not required for oxidase activity in a cell-free system and genetic absence of p40phox does not result in chronic granulomatous disease. p47phox p47phox acts as an adaptor protein facilitating the interaction of Rac and p67phox with the membrane subunits. Mutations in p47phox result in chronic granulomatous disease indicating its physiologic importance. Under physiological conditions, p47phox-mediated translocation of the p67phox–Rac complex to the membrane is a critical step in the activation of the oxidase in PMNs. However, p47phox is not directly involved in the activation of the oxidase since sufficiently high concentrations of p67phox and Rac can activate the enzyme complex in vitro even in the absence of p47phox. p47phox appears primarily to serve in the intact cell to provide a regulatory response element for oxidase assembly induced by extracellular activators, and to act as an adaptor to facilitate binding of p67phox with cyt b558. p67phox In contrast to p47phox and p40phox, p67phox is an essential regulatory component of NADPH oxidase in the cell-free system. The p67phox N-terminus contains elements necessary for Racbinding. The p67phox fragment crystallized with Rac1 contains amino acid repeats that occur in tandem arrays and are involved in a variety of protein– protein interactions. A peptide sequence in p67phox (aa199–210), and perhaps (aa187–193), appears to interact directly with p22phox in cyt b558 to regulate the transfer of electrons from NADPH to the FAD component of gp91phox. p67phox also appears to serve as the bridging molecule that links p47phox with

p40phox. Both p47phox and p67phox contain two src-homology 3(SH3) domains. The C-terminal SH3 domain of p67phox has been shown to interact with the proline-rich domain of p47phox, facilitating the transport of p67phox to the membrane. Rac Rac is a member of the small GTPase protein family and is a key regulator of NADPH oxidase activity in PMNs. Small GTPases are monomeric proteins with molecular mass B20–28 kDa that have been divided into six families: Ras, Rho, Rab, Arf, Ran, and Rad. All members of these families contain consensus GTP-binding motifs and have B30% homology to Ras. Rac is a member of the Rho small GTPase family. Inactive, GDP-bound Rac exists as a cytosolic complex with Rho guanine nucleotide dissociation inhibitor (RhoGDI). Upon activation, Rac translocates to the cell membrane along with the other cytosolic components of NADPH oxidase. At the membrane, Rac translocation is simultaneous with but independent of the translocation of p67phox and p47phox. Rac binds to a specific binding site (aa170–199) of p67phox in an interaction that is strictly GTP-dependent. Translocation of Rac and its binding to p67phox and p22phox is essential for activity of NADPH oxidase. Both Rac1 and Rac2 are effective activators of NADPH oxidase in vitro; in vivo, Rac2 is the activator in PMNs while Rac1 may be more important in other cell types. Homologs of gp91phox

Recently, several novel homologs of gp91phox have been cloned and characterized. These homologs have been designated as Nox proteins (1–5) and Duox1 and 2 (Nox is an abbreviation for NADPH oxidase and Duox for Dual oxidase.) In this nomenclature, gp91phox has been renamed as Nox2. All of the Nox proteins function in electron transfer from NADPH to O2 as described for gp91phox. The Duox enzymes contain both a NADPH oxidase domain as well as a peroxidase domain (hence the name, dual oxidase). The peroxidase domain utilizes H2O2 that is generated by dismutation of O2 d produced by the NADPH oxidase domain. The molecular mass deduced from the mRNA for the corresponding primary translation products is 65–66 kDa for Nox1 to Nox4, 85 kDa for Nox5, and 175–177 kDa for Duox1 and 2. (Extensive glycosylation accounts for the larger size and the observed migration of the proteins on gel electrophoresis.) The several proteins in the Nox family have similar structure characterized by six transmembrane domains with four histidines constituting the conserved heme-binding residues of the cytochromes. Intracellular C-termini

NADPH AND NADPH OXIDASE 81

have conserved binding sites for FAD and NADPH, which are essential for generation of O2 d. Unlike gp91phox, these newly described proteins are not expressed in phagocytes; their cellular and subcellular distribution, biochemical properties, and physiologic functions are not yet fully understood.

Biological Functions of NADPH Oxidase The physiological functions that have been described for NADPH oxidase include microbicidal activity and cell signaling. Microbicidal activity is a function of phagocytic cells while signaling may be an important function in all cell types that express this enzyme. A role for NADPH oxidase in O2-sensing has also been suggested although the experimental evidence for this function gives equivocal support. The NADPH Oxidase as the Major Source of Reactive Oxygen Species in PMNs and Other ‘Professional’ Phagocytic Cells

The term ‘professional’ phagocyte refers to those cells with a primary function to phagocytose microorganisms, particles, damaged cells, and cellular debris. Through this mechanism, PMNs play an essential role in host defense against infection. An important part of this defense is the generation of superoxide anions and their subsequent reaction products, which participate in bactericidal mechanisms. The NADPH oxidase system is the enzymatic source of the superoxide anions that are generated during phagocytosis and phagocytes lacking NADPH oxidase cannot efficiently kill bacteria and fungi. Patients with this inherited defect demonstrate chronic granulomatous disease (described below). The respiratory burst When PMNs, macrophages, and other phagocytic cells engulf bacteria or other particles, they exhibit a rapid but transient increase in oxygen consumption known as the respiratory burst. The burst reflects primarily the increased utilization of oxygen for the generation of superoxide anion via NADPH oxidase activity. The increased superoxide generation is accompanied by increased NADPH consumption and increased activity of the pentose phosphate cycle to regenerate NADPH. Superoxide generated by the oxidase plays an important role in the killing of microorganisms. Thus, NADPH oxidase activity reflects the respiratory burst and constitutes a primary defense mechanism against microbial infections. Mechanism for Od generation As described 2 above, NADPH oxidase is normally quiescent in these cells but is activated with exposure to

microorganisms; upon activation, the cytosolic proteins translocate to the membrane and associate with the membrane-bound subunits. The initial and crucial step in the assembly and activation of the NADPH oxidase is the binding of the small GTPase Rac to p67phox. Subsequent translocation of the P67phox–Rac complex and recruitment of p47phox with binding to p22phox in the cell membrane are necessary for activation. The role of O2 d in microbicidal activity relies largely on generation of H2O2 derived from this anion through a spontaneous reaction that is catalyzed by superoxide dismutase (SOD) (reaction [3]). H2O2 in turn reacts in a reaction catalyzed by myeloperoxidase (MPO) to generate HOCl (reaction [4]): SOD

þ Od 2 þ 2H - H2 O2 MPO

H2 O2 þ Hþ þ Cl - HOCl þ H2 O

½3
½4


Additional toxic products that can be generated include the hydroxyl radical (dOH) and peroxinitrite (ONOO  ) (reactions [5] and [6]). Fe2 þ (or other transition metals) are important catalysts for the formation of dOH. The range of molecules derived by partial reduction of oxygen have been called reactive oxygen species (ROS): Fe2þ

 d Od 2 þ H2 O2 - OH þ OH þ O2

½5


 d Od 2 þ NO-ONOO

½6


O2 d is generated by electron transfer across the cell membrane into the phagolysosome. The phagolysosome is formed by particle engulfment and is external to the cell membrane of the phagocytic cell. Thus, the production of O2 d by the cell membrane NADPH oxidase is into the extracellular milieu. NADPH Oxidase-Derived ROS as Second Messengers

ROS have been implicated in several major intracellular signaling pathways leading to changes in gene transcription and protein synthesis. ROS can function as signaling molecules since they are effective at low concentration and cellular mechanisms for their removal can rapidly terminate their activity. H2O2 appears to be the major effector of ROS-mediated signaling events. In the usual case, NADPH oxidase generates O2 d at the external surface of the cell membrane where it is converted spontaneously or enzymatically (via extracellular SOD) to H2O2, which freely crosses cell membranes. O2 d can cross cell membranes itself also, albeit rather slowly, via anion channels. H2O2 can inhibit expression of

82

NADPH AND NADPH OXIDASE NADPH + O2 NADPH oxidase O2− • SOD H2O2

Low conc.

High conc.

Cytotoxicity

Signaling

Fe2+ NF-
B

p38 MAPK

ERK 1,2

PI3k Akt

JAK2 STATs

Moderate conc.

Redox regulation GSH Mitochondria

•

OH Cytochrome c

DNA damage Lipid peroxidation Protein oxidation

Pro-caspase-9 Caspase-9

AIF

Proliferation Caspase 3,6,7 Necrosis Apoptosis Figure 3 Biological consequences of NADPH oxidase activation on cellular proliferation and survival. Activation of NADPH oxidase in cells generates O2 d, which dismutes to H2O2. H2O2 in cells can function as a signaling molecule leading to cellular proliferation or can result in cell death. At low concentrations, H2O2 serves as a second messenger to activate transcription factors including NF-kB and various kinases (p38 MAPK, ERK, PI3k, Akt, JAK2, STAT), which regulate gene expression and cell growth (left). H2O2 at slightly higher concentrations can induce the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into the cytosol (right) where they trigger the assembly of a caspase activation complex, leading to cell death by apoptosis. H2O2 can also generate toxic radicals such as dOH, especially in the presence of Fe2 þ ; these can damage cellular macromolecules resulting in cell death by necrosis (middle).

protein tyrosine phosphatases (PTP), evolutionarily conserved enzymes that play a central role in transmission of signals from cell-surface receptors to the nucleus. H2O2 can downregulate several transcription factors such as p53, Jun, and Fos also, and can activate nuclear factor kappa B (NF-kB) and the c-jun N-terminal kinase (JNK) pathway (Figure 3). These effects can account for the link that has been observed between NADPH oxidase activity and cellular proliferation. NADPH Oxidase in Vascular Function

It has recently become evident that generation of O2 d via the NADPH oxidase pathway plays an important role in homeostasis of the vasculature, including function of endothelial cells and vascular

smooth muscle. There has been considerable confusion in the literature regarding the relative importance of NADPH versus NADPH as the nucleotide cofactor in these reactions and details of the role of these pathways are currently under investigation. NADPH oxidase in endothelial cells The NADPH oxidase in endothelial cells is similar in composition to the PMN enzyme and contains gp91phox as the O2 d generator. Unlike the situation in PMN, Nox4 is also present, at least in some endothelial cells. Endothelial cells, unlike phagocytes, are thought to generate small quantities of superoxide in the absence of stimulation, indicating partial preassembly of the NADPH oxidase complex. Endothelial cells can respond to appropriate stimuli resulting in the

NADPH AND NADPH OXIDASE 83

equivalent of a respiratory burst with a marked increase in the rate of O2 d production. An example of the endothelial respiratory burst is the increased O2 d generation that accompanies a rapid change in endothelial cell shear stress. Endothelial cells are subjected normally to shear stress due to the flow of blood in the vascular lumen. Altered shear stress can modulate endothelial cell function by initiating a broad range of responses including activation of flow-sensitive ion channels, altered gene expression, and cytoskeletal reorganization. Either abrupt increase (increased flow) or loss (ischemia) of shear stress can result in activation of endothelial membrane-localized NADPH oxidase and superoxide generation followed by ROS-mediated cell-signaling events. This response to ischemia has been described for the pulmonary microvasculature where acute reduction in shear stress does not result in O2 deprivation so that ROS generation can continue during the ischemic episode. NADPH oxidase in smooth muscle cells In contrast to the NADPH oxidase in PMNs and endothelial cells, expression of gp91phox (Nox2) is not detectable in smooth muscle cells. Smooth muscle cells express Nox1 and Nox4, which seem to be the major contributors to ROS generation, and Nox5. Nox4 activation appears to retard proliferation and growth of smooth muscle, but the precise role of the Nox proteins in smooth muscle cell function is not fully defined. It has been suggested that NADPH oxidase has a role in O2 sensing in the pulmonary circulation and other O2-sensitive cells. Oxygen sensing is an important physiological process responsible for regulation of hypoxic pulmonary vasoconstriction, function of the carotid body and the lung neuroepithelial bodies, and release of erythropoietin. Much recent work has demonstrated the role of hypoxia-inducible factor (HIF) and prolyl hydroxylases in sensing of decreased O2 availability but NADPH oxidase has been suggested as an alternative mechanism. Several mechanisms for the possible role of NADPH oxidase have been proposed but none conform precisely with the known characteristics of this enzyme complex. The experimental evidence for the NADPH oxidase hypothesis is largely based on inhibitor studies, which have given equivocal results. Further, deletion of gp91phox or p47phox, either through gene targeting of mice or because of CGD in humans, does not result in decreased response to hypoxia. Although, this does not exclude a role for NADPH oxidase since one of the other Nox isoforms could be involved, a role for NADPH oxidase as a universal O2 sensor appears to be unlikely.

NADPH Oxidase, ROS, and Cell Death

Just as ROS participate in the killing of microorganisms, they can injure host cells through oxidative modification of cellular biomolecules. ROS generated by the NADPH oxidase of PMNs or other cells can cause oxidative stress resulting in cellular lipid peroxidation, protein oxidation, DNA damage, and ATP depletion. Extensive damage results in disruption of cell membrane and other organelles resulting in necrotic cell death (Figure 3). ROS may also trigger cell death by apoptosis, although the precise details are still under investigation, partly due to the difficulty in establishing whether oxidative events are a consequence or cause of cell death (Figure 3). In PMNs, apoptosis is induced through cell surface proteins such as complement receptor (CR) and Fcg receptor (FcgR) ligation with ligands. CR3-mediated phagocytosis promotes apoptosis through a caspasedependent pathway that is modulated by ROS generated by NADPH oxidase. H2O2 at low concentrations can induce survival pathways of cells, while higher concentrations induce cell apoptosis. Thus, survival and apoptotic signaling pathways mediated by ROS appear to be competitively regulated.

NADPH Oxidase in Respiratory Disease Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is an inherited disease characterized by severe recurrent infections due to lack of superoxide generation by PMNs and other phagocytic cells. The disease is caused by deficiency of one of the NADPH oxidase components gp91phox, p22phox, p67phox, or p47phox resulting in failure of the enzyme to generate O2 d. Deficiency of Rac results only in diminished activity of the oxidase, which appears to be stimulus specific. The frequency of infection is not significantly increased with MPO deficiency and CGD does not result. Phagocytes with NADPH oxidase deficiency retain the ability to phagocytose bacteria but the killing mechanisms that rely on O2 d generation are defective. CGD has an estimated incidence of 1 in 250 000 individuals, without any ethnic preference. The disease is more common in males, consistent with the X-chromosomal location of gp91phox. Patients with CGD lack the essential host antimicrobial pathway associated with NADPH oxidase, and develop recurrent and often severe bacterial and fungal infections, especially infection with Staphylococcus aureus and Aspergillus fumigatus. The most serious infection is chronic inflammation of the lung, and intractable pneumonia and septicemia are a frequent cause of death.

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Acute Lung Injury

Acute lung injury (ALI) is a syndrome characterized by widespread impairment of the small blood vessels in the lung resulting in noncardiogenic pulmonary edema and alterations in pulmonary gas exchange. Severe manifestations are termed the acute respiratory distress syndrome (ARDS). Etiology of the syndrome can be classified as either direct injury to the lungs such as due to acid aspiration or an indirect injury such as ALI associated with systemic infection. A consistent finding is the presence of large numbers of inflammatory cells in the lung. It has been suggested that increased ROS generation by activated PMNs possibly combined with decreased antioxidant capacity plays a central role in pathogenesis of ARDS. By this theory, the NADPH oxidase and its activation generate extracellular ROS, which damage normal cellular constituents. ROS generation by endothelium or other lung cells may contribute to the oxidant load. Although this hypothesis has some experimental support, suppression of PMNs has given equivocal results for severity of injury, and antioxidant therapy has not yet proven useful in amelioration of the disease. See also: Acute Respiratory Distress Syndrome. Apoptosis. Leukocytes: Neutrophils. Transcription Factors: NF-kB and Ikb.

Further Reading Babior BM, Lambeth JD, and Nauseef W (2002) The neutrophil nadph oxidase. Archives in Biochemistry and Biophysics 397: 342–344. Bengtsson SH, Gulluyan LM, Dusting GJ, and Drummond GR (2003) Novel isoforms of NADPH oxidase in vascular physiology and pathophysiology. Clinical and Experimental Pharmacology and Physiology 30: 849–854. Bokoch GM and Knaus UG (2003) NADPH oxidases: not just for leukocytes anymore!. Trends in Biochemical Sciences 28: 502–508.

Burridge K and Wennerberg K (2004) Rho and rac take center stage. Cell 116: 167–179. Cai H, Griendling KK, and Harrison DG (2003) The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends in Pharmacological Sciences 24: 471–478. Cross AR and Segal AW (2004) The nadph oxidase of professional phagocytes – prototype of the nox electron transport chain systems. Biochimica et Biophysica Acta 1657: 1–22. Fisher AB, Chien S, Barakat AI, and Nerem RM (2001) Endothelial cellular response to altered shear stress. American Journal of Physiology. Lung Cellular and Molecular Physiology 281: L529–L533. Forman HJ and Torres M (2002) Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. American Journal of Respiratory and Critical Care Medicine 166: S4–S8. Griendling KK, Sorescu D, and Ushio-Fukai M (2000) NAD(P)H oxidase: role in cardiovascular biology and disease. Circulation Research 86: 494–501. Groemping Y, Lapouge K, Smerdon SJ, and Rittinger K (2003) Molecular basis of phosphorylation-induced activation of the NADPH oxidase. Cell 113: 343–355. Hoidal JR, Brar SS, Sturrock AB, et al. (2003) The role of endogenous NADPH oxidases in airway and pulmonary vascular smooth muscle function. Antioxidants and Redox Signaling 5: 751–758. Jones RD, Hancock JT, and Morice AH (2000) NADPH oxidase: a universal oxygen sensor? Free Radical Biology and Medicine 29: 416–424. Lambeth JD (2004) Nox enzymes and the biology of reactive oxygen. Nature Reviews: Immunology 4: 181–189. Patil S, Bunderson M, Wilham J, and Black SM (2004) Important role for Rac1 in regulating reactive oxygen species generation and pulmonary arterial smooth muscle cell growth. American Journal of Physiology: Lung Cellular and Molecular Physiology 287: L1314–L1322. Quinn MT and Gauss KA (2004) Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases. Journal of Leukocyte Biology 76: 760–781. Takeya R and Sumimoto H (2003) Molecular mechanism for activation of superoxide-producing NADPH oxidases. Molecules and Cells 16: 271–277. Vignais PV (2002) The superoxide-generating nadph oxidase: structural aspects and activation mechanism. Cellular and Molecular Life Sciences 59: 1428–1459.

NEONATAL CIRCULATION J A Adams, Mount Sinai Medical Center, Miami Beach, FL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract This article summarizes the development of the neonatal pulmonary circulation. The complexity of such circulation during development and the factors responsible for structural and biochemical growth will be discussed. The fetal circulation will be reviewed from an anatomical and biochemical perspective

highlighting the important mediators responsible for maintenance of the vascular resistance in utero. Transition to the extrauterine environment will be reviewed with emphasis on the balance between vasoconstrictor and vasodilators and the effects of various stressors on this transition. To simplify the understanding of how pathological processes affect the pulmonary vascular system, these pathological processes will be grouped and examples provided based on their type and time of occurrence: (1) induced intrauterine vascular abnormalities, (2) anatomical anomalies, (3) abnormal transition to extrauterine life, and (4) abnormal development of the postnatal circulation. An understanding of the importance of how various diseases affect vascular development depending upon the timing within lung

NEONATAL CIRCULATION 85 development and the biology of the pulmonary vascular endothelium will likely lead to important therapeutic interventions in the near future that will include molecular biology and endothelial conditioning strategies.

walls of the pulmonary artery, veins (vasa vasorum), and nerves. Vasculogenesis and Angiogenesis

Development of the Circulatory System Adaptation to the extrauterine environment requires morphologically appropriate pulmonary vascular development, both arterial and venous, and adequate biochemical and biophysical response of this vasculature. The pulmonary vascular growth closely follows the stages of pulmonary parenchymal growth and development (Figure 1). At 4 weeks of human development, an endodermal lung bud arises from the primitive gut, which is surrounded by mesenchymal vascular network. As gestation progresses, the vessels accompany the developing airways branch synchronously with airways branching. The pulmonary veins arise from the mesenchyme of the lung septa and ultimately join the left atrium. Bronchial arteries arise from the descending aorta, intercostals, subclavian, or internal mammary arteries. There are two classes of bronchial arteries: (1) extrapulmonary, which give off small branches to the esophagus, mediastinal tissue, hiliar lymph nodes, and lobar bronchi and (2) intrapulmonary, which distribute blood to the supporting tissue and structures of the intralobal bronchi (mucous membranes, muscle, perichondrium, secretory glands), pulmonary pleura,

Fetal stage

Airway trachea

Arterial development

Extrapulmonary artery 34 days

Embryonic 0−7 weeks

Lobar arteries 44 days Bronchi

Preacinar arteries 11–23 generations − conventional plus supernumeraries

Pseudoglandular 7−17 weeks Bronchioli

Canalicular 17−27 weeks

Two important processes that contribute to the development of the pulmonary vasculature are vasculogenesis (differentiation and segregation of angioblasts from the mesoderm) and angiogenesis (the sprouting of new vessels from pre-existing vessels or vascular channels). In addition to vasculogenesis and angiogenesis a third process, fusion, is ultimately necessary to connect the angiogenic and vasculogenic vessels and expand the vascular network. Induction of local growth factors such as basic fibroblast growth factor (FGF), transforming growth factor beta (TGF-b), and platelet-derived growth factor (PDGF) are all important for angiogenesis, endothelial proliferation, collagen production, and smooth muscle and cell differentiation. Normal vascular development may also require the expression of angiostatic molecules such as endothelial monocyte activating peptide-2 (EMAP-2), which induces endothelial apoptosis. In addition, other stimuli contribute to both alveolar and vascular growth and development and in part involve cross-talk of paracrine signals between the epithelium and mesenchyme. An example of one such signal is vascular endothelial growth factor (VEGF). The exact stimuli for production of these local

Respiratory bronchioli Alveolar ducts

Saccular/Alveolar 28 weeks − term Alveoli pleura

5−17 weeks Intra-acinar arteries 3−5 generations 18−25 weeks Alveolar duct arteries 2−3 generations 25 weeks−18 months

Acinus

Alveolar capillaries 30 weeks−18 yrs

Figure 1 Stages of lung airway and vascular development. Adapted from Hislop A (2005) Developmental biology of the pulmonary circulation. Paediatric Respiratory Reviews 6: 36, with permission from Elsevier.

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NEONATAL CIRCULATION

factors remain to be elucidated, but they are clearly important in vascular development. Initially the endothelial cells form channels but as development progresses these channels acquire a smooth muscle layer, with the ability to regulate flow and vascular tone. The smooth muscle cells are derived from the adjacent bronchial smooth muscle, and later from the mesenchyme. The smooth muscle cells of the airways, arteries, and veins show progressive and gradual maturation of their cytoskeletal structure. The arterial smooth muscle cells change structurally during development and continue postnatally (Figure 2). Normal postnatal pulmonary arterial development can be divided into three structural stages: Stage I: adaptation to extrauterine life from birth to approximately 4 days. Stage II: structural stabilization that lasts until 3–4 weeks of age. Stage III: growth that continues to adulthood. During stage I, endothelial cells become thinner and show less overlap, vessel wall becomes thinner, and lumen diameter increases. This stage is characterized by dilatation and recruitment which occurs in the first 24 h of life in the nonmuscular and partially muscular precapillary bed. There is an increase in cell length, the cells spread out within the vessel wall to increase lumen diameter and lower vascular resistance. Structural changes during stage II

Adult

Child

Fetus

include the deposit of connective tissue around the cells in order to ‘fix’ themselves to the new wall structure. During stage III, the intrapulmonary arteries increase in size and their wall thickness increases. Cross-sectional area of the precapillary bed is increased to accommodate a larger cardiac output, while maintaining a low vascular resistance. In addition, connective tissue increases and the proportion of the different types of interstitial connective tissue also changes with age. This affects the mechanical properties of the vessel wall, with greater structural stiffness. Additionally, regulation of lung vascular morphogenesis appears to be influenced by oxygen tension. Hypoxia is an important regulator of both vasculogenesis and angiogenesis. In vitro experiments and fetal lung explant studies suggest that exposure to normoxic environments of fetal mammalian cells can be detrimental to embryonic development.

The Fetal Circulation During fetal life, pulmonary vascular resistance (PVR) is greater than systemic vascular resistance. Less than 10% of the combined right and left cardiac output is directed to the pulmonary circulation. The factors responsible for the increase in PVR during fetal life include decreased intrauterine oxygen tension, decrease in vascular cross-sectional area, accumulation of the metabolic products of the

19 yrs 11 yrs 10 yrs 5 yrs 4 yrs 3 yrs 18 mths 10 mths 4 mths 3 days 40 wks 38 wks 36 wks 28 wks Alveolar ducts

Terminal bronchiolus

Respiratory bronchioli

Alveoli

Figure 2 Appearance and extension of the muscle in the wall of intra-acinar arteries as a function of age. Adapted from Haworth SG (1992) Development of the Pulmonary Circulation. In: Polin and Fox Fetal and Neonatal Physiology, vol. 1, p. 672. Philadelphia: Saunders, with permission from Elsevier.

NEONATAL CIRCULATION 87

arachidonic acid pathway, and endothelin. Thus, the fetal pulmonary circulation is a high-resistance, lowflow circuit. The flow of blood during fetal life can be categorized as two parallel circuits. Since fetal pulmonary vascular resistance is high, most of the venous return from the inferior and superior vena cava is shunted via the foramen ovale and the ductus arteriosus to the systemic circulation. Once in the descending

aorta, blood flows via the umbilical arteries to the placenta where oxygenation and removal of CO2 takes place to return via the inferior vena cava through the umbilical vein. The low systemic vascular resistance in the fetus is due to the very low vascular resistance of the placenta. An additional shunt in the liver (ductus venosus) combines umbilical venous blood with systemic venous blood, with return to the right heart (Figures 3–5). Ductus arteriosus

SVC PA

Foramen ovale RA

LA RV

Aorta

Ductus venosus

Umbilical vein

Umbilical arteries

Figure 3 Fetal circulation. Umbilical veins carry oxygenated blood from the placenta, while the two umbilical arteries carry systemic blood to the placenta for oxygenation. LA, left atrium; PA, pulmonary artery; RA, right atrium; RV, right ventricle; SVC, superior vena cava. Adapted from Bloom RS (2002) Delivery room resuscitation of the newborn. In: Fanaroff A and Martin RJ (eds.) Neonatal– Perinatal Medicine, 7th edn., vol. 1, p. 417. St Louis: Mosby, with permission from Elsevier.

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Ductus arteriosus

Left heart

Right heart

Body

Foramen ovale

Figure 4 Diagram of the fetal parallel circulation. Note that the majority of the right ventricular output goes via the ductus arteriosus to supply the brain and the rest of the body. The placenta performs the functions of oxygenation and carbon dioxide removal.

Transition to the Extrauterine Environments and Postnatal Blood Flow At birth, several events and local and systemic factors intervene to change the pulmonary circulation from a high-resistance low-flow circuit to a low-resistance higher-flow one. Further, the systemic circulation changes from a low-resistance to a higher-resistance circuit. Upon clamping the umbilical vessels, the placental circulation is no longer operative, and thus systemic vascular resistance increases (Figure 6). The mechanisms responsible for the postnatal fall in PVR are uncertain but the initial rapid dilatation of the pulmonary vasculature is stimulated by rhythmic ventilation, increase in oxygen tension, and increase in vascular shear stress. The sudden increase in flow and shear stress lead to production of potent vasodilators such as endothelial derived nitric oxide (eNO), prostacyclin (PGI2), and bradykinin. Subsequently, maintenance of low-pressure system in the pulmonary vasculature becomes a balance between vasodilators (eNO) and vasoconstritors (endothelin-I) (Table 1). Functional closure of the ductus arteriosus also occurs over the initial 24–72 h after birth. Factors that are responsible for this initial functional closure

include increased arterial O2 tension, plasma catecholamine levels, suppression of PGI2, and switching off of prostaglandin E receptors. Complete anatomical closure of the ductus can take 2–3 weeks. The transition from fetal to neonatal circulation involves several mechanisms involving mechanical and humoral factors, which act together to promote transition from a parallel circulation to a series one.

Pathophysiology To simplify the understanding of how pathological processes affect the pulmonary vascular system, these pathological processes can be based upon their type and time of occurrence: (1) induced intrauterine vascular abnormalities, (2) anatomical anomalies, (3) abnormal transition to extrauterine life, and (4) abnormal development of the postnatal circulation. Induced Intrauterine Vascular Anomalies

Induced vascular abnormalities have been documented after intrauterine exposure to a host of medications. Prostaglandin inhibition, such as occurs with prenatal administration of nonsteroidal

NEONATAL CIRCULATION 89

Right heart

Left heart

Body

Figure 5 Diagram of the postnatal circulation in series. The ductus arteriosus and foramen ovale are no longer functional, and thus the entire output from the right ventricle goes to the lungs. The left ventricular output supplies the brain and the rest of the body. Oxygenation and carbon dioxide removal is solely achieved in the lungs.

Pulmonary

Systemic

550

250 Left

450 400

200

Ductal shunt

150

Right

100

350 300 250 200

Ventricular output (ml/kg/min)

Resistance (mmHg/l/kg/min)

500

50

150 0

100 Birth

2−6

6−12

12−25

25−54 (hrs)

Age Figure 6 Changes in vascular resistance and ventricular output as a function of age in hours. Note the very high pulmonary vascular resistance and low systemic vascular resistance at birth. The decrease in pulmonary vascular resistance occurs abruptly after birth, and achieves its adult levels early. The increase in systemic vascular resistance also occurs abruptly and decreases over time. Yellow line depicts the right ventricular output and the black line the left ventricular output, changes over age in hours. The difference in ventricular outputs is accounted by the presence of a ductal shunt in the first 24 h of age. Adapted from Nelson N (1999) The onset of respiration. In: Avery G, Fletcher MA, and MacDonald MG (eds.) Neonatology: Pathophysiology and Management of the Newborn, 5th edn., p. 264. Baltimore: Lippincott Williams & Wilkins, with permission.

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Table 1 Mediators of pulmonary vascular tone in fetal and transitional circulation Vasoconstrictors: maintain high fetal PVR

Vasodilators: decrease PVR during transition

Norepinephrine a-Adrenergic stimulation Hypoxia Endothelin Thromboxane Leukotrienes PAF PDF2a

PGI2, PGD2, PGE2 Nitric oxide CGMP Oxygen Adenosine ATP Bradykinin

ATP, adenosine triphosphate; cGMP, cyclic guanosine monophosphate; PAF, platelet-activating factor; PDF, platelet-derived factor; PG, prostaglandin; PVR, pulmonary vascular resistance. Adapted from Dukarm RC, Steinhorn RH, and Morin FC (1996) The normal pulmonary vascular transition at birth. Clinics in Perinatology 23(4): 714, with permission from Elsevier.

anti-inflammatory agents, can produce pulmonary vascular hypoplasia. Thalidomide, now banned for use in pregnancy, is a well-known antiangiogenic agent; exposure of human and animals prenatally leads to pulmonary vascular dysregulation with vascular hypoplasia. Antenatal administration of corticosteroids accelerates lung maturation of type II pneumocytes, thinning of the double capillary loops during sacular and alveolar stages, and partial suppression of the formation of secondary septa. Moreover, corticosteroids may also decrease somatic and lung growth. However, antenatal treatment with corticosteroids in humans does not appear to affect lung function as measured at 7 years of age. Angiotensin converting enzyme inhibitor (ACE-I) drugs during gestation have been associated with high risk of fetal morbidity, intrauterine growth retardation, oligohydramnios, and renal failure. Pulmonary hypoplasia has also been reported and the use of these drugs should be avoided during pregnancy. In general, the above-mentioned drugs administered during pregnancy produce pulmonary vascular dysregulation and dysmorphogenesis. Anatomical Anomalies

Because of the temporal and physical proximity and interaction of lung development on airway and vascular growth, it is not surprising that lung developmental pathologies also affect the pulmonary vasculature. In congenital diaphragmatic hernia (CDH), there is an equal reduction of arteries and airway numbers. These newborns also have pulmonary hypertension with an increase in arterial wall thickness. Newborns with renal agenesis (Potter

syndrome), thoracic dystrophy, and idiopathic pulmonary hypoplasia have a similar reduction in their pulmonary vascular bed. The above pathologies share the commonality of abnormal lung growth. Abnormal Transition to Extrauterine Life

The hallmark of an abnormal transition to extrauterine life is persistent pulmonary hypertension of the newborn (PPHN). This is defined as a failure of the normal pulmonary vascular relaxation at or shortly after birth which results in increased impedance to pulmonary blood flow such that pulmonary vascular pressure exceeds systemic vascular resistance. The end result is that deoxygenated blood is shunted via the foramen ovale and ductus arterious to the systemic circulation. Perinatal stressors that can produce PPHN include hypoxia, hypoglycemia, cold stress, sepsis, and direct lung injury. In the initial course of PPHN the pulmonary vasospasm is dynamic, with labile flow via the pulmonary circuit and right-to-left shunting. However, in time this dynamic vasospasm is ultrastructurally transduced to thickened vascular media with hyperplasia and hypertrophy of the smooth muscle layer and increased extracellular matrix deposition, thereby producing a fixed vascular resistance. While the latter biphasic response is a well-known response, the timeframe of disease progression varies between different pathophysiologies. Hypoxia produces endothelial dysfunction, with dysfunction in the regulation of the endothelin-1/eNO balance in favor of the vasoconstrictor effect of endothelin-1. Chronic hypoxia has been shown to inhibit the postnatal maturation of the pulmonary artery relaxation by both endothelium-dependent and -independent means and impairment in eNO release and expression. Chronic hypoxia also impairs the normal course of cardiovascular transition since the rapid change in smooth muscle cytoskeletal architecture in the vascular media from densely shaped to elongated shape fails to occur. In addition to the hypoxic pulmonary vasoconstriction produced by direct lung injury such as occurs in meconium aspiration or pneumonia, these conditions may alter the cyclooxygenase pathway. This causes increased thromboxane/prostacyclin ratio, upregulation of cyclooxygenase-2, and activation of inducible nitric oxide synthase leading to prolonged production of large amounts of nitric oxide. Inflammatory mediators such as cytokines that are produced during direct lung injury further amplify the pulmonary hypertension by causing vascular smooth muscle contraction and arterial fibrosis. The end result of this complicated cascade is further endothelial

NEONATAL CIRCULATION 91

dysfunction and ultimately vascular remodeling and fixed vascular resistance. Early stages of dynamic pulmonary vasospasm appear to respond to the therapeutic use of inhaled nitric oxide; however, as fixed vascular resistance occurs, inhaled nitric oxide and other therapies are less likely to be successful. It is beyond the scope of this article to discuss therapeutic modulation of the pulmonary vasculature. Abnormal Development of the Postnatal Circulation

The pathologies that comprise abnormal development of the postnatal circulation have a commonality of potentially irreversible vascular remodeling. The substrate is most often an immature lung which has undergone mechanical ventilatory support, exposure to high concentrations of alveolar oxygen, inflammation, volutrauma, and barotrauma. This leads to underdevelopment or maldevelopment of both lung parenchyma and vasculature. An important example of this process is chronic lung disease (CLD) in premature infants or bronchopulmonary dysplasia (BPD). The risk of CLD increases with decreasing birthweight and gestation, with an incidence of greater than 85% in newborns born with 500– 700 g. The developmental timing of the lung injury is a critical factor. In addition to altered pulmonary structure of increased smooth cell proliferation, altered vasoreactivity, and impaired metabolic endothelial function, there is decreased aleveolarization and angiogenesis. It is therefore not surprising that pulmonary hypertension is a common finding in CLD.

Conclusions It is apparent that normal transition to the extrauterine environment requires normal development of the vascular system, adequate biochemical and biophysical response of this vasculature, and the absence of either intrauterine or postnatal insults. Control and modulation of the PVR during deranged conditions remains a formidable research problem. Understanding the complexity of endothelial function and dysfunction and the interdependence of each factor on pulmonary vascular growth (both arterial and venous) will undoubtedly lead to new therapies and interventions. See also: Bronchopulmonary Dysplasia. Fibroblast Growth Factors. Lung Development: Overview. Platelet-Derived Growth Factor. Pulmonary Vascular Remodeling. Transforming Growth Factor Beta

(TGF-b) Family of Molecules. Vascular Endothelial Growth Factor.

Further Reading Bloom RS (2002) Delivery room resuscitation of the newborn. In: Fanaroff A and Martin RJ (eds.) Neonatal–Perinatal Medicine, 7th edn., vol. 1, p. 417. St Louis: Mosby. Boels PJ, Deutsch J, Gao B, and Haworth SG (2001) Perinatal development influences mechanisms of bradykinin-induced relaxations in pulmonary resistance and conduit arteries differently. Cardiovascular Research 51: 140–150. Bourbon J, Boucherat O, Chailley-Heu B, and Delacourt C (2005) Control mechanism of the lung alveolar development and their disorders in bronchopulmonary dysplasia. Pediatric Research 57: 38R–46R. Dakshinamurti S (2005) Pathophysiologic mechanism of persistent pulmonary hypertension of the newborn. Pediatric Pulmonology 39: 492–503. Dukarm RC, Steinhorn RH, and Morin FC III (1996) The normal pulmonary vascular transition at birth. Clinics in Perinatology 23: 711–726. Fanaroff AA and Martin RJ (2002) Neonatal–Perinatal Medicine: Diseases of the Fetus and Infant. St Louis: Mosby. Ghanayem NS and Gordon JB (2001) Modulation of pulmonary vasomotor tone in the fetus and neonate. Respiratory Research 2: 139–144. Hall SM, Hislop A, and Haworth SG (2002) Origin, differentiation, and maturation of human pulmonary veins. American Journal of Respiratory Cell and Molecular Biology 26: 333–340. Haworth SG (1992) Development of the Pulmonary Circulation. In: Polin and Fox Fetal and Neonatal Physiology, vol. 1, p. 672. Philadelphia: Saunders. Hislop A (2005) Developmental biology of the pulmonary circulation. Paediatric Respiratory Reviews 6: 35–43. Kotecha S (2000) Lung growth: implications for the newborn infant. Archives of Disease in Childhood Fetal and Neonatal Edition 82: F69–F74. Lakshminrusimha S and Steinhorn RH (1999) Pulmonary vascular biology during neonatal transition. Clinics in Perinatology 26: 601–619. Nelson N (1999) The onset of respiration. In: Avery G, Fletcher MA, and MacDonald MG (eds.) Neonatology: Pathophysiology and Management of the Newborn, 5th edn., p. 264. Baltimore: Lippincott Williams & Wilkins. Parker T and Abman SH (2003) The pulmonary circulation in bronchopulmonary dysplasia. Seminars in Neonatology 8: 51–62. Perreault T and Coceani F (2003) Endothelin in the perinatal circulation. Canadian Journal of Physiology and Pharmacology 81: 644–653. Polin RA and Fox WW (1992) Fetal and Neonatal Physiology. Philadelphia: Saunders. Stenmark KR and Abman SH (2005) Lung vascular development: implications for the pathogenesis of bronchopulmonary dysplasia. Annual Review of Physiology 67: 623–661. Xue C, Reynolds PR, and Johns RA (1996) Developmental expression of NOS isoforms in fetal rat lung: implications for transitional circulation and pulmonary angiogenesis. American Journal of Physiology: Lung Cellular and Molecular Physiology 270: L88–L100. Ziegler JW, Ivy D, Kinsella JP, and Abman SH (1995) The role of nitric oxide endothelin and prostaglandins in the transition of the pulmonary circulation. Clinics in Perinatology 22: 387–403.

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NEUROFIBROMATOSIS T E King Jr, San Francisco General Hospital, San Francisco, CA, USA A C Zamora, Hospital Universitario ‘Dr Jose E Gonzalez’, Monterrey Nuevo Leon, Mexico & 2006 Elsevier Ltd. All rights reserved.

Abstract Neurofibromatosis (NF) is a relatively common autosomal dominant genetic disorder characterized by neurological and cutaneous lesions. NF-1 (von Recklinghausen’s disease) accounts for approximately 85% of the patients and it is this form that is most likely to demonstrate lung involvement. Respiratory involvement occurs in 10–15% of patients with NF, most commonly in adults. The thorax and lungs can be affected in several ways: cutaneous and subcutaneous neurofibromas on the chest wall that can overlay the lung and appear as lung nodules; kyphoscoliosis; ribbon deformity of the ribs; thoracic neoplasms; diffuse interstitial fibrosis; or bullae.

Introduction Neurofibromatosis (NF) is a relatively common disorder characterized by neurological and cutaneous lesions. There are two major forms: type 1 (NF-1, von Recklinghausen’s disease) and NF-2 (formerly known as bilateral acoustic NF or central NF) (Table 1). NF-1 accounts for approximately 85% of the patients and its prevalence in the population is 1 in 5000. In 30–50% of cases of NF-1 there is no family history; these sporadic cases probably arise from (usually paternal) germ cell mutations. NF-2 is the less common form affecting about 1 in 40 000 persons, with a prevalence within the population of 1 in 210 000. Sporadic gene mutations occur in 50% of cases. NF-2 is characterized by bilateral tumors on the eighth cranial nerve, which cause progressive hearing loss.

Cutaneous lesions are the result of the maldevelopment of neural crest cells. The number of dermal neurofibromas varies from individual to individual. Large plexiform neurofibromas develop along peripheral nerves and involve deeper tissues. Extracutaneous (visceral) involvement may not be apparent during life unless such lesions produce symptoms. In NF-1, the thorax and lungs can be affected in several ways: cutaneous and subcutaneous neurofibromas on the chest wall that can overlay the lung and appear as lung nodules; kyphoscoliosis; ribbon deformity of the ribs; thoracic neoplasms; diffuse interstitial fibrosis; or bullae.

Pathology Neurofibromas are hamartomatous in nature and of multicellular origin; they are composed mostly of Schwann cells, but also contain fibroblasts, mast cells, and macrophages. Vascular histologic changes in NF are commonly described in six types (intimal, advanced intimal, nodular-aneurysmal, periarterial, epithelioid-cell, and pericapillary). Immunohistochemical and electron microscopic observations suggest a smooth muscle cell origin of proliferating spindle-shaped cells. The histopathologic findings in the lung are described below.

Clinical Features NF is extremely variable in its symptoms, signs, intensity, and progression. There is no discrimination in its sexual, racial, ethnic, or national distribution. Table 2 provides the diagnostic criteria for NF-1. These criteria are both highly specific and sensitive in all but the youngest children. The following features confirm a diagnosis of NF-2: bilateral vestibular

Table 1 Classification of neurofibromatosis Variable

Neurofibromatosis type 1

Neurofibromatosis type 2

Inheritance Penetrance Incidence Prevalence Features

Autosomal dominant Complete 1/2600 to 1/4000 1/5000 Neurofibromas, cafe-au-lait macules, learning disabilities, skeletal dysplasia NF-1 chromosome 17q11.2 Neurofibromin GTPase activating protein

Autosomal dominant Complete 1/40 000 1/210 000 Vestibular schwannomas, other schwannomas, meningiomas, ependymomas, cataracts NF-2 chromosome 22 Merlin or schwannomin Cytoskeletal protein

Gene Protein Function

Adapted from Korf BR, Henson JW, and Stemmer-Rachamimov A (2005) Case 13-2005 – a 48-year-old man with weakness of the limbs and multiple tumors of spinal nerves. New England Journal of Medicine 352: 1800–1808.

NEUROFIBROMATOSIS 93 Table 2 Diagnostic criteria for neurofibromatosis type 1 At least two of the following clinical features must be present: 1. Six or more cafe´-au-lait spots/macules
45 mm in prepubertal patients
415 mm in postpubertal patients 2. Two or more neurofibromas of any type or one plexiform neurofibroma 3. Skin fold freckle (groin, axilla, and neck base) 4. Optic glioma (detected by magnetic resonance imaging) 5. Lisch nodules (X2 iris hamartomas) 6. Skeletal dysplasia: a distinctive bony lesion such as sphenoid dysplasia or thinning of the long bone cortex with or without psuedoarthrosis 7. Family history: a first degree relative (parent, sibling, or offspring) with NF-1 based upon the above criteria Adapted from National Institutes of Health Consensus Development Conference (1988) Neurofibromatosis: conference statement. Archives of Neurology 45: 575–578 and Korf BR, Henson JW, and Stemmer-Rachamimov A (2005) Case 13-2005 – a 48-year-old man with weakness of the limbs and multiple tumors of spinal nerves. New England Journal of Medicine 352: 1800–1808.

schwannomas or family history of NF-2 (first degree relative) plus either unilateral vestibular schwannoma in patients o30 years old or any two of the following: meningioma, glioma, schwannoma, and juvenile posterior subcapsular lenticular opacities/juvenile cortical cataract. General Features

The typical order of appearance of clinical features is cafe-au-lait spots, axillary freckling, Lisch nodules, and neurofibromas (Figure 1). Cafe´-au-lait spots are present in 99% of the patients. They may be difficult to distinguish in individuals of African descent. Freckling occurs in areas of skin apposition, especially the axillary and groin areas. Freckling is usually not apparent at birth but often appears during early childhood. Neurofibromas are characteristic tumors of NF that grow especially during puberty and pregnancy. They almost always involve the skin,

(a)

(b)

(c)

(d)

Figure 1 (a) Cafe-au-lait spots: flat; uniformly hyperpigmented macules that range in size from 1 to 60 mm and vary in number from at least six to hundreds. Reproduced from http://www.nfinc.org/what.shtml Neurofibromatosis, Inc., PO Box 18246, Minneapolis, MN 55418, with permission. (b) Neurofibromas: cutaneous neurofibromas are benign and do not carry an increased risk of developing malignant transformation. However, they often represent a major cosmetic problem in adults. (c) Lisch nodules in iris: bilateral, welldefined, dome-shaped, gelatinous elevations from the iris surface, ranging from clear to yellow or brown in color. (d) Freckles in skin fold of axilla. Reproduced from http://www.nfinc.org/what.shtml Neurofibromatosis, Inc., PO Box 18246, Minneapolis, MN 55418, with permission.

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but they may occur in deeper peripheral nerves and nerve roots and in or on viscera and blood vessels innervated by the autonomic nervous system. Multiple cranial nerves may be involved resulting in facial weakness or numbness, visual or auditory disturbances. The tumors may be painful or tender under pressure. Lisch nodules are pigmented iris hamartomas that are present in 94% of patients 6 years old or older. Bony abnormalities include pseudoarthrosis (or false joint), bone dysplasia, short stature (13% have a height 2 standard deviations below the population mean), and scoliosis (10–25% of affected individuals). Neurologic disorders include seizures and neuroimaging abnormalities. Macrocephaly is a common feature. Approximately 40– 60% of children with NF-1 manifest below average performance on standardized IQ tests and demonstrate problems with reading, writing, and use of numbers. NF patients rarely have mental retardation. Systemic arterial hypertension is a frequent finding in adults with NF-1 and may develop during childhood. NF is associated with an increased incidence of malignancy, ranging from malignant tumors of the central nervous system to Wilms’ tumor, rhabdomyosarcoma, juvenile chronic myeloid leukemia, and pheochromocytoma. The most common are central nervous system neoplasms (e.g., astrocytomas and brainstem gliomas) and optic pathway gliomas.

Thoracic Involvement

Table 3 lists diseases such as upper airway, chest wall, mediastinal, thoracic neoplasms, and lung parenchyma.

Table 3 Thoracic manifestations of neurofibromatosis Upper airway Tracheal neurogenous tumors Laryngeal neurogenous tumors Chest wall Subcutaneous neurofibromas Kyphoscoliosis Rib notching (inferior) from intercostal neurofibroma Apical neurofibroma (Pancoast’s syndrome) Mediastinal Neurogenic tumors Plexiform neurofibroma Meningocele Vegal nerve neurofibroma Thoracic neoplasms Malignant degeneration of neurofibromas Metastatic neural tumor Lung parenchyma Bullous lung disease Interstitial lung disease

Upper airway An estimated 5% of patients with NF-1 have an intraoral manifestation of the disease. Discrete neurofibromas may involve the tongue, the larynx, or occur in the cervical region where large tumors of the parapharyngeal space may result in distortion of the airway. These lesions may cause obstruction, and symptoms of dyspnea, stridor, loss or change of voice, or dysphagia. Tracheal and endotracheal neurofibromas are rare. Hoarseness may result from recurrent laryngeal nerve involvement. It has been suggested that radiographic examination of the neck be performed before anesthesia in these patients in order to examine the upper airway and to avoid spinal cord damage during laryngoscopy and tracheal intubation. Thoracic cage Rib abnormalities are common in patients with NF. They have been described as twisted ribbon deformity. Notching of the ribs occurs secondary to erosion by intercostal neurofibromas or a primary defect in bone formation. Vertebral abnormalities Scoliosis is common and occurs in 10–58% of patients with NF. It typically involves the lower cervical and upper thoracic spine in children aged 5–15 years of age, and there is no sex predilection. Although scoliosis is often associated with paravertebral neurofibromas, it is not clear whether the tumors themselves are the cause of the vertebral column distortion. Other possible causes of spinal deformity in NF include osteomalacia, a localized neurofibromatous tumor eroding and infiltrating bone and mesodermal dysplasia. Thoracic neoplasms Cutaneous and subcutaneous neurofibromas on the chest wall can overlay the lung and appear as lung nodules. On the other hand, primary or secondary malignancy in the lung has been overlooked because of confusion caused by overlying cutaneous lesions on X-rays. In 5% of patients, the neurofibromas in NF undergo transformation to malignant degeneration and commonly metastasize to the lungs. Neurofibromas may be found in virtually any location including the mediastinum and account for about 10–20% of primary mediastinal masses in adults (Figure 2). One-third of patients with neurofibromas have NF. Neurofibromas arise from peripheral nerves and nerve sheath. The radiograph features are a round, elliptical, or lobulated paravertebral mass, often one or two interspaces in length. Neurogenic neoplasms that arise in the intercostal nerves can be associated with rib destruction and a chest wall mass. Paraspinal neurofibromas may grow

NEUROFIBROMATOSIS 95

Figure 2 Neurofibroma: mediastinal mass. Photo courtesy of W R Webb, Department of Radiology, University of California  San Francisco.

to an enormous size, giving rise to the classic thoracic ‘dumbbell’ tumor (Figure 3). Plexiform neurofibromas are the hallmark lesion of NF-1. These occur in 30% of NF-1 patients, are usually congenital, and affect long portions of the nerve involved; infiltration of the nerve itself and the surrounding tissue may occur, giving rise to extensive disfiguration. Plexiform neurofibromas are multiple and lobulated, have ill-defined margins with mediastinal fat, and surround great vessels with loss of normally visible fat planes. They can mimic the appearance of extensive mediastinal lymph node enlargement (Figure 4). Malignant progression occurs in 2–16% of patients and is the major cause of morbidity and mortality in NF-1. Meningocele Intrathoracic meningocele has been associated with NF (64% of cases). It represents anomalous herniation of the spinal meninges through an intervertebral foramen or a defect in the vertebral body. It is slightly more common on the right side. Meningocele commonly presents as an incidental finding (60%) or with dyspnea (23%). On chest radiographs, a meningocele is a sharply marginated paravertebral soft tissue mass associated with scoliosis or rib and vertebral defects at the same levels. On computed tomography (CT), it has low attenuation because it contains cerebrospinal fluid. Pulmonary vascular involvement NF can involve vessels throughout the body, such as the retinal, lumbar, mesenteric, renal, and both the intracranial and extracranial arteries. The incidence of vascular lesions has been reported to be 3.6%. Involvement of

Figure 3 Paraspinal neurofibromas. Coronal T1-weighted MRI showing large dumbbell neurofibroma in T2/T3 intervertebral foramen and occupying the apex of the left thoracic cavity and displacing the spinal cord to the right. Reproduced from Hirsch NP, Murphy A, and Radcliffe JJ, Neurofibromatosis: clinical presentations and anaesthetic implications, British Journal of Anaesthesia, 2001, 86, 555564, & The Board of Management and Trustees of the British Journal of Anaesthesia. Reproduced by permission of Oxford University Press/British Journal of Anaesthesia.

Figure 4 Plexiform neurofibromas in NF. Mediastinal widening (large arrows) reflects the presence of multiple neurofibromas along the courses of mediastinal nerves. Extensive intercostals neurofibromas (small arrows) mimic pleural thickening. Photo courtesy of W R Webb, Department of Radiology, University of California  San Francisco.

the pulmonary artery has been reported in a patient with severe pulmonary hypertension. Hemothorax Spontaneous hemothorax occurs more frequently in women and is most often found on the left side. The intercostal artery and the subclavian artery are the most common ruptured

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arteries. Biopsy results reveal neurofibromatous invasion of the vessel wall. The prognosis is poor (50% survival) especially when the blood loss is massive. The recommended treatment is immediate exploratory thoracotomy and ligation, bypass, or replacement of the affected vessel. Diaphragmatic paralysis Involvement of the phrenic nerves, both central (posterior fossa tumor) or peripheral root nerves, by NF has occasionally caused diaphragmatic paralysis. Parenchymal abnormalities Diffuse parenchymal lung disease has an incidence of between 10% and 33% among patients with NF. The number of affected men appears to outnumber affected women but this may be explained by the fact that the largest number of cases comes from studies on patients from Veterans Administration Hospitals. Diffuse interstitial fibrosis and bullae, either alone or in combination, are the best described pulmonary parenchymal lesions.

Figure 5 Chest radiograph shows large lung volumes with upper lobe hyperlucency (right greater than the left) and a reticular pattern predominant at the lung bases. Photo courtesy of W R Webb, Department of Radiology, University of California  San Francisco.

Clinical features NF is usually diagnosed years before the appearance of lung disease. Age at diagnosis ranges from 23 to 72 years with a mean of 50 years for studies. In most patients, the finding of lung disease was incidental, but dyspnea on exertion was the most common complaint. There are no apparent abnormal physical signs on examination. Coexisting NF and pulmonary fibrosis has been reported in a mother and son, but a familial predisposition is rare. Pulmonary function test Generally, lung function testing shows an obstructive pattern, but a restrictive defect may be dominant. A decreased diffusion capacity (DLCO) and hypoxemia can be seen. Radiographic findings Bullous lung disease may occur alone or in combination with diffuse interstitial lung disease. The interstitial changes are usually diffuse, bilateral, basal, and subpleural (Figure 5). Large apical asymmetric thin-walled bullae sometimes occupy a major portion of the hemithorax associated with areas of hypovascularity (Figure 6). Classical honeycombing is rare. Diminished perfusion and ventilation to apices of the lungs have been documented by radionuclide studies in a patient with cutaneous NF. Histopathological findings The pulmonary parenchymal disease is attributed to a mesenchymal defect resulting in primary deposition of collagen. Ultrastructural studies have shown fragmentation of collagen fibers in the lung. The pathologic features of

Figure 6 High-resolution computed tomography (HRCT) shows cysts and bullae often indistinguishable from emphysema. Photo courtesy of W R Webb, Department of Radiology, University of California  San Francisco.

the pulmonary parenchymal lesions, at autopsy or lung biopsy, revealed areas of relatively normal lung alternated with patchy interstitial fibrosis, thickening, and cellularity of the alveolar septa. The findings appear similar to those found in non-specific interstitial pneumonia rather than usual interstitial pneumonia. In more advanced stages, the lesions show more severe fibrosis and obliteration of alveoli and blood vessels with honeycombing. Complications Interstitial lung disease in NF may be severe enough to result in cor pulmonale. Scar

NEUROFIBROMATOSIS 97

carcinoma can develop, especially if the patient is a smoker.

Pathogenesis NF-1 is an autosomal dominant genetic disorder with an incidence of approximately 1 in 2600 to 1 in 4000 individuals (Table 1). Up to 50% of cases are spontaneous. The gene is found on chromosome 17, which functions as a tumor suppressor predisposing to the development of cancer. Mutations at the NF-1 gene result in diminished levels of neurofibromin with resultant development of the wide variety of tumors seen in the disease. The pathogenesis of the pulmonary process remains ill defined.

Animal Models A number of approaches have been taken to develop models for the tumors seen in individuals affected with NF-1. Initial studies focused on the generation of mice with a targeted mutation in the NF-1 gene. Second-generation models included NF-1  /  chimeric mice and NF-1 exon-specific knockout mice. Recently, tissue-specific inactivation of NF-1 has yielded important insights into the function of neurofibromin in specific cell types and the consequence of NF-1 loss on tumorigenesis in NF-1. Similar approaches have been taken for the study of NF-2. Pathologic analyses of a transgenic murine model for NF-2 demonstrated a high incidence of Schwann cell-derived tumors and Schwann cell hyperplasia. This model has been shown to develop spontaneous schwannomas detected by magnetic resonance imaging (MRI). Transgenic mice (Nf1 þ /  ) lacking neurofibromin expression in astrocytes have been developed to closely mimic NF-1. They develop optic nerve and optic chiasm low-grade fibrillary astrocytomas, which resemble their human counterparts in some respects but lack the characteristic microcystic spaces, Rosenthal fibers, and granular bodies.

Management and Current Therapy Genetic counseling is the only preventive approach to NF. No specific treatment is available for the pulmonary parenchymal lung disease. See also: Genetics: Overview. Interstitial Lung Disease: Overview. Oncogenes and Proto-Oncogenes:

Overview. Transgenic Models. Tuberous Sclerosis. Tumors, Malignant: Overview.

Further Reading Bourgouin PM, Shepard JO, Moore EH, et al. (1988) Plexiform neurofibromatosis of the mediastinum: CT appearance. American Journal of J Roentgenology 151: 461–463. Casselman ES and Mandell GA (1979) Vertebral scalloping in neurofibromatosis. Radiology 131: 89–94. Casselman ES, Miller WT, Shu Ren L, et al. (1977) von Recklinghausen’s disease: incidence of roentgenographic findings with a clinical review of the literature. CRC Critical Review of Diagnostic Imaging 9: 387–419. Chaglassian JH, Riseborough EJ, and Hall JE (1976) Neurofibromatous scoliosis. Natural history and results of treatment in thirty-seven cases. Journal of Bone and Joint Surgery of America 58: 695–702. Dolynchuk KN, Teskey J, and West M (1990) Intrathoracic meningocele associated with neurofibromatosis: case report. Neurosurgery 27: 485–487. Friedman JM and Birch PH (1997) Type 1 neurofibromatosis: a descriptive analysis of the disorder in 1,728 patients. American Journal of Medical Genetics 70: 138–143. Gutmann DH and Giovannini M (2002) Mouse models of neurofibromatosis 1 and 2. Neoplasia 4: 279–290. Hirsch NP, Murphy A, and Radcliffe JJ (2001) Neurofibromatosis: clinical presentations and anaesthetic implications. British Journal of Anaesthesia 86: 555–564. Israel-Asselain R, Chebat J, Sors C, et al. (1965) Diffuse interstitial pulmonary fibrosis in a mother and son with von Recklinghausen’s disease. Thorax 20: 153–157. Klatte EC, Franken EA, and Smith JA (1976) The radiographic spectrum in neurofibromatosis. Seminars in Roentgenology 11: 17–33. Korf BR, Henson JW, and Stemmer-Rachamimov A (2005) Case 13-2005 – A 48-year-old man with weakness of the limbs and multiple tumors of spinal nerves. New England Journal of Medicine 352: 1800–1808. Massaro D and Katz S (1966) Fibrosing alveolitis: its occurrence, roentgenographic and pathologic features in von Recklinghausen’s neurofibromatosis. American Review of Respiratory Diseases 93: 934–942. Massaro D, Katz S, Matthews MJ, et al. (1965) von Recklinghausen’s neurofibromatosis associated with cystic lung disease. American Journal of Medicine 38: 233–240. National Institutes of Health Consensus Development Conference (1988) Neurofibromatosis: conference statement. Archives of Neurology 45: 575–578. Riccardi VM (1981) von Recklinghausen neurofibromatosis. New England Journal of Medicine 305: 1617–1627. Ryu JH, Parambil JG, McGrann PS, and Aughenbaugh GL (2005) Lack of evidence for an association between neurofibromatosis and pulmonary fibrosis. Chest 128: 2381–2386. Samuels N, Berkman N, Milgalter E, et al. (1999) Pulmonary hypertension secondary to neurofibromatosis: intimal fibrosis versus thromboembolism. Thorax 54: 858–859. Webb WR and Goodman PC (1977) Fibrosing alveolitis in patients with neurofibromatosis. Radiology 122: 289–293.

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NEUROMUSCULAR DISEASE / Inherited Myopathies

NEUROMUSCULAR DISEASE Contents

Inherited Myopathies Lower Motor Neuron Diseases Acquired Myopathies Peripheral Nerves Upper Motor Neuron Diseases Myasthenia Gravis and Other Diseases of the Neuromuscular Junction

Inherited Myopathies D Gozal and A D Goldbart, University of Louisville, Louisville, KY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Neuromuscular disorders include a diverse group of diseases with different etiologies. Respiratory failure, presenting both in the acute as well as in the chronic forms, is one of the leading causes of death in most of these diseases. The pathological characteristics of this group of disorders can be classified into five major categories that include muscular dystrophies, congenital and metabolic myopathies, disorders of the neuromuscular junction, peripheral neuropathies, and anterior horn cell disease (spinal muscular atrophies). Symptoms vary according to the type of disease and with the severity and age of onset, but usually include feeding and respiratory difficulties as well as skeletal and muscular abnormalities like scoliosis and kyphosis. Intercurrent illnesses may prompt sudden deterioration of the muscular weakness and lead to respiratory failure. The diagnosis is usually made during the first decade of life and in most cases a muscle biopsy is necessary to establish the diagnosis. However, since most of these disorders are genetically determined, gene testing is available or will soon become available. Interventional measures should involve a multidisciplinary team and are currently restricted to primarily supportive therapy including respiratory muscle training and noninvasive ventilation.

Introduction One of the most common causes of death among patients afflicted with inherited myopathies is respiratory insufficiency. Respiratory failure may present either acutely, as a result of pneumonia, or may develop more chronically as a result of progressive ventilatory decompensation. This article will describe the major inherited myopathies presenting respiratory manifestations, and review the etiology, pathology, clinical features, animal models (when present), and current state-of-the-art therapy in patients with Duchenne muscular dystrophy (DMD), myotonic muscular dystrophy (DM), acid maltose,

limb-girdle and, mitochondrial disorders, since this particular group of diseases accounts for the vast majority of the patients seen by most respiratory practitioners. Patients who suffer from the disorders that will be described in this article usually present with feeding and respiratory difficulties, as well as with skeletal and muscular abnormalities such as scoliosis and kyphosis; intercurrent respiratory illnesses may prompt sudden deterioration and lead to respiratory failure. This article will not deal with upper and lower motor neuron as well as the neuromuscular disorders that will be reviewed elsewhere (Neuromuscular Disease: Lower Motor Neuron Diseases; Acquired Myopathies; Myasthenia Gravis and Other Diseases of the Neuromuscular Junction; Peripheral Nerves; and Upper Motor Neuron Diseases). The mechanisms underlying the development of pulmonary manifestations will be reviewed, and will emphasize the alterations in respiratory mechanics, and in respiratory control during sleep and wakefulness. In the last 20 years, the genetic basis for most of the inherited myopathies and muscular dystrophies has been unveiled. However, extensive work will be needed in future years to identify and understand the relationship of modifier genes producing the phenotypic heterogeneity of many of these diseases, and to characterize targets for therapeutic intervention, whether through gene therapy or using pharmacological approaches.

Etiology Duchenne Muscular Dystrophy

DMD, an X-linked inherited disorder, is the most common muscular dystrophy with an estimated incidence of 1 in 3500 male births. DMD is allelic with Becker muscular dystrophy since both are due to deletions or mutations in the X chromosome (Xp21). The dystrophin gene (2.4 Mb), known to be involved

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in DMD, encodes for a 427 kDa muscle-specific protein carrying the same name. As part of a membrane-bound complex of proteins, dystrophin connects the cytoskeleton of the muscle cell to the extracellular matrix. Congenital Muscular Dystrophy

DM is an autosomal dominant multisystemic disease with a highly variable phenotype. Two genetic loci have been associated with the DM phenotype: DM1 on chromosome 19, and DM2 on chromosome 3. Type 1 myotonic dystrophy or DM1 (also known as Steinert’s disease) can present with myotonia and involvement of the brain, eyes, smooth muscle, cardiac conduction, and endocrine systems. For both DM1 and DM2, the molecular basis of the disease involves expansion of an unstable repeat sequence in a noncoding part of a gene, leading to disruption of mRNA metabolism and induction of aberrant splicing, thereby explaining the multisystemic nature of the condition. Acid Maltase Myopathy

Acid maltase deficiency (AMD) is a rare autosomal recessive genetic disorder that results in the deficiency of lysosomal acid a-1,4 glucosidase (a,a-G). Affected individuals lack the ability to degrade glycogen from the lysosome, causing accumulation of glycogen in the lysosomal storage vacuoles in skeletal and cardiac muscle, liver parenchyma, and the nervous system. The gene encoding for a,a-G has been identified, sequenced, and located to the long arm of chromosome 17. Three forms of AMD have been characterized, namely infantile, juvenile, and adult variants. Age of onset, clinical findings, and severity of symptoms reflect the large phenotypic variability of this disease, with the main cause of death among adults with AMD being respiratory failure. Limb-Girdle Muscular Dystrophy

The limb-girdle muscular dystrophy (LGMD) phenotype is defined by involvement of the pelvic and shoulder girdles, generally with an onset in the second or third decade of life, and often with a mild progression. LGMD is divided into six autosomal dominant forms (LGMD1A–F) and 10 autosomal recessive forms (LGMD2A–J) that represent most of the patients, and differ on age of onset, severity, and clinical presentation. Generally, these diseases will present with muscle weakness, frequently with high serum creatine kinase levels, and result in significant morbidity and disability.

Congenital and Metabolic Myopathies (Mitochondrial Disorders)

Genetic disorders of the mitochondrial respiratory chain are probably the most common group of inborn errors of metabolism, affecting at least 1 in 5000 individuals. Mutations in more than 30 mtDNA genes and more than 30 nuclear genes have now been identified as causing human disease. The molecular genetic investigation of mtDNA is not a routine procedure so that the diagnosis may be missed.

Pathology Duchenne Muscular Dystrophy

Striated muscle contraction requires precise and coordinated activity of both the nervous and somatic systems, from excitation of individual myofibers at the neuromuscular junction to the ATP-regulated myosin activity. Throughout muscle contraction, the myofibers must remain intimately connected with the membrane and the extracellular matrix. Without this association, movement would be improperly transmitted and myocytes would risk sustaining damage to the membrane. One function of the dystrophin glycoprotein complex (DGC) is to provide a strong mechanical link from the intracellular cytoskeleton to the extracellular matrix. The DGC is composed of transmembrane, cytoplasmic, and extracellular proteins, and holds both structural and signal transduction properties. Known components of the DGC include dystrophin, sarcoglycans, dystroglycan, dystrobrevins, syntrophins, sarcospan, caveolin-3, and NO synthase. The DGC is best thought of as a mechanosignaling complex with dual mechanical and nonmechanical membrane stabilizing functions. Replicative aging of myogenic (satellite) cells, due to enhanced myofiber turnover, is a common explanation of the progression of DMD. This hypothesis has however been re-examined recently, and a large body of in vitro, animal (mdx mice), and patient data indicates that impaired differentiation, rather than replicative aging, is most likely the leading factor underlying progression of DMD. Myotonic Muscular Dystrophy

Light microscopy of muscles of DM2 patients shows a combined pattern of myopathic and ‘denervationlike’ changes that includes fiber size variation, internal nuclei, small angulated fibers, pyknotic nuclear clumps, and predominant type 2 fiber atrophy (in contrast to type 1 fiber atrophy in DM1 patients). The pathogenic mechanism induced by repeat expansions expressed at the RNA level leads to altered RNA processing and interferes with alternative

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splicing of other genes. For example, in both DM1 and DM2, altered splicing of chloride channel and insulin receptor transcripts leads to myotonia and insulin resistance, respectively. Acid Maltase Myopathy

Most infants afflicted with this condition will develop massive and progressive cardiomegaly before 6 months of age. This is due to glycogen accumulation in cardiac muscle, resulting in a hypertrophic cardiomyopathy, which progresses to a dilated cardiomyopathy. Increasing left ventricular thickness can also lead to obstruction of the left ventricular outflow tract. At autopsy, the heart can be as much as three times its normal size, and also display endocardial fibroelastosis. GAA enzyme assay is usually performed in muscle or cultured skin fibroblasts to establish diagnosis. Prenatal diagnosis involves direct enzyme analysis or mutation analysis that can be performed on cells obtained by either chorionic villus sampling or amniocentesis. Limb-Girdle Muscular Dystrophy

This dystrophic disorder is characterized by necrotic and regenerating fibers, increase in fiber size variation, fiber splitting, and centrally located myonuclei. Successive rounds of degeneration and regeneration of muscle fibers eventually result in necrosis and replacement of muscle with fatty and fibrous tissue. The extreme diversity at the clinical level is mirrored by the evolving molecular picture, with causative mutations in many different genes being linked to components of the myofiber in the contractile apparatus, the nuclear lamina, the sarcolemma, or the cytoplasm. Congenital and Metabolic Myopathies (Mitochondrial Disorders)

The histological and histochemical analysis of the muscle biopsy remains one of the most important diagnostic tests for mitochondrial abnormalities. The Gomori trichrome stain (of the quadriceps preferably) will show the subsarcolemmal collection of mitochondria: the so-called ‘ragged-red fiber’. However, it is much better to evaluate mitochondrial involvement by using specific enzymatic reactions for the mitochondrial enzymes, such as succinate dehydrogenase, which will typically show the subsarcolemmal accumulation of mitochondria, and staining for cytochrome c oxidase, which contains subunits encoded for by both the mitochondrial and nuclear genomes and is useful in the evaluation of mitochondrial myopathies.

Clinical Features Duchenne Muscular Dystrophy

The respiratory problems of DMD patients are traditionally related to the restrictive defect caused by diaphragmatic, intercostal, and accessory respiratory muscle weakness, that leads to ventilatory failure. In addition, cardiomyopathic changes may develop in DMD, involving mainly left ventricular contractility. Elevation of serum creatine kinase may be massive especially during the early phases of the disease, during which extensive skeletal muscle loss occurs. DMD is usually recognized around the time the child starts walking or climbing stairs. Physical examination will reveal the presence of the Gower’s sequence at that time. Lower limbs are affected prior to the onset of upper limb weakness. Muscle pain and muscle enlargement, particularly of the calves, are seen in most of the patients, while macroglossia is not as frequent. Intellectual impairment is another manifestation of the dystrophin gene abnormality. Around the age of 10 years, most of the patients will have ceased to walk. Contractures of the hip joints and knee flexors will develop, and although some patients may retain their muscle bulk through replacement by fat and connective tissue, significant muscular atrophy and wasting will develop in others. Sleep-disordered breathing occurs in up to two-thirds of patients during the early teenage years and numerous studies have shown that, without ventilatory support, the average life expectancy in DMD is around 20 years, with death usually occurring following a respiratory infection or eventually through progressive respiratory muscle fatigue and failure. Myotonic Muscular Dystrophy

In contrast with nondystrophic myotonias, myotonic dystrophies are associated with early and substantial progressive weakness. There are two principal groups within this disease (DM1 with four categories, and DM2) that are clinically similar with the exception of age of onset (later in DM2), the apparent absence of congenital cases of DM2, the pattern of muscle weakness, and the limited evidence for central nervous system involvement in DM2. The phenomenon of anticipation, that is, earlier onset of the disease and more severe course in subsequent generations, is a striking feature in DM1 compared to DM2. Acid Maltase Myopathy

Clinically, Pompe disease encompasses a range of phenotypes. Infantile-onset Pompe disease is uniformly lethal. Affected infants present in the first few

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months of life with severe hypotonia, generalized muscle weakness, and a hypertrophic cardiomyopathy, followed by death from cardiorespiratory failure or respiratory infection, usually by 1 year of age. Juvenile and adult-onset variants are characterized by the absence of severe cardiac involvement and a less severe short-term prognosis. Symptoms may start at any age, and are usually related to progressive dysfunction of the skeletal musculature. With disease progression, patients become confined to a wheelchair and require artificial ventilation. Respiratory failure is the cause of significant morbidity and mortality in this form of the disease. The age of death varies from early childhood to late adulthood, depending on the rate of disease progression and the extent of respiratory muscle involvement. Limb-Girdle Muscular Dystrophy

The term LGMD is used for all the noncongenital forms of muscular dystrophy with progressive proximal weakness that are not caused by a primary dystrophin deficiency. Onset, progression, and distribution of weakness vary considerably among patients according to genetic subtypes. Muscle biopsy usually is necessary to confirm the presence of dystrophic changes, including evidence of degeneration and regeneration. Compared with the dominant forms, autosomal recessive forms are about 10 times commoner. Within autosomal recessive LGMD, calpainopathies, sarcoglycanopathies, dysferlinopathies, and fukutin-related protein (FKRP) mutations are the most important recognizable entities. For formulation of a clinical approach, age at onset and progression, the pattern of weakness and contractures, and the mode of inheritance are important clues to narrow down the differential diagnostic possibilities and pursue further directed histologic and genetic workup. When larger number of patients with LGMD are compared, there seems to be a gradation according to the age at onset and clinical severity. While early-onset Duchenne-like phenotypes tend to be caused by either sarcoglycan or FKRP mutations, calpain mutations more commonly present with juvenile onset and dysferlin mutations in early adulthood. The autosomal dominant types often show milder phenotypes, with onset in the later teens or adulthood. Congenital and Metabolic Myopathies (Mitochondrial Disorders)

A defect of oxidative phosphorylation can be suspected in patients presenting with (1) an unexplained combination of neuromuscular and/or nonneuromuscular symptoms, (2) a progressive course, and

(3) involvement of seemingly unrelated organs or tissues. The clinical symptoms, either isolated or in combination, may occur at any stage, but a frequent feature is the increasing number of organs involved in the course of the disease.

Animal Models Transgenic and viral experiments on mdx mice have offered invaluable information regarding the function of the gene product involved in DMD as well as the dystrophin-associated protein complex; however, the mdx mouse (a mouse X-chromosome mutant) has a mild phenotype despite elevated creatine kinase levels. Hypertrophic feline muscular dystrophy has been less well studied and exhibits similar clinical symptoms to human DMD. Canine X-linked muscular dystrophy models include the golden retriever and the German shorthaired pointer. The use of DMD dog models is largely restricted to comparative pathophysiological experiments and evaluation of DMD treatments. Transgenic DM1 mouse models can study either DM1 disease pathology, or the mechanisms underlying DNA instability. Mouse models for DM2 have not been thus far reported. Several mouse models have been described for the study of glycogen storage disease, including a acid alpha-glucosidase (GAA)-deficient mouse and knockout, GAA-KO/severe combined immunodeficiency disease (SCID) mice (an immune-deficient mouse model of GSD-II). These models offer great potential for assessment of the efficacy of any GAA-expressing vector for use in GSD-II gene therapy. A typical mitochondrial myopathy with ragged-red fibers has been produced in a recombinant mouse model, by disrupting the gene for mitochondrial transcription factor A, Tfam, in skeletal muscle. Two models of optic nerve degeneration were generated by inhibiting the expression of the mitochondrial superoxide dismutase or by a ribozyme-mediated specific inhibition of complex I.

Management and Current Therapy Duchenne Muscular Dystrophy

Patients with DMD should visit a pulmonologist once a year prior to, and twice a year following, confinement to a wheelchair. Objective measures should include: oxyhemoglobin saturation, spirometric measurements of forced vital capacity, forced expiratory volume in 1 s (FEV1), and maximal midexpiratory flow rate, maximum inspiratory and expiratory pressures, and peak cough flow. Annual laboratory studies in patients requiring wheelchair

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for ambulation should include a complete blood count, serum bicarbonate concentration, and a chest radiograph. Percentage ideal body weight and body mass index must be assessed regularly, and a nutritionist should be involved in the management. In areas where full polysomnography is not readily available, overnight pulse oximetry with continuous CO2 monitoring will provide useful information. However, whenever possible the patients should undergo an overnight polysomnography once a year. All individuals with DMD require regular cardiac evaluation with annual electrocardiograms and echocardiograms, starting at least by school age. All patients should be taught strategies to improve airway clearance, and how to employ those techniques early and regularly. Mechanical insufflation–exsufflation appears as a good approach for mucociliary clearance. Tracheostomy should be considered as a last resort, when noninvasive ventilation is not feasible due to severe bulbar weakness or dysfunction. According to a recent American Thoracic Society consensus statement, future research is required to confirm and further define the potential and conflictive pulmonary benefits of oral steroids and many other supportive measures. All patients should also receive pneumococcal immunization and annual influenza vaccines. Desirable goals of end-of-life care for patients with muscular dystrophy include: 1. Treating conditions (pain, dyspnea) that cause distress (palliative care). 2. Attending to the psychosocial and spiritual needs of the patient and their family. 3. Respecting the patient and family’s choices concerning testing and treatment. Myotonic Muscular Dystrophy

In myotonic dystrophy moderate-intensity strength training appears not to be harmful, yet there is insufficient evidence to establish that it affords any benefit. Patients requiring prolonged mechanical ventilation (430 days) experience 25% mortality in their first year, and prolonged ventilation is followed by greater morbidity and developmental delay in children compared to shorter ventilation duration. Others have shown improved quality of life, physical activity and hemodynamics, normalization of blood gases, and slight improvement in other physiological measures, such as the vital capacity and maximal mouth pressures following nocturnal noninvasive ventilatory support. Several anecdotal reports on the use of selenium and vitamin E and amitriptyline in such patients will require further substantiation of their potential benefit.

Acid Maltase Myopathy

Currently, treatment options for Pompe disease are limited to supportive or palliative care. However, treatments that address the underlying cause of the disease are in development, most notably enzyme replacement therapy, which is in clinical trials, and gene therapy, which is still in preclinical stages. Bone marrow transplantation has not been found to be effective because of poor enzyme penetration in muscle tissue; however, newer methods involving mesenchymal stem cell transplantation could be more successful. Infantile-onset patients often have a left lower lobe collapse because of compression of the left mainstem bronchus by the massively enlarged heart. Weakness of the diaphragm in addition to weak abdominal and intercostal muscles also makes it difficult for patients afflicted with Pompe disease to cough, thereby increasing their susceptibility to infection and aspiration. Congenital and Metabolic Myopathies

No effective therapy is currently available for mitochondrial disorders. However, several supportive measures, such as improvements in nutrition, surgical correction of ptosis, seizure control, and correction of lactic acidosis can ameliorate not only the specific manifestation but also improve the quality of life. Creatine, which is routinely used for therapy of many of the mitochondrial disorders, is the substrate for the synthesis of phosphocreatine, an abundant energy storage composite in muscle, heart, and brain, and may be effective in some, but not all mitochondrial diseases. A number of experimental strategies are currently being pursued. These include the introduction of modified genes or gene products into mitochondria via the protein import machinery, and inhibition of replication of mutant mtDNA by sequence-specific antigenomic peptide-nucleic acids. See also: Neuromuscular Disease: Lower Motor Neuron Diseases; Acquired Myopathies; Peripheral Nerves; Upper Motor Neuron Diseases; Myasthenia Gravis and Other Diseases of the Neuromuscular Junction. Neurophysiology: Neurons and Neuromuscular Transmission.

Further Reading American Thoracic Society (2004) Respiratory care of the patient with Duchenne muscular dystrophy: ATS consensus statement. American Journal of Respiratory and Critical Care Medicine 170(4): 456–465. DiMauro S (2004) Mitochondrial diseases. Biochimica et Biophysica Acta 23(1658): 80–88. Harper PS (1989) Myotonic Dystrophy, 2nd edn. London: Saunders.

NEUROMUSCULAR DISEASE / Lower Motor Neuron Diseases 103 Hirschhorn R and Reuser AJJ (2001) Glycogen storage disease type II: acid alpha-glucosidase (acid maltase) deficiency. In: Scriver CR, Beaudet AL, Sly WS, et al. (eds.) The Metabolic and Molecular Bases of Inherited Disease, pp. 3389–3420. New York: McGraw Hill. Kapsa R, Kornberg AJ, and Byrne E (2003) Novel therapies for Duchenne muscular dystrophy. Lancet Neurology 2: 299–310. Kirschner J and Bonnemann CG (2004) The congenital and limbgirdle muscular dystrophies: sharpening the focus, blurring the boundaries. Archives of Neurology 61: 189–199. Kishnani PS and Howell RR (2004) Pompe disease in infants and children. Journal of Pediatrics 144(5 Supplement): S35–S43. Muntoni F and Voit T (2004) The congenital muscular dystrophies in 2004: a century of exciting progress. Neuromuscular Disorders 14: 635–649. Shelton GD and Engvall E (2005) Canine and feline models of human inherited muscle diseases. Neuromuscular Disorders 15: 127–138. Taylor RW, Schaefer AM, Barron MJ, McFarland R, and Turnbull DM (2004) The diagnosis of mitochondrial muscle disease. Neuromuscular Disorders 14: 237–245.

Lower Motor Neuron Diseases B Riley, Nottingham University, Nottingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Lower motor neuron diseases are a heterogeneous group of disorders with multiple etiologies. Amyotrophic lateral sclerosis is a progressive degenerative process that affects both upper and lower motor neurons with degeneration in the cranial nerve nuclei, pyramidal neurons of the motor cortex, and motor neurons in the spinal cord. Consequently, there is often a combination of both upper and lower motor neuron signs. Paralytic shellfish poisoning occurs after eating shellfish that have fed on the marine dinoflagellate Gonyaulax catanella. The major neurotoxin found in the organism was named Saxitoxin. Pufferfish poisoning is caused by tetrodotoxin, which is found in several species of pufferfish, some newts, and the blue-ringed octopus. It is produced by several species of bacteria of the family Vibrionaceae. Saxitoxin and tetrodotoxin block voltage-gated sodium channels affecting both central and peripheral nerves and skeletal muscle. Intrathecal injection of vincristine, a chemotherapeutic agent, can cause destruction of the ascending spinal cord when rostral spread of the drug results in destruction of the anterior horn cells innervating respiratory muscles. Poliomyelitis is an epidemic viral disease, which may result in paralysis and respiratory failure. It is caused by one of three poliovirus serotypes with all the viruses being a member of the entrovirus subgroup, the family Picornavridae. The paralytic form of polio causes respiratory failure. Guillain Barre syndrome is an immunologically mediated postinfectious progressive acute inflammatory demyelinating polyneuropathy that develops after a respiratory infection or diarrhea. A rapidly progressive muscle weakness begins in the lower limbs, extends to the upper limbs, and may cause respiratory failure.

Amyotrophic Lateral Sclerosis Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of unknown etiology. It is alternatively named motor neuron disease (MND) or Lou Gehrig’s disease. The degenerative process affects both upper and lower motor neuronesneurons with degeneration being seen in both the cranial nerve nuclei and the pyramidal neuronesneurons of the motor cortex, in addition to degeneration of the motor neuronesneurons in the spinal cord. Therefore, there is often a combination of both upper and lower motor neuronesneuron signs. Symptoms generally develop insidiously with 70% patients presenting with progressive asymmetrical weakness of the limbs. The small muscles of the hand are frequently affected with wasting accompanied by fasciculation. Alternatively, patients may present with a foot drop, difficult with fine movements, or wasting of the shoulder girdles. Occasionally, pain and cramping may precede weakness. As the disease progresses, weakness becomes more generalized and bulbar dysfunction occurs resulting in aspiration pneumonias. Eventually, the respiratory muscles are affected and respiratory failure occurs. The disease is incurable and approximately 50% of patients die within 5 years of diagnosis, generally as a consequence of respiratory disease. Some patients however, with adequate respiratory therapy, may survive for many years, with an attendant good-quality lifestyle, as their intellectual capacity remains intact. ALS has an incidence of about 2 in 100 000 of the population per year and a prevalence of approximately 5 per 100 000. The male:female ratio is 1.5:1 and in the most common form of the disease, the mean age of onset is 60 years, whereas in the rarer familial form, the mean age of onset is around 50 years. The geographical distribution of the disease remains relatively constant apart from a hot spot in the Western Pacific when it is often associated with Parkinson’s disease and dementia. Etiology

ALS has no known cause. Pathology

Macroscopic examination of the brain and spinal cord is often normal. Microscopically, there is loss of the pyramidal cells of the motor cortex and degenerative changes in the corticospinal tract. Similarly, degenerative changes are seen in the brain stem motor nuclei in the majority of cases. Neurofilaments (spheroids) are found in the proximal axons of spinal motor neuronesneurons and cytoplasmic eosinophilic inclusion bodies (bunina bodies) are seen in the anterior horn cells and degenerating motor nuclei.

104 NEUROMUSCULAR DISEASE / Lower Motor Neuron Diseases Clinical Features

Symptoms generally develop insidiously with 70% patients presenting with progressive asymmetrical weakness of the limbs. The small muscles of the hand are frequently affected with wasting accompanied by fasciculation. Alternatively, patients may present with a foot drop, difficult with fine movements, or wasting of the shoulder girdles. Occasionally, pain and cramping may precede weakness. Where muscle atrophy has started to occur, there may be hyperreflexia in the absence of any change in sensation in the corresponding dermatome. Dysphagia with dribbling and drooling occurs. Very rarely, respiratory failure is the initial presenting feature. Involvement of the bladder and bowels is uncommon. Progressive bulbar dysfunction may result in aspiration pneumonitis, and as the respiratory muscles are affected, respiratory failure may occur. Examination demonstrates a combination of fasciculation, wasting, and weakness and brisk tendon reflexes with extensor plantar responses. Eye movements are normal but there may be weakness of the lower facial muscles. Dysarthria occurs because of both upper and lower motor neuron degeneration in the nerves supplying the bulbar muscles. The tongue may be wasted and show fasciculations. Seventy percent of patients with ALS present with a progressive asymmetrical weakness, muscle fasciculation, and wasting which classically occurs in the small muscles of the hands. Upper limbs are more commonly involved than the legs. Muscle cramps and fasciculation may occur before weakness, but in most cases the weakness occurs first. As the disease progresses, it becomes more generalized and symmetrical, and a mixture of upper and lower motor neurone signs may be seen. Where muscle atrophy has started to occur, there may be hyperreflexia in the absence of any change in sensation in the corresponding dermatome. Similar features are observed in patients (25%) who present with spastic dysarthria (pseudo-bulbar-palsy) which occurs more commonly than a true bulbar palsy. Dysphagia with dribbling and drooling occurs. Very rarely, the initial presenting feature is of respiratory failure. Involvement of the bladder and bowels is uncommon. Progressive bulbar dysfunction may result in aspiration pneumonitis, and as the respiratory muscles are affected, respiratory failure may occur. Examination demonstrates a combination of fasciculation, wasting and weakness, and brisk tendon reflexes with extensor plantar responses. Eye movements are normal but there may be weakness of the lower facial muscles. Dysarthria occurs because of both upper and lower motor neuron degeneration in the nerves

supplying the bulbar muscles. The tongue may be wasted and show fasciculations. Diagnosis is made largely by excluding other causes of the neurological symptoms, together with the use of magnetic resonance imaging (MRI) of the spinal cord and electromyography. A T2-weighted MRI scan shows high-intensity lesions in the motor cortex, internal capsule, brain stem, and corticospinal tracts. Electromyography and nerve conduction studies demonstrate anterior horn cell damage. Pathogenesis

For the majority of ALS patients, the etiology and pathogenesis are unknown. There is however a familial type of ALS which represents about 5% of the total patient population. The family history is suggestive of an autosomal-dominant inheritance pattern. Twenty percent of such families have a mutation of the CU, ZN superoxide dysmutase (SOD1) gene on chromosome 21. A similar mutation is rarely found in the nonfamilial type of ALS, as is a mutation to the neurofilament heavy chain gene. Animal Models

There are no animal models of ALS. Management and Current Therapy

There is no specific treatment for ALS and care is largely supportive aimed at providing help with progressive disability. Treatment is best coordinated in specialist neurological centers using a team approach involving the neurologist, physiotherapist, occupational therapist, speech therapist, and clinical nurse specialist. Support at home and modifications to cope with an increasingly disabled patient may be required. Treatment with the glutamate release inhibitor Riluzole has resulted in increased survival, but this is limited to a few months only. Spasticity is treated with drugs such as Baclofen or Dantrolene. Quinine sulfate is sometimes used for muscle cramping. Excess salivation may be treated with drying agents such as Benzhexole. Concurrent depression and insomnia may be treated by Amitryptyline, which has the additional side effect of decreasing salivation. Progressive deterioration in bulbar function causing dysphagia may make adequate nutrition difficult, and percutaneous endoscopic gastrostomy may be the best way of providing adequate nutrition. Progressive weakness of the respiratory muscles may require the use of nocturnal CPAP or nasal intermittent positive pressure ventilation. Where permanent respiratory failure occurs, permanent tracheostomy may be offered. Death is usually the result of respiratory failure, which may be precipitated by

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aspiration pneumonitis. In patients presenting with bulbar symptoms, the average life expectancy is around 2 years as compared to 5 years in those patients with the onset of peripheral limb features. However, some patients survive for as much as 20 years after the diagnosis.

Paralytic Shellfish Poisoning (Saxitoxin) Paralytic shellfish poisoning may occur after eating shellfish which have fed on the marine dinoflagellate Gonyaulax catanella. The major neurotoxin found in the organism was named Saxitoxin, after the Alaskan butter clam, Saxidomus from which the compound was originally isolated. The rapid reproduction of this organism is responsible for the so-called ‘red tides’. There are other neurotoxins present in red algal tides and not all of them have been isolated. It is thought that the first red algal tide was reported in the biblical book Exodus: ‘‘And all the waters that were in the river were turned to blood. And the fish that were in the river died; and the river stank and the Egyptians could not drink the water of the river.’’ The most common food vectors in humans are mussels and clams. Other neurotoxins present in red algal tides but not all of them have been isolated. Saxitoxin is a water-soluble heterocyclic guanidine. It is stable to heat and steam, and cooking does not diminish its toxicity. Paralytic shellfish poisoning is a problem in both North America and Europe.

Diagnosis is based predominantly on the history but there are electromyographic changes which show prolongation of motor and sensory latencies and a significant decrease in the conduction velocity and amplitude. The mortality of paralytic shellfish poisoning has been reported as ranging from 8% to 20%. Treatment is supportive, and once respiratory failure has occurred, then intubation and ventilation is necessary. Respiratory paralysis develops between 6 and 24 h. If the patient is ventilated, then the prospect for recovery is good.

Pufferfish Poisoning (Tetrodotoxin) Tetrodotoxin is found in several species of pufferfish, some newts, and the blue-ringed octopus. The toxin is in fact produced by several species of bacteria of the family Vibrionaceae, and these bacteria are ingested by the animal. As with Saxitoxin, tetrodotoxin blocks voltage-gated sodium channels affecting both central and peripheral nerves and skeletal muscle. Pufferfish poisoning is most common in Japan where the fish is regarded as a delicacy. It is prepared by specially trained chefs who ensure that the gastrointestinal tract is thoroughly removed prior to serving the dish. Tetrodotoxin is one of the deadliest natural toxins and is not destroyed by washing, cooking, or other food preparation. Failure of preparation may result in the death of the diner.

Etiology

Etiology

Saxitoxin is a water-soluble heterocyclic guanidine. It is stable to heat and steam, and cooking does not diminish its toxicity. Saxitoxin binds reversibly to the voltage-sensitive sodium channels in both the central and peripheral nervous system. As a result, it abolishes the propagation of nerve impulses due to the failure of the sodium shift that occurs during the early phase of an action potential.

Tetrodotoxin is produced by several species of the bacterium Vibronacea and it is the ingestion of these organisms that allows the toxin to accumulate within the fish. The toxin blocks voltage-gated sodium channels in excitable membranes of all tissues including brain, spinal cord, peripheral nerves, cardiac and skeletal muscle. The sodium channels in cardiac muscle are less susceptible to its effects than are other tissues and the hypotension seen in the condition reflects effects on the autonomic nerves. Death from respiratory failure occurs from a combination of effects on the anterior horn cells of respiratory muscles and direct depression of the muscles themselves.

Pathology

There are no macroscopic or microscopic changes to nerves incubated with Saxitoxin. Clinical Features

Initial features are tingling and burning around the mouth, tongue, and face. The remainder of the body gradually becomes involved and the paresthesia changes to numbness. Nausea, vomiting, diarrhea, and epigastric pain are variable findings. As the symptoms progress, weakness and muscle paralysis occur, with death resulting because of respiratory failure.

Pathology

There are no specific pathological findings. Clinical Features

The initial symptoms are numbness of the tongue and lips followed by circum-oral paresthesiaeparesthesias

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and tingling of the extremities. Death may occur within 30 min, depending on the dose, as a result of paralysis of the diaphragm and severe hypotension. In Japan there are between 100 and 200 cases of pufferfish poisoning each year with a mortality of 50%. Pufferfish remains a delicacy in Japan and specific restaurants are licensed to serve the delicacy. There is no specific treatment, and early ventilation and volume support as well as cardiovascular support are required until the tetrodotoxin level has dropped as a result of excretion.

Intrathecal Vincristine Vincristine is a chemotherapeutic agent administered in combination with other drugs such as methotrexateuse for the treatment of malignancy (e.g., lymphoma and leukemia). Despite the well-documented disastrous effects of accidental intrathecal injection, deaths continue to occur. Ascending spinal cord destruction occurs and respiratory failure occurs when rostral spread of the drug results in destruction of the anterior horn cells innervating respiratory muscles. Etiology

Vincristine is a chemotherapeutic agent derived from the periwinkle plant (Vinca rosa). It causes inhibition of the metaphase of mitosis as a result because of vincristine binding to tubulin, which prevents polymerization of the subunits to form microtubules. The neurotoxicity of the drug is mediated by similar microtubule destruction. Clinical Features

Accidental intrathecal injection results in segmental cord destruction with symmetrical loss of motor, sensory, and sympathetic activity in an ascending manner as rostral spread proceeds. A central de-afferenation pain syndrome occurs. Respiratory muscle paralysis occurs when the drug reaches the appropriate level in the cord. Pathogenesis

Histologically, there is a decrease in the neurotubules within the neurons of the spinal cord and anterior horn cells. Neurofibrillary tangles are seen together with diffuse necrosis of all parts of the cord in contact with the cerebrospinal fluid (CSF). Treatment

The only treatment is to insert venticular and lumbar drains, and infuse Ringer’s lactate solution and glutamic acid into the ventricular drain, and

simultaneously drain CSF from the lumbar drain. Very few people survive.

Poliomyelitis Poliomyelitis is an epidemic viral disease, which may result in paralysis and respiratory failure. It is caused by one of three poliovirus serotypes with all the viruses being a member of the entrovirus subgroup, the family Picornavridae. These entroviruses may be found in the gastrointestinal tract and are rapidly inactivated by heat, formaldehyde, chlorine, or ultraviolet light. The word poliomyelitis is derived from the Greek for gray (polio) and myelon (meaning marrow). It is the effect of the virus on the spinal cord that leads to the classic manifestation of paralysis. Polio may lead to a paralytic or nonparalytic form. Twenty-one thousand paralytic cases were reported in the United States in 1952, but the last case of wild virus polio infection in the United States was reported in 1979. It remains a World Health Organization goal to eradicate poliomyelitis by universal vaccination. The paralytic form of polio causing respiratory failure provided the first classical use of both noninvasive ventilation by the ‘iron lung’ and by intermittent positive pressure ventilation via tracheostomy. Polio was first described in England by Michael Underwood in 1789, who described a weakness in the lower limbs, which is recognized as poliomyelitis. The first cases in the United States were reported in 1843. Thereafter, epidemics were reported from all the developing countries in the northern hemisphere with a tendency to occur in summer and autumn. As the epidemics became increasingly severe and the age of the affected patients increased, the death rate also increased. The eradication of polio by vaccination with either the dead vaccine (SALK) or the live attenuated virus vaccine (SABIN) has been extremely successful in North America and Europe. For example, 21 000 paralytic cases were reported in the United States in 1952 but the last case of wild virus polio infection in the United States was reported in 1979. Unfortunately, outbreaks of the disease may still occur in the Indian subcontinent and Africa. It remains a World Health Organization goal to eradicate poliomyelitis by universal vaccination. Etiology

There are three poliovirus subtypes; P1, P2, and P3, with minimal heterotypic immunity between the three serotypes. In other words, immunity to one serotype does not produce significant immunity to the other serotype. The poliovirus is a singlestranded RNA enterovirus.

NEUROMUSCULAR DISEASE / Lower Motor Neuron Diseases 107 Pathology

Initial infection with the virus occurs because of ingestion into the gastrointestinal tract where it initially multiplies in the oropharyngeal and intestinal mucosa. Following this, it enters the central nervous system because of blood-borne spread or along vagal autonomic nerve fibers from the gastrointestinal tract, although it may be isolated from the throat or the stool before the onset of the clinical signs. The virus appears to enter the blood stream after colonization of the local GI tract lymphoid tissue. Replication of the poliovirus then takes place in the motor neurons of the anterior horn and brainstem. Alternatively, the poliovirus may bind to a ‘poliovirus receptor’ found at the motor endplate. This may allow endocytosis and retrograde transport to the anterior horn cells in the spinal cord and brainstem. Acute poliomyelitis is characterized pathologically by severe anterior horn cell inflammation with loss of spinal and bulbar motor neurons.

Clinical Features

The incubation period for poliomyelitis is between 7 and 14 days but may range from 3 to 35 days. The vast majority of polio infections are asymptomatic but infected people are carriers and have no symptoms but continue to shed the virus in stool and are able to transmit the virus to other susceptible individuals. In about 5% of cases, there are minor nonspecific symptoms of virus infection with pyrexia, headache, sore throat, mild nausea, vomiting, abdominal pain, and constipation. The symptoms are often indistinguishable from other viral illnesses. Approximately 2% of infections result in a nonparalytic aseptic meningitis, which may result in neck stiffness, stiffness of the back and/or legs, which follows a few days after the typical prodromal signs. Increased or abnormal sensation may occur. These signs typically last from 2 to 10 days and are followed by complete resolution. Less than 1% of all polio infections result in classical paralytic polio. The initial symptoms of fever, malaise, headache, and gastrointestinal disturbance are similar but then progress to produce signs of more intense meningeal irritation and muscle soreness and pain becomes prominent, particularly in the back and neck. Patients who develop paralysis generally do so some 4–5 days after the meningeal signs have developed. The muscle weakness develops over the next few days with patients complaining of severe pain, spasms, and generalized asymmetric flaccid paralysis will occur in less than 1% of all polio infection. There are no changes in sensation.

Where paralytic polio does occur, it may be subdivided into three types depending on the level of the neuraxsis involved. Spinal polio may occur in around 80% of paralytic cases. It is typically characterized by the asymmetric flaccid paralysis of the legs with loss of deep tendon reflexes. Bulbar polio accounts for approximately 2% of cases and leads to weakness of those muscles innervated by the cranial nerves, resulting in difficulty in swallowing. The most severe form is bulbospinal polio, which is a combination of both the bulbar and the spinal type. The majority of cases of polio resolve spontaneously with the death rate varying with age. Approximately 5% of children with paralytic polio died, whereas in adults the death rate rose to approximately 30%. Once bulbar and spinal paralysis occurred, the death rate was much higher, ranging from 25% to 75%. Laboratory diagnosis rests on the isolation of the virus from the stool or the pharynx, positive serology, or CSF examination. The CSF shows a lymphocytic pleocytosis, a rise in protein and a normal glucose concentration. The virus may be cultured from the CSF or stool. Postpolio syndrome is defined as the development of new muscle weakness and fatigue in skeletal or bulbar muscles, unrelated to any unknown cause, which begins 25–30 years after an acute attack of paralytic poliomyelitis. For the diagnosis to be made there must be documented evidence of acute paralytic poliomyelitis in childhood, partial recovery of motor function and functional stability for at least 15 years, residual asymmetrical muscle atrophy with weakness, areflexia, and normal sensation in at least one limb, and normal functioning of the bladder and rectum. Management and Current Therapy

The majority of cases of polio resolve spontaneously with the death rate varying with age. Approximately 5% of children with paralytic polio died, whereas in adults the death rate was approximately 30%. Once bulbar and spinal paralysis occurred, the death rate was much higher, ranging from 25% to 75%. Bed rest is recommended in the early stages, as exercise does appear to increase the incidence of paralysis in terms of both frequency and severity. Bulbar spinal paralytic polio was classically treated by management of respiratory failure in the iron lung. Where bulbar symptoms are marked, early tracheostomy and supportive ventilation greatly reduce the mortality of the disease. In all other respects, the treatment is limited to supportive measures aimed at improving secretion clearance from the chest, decreasing secondary

108 NEUROMUSCULAR DISEASE / Lower Motor Neuron Diseases

infection, maintaining adequate nutrition, pressure sore prevention, deep vein thrombosis prophylaxis, and avoidance of muscular contractions and wasting.

Guillain–Barre´ Syndrome, Acute Inflammatory Demyelinating Polyradiculopathy Guillain–Barre´ syndrome (GBS) is the most common cause of rapid onset flaccid paralysis causing respiratory failure in western critical care practice. In its classical form it is an immunologically mediated postinfective progressive acute inflammatory demyelinating polyneuropathy that develops after a respiratory infection or diarrhea in about 70% of cases. There is a rapidly progressive muscle weakness that begins most commonly, in the lower limbs and extends to the upper limbs and may cause respiratory failure in about 20% of cases. CSF protein is generally raised at some point in the disease. Therapy is largely supportive together with specific treatment aimed at ameliorating the immune reaction by either plasma exchange or the administration of immunoglobulin G (IgG). Resolution of the disease occurs in the majority of cases over 18–24 months but the overall mortality is around 5–10%. A case of ascending loss of sensation and muscle weakness was first described by James Wardrop in the nineteenth century while Landry described the same symptoms in 10 patients in 1859. Despite these early reports, identification of the disease is commonly ascribed to Guillain, Barre, and Strohl in 1916, who were the first to describe the association with a raised CSF protein. Historically, GBS was considered a single disorder. It now is recognized as a heterogeneous syndrome with several variant forms (i.e., acute inflammatory demyelinating polyneuropathy, acute motor axonal neuropathy, acute motor-sensory axonal neuropathy, and the Miller Fisher variant). The Miller Fisher variant is a triad of ataxia, areflexia, and opthalmoplegia, as well as other cranial nerve abnormalities. Other variants have been described including acute motor-sensory axonal neuropathy where the immune reaction is in the axons themselves rather than the myelin sheathes. GBS is the most common cause of rapid onset flaccid paralysis causing respiratory failure in western critical care practice. In its classical form it is an immunologically mediated postinfective progressive acute inflammatory demyelinating polyneuropathy that develops after a respiratory infection or diarrhea in about 70% of cases. It has an annual incidence of 0.6 to 2.4 cases per 100 000 population, and occurs at all ages and in both sexes.

Etiology

The disease is the result of a cell-mediated immune reaction to myelin or axons. Various antibodies to nerves have been identified including glycolipids and ganglioside GM1 and GQ1b. Various infective and some noninfective triggers of GBS have been described notably influenza, CMV, EBV, HIV, chicken pox, and measles. The strongest association is with Campylobacter jejuni, with around 30% of patients having a history of gastro-enteritis. Pathology

Microscopically, there is a perivenular inflammatory infiltrate most frequently at nerve roots, spinal nerves, and plexuses. Macrophages may be seen phagocytosing the myelin of Schwann cells. Clinical Features

Rapidly progressive muscle weakness, most commonly ascending from the legs to the arms with more marked distal weakness, is a common presentation. Paresthesias and pain may occur before weakness. Respiratory failure occurs in around 20% of cases. Weakness generally progresses over 1–3 weeks but a more rapid onset may occur. Facial and bulbar weakness is common. Sensation may be largely preserved, although loss of proprioception and vibration sense is common. Pain or hyperesthesia may occur and require treatment with simple or narcotic analgesics or gabapentin. In patients who progress to ventilation, autonomic dysfunction may commonly occur with hypertension or hypotension, bradycardia, and tachycardia. Investigations should include lumbar puncture, which will show a raised protein as the only abnormality. Early on in the disease process, less than 10 days, the protein level may be normal. Viral or bacterial serology may identify the infective trigger. Electromyographic studies may show decreased conduction velocity and prolonged latency. The diagnosis is largely clinical and botulism, poliomyelitis, neurotoxicity, myasthenia, porhyria, or severe lead poisoning must be excluded. Pathogenesis

Immune-mediated diseases commonly follow infection. GBS has also been associated with systemic processes, including Hodgkin’s disease, systemic lupus erythematosus, sarcoidosis, and infection with campylobacter, lyme disease, hemophilus influenzae, Epstein–Barr virus, cytomegalovirus, herpes simplex viruses, and mycoplasma.

NEUROMUSCULAR DISEASE / Acquired Myopathies 109 Animal Model

Acquired Myopathies

GBS has no known cause.

B Bozˇicˇ, University of Ljubljana, Ljubljana, Slovenia B Rozman, M Tomsˇicˇ, and T Kveder, University Medical Centre, Ljubljana, Slovenia

Management and Current Therapy

During the acute phase of deterioration, the most important feature of treatment should be regular examination to determine the rate of progression and extent of the weakness. Regular monitoring of respiratory function should be carried out and if the vital capacity falls to less than 1 l, then intubation and ventilation should be undertaken. Intubation should be considered early in those patients who develop bulbar dysfunction, as the risk of aspiration of gastric contents is high. Supportive management to prevent pressure sores, flexion contractures, foot drop, and deep vein thrombosis is essential together with provision of adequate nutrition. Ileus is common and a period of total parenteral nutrition may be required. The main modalities of therapy for GBS are aimed at decreasing the severity of the immune reaction and include plasmapheresis and administration of intravenous immune globulin. Specific treatment is aimed at decreasing the severity of the immune reaction. This may be attempted by plasmaphoreis plasma exchange daily for 7 days, which has been shown to decrease the number of patients requiring ventilation, shorten duration of ventilation, and reduce time to recover motor function. The treatment is most effective if started early, generally within 2 weeks of the onset of symptoms. Alternatively, a daily infusion of IgG at 0.4 g kg
1 for 5 days may be used. Ten percent of patients may relapse after either treatment and some units would then either repeat the treatment or try its alternative. There appears to be no advantage in combining the two treatments. See also: Neuromuscular Disease: Inherited Myopathies. Neurophysiology: Neuroanatomy.

Further Reading (2002) Neurological disease. In: Davidson’s Principles and Practice of Medicine, 19th edn., ch. 20. Churchill Livingstone. DeLisser HM (2005) Guillain-Barre´ syndrome in adults. In: Rose BD (ed.) MA: Wellesley. Evans MH (1972) Mechanism of saxitoxin and tetrodotoxin poisoning. British Medical Bulletin 25: 263. Jackson DV, Wells HB, Atkins JN, et al. (1998) Amelioration of vincristine neurotoxicity by glutamic acid. American Journal of Medicine 84: 1016. Long RR, Sargent JC, and Hammer K (1990) Paralytic shellfish poisoning: a case report and serioelectrical physiological observations. Neurology 40: 1310. Morse EV (1997) Paralytic shellfish poisoning: a review. Journal of the American Medical Association 171: 1178. Neuromuscular disease in intensive care. In: Oh’s Intensive Care Manual, 5th edn., ch. 47, pp. 537–546.

& 2006 Elsevier Ltd. All rights reserved.

Abstract Acquired myopathies are a group of diseases in which the primary pathology causing muscle weakness and wasting lies in the muscles. They can be divided into three major groups: idiopathic inflammatory myopathies (IIMs) and infectious and nonimmune myopathies (metabolic, endocrine, and toxic). The prevalence of IIMs is low, whereas nonimmune myopathies are frequent. Etiological agents for IIMs are unknown, but genetic factors may have a role. A number of causes for toxic, endocrine, and metabolic myopathies have been reported. IIMs, a heterogeneous group of autoimmune rheumatic diseases, are defined by criteria that include clinical features, serological abnormalities, electromyographic changes, and typical histological appearance. Exact diagnostic criteria remain undefined because various proposed criteria have not been properly validated. It is generally believed that the diagnosis should rely on histopathology and immunopathology. In polymyositis and inclusion body myositis, class I MHC antigens expressed on all muscle fibers seem to be the target of CD8 þ autoinvasive T cells. In contrast, the main effector response in dermatomyositis is humoral, directed against endothelial cells of the microvasculature. Corticosteroids alone or in combination with azathioprine, methotrexate, cyclosporine, cyclophosphamide, and mycophenolate are the therapeutic agents of choice. Adequate double-blind controlled clinical trials are missing, but it seems certain that corticosteroids have substantially improved the outcome of patients with IIMs.

Introduction Acquired myopathies are a group of diseases in which the primary pathology causing muscle weakness and wasting lies in the muscles. Acquired myopathies with many different causes can be divided into three major groups: idiopathic inflammatory myopathies (IIMs) and infectious and nonimmune myopathies (metabolic, endocrine, or toxic). IIMs can be roughly classified as polymiositis (PM), dermatomyositis (DM), and inclusion body myositis. The estimated prevalence is 0.5–8.4 per 1 million, whereas the incidence appears to be increasing with apparent racial differences, being the highest in blacks. There are two age peaks of disease onset, 10–15 years and 45–60 years, with a female to male ratio of 2:1. The prevalence of nonimmune myopathies is not known. The chronic form of alcohol-induced muscle disease, which is an example of toxic myopathies, is very frequent and affects more than 2000 subjects per 100 000 population in the Western hemisphere.

110 NEUROMUSCULAR DISEASE / Acquired Myopathies

Etiology The etiology of IIMs is unknown. Genetic factors may play a role. In some rare familial occurrences, associations with certain HLA genes have been found: *

* *

DRB1*03012 alleles with PM and inclusion body myositis, HLA DQA1 0501 with juvenile DM, and tumor necrosis factor 308A polymorphism with photosensitivity in DM.

Sun exposure has been reported as a precipitating environmental factor for the onset of DM. In Mediterranean European countries, myositis patients mostly have DM, whereas in northern countries PM is more pronounced. Other inflammatory myopathies arise as a consequence of parasitic or bacterial infections (pyomyositis). Often, toxic myopathies occur due to a low selenium concentration, chronic alcohol consumption, some intravenous anesthetics, and cholesterol-lowering therapy (statins). Endocrine myopathies usually occur due to hormonal imbalance in hyperthyroidism, myxedema, Cushing’s syndrome, hyperparathyroidism, acromegaly, or osteomalacia. Metabolic myopathies are most often found in hypokalemic patients due to vomiting, diarrhea, polyuria, or nephritis and in patients with hyperaldosteronism.

Pathology IIMs, usually considered a heterogeneous group of autoimmune rheumatic diseases, are defined by a combination of clinical features, serological abnormalities, electromyographic changes, and a typical histological appearance. None of the proposed criteria have been properly validated; therefore, diagnostic criteria remain undefined. For a definite diagnosis of DM/PM, the criteria of Bohan and Peter are most often applied. Using systemic lupus erythematosus and systemic sclerosis patients as control groups, a 73% sensitivity and 99% specificity have been shown. However, it is sometimes impossible to distinguish PM from inclusion body myositis or from certain nonimmune myopathies. Therefore, it is believed that diagnostic criteria should rely on histopathology and immunopathology. Nonimmune myopathies should be considered in the context of many possible metabolic, endocrine, and toxic agents.

Clinical Features Idiopathic Inflammatory Myopathies

The dominant clinical feature of PM is a symmetric proximal muscle weakness. It can be accompanied by

myalgia, tenderness, and, eventually, muscle atrophy. Weakness initially affects the muscles of the shoulders and pelvic girdle. Dysphagia, dysphonia, Raynaud’s phenomenon, and arthralgia may develop, and pulmonary involvement is not rare. The clinical features of DM include all those described for PM and also cutaneous manifestations. Skin involvement varies widely. Juvenile DM differs due to concurrent vasculitis, calcifications, and lipodystrophy. Inclusion body myositis affects primarily older individuals; muscle weakness may be focal, distal, or asymmetric. IIMs are frequently associated with other connective tissue diseases (overlap syndromes) with relevant combined clinical features. IIMs can be associated with malignancies; the most common cancers are those of the ovaries, gastrointestinal tract, lung, breast, and non-Hodgkin’s lymphomas. Levels of serum creatine kinase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and aldolase are usually increased. The most diagnostically sensitive muscle enzyme is creatine kinase, which is always increased in active PM (even up to 50 times) but can be normal in some patients with active DM and in many patients with inclusion body myositis. The serum creatine kinase activity in chronic myositis is considerably lower than that in acute forms of the disease. In acute and subacute forms of PM, there is a considerable increase in aspartate aminotransferase; in the chronic form, there are normal or slightly elevated values. In DM, a mild increase in aspartate aminotransferase is possible, whereas alanine aminotransferase remains normal. Serum and urine myoglobin elevations due to skeletal muscle damage show almost identical patterns to that of serum creatine kinase. Defined autoantibodies, traditionally divided into myositis specific and myositis associated, can be found in approximately half of myositis patients. Specific autoantibodies against aminoacyl-tRNA synthetases (particularly Jo-1), their cognate tRNAs, and signal recognition particle are regularly associated with PM and interstitial lung disease. Antibodies against the components of a nucleosome remodeling complex called Mi-2 are relatively rare but found mostly in patients with DM. It has become clear that autoantibodies against aminoacyl-tRNA synthetases are specific for a defined disease entity of which myositis may only be a part: the antisynthetase syndrome with typical histopathologic features. Autoantibodies to the signal recognition particle are seen exclusively in PM and are specific for immune-mediated necrotizing myopathy. Myositis-associated autoantibodies (i.e., against PM/Scl and U1RNP) are well known and usually point to specific subsets (e.g., scleroderma–polymyositis overlap and mixed connective tissue disease).

NEUROMUSCULAR DISEASE / Acquired Myopathies 111

Needle electromyography features associated with inflammatory myopathies are non-specific but can help to confirm active IIMs. Obligatory examination of proximal and distal muscles usually shows a decreased motor unit potential together with reduced amplitude. There is an increased insertional activity with fibrillation potentials and positive sharp waves. Complex repetitive discharges and myotonic discharges may be seen in inflammatory myopathies as well. The presence of spontaneous activity is useful to distinguish active IIMs from nonimmune myopathies. The main histological finding in PM is infiltration with mononuclear cells (distinguishing PM from toxic, necrotizing, or dystrophic myopathies). Additional muscle biopsy of a muscle different from the one first biopsied should be considered if a patient fulfills the clinical criteria but no inflammation was found in the initial histological preparation. Multifocal lymphocytic infiltrates with CD8 þ cells surround and invade healthy muscle fibers, which express MHC class I antigens. In inclusion body myositis, vacuolated muscle fibers with basophilic granular and abnormal amyloid deposits with Congo red stain can be seen. Due to paraffin embedding of histological preparations, which dissolves the diagnostic red-rimmed granular material making vacuolated fibers unnoticeable, inclusion body myositis is regularly misdiagnosed as PM. In DM, perivascular accumulation of CD4 þ cells may be seen together with perifascicular atrophy. This atrophy, typically observed as 2–10 layers of atrophic fibers, confirms the diagnosis of DM, even in the absence of inflammation. Histopathology of the skin discriminates DM from other papulosquamous disorders but not from cutaneous lupus. Nonimmune Myopathies

Nonimmune myopathies vary from those characterized by muscle pain to those with profound myalgia, cramps, paralysis, and myoglobinuria. Reductions in muscle mass and impaired muscle strength can be observed. In myopathies associated with hyperthyroidism, hyperparathyroidism, and adrenal disorders, serum creatine kinase is usually normal, but it increases 10- to 100-fold in hypothyroidism. In drug-induced myopathies, creatine kinase can be normal (zidovudine and steroids), slightly elevated (diuretics and L-tryptophan), or significantly elevated (lipid-lowering agents, cyclosporine, chloroquine, amiodarone, colchicine, vincristine, and corticosteroids). Needle electromyography can show fibrillation and positive sharp waves (toxic myopathies) or myotonic discharges (iatrogenic, metabolic, or endocrine myopathies). Endocrine myopathies show minimal muscle membrane instability or abnormal spontaneous

activity. Histologically, macrophages, but not lymphocytes, predominate.

Pathogenesis Idiopathic Inflammatory Myopathies

PM, DM, and inclusion body myositis are secondary to an autoimmune process probably induced by a muscle injury. Their association with other autoimmune disorders, autoantibodies, histocompatibility genes, T-cell-mediated myocytotoxicity, or complement-mediated microangiopathy supports the autoimmune hypothesis. Autoantibodies can be found in the sera of most patients, with anti-histidyl-tRNA synthetase (anti-Jo-1) as the most studied. It seems that the formation of anti-Jo-1 autoantibodies is not simply a result of muscle inflammation and is closely linked to the pathophysiology of DM/PM. The ‘antigen-driven’ hypothesis includes (1) direct interaction with viruses’ RNA, (2) molecular mimicry, and (3) anti-idiotypic antibodies’ response against viral proteins. Another model hypothesizes that certain modified self-proteins are recognized as non-self. The immune system, but not a muscle, is proposed to be the site of antigen presentation. Fragments of several tRNA synthetases function as chemokines, which are small chemoattractant proteins that are mainly involved in lymphocyte trafficking. This is an additional factor to overexpressed signal transduction with upregulated cytokines in PM and DM. Muscle fibers are normally negative for major histocompatibility complex (MHC) class I antigens, which are upregulated under some pathological conditions. In PM, almost all muscle fibers strongly express class I MHC molecules, which are targets of CD8 þ (cytotoxic) T cells. Up to 50% of CD8 þ autoinvasive T cells are activated, and the vast majority of CD4 þ and CD8 þ T lymphocytes are memory cells. Activated cytotoxic T lymphocytes contain perforin and granzyme granules, which are directed toward muscle fibers. The major cytotoxic effector mechanism in PM as well as in inclusion body myositis seems to follow the perforin pathway but not the Fas–Fas ligand pathway. Inclusion body myositis exhibits some characteristic features: *

*

*

Inclusions react with antibodies, recognizing phosphorylated t, b-amyloid, ubiquitin, presenilin I, or other proteins that accumulate in the brain of patients with Alzheimer’s disease. MHC I expression is highest in the invaded fibers or near inflammatory exudates. Clonal restriction of T-cell receptors persists in muscles of patients with inclusion body myositis.

112 NEUROMUSCULAR DISEASE / Acquired Myopathies

In DM, the primary target is the endothelium of capillaries, and the main effector response is humoral. Bound antibodies activate complement, and depletion of capillaries may be observed before changes due to inflammation. MHC class I expression is usually restricted to perifascicular fibers, and inflammatory exudates are mainly composed of B and CD4 þ lymphocytes and macrophages. Nonimmune Myopathies

The pathogenesis of nonimmune myopathies depends on the cause. For the majority of them, the pathogenesis is not fully understood. Evidence suggests that changes in the alcoholic muscle disease are not due to dietary deficiencies but, rather, due to direct effects of ethanol or its ensuing metabolites on mRNA levels, resulting in impaired protein synthesis. Increased protein catabolism may be involved in corticosteroid myopathy.

Animal Models Animal models of human myositis include spontaneous, induced, and transgenic models. It is unlikely that any single model would reproduce all features of human diseases. The autoimmune (immunization with muscle antigen and adjuvant) and microbialinduced models (e.g., enteroviruses, coxsackievirus B1, and CBA/J mice infected with Trypanosoma cruzi) have limitations as the models of human disorders, but because they are well characterized, they have value in the study of immune effector mechanisms. Although spontaneous models (myopathy in the SJL/J mice and DM-like syndrome in dogs) are less characterized, they may provide some insight into the induction mechanisms of IIMs. Accumulations of b-amyloid and its precursors in skeletal muscles or chronic deprivation of nerve growth factor in transgenic mice are thought to play important roles in the pathogenesis of inclusion body myositis.

Management and Therapy Corticosteroids alone or in combination with azathioprine, methotrexate, cyclosporine, cyclophosphamide, and mycophenolate are the therapeutic agents of choice. There are no adequate double-blind controlled trials using corticosteroids, but it seems certain that these drugs have substantially improved the outcome of patients with IIMs. Neither steroids alone nor combinations with other immunosuppressive drugs have the capacity to induce full remission in every case. At least one-third of patients with DM or PM end up with mild to severe disability. Older

age and the association with cancer are factors for poor prognosis. Intravenous immunoglobulins, total body irradiation, and thoracic drainage either have not been adequately assessed in double-blind controlled clinical trials or have been shown to be of limited benefit. Plasmapheresis was not found to be helpful. There has been some speculation on the potential therapeutic implications of nonclassical MHC molecule (HLA-G) that relate to special approaches, such as myoblast transplantation, or strategies against inflammation in general. In order to assess the overall effect of myositis on a patient, disease activity and damage indexes need to be developed. Creatine kinase is a good indicator of disease activity (PM and DM), but it is not the absolute target of therapy. An international multidisciplinary consensus study has established the reliability of myositis intention to treat index (MITAX), myositis clinical assessment visual analog scale (MYOACT), and myositis damage index (MDI) as fair to good for most aspects of follow-up. It is expected that the use of these indexes will enhance the consistency, comprehensiveness, and reliability of assessing disease activity and damage in patients with myositis. Nonimmune myopathies are regularly reversible upon withdrawal of the insulting agent or after successful treatment of endocrine disease. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Cell: Vascular; Epithelial. Angiogenesis, Angiogenic Growth Factors and Development Factors. Apoptosis. Autoantibodies. Bronchoalveolar Lavage. Carbon Monoxide. Chemokines. Chest Wall Abnormalities. Complement. Corticosteroids: Therapy. Endothelial Cells and Endothelium. Fibroblasts. Gene Regulation. Immunoglobulins. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Leukocytes: Monocytes; T cells; Pulmonary Macrophages. Lung Imaging. Matrix Metalloproteinases. Myoglobin. Neuromuscular Disease: Inherited Myopathies. Nitric Oxide and Nitrogen Oxides. Physiotherapy. Polymyositis and Dermatomyositis. Progressive Systemic Sclerosis. Pulmonary Effects of Systemic Disease. Pulmonary Fibrosis. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Signal Transduction. Surfactant: Overview. Transgenic Models. Tumor Necrosis Factor Alpha (TNF-a). Tumors, Malignant: Overview. Vasculitis: Overview. Ventilation: Overview. Viruses of the Lung.

Further Reading Amato AA and Griggs RC (2003) Treatment of idiopathic inflammatory myopathies. Current Opinion in Neurology 16(5): 569–575. Bohan A and Peter JB (1975) Polymyositis and dermatomyositis. New England Journal of Medicine 292: 344–347; 403–407.

NEUROMUSCULAR DISEASE / Peripheral Nerves 113 Chinoy H, Ollier WE, and Cooper RG (2004) Have recent immunogenetic investigations increased our understanding of disease mechanisms in the idiopathic inflammatory myopathies? Current Opinion in Rheumatology 16(6): 707–713. Dalakas MC and Hohlfeld R (2003) Polymyositis and dermatomyositis. Lancet 362(9388): 971–982. Figarella-Branger D, Civatte M, Bartoli C, and Pellissier JF (2003) Cytokines, chemokines, and cell adhesion molecules in inflammatory myopathies. Muscle and Nerve 28(6): 659–682. Hengstman GJ, van Engelen BG, and van Venrooij WJ (2004) Myositis specific autoantibodies: changing insights in pathophysiology and clinical associations. Current Opinion in Rheumatology 16(6): 692–699. Hengstman GJD and van Engelen BGM (2004) Polymyositis, invasion of non-necrotic muscle fibres, and the art of repetition. British Medical Journal 329: 1464–1467. Hoogendijk JE, Amato AA, Lecky BR, et al. (2004) 19th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis. Neuromuscular Disorders 14: 337–345. Isenberg DA, Allen E, Farewell V, et al. (2004) International Myositis and Clinical Studies Group (IMACS). International consensus outcome measures for patients with idiopathic inflammatory myopathies. Development and initial validation of myositis activity and damage indices in patients with adult onset disease. Rheumatology (Oxford) 43(1): 49–54. Nagaraju K and Plotz PH (2002) Animal models of myositis. Rheumatic Disease Clinics of North America 28(4): 917–933. Sieb JP and Gillessen T (2003) Iatrogenic and toxic myopathies. Muscle and Nerve 27(2): 142–156. Srinivasan J and Amato AA (2003) Myopathies. Physical Medicine and Rehabilitation Clinics of North America 14: 403–434. Wortman RL (2005) Inflammatory disease of muscle and other myopathies. In: Harris ED, et al. (eds.) Kelley’s Textbook of Rheumatology, 7th edn., pp. 1309–1335. Philadelphia: Elsevier/ Saunders.

Peripheral Nerves H M DeLisser, University of Pennsylvania School of Medicine, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Injury to and/or dysfunction of peripheral nerves may arise from autoimmune processes that target the Schwann cell, myelin, and/ or axon. Because of the dramatic and potentially life-threatening involvement of the respiratory system, the Guillain–Barre´ syndrome (GBS), the prototypic immune-mediated peripheral neuropathy, will be the focus of this article. Required criteria for its diagnosis include progressive weakness of two or more limbs, hyporeflexia or areflexia, and progression for no more than 4 weeks. At least 70% of patients with GBS have a history of a preceding infection and case–control studies have demonstrated significant associations with a number of pathogens, particularly Campylobacter jejuni. The pathogenesis of GBS is currently viewed as an organ-specific, immune-mediated disorder arising from synergistic interactions of humoral and cell-mediated immune responses to as yet incompletely characterized peripheral nerve antigens. The mainstay of treatment continues to be supportive measures and intensive care, but several control clinical

trials have demonstrated that both plasma exchange and intravenous immunoglobulin shorten the time to recovery when used in the early stages of the disease. Overall, the prognosis for recovery is favorable, with mortality below 5% and the majority of patients experiencing little or no disability.

Introduction Classification of Peripheral Nerve Disorders

Disorders of the peripheral nervous system are many and diverse, but may be due to: (1) focal injury of a single nerve trunk, typically from a local process such as compression, entrapment, or trauma (mononeuropathy); (2) simultaneous or sequential involvement of noncontiguous nerve trunks, often a manifestation of multiple nerve infarcts in the setting of a systemic vasculitis (mononeuropathy multiplex); and (3) a relatively homogeneous process affecting many peripheral nerves, with distal nerve involvement being especially prominent (polyneuropathy). Polyneuropathies may be the result of systemic diseases (Table 1), drugs or environmental toxins (Table 2), genetically determined processes, or inflammatory damage. Many of the inflammatory neuropathies are now understood to arise from autoimmune processes that target the Schwann cell, myelin, or axon, or some combination of these structures (Table 3). Because of its dramatic and potentially life-threatening involvement of the respiratory system, the Guillain–Barre´ syndrome (GBS), the prototypic immune-mediated peripheral neuropathy, will be the focus of this article. Furthermore, the study of GBS and related entities has lead to increased understanding of the molecular and cellular mechanisms that lead to inflammatory injury of the peripheral nervous system. Guillain–Barre´ Syndrome

Initially described by three French army physicians in 1916 as an illness characterized by rapidly ascending paralysis, hyporeflexia or areflexia, and elevated cerebrospinal fluid protein with normal cell counts, GBS is now recognized as several disorders characterized by immune-mediated attack on peripheral nerves. At least four subtypes have been recognized: (1) the classic, acute demyelinating type, designated acute inflammatory demyelinating polyneuropathy (AIDP), constituting the majority (75%) of cases in Europe and North America; (2) a pure motor axonal type, identified as acute motor axonal neuropathy (AMAN), the most prevalent form in China, where it occurs in summer outbreaks in children and young adults; (3) a rare axonal form involving both motor and sensory fibers known as acute motor and sensory axonal neuropathy (AMSAN), characterized by

114 NEUROMUSCULAR DISEASE / Peripheral Nerves Table 1 Polyneuropathies associated with systemic diseasesa Systemic diseases

Common Carcinoma (late; due to wasting and weight loss) Critical illness (particularly in sepsis) Diabetes Occasional Carcinoma (demyelinating; may be relapsing) Carcinoma (sensorimotor; mostly lung cancer) HIV infection (late stages) Liver disease (chronic) Lyme disease Lymphoma (including Hodgkins) Malabsorption (sprue, celiac disease) Multiple myeloma (lytic) Multiple myeloma (osteosclerotic) MGUSd IgA IgG IgM Uremia Vitamin deficiency (excluding B12) Vitamin B12 deficiency (neuropathy overshadowed by myelopathy)

Axonal b

Fiber c

Demyelinating

Acute

Subacute

Chronic

Acute

Subacute

Chronic

Sensory vs. motor

–

þ

þ

–

–

–

S4M

– –

7 7

þ þ

– –

– 7

– þ

M4S S, SM4M

–

–

–

þ

þ

7

SM

–

þ

þ

–

–

–

SM

– – – – – – –

7 – 7 þ 7 7 –

þ – þ þ þ þ 7

– – – þ – – –

– – – þ – – 7

– þ – 7 – – þ

S4M S or SM S4M S, SM4M S or SM S, M, or SM SM

– – – 7 – –

7 7 – þ þ 7

þ þ – þ þ þ

– – – – – –

– – 7 – – –

– – þ – – –

SM SM S or SM SM SM S

a Polyneuropathy may be rarely associated with porphyria, hypoglycemia, primary biliary cirrhosis, primary systemic amyloidosis, hypothyroidism, chronic obstructive lung disease, acromegaly, polycythemia and cryoglobulinemia. b þ usually; 7 sometimes; – rare, if never. c S, sensory; M, motor; SM, sensorimotor. d Monoclonal gammopathy of undetermined significance. Adapted from Asbury AK (2005) Approach to the patient with peripheral neuropathy. In: Kasper DL, Braunwald E, Fauci AS, et al. (eds.) Harrison’s Principles of Internal Medicine, 16th edn., p. 2504. New York: McGraw-Hill.

significant sensory involvement and a more severe course and poorer prognosis; and (4) the Miller Fisher syndrome (MFS), characterized by ophthalmoplegia, ataxia, and areflexia, a proportion of which may progress to generalized GBS. With the virtual elimination of poliomyelitis, GBS now represents the most common cause of acute flaccid paralysis in the Western hemisphere, with an incidence of 1.5/10 000 and a slight male predominance. The incidence increases with age, although there is a minor peak among young adults.

Etiology Extensive clinical observations and numerous epidemiological studies have suggested that 70% or more of GBS patients have a history of a preceding infection. Further, serological evidence of an antecedent infection can be found in up to 50% of patients. Consistent with these data are the reports of outbreaks of GBS within communities or associated with

cases of food poisoning. Case-control studies have demonstrated significant associations with a number of pathogens. The most widely studied and the most common antecedent infectious agent in GBS is Campylobacter jejuni, a major cause of acute gastroenteritis in developed countries. Serological evidence of C. jejuni can be found in 30% of patients with GBS and appears to be associated with AMAN variant and a slightly more severe course. Associations have also been reported for cytomegalovirus, Mycoplasma pneumoniae, Haemophilus influenzae, and EpsteinBarr virus. As will be further discussed, it is postulated that molecular mimicry between microbial and selfcomponents trigger an immune response against autoantigens. GBS has followed influenza vaccinations, but the risk, if any, appears to be very small.

Pathology The pathological hallmark of the classic (AIDP) GBS is a multifocal, inflammatory demyelination of

NEUROMUSCULAR DISEASE / Peripheral Nerves 115 Table 2 Polyneuropathies associated with drugs and toxins Systemic diseases

Drugsc Antimicrobial agents: Dapsone (dose-related) Isoniazid Metronidazole Nitrofurantoin Antiretroviral nucluside analogs Cardiovascular drugs: Amiodarone (reversible by decreasing dose) Hydralazine Statins Chemotherapeutic agents: Cisplatin Suramin Taxol Vincristine Miscellaneous drugs: Aurothioglucose Disulifram Misonidazole Phenytoin Toxinsd Acrylamide (flocculant; grouting agent) Arsenic g-Diketone hexacarbons (solvents) Inorganic lead (selective motor neuropathy with prominent wrist drop) Organophosphates (brain and spinal cord also involved) Thallium (rat poison)

Axonal a

Fiber b

Demyelinating

Acute

Subacute

Chronic

Acute

Subacute

Chronic

Sensory vs. motor

– – – – 7

7 7 – 7 þ

þ þ 7 þ þ

– – – – –

– – – – –

– – – – –

M SM S or SM SM S4M

–

–

þ

–

–

þ

SM

– –

7 7

þ þ

– –

– –

– –

S4M S4M

– þ 7 –

þ þ þ þ

þ – 7 þ

– þ 7 –

– þ þ –

– – 7 –

S M4S S4M S4M

7 7 – –

7 þ 7 –

– þ þ þ

þ – – –

þ – – –

– – – –

SM SM S or SM S4M

– 7 – –

7 þ 7 –

þ þ þ þ

– – – –

– – – –

– – þ –

S4M SM SM M4S

–

7

þ

–

–

–

SM

–

þ

þ

–

–

–

SM

þ Usually; 7 sometimes; – rare, if never. S, sensory; M, motor; SM, sensorimotor. c The following drugs may also be toxic to peripheral nerves: allopurinol, amitriptyline, chloramphenicol, colchicine, ethambutal, flecainide, indomethacin, lithium, nitrous oxide, perhexiline maleate, podophyllin, sodium cyanate, thalidomide, L-tryptophan. d The following environmental toxins may also be toxic to peripheral nerves: allyl chloride, buckthorn berry, carbon disulfide, diglycols, dimethylaminoproprionitrile (DMAPN), ethylene oxide, metallic mercury, methyl bromide, polychlorinated biphenyls, styrene, trichloethylene, vacor. Adapted from Asbury AK (2005) Approach to the patient with peripheral neuropathy. In: Kasper DL, Braunwald E, Fauci AS, et al. (eds.) Harrison’s Principles of Internal Medicine, 16th edn., p. 2505. New York: McGraw-Hill. a b

Table 3 Classification of immune-mediated polyneuropathies Guillain–Barre´ syndrome Acute inflammatory demyelinating polyneuropathy (AIDP) Acute motor axonal neuropathy (AMAN) Acute motor sensory axonal neuropathy (AMSAN) Miller Fisher syndrome (MFS) Chronic immune neuropathies Chronic inflammatory demyelinating polyneuropathy (CIDP) Multifocal motor neuropathy (MMN) Multifocal acquired demyelinating sensory and motor (MADSM) neuropathy Antimyelin-associated glycoprotein (MAG) neuropathy Antisulfatide neuropathy, anti-GM2 and GalNac-GD1a neuropathy Gait ataxia late-onset polyneuropathy (GALOP) syndrome

peripheral nerves, accompanied by variable degrees of lymphocytic infiltration. Pathological changes can be detected along the entire peripheral nerve, from the roots to the most distal intramuscular motor nerve. Lesions, however, are most often noted along the ventral roots, proximal spinal nerves, and lower cranial nerves, where they are found primarily at the nodes of Ranvier, accompanied by macrophage accumulation. In severe cases, demyelination may be accompanied by axonal degeneration, presumably the result of ‘collateral’ injury. In contrast to AIDP, pathological findings in autopsy cases of AMAN and AMSAN have revealed axonal degeneration of the motor and/or sensory

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axons with little demyelination or lymphocytic infiltration. Macrophages are present in close proximity to the axons and IgG and complement are detected on the surface of axons, suggesting a macrophagedependent, antibody-mediated attack on antigens of axonal origin.

Clinical Features Clinically, GBS is defined as a rapidly progressive ascending polyradiculopathy, with involvement of cranial nerves in a significant proportion of cases. The diagnostic criteria for typical GBS require: (1) weakness in two or more limbs; (2) parasthesias in hands and feet; (3) hyporeflexia and areflexia in all limbs by the end of the first week; and (4) progression of the weakness, parasthesias, and reflex abnormalities over several days to 1 month. Other causes of an acute neuropathy such as botulism, vasculitis, porphyria, and lead poisoning must be excluded. Supportive features include mild sensory findings, elevated protein in the cerebrospinal fluid (CSF), with a relatively normal cell count and neurophysiological evidence of axonal or demyelinating neuropathy. Electrodiagnostic evidence of a demyelinating process includes a slowed nerve conduction velocity; dispersion of evoked compound action potentials; a nerve conduction block; and marked prolongation of distal latencies. In contrast, axonal neuropathies are characterized by a reduction in the amplitude of evoked compound action potential, with relative preservation of the nerve conduction velocity. With respect to neurophysiological testing, it should be noted that in the very early stages, nerve conduction may be preserved and abnormalities only detected after the disease is further established. Also, in occasional patients neurophysiological testing may be ‘normal’ because the demyelinating lesions are located exclusively proximal and therefore are not accessible to easy neurodiagnostic evaluation. In about 70% of patients, the onset of the typical disease is preceded, often by 1–2 weeks, by a febrile, usually mild, illness. The weakness may begin distally or proximally, but is typically symmetrical. Cranial nerve involvement is observed in approximately 50% of patients, with bifacial weakness the most common finding. About a third of patients will require mechanical ventilation due to diaphragmatic muscle weakness from phrenic nerve involvement. Autonomic dysfunction, manifested by cardiac arrhythmias, hypotension, hypertension, gastroparesis, and urinary retention, occurs in more than half of patients. Pain is a common symptom, occurring in up to 50% of patients with GBS, manifesting either as an aching discomfort of the muscles of the lower

back, buttocks, quadriceps and calves and/or a burning or tingling sensation of the extremities. The nadir of the disease is reached within 4 weeks, followed by a plateau stage of variable duration and eventual recovery in most patients. The pattern and rates of progression differ in AIDP and the AMAN subtypes, with the AMAN demonstrating a rapid progression and an early nadir, while the impairments in patients with AIDP will typically evolve less rapidly. Miller Fisher Syndrome

The Miller Fisher syndrome (MFS) is characterized by the clinical triad of ophthalmoplegia, ataxia, and areflexia. Because of the similarities to GBS, it is often classified as an acute inflammatory neuropathy. The ocular motor nerves (third, fourth, and sixth cranial nerves) are targeted, but other cranial nerves, most notably the facial nerve, may also be involved. Most patients will initially present with diplopia, followed by gait and limb ataxia. The ocular manifestations range from complete ophthalmoplegia with unreactive pupils to external ophthalmoplegia with or without ptosis. Motor strength is usually preserved, although overlap with GBS can occur, with some patients progressing to quadriplegia. Despite this, MFS is usually a relatively benign illness that resolves without treatment. Chronic Inflammatory Demyelinating Polyneuropathy

In many patients, the chronic inflammatory demyelinating polyneuropathy (CIDP) resembles what would appear to be a slowly progressive form of GBS, with symptoms present and evolving for 2 or more months. Most patients demonstrate involvement of both motor and sensory fibers, with evidence of proximal and distal muscle weakness (typically symmetric), hyporeflexia or areflexia, and distal sensory loss of vibration and joint position perception. Facial and respiratory muscle weakness occurs less frequently than in GBS and very few patients progress to respiratory failure. The CSF protein is elevated but, unlike GBS, few patients report a preceding infection or febrile illness. Also, CIDP responds to a wider array of treatments than GBS.

Pathogenesis The pathogenesis of GBS is poorly understood, but is currently best viewed as an organ-specific, immune-mediated disorder arising from synergistic interactions of humoral and cell-mediated immune responses to as yet incompletely characterized

NEUROMUSCULAR DISEASE / Peripheral Nerves 117

peripheral nerve antigens. ‘Molecular mimicry’ has been proposed as a key (but not sole) element in the immunopathogenesis of this disease, with shared carbohydrate epitopes between microbial agents and nerve fibers serving as targets for aberrant crossreactive immune responses. Humoral Immunity

Several observations suggest that humoral factors are critical to the pathogenesis of GBS: (1) deposition of immunoglobulin and complement has been noted on myelinated fibers in nerve biopsies and in CSF obtained from affected patients; (2) circulating antibodies directed against structures on peripheral nerve tissue can be detected in the sera of patients with GBS; and (3) plasma exchange and intravenous immunoglobulin (IVIg) result in clinical improvement. It is proposed that antibodies targeting epitopes on the surface of the axon and/or Schwann cell activate, complement, and subsequently disrupt plasma membrane integrity. This leads to entry of calcium, which in turn activates calcium-sensitive enzymes capable of degrading axonal and myelin proteins. The specific targets of such antibodies remain elusive, but for some variants of GBS, immune responses against gangliosides and other glycolipids are strongly implicated. For example, IgG antibodies against GM1, GD1a, and related gangliosides are frequently present in patients with the post-campylobacter AMAN variant of GBS, and immunization of rabbits with GM1 has produced a model of AMAN. Furthermore, in MFS, anti-GQ1b ganglioside antibody is detected in most patients, decays rapidly with clinical recovery, and is not found in normal or disease control serum samples. GQ1b is enriched in oculomotor nerves, and thus targeting of this antigen would be consistent with the clinical manifestations of this entity. Although circulating antibodies against peripheral nerve antigens or purified myelin have been detected with varying frequency, no characteristic pattern of antiganglioside antibodies has been determined for AIDP. Consequently, the specific role of antiganglioside antibodies in GBS continues to be investigated and debated.

demonstrated in GBS and are consistent with peripheral T-cell activation. However, studies to characterize the nerve antigens to which aberrant T-cell responses are directed, as well as efforts to establish T-cell lines from affected patients have produced conflicting results.

Animal Models Much of the present knowledge about the mechanisms involved in the pathogenesis of immune-mediated demyelination has been derived from studies in animal models of experimental autoimmune neuritis (EAN). In this system, an acute inflammatory polyradiculoneuropathy can be induced in mice, rats, rabbits, and monkeys by active immunization with whole peripheral nerve homogenate, myelin, myelin proteins P0 and P2, or peptides derived from P0 and P2. Adoptive transfer of P0, P0 peptide-specific, P2, and P2 peptide-specific CD4 þ T-cell lines can also induce EAN. Pathologically, EAN is characterized by infiltration of the peripheral nervous system by lymphocytes and macrophages with multifocal demyelination of axons predominantly around venules. The macrophages actively denude the axons of myelin, induce vesicular disruption of the myelin sheath and phagocytose both intact and damaged myelin.

Management and Current Therapy Supportive Therapy

The mainstay of treatment of GBS continues to be supportive measures and intensive care. The elements of this care include: (1) timely initiation of ventilatory support and optimization of its use to prevent respiratory muscle atrophy or fatigue; (2) monitoring for and treatment of cardiovascular disturbances particularly hypotension and bradycardias; (3) prophylaxis for deep venous thrombosis; (4) aggressive nutritional support (preferably enteral); (5) physical and occupational therapy to prevent contractures and disuse syndromes; (6) treatment of pain syndromes; and (7) attentive nursing care. Immunotherapy

Cellular Immunity

The presence of inflammatory cell infiltrates within nerves and the demonstrated role of neuritogenic T lymphocytes in experimental animal models of GBS suggest that disordered cellular immunity may also be critical to the pathogenesis of this disease. Enhanced expressions of antigens, MHC class II and costimulatory molecules, and various proinflammatory cytokines such as TNF-a and IFN-g have all been

In terms of therapy directed at the underlying immune-mediated injury, and ameliorating or reversing the nerve damage, several control clinical trials have demonstrated that both plasma exchange and IVIg shorten the time to recovery when used in the early stages of the disease. There does not appear to be significant differences between IVIg and plasma exchange in mortality, change of disability grade, time to walk unaided, or proportion of patients unable to

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walk at 1 year. Thus, of the two modalities, IVIg is now considered the treatment of first choice due to its efficacy, ease of administration, and low incidence of side effects. Although plasma exchange clearly removes presumed blood-borne factors involved in the pathogenesis of the disease, the mechanisms of action of IVIg administration are less certain, but may include binding of Fc receptors, increased clearance of immunoglobulins, and/or stimulation of remyelination. Unlike CIDP, steroids have not been shown to be helpful in treatment of GBS.

Prognosis

Overall, the prognosis for recovery of patients with GBS is favorable with the majority of patients (65%) experiencing little or no disability. Of the remaining patients, less than 5% will die in the acute stages of the illness, usually from cardiac arrhythmias or pulmonary embolism while those who do survive will be left with persistent deficits. The percentage of patients experiencing chronic impairments has not been significantly altered by the introduction of IVIg or plasma exchange, which both shorten time to recovery but not the proportion of patients attaining a good recovery. Factors associated with a poor outcome include advanced age, rapid progression of symptoms, need for ventilatory support, and severely diminished muscle action potential amplitude on electromyography. Also, a more severe course and poorer prognosis are associated with the AMSAN subtype. See also: Autoantibodies. Myoglobin. Neurophysiology: Neurons and Neuromuscular Transmission.

Further Reading Asbury AK (2005) Approach to the patient with peripheral neuropathy. In: Kasper DL, Braunwald E, Fauci AS, et al. (eds.) Harrison’s Principles of Internal Medicine, 16th edn., pp. 2500– 2510. New York: McGraw-Hill. Asbury AK and Thomas PK (eds.) (1995) Peripheral Nerve Disorders II. Oxford: Butterworth–Heinemann. Bennett CL and Chance PF (2001) Molecular pathogenesis of hereditary motor, sensory and autonomic neuropathies. Current Opinion in Neurology 14: 621–627. Chalela JA (2001) Pearls and pitfalls in the intensive care management of Guillain–Barre´ syndrome. Seminars in Neurology 21: 399–405. Donofrio PD (2003) Immunotherapy of idiopathic inflammatory neuropathies. Muscle and Nerve 28: 273–292. Kieseier BC and Hartung HP (2003) Therapeutic strategies in the Guillain–Barre´ syndrome. Seminars in Neurology 23: 159–168. Kieseier BC, Kiefer R, Gold R, Hemmer B, Willison HJ, and Hartung HP (2004) Advances in understanding and treatment of immune-mediated disorders of the peripheral nervous system. Muscle & Nerve 30: 131–156.

Paparounas K (2004) Anti-GQ1b ganglioside antibody in peripheral nervous system disorders: pathophysiologic role and clinical relevance. Archives of Neurology 61: 1013–1016. Rabinstein AA and Wijdicks EFM (2003) Warning signs of imminent respiratory failure in neurological patients. Seminars in Neurology 23: 97–104. Victor M and Ropper AH (2001) Adams and Victor’s Principles of Neurology, 7th edn. New York: McGraw-Hill. Willison HJ and Yuki N (2002) Peripheral neuropathies and antiglycolipid antibodies. Brain 125: 2591–2625. Winer JB (2001) Guillain–Barre´ syndrome. Molecular Pathology 54: 381–385. Yuki N, Susuki K, Koga M, et al. (2004) Carbohydrate mimicry between human ganglioside GM1 and Campylobacter jejuni lipooligosaccharide causes Guillain–Barre´ syndrome. Proceedings of the National Academy of Sciences 101: 11404–11409.

Upper Motor Neuron Diseases E Servera, J Marı´n, and J Sancho, Valencia University, Valencia, Spain & 2006 Elsevier Ltd. All rights reserved.

Abstract Upper motor neuron diseases are a heterogeneous group of disorders in which a degeneration of motor neurons of the cortex and tronchoencephalic motor nucleus occurs. Clinically, these disorders are characterized by weakness, motor clumsiness, spasticity, and hyperreflexia. The major cause of morbidity and mortality in these disorders is due to the involvement and dysfunction of respiratory muscles. Amyotrophic lateral sclerosis is the most characteristic example of motor neuron disease in which both the upper and lower motor neuron are involved; when only the upper motor neuron is affected, it is called primary lateral sclerosis. Other diseases with upper motor neuron dysfunction are spinal cord injury, multiple sclerosis, and stroke. In Parkinson’s disease, the upper motor neuron is indirectly affected. Respiratory muscle involvement entails alveolar hypoventilation, decreased cough capacity, and the risk of aspiration due to bulbar dysfunction. The use of respiratory muscle aids, noninvasive mechanical ventilation, and manually and mechanically assisted cough can improve survival, quality of life, and avoid hospitalization when respiratory muscles are involved. Moreover, during acute respiratory episodes, the combined use of inspiratory and expiratory muscle aids can avoid endotracheal intubation; this means continuous noninvasive ventilation with adequate respiratory secretions management if different interfaces are correctly used.

Introduction Upper motor neuron diseases are a heterogeneous group of disorders in which a degeneration of motor neurons of the cortex and tronchoencephalic motor nucleus occurs. Clinically, these disorders are characterized by muscular weakness, particularly of the extensor musculature of the upper limb and of the flexor musculature of the lower limb, motor clumsiness,

NEUROMUSCULAR DISEASE / Upper Motor Neuron Diseases 119

spasticity, and hyperreflexia; they may be associated with clonus and pathological reflexes (e.g., Babinski sign). When these disorders affect the corticobulbar way bilaterally, the most characteristic symptom is emotional lability. In these disorders, dysfunction of the respiratory muscles (the inspiratory and expiratory muscles) and the upper airway muscles accounts for the major cause of morbidity and mortality.

Etiology Amyotrophic lateral sclerosis (ALS) is the most characteristic example of motor neuron disease. In ALS, both upper and lower motor neuron diseases are involved. When the dysfunction is selective of the upper motor neuron, it is called primary lateral sclerosis (PLS); this is a sporadic disease with a lower incidence than ALS. Spinal cord injury (SCI) is another important cause of upper motor neuron affection and is the main cause of chronic hypoventilation failure in young people. Other disorders that can affect upper motor neurons are multiple sclerosis (MS) and strokes in which corticospinal and bulbospinal tracts are involved. Another serious disorder in which the upper motor neuron is indirectly affected is Parkinson’s disease (PD). In PD, the excessive inhibition of the talamocortical pathway produces a diminution of the stimulus of motor and premotor cortical areas.

Pathology and Physiopathology Primary Lateral Sclerosis and Amyotrophic Lateral Sclerosis

In PLS, there is a selective degeneration of the Betz motor neuron in lamina V of the cortex and a degeneration of corticospinal tracts. In ALS, the abnormality spreads to the anterior horn of the medulla and bulbar motor centers. In both diseases, the respiratory problems are related to weakness of respiratory muscles; respiratory complications are more severe in ALS than in PLS because in the former both upper and lower motor neurons are affected. Inspiratory muscle weakness produces hypoventilation. In the early stages of the disease, hypoventilation is present only in the rapid eye movement phase of sleep, but worsening gas exchange abnormalities contribute to frequent arousals, reducing sleeping time and sleeping efficiency, and result in daytime symptoms of sleep deprivation. As the disease worsens, hypoventilation extends to total sleeping time and then to the period during which the patient is awake. Moreover, due to inspiratory muscle weakness, the elastic load of the chest wall

gradually increases, which is related to the inability to achieve a full inhalation and contributes to increase the effort required for breathing. This loss of the capacity to perform maximum inflations produces structural changes of the lung and the thoracic cage. Deformities associated with the disease, such as kyphoscoliosis, also contribute to chest wall stiffness and increase the effort required for breathing. Cough capacity decreases due to inspiratory muscle weakness that inhibits a deep inspiration at the beginning of the cough effort, expiratory muscle weakness that inhibits an energetic contraction, unstable upper airway dysfunction, and inability to close the glottis (Figure 1). Involvement of the bulbar muscles produces inadequate respiratory protection and a risk of aspiration. Spinal Cord Injury

In SCI, respiratory muscle impairment depends on the level of the medullary lesion. High cervical cord lesions (C1–C2) and middle cervical cord lesions (C3– C5) cause paralysis of the diaphragm, intercostal, scalene, and abdominal muscles. These lesions result in severe hypoventilation, reduced ability to clear secretions (due to the loss of the ability to hyperinflate the lungs and an ineffective cough), and atelectasis. Low cervical lesions (C6–C8) and upper thoracic cord lesions denervate the intercostal and abdominal muscles but leave the diaphragm and neck muscles intact. Although phrenic nerves remain completely, or at least partially, intact at these levels, the intercostal activity necessary to stabilize the ribcage is lost, resulting in a compromised ventilation. Moreover, cough capacity is decreased due to weakness of expiratory muscles. Prognosis improves as lesions move downwards. When the lesion is lower in the spinal cord, the expiratory muscles, and thus cough capacity, are often more affected than the inspiratory muscles. Multiple Sclerosis

The pathological hallmark of MS is the presence of multiple areas of nerve demyelination, which may vary in size and localization. When MS affects the motor pathway, it causes muscle weakness and spasticity. Respiratory failure is a late manifestation of advanced disease, although acute respiratory failure can occur due to demyelination lesions of the cervical spinal cord or the respiratory centers of the medulla. Patients usually develop the insidious onset of chronic respiratory failure due to weakness of the respiratory muscles, especially the expiratory muscles. This weakness is produced due to demyelination, inactivity, and the effects of treatment with steroids. The respiratory dysfunction in MS may be

120 NEUROMUSCULAR DISEASE / Upper Motor Neuron Diseases

Malnutrition Fatigue

Weakness Bulbar dysfunction

Thoracic cage deformities

Decreased cough capacity

Decreased parenchima

Alveolar hy hypoventilation

Loss of compliance Bronchial obstruction

Sleep disturbances

Figure 1 Factors determining respiratory failure in motor neuron disease. Figure reprinted from Archivos de Bronconeumologı´a 2003; 39(9): 418–427. Permission granted by Ediciones Doyma S.L. Table 1 Patterns of respiratory involvement in multiple sclerosis depending on demyelinating plaque localization

Table 2 Indications for noninvasive mechanical ventilation in neuromuscular diseases

Abnormality

Anatomic localization

Paralysis of voluntary breathing Paralysis of automatic breathing

Bilateral corticospinal tracts, brainstem, or cervical cord Dorsomedial medulla, nucleus ambiguus, and medial lemnisc Upper cervical cord Lower brainstem Lower brainstem Tegmentum of medulla Medulla in region of nucleus tractus solitarius and floor of fourth ventricle

Symptoms: e.g., fatigue, dyspnea, and morning headache Physiologic criteria (one of the following): PaCO2445 mmHg Nocturnal oximetry demonstrating oxygen saturation o88% for 5 consecutive min For progressive neuromuscular disease, maximal inspiratory pressures o60 cmH2O or FVC o50% predicted

Diaphragmatic paralysis Apneustic breathing preserved Paroxysmal hyperventilation Obstructive sleep apnea Neurogenic pulmonary edema

caused by respiratory muscle weakness (hypoventilation and ineffective cough), bulbar dysfunction, obstructive sleep apnea, abnormalities in respiratory control, and paradoxical hyperventilation (Table 1). Stroke

The two most important motor pathways for respiratory control are the corticospinal tracts and the bulbospinal tracts. The former is responsible for voluntary breathing and the latter for automatic breathing. Hemispheric ischemic strokes influence respiratory function to only a modest degree; reduced chest wall movement and diaphragmatic excursion contralateral to the stroke have been reported. Therefore, vascular accidents of the cortex do not cause significant diaphragmatic impairment; however, in capsular stroke with extensive damage of the pyramidal respiratory fibers, the voluntary control of breathing

Source: American College of Chest Physicians; Goldberg AC, Legger P, Hill NS, et al. (1999). Clinical indications for noninvasive positive pressure ventilation in chronic respiratory failure due to restrictive lung disease, COPD and nocturnal hypoventilation. A consensus conference report. Chest 116: 521–534.

can be impaired with inability to change breathing voluntarily in the presence of preserved autonomic respiratory function. Damage to the bulbospinal tract eliminates autonomic control of breathing but leaves voluntary control intact (Ondine’s syndrome). In this situation, severe hypoventilation is produced during resting. Lateral medullary strokes can cause hypoventilation, apneustic breathing, ataxic breathing, and hyperventilation. Parkinson’s Disease

In PD, there is a degeneration of the compact part of the nigra substance resulting in a degradation of the dopaminergic nigrostriatal pathway that produces a dopaminergic denervation of the striatum nucleus. This results in an increase in acid g-aminobutiric inhibitory activity due to an increase in the activity of inner palidus nucleus neurons. The dysfunction of

NEUROMUSCULAR DISEASE / Upper Motor Neuron Diseases 121

Figure 2 (a) Noninvasive mechanical ventilation via mouthpiece and (b) mechanical aids to coughing with the Emerson InExsufflatorTM for a hospitalized patient with ALS and a pulmonary infection. Reproduced from Servera E, Sancho J, Go´mez-Merino E, et al. (2003) Noninvasive management of an acute chest infection for a patient with ALS. Journal of Neurological Science 209: 11–13, with permission from Elsevier.

respiratory muscles results in alveolar hypoventilation, a decrease in cough effectiveness, and upper airway dysfunction. Inspiratory muscle strength decreases with the progression of the disease due to impaired neural drive of the muscles. Inspiratory muscle endurance decreases in early PD due to impaired activation of the motor cortex for the respiratory muscles, a shift in the fibers fatigue-resistant type I and IIa to the more fatigable type IIb, mitochondrial abnormalities, and lack of coordination between inspiratory and

expiratory muscles. Expiratory muscle weakness produces a decrease in cough capacity. Upper airway dysfunction can produce occasional sudden and intermittent airway closure.

Clinical Features Respiratory symptoms in disorders that damage upper motor neurons are related to alveolar hypoventilation, impaired cough capacity, and upper airway dysfunction. Dyspnea, orthopnea, restless sleep,

122 NEUROMUSCULAR DISEASE / Upper Motor Neuron Diseases

nightmares, early morning headaches, and daytime hypersomnolence are related to alveolar hypoventilation. When respiratory impairment progresses, pulmonary hypertension with cardiac failure (cor pulmonale) can be present with increased orthopnea and dyspnea and lower extremity edema. Impaired cough capacity produces retained respiratory secretions that increase airway resistance and alter the respiratory mechanics. Moreover, these retained secretions can produce atelectasis and there is a high risk of superinfection and pneumonia. With the involvement of the upper airway, speaking and swallowing are affected and the risk of aspiration is greater, contributing to the impairment of cough effectiveness. This results in inadequate nutrition, weight loss, drooling, choking episodes and recurrent pulmonary infections due to aspiration episodes. In PD, the upper airway muscle dysfunction with closure can produce stridor, sudden death by apnea, and obstructive sleep apnea. During an acute respiratory episode such as a banal chest cold, respiratory muscle weakness is increased, impairing hypoventilation and cough capacity. In this way, these episodes can lead to acute respiratory failure.

In order to view the drop in pulseoxihemoglobin saturation during sleep, a nocturnal pulse oximetry must be performed. A polysomnographic record is appropriate when obstructive sleep apnea is suspected. To study swallowing disorders related to bulbar dysfunction, videofluoroscopy is performed.

Management of Respiratory Problems Management of respiratory problems in motor neuron disorders must be centered on three key points: alveolar hypoventilation, respiratory secretions, and swallowing disorders. Alveolar Hypoventilation

Noninvasive mechanical ventilation (NIV) has been used successfully to treat hypoventilation in neuromuscular patients (Table 2). NIV provides mechanical ventilation without access to the trachea and relieves dyspnea, and it also improves survival, cognitive function, and quality of life. The most common method of NIV is positive pressure ventilation with a pressure or volume ventilator. Initially, NIV is administered nocturnally and as needed during the daytime. A combination of different interfaces can be used to maintain NIV continuously throughout the

Lung Function Tests

Arterial blood gases show hypoxemia and, with the progression of the disease and impairment of hypoventilation, hypoxemia and hypercapnia. Spirometry and lung volumes show a restrictive pattern with decreased vital capacity and forced expiratory volume in 1 s and total lung capacity. Vital capacity has been shown to be a good prognostic factor; however, maximum insufflation capacity (MIC) is proposed to be a more useful prognostic measurement. MIC is the maximum volume of air that can be stacked in lungs with the glottis closed. MIC lower than 1500 ml indicates great difficulty in attaining an effective cough. Respiratory muscle force tests, such as static maximum pressures at the mouth (PImax and PEmax) and transdiaphragmatic pressure (Tdi), are infrequently used. Measurement of flows generated during a coughing effort has been proposed as an effective and useful parameter of cough capacity because peak flows generated during the expulsive phase of cough (PCF) are responsible for airway secretion removal. PCF lower than 2.7 l s 1 probably results in an ineffective cough, and PCF lower than 4.5 l s 1 in a stable patient indicates a high risk that the cough will become ineffective during an acute chest episode. Another test for measuring cough capacity is gastric pressure during a cough effort (PgaCo); however, this causes discomfort to the patient.

Figure 3 Complete atelectasis due to aspiration solved after 6 h with intensive use of mechanical in-exsufflation.

NEUROMUSCULAR DISEASE / Upper Motor Neuron Diseases 123

day (mouthpiece during day and nasal, oronasal, or lipseal during sleep) (Figure 2). In patients with severe bulbar involvement, NIV is often ineffective and poorly tolerated. When NIV is ineffective or poorly tolerated or refused by the patient, a tracheostomy is mandatory to initiate invasive mechanical ventilation. During acute episodes, continuous NIV can avoid endotracheal intubation with adequate respiratory secretions management and if different interfaces are correctly used. In patients with severe bulbar dysfunction, NIV tends to fail in acute episodes. Respiratory Secretions

When the patient is unable to perform an effective cough effort (PCFo2:70 l s 1 ), there is a high risk of major complications, such as atelectasis, superinfection of retained secretion, pneumonia, increased difficulty in breathing with acute respiratory failure, and failure of NIV. Several approaches are available to increase cough capacity in neuromuscular patients; however,

manually and mechanically assisted techniques have proved effective in removing respiratory secretions. In manually assisted cough, a thoracic and/or abdominal thrust is applied in synchrony with the patient’s cough effort, leading to enhanced expiratory flow rates. The effectiveness of manually assisted cough is greater if the patient’s lungs are insufflated before the highest volume that can be retained with a closed glottis with a manual resuscitator or a portable volume-limited ventilator. This technique requires patient cooperation and good bulbar function to close the glottis. When manually assisted cough cannot generate an effective cough effort, mechanically assisted cough TM with Cough Assist is the best alternative (Figures 3 TM and 4). Cough Assist delivers a deep insufflation using positive pressure followed immediately by an equal negative pressure that produces a forced exsufflation (MI-E). If a thoracic and/or abdominal thrust is applied during the exsufflation cycle, the PCF generated will be greater. MI-E does not require

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Figure 4 Flow–volume loops during coughing show unassisted and assisted peak cough flow (PCF). (a) No respiratory muscle involvement with effective unassisted PCF. (b) Ineffective unassisted PCF. It is effective, however, when manual and mechanical aids are applied: Both have the same values. (c) Ineffective unassisted PCF. Mechanically assisted PCF are clearly greater than manually assisted ones, but both are effective. (d) Severe bulbar dysfunction with ineffective assisted PCF.

124 NEUROMUSCULAR DISEASE / Myasthenia Gravis and Other Diseases

the patient’s collaboration; in severe bulbar dysfunction, however, MI-E can be ineffective due to a dynamic upper airway collapse during the exsufflation cycle (Figure 4(d)). When assisted coughing techniques cannot remove airway secretions, a tracheostomy must be performed in order to gain direct access to them. MI-E can be applied through a tracheostomy to avoid complications related to aspiration with a catheter. Moreover, aspiration with a conventional catheter through a tracheostomy tube fails to enter the main left stem bronchus in 90% of cases, but MI-E, when it is applied through the tracheostomy tubes, is able to clear the central, medium, and small bronchi of both left and right airways. Swallowing Disorders

When bulbar dysfunction produces aspiration, a percutaneous endoscopic gastrostomy must be performed in order to provide adequate nutrition and avoid the risk of respiratory problems related to an incompetent glottis. See also: Arterial Blood Gases. Carbon Dioxide. Neuromuscular Disease: Inherited Myopathies; Lower Motor Neuron Diseases; Acquired Myopathies; Peripheral Nerves; Upper Motor Neuron Diseases; Myasthenia Gravis and Other Diseases of the Neuromuscular Junction. Neurophysiology: Neurons and Neuromuscular Transmission. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Signs of Respiratory Disease: Breathing Patterns. Sleep Disorders: Central Apnea (Ondine’s Curse); Hypoventilation. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation.

Further Reading Bach JR (1994) Update and perspectives on noninvasive respiratory muscle aids. Part 1: The inspiratory aids. Chest 105(4): 1230–1240. Bach JR (1994) Update and perspective on noninvasive respiratory muscle aids. Part 2: The expiratory aids. Chest 105(5): 1538–1544. Bach JR (2004) Noninvasive respiratory muscle aids: intervention goals and mechanisms of action. In: Bach JR (ed.) Management of Patients with Neuromuscular Disease, pp. 211–270. Philadelphia: Hanley & Belfus. Brown LK (1994) Respiratory dysfunction in Parkinson’s disease. Clinical Chest Medicine 15: 715–722. Goldberg AC, Legger P, Hill NS, et al. (1999) Clinical indications for noninvasive positive pressure ventilation in chronic respiratory failure due to restrictive lung disease, COPD and nocturnal hypoventilation. A consensus conference report. Chest 116: 521–534. Gosselink R, Kovacs L, and Decramer M (1999) Respiratory muscle involvement in multiple sclerosis. European Respiratory Journal 13: 449–454.

Hadjikoutis S and Wiles CM (2001) Respiratory complications related to bulbar dysfunction in motor neuron disease. Acta Neurologica Scandinavia 103: 207–213. Laghi F and Tobin MJ (2003) Disorders of the respiratory muscles. American Journal of Respiratory and Critical Care Medicine 168: 10–48. Make BJ, Hill NS, Goldberg AL, et al. (1998) Mechanical ventilation beyond the intensive care unit. Report of a consensus conference of the American College of Chest Physicians. Chest 113(5 supplement): 289S–344S. Mausel JK and Norman JR (1990) Respiratory complications and management of spinal cord injuries. Chest 97: 1446–1452. Miller RG, Rosenberg JA, Gelinas DF, et al. (1999) Practice parameter: the care of the patient with amyotrophic lateral sclerosis (an evidence based review): report of the quality standards subcommittee of the American Academy of Neurology: ALS Practice Parameters Task Force. Neurology 52: 1311–1323. Perrin C, Uterborn J, Da´mbrosio C, et al. (2004) Pulmonary complications of chronic neuromuscular diseases and their management. Muscle & Nerve 29: 5–27. Servera E, Sancho J, and Zafra MJ (2003) Cough and neuromuscular diseases. Noninvasive airway secretions management. Archivos de Bronconeumologı´a 39: 418–427. Servera E, Sancho J, Go´mez-Merino E, et al. (2003) Noninvansive management of an acute chest infection for a patient with ALS. Journal of Neurological Science 209: 11–13. Vingerhoest F and Bogousslavsk J (1994) Respiratory dysfunction in stroke. Clinical Chest Medicine 15: 729–737. Winslow C and Rozovsky J (2003) Effect of spinal cord injury on the respiratory system. American Journal of Physical Medicine and Rehabilitation 82: 803–814.

Myasthenia Gravis and Other Diseases of the Neuromuscular Junction H M DeLisser, University of Pennsylvania School of Medicine, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Processes that target the neuromuscular junction may result in clinical syndromes characterized by muscle weakness. Among these disorders, the most common is myasthenia gravis (MG), an acquired, autoimmune disease in which the acetylcholine receptor is targeted in the majority of affected patients. The underlying cause of MG, however, remains unknown. Fluctuating muscle weakness is the hallmark feature of MG. Ocular, facial, oropharyngeal, axial and limb muscles may be differentially involved, leading to a variety of clinical presentations. Ventialtory support may be required for up to 20% of patients who develop respiratory insufficiency due to inspiratory muscle weakness and/or inability to maintain a patent upper airway. MG must be distinguished from a number of other disorders including, the Lambert–Eaton myasthenic syndrome, botulism, and non-immune genetic congenital myasthenic syndromes. In addition to several general measures, a number of treatments are available for MG with differing onsets, peaks and durations of effects.

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Introduction The neuromuscular junction (NMJ) is a specialized synapse designed to transmit nerve impulses to the muscle via a chemical transmitter, acetylcholine (ACh), stored in synaptic vesicles at the nerve terminal (Figure 1). When an action potential reaches the nerve terminal, voltage-gated calcium channels are activated, resulting in a rise in intracellular calcium that triggers vesicle fusion with the plasma membrane and the release of ACh into the synaptic cleft. Repolarization of the motor nerve terminal results from the opening of voltage-gated potassium channels. Diffusion of ACh across the synaptic cleft is rapid, facilitated by the narrow (B50 nm) space between the nerve and muscle plasma membrane and the high diffusibility of ACh. The matrix of the synaptic cleft is composed of various proteins that regulate the synthesis of postsynaptic proteins and the concentration of acetylcholinesterase (AChE). In hydrolyzing ACh, AChE causes a rapid decline in ACh concentration that acts to limit the activation of the acetylcholine receptor (AChR). After crossing the synaptic cleft, ACh binds to AChR, an ion channel composed of two a-subunits, and one copy each of the b-, d-, and e- or g- subunits. Receptor binding results in the opening of the AChR ion channel, the entry of cations, primarily sodium, into the muscle, and the generation of an endplate potential. When a certain threshold depolarization is reached, voltage-gated sodium channels are

activated, providing entry of more sodium ions, generation of the muscle action potential, and ultimately stimulating muscle contraction. Processes that target the NMJ may result in clinical syndromes characterized by muscle weakness. Among these disorders, the most common is myasthenia gravis (MG), an acquired autoimmune disease in which AChR is targeted in the majority of affected patients. Less commonly, impaired neuromuscular transmission may also result from other acquired conditions such as the Lambert–Eaton myasthenic syndrome or botulism, or non-immune congenital myasthenic syndromes.

Myasthenia Gravis Initial descriptions of the clinical features of MG date back to reports by Wilhelm Erb, Samuel Goldflam, and others from the late nineteenth century of a syndrome of fluctuating weakness, characterized by frequent involvement of bulbar muscles and a tendency for remissions and relapses over time. Hence, MG was initially referred to as Erb’s or Erb–Goldman disease. However, Friedrich Jolly, a prominent neurologist of that period, proposed the term ‘myasthenia gravis pseudoparalytica’ (myo, muscle; asthenia, weakness; gavis, severe) to emphasize the potential involvement of muscles beyond those innervated by cranial nerves. In time, his term (without ‘pseudoparalytica’) emerged as the accepted name for this disease.

Motor nerve terminal ACh VGKC VGCC AChE

AChR MuSK Muscle Figure 1 Neuromuscular Transmission. Neuromuscular transmission involves the entry of calcium through voltage-gated calcium channels (VGCCs) into the motor nerve terminal and the release of acetylcholine (ACh) into the synaptic space. ACh subsequently binds to its postsynaptic receptor (AChR), opening associated ion channels. This leads to a depolarization that, if sufficient, activates voltagegated sodium channels (not shown), resulting in an action potential in the muscle and muscle contraction. ACh is rapidly hydrolyzed by acetylcholinesterase (AChE) and repolarization of the motor nerve terminal results from the opening of voltage-gated potassium channels (VGKCs). Accessory molecules such as the muscle-specific receptor tyrosine kinase (MuSK), which may play a role in the clustering of the AChR, are also present on the postsynaptic surface.

126 NEUROMUSCULAR DISEASE / Myasthenia Gravis and Other Diseases Table 1 Classification of MG patients with AChR-specific antibodiesa Subgroups

Sex (male:female)

Frequent MHC associations

Pathology of thymus

Associated autoantibodies

Early onset (p40 years)

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Thymoma-associated (primarily 40–60 years) Late onset (440 years)

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Epithelial malignancy with lymphocytic infiltrates Normal or atrophied

High frequency of tissuespecific antibodies (e.g., thyroid) Antibodies against ryanodine, titin, and cytokines are common Antibodies against titin and ryanodine detected, particularly over age 60

a

This classification may not apply to non-Caucasian racial groups, seronegative patients or ocular MG.

MG is rare, with an estimated incidence of 4 to 6 new cases per million per year and a prevalence currently of 40–80 per million. Its prevalence has steadily increased over the last 50 years due to improved recognition of the disease; the availability of more sensitive and specific diagnostic tests; increased life spans in affected individuals due to more effective treatments; and increased persons at risk for the disease because of aging populations. Although acquired MG can occur at any age, the peak ages of onset in females are between the teens and the thirties, while in males they are between 50 and 70 years of age. Etiology

Although the autoimmune nature of the disease is well established, the underlying cause of MG remains unknown. However, for affected patients with antibodies against AChR, MG can be divided into three different forms based on age of onset, gender, thymic pathology, MHC associations, and the absence or presence of antibodies specific for antigens other than AChR (Table 1). This suggests that at least three, perhaps partly genetically determined, etiological pathways are available for the development of AChR-specific antibodies and a similar clinical expression of the disease. Further, additional causes may exist for patients with MG, but who lack antibodies against AChR (‘seronegative MG’, see below). Clinical Features

Clinical presentation Fluctuating muscle weakness is the hallmark feature of MG. This weakness may vary from day to day or even hour to hour and fluctuate from muscle to muscle, typically increasing over the course of the day. Ocular, facial, oropharyngeal, axial, and limb muscles may be differentially involved, leading to a variety of clinical presentations

Table 2 Symptoms and signs of myasthenia gravis Eye muscles Diplopia Gaze pareses Photophobia Ptosis Facial muscles Inability to maintain upper eyelid closure against manual efforts to open it (orbicularis oculi weakness) Inability to prevent the escape of air through pursed lips as expanded cheeks are compressed (orbicularis oris weakness) Tongue and posterior pharyngeal muscles Difficulty speaking Weakness in chewing Dysphagia Axial muscles Neck muscle weakness Vocal cord paralysis Respiratory muscle weakness Limb muscles Difficulty raising arms to bathe, dress, brush hair, etc. (proximal upper-extremity weakness) Difficulty walking up stairs or for long distances (proximal lowerextremity weakness)

(Table 2). Deep tendon reflexes and skin sensation are intact. Muscle atrophy is uncommon, but may be noted in isolated muscles. Respiratory insufficiency due to inspiratory muscle weakness and/or the inability to maintain a patent upper airway may develop in up to 20% of MG patients. This is the most serious complication of this disease and when it occurs the patient is said to be in crisis. An inadequate negative inspiratory force (
20 to 0 cmH2O) or a low vital capacity (o10 ml per kg of body weight), particularly when the trend is downward, are evidence of severe inspiratory muscle weakness and indicate the need for ventilatory support. Additionally, expiratory muscle weakness may produce an ineffective cough that predisposes the affected patient to retention of airway secretions, mucus plugging, atelectasis, and pneumonia.

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Owing to the variable nature of the disease, it is not possible at the time of the initial diagnosis to predict the outcome for an individual patient. For generalized MG, the clinical course can be divided into three phases: an initial active stage of 5–7 years characterized by exacerbations and remissions; an inactive, stable second phase lasting up to 10 years; and a final burned-out stage in which there is slow improvement. Confirming the clinical diagnosis Serological testing Antibodies that bind to the AChR are present in 85% of MG patients and are the most specific diagnostic marker for MG. Positive results, however, are less likely with early, mild, or purely ocular disease; consequently repeat testing may be warranted when MG is strongly suspected and initial values are normal. Further, the serum level of AChR binding antibodies varies considerably among patients with similar degrees of weakness. Acetycholine receptor binding antibodies without clinical evidence of MG have been reported in small percentages of patients with autoimmune liver or thyroid diseases, Lambert–Eaton myasthenic syndrome, systemic lupus erythematosus, inflammatory neuropathies, and amyotrophic lateral sclerosis. Antibodies targeting other skeletal muscle proteins have been identified that may also be helpful in the diagnostic evaluation of MG variants (see below). Pharmacological confirmation The most rapid pharmacological test involves the intravenous administration of 2–5 mg edrophonium chloride (tensilon), a short-acting anticholinesterase. A positive test requires a measurable change in a physical finding (e.g., ptosis, gaze paresis, grip strength) or a measure of respiratory function (e.g., vital capacity) a few minutes after injection. Although widely used and generally accepted, the tensilon test is neither absolutely specific nor sensitive for MG and is dependent on the patient giving a maximal effort before and after the drug is administered. Electrophysiological testing Electrophysiological studies may help to confirm an impairment in neuromuscular transmission and exclude other potential confounding conditions such as the Lambert–Eaton myasthenic syndrome or congenital myasthenic disorders. Repetitive nerve stimulation is the most commonly used confirmatory electrophysiological test due to its wide availability and ease of performance. Single-fiber electromyography, a more sensitive study for detecting neuromuscular dysfunction, may also be employed, but requires specialized equipment and training.

Variants of myasthenia gravis Seronegative myasthenia gravis In the 15% of patients that are negative for AChR antibodies (‘seronegative MG’), a proportion will have antibodies to muscle-specific receptor tyrosine kinase (MuSK), a NMJ protein required for the clustering of AChRs. These antibodies have been reported in 25– 72% of patients with seronegative MG, but are not detected in AChR antibody-positive patients. The clinical features of the seropositive and seronegative diseases are similar, although some reports have noted that seronegative patients are more likely to have milder or purely ocular disease. Thymoma-associated myasthenia gravis Thymomas occur in 10–15% of adult patients with MG. Although usually benign and encapsulated, a third or more of thymomas can be invasive as determined at the time of surgery. In addition to elevated serum AChR antibodies, the majority of patients with MG and a thymoma will also have antibodies to titin (a giant filamentous muscle protein) and ryanodine (sarcoplamic reticulum calcium-release channels). Although the role of these antibodies in disease pathogenesis is unclear, the absence of elevated serum titers of titin and ryanodine antibodies argues against the presence of a thymoma.

Conditions to Distinguish from Myasthenia Gravis Lambert–Eaton Myasthenic Syndrome

This is a rare, autoimmune disease involving antibodies against the presynaptic, voltage-gated calcium channels that mediate vesicle fusion with the membrane. Less ACh is released into the synaptic cleft and, as a result, neuromuscular transmission is compromised. Lambert–Eaton myasthenic syndrome (LEMS) can occur as an idiopathic disorder, but 50– 70% of affected patients will present as a paraneoplastic syndrome, most often associated with small cell lung cancer. It may precede the diagnosis of cancer by as much as 2 years. In addition to malignancies, LEMS has been associated with other autoimmune disorders, including Graves’ disease, pernicious anemia, celiac disease, and juvenile diabetes. Patients will most often be older than age 40 and will present with proximal muscle weakness, typically greater in the lower extremities than in the upper extremities, that is exacerbated by heat or exercise. Compared to autoimmune MG, bulbar and ocular involvement is less prominent. Patients with LEMS rarely present with respiratory failure,

128 NEUROMUSCULAR DISEASE / Myasthenia Gravis and Other Diseases

although it may develop later in the course of the disease. Autonomic dysfunction manifested by constipation, blurred vision, dry mouth, or orthostatic hypotension may occur in 75% of patients. With continuous contraction from sustained strength testing, muscle strength may actually appear to increase for a period of time, a finding that may persist for up to 30 s. For LEMS patients, during sustained muscle contraction, calcium concentrations are transiently increased in the presynaptic terminal resulting in more ACh release. The reasons for this phenomenon are obscure, but it is the basis for the Lambert sign where the grip becomes more powerful over several seconds of strength testing. When associated with a cancer, treatment of the malignancy will usually result in significant clinical improvement. Regardless of the presence of a cancer, therapies used for MG (see below) are also employed, but these may be less effective in LEMS. Botulism

This is a paralyzing disease caused by a toxin produced by Clostridium botulinum, an anaerobic, spore-forming, Gram-positive bacteria. This extremely potent toxin blocks the release of ACh from the presynaptic terminal by cleaving proteins needed for the exocytosis of the presynaptic vesicles. There are five recognized forms of botulism: classical (food-borne), infant, wound, hidden, and inadvertent. In the classical form, preformed toxin is ingested in contaminated food, while in infant botulism spores of C. botulinum are ingested and germinate in the intestinal tract. In the past, wound botulism has been rare and almost exclusively seen in patients following surgery and trauma. However, new cases of wound botulism are now being observed in heroin-injecting drug abusers. Hidden (or ‘unclassified’) botulism refers to patients for which a history of contaminated food, wounding, or heroin injection drug use is lacking. These cases are probably adult variants of the infant form. The recently described inadvertent form has been reported in patients who were treated with intramuscular injections of botulism toxin. The clinical features of botulism involve a typical pattern starting with cranial nerve palsies manifested by blurred or double vision and difficulties in speaking or swallowing followed by descending weakness of the limbs, culminating with respiratory failure in severe cases. Dilated and poorly responsive pupils, absence of deep tendon reflexes, and evidence of autonomic dysfunction (constipation, dry mouth, urinary retention) help to distinguish botulism from MG.

Congenital Myasthenic Syndromes

These are genetic disorders of the neuromuscular junction (Table 3) with most due to mutations that reduce the expression or alter the kinetics of the AChR. One of these disorders should be suspected in the following settings: (1) infants with hypotonia and poor development of motor milestones; (2) children with weakness and small underdeveloped muscles; (3) adults with childhood history of neuromuscular difficulties affecting limb, truncal, cranial, or respiratory muscles; and (4) patients with seronegative myasthenia gravis.

Pathogenesis A number of clinical and experimental features provide clear evidence that MG is an antibody-mediated autoimmune disorder: (1) antibodies against AChR are present in 80–90% of affected patients; (2) immunoglobulin G (IgG) is detected at the NMJ, the site of dysfunction; (3) administration of IgG from MG patients to experimental animals reproduces the typical features of the disease; (4) treatments that reduce the serum levels of circulating anti-AChR antibodies improve symptoms; and (5) MG can be induced in animals by immunization with AChR. Autoantibodies

AChR antibodies are polyclonal, vary in light chain compositions and subclass structure, and recognize a heterogeneous epitope repertoire that varies from patient to patient. Most of the pathogenic antibodies target a region of the a-subunit termed the ‘main immunogenic region’ (MIR). Neuromuscular transmission is impaired by these antibodies via three distinct mechanisms: (1) they bind to the AChR, Table 3 Congenital myasthenic syndromes Presynaptic disorders Choline acetyltransferase deficiency Reduced quantal release of ACh with/without synaptic vesicles Lambert–Eaton syndrome-like CMSa Synaptic disorders End-plate acetylcholinesterase deficiency Postsynaptic disorders Primary AchRb deficiencies Reduced AChR expression due to AChR mutations Reduced AChR expression due to plectin deficiency Reduced AChR expression due to rapsyn mutations Primary AChR kinetic abnomalities Fast-channel CMS Slow-channel CMS Mutations of perijunctional sodium channels a b

CMS, congential myasthenic syndrome. AChR, acetylcholine receptor.

NEUROMUSCULAR DISEASE / Myasthenia Gravis and Other Diseases 129

interfering with ACh binding and altering receptor function; (2) they accelerate AChR degradation via antibody-mediated receptor cross-linking and enhanced endocytosis; and (3) destruction of the postsynaptic surface by antibody-mediated complement activation. Of these three processes, complement-mediated damage is probably the most important. The net effect is to reduce the number of functional AChRs at the postsynaptic muscle surface, leading to less end-plate depolarization and reduced muscle action potentials. Autoantibodies against antigens other than AChR have also been found in MG. These include antibodies against skeletal muscle proteins, including titin, ryanodine receptor, myosin, tropomyosin, actinin, and actin. Further, as noted above, a proportion of the seronegative patients will have antibodies against MuSK. The pathogenic significance, however, of these other autoantibodies is still unknown. Cellular Autoimmunity

CD4 þ T-helper cells that are AChR-specific are essential to the activation of B cells and for the production of high-affinity IgG autoantibodies. The majority of the T-cell recognition sites are located in the AChR a-subunit and are distinct from those in the MIR targeted by antibodies. As with antibody production, T-cell responses are polyclonal and vary among patients. The T cell-dependent nature of MG is consistent with its association with HLA antigens (see Table 1). The Thymus in MG Pathogenesis

A number of pathological, immunological, and experimental features point to a role for the thymus in the pathogenesis of MG. Lymphoid follicular hyperplasia and thymomas are common in patients with MG. Thymic tissue from MG patients contain elevated numbers of mature T cells, particularly AChR-reactive T cells, as well as B cells capable of producing AChR antibodies. Proteins antigenically similar to AChR are present in normal and myasthenic thymus that could provide a source of antigen for autosensitization. When fragments of myasthenic thymic tissue are transplanted into severe combined immunodeficiency mice, human antibodies are generated that bind to the AChR. Lastly, thymectomy appears to improve the clinical course of MG patients. Animal Models

Animal models of experimental autoimmune MG (EAMG) can be induced in rats and mice by immunization with Torpedo californica acetylcholine

receptors in complete Freund’s adjuvant. The pathogenesis of mouse EAMG closely resembles that of human MG, while the acute phase of EAMG in rats fails to mimic the pathology of the human disease. Consequently, the murine model is preferred, and even more so given the ever-increasing array and availability of knockout and transgenic mice targeting cytokine and immune response genes. There are, however, strain differences in the development of EAMG in mice, based on MHC (H-2)-linked genes.

Management and Current Therapy In addition to several general measures, a number of treatments are available for MG with differing onsets, peaks, and durations of effects (Table 4). General Measures

Several general approaches are employed in all patients. These include: (1) insuring adequate rest and avoiding overexertion or unnecessary fatigue; (2) a well-balanced diet containing foods rich in potassium; (3) aggressive treatment of infections as they may exacerbate weakness; (4) avoiding or discontinuing drugs known to unmask or exacerbate MG (Table 5); (5) assessing for thyroid dysfunction and vitamin B12 deficiency as manifestations of concomitant autoimmune processes that may increase myasthenic symptoms; and (6) participation in support groups. Anticholinesterases

These medications are the initial therapy for patients with MG. By inhibiting acetylcholinesterase, they increase the amount of ACh available for neuromuscular transmission. Some muscles may improve for a few hours, while others may fail to respond or even weaken on these drugs. The most commonly used acetylcholinesterase inhibitor is pyridostigmine bromide (Mestinon). Anticholinesterases are safe but their use may be limited by abdominal cramps and diarrhea. If significant symptoms are present at pyridostigmine doses of 60–90 mg four times a day, immunomodulating therapy should be added. Table 4 Time to clinical effect of therapies for myasthenia gravis Therapy

Time to clinical effect

Pyridostigmine Plasmapheresis IVIg Prednisone Thymectomy

10–15 min 1–14 days 1–4 weeks 2–8 weeks 6–36 months

130 NEUROMUSCULAR DISEASE / Myasthenia Gravis and Other Diseases Table 5 Drugs that adversely affect myasthenia gravis

Plasmapheresis and Intravenous Immunoglubulin

Drugs that unmask or exacerbate MG Anesthetic agents Antiarrhythmics Procainamide Quinidine Verapamil Intravenous lidocaine Antibiotics Aminoglycosides Tetracyclines Polypeptides (polymyxin B, colistin, colistemethate) Miscellaneous (clindamycin, lincomycin, ciprofloxacin, high-dose ampicillin and intravenous erythromycin) Anticonvulsants Gabapentin Phenytoin Magnesium salts Neuromuscular blocking agents Thyroid preparations Drugs that may induce MG by precipitating an autoimmune reaction Alpha-interferon Chloroquine Interleukin-2 D-Penicillamine Trimethadione Drugs implicated in isolated instances of MG exacerbation a Beta-blockers Cimetidine Citrate Cloroquine Cocaine Diazepam Gemfibrozil Lithium carbonate Radiocontrast media Statins Trihexyphenidyl hydrochloride

These are two available methods for decreasing the level of circulating AChR antibodies in affected patients. Although the effectiveness of plasmapheresis is clearly based on its ability to remove the pathogenic autoantibodies, the mechanism of action of intravenous immunoglobulin (IVIg) in MG is unknown. Both treatments are especially useful when there is an urgent need for a relatively quick response such as impending myasthenic crisis or prior to procedures known to exacerbate MG, such as surgery or irradiation. Patients will usually begin to improve within several days, but the benefit persists only for 3–6 weeks. Current data suggest that plasmapheresis and IVIg are equally effective in MG. Given this and its lower cost, relative ease of administration, and somewhat more favorable side effect profile, IVIg is probably favored over plasmapheresis, particularly if the treatment is to be repeated chronically as an adjunct to managing steroid-dependent refractory MG.

a Not every MG patient is expected to react adversely to these medications.

Immunosuppressive Drug Therapy

High-dose prednisone (50–60 mg daily) is the mainstay of immunosuppressive therapy for moderate to severe disease, particularly in the setting of a myasthenic crisis. Some patients receiving these doses of steroid may initially experience a transient deterioration that in some instances may require ventilatory support. Consequently, prednisone therapy for affected patients is often initiated using low doses given on alternate days and is then gradually increased until symptoms improve or a daily dose of 50–60 mg is reached. For patients who cannot tolerate or fail to respond to prednisone or who require high doses of prednisone for symptom control, azathioprine is often added. As alternatives to azathioprine, there are reports on the use of mycophenolate mofetil (Cellcept), cyclosporine, and cyclophosphamide to treat refractory MG.

Thymectomy for Nonthymomatous MG

This procedure is generally reserved for younger patients, early in the course of their disease, with moderate to severe generalized MG whose symptoms are not controlled with low doses of anticholinesterases or immunosuppressive medications. Although considered a potential option for inducing symptom improvement or disease remission, the benefit of thymectomy in nonthymomatous autoimmune MG has never been conclusively demonstrated. Thus, patients having this surgery may experience some improvement, but the majority will not become symptom-free or sustain a durable remission. Further, when improvement does occur, it may take up to several months, or even years, to achieve its peak effect. See also: Autoantibodies. Neuromuscular Disease: Peripheral Nerves. Neurophysiology: Neurons and Neuromuscular Transmission.

Further Reading Cherington M (2004) Botulism: update and review. Seminars in Neurology 24: 155–163. Christadoss P, Poussin M, and Deng C (2000) Animal models of myasthenia gravis. Clinical Immunology 94: 75–87. Dalakas MC (2004) Intravenous immunoglobulin in autoimmune neuromuscular diseases. Journal of American Medical Association 291: 2367–2375. Darnell RB and Posner JB (2003) Paraneoplastic syndromes involving the nervous system. New England Journal of Medicine 349: 1543–1554. Harper CM (2004) Congenital myasthenic syndromes. Seminars in Neurology 24: 111–123. Hughes BW, Moro De Casillas ML, and Kaminski HJ (2004) Pathophysiology of myasthenia gravis. Seminars in Neurology 24: 21–30.

NEUROPHYSIOLOGY / Overview 131 Keesey JC (2002) Myasthenia Gravis. An Illustrated History. Roseville, CA: Design Group. Keesey JC (2004) Clinical evaluation and management of myasthenia gravis. Muscle and Nerve 29: 484–505. Mareska M and Gutmann L (2004) Lambert–Eaton myasthenic syndrome. Seminars in Neurology 24: 149–153. Marx A, Muller-Hermelink HK, and Strobel P (2003) The role of thymomas in the development of myasthenia gravis. Annals of the New York Academy of Sciences 998: 223–236. Meriggioli MN and Sanders DB (2004) Myasthenia gravis: diagnosis. Seminars in Neurology 24: 31–39.

Newsom-Davis J (2003) Therapy in myasthenia gravis and Lambert-Eaton myasthenic syndrome. Seminars in Neurology 23: 191–198. Saperstein DS and Barohn RJ (2004) Management of myasthenia gravis. Seminars in Neurology 24: 41–48. Vincent A (2002) Unraveling the pathogenesis of myasthenia gravis. Nature Reviews: Immunology 2: 797–804. Vincent A, McConville J, Farrugia ME, et al. (2003) Antibodies in myasthenia gravis and related disorders. Annals of the New York Academy of Sciences 998: 324–335.

NEUROPHYSIOLOGY Contents

Overview Neural Control of Airway Smooth Muscle Neuroanatomy Neuroendocrine Cells Neurons and Neuromuscular Transmission

Overview K M Spyer, G L Ackland, and A V Gourine, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Breathing is controlled by a neuronal network located within the lower brainstem. The basic respiratory rhythm is generated by a population of neurons within the so-called pre-Bo¨tzinger complex and then subsequently shaped, modified, and transmitted to bulbospinal respiratory pre-motor neurons which in turn relay the respiratory pattern to spinal motor neurons innervating inspiratory and expiratory muscles. The brainstem respiratory network receives continuous modulatory inputs from the other parts of the brain and afferent inputs from the periphery. Inputs from the peripheral and central chemoreceptors (which provide information about arterial levels of PO2 and PCO2), from the mechanoreceptors located in the respiratory tract (which provide information about the mechanical status of the lungs and chest), from proprioceptors in muscles, tendons, and joints (which are important for coupling of respiratory and locomotor patterns) as well as from the higher centers of the central nervous system (CNS), such as limbic system, are all essential for adaptive changes (both short- and long-term) in the respiratory motor output to ensure appropriate ventilation of the lungs in variable environmental and physiological conditions. Since breathing is a fundamental rhythmic activity which is essential to support life of an individual, disorders of central nervous respiratory control are critical, resulting in substantial morbidity and mortality.

Introduction Breathing is the vital rhythmic activity which is essential to maintain appropriate rates of oxygen uptake

and CO2 elimination in animals with high metabolic rates. This respiratory rhythm is a coordinated alternation of inspiratory, postinspiratory, and expiratory activities. Their coordination is important, not only for lung ventilation, but also for vocalization, swallowing, and motor control. In mammals, breathing is controlled by a network of neurons located within the lower brainstem – in the medulla and pons (Figure 1). The respiratory neuronal network has to be fully operational before birth and must ensure adaptation and survival of the organism in variable environmental and physiological conditions. In this short overview, we discuss the neurophysiology of the central nervous control of breathing focusing on the anatomy of the brainstem respiratory network, mechanisms of respiratory rhythm generation, and the control of breathing exerted by chemo- and mechanoafferent inputs. We also mention how central nervous control of breathing is modified in some common physiological and pathophysiological states and give a brief overview of common and serious disorders of the central nervous control of respiratory activity.

Central Nervous Control of Breathing Anatomy

The core neuronal circuitry responsible for the generation and shaping of the respiratory rhythm, as well as transmitting this rhythm to the spinal motor neurons controlling the diaphragm and intercostal muscles is located in the lower brainstem (Figure 2).

132 NEUROPHYSIOLOGY / Overview Temperature

Stress, emotion Sleep Limbic system

Thermoreception

Locomotion, exercise

Brainstem respiratory network

Proprioception

H+

PaCO2

PaO2

Central and peripheral chemoreception Spinal respiratory motor neurons Mechanoreception

Ribcage

Diaphragm Figure 1 Regulation of breathing by the central nervous system: main afferent inputs and motor output. Refer to the text for details.

The respiratory outflow originates from a bilaterally organized dorsal respiratory group and ventral respiratory column (VRC) of neurons in the pons and the medulla oblongata. The VRC is essential and sufficient to generate the respiratory rhythm. The VRC extends rostrocaudally in close proximity to the nucleus ambiguus and forms a ‘distributed’ network, involving assemblies of different classes of respiratory neurons that are located in close proximity to one another. The VRC can be divided into several functional compartments, including from rostral to caudal: the retrotrapezoid nucleus (RTN), parafacial respiratory group (pFRG), Bo¨tzinger complex, pre-Bo¨tzinger complex (PBC), rostral and caudal ventral respiratory groups (Figure 2). The functional divisions of the VRC can be identified using various neurochemical markers and physiologically by monitoring central respiratory output following activation and/or blockade of neurons in these regions of the brainstem. Selective lesioning of

the PBC has identified it as the principal kernel for generating respiratory activity. It contains respiratory neurons that appear essential for generation of the respiratory rhythm. The PBC can be visualized by labeling the neurons with antibodies against neurokinin-1, m-opioid receptors, and somatostatin, which are suggested to be specific predominantly for PBC respiratory neurons. Recent evidence suggests that neurons in the RTN/pFRG may also play a role in the generation of the respiratory rhythm (in addition to that in the PBC), while the other functional compartments of the VRC contribute to shaping of this basic rhythm, and these interactions produce a respiratory pattern which is then relayed to spinal respiratory motor neurons. A group of neurons located in the pontine parabrachial and Ko¨lliker–Fuse nuclei (referred as ‘pneumotaxic centre’ in the older literature) (Figure 2) provides input to the VRC and plays an important role in reflex regulation and shaping of respiratory

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K–F

LC PB

RTN/ pFRG BC

NTS

PBC rVRG cVRG

Spinal respiratory motor neurons

PB K–F

Mo5

RTN/pFRG

NTS

7n

PBC rVRG cVRG BC Amb

7

LRt 1 mm

Ventral surface chemosensitive areas From the peripheral chemoreceptors (a)

From the airway mechanoreceptors

(b)

Figure 2 Neuroanatomy of the brainstem respiratory network. Horizontal (a) and saggittal (b) views of the rat brainstem showing locations of the main groups of respiratory neurons in the mammalian central nervous system. 7, facial nucleus; Amb, nucleus ambiguus; BC, Bo¨tzinger complex; cVRG, caudal ventral respiratory group; K–F, Ko¨lliker–Fuse nucleus; LC, locus ceruleus; LRt, lateral reticular nucleus; Mo5, motor trigeminal nucleus; NTS, nucleus tractus solitarii; PB, parabrachial nucleus; PBC, pre-Bo¨tzinger complex; RTN/pFRG, retrotrapezoid nucleus and parafacial respiratory group; rVRG, rostral ventral respiratory group.

activity patterns. The major peripheral afferent inputs to the brainstem respiratory network that influence its activity originate from the arterial chemoreceptors, which are sensitive to both changes in PaO2 and PaCO2, and from mechanoreceptors that provide information about the mechanical status of the respiratory tract. Both arterial chemoreceptor afferents and afferents originating from the pulmonary mechanoreceptors terminate in the dorsal medullary nucleus tractus solitarii (NTS). The initial integration of these afferent inputs that are essential for respiratory control occurs at the level of the NTS. A major role of the dorsal respiratory group of neurons (located within the ventrolateral NTS) is to modulate respiratory activity patterns in relation to the afferent input and contribute in particular to promoting transition from inspiration to expiration. Mechanisms of CO2 chemosensitivity ensure that breathing is always appropriate for the current metabolic rate; therefore, CO2 provides the main tonic drive for ventilation. It is probably not a coincidence that the primary central CO2 chemosensitive areas are located on the ventral surface of the medulla just beneath the VRC in the close proximity to the brainstem respiratory rhythm and pattern generator (Figure 2).

Generation of the Respiratory Rhythm

Ventilation of the lungs is characterized by the respiratory frequency, tidal volume, and minute ventilation which are based on the time spent in inspiration and expiration as well as the rate of the air flow. However, neurophysiological data indicate that the medullary respiratory network oscillates in a three-phased respiratory pattern. These three phases are defined as inspiration, postinspiration (passive expiration), and expiration (active expiration). At least two antagonistic phasic activities are necessary, for example, inspiratory and postinspiratory activities during rapid shallow breathing. These phases are usually supplemented by a third, expiratory activity, which is, however, either weak or absent during quiet breathing. Accordingly, the VRC respiratory neurons are classified according to the timing of their discharge pattern in relation to different phases of the respiratory cycle. The discharge of the phrenic nerve is often used in experiments involving animal models to define respiratory phases. Expiratory neurons have an onset in their discharge after the postinspiratory phase of the phrenic neurogram, and increasing activity during the expiratory phase. Inspiratory neurons exhibit a

134 NEUROPHYSIOLOGY / Overview

steady increase in firing rate during the inspiratory phase of phrenic nerve activity. Preinspiratory neurons also discharge during the inspiratory phase as well as for a short period before that. Postinspiratory neurons discharge with a rapid onset immediately at the end of the inspiratory phase and then show a decrement in firing during the postinspiratory phase. As mentioned above the PBC is believed to contain the principal kernel for generating respiratory rhythm. The vital role of the PBC was demonstrated in experimental animals from evidence showing that the rhythmic respiratory output from the brainstem disappears when the PBC is destroyed. All PBC respiratory neurons which discharge with preinspiratory firing pattern express neurokinin-1 receptor. Selective elimination of about 3/4 of these neurons results in an abnormal (ataxic) breathing pattern. The PBC respiratory network module continues to generate rhythmic activity when isolated within coronal or sagittal brainstem slices prepared from the brains of neonatal rodents. Identification of respiratory pacemaker-like neurons within the PBC supported the pacemaker model for central respiratory rhythm generation – however, the proportion of these neurons within the PBC is rather low. The ‘pacemaker model’ postulates that neurons with voltage-dependent pacemaker properties (they show spontaneous rhythmic oscillations in membrane potential) are responsible for generation of the respiratory rhythm. There is, however, evidence that a similar rhythm remains in slices in which the key ion channels for pacemaker activity were blocked, supporting an alternative model which proposes that respiratory rhythm is generated by neuronal networks exhibiting oscillatory behavior through chemical and electrotonic excitatory synaptic interactions. There is also a third unifying ‘hybrid pacemaker-network model’ for respiratory rhythm generation which suggests that under normal conditions synaptic interactions between respiratory neurons within a network are shaping the influences of the pacemaker inputs. Although there is a significant data indicating that all mammalian breathing patterns, including sighing and gasping, may originate in the PBC, new evidence is emerging which suggests that there might be more than one central respiratory rhythm generator in the mammalian brainstem. Recent studies helped to identify within the VRC – in particular in the RTN/ pFRG – an additional respiratory oscillator, which generates expiratory rhythm. Partial blockade of the PBC respiratory oscillator activity by opiates reveals the input and importance of this more rostral expiratory oscillator. It was proposed that under normal conditions the activities of different respiratory

oscillators are tightly coupled but dominated by the rhythm generated by PBC. The basic respiratory rhythm generated by these oscillators is subsequently shaped, modified, and transmitted to bulbospinal inspiratory (located within the rostral ventral respiratory group) and expiratory (within the caudal ventral respiratory group) premotor neurons which in turn relay the resultant respiratory pattern to spinal motor neurons innervating inspiratory and expiratory muscles. The medullary respiratory network receives continuously modulatory inputs from the other parts of the brain as well as afferent inputs from the periphery and ensures appropriate ventilation of the lungs in variable environmental and physiological conditions. Afferent Inputs

The main peripheral afferent inputs to the brainstem respiratory network that influence its activity originate from the arterial chemoreceptors, sensitive to changes in PaO2 and PaCO2, and from the mechanoreceptors located in the airways (Figure 1). There are also CO2/pH chemoreceptors which are located within the central nervous system and responsible for adaptive changes in breathing to be appropriate for metabolism. Chemosensory inputs Physiological responses to the reduction of oxygen in the arterial blood involve activation of highly specialized oxygen-sensing neurosecretory cells located in the carotid and, in some species, in the aortic bodies. In adult mammals the glomus cells of the carotid body (located strategically in the bifurcation of the common carotid artery) are the major peripheral PaO2 chemosensitive elements. These cells detect changes in the arterial PO2 and transmit this information to the afferent nerve fibers of the carotid sinus nerve. Information about arterial levels of O2 enters the brain within the glossopharyngeal nerve and relayed to second-order neurons in the NTS. Under basal conditions (PaO2 B75– 100 mmHg) the drive from the peripheral O2 chemoreceptors is low. However, when PaO2 decreases below 60 mmHg, chemoreceptor activity and, therefore, ventilation increases exponentially. This ensures appropriate supply of O2 to the brain which is highly sensitive to hypoxia. Central respiratory drive is extremely sensitive to changes in the arterial PCO2, which is maintained at constant level of about 40 mmHg. Indeed, CO2 provides the major tonic drive for ventilation. Even a small increase in PaCO2 results in a profound increase in respiratory activity to restore the normal level of CO2 in the arterial blood. The ventilatory

NEUROPHYSIOLOGY / Overview 135

response to hypercapnia (increase in PaCO2) is largely preserved in experimental animals after denervation of the carotid and aortic bodies – up to 80% of the CO2-evoked respiratory response is mediated by the action of CO2 at the chemoreceptors located within the lower brainstem. The primary central CO2 chemosensors are located on the ventral surface of the medulla oblongata, although other structures of the brainstem have been reported to be chemoreceptive. Whilst the mechanisms responsible for this chemosensitivity remain largely unknown, it is apparent that changes in the pH of the cerebrospinal fluid, that follow changes in PaCO2 represent the adequate stimulus for the central chemosensors. Ventral surface CO2 chemosensitive areas are located just beneath and in a close proximity to the VRC (discussed above). There is evidence that upon stimulation these chemosensors release the purine nucleotide ATP which may act on the VRC respiratory neurons to evoke appropriate changes in breathing. There is also evidence indicating that some respiratory neurons within the VRC could be intrinsically sensitive to CO2. Central CO2 chemosensors appear to be insensitive to physiological changes in PaO2. Mechanosensory inputs Mechanoreceptors are present throughout the entire respiratory tract. They provide information to the brainstem respiratory network about the mechanical status of the lungs and chest, contribute to the control of breathing, and initiate protective reflexes such as cough. Mechanoreceptors in the lungs and airways have both myelinated and unmyelinated nerve fibers, which enter the brain mainly via glossopharengeal and vagus nerves and terminate in the NTS. Slowly adapting pulmonary stretch receptors have large myelinated fibers and are located in the smooth muscles of the airways. These receptors are activated when the lungs inflate and play a critical role in termination of inspiration and prolongation of expiration (Breuer– Hering reflex). The information from the slowly adapting pulmonary stretch receptors is transmitted to the VRC via the NTS second-order relay neurons located within the dorsal respiratory group. Two other groups of mechanoreceptors – rapidly adapting pulmonary stretch receptors and bronchopulmonary C fiber receptors – initiate defensive respiratory reflexes in response to inhaled irritants or rapid lung inflations or deflations. Main physiological stimuli for pulmonary C fiber afferents are probably lung congestion and edema. Other inputs Activity of the brainstem respiratory network is also affected by the inputs arising from the limbic system, which induces modifications of

breathing during stress, effective behavior, etc. Afferent inputs from the proprioceptors in muscles, tendons, and joints stimulate breathing and contribute to the increase in ventilation during exercise. There are also pathways originating from the neocortex (some of them going directly to the spinal motor neurons innervating the respiratory muscles) which allow voluntary control of breathing. Motor Output and Plasticity

The diaphragm and intercostal muscles are innervated by the motor neurons located in the midcervical and thoracolumbar segments of the spinal cord. Activation of phrenic motor neurons innervating the diaphragm and thoracic motor neurons innervating the external intercostal muscles results in inspiration. There is also a population of expiratory motor neurons which innervate internal intercostal muscles and are responsible for active expiration. Activities of the spinal motor neurons are controlled by the premotor respiratory neurons located in the rostral and caudal ventral respiratory groups of the medulla oblongata (discussed above). There are mono- and polysynaptic pathways which transmit respiratory pattern from the brainstem respiratory network to the spinal respiratory neurons. Respiratory motor output is constantly adjusted to ensure appropriate ventilation of the lungs in variable conditions. It exhibits considerable plasticity, both short-term, when changes in the respiratory output outlast the duration of the stimulus (e.g., hypoxic episode) by seconds and minutes, and longterm, when these changes persist for up to several hours. There is evidence that respiratory plasticity can be elicited by hypoxia, hypercapnia, exercise, deafferentation, neural injury, conditioning, and some other stimuli. Several neurotransmitters have been proposed to be responsible for the respiratory neuroplasticity, including serotonin, dopamine, and nitric oxide. For example, serotonin acting within the brainstem as well as at the level of the phrenic motor neurons appears to play a central role in the longterm facilitation of breathing triggered by repeated episodes of hypoxia.

Central Nervous Control of Breathing in Common Physiological and Pathophysiological States Exercise and Locomotion

A coordinated increase in ventilation is crucial to support the metabolic demands of the exercise. Central respiratory drive increases even before the onset of the exercise, independently of inputs from the

136 NEUROPHYSIOLOGY / Overview

respiratory chemoreceptors or muscle proprioceptors. The caudal hypothalamus is believed to be the source of this ‘central anticipatory command’ to the brainstem respiratory centers. The major cortical inputs into the caudal hypothalamus originate from the areas 4 and 6 of the motor cortex mainly associated with locomotion. During exercise (and locomotion in general), respiratory and locomotor patterns are well coupled. Breathing is entrained by the somatic afferent input which is coming to the VRC via the lateral parabrachial nucleus of the pons and causes resetting of the respiratory rhythm. Emotions and Stress

Emotions and stress result in a considerable alteration in the breathing patterns. Areas rostral to the diencephalon exert an inhibitory influence on respiration that is balanced by facilatory drive coming directly from the diencephalon. The cortex receives various afferent inputs, with descending projections originating from the area 4 of the motor cortex to the caudal hypothalamus, VRC, phrenic, and thoracic motor neurons. Inputs from the limbic system to the brainstem regions concerned with central respiratory control evoke changes in breathing activity during stress, effective behavior, etc. Sleep

Activity of the brainstem respiratory network varies with sleep–wake cycles. Sensitivities to changes in the arterial levels of PO2 and PCO2 are reduced during sleep, and the apneic threshold is raised. Alveolar ventilation slightly decreases, which results in an increase in PaCO2 by around 2–5 mmHg. This modest decrease in central respiratory drive may be related to a withdrawal of a wakefulness-associated excitatory drive to the VRC, the sources of which are as yet unknown. Sleep also results in a reduced or abolished nonchemical tonic upper airway tone, which increases upper airway resistance. Sleep stages 3 and 4 are associated with the greatest upper airway resistance, which in some cases can lead to complete upper airway obstruction and profound hypoxia (discussed below). Because sleep is an oscillatory state, the resultant changes in central respiratory control may be recurrent and/or amplified with repeated episodes.

the hypothalamus, mesencephalon, and brainstem – have been shown to be involved in this secondary respiratory decline. In neonatal animals brainstem transection or lesions in the lateral upper pons help to maintain breathing activity that is normally abolished during acute sustained hypoxia. It has been proposed that several brain sites within the thalamus, hypothalamus, pons, and medulla may function as central oxygen sensors. Their physiological role as yet remains largely unclear. A number of neurotransmitters and neuromodulators have been put forward as putative mediators of hypoxic ventilatory decline. GABA, serotonin, adenosine, opioids, nitric oxide, and others have all been implicated. A temporal sequence in the release of these factors in the brainstem (within the VRC) during hypoxia suggests a specific role for each of them in the sequential development of the hypoxic ventilatory response. In addition, there is pharmacological evidence showing that blockade of GABA, serotonin, or adenosine receptors in the lower brainstem reduces the degree of respiratory decline evoked by hypoxia. Anesthesia

The mechanisms of inhibitory actions of anesthetic and analgesic agents on central respiratory control are complex. Anesthetic agents are known to depress excitability of the respiratory neurons. Both inhalational and intravenous anesthetics also reduce hypoxic ventilatory drive when administered even at low, subanesthetic concentrations. Similarly, opiate analgesic agents acting centrally depress the ventilatory response to hypoxia. There is also evidence that opiates may directly inhibit the rhythm generating respiratory neurons in the PBC.

Disorders of the Central Nervous Control of Breathing Disorders of the central nervous control of breathing are relatively common and life threatening. They are predominantly associated with abnormal/altered neurochemistry of central respiratory control and/or reduced sensitivities to changes in the arterial levels of oxygen and carbon dioxide.

Hypoxia

Sudden Infant Death Syndrome

The ventilatory response to hypoxia in neonates and adults is characterized by an initial increase in central respiratory drive, due to an increase in the activity of the peripheral chemoreceptors. This transient increase in breathing is followed by a decline in central respiratory output. Several brain regions – including

Disorders of the central respiratory control associated with substantial morbidity and mortality such as apnea of prematurity, sudden infant death syndrome (SIDS), and congenital central hypoventilation are relatively frequent conditions in infants and children. SIDS is the leading cause of death of infants

NEUROPHYSIOLOGY / Overview 137

between 1 month and 1 year of age in the industrial world. SIDS is mostly associated with infants sleeping face down, where normal ventilation could be compromised by bedding, resulting in systemic hypoxia and hypercapnia. Several central nervous system (CNS) neuropathophysiological theories of SIDS have been proposed. Alterations in the expression of the excitatory amino acid receptors, early postnatal changes in the composition of inhibitory GABAA receptors, impaired catecholaminergic neuronal development, and modification in the serotonin transporter gene expression have all been associated with SIDS, suggesting that abnormal neurochemistry of the central nervous control of breathing is responsible. Significantly reduced chemosensitivities to changes in PaO2 and PaCO2 may also contribute. There is also evidence that incomplete arousal processes from sleep occur in infants who subsequently succumb to SIDS. This is supported by the experimental data demonstrating that intermittent hypoxia induces transient arousal delay. Tobacco smoke is an important environmental factor associated with SIDS, which also reduces the sensitivity of the brainstem respiratory network to hypoxia. Obstructive Sleep Apnea

Obstructive sleep apnea is characterized by repeated upper airway obstruction during sleep causing intermittent hypoxia. Obstructive sleep apnea leads to the increased cardiovascular morbidity, neurocognitive and behavioral deficits, which are confirmed by magnetic resonance imaging showing significant gray matter loss in several regions of the brain. Localized gray matter loss also occurs in normally well-perfused brain regions involved in motor control of the upper airways, suggesting that underlying brain damage may precipitate and/or perpetuate obstructive sleep apnea. The hallmark of obstructive sleep apnea, chronic intermittent hypoxia, has several neurophysiological effects on central respiratory control. Long-term facilitation of respiratory motor output occurs after several short periods of hypoxia. This has been observed experimentally as well as in patients with obstructive sleep apnea, who express ventilatory long-term facilitation when awake, whereas normal awake subjects do not. Congenital Central Hypoventilation Syndrome

Congenital central hypoventilation syndrome or ‘Ondine’s curse’ is a rare autosomal dominant syndrome associated with a serious deficit in central respiratory control during sleep, while voluntary control of breathing is unaffected. Respiratory chemosensitivities to O2 and CO2 are also markedly reduced or absent. Furthermore, arousal responses and dyspneic

sensation during hypercapnia and/or hypoxia are also absent. Recently, genetic studies have associated heterozygous mutations of the homeobox gene Phox2b with congenital central hypoventilation syndrome. Phox2b is a key regulator of the noradrenergic phenotype and autonomic medullary reflex pathways including peripheral chemoreceptors and their afferent visceral pathways.

Summary Breathing is controlled by neuronal network located within the lower brainstem. The basic respiratory rhythm is generated by a population of neurons within the so-called PBC and then subsequently shaped, modified, and transmitted to bulbospinal respiratory premotor neurons which in turn relay the respiratory pattern to spinal motor neurons innervating inspiratory and expiratory muscles. The brainstem respiratory network receives continuous modulatory inputs from the other parts of the brain and afferent inputs from the periphery. Inputs from the peripheral and central chemoreceptors (which provide information about arterial levels of PO2 and PCO2), from the mechanoreceptors located in the respiratory tract (which provide information about the mechanical status of the lungs and chest), from proprioceptors in muscles, tendons, and joints (which are important for coupling of respiratory and locomotor patterns) as well as from the higher centers of the CNS, such as limbic system, are all essential for adaptive changes (both short- and long-term) in the respiratory motor output to ensure appropriate ventilation of the lungs in variable environmental and physiological conditions. Since breathing is a fundamental rhythmic activity which is essential to support life of an individual, disorders of central nervous respiratory control are critical, resulting in substantial morbidity and mortality. See also: Neurophysiology: Neural Control of Airway Smooth Muscle; Neuroanatomy; Neuroendocrine Cells; Neurons and Neuromuscular Transmission. Sleep Apnea: Overview. Sleep Disorders: Hypoventilation. Sudden Infant Death Syndrome.

Further Reading Bissonnette JM (2000) Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 278: R1391–R1400. Burton MD and Kazemi H (2000) Neurotransmitters in central respiratory control. Respiratory Physiology 122: 111–121. Davenport PW and Reep RL (1995) Cerebral cortex and respiration. In: Dempsey JA and Pack AI (eds.) Regulation of Breathing, pp. 365–388. New York: Dekker.

138 NEUROPHYSIOLOGY / Neural Control of Airway Smooth Muscle de Burgh Daly M (1997) Peripheral Arterial Chemoreception and Respiratory–Cardiovascular Integration, Monograph for the Physiological Society. Oxford: Oxford University Press. Duffin J (2004) Functional organization of respiratory neurones: a brief review of current questions and speculations. Experimental Physiology 89: 517–529. Feldman JL and McCrimmon DR (1999) Neural control of breathing. In: Zigmond MJ, Bloom FE, Landis SC, Roberts JL, and Squire LR (eds.) Fundamental Neuroscience, pp. 1063– 1090. San Diego: Academic Press. Feldman JL, Mitchell GS, and Nattie EE (2003) Breathing: rhythmicity, plasticity, chemosensitivity. Annual Review of Neuroscience. 26: 239–266. Gozal D (2004) New concepts in abnormalities of respiratory control in children. Current Opinion in Pediatrics 16: 305–308. Richter DW and Spyer KM (2001) Studying rhythmogenesis of breathing: comparison of in vivo and in vitro models. Trends in Neuroscience 24: 464–472. Smith JC, Butera RJ, Koshiya N, et al. (2000) Respiratory rhythm generation in neonatal and adult mammals: the hybrid pacemaker-network model. Respiratory Physiology 122: 131–147. Speck DF, Dekin MS, Revellette WR, and Frazier DT (eds.) (1993) Respiratory Control – Central and Peripheral Mechanisms. Lexington: The University Press of Kentucky. St-John WM and Paton JF (2004) Role of pontile mechanisms in the neurogenesis of eupnea. Respiratory Physiology and Neurobiology 143: 321–332. Taylor EW, Jordan D, and Coote JH (1999) Central control of the cardiovascular and respiratory systems and their interactions in vertebrates. Physiological Reviews 79: 855–916.

Neural Control of Airway Smooth Muscle Q Gu and L-Y Lee, University of Kentucky Medical Center, Lexington, KY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Airway smooth muscle (ASM) is the primary effector cell responsible for controlling airway caliber and thus the resistance to airflow of the entire tracheobronchial tree. The autonomic nervous system is the principal regulator of the ASM tone and consists of three separate neural pathways. First, parasympathetic (vagal) innervation of ASM is the primary contractile pathway, and acetylcholine causes ASM contraction by activating the M3 muscarinic cholinoceptors. Second, sympathetic innervation of human ASM is relatively sparse, but epinephrine secreted by adrenal medulla can induce ASM relaxation by activating the b2 adrenoceptor. Third, the existence of nonadrenergic, noncholinergic (NANC) innervation of ASM has been demonstrated in various species including humans. The inhibitory NANC fibers are carried in parasympathetic nerves, and vasoactive intestinal peptide and nitric oxide are the putative neurotransmitters. The excitatory NANC mechanism involves the ‘efferent’ functions of vagal bronchopulmonary sensory nerves that release tachykinins upon activation and cause ASM contraction. Bronchoconstriction is an important component of the airway defense reflexes elicited by inhaled irritants. Three subtypes of vagal bronchopulmonary afferents

function as the primary sensors of inhaled irritants: C-fiber afferents (tachykinin-containing afferents), rapidly adapting pulmonary receptors, and laryngeal afferents. Compelling evidence suggests that hypersensitivity of these airway afferents and autonomic nerve dysfunction and/or dysregulation are important contributing factors in the pathogenesis of airway hyperresponsiveness. A better understanding of the mechanisms underlying the neural control of ASM tone should help in the development of novel therapeutic strategies for alleviating bronchospasm occurring in airway inflammatory diseases.

Structure and Function of Airway Smooth Muscle Structure Characteristics

Airway smooth muscle (ASM) is present in the respiratory tract of all mammals, extending from trachea to small airways. ASM is inserted on the free ends of the C-shaped cartilages and forms dense bundles of trachealis in the posterior wall of trachea. Below the carina, ASM bundles encircle the entire airway wall and lie between the mucosa and submucosa (Figure 1). ASM occupies a relatively larger portion of the cross-section area of the airway wall as the airway diameter becomes smaller, reaching a maximum in the terminal bronchioles. Its orientation is transverse in the trachea, and it forms a helical– antihelical pattern in bronchi and distal airways. There are also a smaller number of longitudinally oriented bundles of ASM in human thoracic trachea. In the canine trachea, the ASM cell is elongated and spindle shaped, and it is approximately 250 mm long and 3–5 mm wide, on average, at the level of the nucleus. The perinuclear sacroplasm contains various organelles, such as endoplasmic reticulum, Golgi apparatus, and mitochondria, and the peripheral sacroplasm is occupied primarily by myofilaments (actin and myosin-containing thick and thin filaments). Individual ASM cells are surrounded by an extracellular matrix (ECM) that consists of a complex network of interlacing macromolecules such as reticular fibers, collagenous bundles, and various ECM proteins. A main feature of the ASM cell membrane is the insertion of the contractile apparatus, which is constituted by myofilaments, intermediate filaments, membrane-associated dense bands, and cytosolic dense bodies; adherens junctions form between ASM cells and provide the insertion points for the myofilaments of the contract apparatus. There are also gap junctions between individual ASM cells that allow direct flow of ions and form electrical and metabolic coupling between cells. Physiological Properties and Functions

ASM is the primary effector cell responsible for controlling airway caliber and thus the resistance to

NEUROPHYSIOLOGY / Neural Control of Airway Smooth Muscle 139

SM

C

M M

SM

SM

(a)

(b)

Figure 1 (a) Transverse section of the trachea from a newborn child. H&E/Alcian blue; magnification
9. (b) Transverse section of bronchiole o1 mm in diameter. H&E; magnification
160. C, cartilage; M, mucosa; SM, smooth muscle. A few strands of longitudinal SM are disposed behind the trachealis muscles in (a). The smooth muscle layer is the most prominent feature of the bronchiole that has no cartilaginous support. Reproduced from Wheater PR, Burkitt HG, and Daniels VG (1987) Functional Histology: A Text and Color Atlas. London: Longman/Churchill Livingstone, with permission from Churchill Livingstone.

airflow of the entire tracheobronchial tree. The action of the contractile apparatus in ASM cells is consistent with the energy-dependent relative sliding of adjacent actin and myosin filaments, and the calcium-sensitive regulation of contraction is mediated by a calmodulin/myosin light chain kinase/phosphatase system. The cell junctions of the adherens type between individual ASM cells are numerous, and they enable coordinated slow and sustained contraction of the whole muscle bundle. The ASM contraction overcomes viscous and elastic structural elements in the airway wall and reduces the size of airway lumen. It is believed that the basal ASM tone is regulated to provide an optimal balance between airway resistance and dead space during eupneic breathing. Furthermore, the ASM tone plays an important role in maintaining the airway patency when the transmural pressure acts to collapse the airways (e.g., during hyperventilation and cough), particularly in the small and median-sized ‘membranous’ airways. In response to inhaled irritants, bronchoconstriction resulting from ASM contraction, in conjunction with increased airway secretion and cough reflex, plays an important role in the airway protective function.

Neural Regulation of Bronchomotor Tone The autonomic nervous system, the principal neural pathway regulating ASM tone, consists of two primary sets of efferent nerves: parasympathetic fibers carried by the vagus nerves and sympathetic nerve fibers arising from thoracic and cervical sympathetic ganglia (Figure 2). The parasympathetic fibers originate from the ganglionic neurons in the tracheal and bronchial plexuses that receive input from the

superior and recurrent laryngeal nerves as well as the bronchial and pulmonary branches of the vagus nerves. Some of sympathetic postganglionic fibers reach the tracheal and bronchial ASM as perivascular nerves. The efferent fibers penetrate and innervate the muscle cells mainly from their adventitial surface either singly or in small bundles that run in the connective tissue between the ASM cell bundles in the form of a mesh-like net. One of the prominent features of these terminals is the presence of varicosities accompanied by Schwann cell processes. Varicosities packed with vesicles are located close to ASM cells, with a cleft of 100 nM or less that function as the neuromuscular junction. However, there are no apparent or specialized postjunctional structures in ASM cells. Cholinergic Mechanisms

Parasympathetic innervation of ASM has been demonstrated in all mammalian species including humans and is the primary contractile neural pathway. The preganglionic fibers of the parasympathetic nerve are localized in the brain stem (dorsal vagal nucleus and nucleus ambiguus), and their activities are conducted in the vagus nerve. Upon reaching the airways, the preganglionic fibers form synapses at the parasympathetic ganglia located in the tracheal and bronchial plexuses in the airway walls (Figure 3). These ganglion neurons play an important role in integrating, filtering, and modulating the various input signals and regulate the electrical activity carried by the postganglionic efferent fibers to the ASM (Figure 2) and other effector organs in the airways. Parasympathetic postganglionic nerves release acetylcholine from the varicosities to evoke contraction of ASM by activating

140 NEUROPHYSIOLOGY / Neural Control of Airway Smooth Muscle

Spinal cord

Vagus

Parasympathetic nerve

Sympathetic nerve

Adrenal gland

(−) Blood vessel

(+) M

2

EPi
2

NE 

NO/VIP (iNANC)

(−)

ACh M3

(eNANC) SP, NKA

Sensory nerve

NK-1,2

Smooth muscle cell Figure 2 Neural pathways regulating the airway smooth muscle tone. M3 cholinoceptors mediate the muscle contraction in response to acetylcholine (ACh) released from parasympathetic postganglionic nerves. Prejunctional M2 cholinoceptors mediate autoinhibitory feedback of ACh release. b2 adrenoceptors mediate the muscle relaxation in response to circulating adrenaline. Sympathetic nerves may also modulate the cholinergic neurotransmission. Parasympathetic postganglionic fibers can release inhibitory nonadrenergic, noncholinergic (iNANC) neurotransmitters, such as nitric oxide (NO) and vasoactive intestinal peptide (VIP). The excitatory NANC (eNANC) pathway involves antidromic activation of afferent endings that release tachykinins such as substance P (SP) and neurokinin A (NKA) and cause muscle contraction. Modified from Stewart AG, Fernandes DJ, Koutsoubos V, et al. (2000) Airway smooth muscle. In: Page CP, Banner KH, and Spina D (eds.) Cellular Mechanisms in Airway Inflammation, pp. 263–302. Basel: Birkha¨user Verlag; Canning BJ and Fischer A (2001) Neural regulation of airway smooth muscle. Respiratory Physiology 125: 113–127; Dey R (1994) Airways ganglia. In: Raeburn D and Giembycz MA (eds.) Airway Smooth Muscle: Structure, Innervation and Neurotransmission, pp. 79–101. Basel: Birkha¨user Verlag.

muscarinic cholinoceptors of the M3 subtype, whereas the prejunctional M2 cholinoceptors exert a profound autoinhibitory feedback control of acetylcholine release (Figure 2). Pharmacological agents such as atropine and ipratropium bromide (ATROVENT) prevent bronchospasm evoked by exogenously administered acetylcholine or bronchoconstriction mediated by activation of postganglionic cholinergic nerves. The effect of these antimuscarinic agents is due to their ability to antagonize the actions of acetylcholine at the M3 muscarinic receptor in the ASM. In contrast, the antagonists of the M2 cholinoceptor (e.g., gallamine) have no effect on the ASM contraction evoked by exogenously administered acetylcholine, but they can amplify the bronchospasm generated by activation of the cholinergic pathway; the latter is caused by the antagonizing action of these agents on the neuronal M2 autoreceptor. The basal level of ASM tone is regulated primarily by the activity of cholinergic innervation. Changes in parasympathetic activity at any point along the efferent pathway (e.g., central motor neurons, parasympathetic ganglia neurons, or the neuromuscular junction) can effectively alter the ASM tone and,

consequently, airway lumenal size and airflow to the lung periphery. Adrenergic Mechanisms

Sympathetic–adrenergic nerve fibers have been identified in the airways and lungs; b-adrenoceptor (particularly b2) agonists are known to be potent bronchodilators, and activation of a-adrenoceptors produces bronchoconstriction. However, the extent and importance of sympathetic innervation of ASM are species dependent and appear to be sparse or nonexistent in human bronchial smooth muscles. Norepinephrine is the primary sympathetic neurotransmitter, but its importance and the nature of the adrenoceptors mediating the sympathetic nerveevoked relaxation of the airways have not been clearly defined. Evidence suggests that sympathetic nerves may exert an indirect influence on ASM tone by inhibiting the cholinergic neurotransmission (Figure 2). In addition to norepinephrine, epinephrine secreted by adrenal medulla functions as a hormone and can induce ASM relaxation by activating primarily the b2 adrenoceptor (Figure 2).

NEUROPHYSIOLOGY / Neural Control of Airway Smooth Muscle 141 Nonadrenergic, Noncholinergic Mechanisms

Autonomic neural control of ASM tone cannot be fully explained by the functions of the cholinergic and adrenergic nervous system. For example, striking changes in ASM tone can be induced even in the presence of anticholinergic (atropine) and antiadrenergic (propranolol) agents. The existence of nonadrenergic, noncholinergic (NANC) innervation of ASM is clearly recognized (Figures 2 and 4).

R

P P V

R

V

Figure 3 Tracing of the dorsal aspect of extrinsic and intrinsic nerves of the trachea from the larynx (top) to the carina (bottom) of a newborn ferret. This reconstruction is a tracing of nerves and ganglia that were visible in a montage of photographs taken from the specimen tested histochemically for acetylcholinesterase activity. It includes the vagus nerves (V), the recurrent laryngeal nerves (R), the pararecurrent laryngeal nerves (P), the longitudinal nerve trunks, and the superficial muscle plexus, but it does not show the deep muscle plexus. Magnification
5.5; scale bar ¼ 2 mm. Reproduced from Journal of Comparative Neurology; Baker DG, McDonald DM, Basbaum CB and Mitchell RA; Copyright & 1986, Wiley-Liss. Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

Inhibitory NANC mechanisms Inhibitory NANC (iNANC) innervation has been described in many species, including man, and is considered the primary neural mechanism mediating ASM relaxation (Figure 4(a)). iNANC relaxations are thought to be generated by a combined effect of vasoactive intestinal peptide (VIP), VIP structure-related peptides (e.g., peptide histidine methionine), and nitric oxide (NO). Indeed, VIP, VIP-like peptides, and NO synthase (NOS) have been identified in the parasympathetic ganglia and nerve fibers innervating the ASM. Furthermore, endogenously released VIP-like and NO-like substances can attenuate ASM contraction induced by acetylcholine. However, the precise anatomical pathways of iNANC innervation of the ASM are not clear and may vary between species. Excitatory NANC mechanisms Evidence clearly demonstrates a potent excitatory effect on the ASM involving the ‘efferent’ functions of a specific subtype of bronchopulmonary sensory nerves containing tachykinins (e.g., substance P and neurokinin A). When these afferent endings are activated, the impulses trigger the release of tachykinins either locally or propagating antidromically to other peripheral branches via the axonal ramifications. These sensory neuropeptides can activate neurokinin-1 (NK-1) and NK-2 receptors located on the ASM membrane and produce intense and sustained bronchoconstriction (Figure 4(b)). Indeed, ASM contraction can be induced by chemical or mechanical stimulation of these afferent endings; the bronchoconstrictive response is resistant to atropine or vagotomy, and it is absent in animals whose sensory neuropeptides are depleted. Furthermore, ASM contraction can be completely blocked by antagonists of the NK-1 and NK-2 receptors. This action of tachykinins on ASM is mediated primarily through activation of the NK-2 receptor and consists of both a direct effect on the ASM cell and a facilitation of the cholinergic ganglionic transmission. Although a significant role of tachykinins released locally from these nerve endings in regulating the bronchomotor tone is well established, particularly during airway inflammation, the

RL(cmH2O l –1 sec–1)

142 NEUROPHYSIOLOGY / Neural Control of Airway Smooth Muscle

120 80 40 0

Atropine (1mg kg–1)

2 min

Propranolol (2mg kg–1)

Hexamethonium (2mg kg–1)

5-HT Infusion

(a) Control 2

Hilar bronchus

Control 1

After atropine + propranolol

After atropine + propranolol +CP99994 + SR48968

0.25 g 5 min (b) Figure 4 Experimental records demonstrating the iNANC and eNANC responses. (a) Changes in lung resistance (RL) evoked by mechanical stimulation of the larynx (solid circles) in an anesthetized, artificially ventilated cat. The laryngeal stimulation was applied by gently stroking its dorsal mucosa with a fine probe. Initially, the larynx was stimulated under basal conditions (far left). Thereafter, baseline airway tone was elevated by a continuous infusion of 5-hydroxytryptamine (5-HT). Laryngeal stimulation then evoked a brief bronchoconstriction followed by a more prolonged bronchodilatation; the latter resulted from activation of the iNANC pathway. Reproduced from Szarek JL, Gillespie MN, Altiere RJ, and Diamond L (1986) Reflex activation of the nonadrenergic noncholinergic inhibitory nervous system in feline airways. American Review of Respiratory Disease 133(6): 1159–1162, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission. (b) Responses of isolated guinea pig hilar bronchus to electrical field stimulation (solid circles) that activated both efferent and afferent vagal nerves. Two consecutive control responses were separated by 30 min. The stimulation was repeated after pretreatment with atropine and propranolol to block the cholinergic and adrenergic pathways, respectively. The eNANC contraction was completely abolished by pretreatment with CP99994 (a selective NK-1 receptor antagonist) and SR48968 (a selective NK-2 receptor antagonist). Modified from Wu ZH, Yang QH, Ruan T, and Lee LY (2004) Influence of maturation on contractile response to stimulation of C-fiber afferents in isolated guinea pig airways. American Journal of Physiology: Lung Cellular and Molecular Physiology 287: L168–L175.

extent and importance of its function vary substantially between different species.

Reflex Bronchoconstriction Response to Inhaled Irritants

Bronchoconstriction is an important and effective component of the airway defense reflexes protecting the lung and the rest of the body against inhaled irritants and airborne toxins such as cigarette smoke and acid aerosol. When certain types of sensory nerves in the respiratory tract are activated by chemical irritants, the action potentials generated by these nerves are conducted through the vagus nerves to the brain stem, which immediately elicits reflex bronchoconstriction via the cholinergic efferent pathway accompanied by hypersecretion of mucus, cough, and dyspneic sensation. In addition, activation of these afferents can trigger the local ‘axonal reflex’ that is mediated by the release of tachykinins from these sensory nerve endings and causes sustained contraction of ASM, increased vascular permeability to protein, and chemotaxis (Figure 5). These responses act

collectively to protect the lung by reducing and preventing further penetration of irritants into the deeper regions of the lung. Bronchopulmonary Afferents Involved

The afferent activities that arise from sensory terminals located in the lung and airways are conducted almost exclusively by vagus nerves and their branches (superior and recurrent laryngeal nerves and bronchial and pulmonary branches). These vagal afferent fibers innervate the entire respiratory tract, ranging from the larynx and the trachea to lung parenchyma, and project to the nucleus tractus solitarius in the medulla. The major subsets of these afferents include bronchopulmonary C-fiber afferents (tachykinin-containing afferents), rapidly adapting pulmonary receptors, and laryngeal afferents. Cell bodies of these afferent fibers reside in two adjacent but distinct anatomic structures: nodose and intracranial jugular ganglia. Bronchopulmonary C fibers are small-diameter, slow-conducting, unmyelinated afferents innervating all levels of the respiratory tract, including the alveolar wall, and represent 475% of the afferent fibers in

NEUROPHYSIOLOGY / Neural Control of Airway Smooth Muscle 143 Cigarette smoke, SO2, acid, other inhaled irritants

N

Vagal afferent Vagal efferent

TKs CGRP Mast cell

E

L

Microvessel

TKs, CGRP

Mucous gland

Smooth muscle

Figure 5 Schematic illustration of the distribution pattern of vagal C-fiber afferent endings and their potential interaction with other cell types in airway mucosa. E, erythrocyte; L, leukocyte; TKs, tachykinins; N, neuroepithelial body. Reproduced from Lee LY and Undem BJ (2005) Bronchopulmonary vagal afferent nerves. In: Undem BJ and Weinreich D (eds.) Advances in Vagal Afferent Neurobiology, Frontiers in Neuroscience Series, pp. 279–313. Boca Raton, FL/London: CRC Press/Taylor & Francis.

the pulmonary branch of the vagus nerve. It is well recognized that the afferent activity arising from these C-fiber endings plays an important role in regulating the bronchomotor response to inhaled irritants. Tachykinins, potent constrictors of ASM, are synthesized in the cell bodies of pulmonary C neurons in nodose and jugular ganglia, transported, and stored in the sensory terminals. Immunohistochemical studies have clearly illustrated the presence of C-fiber sensory endings containing tachykinins in the mucosa of all sizes of airways in various species including humans. These nerve endings display extensive axonal arborization that either extends into the space between epithelial cells or forms networklike plexus immediately beneath the basement membrane of the epithelium (Figure 5). The superficial locations of these nerve endings in the airway lumen and their exquisite sensitivity to chemical irritants (e.g., cigarette smoke and acid) are the most prominent characteristics of these afferents, which may explain their role as the primary sensor detecting the chemical irritants and regulating the ASM tone and other airway functions. Indeed, tachykinin-containing nerve profiles have been identified in the close vicinity of a number of other effector cells in the airways, including mucous glands, microvessels, and cholinergic ganglia (Figure 5). Rapidly adapting pulmonary receptors are located along the entire tracheobronchial tree with a higher

density in the larger airways, particularly at the branching points, and can be stimulated by inhaled irritants such as sulfur dioxide, dust, and cigarette smoke. It was their sensitivity to chemical irritants that led to their more publicized name, ‘irritant receptors’. In addition to their well-recognized role in eliciting the cough reflex, stimulation of rapidly adapting receptors by chemical irritants is also believed to induce ASM contraction via the cholinergic reflex. Because of its strategic location, the larynx plays a critical role in the airway defense function. Activation of the ‘irritant receptor-like’ subtype of laryngeal afferents by chemical or mechanical stimulation has been shown to trigger reflex bronchoconstriction. Other nonpulmonary sensory afferents may also be involved in regulating the ASM tone under certain physiological conditions; for example, stimulation of arterial chemoreceptor (e.g., carotid bodies) by hypoxia causes ASM contraction, which is mediated through the respiratory neural circuit in the brain stem and the cholinergic pathway. Effect of Airway Inflammation

Bronchial hyperresponsiveness is known to occur during acute airway inflammation/injury caused by ozone exposure, viral infection, etc., and the involvement of an augmented reflex bronchoconstriction in these pathophysiological conditions has been clearly

144 NEUROPHYSIOLOGY / Neural Control of Airway Smooth Muscle

documented. Indeed, the sensitivity of these sensory nerves mediating the reflex bronchoconstriction can be elevated by certain endogenously released inflammatory mediators. Thus, a given level of inhaled irritant will generate a greater intensity of afferent discharge from these afferents (bronchopulmonary C fibers) during airway inflammation, which consequently causes a more severe bronchoconstriction mediated through both the cholinergic reflex pathway and local release of tachykinin. An example of these autacoids is prostaglandin E2 (PGE2); PGE2 is a potent inflammatory mediator derived from arachidonic acid cyclooxygenase metabolism and released locally from the airway epithelium, a major site of initial assault by the inhaled irritants. Indeed, PGE2 induces reflex bronchoconstriction in asthmatic patients despite its potent, direct dilating effect on ASM. Studies have demonstrated that the PGE2-induced sensitization of airway sensory nerves is mediated through the intracellular cyclic AMP/protein kinase A transduction pathway, which in turn modulates the excitability of various ligand-gated and voltage-sensitive ion channels and enhances the excitability of the neuronal membrane. Similar sensitizing effects generated by several other endogenous substances, such as bradykinin, histamine, adenosine, proton, and nerve growth factor, have also been reported. Extensive and convincing evidence suggests the possible involvement of these autacoids and their sensitizing effects in the manifestation of various pathophysiologic conditions of the airways, such as bronchoconstriction, during airway inflammation.

Airway Smooth Muscle in Respiratory Diseases Asthma is a recurrent disease in which multiple factors interact to generate clinical manifestations of shortness of breath, hyperresponsiveness to non-specific bronchoconstrictor stimuli, cough, and airway inflammation. A prominent pathophysiological feature of asthma is airflow obstruction caused by combinations of ASM contraction, mucosal edema and inflammation, and excessive mucin secretion. Compelling evidence supports the hypothesis that hypersensitivity of airway afferents and autonomic nerve dysfunction and/or dysregulation are important contributing factors in the pathogenesis of airway hyperresponsiveness. It is well documented that the sensitivity of airway afferents, bronchopulmonary C fibers and rapidly adapting airway receptors, is elevated during airway inflammation and injury, which results in a lower threshold of bronchoconstriction via both the

cholinergic pathway and axonal reflexes. In addition, dysfunction of inhibitory M2 muscarinic receptors and the resulting increase in acetylcholine release have been shown to contribute to the airway hyperresponsiveness during acute airway infection/ inflammation. ASM may also be involved in the pathogenesis of asthma due to its changes in properties and functions. In chronic severe asthma, the ASM mass increases in the remodeling process of the airway wall, which appears to be due to an increase in cell number (hyperplasia) as well as an increase in cell size (hypertrophy). The ASM mass increase may represent either a pathologic or an injuryrepair response due to chronic inflammation. The precise mechanisms underlying the ASM proliferation and whether the increase in ASM mass increases the contractile response remain to be determined. It has been shown that ASM may also be a source of a variety of cytokines/chemokines (e.g., IL-1, IL-5, IL-6, IL-8, and RANTES), other proinflammatory mediators (e.g., PGE2), and enzymes (e.g., NOS). These biologically active products may modulate the inflammatory process in asthma and other chronic respiratory diseases, such as chronic bronchitis and emphysema. Furthermore, there may be complex interactions between inflammatory cells, structural cells, and afferent/efferent nerves in the airways. Some of these endogenous substances are known to enhance the airway afferent sensitivity and to augment reflex bronchoconstriction. In view of the important interactive role of ASM and afferent/efferent nerves in both acute and chronic processes of airway inflammation, injury, and repair, a better understanding of the mechanisms underlying the neural control of ASM tone should help to develop novel therapeutic strategies for alleviating bronchospasm occurring in airway inflammatory diseases. See also: Asthma: Exercise-Induced; Extrinsic/Intrinsic. Kinins and Neuropeptides: Tachykinins. Neuromuscular Disease: Peripheral Nerves; Myasthenia Gravis and Other Diseases of the Neuromuscular Junction. Neurophysiology: Neuroanatomy; Neurons and Neuromuscular Transmission.

Further Reading Amrani Y and Panettieri RA (2003) Airway smooth muscle: contraction and beyond. International Journal of Biochemistry and Cell Biology 35: 272–276. Baker DG, McDonald DM, Basbaum CB, and Mitchell RA (1986) The architecture of nerves and ganglia of the ferret trachea as revealed by acetylcholinesterase histochemistry. Journal of Comparative Neurology 246: 513–526. Barnes PJ (1998) Pharmacology of airway smooth muscle. American Journal of Respiratory and Critical Care Medicine 158: S123–S132.

NEUROPHYSIOLOGY / Neuroanatomy 145 Canning BJ and Fischer A (2001) Neural regulation of airway smooth muscle. Respiration Physiology 125: 113–127. Dey R (1994) Airways ganglia. In: Raeburn D and Giembycz MA (eds.) Airway Smooth Muscle: Structure, Innervation, and Neurotransmission, pp. 79–101. Basel: Birkha¨user Verlag. Fryer AD and Jacoby DB (1998) Muscarinic receptors and control of airway smooth muscle. American Journal of Respiratory and Critical Care Medicine 158: S154–S160. Gabella G (1989) Structure of airway smooth muscle. In: Coburn RF (ed.) Airway Smooth Muscle in Health and Disease, pp. 1–13. New York: Plenum. Hall IP (2000) Second messengers, ion channels and pharmacology of airway smooth muscle. European Respiratory Journal 15: 1120–1127. Jordon D (2001) Central nervous pathways and control of the airways. Respiration Physiology 125: 67–81. Lee LY and Pissari T (2001) Afferent properties and reflex functions of bronchopulmonary C fibers. Respiration Physiology 125: 47–65. Lee LY and Undem BJ (2005) Bronchopulmonary vagal afferent nerves. In: Undem BJ and Weinreich D (eds.) Advances in Vagal Afferent Neurobiology, Frontiers in Neuroscience Series, pp. 279–313. Boca Raton, FL/London: CRC Press/Taylor & Francis. Seow CY, Schellenberg RR, and Pare PD (1998) Structural and functional changes in the airway smooth muscle of asthmatic subjects. American Journal of Respiratory and Critical Care Medicine 158: S179–S186. Stephens NL (2002) Airway smooth muscle. Lung 179: 333–373. Stewart AG, Fernandes DJ, Koutsoubos V, et al. (2000) Airway smooth muscle. In: Page CP, Banner KH, and Spina D (eds.) Cellular Mechanisms in Airway Inflammation, pp. 263–302. Basel: Birkha¨user Verlag. Szarek JL, Gillespie MN, Altiere RJ, and Diamond L (1986) Reflex activation of the nonadrenergic noncholinergic inhibitory nervous system in feline airways. American Review of Respiratory Diseases 133(6): 1159–1162. Wheater PR, Burkitt HG, and Daniels VG (1987) Functional Histology: A Text and Color Atlas. London: Longman/Churchill Livingstone. Widdicombe JG and Wells UM (1994) Vagal reflexes. In: Raeburn D and Giembycz MA (eds.) Airway Smooth Muscle: Structure, Innervation, and Neurotransmission, pp. 279–307. Basel: Birkha¨user Verlag. Wu ZH, Yang QH, Ruan T, and Lee LY (2004) Influence of maturation on contractile response to stimulation of C-fiber afferents in isolated guinea pig airways. American Journal of Physiology: Lung Cellular and Molecular Physiology 287: L168–L175.

Neuroanatomy J A Neubauer, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ, USA

intercostals, abdominal, and upper airway). This central neural network receives afferent feedback from sensors that monitor the mechanical effectiveness of lung expansion (mechanoreceptors) and the adequacy of arterial oxygen and carbon dioxide levels (chemoreceptors). The integrated respiratory neural drive is adjusted to accomplish a wide range of reflex activities, from sleep to exercise, as well as voluntary maneuvers such as vocalization or breath holding. The optimization of respiration to meet these various demands is accomplished by the respiratory neural control system, which initiates a respiratory rhythm (central pattern generator located within the brainstem) that is modulated by afferent information from the sensors and other higher brain centers into an integrated neural output to respiratory muscles. A number of clinical disorders produce abnormal breathing patterns. The causes of the neuropathology can be disease linked or genetic. The abnormal pattern that is produced depends on the location of the defect within the respiratory network and can result in failure to breathe or altered breathing patterns.

Neuroanatomy of Respiration in Normal Lung Function Breathing is essential for life. The act of breathing requires a system that functions to coordinate a rhythmic inflation and deflation of the lungs to extract oxygen from the atmosphere and eliminate carbon dioxide. Unlike the heart, which continues to beat in the absence of neural input, respiration ceases without neural drive. Thus, breathing requires a central neural network that innervates respiratory muscles (diaphragm, intercostals, abdominal, and upper airway) plus sensors to monitor the mechanical effectiveness of lung expansion (mechanoreceptors) and the adequacy of arterial oxygen and carbon dioxide levels (chemoreceptors). The integrated respiratory neural drive must be adjusted to accomplish a wide range of activities, from sleep to exercise. It must also be adjusted during swallowing, coughing, yawning, speaking, or singing. In addition, because the muscles of respiration are also involved in posture, they must change their level of tone to adjust to body position. The optimization of respiration to meet these various demands is accomplished by the respiratory neural control system, which initiates a respiratory rhythm (central pattern generator (CPG)) that is modulated by afferent information from the sensors and other higher brain centers into an integrated neural output to respiratory muscles (effectors) (Figure 1).

& 2006 Elsevier Ltd. All rights reserved.

Respiratory Neural Network

Abstract The neuroanatomy that supports the act of normal breathing consists of a central neural network of inspiratory and expiratory neurons that project to spinal and brainstem motor nuclei that ultimately innervate the respiratory muscles (diaphragm,

The principal neural sites that generate respiratory rhythm are located within the brainstem in the medulla and pons. More rostral brain regions (suprapontine) influence this basic breathing pattern. The medullary and pontine centers responsible for

146 NEUROPHYSIOLOGY / Neuroanatomy

Phonation posture state Sleep-wake Emotion Exercise

Respiratory controller

Mechanoreceptors

Motor Neurons Spinal Cranial

Chemoreceptors Peripheral Intracranial

Muscles Thoracic Abdominal

Lung

Laryngeal Pharyngeal

Airway resistance

Autonomic nervous system

PaO2

PaCO2 pH

Ventilation

Nonventilatory factors (metabolism, renal regulation)

Figure 1 Schematic drawing of neural control of ventilation. Output of brain respiratory controller activates spinal and cranial motor neurons innervating thoracic/abdominal and laryngeal/pharyngeal muscles. Thoracic and abdominal muscles control configuration of ribcage and diaphragm, which via mechanical coupling with the lung results in ventilation. The ease with which air enters and leaves the lungs is determined by airway caliber, which is partly controlled by laryngeal and pharyngeal muscles and via autonomic innervation of airway smooth muscle. Ventilation, along with nonventilatory factors, determines blood and brain fluid levels of O2, CO2, and pH. Levels of these regulated variables are transduced into a pattern of afferent activity at peripheral and intracranial chemoreceptors. These signals feed into the respiratory controller, which makes appropriate adjustments in its output to motor neurons, thereby controlling ventilation and regulating PO2, PCO2, pH, and related variables. The respiratory controller also makes adjustments in output based on signals from mechanoreceptors in respiratory muscles and in lung and airways. In addition, higher brain centers influence the respiratory controller to make adjustments appropriate for changes in behavior or state. PaO2 and PaCO2, partial pressures of O2 and CO2 of arterial blood, respectively.

respiratory rhythm generation and patterning (frequency and amplitude of breathing) were delineated by characterizing alterations in ventilation following sequential transections of the brainstem. Automatic control of respiration was found to only require the presence of the pons and medulla since a normal respiratory pattern persisted following transection of the brainstem at this level. Sequential transections through the brainstem gave rise to the concept of three respiratory centers: pneumotaxic, apneustic, and medullary centers. The neurons in these respiratory centers, which show action potentials in phase with either inspiration or expiration (or both), generate a respiratory rhythm using the excitatory and inhibitory amino acids, glutamate and gamma-amino butyric acid (GABA). These neurotransmitters and their receptors are found throughout the brain and so the respiratory rhythm-generating centers are not unique. Other neurotransmitters and neuromodulators modulate

the activity of the CPG, including serotonin, acetylcholine, opioids, nitric oxide, substance P, and somatostatin, which are released from nerves originating from regions outside the CPG. These modulating effects require the presence of the appropriate receptors, which in some cases are unique enough to identify a particular respiratory region (e.g., pre-Bo¨tzinger complex). Pontine respiratory centers Pontine respiratory group (pneumotaxic center) The pontine respiratory group (PRG) is a group of respiratory neurons located bilaterally in the rostral third of the pons within the parabrachialis and Kolliker– Fuse nuclei. Within the PRG there are inspiratory, expiratory, and inspiratory/expiratory (phase-spanning) neurons that transmit information to the medullary respiratory centers. It is believed that the primary function of the PRG is to provide for a smooth transition from inspiration to expiration. The

NEUROPHYSIOLOGY / Neuroanatomy 147

original name of the PRG, pneuomotaxic center, arose because a change in PRG activity alters the rate of the breathing. Together with vagal feedback from the pulmonary stretch receptors, which monitor the adequacy of lung inflation, the PRG functions to vary the point of termination of a breath (inspiratory offswitch). Thus, activation of the PRG leads to rapid shallow breathing, and decreasing PRG output leads to slow deep breaths. If both of the inspiratory offswitches are ablated (i.e., the vagi are cut and the rostral third of the pons is lesioned), there are very prolonged deep breaths separated by short periods of expiration. This pattern of breathing is called apneustic breathing. Apneustic center The apneustic center is functionally defined on the basis that prolonged inspiration (apneusis) occurs when the rostral third of the pons and the vagus nerves are cut. The neuroanatomical source of this strong inspiratory drive within the caudal two-thirds of the pons is unknown, although one hypothesis is that it reflects the influence of the reticular activating system. If the pons is removed entirely so that only the medulla remains intact, a more rhythmic pattern of breathing resumes; hence, the medulla contains the neurons responsible for rhythm generation. Medullary respiratory centers The medullary centers contain respiratory neurons in two well-defined bilateral respiratory regions: the dorsal respiratory group (DRG) and the ventral respiratory group (VRG). If these medullary centers are removed by transecting at the level of the cervical spinal cord, all rhythmic breathing ceases. Dorsal respiratory group The DRG is located in the dorsomedial medulla within the ventrolateral nucleus of the solitary tract (NTS) and contains primarily inspiratory neurons. This subnucleus of the NTS is the site where afferent fibers of the glossopharyngeal and vagus nerves carry afferent sensory information from the peripheral chemoreceptors and lung mechanoreceptors. Sensory feedback is relayed to the DRG, where it synapses on premotor inspiratory neurons that project mostly to spinal motor neurons. Neurotransmission uses primarily the amino acid neurotransmitters glutamate and GABA; however, there is evidence that substance P is important for relaying sensory input and that nitric oxide has an excitatory modulatory influence on these inspiratory neurons. Ventral respiratory group The VRG is a ventrolateral column of respiratory neurons that includes the

nucleus ambiguous, nucleus retroambigulalis, and the pre-Bo¨tzinger and Bo¨tzinger complexes. The VRG contains both expiratory and inspiratory neurons, which either project to other brainstem neurons or function as premotor neurons with projections to the respiratory motor neurons. Expiratory and inspiratory neurons are localized within discrete regions of the VRG, with expiratory neurons located in the caudal and rostral VRG and inspiratory neurons contained in the intermediate region of the VRG. The Bo¨tzinger complex is located at the most rostral end of the VRG and contains neurons that inhibit most inspiratory neurons during expiration. The preBo¨tzinger complex is the site of rhythm-generating inspiratory neurons. Studies have shown that the activity of pre-Bo¨tzinger neurons is increased by substance P and decreased by opioids. This region has also been shown to act as central oxygen chemosensor and function to produce sighs or gasps (autoresuscitation). Spinal motor neurons The motor nuclei for the major muscles of respiration reside in the spinal cord. The phrenic motor nucleus in the cervical region drives the diaphragm. Activation of the intercostal muscles arises from the motor neurons in the thoracic spinal cord and activation of the abdominal muscles is driven from motor neurons in both the lower thoracic and lumbar spinal cord. These motor neurons receive respiratory input from the premotor neurons in the VRG and DRG via glutamate. The spinal motor neurons are modulated by serotonergic fibers from the raphe nucleus, which modulates respiratory output during sleep and is an important adaptive mechanism allowing facilitation of the integrated respiratory output. Cranial motor nuclei Two cranial motor nuclei that have important influences on airway resistance are the vagus and hypoglossus. The vagal nuclei are located bilaterally in the dorsal medulla within the NTS, whereas the hypoglossal motor nuclei are located more dorsomedial to the NTS. These cranial nerves drive the activity of muscles that control the patency of the upper airway (laryngeal, pharyngeal, and tongue muscles). The hypoglossal nucleus has been extensively studied since its loss of tone during sleep makes it an important contributor to obstructive sleep apnea. Serotonergic input from the raphe as well as pontine cholinergic and adrenergic influences contribute to this loss of tone. Suprapontine influences Breathing is modulated by higher brain centers in response to behavior, emotion, state (sleep/wake), fever, exercise, or voluntary

148 NEUROPHYSIOLOGY / Neuroanatomy

Emotion Vocalization Locomotion Sleep

Higher center

PRG

DRG

Premotor VRG

Böt C

MN

Pre-Böt C

Figure 2 Schematic drawing of a saggital view of the brain identifying the major neuroanatomical sites important for the control of breathing. The major sites critical for the generation of a breath are located within the pons and medulla, which contain inspiratory and expiratory premotor neurons that project to spinal (or cranial) motor neurons. Higher centers (suprapontine) project to the pontine and medullary respiratory centers to adjust breathing during behavioral or changes in state. Motor neurons in the spinal cord project to the major muscles of respiration. Bo¨t C, Bo¨tzinger complex; Pre-Bot C, pre-Bo¨tzinger complex; DRG, dorsal respiratory group; VRG, ventral respiratory group; PRG, pontine respiratory group.

efforts. Little is known about the specific neural sites and pathways that modulate respiration during these complex responses. The hypothalamus contains regions that are involved in thermoregulation and locomotion and appear to coordinate the respiration during fever and exercise. Regions of the cortex are involved in many of the behavioral and emotional effects on breathing, including voluntary control. Voluntary control of breathing overrides automatic rhythm generation and facilitates breath holding, coughing, hyperventilation, or other voluntary respiratory maneuvers (Figure 2).

Neuroanatomy of Respiration in Respiratory Diseases A number of diseases and syndromes produce abnormal breathing patterns. The causes of the neuropathology can be disease linked or genetic. The abnormal pattern that is produced depends on the location of the defect within the respiratory network. In some cases, the neuroanatomical location is known, but often the location can only be speculated on based on how the respiratory pattern changes. Complete or Partial Loss of Breathing

Respiratory Rhythm Generation

The act of breathing requires two phases: inspiration and expiration. These two phases reflect a reciprocal activation of inspiratory and expiratory neurons within the medullary respiratory centers. For many years, this rhythmic oscillation of activation of inspiratory neurons followed by activation of expiratory neurons was thought to be due entirely to the membrane properties of the neural network, based on their excitability properties and their ability to reciprocally inhibit each other. This reciprocally inhibiting neural network was thought to have no requirement for a pacemaker even though many other rhythmic motor systems contain pacemakers in their networks. However, evidence showing that the preBo¨tzinger complex contains inspiratory neurons that can initiate a rhythm has led to a hybrid model of respiratory rhythm generation (conditional pacemaker in a network).

An inability to breathe is usually due to some neuropathology resulting in injury to the spinal cord. The spinal injury may be the result of physical trauma or ischemic damage due to impaired blood flow. Depending on what level of the spinal cord is injured, there can be either loss of breathing entirely (complete lesioning of the cervical spinal cord) or loss of some aspects of breathing due to how much the drive to thoracic and abdominal respiratory muscles is affected (thoracic and lumbar spinal cord). This is why patients who suffer from a spinal injury to the high cervical spinal cord require continuous ventilator support. In the case of a syndrome known as Ondine’s curse, the patient loses the automatic control of breathing presumably due to some pathology in the ventrolateral spinal cord responsible for relaying the descending respiratory tracts. These patients must voluntarily control their breathing, which allows them to breathe while they are awake but during sleep they need to be artificially ventilated.

NEUROPHYSIOLOGY / Neuroendocrine Cells 149

Sudden infant death syndrome is another syndrome whose etiology is thought to be related to pathology of the central respiratory neural network causing a cessation of breathing. In this case, the exact site of the neuropathology is not known, although there is evidence that there are abnormalities in the regions of the ventral medulla that could affect the normal pattern of respiratory rhythm generation. Irregular Breathing Patterns

There are a number of abnormal breathing patterns associated with disease processes. Because they can occur at any level of the brain, cerebral strokes can result in patterns that replicate those of early studies used to identify and characterize the neural components of the respiratory neural network. If the stroke occurs in the cortex, a waxing and waning of breathing occurs (Cheyne–Stokes breathing) due to the loss of higher cortical influences. A lesion in the diencephalon produces a rapid shallow breathing (central neurogenic hyperventilation), suggesting that the inhibitory influences on the rate of breathing are removed. A stroke that lesions the rostral third of the pons could produce an apneustic pattern of breathing if there is an impaired function of the PRG. Strokes that produce lesions in the medullary centers could result in patterns of clustered breathing or ataxic breathing depending on how much of the neural circuitry they destroy. Other abnormal breathing patterns are associated with conditions such as congenital hypoventilation syndrome, obstructive sleep apnea syndrome (OSA), and Rett syndrome. The causes of these syndromes are not fully understood but are likely related to genetic factors or, in the case of OSA, a combination of genetic and acquired factors that compromise the upper airway. See also: Breathing: Breathing in the Newborn; Fetal Breathing. Chemoreceptors: Central; Arterial. Diving. Exercise Physiology. High Altitude, Physiology and Diseases. Neuromuscular Disease: Lower Motor Neuron Diseases; Upper Motor Neuron Diseases. Neurophysiology: Neural Control of Airway Smooth Muscle. Obesity. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Sleep Apnea: Overview; Adult; Children; Continuous Positive Airway Pressure Therapy; Drug Treatments; Genetics of Sleep Apnea. Sleep Disorders: Overview; Central Apnea (Ondine’s Curse); Hypoventilation; Upper Airway Resistance Syndrome. Space, Respiratory System. Sudden Infant Death Syndrome. Upper Airway Obstruction. Ventilation: Control.

Further Reading Berger AJ (2001) Central mechanisms of respiratory control. In: Hlastala MP and Berger AJ (eds.) Physiology of Respiration, pp. 134–147. New York: Oxford University Press.

Berger AJ and Bellingham MC (1995) Mechanisms of respiratory motor output. In: Dempsey JA and Pack AI (eds.) Lung Biology in Health and Disease, pp. 71–149. New York: Dekker. Bianchi AL, Denavit-Saubie´ M, and Champagnat J (1995) Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters. Physiological Reviews 75: 1–45. Bonham AC (1995) Neruotransmitters in CNS control of breathing. Respiration Physiology 101: 219–230. Del Negro CA, Wilson CG, Butera RJ, et al. (2001) Unstable breathing rhythms and quasiperiodicity in the pre-Bo¨tzinger complex. Advances in Experimental Medicine and Biology 499: 133–138. Feldman JL, Mitchell GS, and Nattie EE (2003) Breathing: rhythmicity, plasticity, chemosensitivity. Annual Review of Neuroscience 26: 239–266. Feldman JL and Smith JC (1995) Neural control of respiratory pattern in mammals: an overview. In: Dempsey JA and Pack AI (eds.) Lung Biology in Health and Disease, pp. 39–69. New York: Dekker. Funk GD and Ramirez JM (2002) Neural control of breathing. Respiratory Physiology & Neurobiology 131: 1–3. Kinney HC and Filiano JJ (1988) Brainstem research in sudden infant death syndrome. Pediatrician 15: 240–250. Ramirez JM, Zuperku EJ, Alheid GF, et al. (2002) Respiratory rhythm generation: converging concepts from in vitro and in vivo approaches? Respiratory Physiology & Neurobiology 131: 43–56. Rekling JC and Feldman JL (1998) Pre-Bo¨tzinger complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation. Annual Review of Physiology 60: 385–405. Richter DW, Manzke T, Wilken B, and Ponimaskin E (2003) Serotonin receptors: guardians of stable breathing. Trends in Molecular Medicine 9: 542–548. Richter DW and Spyer KM (2001) Studying rhythmogenesis of breathing: comparison of in vivo and in vitro models. Trends in Neuroscience 24: 464–472.

Neuroendocrine Cells E Cutz and H Yeger, The Hospital for Sick Children, Toronto, ON, Canada & 2006 Elsevier Ltd. All rights reserved.

Abstract Pulmonary neuroendocrine (NE) cells are widely distributed within the airway epithelium of mammalian lungs. NE cells are either found as a solitary cell or as part of distinctive innervated cell clusters, neuroepithelial bodies (NEBs), both of which have abundant cytoplasmic dense core vesicles (DCVs), the storage site for amine (serotonin, 5-HT) and peptides (bombesin, gastrin-releasing peptide) that act as neurotransmitters, neuromodulators, and growth factors. During lung development NE cells/ NEBs express a mixed epithelial, neural, and neuroendocrine phenotype and their differentiation is directed by proneural basic helix–loop–helix factors. In fetal lung, these cells appear numerous compared to the adult and are thought to be involved in lung morphogenesis, while postnatally NEBs function as hypoxia-sensitive airway chemoreceptors. Hypoxia activates a membrane-bound molecular complex composed of an oxygensensitive K þ channel coupled to a multicomponent NADPH

150 NEUROPHYSIOLOGY / Neuroendocrine Cells oxidase leading to exocytosis of DCVs with release of 5-HT and other mediators acting locally or via vagal afferents transmitting the hypoxia signal to the brainstem. NEB cells also express multiple neurotransmitter receptors involved in the chemotransmission of hypoxia and other stimuli. The biological complexity and multifunctional nature of neuroendocrine cell systems implies wide-ranging involvement in normal lung functions and in the pathophysiology of a variety of pediatric as well as adult respiratory diseases including lung carcinogenesis.

Introduction Pulmonary neuroendocrine (NE) cells were first described 75 years ago when solitary and clusters of argyrophilic cells, analogous to similar cells in the gastrointestinal tract, were observed within the airway epithelium of human and animal lungs (Figure 1). These cells were thought to belong to a system of diffuse endocrine cells found in both endocrine and nonendocrine tissues. The term NE cell was adopted after the discovery of amine and peptide hormone expression by these cells and the recognition of their neural and endocrine phenotype. Structure

At the light microscopy level, NE cells can now be identified by immunohistochemical methods using antibodies against a variety of neuroendocrine markers (Table 1). Solitary NE cells appear as flaskshaped cells with their apical surface reaching the airway lumen, or as elongated cells with lateral cytoplasmic processes extending along the basement membrane. The solitary NE cells are distributed throughout the epithelium of trachea and bronchi up

to the terminal bronchioles. Neuroepithelial bodies (NEBs) are corpuscular structures composed of 5–15 cells that are localized within the intrapulmonary airways and appear concentrated at airway bifurcations. Pulmonary NE cells/NEBs are relatively numerous in fetal/neonatal lungs compared to the adult. Solitary NE cells and NEBs constitute the neuroendocrine cell system with similar features and are therefore discussed collectively. Ultrastructurally, NE cells/NEBs are characterized by the presence of cytoplasmic dense core vesicles (DCVs), the storage site for amine and peptides (Figure 2(a)). The apical cell membrane forms short microvilli exposed to the airway lumen. The other cell organelles include small mitochondria, rough endoplasmic reticulum, ribosomes, microtubules, and bundles of microfilaments. A well-developed Golgi complex shows a variety of associated vesicles related to the formation of DCVs. In NEBs of rabbit fetal and neonatal lungs two morphological types of nerve endings in contact with the granulated cells are found: afferent-like sensory nerve endings containing numerous small mitochondria (Figure 2(b)); and efferent-like nerve fibers with agranular vesicles, microtubules, and sparse mitochondria (Figure 2(c)). Serial section studies have shown that these two types of nerve endings may be in continuity, an arrangement consistent with local modulation via an axon reflex. Innervation

The nature and origin of intraepithelial nerve endings in contact with NEB cells have been characterized in

Figure 1 Corpuscular NEB (arrow) and solitary pulmonary NE cells (arrowhead) within airway mucosa of newborn infant lung. Grimelius silvernitrate method; Magnification
600. Reproduced from Cutz E et al. (1995) Pulmonary neuroendocrine system: an overview of cell biology and pathology with emphasis on pediatric lung disease. Perspectives in Pediatric Pathology 18: 32–70, with permission from S. Karger AG, Basel.

NEUROPHYSIOLOGY / Neuroendocrine Cells 151 Table 1 Immunohistochemical and molecular markers of NEB cells in mammalian lungsa Amine Serotonin (5hydroxytryptamine, 5-HT) Amine metabolizing enzymes Tryptophane hydroxylase Aromatic amino acid decarboxylase Regulatory peptides Bombesin/gastrin-releasing peptide (GRP) Calcitonin Calcitonin gene-related peptide (CGRP) Cholecystokinin (CCK) Substance P Somatostatin Endothelin Peptide YY Helodermin Pituitary adenyl cyclaseactivating protein Transcription factors Mash-1

Neuroendocrine/ neurosecretory markers Neuron specific enolase (NSE) Protein gene product 9.5 (PGP9.5) Chromogranin A NCAM/MOC-1 Leu 7/NHK Goa Synaptophysin Synaptic vesicle protein 2 (SV2) Synaptobrevin Synaptogamin SNAP-25 Calbindin D28K

Functional proteins NADPH oxidase (gp91phox, p22phox, p47phox, rac2)

a There is marked species variation in the expression of different markers. However, the majority have been identified in NEB cells of human lung and/or experimental animals (i.e., rabbit, rat, hamster, mouse). Reproduced with permission from Cutz E, Fu XW, Yeger H, et al. (2003) Oxygen sensing in pulmonary neuroepithelial bodies and related tumor cell models. In: Lahiri S, Semenza GL, and Prabhakar NR (eds.) Oxygen Sensing Responses and Adaptation to Hypoxia, Lung Biology in Health and Disease, vol. 175, pp. 567– 602. New York: Dekker. Copyright CRC Press, Boca Raton, FL.

the rabbit and rat lung. Although there are species differences, the predominant neural connections innervating NEB are sensory vagal afferents with nerve cell bodies residing in the nodose ganglion. In the rat, two additional components have been demonstrated: calcitonin gene-related peptide (CGRP) immunoreactive nerves derived from the spinal ganglia, and nitrergic nerve endings originating from peribronchial ganglia (Figure 3). The expression of several ionotropic neurotransmitter receptors together with complex innervation probably reflect the multifunctional role of NEB in the lung. Ontogeny and Differentiation

It is thought that pulmonary NE cells/NEB are derived from the foregut endoderm (as has been

demonstrated for gastrointestinal (GI) endocrine cells and islets of Langerhans). In early fetal lungs NE cells/ NEBs are the first cell types to differentiate within primitive airway epithelium, implying that NE cells could promote differentiation of other pulmonary cell types. Several proneural and neurogenic genes belonging to the family of basic helix–loop–helix (bHLH) transcription factors have been identified that are involved in the ontogeny of NE cells/NEBs. The activator of mammalian homolog of achaete-scute complex (Mash-1) in mice and its human counterpart (Hash-1) are essential for pulmonary NE cell development. The disruption of the Mash-1 (Mash-1  /  ) gene in mice resulted in failure of NE cell differentiation in the lung while GI endocrine cells were still present. Interestingly, lung morphogenesis did not appear to be affected by the loss of Mash-1 expression although these mice die shortly after birth due to respiratory failure. Ito and colleagues showed that the differentiation of NE cells was increased in hairy-enhancer-split (Hes-1)deficient mice indicating that Hes-1 acts as a neurogenic gene repressor and that Mash-1 and Hes-1 likely act in concert with Notch/Notch ligand signaling pathways.

Neuroendocrine Cell Functions in the Normal Lung VanLommel and Lauweryns have proposed a dual role for NE cells/NEBs as having a local paracrine or endocrine role during intrauterine lung development and postnatally as airway chemoreceptors. The local paracrine functions are dependent on the synthesis and release of a number of biologically active substances including amine (serotonin, 5-hydroxytryptamine, 5-HT), peptides (e.g., bombesin/ gastrin-releasing peptide (GRP), CGRP, calcitonin, cholecystokinin, etc.) and other neuroendocrine molecules produced by NE cells/NEBs (Table 1). The predominant amine is 5-HT and is found in NE cells/ NEBs throughout the animal kingdom. The classical pharmacological activities of 5-HT in the lung include vasoconstriction, enhanced vascular permeability, and in some species bronchoconstriction. Other biological actions of 5-HT relevant to developing lung are that of a mitogen. The most prominent regulatory peptide identified in NE cells/NEBs of human lung is bombesin, a tetradecapeptide originally isolated from frog skin. This amphibian peptide is structurally related to a 27 amino acid mammalian peptide, GRP. Bombesin and GRP share an identical C-terminal decapeptide, accounting for the cross-reactivity of respective antibodies and for the similarities in their bioactivity.

152 NEUROPHYSIOLOGY / Neuroendocrine Cells

2 µm

∗

∗

∗

Figure 2 (a) Low magnification electron micrograph of bronchiolar NEB in rabbit neonatal lung located at a bifurcation. Apical surface of NEB cells form short microvilli (mv) exposed to airway lumen (AL). Columnar NEB cells with indented nuclei (Nu) contain numerous cytoplasmic dense core vesicles (DCV, arrows), and nerve endings in intercellular spaces (asterisk). The basal aspect of NEB rests on a basement membrane (arrowheads). (b) Afferent-like nerve ending containing numerous small mitochondria (mi). (c) Efferent-like nerve ending with small empty vesicles (ve). Typical DCV visible in adjacent cell cytoplasm (arrows).

Bombesin/GRP immunoreactivity has been demonstrated in NE cells/NEBs of human fetal, neonatal, and postnatal lungs throughout all ages. Expression of bombesin/GRP is developmentally regulated as high levels are detected during intrauterine life and in neonates, with significant decline postnatally. A single gene coding for human GRP has been identified on chromosome 18. Three distinct mRNA species encoding pro-GRP are generated from the primary transcript by alternative splicing. The highly conserved C-terminal region of bombesin and GRP is responsible for high-affinity binding to cell surface receptors mediating the various biological activities of bombesin-like peptides. At least three distinct

bombesin/GRP receptor subtypes have been identified. These receptors belong to a family of seven transmembrane G-protein-coupled cell surface receptors interacting with several intracellular signal transduction pathways. Sunday and colleagues investigated the potential role of bombesin/GRP in developing lung. They showed that the GRP gene was expressed in a proximal-to-distal pattern in human fetal lung in parallel with the growth of the developing airways. Exogenous bombesin was shown to act as a potent growth and differentiation factor for fetal murine lung in utero and for human and other species in lung organ culture. Bombesin-like peptides in vitro and

NEUROPHYSIOLOGY / Neuroendocrine Cells 153 Dorsal motor nucleus? Nucleus ambiguus?

Cholinergic nonnitrergic NTS

Hyperactivity? Hyper-reactivity? Jugular ganglion

DRG (T1−T6)

? Nodose ganglion Purinergic receptors Vagus nerve Myelinated (MBP+)

Adenosine receptors? CGRP+ / SP+ capsaicin sensitive (P2X2?) / P2X3 receptor + CGRP-/capsaicin A(1?) adenosine insensitive NEB receptor + glutamatergic / CB+ CGRP+/SP+ Airway capsaicin sensitive epithelium

CB+ 5HT+ ACh+ CALC+ CGRP+

ATP+ Cholinergic Nitrergic noncholinergic nonnitrergic

?

? BV ?

? ?

?

?

?

Nitrergic noncholinergic

?

+

+ Reflex bronchoconstriction Airway smooth muscle

+

+

+

+

Cholinergic nonnitrergic (purinoreceptor-)?

Airway ganglion Figure 3 Innervation of NEB in rat lung. Schematic representation of possible interactions between purines, nerve terminals, and complex NEB receptors in the rat lung. The center of the scheme represents a pulmonary NEB (colored yellow) and its extensive interactions with nerve terminals. The upper left part shows the vagal afferent (nodose ¼ red neurons; jugular ¼ ochre neurons) and preganglionic parasympathetic efferent (colored lilac) connections; the bottom part shows a postganglionic parasympathetic ganglion of the airway wall (cholinergic neurons ¼ purple; nitrergic neurons ¼ green) and the upper right part shows dorsal root afferents (blue neurons). Another population of neurons intrinsic to the airway wall, located immediately beneath the epithelium, is represented in the right middle part of the scheme, close to the base of the NEB, and shows a nitrergic neuron (colored green) that innervates the NEB. Furthermore, all characterized nerve fiber populations that are important for the present overview are included (color-coded). Known characteristics of the NEB and of the represented neuronal populations are included in the scheme in the same color as the respective structures. 5HT, serotonin; ACh, acetylcholine; BV, blood vessel; CALC, calcitonin; DRG, dorsal root ganglia; NTS, nucleus of the solitary tract. Reproduced from Adriaensen D and Timmermans JP (2004) Purinergic signalling in the lung: important in asthma and COPD? Current Opinion in Pharmacology 4: 207–214, with permission from Elsevier.

154 NEUROPHYSIOLOGY / Neuroendocrine Cells Pulmonary NE cells

Airway epithelium

Type II cell O2 sensor

2

K+

Ca2+

1

5-HT2cR

2

GRP, 5-HT, CGRP

3 KGF, CRF

EGF PDGF

4

3 GRP-R

Mesenchyme Figure 4 Role of NE cells in lung development. Schematic diagram of the proposed role of NE cells and their peptide and amine products (GRP/5HT) in developing lung. The schema indicates that pulmonary NE cells release GRP/5HT (1) when stimulated by hypoxia as modulated through the O2 sensor. 5HT in turn also acts on type II cells (2), which express the serotonin receptor-HT2cR. GRP can act on the lung mesenchyme, which expresses the GRP receptor (GRP-R) and in turn stimulates mesenchymal cells to release epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) (3), which modulate epithelial cell functions, thereby completing the epithelial–mesenchymal interaction (4). Mesenchyme, stimulated by GRP, will release keratinocyte growth factor (KGF) and corticotropin releasing factor (CRF) (3), which can further influence development of type II cells (alveolar compartment). Since NE cells start functioning early during lung development, they are viewed as important modulators of lung organogenesis. Modified from Sunday ME and Cutz E (2000) Role of neuroendocrine cells in fetal and post natal lung. In: Mendelson CR (ed.) Endocrinology of the Lung: Development and Surfactant Synthesis, pp. 299–336. Totowa: Humana Press.

in vivo promoted proliferation of both mesenchymal and epithelial cells in conducting airways and in primitive alveoli, increased branching morphogenesis, and induced differentiation of alveolar type II cells (Figure 4). These effects are directly related to the expression of GRP-preferring receptors widely distributed in the lung parenchyma. In addition to growth factor-like properties, bombesin/GRP-like peptides exhibit a variety of biological and pharmacological activities including constrictor effects in various smooth muscle preparations. Function of NEB as Airway Chemoreceptors

Morphological features consistent with a chemoreceptor function include: (1) preferential location at airway branch points, ideally positioned to monitor airway gas concentration; (2) the presence of amine and peptide neurotransmitters that are released upon hypoxia stimulus; and (3) vagal sensory innervation transmitting hypoxia signal to the central nervous system (CNS). In whole animal studies, Lauweryns and co-workers demonstrated degranulation and release of amine from NEB cells in lungs of rabbit neonates exposed to acute airway hypoxia, and

suggested that NEBs function as hypoxia-sensitive airway chemoreceptors modulated by the CNS. Cellular and Molecular Mechanisms of Oxygen Sensing

Electrophysiological studies have confirmed the role of NEB cells as airway O2 sensors and the actual transducers of hypoxia stimulus. Whole cell patch-clamp studies using cultures of NEBs isolated from rabbit fetal lung have demonstrated the presence of voltageactivated Na þ , Ca2 þ , and K þ currents, a key feature of excitable cells. Hypoxia (PO2 25–30 mmHg) reversibly inhibited the outward K þ current in NEB cells while the inward Na þ and Ca2 þ were unaffected. Under current clamp conditions, hypoxia caused an increase in the slope and frequency of the depolarizing pacemaker potential. Hence, it was confirmed that NEB cells express a hypoxia-sensitive K þ (O2) current similar to that identified in other O2 sensing cells, namely the carotid body (CB) glomus cells, pulmonary artery myocytes, and certain neurons in the brain. The primary event in NEB O2 sensing in response to airway hypoxia is the closure of the O2-sensitive K þ channel. The actual O2 sensor is represented by a

NEUROPHYSIOLOGY / Neuroendocrine Cells 155

heme-linked NADPH oxidase closely associated with an O2-sensitive Kþ channel. Under normoxia, the oxidase tonically generates superoxide from ambient O2, which is then rapidly converted to hydrogen peroxide (H2O2); this in turn modulates O2-sensitive Kþ channel activity. Hypoxia decreases the level of H2O2 production resulting in Kþ channel closure and NEB cell membrane depolarization. This then causes activation of voltage-sensitive Ca2þ channels with influx of extracellular Ca2þ triggering exocytosis of DCVs and release of neurotransmitters (i.e., 5-HT). NADPH oxidase is the principal O2 sensor in NEB cells since mice with oxidase deficiency (OD, gp91phox k/o) show no response to acute hypoxia. Furthermore, neonatal OD mice show abnormal breathing and fail to respond to acute hypoxia challenge when assessed by whole body plethysmography. On the other hand, the CB function and pulmonary artery responses to hypoxia in the same OD mice appear intact, suggesting alternate O2 sensing mechanisms. Chemotransmission of Hypoxia Stimulus

The candidates for neurotransmitters that mediate fast chemosensory transmission of hypoxia stimulus from NEB cells via vagal sensory afferents include 5HT, acetylcholine (ACh), and adenosine triphosphate (ATP). Evidence in support of 5-HT as a transmitter of hypoxia stimulus include in vivo and in vitro studies in the rabbit model as well as recent investigation using carbon fiber amperometry. These studies have shown that hypoxia causes dose-dependent 5-HT release from NEB within the physiologic range expected in the airway (i.e., PO2 o95 mmHg). NEB cells express a 5-HT 3 receptor acting as an autoreceptor with positive feedback and amplification of hypoxia signaling. The evidence for cholinergic mechanisms in NEB includes demonstration of acetycholinesterase activity, the presence of small clear vesicles in efferent-like nerve endings in contact with NEB cells, and immunohistochemical demonstration of vesicular acetylcholine transporter (VChat) activity. NEB cells also express functional nicotinic acetylcholine (nACh) receptors, which are critical in mediating the effects of nicotine derived from smoking. The possible involvement of ATP and purinergic mechanisms is based on the demonstration of ATP in NEB cells and expression of P2/X receptors in nerve endings innervating NEBs as well as on NEB cells proper. Heteromeric P2X2/3 receptors in NEB cells possibly function as autoreceptors involved in modulation of hypoxia chemotransmission and other stimuli. Neuroendocrine Cells in Respiratory Diseases

Abnormalities in the number and distribution of NE cells/NEB have been documented in various pediatric

lung disorders, as well as in adults with a variety of chronic interstitial lung diseases and in lung carcinogenesis. Increased numbers of NE cells/NEBs have been reported in lungs of infants with lung hypoplasia due to diaphragmatic hernia. The proposed mechanisms include compensatory increase related to impaired lung growth and/or failure of neuropeptide release during neonatal adaptation. Significant hyperplasia of bombesin/GRP immunoreactive NE cells/NEBs has been reported in cases of bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants characterized by ongoing airway epithelial injury and repair. Although the precise mechanism is not known, chronic hypoxia and/or release of inflammatory cytokines could stimulate mitogenesis of NE cells/NEBs or recruitment from precursor cell. Infants with BPD exhibit a number of physiological abnormalities (i.e., airway hyperreactivity, pulmonary hypertension, and increased apneic spells) possibly related to NE cells/ NEB hyperplasia and increased levels of pulmonary bombesin/GRP. Hyperplasia of NE cells/NEBs has also been described in congenital central hypoventilation syndrome and in sudden infant death syndrome (SIDS), both disorders characterized by dysfunction in respiratory control. In cases of SIDS there is marked hyperplasia of NE cells/NEBs, especially in lungs of infants whose mothers smoked during pregnancy. In this situation the proliferation of these cells is likely driven via nACh-receptors as demonstrated in several animal models exposed to cigarette smoke or nicotine. Possible dysfunction of NE cells/NEBs has been implicated in the pathogenesis of cystic fibrosis (CF) since these cells express cystic fibrosis transmembrane regulator (CFTR). Experimental data suggest that normal CFTR function is required for O2 sensing and for amine/peptide secretion by NE cells/NEBs, which in turn may affect the composition of pericilliary fluid eventually leading to thickened mucus accumulation and airway plugging. The lung diseases in adults associated with NE cell/ NEB hyperplasia encompass both benign and neoplastic conditions including usual interstitial pneumonitis, eosinophilic pneumonitis, idiopathic pulmonary NE cells hyperplasia, carcinoid tumors, and small cell lung carcinoma (SCLC). The possible mechanisms include interactions of inflammation, cytokines, smoking and hypoxia-activating neurogenic gene (Hash-1) expression, and mitogenic pathways. In SCLC a bombesin/GRP autocrine growth mechanism drives tumor cell proliferation, hence a potential therapy using peptide antagonists is being actively pursued. Presently, there are no specific therapies that target NE cell/NEB dysfunction.

156 NEUROPHYSIOLOGY / Neurons and Neuromuscular Transmission See also: Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Lung Development: Overview. NADPH and NADPH Oxidase.

Further Reading Adriaensen D, Brouns I, Van Genechten J, and Timmermans JP (2003) Functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors. Anatomical Record 270A: 25–40. Adriaensen D and Timmermans JP (2004) Purinergic signalling in the lung: important in asthma and COPD? Current Opinion in Phamacology 4: 207–214. Cutz E, et al. (1995) Pulmonary neuroendocrine system: an overview of cell biology and pathology with emphasis on pediatric lung disease. Perspectives in Pediatric Pathology 18: 32–70. Cutz E (ed.) (1997) Cellular and Molecular Biology of Airway Chemoreceptors. Austin: Landes Bioscience. Cutz E, Fu XW, Yeger H, et al. (2003) Oxygen sensing in pulmonary neuroepithelial bodies and related tumor cell models. In: Lahiri S, Semenza GL, and Prabhakar NR (eds.) Oxygen Sensing Responses and Adaptation to Hypoxia, Lung Biology in Health and Disease, vol. 175, pp. 567–602. New York: Dekker. Cutz E and Jackson A (1999) Neuroepithelial bodies as airway oxygen sensors. Respiratory Physiology 115: 201–214. Cutz E and Jackson A (2001) Airway inflammation and peripheral chemoreceptors. In: Bayard RW and Krous HF (eds.) Sudden Infant Death Syndrome: Problems, Progress and Possibilities, pp. 156–180. London: Arnold. Gosney JR (1992) Pulmonary Endocrine Pathology. Oxford: Butterworth Heinemann. Ito T, Udaka N, Okudela K, et al. (2003) Mechanisms of neuroendocrine differentiation in pulmonary neuroendocrine cells and small cell carcinoma. Endocrine Pathology 14: 133–139. Sunday ME (1997) Neuropeptides and lung development. In: McDonald JE (ed.) Lung Growth and Development. Lung Biology in Health and Disease, vol. 100, pp. 401–494. New York: Dekker. Sunday ME and Cutz E (2000) Role of neuroendocrine cells in fetal and post natal lung. In: Mendelson CR (ed.) Endocrinology of the Lung: Development and Surfactant Synthesis, pp. 299– 336. Totowa: Humana Press. Van Lommel A, Bolle T, Faunes W, and Lauweryns JM (1999) The pulmonary neuroendocrine system: the past decade. Archives of Histology and Cytology 62: 1–16. Youngson C, Nurse CA, Yeger H, and Cutz E (1993) Oxygen sensing in airway chemoreceptors. Nature 365: 153–155.

Neurons and Neuromuscular Transmission E van Lunteren, Case Western Reserve University, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Brain neurons that generate and modulate respiratory rhythms are located predominantly in the medulla and pons. They activate motor neurons located in the spinal cord and cranial nerve

nuclei, which in turn supply respiratory muscles in the thorax, abdomen, and upper airway. Neuromuscular transmission is the process by which motoneuronal activation results in muscle contraction. An action potential is propagated from the motoneuronal cell body down the axon to the nerve terminal. The depolarization opens voltage-gated calcium channels, elevating intracellular calcium concentrations. Acetylcholine is stored in vesicles at the nerve terminal. Vesicle docking requires the generation of the synaptic fusion complex, in which SNARE proteins play key roles. Exocytosis stimulated by elevated calcium levels releases acetylcholine into the synaptic cleft. When two acetylcholine molecules bind to a postjunctional acetylcholine receptor, the cation channel of the receptor opens, depolarizing the cell. If sufficient transmitter is released, the depolarization will initiate an action potential in the muscle cell, eliciting contraction via a cascade of electrophysiological and biochemical events. Acetylcholinesterase terminates the action of acetylcholine, and transmitter is then recycled by reuptake into the nerve terminal. Impairment of neuromuscular transmission can occur during high-intensity and -duration activation in normal neuromuscular junctions or as a result of specific perturbations caused by diseases such as myasthenia gravis and botulism.

Description The respiratory motor system maintains ventilatory homeostasis during rest and exercise and also in response to a variety of environmentally and diseaseinduced perturbations. It is composed of several key elements. At the level of the brain, neuronal groups located primarily in the medulla and pons are responsible for the generation and modulation of the respiratory rhythm and for integrating sensory information. These project to the spinal cord and cranial motor nuclei, where respiratory motoneurons send axons to inspiratory and expiratory muscles of the thorax, abdomen, and upper airways. In the periphery, motoneurons transmit information to the skeletal muscles via the neuromuscular junction (Figure 1). In the mammalian respiratory system, each motoneuron supplies several muscle fibers, and each muscle fiber is supplied by one motoneuron. The motoneuron and the muscle fibers it supplies comprise the motor unit, which forms the cellular basis for the regulation of respiratory muscle contraction. Muscles involved in breathing are a heterogeneous group with respect to anatomic location, size, mechanical action, fiber-type composition, biochemical properties, and contractile performance. During low to moderate levels of activation, transmission from motoneuron to muscle has complete fidelity. As a result, the mechanical output of the muscle depends directly on the firing pattern of each motoneuron and the contractile response of each muscle fiber to that pattern of activation. Neuromuscular transmission has a high safety factor in that considerably more acetylcholine is released than required to ensure muscle fiber contraction. However, high-intensity

NEUROPHYSIOLOGY / Neurons and Neuromuscular Transmission 157

and/or prolonged motoneuronal activation leads to a gradual depletion of transmitter available for release, at which time muscle fiber contraction may not occur each time the motoneuron fires. This occurs in diseases of the neuromuscular junction, in which the

MHCslow

MHC2X

MHC2A

MHC2B

safety factor for transmission is reduced by specific prejunctional and postjunctional perturbations. Each motoneuron sends a single axon from the spinal cord or brainstem to the periphery. Once it reaches the muscle, the axon branches distally to allow innervation of multiple muscle fibers in a localized area. The key elements of the neuromuscular junction proper include the nerve terminal, the synaptic cleft, and the muscle end plate (Figures 1 and 2). Neuromuscular junctions vary among muscle fiber types with respect to size and complexity of structure, including the extent of postjunctional folding (Figure 1). Acetylcholine is released from vesicles in each nerve terminal, diffuses across the synaptic cleft, and binds to acetylcholine receptors on the muscle end plate, thereby effecting neuromuscular transmission. Acetylcholine is then broken down by acetylcholinesterase. This is followed by reuptake of the choline at the nerve terminal and resynthesis of acetylcholine, thereby replenishing presynaptic vesicles.

Figure 1 Neuromuscular junctions of the phrenic nerve and diaphragm. In this confocal image, axons and nerve terminals are labeled red, selected muscle fibers are labeled blue, and motor end plates are labeled green. (Left) Two neuromuscular junctions, a nerve branch with multiple axons, and several muscle fibers. (Right) Motor end plates on diaphragm fibers containing different types of myosin heavy chain (MHC) isoforms, illustrating diversity in motor end plate morphology. Reproduced from Mantilla CB and Sieck GC (2003) Mechanisms underlying motor unit plasticity in the respiratory system. Journal of Applied Physiology 94: 1230–1241, used with permission from The American Physiological Society.

Normal Neurotransmission Acetylcholine Release

Acetylcholine is contained in vesicles within the nerve terminal (Figure 2). Release of acetylcholine

Action potential opens calcium Ca2+ channel

Axon

Calcium channels Nerve terminal

Reserve pool

Recycling of transmitter

Vesicles with acetylcholine Readily releasable pool

Docking

Fusion and exocytosis

Reuptake of choline

Synaptic cleft Acetylcholine receptors Binding of acetylcholine to receptor, and generation of end plate potential

Muscle fiber

Acetylcholine broken down into choline and acetic acid

Acetylcholinesterase

Figure 2 Structure and function of the neuromuscular junction. Release of acetylcholine requires the formation of a synaptic fusion complex for vesicle docking, as well as elevation of intracellular calcium via action potential-mediated opening of voltage-gated calcium channels. Following release, acetylcholine diffuses across the synaptic cleft and binds to acetylcholine receptors, the activation of which depolarizes the muscle cell membrane and elicits an action potential.

158 NEUROPHYSIOLOGY / Neurons and Neuromuscular Transmission

requires docking of the vesicles at the cell membrane, followed by ATP-dependent priming and, finally, fusion of the vesicle with the cell membrane resulting in exocytosis. Docking is a process that requires SNARE proteins: synaptobrevin, SNAP-25, and syntaxin. Synaptobrevin is attached to the synaptic vesicles, whereas syntaxin is attached to the cell membrane, and SNAP-25 is attached to syntaxin. Docking results from the formation of a fusion complex between the synaptobrevin and the other two SNARE proteins. Fusion and exocytosis-mediated acetylcholine release is triggered by calcium entering the cell via predominantly P/Q-type voltage-gated calcium channels. Opening of the calcium channels occurs when an action potential originating in the motoneuronal cell body depolarizes the nerve terminal. Typically, 50–300 vesicles are exocytosed in response to an action potential, with B10 000 acetylcholine molecules released per vesicle. Once released from the nerve terminal, acetylcholine diffuses across the synaptic cleft to the postjunctional side. Acetylcholine Receptors and End Plate Electrophysiology

Skeletal muscle acetylcholine receptors are located predominantly at the neuromuscular junction, with a much lower density found in extrajunctional areas. Within the folded structure of the postjunctional membrane, they are clustered close to the nerve terminal (Figure 2). Acetylcholine receptors are nicotinic rather than muscarinic. Receptors span the cell membrane and consist of five subunits arranged around a central pore (Figure 3). Adult skeletal muscle acetylcholine receptors are composed of two alpha1 subunits and one each of the beta1, epsilon, and delta subunits. The fetal form differs from the adult form by one subunit, with the epsilon subunit being replaced by a gamma subunit. Each skeletal muscle acetylcholine receptor has two acetylcholine binding sites, one at the interface between one of the alpha1 subunits and the delta subunit, and the other at the interface between the other alpha1 subunit and either the epsilon (adult) or gamma (fetal) subunit. Activation of each acetylcholine receptor requires the binding of acetylcholine to both binding sites (Figure 3). The receptor functions as a cation channel, the opening of which leads to influx of sodium ions and efflux of potassium ions. The resultant transient depolarizing of the cell is termed an end plate potential (Figure 4). If sufficient numbers of receptors are activated and the end plate potential is sufficiently large, the resultant depolarization will activate voltage-gated sodium channels. This further

Acetylcholine binding sites Alpha 1

Delta

Epsilon or gamma

Pore through which cations flow Alpha 1

Beta 1

Extracellular Plasma membrane Intracellular

Figure 3 Structure of acetylcholine receptor, which is composed of five subunits surrounding a pore. Binding of two acetylcholine molecules is required to open the ion channel. Acetylcholine binding sites are at the junctions of subunit pairs.

depolarizes the cell and generates an action potential, which then propagates throughout the muscle cell. On the other hand, with insufficient receptor activation, the threshold for action potential generation will not be reached, and the muscle will not contract. Action potential-induced membrane depolarization and charge movement results in the release of calcium from the sarcoplasmic reticulum and, via a cascade of biochemical reactions involving actin and myosin, muscle contraction. Termination of the action potential is due to a combination of sodium channel closure and transient opening of potassium channels, returning membrane potential to resting values and ending muscle contraction due to resequestration of calcium. Acetylcholinesterase

Within the folded membrane topography of the synaptic cleft and end plate, acetylcholinesterase is preferentially located deep within the folds, away from the nerve terminal. Thus, acetylcholine released from the nerve terminal diffuses past the acetylcholine receptors before it reaches the acetylcholinesterase. The neuromuscular junction acetylcholinesterase is a very large protein consisting of three catalytic tetramers covalently linked to a three-stranded collagenlike tail. Localization of the acetylcholinesterase to specific sites within the postjunctional folds results from localized transcription and translation of the catalytic and noncatalytic subunits, localized assembly and secretion, and, finally, localized attachment to elements of the extracellular matrix. Acetylcholine

NEUROPHYSIOLOGY / Neurons and Neuromuscular Transmission 159

ON : 333 ms

OFF : 667 ms Figure 4 End plate potentials recorded from rat diaphragm during intermittent 50 Hz stimulation of the phrenic nerve (train duration 333 ms, with one train per second). Action potentials were prevented with use of m-conotoxin GIIIB, which preferentially blocks muscle over nerve sodium channels. End plate potential size is a direct reflection of acetylcholine release by the nerve terminal. Note the reduction of end plate potential size during each train but good recovery from train to train.

is broken down into choline and acetic acid by hydrolyzing the ester bond. Neurotransmitter Replenishment

After acetylcholine is cleaved by acetylcholinesterase, the choline is taken back up into the nerve terminal by endocytosis and combined with acetylCoA by acetyltransferase to reconstitute acetylcholine. Neurotransmitter replenishment has been modeled functionally, with vesicles being distributed among three pools. The docked pool is located at the membrane surface and is ready for release. The reserve pool resides away from the membrane and replenishes the docked pool. The fused pool, which consists of vesicles exposed to the extracellular fluid during the time between exocytosis and endocytosis, replenishes the reserve pool. Thus, transmitter replenishment is effected by the fused pool replenishing the reserve pool, and the reserve pool in turn replenishing the docked pool. However, there is also a faster pathway by which approximately 20% of the transmitter is recycled directly from the synaptic cleft into the docked pool rather than via the reserve pool.

Neurotransmission in Disease States Failure of Transmission in Normal Neuromuscular Junctions

The safety factor for neurotransmission is defined as the extent to which transmitter release by the nerve terminal exceeds the minimum amount needed for action potential generation at the end plate. It is calculated physiologically from the ratio of the size of the end plate potential to the difference between resting membrane potential and the threshold for action potential generation. It is generally estimated to be 3–5 in mammalian neuromuscular junctions, and it reflects excess release of acetylcholine as well as topographic effects of postjunctional folds and sodium channels within these folds. The safety factor is usually higher in junctions from fast-twitch than slow-twitch muscles or muscle fibers. However, with repeated activation, the rate at which the safety factor diminishes, and hence neurotransmission failure occurs, may be faster in junctions supplying fasttwitch, highly fatigueable fibers than slow-twitch fibers. Failure of transmission can potentially be attributed to two general processes: reductions in transmitter

160 NEUROPHYSIOLOGY / Neurons and Neuromuscular Transmission

release and desensitization of postsynaptic receptors. The former accounts for most, if not all, transmission failure during physiological stimulation frequencies in normal neuromuscular junctions. Reductions in transmitter available in the immediately releasable pool occur when the rate of transmitter replenishment is lower than the antecedent rate of release. Of interest is that high stimulation frequencies not only increase transmitter release but also promote the rate of vesicle replenishment, improving the match between supply and demand. ATP is coreleased in very small amounts with acetylcholine during normal neurotransmission. Adenosine, which is formed from the ATP, has a direct inhibitory effect on acetylcholine release, but this only plays an adjunct role in reducing subsequent transmitter release, and only in limited circumstances. Desensitization of postjunctional receptors can be demonstrated with extremely high stimulation frequencies, paired stimuli, exogenous application of acetylcholine, or in the presence of anticholinesterases. However, its role in neurotransmission failure in the intact organism is probably minimal at best. In addition, failure of axonal conduction can occur at branch points due to depolarization block, which may masquerade as neurotransmission failure during physiological testing. Transmission failure at normal neuromuscular junctions can be demonstrated at frequencies within the physiological range, albeit more so during highrather than low-frequency stimulation, and more so during prolonged rather than brief periods of neuromuscular junction activation. The respiratory motor system differs from limb motor systems by the need to be active for the entire life of the organism. However, each respiratory nerve and muscle is quiescent during a portion of each respiratory cycle, which provides time for transmitter replenishment to occur (Figure 4). Changes in breathing pattern that increase the duty cycle of respiratory muscle activation may worsen neuromuscular transmission by impeding recovery. Conditions associated with intense respiratory neuromuscular junction activation rates include exercise, chemical stimuli such as hypoxia and hypercapnia, and mechanical loading from lung disease. In addition, hypoxia impairs neuromuscular transmission by a direct effect on the neuromuscular junction. Failure of Transmission in Diseased Neuromuscular Junctions

Neuromuscular transmission may be impaired by prejunctional and postjunctional abnormalities. When involvement of the respiratory muscles by neuromuscular junction diseases becomes sufficiently severe, hypercapnic respiratory failure ensues. An example of

a disorder acting at the nerve terminal is Lambert– Eaton myasthenic syndrome. It is characterized by antibodies against the voltage-gated calcium channel of the nerve terminal. The consequential reduction in calcium entry during nerve firing reduces acetylcholine release. On the other hand, repetitive high-intensity nerve activation leads to transient intracellular calcium accumulation, accounting for the phenomenon of force augmentation during repetitive contractions in Lambert–Eaton myasthenic syndrome. An effective pharmacologic treatment for this disorder is the aminopyridines, which block voltage-gated potassium channels, thereby prolonging action potential duration, increasing intracellular calcium concentrations, and augmenting transmitter release. A second nerve terminal disorder is botulism, caused by seven related toxins, types A through G, produced by several clostridium organisms (most commonly Clostridium botulinum). All of the toxins cleave protein components of the neuroexocytosis apparatus (the SNARE proteins), with toxins B, D, F, and G cleaving synaptobrevin; toxins A, C, and E cleaving SNAP-25; and toxin C also cleaving syntaxin. This action prevents docking of the synaptic vesicles to the membrane of the nerve terminal, reducing acetylcholine release. Recovery requires regeneration of new end plates, accounting for the slow recovery phase of this disease. The key treatment modality for botulism is early administration of antitoxin to minimize the severity of neuromuscular junction impairment. Of note, botulinum toxin is used therapeutically via local injection for the management of spastic disorders and hyperhydrosis and for the alleviation of facial wrinkles. Examples of causes of impaired neurotransmission due to events at postjunctional muscle sites are myasthenia gravis as well as pharmacological agents used clinically to produce muscle paralysis. Myasthenia gravis results from antibody-mediated destruction of acetylcholine receptors in the postjunctional membrane, thereby reducing the size of the end plate potential. This leads to failure of action potential generation and muscle contraction, which becomes more prominent with repetitive contractions. In this disorder, there is also postjunctional remodeling, in which the postjunctional folds become more shallow and fewer in number. The major treatment modalities for myasthenia gravis are the anticholinesterases, which prolong the amount of time the acetylcholine is available in the synaptic cleft, as well as various interventions that reduce the intensity of the autoimmune response, such as immunomodulatory drugs, plasmapheresis, and thymectomy. Pharmacologic agents used therapeutically to produce muscle paralysis (other than botulinum toxin)

NITRIC OXIDE AND NITROGEN OXIDES 161

act on acetylcholine receptors. Depolarizing agents activate the receptors and maintain the channels in an open state. As a result, each muscle fiber contracts a single time before becoming paralyzed, which can be seen as fasciculations when observing the muscle. Further contractions are prevented due to the depolarized state of the muscle fibers. Nondepolarizing agents, on the other hand, block binding of acetylcholine to the receptors in a competitive manner, preventing normal opening of the channels and the generation of end plate potentials. See also: Acetylcholine. Neuromuscular Disease: Myasthenia Gravis and Other Diseases of the Neuromuscular Junction. Respiratory Muscles, Chest Wall, Diaphragm, and Other.

Further Reading Arnon SS, Schechter R, Inglesby TV, et al. (2001) Botulinum toxin as a biological weapon: medical and public health management. Journal of the American Medical Association 285: 1059–1070. Boonyapisit K, Kaminski HJ, and Ruff RL (1999) Disorders of neuromuscular junction ion channels. American Journal of Medicine 106: 97–113. Ezure K (2004) Reflections on respiratory rhythm generation. Progress in Brain Research 143: 67–74.

Hemmerling TM and Donati F (2003) Neuromuscular blockade at the larynx, the diaphragm and the corrugator supercilii muscle: a review. Canadian Journal of Anesthesia 50: 779–794. Laghi F and Tobin MJ (2003) Disorders of the respiratory muscles. American Journal of Respiratory and Critical Care Medicine 168: 10–48. Lindstrom JM (2003) Nicotinic acetylcholine receptors of muscles and nerves: comparison of their structures, functional roles, and vulnerability to pathology. Annals of the New York Academy of Sciences 998: 41–52. Mantilla CB and Sieck GC (2003) Mechanisms underlying motor unit plasticity in the respiratory system. Journal of Applied Physiology 94: 1230–1241. Rotundo RL (2003) Expression and localization of acetylcholinesterase at the neuromuscular junction. Journal of Neurocytology 32: 743–766. Ruff RL (2003) Neurophysiology of the neuromuscular junction: overview. Annals of the New York Academy of Sciences 998: 1–10. Sanes JR and Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annual Review of Neuroscience 22: 389–442. Ungar D and Hughson FM (2003) SNARE protein structure and function. Annual Review of Cell and Developmental Biology 19: 493–517. Urbano FJ, Rosato-Siri MD, and Uchitel OD (2002) Calcium channels involved in neurotransmitter release at adult, neonatal and P/Q-type deficient neuromuscular junctions. Molecular Membrane Biology 19: 293–300. Wood SJ and Slater CR (2001) Safety factor at the neuromuscular junction. Progress in Neurobiology 64: 393–429.

NITRIC OXIDE AND NITROGEN OXIDES H Meurs, H Maarsingh, and J Zaagsma, University of Groningen, Groningen, The Netherlands & 2006 Elsevier Ltd. All rights reserved.

Abstract Nitric oxide (NO) is a ubiquitous signaling molecule with a variety of biological functions. Due to its unstable nature, NO is subject to many chemical reactions, which depend on its biological environment and which may be involved in its effects. NO is generated via the oxidation of L-arginine catalyzed by different isotypes of the enzyme NO synthase (NOS), including neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), which are all present in the respiratory tract. Relatively small amounts of NO generated from the constitutive isoforms (nNOS and eNOS) as well as S-nitrosothiols are primarily involved in the regulation of airway and vascular smooth muscle tone, employing both cyclic GMP-dependent and independent pathways. In addition to its effects on smooth muscle tone, large amounts of NO produced by iNOS may have proinflammatory and cytotoxic effects involved in the host defense against microorganisms and malignant cells. Most of these effects may be attributed to the formation of the highly reactive oxidant species peroxynitrite (ONOO
), produced under oxidative

stress conditions during the inflammatory response. The bioavailability of L-arginine to NOS is under important control of arginase, by competition for the common substrate. Induction of arginase during inflammatory reactions may cause a relative deficiency of NO, resulting in reduced inhibition of airway and vascular tone and in production of oxygen radicals by iNOS, which enhances ONOO
formation. Aberrant NO homeostasis is involved in a number of pulmonary diseases, including asthma, COPD, cystic fibrosis, and pulmonary hypertension, which may be monitored by measurement of NO in the exhaled air. Inhalation of NO may be of benefit for the improvement of hemodynamics in pulmonary hypertension.

Introduction Nitric oxide (NO) is one of the most ubiquitous substances in mammalian species, serving as a signaling molecule that is involved in the control of nearly every cellular and organ function in the body, including the lung and the airways. In 1977, exogenous gaseous NO as well as NO generated from various nitro compounds, including nitrovasodilators used for over 100 years, was

162 NITRIC OXIDE AND NITROGEN OXIDES

recognized as having biological activity by activating soluble guanylyl cyclase and inducing smooth muscle relaxation. Ten years later, it was reported that NO represents endothelium-derived relaxing factor (EDRF) in the vasculature, which raised an explosion of research uncovering the major regulatory role of this simple molecule in numerous physiological and pathophysiological processes. The exciting discoveries unraveling the role of NO as a biological signaling molecule were awarded the Nobel prize in Medicine or Physiology to Robert A Furchgott, Louis J Ignarro, and Ferid Murad in 1998. In the lung, NO is produced by a wide variety of cells, including epithelial cells, airway nerves, vascular endothelial cells, and inflammatory cells. Depending on the site of generation, on the amount produced, as well as the redox status of the microenvironment, NO can have many effects in the respiratory tract, including lung development, inhibition of airway and vascular smooth muscle tone, and inflammatory processes involved in the host defense against invading microorganisms and malignant cells. Based on these effects, altered NO homeostasis has been implicated in the pathophysiology of various pulmonary diseases, such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and pulmonary hypertension.

Structure NO is a simple diatomic hydrophobic molecule, which forms a colorless and odorless gas at room temperature and pressure. NO has one unpaired electron and thus is formally a free radical species ðd NOÞ. Due to its radical nature, NO is a highly reactive and unstable molecule capable of undergoing numerous reactions in biological systems, which are of relevance to the physiological and pathophysiological actions of the molecule.

Chemistry of Nitrogen Oxides By redox reactions in the microenvironment, NO may also be present as nitroxyl (NO
) and nitrosium (NO þ ) ions, which are also highly unstable and may be differentially involved in NO-mediated reactions. In biological systems, NO species react with various metals to form nitrosyl (-NO) complexes. The majority of these reactions are with metalloproteins containing iron, particularly in the form of heme. The best-known ferrous heme protein involved in cellular signaling known to interact avidly with NO is guanylyl cyclase, which catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) upon nitrosylation.

Another important target of NO involved in cell signaling are sulfhydryl (
SH) groups in L-cysteine residues of proteins and in glutathione or free Lcysteine itself. Covalent binding of NO to these RSH groups results in the formation of S-nitrosothiols (RSNOs), predominantly by reaction of the RSH groups with the oxidized metabolite of NO, N2O3 (reactions [1]–[3]): 2d NO þ O2 -2d NO2

½1

NO2 þd NO-N2 O3

½2

d

þ R-SH þ N2 O3 -R-SNO þ NO
2 þH

½3

RSNOs are more stable than NO and may function as a reservoir for storing or carrying NO. RSNOs may act as endogenous NO donors in the presence of cuprous ions (reaction [4]), which has been implicated as a mechanism by which RSNOs activate guanylyl cyclase. RS-NO þ CuI -RS
þd NO þ CuII

½4

RSNOs can also undergo transnitrosation (transfer of NO þ ), which may be an important mechanism for transfer of protein-bound NO. Many cysteinecontaining proteins, including enzymes, ion channels, and transcription factors, have been shown to be S-nitrosylated and their function modified, indicating that S-nitrosothiols have important biological activity. As a radical, NO reacts with other chemical species with unpaired electrons. One of the most common reactions of this type is the reaction of NO with O2, which has two unpaired electrons. This generates nitrogen dioxide NO2 (reaction [1]), a radical which may cause a variety of oxidation reactions. NO2 may react with water (reaction [5]) to form the relatively stable inactivation products of NO, nitrite and nitrate.
þ 2d NO2 þ H2 O-NO
2 þ NO3 þ 2H

½5

Nitrite is also formed from the NO2 reaction product N2O3 with water (reaction [6]). þ N2 O3 þ H2 O-2NO
2 þ 2H

½6

In addition, NO readily reacts with oxyhemoglobin or oxymyoglobin to give nitrate and the oxidized hemoproteins, methemoglobin, and metmyoglobin (reaction [7]). HbðFeðIIÞ-O2 Þ þd NO-metHbðFeðIIIÞÞ þ NO
3

½7

Due to high concentrations of oxyhemoglobin in the body, its reaction with NO is considered as a primary metabolic as well as primary inactivation mechanism for NO.

NITRIC OXIDE AND NITROGEN OXIDES 163

Particularly in inflammatory conditions, NO interacts with superoxide radical O2
in a near- diffusion controlled reaction to form the powerful oxidant, peroxynitrite (ONOO
) (reaction [8]). d


NO þd O
2 -ONOO

ONOO
þ Hþ -ONOOH þ ONOOH-d OH þd NO2 -NO
3 þH

½8

Regulation of Production and Activity

½9

NO Synthases

½10

At physiological pH, ONOO
leads to significant formation of the protonated species ONOOH (reaction [9]), which isomerizes to the highly reactive d OH and d NO2 radicals before decomposition to nitrate (reaction [10]). Both ONOO
and ONOOH cause a variety of reactions, including oxidation of thiols, lipid peroxidation, nitration of tyrosine, cleavage of DNA, and nitration or oxidation of guanosine and methionine. In a physiological milieu ONOO
will also react with CO2, which similarly leads to decomposition of the oxidant. As an intermediate ONOOCO2
is formed, which is a less oxidizing but more powerful nitrating agent than ONOO
itself (reaction [11]).


ONOO þ

CO2 -ONOOCO
2 -CO2

þ

NO
3

High concentrations of ONOO
and subsequent reaction products have been implicated in cytotoxicity and tissue damage during host defense reactions and inflammation in the respiratory tract.

NO is synthesized by nitric oxide synthase (NOS) enzymes, in a two-step reaction which converts the amino acid L-arginine into L-citrulline and NO (Figure 1). This reaction involves a five-electron oxidation of a guanidino nitrogen of L-arginine, requiring molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate (NADPH) as cosubstrates, as well as several cofactors such as flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), calmodulin, tetrahydrobiopterin (BH4), and heme (reactions [12] and [13]). L-arginine

þ O2 þ NADPH þ Hþ -

No -hydroxy-L-arginine þ NADPþ þ H2 O

½12

No -hydroxy-L-arginine þ O2 þ NADPH-

½11

L-citrulline

þd NO þ NADPþ þ H2 O

½13

L-arginine

CAT

Plasma membrane NOS Arginase NO

L-arginine L-ornithine

Urea

ASL

L-citrulline

ASS

ODC

L-aspartate

Argininosuccinate Fumarate OAT Putrescine Spermidine Spermine

Pyrrolidine-5-carboxylate L-proline

Figure 1 L-arginine uptake and metabolism in NO-producing cells. L-arginine is transported into the cell via cationic amino acid transporters (CAT) and can be metabolized by nitric oxide synthase (NOS) or arginase. NOS converts L-arginine to NO and L-citrulline. L-citrulline can be recycled to L-arginine in the citrulline-NO cycle, employing the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) with argininosuccinate as intermediate. The products generated by arginase are L-ornithine and urea. L-ornithine is converted by ornithine decarboxylase (ODC) to putrescine, which may be further metabolized to the polyamines spermine and spermidine. L-ornithine can also be converted to pyrrolidine-5-carboxylate by the enzyme ornithine aminotransferase (OAT). Pyrrolidine-5-carboxylate can be metabolized further to L-proline. It should be noted that these pathways are not necessarily coexpressed within the same cell type. Adapted from Ricciardolo FLM, Zaagsma J, and Meurs H (2005) The therapeutic potential of drugs targeting the arginase pathway in asthma. Expert Opinion on Investigational Drugs 14: 1221–1231.

164 NITRIC OXIDE AND NITROGEN OXIDES

Three different NOS isoforms have been identified, all of which are present in the respiratory tract: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS III). These isoforms are different gene products, all of them consisting of a single polypeptide chain comprising two domains: the C-terminal reductase domain containing NADPH, FAD, and FMN-binding sites and the N-terminal oxygenase domain containing the catalytic site involving heme, BH4, and L-arginine-binding sites. The reductase domain catalyzes electron transfer from NADPH via FAD and FMN to the heme that is responsible for reductive oxygen activation and L-arginine oxidation. A consensus sequence binding calmodulin links the reductase and oxygenase domains. Calmodulin binding allows the transfer of NADPH-derived electrons between the flavins and heme. nNOS and eNOS are constitutively expressed and therefore collectively called constitutive NOS (cNOS). These enzymes are primarily dependent on Ca2 þ to bind calmodulin, and produce low (picomolar) amounts of NO in response to depolarization- or receptor-mediated increase in intracellular Ca2 þ . This activation is rapid (within seconds) and short-lived, and the NO produced serves as a diffusible signaling molecule in local transcellular communication. Both cNOS isozymes may be associated with the plasma membrane. eNOS is N-myristoylated and palmitoylated, anchoring the enzyme into the membrane. eNOS is located in caveolae, where it is complexed in an inactive form with caveolin-1. Upon activation by Ca2 þ , eNOS complexes with calmodulin as well as with heat shock protein 90 (hsp90), causing dissociation from caveolin-1. Concomitantly, eNOS is phosphorylated by protein kinase B (Akt/ PKB), which contributes to its activation. Notably, association with hsp90 and Akt/PKB-induced phosphorylation activating eNOS may also occur in response to Ca2 þ -independent processes, such as shear stress. The amino acid terminal of nNOS possesses a PDZ domain, which may interact with membranedocking proteins like the postsynaptic density proteins PSD-95 and PSD-93, and a1-syntropin. In addition, stimulatory interactions with hsp90 and inhibitory interactions with caveolin-1 and caveolin-3 have been described for nNOS. iNOS does not require Ca2 þ elevation for its activation, calmodulin being constitutively bound to the enzyme at normal intracellular Ca2 þ concentration. Instead, iNOS activity is primarily regulated at the transcriptional level. iNOS is induced by various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-a), interleukin-1b (IL-1b) and interferon gamma (IFN-g), endotoxin, or oxidants,

either alone or in combination, often with strong synergism between these agents. Induction of iNOS has been associated with the activation of nuclear factor kappa B (NF-kB), a trancription factor that is implicated in the induction of multiple genes expressed in inflammatory responses. iNOS releases large (nanomolar) quantities of NO starting several hours after exposure to the inducing stimulus, which may sustain during prolonged periods of time (hours to days). Glucocorticoids inhibit the induction of iNOS, which involves inhibition of NF-kB. Of note, the expression of iNOS as well as of proinflammatory cytokines may also be inhibited by NO via inhibition of NF-kB by S-nitrosylation, acting as a negative feedback mechanism. Inhibition of iNOS expression is also induced by Th2 cytokines, such as IL-4, IL-10, and IL-13, and by transforming growth factor beta (TGF-b), involving posttranscriptional mechanisms. Moreover, translation of iNOS is controlled by the bioavailability of L-arginine, with low levels of the amino acid causing decreased translation of the enzyme. In contrast to the paradigm, there is evidence that iNOS may also be constitutively expressed as in airway epithelial cells, whereas cNOS isoforms may be under transcriptional control of various factors, including shear stress, vascular endothelial growth factor, TGF-b, IFN-g and estrogens. L-Arginine Homeostasis and Arginase

The bioavailability of L-arginine is a major determinant of NOS activity. L-arginine is a semi-essential amino acid, which is supplied by the diet and endogenously synthesized from L-citrulline mainly generated in the intestinal mucosa. The conversion of L-citrulline to L-arginine is mediated by successive actions of argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), which predominantly takes place in the kidney. The latter enzymes are also involved in the urea cycle in liver as well as in the citrulline-NO cycle in many, but not all, NO-producing cells, recycling L-citrulline to L-arginine for maintaining NO production (Figure 1). In most cells, L-arginine requirements are met primarily by uptake of extracellular L-arginine via specific cationic amino acid (CAT) transporters. The bioavailability of L-arginine to NOS is also importantly determined by the enzyme arginase, which hydrolyzes L-arginine to L-ornithine and urea (Figure 1). Arginase, classically an enzyme of the urea cycle in liver, is also found in many other cells or tissues that do not express a complete urea cycle, including the lung. Two distinct isoforms of mammalian arginase have been identified, which are

NITRIC OXIDE AND NITROGEN OXIDES 165

encoded by different genes and differ with respect to their tissue distribution, subcellular localization and mode of regulation. Type I arginase is a cytosolic enzyme, mainly expressed in liver and to a limited extent in other tissues, whereas type II arginase is a mitochondrial enzyme predominantly expressed in extrahepatic tissue. The role of extrahepatic arginase has not been fully elucidated, but possible metabolic roles of arginases include regulation of L-arginine availability for NO synthases and production of L-ornithine for the synthesis of polyamines and L-proline (Figure 1), which have been implicated in cell proliferation and fibrotic processes in wound healing. The expression of nonhepatic arginase I and arginase II is differentially regulated by a wide range of stimuli, including the Th2 cytokines IL-4 and IL-13, TGF-b, endotoxin and cAMP-elevating agents, as well as combinations thereof, and oxygen tension. Expression of particularly arginase I is under important control of the transcription factors STAT-6 and C/EBP. Moreover, the activity of both arginases is inhibited by No-hydroxy-L-arginine, an intermediate in the NO synthesis (see reaction [12]), which contributes to a delicate balance between NOS and arginase activity in the control of NO production. Regulation of NO Activity

NO is mainly deactivated by its reactions with O2 and oxyhemoglobin to form nitrite and nitrate (reactions [1], [2], [5], [6], and [7]). It should, however, be noted that nitrite may be converted to NO at low pH, as in the epithelial lining fluid of asthmatics (reactions [14], [15], and [16]) (see below also). þ NO
2 þ H -HNO2

½14

2HNO2 -N2 O3 þ H2 O

½15

N2 O3 -d NO þd NO2

½16

Under physiological conditions, NO reacts with low concentrations of O2
leading to formation of low levels of ONOO
(reaction [8]), which has little or no effect on cellular function and can thus be considered as an inactivation mechanism. Scavenging of O2
by superoxide dismutase (SOD) attenuates this inactivation process. Under pathophysiological conditions, where high concentrations of O2
are formed, subsequently generated high levels of ONOO
may lead to a variety of oxidative reactions and should thus not be seen as a mechanism of inactivation. As indicated above, NO activity is also conveyed through the covalent modification of L-cysteine sulfhydryl groups to form RSNOs (reaction [3]).

Biological Functions In the respiratory tract, isoforms of cNOS are mainly expressed in inhibitory nonadrenergic noncholinergic (iNANC) neurons (nNOS) and in epithelial (eNOS and nNOS) and endothelial (eNOS) cells, and are primarily involved in the neural and paracrine regulation of airway and/or vascular smooth muscle tone (Figure 2). The iNANC nervous system is the most effective bronchodilating neural pathway of the airways, which is principally mediated by the release of nNOS-derived NO upon depolarization. Furthermore, iNANC nerve-induced NO has dilatory effects on blood vessels of the pulmonary circulation and may be involved in inhibition of airway mucus secretion. Airway smooth muscle tone is also importantly regulated by non-neural NO derived from nNOS and eNOS in the airway epithelium. These enzymes are activated by various contractile agonists, including acetylcholine, histamine, bradykinin, and tachykinins, which dampens the contractile response to these agonists (Figure 2). Similarly, endothelial eNOS-derived NO has an important vasodilator role in the bronchial and pulmonary circulation and inhibits microvascular leakage at physiological conditions. In addition, eNOS-derived NO has been shown to inhibit airway inflammation by suppressing the activation of NF-kB, thereby inhibiting the expression of iNOS as well as the production of inflammatory cytokines, and by inhibition of endothelial adhesion and extravasation of leukocytes. There is evidence that both cNOS isozymes may be involved in developmental changes in the lung during fetal life. Under inflammatory conditions, iNOS may be expressed in a variety of pulmonary cells, including epithelial, endothelial, and vascular smooth muscle cells, fibroblasts and various inflammatory cells, such as alveolar macrophages, eosinophils, and neutrophils. Increased iNOS expression causing generation of high NO concentrations has been implicated in non-specific host defense mechanisms against pathogens and malignant cells as well as in chronic inflammatory diseases like asthma and COPD (Figure 2). High NO concentrations are cytotoxic or cytostatic to microorganisms and tumor cells, but may also be detrimental by destruction of healthy cells in the inflamed tissue. Mechanisms involved in cytotoxicity include inhibition of key enzymes of the mitochondrial respiratory chain and energy metabolism, mitochondrial Ca2 þ efflux, activation of apoptotic enzymes, lipid peroxidation, and DNA strand breakage with subsequent activation of poly (adenosine diphosphate-ribose) synthetase (PARS). Most of these mechanisms can be attributed to ONOO
, generated

166 NITRIC OXIDE AND NITROGEN OXIDES Agonist

Cytokine

L-arginine

L-arginine

cNOS

Ca2+

Transcription

iNOS

L-citrulline

L-citrulline

Epithelium

Agonist

Ca2+

Contraction

Inflammation, cytotoxicity

NO

NO

cGMP and KCa channel opening

NO Airway smooth muscle

nNOS

Ca2+

iNANC nerve

Relaxation Figure 2 Role of nitric oxide (NO) in airway reactivity and inflammation. Under basal conditions, small amounts of NO are synthesized by constitutive NO synthase (cNOS) isozymes present in the airway epithelium (endothelial eNOS and neuronal nNOS) and in inhibitory nonadrenergic noncholinergic (iNANC) nerves (nNOS). Activation of the cNOS isozymes by contractile agonists (epithelium) or depolarization (iNANC nerves) causes relaxation of the airway smooth muscle by increasing the production of cGMP and/or opening Ca2 þ -activated K þ (KCa) channels, thereby reducing airway responsiveness to contractile stimuli. Inducible NOS (iNOS) is induced in the epithelium by inflammatory cytokines and produces large amounts of NO. The resulting relaxation of airway smooth muscle is beneficial whereas the proinflammatory and cytoxic actions have detrimental effects. Reproduced from Meurs H, Maarsingh H, and Zaagsma J (2003) Arginase and asthma: novel insights into nitric oxide homeostasis and airway hyperresponsiveness. Trends in Pharmacological Sciences 24: 450–455, with permission from Elsevier.

by concomitant overproduction of NO and O2
under these conditions. As a positive feedforward response, high concentrations of iNOS-derived NO may promote the infiltration and activation of inflammatory cells into the airways, which may similarly involve ONOO
. Moreover, high concentrations of iNOS-derived NO contribute to the increased vascular permeability and mucus secretion observed in airway inflammation. High concentrations of NO also have immunomodulatory effects by inhibiting proliferation and activation of Th1, but not Th2, lymphocytes, which may perpetuate Th2 cell-mediated inflammatory responses in the airways as observed in asthma.

protein kinase (PKG). PKG-induced phosphorylation of target proteins mediate smooth muscle relaxation by reduction of the intracellular Ca2 þ -concentration, activation of hyperpolarizing Ca2 þ -dependent potassium (KCa) channels, and reduction of Ca2 þ -sensitivity of the contractile apparatus. Alternatively, cGMP-independent mechanisms of smooth muscle relaxation include activation of KCa channels and reduction of Ca2 þ sensitivity by S-nitrosylation of the effector molecules. Notably, ONOO
formed during oxidative stress may have a procontractile action, which involves oxidative inactivation of the KCa channel as well as of sarco/endoplasmatic reticulum Ca2 þ -ATPase type 2.

Signaling Mechanisms

Nitrogen Oxides in Repiratory Diseases

There is no conventional receptor for NO. Many of the NO-mediated signaling processes involved in smooth muscle relaxation, modulation of gene transcription, and many other biological processes proceed via chemical nitrosylation of heme or sulfhydryl groups in target proteins. Thus, NO-dependent relaxation of airway and vascular smooth muscle is importantly mediated via binding of NO to the heme prosthetic group of soluble guanylyl cyclase, thereby increasing the production of cGMP, which acts as a second messenger via activation of cGMP-dependent

Altered NO homeostasis has been implicated in the pathophysiology of various respiratory diseases, including asthma, COPD, cystic fibrosis, and pulmonary hypertension. To date, most studies have focused on the pathophysiology of asthma. Asthma

In asthmatic airways, there is marked upregulation of iNOS expression, particularly in the airway epithelium and in inflammatory cells, including macrophages, eosinophils, and neutrophils, which is

NITRIC OXIDE AND NITROGEN OXIDES 167

associated with increased concentrations of NO in exhaled air. Significant correlations between exhaled NO, iNOS expression in airway epithelial and inflammatory cells, airway eosinophilia, and airway hyperresponsiveness (AHR) have been observed in asthmatics, whereas all are reduced after glucocorticosteroid treatment. Moreover, exhaled NO is increased during the allergen-induced late asthmatic reaction, which is associated with induction of iNOS. Based on these observations, the NO concentration in exhaled air is considered to be a useful marker of asthmatic airway inflammation. Moreover, iNOS expression in airway epithelial and inflammatory cells in bronchial biopsies of asthmatic patients has been associated with increased nitrotyrosine expression in these cells. The ONOO
expression was similarly correlated with exhaled NO and AHR, and sensitive to glucocorticoids, suggesting a major role for ONOO
in iNOS-induced asthmatic airway inflammation. However, elevation of expired NO may at least partially be explained by airway acidification of the airway lining fluid in asthmatics, by protonation of nitrite to form nitrous acid, which is converted to NO and NO2 (reactions [14], [15], and [16]). Given the variety of enzymatic and cellular sources of NO in the airways, the different cellular targets and physiological effects of NO, as well as the influence of the local microenvironment on NO homeostasis, the relevance of increased exhaled NO to discrete pathophysiological processes in the airway wall may be difficult to determine. This is strikingly illustrated by a deficiency of bronchodilating cNOS-derived NO as well as of S-nitrosoglutathione (GSNO) in the airways of patients with asthma, both of which may contribute to AHR in these patients. Mechanisms of reduced cNOS activity have implicated reduced protein expression of both nNOS and eNOS, which may occur after repeated allergen exposition. In addition, alterations in L-arginine homeostasis, causing reduced bioavailability of the substrate to cNOS, appear to play a key role in allergen-induced AHR. Recent animal studies have indicated that polycationic proteins, including eosinophil-derived major basic protein, inhibit the cellular uptake by CAT transporters of L-arginine in alveolar macrophages and airway epithelial cells, which may contribute to deficiency of cNOS-derived NO and AHR after the early asthmatic reaction. A second mechanism that might be crucial is reduced bioavailability of L-arginine caused by increased activity of arginase. Types I and II arginases are both expressed constitutively in the airways, particularly in the bronchial epithelium and in fibroblasts from the peribronchial tissue. In guinea pigs, it has been demonstrated that arginase activity is

functionally involved in basal airway responsiveness by limiting cNOS-derived NO production. In different models of asthma, it has been demonstrated that allergen challenge causes a considerable increase in the expression and activity of arginase type I particularly, most likely via Th2 cytokines involved in the asthmatic airway inflammation. Increased arginase activity in allergen-challenged guinea pigs contributed to AHR as well as reduced iNANC relaxation of isolated airway preparations obtained after the early asthmatic reaction. Recent studies in asthmatic patients have also indicated increased expression of arginase I in the airways, particularly in epithelial and inflammatory cells. Moreover, in asthmatics experiencing an exacerbation, a striking reduction in plasma L-arginine levels is measured, which is associated with an increase in serum arginase activity. Recent evidence suggests that reduced L-arginine availability to iNOS, induced by increased arginase activity as well as reduced transport of the amino acid, may lead to synthesis of both NO (via the oxygenase moiety) and O2
(via the reductase moiety and normally suppressed by physiological L-arginine concentrations) by this enzyme. This may effectively contribute to the allergen-induced formation of ONOO
in the airways, and has shown to contribute to the AHR after the late asthmatic reaction. It has been hypothesized that increased synthesis of L-ornithine by arginase may contribute to inflammation-induced airway remodeling in chronic asthma. L-ornithine is a precursor of polyamines and L-proline, which may promote proliferation of structural subepithelial cells (fibroblasts and smooth muscle cells) and collagen deposition, respectively. Together with its NO-dependent effects, this would indicate a central role of arginase in the pathophysiology of acute and chronic asthma (Figure 3). Chronic Obstructive Pulmonary Disease

Using the current single expiratory flow method predominantly monitoring larger airway-derived NO, exhaled NO is often in the normal range or even reduced in mild to moderate COPD, probably due to downregulation of eNOS and iNOS by cigarette smoke. Recently, methods for measuring exhaled NO at multiple expiratory flows have indicated that COPD is associated with elevated alveolar NO, reflecting inflammation of the peripheral lung. This corresponds well with increased expression of iNOS in alveolar walls, small airway epithelium, and vascular smooth muscle, and sputum macrophages of COPD patients. Cigarette smoke imposes considerable oxidative stress on the airways, due, in part, to a considerable increase in activated neutrophils and

168 NITRIC OXIDE AND NITROGEN OXIDES L-arginine

IL-4, IL-13

IFN-
, IL-1, TNF- iNOS

Arginase Agonist L-ornithine

Putrescine

+ urea

NO + O2–

cNOS

L-proline

NO

ONOO –

Spermidine Collagen Spermine

Enhanced smooth muscle contractility, inflammation, cytotoxicity

Proliferation

Airway remodeling Figure 3 Postulated role of arginase in airway hyperresponsiveness, inflammation, and remodeling in asthma. Induction of increased arginase activity by Th2 cytokines reduces L-arginine availability to cNOS and iNOS, thereby reducing the production of bronchodilating NO by these enzymes and inducing the production of superoxide anion (O2
) by iNOS. By the rapid reaction of NO with O2
, peroxynitrite (ONOO
) is formed, which has proinflammatory, cytotoxic, and procontractile actions in the airways. Furthermore, the increased synthesis of L-ornithine by arginase provides a precursor for polyamines (putrescine, spermidine, and spermine) and L-proline, which stimulate cell growth and collagen synthesis, respectively. Therefore, arginase might also be involved in inflammation-induced airway remodeling. Reproduced from Meurs H, Maarsingh H, and Zaagsma J (2003) Arginase and asthma: novel insights into nitric oxide homeostasis and airway hyperresponsiveness. Trends in Pharmacological Sciences 24: 450–455, with permission from Elsevier.

alveolar macrophages. This could account for the strongly increased ONOO
generation observed in sputum macrophages of COPD patients. Moreover, this may render these cells insensitive to glucocorticoids by nitration and inactivation of histone deacetylase 2. Cystic Fibrosis

Even though cystic fibrosis (CF) is an inflammatory disease of the airways, NO levels are paradoxically decreased. The mechanisms leading to low airway NO in CF are not well understood, but may include reduced expression of iNOS, polymorphisms of nNOS and eNOS, reduced substrate availability to NOS, mechanical retention of NO in airway secretions, increased metabolism by oxidative processes associated with the formation of ONOO
, and consumption of NO by denitrifying bacteria. Reduced NO in the airways of CF patients might contribute to microbial infection and colonization. In a murine model of cystic fibrosis, a reduced relaxation of electrical field-stimulated-induced airway smooth muscle contraction was found, which was reversed by L-arginine and NO. This indicates that a relative deficiency of NO due to substrate limitation to cNOS compromises airways relaxation and

contributes to bronchial obstruction. Indeed, in patients with cystic fibrosis a positive correlation was found between pulmonary function and exhaled NO and NO metabolite concentration in sputum. Pulmonary Hypertension

Reduced levels of NO have also been observed in lungs of patients with pulmonary arterial hypertension (PAH). A role for altered NOS expression, in particular of eNOS in endothelial cells, is still controversial. Abnormalities in L-arginine metabolism have been found in patients with primary and secondary forms of hypertension, as reflected by low levels of plasma L-arginine. In infants with neonatal pulmonary hypertension, this may imply an inactive form of carbamoyl-phosphate synthetase, the ratelimiting enzyme of the urea cycle. Increased arginase activity in the serum has been associated with reduced plasma L-arginine levels in patients with primary pulmonary hypertension (PPH) and in patients with sickle cell anemia and associated secondary pulmonary hypertension. Moreover, increased arginase II expression has been observed in patients with PPH. Both L-arginine supplementation and inhalation of NO have shown to be of potential benefit in the treatment of PAH.

NOTCH 169 See also: Arteries and Veins. Asthma: Overview. CD1. Cystic Fibrosis: Overview. Diffusion of Gases. Endothelial Cells and Endothelium. Endotoxins. Environmental Pollutants: Overview. Systemic Disease: Sickle Cell Disease. Vascular Disease.

Further Reading Barnes PJ, Ito K, and Adcock IM (2005) Corticosteroid resistance in chronic obstructive pulmonary disease: inactivation of histone deacetylase. Lancet 363: 731–733. Boucher JL, Moali C, and Tenu JP (1999) Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for L-arginine utilization. Cellular and Molecular Life Sciences 55: 1015–1028. Colasanti M and Suzuki H (2000) The dual personality of NO. Trends in Pharmacological Sciences 21: 249–252. Grasemann H and Ratjen F (1999) Cystic fibrosis lung disease: the role of nitric oxide. Pediatric Pulmonology 28: 442–448. Hammermann R, Hirschmann J, Hey C, et al. (1999) Cationic proteins inhibit L-arginine uptake in rat alveolar macrophages and tracheal epithelial cells. Implications for nitric oxide synthesis. American Journal of Respiratory Cell and Molecular Biology 21: 155–162. Meurs H, Maarsingh H, and Zaagsma J (2003) Arginase and asthma: novel insights into nitric oxide homeostasis and airway hyperresponsiveness. Trends in Pharmacological Sciences 24: 450–455. Miranda KM, Espey MG, Jourd’heuil D, et al. (2000) The chemical biology of nitric oxide. In: Ignarro LJ (ed.) Nitric Oxide,

Biology and Pathobiology, pp. 41–55. San Diego: Academic Press. Morris CR, Poljakovic M, Lavrisha L, et al. (2004) Decreased arginine bioavailability and increased serum arginase activity in asthma. American Journal of Respiratory and Critical Care Medicine 170: 148–153. Morris SM Jr (2004) Recent advances in arginine metabolism. Current Opinion in Clinical Nutrition and Metabolic Care 7: 45–51. Ricciardolo FLM, Sterk PJ, Gaston B, and Folkerts G (2004) Nitric oxide in health and disease of the respiratory system. Physiological Reviews 84: 731–765. Ricciardolo FLM, Zaagsma J, and Meurs H (2005) The therapeutic potential of drugs targeting the arginase pathway in asthma. Expert Opinion on Investigational Drugs 14: 1221–1231. Saleh D, Ernst P, Lim S, Barnes PJ, and Giaid A (1998) Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid. FASEB Journal 12: 929–937. Wu G and Morris SM Jr (1998) Arginine metabolism: nitric oxide and beyond. Biochemical Journal 336: 1–17. Xu W, Kaneko FT, Zheng S, et al. (2004) Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension. FASEB Journal Published online September 13, 2004. Zimmermann N, King NE, Laporte J, et al. (2003) Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis. Journal of Clinical Investigation 111: 1863–1874.

NOTCH R Boyton and D M Altmann, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The Notch family of transmembrane receptors are a highly evolutionarily conserved set of molecules that interact with two key families of ligands, Delta and Serrate/Jagged, to perform a broad range of functions at switch points of cell fate determination. In mammals, there are four Notch receptors (Notch-1 through -4) and five ligands (Jagged-1 and -2 and Delta-1, -3, and -4). The ligand–receptor interaction leads to proteolytic cleavage of the receptor, liberating the cytoplasmic domain for translocation to the nucleus and interaction with transcription pathways. Signals transmitted through Notch affect differentiation, proliferation, cell-cycle arrest, border formation, and apoptosis at all stages of development, from embryonic development to cell fate decisions in differentiated cell lineages. As such, Notch signaling impinges on several body systems. Its effects in diverse biological systems are fundamental and wide reaching, and many of its functions are not yet determined. There are several controversial areas under investigation, including the role of Notch in developmental biology and T-cell immunology, making it an exciting subject for current scientific

debate where there are still many unknowns. Examples of the possible influences of Notch expression in the respiratory system include its effects on the development of lung tissue and on polarization of T-lymphocyte effector function in the immune system. In oncogenesis including lung cancer, Notch may act either as a tumor oncogene or as a tumor suppressor gene, depending on the specific cellular context.

Introduction Notch was initially identified in 1917 in a strain of Drosophila melanogaster (fruit fly) as the gene associated with a phenotype involving notches at the wing margin. Notch family members have now been identified in species spanning from Candida albicans to humans. Notch interactions have a fundamental, conserved role with respect to communication between cells in developing tissue. Notch interactions act as gatekeepers regulating differentiation switch points, such that cells receiving a Notch signal assume one fate and those that do not assume another. Thus, it was demonstrated in the 1930s that Notch mutations could produce a neurogenic phenotype,

170 NOTCH

such that cells destined to become epidermis change fate and turn into neural tissue. It is not just the Notch receptors that are highly conserved in evolution but also their receptors and the signaling pathway by which Notch receptors undergo regulated proteolysis and nuclear translocation to activate transcription. Knockout mouse strains for Notch family members and ligands commonly result in an embryonic lethal phenotype with defects in diverse tissues. Considerable research effort has been devoted to analysis of the consequences of Notch signaling in the developing and mature immune system. Examples of this are the analysis of T-lymphocyte differentiation into T-helper-1 (Th1) or T-helper-2 (Th2) effector cell phenotypes and into effector versus regulatory phenotypes. Notch research in the lung has focused both on the influence of expression on normal development of the lung and on expression and function of Notch in cancer.

the trans-Golgi network by proteases of the Furin family. Notch accumulates at the cell membrane as a heterodimer of an ectodomain and a membrane-linked intracellular domain. Modulation of glycosylation can affect Notch binding to its ligands. For example, the addition of N-acetylglucosamine, mediated by a family of glycosyl transferases termed fringe proteins, is permissive for Notch signaling to Delta ligands but not for signaling to Jagged ligands. Ligand binding leads to two proteolytic cleavage steps. Following ligand binding, Notch becomes sensitive to cleavage by extracellular proteases of the ADAM/TACE/Kuzbanian family. The released, tethered Nic (intracellular Notch) can then be recognized by the g-secretase complex, including presenelin and nicastrin. This cleavage step releases soluble Nic for translocation to the nucleus. There, it binds the transcription factor CSL/RBP-J. With Mastermind, Nic and CSL/RBP-J generate a large, multiprotein complex, recruiting other coactivators.

Structure The Notch gene encodes a 300 kDa transmembrane receptor. The extracellular domain encompasses a variable number of tandem epidermal growth factorlike repeats (up to 36 in various species) and three cysteine-rich Notch/LIN-12 repeats (see Figure 1). The human Notch-1 gene is at 9q34, Notch-2 at 1p13, Notch-3 at 19p13, and Notch-4 at 6p21. Jagged-1 is at 20p12.

Regulation of Production and Activity Notch receptors and their ligands are widely expressed in several cell types and tissues. Notch molecules are processed from a single precursor protein in

Ankyrin repeats

Transactivation domain

Notch interactions impact on a wide variety of developmental cell fate decisions in all species in which they have been studied. Knockout mice have been generated for each of the mammalian Notch ligands and receptors, and all are embryonic or perinatal lethal, reflecting the fundamental role of these interactions in common pathways of cellular differentiation and cell fate branch points. Notch-1-deficient mice die by day 11 of gestation due to defects in somitogenesis and vasculogenesis, and Jagged-2 knockouts succumb to perinatal death with craniofacial and limb defects. Functional experiments have subsequently depended on the use of conditional knockouts as well

Common N-terminal domain DSL (Delta, Serrate, and Lag)

Notch

Pest sequence

Biological Function

Nuclear localization signal LIN EGF-like repeats

Cysteine-rich domain

Jagged

EGF-like repeats

RAM domain

Figure 1 Notch ligand–receptor interaction (interaction between Notch-1 and Jagged) showing generic structural features of intercellular signaling. Notch-3 and -4 lack the indicated transactivation domain. The ligands for the mammalian Notch receptors are Jagged1 and -2 and Delta-1, -3, and -4.

NOTCH 171

as transfection experiments. A large number of studies have investigated Notch interactions in hematopoietic and immune cell differentiation. An example of this is the demonstrated influence of Notch-1 signaling on the development of a normal T-lymphocyte compartment: loss of Notch-1 function leads to poor thymic development, lacking T lymphocytes and with increased B lymphocytes. Once cells have entered the T-lymphocyte lineage, Notch signaling seems to also influence the switch point between the T-cell receptor ab and gd lineages, with higher levels of Notch signaling favoring entry into the ab lineage. Notch ligands and receptors are expressed by peripheral T cells and antigen presenting cells, and antigenic stimulation of naı¨ve T-cells causes substantial upregulation of Notch-1 through -4. Much evidence points to a role for Notch interactions in the T-cell response to antigen. A key focus of research has been to determine the influence of Notch interactions on functional polarization of T-cell responses into the Th1 or Th2 effector programs. Delta-1/Notch-3 interactions may promote Th1 effector function. Furthermore, the Jagged/Notch pathway may permit early IL-4 release, leading to Th2 differentiation. A large number of experiments address the influence of Notch interactions on the decision point of activation versus anergy or tolerance, including the induction of regulatory T cells. Human CD4 þ CD25 þ regulatory T cells express Notch-4. Expression of Jagged-1 on antigen presenting cells causes peripheral T lymphocytes to differentiate into regulatory cells. Analysis of the influence of Notch signaling on lung development has been largely descriptive. Notch regulation is believed to underpin the binary fate determination between endocrine and nonendocrine airway epithelial cells. Notch signaling appears essential for the lung to achieve its normal size: mice carrying a targeted disruption for the downstream hairy enhancer of split (Hes)-1 transcription factor show a 20% reduction in lung length. This is accounted for by a reduction in Clara cells in the small and medium-sized airways. Notch-2 and -3, but not Notch-1, are expressed in lung mesenchyme. Notch4 expression appears to be endothelial specific in the lung. An alternative approach has been to use antisense oligonucleotides for Notch or Jagged family members to manipulate branching morphogenesis and neuroendocrine cell differentiation during murine lung development. Branching of embryonic lung buds increased when they were treated with Notch-1 antisense oligonucleotides, indicating a likely role for Notch at this branch point. Another key developmental switch point over which Notch/Jagged interactions exert a gatekeeper role is vasculogenesis. In detailed analysis of Notch and Jagged expression

during vascular development of mouse embryonic lung, it is found that unlike other organs studied, the developing lung shows a pattern of increasing endothelial cell-specific Notch-1/Jagged-1 expression. This is seen initially on well-formed, larger vessels in the embryonic lung bud and then on finer vascular networks.

Receptors The mammalian Notch receptors are the Serrate homologs Jagged-1 and -2 and Delta-1, -3, and -4.

Notch in Respiratory Diseases It is likely that ongoing studies of the influence of Notch on T-helper-cell differentiation may impinge on the analysis of diseases of lung inflammation. Studies have revealed a complex pattern of Notch influence on Th1/Th2 polarization and on differentiation of regulatory T cells. Thus, although there is hope in the field that manipulation of Notch-governed differentiation switch points may ultimately lead to novel therapeutics capable of modulating, for example, inappropriate Th2 immunity in asthma, such developments are still awaited. Other respiratory disease-related research focuses on the expression of Notch in lung cancer. Much data have been reported on the measurement of Notch ligands and receptors in cancer cell lines. Notch function in lung cancer shows properties of both tumor inhibition and promotion. In non-small cell lung cancer, Notch is believed to have a tumor growth-promoting function. Chromosome 1p alterations are relatively common in lung cancer, and detailed mapping studies of the amplified region at 1p36.12 show the affected region to encompass the NOTCH-2 gene. This is associated with overexpression of Notch-2 in the tumors carrying those amplifications. Although the role of these changes, if any, in tumorigenesis remains to be determined, the findings indicate the possible impact of therapeutic manipulation of Notch signaling. See also: Clara Cells. Leukocytes: T cells. Lung Development: Overview.

Further Reading Amsen D, Blander JM, Lee GR, et al. (2004) Instruction of distinct CD4 T helper cell fates by different Notch ligands on antigen presenting cells. Cell 117: 515–526. Artavanis-Tsakonis S, Rand MD, and Lake RJ (1999) Notch signalling: cell fate control and signal integration in development. Science 284: 770–776. Baron M (2003) An overview of the Notch signalling pathway. Seminars in Cell and Developmental Biology 14: 113–119.

172 NUTRITION IN RESPIRATORY DISEASE Collins BJ, Kleeberger W, and Ball DW (2004) Notch in lung development and lung cancer. Seminars in Cancer Biology 14: 357–364. Dallman MJ, Smith E, Benson RA, and Lamb JR (2005) Notch: control of lymphocyte differentiation in the periphery. Current Opinion in Immunology 17: 259–266. Garnis C, Campbell J, Davies JJ, et al. (2005) Involvement of multiple developmental genes on chromosome 1p in lung tumorigenesis. Human Molecular Genetics 14: 475–482. Lehar SM and Bevan MJ (2004) Polarizing a T cell response. Nature 430: 150–151. Maillard I, Adler SH, and Pear WS (2003) Notch and the immune system. Immunity 19: 781–791. Maillard I, Fang T, and Pear WS (2005) Regulation of lymphoid development, differentiation and function by the Notch pathway. Annual Review of Immunology 23: 945–974.

Nye JS (1999) The Notch proteins. Current Biology 9(4): R118. Radtke F, Wilson A, Mancini JC, and Robson MacDonald H (2004) Notch regulation of lymphocyte development and function. Nature Immunology 5: 247–253. Radtke F, Wilson A, and Robson MacDonald H (2004) Notch signalling in T and B cell development. Current Opinion in Immunology 16: 174–179. Robey E (1999) Regulation of T cell fate by Notch. Annual Review of Immunology 17: 283–295. Robey EA and Bluestone JA (2004) Notch signalling in lymphocyte development and function. Current Opinion in Immunology 16: 360–366. Schweisguth F (2004) Regulation of Notch signalling activity. Current Biology 14: R129–R138.

NUTRITION IN RESPIRATORY DISEASE I Romieu, Instituto Nacional de Salud Publica, Cuernavaca, Morelos, Mexico & 2006 Elsevier Ltd. All rights reserved.

Abstract The role that dietary factors may play in the oxidative process and in inflammatory response has generated growing interest because they may impact the genesis and evolution of lung diseases, particularly lung cancer, chronic obstructive lung disease, and asthma. Dietary factors in this category include fruits and vegetables; antioxidant vitamins such as vitamin C, vitamin E, b-carotene and other carotenoids, vitamin A, and fatty acids; and some minerals, such as sodium, magnesium, and selenium. Intake of fresh fruits and some vegetables appears to have a beneficial effect on lung health and their consumption on a daily basis should be recommended. Antioxidant vitamins, particularly vitamin C and, to a lesser extent, vitamin E, and flavonoids appear to have a protective effect against lung diseases. However, the few existing intervention studies have not been conclusive. Supplementation of vitamin C and other antioxidants may be proposed in subjects who experience additional oxidative stress challenge, such as exposure to high levels of air pollution.

Introduction Several lung diseases have been associated with oxidative stress and linked to oxidant insults such as cigarette smoke, air pollutants, and infections. Consequently, dietary factors and nutrients that play a potentially protective role against the oxidative process and inflammatory response have been implicated as preventive measures for detaining the genesis or evolution of these diseases. These nutrients include fruits and vegetables; antioxidant vitamins such as vitamin C, vitamin E, b-carotene and other carotenoids, vitamin A, and fatty acids; and some minerals, such

as selenium. Other nutrients, such as sodium and magnesium, may play an important role in the activities of airway smooth muscle and enzymatic reactions that affect neuromuscular transmission. Thus, an adequate diet may inhibit, arrest, or even reverse the chain of events caused by toxicity, whereas a deficient diet may increase susceptibility to adverse environmental exposure. This chapter examines the relationship between the most prevalent obstructive lung diseases, including lung cancer, asthma, and chronic obstructive pulmonary disease (COPD), and dietary factors frequently implicated in the genesis or severity of these diseases. The specific topic of food allergy is not addressed here.

Role of Nutrients Antioxidants

Antioxidants in the lung are the first line of defense against oxygen free radicals and include endogenous enzyme systems and nonenzymatic antioxidant compounds. Nonenzymatic antioxidants, such as vitamin C (ascorbic acid), vitamin E (a-tocopherol) b-carotene (a precursor of vitamin A), ubiquinone, flavonoids, and selenium, are present in foods and may therefore play a role in the host’s defense against oxidative lung damage. Vitamin C, a water-soluble vitamin, contributes to antioxidant activity through several mechanisms, including the scavenging of superoxide radical O2. It appears to be the most abundant antioxidant substance in the extracellular fluid lining of the lung and also contributes to the regeneration of membrane-bound oxidized vitamin E. Vitamin C also plays a role in immune function

NUTRITION IN RESPIRATORY DISEASE 173

and is transported into neutrophils and lymphocytes. Vitamin E, a lipid-soluble vitamin, represents the principal defense against oxidant-induced membrane injury in human tissue because of its role in breaking the lipid peroxidation chain reaction. b-Carotene, a precursor to vitamin A, accumulates in tissue membranes, scavenges O2
, and reacts directly with peroxyl free radicals. Flavonoids such as quercetin are scavengers of superoxide anions and peroxyl radicals. Selenium is an essential trace element and plays a role in the detoxification of peroxides and free radicals (Table 1). Omega-3 Polyunsaturated Fatty Acids

Dietary intakes of polyunsaturated fatty acids (PUFAs), primarily found in diets rich in seafood, have been shown to play a role in lung diseases and in the normal aging process of the lungs. Increased intake of omega-3 PUFA can decrease the inflammatory reaction by changing the contents of lipid membranes and other substrates, which are in turn the substrates for eicosanoid production. Briefly, the substitution in the membrane of n-3 PUFA (a-linoleic acid (ALA 18:3n3) and eicosapentaenoic acid (EPA 20:5n-3)) and for n-6 fatty acids (linoleic acid (LA; 18:2n-6), these being the most abundant in the Western diet), leads to the production of fewer inflammatory mediators (prostaglandin E3 (PGE3) instead of PGE2 and leukotriene 5 (LTB5) instead of LTB4). PGE2 has been shown to act on T lymphocytes to reduce the formation of interferon gamma (IFN-g) without affecting the formation of interleukin-4 (IL-4). This may lead to the development of allergic sensitization since IL-4 promotes the synthesis of immunoglobulin E (IgE), whereas IFN-g has the opposite effect. LTB4, a potent stimulator of airway smooth muscle cells, increases postcapillary vascular permeability and mediates pulmonary asthma because of their involvement in vasoconstriction and mucus secretion. The competitive interactions between n-6 PUFA and n-3 PUFA determine the cellular contents of arachidonic acid (AA) and EPA. Higher concentrations of EPA were found in diets low in LA. In the Western diet, 20–25 times more n-6 PUFA is consumed than n-3 PUFA. The change in dietary habits in developed countries during the past few decades may therefore partly explain the increase in the prevalence of asthma. Minerals

Certain minerals, such as sodium and magnesium, have also been implicated in the development of airway diseases because of their effect on the airway smooth muscle cells. The contractile response of airway smooth muscle cells to specific antigens was

demonstrated to be dependent on the level of hyperpolarization resulting from sodium influx. Magnesium has been shown to dilate asthmatic airways in vivo, to inhibit cholinergic neuromuscular transmission, to stabilize mast cells and T lymphocytes, and to stimulate the generation of nitric oxide and prostacyclin. A diet rich in sodium and low in magnesium may therefore constitute a risk factor for airway diseases.

Sources of Specific Nutrients and Reference Daily Intake The concentration of specific nutrients varies widely in different foods. The Recommended Daily Intake (RDI) is defined as the amount of nutrients that most individuals should ingest to achieve an optimal body function and to minimize the risk of disease. Antioxidants

Fresh fruits and vegetables contain considerable amounts of vitamin C (e.g., broccoli, spinach, tomatoes, and citrus fruits) and carotenoids (e.g., carrots, tomatoes, grapefruits, beans, broccoli, oranges, and mangos). Vitamin C is mainly present in citrus fruits and some vegetables. The main carotenoids serving as pro-vitamin A, a- and b-carotene and cryptoxanthin, may be transformed into vitamin A. The richest sources of vitamin E in the human diet are oil products such as mayonnaise, vegetable and seed oil (corn, safflower, and soybean), butter, and eggs. Flavonoids are mainly found in fruits and vegetables (apples, lemons, oranges, potatoes, and cauliflower) as well as tea. Selenium is found in grain, meat, seafood, and certain vegetables. The current RDI requirements for vitamin C are 75 mg day
1 for women (100 mg day
1 for smokers) and 90 mg day
1 for men (125 mg day
1 for smokers), with an upper safety limit of 2000 mg day
1. It is important to note that the RDI for smokers is 35 mg day
1 higher than that for nonsmokers because of the additional oxidant exposure caused by smoking. This recommendation must also apply to subjects exposed to high oxidative factors such as ozone. The RDI for vitamin A is 800–1000 retinol equivalents (RE) day
1 or 4000–5000 IU day
1 (1 RE ¼ 6 mg b-carotene). There is no RDI for b-carotene but the recommended intake is 15 mg day
1. The RDI for vitamin E is 15–20 mg day
1 (a-tocopherol equivalent, 22–30 IU natural), and the Recommended Dietary Allowance is 55–70 mg for selenium. Omega-3 Polyunsaturated Fatty Acids

The major food sources of omega-3 PUFA are fish oils, fish (mostly fatty), and shellfish, and for

174 NUTRITION IN RESPIRATORY DISEASE Table 1 Nutrients linked to obstructive lung disease Nutrient

Mechanisms

Dietary sources

Comments

Vitamin C (ascorbic acid)

Water-soluble antioxidant Scavenges O2
Regenerates oxidized vitamin E Present in neutrophils and lymphocytes

Fruits: papaya, canteloupe, citrus fruits, strawberries Vegetables: cauliflower, broccoli, brussels sprouts, kale, sweet peppers

Humans are unable to synthesize vitamin C. Recommended dietary allowance (RDI): 75 mg day
1 in women and 90 mg day
1 in men Large doses of vitamin C are usually well tolerated (2000 mg day
1). Doses above this range may result in nausea and diarrhea.

Vitamin E (a-tocopherol)

Lipid-soluble antioxidant Reacts with peroxyl radicals to terminate membrane lipid peroxidation

Vegetable and seed oils Eggs Green vegetables

Level of vitamin E in food best correlates with the level of unsaturated fat. RDI: 15 mg day
1 In adults 200–400 mg day
1 is tolerated without adverse effect with the exception of gastrointestinal upset. With doses of 800–1200 mg day
1, antiplatelet effect and bleeding may occur. Doses over 1200 mg day
1 may result in adverse effects such as nausea, headache, fatigue, and diarrhea.

b-Carotene, lycopene, and other carotenoids

Lipid-soluble antioxidants React with peroxyl free radicals, decreasing lipid peroxidation Scavenge O2
Growth regulation of malignant cells (via vitamin A effects)

Red, orange, and yellow fruits and vegetables: sweet potato, carrots, winter squash Green vegetables

There are more than 600 carotenoids. Antioxidant and pro-vitamin A activity (transformed to retinol (vitamin A) by cleavage of central bond) Antioxidant activity of carotenoids may be more important in environmental lung disease than the pro-vitamin A effects. No RDI; recommended intake is 15 mg day
1. Carotenoid toxicity includes carotenodermia (yellowing of the skin) and nausea and diarrhea.

Vitamin A

Lipid soluble Differentiation of epithelial cells Growth regulation of malignant cells Reproduction Vision

Liver Egg yolk Milk fat Fish oils Pro-vitamin A carotenoids

Vitamin A status is very dependent on adequate intake of proteins, calories, and zinc. The synthesis of retinol binding protein (RBP) is Zn dependent. RDI: 4000/5000 IU (or 800/1000 mg) day
1 for men and women Additional 1000 IU recommended during pregnancy and lactation. Toxicity: skin erythema, desquamation, abdominal pain, nausea. In pregnant women, excessive intake (over 18 000 IU) in early pregnancy could lead to birth defects.

Flavonoids (most frequent in food): quercetin, flavone, kaempherol, rutin, hesperidin, hesperitin

Hydrosoluble Scavenging lipid peroxyl radicals Scavenger O2
, HOd , FeðOHÞd3 Bind metal ions

Apples, lemons, oranges Potatoes, cauliflower Tea Skin of tubers and roots

More than 3000 flavonoids have been identified in plants. No RDI

NUTRITION IN RESPIRATORY DISEASE 175 Table 1 (Continued ) Nutrient

Mechanisms

Dietary sources

Comments

Inhibitor of enzymatic systems responsible for free radical production Antimutagenic effect

Red wine

Omega-3 polyunsaturated fatty acids

Decreased leukotriene synthesis Inhibition of PGE2 synthesis Growth regulation of malignant cells

Fish oils Fish and shellfish Soy and canola oil Leafy vegetables

Ratio of n-3:n-6 fatty acids in diet may be more important than absolute consumption levels. No RDI Effective dose: 4 g day
1 for suppression of inflammation

Magnesium

Cofactor in enzyme activation reactions requiring ATP Bronchodilator of airway smooth muscle Inhibits cholinergic neuromuscular transmission Stabilizes mast cells and T lymphocytes

Nuts, legumes Cereal grains Corn, peas, carrots, parsley, spinach, lima beans Brown rice Seafood

RDI: 300–350 mg day
1

Selenium

Cofactor for glutathione peroxidase, which reduces lipid and hydrogen peroxides Detoxification of heavy metals Role in DNA repair

Animal products, especially organ meats Seafood

Foods grown in selenium-poor soils may contribute to selenium deficiency in humans. Vitamins C, E, and A enhance selenium absorption. Mercury and other heavy metals inhibit selenium absorption. RDI: 55–70 mg day
1 Reference dose (daily intake without lifetime adverse effect): 0.05 mg kg
1 day
1 or 350 mg day
1 for men of 70 kg Toxicity: loss of hair and nails

Adapted from Romieu I and Trenga C, Diet and obstructive lung diseases, Epidemiologic Reviews, 2001, vol. 23, No. 2, p. 272, by permission of Oxford University Press.

monounsaturated oils the sources are canola and flaxseed oils and leafy vegetables, whereas omega-6 PUFA is found in polyunsaturated fats, such as vegetable oils (soybean, corn, safflower, and sunflower) as well as margarine, mayonnaise, and food processed with oil. There is no RDI for omega-3 PUFA. Mineral

Magnesium in the diet is obtained principally from cereals, nuts, green vegetables, and dairy products. The RDI for magnesium is 300–350 mg (Table 1).

Epidemiological Studies Concerning Diet and Lung Diseases Methodological Issues

Study design Both observational and experimental studies have been performed to determine the impact of diet on obstructive lung diseases. The type of study

most frequently carried out consists of a cross-sectional design in which dietary intake, airway responsiveness, change in lung functions, or respiratory symptoms are simultaneously assessed. Because of their cross-sectional nature, these studies cannot provide information on the temporal relationship between dietary intake and lung diseases and are therefore more likely to be biased. For instance, subjects with prevalent symptoms may alter their diet, thus altering the nature of the association. In addition, uncertainty exists concerning the biologically relevant exposure window, and it is plausible that dietary agents may act at different stages of the disease process. Cohort studies enroll individuals free of disease and evaluate the incidence of lung disease, thus providing a correct temporal relationship and stronger evidence for a causal relation between diet and lung disease. However, long-term dietary intake assessment is difficult because of changes in nutrient status during follow-up and it is also subject to

176 NUTRITION IN RESPIRATORY DISEASE

misclassification, thus potentially underestimating associations. In experimental studies, patients are randomly assigned to receive a dietary supplement or a placebo and therefore their diet is controlled. These studies are more likely to provide information indicating a true causal relation; however, the results can only be extrapolated to populations that are similar to the one under study. Assessment of dietary intake In observational studies, different methodologies for dietary assessment have been used, and it is important to understand the limitations of such methods for interpreting the results of these studies. Biochemical indicators of dietary intake mostly reflect recent intake and are unable to provide adequate information concerning long-term intake, which represents the most relevant risk factor for chronic disease. In addition, certain factors, such as exposure to tobacco smoke, will decrease the level of vitamin C in serum, and if these effects are not accounted for, indications of dietary intake will be erroneous. Food frequency questionnaires provide a reasonable estimate of long-term dietary intake but are subject to random error and may thus lead to an underestimate of the relationship between dietary factors and lung disease. For some nutrients, such as vitamin E and selenium, relying on dietary assessment methods to classify individuals is problematic since accurate data on food content are limited. The selenium content will vary according to the origin of the food. Also, the quantity of fats or oils, rich in vitamin E, that are ingested is difficult to discern. Finally, because few studies consider more than one nutrient in their analysis, a protective or adverse effect may be attributed to one nutrient or micronutrient, such as vitamin C, when in fact it indicates the effect of another correlated dietary constituent or the effect of an interaction between dietary constituents. When analyzing the effect of foods, it is particularly difficult to exclude the possibility that a certain component of fruit or vegetable may have a truly protective effect.

Lung Cancer Fruits and Vegetables

Lung cancer is the leading cause of cancer-related deaths worldwide. Evidence from multiple observational retrospective and prospective studies strongly suggests that high consumption of fruits or vegetables or both reduces the risk of lung cancer by approximately 25%, with a similar level occurring among current smokers, ex-smokers, and those who

have never smoked. Fruits and vegetables rich in carotenoids seem to play a major role, as do brassica vegetables, rich in glucosinolates and further hydrolyzed in isothiocyanates, which are known for their anticarcinogenic properties. Other Foods

Lung cancer rates are highest in countries that have high fat intake. In several case–control studies, positive associations have been observed between lung cancer and the intake of total fat and saturated fat, particularly among nonsmokers. However, a metaanalysis of eight prospective studies did not indicate an association between dietary fat and lung cancer, suggesting that the results observed in case–control studies may have been due to a combination of selection and recall bias. Antioxidant Nutrients

Dietary intake of carotenoids and b-carotene levels found in blood samples collected for prospective analysis have been inversely associated with risk of subsequent lung cancer in numerous studies. This prompted large primary, randomized chemoprevention trials that used vitamin supplementation as an intervention and subsequently reported an increase in lung cancer incidence in the group receiving high doses of b-carotene. In the a-Tocopherol and b-Carotene Cancer Prevention Study (ATBC Study), participants were randomly assigned 50 mg a-tocopherol, 20 mg b-carotene, both, or a placebo. After 5–8 years of supplementation with 20 mg b-carotene, there was an 8% increase in overall mortality and 18% increase in lung cancer. In the b-Carotene and Retinol Efficacy Trial (CARET), participants were randomly assigned 30 mg b-carotene and 25 000 IU retinyl palmitate (vitamin A) for an average of 4 years. The supplementation group experienced 28% more lung cancer and 17% more deaths than participants not taking the supplements. The Physician Health Study, which assigned 50 mg b-carotene every other day to male physicians in the US for 12 years, demonstrated neither positive nor negative effects with b-carotene supplementation. One hypothesis to explain these results is that high doses of b-carotene may downregulate tumor suppressor genes and upregulate genes of cell proliferation, particularly among smokers. Contrastingly, among subjects assigned to receive the placebo, a high intake of fruits and vegetables was shown to decrease the risk of lung cancer, suggesting that other dietary factors associated with fruit and vegetable intake may be protective and were not present in the supplements provided (e.g., fibers, other

NUTRITION IN RESPIRATORY DISEASE 177

carotenoids or nutrients, and other protective chemicals such as non-pro-vitamin A carotenoids).

Obstructive Lung Diseases Obstructive lung diseases, such as asthma and COPD, have airway narrowing in common, with a consequent increase in effort for breathing. Asthma may be defined as a disease that has reversible airway obstruction as its hallmark, from which unremitting airway obstruction may develop, whereas COPD characteristically involves chronic bronchitis and/or emphysema with or without airway obstruction. Asthmatics who are exposed to chronic irritation may also manifest chronic productive cough and features of both asthma and chronic bronchitis, whereas patients with chronic bronchitis and emphysema may develop increased reactivity of the airway that may or may not be reversible. The interrelationship between these obstructive diseases is reflected in the epidemiological literature; studies have addressed health outcomes ranging from airway hyperresponsiveness to changes in lung function, respiratory symptoms, or reported diseases. There is no perfect way to summarize epidemiological data, given the interrelationship of chronic airflow limitation; therefore, this chapter focuses on (1) asthma-like symptoms, including airway hyperresponsiveness and wheezing, and (2) COPD, changes in lung function, and chronic respiratory symptoms, acknowledging that there is no clear division between these pathologies and that some studies have addressed various health outcomes. Most of the evidence concerning nutritional determinants of the major obstructive airways diseases (COPD and asthma) derives from cross-sectional studies and consistently demonstrates an association between a high intake of dietary antioxidants – (pro-) vitamin A, C, and E and some minerals – and a reduced risk of these disorders. However, cross-sectional data cannot provide information on the temporal relationship between dietary intake and lung diseases. It is plausible that dietary agents may act at different stages of the disease process and the effect of diet on lung development may be an important factor that affects obstructive lung diseases in adulthood. In addition, the difficulty of reconstructing past dietary intake and the potential confounding effect of other lifestyle factors render the interpretation of cross-sectional data difficult. Chronic Obstructive Pulmonary Disease

Chronic obstructive airway disease is a major cause of death and disability among adults in the US and

throughout the world. Factors such as dietary intake may play a role in modulating the impact of environmental exposure on the lung. In assessing the association of chronic obstructive airway diseases and diet, most studies have focused on lung function. Fruits and Vegetables

Fresh fruits and juice have been associated with greater lung function and lower prevalence of cough in several cross-sectional studies. In addition, a reduction in fresh fruit consumption over time has been associated with a more marked decline in forced expiratory volume in 1 s (FEV1) compared to that in subjects who did not modify their diet. Solid fruits such as apples and pears, but especially apples, which are rich in flavonoids, may play a more important role than other types of fruit. A lower incidence of COPD has been observed among subjects with a high intake of solid fruits. Antioxidant Nutrients

There is probably more epidemiological evidence concerning the role of vitamin C in COPD than for any other individual nutrient. Both cross-sectional and longitudinal studies have consistently shown that subjects consuming high levels of vitamin C have greater FEV1 and forced vital capacity than their counterparts and a lower rate of lung function decline in adulthood. Greater FEV1 has also been reported in association with higher vitamin E and carotenoid intake and higher serum levels of these nutrients, but with less consistency. In randomized supplementation trials, there was no evidence that supplementation with a-tocopherol (vitamin E) or b-carotene (carotenoid) reduced the incidence of COPD symptoms, nor did they affect the rate of decline in lung function among smokers and former smokers. However, subjects recruited in these trials either had an extensive past exposure to asbestos (CARET) or were heavy smokers (ATBC), thus making it difficult to generalize these results. Flavonoids are effective antioxidants because of their free radical scavenging properties and because they are chelators of metal ions. A greater intake of flavonoids (catechin, flavonol, and flavone) has been found to be positively associated with increased FEV1 and inversely associated with chronic cough. Omega-3 Polyunsaturated Fatty Acids

Cross-sectional data suggest that omega-3 fatty acid may have a protective effect against COPD and lung function decrement; however, study results are inconsistent and may be biased because of the lack of

178 NUTRITION IN RESPIRATORY DISEASE

control concerning the intake of antioxidants. The only prospective study observed no protective effect for omega-3 fatty acids after adjusting for other nutrients.

Airway Hyperresponsiveness, Wheezing, and Asthma Asthma prevalence has increased substantially during the past 30 years in most developed countries and appears to have increased in developing countries in relation to the degree of affluence of the population. Deficiency in dietary antioxidants and other nutrients such as omega-3 PUFA is one of the factors that may contribute to asthma prevalence. Changes in diet must be taken into account, and these include a radical decrease in the intake of fresh fruits and vegetables, both in the US and in other countries. Antioxidant vitamins and foods rich in antioxidants – fresh fruits and vegetables – are the most strongly implicated in asthma etiology. Fruits and Vegetables

The intake of fresh fruits and vegetables has been consistently shown to have a beneficial effect on the lungs of children. Generally, a high intake of fresh fruits, fresh juice, salads, and green vegetables has been associated with higher FEV1 and lower respiratory symptoms, whereas low intakes of vitamins C, E, and A from fresh fruits, vegetables, and juice were found to be associated with diminished lung function and a high prevalence of respiratory symptoms. Among adults, a high intake of apples rich in quercetin (flavonoid) has been related to a lower risk of asthma. High intakes of other foods, such as carrots, tomatoes, and leafy vegetables, have also been related to low asthma prevalence. Although modification of diet after asthma diagnosis is one possible explanation, the results suggest that foods rich in some antioxidant nutrients, other than vitamin C, may also be beneficial for asthma. Antioxidant Nutrients

Asthma patients have been reported to have lower than normal concentrations of vitamin C in their plasma and blood leukocytes, which suggests that asthma may be associated with chronically lower concentrations of vitamin C. Vitamin C has been shown in several cross-sectional and case–control studies to be associated with a reduced asthma risk. Analyses of the National Health and Nutrition Survey (NHANES II and III) have shown that lower levels of vitamin C in serum

were associated with a greater risk of asthma. Vitamin C intake was also related to a lower risk of respiratory symptoms among adults who had a high prevalence of symptoms related to smoking. Supplementation studies in clinical settings have suggested that high doses of vitamin C (500 mg to 2 g day
1) administered a few hours before a bronchoconstriction challenge may reverse airway hyperreactivity and other symptoms of asthma. However, daily supplementation with vitamin C and magnesium (1 g day
1 vitamin C and 450 mg day
1 magnesium) among asthmatic adults over a 16-week period did not improve the symptom score or lung function in the supplemented group compared to the placebo. The only trials in which the findings suggested that vitamin C, b-carotene, and a-tocopherol may have a protective effect on the lungs were intervention trials of individuals exposed to high levels of ozone. Studies conducted among healthy individuals exposed to high ozone levels showed that supplementation with vitamin C, b-carotene, and vitamin E protected against bronchoconstriction. Similarly, vitamin C and vitamin E appeared to protect against ozonerelated bronchoconstriction and decrease respiratory symptoms among young asthmatics. This protective effect was stronger among subjects who were genetically susceptible (GSTM1 null polymorphism). The specificity of the effect of each individual supplement could not be established because they were given together. Few cross-sectional studies have demonstrated an association between vitamin E intake and asthma reduction. Higher concentrations of vitamin E intake have been associated with a lower prevalence of allergen skin sensitization and lower total serum IgE levels in adults. Supplementing patients with adultonset asthma demonstrated no benefits. Similarly, there is little or no evidence concerning the beneficial effect of increased vitamin A or b-carotene intake among patients with asthma. Selenium also has some antioxidant proprieties. A greater intake of selenium was related to a lower risk of asthma, but supplementation with sodium selenite among patients with intrinsic asthma demonstrated no improvement with regard to their lung function measurements. Although antioxidant nutrients have been shown to reduce the prevalence of wheezing and respiratory symptoms, there is no evidence that such nutrients have an effect on the incidence of asthma. The only prospective study conducted among a large cohort of adult women did not find an association between the intake of vitamin C or other antioxidants and the onset of asthma.

NUTRITION IN RESPIRATORY DISEASE 179 Magnesium

Several cross-sectional studies have reported that magnesium intake has a protective effect on the prevalence of asthma. However, supplementation studies do not confirm these results. Omega-3 Polyunsaturated Fatty Acids

The hypothesis that high fish intake in the diet may reduce susceptibility to chronic airway diseases originated from the observation that the Inuit population has a very low prevalence of asthma, whereas in most other populations asthma rates have been shown to increase with the increased dietary intake of polyunsaturated fats, especially linoleic acid (omega-6). Data from cross-sectional studies have suggested that a high intake of oily fish was related to a lower prevalence of wheezing among children. The risk of current wheezing among children eating oily fish was 3.8 times less than that of children who did not eat oily fish. No study of adults has observed such an effect. Fish intake early in life may protect against allergic rhinitis later in life. Experimental studies of omega-3 PUFA have shown a decrease in airway hyperresponsiveness and inflammatory markers (tumor necrosis factor-a and leukotriene) but little or no effect on symptoms among asthmatic children. Among young elite athletes, omega-3 supplementation appears to reduce postexercise-induced bronchoconstriction and decrease inflammatory marker levels in urine and plasma. Omega-3 PUFA supplementation during pregnancy has been shown to reduce the risk of wheezing in the first 18 months of the offspring’s life and also appears to decrease the risk of atopy among susceptible infants.

Conclusion High intake of fresh fruits and vegetables is beneficial for chronic obstructive lung diseases, as observed for other chronic diseases. Although the antioxidant content of these foods likely plays a major role, the discrepancy between observational and experimental studies indicates that it is difficult to make an extrapolation concerning food intake, which confirms the effect of a specific dietary supplement. Dietary guidelines from the National Institutes of Health that were presented in a report in Healthy People 2000–2010 recommended ‘‘five a day for better health.’’ This proposed the consumption of two to four fruits per day and three to five vegetable portions per day – amounts that should cover the RDIs for important micronutrients. In some circumstances, metabolic, environmental, and genetic factors may lead to individual requirements that differ from the

estimated RDI and justify supplementation. However, there are few data indicating the benefit of nutritional supplementation on lung health, although evidence supports protecting against additional oxidative stress challenges resulting from high levels of air pollution, especially in patients suffering from impaired immune response. See also: Allergy: Overview. Asthma: Overview; Acute Exacerbations. Chronic Obstructive Pulmonary Disease: Overview; Emphysema, Alpha-1-Antitrypsin Deficiency. Environmental Pollutants: Overview; Oxidant Gases; Particulate Matter, Ultrafine Particles. Genetics: Overview. Oxidants and Antioxidants: Antioxidants, Nonenzymatic.

Further Reading Erickson KL, Medina EA, and Hubbard NE (2000) Micronutrients and innate immunity. Journal of Infectious Diseases 182(S1): S5–S10. Fairfield KM and Fletcher RH (2002) Vitamins for chronic disease prevention in adults. Journal of the American Medical Association 287: 3116–3126. Fogarty A and Britton J (2000) The role of diet in the aetiology of asthma. Clinical & Experimental Allergy 30: 615–627. Grievink L, Smit HA, and Brunekreef B (2000) Anti-oxidants and air pollution in relation to indicators of asthma and COPD: a review of the current evidence. Clinical & Experimental Allergy 30: 1344–1354. Institute of Medicine (2000) Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, Carotenoids. Washington, DC: National Academy Press. James MJ, Gibson RA, and Cleland LG (2000) Dietary polyunsaturated fatty acids and inflammatory mediator production. American Journal of Clinical Nutrition 71: S343–S348. Knekt P, Kumpulainen J, Jarvinen R, et al. (2002) Flavonoid intake and risk of chronic diseases. American Journal of Clinical Nutrition 76: 560–568. McKeever TM and Britton J (2004) Diet and asthma. American Journal of Respiratory and Critical Care Medicine 170: 725–729. Neuhouser ML, Patterson RE, Thornquist MD, et al. (2003) Fruits and vegetables are associated with lower lung cancer risk only in the placebo arm of the beta-carotene and retinol efficacy trial (CARET). Cancer Epidemiology Biomarkers & Prevention 12: 350–358. Olick CN, Michaud DS, Stolzenberg-Solomon R, et al. (2002) Dietary carotenoids, serum beta-carotene, and retinol and risk of lung cancer in the alpha-tocopherol, beta-carotene cohort study. American Journal of Epidemiology 156: 536–547. Paolini M, Abdel-Rahman SZ, Sapone A, et al. (2003) Beta-carotene: a cancer chemopreventive agent or a co-carcinogen? Mutation Research 543: 195–200. Potter JD, Finnegan JR, and Guinard JX (2000) 5 a day for better health program evaluation report, NIH Publication No. 01-4904. Bethesda, MD: National Cancer Institute. Romieu I and Trenga C (2001) Diet and obstructive lung diseases. Epidemiologic Reviews 23: 268–287. Smith AH (2001) Chronic obstructive pulmonary disease, asthma and protective effects of food intake; from hypothesis to evidence? Respiratory Disease 2: 261–264. Smith-Warner SA, Ritz J, Hunter DJ, et al. (2002) Dietary fat and risk of lung cancer in a pooled analysis of prospective studies. Cancer Epidemiology Biomarkers & Prevention 11: 987–992.

O OBESITY M H Sanders and R Givelber, University of Pittsburgh, Pittsburgh, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Overweight and obesity conditions are becoming increasingly pervasive in the community with far-reaching health consequences. The respiratory system is susceptible to clinically significant dysfunction that is interactively manifested during wakefulness and sleep. In general, pulmonary mechanics assessed during wakefulness in overweight/obese individuals reflects a restrictive pattern with the greatest impact in those with greater obesity and as the body habitus displays a more central distribution of adiposity. This observation notwithstanding, other etiologies should be excluded before attributing pulmonary restriction to obesity. Overweight/obesity-related alterations in lung volumes may also be associated with systemic hypoxemia due to breathing below the closing volume of small airways and ventilation/perfusion inhomogeneity. Epidemiologic studies have demonstrated an association between overweight/obese conditions and asthma. However, whether or not overweight/obesity increases bronchial hyperresponsiveness remains controversial. Obesity may also indirectly promote nocturnal asthma by predisposing individuals to gastroesophageal reflux. Increased body mass index (BMI) is a major risk factor for obstructive sleep apnea–hypopnea (OSAH) and this represents another pathway through which overweight/obese conditions contribute to cardiovascular disease and possibly type 2 diabetes mellitus. Importantly, sufficient weight reduction often results in marked improvement in OSAH. Greater degrees of obesity may, in some individuals, be associated with hypoventilation with or without concomitant OSAH as well as increased risk for pulmonary artery hypertension and cor pulmonale. Obesity-hypoventilation may initially be evident during sleep and then extend into wakefulness. A high index of suspicion is needed to diagnose nocturnal obesity-hypoventilation in order to initiate noninvasive positive pressure therapy in the short term and reinforce the need for long-term weight reduction through either medical or surgical intervention.

condition is present when the BMI is 25–29.9 and obesity is present when BMI 430. As reviewed by Stein and Colditz in 2004, there was a 40% increase in the prevalence of being overweight and a 110% increase in the prevalence of obesity between 1976– 80 and 1999–2000. Another perspective on the magnitude of the problem is revealed by the National Center for Health Statistics indicating that as of 1999, 34% of US adults were overweight and an additional 27% were obese. The health impact of overweight and obese conditions is readily apparent when one considers the notable number of diseases for which they place patients at increased risk, and thus exacerbate pathophysiologic effects and worsen outcomes. A partial list of these conditions is provided in Table 1. In addition to the substantial toll taken by overweight/obesity on personal health and quality of life, obesity and obesity-impacted disorders extort societal costs through high healthcare resource utilization and lost productivity. As noted by Stein and co-workers, the cost of obesity amounts to at least 117 billion dollars per year. Due to the multifaceted undesirable consequences of overweight/obesity, clinicians should understand the ways in which these conditions adversely influence health and the benefits associated with therapy. In this section, we will focus on the breathing-related consequences of overweight/obesity. These consequences may be conceptualized as those that are clinically manifested during wakefulness and those manifested during sleep (although with consequences that extend into wakefulness).

Obesity and Pulmonary Mechanics Introduction As revealed by the National Health and Nutrition Examination Survey (NHANES) and the Behavioral Risk Factor Surveillance System, excess body weight is increasingly prevalent. Body size is often quantified using the body mass index (BMI) which is calculated as an individual’s weight in kilograms/(height in meters)2. A normal BMI is considered to be present at values between 18.5 and 24.9, an overweight

In 1983, Ray and co-workers reported their assessment of pulmonary function in 43 adult nonsmokers who were also obese. The study population was divided into categories defined by the weight (kg)/ height (cm) ratio. There was no decrease in the vital capacity (VC) until the weight/height ratio was 1.0 or greater. Similarly, total lung capacity (TLC) and functional residual capacity (FRC) were not notably diminished until the weight/height ratio was X1.1. In

182 OBESITY Table 1 Disorders and conditions which are at least in part directly or indirectly attributable to, or exacerbated by overweight and obesity Cardiovascular disorders Systemic hypertension Ischemic heart disease Stroke Pulmonary Restrictive pattern on pulmonary function testing Obstructive sleep apnea–hypopnea Asthma (including nocturnal asthma) Obesity-hypoventilation Metabolic disorders Type 2 diabetes mellitus Dyslipidemia Metabolic syndrome Neoplasms Esophagus Colon and rectum Liver Gallbladder Pancreas Kidney Non-Hodgkin lymphoma Multiple myeloma Stomach Prostate Breast Uterus Cervix Ovary Gall stones Osteoarthritis Menstrual irregularity Reduced quality of life

contrast, expiratory reserve volume (ERV) (expressed as percent of the predicted value) as well as the ratio of the ERV to inspiratory capacity (IC) decreased with increasing degrees of overweight/obesity. There was no noteworthy change in the forced expiratory volume in 1 s (FEV1), FEV1/VC ratio or the diffusing capacity (DLCO) across categories of weight/height ratio. The maximal voluntary ventilation (MVV) tended to be reduced when the weight/height ratio was 1.1 or greater. The authors concluded that their observations were consistent with a mass loading effect on the chest wall with normal lung compliance. The increased DLCO was attributed to increased pulmonary capillary blood volume accompanying obesity. Because the data within each weight/height ratio category were presented as means of the percentage of predicted values, the true effect of obesity

on pulmonary function test (PFT) results may have been obscured. More revealing may be the data comparing PFT results before and after substantial weight loss (an average of about 54 kg). Although still considerably obese, these individuals experienced an increased VC and MVV and a reduction in DLCO. Other studies, such as that of Harik-Khan in 2001, reinforce the adverse effect of increasing BMI on the FVC and FEV1 in asymptomatic neversmokers and the relatively greater effect in men than women. The observations of Harik-Khan and co-workers as well as those of Lazarus and co-workers indicating that the FVC and FEV1 are inversely related to the magnitude of the waist circumference/ hip circumference ratio, provides evidence that the effect of obesity on pulmonary mechanics is at least partly attributable to the distribution of adiposity, with a central distribution conferring greater negative impact. Nonetheless, the impact of central adiposity appears to be more pronounced in men than women. Wise and co-workers examined the clinically important issue of the effect of weight gain in patients with mild-to-moderate chronic obstructive pulmonary disease (COPD) who had either quit smoking, were continuing to smoke, or who were intermittent smokers. Greater weight gain over a 5-year interval was associated with greater decrement in the forced VC (FVC). As noted in studies of populations without COPD, the effect was more pronounced in men than in women, perhaps due to the propensity for central deposition of adipose tissue in men. Similar but smaller decrements with weight gain over time were seen in the FEV1. After adjusting for age, height, baseline FEV1, FVC, and number of cigarettes smoked per day at baseline, in those subjects who quit smoking, the FVC was reduced by approximately 17 and 11 ml kg
1 of weight gained for men and women respectively; the FEV was reduced by approximately 11 and 6 ml kg
1 of weight gained for men and women respectively. The authors concluded that relative to the health benefits of smoking cessation, the impact of weight gain on pulmonary function following smoking cessation is small. Nonetheless, the data indicate that weight gain augments pulmonary dysfunction in patients with even mildto-moderate COPD and steps to avoid weight gain are prudent from this as well as other perspectives. These data reinforced the observations by Sahebjami and co-workers indicating that the BMI is inversely related to the FVC and FEV1, and directly related to the DLCO in patients with COPD. Although the association between overweight/ obesity and reduced lung volumes is apparent from numerous studies, attribution of abnormal lung

OBESITY 183

Obesity and Asthma As outlined in the proceedings of a recent National Heart, Lung, and Blood Institute workshop, there appears to be an epidemiologic association between overweight/obesity and asthma with the former increasing the risk for the latter. The nature of the relationship is controversial in that although a number of studies have observed bronchial hyperresponsiveness in obese individuals, others have not. Potential mechanisms for asthma in obesity include heightened systemic inflammation associated with obesity, reduced bronchial smooth muscle dilation-stretch due to reduced lung volume, reduced adrenal medullary activity, and environmental as well as complex genetic interactions and influences. Although, temporally associated with the hours of sleep, nocturnal asthma is considered in the context of poorly controlled asthma. It is therefore discussed in this section rather than the section below on ‘Sleep-related breathing consequences of obesity’. As discussed by Calhoun in proceedings of the Thomas L Petty 45th Annual Aspen Lung Conference, nocturnal asthma reflects a condition in which there is exaggeration of the magnitude of circadian swings in pulmonary function reflecting overnight worsening. Obesity has been associated with nocturnal asthma. Moreover, Hakala and co-workers demonstrated that the degree of these fluctuations may be reduced after weight reduction. The factors that link obesity and nocturnal asthma have not been identified with certainty. However, potential candidates include nocturnal gastroesophageal reflux, the proinflammatory milieu which characterizes obesity, or further reductions in lung volume during sleep. It has also been speculated that nocturnal asthma is mediated by activation of upper airway reflexes due to upper airway obstruction during sleep.

Sleep-Related Breathing Consequences of Obesity Obstructive Sleep Apnea–Hypopnea

Obstructive sleep apnea–hypopnea is characterized by episodic occlusion (apnea) and partial obstruction

(hypopnea) of the upper airway during sleep with consequent sleep disruption (fragmentation) and oxyhemoglobin desaturation. Patients with this disorder often suffer from daytime hypersomnolence which has substantial social implications apart from reducing productivity. The hypersomnolence can be life-threatening as evidenced by the high rate of vehicular crashes experienced by these patients. Additionally, obstructive sleep apnea patients are at increased risk for cardiovascular disease, glucose intolerance/metabolic syndrome, and diabetes mellitus super-imposed on the risks conferred by obesity. Increasing weight notably increases the frequency of apneas and hypopneas during sleep (Figure 1). Moreover, Young and co-workers demonstrated that an increase in BMI of 1-standard deviation above the mean was associated with an odds ratio of approximately 4.2 for having an apnea þ hypopnea index (average number of apneas þ hypopneas per hour of sleep)X5. As demonstrated by Tishler and coworkers the impact of obesity on the risk of sleep apnea–hypopnea decreases with age (Figure 2). Conceivably, this could reflect a survivor bias in which there was early mortality of heavier individuals with sleep apnea–hypopnea. Although for an individual the BMI is not a good predictor of the apnea þ hypopnea index, the noteworthy improvement in obstructive sleep apnea–hypopnea with weight reduction attests to the pathophysiologic importance of obesity. The weight reduction may be achieved either through dietary modification as illustrated by Smith and co-workers in the only randomized controlled 6 Mean change in AHI, events h−1

function to overweight/obesity in the clinical setting must be by exclusion. Other etiologies of pulmonary restriction must first be ruled out. Overweight/obesity is also associated with arterial hypoxemia. In individuals with reduced FRC, breathing occurs below the closing volume of the small airways. This promotes ventilation/perfusion inhomogeneity and consequent hypoxemia with a widened alveolar–arterial oxygen gradient.

4

2

0

−2 −4

−20% to < −10% (n = 22)

−10% to < −5% (n = 39)

−5% to +5% to +10% to < +5% < +10% +20% (n = 371) (n = 179) (n = 79)

Change in body weight Figure 1 Change in the apnea þ hypopnea index with percent change in body weight. Peppard PE, Young T, Palta M, Dempsey J, and Skatrud J (2000) Longitudinal study of moderate weight change and sleep-disordered breathing. Journal of the American Medical Association 284: 3015–3021.

184 OBESITY

1.1

with improved upper airway stability during sleep as demonstrated by Schwartz and co-workers. Consequently, weight reduction should be strongly recommended as therapy for obstructive sleep apnea– hypopnea in all overweight/obese patients with this disorder.

1.0

Obesity-Hypoventilation Syndrome

1.3

Odds ratio

1.2

0.9

0.8 30

35

40

45

50

55

60

65

70

Baseline age (Y) Figure 2 Odds ratio for incident AHI 410 over 5 years by change in BMI of 1 unit and by age. Tishler PV, Larkin EK, Schluchter MD, and Redline S (2003) Incidence of sleep-disordered breathing in an urban adult population: the relative importance of risk factors in the development of sleep-disordered breathing. Journal of the American Medical Association 289: 2230–2237.

10 5 0 −5 −10 −15 −20 Control Treatment

−25 −30 −35 −40 −45 −50 % Change in weight

% Change in apnea + hypopnea index

Figure 3 Randomized controlled trial of dietary modification/ weight reduction therapy for obstructive sleep apnea. Smith PL, Gold AR, Meyers DA, Haponik EF, and Bleecker ER (1985) Weight loss in mildly to moderately obese patients with obstructive sleep apnea. Annals of Internal Medicine 103: 850–855.

trial of this intervention (Figure 3) or by bariatric surgery as discussed in the meta-analysis by Buchwald and co-workers. The mechanism through which obesity promotes obstructive sleep apnea–hypopnea, be it increased volume of adipose tissue in the upper airway, altered upper airway function by virtue of reduced lung volume, or combinations of these and other factors, has not been conclusively identified. However, it is evident that weight loss is associated

As described in a review by Martin and Sanders, some but not all obese individuals have a partial pressure of carbon dioxide in the arterial blood (PaCO2 ) that exceeds 45 mmHg (hypercapnia) during wakefulness, indicating alveolar hypoventilation. The degree of obesity that is required (in conjunction with PaCO2 445 mmHg) to define obesity-hypoventilation syndrome is not well established although some sources indicate that the BMI should be at least 35. Obesity-hypoventilation syndrome is discussed in the context of sleep-related breathing disorders because elevation of PaCO2 is manifested during sleep before being present during wakefulness. Although it is likely that most patients with obesity-hypoventilation syndrome also have obstructive sleep apnea– hypopnea, a small but measurable minority do not, as demonstrated by Kessler and co-workers. The mechanism(s) responsible for hypoventilation and its variable presentation remain incompletely defined. Candidate etiologies include mechanical loading of the chest wall imposed by obesity, chronic ventilatory muscle fatigue, and reduced ventilatory chemosensitivity. Mechanical impairment alone does not explain the hypoventilation since patients with relatively limited degrees of impairment may exhibit hypoventilation. It is likely that a combination of factors including loading of the chest wall and reduced chemosensitivity predisposes to hypoventilation. The addition of normal physiologic features associated with sleep including the normal increase in upper airway resistance that accompanies sleep and the inhibition of the intercostals and accessory muscles during rapid eye movement (REM) sleep, may heighten the risk for hypoventilation. This may explain why hypoventilation is manifest during sleep before extending into wakefulness. Indeed, hypercapnia during sleep may increase blood bicarbonate and reduce hypercapnic chemoresponsiveness during wakefulness, thereby potentially establishing a vicious cycle. Recognition of obesity-hypoventilation is important since these patients may experience notable hypoxemia during sleep and wakefulness, and are at increased risk for pulmonary artery hypertension and cor pulmonale. Treatment of sleep-related hypoventilation and when present, obstructive sleep apnea–hypopnea using noninvasive positive pressure

OBESITY 185

ventilatory assistance improves arterial oxygen tension and reduces hypercapnia during wakefulness as well as during sleep. On a long-term basis, weight reduction through either medical or surgical intervention is of substantial benefit and may obviate the requirement for ventilatory assistance. See also: Asthma: Overview. Chest Wall Abnormalities. Chronic Obstructive Pulmonary Disease: Overview. Sleep Apnea: Overview. Sleep Disorders: Hypoventilation.

Further Reading Bottai M, Pistelli F, Di Pede F, et al. (2002) Longitudinal changes of body mass index, spirometry and diffusion in a general population. European Respiratory Journal 20: 665–673. Buchwald H, Avidor Y, Braunwald E, et al. (2004) Bariatric surgery: a systematic review and meta-analysis. Journal of the American Medical Association 292: 1724–1737. Carey IM, Cook DG, and Strachan DP (1999) The effects of adiposity and weight change on forced expiratory volume decline in a longitudinal study of adults. International Journal of Obesity and Related Metabolic Disorders 23: 979–985. Chan CS, Woolcock AJ, and Sullivan CE (1988) Nocturnal asthma: role of snoring and obstructive sleep apnea. American Review of Respiratory Disease 137: 1502–1504. Collins LC, Hoberty PD, Walker JF, Fletcher EC, and Peiris AN (1995) The effect of body fat distribution on pulmonary function tests. Chest 107: 1298–1302. Flegal KM, Carrol MS, Ogden CL, and Johnson CL (2002) Prevalence and trends in obesity among US adults, 1999– 2000. Journal of the American Medical Association 288: 1723–1727. Hakala K, Stenius-Aarniala B, and Sovijarvi A (2000) Effects of weight loss on peak flow variability, airways obstruction, and lung volumes in obese patients with asthma. Chest 118: 1315–1321. Harik-Khan RI, Fleg JL, Muller DC, and Wise RA (2001) The effect of anthropometric and socioeconomic factors on the racial difference in lung function. American Journal of Respiratory and Critical Care Medicine 164: 1647–1654. Harik-Khan RI, Wise RA, and Fleg JL (2001) The effect of gender on the relationship between body fat distribution and lung function. Journal of Clinical Epidemiology 54: 399–406. Kessler R, Chaouat A, Schinkewitch P, et al. (2001) The obesityhypoventilation syndrome revisited. Chest 120: 369–376. Lazarus R, Sparrow D, and Weiss ST (1997) Effects of obesity and fat distribution on ventilatory function. Chest 111: 891–898. Martin TJ and Sanders MH (1995) Alveolar hypoventilation: a review for clinicians. Sleep 18: 317–334.

Mokdad AH, Bowman BA, Vinicor F, Marks JS, and Koplan JP (2001) The continuing epidemics of obesity and diabetes in the United States. Journal of the American Medical Association 286: 1195–1200. National Center for Health Statistics, Health E-Stats, prevalence of overweight and obesity among adults: United States, 1999, 2000. Peppard PE, Young T, Palta M, Dempsey J, and Skatrud J (2000) Longitudinal study of moderate weight change and sleep-disordered breathing. Journal of the American Medical Association 284: 3015–3021. Ray CS, Sue DY, Bray G, Hansen JE, and Wasserman K (1983) Effects of obesity on respiratory function. American Review of Respiratory Disease 128: 501–506. Sahebjami H, Doers JT, Render ML, and Bond TL (1993) Anthropometric and pulmonary function test profiles of outpatients with stable chronic obstructive pulmonary disease. American Journal of Medicine 94: 469–474. Schwartz AR, Gold AR, Schubert N, et al. (1991) Effect of weight loss on upper airway collapsibility in obstructive sleep apnea. American Review of Respiratory Disease 144: 494–498. Sherrill DL, Enright PL, Kaltenborn WT, and Lebowitz MD (1999) Predictors of longitudinal changes in diffusing capacity over 8 years. American Journal of Respiratory and Critical Care Medicine 160: 1883–1887. Smith PL, Gold AR, Meyers DA, Haponik EF, and Bleecker ER (1985) Weight loss in mildly to moderately obese patients with obstructive sleep apnea. Annals of Internal Medicine 103: 850–855. Stein CJ and Colditz GA (2004) The epidemic of obesity. Journal of Clinical Endocrinology and Metabolism 89: 2522–2525. Tishler PV, Larkin EK, Schluchter MD, and Redline S (2003) Incidence of sleep-disordered breathing in an urban adult population: the relative importance of risk factors in the development of sleep-disordered breathing. Journal of the American Medical Association 289: 2230–2237. Vaughan RW, Cork RC, and Hollander D (1981) The effect of massive weight loss on arterial oxygenation and pulmonary function tests. Anesthesiology 54: 325–328. Viegi G, Sherrill DL, Carrozzi L, et al. (2001) An 8-year follow-up of CO diffusing capacity in a general population sample of North Italy. Chest 120: 74–80. Weiss ST and Shore S (2004) Obesity and asthma. Directions for research. NHLBI workshop. American Journal of Respiratory and Critical Care Medicine 169: 963–968. Wise RA, Enright PL, Connett JE, et al. (1998) Effect of weight gain on pulmonary function after smoking cessation in the Lung Health Study. American Journal of Respiratory and Critical Care Medicine 157: 866–872. Young T, Palta M, Dempsey J, et al. (1993) The occurrence of sleep-disordered breathing among middle-aged adults. New England Journal of Medicine 328: 1230–1235.

186 OCCUPATIONAL DISEASES / Overview

OCCUPATIONAL DISEASES Contents

Overview Coal Workers’ Pneumoconiosis Hard Metal Diseases – Berylliosis and Others Inhalation Injury, Chemical Asbestos-Related Lung Disease Silicosis

Overview B Nemery, Katholieke Universiteit Leuven, Leuven, Belgium & 2006 Elsevier Ltd. All rights reserved.

Abstract This overview describes the main categories of specific occupational disorders and it covers also how work exposures are potential determinants of common respiratory conditions. Acute inhalation injuries may present as inhalation fever or as acute tracheobronchitis and pneumonitis. Occupational asthma is the most frequent work-related respiratory disease. It may be caused by allergic sensitization to macromolecules of biologic origin or to chemicals of low molecular weight, as well as by (heavy) exposure to workplace irritants. The pneumoconioses are caused by the accumulation of dust particles or fibres in the lungs. They include silicosis, coal workers’ pneumoconioses, asbestosis and other less common pneumoconioses. Chronic beryllium disease is caused by a cell-mediated sensitization to beryllium and resembles sarcoidosis. Hard-metal lung disease is caused by sensitivity to cobalt and resembles hypersensitivity pneumonitis. Extrinsic allergic alveolitis or hypersensitivity pneumonitis is generally caused by sensitization to aerosolized biological antigens. Several types of infections may be related more or less specifically to work. Chronic obstructive lung disease is mainly caused by cigarette smoking, but exposure to dusts and gases contribute to its incidence. Similarly, bronchopulmonary cancer is not only caused by smoking, but also by occupational agents, most notably asbestos. Asbestos is also a cause of nonmalignant and malignant pleural disease.

Introduction In industrially developed countries the frequency and severity of the traditional occupational diseases have decreased in the past decades, thanks to improvements in work legislation and practices. However, working conditions are still far from being acceptable, let alone healthy, in all areas of the world. In many poorer countries, considerable numbers of workers are exposed to serious hazards associated with underground mining, various types of industry (metal, textile, wood, food, electronics, etc.), construction,

and agriculture. In industrially advanced countries, occupational lung diseases still occur as a consequence of working conditions of the past, but they also continue to occur, because occupational risks are still present in many jobs and novel risks emerge with the advent of new technologies. In general, it is difficult, if not impossible, to quantify exactly the contribution of occupation to the burden of respiratory disease. Exact figures of the incidence and prevalence of occupational lung disorders are often not available, even in countries where government agencies exist for reporting and compensating occupational diseases. This is due, in varying proportions, to legal and administrative restrictions in the eligibility for compensation, to a lack of obligation or incentive to notify occupational diseases, and to insufficient awareness, by physicians, of the possible occupational etiology of diseases in general. Thus, underreporting of occupational diseases is most likely to occur for pensioners, who are no longer at work, but whose condition may well be due to their previous job. However, even in working people, diagnoses of occupational disease, such as occupational asthma, are often missed for a variety of reasons. In various countries, schemes have been created for the voluntary reporting of occupational respiratory diseases by pneumologists and/or occupational physicians, for example, the Surveillance of Work Related and Occupational Respiratory Disease (SWORD) system in the UK or the Sentinel Event Notification System for Occupational Risks (SENSOR) Program, initiated by the National Institute for Occupational Safety and Health (NIOSH) in the US. Another, more generic reason for the underreporting of occupational lung disorders is that it is not always so easy to define occupational respiratory disease. Indeed, occupational respiratory diseases are not restricted to a limited range of highly specific occupational diseases, such as silicosis or asbestosis. Most common respiratory diseases, such as bronchial asthma, chronic obstructive pulmonary disease

OCCUPATIONAL DISEASES / Overview 187

(COPD), and bronchopulmonary cancer, are caused by a combination of endogenous and exogenous factors, including occupational exposures. Thus, even though an occupational etiology is often difficult to substantiate and document in individual patients, the attributable fraction of occupational factors in mortality and morbidity from respiratory diseases is far from being negligible. This overview describes briefly the main categories of specific occupational respiratory disorders and it covers also briefly how work exposures are potential determinants of common respiratory conditions.

Acute Inhalation Injuries Inhalation Fever

Inhalation fever is a clinical term used to describe influenza-like reactions that occur following a single exposure to high levels of: *

*

*

metal fumes (mainly zinc), causing metal fume fever; organic dusts (grain, cotton, etc.) and other bioaerosols contaminated with microorganisms and endotoxins and/or mycotoxins, causing the organic dust toxic syndrome (ODTS); and fumes produced by heating plastics (mainly fluorine-containing polymers), causing polymer fume fever.

These inhalation fevers are the clinical expressions of an intense nonallergic pulmonary inflammation, consisting mainly of neutrophils. These reactions are usually self-limited and not associated with much structural damage to the respiratory tract.

such as the reactive airways dysfunction syndrome (RADS).

Occupational Asthma In modern societies, occupational asthma is now considered the most frequent work-related respiratory disease. Occupational asthma is asthma that is causally related to an exposure at work. In addition to asthma that is caused – more or less clearly – by work, many asthmatics also experience a worsening of their asthma by their working circumstances. The latter is called ‘work-aggravated asthma’. The contribution of occupational factors to the causation and the manifestations of bronchial asthma has been recently estimated to amount to 15% of adult asthmatics. Excess risks of asthma are found for farmers, painters, plastic workers, cleaners, spray painters, and agricultural workers. Depending on its pathogenesis, occupational asthma may be categorized into occupational asthma caused by immunologic (allergic) sensitization to a (specific) agent and asthma caused by other mechanisms. Occupational Asthma Caused by Allergic Sensitization

A large number of workplace agents have been shown to be capable of causing sensitization and occupational asthma. These occupational ‘asthmogens’ include: *

*

Toxic Acute Tracheobronchitis and Pneumonitis

Severe injury to the respiratory tract may result from the inhalation of irritant or toxic gases, vapors, or complex aerosols released through explosions, fires, leaks, or spills from industrial installations, transport accidents, and military or terrorist operations. Depending on the nature of the chemical and the intensity of the exposure, there will be rhinitis, pharyngitis, laryngitis, tracheobronchitis, bronchiolitis, and/ or pneumonitis. Chemical pneumonitis is generally associated with noncardiogenic pulmonary edema, and may evolve to acute respiratory distress syndrome (ARDS). Organizing pneumonia with obliterating bronchitis may also be a feature of chemical injury to the terminal airspaces. In survivors of acute inhalation injury, there may be persisting structural lesions or functional sequelae,

*

*

macromolecules of biological origin, that is, (glyco)proteins derived from plants (flour, latex, etc.), animals (farm animals, laboratory animals, seafood, etc.), or microorganisms (enzymes in detergents, baking additives, animal feed, etc.); low-molecular-weight chemicals of natural origin (e.g., wood-derived chemicals); low-molecular-weight synthetic chemicals (diisocyanates and other reactive chemicals, pharmaceutical agents, reactive dyes, etc.); and metallic agents (complex platinum salts, hexavalent chromium, cobalt, etc.).

The mechanisms of sensitization involve IgE antibodies (for macromolecular antigens) or other less well-characterized immunological mechanisms (for chemicals). Occupational Asthma without Evidence of Specific Sensitization

Irritant-induced occupational asthma may be caused either by a single acute inhalation accident (RADS), or through repeated or chronic exposure to excessive

188 OCCUPATIONAL DISEASES / Overview

levels of irritants. The latter is still somewhat controversial. ‘Asthma-like’ disorders without evidence of sensitization are also found in workers exposed to (endotoxin-contaminated) vegetable dusts (e.g., byssinosis in cotton workers, asthma-like syndrome in grain handlers and in swine confinement workers). These may form the basis for the high prevalence of COPD in agricultural workers.

Interstitial Lung Disease Pneumoconioses

Pneumoconioses are diseases of the lung parenchyma caused by the accumulation of dust particles or fibers in the lungs. Although individual susceptibility plays some role, pneumoconiosis is considered to be caused essentially by the progressive accumulation of toxic particles beyond the lung’s normal clearance mechanisms. This leads to inflammation (characterized initially by increased numbers of alveolar macrophages) and various degrees and types of fibrosis, depending on the agent. Silicosis Silicosis has become relatively uncommon in industrialized countries, thanks to dust controls in the workplace. Hazardous exposures to silica (SiO2) may occur in mining, tunnel drilling or stone quarrying, in processing stone or sand, in building and demolition, in foundries, in pottery or ceramic manufacture, in the abrasive use of sand (sandblasting), in the manipulation of calcined diatomaceous earth, and other sometimes unexpected settings. Free crystalline silica (in practice, mainly quartz and cristobalite) is highly fibrogenic and leads to the formation of silicotic noduli. These lead initially to ‘simple silicosis’, characterized radiologically by small discrete opacities, and through coalescence they give rise to larger nodular opacities, characteristic of ‘progressive massive fibrosis’ (PMF). Silicosis is also associated with other conditions, such as COPD, tuberculosis, bronchopulmonary cancer, and collagen disease, including systemic sclerosis. Coal workers’ pneumoconiosis The pathology of coal workers’ pneumoconiosis (CWP) differs from that of silicosis, but both conditions share many clinical features, including the potential for evolution towards PMF. Hence, and because coal miners are also exposed to varying amounts of silica, CWP is sometimes considered as a mixed pneumoconiosis, labeled anthracosilicosis.

Asbestosis Asbestosis (i.e., fibrosis of the lung parenchyma caused by asbestos) is found in patients who were heavily exposed to asbestos fibers (mainly chrysotile, and the serpentines crocidolite, amosite, and tremolite), for example, during the manufacture of asbestos–cement products, friction materials or fireproof textiles, or when using asbestos for heat insulation or fire protection purposes in construction, heating systems, power stations, furnaces, shipyards and railroads, etc. The incidence of asbestosis will continue to decrease in those countries where the use of asbestos has been forbidden. Nevertheless, a risk of asbestosis will remain for those engaged in asbestos removal and waste handling, as well as in developing countries where the use of asbestos is still allowed and poorly regulated. The pathology of asbestosis is very similar to that of idiopathic pulmonary fibrosis, from which it must be distinguished by the presence of asbestos bodies and/or associated asbestos pleural lesions. Less common pneumoconioses Less common pneumoconioses include those caused by nonfibrous silicates (such as talc, kaolin, or mica) or other minerals. Some compounds cause so-called benign pneumoconioses (e.g., siderosis, caused by iron dust), a term implying the lack of serious fibrosis and functional impairment. Some synthetic agents have also been associated with interstitial lung disease. Thus, exposure to polyvinyl chloride (PVC) dust has been shown to cause pneumoconiosis, and heavy exposure to synthetic microfibers (mainly nylon, but also polypropylene and polyethylene) can cause interstitial lung disease (‘flock worker’s lung’). Heavy exposure to aerosolized paints caused the Ardystil syndrome, a severe form of organizing pneumonia, in textile workers in Spain and in Algeria. Chronic Beryllium Disease and Hard-Metal/Cobalt Lung Disease

These diseases are not included among the mineral pneumoconioses here, because their occurrence does not appear to be based as much on dust accumulation, as on individual susceptibility. Chronic beryllium disease Chronic beryllium disease (CBD) or berylliosis is caused by sensitization to beryllium (Be), a light metal that is increasingly used in modern technology. CBD is a granulomatous lung disease and is clinically and pathologically similar to sarcoidosis. CBD is caused by a cell-mediated sensitization to Be, which can be demonstrated by proliferation of lymphocytes from peripheral blood or

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bronchoalveolar lavage upon incubation with Be salts. The HLA-DPb1 Glu69 allele confers a strong genetic susceptibility to develop CBD. Other agents (e.g., talc, zirconium) have been associated with granulomatous lung disease, masquerading as sarcoidosis. Hard-metal lung disease Hard-metal lung disease is a rare disease caused, in susceptible individuals, by exposure to cobalt (Co), as a component of hard metal (a composite based on tungsten carbide) or diamond–cobalt. Clinically the disease resembles hypersensitivity pneumonitis, although little evidence exists for an immune reaction against Co. In its most typical presentation, its pathology is characterized by giant cell interstitial pneumonia (GIP). The pathogenesis of hard-metal lung disease is not clear, but it may be related to the generation of oxidant species from the oxidation of Co. Extrinsic Allergic Alveolitis/Hypersensitivity Pneumonitis

Occupational causes of extrinsic allergic alveolitis or hypersensitivity pneumonitis are quite diverse. The more common etiologies consist of dusts originating from microorganisms (farmer’s lung, humidifier’s lung) or from birds (pigeon breeder’s lung, bird fancier’s lung). However, all environments where there is inhalation of bio-aerosols should be considered as carrying a risk of extrinsic allergic alveolitis. These include mushroom farms, composting installations, wood processing, vegetable storage, machining shops (through the use of machining fluids), etc. Some chemicals, most notably isocyanates, may also cause the condition. Occupational extrinsic allergic alveolitis has been studied most in farmers, in whom the disease is caused by sensitization to (thermophilic) microorganisms that grow in hay or other organic substrates. The frequency of farmer’s lung exhibits a considerable geographic variation, depending on climatic factors and farming practices, and the causative antigens also differ between regions. It is most frequent in the cold humid climates of northern Europe and America or in mountainous areas, such as the Doubs in France.

Occupational Infections Most respiratory infections are community-acquired, but sometimes they may be related to specific occupations. Common viral or, more rarely, bacterial infections may affect those working in crowded environments, schools, hospitals, and other communities. Immune-compromised subjects are at increased

risk of acquiring invasive fungal infections in some work environments. Tuberculosis is a well-recognized risk in health workers, but other categories of workers may be at risk, such as prison guards or social workers. Emerging infections pose a particular threat to hospital workers and their families, as shown by the recent outbreak of severe acute respiratory syndrome (SARS). Workers involved in maintaining (hot) water pipes, reservoirs, pumps, or fountains may be at risk of contracting Legionella pneumonia. Working in wild environments may lead to infections such as histoplasmosis, tularemia, or hantavirus pneumonia. Other zoonoses, such as anthrax, Q fever, ornithosis, avian influenza, affect workers in jobs involving direct or indirect contact with farm animals or birds. Moreover, dissemination of anthrax and other microorganisms by terrorists is a definite threat for various categories of workers, such as postal workers, maintenance workers, law enforcement personnel, and health workers. It is not established to what extent microorganisms and biological contaminants are responsible, together with indoor climate factors and volatile organic compounds, as well as psychosocial factors, for outbreaks of the ‘sick-building syndrome’. This syndrome refers to the occurrence, in a large proportion of the workforce, of non-specific work-related respiratory and other complaints among occupants of some buildings, particularly air-conditioned office buildings.

Chronic Obstructive Pulmonary Disease Although the dominant cause of COPD is cigarette smoking, there is little doubt that occupational exposures to mineral dusts, organic dusts, and irritant gases or vapors contribute to the incidence and the severity of chronic airways disease, including COPD. The quantitative contribution of occupational factors to the burden of COPD morbidity or mortality has been estimated at 15%. The most common respiratory manifestation of exposure to dusts or fumes consists of the presence of chronic bronchitis, that is, ‘industrial bronchitis’, which may or may not be associated with airflow limitation. Several longitudinal studies have shown that exposure to mine dust is associated with a loss of ventilatory function, even in the absence of pneumoconiosis. Other occupations with exposure to mineral dusts (such as building workers) or fumes (such as welders) are probably also at risk of occupationally induced COPD. Exposure to agricultural

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dusts (such as grain dust or vegetable fibers) is also a significant cause of chronic airway disease.

Bronchopulmonary Cancer As is the case for COPD, the most important exogenous factor in causing bronchopulmonary cancer is cigarette smoking. However, numerous epidemiological studies have investigated the role of occupational exposures in causing lung cancer, and despite the many difficulties of such studies, many occupational agents (or jobs) have been identified as definite or probable causes of lung cancer. Thus, asbestos fibers, some chromium(VI) compounds, arsenic, radon gas and its decay products (radon daughters), bis(chloromethyl)ether (BCME), and crystalline silica (occupational exposure) are well-established human lung carcinogens (belonging to category 1 of the International Agency for Research on Cancer (IARC)). Depending on the agent, as well as on methodological aspects, additive or multiplicative modes of interaction have been shown to operate with cigarette smoking. Established carcinogenic processes for the lung include coke production and coal gasification (possibly related to polycyclic aromatic hydrocarbons), iron and steel founding, paint manufacture and painting. Occupational exposure to diesel exhaust and environmental tobacco smoke are also probable causes of lung cancer, although the magnitude of the risk is smaller than that found for the established carcinogenic agents. The contribution of occupation to the causation of lung cancer has been shown to be considerably larger than for most other common cancers. The most frequently quoted estimate is 15% for males and 5% for females, and occupational asbestos exposure is considered the most influential factor.

Pleural Disease Occupational pleural disorders concern almost exclusively those who have had exposure to asbestos fibers (and perhaps also refractory ceramic fibers). Nonoccupational (domestic or environmental) exposures to industrial asbestos fibers or to mineral fibers such as tremolite may also cause such lesions. Asbestos-related pleural plaques are focal areas of essentially noncellular thickening of the (parietal) pleura; they are often bilateral and they may be calcified. They may occur even in people who have had only a light exposure to asbestos. It is generally accepted that the mere presence of asbestos-induced pleural plaques does not lead to symptoms or impairment and that such plaques are not precursors of a malignant evolution. However, pleural plaques

may be considered as fairly specific biomarkers of previous asbestos exposure. Asbestos may also cause nonmalignant pleural effusions, diffuse pleural fibrosis, and round atelectasis. Malignant mesothelioma is a pleural (or pericardial or peritoneal) tumor that is very specific for past asbestos exposure, either occupationally or environmentally. The latency between exposure and the manifestations of the tumor is usually 30 years or more; the tumor carries a very poor prognosis and may occur even after brief or low exposures. The incidence of mesothelioma has paralleled the industrial use of asbestos and its incidence will continue to increase until approximately 2010 to 2020 in most European countries. See also: Acute Respiratory Distress Syndrome. Asthma: Occupational Asthma (Including Byssinosis). CD1. Mesothelioma, Malignant. Occupational Diseases: Coal Workers’ Pneumoconiosis; Hard Metal Diseases – Berylliosis and Others; Inhalation Injury, Chemical; Asbestos-Related Lung Disease; Silicosis.

Further Reading Balmes J (chair), et al. (2003) Occupational contribution to the burden of airway disease: official statement of the American thoracic society. American Journal of Respiratory and Critical Care Medicine 167: 787–797. Bernstein IL, Chan-Yeung M, Malo J-L, and Bernstein DI (eds.) (1999) Asthma in the Workplace, 2nd edn. New York: Dekker. Bourke SJ, Dalphin JC, Boyd G, et al. (2001) Hypersensitivity pneumonitis: current concepts. European Respiratory Journal 18(supplement 32): 81s–92s. Doll R and Peto R (1981) The causes of cancer: quantitative estimates of avoidable risks of cancer in the US today. Journal of the National Cancer Institute 66: 1191–1308. Guidotti TL (chair) et al. (2004) Diagnosis and initial management of nonmalignant diseases related to asbestos. American Journal of Respiratory and Critical Care Medicine 171: 528–530. Heederik D (2000) Epidemiology of occupational respiratory diseases and risk factors. European Respiratory Monographs 15: 429–447. Hillerdal G (2002) Asbestos-related pleural disease including diffuse malignant mesothelioma. European Respiratory Monographs 22: 189–203. Malmberg P and Rask-Andersen A (1993) Organic dust toxic syndrome. Seminars in Respiratory Medicine 14: 38–48. Nemery B, Bast A, Behr J, et al. (2001) Interstitial lung disease induced by exogenous agents: factors governing susceptibility. European Respiratory Journal 18(supplement 32): 30s–42s. Nemery B, Verbeken EK, and Demedts M (2001) Giant cell interstitial pneumonia (hard metal lung disease–cobalt lung). Seminars in Respiratory and Critical Care Medicine 22: 435–447. Newman LS (1998) Metals that cause sarcoidosis. Seminars in Respiratory Infections 13: 212–220. Peto J, Decarli A, La Vecchia C, Levi F, and Negri E (1999) The European mesothelioma epidemic. British Journal of Cancer 79: 666–672. Redlich CA, Sparer J, and Cullen MR (1997) Sick-building syndrome. Lancet 349: 1013–1016.

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& 2006 Elsevier Ltd. All rights reserved.

annual UK rate for the recognition of CWP for state compensation in working and retired miners decreased from about 7% to 1–2%. The overall prevalence of CWP in US coal miners declined from about 12.7% to 3.9% between 1969 and 1988, but there were substantial regional differences. The overall prevalence of simple CWP is 2.8%; the highest rate of 14% occurs in workers with 30 years or more of mining experience. Similar regional differences and similar declines have been noted in the US and other countries.

Abstract

Etiology

Coal workers’ pneumoconiosis (CWP) is defined as the nonneoplastic reaction of the lung to inhaled coal-mine dust. It is characterized by nodular and/or coalescent opacities on chest X-ray. Symptoms are usually limited to dyspnea in advanced stages of the disease. Computed tomography better defines radiological abnormalities. Simple CWP has no significant effect on spirometric measures, whereas lung function in the more advanced stages of progressive massive fibrosis (PMF) shows an obstructive and restrictive pattern. Pathologically, simple CWP is associated with the macular and nodular lesions, whereas complicated CWP is associated with PMF (opacity lesion of 1 cm in diameter or more) and the lesions of rheumatoid pneumoconiosis. No specific treatment affects the course of CWP, though treatment options are available for complications such as tuberculosis and chronic hypoxemia. Supportive care includes bronchodilators in patients with obstructive syndrome, routine vaccination, antibiotics for exacerbations, and pulmonary rehabilitation.

Coal dust is not a mineral of fixed composition and comprises coal and quartz in various proportions. Coal is graded by rank, the rank reflecting its carbon content and thus coal quality and combustibility; anthracite is the highest ranked coal, with a carbon content of around 98%. Lower-ranked coals have carbon contents of around 90–95% carbon. The rank of coal has an influence on the risk of disease (higher-rank coals entail higher risk than lower-rank coals) and the progression of pneumoconiosis. Exposure to coal dust with a quartz concentration greater than 15% is associated with a high risk of a rapidly progressive form of pneumoconiosis that has the characteristics of silicosis. In open mines, dust levels rarely approach those of underground mines. Coal is currently actively mined in the US, UK, Western and Eastern Europe, India, China, South America, Australia, and Africa. There are three groups of factors that are known to influence the character and severity of lung tissue reaction to the mineral dusts. The risk of pneumoconiosis is related to the intensity and years of exposure. However, among a group of workers exposed to the same dust, only a fraction develop pneumoconiosis, because of an individual susceptibility. The nature and properties of each specific dust constitute the third factor under consideration; for each mineral, geometric and aerodynamic properties, chemistry, and surface properties have to be considered. In order to cause pneumoconiosis, particles must be small enough (0.5–5 mm) to reach the respiratory bronchioles and be deposited there.

Schenker MBC (1998) Respiratory health hazards in agriculture. American Journal of Respiratory and Critical Care Medicine 158: S1–S76. Wagner GR (1997) Asbestosis and silicosis. Lancet 349: 1311– 1315.

Coal Workers’ Pneumoconiosis B Wallaert, Hopital A. Calmette, Lille, France

Introduction Pneumoconiosis is defined as the accumulation of dust in the lungs and the reaction of tissues to its presence. The prolonged inhalation of coal-mine dust may result in the development of coal workers’ pneumoconiosis (CWP), silicosis, and industrial chronic bronchitis and emphysema, either singly or in various combinations. CWP is the term generally applied to interstitial disease of the lung resulting from chronic exposure to coal dust, whereas silicosis is due to inhalation of dust containing silica. The pneumoconioses differ in a number of ways from the acute allergic and toxic interstitial diseases that are associated with exposure to organic dusts, principally because of the long latency period (usually 10– 20 years or more) between exposure and recognition of the disease. CWP was first recognized in Scottish miners in 1830. In recent decades, the incidence of CWP has been declining in industrial countries due to improved dust controls, though increased mechanization in the mid-1960s led to a temporary increase in dust levels in some countries. Over the period 1950–80, the

Pathology The lesions of CWP are focal in nature. Simple CWP is associated with the macular and nodular lesions, whereas complicated CWP is associated with progressive massive fibrosis (PMF, opacity lesion of 1 cm in diameter or more) and the lesions of rheumatoid pneumoconiosis (Caplan’s syndrome). On gross

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examination the pleural surfaces in patients with coal dust exposure show an irregular pattern of bluish black pigmentation, often outlining the pleural lymphatics when dust exposure is fairly mild, but sometimes completely blackening the pleura when exposure has been heavy. Peribronchial, hilar, and paratracheal lymph nodes are enlarged, black, and firm. Microscopically, the lymph nodes show large numbers of pigmented histiocytes and a variable degree of fibrosis. The initial lesions in the lung are the coal dust macules, which correspond macroscopically to focal areas of black pigmentation. Microscopically, the macule is composed of macrophages laden with coal dust within the walls of the respiratory bronchioles and adjacent alveoli (Figure 1). Focal emphysema around the coal dust macule is sufficiently common to be considered as an integral part of the lesion of simple CWP. Focal emphysema is a form of centriacinar emphysema. Nodules may be the only lesions in the lung, but are more commonly seen against a background of macules. They are not confined to the respiratory bronchioles, but are also seen in the subpleural and peribronchial connective tissues. Nodules have round or irregular borders and may be surrounded by enlarged airspaces. There is a tendency for nodules to cluster and eventually coalesce to produce PMF. Nodules may be calcified. Histologically, they consist of dust-laden macrophages in a fibrotic stroma composed of collagen and reticulin (Figure 2). Nodules centered on respiratory bronchioles probably develop through progressive enlargment and collagenization of pre-existing macules.

PMF almost invariably occurs against a background of severe simple CWP (usually of nodular type) and is usually bilateral. PMF lesions appear as black fibrotic masses that may be round, oval, or irregular in shape; they are well demarcated from the surrounding lung but are not encapsulated. Satellites nodules are usually found in the adjacent lung. The lung and bronchovascular rays become markedly distorted; a characteristic feature of PMF is the tendency of the lesion to transgress normal anatomic boundaries, with fissures, bronchi, and vessels becoming obliterated as the lesion progresses. Microscopically, the lesions are composed of bundles of haphazardly arranged hyalinized collagen fibers and/ or reticulin fibers and coal dust. Dust particles near the periphery of the lesion are mainly found within macrophages, whereas in the center, the dust tends to lie free in clefts and cavities. Areas of liquefactive necrosis containing fragments of degenerating collagen and cholesterol crystals are frequently observed. A giant cell reaction may be seen in association with necrosis cells and collagen degradation. Miners with PMF have more severe emphysema than do those with less severe categories of CWP. All types of emphysema may be observed.

Pathogenesis The pathogenesis of pneumoconiosis is similar to that of all interstitial lung diseases. There is a chronic inflammatory state (alveolitis) in which inflammatory cells are activated and damage the pulmonary architecture. This produces fibrotic scarring. Inorganic particles are phagocytosed by alveolar

Figure 1 Macular lesion of coal workers’ pneumoconiosis. This coal macule consists of collections of macrophages that are laded with coal dust and extend into the connective tissue surrounding the respiratory bronchioles. Courtesy of Prof. M C Copin, Lille, France.

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Figure 2 Nodular lesion of coal workers’ pneumoconiosis. This early nodular lesion shows a smooth, sharp border, dust laden macrophages and laminated collagen deposition within the interstitium of the lung. The small central area is pale and rich in collagen, and the periphery contains a varying amount of fibrogenic dust. Courtesy of Prof. M C Copin, Lille, France.

macrophages, causing activation and the release of inflammatory mediators such as cytokines and arachidonic acid metabolites. The mediators in turn induce the recruitment of other inflammatory cells within the alveolar wall and on the alveolar epithelial surface. The alveolitis is dominated by alveolar macrophages. Toxic oxygen derivatives and proteolytic enzymes are released by the inflammatory cells, which cause cellular damage and disruption of the extracellular matrix. The inflammatory phase is followed by a reparative phase in which growth factors stimulate the recruitment and proliferation of mesenchymal cells and regulate neovascularization and re-epithelialization of injured tissues. During this phase, abnormal or uncontrolled reparative mechanisms may result in the development of fibrosis. Tumor necrosis factor alpha (TNF-a) seems to play a key role in the recruitment of inflammatory cells induced by toxic dusts. In addition, neutrophils recruited in the area of inflammation may contribute to the alveolitis, and respiratory and endothelial cells may play a further role by releasing inflammatory mediators like MIP-2, IL-8, and ICAM-1. Several studies demonstrate a key role of transforming growth factor beta (TGF-b) in the pathogenesis of lung fibrosis, and elevated TGF expression has been observed in CWP. Thus, increased levels of TNF-a and IL-1 have been observed in CWP. Several human studies demonstrated that alveolar macrophages of individuals with CWP spontaneously release significant amounts of growth factors such as platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and fibroblast growth factor (FGF). These mediators are involved

in the proliferative response of type II epithelial cells that occurs in PMF. Lastly, recent studies have also demonstrated an association between apoptosis and inflammation supporting a potential role for IL-1bdependent nitric oxide-mediated apoptosis in the development of pneumoconiosis.

Clinical Features Diagnosis

The diagnosis of CWP can be made using radiologic as well as pathologic criteria. CWP is usually first recognized from the plain chest radiograph, which is critical also in evaluating disease progression. Requirements for the diagnosis include a history of significant exposure, radiographic features consistent with these illnesses, and the absence of illnesses that may mimic these diseases (primarily infections with a predominantly miliary radiographic pattern, such as tuberculosis, fungal infections, or sarcoidosis). The radiographic appearances are most usefully described by the coding system devised for standard films of pneumoconiosis under the auspices of the International Labour Organization (ILO) (Table 1). In clinical practice, simple CWP is characterized by small rounded opacities (nodules) rather than small irregular opacities, though the latter may be seen in much lesser profusion. Surgical biopsy is very rarely required in the investigation of CWP. However, biopsy material (whether surgical, transbronchial, or from percutaneous fine needle aspiration) may be needed in special circumstances; for example, when there is uncertainty over the nature of conglomerate lesions in

194 OCCUPATIONAL DISEASES / Coal Workers’ Pneumoconiosis Table 1 International Labour Organization radiographic classification of pneumoconioses Small opacities (o10 mm in diameter) are defined by their average shape, size, and profusion: Rounded (nodular): p for diameters up to 1.5 mm q for diameters1.5–3 mm r for diameters 3–10 mm Irregular (reticular): s if fine t if medium u if coarse Profusion of these opacities is scored in four major categories defined by the standard films (0–3): Category 0 – small opacities are absent or less profuse than in category 1 Category 1 – small opacities are present but few in number Category 2 – small opacities are numerous Category 3 – small opacities are very numerous and obscure the normal radiographic markings Conglomeration, or coalescence, of these opacities to produce one or more opacities with diameters greater than 10 mm defines ‘complicated’ pneumoconiosis or progressive massive fibrosis Complicated pneumoconiosis (PMF) is divided into categories A, B, and C based on the size of the large opacities: A – sum of diameters of lesions is not more than 50 mm B – total area occupied by lesions is not greater than the area of the right upper lobe C – total area occupied by lesions is greater than the area of the right upper lobe

PMF or the nodular lesions in simple CWP (is lung cancer present?). Symptoms and Signs

Simple CWP and category A complicated CWP are not associated with respiratory symptoms. Breathlessness and cough in coal miners are usually a consequence of cigarette smoking. However, coalmine dust may itself cause chronic bronchitis and chronic obstructive pulmonary disease (COPD). By contrast, complicated pneumoconiosis (PMF) at categories B and C may present with undue breathlessness and productive cough. Melanoptysis is the result of necrosis within the conglomerate, coal-containing lesions that characterize PMF. Progressive undue exertional dyspnea is usually the dominant symptom, but rarely there may be breathlessness at rest. There are no specific abnormal physical signs in CWP, but when complicated CWP is very advanced the signs of emphysema or fibrotic lobar shrinkage may be detected. Finger-clubbing and fine inspiratory crackles are not features of the disease and if these are present, another explanation should be sought (especially hypersensitivity pneumonitis, idiopathic-like

pulmonary fibrosisy Only in a small proportion of severe cases of complicated disease does CWP evolve to produce chronic respiratory failure and cor pulmonale. When this does occur, extensive and multifocal conglomerate lesions are to be expected, generally with emphysema and concomitant disabling chronic airway obstruction. Associated Disorders

Emphysema and bronchitis There is a clear relationship between coal-mine dust and the development of emphysema. A further association with complicated CWP, if manifested by bullous emphysema, is spontaneous pneumothorax, though this is not likely to occur excessively in simple pneumoconiosis. The advanced stages of complicated CWP are additionally associated with recurrent episodes of acute and subacute bronchitis, as well as a regular productive cough. Persistent productive cough, shortness of breath, and wheezing are also common in coal miners in the absence of CWP; a significant dose–response relationship between coal-mine dust exposure and chronic bronchitis has been observed. Autoimmune diseases The autoimmune disorders rheumatoid disease and progressive systemic sclerosis may be associated with CWP. The latter, however, is more clearly associated with silicosis, and it is not clear whether it occurs with greater incidence than expected when there is occupational exposure to coal alone. The association of rheumatoid disease with CWP is known as Caplan’s syndrome. The diagnosis is suggested by the association of coal dust exposure, rheumatoid arthritis, and multiple well-defined, large, rounded opacities (nodules with diameter 10 mm) on the chest radiograph. Spontaneous disappearance is common, with or without initial cavitation, and new nodules commonly emerge in different locations. If the nodules become superimposed, suggesting conglomerate masses, PMF may be simulated, but often the radiological appearances of simple CWP are absent and there is a need to consider the possibility of primary lung tumor. There is no evident relationship between the severity of the rheumatoid disease and the extent of Caplan’s syndrome. Pathologically, the Caplan nodule is generally bigger than the nodule of complicated CWP, smoother in outline, and distributed at random within the lung fields. Caplan nodules histologically show a necrotic center with a peripheral palisade of histiocytes and giant cells. The Caplan nodule is indistinguishable pathologically from necrobiotic nodules of rheumatoid disease unassociated with coal dust exposure; it is more likely to cavitate,

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thereby producing a concentric ring pattern, and so is also known as a necrobiotic nodule. Infections CWP has also been linked with a number of specific infections, the most prominent of which historically has been tuberculosis. The association observed with coal mining (and hence CWP) in some countries appears to have been a consequence only of close contact during long hours of work in the confined mine environment. Nontuberculous mycobacteria, on the other hand, may infect lungs damaged by CWP with greater than usual frequency, and so CWP does appear to increase the risk for infection with opportunistic organisms. The development of mycobacterial infection in a patient with CWP may be symptomless, at least in its early course, and it may be difficult or impossible to distinguish it radiographically from PMF lesions since both characteristically begin in the apices. Similarly, it may be difficult to attribute changes in the radiographic appearances to advancing infection or progressive PMF. Other opportunistic infections have been reported in association with CWP, and Aspergillus species have been noted to colonize cavities in conglomerate lesions of complicated CWP.

Figure 3 Chest X-ray of simple coal workers’ pneumoconiosis showing a diffuse distribution of small rounded opacities, more prominent in the upper zones than the lower zones.

Lung cancer There is no evidence of a causal relationship between CWP and carcinoma of the lung, though there is strengthening evidence linking silica exposure with lung cancer. In 1996 International Agency for Research on Cancer (IARC) reclassified crystalline silica as a group I carcinogen stating that: ‘‘sufficient evidence of carcinogenicity indicates that there is a causal relationship between the exposure and human cancer.’’ Chest Radiology

The radiographic pattern of simple CWP is typically one of small rounded opacities (Figure 3), which appear first in the upper zones. The middle and lower zones become involved as the number of opacities increases. The nodules increase in profusion with increasing dust exposure; a change in profusion after dust exposure has ceased is very unusual. Calcification of the nodules may occur (10–20% of cases). Complicated pneumoconiosis is defined as a lesion of 1 cm or greater in longest diameter. The large opacities are usually predominant in the upper lobes (Figure 4), may be uni- or bilateral, and are symmetrically or asymmetrically distributed. The pattern of change in size is variable and unpredictable (Figure 5). Most PMF occurs on a background of simple pneumoconiosis, but this is not invariably so, and it may occur after dust exposure has ceased. Cavitation can develop

Figure 4 Chest X-ray showing progressive massive fibrosis, with multiple nodular opacities and discrete masses in the upper zones.

within a PMF lesion, and occasionally there is a dense peripheral arc or rim at its lower pole, which represents calcification. Dense calcification with the lesion is also sometimes seen. PMF is often associated with bullous emphysema and fibrotic scarring leading to distortion of the lung, and shift of the trachea and mediastinum to the affected side. Irregular, mainly basal, opacities may also be seen on standard radiographs. Large nodular opacities can be rheumatoid nodules of Caplan’s syndrome, and lung cancer must be kept in mind. Eggshell calcification as well

196 OCCUPATIONAL DISEASES / Coal Workers’ Pneumoconiosis Table 2 Differential diagnosis of coal workers’ pneumoconiosis based on nodular pattern on chest X-rays Infectious Tuberculosis Bacteria (staphylococcus, salmonella) Fungi Viruses (rubeola, varicella-zoster) Parasites (Schistosoma, Pneumocystis, Toxoplasma, Histoplasma) Immunologic Sarcoidosis Wegener Rheumatoid nodules Alveolar microlithiasis Gaucher’s disease Neoplastic Hematogenous metastases Hodgkin’s disease and lymphoma

Figure 5 Chest X-ray complicated CWP or PMF, stage C. The left mass has undergone necrosis. Note basal overdistension.

as pleural effusion are uncommon in CWP. The differential diagnosis of CWP is listed in Table 2. Computed tomography (CT) scans can show parenchymal lesions, which are not visible on normal chest radiographs. CT scans have greater sensitivity than plain radiographs in detecting simple CWP, but there is less obvious benefit for the detection of complicated pneumoconiosis. There is a posterior and right-sided predominance in the upper zones. Nodules are usually observed against a background of parenchymal micronodules and are generally associated with subpleural micronodules (Figure 6). Coalescence of subpleural micronodules produces ‘pseudoplaques’. Two categories of lesions can be observed in PMF: lesions with irregular borders that are associated with disruption of the pulmonary parenchyma and lead to typical scar emphysema (Figure 7); and lesions with regular borders that are not associated with scar emphysema (Figure 8). When the lesions are larger than 4 cm in diameter, irregular areas of aseptic necrosis can be observed with or without cavitation (Figure 9). CT, particularly high-resolution images, may additionally distinguish other pathological processes whether they are related to CWP or not. Focal lung emphysema indicates distension of the bronchioles within macules and is considered an integral part of the lesions of CWP. On the other hand, two major forms of emphysema occurring in coal workers can be detected on CT: bullous changes around PMF lesions referred to as paracicatricial or scar emphysema; and nonbullous emphysematous lesions

Inhalational Berylliosis Aluminum pneumoconiosis Other pneumoconiosis due to inorganic dusts Cardiovascular Hemosiderosis due to chronic postcapillary hypertension Thromboembolism Embolism from various particles

defined as centrilobular emphysema (Figure 10). Lesions of diffuse pulmonary fibrosis can be detected on high-resolution CT as honeycombing or areas of ground-glass attenuation (Figure 11). Two specific etiologies of fibrosis of coal miners should be considered: a direct effect of deposited coal or silica particles; or an indirect effect due to an association with scleroderma. Lung Function Testing

Simple CWP does not have a significant effect on spirometric measures, when prior dust exposure is taken into account and when smoking habits are also considered. Studies of lung function in the more advanced stages of PMF have shown an obstructive and restrictive pattern, compliance is usually somewhat decreased, diffusing capacity is usually reduced, and a slight reduction in arterial oxygen tension on effort may be observed. Oxygen desaturation is not present at rest or on moderate effort in the nonconglomerate stages of disease but cardiopulmonary exercise testing can add significant information in patients with early disease and mild impairment on resting studies. As in the case of radiographic progression, the changes in pulmonary function are more likely to occur in workers who have had intense exposure to

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Figure 6 High-resolution CT (HRCT) scan obtained at the level of the upper lobes showing bilateral parenchymal micronodules and coalescence in the right upper lobe. Note the presence of subpleural micronodules and pseudoplaques.

Figure 7 HRCT scan showing bilateral upper lobe masses consistent with progressive massive fibrosis. Note the background of nodules associated with bullous changes around PMF lesions referred to as paracicatricial emphysema.

dust. In addition it must be pointed out that miners who did not have CWP on chest radiography may exhibit lower FEV1 than controls, suggesting that coal dust may induce chronic obstructive pulmonary disease. In PMF, lung function depends on the extent of the lesions and associated emphysema. Ultimately, hypoxemic acute respiratory failure may occur; their long-term prognosis is poor.

Biological Tests

There are no specific biological features for pneumoconiosis. However, immunological abnormalities are now well described due to the presence of positive circulating antinuclear antibodies and rheumatoid factor. Serum immunoglobulins IgA and IgG have significantly raised levels in miners with

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Figure 8 HRCT scan showing right upper lobe mass with regular borders without emphysema.

Figure 9 HRCT scan showing bilateral masses with necrosis and cavitation of the right mass. Note bilateral emphysema.

pneumoconiosis, compared to non-miners. Finally, increased serum angiotensin-converting enzyme levels were observed in 45% of pneumoconiotic coal miners, whatever the radiological classification of pneumoconiosis. Bronchoalveolar lavage usually demonstrated an increase in cell numbers without change in differential cell count, in contrast to a number of other interstitial disorders of the lung.

directly due to complicated pneumoconiosis, especially in miners and ex-miners with category B or C complicated CWP. The rate of progression to PMF appears to be influenced chiefly by the age at which the miner begins to show radiographic changes of CWP reflecting individual susceptibility and the level of cumulative exposure.

Prognosis

Management and Current Therapy

Simple CWP is not associated with premature mortality; approximately 4% of deaths in coal miners are

No specific treatment affects the course of CWP, though treatment options are available for

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Figure 10 HRCT scan showing small bullae and low attenuation, without PMF lesions, defined as centrilobular (non-paracicatricial) emphysema. Courtesy of Prof. M Remy-Jardin, Lille, France.

Figure 11 HRCT scan obtained at the level of lower lobes in a patient with simple pneumoconiosis and systemic sclerosis showing ground-glass attenuation and honeycombing in the subpleural zone.

complications such as tuberculosis and chronic hypoxemia. Supportive care includes bronchodilators in patients with obstructive syndrome, routine vaccination, antibiotics for exacerbations, and pulmonary rehabilitation. When a miner is found to have CWP, further dust exposure should be excluded. Simple pneumoconiosis does not necessarily require complete exclusion from mining, especially in older subjects; however, if

PMF is detected, further exposure to coal dust should be avoided. Pulmonary function tests give additional information since the development of an obstructive ventilatory defect (due to dust exposure) may occur in the absence of CWP. In all patients who smoke, advice and support in smoking cessation should be given. If a physician concludes there is disablement from CWP, the patient should then be given advice on how to seek compensation.

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The prevention of pneumoconiosis depends on keeping exposure to concentrations of ambient dust at levels known to be associated with minimal and acceptable risk. Dust control is achieved primarily by ventilation, though water sprayed at points of dust generation is a useful technique for dust suppression. When each process is limited by practical constraints in unusual situations, the miner can be provided with respiratory protection equipment or the duration of exposure can be limited. The effectiveness of such measures should be monitored by regular assessment of dust concentrations, and regular clinical and radiological surveillance of the workforce. Static samplers are commonly used at the coalface and other potential sites of exposure to monitor exposure levels, the respirable particles of 1–7 mm diameter being collected by size-selective, gravimetric elutriators. Personal samplers worn by individual miners can also be used when necessary. Surveillance allows early recognition of workers with simple pneumoconiosis, who are likely to be those with greatest susceptibility, so that ongoing exposure can be restricted (perhaps by transfer to jobs with lower exposure) and the risk of future disablement from PMF reduced. Permissible levels of respirable dust vary among countries, though they are often based on the same epidemiologic studies. The following standards are currently in use: * * * *

US: 1 mg m
3 UK: 3.8 mg m
3 Australia: 3 mg m
3 Germany: 4 mg m
3

Variability in individual susceptibility is likely to be an important determinant for CWP, as is the case for most occupational disorders, and a number of predictive factors may be useful in identifying miners with higher than average risk: initially expiratory wheezes, an obstructive pattern of lung function, and many micronodules on CT scans, suggesting a predictive value for the initial CT abnormalities, and for these early symptoms and lung function abnormalities. An alternative approach for the future might involve genetic screening, which may provide markers of undue individual susceptibility. TNF-a is known to be important in the development of fibrosis due to silica exposure, and is probably relevant to the development of PMF in coal miners. A recent study showed a polymorphism in the promoter of the TNF-a gene in coal miners with a predominance of the genotype A308 in miners with PMF, compared to simple pneumoconiosis.

A further study has shown an increased plasma level of soluble TNF-a receptor in coal miners with pneumoconiosis. In any event, control of exposure levels alone is likely to prevent most cases of disabling PMF, and it has been predicted that an exposure concentration over 35 working years that does not exceed an average of 4.3 mg m
3 is associated with a probability for the development of category 2 or more CWP of no more than 3.4%. This represents a dramatic reduction in risk over the last 50 years. See also: Bronchiectasis. Bronchiolitis. Chronic Obstructive Pulmonary Disease: Emphysema, Alpha1-Antitrypsin Deficiency; Emphysema, General. Lung Anatomy (Including the Aging Lung). Occupational Diseases: Silicosis. Panbronchiolitis. Trauma, Chest: Subcutaneous Emphysema. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Begin R, Cantin A, and Masse´ S (1989) Recent advances in the pathogenesis and clinical assessment of mineral dust pneumoconioses: asbestosis, silicosis and coal pneumoconiosis. European Respiratory Journal 2: 988–1001. Brichet A, Salez F, Lamblin C, and Wallaert B (1999) Coal workers’ pneumoconiosis and silicosis. Occupational Lung Disorders, Monograph 11. European Respiratory Monograph 4: 136–157. Coggon D and Newmann-Taylor A (1998) Coal mining and chronic obstructive pulmonary disease: a review of the evidence. Thorax 53: 398–407. Green FHY (1998) Coal workers’ pneumoconiosis and pneumoconiosis due to other carbonaceous dusts. In: Churg A and Green FHY (eds.) Pathology of Occupational Lung Disease, 2nd edn., pp. 129–208. Williams and Wilkins. International Agency for Research on Cancer (1996) Silica, Some Silicates, Coal Dust and Para-amid Fibrils. Lyon: IARC. Morgan WKC and Lapp NL (1976) Respiratory disease in coal miners. State of the art. American Review of Respiratory Diseases 113: 531. National Institute for Occupational Safety and Health (1995) Criteria for a recommended standard: occupational exposure to respirable coal mine dust. National Institute for Occupational Safety and Health Publication 95–106: 1–336. Parkes WR (1994) Pneumoconiosis associated with coal and other carbonaceous materials. In: Parkes WR (ed.) Occupational Lung Disorders, 3rd edn., pp. 366–368. London: Butterworths. Remy-Jardin M, Remy J, Farre I, and Marquette CH (1992) Computed tomography evaluation of silicosis and coal workers’ pneumoconiosis. Radiologic Clinics of North America 30: 1155–1176. Rom WM and Crystal RG (1991) Consequences of chronic inorganic dust exposure. In: Crystal RG, West JB, et al. (eds.) The Lung: Scientific Foundations. New York: Raven Press. Schins RPF and Borm PJ (1999) Mechanisms and mediators in coal dust induced toxicity: a review. Annals of Occupational Hygiene 43: 7–33. Seaton A (1995) Coal workers’ pneumoconiosis. In: Morgan WK and Seaton A (eds.) Occupational Lung Diseases, 3rd edn., pp. 374–406. London: Saunders.

OCCUPATIONAL DISEASES / Hard Metal Diseases – Berylliosis and Others 201 Vanhee D, Gosset P, Boitelle A, Wallaert B, and Tonnel AB (1995) Cytokines and cytokine network in silicosis and coal workers’ pneumoconiosis. European Respiratory Journal 8: 1–9.

Hard Metal Diseases – Berylliosis and Others L A Maier and T Barnes, National Jewish Medical and Research Center, Denver, CO, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Exposure to metals in the workplace can result in a spectrum of lung diseases from a self-limited metal-fume fever to asthma to interstitial lung disease. Berylliosis, or chronic beryllium disease (CBD), is an immune-mediated pulmonary granulomatous disease that results from the inhalation of respirable particulate beryllium. Of those workers exposed, some will develop an immune sensitivity to beryllium without disease known as beryllium sensitization (BeS), while others will develop the chronic disease. Increased risk of disease is evident in machinists producing a fine beryllium aerosol, and in individuals with a specific human leukocyte antigen class II HLA-DPB1 with a glutamic acid residue at amino acid position 69. It is likely that gene-by-environment interactions are important in disease pathogenesis, although this has not been well defined to date. The cell-mediated (type IV) hypersensitivity reaction in which beryllium probably functions as a hapten, results from the interaction of class II restricted antigen-presenting cells and CD4 þ cells. Release of the cytokines tumor necrosis factor alpha (TNF-a) and interferon gamma (IFN-g) promotes proliferation and local accumulation of beryllium-specific CD4 þ T cells leading to progressive granuloma formation and end-stage fibrosis. The proliferative response to beryllium is assessed in a diagnostic test, the beryllium lymphocyte proliferation test (BeLPT), which detects BeS, a precursor to CBD, and CBD at an early stage, and differentiates it from other lung diseases. Individuals with BeS have no evidence of pulmonary impairment, while individuals with CBD may have a variable manifestation. The disease presentation ranges from asymptomatic individuals without evidence of radiographic or physiologic impairment to those with severe disabling symptoms and physiology along with chest radiographic abnormalities consistent with an interstitial disease. Although there is no cure for CBD, standard of care includes removal from exposure and use of immunosuppressant agents, such as corticosteroids. To reduce the burden of disease, exposure to beryllium should be reduced to levels below the current standards and the BeLPT should be used in medical surveillance to detect BeS and CBD at an early stage, before irreversible impairment occurs.

Introduction Metals are encountered in many industries today and can result in a spectrum of lung diseases. Beryllium (Be) is used in numerous high technological settings

because of its light weight, stiffness, corrosion resistance, and conduction properties. Upon the introduction of beryllium use in industrial settings in the early part of the twentieth century, cases of the granulomatous lung disease, chronic beryllium disease (CBD), became apparent. It is frequently misdiagnosed as sarcoidosis, a granulomatous lung disease of unknown etiology. A test, the beryllium lymphocyte proliferation test (BeLPT), can differentiate CBD from other granulomatous lung diseases. The BeLPT also detects beryllium sensitization (BeS), in which individuals have been exposed to Be and have developed a cell-mediated immune response in the blood, but do not have evidence of granulomatous lung disease. We now understand that inhalation of Be leads to a cell-mediated (delayed, type IV) hypersensitivity reaction that initially results in BeS, and later (in some) in the formation of noncaseating granulomas or CBD, and in a subset, pulmonary fibrosis and death. This cell-mediated immune response is dependent on the interaction of human leukocyte antigen (HLA) class II antigen-presenting cells (APCs) and CD4 þ T cells. In fact, an HLADPB1 with a glutamic acid at amino acid position 69 (Glu69) is found in the vast majority of BeS and CBD patients studied, indicating that Glu69 modifies susceptibility to Be-related health effects. This article will focus on our current understanding of the etiologic factors, pathology, clinical features, and pathogenesis of CBD and BeS, and will briefly describe other metal induced lung diseases, such as asthma and interstitial lung disease (ILD).

Chronic Beryllium Disease and Beryllium Sensitization Etiology

Beryllium exposure and CBD Soon after Be was first used in industry, outbreaks of the granulomatous lung disease CBD became apparent. Initially, Be was primarily used in the manufacture of nuclear weapons. Exposure to Be now occurs in numerous other industries, including aerospace, automotive, electronics, jewelry, metal production such as Be and other precious metals, military, nuclear energy, recycling, and telecommunications. Cases of CBD have been reported from all continents. Estimates of workers exposed are unclear, ranging from 200 000– 800 000 workers in the US alone. These numbers likely underestimate the workers currently and previously exposed. Exposure to respirable particulate from pure Be metal, beryllium oxide, beryllium ceramic, and beryllium alloys, including beryllium– aluminum, beryllium–nickel, and beryllium–copper

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alloys containing as little as 2% Be, can produce BeS and disease. Rates of BeS and CBD vary by workplace and job task, from 2–16% of exposed workers. Of those who are sensitized to Be, the rate of CBD also varies with between 30% and 100% of workers with an abnormal BeLPT demonstrating evidence of CBD. Numerous studies have demonstrated an increased risk of BeS and CBD in workers machining Be compared to those not involved in machining. Machining Be has been associated with higher exposures. However, BeS and CBD also occur in workers with apparent trivial exposure, including secretaries, security guards, and short-term contract workers. Risk of Be disease has not been limited to the workplace, as cases of CBD have been detected in residents of communities surrounding Be facilities, and household contacts of Be workers. In an effort to reduce exposure to Be and cases of CBD, the US Occupational Safety and Health Administration adopted an 8 h time-weighted average exposure standard of 2 mg per m3, which is used in many other countries. This standard does not protect workers, as exposures at or below this standard result in BeS and CBD. It is unknown if there is an exposure level at which CBD or BeS does not occur. Recent studies indicate that CBD and BeS can occur at levels less than 0.1 mg per m3. To reduce exposures in the community, a 24 h exposure standard was set at 0.01 mg per m3 averaged over a 30-day period. Whether this exposure standard is protective for the community is also unclear. Genetic influences in CBD and BeS Although some evidence indicates a dose–response relationship in certain cases, there are other factors modifying this relationship, which likely includes genetic susceptibility. There are numerous lines of evidence supporting a genetic predisposition to CBD, including reports of family members with disease and animal models with varying susceptibility based on varying HLA background. Early studies of the immunopathogenesis of CBD indicated that this was an HLA class II-mediated disease, as antibodies to class II could block the proliferative response to Be, while those to class I could not. In a landmark article, Richeldi and co-workers observed an increase in the frequency of an HLA-DPB1 variant associated with CBD with a glutamic acid at amino acid position 69 (Glu69) compared to Be-exposed nondiseased workers. A number of other studies have since confirmed a genetic association between Glu69 and CBD, and have shown an association with BeS at a similar frequency of about 80–85% compared to 30–40% of the controls. This suggests that Glu69 is a marker of the immune response to Be and not specifically a

disease. Because of the high prevalence of the Glu69 variant in nondiseased workers, this genetic test has a low positive predictive value and thus is not a good screening test to be used to detect CBD or BeS clinically at this time. Other genetic factors are likely important in the development of BeS and CBD, although these have been less well studied. Studies to date suggest that a tumor necrosis factor (TNF) promoter polymorphism associated with higher Be-stimulated TNF-a is also associated with CBD and BeS. Defining additional genetic susceptibility factors will help illuminate the pathogenesis of BeS and disease. In addition, the combination of exposure and genetic variables and their interaction will likely be most important in defining disease risk. Pathology

Today, lung pathology in CBD is usually obtained by bronchoscopy via a transbronchial biopsy, although occasionally it can be done by lung or lymph node biopsy. The pathologic hallmark of CBD is the noncaseating granuloma (Figure 1). The granuloma of CBD is indistinguishable from those of other noninfectious granulomatous lung diseases, such as sarcoidosis or hypersensitivity pneumonitis. In addition to the granuloma, other pathologic features of CBD include a mononuclear cell interstitial infiltrate and fibrosis. While a finding of granulomas is frequently used to make a diagnosis of CBD, open-lung biopsy pathology review from a large number of CBD cases revealed poorly formed or no granulomas present in up to 50% of cases. The absence of granulomas in

Figure 1 A low-power (10X) view of granulomatous inflammation from a patient with chronic beryllium disease reveals typical noncaseating granulomas with multinucleated giant cells and a core of epithelioid cells, macrophages, and lymphocytes, surrounded by a rim of dense lymphocytes. The surrounding tissue reveals evidence of a mononuclear cell interstitial infiltrate and thickened alveolar septae.

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the correct clinical setting does not exclude a diagnosis of CBD. Pathogenesis

Innate immune response It is thought that the initial host response to Be inhalation results from its direct chemical toxicity. The known toxic effects of Be in the lung include alveolar epithelial cell injury, vascular epithelial cell injury, changes in alveolar–capillary permeability, impairment of vascular endothelial functions, and changes in complement activity. In animal models, acute Be exposure is followed by an alveolar inflammatory response characterized by the infiltration of mononuclear cells. Acquired immune response Alveolar inflammation provides an influx of APCs including macrophages and dendritic cells. The interaction of Be-presenting APCs and naive T cells results in the clonal expansion of T cells that recognize the Be/major histocompatibility complex (MHC) (Figure 2). Specifically, antibodies to HLA-DP can block Be-stimulated proliferation and cytokine production, demonstrating the MHC class II restriction of this response. The majority of the T cells are CD4 þ , T-helper-1 (Th1). Analysis of the T-cell receptor (TCR) a- and b-chain genes expressed by CBD bronchoalveolar lavage (BAL) CD4 þ T cells indicates an oligoclonal expansion of a subset of T cells in the lungs of CBD subjects persisting over time. These findings suggest that there are specific clonal T cells responding to Be over time, likely due to persistent antigen stimulation. The exact nature of the beryllium antigen remains unclear. The small size of the Be atom (ionic radius, 0.31 A˚) would appear to preclude its function as a stand-alone antigen. Modeling of the DPB1 residues thought to be important in antigen binding indicate that they may be buried deep within the peptidebinding groove. Thus, Be may act as a hapten in concert with an additional peptide, although the identity of this peptide is unknown. Computer modeling indicates that beryllium clusters bound to several carboxylate-rich regions within the peptide-binding cleft serve as an integral part of the antigen, and may even act solely as antigen. Another study, based on binding competition, indicates that Be may well interact directly with the highly important HLA-DPB1 Glu69 residue. Whatever the nature of the beryllium antigen, the diverse TCR Vb repertoire among Be-specific CD4 þ T cells suggests that a variety of HLA/ beryllium complexes are recognized by CD4 þ T cells. Once activated, the Be-specific CD4 þ Th1 cells proliferate and this response detected in the blood

is used to diagnose BeS. During progression from BeS to CBD, it is likely that additional Be-specific CD4 þ Th1 cells along with other inflammatory cells are recruited to the lung. This process is poorly understood, but is presumably mediated by chemokines and cytokines (e.g., RANTES, MIP1-a, MCP-1, IL-8) and results in the upregulation of various adhesion molecules on both leukocyte and endothelial cells (e.g., ICAM-1). It is interesting to note that T cells have been shown to migrate in direct response to in vitro Be-stimulation. Examination of CBD BAL CD4 þ T cells reveals a surface-marker pattern indicative of T-effector memory (TEM) cells – a terminally differentiated subpopulation of Tmemory cells that express CD45RO, lack the lymph node-homing receptors CD62L and CCR7, and immediately secrete cytokine following antigen-specific stimulation. Cytokine secretion Analysis of both unstimulated ex vivo CBD BAL cells and Be-stimulated in vitro CBD BAL cells indicates that the mRNA and protein levels for several important cytokines are elevated following Be exposure. In addition to the Th1-type cytokines IL-2, IFN-g, and TNF-a, cytokines with elevated levels also include IL-6, IL-10, IL-18, and granulocyte-macrophage colony-stimulating factor. The levels of IL-1b, IL-4, IL-7, and IL-12 have been measured but are unresponsive to Be-stimulation. The Th1 cytokine response to Be is only minimally affected by the in vitro modulation of IL-2 (a strong T-cell proliferation signal), IL-4 (a Th2 cytokine), or IL-10 (a potent downregulator of proinflammatory cytokine synthesis). Granuloma formation Granuloma formation is complex and not fully understood. It involves a variety of mechanisms acting in concert to bring about an inflammatory lesion that is able to contain and destroy intracellular pathogens. The cytokines IFN-g, TNF-a are known to play critical roles, and are important in CBD pathogenesis. IFN-g promotes a number of responses relevant to granuloma formation, including macrophage activation, T-cell adhesion and Th1 differentiation. TNF-a also promotes adhesion molecule upregulation as well as macrophage activation and cellular apoptosis. Apoptosis and antigen persistence Central to our understanding of inflammatory granuloma formation is the persistence of antigen. Studies have shown that Be persists within the lungs of affected individuals for many years after exposure has ceased. A possible explanation for the persistence of Be is its

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Be CD28 Naive T cell

TCR

B7 Be Be Ag

Be/Ag processing HLA II

APC

CD4

(1)

Be CD45RO Effector memory T cell

TCR

Be/Ag processing Be Be Ag

HLA II

APC

CD4

(2)

IL-2 IFN- TNF-


IL-6 TNF-


(3) Figure 2 This figure outlines the interaction of beryllium-antigen presenting cells and beryllium-specific CD4 þ Th1 cells, which leads to the formation of noncaseating granulomas, granulomatous inflammation, and pulmonary fibrosis in CBD. (1) Beryllium is most likely internally processed by the antigen-presenting cells (APC) prior to presentation on the cell surface. Initial presentation leads to the activation of naive T cells specific for beryllium. Following T-cell differentiation and proliferation, beryllium-specific CD4 þ Th1 cells return to the lung. (2) After additional beryllium stimulation, these effector memory cells proliferate and secrete various cytokines including IFN-g and TNF-a. (3) In a manner that is not well understood, IFN-g and TNF-a ,and a persistent antigen result in granuloma formation.

ability to induce apoptosis of the lung macrophage, which has been observed in recent studies. Pulmonary fibrosis Eventually, the granuloma may become encapsulated in a fibrotic rim. Little is known about the mechanisms that govern the transition from granulomatous inflammation to increasing fibrosis in CBD although a number of preliminary studies have identified the possible involvement of mast cells and/ or basic fibroblast growth factor.

Animal Models

A good animal model for CBD that mimics all aspects of disease, including BeS and granuloma formation, has not been identified. Dogs, following inhalation of Be, develop granulomatous lesions within the lung that include macrophage and epithelial cell hyperplasia, granulomas, fibrosis, and lymphocytic infiltrates, along with an immune response to Be in both blood and lung as determined by BeLPT, but the granulomatous

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lesions and Be-specific response resolves over time. Cynomolgus monkeys exposed to Be also exhibit granulomatous lesions and Be-specific immunity, although the long-term response is unknown. Following inhalation of Be, certain mice strains (A/J and C3H/ HeJ) demonstrate granulomatous lesions with interstitial infiltrates of CD4 þ lymphocytes and minimal to mild interstitial fibrosis, but without a Be-specific immune response. Attempts to produce granulomatous lesions in other mouse strains (BALB/c and C57BL6/J) have been unsuccessful suggesting that differences in the MHC may play an important role in how mice respond to Be. Clinical Features

BeLPT As indicated above, the diagnosis of BeS and CBD rely on the demonstration of an abnormal immune response to Be with an abnormal BeLPT. This test measures blood or BAL cells’ ability to proliferate (or incorporate tritiated thymidine) in the presence of Be. Cells from normal or unexposed individuals do not respond to Be on this test. The blood test is highly specific, with a low rate of false positives, and provides good sensitivity for BeS and CBD. It is affected by smoking, which can cause false negative results. The blood BeLPT has found wide application in medical surveillance of beryllium industries to detect CBD at an early stage of disease and to detect BeS. The BeLPT has helped provide the correct diagnosis of cases which were misdiagnosed as other lung diseases such as asthma, sarcoidosis, and silicosis. Finally, the BeLPT can help identify high-risk exposures and job tasks associated with BeS and CBD that may require engineering controls or other measures to reduce exposure and potentially reduce disease burden. BeS The use of the BeLPT has defined a population of workers who develop a cell-mediated immune response to Be, but do not have clinical or pathological features attributable to CBD. They are usually asymptomatic without evidence of radiographic or physiologic abnormality. Longitudinal studies indicate that BeS progresses to CBD at a rate of approximately 8% per year of follow-up. It is unclear if all individuals with BeS will eventually develop CBD. However, BeS individuals require follow-up to determine if they have progressed to CBD. CBD The current diagnosis of CBD requires the demonstration of an abnormal immune response to Be, usually with the BeLPT, or with a BAL LPT in conjunction with evidence of granulomatous inflammation on lung biopsy. Bronchoscopy with BAL is

the test of choice to confirm a diagnosis of CBD. This test allows evaluation of the BAL cells, their response to Be, and provides an opportunity to obtain lung biopsies using a lower-risk procedure than surgery. As part of the evaluation of the BAL cells, a cell count is assessed and may reveal increased numbers of white cells, with lymphocyte predominance. In addition to a cell count, the cells’ response to Be is assessed with a BAL LPT. The BAL abnormalities correlate with disease severity on average. The BAL LPT may be abnormal in some cases when the blood BeLPT is normal and thus helps confirm a diagnosis of CBD. In less severe disease, the BAL cell count and BAL LPT may be normal. A search for other diagnostic tests, which do not require the patient to undergo a bronchoscopy with BAL and transbronchial biopsies, is underway. CBD can occur within weeks after first exposure or years after exposure cessation. Today many individuals are referred for evaluation for CBD after BeS is detected through workforce medical surveillance with the BeLPT. Some are asymptomatic without radiographic or physiologic abnormality. Others present with significant non-specific respiratory symptoms, including cough, chest tightness or shortness of breath, and/or systemic symptoms of fatigue, night sweats, and weight loss. Physical exam may be normal or may reveal dry crackles on auscultation, lymphadenopathy, or skin changes. With more severe disease cyanosis, clubbing and findings of pulmonary hypertension, or cor pulmonale may be apparent. Individuals with CBD may demonstrate pulmonary function abnormalities consisting of an obstructive, restrictive, or a mixed process, or an isolated gas exchange abnormality with a reduced diffusion capacity for carbon monoxide (DLCO) on pulmonary function tests (PFTs). Exercise testing is the most sensitive indicator of a physiologic abnormality in CBD sometimes revealing defects in physiology when the complete PFTs are normal. The most common exercise defects present include reduced exercise capacity, decreased oxygen consumption, an abnormal fall in oxygen levels with exercise, widening alveolar–arterial gradient, and ventilatory limitation to exercise. Early on, a high physiologic dead space-totidal volume ratio (Vd/Vt) may be the only abnormality noted on exercise testing. Chest radiographs may be normal in CBD. When significant physiologic disease is present, the chest radiograph will usually reveal interstitial abnormalities consisting of ground-glass opacifications (Figure 3(b)), centrilobular nodularity (Figure 3(a)), and/or fibrosis (Figure 4). In more severe disease, consolidation of nodules or conglomerate masses may be evident and, eventually, frank honeycombing. High-resolution

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Figure 3 High-resolution computed tomography scans of patients with chronic beryllium disease reveal (a) scattered parenchymal and subpleural nodularity, with coalescence of nodules and formation of a conglomerate mass, with some calcification, along with septal thickening and fibrosis, and (b) diffuse areas of patchy ground-glass opacification, bronchial-wall thickening, and few linear opacities.

of respiratory failure. By detecting CBD at an earlier stage and following the disease progression closely, we are able to intervene with therapy earlier and halt the progression of the disease. Management and Current Therapy

Figure 4 Posteroanterior chest radiograph of a patient with chronic beryllium disease reveals reticulonodular densities apparent in all lung fields with evidence of fibrosis, traction bronchiectasis, and volume loss, along with enlarged pulmonary arteries consistent with pulmonary hypertension.

computed tomography (HRCT) is more sensitive for abnormalities of CBD than routine chest radiography. Other non-specific findings may be present such as bronchial wall thickening, although this is not pathognomic of CBD. If an individual is unable to undergo bronchoscopy for medical reasons, findings of radiographic abnormalities consistent with CBD either on HRCT or routine chest radiography and an abnormal BeLPT maybe used to establish a diagnosis of probable CBD. The natural history of CBD varies. Some individuals remain stable for years while others develop a progressive course. Once disease progression begins, the untreated disease usually results in severe respiratory impairment, sometimes with the development

Unfortunately there is no cure for CBD or BeS. Removal from exposure or at least minimization of exposure is recommended for workers with BeS and CBD. Individuals with BeS should be informed of their risk of progression to CBD and should be followed clinically. We recommend every two-year follow-up with tests to assess pulmonary abnormalities, including a history and physical exam, chest radiograph with International Labour Organization B reading to assess the extent of radiographic abnormality, PFTs to include a DLCO, and exercise testing. A bronchoscopy with BAL is often performed as part of this evaluation to determine definitively if the individual has progressed to CBD, depending on the individuals’ clinical presentation. Removal from exposure and clinical follow-up, usually at least yearly, with testing to assess organ impairment and the need for therapy are recommended for cases of CBD. The tests listed above in the follow-up of BeS are also obtained in CBD, with the exception of the bronchoscopy. Evidence of physiologic pulmonary impairment is an indication for medical therapy. Other indications for therapy include severe disabling symptoms, such as debilitating cough, progressive decline or significant abnormality in PFTs, exercise tolerance, gas exchange or other physiologic indicators, and/or evidence of pulmonary hypertension and cor pulmonale. Corticosteroids are the first line agents used to treat

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impairing disease. The goal of therapy is to reduce the inflammatory response to Be. As in the treatment of sarcoidosis, corticosteroids are usually initiated daily or every other day at 40 mg per day for 3–6 months. The response to corticosteroids is assessed and the dose is tapered to a minimum dose required to maintain objective and symptomatic improvement. Corticosteroids are usually continued lifelong, because cessation of therapy usually results in disease relapse. Numerous case reports and clinical experience indicate that corticosteroids result in physiologic, radiographic, and symptomatic improvement. Unfortunately, corticosteroid side effects and medication failure do occur, occasionally necessitating the use of other immunosuppressive agents, such as methotrexate, to treat CBD. Other supportive measures, including supplemental oxygen, use of inhalers, and diuretics may be needed to treat CBD, depending on the severity of disease. Lung transplantation has been used for more severe cases of CBD. Routine preventive measures such as smoking cessation, yearly flu shot and periodic pneumococcal pneumonia vaccination, and regular exercise should be recommended for most cases of CBD. The best option to reduce Be disease burden is to prevent disease or detect it at an earlier stage to limit the development of severe disease. Workers and healthcare providers should be educated about the risk of Be exposure, so that clinical disease can be recognized early on. The BeLPT should be used in workplace medical monitoring to detect BeS and CBD at early stages. Medical surveillance information should also be used in combination with exposure monitoring to identify areas of high risk. In addition, general efforts should be made to reduce exposure to Be in the workplace, as currently recommended exposure limits do not adequately protect workers. Hopefully new, more protective exposure standards will be put in to place to help reduce exposure and resultant Be disease.

Hard-Metal Lung Disease Hard metal refers to a manmade material composed of tungsten carbide combined with small amounts of cobalt acting as a binder. It is 90–95% as strong as diamond. After this substance was used in industrial settings in Europe, cases of interstitial lung disease (ILD) were noted. Subsequently, it was determined that cobalt itself and not tungsten carbide, is the pathologic exposure resulting in ILD and, more commonly, in asthma. The production of hard metal, the maintenance and resharpening of hard metal/carbide tools, and the manufacture and use of diamond tools,

which are composed of up to 90% cobalt, result in ILD and asthma. The term hard-metal lung disease usually refers to the ILD, and not asthma. The pathogenesis of hardmetal lung disease is not well understood. Immunologic mechanisms are postulated to be important in the development of this disease process, although the exact mechanisms have not been elucidated. The clinical presentation of hard-metal lung disease varies: some workers may present with a more subacute form while others may manifest a more insidious progressive form of the disease. CT findings in hardmetal lung disease include ground-glass opacities, fibrotic changes similar to usual interstitial pneumonitis (UIP), and those suggestive of non-specific interstitial pneumonitis (NSIP). The diagnosis of the disease usually hinges on BAL evaluation or pathologic evaluation of a lung biopsy. Frequently, the diagnosis will be raised by the pathologist and then confirmed by the clinician. The BAL cells may reveal cannabilistic multinucleated giant cells. If present, these are diagnostic of hard-metal lung disease and eliminate the need for a lung biopsy. The classic pathologic findings of hard-metal lung disease are the presence of giant cells in the lung or giant cell interstitial pneumonitis. Unfortunately, not all patients with hard-metal lung disease have GIP and the absence of giant cells does not exclude this disease process. A clinical suspicion and determination of exposure to cobalt is needed to diagnose hardmetal lung disease. Once diagnosed, it is imperative to remove workers from any further exposure. Ongoing exposure is likely to result in progression of disease and even death in some cases. Clinical experience suggests that steroids may result in clinical, functional, and radiographic improvement in some cases.

Other Metal-Induced Lung Diseases As is exemplified in the case of cobalt and Be above, a diagnosis of occupational lung disease will impact the patient’s therapy and potentially prognosis, usually necessitating removal from work and resulting in medicolegal implications. Exposure to metals may result in a variety of occupational lung diseases from asthma (Table 1) to ILD (Table 2) and cancer (Table 3). The types of ILD due to metals vary from granulomatous inflammation to interstitial fibrosis (Table 2). Benign pneumoconiosis may result from exposure to antimony, barium, iron (siderosis), and tin (stannosis) in which radiographic abnormalities are present, but there is no evidence of physiologic lung impairment. Exposure to fumes from some metals, including cadmium, chromium, iron, manganese,

208 OCCUPATIONAL DISEASES / Inhalation Injury, Chemical Table 1 Metals causing asthma

Further Reading

Aluminum (potroom asthma) Chromium Cobalt Nickel Platinum Tungsten carbide Zinc

Finch GL, Hoover MD, Hahn FF, et al. (1996) Animal models of beryllium-induced lung disease. Environmental Health Perspectives 104: 973–979. Fontenot AP, Newman LS, and Kotzin BL (2001) Chronic beryllium disease: T cell recognition of a metal presented by HLADP. Clinical Immunology 100: 4–14. Glazer CS and Newman LS (2004) Occupational interstitial lung disease. Clinics in Chest Medicine 25: 467–478. Hardy HL (1965) Beryllium poisoning: lessons in control of man-made disease. New England Journal of Medicine 273: 1188–1199. Kreiss K, Mroz MM, Newman LS, Martyny J, and Zhen B (1996) Machining risk of beryllium disease and sensitization with median exposures below 2 mg/m3. American Journal of Industrial Medicine 30: 16–25. Kreiss K, Mroz MM, Zhen B, Martyny J, and Newman LS (1993) Epidemiology of beryllium sensitization and disease in nuclear workers. American Review of Respiratory Disease 148: 985– 991. Maier LA (2002) Clinical approach to chronic beryllium disease, and other non-pneumoconiotic interstitial lung diseases. Journal of Thoracic Imaging 17: 273–284. Maier L, Martyny J, Mroz M, et al. (2002) Genetic and environmental risk factors in beryllium sensitization and chronic beryllium disease. Chest 121: 81S–82S. Maier LA and Newman LS (1998) Beryllium disease. In: Rom WN (ed.) Environmental and Occupational Medicine, 3rd edn., pp. 1017–1031. Philadelphia, PA: Lippincott–Raven. Martyny J, Maier L, and Newman LS (2003) Beryllium related industries. In: Greenberg MI (ed.) Occupational, Industrial and Environmental Toxicology, 2nd edn., pp. 468–481. Philadelphia, PA: Harcourt Health Sciences. McCanlies EC, Kreiss K, Andrew M, and Weston A (2003) HLADPB1 and chronic beryllium disease: a HuGE review. American Journal of Epidemiology 157: 388–398. Richeldi L, Sorrentino R, and Saltini C (1993) HLA-DPb1 glutamate 69: a genetic marker of beryllium disease. Science 262: 242–244. Sawyer RT, Maier LA, Kittle LA, and Newman LS (2002) Chronic beryllium disease: a model interaction between innate and acquired immunity. International Immunopharmacology 2: 249– 261. Sterner JH and Eisenbud M (1951) Epidemiology of beryllium intoxication. Archives of Industrial Hygiene and Occupational Medicine 4: 123–151. Tinkle SS, Kittle LA, Schumacher BA, and Newman LS (1997) Beryllium induces IL-2 and IFN-gamma in berylliosis. Journal of Immunology 158: 518–526.

Table 2 Metals causing interstitial lung disease Aluminum: granulomatous inflammation Antimony: benign pneumconiosis Barium: benign pneumconiosis Beryllium (CBD): granulomatous inflammation Copper: granulomatous inflammation Cobalt (hard metal lung disease): giant cell interstitial pneumonitis Iron: benign pneumoconiosis Mercury: interstitial fibrosis post acute pneumonitis Tin: benign pneumoconiosis Titanium: granulomatous inflammation and interstitial fibrosis Zirconium: granulomatous inflammation

Table 3 Metals causing cancer Metal

Types of respiratory tract malignancy

Aluminum Arsenic Beryllium Cadmium Hexavalent chromium compounds Iron Nickel

Lung Lung Lung Lung and nasopharyngeal Lung Lung Nasopharyngeal, laryngeal and lung

magnesium, nickel, titanium, and zinc can cause metal-fume fever, a self-limited febrile response occurring within hours of exposure. Finally, exposure to cadmium and vanadium can cause emphysema. This discussion highlights the ability of metals to induce a variety of lung diseases, and need to exclude disease of known etiology in the diagnosis of these diseases, before they can be considered idiopathic. See also: Allergy: Allergic Reactions. Asthma: Occupational Asthma (Including Byssinosis). Bronchoalveolar Lavage. Bronchoscopy, General and Interventional. Genetics: Gene Association Studies. Hypoxia and Hypoxemia. Interstitial Lung Disease: Overview. Leukocytes: T cells; Pulmonary Macrophages. Occupational Diseases: Overview. Pulmonary Fibrosis. Tumor Necrosis Factor Alpha (TNF-a).

Inhalation Injury, Chemical B Nemery, Katholieke Universiteit Leuven, Leuven, Belgium & 2006 Elsevier Ltd. All rights reserved.

Abstract Inhalation injury may occur as a result of exposure to gases, vapors, or particulates in the workplace, at home or in the community. The nature and severity of disorders caused by

OCCUPATIONAL DISEASES / Inhalation Injury, Chemical 209 inhaled agents depend on the type of chemical and the amount inhaled. Inhaled agents that have a high solubility in water mainly affect the upper respiratory tract, whereas agents with low water solubility can reach the distal airways and alveoli. With aerosols, the degree of penetration depends mainly on the size of the particles, with the smallest particles having the greatest probability of reaching the distal airways. The clinical presentation of inhalation injury ranges from self-limited inhalation fever (metal fume fever, organic dust toxic syndrome, polymer fume fever) to life-threatening chemical pneumonitis with lung edema and evolution to acute respiratory distress syndrome. Following acute inhalation injury, various structural lesions and functional sequelae may persist.

Introduction Although many respiratory diseases result from damage caused by inhaling chemicals – for example, those present in cigarette smoke – the term ‘inhalation injury’ (‘or chemical-induced lung injury’) is usually restricted to airway or lung injury that results from acute exposure to high amounts of gases, vapors, or particulates. Such exposures occur mainly as a result of workplace accidents, but they may also occur at home or in the community, for instance as a result of fires and explosions, volcanic eruptions, industrial disasters, accidents involving trains or trucks transporting chemicals, as well as warfare or terrorism. Epidemiologic data about the occurrence of chemical-induced inhalation injury are few, and much knowledge in this area is based on anecdotal reports of accidental inhalation injuries in one or more individuals and by more or less well-defined chemicals. Poison control center data indicate that symptomatic inhalational exposures are common, particularly in the domestic environment, but severe acute and persistent morbidity is uncommon. In the SWORD (Surveillance of Work Related and Occupational Respiratory Disease) voluntary surveillance scheme in the UK, less than 10% of reported cases of occupational lung disease between 1990 and 1997 concerned inhalation accidents. However, inhalation accidents may take catastrophic proportions and involve hundreds of victims, as occurred after the explosion of a tank containing methyl isocyanate in Bhopal in 1984. Mass casualties with inhalation injuries may also result from chemical warfare, as in World War I or the Iran–Iraq war, and from conventional warfare or terrorist actions involving explosions, fires, and building destructions, as in the collapse of the World Trade Center on 11 September 2001. The clinical presentation and severity of inhalation injury ranges from self-limited inhalation fever to life-threatening chemical pneumonitis with lung edema and evolution to acute respiratory distress

syndrome (ARDS) and multiorgan failure. Following inhalation injury, the lesions may heal completely or there may be persisting structural or functional sequelae.

Etiology: General Considerations The nature and severity of the disorders caused by inhaled agents depend on the type of chemical and the amount inhaled. Inhalation injury may result from a great variety of specific substances, such as simple gases (sulfur dioxide, nitrogen dioxide, ozone, etc.), vapors or aerosols of inorganic compounds (acids, metallic agents, minerals, etc.), or organic chemicals (acids, aldehydes, isocyanates, solvents, pesticides, etc.). However, exposures often consist of complex mixtures of many diverse chemicals, which are present as gases and particles, as is the case for the smoke resulting from burning wood, plastics, or other materials. As a general rule, gaseous irritants that have a high solubility in water mainly affect the upper respiratory tract, causing rhinitis, pharyngitis, laryngitis, tracheitis, or bronchitis (Figure 1). Such water-soluble irritants are easily trapped in the aqueous surfaces of the upper respiratory tract and the eyes, where they cause rapid irritation thus leading the subject to avoid further exposure. In contrast, poorly water-soluble gases are not so well scrubbed by the upper respiratory tract and they can more easily reach the deep lung, where they may lead to pulmonary edema, usually after a latency of several hours. Moreover, as they cause much less sensory irritation, significant exposure to such gases may be tolerated without much trouble and even go relatively unnoticed. Consequently, such insoluble gases are more hazardous. Gases of intermediate solubilities mainly affect the upper respiratory tract and large bronchi, but high or prolonged exposures to these agents may also injure the lung and cause chemical pneumonitis. The latter may, in fact, also occur when massive quantities of highly water-soluble gases are inhaled. With aerosols, the degree of penetration into the respiratory tract depends mainly on the size of the particles, with the smallest particles (o5 mm aerodynamic diameter) having the greatest probability of reaching the distal airways and alveoli (Figure 2). Such small particles are often produced by combustion processes or by condensation of vapors. The particles may be toxic by themselves (e.g., cadmium oxide, CdO) or they may carry irritants, such as sulfates or aldehydes adsorbed onto soot particles. Chemical-induced lung injury may also be caused by routes other than inhalation. Thus, the lungs may be damaged by the aspiration of ingested liquids,

210 OCCUPATIONAL DISEASES / Inhalation Injury, Chemical

Water solubility

+++

Conducting zone

Trachea

z

→

Irritation

NH3, SO2, HCl, R-CHO, etc. rapid

0 1

BR BL

2 3 4

++

Cl2, CH3NCO, etc.

17 18 19 20 21 22 23

+

O3, NO2, COCl2, etc.

Transit. & Resp. Z.

TBL RBL AD AS

slow

Lipid soluble

Solvents, anesthetics C6H6, CHCl3, F3C-CHBrCl, etc.

Figure 1 The site of respiratory uptake of inhaled gases depends on their solubility in water. Gases with high water solubility ( þþþ ) generally cause rapid irritation and mainly affect the upper respiratory tract; gases with intermediate solubility ( þþ ) mainly affect the large airways; gases with low water solubility ( þ ) cause little sensory irritation and mainly affect the distal airways and alveoli, usually after a latency of several hours (slow); and gases with high lipid solubility (and low reactivity) pass readily into the blood. Reproduced from Medical Radiology – Diagnostic Imaging. Volume Imaging of Occupational and Environmental Disorders of the Chest, 2005, Chemical-induced lung injury and its long-term sequelae, Nemery B, figure 2.3.1., Berlin: Springer (in press), with kind permission of Springer Science and Business Media.

such as solvents or fuels. Some chemicals, most notably agrichemicals such as paraquat or cholinesterase inhibitors, may cause lung injury following ingestion or dermal absorption. Toxic pneumonitis may also result from inhaling or injecting illegal drugs or as a side effect from various drugs, but this will not be covered here.

Clinical Presentations and Their Specific Etiologies Inhalation Fever

Inhalation fever is the name given to cover a group of flu-like clinical syndromes, such as metal fume fever, the organic dust toxic syndrome (ODTS), and polymer fume fever: 1. Metal fume fever is caused by acute exposure to high amounts of some metallic fumes, most notably those emitted when heating zinc. 2. Organic dust toxic syndrome (ODTS) is caused by the inhalation of large quantities of agricultural

and other dusts of biologic origin. Such bio-aerosols are often heavily contaminated with microorganisms which produce toxins (e.g., bacterial endotoxin, mycotoxins) that are believed to contribute to the pathogenesis of the syndrome. The syndrome is also known by various other names, such as mycotoxicosis. 3. Polymer fume fever is a less common cause of inhalation fever. It occurs after exposure to the fumes that are produced when fluorine-containing polymers, such as polytetrafluoroethylene (PTFE, also known as Teflon) are heated above 3001C, and possibly also fumes evolving from heating other plastics. The clinical features of the inhalation fevers are best described as those of a beginning influenza. The actual exposure may or may not have been experienced as irritant or troublesome for the eyes and respiratory tract. Some 4–8 h after the exposure, the subject begins to feel unwell with fever, chills, headaches, malaise, nausea, and muscle aches. In the case of metal fume fever, there may be a metallic taste in the

OCCUPATIONAL DISEASES / Inhalation Injury, Chemical 211

Air velocity

Particle size

+++

Conducting zone

Trachea

z

1 BR BL

Impaction

>5 µm

Sedimentation

1−5 µm

0 ++

2 3 4 +

Transit. & Resp. Z.

TBL RBL AD AS

17 18 19 20 21 22 23

<0.2 µm 0

Diffusion <<0.1 µm

Figure 2 The site of deposition of inhaled particles depends on their size and air velocity in the respiratory tract. Particles with high aerodynamic diameter deposit, by impaction, in the upper respiratory tract; smaller particles deposit, mainly by sedimentation, in the large airways; and the smallest particles deposit, by Brownian diffusion, in the distal airways. Reproduced from Medical Radiology – Diagnostic Imaging. Volume Imaging of Occupational and Environmental Disorders of the Chest, 2005, Chemical-induced lung injury and its long-term sequelae, Nemery B, figure 2.3.2., Berlin: Springer (in press), with kind permission of Springer Science and Business Media.

mouth. Respiratory symptoms are usually mild and consist mainly of cough and/or sore throat, but occasionally subjects may have more severe responses with dyspnea. The body temperature may rise as high as 39–401C, but there are also less full-blown inhalation ‘fevers’, even without fever, but with malaise, headache or systemic symptoms. Diagnosis The diagnosis of inhalation fever rests essentially on the exposure history and the clinical condition, and when these clearly point to inhalation fever, no sophisticated investigations are required. In general, chest auscultation and chest X-ray are normal, but in the more severe cases, which are more likely to seek medical attention, crackles may be heard and there may be transient infiltrates on chest X-ray (in the latter circumstances, one would be justified to challenge the diagnosis of inhalation fever and consider that there is a real chemical pneumonitis). Although pulmonary function is often within normal limits, reductions in vital capacity and FEV1 are perhaps more common than generally accepted; in severe cases there may be a decrease in transfer factor and arterial hypoxemia. Increased peripheral blood leukocytosis, with a rise in

neutrophils, is a consistent finding until 24 h after the exposure; other blood tests should be normal, except probably for indices of an inflammatory response. Bronchoalveolar lavage studies have shown very pronounced and dose-dependent increase in polymorphonuclear leukocytes (between 19% and 63% of cells) on the day after exposure to zinc fumes or organic dust. It is important not to confuse inhalation fever with other more serious conditions, including chemical pneumonitis, which in its early phases could be mistaken for inhalation fever. A differential diagnosis must also be made with various types of infectious pneumonias or with acute extrinsic allergic alveolitis (EAA), in which fever, malaise and systemic symptoms occur. This is particularly relevant in the case of exposure to bio-aerosols, which is a common cause of acute EAA. Pathogenesis The pathogenesis of metal fume fever and ODTS is considered to be based on a non-specific, that is, nonallergic activation of macrophages or pulmonary epithelial cells with the local and systemic release of pyrogenic and chemotactic mediators. The mechanism of polymer fume fever is unknown and

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the exact components of the fume that cause the toxicity are also unknown. In principle, inhalation fever is a self-limited syndrome and recovery normally takes place after a night’s rest. Tolerance exists against re-exposures occurring shortly after a bout of metal fume fever or ODTS, but it seems that this feature is less typical of polymer fume fever. Adequate occupational hygiene should be instated to prevent the occurrence of inhalation fever. Acute Chemical Pneumonitis

The response to acute chemical injury in the respiratory tract is rarely compound specific. Following exposure to water-soluble irritants, there may be pronounced signs of upper airway irritation, with severe cough, hoarseness, stridor or wheezing, retrosternal pain, discharge of bronchial mucus, possibly with blood, mucosa tissue, and soot. Death may occur as a result of laryngeal edema. If the lung parenchyma is also involved, noncardiogenic pulmonary edema may develop over the course of several hours. It is important to realize that victims of serious inhalation accidents may feel perfectly well and walk into the infirmary or an emergency room, or even go home following the inhalation, and then experience progressive dyspnea, shallow breathing, cyanosis, frothy pink sputum, and eventually ventilatory failure. A clinical picture of ARDS may thus develop gradually over 4–72 h, even after a period of clinical improvement. In the days that follow severe acute inhalation injury, pulmonary infectious complications may also occur. Depending on the circumstances of the accident, there may be thermal or chemical facial burns, as well as signs of mucosal irritation and edema, and even hemorrhage and ulcerations in the air passages. Auscultation of the chest may or may not be abnormal, with wheezing, rhonchi, or crepitations. Pulmonary function can be used to monitor ambulatory subjects who have been exposed. Arterial blood gases show varying degrees of hypoxemia and respiratory acidosis, depending on the severity of the injury. The chest radiograph is usually normal if only the conducting airways are involved, but there may be signs of peribronchial cuffing. After exposure to deep lung irritants the chest radiograph is unremarkable in the first hours after presentation, but signs of interstitial and alveolar edema may become visible and, with time, patchy infiltrates, areas of atelectasis and even ‘white lungs’ may develop. These changes may be due to tissue damage and organization or they may reflect superimposed infectious (broncho) pneumonia.

The pathology of acute toxic pneumonitis is that of diffuse alveolar damage with epithelial disruption, interstitial and alveolar edema, hemorrhage and formation of hyaline membranes, and, depending on the stage of the lesions, varying degrees of infiltration by polymorphonuclear leukocytes, hyperplasia of the alveolar and bronchiolar epithelium, and interstitial or intra-alveolar fibrosis. In some instances, particularly in the later stages of chemical pneumonitis, there may be pathological (and radiological) features of organizing pneumonia (OP) or BOOP (bronchiolitis obliterans organizing pneumonia), as it is called by some. However, the body of evidence linking toxic exposures to the development of (BO)OP is not very large. Inhalation of nitrogen dioxide (NO2), as in silo filler’s disease, is the best documented cause of bronchiolitis obliterans. Following resolution of the acute pulmonary edema caused by massive exposure to NO2, a relapse in the clinical condition may occur after 2–6 weeks with dyspnea, cough, fine crackles, a radiographic picture of miliary nodular infiltrates, arterial hypoxemia, and a restrictive or mixed impairment, with low diffusion capacity. The site of cellular damage caused by NO2 is the centriacinar region, and the relapse phase has been attributed to bronchiolar scarring with peribronchiolar and obliterating fibrosis of the bronchioli. It is not clear whether these instances of (BO)OP result from the ‘normal’ repair of a particularly severe form of epithelial injury (i.e., with disruption of the basement membrane), or whether they result from an abnormal pattern of cellular proliferation in this critical region of the lung. Several anecdotal reports have reported that bronchiolitis obliterans could be caused by other inhaled agents, but in some instances, most notably after inhalation of water-soluble agents (sulfur dioxide (SO2) and ammonia (NH3)), the disease appears to consist of a constrictive bronchiolitis obliterans, rather than the ‘classical’ organizing bronchiolitis obliterans. As indicated above, a large number of inhaled agents can cause inhalation injury (Table 1). In the following paragraphs, specific categories of chemicals are briefly highlighted. Irritant gases and organic chemicals Highly water-soluble gases In general, gases with high water solubility, such as NH3, SO2, hydrocholoric acid (HCl), formaldehyde (HCHO), or acetic acid (CH3COOH), which also have good warning properties, only cause mild upper airway irritation, unless the exposure concentration (or duration) has been considerable. The latter may occur following explosions or major accidents in mines or chemical installations.

OCCUPATIONAL DISEASES / Inhalation Injury, Chemical 213 Table 1 Possible causes of toxic pneumonitis Cause

Examples

Irritant gases

High water solubility: NH3, SO2, HCl, etc. Moderate water solubility: Cl2, H2S, etc. Low water solubility: O3, NO2, COCl2, etc.

Organic chemicals

Organic acids: acetic acetic, etc. Aldehydes: formaldehyde, acrolein, etc. Isocyanates: methyl isocyanate (MIC), toluene diisocyanate (TDI) Amines: hydrazine, chloramines, etc. Tear gas (CS) and mustard gas Organic solvents, including some leather treatment sprays Some agrichemicals (paraquat, cholinesterase inhibitors)

Metallic compounds

Mercury vapors Metallic oxides: CdO, V2O5, MnO, Os3O4, etc. Halides: ZnCl2, TiCl4, SbCl5, UF6, etc. Ni(CO)4 Hydrides: B2H5, LiH, AsH3, SbH3

Complex mixtures

Fire smoke Pyrolysis products from plastics Solvent mixtures Spores and toxins from microorganisms

Intermediate water-soluble gases The same considerations apply to gases with intermediate water solubilities. Thus, accidental release of chlorine (Cl2) is probably one of the most frequent causes of inhalation injury, not only in industry, but also in the community as a result of transportation accidents or the use of chlorine for the disinfection of swimming pools. An important cause of toxic inhalation in the domestic setting is that which results from the mixing of bleach (NaClO) with acids, thus leading to the release of gaseous chlorine, or with ammonia, thus leading to the release of volatile chloramines (including trichloramine, NCl3). Hydrogen sulfide (H2S), which is formed by the putrefaction of organic material in sewage drains, manure pits, or ship holds, and is also a frequent contaminant in the petrochemical industry, has special properties as an irritant gas because it does not only cause mucosal irritation, but it also leads to chemical asphyxia by mechanisms that are somewhat similar to those of cyanide. Victims who survive massive inhalation of H2S may exhibit (hemorrhagic) pulmonary edema, as well as pneumonia in the days following the event. Methyl isocyanate (CH3CNO, MIC) has gained notoriety as the chemical that caused the highest number of casualties in a single accident, when it was released from a tank in a pesticide factory in Bhopal, India, in December 1994.

Poorly water-soluble gases As indicated above, the poorly water-soluble gases are the most hazardous ones because they are hardly noticed and they can penetrate down to the distal airways and cause delayed noncardiogenic pulmonary edema. NO2 is a reddish-brown gas, heavier than air, and is often incorrectly referred to as ‘nitrous fumes’. It may be encountered in a wide variety of occupational settings. A classical risk in agriculture is that of ‘silo filler’s disease’ (not to be confused with silo unloader’s syndrome’, which is a form of the organic dust syndrome). Silo filler’s disease occurs because NO2 is produced within a few days of fermentation of the silage, thus, posing a risk of fatal inhalation injury for anyone entering the silo. Fatally high quantities of NO2 may also be produced when special jet fuels explode, when tanks of nitric acid (HNO3) explode, when materials containing high quantities of nitrogen are burned in fires, or when nitric acid reacts with metals, wood, or other cellulose materials. Outbreaks of acute respiratory illness in players and spectators attending ice hockey matches have been attributed to NO2 as a result of malfunctioning ice resurfacing machines. Phosgene (carbonyl chloride, COCl2) is a well-known cause of pulmonary edema, since it was used in chemical warfare during World War. This chemical is used in chemical syntheses, notably in the manufacture of isocyanates, and it may be produced by thermal or ultraviolet decomposition of chlorine-containing chemicals, such as methylene chloride or trichloroethylene. Intentionally initating agents Some chemicals are made intentionally to cause respiratory irritation. The most common lachrymating agents, known as tear gases (although they are in fact aerosoldispersed chemicals), used in riot control operations or as personal antiharassment weapons, are ortho-chlorobenzylidene malononitrile (CS) and a-chloroacetophenone (CN). The action of these agents is usually short-lived and limited to the mucous membranes of the eyes and upper respiratory tract, but their use in confined spaces (e.g., prison cells) may lead to more serious lung damage. Lungdamaging chemical warfare agents include the choking agents (chlorine, phosgene, diphosgene, and chloropicrin) and the vesicants (or blister agents). Among the latter, mustard gas (sulfur mustard) caused severe bronchopulmonary damage in soldiers during the Iran–lraq war. Solvents Exposure to organic solvents is only rarely a cause of toxic pneumonitis. However, acute exposure to very high concentrations of solvent vapors in confined spaces (e.g., in chemical tanks) may be a

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cause of chemical pneumonitis and pulmonary edema, often in victims who have been unconscious. Pneumonia and respiratory distress syndrome caused by loss of alveolar surfactant may also result from the aspiration of intentionally (e.g., by fire eaters) or unintentionally (e.g., from siphoning petrol) ingested solvents or fuels. A special mention should also be given to cases of severe acute respiratory illness caused, often in the domestic environment, by acute exposure in confined spaces to fluorocarbon-containing water-proofing sprays and leather conditioners. Agrichemicals Some agrichemicals may cause toxic pneumonitis after noninhalatory exposure. The best known of these agents is the herbicide paraquat which exerts a selective toxicity for the pulmonary epithelium. Depending on dose, paraquat causes either multiorgan failure or a delayed pulmonary fibrosis, after ingestion or, more rarely, after dermal exposure. Poisoning by cholinesterase inhibitors, such as the organophosphate or carbamate insecticides, is also associated with significant respiratory symptoms such as bronchospasm, bronchorrhea, respiratory depression, and sometimes also (delayed) pulmonary edema. Metallic compounds In general, the principles governing the site and type of damage caused by inhaled agents also apply to metallic compounds, many of which may cause lung injury. Metallic compounds may be inhaled as fumes (i.e., generally as oxides), very fine particles, or as salts. Cadmium pneumonitis is perhaps the best-documented example of metal-induced acute pneumonitis. Cadmium may be liberated, often unknowingly to the worker, from the welding or burning of cadmium-containing alloys and cadmium-plated metal, from the use of hard solders, or from the smelting of zinc or lead (or scrap metal), which often contain significant levels of contaminating cadmium. As with other pneumotoxic agents, exposure to toxic levels of cadmium fumes does not necessarily lead to immediate respiratory symptoms, but symptoms of pneumonitis may start many hours after the exposure. Severe chemical pneumonitis may also result from exposure to high levels of mercury vapors, for example, when refining gold or silver (using amalgams) in confined spaces. Vanadium pentoxide (V2O5) may be present in significant quantities in slags from the steel industry (ferrovanadium) and, because some fuel oils contain high quantities of vanadium, in furnace residues from oil refineries or in soot from oil-fired boilers. Dust containing V2O5 may cause upper and lower

airway irritation: rhinitis with sneezing and nose bleeds, pharyngitis, acute tracheobronchitis, with cough, wheeze, and (possibly) airway hyperreactivity (‘boilermakers’ bronchitis), as well as possibly bronchopneumonia. Exposure to high levels of oxides of beryllium, cobalt, manganese, or osmium may cause airway irritation and even bronchopneumonia. Cases of adult respiratory distress syndrome, some with a protracted course, have been reported in military or civilian personnel accidentally exposed to smoke bombs which liberate zinc chloride (ZnCl2). Accidental exposure, for example, as a result of explosions, burst pipes, or leaks in chemical plants, to antimony trichloride (SbCl3) and antimony pentachloride (SbCl5), Zirconium tetrachloride (ZrCl4), titanium tetrachloride (TiCl4), and uranium hexafluoride (UF6) may also lead to severe and even fatal inhalation injury. Nickel carbonyl (Ni(CO)4) is a volatile liquid of very high toxicity for the lungs and brain. Lithium hydride (LiH), phosphine (hydrogen phosphide (PH3), used as a doping agent for the manufacture of silicon crystals, or released from aluminum phosphide grain fumigants or zinc phosphide rodenticides), hydrogen selenide (SeH3), and diborane (B2H5, used as high-energy fuel) have also been reported to cause acute inhalation injury with, possibly, pulmonary edema. New technologies, such as those involving the thermal spraying of metals, may also prove to be particularly hazardous. Complex mixtures Probably the commonest cause of toxic pneumonitis is smoke inhalation caused by domestic, industrial, or other fires. Respiratory morbidity is often the major complication in burn victims. It may be caused by direct thermal injury (particularly if hot vapors have been inhaled), but more generally the lesions are caused by chemical injury. The composition of smoke is highly complex and variable depending on the materials that are involved and the stages of the fire. The toxic components of smoke are numerous and involve gaseous asphyxiants (CO, HCN) and irritants, as well as particulates. Of particular concern are conditions which involve the burning or pyrolysis (e.g., caused by overheating) of plastics, such as polyurethanes, polyacrylates, and other polymers which are known to give off numerous, generally poorly characterized but potentially highly toxic chemicals. Subacute Toxic Pneumonitis

Although the concept of chemical-induced lung injury is used only for disorders resulting from a single,

OCCUPATIONAL DISEASES / Inhalation Injury, Chemical 215

acute exposure to a toxic chemical, the term ‘subacute toxic pneumonitis’ may be used to refer to conditions of lung injury caused by repeated peaks of toxic exposures or a more prolonged toxic exposure over weeks to months. The alveolar proteinosis caused by heavy exposure to silica (acute silicoproteinosis) and possibly by other agents would qualify for this entity. Similarly, some instances of exogenous lipoid pneumonitis seem to correspond well to the concept of subacute toxic pneumonitis. The pulmonary hemorrhagic syndrome associated with exposure to trimellitic anhydride, and possibly to methylene diphenyl diisocyanate, is possibly also a form of toxic response, but in this instance the role of specific immunological mechanisms is likely. The Ardystil syndrome represents the most convincing example up to now of subacute toxic pneumonitis. This outbreak of severe respiratory disease occurred in 1992 in Spain, and involved several workers from factories where textiles were airsprayed with dyes. Most of the affected workers had worked in a factory named Ardystil, hence the name given to the disease. Six subjects died from the disease over the course of a few months. A similar, though smaller, outbreak was also reported from Algeria. The clinical features of the disease included cough, epistaxis, dyspnea, and chest pain, as well as crackles on auscultation. Radiology showed patchy infiltrates in two-thirds of patients and a micronodular pattern in one-third of the studied patients. There was a restrictive functional impairment and rapid progression to irreversible respiratory failure, despite corticosteroid treatment, in several patients. The pathology was characterized by organizing pneumonia. Another recently described form of subacute toxic lung injury is ‘popcorn worker’s lung’. This severe lung disease, characterized as bronchiolitis obliterans, occurred in subjects occupationally exposed to vapors of butter flavoring (containing diacetyl) in factories making microwave-popcorn. Possible Sequelae of Acute Inhalation Injury

Following acute inhalation injury, there is often complete recovery. However, this is certainly not always the case. The experience with ARDS caused by etiologies other than acute inhalation injury suggests that a substantial proportion of patients recover with more or less severe dyspnea and functional impairment, often a reduced diffusing capacity. However, it is still not well known whether and how frequently pulmonary fibrosis occurs after diffuse lung injury of toxic origin. The possible occurrence of residual lesions in the airways is better documented, even

though the evidence is also often based on single instances only. Thus, various chronic sequelae such as constrictive bronchiolitis, bronchiectases, and other bronchial lesions, such as strictures or polyps, have been reported to result from acute inhalation injury, depending on the severity of the initial damage and perhaps also depending on treatment modalities, although very little hard data, let alone controlled studies, are available regarding the latter issue. Moreover, even in the absence of structural sequelae, which may be identified by imaging studies or through bronchoscopy, or in the absence of significant defects in basal spirometry, a state of permanent non-specific bronchial hyperreactivity may be observed. This condition of adult-onset, nonallergic asthma has been named reactive airways dysfunction syndrome (RADS) and occurs in a number of survivors of (severe) airway injury. The incidence and mechanisms giving rise to postinhalation asthma and irritant-induced asthma still remain to be elucidated. Recent observations in firefighters and other personnel involved in rescue operations during and following the collapse of the World Trade Center on 11 September 2001, suggest that RADS may occur in a high proportion of exposed subjects, even without the occurrence of clinically serious injury.

Management and Current Therapy Immediate Treatment

At the scene of the accident, appropriate medical intervention includes removal from exposure, resuscitation, and supportive treatment. In some instances, emergency personnel must also be protected from chemicals that remain present on victims or their clothes and decontamination procedures must be available. For some types of exposures, asymptomatic persons must remain under observation for 24 h. There is anecdotal evidence, as well as physiological ground, that persons at risk of developing pulmonary edema should not exercise, nor should they be overfilled by intravenous fluids. It seems to be common practice to administer oxygen to any victim of an inhalation accident. However, oxygen treatment should probably be given sparingly and only as required by the level of arterial oxygen saturation, because oxygen has a considerable pulmonary toxicity of its own and it is conceivable that adding an oxidant stress to an already damaged and inflamed lung may hamper the resolution of the damage. It must be recognized, however, that few studies are available on the subject, except in the case of paraquat intoxication, where it is well known that there is synergy between paraquat and oxygen.

216 OCCUPATIONAL DISEASES / Asbestos-Related Lung Disease Continuing Management

Further Reading

The further management of acute inhalation injury will be governed by the severity of the patient’s condition and will involve intensive care treatment with intubation and artificial ventilation, as required. Antibiotics are only to be given if there are signs of infection. In victims of smoke injury, bronchoscopic removal of soot from the airways may be necessary. The administration of (systemic) corticosteroids is probably justified to prevent complications arising from (excessive) inflammation, such as bronchiolitis obliterans, although there are no controlled studies on this issue. The available data concerning NO2induced bronchiolitis obliterans suggest that this complication does not occur when steroids have been administered. Moreover, ‘classical’ organizing pneumonia is known to resolve rapidly with corticosteroids treatment. Consequently, in the present state of knowledge, it seems advisable to administer corticosteroids to victims of severe inhalation injury. It is not known whether inhaled steroids should be given preventively in victims of less severe inhalation injuries and whether this prevents the occurrence of irritant-induced asthma or RADS.

Bismuth C and Half AH (1995) Paraquat Poisoning: Mechanisms, Prevention, Treatment. New York: Dekker. Blanc P and Boushey HA (1993) The lung in metal fume fever. Seminars in Respiratory Medicine 14: 212–225. Das R and Blanc PD (1993) Chlorine gas exposure and the lung: a review. Toxicology and Industrial Health 9: 439–455. Douglas WW and Colby TV (1994) Fume-related bronchiolitis obliterans. In: Epler GR (ed.) Diseases of the Bronchioles, pp. 187–213. New York: Raven Press. Douglas WW, Hepper NGG, and Colby TV (1989) Silo filler’s disease. Mayo Clinic Proceedings 64: 291–304. Kreiss K, Gomaa A, Kullman G, et al. (2002) Clinical bronchiolitis obliterans in workers at a microwave-popcorn plant. New England Journal of Medicine 347(5): 330–338. Loke JS (2000) Thermal lung injury and acute smoke inhalation. In: Fishman AP (ed.) Pulmonary Diseases and Disorders, 3rd edn., vol. 1, pp. 989–1000. New York: McGraw-Hill. Moya C, Anto JM, Newman Taylor AJ, and Collaborative Group (1994) Outbreak of organizing pneumonia in textile printing sprayers: collaborative group for the study of toxicity in textile aerographic factories. Lancet 344(8921): 498–502. Nemery B (1990) Metal toxicity in the respiratory tract. European Respiratory Journal 3: 202–219. Nemery B (1996) Late consequences of accidental exposure to inhaled irritants: RADS and the Bhopal disaster. European Respiratory Journal 9: 1973–1976. Nemery B (2002) Toxic pneumonitis. In: Hendrick DJ, Burge PS, Beckett W, and Churg A (eds.) Occupational Disorders of the Lung: Recognition, Management, and Prevention, pp. 201–219. London: Saunders. Nemery B (2003) Reactive fallout of World Trade Center dust. American Journal of Respiratory and Critical Care Medicine 168(1): 2–3. Nemery B (2005) Chemical-induced lung injury and its long-term sequelae. In: Gevenois PA and De Vuyst P (eds.) Medical Radiology – Diagnostic Imaging, Imaging of Occupational and Environmental Disorders of the Chest. Berlin: Springer (in press). (The present chapter is a shortened and updated version of this article.) Olson KR and Shusterman DJ (1993) Mixing incompatibilities and toxic exposures. Occupational Medicine 8: 549–560. Rask-Andersen A (1996) Inhalation fever. In: Harber P, Schenker MB, and Balmes JR (eds.) Occupational and Environmental Respiratory Disease, pp. 243–258. St Louis: Mosby. Shusterman DJ (1993) Polymer fume fever and other fluorocarbon pyrolysis-related syndromes. Occupational Medicine 8: 519–531.

Documentation

An important, but in practice often neglected, aspect concerns the documentation of the lesions and their severity in victims of inhalation injury. This then leads to difficult medicolegal problems when victims seek compensation, sometimes many months or years after the event. It is, therefore, important that physicians treating victims in the early days after the incident document accurately the clinical condition and all relevant data in these patients. Documentation of the damage by bronchoscopy and high-resolution computed tomography may be justified. Repeated measurements of ventilatory function and arterial blood gases must be carried out, and victims of acute inhalation injury should never be discharged without a comprehensive assessment of their pulmonary function. It is also important to consider that further accidents must be prevented by adequate technical and administrative measures, which must be elaborated on the basis of a comprehensive accident report and analysis. See also: Interstitial Lung Disease: Cryptogenic Organizing Pneumonia; Hypersensitivity Pneumonitis. Laryngitis and Pharyngitis. Occupational Diseases: Overview. Oxidants and Antioxidants: Oxidants. Reactive Airways Dysfunction Syndrome. Upper Respiratory Tract Infection.

Asbestos-Related Lung Disease A R Brody, Tulane University Health Sciences Center, New Orleans, LA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Asbestos is a naturally occurring mineral that is mined in several parts of the world. This fibrous substance has been useful in

OCCUPATIONAL DISEASES / Asbestos-Related Lung Disease 217 many commercial products since the turn of the twentieth century. Unfortunately, when the fibers are inhaled, they can cause asbestosis (scar tissue in the gas-exchange region of the lung), pleural scarring, lung cancer, and mesothelioma (a neoplasm of the parietal, visceral, or peritoneal mesothelial lining). Scar tissue forms as a consequence of inhaled fibers being deposited on the alveolar epithelium of the gas exchange surfaces. Fibers small enough to pass through the conducting airways can be intercepted by the bronchiolar–alveolar duct (BAD) bifurcations, which are the primary sites for scar tissue development because of the localized dose response. If occupational exposure continues, numerous fibers are translocated by alveolar epithelial cells to the underlying interstitial and lymphatic spaces. There are repeated injuries to the epithelial cells, interstitial fibroblasts and myofibroblasts, and lung macrophages are attracted to the sites of fiber deposition by asbestos-activation of the 5th component complement. All of these cells synthesize and release a panoply of peptide growth factors that cause the epithelial, mesenchymal, and endothelial cell populations to go through multiple rounds of cell division, and the fibroblasts and myofibroblasts elaborate a scar tissue matrix that spreads from the original BAD junctions and is recognized clinically as asbestosis after a latency of decades. Fibers transported to the pleura by lymphatics can induce the same process, causing pleural scarring. Lung cancer and mesothelioma occur when the fibers cause errors in genes that control cell growth by binding to DNA and/or generating reactive oxygen molecules. All the asbestos varieties cause all of the asbestos-related diseases, and cigarette use synergizes with asbestos to dramatically increase the risk of developing lung cancer but has no effect on the incidence of mesothelioma. Inhaled asbestos is the only known environmental cause of mesothelioma. There are no effective treatments for any of the asbestos-induced diseases. As the use of asbestos tapers off worldwide, these diseases will eventually disappear, but many tons of asbestos are still being shipped and utilized, primarily in developing countries where the asbestosinduced diseases remain prevalent.

Introduction Asbestos is a naturally occurring mineral that is mined in several corners of the world, including Quebec, the US, South Africa, and Russia. There are two groups of commercially useful asbestos (Table 1). Table 1 Most commonly used asbestos types Commercial name

Mineral group

Chemical formula

Chrysotile (white asbestos) Crocidolite (blue asbestos) Amosite (brown asbestos)

Serpentine

(MgFe)6(OH)8Si4O10

Amphibole

Na2Fe2 3 þ Fe3 2 þ (OH)2Si8O22

Amphibole

Mg7(OH)2Si8O22

Fe7(OH)2Si8O22 Modified from Bignon J, Peto J, and Saracci R (eds.) (1989) NonOccupational Exposure to Mineral Fibres, No. 90, p. 5. Lyon, France: IARC Scientific Publications.

One variety, named chrysotile, constitutes approximately 95% of the world’s use. The amphibole group is comprised of several fiber types, most notably crocidolite, amosite, anthophyllite, and tremolite, which together make up the remaining 5% of commercial use. The asbestos ore is crushed and milled into clumps and bundles of fibers. The resulting fibrous substance has been used since around the turn of the twentieth century for a wide variety of useful products, including cement for water pipes, construction materials, textiles, insulation, and fireproofing. Asbestos-induced diseases develop when individuals are exposed to the microscopic fibers released during mining, manufacturing, and commercial activities (Figure 1). Others have been exposed to fibers in para-occupational settings in buildings (e.g., as elevator repairmen or janitors) and at home while washing the contaminated clothing of asbestos workers. There are two types of diseases caused by inhaled asbestos: fibrotic and neoplastic. All of the asbestos fiber types cause all of the diseases. Fibrotic disease connotes the elaboration of scar tissue, and the disease asbestosis is, by definition, scar tissue in the lung caused by inhaled asbestos fibers, whereas pleural plaques and pleural fibrosis develop when scar tissue is deposited along the parietal or visceral pleural membranes. Neoplastic disease means cancer primarily of epithelial or mesothelial cell origin. Thus, lung cancer develops from a cell of the epithelium of the conducting airways, whereas mesothelioma develops from the mesothelial cell linings of the parietal or visceral pleura. Asbestos exposure is known to increase the incidence of mesothelioma of the peritoneal cavity, and some epidemiological studies have shown increased prevalence of cancers of the gastrointestinal tract in asbestos-exposed individuals.

Fibrotic Diseases Asbestosis

Asbestosis typically arises with a long latency of 20– 40 years after exposure to high concentrations of fibers in occupational settings. The disease begins when inhaled fibers, small enough to pass through the conducting airways, are deposited on the type I alveolar epithelium. The primary sites of deposition are the bronchiolar–alveolar duct (BAD) bifurcations that extend from the ends of the terminal bronchioles (Figure 2). This observation was initially made in rats exposed to high concentrations of fibers for 1 h and then sacrificed immediately following the brief exposure period. Multiple and extended exposure protocols result in the same deposition pattern, and quantitating fiber numbers on the alveolar surfaces

218 OCCUPATIONAL DISEASES / Asbestos-Related Lung Disease

Mg

Si

100 µm (a)

(b)

10 µm (c) Figure 1 (a) Chrysotile asbestos fibers as seen under a scanning electron microscope (SEM). The fibers typically are found in bundles that may be hundreds of micrometers long (arrow) and as individual fibrils about one-tenth of 1 mm in diameter (arrowheads). When these fibers break apart, they can be aerosolized and inhaled. (b) When an electron beam strikes the chrysotile fibers, electrons are released that exhibit energies specific for the elements from which they emanated. Here an X-ray energy spectrometer has been used to analyze the nature of the electrons and shows two elements that have released X-rays, magnesium (Mg) and silicon (Si), the elements known to comprise chrysotile asbestos, thus confirming its identity. (c) An asbestos body recovered from the lung of an insulator as seen by SEM. The asbestos core (arrow) of the body was proven by X-ray analysis to be amosite, which is surrounded by clumps of proteinaceous iron (arrowheads). These bodies are found commonly in the lungs of individuals with asbestosis (see Figure 4).

showed that there were more fibers initially deposited on the first BAD bifurcation than on the second BAD, more on the second than the third, and so on. This results in a localized dose–response, which translates into the earliest appearing and most severe fibrotic lesions developing at the first BAD junctions in experimental animals (Figure 3), as well as in

humans (Figure 4). While investigators can readily follow the anatomic details of disease development in animal models, the disease in humans usually is observed at only one point in time, many years after exposure is initiated. The fundamental molecular mechanisms that mediate the disease processes in exposed individuals must be gleaned from the animal

OCCUPATIONAL DISEASES / Asbestos-Related Lung Disease 219

BAD AD

AD Br

(a)

(b)

CC

CC

Ep Ci AM

CC

AM AM AD

(c)

(d) Epll AM

AM

Epl (e) Figure 2 (a) This section of normal lung tissue from a rat shows terminal bronchioles (Br) and entrances to the alveolar ducts (arrows) where asbestos fibers that pass through the conducting airways deposit. The thin visceral pleura surrounds the outside of the lung. (b) A bronchiolar–alveolar duct (BAD) bifurcation immediately after 1 h of exposure to an aerosol of chrysotile asbestos. Some of the fibers (arrows) remain on the thin alveolar epithelium, while other thin fibers (arrowheads) are already partially or wholly covered by this type I alveolar epithelium. This BAD bifurcation has intercepted a number of asbestos fibers that were passing down the alveolar ducts (ADs). (c) Shortly after initial fiber deposition, alveolar macrophages (AMs) begin to accumulate at the sites of fiber deposition as a result of activation of the 5th component of complement by the inhaled fibers (arrows). Clara cells (CCs) of the BAD surface and the alveolar epithelium (Ep) are observed. (d) This single AM was caught by SEM moving on its way from the alveolar epithelium that covers the surface of the AD and on to the ciliated (Ci) bronchiolar airway. The macrophage contains a number of asbestos fibers, one of which is randomly oriented to form a ‘pointer’ at the leading edge of cell (arrowheads), thus dramatically indicating the direction of fiber clearance by macrophages up the mucociliary escalator. (e) AMs like these normally reside on the type I alveolar epithelium (EpI). The AM in the background is observed in front of a type II epithelial cell (EpII) and exhibits membrane ruffling but no direction. The AM in the foreground is stretched toward an organic sphere (arrowhead), possibly a pollen grain or bacterium, that has attracted the cell, which will engulf the particle and transport it to the mucociliary escalator as demonstrated above by a macrophage carrying asbestos fibers.

models. Thus, the histopathologic picture of early asbestosis in humans exhibits a pattern of fibrogenesis, i.e., scar tissue formation, along the respiratory bronchioles and alveolar ducts closest to the ends of

the terminal bronchioles, so-called stage 1 asbestosis (Figure 4). The disease first becomes prominent in this pattern because of the increased fiber deposition at BAD bifurcations as described above. As the

220 OCCUPATIONAL DISEASES / Asbestos-Related Lung Disease

AM

Br

AM CT IM CT

AD (a)

(b) Pseudocolor image bifurcation after asbestos exposure: BrdU staining

AS Epl En

Epl

Labeled cell Unlabeled cell

Ca

AD

(c)

(d) Ep AS Epl Co

Co

Eo

En

Fi

(e)

(f)

Figure 3 (a) Histopathology shows that 3 days of exposure to asbestos induces the development of prominent fibrogenic lesions (arrow and box) at the bifurcations and along the alveolar duct (AD) walls. The lesion in the box is magnified for further analysis in part b. Reproduced with permission from Liu et al. American Journal of Pathology 149: 205. (b) Alveolar macrophages (AMs) and interstitial macrophages (IM) stained with an antimacrophage antibody (brown stain) have surrounded and infiltrated the developing lesion. The macrophages, epithelial cells, and mesenchymal cells (arrowheads) release growth factors that induce cell proliferation (see part c) and connective tissue (CT) production, the hallmark of asbestosis. (c) This lesion at an AD bifurcation is from an asbestos-exposed rat treated with bromodeoxyuridine (BrdU), a thymidine analog that is taken up by cells that are dividing. Numerous epithelial cells (arrowheads) and interstitial cells (arrowheads) have incorporated BrdU in the developing fibrogenic lesion. (d) This shows the alveolar– capilllary membrane through which oxygen–carbon dioxide exchange takes place between the airspace (AS) and capillary (Ca) bed. Transmission electron microscopy (TEM) has shown that inhaled asbestos fibers, which deposit on the type I alveolar epithelium (EpI) are phagocytized (see Figure 2(d)) and transported to the interstitium (see part e), and vascular spaces (see part f). Here, inhaled chrysotile fibers (arrowheads) are embedded in the epithelium within hours after deposition. The endothelium (En) that lines a capillary (Ca) is separated from the EpI by a basement membrane. (e) TEM shows an interstitial chrysotile asbestos fiber (arrowheads) embedded in a collagen (Co) matrix. The fiber was transported through the overlying epithelium (Ep), and the collagen is produced by interstitial fibroblasts (Fi). (f) Some asbestos fibers (arrowhead) are translocated from the AS through the EpI, through the basement membrane (arrow) and through the endothelium (En) to the capillary bed. In this TEM, an eosinophil (Eo) is passing through the capillary, certifying the location of the fiber. Once fibers reach the interstitium and capillary bed, they can be transported to any other anatomic site in the body by blood or lymphatic flow.

disease progresses, both during repeated exposures and in some cases after all exposure ceases, scar tissue deposition becomes more diffuse around the affected regions, eventually coalescing to form

asbestosis stages 2–4 (Figure 4), which can be seen on X-rays. This causes shortness of breath and a restrictive pattern of pulmonary function, which are characteristic of clinical asbestosis. It typically takes

OCCUPATIONAL DISEASES / Asbestos-Related Lung Disease 221

Br AS AS Br Br (a)

(b)

AS

CT Pa

Pl

CT AS (c)

(d)

Figure 4 (a) Light micrograph of normal lung tissue shows a bronchiole surrounded by thin-walled alveolar spaces (AS). (b) In asbestosis in humans, the early fibrogenic lesions (arrows) develop initially at the ends of the terminal bronchioles (Br) and along the alveolar ducts as demonstrated in the animal models (see Figure 3(b)). (c) Advanced clinical asbestosis is marked histologically by alveolar walls dramatically thickened with interstitial cells and connective tissue (CT). Additional scar tissue is marked by an arrow. A classic appearing interstitial asbestos body (arrowheads) is recognized by light microscopy (see Figure 1(c)). (d) A thickened and scarred pleura (Pl) is a common finding in asbestos-exposed individuals. This pleural scar is approximately 35 times thicker than a normally thin pleura. Fibrotic lung parenchyma (Pa) is observed to the left of the pleura and the pleural space is to the far right.

decades for the clinical picture to emerge, and during that time a series of cellular, biochemical, and molecular events takes place in rapid and continued succession in the bronchiolar–alveolar walls, resulting in increased numbers of fibroblasts and myofibroblasts and the production of scar tissue by these cells. An outline of the process that culminates in clinical asbestosis is as follows: 1. Inhaled fibers with a wide variety of lengths (from less than a micrometer to hundreds of micrometers long) and a range of diameters from 0.1 mm to about 5 mm are deposited on type I epithelial cells of the BAD junctions (Figure 2). 2. A proportion of the fibers (approximately 20%) are enveloped (phagocytized) by type I epithelial cells and actively transported to several interstitial locations (Figures 2 and 3): (a) the basement membranes; (b) the vascular or lymphatic space; and (c) the interstitial extracellular matrix. 3. While on the epithelial cell surfaces, the fibers damage the plasma membranes by lipid peroxidation and the generation of reactive oxygen species, resulting in a breach of the interepithelial tight junctions and increased fluid and protein in the alveolar spaces.

4. While on the epithelial surfaces, some fibers convert the third component of alveolar complement protein to C5a, a potent chemoattractant for macrophages, thus causing the accumulation of these cells at the sites of original fiber deposition (Figure 2). 5. Fibers are phagocytized by epithelial and endothelial cells, by alveolar and interstitial macrophages, and by interstitial mesenchymal cells. 6. The preceding events result in the expression of genes that code for cytokines and growth factors known to control cell growth, chemotaxis, and extracellular matrix production (scar tissue production). Examples of the molecules induced by asbestos exposure are: (a) platelet-derived growth factor (PDGF), the most potent inducer of mesenchymal cell growth yet discovered; (b) transforming growth factor beta (TGF-b), the most potent inducer of extracellular matrix synthesis by mesenchymal cells; (c) transforming growth factor alpha (TGF-a), a strong mediator of epithelial cell mitosis; and (d) tumor necrosis factor alpha (TNFa), a so-called master cytokine that has potent pleiotropic effects and strongly influences the expression of other factors such as PDGF and TGF-b. When mice have the genes that code for both membrane receptors of TNF-a knocked out,

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the animals exhibit reduced expression of PDGF and TGF-b1 and they are protected from the fibrogenic effect of inhaled asbestos and silica. 7. The epithelial and mesenchymal cell populations go through multiple rounds of cell division (Figure 3), increasing in number in regions where the fibers are deposited and moved about. The fibroblasts and myofibroblasts synthesize and release scar tissue, focally at first (microscopic or subclinical asbestosis) and then in a diffuse pattern that is recognized as clinical asbestosis (Figure 4). The most severe cell damage results in scarring within the alveolar spaces as the mesenchymal cells migrate from the interstitium and elaborate a collagen matrix (Figure 4). Asbestos fibers exert their toxic properties primarily by binding to cell membranes and generating reactive oxygen species (ROS). Chrysotile asbestos binds to membranes through its positive surface charge. This charge results from the copious magnesium ions that combine with hydroxyl compounds to form the bulk of the fibers as a magnesium hydroxide, which alternates with layers of silicon dioxide. The sheets of Mg6OH8 and Si4O10 roll into the miniature hollow cylinders that are chrysotile asbestos fibers. These fibers have no detectable iron as a component of the chemical formula, but it has been shown that iron as Fe2O3 or FeO readily accumulates within the chrysotile fibers from natural geologic sources or from biological fluids. Iron can comprise from 1% to 10% of this asbestos type (Table 1). Amphibole fibers are more solid and plank-like. They are predominantly crystalline silicon dioxide (SiO2), which imparts a negatively charged surface, and all the amphibole varieties exhibit a considerable proportion of iron (15–40%) in their chemical formulae. X-ray energy spectrometry in combination with scanning or transmission electron microscopy (Figure 1) is an established method used to identify the precise types of asbestos fibers found in human lung tissue. The technique of quantifying the so-called lung burden of asbestos fibers in autopsy samples of exposed workers and nonoccupationally exposed controls has provided important information. These studies show that amphibole fibers are retained for longer periods of time in the lung than chrysotile fibers; when lung tissue is digested or converted to ash decades after exposure has ceased, the majority of fibers remaining are amphiboles, even if the predominant exposures decades earlier were to chrysotile. Animal studies have confirmed these findings by showing that as the fibers break down and become smaller, they are more readily cleared from the lungs

by macrophages and lymphatic drainage pathways. Chrysotile fibers, because of the more soluble magnesium hydroxide component, break down more rapidly. This could explain how small chrysotile fibers accumulate in pleural tissues and in lymph nodes. Of course it is not possible to follow the pathobiological actions of chrysotile fibers from inhalation to passage through the anatomic compartments of human lungs, where they induce fibrogenesis (asbestosis) and neoplastic disease. Thus, the lungs of an individual who was occupationally exposed to chrysotile asbestos may contain relatively few of these fibers decades later, compared to the millions of amphibole fibers that were a contaminant of chrysotile or were inhaled independently. This finding does not allow the conclusion that the amphibole fibers cause disease while the chrysotile fibers do not. Conclusions on cause of disease must be drawn from knowledge of what the individual was originally exposed to, not only on what remains in the lung. However, these two pieces of information may very well correlate and then a very useful quantitative analysis can be made. When positively charged chrysotile asbestos fibers bind to cell membranes, they do so by adhering to negatively charged sialic acid complexes found universally on the membranes. Negatively charged amphibole fibers appear to bind to positively charged protein complexes, but this mechanism has not been as clearly elucidated. It is known that fiber binding to cell membranes causes gene expression, arachidonic acid release, and rearrangement of vital components such as growth factor receptors and ion channels, and lipid peroxidation of the membranes may ensue, adding to cellular dysfunction. While cytotoxicity has been readily produced in vitro by treating a variety of lung cells with asbestos fibers, it is unlikely that cell death plays a large role in the pathobiological effects leading to disease. This conclusion is drawn from several findings: 1. The predominant inflammatory cell type in experimental and human asbestosis is the macrophage, whereas a cytotoxic response engenders the accumulation of polymorphonuclear leukocytes (PMN). PMNs certainly are identified consequent to asbestos exposure in animals and man, but high, long-term exposure or intratracheal administration of fibers are required in the model systems, and in humans there could be superimposed infection and other ongoing processes that require infiltration of PMNs. 2. Extensive in vitro experiments with a variety of lung cell types and organ cultures show little toxicity unless very large numbers of fibers are used,

OCCUPATIONAL DISEASES / Asbestos-Related Lung Disease 223

that is, many more than a cell would confront in situ, even at high concentration exposures. 3. Both light and electron microscopy studies show chrysotile and amphibole fibers phagocytized by lung cells (as described above), and translocation of the fibers to other cells and lung compartments ensues without cell death. The abnormalities and consequent disease processes are caused by the biochemical and molecular injuries described herein, rather than by outright cell death. As asbestos fibers are distributed throughout the lung and pleura, they leave indelible marks that are recognized by light and electron microscopy. For example, a small proportion of the larger inhaled fibers form ‘asbestos bodies’. These are fibers (usually amphiboles) around which has accumulated proteinaceous iron from biological sources, forming clumps or beads (Figures 1 and 4) that appear amber in a typical histopathology slide, or bright blue when stained for the presence of iron. Asbestos bodies are nontoxic and provide a good marker of occupational exposure, even though only about 10% of inhaled fibers form bodies. Another mark of exposure is the millions of uncoated fibers ranging in size from fragments that are barely fibers to those hundreds of micrometers long (Figures 1 and 2). Fibers longer than 15 mm are cleared very slowly, if at all, regardless of the fiber type. Even normal lungs from individuals in the general population can contain millions of asbestos fibers. These fibers, inhaled a few at a time from the ambient environment as asbestos-containing products break down, are unlikely to accumulate in sufficient numbers to cause disease in the average person. Finally, we see evidence of disease in the increased extracellular matrix in the interstitium of the alveolar walls and pleural membranes (see above), and increased genetic errors in the epithelial cells of the conducting airways and mesothelial cells of the pleural and peritoneal surfaces (see below). Who will finally develop clinical disease is dependent on the genetic susceptibilities of that individual together with the dose of asbestos they received. Pleural Scarring

A proportion of the fibers that reach the interstitial lymphatic spaces are translocated to the pleural space and membrane linings, visceral and parietal, by lymphatic fluid flow. There, the fibers induce a sequence of events similar to those described above so that the population of fibroblasts that reside in the submesothelial interstitium multiplies and elaborate collagenous scar tissue, resulting in pleural plaques (if the scarring is focal and circumscribed) or pleural fibrosis (if the scarring is diffuse) (Figure 4). This

process can be an early marker of asbestos exposure in some individuals and may cause some decrements in pulmonary function.

Neoplastic Diseases A carcinogen, by definition, is a cancer-causing agent. All asbestos varieties are carcinogens. Cigarette smoke contains numerous separate carcinogenic substances. To act as a carcinogen, the substance must cause DNA damage that is passed on to succeeding generations of the cell type. The two most likely mechanisms through which asbestos influences alterations of DNA are by direct binding or by elaborating ROS. The production of ROS by asbestos fibers is well documented, and a number of carcinogens, including components of cigarette smoke, have been shown to cause specific gene mutations through the release of ROS. There are a number of DNA sequences classified as growth-control genes. These genes may code for positive growth control proteins in the cell cycle, and a number of the proteins may be powerful suppressors of cell division. Other genes are known to control the cell cycle such that rapid progression to cell death (so-called apoptosis) will ensue following DNA damage from a carcinogen. For a lung cancer or mesothelioma to develop, the carcinogen must reach the appropriate target cell for that tumor and cause errors in a number of genes that control cell growth. Lung Cancer

Lung cancer connotes a primary neoplasm arising from an epithelial or mesenchymal cell type in the lung. Most commonly associated with cigarette use and asbestos exposure is bronchogenic carcinoma (BrCa), which is a cancer that arises from an epithelial cell type in a conducting airway. Smoking cigarettes causes the greatest risk of developing a BrCa, but in combination with asbestos exposure, that risk is multiplied. In the 1950s, 1960s, and 1970s, when clinical asbestosis from heavier exposures was common and most asbestos workers smoked cigarettes, the increased risk of exposure to the two carcinogenic substances became apparent and the synergistic effects were documented. In more recent years, lung cancers in asbestos workers who smoke remain common, but the signs and symptoms of clinical asbestosis are observed less frequently. This situation has made attribution of some cancer cases to asbestos exposure more difficult to ascertain in cigarette smokers. However, a series of epidemiologic studies has demonstrated that the risk of developing a bronchogenic carcinoma is increased significantly in

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asbestos-exposed cigarette smokers, even if there is no clinical presentation of asbestosis. The risk of getting lung cancer in asbestos-exposed cigarette smokers is enhanced because the target cells that comprise the mucociliary epithelium undergo a series of phenotypic and molecular changes. For example, cigarette smoking causes regions of the airway epithelium to exhibit squamous metaplasia or a more advanced dysplasia. These conditions are considered preneoplastic, and when inhaled asbestos fibers are deposited on an altered epithelium, the fibers are more likely to be taken up by the airway epithelium and retained in the lung. If the fibers (themselves capable of causing DNA damage as discussed above) have additional smoke-derived carcinogenic materials bound to their highly charged surfaces, the likelihood of developing lung cancer would be increased in a synergistic fashion as the combined carcinogens are delivered efficiently time after time to the target cell populations. The tumor arises when one of these airway epithelial cells, with a series of errors in a number of genes that control cell growth, loses control of normal growth and is manifested as a lung cancer. Mesothelioma

Mesothelioma is a cancer that arises from the mesothelial cells that line the pleural and peritoneal cavities. This lining is a single cell sheet that is spread over the entire visceral and parietal pleural surfaces. The cells provide an airtight surface that eliminates friction between the pleural membranes and limits the normal excursions of the lung. Why the mesothelial linings are so sensitive to the carcinogenic effects of asbestos fibers remains unknown. Mesothelioma is a deadly tumor with an expected survival time of less than 2 years after the diagnosis is made; the latency from first exposure to the time of diagnosis can range from as little as 10 years to more than 50 years, with a median latency of about 35 years in most studies. Inhaled asbestos is the only known environmental cause of mesothelioma in North America. Epidemiology has shown that cigarette use does not increase the risk of developing malignant mesothelioma (MM). Malignant mesothelioma is a ‘signal tumor’, meaning that upon diagnosis, the treating physician is likely to ask ‘‘Where were you exposed to asbestos?’’ The amphibole fibers appear, on a fiber for fiber basis, to be more potent than chrysotile, as inducers of mesothelioma. The degrees of difference in potency have not been established since all the asbestos varieties cause MM, and it is the accumulation of inhaled fibers, over time, that confers the risk of developing

genetic errors (as discussed below). In Turkey, an asbestos-like zeolite called erionite has been used in the past in the construction of homes, and this has resulted in a large number of cases of MM. Approximately 85–90% of all mesothelioma victims have a documented exposure to asbestos. As a general principle, MM is a dose-responsive disease like the other asbestos-induced diseases defined above. However, there are many cases of low, brief exposures that have caused MM. Thus, no threshold or safe dose of asbestos has been established for preventing mesothelioma. Animal models of MM are difficult to work with because high concentrations of fibers are required for an extended period of time to produce significant numbers of tumors in the short-lived subjects. Thus, most investigators use intraperitoneal injection models or cells from human MM to pursue an understanding of the molecular mechanisms that lead to disease. Like most other cancers, MM has been slow to reveal the combinations of genetic errors that mediate disease development. For example, all of the asbestos varieties and fiber lengths have been shown to produce aneuploidy by interfering with chromosome separation during mitosis (Figure 5). There is evidence of DNA binding to fiber surfaces and disruption of microtubules required for normal chromosome migration. These events result in non-specific damage such as chromosomal aberrations, micronuclei formation, lagging chromosomes, and other characteristics of DNA damage. More recently, a number of genes that code for tumor suppressor proteins, cell-cycle modulations, and transcription factors have

Figure 5 These rat pleural mesothelial cells were treated in vitro with chrysotile asbestos fibers. The two cells on the left are ‘tied’ together by a chrysotile fiber (arrow) that has bound a fragment of DNA (arrowhead) and caused aneuploidy. All the asbestos varieties cause genetic damage of the kind shown here. Reproduced from Yegles et al. (1993) Induction of metaphase and anaphase/telophase abnormalities by asbestos fibers in rat pleural mesothelial cells in vitro. American Journal of Respiratory Cell and Molecular Biology 9: 186–191, Official Journal of the American Thoracic Society, & American Lung Association, with permission.

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exhibited alterations. Some genetic alterations are induced by asbestos in animal models or cell lines and these, along with other genetic errors, are found with consistency in human mesotheliomas. For example, chrysotile asbestos has been shown to downregulate the expression of the cell-cycle control protein p21 and the integrin-associated gene BigH3. Also, there are a series of genes that consistently appear in model systems and human tumors and are under intense investigation to determine whether or not they play a role in the development of MM. These include the neurofibromatosis gene NF2, the genes encoding the tumor suppressor proteins’ retinoblastoma (RB) and p53, and homozygous deletions of the genes that code for p14 and p16, which result in abnormal regulation of the cell growth pathways controlled by RB and p53. Asbestos fibers also disrupt intracellular signal transduction pathways at several levels including the dysregulation of receptors for peptide growth factors, the altered expression of oncogenes that control cell growth and migration, and the phosphorylation of kinases that signal through proteins that confer cell survival. All of these genotoxic, metabolic, and biochemical injuries could play a role in the development of lung cancer and mesothelioma, but the sequence of events that are necessary and sufficient to induce a malignancy have not been established at the time of this writing. An interesting contributor to the development of MM at the molecular level could be the simian virus (SV)40. This is a well-described oncogenic DNA virus that causes immortalization and neoplastic transformation of a variety of cell types. SV40, which is believed to have contaminated samples of the polio vaccine administered decades ago, has been detected in human mesothelial tumors in the US and Europe. Whether or not it is a causative factor in tumor development remains unclear, but SV40 does cause mesotheliomas in a hamster model, and it induces malignant transformation of human mesothelial cells in vitro. However, it is clear that the vast majority of individuals with MM have been exposed to asbestos, and this is the defining epidemiologic principle, not the presence of SV40. Thus, malignant mesothelioma is caused by asbestos, but in some individuals, SV40 could act as a co-carcinogen, making the occurrence of the disease more likely. Malignant mesothelioma has long been recognized as a tumor highly resistant to therapy, as indicated by the short survival times reported. Investigators, including surgeons and basic molecular biologists, are approaching MM with aggressive and innovative therapies. A feature of most malignant tumors, including MM, is their resistance to apoptosis. This so-called genetically programed cell death is a process

that is essential for preventing the multiplication of cells with genetic damage, thus preventing the accumulation of cells that are likely to become neoplastically transformed. In one approach, investigators have used a death ligand named TRAIL (TNF-a-related apoptosis-inducing ligand) to treat human mesothelial cells. TRAIL induces apoptosis in normal cells, but not in malignant mesothelial cells. However, further treatment of the cells with DNA-damaging agents caused the tumor cells to be sensitive to TRAIL-induced apoptosis. The findings have raised the possibility that TRAIL, in combination with agents that further damage specific genes, could be used to control tumor growth by apoptosis. In another approach, investigators are asking if TGF-b can be used as a therapeutic tool. This is the same molecule described above as a potent mediator of lung fibrogenesis consequent to asbestos exposure. Among the various functions proven for TGF-b is its role as a local and systemic immunosuppressant. TGF-b is produced in large amounts by a number of tumor cell types, including those of MM. Release of TGF-b into the tumor environment appears to be one of the cancer cell’s evolutionary successes in avoiding detection by the immune system, along with the cells’ abilities to escape the apoptotic mechanisms and a number of growth control check points. Thus, a soluble circulating type II receptor for TGF-b1 and TGF-b3 was used to treat malignant mesothelioma in a mouse model. Tumors regressed in these animals, but the soluble receptor had no effect on mesothelial tumors that did not produce TGF-b. The investigators propose that human mesotheliomas could be screened and the soluble receptor used as a treatment in those individuals with tumor positive for TGF-b production. Unfortunately, the more conventional chemotherapeutic approaches have shown little promise for MM. Commonly used drugs like cisplatin, doxorubicin, and gemcitabine provide only small effects, although recent clinical trials of these drugs, along with interferons and in combinations, do offer some hope. More optimistically, a combination of aggressive surgery, with direct applications of drugs to the tumor, has shown some success. Hundreds of patients with MM have undergone ‘extrapleural pneumonectomy’ or radical surgical resection to remove the bulk of the tumor, followed by trials of high-dose cisplatin, other experimental drugs, and photodynamic therapy. These combinational approaches have increased the survival period for many individuals.

Conclusion As the use of asbestos tapers off worldwide, the asbestos-induced diseases will eventually disappear.

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However, thousands of tons of asbestos are still being shipped and used largely in developing countries where the asbestos-related diseases are prevalent. As our understanding of the biochemical and molecular mechanisms of asbestos-induced diseases expands, societies should expend considerable resources on developing effective therapies as well as on preventing exposure to the fibers. Success with these approaches will be directly applicable to fibrogenic and neoplastic diseases with other etiologies. See also: Fibroblasts. Leukocytes: Pulmonary Macrophages. Particle Deposition in the Lung. Pulmonary Fibrosis.

Further Reading Bignon J, Peto J, and Saracci R (eds.) (1989) Non-Occupational Exposure to Mineral Fibres, No. 90, p. 5. Lyon, France: IARC Scientific Publications. Brody AR (1997) Asbestos. In: Roth RA (ed.) Comprehensive Toxicology, vol. 8(25), pp. 393–413. New York: Elsevier Science. Brody AR, George G, and Hill LH (1983) Interactions of chrysotile and crocidolite asbestos with red blood cell membranes: chrysotile binds to sialic acid. Laboratory Investigations 49: 468–475. Brody AR, Hill LH, Adkins B, and O’Connor RW (1981) Chrysotile asbestos inhalation in rats: deposition pattern and reaction of alveolar epithelium and pulmonary macrophages. American Review of Respiratory Diseases 123: 670–679. Consensus Report (1997) Asbestos, asbestosis, and cancer: the Helsinki criteria for diagnosis and attribution. Scandinavian Journal of Work and Environmental Health 23: 311–316. Hesterberg TW, Butterick CJ, Oshimura M, Brody AR, and Barrett JC (1986) Role of phagocytosis in cytogenetic effects and cell transformation induced by asbestos and short and long glass fibers. Cancer Research 46: 5795–5802. Knudson A (1995) Asbestos and mesothelioma: genetic lessons from a tragedy. Proceedings of the National Academy of Sciences, USA 92: 10819–10820. Lasky JA, Ortiz LA, and Brody AR (2005) Chronic lung injury and fibrosis: potential roles for TNF-a, PDGF, CTGF, and TGF-b. In: Notter RH, Holm BA, and Finkelstein JN (eds.) Lung Injury: Mechanisms, Pathophysiology, and Therapy, Vol. 196, pp. 175– 226. New York: Dekker. Liu J-Y, Brass DM, Hoyle GW, and Brody AR (1998) TNF-a receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers. American Journal of Pathology 153: 1839–1847. Liu et al. American Journal of Pathology 149: 205. Morris GF and Brody AR (1998) Molecular mechanisms of particle-induced lung disease. In: Rom WR (ed.) Environmental and Occupational Medicine, 3rd edn., pp. 305–333. Philadelphia: Lippincott-Raven Publishers. Mossman B and Churg A (1998) State of the art: mechanisms in the pathogenesis of asbestosis and silicosis. American Journal of Respiratory and Critical Care Medicine 157: 1666–1680. Peto J, Declari A, LaVecchia C, Levi F, and Negri E (1999) The European mesothelioma epidemic. British Journal of Cancer 79(3/4): 666–672.

Pociask DA, Sime PJ, and Brody AR (2004) Asbestos-derived reactive oxygen species activate TGF-b1. Laboratory Investigation 84: 1013–1023. Rom WN, Travis WD, and Brody AR (1991) Cellular and molecular bases of the asbestos-related diseases. American Review of Respiratory Diseases 143: 408–422. Schwartz DA, Galvin JR, Dayton CS, et al. (1990) Determinants of restrictive lung function in asbestos-induced pleural fibrosis. Journal of Applied Physiology 68: 1932–1937. Selikoff IJ and Lee DHK (1978) Asbestos and Disease. New York: Academic Press. Sluyser M (1991) Asbestos-related cancer. In: Sluyser M (ed.) Ellis Horwood Series in Biochemistry and Biotechnology. Chichester: Ellis Horward Limited. Warheit DB, Hill LH, George G, and Brody AR (1986) Time course of chemotactic factor generation and the macrophage response to asbestos inhalation. American Review of Respiratory Diseases 134: 128–133. Warshamana GS, Pociask DA, Sime PJ, Schwartz DA, and Brody AR (2002) Susceptibility to asbestos-induced and TGF-b1-induced fibroproliferative lung disease in two strains of mice. American Journal of Respiratory Cell and Molecular Biology 27: 705–713. Wilkinson P, Hansell DM, Janssens J, et al. (1995) Is lung cancer associated with asbestos exposure when there are no small opacities on the chest radiograph? Lancet 345: 1074–1078. Yegles et al. (1993) Induction of metaphase and anaphase/telophase abnormalities by asbestos fibers in rat pleural mesothelial cells in vitro. American Journal of Respiratory Cell and Molecular Biology 9: 186–191.

Silicosis G S Davis, University of Vermont, Burlington, VT, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Silica (silicon dioxide, SiO2) is an abundant mineral that forms the major component of most rocks and soil. Silicosis is a chronic diffuse interstitial fibronodular lung disease caused by the inhalation of dust containing free crystalline silica. Workers may be exposed to silica where the use of power machinery generates numerous fine particles from stone. Exposures can be grouped broadly into trades where rock is drilled or removed from the earth, trades where silica-bearing rock is worked to produce stone products, jobs utilizing abrasives containing silica, and sites where finely powdered silica is used as an additive in manufacturing. Prolonged exposure to silica, usually over decades, is required to cause silicosis. The patient reports progressive shortness of breath and cough. Physical examination may be normal, or end-inspiratory crackles (rales) or wheezes may be heard over the mid-lung areas. Restrictive pulmonary function or a mixed pattern of obstruction and restriction appear with advanced disease. The chest radiograph shows an early diffuse pattern of small rounded opacities, predominantly in the upper lobes, enlargement of hilar and mediastinal lymph nodes, and later progression to coalescent opacities (progressive massive fibrosis) and compensatory emphysema. The characteristic pathology of silicosis is the silicotic nodule, with whorled collagen centrally, surrounding macrophages, lymphocytes, and fibroblasts, numerous birefringent polarizable silica particles,

OCCUPATIONAL DISEASES / Silicosis 227 and fibrosis. Silicosis patients are at risk for difficult to eradicate tuberculosis. Silica exposure carries a slightly increased risk for lung cancer. There is no effective treatment for silicosis; thus, it must be prevented before it occurs. The prevalence of silicosis has been decreasing in developed countries through industrial hygiene measures to reduce airborne dust levels, but silicosis remains common in the developing nations.

Introduction Silica is a naturally occurring abundant mineral that forms the major component of most rocks and soil. Silicosis is a chronic diffuse interstitial fibronodular lung disease caused by long-term inhalation of dust containing free crystalline silica. It is considered a traditional pneumoconiosis, or mineral dust lung disease. Most clinically significant exposure to silica occurs through occupations or trades working with stone, sand, or silica powder. Incidental environmental or household exposure rarely, if ever, causes silicosis. The inhalation of silica can be associated with other adverse health effects, including chronic bronchitis (industrial bronchitis), chronic obstructive pulmonary disease (COPD), tuberculosis, scleroderma and other rheumatologic conditions, and an increased risk for lung cancer.

earth is fluxed in processing some crystalline cristobaite is formed. The mineral must be in crystalline form to cause silicosis, since amorphous silica (glass) is essentially innocuous in crushed, powdered, or fibrous form. Among the crystalline polymorphs, quartz is the most abundant, and is the form usually associated with human disease. All of the crystalline forms have the same chemical formula, and all but stishovite assume the shape of a tetrahedron with four oxygen atoms shared by two silicon atoms. Natural stone varies in its silica content. Beach sand is pure quartz, while granite contains 10–15% crystalline free silica. Amorphous noncrystalline forms of silica occur in nature as diatomaceous earth (the skeletons of marine organisms) and as vitreous silica or volcanic glass. The surface properties of the particles appear to be critical in conferring the biological properties to crystalline silica. The biological activity of silica is attributable to silanol groups and other chemically reactive species on the crystal surface, and not to its physical shape or sharp edges. The particles must also be small enough to be resipirable (0.2–10 mm aerodynamic diameter) in order to reach the distal airspaces of the lung.

Source and Exposure

Structure and Physicochemical Properties The element silicon (Si) is abundant and widely distributed, comprising approximately 25% of the earth’s crust. Silicon is usually complexed with oxygen to form silica (silicon dioxide, SiO2) or with other anions and oxygen to form various silicates. Silica (SiO2) occurs in several crystalline forms and in noncrystalline amorphous forms as glass (Table 1). The crystalline and amorphous glass forms are interconvertible upon exposure to appropriate conditions of heat, pressure, and cooling. For example, window glass (amorphous silica) is manufactured from quartz sand by melting and then cooling the crystalline form; when amorphous diatomaceous

Table 1 Mineral forms of silicon dioxide (SiO2) Mineral name

Natural occurence

Crystalline form

Biological activity

Quartz Cristobalite Trydamite Stishovite Coesite Glass

Abundant Rare (or man-made) Uncommon Uncommon Uncommon Rare (or man-made)

Crystalline Crystalline Crystalline Crystalline Crystalline Amorphous

Moderate High High Low Low Very low

Workers may be exposed to silica dust in a variety of industrial settings where the use of power machinery generates numerous fine particles. These can be grouped broadly into trades where rock is drilled or removed from the earth (mining, quarrying, tunnel drilling), trades where silica-bearing stone is worked to produce other products (sculpting, cutting), jobs utilizing abrasives containing silica (tool grinding, sand blasting) or where abrasives are directed at a stone surface (building cleaning), and occupations where powdered silica is made or used as a raw material or additive in manufacture (glass, paint, plastics). Table 2 lists some of the occupations where substantial exposure to silica may occur. A common theme involves drilling and blasting through silica-containing rock to extract minerals (mining) or stone (quarrying) or to create passageways (tunneling), and where the work takes place in a confined space that may be poorly ventilated. Crafting stone for monuments, tombstones, or sculpture can generate substantial airborne dust, particularly when power tools rather than hand-held hammers and chisels are used to carve the stone. Abrasive blasting using sand for grit was an important cause of silicosis until recently. Sand blasters would clean iron boilers, oil drilling equipment, and

228 OCCUPATIONAL DISEASES / Silicosis Table 2 Occupations with exposure to silica Extractive and construction silica exposures

Fabricating industries with silica exposures

Hard rock mining Tunnel drilling

Silica flour production Diatomaceous earth production Glass manufacture Plastics manufacture Paint manufacture Ceramic composite manufacture Plastic resin manufacture

Stone quarrying Stone carving and sculpture Stone crushing Tool grinding and sharpening Building construction and demolition (concrete) Road and concrete repair Abrasive blasting

Pottery fabrication Metal foundry casting

other industrial parts using an air-driven stream of quartz sand. The work often took place in an enclosed and poorly ventilated space. Impact with the target could generate high concentrations of freshly fractured respirable silica particles. Sand has been replaced by less toxic abrasives such as carbonyl iron and other noncrystalline materials, and the use of sand for abrasive blasting is now regulated in most industrialized nations. The cleaning of stone buildings can still cause silica exposure when nonsilicaceous abrasive is blasted against silica-bearing stone. Sharpening tools or cleaning sand-cast foundry parts with silica-containing abrasive wheels can produce substantial exposures. Silicosis caused by mining, quarrying, tool grinding, and similar activities has been well recognized since antiquity. These exposures can usually be reduced to safe levels by vigilant industrial hygiene measures that involve efficient air extraction, spraying water on cutting surfaces, abrasive blasting in isolation booths, and the use of respiratory protective devices or independent clean air sources when needed. The development and implementation of control measures and the government regulation of these exposures to silica represent one of the major industrial hygiene success stories of the past halfcentury. It is a major reason for the progressive decline in the number of cases and deaths due to silicosis in the US and other industrialized countries. Unfortunately, silicosis is still very common in developing nations where extractive industries are growing and power equipment is being used more widely, but worker protection and industrial hygiene remains limited. Modern industry offers new types of exposures to silica that require different dust control measures than the traditional trades. Pure quartz sand is melted or fired to produce glass and ceramics. Lower purity silica sand is used for foundry casting of iron,

steel, aluminum, copper, and other metals. Workers involved in the production of abrasives, glass sand, and particularly silica powder may experience significant exposure and develop silicosis. Powdered silica, referred to as ‘silica flour’, is produced by grinding quartz sand into very small particles. In the finest grades of silica flour, more than 75% of the particles are respirable at less than 10 mm and are 99% pure quartz. Silica flour is used widely as an additive, absorbent, bulking agent, or abrasive in many products such as paints, plastics, toothpastes, and detergents. The additive is supplied in bags of dry powder, and workers who add this powder to product mixtures may receive brief but intense exposure to highly respirable silica dust. The critical elements in all of these trades are (1) the free crystalline silica content of whatever dust aerosol may be generated, (2) the concentration of particles in the air, and (3) the duration of exposure. The effects of the exposure will be modified by the general health, genetic susceptibility, and personal work habits of each individual. Permissible Exposure Limits to Airborne Silica

The US Occupational Safety and Health Administration (OSHA) and the Mine Safety Administration (MSA) have established a permissible exposure limit (PEL) for crystalline quartz of less than 0.10 mg m
3. The PEL for crystobalite and tridymite is 0.05 mg m
3. These values are calculated for the respirable fraction and as a time weighted average (TWA) across an 8 h work day. The US National Institute of Occupational Safety and Health (NIOSH) has recommended a more stringent standard for quartz, a PEL of 0.05 mg m
3. The UK Health and Safety Executive (HSE) specifies a transient peak maximum exposure limit (MEL) of 0.3 mg m
3. European and international regulatory and advisory groups have offered similar standards, and similar data to support them. Epidemiology of Silicosis

Lung disease caused by inhaled stone dust is ancient problem. Hippocrates noted respiratory difficulties in rock miners, pre-Columbian mummies in the Andes Mountains of Peru showed evidence of silicosis, and silicosis was common in the early nineteenth century among the tool grinders of Sheffield, England. The late nineteenth and early twentieth centuries saw increasing recognition of the cause and consequences of these diseases. Epidemiologic studies in many industries demonstrated clear relationships between the intensity of exposure, the duration of exposure, and the extent of disease. Workers and their labor

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unions, management groups, and government regulatory agencies worked to develop effective means to reduce dust exposure, monitor airborne dust levels, survey workers for lung disease, and ultimately decrease silicosis. In 1980 deaths related to silicosis totaled approximately 450 year
1 in the US, and have declined progressively over the past two decades. Deaths due primarily to silicosis now involve fewer than 100 year
1 cases with a similar number due to another primary disease but with silicosis as a contributing cause. Significant exposure to silica continues in the industrialized nations despite well-recognized PEL standards. Compliance with these limits varies among industries, but most surveillance reports fall below action levels. In the Vermont and Georgia granite industries most exposures now fall below the 0.10 mg m
3 PEL, but up to 15% of air samples exceed this level, and some jobs may reach exposures eightfold above the standard. In the slate production industry in Alta, Norway measurements of outdoor air and samples within quarry sheds ranged up to fourfold above the standard. Silica flour production workers in the US were identified as a group at high risk during the early 1980s, and in 1986 up to 30% of environmental samples still exceeded the specified PEL. In France exposures ranged from half to sixfold above the standard among 10 worker cohorts in various industries. A cross-sectional survey of silica-exposed American factory workers showed a slight excess loss of pulmonary function (forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1)) beyond that attributable to tobacco smoking, and the loss was directly related to the intensity and duration of silica exposure. European estimates suggest slight excess mortality (6–10 deaths per 1000 workers) attributable to silica. Both US and European surveys of construction trades have shown frequent exposures above permissible limits and rates of pneumoconiosis that are second only to those of miners and quarrymen. Surveys in mining and manufacturing industries in developing nations show high airborne dust levels where the use of power machinery has preceded workplace hygiene, and a high prevalence of advanced silicosis among the workers. These recent diverse international experiences clearly show that while the overall prevalence of silicosis is decreasing in developed nations, exposure to respirable silica dust remains a problem in many industries. The establishment of apparently safe air-quality standards by governmental regulatory agencies is not sufficient. These standards must be implemented by industries and by workers to create healthy work environments. The standards must be enforced by governments to assure worker safety.

Developing nations confront an even greater problem with less well-established governmental regulations, a lower level of worker awareness, and limited technological and financial resources. These exposures may cause excess mortality due to silicosis, and many more instances of substantial respiratory impairment. Silicosis in modern industry may not yet be a disease of the past.

Clinical Consequences Silicosis presents typically as a chronic diffuse lung disease with numerous small nodules, and evolves slowly over years or decades. This form is deemed ‘chronic simple’ silicosis and may progress to more extensive disease with coalescence of the nodules into conglomerate masses with surrounding fibrosis and traction emphysema. Extremely high levels of exposure can cause an acute and usually fatal form of silicosis accompanied by an outpouring of alveolar surfactant lipids and proteinaceous debris, a rare condition known as acute silicosis or silicoproteinosis. An intermediate form, termed accelerated silicosis, can develop in 2–5 years if exposure is intense. Symptoms and Exposure History

The patient with silicosis reports gradually progressive shortness of breath with exertion and cough, sometimes with clear or white sputum. The symptoms evolve slowly over years with little variation from day to day. Long exposure is required to cause illness and the disease progresses slowly; thus most workers with clinically evident silicosis are 40 years of age or older. Cough and sputum production are frequent, and many workers have associated bronchitis. Since many workers exposed to silica also smoke tobacco, it may be very difficult to distinguish the symptoms of silicosis from those of COPD. The work history is essential in establishing the exposure that may have led to silicosis. Years of exposure to airborne silica are usually required, and silicosis may progress long after exposure has ended because of the effects of the mineral retained in the lung. It is critical to obtain a complete past work history and to obtain as much detail as possible about what tasks were performed. This Information may be essential both to establish a secure diagnosis of silicosis and to qualify the worker for compensation. Physical Examination Findings

Physical examination may be normal, or high-pitched end-inspiratory crackles (rales) may be heard over the mid-lung areas. Emphysema or overdistension may surround zones of fibrosis leading to diminished

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Figure 1 Chest radiographs of simple silicosis. The frontal chest X-ray (a) demonstrates small rounded opacities throughout the mid lung zones. Larger opacities are evident at the left apex. The hilar lymph nodes are enlarged. The lateral view (b) also highlights the prominent hilar shadows and the larger opacities. The flattened diaphragms and hyperlucent retrostemal and posterior airspaces suggest concomitant emphysema. Radiology images courtesy of JS Klein, University of Vermont.

breath sounds with advanced disease. Silicotic lesions within or adjacent to airway walls can cause narrowing, and produce end-expiratory wheezing and prolonged expiration. Digital clubbing is very uncommon in silicosis. Signs of cor pulmonale (accentuated second heart sound, peripheral edema) may be late manifestations of advanced disease. In general, the physical examination in silicosis is less impressive than one might expect from the appearance of the chest radiograph or the abnormalities of pulmonary function. Pulmonary Function Testing

Pulmonary function may be normal in early or simple silicosis, particularly if testing is limited to spirometry. A mixed pattern of obstruction and reduction in lung volumes appears in workers with more advanced disease. This pattern may be confused with the abnormalities produced by COPD if the worker is a tobacco smoker. Measurements of gas transfer (diffusing capacity) are often abnormal. Oxygen saturation may be normal at rest, but decline with exertion. Progressive massive fibrosis produces severe restriction, loss of pulmonary compliance, and hypoxemia. Chest Radiology

The characteristic chest X-ray abnormalities of silicosis include a diffuse pattern of small rounded opacities, predominantly in the upper lobes of the lung, and enlargement of hilar and mediastinal lymph nodes with peripheral calcification. Specific categories and descriptions of radiographs that relate to silicosis are included in the scheme for classification of radiographs of the pneumoconiosis developed

by the International Labour Organization. Simple silicosis appears as small rounded opacities (type p, profusion 1/1–2/1). More extensive disease involves enlargement in nodule size and number (types p–q, profusion 1/2–2/2) (Figure 1). Peripheral ‘eggshell’ calcification of the hilar nodes is quite characteristic of silicosis, but most patients do not show this finding. Silicotic nodules in the lung parenchyma may calcify as well. The extent of radiographic abnormality roughly parallels the degree to which pulmonary function tests are decreased. With advanced disease, the discrete rounded opacities coalesce to form large irregular-shaped masses, thus the label of progressive massive fibrosis (Figure 2). The imaging appearances of silicosis are quite distinctive, and offer a specific diagnosis if a compatible exposure history is present. All of these abnormalities can be visualized much more clearly with high-resolution computed tomography (HRCT) of the chest (Figure 3). The patterns of silicosis found with HRCT are distinct, although some features may resemble sarcoidosis. Usually the patients with silicosis will be older, the extent of hilar lymphadenopathy is not as great, and the parenchymal opacities will not follow as axial a pattern along bronchovascular structures as with sarcoidosis, and the dystrophic calcifications will be more extensive in silicosis. Pathology of Silicosis

The characteristic lesion of silicosis is the silicotic nodule. The typical silicotic nodule is composed of whorled collagen and reticulin centrally, with surrounding macrophages, lymphocytes, fibroblasts, and fibrosis (Figure 4(a)). The nodules usually are

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Figure 2 Chest radiograph of progressive massive fibrosis. The radiograph of an iron-foundry worker with advanced silicosis shows changes dubbed ‘progressive massive fibrosis’, with large coalescent opacities in the upper and mid-lung zones. There is substantial compensatory emphysema surrounding the coalescent masses. The hilar lymph nodes are greatly enlarged. Radiology image courtesy of JS Klein, University of Vermont.

located near or around respiratory bronchioles. The smallest (or earliest) lesion is a collection of macrophages with dust particles concentrated within it. The developing silicotic nodule shows a distinct architecture, with large macrophages and silica particles abundant at the center. As the nodules enlarge, whorled concentric layers of collagen appear at the center of the lesions. Macrophages and many lymphocytes surround the central core, and the dust may move to the periphery as the lesions expand. Epithelioid giant cells are a variable feature. Granulomas are found occasionally. Diffuse fibrosis extending outwards from the conglomerate silicotic masses can be seen with advanced disease. Emphysema may develop around these lesions as they coalesce and contract. The histopathology of silicosis is quite distinct, and usually offers a definitive diagnosis. The inhaled dust is cleared from the lung by removal of the bronchial mucociliary escalator, or is translocated from the airspaces via lymphatics to central lymph nodes and peripheral subpleural lymphoid aggregates. Silicotic nodules and abundant dust can be found within the hilar and mediastinal lymph nodes, and sometimes within supraclavicular nodes as secondary lymphatic drainage sites. Dystrophic ossification occurs frequently in silicotic nodules producing the calcifications seen on chest radiographs.

Figure 3 Computed tomography of silicosis. High-resolution computed tomogram displayed with a bone algorithm ((a): lung windows) shows numerous small rounded opacities, coalescent opacities, and subpleural nodules. The mediastinal views (b) show extensive calcifications with the enlarged hilar and mediastinal lymph nodes. Radiology images courtesy of JS Klein, University of Vermont.

Silica particles appear as weakly birefringent crystalline material when lung tissue is examined under polarized light microscopy (Figure 4(b)). The smallest particles, those less than 0.5 mm, may be very difficult to see, but larger particles and aggregates of small ones can usually be detected easily. Mixed silicates, talc, and other minerals are also birefringent under polarized light, and may be confused with silica. However, they are usually more brightly birefringent. Special techniques of mineral analysis can be used to determine whether and how much silica is present, in lung tissue. Scanning electron microscopy (SEM) with back-scattered X-ray energy dispersive spectrometry (XES) will detect particles too small to visualize in the polarizing light microscope. Paraffinembedded lung tissue sections can be examined by SEM, electron-dense particles identified, and their relative composition of medium-to-high atomic number elements determined. Only Si would be recorded from particles of pure quartz, while Mg, Al,

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Figure 4 Pathology of silicosis. A silicotic nodule seen in lung tissue (a) shows central whorls of collagen with pigmented dust, surrounded by a rim of mononuclear cells and fibroblasts. Viewed with polarized light microscopy (b), birefringent particles are seen within a silicotic lesion. Pathology images courtesy of KJ Butnor, University of Vermont.

Fe, and other elements would be identified in mixed silicates, talc, or asbestos fibers. Individuals with no occupational exposure have small numbers of silica and silicate particles in their lungs from normal exposure to environmental dusts. Clinically significant silicosis always involves exposure to large quantities of inhaled dust, and thus silica particles are always abundant in their lung tissues. Although rarely needed, larger specimens of lung tissue can be ashed and subjected to X-ray diffraction analysis for quantitative reporting of the amounts of specific minerals present. Laboratory Tests

Laboratory blood tests are not particularly helpful in silicosis. Elevation of serum immunoglobulin levels, circulating immune complexes, rheumatoid factor, and antinuclear antibodies are sometimes observed but are neither sensitive nor specific. Other laboratory abnormalities usually are not found.

The radiographic changes are similar in both silicosis and tuberculosis, with progression of densities and infiltrates in upper lung zones. Tuberculosis with silicosis may be indolent and progress slowly, with few organisms shed, and barely positive sputum smears or cultures. Silicosis predisposes to infection with both typical Mycobacterium tuberculosis and ‘atypical’ nontuberculous mycobacteria. Both the species and drug sensitivities should be confirmed if mycobacteria are isolated from the sputum of a silica-exposed individual. Patients with silicotuberculosis usually respond well to conventional antituberculous therapy with clinical improvement and radiographic stabilization, but complete eradication of organisms may be very difficult. Reactivation following an apparent cure is common in silicotuberculosis. Lifelong antituberculosis chemotherapy is sometimes recommended for individuals with tuberculosis and silicosis. Tissue Diseases of Connective

Tuberculosis

An association between silicosis and increased susceptibility to tuberculosis has been demonstrated in hard rock miners, coal miners, granite workers, and other industrial groups. Workers with established silicosis appear to be more susceptible than the general population to developing active tuberculosis when exposed, and to a more chronic persistent form of tuberculosis once infected. The combined disease is sometimes referred to as silicotuberculosis.

An association between silica exposure and rheumatoid arthritis, systemic lupus erythematosus, and other connective tissue diseases has been proposed. Prevalence studies have sometimes (but not always) found an association between these conditions. The link is strongest for workers with overt silicosis. Workers who have silicosis and then develop rheumatoid arthritis may evidence Caplan’s syndrome, large pulmonary nodules that undergo central necrosis. Epidemiologic studies have revealed

OCCUPATIONAL DISEASES / Silicosis 233

convincing evidence for an excess prevalence of scleroderma (progressive systemic sclerosis) among workers exposed to silica, with features of scleroderma similar to those seen in nonoccupational settings. Lung Cancer

Workers with a dust exposure that is substantial enough to cause silicosis are at increased risk to develop lung cancer (bronchogenic carcinoma), and the relative risk may approach four times that of controls. Recent surveys of North American workers at plants producing quartz sand and of diatomaceous earth workers in California support the conclusion that lung cancer risk is slightly increased independently of tobacco smoking for individuals exposed to silica. Surveys of granite workers have varied in whether or not an increased cancer risk was found. Silica has been designated as a ‘known human carcinogen’ by the International Agency for Research on Cancer (IARC). This conclusion is based on extensive epidemiologic data from various countries and on substantial animal exposure experimentation. These conclusions and their-impact for human health remain controversial, however. For nonsmokers with levels of silica exposure below the recommended threshold limits, lung cancer remains rare, and the relative risks appear to be similar to or slightly above those for control populations (relative risk 1.0–1.5). For workers with silicosis who are smokers the cancer risk may be increased significantly.

Mechanisms of Disease The mechanisms by which silica triggers its characteristic pathological responses have been elucidated over the past half-century, but many details are not yet defined. Reactive moieties on the silica tetrahedral crystal surface (silanol groups) and reactive oxygen species generated by them appear to interact with biological membranes on the surface of cells and within cells after phagocytosis of the particles. Phagocytosis of silica particles through Toll-like receptors may also cause macrophage activation. These interactions cause damage or death to some macrophages and epithelial cells, and activation or stimulation of many others. Apoptosis of silica-bearing macrophages mediated through the Fas/Fas-ligand pathway appears to be a key step in the disease process. Silicosis persists as a chronic innate immune–inflammatory response. Chemokines may be liberated from the alveolar and bronchiolar epithelium and from pulmonary macrophages. These chemokines

recruit and activate lymphocytes and other cell types. Cytokines such as tumor necrosis factor alpha (TNFa), interleukin-1 (IL-1), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (ILGF) stimulate fibroblast influx and proliferation. Subsequently other cytokines such as transforming growth factor beta (TGF-b) may drive excess collagen production and downregulate matrix removal. Cytokines such as TNF-a, IL-1, and interferon gamma (IFN-g) stimulate the formation of the granuloma-like silicotic nodules. The lymphocytes that are prominent in this response are a mixture of CD4 þ and CD8 þ T cells and natural killer (NK) cells, and manifest a TH-1like phenotype by producing substantial IFN-g but little IL-4 or IL-13. The lymphocytes are polyclonal, and the response does not appear to be major histocompatibility complex (MHC) restricted. A key aspect of this pathogenetic process is probably the persistence of the causative agent, silica, at sites of active disease. Ultimately, the sites of active inflammation transition to fibrosis and, at some locations, to dystrophic calcification. Recent molecular genetic surveys have found a slightly increased risk for silicosis among individuals carrying a particular single nucleotide polymorphism (SNP) for interleukin-1 receptor antagonist (IL1RA), and increased severity for silicosis among those with the disease who carry several different SNPs for TNF-a. These findings suggest the validity of the animal and human research highlighting the roles of these cytokines, and focus attention on individual susceptibility as a modifying factor in the severity of silicosis among workers with substantial dust exposure.

Carborundum Silicon carbide (SiC) or carborundum is a synthetic abrasive manufactured through the fusion of highgrade silica sand and finely ground carbon (petroleum coke) in an electric furnace at high temperature (1600–25001C). The process generates both particles and fibers of silicon carbide as well as quartz and cristobalite silica, all of which may become airborne in the workplace during manufacture. The process for making silicon carbide as an industrial abrasive was patented in 1893 by the Pennsylvania inventor Edward G Acheson who dubbed the material ‘carborundum’. It is credited with a key role in making possible the mass production of interchangeable metal parts. Silicon carbide particles with sharp edges have strong abrasive properties and excellent durability. When glued or sintered onto wheels, disks, cloth, or sheets of paper, carborundum is

234 OCCUPATIONAL DISEASES / Silicosis

widely used in many grinding, shaping, and finishing applications. Silicon carbide can also be formed as fibers with excellent heat stability, thermal conductivity, and electrical current density, and these man-made fibers have broad applications in electronic devices and friction products. The noncrystalline silicon carbide fibers are a form of refractory ceramic fibers, or man-made vitreous fibers, and are used primarily for thermal insulation at high temperatures. Workers exposed to silicon carbide during its manufacture or who use carborundum abrasives extensively may be subject to diffuse interstitial pulmonary fibrosis or to a lung disease that resembles silicosis. Studies among Canadian and Norwegian silicon carbide production workers have suggested two- to fourfold increased mortality from nonmalignant respiratory disease and from lung cancer in proportion to the intensity of exposure, after adjustment for tobacco smoking. Two chest radiograph surveys of silicon carbide manufacturers showed a low fraction of workers with small rounded opacities or reticulonodular densities in low profusion. Small case series have described lung biopsy or autopsy findings, including large amounts of black material within fibrotic alveolar septae and abundant macrophages laden with particles. In several cases numerous black fibers were present, and ferruginous bodies had formed around some of them. When lung tissues were subjected to crystallographic and elemental analysis virtually all particles or fibers were amorphous (noncrystalline) silicon carbide, with very small fractions of crystalline quartz or cristobalite. The particle and fiber burdens approached those found in coal miners with progressive massive fibrosis (PMF) or workers exposed to asbestos. Experiments in which silicon carbide particles, quartz, asbestos, or silicon carbide fibers were instilled into the lungs of sheep showed essentially no biological effect but long persistence of the silicon carbide particles. In contrast, the silicon carbide fibers were cleared from the lung more effectively, but did produce a sustained nodular fibrosing alveolitis similar to that caused by the crocidolite asbestos. OSHA has recommended a PEL for silicon carbide of 5 mg m
3 respirable fraction limit (a high value appropriate for a nuisance dust), but this level is not enforced. These human and experimental reports suggest that silicon carbide may cause fibrotic lung disease, and that the fibers may be more toxic than the particulate form. The human studies are limited by the relatively small numbers of individuals surveyed, and the confounding effects of concomitant exposures to tobacco smoke, adsorbed hydrocarbons, and possibly crystalline silica. Considering the wide use of

carborundum abrasives for more than a century, it is likely that a major health problem caused by these particles would have been noticed. More subtle lung diseases, or an increased risk for lung cancer, caused by the silicon carbide ceramic vitreous fibers remains a concern and a focus for continued surveillance.

Management and Treatment There is no effective treatment for silicosis; it must be prevented by industrial hygiene measures. Many workers with abnormal chest radiographs and mild simple nodular silicosis experience few symptoms and mild impairment, while individuals with PMF are usually quite ill. Treatment should be supportive and focused on the individual patient. Helpful measures may include oxygen therapy during exertion or at rest if desaturation occurs, exercise training and rehabilitation if the patient is deconditioned, smoking cessation, primary therapy for COPD if present, antibiotic therapy for intercurrent respiratory infections, and surveillance and treatment of mycobacterial infection. Lung transplantation is an option for relatively young patients with severe disease. Several therapies for silicosis have been attempted but remain unproven. Corticosteroid treatment has been reported in a small number of patients with relatively acute, severe disease, and some short-term improvement was noted. However, there is no evidence for a long-term benefit in chronic silicosis. The Chinese natural medicine tetandrine has been evaluated in some patients, but toxicity and efficacy remain uncertain. Whole-lung lavage has been reported to show limited improvement in accelerated silicosis, but the long-term effectiveness is not known. Compensation for silicosis as an occupational lung disease is available in many countries, thus establishing a definitive diagnosis may be an important issue for some workers. The diagnosis usually is based on the combination of compatible chest X-ray abnormalities linked with a substantial appropriate exposure history. HRCT of the chest will substantiate the radiographic changes in more detail. Lung biopsy and mineral analysis of the tissue are almost never needed to confirm silicosis unless the radiographic findings are atypical or the exposure history is doubtful. The best strategy for reducing the health burden of silicosis is prevention through the reduction of airborne dust in the workplace. High-efficiency particulate filters on tightly fitting facemasks are needed in order to remove the fine respirable dust from the inhaled air. A simple paper dust mask is not sufficient. Many jobs with silica exposure involve heavy manual labor with high minute ventilation. It is not practical for a worker to wear a closed mask for this

OCCUPATIONAL DISEASES / Silicosis 235 Table 3 Strategies to reduce silica exposure in the workplace Strategy

Example

Increase fresh air at work site

Increased air exchanges in the mine tunnel Vacuum air extractor at the stone chisel face Spray water on the tool–stone contact point Substitute nonsilica abrasives in blasting Perform sand blasting in closed glove booths Use individual positivepressure air hoods Use tight-fitting efficient dust masks

Remove dust from the generation point Decrease the generation of dust Reduce the use of crystalline silica Isolate the worker from dust generation site Isolate the worker from dusty air Clean the inspired air

type of labor for a working lifetime. Thus effective industrial hygiene can be achieved best by measures that decrease the generation of dust and remove it from the air, rather than by respiratory protection of the individual worker. Some of the strategies that are applied in industry to reduce exposure to silica are listed in Table 3. Most work sites combine these approaches in order to decrease the net exposure. The other key elements in workplace safety are surveillance of the air for dust content and surveillance of the workers for evidence of disease. Air quality can be monitored by sampling from a central site within the facility or by collecting air near the face of an individual worker using a personal dust sampler. Measured quantities of air are drawn through small pore filters or other devices to entrap the particles. The filters are analyzed to determine the silica fraction and the weight of respirable particles per cubic meter of air. The results can then be compared with the PEL. Some industries with substantial silicosis risk perform periodic medical evaluations of their workers, utilizing respiratory symptom questionnaires, spirometry, and chest radiographs. Both air-quality monitoring and medical surveillance may be mandated or performed by governmental regulatory agencies.

Conclusions Knowledge about silicosis has increased tremendously over the last 150 years, bringing understanding about the nature of the dust that causes it, how to reduce exposure in the workplace, and how to recognize the disease when it occurs. The pathogenetic mechanisms of how crystalline mineral particles trigger chronic inflammatory and fibrotic responses in lung tissue have also been clarified, although many details remain unknown. Despite these advances,

silicosis is not entirely a disease of the past. A substantial number of cases appear each year in the industrialized nations despite adequate regulations and technology to prevent them. In the developing nations the challenge is even greater, as the rewards for work are high and both the knowledge and the resources for worker safety may be in short supply. Effective treatments are not available to remove the dust from the lung after it has been inhaled, to render the dust nontoxic, or to blunt the tissue response to silica. Thus the health impact of silicosis must be prevented before it occurs. Continuing attention to the details of workplace safety to reduce the levels of respirable crystalline silica particles in the air is essential to the prevention of silicosis. See also: Extracellular Matrix: Collagens. Occupational Diseases: Coal Workers’ Pneumoconiosis. Oxidants and Antioxidants: Oxidants. Particle Deposition in the Lung.

Further Reading Akira M (2002) High-resolution CT in the evaluation of occupational and environmental disease. Radiologic Clinics of North America 40(1): 43–59. Anonymous (2004) Changing patterns of pneumoconiosis mortality: United States, 1968–2000. MMWR. Morbidity and Mortality Weekly Report 53(28): 627–632. Beckett WC, Abraham JL, Becklake MR, et al. (1997) Adverse effects of crystalline silica exposure: American Thoracic Society Statement. American Journal of Respiratory and Critical Care Medicine 155: 761–765. Brody AR (1997) Occupational lung disease and the role of peptide growth factors. Current Opinion in Pulmonary Medicine 3(3): 203–208. Corbett EL, Mozzato-Chamay N, Butterworth AE, et al. (2002) Polymorphisms in the tumor necrosis factor-alpha gene promoter may predispose to severe silicosis in black South African miners. American Journal of Respiratory and Critical Care Medicine 165(5): 690–693. Craighead JE, Kleinerman J, Abraham JL, et al. (1998) Diseases associated with exposure to silica and nonfibrous silicate minerals: Silicosis and Silicate Disease Committee. Archives of Pathology & Laboratory Medicine 112: 673–720. Davis GS (2002) Silicosis. In: Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) Occupational Disorders of the Lung: Recognition, Management, and Prevention, pp. 105–127. London: Saunders/Harcourt Publishers. Davis GS, Holmes CE, Pfeiffer LM, and Hemenway DR (2001) Lymphocytes, lymphokines, and silicosis. Journal of Environmental Pathology, Toxicology and Oncology 20(Supplement 1): 53–65. Davis GS, Pfeiffer LM, and Hemenway DR (2000) Interferongamma production by specific lung lymphocyte phenotypes in silicosis in mice. American Journal of Respiratory Cell and Molecular Biology 22(4): 491–501. Durand P, Begin R, Samson L, et al. (1991) Silicon carbide pneumoconiosis: a radiographic assessment. American Journal of Industrial Medicine 20: 37–47.

236 ONCOGENES AND PROTO-ONCOGENES / Overview Graham WG, Vacek PM, Morgan WK, Muir DC, and Sisco-Cheng B (2001) Radiographic abnormalities in long-tenure Vermont granite workers and the permissible exposure limit for crystalline silica. Journal of Occupational and Environmental Medicine 43(4): 412–417. Hughes JM, Weill H, Rando RJ, et al. (2001) Cohort mortality study of North American industrial sand workers. II. Case-referent analysis of lung cancer and silicosis deaths. Annals of Occupational Hygiene 45(3): 201–207. Infante-Rivard C, Dufresne A, Armstrong B, Bouchard P, and Theriault G (1994) Cohort study of silicon carbide production workers. American Journal of Epidemiology 140(11): 1009–1015. International Agency for Research on Cancer (1987) Silica and some silicates. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans 42: 39–143. International Labor Organization (1981) Classification of radiographs of the pneumoconioses. Medical Radiography and Photography 57: l–17. Mossman BT and Churg A (1998) Mechanisms in the pathogenesis of asbestosis and silicosis. American Journal of Respiratory and Critical Care Medicine 157(5 Pt 1): 1666–1680.

National Institute for Occupational Safety and Health (2002) Health Effects of Occupational Exposure to Respirable Crystalline SilicaNIOSH Hazard Review. London: NIOSH. Talini D, Paggiaro PL, Falaschi F, et al. (1995) Chest radiography and high resolution computed tomography in the evaluation of workers exposed to silica dust: relation with functional findings. Journal of Occupational and Environmental Medicine 52(4): 262–267. Wiles FJ, Baskind E, Hessel PA, Bezuidenhout B, and Hnizdo E (1992) Lung function in silicosis. International Archives of Occupational and Environmental Health 63: 387–391. Yucesoy B, Vallyathan V, Landsittel DP, et al. (2001) Association of tumor necrosis factor-alpha and interleukin-1 gene polymorphisms with silicosis. Toxicology and Applied Pharmacology 172(1): 75–82.

Relevant Website http://www.cdc.gov – National Institute for Occupational Safety and Health.

ONCOGENES AND PROTO-ONCOGENES Contents

Overview jun Oncogenes MYC RAS

Overview R Salgia and O Abidoye, University of Chicago, Chicago, IL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Many cancer cells contain genetic mutations and alterations that are responsible for tumorigenesis. Dominant molecular alterations lead to the formation of genes known as oncogenes and proto-oncogenes. The activities of these genes cause critical modifications in the cell cycle necessary for tumorigenesis. These include abnormalities in self-sufficiency of growth signals; evading apoptosis; insensitivity to antigrowth signals; limitless replicative potential; sustained angiogenesis; and tissue invasion and metastases. A few that are recognized for their role in lung cancer, include the c-Met, and c-Kit receptor tyrosine kinases, as well as the ERBB, the RAS, the MYC, and the BCL-2 genes. The c-Met and c-Kit proto-oncogenes act through the modulation of growth factors and their cognate receptors in lung cancer, and also through the production of autocrine and paracrine growth stimulation loops. The ERBB proto-oncogenes are also a group of transmembrane receptor tyrosine kinases which, together with their ligands, constitute a potential growth

stimulatory loop, particularly for non-small cell lung cancer (NSCLC). The RAS proto-oncogenes regulate processes in signal transduction pathways. The MYC proto-oncogenes encode nuclear products which target the RAS signal transduction. The BCL-2 proto-oncogene protects against apoptosis and its expression is higher in small cell lung cancer (75–95%) than in NSCLC. Recent advances in the understanding of the oncogenes and proto-oncogenes involved in lung cancer biology have led to the development of numerous molecularly targeted therapies, which are beginning to play a great role in the treatment and prevention of lung cancer.

Introduction The two major types of lung cancer include small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Molecular biological studies have shown that lung cancers develop through sequential morphological steps, with the accumulation of multiple genetic and epigenetic alterations affecting dominant oncogenes/proto-oncogenes and alteration/deletion of tumor suppressor genes. The presence, amplification, and/or overexpression of proto-oncogenes/oncogenes

ONCOGENES AND PROTO-ONCOGENES / Overview 237 Increased expression Proto-oncogene

Proto-oncogene

Oncogene

Point mutation

Altered protein leading to altered function with activated growth

Overexpression of protein leading to activated growth

Figure 1 Overview of activity of oncogenes and proto-oncogenes.

Table 1 Molecular and genetic abnormalities in lung cancer Gene oncogene

c-Kit c-Met K-RAS MYC ERBB-2 BCL-2

Mutation

Expression/signal transduction Overexpression/signal transduction Point mutation (codon 12) DNA amplification overexpression Increased expression Expression of protein

can correlate with development of disease, response to treatment, and prognosis or survival. The wellidentified oncogenes in lung cancer include the RAS, MYC, and BCL-2 families, as well as the receptor tyrosine kinase (RTK) proto-oncogenes which include c-erbB family, c-Kit, c-Met, as well as Eph (Figure 1). Though standard chemotherapy and radiation continues to be the mainstay of treatment in lung cancer, recent growth in understanding of these proto-oncogenes/oncogenes led to development of novel therapeutic agents which target specific genes and the pathways they affect (Table 1).

The RAS Family This family of oncogenes includes H-RAS, K-RAS, and N-RAS. The RAS proteins have a molecular weight of 21 kDa and a high homology to large G proteins. They play a key role in signal transduction and cellular proliferation. This occurs through downstream activation of serine/threonine kinases, such as

Abnormal expression frequency NSCLC

SCLC

40% Not reported 30% 10% 25% 25%

30–40% Not reported Not reported 10–40% Not reported 55–90%

RAF, and mitogen-activated protein kinases (MAPKs) (ERK1 and ERK2), which translocate to the nucleus and activate transcription factors (Figure 2). Mutation of the K-RAS oncogene is seen in 30% of cases of NSCLC, and this along with their overexpression correlates with decreased survival, especially with resectable disease. Farnesyl transferase inhibitors (FTIs) inhibit posttranslational modification of the K-RAS proteins leading to inhibition of cell proliferation and derangement of the cell cycle of NSCLC cell lines. Three FTIs currently in clinical trials include R115777 (tipifarnib, Johnson and Johnson) and SCH66336 (lonafarnib, Schering-Plough) which are orally bioavailable, and BMS214662 (Bristol-Myers Squibb) which is administered intravenously.

The MYC Family The MYC family of oncogenes includes c-MYC, N-MYC, and L-MYC. The most identified family member in lung cancer is c-MYC. These genes all show amplification and/or overexpression in SCLC

238 ONCOGENES AND PROTO-ONCOGENES / Overview

EGFR

Oncogenes

ERBB2

BAX

BAX

BCL-2

BCL-2

BAX

MYC

RAS

p14ARF

BCL-2

p16INK4a

p16INK4B

p53

Cell apoptosis

RB

Cell cycle arrest Figure 2 Pathways of tumorigenesis in lung cancer involving the RAS and MYC oncogenes. Oncogene pathways in lung cancer. The p14ARF-p53 and p16INK4A-RB pathways are functionally linked and are involved in the pathogenesis of lung cancer. This is largely through their various components which serve as checkpoints and growth inhibitory pathways. Several oncogenes and growth factors are linked to these pathways. Note that many of the components are also inactivated in lung cancer.

cell lines, but c-MYC is the only member with activity that correlates with prognosis and clinical outcomes. The structure consists of three exons and two introns, measuring 5 kb and located on chromosome 8q24. It is divided into three functional regions. The c-MYC oncogene activity has been observed in genetic pathways controlling progression through the G1 phase of the cell cycle. The end processes of MYC gene amplification and/or overexpression in lung cancer play a pivotal role for the direct stimulation or repression of several distinct components of the cell cycle. Some novel agents for this oncogene downregulate gene overexpression with antisense oligodeoxyribonucleotides. This acts by interruption of translation of messenger RNA into protein, thereby blocking gene expression. Other agents under investigation include LC-1 (a monoclonal antibody with activity against the lung cancer cell line SPC-A-1) and gene therapy with the herpes simplex virus thymidine kinase (HSV-TK) gene.

The BCL-2 Family The B-cell leukemia/lymphoma 2 (BCL-2) oncogene is located on the human chromosome 18q21. The

Figure 3 BCL-2 and BAX in the regulation of apoptosis. Complexes of BCL-2 and BAX in the regulation of apoptosis. Under normal conditions, BCL-2 and BAX occur as heterodimers. In certain milieus, there is decreased transcription of BCL-2 and transcription of BAX. This supports formation of BAX homodimers, leading to cell apoptosis.

gene contains three exons and extends over at least 370 kb. BCL-2 functions as a negative regulator of cell death, prolonging the survival of noncycling cells, and inhibits apoptosis leading to prolonged cellular survival without increasing cellular proliferation. Overexpression of this gene has been reported in 22–56% of lung cancers, with a higher expression seen in SCLC (75–95%) than in NSCLC (Figure 3). Oblimersen sodium (Genasense, Genta-Aventis) is a BCL-2 antisense oligodeoxynucleotide undergoing phase II clinical trials (CALGB 30103) in SCLC. It is also being evaluated for efficacy as second-line therapy in NSCLC.

The Receptor Tyrosine Kinases RTK proto-oncogenes identified in the pathogenesis of lung cancer include c-erbB, c-Kit, c-Met, and Eph. Their molecular structure involves an N-terminal extracellular ligand binding domain, a hydrophobic transmembrane domain, and a C-terminal intracellular tyrosine kinase domain. The erbB Family (EGFR and HER2/neu)

The erbB family consists of EGFR (HER1), HER2, HER3, and HER4; other names for these receptors are erbB-1, erbB-2, erbB-3, and erbB-4, respectively. This family plays an important role in local tumor growth. This phenomenon requires growth-regulatory proteins such as epidermal growth factor receptor (EGFR) or proto-oncogene erb-B2.

ONCOGENES AND PROTO-ONCOGENES / Overview 239

Epidermal growth factor receptor EGFR is overexpressed in 40–80% of NSCLC, and its overexpression is associated with a poor prognosis. In SCLC, EGFR overexpression is rare. EGFR inhibition appears to lead to decreased cell proliferation, increased apoptosis, and reduced angiogenesis. EGFR can be mutated in the tyrosine kinase domain or amplified in some of the NSCLC. These findings have significant prognostic and therapeutic implications. RTK inhibitors have been widely studied in NSCLC. Two agents which have undergone FDA approval, and are currently in phase III clinical trials are gefitinib (Iressa, AstraZeneca) and erlotinib (Tarceva, Genetech). Gefitinib was the first targeted therapy to be approved by the Food and Drug Administration (FDA) for use in lung cancer. It has been studied in NSCLC alone and in combination with cytotoxic chemotherapy. Phase II testing (IDEAL 1 and IDEAL 2) using gefitinib as monotherapy in patients with refractory NSCLC demonstrated a substantial response. Despite the negative phase III trials, several clinical trials have observed that certain subgroups of patients had striking responses to gefitinib. Both the IDEAL 1 and IDEAL 2 trials noted increased responses to gefitinib among females, adenocarcinoma histology, nonsmokers, and patients of Asian descent. A retrospective review of patients with NSCLC revealed that patients who were never smokers and had adenocarcinoma histology with bronchioloalveolar features were more likely to respond to gefitinib. Recent studies indicate that gefitinib might also have antitumor activity in brain metastases from lung cancer. From the most recent ISEL trial of gefitinib in a phase III setting, no overall benefit of survival was seen. Erlotinib (OSI 774, Tarceva) is another EFGR tyrosine kinase inhibitor that has recently been approved by the FDA for the treatment of patients with NSCLC. Currently, the data generated from the erlotinib trials appear similar to the trials with gefitinib. Monoclonal antibodies against the extracellular domain of EGFR provide another avenue to inhibit the activity of EGFR. Cetuximab (Erbitux, BristolMyers) is a human/mouse chimeric antibody that binds EGFR and competitively inhibits EGF RTK activation. Several phase I/II studies demonstrated activity of cetuximab in previously untreated NSCLC, with response rates as high as 53%. HER2/neu The HER2/neu gene is localized on the long arm (q21) of chromosome 17. This gene codes for a 185 kDa receptor-type tyrosine protein kinase (p185 neu or c-erbB-2) similar to EGFR. This 1255 amino acid transmembrane glycoprotein is composed

of three domains: an extracellular factor-binding domain, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. In NSCLC, 25–30% of tumors overexpress HER2/neu, while a smaller percentage of patients with SCLC overexpress HER2/neu. This gene is involved in regulation of normal cell growth and differentiation, and can be associated with multiple signal transduction pathways. Overexpression of HER2/neu in lung cancer has shown a correlation with poor prognosis, and recently in some adenocarcinomas, HER2/ neu mutations in the tyrosine kinase domain have been detected. Trastuzumab (Herceptin, Genentech) is a humanized monoclonal antibody that targets the HER2/neu receptor. It has recently been FDA approved and is currently in several nonrandomized phase II trials in combination with cytotoxic chemotherapy. Vascular endothelial growth factor The vascular endothelial growth factor (VEGF) and its tyrosine kinase receptor (VEGFR) are the principal molecules involved in endothelial cell proliferation, formation of new blood vessels, and vascular permeability. Increased levels of VEGF are associated with a worse prognosis in many cancers, including NSCLC. Angiogenesis is an essential mechanism for tumor growth and metastasis, and its inhibition is an attractive target for investigators. Bevacizumab (Avastin, Genentech) is a humanized monoclonal antibody against VEGF, recently approved as first-line treatment in metastatic colon cancer. Phase II trials have shown that bevacizumab in combination with standard chemotherapy in NSCLC leads to higher response rates with an increase in survival. Recently, it has been shown that combination of bevacizumab with carboplatin and paclitaxel leads to survival benefit in first-line therapy for NSCLC. VEGF receptor tyrosine kinase inhibitors act by inhibiting VEGF signaling. Several of these agents are in development and being tested in phase I/II trials; these include vatalanib (PTK787, Novartis), SU6668 (Sugen), and ZD6474 (AstraZeneca). Other agents under investigation include endogenous angiogenesis inhibitors. The Kit Family

The c-Kit is a type III RTK similar to c-Fms, Flt3, and platelet-derived growth factor (PDGF). It is located on chromosome 4 (4q11–q12), which is in close proximity to the PDGF receptor. It is a 145 kDa protein containing five extracellular immunoglobulinlike domains, a single transmembrane domain, and a cytoplasmic domain with a split kinase domain and

240 ONCOGENES AND PROTO-ONCOGENES / Overview

a hydrophilic kinase insert sequence. The ligand for c-Kit is the stem cell factor (SCF), also known as the steel factor. This ligand supports growth and survival of immature hematopoietic cells of multiple lineages. This RTK proto-oncogene is expressed in numerous normal tissues, but is overexpressed in 30–40% of SCLC tumors. Imatinib (Gleevec, Novartis) is a tyrosine kinase inhibitor with activity against c-Kit and PDGFR. Imatinib was developed as a target against the BCR/ ABL oncogene, a fusion protein found in chronic myelogenous leukemia, and it has also shown activity in SCLC cell lines, where it inhibits tumor growth by inactivating c-Kit. Recent phase II trials failed to demonstrate a clinical response in patients, but further study targeting specific c-Kit expression is warranted. Also, it would be more useful to do combinations with cytotoxic chemotherapy.

c-Met can be mutated in the juxtamembrane domain or the semaphoring domain in some of the SCLC and NSCLC. Expression of c-Met has been shown to correlate with higher pathologic tumor stage and worse prognosis. NSCLC and SCLC cell lines have been shown to express c-Met, as well as 72% of NSCLC adenocarcinomas and 38.5% of NSCLC squamous carcinomas. The development of small molecules that inhibit c-Met is currently in preclinical stages. SU11274 (Sugen) and PHA665752 (Sugen) are two c-Met inhibitors under investigation. In lung cancer cell lines, SU11274 induces cell-cycle arrest and apoptosis in cell lines that overexpress c-Met and Tpr/Met. PHA665752 inhibits c-Met phosphorylation, and the phosphorylation of c-Met’s downstream effectors, and is believed to have activity in the inhibition of tumor growth.

The Met Family

Eph

c-Met is a disulfide-linked heterodimeric RTK with an extracellular alpha chain and beta subunit that contains the ligand-binding site, a single transmembrane domain, and an intracellular tyrosine kinase catalytic domain. A single precursor weighing 170 kDa is cleaved and glycosylated to obtain both of the subunits. c-Met is activated by its ligand hepatocyte growth factor (HGF) (also known as the scatter factor), which causes phosphorylation at multiple sites, with the major site located at Y1230, Y1234, and Y1235. HGF stimulation of c-Met can lead to proliferation, survival, motility, invasion of extracellular matrix, and formation of tubules.

The Eph receptors represent the largest family of receptor protein tyrosine kinases and interact with ligands called ephrins. The Eph receptors and ephrins are divided into the two subclasses, A and B, based on their sequence homologies, structures, and binding affinities. Eph receptors contain a single transmembrane-spanning domain, with an extracellular region comprised of a ligand-binding domain and an intracellular region comprised of a juxtamembrane domain, a tyrosine kinase domain, sterile alpha motif (SAM), and PDZ binding motif (PSD-95 postsynaptic density protein, disks large, and zona occludens tight junction proteins). Ephrin A ligands are bound

Ephrin A

Cell membrane EPH Kinase Glob

Cys

FNIII

FNIII

SAM

Ephrin B

Figure 4 Structure of the Eph receptor. FNIII, fibronectin type III; Glob, globular domain; SAM, sterile alpha motif.

ONCOGENES AND PROTO-ONCOGENES / jun Oncogenes 241

by EphA-type receptors, while ephrin B ligands contain a transmembrane domain and a short cytoplasmic region and are bound by EphB-type receptors (Figure 4). Eph receptor stimulation by their ephrin ligands mediates angiogenesis, cell segregation, and cell attachment, shape, and motility. Recent studies have demonstrated upregulation/downregulation of specific Eph receptors and their ephrin ligands in lung cancer, as well as correlation between levels of expression and stage of disease. High expression of EphA2 in NSCLC appears to favor the development of metastasis to the brain while low expression serves as a predictor of disease-free survival or contralateral lung metastasis. The Eph1B receptor is believed to affect SCLC behavior through ephrin-B ligand expression. Recent development of targeted therapies includes antibody targeting of EphA2 as well as monoclonal antibodies to mimic ephrin A1. Animal model studies have been successful in demonstrating ability of these antibodies to inhibit tumor angiogenesis and cell growth; however, to date this effect has not been demonstrated in human models. See also: Hepatocyte Growth (Scatter) Factor. Oncogenes and Proto-Oncogenes: jun Oncogenes; MYC; RAS. Oxidants and Antioxidants: Oxidants. Signal Transduction. Transcription Factors: ATF; NF-kB and Ikb. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumors, Malignant: Lymphoma.

Further Reading ASCO (2005) Annual meeting proceedings. Journal of clinical Oncology (supplement) 23: 165. Fong KM, Sekido Y, Gazdar AF, and Minna JD (2003) Lung cancer 9: Molecular biology of lung cancer: clinical implications. Thorax 58(10): 892–900. Gazdar AF and Carbone DP (1994) The Biology and Molecular Genetics of Lung Cancer. Austin, TX: RG Landes (Medical Intelligence Unit). Haura EB, Sotomayor E, and Antonia SJ (2003) Gene therapy for lung cancer. Molecular Biotechnology 25(2): 139–148. Sanchez-Cespedes M (2003) Dissecting the genetic alterations involved in lung carcinogenesis. Lung Cancer 40(2): 111–121. Sattler M and Salgia R (2003) Molecular and cellular biology of small cell lung cancer. Seminars in Oncology 30(1): 57–71. Talbot SG, O-Charoenrat P, Sarkaria IS, et al. (2004) Squamous cell carcinoma related oncogene regulates angiogenesis through vascular endothelial growth factor-A. Annals of Surgical Oncology 11(5): 530–534. Valle RP, Chavany C, Zhukov TA, and Jendoubi M (2003) New approaches for biomarker discovery in lung cancer. Expert Review of Molecular Diagnostics 3(1): 55–67. Wistuba II and Gazdar AF (2003) Characteristic genetic alterations in lung cancer. Molecular Medicine 74: 3–28.

jun Oncogenes P K Vogt and A G Bader, The Scripps Research Institute, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The discovery of oncogenic transcription factors brought into focus an important aspect of oncogenesis, deregulated transcription. Transcriptional controls are the principal determinant of gene expression profiles that characterize specific cellular phenotypes in development and differentiation. Jun is a transcriptional regulator with strong oncogenic potential. It plays an important role in the cell cycle and promotes cell proliferation. Jun activity is tightly controlled in the normal cellular context. With that control absent, Jun can induce cancer in the animal and likely contributes to the development of malignancies in man, including pulmonary disease.

The jun Oncogene The jun gene was originally identified as the oncogenic element of the avian sarcoma virus 17, a retrovirus that readily causes fibrosarcomas in chickens (ASV17; ‘jun’ is a truncated form of ju-nana, Japanese for 17). This viral jun (v-jun) is derived from the genome of the avian host and inserted into the retroviral genome as a result of recombination between virus and cell. The N-terminus of the v-Jun protein is joined to viral sequences, and in the infected cell vJun is expressed as a hybrid Gag–Jun fusion protein (Figure 1). Besides being fused to Gag, the v-Jun protein differs from the cellular c-Jun protein by (1) the N-terminal addition of nine amino acids encoded by the 50 untranslated region of c-Jun, (2) an internal deletion of 27 amino acids defining the delta domain (d), and (3) two point mutations, altering serine 222 to phenylalanine and cysteine 248 to serine. These structural distinctions of v-Jun and its high expression level that is controlled by the active retroviral promoter are the underlying cause for the potent tumorigenicity of v-Jun.

Jun is a Transcription Factor The structural and biochemical properties of Jun are that of a transcription factor. Jun is primarily an activator of transcription, but in certain cellular and genomic contexts can also function as a transcriptional repressor. There are two other members of the Jun protein family, JunB and JunD. Their roles of transcription are highly dependent on the cellular environment. The domain structure of Jun includes two N-terminal activation regions (A1 and A2) and a C-terminal bZIP structure consisting of a basic region and a leucine zipper (Figure 1). The basic region

242 ONCOGENES AND PROTO-ONCOGENES / jun Oncogenes 222 248

1


A1

bZIP A2 Ser Cys

A1

A2

220

1 ∆Gag

310 c-Jun 512 v-Jun

bZIP

Phe Ser Figure 1 The primary structure of the chicken c-Jun and v-Jun proteins. Positions of amino acids are shown in the figure. Red bars indicate structural differences between c-Jun and v-Jun. Various protein domains are color-coded: yellow: bZIP, basic region and leucine zipper; blue: transactivation domains A1 and A2; dark gray: nine additional amino acids encoded by the 50 untranslated region of chicken c-jun; green: ASV17 viral Gag protein sequences.

Table 1 bZIP protein families and their members Protein family

Name

Protein members Homodimers

Jun

Fos ATF CREB C/EBP Maf PAR

v-jun avian sarcoma virus 17 oncogene homolog FBJ murine osteosarcoma viral oncogene homolog Activating transcription factor cAMP responsive element binding protein CCAAT/enhancer binding protein Avian musculoaponeurotic fibrosarcoma virus homolog Proline and acidic rich

Homo- and Heterodimers

Heterodimers

c-Jun, JunD, JunB

ATF1, ATF6

ATF2, ATF4, ATF7

c-Fos, FosB, Fra-1, Fra-2, JDP1, JDP2 ATF3

CEBPa, CEBPb, CEBPd, CEBPe, CEPBg MafF, MafG, MafK

c-Maf, MafB, NRL

CREB1, CREB3, CREM, CREB-H, CREB-L1

DBP, TEF, HLF

Jun

Leucine zipper

Fos

sic

Ba ion

reg

of the bZIP domain forms the DNA contact surface, and the leucine zipper structure mediates dimerization of the Jun and related proteins. The bZIP region is emblematic for a large class of transcriptional regulators encompassing the families of Jun, Fos, activating transcription factor (ATF), cAMP responsive element binding protein (CREB), CCAAT enhancer binding protein (C/EBP), avian musculoaponeurotic fibrosarcoma virus homolog (Maf), and proline and acidic rich (PAR) proteins (Table 1). For these proteins, dimerization is a prerequisite for DNA binding and hence for transcriptional activity. Some bZIP proteins form only homodimers, others require heterodimerization, and a third group is able to homo- and heterodimerize. These dimerization specificities are encoded in the amino acid sequences of the leucine zippers. The crystal structure of the bZIP domains of the Jun ortholog in yeast, GCN4, and of the Jun–Fos heterodimer bound to DNA shows a pair of continuous a-helices that form a Y (Figure 2). The branches mark the N-terminal region of the structure and contain the basic regions that reach into the major grove of the target DNA whereby arginine and lysine residues interact with the DNA backbone. Further

DNA

TGACTCA Figure 2 Schematic of AP-1 bZIP structure on DNA. The leucine zipper domains of the Jun (blue) and Fos (green) proteins facilitate dimerization, the basic regions make direct contacts with the DNA duplex. The classic DNA-binding site recognized by the Jun–Fos dimer is TGACTCA.

N-terminally positioned residues, one asparagine, alanines and other amino acids, make specific contacts with DNA base pairs and are responsible for the recognition of the DNA target sequence. The stem of the Y consists largely of the leucine zipper dimerization region that extends away from the DNA at an angle of almost 901. In the leucine zipper, every seventh position is a leucine or an alternative

ONCOGENES AND PROTO-ONCOGENES / jun Oncogenes 243

hydrophobic residue (heptad repeat). Since the ahelix has a periodicity of 3.5 residues per turn, every seventh amino acid is in the same structural environment. Leucine zipper helices are amphipathic with opposing hydrophobic and hydrophilic faces oriented along the long axis of the helix. In the dimer structure, the hydrophobic faces containing the leucines form the contact surface. Nonleucine residues within the proximity of the hydrophobic core determine the stability of the dimer and the compatibility of dimerization partners. The heterodimer of Jun and Fos is the classic form of the functional Jun. It is also known as activator protein 1 (AP-1). AP-1 preferentially binds to the pseudopalindromic DNA sequence TGA C/G TCA. Transcription of genes regulated by AP-1-binding sites is stimulated by 12-O-tetradecanoyl 13-phorbol acetate (TPA), an activator of protein kinase C that works through AP-1. The AP-1-binding sequence is therefore also known as the TPA response element (TRE). Since Jun proteins are able to dimerize with various bZIP proteins, the definition of AP-1 proteins has been extended to any dimer that involves at last one Jun protein. The dimerization partners of Jun can modulate the specificity of DNA-binding, and some Jun heterodimers interact preferentially with variant binding sites. Jun-ATF2 for instance binds to the cAMP response element TGACGTCA or TTACGTCA.

Regulation of AP-1 Activity The regulation of AP-1 activity occurs at several levels. The first level is transcriptional. The jun genes and genes encoding proteins of the AP-1 complex are regulated individually. Whereas some are expressed constitutively, others become activated only upon certain stimuli. Some are tissue specific, others are found ubiquitously. Members of the jun and fos gene families belong to the class of ‘immediate-early’ genes that are highly activated in response to mitogenic signals during the cell cycle in the transition from G1 into the S phase. Yet, the expression profiles

CKII

JNK T91 T93

1


A1 S63 S73 JNK ERK

of these genes are distinct. c-fos mRNAs are rapidly elevated within 30 min after growth stimulation, followed by c-jun, junB, fra-1, and fra-2. In contrast, junD mRNAs are expressed at steady-state levels during the cell cycle. The second level of regulation occurs at the stage of mRNA and protein turnover. Specific protein domains or sequences within the mRNA determine the stability and half-life of these molecules. c-jun and c-fos mRNAs contain a series of AU-rich elements (AREs) with the sequence AUUUA or variations thereof in the 30 untranslated region. AREs facilitate the interaction with the AU-rich binding protein AUF-1 that induces rapid degradation of the target mRNA. Similar to mRNAs, Jun and Fos proteins are short-lived and rapidly degraded by the 26 S proteasome. c-Jun is a moderately labile protein with a half-life of 2 h; JunD is much more stable. Protein stability is regulated by N-terminal Jun protein sequences and the degree of phosphorylation mediated by various kinases. Transient transcriptional stimulation followed by rapid mRNA and protein turnover generates a distinct peak of Jun expression in the first hour after induction that gives way to basal levels shortly thereafter. A third level of control is achieved by posttranslational modification. Jun activity is regulated by protein kinases. c-Jun becomes phosphorylated by extracellular-signal-regulated kinases (ERKs) and Jun amino-terminal kinases (JNKs, also referred to as stress-activated protein kinases or SAPKs). A map of c-Jun phosphorylation sites is shown in Figure 3. ERKs and JNKs belong to the class of mitogen-activated protein kinases (MAPKs) that represent the terminal members of a series of kinases transducing signals from the plasma membrane to intracellular effectors. Phosphorylation of human c-Jun occurs in the transactivation domain A1 at serines 63 and 73, as well as threonines 91 and 93. The phosphorylation of residues 63 and 73 is mediated by JNKs or ERKs; phosphorylation of amino acids at positions 91 and 93 is responsive to JNKs only. The C-terminally located transactivation domain A2 is targeted ERK

T231 

S243

A2

331 bZIP

T239 GSK3

Figure 3 Map of phosphorylation sites of human c-Jun. Positions of amino acids are indicated by numbers. See text for explanations. JNK, Jun N-terminal kinase; ERK, extracellular regulated kinase; CKII, casein kinase II; GSK3, glycogen synthase kinase 3.

244 ONCOGENES AND PROTO-ONCOGENES / jun Oncogenes

by casein kinase II (CKII), glycogen synthase kinase 3 (GSK3), and ERKs at three major phosphorylation sites: threonine 231, threonine 239 and serine 243. The phosphorylation of A1 and A2 has opposite effects on Jun activity. Phosphorylation of A1 increases the transcriptional activity and the half-life of c-Jun, phosphorylation of A2 negatively controls Jun by decreasing DNA binding. The identity of the phosphatases responsible for dephosphorylation of sites in A2 remains to be determined. In a simplified model, phosphorylation of c-Jun in A1 precedes and stimulates the dephosphorylation of sites in A2 which activates DNA binding. The phosphorylation sites in A1 are functionally linked to the two domains d and e (Figure 3). The d domain is a docking platform to recruit JNKs for subsequent phosphorylation. In addition, the d domain has an inhibitory function, as does the e domain. Both domains interact with histone deacetylases (HDACs) and other transcriptional co-repressors that dissociate from c-Jun upon phosphorylation of A1. At the final stage of control, AP-1 activity is regulated by the composition of the protein dimer and AP-1 interacting proteins. Jun forms homo- or heterodimers with other members of the bZIP family. The identity of the dimer depends on the availability of and the preference for the individual dimerization partners. The composition of the dimer determines the preference for the particular DNA-binding site, and as a consequence the regulation of an individual set of target genes. In addition, the AP-1 dimer together with DNA is able to form a quarterny complex with ancillary proteins. Proteins known to interact with AP-1 include members of the Ets and Smad transcription factor families, the octamer binding proteins (Oct), nuclear factor of activated T cells (NFAT), and nuclear factor kappa B (NF-kB) proteins. Binding to these cofactors alters DNA-binding specificity of the AP-1 protein complex.

Oncogenicity of Jun Jun and its partner proteins affect numerous biological processes in embryonic development, programmed cell death, proliferation, and tumorigenesis. Genetic knockout of the c-jun or junB genes in mice results in early embryonic death, demonstrating the importance of Jun in cellular biology (Table 2). It induces proliferation by stimulating the transition from the G1 into the S phase of the cell cycle. c-Jun transcriptionally activates the expression of cyclin D1, a positive regulator of proliferation, and represses expression of p53, a cell-cycle inhibitor. Most members of the Jun and Fos families are inherently oncogenic. The viral proteins, v-Jun and v-Fos, as well as c-Jun, c-Fos, FosB,

Table 2 Analysis of jun and fos knockout mice Gene

Phenotype

Affected organ

c-jun junB junD c-fos fosB fra-1

Embryonic lethality Embryonic lethality Male sterlity Osteopetrosis Nurturing defect Embryonic lethality

Liver Extra-embryonic tissue, placenta Testis Bone Brain Extra-embryonic tissue, placenta

Fra-1, and Fra-2 are transforming in cell culture; vJun, v-Fos, c-Fos, and Fra-1 cause tumors in the animal. In contrast, JunB and JunD have antioncogenic activity. The effect of structure–function changes on oncogenicity is illustrated by a comparison of v-Jun and c-Jun (Figure 1). Mutational analyses show that the lack of the d domain and the two point mutations within the A2 and bZIP domains in v-Jun enhance oncogenicity of Jun. N-terminal Gag sequences of vJun are dispensable for oncogenic activity. In human cJun, phosphorylation of serine 243 negatively affects DNA binding. The corresponding residue in chicken c-Jun is serine 222 that is altered to phenylalanine in v-Jun. This mutated residue can no longer be phosphorylated and, hence, contributes to enhanced DNAbinding activity in v-Jun. Deletion of the d domain enhances the transcriptional activity and oncogenic potential of c-Jun. Jun lacking the d domain is free of transcriptional co-repressors and bypasses intracellular controls by JNKs and ERKs. The present understanding of Jun views oncogenesis as a deregulation of transcriptional control. Several studies have attempted to identify target genes that are either up- or downregulated by oncogenic Jun and whose differential expression is an essential feature of oncogenic transformation. The relevance for the oncogenic process is judged by the partial transforming activities of upregulated targets and by the antioncogenic activities of downregulated targets (Table 3). Cells with the characteristics of Jun-transformed cells cannot be generated by the differential expression of any single target. Jun-induced oncogenicity is therefore likely the result of a combined effect originating in the differential expression of several targets.

Jun in Human Cancer The identification of Jun as the tumorigenic component of a chicken retrovirus established a fundamental role of transcription in cancer. Yet, there is no genetic evidence that Jun is directly involved in human cancer. There are no activating Jun mutations, nor have any gene amplifications or chromosomal translocations involving jun been found. Nevertheless, indirect

ONCOGENES AND PROTO-ONCOGENES / jun Oncogenes 245 Table 3 v-Jun target genes that affect the cellular phenotype Gene

Name

Function

Upregulated genes contributing to oncogenic cell transformation when overexpressed HB-EGF Heparin-binding epidermal growth factor-like Growth factor growth factor Jac Jun-activated gene in chicken Unknown Toj3 Target of Jun Nucleolar protein of unknown function Downregulated genes interfering with oncogenic cell transformation when overexpressed Sparc Secreted protein, acidic and rich in cysteine Extracellular matrix protein Marcks Akap12 a

Myristylated alanine-rich protein C kinase substrate A-kinase anchor protein 12

Cellular phenotype Focus formation and colony formationa Colony formation Colony formation

Scaffolding protein

Reduced tumor growth induced by v-Jun Reduced colony formation

Scaffolding protein

Reduced colony formation

Focus and colony formation assays are tools to measure anchorage-independent growth, a hallmark of cancer cells.

evidence suggests that Jun may participate as an essential component in human tumors. Jun is able to transform mammalian cells in cooperation with another activated oncoprotein, such as Ras or Src. Exposure to cigarette smoke and asbestos, and more specifically reactive oxygen species (ROS), upregulate AP-1 expression and AP-1 DNA-binding activity which contributes to the progression of pulmonary disease. Transgenic expression of Fra-1 induces lung tumors in mice. Deletion of c-jun in mouse hepatocytes does not interfere with normal cellular function, but prevents the occurrence of chemically induced hepatocellular carcinomas. Similarly, dominant negative Jun mutants attenuate the growth of various human tumor cell lines and interfere with oncogenic cell transformation linked to the Ras pathway. c-Jun levels are elevated in tumor cells from patients with pancreatic cancer, acute myeloid leukemia, Hodgkin lymphomas, or anaplastic large cell lymphomas.

Acknowledgments This is TSRI manuscript number 16993-MEM. This work was supported by grants from the National Cancer Institute, National Institutes of Health. See also: Apoptosis. Cell Cycle and Cell-Cycle Checkpoints. DNA: Structure and Function. Gene Regulation. Oncogenes and Proto-Oncogenes: Overview; MYC; RAS. Signal Transduction. Transcription Factors: Overview; AP-1; ATF; PU.1.

Further Reading Dunn C, Wiltshire C, MacLaren A, and Gillespie DA (2002) Molecular mechanism and biological functions of c-Jun N-terminal kinase signalling via the c-Jun transcription factor. Cell Signalling 14: 585–593.

Eferl R and Wagner EF (2003) AP-1: a double-edged sword in tumorigenesis. Nature Reviews: Cancer 3: 859–868. Ellenberger TE, Brandl CJ, Struhl K, and Harrison SC (1992) The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the proteinDNA complex. Cell 71: 1223–1237. Glover JN and Harrison SC (1995) Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature 373: 257–261. Hartl M, Bader AG, and Bister K (2003) Molecular targets of the oncogenic transcription factor jun. Current Cancer Drug Targets 3: 41–55. Maki Y, Bos TJ, Davis C, Starbuck M, and Vogt PK (1987) Avian sarcoma virus 17 carries the jun oncogene. Proceedings of the National Academy of Sciences, USA 84: 2848–2852. Morton S, Davis RJ, McLaren A, and Cohen P (2003) A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun. EMBO Journal 22: 3876–3886. O’Shea EK, Klemm JD, Kim PS, and Alber T (1991) X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254: 539–544. Reddy SPM and Mossman BT (2002) Role and regulation of activator protein-1 in toxicant-induced responses of the lung. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L1161–L1178. Shaulian E and Karin M (2002) AP-1 as a regulator of cell life and death. Nature Cell Biology 4: E131–E136. Sprowles A and Wisdom R (2003) Oncogenic effect of delta deletion in v-Jun does not result from uncoupling Jun from JNK signaling. Oncogene 22: 498–506. Vinson C, Myakishev M, Acharya A, et al. (2002) Classification of human B-ZIP proteins based on dimerization properties. Molecular Cell Biology 22: 6321–6335. Vogt PK (2002) Fortuitous convergences: the beginnings of JUN. Nature Reviews: Cancer 2: 465–469. Wagner EF (ed.) (2001) AP-1. Oncogene 20: 2333–2497. Weiss C and Bohmann D (2004) Deregulated repression of c-Jun provides a potential link to its role in tumorigenesis. Cell Cycle 3: 111–113. Weiss C, Schneider S, Wagner EF, et al. (2003) JNK phosphorylation relieves HDAC3-dependent suppression of the transcriptional activity of c-Jun. EMBO Journal 22: 3686– 3695. Wisdom R (1999) AP-1: one switch for many signals. Experimental Cell Research 253: 180–185.

246 ONCOGENES AND PROTO-ONCOGENES / MYC

MYC R Salgia and O Abidoye, University of Chicago, Chicago, IL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The MYC genes are a family of oncogenes and proto-oncogenes which play a role in the development of human tumors. Members of this family include the c-MYC, N-MYC, and the L-MYC genes. The importance of the MYC gene in lung cancer has been observed through recent studies demonstrating their role in tumorigenesis, apoptosis, metastasis, and drug resistance. Overexpression and amplification of MYC has been clearly identified in lung cancer. The MYC oncogene is believed to encode DNA through the formation of a complex called MYCMax. This complex is thought to lead to alterations in gene expression and consequently cell proliferation and differentiation. The amplification/overexpression of this oncogene, specifically the c-MYC oncogene, has also been observed to correlate with patient prognosis, particularly in small cell lung cancer. The c-MYC oncogene may have activity through two pathways that control progression through the G1 phase of the cell cycle. Overexpression of MYC induces the activity of certain transcription factors as well as the activation of cyclin kinases, leading to the suppression of growth inhibition factors. Lung cancer as a disease is characterized by early metastasis, a rapid tumor cell proliferation, with initial response to therapy followed by a rapid decline in sensitivity, leading to a more aggressive course of disease than its counterparts. The discovery of the MYC genes has led to new developments in novel targeted therapy in lung cancer.

Introduction The MYC genes are a family of oncogenes and protooncogenes which share a homology with retroviruses, code for nuclear binding proteins with known short half-lives, as well as share a common structure. The importance of this oncogene family lies in its function to encode a group of nuclear phosphoproteins that are believed to play a role in cell growth and in the development of human tumors, including lung cancer. Overexpression and amplification of MYC have been identified in lung cancer cells. Although this is a rare event that may be induced by prior chemotherapy exposure, overexpression has been reported in a substantial portion of small cell lung cancer (SCLC) and in a subset of non-small cell lung cancer (NSCLC). The three identified oncogenes which constitute the family of MYC oncogenes are the c-MYC, N-MYC, and the L-MYC genes. Recent focus has been placed on the study of this family of oncogenes and their role in tumorigenesis. This will lead to better understanding of the pathogenesis of cancer and the development of novel targeted agents for treatment.

Historically, knowledge of the MYC gene evolved from studies conducted on the defective avian retrovirus known as the Myelocytomatosis virus, MC29. The MC29 virus was known to cause leukemias, carcinomas, and sarcomas in chicken. Studies then showed that the virus carried incomplete retroviral gene sequences along with a hybrid-transforming gene termed the v-myc gene. The v-myc gene consisted of a 1568 base pair (bp) sequence from the retroviral gag gene. In addition, this was demonstrated to share a greater than 99% nucleotide sequence and amino acid homology with another gene of a 2735 bp sequence termed the chicken myc gene or c-myc gene. This suggested that the hybrid gene of the retrovirus MC29 was generated by inaccurate recombination of the retroviral sequences. The human MYC gene was later cloned from human DNA, and shown to play a central role in coding DNA binding proteins which help regulate cell growth and differentiation. Subsequent studies have identified the role of this gene in tumorigenesis in cancers such as Burkitt’s lymphoma, retinoblastoma, and recently lung cancer.

Structure The human MYC gene consists of three exons and two introns, measures approximately 5 kb and is located on chromosome 8q24. It contains 439 amino acids which are divided into three functional regions (Figure 1). Region I (amino acids 1–206) contains the transcriptional activation domain that influences gene expression. Region II (amino acids 206–321) contains a nonsequence-specific DNA-binding domain. Region III (amino acids 321–439) incorporates a basic region/helix–loop–helix/leucine zipper construct (amino acids 350–439) and is found in proteins involved in gene expression during cell growth and differentiation.

Function The MYC family oncogenes share many structural and functional similarities including a helix–loop– helix–leucine zipper in the carboxyl terminus of the protein known as MYC associated factor X or Max, as well as homology at regions I and II in the N-terminal transactivation domain. The C-terminal region has been identified to compete with other nuclear factors to form heterodimers with Max in order to activate and repress transcription. The N-terminal transactivation domain has been reported to bind a number of proteins such as the pRb-related family member, a-tubulin, and microtubules. Each of these

ONCOGENES AND PROTO-ONCOGENES / MYC 247 Transactivation

I

TAD

DNA binding

II

1

A 143

NLS

Dimerization

B

HLH

ZIP 439

(a)

B 1

HLH

ZIP 151

(b) Figure 1 Structural and functional domain of (a) c-MYC protein and (b) MAX protein. The c-MYC proteins contain a transcriptional activation domain TAD (amino acids 1–143), a basic region B (amino acids 355–368), a helix–loop–helix motif HLH (amino acids 368– 410), and a leucine zipper domain ZIP (411–439). The two sections within TAD represent the MYC homology Box I and II (amino acids 45–63 and 129–141 respectively), which are two conserved regions ubiquitous to all MYC oncogenes. A represents the highly acidic region, and NLS represents the nuclear localization signal. Max dimerizes with MYC through the HLH–ZIP domain.

interactions has been proposed to influence MYC regulation of cell-cycle progression. The cell-cycle progression through the G1/S boundary is regulated by the retinoblastoma (pRb) protein pathway, which includes cyclin-dependent kinases (CDK2, CDK4 and CDK6), D- and E-cyclins, CDK inhibitors (CDKIs, p15Ink4b, p16Ink4a, p21Waf1, and p27Kip1), and Rb-bound partners, including the E2F transcription factor family. This pathway appears to modify cell-cycle progression to prevent neoplastic growth. In all eukaryotic cells, cyclin-mediated Rb hyperphosphorylation releases E2F-1 which then acts to transactivate genes required for DNA synthesis. This pathway is deregulated in all lung cancer cell lines and lung tumor tissues. Other prerequisite genetic changes for tumor development may evolve from the deregulation of MYC family members. The c-MYC oncogene has been proposed to both stimulate and inhibit specific components of cell-cycle machinery and has been implicated in two distinct genetic pathways controlling progression through the G1 phase of the cell cycle (Figure 2). In one pathway, overexpression of MYC induces the activity of the E2F/DP family of transcription factors. E2F/DP are inactive when bound to the Rb family of proteins; however, following cdk/cyclinmediated pRb phosphorylation, E2F/DP is released from pRb in its transcriptionally active form. Thus, E2F-dependent transcription may be another indirect consequence of MYC regulated cdk activity. In other studies, MYC was also observed to regulate the promoter activity of E2F-2 gene and overexpression of

MYC resulted in overexpression of E2F proteins, demonstrating that induction of E2F activity might be a direct pathway of MYC. Most recent data, however, show that ectopic expression of MYC, like cyclin E, can rapidly induce S-phase entry and DNA replication in cells deprived of E2F function, suggesting that the pRb/E2F pathway does not represent the only physiological pathway for cell cycle progression. Therefore, the MYC and CDK/cyclin pathway may be capable to promote the G1/S transition in parallel with the pRb/E2F pathway. In another pathway, MYC expression rapidly induces activation of cyclin E-cdk2 kinase activity with the simultaneous release of p27Kip1 from cdk2 complexes and these events appear to be required for MYC-driven cell proliferation. The released p27Kip1 is then sequestered by cyclin D–cdk4 which leads to the formation of an active cyclin D–cdk4–p27Kip1 complex. Direct transactivation of cyclin D and cdk4 genes by MYC may also serve to promote sequestration of p27Kip1 and formation of both active cyclin E and D complexes. MYC has also been reported to suppress expression of the p15Ink4b gene, which functions to inactivate cyclin D–cdk4 complex, leading to further sequestration of p27Kip1 by these complexes. The p27Kip1 gene is consequently degraded with the help of the Cul-1 gene which might function as a downstream target for MYC. Therefore, expression of MYC modulates the physiological levels of p27Kip1 and controls the G1 progression by suppressing the growth inhibitory function of p27Kip1 and thus, activating cyclin E and D complexes.

248 ONCOGENES AND PROTO-ONCOGENES / MYC

p16

M

p15

p21

p27

pRB/E2F

G1 Cyclin D Cdk 4 or 6 Cyclin E Cdk 2

G2

S

Figure 2 Cell cycle showing the interactions of cyclins, cyclin-dependent kinases (cdk) and cdk inhibitors, cdkl (p16, p15, p21, and p27) along with MYC activated and repressed pathways affecting G1 and S progression in cells. MYC amplification/overexpression leads to loss of cdkI’s leading to inactivation of pRb through phosphorylation, release of E2F and progression from G1 to S phase. MYC also induces activity of E2F causing activation of E2F transcription of DNA synthetic proteins. Red lines represent pathways affected by MYC.

The p16/cyclin D1/cdk4/pRb/E2F pathway is targeted for mutational and epigenetic inactivation essentially in all lung cancers. Overexpression of MYC may further augment the cell-cycle progression in these lung tumors by MYC-mediated transcriptional activation of E2F family members. pRb (–) cells selectively deprived of either E2F or MYC are unable to proliferate, suggesting that deregulated activities of both the Rb and MYC pathways are required for progression and thus proliferation of tumor cells. These observations imply that in lung cancer both the pRb/E2F and MYC regulated pathways need to converge on cyclin E, a key promoting enzyme necessary for the entry into the S phase. Cyclin E is overexpressed in 41% of NSCLC and the low amount of its inhibitor p27Kip1 in lung cancer correlates with poor prognosis. Although these pathways appear to operate autonomously, significant cross-talk exists between Rb-E2F and MYC. The concept emerging from these recent discoveries is that overexpression of MYC in lung cancer may be a driving factor for the direct stimulation or

repression of several distinct components of the cell cycle. The absence of one of the tumor suppressor genes in lung cancer, such as pRb or p16Ink4a, combined with overexpression of MYC which activates E2F and cyclin E-cdk2, a key G1/S promoting complex essential for S-phase entry, results in loss of cell-cycle arrest and uncontrolled tumor growth. Although the components of the pRb pathways, that is, cyclins, cdk and cdkI, are the logical molecular targets for the development of specific inhibitors of G1/ S transition, recent data point to MYC oncogenes as another potential important molecular target for the development of novel drugs that can specifically regulate MYC-related cell-cycle activity in lung cancer.

Role of MYC Oncogene in Lung Cancer In lung cancer, amplification of c-MYC, N-MYC, or L-MYC genes is seen in up to 33% of cell lines and 15% of all lung tumors and usually observed in patients previously treated with chemotherapy, suggesting that this is a late event in the development of

ONCOGENES AND PROTO-ONCOGENES / MYC 249

the cancer. Increased MYC expression is seen in over 80% of cell lines and 45% of tumors, often in the absence of gene amplification. In the majority of such cases, the mechanism leading to overexpression remains unknown. One possible pathway might be through mRNA stabilization. The c-MYC, N-MYC, and L-MYC oncogenes have all shown amplification and/or overexpression in SCLC tumor lines. However, the c-MYC oncogene was the first seen to have altered expression in SCLC cell lines and has been the most frequently observed. Cell lines of SCLC with a c-MYC amplification are pared to those without the c-MYC amplification. The c-MYC oncogene is the only member of the MYC family whose amplification/overexpression can be correlated with a patient’s prognosis. One study demonstrated a c-MYC amplification/overexpression in up to 23% of selected patients with SCLC. Other studies later showed direct correlations with survival time, altered cell morphology, tumor doubling time, and the number of double minute chromosomes. Although amplification/overexpression of L-MYC and N-MYC also occurs in SCLC, no correlation with clinical outcomes are yet been defined.

Role of MYC Oncogene in Current and Future Lung Cancer Therapies Through the functions of gene amplification and overexpression, it has been shown that the MYC oncogene can play a role in cell apoptosis as well as metastasis and drug resistance. Currently, the role in cell apoptosis is still unclear, along with their role in immortalizing tumor cells through their atypical expression and amplification during tumorigenesis. Despite this, recent advances in the understanding of gene regulation and overexpression have opened the door for development of new anticancer agents targeted towards specific molecular targets. Several agents are currently being investigated to determine their potential efficacy as anticancer agents by targeting functions of the MYC oncogene. One of these therapies involves the downregulation of gene overexpression through antisense technology. This is possible through the development of synthetic analogs known as antisense oligodeoxyribonucleotides (ASODNs). These agents bind to the RNA transcript of target genes in a sequence-specific manner, which leads to an interruption of translation of messenger RNA into protein, thereby blocking gene expression. Another agent which has been investigated is LC-1, a monoclonal antibody that recognizes lungcancer-associated common antigens, with activity against a lung adenocarcinoma cell line SPC-A-1.

After transfection, LC-1 expression was observed and RT-PCR analysis showed that c-MYC expression was downregulated in these SPC-A-1 cells. It is felt that the antigens recognized by LC-1 are involved in a growth-stimulating pathway, and the antibody blocking of the antigen function shuts down the pathway and thus downregulates the expression of c-MYC and growth of the cells. Other studies of gene therapy in the treatment of lung cancer involving MYC overexpression have shown selective sensitivity to ganciclovir (GCV) by transducing the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the MYC– Max response elements (a core nucleotide sequence, CACGTG). This construct (MYCTK) may represent a novel treatment against chemo-radio-resistant SCLC.

Conclusions Over recent years, the role the oncogenes have played in the development and proliferation of tumor cells has become a greater focus of study, which has led to better understanding to tumor characteristics as well as the development of novel targeted therapies in cancer treatment. The MYC family of oncogenes, which includes, c-MYC, N-MYC, and L-MYC, are important for their role in the transcription which leads to cell proliferation, apoptosis, and the development of tumors. In lung cancer, the amplification and overexpression of MYC has become an identified pathway of tumorgenesis and cell proliferation, and now offers a potential molecular target for novel target therapies in the treatment of lung cancer. See also: Gene Regulation. Genetics: Overview. Oncogenes and Proto-Oncogenes: Overview. Tumors, Malignant: Overview; Lymphoma; Rare Tumors.

Further Reading Dang CV (1999) c-Myc target genes involved in cell growth, apoptosis, and metabolism. Molecular and Cellular Biology 19(1): 1–11. Dang CV and Lee LA (1996) C-Myc function in neoplasia. In: Molecular Biology Intelligence, pp. 1–196. New York: Chapman and Hall. Drayton S, Brookes S, Rowe J, and Peters G (2004) The significance of p16INK4a in cell defenses against transformation. Cell Cycle 3(5): 611–615. Gogali A, Charalabopoulos K, and Constantopoulos S (2004) Integrin receptors in primary lung cancer. Experimental Oncology 26(2): 106–110. Kijima T, Maulik G, and Salgia R (2003) Molecular alterations in lung cancer. Impact on prognosis. Methods in Molecular Medicine 75: 29–38.

250 ONCOGENES AND PROTO-ONCOGENES / RAS Murray N, Salgia R, and Fossella FV (2004) Targeted molecules in small cell lung cancer. Seminars in Oncology 31(1 supplement 1): 106–111. Pisick E, Jagadeesh S, and Salgia R (2003) Small cell lung cancer: from molecular biology to novel therapeutics. Journal of Experimental Therapeutics & Oncology 3(6): 305–318. Prochownik EV (2004) c-Myc as a therapeutic target in cancer. Expert Review of Anticancer Therapy 4(2): 289–302. Rosenwald IB (2004) The role of translation in neoplastic transformation from a pathologist’s point of view. Oncogene. 23(18): 3230–3247. Salgia R and Skarin AT (1998) Molecular abnormalities in lung cancer. Journal of Clinical Oncology 16: 1207. Sattler M and Salgia R (2003) Molecular and cellular biology of small cell lung cancer. Seminars in Oncology 30(1): 57–71. Wanzel M, Herold S, and Eilers M (2003) Transcriptional repression by Myc. Trends in Cell Biology 13(3): 146–150.

RAS K Junker, University Hospital Bergmannsheil, Bochum, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract The three human ras genes, H-ras (the oncogene of Harvey % murine sarcoma virus Ha-MuSV), K-ras (the oncogene of Kirs% ten murine sarcoma virus, Ki-MuSV), and N-ras (detected in neuroblastoma cell lines) code for similar membrane-bound proteins (p21ras) that bind and hydrolyze GTP. After posttranslational modification and anchoring to the cytoplasmic membrane, RAS proteins can display their functional activity in signal transduction. Several genetic changes in oncogenes and tumor suppressor genes have been identified in the multistep carcinogenesis of lung cancer, including ras mutations, which were detected in non-small cell carcinomas (NSCLC), rarely in carcinoids, but not in small cell lung cancer (SCLC). Single point mutations of ras genes suffice to induce oncogenic transformation, resulting in a continuously activated RAS protein, with increased cell proliferation, increased angiogenesis, and reduced apoptosis. ras gene mutations have been identified in codons 12, 13, 59, and 61, those in codons 12 and 61 occurring most frequently. In NSCLC, the prominent mutation is detected in position 1 of codon 12 of the K-ras gene. RAS-targeted therapy approaches have been developed, intended to control or reduce tumor proliferation. Several agents, aiming at different targets, are in clinical trials, the farnesyl transferase inhibitors being in the most advanced stage of development.

Introduction The name ‘ras’ is an acronym derived from ‘rat sar% coma’. About 50 currently known ras-related genes form the ras superfamily. There are three functional human ras genes: H-ras (the oncogene of Harvey murine sarcoma virus Ha-MuSV), K-ras (the% oncogene of Kirsten murine sarcoma virus, Ki-MuSV), %

and N-ras (detected in neuroblastoma cell lines). % chromosomes, but code They are located on different for similar membrane-bound proteins (p21ras) that bind and hydrolyze GTP. Mutations of these genes may be caused by chemical or physical carcinogens and are identified in many different tumors, with the highest frequencies found in pancreatic, colorectal, and lung cancer.

Structure Genes

The ras genes are evolutionarily highly conserved. Among different species, they are characterized by high homology. Whereas N-ras has been assigned to the short arm of human chromosome 1 (1 p22–p32), H-ras and Kras have been assigned to the short arms of chromosome 11 (11 p15.1–p15.5) and 12 (12 p12.1–pter), respectively. The ras gene region comprises 189 amino acids. The p21ras coding sequences of each of these genes are equally distributed among four exons, except for the K-ras gene that includes two alternative fourth coding exons (4A and 4B), differing only in the C-terminal 25 amino acids of the corresponding proteins. K-ras contains larger introns (exons and introns: 3.2 kb) as compared with H-ras (1.2 kb) and N-ras (2.0 kb). Functional differences of the four isoforms (H-RAS, N-RAS, two K-RAS isoforms) are not known. Proteins

The corresponding RAS proteins (p21ras) are ubiquitously located in different tissues. They reveal a molecular mass of 21 kDa and consist of 189 amino acids, except for one K-RAS isoform (K-RAS B) with 188 amino acids. The first 85 amino acids comprise a highly conserved region. In the next region of 80 amino acids, the structure of different RAS molecules diverges slightly, whereas the rest of the proteins, except for the last four amino acids, are formed by a highly variable region (amino acids 165–185). p21ras comprises a central, six-stranded b-sheet and five helices with 10 loops (loop ¼ L). L2 includes the so-called effector domain (amino acids 32–40) that may be involved in the interaction of RAS proteins with cellular targets and is also essential for the stimulation of GTPase by GTPase-activating protein (GAP). L1 contains glycine 12 (Gly12), the most frequent site of mutations in human tumors, and L4 glutamine 61 (Glu61), another important site of mutation.

ONCOGENES AND PROTO-ONCOGENES / RAS

Regulation of Production and Activity Synthesis and Posttranslational Modification

RAS proteins play an essential part in cellular growth signal transduction. They are synthesized in the cytoplasm on free ribosomes as hydrophilic precursor proteins (pro-p21). To reach a functionally active state and to be converted into lipophilic molecules that anchor to the cell membrane, RAS proteins need posttranslational modification. This modification comprises four steps: prenylation, proteolysis, carboxymethylation, and palmitoylation. Prenylation is the covalant binding of farnesyl or geranyl groups to the terminal cysteine residue at the carboxyl end of a cellular protein. Farnesylation, which is the rate-limiting step of posttranslational modification, is catalyzed by the enzyme farnesyl protein transferase (FPTase). Alternatively, a geranylgeranyl group can be transferred to some RAS proteins by protein geranylgeranyl transferase (GGTase), particularly when FTPase is therapeutically inhibited by farnesyl transferase inhibitors (FTIs). Functional Activity

After posttranslational modification and anchoring to the cytoplasmic membrane, RAS proteins can display their functional activity in signal transduction. They cycle between an inactive GDP-bound and an active GTP-bound form. The intrinsic GTPase activity of wild-type RAS enables the protein to hydrolyze GTP, thereby returning to an inactive state. Mutated RAS protein, however, has lost its GTPase activity. As a consequence of this, mutated ras remains in its active state for a longer period of time, with continuous activation of the corresponding signal transduction pathways. Under physiological conditions, RAS inactivation is tightly regulated by proteins, collectively termed GTPase activating proteins (RAS GAPs or GAPs), which stimulate the hydrolysis of GTP. On the other hand, guanine nucleotide exchange effectors (GNEFs) promote the formation of active GTPbound RAS protein.

Biological Function Receptors and Pathways Upstream of RAS

Several classes of receptors are involved in RAS signal transduction, such as the integrins and the receptor tyrosine kinases (RTKs) as well as the serpentine receptors and other transmembrane receptors. RTKs consist of an extracellular ligandbinding domain, a transmembrane-spanning region, and a cytoplasmic tyrosine kinase domain. They

251

include growth factor receptors like the epidermal growth factor receptor (EGFR). EGFR can be externally stimulated through binding of the corresponding ligand’s epidermal growth factor (EGF), transforming growth factor alpha (TGF-a), or amphiregulin (AR), which in turn causes intracellular dimerization and autophosphorylation of the receptor molecule. This is followed by binding to adaptor proteins that link activated EGFR to other proteins such as son of sevenless (SOS, belonging to the GNEFs). Binding of SOS to RAS leads to conformational changes and its activation by binding to GTP. Pathways Downstream of RAS

Several cytoplasmic pathways downstream of RAS have been identified. The best-characterized signal transduction cascade is the RAS/RAF pathway, starting with the activation of RAS. In a multistep process, activated RAS recruits and activates the cytoplasmic serine/threonine kinase RAF-1 (RAS-activated factor 1). Activated RAF-1 phosphorylates mitogen-activated protein kinase kinase (MAPKK, also termed MEK), which in turn phorphorylates MAPK, also known as extracellular signal-regulated kinase (ERK1/ERK2). In the nucleus, MAPK activates transcription factors, including ELK-1, FOS, JUN, and MYC. Finally, these signals induce the expression of D-type cyclins, which form complexes with cyclin-dependent kinases (CDKs) and, thus, lead to cell-cycle progression (Figure 1). However, the RAS/RAF/MAPK cascade is not the only way to cell-cycle progression and cell proliferation. Each component of this pathway can be induced by different upstream proteins and may by itself activate other targets. There are, for example, interrelations with CDK inhibitor p21WAF1/ZIP1 and with P53.

RAS in Respiratory Diseases ras Mutations in Lung Cancer

ras genes are expressed in most human cells, but more strongly in some than in others. Single point mutations of these genes suffice to induce oncogenic transformation, resulting in a continuously activated RAS protein, with increased cell proliferation, increased angiogenesis, and reduced apoptosis. Several genetic changes in oncogenes and tumor suppressor genes have been identified in the multistep carcinogenesis of lung cancer, including ras mutations, which were detected in NSCLC, rarely in carcinoids, but not in SCLC. In squamous cell carcinomas, ras mutations are seen as a late event

252 ONCOGENES AND PROTO-ONCOGENES / RAS EGFR, HER2/neu

Inactive RAS - GDP

GTPaseactivating proteins (GAPs)

Guanine nucleotide exchange factors (GNEFs)

Active RAS - GTP

Signal transduction RAF-1

(RAS-activated factor 1)

MAPKK

(Mitogen-activated protein kinase kinase; MEK)

MAPK

(Mitogen-activated protein kinase; ERKs)

ELK-1 FOS JUN Transcriptional activation

MYC Cyclin D1

Proliferation Apoptosis Angiogenesis

Figure 1 Schematic representation of RAS-mediated signal transduction cascade and following transcriptional activation.

in carcinogenesis, whereas they represent an earlier genetic change in the carcinogenesis of pulmonary adenocarcinoma. ras gene mutations have been identified in codons 12, 13, 59, and 61, with those in codons 12 and 61 occurring most frequently. In NSCLC, the prominent mutation is detected in position 1 of codon 12 of the K-ras gene (wild-type nucleotide sequence GGT), where guanine (G) is predominantly replaced by thymine (T) (G-T transversion), representing a ‘hot spot’ of ras point mutation. In lung cancer, H-ras or N-ras mutations are identified only in exceptional cases. In general, pulmonary adenocarcinomas reveal a higher frequency of ras mutations than squamous cell carcinomas of the lung, with a wide range (15–60%). ras mutations are also described in a large proportion of large cell carcinomas. Several studies have shown a strong association between the occurrence of K-ras mutations in pulmonary adenocarcinomas and smoking history; occurrence has been shown to be four times higher in smokers or ex-smokers compared with those who have never smoked. The most frequent single base substitutions in NSCLC G to T and G to A can be caused by benzo(a)pyren and N-nitroso compounds, both well-known components of tobacco smoke. These carcinogens might therefore be responsible for the development of ras gene mutations in human lung cancer.

In lung adenocarcinomas or NSCLC in general, K-ras mutations have been demonstrated to be associated with shorter survival. Even after complete tumor resection, the presence of K-ras mutations was a significant predictor for poor progressionfree survival. A clear association between the occurrence of K-ras mutations and the presence of metastases or the stage of the disease could not be established. ras gene mutations have also been identified in sputum or bronchoalveolar lavage fluid from patients with NSCLC, possibly serving as additional information in the diagnosis of lung cancer. But, up to now, corresponding analyses have not been carried out in daily routine. Obviously, overexpression of a normal RAS protein is also associated with tumorigenic transformation. But this appears to be a rare event compared with transformation as a result of ras mutation. RAS overexpression, as can be demonstrated by immunohistochemistry, has to be seen as an adaptation process and does not show a clear correlation with the occurrence of ras mutations. There is a higher incidence of lung cancer in patients with pulmonary fibrosis. In these patients, expression of RAS protein in type II pneumocytes and mutations in codon 12 of the K-ras gene may contribute to the induction of lung cancer and might be seen as an early event in lung carcinogenesis.

ONCOGENES AND PROTO-ONCOGENES / RAS Table 1 Potential RAS-targeted therapy approaches *

*

* * *

Inhibition of RAS protein expression (e.g., antisense oligonucleotides) Inhibition of RAS anchoring to the cell membrane (e.g., farnesyl transferase inhibitors, FTIs) Inhibition of RAS trafficking (inhibition of heat shock proteins) Restoration of GTPase activity of mutated RAS Inhibition of downstream effectors of RAS (e.g., inhibition of RAF or MAPKK)

Inhibitors of RAS Signaling Against the background of an improved understanding of the complex molecular interactions of RAS, RAS-targeted therapy approaches (Table 1) have been developed that are intended to control or reduce tumor proliferation. Several agents, aimed at different targets, are in clinical trials, the FTIs being at the most advanced stage of development. Farnesyl Transferase Inhibitors

FTIs were developed to inhibit posttranslational modification of RAS proteins, thus reducing cell proliferation. Most FTIs made to date inhibit FTase activity with high selectivity in vitro and in cultured cells. In animal models, several studies have demonstrated a combination therapy of FTIs with other cytotoxic agents or radiation to be more effective than monotherapy with FTIs. FTIs also inhibited the growth of human SCLC and NSCLC cell lines in vitro and in xenograft models. To determine whether the corresponding FTIs show any activity against lung cancer, three phase II trials have been performed in the US: R115777 and L-778,128 in patients with previously untreated NSCLC, and R115777 in patients with sensitive recurrent SCLC. But in all three studies, no objective responses were observed. In a phase I/II trial, 15% partial responses have been observed with a combination of another FTI, SCH66336 (lonafarnib), with paclitaxel for patients with texane refractory/resistant NSCLC. Up to now there have been no final reports on randomized phase III trials with combination therapy including FTIs. Most of the earlier studies on FTIs were based on the assumption that tumors with K-ras mutation would show special sensitivity to FTI therapy. However, current data indicate that tumors with K-ras mutations can be even more resistant to FTIs, whereas the sensitivity to these substances seems to be independent of ras mutational status. It is now thought that FTIs have the potential to influence several signaling pathways as well as the farnesylation of other proteins. The real target of FTIs – whether wild-type ras or some other farnesylated

253

proteins – and their effect on combination therapy with standard cytotoxic agents still remains to be defined in ongoing studies. A closer look at the signal transduction cascade leading from transmembrane tyrosine kinase receptors like EGFR via RAS activation to the induction of transcription factors such as ELK-1, FOS, JUN, or MYC may give rise to the assumption that ras mutation could be a negative predictor for the effectiveness of EGFR inhibition. However, up to now, no correlation has been demonstrated in vitro between the presence of activating K-ras mutations and relative resistance to an EGFR tyrosine kinase inhibitor. See also: Angiogenesis, Angiogenic Growth Factors and Development Factors. Apoptosis. Cell Cycle and Cell-Cycle Checkpoints. DNA: Structure and Function. Epidermal Growth Factors. Gene Regulation. Genetics: Overview. G-Protein-Coupled Receptors. Oncogenes and Proto-Oncogenes: Overview; jun Oncogenes; MYC. Signal Transduction. Transcription Factors: Overview. Tumors, Malignant: Overview; Bronchogenic Carcinoma; Chemotherapeutic Agents. Vascular Endothelial Growth Factor.

Further Reading Barbacid M (1987) ras genes. Annual Review of Biochemistry 56: 779–827. Broermann P, Junker K, Brandt B, et al. (2002) Trimodality treatment in stage III non-small cell lung cancer: prognostic impact of K-ras mutations after neoadjuvant therapy. Cancer 94: 2055–2062. Caponigro F, Casale M, and Bryce J (2003) Farnesyl transferase inhibitors in clinical development. Expert Opinion in Emergency Drugs 12(6): 943–954. Crul M, de Klerk GJ, Beijnen JH, and Schellens JH (2001) Ras biochemistry and farnesyl transferase inhibitors: a literature survey. Anticancer Drugs 12(3): 163–184. Ghobrial IM and Adjei AA (2002) Inhibitors of the ras oncogene as therapeutic targets. Hematology and Oncology Clinics of North America 16(5): 1065–1088. Hesketh R (1994) The Oncogene Handbook. London: Academic Press. Husgafvel-Pursiainen K, Ridanpaa M, Anttila S, and Vainio H (1995) p53 and ras gene mutations in lung cancer: implications for smoking and occupational exposures. Journal of Occupational and Environmental Medicine 37(1): 69–96. Johnson BE and Heymach JV (2004) Farnesyl transferase inhibitors for patients with lung cancer. Clinical Cancer Research 10: 4254–5457. Malumbres M and Barbacid M (2003) RAS oncogenes: the first 30 years. Nature Reviews: Cancer 3(6): 459–465. Peters G and Vousden KH (1997) Oncogenes and Tumour Suppressors. Oxford: Oxford University Press. Sebti SM and Adjei AA (2004) Farnesyl transferase inhibitors. Seminars in Oncology 31(supplement 1): 28–39. Suzuki T, Nakagawa T, Endo H, et al. (2003) The sensitivity of lung cancer cell lines to the EGFR-selective tyrosine kinase inhibitor ZD1839 (‘Iressa’) is not related to the expression of EGFR or HER-2 or to K-ras gene status. Lung Cancer 42(1): 35–41. Vachtenheim J (1997) Occurrence of ras mutations in human lung cancer. Minireview. Neoplasma 44(3): 145–149.

254 ONCOSTATIN M

ONCOSTATIN M D Knight, St Paul’s Hospital, Vancouver, BC, Canada & 2006 Elsevier Ltd. All rights reserved.

Abstract Oncostatin M (OSM) is a multifunctional cytokine secreted as a 28 kDa glycoprotein primarily from activated T lymphocytes and macrophages. OSM belongs to the interleukin-6 family that also includes interleukin-11, leukemia inhibitory factor, ciliary neurotrophic factor, cardiotrophin-1, ‘novel neurotrophin-1/B cell-stimulating factor-30 /cardiotrophin-like cytokine’, IL-27, and IL-31. These cytokines are grouped together on the basis of weak sequence homology and shared use of the signal transducing subunit gp130 as part of their receptor complexes. Accordingly, oncostatin M shares properties with all members of this cytokine family, but is most closely related in structure and function to leukemia inhibitory factor. Oncostatin M acts on a variety of cells and displays functions involved in regulating gene activation, cell growth, and inflammatory processes. Despite these functions, investigations into its role in respiratory disease have been limited. Those studies that have been performed suggest that oncostatin M is a potent inducer of antiproteases, is anti-inflammatory, and promotes the deposition of extracellular matrix proteins. Thus, it is likely that in the lung, oncostatin M may play a prominent role in normal wound healing as well as remodeling and pathological fibrosis.

Introduction Oncostatin M (OSM) is a multifunctional polypeptide cytokine. It was isolated in 1986 from lymphoma cells and is characterized by its ability to inhibit the proliferation of melanoma cells, but not normal cells. OSM is a member of the IL-6/gp130 family of cytokines, which also includes interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), ‘novel neurotrophin-1/B cell-stimulating factor-30 (NNT-1/BSF-3), also known as cardiotrophin-like cytokine (CLC), as well as IL-27 and, more recently,

STAT

AP-1

IL-31. OSM shares structural and functional similarities with all members of this family of proteins, but is most closely related in structure and function to LIF. OSM acts on a wide variety of cells and as a consequence elicits a wide array of biological responses, leading to speculation that it may play a role in treating inflammatory or degenerative diseases, stimulating erythropoiesis, regulating tissue remodeling, and promoting healing after injury.

Structure The human OSM gene is located on chromosome 22q12, immediately adjacent to the gene for LIF. The coding region of the OSM gene is covered by 3 exons of 86, 143, and 1633 bp, respectively (Figure 1). The minimal promoter region necessary for full activity of the OSM gene resides within 304 bp upstream of the transcription initiation site. The translated protein is secreted as a variably glycosylated monomer of 28 kDa. Sequence analysis of its complementary DNA (cDNA) shows that the cytokine is initially synthesized as a 252 amino acid precursor polypeptide. The mature protein, containing 196 amino acids, results from the removal of a 25 N-terminal amino acid leader sequence and a 31 C-terminal peptide. The mature protein contains five cysteine residues, which form two intermolecular disulfide bridges (C6-C127 and C49-C167) to give the characteristic four-helix bundle configuration of these cytokines.

Regulation of Production and Activity The actions of OSM suggest an involvement in limiting tissue damage subsequent to inflammatory damage, regulating tissue repair and coordinating the return to tissue homeostasis. Thus, OSM may be

G/C rich STAT

C-EBP 86

143

1633

Exon I

Exon II

Exon III

304 bp promoter Figure 1 The gene structure of OSM. The coding region of the OSM gene consists of 3 exons of 86, 143, and a third much larger exon of 1633 bp. The minimal promoter region necessary for full activity of the OSM gene is contained within 304 bp upstream of the transcription initiation site. The 50 proximal region of the promoter lacks a TATA box, but does contain a consensus CCAAT sequence. This region also contains a C/EBP consensus sequence at around
45 bp and a G/C rich region at
60, each of which can drive basal promoter activity.

ONCOSTATIN M 255

classified as an anti-inflammatory cytokine. The expression of OSM by inflammatory cells such as T lymphocytes, monocyte/macrophages, neutrophils, and dendritic cells supports the notion that OSM would be present at sites of inflammation. Little is known about the degradation and removal of OSM, although the cytokine has been shown to be bound and sequestered by collagen types I, III, IV, and VI in vitro. Other proteins present in the extracellular matrix, such as fibronectin and laminin, did not bind OSM, suggesting that the effect is specific for collagen. Similarly, other members of the IL-6/gp130 family did not bind to collagens or interfere with OSM binding to collagens, suggesting the effect is specific for OSM. It is likely that binding of OSM to interstitial collagens may be a mechanism to regulate the local bioavailability and activity of this cytokine in an analogous manner to that shown for other growth factors such as transforming growth factor (TGF)-b.

Biological Functions OSM exhibits a multitude of biological activities, depending on cell type (Figure 2). It inhibits the

growth of several tumor cell lines including melanoma and lung carcinomas. OSM can also inhibit the proliferation of murine M1 myeloid leukemic cells and induce their differentiation into macrophage-like cells, a function shared by LIF, granulocytemacrophage colony-stimulating factor (GM-CSF), and IL-6. Conversely, OSM is a potent growth factor for HIV-related Kaposi sarcomas, promoting a change in morphology and promoting the growth of these cells in soft agar. In patients with multiple myeloma, OSM is frequently found in the serum and has been associated with serum IL-6 and progressive disease. Oncostatin M also influences the growth of cultured endothelial cells and regulates the production of plasminogen activator by these cells. It has been shown to induce the expression of granulocyte colony-stimulating factor (G-CSF) and GMCSF by human endothelial cells and may thus play a role in the regulation of myelopoiesis. In human endothelial cells, OSM induces the synthesis of IL-6, and this activity is augmented with TGF-b but not with interferon gamma (IFN-g). In hepatocytes, OSM induces the synthesis of acute phase proteins

T lymphocyte Macrophage

Neutrophil

OSM OSMR

Local effects of OSM Myofibroblast Alveolus

Epithelium Basal cell

Basement membrane

Fibroblasts • Survival and proliferation • Differentiation • Collagen production • Glycosaminoglycans production • IL-6, MCP-1, TIMP • MMP′s Endothelium Epithelium • IL-6, endothelin-1 • NO • Adhesion molecules • PGE2

Fibroblast

Endothelium Figure 2 Diagrammatic representation of the cells that produce OSM and of the systemic and local effects of OSM in the body. IL6, interleukin-6; MCP-1, monocyte chemoattractant protein 1; TIMP, tissue inhibitor of metalloproteinase; MMPs, matrix metalloproteinase; PGE2, prostaglandin E2.

256 ONCOSTATIN M

and enhances the uptake of low-density lipoprotein (LDL) and induces the expression of LDL receptors. OSM is a potent stimulus for the synthesis and release of IL-6 and the CC chemokine monocyte chemotactic protein 1 (MCP-1). A growing number of reports have demonstrated that OSM displays profibrotic activities in various tissue/disease models. For instance, OSM has been found to stimulate the growth of mouse synovial fibroblasts and the growth of human dermal fibroblasts in vitro. OSM also promotes the production of collagen and glycosaminoglycans in dermal fibroblasts. In addition, transgenic mice engineered to overexpress OSM in the pancreas display extensive fibrosis, characterized by excessive collagen deposition. In contrast to its anabolic properties in these cells, OSM seems to have a catabolic effect in chondrocytes causing increased collagen degradation, a characteristic feature of bone degenerative diseases such as arthritis. OSM has also been identified as an important cytokine in inflammatory diseases, such as inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis and is a key component in regulating cellular invasiveness. There have been conflicting reports suggesting that OSM is either a proinflammatory or anti-inflammatory mediator. For instance, OSM is a potent inhibitor of tumor necrosis factor (TNF)-a release following lipopolysaccharide (LPS) treatment in vivo. Although OSM does not inhibit IL-1 directly, it has been found to inhibit IL-1-induced expression of IL-8 and GM-CSF by synovial and lung fibroblasts in vitro. OSM also acts synergistically with IL-1 to induce Cox-2 expression and prostaglandin E2 (PGE2) release in human airway smooth muscle cells. Furthermore, OSM may be an important cofactor in the release of PGE2 and nitric oxide (NO) from lung epithelial cells and may play a role in defense against exogenous proteases such as those derived from house-dust mites. When expressed in bacteria, OSM is proinflammatory, inducing the expression of adhesion molecules, such as P-selectin and E-selectin, on human umbilical vein endothelial cells in vitro and inducing inflammatory infiltrate in vivo. In contrast, others have studied the effects of OSM on endothelial cells using highly purified mammalian protein or yeast-derived protein. These in vitro and in vivo studies did not support the earlier findings. To date, no studies have reported the effect of OSM on the expression of adhesion molecules in human lung tissue.

Table 1 Phenotype of OSM and OSMR mutant mice OSM

TG (targeted)

OSM

KOa

OSMR OSMR

TGb KO

a b

Lung fibrosis, pancreatic fibrosis, abnormal spermatogenesis, extrathymic T-cell development Develop normally, significantly reduced nociceptive responses to acute thermal, mechanical, chemical, and visceral pain. Not examined Develop normally, regulated hematopoiesis in the spleen and bone marrow, enhanced liver injury and degradation of hepatic ECM, reduced responses to IL-6

Knockout mouse. Transgenic mouse.

Genetically Engineered Mice To date, murine models of transgenically overexpressed OSM have not been developed. Targeted expression of OSM fusion genes containing tissuespecific promoters causes abnormalities in bone growth and spermatogenesis, induce fibrosis in the lung as well as the pancreas, and disrupt normal lymphoid tissue development (Table 1). Mice deficient in OSM appear to develop normally. Limited studies using these mice suggest that OSM plays an important role in hematopoiesis, nociception, and wound repair.

OSM Receptors Two types of functional OSMR receptor complexes (OSMR) are present in humans. The type I receptor, which is identical to the receptor for LIF, consists of the LIFRb chain and gp130. A second receptor, specific for OSM consisting of gp130 and the OSMRb chain has been identified and termed the type II receptor. Many overlapping effects of OSM and LIF are likely to be mediated by the shared type I receptor, whereas specific responses manifest by OSM are mediated by the type II receptor. The human OSMR is unique among the IL-6 family in that OSM binds directly to gp130, rather than its own receptor subunit. The genes encoding OSMbR and gp130 have both been mapped to chromosome 5. Of note, gp130 has been mapped to chromosome 5q11, an area associated with a number of other candidate asthma genes. The gene for OSMR is at 5p12. The promoter region and complete coding sequence for the gp130 gene has been cloned and characterized and sequence

ONCOSTATIN M 257

OSMR


gp130

Jak

P767

Grb2

P

Shc

P861

Sos

P812

P

STAT -3

P

STAT-3 Ser P

P

STAT-3 Ser P

P904 P914

Ras Raf ?-

MEK MAPK

Cell cycle progression

Transcription /differentiation

Figure 3 Signaling pathways activated by binding of OSM to its receptor. Following binding of OSM to gp130, the OSMR is recruited to generate a high-affinity receptor complex. gp130 has no intrinsic tyrosine kinase activities, so the cytoplasmic janus kinases (JAKs) are recruited and activated, which results in activation and phosphorylation of gp130. The subsequent activation of intracellular signaling is dependent on specific phosphotyrosine residues on gp130. Unlike other IL-6 cytokines, OSM does not activate MAPK via recruitment of shp-2 via phosphorylation of Tyr759. However, OSM does activate MAPK pathway via recruitment of adapter protein shc, which binds Tyr861. In contrast, members of the STAT transcription factors dock to several membrane distal phosphotyrosine residues (Tyr767, 812, 904 914 , ). Once phosphorylated, STAT proteins form dimers, translocate to the nucleus, and transactivate target genes that normally lead to differentiation.

information is available. While the coding region of the OSMbR gene has also been cloned, the promoter for this gene has yet to be defined. Binding of OSM to its receptor simultaneously initiates at least two intracellular signaling pathways (Figure 3): the janus kinase (JAK)/signal transducers and activators of transcription (STATs) and the Ras/mitogen-activated protein kinase (MAPK) pathways. Gene-targeting strategies have revealed that signaling by the JAK/STAT and MAPK pathways is tightly regulated, suggesting that balanced signaling from each pathway is critical for the physiological responses. Transgenic mice overexpressing the OSMR have not been developed. Analysis of OSMR-deficient mice shows that while they are viable and fertile, hematopoiesis is compromised. These mice also exhibit impaired wound healing.

OSM in Respiratory Diseases OSM is produced in cell types that are known to increase in number and activity in asthma, including

monocytes and macrophages, dendritic cells, activated T cells, and neutrophils. In contrast to other members of this family, there is no evidence to suggest that OSM is produced in resident cells, such as epithelial cells, fibroblasts, or airway smooth muscle. In the lungs, therefore, it seems that OSM is primarily produced by infiltrating cells in response to injury. Oncostatin M induces eotaxin expression in murine lung fibroblasts and is capable of regulating eosinophilic inflammation in an in vivo model, supporting a role for OSM in pulmonary inflammatory diseases that involve eosinophil recruitment such as eosinophilic pneumonia. OSM is a potent inducer of specific antiproteases, including a1-antitrypsin and a1-proteinase inhibitor from human alveolar epithelial cells. OSM has also been shown to be a potent inducer of the acute phase proteins, serum amyloid A, and a1-glycoprotein. Excess extracellular matrix (ECM) deposition in the airway wall and epithelial basement membrane is at least partly responsible for the characteristic feature of airway remodeling that occurs in asthmatic patients. It is well established that myofibroblasts

258 OXIDANTS AND ANTIOXIDANTS / Antioxidants, Enzymatic

situated immediately beneath the basement membrane are the major source of collagen and other ECM proteins. OSM stimulates the proliferation and induces collagen production by human lung fibroblasts. More recently, adenoviral-mediated overexpression of OSM in the lungs was shown to induce collagen production in the lung and in the peribronchial regions. Interestingly, this fibrotic response was not observed until several days after administration, suggesting that OSM is important in wound repair, rather than the immediate inflammatory response. OSM is also a potent inducer of tissue inhibitor of metalloproteinase (TIMP)-1 expression in human lung fibroblasts in vitro, which would further amplify the accumulation of collagen and other extracellular matrix proteins. Based on these findings, it is likely that different types of inflammation may have differential effects on the expression/activity of OSMR in the airways. The specific inflammatory response following activation of OSM will be influenced by the tissue distribution of OSMbR, coupled with a specific set of OSM inducible genes. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Cell: Vascular; Epithelial. Apoptosis. Asthma: Overview. Chemokines. Chemokines, CXC: IL-8. Dendritic Cells. Eotaxins. Extracellular Matrix: Collagens. Fibroblasts. Gene Regulation. Interleukins: IL-1 and IL-18; IL-6; IL-23 and IL-27. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Neutrophils; Pulmonary Macrophages. Lipid

Mediators: Overview. Myofibroblasts. Proteinase Inhibitors: Alpha-2 Antiplasmin. Stem Cells.

Further Reading Bravo J and Heath JK (2000) Receptor recognition by gp130 cytokines. EMBO Journal 19: 2399–2411. Grant SL and Begley CG (1999) The oncostatin M signalling pathway: reversing the neoplastic phenotype? Molecular Medicine Today 5: 406–412. Heinrich PC, Behrmann I, Haan S, et al. (2003) Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochemical Journal 374: 1–20. Heinrich PC, Behrmann I, Muller-Newen G, Schaper F, and Graeve L (1998) Interleukin 6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochemical Journal 334: 297–314. Knight DA, Ernst M, Anderson GP, Moodley YP, and Mutsaers SE (2003) The role of gp130/IL-6 cytokines in the development of pulmonary fibrosis: critical determinants of disease susceptibility and progression? Pharmacology and Therapeutics 99: 327–338. Mosley B, Imus CD, Friend D, et al. (1996) Dual Oncostatin M receptors. Journal of Biological Chemistry 50: 32636–32643. Nicola NA (1994) Cytokine pleiotropy and redundancy: a view from the receptor. Stem Cells 12(supplement 1): 3–12. Rose TM and Bruce AG (1991) Oncostatin M is a member of a cytokine family that includes leukemia-inhibitory factor, granulocyte colony-stimulating factor, and interleukin 6. Proceedings of the National Academy of Sciences, USA 88: 8641–8645. Thoma B, Bird TA, Friend DJ, Gearing DP, and Dower SK (1994) Oncostatin M and leukemia inhibitory factor trigger overlapping and different signals through partially shared receptor complexes. Journal of Biological Chemistry 269: 6215–6222. Zarling JM, Shoyab M, Marquardt H, et al. (1986) A growth regulator produced by differentiated histiocytic lymphoma cells. Proceedings of the National Academy of Sciences, USA 83: 9739–9743.

OXIDANTS AND ANTIOXIDANTS Contents

Antioxidants, Enzymatic Antioxidants, Nonenzymatic Oxidants

Antioxidants, Enzymatic I Rahman, University of Rochester Medical Center, Rochester, NY, USA S K Biswas, Dr Ambedkar College, Nagpur, India & 2006 Elsevier Ltd. All rights reserved.

Abstract The lungs have the richest oxygen supply compared with other organs of the body. Therefore, lungs are always vulnerable to

attack by free radicals and reactive oxygen species (ROS). Regulation of the oxidation/reduction (redox) state is critical for cell activation, proliferation and viability, and other organ functions. The ROS generated in the lungs are encountered and neutralized by a battery of protein and nonprotein molecules known as antioxidants. The lung antioxidant defense system is comprised of enzymatic antioxidants such as superoxide dismutases (three types that transform superoxide radicals into hydrogen peroxide), catalase (degrades hydrogen peroxide into water), glutathione peroxidases (mainly transform organic hydroperoxides into less toxic organic hydroxides with the help of glutathione), and other accessory enzymes such as thioredoxins, peroxiredoxins,

OXIDANTS AND ANTIOXIDANTS / Antioxidants, Enzymatic 259 and glutaredoxins. The nonenzymatic antioxidant defense system is chiefly comprised of low-molecular-weight compounds such as vitamins (vitamins C and E), thiols (mainly glutathione), uric acid, b-carotene, etc., either supplemented through dietary sources or synthesized in the body. In several inflammatory conditions of the lungs the levels of these antioxidants have been found to be reduced either due to impaired synthesis or inactivation. Since antioxidants are the main weapon for defense of the lungs, any impairment in their status would lead to several injurious and acute or chronic inflammatory lung conditions.

Introduction The lung has a uniquely large epithelial surface area that is exposed to oxygen and the external environment through direct exchange of gaseous material. Therefore, lungs are prone to attack by a wide variety of external oxidants including particulate matter and infectious agents. However, the lung airways and alveolar septa are adequately designed to ward off such oxidant challenges under normal exposures to the environment. The first line of antioxidant defense of the lungs is provided by the lining of the airways and interstitial fluids. These lung fluids are armed with a wide variety of protein (enzymes) and nonprotein organic molecules, collectively known as antioxidants. These soluble antioxidants are responsible for protecting the lungs from oxidant-mediated lung injuries. The antioxidant system has been one of the major defense mechanisms adopted by lungs throughout evolution.

What Are Antioxidants and Oxidants? Classically, an antioxidant has been defined as a substance that when present at low concentrations compared to that of an oxidizable substrate significantly delays or reduces oxidation of the substrate by reactive oxygen species (ROS) and other free radicals. Antioxidants are so called because of their ability to combat the process of oxidation. Oxidants (or free radicals), on the other hand, are chemical species that contain one or more unpaired electrons and therefore have the ability to donate electrons or abstract electrons from otherwise electronically stable chemical species. On gain or loss of an electron, such stable compounds become unstable and often trigger an undesired set of reactions leading to various chemical reactions including membrane peroxidation of lipids.

Sources of Free Radicals Free radicals are produced in cells by enzymatic or nonenzymatic electron transfer reactions. The

sources of oxidants or free radicals fall into two groups: (1) endogenous sources such as autoxidation of biomolecules, enzymatic oxidation, respiratory burst by phagocytic cells, subcellular organelles, the mitochondrial respiratory chain, and intracellular enzymes such as NADPH oxidase, xanthine oxidase, etc.; and (2) exogenous sources such as drugs, radiation, tobacco smoke, transition metal ions, ischemia reperfusion injury, inorganic particles, for example, asbestos, quartz, and silica, gases such as ozone, and other conditions such as fever, excess glucocorticoid therapy, and photochemical air pollutants, for example, pesticides, solvents, anesthetics, and exhaust fumes. Various sources of free radicals and the general mechanism leading to lung inflammation are illustrated in Figure 1.

Lung Antioxidants Antioxidants function by delaying or blocking free radical processes and by eliminating toxic ROS and their products generated during cellular homeostasis. The antioxidant system in the lung is very complex and has primarily evolved to protect normal cellular homeostasis against the backdrop of an oxygen-rich environment. Antioxidants in the lungs can be divided into two broad categories: enzymatic and nonenzymatic. Nonenzymatic Antioxidants of the Lung

These are low-molecular-weight compounds such as vitamins (vitamins C and E), b-carotene, uric acid, and glutathione (GSH) (a tripeptide (L-g-glutamyl-Lcysteinyl-L-glycine) that contain a thiol (sulfhydryl) group, and retinoid such as retinol and coenzyme Q10 (CoQ10), also known as ubiquinone-50. Albumins, having one sulfhydryl group per molecule, are other antioxidants of biological significance. Enzymic Antioxidants of the Lung

The major enzymic antioxidants of the lungs are superoxide dismutases (SODs), catalase (CAT), and glutathione peroxidase (GPx). Recently, heme-oxygenase-1 (HO-1) and low-molecular-weight redox proteins such as thioredoxins, peroxiredoxins, and glutaredoxins have also been found to play a crucial role in the antioxidant defense system (Figures 2 and 3). Superoxide dismutase Superoxide dismutase is an endogenously produced intracellular enzyme present virtually in every cell of the body. SOD has been shown to play an important role in protecting cells and tissues against oxidative stress. Three types of SOD have been identified (see below). All the forms

260 OXIDANTS AND ANTIOXIDANTS / Antioxidants, Enzymatic

External sources Cigarette smoke Radiation Carcinogens, drugs Hyperoxia, ozone

Cellular sources Inflammatory cells Fibroblasts Endothelial cells Respiratory chain Xanthine and NADPH oxidase

NO

CuZnSOD MnSOD ECSOD H2O

Catalase GPx

Fe2+ H2O2

OH• + NO2

Cell damage Figure 1 Cellular generation of ROS and antioxidant defense system. Intracellular reactive oxygen species: Od
2 , superoxide anion; NO, nitric oxide; H2O2, hydrogen peroxide; OHd, hydroxyl radical; NO2, nitrogen dioxide; ONOO
, peroxynitrite.

O2•−+ 2 H+ 2 H2O2 ROOH + 2 GSH ONOO− + 2 GSH H2O2 + Trx(SH)2

Superoxide dismutase Catalase Glutathione peroxidase Peroxiredoxin VI Glutathione peroxidase Peroxiredoxin (most)

GSSG + NADPH + H+

Glutathione reductase

TrxS2 + NADPH + H+

Thioredoxin reductase

H2O2 + O2 O2 + 2 H2O ROH + GSSG + H2O ONO− + GSSG + H2O 2H2O + TrxS2 2 GSH + NADP+ Trx(SH)2 + NADP+

Figure 2 Enzymatic reactions in glutathione and thioredoxin redox systems.

of SOD act by a common mechanism of dismutation of the superoxide radical ðOd
2 Þ to the less potent hydrogen peroxide (H2O2) as shown in the following equation: þ 2Od
2 þ 2H þ SOD-H2 O2 þ O2

The reaction is pseudo first order and almost diffusion limited (Michaelis–Menten constant 4109 m
1 s
1). SOD is considered fundamental in the process of eliminating ROS by reducing (adding an electron) to superoxide ðOd
2 Þ to form H2O2. Catalase and glutathione peroxidase then reduce H2O2 to H2O. Increased levels of H2O2 slowly inactivate

CuZnSOD. Therefore, catalase and glutathione peroxidase, by reducing H2O2, conserve SOD; and SOD, by reducing superoxide, in turn conserves catalase and glutathione peroxidase. Through this feedback loop, a steady low-level state of SOD, glutathione peroxidase, and catalase, as well as superoxide and H2O2 are maintained, which keeps the entire system in a fully functioning state. Three major forms of SOD are reported based on their structure, localization, inducibility, and metal ion requirements. Copper–zinc superoxide dismutase SOD) CuZnSOD is a homodimeric

(CuZnprotein

OXIDANTS AND ANTIOXIDANTS / Antioxidants, Enzymatic 261

Mito

ER NADH/NADPH

O2• −

Oxidases

O2• −

ECSOD

CuZnSOD MnSOD ECSOD

H2O2

H2O2 CAT Trx Prx Grx GPx H2O

eGPx GSH

GSH

Prx

GR GSSG

(Intracellular)

H2O

(Extracellular)

Figure 3 Enzymatic degradation of reactive oxygen species. Superoxide anion is dismutated by SODs and H2O2 by various enzymatic antioxidants.

(mol. wt 32.5 kDa) localized in the cytosol that requires both Cu and Zn at its active site for its activity. This enzyme is inducible and is expressed in the type II alveolar and bronchial epithelial cells, type II pneumocytes, fibroblasts, alveolar macrophages, and capillary endothelium of the lungs. In the bronchial epithelium, CuZnSOD is highly expressed in ciliated epithelial cells. Although the most abundant form of SOD, it is less inducible than the mitochondrial variety (discussed in the next section). CuZnSOD gene expression is altered during hypoxia and other oxidative states. Manganese superoxide dismutase (MnSOD) MnSOD is considered to be one of the most important antioxidant components of a cell. It is a homotetrameric enzyme (mol. wt 88 kDa) and requires manganese at its active center. MnSOD constitutes about 10–15% of the total SODs and is localized in the mitochondria of type II pneumocytes, alveolar macrophages, and bronchial epithelium in rats and at least in the bronchial epithelial cells of human lungs. MnSOD mRNA is predominantly expressed in cells of the airway walls, the septal tips of alveolar ducts, and in arteriolar walls located adjacent to airways. Altered cellular redox states, inflammatory cytokines such as interleukin-1 and -6 (IL-1 and IL-6), interferon-g, tumor necrosis factor alpha (TNF-a), cigarette smoke, and hyperoxia induce MnSOD gene expression. MnSOD has been reported to be crucial for survival and even 50% expression has been found to provide resistance to hyperoxia. Mice with

depleted MnSOD or a knocked out MnSOD gene have been found to survive for only 2–3 weeks. However, studies of overexpression of this antioxidant enzyme in transgenic mice have yielded inconclusive results as to the ability of these animals to resist oxygen toxicity. On the other hand, administration of SOD to reperfused tissues following hyperoxia has been found to be protective against oxygen toxicity. Extracellular superoxide dismutase (ECSOD) ECSOD is the major extracellular SOD of the pulmonary fluids and interstitial spaces of the lungs, which is the major organ expressing this enzyme in both rats and humans. ECSOD is abundant in blood vessels and airways. It accounts for about 70% of the total SOD in certain pulmonary and systemic blood vessels. It is a secretory, tetrameric glycoprotein (mol. wt 135 kDa) and contains Cu and Zn in its active site. Characteristically, ECSOD exhibits heterogeneous affinity for heparin, regulates nitric oxide (NOd ) bioavailability, and modulates NO levels. The expression of ECSOD is induced by IFN-g and depressed by TNF-a, transforming growth factor beta (TGF-b), and IL-1a in cultured fibroblasts. Alveolar macrophages and neutrophils produce large amounts of ECSOD on antigenic stimulation by lipopolysaccharides (LPS). Transgenic mice overexpressing ECSOD have been found to be resistant to hyperoxia and virus-mediated and posthemorrhage lung damage. Although animals expressing low levels of this enzyme may be otherwise healthy, they are

262 OXIDANTS AND ANTIOXIDANTS / Antioxidants, Enzymatic cys−gly +
-glutamic acid

Extracellular GSH

Membrane γ-GT

Membrane

GSH biosynthesis GSH

GSH synthetase
-glu-cys SH glycine + ATP


GCL cys-SH + ATP


-Glutamic acid Thioether conjugates

GST Lipid peroxide H2O2

GSH GPx

Lipid-OH H2O

NADP G-6P-D

GR

NADPH

GSSG

Glucose 6-PO4 HMP shunt 6-phosphogluconate

GSH redox cycle Figure 4 Glutathione biosynthesis and its redox cycle in lung cells. The steps involved in de novo GSH synthesis and breakdown of extracellular GSH are shown. Glutathione converts hydrogen and lipid peroxides to nontoxic hydroxy fatty acids and/or water. Glutathione disulfide (GSSG) is subsequently reduced to glutathione in the presence of NADPH and glutathione reductase, which are linked with hexose monophosphate shunt. cys, cysteine; gly, glycine.

more susceptible to hyperoxic injury. ECSOD in conjunction with GPx constitute a major first-line defense against inhaled oxidants. Catalase This antioxidant enzyme is a homotetrameric protein (mol. wt. 240 kDa) that catalyzes the conversion of hydrogen peroxide to water and oxygen: 2H2 O2 -2H2 O þ O2 Catalase is present in most aerobic cells in animal tissues and is especially concentrated in the liver and erythrocytes. The brain, heart, and skeletal muscle contain only low amounts. Catalase and glutathione peroxidase seek out hydrogen peroxide and convert it to water and diatomic oxygen. Catalase is found in peroxisomes and the cytoplasm and is especially localized in the alveolar type II pneumocytes and macrophages. It is considered to be the most important antioxidant enzyme consuming exogenous H2O2 in rat type II pneumocytes. Together with the SODs and GSH and related enzymes, it forms one of the major intracellular antioxidant systems protecting against oxidative stress. Catalase is not as effective antioxidant as glutathione peroxidase, which can detoxify a greater number of peroxide radicals including organic and lipid hydroperoxides. Glutathione and its redox recycling The synthesis of glutathione requires the presence of two enzymes and the amino acids glycine, cysteine, and glutamate,

with cysteine being the rate-limiting substrate. The tripeptide GSH is formed by the consecutive actions of g-glutamylcysteine ligase (GCL), formerly called g-glutamylcysteine synthetase (g-GCS) and glutathione synthetase (Figure 4). In general, the activity of GCL determines the rate of glutathione synthesis. The reaction catalyzed by GCL is feedback-inhibited by GSH. The mammalian GCL holoenzyme is a heterodimer consisting of a heavy catalytic subunit (GCLC-HS 73 kDa) and a light modifier subunit (GCLM-LS 30 kDa). Although the heavy subunit contains all of the catalytic activity, GCL activity can be modulated by the association of the heavy subunit with the regulatory light subunit. It has been proposed that the regulatory properties of GCLM are mediated by a disulfide bridge between the subunits that would allow conformational changes in the active site depending on the oxidative state of the cell. This implies that the potential for increasing the rate of GSH synthesis exists under conditions of GSH depletion. It is interesting to note that increased GSH levels are known to inhibit GCL by feedback mechanisms, further emphasizing the importance of redox balance of a cell. The GSH redox system is crucial in maintaining intracellular GSH/GSSG homeostasis, which is critical to normal cellular physiological processes, and represents one of the most important antioxidant defense systems in lung cells. This system uses GSH as a substrate in the detoxification of peroxides such as hydrogen peroxide and lipid peroxides, a reaction that involves glutathione peroxidase. This reaction

OXIDANTS AND ANTIOXIDANTS / Antioxidants, Enzymatic 263

generates oxidized GSH (GSSG), which is then reduced to GSH by glutathione reductase in a reaction requiring NADPH; this is generated by the hexose monophosphate (HMP)-shunt pathway. Glutathione peroxidase GPxs are a family of selenium-dependent and-independent antioxidant enzymes and can be divided into two groups: cellular and extracellular. In general, GPx is a tetrameric protein (mol. wt. 85 000 kDa). It requires four atoms of selenium (Se) bound as seleno-cysteine moieties that confer the catalytic activity. Glutathione peroxidase reduces H2O2 to H2O by oxidizing GSH as shown in eqn [1]. Re-reduction of GSSG is then catalyzed by glutathione reductase through the glutathione cycle (eqn [2]): H2 O2 þ 2GSH-GSSG þ 2H2 O

½1

GSSG þ NADPH þ Hþ -2GSH þ NADPþ

½2

Three genetically distinct, selenium-requiring GPx and one selenium independent classical GPx (cGPX) have been identified in a wide variety of cells. These enzymes are ubiquitously present in the cytosol of most cells. Other varieties include phospholipid hydroperoxide GPx and gastrointestinal GPx. An extracellular form of selenium-dependent GPx (eGPx) has also been reported in plasma and milk, which is secreted in vitro by different cells and cell lines. The alveolar epithelial lining fluid (ELF) contains a very large amount of both eGPx and cGPx in consonance with a very high level of GSH. Primary bronchial epithelial cells, alveolar macrophages, and other lung cell lines can synthesize cGPx and eGPx and also secrete eGPx. Under hyperoxic conditions, GPxs have been found to render a protective effect. The GPx genes are induced by hyperoxia or a combination of hyperoxia and inflammatory cytokines such as TNF-a. The alveolar ELF of normal individuals contains high levels of GSH, and the levels of both cGPx and eGPx are decreased after exposure to O3, but not NO2. Also glutathione reductase (GR) and NADPH, which are important for maintenance of GSH in its active (reduced) form, are found in ELF. Heme oxygenases Heme oxygenase (HO-1; previously known as heatshock protein 32, HSP32) is a member of the heatshock family of proteins that play a protective role in inflammation. HO-1 catalyzes the reaction whereby the heme molecule is degraded into bile pigments (biliverdin), generating carbon monoxide (CO) and iron in the process. HO-1 is induced by many stimuli such as hyperoxia, hypoxia, LPS, and oxidative stress. It is suggested that HO might

have important antioxidant properties and may be significant in protecting lung cells against exposure to oxidants. Three isoforms of HO (HO-1, HO-2, and HO-3) have been characterized. HO-1 alone is inducible whereas HO-2 and HO-3 are constitutive. HO-1 is expressed by airway epithelial cells, alveolar macrophages, and bronchial epithelial and inflammatory cells. HO-1 has a potential anti-inflammatory role and has been found to be protective against hyperoxic lung injury in rats. Many factors such as hyper- and hypoxia, GSH depletion, cytokines, LPS, and oxidative stress have been shown to induce HO-1 expression. On the other hand, TGF-b1 downregulates HO-1 expression. HO-1 expression has been found to be increased in lungs and lung inflammatory cells in a number of diseases including chronic obstructive pulmonary disease (COPD) and asthma. Overexpression of HO-1 protects cells against hyperoxia in vitro. HO-1 also protects against oxidative stress in lungs of rats infused with LPS. Although the mechanism of antioxidant protection by HO-1 is as yet unknown, the release of iron and subsequent chelation and induction of ferritin may provide at least part of protection. Ferritin has been shown to be increased concomitantly with the HO-1-dependent release of iron. In addition, carbon monoxide and bile pigments generated as a result of HO-1 action on heme have also been shown to possess free radical scavenging properties. Thioredoxins (Trxs) The redox status of proteins outside and within the cells is maintained by different mechanisms. While the proteins present on the extracellular face of a cell are stabilized by disulfide bonds (S-S) between two protein molecules, the proteins within the cells are redox stabilized by free sulfhydryl (-SH) moieties. Regulation of such redox states of proteins is carried out by a special class of proteins known as thioredoxins (Trx, mol. wt. 10–12 kDa). These are the major ubiquitous disulfide reductases belonging to the flavoprotein family responsible for maintaining proteins in their reduced states. Trxs are dithiol [(SH)2]-disulfide oxidoreductases and catalyze reduction of disulfide to their corresponding sulfhydryls. The Trx system is comprised of Trx and thioredoxin reductase (TrxR) components, which need NADPH for their function. Mammalian TrxRs are selenoenzymes that reduce oxidized thioredoxin and other protein disulfides. Trxs of lower and higher animals are quite different, mammalian thioredoxins being closely related to the enzyme glutathione reductase. Compared to bacteria, yeasts, and plants, which have multiple Trxs, only one form has been identified in the cytosol of human cells. It has been noted that a truncated

264 OXIDANTS AND ANTIOXIDANTS / Antioxidants, Enzymatic

oxidized form of Trx may be the secretory form of this peptide in humans. Trxs are involved in cell proliferation, reduction of ribonucleotide reductase, thioredoxin peroxidase, thiol–dithiol exchange between cysteine residues of key transcription factors, and protection against exogenous oxidants. In healthy lungs, Trxs and TrxR are expressed in bronchial and alveolar macrophages. Their distribution may be altered in diseases like usual interstitial pneumonia (UIP), desquamative interstitial pneumonia (DIP), UIP associated with collagen diseases, sarcoidosis, allergic alveolitis, granulomatous disease, and other lung disorders. Thioredoxin reduces the viscosity of sputum in cystic fibrosis patients. Exogenous thioredoxin has been shown to reduce ischemia reperfusion in the lungs. Peroxiredoxins (Prxs) These are a recently identified class of antioxidant enzymes and are more associated with cellular signaling mechanisms during oxidative stress. Peroxiredoxins act by decomposition of H2O2 both in vivo and in vitro. Recently, six different types of peroxiredoxins (Prx I–VI) have been characterized in human lungs. Prx I (mol. wt. 22 kDa) is predominantly present in bronchial epithelial, alveolar macrophage cells and a lesser degree of expression is seen in alveolar epithelial and vascular endothelial cells. There is a low level of expression of Prx II (mol. wt. 22 kDa) in alveolar type II cells and it has not been demonstrated in vascular endothelial cells of the lung. Prx III (mol. wt. 28 kDa), Prx V (mol. wt. 17 kDa), and Prx VI (mol. wt. 25 kDa) are the most strongly expressed variety in lungs, especially in bronchial epithelial, alveolar epithelial and macrophage cells. They are scantly expressed by the vascular endothelial cells of the lungs. Prx IV (mol. wt. 31 kDa) is expressed to a very low extent by all cells of the lungs. Glutaredoxin (Grx) Grxs are thiol–disulfide oxidoreductases requiring GSH for their catalytic activity. Grxs have a profound antioxidant capacity and are abundantly present in lungs. Grxs catalyze the reduction of protein disulfide to their respective sulfhydryls by donating reducing equivalents to the oxidized proteins. The oxidized Grx in turn is reduced by transfer of reducing equivalents from GSH as shown below: Prot-S-SProt þ Grx SHðredÞ -2Prot-SH þ Grx SðoxdÞ Grx SðoxdÞ þ 2GSH-Grx SHðredÞ þ GSSG Two types of Grx, Grx1 and Grx2, are identified in cultured human alveolar epithelial A549 cell lines.

Grx1 is predominantly expressed in lung homogenates and alveolar macrophages and to a lesser extent in bronchial epithelium. Grx1 can be located in the plasma membrane, vacuoles, and the nucleus of cells. In pulmonary sarcoidosis, allergic alveolitis, and fibroid lung diseases the expression of Grx is markedly reduced further emphasizing the protective role of this protein.

Antioxidant Enzymes in Lung Diseases Alterations in the status of both enzymatic and nonenzymatic antioxidants have been reported in various lung diseases. In this section, the antioxidant status in some of the major categories of lung disease is reviewed briefly. Smokers, Asthma, and Chronic Obstructive Pulmonary Disease

A general decrease in the levels of nonenzymatic oxidants such as vitamins E and C, GSH, b-carotene has been reported in bronchoalveolar lavage fluid (BALF), ELF, bronchial and alveolar epithelial cells, alveolar macrophages, and other inflammatory cells of the lungs. In contrast, the status of antioxidant enzymes has been found to differ among lung conditions and the cell type involved. All three forms of SOD (CuZnSOD, ECSOD, and MnSOD) have generally been found to be elevated under most inflammatory conditions of the lungs in almost all types of cells involved in a particular pathology, thus indicating the cell’s commitment towards defense against oxidative challenge. Catalase, on the other hand, was either found to be elevated or unchanged in inflammatory situations. SOD and catalase were increased while GPx was unaltered in the BALF of smokers. However, prolonged exposure to cigarette smoke led to an increased expression of GPx and a transient increase in the expression of MnSOD. MnSOD is elevated in the alveolar epithelium of cigarette smokers, without any significant changes in CuZnSOD and ECSOD. Furthermore, the activities of CuZnSOD, glutathione-S-transferase (GST), and glutathione peroxidase (GPx) were all decreased in alveolar macrophages from elderly smokers. Glutathione Stransferase P1 (GSTP1) has been shown to act as a protective enzyme against cigarette smoke in the airway cells. Similarly, HO-1 is induced in alveolar spaces of smokers suggesting that oxidative stress due to cigarette smoke may increase the gene expression of HO-1 leading to increased levels of exhaled CO. Similarly, HO-1 is induced in alveolar macrophages of asthmatics. HO-1 has been implicated in the regulation of cell injury and cell death

OXIDANTS AND ANTIOXIDANTS / Antioxidants, Enzymatic 265

and, in particular, modulation of apoptosis in human endothelial cells and monocytes. Asthmatics exhibit either reduced or unchanged levels of antioxidant levels in peripheral blood cells. The activity of CuZnSOD has been found to be decreased in asthmatic airway epithelium and, in agreement with this, total SOD activity is reduced in lung cells of asthma patients. The increased levels of glutathione following exposure to cigarette smoke condensate has been shown to be due to transcriptional upregulation of the gene for GSH synthesis, gamma-glutamylcysteine synthetase (GCL), by components within cigarette smoke. These events are likely to account for the increased levels of glutathione seen in the epithelial lining fluid in chronic cigarette smokers. However, the injurious effects of cigarette smoke may occur repeatedly during and immediately after cigarette smoking when the lung is depleted of antioxidants, including glutathione. Diminished immunostaining of GCL has been shown in the bronchial and airway epithelial cells of smokers implicating the decreased GCL enzymatic activity. It can be concluded from the above discussion that the status of different antioxidants in varied lung diseases do not follow uniformity of expression and activity and their interpretation might be quite complex. Interstitial Lung Diseases

Idiopathic pulmonary fibrosis (IPF), asbestosis, and granulomatous lung diseases such as sarcoidosis and allergic alveolitis are a consequence of excessive ROS challenge to the lung interstitium. A significantly high level of Od
2 is generated by alveolar macrophages in these diseases. A corresponding increase in the protein levels of CuZnSOD has been recorded in these diseases. The level of this antioxidant enzyme has been found to be proportional to the severity of the disease. However, MnSOD levels were relatively higher than CuZnSOD and there was a greater production of MnSOD and catalase in various interstitial diseases. Treatment of rats with polyethylene glycol (PEG)-conjugated catalase has been shown to reduce fibrotic lung injuries, further emphasizing the protective role of these antioxidant enzymes in such diseases. A fourfold deficiency in glutathione levels in the BALF of patients with IPF has been detected suggesting an oxidant/antioxidant imbalance as a mechanism of tissue injury in IPF. The expression of Grx1, Prx, Trx, and TrxR was decreased in alveolar epithelial type II cells and alveolar macrophages from IPF, sarcoidosis, and

allergic alveolitis patients compared with controls, and the bronchial epithelium showed no Grx1 immunoreactivity. Decreased expression of Grx1 suggests the impairment of this system both in inflammatory and fibrotic lung diseases, consistent with the downregulation of Grx1 by TGF-b in vitro. Adult Respiratory Distress Syndrome

It has been postulated that an imbalance between the antioxidant to oxidant ratio plays an important role in the pathogenesis of adult respiratory distress syndrome (ARDS). The alveolar lining fluid of patients with ARDS shows decreased levels of GSH and other nonenzymatic antioxidants such as vitamins E and C. Elevated levels of H2O2 indicate lower levels of catalase and GPx in ARDS. SOD levels are generally increased in the inflammatory cells and are either unaltered or increased in the lung lining cells. Higher activity/expression of SOD indicates an increased generation of H2O2, which due to diminished catalase or GPx activity may lead to lung injury. In general, however, the antioxidant levels of serum from patients with ARDS are higher compared with their levels in the pulmonary fluids. Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is a chronic lung disease observed in infants whose lungs are exposed to high oxidative stress. Various factors such as barotrauma (pressure-dependent injury), volutrauma (lung volume-dependent injury), prematurity, and oxygen toxicity in the infant lungs are responsible for this condition. In preterm infants, the levels of CuZnSOD, MnSOD, catalase, and GPx are normally lower than observed at full term. Therefore, preterm infants are more susceptible to oxygen toxicity during this stage. In response to hyperoxia, human BPD patients exhibit higher levels of MnSOD staining in type II pneumocytes and alveolar macrophages. Treatment of BPD infants with surfactants, gentler ventilation, antenatal steroids, antioxidants, and antioxidant enzyme supplementation has yielded promising results, thus emphasizing the role of oxidative stress in the pathogenesis of BPD. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Bronchopulmonary Dysplasia. Chronic Obstructive Pulmonary Disease: Overview; Smoking Cessation. Environmental Pollutants: Oxidant Gases. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Occupational Diseases: Asbestos-Related Lung Disease. Oxidants and Antioxidants: Antioxidants, Nonenzymatic. Systemic Disease: Sarcoidosis.

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Further Reading Arner ES and Holmgren A (2000) Physiological functions of thioredoxin and thioredoxin reductase. European Journal of Biochemistry 267: 6102–6109. Avissar N, Finkelstein JN, Horowitz S, et al. (1996) Extracellular glutathione peroxidase in human lung epithelial lining fluid and in lung cells. American Journal of Physiology 270: L447–L455. Cantin AM, North SL, Hubbard RC, and Crystal RJ (1987) Normal alveolar epithelial lining fluid contains high levels of glutathione. Journal of Applied Physiology 63: 152–157. Comhair SA and Erzurum SC (2002) Antioxidant responses to oxidant-mediated lung diseases. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L246–L255. Heffner JE and Repine JE (1989) Pulmonary strategies of antioxidant defense. American Journal of Respiratory Diseases 140: 531–554. Kinnula VL and Crapo JD (2003) Superoxide dismutases in the lung and human lung diseases. American Journal of Respiratory and Critical Care Medicine 167: 1600–1619. Lehtonen ST, Markkanen PM, Peltoniemi M, Kang SW, and Kinnula VL (2005) Variable over-oxidation of peroxiredoxins in human lung cells in severe oxidative stress. American Journal of Physiology: Lung Cellular and Molecular Physiology 288: L997–L1001. Marklund SL (1984) Extracellular superoxide dismutase in human tissues and human cell lines. Journal of Clinical Investigation 74: 1398–1403. McCord JM and Fridovich I (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). Journal of Biological Chemistry 244: 6049–6055. Meister A and Anderson ME (1983) Glutathione. Annual Review of Biochemistry 52: 711–760. Morse D and Choi AMK (2002) Heme oxygenase-1. The ‘Emerging Molecule’ has arrived. American Journal of Respiratory Cell and Molecular Biology 27: 8–16. Otterbien L and Choi AMK (2000) Heme oxygenase: colors of defense against cellular stress. American Journal of Physiology 279: L1029–L1037. Peltoniemi M, Kaarteenaho-Wiik R, Saily M, et al. (2004) Expression of glutaredoxin is highly cell specific in human lung and is decreased by transforming growth factor-beta in vitro and in interstitial lung diseases in vivo. Human Pathology 35: 1000– 1007. Rahman I and MacNee W (1999) Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease. American Journal of Physiology 277: L1067–L1088.

Antioxidants, Nonenzymatic A Papi and M Chicca, Universita` di Ferrara, Ferrara, Italy A Pandit, Royal Glamorgan Hospital, Llantrisant, UK G Caramori, University of Ferrara, Ferrara, Italy & 2006 Elsevier Ltd. All rights reserved.

Abstract Oxidant generation is part of the normal metabolism of many types of cells. To protect itself against exposure to noxious oxidants the lung has a well-developed antioxidant system. When

an imbalance occurs between oxidants and antioxidants in favor of oxidants, oxidative stress is said to occur. Many experimental and clinical data suggest that an imbalance between oxidants and antioxidants (enzymatic and/or nonenzymatic) play a role in the pathogenesis of several respiratory disorders. Here we will review the nonenzymatic antioxidant systems of the lower airways. Most of the clinical data currently available refer to the glutathione system. Some studies have been recently performed on other nonenzymatic antioxidants such as vitamin C and vitamin A.

Introduction Antioxidants in the Lower Airways: Integrated Mechanisms of Enzymatic and Nonenzymatic Systems

Humans have a network of sophisticate cellular defenses that are collectively termed antioxidants to overcome free radical toxicity. The imbalance between reactive oxygen species or reactive nitrogen species and antioxidants is termed oxidative stress. Redox homeostasis is maintained by antioxidants, substances that are able to compete at relatively low concentrations with oxidizable substrates, thus inhibiting or delaying oxidation of these substrates. Antioxidative protective systems in the lower airways of humans are complex. Antioxidants may be either enzymes such as superoxide dismutases, catalases, and glutathione peroxidase, or nonenzymatic compounds such as ascorbic acid (ascorbate, vitamin C), uric acid, vitamin E, vitamin A, trace elements, particularly selenium, and thiols, particularly (reduced) glutathione. Superoxide dismutases (SODs) are important antioxidant enzymes able to decompose superoxide radicals to hydrogen peroxide and considered to play an important role in the antioxidant defense of the lung. There are three SODs acting in different cellular compartments: the intracellular copper–zinc SOD (CuZnSOD), the mitochondrial manganese SOD (MnSOD), and the extracellular SOD (ECSOD). All three have been detected in lung cells, but only ECSOD shows higher expression in the lung than in other tissues. Other antioxidant enzymatic systems active in the lung include thioredoxin peroxidases (peroxiredoxins), lactoperoxidase, glutathione peroxidase, and glutaredoxins which are markedly expressed in the airways and in alveolar macrophages and able to consume hydrogen peroxide, as well as the catalases, regulating the glutathione redox state. In the lung, a thin overlying layer of fluid, the so-called respiratory tract lining fluid (RTLF) serves as the first line of defense against oxidant-mediated injury. RTLF is rich in enzymatic (e.g., superoxide dismutase, catalase) and nonenzymatic (e.g., glutathione, ascorbic acid, uric acid, vitamin E) antioxidants.

OXIDANTS AND ANTIOXIDANTS / Antioxidants, Nonenzymatic Nonenzymatic Antioxidants in the Lower Airways

Among nonenzymatic antioxidants, glutathione and its redox enzymes appear to have an important protective role in the lower airspaces and intracellularly in epithelial cells of the lower airways where it is present in high concentrations. Glutathione is abundantly present in RTLF in concentrations approximatively 100 times greater than those present in plasma. Again there is a marked interindividual variability of RTLF composition in normal subjects. Glutathione is an essential tripeptide present in most eukaryotic cells. Because of its sulfhydryl group, reduced glutathione (GSH) is a versatile molecule capable of protecting cells against oxidants and toxic xenobiotics. However, it also plays key roles in multiple metabolic pathways, such as the synthesis of certain leukotrienes, proteins, and DNA precursors as well as the activation of enzymes, the regulation of immune responses and others. Not only is GSH synthesized by cells for local use but it also participates in an elaborate intercellular exchange process regulated by the gamma-glutamyl cycle. Extracellular GSH in plasma and in bronchoalveolar lavage (BAL) fluid is thus subject to variations according to the degree of expression of gamma-glutamyl cycle enzymes and the rate of consumption of GSH by electrophilic molecules. BAL has allowed us to observe many of these variations of GSH within the extracellular environment of the normal and diseased human lung. The synthesis of GSH requires the presence of two enzymes and the amino acids glycine, cysteine, and glutamate with cysteine being the ratelimiting substrate. The tripeptide GSH is formed by the consecutive actions of gamma-glutamylcysteine synthetase (g-GCS) and glutathione synthetase. The rate-limiting enzyme in GSH synthesis is g-GCS. The human g-GCS enzyme is a heterodimer consisting of a heavy subunit (g-GCS-HS, 73 kDa) and a light subunit (g-GCS-LS, 30 kDa). The human g-GCS heavy and light subunits are regulated by the transcription factor activating protein 1 (AP-1) and antioxidant response elements (ARE) and are modulated by oxidants and growth factors, in airway epithelial cells. In general, the activity of g-GCS determines the rate of GSH synthesis. The reaction, catalyzed by g-GCS, is feedback-inhibited by GSH. Although the heavy subunit contains all of the catalytic activity, g-GCS activity can be modulated by the association of the heavy subunit with the regulatory light subunit. The mechanism of adaptive response to oxidative stress in lower airway epithelial cells is not known. It has been shown that the expression of the g-GCS-HS gene is regulated by different regulatory signals in response to diverse

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stimuli including oxidants in specific cells. For example, the regulatory region of human g-GCS-HS gene is regulated by a putative AP-1 binding sequence in A549 (alveolar-like) cells, a distal ARE containing a phorbol myristate acetate (PMA)-responsive element (TRE/AP-1). These transcription factors are activated by oxidative stress in lower airway epithelial cells. The modulation of gamma-glutamyl transpeptidase (g-GT) may be another way for the regulation of intracellular GSH levels in airway epitheial cells. Gamma-GT cleaves extracellular GSH into its constituent amino acids and leads to the resynthesis of intracellular GSH rather than by direct intact cellular GSH uptake. Thus, g-GT acts as a salvage enzyme for cellular GSH synthesis. The lower airway epithelium has been shown to have high levels of g-GT activity and utilizes extracellular GSH from the alveolar lining fluid. Expression of g-GT is increased in lung epithelial cells by oxidants suggesting that g-GT might play a role in the adaptive response against oxidative stress in bronchial epithelial cells. Although acting with low efficiency, free amino acids rich in thiol (sulfhydryl) groups, such as cysteine and methionine, constitute a major nonenzymatic antioxidant reservoir in the lungs due to their high intercellular concentration. They probably act by replenishing intracellular stores of glutathione. N-acetylcysteine (NAC) is a sulfydryl-containing compound able to interact with free radicals with formation of NAC thiol radicals, with NAC disulphide as the major nonoxidizing end product. Ascorbic acid functions as a reducing cofactor in a number of reactions by transferring electrons to enzymes that provide reducing equivalents. Uric acid (urate) serves as a potent antioxidant by means of radical scavenging and reducing activities. The antioxidant action of urate is also partly dependent by its stabilizing activity on acid ascorbic through the formation of a stable, noncatalytic urate–iron complex which inhibits iron-catalyzed oxidation of ascorbate in biological fluids. The mechanism of action of b-carotene is still unknown. Vitamin A exerts most of its cellular effects through binding to specific multiple nuclear receptors and modulating gene expression. Vitamin E (which includes eight naturally occurring tocopherols, the most active form being RRR-atocopherol) has antioxidant properties through its free-radical scavenging activity. Oxidized vitamin E reacts with other antioxidant compounds, such as ascorbic acid, to regenerate Vitamin E. Selenium is a trace element. There are at least 25 selenoproteins, each one containing one or more molecules of selenium in the form of the amino acid

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selenocysteine. In this way selenium is essential for the function of many antioxidant enzymes including glutathione peroxidase and thioredoxin reductase. Coenzyme Q10 (ubiquinone), a vitamin-like substance, represents an important member of the antioxidative potential in humans. This substance plays an important role in the production of cell energy and the scavenger activity of free oxygen radicals. The highest concentrations of coenzyme Q10 are present in tissues and organs with a high need of energy consumption.

Nonenzymatic Antioxidant Defenses in Inflammatory Respiratory Diseases Some controlled studies suggest that there is a deficiency of nonenzymatic antioxidants in the lungs of patients with inflammatory respiratory diseases. Asthma

Several data indicate that oxidative stress is enhanced in asthma, and that such redox imbalance is not confined within the lungs. Recently, many direct and indirect markers of oxidative stress have been found increased in urine, plasma, sputum, BAL, and lung tissues from asthmatic patients. Their level is often related to the severity and inversely related to the degree of stability of the disease. Controlled studies also suggest that in the lungs and/or in the systemic circulation of patients with asthma, there is a deficiency of antioxidants along with an activation of the pathways protecting lung cells from the oxidant-mediated damage. The levels of the oxidant nitric oxide (NO) are generally low in the lower airways as NO produced by endogenous NO synthases (NOSs) is complexed with reducing agents like thiols, including glutathione, to form a major reservoir of NO-related bioactivity in the form of S-nitrosothiols (SNOs), which are relatively resistant to oxidants. Interestingly, airway levels of the endogenous bronchodilator S-nitrosoglutathione (GSNO) are reduced in children with near-fatal asthma. It has been hypothesized that GSNO could be broken down by oxidants in the asthmatic lung and that its catabolism could have an inhibitory effect on airway smooth muscle relaxation. Peroxynitrite inhibitory activity, a not fully characterized antioxidant mechanism, is reduced in sputum of patients with stable asthma and its level is positively related to the level of pulmonary function and negatively related to the degree of sputum eosinophilia. A link between asthma and selenium deficiency has also been hypothesized. However, a

controlled trial did not show any significant benefit of short-term selenium supplementation in patients with intrinsic asthma. The antioxidant vitamin E inhibits immunoglobulin (IgE) responses to allergic stimuli in animals; dietary vitamin E levels are inversely related to the incidence of asthma. However, there is a lack of controlled clinical studies on the role of vitamin E supplementation in the prevention of asthma. Among antioxidant vitamins, ascorbic acid has been studied for its possible inverse relationship with increased risk of asthma. Accordingly, a study monitoring lung functions in a small group of patients with exercise-induced asthma demonstrated a beneficial effect of ascorbate supplementation. To date, evidence from randomized controlled trials is insufficient to recommend a specific role for vitamin C in the treatment of asthma. Nevertheless, by studying the effect of administering a vitamin E/vitamin C combination in asthmatic children exposed to ozone it has been recently found that the antioxidants effectively protected against ozone-mediated deterioration of lung function. Similarly, the beneficial role of carotenoids has been suggested in exercise-induced asthma. Interestingly, antioxidant status of RTLF (measured as ascorbate, urate, and vitamin E levels in BAL) seems not to be hampered in asymptomatic asthmatic children, as compared to healthy controls, although there was evidence of protein oxidation which was also well correlated with marked eosinophilia. On the other hand, marked reduction of the plasma total antioxidant capacity occurs during asthma exacerbations, suggesting that the redox milieu may play a strong regulatory role in the process. Taken together these data suggest that antioxidant defense impairment is associated to the inflammation profile of the disease. This derangement involves nonenzymatic antioxidant system, whose impairment correlates in some studies with the degree of inflammation. Chronic Obstructive Pulmonary Disease

Recent studies indicate that many antioxidant mechanisms, not only within the respiratory system, are impaired in chronic obstructive pulmonary disease (COPD). Preliminary studies suggest that the plasma vitamin E and red blood cell SOD levels are low in patients with COPD. Short-term exogenous supplementation with vitamin E in COPD patients does not have any significant effect on lung function but reduces plasma levels of the oxidant malondialdehyde, while long-term studies have shown no benefit from

OXIDANTS AND ANTIOXIDANTS / Antioxidants, Nonenzymatic

supplementation with vitamin E on symptoms (cough, sputum, dyspnea). Conversely, recent studies of lung function decline in adults suggest that daily intake of vitamin C at levels slightly exceeding the current recommended dietary allowance (60 mg day 1 for nonsmokers and 100 mg day 1 for smokers) may have a protective effect. However, it remains to be proven whether this approach will decrease significantly the rate of pulmonary function decline that is typical of the disease, measured as forced expiratory volume in 1 s (FEV1) in smokers with COPD. Alveolar macrophages obtained from BAL of smokers with COPD have an intracellular thiol deficiency and this reduction relates with the impairment in lung function. Interestingly, a diminished immunoreactivity for the enzyme g-GCS, critical for the resynthesis of glutathione, has been described in the bronchial mucosa of the same group of subjects. Recent studies have documented that peroxynitrite inhibitory activity is reduced in sputum of patients with stable COPD and that its level is positively related to the level of pulmonary function and negatively related to the degree of sputum neutrophilia. Therefore, similarly to what is observed in asthma, both clinical and experimental data indicate that a decrease of the antioxidant protection in the respiratory system, particularly of nonenzymatic compounds, occurs in COPD even in stable conditions. Such alteration is associated to the lung function impairment and the inflammatory process characteristic of COPD. COPD exacerbations are also characterized by antioxidant defense impairment. Indeed, a marked reduction of the plasma total antioxidant capacity and of the erythrocyte glutathione peroxidase activity has been described during COPD exacerbations, suggesting that a further consumption of the antioxidant shield occurs during the episodes of superimposed acute inflammation that characterizes exacerbations. Interstitial Lung Diseases

The pathogenesis of interstitial lung diseases (ILDs) is known to be associated with systemic and lung oxidative stress. GSH concentration is reduced in the sputum of patients with idiopathic fibrosing pneumonia (IFP). IFP patients have significantly lower levels of total GSH compared to sarcoidosis patients or controls in their BAL. The rate-limiting enzyme of GSH synthesis, g-GCS, is widely expressed in regenerating epithelium but is low in the fibrotic areas of usual interstitial pneumonia (UIP), probably because of enzyme downregulation.

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The concentrations of total and reduced glutathione in BAL are decreased in patients with acute symptomatic farmer’s lung and increased in asymptomatic farmers. These findings suggest that the individual ability to upregulate GSH in the alveolar space in response to an inflammatory stimulus may have implications for the development of symptomatic farmer’s lung. Antioxidant enzymes, such as catalase, glutathione peroxidase, and SOD, are increased in coal miners with clinically evident radiographic coal workers’ pneumoconiosis. This upregulation of antioxidant defenses is not found in coal miners in the absence of clinically evident radiographic disease. In contrast glutathione levels are increased in BAL from patients with chronic beryllium disease compared with smokers and nonsmoker controls. Increases in GSH are correlated with extracellular glutathione peroxidase, indicating similar inducing mechanisms for these antioxidants. Drug-Induced Pulmonary Toxicity

Delayed pulmonary toxicity syndrome after highdose chemotherapy (HDC) and autologous hematopoietic support occurs in up to 64% of women with advanced-stage breast cancer. Using a similar but nonmyeloablative HDC treatment regimen in mice, both immediate and persistent lung injury coincide with marked decreases in lung tissue glutathione reductase activity and are accompanied by increases in lung oxidized glutathione, BAL lipid peroxidation, and BAL total cell counts. Bronchiolitis Obliterans Syndrome

Bronchiolitis obliterans syndrome (BOS) is the most serious long-term sequel of lung or heart–lung transplantation. Neutrophilia in the lower airways is a prominent feature of BOS and neutrophils are capable of releasing large quantities of oxidants which may be involved in the pathogenesis of BOS. Myeloperoxidase activity in the BAL and oxidized methionine residues in BAL-derived proteins are elevated in BOS. In addition, the concentration of reduced glutathione in BAL is decreased in BOS whereas the proportion of oxidized glutathione is increased. Reduced glutathione levels in BAL are positively correlated with FEV1 level. Lung Damage During HIV Infection

Concentrations of glutathione are decreased in the BAL of human immunodeficiency virus (HIV) seropositive subjects. Evidence exists that HIV seropositive individuals may be at increased risk for the development of precocious pulmonary emphysema

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and it has been suggested that the deficit of glutathione may contribute to its pathogenesis. In the setting of HIV, emphysema is more prominent and lung GSH concentrations are higher in the upper lobes. Acute Respiratory Distress Syndrome

In the acutely inflamed lung of patients with acute respiratory distress syndrome (ARDS), oxidant stress occurs within the alveolar compartment. BAL oxidized glutathione is increased in ARDS, thus indicating oxidant stress. Reduced glutathione levels in patients does not change significantly, whereas BAL levels of vitamins A, C, and E and uric acid are significantly elevated in ARDS. However, interestingly, plasma levels of vitamins A, C, and E and selenium are decreased in ARDS suggesting their use in the inflammed lungs.

Considerations on Therapeutic Implications There is a paucity of data, often contradictory, on the effect of drugs on the oxidant–antioxidant balance in respiratory diseases. In particular it is still unknown whether the clinical efficacy of the treatment is somehow linked to the pharmacological modulation of the nonenzymatic antioxidant balance. However, studies of nonenzymatic antioxidants have led to a better understanding of physiopathology of many inflammatory respiratory diseases and have stimulated investigations of novel approaches for their treatment. A randomized clinical trial on NAC in patients with COPD has been recently completed. The results indicate that oral administration of once-daily 600 mg NAC in combination with standard therapy for 3 years did not influence the rate of exacerbations and of decline in lung function, whereas it reduced hyperinflation in COPD patients. Nonenzymatic antioxidant therapy may be useful in diseases with impaired oxidant–antioxidant balance such as pulmonary fibrosis. In an animal model of bleomycin-induced lung fibrosis, oral administration of NAC significantly increased total glutathione and taurine levels in BAL and partially decreased the augmented collagen deposition in bleomycinexposed rats. NAC failed to inhibit the bleomycininduced increases in lung wet weight and in cell counts and protein levels of BAL. Interestingly, however, in the same model, NAC administration by inhalation attenuated the cellular infiltration in both BAL and alveolar tissues, the increase of proinflammatory chemokines and of lipid hydroperoxide, and the fibrotic changes.

In patients with IFP, total GSH levels in the BAL increased after 6 and 12 months following therapy with immunosuppressive drugs such as glucocorticoids, methotrexate, or cyclosporin A, clearly indicating the crucial role of the active inflammatory process in on antioxidant defense consumption. In a small uncontrolled trial the administration of high-dose oral NAC significantly elevated BAL GSH levels in patients with fibrosing alveolitis, indicating an antioxidant effect at the alveolar surface. These biochemical changes were accompanied by an improvement of lung function test in patients under maintenance immunosuppression. A large randomized clinical trial on high doses of NAC in patients with IPF has recently been carried out. The preliminary results (reported only in the form of abstract) suggest that high-dose oral NAC in combination with standard therapy in a 12-month period has a positive effect on lung function, even if it does not affect radiological and arterial blood gas values. In an animal model of delayed pulmonary toxicity syndrome after high-dose chemotherapy and autologous hematopoiesis, a single supplemental dose of glutathione prevents early lung injury at 48 h (but does not have lung-protective effects at 6 weeks), whereas single doses of other thiol-sparing agents are not effective. In HIV seropositive subjects after 3 days of twicedaily doses each of 600 mg aerosolized reduced glutathione, total glutathione levels in the BAL increased and remained in the normal range for at least 3 h after treatment. Strikingly, even though 495% of the glutathione in the aerosol was in its reduced form, the percentage of oxidized glutathione in epithelial lining fluid increased from 5% before treatment to about 40% 3 h after treatment, probably reflecting the use of glutathione as an antioxidant in vivo. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. CD1. Cystic Fibrosis: Overview. Drug-Induced Pulmonary Disease. Human Immunodeficiency Virus. Interstitial Lung Disease: Overview; Cryptogenic Organizing Pneumonia. Nutrition in Respiratory Disease. Oxidants and Antioxidants: Antioxidants, Enzymatic; Oxidants. Oxygen Toxicity.

Further Reading Abushamaa AM, Sporn TA, and Folz RJ (2002) Oxidative stress and inflammation contribute to lung toxicity after a common breast cancer chemotherapy regimen. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L336–L345. Beeh KM, Beier J, Haas IC, et al. (2002) Glutathione deficiency of the lower respiratory tract in patients with idiopathic pulmonary fibrosis. European Respiratory Journal 19: 1119–1123.

OXIDANTS AND ANTIOXIDANTS / Oxidants 271 Behr J, Degenkolb B, Beinert T, Krombach F, and Vogelmeier C (2000) Pulmonary glutathione levels in acute episodes of Farmer’s lung. American Journal of Respiratory and Critical Care Medicine 161: 1968–1971. Behr J, Maier K, Braun B, Schwaiblmair M, and Vogelmeier C (2000) Evidence for oxidative stress in bronchiolitis obliterans syndrome after lung and heart-lung transplantation: The Munich Lung Transplant Group. Transplantation 69: 1856–1860. Cantin AM (2004) Potential for antioxidant therapy of cystic fibrosis. Current Opinion in Pulmonary Medicine 10: 531–536. Caramori G and Papi A (2004) Oxidants and asthma. Thorax 59: 170–173. Decramer ML, Rutten-van Molken M, Dekhuijzen PN, et al. (2005) Effects of N-acetylcysteine on outcomes in chronic obstructive pulmonary disease (bronchitis randomized on NAC cost-utility study, BRONCUS); a randomized placebo-controlled trial. Lancet 365: 1552–1560. Diaz PT, Wewers MD, King M, et al. (2004) Regional differences in emphysema scores and BAL glutathione levels in HIV-infected individuals. Chest 126: 1439–1442. Diwadkar-Navsariwala V and Diamond AM (2004) The link between selenium and chemoprevention: a case for selenoproteins. Journal of Nutrition 134: 2899–2902. Luppi F, Cerri S, Beghe B, Fabbri LM, and Richeldi L (2004) Corticosteroid and immunomodulatory agents in idiopathic pulmonary fibrosis. Respiratory Medicine 98: 1035–1044. Montaldo C, Cannas E, Ledda M, et al. (2002) Bronchoalveolar glutathione and nitrite/nitrate in idiopathic pulmonary fibrosis and sarcoidosis. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 19: 54–158. Psarras S, Caramori G, Contoli M, Papadopolous N, and Papi A (2005) Oxidants in asthma and chronic obstructive pulmonary disease. Current Pharmaceutical Design 11: 1–10. Rahman I and MacNee W (2002) Oxidative stress and adaptive response of glutathione in bronchial epithelial cells. Clinical and Experimental Allergy 32: 486–488. Schmidt R, Luboeinski T, Markart P, et al. (2004) Alveolar antioxidant status in patients with acute respiratory distress syndrome. European Respiratory Journal 24: 994–999. Wu G, Fang YZ, Yang S, Lupton JR, and Turner ND (2004) Glutathione metabolism and its implications for health. Journal of Nutrition 134: 489–492.

Oxidants A Shukla and B T Mossman, University of Vermont, Burlington, VT, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract A group of reactive intermediates including electrophiles and free radicals are known as oxidants and are capable of damaging cellular constituents during their generation in normal physiological or pathophysiological processes. The cellular consequences of this damage include enhanced mutation rates, altered cell signaling, and many other damaging events. In many cases, the initially generated reactive intermediates convert cellular constituents into second-generation reactive intermediates capable of inducing further damage. Cells have adapted to the existence of reactive intermediates by the evolution of defense

mechanisms called antioxidants that either scavenge these intermediates or repair the damage they cause. Oxidative stress is thought to be a critical process in inflammatory and injurious events that occur in many lung diseases like asthma, chronic obstructive pulmonary disease, adult respiratory distress syndrome, and lung cancer. Activated phagocytic cells that are normally resident in the lung and those recruited following lung injury can cause generation of oxidant products. An understanding of the correlation between oxidative stress and initiation of lung diseases needs to be established.

Introduction Free molecular oxygen probably appeared on the earth’s surface some 2
109 years ago as a result of photosynthetic microorganisms acquiring the ability to split water. It is the most abundant element in the earth’s crest, and the second most abundant element in the biosphere. Oxygen is an unusual molecule in that it has two unpaired electrons, with parallel spins. It is therefore a biradical. To overcome spin restriction, oxygen prefers to accept electrons one at a time, and the sequential addition of electrons leads to the formation of oxidants or reactive oxygen intermediates (ROI) that also include free radicals. The term reactive oxygen species (ROS) is also used to describe free radicals and those molecules that themselves get easily converted to free radicals or are powerful oxidizing agents. These include superoxide anion (O2 d), hydrogen peroxide (H2O2), hydroxyl radical (OHd), products of myeloperoxidase, and reactive nitrogen species (NOd, N2O4, ONOO  ). The human respiratory tract stretches from the nasal and oral cavity to the alveoli or airspaces of the lung, making the total surface area that is directly exposed to the external environment comparable to that of a tennis court. Since the lung is constantly exposed to many atmospheric pollutants including tobacco smoke, fuel emissions, environmental particulates, ozone, and nitrogen dioxide (NO2), and given the natural oxidizing nature of the atmosphere (21% O2), the lung is always at risk of oxidative injury. ROS are produced continuously in living cells in numerous biological processes as part of normal activity. In rats, an average of about 1012 oxygen molecules are processed by each cell daily under basal conditions, and the leakage of partially reduced oxygen molecules is about 2%. Human cells under basal conditions produce about one-tenth the ROS of rats or 2
109 O2 d and H2O2 molecules per cell per day. Oxidants are found intracellularly and extracellularly and may be produced endogenously or exogenously, that is, from the environment. Airways are unique tissues in both their exposure to high levels of environmental oxidants and their unusually high concentration of extracellular

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antioxidants. In the resting state, the balance between antioxidants and oxidants is sufficient to prevent the disruption of normal physiologic functions; however, either increases in oxidants or decreases in antioxidants can disrupt this balance. The state of imbalance towards oxidants is collectively referred to as oxidative stress and is associated with diverse airway pathologies.

Regulation of Production and Activity More than 90% of all the oxygen we breathe undergoes a concerted tetravalent reduction to produce water in a reaction catalyzed by cytochrome oxidase in the mitochondrial electron transport chain. Oxygen (O2) can also be reduced via a nonenzymatic pathway through four successive one-electron (e  ) reductions: O2 þ 4Hþ þ 4e -2H2 O Cytochrome oxidase is the terminal electron acceptor in the respiratory chain and must donate its reducing equivalents to oxygen to allow continued electron transport. Otherwise, ATP production cannot continue. Thus, a major role for oxygen in all aerobic organisms is simply to act as a sink or dumping ground for electrons. The tetravalent reduction of oxygen by the mitochondrial electron-transport chain is considered a relatively safe process. Nonetheless, the electron carriers catalyze alternating oneelectron oxidant reduction reactions, and they can react with O2 to generate ROS such as O2 d. Mitochondria are the major intracellular sites of O2 d generation under physiological conditions. Other endogenous sources that contribute to the generation of ROS in the lung probably include the molybdenum hydroxylases (xanthine dehydrogenase/ oxidase (XDH/XO) and aldehyde oxidase), the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH) oxidoreductase (including phagocytic cell oxidase, cytochrome P-450, and nitric oxide synthase) and arachidonic acid metabolizing enzymes (such as cyclooxygenase). The NADPH oxidase enzymatic system that is found in neutrophils, monocytes, and macrophages is called NADPH oxidase of phagocytes. This oxidase activity is low or nonexistent in resting cells. Stimulation of this oxidase is the process by which phagocytes transform this latent activity to an expressed activity, and the process of generation of ROS by this process is called the respiratory burst. This enzyme is a multisubunit complex that generates O2 d in one electron reduction of O2 using

electron supplied by NADPH. The oxidase consists of two membrane proteins gp91 and p22, that together form a unique cytochrome with a redox midpoint potential of  245 mM and a reduced minus oxidized difference spectrum of 558, and consists of several cytosolic components, including p47, p67, a small G protein, and p40-phox. In phagocytes, an assembly of the cytosolic proteins with membrane components activates the oxidase. Heritable defects of gp91, p22, p47, or p67 are the basis for chronic granulomatous disease, a disorder of white blood cell function characterized by recurrent, severe bacterial and fungal infections. Production of ROS is a regulated process closely linked to phagocytosis and secretion of enzymes. The first regulatory step in physiologic stimulation of phagocyte O2 d production is the specificity of receptor binding. The respiratory burst is similar to other receptormediated processes, which are characterized by a receptor occupancy requirement, inactivation and recycling of receptors, and cross desensitization by stimuli. Although stimuli can produce differing amounts of O2 d generation, the same oxidase activity is apparently involved. One of the first events after binding appears to be depolarization of plasma membrane potential, as demonstrated by both fluorescent dye and microelectrode impalement techniques. A transient increase in free cytosolic Ca2 þ and binding to calmodulin appears to occur next in receptor-mediated respiratory burst stimulation. Contributions from internal stores and extracellular Ca2 þ may reflect increases in cytosolic Ca2 þ . There is evidence that an enzyme complex similar to the phagocytic cell oxidase is present and exerts a variety of functions in nonphagocytic cells, including vascular endothelial cells, smooth muscle cells, fibroblasts, and the carotid body. Both endothelial and vascular smooth muscle cells contain a nonphagocytic membrane-bound oxidase (NOX) that utilizes NADH and NADPH as substrates for electron transfer to O2, similar to the NADPH oxidase of neutrophils. There are, however, important differences between the nonphagocytic and phagocyte oxidases. These include the delayed time course for activation, low output, and, in some cases, the preference for NADH over NADPH in NOX. The NADPH oxidase system and its regulation in vascular cells are different from those in phagocytes. The main pathway for modulation in vascular cells appears to be via gp91, which is upregulated in response to lung injury. The role of NADPH oxidase may not be limited to its ability to generate toxic oxidants but it may also function as an oxygen sensor and, possibly, as an iron uptake system. A third function of the vascular NADPH oxidase central to the pathobiochemistry of

OXIDANTS AND ANTIOXIDANTS / Oxidants 273

acute lung injury is to serve as a ferric reductase in a transferring-independent iron uptake system. XDH/XO, rate-limiting enzymes in purine metabolism, are members of a molybdenum-containing hydroxylase and a homodimer with a subunit molecular weight of about 150 000. Each subunit contains four redox active centers: two iron–sulfur, one FAD, and one molybdopterin. XDH catalyzes the final two reactions of purine metabolism to produce uric acid. XDH can be readily converted to XO by reversible sulfhydryl oxidation or by irreversible proteolytic modification. XO uses molecular oxygen as its electron carrier and produces ROI, either O2 d or H2O2. XDH/XO gene expression is regulated in a cell-specific manner and is markedly affected by inflammatory cytokines, steroids, and physiologic events such as hypoxia. Hypoxia not only transcriptionally regulates XDH/XO but also enhances enzyme activity by as yet undefined posttranslational processes that do not involve conversion of XDH to XO. The evidence that human XDH/ XO can undergo activation–deactivation cycles at the molybdenum redox center further emphasizes the potential importance of posttranslational regulation of XDH/XO. O2 d is a relatively unstable molecule with a halflife of only milliseconds. It does not easily cross cell membranes because of its charge. However, O2 d reacts with proteins that contain transition metal prosthetic groups, such as heme moieties or iron–sulfur clusters. These reactions may damage amino acids, resulting in loss of protein/enzyme function. Most of O2 d generated in vivo undergoes a nonenzymatic or superoxide dismutase (SOD)-catalyzed reaction, resulting in the nonradical H2O2 molecule: d þ Od 2 þ O2 þ 2H -H2 O2 þ O2

H2O2 can also be directly produced by several oxidase enzymes, including XO, monoamine oxidase, and amino acid oxidase. H2O2 can be oxidized by eosinophil-specific peroxidase (EPO) and neutrophilspecific peroxidase (MPO) using halides (X  ) as a cosubstrate to form the potent oxidant hypohalous acids (HOX) and other reactive halogenating species: H2 O2 þ X þ Hþ -HOX þ H2 O X ¼ Br or Cl Much of the damage done by O2 d and H2O2 in vivo is due to their production of OHd in a series of transition ion-catalyzed reactions. One such example is the iron-catalyzed Haber–Weiss reaction in which Fe3 þ is reduced to Fe2 þ , followed by the Fenton reaction in which the Fe2 þ catalyzes the

transformation of H2O2 into OHd: 3þ 2þ Od þ O2 2 þ Fe -Fe 2þ

H2 O2 þ Fe -Fe

3þ

Haber2Weiss reaction

þ OH þ OHd Fenton reaction

An alternative pathway for OHd formation in vivo may involve MPO and EPO. Under physiological concentrations of halides, MPO produces hypochlorous acid (HOCl), and EPO produces hypobromous acid (HOBr). Studies of OHd with spin trapping agents and chemical traps have demonstrated that hypohalous acids can generate OHd after reacting with O2 d. OHd can also react with different macromolecules such as protein, DNA, and lipids (Figure 1).

Biological Functions Oxidants and Cell Activation

Lung exposure to oxidant-generating air pollutants or particulates can induce direct or indirect oxidant stress resulting in inflammation and/or enhanced formation of intracellular oxidants that play key roles in the cellular responses to active environmental or inflammatory stimuli. This process may result in the activation of macrophages, which generate large amounts of oxidants and nitric oxide (NO) as well as cytokines such as tumor necrosis factor alpha (TNF-a). TNF-a and extracellular oxidants can activate airway epithelial cells to induce proinflammatory genes, such as NOS-2, COX-2, intercellular adhesion molecule (ICAM-1), interleukin-6 (IL-6), IL-8, MIP1, and granulocyte-macrophage colony-stimulating factor (GM-CSF), which collectively promote the recruitment and activation of inflammatory cells. In addition, they also enhance mucin secretion from respiratory epithelial cells. This process appears to involve diverse signaling pathways including phospholipase C, protein kinase C, mitogen-activated protein-kinase (MAPK), and activation of nuclear factor kappa B (NF-kB). Increased mucin secretion may represent a protective mechanism to promote mucociliary clearance of inhaled particulates and to recruit protective antioxidant, anti-acid, and antiproteolytic substances to airway surfaces. However, it could also hinder respiratory airflow under conditions of reduced ciliary beating, a consequence of oxidant injury to ciliated cells. Oxidants and Aging

A variety of evidence suggests that oxidants participate in normal aging and in age-related disease maladies such as cancer, atherosclerosis, and neurodegeneration. Also, there is evidence that links

274 OXIDANTS AND ANTIOXIDANTS / Oxidants

Inhaled pollutants

Oxidants O2

.

−

O2

SOD

H2O2 Reactive metabolites

NAD(P)H oxidase EPO MPO Phagocytic cell

Proteins, enzymes oxidation Lipid peroxidation, Cell membrane damage DNA strand breaks

HOBr HOCl

Br −, Cl −

Rise in intracellular Ca2+

ELF

Cell damage Mucin

Epithelial cell

Phagocytic cell

Goblet cell

Figure 1 Illustrative representation of oxidative events occurring in lung. ELF, epithelial lining fluid; EPO, eosinophil peroxidase; MPO, myeloperoxidase; SOD, superoxide dismutase.

associations between cellular responses to oxidants with mechanisms that regulate longevity. Mammalian cells execute a stress response mechanism following exposure to various external stimuli such as endotoxins, inflammatory agents, heat, xenobiotics, and oxidants. This involves a rapid and specific activation and expression of stress proteins such as ubiquitin, heat shock proteins (hsp-27, -70, -90), and heme oxygenase. This stress response appears to serve as an important defense mechanism against agents such as oxidants that induce acute lung injury. An important feature of the stress response is the prioritization of gene expression in favor of stress proteins and selective inhibition of nonstress protein gene expression. For example, hsp-70 and heat shock factor inhibit the activation of NF-kB that results in reduced expression of proinflammatory genes such as iNOS or pro-IL-1b. An inhibition of proinflammatory gene expression may contribute to the protective mechanisms of the stress response. Forkhead proteins are a family of transcription factors that appear to regulate longevity in simple organisms like Caenorhabditis elegans. DAF-16 appears to be the Forkhead homolog in worms that is most tightly linked to life span. Adult worms with an increase in DAF-16 activity live longer than control worms and appear to possess augmented stress resistance, including resistance to ROS. Other studies have also demonstrated that mammalian Forkhead

proteins appear to regulate the transcription antioxidant proteins that scavenge O2 d and H2O2. Another protein that is known to regulate aging in mice is p66shc. Mice lacking p66shc have an increase in life span of around 30%. Studies have demonstrated that cells from p66shc  /  mice have reduced intracellular levels of ROS. Another link between aging and oxidants comes from studies with the mammalian homolog of Sir2, a protein previously implicated in gene silencing and aging in yeast. Sir2 possesses an NAD-dependent histone deacetylase activity, and three studies have demonstrated that the tumor suppressor gene p53 is a novel and important target of Sir2-dependent deacetylation. It has also been reported that this Sir2-dependent activity protects cells against death following oxidative stress. Oxidants and Cell Signaling

Studies have demonstrated that receptor binding of numerous peptide growth factors stimulate ROS production and that this oxidant burst is required for certain aspects of downstream signaling. Oxidants have also been demonstrated to affect activation of growth factor receptors, such as epidermal growth factor (EGF). Environmental or inflammatory oxidants, as well as particulates that contain redox-active transition metals on their surfaces, lead to the activation of the MAPK family, including

OXIDANTS AND ANTIOXIDANTS / Oxidants 275

extracellular signal regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38, to variable degrees. Activation of members of the MAPK family leads to the transactivation of transcription factors such as c-Jun, ATf2, and ELK-1, activating the early response genes c-fos and c-jun, that eventually result in expression of genes regulating a battery of distinct cellular events including apoptosis, proliferation, morphologic transformation, and differentiation. Moreover, these same signaling pathways are also involved in stress responses and the activation of hsps. Although oxidants have commonly been associated with cell toxicity or injury, it has become increasingly clear that oxidants represent an integral component of various cell signal transduction systems, and are incriminated in cellular pathophysiologic activities. For example, large amounts of oxidants are produced by NADPH oxidase in inflammatory/immune cells, whereas other cell types (vascular smooth muscle cells, chondrocytes, fibroblasts) produce oxidants at considerably lower levels using similar NAD(P)H oxidase systems. Thus, the stimulation of various cell types with diverse ligands has been demonstrated to generate intracellular oxidants that appear to be important in the stimulation of signal transduction pathways. Considerable progress has been made in identifying the specific intracellular targets of oxidant signaling. Redox-sensitive molecular targets usually contain highly conserved cysteine residues, and their oxidation and formation of disulfide links are crucial events in redox signaling. Such molecular targets include transcription factors, ion channels, and signaling molecules such as Ras or protein tyrosine phosphatases (PTPs), thereby modulating MAPK signaling pathways. Given that oxidant bursts are often associated with an increase in tyrosine phosphorylation, one important event for ROS-mediated effects is alteration of the tyrosine kinase/phosphatase balance. PTPs are particularly attractive candidates because they contain reactive cysteine residues within their active sites. It has been reported that exogenous oxidants or oxidants generated by binding of peptide growth factors can reversibly oxidize and hence inactivate PTPs. Oxidized inactive PTPs presumably undergo spontaneous or enzymatic reduction leading to a restoration of the enzyme activity. Such a reductive-restoration process might involve a protein–glutathione intermediate. Intracellular glutathione (GSH) and thioredoxin (TRX), and the accompanying enzymes glutaredoxin and TRX reductase, are of central importance in the regulated control of such redox signaling pathways by reducing disulfide bridges or oxidized cysteine residues. In the

lung, the same signaling pathways can also be activated by various oxidant-generating airborne pollutants (asbestos, metals, particulates, etc.) as well as by inflammatory oxidants. The cellular responses depend upon the degree or duration of the activating stimulus. While almost all of the classic oxidases generate O2 d, most aspects of signaling have been linked to the more stable derivative, H2O2. For example, T-cell stimulation leads to the generation of both O2 d and H2O2, and distinct intracellular pathways are activated by either oxidant. Integrin-mediated cell shape changes and subsequent downstream signaling events often require intracellular oxidant generation, and the source of these oxidants is the mitochondria. The knowledge that oxidants released from mitochondria might have a signaling function provides an interesting twist in the field of redox signal transduction. Oxidants and Transcription Factors

Oxidants are involved both in regulation as well as binding of transcription factors. Many environmental stresses are known to cause activation of NF-kB by the intermediate formation of H2O2 that promotes either tyrosine phosphorylation or increased ubiquitination and degradation of the inhibitory component, IkB kinase. The binding of NF-kB to DNA also involves redox-sensitive mechanisms, as the DNA-binding domain contains a highly conserved cysteine residue that needs to be reduced to allow binding. Activation of NF-kB results in rapid activation of expression of an array of genes involved in inflammation, infection, and stress and also has been linked to cellular protection from apoptotic death induced by TNF-a, irradiation, and chemotherapeutic agents via formation of oxidants. Activator protein 1 (AP-1), which is composed of homodimers or heterodimers of Fos and Jun proteins, can also be activated by oxidants. DNA binding of these proteins is regulated by the reduction–oxidation of a single conserved cysteine residue (Lys-Cys-Arg) in their DNA-binding domains. Hypoxia-induced heterodimeric transcription factor 1 (HIF-1) is also susceptible to oxidant-sensitive degradation. Furthermore, DNA binding of HIF-1 is controlled by redox mechanisms similar to those of the NF-kB system. Thus oxidants can activate various transcription factors, and higher concentrations of these molecules in the nuclear compartment can prevent their ability to bind to DNA. Since AP-1 and NF-kB play key roles in proliferation and inflammation, respectively, the oxidant-induced regulation of these transcription factors is an attractive target for selective therapeutic intervention. Figure 2 shows the

276 OXIDANTS AND ANTIOXIDANTS / Oxidants Oxidants

Exogenous (air pollutants) Reaction with DNA, protein, and lipid

Endogenous (mitochondrial, phagocytosis, XDH / XO)

First-generation reactive oxygen species Second-generation reactive oxygen species Signaling pathways (PKCs, I
B, MAPK)

Asthma

Transcription factors (AP-1, NF-
B, NFAT) Gene transcription Apoptosis

Transformation

are thought to be the result of nonenzymatic reactions between ROS/RNS and protein lipids or DNA. Measurements of lipid peroxidation products and oxidation products in proteins or DNA from lung or epithelial lining fluid (ELF), as indicators of antioxidant status, and direct measurement of oxidants have in many cases indicated oxidative stress within the respiratory tract of patients with such diverse lung diseases as asthma, acute lung injury, lung fibrosis, pneumoconiosis, emphysema, allograft rejection, and lung cancer. In this section, we will discuss in brief some concepts regarding involvement of oxidative stress in selected respiratory tract diseases.

Proliferation

Lung diseases Figure 2 A hypothetical schema showing oxidant-dependent signal transduction and transcription factor activation leading to various lung diseases. AP-1, activator protein 1; IkB, inhibitory kappa B; MAPK, mitogen-activated protein-kinase; NF-kB, nuclear factor kappa B; NFAT, nuclear factor of activated T cell; PKC, protein kinase C.

regulation of cell signaling pathways and transcription factors by oxidants. Caspases, the cysteine protease executioners of the apoptotic response, can also be modified by oxygen radicals. These enzymes catalyze the proteolysis of proteins necessary for cell survival, using a nucleophilic cysteine residue in the active site to stimulate cleavage of target proteins at consensus aspartate residues. Several studies have shown that inactivation of caspase 3 by free radicals occurs presumably because of modification of its catalytic cysteine residues. The modification of caspase 3 by various agents represents a potential method for regulating its activity and thereby preventing apoptosis.

Oxidants in Respiratory Diseases In many airway diseases, the delicate balance between ROS/RNS and antioxidants is in disequilibrium because of the excess production of ROS/RNS or depletion of antioxidants. Because direct measurement of ROS/RNS is technically difficult in vivo, there are few reports of direct measurements of ROS/ RNS in airway diseases. Instead, many investigators report footprints of oxidative stress. These footprints

This condition manifests as a widespread narrowing of the bronchial airways, which changes in severity over time and leads to cough, wheezing, and difficulty in breathing. Asthmatic patients show decreased antioxidant status and increased indices of oxidative stress in their lung. Studies with isolated animal tracheal or airway tissues have indicated that oxidants can influence airway smooth muscle homeostasis in favor of increases in contractile tone. Levels of H2O2 and CO (a product of heme oxygenase) are found to be elevated in the exhaled breath of asthmatic patients, suggesting activation of oxidant generation and the stimulation of a stress response in the respiratory tract. There are some studies indicating that deficiencies in dietary antioxidant substances are related to increased incidences of atopy, airway hyperresponsiveness, and asthma. The significance of inflammatory oxidants in the etiology of asthma is not yet established, but is an area of intense investigation. Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by decreased compliance of the lung with changes in transpulmonary pressure that evoke smaller than normal changes in lung volume. This disease is thought to arise as a response to persistent lung injury and inflammation. Increased oxidant burden and decreased glutathione levels (the major antioxidant in lung lining fluid) have been reported in this disease. The increased oxidant burden appears to come from the accumulation of inflammatory cells, which includes activated alveolar macrophages and neutrophils in the lower respiratory tract. The oxidants generated from these cells cause epithelial cell injury that is a characteristic of this condition. IPF patients have also been shown to release increased amounts of ROS in lavage samples. These ROS react

OXIDANTS AND ANTIOXIDANTS / Oxidants 277

with myeloperoxidases in the ELF to produce the highly toxic hypohalide ion (HOCl  ). Oxidants may also promote fibrosis indirectly through the enhanced release of cytokines. Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) includes chronic bronchitis and emphysema, the two most common causes of respiratory failure. COPD is characterized by varying degrees of airway inflammation, smooth muscle hyperreactivity, and mucus overproduction. The destruction of the wall structures of respiratory bronchioles, alveolar ducts, and alveoli all contribute to an accelerated decline in respiratory gas conducting and exchange functions. The important events in the pathogenesis of COPD are considered to be lung inflammation, an increased oxidant burden, and a protease–antiprotease imbalance in the lungs. There is ample evidence that oxidant stress occurs in patients with COPD, as indicated by elevated levels of exhaled H2O2 or NO, reduced ELF antioxidant status, and increased levels of lipid oxidation products such as isoprostanes. Cigarette smoke is the predominant etiological factor in COPD, increasing the oxidant burden significantly. This is because one puff contains approximately 1014 free radicals, and many of these radicals such as tar semiquinone, which can generate H2O2 by the Fenton reaction, are relatively long lived. Acute Lung Injury/Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a common response of the lung to diverse insults including sepsis, endotoxemia, trauma, aspiration, and pneumonia and is characterized by diffuse inflammation in the lung parenchyma. In its initial phases, acute lung injury is characterized by an acute inflammatory response with recruitment and activation of alveolar macrophages and neutrophils. ARDS patients have increased production of oxidants in their lungs as indicated by an increased concentration of H2O2 in expired air, a deficiency of glutathione and increases in the oxidized form in alveolar ELF, and potentially toxic levels of peroxynitrite. In addition, urinary F2 isoprostanes, vasoconstrictors that are markers of nonenzymatic lipid oxidation, are significantly elevated in ARDS subjects. Lung Cancer

A multistage cancer process can be influenced by environmental and endogenous oxidants at several steps. Oxidative stress has been shown to play an

important role in lung cancer as many carcinogens are free radicals, stimulate the generation of oxidants, or are the products of biological free radical reactions. Free radicals or their metabolites generated from different carcinogens may initiate carcinogenesis by direct DNA damage. In addition, byproducts of lipid peroxidation, including malondialdehyde and lipid peroxides are mutagenic and may be carcinogenic. Other than those diseases discussed above, there are many lung diseases like cystic fibrosis, infant respiratory distress syndrome, pneumonia, bronchopulmonary dysplasia, lung transplantation, primary isograft failure, mineral dust pneumoconiosis, radiation toxicity, etc., where oxidative stress plays a significant role. Since oxidative stress-related diseases are caused by an oxidant/antioxidant imbalance involving an excess of oxidants, it seems logical that these diseases might be prevented or treated by quenching excess oxidants. See also: Acute Respiratory Distress Syndrome. Apoptosis. Asthma: Overview. Cell Cycle and CellCycle Checkpoints. Chronic Obstructive Pulmonary Disease: Overview. Environmental Pollutants: Overview; Diesel Exhaust Particles; Oxidant Gases; Particulate Matter, Ultrafine Particles; Particulate and Dust Pollution, Inorganic and Organic Compounds. Hyperbaric Oxygen Therapy. Hypoxia and Hypoxemia. Interstitial Lung Disease: Overview. Nitric Oxide and Nitrogen Oxides. Occupational Diseases: Overview; Coal Workers’ Pneumoconiosis; Hard Metal Diseases – Berylliosis and Others; Inhalation Injury, Chemical; Asbestos-Related Lung Disease; Silicosis. Oncogenes and Proto-Oncogenes: Overview. Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic. Oxygen Therapy. Radiation-Induced Pulmonary Disease. Signal Transduction. Transcription Factors: Overview. Tumors, Malignant: Overview.

Further Reading Andreadis AA, Hazen SL, Comhair SAA, and Erzurum SC (2003) Role of reactive oxygen and nitrogen species (ROS/RNS) in lung injury and diseases. Oxidative and nitrosative events in asthma. Free Radical Biology and Medicine 35: 213–225. Comhair SAA and Erzurum SC (2002) Antioxidant response to oxidant-mediated lung diseases. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L246–L255. Finkel T (2003) Oxidant signals and oxidative stress. Current Opinion in Cell Biology 15: 247–254. Forman HJ and Thomas MJ (1986) Oxidant production and bactericidal activity of phagocytes. Annual Review of Physiology 48: 669–680. Kelly FJ, Dunster C, and Mudway I (2003) Air pollution and the elderly: oxidant/antioxidant issues worth consideration. European Respiratory Journal 21: 70–75.

278 OXYGEN THERAPY Lang JD, McArdle PJ, O’Reilly PJ, and Matalon S (2002) Oxidant–antioxidant balance in acute lung injury. Chest 122: 314S–320S. Marnett LJ, Riggins JN, and West JD (2003) Endogenous generation of reactive oxidants and electrophiles and their reactions with DNA and protein. Journal of Clinical Investigation 111: 583–593.

Sanders KA, Huecksteadt T, Xu P, Sturrock AB, and Hoidal JR (1999) Regulation of oxidant production in acute lung injury. Chest 116: 56s–61s. van der Vliet A and Cross CE (2000) Oxidants, nitrosants, and the lung. American Journal of Medicine 109: 398–421.

OXYGEN THERAPY C Lewis, Papworth Hospital, Cambridge, UK J Kolbe, University of Auckland, Auckland, New Zealand & 2006 Elsevier Ltd. All rights reserved.

Abstract Oxygen therapy is used widely in both respiratory and emergency medicine. Acute oxygen therapy is used empirically in cardiorespiratory arrest, hypotension, or respiratory distress. Acute oxygen is also used where hypoxia or respiratory failure is present in conditions such as pneumonia, exacerbation of airways disease, and pulmonary embolus. High inspired oxygen fractions may speed resolution of pneumothorax. Use in acute heart failure is now supplemented by continuous positive airways pressure, but in acute myocardial infarction is controversial. Use of chronic oxygen therapy in a domiciliary setting has increased since the landmark studies carried out in the 1980s, and our knowledge of the benefits of such therapy continues to draw heavily on studies of patients with chronic obstructive pulmonary disease (COPD). Long-term oxygen therapy for 15 h day
1 or more improves survival in chronically hypoxic COPD patients, and probably also improves quality of life. The use of nocturnal oxygen in patients with isolated overnight hypoxia is controversial. Ambulatory oxygen is increasingly utilized with studies showing improvement in exercise tolerance, reduced dyspnea, and improved short-term quality of life. Finally, the use of short-burst or intermittent oxygen therapy as required for relief of dyspnea does not appear justified based on current evidence.

History Oxygen was first discovered by the English chemist Joseph Priestley in 1774, but was used only sporadically as a medical treatment during the nineteenth century. Oxygen therapy came into more widespread use during and following World War I, after which appropriate delivery methods were invented for hospital use and the need for continuous supply became appreciated and available. Domiciliary oxygen therapy has been utilized for the last 50 years, initially by cylinder and subsequently by concentrator, the technology for which was invented in the 1950s but has been refined since. Initially, the benefits of domiciliary oxygen were suggested by case series and small uncontrolled studies in chronic obstructive

pulmonary disease (COPD). However, use of such therapy gained impetus following landmark randomized controlled trials carried out in the 1980s showing mortality benefits in chronically hypoxemic COPD patients. Subsequent to this, concentrators became more widely available for prescription. Further studies to widen the criteria for domiciliary oxygen therapy in COPD to less hypoxemic patients have not shown such mortality benefits. More recently, attention is focusing on the clinical utility of oxygen therapy during exercise and for relieving dyspnea, in part aided by the development of robust tools to measure health-related quality of life (HRQL).

Uses in Respiratory Medicine Acute Oxygen Therapy

In any acutely ill patient, once the airway has been secured, oxygen therapy is administered with or without artificial ventilation to prevent hypoxemia, ensure adequate oxygen delivery in conjunction with an adequate circulation, and prevent tissue hypoxia. The consequences of acute hypoxemia are described elsewhere. Clinical symptoms and signs of hypoxemia are non-specific and include tachypnea, altered conscious state, and cardiac arrhythmias. The clinical sign of central cyanosis has poor intra- and interobserver reproducibility and is even harder to interpret in the presence of anemia or polycythemia. Hypoxemia is best assessed by analysis of arterial blood gases (ABG). Oxygen saturation can be noninvasively measured and continuously monitored using cutaneous pulse oximetry. However, oximetry interpretation must be tempered by knowledge of the accuracy of pulse oximeters (73%) and the fact that it gives no information about pH or pCO2. Oxygen is indicated acutely in any patients with saturations of o90% on air or a pO2 of o8 kPa, but is often administered empirically in cardiorespiratory arrest, hypotension, or respiratory distress. As for any other drug, the dose of oxygen and administration method should be explicitly documented.

OXYGEN THERAPY 279

Oxygen therapy is usually first administered by face mask. Use of a low-flow mask (4–15 l min
1) enables an inspired oxygen fraction (FiO2) of up to 60% to be achieved. Flow rates below 4 l min
1 should not be used with these face masks, as there is a risk of rebreathing of exhaled CO2. The use of a mask with a non-rebreathing valve and reservoir circuit achieves a higher FiO2, although rarely approaches 100%. A more reliable FiO2 can be achieved with the use of a high-flow venturi valve, which blends a jet of oxygen with high flow rates of room air (Figure 1). These systems are not influenced by variations in the patient’s respiratory rate, and avoid rebreathing of exhaled CO2. Humidification of prolonged mask oxygen therapy is recommended to aid comfort and prevent drying and retention of secretions. Nasal cannulae are more comfortable and allow eating and talking, and humidification is not required. However, they cannot deliver a high or a reliable FiO2 as this depends on mixing of oxygen with inspired air in the nasopharynx (FiO2B20 þ (4  flow)). Respiratory emergencies commonly requiring acute oxygen therapy include pneumonia, asthma, pulmonary embolus, and exacerbations of chronic airflow limitation. In some patients with chronic hypercapnic respiratory failure, administration of a high FiO2 causes acute hypoventilation, worsening hypercapnia, and acute-on-chronic respiratory acidosis. Although traditionally thought to be due to abolition of the hypoxic respiratory drive on which chronically hypercapnic patients rely to maintain

ventilation, other mechanisms for the rise in pCO2 with high administered FiO2 include the Haldane effect and worsening of V/Q mismatch by loss of hypoxic vasoconstriction to more poorly ventilated areas of lung. Thus, the rise in pCO2 on initiation of oxygen therapy that occurs in some patients with chronic respiratory disease is not necessarily clinically important, is sometimes overstated, and can lead to inappropriate withholding of adequate oxygen therapy. In practice, patients with exacerbations of chronic respiratory disease should receive a controlled FiO2 of 24–28% aiming for saturations of 90%, unless in extremis when mechanical ventilation should be urgently considered. Patients should be carefully monitored for respiratory distress, respiratory rate, and level of consciousness. ABG should be measured after 30 min to help titrate therapy, and ideally rechecked after each change in FiO2. Patients who develop worsening acidosis (rather than merely an increase in pCO2) should be considered for noninvasive ventilation (NIV), which has superseded use of pharmacological respiratory stimulants. Other complications of high FiO2 administration are discussed elsewhere. It is important to remember that whilst oxygen therapy can correct hypoxemia, it does not improve ventilation. If adequate oxygenation is not achieved the addition of continuous positive airways pressure (CPAP) should be considered. If respiratory effort and pH fall or pCO2 rises, NIV or mechanical ventilation should be instituted urgently.

Figure 1 High-flow venturi mask and valve to provide 60% FiO2 set to 12 l min
1 of oxygen; valves to provide (from bottom left) FiO2 of 24% at 3 l min
1, 28% at 6 l min
1, 31% at 8 l min
1, 35% at 10 l min
1 and 40% at 12 l min
1 of oxygen respectively.

280 OXYGEN THERAPY

In acute heart failure, evidence supports the use of CPAP with oxygen, although the use of NIV remains controversial. Oxygen is often used routinely in myocardial infarction although this is not evidencebased. Oxygen at high FiO2 may speed resolution of pneumothoraces by increasing the diffusion gradient for nitrogen between the pleural place and the lung. Hyperbaric oxygen therapy is used for acute carbon monoxide poisoning, decompression sickness, and in some severe anaerobic infections. Oxygen may be used at high altitude. Some patients with respiratory disease flying on commercial aircraft require oxygen, as at cruising altitude the cabin is usually pressurized to an altitude equivalent of approximately 2500 m. Because of the reduced PiO2 and the shape of the oxyhemoglobin saturation curve, patients with adequate oxygen saturations at sea level may exhibit desaturation during flight, although the clinical importance of this is largely unknown. Prediction of desaturation during flight has proved difficult but can be assisted by a hypoxic challenge at sea level.

Chronic Oxygen Therapy

The use of domiciliary oxygen therapy is almost entirely based on studies carried out in COPD. The results of these studies are often extrapolated to guide prescription of domiciliary oxygen in other diseases causing chronic hypoxemia for which no evidence base exists. Current indications for domiciliary oxygen therapy in COPD are given in Table 1, with similar indications often pragmatically used in other diseases. Long-term oxygen therapy The current prescription of long-term oxygen therapy (LTOT) is largely based on two landmark studies carried out on hypoxic COPD patients, the Medical Research Council (MRC) and Nocturnal Oxygen Therapy Trial (NOTT) trials. The MRC study showed that use of LTOT for 15 h day
1 improved survival compared with controls. The NOTT study showed that ‘continuous’ use of LTOT (average 17.7 h day
1) improved long-term survival compared with

Table 1 Indications for domiciliary oxygen: current guidelines

Long-term oxygen therapy (LTOT)

National Institute for Clinical Excellence (UK), 2004

American Thoracic Society/ European Respiratory Society, 2004

Thoracic Society of Australia and New Zealand, 2005

Stable pO2 o7.3 kPa

Stable COPD with pO2 of o7.3 kPa; pO2 of 7.3–7.8 kPa and signs of tissue hypoxia (pulmonary hypertension, erythrocytosis, edema, or impaired mental status) Increase dose by 1 l min
1 during sleep and exercise or titrate individually using oximetry

Stable daytime resting pO2 of o7.3 kPa; pO2 7.4–7.8 kPa with evidence of hypoxic organ damage (right heart failure, polycythemia, pulmonary hypertension) Increase dose by 1 l min
1 during sleep and exercise

Stable pO2 of 7.3–7.9 kPa and polycythemia, desaturation o90% for 430% of the night, peripheral edema or pulmonary hypertension Exercise/sleep doses not discussed Nocturnal

Not discussed / recommended

Indicated if sleep desaturation not corrected by CPAP

May be indicated if nocturnal saturations o88%, for 41/3 of night or if hypoxic organ damage

Ambulatory

Patients on LTOT who wish to continue with oxygen therapy outside the home Exercise desaturation if shown to have an improvement in exercise capacity or reduction in dyspnea with oxygen therapy

Patients on LTOT unless unable/ unwilling to be mobile

LTOT patients if mobile

Indicated for exercise desaturation

May improve HRQL in patients who desaturate o88% during exercise; rapid improvements in exercise capacity and dyspnea should be demonstrated

Consider for episodes of severe dyspnea not relieved by other treatments Improvement in dyspnea with therapy should be documented

Not discussed/recommended

Not recommended unless hypoxic and life expectancy less than 3 months

Short-burst or intermittent

OXYGEN THERAPY 281

nocturnal oxygen alone. The mechanism of this mortality benefit is uncertain, although it is most likely that LTOT attenuates the adverse effects of chronic hypoxia on the pulmonary vasculature, with subsequent prevention of pulmonary hypertension and right heart failure. Current recommendations for LTOT are described in Table 1. ABG should be measured at rest on two occasions at least 3 weeks apart during clinical stability. Chronic hypoxia may adversely affect neuropsychiatric and higher function, and the NOTT study showed modest improvements in these parameters with LTOT. Placebo-controlled studies of LTOT with HRQL outcome measures would now be considered unethical, but well-performed nonrandomized studies have shown that LTOT improves HRQL. LTOT is usually supplied by a concentrator, which produces oxygen from atmospheric air by molecular sieving. Concentrators are extremely effective at rates of up to 4 l min
1, after which the FiO2 emitted falls below 90%. Nasal prongs are usually employed and long tubing allows ambulation within the home. LTOT should aim to raise the resting pO2 to above 8 kPa or the saturation to 490%. In clinically stable patients the development of hypercapnia and acidosis is much less common than with acute oxygen therapy, but if problematic, low-flow meters can be used to titrate to the lowest flow possible to obtain acceptable saturation. Some authorities recommend adjustment of dose for exercise and sleep as well as provision of ambulatory oxygen (Table 1). LTOT should be used for at least 15 h day
1, and the NOTT study suggests that benefits are maximized by use for as long as possible in each 24 h period. It is recommended that patients on LTOT be reviewed at least annually, and flow rates adjusted based on resting oximetry. Current smokers were excluded from the landmark studies and thus the benefits of LTOT are uncertain in this group. Prescription of LTOT to current smokers is usually not recommended for safety reasons. Nocturnal oxygen therapy Approximately 30–40% of COPD patients without daytime hypoxemia exhibit nocturnal desaturation, predominantly caused by reduction in minute ventilation during sleep. Desaturation may cause transient rises in pulmonary artery pressure (PAP), repeated episodes of which have in turn been hypothesized to cause sustained pulmonary hypertension and right heart disease. Portable battery-operated pulse oximeters allow adequate assessment of COPD-related overnight desaturation, and formal polysomnography is not necessary unless the clinical suspicion of coexistent sleep-disordered breathing is high. Nocturnal oxygen therapy (NOT), administered in a similar fashion to

LTOT to prevent desaturation, could potentially prevent the development of such a complications. An early study found that in suitable patients, NOT resulted in a small reduction in PAP compared to a small rise with placebo. However, a subsequent larger study found no difference in PAP or mortality between NOT treated patients, untreated controls, or patients without nocturnal desaturation. Episodes of nocturnal desaturation cause brief arousals from sleep but it remains unclear whether sleep quality is impaired by nocturnal hypoxemia in COPD or whether NOT improves short-term sleep quality. The effects of nocturnal desaturation and NOT on longer-term sleep quality and HRQL have not been studied. Thus, prescription of NOT to patients with isolated nocturnal desaturation remains controversial (Table 1). Ambulatory oxygen therapy Oxygen therapy during exercise in COPD improves oxygen delivery to tissues, reduces minute ventilation at given workload, prevents desaturation, and reduces the amount of dynamic hyperinflation. A number of studies have shown that oxygen therapy delivered during exercise in COPD improves performance and reduces breathlessness. There is no universally agreed method of assessing exertional desaturation in COPD, but the 6 min walk is most commonly used as it is easy to perform, well standardized, and reproducible after three attempts. In patients without resting hypoxemia, desaturation below 88% is considered significant and an indication for oxygen therapy during exercise by most authorities (Table 1). In patients being assessed for supply of ambulatory oxygen, it is suggested that an improvement in walking distance and/or a reduction in breathlessness during the 6 min walk should also be demonstrated. Ambulatory systems have been developed to attempt to deliver oxygen during exercise in the domiciliary setting. In practice, this usually involves patients carrying small cylinders in a portable backpack during exertion. An alternative is to use a cylinder in a small carrying device mounted on wheels and moved by the patient during exertion. Liquid oxygen systems are lighter to carry, but are more expensive and difficult to maintain. The weight of ambulatory oxygen equipment could unfortunately attenuate any clinical benefit obtained. There is conflicting evidence as to whether ambulatory oxygen improves HRQL. The largest study to date reported improvements at 6 weeks compared with placebo. However, there was no significant correlation between this acute response to oxygen and longer-term HRQL, raising doubts about the validity of basing prescription of ambulatory oxygen on the acute response and suggesting that

282 OXYGEN TOXICITY

patients should be monitored closely to ensure ongoing clinical benefit. It is not known whether ambulatory oxygen alters disease progression in patients with isolated exertional desaturation. Short-burst oxygen therapy (SBOT) SBOT refers to oxygen used intermittently for relief of breathlessness, often before and after exertion. It is usually supplied by stationary cylinders. There is generally no requirement for the patient to be hypoxic or demonstrate exertional desaturation. Thus, SBOT is the most controversial form of domiciliary oxygen therapy, as the physiological rationale for such use is questionable and psychological factors may be involved. Studies examining the acute response to SBOT, utilizing gymbased field tests and subjective measures of dyspnea such as visual analog scales, have not demonstrated convincing and reproducible benefits for SBOT over placebo for reducing dyspnea. Furthermore, availability of SBOT to nonhypoxic COPD patients at hospital discharge was not associated with long-term differences in HRQL or acute healthcare utilization. Therefore, the use of SBOT for relief of dyspnea is not supported by current evidence, despite widespread expenditure on the provision of oxygen cylinders for such use in many countries. See also: Acute Respiratory Distress Syndrome. Chronic Obstructive Pulmonary Disease: Overview. High Altitude, Physiology and Diseases. Hyperbaric Oxygen Therapy. Hypoxia and Hypoxemia. Oxygen Toxicity. Pneumothorax. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation; Ventilator-Associated Pneumonia.

Further Reading American Thoracic Society and European Respiratory Society (2004) Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position statement. European Respiratory Journal 23: 932–946.

British Medical Journal (1998) ABC of oxygen series. British Medical Journal 317: 798–801, 871–874, 935–938, 1063–1065, 1140–1143, 1213–1216, 1302–1306, 1370–1373. Chaouat A, Weitzenblum E, Kessler R, et al. (1999) A randomized trial of nocturnal oxygen therapy in chronic obstructive pulmonary disease patients. European Respiratory Journal 14: 1002–1008. Eaton T, Garrett JE, Young P, et al. (2002) Ambulatory oxygen improves quality of life in COPD patients: a randomized controlled study. European Respiratory Journal 20(2): 306–313. Fletcher EC, Luckett RA, Goodnight-White S, et al. (1992) A double-blind trial of nocturnal supplemental oxygen for sleep desaturation in patients with chronic obstructive pulmonary disease and a daytime PaO2 above 60 mm Hg. American Review of Respiratory Disease 145: 1070–1076. Gorecka D, Gorzelak K, Sliwinski P, et al. (1997) Effect of longterm oxygen on survival in patients with chronic obstructive pulmonary disease with moderate hypoxemia. Thorax 52: 674–679. Medical Research Council Working Party (1981) Long term domiciliary oxygen therapy in chronic hypoxic cor pulmonale complicating chronic bronchitis and emphysema. Lancet 681–686 National Institute for Clinical Excellence (2004) Chronic Obstructive Pulmonary Disease: Management of Chronic Obstructive Pulmonary Disease in Adults in Primary and Secondary Care. London: National Institute for Clinical Excellence. Nocturnal Oxygen Therapy Trial Group (1980) Continuous or nocturnal oxygen therapy in hypoxaemic chronic obstructive lung disease: a clinical trial. Annals of Internal Medicine 93: 391–398. Pepin J-L and Levy P (2003) Principles of oxygen therapy. In: Gibson GJ, Geddes DM, Costabel U, et al. (eds.) Respiratory Medicine, 3rd edn, pp. 502–521. Philadelphia: Saunders. Royal College of Physicians (1999) Domiciliary Oxygen Therapy Guidelines: Clinical Guidelines and Advice for Prescribers. London: Royal College of Physicians. Thoracic Society of Australia and New Zealand (2005) Adult domiciliary oxygen therapy: position statement. Medical Journal of Australia 182: 621–626.

Relevant Websites http://www.ersnet.org – European Respiratory Society. http://www.nice.org.uk – National Institute for Clinical Excellence. http://www.rcplondon.ac.uk – Royal College of Physicians of London. http://www.thoracic.org.au – Thoracic Society of Australia and New Zealand.

OXYGEN TOXICITY M-F Tsan, Georgetown University, Washington, DC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Oxygen toxicity is an iatrogenic illness caused by a high partial pressure of inspired oxygen during the course of oxygen

therapy. Oxygen is toxic because of its propensity to undergo univalent reduction leading to the generation of reactive oxygen species. Oxygen toxicity occurs when reactive oxygen species overwhelm the natural antioxidant defense system. As the lung is exposed directly to the highest partial pressure of inspired oxygen, it is the primary target organ for oxygen-induced injury. The pathogenesis of pulmonary oxygen toxicity involves complex interaction between reactive oxygen species and the antioxidant defense system as well as between reactive oxygen species and host response. The clinical syndrome of pulmonary

OXYGEN TOXICITY 283 oxygen toxicity is that of tracheobronchitis and acute respiratory distress syndrome. As there is no specific therapy for pulmonary oxygen toxicity, prevention remains the cornerstone of management.

Introduction Since the discovery of molecular oxygen by Joseph Priestley in 1775, there have been concerns regarding the potential toxicity of this molecule. In 1899, Lorrain-Smith first documented the pulmonary pathological effects associated with prolonged exposure to an increased oxygen tension. Now it is well recognized that oxygen at sufficiently high concentrations is toxic to all cells. Oxygen is toxic because of its propensity to undergo univalent reduction resulting in incompletely reduced chemical states known as reactive oxygen species (ROS). As the lung is exposed directly to the highest partial pressure of inspired oxygen, it is the primary target organ for oxygen-induced toxicity. The degree of pulmonary oxygen toxicity is proportional to the partial pressure of oxygen and the duration of exposure. Hyperbaric oxygen markedly accelerates the toxic effects of oxygen, and when the oxygen partial pressure is 2.8 atmospheres absolute (ATA) or higher, generalized seizures due to central nervous system (CNS) toxicity may occur prior to the development of serious pulmonary oxygen toxicity. Thus, while oxygen is an important therapeutic modality for patients with severe hypoxemia and a number of other clinical situations (see Hyperbaric Oxygen Therapy. Oxygen Therapy), prolonged exposure to a high partial pressure of oxygen causes primarily pulmonary injury and may exacerbate the pre-existing lung pathology.

Etiology Oxygen toxicity is an iatrogenic illness caused by a high partial pressure of oxygen during the course of oxygen therapy, and is caused by an excessive amount of ROS that overwhelms the natural antioxidant defense systems, that is, the oxygen radical hypothesis as proposed by Gershman and colleagues in 1954.

Reactive Oxygen Species The superoxide anion radical (O2
) is the first ROS in the univalent reduction pathway of O2 to water. While in the normoxic environment only 0.1–1.0% of the electrons transferred to O2 result in O2
production in most cells, the production of O2
is markedly accentuated under hyperoxic conditions. Spontaneous or enzymatic (via superoxide dismutase

(SOD)) dismutation of O2
produces hydrogen peroxide (H2O2)
þ O
2 þ O2 þ 2 H -H2 O2 þ O2

Hydrogen peroxide can also be produced directly from O2 via enzymatic bivalent reduction. Superoxide can act as an oxidant or a reductant. However, under the intracellular reducing environment, it functions primarily as an oxidant. Oxidation of the [4Fe–4S] cluster-containing dehydratases such as aconitase and 6-phosphogluconate dehydratase by O2
inactivates the enzymes and releases Fe(II) from the cluster. The released Fe(II) can then reduce H2O2 in a Fenton-type reaction producing hydroxyl radical (OHd) FeðIIÞ þ H2 O2 -FeðIIIÞ þ HOd þ OH
In addition, O2
reacts with nitric oxide (NOd) at a diffusion-limited rate producing peroxynitrite (ONOO
) d
O
2 þ NO -ONOO

The produced HOd and ONOO
are among the most potent oxidants in biological systems and are capable of oxidizing DNA, proteins, and lipids leading to tissue injury (see Oxidants and Antioxidants: Oxidants). Superoxide, because of its negative charge, does not diffuse across biological membranes readily. Thus, O2
is compartmentalized where it is produced. In the cytoplasm, O2
can be generated either nonenzymatically via autoxidation of small molecules such as thiols, hydroquinones, and catecholamines, or enzymatically by various oxidases and dehydrogenases such as xanthine oxidase and flavoprotein dehydrogenase. The mitochondrial electron transport chain is a major site of O2
production mainly via autoxidation of ubisemiquinone. The microsomal electron transport system present in the endoplasmic reticulum is also a source of O2
. In phagocytic cells such as neutrophils and macrophages, the NADPH oxidase present in the plasma membrane, when activated, produces large amounts of O2
into the phagosomes and extracellular space. The peroxisomes are an important source of intracellular H2O2. During hyperoxic exposure, O2
production is increased in the lung, initially from lung parenchymal cells and subsequently from infiltrating neutrophils and macrophages.

Antioxidant Systems Cellular defense against ROS-mediated injury includes both enzymatic and nonenzymatic antioxidant

284 OXYGEN TOXICITY

systems (see Oxidants and Antioxidants: Antioxidants, Enzymatic. Oxidants and Antioxidants: Antioxidants, Nonenzymatic). While the nonenzymatic antioxidants such as reduced glutathione (GSH), a-tocopherol (vitamin E), and ascorbic acid (vitamin C) serve an important role in cellular antioxidant defense, the enzymatic antioxidant system plays the major role in the cellular defense against oxygen toxicity. Superoxide dismutase, catalase, and GSH peroxidase constitute the main enzymatic antioxidant system. Superoxide dismutase is a family of enzymes that catalyze the dismutation of O2
to H2O2 and O2. By rapidly eliminating O2
, SOD reduces the cellular and tissue concentration of O2
, and prevents the production of OHd and ONOO
. Three forms of SOD, with distinct distribution and metal components, exist in mammalian tissues: CuZnSOD (SOD1), MnSOD (SOD2), and extracellular SOD (ECSOD, SOD3). The Cu and Zn containing SOD, CuZnSOD and ECSOD, constitute approximately 85–90% of the activity, while MnSOD constitutes approximately 10–15% of the activity in most tissues. In mammalian lungs, the level of ECSOD is quite variable among different species ranging from less than 1% (rat, cat, and dog) to about 10% (mouse and human) of the total SOD activity. The intracellular CuZnSOD is the major SOD isozyme and is located in the cytosol, nucleus, and cellular organelles such as peroxisomes. In addition to its dismutase activity, CuZnSOD has non-specific peroxidase activity, leading to potential production of HOd. It can also catalyze the nitration of tyrosyl residues by peroxynitrite. These non-specific activities make CuZnSOD potentially dangerous, especially when it is overexpressed such as in Down’s syndrome patients with trisomy 21 (CuZnSOD is located on chromosome 21), or mutated resulting in enhanced peroxidase activities such as in patients with familial amyotrophic lateral sclerosis (Lou Gehrig disease). Manganese SOD is an inducible enzyme located in mitochondria. Induction of MnSOD has been demonstrated following exposure to hyperoxia, irradiation, paraquat, tumor necrosis factor (TNF), interleukin-1 (IL-1), or endotoxin (lipopolysaccharide). Extracellular SOD is a secreted, Cu- and Zn- containing SOD distinct from intracellular CuZnSOD. It is the dominant SOD isoform in the plasma and interstitium. Catalase, which catalyzes the conversion of H2O2 to O2 and water, is located mainly in peroxisomes. Glutathione peroxidase, a selenium-containing protein, is present in the cytoplasm. It scavenges alkyl hydroperoxide and H2O2 in the presence of GSH as a cofactor. The reduced GSH is then regenerated from

the oxidized GSH (GSSG) through GSH reductase. Thus, mammalian tissues are equipped with both enzymatic and nonenzymatic antioxidant systems to dispose of intracellular as well as extracellular ROS. Pulmonary oxygen toxicity occurs when hyperoxiainduced ROS overwhelms the lung antioxidant defense systems.

Pathology The pathological changes of the lung resulting from oxygen-induced damage consist of destructive and reparative processes, and are dependent on the inspired partial pressure of oxygen and the duration of exposure. Continuous exposure to a lethal dose of oxygen, for example, 100% O2 at 1 atmosphere, leads to death of all animal species including human. However, there is considerable variation among species in susceptibility to oxygen toxicity. For example, rats die in 3 days, mice in 4–5 days, baboons in 5–7 days, and humans probably in around 7 days. Upon exposure to a lethal dose of oxygen, there is an initial phase where no pathological changes are detectable. Undoubtedly, biochemical alterations do occur during this period. This initial phase is followed by an inflammatory phase with the accumulation of platelets in the pulmonary capillary beds, and influx of neutrophils into the lung interstitium. The release of inflammatory mediators and ROS by infiltrating neutrophils markedly accelerates the endothelial injury resulting in interstitial and perivascular edema. This inflammatory phase is followed shortly by apoptotic and necrotic cell death leading to a massive destruction of endothelial and type I alveolar epithelial cells with fibrous exudation, hyaline membrane formation, intra-alveolar hemorrhage, and diffuse alveolar damage typical of the acute respiratory distress syndrome. This destructive phase usually leads to death with no evidence of reparative process, unless it is interrupted by termination of oxygen exposure. Exposure to a sublethal dose of oxygen, for example, 85% O2, leads to a similar pulmonary response. However, the initial phase lasts longer, and the inflammation and destruction are less intense. In addition, the destructive phase is superimposed with reparative processes including proliferation of interstitial cells, predominantly fibroblasts, and accumulation of monocytes and macrophages. There is also proliferation of type II alveolar epithelial cells. Continuation of oxygen exposure may lead to pulmonary fibrosis with the deposition of collagen in the interstitium and an increase in the thickness of alveolar interstitial space and the number of fibroblasts. This

OXYGEN TOXICITY 285

fibrotic reaction may also occur during the recovery period after acute, intense exposure to hyperoxia. However, total resolution of pulmonary oxygen toxicity is possible if the initial hyperoxic exposure is not overwhelming.

Clinical Features Studies of healthy volunteers have revealed that within 6 h of exposure to 90–95% O2, tracheitis develops with an impaired tracheal clearance of mucus. Following exposure to 100% O2 for 6–12 h, there was no change in alveolar–arterial oxygen gradient, pulmonary arterial pressure, total pulmonary resistance, cardiac output, or pulmonary extravascular water volume, and there were no symptoms or radiological abnormality. The earliest manifestations of pulmonary oxygen toxicity in volunteers exposed to 100% O2 for 12–24 h were substernal chest pain and nonproductive cough. In addition, systemic symptoms such as malaise, nausea, anorexia, and headache occurred. At 25 h of 100% O2 exposure, pulmonary vital capacity was reduced. At 48 h of 98% O2 exposure, decrements in static lung compliance and carbon monoxide diffusing capacity were seen. The longest reported exposure of a volunteer to 100% O2 was 110 h, and the subject developed severe dyspnea, markedly impaired pulmonary function, and acute respiratory failure. Thus, the clinical picture of acute pulmonary toxicity due to 100% O2 exposure at 1 atmosphere (1 ATA) is that of tracheobronchitis initially and acute respiratory distress syndrome subsequently. Exposure to hyperbaric oxygen increases the toxicity exponentially. At 2 ATA, pulmonary symptoms may develop as early as 3 h after exposure. At 3 ATA, generalized convulsions may occur in 3 h. These convulsions are usually self-limited and without evidence of lasting side effects when the inspired O2 fraction is decreased to normal levels. On the other hand, decreasing the oxygen concentration markedly decreases the oxygen toxicity. While exposure to 75% O2 at 1 atmosphere for 24 h produces subjective and objective indications of pulmonary oxygen toxicity, exposure to 55% O2 at 1 atmosphere for multiple days produces no significant impairment of pulmonary functions. Preterm and term neonates treated with high concentrations of oxygen and positive pressure ventilation for respiratory distress syndrome develop bronchopulmonary dysplasia, a chronic, debilitating lung disease. In the past, less discriminate use of high concentrations of inspired oxygen also caused retrolental fibroplasias leading to blindness in premature infants.

Diagnosis

The diagnosis of pulmonary oxygen toxicity depends on the correct association of non-specific symptoms and abnormal pulmonary functions in a proper clinical setting. As pulmonary oxygen toxicity develops insidiously after a variable period of exposure to a high partial pressure of oxygen, early detection requires a high degree of suspicion. Development of substernal chest pain on inspiration, tachypnea, or cough in a patient receiving oxygen therapy should alert the physician to the possibility of oxygen toxicity. Patients previously treated with bleomycin within the last 6 months, may be more susceptible to oxygen toxicity. Likewise, oxygen toxicity may exacerbate the pre-existing lung pathology. Physical examination may reveal signs of pulmonary interstitial or alveolar edema. Pulmonary function tests may show decreases in vital capacity, lung compliance, or carbon monoxide diffusing capacity as well as a widening of alveolar– arterial oxygen gradient. Serial measurements of vital capacity may be useful in monitoring patients with oxygen toxicity. The chest radiographic findings may show increased densities consistent with interstitial and alveolar edema, and diffuse alveolar damage.

Pathogenesis While considerable evidence supports the theory that ROS are responsible for oxygen toxicity, the pathogenesis of pulmonary oxygen toxicity involves complex interactions between ROS and the host. Reactive Oxygen Species and Antioxidant Enzymes

In order to cause tissue injury, the amount of ROS produced has to overwhelm the natural antioxidant defense, particularly the enzymatic antioxidant system. During hyperoxia, ROS are produced intracellularly from mitochondria, nucleus, cytoplasm, and cytoplasmic organelles, as well as extracellularly from infiltrating neutrophils. Studies using transgenic and knockout mice in which a specific antioxidant enzyme gene is enhanced or inactivated have provided important insights into the relative contribution of various ROS from different sources (Table 1). CuZnSOD and cytoplasmic ROS Homozygous CuZnSOD knockout (Sod1
/
) mice with a complete absence of CuZnSOD activity developed normally into adulthood and showed no evidence of overt oxidative damage. Upon exposure to 100% O2, Sod1
/
mice showed a survival curve virtually identical to those of the heterozygous (Sod1
/ þ ) and wild-type (Sod1 þ / þ ) littermates. These results

286 OXYGEN TOXICITY Table 1 Role of antioxidant enzymes in the host defense against pulmonary oxygen toxicity (gene knockout and transgenic mice studies) Antioxidant enzyme

Genotype

Phenotype

Resistance to hyperoxia

CuZnSOD

Sod1
/
Transgenic

Normal Down’s syndorme-like abnormalities

No change Variable

Sod2
/


Abnormal (die within 10–18 days)

Decreased

Sod2
/ þ Transgenic

Normal Normal

No change Increased

Sod3
/
Transgenic

Normal Normal

Decreased Increased

Cat
/
GPx-1
/
Transgenic

Normal Normal Normal

No change No change No change

MnSOD

ECSOD

Catalase GSH Peroxidase

indicate that CuZnSOD, despite being the major intracellular SOD isoform, is not required for normal development and survival in mice, and that O2
produced in the cytoplasm plays a limited role in the pathogenesis of pulmonary oxygen toxicity. A boy with partial monosomy 21 was found to have a markedly increased sensitivity to pulmonary oxygen toxicity. Since the Sod1 gene is located on chromosome 21 and the boy had a 55% reduction in the CuZnSOD activity of his red blood cells, it was suggested that the reduced CuZnSOD activity was responsible for his susceptibility to pulmonary oxygen toxicity. However, the missing piece of chromosome contains many genes other than Sod1. Thus, based on the observations made in Sod1
/
mice, the increased O2 sensitivity may be due to something other than the reduced level of CuZnSOD activity. CuZnSOD-transgenic mice on a CBYB6 X B6D2 genetic background showed increased levels of lung CuZnSOD (100%), GSH peroxidase, and glucose 6phosphate dehydrogenase activities, but not MnSOD or catalase activity at the age of 2.5 months. However, at 5.5 months, only an increase in CuZnSOD activity (150%) was noted. Upon exposure to 100% O2 at a reduced atmospheric pressure of 630 Torr (0.83 ATA), young (2.5 months) female, but not

Remarks

CBY6XB6D2 background: increased resistance to 100% O2 in young (2.5 months) female mice at 630 Torr and young male mice at 760 Torr B6C3 background: no change in resistance to hyperoxia Severe mitochondrial injury (in room air) in metabolically active tissues, e.g., myocardium and central nervous system neurons Decreased resistance to hyperoxia at 50% or 80% O2 SP-C promoter with expression of transgene in type II alveolar and nonciliated bronchiolar epithelial cells; marked protection at 95% O2 b-Actin promoter with expression of transgene in most lung cells; no protection at 100% O2, slight protection at 90% O2 SP-C promoter with expression of transgene in type II alveolar and nonciliated bronchiolar epithelial cells; marked protection at 100% O2 and reduced numbers of infiltrating neutrophils

male, transgenic mice showed a significant survival advantage over control mice. However, no survival advantage was noted in the old (5.5 months) transgenic mice of either sex. In contrast, at sea level (760 Torr, 1 ATA), only a slight survival advantage was noted in young male, but not female, transgenic mice. On the other hand, Sod1-transgenic mice on a B6C3 background with an 80% increase in lung CuZnSOD activity showed no increased resistance to pulmonary oxygen toxicity. MnSOD and mitochondrial ROS Homozygous MnSOD knockout (Sod2
/
) mice with no detectable MnSOD activity and no compensatory increase in CuZnSOD activity died 10–18 days after birth, and exhibited severe mitochondrial injury in metabolically active tissues such as myocardium and CNS neurons. These mice also showed a markedly increased sensitivity to oxygen toxicity even at an O2 concentration of 50% or 80%. Thus, MnSOD, despite being the minor intracellular SOD isoform, is essential for the survival of animals, and O2
produced in the mitochondria plays a pivotal role in the pathogenesis of pulmonary oxygen toxicity. The Sod2-transgenic mice generated using a human surfactant protein C (SP-C) promoter to

OXYGEN TOXICITY 287

overexpressing MnSOD in type II alveolar epithelial cells and nonciliated bronchiolar epithelial cells survived significantly longer than nontransgenic littermates in 95% O2 and showed less O2-induced pulmonary injury. On the other hand, transgenic overexpression of the Sod2 gene in all lung cells using a human b-actin promoter showed no survival advantage over nontransgenic littermates in 100% O2, and only a slight survival advantage in a 90% O2 environment. These results suggest that type II alveolar epithelial cells and nonciliated bronchiolar epithelial cells are important targets for oxygen toxicity. ECSOD and extracellular ROS Homozygous ECSOD knockout (Sod3
/
) mice with no detectable ECSOD activity were healthy for up to 14 months of age. However, when exposed to a lethal dose of O2, these mice developed severe lung injury and had a shortened survival as compared to wildtype (Sod3 þ / þ ) littermates. Thus, extracellular O2
mostly generated by infiltrating neutrophils during hyperoxic exposure plays an important role in the pathogenesis of pulmonary O2 toxicity. Transgenic mice expressing human Sod3 under the control of a human b-actin promoter showed no increase in ECSOD activity in the lungs, liver, and spleen where baseline ECSOD levels were high. However, significant increases in ECSOD activity were noted in the brain, heart, and muscle where baseline levels of the enzyme activity were low. Exposure of these transgenic mice to hyperbaric oxygen (100% O2 at 6 ATA for 25 min) paradoxically resulted in more brain toxicity and higher mortality as compared to nontransgenic littermates. Since inhibition of NOd synthase activity attenuated hyperbaric O2 toxicity, it was suggested that NOd is an important mediator of hyperbaric O2 neurotoxicity and ECSOD increases O2 neurotoxicity by inhibiting O2
-mediated scavenging of NOd. Transgenic mice expressing human Sod3 driven by the SP-C promoter, on the other hand, showed a threefold increase in lung ECSOD activity, with most of the human ECSOD being located in type II alveolar epithelial cells and nonciliated bronchiolar epithelial cells. A significant amount of human ECSOD was present in the epithelial lining fluid layer. These Sod3-transgenic mice were more resistant to 100% O2 toxicity, consistent with the important role of extracellular O2
produced mostly by infiltrating neutrophils in the pathogenesis of pulmonary oxygen toxicity. GSH peroxidase or catalase and H2O2 Homozygous catalase knockout (cat
/
) mice and GSH

peroxidase-1 knockout (GPx-1
/
) mice were no more sensitive, and GSH peroxidase-1 transgenic mice were no more resistant than control mice to 100% O2 toxicity. Thus, either catalase or GSH peroxidase alone is sufficient to dispose of H2O2 produced under hyperoxic conditions. In addition, the combination of GSH peroxidase and catalase is able to remove the increased amount of H2O2 produced in SOD transgenic mice. Reactive Oxygen Species and Host Response

Reactive oxygen species are capable of inducing the activation of intracellular signal transduction pathways and redox-sensitive transcription factors leading to subsequent gene expressions. These oxidant stress responses have protective as well as detrimental effects on the host and play an important role in the pathogenesis of pulmonary oxygen toxicity. Mitogen-activated protein-kinase (MAPK) signaling pathways Hyperoxic exposure activates all three MAPK signal transduction pathways in the lung, including the extracellular signal-regulated kinase (ERK) pathway, the c-Jun NH2-terminal kinase (JNK, or stress-activated protein kinase) pathway, and the p38 MAPK pathway. Homozygous JNK knockout (Jnk1
/
or Jnk2
/
) mice are more sensitive to oxygen toxicity than their wild-type littermates, suggesting that the JNK pathway plays a protective role against oxygen toxicity. The downstream elements of the JNK pathway responsible for the protection are not clear. Likewise, homozygous MKK3 knockout (mkk3
/
) mice are more sensitive to oxygen toxicity than their wild-type littermates. Mitogen-activated proteinkinase-kinase-3 (MKK3) is the major upstream kinase regulating the activation of the p38 MAPK pathway. Following hyperoxic exposure, the mkk3
/
mice produced increased amounts of proinflammatory cytokines, TNF-a, IL-1, and IL-6. Whether the increased cytokine production is responsible for its increased sensitivity to oxygen toxicity is not clear. Thus, both the JNK and MKK3/p38 MAPK pathways play a protective role against oxygen toxicity. Redox-sensitive transcription factors Three redoxsensitive transcription factors, nuclear factor kappa B (NF-kB), activator protein-1 (AP-1) and NF-E2related factor 2 (NRF2), have been shown to be activated in the lung under hyperoxic conditions. NF-jB The activation of NF-kB leads to the production of proinflammatory cytokines such as TNF-a, IL-1, and IL-8. The production of IL-8, a

288 OXYGEN TOXICITY

potent chemotactic factor, may in part be responsible for the influx of neutrophils leading to the intense pulmonary inflammatory response during hyperoxic exposure. TNF-a induces expression of endothelial adhesive molecules, E-selectin, contributing to the recruitment of neutrophils. TNF-a may also induce apoptosis. On the other hand, TNF-a and IL-1 can selectively induce the protective antioxidant enzyme, MnSOD. Thus, the activation of NF-kB has protective as well as detrimental effects. AP-1 The activation of AP-1 (Jun and Fos) transcription factor leads to the induction of heat shock proteins such as Hsp32. Hsp32 is the inducible heme oxygenase-1 (HO-1). It functions in the degradation of heme as well as in the cytoprotection against oxidative stress. It has been shown that adenovirus-mediated gene transfer of HO-1 to rat lungs renders the animals more resistant to oxygen toxicity, suggesting that HO-1 plays a protective role against oxygen toxicity. HO-1 knockout mice have been produced, but whether these HO-1-deficient mice are more sensitive to oxygen toxicity has not been reported. Heme oxygenase catalyzes the degradation of heme to equimolar quantities of biliverdin, carbon monoxide (CO), and iron. It has been suggested that CO may be responsible for HO-1-mediated protection against oxygen toxicity. However, contradictory results have been reported on the protective effect of CO against oxygen toxicity. The reported protection against oxygen toxicity requires pre-exposure to CO prior to oxygen exposure and the final inspired O2 concentration in the CO-treated animals appears to have been lower than that of the control animals. Thus, the mechanism responsible for HO-1-mediated protection against oxygen toxicity remains unclear. NRF2 NRF2 is a cap‘n’collar basic leucine zipper transcription factor. It plays an important role in regulating antioxidant response element (ARE)-mediated gene transcription. Homozygous NRF2 knockout (Nrf2
/
) mice are more sensitive to oxygen toxicity than their wild-type (Nrf þ / þ ) counterparts. Upon hyperoxic exposure, the expression of NRF2 and phase 2 detoxifying enzymes such as NAD(P)H:quinone oxidoreductase 1, GSH-S-transferase, UDP glycosyl transferase, GSH peroxidase-2, and HO-1 were markedly induced in Nrf þ / þ mice. The increased sensitivity of Nrf2
/
mice to oxygen toxicity was associated with significantly reduced levels of these phase 2 detoxifying enzymes. Thus, the protective effect of NRF2 against oxygen toxicity is likely mediated through the transcriptional activation of these phase 2 detoxifying enzymes in the lung.

In summary, the pathogenesis of pulmonary oxygen toxicity involves complex interactions between ROS and the antioxidant defense system, as well as between ROS and host response to oxidant stress (Figure 1).

Animal Models Much of our understanding of the pathogenesis of pulmonary oxygen toxicity comes from studies using animal models including, but not limited to, mouse, rat, rabbit, dog, pig, monkey, and baboon. Mouse and rat are the most frequently used animal models. Because of their sensitivity to oxygen toxicity and their small size, studies can be performed within a short period of time provided a sufficient number of animals is used. In addition, the mouse model has the unique advantage in that genetic manipulation is possible, providing invaluable information that cannot be obtained elsewhere. However, there is considerable species difference among various animal models. Thus, information obtained from animal models may not be applicable to humans. Monkey and baboon are phytogenetically closer to human; nonetheless, the final confirmation must come from observations made in humans.

Management and Current Therapy There is as yet no specific therapy for pulmonary oxygen toxicity. Prevention remains the cornerstone of management. The goal of oxygen therapy is to ensure the adequacy of tissue oxygenation with minimal toxicity. Thus, oxygen should be administered at the lowest possible concentrations that achieve therapeutic efficacy and for as short a period as possible. The patients should be closely monitored for adequate oxygenation through measurements of arterial oxygen tension or the use of pulse oximetry, and for signs and symptoms of oxygen toxicity such as cough, substernal chest pain, and tachypnea. In critically ill patients and those requiring assisted ventilation, these symptoms and signs may be impossible to detect. In general, inspired oxygen concentrations of up to 50% are believed to be safe for long periods of time, whereas oxygen concentrations above 60% are clearly toxic. Animal studies have shown that administration of surfactants, exogenous antioxidant enzymes such as liposome-encapsulated or polyethylene glycol-conjugated SOD and/or catalase, or activation of endogenous antioxidant enzymes may enhance oxygen tolerance and prevent the development of oxygen toxicity. However, these approaches remain experimental and there is no evidence that they work in humans.

OXYGEN TOXICITY 289 Hyperoxia

Lung

Reactive oxygen species (ROS) Antioxidants ROS

ROS

NRF2

MAPK

ERKs

JNK

p38

Phase-2 detoxifying enzymes

AP-1

NF-
B

HO-1

TNF- IL-1 IL-8

MnSOD

Neutrophils

Apoptosis and necrosis of pulmonary endothelial and epithelial cells Figure 1 The pathogenesis of pulmonary oxygen toxicity. Upon exposure to hyperoxia, reactive oxygen species (ROS) are produced in the lung in quantities that overwhelm the pulmonary antioxidant defense system. In addition to causing direct injury to the cells, ROS activate signal transduction pathways and transcription factors leading to both protective and detrimental effects. Together, they lead to apoptotic and necrotic death of pulmonary endothelial and epithelial cells.

Once the development of oxygen toxicity is suspected, the concentration of oxygen should be reduced or the therapy discontinued whenever possible to prevent further lung injury. However, it is important to bear in mind that the detrimental effects of severe tissue hypoxia may far outweigh those of pulmonary oxygen toxicity. The clinician should judiciously balance the benefits and risks of oxygen therapy. See also: Acute Respiratory Distress Syndrome. Hyperbaric Oxygen Therapy. Oxidants and Antioxidants: Antioxidants, Nonenzymatic; Oxidants. Oxygen Therapy.

Further Reading Cho HY, Jedlicka AE, Reddy SPM, et al. (2002) Role of NRF2 in protection against hyperoxic lung injury in mice. American Journal of Respiratory Cell and Molecular Biology 26: 175–182. Clark JM and Lambertsen CJ (1971) Pulmonary oxygen toxicity: a review. Pharmacological Review 23: 37–133. Clayton CE, Carraway MS, Suliman HB, et al. (2001) Inhaled carbon monoxide and hyperoxic lung injury in rats. American Journal of Physiology: Lung Cellular and Molecular Physiology 281: L949–L957.

Crapo JD (1986) Morphologic changes in pulmonary oxygen toxicity. Annual Review of Physiology 48: 721–731. Deneke SM and Fanburg BL (1980) Normobaric oxygen toxicity of the lung. New England Journal of Medicine 303: 76–86. Fridovich I (1999) Fundamental aspects of reactive oxygen species, or what’s the matter with oxygen? Annals of New York Academy of Science 893: 13–18. Kinnula VL, Crapo JD, and Raivio KO (1995) Generation and disposal of reactive oxygen metabolites in the lung. Laboratory Investigation 73: 3–19. Mantell LL and Lee PJ (2000) Signal transduction pathways in hyperoxia-induced lung cell death. Molecular Genetics and Metabolism 71: 359–370. Morse D, Ottenbein LE, Watkins S, et al. (2003) Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice. American Journal of Physiology: Lung Cellular and Molecular Physiology 285: L250–L257. Ottenbein LE, Ottenbein SL, Ifedigbo E, et al. (2003) MKK3 mitogen-activated protein kinase pathway mediates carbon monoxide-induced protection against oxidant-induced lung injury. American Journal of Pathology 163: 2555–2563. Tsan MF (2001) Superoxide dismutase and pulmonary oxygen toxicity: lessons from transgenic and knockout mice (review). International Journal of Molecular Medicine 7: 13–19. Wispe JR and Roberts RJ (1987) Molecular basis of pulmonary oxygen toxicity. Clinics in Perinatology 14: 651–666.

290 OXYGEN–HEMOGLOBIN DISSOCIATION CURVE

OXYGEN–HEMOGLOBIN DISSOCIATION CURVE R M Winslow, University of California, San Diego, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The oxygen equilibrium curve of red blood cells is the relationship between the concentration of oxygen in solution and the fractional saturation of hemoglobin. Hemoglobin is a complex protein, made up of four subunits, each one with an iron-containing pyrrole group, heme. At the center of each heme group is an iron atom, the site of ligation of one oxygen molecule. Thus each molecule of hemoglobin can reversibly bind four molecules of oxygen. The mechanism of oxygen binding and release by hemoglobin is a result of interaction of the peptide subunits. The simplest model of this interaction involves two fundamental conformations, corresponding to a low-affinity deoxyhemoglobin (T or tense) and a high-affinity oxyhemoglobin (R or relaxed). As hemoglobin binds oxygen molecules sequentially, the conformation shifts from T to R, thereby increasing the affinity, a process called ‘cooperativity’. The physiologic consequence of cooperativity is that oxygen is bound to hemoglobin until red blood cells reach tissue capillary beds where it is released and available to participate in oxidative phosphorylation.

Introduction Heme-containing proteins, common to almost all higher organisms, are oxygen transporters that make multicellular life possible. Without such a transporter, oxygen availability would be limited to simple diffusion from the environment to sites of metabolism. Thus the problem that hemoglobin solves in mammals is to transport oxygen from the lung to sites of tissue metabolism without release prior to arrival in capillary networks. This is possible because of a sigmoid (cooperative) oxygen binding curve and diffusion barriers that surround the red blood cell. Hemoglobin is a tetrameric protein contained in the red blood cell. Each subunit of approximately 16.1 kDa molecular weight contains a tetrapyrrole prosthetic group with an iron atom that binds a single molecule of oxygen when the iron is in the reduced ferrous (Fe2 þ ) state. Maintenance of ferrous iron is essential to the function of hemoglobin as an oxygen carrier, and enzyme systems are contained within the red cell to recycle oxidized ferric (Fe3 þ ) iron back to the reduced state. When iron is oxidized, an electron is lost which can promote the formation of harmful oxidative molecules. Life can be sustained, albeit at a minimal level, by as little as 5 g dl
1 of hemoglobin. In a 70 kg human, this corresponds to approximately 250 g of hemoglobin. If hemoglobin were not contained in the red cell, its

rate of turnover would be so high that the body could not replace it fast enough to maintain a minimum level. Thus, the intracellular location of hemoglobin maintains reduced hemoglobin, protects hemoglobin from breakdown and clearance, prevents tissue damage by toxic oxidants, and promotes the release of oxygen in capillaries rather than in precapillary vessels.

The Oxygen Equilibrium Curve The relationship between the fractional saturation of hemoglobin with oxygen and the oxygen physically dissolved in solution under equilibrium conditions is the familiar oxygen equilibrium curve (OEC) of hemoglobin (Figure 1). The concentration of oxygen in solution is a function of both the partial pressure of O2 ðPO2 Þ in the gas phase in equilibrium with the solution and the solubility coefficient, a : ð½O2  ¼ aPO2 ; a ¼ 2:14 ml dl
1 at 1 atm O2 pressure). Since a does not vary appreciably under physiologic conditions, oxygen concentration is commonly denoted simply as PO2 . The position of the OEC is often represented by the value P50, the PO2 at which the molecule is half-saturated with oxygen. If oxygen affinity increases, the OEC shifts left (reduced P50). If oxygen affinity decreases, the OEC shifts right (increased P50). The principal physiologic effectors of hemoglobin function within the red cell (the molecules that cause such shifts) are H þ , 2,3-diphosphoglycerate (2,3-DPG), and CO2. Other effectors, Cl
and adenosine triphosphate (ATP) also decrease O2 affinity, but their physiologic roles are minor. The OEC is very sensitive to temperature. The sigmoid shape of the hemoglobin OEC allows the normal loading and unloading of oxygen under physiologic conditions. Myoglobin, isolated a and b chains of hemoglobin, and ab subunits bind oxygen with such high affinity that no effective oxygen release could occur at tissue sites. This is illustrated in Figure 1, which compares the equilibrium curves for hemoglobin and myoglobin under identical conditions. If the oxygen tension in the human alveolus is about 90 mmHg and in resting tissues about 40 mmHg, it is apparent from the figure that at 40 mmHg, hemoglobin can release about 23% of its oxygen load. If myoglobin were the respiratory pigment in blood, PO2 in the tissues would have to reach about 16 mmHg before an equivalent amount of O2 could be released. Since the oxygen affinity of a or b hemoglobin chains (or of ab dimers)

OXYGEN–HEMOGLOBIN DISSOCIATION CURVE 291 A

1 D

C

0.8 Saturation (fraction)

E B

Effectors

0.6

2,3−DPG H+ CO2 Cl−

0.4

Temperature P50 0.2

0 0

100

50

150

PO (mmHg) 2

Figure 1 The normal whole-blood oxygen equilibrium curve. P50 is the PO2 at which hemoglobin is half-saturated with O2. The principal effectors that alter the position and shape of the curve are indicated. Note that while both the hemoglobin and myoglobin curves allow saturation of about 95% at normal PO2 (A, C), hemoglobin unloads 23% of its O2 (B) and myoglobin only 7% (D) at 40 Torr. In order for myoglobin to deliver 23% of bound O2, a venous PO2 of about 16 Torr would result in this example (point E). Reproduced with permission from Winslow RM and Vandegriff KD (1997) Oxygen–hemoglobin dissociation curve. In: Crystal RG, et al. (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 1625–1632. Philadelphia: Lippincott Williams & Wilkins, & 1997, Lippincott Williams & Wilkins.

is approximately that of myoglobin, we can see that the tetrameric structure of hemoglobin is necessary for its suitably low oxygen affinity. Although hemoglobin also acts as a buffer inside the red cell, its main function is the reciprocal transport of O2 and CO2 between tissues and lungs. The molecular properties underlying these functions are the cooperative binding of oxygen to heme sites, the Bohr effect, and the interactions of small molecules which alter oxygen binding. All these properties are closely interrelated with extraordinary intricacy.

relates hemoglobin saturation (Y) to oxygen tension (p). The constant k is the y-axis intercept. When plotted according to this equation, the major portion of the OEC falls on a straight line with slope (n) proportional to the degree of cooperativity. It was found that n for hemoglobin was about 3 and for myoglobin, devoid of cooperativity, 1. This mathematical model provides no information about the mechanism of O2 binding, but the use of Hill’s parameter, n, as an index of cooperativity has persisted in the literature. Partially Liganded Intermediates: Adair Model

Equilibrium Models of Oxygen Binding Empirical Description: Hill Model

Early physiologists observed the sigmoid nature of the OEC and concluded that oxygen binding increased during progressive oxygenation. They felt that this reflected interaction between heme sites so that oxygen binding to one site made binding at the next site more likely. They called this phenomenon ‘cooperativity’. Hill’s empirical equation Y ¼ kpn 1
Y

½1

A more general approach to the quantitation of the oxygenation of hemoglobin was made by Adair, who recognized that hemoglobin was made up of four subunits, each of which could bind a single O2 molecule. He derived equations for the stepwise oxygenation of hemoglobin: HbðO2 Þi þ O2 -HbðO2 Þiþ1

½2

where i ranges from 0 to 3 and the association equilibrium constants for the reactions could be represented: ki ¼

HbðO2 Þiþ1 ½HbðO2 Þ½O2 

½3

292 OXYGEN–HEMOGLOBIN DISSOCIATION CURVE

Combining the four resulting equations, we obtain the general Adair equation: Y¼

a1 p þ 2a2 p2 þ 3a3 p3 þ 4a4 p4 4ð1 þ a1 p þ a2 p2 þ a3 p3 þ a4 p4 Þ

Unless the affinities of the a and b chain O2 binding sites are identical, the scheme proposed by Adair is an oversimplification since a variety of intermediate molecules is possible (Figure 3). Low-temperature quenching techniques have been developed by Perrella and co-workers which verify the presence and amounts of partially-liganded intermediates. The intermediates shown in Figure 3 are, in fact, present, and their concentrations are in general agreement with the predictions from the Adair model.

½4

where Y is fractional saturation of hemoglobin with oxygen, p is the partial pressure of oxygen, and the as are related to the equilibrium constants (k, eqn [3]) such that k1 ¼ a1 and for the remaining as, ki ¼ ai =ai
1 . The new parameters (as) are called ‘Adair parameters’. One difficulty with the Adair scheme is that it is too general: unique values of the as are difficult to determine experimentally and, therefore, the ks nearly impossible because of the high degree of interdependence among the parameters. In addition, the model is structure-independent, and thus is of little help in conceptualizing structure–function relationships. Nevertheless, it is possible to describe the OEC by the Adair parameters, and this has practical utility in various calculations and models, even if the values are not unique. Using experimentally derived values for the Adair parameters of human blood, the concentration of each of the partially liganded intermediates can be calculated. The concentration of Hb(O2)3 is very low at all saturation levels (Figure 2). This is consistent with the notion that molecules undergo a conformational switch when the third O2 binds, such that binding of the fourth O2 is almost instantaneous.

Allosteric Effectors: Two-State Model

Since the elucidation of the exact structure of hemoglobin, several mechanistic theories for the influence of the allosteric effectors (e.g., H þ , 2,3-DPG, CO2, Cl
) have been proposed. Monod et al. proposed the most successful of these, based on the structural work of Perutz, who described two conformations for hemoglobin, corresponding to deoxy (T state) and oxy (R state) hemoglobin. R and T states are high and low oxygen affinity hemoglobin conformations respectively. According to the two-state model, the R and T state conformers have unique oxygen affinities. The equilibrium constants for their reactions with oxygen are KR and KT. The ratio of these constants, c¼

KR KT

½5

1 Saturation 0.8

Hb(O2)4

Fraction

0.6 Hb 0.4 Hb(O2)

Hb(O2)3

0.2

Hb(O2)2

0 0

20

40

60

80

100

PO (mmHg) 2

Figure 2 Partially liganded O2 intermediates calculated from the ‘Adair’ parameters for the stepwise oxygenation of hemoglobin [eqn 4]. Note that the triply ligated species, Hb(O2)3, is predicted to be present in very small quantities. Reproduced with permission from Winslow RM and Vandegriff KD (1997) Oxygen–haemoglobin dissociation curve. In: Crystal RG, et al. (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 1625–1632. Philadelphia: Lippincott Williams & Wilkins, & 1997, Lippinwitt Williams & Wilkins.

OXYGEN–HEMOGLOBIN DISSOCIATION CURVE 293

∗

1

∗

1

1

2 1

1

2

2 1

2

1∗

2

2

1

2

2 ∗

∗

1

1

2 ∗

2

∗ 1

1∗

2

2

1

1∗

2

∗ 2

∗

1

1∗

2 ∗

2

∗ 1

∗

2

2 ∗

∗  ∗ 2 2  ∗2

2 ∗

1

Figure 3 The possible structural varieties of partially liganded oxygenation intermediates, assuming a and b heme sites have unique affinities for O2. In this case, the T–R transition would occur as the third O2 is bound so the fourth binds almost simultaneously. Reproduced with permission from Winslow RM and Vandegriff KD (1997) Oxygen–haemoglobin dissociation curve. In: Crystal RG, et al. (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 1625–1632. Philadelphia: Lippincott Williams & Wilkins, & 1997, Lippincott Williams & Wilkins.

is about 0.01 for human hemoglobin; that is, the affinity of the R conformer is about 100 times greater than that of the T conformer. The essence of the allosteric theory is that the equilibrium between the two conformations in the deoxy state, HbR 2HbT

½6

given by the allosteric constant, L¼

½HbT ½HbR

½7

is influenced by a number of other molecules or ‘allosteric effectors’. These small molecules react with hemoglobin at nonheme sites in such a way as to stabilize the T conformation. Thus, the overall reactivity of a mixture of R and T hemoglobin molecules toward oxygen will depend on the position of the R–T equilibrium. Monod, Wyman, and Changeaux related the allosteric constant L to PO2 (p) and fractional saturation of hemoglobin with oxygen (Y): Y¼

LKT pð1 þ KT pÞ3 þ KR pð1 þ KR pÞ3 4

Lð1 þ KT pÞ þ ð1 þ KR pÞ

4

½8

Of fundamental importance in this model is that the T state is physically constrained by salt and hydrogen bonds to a much greater degree than the R state. With successive oxygenation of the heme groups, some of these bonds break, the stability of the T structure

decreases, and as a result the molecules undergo a transition to the R state with the release of the constraints and an increase in oxygen affinity. For deoxyhemoglobin the equilibrium is almost entirely on the T side. It is the shift from T to R during oxygenation which accounts for cooperativity. For normal hemoglobin, most of the molecules probably shift from T to R between binding of the second and third oxygen molecules; hence the OEC is steepest in its middle portion (maximal value of Hill’s parameter, n, see eqn [1] and Figure 1). Although the two-state model may not apply in all circumstances, it has been very useful in explaining certain properties of some abnormal (mutant) and chemically modified hemoglobins. Many of them can be viewed as structural alterations which affect the value of L by favoring either the T (low-affinity) or R (high-affinity) conformation.

Allosteric Effectors The Bohr Effect (H þ and CO2)

Bohr et al. observed that the position of the blood OEC along the abscissa was affected by changes in PO2 . That is, when pH was lowered (or PCO2 raised), the oxygen affinity of the blood decreased (increased P50). The separate effects of H þ and CO2 are qualitatively similar, but the effect of H þ (pH Bohr effect) is much stronger than the CO2 effect in lowering oxygen affinity.

294 OXYGEN–HEMOGLOBIN DISSOCIATION CURVE

According to the two-state model of hemoglobin function, H þ shifts the R–T equilibrium toward T, stabilizing that structure. The stereochemical interpretation of this phenomenon is that H þ participates in opening and closing salt bridges involving the C-terminal residues. The pKs of the imidazoles of histidine HC3(146)b and of valine Nal(1)a are lowered in oxyhemoglobin as a result of the rupture of the salt bridges in which they participate. This change in pK leads to a release of protons during the transition from T to R. Experiments with mutant and chemically modified hemoglobins suggest that these groups account for about two-thirds of the alkaline Bohr effect. 2; 3-DPG

The red cell glytolytic intermediate 2,3-DPG (and, to a lesser extent, ATP) is an allosteric effector of hemoglobin. Hyperventilation, as in such as anemia or hypoxia, lowers blood pH and favors accumulation of 2,3-DPG which, in turn, lowers oxygen affinity by its shift of the R–T equilibrium toward T. 2,3-DPG can bind to deoxyhemoglobin in a molar ratio 1 : 1 and by doing so, further constrain the T conformation, lowering oxygen affinity. Salt bridges involving the free amino groups of valine NA1(1)b and the imidazoles of histidine H21(143)b are probably the most important binding sites. In deoxyhemoglobin, 2,3-DPG lies in the central cavity of the molecule and is coordinated to the groups mentioned above, plus the a-amino groups of lysine EF6(82)b. On transition to the R structure, the valines move apart, the H helices move together, and 2,3-DPG drops out. Like the effects of the other salt bridges in the deoxy structure, 2,3-DPG increases the alkaline Bohr effect. CO2

Carbon dioxide (CO2) can bind to the a-amino groups of the N-terminal amino acids with a resultant decrease of oxygen affinity. This binding is diminished in the presence of 2,3-DPG, since both 2,3-DPG and CO2 can bind to the b chain N-terminal amino group. Such competition does not occur for the a chain N-terminus, since 2,3-DPG does not bind there.

The OEC and Oxygen Transport It is clear that the oxygen-binding behavior of hemoglobin can affect overall oxygen transport, because certain genetic alterations in the molecule may lead to polycythemia. Furthermore, an extreme left-shift in the OEC caused by severe respiratory alkalosis was an essential feature in oxygen transport on the

summit of Mount Everest. The effect of reduced O2 affinity has been shown by a number of authors to improve O2 delivery to tissues when arterial oxygenation is adequate. Experimental demonstration that changes in the position or shape of the OEC may affect oxygen transport in humans has been difficult, and therefore theoretical studies of the problem have been based on simplified models of hemoglobin oxygenation. Other known red cell properties that affect oxygen transport include the rates of binding of physiologic ligands (O2, CO2, H þ , 2,3-DPG), buffering capacity, the barrier to diffusion presented by the red cell membrane, the layer of unstirred plasma immediately surrounding it, and the hematocrit-dependent blood viscosity. O2 and CO2 Diffusion

Oxygen, CO2, and hemoglobin interact in a very complex way in blood to regulate the position and shape of the OEC. This interaction can be appreciated by a classical analysis of the diffusive transfer of O2 and CO2 in the pulmonary capillary. The actual mechanisms are discussed in greater detail elsewhere in this encyclopedia. The flux of O2 into the pulmonary capillary blood is proportional to the diffusing capacity of the lung and the difference in alveolar and capillary O2 concentrations: dðO2 Þ 100 DLO2 ¼  PAO2
PcO2  dt Vc 60

½11

where Vc is the volume of capillary blood, DLO2 is the diffusing capacity for O2 in the lung, PAO2 is the alveolar PO2, and PcO2 is the pulmonary capillary PO2 . The components of DLO2 are: 1 DLO2

¼

1 DMO2

þ

1 YO2  Vc

½12

where DMO2 is the membrane component of the diffusing capacity YO2 is the reaction rate with hemoglobin. YO2 is dependent on the hemoglobin concentration, and the rate of combination with O2. Changes in total CO2 follow an equation analogous to that for O2: dðCO2 Þ 100 DLCO2 ¼  ðPACO2
PcCO2 Þ  dt Vc 60

½13

where the definition of DLCO2 is parallel to that of DLO2 . CO2 affects O2 affinity of blood in two ways: as an allosteric effector of the hemoglobin–O2 reaction, and by its effect on intracellular pH (the

OXYGEN–HEMOGLOBIN DISSOCIATION CURVE 295

Bohr effect). The second is of greater physiologic importance. This brief overview description of the major acid– base reactions that occur in the blood demonstrate the multitude of effects that determine the actual invivo shape and position of the OEC. H þ , perhaps the strongest physiologic effector, is regulated by PCO2 and the buffering capacity of hemoglobin. Buffering by hemoglobin, in turn, is determined by hemoglobin concentration, and the degree of O2 saturation. O2 saturation is determined by the PO2 and O2 affinity of hemoglobin, and so on. Exact quantitation of these multiple effects in the determination of gas exchange by blood is extremely difficult. 2;3-DPG/Hb Molar Ratio

An increase in 2,3-DPG is usually thought to augment O2 delivery by a right shift of the OEC, but probably only in normoxia (sea level). At altitude, because of the diffusion limitation of O2 uptake in the lung, decreased O2 affinity seems to limit uptake. Whether or not this is offset by tissue unloading remains to be seen, because too few data are available to indicate how low venous PO2 can drop under hypoxic conditions. At this point it seems that the 2,3-DPG effect is a sea-level mechanism, not suited to O2 delivery at altitude. In addition to its role as an allosteric effector of the hemoglobin–O2 reactions, 2,3-DPG also lowers intracellular pH because of its effect on the Donnan equilibrium. Thus it also contributes to the Bohr effect. See also: Acid–Base Balance. Carbon Dioxide. Carbon Monoxide. Hemoglobin. Hyperbaric Oxygen Therapy. Myoglobin. Oxygen Therapy. Ventilation: Overview.

Further Reading Adair G (1925) The osmotic pressure of hemoglobin in the absence of salts. Proceedings of the Royal Society of London 109: 292– 300. Antonini E and Brunori M (1971) Hemoglobin and Myoglobin in Their Reactions with Ligands. New York: Elsevier. Arnone A (1972) X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhemoglobin. Nature 237: 146–149. Bencowitz H, Wagner P, and West J (1982) Effect of change in P50 on exercise tolerance at high altitude: a theoretical study. Journal of Applied Physiology 53(6): 1487–1495. Benesch R and Benesch RE (1967) The effect of organic phosphates from the human erythrocyte on the allosteric properties of hemoglobin. Biochemical and Biophysical Research Communications 26(2): 162–167. Bohr C, Hasselbalch K, and Krogh A (1904) U¨ber einen in biologischer Beziehung wichtigen Einfluss, den die Kohlensaure-

spannung des Blu¨tes auf dessen Saurstoffbindung. Scand Arch Physiol 16: 402–412. Bolton W and Perutz M (1970) Three dimensional fourier synthesis of horse deoxyhemoglobin at 2 Angstrom units resolution. Nature 228: 551–552. Hill A (1910) The possible effects of the aggregation of the molecules of hemoglobin on its oxygen dissociation curve. Journal of Physiology 40: 4–7. Kilmartin J, Fogg J, and Rossi-Bernardi L (1975) Identification of the high and low affinity CO2-binding sites of human haemoglobin. Nature 256: 759–761. Kilmartin J and Rossi-Bernardi L (1973) Interaction of hemoglobin with hydrogen ions, carbon dioxide, and organic phosphates. Physiological Reviews 53(4): 836–890. Lenfant C and Sullivan K (1971) Adaptation to high altitude. New England Journal of Medicine 284(23): 1298–1309. Monod J, Wyman J, and Changeaux J (1965) On the nature of allosteric transitions: a plausible model. Journal of Molecular Biology 12: 88–118. Neville J (1976) Altered haem–haem interaction and tissue-oxygen supply: a theoretical analysis. British Journal of Haematology 34: 387–395. Perrella M and Rossi-Bernardi L (1981) Measurement of CO2 equilibria: the chemical–chromatographic methods. In: Antonini E, Rossi-Bernardi L, and Chiancone E (eds.) Methods in Enzymology, pp. 487–495. New York: Academic Press. Perrella M and Rossi-Bernardi L (1994) Hemoglobin-liganded intermediates. In: Everse J, Vandegriff K, and Winslow R (eds.) Methods in Enzymology, pp. 445–460. New York: Academic Press. Roughton F, DeLand E, Kernohan J, Severinghaus J (1972) Some recent studies of the oxyhemoglobin dissociation curve of human blood under physiological conditions and the fitting of Adair equation to the standard curve. In: Astrup P and Rorth M (eds.) pp. 73–83. Copenhagen: Munksgaard. Roughton F and Forster R (1957) Relative importance of diffusion and chemical reaction rates in determining rate of exchange of gases in the human with specific reference to true diffusing capacity of pulmonary. Journal of Applied Physiology 11(2): 290– 302. Samaja M and Winslow RM (1979) The separate effects of H þ and 2,3-DPG on the oxygen equilibrium curve of human blood. British Journal of Haematology 41(3): 373–381. Wagner P (1977) Diffusion and chemical reaction in pulmonary gas exchange. Physiological Reviews 57(2): 257–312. Willford D, Hill E, and Moores W (1982) Theoretical analysis of optimal P50. Journal of Applied Physiology 52: 1043–1048. Winslow R and Anderson W (1982) The hemoglobinopathies. In: Stanbury JB, et al. (eds.) The Metabolic Basis of Inherited Disease, 5th edn., pp. 1666–1710. New York: McGraw-Hill. Winslow R, Samaja M, and West J (1984) Red cell function at extreme altitude on Mount Everest. Journal of Applied Physiology 56(1): 109–116. Winslow RM (1985) A model for red cell O2 uptake. International Journal of Clinical Monitoring and Computing 2(2): 81–93. Winslow RM, Samaja M, Winslow NJ, Rossi-Bernardi L, and Shrager RI (1983) Simulation of continuous blood O2 equilibrium curve over physiological pH, DPG, and PCO2 range. Journal of Applied Physiology 54(2): 524–529. Winslow RM, Swenberg ML, Berger RL, et al. (1997) Oxygen equilibrium curve of normal human blood and its evaluation by Adair’s equation. Journal of Biological Chemistry 252(7): 2331–2337. Winslow RM and Vandegriff KD (1997) Oxygen–haemoglobin dissociation curve. In: Crystal RG, et al. (eds.) The Lung: Scientific

296 OXYGEN–HEMOGLOBIN DISSOCIATION CURVE Foundations, 2nd edn., pp. 1625–1632. Philadelphia: Lippincott Williams & Wilkins. Woodson R, Wranne B, and Detter J (1973) Effect of increased blood oxygen affinity on work performance of rats. Journal of Clinical Investigation 52: 2717–2724.

Glossary

Cooperativity – the increase of O2 affinity with successive ligation of O2 molecules Donnan equilibrium – the equilibrium characterized by unequal distribution of diffusable ions between two solutions separated by a membrane which is impermeable to at least one of them

a2-cooperon model – a model for O2 binding to hemoglobin that specifies O2 binds preferrentially to a subunit

Heme – an iron-containing tetrapyrrole prosthetic group that binds one molecule of oxygen

Adair equation – a generalized formula to characterize binding of four molecules of O2 to hemoglobin

Myoglobin – a protein that stores O2 in muscle. Approximately the size and shape of a single hemoglobin subunit

Adair parameter – the four parameters that define the shape of the OEC according to the Adair equation

O2 affinity – the affinity of hemoglobin for oxygen, characterized by P50, the PO2 at which hemoglobin is half-saturated with O2

Allosteric effectors – small molecules such as H þ , CO2, and 2,3-DPG that influence O2 binding although they interact with hemoglobin at non-O2 binding sites Blood substitutes – solutions of O2 carriers designed to be used instead of blood Bohr effect – the shift of the OEC to the right (higher P50, lower affinity) caused by reduced pH (increased H þ ) Combinatorial switching model – a model for O2 binding to hemoglobin that specifies the discreet partially liganded intermediates

Hill parameter (n) – an index of cooperativity

Oxidation – loss of an electron. In the case of iron, Fe3 þ is the oxidized state, Fe2 þ the reduced state Prosthetic group – a non-protein group, contained within a protein. Heme is a prosthetic group Two-state model – a model for O2 binding to hemoglobin that specifies two protein conformations, T (deoxy, Tense) and R (oxy, Relaxed) a chain – one of the peptide subunits of hemoglobin b chain – one of the peptide subunits of hemoglobin

P PANBRONCHIOLITIS G R Epler, Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Diffuse panbronchiolitis is an inflammatory disease of the respiratory bronchioles with symptoms of chronic productive cough, sinusitis, and shortness of breath. The chest radiograph shows diffuse nodular opacities and the high-resolution chest computed tomography (CT) scan shows centrilobular small nodules with branching. Pulmonary function generally shows irreversible airflow obstruction. Airway infections have been the primary cause of morbidity and mortality in this disorder and may be the basis of the inflammatory response. Low-dose, longterm macrolide therapy has had an enormous impact on 10-year survival rates, which have increased from less than 15% to more than 90%. The mechanism of action of macrolides is related to decreasing mucus production, decreasing lymphocytes, and alteration of the cytokine balance. Treatment with macrolides has resulted in virtually complete cure of this serious and disabling small airway disorder.

Introduction Diffuse panbronchiolitis was first characterized in a group of 82 patients by Professor Homma in 1983. Patients have symptoms of chronic cough with sputum production, shortness of breath, and chronic sinusitis. Sputum culture grew Pseudomonas aerugionosa in more than 60% of patients, and Hemophilus influenzae, Klebsiella pneumonia, and

(a)

Streptococcus pneumoniae in a smaller percentage of patients. Continuous detection of P. aerugionosa in the sputum indicated that the patient had developed the terminal stage of the disease. In 1983, the outcome of this disorder was poor: 5-year survival was 70% and 10-year survival was only 30%. The discovery of long-term, low-dose erythromycin and other macrolides as a therapeutic agent had a dramatic improvement in survival of diffuse panbronchiolitis with an increase in the 10-year survival to more than 90%.

Epidemiology The prevalence of diffuse panbronchiolitis in Japan is about 11/100 000 with a slight male predominance. The disorder is largely restricted to Japan, although it has been reported in other Asian countries (Taiwan, Korea, China, Malaysia, Thailand, and Singapore) as well as the US, Canada, South America, and Europe. An association with specific human leukocyte antigens (HLAs) has been reported in the past, and now genes designated as C6orf37 and C6orf37OS have been identified as candidate genes for diffuse panbronchiolitis.

Pathological Findings The lesion of diffuse panbronchiolitis is limited to the respiratory and terminal bronchioles (Figure 1(a)).

(b)

Figure 1 (a) Low power micrograph shows a respiratory bronchiole distorted by an interstitial infiltrate rich in foamy macrophages with some associated chronic inflammatory cells. (b) Detail shows the prominent interstitial foamy macrophages. Courtesy of Thomas V. Colby MD, Department of Pathology, Mayo Clinic, Scottsdale, Arizona, USA.

298 PANBRONCHIOLITIS

Infiltration of lymphocytes, plasma cells, and histiocytes is seen in the thickened walls of the terminal and respiratory bronchioles. A common and distinctive feature of diffuse panbronchiolitis is peribronchiolar inflammation with interstitial foam cells (Figure 1(b)). Alveolar spaces may be overinflated. Neutrophils and activated T lymphocytes may occur in the airspaces.

Pathogenesis There are different types of inflammatory response among airway diseases that are characterized by different cascades of inflammatory cells and cytokines. For example, the inflammation of diffuse panbronchiolitis occurs as a result of chronic lower respiratory tract infection while the inflammation of chronic obstructive pulmonary disease is related to cigarette smoking. The pathogenesis of diffuse panbronchiolitis is related to neutrophilic infiltration, T lymphocytes, and cytokine interleukin-8 (IL-8). Patients with diffuse panbronchiolitis have a higher number of lavage lymphocytes and a decreased CD4/ CD8 ratio compared to individuals with bronchiectasis and healthy volunteers. Additional studies of pathogenesis indicate that bronchial lavage defensin-2 is increased in patients with active diffuse panbronchiolitis, but there is a significant decrease after macrolide treatment. Defensins are single-chain antimicrobial peptides that act as a host defense against infection. The decrease in defensin-2 may be due to improved chronic inflammation or macrolide inhibition of this peptide. The lavage concentration of defensin reflects the degree of inflammation in patients with diffuse panbronchiolitis by increased levels during active disease and decreased levels after treatment or during quiescence of the disorder. Diffuse panbronchiolitis is associated with goblet cell metaplasia and increased sputum production causing airway mucus plugging especially in the peripheral airways. The cause of the goblet cell metaplasia and degranulation may be related to neutrophilic inflammation that induces epidermal growth factor receptor expression in the bronchiolar epithelium. This growth factor is induced by inflammation of the airway epithelial cells and causes increased goblet cell activity and sputum production. Mannose-binding lectin is a pattern recognition receptor and a major component of the immune system. This lectin binds to mannose or glucosamine sugar chains present on Gram-positive or Gramnegative bacteria, yeast, and some viruses, which enables the phagocytosis of these infectious organisms. Polymorphisms of the lectin gene at codon 57

are higher among individuals with diffuse panbronchiolitis than patients with other lung diseases and healthy subjects. Low mannose-binding lectin levels result in recurrent respiratory infections and the chronic inflammation seen in patients with diffuse panbronchiolitis.

Clinical Characteristics There is a slight male predominance. The disorder develops during teenage years through to 40 years of age. Chronic cough with sputum production often exceeding 50 ml per day is the initial symptom. Shortness of breath occurs later. Crackles may occur in up to 90% of patients and wheezes are commonly heard by lung auscultation. Chronic paranasal sinusitis is a distinguishing feature occurring in 80–90% of patients. There is no relationship to cigarette smoking as the frequency of smokers and nonsmokers reflects background rates. The most common laboratory finding is persistent increase in cold agglutinins. Other laboratory features include increased leukocyte count, erythrocyte sedimentation rate, serum IgA, and a positive rheumatoid factor.

Radiographic Imaging The chest X-rays may be normal during the early stage. The majority of films show bilateral small nodular opacities diffusely throughout the lungs (Figure 2(a)). In the later stage of the disease, the films show ring-shaped opacities that suggest bronchiectasis. The high-resolution chest computed tomography (HRCT) films (Figure 2(b)) show centrilobular small nodules with branching suggesting the ‘tree-in-bud’ pattern that shows improvement after successful treatment with the macrolides (Figure 2(c)). Expiratory films often show peripheral air trapping. Bronchiectasis and large cysts connected with dilated proximal bronchi occur late in the disease process.

Pulmonary Function Testing Pulmonary function shows airflow obstruction that is sometimes severe without response to bronchodilator inhalation. The diffusing capacity is usually decreased. Hypercapnia occurs late leading to pulmonary hypertension and cor pulmonale.

Macrolide Treatment and Outcome The use of low-dose, long-term macrolide antibiotics has had a dramatic and life-saving effect on the outcome of this disease. In patients with P. aeruginosa infection, the 10-year survival has improved from

PANBRONCHIOLITIS

(a)

299

(b)

(c) Figure 2 (a) Chest X-ray showing diffuse small nodular opacities in the mid and upper lungs. (b, c) High-resolution chest computed tomography (HRCT) showing thickened airways before macrolide treatment and resolution after treatment. Courtesy of Yuji Saito MD, Fujita Health University, Toyoake, Japan.

less than 15% to more than 90%. Response to therapy usually occurs at 2–3 months and the duration of treatment is generally 2 years. There may be recurrence. The new macrolides clarithrymocyin, roxithromycin, and 14-membered ring macrolides are as effective and sometimes more effective than the traditional macrolides. The 16-membered ring macrolides do not appear to be effective. Current guidelines indicate that the first choice for treatment is still erythromycin (400 or 600 mg daily) and the second choice is clarithromycin (200 or 400 mg orally). After treatment, there is normalization of oxygenation, the forced expiratory volume in 1 s (FEV1) greatly increases, the lavage neutrophils decrease, and the lavage lymphocytes decrease. The mechanism of action of the macrolides is related to the anti-inflammatory component, which inhibits mucin secretion and decreased sputum volume as well as decreasing T lymphocytes. Clarithromycin has been shown to inhibit the twitching motility of P. aeruginosa. The macrolides may cause a shift from

Th1 cytokines to Th2 cytokines. After treatment, the lavage IL-4, IL-5, and IL-13 levels are increased and the lavage IL-2 and interferon gamma levels are decreased. In addition to diffuse panbronchiolitis, the macrolides have been shown to be effective in bronchiectasis, chronic bronchitis, sinobronchial syndromes, and for cystic fibrosis. The mechanism of action for these disorders includes inhibition of neutrophil influx, release of proinflammatory cytokines, protection of the epithelium from bioactive phospholipids, and improvement of transport of airway secretions. In summary, diffuse panbronchiolitis is an inflammatory disease of the respiratory bronchioles with symptoms of chronic productive cough, sinusitis, and shortness of breath. Airway infections have been the primary cause of morbidity and mortality in this disorder and may be the basis of the inflammatory response. Treatment with macrolides has essentially resulted in patients being cured of this serious and disabling small airway disorder.

300 PARTICLE DEPOSITION IN THE LUNG See also: Bronchiectasis. Bronchiolitis. Chemokines, CXC: IL-8. Defensins. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia.

Further Reading Epler GR and Colby TC (2001) Bronchiolitis obliterans and other bronchiolar airflow disorders. In: Campbell GD and Payne DK (eds.) Bone’s Atlas of Pulmonary and Critical Care Medicine, 2nd edn., pp. 65–76. Philadelphia: Lippincott Williams & Wilkins. Gomi K, Tokue Y, Kobayashi T, et al. (2004) Mannose-binding lectin gene polymorphism is a modulating factor in repeated respiratory infections. Chest 126: 95–99. Hiratsuka T, Mukae H, Iiboshi H, et al. (2003) Increased concentrations of human b-defensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis. Thorax 58: 425–430. Homma H, Yamanaka A, Taniomoto S, et al. (1983) Diffuse panbronchiolitis: a disease of the transitional zone of the lung. Chest 83: 63–69. Kadota J, Mukae H, Fujii T, et al. (2004) Clinical similarities and differences between human T-cell lymphotropic virus type 1associated bronchiolitis and diffuse panbronchiolitis. Chest 125: 1239–1247. Kadota J, Mukae H, Ishii H, et al. (2003) Long-term efficacy and safety of clarithromycin treatment in patients with diffuse panbronchiolitis. Respiratory Medicine 97: 844–850.

Kim JH, Jung KH, Han JH, et al. (2004) Relation of epidermal growth factor receptor expression to mucus hypersecretion in diffuse panbronchiolitis. Chest 126: 888–895. Kudoh S, Azuma A, Yamamoto M, et al. (1998) Improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin. American Journal of Respiratory and Critical Care Medicine 157: 1829–1832. Matsuzaka Y, Tounai K, Denda A, et al. (2002) Identification of novel candidate genes in the diffuse panbronchiolitis critical region of the class I human MHC. Immunogenetics 54: 301–309. Mukae H, Kadota J, Kohno S, et al. (1995) Increase in activated CD8 þ cells in bronchoalveolar lavage fluid in patients with diffuse panbronchiolitis. American Journal of Respiratory and Critical Care Medicine 152: 613–618. Park SJ, Lee YC, Rhee YK, and Lee HB (2004) The effect of longterm treatment with erythromycin on Th1 and Th2 cytokines in diffuse panbronchiolitis. Biochemical Biophysical Research Communications 324: 114–117. Poletti V, Chilosi M, Casoni G, and Colby TV (2004) Diffuse panbronchiolitis. Sarcoidosis Vasculitis and Diffuse Lung Diseases 21: 94–104. Rubin BK and Henke MO (2004) Immunomodulatory activity and effectiveness of macrolides in chronic airway disease. Chest 125(supplement 2): 70S–78S. Tamaoki J (2004) The effects of macrolides on inflammatory cells. Chest 125(supplement 2): 41S–51S. Wozniak DJ and Keyser R (2004) Effects of subinhibitory concentrations of macrolide antibiotics on Pseudomonas aeruginosa. Chest 235(supplement 2): 62S–69S.

PARTICLE DEPOSITION IN THE LUNG C Darquenne, University of California – San Diego, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The deposition of aerosol in the lung is an important issue regardless of whether the aerosol results from airborne pollutants or aerosol therapy. The specific pattern of deposition of airborne pollutants is a determinant in the development of pulmonary diseases since this pattern determines the local doses in the airspaces and the subsequent redistribution and clearance of the deposited particles. Aerosol therapy allows drugs to be directly administered to injured lung tissues, where they can act more efficiently and rapidly than when administered by injection or ingestion. Deposition of inhaled aerosols can also be used as a diagnostic tool to noninvasively detect changes in the dimensions of airways and alveoli that are critically associated with the development of pulmonary pathologies such as emphysema, asthma, or bronchitis. The deposition of inhaled particles in the lung is primarily governed by the mechanisms of inertial impaction, gravitational sedimentation, Brownian diffusion, and, to a lesser extent, the mechanisms of electrostatic precipitation and interception. Deposition is minimal for particles in the size range 0.4–0.7 mm that have minimal intrinsic mobility. Deposition of larger particles results from significant gravitational and inertial transport,

whereas deposition of smaller particles is mainly due to diffusive transport. Apart from particle size, several factors affect particle deposition, including particle characteristics, physiological and environmental factors, and lung anatomy.

Description The lung is a prime target for a wide variety of airborne particles. When inhaled, these particles are transported into the lung, where they can eventually deposit onto airway and/or alveolar walls. The specific pattern of particle deposition throughout the respiratory tract is an important factor in the development of pulmonary diseases since this pattern determines the local doses in the airspaces and the subsequent redistribution and clearance of the deposited particles. Airborne particles can accumulate in offices, industrial areas, and residential atmospheres in concentrations large enough to pose a potential health hazard to people. Aerosols are also encountered in medical applications for therapeutic, diagnostic, and experimental purposes. Aerosol therapy allows drugs to be directly administered to injured lung tissues, where they can

PARTICLE DEPOSITION IN THE LUNG 301

act more efficiently and rapidly than if they were administered by injection or ingestion. Deposition of inhaled aerosols can also be used as a diagnostic tool to noninvasively detect changes in the dimensions of airways and alveoli that are critically associated with the development of pulmonary pathologies such as emphysema, asthma, or bronchitis. These diagnostic techniques are based on the assumption that the rate of particle loss by gravitational sedimentation during breath-holding periods is directly related to the airspaces containing the particles. The aerosol bolus technique can be used to study regional deposition. It uses monodisperse particles confined to a small volume injected into an inspiration of pure air. Depending on the position of the bolus in the inspired air, particles may penetrate to different volumetric depths in the lung. Deposition is then determined by the difference between the number of inspired particles and that expired during the subsequent expiration. Finally, the deposition of radiolabeled or fluorescent aerosols can also be used as an alternative means to visualize ventilation distribution in different parts of the lung. This latter technique is based on the assumption that the aerosol deposited in different lung units is in direct proportion to the ventilation in these units.

Deposition Mechanisms There are several mechanisms by which particles can be deposited in the respiratory tract (Figure 1). The three main mechanisms are inertial impaction, gravitational sedimentation, and Brownian diffusion. Deposition may also occur by other less important mechanisms, such as electrostatic precipitation and, in the case of elongated particles (fibers), interception. Inertial Impaction

Inhaled particles follow a complex path through the respiratory tract. Each time the flow changes direction, the momentum of the particles tends to keep them on their existing trajectories, causing them to deviate from the air streamlines and to eventually impact on airway walls. The deviation of the particle from the air streamline is greater the larger the particle mass and the greater the flow rate. Inertial impaction is the primary mechanism of deposition for particles larger than 5 mm in diameter and is an important deposition mechanism for particles as small as 2 mm. The most likely sites of deposition by inertial impaction are in the complex geometry of the upper respiratory tract (that extends from the nasal and mouth cavities to the entrance of the trachea) and at the conducting airway bifurcations.

Upper respiratory tract Impaction

Conducting airways

Impaction

− Electrostatic +− −

precipitation

Sedimentation Interception

Diffusion Diffusion

Acinus

Figure 1 Mechanisms of deposition of inhaled particles in the respiratory tract. The mechanisms of deposition are not restricted to the zone of the lung as shown in the figure. For example, deposition by sedimentation can also occur in the acinus (see text for details).

The probability that a particle will diverge from the streamlines of the carrier gas can be expressed as a function of the Stokes’ number (Stk), defined by Stk ¼ ðrp dp2 uÞ=ð18mdÞ

½1


where dp and rp are the particle diameter and density, respectively; u and m are the linear velocity and dynamic viscosity of the carrier gas, respectively; and d is a characteristic length equal to the diameter of the airspace. The higher the Stokes’ number, the more efficient the inertial transport and the more likely that particles will deposit by inertial impaction. Gravitational Sedimentation

The mechanism of gravitational sedimentation refers to the settling of particles under the action of gravity. Particles reach their terminal settling velocity when the gravitational force equals the opposing viscous resistive forces of the air. Particles in the respirable range (o10 mm) reach their terminal velocity in less than 1 ms, a negligible fraction of the typical airflow

302 PARTICLE DEPOSITION IN THE LUNG

transit time in any generation of the respiratory tract. The terminal settling velocity for a spherical sphere is expressed by vs ¼

rp dp2 18m

g

½2


Charged particles then become electrostatically attracted to the airway walls, and as a consequence, deposition of charged particles may be greater than that of neutral particles. Deposition by electrostatic precipitation is small, however, contributing to less than 10% of overall deposition.

where g is gravitational acceleration. The probability that a particle will deposit by gravitational sedimentation is proportional to vs. Therefore, probability increases with increasing particle size and density. Such probability also increases with increasing residence time in the airways. Although deposition by gravitational sedimentation can also occur in the upper respiratory tract, it is most important in the small airways and alveoli. Gravitational sedimentation is the dominant mechanism of deposition for particles with a diameter ranging between 0.5 and 5 mm. Larger particles deposit mainly by inertial impaction, whereas smaller particles are predominantly affected by Brownian diffusion.

Interception

Brownian Diffusion

Most of the studies investigating total deposition in human lungs have been performed on healthy subjects with monodisperse, neutral, nonhygroscopic particles. All studies show the same trend in the extent of deposition as a function of particle size. A typical curve is shown in Figure 2. Particles of unit density in the size range 0.4–0.7 mm present minimum intrinsic mobility and none of the mechanisms of impaction, sedimentation, and diffusion are effective in particle transport. Consequently, these particles have minimum deposition in the respiratory tract because their intrapulmonary trajectories have the smallest deviations from the air streamlines. For

DB ¼ ðckTÞ=ð3pmdp Þ

½3


where k is Boltzmann’s constant, T is the absolute temperature, and c is the Cunningham correction factor that accounts for the decreased air resistance caused by slippage when the particle diameter comes close to the mean free path of the gas molecules so that the particle no longer moves as a continuum in the gas but as a particle among discrete gas molecules. Unlike impaction and sedimentation, which increase with increasing particle size, deposition by Brownian diffusion increases with decreasing particle size and becomes the dominant mechanism of deposition for particles less than 0.5 mm in diameter. Deposition by Brownian diffusion occurs mainly in the acinar region of the lung, although for very small particles (dpo0.01 mm), deposition by diffusion is also significant in the nose, mouth, and pharyngeal airways. Electrostatic Precipitation

Deposition by electrostatic precipitation results from charged particles inducing image charges of opposite sign onto the surfaces of the airways that are electrically conducting while normally uncharged.

Particle Deposition in the Healthy Lung

100 Total deposition, DE (%)

Deposition by Brownian diffusion results from the random motions of the particles caused by their collisions with gas molecules. The diffusion rate is proportional to the Brownian diffusion coefficient

Interception refers to the situation in which, in the absence of the deposition mechanisms described previously, the center of gravity of an elongated particle is in the gas phase while one of its ends touches an airway or alveolar wall. Such a situation is more likely to occur when the dimensions of the particle are comparable to those of the airspaces. Deposition by interception is usually significant only for particles such as fibers for which the ratio between length and diameter is large. Interception is more likely to occur in small airways and alveoli.

80 60 40 20 0 0.001

0.01 0.1 1 Particle diameter, dp (µm)

10

Figure 2 Typical curve showing the effect of particle size on total deposition in the human lung. Total deposition is expressed as a percentage of the total number of inhaled particles.

PARTICLE DEPOSITION IN THE LUNG 303

particles larger than 0.7 mm in diameter, total deposition increases with increasing particle size because of increased gravitational and inertial transport. For particles smaller than 0.4 mm in diameter, total deposition increases with decreasing particle size because of increased diffusive transport. Although particle size is probably the major factor affecting particle deposition in the lung, several other factors influence not only the total deposition of particles but also their regional deposition. These factors can be classified into four main categories: particle characteristics, physiological factors, lung anatomy, and environmental factors. Apart from its size, a particle is characterized by its density, shape, electrostatic charge, and hygroscopic properties. Particle density affects deposition by both impaction and sedimentation but has no effect on diffusive transport. Shape can have a profound effect on the aerodynamic behavior of the particles and therefore on deposition efficiency. Because the relative humidity in most of the lung is close to 100%, hygroscopic growth may significantly alter total and also regional deposition compared to the patterns observed with nonhygroscopic stable particles. Physiological factors affecting deposition include breathing patterns, breathing frequency, tidal volume, functional residual capacity, airflow dynamics, and breathing pathway (oral versus nasal). Increasing airflow rates increase the efficiency of the velocitydependent deposition mechanism (impaction) and decrease that of time-dependent deposition mechanisms (sedimentation and diffusion). Increasing tidal volumes allow particles to reach more distal regions of the lung, where deposition by diffusion and sedimentation are likely to occur, therefore increasing their relative contribution to overall deposition compared to deposition by impaction. During exercise, both flow rates and tidal volumes are increased, leading to higher deposition by impaction in the large airways and by sedimentation and diffusion in the small airways and alveoli. Finally, the nasal route is more efficient at filtering particles than the oral route. Therefore, mouth breathers will tend to deposit more particles in their lungs than nose breathers. Anatomical factors such as airway length, airway diameter, branching angles, and alveolar size also play a key role in particle deposition. Because of intersubject variability, there is a large coefficient of variation in deposition among normal healthy subjects breathing in the same manner. Even within the same individual, the dimensions of the respiratory tract change with lung volume, age, and pathological processes. Gender differences in lung structure also cause variations in deposition between men and women. For example, the average female thorax is

smaller than in men and the volume of the conducting airways is only approximately 75% that in men. Deposition in the tracheobronchial tree is therefore higher in women than in men. However, when these volume differences are combined with smaller resting minute ventilation and flow rates observed in women, total deposition is lower in women than in men mainly because of a smaller penetration of the aerosol in the alveolar region of the female lung. Airway geometry and breathing conditions evolve from birth to adulthood. These changes affect particle deposition throughout childhood. Although there are few studies on particle deposition in children, they all show that deposition in children tends to be higher than in adults by an average factor of 1.5. More important, the number of particles deposited per surface area is increased by a factor of 4 or 5 in children because of their smaller lung surface area compared to that of adults. Finally, environmental factors such as temperature and humidity are likely to affect particle deposition. However, there is little information on the effect of these factors on particle deposition, although ambient humidity can greatly affect hygroscopic particles and therefore their deposition efficiency. Another environmental factor is gravity. Studies performed in the absence of gravity during parabolic flights have shown that deposition in the size range 0.5–3 mm was greatly reduced compared to deposition in normal gravity. These studies also suggested that although reduced, particle deposition in the periphery of the lung is enhanced compared to that with normal gravity.

Particle Deposition in the Diseased Lung There are many factors that alter the deposition pattern of inhaled particles in the diseased lung. These factors, which include airway obstruction, alteration in alveolar dimensions, and changes in breathing patterns, influence the distribution of inspired particles and therefore their potential site of deposition. Obstruction of airways observed in bronchitis, cystic fibrosis, lung cancer, and emphysema tends to divert most of the flow to nonobstructed healthy airways that are increasingly exposed to inspired particles, reducing aerosol exposure and therefore deposition in the alveolar spaces subtended by the obstructed airways. Narrowing of the airways by inflammation, mucus, or bronchial constriction increases the local velocities of the airflow, enhancing deposition by inertial impaction even for small particles. Most lung diseases seem to increase central deposition at the expense of alveolar deposition. Positive correlations have been found between the depth of deposition

304 PECTUS EXCAVATUM

within the respiratory tract and the forced expiratory volume, suggesting decreased peripheral deposition in patients with airway obstruction. See also: Aerosols. Bronchodilators: Anticholinergic Agents; Beta Agonists. Cilia and Mucociliary Clearance. Corticosteroids: Therapy. Environmental Pollutants: Overview; Diesel Exhaust Particles; Particulate Matter, Ultrafine Particles. Eotaxins. Occupational Diseases: Overview; Coal Workers’ Pneumoconiosis; Hard Metal Diseases – Berylliosis and Others; Asbestos-Related Lung Disease; Silicosis. Space, Respiratory System.

International Commission on Radiological Protection (1994) Human respiratory tract model for radiological protection: a report of the task group of the International Commission on Radiological Protection. ICRP Publication 66. Annals of the ICRP 24: 36–53, 231–299. Morrow PE (1986) Factors determining hygroscopic aerosol deposition in airways. Physiological Reviews 66: 330–376. Schulz H, Brand P, and Heyder J (2000) Particle deposition in the respiratory tract. In: Gehr P and Heyder J (eds.) Particle–Lung Interactions, pp. 229–290. New York: Dekker.

Nomenclature c

Cunningham correction factor (dimensionless)

d

airway diameter (cm)

DB

coefficient (cm2 s
1)

dp

particle diameter (cm)

g

gravitational acceleration ( ¼ 981 cm s
2)

k

Boltzmann’s constant ( ¼ 1.38  10
16 g cm2 s
2 K
1)

Stk

Stokes’ number (dimensionless)

T

absolute temperature of air (K)

u

mean linear velocity in airway (cm s
1)

vs

terminal settling velocity (cm s
1)

m

dynamic viscosity of air (g cm
1 s
1)

rp

particle density (g cm
3)

Further Reading Blanchard JD (1996a) Aerosol bolus dispersion and aerosol-derived airway morphometry: assessment of lung pathology and response to therapy, Part 1. Journal of Aerosol Medicine 9: 183–205. Blanchard JD (1996b) Aerosol bolus dispersion and aerosol-derived airway morphometry: assessment of lung pathology and response to therapy, Part 2. Journal of Aerosol Medicine 9: 453–476. Brain JD and Valberg PA (1979) Deposition of aerosol in the respiratory tract1–3. American Review of Respiratory Disease 120: 1325–1373. Brain JD, Valberg PA, and Sneddon S (1985) Mechanisms of aerosol deposition and clearance. In: Newhouse MF and Dolovich MB (eds.) Aerosols in Medicine: Principles, Diagnosis and Therapy, pp. 123–147. New York: Elsevier. Darquenne C and Paiva M (1998) Gas and particle transport in the lung. In: Hlastala MP and Robertson HT (eds.) Complexity in Structure and Function of the Lung, pp. 297–323. New York: Dekker. Heyder J, Gebhart J, Rudolf G, Schiller CF, and Stahlhofen W (1986) Deposition of particles in the human respiratory tract in the size range 0.0005–15 mm. Journal of Aerosol Science 17: 811–825.

of

Brownian

diffusion

PECTUS EXCAVATUM E W Fonkalsrud and C B Cooper, UCLA School of Medicine, Los Angeles, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pectus excavatum (PE) is one of the most common major anomalies of childhood. Males are affected six times more often than females. PE is usually recognized in infancy, becomes more severe with adolescent growth, and remains constant throughout adult life. Symptoms are infrequent during early childhood, but become increasingly severe during adolescence causing fatigue, dyspnea, decreased endurance, chest pain, tachypnea, and tachycardia. The heart is variably deviated into the left chest causing reduction in stroke volume. Pulmonary expansion is confined, causing a restrictive defect. Repair is recommended for symptomatic patients who have an elevated pectus severity index. Open repair with

minimal cartilage resection is usually performed after the age of 10 years, optimally between 12 and 19 years. Adults can be repaired with similarly good results. The open technique removes minimal cartilage routinely using a temporary internal support bar for 6 months. Pain is mild and complications are rare; 97% of patients experience a good to excellent result. A new minimally invasive Nuss repair avoids cartilage resection and takes less operating time, but is associated with severe pain, longer hospitalization, and more complications, with the bar remaining for 2 or more years. This technique is less applicable to older patients and those with asymmetric deformities.

Introduction Pectus excavatum (PE) is the most common congenital malformation of the thorax and is characterized

PECTUS EXCAVATUM 305

by a depression of the body and occasionally the manubrium of the sternum. The depression commonly extends inferiorly from the level of the second costal cartilage, with the deepest point at the junction with the xiphoid. Four to six of the seven costal cartilages which attach to the sternum depress posteriorly. More than half of the deformities are asymmetric with the concavity being deeper on the right side, and the sternum tilted downward to the right. The heart is variably deviated into the left chest. The bony ribs lateral to the costochondrial junctions on the right are often depressed downward into the concavity in asymmetric deformities. The chest wall characteristically has a decreased anterior to posterior diameter.

Etiology The pathogenesis of PE is unclear; however, many believe that an unbalanced overgrowth of the costal cartilages particularly during the early adolescent years of rapid skeletal growth pushes the sternum posteriorly (excavatum) or anteriorly (carinatum), or causes the cartilages to buckle outward. PE is inherited through either parent, although not clearly as a recessive trait. The anomaly occurs in approximately one in 300 Caucasian male births, but is six times less frequent in females. PE is uncommon in Blacks, Latinos, and Asians. Resected cartilage segments occasionally show a disorderly arrangement of cells. Other malformations may coexist, especially musculoskeletal anomalies including scoliosis (approximately 60%) and rarely Marfan’s syndrome, Ehlers–Danlos syndrome, Klippel–Feil syndrome, clubfoot or syndactylism. PE is usually apparent soon after birth, progresses slowly during childhood, becomes more pronounced during adolescence, and persists throughout adulthood. Regression rarely occurs spontaneously.

Clinical Features Symptoms are infrequent during early childhood, apart from the concern about cosmetic appearance. During adolescent years when the deformity characteristically becomes more severe and patients become more involved in competitive sports, dyspnea, reduced endurance, anterior chest discomfort, tachycardia, palpitations, and tachypnea often limit participation in physical activities. With severe sternal depression, the heart is deviated into the left chest which causes varying degrees of reduction in stroke volume and cardiac output. Reduced intrathoracic volume limits pulmonary expansion during inspiration. Approximately one-fourth have an

increased frequency of respiratory infections or asthma. The deformity clearly is physiologically restrictive and is not merely a cosmetic concern.

Evaluation The severity of PE deformities can be calculated by dividing the inner width of the chest at the widest point by the distance between the posterior surface of the sternum and the anterior surface of the spine as determined on computed tomography (CT) scans or chest radiographs (Figure 1). The mean severity index for normal persons is 2.52; the mean index of patients who undergo PE repair is 4.3. Other methods for determining the severity of PE deformities are more complex, less helpful, and often more expensive. Despite the diminished volume of the thorax in patients with severe PE, standard pulmonary function tests at rest are often either normal or show a mild restrictive defect. It is difficult to obtain reliable measurements in patients under 8 years of age. Most symptomatic PE patients will use more extensive phrenic excursions which utilize more energy during exercise than persons with a normal chest; this may explain in part the early fatigue and tiredness with physical activity. Many studies performed over the past four decades have failed to document consistent improvement in pulmonary function at rest after surgical repair of PE, despite considerable symptomatic improvement. Studies performed under exercise condition, however, have shown that the maximum voluntary ventilation, maximum oxygen utilization, total lung capacity, and total exercise time are improved significantly after repair of severe PE. Electrocardiographic abnormalities are common in PE patients, consisting primarily of right axis deviation and depressed ST segments, which reflect rotation of the heart within the thorax rather than an intrinsic abnormality. Compression of the right ventricular outflow tract may occasionally cause a functional systolic cardiac murmur along the upper left sternal border. Echocardiograms may demonstrate mitral valve prolapse in approximately 15%, particularly in patients with Marfan’s syndrome. Measurements of cardiac output using right ventricular catheterization have demonstrated diminished stroke volume and cardiac output in PE patients during upright exercise. These findings are not demonstrable in patients tested supine without exercise and may account for some of the reports of no physiologic impairment in PE patients. Angiocardiograms may show compression of the right ventricular outflow tract and right ventricle. In occasional patients this compression is reflected

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Figure 1 CT scan of chest from 21-year-old male with very severe PE (severity index 14.2). The heart is deviated markedly into the left chest by the depressed sternum, with compression of the right ventricle. The volume of the lungs is reduced from compression by the displaced heart, and the very reduced volume of the thoracic cavity.

physiologically in right ventricular catheterization measurements and pressure waves similar to those of constrictive pericarditis. Cardiac output and stroke volume have been demonstrated to be considerably improved after repair. These observations support the hypothesis that both a restrictive cardiac stroke volume and the increased work of breathing described in several PE patients may be ameliorated by operative repair of the anomaly, thus improving exercise tolerance in most patients.

Treatment In past years, most surgeons recommended that children with moderate to severe PE undergo repair between the ages of 3 and 10 years, despite the absence of symptoms. Although technically easier than in older patients, early repair of PE using techniques that resect a major portion of the deformed costal cartilages has been discouraged during recent years because this may interfere with the rib growth plates and produce a narrow chest, which may cause constriction and reduce the thoracic volume. Furthermore, accelerated costal cartilage growth during adolescent years often causes a recurrent deformity. The optimal age for repair appears to be from 12 through 19 years, although adults with persistent PE extending into the fifth decade have achieved good results following repair. The use of autologous tissue, silastic, or other prosthetic materials to fill a severe

PE deformity provides no physiologic benefit to the patient, often migrates causing further cosmetic deformity, and has little place in the primary management of PE deformities.

Surgical Repair Open repair for PE may be performed using modifications of the original procedures described by Ravitch, Welch, and others, with minimal cartilage resection as described recently, or by a minimally invasive correction of the deformity without costal cartilage resection as described by Nuss. Maintenance of the elevated sternum in the corrected position by external traction using harnesses or other cumbersome devices has almost universally been abandoned in favor of various methods of internal fixation. The use of autologous tissue or other supporting techniques has not been consistent in providing long-term stability of the chest wall in the desired position. Internal support of the anterior chest with a temporary steel strut after repair of PE minimizes the occurrence of postoperative respiratory distress caused by paradoxical chest wall motion, reduces pain, permits early ambulation, permits deeper respirations, reduces hospitalization and cost, and maximizes the extent to which the defect is permanently corrected. Open repair of PE with minimal cartilage resection is performed under general anesthesia with

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endotracheal intubation. Through a chevron inframammary incision with short midline extension superiorly, the pectoralis muscles are reflected laterally and the abdominal muscles are mobilized inferiorly just sufficient to expose the deformed costal cartilages. Short incisions are made through the perichondrium of the deformed cartilages adjacent to the sternum and laterally at the level where the chest wall assumes a near-normal contour. Short segments of cartilage are resected medially and laterally while carefully preserving the perichondrium. The xiphoid and the lower two perichondrial sheaths are detached from the lower sternum and the retrosternal space is mobilized with cautery for 4–5 cm. A transverse wedge osteotomy is made across the anterior table of the sternum at the level where the sternum angles to depress posteriorly. The posterior table of the sternum is gently fractured at the osteotomy without detachment and the lower end of the sternum is then twisted and elevated to the desired position. A thin stainless steel strut is placed across the lower anterior chest posterior to both the sternum and costal cartilages on each side to elevate the anterior chest to the desired level. Additional short segments of cartilage are resected as necessary to assure that the costal cartilages barely touch the sternum and ribs laterally without pressure. The xiphoid and costal cartilages are sutured back to the sternum and the lateral ends of the costal cartilages are sutured to the ribs laterally to provide immediate stability. The pectoralis and abdominal muscles are approximated together and the skin is closed with subcuticular stitches. Postoperative pain is mild for most patients and is managed with intravenous patient-controlled analgesics for 48 h and then by oral non-narcotic medications thereafter. The mean duration of operation is less than 3 h. Hospitalization is rarely more than 3 days. Over 90% of patients return to school or work within 2 weeks. The chest should be protected from direct trauma, twisting movements of the chest, or rapid elevation of the arms in sporting activities for 4 months. The strut is removed 6 months following repair on an outpatient basis, the removal rarely taking more than 15 min. A new minimally invasive technique for repair of PE (MIRPE) has been reported for preschool children. The MIRPE avoids cartilage resection and sternal osteotomy by placing a preformed convex steel bar through the anterior chest between the sternum and heart through bilateral thoracic incisions. The bar is placed with the concave side anteriorly, and then it is turned over with pliers so that the concavity faces posteriorly, thereby raising the sternum and anterior chest to the desired position. The bar is

secured to the lateral chest wall with heavy sutures. The operation rarely takes more than 90 min, and blood loss is rarely more than 80 ml. The bar is left in position for 2 or more years depending on the age of the patient and the severity of the deformity. Removal of the bar is technically more complex than removal of the strut used with the open repair.

Results Following Repair Long-term follow-up following open repair indicates that 97% of patients have experienced a very good to excellent result. There is virtually no mortality associated with repair of PE, and complications are uncommon and usually of minor severity such that treatment is rarely necessary. There is a tendency for mild to moderate recurrence of PE in approximately 15% of patients who are repaired during the first 8 years of life as a result of accelerated costal cartilage growth during the adolescent period of rapid skeletal growth. Recurrence is uncommon (less than 2%) in patients repaired at age 13 or older. Reports of favorable clinical experience with the MIRPE have included primarily young children. In recent years, the MIRPE has been extended to older patients although the complications of postoperative pain are higher, since the force necessary to elevate the chest frequently exceeds 20 kg in adolescents and adults. The MIRPE is unlikely to produce a good result in patients with asymmetric PE. Reports comparing the open repair and the MIRPE indicate that although the operating time is shorter, the length of hospitalization, severity of postoperative pain, complication rate, need for reoperations, and limitation in activity are worse than with the open repair. Longterm follow-up through adolescent years will be necessary to make a valid comparison between the two techniques. The open repair is very versatile, and applicable to patients of all ages and with all types of deformities including those with recurrent defects. Following repair, patients experience improvement in exercise tolerance with much less dyspnea, and increased endurance in most cases. Almost all patients with activity-induced chest pain will note improvement within 4 months. Tachycardia and/or palpitations are frequently reduced. Asthmatic patients often experience fewer episodes of wheezing and a decreased requirement for medications. Almost all patients indicate marked improvement in selfimage and quality of life (Figures 2 and 3). Many patients who do not undergo repair of severe PE in childhood will experience worsening symptoms in adult life. Those adults who have symptoms and activity limitations related to the PE deformity can undergo repair with low morbidity,

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Figure 2 (a) 17-year-old male with a pectus severity index of 5.6; (b) postoperative appearance 2 months after removal of the sternal bar.

Figure 3 (a) 15-year-old boy with a pectus severity index of 4.9; (b) chest appearance 7 months after operation.

short limitations of activity, and a high frequency of symptomatic improvement using the open technique for repair. See also: Chest Wall Abnormalities. Extracellular Matrix: Elastin and Microfibrils. Surgery: Pectus Carinatum, Poland’s Syndrome, Cleft Sternum, and Acquired Restrictive Thoracic Dystrophy.

Further Reading Beiser GD, Epstein SE, Stampfer M, et al. (1972) Impairment of cardiac function in patients with pectus excavatum, with

improvement after operative correction. New England Journal of Medicine 287: 267–272. Bevegard S (1962) Postural circulatory changes at rest and during exercise in patients with funnel chest, with special reference to factors affecting stroke volume. Acta Medica Scandinavica 171: 695–713. Cahill JL, Lees GM, and Robertson HT (1984) A summary of preoperative and postoperative cardiorespiratory performance in patients undergoing pectus excavatum and carinatum repair. Journal of Pediatric Surgery 9: 430–433. Fonkalsrud EW (2003) Current management of pectus excavatum: editorial update. World Journal of Surgery 27: 502–508. Fonkalsrud EW (2004) Open repair of pectus excavatum with minimal cartilage resection. Annals of Surgery 240: 1–5.

PEDIATRIC ASPIRATION SYNDROMES 309 Fonkalsrud EW, Beanes S, Hebra A, et al. (2002) Comparison of minimally invasive and modified Ravitch pectus excavatum repair. Journal of Pediatric Surgery 37: 413–417. Fonkalsrud EW and Reemtsen B (2002) Force required to elevate the sternum of pectus excavatum patients. Journal of the American College of Surgeons 195: 575–577. Haller JA, Kramer SS, and Lietman SA (1987) Use of CT scans in selection of patients with pectus excavatum surgery: a preliminary report. Journal of Pediatric Surgery 22: 904–906. Haller JA Jr and Loughlin GM (2000) Cardiorespiratory function is significantly improved following corrective surgery for severe pectus excavatum: proposed treatment guidelines. Journal of Cardiovascular Surgery: Torino 41: 125–130. Malek MG, Fonkalsrud EW, and Cooper CB (2003) Ventilatory and cardiovascular responses to exercise in patients with pectus excavatum. Chest 124: 870–882.

Martinez D, Juame J, Stein T, et al. (1990) The effect of costal cartilage resection on chest wall development. Pediatric Surgery International 5: 170–173. Nuss D, Kelly RE Jr, Croitoru DP, et al. (1998) A 10-year review of a minimally invasive technique for correction of pectus excavatum. Journal of Pediatric Surgery 33: 545–552. Ravitch MM (1949) Operative technique of pectus excavatum repair. Annals of Surgery 129: 429–444. Shamberger RC (1996) Congenital chest wall deformities. Current Problems in Surgery 33: 469–552. Shamberger RC and Welch KJ (1988) Cardiopulmonary function in pectus excavatum. Surgery Gynecology & Obstetrics 166: 383–391. Welch KJ (1958) Satisfactory surgical correction of pectus excavatum deformity in childhood: a limited opportunity. Journal of Thoracic Surgery 36: 697–713.

PEDIATRIC ASPIRATION SYNDROMES C A Sherrington, Princess Margaret Hospital, Perth, WA, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Aspiration lung disease (ALD) occurs when substances normally confined to the gastrointestinal tract enter the lung and cause disease. Aspiration is frequently considered as a primary or contributory cause of many chronic respiratory disorders in children, albeit without firm scientific evidence in the majority of cases. Pathogenesis of acid aspiration lung disease is well understood whereas understanding of nonacid aspiration is limited. Numerous tests exist for diagnosis of aspiration. Tests for dysphagic aspiration are generally well studied, sensitive, and specific. Tests for reflux aspiration are generally poorly understood. Lack of specific diagnostic methods has lead to a poor understanding of the role of aspiration in respiratory disease. Diagnosis of aspiration in the individual patient generally relies upon identification of a classic pattern of respiratory disease, along with features suggestive of aspiration events and evidence for failure of the protective mechanisms against aspiration. Management may include simple changes to feeding techniques, tube feeding, antibiotics, supportive strategies, and surgery including fundoplication.

Introduction Aspiration lung disease (ALD) occurs when substances normally confined to the gastrointestinal tract enter the lung and cause disease. ALD is a broad term covering several different processes, with varying pathophysiology dependent upon the nature, volume and pattern of aspiration events (AE), and the host response. ALD may be the primary cause of a given lung disease, or only an exacerbating factor. Diseases where aspiration may have a role include infective pneumonia, suppurative bronchitis, chemical pneumonitis, interstitial lung disease, cough, asthma,

cystic fibrosis, apparent life threatening events, and others. Aspiration was recognized by Hippocrates over 2000 years ago. John Hunter conducted the first experiment in 1781 when he showed that brandy kills cats by entering the lungs rather than the stomach. Ironically, in 1848 the first death following chloroform anesthesia was probably secondary to aspiration of brandy used to resuscitate the patient from anesthesia. More recently the medical recognition of aspiration began in the 1930s, largely in the area of obstetric anesthesia. Whilst knowledge of basic pathophysiology of acid injury is advanced, diagnostic methods are generally poor, resulting in limited knowledge of the role of aspiration in specific lung diseases, the overall disease burden, and treatment.

Etiology, Pathogenesis, and Pathology The significant risks for aspiration inherent in the shared aerodigestive tract are countered by a series of structural, chemical, and immune defenses, as well as a complex series of reflexes triggered by sensory receptors throughout the respiratory and upper intestinal tracts. Failure of one component of this protective system is unlikely to result in serious ALD as there is significant reserve capacity. Swallowing is a complex series of reflexes associated with closure of the larynx at several levels. Prevention of reflux of stomach contents into the airway is maintained by several physical barriers including the lower esophageal sphincter, esophagus, upper esophageal sphincter, and larynx. Sensory receptors in the esophagus, pharynx, larynx, and lower respiratory tract detect fluids, solids, and acids, and trigger both primary and secondary esophageal peristalsis and rapid

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increases in closing pressures at each of the above sphincters. If foreign substances enter the lung they are removed by cough, mucociliary clearance, buffering, transcellular transportation, phagocytosis, and immune mechanisms. Pathogenesis of Acid Aspiration Lung Disease

The pathogenesis of acid-induced lung injury has been extensively studied in the Sprague–Dawley rat, using 0.1 M hydrochloric acid instilled into the airway. In pure acid injury, lung injury does not occur where pH is above 2.5 and is maximal at pH 1.5. Larger volumes affect larger areas and have increased mortality. Lung damage occurs both in areas directly in contact with acid as well as distant areas. After instillation, acid reaches the pleural surface of the lung within 18 s and is neutralized within 30 s. Direct tissue damage by acid is probably limited, with the majority of damage occurring secondary to an inflammatory cascade triggered by release of preformed cytokines from aciddamaged cells. Leukotriene B4 and Thromboxane A2 cause direct damage and stimulate the synthesis of other cytokines, such as interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-a), and IL-8. IL-8 is the most important stimulus for neutrophil recruitment. Neutrophils are the major mediators of injury, via release of reactive oxygen species, elastase, other proteases, and other toxic substances. These substances result in direct damage to the endothelial wall, leakage of protein-rich fluid into the alveolar spaces, damage and inhibition of antiproteases, functional surfactant abnormalities, and release of eicosanoids further stimulating the inflammatory process. Pathologically the earliest changes are seen using electron microscopy 1 h after acid instillation. Infiltration by neutrophils accompanied by protein-rich fluid is first seen at 4 h and increases over the next 72 h with hemorrhage, edema, mucosal sloughing, and extensive alveolar consolidation. Resolution of damage takes 2–3 weeks and is associated with patchy parenchymal and pleural scars, bronchiolitis obliterans, and atypical bronchial regeneration. Less is known about the pathogenesis of nonacidic lung injury. Acute inflammatory changes are seen after instillation of food particles, particularly if instilled fluids are hypertonic (e.g., high carbohydrate concentration). Aspiration of bile seems particularly toxic. Clinical evidence strongly suggests that ventilator associated pneumonia results from aspiration of stomach contents laden with microorganisms. Infection is important in other forms of ALD, but its exact role is unclear. Pathologically nonacidic lung injury seems to have a more chronic inflammatory component, including the presence of giant cells.

Clinical Features Clinical features of aspiration are poorly defined, with little information regarding sensitivity and specificity of individual signs and symptoms. The clinical syndrome of ALD has been derived from extrapolation of the clinical features of massive reflux aspiration (see below) combined with features suggestive of failure of natural protective mechanisms against aspiration. Features suggestive of aspiration include coughing or choking with feeds, or whilst vomiting or supine, weak or wet cough, abnormal gag reflex, rapid onset chest x-ray (CXR) changes, unexplained hypoxia, and suggestive comorbidities including vocal cord palsy, neurological deficits, swallowing disorders, and gastroesophageal reflux (GER). Massive Reflux Aspiration (Mendelson Syndrome)

The syndrome of massive reflux aspiration is perhaps the most clearly defined. Acute respiratory distress occurs within minutes to hours of aspiration of a large volume of gastric contents. The AE is frequently witnessed. If conscious, the patient commonly has a combination of cough, wheeze, apnea, or stridor. The syndrome rapidly progresses over hours with increasing respiratory distress, crackles, fever, and impaired gas transfer. Sputum becomes pink and frothy owing to developing pulmonary edema and shows a prominent neutrophilic infiltrate. The CXR shows patchy areas of atelectasis, alveolar opacification, and consolidation that rapidly worsen. Subsequently, the patient may rapidly deteriorate and die, rapidly recover, or initially improve and then deteriorate due to either acute respiratory distress syndrome or infection. The mortality of a witnessed massive reflux AE is around one in three. Increased mortality is associated with early shock or apnea, involvement of more than two lobes on CXR, gastric pH below 1.75, and development of acute respiratory distress syndrome or infection. People who survive a single massive reflux AE usually have complete recovery of pulmonary function and CXR appearances. Aspiration in Other Lung Diseases

Aspiration has been suggested as being important in many different lung diseases but is proven to be relevant in few. ALD is probably important in people with severe spastic quadriparetic cerebral palsy where respiratory conditions are the major cause of death. Dysphagia is present in over 90% and dysphagic aspiration (DA) is detected in around 50% using videofluoroscopy (VFS). Studies in asthmatic subjects have repeatedly shown a clear association between asthma and GER in both adults and children, with increased prevalence of pathological GER,

PEDIATRIC ASPIRATION SYNDROMES 311

hiatus hernia, and symptomatic heartburn, and improvement in asthma following effective acid suppression in some subgroups. Whether this is due to microaspiration or reflexic bronchospasm from esophageal acid contact is unknown. The prevalence of aspiration in neonatal and pediatric intensive care seems high but varies widely between studies and has generally not been correlated with outcome. Aspiration as a cofactor in cystic fibrosis is controversial with one study showing reflux aspiration (RA) in 36%. Aspiration events are frequently seen in children with tracheostomy; however, the clinical relevance of this aspiration is not clear. Foreign Body Aspiration

Aspiration of large chunks of solid matter usually presents with sudden onset of a fit of coughing and wheeze, with or without a history of choking on a solid substance. Physical signs include wheeze, asymmetrical breath sounds, focal bronchial breathing, and occasionally mediastinal shift. The CXR may show focal atelectasis, unilateral hyperinflation owing to a ball-valve effect, or be completely normal. Removal is undertaken by bronchoscopy.

Diagnosis The diagnosis of ALD is suggested by fluctuating chronic lower respiratory tract disease of otherwise unknown etiology, combined with evidence of failure of protective mechanisms against aspiration, and clinical features suggesting AE. Many diagnostic tests have been suggested, but few are truly useful, and ultimately the diagnosis is usually made clinically. The most common erroneous assumption is that demonstration of any AE is both abnormal and clinically relevant. This is partly the result of limited data in control subjects. Diagnostic methods can be divided into three main groups: firstly methods that define a clinical syndrome suggestive of ALD, secondly methods that directly demonstrate AE, and finally, methods that detect foreign substances within the lung. The clinical aspects of diagnosis including history and examination have been reviewed above. Whilst CXR is always part of the standard evaluation, it has poor sensitivity and specificity. No single CXR appearance can be considered pathognomonic or even highly suggestive of aspiration, although disease that is more prevalent in the dependent lobes is suspicious, particularly if of rapid onset. Esophageal pH monitoring in reality detects only a failure of one of the protective mechanisms and is poorly specific and sensitive for ALD. Diagnosis of DA using either VFS or fiberoptic endoscopic evaluation of swallowing (FEES) is

relatively well studied, with both techniques having good sensitivity, specificity, and some evidence for correlation with clinical outcome. Both allow a sensitive assessment of AE along with a more general assessment of dysphagia. Both have been studied in normal controls where aspiration is rare during swallowing. Aspiration events detected using VFS are associated with clear increased risk of pneumonia, particularly where aspiration of thickened fluids and solids occur. Studies in children have shown both methods are useful, although data from normal controls and evidence of correlation with outcome are not available. Other methods of directly detecting DA are inferior, including nuclear medicine swallowing studies (poor sensitivity, practical limitations), salivagram (salivary aspiration present in 50% of normal subjects), and barium swallow (poor sensitivity). Methods for direct detection of RA are generally poor. Milk scan (radionuclide reflux aspiration study) is frequently used but has significant limitations. There is almost no standardization, no useful data in control subjects, no studies aiming to differentiate aspirators from others with nonaspiration lung disease, and only one study that attempts to determine the clinical relevance of a positive result. The sensitivity of milk scan is probably low at around 20%. Several methods exist that detect foreign substances within airway fluid as evidence of a past AE. Detection of lipid-laden macrophages (LLM) is probably the best studied. Its main limitation is poor specificity, particularly when used for clinical diagnosis outside of the research setting. LLM are increased in many chronic lung diseases unrelated to aspiration, including essential hemoptysis, cancer, bronchiectasis, and interstitial fibrosis. Detection of food carbohydrates has been used in intubated adult and neonatal subjects. Glucose detection is both non-specific and poorly sensitive and has little role. Lactose detection is intrinsically more specific but relies upon the use of food containing lactose. Different dyes have been added to enteral feeds in intubated subjects, but the method is insensitive. Several other methods exist, including enzymatic detection of pepsin and immunologic detection of foreign proteins, but they remain experimental.

Management and Current Therapy Most therapy for aspiration is based upon theoretical and anecdotal evidence rather than randomized trials. Therapy aims to decrease the frequency, volume, and toxicity of aspirate, improve pulmonary clearance, and treat infection. Food with high osmolality or carbohydrate concentration or with solid particulate matter should be avoided. Physiotherapy,

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broad-spectrum antibiotics, and standard supportive care are frequently used during exacerbations. DA may respond to changes to feeding (consistency, volume, posture, timing), including the use of tube feeding (nasogastric or gastrostomy) if simple measures fail. Salivary aspiration may respond to positional changes, anticholinergics or surgical repositioning or closure of duct openings. RA if minor and infrequent may respond to alterations in feeding, including small frequent feeds, continuous gastric feeds, or jejunal feeding. Reflux may respond to the use of thickeners, changes to the resting posture (prone or right lateral), and prokinetic agents. Proton pump inhibitors are frequently used to decrease the acidity of aspirated gastric contents, although there is no evidence that this is effective, and they may increase the risk for infection. Where lung disease is severe and the diagnosis of RA seems reasonably likely then fundoplication is indicated. This may dramatically improve lung function and improve survival. Frequently however, RA is not the only factor leading to lung disease, and continuation of symptoms after fundoplication is common. Tracheostomy is sometimes suggested as a physical barrier to aspiration, and to allow suction of aspirate but there is little evidence of efficacy, and it may make things worse. Complete surgical separation of the airway and gastrointestinal tract (e.g., laryngotracheal diversion) is occasionally needed for severe recurrent aspiration. See also: Acute Respiratory Distress Syndrome. Aquaporins. Asthma: Overview. Atelectasis. Bronchoalveolar Lavage. Chemokines, CXC: IL-8. Cilia and Mucociliary Clearance. Complement. Cystic Fibrosis: Overview. Defense Systems. Gastroesophageal Reflux. Infant Respiratory Distress Syndrome. Interleukins: IL-1 and IL-18. Interstitial Lung Disease: Overview. Laryngitis and Pharyngitis. Leukocytes: Pulmonary Macrophages. Lung Abscess. Lung Imaging. Particle Deposition in the Lung. Pediatric Pulmonary Diseases. Pneumonia: Overview and Epidemiology. Pulmonary Edema. Pulmonary Fibrosis.

Surfactant: Overview. Tracheostomy. Ventilation, Perfusion Matching.

Further Reading Bauer ML, Figueroa-Colon R, Georgeson K, and Young DW (1993) Chronic pulmonary aspiration in children. Southern Medical Journal 86: 789–795. Bauer ML and Lyrene RK (1999) Chronic aspiration in children: evaluation of the lipid-laden macrophage index. Pediatric Pulmonology 28: 94–100. Corwin RW and Irwin RS (1985) The lipid-laden alveolar macrophage as a marker of aspiration in parenchymal lung disease. American Review of Respiratory Diseases 132: 576–581. Ewig S and Torres A (2002) Prevention and management of ventilator-associated pneumonia. Current Opinion in Critical Care 8: 58–69. Gleeson K, Eggli DF, and Maxwell SL (1997) Quantitative aspiration during sleep in normal subjects. Chest 111: 1266–1272. Harding SM (1999) Gastroesophageal reflux and asthma: insight into the association. Journal of Allergy and Clinical Immunology 104: 251–259. Johnson JL and Hirsch CS (2003) Aspiration pneumonia: recognizing and managing a potentially growing disorder. Postgraduate Medicine 113: 99–102, 105–106, 111–112. Leder SB and Karas DE (2000) Fiberoptic endoscopic evaluation of swallowing in the pediatric population. Laryngoscope 110: 1132–1136. Matthay MA and Rosen GD (1996) Acid aspiration induced lung injury: new insights and therapeutic options. American Journal of Respiratory and Critical Care Medicine 154: 277–278. McVeagh P, Howman-Giles R, and Kemp A (1987) Pulmonary aspiration studied by radionuclide milk scanning and barium swallow roentgenography. American Journal of Diseases of Children 141: 917–921. Mendelson CL (1946) The aspiration of stomach contents into the lungs during obstetric anesthesia. American Journal of Obstetrics and Gynecology 52: 191–205. Orenstein SR (2001) An overview of reflux-associated disorders in infants: apnea, laryngospasm, and aspiration. American Journal of Medicine 111(supplement 8A): 60S–63S. Sasaki CT and Weaver EM (1997) Physiology of the larynx. American Journal of Medicine 103(supplement 5A): 9S–18S. Schmitt J, Holas M, Halvorson K, and Reding M (1994) Videofluoroscopic evidence of aspiration predicts pneumonia and death but not dehydration following stroke. Dysphagia 9: 7–11. Silver KH and Van Nostrand D (1994) The use of scintigraphy in the management of patients with pulmonary aspiration. Dysphagia 9: 107–115.

PEDIATRIC PULMONARY DISEASES P D Sly, Institute for Child Health Research, Perth, WA, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Pediatric respiratory diseases largely occur as the result of the interaction between an underlying genetic predisposition, an

environmental exposure, and the developmental stage of the child at which the exposure occurs. It is the developmental context that determines the clinical features of pediatric pulmonary diseases and differentiates them from adult respiratory diseases. The developmental stage at which a disease occurs is frequently a major determinant of the long-term sequelae. Lung development is a continuous process from embryo to adolescence suggesting that children may be more vulnerable to the effects of respiratory insults than adults whose lung growth is

PEDIATRIC PULMONARY DISEASES 313 complete. While all pediatric respiratory diseases may have components of genetic susceptibility and environmental exposure, some classification of etiology based on the most apparent causative factors is useful. These include: congenital – where the primary insult occurs in utero and abnormalities are apparent in early life; developmental – where the clinical features of the illness are primarily determined by the developmental stage at which the insult occurs; infectious – where the illness is due to an infectious agent and the clinical response is largely determined by that agent; genetic – where a genetic abnormality or predisposition is the primary cause of the illness; and mixed – where there is one or more primary factor.

The developmental stage at which a disease occurs is frequently a major determinant of the long-term sequelae. Lung development is a continuous process from embryo to adolescence suggesting that children may be more vulnerable to the effects of respiratory insults than adults whose lung growth is complete. Observations from animal studies support this and epidemiological studies suggest that in utero exposure to the products of tobacco smoke have greater consequences than postnatal exposure to environmental tobacco smoke.

Introduction Pediatric respiratory diseases largely occur as the result of the interaction between an underlying genetic predisposition, an environmental exposure, and the developmental stage of the child at which the exposure occurs. It is the developmental context that determines the clinical features of pediatric pulmonary diseases and differentiates them from adult respiratory diseases. Development of the human lung begins in the embryo and continues until the age of 18–20 years. Cellular differentiation and formation of the primary lung structures occur during fetal development, but the majority of growth and maturation of the lung occurs postnatally through the processes of branching morphogenesis and alveolarization. The major antenatal and postnatal developmental milestones are summarized in Table 1. Childhood is a time of major growth and development of the respiratory system. The lungs are immature at birth and contain approximately 30% of the adult complement of alveoli. Collateral ventilation between adjacent lung units is essentially absent in the infant’s lung, aiding the development of widespread airway obstruction and hyperinflation, as seen during acute viral bronchiolitis. Table 1 Normal development of the lungs Prenatal Airway branching to terminal bronchioles complete by 16 weeks Functional smooth muscle by 8–10 weeks, respiratory bronchioles by 26 weeks Cartilage complete by 28 weeks Blood vessels complete by 17 weeks Lamellar bodies in type II cells by 24 weeks 30–50% of alveoli present by term Postnatal Increase in airway size parallels somatic growth Rapid increase in smooth muscle at an early age Alveolarization continues until at least 2 years Maturation of microvasculature during postnatal phase of alveolar development Lung volume approximately double from birth to 18 months and again to 5 years Lung growth continues until approximately 18 years in females and 20–23 years in males

Etiology While all pediatric respiratory diseases may have components of genetic susceptibility and environmental exposure, some classification of etiology based on the most apparent causative factors is useful. These include: congenital – where the primary insult occurs in utero and abnormalities are apparent in early life; developmental – where the clinical features of the illness are primarily determined by the developmental stage at which the insult occurs; infectious – where the illness is due to an infectious agent and the clinical response is largely determined by that agent; genetic – where a genetic abnormality or predisposition is the primary cause of the illness; and mixed – where there is one or more primary factor. These classifications will be somewhat arbitrary and could be subject to argument; however, they serve as a framework for understanding how pediatric respiratory diseases develop.

Pathology The pathological features of pediatric pulmonary diseases can also be considered according to the classification system developed above. Congenital respiratory diseases are those that arise from some intrauterine insult resulting in an alteration in or disruption of normal organogenesis. The timing of the insult can be determined, to some degree, from the effects on lung development; for example, airway branching is essentially complete down to the terminal bronchioles by 16 weeks of gestation; thus, abnormalities of airway development, for example, vascular rings, must occur before this milestone has been reached. Congenital abnormalities of the lung are discussed in more detail in Lung Development: Congenital Parenchymal Disorders. One condition that is not usually thought of as being a congenital lung disease but fulfills the above criteria is the lung disease associated with extreme prematurity. Infants born at 22– 24 weeks of gestation frequently survive. These infants are born before alveolarization is thought to have begun and have a lung disease consistent with alveolar

314 PEDIATRIC PULMONARY DISEASES

hypoplasia (see Bronchopulmonary Dysplasia. Lung Development: Overview). In addition, they are born before lamella bodies develop in the alveolar type 2 cells and have profound surfactant deficiency (see Bronchopulmonary Dysplasia. Epithelial Cells: Type II Cells. Surfactant: Overview). Developmental respiratory diseases occur when the developmental stage of the child determines the clinical features. This may be due to a variety of factors, including the fact that normal anatomical structures have not yet developed, the small size of the airways, increased compliance of the chest wall, or the immaturity of the immune system. Classic examples include acute viral bronchiolitis (see Bronchiolitis), where the classic features of widespread hyperinflation and fine crackles (see Signs of Respiratory Disease: Lung Sounds) are due to the lack of collateral ventilation between adjacent lung units, and not primarily a feature of the causative virus; croup (see Croup), where viral-induced swelling in the subglottic region produces the characteristic stridor because of the relatively small size of the tracheal airway in toddlers, whereas the same infection is unlikely to cause stridor in a normal adult or older child; obstructive sleep apnea in young children (see Sleep Apnea: Children) where the small size of the pharyngeal airway is a major determinant of the severity of the obstruction, and removal of the tonsils and adenoids frequently relieves the obstruction sufficiently well until the child grows; and infant respiratory distress syndrome (see Infant Respiratory Distress Syndrome) where the compliant chest wall in the early newborn period does not provide sufficient elastic recoil to counterbalance the increased stiffness of the lungs due to delayed clearance of fetal lung liquid at birth (see Breathing: Fetal Lung Liquid). The adverse effects of environmental tobacco smoke, air toxics, and pollution can also be considered to be developmental diseases (see Environmental Pollutants: Overview; Diesel Exhaust Particles) in children as the growing lung is more susceptible to effects of these agents than an adult lung. Under this classification, infectious diseases are those where the clinical features are more related to the causative agent than to the age of the patient. Examples include pneumonia (see Pneumonia: Overview and Epidemiology; Atypical; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens); Nosocomial; Parasitic; Mycobacterial; Viral; The Immunocompromised Host); laryngitis and pharyngitis (see Laryngitis and Pharyngitis); and epiglottitis (see Laryngitis and Pharyngitis). The archetypal genetic pulmonary disease in pediatrics is cystic fibrosis (see Corticosteroids: Therapy. Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene). While the correlation between CF

genotype and clinical phenotype is not as close as had been hoped, much of the variability in severity of illness can be attributed to the influence of ‘modifier genes’, especially those promoting pulmonary inflammation. A good example of a mixed pediatric pulmonary disease is atopic asthma (see Allergy: Overview. Asthma: Overview), which primarily develops in those with appropriate genetic predisposition and who are exposed to environmental insults in early life (i.e., at the appropriate developmental stage). At least three wheezing syndromes are recognized in children and while these are relatively easily separated in epidemiological studies, determining which category an individual wheezy child best fits is more difficult. Wheezing is common in the first year of life. The majority of these children do not wheeze after 2–3 years of age and are classified as having transient infantile wheeze. This condition is not associated with a family history of asthma or atopy, and the major risk factors are young maternal age and maternal smoking during pregnancy. Wheezing associated with viral infections is very common in children and the majority of asthma exacerbations at any age are viral-induced (see Asthma: Acute Exacerbations). However, approximately 60–70% of children with a diagnosis of asthma lose their symptoms by adolescence and these children are thought to predominantly have viral-associated wheeze. This form of asthma is less likely to be associated with a positive family history or with atopy. The third wheezing syndrome is atopic asthma, which may start early in life or may begin in midchildhood. Children with atopic asthma typically have a positive family history or asthma/atopy and have other atopic features. This type of asthma is more likely to persist into adult life.

Clinical Features Many of the clinical features of pediatric respiratory diseases are primarily determined by the state of physiological development of the child’s respiratory system at the time of the illness. The main clinical features include: Tachypnea

The normal respiratory rate decreases with age from 40–60 breaths per minute in the newborn; 30–50 in infants aged 1 week to 3 months; 20–40 in children aged 3–24 months; 14–14 in children aged 2–10 years before reaching the adult rate of 12–20 by approximately 10 years of age (see Breathing: Breathing in the Newborn. Signs of Respiratory Disease: Breathing Patterns). Sleeping respiratory rate is one of the earliest and most reliable signs of respiratory

PEDIATRIC PULMONARY DISEASES 315

disease in infants and young children; however, it gives no clues as to etiology. Sternal Retractions

Sternal retractions occur during normal tidal breathing in infants during the first weeks after birth. This is due to the chest wall being relatively more compliant than the lungs to facilitate passage through the birth canal. Sternal retractions become less common as the chest wall stiffens (see Chest Wall Mechanics). However, any pulmonary disease that is associated with upper airway obstruction, for example, croup (see Croup), or with increased stiffness of the lungs, for example, lower respiratory infections (see Pneumonia: Overview and Epidemiology; Viral), is likely to result in sternal retractions. These retractions become much less common as children grow and their chest-wall stiffness increases. Intercostal Retractions

Intercostal retractions occur when the more compliant intercostal regions are sucked inward during inspiration. This is most commonly seen in conjunction with hyperinflation where the ribs become more horizontal and in situations where increasingly negative pressures are generated during inspiration. Developmentally, intercostal retractions are more likely in older children with stiffer chest walls. Harrison’s Sulci

Harrison’s sulci refer to a flaring of the lower ribcage with a relative indentation that occurs in the area of the insertion of the diaphragm anterolaterally (see Chest Wall Abnormalities. Signs of Respiratory Disease: General Examination). They are frequently associated with pectus carinatum, which visually accentuates the sulci. These are signs of long-standing hyperinflation with flattening of the diaphragm, so that the major contractile force of the diaphragm is acting horizontally on the ribcage rather than vertically. Harrison’s sulci are much more common in children, partly due to the relatively greater importance of the diaphragm as a respiratory muscle in early childhood, and partly due to the hyperinflation occurring at a time before the chest wall stiffens. Pigeon Chest Deformity (Pectus Carinatum)

Pectus carinatum refers to an anterior displacement of the upper sternum associated with long-standing hyperinflation and frequently with Harrison’s sulci (see Chest Wall Abnormalities. Signs of Respiratory Disease: General Examination). Again, this is more common when the hyperinflation occurs in children before the chest wall has fully stiffened.

Stridor

Stridor, by definition, is a respiratory noise occurring predominantly in inspiration due to obstruction occurring primarily in the extrathoracic portion of the trachea or laryngeal structures (see Signs of Respiratory Disease: General Examination; Lung Sounds). The two most common causes of stridor in children are acute laryngotracheobronchitis, (croup) (see Croup) and laryngomalacia (see Laryngitis and Pharyngitis). Stridor is more common in children due to the smaller size of the trachea where a relatively modest mucosal swelling can result in sufficient airway tracheal narrowing to limit flow during inspiration. Subglottic stenosis is a less common cause of stridor that may arise from congenital lesions; secondary to prolonged or difficult intubation, usually in infants born prematurely; or from subglottic cysts or hemangiomata (see Lung Development: Congenital Parenchymal Disorders. Laryngitis and Pharyngitis). Stridor with an expiratory component implies more severe obstruction, such that flow limitation is also present in the extrathoracic trachea during expiration or an obstruction extending to the intrathoracic portion of the trachea. Wheeze

Wheeze is a frequent clinical sign of lower respiratory problems in infants and young children (see Signs of Respiratory Disease: General Examination; Lung Sounds. Symptoms of Respiratory Disease: Cough and Other Symptoms). Wheeze implies the presence of flow limitation of intrathoracic airways during expiration. Wheeze with an inspiratory component can occur with more severe airway obstruction. As children have smaller and more compliant airways, sufficient airway narrowing results in expiratory flow limitation and wheeze during tidal breathing more easily than in adults. Cough

Cough is a common feature of many respiratory illnesses in both children and adults (see Symptoms of Respiratory Disease: Cough and Other Symptoms). Chronic or recurrent cough, especially occurring at night, is very common in young children. Recent studies have demonstrated increased cough-receptor sensitivity in many of these children. Similar studies have not been conducted in adults and it remains to be established whether this is a developmental problem confined to early childhood. Crackles

Crackles are fine noises heard by ausculatation at end of an inspiration (see Signs of Respiratory Disease: General Examination; Lung Sounds). They occur

316 PERIPHERAL GAS EXCHANGE

commonly in infants with acute viral bronchiolitis where they are thought to arise due to the ‘popping open’ of fluid menisci in bronchioles. The lack of effective collateral ventilation between adjacent lung units in the first year of life is thought to contribute to crackles in this age group. Crackles are also heard with interstitial lung diseases in both children and adults, pulmonary fibrosis in adults, and in inflammatory diseases involving small peripheral airways, such as exacerbations of cystic fibrosis lung disease.

Animal Models Animal models have been used to investigate a number of pediatric pulmonary diseases. Lung growth and development, in particular the adverse effects of drugs such as corticosteroids, has been studied using rats, mice, rabbits, lambs, and primates. Rodents are born without alveoli and develop them while breathing air, a situation very different from that occurring in humans. Recent studies using primates have provided fascinating insights into the effects of air quality and allergen exposure on lung development; however, these studies are expensive and have been limited in scope. Animal models have also been used to study the mechanisms underlying complex diseases such as asthma. However, here the vast majority of studies have been undertaken in adult animals with mature immune systems – a situation that does not mimic the human counterpart. Studies on the effects of early life respiratory infections on subsequent respiratory diseases are attracting considerable interest. However, most of these have been conducted in small animals with relatively crude measurements of lung function. See also: Allergy: Overview. Asthma: Overview; Acute Exacerbations. Breathing: Breathing in the Newborn; Fetal Lung Liquid. Bronchiolitis. Bronchopulmonary Dysplasia. Chest Wall Abnormalities. Corticosteroids: Therapy. Croup. Cystic Fibrosis: Cystic Fibrosis

Transmembrane Conductance Regulator (CFTR) Gene. Environmental Pollutants: Overview; Diesel Exhaust Particles. Epithelial Cells: Type II Cells. Infant Respiratory Distress Syndrome. Laryngitis and Pharyngitis. Lung Development: Overview; Congenital Parenchymal Disorders. Pneumonia: Overview and Epidemiology; Atypical; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens); Nosocomial; Parasitic; Mycobacterial; Viral; The Immunocompromised Host. Signs of Respiratory Disease: Breathing Patterns; General Examination; Lung Sounds. Symptoms of Respiratory Disease: Cough and Other Symptoms. Sleep Apnea: Children. Surfactant: Overview.

Further Reading Jobe AH, et al. (1998) Fetal versus maternal and gestational age effects of repetitive antenatal glucocorticoids. Pediatrics 102(5): 1116–1125. Jobe AH, et al. (2000) Effects of antenatal endotoxin and glucocorticoids on the lungs of preterm lambs [see comment]. American Journal of Obstetrics and Gynecology 182(2): 401–408. Martinez FD (1997) Definition of pediatric asthma and associated risk factors. Pediatric Pulmonology 15(supplement): 9–12. Martinez FD, et al. (1995) Asthma and wheezing in the first six years of life. New England Journal of Medicine 332(3): 133–138. Robinson MJ and Roberton DM (eds.) (2003) Practical Paediatrics, 5th edn., Edinburgh: Churchill. Silverman M (ed.) (2002) Childhood Asthma and Other Wheezing Disorders, 2nd edn., London: Arnold. Sly PD, Turner DJ, and Hantos Z (2004) Measuring lung function in murine models of pulmonary disease. Drug Discovery Today: Disease Models 1(3): 337–343. Stein RT, et al. (1999) Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years [see comment]. Lancet 354(9178): 541–545. Taussig LM and Landau LI (eds.) (1999) Pediatric Respiratory Medicine. St Louis: Mosby. Tyler WS, Tyler N, Magliano DJ, et al. (1991) Effects of ozone inhalation on lungs of juvenile monkeys: morphometry after a 12-month exposure and following a 6-month post exposure period. In: Berglund R, Lawson D, and Mckee D (eds.) Tropospheric Ozone and the Environment, pp. 151–160. Pittsburgh PA: Air and Waste Management Association.

PERIPHERAL GAS EXCHANGE P D Wagner, University of California – San Diego, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Gas exchange in any tissue or organ requires O2 delivery (and CO2 removal) along a transport pathway involving, sequentially, the lungs, heart, blood, circulation, and tissues. Convective

factors governed mostly by the lungs, heart, blood, and circulation determine how much O2 reaches each tissue, while diffusion underlies tissue O2 unloading and transport to mitochondria. O2 consumption may be limited by these transport processes, or alternatively may be set by tissue metabolic demand. The maximal amount of O2 available to mitochondria is not determined uniquely by any one step in the transport pathway, but by all in an integrated manner. Because the pathway steps are arranged in series, poor function of any single step significantly reduces maximal O2 transport, while greater than

PERIPHERAL GAS EXCHANGE 317 normal function of any such step increases overall transport only slightly. When considering central and peripheral limitations to O2 transport, it is evident that convective O2 delivery to tissues depends in part on peripheral factors (regional vascular conductances), while diffusive unloading in the periphery depends in part on central factors (especially blood flow). In both health and disease, central and peripheral factors contribute to limitations on O2 transport, at least in the area of muscle function during exercise, where the O2 transport system is often stretched to its limits.

In fact, the serial linkage among these transport processes is such that the performance of any one step affects the function of the others. In turn, this means that no single step in the O2 transport chain uniquely determines O2 transport capacity; each step will affect O2 transport. Since identical processes govern the elimination of CO2 produced in the tissues, and the exchange of other gases as well, this article will focus only on O2.

The O2 Transport Pathway This encyclopedia focuses on the lungs, but for O2 to reach the mitochondria of the many tissue cells throughout the body, it must be transported not only from the air to the pulmonary capillary blood, but also from there through the circulation to the various tissue vascular beds. In these beds, O2 must then leave the circulation and diffuse to the mitochondria. The O2 transport pathway therefore involves several organs and tissues and a variety of associated transport processes. Figure 1 illustrates the entire pathway in diagrammatic form. O2 uptake by the lungs begins with ventilation, a largely convective process (see Ventilation: Overview). This delivers O2 from the air to the alveoli in the lungs. Next, diffusion across the pulmonary blood–gas barrier moves O2 from the alveoli into the capillary blood, where O2 rapidly combines with hemoglobin (see Diffusion of Gases. Hemoglobin). The heart and circulation are then responsible for convective movement of O2 in the blood around the body to the microvascular beds of the many tissues and organs where it is to be used. Finally, for it to support metabolism, O2 must diffuse out of the microvasculature into the mitochondria. O2 transport to tissues therefore requires the integrated effort of the lungs, heart, and blood vessels, blood and tissues together acting like a bucket brigade – a serially linked set of transport functions. Structures

Functions Ventilation

Lungs

Diffusion Capillaries Heart, blood, circulation

Perfusion

Capillaries

Diffusion

Tissues Figure 1 A model of the entire O2 transport pathway from the air down to the tissues, encompassing the lungs, blood, heart, circulation, and tissues in a serially linked system. The major structure, function, and process at each step are indicated.

O2 Transport to the Tissue/Organ Vasculature: Convection Pulmonary gas exchange has been considered extensively in the article Ventilation, Perfusion Matching. The end result of this process is arterial blood with a particular level of O2, expressed as PO2 (PaO2 ), O2 saturation (SaO2 ), and O2 concentration (CaO2 ). The rate at which this O2 is delivered to the tissue vascular beds is the product of CaO2 and the rate of ˙ ) to the tissue(s) in question. This is blood flow (Q ’ O ) and is a convective called systemic O2 delivery (Q 2 process. It is expressed as follows: ’
CaO ’O ¼Q Q 2 2

½1

’ O is the rate of ˙,Q If cardiac output is used for Q 2 ˙ were blood O2 delivery to all tissues of the body; if Q ’ O would define O2 flow to an exercising muscle, Q 2 delivered to that muscle. If the normally negligible contribution of physically dissolved O2 to CaO2 is ignored, and since 1 g hemoglobin (Hb) can bind 1.39 ml O2, we can restate eqn [1] as follows: ’
1:39
½Hb
SaO ’O ¼Q Q 2 2

½2

’ O is the It is very important to realize that while Q 2 amount of O2 delivered to the tissue vascular bed(s), not all of this O2 can be extracted from the blood and find its way to the mitochondria. Thus, eqn [2] does not define how much O2 is actually available to the cells. Equation [2] is however helpful in laying out the factors that determine O2 delivered to the tissues. It can be seen that cardiovascular factors that determine ’ hematological factors that determine [Hb], and Q, (mostly pulmonary) factors that determine SaO2 all ’ O in a linked, multiplicative fashion. Clearly, affect Q 2 no single component of the O2 transport system sets ’ O ; there is no single limiting factor to its value. A Q 2 fall in any of the three components will reduce max’ O , defining the classic causes of insufficient imal Q 2 tissue oxygenation: ‘stagnant’, ‘anemic’, and ‘hypoxic’ forms of ‘hypoxia’, respectively. The importance of ’ O is that there are many circumstances considering Q 2 in which O2 delivery is thought to be limiting tissue

318 PERIPHERAL GAS EXCHANGE

O2 Transport from the Tissue/Organ Vasculature to the Mitochondria: Diffusion The process by which O2 moves from Hb (in the red cells flowing through tissue vascular beds) to tissue cell mitochondria has long been known to be one of simple diffusion. Microvascular PO2 is higher than that in tissue cells due to ongoing cellular metabolic use of O2, creating a PO2 gradient that drives movement of O2 from Hb to mitochondria. The factors that determine how much O2 can move to the cells (V’ O2 ) are in principle the same as in the lungs (see Diffusion of Gases. Ventilation, Perfusion Matching). Diffusive flux for O2 will be greater: (1) the greater the capillary–tissue cell interfacial area (A); (2) the shorter the distance from red cells to mitochondria (d); and (3) the larger the PO2 difference between capillaries (PCAPO2) and mitochondria (PMITOO2). Thus: V’ O2 ¼ k
A
ðPCAP O2  PMITO O2 Þ=d ¼ D
ðPCAP O2  PMITO O2 Þ

½3

Here k is the diffusion coefficient per unit area and distance; D ¼ kA/d and represents the total O2 conductance, or ‘diffusing capacity’. For many years, focus was on diffusion distance as the key element regulating O2 availability, but more recent research suggests that the capillary–tissue contact area is more important, at least in skeletal muscle, where myoglobin probably mitigates the issue of distance by facilitating O2 diffusion within myocytes. The size of the PO2 gradient is limited. Thus, upstream microvascular PO2 is limited by arterial PO2 and downstream mitochondrial PO2 is limited by the minimal value required to support oxidative

5 O2 consumption (l min−1)

O2 availability and thus tissue function. Well-known examples include exercise, ascent to altitude (which affects many tissues and organs, not just the muscle during exercise), hypoxemia from pulmonary disease, reduced perfusion in heart failure, various anemias, and, possibly, multiple organ failure syndromes. While in eqn [2], [Hb] and SaO2 will be the same for blood reaching all tissues and organs, two complex issues that affect regional tissue perfusion and ’ O are: (1) how cardiac output is regulated; thus Q 2 and (2) how cardiac output is distributed among the many body tissues and organs. These processes must optimize not only O2 transport but also maintain blood pressure and vital organ perfusion in the face of competing demands for blood flow and O2. These topics are well beyond the scope of this chapter, but suggestions for reading are included in the ‘Further reading’ section.

4 3 2 1

Mean capillary P O2 Muscle venous P O2

0 0

10

20 30 P O2 (mmHg)

40

Figure 2 Variation in leg V’ O2 MAX in trained normal subjects as inspired O2 levels (F IO2 ) are acutely altered. V’ O2 MAX changes with F IO2 in proportion to the PO2 gradient between the muscle capillaries and mitochondria, compatible with a limitation on V’ O2 MAX set by O2 tissue diffusion. The gradient may be represented by either muscle venous or mean capillary PO2 . Reproduced from Roca J, Hogan MC, Story D, et al. (1989) Evidence for tissue diffusion limitation of V’ O2 max in normal humans. Journal of Applied Physiology 67: 291–299, with permission from American Physical Society.

phosphorylation, thought to be about 1–3 mmHg. It is worth noting that the right shift of the O2Hb curve (that occurs as CO2 is loaded from the tissues to the blood) assists O2 unloading by helping to maintain the PO2 gradient even as O2 continues to leave the blood. In the lungs of normal subjects (other than exceptional athletes), there is evidence for complete diffusive equilibration between alveolar and endcapillary PO2 as O2 is taken up, even during heavy exercise. However, in these same subjects (perhaps except for the very sedentary), the unloading of O2 in the tissues is often limited by the diffusive process. Put simply, under such conditions, tissue capillary blood cannot have all its O2 unloaded because this would eliminate the red cell to mitochondrion PO2 gradient necessary to drive O2 flux in the first place. Therefore, at high rates of O2 utilization, some O2 must remain behind in the blood draining the tissues to provide the driving gradient for diffusion. A good example is in skeletal muscles during exercise. Figure 2 shows how maximal exercise capacity is linearly and proportionally related to the O2 diffusion gradient as arterial PO2 is varied by changing inspired O2 levels in normal athletic subjects.

Limits to Tissue O2 Transport and Utilization Limitation of O2 Utilization: O2 Supply Versus O2 Demand

In any tissue or organ, there are physiological limits to O2 transport. They are described by eqns [2] and

PERIPHERAL GAS EXCHANGE 319

40 35 30 VO2 (µmol min−1)

2 mmHg, mitochondrial O2 consumption is reduced as PO2 falls (supply limitation). However, when PO2 is higher than about 2 mmHg, O2 consumption fails to increase further, implying that demand has been more than met by supply (demand limitation). Graphical Analysis of O2 Supply and Demand Limitation

Resting human metabolic O2 consumption (V’ O2 ) is about 300 ml min  1. By far the greatest stress on the O2 transport system comes during heavy exercise when V’ O2 rises to about 3 l min  1 in sedentary normal subjects and to as much as 5 l min  1 in elite athletes. During maximal exercise, the convective and diffusive transport processes, and especially their integration, can be conveniently depicted on a diagram, as shown in Figure 4. This figure depicts tissue O2 consumption on the ordinate and tissue venous PO2 on the abscissa. It can be shown that average PO2 along the tissue capillary is proportional to that in the exiting tissue venous blood, so that the average O2 diffusion gradient can be represented by tissue venous PO2 in this diagram. In addition, mitochondrial PO2 is low enough, compared to mean capillary values, so that it can be neglected. As a result, eqn [3], describing V’ O2 as a function of the diffusive process, can be plotted as a straight line through the origin having a slope proportional to D. Convection: VO2 = Q × [CaO2− C vO2] Diffusion: O2 consumption (ml min–1)

[3] applied to that tissue/organ, and reflect capacity for both the convective and diffusive transport components of the O2 transport pathway. There are also limits on how much O2 can be used by the tissue/ organ (set by biochemical limits to oxidative phosphorylation). To understand tissue oxygenation, one must therefore know, for a particular tissue/organ and under the desired study conditions, whether maximal ATP production is being limited by O2 transport (‘supply’) or by O2 utilization (‘demand’). This is not always easy to establish, especially in intact humans. Most simply, if an experimental increase in O2 transport leads to an increase in O2 used, one would infer that ‘supply’ limitation is operating. Failure to increase O2 used as supply is augmented points to ‘demand’ limitation. Unfortunately, some methods for increasing O2 supply have the potential to increase demand at the same time. Sympathomimetic drugs used in critically ill patients in intensive care units are a typical example of this problem. This is unfortunate, because if in an ill patient, metabolism were shown to be O2 supply limited, it would make sense to search for therapeutic maneuvers to increase O2 availability, unless the therapy also increased O2 demand. However, if metabolism were demand-limited, increasing O2 supply may not be beneficial, and may even aggravate cell tissue damage. In recent years, this conundrum has been a topic of interest in critically ill patients. The classical relationship between mitochondrial PO2 (reflecting supply) and O2 consumption (reflecting demand) shown in Figure 3 illustrates the supply– demand issue well. When PO2 is less than about

25 20

VO2 = D × k × P vO2 (a)

4000 VO max (a) 2 3000 VO2max (b) 2000 1000

Diffusion

(b)

Convection

0

15

0 10 5 0 0.0

0.5

1.0

1.5 2.0 PO2 (mmHg)

2.5

3.0

Figure 3 Relationship between O2 consumption (ordinate) and PO2 (abscissa) in an isolated mitochondrial preparation. Below about 2 mmHg PO2 , O2 consumption is O2 supply limited, while at higher PO2 values, V’ O2 is independent of PO2 , and thus not limited by O2 supply. Reproduced from Wilson DF, Erecinska M, Drown C, and Silver IA (1977) Effect of oxygen tension on cellular energetics. American Journal of Physiology 233: C135–C140, with permission from American Physical Society.

10 20 30 40 50 60 70 80 90 100 Muscle venous PO2 (mmHg)

Figure 4 Diagram bringing together the diffusive and convective components of peripheral O2 transport. V’ O2 is on the ordinate, tissue venous PO2 on the abscissa. The straight line of positive slope reflects the laws of diffusion, indicating that V’ O2 is proportional to the diffusion gradient for O2 (related to venous PO2 ). The curved line of negative slope reflects mass balance, plotting the Fick principle equation relating V’ O2 to blood flow and arteriovenous O2 concentration difference. Their point of crossing indicates the maximal possible value of V’ O2 that the O2 transport chain as a system can support. (a) Maximal mitochondrial V’ O2 exceeds O2 supply; the latter limits V’ O2 MAX . (b) Maximal mitochondrial V’ O2 is less than O2 supply; V’ O2 MAX is set by maximal mitochondrial V’ O2 .

320 PERIPHERAL GAS EXCHANGE

Then, using principles of mass balance, V’ O2 can also be expressed by the well-known Fick principle: ’
½CaO  CvO  V’ O2 ¼ Q 2 2

½4

where CvO2 is tissue venous O2 concentration. CvO2 is uniquely related to venous PO2 by the known O2Hb dissociation curve (see Hemoglobin). Thus, eqn [4] can also be plotted in Figure 4, and appears as an inverted O2Hb dissociation curve decreasing in value as venous PO2 rises. Both relationships describe O2 flux, and the only point on the diagram where both are simultaneously satisfied is their point of intersection. This point thus indicates both, maximal O2 available to mitochondria (ordinate) and the necessarily unique value of PvO2 that enables that maximal V’ O2 (abscissa). Most importantly, this diagram shows the integration among all parts of the O2 transport system: eqn [3] depends on tissue diffusional conductance; eqn [4] on heart, blood, and lungs as described above in eqn [2]. If any of these transport factors were to change, the crossing point of the two lines would also change, shifting maximal V’ O2 . This analysis presumes that mitochondrial V’ O2 is indeed limited by O2 supply, not demand. In other words, maximal possible demand exceeds the V’ O2 at the crossing point, as for example, dashed line (a) in the figure. If maximal V’ O2 was not limited by O2 supply, but rather by demand, it would be lower than indicated by the crossing point in Figure 4 (e.g., as in dashed line (b)). The tissue venous PO2 would be the PO2 where the curved Fick principle line and line (b) intersect. This venous PO2 represents the only point on the figure where eqn [4] (i.e., mass conservation) is satisfied under these conditions. Mathematical Analysis of O2 Supply Limitation

Another way to analyze the interaction between the convective and diffusive elements of O2 unloading in peripheral capillaries is to use calculus to determine the equation describing the time course of O2 unloading along the capillary. This can be done in elegant algebraic form if one is willing to approximate the O2Hb dissociation curve by a straight line of slope b. When this is done, one finds that under the presumption of supply, not demand, limitation, maximal O2 consumption can be expressed as ’
f1  exp ½D=ðb
QÞg ’ V’ O2 ¼ PaO2
b
Q ½5 ’ is in fact the same In this equation, PaO2
b
Q ’ O in eqn [1]. This is because CaO ¼ PaO
b. as Q 2 2 2 The exponential term in curly brackets gives the fraction of O2 delivered to the tissue vasculature that can be extracted and transported by diffusion to

the mitochondria. D is the diffusional conductance for O2. This equation provides great insights into the interplay between convective and diffusive factors in tissue O2 unloading. Most importantly, extraction ˙ ), not just on D itself. depends on the ratio D/(b
Q ˙ An increase in either b or Q without a similar increase in D would reduce the fractional extraction of O2 from the blood. Also of importance, both b and ˙ appear in the numerator in front of the curly Q ˙ brackets as well. Hence, an increase in either b or Q would have two opposing effects: increasing convective O2 delivery but decreasing diffusive O2 extraction. These offsetting effects mean that changes in V’ O2 will always be relatively less than changes in ˙ alone. either b or Q Examination of eqn [5] also leads to the conclu˙ (or b) is very low, V’ O becomes sion that when Q 2 ˙ or (b). However, as Q ˙ invery dependent on Q creases, the offsetting effects mentioned above balance each other to a greater extent. Ultimately, ˙ will not increase O2 availabilfurther increases in Q ity as offsetting effects become essentially equal. This behavior is typical of that seen in a linked ‘bucket brigade’ system such as the O2 transport chain, and turns out to be true in principle for any link in the chain: the weakest link (if substantially weaker than all others) dictates maximal function of the entire pathway. This also means that for an increase in function of the whole O2 pathway, it is more effective to have modest, matched, increments in every step of the pathway than a large increase in just one. It is thus no surprise that exercise training, as one example of an adaptable O2 transport system, is accompanied by increases in function of most steps in the transport pathway. Other Factors Affecting O2 Transport in Tissues

The above discussion highlights the two principal determinants of tissue O2 availability – overall tissue perfusion and the extent of diffusion from red cells to mitochondria. There are additional factors that may interfere with cellular O2 delivery, but how important they are varies from tissue to tissue and is difficult to quantify. One such factor is direct arteriovenous diffusion of O2 between thin-walled arterioles and venules that lie in close proximity to one another. This is however not felt to be a very important limitation to O2 transport. Another is tissue vascular shunting, the diversion of blood through vascular channels that directly connect arterioles and venules, bypassing the tissue cells. This may be important in certain tissues

PERIPHERAL GAS EXCHANGE 321

such as the skin, but appears less important in, for example, exercising skeletal muscle. However, possibly more important is perfusion/metabolism inequality. This may occur both between organs and within organs, and acts much like ventilation/perfusion inequality in the lungs (see Ventilation, Perfusion Matching), impairing the transport of O2 between the tissue vasculature and the mitochondria. It remains very hard to assess experimentally, and therefore its role remains uncertain in both health and disease. When such inequality exists, tissue regions that are underperfused in relation to their metabolic O2 needs cannot get the O2 they require while those that are overperfused, receive more O2 than they can use. Tissue dysfunction may then occur in the underperfused regions, even if overall O2 delivery to the tissue appears normal. Early data from initial methods to examine such inequality has revealed modest levels of heterogeneity that would be predicted to have only minor effects on tissue function, at least in exercising skeletal muscles.

Limits to O2 Transport/Utilization in Health and Disease Central and Peripheral Limitations

It has become popular to ask whether maximal O2 utilization, for example during exercise, is limited by ‘central’ or ‘peripheral’ factors. Most workers define central factors as those pertaining to the lungs, blood, and cardiovascular system, while peripheral factors are thought of as those affecting muscle tissue O2 extraction. This has a certain logic and appeal, since O2 ’ O in eqn [2]) and O2 extraction can be delivery (Q 2 separately measured. However, there are several problems with this concept, both in theory and experimentally. The first is that while systemic O2 delivery (eqn [2]) at first sight seems to separate central and ˙ , Hb, and SaO are supposperipheral factors since Q 2 edly centrally determined by the heart, blood, and lungs, this is an oversimplification. Most importantly, when interested in specific tissues, it is blood flow to those tissues that is important. Peripheral vascular factors that affect between-organ blood flow distribution thus are included implicitly in the supposedly central component. Regarding the ‘peripheral’ component, as eqn [5] shows, extraction is as dependent ˙ ) as on the diffusion conductance on blood flow (Q ˙ is in part determined by cardiac function, (D). Since Q extraction cannot be taken as a simple index of peripheral function. Thus, the so-called ‘central’ factors affect the so-called ‘peripheral’ factors, and vice versa. To separate central from peripheral factors, one needs to determine D (eqn [5]) per se, not just

extraction. This can be done using the graphical analysis shown in Figure 4. Proper assessment of central function really requires not just tissue/organ blood flow measurements but also cardiac output as well. Proper assessment of peripheral function requires measuring not just extraction, but also diffusional conductance, D, as well. The most closely studied system examining limits to O2 transport and utilization in humans is the skeletal muscle during exercise. While there is interest in other organs and tissues, the muscles collectively have, by far, the highest ability to consume O2 than any organ system. This makes exercise an attractive model for studying O2 transport limitations. Moreover, maximal muscle O2 utilization varies greatly among individuals, posing many interesting questions, and impaired muscle function is a hallmark of many chronic diseases, lending clinical significance. O2 Transport/Utilization Limitation in Sedentary Normal Subjects

When sedentary normal subjects exercise maximally, achieving peak V’ O2 values of about 3 l min  1, evidence suggests that they are not limited in their exercise capacity by limits to O2 supply. When given high concentrations of O2 to breathe, exercise capacity is not increased. Similarly, modest reduction in inspired O2 levels does not cause a fall in exercise capacity. This implies that because O2 availability does not affect exercise, peak V’ O2 is limited by metabolic capacity to consume O2. O2 Transport/Utilization Limitation in Athletic Normal Subjects

When sedentary subjects undergo several weeks of intense exercise training, they become sensitive to inspired O2 levels. Thus, exercise capacity increases with hyperoxia and falls with hypoxia. This is also observed in competitive athletes. In contrast to the untrained state, this suggests that in trained subjects, functional limits to the O2 transport system determine maximal exercise capacity. The implication is that adaptation to exercise training augments cellular metabolic potential more than O2 transport. O2 Transport/Utilization Limitation in Patients with Chronic Cardiopulmonary Diseases

It has long been held that in severe chronic lung disease, the ability to ventilate is the factor limiting exercise intensity. Similarly, in patients with severe chronic heart failure, exercise has been considered limited by poor cardiac function alone; in patients with chronic renal failure, the low Hb concentration is blamed for reduced exercise capacity. In support of

322 PERIPHERAL GAS EXCHANGE

this idea, acute administration of O2 often improves exercise capacity (that is, while the extra O2 is being given), suggesting that the muscles could perform better even in the face of continuing primary organ failure if more O2 was available. In fact, skeletal muscle may function at normal or near-normal levels if the limiting effect of the failed central organ can be removed. Experimentally, this can be achieved by studying the exercise capacity of small muscle groups. The concept is simply that a small muscle group can be supported by even a failing heart, lung, or kidney, even while more conventional exercise by large muscle groups cannot. Treadmill and cycle exercise involve large groups of muscles, and usually take the failed heart or lungs to their limits of performance before maximal muscle function is reached. Thus, a small muscle group, such as the knee-extensors of one leg, may be able to perform at (near) normal levels when exercised alone. However, when treadmill or cycle exercise is undertaken on the same day, the same patient with cardiopulmonary disease will show clear exercise impairment. Such results show that it is undoubtedly true that the lungs (via limited ventilation and gas exchange impairment), heart (via limited blood flow), and kidneys (via low Hb levels) play major roles in exercise limitation in diseases of those organs, and that this is, at least in part, due to impaired systemic O2 availability. But it is now accepted that additional factors may contribute. There is evidence that not all patients with advanced cardiopulmonary or renal disease have normal skeletal muscles. They certainly exhibit the manifestations of detraining (low peak V’ O2 , early blood lactate appearance, and reduced oxidative enzyme capacity), as might be expected of very sedentary individuals. Exercise training applied to small muscle groups can improve O2 transport and treadmill or cycle exercise capacity even when the original cardiopulmonary failure remains. Training of larger muscle groups can produce effects such as delayed blood lactate appearance, also improving exercise performance. Possibly, there are myopathic changes in some patients that contribute to exercise limitation over and above those of detraining, even in those patients with normal skeletal muscle mass. However, this is still a subject of considerable debate among the research community. Severe loss of muscle mass is sometimes seen in advanced diseases of the heart and lungs, and while we do not fully understand the reasons for this, it has been proposed that this may be a systemic manifestation of cardiopulmonary disease resulting from increased levels of circulating inflammatory cytokines such as tumor necrosis factor alpha (TNF-a) and interleukins. Clearly this is a myopathic

manifestation of these diseases and must contribute to exercise limitation. In spite of such possibilities, improving O2 availability through muscle training does lead to improved exercise capacity even while the primary cardiopulmonary disease is present. Even small improvements have clinical significance in patients whose exercise ability is severely impaired to begin with. Heart and/or lung transplantation form a natural model to study the consequences of cardiopulmonary disease on O2 transport and exercise. Surprisingly, well after recovery from the surgery itself, lung transplant patients show incomplete restoration of exercise capacity despite normalization of ventilation and pulmonary gas exchange. Typically, patients are capable of no more than 50% of what an agematched normal subject can achieve. This commonly found result suggests that severe heart or lung disease has systemic effects on O2 transport and utilization that give rise to a longstanding peripheral component to exercise limitation that persists even after transplantation. While not fully understood, it is known that immunosuppressive drugs required to control organ rejection after transplant are myopathic and may be more likely responsible for this outcome. In summary (in the absence of muscle wasting), it remains unclear whether severe cardiopulmonary diseases per se affect muscles beyond the effects of detraining. Even if they do, regular exercise will improve exercise capacity to some extent and usually will improve quality of life. See also: Diffusion of Gases. Hemoglobin. Ventilation, Perfusion Matching. Ventilation: Overview.

Further Reading Hogan MC, Bebout DE, and Wagner PD (1991) Effect of increased Hb–O2 affinity on VO2max at constant O2 delivery in dog muscle in situ. Journal of Applied Physiology 70: 2656–2662. Krogh A (1919) The number and distribution of capillaries in muscle with calculations of the pressure head necessary for supplying the tissue. Journal of Physiology (London) 52: 409–415. Maltais F, Jobin J, Sullivan MJ, et al. (1998) Metabolic and hemodynamic responses of lower limb during exercise in patients with COPD. Journal of Applied Physiology 84: 1573–1580. Maltais F and LeBlanc P (2000) Peripheral muscle dysfunction in chronic obstructive pulmonary disease. Clinical Chest Medicine 21: 665–677. Maltais F, LeBlanc P, Simard C, et al. (1996) Skeletal muscle adaptation to endurance training in patients with chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 154: 442–447. Maltais F, LeBlanc P, Whittom F, et al. (2000) Oxidative enzyme activities of the vastus lateralis muscle and the functional status in patients with COPD. Thorax 55: 848–853. Maltais F, Simard AA, Simard C, Jobin J, Desgagnes P, and LeBlanc P (1996) Oxidative capacity of the skeletal muscle and lactic acid kinetics during exercise in normal

PERMEABILITY OF THE BLOOD–GAS BARRIER subjects and in patients with COPD. American Journal of Respiratory and Critical Care Medicine 153: 288–293. Piiper J, Meyer M, and Scheid P (1984) Dual role of diffusion in tissue gas exchange: blood–tissue equilibration and diffusion shunt. Respiratory Physiology 56: 131–144. Piiper J and Scheid P (1981) Model for capillary–alveolar equilibration with special reference to O2 uptake in hypoxia. Respiratory Physiology 46: 193–208. Piiper J and Scheid P (1983) Comparison of diffusion and perfusion limitations in alveolar gas exchange. Respiratory Physiology 51: 287–290. Richardson RS, Noyszewski EA, Kendrick KF, Leigh JS, and Wagner PD (1995) Myoglobin O2 desaturation during exercise: evidence of limited O2 transport. Journal of Clinical Investigation 96: 1916–1926. Richardson RS, Tagore K, Haseler L, Jordan M, and Wagner PD (1998) Increased VO2max with a right shifted Hb–O2 dissociation curve at a constant O2 delivery in dog muscle in situ. Journal of Applied Physiology 84: 995–1002.

323

Roca J, Hogan MC, Story D, et al. (1989) Evidence for tissue diffusion limitation of V’ O2 max in normal humans. Journal of Applied Physiology 67: 291–299. Rowell LB (1993) Human Cardiovascular Control. New York: Oxford University Press. Scheid P, Hlastala MP, and Piiper J (1981) Inert gas elimination from lungs with stratified inhomogeneity: theory. Respiratory Physiology 44: 299–309. Wagner PD (1996) Determinants of maximal oxygen transport and utilization. Annual Review of Physiology 58: 21–50. Weibel ER (1984) The Pathway for Oxygen. Structure and Function in the Mammalian Respiratory System. Cambridge, MA: Harvard University Press. Williams TJ, Patterson GA, McClean PA, Zamel N, and Maurer JR (1992) Maximal exercise testing in single and double lung transplant recipients. American Review of Respiratory Diseases 145: 101–105. Wilson DF, Erecinska M, Drown C, and Silver IA (1977) Effect of oxygen tension on cellular energetics. American Journal of Physiology 233: C135–C140.

PERMEABILITY OF THE BLOOD–GAS BARRIER R M Effros, Harbor–UCLA Medical Center, Torrance, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Two continuous membranes separate the blood and gas phases of alveoli: the endothelium and the epithelium. Gases readily dissolve in cellular membranes of both layers and rapidly exchange between blood and air. Carbon monoxide is generally used to monitor gaseous diffusion. Electrolytes and water can cross intercellular junctions, but water molecules can also traverse a variety of specific water channels (aquaporins) in cell membranes. The endothelium restrains the movement of proteins but is relatively permeable to electrolytes. Transcapillary edema formation is governed by hydrostatic and protein osmotic gradients (Starling forces). The epithelium resists passive movement of both electrolytes and proteins, and airspace edema formation is primarily restrained by osmotic buffering of electrolytes. Capillary injuries, including elevations in capillary pressures, can result in leakage of proteins and red cells into the interstitium, from where they can make their way into the airspaces if the epithelium is ruptured by increases in interstitial pressure. Edema fluid is normally reabsorbed from the airspaces by active sodium transport. Uncertainty persists regarding the osmolality of airway fluid, and methods of collecting respiratory fluid (bronchoalveolar lavage and exhaled breath condensates) have been complicated by uncertainty regarding dilution. Increased epithelial permeability to solutes can be detected by accelerated clearance of radioaerosols from the lungs.

Description Gases

Since most gas exchange occurs in the alveoli, this discussion of gas exchange is primarily limited to the

permeability of the alveoli. The tissue barriers that separate blood from air in the alveoli (Figure 1) are arranged to maximize gas exchange and minimize fluid movement into the airspaces. Exchange of O2 and CO2 is enhanced by the arrangement of capillaries and epithelial cells, with extremely attenuated endothelial and epithelial membranes facing the alveolar gas. The movement of gases across this barrier is accomplished by passive diffusion – that is, random movement of gas molecules from compartments in which they are more numerous to those in which they are less numerous. The rate of diffusion is limited by the resistance of the membranes to gas movement, the molecular weight of the gaseous molecules, the solubility of the gases in the barrier, and the area and thickness of the barrier. The high solubility of most gases in the cell membrane lipids is responsible for the remarkable efficiency of gas exchange in the lungs. Similarly, lipophilic solutes instilled into the lungs (e.g., the alcohols, nicotine, and acetone) also diffuse rapidly between the airspace and blood compartments. Equilibration of inert gases between the airspaces and capillaries is virtually complete well before the blood has traversed the pulmonary microcirculation. However, equilibration of oxygen between the airways may not be complete, particularly in the presence of disease, because of the large binding capacity of hemoglobin for oxygen, which provides a large ‘sink’ for this gas. As described elsewhere, the higher affinity of hemoglobin for CO makes it easier to use this indicator for evaluating gaseous diffusion because it can be assumed that PCO in the capillary remains negligible. Diffusion of

324 PERMEABILITY OF THE BLOOD–GAS BARRIER Nucleus Basement membranes Narrow tight junctions

Electrolytes and water

Vesicles O2, CO2, CO, & other gases

RBC

Type 1 pneumocyte Alveolus Type 2 pneumocyte

Water Sodium transport ELF with surfactant

Capillary

Aquaporin-5

Interendothelial junction Water Aquaporin-1 Lymphatic Figure 1 Transport of gas, water, and solutes in the alveolus. Gases (yellow) and lipid-soluble solutes diffuse directly through the lipid in the cell membranes between the air and blood compartments. Water is transported through aquaporins and intercellular junctions. Small lipophobic solutes (primarily electrolytes) diffuse more rapidly through the interendothelial junctions than the narrow tight junctions of the epithelial cells. Proteins are transported into cells by a variety of vesicular processes. Protein that leaks into the interstitium is collected in the lymphatics and returned to the systemic blood. ELF, epithelial lining fluid; RBC, red blood cell.

CO2 out of the lungs is facilitated by high solubility in lipid as well as carbonic anhydrase in red cells and on the endothelial surface. Solutes and Water

Whereas gases readily traverse both the endothelium and the epithelium, this is not true for electrolytes and other solutes that are insoluble in the lipids of the cell membranes. Furthermore, the sites of solute exchange in the airspaces and airways are less well defined. However, in general, the properties of the endothelium and epithelium are distinctly different. Much of this difference is related to the structure of the intercellular junctions that join adjacent cells. These are much more tenuous in the endothelium than in the epithelium. Interendothelial junctions are in the form of a discontinuous barrier, whereas a complex series of strands seals the epithelial junctions. Passive solute and water movement may occur through intercellular junctions of these membranes, but selective water channels (aquaporins) in the cell membranes are also available for water transport. Most of the alveolar epithelial surface is normally covered by flat alveolar type I cells that are well adapted for gas exchange. Type II cells are more numerous but are relatively spherical and cover a much

smaller fraction of the surface. Type II pneumocytes act as precursors for type I cells and may replace the latter after lung injury. They produce surfactant, which is vital for reducing surface tension, thereby avoiding collapse of airspaces. Many other cells and glands can be found in the airways. A thin (o1– 35 mm) layer of fluid (the epithelial lining fluid (ELF)) covers the epithelial membranes. Despite important advances in understanding how fluid is reabsorbed from the lungs, uncertainty persists concerning the normal osmolality of ELF. Solute and Water Movement across the Blood–Gas Barrier

The forces responsible for passive solute and water movement of solutes across the blood–gas barriers can be understood in terms of the following equation: ! X X JV ¼ Kf S DP
sp Dpp
ss Dps p

s

The rate of filtration across each membrane (JV) is determined by the filtration coefficient (Kf) multiplied by the surface area of the membrane (S) and the difference between the hydrostatic pressure difference across the membrane, DP, and the osmotic

PERMEABILITY OF THE BLOOD–GAS BARRIER

forces that tend to restrain the movement of fluid. The osmotic forces can be divided into those exerted by individual proteins (spDpp) and those exerted by small molecules (ssDps), primarily the electrolytes. The values of Dp (osmotic pressure) are determined by the difference in solute concentrations on either side of the membrane, whereas s, the reflection coefficient, is related to the rate at which the molecule leaks across the membrane. Endothelial membranes restrain the movement of proteins (spE1.0) but are extremely permeable to small molecules (ssE0.0) that leak through the interendothelial junctions, and flows of fluid are therefore promoted by hydrostatic pressures and restrained by the tendency for Dpp to increase with filtration (Starling hypothesis). The basement membranes below the cells may help restrict protein movement. Interstitial fluid and protein can also be removed by the pulmonary lymphatics. The interepithelial junctions resist the movement of both large and small molecules (spE1.0, ssE1.0). Because the molar concentrations of electrolytes are more than 100 times greater than those of proteins, protein differences probably play a minor role in the rate of fluid movement across membranes. Furthermore, edema formation related to increased hydrostatic pressure differences across intact epithelial membranes is severely limited by increases in electrolyte concentration differences that would accompany shifts of solute-free water (e.g., through aquaporins). For example, if the flow of water into the airspaces reduced the concentration of Na þ and Cl
in the airspace by 1 mmol l
1, an osmotic pressure gradient of 40 mmHg would be produced, which would inhibit any further movement of water in this direction. This phenomenon is referred to as ‘osmotic buffering’. Whereas reabsorption of fluid from the pulmonary interstitium may be promoted by increased plasma protein concentrations and lymphatic drainage, reabsorption of fluid from the airspaces is primarily related to active transport of sodium, which can increase protein concentrations well above those in the plasma, illustrating the relatively modest effect that protein concentration gradients have across the pulmonary epithelium. However, absorption of protein from the distal airspaces is much slower than that of electrolytes, and accumulation of protein in the airspaces could slow fluid clearance, particularly if precipitated layers of protein are formed on the epithelial surfaces (hyaline membrane changes). Protein molecules are cleared from the airspaces more slowly than smaller molecules. Some of the clearance of specific protein molecules is mediated by specific receptors and transport can occur through a variety of endocytic mechanisms, but a fraction of the protein may be degraded by proteolytic enzymes.

325

Radioaerosol clearances (usually containing 99mTcdiethylenetriamine pentaacetate (99mTc-DTPA)) have been used clinically to detect increased leakage of solutes across the epithelium (Table 1). Similarly, injected radiotracers (usually including labeled albumin and/or urea) have been used to detect alterations in endothelial permeability. Bronchoalveolar lavage (BAL) during bronchoscopy is commonly used to measure alterations in the constituents of the fluid that covers the ELF. These could presumably be altered by changes in the permeability of the blood–gas barrier. Exhaled breath condensates (EBCs) have been collected by breathing into a cooled condenser to provide similar information in a convenient, noninvasive manner (Table 2). Both approaches have been complicated by uncertainty concerning the dilution of ELF by instilled saline during BAL and by water vapor in EBCs. BAL typically dilutes ELF by a factor of approximately 100, whereas

Table 1 Accelerated clearance of radioaerosols Condition

Species

Adult respiratory distress syndrome a-Thrombin a-Naphthyl thiourea Amiodarone Asbestos Bleomycin Bullous emphysema Burns, pulmonary Cigarettes Cocaine (inhaled crack) Coal workers’ pneumoconiosis Cytotoxic agents Distension of lungs Exercise Extrinsic allergic alveolitis Fire fighting Hemorrhagic fever and renal syndrome Histamine Hydrochloric acid Hypotonic ‘fog’ Idiopathic pulmonary fibrosis Inverse ratio ventilation Neonatal respiratory distress Oleic acid Oxygen 450% Ozone Paraquat Pneumocystis carinii pneumonia Progressive systemic sclerosis Radiation pneumonitis Renal failure (slowed by dialysis) Sarcoidosis Sauropus androgynus vegetable Silicosis Smoke (wood) Surfactant deficiency Systemic lupus erythematosis

Human Sheep Rat Human Human Human Human Human Human, rat Human Human Human Various Human Human, rat Human Human Human Dog Human Human Dog Human Dog, rabbit Human Human Human Human Human Human Human Human Human Human Dog Rabbit Human

326 PERMEABILITY OF THE BLOOD–GAS BARRIER Table 2 Exhaled breath condensatesa Marker

Disorder

H2O2 Leukotrienes 8-Isoprostane Nitrothiols/nitrotyrosine Nitrite/nitrate Acid Aldehydes Isoprostanes Adenosine Endothelin Vibronectin Glutathione Interleukins-4,6,8 Interferon-g Hepatocyte growth factor

Asthma Chronic obstructive lung disease Smokers Acute lung injury Cystic fibrosis Obstructive sleep apnea Surgery Idiopathic pulmonary fibrosis Dry air Ozone exposure Carcinoma Bronchiectasis Ciliary dyskinesia Pneumonia

a

Markers of inflammation and disorders are listed separately and columns are not related.

droplets of ELF are diluted by approximately 10 000 in EBCs.

Abnormalities of the Blood–Gas Barrier in Respiratory Diseases Gases

Impaired diffusion of oxygen to the capillary blood can be associated with hypoxemia, which should respond to increased airspace oxygen tensions (unlike anatomical shunts, which are remote from ventilated airspaces). Airspace edema may also result in retention of CO2. The diffusing capacity of CO is frequently decreased in many restrictive, obstructive, and pulmonary vascular diseases. Solutes and Water

Edema formation in the lungs is most commonly caused by increases in intracapillary pressures. These increases are frequently caused by high left atrial pressures in patients with congestive heart failure. Edema initially collects in the interstitium and then tends to track into the perivascular and peribronchial compartments, a phenomenon that can be detected on X-ray. Provided these pressures do not damage the endothelium, thereby decreasing sp, the protein concentrations of the edema fluid remain relatively low (transudative edema). Decreases in plasma protein concentrations (e.g., due to nephrotic syndrome or severe liver disease) should increase the incidence for pulmonary edema, but it is more frequently detected in congestive heart failure. Nor has it been convincingly demonstrated that increasing serum

albumin concentration can remove fluid from the interstitium of the lungs or airspaces. Early vascular congestion may be associated with wheezing but relatively mild hypoxia. More severe increases in intravascular pressures or other disease processes may result in leakage of proteins (decreased sp), and even red cells, into the interstitium. Although most epithelial barriers can withstand relatively large increases in intraluminal pressures, even minor increases in interstitial pressure can be associated with rupture of the membranes and leakage of fluid into the airspaces. It has been suggested that this leakage might occur at a site in the respiratory bronchioles, but a corresponding channel has not been identified. The entry of fluid into the airspaces is characterized by increasing dyspnea with rales and wheezing, abnormalities in arterial blood gases (hypoxia and, if ventilation is impaired, hypercapnia), and radiological evidence for alveolar edema. Injury to the pulmonary epithelium is a common consequence of a wide variety of diseases, including very elevated airway pressures (e.g., by ventilators), and may be manifested by the formation of hyaline membranes in the alveoli and the movement of protein, blood, inflammatory cells, and mediators into the airspaces. Increased clearance of 99mTc-DTPA from the lungs has been observed in a wide variety of disorders that are associated with injuries at various sites in the pulmonary epithelium (Table 1). Bronchoalveolar lavage is frequently used to detect inflammatory and neoplastic cells, microorganisms, and inflammatory mediators in the lungs. Increases have been observed in the concentrations of many inflammatory mediators in samples of exhaled breath condensates (Table 2) collected from patients with asthma and other inflammatory lung disorders. However, these abnormalities may also be due to an increase in the number and/or volume of respiratory droplets that reach the condenser rather than a corresponding increase in concentrations in ELF. It has been suggested that the electrolyte concentrations of EBCs can be used to estimate the dilution of ELF by water vapor. Contamination of EBCs by oral gases (especially NH4þ , which represents the principal cation in EBCs) and salivary, nasal, and even gastric droplets is another important problem. Acidification of EBCs has also been described in asthma and other airway illnesses, but this change could be related to aerosolization of gastric fluid rather than an alteration in ELF pH. Gastroesophageal reflux is particularly prominent in patients with airway obstruction and coughing, and the presence of even a few gastric droplets (pH 1–2) could result in acidification of EBCs.

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS 327 See also: Adhesion, Cell–Cell: Vascular; Epithelial. Aquaporins. Arterial Blood Gases. Bronchoalveolar Lavage. Carbon Dioxide. Carbon Monoxide. Caveolins. Diffusion of Gases. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes. Fluid Balance in the Lung. Ion Transport: Overview. Peripheral Gas Exchange. Surfactant: Overview.

Glossary ELF – epithelial lining fluid. Attenuated layer of fluid which covers the epithelial surface BAL – bronchoalveolar lavage. Lungs are lavaged with saline to recover cellular, microbial, and solute constituents of ELF EBC – exhaled breath condensate

Further Reading

Aquaporins – water channels in cell membranes

Berthiaume Y, Folkesson HG, and Matthay MA (2002) Lung edema clearance: 20 years of progress. Invited review: alveolar edema fluid clearance in the injured lung. Journal of Applied Physiology 93: 2207–2213. Boitano S, Safdar Z, Welsh DG, Bhattacharya J, and Koval M (2004) Cell–cell interactions in regulating lung function. American Journal of Physiology: Lung Cellular and Molecular Physiology 287: L455–L459. Borok Z and Verkman AS (2002) Lung edema clearance: 20 years of progress. Invited review: role of aquaporin water channels in fluid transport in lung and airways. Journal of Applied Physiology 93: 2199–2206. Effros RM and Chang HK (eds.) (1994) Fluid and Solute Transport in the Airspaces of the Lungs, Lung Biology in Health and Disease Series (Lenfant C, executive ed.). New York: Dekker. Effros RM, Dunning M, Biller J, and Shaker R (2004) The promise and perils of exhaled breath condensates. American Journal of Physiology: Lung Cellular and Molecular Biology 287: L1073– L1080. Kim K-J and Malik AB (2003) Protein transport across the lung epithelial barrier. American Journal of Physiology: Lung Cellular and Molecular Physiology 284: L247–L259. Rottoli P and Bargagli E (2003) Is bronchoalveolar lavage obsolete in the diagnosis of interstitial lung disease? Current Opinion in Pulmonary Medicine 9: 418–425. Weibel ER (1973) Morphological basis of alveolar–capillary gas exchange. Physiological Reviews 53: 419–495. West JB, Mathieu-Costello O, Jones JH, et al. (1993) Stress failure of pulmonary capillaries in racehorses with exercise-induced pulmonary hemorrhage. Journal of Applied Physiology 75: 1097–1109. Wright EM (1983) Solute and water transport across epithelia. American Review of Respiratory Disease 127(5 Pt 2): S3–S8.

Basement membranes – proteinacious membrane which supports the endothelial and epithelial membranes 99m

Tc-diethylenetriamine pentaacetate (99mTc-DTPA) – an extracellular indicator that is frequently incorporated in radioaerosol studies of pulmonary epithelial permeability

Nomenclature JV

flow of fluid across the membrane (e.g., ml min
1)

Kf

filtration coefficient, e.g., (ml min
1)/ (mmHg difference  cm2 surface area)

S

surface area of membrane

s

index of leakiness of membrane to solute (dimensionless). If s ¼ 1, no leakage occurs; if s ¼ 0, then the solute readily leaks across the membrane and can exert no osmotic pressure

DP

hydrostatic pressure difference across the membrane (e.g., mmHg)

Dp

osmotic pressure which would be exerted by the concentration difference of a solute across the membrane if the membrane were completely impermeable to the solute

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS J Roman-Rodriguez, C M Hart, and S W Han, Emory University School of Medicine, Atlanta, GA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Peroxisome proliferator-activated receptors (PPARs) are members of the ligand-activated nuclear hormone receptor

superfamily of transcription factors that includes receptors for steroids, thyroid hormone, retinoic acid, and vitamin D. Upon activation in the cytoplasm, PPARs heterodimerize with retinoid X receptors (RXR) and form a complex that translocates to the nucleus and regulates gene expression. PPARs are thought to play roles in diverse physiological processes ranging from lipid metabolism to inflammation, and have been implicated in diseases such as cancer, atherosclerosis, and diabetes. Although information about the function of PPARs in lung is scarce, data

328 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS implicating these molecules in key processes in lung biology are rapidly emerging. In lung, PPARs are expressed by recruited immune cells (e.g., monocytes) and in resident cells such as tissue macrophages, bronchial and alveolar epithelial cells, endothelium, and airway smooth muscle, among other cell types. Of the three PPAR forms (PPAR-a, PPAR-b/d, PPAR-g), PPAR-g is the best studied, and it is believed to regulate cellular differentiation and proliferation. Several studies suggest that PPAR-g serves to downregulate lung inflammation, promotes vascular function, and acts as a tumor suppressor in lung and other carcinomas.

about the function of PPARs in lung is scarce, data implicating these molecules in key processes in lung biology are rapidly emerging. PPARs are characterized by three functional domains: the N-terminal domain (a site for functional regulation by phosphorylation), the DNA-binding domain, and the ligand-binding domain. Upon activation in the cytoplasm, PPARs heterodimerize with retinoid X receptors (RXR) and form a complex that translocates to the nucleus and regulates gene expression (Figure 1). Ligand binding to PPARs appears to trigger conformational changes that permit their dissociation from co-repressors and favor their association with coactivators (e.g., steroid receptor coactivator-1 and p300). The heterodimer–coactivator complex binds to specific response elements (termed PPREs) in the promoter regions of target genes that consist of a direct repetition of the consensus A-GG-T-C-A half site spaced by one or two nucleotides (DR1 or DR2) (Figure 1A). The coactivator proteins possess or recruit histone acetyltransferase activity to the transcription start site. Acetylation of histone proteins alters chromatin structure, facilitating the binding of RNA polymerase and the initiation of transcription. In addition to their stimulatory effects on gene transcription, PPARs can repress gene expression in a DNA-binding-dependent manner through the recruitment of co-repressors to

Introduction Since their discovery in 1990, peroxisome proliferator-activated receptors (PPARs) have captured the attention of investigators interested in learning about the intracellular pathways that control signal transduction and gene transcription. PPARs were originally cloned in an attempt to identify the molecular mediators of peroxisome proliferation in the liver of rodents. Today, PPARs are recognized as versatile members of the ligand-activated nuclear hormone receptor superfamily of transcription factors that includes receptors for steroids, thyroid hormone, retinoic acid, and vitamin D. PPARs are considered to play key roles in diverse physiological processes ranging from lipid metabolism to inflammation, and have been implicated in diseases such as cancer, atherosclerosis, and diabetes. Although information

Activation

PPAR-
agonists Ligandbinding domain

CoAct

RXR

PPAR RXR

CoRep DNAbinding domain

A

PPRE

PPAR-
B C

Jun Fos

Repression

p65 p50

pCREB STAT1 STAT3

Smads

CoRep

RXR

PPAR PPRE

Trans-repression Figure 1 Differential regulation of gene expression by PPAR-g. Upon activation in the cytoplasm, PPARs heterodimerize with retinoic X receptors (RXR) and form a complex that translocates to the nucleus and regulates gene expression. Ligand binding to PPARs permits their dissociation from co-repressors (CoRep) and favors their association with coactivators (CoAct). The heterodimer–coactivator complex binds to specific response elements (termed PPREs) in the promoter regions of target genes (A). PPAR-g can also repress gene expression in a DNA-binding-dependent manner through the recruitment of co-repressors to unliganded PPARs (B) or in a DNA-binding-dependent manner by interfering with key transcription factors (C) (not all PPARs act in the same fashion and ligands of other PPARs (e.g., PPAR-a) might stimulate gene transcription through the activation of transcription factors).

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS 329

unliganded PPARs (Figure 1B) or in a DNA-bindingindependent manner by interfering with key transcription factors (Figure 1C). Three subtypes of PPARs have been identified and cloned: PPAR-a, PPAR-b/d, and PPAR-g. These subtypes are distinguished by their tissue distribution, and to a lesser degree, by their ligand specificity. Most studies defining the role of PPARs in lung structure and function have examined PPAR-g. Therefore, for the most part, this review focuses on the role of PPAR-g in lung health and disease. PPARs are activated by structurally diverse ligands. PPAR-g is activated by the synthetic thiazolidinedione (TZD) class of insulin-sensitizing agents such as troglitazone, rosiglitazone, and pioglitazone, and certain nonsteroidal anti-inflammatory drugs (e.g., indomethacin, ibuprofen, fenoprofen, flufenamic acid). Natural ligands for PPAR-g include prostanoids, prostaglandin D2, 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2), and certain polyunsaturated fatty acids. These reagents have been used to elucidate the role of PPAR-g in cellular functions both in vitro and in vivo. However, several caveats should be taken into consideration when interpreting such studies. First, the natural ligands that regulate PPARs in vivo have not been determined. Second, not all PPAR-g ligands exert their effects through PPAR-g since there is strong evidence for the activation of PPAR-g-independent signals, particularly with the natural ligand 15d-PGJ2. Third, high affinity ligands for PPAR-g (e.g., the TZDs) may exert partial agonist/antagonist activity. The latter might be due to the fact that individual TZDs induce different PPAR-g conformations that influence the recruitment of different coactivator/co-repressor molecules. Thus, the activity of the PPAR-g transcriptional complex is influenced by the context of a given gene and its promoter, and by the relative availability of pertinent coactivator/corepressor molecules in the cell or tissue of interest.

PPARs in Lung In lung, PPARs are expressed by recruited immune cells (e.g., monocytes) and in resident cells such as tissue macrophages, bronchial and alveolar epithelial cells, endothelium, and airway smooth muscle, among other cell types. In developing murine lungs, PPAR-g expression was found to increase as the lung matured. Although the exact roles PPAR-g and other PPARs play in fetal and adult lungs are unknown, their wide distribution and their ability to regulate cellular proliferation and differentiation as well as apoptosis in vitro suggest important functions for PPARs in lung homeostasis. The factors that regulate PPAR expression in lung also remain unelucidated.

In this regard, it has been reported that agents known for their ability to stimulate alveolar epithelial type II cell differentiation (e.g., dexamethasone, retinoic acid, cAMP, growth factors) stimulate PPAR-g expression (Figure 2). Several studies performed in lung tissues and in isolated primary lung cells have begun to unveil potential roles for PPARs in lung including surfactant homeostasis. Specifically, it was found that 15d-PGJ2 and 9-hydroxyoctadecanoic acid upregulate PPAR-g and downregulate surfactant protein B expression in cultured cells and in fetal lung explants. In other work, PPAR-g agonists were shown to inhibit elastin production and a-smooth muscle actin expression in fibroblasts, which suggests a role in myofibroblast differentiation. In cultured bronchial epithelial cells, PPAR-g agonists exert antiproliferative effects and inhibit the expression of matrix metalloproteinase-9. In human airway smooth muscle cells, PPAR-g agonists inhibit proliferation induced by several stimulants. This and other information discussed below suggest that, at baseline, PPARs might help maintain homeostasis by inhibiting inflammation, and perhaps tissue remodeling, and that their downregulation might predispose to disease. This is consistent with data showing decreased PPAR-g expression in the alveolar macrophages of humans with alveolar proteinosis and in the vascular lesions of subjects with pulmonary hypertension. PPARs, Airways, and Modulation of Inflammation

Despite their demonstrated role in cellular proliferation and differentiation, it is the anti-inflammatory activities of PPARs that have attracted much attention. Using a model of paw edema induced by subplantar injection of carrageenan, it was found that rosiglitazone inhibited the edema in a dose-dependent fashion. Rosiglitazone also attenuated polymorphonuclear cell infiltration of the pleural cavity, neutrophil infiltration of lung tissue, and lipid peroxidation after injection of carrageenan into the pleura. A general exploration of the potential pathways involved in these effects revealed decreased tissue staining for intercellular adhesion molecule-1 and p-selectin, which could explain the reduction in immune cell recruitment into the lung. Reductions in nitric oxide levels and the activity of inducible nitric oxide synthase, and decreases in levels of tumor necrosis factor alpha and interleukin1b were also detected. PPAR-g agonists have also been shown to ameliorate airway neutrophilia and the associated expression of chemoattractants in animals exposed to aerosolized endotoxin. The documentation of the anti-inflammatory effects of PPAR-g ligands has led to investigations testing

330 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS Endothelial cells

PPAR-
agonists Fibroblasts

Decreased proliferation

CoRep or CoAct

Lung carcinoma cells

PPAR RXR RXR

Decreased matrix production

Bronchial−epithelial cells

Decreased proliferation

Decreased proliferation

Monocyte/ macrophages

Alveolar type II epithelial cells Airway smooth muscle

Inhibition of cytokine production

Decreased proliferation and migration

Increased differentiation and surfactant production Figure 2 Effects of PPAR-g agonists on lung cells. PPAR-g is expressed in most lung cell types and it has been implicated in the regulation of surfactant production. PPAR-g induction inhibits matrix expression in fibroblasts, reduces cytokine expression in monocyte/ macrophages, inhibits the proliferation and migration of smooth muscle cells, and reduces the proliferation and expression of intercellular adhesion molecules in endothelial cells. In lung carcinoma cells, PPAR-g is believed to inhibit tumor growth.

their potential role in experimental models of asthma. In a murine model of asthma induced by sensitization and airway challenge with ovalbumin, PPAR-g was found to be expressed mainly in airway epithelium after antigen sensitization. The PPAR-g agonist ciglitazone (administered by nebulization or by means of gavage) decreased airway hyperresponsiveness, basement membrane thickness, mucus production, collagen deposition, and transforming growth factor beta-1 synthesis. PPAR-g agonists were also shown to decrease the proliferation of antigen-specific T cells and to increase the production of the immunomodulatory cytokine interleukin-10. These and other studies suggest that PPARs, especially PPAR-g, exert anti-inflammatory roles in lung and other organs. As such, their activation might serve to counter inflammatory responses induced by injury, and their dysregulation might promote uncontrolled tissue damage due to unopposed inflammation and, perhaps, tissue remodeling. Prolonged leukotriene B4-induced inflammatory responses in PPAR-a-deficient mice suggest potential anti-inflammatory roles for PPAR-a as well. How PPAR agonists inhibit inflammation is the subject of intense investigation. Mounting evidence indicates that PPAR-g inhibits the expression of proinflammatory mediators through effects on potent transcription factors such as nuclear factor kappa B (NF-kB), STAT1,

and activation protein-1 (AP-1). Similar observations have been made for the transcription factors Smad3 and cAMP element binding protein (CREB). PPARs in the Lung Vasculature

Several studies point to a potential protective role for TZDs in vascular disease and suggest that PPAR-g activation in the vascular wall has predominantly antiatherogenic effects. These studies are supported by limited in vivo data in animal and human subjects indicating that TZD therapy is associated with improved endothelial function. However, the role of PPARs in the pulmonary vasculature remains to be defined. The abundance of immunoreactive PPAR-g has been found to be greater in type II epithelial cells than in type I epithelial cells, bronchial epithelial cells, or endothelial cells in the lung. However, despite relatively lower expression of PPAR-g in endothelial compared to epithelial cell compartments, the anti-inflammatory effects of PPAR-g activation appear to extend into the vasculature. In the rat model of sepsis induced by cecal ligation and perforation, treatment with the PPAR-g ligands 15d-PGJ2 or ciglitazone reduced sepsis-mediated hypotension, vascular injury, and pulmonary neutrophil infiltration and resulted in significant improvements in survival. In this study, PPAR-g ligands attenuated sepsis-induced

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS 331

reductions in lung PPAR-g expression and reduced markers of vascular inflammation including nitrotyrosine staining and poly(ADP-ribose) synthetase activation in thoracic aorta, although the relative contributions of pulmonary versus systemic vascular effects of PPAR-g activation were not defined. Other studies have demonstrated that PPAR-g ligands attenuate vascular injury and dysfunction in zymosaninduced sepsis and in ischemia–reperfusion-related lung injury. The role of PPAR-a or -d in the regulation of pulmonary vascular responses has not been examined. Based on the ability of PPAR-g to regulate vascular smooth muscle cell migration and proliferation, apoptosis, and angiogenesis, several investigators examined PPAR-g expression in lung tissue from patients with primary and secondary forms of pulmonary hypertension and from normal lung tissue. PPAR-g expression was evident in alveolar wall structures and small vessel endothelium in normal lung, and its expression was attenuated in these structures from patients with pulmonary hypertension. Interestingly, PPAR-g expression was also reduced in the complex vascular lesions comprised of proliferating endothelial cells in an experimental rat model of pulmonary hypertension. In ECV304 cells, a human endothelial-like cell line, shear stress decreased PPAR-g expression suggesting that altered blood flow in hypertensive pulmonary vessels could account for reduced PPAR-g expression and the proliferative endothelial lesions in this disease. Supporting this concept were additional data that overexpression of PPAR-g in ECV304 cells reduced, while dominant-negative PPAR-g enhanced, the in vitro angiogenic potential of this endothelial cell line. Although a causal relationship between reduced PPAR-g and endothelial proliferation, vascular obstruction, and pulmonary hypertension remains to be established, recent evidence that PPAR-g activation stimulates endothelial nitric oxide release provides additional potential mechanistic links between PPAR-g and pulmonary hypertension.

Several studies have implicated PPAR-g in lung cancer as well. PPAR-g is expressed in small cell and non-small cell lung carcinomas (NSCLC), and PPAR-g agonists induce growth arrest and induce changes associated with differentiation as well as apoptosis in a variety of lung carcinoma cell lines. For example, PPAR-g agonists have been found to inhibit the growth of A549 adenocarcinoma cells due to G0/G1 cell-cycle arrest through the downregulation of G1 cyclins D and E; this was related to sustained Erk 1/2 activation. Most notably, the treatment of NSCLC tumor-bearing SCID mice with a PPAR-g ligand inhibited tumor growth and metastasis. PPAR-g agonists have also been shown to inhibit lung carcinoma cell proliferation through inhibition of cyclooxygenase-2 and reduction in the expression of prostaglandin E2 receptors. In primary NSCLC, the expression of PPAR-g was correlated with tumor histological type and grade, and decreased PPAR-g expression was correlated with poor prognosis. Thus, it has been postulated that PPAR-g mRNA levels may serve as a prognostic marker in lung carcinoma in addition to playing important roles in lung carcinogenesis. However, the first clinical trials to make use of the antineoplastic effects mediated by PPAR-g have shown conflicting results. In summary, although their exact role in lung remains undefined, PPARs are believed to regulate cellular differentiation and proliferation, and studies performed with agonists of PPAR-g, the best studied of the PPARs, suggest that this form of PPAR serves to downregulate lung inflammation, promotes vascular function, and acts as a tumor suppressor in lung carcinomas. The availability of PPAR-g agonists in the clinical arena will aid in investigations directed at studying the true role of PPARs in lung health and disease.

PPARs in Lung Cancer

Further Reading

In view of its ability to inhibit cellular proliferation and promote differentiation, investigations have been carried out to define the role of PPARs in tumors. This work has revealed that PPAR-g agonists inhibit the proliferation of human breast, prostate, colon, and pituitary cancer cells in vitro. Naturally occurring somatic mutations in the gene encoding PPAR-g have been found in sporadic colorectal carcinomas. These findings support a role for PPAR-g as a potential tumor suppressor.

Amershina S, Golpon H, Cool CD, et al. (2003) Peroxisome proliferators-activated receptor gamma (PPARg) expression is decreased in pulmonary hypertension and affects endothelial cell growth. Circulation Research 92: 1162–1169. Asada K, Sasaki S, Suda T, Chida K, and Nakamura H (2003) Antiinflammatory roles of peroxisome-activated receptor gamma in human alveolar macrophages. American Journal of Respiratory and Critical Care Medicine 169: 195–200. Calnek DS, Mazella L, Roser S, Roman J, and Hart CM (2003) Peroxisome proliferator-activated receptor gamma ligands increase release of nitric oxide from endothelial cells. Arteriosclerosis Thrombosis and Vascular Biology 23: 52–57.

See also: Lipid Mediators: Overview; Prostanoids. Smooth Muscle Cells: Vascular. Transcription Factors: NF-kB and Ikb.

332 PHYSIOTHERAPY Clark RB (2002) The role of PPARs in inflammation and immunity. Journal of Leukocyte Biology 71: 388–400. Cuzzocrea S, Pisano B, Dugo L, et al. (2004) Rosiglitazone, a ligand of the peroxisome proliferators-activated receptor-g, reduces acute inflammation. European Journal of Pharmacology 483: 79–93. Hammad H, de Heer HJ, Soullie T, et al. (2004) Activation of peroxisome proliferators-activated receptor-gamma in dendritic cells inhibits the development of eosinophilic airway inflammation in a mouse model of asthma. American Journal of Pathology 164: 263–271. Honda K, Marquillies P, Capron M, and Dombrowicz D (2004) Peroxisome proliferators-activated receptor gamma is expressed in airways and inhibits features of airway remodeling in a mouse asthma model. Journal of Allergy and Clinical Immunology 113: 882–888. Inoue K, Kawahito Y, Tsubouchi Y, et al. (2002) Expression of peroxisome proliferator-activated receptor (PPAR)-g in human lung cancer. Anticancer Research 21: 2471–2476. Ito K, Shimada J, Kato D, et al. (2004) Protective effects of preischemic treatment with pioglitazone, a peroxisome proliferatorsactivated receptor-g ligand, on lung ischemia–reperfusion injury in rats. European Journal of Cardio-Thoracic Surgery 25: 530–536.

Keshamouni VG, Reddy RC, Arenberg DA, et al. (2004) Peroxisome proliferators-activated receptor-g activation inhibits tumor progression in non-small-cell lung cancer. Oncogene 23: 100–108. Marx N, Duez H, Fruchart JC, and Staels B (2004) Peroxisome proliferators-activated receptors and atherogenesis: regulators of gene expression in vascular cells. Circulation Research 94: 1168–1178. Michalik L, Desvergne B, and Wahli W (2004) Peroxisome proliferators-activated receptors and cancers: complex stories. Nature Reviews 4: 61–70. Rangwala S and Lazar MA (2004) Peroxisome proliferators-activated receptor g in diabetes and metabolism. Trends in Pharmacological Sciences 25: 331–334. Ricote M, Li AC, Wilson TM, Kelly CJ, and Glass CK (1998) The peroxisome proliferators-activated receptor-gamma is a negative regulator of macrophage activation. Nature 391: 79–82. Zingarelli B, Sheehan M, Hake PW, et al. (2003) Peroxisome proliferators activator receptor-gamma ligands, 15-deoxyD(12,14)-prostaglandin J2 and ciglitazone, reduce systemic inflammation in polymicrobial sepsis by modulation of signal transduction pathways. Journal of Immunology 171: 6827– 6837.

PHYSIOTHERAPY A M Yohannes, Manchester Metropolitan University, Manchester, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Physiotherapy has long been utilized in the treatment of patients with respiratory problems. In the late 1950s, breathing exercises were recommended as a treatment for patients with chronic chest diseases but this suggestion was short-lived, as the efficacy of these exercises was inconclusive. Until the late 1970s, chest physiotherapy was a passive treatment on the part of the patient and the physiotherapist carried out manual chest physiotherapy techniques including percussion, vibrations, and shaking with gravity-assisted positions. The development of the active cycle of breathing technique (breathing control, lower thoracic expansion exercises, and forced expiratory technique), autogenic drainage, and adjunct physiotherapy aids in the removal of secretions. Procedures such as intermittent positive breathing techniques, have enhanced the treatment of chest clearance for patients with acute or chronic respiratory problems. Chest physiotherapy is routinely employed as a prophylactic measure prior to major surgery and postoperatively to prevent respiratory complications such as atelactasis and pneumonia. However, most of the systematic reviews that examined these techniques were inconclusive in their findings. To ascertain the benefits of these techniques in terms of reducing respiratory morbidity and healthcare usage and to improve the quality of life for patients with chronic respiratory problems, well-controlled clinical trials are needed.

Introduction Respiratory disease has a substantial impact on the health of the population at all ages and every level of

morbidity. Acute upper respiratory tract infections, including chronic lung diseases, are common causes of visits to general practitioners; they are a common cause of hospital admissions during the winter months, particularly in the elderly. Physiotherapy is the art and science of utilizing a variety of modalities to treat by using hands in order to maximize physical function. Chest physiotherapy assists the clearance of secretions and reduces breathlessness to improve lung function and reduce morbidity and mortality. Excessive bronchial secretion retention may contribute to the development of symptoms such as airflow obstruction, wheeze, shortness of breath, fatigue, and cough. Chest physiotherapy is defined as a combination of several mucus-clearance techniques to treat patients with acute or chronic respiratory problems by assisting mucus transport in order to improve lung function. These techniques can be used in a hospital ward setting, either in isolation or with mechanical devices, to treat patients with acute exacerbations, including those on life support machines in critical situations in the intensive care unit. Impaired clearance of the airways may lead to the development of respiratory infection, leading to acute infective bronchitis or, in more severe cases, the development of atelactasis and consolidation (pneumonia). These factors may contribute to poor gaseous exchange, decrease in ventilation perfusion, and breathlessness due to airflow limitation. If they

PHYSIOTHERAPY 333

are not adequately treated and managed, persistent infection may lead to chronic lung disease, for example, bronchiectasis. The aims of chest physiotherapy are * * * * * *

*

to reduce breathlessness, to remove excess bronchial mucus secretions, to increase exercise tolerance, to prevent respiratory complications postsurgery, to improve general fitness, to enhance gaseous exchange and reduce the work of breathing, and to encourage self-management for those with chronic respiratory problems.

Preoperative Physiotherapy It has been reported that pulmonary complications are common after cardiothoracic surgery and may increase the length of hospital stay, resulting in increased healthcare expenditure. The most common complications may include atelactasis (collapse of an area of lung) and pneumonia. Chest physiotherapy is, generally, prescribed as a prophylactic measure for patients admitted for major surgery, to ensure that the lung fields are clear prior to surgery. It also enables the therapist to assess the patient’s respiratory effort, to teach the patient chest clearance and effective coughing techniques, and also determine exercise tolerance. Indeed, performing an appropriate chest assessment will help the therapist to design and implement appropriate treatment strategies postoperatively (see Table 1). Systematic reviews that investigated the benefits of preoperative conventional chest physiotherapy techniques versus other adjunct chest physiotherapy aids have reported conflicting findings. Studies that compared incentive spirometry versus deep breathing

exercises after abdominal surgery showed that breathing exercises have some benefits in reducing respiratory complications. However, other systematic reviews have reported conflicting findings. Indeed, no significant difference was reported in those studies that compared deep breathing exercises and incentive spirometry or continuous positive airway pressure or intermittent positive pressure breathing. Thus the usefulness of chest physiotherapy in the prevention of pulmonary complications after cardiothoracic surgery remains inconclusive.

Postoperative Physiotherapy The effects of anesthesia may compromise the mucociliary clearance system, which is a physical defense mechanism protecting the lungs from damage. In addition, the patient’s fear of pain and rupture of the incision, especially a high incision on the upper abdomen, may compromise the function of the diaphragm. For those patients with routine abdominal surgery, the treatment should focus on breathing exercises to remove excess sputum secretions, encourage effective coughing with or without support, and lower thoracic-expansion exercises to increase ventilation and perfusion in the bases of the lung in order to prevent atelectasis and pneumonia. Early ambulation with the support and encouragement of the physiotherapist may assist in the prevention of deep vein thrombosis. The various chest physiotherapy techniques are discussed in terms of their efficacy, indications, contraindications, and evidence from systematic reviews. An essential requirement for any of the techniques described below, if the patient is in pain, is administration of adequate analgesia prior to chest physiotherapy treatment.

Table 1 Pre- and postoperative physiotherapy treatment Respiratory system Preoperative physiotherapy Assess the lung fields which are clear and free from excess sputum retention and crackles Teach the patient breathing exercises and effective coughing techniques Prepare the patient physically and psychologically for the operation Assess both upper and lower limb strength and mobility Postoperative physiotherapy Assess the chest for sputum retention Check for drains and tubes Monitor the arterial blood gases and oxygen saturation Teach breathing exercises routinely two to three times per day to keep the airways clear For those with sputum retention, encourage effective coughing techniques supporting the wound

Circulatory system Check the patient for circulatory problems including deep vein thrombosis Teach ankle exercises – dorsal flexion, plantar flexion

Advice on posture and positioning Instruct and monitor ankle exercises Bed mobility Encourage walking at the bedside and within the ward

334 PHYSIOTHERAPY

Active Cycle Breathing Techniques Active cycle breathing techniques (ACBT) is a cycle of techniques of breathing control, lower thoracicexpansion exercises, and the forced expiration technique, modifiable to each individual patient. Indications. ACBT is appropriate for mobilizing and removing excess bronchial secretions in order to improve lung function without inducing hypoxemia. Breathing Control

Breathing control (BC) is normal tidal breathing using the lower part of the chest with relaxation of the upper chest and shoulders. The aims of this technique are to encourage relaxation, ease breathlessness, and enhance the normal breathing pattern. The patient should be comfortable and well supported in an upright sitting or half-lying position. However, this can be modified according to the patient’s needs. The technique requires minimal effort from the patient. The therapist may place one hand lightly on the upper abdomen and encourage breathing through the nose, so that the air is warmed and filtered. As the patient breathes in, the hand should be felt to rise up and out; as the patient breathes out, the hand sinks down and in. The technique should be performed at the patient’s own pace. BC technique can be used as many times as possible on its own or as part of ACBT to encourage normal breathing. This technique is indicated for a patient with shortness of breath, hyperinflation, and those experiencing panic attacks/anxiety or for a patient who has had abdominal surgery and is unable to use the lower part of the chest due to pain. No contraindications for usage of this technique have been reported so far. Lower Thoracic-Expansion Exercises

Lower thoracic-expansion exercises (LTEE) are indicated for patients with poor expansion of the lungs due to collapse of a particular segment of the lung (atelectasis), possibly due to fear of pain and copious sputum retention. LTEE helps to increase collateral ventilation so that the air behind the secretions may help to mobilize secretions from the periphery of the lung to the central airways. Thoracic-expansion exercises are deep breathing exercises emphasizing inspiration. Patients should be comfortably supported with pillows in order to relax the upper chest. The technique can be applied singlehanded at the affected lobe where the movement of the chest is to be encouraged or by placing both hands bilaterally at the lower bases of the lung, approximately at the level of the eighth ribs. Instructions include breathing in slowly and deeply and filling up the lungs with air and expanding the lower

chest. The technique can be performed two to three times and interspersed with breathing control and rest. This technique can be combined with a 3-second ‘inspiratory hold’ after full inspiration before the passive relaxed expiration. The benefit of a 3-second hold at full inspiration has been claimed to decrease collapse of lung tissue. Care has to be taken for a patient with severe dyspnea. Further additional lung volume can be achieved by using a ‘sniff’ maneuver at the end of a deep inspiration with the purpose of recruiting collateral ventilation. Customarily, the therapists have employed these techniques two or three times per session; however, evidence of their efficacy is lacking. Precautions are required for a patient with severe shortness of breath. No more than three or four deep breaths should be performed at a time, otherwise it may lead to dizziness. However, the technique can be interspersed with rest and in conjunction with breathing control. Additional techniques such as chest percussion and vibration can be incorporated during chest-expansion exercises to mobilize secretions. Forced Expiratory Technique

Forced expiratory technique (FET) is indicated for patients with excessive sputum retention. The technique comprises one or two forced expirations (huffs) and breathing control. There are two types of huffs. (1) A huff using a mid-to-low lung volume (‘small breath in’) helps to move peripherally situated secretions to proximal larger airways. It is performed from mid-lung with a medium-sized breath and with the mouth and glottis open. (2) A huff employing higher lung volumes (‘a deep breath in’) can be used to clear secretions from the larger airways. However, FET is a difficult technique for some patients to grasp so patients should be taught in a simplified manner, for example, blowing in a tube or cottonwool ball in order to learn the correct FET technique. A reasonable amount of time needs to be spent with the adult patient to execute the technique effectively. Teaching children the FET technique using a peak flow mouthpiece or blowing games is useful. Normally, two or three FETs are adequate within a session interspersed with breathing control. The advantage of this technique is that it can be used at different levels of lung volumes and may assist the patients who produce large amounts of sputum. Excessive use of FET may, however, lead to bronchospasm. The FET technique uses the principle of the equal pressure point, which is the point where the pressure within the airways is equal to the pleural pressure.

PHYSIOTHERAPY 335

The downstream of the equal pressure point towards the mouth, and the dynamic squeezing of airways allows secretions to be mobilized to the upper airways and is cleared by a huff from high lung volumes. This technique can be repeated a couple of times. The cough is a natural protection mechanism for airway clearance. It comprises of deep inspiration followed by short and sharp expiration that leads to closure of the glottis. When the expiratory muscles contract, causing a high increase in intrapulmonary and abdominal pressure, there is a sudden expulsion of air as the glottis open rapidly. This helps to expectorate the sputum from the airways. Excessive continuous coughing may induce bronchospasm so care is needed. ACBT is a successful, comfortable, and safe method of bronchial chest clearance technique for the expectoration of secretions and is widely used by physiotherapists to treat patients with respiratory problems. The whole ACBT cycle should be performed two or three times depending on the amount and the location of secretions. There are no contraindications for this technique. The ACBT cycle sequence may be employed in different combinations. For example, for a patient who is anxious and with overinflation, emphasis will be on breathing control, whereas for a patient with sputum retention, treatment may focus on lower thoracic-expansion exercises and forced expiratory technique, interspersed with breathing control (see Figure 1).

Autogenic Drainage Technique Autogenic drainage (AD) aims to maximize airflow within the airways to enhance the clearance of mucus and improve ventilation. The AD technique is not used as widely as the active breathing cycle technique and is mostly employed in Germany, Netherlands, and Belgium. It has been claimed that AD improves mucus clearance from peripheral to central airways due to changes in airway caliber and breathing at different lung volumes during expiration. Chevaillier originally described the three phases of AD as ‘unstick’, ‘collect’, and ‘evacuate’. Breathing at low lung volume may aid in the mobilization of secretions (‘unstick’) from the periphery to the central airways, breathing at mid-lung volume tends to collect the mucus in the middle range, and breathing at high lung volume may assist in the expectoration of sputum (‘evacuate’) from the larger airways. Several studies have commented that it is difficult for a patient to learn autogenic drainage and it requires a considerable amount of effort and cooperation from

BC BC

FET

TEE Huff BC (a) BC BC

TEE

Huff FET Huff

BC

(b) BC FET

TEE

BC Huff

BC

BC TEE Huff (c)

FET

BC

Figure 1 Examples to demonstrate the flexibility of the active cycle of breathing techniques. BC, breathing control; FET, forced expiratory technique; TEE, thoracic-expansion exercises.

the patient to participate in the treatment. The longterm benefits and its usage in clinical practice require further investigation.

Postural Drainage Postural drainage is indicated when the volume of secretions is greater than 30 ml day 1, when the patient is facing difficulty actively removing secretions, and conditions with excess sputum production prior to utilizing chest-clearance techniques. Technique. Secretions can be drained, as part of self-management, from the affected part of the lobe or segment using gravity-assisted drainage positions. The gravity-assisted drainage positions are based on the anatomy of the bronchial tree (see Table 2). However, in a patient with severe chronic obstructive pulmonary disease, these positions may not be tolerable and should be tailored and modified to the individual lobe in a way the patient can tolerate. The duration of treatment may range from 10 to 20 min. The affected area of the lung should be positioned in an uppermost position with or without a head-down

336 PHYSIOTHERAPY Table 2 Gravity-assisted drainage positions Lobe Upper lobe

Position 1 2

Apical bronchus Posterior bronchus (a) Right

2

Sitting upright

3

(b) Left

4

3

Anterior bronchus

5

Lying on the left side horizontally turned 451 on to the face, resting against a pillow, with another supporting the head Lying on the right side turned 451 on the face, with three pillows arranged to lift the shoulders 30 cm from the horizontal Lying supine with the knees flexed

Lingula

4 5

Superior bronchus Inferior bronchus

7 7

Lying supine with the body a quarter turned to the right maintained by a pillow under the left side from shoulder to hip. The chest is tilted downwards to an angle of 151

Middle lobe

4 5

Lateral bronchus Medial bronchus

9 9

Lying supine with the body a quarter turned to the left maintained by a pillow under the right side from shoulder to hip. The chest is tilted downwards to an angle of 151

Lower lobe

6 7

Apical bronchus Medial basal (cardiac) bronchus Anterior basal bronchus Lateral basal bronchus Posterior basal bronchus

6 8

Lying prone with a pillow under the abdomen Lying on the right side with the chest titled downwards to an angle of 201 Lying supine with the knees flexed and the chest tilted downwards to an angle of 201 Lying on the opposite side with the chest tilted downwards to an angle of 201 Lying prone with a pillow under the hips and the chest tilted downwards to an angle of 201

8 9 10

10 11 12

tip for drainage of secretions. During this time, other techniques such as percussion, vibration, and shaking can be incorporated as part of the treatment to loosen sticky secretions. For different gravity-assisted positions (see Figures 2–14). Contraindications to the head-down position may include: * * * * * * * * * * * * * *

Hypertension Severe dyspnea Recent surgery Severe hemoptysis Nose bleeds Advanced pregnancy Esophagus hiatus hernia Cardiac failure Cerebral edema Aortic aneurysm Head or neck trauma/surgery Mechanical ventilation Epileptic seizure Desaturation observed (whatever the reason) when the procedure is performed.

During head-down position, it is important to monitor cardiovascular and respiratory parameters

including heart rate, respiratory rate, blood pressure, and oxygen saturation measured with oximetry. Postural drainage immediately after meals should be avoided. The patient’s condition pre- and posttreatment should be monitored with a stethoscope to ensure that the affected area is clear.

Relaxed Positions Patients with severe dyspnea expend more energy and effort in daily activities. They may derive some benefit by adopting relaxed positions so that the abdominal content is not pressing on the diaphragm resulting in apical breathing with excessive usage of the accessory breathing muscles, especially at rest or after mild exercise. These positions may optimize the length and tension status of the diaphragm to improve its function, and also assist relaxation of the accessory muscles to reduce breathlessness. These relaxed positions may be helpful as selfmanagement for the patient (by performing breathing control to ease relaxation of the accessory muscles and allow movement of the lower chest). They may assist to overcome the impact of breathlessness

PHYSIOTHERAPY 337

Figure 4 Posterior segment left upper lobe.

Figure 2 Apical segments upper lobes.

Figure 3 Posterior segment right upper lobe.

in daily activities (see Figures 15–19): 1. Upright sitting or relaxed sitting with forward leaning (Figures 15(a) and 15(b)) 2. Forward lean standing (Figure 16) 3. Relaxed standing (Figures 17(a) and 17(b)) 4. Forward lean sitting (Figure 18) 5. Forward kneeling (Figure 19) The therapists have prescribed these positions as a part of chest physiotherapy treatment for patients with chronic chest diseases. However, to date,

Figure 5 Anterior segments upper lobes.

Figure 6 Apical segments lower lobes.

the effects of these positions to improve functional activities or quality of life have not been conclusively proved in controlled clinical trials.

Manual Hyperinflation in Airway Clearance Manual hyperinflation is one of the chest physiotherapy techniques that is used in intensive care in intubated patients. It involves using an ambu bag in order to produce a slow deep inspiration, inspiratory

338 PHYSIOTHERAPY

Figure 10 Anterior basal segments. Figure 7 Lingula.

Figure 11 Lateral basal segment right lower lobe. Figure 8 Right medial basal and left lateral basal segments lower lobes.

Figure 12 Posterial basal segments lower lobes. Figure 9 Right middle lobe.

pause, and unobstructed expiration. The goals of physiotherapy treatment are to remove secretions, resolve atelectasis, and improve ventilation. Contraindications may include cardiovascular instability, barotraumas, severe bronchospasm, undrained pneumothorax, raised intracranial pressure,

and a high level of positive end expiratory pressure 410 cmH2O. A recent review of the usage of manual hyperinflation in airway clearance remains inconclusive. These techniques, however, have been used widely in intensive care units for many years. Future studies are needed to evaluate the correct dosage, patient position, and level of pressures and volumes.

PHYSIOTHERAPY 339

Manual Chest Physiotherapy Percussion

Figure 13 Assisted treatment in high side lying.

Figure 14 Positioning: (a) sitting upright; (b) slumped sitting.

Figure 15 Relaxed sitting.

Percussion or clapping is a synonymous term to describe the rhythmic clapping on the chest wall with relaxed wrist and cupped hand, creating an energy wave that is transmitted to the airways. Indications. Tenacious secretions, when the patient is unable to expectorate on their own. Technique. It is performed using a cupped hand with a rhythmical flexion and extension action of the wrist. It requires usage of both hands. Depending on the area of the chest, it may be more appropriate to use one hand to treat a specific lobe. For the infant,

340 PHYSIOTHERAPY

chest clapping is performed using two or three fingers of one hand. Chest clapping should be performed over a layer of clothing to avoid sensory stimulation of the skin. To

reduce any adverse consequence, the technique should be performed for 30 s, and interspersed with 3–4 lower thoracic-expansion exercises. It is essential to be cautious of vigorous and rapid chest clapping as this could lead to breath-holding and may induce bronchospasm in a patient with hyperreactive airways. Contraindications. Severe osteoporosis and hemoptysis. Shaking

Shaking is the use of coarse oscillations produced by the therapist’s hands compressing and releasing the chest wall and applied during expiration phase only.

Figure 16 Forward lean standing.

Figure 17 Relaxed standing.

Figure 18 Forward lean sitting.

PHYSIOTHERAPY 341

secretions and using mechanical aids to stimulate lung function. Suction

Figure 19 Forward kneeling.

Indication. Sputum retention. Technique. It should be performed following ‘a deep breath in’, during the expiratory phase. The hands are placed on the affected lobe in the directions of the ribs to mobilize secretions. Care must be taken for patients using long-term steroids and patients with osteoporosis, bony metastasis, and severebronchospasm. Shaking is contraindicated over a recent rib fracture or surgical incision. Evidence for its clinical use is sparse. Vibrations

Vibrations are fine oscillations applied to the chest wall by the therapist’s hands and carried out during expiration after a deep breath in. The therapist should keep firm contact and direct the force inwards towards the center of the patient’s chest. Indication. Sputum retention. Contraindications. A recent rib fracture or surgical incision. Anecdotal evidence suggests that vibration has some benefit in the short term but there is a lack of evidence of the long-term benefits of this technique. A recent Cochrane review that investigated the benefits to bronchial hygiene of the use of percussion, vibration, and shaking concluded that there is not enough evidence to refute or support the efficacy of these techniques in treating patients with COPD and bronchiectasis. As most of the studies were small in sample size, had poor methodological designs, and a lack of sensitive outcome measures, it would be difficult to generalize the findings. Future studies should focus on larger sample sizes, with controlled randomized clinical trials being the best way forward.

Intensive Care Unit The role of the physiotherapist in the intensive care unit is to treat intubated patients by clearing chest

Indication. Excessive sputum production (plugging), inability to cough effectively. Technique. Suction catheter should be sterile to prevent cross-infection. In practice, disposable catheters are used. It is good clinical practice to explain the procedure to the patient, if conscious, before carrying out the suction. Ensure the catheter is positioned so as not to damage the airway mucosa. The duration of treatment should be limited to 10–15 s. Suction should be applied constantly while removing the catheter. Saline can be used as an aid to suctioning to assist in the clearing of secretions. The extracted sputum should be sent to the laboratory for microbiological assessment in order to prescribe appropriate antibiotics. Contraindication. Severe hemoptysis, severe bronchospasm, and undrained pneumothorax. The physiotherapist may also be involved in the treatment of this patient group to maintain full range of movements of both upper and lower limb extremities by performing passive and active assisted exercises in order to maintain soft tissue length and function and also to reduce risks of developing edema and deep vein thrombosis in the lower limbs. However, the evidence of preventing or reducing deep vein thrombosis requires further investigation.

Exercise Therapy Exercise therapy is a fundamental part of chest physiotherapy. Patients with chronic chest diseases should be encouraged routinely to be involved in an aerobic exercise program in order to improve general fitness, and increase exercise tolerance and functional activities. Physiotherapists are in a unique position to prescribe appropriate exercise programs as part of medical treatment for patients admitted in hospital with acute exacerbations of chronic lung diseases. The exercise regime should be tailored to the individual patient’s hobbies (if possible) and to baseline functional abilities. The purpose of the physiotherapy treatment is to maximize the patient’s independent function and increase walking endurance. The exercise program will need to determine the intensity, type of exercise, setting, and patient compliance in order to monitor the efficacy of the program.

342 PHYSIOTHERAPY Ground-Based Walking Exercise

Walking improves both cardiovascular fitness, increases exercise tolerance, and stimulates psychological well-being. It is simple to perform, safe, and able to be incorporated into the daily routine of the patient. The exercise can be done at home and does not require special equipment. In simplistic terms, patients with chronic chest problems can monitor their own progress by, for example, monitoring how many meters or miles they have covered or for how long they have walked per session. There is no clinical guideline as to how many times per week ground-based walking exercises will be required in order to produce a significant improvement in the patient’s functional activities and quality of life. Current customary clinical practice in the prescription of ground-based exercise by physiotherapists is two to three times per week for a duration of approximately 30 min. This self-management exercise program requires future investigation to find out if it has any benefits in terms of reducing exacerbations of chronic respiratory disease. Upper Limb Aerobic Exercise Training

Dyspnea on exertion is a primary problem for patients with chronic chest problems. Patients very often have difficulties in performing overhead activities, for example, lifting light objects from a shelf or dusting. This often leads to fear and avoidance of these activities and in turn to atrophy by disuse and weakness of muscles in the upper limbs. This exacerbates and promotes inactivity which may lead to a ‘vicious circle’. Studies have reported that aerobic exercise training in patients with chronic chest problems improves exercise tolerance and reduces metabolic and ventilatory requirement. The following exercises can be prescribed by the therapists after discharge (for home ‘use’). Unweighted arm exercise with repetitive bilateral shoulder flexion and abduction from neutral position synchronized with breathing. The level of intensity of the exercise program can be determined by assessing the patient to see how many times he/she can perform the exercise. The frequency and intensity of the exercise program can be increased over time. For those who are capable of weight-lifting, the exercise can be prescribed using light weights that can be increased suitably. Lower Limb Aerobic Exercise Training

Patients with chronic chest problems, especially older patients, spend substantial amount of their time indoors leading a sedentary lifestyle. This may lead to physical deconditioning and weakness of the lower

limb muscles, for example, reduced strength and endurance of the quadriceps muscle. Quadricepsstrengthening exercise should then be incorporated with the treatment program. Step aerobics have been shown to improve general fitness. For those with severe chest diseases, graduated stair-climbing several times a day may improve general fitness and quadriceps function. The home exercise program can be devised using the bottom of the step and climbing up and down by holding a banister or stair rail. The intensity and duration of the exercise program should be determined by the patient’s exercise tolerance and fatigability. However, patients should be assessed prior to the exercise program for other medical problems such as dizziness. If the patient is unable to perform the exercise mentioned above, this can be substituted by unweighted straight-leg raising when lying and knee extension when sitting. Stationary Cycling

Stationary cycle can be used at home or in a local gymnasium. This may provide more controlled exercise than ground-based walking. This form of exercise is helpful in determining the level of exercise intensity achievable. Hence it can be used as a selfmonitored objective measure to determine the progress of exercise capacity. All these exercise training modalities should be considered both in the clinical setting and as a selfmanagement program at home to improve cardiorespiratory function and exercise tolerance for this patient group.

Conclusion Several chest physiotherapy techniques and adjunct modalities are available in current clinical practice to treat patients with acute and chronic chest diseases. Chest physiotherapy may be effective in improving mucus transport. Whether it has any benefits on pulmonary function in the long term is not clear. In addition, evidence from systematic reviews suggests no single technique is superior than the other. The findings from research studies are inconclusive. Treatment for individual patients has to be ‘tailormade’ in light of the patient’s clinical findings, for example, arterial blood gases and oxygen saturation, in order to be effective. Further studies are required to determine the efficacy and benefits of these physiotherapy techniques and modalities in short, medium, and long term in reducing length of hospital stay, healthcare utilization, and impact on quality of life and self-management programs.

PLATELET-DERIVED GROWTH FACTOR 343 See also: Atelectasis. Exercise Physiology. Symptoms of Respiratory Disease: Cough and Other Symptoms. Ventilation: Control.

Further Reading Denehy L (1999) The use of manual hyperinflation in airway clearance. European Respiratory Journal 14: 958–965. Harden B (2004) Emergency Physiotherapy. London: Church Livingstone. Hough A (2001) Physiotherapy in Respiratory Care. An EvidenceBased Approach to Respiratory and Cardiac Management, 3rd edn. Chelthenham: Nelson Thornes. Jones AP and Rowe BH (2000) Bronchopulmonary hygiene physical therapy for chronic obstructive pulmonary disease and bronchiectasis (Cochrane Review). Cochrane Database of systematic Reviews (2): CD000-045.

Plasminogen Activator and Plasmin

Lapin CD (2002) Airway physiology, autogenic drainage and active cycle of breathing. Respiratory Care 47: 778–785. Pasquina P, Tramer MR, and Walder B (2003) Prophylactic respiratory physiotherapy after cardiac surgery: systemic review. British Medical Journal 327: 1379–1381. Pryor JA (1999) Physiotherapy for airway clearance in adults. European Respiratory Journal 14: 1418–1424. Pryor JA and Prasad SA (eds.) (2002) Physiotherapy for respiratory and cardiac problems. In: Adults and Paediatrics, 3rd edn. London: Churchill Livingstone. van der Schans CP, Postma DS, Koeter GH, and Rubin BK (1999) Physiotherapy and mucus transport. European Respiratory Journal 13: 1477–1486. West JB (2001) Pulmonary Physiology and Pathophysiology. An Integrated Case-Based Approach. Philadelphia: Lippincott Williams & Wilkins. Yohannes AM (2001) Pulmonary rehabilitation and outcome measures in elderly patients with chronic obstructive pulmonary disease. Gerontology 47: 241–245.

see Fibrinolysis: Plasminogen Activator and Plasmin.

PLATELET-DERIVED GROWTH FACTOR J C Bonner, CIIT Centers for Health Research, Research Triangle Park, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Platelet-derived growth factor (PDGF) isoforms are polypeptide mediators that play a major role in stimulating the replication, survival, and migration of mesenchymal cells during the pathogenesis of fibrotic diseases. PDGF is secreted by a variety of cell types including epithelial cells, macrophages, and fibroblasts as a response to injury, and many proinflammatory cytokines mediate their mitogenic effects via the autocrine release of PDGF. PDGF-A and PDGF-B chain dimeric isoforms (PDGF-AA, PDGF-AB, and PDGF-BB) play important roles in the pathogenesis of fibrosis. These isoforms promote myofibroblast proliferation and chemotaxis, but also serve other functions including stimulation of collagen production and promotion of cell adhesion. Less is known regarding the significance of the more recently discovered PDGF-C and PDGF-D chain isoforms. The biological activity of PDGF is determined by the relative expression of PDGF a-receptors and b-receptors on the cell surface. These receptors are induced during lung fibrogenesis, thereby amplifying biological responses to PDGF isoforms. PDGF action is further modulated in the extracellular milleau by binding proteins, matrix molecules, and proteases.

Introduction In the 1970s, Ross and colleagues discovered platelet-derived growth factor (PDGF) in the search for a

serum factor that stimulated the growth of arterial smooth muscle cells during the pathogenesis of atherosclerosis. Several groups subsequently purified this major PDGF on the basis of its ability to stimulate the growth of smooth muscle cells and other mesenchymal cells. In the late 1980s, the genes encoding the classical PDGFs (PDGF-A and PDGF-B) along with two receptor genes (PDGF a-receptor – PDGFRa and PDGFb-receptor – PDGFRb) were cloned. More recently, two novel PDGFs (PDGF-C and PDGF-D) were discovered that are proteolytically activated in the extracellular microenvironment. PDGF and its receptors have been shown to play critical roles in the normal processes of development and tissue repair as well as in the pathogenesis of diseases such as cancer, atherosclerosis, and fibrotic diseases.

Structure The genes encoding the four PDGF polypeptide chains are located on four different chromosomes. The human PDGF-A and PDGF-B genes are located on chromosomes 7 and 22, whereas the PDGF-C and PDGF-D genes are located on chromosomes 4 and 11, respectively. All four PDGFs are synthesized and assembled as disulfide-linked dimeric polypeptides in the endoplasmic reticulum (ER) as inactive precursors, which are then proteolytically processed

344 PLATELET-DERIVED GROWTH FACTOR RRKR86 PDGF-A 87

196/211

RGRR81 PDGF-B 82

190 241 RKSR234

PDGF-C 50

160 235

345

RKSK257 PDGF-D 56

167 258

370

Figure 1 Structure of the four PDGFs and proposed sites for proteolytic activation. PDGF-A and PDGF-B chains are proteolytically activated in the exocytic pathway, while cleavage of the CUB domains in PDGF-C and PDGF-D chains is mediated by extracellular proteases. Adapted from Fredriksson L, Li H, and Eriksson U (2004) The PDGF family: four gene products form five dimeric isoforms. Cytokine & Growth Factor Reviews 15: 197– 204.

to form biologically active PDGFs (Figure 1). In the ER, the classical PDGF chains (PDGF-A and PDGFB) dimerize to form PDGF-AA, PDGF-AB, and PDGF-BB isoforms. These A and B chain dimers are cleaved and activated through the exocytic pathway within the trans-Golgi apparatus. In addition, the matrix-bound forms of PDGF-A and PDGF-B, which contain a retention motif, may require proteolytic cleavage to be released from the extracellular matrix. In contrast to the classical PDGFs, the more recently discovered PDGF-C and PDGF-D combine to form PDGF-CC and PDGF-DD homodimers, and are cleaved and activated extracellularly. The five PDGF dimeric isoforms have the ability to bind and dimerize two cell-surface receptor tyrosine kinases, termed PDGFRa and PDGFRb, to form PDGFRaa, PDGFRab, and PDGFRbb dimeric complexes.

Regulation of Production and Activity All PDGF isoforms are generally regarded as potent mitogens and chemoattractants for fibroblasts, myofibroblasts, and smooth muscle cells that drive the recruitment and replication of these cells at sites of tissue injury. The expression of the PDGFs at the cellular level has been established in a variety of tissues, particularly for PDGF-A and PDGF-B. Both classical forms of PDGFs (PDGF-A and PDGF-B)

along with their receptors (PDGFRa and PDGFRb) are expressed in nonoverlapping patterns that suggest a paracrine mode of signaling. For example, during embryonic development PDGF-A and PDGFRa are expressed at sites of epithelial–mesenchymal cell interaction. PDGF-A is expressed primarily by the epithelium, whereas PDGFRa is expressed on mesenchymal cells. During the progression of airway fibrosis in mice, a similar pattern exists where PDGFA is upregulated in the airway epithelium and serves as a paracrine signal for myofibroblasts and smooth muscle cells adjacent to the epithelium. PDGF and its receptors are regulated by a variety of endogenous and exogenous factors. Endogenous factors such as cytokines (interleukin-1beta (IL-1b)), growth factors (transforming growth factor beta 1 (TGF-b1)), and lipid mediators (postaglandin E (PGE2)) have been reported to upregulate PDGF-A or to control the expression of PDGFRa on lung myofibroblasts. Exogenous factors also influence PDGF receptor expression. PDGF-A and its receptor (PDGFRa) are induced in vitro following exposure to asbestos fibers, and induction of the PDGFRa occurs in vivo during lung fibrogenesis caused by exposure to asbestos or metal-induced oxidative stress. In the lung interstitial compartment where myofibroblasts reside, autocrine activation of PDGF-A and its receptor could be significant in stimulating myofibroblasts to proliferate and deposit collagen. PDGF-C, a more recently discovered ligand for the PDGFRa, may also be important in the development of lung fibrosis. Significant increases in PDGF-C mRNA expression occur following bleomycin-induced lung injury in mice, whereas PDGF-D mRNA expression is downregulated. Interestingly, PDGF-C expression is not upregulated in lungs of BALB/c mice that are resistant to bleomycin-induced lung fibrosis. The expression of PDGF-B is increased by interferon gamma (IFN-g) in human alveolar macrophages. While IFN-g mediates many of its actions through the Stat1 signaling pathway, studies with fibroblasts from Stat1-deficient mice demonstrate that PDGF-A mRNA expression by IFN-g is mediated via STAT1-independent signaling. IL-1b stimulates mitogenesis of fibroblasts and smooth muscle cells in vitro through PDGF-AA release. In vivo, the overexpression of IL-1b in rats via adenoviral transfer increases several profibrotic mediators, including PDGF. Recently, IL-13, a major cytokine that mediates the pathogenesis of asthma, stimulates PDGF-AA production through a STAT6dependent mechanism. These studies emphasize that cytokines elevated early following injury or allergic responses in the lung trigger downstream events that culminate in the proliferation of myofibroblasts through PDGF-dependent mechanisms. While the

Matrix

PLATELET-DERIVED GROWTH FACTOR 345

Collagen and HSPGs SPARC

Soluble


2M

PDGF-BB

PDGF-AB

PDGF-AA PDGF-CC

PDGF-DD

PDGFR


Cell

PDGFR

Figure 2 Regulation of PDGF by extracellular binding proteins and extracellular matrix molecules. PDGF-B chain isoforms bind a2macroglobulin (a2M), which, when activated by proteases, is able to bind its receptor termed LRP and mediate PDGF-B chain clearance and degradation. PDGF-B chain also binds the glycoprotein SPARC, which is associated with the extracellular matrix. PDGFA and PDGF-B (long forms with extended carboxyl terminus) interact with heparin-sulfate proteoglycans (HSPGs) and collagens in the extracellular matrix. The selectivity of the five different PDGF isoforms to bind PDGFRa and PDGFRb is also shown. The possible interactions of PDGF-CC and PDGF-DD with a2 M and matrix components are not known.

biologic responses to PDGF isoforms are determined primarily by the expression of their high-affinity cell-surface signaling receptors, considerable modulation of growth factor action is achieved through interaction with extracellular proteins that represent low-affinity docking sites or reservoirs for growth factors (Figure 2). These proteins include the proteinase inhibitor alpha-2-macroglobulin (a2M), and several components of the extracellular matrix including secreted protein and rich in cysteine (SPARC), glycosaminoglycans, and collagen.

Biological Functions In human disease and in animal models, PDGF expression correlates with the proliferation of the myofibroblast population that ultimately contributes to the production of extracellular matrix proteins such as collagen, fibronectin, and glycosaminoglycans. While the expression of these extracellular matrix proteins is due mainly to TGF-b1, there is also evidence that PDGF plays a role in stimulating the expression of matrix molecules by myofibroblasts. PDGF isoforms function primarily as mitogens and

chemoattractants. PDGF-stimulated cell proliferation is mediated via ligand-dependent dimerization and phosphorylation of PDGFRa and/or PDGFRb, which results in the activation of Ras and the downstream mitogen-activated protein (MAP)kinase signaling pathway to promote cell proliferation. PI3 kinase is also important in mediating cell proliferation, as well as other biologic responses to PDGF including migration, cell attachment, and collagen deposition. PDGF also activates STAT1 and STAT3 that are involved in growth arrest and cell survival, respectively. Animal models of lung injury have been invaluable in defining a role for PDGF in the progression of fibrosis. PDGF-B mRNA is increased in murine models of chronic hyperoxia, bleomycin-induced lung injury, and asbestos-induced pulmonary fibrosis. Genetic studies in mice have provided great insight into the physiological functions of PDGF. Postnatally surviving PDGF-A knockouts display a lung emphysema-like phenotype due to complete failure of alveolar septum formation. A similar phenotype is observed in genetically partially rescued PDGFRa null mutants and in PDGFRa signaling deficient mice. These genetic studies showed

346 PLATELET-DERIVED GROWTH FACTOR

that PDGF-A and its receptor, PDGFRa, both play critical roles in the emergence of a myofibroblast phenotype in the lung and the subsequent deposition of elastin which is necessary for alveolar septum formation. Both PDGF-B and PDGFRb null mutants die at late gestation from widespread microvascular bleeding caused by a severe deficit of vascular smooth muscle cells. Transgenic mice have been generated that express PDGF-A or PDGF-B in the pulmonary epithelium. PDGF-A overexpression leads to overproliferation of mesenchymal cells which compromises expansion of the distal airspaces. PDGF-B overexpression leads to a less dramatic but more pleiotrophic response, including the formation of emphysematous, fibrotic, and inflammatory lesions.

PDGF in Respiratory Diseases In the 1980s, the c-sis proto-oncogene (i.e., the gene encoding PDGF-B) was shown to be upregulated in alveolar macrophages from patients with idiopathic pulmonary fibrosis (IPF) compared to macrophages from normal individuals. Immunohistochemistry and in situ hybridization have been used to demonstrate that PDGF-B and PDGF-A mRNAs are present in macrophages from IPF patients but not in control lung specimens. PDGF is also overexpressed in other fibrotic lung diseases. PDGF expression is associated with the development of obliterative bronchiolitis that occurs in many patients following lung transplantation. Elevated levels of PDGF in bronchoalveolar lavage fluid have also been documented in patients with acute lung injury and in patients with Hermansky–Pudlak syndrome. PDGF expression is elevated in fibrotic diseases such as asbestosis and coal workers’ pneumoconiosis caused by the inhalation and deposition of fibrogenic particles in the lung. Therefore, it is clear that PDGF isoforms are increased prior to the development of a variety of lung fibrotic diseases with different etiologies and play a central role in the development of these diseases. Effective strategies for the treatment of fibrotic diseases are lacking in most instances. Corticosteroid and immunosuppressive therapy, although widely used, does not prevent myofibroblast proliferation and extracellular matrix deposition in the majority of patients with lung fibrosis. In fact, the corticosteroid dexamethasone stimulates the proliferation of fibroblasts obtained from the airways of mild asthmatics. Moreover, dexamethasone increases PDGF-B production by human alveolar macrophages, and upregulates the PDGFRa in rat lung myofibroblasts. These findings suggest that corticosteroids might actually promote interstitial lung fibrosis in IPF or

contribute to airway wall fibrosis and remodeling in chronic asthma. IFN-g has been shown to reduce PDGF-stimulated lung myofibroblast proliferation, yet stimulates the production of PDGF-B from alveolar macrophages. Clinically, IFN-g has been shown to be ineffective in the treatment of fibrotic lung disease. IFN-g dramatically enhances DNA synthesis in response to PDGF in hepatic stellate cells and renal mesanglial cells, indicating that IFN-g could contribute to mesenchymal cell proliferation and fibrosis in the liver and kidney. In fact, transgenic mice overexpressing IFN-g in the liver exhibit histologic features of chronic hepatitis. Therefore, in general there is no conclusive evidence that IFN-g therapy is effective in treating fibrosis, and in some fibrotic diseases IFN-g could excacerbate fibrogenesis. Inhibition of PDGFstimulated myofibroblast proliferation in animal models has been shown to decrease fibrosis. Pirfenidone is an antifibrotic agent that has been shown in experimental animals to reduce fibrosis in a variety of tissues including lung, liver, and kidney. Pirfenidone significantly inhibits PDGF-stimulated hepatic stellate cell proliferation through inhibition of the Na þ /H þ exchanger. Pirfenidone has also been demonstrated to reduce the production of PDGF-A and PDGF-B by alveolar macrophages and reduce bleomycin-induced lung fibrosis in hamsters. The antifibrotic agent pentoxifylline also reduces hepatic fibrosis by reducing PDGF-induced proliferation of hepatic stellate cells. In addition, canrenone, an antialdosteronic drug, reduces PDGF-induced hepatic stellate cell proliferation through inhibition of PDGF-induced PI3 kinase activation and inhibition of Na þ /H þ exchanger. The development of nuclease-resistant, high-affinity oligonucleotide aptamers against the PDGF-B chain has also been used to inhibit renal scarring in a rat model of renal fibrosis and may offer a novel antifibrotic therapy. Recently, the natural anticoagulant activated protein C (APC) was found to inhibit bleomycin-induced lung fibrosis in mice in part through inhibition of PDGF production. Perhaps the most promising drugs for treating fibrosis are the PDGF receptor tyrosine kinase inhibitors. Administration of the PDGFR-selective tyrosine kinase inhibitor AG1296, a tyrphostin analog, has been shown to reduce pulmonary fibrosis in a rat model of metal-induced lung injury. Imatinib, also known as Gleevec (STI571), is a PDGF receptor tyrosine kinase inhibitor that has been shown to ameliorate chronic allograft nephropathy in rats. In patients with chronic myelogenous leukemia, Gleevec treatment resulted in marked regression of bone marrow fibrosis. Taken together, these animal and preliminary clinical studies indicate that inhibition of PDGF receptor tyrosine kinases could offer a viable

PLATELETS 347

treatment strategy for fibrotic diseases in a variety of tissues. See also: Extracellular Matrix: Elastin and Microfibrils. Fibroblasts. Hepatocyte Growth (Scatter) Factor. Insulin-Like Growth Factors. Interleukins IL-1 and IL-18; IL-5; IL-13. Mesothelial Cells. Myofibroblasts. Occupational Diseases: Coal Workers’ Pneumoconiosis; Asbestos-Related Lung Disease. Pulmonary Fibrosis. Signal Transduction. Signs of Respiratory Disease: Clubbing and Hypertrophic Osteoarthropathy. Smooth Muscle Cells: Vascular.

Further Reading Bergsten E, Uutela M, Li X, et al. (2001) PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor. Nature Cell Biology 3: 512–516. Betscholtz C (2004) Insight into the physiological functions of PDGF through genetic studies in mice. Cytokine & Growth Factor Reviews 15: 215–228. Bonner JC (2004) PDGF and its receptors in fibrotic diseases. Cytokine & Growth Factor Reviews 15: 255–273. Bonner JC, Lindroos PM, Rice AB, Moomaw CR, and Morgan DL (1998) Induction of PDGF receptor-alpha in rat myofibroblasts during pulmonary fibrogenesis in vivo. American Journal of Physiology 274: L72–L80.

Fredriksson L, Li H, and Eriksson U (2004) The PDGF family: four gene products form five dimeric isoforms. Cytokine & Growth Factor Reviews 15: 197–204. Heldin CH, Eriksson U, and Ostman A (2002) New members of the platelet-derived growth factor family of mitogens. Archives of Biochemistry and Biophysics 398: 284–290. Heldin C-H and Westermark B (1999) Mechanism of action and in vivo role of platelet-derived growth factor. Physiological Reviews 79: 1283–1316. Hoyle GW, Li J, Finkelstein JB, et al. (1999) Emphysematous lesions, inflammation, and fibrosis in the lungs of transgenic mice overexpressing platelet-derived growth factor. American Journal of Pathology 154: 1763–1775. Ingram JL, Rice AB, Geisenhoffer K, Madtes DK, and Bonner JC (2004) Interleukin-13 and IL-1beta promote lung fibroblast growth through coordinated up-regulation of PDGF-AA and PDGFR-alpha. FASEB Journal 18: 1132–1134. Li J and Hoyle GW (2001) Overexpression of PDGF-A in the lung epithelium of transgenic mice produces a lethal phenotype associated with hyperplasia of mesenchymal cells. Developmental Biology 239: 338–349. Li X, Ponten A, and Aase K (2000) PDGF-C is a new proteaseactivated ligand for the PDGF alpha-receptor. Nature Cell Biology 2: 302–309. Wang YZ, Zhang P, Rice AB, and Bonner JC (2000) Regulation of interleukin-1beta-induced platelet-derived growth factor receptor-alpha expression in rat pulmonary myofibroblasts by p38 mitogen-activated protein kinase. Journal of Biological Chemistry 275: 22550–22557.

PLATELETS C P Page and S C Pitchford, King’s College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Clinical evidence exists demonstrating changes in platelet behavior and function in inflammatory diseases of the respiratory tract, in particular in asthma, rhinitis, acute lung injury, and chronic obstructive pulmonary disease. Despite being anucleate, platelets participate in the pathogenesis of these disorders in a manner distinct from their effects in thrombosis and hemostasis. These include the expression of adhesion molecules, the release of proinflammatory mediators, tissue-degrading enzymes, cationic proteins, and the ability to undergo chemotaxis. Platelets can themselves be activated by mediators released by other inflammatory cells. There is now substantial experimental evidence that supports a role for platelets in leukocyte recruitment to inflamed tissue, airways hyperresponsiveness, lung remodeling and repair, and pulmonary hypertension, processes that occur in respiratory diseases.

Introduction Platelets are anuclear cytoplasmic fragments of the larger precursor megakaryocytes that mature in the

bone marrow; their concentration in the circulation is around (150–400)  109 cells l 1. Platelets are thus a 100-fold more numerous than leukocytes within the circulation. Platelet production from megakaryocytes is poorly understood, but the process of fragmentation may be regulated in the pulmonary circulation. The morphology of platelets is surprisingly complex for a cell with no nucleus, and the structure has been split into three main regions: (1) the peripheral zone, the outermost covering, allows platelets to interact with other cells, tissues, and the vessel wall through presentation of mediators, coagulant proteins, and adhesion molecules; (2) the sol–gel matrix of the platelet cytoplasm provides the contractile machinery involved in platelet shape change, chemotaxis, and migration through inflamed tissues; and (3) the organelle zone is dispersed in the cytoplasm, which is organized into distinct granules (alpha), electron dense bodies, mitochondria, peroxisomes, and lysosomes. Upon appropriate activation, platelets release proinflammatory mediators, chemokines, and enzymes from these organelles. A role for platelets as inflammatory cells has become increasingly accepted and their involvement in

348 PLATELETS

the pathogenesis of atherosclerosis, rheumatoid arthritis, and inflammatory bowel disease is supported by recent literature. Indeed, evidence now exists demonstrating the involvement of platelets in inflammatory diseases of the lung, including asthma, rhinitis, acute lung injury (ALI), and chronic obstructive pulmonary disease (COPD). Numerous studies have revealed alteration in platelet function in patients with inflammatory diseases. These alterations appear to be distinct from the well-characterized involvement of platelets in thrombosis and hemostasis, but it is now generally accepted that platelets participate in immune responses as well as in tissue injury and repair. Platelet activation by proinflammatory mediators, the platelet secretome and proteome, interactions with other cell types involved in inflammation, and the presence of platelets in inflamed tissue support the concept that platelets participate in inflammation.

Platelet Production in the Lung There is controversy about the site of platelet production. Platelets could possibly be formed by: (1) budding from megakaryocytes within the bone marrow; (2) platelet production from the demarcation membrane system (a network of smooth membrane channels formed via invagination of the plasma membrane); or (3) the physical fragmentation of megakaryocytes within the lung circulation. The process of platelet formation has been observed to occur in the pulmonary circulation as megakaryocytes are too large to pass through the lungs without fragmentation. This process results in platelets with varying volumes, density, and reactivity that may have functional consequences in inflammation. Recruitment of megakaryocytes and circulating platelets to the lung has been observed in asthmatic episodes and in patients who have died of status asthmaticus, suggesting that alterations in their reactivity may influence disease progression.

Platelets in Asthma and Rhinitis The participation of platelets in the pathogenesis of asthma and rhinitis has been documented for a number of years. Platelet activation occurs during antigen-induced airway reactions in asthmatic subjects and this altered functionality is manifested as heightened platelet activation in vivo. Platelets from the same allergic patients are found to be refractory to a variety of stimuli ex vivo, possibly resulting from platelet overstimulation or exhaustion. Platelet exhaustion, reported as an inability of noradrenaline and adenosine diphosphate (ADP) to induce full,

second phase aggregation of platelets, has been correlated with increased serum immunoglobulin E (IgE) in asthmatic patients. Full aggregation of platelets in vitro returns when studies are repeated outside of the allergy (pollen) season. This phenomenon is accompanied by a defective release of 5-hydroxytryptamine (5-HT), platelet factor-4 (PF-4), and a decrease in arachidonic acid metabolism from platelets of atopic asthmatics or patients with rhinitis. This alteration in platelet function has been associated with bronchial hyperresponsiveness that accompanies nocturnal asthma. Furthermore, platelets from atopic asthmatics and subjects with rhinitis become desensitized to platelet-activating factor (PAF) in vitro. These observations suggest the possibility of platelet desensitization as a result of chronic activation in vivo. The phenomenon of platelet activation due to allergen-induced anaphylaxis has been shown to be beyond the control of agents that stimulate cAMP and metabolites of the arachidonate pathway. Platelet responses to allergens thus differ from those that stimulate aggregation (Figure 1). These responses are accompanied by a prolonged bleeding time, increased platelet mass and volume, and decreased platelet survival time in stable atopic asthmatics and sufferers of rhinitis. Accelerated platelet consumption correlates with accelerated regeneration of the platelet population. These phenomena can be corrected by treatment of asthmatic patients with glucocorticoids. Large numbers of pulmonary megakaryocytes and platelets have been obtained at autopsy from patients who have died from ‘status asthmaticus’. This event is accompanied by bone marrow karyopoiesis and thrombopoiesis. Platelet recruitment and activation within the lungs during the early and late phase response to allergen is also accompanied by a reduction in circulating platelet numbers. Circulating platelet– platelet, and platelet–leukocyte aggregates have also been detected in patients with spontaneous asthma attacks. These aggregates occur in a biphasic manner, the first peak occurring after allergen challenge. Interestingly, CD11b, an activation marker on the surface of leukocytes, also increases on leukocytes attached to platelets after allergen challenge, but not on freely circulating leukocytes unattached to platelets. This observation suggests that platelets ‘prime’ leukocytes for efficient rolling and adhesion to inflamed vascular endothelium. In this regard, experimental models of asthma support this concept and provide evidence for a requirement of platelets in pulmonary eosinophil and lymphocyte recruitment in rabbits, guinea pigs, and mice. As is the case in leukocyte recruitment in other inflammatory diseases,

PLATELETS 349 Megakaryocyte budding from the bone marrow with fragmentation in the pulmonary vasculature

Circulating resting platelets

Platelet function in response to inflammatory stimuli

Platelet function in response to proaggregatory stimuli Primary aggregation

No secondary phase aggregation

Secondary aggregation

Primary and secondary phase aggregation, associated with the release of ADP, TXA2, thrombin

Inflammatory mediator release, adhesion molecule expression Interactions with other inflammatory cells Chemotaxis

Tissue damage AHR Tissue remodeling

Thrombus formation

Figure 1 Dichotomy of platelet function. Depending on the type of stimulus involved, platelets may be activated by prothrombotic mediators (arrows pointing right), or proinflammatory mediators (arrow pointing left). The type of stimulus in turn therefore dictates the ultimate function of the platelet.

1 µm

P P

RBC (a)

(b)

Figure 2 (a) Evidence of platelet recruitment to the lungs. A platelet (P) passing through a gap (arrow) between endothelial cells of a postcapillary venule. Specimen taken from a right upper lobe bronchus of a subject with mild nonatopic asthma (13 000). Courtesy of A Laitinen and L Laitinen. (b) In allergen-exposed mice, individual platelets (red, integrin alpha II beta (CD41) positive) can be seen attached to eosinophils (green, major basic protein (MBP) positive) in the parenchyma of the lung (1000).

platelet P-selectin is of particular importance in this process. This mechanism has been shown to play a role in platelet–eosinophil interactions in asthma. Platelets have also been observed to undergo diapedesis and localize to airway walls in sections of lung from asthmatic patients, allergen-sensitized and challenged mice, rabbits, and guinea pigs. Platelets may also contribute to the pathogenesis of respiratory diseases via processes independent of

leukocyte involvement (Figure 2). Often, platelets are the first cell type observed to undergo diapedesis into the extravascular tissues following antigen exposure. This effect is mimicked by exposure of animals to PAF. Platelets have also been found in apposition to areas of bronchial smooth muscle (underneath the epithelium) and in areas of eosinophil infiltration. Furthermore, platelets accumulate in the lungs and degranulate following antigen challenge in sensitized

350 PLATELETS

animals, an event that precedes histamine release from mast cells. The close association of platelets with leukocytes in asthmatics, both in circulating blood and in lung tissue, has led to the hypothesis that activated platelets may enhance or direct leukocyte migration into the lung (Figure 2). Indeed, platelets may be selectively recruited to and activated at sites of allergic inflammation by specific cytokines. Cytokines including stromal cell-derived factor-1 (SDF-1), macrophagederived chemokine (MDC), and thymus and activation-regulated cytokine (TARC) activate platelets via their receptors. MDC and TARC expression is induced by the T-helper-2 (Th2) cytokines interleukin (IL)-4 and IL-13, and MDC has been shown to be involved in airways hyperresponsiveness (AHR) and lung inflammation. It is now clear that platelets affect lung function in experimental models of asthma. The intravenous administration of PAF and other platelet agonists in guinea pigs induces bronchospasm, and an accumulation of platelets in the lung. On the other hand, platelet depletion results in the abolition of acute bronchoconstriction in response to inhaled allergen. Platelet depletion has also been shown to protect against anaphylaxis in allergic guinea pigs. The inhibition of bronchoactive agents released by platelets abrogates the resulting bronchospasm in such models. Raised levels of platelet-derived mediators such as the chemokines beta thromboglobulin (b-TG) and PF-4 are observed in plasma and bronchoalveolar lavage (BAL) fluid of atopic individuals during allergen exposure and are often used as markers of platelet activation in inflammation. PF-4 has been shown to upregulate IgG and IgE receptors on eosinophils, induce the release of histamine from basophils, act as a chemokine for neutrophils, monocytes, fibroblasts and eosinophils, and induce hyperresponsiveness to inhaled methacholine in rats. PF-4 is therefore of relevance in the pathogenesis of asthma. Other platelet mediators have been observed in atopic patients after allergen provocation and may influence bronchospasm. These include RANTES (regulated upon activation normally T-cell expressed and secreted), P-selectin, 5-HT, adenosine, histamine, and platelet-derived growth factor (PDGF). Drug induced 5-HT uptake by platelets has been shown to reduce the clinical severity in asthmatic patients. Human platelets may also induce the release of histamine from mast cells and basophils through IgE-dependent mechanisms, as well as following activation with thrombin. Platelet-derived histaminereleasing factor (PDHRF) is a powerful eosinophil chemoattractant, and can cause early and late phase airway obstruction. Other substances released by

platelets in allergic reactions are products of arachidonic acid and phospholipid metabolism. Thromboxane A2 (TXA2), for example, is a potent vasoconstrictor and smooth muscle spasmogen and therefore may act to enhance AHR. Prostaglandin E2 (PGE2) acts as a vasodilator and a primer of naive T cells into a Th2 class. Hydroxyeicosatetraenoic acid (12-HETE) produced by a platelet-specific enzyme (12-lipoxygenase) has chemotactic activity for eosinophils. 12-HETE and arachidonic acid may be taken up by neutrophils to produce 12, 20-diHETE and 5-HETE, respectively; neutrophils require the presence of activated platelets to produce these chemoattractants. 12-HETE also stimulates leukocyte 5-lipoxygenase thus increasing the production of leukotrienes, which induce bronchospasm, mucus hypersecretion, leukocyte chemotaxis, and increased vascular permeability. PAF is produced by a number of inflammatory cells and platelets. This mediator has been implicated in eosinophilia and platelet accumulation in the lungs. Evidence of Platelet Activation by Allergic Stimuli

Production of antigen-specific IgE in response to allergen provocation is a fundamental hallmark of atopic diseases. The cross-linking of antigen to IgE on the surface of mast cells is believed to provide the stimulus for mast cell degranulation in early phase allergic reactions. Interestingly, platelets from atopic individuals with asthma or rhinitis can be activated via IgE as they express both the high-affinity (10– 9 M) and low-affinity (10–7 M) receptors for IgE on the surface membrane. Activation via FceRI results in the release of 5-HT, RANTES, and free radical oxygen species, and activation can be inhibited by drugs such as nedocromil sodium, disodium cromoglycate, and cetirizine. Platelets from asthmatic patients and allergic mice have been observed to undergo chemotaxis in response to allergen exposure, via plateletbound, antigen-specific IgE. This phenomenon is reciprocated in vivo as platelets migrate through lung tissue towards the airway wall in response to allergen exposure. IgE also binds to between 20% and 30% of platelets from normal individuals. This binding affinity can be as high as 50% of platelets from patients with allergies. Platelets from atopic individuals are characterized by a much greater IgE content stored in a-granules compared to nonatopic individuals. Mediators Released by Platelets that Potentially Stimulate Airway Remodeling

In bronchial asthma, chronic inflammation may contribute to airway remodeling. Recent evidence

PLATELETS 351

suggests that platelets are necessary for airway wall remodeling in a murine model of chronic allergic inflammation. Platelets may directly affect bronchial smooth muscle growth, myofibroblast proliferation, and subepithelial fibrosis. These responses may involve platelet products including PDGF, epidermal growth factor (EGF), insulin-like growth factor (IGF), and transforming growth factor beta (TGFb). Interestingly, the major product of arachidonic acid metabolism in platelets, TXA2, is known to induce the proliferation of smooth muscle cells and induce angiogenesis. Antibodies against PDGF-b inhibit both AHR and airway wall thickening, but not cellular infiltration in murine models of asthma. TGF-b increases smooth muscle cell mitogenesis in culture, and it has been suggested that it also increases airway obstruction by participating in subepithelial fibrosis via its chemotactic properties for fibroblasts and neutrophils. Platelets may alter the composition of the extracellular matrix by releasing lysosomal matrix-metalloproteinases found in BAL fluid after allergen challenge in asthmatics. These

effects are germane to the pathogenesis of asthma as they may facilitate inflammatory cell diapedesis and stimulate tissue remodeling.

Platelets in ALI Platelets may play a crucial role in ALI induced by endotoxemia. Platelets are recruited to the inflamed lung and are sequestered in alveolar capillaries before neutrophil influx. Platelets contribute to ALI by directly damaging alveolar-capillary membranes through the release of oxygen radicals; and vasoactive mediators such as 5-HT, TXA2, leading to pulmonary hypertension and culminating in edema. Platelets also interact with the endothelium and form complexes with neutrophils in the pulmonary microcirculation during systemic inflammation. Platelets therefore also contribute to ALI by promoting neutrophil-induced pulmonary damage by facilitating neutrophil chemotaxis and activation with the release of PF-4, TXA2, and products of the lipoxygenase pathway.

Free radical and cationic protein release by platelets and leukocytes, leading to damaged airway epithelium

Mucus hypersecretion leading to obstruction of airway lumen

Subepithelial fibrosis

Smooth muscle hyperplasia, hypertrophy

Enhanced leukocyte recruitment through heterotypic complexes ‘priming’ leukocytes for efficient endothelial adhesion

Extracellular matrix degradation

Platelet activation leading to mediator release and platelet chemotaxis Eosinophil, lymphocyte, neutrophil, platelet chemotaxis Increased pulmonary hypertension and vascular permeability leading to edema

Blood vessel

Figure 3 Putative role of platelets in respiratory diseases. Adapted from Pitchford SC and Page CP (2002) Platelets and allergic diseases. In: Gresele P, Page C, Fuster V, and Vermylen J (eds.) Platelets in Thrombotic and Non-Thrombotic Disorders, pp. 852–868. Cambridge: Cambridge University Press.

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Platelets in COPD

Further Reading

The involvement of platelets in COPD is less well known than the involvement of platelets in asthma. However, the occurrence of platelet hyperreactivity has been demonstrated in ex vivo studies where platelets had an increased sensitivity to various agonists and elevated levels of plasma b-TG. These reports reflect the occurrence of in vivo platelet activation as measured by increased synthesis of TXA2 in patients with COPD, and the administration of a TXA2 antagonist was beneficial in improving respiratory distress in patients with chronic pulmonary emphysema. There is also evidence that in COPD patients, local hyperreactivity of platelets in pulmonary vessels is associated with the induction of pulmonary hypertension, since a significant increase in platelet aggregates and b-TG was observed in COPD patients with secondary pulmonary hypertension. A feature of platelet activation associated with patients with asthma, cardiovascular disease, and rheumatoid arthritis is the presence of circulating platelet– leukocyte complexes, and these actions are platelet P-selectin dependent. Interestingly, increased levels of soluble P-selectin of platelet origin have been reported in COPD patients, suggesting that platelets may play a role in neutrophil recruitment in this circumstance.

Davi G, Basili S, Vieri M, et al. (1997) Enhanced thromboxane biosynthesis in patients with chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 156: 1794–1799. Eichhorn ME, Ney L, Massburg S, et al. (2002) Platelet kinetics in the pulmonary microcirculation in vivo assessed by intravital microscopy. Journal of Vascular Research 39: 330–339. Ferroni P, Basili S, Martini F, et al. (2000) Soluble P-selectin as a marker of platelet hyperactivity in patients with chronic obstructive pulmonary disease. Journal of Investigative Medicine 48: 21–27. Gresele P, Dottorini M, Selli ML, et al. (1993) Altered platelet function associated with the bronchial hyperresponsiveness accompanying nocturnal asthma. Journal of Allergy and Clinical Immunology 91: 894–902. Heffner JE, Sahn SE, and Repine JE (1987) The role of platelets in the adult respiratory distress syndrome. Culprits or bystanders? American Review of Respiratory Diseases 135: 482– 492. Joseph M, Capron A, Ameisen JC, et al. (1986) The receptor for IgE on blood platelets. European Journal of Immunology 16: 306–312. Maccia CA, Gallagher JS, Ataman G, et al. (1977) Platelet thrombopathy in asthmatic patients with elevated immunoglobulin e. Journal of Allergy and Clinical Immunology 59: 101–108. Mathur A and Martin JF (2002) Platelet heterogeneity. In: Gresele P, Page C, Fuster V, and Vermylen J (eds.) Platelets in Thrombotic and Non-Thrombotic Disorders, pp. 70–79. Cambridge: Cambridge University Press. Metzger WJ, Sjoerdsma K, Richerson HB, et al. (1987) Platelets in bronchoalveolar lavage from asthmatic patients and allergic rabbits with allergen-induced late phase responses. Agents Actions 21(supplement): 151–159. Momi S and Gresele P (2002) Platelets and chemotaxis. In: Gresele P, Page C, Fuster V, and Vermylen J (eds.) Platelets in Thrombotic and Non-Thrombotic Disorders, pp. 393–411. Cambridge: Cambridge University Press. Page CP, Paul W, and Morley J (1984) Platelets and bronchospasm. International Archives of Allergy and Applied Immunology 74: 347–350. Pitchford SC, Momi S, Giannini S, et al. (2005) Platelet P-selectin is required for pulmonary eosinophil and lymphocyte recruitment in a murine model of allergic inflammation. Blood 105: 2074–2081. Pitchford SC and Page CP (2002) Platelets and allergic diseases. In: Gresele P, Page C, Fuster V, and Vermylen J (eds.) Platelets in Thrombotic and Non-Thrombotic Disorders, pp. 852–868. Cambridge: Cambridge University Press. Pitchford SC, Riffo-Vasquez Y, Sousa A, et al. (2004) Platelets are necessary for airway wall remodelling in a murine model of chronic allergic inflammation. Blood 103: 639–647. Slater DN, Trowbridge EA, and Martin JF (1983) The megakaryocyte in thrombocytopenia: a microscopic study which supports the theory that platelets are produced in the pulmonary circulation. Thrombosis Research 31: 163–176.

Therapeutic Considerations Despite recent advances revealing platelet participation in the pathogenesis of asthma, ALI, and COPD, detailed investigations of platelet-dependent mechanisms of disease are in their infancy (Figure 3). This may explain why no specific therapy based on inhibition of platelet function has been developed for the treatment of these respiratory diseases. Further investigation of the role of platelets in airway inflammation and remodeling may, however, yield novel therapeutic approaches in the future. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Matrix: Focal Contacts and Signaling. Allergy: Allergic Rhinitis. Asthma: Overview. Bronchoalveolar Lavage. CD1. Chemokines, CC: RANTES (CCL5). Epidermal Growth Factors. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

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PLEURAL EFFUSIONS Contents

Overview Pleural Fluid, Transudate and Exudate Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube Parapneumonic Effusion and Empyema Malignant Pleural Effusions Postsurgical Effusions Pleural Fibrosis Chylothorax, Psuedochylothorax, LAM, and Yellow Nail Syndrome Hemothorax

Overview R W Light, Vanderbilt University, Nashville, TN, USA Y C G Lee, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Pleural effusions are common in clinical practice, affecting 3000 patients per million population each year. Pleural effusions develop when the rate of pleural fluid formation exceeds that of its drainage. A pleural effusion should be considered in any patient with dyspnea, and the diagnosis is usually made on plain radiographs or by ultrasonography. Establishing the underlying cause of a pleural effusion is often challenging because a wide range of local and systemic diseases can lead to pleural effusion formation. An accurate diagnosis often requires detailed clinical data, appropriate radiological imaging, and laboratory analyses of the pleural fluid (and/or tissue). Pleural fluid samples should be analyzed to determine whether it is a transudate or exudate. A transudate usually results from cardiac failure or hepatic cirrhosis, whereas parapneumonic effusions, tuberculosus, and malignancy are the most common cause of exudates. Pleural biopsy may increase diagnostic yield, especially in tuberculous pleuritis. Thoracoscopic or computed tomography-guided pleural biopsy may provide histologic confirmation of malignant pleural effusions. Up to 20% of pleural effusions may remain undiagnosed, and it is hoped that advances in molecular medicine will improve the diagnostic sensitivity for various pleural diseases. Management of pleural effusions should aim at controlling the underlying cause and evacuation of the fluid for relief of dyspnea. More definitive approaches (e.g., pleurodesis) should be considered for patients with recurrent symptomatic effusions.

A pleural effusion refers to an abnormal accumulation of fluid in the pleural cavity. Pleural effusion is an important clinical problem, affecting more than 750 000 people in the United States annually, and is frequently a diagnostic and management challenge. Pleural effusion is often a result of local pleuropulmonary diseases but can also complicate many systemic pathologies. There are more than 50 causes

of exudative effusions and depending on the etiology, differing management strategies are indicated. Hence, establishing a definitive cause of a pleural effusion is important but can be difficult. Pleural effusions are often overlooked in the acute medical setting, resulting in delay in diagnosis and appropriate therapies. Definitive therapeutic interventions are frequently needed to control the recurrence of the effusion and the associated dyspnea.

Pathophysiology In the healthy state, the pleural cavity contains a small amount of fluid (o10 ml in a 70 kg man) that serves as a lubricant to facilitate the gliding of the visceral pleura over the parietal pleural membrane (see Pleural Effusions: Pleural Fluid, Transudate and Exudate. Pleural Space). This fluid is a transudate and contains mainly macrophages. Normally, fluid exits the pleural cavity via the stomata on the parietal pleura, which empty into lymphatic channels. Pleural effusions develop when there is increased fluid formation or reduced fluid removal or both. Transudative effusions, most commonly due to cardiac failure or hepatic cirrhosis, accumulate from imbalances in hydrostatic and oncotic pressures, such that excessive pleural fluid formation saturates the drainage capacity of the pleural cavity. Pleural inflammation, lymphatic disruptions, or malignancy underlie the development of most exudative effusions. Exudates accumulate due to increased vascular permeability. In many cases, decreased fluid resorption also contributes to the accumulation of the pleural fluid. This can be a result of blockage of the lymphatic stomata in the parietal pleura (e.g., by metastatic carcinomas) or obstruction of the downstream lymphatic flow (e.g., by regional lymphadenopathy). Occasionally, fluid can enter the pleural cavity from extrapleural sites. Transdiaphragmatic migration of

354 PLEURAL EFFUSIONS / Overview

ascitic fluid into the pleural cavity is well described. This occurs because the intraperitoneal pressure is less negative than the intrapleural pressure, and diaphragmatic pores exist in many individuals that permit transdiaphragmatic movement of the ascitic fluid. Blood from traumatic laceration of blood vessels, cerebrospinal fluid from a dural–pleural fistula, or urine from a renal tract fistula can accumulate in the pleural cavity. Puncture of central veins by venous catheters can lead to subsequent pleural accumulation of fluids administered intravenously (e.g., parental nutrition).

Symptoms and Signs of Pleural Effusions Patients with pleural effusions can be asymptomatic but often present with breathlessness, especially during exercise. Dyspnea results mainly from altered diaphragmatic and chest wall mechanics rather than from compression of the lung. Underlying lung diseases often contribute to the breathlessness. Pleuritic chest pain, if present, indicates inflammation of the parietal pleura (but not the visceral pleura, which contains no pain fibers). Pleural effusions can be bilateral or unilateral (in the latter case, 60% are right-sided). The size of the effusion and rate of fluid accumulation vary markedly. A pleural effusion, especially when reaching a moderate size (e.g. 4500 ml), usually yields characteristic physical signs of percussion (stony) dullness, reduced fremitus, and the absence of breath sounds. Occasionally, an elevated hemidiaphragm or subphrenic fluid collections will give similar signs.

Diagnosing a Pleural Effusion Detection of Pleural Fluid

Pleural effusions are commonly first detected on chest radiographs (Figure 1). However, ultrasound and computed tomography (CT) scanning have superceded chest (plain and decubitus) radiographs as the most sensitive radiological modalities to detect and locate small pleural effusions (e.g., for thoracentesis (pleural fluid aspiration)). This is because detecting (and estimating the size of) pleural effusions on radiographs can be difficult, especially in the presence of atelectasis, lung consolidation, pleural thickening, or an elevated hemidiaphragm. Although thoracentesis can be safely performed at the bedside for large effusions, the threshold for performing image-guided thoracentesis should be low, especially if the effusion is small and/or loculated, the radiological features are atypical, and the patient is at high risk (e.g., those with poor lung reserve).

Figure 1 Chest radiograph of a large unilateral right-sided pleural effusion. Notice the typical meniscus sign, suggesting that opacity is likely a result of pleural fluid.

Investigating the Underlying Cause of Pleural Effusions

Diagnosing the underlying cause of a pleural effusion is challenging for a multitude of reasons. The range of diseases that can cause a pleural effusion is wide (Table 1). Not uncommonly, the effusion is a complication of systemic disorders or diseases of a distant organ. In most cases, no pathognomic test of the pleural fluid exists to provide a definitive diagnosis. Hence, it must be emphasized that the (detailed) clinical history, radiological imaging, and biochemical/ microbiological/cytological analyses of the pleural fluid (and/or tissue) must be assimilated to accurately establish the cause of an effusion. Even for a given radiological and biochemical result, the most likely cause of a pleural effusion varies with the age (e.g., malignancy in an elderly patient), geographic location (e.g., tuberculous (TB) pleuritis in endemic countries), and individual risk factor (e.g., HIV status) of the patient. Pleural Fluid Analysis

Pleural fluid analysis is crucial in the diagnosis of most pleural effusions (see Pleural Effusions: Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube). Thoracentesis is generally safe. Complications, which are rare, include vasovagal syncope, bleeding, infection, and iatrogenic pneumothorax. Few absolute contraindications exist; however, patients with a

PLEURAL EFFUSIONS / Overview 355 Table 1 Causes of pleural effusions Transudates Common Congestive heart failure Hepatic cirrhosis Nephrotic syndrome Hypoalbuminemia from other causes Uncommon Constrictive pericarditis Transdiaphragmatic migration of peritoneal dialysate Effusions secondary to trapped lungs Superior vena caval obstruction Hypothyroidism Exudates Common Parapneumonic effusions Tuberculous pleuritis Malignancy Hemothorax (e.g., traumatic) Pulmonary embolism Rheumatological (especially rheumatoid arthritis, systemic lupus erythematosus) Pancreatic (acute pancreatitis, pancreatic pseudocyst) Secondary to subdiaphragmatic inflammation (e.g., hepatic abscess) Uncommon Drug-induced Lymphatic disruption (chylothorax, pseudochylothorax, lymphangioleiomyomatosis, yellow nail syndrome) Esophageal perforation Misplacement of central venous catheter Sarcoidosis Benign asbestos pleural effusion Hypothyroidism Ovarian hyperstimulation syndrome Postirradiation Fungal/parasitic infection Meig’s syndrome Cerebrospinal fluid leak Modified from Sahn SA and Heffner JE (2003) Pleural fluid analysis. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 191–209. London: Arnold.

coagulopathy and those with underlying cystic lung diseases are at higher risk of bleeding or pneumothorax. Premedication is not generally required because vasovagal reactions are uncommon (0.6%). Drainage of a large volume of fluid (e.g., 41 l) may lead to re-expansion pulmonary edema. Most patients develop a cough (with or without chest pain) after the removal of a large quantity of pleural fluid, at which time the procedure should be discontinued. Pleural manometry or measurement of elastance can be used as a guide during fluid removal. The first question when investigating an effusion is whether it is a transudate or exudate. Light’s criteria provide the best biochemical criteria to diagnose exudative effusions. A pleural effusion is an exudate if

any of the following criteria are met, and it is a transudate if it meets none of them: * *

*

Pleural fluid: Serum protein ratio 40.5 Pleural fluid: Serum lactate dehydrogenase (LDH) ratio 40.6 Pleural fluid LDH more than two-thirds of the upper limit of normal serum LDH

If the pleural fluid is a transudate, additional tests are not needed, provided it is in keeping with the clinical picture. For exudates, additional tests on the pleural fluid are often required. Light’s criteria can misidentify some transudates as exudates. Differential pleural fluid leukocyte counts can help direct investigations, as can pleural fluid pH and glucose. If clinically appropriate, the fluid should be sent for Gram staining (preferably from cell blocks of the fluid), and bacterial and mycobacterial culture. Cytological examination should be performed if malignancy is suspected. Currently, tumor markers or cytokine levels in pleural fluid have limited clinical value. Specific tests (e.g., pleural fluid hematocrit, amylase, lipid profile, and adenosine deaminase (ADA)) should be ordered when clinically indicated.

Pleural Tissue Analysis Closed pleural biopsy (e.g., with Abram’s or Cope’s needles) remains valuable if the clinical suspicion of tuberculosis is high because the pleura is diffusely involved in TB pleuritis. For malignant pleural effusion, the pleural lesions are usually patchy, and biopsy under direct vision (e.g., during thoracoscopy; Figure 2) or CT-guided cutting needle biopsy are recommended over (blinded) closed pleural biopsy. Complications of pleural biopsy include local pain, pneumothorax, vasovagal reaction, and bleeding. With advances of thoracoscopy, open (thoracotomy) biopsy is seldom necessary.

Management of Common Types of Pleural Effusions General Principles

Management of a pleural effusion depends on its specific etiology. However, several general principles may apply: 1. Treating the underlying condition: in many cases, control of the underlying disease (e.g., cardiac failure) will stop the effusion from reaccumulating. In other cases (e.g., malignant effusions), the underlying disease cannot be controlled, and

356 PLEURAL EFFUSIONS / Overview Common Pleural Effusions

Figure 2 Thoracoscopic view showing malignant deposits in the parietal pleura. Courtesy of Dr Robert J O Davies, Oxford University, Oxford, UK.

management is directed toward minimizing fluid accumulation and the associated symptoms. 2. Evacuation of pleural fluid: drainage of the effusion is useful if the patient is symptomatic. A therapeutic thoracentesis may be adequate in some cases. Large effusions are better drained by a pleural catheter (chest tube). Small-bore catheters are more comfortable and are as efficient as large-bore chest tubes. Indwelling small-bore catheters also allow ambulatory drainage. Pleuroperitoneal shunts can move fluid from the pleural cavity to the peritoneal cavity, where the fluid is reabsorbed. 3. Pleurodesis: pleurodesis, the iatrogenic induction of pleural symphysis to obliterate the pleural cavity, can be performed to control recurrent symptomatic effusions. Pleurodesis is achieved by inducing pleural inflammation either by instillation of a chemical agent intrapleurally or by mechanical abrasion during surgery. Talc followed by tetracycline derivatives and bleomycin are the most commonly used pleurodesing agents. OK-432, silver nitrate, and quinacrine are used in some areas of the world. Talc is generally regarded as more effective, but talc pleurodesis induces systemic and lung inflammation, which in severe cases, can result in acute respiratory failure and, rarely, death. In terminally ill patients, serial thoracenteses and the use of oxygen and narcotic agents to control the dyspnea may be more appropriate than pleurodesis.

Congestive heart failure Congestive heart failure (CHF) is the most common cause of pleural effusions among general medical admissions. Approximately 50–75% of patients admitted with CHF have pleural effusions (commonly bilateral). Experimental studies show that in CHF the pleural space acts as an exit route for the interstitial fluid (edema) of the lung. A compatible clinical history in conjunction with a transudative effusion secures the diagnosis. Occasionally, pleural fluid from CHF may appear as ‘exudates’ with Light’s criteria, especially in patients receiving diuretics. A normal plasma-to-pleural fluid protein gradient of 43.1 g dl 1 indicates that the effusion was formed as a transudate, even if it is categorized as an exudate by Light’s criteria. Management of the underlying CHF should control the effusions (see Pulmonary Edema). If the effusion is large, drainage of the fluid may provide prompt symptomatic improvement. Pleurodesis is not usually required. Parapneumonic effusions and empyema Up to 50% of patients with pneumonia develop a parapneumonic effusion, which must be managed promptly to avoid evolution into empyema. Appropriate antibiotics should be initiated early. Thoracentesis should be performed to assess the characteristics of the pleural fluid, which dictate the subsequent management of the effusion. Patients with pneumonia should have a radiological follow-up to ensure no subsequent accumulation of a parapneumonic effusion, even if none was evident at initial presentation. Empyema occurs if the pleural fluid is infected, and it has significant morbidity and mortality (up to 20%). The pleural fluid is characterized by neutrophilia, low pH and glucose, and may appear as frank pus. Empyemas are often loculated, prohibiting complete drainage. A chest tube should be inserted promptly, preferably under image guidance. If the fluid cannot be satisfactorily drained, surgical drainage by video-assisted thoracoscopic surgery or thoracotomy should be considered. Tuberculous effusions The incidence of TB pleuritis varies significantly worldwide. In endemic countries, TB is the most common cause of a pleural effusion. TB pleural effusions are often difficult to diagnose for several reasons. TB pleuritis develops as a delayed hypersensitivity reaction to the mycobacterial proteins, and the number of organisms in the pleural space is often low. Hence, pleural fluid and pleural tissue culture are often negative. Also, the diagnosis

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of TB is easily overlooked because in 450% of patients TB pleuritis represents a primary infection, without disease activity elsewhere. TB pleural fluid is typically a lymphocytic exudate with (usually but not inevitably) normal glucose and pH. Pleural biopsy is sensitive (480%) for diagnosing TB because the presence of caseating granulomata is indicative of TB pleuritis. Pleural fluid ADA measurement is useful: a high level is very suggestive of TB, although false positives can occur with lymphoma, metastatic malignancies, or rheumatoid pleural effusions. Without treatment, TB effusions usually resolve spontaneously. Left untreated, approximately 60% of patients develop TB in other organ systems within a few years. Standard anti-TB regime is recommended. Both systemic corticosteroids and pigtail drainage of the effusion can improve symptoms but do not reduce the risk of subsequent pleural fibrosis, a known complication of TB pleuritis (see Pleural Effusions: Pleural Fibrosis). Malignant pleural effusions Malignant pleural effusion (MPE) accounts for approximately one-fifth of all pleural effusions and affects many patients with breast (up to 50%) and lung carcinomas (25%). More than 90% of patients with pleural mesothelioma, an endemic disease with 250,000 deaths expected in Western Europe over the next 35 years, present with a pleural effusion (see Mesothelioma, Malignant). In up to 20% of patients with MPE, pleural effusion is the first presentation of malignancy. MPE should only be diagnosed with histological or cytological confirmation. CT scanning or magnetic resonant imaging may provide supportive (but not definitive) evidence that suggests an MPE. Thoracoscopy can be used to obtain pleural biopsy under direct vision. If pleural abnormalities are identifiable on CT scans, CT-guided biopsy is a useful alternative. If the patient has significant symptomatic benefit from fluid drainage, pleurodesis should be considered. Chemical pleurodesis is as effective as surgical pleurodesis for MPEs and avoids the need for anesthesia. An alternative to pleurodesis is the insertion of an indwelling catheter. In patients who fail pleurodesis, repeated thoracenteses, pleuroperitoneal shunting, or indwelling small-bore catheter drainage can be considered. If the patient does not have symptomatic improvement from removal of the pleural effusion (e.g., if the lung is trapped), pleurodesis is not indicated. Pulmonary embolism Approximately 50% of patients with pulmonary embolism have a pleural effusion on CT scanning, and this diagnosis should

be considered in patients with undiagnosed pleural effusions. The biochemistry of the pleural fluids that complicate pulmonary emboli is often non-specific but is usually in the exudative range. These effusions are often small and do not require intervention. Undiagnosed pleural effusions Referral to a specialist respiratory unit is advisable if the effusion remains undiagnosed after the initial workup, especially if the effusion recurs, has atypical radiological or biochemical features, or if the patient is unwell. Up to 20% of pleural effusions remain undiagnosed despite thorough investigations. Most of them are benign and eventually resolve spontaneously.

Conclusion Pleural effusions are common challenges in clinical practice. Identifying the underlying cause depends on obtaining appropriate clinical history and radiological and laboratory analyses. Advances in molecular medicine may aid the diagnosis of pleural effusions in the future. Management strategies include treating the underlying cause and/or drainage of the pleural fluid. A better understanding of the pathophysiology of fluid formation is needed to develop novel approaches to control fluid accumulation. See also: Mesothelial Cells. Mesothelioma, Malignant. Pleural Effusions: Pleural Fluid, Transudate and Exudate; Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube; Parapneumonic Effusion and Empyema; Malignant Pleural Effusions; Postsurgical Effusions; Pleural Fibrosis; Chylothorax, Psuedochylothorax, LAM, and Yellow Nail Syndrome; Hemothorax. Pleural Space.

Further Reading Antunes G, Neville E, Duffy J, and Ali N (2003) BTS guidelines for the management of malignant pleural effusions. Thorax 58(supplement 2): 29–38. Davies CWH, Gleeson FV, and Davies RJO (2003) The British Thoracic Society guidelines on the management of pleural infection. Thorax 58(supplement 2): 18–28. Evans AL and Gleeson FV (2004) Radiology in pleural disease: State of the art. Respirology 9: 300–312. Lee YCG and Davies RJO (2004) Management of pleural effusions in acute medical settings: A practical guide. CPD Journal of Acute Medicine 3: 26–32. Lee YCG and Light RW (2004) Management of malignant pleural effusions. Respirology 9: 148–156. Light RW (2000) Talc should not be used for pleurodesis. American Journal of Respiratory and Critical Care Medicine 162: 2024–2026. Light RW (ed.) (2001) Pleural Diseases. Baltimore: Lippincott, Williams & Wilkins. Light RW (2002) Clinical practice. Pleural effusion. New England Journal of Medicine 346(25): 1971–1977.

358 PLEURAL EFFUSIONS / Pleural Fluid, Transudate and Exudate Light RW and Lee YCG (eds.) (2003) Textbook of Pleural Diseases. London: Arnold. Light RW and Lee YCG (2005) Pneumothorax, chylothorax, hemothorax and fibrothorax. In: Murray J, Nadel J, Mason R, and Broaddus VC (eds.) Textbook of Respiratory Diseases, 4th edn., pp. 1961–1988. Philadelphia: Saunders. Maskell NA and Butland RJ (2003) BTS guidelines for the investigation of a unilateral pleural effusion. Thorax 58(supplement 2): 8–17. Sahn SA and Heffner JE (2003) Pleural fluid analysis. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 191–209. London: Arnold. West SD, Davies RJO, and Lee YCG (2004) Pleurodesis for malignant pleural effusions: Current controversies and variations in practices. Current Opinion in Pulmonary Medicine 10: 305–310.

Pleural Fluid, Transudate and Exudate Y C G Lee, University College London, London, UK M Noppen, University Hospital AZ-VUB, Brussels, Belgium R W Light, Vanderbilt University, Nashville, TN, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pleural effusion is a common clinical problem. In the normal state, the pleural cavity is bathed in a small volume of physiologic pleural fluid containing mainly macrophages and lymphocytes. The volume of the pleural fluid can increase dramatically with most pathologic conditions affecting the pleura. The pleural effusion will alter the respiratory mechanics, commonly resulting in dyspnea. It is useful to differentiate the pleural effusion into transudates and exudates. Traditionally, such differentiation is made using Light’s criteria, based on the protein and lactate dehydrogenase levels in pleural fluid and serum. Transudates occur as a result of altered hydrostatic and/or oncotic pressures and are usually secondary to congestive cardiac failure or hepatic cirrhosis. Exudates develop as a result of plasma extravasation, which is at least in part due to pleural or pulmonary inflammation. Evidence suggests that cytokines, such as vascular endothelial growth factor, play a role in exudative effusion formation. Parapneumonic effusion, malignant effusion, and tuberculous pleuritis are the most common causes of exudative effusions worldwide.

Pleural Fluid Formation in the Normal State In the healthy state, the pleural cavity – which is extremely thin (10 mm) but has a large surface area (1–2 m2) – contains a small amount of fluid that serves as a lubricant to facilitate the gliding of the visceral pleura over the parietal pleural membrane. This fluid is a transudate and contains mainly macrophages.

Most of the knowledge on the volume, composition, and dynamics of normal pleural fluid has been obtained in animal studies, and very few human studies are available. Pleural fluid volume assessment in normal circumstances can be performed using direct measurements (e.g., gentle aspiration after pleural puncture, pleural catheterization, or even thoracotomy) or using indirect techniques (e.g., pleural lavage). The normal physiologic pleural fluid is formed by filtration from systemic vessels and occurs predominantly at the less dependent region of the pleural cavity, where the blood vessels are closest to the mesothelial surface. The systemic blood supply from the intercostal arterial circulation of parietal pleura is believed to be the principal source of this fluid. In humans, the bronchial circulation of the visceral pleura is not likely to contribute significantly because the human visceral pleura is thick and the microvascular pressure for fluid filtration in the bronchial circulation is low (relative to that of the parietal intercostal circulation). Water filters into the pleural space according to the net hydrostatic–oncotic pressure gradient. Water and small molecules (p4 nm) pass freely between the mesothelial cells. Larger molecules can be actively transported through the mesothelial cells via a transcytoplasmatic pinocytic mechanism. This mechanism probably also contributes to liquid and protein exchange in the pleural space, although its overall contribution is unclear. Direct measurements of normal pleural fluid volume in various animal models (e.g., rabbits, dogs, and sheep) consistently show volumes between 0.04 and 0.12 ml kg 1 per pleural space. Differences in measurement results are caused by the various methodologies used and by the fact that the fluid volume adherent to the lung surface has not always been included. In humans, one direct measurement study by Yamada (puncture of the pleural space in the 9th or 10th intercostal space at the posterior axillary line) yielded some fluid in one-third of normal individuals. Interestingly, after exercise, up to two-thirds of individuals had detectable pleural fluid. Most often, only some foam was retrieved, but in some individuals up to 20 ml of fluid was collected. In another study, a pleural lavage procedure was performed in normal subjects undergoing thoracoscopy for treatment of sympathetic disorders. Estimation of the volume of the original pleural fluid present was performed using the urea dilution method, based on the principles of dilution and mass conservation. The volume of the original fluid present in each hemithorax was 0.1370.06 ml kg 1 body mass. The biochemical composition of normal pleural fluid resembles that of other interstitial fluids. The

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concentration of small molecules (e.g., glucose and urea) is similar to that of serum, whereas the concentration of macromolecules such as protein or lactate dehydrogenase is less than half of that found in serum (transudate by Light’s criteria). In normal pleura, the production of physiologic pleural fluid is estimated to be approximately 0.01 ml kg 1h 1 in sheep and 0.02 ml kg 1h 1 in rabbits, which extrapolates to 17 ml day 1 in a 70-kg man. The half-life of fluid turnover in sheep and rabbits is 6–8 h. Fluid exits the pleural cavity via the stomata (diameter, 2.5–10 mm) on the parietal pleura, which empty into lymphatic channels by bulk flow such that liquid and protein are evacuated at the same rate. Hence, the protein concentrations of the residual pleural fluid remain constant. The drainage capacity in normal pleura is large (approximately 0.2– 0.3 ml kg 1h 1) and can increase up to 30-fold over the normal rate of drainage after a pleural fluid load. The cellular composition of normal pleural fluid has mainly been studied in animal models. Total white blood cell counts varied between 12167800 and 24427595 cells ml 1, mostly consisting of macrophages/monocytes. Other free cell populations included mesothelial cells and lymphocytes. Using the direct measurement method, Yamada and colleagues found that in physiologic human pleural fluid, the total white blood cell count was 4500 cells ml 1, with a differential cell count of 53.7% monocytes/macrophages, 10.2% lymphocytes, 3.6% granulocytes, and 3% mesothelial cells (and 29.5% deteriorated cells of unclear classification). In a human pleural lavage study, the concentration of white blood cells in the physiologic pleural fluid was 1716 cells ml 1. In nonsmoking subjects, the leukocytes consisted of 75% macrophages (median, interquartile range (IR), 64–80%), 23% lymphocytes (IR, 18–36%) with a CD4:CD8 ratio of 0.75 (IR, 0.6–1%), and 1% free mesothelial cells (IR, 0–2%). Neutrophils and eosinophils were only sporadically seen. Interestingly, there was a small but significant increase in neutrophils in smokers. Mesothelial cells, macrophages, lymphocytes, eosinophils, and neutrophils are thought to play a role in the defense of the pleural space and may be involved in the generation of a variety of inflammatory responses. Pleural cells are essential participants in the development of pleural pathologies, including inflammatory and metastatic diseases.

Pleural Fluid Formation in Pathologic States A pleural effusion usually refers to an abnormal accumulation of pleural fluid. The pleural space is

under subatmospheric pressure and can accommodate a large volume of fluid, partly through alteration of the chest wall and diaphragm mechanics and through compression of the underlying lung. Pleural fluid accumulates when the rate of pleural fluid formation exceeds the rate of pleural fluid removal (Table 1). Most effusions develop from both increases in pleural fluid entry and decreases in fluid exit rates. In the presence of the normal fluid absorption capacity, fluid formation has to increase by more than 30-fold, and remain at that rate, to create an effusion. On the other hand, decreased removal of the fluid alone is unlikely to result in significant accumulation of pleural fluid, given that the normal rate of pleural fluid formation is only approximately 17 ml day 1. Hence, often abnormalities in production as well as absorption mechanisms are present in patients with pleural effusions. The most common cause of increased pleural fluid formation is increased interstitial fluid in the lung, which can result either from changes in the pressure gradients producing a transudate with a low protein concentration or from changes in vascular permeability producing an exudate with a high protein level. Twenty percent of interstitial fluid leaves the lung via the pleural space. Starling’s equation dictates the movement of fluid across the pleural capillaries, and therefore increased intravascular pressures, increased pleural fluid protein levels, and decreased intrapleural pressures can all contribute to pleural fluid formation. Increased pleural fluid formation also results from pleural inflammation. Exudates form from a vascular bed with a high leakiness to protein. Inflammation or

Table 1 General causes of pleural effusions Increased pleural fluid formation Increased interstitial fluid in the lung Left ventricular failure, pneumonia, and pulmonary embolus Increased intravascular pressure in pleura Right or left ventricular failure, superior vena cava syndrome Increased permeability of the capillaries in the pleura Pleural inflammation Increased levels of VEGF Increased pleural fluid protein level Decreased pleural pressure Lung atelectasis or increased elastic recoil of the lung Increased fluid in peritoneal cavity (if diaphragm communications exist) Ascites or peritoneal dialysis Disruption of the thoracic duct (chylothorax) Disruption of blood vessels in the thorax (hemothorax) Decreased pleural fluid absorption Obstruction of the lymphatics draining the parietal pleura Elevation of systemic vascular pressures Superior vena cava syndrome or right ventricular failure

360 PLEURAL EFFUSIONS / Pleural Fluid, Transudate and Exudate

injury to the vascular bed enhances the vascular permeability, resulting in the accumulation of fluids of high protein concentrations (exudates). Pleural effusion can also occur from transdiaphragmatic migration of ascites, from disruption of the thoracic duct (chylothorax) or an intrathoracic blood vessel (hemothorax), or from a fistula draining other body fluids from extrapleural organs (e.g., urinothorax from a renal fistula). The most common cause of decreased pleural fluid absorption is obstruction of the lymphatics draining the parietal pleura. This can occur in the parietal pleura if the pleura is inflamed, such as with tuberculous pleuritis or parapneumonic effusion, or in the lymph nodes, such as with malignant effusions when the nodes are involved with tumor.

Transudate and Exudate When faced with a patient with abnormal pleural fluid accumulation, a useful first step is to determine if the effusion is a transudate or exudate. This helps to provide insight into the etiology (and differential diagnosis) of the pleural effusion. Transudative effusions, most commonly due to cardiac failure or hepatic cirrhosis, accumulate from imbalances in hydrostatic and oncotic pressures, such that excessive pleural fluid formation saturates the drainage capacity of the pleural cavity. Exudates accumulate due to increased vascular permeability, often (but not necessarily) with a concomitant reduction in fluid reabsorption. Parapneumonic pleural effusions, tuberculous pleuritis, and malignant pleural effusions account for most of the exudative effusions. Most commonly, differentiation between transudative and exudative effusions is made using Light’s criteria. A pleural effusion is an exudate if it satisfies any of the following criteria. Conversely, a transudate is one that meets none of the criteria: * *

*

pleural fluid:serum protein ratio 40.5; pleural fluid LDH more than two-thirds of the upper limit of normal serum LDH; and pleural fluid:LDH ratio 40.6.

Light’s criteria have a high sensitivity for the diagnosis of exudates. False positives are known to occur. In particular, patients with transudative effusions who are on diuretics may have a pleural:serum protein ratio 40.5. In such a situation, the protein gradient (serum–pleural fluid protein) 43.1 g dl 1 would define a true transudate. The diagnosis of pleural effusions and their clinical presentation and management are discussed elsewhere in this book.

Immunologic Basis of Pleural Effusion Formation Increased fluid formation is the principal underlying abnormality in the genesis of most exudative effusions. None of the available strategies for managing recurrent effusions, such as pleurodesis or pleurectomy, targets directly against pleural fluid formation. Various molecules have been proposed as having a pathogenic role in pleural effusion formation, of which vascular endothelial growth factor (VEGF) has attracted the most attention. There is compelling evidence that VEGF promotes angiogenesis and tumor growth, and various anti-VEGF strategies are in clinical trials. VEGF is a potent inducer of vascular permeability and can also enhance permeability in the mesothelial monolayer. In vivo, it induces vasodilatation and can increase the capillary and venular leakage by opening the endothelial intercellular junctions and by inducing the development of fenestrae in endothelia. Additionally, VEGF enhances the activity of vesicular vacuolar organelles, through which macromolecules extravasate, and promotes active transendothelial transport. VEGF is present in significant quantities in pleural and peritoneal effusions of varying etiologies, especially in malignant effusions, empyemas, ovarian hyperstimulation syndrome, and Meigs’ syndrome. Local pleural or peritoneal production of VEGF from residential mesothelial cells and infiltrating cells, such as inflammatory cells and malignant cells, is believed to be the main source of VEGF in exudative effusions. VEGF production can be stimulated by various cytokines, among which transforming growth factorb appears to be the most potent and consistent, both in vitro and in vivo. Staphylococcus aureus, as well as the resultant hypoglycemia and acidosis, can stimulate mesothelial release of VEGF and may explain why VEGF levels are high in empyema. Animal experiments have confirmed the essential role of VEGF in malignant pleural effusion and in ascites accumulation. Promising results are accumulating on the use of VEGF inhibition (e.g., neutralizing anti-VEGF antibodies, VEGF receptor phosphorylation blockade, and antibodies against KDR/Flk-1 receptors) in reducing vascular permeability and malignant effusion formation in animal studies. If these results can be reproduced in clinical settings, anti-VEGF therapy would represent a novel management strategy for recurrent pleural effusions.

Animal Models for Pleural Effusion Studies Many animal models have been employed to study the mechanisms of pleural fluid formation and

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absorption and the associated physiologic changes in the pleural cavity. Transudate formation from heart failure has been studied by creating volume overload in anesthetized ventilated sheep. Fluid transferred from lung parenchyma into the pleural space was collected by wrapping a bag around the exposed lung. Such experiments confirmed that the pleural space provided an important route of clearance of pulmonary edema. A murine model of renal failure and fluid overload has been described by ligating the renal vessels followed by intraperitoneal instillation of isomolar saline. A measurable amount of pleural fluid can be collected after 3 h. This model has been used to study the role of aquaporin water channels in pleural fluid transport, but it can easily be extended to study other aspects of pleural effusion secondary to renal failure. Removal of pleural fluid and particulates from the pleural space has been studied in sheep, rabbits, and, occasionally, dogs using radioactive- or fluorescent isothiocyanate-labeled tracers to follow the protein efflux and reabsorption. Potential drawbacks of these animal studies include the variations in the anatomy and probable differences in the pleural fluid exchange mechanisms between humans and animals. In humans, effusions usually accumulate secondary to various pleural pathologies. Specific animal models have been used to simulate human pleural diseases, including models for malignant effusions (commonly by intrapleural implantation of cancer cells in athymic nude mice) and bacterial empyema (usually in rabbits using Pasteurella multocida). Direct injection of carrageenan into the pleural cavity of animals (e.g., mouse, rat, or rabbit) has often been employed to generate pleural inflammation and exudative pleural effusions for investigations. Although less commonly used, animal models have also been designed for tuberculous pleuritis, rheumatoid pleuritis, effusions from esophageal rupture, etc.

Physiologic Sequelae of Pleural Fluid When pleural fluid is present, its volume must be compensated for by an increase in the size of the thoracic cavity, a decrease in the size of the lung or heart, or a combination of these. The presence of pleural fluid usually leads to an increase in pleural pressure. As a result of this increase, the distending pressures of the lung and heart are decreased while the distending pressure of the thoracic cavity is increased. Effects on Pulmonary Function

When a thoracentesis is performed on a patient with a pleural effusion, the forced vital capacity and the

forced expiratory volume in 1 s (FEV1) will each increase, on average, approximately 200 ml for every liter of pleural fluid removed. The increase in the total lung capacity is almost twice the increase in FEV1. The explanation for this relatively low increase in the pulmonary function test results is that in addition to the lung increasing in size, the thorax decreases in size and the diaphragm moves up after fluid evacuation. However, there is much interindividual variation. The increase in pulmonary function is greater in patients with higher initial pleural pressure and in patients with smaller changes in the pleural pressure as fluid is removed. Effects on Blood Gases

Most patients with more than minimal pleural effusions have abnormal arterial blood gases, primarily a low PaO2. However, the arterial blood gas results do not improve much and may even deteriorate when a thoracentesis is performed. The main mechanism underlying the arterial hypoxemia is an intrapulmonary shunt, which does not change significantly after thoracentesis. Effects on the Diaphragm

The presence of pleural fluid profoundly affects the diaphragm because of the weight of the fluid on the diaphragm. The degree of dyspnea with a pleural effusion is related to its effect on the diaphragm. The patient usually is not dyspneic if the diaphragm is domed and is functioning normally, the patient usually has some dyspnea if the diaphragm is flattened and does not move with respiration, and the patient usually has severe dyspnea if the diaphragm is inverted. Effects on the Heart

In spontaneously breathing dogs, right ventricular diastolic collapse begins when the mean pleural pressure is increased by 5 mmHg. If the mean pleural pressure is increased by 15 mmHg, the stroke volume decreases by approximately 50%, and the cardiac output decreases by 33%. Although there have been few studies on the effects of pleural fluid on the heart in humans, there have been several case reports in which the presence of a pleural effusion has compromised cardiac output.

Summary The abnormal accumulation of pleural fluid is a common clinical presentation and can occur in association with a wide range of pulmonary and extrapulmonary diseases. In health, the pleural cavity

362 PLEURAL EFFUSIONS / Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube

contains a small amount of fluid for lubrication. The volume of the pleural fluid can increase dramatically with various pleural diseases. The respiratory mechanics are altered to accommodate the extra volume of fluid, resulting in dyspnea. Categorizing the effusion into transudates and exudates using Light’s criteria may provide insight on the etiology and pathogenesis of the effusion. Congestive cardiac failure, hepatic cirrhosis, and renal failure are the most common causes of transudative effusions, whereas infective (including tuberculosis) and malignant pleural diseases account for a majority of exudative pleural effusions. Research on the immunologic basis of pleural vascular hyperpermeability may provide novel strategies for future management of recurrent exudative effusions. See also: Mesothelial Cells. Pleural Effusions: Overview; Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube; Parapneumonic Effusion and Empyema; Malignant Pleural Effusions; Pleural Fibrosis; Chylothorax, Psuedochylothorax, LAM, and Yellow Nail Syndrome; Hemothorax. Pleural Space. Signs of Respiratory Disease: Lung Sounds. Vascular Endothelial Growth Factor.

Nahid P and Broaddus VC (2003) Liquid and protein exchange. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 35–44. London: Arnold. Noppen M (2001) Normal volume and cellular contents of pleural fluid. Current Opinion in Pulmonary Medicine 7: 180–182. Noppen M (2004) Pleural lavage as a diagnostic and research tool. Lung Biology in Health and Disease 186: 175–182. Noppen M, DeWaele M, Li R, et al. (2000) Volume and cellular content of normal pleural fluid in humans examined by pleural lavage. American Journal of Respiratory and Critical Care Medicine 162: 1023–1026. Wiener-Kronish JP, Broaddus VC, Albertine KH, et al. (1988) Relationship of pleural effusions to increased permeability pulmonary edema in anesthetized sheep. Journal of Clinical Investigation 82: 1422–1429.

Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube J E Heffner, Medical University of South Carolina, Charleston, SC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Further Reading Broaddus VC and Araya M (1992) Liquid and protein dynamics using a new minimally invasive pleural catheter in rabbits. Journal of Applied Physiology 72: 851–857. Broaddus VC, Wiener-Kronish JP, and Staub NC (1990) Clearance of lung edema into the pleural space of volume-loaded anesthetized sheep. Journal of Applied Physiology 68: 2623–2630. Ferrara N (2000) VEGF: an update on biological and therapeutic aspects. Current Opinion in Biotechnology 11: 617–624. Gourgoulianis KI (2004) Physiology of the pleura. Lung Biology in Health and Disease 186: 45–52. Grove CS and Lee YCG (2002) Vascular endothelial growth factor: the key mediator in pleural effusion formation. Current Opinion in Pulmonary Medicine 8: 294–301. Lee YCG (2003) Experimental models for pleural diseases. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 149–166. London: Arnold. Lee YCG and Lane KB (2001) The many faces of transforming growth factor beta in pleural diseases. Current Opinion in Pulmonary Medicine 7: 173–179. Lee YCG and Lane KB (2003) Cytokines in pleural diseases. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 63–89. London: Arnold. Lee YCG, Malkerneker D, Thompson PJ, Light RW, and Lane KB (2002) Transforming growth factor-b induces vascular endothelial growth factor elaboration from pleural mesothelial cells in vivo and in vitro. American Journal of Respiratory and Critical Care Medicine 165: 88–94. Light RW (2001) Physiology of the pleural space. In: Light RW (ed.) Pleural Diseases, 4th edn., pp. 8–20. Baltimore: Lippincott Williams & Wilkins. Light RW (2003) Physiological effects of pleural air or fluid. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 45–55. London: Arnold.

Analysis of pleural fluid assists the diagnosis of intrathoracic and systemic disorders that cause pleural effusions. Nearly 75% of patients with pleural effusions gain either a definitive or presumptive diagnosis after a systematic analysis of pleural fluid. The need for further diagnostic studies depends on whether pleural fluid is classified by pleural fluid analysis as exudative or transudative in nature. A Bayesian approach to discriminating between exudative and transudative effusions increases diagnostic accuracy. Patients with exudative effusions may benefit from pleural biopsy if the diagnosis remains uncertain after thoracentesis. Infected pleural fluid or large, symptomatic effusions benefit from chest tube drainage. Biopsy can be performed by closed needle, image-guided needle, open surgical, and thoracoscopic techniques. The diagnostic yield of each of these procedures varies in different clinical settings. Chest tubes represent the primary technique for draining symptomatic effusions and performing chemical pleurodesis. The type and caliber of chest tube varies depending on the etiology and physical properties of the effusion.

Introduction Thoracentesis and pleural fluid analysis represent the cornerstones of evaluating patients with undiagnosed pleural effusions. Pleural fluid analysis provides a definitive or presumptive diagnosis in 75% of patients with pleural effusions who undergo thoracentesis. Patients with exudative effusions who remain undiagnosed after pleural fluid analysis can undergo pleural biopsy to establish a diagnosis. An accurate diagnosis guides decisions for selecting patients for chest tube placement and pleural fluid drainage.

PLEURAL EFFUSIONS / Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube 363

Thoracentesis Thoracentesis is performed as a therapeutic or diagnostic procedure. The site for insertion of a needle or catheter into the chest is commonly selected by chest percussion. Increasing evidence suggests that realtime ultrasonography should guide thoracentesis to decrease risks of puncturing intrathoracic and intraabdominal organs. No absolute contraindications exist for thoracentesis, although caution is advised in performing the procedure in the settings of bleeding diatheses, small volumes of pleural fluid, skin infections near the thoracentesis site, and positive pressure ventilation. Therapeutic thoracentesis removes pleural fluid to improve dyspnea for patients with large effusions. The presence of a free-flowing effusion without loculations and radiographic evidence of shift of mediastinal structures away from the effusion identify patients likely to respond to therapeutic thoracentesis. A small-gauge catheter is inserted through the intercostal space into the pleural space with removal of fluid by a vacuum bottle or gravity system. Thoracentesis performed by repeatedly drawing aliquots of fluid out of the chest with a syringe can create a large degree of intrapleural negative pressure and remove pleural fluid beyond the ability of the lung to expand against the chest wall, which risks reexpansion pulmonary edema. Diagnostic thoracentesis is performed when the etiology of a pleural effusion remains uncertain after an initial clinical evaluation. Patients who present with clearcut evidence of conditions known to cause incidental pleural effusions, such as congestive heart failure, do not require a diagnostic thoracentesis. Thoracentesis should be performed if patients do not experience resolution of the effusion after treatment of the primary condition. Diagnostic thoracentesis is performed by inserting a small-gauge needle attached to a syringe or a catheter into the pleural space. Sufficient fluid is obtained to send for hematologic, cytologic, and microbiologic studies (Table 1). The specific studies ordered vary on the basis of the patient’s clinical presentation and suspected cause of the effusion. Complications of thoracentesis are listed in Table 2. With proper technique and ultrasonographic guidance, the procedure is well tolerated with rare instances of life-threatening problems. Patients managed in the intensive care unit with positive pressure ventilation have a low risk pneumothorax from thoracentesis, but the pneumothorax may be a tension pneumothorax when it does occur. Chest radiographs are not needed after thoracentesis unless patients develop signs of intrathoracic complications.

Table 1 Routine tests for initial pleural fluid analysis Examination of gross appearance Color Opacity Viscosity Odor Particulates Chemistry Glucose Lactate dehydrogenase pH Cell analysis Erythrocyte count Nucleated cell count Nucleated cell differential Cytology (when malignancy suspected) Cytological analysis Microbiology (when infection suspected) Aerobic and anaerobic cultures Gram stain Fungal stains and cultures (when fungal disease suspected) Mycobacterial stains and cultures (when tuberculosis suspected)

Table 2 Complications of thoracentesis Bleeding (intrathoracic or intra-abdominal) Pneumothorax Pleural space infection Puncture or laceration to the diaphragm, liver, or spleen Seeding of the needle tract with cancer cells from intrathoracic tumors Chest wall pain at the needle insertion site Intrapleural retention of a sheered thoracentesis catheter Vasovagal reactions

Pleural Fluid Analysis An organized approach to pleural fluid analysis provides a definite or probable diagnosis of the cause of a pleural effusion in the majority of patients. The analysis begins with gross inspection of the fluid followed by determination of the exudative or transudative nature of the effusion. Exudative and transudative effusions present different differential diagnoses and influence the subsequent patient evaluation. Gross Appearance

Certain gross appearances of pleural fluid can provide a specific diagnosis or narrow the list of potential diagnoses. The fluid is placed into a plastic vial and tilted to note its viscosity and transilluminated to observe its color and turbidity. The clinical significance of various gross appearances is listed in Table 3.

364 PLEURAL EFFUSIONS / Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube Table 3 Gross characteristics of pleural fluid Appearance

Suggested diagnosis

Purulent Anchovy paste Putrid smelling Ammonia smelling Bloody

Empyema Amoebic liver abscess Anaerobic empyema Urinothorax Malignancy, chest trauma, postcardiac injury, pulmonary infarction, benign asbestos pleurisy Chylous effusion, chyliform effusion, extravascular migration of central venous catheter used for infusion of lipid products Biliothorax Rheumatoid pleurisy

Milky

Green Yellow-green or ‘fluorescent’ green Appearance of intravenous fluid Extravascular migration of central venous catheter

Chemical Tests

The measurement of pleural fluid protein, lactate dehydrogenase (LDH), glucose, pH, and amylase assist the initial evaluation of pleural effusions. Protein, LDH, and exudative versus transudative effusions Pleural fluid and blood are sent for measurement of protein and LDH to allow classification of the pleural fluid as an exudative or transudative effusion. Exudative pleural effusions most often develop as a consequence of malignant or inflammatory alteration of the permeability of pleural and vascular membranes or obstruction of pleural lymphatics that drain the pleural space. These conditions promote intrapleural accumulation of fluid that has a high concentration of high molecular weight compounds, such as protein and LDH. Exudative pleural fluid can also accumulate by the flow of protein-rich fluid from adjacent body compartments, as occurs in patients with pancreatitis. Transudative effusions develop when an intravascular increase in hydrostatic or decrease in oncotic pressure promotes transudation of fluid from intrathoracic vessels into the pleural space. Transudative effusions also develop with migration of transudates from the mediastinum (extravascular migration of central venous catheters), retroperitoneum (urinothorax), peritoneum (ascites), or central subarachnoid space (cerebrospinal fluid). Transudative effusions have low concentrations of high molecular weight compounds. Table 4 shows the differential diagnosis of exudative and transudative effusions. Light’s criteria represent the classic approach for discriminating between exudative and transudative effusions. This clinical rule assesses three criteria: the

Table 4 Common conditions associated with transudative or exudative effusions Transudates Congestive heart failure Hepatic hydrothorax Nephrotic syndrome Peritoneal dialysis Hypoalbuminemia Urinothorax Atelectasis Constrictive pericarditis Trapped lung Superior vena caval obstruction Pulmonary embolism (may also be exudative) Exudative effusions Intrapleural infections Drug induced Pulmonary embolism Esophageal perforation Malignancy Pancreatitis Collagen vascular disease Benign asbestosis pleurisy Radiation pleurisy Uremic pleurisy Sarcoidosis Postcardiac injury syndrome Hemothorax Chylothorax Pseudochylous effusion Vasculitis Hypothyroidism Ovarian hyperstimulation syndrome Yellow nail syndrome Lymphangiomyomatosis Lymphangiectasia Subphrenic inflammatory or neoplastic process

pleural fluid LDH (4two-thirds the upper limits of normal for serum LDH), pleural fluid-to-serum LDH ratio (40.6), and the pleural fluid-to-serum protein ratio (40.5). Fulfillment of any one of the three criteria supports the exudative nature of the effusion. Light’s criteria have greater than 95% diagnostic accuracy when applied to large groups of patients. Two of Light’s criteria, pleural fluid LDH and pleural fluidto-serum LDH ratio, correlate closely with each other because they are mathematically coupled and share an identical element (pleural fluid LDH). Therefore, the diagnostic accuracy of Light’s criteria is not diminished significantly by excluding either the pleural fluid LDH or the pleural fluid-to-serum LDH ratio from consideration (abbreviated Light’s criteria). Other clinical rules perform as well as Light’s criteria and have some clinical advantages because they do not require serum blood tests. For instance, patients with any one of the following criteria are likely to have an exudative effusion: pleural fluid protein 43.0 g dl 1, pleural fluid cholesterol 445 mg dl 1,

PLEURAL EFFUSIONS / Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube 365

or pleural fluid LDH 4two-thirds the upper limits of normal for a serum LDH. Increasing any of these criteria into multiple-criteria rules, as is done with Light’s criteria, increases the sensitivity for detecting exudative effusions but decreases the specificity. As with all diagnostic rules that use a binary approach (i.e., exudative effusion is either ‘present’ or ‘absent’), the criteria discussed above perform less accurately when patients have test results near any of the criteria’s cut-off points. For instance, when any one of the three Light’s criteria has a result near its cut-off point, the diagnostic accuracy of the Light’s criteria decreases to 75%. Therefore, it has been suggested that physicians use a Bayesian approach to categorize effusions as exudates or transudates wherein the pretest probability of an exudate is estimated with calculation of the post-test probability using likelihood ratios. Several reports have published multilevel likelihood ratios and formulas for calculating continuous likelihood ratios for several pleural conditions (see ‘Further reading’). Glucose A pleural fluid glucose o60 mg dl 1 occurs most often in patients with rheumatoid pleurisy, pleural infections, malignant effusions, tuberculous pleuritis, lupus pleuritis, or esophageal rupture. Some experts recommend the use of pleural fluid glucose to guide the selection of patients with parapneumonic effusions for chest drainage because patients with glucose values o60 mg dl 1 are unlikely to respond to antibiotic therapy alone. Existing data do not, however, strongly support this recommendation. pH Pleural fluid pH values range from 7.30 to 7.45 and 7.40 to 7.55 in exudative and transudative effusions, respectively. A pleural fluid pH below 7.30 suggests the presence of rheumatoid pleurisy, pleural infections, malignant effusions, tuberculous pleuritis, lupus pleuritis, or esophageal rupture. For patients with parapneumonic effusions, a pH o7.20 increases the probability that the fluid requires drainage, although the diagnostic accuracy of pH in this setting is only weakly established. Some experts use pleural fluid pH to select patients with malignant pleural effusion for pleurodesis because an indirect association exists in populations of patients between pH and the probability of a successful pleurodesis and duration of patient survival after pleurodesis. However, this association is not sufficiently strong to allow its use for predicting the clinical courses of individual patients. Amylase A pleural fluid-to-serum amylase 41.0 is found most often in patients with acute or chronic pancreatitis, esophageal rupture, and malignant

pleural effusions. A high ratio can also rarely occur in patients with parapneumonic effusions, cirrhosis, and ectopic pregnancies. Chronic pancreatic effusions typically have pleural fluid amylase concentrations 4100 000 IU l 1. A pleural fluid amylase is obtained only when pancreatic or esophageal disorders are suspected because the frequency of an elevated amylase in patients suspected with malignant disease is insufficiently common to warrant its routine assay. Both malignant pleural effusions and effusions due to a ruptured esophagus are rich in the salivary amylase isoform. Lipid tests Clinical suspicion of a chylous or chyliform effusion warrants assay of pleural fluid for triglycerides and cholesterol. A triglyceride content 4110 mg dl 1 supports the diagnosis of a chylous effusion and a content o50 mg dl 1 excludes the diagnosis. Intermediate values require lipoprotein analysis of pleural fluid to detect chylomicrons, which confirm the diagnosis. Extravasation of lipid-containing parenteral nutrition into the pleural space results in a pleural fluid-to-serum glucose ratio 41. A pleural fluid cholesterol concentration 4200 mg dl 1 with a low triglyceride level supports the diagnosis of a chyliform effusion. A combined elevation in both cholesterol and triglyceride levels warrants measurement of chylomicrons to discriminate between chylous and chyliform effusions. Adenosine deaminase Patients with tuberculous pleuritis have an increased pleural fluid concentration of adenosine deaminase (ADA) usually above 45–60 U l 1. Assay of the isoenzyme ADA-2 further increases diagnostic accuracy. Elevated pleural fluid concentrations of ADA can also occur in patients with rheumatoid pleurisy, pleural infections, and pleural malignancies that include mesothelioma, pleural carcinomatosis, and intrapleural spread of hematologic malignancies. Hematological Studies

Red blood cells Grossly bloody effusions with pleural fluid erythrocyte counts 4100 000 ml 1 suggest malignancy, trauma, benign asbestos pleural effusion, postcardiac injury syndrome, or pulmonary infarction as the cause of the effusion. A hemothorax as defined by a pleural fluid-to-serum hematocrit ratio 40.50 occurs as a result of accidental or iatrogenic chest trauma. White blood cells The number of white blood cells in pleural fluid is never diagnostic although acute

366 PLEURAL EFFUSIONS / Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube

inflammatory conditions have higher white cell concentrations – usually above 10 000 ml 1. Nucleated cell differential Lymphocytosis 450% nucleated cells suggests the presence of lymphoma, chronic rheumatoid pleurisy, yellow nail syndrome, chylothorax, tuberculous pleuritis, uremic pleurisy, sarcoidosis, acute rejection of a transplanted lung, postcardiac surgery effusion (42 months after surgery), trapped lung, or malignant pleural effusion. These conditions often have lymphocyte counts 485% of nucleated cells except for malignant effusions, which are typically in the 50–70% range. Most pleural lymphocytes are T-type cells; the presence of a predominance of B-type cells by flow cytometry suggests lymphoma or chronic lymphocytic leukemia as the cause of the effusion. Pleural fluid eosinophilia (410% nucleated cells) suggests a benign condition characterized by blood or air in the pleural space or a narrow differential of pulmonary infections. Neoplastic etiologies, however, cannot be excluded by the presence of pleural eosinophilia. The most common diagnoses include pneumothorax, hemothorax, Hodgkin’s disease, carcinoma, benign asbestos pleurisy, parasitic disease, pleural infections, or drug-induced effusions. Mesothelial cells Pleural fluid mesothelial cells 45% markedly decrease the likelihood of tuberculous pleurisy. Plasma cells and basophils The presence of high concentrations of plasma cells suggests the presence of multiple myeloma with pleural involvement. Basophils 410% suggest leukemic invasion of the pleural space. Cytopathology

Cytopathologic studies can detect malignant cells in 60–80% of patients with malignant pleural effusions and 20% of patients with mesothelioma. The diagnostic yield of pleural fluid cytometry depends on the cell type of the underlying tumor, the extent of pleural invasion, the volume of fluid removed for analysis, and the number of thoracenteses performed. Cytology can also detect food particles, which confirms the presence of a ruptured esophagus.

diagnostic value of these tests varies widely depending on the etiologic pathogen, the extent of pleural infection, the presence of antimicrobial therapy, and the timing of thoracentesis. Immunologic Studies

Patients with lupus pleuritis tend to have increased pleural fluid antinuclear antibody (ANA) titers (41:160–320), pleural fluid-to-serum ANA ratios 41, and decreased pleural fluid complement levels. Patients with rheumatoid pleurisy often have pleural fluid rheumatoid factor titers 41:320, pleural fluid-to-serum rheumatoid factor ratios 41, and decreased pleural fluid complement levels. However, limited data support the diagnostic value of pleural fluid immunologic studies making their application to individual patients uncertain.

Pleural Biopsy Patients with undiagnosed lymphocytic-predominant, exudative pleural effusions or pleural-based masses should undergo pleural biopsy to establish a specific diagnosis. The available biopsy techniques include closed needle biopsy, CT-guided needle biopsy, thoracoscopy, and open pleural biopsy. Closed Needle Biopsy

Closed needle biopsy using a Cope, Raja, or Abrams needle has its highest diagnostic accuracy for patients with tuberculous pleuritis, 90% of whom have positive biopsy histopathologic or culture results. Closed needle biopsy has limited value for the diagnosis of other causes of exudative effusions because of a low sensitivity. The small pleural sample size may complicate the differentiation of pleural carcinomas from mesotheliomas. CT-Guided Percutaneous Needle Biopsy

CT-guided percutaneous biopsy has a high diagnostic accuracy for pleural-based masses and pleural thickening. It may fail to diagnose mesothelioma because of the need for large biopsy specimens to confirm this diagnosis. Patients with mesothelioma may benefit from postprocedure chest wall radiation to prevent malignant cell seeding of the needle track. Thoracoscopy

Microbiology

Pleural fluid from patients with suspected pleural infections is submitted for Gram stain, aerobic and anaerobic culture, and special studies that would include acid-fast smears and culture and special smears and cultures for fungal and parasitic pathogens. The

Medical and video-assisted thoracoscopic surgery (VATS) can assist the evaluation of undiagnosed pleural effusions by allowing biopsy of pleural lesions under direct vision. Both procedures share the same diagnostic accuracy (Table 5) and most centers prefer the less invasive medical thoracoscopy

PLEURAL EFFUSIONS / Parapneumonic Effusion and Empyema 367 Table 5 Diagnostic accuracy of thoracoscopy

Further Reading

Patients with pleural effusions who remain without a specific diagnosis after thoracentesis 70–100% Mesothelioma 60–75% Pleural carcinomatosis 95% Tuberculous pleuritis 95%

Astoul P (2004) Medical thoracoscopy. In: Bouros D (ed.) Pleural Disease, pp. 183–208. New York: Dekker. Baumann MH (2004) Chest tubes. In: Bouros D (ed.) Pleural Disease, pp. 153–174. New York: Dekker. Heffner JE, Brown LK, and Barbieri C (1997) Diagnostic value of tests that discriminate between exudative and transudative pleural effusions. Chest 111: 970–979. Heffner JE, Brown LK, Barbieri C, et al. (1995) Pleural fluid chemical analysis in parapneumonic effusions. A meta-analysis. American Journal of Respiratory and Critical Care Medicine 151: 1700–1708. Heffner JE, Highland K, and Brown LK (2003) A meta-analysis derivation of continuous likelihood ratios for diagnosing pleural fluid exudates. American Journal of Respiratory and Critical Care Medicine 167: 1591–1599. Light RW (1995) Pleural Diseases, 3rd edn. Baltimore: Williams & Wilkins. Sahn SA (1995) Pleural diagnostic techniques. Current Opinion in Pulmonary Medicine 1: 324–330. Sahn SA and Heffner JE (2003) Pleural fluid analysis. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 191–209. London: Arnold.

when it is available. Thoracoscopy is preferred over closed needle biopsy when malignancy is the most likely cause of an undiagnosed pleural effusion. Conversely, thoracoscopy is rarely needed in patients with suspected tuberculous effusions because of the high diagnostic yield of closed needle biopsy. Open Pleural Biopsy

Since the advent of thoracoscopy, open pleural biopsy is not commonly performed except for patients with mesothelioma who may remain undiagnosed after the performance of less invasive biopsy procedures.

Chest Tube For patients with pleural effusions, chest tubes are indicated to drain hemothoraces, infected pleural fluid, symptomatic chylothoraces, and malignant pleural effusions to palliate dyspnea or prepare for pleurodesis. The clinical setting determines tube size required. Malignant pleural effusions can be drained with small-bore (X8 Fr) percutaneous catheters. Infected pleural fluid requires larger tubes (X28 Fr) as determined by the viscosity of the fluid. Smaller (X10 Fr) catheters placed by image guidance can assist the drainage of infected pleural loculums. Hemothoraces require large tubes (32–40 Fr) to drain blood clots and assess the rate of intrathoracic hemorrhage. No contraindications exist for chest tube drainage although elective insertions for pleurodesis should be delayed in patients with bleeding diatheses. Complications include misplacement outside of the pleural space, lung perforation, re-expansion pulmonary edema, and perforation of other structures, which include the diaphragm, liver, and spleen. See also: Pleural Effusions: Overview; Pleural Fluid, Transudate and Exudate; Parapneumonic Effusion and Empyema; Malignant Pleural Effusions; Postsurgical Effusions; Pleural Fibrosis; Chylothorax, Psuedochylothorax, LAM, and Yellow Nail Syndrome; Hemothorax. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens. Tumors, Malignant: Bronchogenic Carcinoma; Lymphoma; Metastases from Lung Cancer.

Parapneumonic Effusion and Empyema S J Chapman and R J O Davies, Oxford Radcliffe Hospital, Headington, Oxford, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Development of a sterile, exudative pleural effusion (simple parapneumonic effusion) is a common complication of bacterial pneumonia, and the majority of cases resolve with antibiotic treatment alone. A minority become secondarily infected (complex parapneumonic effusions), and require chest drainage for clinical resolution. Infected pleural fluid is characterized biochemically by a low pH and glucose, and an elevated lactate dehydrogenase. Pleural infection is associated with activation of the coagulation cascade, and the subsequent deposition of fibrin within the pleural space results in the formation of septations and impedes drainage. Ongoing infection leads to the accumulation of pus in the pleural cavity (empyema). Treatment of pleural infection comprises appropriate antibiotics, chest drainage, nutritional support, and in some cases surgery; intrapleural fibrinolytics are ineffective. Tuberculous pleural effusions develop as a result of a delayed hypersensitivity reaction to mycobacteria in the pleural space. Diagnosis is based on mycobacterial culture of pleural fluid or pleural tissue, or may be inferred from the finding of granulomas on pleural biopsy. Treatment of tuberculous pleurisy involves the same antimicrobial regimen as that used for pulmonary tuberculosis. The use of steroids to treat tuberculous pleurisy is controversial, although they may lead to symptomatic benefit by hastening pleural fluid absorption.

Introduction Pleural empyema was first described by Hippocrates in 500 BC, and remains a common clinical problem.

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Pleural infection affects approximately 60 000 individuals each year (in the US and UK), with a mortality rate of 15%. Tuberculous pleural effusion causes significant morbidity worldwide, and it may be a more frequent manifestation of tuberculosis in cases of primary infection or HIV co-infection.

Etiology The bacteriology of pleural infection is varied, and there are notable differences between community- and hospital-acquired infections. The commonest organisms responsible for community-acquired pleural infection in the UK are Streptococcus intermedius (approximately 30% of cases), Streptococcus pneumoniae (14% of cases), and staphylococci (12%). Anaerobes are additionally present in nearly a fifth of the cases. Hospital-acquired infection is characterized by a greater proportion of antibiotic-resistant organisms: infection is most commonly due to Staphylococcus aureus (representing 50% of cases, of which nearly three-quarters are methicillin-resistant MRSA), enterobacteria (20%), or enterococci (12%). Pleural infection classically develops as a complication of pneumonia, but may also complicate trauma or iatrogenic procedures. Risk factors for the development of pleural infection include diabetes mellitus, alcohol abuse, gastroesophageal reflux, and intravenous drug abuse. Anaerobic infection may be associated particularly with aspiration and poor dental hygiene. Up to one-third of patients with pleural infection do not have any identifiable risk factors.

Pathology A classification scheme for pleural infection is presented in Figure 1. Initial formation of a pleural effusion is necessary for the development of pleural infection. Sterile, exudative pleural effusions (simple parapneumonic effusions) complicate about 60% of cases of pneumonia, but most resolve following treatment with antibiotics alone. A minority become secondarily infected (complicated parapneumonic effusions) and require drainage for resolution. Bacterial metabolism and neutrophil phagocytosis in the pleural space lead to lactic acid production and increased glucose utilization. This results in the characteristic biochemical features of pleural infection: a reduced pleural fluid pH (o7.2), reduced glucose (o35 mg dl 1), and elevated lactate dehydrogenase (LDH; 41000 IU l 1). The development of infection is associated with activation of the coagulation cascade and inhibition of fibrinolysis within the pleural space. The subsequent production of fibrin forms septations in the pleural cavity, which impede fluid

drainage. Ongoing infection eventually leads to the accumulation of pus in the pleural space (empyema). After a variable time interval, pleural infection enters an ‘organizing’ stage characterized by fibroblast proliferation and the development of a solid fibrous pleural peel. This inhibits lung reexpansion and usually necessitates surgical thoracotomy and decortication. Most pleural effusions due to tuberculosis are a result of a delayed hypersensitivity reaction to mycobacteria in the pleural space, and are characterized by a lower pleural bacterial load than nonmycobacterial infection. Occasionally a true tuberculous empyema develops, when pleural fluid is purulent and smear-positive for acid-fast bacilli.

Clinical Features Pleural infection commonly presents with symptoms of pneumonia, such as fever, cough, chest pain, and breathlessness, and physical signs of a pleural effusion. Patients may present in a more indolent manner with non-specific symptoms such as weight loss. In such cases the diagnosis is often unsuspected. There are no clinical features that reliably indicate the diagnosis or the need for drainage of pleural fluid, and a low threshold for further investigation in the form of diagnostic pleural aspiration is advisable. Pleural aspiration is the single most useful diagnostic test. The finding of frankly purulent pleural fluid and/or the presence of organisms on pleural fluid Gram stain or culture is almost diagnostic of pleural infection, and necessitates drainage of the pleural cavity. A pleural fluid pH of o7.2, in the appropriate clinical setting, is highly suggestive of pleural infection and is also an indication for chest tube drainage. Other pleural fluid biochemical measurements such as reduced glucose (o35 mg dl 1) or elevated LDH (41000 IU l 1) are suggestive of pleural infection, but are a little less diagnostically useful than pH. Note, however, that pleural infection is not the only cause of a reduced pleural fluid pH, and consideration should be given to other causes such as pleural malignancy, rheumatoid pleuritis, tuberculous pleural effusion, esophageal rupture, and lupus pleuritis. Culture of pleural fluid provides a bacteriological diagnosis in approximately 60% of cases. Blood cultures are positive in a minority of cases, but in these patients they are often the only diagnostic microbiological test, and their routine use is therefore recommended. The chest radiograph appearance in pleural infection is variable, but typically shows a loculated pleural effusion (Figure 2). The appearance may be

PLEURAL EFFUSIONS / Parapneumonic Effusion and Empyema 369

Pneumonia (subpleural infection)

Simple parapneumonic effusion

Exudative stage Pleural fluid sterile with clear appearance Pleural fluid biochemistry: normal pH, glucose, and LDH Majority resolve with antibiotic treatment alone

Complicated parapneumonic effusion

Pleural fluid infected but not yet purulent, with clear or turbid appearance Pleural fluid biochemistry: pH < 7.2, glucose < 35 mg dl–1 and LDH > 1000 IU l−1 Pleural fluid Gram stain and/or culture may be positive Chest tube drainage required for resolution

Empyema

'Healing' empyema

Fibrinopurulent stage Pleural fluid purulent Pleural fluid Gram stain and/or culture may be positive Chest tube drainage required for resolution

Organizing stage Formation of thick, inelastic pleural peel Majority require thoracotomy and decortication for resolution

Figure 1 Classification and pathophysiology of pleural infection. LDH, lactate dehydrogenase.

rounded and mass-like, and can be confused with pleural or lung malignancy. Ultrasound scanning typically shows a homogeneous echogenic effusion, and is beneficial in guiding aspiration of small or loculated pleural effusions. On contrast-enhanced computed tomography, pleural infection is characteristically manifest by a thickened, enhancing pleura with increased attenuation of extrapleural subcostal fat. Computed tomography may be useful in guiding drainage procedures in patients with extensive loculated effusions, and may also rarely identify a proximal endobronchial-obstructing lesion. Tuberculous pleural effusions may be asymptomatic, or present with weight loss, chest pain, or fever. They are exudates with protein levels often greater than 5 g dl 1. Pleural fluid glucose and pH values are

moderately depressed in a minority of patients. A marked pleural fluid lymphocytosis is characteristic. The diagnosis is based on mycobacterial culture of pleural fluid or pleural tissue, or it may be inferred from the finding of granulomas on pleural biopsy in the appropriate clinical setting. Pleural tissue is typically obtained using an Abrams’ closed biopsy technique. Reported sensitivities are 10–47% for pleural fluid culture, 39–84% for pleural biopsy histology, and 56–82% for pleural biopsy culture. Culture in addition to histology of pleural biopsy specimens increases, the diagnostic yield when compared to histology alone, and the sensitivity of pleural tissue culture increases with the number of biopsies taken. The diagnostic value of pleural fluid biochemical markers such as adenosine deaminase (ADA) is

370 PLEURAL EFFUSIONS / Parapneumonic Effusion and Empyema

Figure 2 Posterior-anterior chest radiographs in empyema. (a) Pleural infection presenting as a moderate-sized left pleural effusion. (b) A rounded left pleural opacity due to empyema.

affected by both the local prevalence of tuberculosis and the likelihood of an alternative diagnosis. Pleural fluid ADA levels are high in conditions such as infection and malignancy, as well as in pleural tuberculosis. In areas where tuberculosis is prevalent, a raised ADA value is highly sensitive and specific, especially in young patients in whom empyema has been excluded. An elevated ADA level is less useful in the elderly and in regions where tuberculosis is uncommon, because the possibility of an alternative diagnosis is increased; a low ADA value may still be valuable, however, as it suggests, the diagnosis is unlikely to be tuberculosis.

Pathogenesis The development of empyema involves a progression from a sterile, simple parapneumonic effusion to a complicated parapneumonic effusion characterized by bacteria invasion and fibrin deposition within the pleural space (Figure 1). The initial formation of a sterile effusion (‘exudative’ stage) is mediated by an increase in vascular permeability following neutrophil migration into the pleural space and the production of proinflammatory cytokines, including interleukin-8 and tumor necrosis factor alpha (TNFa). The pleural mesothelial cell is thought to play an active role in this inflammatory process: it is capable of phagocytosis and nitric oxide production, expresses adhesion molecules that facilitate migration of neutrophils and mononuclear cells into the pleural cavity, and releases proinflammatory cytokines. Further increases in pleural mesothelial permeability allow bacteria into the pleural space, and activation of the coagulation system with inhibition of fibrinolysis occurs (‘fibrinopurulent’ stage).

The relationship between inflammation and coagulation/fibrinolysis within the pleural space is complex and poorly understood. Infected pleural fluid is characterized by elevated levels of plasminogen activator inhibitor-2 and depressed levels of tissue plasminogen activator; release of plasminogen activator inhibitors from pleural mesothelial cells has been demonstrated to occur following stimulation with TNF-a. Fibrin strands within the pleural cavity may subsequently act as a framework for the proliferation of fibroblasts, leading to the formation of an inelastic pleural peel (‘organizing’ stage). Pleural mesothelial cells may again play a key role in this stage, and have been demonstrated to release growth factors for fibroblasts, including transforming growth factor beta (TGF-b) and platelet-derived growth factor.

Animal Models The most extensively studied animal model of pleural infection involves the introduction of Pasteurella multocida into the rabbit pleural space, with administration of antibiotics to prevent death from overwhelming sepsis. This model has been used to study the penetration of systemically administered antibiotics into the pleural space during empyema, and has demonstrated that metronidazole and penicillin penetrate to a much greater degree than gentamicin. Investigation of pleural drainage techniques in this rabbit model has suggested that therapeutic thoracentesis may be as effective as chest tube placement, although this has not yet been examined in humans. More recently, pleural fluid TGF-b1 levels in the rabbit model have been correlated with microscopic pleural thickness and fibroblast number within the visceral pleura, and intrapleural injection of

PLEURAL EFFUSIONS / Parapneumonic Effusion and Empyema 371

monoclonal antibody to TGF-b appeared to halt empyema progression and pleural fibrosis. This research raises the possibility that inhibition of TGF-b may have a future therapeutic role in pleural infection.

Management and Current Therapy Key components of the treatment of complicated parapneumonic effusion and empyema are the use of appropriate antibiotics, provision of nutritional support, and drainage of infected pleural fluid. Initial, empirical antibiotic treatment should be administered intravenously and address the likely organisms based on location of acquisition of infection. Possibilities for community-acquired empyema include a second generation cephalosporin (e.g., cefuroxime) or aminopenicillin intravenously, in addition to therapy for anaerobic infection (e.g., metronidazole). The combination of ciprofloxacin and clindamycin is suitable for patients allergic to both penicillin and cephalosporins. Treatment of hospitalacquired empyema requires coverage of both Grampositive and Gram-negative organisms, some of which are antibiotic-resistant. An appropriate regimen for patients with culture-negative hospital-acquired empyema is vancomycin (to treat MRSA) and meropenem (which has a broad spectrum of activity, including anaerobic efficacy). Antibiotic choice should be rationalized with the results of blood and pleural fluid cultures and sensitivities, when available; coexisting anaerobic organisms are frequently resistant to culture, however, and additional empirical anaerobic cover should be considered. The optimal duration of antibiotic treatment is unknown, although it is likely to be at least 3 weeks. Malnutrition is common in the setting of empyema, and a low serum albumin is associated with a poor prognosis. Many patients benefit from supplementary nasogastric feeding, and parenteral nutrition may sometimes be required. Indications for chest tube drainage are frankly purulent pleural fluid, identification of organisms on pleural fluid Gram stain or culture, or pleural fluid pH o7.2 in the clinical setting of a pneumonic illness. The optimal chest tube size remains controversial; small-bore (12–14 French) drains are adequate for the majority of cases, and regular saline flushes and use of suction helps prevent their occlusion. Infected pleural fluid frequently becomes loculated, and the administration of intrapleural fibrinolytic agents to dissolve fibrous adhesions has previously been advocated. The UK Multicenter Intrapleural Streptokinase Trial (MIST) randomized 454 patients to receive the fibrinolytic streptokinase or placebo, and demonstrated no difference in mortality or need for surgery between

the two groups. The routine use of intrapleural fibrinolytics is therefore no longer recommended. Intrapleural administration of agents that reduce pus viscosity, such as deoxyribonuclease (DNase), may facilitate drainage; however, such agents have not yet been evaluated in a randomized trial. Approximately 15% of patients with pleural infection fail to improve following treatment with chest tube drainage and antibiotics, and require a surgical procedure for fluid drainage. Surgical approaches for empyema include thoracotomy and decortication, video-assisted thoracoscopic surgery (VATS), and rib resection with open thoracic drainage. VATS allows the disruption of pleural adhesions and the drainage of residual fluid, and is increasingly used as an alternative to thoracotomy. Thoracotomy and decortication are indicated for the treatment of empyema in the ‘organizing’ stage, where the visceral pleura is thickened. Rib resection with open thoracic drainage may be performed under local anesthesia, and is used in patients who are unable to tolerate general anesthesia. Adverse prognostic factors in pleural infection include age 465 years, serum urea 47.0 mmol l 1, serum albumin o25 g l 1, diastolic blood pressure o70 mmHg, and acquisition of infection in hospital. Approximately two-thirds of patients with four or more of these criteria die within 12 months; patients with two or fewer criteria, on the other hand, generally have an excellent prognosis. Interestingly, the purulence of pleural fluid and size of pleural collection judged on chest radiograph do not appear to affect outcome. Treatment of tuberculous pleurisy involves the same antimicrobial regimen as that used for pulmonary tuberculosis. Although tuberculous effusions frequently resolve spontaneously in the absence of treatment, up to 65% of untreated patients develop pulmonary tuberculosis within 5 years. The use of steroids to treat tuberculous pleurisy is controversial, although they may lead to symptomatic benefit by hastening pleural fluid absorption. Up to 50% of treated patients later develop pleural thickening, although significant functional impairment is rare. Treatment with steroids or drainage of the effusion does not appear to prevent this complication. See also: Coagulation Cascade: Fibrinogen and Fibrin. Fibrinolysis: Overview. Lung Imaging. Mesothelial Cells. Pleural Effusions: Overview; Pleural Fluid, Transudate and Exudate; Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Nosocomial; Mycobacterial. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

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Further Reading

Introduction

Antony VB and Mohammed KA (1999) Pathophysiology of pleural space infections. Seminars in Respiratory Infections 14(1): 9–17. Colice GL, Curtis A, Deslauriers J, et al. (2000) Medical and surgical treatment of parapneumonic effusions. An evidence-based guideline. Chest 18: 1158–1171. Davies CW, Kearney SE, Gleeson FV, and Davies RJ (1999) Predictors of outcome and long-term survival in patients with pleural infection. American Journal of Respiratory and Critical Care Medicine 160: 1682–1687. Davies CWH, Gleeson FV, and Davies RJO (2003) On behalf of the BTS Pleural Disease Group, a sub-group of the BTS Standards of Care Committee. BTS guidelines for the management of pleural infection. Thorax 58(supplement II): ii18–ii28. Ferguson AD, Prescott RJ, Selkon JB, Watson D, and Swinburn CR (1996) Empyema subcommittee of the Research Committee of the British Thoracic Society. The clinical course and management of thoracic empyema. Quarterly Journal of Medicine 89: 285–289. Heffner JE, Brown LK, Barbieri C, and DeLeo JM (1995) Pleural fluid chemical analysis in parapneumonic effusions. A metaanalysis. American Journal of Respiratory and Critical Care Medicine 151: 1700–1708. Maskell NA, Davies CW, Nunn AJ, et al. (2005) U.K. Controlled trial of intrapleural streptokinase for pleural infection. New England Journal of Medicine 352(9): 865–874. Matchaba PT and Volmink J (2003) Steroids for treating tuberculous pleurisy (Cochrane review). In: The Cochrane Library, issue 3. Oxford: Update Software. Valdes L, Pose A, San Jose E, and Martinez-Vazquez JM (2003) Tuberculous pleural effusions. European Journal of Internal Medicine 14: 77–88.

Malignant pleural effusions are effusions that arise from neoplastic involvement of the pleural surface. Malignant pleural effusions are a common clinical problem in patients with various malignancies and produce significant morbidity in the majority of affected patients. The worldwide incidence of malignant pleural effusions is unknown, but the incidence in the US is estimated to be greater than 200 000 cases per year. Malignant pleural effusions are one of the leading causes of exudative effusions. Between 42% and 77% of exudative effusions are secondary to malignancy. The diagnosis of a malignant pleural effusion is the first indication of a malignancy in 13% of patients with a malignant pleural effusion. The majority of patients with malignant pleural effusion will experience dyspnea. Dyspnea may cause limitations in ability to perform activities of daily living in a substantial proportion of patients. In addition, patients may have symptoms of cough and chest pain.

Malignant Pleural Effusions V B Antony and M A Jantz, University of Florida, Gainesville, FL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Malignant pleural effusions result from neoplastic infiltration of the pleural surface. Malignant pleural effusions most commonly arise from lung carcinoma, breast carcinoma, and lymphoma. The true incidence of malignant pleural effusion is unknown but up to 15% of patients with lung cancer and 11% of patients with breast cancer will have a malignant effusion at some time during the course of disease. Most patients will have varying degrees of dyspnea at presentation, which is the main symptom related to the effusion. On chest radiograph and computed tomography, there will be evidence of an effusion as well as potential parenchymal lesions consistent with a lung primary tumor or parenchymal metastases. Diagnosis is established by demonstration of malignant cells in the pleural fluid or pleural tissue. Malignant pleural effusions result from metastases to the visceral pleural surface with secondary seeding of the partial pleural surface. In addition to treatment of the primary tumor, management consists primarily of palliation of dyspnea via pleurodesis by means of a chest tube-administered sclerosant agent or talc poudrage by way of medical thoracoscopy.

Etiology Nearly all neoplasms have been reported to involve the pleura. Malignant pleural effusions most commonly arise from metastatic carcinomas outside the pleura, but can also develop from primary pleural neoplasms such as mesothelioma. Lung carcinoma is the most common malignancy causing a malignant pleural effusion in most studies and accounts for approximately one-third of all malignant effusions. Malignant pleural effusions are present in 7–15% of patients with lung cancer at some time during the course of disease. Breast carcinoma is the second most common neoplasm producing a malignant effusion with about 25% of malignant effusions being due to breast cancer. Between 7% and 11% of patients with breast carcinoma will develop a malignant pleural effusion during the course of their disease. Lymphomas, including Hodgkin’s disease and non-Hodgkin’s lymphoma, account for approximately 10% of malignant pleural effusions. In some cases, pleural effusion may be the only evidence of malignancy in a patient. Adenocarcinoma is the most common histological type for malignant pleural effusion with an unknown primary tumor. In some cases the patient will have a known malignancy and a pleural effusion but malignant cells in the pleural fluid or pleural tissue cannot be demonstrated. These effusions, termed ‘paramalignant effusions’ by Sahn, are not the direct result of pleural involvement with tumor but are related to effects of the primary tumor or complications of therapy for

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the primary tumor. Unlike patients with malignant pleural involvement, some patients with lung carcinoma and paramalignant effusions may still be candidates for surgical resection. The pathogenesis of paramalignant effusions is discussed below.

Pathology The pathology of malignant pleural effusions will be similar to the underlying malignancy causing the pleural effusion.

Clinical Features Dyspnea is the most common presenting symptom in patients with malignant pleural effusions. More than half of patients will have some degree of dyspnea. The pathogenesis of dyspnea from a pleural effusion has not been completely elucidated, but several factors may be involved including decreased chest wall compliance, decreased ipsilateral lung volume, contralateral shifting of the mediastinum, and reflex stimulation from the lung parenchyma and chest wall. Cough is the second most common symptom related to malignant pleural effusions. Because malignant pleural effusions represent an advanced stage of the malignancy, patients may also have systemic manifestations such as weight loss, anorexia, and malaise. Some patients may experience chest pain, which is described as dull and aching. Chest pain is more common in patients with malignant mesothelioma than other types of malignant effusions. The presence of chest pain usually signifies parietal pleural infiltration and possible chest wall involvement. On examination, patients with a moderate to large malignant pleural effusion will have findings consisting of decreased breath sounds and dullness to percussion over the effusion. Adenopathy and cachexia may also be noted. Radiological Findings

At presentation, most patients with malignant pleural effusions have moderate to large effusions on chest radiograph. These effusions will have volumes ranging from 500 to 2000 ml (Figure 1). About 10% of patients will have massive pleural effusions, defined as producing total or near-total opacification of the hemithorax. It should be noted that malignancy is the most common cause of a massive pleural effusion. About 15% of patients will have effusions o500 ml in volume, and most of these patients will be relatively asymptomatic. When evaluating the chest radiograph of a patient with a malignant pleural effusion, particular attention

Figure 1 Chest radiograph of patient with moderate pleural effusion due to breast carcinoma.

Figure 2 Chest radiograph of patient with large pleural effusion due to lung carcinoma. Note the contralateral shift of the mediastinum.

should be given to the presence or absence of mediastinal shift. In the patient with a large or massive effusion, contralateral mediastinal shift would be expected (Figure 2). If the mediastinum is midline or is shifted ipsilaterally, involvement of the ipsilateral mainstem bronchus by endobronchial tumor producing concurrent atelectasis should be suspected (Figure 3). Other causes for a lack of contralateral mediastinal shift include fixation of the mediastinum by tumor or lymph node involvement, malignant mesothelioma with extensive pleural involvement, extensive visceral pleural involvement by a primary lung carcinoma or metastatic malignancy, and extensive tumor infiltration of the ipsilateral

374 PLEURAL EFFUSIONS / Malignant Pleural Effusions

Figure 3 Chest radiograph of patient with massive pleural effusion due to lung carcinoma. Note the lack of mediastinal shift due to mainstem bronchial obstruction.

involvement by endobronchial tumor. The presence of pleural plaques suggests prior asbestos exposure and possible mesothelioma as the etiology of the effusion. As part of the evaluation of a patient with a suspected or known malignancy, a chest CT scan may demonstrate pleural effusions that are too small to be detected on chest radiographs. Ultrasonography may aid in identifying pleural lesions in patients with malignant effusions and is also useful in guiding thoracentesis in patients with small effusions and thus reducing complications of thoracentesis such as pneumothorax. Magnetic resonance imaging (MRI) has a limited role in the evaluation of malignant pleural effusions. MRI may be helpful in evaluating the presence of and extent of chest wall involvement by tumor. The utility of positron emission tomography (PET) in the evaluation of malignant pleural effusions has yet to be clearly defined. Malignant pleural effusions may show abnormal uptake on PET imaging although the accuracy of PET in diagnosing an effusion as malignant is unknown. PET has been reported to be helpful in evaluating the extent of disease in malignant mesothelioma. Pleural Fluid Analysis

Figure 4 Chest radiograph of patient with trapped lung due to lung carcinoma. Note the lack of lung expansion following chest tube placement and apparent pneumothorax.

lung parenchyma, which radiographically mimics a large effusion. With ipsilateral mediastinal shift in the setting of a large effusion, the possibility of trapped lung should be considered. The diagnosis is confirmed when there is lack of radiographic expansion following large volume thoracentesis or chest tube thoracostomy (Figure 4). Computed tomography (CT) is usually performed in the evaluation of a patient with a potential malignant pleural effusion. In addition to providing further evaluation of the pleural space, CT scans also provide information about mediastinal lymph node involvement, pulmonary parenchymal involvement by the primary tumor or metastases, and airway

Malignancy should be considered and a diagnostic thoracentesis performed in any patient with a unilateral effusion or bilateral effusions, with a normal heart size on chest radiograph. Thirty to fifty milliliters of pleural fluid is adequate for diagnostic studies. The pleural fluid should be sent for the following studies: nucleated cell count and differential, total protein, glucose, lactate dehydrogenase (LDH), pH, and cytology. The utility of measurement of pleural fluid amylase is debatable. Malignant pleural effusions can be serous, hemorrhagic, and grossly bloody in appearance. The malignant pleural effusion is almost always an exudate. A small number, however, may be transudates and are usually paramalignant effusions rather than true malignant effusions. The LDH criterion for an exudate is almost always present in malignant effusions while the pleural fluid protein to serum ratio may be o0.5 in some effusions. The nucleated cell count is typically low (o3000 cells ml 1) and is composed mainly of lymphocytes, macrophages, and mesothelial cells. About half the time the cell count will be lymphocyte predominant with a differential of 50–70% lymphocytes. In lymphomatous pleural effusions, the lymphocytes are typically 480% of the nucleated cells. The presence of neutrophils or eosinophils is much less common but does not exclude a malignant pleural effusion.

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Approximately one-third of malignant effusions have a pleural fluid pHo7.30 and glucose o60 mg dl 1 at presentation. The cause of these low-pH, low-glucose malignant effusions appear to be increased tumor mass within the pleural space. The increased tumor burden results in decreased glucose transfer into the pleural space and decreased efflux of acidic byproducts of glucose metabolism out of the pleural space. Malignant effusions with a low pH and low glucose concentration have been noted to have higher diagnostic yields on cytology and to be associated with worse survival and lower response to pleurodesis. A recent meta-analysis by Heffner and colleagues found that, while pleural fluid pH was an independent predictor of survival, it had insufficient predictive accuracy for selecting patients for pleurodesis procedures on the basis of estimated survival. Diagnosis

Pleural fluid cytology Pleural fluid cytology is the least invasive method for obtaining a diagnosis of a malignant pleural effusion. Diagnostic yields for pleural fluid cytology range from 60% to 90%. Pleural fluid cytology is most often positive with adenocarcinomas and is positive less often with squamous cell carcinoma, sarcoma, and mesothelioma. It does not appear that sending a large volume of pleural fluid for analysis increases the yield of diagnosis compared with sending just 20–50 ml. Some clinicians have recommended the routine use of cell blocks plus cytology smears while others suggest this is not cost effective. The use of current tumor markers for the diagnosis of malignant pleural effusion does not appear to be helpful although some tumor markers such as carcinoembryonic antigen (CEA), Leu-1, mucin, and surfactant protein may be helpful in distinguishing between adenocarcinomas and reactive mesothelial cells or mesothelioma. In cases where non-Hodgkin’s lymphoma is suspected, flow cytometry analysis of the pleural fluid may be helpful in establishing a diagnosis. Closed pleural biopsy Closed pleural biopsies are less sensitive than pleural fluid cytology for the diagnosis of malignant pleural effusion. Diagnostic yields range from 40% to 75%. Studies have shown that 7–12% of patients with malignant effusion may be diagnosed with closed pleural biopsy when fluid cytology is negative. The reasons for the low yield of closed pleural biopsy include the blind nature of the procedure, early stage of disease with minimal pleural involvement, initial lesions being located along the mediastinal and diaphragmatic pleura which is not sampled during blind biopsy, and operator inexperience.

Recently, there has been interest in the use of CTguided cutting-needle biopsy for diagnosis of malignant pleural effusions with one study demonstrating higher yields than blind closed pleural biopsies. Thoracoscopy and video-assisted thoracic surgery The diagnostic yields for thoracoscopy and video-assisted thoracic surgery (VATS) range from 95% to 100%. Medical thoracoscopy has an advantage over VATS in that it can be performed in an endoscopy suite under local anesthesia and conscious sedation. By allowing direct visualization of the pleural cavity, biopsies can be obtained from suspicious areas under direct view (Figures 5–7). This is particularly advantageous in earlier disease when there is patchy pleural involvement by the malignancy. The reasons for false-negative thoracoscopies include presence of adhesions (often a consequence of repeated

Figure 5 Thoracoscopic view of patient with patchy pleural involvement by metastatic breast carcinoma.

Figure 6 Thoracoscopic view of patient with patchy pleural involvement by metastatic sarcoma. A large parenchymal lung mass due to sarcoma is also visible superiorly.

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removing 500 ml of fluid are highly suggestive of a trapped lung.

Pathogenesis Malignant Pleural Effusions

Figure 7 Thoracoscopic view of patient with extensive pleural involvement by lung carcinoma. Diaphragmatic involvement at the left aspect of the image and lung involvement at the superior aspect of the image is also visible.

thoracenteses) that prevent complete examination, insufficient and nonrepresentative tissue samples, and operator inexperience. Pleurodesis via talc poudrage may be performed at the time of thoracoscopy when the diagnosis of malignancy has been established. In addition, thoracoscopy is helpful in distinguishing malignant from paramalignant effusions and identifying those patients with lung carcinoma that may be candidates for surgical resection. VATS procedures usually require general anesthesia and single lung ventilation. VATS may be necessary if the pleural space contains multiple adhesions that would make performing medical thoracoscopy unsafe. Pleurodesis procedures may also be performed following the diagnostic portion of the VATS procedure. Bronchoscopy The diagnostic yield of bronchoscopy is low in patients with undiagnosed pleural effusions and is not routinely recommended. Bronchoscopy may be helpful when there is a large effusion with ipsilateral mediastinal shift or if the patient has hemoptysis suggesting an endobronchial lesion. Bronchoscopy should be considered to exclude endobronchial obstruction when there is absence of lung expansion after therapeutic thoracentesis. Trapped lung A trapped lung is suggested by failure of the lung to expand completely after removal of the majority of fluid via thoracentesis. Although not widely performed, pleural manometry may also suggest the diagnosis of trapped lung. An initial pleural fluid pressure o 5 cmH2O, a decrease in pleural fluid pressure to o 20 cmH2O after 1 l is removed, or a pleural fluid elastance of 419 cmH2O while

Autopsy studies suggest that most pleural metastases arise from tumor emboli to the visceral pleural surface followed by secondary seeding of the parietal pleura. In the case of mesothelioma, the tumor originates on the parietal pleural surface and then spreads to the visceral pleural surface. With lung carcinoma, breast carcinoma, and chest wall neoplasms, there may be direct tumor invasion into the pleural space. There can also be hematogenous spread to the parietal pleura from extrapulmonary primaries as well as lymphatic involvement. Interference with the lymphatic drainage system anywhere between the parietal pleura and the mediastinal lymph nodes can result in pleural effusion formation. Pleural effusions from malignancy develop primarily due to increased pleural membrane and vascular permeability with resultant plasma leakage. Reduced pleural fluid outflow results from tumor blockage of parietal pleural stomas or lymphatic obstruction due to mediastinal node involvement. The mechanisms of pleural metastasis have not been clearly defined but include processes such as cell adhesion, migration, propagation, and angiogenesis. Angiogenesis is critical for the ability of malignant cells to develop blood vessel formation to provide substrate for tumor growth. Malignant cells can produce multiple cytokines including vascular endothelial growth factor (VEGF) and basic-fibroblast growth factor (bFGF). These cytokines are angiogenic and increase the permeability of the pleural tissue allowing for development of new capillaries and tumor growth. In addition, malignant cells can produce a variety of autocrine growth factors. Paramalignant Pleural Effusions

As noted, a paramalignant pleural effusion is an effusion that is related to effects of the tumor or complications of therapy, but malignant cells are not present in the pleural fluid or pleural tissue. The local effects of tumor producing a paramalignant effusion include lymphatic obstruction, endobronchial obstruction with pneumonia, endobronchial obstruction with atelectasis, trapped lung, chylothorax, superior vena cava syndrome, and malignant pericardial effusion. Systemic effects of the malignancy may cause an effusion via pulmonary embolism related to a hypercoagulable state as well as hypoalbuminemia due to anorexia and cachexia. Complications of

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radiation therapy, including radiation pleuritis, mediastinal fibrosis, and constrictive pericarditis, may also produce an effusion. Lastly, certain chemotherapeutic agents, such as methotrexate, procarbazine, cyclophosphamide, and docetaxel, may cause pleural effusions.

lack of lung expansion suggests trapped lung due to extensive pleural tumor infiltration or mainstem bronchial obstruction. In some patients with significant dyspnea and a large effusion with contralateral mediastinal shift, some clinicians may elect to proceed directly to chest tube drainage and chemical pleurodesis or thoracoscopy with talc poudrage.

Trapped Lung

In the setting of malignancy, a trapped lung occurs when the visceral pleura becomes encased with tumor or a fibrous peel. This prevents lung expansion to the chest wall, and in the area where the lung cannot expand, the pleural pressure becomes more negative. The negative pleural pressure results in fluid movement from the interstitium into the pleural space to produce a volume of fluid that reflects the steady state of fluid formation and resorption. If pleural fluid is removed, the same volume will accumulate within 48–72 h. A similar pathophysiology occurs when there is endobronchial obstruction by tumor.

Chemotherapy

Malignant pleural effusions that are likely to be chemotherapy responsive include small cell lung carcinoma, breast carcinoma, and lymphoma. Effusions due to prostate, ovarian, thyroid, and germ-cell tumors may also be chemotherapy responsive. For patients with these malignancies who have no symptoms or mild symptoms, a trial of chemotherapy with observation of the effusion is reasonable. For more symptomatic patients, therapeutic thoracentesis followed by a trial of chemotherapy may be warranted. Malignant pleural effusions due to neoplasms other than those mentioned above are unlikely to be controlled by chemotherapy alone.

Animal Models

Radiation Therapy

Tumor cells are injected, either intravenously or intrapleurally, into animals that usually have immunological deficiencies to induce disseminated malignancy with pleural effusion. Yano and co-workers have developed a model of malignant pleural effusion in athymic BALB/c nude mice. The human lung adenocarcinoma cell line PC14PE6 and the squamous cell line H226 are injected into the lateral tail vein with subsequent development of lung tumors and pleural effusions. Ohta and colleagues have produced a model of malignant pleural effusion in rats. In this model, PC-14 adenocarcinoma cells are injected into the thoracic cavity with resultant disseminated malignancy and pleural effusion.

In general, radiation therapy of the hemithorax is contraindicated for management of malignant pleural effusions. The adverse effects of radiation pneumonitis outweigh the possible benefits of therapy. Radiation therapy to the mediastinum may be helpful in management of pleural effusions due to mediastinal lymph node involvement by lymphoma or small cell carcinoma.

Management and Current Therapy For patients with malignant pleural effusion who are symptomatic, palliative therapy should be considered. Therapeutic thoracentesis should be performed in dyspneic patients with malignant pleural effusions to determine its effects on breathlessness and rate of recurrence. Rapid recurrence of the effusion suggests the need for immediate therapy. In the absence of recurrence, observation may be warranted. If dyspnea is not relieved, other causes such as lymphangitic carcinomatosis, endobronchial obstruction, thromboembolism, and tumor embolism should be considered. Lung expansion following therapeutic thoracentesis should be assessed. The

Therapeutic Thoracentesis

Repeated therapeutic thoracentesis may be appropriate therapy in patients with far advanced disease, poor performance status, and a short life expectancy. For other patients who have recurrence of the effusion and symptoms following initial thoracentesis, we favor chest tube drainage and chemical pleurodesis or thoracoscopy and talc poudrage as repeated thoracentesis may produce multiple adhesions that limit the success of pleurodesis procedures. In addition, a delay in performing a pleurodesis procedure may allow for development of extensive pleural tumor burden, which decreases the effectiveness of a pleurodesis procedure, or the development of a trapped lung, which precludes performing a pleurodesis procedure. The volume of fluid that can be safely removed from the pleural space during a therapeutic thoracentesis is unknown. Monitoring of pleural pressure during thoracentesis may be performed with removal of fluid as long as the pleural fluid pressure does not

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fall below 20 cmH2O. As most clinicians do not measure pleural pressure during therapeutic thoracentesis, it is recommended that only 1–1.5 l be removed at one time and the procedure be terminated if the patient develops significant cough, chest tightness, or dyspnea. In patients without contralateral mediastinal shift, the possibility of a precipitous fall in pleural pressure is increased, and either pleural pressure should be monitored during thoracentesis or a smaller volume of fluid (300–500 ml) should be removed. Chest Tube Drainage and Chemical Pleurodesis

Patients with recurrence of effusion following initial thoracentesis or highly symptomatic patients with large effusions at presentation should be considered for pleurodesis. Following chest tube drainage, complete lung expansion should be demonstrated, as pleurodesis will not be successful without apposition of the visceral and parietal pleural surfaces. A variety of sclerosant agents aimed at achieving pleurodesis have been utilized. The most studied agents include talc, tetracycline and doxycycline, and bleomycin. Of these, talc is the most effective with success rates reported from 88% to 100% with a mean of 90%. Doxycycline has had success rates varying from 65% to 100% with a mean of 76%. Tetracycline is no longer available as an intravenous preparation and thus is not available in a form suitable for intrapleural administration. Lastly, bleomycin has been reported to have success rates of 58–85% with a mean of 61%. Bleomycin is by far the most expensive of these agents. For talc slurry, the recommended dose is 3–5 g mixed in 50 ml of normal saline with most studies using a dose of 5 g. The most common adverse reactions of talc pleurodesis are fever (16%) and chest pain (7%). Acute pneumonitis and respiratory failure have been noted following talc slurry pleurodesis and talc poudrage with an estimated incidence of 1%. The mechanism of acute pneumonitis is unclear but the dose of talc and physical characteristics of the talc, including size and type, may be important determinants for the development of this complication. To reduce the risk of this reaction, no more than 4 g of talc should be used, the majority of talc particles in the preparation should be 430 mm, and talc pleurodesis should not be performed in patients with severe underlying pulmonary disease. For doxycycline, the recommended dose is 500 mg mixed with 50–100 ml of sterile saline. Chest pain occurs in 40% and fever in 31% of patients treated with doxycycline. With bleomycin, most studies have used 60 IU mixed with 50–100 ml of normal saline.

Chest pain and fever have been noted in 28% and 24% of patients, respectively, following bleomycin pleurodesis. Traditionally, 24–32 French chest tubes have been used for pleurodesis procedures although more recently 10–14 French chest tubes have been successfully utilized. For talc slurry pleurodesis, however, some clinicians still favor larger bore chest tubes due to concern for tube occlusion. Historically, introduction of the sclerosant agent for pleurodesis has been performed when the lung is fully expanded on chest radiograph and there is o150 ml day 1. More recent studies, however, suggest that pleurodesis can be performed as soon as the lung is re-expanded, regardless of chest tube output. Following instillation of sclerosant, the chest tube should be clamped for 1 h. Patient rotation is not necessary after administration of doxycycline but may be considered for talc slurry. The chest tube may be removed 24–48 h after sclerosant administration unless there is excessive chest tube drainage (4250 ml day 1). Repeat pleurodesis may be considered if there is continued high fluid output. Based on animal studies, systemic administration of corticosteroids may decrease the efficacy of pleurodesis and should be discontinued or the dosage reduced if possible. Thoracoscopy and Talc Poudrage

Talc poudrage involves insufflation of talc in the form of a dry powder through the thoracoscope under direct visualization (Figure 8). Talc poudrage can be performed during medical thoracoscopy using conscious sedation and can be performed in the same setting as diagnostic thoracoscopy when the diagnosis of malignancy is established. The success rate for talc poudrage is 490% with a perioperative mortality rate o0.5%. Although the use of thoracoscopy

Figure 8 Thoracoscopic view of pleural surface following talc poudrage for malignant effusion due to lung carcinoma.

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with talc poudrage is strongly favored over chest tube drainage and talc slurry by some clinicians, one small and one large randomized trial suggest that success rates are similar. Long-Term Indwelling Pleural Catheter Drainage

Recently, the use of a long-term tunneled pleural catheter has been described. The catheter may be placed as an outpatient procedure. Through a oneway valve system, the patients connect themselves to a drainage bottle that removes 500–1000 ml of fluid each day or every other day. Spontaneous pleurodesis with the catheter in place has been reported in 21– 46% of patients. Catheter malfunction and pleural space infection has been reported in a small number of patients. The use of tunneled pleural catheters may be helpful in the management of patients with trapped lung who are not candidates for a pleurodesis procedure and for those patients who fail a pleurodesis procedure.

Pollak JS (2002) Malignant pleural effusions: treatment with tunneled long-term drainage catheters. Current Opinion in Pulmonary Medicine 8: 302–307. Rodriguez-Panadero F (2003) Effusions from malignancy. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 297– 309. London: Arnold. Rodriguez-Panadero F (2004) Pleurodesis. In: Bouros D (ed.) Pleural Disease, pp. 479–503. New York: Dekker. Sahn SA (1998) Malignancy metastatic to the pleura. Clinics in Chest Medicine 19: 351–361. Sahn SA (2004) Malignant pleural effusions. In: Bouros D (ed.) Pleural Disease, pp. 411–438. New York: Dekker. Villanueva AG and Beamis JF Jr (2004) Medical thoracoscopy: diagnosis of pleural pulmonary disorders. In: Beamis JF Jr, Mathur PN, and Mehta AC (eds.) Interventional Pulmonary Medicine, pp. 431–449. New York: Dekker. Wohlrab JL and Read CA (2004) Medical thoracoscopy: therapy for malignant conditions. In: Beamis JF Jr, Mathur PN, and Mehta AC (eds.) Interventional Pulmonary Medicine, pp. 451– 468. New York: Dekker.

Postsurgical Effusions

Pleuroperitoneal Shunts

R W Light, Vanderbilt University, Nashville, TN, USA

Pleuroperitoneal shunts may be considered as an alternative therapy in patients with a trapped lung or who fail pleurodesis. Shunt occlusion rates vary from 12% to 25% and typically require replacement of the shunt. Often the pressure gradient between the pleural and peritoneal space is low, and the shunt requires manual compression of the pump chamber by the patient, sometimes over 400 times per day.

& 2006 Elsevier Ltd. All rights reserved.

Parietal Pleurectomy

Parietal pleurectomy provides an effective method for controlling malignant pleural effusions. The procedure carries significant morbidity and perioperative mortality rates of 10–13%. This therapy should be reserved for patients who have failed pleurodesis, have a good performance status, and have an expected survival of more than 6 months. See also: Mesothelial Cells. Mesothelioma, Malignant. Pleural Effusions: Overview; Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube.

Further Reading Antony VB, Loddenkemper R, Astoul P, et al. (2000) Management of malignant pleural effusions. American Journal of Respiratory and Critical Care Medicine 162: 1987–2001. Antunes G, Neville E, Duffy J, and Ali N (2003) BTS guidelines for the management of malignant pleural effusions. Thorax 58(supplement II): ii29–ii38. Lee YCG and Light RW (2004) Management of malignant pleural effusions. Respirology 9: 148–156.

Abstract The majority of patients who undergo coronary artery bypass graft surgery develop a pleural effusion that is usually predominantly left sided and small. Approximately 10% of patients develop a pleural effusion that occupies more than 25% of the hemithorax and the main symptom is dyspnea. The pleural effusions usually disappear after one to three therapeutic thoracenteses. The postcardiac injury syndrome occurs after various types of trauma to the heart, including blunt trauma, myocardial infarction, and cardiac surgery. It is characterized by the development of fever, pleuropericarditis, and parenchymal pulmonary infiltrates in the weeks posttrauma. The primary symptoms are chest pain and fever. The primary treatment is the administration of anti-inflammatory agents, and sometimes corticosteroids are required. After abdominal surgery, the incidence of pleural effusion is approximately 50%. Most effusions are small, but approximately 10% of patients develop moderate to large effusions. The majority of patients postliver transplantation develop a pleural effusion. The effusions occupy more than 25% of the hemithorax in one-fourth of patients and usually are right sided or, if bilateral, larger on the right. There is increased pleural fluid production immediately after lung transplant because the lymphatics draining the pulmonary interstitial fluid are transected. Pleural effusions are not apparent immediately postoperatively because chest tubes are in place. Pleural effusions occurring more than a few weeks after lung transplantation are likely to be due to a complication of the transplant, such as acute rejection, chronic rejection, pulmonary infection, or lymphoproliferative disease.

Introduction Increasingly more surgical procedures are being performed. Some of these surgical procedures are

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associated with the development of a pleural effusion. In this article, the pleural effusions that occur after coronary artery bypass graft (CABG) surgery, abdominal surgery, liver transplantation, lung transplantation, and cardiac trauma are discussed.

Etiology The majority of patients who undergo CABG surgery will develop a small pleural effusion that is usually left sided or bilateral in the first few days postoperatively. In the majority of cases, the pleural effusion will gradually disappear. However, in some patients the effusion will continue to enlarge. If the effusion increases in size during the first few weeks after surgery, it is usually bloody, with a mean pleural fluid hematocrit of approximately 20%. A smaller percentage of patients will develop a large pleural effusion more than 30 days after surgery. The incidence of larger pleural effusions (occupying more than 25% of the hemithorax) is higher if patients receive an internal mammary artery bypass than if they receive a saphenous vein bypass. This is probably due to the fact that harvesting the internal mammary artery produces more trauma to the pleura. Patients who receive topical hypothermia at the time of surgery with iced slush are more likely to develop a pleural effusion. The postcardiac injury syndrome (PCIS), also known as Dressler’s syndrome, is characterized by the development of fever, pleuropericarditis, and parenchymal pulmonary infiltrates in the weeks following trauma to the pericardium or myocardium. Noncomplicated PCIS is defined as the presence of a temperature 4100.51F, patient irritability, pericardial fraction rub, and a small pericardial effusion with or without pleural effusion following cardiac trauma. Complicated PCIS is defined as a noncomplicated PCIS plus the need for hospital readmission with or without the need for pericardiocentesis or thoracentesis. PCIS has been reported following myocardial infarction, cardiac surgery, blunt chest trauma, percutaneous left ventricular puncture, pacemaker implantation, and angioplasty. Pleural effusions are also common after several other surgical procedures. The incidence of pleural effusions is as follows: postabdominal surgical procedures, B50%; postliver transplantation (B70%); and postlung transplantation, B50%.

Pathology There have been limited studies on the pathology of the pleura in patients who have pleural effusions post-CABG surgery. In pathology specimens obtained

within the first 6 months of surgery, there is a predominance of inflammation, with dense cellular infiltrates, especially lymphocytes, and relatively little pleural fibrosis. Immunohistochemical studies demonstrate a mixed population of lymphocytes with a predominance of B cells. As the interval between surgery increases, the biopsy samples show progressively less inflammation, decreased density of the cellular infiltrate, and increased amounts of fibrosis. The fibrous component of the thickened pleura in the later stages is typically paucicellular but does contain collections of entrapped cells that are cytokeratin immunoreactive, compatible with mesothelial cells. Since the diagnosis of PCIS is based on clinical grounds, there are no studies describing the pathologic characteristics of this syndrome. Similarly, there are no pathologic studies of patients who have pleural effusions secondary to abdominal surgery, liver transplantation, or lung transplantation.

Clinical Features Most patients with post-CABG pleural effusions are asymptomatic. When patients are symptomatic, the most common symptom is dyspnea. Chest pain and fever are distinctly unusual. The chest radiograph demonstrates a pleural effusion that is usually unilateral on the left or, if bilateral, larger on the left. The pleural fluid from both the early (occurring within 30 days) and late (occurring after 30 days) post-CABG effusions is an exudate. The pleural fluid from the early effusion is bloody, with a median red blood cell count of 4700 000 cells mm 3. The mean eosinophil percentage is nearly 49%, and the median pleural fluid lactate dehydrogenase (LDH) is approximately twice the upper limit of normal for serum. The pleural fluid from the late-appearing post-CABG effusions is usually a clear yellow exudate that contains predominantly lymphocytes. The median level of pleural fluid LDH is approximately the upper normal limit for serum. The pleural fluid glucose is not reduced, and even though the fluid is lymphocytic, the pleural fluid adenosine deaminase level is less than 40 IU l 1. The clinical manifestations of PCIS include fever, chest pain, pericarditis, pleuritis, and/or pneumonitis. Following cardiac surgery, PCIS is seen at an average of 3 weeks, but it can occur any time between 3 days and 1 year after surgery. Following myocardial infarction, the symptoms usually develop in the second or third week. The chest pain often precedes the onset of fever and varies from crushing and agonizing, mimicking myocardial ischemia, to a dull ache to pleuritic chest pain. A pericardial friction rub is present in almost all patients. Laboratory tests

PLEURAL EFFUSIONS / Postsurgical Effusions 381

reveal a peripheral leukocytosis and an elevated erythrocyte sedimentation rate in most patients. Approximately 80% of patients will have a pleural effusion, and the effusions are bilateral in approximately 50%. The pleural fluid is an exudate with a normal pH and normal glucose. In patients postabdominal surgery, the majority of the pleural effusions are small, with the thickness of the fluid measuring less than 10 mm on decubitus radiograph or ultrasound examination. Approximately 10% of patients undergoing abdominal surgery develop effusions that are more than 10 mm thick on the decubitus radiograph. These effusions are usually asymptomatic. The larger effusions are particularly common after splenectomy. The pleural fluid is an exudate in the majority of patients. The majority of patients develop a pleural effusion postliver transplantation. The effusion occupies more than 25% of the hemithorax in one-fourth of patients receiving liver transplantation. These pleural effusions are usually unilateral on the right or bilateral with more fluid on the right than on the left. When the effusions are large, they can produce shortness of breath. There has been no systemic analysis of the characteristics of the pleural fluid in these patients.

splenectomy, when damage to the diaphragm is common. It is thought that the pleural effusion following liver transplantation is due to injury or irritation of the right hemidiaphragm caused by the extensive right upper quadrant dissection and retraction. The pathogenesis of the pleural fluid that accumulates after lung transplantation is related to the transaction of the lymphatics that drain the lung at the time of transplantation. Normally, 80% of the fluid that enters the interstitium of the lung is cleared from the lung via the lymphatics, whereas 20% is cleared through the pleural space. However, in the transplanted lung, almost all the fluid that enters the lung exits through the pleural space because of the transaction of the lymphatics. The continuity of the lymphatics is restored within 2–4 weeks after lung transplantation.

Animal Models There are no animal models for pleural effusions that occur post-CABG surgery or after cardiac injury. Likewise, there are no animal models for pleural effusions that occur after abdominal surgery, liver transplantation, or lung transplantation.

Management and Therapy Pathogenesis The pathogenesis of the early post-CABG pleural effusion is probably pleural inflammation due to the trauma of surgery. The presence of lymphocytes in the pleural fluid of the late post-CABG pleural effusion coupled with the lymphocytes in the pleural biopsy specimens suggests that it is due to an immunological reaction. It is possible that it represents a limited form of PCIS. The pathogenesis of PCIS is not completely known, but apparently it has an immunologic basis. When the myocardium or pericardium are injured, cellular components including autoantigens are released. These autoantigens can provoke an autoimmune antibody reaction by anti-myocardial antibodies. There appears to be a close relationship between the occurrence of PCIS and the development of anti-myocardial antibodies in patients undergoing surgical procedures involving the pericardium but not in patients with acute myocardial infarction. There is also a seasonal variation in the occurrence of PCIS, with the highest incidence corresponding to the time of the highest prevalence of viral infection in the community. The pathogenesis of pleural effusions after abdominal surgery is probably related to the trauma of surgery. Pleural effusions are more likely to develop after upper abdominal surgery and especially after

When a patient is seen post-CABG surgery with a pleural effusion, no therapy is recommended if the patient is asymptomatic and the effusion is typical of a post-CABG pleural effusion (e.g., left side unilateral or larger on the left). If the patient has fever or chest pain, or is short of breath, a therapeutic thoracentesis should be performed. The thoracentesis should relieve the shortness of breath and the pleural fluid analysis should demonstrate whether the pleural effusion is due to congestive heart failure or a pleural infection or a chylothorax. There have been no controlled studies evaluating adjunctive therapies such as anti-inflammatory agents, diuretics, and corticosteroids, and they are not recommended. The pleural effusion will resolve completely in the majority of patients with one to three therapeutic thoracenteses. Rarely, a patient will develop a thin fibrous membrane over the visceral pleura that will produce a trapped lung. In this situation, removal of the fibrous membrane with thoracoscopy or thoracotomy will result in resolution of the effusion. Such invasive therapy should not be tried until the patient has had at least three therapeutic thoracenteses with recurrence of the pleural effusion. The management of PCIS involves the administration of anti-inflammatory agents such as aspirin or indomethacin. In more severe forms of the syndrome, corticosteroids may be necessary. Single episodes

382 PLEURAL EFFUSIONS / Pleural Fibrosis

of PCIS are usually self-limited, but recurrences are frequent. Recurrent episodes become progressively more resistant to treatment with nonsteroidal antiinflammatory agents but usually respond to corticosteroid therapy. The management of pleural effusions occurring after abdominal surgery depends on the size of the effusions. If the effusion is small, no diagnostic or therapeutic interventions are warranted. If the effusion is more than minimal, a diagnostic thoracentesis should be performed to rule out pleural infection. If the effusion develops more than 72 h after surgery, pulmonary embolism and subphrenic abscess should be considered. The natural history of a pleural effusion after liver transplantation is to increase in size during the first 3 postoperative days and then gradually resolve over several weeks to months. Pleural effusions that increase in size after the first 3 days are likely associated with subdiaphragmatic pathology such as hematoma, biloma, or abscess and, accordingly, such patients should be evaluated for subdiaphragmatic pathology. If the patient becomes short of breath from the effusion, a therapeutic thoracentesis should be performed. One report suggested that pleural effusions that occur after liver transplantation can be largely prevented if a fibrin sealant is sprayed on the undersurface of the diaphragm around the insertion of the liver ligaments at the time of transplantation. Immediately postlung transplantation, pleural effusions are usually not evident because patients have a chest tube. The mean output of pleural fluid is approximately 400 ml per day on day 1 and decreases gradually to 200 ml per day on day 4. The pleural fluid on day 1 is a bloody neutrophil-predominant exudate. Patients who develop complications postlung transplantation are also likely to have a pleural effusion. Pleural effusions occur in more than 50% of patients who develop acute rejection, chronic rejection, pulmonary infection, and lymphoproliferative disease. Treatment of early pleural effusions with lung transplantation consists of continuation of the tube thoracostomy. Management of late pleural effusions is dependent on the underlying complication causing the effusions. See also: Pleural Effusions: Overview; Pleural Fluid, Transudate and Exudate; Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube. Surgery: Transplantation.

Golfieri R, Giampalma E, Morselli Labate AM, et al. (2000) Pulmonary complications of liver transplantation: radiological appearance and statistical evaluation of risk factors in 300 cases. European Radiology 10: 1169–1183. Lee YC, Vaz MAC, Ely KA, et al. (2001) Symptomatic persistent post-coronary artery bypass graft pleural effusions requiring operative treatment. Clinical and histologic features: Chest 119: 795–800. Liem KL, ten Veen JH, Lie KI, et al. (1979) Incidence and significance of heart-muscle antibodies in patients with acute myocardial infarction and unstable angina. Acta Medica Scandinavica 206: 473–475. Light RW (2001) Pleural effusions following cardiac injury and coronary artery bypass graft surgery. Seminars in Respiratory and Critical Care Medicine 22: 657–664. Light RW (2001) Pleural effusion secondary to diseases of the heart. In: Pleural Diseases, 4th edn., pp. 241–246 Baltimore: Lippincott Williams & Wilkins. Light RW and George RB (1976) Incidence and significance of pleural effusion after abdominal surgery. Chest 69: 621–626. Light RW, Rogers JT, Cheng D-S, and Rodriguez RM; Cardiovascular Surgery Associates (1999) Large pleural effusions occurring after coronary artery bypass grafting. Annals of Internal Medicine 130: 891–896. Light RW, Rogers JT, Moyers JP, et al. (2002) Prevalence and clinical course of pleural effusions at 30 days after coronary artery and cardiac surgery. American Journal of Respiratory and Critical Care Medicine 166: 1563–1566. Miller RH, Horneffer PJ, Gardner TJ, Rykiel MF, and Pearson TA (1988) The epidemiology of the postpericardiotomy syndrome: a common complication of cardiac surgery. American Heart Journal 116: 1323–1329. Mott AR, Fraser CD Jr, Kusnoor AV, et al. (2001) The effect of short-term prophylactic methylprednisolone on the incidence and severity of postpericardiotomy syndrome in children undergoing cardiac surgery with cardiopulmonary bypass. Journal of the American College of Cardiology 37: 1700–1706. Nielsen PH, Jensen SB, and Olsen AD (1989) Postoperative pleural effusion following upper abdominal surgery. Chest 96: 1133– 1135. Sadikot RT, Rogers JT, Cheng D-S, et al. (2000) Pleural fluid characteristics of patients with symptomatic pleural effusion after coronary artery bypass graft surgery. Archives of Internal Medicine 160: 2665–2668. Spizarny DL, Gross BH, and McLoud T (1993) Enlarging pleural effusion after liver transplantation. Journal of Thoracic Imaging 8: 85–87. Uetsuji S, Komada Y, Kwon AH, et al. (1994) Prevention of pleural effusion after hepatectomy using fibrin sealant. International Surgery 79: 135–137.

Pleural Fibrosis J T Huggins and S A Sahn, Medical University of South Carolina, Charleston, SC, USA & 2006 Elsevier Ltd. All rights reserved.

Further Reading

Abstract

Engle MA, Zabriskie JB, Senterfit LB, and Ebert PA (1975) Postpericardiotomy syndrome: a new look at an old condition. Modern Concepts of Cardiovascular Disease 44: 59–64.

The response of the mesothelial cell and basement membrane to injury is vital in determining whether there is normal healing or pleural fibrosis. The formation of a fibrinous intrapleural matrix

PLEURAL EFFUSIONS / Pleural Fibrosis 383 is critical to the development of pleural fibrosis. This matrix is the result of disordered fibrin turnover, whereby fibrin formation is upregulated and fibrin dissolution is downregulated. Transforming growth factor beta and tumor necrosis factor alpha are important cytokines that facilitate formation of the fibrin matrix. A complete understanding of the pathogenesis of pleural fibrosis and why dysfunctional pleural space remodeling occurs in some and not in others remains unknown. Clinically significant pleural fibrosis requires involvement of the visceral pleura, while isolated parietal pleural fibrosis does not cause restriction or respiratory impairment. The vast majority of diseases causing visceral pleural fibrosis include asbestos-associated diffuse pleural thickening, coronary bypass graft surgery, tuberculous pleurisy, rheumatoid pleurisy, uremic pleurisy, hemothorax, radiation, and empyema. Systemic and intrapleural corticosteroids administered during the initial presentation of rheumatoid pleurisy anecdotally may have decreased the incidence of pleural fibrosis. Several randomized control trials of corticosteroids in tuberculous pleurisy have not shown efficacy in reducing residual pleural fibrosis. Decortication is effective in treating symptomatic patients regardless of the cause of pleural fibrosis as long as chronicity has been documented and significant underlying parenchymal disease has been excluded.

Introduction Pleural fibrosis can result from a vast array of inflammatory processes, including dust exposure (asbestos), immunologic disease (rheumatoid pleurisy), infection (bacterial empyema, tuberculous pleurisy), improperly drained hemothorax, radiation, malignancy, postcoronary artery bypass surgery (post-CABG), and uremic pleurisy. The response of the mesothelial cell to injury and its ability with the underlying basement membrane to maintain integrity is crucial in determining whether there is normal or dysfunctional healing (pleural fibrosis). Trapped lung and fibrothorax are two unique and rare clinical entities that can be a consequence of pleural fibrosis. Trapped lung presents as a chronic, unilateral, pleural effusion that is the result of a remote inflammatory process. It is characterized by the inability of the lung to expand and fill the thoracic cavity due to a restrictive, fibrous visceral pleural peel. The resultant negative pressure pleural space becomes filled with low-protein pleural fluid that persists due to the hydrostatic disequilibrium. A prerequisite for the development of a trapped lung is remote pleural inflammation or hemothorax. The pleural effusion must persist long enough to allow fibrous tissue to develop on the visceral pleura while the pleural surfaces remain separated. Fibrothorax represents the most severe form of pleural fibrosis. This term is reserved for an intense fibrotic response of the visceral pleura leading to fusion of both the visceral and parietal membranes. As a result, there is contracture of the involved hemithorax and reduced mobility of the lung and thoracic

cage due to progressive pleural fibrosis and symphysis of the pleural membranes. As the fibrothorax matures, the intercostal spaces narrow, the size of the ipsilateral hemithorax diminishes, and the mediastinum is displaced ipsilateral. Pleural fibrosis becomes clinically important when it involves the visceral pleural surface. Isolated parietal pleural fibrosis, as with asbestos-related pleural plaques, does not result in respiratory symptoms or clinically relevant decrements in lung function. In contrast, the development of visceral pleural fibrosis can lead to lung entrapment resulting in dyspnea and, in some patients, respiratory failure.

Etiology Asbestos

Pleural fibrosis is the most common radiographic finding among those exposed to asbestos. The two forms of asbestos-induced pleural fibrosis are parietal pleural plaques and diffuse pleural thickening. The development of pleural fibrosis is dependent on the duration and cumulative dose of asbestos exposure. Pleural plaques, localized fibrous lesions of the parietal pleura, are the most common manifestation of asbestos exposure. Because of their parietal pleural location, they are not typically associated with respiratory symptoms or pulmonary impairment. Dyspnea or restrictive physiology implies asbestosis, even with a normal chest radiograph, or an alternative diagnosis. Pleural plaques appear on average 20– 30 years after initial exposure to asbestos, with the incidence varying with asbestos fiber type and duration of exposure. The term diffuse pleural thickening is often used interchangeably with two different presentations: (1) diffuse thickening involving the majority of the pleura leading to encasement of the lung; and (2) limited thickening involving at least one-fourth of the visceral pleura. Diffuse pleural thickening primarily involves the visceral pleura, is usually unilateral, and commonly involves the costophrenic recess. Diffuse pleural thickening is thought to be the sequela of an asbestos-related exudative pleural effusion. It could also be the result of repeated bouts of asbestos-related pleurisy whereby a fibrinous scaffold is laid down, matures, and organizes into dense collagenous tissue. Pleural adhesions and fusion of the visceral and parietal pleura commonly occur with diffuse pleural thickening. Rheumatoid Pleurisy

Pathological studies have demonstrated that pleural effusion and pleural fibrosis occur in 50% of patients

384 PLEURAL EFFUSIONS / Pleural Fibrosis

with rheumatoid arthritis. The degree of pleural fibrosis ranges from small fibrous plaques to extensive fibrosis of the pleura. Those with a protracted course of rheumatoid pleurisy may develop significant pleural fibrosis and trapped lung. Tuberculous Pleurisy

Tuberculous pleurisy is the most frequent extrapulmonary manifestation of tuberculosis. Residual pleural thickening is the most common sequela following pleural tuberculosis, and has been reported in 20– 50% of cases. Coronary Artery Bypass Surgery

Left-sided exudative pleural effusions following CABG are common. The incidence of pleural effusions is higher with internal mammary artery grafts than with saphenous vein grafts. The natural history of these chronic effusions is gradual resolution. However, in a subgroup of patients, effusions may persist for months following CABG. Pleural biopsy specimens from patients undergoing thoracoscopy for persistent post-CABG effusions show an intense lymphocytic pleuritis within the first few months following surgery. With time, there is a reduction in the cellular inflammation and an increase in pleural fibrosis leading to the development of a trapped lung. The true incidence of trapped lung following CABG is unknown. However, due to the vast number of these surgical procedures performed each year, CABG is the most common cause of trapped lung encountered today. Fibrosing Uremic Pleuritis

Fibrinous pleuritis has been found in 20% of uremic patients at autopsy. Spontaneous resolution of uremic pleurisy typically occurs in 4–6 weeks leaving clinically unimportant pleural thickening in most patients. In rare cases, the pleural fluid becomes gelatinous and a thick fibrous pleural peel develops (Figure 1). Hemothorax

Diffuse pleural thickening producing a fibrothorax is a late complication of a hemothorax. Fortunately, this complication occurs in less than 1% of patients even if residual blood is not removed from the pleural space. Fibrothorax appears to be more common with a hemopneumothorax or when pleural space infection complicates management. Cryptogenic Fibrosing Pleuritis

This disease entity was originally described in four patients who developed progressive bilateral pleural fibrosis following exudative pleural effusions. An

Figure 1 Chest radiograph showing bilateral pleural thickening and pleural effusions in a patient with uremic pleuritis.

extensive evaluation failed to reveal an attributable cause of the pleural fibrosis; hence the term, bilateral cryptogenic fibrosing pleuritis. All four cases plus two of our cases were HLA-B44 positive suggesting a genetic link with this disease. Trapped Lung and Fibrothorax

Conditions most often associated with trapped lung are complicated parapneumonic effusion, empyema, and postcardiac surgery, especially with internal mammary artery grafting. Other conditions associated with trapped lung include uremic pleuritis, rheumatoid pleuritis, tuberculous pleurisy, and a complicated hemothorax. The causes of fibrothorax are similar to those that cause trapped lung. The primary causes of fibrothorax include hemothorax, bacterial empyema, and tuberculous empyema. Uremia and rheumatoid pleurisy have also been associated with fibrothorax.

Pathology Pleuritis with pleural effusion frequently develops in a wide variety of disorders with most of the pleural changes regressing spontaneously without intervention. How these pleural changes regress is not completely understood. The histology of asbestos-pleural plaques reveals a hypocellular lesion comprised of dense collagen

PLEURAL EFFUSIONS / Pleural Fibrosis 385

bundles arranged in a ‘basket-weave’ configuration. Rarely, mild inflammation may be observed; however, the mesothelial cell layer overlying the plaque appears normal. Noninvasive ways of grading pleural thickening by computed tomography have been proposed by Copley and co-workers. Quantification of pleural fibrosis by their method has been correlated to the severity of physiologic impairment.

Clinical Features Standard chest radiographs are the most common diagnostic modality used to detect focal and diffuse pleural fibrosis. Asbestos-pleural plaques appear as well-demarcated lesions along the midlateral chest wall (Figure 2). The costophrenic angles and apices are typically spared. Plaques may also be found over the dome of the diaphragm and on the pericardium. Occasionally, it may be difficult to distinguish plaques from muscle or extrapleural fat. Computed tomography can differentiate plaques from extrapleural fat causing an increased radiographic density. The radiographic appearance of diffuse pleural thickening associated with asbestos is a smooth, uninterrupted pleural density extending over at least one-fourth of the chest wall. The posterior and posteromedial pleura overlying the lower lobes are more commonly involved. Nodularity and pleural thickening greater than 1 cm are radiographic signs suggestive of malignant mesothelioma or pleural metastasis.

Figure 2 Computed tomography of a patient with benign asbestos pleural plaques. In addition, note rounded atelectasis and crow’s feet.

For nonasbestos-related diffuse pleural thickening, the radiograph can provide helpful clues as to the underlying etiology. For example, in patients with chronic tuberculous empyema, unilateral parietal and visceral calcifications and remote parenchymal disease is often seen. Because of their parietal pleural location, asbestospleural plaques are not typically associated with respiratory symptoms. Dyspnea or restrictive physiology implies asbestosis, even with a normal chest radiograph, or an alternative diagnosis. In contrast, diffuse pleural thickening regardless of etiology can cause dyspnea of varying severity to overt respiratory failure. Restriction on pulmonary function testing is often noted. The diagnosis of trapped lung requires documentation of chronicity and the absence of malignancy or active pleural inflammation. The pleural fluid resulting from a trapped lung has lactate dehydrogenase and total protein values in the transudative range. Findings that suggest a trapped lung are an initial negative mean pleural liquid pressure, increased pleural space elastance (a change of 425 cmH2O pressure after removing 1 l of pleural fluid), failure of lung expansion after thoracentesis or tube drainage, and the demonstration of a visceral pleural peel on an air-contrast chest computed tomography. Trapped lung is confirmed when the lung expands to the chest wall following decortication.

Pathogenesis Pleuritis with pleural effusions develops in patients with a number of diseases. The majority of these pleural insults regress, either spontaneously or with medications, without the need for pleural space drainage. How pleural inflammation resolves in some and pleural fibrosis develops in others remains unclear. In experimental models of pleuritis, the mesothelial cells proliferate and become reactive in response to injury. These mesothelial cell changes appear to assist in the removal of fibrin and inflammatory debris and in repair of the pleural surface. The degree of mesothelial cell and basement membrane injury and regeneration are pivotal in determining if pleural fibrosis occurs. In the normal pleural space, a matrix does not exist. For pleural fibrosis to develop, a matrix must form to allow the fibrotic process to continue. This matrix is a product of plasma coagulation substrates that are released into the pleural space by the inflammatory response. Tissue factor is the major procoagulant that initiates matrix formation. Tissue factor, released from mesothelial cells, fibroblasts, and macrophages, acts upon other procoagulant

386 PLEURAL EFFUSIONS / Pleural Fibrosis

substrates, resulting in the conversion of fibrinogen to fibrin, which is pivotal to the creation of the matrix. Tissue factor pathway inhibitor (TFPI), an inhibitor of tissue factor, is also elaborated by mesothelial cells and lung fibroblasts in response to injury. The balance between tissue factor and TFPI is important in the production and persistence of pleural fibrosis. When intrapleural generation of tissue factor exceeds the formation of TFPI, the procoagulant state that exists favors fibrin formation. Concomitantly, fibrinolysis is downregulated by the increased expression of plasminogen activator inhibitor-1 (PAI-1), allowing for the formation of the fibrin matrix. Therefore, the pathogenesis of pleural fibrosis can be considered a disorder of fibrin turnover. An early response to mesothelial cell injury is the elaboration of chemokines, which facilitate phagocytic cell migration intrapleurally. Vascular endothelial growth factor (VEGF) is one of the chemokines that is upregulated in malignancy, empyema, and tuberculous pleurisy. VEGF not only promotes angiogenesis but is also responsible for increasing vascular permeability, which allows for the efflux of procoagulant fluid intrapleurally. Both cellular and extracellular proteins are responsible for perpetuating pleural inflammation. One of these protein mediators is hyaluronan, which emulates the inflammatory process by attracting mononuclear cells and malignant cells in pleural metastases. Type I and IV collagen and fibronectin released by mesothelial cells may also participate in sustaining inflammation through recruitment of inflammatory cells and maintenance of vascular permeability. The mesothelial cells not only are vital in initiating inflammation but also in regulating the duration of the inflammatory response. Apoptosis of inflammatory cells is inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF), which is elaborated by mesothelial cells. GM-CSF will also inhibit programmed cell death of neutrophils, monocytes, and lymphocytes during pleural space inflammation. The inflammation may resolve ‘normally’ with an intact mesothelium without fibrosis/remodeling; however, pleural space remodeling may occur. How pleural space remodeling occurs remains unknown, although some insight into this process has been made with the talc pleurodesis model. Intrapleural instillation of talc rapidly upregulates basic fibroblast growth factor (b-FGF) in pleural fluid, while in vitro, talc stimulates b-FGF release from mesothelial cells. An inverse correlation was observed between tumor mass and b-FGF levels indicating that patients with larger tumor burdens had lower b-FGF levels and were more likely to have failed pleurodesis. Therefore, for talc pleurodesis to be successful,

mesothelial cells must be intact and stimulated to produce b-FGF, which leads to pleural fibroblast proliferation and subsequent pleural fibrosis. Another cytokine that plays a pivotal role in the development of pleural fibrosis is transforming growth factor beta (TGF-b). TGF-b, which is considered to be the most potent profibrotic cytokine, has generated recent attention for its role in promoting pleural fibrosis. Intrapleural instillation of TGF-b induces rapid pleural fibrosis in an experimental animal model. Not only is TGF-b a potent chemoattractant for fibroblasts, it also plays a role in matrix production by upregulation of collagen formation and matrix remodeling. TGF-b has been shown to inhibit plasminogen activator and stimulate plasminogen activator inhibitor secretion from pleural mesothelial cells. TGF-b appears to be an important link between disordered fibrin turnover and the development of pleural fibrosis (Figure 3).

Animal Models Most animal models of pleural inflammation utilizing irritants are a method of investigating fibrothorax; however, there are animal models more relevant to investigating the common pathological pleural fibrotic diseases seen in humans. Asbestos-induced pleural disease and tuberculous pleuritis are two examples of diseases studied in several mammalian species that resemble human diseases. In tuberculous pleurisy, animals are immunized to BCG several weeks prior to intrapleural instillation injection of BCG. The development of pleurisy with well-defined stages of inflammation is consistently observed. The earliest stage of pleuritis is the development of a granulocytic exudative pleural effusion, which rapidly changes to a predominance of mononuclear leukocytes. With resolution of the pleural effusion, there is a residue of pleural adhesions and fibrosis. In a model of asbestos-induced pleural effusion, chrysotile asbestos was injected into the pleural space of rabbits. The initial response to the intrapleural asbestos fibers was a granulocytic influx with localization of asbestos fibers into granulomatosis pleural plaques. In neutropenic rabbits, a granulocytic response to asbestos fibers was absent and plaque formation did not develop. Atypical mesothelial cells invading the subpleural connective tissue was noted at 10 months.

Management and Current Therapy Appropriate management of pleural fibrosis requires an understanding that clinically significant pleural

PLEURAL EFFUSIONS / Pleural Fibrosis 387

Lung

Pleural space

Lung fibroblast

↓ Fibrinolysis

Recruitment of fibroblast

↓ PAI-1

Fibrin neomatrix

Plasma coagulation substrates

↓ TFPI

↑ TGF-


Collagen deposition

Pleural injury

Parietal pleura

Damage of mesothelium and basement membrane

↑ VEGF (capillary leak)

Chest wall

Mesothelial cells become reactive to injury

Figure 3 Pathogenic model of pleural fibrosis. TGF-b, transforming growth factor beta; PAI-1, plasminogen activator inhibitor-1; TFPI, tissue factor pathway inhibitor; VEGF, vascular endothelial growth factor.

fibrosis requires involvement of the visceral pleura. Patients with isolated parietal pleural fibrosis, such as asbestos pleural plaques, do not develop respiratory symptoms. In contrast, visceral pleural fibrosis can lead to significant restriction, dyspnea, and respiratory failure. The use of steroids to reduce or prevent pleural fibrosis has been limited to tuberculous and rheumatoid pleurisy. Some have advocated systemic corticosteroids in the management of tuberculosis pleurisy to limit the degree of pleural inflammation and resultant pleural fibrosis. Although most agree that residual pleural thickening following tuberculous pleurisy has little or no effect on lung function, several randomized trials of corticosteroids have been performed. Based on these studies, there is insufficient evidence to support the use of corticosteroids to prevent or diminish the development of pleural fibrosis in tuberculous pleural effusions. However, systemic

corticosteroids will result in more rapid resolution of the effusion and improvement in symptoms. Those with a protracted course of rheumatoid pleurisy may develop significant pleural fibrosis and trapped lung. There are no controlled studies that have evaluated the effectiveness of corticosteroids in the management of rheumatoid pleurisy. Single case reports and small series using systemic and intrapleural corticosteroids have described variable success. Decortication is an effective strategy for treating symptomatic patients regardless of cause when significant underlying parenchymal disease has been excluded. For patients with severe respiratory compromise, decortication is the only effective treatment of a trapped lung or fibrothorax. The timing of decortication is important, since pleural thickening in some patients typically resolve over several months. Therefore, only when pleural fibrosis has been stable

388 PLEURAL EFFUSIONS / Chylothorax, Pseudochylothorax, LAM, and Yellow Nail Syndrome

or progressive over a 6-month period should decortication be considered. See also: Fibrinolysis: Plasminogen Activator and Plasmin. Fibroblast Growth Factors. Occupational Diseases: Asbestos-Related Lung Disease. Pleural Effusions: Hemothorax. Pulmonary Fibrosis. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumor Necrosis Factor Alpha (TNF-a). Vascular Endothelial Growth Factor.

Further Reading Antony V (2003) Pleural inflammation. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 56–62. New York: Arnold. Copley SJ, Wells AV, Rabus MB, et al. (2001) Functional consequences of pleural disease evaluated with chest radiography and CT. Radiology 220: 237–243. Doelken P and Sahn SA (2001) Trapped lung. Seminars in Respiratory and Critical Care Medicine 22: 631–635. Idell S (1995) Coagulation, fibrinolysis and fibrin deposition in lung injury and repair. In: Phan SH and Thrall RS (eds.) Pulmonary Fibrosis, vol. 80, pp. 743–776. New York: Dekker. Lee YCG and Lane KB (2003) Cytokines in pleural diseases. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 63–89. New York: Arnold. Nishumura SL and Broaddus C (1998) Asbestos-induced pleural disease. Clinical Chest Medicine 19: 311–329. Sahn SA (1988) State of the art. The pleura. American Review of Respiratory Disease 138: 184 –234. Schwartz DA and Peterson MW (1998) Asbestosis and asbestosinduced pleural fibrosis. In: Schwartz MI and King TE (eds.) Interstitial Lung Disease, pp. 351–366. Hamilton, ON: Decker.

Chylothorax, Pseudochylothorax, LAM, and Yellow Nail Syndrome I Kalomenidis, Athens University, Athens, Greece Y C G Lee, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Chylothorax and pseudochylothorax are pleural effusions with high lipid contents. Chylothorax is a triglyceride-rich effusion caused by the disruption of the thoracic duct, most commonly by trauma or mediastinal tumors. Pseudochylothorax is a chronic, cholesterol-rich pleural effusion, usually secondary to tuberculous pleuritis. Pleural fluid lipid analysis is needed to establish the diagnoses. The initial management strategies for chylothorax include nutrition support, reduction of chyle flow, and drainage of the pleural space for symptomatic relief. If the chylothorax persists, thoracic duct embolization, surgical repair, or pleurodesis may be considered, depending on the etiology of the chylothorax. Lymphangioleiomyomatosis is a progressive

cystic lung disease often complicated by recurrent chylous effusions and/or pneumothoraces. Yellow nail syndrome is a rare disorder of unknown etiology characterized by yellow nails, lymphoedema, and recurrent lymphocytic pleural effusions, which may require pleurodesis.

Chylothorax Chylothorax, the accumulation of chyle in the pleural cavity, usually develops secondary to disruption of the thoracic duct along its intrathoracic route. The thoracic duct drains chyle (lymph-rich in chylomicrons from dietary fat) from the abdomen through the diaphragm and into the posterior mediastinum before terminating in the region of the left jugular and subclavian veins. The thoracic duct drains approximately 2 l lymphs a day, but the flow rate varies with diet and increases significantly after ingestion of fat. Chyle is rich in T lymphocytes and, typically, has an electrolyte profile similar to serum and a protein concentration higher than 3 g dl 1. Loss of chyle can lead to immunodeficiency, dehydration, and nutritional depletion. Chyle in the pleural space seldom elicits pleural inflammation or fibrosis, and secondary infection of chylous effusion is rare. Pathogenesis and Etiology

Chylothorax most frequently results from trauma (including surgery) or tumor infiltration of the thoracic duct (Table 1). Occasionally, chylothorax can accumulate when chylous ascites migrate through the diaphragm into the pleural space. Common causes of chylous ascites include traumatic or malignant disruption of intra-abdominal lymphatic vessels and cirrhosis. Malignancy and trauma (including surgery) are by far the leading causes of chylothorax (Table 1). Lymphoma accounts for 75% of the chylothorax cases due to malignancy. As such, lymphoma needs to be excluded in all patients with chylothoraces in whom no other causes can be identified. Cardiothoracic surgery, especially involving the posterior mediastinum, can lead to postoperative chylothorax (up to 4% in an eosophagectomy series). Nonsurgical thoracic trauma (both penetrating and nonpenetrating) is a recognized cause of chylothorax. Lymphatic abnormalities (e.g., lymphangioleiomyomatosis and benign mediastinal disorders; Table 1) are less common etiologies. A chylothorax is termed idiopathic when no cause is found. Chylothorax can affect people of all ages. Fetal chylothorax, although uncommon, is a recognized cause of developmental abnormalities and can be fatal. Chylothorax is also the most common type of

PLEURAL EFFUSIONS / Chylothorax, Pseudochylothorax, LAM, and Yellow Nail Syndrome 389 Table 1 Etiologic classification of chylothorax Malignacy Lymphoma, metastatic carcinoma, Kaposi’s sarcoma Trauma Iatrogenic Eosophagectomy, esophagoscopy, cardiac surgery, lung resection (lobectomy or pneumonectomy), heart and lung transplantation, aortic surgery, stellate ganglion blockade, thoracic sympathectomy, high traslumbar aortography, urological laparoscopy, spinal surgery, repair of congenital diaphragmatic hernia, central venous catheter placement Nonsurgical trauma Penetrating or closed trauma involving the thorax or the neck, spinal fracture Minor trauma Weight lifting, straining, coughing, vomiting, childbirth, yawning Miscellaneous Lymphangioleiomyomatosis, tuberous sclerosis, intestinal lymphangiectasis or reticular hyperplasia, yellow nail syndrome, superior vena caval or subclavian vein thrombosis/obstruction, constrictive pericarditis, filariasis, sarcoidosis, tuberculosis, benign tumors, retrosternal goiter, mediastinal fibrosis, lymphangitis of the thoracic duct, amyloidosis, Gorham’s syndrome Idiopathic

neonatal pleural effusion. It may represent a fetal chylothorax that persisted or can result from thoracic duct rupture during delivery, developmental abnormalities of the thoracic duct, or increased venous pressure from congenital heart diseases. Clinical Manifestations and Diagnosis

The main symptom of chylothorax is dyspnea on exertion. Pleuritic chest pain and fever, commonly associated with other exudative pleural effusions, are rare because most disease processes causing chylothoraces involve the mediastinum and/or the lymphatics but not the pleura. Neonatal chylothorax usually presents with respiratory distress, most commonly (75%) within the first week of life. Chylothorax may be unilateral or bilateral (Figure 1). The thoracic duct ascends along the right side of the vertebrae and crosses over to the left between the level of the T4 and T6 vertebrae. Although this route is the typical one, anatomic variations are common among individuals. Rupture of the duct when it ascends to the right of the vertebral column tends to produce right-sided chylothoraces, and interruption of the duct after it crosses over the midline tends to result in left-sided chylothoraces. Pleural fluid from chylothoraces has a characteristic milky or turbid appearance due to its high lipid content. Empyema can also appear turbid from

Figure 1 (a) A posteroanterior radiograph of a patient who presented with superior vena cava syndrome and bilateral pleural effusions. The patient has had a pacemaker for 16 years. Twelve years ago, a new pacemaker was inserted from the left side. The wire of the old pacemaker is seen on the patient’s right side (arrow). A diagnostic thoracentesis revealed a milky pleural fluid with triglyceride 4500 mg dl 1, a pleural fluid:serum triglyceride ratio 41, and a pleural fluid:serum cholesterol ratio o1 at both hemothoraces. (b) Venography revealed thrombotic obstruction of the superior vena cava extending to the proximal part of the subclavian veins (arrows). Diagnosis: bilateral chylothorax due to obstruction of the superior vena cava.

suspended cellular debris and bacteria. However, pleural fluid from empyemas will become clear upon centrifugation. If cloudiness of the pleural fluid persists after centrifugation, it indicates a high lipid content, either due to chylomicrons (as in chylothorax) or cholesterol and lecithin–globulin complexes (as in pseudochylothorax). It must be remembered that pleural fluid in chylothorax may not always look

390 PLEURAL EFFUSIONS / Chylothorax, Pseudochylothorax, LAM, and Yellow Nail Syndrome

milky, especially if the patient is fasting, which can significantly reduce the lipid content in the chyle. Likewise, the effusion remains clear in neonatal chylothorax until milk feeding begins. In patients with cirrhosis, the pleura fluid may be a transudate as a result of dilution by transudative cirrhotic ascitic fluid. A high index of suspicion is therefore required for the diagnosis of chylothorax. The clinical presentation is helpful to differentiate chylothorax from pseudochylothorax. The former is usually a relatively acute condition with a normal pleural surface; the latter is a chronic effusion (often of years) commonly associated with a markedly thickened, or even calcified, pleura. The demonstration of chylomicrons in lipoprotein analysis of the pleural fluid provides a definitive diagnosis of chylothorax. Patients with chylothoraces usually have a fluid triglyceride level 4110 mg dl 1 (1.24 mmol l 1), a fluid:serum triglyceride ratio 41, and a fluid:serum cholesterol ratio o1. A triglyceride content o50 mg dl 1 usually excludes the diagnosis. However, it should be noted that the fluid triglyceride levels in chylous effusions may be low after fasting, producing false or negative results. An intermediate triglyceride content (between 50 and 110 mg dl 1) may require lipoprotein analysis of pleural fluid for chylomicrons. Lipoprotein analysis may also aid the differentiation between a chylothorax and a pseudochylothorax, if the pleural fluid cholesterol and triglyceride levels are both elevated. Every patient with a nontraumatic chylothorax should undergo computed tomography (CT) scanning of the thorax and abdomen to search for lymphadenopathy or pulmonary parenchymal abnormalities. Bipedal lymphangiography or lymphoscintigraphy (e.g., with Tc-99 human serum albumin) can be performed preoperatively to locate the site of chyle leak if surgical repair is to be undertaken. Pleural biopsy or thoracoscopy is not indicated since the pleura is usually normal in patients with a chylothorax. Neonates with chylothoraces often (50%) have symptoms within the first 24 hours and the chylothoraces are either right-sided or bilateral. Ultrasonography is essential in the diagnosis of fetal chylothorax. Treatment

The aims of management of patients with chylothorax are (Table 2): 1. To maintain nutrition and reduce the chyle flow: prolonged loss of chyle will rapidly render the patient malnourished and immunocompromised. Parenteral hyperalimentation or a low-fat diet with

Table 2 Management of chylothorax Measures to maintain nutrition and reduce the flow of chyle through the thoracic duct Total parental nutrition or diet with medium-chain triglyceride Octreotide Measures to relief dyspnea by inhibiting the reaccumulation of chyle in the pleural space Thoracentesis or tube thoracostomy Pleuroperitoneal shunting Pleurodesis Treatment of the underlying defect Thoracic duct embolization Ligation of the thoracic duct (video-assisted thoracoscopic surgery or thoracotomy) Radiotherapy (in the case of mediastinal lymphoma) Modified from Light RW and Lee YCG (2005) Pneumothorax, chylothorax, hemothorax and fibrothorax. In: Murray J, Nadel J, Mason R and Broaddus VC (eds.) Textbook of Respiratory Diseases, 4th edn., pp. 1961–1988. Philadelphia: Saunders.

most fats in the form of medium-chain triglycerides (which are absorbed directly into the blood) are recommended because they provide nutritional support without stimulating chyle flow. Input from an experienced dietitian is encouraged. Reducing chyle flow not only decreases nutritional loss but also aids in the closure of the thoracic duct leak. Octreotide, a somatostatin analog, has been reported to reduce chyle leak and hasten closure of thoracic duct, possibly by reducing intestinal fat absorption and increasing fecal fat excretion. 2. To remove chylous pleural fluid to relieve dyspnea: thoracentesis or tube thoracostomy can provide symptomatic relief. If the leak persists, pleuroperitoneal shunts can be considered because they allow ‘recycling’ of the lymph and thus minimize loss of nutrition and lymphocytes. Pleuroperitoneal shunts are contraindicated in the presence of ascites. In patients with ongoing chyle leak, pleurodesis can be considered. 3. Treatment of the underlying thoracic duct defect: in patients with traumatic chylothorax, the defect frequently closes spontaneously with supportive care. If the chyle leak persists, especially those draining in high volumes despite total parental nutrition, a definitive repair option should be considered: Percutaneous thoracic duct embolization: if available, lymphatic embolization should be attempted before more invasive procedures (e.g., surgery) are performed. With this technique, the duct is cannulated transabdominally under fluoroscopic guidance and embolized with devices such as microcoils. If feeding lymphatic vessels are too small, occlusion of the vessels by needle disruption can reduce the chyle flow. A response rate of 74%

PLEURAL EFFUSIONS / Chylothorax, Pseudochylothorax, LAM, and Yellow Nail Syndrome 391

and good safety profile have been reported in case series. Surgical repair: video-assisted thoracoscopic surgery or thoracotomy can help locate the site of leakage and the thoracic duct can be ligated. A pleurodesis may also be performed during the same operation. Treating the underlying cause of the chylothorax: in patients with a chylothorax from malignant disruption of the thoracic duct, radiotherapy has been shown to be effective in approximately two-thirds of patients with lymphoma and half of those with metastatic carcinoma. Should radiotherapy fail, other treatment options listed previously should be considered. For chylothorax secondary to central venous thrombosis, fibrinolytic therapy to unblock the venous drainage can improve the chylothorax. Likewise, transjugular intrahepatic portosystemic shunting has been successfully used to treat chylothorax and chyloascites from cirrhosis. Special consideration for fetal or neonatal chylothorax: congenital chylothorax in utero should be drained under ultrasound guidance and maternal dietary fat restriction initiated. If the chylothorax reaccumulates, pleuroamniotic shunting or pleurodesis have been shown to be useful. If the congenital chylothorax is diagnosed after birth, repeated thoracentesis should be performed to prevent respiratory failure. Octreotide may be beneficial. If these measures fail, a pleuroperitoneal shunt or thoracic duct ligation should be considered.

Pseudochylothorax Pseudochylothorax or chyloform pleural effusion is rare and is characterized by a high content of cholesterol or lecithin–globulin complexes in the pleural fluid. Pathogenesis and Etiology

The leading causes of a pseudochylothorax are tuberculous pleuritis (54%) and rheumatoid pleuritis (9%). Other, rare, etiologies include paragonimiasis, trauma, and surgery. Many patients with tuberculosis (TB)-related pseudochylothoraces have been treated with artificial pneumothorax for pulmonary TB and have chronic pleural effusions secondary to the atelectatic lung. Pleural fluid from TB-related pseudochylothoraces is usually culture negative. The exact pathogenesis of pseudochylothorax is poorly understood. The typical patient has a longstanding exudative effusion and a thickened pleura.

Cholesterol may accumulate in the pleural cavity from plasma extravasation during the initial inflammatory phase of the underlying pleural disease. Alternatively, it can arise from degeneration of erythrocytes and leukocytes within the pleural space. The grossly thickened pleura may contribute to the retention of cholesterol by blocking its exit from the pleural parietal lymphatics. Clinical Manifestations and Diagnosis

The pleural effusion is usually, although not inevitably, chronic (mean duration, 5 years). Most patients are asymptomatic. Exertional dyspnea due to restrictive pulmonary function defect or symptoms associated with the underlying disease may also be present. The pleural fluid is milky or turbid and cannot be differentiated from that of chylothorax or empyema visually. In pseudochylothorax, the pleural fluid remains turbid after centrifugation, and the pleural fluid cholesterol level is usually 4250 mg dl 1 (6.45 mmol l 1) and the fluid:serum triglyceride ratio 41. Microscopic examination of the fluid may reveal cholesterol crystals with a rhomboid appearance (Figure 2); if present, it is considered diagnostic of pseudochylothorax. A CT scan of the thorax commonly demonstrates thickened or calcified pleura and often a layering of fat in nondependent sites of the hemithorax. Treatment

Since the majority of pseudochylothoraces are due to TB, it is reasonable to initiate antituberculous chemotherapy in patients with a positive Mantoux test and a history compatible with previous tuberculous infection who have not received appropriate therapy. A therapeutic thoracentesis may help alleviate dyspnea but can be difficult because the pleura is exceedingly thick and the intrapleural pressure strongly negative. As such, the effusions often reaccumulate. Decortication should only be considered in severely symptomatic patients in whom the underlying lung is believed to be functional.

Lymphangioleiomyomatosis Lymphangioleiomyomatosis (LAM) affects predominantly females of reproductive age and is characterized by progressive cystic lung disease complicated by recurrent pneumothoraces and chylous effusions. Details of its pathophysiology can be found elsewhere in this encyclopedia (see Interstitial Lung Disease: Lymphangioleiomyomatosis). The majority of patients with LAM (60–81%) will develop a pneumothorax during the course of their

392 PLEURAL EFFUSIONS / Chylothorax, Pseudochylothorax, LAM, and Yellow Nail Syndrome

Figure 2 Pleural fluid from a patient with pseudochylothorax. (a) The milky appearance of the pleural fluid persisted after the centrifugation. (b) The presence of cholesterol crystals in microscopic examination of the pleural fluid established the diagnosis.

disease. Chylothorax is also common and affects up to 30% of patients. Chylothorax is thought to be the result of lymphatic obstruction (from perilymphatic smooth muscle proliferation) and of infiltration of the mediastinal lymph nodes by immature smooth muscle cells. Occasionally, chylothorax accumulates secondary to transdiaphragmatic flow of chylous ascites. LAM should be considered in every woman of reproductive age who presents with a pneumothorax or chylothorax. A CT scan is usually diagnostic for LAM (Figure 3). For the chylothoraces, simple thoracentesis for symptomatic relief is adequate in some patients, but most eventually require pleurodesis or thoracic duct ligation. Because up to 19% of patients with LAM may proceed to lung transplantation, the management of their chylothoraces (and pneumothoraces) should be made in conjunction with the transplant team. Increased bleeding during lung transplant, and a higher resultant mortality, has been described in patients with LAM. This is presumed to be from pleural adhesions, which are often highly vascularized, from associated pleural diseases.

Yellow Nail Syndrome Yellow nail syndrome (YNS) is a rare entity characterized by yellow nails, lymphedema, and recurrent pleural effusions. The complete triad is seen only in

one-third of patients, and each manifestation of the syndrome may appear at a different time. Therefore, the presence of two of the three symptoms is considered sufficient to establish the diagnosis. YNS has been associated with malignancy and autoimmune diseases. A case of spontaneous resolution of yellow nails after cancer treatment has been reported. The clinical onset varies from birth to late adult life. Nail changes are often the earliest and most common symptom: The nails are slow growing, excessively curved, with a yellowish discoloration. Pleural effusions are present in up to 36% of patients, are often bilateral, and tend to recur. The pleural fluid is usually lymphocytic, exudative, and has a relatively low lactate dehydrogenase (LDH) level. Chylothorax and chylous ascites have been reported. Bronchiectasis, rhinosinusitis, and, rarely, pericardial effusion may also occur with YNS. The pathogenesis of YNS is unknown. It is believed that the main defects involve anatomical (congenital hypoplasia) or functional abnormalities of the lymphatic vessels. Pleural effusions are thought to result from impaired drainage of the pleural space by hypoplastic lymphatic vessels. It is assumed that the pathogenesis involves a ‘triggering event’, such as an inflammatory pleuritis, that increases pleural fluid production, which overwhelms the draining capacity of marginally effective lymphatics. The optimal treatment for YNS is unknown. Symptomatic effusions should be drained. Since

PLEURAL EFFUSIONS / Hemothorax 393 Hillerdal G (1997) Chylothorax and pseudochylothorax. European Respiratory Journal 10: 1157–1162. Hillerdal G (2003) Effusions from lymphatic disruptions. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 362– 369. London: Arnold. Light RW (ed.) (2001) Chylothorax and pseudochylothorax. In: Pleural Diseases, 4th edn., pp. 327–343. Philadelphia: Lippincott Williams & Wilkins. Light RW and Lee YCG (2005) Pneumothorax, chylothorax, hemothorax and fibrothorax. In: Murray J, Nadel J, Mason R, and Broaddus VC (eds.) Textbook of Respiratory Diseases, 4th edn., pp. 1961–1988. Philadelphia: Saunders. Perkett EA (2003) Pediatric pleural diseases. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 475–480. London: Arnold. Romero S (2000) Nontraumatic chylothorax. Current Opinion in Pulmonary Medicine 6: 287–291. Ryu JH, Doerr CH, Fisher SD, et al. (2003) Chylothorax in lymphangioleiomyomatosis. Chest 123: 623–627. Strausser JL and Flye MW (1981) Management of nontraumatic chylothorax. Annals of Thoracic Surgery 31: 520–526. Sullivan EJ (1998) Lymphangioleiomyomatosis: a review. Chest 114: 1689–1703.

Hemothorax J John and S Idell, University of Texas Health Center, Tyler, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract

Figure 3 (a) A posteroanterior radiograph of a woman with LAM demonstrating hyperinflation, diffuse reticular shadows, and small bilateral pleural effusions. (b) CT scan of the right hemithorax with the characteristic cystic lesions of the lung parenchyma and a pleural effusion. Diagnostic thoracentesis revealed a chylothorax. Kindly provided by Dr Gougoulianis, University of Thessaly, Larisa, Greece.

pleural effusions typically reaccumulate, the majority of patients will require a pleurodesis. See also: Interstitial Lung Disease: Lymphangioleiomyomatosis. Pleural Effusions: Overview; Pleural Fluid Analysis, Thoracentesis, Biopsy and Chest Tube.

Further Reading Al-Zubairy SA and Al-Jazairi AS (2003) Octreotide as a therapeutic option for management of chylothorax. Annals of Pharmacotherapy 37: 679–682. Browse NL, Allen DR, and Wilson NM (1997) Management of chylothorax. British Journal of Surgery 84: 1711–1716. Cope C (2004) Management of chylothorax via percutaneous embolization. Current Opinion in Pulmonary Medicine 10: 311–314.

Hemothorax is a collection of bloody fluid in the pleural space with a hematocrit at least 50% of the peripheral blood. Hemothorax is most commonly caused by trauma and rarely due to malignancy or pulmonary embolism. Hemothoraces are commonly encountered in clinical practice and may need to be drained depending on their size and effect on gas exchange. Diagnosis hinges on the clinical setting and confirmation by pleural fluid analysis. Apposition of the parietal and visceral pleura by drainage may arrest intrapleural bleeding via tamponade. Thoracoscopy and thoracotomy may be required if drainage alone is unsuccessful. The pathogenesis of large hemothoraces can involve progressive organization and fibrosis leading to lung restriction, arguing for the removal of this fibrotic encasement. Intrapleural thrombolytic therapy is an alternative approach to address organizing hemothoraces.

The pleural space is a potential compartment that separates the lung from the ribcage, the diaphragm, and mediastinum. It is lined by the parietal and visceral pleura and normally contains a thin layer of fluid measuring approximately 5–15 ml. A hemothorax is typically a bloody exudative effusion. Laennec first described spontaneous hemothorax in 1819. A pleural fluid hematocrit representing greater than 50% of the peripheral hematocrit defines the presence of a hemothorax. Blood-tinged pleural fluids containing lesser amounts are termed hemorrhagic effusions.

394 PLEURAL EFFUSIONS / Hemothorax

Etiology The most common cause of hemothorax is trauma. Malignancies including malignant mesothelioma and pulmonary embolism cause hemothoraces less commonly. Rare causes include bleeding diatheses, catamenial hemopneumothorax, aortic dissection, postcardiac injury syndrome (PCIS), benign asbestos pleural effusion (BAPE), and uremia. Trauma may be penetrating or nonpenetrating. A fractured rib may lacerate the pulmonary parenchyma and lacerate vessels from the pulmonary or bronchial circulation. Laceration of the subclavian vessels during central line placement or of the intercostal vessels during thoracentesis may be iatrogenic causes of hemothorax. Lung and breast cancer along with lymphoma constitute the most common causes of a malignant effusion and may be associated with hemothorax. Pulmonary embolism must be considered in any effusion, including hemothorax, where the cause is unclear. Hemothorax present for more than 1 year is unlikely to be malignant. It may be due to recurrent embolism or a rare cause of hemothorax.

Pathology The pathologic hallmark of hemothorax is the presence of blood in the pleural space. Blood that enters the pleural space may be diluted by pleural fluid that is already present or results due to exudative pleuritis, which can decrease the pleural fluid hematocrit. Over the course of days, such collections may clot, organize, and ultimately remodel with scar formation.

Clinical Features Clinical presentation may be due to the presence of an effusion, blood loss into the pleural space, or reflect the etiology of the hemothorax. Symptoms due to effusion are detailed in Pleural Effusions: Overview. Patients may be asymptomatic, but most present with chest pain, cough, or shortness of breath. The quality of pain may be pleuritic. Pain may be referred to the ipsilateral shoulder if the diaphragmatic pleura on the same side is involved. Cough is usually nonproductive and attributable to reflex stimulation of receptors in the atelectatic lung segments. Shortness of breath depends on the volume of effusion and the presence of underlying lung or cardiovascular disease. Fever may occur due to embolism, malignancy, infection of a hemothorax, or underlying pneumonia. PCIS occurs with recent cardiac surgery and presents with fever, dyspnea, and chest pain. Catamenial hemothorax is usually right sided and occurs during menses. Patients with a bleeding diathesis may bleed from other sites.

A history of trauma or a thoracic procedure is of diagnostic importance, as is that of underlying malignancy. In particular, malignant mesothelioma should be considered with a history of remote, more than 20 years previous, asbestos exposure. In the first two decades after exposure, the most common entity attributable to asbestos is BAPE. Patients with pulmonary embolism may present with pleuritic pain and dyspnea that is out of proportion to the volume of the effusion. Blood loss may be associated with dizziness and fatigue. Pneumonias are a rare cause of hemothorax. Hemothorax is rarely associated with inhalational anthrax, tuberculosis, tularemia, plague, and other pneumonias, especially if associated with dissemination and in an immunocompromised host. On examination, the patient may be tachycardic or tachypneic, depending on the volume of the hemothorax. Extensive blood loss may cause orthostasis and shock. Hemolysis of the lost blood may cause jaundice. The trachea may be shifted to the contralateral side in the presence of a large hemothorax. Breath sounds and vocal resonance will be diminished on auscultation. Patients with trauma may have a flail chest. Patients with malignancy may have cachexia, clubbing, or lymphadenopathy. PCIS may be associated with a pleuropericardial rub if the effusion is small. On chest X-ray, hemothorax, if not loculated, will obliterate the posterior and lateral costophrenic angles with approximately 200 and 500 ml collection of fluid, respectively. Larger effusions may shift the mediastinum to the contralateral side. If the mediastinum does not shift, then one should suspect fixation due to carcinoma or atelectasis of the underlying lung. Smaller amounts of fluid may be detected on a lateral decubitus radiograph. Chest trauma may be associated with a fractured rib, pneumothorax, pneumomediastinum, pulmonary contusion, or subcutaneous emphysema. Hemothorax from pulmonary embolism is usually small and seen on the initial radiograph. Hemothorax associated with malignancy may occur with radiographic evidence of a mass, adenopathy, bronchial obstruction, or atelectasis. Patients with hemothorax and malignant mesothelioma may exhibit pleural thickening or the radiographic suggestion of lung encasement in association with hemothorax. A computed tomography (CT) scan of the chest will delineate the previously mentioned radiographic features in three dimensions and at a higher resolution. Contrast will enhance hemothoraces in a CT scan. Magnetic resonance imaging may likewise indicate the presence of blood. Ultrasound of the thorax can be used to localize, quantitate, and show loculation of a hemothorax. Laboratory evaluation may reveal the presence of anemia, suggestion of a bleeding diathesis

PLEURAL EFFUSIONS / Hemothorax 395

on coagulation profile, or other findings referable to the specific underlying cause of the hemothorax.

Pathogenesis Traumatic hemothorax is associated with vascular disruption. Hemothorax in malignancy may be due to tumor invasion into blood vessels and increased vascular permeability. Hemorrhage in embolism is due to ischemia of the visceral pleura that is likewise associated with increased vascular permeability and red cell diapedesis. PCIS is an immunological response to trauma or infarction of the myopericardium. Catamenial hemothorax occurs from ectopically situated endometrial tissue in the pleural space that ‘hemorrhages’ during menses. Hemothoraces that are sizeable and persist in the pleural space can organize. In this process, the intrathoracic blood clots. The coagulum forms a transitional neomatrix that is invaded by inflammatory cells and fibroblasts in a process that recapitulates wound healing. Ultimately, formation of mature scar tissue can occur, leading to clinical sequelae attributable to restriction of the underlying lung (Figure 1).

Animal Models Diverse models of pleural effusion have been used to determine the volume of pleural fluid that is amenable to detection by various radiological techniques. A swine model of penetrating trauma has evaluated the efficacy of different aspiration systems for evacuating a hemothorax.

Management and Current Therapy Diagnosis of a hemothorax is usually established by thoracentesis. Ultrasound or CT guidance may be obtained for thoracentesis, especially if the fluid is loculated. Because hemothorax is exudative, the lactate dehydrogenase and protein content is elevated in pleural fluid. A reddish tint by itself has no diagnostic significance, but at counts of 100,000 red cells per milliliter the fluid is frankly hemorrhagic. Pleural fluid, which is bloody but then clears, is due to a traumatic thoracentesis. Fluid from a traumatic tap Trauma, Malignancy or Other Pleural Insult ⇓ Hemothorax ⇓ Coagulation and Formation of a Fibrinous Transitional Matrix ⇓ Organization and Remodeling of the Transitional Matrix ⇓ Pleural Scar Formation Figure 1 Pathogenesis of fibrothorax.

will clot immediately and will have a clear supernatant. The supernatant will be reddish in a hemothorax if there has been time for hemolysis to occur. Bloody pleural aspirates should be submitted for hematocrit estimation. Pleural fluid hematocrit o1% is a non-specific finding. Hematocrit between 1 and 25% of the hematocrit of peripheral blood is consistent with trauma, embolism, or malignancy. Pleural fluid hematocrit is at least 50% of the peripheral blood in a hemothorax. A hemorrhagic effusion in the absence of trauma should heighten the suspicion for malignancy. However, a pleural fluid hematocrit 450% of the peripheral hematocrit is unusual in malignancies. Hematocrit similar to peripheral blood is consistent with trauma. Treatment of a hemothorax depends largely on its etiology (Figure 2). Patients with a large hemothorax, especially after trauma, may be unstable and require resuscitation. It is not mandatory that a hemothorax be evacuated because blood is usually reabsorbed from the pleural space. However, tube thoracostomy is indicated if intrathoracic bleeding is 1.5 l per day; 100–200 ml h 1 for 12 h; necessitates blood transfusion or causes hemodynamic instability. Chest tube drainage helps quantitation of the bleeding and allows the parietal and visceral layers of the pleura to approximate and tamponade the bleeding site. Tamponade by approximation of the lung with the chest wall following pleural drainage of blood may be useful, especially with bleeding from the bronchial or intercostal vessels under systemic pressure. The hematocrit is monitored serially and blood transfused depending on the severity of blood loss and the accompanying hemodynamic status. Blood from the pleural space in nonpenetrating trauma may be autotransfused. Stable hemothoraces may be monitored or aspirated depending on the clinical scenario and may need tube placement only when there is nonresolution or suspicion of pleural infection. In approximately 10% of trauma patients, there will be increasing opacification of the hemithorax at 48 h. CT scan will help to resolve whether the opacification is due to parenchymal contusion, clotted blood in the pleural space, or fibrothorax. Thoracoscopy or thoracotomy may be needed to control bleeding in an acute setting. In a subacute setting, thoracoscopy is performed when there is 500 ml of clotted blood or opacification of one-third of the hemithorax or fibrin encasement. Angiographic embolization of both the bronchial and the intercostal circulation has been achieved in certain centers, especially in patients who are not surgical candidates. Thoracotomy is undertaken if there is bronchial disruption, failure of thoracoscopy to contain the bleeder, bleeders are located near the central portions of the lung or in the

396 PLEURAL EFFUSIONS / Hemothorax Traumatic hemothorax

Bleeding > 1.5 l d–1 or 100−200 ml h–1 or hypotension or transfusion

Stable effusion No hypotension No transfusion

Monitor and aspirate

Nonresolution

Resolution

Tube placement

Nonresolution

Resolution

CT chest

.500 ml blood clot, Opacification of .1/3 of the hemithorax

Parenchymal contusion, <500 ml clotted blood, Opacification of <1/3 of the hemithorax

Thoracoscopy

Nonresolution

Resolution

Monitor to resolution

Thoracotomy Figure 2 Treatment of hemothorax.

mediastinum, or failure of tube thoracotomy to adequately drain the pleural space and re-expand the lung. Thoracoscopy obviates the need for thoracotomy in 60% of patients. Adequate drainage of large hemothoraces is essential to prevent development of a fibrothorax. Thrombolytic therapy has been used to clear large organizing hemothoraces but should not be used in the setting of known or recent intrapleural hemorrhage or in the presence of other contraindications to the use of thrombolytic therapy, as described in the American Heart Association’s Advanced Cardiac Life Support guidelines. Mortality and length of stay in the intensive care unit and ventilator time increase with delayed evacuation of hemothorax and formation of fibrothorax or empyema. In selected circumstances, caveats apply. In patients with hemothorax and malignancy, radiation to the

hemithorax usually is contraindicated and chemotherapy is usually not very effective. Hemothorax associated with embolism is not a contraindication for anticoagulation. The hemothorax usually resolves spontaneously but can persist beyond 10 days when associated with pulmonary infarction. Recurrence of effusion in an embolism usually suggests re-embolization, an alternative diagnosis, or hemorrhage due to anticoagulation. If the hemothorax increases in size and the hematocrit is more than 50% that of the peripheral blood, then bleeding is likely due to anticoagulation. Anticoagulation should then be discontinued and chest tube drainage instituted. An infected hemothorax is treated by drainage and antibiotics. Catamenial hemothorax is treated with hormonal manipulation. PCIS is usually self-limited and may be treated with aspirin or nonsteroidal

PLEURAL SPACE 397

anti-inflammatory drugs. If the effusion is large and causes dyspnea, then it must be evacuated. Steroids may be added to limit inflammation and preclude graft occlusion. See also: Mesothelioma, Malignant. Pleural Effusions: Overview; Pleural Fluid, Transudate and Exudate; Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube; Malignant Pleural Effusions; Postsurgical Effusions; Pleural Fibrosis. Pulmonary Thromboembolism: Pulmonary Emboli and Pulmonary Infarcts.

Further Reading Abdel-Razek H, Qari M, Kristensen J, et al. (2004) GCC Thrombosis Study Group: Guidelines for diagnosis and treatment of deep venous thrombosis and pulmonary embolism. Methods in Molecular Medicine 93: 267–292. Ahmed N and Jones D (2004) Video-assisted thoracic surgery: State of the art in trauma care. Injury 35: 479–489. Beckh S, Bo¨lcskei PL, and Lessnau K-D (2002) Real-time ultrasonography. A comprehensive review for the pulmonologist. Chest 122: 1759–1773.

British Thoracic Society Standards of Care Committee Pulmonary Embolism Guidelines Development Group (2003) British thoracic society guidelines for the management of suspected acute pulmonary embolism. Thorax 58: 470–484. Chan O and Hiorns M (1996) Chest trauma. European Journal of Radiology 23: 23–24. Cugell DW and Kamp DW (2004) Asbestos and the pleura. A review. Chest 125: 1103–1117. Gallardo X, Castaner E, and Mata JM (2000) Benign pleural diseases. European Journal of Radiology 34: 87–97. Goldhaber SZ (2004) Pulmonary embolism. Lancet 363: 1295– 1305. Idell S (1995) Coagulation, fibrinolysis and fibrin deposition in lung injury and repair. In: Phan SH and Thrall RS (eds.) Lung Biology in Health and Disease – Pulmonary Fibrosis, pp. 743– 776. New York: Dekker. Landreneau RJ, Keenan RJ, Hazelrigg SR, Mack MJ, and Naunheim KS (1995) Thoracoscopy for management of empyema and hemothorax. Chest 109: 18–24. Parker C and Neville E (2003) Lung cancer 8. Management of malignant mesothelioma. Thorax 58: 809–813. Ziedalski TM, Sankaranarayanan V, and Chitkara RK (2004) Thoracic endometriosis. A case report and literature review. Journal of Thoracic and Cardiovascular Surgery 127: 1513– 1514.

PLEURAL SPACE Y C G Lee, University College London, London, UK C A Clelland, John Radcliffe Hospital, Oxford, UK N M Rahman, Oxford Centre for Respiratory Medicine, Oxford, UK

recruited efficiently during pleural inflammation or infection. Cancer cells can invade the pleura and often lead to increased vascular permeability and accumulation of malignant effusions.

& 2006 Elsevier Ltd. All rights reserved.

Anatomy, Histology, and Structure Abstract

Introduction

The pleural cavity consists of a double-layered membrane lining the inside of the thoracic cavity (parietal pleura) and the outside of the lung surface (visceral pleura). Each pleural membrane consists of a layer of mesothelial cells lined with a brush border of microvilli, and several noncellular layers. The structure of the mesothelial cell and the noncellular layers are variable according to the underlying tissue and the amount of movement of the chest wall. Absorption of pleural fluid occurs via the parietal pleura through direct communications (stomata) between the pleural space and a complex underlying lymphatic network. These allow passage of large molecules, including cells, between the pleural space and the systemic circulation via the parietal lymphatics. A constant layer of pleural fluid is maintained between the visceral and parietal pleura, allowing smooth lung movement and elasticity within the thoracic cage. Fluid may accumulate within the pleural space in pathologic states via a variety of mechanisms which either increase production of pleural fluid or decrease its absorption, or both. Air may enter the pleural space (pneumothorax) from the lung via rupture of blebs through the visceral pleura; or from outside the chest cavity when the pleural space is penetrated accidentally or iatrogenically. The mesothelial cell is multipotent and is the predominant cell type in the pleural cavity. In the normal state, there are few leukocytes in the pleural cavity, but inflammatory cells can be

The pleura is an intriguing tissue with significant interspecies variation: its precise structure and function is not fully understood. In humans the pleural cavities are separated by the mediastinum. Many other species, such as the mouse, do not have a complete mediastinal separation such that fluid and air can freely move between the left and right pleural cavities. Some large animals such as the bison (American buffalo) are also known to have a single pleural cavity. On the other hand, the elephant does not have a pleural cavity. While the fetal elephant has a normal pleural space, in late gestation a sheet of dense connective tissue replaces the parietal pleura and the two pleural membranes are separated by loose connective tissue that allows sliding of the lung over the chest wall. Such variations in the pleural structure in different species have not been explained. This article describes the anatomy and function of the pleural cavity in humans.

398 PLEURAL SPACE Macroscopic Anatomy

The pleura consists of a double-layered serous membrane overlying the inner surface of the thoracic cage and the outer surface of the lung. Between these two delicate membranes lies the pleural cavity, a sealed space maintained B10–20 mm across. In humans, the left and right pleural cavities are separated from each other and from the pericardial space. The pleural cavities contain the visceral pleura, overlying the entire lung surface, and the parietal pleura, overlying the inner surface of the entire thoracic cage, including the mediastinum and diaphragm. The area of the entire pleura is estimated to be 2000 cm2 in an average adult male. The two pleural membranes coalesce at the lung hilae, where they are penetrated by the major airways and pulmonary vessels. The visceral pleura tightly adheres to the lung surface throughout the thorax and extends deep within the interlobar fissures. The parietal pleura is anatomically divided into four parts: *

* *

*

costal pleura – overlying ribs, intercostal muscles, costal cartilage, and sternum; mediastinal pleura; cervical pleura – extending above the 1st rib by 2–3 cm over the medial end of the clavicle; and diaphragmatic pleura.

The inferior boundary of the parietal pleura mirrors the lower border of the thoracic cage, but may extend beyond the costal surface, specifically at the right lower sternal region and at the posterior junction of ribs and vertebra bilaterally. Several structures (e.g., the hilae and sometimes the great veins) within the thoracic cavity acquire a double layer of parietal pleura during embryological development. Such a double layer is pulled into the thorax by the developing lung, and extends from the lung hilum vertically downward to the diaphragm on both sides – these form the pulmonary ligaments. They are of importance surgically as they may contain lymphatics, tumor, or vessels. Their presence may prevent torsion of the lower lobes. Microscopic Anatomy

Layers of the pleural membrane Both visceral and parietal pleura in humans are approximately 40 mm thick. Between pleural surface and underlying tissue, five layers are identified histologically, consisting of a single-cellular layer and four subcellular layers, as follows: 1. a monolayer of mesothelial cells; 2. the basal lamina and a thin connective tissue layer;

3. a thin superficial elastic layer, often merged with the second layer; 4. a loose connective tissue layer, containing nerves, blood vessels, and lymphatics; and 5. a deep fibroelastic layer, often fused to the underlying tissue (Figure 1). Mesothelial cells Mesothelial cell is the predominant cell type in the pleural cavity (see Mesothelial Cells). No significant differences have been identified between the mesothelial cells in the visceral or parietal pleura, or among those in the pleural, peritoneal, or pericardial cavities. Mesothelial cells can vary in shape (from flat to cuboidal) and in size, ranging from approximately 10 to 50 mm in diameter and from 1 to over 4 mm in thickness. Under electron microscopy, multiple pinocytic vesicles can be identified within the mesothelial cells, implying secretion and absorption processes. Evidence of metabolic activity is apparent, with abundant polyribosomes, glycogen granules, and mitochondria. Each cell is covered at the pleural surface with a carpet of microvilli, 0.1 mm in diameter and 3 mm long (Figure 2). Their density varies according to position within the thoracic cavity, with a high density in the inferior parts of the thorax (i.e., the most actively moving parts of the lung) and a higher density in visceral than in parietal pleura at the same thoracic level. Mesothelial cells are adherent to one another at the apical surface via tight junctions. At the basal surface, the cells are more loosely associated, although the basal portions are often seen to overlap. The cells slide over one another during the respiratory cycle; therefore, at full inspiration the overlap disappears completely. Mesothelial cells are multipotent with functions in extracellular matrix synthesis, phagocytosis, inflammatory cytokine production, etc. A detailed discussion of the biology of the mesothelial cells can be found in Mesothelial Cells (see also Tables 1 and 2). Differences in pleural microstructure The parietal pleura demonstrates subtle alterations in its cellular and noncellular structure according to position within the thoracic cavity, reflecting structure of the underlying tissue and activity of the mesothelial cells. In apical areas, where the pleura is statically expanded with relatively little movement, mesothelial cells are flattened with few microvilli and the subcellular layers are poorly developed and hence difficult to differentiate. In lower thoracic areas, where the majority of chest-wall movement occurs, mesothelial cells are cuboidal, more numerous per unit

PLEURAL SPACE 399

Figure 1 (a) Visceral pleura showing a surface layer of mesothelium with underlying connective tissue including a layer of elastic tissue that is better displayed in (b), an elastic van Gieson stain. (c) The parietal pleura also has a covering of mesothelium and there is generally more underlying connective tissue here than in the visceral pleura.

area, have a high density of microvilli, and welldefined thick layers of collagen and elastic tissue. Pleura overlying rigid tissues – such as rib, central diaphragm, and intercostal muscles – contains flat mesothelial cells, few microvilli, and thin subcellular layers. The fifth fibroelastic layer is fused with the underlying structure, such as the periosteum. Where pleura overlies looser substructures, such as the mediastinum, mesothelial cells are cuboidal and prominent, subcellular layers are well defined, and the fifth fibroelastic layer is often absent. The fourth loose connective tissue layer is often merged with the underlying interstitial tissue.

the lung via bronchovascular bundles. There are more lymphatic vessels in the dependent parts of the lung, associated with higher intravascular pressures. Lymphatic plexuses within the parietal pleura are present in intercostal spaces and over the diaphragm, but absent over ribs. The costal pleura drains anteriorly into the internal mammary nodes and posteriorly into the intercostal lymph nodes posterior to the rib heads. Pleura from the lung apex drain into the cervical chain, while pleura lining the diaphragm drain into the anterior and posterior mediastinal nodes. Blood Supply

Lymphatic Drainage, Blood Supply, and Innervation Pleural Lymphatics

The visceral pleural lymphatic system consists of a superficial network of lymphatic capillaries and collecting vessels, forming a network along the pleural base of the secondary pulmonary lobule. Lymph flows from lymphatic capillaries toward the hilae of

The parietal pleura is supplied by the systemic arteries, according to region. The costal pleura is supplied by the intercostal and internal mammary arteries and the mediastinal pleura by the bronchial, upper diaphragmatic, internal mammary, and mediastinal arteries. The cervical pleura is supplied by the subclavian artery, whereas the internal mammary artery and aorta (via posterior mediastinal and inferior phrenic arteries) supply the diaphragmatic pleura.

400 PLEURAL SPACE

drainage of the visceral pleura is mostly via the pulmonary veins. Innervation

It is noteworthy that only the parietal, but not the visceral, pleura contains pain-carrying fibers. The parietal pleura is supplied by the intercostal nerves; any pain will be referred to the chest wall with the corresponding innervation. The exception is the central diaphragm, supplied by the phrenic nerve, resulting in referred pain to the ipsilateral shoulder. The visceral pleura is innervated by the vagus and sympathetic trunk and contains no pain-carrying fibers. Hence, pleuritic pain implies involvement of the parietal pleura.

Transport from the Pleural to Systemic Compartments

Figure 2 At electron microscopy the mesothelial cell surface is covered by long slender microvilli with high length/diameter ratios and desmosomes (arrowed) are present. Table 1 Maturation of mesothelial cells

Surface mesothelium Proliferating submesothelial cells Resting submesothelial cells

Low MW CK

High MW CK

Vimentin

þ þ

þ 

 þ





þ

The Pleurolymphatic Communication

MW, molecular weight. Table 2 Immunoprofile of mesothelial cells vs. epithelial cells pan CK BerEP4 MOC31 CK5 Calretinin Mesothelial cells Epithelial cells

þ þ

Small particles of o4 nm in size (including water) may pass freely between cells of the mesothelial monolayer, whereas moderate-sized particles are slowly but actively transported across the mesothelial cell by pinocytosis (e.g., carbon at 20 nm or ferritin at 11 nm). Large particles of 41000 nm may become trapped within mesothelial cells and are unable to cross the basement membrane. Studies using radioactive-labeled red blood cells and protein molecules have shown that large molecules can move from the pleural space into the systemic circulation rapidly. This implies the existence of a transport mechanism between pleura and systemic circulation which is now known to be mediated by the parietal lymphatic system.

 þ

 þ

þ 

þ 

Venous drainage follows arterial supply into the azygos vein and thence into the superior vena cava. The diaphragmatic pleura drains via the inferior phrenic veins into the inferior vena cava. The arterial supply of the visceral pleura in humans is controversial. In animals with thick visceral pleura similar to that of humans, the bronchial arteries provide the blood supply to the visceral pleura. This is believed to be the case for the visceral pleura in humans, although supply of the lung apex and its convex surface is debated. Venous

The parietal pleura plays the major role in the formation and removal of pleural fluid. Direct communications, known as stomata, exist between the pleural space and the underlying lymphatic network, allowing removal of large particles from the pleural space. Stomata are unique to the parietal pleura. The stomata are openings (2–6 mm in diameter) in the continuous sheet of overlying mesothelial cells, and tend to cluster in groups of 10 or more. On average, there are 100 stomata per cm2 of pleura, but a higher density has been observed in the anterior lower chest, the mediastinum and the diaphragm. In specimens of parietal pleura from older animals, large openings of up to 10
50 mm are seen where stomata are normally present, thought to represent areas where stomata have coalesced. Underlying each stoma is a network of interwoven connective tissue bundles – the membrana cribriformis. This network in turn forms the roof of a dilated lymphatic space, the lacuna. The lacunae

PLEURAL SPACE 401

drain into lymphatic vessels via a system of valves to ensure one way flow. During inspiration, the stomata become stretched to over 10 mm allowing passage of large structures, and the lacunae similarly stretch open and fill. During expiration, the lacunae are compressed and empty into lymphatic capillaries, with unidirectional flow maintained by valves. Present in association with some collections of stomata are modified cuboidal mesothelial cells, surrounded by aggregates of immune cells, including lymphocytes, plasma cell, and mononuclear cells, around a central lymphatic vessel. These are known as Kampmeier’s foci, visible on electron microscopy as elevated mounds. Mesothelial cells in these areas are in a state of activation, and the foci are thought to be analogous to Peyer’s patches in the gut, as a mechanism of local host defense.

The Pleura in Health Normal Function and Content of the Pleura

The pleural space is a real rather than potential space with experiments in sheep demonstrating no direct contact of the parietal and visceral pleura. The pleura permits friction-free movement of the lungs within the relatively rigid thorax, and facilitates the development of positive and negative intrapleural pressure during the respiratory cycle. Mesothelial cells, with its carpet of microvilli, vastly increase surface area and allow secretion and maintenance of a film of glycoproteins and hyaluronic acid, acting as a lubricating fluid layer between the two pleural surfaces. The composition of the pleural fluid as well as its formation and turnover, in health and in disease states, has been detailed in Pleural Effusions: Pleural Fluid, Transudate and Exudate. Pleural structure alters according to position within the thoracic cavity, reflecting specialization in response to different degrees of mechanical stress and different underlying tissues.

Pleural Disorders Pneumothorax

Stretched-out visceral pleura in the statically expanded lung apices are prone to bleb and bullae formation, even in nonsmoking individuals. These are thought to be an important factor in predisposition to primary spontaneous pneumothorax. Due to the anatomical boundaries of the parietal pleura, iatrogenic pneumothorax may result from insertion of subclavian central venous catheters, damaging the pleura above the first rib. Due to pleural extension below the costal margin inferiorly, pneumothorax may occur during attempted posterior access to upper abdominal organs.

Pleural Inflammation and Fibrosis

The pleural cavity is frequently invaded by undesirable agents (e.g., bacteria), but the pleural cavity is not under close surveillance by polymorphonuclear cells. In the normal state, there is only one leukocyte in the pleural fluid to every 10 million mesothelial cells in the pleura. Mesothelial cells, therefore, form the first line of defense in the pleural cavity. Experimental evidence suggests that they are capable of releasing chemokines (e.g., interleukin 8) promptly and effectively to recruit inflammatory cells (e.g., neutrophils), to initiate appropriate immune responses to eradicate the pathogens. Chronic inflammation develops with many pleural diseases and results in pleural fibrosis and thickening. While fibroblasts are conventionally regarded as the main cellular source of collagen and fibrosis, mesothelial cells are capable of producing collagen and are likely to hold an important role in pleural fibrosis. Under appropriate circumstances, mesothelial cells can transform into fibroblast-like cells (a process known as epithelial–mesenchymal transformation). The causes and pathology of pleural fibrosis are detailed in Pleural Effusions: Pleural Fibrosis. Pleural Effusion

A wide range of pulmonary or systemic diseases can affect the pleura, and commonly lead to the development of pleural effusions (see Pleural Effusions: Pleural Fluid, Transudate and Exudate). Pleural Malignancy

Cancer invasion of the pleural cavity is common in clinical practice, and often results in development of malignant pleural effusions (see Pleural Effusions: Malignant Pleural Effusions). Metastatic pleural disease accounts for most cases, with lung and breast being the most common primary cancer sites. Pleural mesothelioma, a primary pleural malignancy, often develops as a result of asbestos exposure. Metastatic carcinoma generally deposits first in the visceral pleura and migrates eventually onto the parietal surface. This contrasts mesothelioma, which originates from the parietal pleura before spreading to the visceral pleura. The pathology of pleural malignancies is further described in Pleural Effusions: Malignant Pleural Effusions.

What Is the Role of the Pleural Space? Interestingly, there is little evidence to support any essential role of the pleural space in humans. Although the pleural space can act as an escape route

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for unwanted substances (such as excess fluid in pulmonary edema) in the lung parenchyma, it is unlikely that the pleural space exists solely for such purpose. Studies on pleurodesis, the iatrogenic obliteration of the pleural space to prevent recurrent effusions or pneumothoraces, showed no significant limitations to pulmonary functions. Patients who received talc pleurodesis over 22 years ago for spontaneous pneumothoraces showed a higher incidence of pleural thickening but minimal restrictive changes in lung functions. Several small studies in humans and animals also revealed no significant impairment in lung volumes and gaseous exchange at rest or during exercise following pleurodesis. The fact that obliteration of the pleural space does not affect health and functioning supports the belief that the pleural space in humans serves no significant role. Why the pleural space exists in the first place and why it is preserved through evolution remain unanswered. See also: Mesothelial Cells. Mesothelioma, Malignant. Pleural Effusions: Overview; Pleural Fluid, Transu-

Pneumoconiosis

date and Exudate; Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube; Parapneumonic Effusion and Empyema; Malignant Pleural Effusions; Postsurgical Effusions; Pleural Fibrosis; Chylothorax, Psuedochylothorax, LAM, and Yellow Nail Syndrome; Hemothorax. Pneumothorax.

Further Reading Jones JS (2001) The pleura in health and disease. Lung 179: 397– 413. Lee KF and Olak J (1994) Anatomy and physiology of the pleural space. Chest Surgery Clinics of North America 4(3): 391–403. Lee YCG and Light RW (2001) How does pleurodesis affect lung function? Journal of Respiratory Diseases 22: 706. Miserocchi G (1997) Physiology and pathophysiology of pleural fluid turnover. European Respiratory Journal 10: 219–225. Mutsaers SE (2002) Mesothelial cells: their structure, function and role in serosal repair. Respirology 7: 171–191. Peng MJ and Wang NS (2003) Embryology and gross structure. In: Light RW and Lee YCG (eds.) Textbook of Pleural Disease. pp. 3–17. Arnold Publishers. Sahn SA (1988) State of the art: the pleura. American Review of Respiratory Diseases 138: 184–231. Wang NS (1998) Anatomy of the pleura. Clinics in Chest Medicine 19: 229–240.

see Occupational Diseases: Coal Workers’ Pneumoconiosis.

PNEUMONIA Contents

Overview and Epidemiology Atypical Community Acquired Pneumonia, Bacterial and Other Common Pathogens Fungal (Including Pathogens) Nosocomial Parasitic Mycobacterial Viral The Immunocompromised Host

Overview and Epidemiology R G Wunderink and G M Mutlu, Northwestern University Feinberg School of Medicine, Chicago, IL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pneumonia is the infection of the distal lower respiratory tract, principally the alveolar space, including the small bronchi and

bronchioles. Pneumonia results from the proliferation of microorganisms at these sites in combination with the host response to the presence of microorganisms. A variety of pneumonia syndromes can be defined based on microbial etiology, underlying host defenses, clinical presentation, and site of acquisition. Because of the non-specific nature of the signs and symptoms, the diagnosis of pneumonia rests disproportionately on the presence of an infiltrate on chest radiograph. The frequent lack of a microbiologic diagnosis and the non-specificity of signs, symptoms, and radiographic infiltrates leads to frequent consideration of pneumonia in the differential diagnosis of many other pulmonary diseases. The etiologic spectrum of all

PNEUMONIA / Overview and Epidemiology 403 types of pneumonia always includes the common communityacquired pathogens, with the spectrum broadening in response to either increasing immunocompromise or greater exposure to broad-spectrum antibiotics. While new or previously unrecognized pathogens can play a role in management decisions, the major issue in pneumonia is the appearance of antibiotic resistant strains of known common bacterial pathogens. Lack of even minimally effective agents is more the issue for nonbacterial pneumonias. Immunomodulation, in the form of less immunosuppressive cancer and transplant chemotherapy, immunization, or specific immunomodulatory drugs holds some hope for improving pneumonia outcome in the future.

Definitions Pneumonia is the infection of the distal lower respiratory tract, principally the alveolar space, including the small bronchi and bronchioles. Pneumonia results from the proliferation of microorganisms at these sites in combination with the host response to the presence of microorganisms. Proliferation alone does not necessarily result in pneumonia, since many patients on chronic mechanical ventilation will have quantitative colony counts thought to be diagnostic of pneumonia in other settings without any clinical manifestations of pneumonia. Presence of microorganisms without a corresponding host immune reaction is designated as colonization. Infection at the alveolar level distinguishes pneumonia from other respiratory tract infections with which it may be confused clinically. Infections of the conducting airways below the vocal cords are designated as tracheitis, bronchitis, or bronchiolitis, depending on the main area of involvement. These are at times combined as lower respiratory tract infections (LRTIs) to distinguish them from upper respiratory infections (URIs). The latter, which include laryngitis, pharyngitis, and rhinitis, are generally more common, more often viral, and generally less serious. Diphtheria, acute epiglottitis, and pertussis are some of the exceptions to these general rules that suggest careful distinction and accurate diagnosis are critical to clinical care. Even within the designation of pneumonia a variety of pneumonia syndromes occur (Table 1). These can be classified in a number of ways based on microbial etiology, underlying host defenses, and clinical presentation, among others. The most common initial grouping is by patient location at time of acquisition, that is, community-acquired or nosocomial pneumonia. Recently, a third category has been added: healthcare-associated pneumonia (HCAP) (Table 2). This category was needed to designate patients with pneumonia developing outside the hospital but whose pathogenesis, causative agents, and antibiotic resistance patterns were more consistent with nosocomial pathogens. The majority of these

Table 1 Pneumonia syndromes 1. Community-acquired pneumonia a. Typical (normal host) i. Bacterial ii. Viral iii. Fungal iv. Mycobacterial v. Parasitic/other b. Immunocompromised i. Acquired immunodeficiency syndrome ii. Other c. Aspiration pleuropneumonia/lung abscess 2. Healthcare-associated pneumonia (HCAP) a. Nursing home pneumonia b. Other 3. Nosocomial pneumonia a. Ventilator-associated b. Immunocompromised i. Transplant-related ii. Chemotherapy-related

Table 2 Healthcare-associated pneumonia Antibiotic therapy in the preceding 3 months Hospitalization for at least 2 days in the preceding 90 days Residence in a nursing home or extended care facility Home infusion therapy (including antibiotics) Chronic dialysis within 30 days Home wound care Immunocompromised state (disease or immunosuppressive therapy)

patients are from nursing homes but the group also includes chronic dialysis patients or others with frequent or recent contact with the medical system. Within each of these categories, patients with compromised immunity are usually discussed separately, since the spectrum of causative etiologies is much larger and therefore the diagnostic and therapeutic approaches differ from that of patients with overtly normal host immunity. Most of these pneumonia syndromes have separate entries in this encyclopedia.

Pathology The common component to the pathology of pneumonia is an inflammatory response within the lung parenchyma. The pattern of inflammatory response will vary by causative etiology. Mycobacterial and fungal infections result in a granulomatous response characterized by histiocytes and mononuclear cell proliferation. Most bacterial pneumonias result in a neutrophilic alveolitis while many viruses and obligate intracellular agents generate a lymphocytic alveolitis. The key to the development and pattern of pneumonia is the alveolar macrophage. The overwhelming

404 PNEUMONIA / Overview and Epidemiology

majority of microorganisms that reach the alveolar level are killed and/or cleared by the alveolar macrophage without inducing an inflammatory response. Simply to get to the alveolar level means that the microorganism has avoided all the mechanical and other host defense mechanisms of the upper respiratory tract and the conducting airways. These include the branching airway pattern and the mucociliary clearance mechanisms, mucosal immunoglobulins, complement, surfactant, lactoferrin, defensins, and other antimicrobial peptides. Given the microbial and particle challenge presented to the respiratory tract on a daily basis, these mechanisms are highly efficient at preventing an overwhelming challenge to the alveolar macrophage. However, either because of the virulence or the volume of microorganisms that it is exposed to, the alveolar macrophage can be overwhelmed. The response is release of a variety of mediators that elicit an inflammatory response appropriate for the particular challenge. The inflammatory response, rather than shear numbers of microorganisms, is responsible for the radiographic changes of pneumonia while the mediators released as part of the inflammatory response result in many of the clinical manifestations, such as fever and leukocytosis. Microorganisms can reach the alveolar level through two major mechanisms: aerosol deposition and aspiration of an infected bolus. The former is the mechanism for most viral, mycobacterial, and fungal infections while many of the bacterial pneumonias are a result of aspiration. Aspiration pleuropneumonia is a specific variant of aspiration pneumonia that is characteristically polymicrobial with prominent involvement of anaerobic bacteria. In this disorder, a large volume aspiration episode, usually from a seizure or alcoholic stupor, occurs days to weeks before presentation with the signs and symptoms of pneumonia. The aspirated bolus is usually enriched with anaerobic microorganisms by the presence of severe gingivitis, which explains the fact that this syndrome is very rare in the edentulous. The presence of anaerobes often results in a lung abscess and corresponding severe pleural reaction. Only rarely do two other mechanisms allow microorganisms to appear at the alveolar level. The first is hematogenous spread. While this clearly can occur with tricuspid endocarditis, the bronchocentric (rather than vasocentric) pathology of most bacterial pneumonias suggests that this is rare even in immunocompromised patients with other sites of infection. The second mechanism is direct spread from a contiguous site of infection, such as infradiaphragmatic infections or primary empyema.

The mechanism by which microorganisms reach the lower respiratory tract has important implications for prevention and infection control.

Diagnosis The diagnosis of pneumonia rests disproportionately on the presence of an infiltrate on chest radiograph. Pneumonia should be considered in any patient who has newly acquired respiratory symptoms (cough, sputum production, dyspnea, and chest pain), especially if accompanied by fever and auscultatory findings of abnormal breath sounds, particularly crackles. In the elderly and those with an inadequate immune response, pneumonia may present with nonrespiratory symptoms such as delirium, fatigue, failure to thrive, worsening of an underlying chronic illness, or a fall. In elderly patients, fever may be absent but tachypnea is usually present, along with an abnormal physical examination of the chest. Unfortunately, none of these symptoms is unique to pneumonia. Many of both symptoms and clinical signs can occur with other LRTIs. Hence the dependence on an abnormal chest radiograph for an accurate diagnosis. Unfortunately, the plain chest radiograph is not a reliable standard on which to base a diagnosis. Significant inter- and intraobserver variability exists in interpretation. The problem is particularly acute in patients suspected of ventilator-associated pneumonia (VAP) where not only multiple radiographic mimics of pneumonia exist but also simply changing ventilator settings will alter the radiograph. In patients with underlying pulmonary disease, differentiation between an infectious process and the deterioration of the underlying pulmonary disease is also often difficult. Occasionally, patients with pneumonia may have chest radiographs interpreted as normal. Most of these false-negative readings represent subtle findings overlooked in a routine interpretation. Interpretation can be improved by obtaining a standard upright posteroanterior (PA) and lateral views whenever possible. Use of more sensitive techniques, such as computed tomography (CT), can often detect these infiltrates. Occasionally, severe dehydration or lack of circulating neutrophils will prevent the radiographic changes of pneumonia until the underlying deficit is corrected. Despite its shortcomings, the radiograph can be useful in differentiating pneumonia from other conditions that may clinically mimic it. Moreover, the radiographic findings may suggest specific etiologies or conditions, including lung abscess or tuberculosis. The radiograph can also identify coexisting

PNEUMONIA / Overview and Epidemiology 405 Table 3 Differential diagnosis of pneumonia Community-acquired

Nosocomial

Pulmonary embolus/infarction Congestive heart failure Malignancy Bronchogenic with airway obstruction Bronchoalveolar cell Acute respiratory distress syndrome (ARDS) – nonpulmonary source Drug-induced lung disease Radiation pneumonitis Vasculitis Systemic lupus erythematosis Wegener’s granulomatosis Hypersensitivity pneumonitis Acute eosinophilic pneumonia Mucus plugs Graft-vs.-host disease

Atelectasis Atypical pulmonary edema Acute respiratory distress syndrome (ARDS)/diffuse alveolar damage Aspiration pneumonitis Lung contusion Drug-induced lung disease Pleural effusion Pulmonary hemorrhage Pulmonary infarction Bronchiolitis obliterans with organizing pneumonia (BOOP) Acute transplant rejection

conditions, such as bronchial obstruction or pleural effusion, and may be helpful in evaluation of the severity of illness by identifying multilobar involvement. The diagnosis of pneumonia is actually a two-step process. The first is determining that pneumonia is indeed present. However, for optimal antimicrobial management an etiologic diagnosis is also needed. Demonstrating the presence of a microorganism in culture or in stains of pathologic specimens are the classic methods to demonstrate the causative agent. Once again these methods are woefully inadequate for diagnosing pneumonia. The reason varies depending on the type of pneumonia. In communityacquired pneumonia (CAP), many of the causative microorganisms are not easily grown in culture and even with the most aggressive culture and extensive use of other diagnostic modalities, the etiologic diagnosis remains unknown in the majority of cases. In contrast, for nosocomial pneumonia, especially VAP, the high incidence of colonization frequently results in many false-positive cultures and polymicrobial cultures in monomicrobial pneumonias. For some of the chronic pneumonias such as tuberculosis and endemic fungal disease, the slow growth characteristics delay a definitive microbiologic diagnosis for weeks. The net result is that the antimicrobial treatment of pneumonia is often empiric. Clearly, new nonculturebased diagnostic methods are needed. This lack of a microbiologic diagnosis and the non-specificity of signs, symptoms, and radiographic infiltrates leads to frequent consideration of pneumonia in the differential diagnosis of many pulmonary diseases. Essentially any disorder that can cause signs or symptoms of inflammation and an abnormal radiographic infiltrate can be mistaken for pneumonia. The differential diagnosis varies somewhat

among the various CAP syndromes. Table 3 lists many of the entities in the differential diagnosis but is by no means exhaustive. The differential diagnosis expands further if prior radiographs and clinical information is not known. Bronchitis or even URIs can be mistaken for pneumonia in patients with previously unknown chronic infiltrates. Common combinations of disorders can also mimic pneumonia. For example, congestive heart failure can often be complicated by influenza. If the patient also has chronic obstructive pulmonary disease (COPD), asymmetric infiltrates may be seen. This problem is worst in patients with suspected VAP because of the poor radiographic technique and multiple other causes for fever or leukocytosis in the critically ill. Presence of pneumonia and a putative etiology is often assumed by an ‘appropriate’ response to antibiotics. However, many of the diseases in the differential may be self-limiting or respond to adjuvant therapy for pneumonia and therefore falsely suggest that the diagnosis is pneumonia. Conversely, 15% of CAP patients and up to 40–50% of cases of VAP due to specific etiologies do not respond even to specific therapy.

Epidemiology Pneumonia remains a common and serious illness despite the availability of potent antimicrobials and effective vaccines. In the US, pneumonia is the seventh leading cause of death and the number one cause of death from infection. A similar annual rate of approximately 50–60 cases per 100 000 is probably the case in most developed countries. However, data regarding its incidence are suboptimal because pneumonia is not a notifiable disease. The US data

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reflects a combination of pneumonia and influenza and there is a disproportionately high number of cases of CAP because of the reporting bias. Data on deaths due to other diseases may also be mixed up with those due to pneumonia. For example, before effective antiretroviral therapy became available, many deaths from the human immunodeficiency virus were attributed to pneumonia. Septicemia has now become the 10th leading cause of death in the US. Other data suggest that the source of infection in approximately 40% of septic shock patients is pneumonia. Therefore, there is both under-reporting and over-reporting of deaths from pneumonia. At the beginning of the twentieth century, pneumonia and tuberculosis were the leading causes of death in the US and most other developed nations. Deaths from heart disease did not exceed those of pneumonia in the US until the 1920–30s and deaths from pneumonia were exceeded by deaths from all malignancies by the 1930–40s. Death from pneumonia was rapidly falling even before effective antibiotics were discovered. Influenza pandemics would occasionally cause significant spikes in pneumonia mortality. This falling mortality without antibiotics represents improvements in sanitation and other public health measures and is paralleled by similar decreases in tuberculosis mortality. However, once effective antimicrobial therapy became routinely available, the parallels with tuberculosis disappeared. Effective antituberculous therapy has resulted in a persistent and progressive fall in mortality rates, such that tuberculosis is no longer in the top 25 causes of death and the age-adjusted mortality rate in the US is only 0.3 cases per 100 000 population. In contrast, after an initial increase in the slope of decline in pneumonia mortality, the age-adjusted mortality for pneumonia in the US reached a plateau and has not fallen significantly since approximately 1955. This persistent mortality suggests that while adequate antibiotic therapy is necessary, antibiotics alone are not sufficient to further decrease pneumonia mortality. Community-Acquired Pneumonia

The overall incidence of cases of CAP is approximately 10–12 per 1000 persons per year; however, this varies considerably with age, sex, race, and socioeconomic condition. The incidence of CAP is highest among the oldest and youngest members of the population. In the US, the highest incidence is in children younger than 4 years of age (12–18 cases per 1000 persons per year), which is similar to the rate reported in older individuals (age X60) in Europe (20 cases per 1000 persons per year). Pneumonia

accounts for nearly half of all deaths resulting from infectious disease in the geriatric population, and 90% of all deaths from respiratory tract infection occur in persons older than 64 years. However, while the case fatality rates are increased in both the very young and the elderly, more than 50% of deaths from pneumococcal pneumonia occur in patients aged 18–65 years. Approximately 4 million cases of CAP are estimated to occur annually in the US, resulting in 600 000 hospitalizations per year. Despite its frequency, pneumonia is the cause of acute LRTI symptoms in only a small minority of patients from the community. In one prospective study of LRTIs requiring antibiotics in adults seeking attention from a physician, only 12% of patients were found to have pneumonia. The majority (up to 80%) of patients with CAP are treated as an outpatient with a mortality rate of less than 1% in these patients. The need for hospitalization varies widely depending on characteristics of the healthcare system and physician preference. The incidence has ranged from 15% to 42% in both European and North American studies. The frequency of admission of patients at low risk of death by various prediction models varies greatly and physicians in general overestimate the risk of dying from CAP. Among patients who have pneumonia of sufficient severity to require admission to hospital, reported mortality rates range from 4% to as high as 37% and increase with increasing age. Generally, the higher mortality rates are found in the US where greater use of outpatient treatment and more aggressive treatment of terminally ill patients results in an overall sicker population of patients admitted for CAP. Admission to an intensive care unit (ICU) also varies dramatically by country and individual hospital. Generally, a higher percent of patients with CAP are admitted to the ICU in North America (415%) than in Europe (5–10%). In hospitals or areas with limited ICU resources/beds, a much higher number of CAP patients admitted to the ICU have either vasopressor-dependent septic shock or respiratory failure. The associated mortality is inversely proportional to the number admitted, mainly because the mortality in ventilated or septic shock patients is much greater. Mortality is as high as 50% in ICUs where nearly all patients are ventilated, while mortality decreases to 20–30% in ICUs where less than half of patients with severe CAP are ventilated. Healthcare-Associated Pneumonia

Defining HCAP as a new entity has complicated the epidemiology of CAP, since studies that did not

PNEUMONIA / Overview and Epidemiology 407

distinguish this subgroup would have classified these patients as CAP. While clearly recognized as being distinct from usual CAP, no extensive study of HCAP has been published. The majority of HCAP patients are from nursing homes or other chronic care facilities and a few epidemiologic studies have focused on this group of pneumonia patients. Whether other types of HCAP, for example, in patients receiving long-term home parenteral antibiotics or chronic hemodialysis patients, have a similar epidemiology to nursing home patients is unclear. Nursing home-acquired pneumonia (HAP) is the most common form of HCAP. Pneumonia has the highest mortality rate for any nursing home-acquired infection and frequently results in the transfer of cases to the hospital for management. The overall incidence of nursing home-acquired pneumonia varies from 0.3 to 2.5 episodes per 1000 days of resident care.

plateaus, such that patients undergoing long-term mechanical ventilation have substantially lower daily attack rates. Among nosocomial infections, pneumonia has the highest morbidity and mortality. Mortality in such patients is high, being estimated at 30–70% in different series. Only one third to one half of HAP deaths are thought to be due to the infection itself, the remainder of deaths being the result of comorbid disease. By contrast, in the setting of VAP, attributable mortality of VAP is on average lower, being only 27–33% in some studies. In fact, some authors have estimated that the development of VAP may not increase mortality at all after correction for confounding factors that independently influence mortality. While the attributable mortality of VAP may be debatable, development of VAP consistently increases length of stay in survivors by an average of 7–9 days per patient.

Nosocomial Pneumonia

Other Pneumonia Syndromes

Nosocomial pneumonia includes both hospital-acquired and ventilator-associated pneumonias. Hospital-acquired pneumonia is defined as pneumonia that occurs at least 48 h after admission to hospital and VAP is defined as pneumonia that occurs more than 48 h after intubation. Hospital-acquired pneumonia occurs in about 5–10 per 1000 hospitalized patients and complicates the course of as many as 20% of patients undergoing surgery. The incidence is significantly higher in patients undergoing mechanical ventilation, occurring in 10–25% of these patients. Crude rates for VAP range from 1% to 3% per day of intubation and mechanical ventilation. The cumulative incidence increases at this rate for approximately 3–4 weeks then

The epidemiology of other types of pneumonia is not as well defined. Pneumonia in the immunocompromised patient is dependent on the type of immunocompromise. Epidemiologic cues for other less common forms of CAP are listed in Table 4. Further information on the epidemiology of these types of pneumonia and specific microorganisms can be found elsewhere in this encyclopedia.

Etiology The etiology of pneumonia depends on several factors including patient’s immune status (i.e., immunocompetent vs. immunocompromised), recent antibiotic use, and location of the patient when pneumonia

Table 4 Conditions related to specific pathogens in patients with CAP Condition

Commonly encountered pathogens

Alcoholism COPD/smoker

Streptococcus pneumoniae ( þ DRSP), anaerobes, Gram-negative bacilli, tuberculosis Streptococcus pneumoniae, Hemophilus pneumoniae, Branhamella catarrhalis, Legionella spp. Anaerobes Histoplasma capsulatum Chlamydia psittaci, Histoplasma capsulatum, Cryptococcus neoformans, Influenza Francisella tularensis Coccidioidomycosis, Yersinia pestis Paracoccidiomycoses Burkholderia pseudomallei Coxiella burnetii (Q fever) Anaerobes Staphyloccus aureus, anaerobes, tuberculosis, Pneumocystis carinii DRSP, Pseudomonas aeruginosa

Poor dental hygiene Exposure to bats Exposure to birds Exposure to rabbits Travel to Southwest US Travel to Mexico and South America Travel to Southeast Asia Exposure to farm animals or cats Suspected large-volume aspiration Injection drug use Recent antibiotic therapy Check in CAP guidelines. DRSP, drug-resistant S. pneumoniae.

408 PNEUMONIA / Overview and Epidemiology Table 5 Common etiologies for pneumonia Community-acquired pneumonia (acute) Streptococcus pneumoniae Legionella spp. Hemophilus pneumoniae Chlamydophila pneumoniae Staphylococcus aureus Mycoplasma pneumoniae Klebsiella pneumoniae Influenza Respiratory syncytial virus Community-acquired pneumonia (subacute) Mycobacteria tuberculosis Histoplasmosis Blastomycoses Coccidiomycoses Nosocomial pneumonia (all acute CAP organisms plus) Pseudomonas aeruginosa Acinetobacter spp. Enterobacteriaceae Immunocompromised (all CAP and nosocomial plus) Pneumocystis jerovecii (carinii) Cytomegalovirus Herpes virus Varicella Aspergillus spp. Other fungi Nocardia asteroids Healthcare-associated pneumonia (HCAP) All of CAP and HAP

occurred (community acquired vs. nosocomial) (Table 5). However, the causative spectrum always includes the pathogens most frequently associated with acute CAP. VAP in patients recently admitted to the hospital and not receiving antibiotics will mimic the spectrum of acute CAP. Many pneumonias in immunocompromised patients are also caused by the typical CAP pathogens. Two factors combine to shift the spectrum away from that of typical CAP: (1) worsening immunocompromise; and (2) exposure to broad-spectrum antibiotic therapy, especially when prolonged. An example of the former is human immunodeficiency virus (HIV) disease. When helper T-lymphocyte counts (cluster determinant or CD4) are 4500 cells/mm3, usual CAP pathogens and tuberculosis are common causes of pneumonia. Once the CD4 count falls below 200 cells/mm3, opportunistic infections including Pneumocystis jerovecii and CMV become more common. Exposure to broad-spectrum antibiotics is enough to shift the spectrum of HCAP toward that of nosocomial pneumonias.

The etiologic spectrum of pneumonia continues to slowly expand. Roughly every decade a new pathogen is added to the spectrum. Most of these newly recognized pathogens are a result of either a change in host immunity, such as the HIV epidemic or bone marrow/stem cell transplants, or improved diagnostic testing. The latter advance in medical practice often provides an explanation for previously enigmatic cases of pneumonia. Many of these new pathogens are first recognized in conjunction with epidemics. The classic example is legionnaire’s disease. After the initial well-publicized epidemic led to identification of the genus, multiple species were subsequently defined and testing of samples from prior epidemics and random cases retrospectively demonstrated the importance of Legionella spp. for pneumonia. Hantavirus pulmonary syndrome was recognized approximately a decade later. The latest example is the severe acute respiratory syndrome (SARS) epidemic, which first appeared in southeastern Asia in late 2002. The SARS pathogen was subsequently identified as a coronavirus by the World Health Organization (WHO) laboratory network in April 2003. The SARS coronavirus (SARS-CoV) is only distantly related to previously sequenced coronaviruses and is not believed to have circulated in humans previously. SARS-CoV is postulated to be a previously unknown animal coronavirus that mutated and developed the ability to infect humans. The high transmission rates, especially in healthcare settings and households, suggest that little native immunity to this virus existed previously.

Treatment While new or previously unrecognized pathogens can play a role in management decisions, the major issue in pneumonia is the appearance of antibiotic resistant strains of known common bacterial pathogens. The emergence of antibiotic-resistant isolates has truly reached epidemic proportions for CAP and VAP. Multidrug-resistant pathogens are the norm for VAP. The emergence of drug-resistant Streptococcus pneumoniae (DRSP) is an increasingly common problem worldwide, with more than 40% of all pneumococci in some areas falling into this category by current definitions. Controversy exists, however, about the clinical relevance of in vitro resistance in the absence of meningitis, and whether new therapeutic approaches are required and whether resistance influences the outcome of CAP. When resistance to penicillin is present, there is often in vitro resistance to other agents. Methicillin-resistant Staphylococcal aureus, previously thought to be exclusively a

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HAP pathogen, is now making an appearance as a cause of CAP. The common denominator in development of antibiotic resistance is antibiotic over usage. Unfortunately, development of new antimicrobials is slowing and so far has only been successful in fighting a rearguard action against the advance of antibiotic resistance. Some benefit accrues from shortening the course of appropriate antibiotic use, such as for suspected VAP or with surgical prophylaxis. However, avoiding even starting antibiotics for nonbacterial infections, such as outpatient viral URIs, is the strategy most likely to slow this trend. In contrast to bacterial and a few other types of pneumonia (tuberculous, P. jeroveci), the lack of even minimally effective agents is more the issue for nonbacterial pneumonias. For Aspergillus, nontuberculous mycobacteria, respiratory syncytial virus (RSV), CMV, SARS, and many other viral agents, cure of pneumonia really depends on reconstitution of the patient’s immune system. Clearly more research is needed in this area. Given the resistance issues with antibiotics and lack of effective treatment for some types of pneumonia, immunomodulatory treatment is an increasingly important consideration for treatment of pneumonia. Immune modulation can take three pathways. One is the development and use of less immunosuppressive treatments for malignancies and transplant patients. Important advances in this area have already resulted in renal transplant patients returning to a near normal response to bacterial pneumonia and infrequent occurrence of opportunistic pathogens causing pneumonia. The second route is immunization. Childhood immunization has a long track record of effectively preventing respiratory infections with pertussis and diphtheria. The only infections seen with these pathogens in developed societies are when vaccination has not been provided. Recently, the use of the Haemophilus influenzae and protein conjugate pneumococcal vaccines have nearly eliminated meningitis from these bacteria and significantly reduced the incidence of respiratory tract infections. Not only has the conjugate pneumococcal vaccine decreased infection in children but it has also resulted in a decrease in invasive infections in the child’s adult caregivers. Even the polysaccharide pneumococcal vaccine appears to decrease the rate of invasive disease, even if it does not decrease overall pneumonia rates. The third immunomodulatory strategy is specific immunomodulation for established infection. The use of the colony-stimulating factors filgrastim and sargramostim for neutropenic patients is one example. The recent release of drotrecogin alfa activated (activated protein C) for

severe sepsis offers some hope to improve the mortality of bacterial CAP, especially pneumococcal CAP.

Conclusion Pneumonia is both a common and a significant problem in clinical medicine. While certain aspects of pathogenesis and management are common to all forms of pneumonia, significant differences also exist. These differences are discussed further in other articles specific to the different pneumonia syndromes, pathogens, molecules involved in host immunity, and diseases in the differential diagnosis.

Acknowledgments This work was supported in part by the American Heart Association, American Lung Association, and American Lung Association Metropolitan Chicago. See also: Antiviral Agents. Atelectasis. Bronchiectasis. Bronchiolitis. Coagulation Cascade: Protein C and Protein S. Colony Stimulating Factors. Defensins. Drug-Induced Pulmonary Disease. Granulomatosis: Wegener’s Disease. Human Immunodeficiency Virus. Immunoglobulins. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia; Hypersensitivity Pneumonitis. Laryngitis and Pharyngitis. Leukocytes: Neutrophils; Pulmonary Macrophages. Lung Abscess. Pleural Effusions: Parapneumonic Effusion and Empyema. Pneumonia: Atypical; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens); Nosocomial; Parasitic; Mycobacterial; Viral; The Immunocompromised Host. Pulmonary Thromboembolism: Pulmonary Emboli and Pulmonary Infarcts. Radiation-Induced Pulmonary Disease. Surgery: Transplantation. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome; Eosinophilic Lung Diseases. Tumors, Malignant: Bronchogenic Carcinoma; Chemotherapeutic Agents. Upper Respiratory Tract Infection. Vaccinations: Bacterial, for Pneumonia; Viral. Vasculitis: Overview. Ventilation, Mechanical: Ventilator-Associated Pneumonia. Viruses of the Lung.

Further Reading American Thoracic Society/Infectious Disease Society of America (2005) Guidelines for the management of adults with hospitalacquired, ventilator-associated, and healthcare-associated pneumonia. American Journal of Respiratory and Critical Care Medicine 171(4): 388–416. BTS (2001) Guidelines for the management of community acquired pneumonia in adults. Thorax 56 (supplement 4): IV1-64. Chakinala MM and Trulock EP (2005) Pneumonia in the solid organ transplant patient. Clinics in Chest Medicine 26(1): 113–121.

410 PNEUMONIA / Atypical Chastre J and Fagon JY (2002) Ventilator-associated pneumonia. American Journal of Respiratory and Critical Care Medicine 165(7): 867–903. Craven DE, Palladino R, and McQuillen DP (2004) Healthcareassociated pneumonia in adults: management principles to improve outcomes. Infectious Disease Clinics of North America 18(4): 939–962. Fagon JY, Chastre J, Wolff M, et al. (2000) Invasive and noninvasive strategies for management of suspected ventilator-associated pneumonia. A randomized trial. Annals of Internal Medicine 132(8): 621–630. Hubmayr RD, Burchardi H, Elliot M, et al. (2002) Statement of the 4th International Consensus Conference in Critical Care on ICU-Acquired Pneumonia – Chicago, IL, May 2002. Intensive Care Medicine 28(11): 1521–1536. Koivula I, Sten M, and Makela PH (1994) Risk factors for pneumonia in the elderly. American Journal of Medicine 96(4): 313–320. Mandell LA (2004) Epidemiology and etiology of community-acquired pneumonia. Infectious Disease Clinics of North America 18(4): 761–776. Mandell LA, Bartlett JG, Dowell SF, et al. (2003) Update of practice guidelines for the management of community-acquired pneumonia in immunocompetent adults. Clinical Infectious Diseases 37(11): 1405–1433. Niederman MS, Mandell LA, Anzueto A, et al. (2001) Guidelines for the management of adults with community-acquired pneumonia. Diagnosis, assessment of severity, antimicrobial therapy, and prevention. American Journal of Respiratory and Critical Care Medicine 163(7): 1730–1754. Tablan OC, Anderson LJ, Besser R, Bridges C, and Hajjeh R (2004) Guidelines for preventing health-care-associated pneumonia, 2003: recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee. Morbidity and Mortality Weekly Report. Recommendations and Reports 53(RR-3): 1–36. Whitney CG, Farley MM, Hadler J, et al. (2003) Decline in invasive pneumococcal disease after the introduction of proteinpolysaccharide conjugate vaccine. New England Journal of Medicine 348(18): 1737–1746.

Relevant Website www.brit-thoracic.org.uk – The British Thoracic Society is formed by the amalgamation of British Thoracic Association and Thoracic Society. It includes medical practioners, nurses, scientists, and any professional with an interest in respiratory disease.

Atypical J P Lynch III, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA N M Clark, University of Illinois – Chicago, Chicago, IL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract ‘Atypical pneumonia’ refers to a clinical syndrome associated with pneumonia (typically mild, nonlobar) and diverse upper respiratory tract and extrapulmonary manifestations. Clinical features overlap with bacterial pneumonia, and co-infection

with both typical (e.g., Streptococcus pneumoniae or other bacteria) and atypical pathogens may occur. ‘Atypical’ pathogens include Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. In large epidemiological studies, Mycoplasma pneumoniae has been implicated in 2–18% of community-acquired pneumonias; Chlamydia pneumoniae, in 2–8%; Legionella sp., 1–4%. Atypical pathogens lack cell walls and are resistant to b-lactam antibiotics but are usually susceptible to tetracyclines, macrolides, ketolides, and fluoroquinolone antibiotics. In this article, we also review other unusual causes of pneumonia which are transmitted by insects or vectors (e.g., Rocky Mountain spotted fever, cat scratch fever, Q fever, ehrlichiosis, Lyme disease, and tularemia). These diverse organisms are not found on Gram stain, and diagnosis requires special culture techniques or serological assays. We review the salient clinical and laboratory features of these various disorders, and discuss diagnostic and therapeutic strategies.

Introduction In 1938, Hobart Reimann described a group of patients with an initial mild respiratory illness, often accompanied by headache and sore throat, progressing to pneumonia without sputum production. Because this was a picture very different from the well-known presentation of acute pneumococcal pneumonia, Reimann coined the term ‘atypical pneumonia’ to describe these cases. His observation was confirmed by subsequent similar reports and it was noted that the etiologic agents for these illnesses were unidentifiable on Gram stain and not recovered by culture methods used at the time. The evolution of diagnostic techniques eventually led to the identification of many of the causative pathogens of atypical pneumonia, including the most significant ones, Mycoplasma, Chlamydia, and Legionella. Clinical features of atypical and typical community-acquired pneumonia (CAP) overlap, and the etiology of CAP cannot be determined on demographic, clinical, or radiologic features. However, the term ‘atypical pneumonia’ remains a useful one and describes a clinical picture characterized by more pronounced systemic than respiratory symptoms, bilateral patchy or interstitial infiltrates on chest radiographs, and negative results on sputum Gram stain and routine cultures. Atypical pathogens are being isolated with increasing frequency in CAP and may occur as coinfecting organisms with typical bacterial pathogens. Recent expert consensus statements recommend that empirical therapy for CAP should include coverage for atypical pathogens. In this article, we discuss many of the important microbes that cause atypical pneumonia, including epidemiology, clinical presentation, and therapy (see Table 1). Additional pathogens that can induce an ‘atypical pneumonia’ picture include viruses (e.g., influenza, adenovirus,

PNEUMONIA / Atypical 411 Table 1 Summary of atypical pneumonia agents Organism

Transmission

Incubation

Treatment

Mycoplasma pneumoniae

Person to person

3 weeks

Chlamydia pneumoniae

Person to person

3 weeks

Chlamydia psittaci

Birds and domestic animals Ticks Ticks Ticks Cattle, sheep, goats, cats

5–15 days

Bartonella henselae Borrelia burgdorferi

Cats Ticks

3–10 days 1 week

Francisella tularensis

Rodents, rabbits, hares

2–10 days

Macrolides, tetracyclines, fluoroquinolones, ketolides Macrolides, tetracyclines, fluoroquinolones, ketolides Tetracyclines; erythromycin as alternative Tetracyclines or chloramphenicol Tetracyclines Tetracyclines Tetracyclines; combination therapy with tetracyclines and hydroxychloroquine, rifampin, or fluoroquinolone for endocarditis Macrolides and tetracyclines Tetracyclines or amoxicillin; ceftriaxone for neurologic disease Streptomycin

Rickettsia rickettsii Ehrlichia chaffeensis Anaplasma phagocytophila Coxiella burnetii

respiratory syncytial virus, severe acute respiratory syndrome (SARS)-associated coronavirus, and cytomegalovirus), certain fungi such as Histoplasma capsulatum and Pneumocystis jiroveci (formerly P. carinii). A discussion of these diverse pathogens is beyond the scope of this article.

Mycoplasma pneumoniae Mycoplasma pneumoniae, tiny (125–150 nm) intracellular bacteria that lack cell walls and do not stain with the Gram stain, are implicated in 2–18% of CAP. Epidemiology

M. pneumoniae is transmitted from person-to-person by infected respiratory droplets. The incubation period following exposure averages 3 weeks. Infections due to M. pneumoniae are endemic in densely populated areas, with cyclic increases that lead to epidemics. Attack rates are highest in children and adolescents and only 2–4% of infections occur in adults older than 65 years. More severe cases may be found in immunosuppressed patients or those with sickle cell disease or asplenia. Clinical Features

Illness due to M. pneumoniae is usually mild and selflimited. Most common symptoms include indolent onset, low-grade fever, malaise, headache, upper respiratory symptoms (e.g., rhinorrhea, sinusitis, and pharyngitis), and bronchitis. A protracted, hacking cough may occur. Shaking chills are rare. Pneumonia

2–14 days 1 week 1 week 2–14 days

develops in 3–10% of infected patients, but is rarely severe. However, co-infection with bacterial pathogens occurs in one-third or more of patients. Chest findings on physical examination are usually minimal. Chest radiographs typically demonstrate peribronchial thickening and interstitial infiltrates; frank consolidation is uncommon. Pleural effusions are noted in 5–20% of patients, but are typically small; empyema is rare. M. pneumoniae may elicit wheezing in nonasthmatics, but whether it has a pathogenic role in asthma is controversial. Extrapulmonary features may include hemolysis, skin rash, and arthralgias; gastrointestinal complaints are unusual. In children, bullous or hemorrhagic myringitis is noted in 5–18% of cases with Mycoplasma infections. Rare manifestations of Mycoplasma infections include involvement of the central nervous system (CNS) (1– 7%) or heart (1%), and arthritis (o1%). Fatalities are rare. Diagnosis

IgM antibodies to the I antigen on erythrocyte membranes produce a cold agglutinin response in 50–65% of patients with infections due to M. pneumoniae, but cold agglutinin responses are nonspecific. Peripheral blood leukocyte count is normal in 75–90% of cases. Culturing M. pneumoniae is difficult as it requires 2–3 weeks, and is rarely performed. Confirmation of the diagnosis of the infection usually relies upon detecting complement-fixing (CF) antibodies. Positive results are defined by more than fourfold rise in antibody titer (acute to convalescent) and single titer X1:32. Antibody titers rise 7–10 days after infection, and peak at 3–4 weeks. Newer

412 PNEUMONIA / Atypical

immunoassays may be more sensitive and specific than CF serologies. The IgM capture test is optimal to diagnose M. pneumoniae in children. An enzymelinked immunoassay that detects IgG is preferred for adults as they may not elaborate an IgM response. As results are not available for 3–4 weeks, serologies are performed infrequently in clinical practice. However, they are invaluable for epidemiological investigations and tracking epidemics. Polymerase chain reaction (PCR) assays can detect genomic DNA in nasopharyngeal secretions and are highly specific and sensitive for M. pneumoniae in patients with respiratory tract infections. However, PCR is infrequently used, except for research epidemiologic studies. Treatment

Penicillin and cephalosporin antibiotics are ineffective against M. pneumoniae due to the lack of a cell wall. Tetracyclines, macrolides, ketolides, and fluoroquinolone antibiotics are active in vitro against M. pneumoniae. Resistance to macrolides may occur due to point mutations in the 23S ribosomal gene, but is rare. Doxycycline (100 mg b.i.d.) is efficacious and cost-effective. A 7–10 days course of therapy is adequate. Rare immunological complications of M. pneumoniae infections such as hemolytic anemia or CNS disease may require concomitant treatment with corticosteroids or even plasmapheresis.

Chlamydia Species Chlamydia species are small, Gram-negative obligate intracellular parasites of animals and humans. Three species of Chlamydia cause human disease including: C. trachomatis (the cause of trachoma, oculogenital infection, and lymphogranuloma venereum); C. pneumoniae (a cause of respiratory infection); and C. psittaci (an avian pathogen for which man is an incidental host). The discussion in this article is limited to C. pneumoniae and C. psittaci. Chlamydia pneumoniae

C. pneumoniae, formerly termed TWAR (after the laboratory designations of the initial isolates), was first identified as a respiratory pathogen in 1983. Serological studies implicate C. pneumoniae as the cause of 2–8% of community-acquired pneumonias. Epidemiology Humans are the only known reservoir of C. pneumoniae. Transmission of infection is via respiratory secretions and the incubation period may be prolonged (up to several weeks). Seropositivity rates rise quickly during childhood

after age 5, with antibodies present in 50% of individuals by age 20, and a continued gradual rise in seropositivity till the age of 80. Epidemics have been described in military barracks, schools, and nursing homes. Infections due to C. pneumoniae are a significant cause of morbidity among the elderly. Clinical features The vast majority (90%) of infections due to C. pneumoniae are asymptomatic or associated with mild, upper respiratory tract symptoms (headache, laryngitis, pharyngitis, sinusitis). Bronchitis or pneumonia due to C. pneumoniae is usually mild and self-limited. However, severe (sometimes fatal) CAP has been described. Chest radiograph findings are non-specific but patchy, subsegmental infiltrates are most common and severe, multilobar involvement is rare. As with other atypical pneumonias, blood leukocyte counts are usually normal. Uncommon extrapulmonary manifestations of C. pneumoniae infections include meningoencephalitis, Guillain–Barre syndrome, arthritis, and myocarditis. Fatalities are rare. Diagnosis Isolation of C. pneumoniae in cultures is difficult, and is not performed in most clinical laboratories. The diagnosis is usually made by seroconversion. The CF antibody test is most often used, but is non-specific, since this detects antigens shared among all Chlamydia species. A fourfold rise in CF antibodies against Chlamydia or a single titer of X1:64 is considered evidence of acute infection. Microimmunofluorescence (MIF) is more specific, but is technically difficult and is performed only in research laboratories. Criteria for the diagnosis of acute C. pneumoniae by MIF include a single IgM titer of X1:16, a fourfold rise in IgG titer (acute to convalescent), or a single IgG titer of X1:512. In primary infections, IgM is evident at 3 weeks after the onset of symptoms, followed by the IgG response at 6–8 weeks. The immunologic response to infection with C. pneumoniae appears incompletely protective as re-infection can occur. Re-infections are associated with IgG responses (not IgM) after 1–2 weeks. Other diagnostic techniques for detecting Chlamydia infection include immunoassay (EIA), direct fluorescent antibody, and PCR. PCR can be applied to nasopharyngeal swabs, sputum, or bronchoalveolar lavage (BAL) fluid and is more sensitive than cultures. Treatment Tetracyclines, macrolides, ketolides, and fluoroquinolone antibiotics are active in vitro against C. pneumoniae, and are recommended for treating symptomatic infections. Given the mild, self-limited

PNEUMONIA / Atypical 413

nature of the disease, there are limited data on the optimal agents or duration of therapy. Most experts recommend treating for 10–14 days, as responses to therapy may be slow.

Treatment Doxycycline (100 mg b.i.d. for 10–14 days) is the treatment of choice for infections due to C. psittaci. Other options include macrolides, chloramphenicol, or fluoroquinolones.

Chlamydia psittaci

Rickettsial and Related Diseases

C. psittaci is a rare cause of pneumonia that occurs following exposure to infected birds. The pneumonia caused by C. psittaci is termed psittacosis, derived from the Greek word for parrot, but is more properly designated an ‘ornithosis’ as all birds, not just parrots, can carry and spread the infection.

Epidemiology Birds are the primary carriers of psittacosis and humans may be infected via aerosolized droplets from infected birds. Birds may be asymptomatic or obviously ill when infected with C. psittaci. Rarely, humans acquire infection from other animals (e.g., sheep, goats, cattle, horses, cats, and dogs). Psittacosis is an occupational hazard of veterinarians, aviary or pet shop employees, zoo personnel, farmers, and workers in poultry-processing plants.

The order Rickettsiales includes the families Rickettsiaceae and Ehrlichiaceae, and the family Rickettsiaceae formerly included organisms of the genus Coxiella and Rochalimaea (now known as Bartonella). Within these groups are a great number of human pathogens that are maintained in nature by both mammals and arthropod vectors. In this review, we restrict our discussion to a few select species including: Rickettsia rickettsii (the cause of Rocky Mountain spotted fever (RMSF) and the most common rickettsial disease in the US), Ehrlichia chaffeensis (the cause of Ehrlichiosis in the US), Coxiella burnetti (the cause of Q fever), and Bartonella henselae (the causative agent for cat scratch fever). In general, the diagnosis of infections due to these agents is based on epidemiologic exposure history and serologic testing. Rocky Mountain Spotted Fever

Clinical features The incubation period of psittacosis ranges from 5 to 15 days. Clinical manifestations are varied, but include an abrupt onset of fever, headache (often severe), a flu-like syndrome, malaise, myalgias, anorexia, and a dry cough. The clinical course ranges from a mild illness to fulminant infection with multisystemic involvement. Pneumonia is common (475%), but is usually mild and dyspnea occurs in o20% of infected patients. Fatal respiratory failure is a rare complication. Unusual extrapulmonary organ manifestations include CNS involvement, endocarditis or myocarditis, proteinuria, pancreatitis, hemolysis, and hepatosplenomegaly.

Diagnosis The diagnosis is suggested by a history of contact with birds. Cultures are difficult and dangerous and not recommended except in specialized laboratories. Paired acute and convalescent antibody tests are the mainstay of diagnosis. CF assays are most often used, but cannot differentiate C. psittaci from other Chlamydia species. The MIF assay is specific for C. psittaci but is available only in specialty laboratories. A fourfold rise in antibody titer or an IgM antibody X1:16 are considered diagnostic. Other techniques (e.g., monoclonal antibody and PCR) have been developed, but are not readily available.

Rickettsiae are Gram-negative coccobacillary obligate intracellular parasites and are spread by arthropod vectors (e.g., lice, fleas, mites, ticks). RMSF is the most severe and most often reported rickettsial illness in the US. It is caused by the bite of a tick infected with Rickettsia rickettsii and is a potentially lethal disease, particularly in young children. Epidemiology RMSF is a misnomer as its distribution is not restricted to the Rocky Mountain region of the US. It occurs not only in the northern Rocky Mountain states, but also in the southeastern and south-central states, Cape Cod MA, Long Island NY, Mexico, Central America, and Latin America. The incubation period is 2–14 days following a tick bite. Most cases occur in the spring or summer. Approximately 1000 cases are reported annually in the US. Clinical features Initial symptoms include fever (495%), headache (90%), myalgias (80%), and nausea (60%). Abdominal pain may be a predominant feature. A typical rash develops between the 3rd to 5th day of the illness. The rash starts on the ankles and wrists, and spreads centrally to the palms and soles. A maculopapular eruption evolves to petechiae and a vaculitic rash. Damage to small blood vessels may result in leakage of plasma, reduced blood

414 PNEUMONIA / Atypical

volume, and shock. With severe cases, bleeding, gangrene of the digits, thrombocytopenia, seizures, CNS signs, or renal failure may develop. The lung is not a primary target for Rickettsiae rickettsii. However, pulmonary infiltrates are present in one-third of cases; acute respiratory distress syndrome (ARDS) occurs in fewer than 10% of cases. Diagnosis The diagnosis of RMSF is usually based upon the constellation of clinical features in the appropriate epidemiological setting. The diagnosis is usually confirmed serologically by indirect fluorescent antibody (IFA), which is available in state health departments or reference laboratories. Titers 41:64 are suggestive. Titer rises occur 7–14 days after the onset of illness, so treatment must be initiated prior to results of serologies. Biopsies of skin lesions (direct IF or immunoenzyme methods) establish the diagnosis in two-thirds of cases. Treatment Tetracycline or chloramphenicol are preferred therapies. Prompt initiation of therapy is critical to avert serious complications and mortality. Delay 45 days of initiation of appropriate antimicrobial therapy has been associated with an increased mortality rate (23% versus 6% with early treatment). Ehrlichiosis

Ehrlichiae are Gram-negative obligate intracellular bacteria that grow within membrane vacuoles in human and animal leukocytes. The two most important human ehrlichial diseases are human monocytic ehrlichiosis (HME), caused by Ehrlichia chaffeensis, and human granulocytic ehrlichiosis (HGE), caused by Anaplasma phagocytophila (previously known as E. equi, E. phagocytophila, and the agent of HGE). HME was first recognized in 1987; HGE was described in 1994. Epidemiology E. chaffeensis is transmitted to man through tick bites. White-tailed deer are the principal animal hosts for E. chaffeensis while deer and the white-footed mouse are the principal animals hosts for A. phagocytophilia. As with other tick-borne diseases, infections are most common in spring and summer. Tick bites, exposure to wildlife, and golfing have been implicated as risk factors for the disease. Clinical features After a median of one week of infection with Ehrlichia, characteristic symptoms may develop including fever, headache, nausea, vomiting, myalgias, and anorexia. A maculopapular rash is present in 20–36% of patients with HME but is rare in HGE. Leukopenia and thrombocytopenia are

present in 50–90% of cases. Pulmonary infiltrates are present on chest radiographs in 40–75% of patients; ARDS occurs in 11–18%. Mortality rates range from 2 to 5% for HME and 7–10% for HGE. Diagnosis Culture of Erhlichia is extremely difficult. Examination of peripheral blood or buffy coat may reveal characteristic clusters of intraleukocytic inclusions (morulae). Morulae (Latin for ‘mulberries’) are seen in the cytoplasm of neutrophils in patients with HGE and in mononuclear cells in patients with HME. IFA tests are available in all state health departments for E. chaffeensis. The Centers for Disease Control (CDC) case definition of HME includes fourfold change in antibody titer (acute to convalescent) with a minimum titer of 1:64. IFA tests for HGE are available only in a few research laboratories. PCR tests are available for both HME and HGE. Treatment Controlled studies are lacking, but tetracyclines are effective. Activity of chloramphenicol is variable. Rifampin displays in vitro activity against Ehrlichia and may have clinical utility. Doxycycline (100 mg b.i.d. for 10–14 days) is the preferred therapy. Coxiella burnetii (Q fever)

Coxiella burnetii is a pleomorphic Gram-negative coccobacillus which causes Q fever, an acute febrile illness which may be complicated by pneumonia or hepatitis, or a chronic febrile illness which typically manifests as endocarditis. Epidemiology Infection results from inhalation of organisms through exposure to infected livestock or unpasteurized milk. Q fever is endemic in some rural areas worldwide, but is rare in the US. Pneumonia due to acute Q fever can account for up to 7% of CAP in some regions (e.g., Nova Scotia, Switzerland, Spain, and Israel). C. burnetii is a potential bioterrorism agent, and is reportable in the US. Clinical features Q fever is generally a mild illness; up to 50% of infected persons are asymptomatic. Manifestations are protean, but three syndromes are common: a self-limited, flu-like illness; pneumonia; and hepatitis. Hepatitis is more common in younger patients; pneumonia, in older immunocompromised patients. Severe pneumonia is rare and mortality is low (0.5–1.5%). Other manifestations of Q fever include rash (10%), myocarditis or pericarditis (1%), and meningitis or encephalitis (1%). Chronic forms of Q fever (lasting 46 months) occur in 1% of

PNEUMONIA / Atypical 415

infected patients. In this context, endocarditis is the most common manifestation. Pathogenesis In chronic Q fever, high levels of antibodies and immune complexes may play a role in pathogenesis, eliciting tissue injury in the heart, arteries, bone, or liver. Diagnosis Blood leukocyte counts are normal in 75% of cases; thrombocytopenia occurs in 25%. Liver enzymes are elevated in up to 85% of patients with acute Q fever. Serological studies (IgG and IgM antibodies against C. burnetii) are employed to confirm the diagnosis. Detectable serum IgM antibodies may be present within 7–15 days of onset of symptoms, and are present in 90% by the third week. Antibody titers subside over 12 months. Persistent or recrudescent antibodies suggest chronic infection. Treatment Acute Q fever is usually mild, and spontaneously resolves within 2 weeks. Doxycyline (100 mg b.i.d. for 14 days) is the treatment of choice for patients with severe or persistent symptoms, or with impaired immune systems. The efficacy of erythromycin is controversial. Fluoroquinolones appear to be active. Anecdotal responses have been noted with lincomycin, cotrimoxazole, and chloramphenicol. For Q fever endocarditis, optimal therapy has not been elucidated, but combination therapy with at least two antimicrobials is recommended. In this context, doxycycline combined with hydroxychloroquine, a fluoroquinolone, or rifampin may be used. Bartonella henselae

Bartonella henselae can cause cat scratch disease (CSD) in normal hosts and bacillary angiomatosis, bacillary peliosis, and persistent bacteremia in human immunodeficiency virus (HIV)-infected patients. Epidemiology Domestic cats are the natural reservoir for Bartonella henselae – associated disease in humans. Illness may follow recent contact with domestic cats, usually involving a scratch or bite. Clinical features Typical features of CSD include regional lymphadenopathy and fever; systemic illness may ensue. In immunocompromised patients, involvement of liver, spleen, or visceral lymph nodes is common. Lung involvement is uncommon. Treatment In normal hosts, CSD usually resolves without therapy. Macrolides and doxycycline have excellent in vitro activity, and are recommended

for nonresolving infections or in immunocompromised hosts.

Other Tick-Borne Diseases Lyme Disease

Borrelia burgdorferi, a spirochete transmitted to man via tick bites, is the cause of Lyme disease, the most common vector-borne disease in the US and Europe. Epidemiology B. burgdorferi is transmitted by infected ticks, which are often carried by small mammals residing in wooded areas or parks. Human infections are most common in spring and summer, when outdoor activities are at a peak. The annual incidence of Lyme disease has been increasing in the US; nearly 41 000 cases of Lyme disease were reported to the Centers for Disease Control and Prevention during 2001–02. Ninety-five percent of these cases were from 12 states: Connecticut, Delaware, Rhode Island, Maine, Maryland, Massachusetts, Minnesota, New Jersey, New Hampshire, New York, Pennsylvania, and Wisconsin. Clinical features Within 1 week of infection by B. burgdorferi, a characteristic macular rash, erythema migrans, develops at the site of the tick bite in 80% of patients. Early symptoms include fever, malaise, fatigue, headache, arthralgias, and myalgias. Respiratory involvement is unusual, but nonproductive cough can occur. Secondary skin lesions often develop within days of the initial EM lesion but resolve spontaneously within 4 weeks. However, disseminated disease with involvement of the joints, heart, or nervous sytem can occur weeks to months later. In the US, 15% of untreated patients developed neurologic abnormalities; cardiac complications were reported in 8%. Diagnosis The diagnosis of Lyme disease is confirmed serologically. IgM antibodies against B. burgdorferi appear 3–6 weeks after infection and can be detected by ELISA or immunofluorescence assay. Positive results should be confirmed by Western blotting. Treatment Oral tetracyclines or amoxicillin for 3–4 weeks are preferred therapy for Lyme disease. For most patients with Lyme disease, oral therapy is as effective as intravenous (IV) ceftriaxone. However, when meningitis or encephalopathy are present, IV ceftriaxone is recommended because of superior penetration into cerebrospinal fluid (CSF).

416 PNEUMONIA / Atypical Tularemia

Tularemia is a zoonosis caused by Francisella tularensis, a small, Gram-negative coccobacillus found in rabbits, hares, rodents, and ticks. Epidemiology Transmission to humans occurs via contact with infected animals or tick bites. Risk factors include: hunting, cleaning, or handling animal skins; ingesting poorly cooked wild animal meat; tick bites; laboratory exposure; contact with contaminated water, soil, or hay. An epidemic of primary pneumonic tularemia in Martha’s Vineyard was linked to cutting brush and lawn-mowing. Most cases of tularemia occur in the summertime. Approximately 200–300 cases are reported annually in the US, predominantly in southern states. Clinical features Clinical features vary from an asymptomatic infection to septic shock and death. Fever, chills, headache, and malaise develop after an incubation period of 2–10 days. Manifestations depend upon the route of inoculation, dose of the inoculum, and virulence of the organism. Type A F. tularensis, found predominantly in North America, is more virulent than type B which is found predominantly in Asia and Europe. Ulceroglandular tularemia is the most common form, occurring in 60–80% of cases. A single erythematous lesion with central ulceration is present at the site of the bite or inoculation; tender regional lymphadenopathy may also be present. Five other main forms of tularemia include glandular (lymphadenopathy), typhoidal (fever, chills, headache, abdominal pain), primary oropharyngeal, primary pneumonic, and oculoglandular. Pneumonia can occur via airborne or hematogenous routes. Primary pneumonic tularemia results from inhalation of F. tularensis; it is the most severe form, with mortality up to 60% in the absence of treatment. Radiographic features of tularemic pneumonia include unilateral or bilateral infiltrates, hilar adenopathy, pleural effusions, and cavitary lesions. Rare complications of tularemia include pericarditis, meningitis, ARDS, rhabdomyolysis, and acute renal failure. Mortality associated with tularemia is 2–4%. Diagnosis The diagnosis of tularemia is made on clinical grounds (i.e., exposure history, lack of response to b-lactam antibiotics). Cultures of blood, body fluids, or tissue are insensitive, as the organism is fastidious. The diagnosis is confirmed by serologic testing (a single titer of X1:16, or a fourfold rise in agglutinating antibodies, or ELISA to F. tularensis).

Treatment Streptomycin (10 mg kg 1 intramuscularly b.i.d. for 7–10 days) is highly efficacious (97% cure rate, with no relapses) and is the treatment of choice. Improvement is prompt, usually within 48 h of initiation of streptomycin therapy. Tetracyclines, gentamicin, and chloramphenicol may be effective, but clinical cure rates are lower (77–88%) and relapse rates higher (6–21%). Favorable responses have been noted with macrolides and fluoroquinolones; b-lactams are ineffective. See also: Pneumonia: Overview and Epidemiology; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens); Nosocomial; Mycobacterial. Signs of Respiratory Disease: Lung Sounds. Stem Cells. Systemic Disease: Sickle Cell Disease.

Further Reading (2004) Centers for Disease Control and Prevention. MMWR 53(17): 365–369. Abele-Horn M, Busch U, Nitschko H, et al. (1998) Molecular approaches to diagnosis of pulmonary diseases due to Mycoplasma pneumoniae. Journal of Clinical Microbiology 36: 548–551. Bakken JS and Dumler JS (2000) Human granulocytic ehrlichiosis. Clinical Infectious Diseases 31: 554–560. Bakken JS, Dumler JS, Chen SM, et al. (1994) Human granulocytic ehrlichiosis in upper Midwest United States: a new species emerging? Journal of the American Medical Association 272: 212–218. Bourke SJ and Lightfoot NF (1995) Chlamydia pneumoniae: defining the clinical spectrum of infection requires precise laboratory diagnosis. Thorax 50(supplement 1): S43–S48. Clyde WA Jr (1993) Clinical overview of typical Mycoplasma pneumoniae infections. Clinical Infectious Diseases 17: S32–S36. Dattwyer RJ, Luft BJ, Kunkel MG, et al. (1997) Ceftriaxone compared with doxycycline for the treatment of acute disseminated Lyme disease. New England Journal of Medicine 337: 289–294. Dominguez A, Minguell S, Torres J, et al. (1996) Community outbreak of acute respiratory infection with Mycoplasma pneumoniae. European Journal of Epidemiology 12: 131–134. Dorigo-Zetsma JW, Verkooyen RP, van Helden HP, et al. (2001) Molecular detection of Mycoplasma pneumoniae in adults with community-acquired pneumonia requiring hospitalization. Journal of Clinical Microbiology 39: 1184–1186. Eckman MH, Steere AC, Kalish RA, and Pauker SG (1997) Costeffectiveness of oral as compared with intravenous antibiotic therapy for patients with early Lyme disease or Lyme arthritis. New England Journal of Medicine 337: 357–363. Enderlin G, Morales L, Jacobs RF, and Cross JT (1994) Streptomycin and alternative agents for the treatment of tularemia: review of the literature. Clinical Infectious Diseases 19: 42–47. Eng TR, Harkess JR, Fishbein DB, et al. (1990) Epidemiologic, clinical, and laboratory findings of human erhlichiosis in the United States, 1998. Journal of the American Medical Association 264: 2251–2258. Evans ME, Gregory DW, and Schaffner W (1985) Tularaemia: a 30-year experience with 80 cases. Medicine (Baltimore) 64: 251–269. Faul JL, Doyle RL, Kao PN, and Ruoss SJ (1999) Tick-borne pulmonary disease. Update on diagnosis and management. Chest 116: 222–230.

PNEUMONIA / Community Acquired Pneumonia, Bacterial and Other Common Pathogens 417 Feldman KA, Enscore RE, Lathrop SL, et al. (2001) An outbreak of primary pneumonia tularemia on Martha’s Vineyard. New England Journal of Medicine 345: 1601–1606. Fix AD, Strickland GT, and Grant J (1998) Tick bites and Lyme disease in an endemic setting: problematic use of serologic testing and prophylactic antibiotic therapy. Journal of the American Medical Association 279: 206–210. Foy HM, Kenny GE, McMahan AM, and Grayston JT (1970) Mycoplasma pneumoniae pneumonia in an urban area. Five years of surveillance. Journal of the American Medical Association 214: 1666–1672. Gikas A, Kofteridis DP, Manios A, et al. (2001) Newer macrolides as empiric treatment for acute Q fever infection. Antimicrobial Agents and Chemotherapy 45: 3644–3646. Gleason PP (2002) The emerging role of atypical pathogens in community-acquired pneumonia. Pharmacotherapy 1(pt. 2): 2S–11S. Gray GC, Witucki PJ, Gould MT, et al. (2001) Randomized, placebo-controlled clinical trial of oral azithromycin prophylaxis against respiratory infections in a high-risk, young adult population. Clinical Infectious Diseases 33: 983–989. Hammerschlag MR (2001) Mycoplasma pneumoniae infections. Current Opinion in Infectious Diseases 14: 181–186. Helmick CG, Bernard KW, and D’Angelo LJ (1984) Rocky Mountain spotted fever: clinical, laboratory, and epidemiological features of 262 cases. Journal of Infectious Diseases 140: 480–488. Kauppinen MT, Herva E, Kujala P, et al. (1995) The etiology of community-acquired pneumonia among hospitalized patients during a Chlamydia pneumoniae epidemic in Finland. Journal of Infectious Diseases 172: 1330–1335. Kirk JL, Sexton DJ, Fine DP, and Muchmore HG (1990) Rocky Mountain spotted fever. A clinical review based on 48 confirmed cases (1943–1986). Medicine (Baltimore) 69: 35–45. Kirkland KB, Wilkinson WWE, and Sexton DJ (1995) Therapeutic delay and mortality in cases of Rocky Mountain spotted fever. Clinical Infectious Diseases 20: 1118–1121. Marrie TJ, Peeling RW, Fine MJ, et al. (1996) Ambulatory patients with community-acquried pneumonia: the frequency of atypical agents and clinical course. American Journal of Medicine 101: 508–515. Moroney JF, Guevara R, Iverson C, et al. (1998) Detection of chlamydiosis in a shipment of pet birds, leading to an outbreak of clinically mild psittacosis in humans. Clinical Infectious Diseases 26: 1425–1429. Oldach DW, Gaydes CA, Mundy LM, and Quinn TC (1993) Rapid diagnosis of Chlamydia psittaci pneumonia. Clinical Infectious Diseases 17: 338–343. Pereyre S, Guyot C, Renaudin H, et al. (2004) In vitro selection and characterization of resistance to macrolides and related antibiotics in Mycoplasma pneumoniae. Antimicrobial Agents and Chemotherapy 48: 460–465. Raoult D and Marrie T (1995) Q fever. Clinical Infectious Diseases 20: 489–496. Raoult D, Tissot-Dupont H, Foucault C, et al. (2000) Q fever 1985–1998. Clinical and epidemiologic features of 1,383 infections. Medicine (Baltimore) 79: 109–123. Salgo MP, Telzak EE, Currie B, et al. (1988) A focus of Rocky Mountain spotted fever within New York city. New England Journal of Medicine 318: 1345–1348. Spach DH, Liles WC, Campbell GL, et al. (1993) Tick-borne diseases in the US. New England Journal of Medicine 329: 936– 947. Taylor-Robinson D and Bebear C (1997) Antibiotic susceptibilities of Mycoplasma and treatment of mycoplasmal infections. Journal of Antimicrobial Chemotherapy 40: 622–630.

Tuuminen T, Palomaki P, and Paavonen J (2000) The use of serologic tests for the diagnosis of chlamydial infections. Journal of Microbiolical Methods 42: 265–279. Verweij PE, Meis JF, Eijk R, et al. (1995) Severe human psittacosis requiring artificial ventilation: case report and review. Clinical Infectious Diseases 20: 440–442.

Community Acquired Pneumonia, Bacterial and Other Common Pathogens M S Niederman, State University of New York, Stony Brook, NY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Community-acquired pneumonia (CAP) is the number one cause of death from infectious diseases in the US, and the patient population that is affected is becoming increasingly more complex due to the presence of chronic illness which is commonly managed in outpatients who are at risk for pneumonia. The number one pathogen causing CAP is pneumococcus, which is commonly resistant to multiple antibiotics, thus complicating management. Other common pathogens include atypical organisms (Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae), Hemophilus influenzae, enteric Gram-negatives (especially in those with chronic illness and aspiration risk factors), and Staphylococcus aureus. Successful management requires careful assessment of disease severity so that a site-of-care decision can be made (outpatient, inpatient, intensive care unit), appropriate samples for diagnostic testing collected, and antibiotic therapy initiated in a timely and accurate fashion. Initial antibiotic therapy is empiric, but even with extensive diagnostic testing, less than half of all patients have an etiologic pathogen identified. All patients with CAP require therapy for pneumococcus, atypical pathogens, and other organisms, as dictated by the presence of specific risk factors. Because pneumonia has both short-term and long-term impact on mortality, it is also important to focus on prevention of this illness, which requires smoking cessation, and giving at-risk individuals both pneumococal and influenza vaccines.

Introduction Pneumonia, a respiratory infection of the alveolar space, can vary from a mild outpatient illness to a severe illness necessitating hospitalization and intensive care. It is the sixth leading cause of death in the US and the number one cause of death from infectious diseases. When the infection occurs in patients who are living in the community it is termed community-acquired pneumonia (CAP), while it is called nosocomial pneumonia if it arises in patients who are already in the hospital. Presently, the distinction between community-acquired and nosocomial infection is less clear because the ‘community’ includes

418 PNEUMONIA / Community Acquired Pneumonia, Bacterial and Other Common Pathogens

complex patients such as those who have recently been hospitalized, those in nursing homes, and those with chronic diseases who are commonly managed in such facilities as dialysis centers or nursing homes. When this latter group develops pneumonia, it has been termed healthcare associated pneumonia (HCAP) and this illness shares clinical features with CAP, but the etiologic pathogens may overlap with those seen in more traditional nosocomial pneumonia. Thus, the relationship between bacteriology and the site of origin of infection is a reflection of several factors, including: the types of patients who develop the illness, their host defense related predisposition to infection with specific pathogens, and their environmental exposure to certain organisms. This discussion focuses on pneumonia arising out of the hospital in immune-competent individuals, but excludes discussion of patients with human immunodeficiency virus (HIV) infection or traditional immune suppression (cancer chemotherapy, immune suppressive medications). In 1994, over 5.6 million people were diagnosed with CAP in the US, but the majority, 4.6 million, were treated out of the hospital. Although the majority of cases of CAP are managed in the outpatient setting, the greater part of the cost of treatment is focused on hospitalized patients. Those who are admitted to the hospital commonly tend to be older and have a high frequency of comorbid illness. In the US, the population of elderly patients is increasing, and those aged over 65 make up about one-third of all CAP patients, but this group accounts for more than half of the cost because of the frequent need for elderly CAP patients to be hospitalized. The reported mortality of CAP varies with the population being evaluated, ranging from less than 5% among outpatients, to 12% among all hospitalized CAP patients, but rising to over 30% among those admitted to the intensive care unit (ICU). While most studies have focused on the in-hospital mortality of CAP, one recent evaluation of CAP patients over the age of 65 found that while the short-term risk (in-hospital mortality) of illness was an 11% death rate, the mortality rate at 1 year was over 40%, emphasizing that for many patients, CAP is a marker of underlying serious comorbidity, and a predictor of poor outcome, even after hospital discharge, for a variety of reasons. These findings add to the emphasis on pneumonia prevention, especially through the use of available vaccines. The complexity of CAP management is also increasing because the etiologic pathogens are changing. Historically, CAP was regarded as a bacterial illness caused by one pathogen, Streptococcus pneumoniae. Today, the number of etiologic pathogens

has mushroomed to include not only bacteria, but also viruses (influenza, severe acute respiratory syndrome (SARS)), fungi, and a number of other recently identified organisms (such as Legionella, Chlamydophila pneumoniae). Not only is the number of pathogens expanding, but our ability to treat is being challenged by the rising frequency of resistance among bacteria to a wide range of commonly used, and often overused, antimicrobial agents.

Pathology and Pathogenesis Pneumonia is an infection of the gas-exchanging units of the lung, most commonly caused by bacteria, but occasionally caused by viruses, fungi, parasites, and other infectious agents. In the immunocompetent individual, it is characterized by a brisk filling of the alveolar space with inflammatory cells and fluid. If the alveolar infection involves an entire anatomic lobe of the lung, it is termed ‘lobar pneumonia’, and multilobar illness can be present in some instances. When the alveolar process occurs in a distribution that is patchy, and adjacent to bronchi, without filling an entire lobe, it is termed as ‘bronchopneumonia’. Pneumonia occurs when a patient’s host defenses are overwhelmed by an infectious pathogen. This can happen because the patient has an inadequate immune response, often as a result of underlying comorbid illness (congestive heart failure, diabetes, renal failure, chronic obstructive lung disease, malnutrition), because of anatomic abnormalities (endobronchial obstruction, bronchiectasis), as a result of acute illness-associated immune dysfunction (as can occur with sepsis or acute lung injury), or because of therapy-induced dysfunction of the immune system (corticosteroids, endotracheal intubation). Pneumonia can also occur in patients who have an adequate immune system, if the host defense system is overwhelmed by a large inoculum of microorganisms, which can occur in a patient with massive aspiration of gastric contents. In patients outside the hospital, a normal immune system can be overcome by a particularly virulent organism, to which the patient has no pre-existing immunity (such as certain bacteria or viruses) or to which the patient has an inability to form an adequate acute immune response. Bacteria can enter the lung via several routes, but aspiration from a previously colonized oropharynx is the most common way that organisms lead to pneumonia. Although most pneumonias result from microaspiration, patients can also aspirate large volumes of bacteria if they have impaired neurologic protection of the upper airway (stroke, seizure), or if they have intestinal illnesses that predispose to vomiting. Other routes of entry include inhalation,

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which applies primarily to viruses, Legionella pneumophila and Mycobacterium tuberculosis; hematogenous dissemination from extrapulmonary sites of infection (right-sided endocarditis); and direct extension from contiguous sites of infection (such as liver abscess). Keeping these concepts in mind, it is easy to understand why previously healthy individuals develop infection with virulent pathogens such as viruses, L. pneumophila, Mycoplasma pneumoniae, C. pneumoniae, and S. pneumoniae. On the other hand, chronically ill patients can be infected by these organisms, as well as by organisms that commonly colonize patients, but only cause infection when immune responses are inadequate. These organisms include enteric Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter spp.) and fungi. Recent studies have evaluated the normal lung immune response to infection and have shown that it is generally ‘compartmentalized’, and thus most patients with unilateral pneumonia have an inflammatory response that is limited to the site of infection, not spilling over to the uninvolved lung or to the systemic circulation. In patients with localized pneumonia, tumor necrosis factor (TNF) and interleukins 6 and 8 (IL-6, IL-8) levels were increased in the pneumonic lung and generally not increased in the uninvolved lung or in the serum. In patients with severe pneumonia, the immune response is characterized by a spillover of the immune response into the systemic circulation, reflected by increases in serum levels of TNF and IL-6. It remains uncertain why localization does not occur in all individuals, and why some patients develop diffuse lung injury (acute respiratory distress sydrome, ARDS) or systemic sepsis as a consequence of pneumonia. Recent studies have suggested that genetic polymorphisms may explain some of these differences, with patients who have certain inherited patterns of immune response being more prone than others to severe forms of pneumonia, and even mortality from this illness. For example, certain genetic polymorphisms are associated with a greater risk of death from sepsis but not CAP. Specifically, TNF-308 polymorphisms increase sepsis mortality, but not mortality from CAP. In addition, CAP severity is increased with genetic changes in the IL-10–1082 locus, which are often present along with changes in the TNF-308 locus. Another genetic change associated with an increased risk of septic shock from CAP is a modification in heat shock protein 70-2. Currently, we are aware of the large number of genes that can affect the severity and outcome of CAP, but much more must be learned to put these findings into a true clinical context.

Etiology Etiologic Pathogens

Even with extensive diagnostic testing, an etiologic agent is defined in only about half of all patients with CAP, pointing out the limited value of diagnostic testing, and the possibility that we do not know all the organisms that can cause CAP. For example, in the past three decades, a variety of new pathogens for this illness have been identified, including L. pneumophila, C. pneumoniae, hantavirus, and the SARS virus (a coronavirus). In addition, antibiotic resistant variants of common pathogens such as S. pneumoniae (pneumococcus) have become increasingly common and are referred to as drug-resistant S. pneumoniae (DRSP). For all patients with CAP, pneumococcus (including DRSP) is the most common pathogen, and some studies have suggested that it may be responsible for many of the patients with no established etiologic diagnosis, using standard diagnostic methodology. Recent studies have also suggested that atypical pathogens (M. pneumoniae, C. pneumoniae, and L. pneumocphila) are common in patients with CAP, often as copathogens, along with bacterial organisms. Viruses may be present in up to 20% of all patients, but specialized diagnostic testing, usually involving acute and convalescent titers, is needed, thus explaining the ordinary low frequency of documenting these organisms. Hemophilus influenzae is a common organism in patients who smoke cigarettes, and in those with chronic obstructive lung disease. Enteric Gramnegatives are not common causes of CAP, but can be present in up to 10% of hospitalized patients, particularly those with advanced age, comorbid illness, or a high likelihood of aspiration. In general, certain specific patients are at risk for infection with specific pathogens, in different frequencies. Table 1 summarizes the common pathogens causing CAP in both outpatients and inpatients. The classification is based on the severity of illness and the presence of clinical risk factors for specific pathogens, referred to as modifying factors. Patients with severe CAP may have a slightly different spectrum of organisms than less severely affected individuals, being commonly infected with pneumococcus, atypical pathogens, enteric Gram-negatives (including P. aeruginosa), Staphylococcus aureus, and H. influenzae. The modifying factors that increase the risk of infection with DRSP are: age over 65 years, beta-lactam therapy within the past 3 months, alcoholism, immune suppressive illness (including therapy with corticosteroids), multiple medical comorbidities, and exposure to a child in a day care setting. The modifying factors for enteric Gram-negatives include: residence in a nursing home,

420 PNEUMONIA / Community Acquired Pneumonia, Bacterial and Other Common Pathogens Table 1 Common Pathogens Causing CAP in specific patient populations (in order of decreasing frequency) Outpatient, no cardiopulmonary disease or modifying factors S. pneumoniae, M. pneumoniae, C. pneumoniae (alone or as mixed infection), H. influenzae, respiratory viruses, others (Legionella sp., M. tuberculosis, endemic fungi) Outpatient, with cardiopulmonary disease and/or modifying factors All of the above plus: DRSP, enteric Gram-negatives, and possibly anaerobes (with aspiration) Inpatient, with cardiopulmonary disease and/or modifying factors S. pneumoniae (including DRSP), H. influenzae, M. pneumoniae, C. peumoniae, mixed infection (bacteria plus atypical pathogen), enteric Gram-negatives, anaerobes (aspiration), viruses, Legionella sp., others (M. tuberculosis, endemic fungi, Pneumocystis jerovici) Inpatient, with no cardiopulmonary disease or modifying factors All of the above, but DRSP and enteric Gram-negatives are not likely Severe CAP, with no risks for P. aeruginosa S. pneumoniae (including DRSP), Legionella sp., H. influenzae, enteric Gram-negative bacilli, S. aureus, M. pneumoniae, respiratory viruses, others (C. pneumoniae, M. tuberculosis, endemic fungi) Severe CAP, with risks for P. aeruginosa All of the pathogens above, plus P. aeruginosa

Table 2 Clinical associations with specific pathogens Condition

Commonly encountered pathogens

Alcoholism

S. pneumoniae (including DRSP), anaerobes, Gram-negative bacilli (possibiy K. pneumoniae) S. pneumoniae, H. influenzae, Moraxella catarrhalis, Legionella

Chronic obstructive pulmonary disease (COPD)/current or former smoker Residence in nursing home Poor dental hygiene Bat exposure Bird exposure Rabbit exposure Travel to Southwest US Exposure to farm animals or parturient cats Travel to Southeast Asia Suspected bioterrorism Endobronchial obstruction Post influenza pneumonia Structural disease of lung (bronchiectasis, cystic fibrosis, etc.) Recent antibiotic therapy

underlying cardiopulmonary disease, multiple medical comorbidities, and recent antibiotic therapy. The risk factors for P. aeruginosa infection are: structural lung disease (bronchiectasis), corticosteroid therapy (410 mg day 1 prednisone), broad-spectrum antibiotic therapy for 47 days in the past month, and malnutrition. Table 2 shows that certain clinical conditions are associated with specific pathogens, and these associations should be considered in all patients when obtaining a history. Streptococus pneumoniae

As mentioned, S. pneumoniae is the most common pathogen for CAP, in any patient population,

S. pneumoniae, Gram-negative bacilli, H. influenzae, S. aureus, anaerobes, C. pneumoniae; consider M. tuberculosis Anaerobes Histoplasma capsulatum Chlamydia psittaci, Cryptococcus neoformans, H. capsulatum Francisella tularensis Coccidioidomycosis; hantavirus in selected areas Coxiella burnetii (Q fever) M. tuberculosis, Burkholderia pseudomallei, SARS virus Anthrax, smallpox, pneumonic plague Anaerobes S. pneumoniae, S. aureus, H. influenzae P. aeruginosa, P. cepacia, or S. aureus Pneumococcus resistant to the class of agents to which the patient was recently exposed

possibly even among those without an etiology recognized by routine diagnostic testing. In one study, transthoracic needle aspirates were used to define the etiology of CAP in patients with no identified organisms by conventional diagnostic testing, using a polymerase chain reaction (PCR) probe analysis of the samples, and in half of the patients in whom the needle provided a diagnosis when other methods had failed, pneumococcus was identified. The organism is a Gram-positive, lancet-shaped diplococcus, of which there are 84 different serotypes, each with a distinct antigenic polysaccharide capsule, but 85% of all infections are caused by one of 23 serotypes, which are now included in a vaccine. Infection is most common in the winter and early

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spring, which may relate to the finding that up to 70% of patients have a preceding viral illness. The organism spreads from person to person, and commonly colonizes the oropharynx before it leads to pneumonia. Pneumonia develops when colonizing organisms are aspirated into a lung that is unable to contain the aspirated inoculum. Infection is more common in the elderly; those with asplenia, multiple myeloma, congestive heart failure, or alcoholism; after influenza; and in patients with chronic lung disease. In patients with HIV infection, pneumococcal pneumonia with bacteremia is more common than in healthy populations of the same age. The classic radiographic pattern is a lobar consolidation, but bronchopneumonia can also occur, and in some series, this is the most common pattern. Bacteremia is present in up to 20% of hospitalized patients with this infection, but the impact of this finding on mortality is uncertain. Extrapulmonary complications include meningitis, empyema, arthritis, endocarditis, and brain abscess. Since the mid-1990s, antibiotic resistance among pneumococci has become increasingly common, and penicillin resistance, along with resistance to other common antibiotics (macrolides, trimethoprim/sulfamethoxizole, selected cephalosporins), is present in over 40% of these organisms. Fortunately, in the US, a large number of penicillin-resistant organisms are of the intermediate type (penicillin minimum inhibitory concentration, or MIC, of 0.1–1.0 mg l 1) and not of the high-level type (penicillin MIC of 2.0 mg l 1 or more). It is difficult to show a clinical impact of in vitro resistance on outcomes such as mortality in large numbers of patients, but most experts believe that organisms with a penicillin MIC of X4 mg l 1, still an uncommon finding, can lead to an increased risk of death. In an early study of the topic, there was no impact of resistance on mortality, after adjusting for severity of illness in a population with nearly a 30% frequency of in vitro resistance. More recently, some studies have shown that resistance can affect outcome. In a group of patients with pneumococcal bacteremia, of which more than half were HIV-positive, high-level resistance was a predictor of mortality. Other investigators did not find an increased risk of death from infection with resistant organisms, but did find an enhanced likelihood of suppurative complications (empyema), and a more prolonged length of stay in hospital. These conflicting data may have been the result of studying relatively few patients, many of whom did not have high levels of in vitro resistance. One large study evaluated more than 5000 patients with pneumococcal bacteremia and CAP and found an increased mortality for patients

with a penicillin MIC of at least 4 mg l 1 or greater, or with a cefotaxime MIC of 2.0 mg l 1 or more. However, this increased mortality was only present if patients who died in the first 4 days of therapy were excluded from analysis. Fortunately, very few organisms are currently at this level of resistance, which may explain the conflicting findings in various studies. More recently, another study using both a cohort and matched control methods found that severity of illness, and not resistance or accuracy of therapy, was the most important predictor of mortality. In some studies, severity of illness was greater in patients without resistant organisms, implying a loss of virulence among organisms that become resistant, a finding echoed in other studies that have found that the absence of invasive illness is a risk factor for pneumoccal resistance. There are also conflicting data on the impact of discordant therapy in patients infected with DRSP. In one study of bacteremic infection, the only antibiotic associated with a poor outcome, in the presence of in vitro resistance, was cefuroxime. In another study, discordant therapy in general was associated with an increased risk of mortality, as was multilobar illness, underlying chronic obstructive pulmonary disease (COPD), and hospitalization within the past 3 months, but these latter factors did not have as much of a risk of death as did discordant therapy. Also in this study, discordant therapy was less likely if patients were treated with ceftriaxone or cefotaxime, compared to other therapies. Macrolide-resistant pneumococcus is also occurring with increasing frequency, and up to 40% of organisms may be resistant to these agents in vitro. However, it is important to understand that most macrolide resistance in the US is low level, and due to an efflux mechanism, and thus it is a type of resistance that may not be clinically relevant, because local concentrations of macrolides at respiratory sites of infection may be adequate for effective therapy. However, some resistance is higher level and due to an inability of the antibiotic to bind to its ribosomal site of action, and this form of resistance may be much higher level and more likely to be clinically relevant. There are however, very few reports of macrolide failures in CAP, especially considering the widespread use of these agents. Reports of breakthrough bacteremia have appeared, but have been due to organisms that were resistant by either the efflux or ribosomal mechanism. It is likely that higher levels of resistance will become more likely in the future, and the impact on outcome is likely to be more apparent. Resistance of pneumococcus has even been reported to the quinolones, which are ordinarily a

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reliable class of antibiotics for these organisms. In general, one important risk factor for resistance is repeated use of these agents in the same patient. In fact, pneumococcal resistance to beta-lactams (penicillins and cephalosporins), macrolides, and quinolones is more likely if a patient has received the same agent in the recent past. It remains uncertain how long after antibiotic exposure that there is still risk of resistance, but 3–6 months has been reported for beta-lactams, up to 6 months for macrolides, and up to 12 months for quinolones. With these data in mind, new guidelines have suggested that CAP patients should not receive the same antibiotic as in the recent past, with a relatively arbitrary cutoff of defining this time interval as ‘within the past 3 months’.

Atypical Pathogens Originally the term ‘atypical’ was used to describe the unusual clinical features of infection with certain organisms, but recent studies have suggested that the term does not accurately describe a unique clinical pneumonia syndrome related to specific pathogens. However, the term has been retained to refer to a group of organisms which includes M. pneumoniae, C. pneumoniae, and Legionella, and this group of organisms cannot be reliably eradicated by betalactam therapy (penicillins and cephalosporins), but must be treated with a macrolide, ketolide (telithromycin is the only one currently available), tetracycline, or a quinolone. Some recent studies have shown that these infections are common in patients of all ages, not just young and healthy individuals as originally described, and these organisms have even been reported among the elderly in nursing homes. In addition, they can occur as either primary pathogens, or may be part of a mixed infection, along with traditional bacterial pathogens. When mixed infection is present, it may lead to a more complex course and a longer length of stay than if a single pathogen is present. There may be a particular synergy between C. pneumoniae and pneumococcus, with either sequential, or mixed infection with C. pneumoniae leading to a more severe course for pneumococcus. The frequency of atypical pathogens can be as high as 60% of all CAP patients, in some series, with as many as 40% of patients having mixed infection identified. Although these high incidence numbers have been derived with serologic testing, which is of uncertain accuracy, the importance of these organisms is suggested by studies of inpatients, which have shown a reduced mortality and length of stay when patients receive empiric therapy that accounts for these organisms, compared to regimens that do not

account for these organisms. In fact, several studies of patients with pneumococcal bacteremia have even suggested a mortality benefit to combination therapy that provides coverage for atypical pathogens, as well as pneumococcus. Atypical organism pneumonia may not be a constant phenomenon, and the frequency of infection may vary over the course of time and with geographical location. In one study, the benefit of providing empiric therapy directed at atypical pathogens was variable, being more important in some calendar years than in others. The incidence of Legionella infection among admitted patients has varied from 1% to 15% or more, and is also a reflection of geographic and seasonal variability in infection rates, as well as a reflection of the extent of diagnostic testing. For Legionella to be identified, it is necessary to collect both acute and convalescent serologic studies. In patients with severe CAP, atypical pathogens can be present in almost 25% of all patients, but the responsible organism may vary over time. In one series, Legionella was the most common atypical pathogen leading to severe CAP, but in the same hospital a decade later, it had been replaced by Mycoplasma and Chlamydophila infection. L. pneumophila is a small, weakly staining, Gramnegative bacillus first characterized after an epidemic in 1976, and can occur either sporadically or in epidemic form. At present, although multiple serogroups of the species L. pneumophila have been described, serogroup 1 causes the most cases of pneumonia. The other species that commonly causes human illness is Legionella micdadei. Legionella is a water-borne pathogen and can emanate from air-conditioning equipment, drinking water, lakes and river banks, water faucets, saunas, and shower heads. Infection is more common in the summer and early fall, and is generally caused by inhalation of an infected aerosol generated by a contaminated water source. When a water system becomes infected in an institution, endemic outbreaks may occur, as has been the case in some hospitals, particularly in patients who are receiving corticosteroid therapy. In its sporadic form, Legionella may account for 7–15% of all cases of CAP, being a particular concern in patients with severe forms of illness. As mentioned, it is very difficult to use clinical features to predict the microbial etiology of CAP; however, the classic Legionella syndrome is characterized by high fever, chills, headache, myalgias, and leukocytosis. The diagnosis is also suggested by the presence of a pneumonia with preceding diarrhea, along with early onset of mental confusion, hyponatremia, relative bradycardia, and liver function abnormalities, but this syndrome is usually not

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present. Symptoms are rapidly progressive, and the patient may appear to be quite toxic, so this diagnosis should always be considered in patients admitted to the ICU with CAP. M. pneumoniae can cause CAP year-round, with a slight increase in the fall and winter. All age groups are affected, and although it is common in those less than 20 years of age, it is also a common cause of CAP, even in older adults. Respiratory infection occurs after the organism is inhaled and then binds via neuraminic acid receptors to the airway epithelium. An inflammatory response with neutrophils, lymphocytes, and macrophages then follows, accompanied by the formation of immunoglobulin M (IgM) and then IgG antibody. Some of the observed pneumonitis may be mediated by the host response to the organism rather than by direct tissue injury by the Mycoplasma. Up to 40% of infected individuals will have circulating immune complexes. Although Mycoplasma causes pneumonia, infection is often characterized by its extrapulmonary manifestations. Up to half of patients will have upper respiratory tract symptoms, including sore throat and earache (with hemorrhagic or bullous myringitis). Pleural effusion is quite common, being seen in at least 20% of patients with pneumonia, although it may be small. Other extrapulmonary manifestations include neurologic illness such as meningoencephalitis, meningitis, transverse myelitis, and cranial nerve palsies. The most common extrapulmonary finding is an IgM autoantibody that is directed against the I antigen on the red blood cell and causes cold agglutination of the erythrocyte. Although up to 75% of patients may have this antibody and a positive Coombs’ test, clinically significant autoimmune hemolytic anemia is uncommon. Other systemic complications include myocarditis, pericarditis, hepatitis, gastroenteritis, erythema multiforme, arthralgias, pancreatitis, generalized lymphadenopathy, and glomerulonephritis. The extrapulmonary manifestations may follow the respiratory symptoms by as long as 3 weeks. Gram-Negative Bacteria

The most common Gram-negative organism causing CAP is H. influenzae (see below). Enteric Gram-negatives are generally not common in CAP, unless the patient is elderly and has chronic cardiac or pulmonary disease, has healthcare associated pneumonia, or is alcoholic. In these patients, organisms such as E. coli and K. pneumoniae can be found. P. aeruginosa is an uncommon cause of CAP, but can be isolated from patients with CAP and bronchiectasis, and in those with severe forms of CAP, particularly in the elderly patient aged over 75.

Controversy has persisted about how commonly enteric Gram-negative bacteria are a cause of CAP. However, in one large study of hospitalized CAP patients, careful diagnostic testing identified Gram-negative enterics in 11%, with more than half of these being P. aeruginosa. Identified risk factors for Gramnegative infection were: probable aspiration, previous hospital admission within 30 days of admission, previous antibiotics within 30 days of admission, and presence of pulmonary comorbidity. Risk factors for P. aeruginosa were pulmonary comorbidity and previous hospitalization. Infection with a Gramnegative increased the chance of dying by more than threefold, with a mortality rate of 32%, and these patients also need ICU admission and mechanical ventilation more often than patients with other organisms. Anaerobes

These organisms have always been a concern in patients with poor dentition who aspirate oral contents, and those at risk have been patients with neurologic or swallowing disorders, as well as individuals who abuse alcohol and opiate drugs. Several recent studies have questioned whether these organisms are really common in patients with risk factors for aspiration. In one evaluation of residents of longterm care facilities who had severe aspiration pneumonia (defined by the presence of risk factors for oropharyngeal aspiration), requiring ICU admission, bacteriology was determined by protected bronchoalveolar lavage (BAL) within 4 h of admission. When a pathogen was identified, the organisms were: enteric Gram-negatives in 49%, anaerobes in 16%, and S. aureus in 12%. Many of the anaerobes were recovered with aerobic Gram-negatives, and their presence did not correlate with oral hygiene, but the presence of Gram-negatives did correlate with functional status, being more common in patients who were totally dependent. Many patients received inadequate therapy for anaerobes, yet most recovered, raising a question about whether infection with these organisms really needs to be treated. These findings suggest that anaerobes may not always be pathogens, but may be colonizers in the institutionalized elderly, including those with aspiration risk factors. Haemophilus influenzae

This Gram-negative coccobacillary rod can occur in either a typable, encapsulated form or a nontypable, unencapsulated form. The encapsulated organism can be one of seven types, but type B accounts for 95% of all invasive infections. Encapsulated organisms require a more elaborate host response, and

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thus they are more virulent than unencapsulated organisms. However several studies have shown that in adults, particularly those with COPD, infection with unencapsulated bacteria is more common than infection with encapsulated organisms. The organism may cause bacteremic pneumonia in some patients, particularly in those with segmental pneumonias as opposed to those with bronchopneumonia. The encapsulated type B organism is more common in patients with segmental pneumonia than in those with bronchopneumonia. Most patients with this infection have some underlying illness such as alcoholism, smoking history, or COPD. Staphylococcus aureus

CAP can also be caused by S. aureus, which can lead to severe illness and to cavitary lung infection. This organism can also seed the lung hematogenously from a vegetation in patients with right-sided endocarditis or from septic venous thrombophlebitis (from central venous catheter or jugular vein infection). When a patient develops post influenza pneumonia S. aureus can lead to secondary bacterial infection, along with pneumococcus and H. influenzae. Most recently, there have been reports of CAP caused by methicillin-resistant S. aureus (MRSA), and if this becomes a more common occurrence, it may change the way this disease is treated empirically. To date, most patients with MRSA CAP have had a severe necrotizing pneumonia, generally following influenza or another viral infection. The organism responsible has had a specific virulence factor, the Panton–Valentine leukocidin, and all the organisms causing pneumonia appear to be genetically related. Viruses

The incidence of viral pneumonia is difficult to define, but during epidemic times, influenza should be considered, and it can lead to a primary viral pneumonia, or to secondary bacterial pneumonia. One careful study of over 300 non-immune-compromised CAP patients collected paired sera for respiratory viruses, and found that 18% had viral pneumonia, with about half being pure viral infection and the others being mixed with bacterial pneumonia. Patients with congestive heart failure were at more risk of pure viral pneumonia than they were of pneumococcal infection. Influenza (A more than B), parainfluenza, and adenovirus were the most commonly identified viral causes of CAP. While influenza A and B still remain the most common causes of viral pneumonia, vigilance to new agents is essential as evidenced by the recent experience with SARS, which demonstrated the potential

of epidemic, person-to-person spread of virulent respiratory viral infection. Continued concern about epidemic viral pneumonia remains, with the current worry being focused on avian influenza, and bioterrorism with agents such as smallpox.

Clinical Features Symptoms and Physical Findings

Patients with an intact immune system who develop CAP generally have respiratory symptoms such as cough, sputum production, and dyspnea, along with fever and other complaints. Cough is the most common finding, and is present in up to 80% of all patients, but is less common in those who are elderly, those with serious comorbidity, or individuals coming from nursing homes. The elderly generally have fewer respiratory symptoms than a younger population, and the absence of clear-cut respiratory symptoms and an afebrile status have themselves been predictors of an increased risk of death. This may be the consequence of nonrespiratory presentations being an indication of an impaired immune response, as well as a factor leading to delayed presentation to medical attention and recognition of the correct diagnosis. Pleuritic chest pain is also commonly seen in patients with CAP, but its absence has been identified as a poor prognostic finding. In the elderly patient, pneumonia can have a nonrespiratory presentation with symptoms of confusion, falling, failure to thrive, altered functional capacity, or deterioration in a pre-existing medical illness, such as congestive heart failure. In general, overall symptoms are less prominent in those above age 65 than in those who are younger. Patients with advanced age generally also have a longer duration of symptoms such as cough, sputum production, dyspnea, fatigue, anorexia, myalgia, and abdominal pain than younger patients. Studies have found no association between the type of etiologic microorganisms and the clinical presentation of CAP, except for pleuritic chest pain, which is likely to be more common in pneumonia caused by bacterial pathogens such as S. pneumoniae than in nonbacterial pneumonia. Delirium or acute confusion can be more frequent in the elderly patients with pneumonia than in age-matched controls who do not have pneumonia. Very few elderly patients with pneumonia are considered well nourished, with kwashiorkor-like malnutrition being the predominant type of nutritional defect, and the one associated with delirium on initial presentation. Physical findings of pneumonia include tachypnea, crackles, rhonchi, and signs of consolidation

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(egophony, bronchial breath sounds, dullness to percussion). Patients should also be examined for signs of pleural effusion. In addition, extrapulmonary findings should be sought to rule out metastatic infection (arthritis, endocarditis, meningitis), or to add to the suspicion of an ‘atypical’ pathogen such as M. pneumoniae or C. pneumoniae. One of the most important evaluations in any patient suspected of having pneumonia is a measurement of respiratory rate. In the elderly, an elevation of respiratory rate may be the initial presenting sign of pneumonia, preceding other clinical findings by as much as 1–2 days. In prospective evaluations in a long-term care setting, most patients who were diagnosed with lower respiratory tract infection had a respiratory rate above the normal range of 16– 25 min 1, and in general the elevated rate preceded other clinical findings. Although this finding is certainly not specific, it appears to be a very sensitive indicator of the presence of respiratory infection. In general, tachypnea is the most common finding in elderly patients with pneumonia, being present in over 60% of all patients, and being present more often in the elderly than in younger patients with pneumonia. Measurement of respiratory rate not only has diagnostic value, but also prognostic significance. In evaluating patients with CAP, the finding of a respiratory rate 430 min 1 is one of several factors associated with increased mortality. Typical versus Atypical Pneumonia Syndromes

In the past, the clinical and radiographic features of CAP have been characterized as fitting into a pattern of either ‘typical’ or ‘atypical’ symptoms, and the pattern was used to predict a specific etiologic agent. Recent studies have shown that this approach is not highly accurate, and there is only a weak relationship between clinical features and the etiologic pathogen, primarily because host as well as pathogen factors play a role in defining patient symptoms. The typical pneumonia syndrome is characterized by sudden onset of high fever, shaking chills, pleuritic chest pain, lobar consolidation, a toxic appearing patient, with the production of purulent sputum. Although this pattern has been attributed to pneumococcus and other bacterial pathogens, these organisms do not always lead to such classic symptoms, especially in the elderly, as discussed above. The atypical pneumonia syndrome, which is characterized by a subacute illness, nonproductive cough, headache, diarrhea, or other systemic complaints, is usually the result of infection with M. pneumoniae, C. pneumoniae, Legionella sp., or viruses. However, patients with impaired immune responses (especially

the elderly with chronic illness) may present in this fashion, even with bacterial pneumonia. Clinical features have been shown to be only about 40% accurate in differentiating pneumococcus, M. pneumoniae, and other pathogens from one another. In addition, careful comparisons of patients with S. pneumoniae, H. influenzae, L. pneumophila, and C. pneumoniae have shown no significant differences in their clinical presentations. The limitations of clinical features in defining the microbial etiology also apply to evaluations of radiographic pattern. Clinical Assessment of Severity of Pneumonia

One of the most important patient assessments is to define severity of illness, which can be used to guide decisions about whether to hospitalize a patient, and if so, whether to admit the patient to the ICU. While a number of prediction models have been developed to predict severity of illness and to guide the admission decision, no rule is absolute and the decision to admit a patient should be based on social as well as medical considerations, and remains an ‘art of medicine’ determination. In general the hospital should be used to observe patients who have multiple risk factors for a poor outcome, those who have decompensation of a chronic illness, or those who need therapies not easily administered at home (oxygen, intravenous fluids, cardiac monitoring). Risk factors for a poor outcome include: a respiratory rate X30 min 1, age X65 years, systolic blood pressure o90 mmHg, diastolic blood pressure p60 mmHg, multilobar pneumonia, confusion, blood urea nitrogen 419.6 mg dl 1, PaO2 o60 mmHg, PaCO2 450 mmHg, respiratory or metabolic acidosis, or signs of systemic sepsis. The two best-studied and widely regarded prediction rules for pneumonia severity are the pneumonia severity index (PSI) and the CURB-65 rule, a modification of a prognostic model developed by the British Thoracic Society. The PSI uses a number of demographic and historical findings, physical findings, and laboratory data to assign a score to each patient, and the score is used to categorize patients into one of five classes, each with a different risk of death. This tool has worked very well to define mortality risk, but has had variable success in predicting need for admission, is cumbersome to use, and does not discriminate very well among the most severely ill patients. The CURB-65 rule is simpler, using only five assessments: confusion, urea47 mmol l 1 , respiratory % systolic % pressureo90 mmHg rateX30%min 1 , blood % or p60 mm diastolic, and age X65 years. Each of the five criteria is scored and as the score rises from 0 to 5, mortality risk rises. In recent studies, both tools

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have worked equally well to identify patients at low risk of dying, but the CURB-65 has been more discriminating in recognizing patients who need ICU care (score of at least 3) and who have the highest risk of death. A major difference between the two models is that the PSI weights advanced age and chronic illness very heavily, while the CURB-65 model includes age as only one of several risk factors, and comorbid illness is not measured, but instead most of the score is based on acute physiologic abnormalities. None of the prediction models includes social factors in the scoring system, and clearly these issues need to be included in patient assessment, paying attention to whether the patient has a stable home environment for outpatient care, an ability to take oral medications, the absence of acute alcohol or drug intoxication, and stability of other acute and chronic medical problems. There is no specific rule for who should be admitted to the ICU, but in general the ICU is used for approximately 10% of all CAP patients, and this population has a mortality rate of at least 30%, compared to a mortality rate of 12% for all admitted patients, and a 1–5% mortality rate for outpatients. There is some debate about the benefit of ICU care for patients with CAP, but the benefit seems most certain if patients are admitted early in the course of severe illness, making assessment of mortality risk an important clinical assessment. Criteria for ICU admission, in addition to need for mechanical ventilation and septic shock, are the presence of at least two of the following: PaO2/FiO2 ratio o250, multilobar infiltrates, systolic blood pressure o90 mmHg. Radiographic Features

The entry point into most treatment algorithms for CAP is the presence of a new radiographic infiltrate, but not all patients with this illness will have this finding when first evaluated. Even when the radiograph is negative, if the patient has appropriate symptoms and focal physical findings, pneumonia may still be present. In one study, nearly 50 patients with clinical signs and symptoms of CAP were evaluated with both a chest radiograph and a high-resolution computed tomography (CT) scan of the chest and there were almost 20% of patients identified by CT scan to have pneumonia who had a negative chest radiograph. In addition, more extensive abnormalities were found on CT scan in many patients than were present on the chest radiograph. The findings of this study confirm the need to repeat the chest film after 24–48 h in certain symptomatic patients with an initially negative chest film. The reason for an initially negative chest film is not clear, but some

studies have suggested that febrile and dehydrated patients can have a normal radiograph when first admitted, although the idea of hydrating a pneumonia is in the realm of ‘conventional wisdom’ and anecdotal reports. Although a variety of radiographic patterns can be seen in pneumonia, and radiographic findings cannot generally be used to predict microbial etiology in CAP, there are certain patterns that have been associated with specific pathogens. Focal consolidation can be seen with infections caused by pneumococcus, K. pneumoniae (with upper lobe consolidation and the classic bulging down of the upper lobe fissure), aspiration (especially if in the lower lobes or other dependent segments), S. aureus, H. influenzae, M. pneumoniae, and C. pneumoniae. Interstitial infiltrates should suggest viral pneumonia as well as infection due to M. pneumoniae, C. pneumoniae, Chlamydia psittaci, and Pneumocystis jerovici. Lymphadenopathy with an interstitial pattern should raise concerns about anthrax, Francisella tularensis, and C. psittaci, while adenopathy can be seen with focal infiltrates in tuberculosis, fungal pneumonia, and bacterial pneumonia. Cavitation can be the result of an aspiration lung abscess, or infection with S. aureus, aerobic Gram-negatives (including P. aeruginosa), tuberculosis, fungal infection, nocardia, and actinomycosis. Pleural effusion may appear on the initial chest radiograph and if present, it is necessary to distinguish empyema from a simple parapneumonic effusion by sampling the pleural fluid. Pneumococcal pneumonia is the infection most commonly complicated by effusion (36–57% of patients), but other pathogens causing effusion include H. influenzae, M. pneumoniae, Legionella, and tuberculosis.

Diagnostic Testing Once the clinical evaluation suggests the presence of pneumonia, the diagnosis should be confirmed by chest radiograph. Although some patients may have clinical findings of pneumonia (focal crackles, bronchial breath sounds), and a negative chest radiograph, the need for antibiotic therapy of CAP has been established in studies of patients with a radiographic infiltrate. In some populations, such as the elderly and chronically ill, the clinical diagnosis is difficult, and for these individuals, a chest radiograph is essential to define the presence of parenchymal lung infection. Although a radiograph is recommended in all outpatients and inpatients, it may be impractical in some settings outside of the hospital. A chest radiograph not only confirms the presence of pneumonia, but can be used to identify complicated illness and to grade severity of disease, by noting

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such findings as pleural effusion and multilobar illness. As mentioned above, there is no specific radiographic pattern that can be used to define the etiologic pathogen of CAP, but certain findings can be used to suggest specific organisms, such as anaerobes if a cavitary infiltrate is found, or tuberculosis if a posterior upper lobe infiltrate is present. Even with extensive testing, most patients do not have a specific pathogen identified, and many who do, have this diagnosis made days or weeks later, as the results of cultures or serologic testing become available. In addition, recent studies have emphasized the mortality benefit of prompt administration of effective antibiotic therapy, with a goal of administering intravenous antibiotics within 4 h of admission to the hospital, for those with moderate to severe illness. Thus, therapy should never be delayed for the purpose of diagnostic testing, and the diagnostic workup should be streamlined, with all patients receiving empiric therapy based on predicting the most likely pathogens, as soon as possible. Recommended testing for outpatients is limited to a chest radiograph and pulse oximetry, if available, with sputum culture being considered in patients suspected of having an unusual or drug-resistant pathogen. For admitted patients, diagnostic testing should include a chest radiograph, assessment of oxygenation (pulse oximetry or blood gas, the latter if retention of carbon dioxide is suspected), and routine admission blood work. If the patient has a pleural effusion, this should be tapped and the fluid sent for culture and biochemical analysis. Although blood cultures are positive in only 10– 20% of CAP patients, they can be used to define a specific diagnosis and to define the presence of drugresistant pneumococci. One concern with blood cultures is that they be limited to patients with a reasonable likelihood of being positive. If low-risk patients routinely have blood cultures, it is possible that the frequency of false positives may exceed the true positives, and lead to inaccurate and unnecessary therapy. One study of a large Medicare database found that predictors of bacteremia, among admitted patients, were: absence of prior antibiotics, comorbid liver disease, systolic blood pressure o90 mmHg, fever o35 or 4401C, pulse 4125 min 1, blood urea nitrogen 430 mg dl 1, serum sodium o130, white blood cell count o5000 or 420 000. Based on these findings, blood cultures will have the greatest yield of true positive results in patients who have at least one of these risk factors above, or if none, when there is also no history of receiving antibiotics prior to admission. Sputum culture should be limited to patients suspected of infection with a drug-resistant or unusual

pathogen. The role of Gram’s stain of sputum to guide initial antibiotic therapy is controversial, but this test has its greatest value in guiding the interpretation of sputum culture, and can be used to define the predominant organism present in the sample. The role of Gram’s stain in focusing initial antibiotic therapy is uncertain since the accuracy of the test to predict the culture recovery of an organism such as pneumococcus depends on the criteria used. Investigators have shown the practical limitations of the test, because fewer than half of all patients can even produce a sputum sample, only about half of these are valid, and very few are diagnostic, and thus it is uncommon to choose an antibiotic directed to the diagnostic result. Even if Gram’s stain findings are used to focus antibiotic therapy, this would not allow for empiric coverage of atypical pathogens which might be present with pneumococcus, as part of a mixed infection. In spite of these limitations, Gram’s stain can be used to broaden initial empiric therapy by enhancing the suspicion for organisms that are not covered in routine empiric therapy (such as S. aureus being suggested by the presence of clusters of Gram-positive cocci, especially during a time of epidemic influenza). Routine serologic testing is not recommended. However, in patients with severe illness, the diagnosis of Legionella can be made by urinary antigen testing, which is the test that is most likely to be positive at the time of admission, but a test that is specific only for serogroup 1 infection. Commercially available tests for pneumococcal urinary antigen have been developed, but their role in the clinical management of CAP is still being defined. Bronchoscopy is not indicated as a routine diagnostic test, and should be restricted to immune-compromised patients, and to selected individuals with severe forms of CAP.

Therapy The initial therapy for patients with CAP should be focused on the provision of antibiotics and supportive care. Antibiotics are given on a empiric basis, since it is virtually impossible to rely on clinical or laboratory data to provide an exact etiologic pathogen, at the time of initial diagnosis, and thus therapy must be focused on the pathogens most likely to be present for a given type of patient. Supportive care includes oxygen as needed, hydration, possibly chest physiotherapy, as well as bronchodilators and expectorants. For more severely ill patients, it may be necessary to support the blood pressure with pressors, use corticosteroids for possible relative adrenal insufficiency, and provide other therapies directed at signs of sepsis (such as drotrecogin alpha in selected

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patients). There may also be benefit from the routine use of corticosteroids in severe pneumonia, for unclear reasons, because some studies have shown a survival benefit to this intervention. Antibiotic Therapy

Initial empiric therapy is selected by categorizing patients on the basis of place of therapy (outpatient, inpatient, ICU), severity of illness and the presence or absence of cardiopulmonary disease or specific modifying factors that make certain pathogens more likely. By using these factors, a set of likely pathogens can be predicted for each type of patient (Table 1), and this information can be used to guide therapy. If a specific pathogen is subsequently identified by diagnostic testing, then therapy can be focused. In choosing empiric therapy of CAP, certain principles should be followed (Table 3). Empiric therapy for outpatients with no cardiopulmonary disease or modifying factors should be with a new oral macrolide (azithromycin or clarithromycin) or a tetracycline. Although erythromycin has been used for these patients, its value is limited by its lack of coverage of H. influenzae, and a higher frequency of intestinal complications (nausea, vomiting) than with the newer macrolides. Therapy with an antipneumococcal quinolone (gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin) is not necessary in these outpatients, because they are not at risk for organisms such as DRSP and enteric Gram-negatives. However, outpatients with cardiopulmonary disease

and/or modifying factors, should not receive macrolide monotherapy, but should be treated with either a selected oral beta-lactam (Table 3) with a macrolide or with monotherapy using an oral antipneumococcal quinolone (gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin) alone. The ketolide telithromycin can also be used in this population as oral monotherapy for patients at risk for DRSP, but with no risk factors for aspiration or for enteric Gramnegatives. For the non-ICU inpatient, therapy can be with an intravenous macrolide (azithromycin) alone, provided that the patient has no underlying cardiopulmonary disease, and no risk factors for infection with DRSP, enteric Gram-negatives, or anaerobes. Although very few patients of this type are admitted to the hospital, macrolide monotherapy has been documented to be effective in this population. The majority of inpatients will have cardiopulmonary disease and/or modifying factors, and they can be treated with either a selected intravenous beta-lactam (Table 3) combined with a macrolide, or they can receive an intravenous antipneumococcal quinolone (gatifloxacin, levofloxacin, moxifloxacin) alone. From the available data, it appears that either regimen is therapeutically equivalent, and although not proven, it may be useful to use these two types of regimens interchangeably, striving for ‘antibiotic heterogeneity’, selecting an agent in a different class from what the patient received in the past 3–6 months. Although oral quinolones may be as effective as intravenous quinolones for admitted patients

Table 3 Principles of antibiotic therapy Administer initial antibiotic therapy within 4 h of arrival to the hospital Treat all patients for pneumococcus and for the possibility of atypical pathogen coinfection: use either a macrolide alone (selected patients with no cardiopulmonary disease or modifying factors) or for those outpatients with cardiopulmonary disease or modifying factors: use monotherapy with a quinolone, a ketolide (if no risk factors for enteric Gram-negatives), or the combination of a selected beta-lactam with a macrolide or ketolide or tetracycline Limit macrolide monotherapy to outpatients or inpatients with no risk factors for DRSP, enteric Gram-negatives, or aspiration Limit ketolide monotherapy to outpatients with risk factors for DRSP, but no risk factors for enteric Gram-negatives Provide initial therapy for hospitalized patients with an intravenous agent, or if oral only, use a quinolone For patients at risk for DRSP, acceptable oral beta-lactams are: cefpodoxime, cefuroxime, high-dose ampicillin (3 g day 1) or amoxicillin/clavulanate (up to 4 g day 1) For inpatients at risk for DRSP, the selected acceptable intravenous beta-lactams include: ceftriaxone, cefotaxime, ertapenem, ampicillin/sulbactam Limit antipseudomonal therapy to patients with risk factors: Antipseudomonal agents include: beta-lactams such as cefepime, imipenem, meropenem, piperacillin/tazobactam; quinolones such as ciprofloxacin or high-dose levofloxacin (750 mg day 1); and aminoglycosides such as amikacin, gentamicin, or tobramycin; aztreonam is a monobactam that is also active, but cannot be used alone The new antipneumococcal quinolones, in order of decreasing antipneumococcal activity are: gemifloxacin (oral only), moxifloxacin (oral and intravenous), gatifloxacin (oral and intravenous), levofloxacin (oral and intravenous) Never use monotherapy for patients with severe CAP. If no pseudomonal risk factors use a selected beta-lactam (above) plus a macrolide or antipneumococcal quinolone. For those with pseudomonal risk factors, use an antipseudomonal beta-lactam (above) plus either ciprofloxacin/high-dose levofloxacin or the combination of an aminoglycoside with either a macrolide or antipneumococcal quinolone (above) Vancomyin and linezolid should be used rarely and only in those with severe CAP and either meningitis (vancomycin) or severe necrotizing pneumonia after influenza (either agent)

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with moderately severe illness, most admitted patients should receive initial therapy intravenously to be sure that the medication has been absorbed. Once the patient shows a good clinical response, oral therapy can be started. Selected inpatients with mild to moderate disease can initially be treated with the combination of an intravenous beta-lactam and an oral macrolide, switching to exclusively oral therapy once the patient shows a good clinical response. In the ICU population, all individuals should be treated for DRSP and atypical pathogens, but only those with appropriate risk factors (above) should have coverage for P. aeruginosa. Since the efficacy, dosing, and safety of quinolone monotherapy has not been established for ICU-admitted CAP patients, the therapy for such patients, in the absence of pseudomonal risk factors, should be with a selected intravenous beta-lactam, combined with either an intravenous macrolide or an intravenous quinolone. For patients with pseudomonal risk factors, therapy can be with a two-drug regimen, using an anti-pseudomonal beta-lactam (cefepime, imipenem, meropenem, piperacillin/tazobactam) plus ciprofloxacin or highdose levofloxacin, or alternatively, with a three-drug regimen, using an anti-pseudomonal beta-lactam plus an aminoglycoside plus either an intravenous nonpseudomonal quinolone or macrolide. The antipneumococcal quinolones are being widely used in both inpatients and outpatients as monotherapy because as a single drug, given once daily, it is possible to cover pneumococcus (including DRSP), Gram-negatives, and atypical pathogens. Quinolones penetrate well into respiratory secretions, and are highly bioavailable, achieving the same serum levels with oral or intravenous therapy, thereby allowing moderately ill outpatients to be managed effectively with oral antibiotics. There are differences among the available agents in their intrinsic activity against pneumococcus, and, based on MIC data, these agents can be ranked from most to least active as: gemifloxacin (available only in oral form), moxifloxacin, gatifloxacin, and levofloxacin. Some data suggest a lower likelihood of both clinical failures and the induction of pneumoccal resistance to quinolones, if the more active agents are used in place of the less active agents. In addition to the general approach to therapy outlined above, there are several other therapeutic issues in the management of CAP, which are highlighted in Table 3. These include the need for timely administration of initial antibiotic therapy, the limited use of therapy directed at methicillin-resistant S. aureus, the need for routine atypical pathogen coverage for all patients, and the emphasis on using highly active agents in all patients with risk factors

for infection with DRSP. The reason for this last recommendation is because if a patient is at risk for infection with DRSP, use of a highly active agent is not only likely to minimize the risk of treatment failure, but may also rapidly and reliably eradicate pneumococcal organisms that have low levels of resistance, so that there is less selection pressure for emergence of organisms with high level of resistance. Response to Therapy

The majority of outpatients and inpatients will respond rapidly to the empiric therapy regimens suggested above, with clinical response usually occurring within 24–72 h. Clinical response for inpatients is defined as improvement in symptoms of cough, sputum production, and dyspnea, along with ability to take medications by mouth, and an afebrile status for at least two occasions 8 h apart. When a patient has met these criteria for clinical response, it is appropriate to switch to an oral therapy regimen and to discharge the patient, if he is otherwise medically and socially stable. Radiographic improvement lags behind clinical improvement, and in a responding patient, a chest radiograph is not necessary until 2–4 weeks after starting therapy. If the patient fails to respond to therapy in the expected time interval, then it is necessary to consider infection with a drug-resistant or unusual pathogen (tuberculosis, anthrax, C. burnetii, Burkholderia pseudomallei, Pasteurella multocida, endemic fungi, or hantavirus); a pneumonic complication (lung abscess, endocarditis, empyema); or a noninfectious process that mimics pneumonia (bronchiolitis obliterans with organizing pneumonia, hypersensitivity pneumonitis, pulmonary vasculitis, bronchoalveolar cell carcinoma, lymphoma, pulmonary embolus). The evaluation of the nonresponding patient should be individualized but may include CT scanning of the chest, pulmonary angiography, bronchoscopy, and occasionally open lung biopsy. Prevention

Prevention of CAP is important for all groups of patients, but especially the elderly, who are at risk for both a higher frequency of infection and a more severe course of illness. Appropriate patients should be vaccinated with both pneumococcal and influenza vaccines, and cigarette smoking should be stopped in all at-risk patients. Even for the patient who is recovering from CAP, immunization while in the hospital is appropriate to prevent future episodes of infection, and the evaluation of all patients for vaccination need and the provision of information about

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smoking cessarion are now performance standards used to evaluate the hospital care of CAP patients. Pneumococcal vaccine Pneumococcal capsular polysaccharide vaccine can prevent pneumonia in otherwise healthy populations, as was initially demonstrated in South African gold miners and American military recruits. The benefits in those of advanced age or with underlying conditions in nonepidemic environments are less clearly defined. In immunocompetent patients over the age of 65, effectiveness has been documented to be 75%. The vaccine efficacy has ranged from 65% to 84% in patients with diabetes mellitus, coronary artery disease, congestive heart failure, chronic pulmonary disease, and anatomic asplenia. Its effectiveness has not been proven in immune-deficient populations such as those with sickle cell disease, chronic renal failure, immunoglobulin deficiency, Hodgkin’s disease, lymphoma, leukemia, and multiple myeloma. A single revaccination is indicated in a person who is aged over 65 years who initially received the vaccine 46 years earlier and was less than 65 years old on first vaccination. If the initial vaccination was given at age 65 or older, repeat is only indicated (after 6 years), if the patient has anatomic or functional asplenia, or has one of the immune compromising conditions listed above. The available pneumococcal vaccine is widely underutilized, and the 23-valent pneumococcal vaccine carries the pneumococcal serotypes causing the majority of clinical infection seen in the US. A proteinconjugated pneumococcal vaccine has been licensed and it appears more immunogenic than the older vaccine, but it contains only seven serotypes, and is recommended for healthy children, and has not yet been shown to be effective in adults for preventing both pneumococcal bacteremia and hospitalization for pneumonia. Hospital-based immunization for most admitted patients could be highly effective, since over 60% of all patients with CAP have been admitted to the hospital, for some indication, in the preceding 4 years, and hospitalization could be defined as an appropriate time for vaccination. Influenza vaccination The current vaccine includes three strains: two influenza A strains (H3N2 and H1N1) and one influenza B strain. Vaccination is recommended for all patients aged over 65, and to those with chronic medical illness (including nursing home residents), and to those who provide healthcare to patients at risk for complicated influenza. It is given yearly, usually between September and mid November (in the northern hemisphere). When the vaccine matches the circulating strain of influenza, it

can prevent illness in 70–90% of healthy persons aged over 65. For older persons with chronic illness, the efficacy is less, but the vaccine can still attenuate the influenza infection and lead to fewer lower respiratory tract infections and the associated morbidity and mortality that follow influenza. See also: Endotoxins. Leukocytes: Neutrophils. Pleural Effusions: Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube; Parapneumonic Effusion and Empyema. Pneumonia: Overview and Epidemiology; Parasitic. Stem Cells. Systemic Disease: Sickle Cell Disease. Ventilation, Mechanical: Ventilator-Associated Pneumonia.

Further Reading Arancibia F, Bauer TT, Ewig S, et al. (2002) Community-acquired pneumonia due to Gram-negative bacteria and Pseudomonas aeruginosa: incidence, risk and prognosis. Archives of Internal Medicine 162: 1849–1858. El-Solh AA, Pietrantoni C, Bhat A, et al. (2003) Microbiology of severe aspiration pneumonia in institutionalized elderly. American Journal of Respiratory and Critical Care Medicine 167: 1650–1654. Ewig S, De Roux A, Bauer T, et al. (2004) Validation of predictive rules and indices of severity for community acquired pneumonia. Thorax 59: 421–427. Feikin DR, Schuchat A, Kolczak M, et al. (2000) Mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance, 1995–1997. American Journal of Public Health 90: 223–229. File TM and Niederman MS (2004) Antimicrobial therapy of community-acquired pneumonia. Infectious Disease Clinics of North America 18: 993–1016. Fine MJ, Auble TE, Yealy DM, et al. (1997) A prediction rule to identify low-risk patients with community-acquired pneumonia. New England Journal of Medicine 336: 243–250. Gleason PP, Meehan TP, Fine JM, Galusha DH, and Fine MJ (1999) Associations between initial antimicrobial therapy and medical outcomes for hospitalized elderly patients with pneumonia. Archives of Internal Medicine 159: 2562–2572. Houck PM, Bratzler DW, Nsa W, Ma A, and Bartlett JG (2004) Timing of antibiotic administration and outcomes for Medicare patients hospitalized with community-acquried pneumonia. Archives of Internal Medicine 164: 637–644. Houck PM, MacLehose RF, Niederman MS, and Lowery JK (2001) Empiric antibiotic therapy and mortality among Medicare pneumonia inpatients in 10 Western states: 1993, 1995, 1997. Chest 119: 1420–1426. Kaplan V, Clermont G, Griffin MF, et al. (2003) Pneumonia: still the old man’s friend? Archives of Internal Medicine 163: 317– 323. Lieberman D, Schlaeffer F, Boldur I, et al. (1996) Multiple pathogens in adult patients admitted with community-acquired pneumonia: a one year prospective study of 346 consecutive patients. Thorax 51: 179–184. Lim WS, van der Erden MM, Laing R, et al. (2003) Defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study. Thorax 58: 377–382. Martinez JA, Horcajada JP, Almela M, et al. (2003) Addition of a macrolide to a b-lactam-based empirical antibiotic regimen is

PNEUMONIA / Fungal (Including Pathogens) 431 associated with lower in-patient mortality for patients with bacteremic pneumococcal pneumonia. Clinical Infectious Diseases 36: 389–395. Metlay JP and Fine MJ (2003) Testing strategies in the initial management of patients with community-acquired pneumonia. Annals of Internal Medicine 138: 109–118. Metersky ML, Ma A, Bratzler DW, and Houck PM (2004) Predicting bacteremia in patients with community-acquired pneumonia. American Journal of Respiratory and Critical Care Medicine 169: 342–347. Niederman MS, Mandell LA, Anzueto A, et al. (2001) Guidelines for the management of adults with community-acquired lower respiratory tract infections: diagnosis, assessment of severity, antimicrobial therapy and prevention. American Journal of Respiratory and Critical Care Medicine 163: 1730–1754. Syrjala H, Broas M, Suramo I, Ojala A, and Lahde S (1998) High resolution computed tomography for the diagnosis of community-acquired pneumonia. Clinical Infectious Diseases 27: 358–363.

Fungal (Including Pathogens) R Boyton and D M Altmann, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Fungal infection generally originates from an exogenous, environmental source acquired by inhalation, ingestion, or trauma. Fungi are rarely associated with significant disease in the normal host, although many more cause serious disease in the immunocompromised host. Opportunistic fungal infections have become increasingly common, especially in AIDS patients, and constitute a major cause of morbidity and mortality in this group. Pathogenicity depends on the interplay between components of the host immune system and specific features of the fungal strain. Considerable efforts are underway to conduct genetic characterization of fungal virulence and host susceptibility factors in disease. Genome projects have been undertaken for a number of the key fungal pathogens. Here we consider the etiology, pathology, clinical features, management, and molecular mycology of Blastomycosis, Coccidioidomycocis, Histoplasmosis, Paracoccidioidomycosis, Aspergillosis, Candidosis, Cryptococcosis, and Mucormycosis.

Introduction Fungi are a diverse group of eukaryotic organisms that have cell walls and no chlorophyll. They exist in nature as parasites or saprobes, dependent on living or dead organic matter for nutrition. About 250 000 species of fungi have been described, but less than 200 have been associated with human disease. Fungal infection usually originates from an exogenous, environmental source acquired by inhalation, ingestion, or trauma. Very few fungi cause significant disease in the normal host, but many more can cause disease in the immunocompromised host. Thus,

pathogenicity can be considered to depend on the interplay between aspects of the host immune system and specific features of the fungal strain. Considerable efforts are underway to conduct genetic characterization of fungal virulence and host susceptibility factors in disease. Genome projects have been undertaken for a number of key pathogens (see ‘Relevant websites’). Systemic mycoses often start in the lung, but may spread to other organs. They are usually acquired by inhaling spores of organisms growing as saprobes in the soil or decomposing organic material, or as plant pathogens. The organisms that cause systemic fungal infection in humans can be divided into two groups, true pathogens and opportunists. True pathogens, able to invade and grow in tissues of a normal host, include Histoplasma capsulatum, Coccidoides immitis, Blastomyces dermatidis and Paracoccidioides braziliensis (Table 1). In general, infections with true pathogenic fungi are asymptomatic or mild, of short duration, occur in regions endemic for the fungus, and follow inhalation of spores from the environment. Individuals either recover from infection with protective immunity to re-infection or sometimes develop chronic granulomatous disease. In the immunocompromised host, however, true pathogenic fungi may cause significant, relapsing, life-threatening disease that is difficult to treat. Opportunist fungi, such as Aspergillus fumigatus, are less virulent and less well-adapted organisms that are only able to invade the tissues of a debilitated or immunocompromised host. Resolution of infection does not confer protection and re-infection or reactivation may occur. Many of these organisms are ubiquitous saprobes found in the soil, decomposing organic Table 1 Pathogens that cause fungal pneumonia according to the immunological status of the host Normal host Blastomyces dermatidis Coccidoides immitis Histoplasma capsulatum Paracoccidioides braziliensis Normal and immunocompromised host Aspergillus spp. Cryptococcus neoformans Geotrichum spp. Penicillium marneffei Sporothrix schenckii Immunocompromised host Candida spp. Fusarium spp. Hansenula spp. Mucormycosis Penicillium spp. Pneumocystis jiroveci Pseudoallescheria boydii

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matter, or in the air. Diseases caused by opportunistic fungi in the immunocompromised host include aspergillosis, candidosis, cryptococcosis, and mucormycosis and pneumocystis pneumonia (Table 1). Pneumocystis pneumonia will not be discussed here as it is covered in the article Pneumonia: The Immunocompromised Host of this encyclopedia. Effective phagocytic function is important in host defense to fungal infection, with key roles also demonstrated for macrophage, neutrophil, and T-cell function. Endothelial, alveolar epithelial, natural killer, and dendritic cells are also involved in infection. Pathogen-associated molecular patterns (PAMP) and pattern recognizing receptors (PPR) such as tolllike receptors TLR-2 and TLR-4 may influence the inflammatory response, dominant cytokine profile, and T-cell helper response to infection. An interesting point with respect to potential development of vaccines for use in immunocompromised hosts is that experimental vaccination studies for Blastomyces and Histoplasma suggest that it may be possible to generate effective immunity with an intact CD8 Tcell compartment, in the absence of any functional CD4 T cells. A number of projects to develop vaccines against a wide range of fungi are underway. A wide variety of experimental, fungal vaccine approaches are being attempted using formaldehydekilled cells, glycoproteins, peptides, or DNA vaccines as immunogens. In addition to the immunological status of the host, the geographical location and travel history are important diagnostic factors (Table 2). For example, C. immitis only occurs in alkaline soils in parts of

North America and Mexico where there are hot summers and few cold winter periods. The majority of cases of coccidioidomycosis, therefore, occur in these regions. By comparison, Cryptococcus neoformans, found in soil contaminated by pigeon droppings, causes cryptococcosis that is seen worldwide. There has been a dramatic increase in the range and number of reported fungal infections due to a combination of improved recognition and an increasing population of susceptible, debilitated, and/ or immunocompromised individuals. Furthermore, the true incidence is probably greater than actually reported because many fungal infections will go undiagnosed. Amphotericin B remains an effective treatment option for the majority of fungal infections. In addition, there are also newer antifungal agents that are easier to administer and/or better tolerated.

Histoplasmosis Etiology and Pathology

Histoplasmosis is caused by a diamorphic fungus called H. capsulatum that grows as a mold in nature and in a yeast phase producing round or oval cells 2–3  3–4 mm in diameter in human infection. The infective propagules are microcondia (2–5 mm in diameter) and macrocondia (8–14 mm in diameter). It is found in the US and Canada, South America, Africa, Australia, and East Asia. Areas with high prevalence of exposure are the valleys of the Mississippi and Ohio rivers in the US. The mold grows in areas where

Table 2 Pathogens that cause fungal pneumonia according to geographical location, travel history, and risk factors Geographical location

Risk factors Men4women Areas subject to flooding

Aspergillosis

North America Central and South America Africa Southwest US North Mexico Central US Canada Africa Australia East Asia South and Central America Mexico Worldwide

Candidosis Cryptococcosis Mucormycosis

Worldwide Worldwide Worldwide

Blastomycosis

Coccidiidomycosis Histoplasmosis

Paracoccidioidomycosis

Laboratory staff Soil contaminated by bird or bat feces COPD

Men4women Corticosteroid-dependent asthma Cystic fibrosis/bronchiectasis Pre-existing lung cavity Building sites, pepper, pot plants Exposure to bird guano Diabetes with metabolic acidosis Renal failure

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there are accumulations of bird or bat excreta in the soil and outbreaks of histoplasmosis infections have been linked to activities that generate an aerosol from the soil such as building work. Having entered the lung, the microcondia develop into a yeast phase and produce fungal heat shock protein (HSP 70) and several virulence determinants. H. capsulatum then becomes an intracellular pathogen of macrophages. It uses different host receptors for binding to macrophages (beta 2 integrins) than it uses for binding to dendritic cells (the fibronectin receptor). The infection is controlled by a T-cell response in normal hosts at day 10–14. Typically, there is a noncaseating granulomatous response. However, in immunocompromised individuals such as those with HIV infection, the fungal infection may progress. Reactivation may occur in immunosuppressed individuals with lymphoma or organ transplantation. Clinical Features

Most normal hosts exposed to inhaled spores are asymptomatic. However, the disease may present clinically with acute pulmonary histoplasmosis, chronic pulmonary histoplasmosis, or disseminated histoplasmosis depending on the host. About 1% of individuals develop an acute pulmonary histoplasmosis after an incubation period of 7–21 days. They usually present with non-specific symptoms of fever, headache, myalgia, loss of appetite, cough, and chest pain although a minority of patients (about 10%) develop arthralgia and erythema nodosum. The chest radiograph shows patchy or micronodular pulmonary infiltrates, hilar node enlargement, and occasionally pleural effusion (Figure 1). Most individuals recover without treatment after about one to three weeks. The chest radiograph may show a residual round nodule called a histoplasmoma. Chronic pulmonary histoplasmosis is a slow progressive disease that commonly occurs in patients with underlying chronic obstructive airways disease. Presenting symptoms include increased cough, fever, weight loss, malaise, and pleuritic chest pain of several weeks or months duration. The chest radiograph shows interstitial infiltrates in the apical segments of the upper lobes of the lung. Cavitation can occur resulting in hemoptysis, bacterial infection, abscess formation, and death. This occurs more commonly in the right upper lobe, but is bilateral in about a quarter of cases with infection eventually spreading to the lower lobes. Pleural effusion is not common, but pleural thickening adjacent to the lesion is. Disseminated histoplasmosis is a progressive disease, often lethal if left untreated, developing in

Figure 1 Histoplasmosis in a 30-year-old man with a three week history of progressive shortness of breath and fever. Chest radiograph showing bilateral pulmonary infiltrates. Reprinted by permission of Wheat LJ, Dewey C, Allen SD, Blue-Hnidy D, and Loyd J (2004) Pulmonary histoplasmosis syndromes: recognition, diagnosis, and management. Pulmonary fungal infections. Seminars in Respiratory and Critical Care Medicine 25: 129–144.

immunosuppressed individuals or predisposed groups such as infants and the elderly. Infants and immunocompromised individuals present unwell and feverish with weight loss, malaise, and loss of appetite of over 3 weeks duration. There is hepatosplenomegaly, anemia, and mucosal lesions. The chest radiograph shows diffuse interstitial infiltrates. In the normal host, disseminated histoplasmosis follows a more chronic and indolent course, with mucosal ulceration, meningitis or focal cerebral lesions, hepatic infection, adrenal gland destruction and rarely, endocarditis. Often in these cases the chest radiograph is normal. Management and Current Therapy

The key to definitive diagnosis is culture of H. capsulatum although identification can take up to 6 weeks. In disseminated disease, about three quarters of blood cultures, bone marrow cultures, lung lavage cultures, and a third of liver and urine cultures are positive in patients with HIV infection. Chronic pulmonary histoplasmosis often requires multiple cultures and acute pulmonary histoplasmosis is diagnosed in about 10% of cases by culture. Skin testing with histoplasmin is unreliable for diagnostic purposes as it reflects exposure and not disease.

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A fourfold rise in titer of serological tests from sequential specimens is a useful indication of active infection. Other useful tests include immunodiffusion, radioimmunoassay, and enzyme-linked immunosorbent assay (ELISA). Patients with the acute self-limiting disease usually recover completely without specific treatment. Untreated disseminated disease usually results in death and chronic pulmonary disease usually progresses if untreated. Treatment is usually with oral itraconazole or amphotericin B. Unless AIDS patients receive long-term therapy, they can relapse within a year of curative treatment.

Coccidioidomycosis Etiology and Pathology

Coccidioidomycosis is caused by infection with the dimorphic fungus called Coccidioides immitis. It is found growing in the soil in Southwestern US and parts of Central and South America as a mold with septate hyphae (Table 2). Some hyphae form arthroconidia that are resistant to drying. In the body, inhaled arthroconidia form spherules that are 50– 100 mm in diameter and mature to release endospores (2–3 mm in diameter). The endospores mature to form new spherules. The disease affects people who live in endemic areas as well as visitors and laboratory workers. The inhaled arthroconidia initially provoke attack by neutrophils that is followed by a granulomatous reaction. Clinical Features

The majority of individuals exposed to C. immitis arthrospores (about 80%) are asymptomatic. After an incubation period of one to four weeks, the remainder of exposed individuals will develop a mild to moderate flu-like illness that resolves spontaneously. Patients commonly present with fever, pleuritic chest pain and sometimes with cough, malaise, headache, myalgia, and loss of appetite. About half of the symptomatic patients develop a diffuse erythematosus maculopapular rash on the trunk and limbs and about a third will develop erythema nodosum or erythema multiforme accompanied by arthralgia and iritis. Chest radiographs show a patchy bronchopneumonia, sometimes with associated ipsilateral hilar adenopathy and pleural effusion. Persistent pulmonary infection occurs in a minority of cases (about 5%) with the presence of infiltrates, nodules, and cavitation. Disseminated coccidioidomycosis occurs in less than 1% of infected individuals. It is progressive, often results in death if untreated and tends to occur in immunosuppressed or predisposed

groups. It can occur following the initial infection or more commonly, following reactivation, in an immunosuppressed individual. There is widespread dissemination, with multiorgan disease such as skin lesions, arthritis, osteomyelitis, and meningitis. Other sites of infection include lymph nodes, spleen, liver, and adrenal glands. Management and Current Therapy

In endemic areas, a diagnosis of coccidioidomycosis may be suggested on the basis of clinical findings. In order to establish a clear diagnosis, the presence of spherules in biopsy or aspirated material and/or sputum is required. The coccidioidin skin test is an indicator of exposure. In the first 4 weeks of illness antibodies against C. immitis can be detected in the serum by immunodiffusion. The standard diagnostic test is a complement fixation test to detect IgG antibodies that develop 4–12 weeks after the onset of symptoms. Culture of C. immitis needs to be carried out with care, as there is danger of infection of laboratory staff by the concentrated, easily dispersed, and highly infectious arthrospores. The exoantigen test allows rapid identification. Treatment depends on the type of disease. Amphoterecin B is usually used in rapidly progressive disease. For uncomplicated primary infections, cavitatory disease, or disseminated disease of slow onset, either itraconazole or fluconazole may be used. Fluconazole is usually used as a first-line treatment for meningitis.

Blastomycosis Etiology and Pathology

Blastomycosis is caused by infection with the diamorphic fungus B. dermatitidis. In nature it grows as mycelium (2–3 mm diameter) with hyphae that bear conidia, the infective agent. In tissue it grows as yeast (8–15 mm diameter) and reproduces by budding. The disease occurs in Southcentral and Southeastern regions of North America, Central and South America, and parts of Africa. It affects men about ten times more commonly than women, and is associated particularly with areas close to water or those subject to flooding. If infection fails to be contained by the initial neutrophil response, T cell recruitment ensues, and granulomas form. The infection is usually confined to the lung and hilar nodes. Once introduced into a host, the fungus undergoes a temperatureinduced phase transition to the pathogenic yeast form. This leads to generation of a 120 kDa protein designated Blastomyces adhesin 1 (BAD1). BAD1 acts both as an adhesin and immune modulator and is an

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essential virulence factor of B. dermatitidis. Released BAD1 promotes adherence of the yeast to macrophages. Phagocytosis is also promoted by binding of CR3 and CD14 receptors. Clinical Features

The majority of individuals exposed to B. dermatitidis conidia by inhalation are asymptomatic. The incubation period is between 3 weeks and 100 days, depending on the host and the intensity of exposure. In most cases, symptoms develop slowly with cough, fever, malaise, and weight loss. There is no typical pattern on chest radiographs. Pleural effusion and solitary nodules are uncommon. Occasionally cavitation is seen, but calcification is rare. In disseminated disease there are widespread skin lesions, as well as bone, prostate, adrenal, liver, and central nervous system (CNS) infection. Blastomycosis is not often seen in AIDS patients. Management and Current Therapy

The diagnosis is made by microscopic examination of sputum, pus, bronchial washings, or other clinical material. Characteristic features are a thick refractile wall and single daughter cell attached on a broad base. The definitive diagnosis depends on isolation of the fungus in culture. Serological tests are of limited value because of the lack of sensitivity and specificity. Amphotericin B is the treatment of choice for patients with life-threatening infection and/or meningitis. Oral itraconazole is the first-line treatment for patients with the more indolent forms of blastomycosis.

Paracoccidioidomycosis Etiology and Pathology

Paracoccidioidomycosis is caused by the infection with the diamorphic fungus, Paracoccidioides braziliensis. In nature it grows as mycelium, in tissue as a yeast. The yeast has a characteristic form of budding, with multiple buds encircling the mother cell. The development of the yeast phase is partially controlled by an estrogen receptor. Paracoccidioidomycosis infections in women are rare. The disease occurs in South and Central America in regions classified as tropical or subtropical mountain forests. Clinical Features

In most cases the onset of symptoms is slow with cough, fever, malaise, and weight loss. Chest radiographs show bilateral infiltrates. Fibrosis and cavitation can occur in long-standing cases. The disease

commonly affects the skin: cutaneous lesions are found on the face around the mouth and nose. Painful ulcerating lesions develop on the gums, lips, tongue, or palate. Lymphadenitis is common in young patients. Widespread disseminated infection can occur affecting the small or large intestine, liver, adrenal gland, bone, joints, or CNS. Management and Current Therapy

Microscopy of sputum or other clinical material can allow diagnosis if the characteristic large round cells with multiple buds are seen, but isolation of P. braziliensis in culture and/or rapid identification by an exoantigen test are required for a definitive diagnosis. Serological tests such as a complement fixation and immunodiffusion are useful, although cross-reactivities with other pathogens can be a confounding factor. All patients should be treated with antifungal agents, as spontaneous resolution does not occur. Furthermore, long-term follow-up is required because of the high incidence of relapse. Itraconazole or, where oral absorption is problematic, amphotericin B and sulfadiazine can be used.

Aspergillosis Etiology and Pathology

Aspergillosis is caused by infection due to molds belonging to the genus Aspergillus. Most human aspergillus infections are caused by A. fumigatus, but A. flavus, A. nidulans, A. niger, A. terreus, A. clavatus, A. niveus, and A. oryzae can also cause disease of the lung and sinuses. These molds are widespread in the environment, found in soil, dust, and decomposing organic matter. Most infections follow inhalation of spores released into the air. The spores are 2–3 mm in diameter. Pathogenicity depends on the immune status of the patient and the fungal strain. Like other fungal genomes that have been analyzed, virulence is under polygenic control. Virulence genes are diverse and include those encoding cell wall components, those involved in evasion of the host immune response, and those involved in detoxifying systems for reactive oxygen species. There are three major categories of pulmonary disease caused by Aspergillus. Allergic aspergillosis, including allergic bronchopulmonary aspergillosis (ABPA), allergic Aspergillus sinusitis, and allergic alveolitis are all associated with disease pathogenesis resulting from the host allergic response to the organism. Colonizing aspergillosis including aspergilloma is found in pre-existing scarred or cavitated areas of lung. Invasive and disseminated aspergillosis is where disease results from fungal

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invasion itself, usually in the context of an impaired host immune response. Here, the discussion will be limited to aspergilloma, invasive and disseminated aspergillosis. Aspergillomas occur where Aspergillus colonization of the airways is found in damaged lung produced by another disease, a common example being tuberculosis (TB) where a ball of fungus forms in a pre-existing cavity. Any cavitating lung disease such as pneumonia, lung abscess, sarcoidosis, pulmonary infarction, pneumoconiosis, carcinoma, fibrosing alveolitis, upper lobe fibrosis caused by ankylosing spondylitis, and histoplasmosis can predispose to aspergilloma. Aspergillomas are more common in the upper lobes, because these are the sites most frequently affected by pulmonary TB. The ball contains fibrin, inflammatory cells, fungal hyphae, and is surrounded by a fibrous wall. The cavity wall is not invaded by the fungus in uncomplicated aspergilloma, but in invasive aspergillosis the wall and surrounding lung tissue become invaded and destroyed by the fungus. Invasive aspergillosis, although not common, is a serious pulmonary fungal infection. There are three patterns of disease: angioinvasive aspergillosis, airway-invasive aspergillosis, and chronic necrotizing aspergillosis. The first two patterns are rapidly progressive and can result in lung damage and death in a matter of days. Chronic necrotizing aspergillosis is a more indolent infection that occurs over weeks, months, or years. Invasive aspergillosis can occur in previously healthy patients but is more common in immunocompromised patients or those with a debilitating disease such as diabetes mellitus, malnutrition, alcoholism, or prolonged corticosteroid therapy. There has been an increase in the incidence of invasive aspergillosis due to the marked increase in numbers of immunocompromised/neutropenic individuals such as those on chemotherapy for malignancy, immunosuppressive drugs for autoimmune disease, bone marrow and organ transplant recipients, and individuals with AIDS. The incidence increases with the number of granulocytopenic days. Nosocomial invasive aspergillosis occurs in immunocompromised individuals with recognized sources of infection being building sites, pepper, and pot plants. Angioinvasive aspergillosis occurs in immunocompromised patients with a severe neutropenia and is characterized histologically by invasion and occlusion of small to medium pulmonary arteries by fungal hyphae, leading to the formation of necrotic pulmonary nodules or pleural-based wedge-shaped infarcts. Airway-invasive aspergillosis is less common and is characterised by the invasion of Aspergillus organisms deep to the airway basement

membrane, with a predominantly neutrophilic infiltrate. It is more common in lung transplant recipients and HIV infection. There may be tracheobronchitis, bronchiolitis, and/or bronchopneumonia. Chronic necrotizing aspergillosis is uncommon and tends to be seen in individuals with milder forms of immunosuppression and/or an underlying debilitating disease. This is a slowly evolving process with focal, often cavitating lesions, areas of consolidation, and/ or multiple nodular opacities. Clinical Features

Invasive aspergillosis usually presents with nonspecific symptoms of fever, nonproductive cough, pleuritic chest pain, and breathlessness. Hemoptysis is a late feature of disease and can be massive and result in death. Neutropenic patients may present with pneumothorax. Management and Current Therapy

The diagnosis is made on the basis of clinical history, radiology, serology, and mycology examination. The chest radiograph may be normal, show infiltrates, nodular opacities, or consolidation. Computed tomography (CT) is the investigation of choice. The characteristic changes in angioinvasive disease are nodules surrounded by a halo of ground-glass attenuation (halo sign) or pleural-based, wedge-shaped areas of consolidation (Figure 2). Air-crescents are seen 2–3 weeks into treatment as the neutrophil count recovers (Figure 3). Airway-invasive aspergillosis presents with peribronchial nodular opacities and consolidation. There may be a ‘tree-in-bud’ appearance (Figure 4). Chronic necrotizing aspergillosis

Figure 2 Angioinvasive aspergillosis in a 68-year-old man with neutropenia. CT thorax shows a nodule surrounded by a halo of ground-glass attenuation (halo sign). Reproduced from Franquet T, Gimenez A, and Hidalgo A (2004) Imaging of opportunistic fungal infections in immunocompromised patient. European Journal of Radiology. 51: 130–138, with permission from Elsevier Ireland Ltd.

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Figure 3 Angioinvasive aspergillosis in a 54-year-old man with neutropenia. CT thorax shows bilateral nodules and cavitation with the presence of an air-crescent. Reproduced from Franquet T, Gimenez A, and Hidalgo A (2004) Imaging of opportunistic fungal infections in immunocompromised patient. European Journal of Radiology. 51: 130–138, with permission from Elsevier Ireland Ltd.

Figure 4 Acute airway-invasive aspergillosis with nodules and peribronchial thickening and tree-in-bud pattern. Reproduced from Buckingham SJ and Hansell DM (2003) Aspergillus in the lung: diverse and coincident forms. European Radiology 13: 1786–1800, with kind permission of Springer Science and Bussiness Media.

disease, and diffuse rather than focal disease. The identification of Aspergillus in BAL from an immunocompromised patient in the absence of chronic lung disease (where colonization is common) is highly predictive of invasive disease. Bronchoscopy plus BAL, fine needle aspiration, and thoracotomy can be considered where the investigations already described are not conclusive. However, often the patient is too unwell to undergo invasive procedures and empirical therapy is started, particularly if the patient is neutropenic. Left untreated, acute invasive pulmonary aspergillosis is rapidly fatal, and it is wise to consider empirical treatment in patients at high risk where initial investigations are consistent with the diagnosis. Amphotericin B is the usual first-line therapy to achieve remission followed by consolidation therapy with itraconazole. A recent randomized trial, comparing amphotericin with voriconazole, placed voriconazole as an alternative treatment. The availability of voriconazole, lipid preparations of amphotericin, and caspofungin (an echinocandin) has raised the possibility of combination therapies. Initial nonrandomized studies have indicated that combination therapy may be beneficial, but further randomized controlled trails are required to establish if this is indeed the case. The risk of massive hemoptysis is greatest in central lesions next to great vessels and is most likely to occur during neutrophil recovery. Emergency surgery is indicated where there is hemoptysis and angioinvasive disease, lesions that are enlarging or close to major vessels, and/or high risk of severe hemoptysis. It has been shown that the combination of early CT, serology, azole therapy, and surgery in selected patients with acute leukemia and acute invasive aspergillosis significantly reduced mortality.

Candidosis presents with non-specific CT findings including areas of peripheral consolidation, multiple nodules, cavitation, and mycetoma formation. An ELISA for galactomannan in serum and the 1-3, b-D-glucan detection assay are useful in the early detection of invasive aspergillosis. Used as screening tests in high-risk patients, they have a high sensitivity and specificity for the diagnosis. Aspergillus precipitins and other antibody tests are unhelpful in the context of acute disease, but are often positive in chronic necrotizing aspergillosis. Cough is usually nonproductive and sputum samples are rarely useful in the diagnosis of early disease. Bronchoalveolar lavage (BAL) is more likely to be positive in airway-invasive than angioinvasive

Etiology and Pathology

Candidosis is caused by infection due to organisms belonging to the genus Candida and its distribution is worldwide. Candida albicans most commonly causes human infection, but the impact of other members of the genus is rising. Candida is commonly found in the mouth and gastrointestinal tract of normal individuals. Candidosis occurs in debilitated individuals who are infected by their own reservoir or organisms. The infection can be superficial causing infection of the mouth, vagina, penis, skin, and nails, or deep resulting in infection of the gastrointestinal tract (especially the esophagus), lung, CNS, heart, kidney, lower urinary tract, liver and spleen, bone,

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muscle, and lower genital tract in pregnancy (causing intrauterine infection). Infection may be confined to one organ or become widespread (disseminated candidosis). Deep infections affecting the gastrointestinal tract, liver and spleen, and lungs tend to occur in immunosuppressed individuals such as patients infected with HIV or rendered neutropenic due to underlying malignancy or its treatment. Surgical and burns patients are also at risk of deep infections. Clinical Features, Management, and Current Therapy

Pulmonary candidosis is rarely diagnosed during life. Cultures taken at bronchoscopy are frequently contaminated and grow Candida species. The diagnosis of pulmonary candidosis, therefore, requires histological evidence of invasive fungal disease in the lung tissue. Pulmonary infection is nearly always associated with disseminated disease. The clinical and radiological presentation is non-specific. Hematogenous dissemination results in diffuse nodular infiltrates in both lungs. Endobronchial infection causes local or diffuse bronchopneumonia. Newborn infants can become infected following aspiration of organisms from the mouth. A diagnosis of deep candidosis is often difficult to make because the clinical presentation is non-specific and microbiological and serological tests difficult to interpret. Detection of the typical blastospore or filamentous forms of Candida species in stained tissue sections is diagnostic. Culture of the genus Candida from blood, closed sites, or macronodular cutaneous lesions of the skin provide evidence of deep infection. For deep candidosis, combination therapy is recommended with drugs such as amphotericin B, flucytosine, itraconazole, and fluconazole. Newer antifungal combinations are currently being investigated using voriconazole and caspofungin.

Cryptococcosis Etiology and Pathology

Cryptococcosis is caused by infection with members of the genus, Cryptococcus. They are encapsulated, budding yeasts. The pathogens of clinical interest are the two varieties of C. neoformans, C. neoformans var. neoformans and C. neoformans var. gattii. These two varieties differ in their serotypes, biochemical properties, epidemiology, and ecology. For example, while neoformans have a worldwide distribution, gattii is restricted to tropical areas including Africa and East Asia. Following a major initiative in recent years, the 20 Mb C. neoformans genome was recently published in full. The genome contains around

6500 intron-rich gene structures, encoding a transcriptome abundant in alternatively spliced and antisense messages. It is rich in transposons, the presence of which may drive karyotype instability and phenotypic variation. Clinical isolates in culture and tissue demonstrate spherical, budding, encapsulated yeast cells. The hallmark of C. neoformans is its capsule, visualized by Indian ink staining. Cryptococcosis is found worldwide. The major source of C. neoformans var. neoformans is thought to be pigeon droppings, whereas major source of C. neoformans var. gattii is the red gum tree, Eucalyptus camaldulensis. The fact that Cryptococcus is prevalent in nature, yet cryptococcosis is rare except in the immunocompromised suggests that most individuals can generate effective immune responses to it and that such infections must generally be asymptomatic. An intact, functional T-cell adaptive immune response is required to control infection. Cryptococcosis follows inhalation of the small, minimally encapsulated yeast cells. The first line of innate immunity encountered is alveolar macrophages. Uptake and phagocytosis by the alveolar macrophages is facilitated by receptor recognition of mannose-rich determinants on the yeast cell walls. Strains of C. neoformans vary in the extent to which they become engulfed by alveolar macrophages, depending on variables such as the size of the yeast particle and the thickness of the capsule. Clinical Features, Management, and Therapy

Cryptococcosis most commonly affects the lung, but this infection is often transient and asymptomatic. Symptoms of pulmonary infection include cough, chest pain, mucoid sputum production, weight loss, fever, night sweats, and malaise. Common radiological findings are a reticular or reticulonodular interstitial pattern. Less commonly, there may be ground-glass attenuation, airspace consolidation, and multiple small nodules. Cavitating lesions may be seen. It is common for cryptococcosis to present as disseminated infection with the CNS being the usual site of involvement. All patients with pulmonary cryptococcosis should be investigated for disseminated infection. The skin, bone, liver, adrenal glands, and eye are also affected. Cryptococcosis is one of the most common life-threatening fungal infections in patients with HIV infection with most cases occurring when the CD4 cell count falls below 50 mm 3. Encapsulated C. neoformans cells can be identified by microscopy of cerebrospinal fluid (CSF) and other host fluids or secretions mounted in Indian ink. It can be cultured from CSF, blood, sputum, urine, and BAL.

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Serological tests are helpful since high antibody titers against cryptococcal polysaccharide antigens in the serum, urine, and CSF are diagnostic of C. neoformans infection. All immunocompromised patients with cryptococcosis should be treated with an antifungal agent. The drugs of choice for cryptococcal pneumonia are amphotericin B and fluconazole. The choice and length of therapy depends on the underlying immune status of the host.

Mucormycosis Etiology and Pathology

Mucormycosis is caused by the infection due to ubiquitous, saprophytic molds belonging to the order Mucorales of the class Zygomycetes. Human infections are most commonly caused by Rhizopus species and are worldwide in distribution. These molds are ubiquitous, and can be isolated from soil or decomposing organic matter. Infection follows inhalation of spores released into the air, and the lung and nasal sinuses are common initial sites of infection. Infection can also follow ingestion or traumatic inoculation into the skin. Mucormycosis is an opportunistic infection seldom seen in normal individuals. Diabetic patients with a metabolic acidosis, immunocompromised patients or those on immunosuppressive agents with neutropenia, and patients on iron-chelating therapy such as deferroxamine especially in renal failure are all susceptible to infection. Clinical Features

Mucormycosis most commonly affects the nasal mucosa and sinuses, but can also infect the lung, gastrointestinal tract, skin, or become disseminated. Rhinocerebral mucormycosis begins in the upper airways and then spreads to the orbit, face, palate, and brain. Untreated, it is rapidly fatal and is seen in patients with acidosis (particularly uncontrolled diabetes), patients with leukemia, and organ transplant recipients. The initial presentation involves unilateral headache, nasal and sinus congestion/pain, nasal discharge, and fever. There may be periorbital, perinasal swelling, ptosis, proptosis, loss of vision, frontal lobe necrosis and abscess formation, nasal septum or palatal perforation. The disease is rapidly progressive. A diagnosis of pulmonary mucormycosis should be considered in immunocompromised patients presenting with fever and progressive lung infiltrates despite broad-spectrum antibacterial treatment. Gastrointestinal disease is not common and is seen in malnourished infants and children. Necrotic ulcers occur in the stomach, colon, and ileum that perforate causing peritonitis and septic shock. Cutaneous disease is

seen in patients with burns. Disseminated disease is usually seen in neutropenic patients with pulmonary infection or via the gastrointestinal tract, burns, or other skin lesions. Management and Therapy

This is a rapidly progressive, aggressive infection with a high mortality. Radiographic manifestations are non-specific and include consolidation, cavitation, nodules, and masses. Lesions are most often unifocal affecting the upper lobes. The air-crescent sign may be seen. Microscopic examination in clinical material taken from necrotic lesions helps to establish a diagnosis. Culture of the organisms is less helpful because Mucorales are common contaminants. There are no routine serological tests. Treatment includes management of the underlying condition, urgent surgical removal of infected, necrotic material, and antifungal therapy. High-dose amphotericin B is used. See also: Asthma: Allergic Bronchopulmonary Aspergillosis. Defense Systems. Pneumonia: The Immunocompromised Host.

Further Reading Al-Abdely HM (2004) Management of rare fungal infections. Current Opinion in Infectious Diseases 17: 527–532. Bethlem EP, Capone D, Maranhao B, Carvalho CR, and Wanke (1999) Paracoccidioidomycosis. Current Opinion in Pulmonary Medicine 5: 319–325. Bradsher RW, Chapman SW, and Pappas PG (2003) Blastomycosis. Infectious Disease Clinics of North America 17: 21–40. Buckingham SJ and Hansell DM (2003) Aspergillus in the lung: diverse and coincident forms. European Radiology 13: 1786– 1800. Chiller TM, Galgiani JN, and Stevens DA (2003) Coccidioidomycosis. Infectious Disease Clinics of North America 17: 41–57. de Camargo ZP and de Franco MF (2000) Current knowledge on pathogenesis and immunodiagnosis of paracoccidioidomycosis. Revista Iberoamericana de Micologia 19: 41–48. Franquet T, Gimenez A, and Hidalgo A (2004) Imaging of opportunistic fungal infections in immunocompromised patient. European Journal of Radiology 51: 130–138. Haynes K (2001) Virulence in candida species. Trends in Microbiology 9: 591–596. Latge JP (1999) Aspergillus fumigatus and aspergillosis. Clinical Microbiology Reviews 12: 310–350. Magee PT, Gale C, Berman J, and Davis D (2003) Molecular genetic and genomic approaches to the study of medically important fungi. Infection and Immunity 71: 2299–2309. Mitchell TG and Perfect JR (1995) Cryptococcosis in the era of AIDS – 100 years after the discovery of Cryptococcus neoformans. Clinical Microbiology Reviews 8: 515–548. Mukherjee PK, Sheehan DJ, Hitchcock CA, and Ghannoum MA (2005) Combination treatment of invasive fungal infections. Clinical Microbiology Reviews 18: 163–194.

440 PNEUMONIA / Nosocomial Rementeria A, Lopez-Molina N, Ludwig A, et al. (2005) Genes and molecules involved in aspergillus fumigatus virulence. Revista Iberoamericana Micologia 22: 1–23. Rulnke M (2004) Mucosal and systemic fungal infections in patients with AIDS. Prophylaxis and treatment. Drugs 64: 1163–1180. Silveira F and Paterson DL (2005) Pulmonary fungal infections. Current Opinion in Pulmonary Medicine 11: 242–246. Wheat LJ (2003) Current diagnosis of histoplasmosis. Trends in Microbiology 11: 488–494. Wheat LJ, Dewey C, Allen SD, Blue-Hnidy D, and Loyd J (2004) Pulmonary histoplasmosis syndromes: recognition, diagnosis, and management. Pulmonary fungal infections. Seminars in Respiratory and Critical Care Medicine 25: 129–144. Wheat LJ and Kauffman CA (2003) Histoplasmosis. Infectious Disease Clinics of North America 17: 1–19.

Relevant Websites http://www-sequence.Stanford.edu – C. albicans http://www.tigr.org – C. neoformans, A. fumigatus, C. immitis http://www.fgsc.net – A. fumigatus http://www.ncbi.nlm.nih.gov – A. fumigatus http://www.genome.wustl.edu – H. capsulatum

Nosocomial A Torres, M Valencia, and J Sellares, Hospital Clı´nic de Barcelona, Barcelona, Spain & 2006 Elsevier Ltd. All rights reserved.

Abstract Pneumonia is defined as the presence of microorganisms in the pulmonary parenchyma leading to the development of inflammatory response by the host which may be localized in the lung or may extend systemically. Nosocomial pneumonia (NP) is an infectious process which develops within 48 h after admission to the hospital and in patients nonintubated at the time of hospitalization. Most epidemiological and clinical studies on NP have been focused on critically ill patients and those receiving mechanical ventilation (MV). NP is the second most common hospital-acquired infection and is the leading cause of death among this type of infection. There are several modifiable risk factors for NP, such as antibiotic use, the body position, and stress ulcer prophylaxis. The diagnostic approach begins with the clinical suspicion. Then, quantitative cultures of respiratory secretions should be obtained through tracheobronchial aspirate or bronchoscopic samples. Prompt and adequate antimicrobial treatment is mandatory and this may be based on practice guidelines.

Introduction Pneumonia is defined as the presence of microorganisms in the pulmonary parenchyma leading to the development of inflammatory response by the host

which may be localized in the lung or may extend systemically. Nosocomial pneumonia (NP) is an infectious process which develops within 48 h after admission to the hospital and in patients nonintubated at the time of hospitalization. Ventilator-associated pneumonia is considered a subgroup of NP and is an infectious pulmonary process which develops 48 h after the presence of an artificial airway and mechanical ventilation (MV). Since a large proportion of the patients who develop NP are intubated and receive MV, most epidemiological and clinical studies on NP have been focused on critically ill patients and those receiving MV. From a clinical point of view, NP is of great importance not only because of the consequences of the morbimortality but also due to the high costs which the development of this disease carries.

Epidemiology Despite the large amount of data available on the epidemiology of ventilator-associated pneumonia (VAP), the results provided by the different studies published vary widely. This may be due to the lack of a standardized diagnostic approach and the different populations studied. Moreover, although VAP has been well defined, disagreement as to the final diagnosis may be attributed to: focal areas of the lobe that may be missed, negative microbiological studies despite the presence of inflammation in the lung, and pathologists may disagree in their conclusions. The point of time at which VAP develops has important implications in the etiology, treatment, and diagnosis of this disease. VAP has classically been determined as early-onset pneumonia which occurs during the first 4 days after hospital admission and late-onset pneumonia which develops 5 or more days after admission. Nosocomial pneumonia is the second most common hospital-acquired infection and is the leading cause of death among this type of infection. The incidence of NP ranges from 4 to 50 cases per 1000 admissions in community hospitals and general medical wards to up to 120 to 220 cases per 1000 admissions in some intensive care units (ICUs) or among patients requiring MV. The EPIC study, a large 1-day point prevalence study of pneumonia, was carried out in 1417 ICUs and included 10 038 patients. The prevalence of ICU-acquired infections was of 21%; 47% of these patients had pneumonia, of nosocomial origin in 10%. In a large, prospective cohort study including 1014 patients with MV, 177 (18%) developed VAP. The incidence of this disease was found to be 24% (78/322) in a Spanish study on VAP risk factors. A recent study reported a mean

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incidence of hospital-acquired pneumonia in nonICU patients of 371.4 cases per 1000 hospital admissions. Most patients were in medical wards (64.2%), had severe underlying diseases, and had been in the hospital for more than 5 days. The overall incidence of VAP varies from 8% to 28%. A prospective Italian study on VAP including 724 critically ill patients who had received prolonged ventilatory support after admission reported an incidence of 23%. This rate rose from 5% in patients receiving MV for 1 day to 69% in those receiving MV for more than 30 days. In a study including 567 patients receiving MV evaluated with invasive procedures the rate of VAP was 9%. In this latter study, the cumulative risk of pneumonia was estimated to be 7% at 10 days and 19% at 20 days after initiation of MV, thereby showing the classical incremental risk of pneumonia of 1% per day. However, in a large series of 1014 patients receiving MV, the rate of VAP was 18%, and although the cumulative risk for developing VAP increased over time, the daily hazard rate decreased after day 5. The risk per day was determined to be 3% on day 5, 2% on day 10, and 1% on day 15. Several studies have reported the rate of mortality in VAP to range from 24% to 76%. Patients with VAP in the ICU receiving MV may have a 2- to 10fold greater risk of death than patients without this complication. In a study of 78 episodes of NP detected in 322 consecutive patients with MV, the overall mortality rate was 23%. Similarly, the Neumos 2000 study group reported a mortality of 26% (pneumonia-attributed 13.9%) in 186 non-ICU patients with hospital-acquired pneumonia. The mortality of patients with NP was higher (33%) when compared with rates of patients without NP (19%, po0:01). In a previous study the same group described an overall mortality rate of 36%. On stepforward logistic regression analysis the identification of ‘high-risk’ microorganisms (Pseudomonas aeruginosa, Enterobacteriaceae, and other Gram-negative bacilli, Enterococcus faecalis, Staphylococcus aureus, Candida spp., Aspergillus spp., and episodes of polymicrobial pneumonia), bilateral involvement on chest X-ray, the presence of respiratory failure, inappropriate antibiotic therapy, age over 60 years, or an ultimately or rapidly fatal underlying condition were factors which were found to independently worsen the prognosis. The increased ratios of risk of mortality in patients with VAP varies from 1.7 to 4.4. Although several studies have shown that VAP is a severe disease, the controversy as to attributing mortality to NP continues. However, several studies have shown NP to be an independent prognostic factor. Groups of patients in which the attributable mortality

is increased include: patients undergoing cardiac surgery, those with acute lung injury, and immunocompromised patients. In contrast, in patients with lifethreatening medical conditions such as cardiac arrest in young patients with no underlying disease and those admitted due to trauma, NP does not seem to significantly increase the mortality.

Etiopathogenesis For pneumonia to develop virulent microorganisms must invade the lung parenchyma. This may occur either as the result of a defect in defense mechanisms of the host or by an overwhelming inoculum. The normal human respiratory tract has a variety of defense mechanisms such as anatomic barriers, cough reflexes, cell-mediated and humoral immunity, and a dual phagocytic system involving both alveolar macrophages and neutrophils. Virulent microorganisms can reach the alveolar space in several ways: colonization of the upper airway by potentially pathogenic microorganisms and posterior microaspirations, macroaspirations of gastric material and the gastropulmonary route, contaminated respiratory care equipment such as condensates in ventilator tubing, fibrobronchoscopes, tracheal suctioning material, nebulizers, etc., the hematogenous route, and direct dissemination from contiguous sites such as the pleura, the pericardium, or the abdomen. Oropharyngeal and tracheal colonization play a central role in the pathogenesis of VAP. Early colonization (within the first 24 h of MV) has been described in patients who are intubated and in those with MV, varying from 80% to 89%. One study demonstrated that 45% of 213 patients admitted to a medical ICU became colonized with aerobic Gramnegative bacilli by the end of 1 week in the hospital. Among the 95 colonized patients, 23% developed NP while only 4 out of the 118 noncolonized patients developed pneumonia. The composition of the colonizing tracheal flora is of note. Pseudomonas spp. have an increased affinity to ciliated tracheal epithelial cells and these microorganisms are not usually present in the oropharynx. Adherence of Pseudomonas increases in desquamated epithelium following influenza virus infection, tracheostomy, or repeated tracheal suctions in intubated patients. In a study including 86 patients receiving MV, oropharyngeal colonization, which was detected either on admission or from subsequent samples, was a predominant factor of NP compared with gastric colonization. Oropharyngeal colonization with Acinetobactor baumanii yielded an estimated 7.45fold increased risk of pneumonia compared with patients who had not yet or who were not identically

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colonized (p ¼ 0:0004). DNA genomic analysis demonstrated that an identical strain was isolated from oropharyngeal or gastric samples and bronchial samples in all but three cases of pneumonia due to S. aureus.

Risk Factors There is a great deal of data concerning risk factors in VAP. These factors are important since they may contribute to the development of effective prevention programs by indicating which patients may be most likely to benefit from prophylaxis against pneumonia. We herewith discuss the most relevant risk factors for endogenous infection in VAP, although data on many of these factors continue to be controversial. Antimicrobial Agents

The distribution of microorganisms, especially potentially resistant bacteria, differs in patients who have or have not received prior antibiotic therapy. Likewise, previous antibiotic administration may influence the development of VAP in two different ways: its use may be associated with a protective effect against early-onset pneumonia, while, on the other hand, it may be associated with an increased risk of late-onset pneumonia. To determine the baseline and time-dependent risk factors for VAP, 1014 patients were evaluated. On multivariate analysis independent predictors of VAP were a primary diagnosis on admission of burns (risk ratio 5.09), trauma (risk ratio 5.00), central nervous system disease (risk ratio 3.40), respiratory disease (risk ratio 2.79), cardiac disease (risk ratio 2.72), mechanical ventilation within the previous 24 h (risk ratio 2.28), witnessed aspiration (risk ratio 3.25), and paralytic agents (risk ratio 1.57). Exposure to antibiotics conferred protection (risk ratio 0.37), but this effect attenuated over time. Another study evaluated the risk factors for VAP within the first 8 days of MV in 83 consecutive intubated patients undergoing continuous aspiration of subglottic secretions (CASS). Multivariate analysis showed the protective effect of antibiotic use (risk ratio 0.10) whereas failure of the CASS technique (risk ratio 5.29) was associated with a greater risk of pneumonia. In addition, the use of systemic prophylaxis with cefuroxime before intubation on the incidence of VAP was evaluated in 100 patients, 50 of whom received one dose of 1.5 g of cefuroxime intravenously at the time of intubation and a second dose 12 h later. The global incidence of early-onset VAP was 37% (n ¼ 37): 12 (24%) in the cefuroxime groups and 25 (50%) in the control group

(p ¼ 0:007). All these studies demonstrate the protector effect of antibiotic therapy in early VAP caused by endogenous flora. In a prospective study in 277 patients with MV, the following four factors were found to be associated with VAP: index of systemic organ failure X3 (odds ratio 10.2), age X60 years (odds ratio 5.1), previous antibiotics (odds ratio 3.1), and supine head position within the first 24 h of MV (odds ratio 2.9). In addition to the importance of antibiotic therapy as a risk factor for VAP, the influence of antibiotics on the etiology of this disease is also worthy of mention. In a prospective study, 129 consecutive episodes of VAP were studied to evaluate the influence of prior antibiotic administration on the etiology and mortality of VAP. The rate of VAP caused by Gram-positive cocci or Haemophilus influenzae was statistically lower (po0:05) in patients who had received antibiotics previously while the rate of VAP caused by P. aeruginosa was statistically higher (po0:01). Step-forward logistic regression analysis only determined previous antibiotic use (odds ratio 9.2) to significantly influence the risk of death in VAP. Likewise, a study using logistic regression analysis demonstrated that three variables remained significantly associated with potentially resistant microorganisms as a causative etiology in VAP: duration of MVX7 days (odds ratio 6.0), prior antibiotic use (odds ratio 13.5), and prior use of broad-spectrum drugs (third generation cephalosporin, fluoroquinolone, and/or imipenem) (odds ratio 4.1). All of these studies have demonstrated that antimicrobial therapy has a bimodal effect on the development of VAP. Antibiotics protect against early-onset pneumonia, especially against pneumonia caused by endogenous flora, but they are also responsible for the selection of resistant microorganisms causing late-onset pneumonia such as P. aeruginosa and methicillin-resistant S. aureus (MRSA). Body Position

It has been demonstrated that up to 50% of healthy adults aspirate at night. However, in these subjects it is of no clinical significance since lung defense mechanisms remain intact. Body position is important in gastric reflux and tracheal aspiration. After instillation of a colloid with technetium via nasogastric tube, where patients were placed in a recumbent position, there was a significant reduction in the radioactivity of tracheal secretions compared with patients in the supine position. Moreover, another randomized study looked at the impact of body position on the development of VAP. Thirty-nine patients were placed in a semirecumbent (451) or supine (01) body position. Microbiologically confirmed

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pneumonia developed in 5% of the patients in the first position and in 23% of those in the latter (p ¼ 0:018). Gastric Colonization and Stress Ulcer Prophylaxis

Low gastric pH prevents bacterial growth in the gastric chamber and bacterial migration from the small bowel. The relationship between gastric pH and gastric colonization has been well established in several studies. The use of prophylactic agents for stress ulcers which alter the gastric pH may increase gastric colonization and the rates of VAP, although this remains to be demonstrated. A meta-analysis done in 1991 found that antacids and H2 antagonists were significantly more effective in preventing stress bleeding in treated versus untreated patients. Sucralfate was superior to H2 antagonists. Patients treated with antacids or H2 antagonists showed a significantly higher risk for the development of NP; and a posterior study demonstrated a trend toward lower clinically important bleeding with H2 antagonists and antacids than sucralfate with a trend toward an increased risk of pneumonia associated with H2 antagonists compared with no prophylaxis and a significantly higher risk compared to sucralfate. Finally, another meta-analysis concluded that ranitidine is not effective in the prevention of gastrointestinal bleeding and may increase the risk of pneumonia. Studies on sucralfate do not provide conclusive results. Currently, there are not enough data to conclude anything one way or the other.

Diagnosis The first problem in the diagnosis of NP and VAP is the lack of a ‘gold standard’ for comparing the different techniques used to confirm suspicion of an infectious process. Despite the use of histology in pulmonary biopsy and cultures of pulmonary tissue in the immediate post-mortem period, the use of these techniques in parenchymatous infections has not been unequivocally demonstrated. Suspicion of NP depends on the finding of new and persistent infiltrates on chest X-ray in association with some clinical signs and symptoms (fever or hypothermia, purulent secretions, and leukocytosis or leukopenia). Based on histology and microbiological cultures of post-mortem pulmonary biopsies, one study demonstrated that the presence of radiological findings plus two or more clinical criteria showed a sensitivity and specificity of 69% and 75%, respectively. In recent years the clinical pulmonary infection score (CPIS) has been widely used. This score combines different clinical, radiological, physiological,

laboratory, and microbiological parameters to increase the specificity of the clinical diagnostic approach. A score greater than 6 demonstrates a good correlation with the presence of pneumonia. The results of different studies on the diagnostic performance of this score are contradictory with values of sensitivity and specificity of around 77% and 42%, respectively. Some studies have been aimed at increasing the diagnostic yield of this score with the addition of Gram staining of secretions of the lower respiratory tract. The diagnostic tests performed on suspicion of VAP have two objectives: the first is to determine whether the patient really has an infectious pulmonary process as indicated by the signs and symptoms leading to the use of these tests, and the second is to isolate the causative microorganisms of the disease. For many years the diagnostic performance of the different techniques used to confirm suspicion of VAP has been under debate. At present, the profitability of noninvasive tests such as tracheobronchial aspirate (TBAS) and invasive bronchoscopic tests such as bronchoalveolar lavage (BAL) and the protected specimen brush (PSB) as well as the advantages and disadvantages of each have been well established. As a general rule, the TBAS has a very good sensitivity with a specificity a little lower than the invasive tests when using a quantitative culture of the respiratory secretions obtained by this method. In different studies the sensitivity of this test varies from 38% to 100% with a specificity of 14% to 100%. A negative TBAS culture in a patient who has not received antibiotic treatment has a high negative predictive value for the presence of VAP. In one study on the diagnostic value of this technique, the negative predictive value was found to be 72% in 102 patients evaluated with this technique and with invasive methods. The sensitivity of PSB and BAL are 33% to 100% and 42% to 93%, respectively, and the specificity is 50% to 100% and 45% to 100%. On the other hand, the current controversy lies in the role which invasive and noninvasive methods have in the prognosis and use of antibiotics in these patients. In a pilot study with 51 patients, bronchoscopic methods led to a greater change in initial antibiotic treatment (42% vs. 16%; po0:05) with no significant differences in regard to either global or attributable mortality or morbidity. This study was limited by its small sample size and the lack of a standard treatment protocol in the invasive group. Another study demonstrated a greater number of antibiotic changes with invasive techniques with no clear influence on mortality, length of ICU stay, and days on MV. Yet another study on a group of 765 patients with suspicion of VAP (39 noninvasive and 37 invasive) concluded that

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the diagnostic performance of both techniques in VAP is similar, as were mortality at 30 days, days on MV, and length of ICU stay. The costs of invasive studies were clearly greater. One study reported positive results with respect to a decrease in mortality on day 14 with the sequential organ failure assessment (SOFA) score on days 3 and 7, and a reduction in the use of antibiotics and the number of antibiotic-free days with the invasive technique. Nonetheless, the study was limited by the use of qualitative cultures of

the tracheal aspirates which thereby limited comparison with the remaining studies. In summary, we suggest the following diagnostic approach in NP (Figure 1). First, clinical suspicion of pneumonia should be based on classical clinical criteria or a CPIS46. Respiratory secretions should be collected at this time by tracheobronchial aspirate or bronchoscopy for obtaining quantitative cultures. Re-evaluation should be made at 48–72 h and the decision as to whether to continue antimicrobial

New and persistent infiltrate on CXR, plus two of the following: fever or hypothermia, leukocytosis or leukopenia and purulent secretions or CPIS > 6

Obtain lower respiratory tract sample (TBAS, BAL, or PSB) for quantitative culture and microscopy

Begin empiric antimicrobial therapy based on local microbiologic data and the presence of the following criteria

Late-onset pneumonia or risk factors for multidrug resistant microorganisms

Yes

No

Antipseudomonal cephalosporin or antipseudomonal carbapenem or
-lactam/
-lactamase inhibitor plus antipseudomonal fluoroquinolone or aminoglycoside plus linezolid or vancomycin

Ceftriaxone or levofloxacin, moxifloxacin, or ciprofloxacin or ampicillin/sulbactam or ertapenem

Day 3 : Check cultures and assess clinical response with clinical parameters, laboratory results, CRX or CPIS

Criteria of nonresponse: • Failure to improve the PaO2/FiO2 or need for intubation and mechanical ventilation • Persistence of fever or hypothermia • Worsening of the pulmonary infiltrates by >50% • Development of septic shock or MODS Figure 1 Algorithm for management of patients with nosocomial pneumonia. CRX, chest X-ray; CPIS, clinical pulmonary infection score; TBAS, tracheobronchial aspirate; BAL, bronchoalveolar lavage; PSB, protected specimen brush; PaO2, oxygen arterial pressure; FiO2, inspired fraction of oxygen; MODS, multiple organ dysfunction syndrome.

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treatment should be based on the probability of pneumonia, the results of the cultures, and the presence of an alternative diagnosis.

Treatment When deciding to treat a suspected episode of ICUacquired pneumonia, several aspects should be taken into account. The main foundations for the treatment of NP are discussed below. The importance of early initiation of an adequate antimicrobial therapy for the treatment of VAP has been emphasized in recent years in several studies. A study of 430 patients with VAP found a higher attributable mortality (24.7% vs. 16.3%; p ¼ 0:039) and a higher incidence of septic shock and gastrointestinal bleeding among patients receiving inadequate initial treatment. With mini-BAL fluid cultures, it has been reported that inappropriate antibiotic therapy was associated with an odds ratio for death of 3.28. Empiric antimicrobial therapy may be inadequate as a consequence of the presence of unexpected species not covered by the initial antibiotic schedule but this may be mainly due to unanticipated resistance. In most of the previously mentioned studies, a large proportion of the episodes of inadequate antimicrobial treatment were attributed to potentially resistant Gram-negative bacteria (especially P. aeruginosa, Acinetobacter spp., and Enterobacter spp.) or MRSA. Another important aspect to take into account is whether the modification of the initial inadequate therapy according to microbiological results improves the outcome of the patient. Studies addressing this issue did not find any improvement in mortality with this strategy. It has been shown that therapeutic changes made after bronchoscopy led to more patients (n ¼ 42) receiving adequate therapy. Nonetheless, the mortality in this group was comparable to the mortality reported among patients who continued to receive inadequate therapy (n ¼ 23). In another study, bronchoscopic results led to a change in antibiotic treatment in 27 cases (23.9%) considered to receive inadequate initial treatment. Despite clinical resolution in 17 of these cases (62.9%), the mortality was higher compared to patients with initial adequate therapy. A high prevalence (73.3%) of inadequate initial antibiotic therapy was found in a study of 130 patients with VAP. In this study the mortality of patients in whom the antibiotic therapy had been started or changed based on the results of mini-BAL culture was significantly higher compared to patients with unchanged or discontinued treatment (60.8%, 33.3%, and 14.3%, respectively).

The results of these studies demonstrate the need for early initiation of broad-spectrum empirical antimicrobial therapy on suspicion of VAP. The American Thoracic Society (ATS) published its first guidelines for the management of hospital-acquired pneumonia in 1995. Soon afterwards a different classification (Trouillet) was suggested for the prediction of pathogens and the selection of antibiotic treatment based on the use or not of previous antibiotic use and the duration of MV. These classifications provide a different rationale for the prediction of microbial etiology with the aim of aiding clinicians in prescribing the appropriate initial empiric therapy. In a prospective study, we recently evaluated the level of bacterial coverage and validated the adequacy of the antibiotic strategy proposed by the 1996 ATS guidelines and the Trouillet framework. Both classifications were found to be to effective in predicting the pathogen involved (91% and 83%, respectively). However, taking the in vitro sensitivity of the pathogens isolated into account, the adequacy of the antibiotic treatment proposed by these classifications was found to be rather lower (79% for ATS and 80% for Trouillet). The microorganisms involved in treatment inadequacy were multiresistant P. aeruginosa, A. baumanii, S. maltophilia, and MRSA. These findings underline the importance of considering additional parameters such as local microbial epidemiology and more accurate models of prediction of resistance to improve the level of coverage and the appropriateness of antibiotic treatment. The ATS recently published the new guidelines for the management of adults with hospital-acquired pneumonia and, contrary to the previous guidelines, the severity of pneumonia does not play an important role in the decision-making as to the initial empiric treatment to be implemented (Table 1). Regardless of the severity of pneumonia, patients with risk factors for infection with multidrug resistant microorganisms or with hospital admission greater than 5 days should receive empiric broad-spectrum antibiotic therapy which adequately covers infection by P. aeruginosa. The different schedules recommended for this group include: antipseudomonic cefalosporin or carbapenem or piperacillin/tazobactam associated with an aminoglycoside or an antipseudomonic fluoroquinolone. Linezolide or vancomycin should be associated in cases with suspicion of MRSA infection or hospitals with a high incidence. Patients who do not fulfill the previously mentioned characteristics should receive empiric treatment with schedules which cover the ‘core’ microorganisms such as S. pneumoniae, H. influenzae, MRSA, and antibioticsensitive aerobic Gram-negative bacilli. The drugs of

446 PNEUMONIA / Nosocomial Table 1 Etiology of nosocomial pneumonia Patients with no risk factors for multidrug resistant pathogens, early onset, and any severity

Patients with late-onset pneumonia or risk factors for multidrug resistant pathogens and any severity

Streptococcus pneumoniae Haemophilus influenzae Methicillin-sensitive Staphylococcus aureus (MSSA) Enteric Gram-negative bacilli Escherichia coli Klebsiella pneumoniae Enterbacter spp. Proteus spp. Serratia marcescens

The same as the previous group, plus Pseudomonas aeruginosa Klebsiella pneumoniae (extended spectrum b-lactamases) Acinetobacter spp. Methicillin-resistant Staphylococcus aureus (MRSA) Legionella pneumophila

choice include: ceftriaxone or a fluoroquinolone or b-lactam/b-lactamase inhibitor or ertapenem. To date, the use of combined antibiotic therapy is still recommended in the treatment of VAP on suspicion of P. aeruginosa or other potentially resistant pathogens. Previous studies on bacteremic infections caused by P. aeruginosa and Klebsiella demonstrated a higher mortality associated with the use of initial empiric monotherapy compared to combined therapy. However, these studies are limited in that they were performed when less potent b-lactams were used. Further trials are needed to clarify this issue. For now, the use of monotherapy should be limited to the treatment of severe NP in patients without risk factors for potentially resistant pathogens. The length of antimicrobial treatment is also under debate. In the ATS guidelines the experts recommend that the length of treatment be shortened from the traditional 14 to 21 days to short periods of 7 to 10 days. The latter shorter treatment is recommended in the treatment of S. aureus and H. influenzae pneumonia. However, in specific situations such as multilobar involvement, malnutrition, cavitation, Gram-negative necrotizing pneumonia, and/or isolation of P. aeruginosa or Acinetobacter spp. infection, 14- to 21-day therapy should be initiated. Nonetheless, recent evidence has suggested that short treatment is as effective as longer treatment in VAP. A recent study evaluated 401 patients with VAP, 197 of whom were randomized to receive short (8-day) treatment and 204 a long (15-day) course of antibiotic treatment. No differences were observed in the mortality rate (18.8% vs. 17.2%) or in the recurrence of pulmonary infection (28.9% vs. 26%) on comparing the two groups of patients. However, those treated with a short course of antibiotics had significantly more antibiotic-free days (1377.4 vs. 8.775.2 days, po0:001). The possibility of providing adequate treatment with shorter courses of antibiotics will not only reduce healthcare costs but will also have favorable consequences on microbial ecology by reducing the selection pressure for resistance.

Conclusion Nosocomial pneumonia is the leading cause of death among hospital-acquired infections. Its incidence ranges from 4 to 50 cases per 1000 admissions in community hospitals and general medical wards. Aspiration of colonized pharyngeal secretions is considered the most important etiopathogenic mechanism of NP. Risk factors to develop NP include previous use of antibiotics, body position, prophylaxis of stress ulcer, and duration of hospital admission. The diagnostic approach should start with the criteria of clinical suspicion exposed above and followed by quantitative cultures of respiratory secretions obtained through tracheobronchial aspirate or bronchoscopic samples. The initial empiric antimicrobial therapy is based on the duration of hospital admission and the presence of risk factor of multiresistant microorganisms. See also: Bronchoalveolar Lavage. Pleural Effusions: Parapneumonic Effusion and Empyema. Pneumonia: Overview and Epidemiology; The Immunocompromised Host. Surgery: Transplantation. Ventilation, Mechanical: Ventilator-Associated Pneumonia.

Further Reading Chastre J and Fagon J (2002) Ventilator-associated pneumonia. American Journal of Respiratory and Critical Care Medicine 165: 867–903. Craven DE, De Rosa FG, and Thornton D (2002) Nosocomial pneumonia: emerging concepts in diagnosis, management, and prophylaxis. Current Opinion in Critical Care 8: 421–429. Hernandez G, Rico P, Diaz E, and Rello J (2004) Nosocomial lung infections in adult intensive care units. Microbes and Infection 6(11): 1004–1014. Kollef MH (2004) Prevention of hospital-associated pneumonia and ventilator-associated pneumonia. Critical Care Medicine 32: 1396–1405. Torres A (ed.) (2003). Hospital-acquired pneumonia: the European perspective. Infectious Disease Clinics of North America 17(4). Vincent JL (2004) Ventilator-associated pneumonia. The Journal of Hospital Infection 57(4): 272–280.

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Parasitic T B Nutman and K R Talaat, National Institutes of Health, Bethesda, MD, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Parasites are eukaryotic organisms that live within another organism or host. They are broadly divided into two groups: single-celled protozoa and multicellular helminths. Most parasites have complex life cycles with multiple stages, often requiring multiple host species to complete their life cycle. Humans acquire infection with parasitic organisms in a number of different ways. Some are ingested through a fecal-oral spread, others transmitted by insect vectors, still others by penetrating through the skin of the host from contaminated soil or water. This article focuses on the major parasitic infections that affect the lungs. Few parasites specifically go to the lungs as their final target organ, with the exception of Paragonimus, the lung fluke. For the majority of the organisms that cause pulmonary disease, either the lungs are secondary sites of infection (e.g., amebiasis and toxoplasmosis), or the parasites migrate through the lung en route to another organ system (e.g., Strongyloides, Ascaris). In some infections, pulmonary symptoms are due to a hypersensitivity immune response to parasite antigen as seen in the tropical pulmonary eosinophilia syndrome.

Introduction Parasites are eukaryotic organisms that live within another organism or host. They are broadly divided into two groups: single-celled protozoa and multicellular helminths. Whereas protozoa are often intracellular, helminths are characteristically extracellular. Most parasites have complex life cycles with multiple stages, often requiring multiple host species to complete their life cycle. These host species could be another mammal (e.g., dogs and sheep for Echinococcus), an invertebrate host (crustaceans for Paragonimus sp.), or an insect vector such as Anopheline mosquitoes for the malaria parasites. For most of the infections being described in this article, humans are the definitive hosts (in which the adult or sexual stage (if there is one) is found). For other infections, humans are the intermediate hosts or the incidental dead-end hosts of a parasite that usually infects other species (e.g., Toxocara). Infection with parasitic organisms is acquired in a number of different ways. Some are ingested and are spread person to person or through fecal/oral or fecal/soil/oral transmission (i.e., Ascaris). Others require an insect vector for transmission. Still others have intermediate species in which they complete part of their life cycles. Some organisms can enter from a contaminated environment by penetrating the skin (e.g., Strongyloides, hookworm).

Some of these organisms have worldwide distribution (e.g., Ascaris, Toxoplasma), but most have geographic foci of endemicity. Often, these foci are in regions of the world with limited resources and where free and easy access to clean water and sanitation is not available. In industrialized countries, these infections are most commonly encountered in immigrants or returning travelers. Once thought exotic, parasitic diseases account for an increasing presence in industrialized countries because of returning travelers, immigration, and mass movements of people as a result of political or socioeconomic upheavals. An awareness of the clinical manifestations and management of these diseases is therefore crucial for both primary care physicians and specialists. By no means comprehensive, this article will briefly examine those parasites most likely to cause pulmonary disease.

Entamoeba histolytica Entamoeba histolytica, the causative organism of amebiasis, is a ubiquitous protozoan. It is more common in developing countries, especially in areas of low socioeconomic resources where access to adequate sanitation is not available. Cysts are ingested in fecally contaminated food and water. Excystation occurs in the small intestine, where the larvae divide into trophozoites that migrate from the intestine to the liver through the portal circulation. The clinical spectrum of E. histolytica infection is very broad. Only about 10% of people infected with E. histolytica develop disease; others can spontaneously clear their asymptomatic infection within 1 year of infection. The most common site for extraintestinal infection with E. histolytica is the liver. Liver disease occurs in 3–9% of cases of amebiasis. Lung disease occurs in 2–7% of those with invasive amebiasis. Most commonly, lung disease develops from extension of a liver abscess. It occurs by direct extension, insidiously from the liver through the diaphragm; by rupture, involving the pleura (leading to pleurisy, effusion, or empyema); or with development of pulmonary consolidation, lung abscess, or bronchohepatic fistula. Up to 40% of patients with liver abscess develop pulmonary complications. The most common is reactive pleuritis secondary to the hepatic abscess. Clinically, patients can complain of right upper quadrant pain with pleuritic chest pain. They frequently have normal liver enzymes. A pleural rub is common in patients with amoebic liver abscess. The fluid in the pleural space is exudative and sterile. If the abscess extends into the pleural cavity, an amoebic empyema can form. The onset of this can be insidious or acute.

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Patients have fever, right upper quadrant and chest pain, and a dry cough. Rupture into the pleural cavity is often signaled by abrupt exacerbation of pain with rapidly progressive respiratory distress, possibly sepsis and shock. If invasion into a major bronchus occurs, hemoptysis can develop containing the anchovy paste-like pus coming from the amoebic abscess. Physical examination findings of amoebic lung disease often include hepatic enlargement and tenderness, dullness to percussion at the right base with decreased or absent breath sounds, and a pleural rub with occasional crepitation. If the lung disease is chronic, cachexia and clubbing can sometimes be found. Chest X-ray abnormalities occurred in 57% of patients with amoebic liver disease. Untreated, the mortality of invasive amebiasis is over 80%. The diagnosis of invasive amebiasis is made by identifying the organism, by serology or by polymerase chain reaction (PCR). Treatment is with metronidazole and paromomycin or dioxanide furoate.

Toxoplasma gondii Toxoplasma gondii is an obligate intracellular parasite in which humans are the incidental host. It is most frequently acquired by ingestion of cysts excreted in cat feces. Once inside the host, the organism can invade any cell of the body, most commonly the brain, lymph node, heart, and lung. The vast majority of immunocompetent persons who are infected is clinically asymptomatic. Immunosuppressed individuals can have more fulminant toxoplasmosis, either during primary infection or with reactivation of latent infection. In the lung, T. gondii infection can cause a necrotizing pneumonia with nodule formation, diffuse alveolar damage, and interstitial pneumonitis. Edema develops in the alveolar capillary interface with lymphocytic infiltration. AIDS patients with Toxoplasma pneumonia present with cough, dyspnea, and fever. Toxoplasmosis is diagnosed by serology or by identifying tachyzoites in bronchioalveolar lavage (BAL) smears. Pyrimethamine with folinic acid and sulfadiazine is the treatment of choice for Toxoplasma pneumonia.

Paragonimus Few helminths specifically go to the lungs as their final target organ, the notable exception being Paragonimus, or the lung fluke. Found throughout the world, but primarily in Asia, paragonimiasis is acquired by ingestion of raw, undercooked or pickled crabs or other crustaceans. Once ingested the larvae penetrate through the intestines and migrate through the diaphragm and pleura into the lungs. There,

worm pairs encyst together near bronchial passages and mature into adult worms that lay eggs. Paragonimiasis is rarely a serious or fatal infection. The symptoms depend on the location of the worms and their developmental stage. Shortly after infection, patients may complain of diarrhea or abdominal pain or discomfort, although most people are asymptomatic initially. The initial penetration of the larvae through the diaphragm and pleura can cause pleuritic chest pain and pneumothoraces. Symptoms of paragonimiasis include cough, hemoptysis, and chest pain. The hemoptysis is usually of rusty or chocolate sputum; large amounts of frank bright red blood are unusual. Pleural effusions can form and, if large, can result in dyspnea. Chronic infection is often mistaken for tuberculosis, especially in areas where paragonimiasis is less prevalent; however, in some instances, tuberculosis and paragonimiasis can coexist. The physical exam is usually normal, although rales may be heard. Rarely, clubbing of the fingers is seen. Radiographically, nodules, pneumothoraces and interstitial infiltrates, effusions, cavities, or ring cysts resembling bronchiectasis can be seen. Parenchymal consolidation is seen in the majority of patients. The infiltrates can be transient, especially in acute infection as the worms migrate. CT can show the migration tracks of the worms as linear opacities and later as cysts within the parenchymal consolidations (see Figure 1). The chest X-ray can be normal in 10–20% of cases. Once suspicion of paragonimiasis has been aroused by a history of handling or eating freshwater crabs or crayfish, isolating Paragonimus eggs in the sputum or stool make the diagnosis. Treatment is with praziquantel.

Intestinal Roundworms: Ascaris, hookworm, Strongyloides Roundworms or nematodes can be classified broadly into those inhabiting the intestine, which are by far the most common, and those infecting deep tissue. It is estimated that there are more than 1 billion cases of ascariasis worldwide. With the exception of Strongyloides stercoralis, intestinal nematodes do not multiply within humans, and finding either eggs or parasites, usually in the stool, makes the diagnosis. Strongyloides, through its autoinfection cycle, can multiply and disseminate widely in an immunocompromised individual (hyperinfection syndrome). Pulmonary disease in intestinal helminth infections is relatively uncommon but can occur during the stage of larval migration through the lungs en route to the intestine. When apparent in immunocompetent hosts, pulmonary disease usually occurs as

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Figure 1 Computed tomography image of Paragonimus westermani infection in a 46 year Korean man showing a cystic lesion within an infiltrate. The patient also developed a pulmonary effusion. Courtesy of Myoung-don Oh, Seoul National University College of Medicine, South Korea.

a Loeffler’s syndrome with cough, asthma, fever, patchy pulmonary infiltrates on chest radiographs, and eosinophilia. Ascaris, hookworm, and Strongyloides are the most common causes of this syndrome among the intestinal helminths. Ascariasis is the most common cause worldwide of transient eosinophilic pulmonary infiltrates. In some areas, the pneumonitis occurs at specific times of the year depending on the climate. Infection is acquired by ingestion of eggs on contaminated foods or from soil. Symptoms develop 9–12 days after ingestion of the eggs and can last from 2 to 3 weeks. Larval migration through the lungs leads to pneumonitis, which can manifest as a nonproductive cough, asthma, burning substernal pain that is pleuritic in nature, dyspnea, and occasionally mild hemoptysis, rales, or wheezes. These may be associated with an urticarial rash or low-grade fever. Rarely, an adult worm can ectopically migrate into the lungs or other viscera and get ‘lost’. Human hookworm disease is caused primarily by either Ancylostoma duodenale or Necator americanus. Larvae enter the skin and then migrate to the lungs. From there, they crawl up the bronchial tree to the epiglottis, where they are swallowed. Once in the small intestine, the adult worm develops and starts shedding eggs. The migration of the larvae through the lungs may cause a Loeffler’s syndrome similar to that in ascariasis. Patients may have a mild cough, or the symptoms may be more severe, with associated dyspnea and wheezing. Symptoms generally last B2 weeks; in heavy infections, they can last up to 3 months. Chest X-ray can show transient, migratory opacities, especially in the hilar areas, with spontaneous clearing.

Strongyloides infection is also acquired when larvae penetrate through skin on contact with fecally contaminated soil or water. In strongyloidiasis, the pulmonary symptoms are predominantly cough, dyspnea, and bronchospasm during the migratory phase, although persistent asthma due to Strongyloides infection has been reported. Patients with chronic lung disease may retain the larvae in the lungs, where they molt into adults and complete their entire life cycle, leading to worsening lung disease with chronic cough and bronchospasm. In disseminated disease, intense worm burdens or inflammatory response can cause acute respiratory distress syndrome (ARDS), alveolar hemorrhage, or bacterial superinfection of the lungs. During the initial, pulmonary phase of infection, intestinal roundworm infections are difficult to diagnose. Once the mature worms have started shedding eggs in the intestines, these eggs (for Ascaris and hookworm), or larvae (for Strongyloides), can be identified in the stool. Anthelmintic treatment of intestinal infections probably has no effect on larval stages in the lungs; however, pulmonary disease is usually self-limited and does not require specific treatment, except for symptomatic relief. To treat intestinal disease, albendazole or mebendazole can be used for ascariasis and hookworm, and ivermectin is the treatment of choice for strongyloidiasis.

Lymphatic Filariasis Filariae are a family of tissue-dwelling roundworms that infect adults in the tropics and subtropics. The major pulmonary manifestation of these organisms is tropical pulmonary eosinophilia (TPE) seen in

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infection with the lymphatic filariae, Wuchereria bancrofti and Brugia malayi. TPE is felt to be secondary to increased immune responsiveness to the parasites, and is really a systemic syndrome, with the pulmonary component being the most significant. Initially, patients complain of vague systemic symptoms, including low-grade fevers and myalgias. Then pulmonary symptoms start, and these include cough (which can be paroxysmal) and wheezing, both being worse at night, dyspnea, and occasionally chest pain. This can often be mistaken for asthma, and patients may be incorrectly treated for more common disorders. Pulmonary function is abnormal in these patients. As the disease progresses, the symptoms can spontaneously remit and recur and gradually worsen, with a decrease in the wheezing but an increase in dyspnea. This eventually leads to chronic lung disease with dyspnea and hypoxia at rest, which in later stages is indistinguishable from any cause of interstitial pulmonary fibrosis. Rarely, TPE can present as cor pulmonale. The diagnosis of TPE requires that a patient meets both clinical and laboratory criteria with marked elevations of peripheral eosinophil and IgE levels. Most patients respond dramatically to treatment with diethylcarbamazine.

Toxocara Visceral larva migrans (VLM) or toxocariasis is a zoonotic infection usually caused by dog or cat ascarids of the Toxocara genus. The eggs are ingested and then hatch into larvae, which penetrate the intestine and start migrating. Because humans are not the definitive host, the larvae cannot mature, and so continue migrating for months or years. Most people who are infected with Toxocara spp. are asymptomatic. Symptoms depend on the extent and frequency of infection, the distribution of larvae in tissues, and the inflammatory response of the host. There are two clinical syndromes of infection: VLM and ocular larval migrans (OLM). The symptoms of VLM are related to the organ invaded, most commonly the liver, lung, or other thoracic or abdominal organ. VLM usually affects children o5 years old, although it can affect adults. In children with VLM 33–86% had pulmonary symptoms: most commonly, chronic cough, which may be paroxysmal and worse at night, wheezing, and pulmonary infiltrates. On exam, 65% had hepatomegaly, 43% had an abnormal lung exam (wheezes or rales). VLM is diagnosed by serology and treated with albendazole.

Echinococcus Humans can develop lung disease after being incidentally infected with Echinococcus sp., which are

usually parasites of canines, sheep, and other domestic ungulates. Infection with Echinococcus is always asymptomatic for months to decades. Cysts are commonly found when symptoms develop or on routine or screening imaging. Symptoms develop because of one or more of three processes: (1) mechanical pressure or deformation of tissues, or nearby vascular structures; (2) rupture or leakage of the cyst; or (3) superinfection. The liver is the organ most commonly affected in 60% of cases, and the lungs are affected in 20–30% of cases. Pulmonary symptoms have been reported in 67–89% of patients with lung cysts. Most commonly, symptoms are cough, fever, and chest pain. Occasionally, hemoptysis can occur, and rarely, biliptysis, pneumothorax, or pleuritis. With rupture of the cyst into a bronchus, hydatid vomica of cystic contents can occur: clear, salty/peppery fluid with or without hemoptysis. The fluid can be purulent if superinfected. With dissemination of the antigenic fluid, respiratory failure or anaphylaxis can occur. One-third of diagnosed cases of pulmonary hydatid cyst show ruptured or infected cysts. Diagnosis is made by clinical history and radiologic findings. Early surgical intervention promptly after diagnosis can help prevent complications. Perioperatively, or for unresectable lesions, medical therapy with anthelmintics (albendazole) should also be used. See also: Allergy: Allergic Reactions. Asthma: Overview. Human Immunodeficiency Virus. Leukocytes: Eosinophils. Pneumonia: Overview and Epidemiology; The Immunocompromised Host. Systemic Disease: Eosinophilic Lung Diseases.

Further Reading Chu E, Whitlock WL, and Dietrich RA (1990) Pulmonary hyperinfection syndrome with Strongyloides stercoralis. Chest 97: 1475–1477. DeFrain M and Hooker R (2002) North American paragonimiasis: case report of a severe clinical infection. Chest 121(4): 1368– 1372. Dogan R, Yuksel M, Cetin G, et al. (1989) Surgical treatment of hydatid cysts of the lung: report on 1055 patients. Thorax 44(3): 192–199. Gottstein B and Reichen J (2002) Hydatid lung disease (echinococcosis/hydatidosis). Clinics in Chest Medicine 23(2): 397– 408. ix. Huntley CC, Costas MC, and Lyerly A (1965) Visceral larva migrans syndrome: clinical characteristics and immunologic studies in 51 patients. Pediatrics 36: 523–536. Ibarra-Perez C (1981) Thoracic complications of amebic abscess of the liver. Report of 501 cases. Chest 79: 672–677. Mandell GL, Bennett JE, and Dolin R (eds.) (2000) Principles and Practice of Infectious Diseases, 5th edn. Philadelphia: Churchill Livingstone. Meehan AM, Virk A, Swanson K, and Poeschla EM (2002) Severe pleuropulmonary paragonimiasis 8 years after emigration from

PNEUMONIA / Mycobacterial a region of endemicity. Clinical Infectious Diseases 35(1): 87–90. O’Lorcain P and Holland CV (2000) The public health importance of Ascaris lumbricoides. Parasitology 121(supplement): S51–S71. Ottesen EA and Nutman TB (1992) Tropical pulmonary eosinophilia. Annual Review of Medicine 43: 417–424. Sarinas PSA and Chitkara RK (1997) Ascariasis and hookworm. Seminars in Respiratory Infections 12(2): 130–137. Shamsuzzaman SM and Hashiguchi Y (2002) Thoracic amebiasis. Clinics in Chest Medicine 23(2): 479–492. Stanley SL Jr (2003) Amoebiasis. Lancet 361(9362): 1025–1034. Talaat KR and Nutman TB (2005) Parasitic infections. In: Murray JF, Nadel JA, Mason RJ, and Broaddus VC (eds.) Textbook of Respiratory Medicine, 4th edn. Philadelphia: Elsevier. Tor M, Atasalihi A, Altunas N, et al. (2000) Review of cases with cystic hydatid lung disease in a tertiary referral hospital located in an endemic region: a 10 years’ experience. Respiration 67(5): 539–542.

Mycobacterial N W Schluger, Columbia University College of Physicians and Surgeons, New York, NY, USA J Burzynski, New York City Department of Health, New York, NY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Tuberculosis has probably been the cause of more deaths than any other infectious disease. Although the disease is almost always treatable with a handful of antibiotics taken once a day, it continues to cause tremendous suffering and is a leading cause of death. Despite the recent attention and efforts at control in the short term, rates are likely to increase. In many parts of the world, current tuberculosis control programs are ineffective and the number of tuberculosis cases is rising. A program of directly observed therapy short (DOTS) course has been widely accepted as a standard of care but less than half of all persons with tuberculosis receive treatment with directly observed therapy. In many countries with high disease burden, coinfection with human immunodeficiency virus (HIV) has contributed to increasing rates of tuberculosis. Tuberculosis is the leading cause of death from an opportunistic infection in persons with acquired immunodeficiency syndrome (AIDS) worldwide.

Epidemiology One-third of the world’s population is infected with tuberculosis and infection leads to over eight million cases of active disease and two million deaths per year. Case rates of active disease vary greatly by geographic region and by country and are influenced by many factors including varying case definitions and completeness of reporting. The highest incidence rates are in sub-Saharan Africa; China and India have the highest number of total cases. Case rates in

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the US and Western Europe trended downwards for most of the twentieth century and are currently at the lowest rate in recorded history. A thorough understanding of the epidemiology of tuberculosis is essential in establishing effective public health control measures. Clinicians’ understanding of the epidemiology and the ability to suspect and establish the diagnosis is heavily influenced by an understanding of a patient’s epidemiologic risk factors. Case rates are highest in young adults as a result of increased social interaction and an initial opportunity for exposure to someone else with an infectious form of the disease. At the extremes of age, young children and the elderly are also especially prone to developing active disease. Children who are infected after an exposure to an active case have a much higher rate of progression to active disease. While overall, 5–10% of infected contacts progress to active disease after an exposure to an active case, 43% of infants under 1 year of age and 24% of children 1–5 years old develop active disease after recent infection. The elderly have a higher case rate as a result of reactivation disease. With the advances of age, a weakened immune system can no longer control dormant bacteria from a previous infection and active disease develops. In low incidence countries like the US and most of the countries of Western Europe, tuberculosis cases are more common in men than in women but now in some countries with high rates of HIV infection, tuberculosis case rates are higher in women than in men. Certain host characteristics are associated with disease progression. Clinicians have long noticed more cases of pulmonary disease associated with tall, lean persons. Poor nutrition and being underweight are also risk factors. Certain medical conditions in the host are associated with an increased risk of tuberculosis disease, including HIV infection, diabetes mellitus (particularly when poorly controlled), end-stage renal disease, and silicosis. These conditions that have an effect on the response of the immune system all have well-documented associations with active tuberculosis disease. The use of certain medications that affect host defenses including corticosteroids, blockers of tumor necrosis factor (TNF), and immunosuppressive medications used for patients with organ transplantation also pose significant risks for developing active tuberculosis. Nothing has had more impact on tuberculosis epidemiology than HIV. HIV coinfection is the greatest risk factor for reactivation of latent infection into clinical tuberculosis disease. HIV-positive persons

452 PNEUMONIA / Mycobacterial

infected with tuberculosis have a 10% per year rate of progression to active tuberculosis. The HIV epidemic has contributed enormously to increasing tuberculosis rates in countries with high rates of both diseases that overlap. In sub-Saharan Africa where tuberculosis rates are increasing, 31% of new cases and 34% of deaths are associated with HIV.

Transmission and Pathogenesis The management and control of tuberculosis is usually the responsibility of public health departments. The disease is most often transmitted from person to person through airborne transmission. The only important exception is bovine tuberculosis, which can be transmitted through the milk of infected cows and is seen in parts of the world where milk and milk products are consumed raw without pasteurization. When individuals with pulmonary disease cough and produce enough force to produce respiratory droplets filled with tubercle bacilli, the bacilli remain suspended in the air and can be inhaled by anyone else sharing the air space. Public health practitioners have the responsibility to reduce the possibility of the interaction of the tubercle bacilli with a susceptible population. Primary tuberculosis infection occurs in persons who do not have previous exposure but then are exposed and inhale the tubercle bacilli in the form of respiratory droplets into their lungs. These respiratory droplets are small and are able to evade the barrier defenses of the lung and can travel to the terminal alveoli. The bacilli multiply in the alveolar macrophages of the lung. The location where they are concentrated is known as the Ghon focus. This focus is drained by lymphatics to the hilar lymph nodes, and the focus and the draining hilar lymph nodes together are known as the Ghon complex. Bacilli may spread from the hilar lymph nodes through the blood to any organ in the body. Once bacilli enter the lungs and start to multiply, a complex interaction between the bacteria and the host immune system is initiated. This interaction most often results in arrest of the infection and the tubercle bacilli is trapped in a state where it is inactive and becomes dormant. This is the outcome in at least 90% of infected persons. Such persons have latent tuberculosis infection and are asymptomatic. If the tubercle bacilli are not adequately controlled by the immune system they will continue to multiply and may cause symptoms and clinical illness that is called tuberculosis disease. Through a complex interplay of macrophages and lymphocytes mediated through chemokines and

cytokines, particularly interferon gamma, the bacteria are eliminated, suspended in a latent state, or progress and continue to replicate. The process starts when the bacillus reaches the alveolus and it is taken up by alveolar macrophages. Within the phagosomal vacuole of the macrophage the bacilli replicate and ultimately destroy the cell with release of more bacilli. The interaction of the bacilli and macrophage results in the release of cytokines and chemokines that attract other cells including more macrophages, dendritic cells, peripheral blood monocytes, lymphocytes, and neutrophils. When these cells respond and control an infection they form a familiar pattern known as a granuloma. The granuloma is the cardinal feature of the initial host response to infection and is the hallmark of latent infection. For most hosts, viable bacteria may remain suspended in the granuloma for a prolonged period, perhaps for the life of the host. Many factors are involved in maintaining the integrity of the granuloma and the latent state. The importance of TNF for maintaining the latent state of infection was demonstrated in humans when patients who were given infliximab, a monoclonal antibody to TNF, later developed an unexpectedly high rate of active tuberculosis. Certainly T lymphocytes have an important role in controlling bacterial replication and disease progression. The CD4 cell depletion that characterizes patients with HIV infection is a critical reason for the much greater rates of progression from latent tuberculosis infection to active disease in coinfected persons.

Latent Tuberculosis Infection Latent tuberculosis infection has produced a huge reservoir of infected persons that may later develop tuberculosis disease. The two billion persons with latent tuberculosis infection, that is persons who have been infected with tuberculosis and harbor the tubercle bacilli, are a huge reservoir of potential active cases for the future. Clinically, this group is characterized by evidence of tuberculosis infection through a positive reaction to tuberculin skin testing (TST), a normal chest X-ray, and the absence of signs or symptoms of active tuberculosis. From this group will emerge active tuberculosis cases in the future. While the distinction between latent tuberculosis infection and clinically active disease may be difficult to make at times in some patients, biologically a distinction between the two states can be appreciated if considering the difference of the burden of bacteria in the two populations. In latent tuberculosis infection the tuberculosis bacillus is quiescent,

PNEUMONIA / Mycobacterial

captured, and maintained in the confines of the macrophage. The total number of bacilli in an infected person is in the range of 102. The situation in a person with cavitary pulmonary disease is distinctly different, with the estimated number of bacilli from just a medium-sized cavity being 108.

Active Pulmonary Disease Although tuberculosis can infect any organ in the body, it is chiefly a pulmonary disease. Prior to the HIV era, 80–90% of tuberculosis cases had clinical pulmonary involvement. Pulmonary disease will be the emphasized in this article. Pulmonary disease in tuberculosis has classically been divided into three categories: (1) primary; (2) reactivation; and (3) miliary. Primary Tuberculosis

Traditionally, tuberculosis is classified as primary or secondary depending on the time between the initial infection with Mycobacterium tuberculosis and the appearance of clinical disease. Usually disease is considered primary if the time from initial infection to clinical symptoms is less than 5 years. After someone is exposed to a person with active tuberculosis and inhales droplet nuclei with bacilli, if the bacilli are deposited in the alveoli they are ingested by alveolar macrophages of the immune system. If the initial efforts of the immune system do not control the infection the bacilli will cause a lesion in the lung. Grossly these lesions will have the appearance of a chalky substance that is termed caseation. The primary lesion together with draining lymphatics and involved lymph nodes is known as the Ghon complex. If the initial focus continues to progress it is called a progressive primary complex and usually presents clinically. Historically, it has been taught that radiographic characteristics of primary tuberculosis are the involvement of lower and mid-lung zones and pleural effusions and adenopathy are more frequent. However, recent studies using tools of molecular epidemiology suggest that radiographs do not accurately reflect the time elapsed between acquisition of infection and development of active disease. Symptoms are a manifestation of factors produced by the response of the immune system and may include fever, cough, poor weight gain, or weight loss. Pleural effusions typically present clinically with pleuritic chest pain. In general, symptoms are less protracted and less severe. Some patients, particularly children, may be asymptomatic. The clinical presentations of symptoms and chest X-ray findings that are classified as primary

453

tuberculosis disease have been thought to reflect the development of disease shortly after an exposure with recent infection. Young children usually present with clinical findings of primary disease and were necessarily recently exposed. Persons coinfected with HIV more frequently present with findings typical of primary disease. However, whether this is due to recent exposure with rapid progression of disease or whether it represents reactivation of a long-standing infection in the setting of an abnormal immune response has not been clear. As noted above, a recent epidemiologic study using molecular technology has suggested that these typical clinical symptoms and X-ray findings may not necessarily be the result of recent infection but may more commonly reflect the underlying immune status of the patient. The clinical presentation and X-ray findings of primary tuberculosis are more common in persons coinfected with HIV. Reactivation Tuberculosis

Reactivation disease has traditionally been described as occurring long after an exposure to a case of active pulmonary tuberculosis. After initial infection, as the immune system struggles for control, bacilli may enter the bloodstream and travel to any site in the body. One outcome is a fibronodular lesion in the apices of the lungs that contain viable bacilli, but the majority of patients maintain normal chest X-rays. Most patients will never know that they were infected and will remain symptom free. However, in a small percentage of infected patients (maybe 5– 10%), by mechanisms that are not entirely clear, the bacillus escapes effective control and begins to multiply, possibly many years after the initial infection. If the focus of the disease is the lungs, patients often develop symptoms such as fever, cough, night sweats, malaise, and weight loss. The range of symptoms is quite variable. Some patients may be severely ill with dramatic symptoms, a syndrome termed ‘galloping consumption’. Other patients may present for medical attention after months of low-grade symptoms even with extensive disease by X-ray. The cough is usually productive and may be blood tinged in up to a third of patients. Sometimes massive hemoptysis develops. Typical radiographic findings are upper lobe infiltrates or posterior segment lower lobe infiltrates. The infiltrates are typically cavitary and accompanied by lung retraction. As mentioned in the section on primary disease, reactivation tuberculosis may be more a reflection of the host immune status than a reflection of the time involved from original infection to disease progression.

454 PNEUMONIA / Mycobacterial Miliary Tuberculosis

The term miliary tuberculosis is used to describe a characteristic pattern on X-ray demonstrating the unfettered hematogenous spread of the tubercle bacillus. The appearance on chest X-ray is one of a vast number of tiny nodules throughout the lungs that were once described to have the appearance of millet seeds. The term miliary tuberculosis is often used interchangeably with the term disseminated disease reflecting the pathophysiology of the spread of the organism to many sites in the body. The pathophysiology may sometimes be a part of the bacillemia of primary tuberculosis after a recent exposure to an active case or it may be a presentation of reactivation disease after an exposure many years in the past as massive numbers of bacilli from a site of active replication gain access to the vascular system. It is likely that it is not time from exposure but the immune status of the patient that is the key determinant of which patients develop miliary tuberculosis. Before the HIV epidemic, miliary tuberculosis was relatively rare, being found in 1–2% of reported cases. It was most often seen in young children and the elderly reflecting less developed or impaired cellular immunity in those groups. Because it is more common in HIV-infected persons, its incidence has increased as a percentage of reported cases in recent years. Clinical presentations range widely from minimal symptoms to overwhelming illness with acute respiratory distress syndrome (ARDS). Symptoms usually include fever, weakness, anorexia, and weight loss. Cough and dyspnea are also frequent with diffuse pulmonary disease. Often patients with miliary tuberculosis have scant sputum production and the sputum is frequently smear negative for acid-fast bacilli (AFB). The tuberculin skin test more often produces false-negative results than in other forms of the disease. Anemia and elevated liver transaminase values are frequent but non-specific. If the chest X-ray appearance is not typical or disseminated tuberculosis is suspected without a characteristic appearance on chest X-ray, high resolution computed tomography may provide a characteristic pattern. The diagnosis is often difficult to establish and therefore a bronchoscopy with biopsy or biopsy of other involved organs may be necessary as part of the evaluation. Meningeal disease should be excluded during the diagnostic workup.

Diagnosis Sputum smear microscopy is the principal mode of diagnosis in most countries with a high burden of

tuberculosis. It is the most efficient way of identifying sources of infectious tuberculosis and confirming the diagnosis in suspected persons. Expectorated sputum is stained for AFB through a variety of methods; Ziehl-Neelson, Kinyoun, and fluorochrome stains can be used. Processing the sputum for AFB staining can be done with relative ease and with a good laboratory can be reliably interpreted. However, the sensitivity of sputum smear for the diagnosis of pulmonary tuberculosis ranges from 34% to 80%. A major disadvantage of relying on sputum smears for diagnosis is that a large number of pulmonary cases will not be detected using only this method. The sputum smear is more effective in detecting disease in cases of cavitary pulmonary disease in patients with a forceful cough. It is less sensitive for noncavitary cases or in patients such as children who do not have a forceful cough. A particular concern in regions where patients with tuberculosis have a high rate of HIV coinfection is that these patients are less likely to have cavitary chest X-rays and are less likely to produce AFB smear-positive sputum. Sputum smears are not useful in cases that are extrapulmonary only. When resources are not limited, three sputum samples are collected from patients with suggestive radiological or clinical evidence of active tuberculosis. Three specimens are collected on three separate days and are submitted to the laboratory for AFB smear and mycobacteriologic culture. A positive culture for tuberculosis is the gold standard for diagnosis. Cultures have a high sensitivity ranging from 80% to 93% and a high specificity of 98%. Two types of methods are used: solid media or liquid media. Solid media have long been used as the standard for culturing mycobacteria but growth is slower than with liquid media. A culture may become positive within 4 weeks but cultures are usually held for 8 weeks with no growth before they can be classified as negative. Liquid media can detect the growth of mycobacteria within 2 weeks. Both liquid and solid media are used for drug susceptibility testing although only solid media is used for testing second line medications. The nucleic acid amplification assays (NAAs) are a more recent technology that allow the detection of tuberculosis directly from clinical specimens. These assays are rapid, sensitive, and specific. The sensitivity of the assays currently in use is about 80% but it is higher with smear-positive samples than with smear-negative samples. The specificity is 98–99%. The nucleic acid amplification tests are particularly useful for the proper classification of patients with sputum specimens that are AFB positive. Patients with a negative NAA can be removed more rapidly

PNEUMONIA / Mycobacterial

from hospital isolation and patients with a positive NAA can be promptly started on therapy.

Treatment Drug-Susceptible Tuberculosis

Medical care providers along with public health departments have the responsibility for ensuring their patients complete therapy. One method to assist and ensure completion of therapy is directly observed therapy (DOT). DOT is a service provided by healthcare departments in which a trained public health worker watches the ingestion of each dose of medication taken to complete therapy. Many studies have demonstrated that DOT increases tuberculosis treatment completion rates and improves outcomes. DOT is the standard of care for patients with tuberculosis and should be offered to all patients receiving medical therapy. Effective antibiotics for tuberculosis first became available in the 1940s with the introduction of streptomycin. This was followed within a few years by two more antituberculosis medications, para-aminosalicylic acid and isoniazid. The discovery and widespread use of these drugs dramatically changed the lives of millions of people. The tuberculosis sanatorium, which offered patients the comfort of bed rest and helped diminish tuberculosis transmission in the community, long the only hope for many patients, soon became obsolete. A cure was now possible for most patients. However, it was recognized early that without prolonged therapy, patients had a high rate of relapse after therapy was discontinued. The minimum effective treatment duration was 18 months. Shorter length of treatment was not possible until trials conducted by the British Medical Research Council in the 1970s demonstrated that isoniazid and rifampin given for 9 months was effective with low rates of relapse. It was later shown that with the addition of pyrazinamide treatment regimens could be shortened to 6 months. Because a small percentage of naturally occurring tubercle bacilli will have inherent resistance to various tuberculous medications, at least two medications to which the bacilli is susceptible should always be used. It is a central tenet of tuberculosis therapy not to add a single drug to a failing regimen as it can lead to further resistance and treatment failure. Current recommendations are to use a regimen of isoniazid, rifampin, pyrazinamide, and ethambutol. Ethambutol is included in the regimen in the event that drug resistance is later detected from the tuberculosis culture. If the offending organism is known to be susceptible to all antituberculous medications

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then ethambutol can be excluded. Pyrazinamide should be used for the first 2 months, and isoniazid and rifampin should be given throughout therapy. Most patients should receive treatment for a total of 6 months. The standard 6 month regimen is associated with low rates of relapse. Patients with cavitary chest X-rays who remain sputum culture positive for tuberculosis after 2 months of therapy have high rates of relapse. Current guidelines recommend extending therapy with isoniazid and rifampin for a total of 9 months of treatment for such patients. Tuberculosis treatment is usually given daily at the initiation of therapy. After 2 months of therapy most patients are receiving only two medications, INH and rifampin. These medications can be given on an intermittent schedule, either two or three times a week. One widely accepted regimen starts with daily therapy and switches to intermittent therapy after the first 2 weeks. Intermittent therapy is easier for patients, is associated with fewer side effects, and reinforces the message of the importance of DOT as all doses are taken with observation. The treatment of HIV-related tuberculosis follows the same recommendations as for those who are not HIV infected. Most patients with HIV-related pan-susceptible tuberculosis can be successfully treated with 6 months of therapy. Some studies have suggested a higher rate of relapse for HIV-infected tuberculosis patients; however, it is not clear that prolonging therapy will substantially lower relapse rates. Patients with extrapulmonary tuberculosis in addition to pulmonary tuberculosis or with isolated extrapulmonary tuberculosis can be treated with standard regimens. Corticosteroids are used in the treatment of severe central nervous system (CNS) disease and pericarditis. The recommendation for CNS-related tuberculosis is to extend therapy for a total of 9–12 months. Multidrug-Resistant Tuberculosis

By definition multidrug-resistant tuberculosis strains have laboratory evidence of resistance to isoniazid and rifampin. Most strains have additional resistance to the other first line drugs pyrazinamide, ethambutol, and streptomycin and also some of the second line drugs. Multidrug-resistant tuberculosis is transmitted in a similar manner and but may be less virulent than susceptible tuberculosis. However, it can cause serious disease and has a much slower response to available therapy. Without isoniazid and rifampin the treatment regimen is chosen from the remaining drugs to which the organism is susceptible. These regimens usually include at least one

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injectible medication like streptomycin, or if the organism is resistant to streptomycin, then capreomycin or amikacin are chosen. If possible, at least three other oral medications should be used along with the injectable. Duration of therapy is at least 18 months; the injectable should be included in the regimen for at least the first 6 months of therapy if tolerated. For patients who have localized disease and do not have culture conversion by 2 months of therapy, consideration should be given to surgical removal of the diseased lung. Treatment regimens for patients coinfected with HIV are the same although therapy should be extended to 24 months. Patients coinfected with HIV and MDRTB have a high mortality rate. See also: Acute Respiratory Distress Syndrome. Bronchiectasis. Bronchoalveolar Lavage. Human Immunodeficiency Virus. Pneumonia: Overview and Epidemiology; Atypical. Pulmonary Fibrosis. Systemic Disease: Eosinophilic Lung Diseases. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading American Thoracic Society (2000) Targeted tuberculin testing and treatment of latent tuberculosis infection. American Journal of Respiratory and Critical Care Medicine 161: S221– S247. American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America (2003) Treatment of tuberculosis. American Journal of Respiratory and Critical Care Medicine 167: 603–662. Brodie D and Schluger NW (2005) The diagnosis of tuberculosis. Clinics in Chest Medicine 26: 247–271. Cole ST, Brosch R, Parkhill J, et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393: 537–544. Corbett EL, Watt CJ, Walker N, et al. (2003) The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Archives of Internal Medicine 163: 1009– 1021. Dye C, Watt CJ, Bleed DM, Hosseini SM, and Raviglione MC (2005) Evolution of tuberculosis control and prospects for reducing tuberculosis incidence, prevalence, and deaths globally. JAMA 293: 2767–2775. Flynn JL and Chan J (2005) What’s good for the host is good for the bug. Trends in Microbiology 13: 98–102. Frieden TR and Munsiff SS (2005) The DOTS strategy for controlling the global tuberculosis epidemic. Clinics in Chest Medicine 26: 197–205. Geng E, Kreiswirth B, Burzynski J, and Schluger NW (2005) Clinical and radiographic correlates of primary and reactivation tuberculosis: a molecular epidemiology study. JAMA 293: 2740– 2745. Smith I (2003) Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clinical Microbiology Reviews 16: 463–496. Tufariello JM, Chan J, and Flynn JL (2003) Latent tuberculosis: mechanisms of host and bacillus that contribute to persistent infection. Lancet Infectious Diseases 3: 578–590.

Viral D S Hui and P K S Chan, Chinese University of Hong Kong, Hong Kong, People’s Republic of China & 2006 Elsevier Ltd. All rights reserved.

Abstract Viral infection accounts for a substantial proportion of cases of acute pneumonia especially among young children and the elderly, the immunocompromised, and those with comorbidities. Influenza A and respiratory syncytial virus are by far the most common causes of viral pneumonia followed by adenovirus, parainfluenza virus types 1, 2 and 3, and influenza B. Other less common agents include picornaviruses, varicella-zoster virus, herpes simplex virus, cytomegalovirus, and hantavirus. The newly identified human metapneumovirus also plays a role. Zoonotic infections caused by severe acute respiratory syndrome-associated coronavirus and avian influenza A/H5N1 are examples of acute ‘atypical’ pneumonia with epidemic and pandemic potentials. In general, there are no reliable clinical or radiological features to distinguish viral from other causes of pneumonia. Respiratory viruses often show seasonality and a predilection for certain host groups. These epidemiological features are helpful in gauging the differential diagnoses. Confirmation of infection relies on laboratory investigations based on conventional approaches including direct viral antigen detection by specific monoclonal antibodies, virus isolation, and serology, as well as modern molecular approaches to amplify viral nucleic acid present in clinical specimens. The available spectrum of antiviral agents and the window for effective application are narrow. Treatment of viral pneumonia is primarily supportive. Vaccines for general use are only available for influenza A and B.

Introduction Acute pneumonia is an important cause of morbidity and mortality worldwide. While the majority of cases of acute community acquired pneumonia (CAP) are due to bacteria, especially Streptococcal pneumoniae and less commonly the atypical organisms, respiratory tract viruses also contribute to a substantial proportion. More importantly, in general, viral pneumonia has a higher outbreak potential. A wide spectrum of viruses can cause viral pneumonia (Table 1, Figure 1). The relative importance of these viruses depends on the host’s age, immune status, comorbidity, and the local epidemiological determinants such as seasonality.

Influenza The Virus

Influenza viruses belong to the genus Orthomyxovirus within the family Orthomyxoviridae. This enveloped virion contains eight (influenza A and B) or seven (influenza C) segments of single, negative-stranded RNA. The pleomorphic envelope is composed of two

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Table 1 Viruses associated with acute pneumonia Children

Adults

Immunocompromised a

Common Respiratory syncytial virus Parainfluenza virus types 1, 2, 3 Influenza A Uncommon Adenovirus Human metapneumovirus Influenza B Measles Rhinovirus Enterovirus Hantavirus

Common Influenza A Influenza B Uncommon Adenovirus Respiratory syncytial virus Parainfluenza virus types 1, 2, 3 Rhinovirus Enterovirus Human metapneumovirus Hantavirus Varicella-zoster virus Measles SARS-associated coronavirus

Cytomegalovirus Human herpes simplex virus 6 Herpes simplex virus

a

In addition to respiratory viruses seen for the age group.

Picornaviridae Rhinovirus Enterovirus

Coronaviridae SARS-coronavirus Coronavirus NL63

Animal virus family

Adenoviridae Adenovirus

Paramyxoviridae Parainfluenza virus Respiratory syncytial virus Human metapneumovirus

Orthomyxoviridae Influenza A, B, C Figure 1 Electron micrographs of the major human respiratory virus families.

major antigenic glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Major changes in the antigenic protein structure of HA (i.e., antigenic shift) may occur and lead to a pandemic. Minor changes involving a small number of amino acid substitutions (i.e., antigenic drift) lead to an epidemic of influenza. Antigenic shift is rare, whereas significant antigenic drift occurs once every few years. Altogether, 16 different subtypes of HA and nine different subtypes of NA have been identified from influenza A viruses from various animal species (Table 2). Currently, two subtypes of influenza A, A/H1N1 and A/H3N2, are in human circulation. Introduction of a new subtype

into humans can result in a large-scale pandemic with high morbidity and mortality. This is because of the naive immunity and the absence of an effective vaccine in the initial phase of outbreak. Three influenza pandemics occurred in the twentieth century. The most severe of these was ‘Spanish flu’ A/H1N1, which occurred in 1918–19 with a global mortality of at least 20–25 million people. It was estimated that 50% of the global population became infected, half of which suffered clinical illnesses. The A/H2N2 pandemic that occurred between 1957 and 1958 caused about 70 000 deaths in the US. In 1968, a new type (A/H3N2) emerged and remains in circulation today.

458 PNEUMONIA / Viral Table 2 Hemagglutinin (HA) and neuraminidase (NA) subtypes of influenza A circulating in human and other animals Subtype

Hemagglutinin H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 Neuraminidase N1 N2 N3 N4 N5 N6 N7 N8 N9

Host species Human

Bird

Pig

O (Major circulating subtype) O O (Major circulating subtype)

O O O O O O O O O O O O O O O O

O

O (No efficient human-to-human transmission) O (Isolated outbreaks) O (Isolated cases)

O (Major circulating subtype) O (Major circulating subtype)

O O O O O

O (Isolated outbreaks)

O O O

Human Influenza

Influenza A viruses infect humans, pigs, birds, horses, and other species, whereas influenza types B and C only infect humans. In humans, influenza is mainly transmitted by respiratory droplets. The incubation period ranges from 1 to 4 days. The onset of illness is remarkably abrupt with fever, headache, photophobia, shivering, malaise, myalgia, nausea, dry cough, hoarseness of voice, and nasal congestion. Fever is usually continuous and lasts for about 3 days. A biphasic fever pattern may occur. Most patients recover after 1 week. Pneumonia in patients with influenza can be a primary viral pneumonia sometimes complicated by secondary bacterial infections, often due to Staphyloccocus aureus, Streptococcus pneumoniae, or Haemophilus influenzae. Both forms of pneumonia are relatively uncommon for infection with usual human influenza subtypes. Pneumonia is mainly seen in immunocompromised hosts and the elderly with preexiting cardiopulmonary diseases or chronic illnesses. Avian Influenza

Birds, particularly aquatic and migratory species, are natural hosts of all 16 HA subtypes of influenza A

O

Horse

O

O

O O

O O

virus, whereas only a few subtypes have established transmissible infections in humans, pigs, and other mammals (Table 2). In 1997, 18 people in Hong Kong were infected with the avian influenza A/H5N1 virus, resulting in six deaths. Subsequent serological studies have shown that human-to-human transmission can occur although the transmission efficiency is low. In 2003, there were another two human cases of H5N1 probably imported from Fujian, mainland China, to Hong Kong. The highly pathogenic avian influenza virus H5N1 has caused further disease outbreaks in poultry in multiple East Asian countries since late 2003. The virus caused fatal human infections in Thailand, Vietnam, and Cambodia. From January 2004 to mid-June 2005, at least 107 cases of human avian H5N1 occurred in Vietnam, Thailand, and Cambodia with reported mortality rates ranging from 46% to 100%. A series of genetic reassortment events have been demonstrated to be traceable to the precursor of the A/H5N1 virus that caused the initial human outbreak in 1997, and the subsequent avian outbreaks in 2001 and 2002 in Hong Kong. These events gave rise to a dominant A/H5N1 genotype (Z) in chickens and ducks that was responsible for the regional outbreak

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in 2003–04. It appears that domestic ducks in southern China play a central role in the generation and maintenance of this virus. Wild birds may have contributed to the increasingly widespread distribution of the virus in Asia. A/H5N1 viruses with pandemic potential have become endemic in birds in the region and are not easily eradicated. The greatest concern is that there will be reassortment between the current avian A/H5N1 strain and circulating human or porcine influenza viruses, producing a novel, virulent, and readily transmissible human strain. This ecological feature of influenza poses a tremendous threat to public and veterinary health in the region and the world. The clinical features and spectrum of human A/H5N1 cases are shown in Table 3 and Figure 2. High fever, cough, and dyspnea are the major symptoms at presentation but gastrointestinal symptoms such as diarrhea are also common. The chest radiographs of a human case are shown in Figure 3. Many patients progress to multiorgan failure involving the lungs, liver, and kidneys with evidence of hemophagocytosis. The primary lesion of experimental A/H5N1 virus in macaques was a severe necrotizing broncho-interstitial pneumonia similar in character and severity to that found in influenza viral pneumonia in humans. Recent reports have indicated that some cases may present with gastrointestinal symptoms initially without any respiratory symptoms, and with rapid progression to encephalitis or

Table 3 Symptoms of human influenza A/H5N1 infection Symptoms

Frequency (%)

Fever Cough Dyspnea Sore throat Myalgia Diarrhea Vomiting Abdominal pain Conjunctivitis

100 100 100 75 42 42–70 25 17–90 0

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multiorgan dysfunction syndrome and subsequent death. Asymptomatic infection may occur and this is highlighted by the fact that 10% of poultry workers had positive serology to A/H5N1 and at least one worker who had participated in culling of poultry in Hong Kong in 1997 showed evidence of seroconversion without any symptoms. Treatment

There are two classes of antiviral compounds known to be effective against influenza, namely the M2-ion channel blockers (amantadine and rimantadine) and the NA inhibitors (oseltamivir and zanamivir). Antiviral administration is recommended to reduce morbidity and mortality during a pandemic. The A/H5N1 viruses isolated from recent (2004–05) human cases are resistant to M2-ion channel blockers. This resistance may be retained in a future pandemic strain. The current World Health Organization (WHO) and the US Center for Disease Control guidelines recommend NA inhibitors as the preferred drugs for use during an influenza pandemic. Oseltamivir, in the form of a capsule or oral suspension, is readily absorbed from the gut, and thus has a high bioavailability (at least 75%). Zanamivir, in the form of oral inhalation, has a low systemic bioavailability (o20%). NA inhibitors should ideally be administered within 48 h of the onset of symptoms. The dosage recommendations for usual human influenza strains are shown in Table 4. The future pandemic strain may require a higher dosage and a longer duration of treatment. Zanamivir has a low systemic distribution but dosage adjustment for age or renal function is not required. Oseltamivir has a better systemic distribution, which may be an advantage in case a pandemic strain causes significant extrapulmonary infection. A trivalent influenza vaccine targeting the circulating A/H1N1, A/H3N2, and B subtypes is available. Annual vaccination 6–8 weeks before seasonal peak is recommended for those at risk and for healthcare workers.

Fever, URTI, GIT May have no initial respiratory symptom

No symptom

Mild

Pneumonia, ARDS, MODS, encephalitis, death

Severe

Figure 2 Human influenza A/H5N1: clinical spectrum of disease. URTI, upper respiratory tract infection; GIT, gasterointestinal tract; ARDS, acute respiratory distress syndrome; MODS, multiorgan dysfunction syndrome.

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Figure 3 (a–d) Chest radiographs of a human case of A/H5N1 infection. This 6-year-old girl had previously been in contact with sick poultry. She had fever for 8 days before admission to hospital. On admission, she developed severe respiratory failure, with a total white blood cell count of 2.4  109 l 1, lymphocyte count of 0.5  109 l 1, platelet count of 127  109 l 1, ALT 246 IU l 1, and AST 1379 IU l 1. She was treated with oseltamivir on admission. She died 3 days later of multiorgan failure.

Table 4 Regimen for neuraminidase inhibitors for influenza Treatment Oseltamivir Adults & adolescents 13 years of age or above Children between 1 and 12 years of age 15 kg 16–23 kg 24–40 kg 440 kg Zanamivir Adults & children 5 years of age or above Prophylaxis Oseltamivir Adults & adolescents 13 years of age or above Community outbreak Close contacts Children below 13 years of agea Health care and essential service workers during pandemicb Zanamivir Healthy young adults (age not specified, not licensed for prophylaxis in some countries) a

75 mg twice a day  5 days 30 mg 45 mg 60 mg 75 mg

twice twice twice twice

a a a a

day day day day

   

5 5 5 5

days days days days

10 mg (2 inhalations) twice a day  5 days

75 mg once daily up to 6 weeks 75 mg once daily  7 days 75 mg once daily

10 mg (2 inhalations) once daily, up to 1 month if exposure risk is long

The safety and efficacy of oseltamivir as a prophylactic agent for children under 13 years of age have not been established. However, it may be considered for prophylactic use in this group when the benefits are expected to outweigh the risks. b Duration of prophylaxis should be determined by the intensity and duration of exposure. Safety and efficacy of prophylactic administration of oseltamivir have been demonstrated for continued use of the drug for up to 6 weeks.

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461

Respiratory Syncytial Virus

Treatment

The Virus

Supportive care is most important. Ribavirin, a synthetic nucleoside, has an antiviral effect on RSV in vitro. It is administered by small particle aerosol into an oxygen tent or via a ventilator. However, the clinical benefits and the indications are not entirely clear. RSV immunoglobulin administered once a month has clinical benefit in preventing severe RSV disease in high-risk children with pulmonary or cardiac disease.

Respiratory syncytial virus (RSV) is an enveloped, nonsegmented negative-stranded RNA virus classified as a member of the genus Pneumovirus within the family Paramyxoviridae. Under electron microscopy, the virions appear as pleomorphic spherical particles ranging from 150 to 300 nm in diameter. The morphology is similar to other paramyxoviruses including measles, mumps, and parainfluenza viruses (PIVs). RSV has two antigenic subgroups, A and B, with greater than 90% amino acid homology. The role of strain variation in the epidemiology, severity, and clinical impact of an RSV outbreak remains unclear. Annual seasonal epidemics of RSV occur worldwide. A clear seasonality is seen in the temperate zone with peaks at fall, winter, or spring. In the tropical and subtropical areas, epidemics occur in rainy seasons. Clinical Manifestations

RSV is the single most important cause of hospitalization for respiratory tract infections in babies and young children worldwide. Primary infection occurs in children aged 6 weeks to 2 years old. Rhinorrhea, sneezing, decrease in appetite, low-grade fever, and cough are common presenting symptoms. Tachypnea, diffuse rhonchi, and wheeze indicating involvement of the lower respiratory tract may follow. Bronchiolitis, pneumonia, and otitis media are common complications. Most children recover after 7–12 days. In severe cases, the chest radiograph shows features of interstitial pneumonia with hyperexpansion, segmental or lobar consolidation, and peribronchial thickening; whereas pleural effusion and bacterial superinfection are unusual. Severe illness often occurs in infants less than 9 months old, particularly in those with underlying cardiac or respiratory disease. Premature infants may present with apneic spells. Paradoxically, lower respiratory tract involvement is not common in newborns, reflecting the protective effects of maternal antibodies. Most studies reveal that a substantial proportion of children with severe RSV infection have later decreased pulmonary function, recurrent cough, and wheezing. Repeated RSV infections in older children are usually less severe. It is now realized that RSV infection occurs commonly in healthy adults with a presentation ranging from asymptomatic to severe lower respiratory tract infection. For older persons and the immunocompromised individual, RSV can result in severe pneumonia and acute respiratory distress syndrome (ARDS).

Parainfluenza The Virus

PIVs belong to the family Paramyxoviridae. There are 5 types: PIV1, PIV2, PIV3, PIV4a, and PIV4b. The pleomorphic, enveloped virion contains a nonsegmented, negative, single-stranded RNA genome. The surface glycoprotein, hemagglutinin neuraminidase (HN), is responsible for attachment to the sialic acid on the host cell to initiate the infection cycle. HN together with another enveloped protein, the fusion protein (F), mediate fusion between the viral envelope and host cell plasma membrane. Clinical Manifestations

PIVs are the second leading cause, after RSV, of hospitalization for respiratory tract illnesses in young children. PIVs are transmitted by droplets and produce a spectrum of clinical illnesses ranging from mild upper respiratory tract infection and pharyngitis to otitis media, bronchiolitis, and severe pneumonia. In addition, a syndrome (croup) characterized by croupy cough, inspiratory stridor, hoarse voice, and difficulty in inspiration caused by acute laryngotracheobronchitis is a hallmark presentation of PIVs. Croup is mainly seen in young children, but presentation in adults has been documented. Pneumonia is uncommon in healthy children but fatal pneumonia may occur in those with severe combined immune deficiency, bronchopulmonary dysplasia, prematurity, congenital heart disease, and following bone marrow transplantation. Bronchiolitis obliterans organizing pneumonia has been reported as a long-term sequela. Extrapulmonary infection affecting the central nervous system has been documented. Treatment

Ribavirin, a broad-spectrum antiviral agent, has inhibitory effects in vitro, and has been used to treat PIV pneumonia in immunosuppressed patients. Zanamivir, a sialic acid analog approved for the treatment of influenza, also has inhibitory effects on

462 PNEUMONIA / Viral

PIVs in vitro. However, the clinical benefit of these agents remains to be evaluated. There are no PIV vaccines available yet.

Adenovirus The Virus

Adenoviruses (AdVs) are nonenveloped viruses with an icosahedral capsid containing a linear doublestranded DNA genome. There are more than 40 different serotypes of human AdVs within the family Adenoviridae. Serotypes associated with lower respiratory tract infections are AdV 3, AdV 4, AdV 7, and AdV 21, and less commonly AdV 1, AdV 2, AdV 5, AdV 14, and AdV 35. Clinical Manifestations

In addition to respiratory illnesses, AdVs also cause acute conjunctivitis and keratoconjunctivitis, gastroenteritis, hemorrhagic cystitis, meningitis, encephalitis, skin eruptions, genital tract infection, hepatitis, myocarditis, pericarditis, pancreatitis, rhabdomyolysis, and polyarthritis. The routes of transmission for AdVs are multiple including direct contact, droplet aerosol, fecal-oral, and water-borne. AdVs being nonenveloped viruses are more resistant to the adverse environment, which allows them to be more readily spread via fomites, such as ocular instruments. AdVs account for about 5% of all childhood respiratory illnesses requiring hospitalization. The major presenting clinical features include fever, pharyngitis, tonsillitis, and cough. Lower respiratory tract involvement with pulmonary infiltration occurs in less than half of the hospitalized children. Hilar lymphadenopathy is more commonly observed in chest radiographs compared with other respiratory viruses. Outbreaks of acute respiratory disease due to infection with AdV 3, AdV 4, AdV 7, AdV 14, and AdV 21 in military recruits have been reported from different parts of the world. Most cases presented at the third week of training with fever, malaise, nasal congestion, headache, sore throat, and cough. Around 10% developed patchy pulmonary infiltrates. However, similar outbreaks seldom occur in other crowded situations such as dormitories. AdVs are frequently isolated from patients with pertussis syndrome, which is primarily caused by Bordetella pertussis. There may be a role for AdVs in this syndrome. AdVs may establish latency in adenoidal and tonsillar tissues following primary infections in childhood. Reactivations during immunosuppression may

account for a proportion of the severe adenoviral diseases observed in immunocompromised patients. The overall fatality rate for adenoviral pneumonia, which often has multiorgan dissemination, in the immunocompromised patients is 60%. Treatment

A few antiviral compounds have been used for treating adenoviral diseases. Successful experience has been described for ribavirin in treating pneumonia, hemorrhagic cystitis, and hepatitis in transplant recipients. Cidofovir, a broad-spectrum compound for DNA viruses, has been reported to be successful in treating adenoviral diseases in pediatric hematopoietic stem cell transplant recipients. However, effectiveness of any antiviral compounds demonstrated by randomized controlled trials is not yet available. A live oral vaccine has been used for US military recruits, but production of the vaccine ceased in 1996. AdV vaccines are not available for the general population.

Human Metapneumovirus The Virus

The genus Metapneumovirus is classified within the family Paramyxoviridae. Human metapneumovirus (hMPV) is the only known human virus in this genus. It was first recognized in 2001. hMPV shares basic properties with RSV, which belongs to the same subfamily, the Pneumovirinae. Clinical Manifestation

The transmission of hMPV is probably similar to that of RSV, that is, mainly by droplets and fomites. hMPV is ubiquitous with infections occurring commonly in young children. Annual seasonal peaks occur in winter months in the moderate climate zones, but occur in late spring or early summer in the subtropics. Virtually all children have serological evidence of infection by the age of 5 years. Most infections are mild with cough, rhinorrhea, fever, and exacerbation of asthma. hMPV accounts for 2–12% of lower respiratory tract illnesses in children, and just a few percent in adults. Coinfection with RSV has been postulated to cause more severe disease. In adults, hMPV predominantly causes a mild upper respiratory tract infection. Infection in the frail elderly and the immunocompromised produces more severe disease. Fatal pneumonia associated with hMPV has been reported in children and adults with leukemia, as well as in a previously healthy adult.

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Hantavirus The Virus

The genus Hantavirus is classified within the family Bunyaviridae. Hantaviruses are lipid-enveloped, single-stranded RNA viruses. Rodent species are the natural host and source of human infections for hantaviruses. Hantaviruses are associated with a range of nephritic and hepatic diseases in Asia and Europe. The respiratory form of disease manifestation, hantavirus pulmonary syndrome (HPS), is mainly seen in America. Clinical Manifestations

Hantavirus infections are associated with domestic, occupational, or recreational activities that bring humans into contact with infected rodents, usually in rural settings. Transmission can occur when dried materials contaminated by rodent excreta are disturbed and inhaled, directly introduced into broken skin or conjunctivae, or, possibly, when ingested in contaminated food or water. Persons visiting laboratories where infected rodents were housed have been infected after only a few minutes of exposure to the animal holding areas. HPS typically starts with an influenza-like illness with high fever, myalgia, and asthenia. After a prodromal period of 2–7 days, dyspnea, respiratory failure, and hemodynamic instability appear. Radiologically, HPS presents as pulmonary interstitial edema with or without rapid progression to airspace disease, which has a central or bibasal distribution. Pleural effusion is a common feature. Occasionally, pulmonary manifestations may occur during the oliguric phase of renal failure, and present as pulmonary edema and cardiomegaly with or without pleuropericardial effusion. All hantaviruses known to cause HPS in the US are carried by the New World mice and rats (family Muridae, subfamily Sigmodontinae). The subfamily Sigmodontinae contains at least 430 species of mice and rats, which are widespread in North and South America. These wild rodents are not generally associated with urban environments. However, some species (e.g., deer mouse and white-footed mouse) may enter human habitation in rural and suburban areas. The association of hantaviruses with rodent reservoirs warrants recommendations to minimize exposure to wild rodents. The mortality for HPS treated with intravenous ribavirin (47.7%) appeared slightly lower than when treated conservatively (63.4%). The use of methylpredniso lone was associated with a dramatic decrease in mortality to 13.3%.

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Severe Acute Respiratory SyndromeAssociated Coronavirus The Virus

Severe acute respiratory syndrome (SARS) is a newly emerged disease due to infection with a novel coronavirus (SARS-CoV) that was first recognized in 2003. This was eradicated in the summer of 2004 but it is not known if it will return. SARS-CoV is a new member of the family Coronaviridae. CoVs are enveloped viruses with a large, positive, single-stranded genome. SARS-CoV appears to have originated from the wild animal reservoir in mainland China. A CoV almost identical to that in SARS patients was identified from masked palm civets (Paguma larvata) and the raccoon dog (Nyctereutes procyonoides). There was also a much higher sero-prevalence of SARSCoV among wild animal handlers compared with controls in Guangdong, southern China. Clinical Manifestations

The route of transmission of SARS-CoV was predominantly by close person-to-person contact via respiratory droplets. There was evidence to suggest that SARS might have spread by airborne transmission in a rapidly evolving community outbreak in a private residential complex in Hong Kong. Air samples obtained from a room occupied by a SARS patient, and swabs taken from frequently touched surfaces in rooms and in the nurses’ station in a hospital in Toronto tested positive for SARS-CoV RNA by polymerase chain reaction. The estimated mean incubation period was 4.6 days, and the mean time from symptom onset to hospitalization varied between 2 and 8 days during the outbreak in 2003. In adults, the major clinical features included persistent fever, chills/rigor, myalgia, malaise, dry cough, headache, and dyspnea. Watery, non-bloodstained diarrhea was common at presentation or developed subsequently within the first 2 weeks. Respiratory failure was the major complication of SARS. At least half of the patients required supplemental oxygen during the acute phase. About 20% of patients progressed to ARDS requiring invasive mechanical ventilatory support. The presentation of SARS in children was similar to upper respiratory infections due to other viruses but the clinical course of SARS in children was mild. Lymphopenia, low-grade disseminated intravascular coagulation (thrombocytopenia, prolonged activated partial thromboplastin time, elevated DDimer), elevated lactate dehydrogenase (LDH), and creatinine phosphokinase (CPK) were common laboratory features of SARS. However, a retrospective

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Figure 4 Serial chest radiographs of a 33-year-old male with severe acute respiratory syndrome who presented with fever, cough, and mild dyspnea. On day 2 of illness onset, there was mild right lower zone infiltrate (a). He developed acute respiratory distress syndrome (ARDS) on day 8 (b). Following pulsed steroid and intensive care support, there was marked improvement on day 14 (c).

study in Toronto has shown that all laboratory variables except the absolute neutrophil count de monstrated fair to poor discriminatory ability in distinguishing SARS from other causes of CAP. Radiographic features of SARS resembled those found in other causes of CAP (Figure 4). The more distinctive radiographic features of SARS included the predominant involvement of lung periphery and the lower zones. Cavitation, hilar lymphadenopathy, and pleural effusion were characteristically absent. Ground-glass opacification, sometimes with consolidation, and interlobular septal and intralobular interstitial thickening with predominantly a peripheral and lower lobe involvement were commonly revealed by high-resolution computed tomography. Treatment

Owing to the limited understanding of this new disease, treatment for SARS was empirical in the 2003 outbreak. Ribavirin in combination with a protease inhibitor (lopinavir/ritonavir) as initial therapy was associated with a lower death rate than treatment

with ribavirin alone. Systemic steroid may have played a role in minimizing the immune-mediated lung injury that occurs in the later phase of the disease whereas the use of interferon alfacon-1 plus corticosteroids was associated with improved oxygen saturation, more rapid resolution of radiographic lung opacities, and lower levels of CPK in SARS patients. Successful examples of using convalescent plasma with favorable outcome have been reported.

Picornaviruses The Virus

Picornaviridae is a large virus family that includes the two genera Rhinovirus and Enterovirus, which are associated with respiratory tract infections. Both rhinoviruses and enteroviruses are small nonenveloped, single, positive-stranded RNA viruses. These viruses are more resistant to adverse environmental conditions. They are transmitted more readily by direct contact and fomites compared with other respiratory viruses.

PNEUMONIA / Viral Clinical Manifestations

Rhinovirus infection is the most important cause of acute nasopharyngitis often referred to as the ‘common cold’. Repeated rhinovirus infections are common and several episodes can occur in the same individual within a year. This is because of the large number of serotypes, more than a hundred, and the type-specific immunity. Enteroviruses also contribute to respiratory tract infections. The majority of respiratory tract infections caused by picornaviruses are self limiting. Significant morbidity occurs in a few settings. Complications include otitis media, sinusitis, and exacerbation of asthma, and other chronic respiratory disorders (e.g., chronic obstructive pulmonary disease (COPD)). Lower respiratory tract involvement with or without secondary bacterial pneumonia are more common in the elderly and those with COPDs. Other high-risk groups prone to severe, potentially fatal lower respiratory infections due to picornaviruses include infants with bronchopulmonary dysplasia, patients with cystic fibrosis, severe immunosuppression associated with bone marrow or organ transplantation, or infection with human immunodeficiency virus. Treatment

Interferon has been investigated for treating the common cold, but its high cost and the adverse effect of local bleeding preclude its general use. Recent data suggest a potential therapeutic application for the soluble form of rhinovirus receptor intercellular adhesion molecule 1.

Measles The Virus

The measles virus is a member of the genus Morbillivirus within the family Paramyxoviridae. It is an enveloped virus with a nonsegmented, negative, single-stranded RNA genome. Clinical Manifestations

Measles was a ubiquitous common childhood febrile rash illness before the vaccine era. Typical clinical presentations include fever, conjunctivitis, malaise, sneezing, congestion, rhinitis, and cough. The pathognomonic Koplik’s spots can be seen in the buccal mucosa. The distinctive maculopapular rash begins at the ears and forehead, and then spreads to face, neck, trunk, and the extremities. When the rash reaches its peak, respiratory symptoms start to subside. Complications such as otitis media and bacterial pneumonia are relatively rare.

465

Giant cell pneumonia is a life-threatening complication of measles seen in the immunocompromised patients. It is characterized by giant cell formation and squamous metaplasia of bronchiolar epithelia. Atypical measles occurs in individuals who have received incomplete measles vaccination prior to the exposure to natural infection. The pattern of exanthema is different. A skin rash first develops in the extremities and then spreads, often becoming purpuric, vesicular, or urticarial. Most patients develop lobular or segmental pneumonia with pleural effusion. Recovery of respiratory symptoms is slow. Chest radiographic abnormalities remain detectable for months. The pathogenesis probably has an immune-mediated component resulting from the preexisting low-level measles antibodies.

Herpesvirus The Virus

The virus family Herpesviridae comprises eight members that can cause infections in humans. They are herpes simplex virus (HSV) types 1 and 2, varicella-zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV-7), and Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8). All human herpesviruses, except KSHV/HHV-8, are ubiquitous. Primary infections are followed by life-long latency with episodic reactivations. Clinical Manifestations

The respiratory tract is not the primary site of infection for herpesviruses. Herpesviral pneumonia is rare in healthy individuals. The risk of varicella pneumonia is about 1 in 200 000 in healthy children and about 1 in 200 in healthy adults with primary VZV infection (chickenpox). In pregnant women, the incidence increases to 9% and it is more common in smokers. VZV pneumonia occurs 1–6 days after the onset of rash. Clinical features include dry cough, dyspnea, tachypnea, chest pain, hemoptysis, and cyanosis. Treatment with high-dose intravenous acyclovir is indicated. Pneumonia is the most common cause of VZV-associated death. Most herpesviral pneumonia occurs in severely immunocompromised patients. HSV pneumonia can be part of the potentially fatal disseminated infec tion that occurs in newborns and heavily immunosuppressed transplant recipients. Treatment with acyclovir is indicated. CMV pneumonitis is a lifethreatening complication seen in allograft recipients with a mortality of 80–90%. Paradoxically, acquired immunodeficiency syndrome (AIDS) patients do not

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develop severe CMV pneumonitis despite the fact that other severe CMV diseases (enteritis, retinitis, adrenalitis) are common. This can be explained by the pathogenesis of CMV pneumonitis, which has an immune-mediated component. Treatment for CMV pneumonitis requires a combination of an antiviral (ganciclovir) and immunoglobulin (CMV hyperimmune globulin or human normal immunoglobulin). HHV-6 has also been associated with severe fatal pneumonitis in hemopoietic stem cell transplant recipients. HHV-6 is susceptible to ganciclovir, foscarnet, and cidofovir in vitro. The optimal treatment for HHV-6 pneumonitis is not clear. See also: Antiviral Agents. Pediatric Pulmonary Diseases. Pneumonia: Overview and Epidemiology; Community Acquired Pneumonia, Bacterial and Other Common Pathogens. Stem Cells. Surfactant: Surfactant Protein D (SP-D). Vaccinations: Viral. Viruses of the Lung.

Further Reading Chan PK (2002) Outbreak of avian influenza A (H5N1) virus infection in Hong Kong in 1997. Clinical Infectious Diseases 34(supplement 2): S58–S64. de Jong MD, Bach VC, Phan TQ, et al. (2005) Fatal avian influenza A (H5N1) in a child presenting with diarrhea followed by coma. New England Journal of Medicine 352: 686–691. Falsey AR, Hennessey PA, Formica MA, et al. (2005) Respiratory syncytial virus infection in elderly and high risk adults. New England Journal of Medicine 352: 1749–1759. Henrickson KJ (2003) Parainfluenza viruses. Clinical Microbiology Reviews 16: 242–264. Hui DS and Sung JJ (2004) Treatment of severe acute respiratory syndrome. Chest 126: 670–674. Lee N, Hui DS, Wu A, et al. (2003) A major outbreak of severe acute respiratory syndrome in Hong Kong. New England Journal of Medicine 348: 1986–1994. Li KS, Guan Y, Wang J, et al. (2004) Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430: 209–213. Mandell LA, Barlett JG, Dowell SF, et al. (2003) Update on practice guidelines for the management of community acquired pneumonia in immunocompetent adults. Clinical Infectious Diseases 37: 1405–1433. Nicholson KG, Wood JM, and Zambon M (2003) Influenza. Lancet 362: 1733–1745. Ogra PL (2004) Respiratory syncytial virus: the virus, the disease and the immune response. Paediatric Respiratory Review 5(supplement A): S119–S126. Peiris JS, Guan Y, and Yuen KY (2004) Severe acute respiratory syndrome. Nature Medicine 10(supplement 12): S88–S97. Peters CJ, Simpson GL, and Levy H (1999) Spectrum of hantavirus infection: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Annual Review of Medicine 50: 531– 545. Savolainen C, Blomqvist S, and Hovi T (2003) Human rhinoviruses. Paediatric Respiratory Review 4: 91–98. Tran TH, Nguyen TL, Nguyen TD, et al. (2004) Avian influenza A (H5N1) in 10 patients in Vietnam. New England Journal of Medicine 350: 1179–1188.

Ungchusak K, Auewarakul P, Dowell SF, et al. (2005) Probable person-to-person transmission of avian influenza A (H5N1). New England Journal of Medicine 352: 333–340.

The Immunocompromised Host M B Feinstein, Memorial Sloan-Kettering Cancer Center, New York, NY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pneumonias in the immunocompromised host can be caused by virulent organisms, which also infect normal patients, or atypical organisms not ordinarily seen in the healthy population. The type of pneumonia that develops in the immunocompromised host is determined by whether the immune defect is secondary to abnormalities in neutrophils, T lymphocytes, or B lymphocytes. Although coughing, fever, and shortness of breath develop commonly, pneumonia may be clinically silent or have unusual radiographic findings. Therefore, diagnosis often requires an open mind toward unusual organisms that may exist, as well as information acquired by sputum analysis, bronchoscopy, and/or surgical lung biopsy. Immunofluorescence and other organ-specific techniques may also be helpful in some patients. Antibiotics should be guided by cultures and other microbiology data; however, when such data are not available, they must be administered empirically.

Introduction Immunocompromised patients are those susceptible to infectious agents of otherwise little virulence. Such patients are predisposed to develop infections from atypical organisms that do not cause disease in healthy subjects as well as from highly virulent pathogens that do. There are many ways in which patients can become immunocompromised, each associated with its own spectrum of potential pathogens. The lung is a frequent target of opportunistic infections. This article reviews the spectrum of pulmonary diseases in the immunocompromised host, including mechanisms of immunodeficiency, types of opportunistic infections, and available diagnostic modalities.

Etiology There exist three general mechanisms by which the immune system can be compromised. Each results from abnormalities of a different cell type, rendering the host susceptible to specific types of opportunistic infections (Table 1). Occasionally, more than one mechanism may be present in the same patient. They include neutrophil dysfunction or deficiency, T-cell dysfunction or deficiency, and B-cell dysfunction or deficiency.

PNEUMONIA / The Immunocompromised Host 467 Table 1 Types of immunodeficiencies Type of immune defect

Disease or condition

Neutrophil dysfunction

Cytotoxic chemotherapy Leukemia Aplastic anemia Myelofibrosis HIV infection Che´diak–Higashi syndrome Chronic granulomatous disease Job’s syndrome Malnutrition

T-cell dysfunction

AIDS Hodgkin’s disease T-cell lymphoma Thymic aplasia or hypoplasia Mucocutaneous candidiasis Purine nucleoside phophorylase deficiency DiGeorge syndrome Nude syndrome

B-cell dysfunction

Splenectomy Chronic lymphocytic leukemia Non-Hodgkin’s lymphoma Multiple myeloma Dysglobulinemia Agammaglobulinemia Transient hypogammaglobulinemia of childhood

Neutrophil Dysfunction

Neutropenia, usually defined as an absolute neutrophil count below 1000 cells mm 3, is the most common cause of neutrophil dysfunction and most often results from acute leukemia or from chemotherapy that affects the bone marrow. The risk of infection is directly related to length of neutropenia. Typical pathogens include both Gram-positive and Gramnegative bacteria, particularly Gram-negative bacteria normally confined to the gastrointestinal tract. However, as the length of neutropenia increases, patients are also uniquely susceptible to opportunistic fungi against which neutrophils offer the primary defense. Such fungi, which include Aspergillus, are relatively rare among other types of immunodeficiency. T-Cell Dysfunction

T-cell dysfunction, or a defect in cellular immunity, is the most common immunodeficiency in humans. The primary defect in AIDS, T-cell dysfunction is also associated with a number of other systemic illnesses, such as diabetes and renal failure; T-cell depletion performed in the context of hematologic transplantation (bone marrow and stem cells transplants); and

a number of medications, such as prednisone and cyclosporine. Organisms prevalent in patients with T-cell dysfunction reflect a broad spectrum but are often intracellular or multicellular. Legionella (the organism that causes Legionnaires’ disease), Strongyloides, as well as some viruses, such as cytomegalovirus and herpes species, are seen relatively commonly. Two infections associated with HIV infection, tuberculosis and pneumonia due to Pneumocystis, are directly associated with the type of T cells called CD4 cells. B-Cell Dysfunction

B-cell dysfunction is a defect that usually results from inadequate or inappropriate antibody production, resulting in susceptibility to bacteria with capsules. Frequent sites of infection include the sinuses, middle ear, and lung. B-cell dysfunction occurs in such conditions as lymphoma, prednisone treatment, and splenectomy, as well as in conditions associated with abnormal antibody production, including multiple myeloma.

Clinical Features The presentation of pneumonia in the immunocompromised host can vary greatly and is largely contingent on both the underlying immune dysfunction and the specific organism involved. The clinical features of pneumonia are reviewed here in the context of organism type. Fungal Infection

Aspergillus Aspergillus is a fungal species ubiquitous in the soil, but it is also found in water and decaying matter. Although the species Aspergillus fumigatus and Aspergillus flavus account for the majority of infections in the US, a number of other species exist and occasionally cause human disease (Figure 1). Classically a disorder of neutrophil dysfunction, an increasing number of HIV patients are also developing Aspergillus infection. This may be secondary to poorly understood defects in B lymphocytes as well as cytokine effects that occur during the late stages of this disease. Aspergillus can adversely affect the immunocompromised lung in a number of ways. Aspergillomas, also called fungus balls, are masses of intertwined hyphae that grow in lung cavities. Although fungus balls can develop in either the immunocompetent or the immunocompromised host, their consequences in these two populations are different. In the immunocompetent host, aspergillomas develop when the fungus colonizes a cavity formed by a pre-existing

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Figure 2 CT scan of a patient with pneumonia from Aspergillus fumigatus. Ground glass opacities are present surrounding the pulmonary infiltrates; this is sometimes referred to as the halo sign.

Figure 1 Aspergillus fumigatus organism seen on silver stain. In the tissues, Aspergillus appears as septate filaments of relatively constant width of 5–10 mm. Classically, these filaments branch at acute angles less than 451. Reproduced from Travis WD, Colby TV, Koss MN, et al. (2002) Non-Neoplastic Disorders of the Lower Respiratory Tract, American Registry of Pathology, Washington, DC, with permission from American Registry of Pathology.

lung condition. In contrast, in the immunocompromised host, Aspergillus invades and causes necrosis of normal pulmonary tissue. Here, the resulting cavities are usually due to the necrotizing fungal infection and not any pre-existing lung disease. Invasive aspergillosis can be clinically silent; symptoms, if present, are typically non-specific, including cough, sputum production, and low-grade fever. Coughing may be productive of blood, a symptom known as hemoptysis. The diagnosis often rests on characteristic radiographic findings in the right clinical setting. Patients with invasive aspergillosis typically have single or multiple focal nodular infiltrates that, if untreated, will slowly progress. A ‘halo sign’, or a ground glass pattern surrounding a mass-like infiltrate, is highly suggestive of fungal pneumonia, as is the ‘air-crescent sign’ (Figure 2). The crescent develops due to necrosis of the lung tissue surrounding the infiltrate as neutropenia resolves. Although the yield for bronchoscopy with biopsy is approximately 50%, this procedure is often complicated

by low platelet counts, which renders any biopsy problematic. Serological studies have historically been unhelpful, but assays of galactomannan, a polysaccharide Aspergillus cell wall component, may be useful in detecting systemic infection. Aspergillus tracheobronchitis, characterized by the presence of Aspergillus organisms in the airway mucosa deep to the basement membrane, is found only in the most profoundly immunocompromised patients, particularly those with advanced AIDS and prolonged neutropenia. Clinical findings may mimic acute bronchitis and pneumonia. Chest radiography is usually normal, although tracheal or bronchial wall thickening is occasionally seen on computed tomography (CT) scans. The diagnosis of Aspergillus tracheobronchitis is often made by bronchoscopy, in which a cheesy material can be seen on airway inspection. Response of patients to antifungal antibiotics is usually poor. Candida Candida is ubiquitous as part of the normal flora of the skin and mucus membranes. Although thrush and vaginal infections are relatively common, particularly in the HIV population, pulmonary candidiasis is quite rare. Proving a ‘true’ Candida pulmonary infection can be difficult because the presence of Candida in most respiratory cultures reflects contamination from the oropharynx. Therefore, the diagnosis often requires lung biopsy. Cryptococcus Cryptococcus neoformans is a budding encapsulated yeast found throughout the world (Figure 3). Although it grows best in desiccated bird feces, most patients with cryptococcal infection do not report any history of bird exposure.

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Figure 3 Cryptococcus neoformans typically appears in tissue as single yeast organisms with a characteristic capsule. The capsule appears bright red on mucicarmine stain (shown here) and as a halo with hematoxylin and eosin. Silver stains are commonly employed to identify the yeast but do not show the capsule. Reproduced from Travis WD, Colby TV, Koss MN, et al. (2002) Non-Neoplastic Disorders of the Lower Respiratory Tract, American Registry of Pathology, Washington, DC, with permission from American Registry of Pathology.

Cryptococcus predominantly causes clinical disease among patients with abnormal T-cell function. Although the lungs serve as the portal of entry, the most common form of systemic disease is meningitis. Patients with cryptococcal lung disease are usually asymptomatic, but they may develop fever, cough, chest pain, or malaise. Radiographic findings usually consist of pulmonary nodules or masses (either solitary or multiple), diffuse infiltrates, fluid around the lungs, or enlarged lymph nodes. The detection of cryptococcal infection often results from the detection of the serum cryptococcal polysaccharide capsular antigen. Although this test is highly effective at identifying active disease, it does not discriminate the site of infection. Thus, patients often require a second diagnostic procedure, such as a biopsy or sputum examination. Pneumocystis Pneumocystis carinii, also known as Pneumocystis jiroveci, was first described by Dr Carlos Chaga in 1909 from autopsy studies of starved South American males (Figure 4). This organism has been reclassified as a fungus. Multiple strains are known to exist, each one capable of infecting a single specific species. For instance, human derived P. carinii can only infect humans, whereas rat-specific P. carinii can only infect rats. Pneumocystis pneumonia remained a rare entity until the AIDS epidemic of the 1980s, when it quickly became the most common AIDS-defining illness.

Figure 4 Pneumocystis jiroveci organisms as seen on silver stain. Reproduced from Travis WD, Colby TV, Koss MN, et al. (2002) Non-Neoplastic Disorders of the Lower Respiratory Tract, American Registry of Pathology, Washington, DC, with permission from American Registry of Pathology.

Prior to routine prophylaxis use, 75% of AIDS patients developed Pneumocystis pneumonia at some point in the course of their illness. Its frequency is inversely related to CD4 levels, rarely occurring in patients with CD4 counts exceeding 200 mm 3. Yet despite intensive study, its mode of transmission remains largely unknown; there is evidence supporting both the reactivation of a latent infection and personto-person spread. Breathlessness, cough, and fever are the most common clinical manifestations associated with Pneumocystis. The classic chest radiograph shows bilateral infiltrates. However, multiple atypical findings have also been described, including focal infiltrates, thin-walled cysts, pulmonary nodules, pneumothorax, and lung cavities. Among patients without HIV, the incidence of Pneumocystis has increased during the past 20 years with the more common use of chemotherapy and immunosuppressive medications, especially systemic steroids, for cancer, organ transplantation, and various inflammatory conditions. There exists a clinical dichotomy between HIV- and non-HIV-infected patients. Whereas HIV patients may present with an indolent febrile illness, symptoms having been present for 1 month or more, non-HIV patients often present with an acute pneumonitis. In less than 5% of cases, Pneumocystis can infect organs other than the lungs, notably the ear, eye, intestines, and thyroid. Protozoa Infection

Strongyloides Strongyloides stercoralis is a parasite endemic to the southeastern US, the Caribbean, Central America, and Central Africa (Figure 5). Up to

470 PNEUMONIA / The Immunocompromised Host Virus Infection

Figure 5 Larvae of Strongyloides stercoralis in the lung. Reproduced from Travis WD, Colby TV, Koss MN, et al. (2002) NonNeoplastic Disorders of the Lower Respiratory Tract, American Registry of Pathology, Washington, DC, with permission from American Registry of Pathology.

6% of US servicemen returning from Vietnam were colonized. Among the immunocompromised, especially those patients with HIV or receiving high doses of glucocorticoids, S. stercoralis is associated with a ‘hyperinfection syndrome’, often characterized by respiratory failure and sepsis. Travel to one of the endemic regions can usually be identified by history. However, diagnosis rests on detection of the organism in sputum, stool, or small bowel aspirate or by examination of fluid obtained at bronchoscopy. Unfortunately, the diagnosis is often established late in the disease, resulting in a high mortality rate. Toxoplasma Toxoplasma gondii is a well-recognized parasite in AIDS patients and transplant recipients. Infection among the immunocompromised can be devastating; in bone marrow transplant recipients, mortality rates exceeding 90% have been reported. Toxoplasmosis can cause mass lesions in the brain, resulting in fever, headache, confusion, seizures, or focal findings on neurological exam. Blurred vision may result from retinal involvement. In the lung, T. gondii may cause clinical pneumonia and bilateral pulmonary infiltrates.

Cytomegalovirus Cytomegalovirus (CMV), a member of the herpesvirus family, is the most common and worrisome virus capable of infecting the immunocompromised host. Associated with cellular immunity, CMV lung infections occur most commonly among hematologic and solid organ transplantation recipients. During the 1980s, CMV pneumonia occurred in up to 20% of all bone marrow transplant recipients. Nearly half of these patients died. Fortunately, the potential impact of CMV pneumonia is decreasing through the use of CMV serum antigen screening and prophylaxis with ganciclovir in selected patients. The symptoms of CMV pneumonitis are nonspecific and include fever, breathlessness, and nonproductive cough. Diffuse bilateral interstitial infiltrates are the most common radiographic findings. However, pulmonary nodules and focal consolidation have also been described. In hematologic transplant recipients, CMV infection does not occur during the period directly following transplant, when the bone marrow is not yet producing adequate numbers of white blood cells. Recipients of solid organ transplants typically manifest CMV infection approximately 6 weeks after transplant. Both the incidence and the severity of CMV pneumonia are higher among lung transplants than those of other solid organs and have been associated with chronic graft rejection. Influenza Influenza is a disease of the immunocompetent and immunocompromised alike and rarely can progress to pneumonia. Although the virus can cause pneumonia, the high mortality rates of prior influenza pandemics, such as the one in 1918, were secondary to bacterial pneumonias, particularly with Staphylococcus aureus and Streptococcus pneumoniae. Bacterial pneumonia is the most common cause of death among influenza patients. Primary influenza pneumonia predominantly affects patients with altered cell-mediated (i.e., T cell) immunity. The exact prevalence of influenza among immunocompromised patients is not clear, possibly because of difficulties in diagnosis. In one review of viral respiratory infections at a major bone marrow transplantation center, influenza pneumonia occurred in less than 10% of patients in whom the virus was isolated. The diagnosis of influenza is complicated by the fact that the most common symptoms, such as fever, headache, muscle aches, and malaise, are non-specific. Similarly, influenza pneumonia often cannot be clinically differentiated from other known causes of pneumonia. Immunofluorescent

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staining from nasopharyngeal washings is the most rapid method of diagnosis. However, influenza can also be cultured from respiratory secretions, such as sputum, within 72 h. Respiratory syncytial virus Respiratory syncytial virus (RSV), one of the viruses associated with the common cold, can cause a progressive and potentially fatal pneumonia among infants younger than 6 months old and the severely immunosuppressed. Associated with communal outbreaks during the late fall, winter, and early spring, RSV is particularly dangerous to bone marrow and stem cell transplant recipients, of whom up to two-thirds with pneumonia eventually succumb to their illness. Most patients who develop RSV pneumonitis will first develop an upper respiratory tract illness with typical ‘cold’ symptoms, including nasal congestion, pharyngitis, cough, and low-grade fever. Symptoms may migrate down the respiratory tree, with ensuing bronchitis, pneumonia, and/or respiratory failure. Radiographs often show diffuse, bilateral infiltrates. Although RSV grows well in several human cell lines, diagnosis often results by the detection of viral antigen from nasopharyngeal secretions and/or bronchoscopy fluid. Other viruses Pneumonia secondary to herpes simplex virus generally results from the aspiration of organisms from the oropharynx or by the extension of herpetic tracheobronchitis. Herpetic infection was initially a significant complication of allogeneic bone marrow transplantation, occurring in the immediate posttransplant period, but its importance in this group has declined with the use of prophylaxis with the antiviral antibiotic acyclovir. The chest radiograph most often shows focal infiltrates. Bilateral infiltrates and respiratory failure may also occur. Parainfluenza and human herpesvirus 6 (HHV-6) are rare causes of pneumonia. In contrast to most respiratory viruses, such as RSV and influenza, which are prevalent during the winter months, parainfluenza may cause infection year-round. HHV-6, which classically causes infection after hematologic or solid organ transplant, may account for some cases of pneumonitis previously classified as idiopathic interstitial pneumonia.

of infections. However, the variety of potential bacterial infections broadens significantly once the immune system has been impaired, with specific pathogens often associated with specific defects in the immune system. Patients with defects in B-cell dysfunction are classically susceptible to encapsulated organisms, such as S. pneumoniae. Patients with neutropenia, in contrast, are also at risk for enteric Gram-negative bacteria. To some extent, whether or not patients are institutionalized also determines the types of organisms to which they are susceptible. Chronically hospitalized patients, or those in nursing homes, have a higher prevalence of organisms resistant to antibiotics. This situation is exacerbated among patients receiving mechanical ventilation, in whom medical hardware, such as endotracheal tubes, bypasses endogenous upper airway defenses against bacterial infection. In HIV, the risk of bacterial pneumonia is inversely proportional to the degree of immune dysfunction. It is noteworthy that some prophylactic regimens against organisms known to infect HIV patients, such as Pneumocystis, are also found to effectively prevent bacterial pneumonia. Nocardia Seen in a variety of immunodeficient states, pneumonia from Nocardia has been described in patients with HIV and hematologic malignancies, as well as those receiving systemic steroids. Nocardia asteroides accounts for more than 80% of pulmonary and disseminated infections (Figure 6). Nocardia, which is ubiquitous in the soil, is a Gram-positive rod with a complex branching structure that

Bacterial Infection

Although respiratory infections in the immunocompetent are relatively common, the spectrum of organisms that cause clinical disease is fairly narrow. Such organisms as S. pneumoniae, Hemophilus influenza, and Moraxella catarrhalis cause the majority

Figure 6 CT scan of a patient with pneumonia from Nocardia asteroides. Pulmonary nodules can be quite large and cavitate.

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appears lacy and beaded on either Gram or AFB stains. It also stains strongly with the silver stain used for fungi. Usually, patients with nocardiosis will present in a subacute manner, with symptoms of fever, fatigue, weight loss, and cough. Occasionally, infection can disseminate, spreading hematogenously to the central nervous system or directly invading the pleura. Nocardia does not respect tissue planes. Skin infection can occur, with or without concurrent sinus tracts. Radiographically, early infection often manifests as a bronchopneumonia. However, a number of ‘atypical’ findings are also possible, including one or more pulmonary nodules, which can cavitate, as well as the accumulation of fluid around the lungs. Diagnosis of pulmonary nocardiosis is difficult and requires a high index of suspicion. Nocardia will eventually grow on the standard medium for bacterial cultures; however, growth is slower than that of standard bacteria and cultures may be inadvertently discarded before growth is evident. Therefore, if Nocardia is suspected, cultures should be held longer to ensure that this diagnosis is not missed. Legionella Legionella, the bacteria responsible for Legionnaires’ disease, is a common cause of pneumonia that is extremely difficult to culture. Its importance as a potential nosocomial pathogen, especially among transplant patients, is supported by its frequent colonization of water systems in hospitals and other institution. Therefore, clinical cases often occur in clusters. Clinical presentation may be protean, varying from indolent fevers to fulminant respiratory failure. More than 50% of patients with Legionella pneumonia have extrapulmonary symptoms, such as diarrhea, abdominal pain, or headache. Chest radiographs sometimes lag behind the acute illness, and approximately half of all infections are associated with fluid around the lung. Cavitary lesions have been reported, particularly among immunocompromised patients. The diagnosis requires a high clinical suspicion and confirmation of infection via several available diagnostic tests. The most sensitive of these is the urinary antigen against Legionella pneumophila serogroup 1 and the indirect fluorescent antibody against serum anti-Legionellaspecific antibodies. Although direct fluorescent antibody labeling is the most rapid means of diagnosis, its usefulness is limited by the expertise required to perform it as well as potential cross-reactivity with other bacteria. Mycobacteria The genus Mycobacterium encompasses a broad variety of species, of which tuberculosis is the most notorious; however, it also includes a

number of other potential pathogens. The relevance of tuberculosis as a public health hazard has again risen to the forefront of public attention with the AIDS epidemic and evolution of multiresistant bacteria. AIDS patients are at markedly increased risk for both reactivation tuberculosis and primary tuberculosis. The diagnosis of tuberculosis in AIDS is further complicated by the fact that many patients do not display the ‘classic’ radiographic and clinical findings of upper lobe infiltrates. Moreover, the risk of extrapulmonary tuberculosis increases with the degree of immunosuppression. Mycobacterium hemophilum may account for numerous clinical situations in which Mycobacterialike bacteria are detected by staining but cultures remain negative. Isolation of this organism is simple but not routine, requiring that standard mycobacterial medium be supplemented with iron. Conditions associated with these bacteria include solid organ transplants, AIDS, and bone marrow transplantation. Skin lesions, usually subcutaneous nodules, are the most common manifestation of infection, followed by lung disease, arthritis, and osteomyelitis. Radiographic findings are non-specific; pulmonary nodules, cavities, and focal infiltrates have all been reported. Because of special culture requirements, a high clinical suspicion is required for diagnosis. A number of other mycobacteria have been associated with pulmonary disease in the immunocompromised host. Mycobacterium avium complex (MAC), consisting of two species (Mycobacterium avium and Mycobacterium intracellulare), can cause a slow smoldering illness characterized by chronic cough and dyspnea as well as fine infiltrates, most evident anteriorly. MAC affects predominantly the elderly and those with pre-existing structural lung disease. Although clinical findings of Mycobacterium kansasii are often indistinguishable from MAC, these bacteria respond better to standard antituberculous antibiotics. Mycobacterium fortuitum, one of the so-called ‘rapidly growing mycobacteria’, is usually evident within 2–5 days by standard mycobacterial culture and can cause infection after coronary artery bypass surgery. Mycobacterium xenopi and Mycobacterium scrofulaceum both cause disease within the lung and mediastinal lymph nodes, and they have been associated with hospital outbreaks among immunocompromised hosts.

Management and Therapy Diagnosis

Early diagnosis and treatment of pneumonia in the immunocompromised host can pose a significant

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challenge. Because the spectrum of potential pathogens is usually broad, diagnosis requires an understanding of the underlying immune defect as well as an open mind toward unusual organisms that may exist. Some organisms require specific testing. To find Cryptococcus, for example, it is necessary to look for serum cryptococcal antigen and/or stain tissue with India ink. RSV, parainfluenza, and influenza are often diagnosed by immunofluorescence. Although not always optimal, a number of diagnostic procedures may be useful. Sputum, when available, is a cheap and easy biological specimen for examination. Its value is greater when the identified organisms are not normally present in the oropharynx. Sputum cultures growing Cryptococcus or Nocardia, for example, are almost always pathogenic, whereas cultures growing Candida commonly represent colonization of the upper respiratory tract. Fiberoptic bronchoscopy is thought to have a higher diagnostic yield and may be particularly helpful for some organisms, such as Pneumocystis and M. tuberculosis, when not found in the sputum. Transthoracic needle aspiration and surgical lung biopsy avoid the risk of oropharyngeal contamination. Therapy

A number of antibiotics have been used to treat pneumonia in the immunocompromised host. Although it is preferable to obtain specific diagnostic data to guide therapy, patients may be quite ill, may not tolerate any diagnostic procedure (i.e., lung biopsy in a patient with severe thrombocytopenia), or may have already had an unremarkable workup. As such, treatment is often performed empirically against organisms in which there is a high clinical suspicion. Amphotericin Amphotericin B deoxycholate has traditionally been the mainstay of therapy against Aspergillus infections in the lung, but its usefulness has been limited by numerous side effects. Three lipid formulations have also been developed: amphotericin B lipid complex, amphotericin B cholesteryl sulfate, and liposomal amphotericin B. Although much more expensive, these agents have an improved side effect profile and accumulate at higher tissue concentrations within the lung. These formulations were found to be as efficacious but not superior to amphotericin B deoxycholate. Triazole compounds, such as voriconazole and itraconazole, are also reasonable alternatives to amphotericin and can be administered orally. Trimethoprim/sulfamethoxazole Trimethoprim/sulfamethoxazole (TMP-SMX) is the mainstay of therapy

for Pneumocystis and Nocardia. A typical course of Pneumocystis therapy among HIV patients is 3 weeks with the addition of prednisone for those whose oxygen level is sufficiently low. For patients without an active infection but who remain at risk, TMP-SMX is also employed as prophylaxis against Pneumocystis. Among HIV-infected patients, prophylaxis is recommended under the following conditions: T-cell CD4 count less than 200, unexplained fever for 2 or more weeks, or a prior episode of Pneumocystis pneumonia. Prophylaxis is also commonly provided to the immunocompromised non-HIV patient, although the indications are much less clear. In general, patients with significantly impaired T-cell function should be strongly considered, such as those suffering from hematologic malignancies as well as those receiving chronic systemic steroids (i.e., more than 20 mg prednisone daily for 1 month) or certain chemotherapy agents, particularly fludarabine. The usefulness of TMP-SMX among hematologic transplant patients is limited by its associated bone marrow suppression. Intravenously, pentamidine is probably equally as effective to treat acute pneumonia. Moreover, aerosolized pentamidine can be used for prophylaxis. For those intolerant of both medications, other regimens that may be effective include clindamycin/primaquine, dapsone/trimethoprim, or atovaquone. Gancyclovir Treatment of CMV pneumonia includes intravenous ganciclovir, usually in combination with intravenous immunoglobulin. Because ganciclovir is associated with bone marrow suppression, often precluding its use in hematologic transplant recipients, foscarnet is often utilized as an alternative. Anti-influenza drugs Four drugs are approved for the treatment of influenza infection: zanamivir, oseltamivir, amantadine, and rimantadine. The neuraminidase inhibitors zanamivir and oseltamivir are active against both influenza A and B. Amantadine and rimantadine only work against influenza A. When given during the first 48 h of influenza infection, all four agents decrease the duration of illness by 1 or 2 days. To what extent these agents are effective in influenza pneumonia is not known. The most effective treatment method, however, is prevention, which can be achieved through strict adherence to annual influenza vaccines. The vaccine now exists in both an inactivated form, administered intramuscularly, and a live/attenuated form, administered nasally. The live/attenuated preparation should not be given to the immunocompromised patient. Moreover, individuals receiving this preparation should avoid contact with the immunocompromised for at least 21 days.

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Ribavirin Therapy against RSV with aerosolized ribavirin is proven to be helpful in infants, and it remains the drug of choice in adults, usually in combination with intravenous immunoglobulin. However, its effectiveness in adults is uncertain. Ribavirin may be most promising early in RSV disease, when it may attenuate the progression of upper airway disease to severe pneumonia and death. See also: Antiviral Agents. Bronchoscopy, General and Interventional. Human Immunodeficiency Virus. Pneumonia: Overview and Epidemiology; Atypical; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens); Parasitic; Viral. Viruses of the Lung.

Further Reading Breuer R, Lossos IS, Berkman N, and Or R (1993) Pulmonary complications of bone marrow transplantation. Respiratory Medicine 87(8): 571–579. Conces DJ Jr (1998) Bacterial pneumonia in immunocompromised patients. Journal of Thoracic Imaging 13(4): 261–270. Franquet T, Muller NL, Gimenez A, et al. (2001) Spectrum of pulmonary aspergillosis: histologic, clinical, and radiologic findings. Radiographics 21(4): 825–837. Hertz MI, Englund JA, Snover D, Bitterman PB, and McGlave PB (1989) Respiratory syncytial virus-induced acute lung injury in adult patients with bone marrow transplants: a clinical approach and review of the literature. Medicine (Baltimore) 68(5): 269–281.

Kim EA, Lee KS, Primack SL, et al. (2002) Viral pneumonias in adults: radiologic and pathologic findings. Radiographics 22: S137–S149. Kovacs JA, Hiemenz JW, Macher AM, et al. (1984) Pneumocystis carinii pneumonia: a comparison between patients with the acquired immunodeficiency syndrome and patients with other immunodeficiencies. Annals of Internal Medicine 100(5): 663– 671. Menendez R, Cordero PJ, Santos M, Gobernado M, and Marco V (1997) Pulmonary infection with Nocardia species: a report of 10 cases and review. European Respiratory Journal 10(7): 1542– 1546. Rosenow EC III, Wilson WR, and Cockerill FR III (1985) Pulmonary disease in the immunocompromised host. 1. Mayo Clinic Proceedings 60(7): 473–487. Saag MS, Graybill RJ, Larsen RA, et al. (2000) Practice guidelines for the management of cryptococcal disease. Infectious Diseases Society of America. Clinical Infectious Diseases 30(4): 710–718. Santamauro JT, Aurora RN, and Stover DE (2002) Pneumocystis carinii pneumonia in patients with and without HIV infection. Comprehensive Therapy 28(2): 96–108. Torres A and el-Ebiary M (1998) Invasive diagnostic techniques for pneumonia: protected specimen brush, bronchoalveolar lavage, and lung biopsy methods. Infectious Disease Clinic of North America 12(3): 701–722. Travis WD, Colby TV, Koss MN, et al. (2002) Non-Neoplastic Disorders of the Lower Respiratory Tract. Washington, DC: American Registry of Pathology. Wilson WR, Cockerill FR III, and Rosenow EC III (1985) Pulmonary disease in the immunocompromised host. 2. Mayo Clinic Proceedings 60(9): 610–631.

PNEUMOTHORAX C Strange and J T Huggins, Medical University of South Carolina, Charleston, SC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pneumothorax results from the rupture of either the visceral or parietal pleural membranes allowing air to enter the pleural space. Pneumothorax is classified as spontaneous, iatrogenic, and traumatic. Spontaneous pneumothorax is divided into primary (absence of clinical lung disease) and secondary (presence of clinical lung disease). Pneumothorax can present as a life-threatening event requiring prompt diagnosis and appropriate management. The pathogenesis of a spontaneous pneumothorax occurs with either bleb rupture or from alveoli overdistension resulting in disruption of the alveolar wall. Air dissects directly into the pleural space, or more commonly, along the pulmonary interstitium into the mediastinal compartment with secondary rupture into the pleural space resulting in a pneumothorax. The initial management of pneumothorax depends on the existence and severity of underlying pulmonary

disease, the degree of pulmonary symptoms on presentation, risk of a tension pneumothorax, the existence of a persistent air leak, and selection of the therapeutic procedure to re-inflate the lung. Strategies to prevent pneumothorax recurrence should be considered in the majority of patients with secondary spontaneous pneumothorax after first pneumothorax occurrence.

Introduction A pneumothorax is defined by the presence of air in the pleural space. A pneumothorax indicates that either the visceral or parietal pleural membranes have been disrupted to allow airflow from the lung or from the atmosphere. Pneumothoraces occur in a variety of clinical settings and affect a heterogeneous population of patients. Pneumothorax can present as a life-threatening event requiring a prompt diagnosis and thoughtful management approach.

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Etiology

Epidemiology

Pneumothoraces are classified as spontaneous, traumatic, or iatrogenic (Table 1). Spontaneous pneumothorax occurs in the absence of traumatic injury. Traumatic pneumothorax occurs in the setting of either blunt or penetrating chest trauma or barotrauma secondary to mechanical ventilation. Iatrogenic pneumothorax is associated with procedures that directly injure the pleural membranes. Spontaneous pneumothorax can be further subdivided into primary spontaneous pneumothorax (PSP) and secondary spontaneous pneumothorax (SSP). PSP occurs in patients without clinically apparent parenchymal lung disease, while SSP occurs in patients with a wide variety of focal and diffuse parenchymal lung diseases.

The incidence of PSP in men is approximately 12 per 100 000 population per year and in women is 3 per 100 000 population per year. Smoking significantly increases the risk for pneumothorax and most series suggest that 90% of affected patients smoke. The age of presentation is early adulthood, and patients typically have an esthetic body build. Although CT scans have demonstrated emphysema-like changes in this population, pneumothoraces are still characterized as primary in the absence of chronic cough or dyspnea. The prevalence of SSP is highly variable and dependent upon the underlying lung condition. Moreover, the underlying lung condition impacts the risk for pneumothorax recurrence, which affects the clinician’s management decisions after the first occurrence. Unlike PSP, the incidence of SSP peaks later in life and parallels the age distribution of common chronic lung diseases such as emphysema. However, patients with HIV infection, cystic fibrosis (CF), and lymphangioleiomyomatosis (LAM)-associated pneumothoraces present at an earlier age. The incidence and recurrence rates for SSP are highly variable and differ depending upon the underlying lung disease (Table 2). The incidence of SSP in emphysema is o1% with a recurrence rate of 40% that usually occurs within the first 6 months following initial pneumothorax. In CF, the incidence of pneumothorax ranges from 6% to 20%. When a pneumothorax is managed conservatively in CF patients (observation, needle aspiration, or chest tube drainage), the recurrence rate approaches 70%. During the course of HIV infection, the incidence of pneumothorax is 2–6%. Active or prior infection with Pneumocystis carinii is the strongest independent risk factor for the development of pneumothorax and accounts for more than 80% of the cases. The reported incidence of SSP in patients with a history of P. carinii infection varies from 4% to 30%, with pneumothorax recurrence rates as high as 65%. In patients with pulmonary Langerhans’ cell histiocytosis, the incidence of spontaneous pneumothorax ranges from 10% to 28%. In 1–2% of cases,

Table 1 Classification and causes of pneumothorax Spontaneous Primary Absence of lung disease Secondary Clinical presence of lung disease Airway diseases COPD Cystic fibrosis Status asthmaticus Interstitial lung diseases LAM Langerhans’ cell histiocytosis Sarcoidosis (Scadding radiographic stage IV) Usual interstitial pneumonitis Pulmonary infections Pneumocystis carinii Necrotizing pneumonia Tuberculosis Lung abscess Thoracic endometriosis (cetamenial pneumothorax) Iatrogenic Barotrauma Mechanical ventilation Procedural related Central venous catheter placement Thoracentesis Endotracheal intubation Cardiopulmonary resuscitation Bronchoscopy Nasogastric tube placement Tracheostomy Trauma Blunt chest trauma Penetrating chest trauma Esophageal rupture Adapted from Sahn SA and Heffner JE (2000) Spontaneous pneumothorax. New England Journal of Medicine 342: 868–874, with permission.

Table 2 Pneumothorax incidence and recurrence rates for various forms of lung disease Disease Chronic obstructive pulmonary disease Cystic fibrosis AIDS/pneumocystis pneumonia Langerhans’ cell histiocytosis Lymphangioleiomyomatosis

Incidence (%) o1 6–20 4–30 10–28 60–80

Recurrence (%) 40 70 65 25–58 80

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spontaneous pneumothorax occurs simultaneously bilaterally. The rate of recurrent ipsilateral spontaneous pneumothorax is reported to be 25–58%. In patients with LAM, the incidence of pneumothorax is 60–81% during the overall clinical course. Pneumothorax recurrence rate is approximately 80%, and simultaneous bilateral pneumothoraces are reported. Although the prevalence of SSP is highest among the chronic obstructive pulmonary disease (COPD) population, SSP occurs at the highest frequency in LAM when the total number of cases of each lung disease is considered.

Clinical Features The degree of respiratory compromise depends not only on the size of the pneumothorax but also on the severity of the underlying cardiopulmonary disease. Patients with preserved cardiopulmonary function can potentially tolerate large pneumothoraces and have minimal symptoms. Conversely, in the setting of severe cardiopulmonary impairment, extreme dyspnea and life-threatening respiratory failure will occur with small pneumothoraces. Most patients with a pneumothorax will present with sudden onset of pleuritic chest pain and dyspnea. With small pneumothoraces, physical examination is usually normal with the exception of mild tachypnea and tachycardia. A large pneumothorax produces hyperresonance, decreased breath sounds, and decreased tactile fremitus. The presence of significant hypoxemia, hypercapnia, and severe respiratory compromise is more common in SSP than PSP. The development of hypotension and contralateral tracheal deviation is indicative of a tension pneumothorax. A tension pneumothorax occurs when intrapleural pressure exceeds atmospheric pressure throughout the respiratory cycle. A tension pneumothorax is an acute cardiopulmonary emergency and, if unrecognized, can progress to cardiovascular collapse and subsequent death. The diagnosis of tension pneumothorax is a clinical diagnosis, and radiographic findings of tension, such as contralateral mediastinal shift, tracheal deviation, and ipsilateral diaphragmatic depression, may be absent on chest radiographs. A standard upright posterior–anterior and lateral chest radiograph is often the initial diagnostic study to confirm the presence of a pneumothorax. In the normal pleural space, air and fluid distribute according to gravitational effects. Air accumulates in the apex and superior portion of the lung on erect radiographs. Documenting intrapleural air and a thin (o1 mm thick) visceral pleural line displaced away from the chest wall confirms the diagnosis (Figure 1).

Figure 1 Posterior–anterior chest radiograph of a 35-year-old smoker who presented with acute onset of pleuritic chest pain and dyspnea. A right-sided pneumothorax is visualized. Table 3 Radiographic signs of a pneumothorax in the mechanically ventilated patient Pneumothorax Hyperlucency in the anteriomedial and subpulmonic recesses (most common) Deep sulcus sign Depression of hemidiaphragm Visualization of visceral pleural line (rare) Tension pneumothorax Depression of hemidiaphragm Widening of intercostal spaces Increased volume of the hemithorax Tracheal deviation Contralateral mediastinal shift

The detection of pneumothorax in the mechanically ventilated critically ill patient in an intensive care unit is often problematic. In these patients, radiographs are often taken in the supine or semierect position. In this situation, gas migrates along the anterior surface of the lung, creating a hyperlucency in the anteriomedial and subpulmonic recesses. Other indirect signs of a pneumothorax on a supine radiograph include a deep sulcus sign, increased volume of the hemithorax, depression of the hemidiaphragm, contralateral tracheal deviation, and widening of the intercostal spaces (Table 3). Chest computed tomography (CT) remains the gold standard in identifying and excluding small

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pneumothoraces. Chest CT is helpful in differentiating a pneumothorax from bullous disease, which can be misinterpreted on X-rays. Thoracic ultrasonography has emerged as an effective modality for the rapid detection of pneumothorax and has sensitivity and specificity of detecting a pneumothorax that is comparable to chest CT. The advantages of chest sonography compared to CT include the ability to perform quick bedside examinations with less expense. The disadvantages of sonography include a strong dependence on operator expertise, a restricted field of view owing to the bony structures of the thoracic cage, and an inferior imaging of lung parenchyma and mediastinal structures.

Pathogenesis The defining feature of PSP is the absence of clinically apparent lung disease; the majority of these patients have subpleural or apical blebs noted during surgery or on chest CT. Some speculate that PSP occurs with bleb rupture directly into the pleural space, or air dissects along the pulmonary interstitium into the mediastinum with secondary rupture into the pleural space. However, it remains unclear whether the detection of these blebs should guide decisions for surgery to prevent recurrence after the first episode of PSP. In SSP, airway obstruction, regardless of predisposing cause, increases the risk of pneumothorax from alveolar overdistension. For traumatic pneumothoraces, the cause of pneumothorax following penetrating injury is obvious. In blunt chest trauma, the mechanisms are multifold and include acute increases in airway pressure that rupture alveoli or result from lung laceration and disruption of the visceral pleural membrane.

Animal Models Currently, there are no animal models for PSP and SSP.

Management and Current Therapy The initial management of pneumothorax depends on the existence and severity of underlying pulmonary disease, the degree of pulmonary symptoms on presentation, the presence or risk of a tension pneumothorax, the existence of a persistent air leak, and selection of the therapeutic procedure to re-inflate the lung. Moreover, consideration should be given to the situation in which pneumothorax occurs; interventions have to be considered in the majority of SSPs to prevent recurrences. The specific

approaches used to treat pneumothoraces are discussed below. Figure 2 is a management algorithm for the various forms of pneumothorax. Observation

Some patients who are clinically stable with a small PSP may be observed without a pleural drainage procedure. Reliable patients who have quick access to medical care may be discharged home after a pneumothorax has been observed to remain stable for several hours. Approximately 1.25% of the intrapleural gas is reabsorbed every 24 h. High concentrations of inspired oxygen displace nitrogen from the blood and increase the gradient for nitrogen from the pleural space to be absorbed by the vascular space. Therefore, the administration of high flow oxygen can accelerate the reabsorption rate of intrapleural gas by several-fold. A large pneumothorax, signs of tension, and the development of an SSP should not be observed. Furthermore, pleurodesis should be considered in most patients who develop an SSP because of the risk of recurrence and the increased mortality associated with a pneumothorax in this setting. Catheter Aspiration

This technique involves the placement of a smallcaliber catheter into the pleural space with an attached syringe to withdraw the air. Simple aspiration is highly effective in the management of PSP of limited size. Simple aspiration has no utility in the management of a traumatic pneumothorax or in critically ill patients. Tube Thoracostomy

The placement of a chest tube for drainage of air from the pleural space is the definitive acute care modality in the management of a pneumothorax. Tube thoracostomy is indicated for patients who are critically ill, have traumatic pneumothoraces, have SSP in which pleurodesis might occur, and have PSP that fails simple aspiration. Various sizes and types of tube are used for pleural drainage. In the absence of large air leaks, patients can be managed with a small chest tube (7–14 Fr) placed in the midclavicular or anterior axillary line. These tubes can be placed with or without a Seldinger technique. Larger bore chest tubes (20–24 Fr) can be placed for patients whose lungs fail to reexpand because of the presence of a large air leak. Larger bore chest tubes (20–36 Fr) are indicated for initial drainage of a traumatic pneumothorax and for those who develop a pneumothorax while on positive pressure ventilation. In these clinical

478 PNEUMOTHORAX

Patient with pneumothorax

Primary spontaneous pneumothorax

Secondary spontaneous pneumothorax

Asymptomatic PTX size > 3 cm from lung apex Observation

Latrogenic pneumothorax

Asymptomatic PTX size < 3 cm from lung apex Observation

1. High flow O2 2. Tube thoracostomy with pleural drainage or Heimlich valve or 3. Catheter aspiration

Ipsilateral recurrent pneumothorax

Traumatic pneumothorax

Asymptomatic PTX size > 3 cm from lung apex 1. HIgh flow O2 2. Tube thoracostomy with pleural drainage or Heimlich valve or 3. Catheter aspiration

1. High flow O2 2. Tube thoracostomy with pleural drainage or Heimlich valve 3. Surgery + talc/abrasions after 1st episode

Tube thoracostomy with pleural drainage unit Hemopneumothorax requires large bore (36 Fr) chest tube

Surgery with stapling of blebs > 2 cm + talc/abrasion (pneumothorax prevention is performed by some experts after 1st episode of PSP)

Figure 2 Management algorithm for the various forms of pneumothorax.

settings, large air leaks are often present and may exceed the capacity of small-caliber tubes to effectively drain the space and prevent the risk of tension. Moreover, the risk of a hemopneumothorax is high in trauma, requiring larger chest tubes to drain and monitor intrapleural bleeding. For hospitalized patients, the chest tube is connected to an underwater drainage unit to establish a one-way seal. These drainage units contain three elements connected in series. These elements are a suction chamber that allows the adjustment of any negative pressure applied to pleural space, an underwater seal that prevents atmospheric air from entering the pleural space and allows continuous monitoring of an air leak, and a fluid collection chamber. Underwater drainage units are indicated for patients with a pneumothorax with large air leak,

or with significant volumes of pleural fluid that would clog a Heimlich valve. A chest tube should initially be placed to water seal without suction. The application of suction has been shown to perpetuate a bronchopleural fistula and delay removal of the tube thoracostomy. Chest tube suction is indicated when the lung does not fully expand on imaging. The chest tube can be safely pulled after the resolution of the air leak and radiographic demonstration of normal lung re-expansion. Surgery and Pleurodesis

Surgery for patients with a pneumothorax has several objectives: managing a persistent air leak that fails to resolve with chest tube drainage and performing pleurodesis with or without resection of blebs to

POLYMYOSITIS AND DERMATOMYOSITIS 479

prevent pneumothorax recurrence. Several techniques have been developed to accomplish these objectives. Open thoracotomy allows for the resection or stapling of apical blebs followed by a mechanical pleural abrasion or parietal pleurectomy to create a pleural symphysis and prevent pneumothorax recurrence. Pneumothorax recurrence rates are less than 1%. However, with the significant postoperative morbidity associated with an open thoracotomy, less invasive techniques have been developed. The application of video-assisted thoracic surgery (VATS) allows for an endoscopic approach in the management of pneumothorax. This technique allows for the endoscopic stapling of apical blebs and partial pleurectomy. Pleurodesis can be accomplished by either pleural abrasion or insufflation of talc. VATS is an acceptable technique in preventing pneumothorax recurrence for patients with PSP and SSP. Currently, VATS is the surgical procedure of choice in the management of SSP. Although there is no current data to indicate that bullectomy must be performed when talc is administered by poudrage, less successful procedures may be performed in patients at prohibitive risk for general anesthesia. The timing for VATS in the management of pneumothorax remains a matter of debate. Most authors agree that pneumothorax prevention is cost justified in SSP after the first pneumothorax occurrence. However, patient preferences and underlying lung diseases will influence this recommendation. In the management of PSP, most experts recommend VATS only after ipsilateral recurrence. Pleurodesis should not be withheld in patients who might need lung transplantation. Although surgery is technically more difficult after pleurodesis, the consequences of pneumothorax in this fragile subset of patients can be catastrophic. The time that is required from the pleurodesis procedure until an effective pleural symphysis has occurred remains unknown but is estimated at approximately 2 weeks. Therefore, the time from pneumothorax occurrence to safe resumption of air travel remains controversial

and depends on whether pleurodesis was performed and type of lung disease. In general, individuals with previous pneumothoraces should be discouraged from diving. See also: Asthma: Acute Exacerbations. Hypoxia and Hypoxemia. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis; Lymphangioleiomyomatosis. Pleural Space. Signs of Respiratory Disease: Lung Sounds. Trauma, Chest: Bronchopleural Fistula; Subcutaneous Emphysema.

Further Reading Baumann MH, Strange C, Heffner JE, et al. (2001) Management of spontaneous pneumothorax: an American College of Chest Physicians Delphi consensus statement. Chest 119: 590–602. Boutin C, Astoul P, Rey F, and Mathur PN (1995) Thoracoscopy in the diagnosis and treatment of spontaneous pneumothorax. Clinics in Chest Medicine 16: 497–503. Dines DE, Clagett OT, and Payne WS (1970) Spontaneous pneumothorax in emphysema. Mayo Clinic Proceedings 45: 481–487. Dulchavsky SA, Schwartz KL, Kirkpatrick AW, et al. (2001) Prospective evaluation of thoracic ultrasound in the detection of pneumothorax. Journal of Trauma 50: 201–205. Gilbert TB and McGarth GJ (1994) Tension pneumothorax: aetiology, diagnosis, pathophysiology, and management. Journal of Intensive Care Medicine 9: 139–150. Henry M, Arnold T, and Harvey J (2003) BTS guidelines for the management of spontaneous pneumothorax. Thorax 58(supplement 11): 39–52. Miller AC (2003) Spontaneous pneumothorax. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 445–463. London: Arnold. Sahn SA and Heffner JE (2000) Spontaneous pneumothorax. New England Journal of Medicine 342: 868–874. Schramel FM, Postmus PE, and Vanderschueren RG (1997) Current aspects of spontaneous pneumothorax. European Respiratory Journal 10: 1372–1379. Schramel FM, Sutedja TG, Braber JC, van Mourik JC, and Postmus PE (1996) Cost-effectiveness of video-assisted thoracoscopic surgery versus conservative treatment for first time of recurrent spontaneous pneumothorax. European Respiratory Journal 9: 1821–1825. Strange C and Jantz MA (2004) Pneumothorax. In: Demosthenes B (ed.) Pleural Disease (Lung Biology in Health and Disease), vol. 186, pp. 661–667. New York: Dekker.

POLYMYOSITIS AND DERMATOMYOSITIS J H Ryu, Mayo Clinic, Rochester, MN, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Polymyositis (PM) and dermatomyositis (DM) are idiopathic inflammatory myopathies of autoimmune origin involving the

skeletal muscles. Pulmonary involvement occurs and is a source of morbidity and mortality. The main forms of pulmonary involvement include interstitial lung disease, respiratory muscle weakness, and aspiration pneumonia. Non-specific interstitial pneumonia, organizing pneumonia, usual interstitial pneumonia, diffuse alveolar damage, and lymphocytic interstitial pneumonia are the main histologic patterns found. Anti-Jo-1 antibody (anti-histidyl-tRNA synthetase) is commonly found

480 POLYMYOSITIS AND DERMATOMYOSITIS in patients with PM/DM-associated interstitial lung disease. Progressive interstitial lung disease and respiratory muscle weakness can both cause respiratory failure. Aspiration pneumonia is related to weakness of the pharyngeal and upper esophageal muscles. Uncommon pulmonary manifestations include pleural effusion and pleuritis, pulmonary hypertension, and vasculitis. Corticosteroid therapy is the first line treatment and is generally effective in controlling both the myositis and the pulmonary involvement. However, in some patients PM/DM deteriorates despite corticosteroids and immunosuppressive therapy. Better understanding of the pathogenetic mechanisms in PM/DM will lead to more specific, target-directed therapy.

Diagnosis

Idiopathic inflammatory myopathies represent a heterogeneous group of autoimmune syndromes involving the skeletal muscle. The common traits of these disorders include muscle weakness and inflammation. Three major forms of idiopathic inflammatory myopathy include polymyositis (PM), dermatomyositis (DM), and inclusion-body myositis. Each of these entities has distinctive clinical and histopathologic features but the cause remains unknown. Idiopathic inflammatory myopathies are associated with systemic complications including involvement of the lungs. Pulmonary complications in idiopathic inflammatory myopathies are common causes of morbidity and mortality. This article focuses on lung involvement that occurs in patients with PM and DM. The prevalence of inflammatory myopathies has been estimated to range from 0.6 to 6 per 100 000 and estimated incidence has varied from 0.1 to 0.9 per 100 000 per year. There is meager data on the relative incidence of the different forms of inflammatory myopathies. DM may occur in children and adults, whereas PM occurs mainly after the second decade of life. Both DM and PM are more common in females. In all age groups, DM is more common than PM.

The clinical diagnosis of PM and DM is confirmed by three tests: serum muscle enzyme level, electromyography, and muscle biopsy. The most sensitive muscle enzyme test is the serum creatine kinase (CK) level which is usually elevated in patients with active PM or DM, but may occasionally be normal. Electromyographic results are usually abnormal, but the findings are non-specific and may be patchy. The definitive diagnosis of inflammatory myositis requires a muscle biopsy obtained for conventional light microscopic examination as well as immunohistochemical and electron microscopic studies. Pathologic features common to both disorders include necrosis as well as various stages of regeneration involving the muscle fibers. These changes are accompanied by inflammation involving predominantly mononuclear cells and an increased amount of connective tissue. However, there are some microscopic, immunohistochemical, and ultrastructural findings in the muscle biopsy that distinguish PM from DM. Magnetic resonance imaging or computed tomographic scanning can sometimes be helpful, for example, selecting an appropriate biopsy site, but their diagnostic accuracy has not been adequately evaluated. The most common autoantibodies in PM/DM are antisynthetase antibodies directed against various aminoacyl t-RNA synthetases. The best-known antisynthetase antibody is anti-Jo-1 (anti-histidyl tRNA synthetase), which is the most common type detected in patients with PM/DM-associated interstitial lung disease (ILD). The diagnostic and prognostic value of anti-Jo-1 in patients with PM/DM has not been adequately evaluated. Serum level of KL-6, a mucinlike high-molecular-weight glycoprotein, tends to be elevated in patients with PM/DM-associated ILD and may be a useful marker for assessing the activity of ILD.

Etiology

Pathogenesis

The cause of PM/DM is unknown, but these diseases are likely multifactorial in origin involving both genetic predisposition and acquired factors. The role of genetic factors is suggested by occasional familial occurrences, a higher frequency of other autoimmune disorders in the first-degree relatives of these patients, and linkage to certain human leukocyte antigens (HLA). Multiple genes, for example, immune response genes, likely contribute to susceptibility to these disorders. Exposure to certain environmental toxins, drugs, or infectious agents may play a role in some cases. PM-like syndrome has been described in chronic graft-versus-host disease.

Pathogenesis of PM and DM likely involves autoimmune responses, but target antigens have not been identified. In DM, the primary target antigen may be the endothelium of the endomysial capillaries. A high percentage of CD4 þ T cells and B cells are seen in the perivascular inflammation. In PM, certain CD8 þ T cells are clonally expanded in the muscle, possibly driven by an autoantigen. The autoimmune nature of this process is suggested by the association with autoantibodies, other autoimmune disorders, histocompatibility genes, and various T-cell products. A variety of inflammatory chemokines and proinflammatory cytokines including interleukin (IL)-1, IL-2,

Introduction

POLYMYOSITIS AND DERMATOMYOSITIS 481

IL-6, IL-10, tumor necrosis factor alpha (TNF-a), and transforming growth factor beta have been described to be upregulated in PM and DM. Although antisynthetase antibodies are more commonly found in patients with PM/DM-associated ILD compared to those without ILD, the pathogenesis of ILD in these patients remains unknown. The development of ILD in these patients does not appear to correlate with the extent and severity of the muscle or skin disease.

Animal Models A completely satisfactory animal model of PM or DM has yet to be developed. Some animal models for PM have been produced experimentally in guinea pigs, rats, and mice by immunization with homogenates of muscle or muscle protein preparations such as skeletal myosin or C protein. This process has been shown to produce experimental autoimmune myositis with infiltration of T cells into muscle and appearance of necrotic as well as regenerating muscle fibers.

Clinical Features Muscle Weakness

Patients with PM and DM usually present with varying degrees of muscle weakness that is progressive over several weeks to months. Muscle weakness tends to be more severe in the shoulder and pelvic girdle muscles resulting in difficulty performing certain tasks, such as rising from a chair, climbing stairs, or combing hair. Myalgias are relatively uncommon. As the disease advances, dysphagia and respiratory muscle weakness may occur, but sensation remains normal. Skin Involvement

DM is recognized by the characteristic skin rash that often precedes muscle weakness. These skin manifestations include the heliotrope rash on the upper eyelids, Gottron rash on the knuckles, and an erythematous rash on the face, neck, and upper chest and shoulders (Shawl sign). In some patients, the skin rash may be the dominant manifestation and muscle strength may appear normal (amyopathic dermatomyositis). Subcutaneous calcinosis can occasionally be seen, particularly in juvenile DM, and may result in pain, ulcerations, and infections. Other organs including the gastrointestinal tract, joints, heart, and lungs can be involved in patients with PM/DM. In addition, features of PM/DM can

overlap with other autoimmune and connective tissue diseases in some patients. Lung Involvement in PM/DM

The main forms of pulmonary involvement in PM/ DM include ILD, respiratory muscle weakness, and aspiration pneumonia (Table 1). The reported prevalence of pulmonary involvement in PM/DM has varied from 5% to 80%, depending largely on the method of detection, the diagnostic criteria, and the study population. For example, the prevalence of ILD in retrospective studies has ranged from 5% to 9%. However, the use of high-resolution computed tomography (HRCT) in prospective surveys has yielded a prevalence as high as 80%. Women with PM/DM are at a higher risk of developing ILD and the mean age at presentation is 50 years. Less common respiratory manifestations of PM/DM include pulmonary hypertension, vasculitis, and pleural involvement. The clinician needs to keep in mind that a respiratory problem occurring in a patient with a known systemic disease may not only be a manifestation related to the underlying disease, but could also be a complication resulting from treatment, or an unrelated separate disease process. A broad perspective needs to be maintained at the outset of this diagnostic evaluation. In those patients with suspected ILD, the clinician needs to consider the need for lung biopsy to identify the underlying histologic pattern. Assessment of the tempo of the pulmonary disease, radiologic pattern seen on HRCT, and the clinical context will be helpful in reaching this decision. For example, bronchoscopy with bronchoalveolar lavage may suffice if the main concern is possible opportunistic infection. On the other hand, distinguishing different forms of interstitial pneumonias that can occur in patients with PM/DM generally requires a surgical lung biopsy. In many cases, however, identifying the specific histologic pattern of interstitial pneumonia may not necessarily alter treatment decisions since corticosteroid therapy is usually Table 1 Respiratory manifestations of PM and DM Interstitial lung disease Non-specific interstitial pneumonia Organizing pneumonia Usual interstitial pneumonia Lymphocytic interstitial pneumonia Diffuse alveolar damage Aspiration pneumonia Respiratory muscle weakness Others Pleuritis and pleural effusion Pulmonary vasculitis Pulmonary hypertension

482 POLYMYOSITIS AND DERMATOMYOSITIS

the primary mode of treatment for patients with PM/DM. Interstitial Lung Disease

ILD is probably the most common form of pulmonary involvement in PM/DM. ILD is more common in patients with detectable autoantibodies to tRNA synthetases or a mucin-like glycoprotein (KL-6) in the serum. In some patients the ILD precedes the muscle or skin manifestation. There appears to be no correlation between the extent and severity of the muscle or skin disease and the development of ILD. Typical presenting manifestations in patients with PM/ DM-associated ILD include progressive exertional dyspnea, nonproductive cough, and bibasilar crackles. Occasionally, the respiratory symptoms may be abrupt in onset and severe, resembling acute respiratory distress syndrome. Digital clubbing is rare. Anti-Jo-1 antibody (anti-histidyl-tRNA synthetase), the most common type of antisynthetase autoantibody in patients with PM/DM, is detected more commonly in patients with associated ILD (50–70%) than in those without (10–15%). Some patients may present with ILD associated with anti-Jo-1 antibody in the absence of muscle involvement. In patients with PM/DM-associated ILD, the most common histologic pattern seen in the lung is non-specific interstitial pneumonia (NSIP). Histologic features of NSIP are characterized by a uniform-appearing, cellular interstitial pneumonia with a lymphoplasmacytic infiltrate within alveolar septa. Varying amounts of fibrosis are admixed with the chronic inflammation. In contrast to usual interstitial pneumonia (UIP), in NSIP, fibroblastic foci are rare and microscopic honeycombing is absent. The NSIP pattern of lung injury can be seen in patients with other connective tissue diseases as well as in many other clinical contexts. Less common forms of ILD seen in PM/DM include organizing pneumonia, UIP, diffuse alveolar damage (DAD), and lymphocytic interstitial pneumonia (LIP). Subclassification of ILD into these histologic patterns is valuable in predicting response to therapy and prognostication. Pulmonary vascular inflammation is uncommonly seen and includes cases of pulmonary capillaritis. Pleuritis is also relatively uncommon. Chest radiography on patients with PM/DM-associated ILD usually reveals reduced lung volumes and bilateral interstitial opacities, more prominent in the lower lung fields. Consolidation is less commonly seen and honeycombing is unusual. Findings on HRCT will generally reflect the underlying histopathologic pattern of ILD. Main findings on HRCT for those patients with NSIP pattern are reticular

Figure 1 High-resolution computed tomography of the chest of a 38-year-old woman with dermatomyositis and non-specific interstitial pneumonia. Ground-glass opacities and reticular densities with peripheral predominance are seen as well as associated bronchiolectasis. Non-specific interstitial pneumonia was diagnosed by a surgical lung biopsy prior to the diagnosis of dermatomyositis.

Figure 2 Computed tomography of the chest in a 67-year-old woman who presented with patchy areas of consolidation, ground-glass opacities, and pleural effusions. Lung biopsy demonstrated organizing pneumonia and polymyositis was eventually diagnosed.

and/or ground-glass opacities present bilaterally with or without consolidation (Figure 1). Traction bronchiectasis may also be present but honeycombing is usually absent. Patients with organizing pneumonia (Figure 2) or DAD (especially during organizing phase) will have predominantly consolidative opacities on HRCT. Those with the UIP pattern of lung injury will characteristically manifest subpleural peripheral reticular opacities with or without honeycombing. Lymphocytic interstitial pneumonia is characterized by poorly defined centrilobular nodules, ground-glass opacities, and scattered thinwalled cysts. Pleural effusions may be seen in up to 20% of patients with PM/DM-associated ILD and are generally small in size.

POLYMYOSITIS AND DERMATOMYOSITIS 483

Pulmonary function testing in patients with PM/ DM-associated ILD usually demonstrates restrictive abnormalities with reduced lung volumes and diffusing capacity as well as evidence of abnormal gas exchange. Bronchoscopy is generally nondiagnostic in patients with PM/DM-associated ILD. Transbronchial biopsy may yield evidence of interstitial inflammation and fibrosis but is unlikely to characterize fully the underlying histologic pattern. Bronchoalveolar lavage generally demonstrates lymphocytosis in the bronchoalveolar lavage fluid for those with NSIP and organizing pneumonia but can yield variable findings in those patients with other histologic patterns. Respiratory Muscle Weakness

Involvement of respiratory muscles by progressive inflammatory myopathy can result in hypoventilation and occasionally respiratory failure. Isolated diaphragmatic weakness without peripheral muscle involvement has been described as well. Rarely, laryngeal involvement may occur. In patients with respiratory muscle weakness, chest radiography reveals reduced lung volumes with diaphragmatic elevation and discoid basilar atelectasis. Pulmonary function testing demonstrates changes of a restrictive abnormality with reduced maximal respiratory pressures. Aspiration Pneumonia

Dysphagia and reflux resulting from myopathy of the striated muscles of the hypopharynx and upper esophagus can predispose to aspiration. Impaired cough from respiratory muscle weakness may contribute to increased risk of aspiration for patients with PM/DM. Aspiration pneumonia has been reported to occur in 15–20% of patients with PM/DM. The diagnosis of aspiration pneumonia is usually made based on the radiologic findings and the clinical context. Respiratory muscle weakness is usually suggested by clinical and radiographic findings. Reduced maximal respiratory pressures will confirm this diagnosis. Chest radiography and CT typically demonstrate patchy consolidation in the dependent portions of the lungs. Biopsy confirmation is sought only if presenting clinical or radiologic features are atypical. Other Respiratory Manifestations

Several other respiratory manifestations have been associated with PM/DM. Pulmonary hypertension can rarely be a direct manifestation of PM/DM but is seen more commonly secondary to progressive

ILD or chronic ventilatory insufficiency. Few cases of pulmonary vasculitis including pulmonary capillaritis with associated diffuse alveolar hemorrhage have been reported in patients with PM/DM. The use of immunosuppressive agents such as methotrexate and cyclophosphamide can be associated not only with opportunistic pneumonias due to impairment of host defenses, but also drug-induced lung diseases. Pleural manifestations have included pleural effusions, pleuritis, and occasionally spontaneous pneumothorax. DM, more so than PM, can be associated with cancer including lung cancer.

Current Therapy The goal of therapy in PM and DM is to improve muscle strength and to ameliorate extramuscular manifestations. There have been very few controlled clinical trials. The treatment of inflammatory myopathies remains largely empirical, using agents that are nonselective in their effects on the immune system. Prednisone is the first-line drug and early initiation of therapy leads to better outcome. The initial dose of prednisone is usually 1 mg kg
1 day
1 with slow tapering to an alternate-day dosing over the following several months. Approximately twothirds of patients will demonstrate a response to initial corticosteroid therapy. Large doses of intravenous corticosteroids, for example, 0.5–1 g methylprednisolone or equivalent given daily for 3 days, have been used in the initial management of severe cases. Addition of another immunosuppressive drug is commonly needed for steroid-sparing effect or for additive therapeutic effects in controlling the myositis. Azathioprine (2–3 mg kg
1 day
1) or methotrexate (up to 25 mg weekly) are commonly used for this purpose. In patients with aggressive disease, a combination of prednisone and another immunosuppressive drug can be started from the outset. Other treatment options include cyclophosphamide, cyclosporine, chlorambucil, mycophenolate mofetil, leflunomide, tacrolimus, anti-TNF agents (etanercept, infliximab), immune modulators (eculizumab, rituximab), plasmapheresis, total lymphoid irradiation, intravenous immunoglobulins, and autologous hemopoietic stem cell transplantation. None of these treatment modalities has been adequately tested in controlled clinical trials. In addition, relative efficacy of these agents used alone or in various combinations has not been clarified. Each patient needs to be individually assessed in regard to presenting manifestations, comorbid factors, and potential risks associated with the use of these agents. Rehabilitative measures including a regular exercise program may help to prevent or reverse impaired muscle function

484 POLYMYOSITIS AND DERMATOMYOSITIS

and exercise tolerance. Better understanding of pathogenetic mechanisms will lead to more specific, target-directed therapy in the future. Management of respiratory involvement in patients with PM/DM depends on the type of pulmonary complication, the activity of the underlying PM/ DM, and comorbid factors. In many cases, treatment of PM/DM itself, as outlined above, is the most important component in managing the pulmonary complication. For example, treatment of ILD associated with PM/DM involves corticosteroid and immunosuppressive therapy used to treat the underlying disease. The most common regimens used in managing patients with PM/DM-associated ILD are prednisone alone or in combination with azathioprine. The improvement in response to treatment is seen over the course of 4–6 months. This response to treatment in patients with PM/DM-associated ILD partly depends on the histologic pattern of lung injury. Patients with NSIP, organizing pneumonia, or LIP respond better to corticosteroid therapy and have a more favorable prognosis compared to those with UIP. Those patients with the DAD pattern have the worst prognosis. Thus, subclassification of histologic findings in patients with PM/DM-associated ILD appears to be clinically useful in gauging expected response to treatment and prognosis. The serum CK level usually parallels the disease activity in PM/DM and should be monitored at regular intervals. In addition, reliable functional measures of muscle strength and endurance can help monitor the activity of PM/DM and response to therapy. Pulmonary function studies, chest radiographs, and HRCT of the chest provide objective reassessment of pulmonary involvement in PM/DM. Other management options to be considered for patients with PM/DM and pulmonary involvement include ventilatory support and lung transplantation. In patients with respiratory failure from severe ILD or respiratory muscle weakness, invasive or noninvasive ventilatory support may be needed to survive an episode of exacerbation in their disease. Lung transplantation may be an option for those patients with severe pulmonary fibrosis resulting from PM/ DM-associated ILD, in the absence of significant comorbid factors that may contraindicate this procedure and if the underlying PM/DM is under control.

Prognosis The 5-year survival rate after diagnosis for patients with PM/DM overall is approximately 80%. The most common causes of death in these patients are cancer and pulmonary complications. At least a third of the patients with PM/DM are left with mild to

severe muscle weakness. Pulmonary causes of death in patients with PM/DM include progressive ILD, ventilatory failure due to respiratory muscle weakness, recurrent aspiration pneumonia, pneumonias related to immunosuppressive therapy, and occasionally progressive pulmonary hypertension. The natural history of untreated ILD in PM/DM is not entirely known, but 5-year survival rates have been reported to be 50–60% for patients with PM/DM-associated ILD. Incomplete resolution of pulmonary opacities with residual bibasilar linear opacities is commonly observed in these patients. DM, more so than PM, is associated with an increased risk of cancer. This excess risk of cancer appears to be highest around the time of diagnosis. Malignancies of the lungs, ovaries, breasts, and stomach are reported most frequently. See also: Interstitial Lung Disease: Cryptogenic Organizing Pneumonia. Pulmonary Effects of Systemic Disease. Pulmonary Fibrosis. Respiratory Muscles, Chest Wall, Diaphragm, and Other.

Further Reading American Thoracic Society/European Respiratory Society (2002) International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. American Journal of Respiratory and Critical Care Medicine 165: 277–304. Bandoh S, Fujita J, Ohtsuki Y, et al. (2000) Sequential changes of KL-6 in sera of patients with interstitial pneumonia associated with polymyositis/dermatomyositis. Annals of the Rheumatic Diseases 59: 257–262. Bonnefoy O, Ferretti G, Calaque O, et al. (2004) Serial chest ct findings in interstitial lung disease associated with polymyositis– dermatomyositis. European Journal of Radiology 49: 235–244. Braun NM, Arora NS, and Rochester DF (1983) Respiratory muscle and pulmonary function in polymyositis and other proximal myopathies. Thorax 38: 616–623. Cottin V, Thivolet-Bejui F, Reynaud-Gaubert M, et al. (2003) Interstitial lung disease in amyopathic dermatomyositis, dermatomyositis, and polymyositis. European Respiratory Journal 22: 245–250. Dalakas MC and Hohlfeld R (2003) Polymyositis and dermatomyositis. Lancet 362: 971–982. Douglas WW, Tazelaar HD, Hartman TE, et al. (2001) Polymyositis–dermatomyositis-associated interstitial lung disease. American Journal of Respiratory and Critical Care Medicine 164: 1182–1185. Engel AG and Hohlfeld R (2004) Inflammatory myopathies. In: Engel AG and Franzini-Armstrong C (eds.) Myology: Basic and Clinical, 3rd edn., pp. 1321–1366. New York: McGrawHill. Fathi M, Dastmalchi M, Rasmussen E, et al. (2004) Interstitial lung disease, a common manifestation of newly diagnosed polymyositis and dermatomyositis. Annals of the Rheumatic Diseases 63: 297–301. Hill CL, Zhang Y, Sigurgeirsson B, et al. (2001) Frequency of specific cancer types in dermatomyositis and polymyositis: a population-based study. Lancet 357: 96–100.

PRIMARY CILIARY DYSKINESIA 485 Ikezoe J, Johkoh T, Kohno N, et al. (1996) High-resolution CT findings of lung disease in patients with polymyositis and dermatomyositis. Journal of Thoracic Imaging 11: 250–259. Marie I, Hatron PY, Hachulla E, et al. (1998) Pulmonary involvement in polymyositis and in dermatomyositis. Journal of Rheumatology 25: 1336–1343. Mastaglia FL, Garlepp MJ, Phillips BA, and Zilko PJ (2003) Inflammatory myopathies: clinical, diagnostic and therapeutic aspects. Muscle & Nerve 27: 407–425.

Potassium Channels

Schwarz MI, Sutarik JM, Nick JA, et al. (1995) Pulmonary capillaritis and diffuse alveolar hemorrhage: a primary manifestation of polymyositis. American Journal of Respiratory and Critical Care Medicine 151: 2037–2040. Tazelaar HD, Viggiano RW, Pickersgill J, and Colby TV (1990) Interstitial lung disease in polymyositis and dermatomyositis: clinical features and prognosis as correlated with histologic findings. American Review of Respiratory Diseases 141: 727–733.

see Ion Transport: Potassium Channels.

PRIMARY CILIARY DYSKINESIA H Mitchison, The Royal Free Hospital, London, UK M Salathe, University of Miami, Miami, FL, USA M Leigh and J L Carson, University of North Carolina, Chapel Hill, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Primary ciliary dyskinesia (PCD) is caused by ultrastructural ciliary defects that lead to abnormal ciliary beating and, subsequently, mucociliary dysfunction. PCD presents clinically with bronchiectasis, sinusitis, and, in up to 50% of cases, situs inversus. The ultrastructural defects of cilia are diverse but include in many cases outer and/or inner dynein arms. Recent advances have shown that ciliary defects in the embryonic node during development are responsible for the random right–left axis determination in these patients. Genetic approaches have elucidated at least some of the heterogeneous molecular defects underlying PCD. This article summarizes the current knowledge about this disease with respect to clinical manifestations, laboratory diagnosis and pathogenesis, situs inversus, genetics, and therapeutic considerations.

A report of a patient with the seemingly disparate symptoms of bronchiectasis and situs inversus 100 years ago is likely the first account of primary ciliary dyskinesia (PCD). Kartagener refined the description of the syndrome to include chronic sinusitis. However, only approximately 30 years ago, Afzelius and co-workers identified absent axonemal dynein arms in motile cilia with the ‘9 þ 2’ microtubular arrangement of the airway epithelium and in sperm flagella as the cellular defect leading to what had come to be known as Kartagener’s syndrome or immotile cilia syndrome. Recent studies have demonstrated considerable heterogeneity of dynein arm morphology at the ultrastructural level among patients with this syndrome. Moreover, half of patients with clinical

symptoms and ciliary ultrastructural defects do not exhibit situs inversus. Thus, the term PCD is currently used to describe individuals with congenital abnormalities of cilia and flagella and the clinical symptoms of bronchiectasis and chronic sinusitis. Kartagener’s syndrome, which in addition to bronchiectasis and sinusitis includes situs inversus, is thus considered a subset of PCD. The overall incidence of PCD is 1 in 20 000, with enrichment in certain populations. PCD is usually an autosomal recessive disorder, but unusual cases of PCD with apparent dominant or X-linked inheritance pattern have also been reported.

Clinical Manifestations Many clinical features of PCD reflect abnormal ciliary beating leading to impaired mucociliary clearance. Symptoms of mucociliary dysfunction in the nose, sinuses, and middle ear are recurrent or persistent rhinitis, sinusitis, and otitis media. Chronic productive cough is the major symptom of mucociliary dysfunction in the lower airways, and this chronic bronchitis can lead to bronchiectasis. Neonatal respiratory problems, situs inversus, and male infertility, common in PCD, are most likely linked to ciliary or flagellar dysfunction. Chronic nasal congestion is common and often present from early infancy, with little or no seasonal variation. Almost all PCD patients have chronic sinusitis, radiographically demonstrated by mucosal thickening, cloudiness, and/or opacification of all paranasal sinuses. Nasal polyps occur in approximately one-third of patients and may be apparent in early childhood. Almost all patients have chronic otitis media that is much more prominent in early childhood. At the time of diagnosis, most patients

486 PRIMARY CILIARY DYSKINESIA

have either chronic tympanic membrane perforations or have had multiple tympanostomies with insertion of ventilation tubes. Many have conductive hearing loss. Chronic productive cough is an almost universal feature of PCD. The cough is most apparent in the early morning but may occur at night or in association with exercise. In many children, exercise tolerance is normal but becomes impaired with advancing obstructive airway disease. Findings on chest examination are variable, with localized crackles that may or may not clear following forceful cough. Wheezing is relatively uncommon. Chronic airway infection ultimately results in bronchiectasis. Cultures of sputum or bronchoscopic aspirates may yield Hemophilus influenzae, Staphylococcus aureus, Streptococcus viridans, and Streptococcus pneumoniae. Patients with long-standing disease may have chronic infection with Pseudomonas aeruginosa. Pulmonary function tests may be normal early but typically demonstrate mild to moderate obstruction that becomes more severe in adulthood. Bronchodilator responsiveness is variable. Longitudinal analyses of children with PCD suggest that lung function may remain stable over relatively long periods of time. Most patients have a history of transient respiratory problems in the first days of life, with tachypnea, cough, increased secretions, and hypoxemia. The etiology for these respiratory problems is often unexplained or attributed to aspiration pneumonitis or neonatal pneumonia. The possibility of PCD is rarely considered except in cases with situs inversus, persistent atelectasis, persistent pneumonia, or a family history of PCD. The high occurrence of transient neonatal respiratory distress in PCD patients suggests that ciliary activity may be important for clearing fluid during the transition from a fluid-filled fetal lung to an air-filled neonatal lung. Situs inversus occurs in up to 50% of patients with PCD. Typically, all viscera in the chest and abdomen are transposed (situs inversus totalis). Male infertility is common and attributable to impaired sperm motility since ultrastructural and functional ciliary defects are mirrored in sperm flagella. However, some patients with PCD may have absent dynein arms in their cilia but normal dynein arms in sperm with normal motility. The occurrence of ciliary defects in cells lining the fallopian tubes has led to speculation that infertility and ectopic pregnancies could be increased in women with PCD, but this area has not been examined systematically. Hydrocephalus or dilated ventricles have been reported in a few patients with PCD. Ultrastructural and functional defects in ventricular ependymal cilia

provide a theoretical basis for this association, a hypothesis supported by hydrocephalus observed in knockout mouse models.

Laboratory Diagnosis and Pathogenesis Although a variety of heterogeneous ciliary ultrastructural lesions have been described among patients with PCD, a few studies have suggested that clinical symptoms suggestive of PCD are not always accompanied by evidence of an ultrastructural ciliary defect. This could be due to specimen processing or as yet uncharacterized or poorly understood ciliary defects not amenable to conventional electron microscopic inspection. Despite such reports, electron microscopic documentation of ciliary defects remains the standard for diagnosing PCD. In a recent study, 43% of patients exhibited abnormalities (absence or dysmorphology) of the outer dynein arm, 29% of the inner dynein arm, and 24% of both arms. The remaining 4% exhibited anomalies of the central microtubular pairs, radial spokes, or nexin links. A valid diagnosis of PCD thus may be indicated by the absence of both dynein arms, by the absence of either the inner or the outer dynein arm, or by consistent evidence of dynein arm dysmorphology (Figure 1). PCD in association with an absence of radial spokes or the transposition of an outer ODA IDA

(a)

(b)

(c)

(d)

0.15 µm

Figure 1 (a) Cross-section of ciliary axoneme from a healthy human subject. ODA, outer dynein arm; IDA, inner dynein arm. (b) Cross-section of ciliary axoneme from a PCD patient with an ODA defect. (c) Cross-section of ciliary axoneme from a PCD patient with an IDA defect. (d) Cross-section of ciliary axoneme from a PCD patient with absent IDA and ODA.

PRIMARY CILIARY DYSKINESIA 487

peripheral microtubular doublet to occupy the locus of a central microtubular doublet yielding an 8 þ 2 axonemal configuration has also been described. In the normal cilium, the bending motility of the axoneme mediated by ATPase-driven microtubular sliding is a highly ordered event. In contrast, the disorganization of the PCD axoneme yields a beat pattern that ranges from complete immotility to a vigorous motility that may appear virtually normal to an untrained observer. Thus, assessment of motility to aid the laboratory diagnosis and pathogenesis of PCD has been problematic, although it has been used successfully in specialized centers. Efforts to simply evaluate ciliary clearance by assessing the time required for a patient to taste a drop of saccharin placed on the nasal turbinate have fallen into disrepute. To date, the only definitive relationship between ciliary motility and ultrastructural integrity in PCD has been the evidence that cilia with no axonemal dynein arms reveal no motility. Other phenotypes seem to exhibit some, albeit dysfunctional, motion. Ultrastructural evidence has shown that the central microtubular pairs of cilia serve as vectors indicating the direction of beat (airway cilia with absent central pair beat in a circular pattern), and that in PCD the beating direction of adjacent cilia is disoriented relative to one another. Although this feature may help to identify patients with PCD, it also has been observed, at least focally, among individuals with transient upper respiratory infections and thus should not be considered an index lesion.

Situs Inversus The reasons for situs inversus totalis in up to half of the patients with PCD remained uncertain until recently (and continues to be controversial). The first clear indication that situs inversus was related to abnormal ciliary function was the cloning of an axonemal dynein heavy-chain gene, left–right dynein, found to be mutated in a strain of mice with a 50% incidence of situs inversus. This murine gene was expressed in the embryonic node at embryonic day 7.5 – the location and time of right–left axis determination. Cilia were found in the embryonic node at this developmental time as well, but ultrastructural analysis revealed them to be of the ‘9 þ 0’, usually nonmotile, variety. Thus, it came as a surprise when it was shown that embryonic node cilia are in fact motile, despite their ‘9 þ 0’ configuration. With an unusual circular motion, their motility was different from that of ‘9 þ 2’ cilia, but their beating was critical for correct left–right axis determination. Immotile embryonic node cilia were associated with a 50% chance of developing situs inversus. The flow across

the embryonic node created by motile cilia seems to be sensed by nonmotile, flow-sensing cilia, localized in the periphery of the embryonic node. The bending of these cilia elicits a calcium signal on only one side of the node. This signal could cause the appropriate, unilateral expression of genes that ultimately determine the correct left–right axis. Abnormalities in motile or nonmotile cilia of the embryonic node will lead to random axis determination and thus to situs inversus in 50% of cases. Several mouse models of random left–right axis determination are used for further investigation of these issues, but their detailed description is beyond the scope of this article. Ciliary defects can also affect primary or nonmotile cilia (with the ‘9 þ 0’ microtubule arrangement). Since many cell types in the body express nonmotile cilia, PCD can also be associated with disorders that reflect nonmotile ciliary dysfunction other than situs inversus, such as cystic kidney disorder and retinitis pigmentosa.

Genetics Genetic approaches to investigate the heterogeneous molecular defects underlying PCD have focused on identification of disease-causing genes using genetic linkage analysis (positional cloning) and candidate gene analysis. Although a genetic linkage of PCD families to the HLA locus on chromosome 6 was identified, candidate gene analysis in this region has not revealed the causative gene. Linkage analysis using inbred families and homozygosity mapping has identified four PCD loci: DNAH5 on chromosome 5p15, CILD2 on 19q, and additional loci on 16p12 and 15q13–15. Selection of candidate genes for mutational analysis has also proven successful, with identification of mutations in DNAI1 on chromosome 9p13–p21 and DNAH11 on 7p15. However, genetical heterogeneity can even be found within groups of families with the same ultrastructural defect. Mutations in genes encoding two different axonemal outer dynein arm components (DNAI1 and DNAH5) have been shown to cause PCD in patients lacking outer dynein arms. Mutations in the DNAI1 and DNAH5 genes are proposed to account for approximately 24% of PCD cases overall and presumably for a larger percentage of the subgroup of PCD cases associated with outer dynein arm deficiencies. Six DNAI1 mutations have been reported, and these account for mutations in 6 of 47 PCD families screened so far. One mutation, a T insertion predicted to cause a splice-site mutation (219 þ 3insT), occurs more frequently. The relative frequency of this allele may indicate a mutation hotspot in the DNAI1 gene or a population founder effect in PCD.

488 PRIMARY CILIARY DYSKINESIA

Ten DNAH5 mutations have been reported. Of 25 PCD families compatible for linkage to the DNAH5 gene, mutations were found in 8. These are mostly premature truncation mutations predicting loss of motor- and microtubule-binding sites from the protein. Two missense mutations also occur, both located in functionally conserved amino acids. No apparent phenotype differences occur between patients with combinations of truncation or missense mutations. However, a patient homozygous for a splice-site mutation (IVS74-1G4C) has been reported with a partial outer dynein arm deficiency and 54% shortened outer dynein arms, contrasting with patients homozygous for two truncation mutations who displayed the complete absence of outer dynein arms, suggesting a genotype–phenotype correlation. Defects in another axonemal heavy-chain dynein, DNAH11, were identified in a patient with Kartagener’s syndrome and normal cilia ultrastructure. DNAH11 is the human homolog of the mouse left– right dynein gene. DNAH11 mutations are therefore associated with situs inversus and possibly a minority of PCD cases. Mutational analysis on candidate genes encoding other axonemal structural components has excluded the dynein genes for the heavy chain DNAH9, the intermediate chain DNAI2, the light chain TCTEX2, and the gene encoding the central complex protein, hPF20 as major causes of PCD. In one report, an absence of DNAH7 protein in cilia from a PCD patient was shown but no coding mutations were found, suggesting the likely involvement of another gene as the primary defect. Other candidate genes involved in ciliary function have also been investigated, including the genes for the transcription factor FOXJ1 and DNA polymerase lambda (DPCD). Genetic work is aided by the selection of candidate disease genes with a conserved homolog in Chlamydomonas reinhardtii, a biflagellated eukaryotic unicellular alga and an established ciliary model organism. Dysmotile Chlamydomonas strains with axonemal defects similar to those of PCD patients have been and will be valuable in identifying genes associated with PCD. Furthermore, advances in proteomics and comparative bioinformatics provide comprehensive sets of ciliary genes and proteins, which comprise excellent new PCD candidates.

Therapeutic Considerations No specific therapeutic modalities are available to correct ciliary dysfunction in PCD. Management should include aggressive measures to enhance clearance of mucus, prevent respiratory infections, and treat bacterial infections in the airways, sinuses, and

middle ear. Few clinical trials have been conducted because PCD is rare and most centers follow only a few PCD patients. Approaches to enhance mucus clearance from the lung in PCD include chest percussion with postural drainage, mechanical oscillatory Vest percussion, vigorous aerobic exercise, and other maneuvers to encourage cough and deep breathing. Bronchodilators such as albuterol may aid mucus clearance in patients who are bronchodilator responsive. Inhaled corticosteroids have been used, but the role of antiinflammatory agents has not been defined. Measures to prevent respiratory tract infection and irritation should be considered, including routine immunizations (for pertussis, measles, H. influenzae type b, S. pneumoniae, and influenza) and preventive counseling to avoid exposure to respiratory pathogens, tobacco smoke, and other irritants. Prompt institution of antibiotic therapy for bacterial infections (bronchitis, sinusitis, and otitis media) can prevent or delay irreversible damage. Sputum culture results can direct appropriate antimicrobial therapy. In some patients, symptoms recur within days to weeks after completing a course of antibiotics. This subgroup may benefit from extended use of a broad-spectrum antibiotic. If detected and treated early, P. aeruginosa colonization of the airways can be eradicated; however, long-standing pseudomonal infection of bronchiectatic airway is unlikely to clear, even with long-term intravenous antibiotic therapy. Use of tympanostomy with ventilation tube placement may benefit children with chronic ear infections and conductive hearing impairment. Nasal polypectomy and/or sinus drainage may provide short-term relief of symptoms in severe sinusitis without response to antibiotic therapy. However, long-term benefits are unclear. Lobectomy should be considered only in special cases. In patients with end-stage lung disease, lung transplantation has been performed successfully.

Prognosis Chronic lung disease with bronchiectasis may progress to severe disability and eventually respiratory failure. The rate of disease progression is variable. A number of individuals have experienced a normal or near-normal life span. Better diagnostic tools are needed to facilitate earlier diagnosis and institution of therapy, thereby improving the overall health and prognosis for these patients. See also: Bronchiectasis. Symptoms of Respiratory Disease: Cough and Other Symptoms.

PRIMARY MYELOFIBROSIS 489

Further Reading Afzelius BA (1976) A human syndrome caused by immotile cilia. Science 193: 317–319. Avidor-Reiss T, Maer AM, Koundakjian E, et al. (2004) Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis. Cell 117: 527–539. Bush A and O’Callaghan C (2002) Primary ciliary dyskinesia. Archives of Disease in Childhood 87: 363–365. Dutcher SK (1995) Flagellar assembly in two hundred and fifty easy-to-follow steps. Trends in Genetics 11: 398–404. El Zein L, Omran H, and Bouvagnet P (2003) Lateralization defects and ciliary dyskinesia: lessons from algae. Trends in Genetics 19: 162–167. Ibanez-Tallon I, Heintz N, and Omran H (2003) To beat or not to beat: roles of cilia in development and disease. Human Molecular Genetics 12(special issue no. 1): R27–R35. Kartagener M (1933) Zur Pathogenese der Bronchiektasien. Beitra¨ge zur Klinik der Tuberkulose 83: 489–501. Leigh MW (1998) Primary ciliary dyskinesia. In: Chernick V and Boat TF (eds.) Kendig’s Disorders of the Respiratory Tract in Children, pp. 819–825. Philadelphia: Saunders.

McGrath J, Somlo S, Makova S, Tian X, and Brueckner M (2003) Two populations of node monocilia initiate left–right asymmetry in the mouse. Cell 114: 61–73. Meeks M and Bush A (2000) Primary ciliary dyskinesia (PCD). Pediatric Pulmonology 29: 307–316. Nonaka S, Tanaka Y, Okada Y, et al. (1998) Randomization of left–right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 95: 829–837. Noone PG, Leigh MW, Sannuti A, et al. (2004) Primary ciliary dyskinesia: diagnostic and phenotypic features. American Journal of Respiratory and Critical Care Medicine 169: 459– 467. ¨ ber einen Fall von Bronchiektasie bei einem Siewert A (1904) U Patienten mit situs inversus viscerum. Berliner klinische Wochenschrift 41: 139–141. Sleigh MA (1981) Primary ciliary dyskinesia. Lancet 2: 476. Spiden S and Mitchison HM (2001) Homozygosity mapping as an approach for identifying genes involved in primary ciliary dyskinesia. In: Salathe M (ed.) Cilia and Mucus: From Deve lopment to Respiratory Defense, vol. 10, pp. 109–117. New York: Dekker.

PRIMARY MYELOFIBROSIS C J McNamara, The Royal Free Hospital, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Chronic idiopathic myelofibrosis refers to an acquired clonal hematopoietic stem cell disorder that is characterized by hyperplasia of progenitor cells in the bone marrow, reactive bone marrow fibrosis, characteristic changes in the blood and extramedullary hematopoiesis (EMH). Complications include thrombotic disease, bleeding and leukemic transformation. Respiratory complications are less common and include thromboembolic disease, pulmonary hypertension and EMH in the lung. The long-term prognosis is poor. Current treatments are largely palliative; however, experimental treatments targeted at the underlying pathogenesis are likely to become available shortly.

Introduction Chronic idiopathic myelofibrosis (CIMF) is an acquired clonal hematopoietic stem cell disorder that is characterized by hyperplasia of progenitor cells in the marrow, including dysplastic megakaryocytes and clonal monocytes, which secrete growth factors that lead to varying degrees of fibrosis, osteosclerosis, and new vessel formation in the bone marrow. Extramedullary hematopoiesis (EMH), also known as agnogenic myeloid metaplasia, is usually present and refers to ectopic hematopoiesis that occurs most often in the liver and spleen but may be seen in any organ, including the lungs and pleura. This is most likely a

consequence of the replacement of normal hematopoietic tissue by collagen fibrosis, the abnormal mobilization of hematopoietic progenitors into the peripheral blood and their localization in other organs.

Pathology The diagnosis of CIMF is based on morphologic findings and the exclusion of other pathologies known to cause fibrosis of the marrow (see Table 1). It is important to be rigorous in the application of diagnostic criteria as marrow fibrosis and EMH are not specific for CIMF. Bone marrow fibrosis may also be seen in other myeloproliferative disorders, such as polycythemia vera and essential thrombocythemia, as well as in association with metastatic bone marrow tumors. Examination of the blood film frequently suggests the diagnosis of CIMF. Patients in the fibrotic phase of the illness are usually anemic and have characteristic changes on the blood film, such as leukoerythroblastosis (a left-shift in the granulocyte count and nucleated red cells, which are normally absent in the blood) and marked red cell anisopoikilocytosis that includes tear drop cells (see Figure 1). These findings are suggestive but not diagnostic of CIMF. They may also be seen in conditions that replace normal bone marrow that may or may not have associated marrow fibrosis.

490 PRIMARY MYELOFIBROSIS Table 1 Morphologic findings Prefibrotic stage

Fibrotic stage

Bone marrow Minimal/absent reticulin fibrosis Cellularity increased Abundant atypical megakaryocytes with morphologic atypia

Bone marrow Established reticulin fibrosis Cellularity reduced Abundant atypical megakaryocytes with Morphologic atypia Osteosclerosis

Peripheral blood No or mild leukoerythroblastosis Mild red cell anisopoikilocytosis with few tear drop poikilocytes

Peripheral blood Leukoerythroblastosis Marked red cell anisopoikilocytosis with prominent tear drop poikilocytosis

Figure 1 Leukoerythroblastic blood film from a patient with CIMF demonstrating tear drop red cells and a nucleated red blood cell (Wright’s stain,
400).

Figure 2 Silver staining of trephine biopsy demonstrating increased marrow reticulin (
200).

The prefibrotic phase of CIMF is typically associated with a leukocytosis (median white cell count 15
109 l  1 range (1.6–88.3)
109 l  1) and the platelet count is elevated in half of the patients.

Figure 3 End-stage CIMF marrow demonstrating replacement of normal hematopoiesis by fibrous tissue and fibroblasts (Hematoxylin–eosin stain,
200).

The median number of CD34 þ hematopoietic progenitor cells circulating in the periphery is increased in the majority of patients. The bone marrow may be difficult to aspirate because of the fibrotic changes, resulting in a ‘dry tap’. A trephine biopsy is required in all cases to establish the diagnosis. Silver or trichrome staining of the marrow trephine identifies an increase in type III collagen in all patients in the fibrotic phase of the disease (Figure 2). Collagen fibrosis is not present in the prefibrotic or cellular phase, making diagnosis difficult. The end stage, densely fibrotic marrow typically has little granulopoiesis or erythropoiesis but numerous dysplastic megakaryocytes distributed throughout the areas of fibrosis (Figure 3). Immunostaining of the marrow identifies increased marrow vascularity only in the advanced fibrotic phase.

Clinical Features The annual incidence of CIMF is 0.5–1.5 per 100 000. The median age at diagnosis is more than 60 years of age with an equal sex distribution.

PRIMARY MYELOFIBROSIS 491 Table 2 Clinical and laboratory findings Prefibrotic stage

Fibrotic stage

Splenomegaly and/or hepatomegaly: absent or only mild enlargement Mild anemia Mild leukocytosis Mild thrombocytosis

Splenomegaly (90%) and/or hepatomegaly (50%) Moderate to severe anemia Leukocytosis or leukopenia Thrombocytosis or thrombocytopenia

Clinical and laboratory features depend largely on whether the patient is in the early/prefibrotic (20–30%) or the fibrotic phase (70–80%) of the illness at the time of presentation (see Table 2). Up to one-third of patients are detected while asymptomatic, either on a routine blood film or after the finding of hepatomegaly or splenomegaly on physical examination. Splenomegaly is present in most cases. Patients may develop abdominal pain and distension due to splenic enlargement, which can be immense. Impingement of the spleen on adjacent structures may cause local symptoms. Splenic infarction may produce severe left upper quadrant pain that may be confused for respiratory or other abdominal pathologies. The fibrotic phase is characterized by progressive pancytopenia secondary to bone marrow failure. Symptoms related to cytopenias appear, particularly anemia requiring transfusion. Ineffective erythropoiesis and sequestration of red cells and platelets in an enlarged spleen exacerbate the cytopenias. Constitutional symptoms are a common feature in CIMF and include fatigue, weight loss, fever, and sweats. Along with symptomatic organomegaly these symptoms account for the poor quality of life of many CIMF patients. Hematologic Complications

Thrombotic complications are the most important causes of morbidity and mortality in CIMF with a combined rate of 45.6 complications per 100 patient years. This may manifest as deep venous thrombosis (DVT) or pulmonary embolism (PE). In addition, thrombosis at unusual sites, including the hepatic and portal veins as well as the cerebral venous sinuses, may occur, producing distinct clinical syndromes. Patients are at equal risk of arterial thrombosis, the most common sites being the vessels of the limbs or the central nervous system and, less commonly, the coronary or mesenteric arteries. The cause of the prothrombotic state seen in CIMF is not entirely understood. Paradoxically, bleeding may also complicate CIMF, but this is less often fatal. Qualitative platelet

function abnormalities are well documented and hemorrhage may occur even when the platelet count is elevated. However, these abnormalities are usually of little clinical significance. When bleeding does occur, it is most often of the type affecting small vessels in the skin and mucosal sufaces. Antiplatelet agents used to ameliorate the thrombotic tendency may contribute to the bleeding tendency. Leukemic transformation of CIMF occurs in up to 20% of patients within 10 years of diagnosis and portends a very poor prognosis. This event is often heralded by worsening cytopenias, constitutional symptoms, and the appearance of myeloblasts in the peripheral blood. Changes in hepatic vascular compliance can lead to portal hypertension and gastroesophageal varices. Iron overload may develop in patients requiring multiple transfusions because of anemia. Hyperuricemia may also occur due to ineffective hematopoiesis and increased cell turnover. Respiratory Complications

These occur infrequently as the main clinical feature of CIMF. Pulmonary infections may occur, secondary to immunosuppression related either to bone marrow failure or from treatment of the disease. EMH can involve any organ, including the lung and pleura. Consolidation of lung parenchyma and pleural effusions may occur as a result. Leukemic infiltration of the lung has also been documented. There are reports of pulmonary hypertension (PHT) occurring in CIMF patients. Potential causes include occlusion of pulmonary arteries by circulating megakaryocytes, thromboembolic phenomenon, extramedullary hematopoiesis infiltrating pulmonary parenchyma, and in situ pulmonary thrombosis secondary to chronic disseminated intravascular coagulation. Portal hypertension, which is a well-recognized complication of CIMF, may contribute to PHT. Thrombocytosis and accelerated EMH may complicate splenectomy and increase the risk of pulmonary complications. However, whether splenectomy predisposes CIMF patients to PHT remains controversial. Pulmonary edema may occur as congestive cardiac failure and other cardiovascular complications are seen with increased frequency in CIMF. Chemotherapeutic agents used in CIMF may also be associated with lung injury.

Pathogenesis The underlying molecular basis for CIMF is unknown. The clonal nature of the proliferating progenitor cells is demonstrated by cytogenetic analysis and

492 PRIMARY MYELOFIBROSIS

X-chromosome studies. Cytogenetic abnormalities occur in less than 60% of patients and none of these are specific for CIMF. Gene expression studies have identified abnormalities in cell signaling pathways, such as the JAK/STAT pathway, offering hope of novel, targeted therapeutic strategies in the future. The observed bone marrow fibrosis is a reactive phenomenon, mediated by fibrogenic cytokines secreted by abnormal, dysplastic megakaryocytes or monocytes. Specific cytokines and growth factors, such as transforming growth factor beta 1 (TGF-b1), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF), are likely to have an important role in the development and progression of CIMF by stimulating polyclonal fibroblasts to synthesize extracellular matrix and cell adhesion proteins. They also promote the transcription of proteases that inhibit enzymes involved in extracellular matrix degradation. The importance of the megakaryocyte in disease pathogenesis is highlighted by murine experiments where mice given marrow grafts containing cells infected with the megakaryocyte growth factor, thrombopoietin, develop myelofibrosis.

Management and Current Therapy Current therapies for CIMF are unsatisfactory and largely palliative. Allogeneic hematopoietic stem cell transplantation offers the only possibility of cure but is restricted to a small number of patients because of advanced age and comorbidity in the majority of patients. Only 10% of patients are less than 45 years of age. The increasing use of nonmyeloablative conditioning regimens may make this treatment more accessible to CIMF patients. More than one-half of patients require therapy to control elevated blood counts and reduce extramedullary hematopoiesis. Currently, the most frequently used treatment is the ribonucleotide reductase inhibitor hydroxyurea. Treatment of anemia includes the use of erythropoietin, corticosteroids, and androgen preparations. Splenectomy may be helpful in patients with symptoms from organ enlargement or those with cytopenias due to splenic sequestration blood components. The success of this procedure varies according to the indication and is associated with the potential for significant morbidity and mortality. This is mainly related to postsplenectomy thrombocytosis, hepatic enlargement, and thrombosis. In those patients who are not fit for a surgical procedure, splenic irradiation may alleviate symptoms for a short period. Radiotherapy may also be useful in the management of extramedullary hematopoiesis that produces symptoms in strategic locations.

Prophylaxis of thrombotic disease is a cornerstone of management in CIMF. Aspirin is used as primary prophylaxis against thrombotic episodes in patients with elevated platelet counts. Warfarin anticoagulation is reserved for the treatment and secondary prophylaxis of thrombosis. Managing PHT is similar for other patients without CIMF. There may, however, be additional benefit in reducing the platelet count by plateletpheresis or cytoreductive therapy. Antiplatelet therapy may not be effective in preventing PHT. Among the novel investigational agents presently available, thalidomide has been shown to produce a demonstrable improvement in splenomegaly and blood counts in a variable proportion of patients. Thalidomide inhibits the activity of fibrogenic cytokines in vitro. Newer agents targeting dysregulated cytokine production are likely to become available shortly.

Prognosis The median survival for CIMF patients is approximately 5 years. However, the rate of marrow fibrosis and the natural history of the disease are quite variable. CIMF most often pursues a chronic progressive course. Causes of death include fatal thrombosis, hemorrhage, leukemic transformation, bone marrow failure, and cardiac failure. The prognosis for patients with PHT is poor, with a median survival of only 18 months. Other adverse prognostic factors include advanced age, anemia, and male gender. There is intense interest in prognostic scoring models to predict outcome and facilitate decision making particularly when aggressive therapies with appreciable morbidity and mortality, such as bone marrow transplantation, are being considered. See also: Fibroblasts. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Tumors, Malignant: Overview. Vascular Disease.

Further Reading Arora B, Sirhan S, Hoyer JD, Mesa RA, and Tefferi A (2005) Peripheral blood CD34 count in myelofibrosis with myeloid metaplasia: a prospective evaluation of prognostic value in 94 patients. British Journal of Haematology 128: 42–48. Barbui T, Cortellazo S, Viero P, et al. (1983) Thrombohaemorrhagic complications in 101 cases of myeloproliferative disorders: relationship to platelet number and function. European Journal of Cancer and Clinical Oncology 19: 1593–1599. Barosi G, Viarengo G, Pecci A, et al. (2001) Diagnostic and clinical relevance of the number of circulatory CD34-positive cells in myelofibrosis with myeloid metaplasia. Blood 98: 3249–3255.

PROGRESSIVE SYSTEMIC SCLEROSIS 493 Cervantes F, Barosi G, Demory JL, et al. (1998) Myelofibrosis with myeloid metaplasia in young individuals: disease characteristics, prognosis factors and identification of risk groups. British Journal of Haematology 102: 684–690. Chagraioui H, Kamura H, Tulliez M, Vainchenker W, and Wendling F (2002) Prominent role of TGF-b1 secreted by hematopoietic cells in bone marrow fibrosis induction. Blood 100: 3495–3503. Garcia-Manero G, Schuster S, Patrick H, and Martinez J (1999) Pulmonary hypertension in patients with myelofibrosis secondary to myeloproliferative diseases. American Journal of Hematology 60: 130–135. Jaffe ES, Harris NL, Stein H, and Hardiman JW (eds.) (2001) The World Health Organization Classification of Neoplasms of the Hematopoietic and Lymphoid Tissues. Lyon: IARC Press. Kvasnicka HM, Thiele J, Werden C, Zankovich R, Diehl V, and Fischer R (1997) Prognostic factors in idiopathic osteomyelofibrosis. Cancer 80: 708–719. Le Basse-Kerdiles MC and Martyre MC (1999) Dual implication of fibrogenic cytokines in the pathogenesis of fibrosis and myeloproliferation in myeloid metaplasia with myelofibrosis. Annals of Hematology 78: 437–444. Mesa RA, Li CY, Ketterling RP, Schroeder GS, Knudson RA, and Tefferi A (2005) Leukemic transformation in myelofibrosis with

myeloid metaplasia: a single-institution experience with 91 cases. Blood 105: 973–977. Tefferi A, Mesa RA, Schroeder G, et al. (2001) Cytogenetic findings and their clinical relevance in myelofibrosis with myeloid metaplasia. British Journal of Haematology 113: 763–771. Tefferi A, Silverstein MN, and Noel P (1995) Agnogenic myeloid metaplasia. Seminars in Oncology 22: 327–333. Thiele J, Kvasnicka HM, Boeltken B, et al. (1999) Initial (prefibrotic) stages of idiopathic (primary) myelofibrosis (IMF) – a clinicopathological study. Leukemia 13(11): 1741–1748. Thiele J, Zankovich R, Steinberg T, Fischer R, and Diehl V (1989) Agnogenic myeloid metaplasia (AMM) – correlation of bone marrow lesions with laboratory data: a longitudinal clinicopathological study on 114 patients. Hematological Oncology 7: 327–343. Visani G, Finelli C, Castelli U, et al. (1990) Myelofibrosis with myeloid metaplasia; clinical haematological parameters, predicting survival in a series of 133 patients. British Journal of Haematology 75(1): 4–9. Ward HP and Block MH (1971) The natural history of agnogenic myeloid metaplasia in a critical evaluation of its relationship with the myeloproliferative syndrome. Medicine 50: 357–420.

PROGRESSIVE SYSTEMIC SCLEROSIS M M Freemer, University of California, San Francisco, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The pulmonary manifestations of progressive systemic sclerosis (PSS) are important determinants of a patient’s prognosis. Although the etiology and pathogenesis of PSS are unknown, it is recognized that this is a complex disease; it is likely that different genetic susceptibilities and environmental exposures lead to the disease in different individuals. Individuals with diffuse PSS tend to develop interstitial pneumonia, most commonly fibrotic non-specific interstitial pneumonia. Isolated pulmonary hypertension is frequently seen in individuals with limited scleroderma. Screening for these forms of lung disease with high-resolution computed tomography, pulmonary function tests, and echocardiography is important, given the availability of therapeutic options.

Scleroderma, literally hard (sclero) skin (derma), is a systemic disease with the potential for destructive visceral involvement in addition to disfiguring cutaneous lesions. Descriptions of the skin changes in systemic sclerosis date back to Hippocrates. However, it was not until the twentieth century that Goetz described the visceral manifestations of the disease, providing the term ‘systemic sclerosis’. Progressive systemic sclerosis (PSS) has been reported throughout the world, but the prevalence of the disease remains

unknown. In the United States, the prevalence is estimated to be 300 000. PSS affects women more commonly than men at a ratio of approximately 4:1. Although people of any age may be affected, the onset of disease usually occurs in the fourth through sixth decades of life. Studies vary in the reported prevalence of lung disease in PSS (given differing definitions of pulmonary involvement); however, using the most sensitive diagnostic modalities available, as well as evidence from autopsy studies, most (B70%) PSS patients have lung disease. The pulmonary manifestations have been shown to vary with ethnicity; in blacks and Asians, there is a higher prevalence of lung disease which is also more severe and accelerated than in Caucasians. The two primary forms of lung disease, fibrotic interstitial pneumonia and pulmonary hypertension, play a critical role in PSS patients’ prognosis. In fact, lung disease increases the risk of death at least twofold.

Etiology Genetic Contribution

The cause of PSS is unknown but likely involves both genetic predisposition and environmental exposures. The strongest evidence for a genetic contribution to the development of PSS is found in the Choctaw

494 PROGRESSIVE SYSTEMIC SCLEROSIS

Native Americans in Oklahoma, in whom the prevalence of the disease is more than double that in the general population. The first-degree relatives of any individual with systemic sclerosis have a 13-fold increased relative risk of disease. However, given the disease is so rare, familial cases account for less than 2% of all PSS cases. Environmental Contribution

Environmental exposures that have been considered possible etiologic agents in systemic sclerosis include retroviruses, cytomegalovirus (CMV), organic solvents, vinyl chlorine, silica dust, L-tryptophan, toxic oil, silicone, pesticides, and drugs (e.g., appetite suppressants, cocaine, and carbidopa).

Pathology Interstitial Pneumonia

The hallmark pathologic features of PSS are fibrosis and vascular disease. The lung is no exception, with fibrotic interstitial pneumonias accounting for the vast majority of the primary parenchymal lung disease in PSS. Studies on multiple ethnic groups that applied the current American Thoracic Society and European Respiratory Society classification system for interstitial pneumonias demonstrated that although the overwhelming majority of cases are fibrotic non-specific interstitial pneumonia (NSIP), approximately 20% of the NSIP cases were cellular. In addition to the cases of NSIP, a usual interstitial pneumonia (UIP) pattern was also seen in other PSS patients. There is insufficient evidence to determine whether the prognosis of patients varies with the histopathologic pattern (UIP or NSIP). Vascular Disease

The other primary lung disease in PSS is a vasculopathy. Although it remains contested, there is evidence that the pulmonary circulation in PSS undergoes locally mediated arterial vasoconstriction as visibly occurs in the peripheral circulation with Raynaud’s phenomenon (a characteristic feature of PSS).

PSS patients with isolated pulmonary hypertension develop vascular changes that resemble those in other forms of secondary pulmonary hypertension: arteriolar thickening, medial hypertrophy, and concentric intimal proliferation (onion skinning). In addition to isolated pulmonary hypertension (in the absence of interstitial pneumonia), other PSS patients develop interstitial lung disease with secondary pulmonary hypertension.

Clinical Features The criteria for the diagnosis of PSS were developed in 1980; however, 8 years later an expert panel proposed subclassification into two patterns of disease, limited and diffuse (Table 1). Although the utility of the distinction between limited and diffuse disease remains controversial, it is nevertheless relevant to the consideration of the anticipated pulmonary manifestations in different patient subgroups. Diffuse Progressive Systemic Sclerosis

Disease manifestations In patients with diffuse disease, both the skin and the viscera are involved; the former includes the trunk and acral regions, whereas the latter may include the lungs, kidneys, gastrointestinal tract, and heart. In patients with diffuse disease, PSS progresses relatively rapidly. For example, Raynaud’s phenomenon occurs within 1 year of the cutaneous lesions. Within the first 3 years of disease onset, most PSS patients who will develop interstitial lung disease have already done so. The symptoms of pulmonary disease in these patients are non-specific, such as cough and dyspnea. On serologic evaluation, patients with diffuse disease may be positive for antitopoisomerase antibodies. The pathologic pattern seen most frequently in diffuse PSS patients is fibrotic interstitial pneumonia (NSIP or UIP). Pulmonary hypertension may develop as a complication of progressive lung disease. Disease evaluation In diffuse PSS patients with cough or dyspnea, high-resolution computed tomography (HRCT) is the most appropriate imaging

Table 1 Comparison of limited and diffuse PSS features Feature

Limited systemic sclerosis

Diffuse systemic sclerosis

Pulmonary disease Time of lung disease onset

Primary pulmonary hypertension Late in PSS; more than 5 years of disease duration Anticentromere (70% of patients) Decreased or diminishing diffusing capacity

Fibrotic interstitial lung disease Early in PSS; usually before 5 years of disease duration Antitopoisomerase (30% of patients) Decreased forced vital capacity

Associated antibodies Physiologic predictors of disease progression

PROGRESSIVE SYSTEMIC SCLEROSIS 495

modality, given the reduced sensitivity of chest radiography (Figure 1). In patients with respiratory symptoms, areas of ground-glass opacification on HRCT, and restrictive or diffusion deficits on physiologic testing, bronchoalveolar lavage (BAL) may serve three purposes. The first purpose is diagnostic – to assess the presence of ‘alveolitis’ (an elevated percentage of neutrophils or eosinophils in comparison to standardized normal values), which is an indicator of active disease that will progress in the absence of therapy. In addition, BAL eosinophilia has been shown to be associated with diminished survival. Finally, if clinically indicated, BAL may help to exclude infection as the cause of the patient’s symptoms and radiographic abnormalities. Further evaluation with pulmonary function testing is also useful prognostically; an abnormal forced vital capacity at initial evaluation is associated with a higher mortality rate. The utility of lung biopsy in patients with PSS has not been adequately examined.

can progress to right heart failure, with dyspnea as their only respiratory symptom. They are very commonly anticentromere positive. Given the high mortality associated with pulmonary hypertension, PSS patients should be screened regularly with echocardiograms to assess pulmonary artery pressures. Diminished diffusing capacity on pulmonary function testing predicts the development of pulmonary hypertension.

Limited Progressive Systemic Sclerosis

Pathogenesis

In patients with limited disease, the cutaneous involvement remains distal to the elbows and knees, although it may involve the face. In these patients, PSS progresses relatively slowly. For example, Raynaud’s phenomenon has usually been present for years prior to the cutaneous lesions. Isolated pulmonary hypertension occurs late in those with limited disease. Some patients with limited disease have CREST, with characteristic systemic findings (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias). Patients with limited disease (including those with CREST)

The pathogenesis of PSS remains unclear, and the explanation for pulmonary involvement has also not been elucidated. A central question in the pathogenesis of PSS (as well as other autoimmune diseases) is why autoantibodies are formed. Endogenous proteins for which autoantibodies have been found in systemic sclerosis patients include nucleolar antigens, RNA polymerases, centromeres, and fibrillin. With respect to lung disease, two specific autoantibodies are of interest. Anticentromere antibody has been associated with limited PSS and may be considered

Figure 1 Typical HRCT findings in PSS interstitial lung disease. Bilateral, basilar predominant areas of ground-glass opacification associated with reticulations are typical HRCT findings in PSS.

Other Disease Manifestations that Affect the Lung

Aspiration pneumonia is a common finding in PSS patients due to the presence of esophageal dysmotility (Figure 2). A relatively rare primary pulmonary manifestation of PSS is vasculitis with pulmonary hemorrhage. Similarly, restrictive disease secondary to thoracic skin tightening is also quite infrequent. Notably, the incidence of lung cancer is elevated in PSS patients.

Figure 2 Typical pathologic findings in non-specific interstitial pneumonia. This lung biopsy demonstrates a temporally and spatially homogeneous pattern of interstitial thickening and inflammation. In many areas, aggregates of lymphocytes can be seen, and germinal centers may be present.

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‘protective’ against interstitial lung disease. Although antitopoisomerase can be seen in those with diffuse disease and interstitial lung disease, this autoantibody is less common (occurring in approximately 30% of patients with diffuse PSS). Autoantibody Formation

Although definitive evidence for the mechanisms by which these autoantibodies develop is not available, several hypotheses exist. The importance of microchimerism for the development of autoimmunity is an area of active investigation. Microchimerism is the concept that the presence of HLA-disparate cells within an individual results in the formation of antibodies. The source of HLA-disparate cells in individuals with autoimmune disease may include fetal cells (in women who have been pregnant), maternal cells, twin sibling cells, or transfused or transplanted cells. Molecular mimicry is an alternative concept that an antibody formed against an exogenous antigen cross-reacts with an endogenous (self-) antigen with resultant autoantibodies. Similarly, it is possible that autoantibodies develop when proteins are changed in some way. Such alteration may include damage caused by the environmental exposures noted previously. Immune System Dysfunction

In addition to autoantibody formation, immune system dysfunction also likely contributes to the development of PSS. In the skin, T-cell infiltration, predominantly CD4 þ T cells, precedes fibrosis. In the lung, as assessed by BAL, CD8 þ T cells predominate, with an elevated CD8:CD4 ratio. The generally accepted hypothesis is that these cells produce inflammatory cytokines that lead to increased collagen deposition by fibroblasts. In PSS, it remains controversial whether the predominant abnormality is in B-cell or T-cell function, and the T-cell response does not appear to be a purely Th1 or Th2 response. Vasculopathy

In addition to the importance of the immune system, PSS patients also have a vasculopathy. The cause of vascular injury has not been established. Although the vascular damage may result from the deposition of immune complexes, other potential contributing factors include dysregulation of endothelial cell apoptosis, endothelial cell proliferation, and release of von Willebrand factor. Genetic Polymorphisms

Interestingly, several genetic association studies in PSS patients indicate that specific genes may

influence the pathogenetic pathway of PSS at many levels, including the development of autoimmunity, tissue fibrosis, and vasculopathy. For example, a variety of HLA regions, which may influence autoantibody formation, have been shown to be associated with PSS in different ethnic groups. Genomewide screening in the Choctaw population revealed genetic polymorphisms associated with systemic sclerosis, including fibrillin-1; secreted protein, acidic and rich in cysteine (SPARC); and topoisomerase-1. Alterations in the proteins encoded by these polymorphic genes may be involved in the development of antibodies to these proteins. Other genes that have been associated with PSS include polymorphisms of the genes for transforming growth factor beta, tumor necrosis factor, interleukin-1A (IL-1A), and IL-4. Such polymorphisms may affect an individual’s response to antigens. Finally, evidence that PSS patients can have alterations in transcriptional factors for collagen and fibronectin may explain the aberrant amount of protein production. The manner in which environmental exposures, individuals’ genetic susceptibility, T- and B-cell activity, as well as vascular disease interact to produce the heterogeneous disease that we call PSS is unknown. An attempt to relate these possible components of the pathogenesis of PSS is represented in Figure 3.

Animal Models There are several animal models for PSS. One genetic model is the tight-skinned mouse (TSK1) that carries a duplication of exons in the fibrillin gene. A second tight-skinned mouse (TSK2) has also been described that develops skin fibrosis. The TSK2 mutation has been mapped to chromosome 1. Unfortunately, although TSK mice have lung disease, it resembles emphysema rather than the interstitial disease seen in PSS patients. Another animal model was discovered at the University of California at Davis in chickens (UCD L200) that are born with missing combs. These animals rapidly develop severe systemic disease, including pulmonary fibrosis, which is often present at 1 week of age. Other animal models of PSS pulmonary disease are those used in many models of pulmonary fibrosis, such as bleomycin.

Management and Current Therapy Although the patterns of disease manifestations in PSS patients with diffuse versus limited disease can be useful in anticipating an individual’s course, many patients may be difficult to classify and any PSS patient can develop any of the pulmonary disease

PROGRESSIVE SYSTEMIC SCLEROSIS 497

Genetic predisposition: (polymorphisms of) Fibrillin-1 (FBN1) Secreted protein, acidic and rich in cysteine (SPARC) Topoisomerase-1 Transforming growth factor beta (TGF-
)* Tumor necrosis factor (TNF)* Interleukin 1A (IL-1A)* Interleukin 4 (IL-4)* HLA* Transcription of fibronectin and collagen*

B cells

Fibroblast proliferation

Microchimerism Molecular mimicry

Vascular injury: ? Endothelial cell proliferation ? Dysregulation of apoptosis ? von Willebrand's factor

Autoantibody formation

Environmental exposures: Retroviruses Cytomegalovirus (CMV) Organic solvents Vinyl chlorine Silica dust L-tryptophan Toxic oil Silicone Pesticides Drugs (e.g., appetite suppressants, cocaine, carbidopa)

Cytokine release ?Th1 vs. Th2

T cells ? Role of CD8 cells in lung ? Role of CD4 cells in skin

Figure 3 Proposed pathogenesis of systemic sclerosis. The pathogenesis of scleroderma is unknown. Any proposed pathway must explain both the development of fibrosis and the vascular disease, although in an individual patient the pulmonary involvement is usually either fibrotic or vascular disease. There are many proposed mechanisms for the development of autoantibodies, including genetic susceptibility, environmental exposures, molecular mimicry, and microchimerism. Although B cells play a role in antibody production, it remains unclear whether scleroderma is primarily a B- or T-lymphocyte-mediated disease. In either case, it appears that the cytokines that are released favor fibroblast proliferation. Cytokines may also have a role in endothelial cell proliferation that results in vascular injury or abnormal endothelial cell apoptosis. *Alterations in these genes may cause alterations in the resultant proteins and, therefore, lead to antibody production. However, alterations in these genes (or their expression) may also be more important in other steps of the proposed pathogenetic pathway, such as cytokine or T-cell function.

manifestations. Moreover, given the availability of efficacious treatment options for PSS lung disease, screening is appropriate. Therefore, the management of PSS patients should involve screening for pulmonary disease including HRCT, pulmonary function tests, and echocardiography. Serial screening with echocardiography, for example, has been shown to be useful to detect incident disease. In addition, an evaluation for esophageal dysmotility is important for the prevention of aspiration pneumonia. Treatment for Interstitial Pneumonia

PSS patients with interstitial pneumonia with demonstrated physiologic deficits, abnormal HRCT findings (ground-glass and reticulations), symptoms, and BAL evidence of alveolitis should receive therapy. There is only retrospective evidence that the combination of cyclophosphamide and prednisone can increase survival in these individuals. The results

of a multicenter randomized, placebo-controlled trial of this combination therapy are anticipated. Additional investigations are needed to determine the necessary duration of therapy, the efficacy of cyclophosphamide in comparison to other agents, and the potential benefit of combinations of therapeutic agents. Treatment for Pulmonary Hypertension

Treatment for pulmonary hypertension has been revolutionized with the advent of outpatient-based pulmonary vasodilators. Patients with PSS and pulmonary hypertension benefit from (parenteral) prostacyclin therapy as well as (oral) endothelin inhibitors in terms of their functional status. Patients now have alternative choices of treatment, including phosphodiesterase inhibitors such as sildenafil. Trials comparing these agents and/or using them in combination with cytotoxic agents remain to be performed.

498 PROTEASOMES AND UBIQUITIN See also: Alveolar Hemorrhage. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Pulmonary Effects of Systemic Disease. Vascular Disease. Vasculitis: Overview.

Further Reading Black CM and Du Bois R (1996) Organ involvement: pulmonary. In: Clements PJ and Furst DE (eds.) Scleroderma, pp. 299–331. Philadelphia: Williams & Wilkins. Bolster MB and Silver RM (1999) Assessment and management of scleroderma lung disease. Current Opinion in Rheumatology 11(6): 508–517. Bouros D, Wells AU, Nicholson AG, et al. (2002) Histopathologic subsets of fibrosing alveolitis in patients with systemic sclerosis and their relationship to outcome. American Journal of Respiratory and Critical Care Medicine 165: 1581–1586. Bryan C, Knight C, Black CM, and Silman AJ (1999) Prediction of five-year survival following presentation with scleroderma. Arthritis and Rheumatism 42(12): 2660–2665. D’Angelo WA, Fries JF, Masi AT, and Schulman LE (1969) Pathologic observations in systemic sclerosis (scleroderma): a study of fifty-eight autopsy cases and fifty-eight matched controls. American Journal of Medicine 46: 428–440.

Freemer MM and King TE (2003) Connective tissue diseases. In: Schwarz MI and King TE (eds.) Interstitial Lung Disease, pp. 553–562. New York: Decker. Kim EA, Lee KS, Johkoh T, et al. (2002) Interstitial lung diseases associated with collagen vascular diseases: radiologic and histopathologic findings. Radiographics 22: S151–S165. Latsi PI and Wells AU (2003) Evaluation and management of alveolitis and interstitial lung disease in scleroderma. Current Opinion in Rheumatology 15(6): 748–755. Steen V (2003) Predictors of end stage lung disease in systemic sclerosis. Annals of Rheumatic Disease 62: 97–99. Steen V and Medsger TA (2003) Predictors of isolated pulmonary hypertension in patients with systemic sclerosis and limited cutaneous involvement. Arthritis and Rheumatism 48(2): 516–522. Steen VD and Medsger TA (2000) Severe organ involvement in systemic sclerosis with diffuse scleroderma. Arthritis and Rheumatism 43(11): 2437–2444. Subcommittee for Systemic Sclerosis Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee (1980) Preliminary criteria for the classification of systemic sclerosis (systemic sclerosis). Arthritis and Rheumatism 23: 581–590. White B, Moore WC, Wigley FM, Xiao HQ, and Wise RA (2000) Cyclophosphamide is associated with pulmonary function and survival benefit in patients with scleroderma and alveolitis. Annals of Internal Medicine 132(12): 947–954.

PROTEASOMES AND UBIQUITIN D Attaix, Institut National de la Recherche Agronomique, Ceyrat, France & 2006 Elsevier Ltd. All rights reserved.

Abstract The ubiquitin–proteasome-dependent pathway degrades most cell proteins and is involved in the control of many major biological functions. The ubiquitination/deubiquitination system is a complex machinery responsible for the specific tagging and proofreading of substrates degraded by the 26S proteasome, but ubiquitination itself also serves other functions. The formation of a polyubiquitin degradation signal is usually required for proteasome-dependent proteolysis. Hierarchical families of enzymes, which may comprise hundreds of members to achieve high selectivity, control this process. Polyubiquitinated substrates are recognized by the 26S proteasome and degraded into peptides. The 26S proteasome also recognizes and degrades some nonubiquitinated proteins, and several proteasome populations participate in protein breakdown. In fact, mammalian cells contain multiple ubiquitin- and/or proteasome-dependent pathways. The role of these systems in respiratory diseases is still largely unknown. The proteasome is activated in mechanical ventilation-associated diaphragmatic atrophy and possibly in patients with chronic obstructive pulmonary diseases who exhibit muscle wasting. More importantly, the ubiquitin–proteasome system is responsible for the breakdown of the hypoxia-inducible factor-1a subunits, a transcription factor that may regulate the expression of up to 5% of the human genome.

Introduction A large amount of information on the characterization and regulation of proteolysis has been obtained over the last two decades. In particular, we now know that the ubiquitin–proteasome pathway is the major nonlysosomal process responsible for the breakdown of most short- and long-lived proteins in mammalian cells. In addition, the pathway controls various major biological events (transcriptional control, cell-cycle progression, oncogenesis, etc.) via the breakdown of specific protein substrates. There are two main steps in the pathway: (1) covalent attachment of a polyubiquitin chain to the substrate (i.e., polyubiquitination); and (2) specific recognition of this signal, and degradation of the tagged protein into peptides by the 26S proteasome (Figure 1).

Ubiquitination The 76 amino acid polypeptide ubiquitin was first characterized in 1980 as the critical component for the ATP-dependent proteolysis in reticulocytes. Ubiquitin prevails in all eukaryotes and is highly conserved among species. Ubiquitination is defined

PROTEASOMES AND UBIQUITIN 499

Free Ub

Ub

ATP

AMP

Ub

E1

DUBs

E2

Ub

E2-interacting domain

E3

PolyUb substrate

Substrate-binding site

Ub Ub Ub

Lid: PolyUb recognition Base: PolyUb recognition 19S and ATP hydrolysis

Ub

26S proteasome

20S

Peptidase activities

19S

Peptides Figure 1 Ubiquitin (Ub) is first activated by the ubiquitin-activating enzyme (E1) and transferred on one ubiquitin-conjugating enzyme (E2). The E2 with or without a ubiquitin–protein ligase (E3), which possesses a substrate-specific binding site, polyubiquitinates the target protein. The polyubiquitin degradation signal is then recognized by several subunits of the 19S complex of the 26S proteasome and recycled by the deubiquitinating enzymes (DUBs) into free ubiquitin, while the substrate is unfolded and injected into 20S proteolytic core of the 26S proteasome and cut into peptides.

as the covalent attachment of ubiquitin to a protein substrate. This is a multiple step process. In brief, ubiquitin is initially activated in the presence of ATP to a high-energy thiol ester intermediate by the ubiquitin-activating enzyme (E1) (Figure 1). E1 then transfers ubiquitin to one of the ubiquitinconjugating enzymes (E2s), which also forms a thiol ester linkage between the active site cysteine and

ubiquitin. E2s and/or ubiquitin-protein ligases (E3s), which play a role in the selection of proteins for conjugation, bind the first ubiquitin molecule to protein substrates via an isopeptide bond between the activated C-terminal glycine residue of ubiquitin and the e-amino group of a lysine residue of the substrate. The resulting monoubiquitinated protein is usually not targeted for degradation by the proteasome.

500 PROTEASOMES AND UBIQUITIN

Typical monoubiquitinated conjugates are receptors or basic proteins such as histones. Alternatively, E2s and/or E3s catalyze the formation of polyubiquitinated conjugates. This is usually achieved by transfer of additional activated ubiquitin moieties to Lys48 of the preceding conjugated ubiquitin molecule. The ubiquitin-conjugating system is hierarchical. In mammals, there is a single E1, at least 30–40 E2s, and several hundred E3s. Ubiquitination Machinery

The enzymes of the ubiquitin system are present in both the cytosol and nucleus. Only E1 and some E2s can form a polyubiquitin degradation signal, but usually this process also requires one E3. A given E2 interacts with a limited number of E3s (and vice versa), which in turn recognize their specific protein substrates (Figure 1). In addition, several substrates are known to be ubiquitinated by different combinations of E2s and E3s. This results in a wide range of ubiquitination pathways, which are specific for a given protein or a class of substrates. E3s are responsible for the selective recognition of protein substrates and therefore play a key role in the ubiquitin pathway. To date, all known E3s are described as HECT (homologous to E6-AP C-terminus) domain E3s, RING (really interesting new genes) finger E3s, and U-box-containing E3s. The first major group of E3s corresponds to enzymes of the HECT domain family. The N-terminus region of HECT E3s is responsible for substrate recognition, and the HECT domain itself in the C-terminus region mediates E2 binding and ubiquitination of the target protein via thiol ester linkage formation with ubiquitin. Mammalian genome sequencing projects have identified numerous potential uncharacterized HECT E3s. Most E3s are RING finger E3s. The RING finger structure is defined by eight cysteine and histidine residues that coordinate two zinc ions. There are several hundred cDNAs encoding RING finger proteins in the GenBank database. RING finger E3s are either monomeric proteins (i.e., the N-end rule enzyme E3a that binds to proteins bearing basic or bulky hydrophobic N-terminal amino acid residues) or multiple subunit complexes. These complexes form at least three distinct E3 families called the cyclosome or APC (anaphase-promoting complex), the SCF (Skp1-Cdc53-F-box protein family), and the VCB-like (Von Hippel–Lindau tumor suppressorElonginC/B) E3s. All these complexes contain a catalytic core and substrate-specific adapter proteins. For example, in the huge SCF E3 family the catalytic core is formed by three subunits that include the

RING finger subunit and an E2. The adapter protein Skp1 recruits F-box proteins, which themselves recruit specific protein substrates through protein– protein interaction domains such as leucine-rich repeats or WD-40 domains. Only a couple of U-box E3s have been characterized so far. The U-box domain has a three-dimensional structure close to that of the RING finger.

Signals that Target Substrates for Ubiquitination and Proteolysis Very importantly, ubiquitination is not only a degradation signal, but also directs proteins to a variety of fates, which include roles in ribosomal function, DNA repair, protein translocation, and modulation of structure or activity of the target proteins. For example, many monoubiquitinated proteins are targeted for endocytosis. By contrast, and in order to be efficiently degraded by the 26S proteasome, most substrates must be bound to a polyubiquitin degradation signal that comprises at least 4 (and up to 20–30) ubiquitin moieties. The formation of the polyubiquitin degradation signal is determined by short regions in the primary sequence of the targeted protein. This includes the nature of the N-terminal amino acid of the substrate (N-end rule pathway), phosphorylation and/or dephosphorylation events, and a very degenerate 9 amino acid motif called the ‘destruction box’, which is a crucial signal for the ubiquitination and breakdown of mitotic cyclins and other cell-cycle regulators. The 26S proteasome, however, is not an absolute ubiquitin-dependent proteolytic enzyme, as it also degrades a growing number of nonubiquitinated substrates. The precise mechanisms by which such nonubiquitinated substrates can be recognized by the 26S proteasome are unclear.

Deubiquitination Eukaryotic cells also contain DUBs (deubiquitinating enzymes), which are encoded by the UCH (ubiquitin C-terminal hydrolase) and the UBP (ubiquitin-specific processing protease) gene families. UCHs are relatively small proteins (o40 kDa) and only four isoforms have been characterized in the human genome. UCHs mainly hydrolyze small amides and esters at the C-terminus of ubiquitin. In contrast, UBPs are 50–250 kDa proteins and constitute a large family; there are at least 63 distinct human UBPs. UBPs are involved in several biological processes, including the control of growth, differentiation, and genome integrity. In proteasome-dependent proteolysis, the

PROTEASOMES AND UBIQUITIN 501

putative major roles of DUBs are to maintain free ubiquitin levels by processing the products of ubiquitin genes, which all encode fusion proteins, and to recycle polyubiquitin degradation signals into free monomers. DUBs also deubiquitinate substrates erroneously tagged for degradation (proofreading), and keep 26S proteasomes free of polyubiquitin chains that can interfere with the binding of another substrate.

Proteasomes The second major step in the ubiquitin–proteasome pathways is the degradation of polyubiquitinated proteins by the 26S proteasome, which is formed by the binding of two 19S regulatory complexes with the 20S proteasome (Figure 1). Proteasomes are particles responsible for the major neutral proteolytic activity in mammalian cells. They represent up to 1% of soluble proteins, but their abundance varies among cell types. Usually the proteasome concentration is proportional to the rate of tissue-protein turnover. Accordingly, proteasomes are rather abundant in lungs. Proteasomes are present in both the nucleus and the cytosol, but some particles are also associated with the endoplasmic reticulum and the cytoskeleton. Proteasomes were first discovered in liver cells and reticulocytes in 1979; since then, they have been identified by numerous groups in various tissues. The term ‘proteasome’ has only prevailed since 1988; prior to that, over 21 different names were used for this structure. 20S Proteasome

The mammalian 20S proteasome is a cylindrical particle composed of four stacked rings of subunits, with each ring containing seven different subunits (Figure 2). The outer rings are composed of a-subunits, and the two inner rings of b-subunits, which contain the catalytic sites inside the particle. Thus, the 20S proteasome is a self-compartmentalizing protease, as substrates must enter the catalytic chamber in order to be degraded into peptides. In eukaryotes, the 20S proteasome contains at least two chymotrypsin-like, two trypsin-like, and two caspase-like active sites, which are allosterically regulated. Proteasomes hydrolyze most peptide bonds and generate peptides that are typically 3–22 amino acids long and do not conserve biological properties, except for antigen presentation. Very importantly the 20S proteasome is the proteolytic core of a modular system in which peptidase activities can be modulated by the binding of regulatory complexes (see below). The binding of these

Figure 2 The structure of the mammalian 20S proteasome at 2.75 A˚ resolution. This lateral view shows the four rings of a external and b internal subunits.Reproduced from Unno M, Mizushima T, Morimoto Y, et al. (2002) MMDB: Entrez’s 3D-structure database. Structure (Cambridge) 10: 609–618.

complexes induces conformational changes in asubunits that open the gate separating the catalytic chamber of the 20S proteasome from the intracellular environment. Furthermore, there are immunoproteasomes in which three catalytic b subunits are replaced by three distinct b subunits, so that catalytic properties are altered to generate more efficiently antigenic peptides. Thus, there are different populations and subtypes of proteasomes in a given tissue that differ by their catalytic properties. The 19S Complex

The 19S complex (or PA700) stimulates both peptidase and proteolytic activities of the 20S proteasome. This complex contains about 18 different subunits, including at least two polyubiquitin chain receptors, and can be topologically defined by two subcomplexes called the base and the lid (Figure 1). In humans the base contains six ATPases and three non-ATPase subunits, while the lid only contains non-ATPase subunits. The ATPases provide energy for the assembly of the 26S proteasome, the recognition of the polyubiquitin degradation signal, the breakdown of ubiquitinated proteins into peptides, the gating of the proteasome channel, the unfolding of protein substrates and their injection into the catalytic chamber of the proteasome, and finally for peptide release. Other Proteasome Activators

The 11S regulator (or PA28ab) modulates only 20S proteasome peptidase activities. PA28 particles play a role in antigen presentation, by generating peptides

502 PROTEASOMES AND UBIQUITIN

for MHC class I molecules. Other proteasome activators called PA28g and PA200 play a role in growth control and DNA repair, respectively. Finally, there are hybrid proteasomes, in which one 11S regulator and one 19S complex bind simultaneously to a 20S proteasome. Such complexes are induced by interferon-g and play a role both in antigen presentation and in the breakdown of some proteins.

Regulation of Activity The activity of the ubiquitin system is first regulated by the equilibrium between ubiquitination and deubiquitination rates of protein substrates. Proteasome activities are controlled by a variety of subtle mechanisms that include the synthesis and processing of proteasome subunits, the assembly of 20S and 26S proteasomes, posttranslational modifications (e.g., phosphorylation of some 20S and 19S subunits), the binding of proteasome activators and inhibitors, the replacement of catalytic subunits, etc. These processes are influenced by complex signaling pathways, which may implicate mediators such as hormones and cytokines.

transduction, receptor downregulation, and the control of inflammation. In particular, recent studies have shown that hypoxia-inducible factor-1a (HIF-1a) subunits are normally degraded in a ubiquitin- and proteasome-dependent fashion in the presence of oxygen. In hypoxia, HIF-1a proteolysis is blocked and this transcription factor becomes a major player in a widespread oxygen-sensing and signal transduction mechanism. The HIF-1 activation cascade includes protein phosphorylation, nuclear translocation, DNA binding, aryl hydrocarbon receptor nuclear translocator (ARNT) dimerization, recruitment of general and tissue-specific transcriptional cofactors, and target gene transactivation. Indeed, it has been estimated that HIF-1 may regulate the expression of up to 5% of the human genome. Finally, the activation of the ubiquitin–proteasome system plays a key role in muscle wasting. Accordingly, the proteasome is activated in mechanical ventilation-associated diaphragmatic atrophy. Patients with chronic obstructive pulmonary diseases (COPDs) who exhibit muscle wasting may have similar adaptations.

Forthcoming Applications Biological Roles of the Ubiquitin–Proteasome System in the Respiratory Tract The role of the ubiquitin–proteasome pathways in the respiratory tract and respiratory diseases is still largely unknown. However, a number of functions can be assigned to the pathway based on the known roles of the system in other cells and tissues (Table 1). First, the pathway should be responsible for housekeeping functions (basal protein turnover, elimination of abnormal (miscoded, misfolded, oxidized) proteins, etc). Second, the system should play a critical immunological role by generating peptides for class I antigen presentation. Third, the system is involved in many key biological functions such as transcriptional control, cell-cycle progression, oncogenesis, development and differentiation, signal

Not surprisingly, there has been a growing number of studies on the ubiquitin–proteasome system in the respiratory tract and related topics since 1999. In addition, studies on HIF-1 are leading to surprisingly broad applications since drugs that target this system may lead to better and new therapies for cancer, heart attack, stroke, and inflammation. Furthermore, clinical trials in this area are beginning to emerge. For example, Bortezomib is a novel proteasome inhibitor produced by Millennium Pharmaceuticals (Cambridge, MA) that inhibits the growth of lung cancer cell lines in vitro and in vivo in athymic nude mouse xenografts. Bortezomib is currently in Phase II trials in lung cancer patients. The proteasome system also plays a vital role in controlling inflammation (e.g., via the breakdown of inhibitor kB)/ nuclear factor kB), which is key to many respiratory diseases (e.g., asthma and COPD). Thus, it has been

Table 1 Major roles of the ubiquitin–proteasome system in the respiratory tract Housekeeping functions

Immunological role

Regulatory functions

Production of energy

Basal protein turnover Elimination of abnormal proteins

Class I antigen presentation

Cell cycle Transcription Development and differentiation Oncogenesis Signal transduction Control of inflammation

Muscle wasting in COPD patients?

PROTEINASE INHIBITORS / Alpha-2 Antiplasmin 503

suggested that proteasome inhibitors could also be useful in the treatment of such diseases, but there are still no clinical trials in this area. See also: Cell Cycle and Cell-Cycle Checkpoints. Chronic Obstructive Pulmonary Disease: Overview. Gene Regulation. Hypoxia and Hypoxemia. Proteinase Inhibitors: Antichymotrypsin; Cystatins; Secretory Leukoprotease Inhibitor and Elafin. Transcription Factors: Overview. Tumors, Malignant: Overview.

Further Reading Attaix D and Briand Y (2003) The proteasome in the regulation of cell function. International Journal of Biochemistry and Cell Biology 35/5: 545–755. Attaix D, Combaret L, Kee AJ, and Taillandier D (2003) Mechanisms of ubiquitination and proteasome-dependent proteolysis in skeletal muscle. In: Zempleni J and Daniel H (eds.) Molecular Nutrition, pp. 219–235. Oxon: CAB International. Bunn PA Jr (2004) The potential role of proteasome inhibitors in the treatment of lung cancer. Clinical Cancer Research 10: 4263s–4265s.

Coux O, Tanaka K, and Goldberg AL (1996) Structure and functions of the 20S and 26S proteasomes. Annual Review of Biochemistry 65: 801–847. Glickman MH and Ciechanover A (2002) The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction. Physiological Reviews 82: 373–428. Hilt W and Wolf D (eds.) (2001) Proteasomes: The World of Regulatory Proteolysis. Austin: RG Landes & Co. Marx J (2004) Cell biology. How cells endure low oxygen. Science 303: 1454–1456. (This paper nicely summarizes recent findings on HIF-1 leading to possible new strategies for treating various diseases.) Peters JM, Harris JR, and Finley D (eds.) (1998) Ubiquitin and the Biology of the Cell, p. 472. New York: Plenum. Pickart CM (2001) Mechanisms underlying ubiquitination. Annual Review of Biochemistry 70: 503–533. Unno M, Mizushima T, Morimoto Y, et al. (2002) MMDB: Entrez’s 3D-structure database. Structure (Cambridge) 10: 609– 618. Voges D, Zwickl P, and Baumeister W (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annual Review of Biochemistry 68: 1015–1068. Zwickl P and Baumeister W (eds.) (2002) The Proteasome-Ubiquitin Protein Degradation Pathway, p. 213. Berlin: Springer.

PROTEINASE INHIBITORS Contents

Alpha-2 Antiplasmin Antichymotrypsin Cystatins Secretory Leukoprotease Inhibitor and Elafin

Alpha-2 Antiplasmin H R Lijnen, University of Leuven, Leuven, Belgium & 2006 Elsevier Ltd. All rights reserved.

Abstract Alpha-2 antiplasmin (AP), the main physiologic plasmin inhibitor in mammalian plasma, is a 70 kDa single chain serpin (serine proteinase inhibitor) with reactive site peptide bond Arg–Met. It inhibits plasmin very rapidly following formation of an inactive 1:1 stoichiometric complex. The high reaction rate requires the presence of a free active site and free lysine-binding sites(s) in plasmin. The pathophysiologic relevance of AP is evidenced by the finding that homozygous-deficient patients show a bleeding tendency; heterozygotes, in contrast, frequently have no or only mild bleeding complications. Homozygous AP-deficient mice have been generated which display normal fertility, viability, and development. Several in vivo studies confirmed that these mice have an enhanced endogenous fibrinolytic capacity without however showing overt bleeding. These findings suggest that the main role of AP is in regulating plasmin activity in the circulating blood and in controlling intravascular fibrinolysis.

In patients with respiratory diseases, including the adult respiratory distress syndrome, depressed fibrinolytic activity, partially as a result of enhanced AP levels, is a consistent finding, leading to disturbance of the hemostatic balance favouring fibrin deposition.

Introduction The fibrinolytic (plasminogen/plasmin) system contains a proenzyme, plasminogen, that is converted to the active enzyme plasmin, which degrades fibrin, by tissue-type (tPA) or urokinase-type (uPA) plasminogen activator. Inhibition of the fibrinolytic system may occur at the level of plasmin (by a2-antiplasmin, AP) or at the level of plasminogen activators (by plasminogen activator inhibitors) (Figure 1). For a long time, it was accepted that there were two functionally important plasmin inhibitors in human plasma: an immediately reacting and a slower reacting one identical to a2-macroglobulin and a1-antitrypsin, respectively. About 30 years ago, another

504 PROTEINASE INHIBITORS / Alpha-2 Antiplasmin Tissue-type plasminogen activator Urokinase-type plasminogen activator

Plasminogen activator inhibitor-1 Plasminogen activator inhibitor-2 Plasminogen

Plasmin
2-Antiplasmin Fibrin

Fibrin degradation products

Figure 1 Schematic representation of the fibrinolytic system.

A-chain of plasmin S

S 561 Arg–OH Plg. act.

791 HO–Asn

S

Lys–H 77

S

Val–H 562 B-chain of plasmin

LBS 741 Ser HO–Lys 464

Lys 448

Lys 441

Complementary LBS

Met – Arg Cys 377 376 Reactive site H-Met Asn Gln Cys 1 13 14

Cys
2-Antiplasmin Cys

Fibrin-binding site Figure 2 Schematic representation of the plasmin-a2-antiplasmin complex. LBS, high-affinity lysine-binding site in plasmin; Plg. act., site of cleavage in plasminogen for plasminogen activators.

plasmin inhibitor, AP or a2-plasmin inhibitor, was identified. On activation of plasminogen in plasma, this inhibitor preferentially binds plasmin. Complete activation of plasminogen (concentration about 2 mM), resulting in saturation of AP (concentration about 1 mM), must occur before excess plasmin is neutralized by the other inhibitors. AP, like many other plasma proteinase inhibitors, has a broad in vitro inhibitory spectrum, but its physiologic role as an inhibitor of proteinases other than plasmin seems negligible.

Structure Protein Structure

AP is a single-chain protein of 70 kDa containing about 13% carbohydrate (attached at Asn99, Asn268, Asn282, and Asn289). The molecule consists of 464 amino acids and contains one Cys43–Cys116 disulfide bond and possibly two unpaired cysteines (Cys76 and Cys125) (Figure 2). It belongs to the serine proteinase inhibitor family (serpins). The method of choice for the purification of AP consists of chromatography on

PROTEINASE INHIBITORS / Alpha-2 Antiplasmin 505

insolubilized plasminogen fragments that contain kringles 1–3 with a high-affinity lysine-binding site (LBS; see below). Two molecular forms are present in about equal amounts in purified preparations: a native 464-residue-long inhibitor with N-terminal methionine (Met1–AP) and a 12-amino acid shorter form with N-terminal asparagine (Asn13–AP). It is not known whether Asn13–AP is present in the circulating blood or whether it is generated in vitro. The N-terminal Gln14-residue of AP can cross-link to Lys303 in the Aa-chains of fibrin(ogen) in a process that requires Ca2 þ and that is catalyzed by activated coagulation factor XIII. Asn13–AP is more efficiently cross-linked to fibrin than Met1–AP. Mechanism of Interaction with Plasmin

The time course of the inhibition of plasmin by AP is compatible with a kinetic model composed of two successive reactions: a fast, reversible second-order reaction followed by a slower, irreversible first-order transition. The model can be represented by the following scheme: k1

k2

P þ AP $ PAP - PAP0 k
1

where P is plasmin, AP is a2-antiplasmin, PAP is a reversible inactive 1:1 stoichiometric complex, and PAP0 is an irreversible inactive complex. The second-order rate constant (k1 ¼ 2 to 4  107 M
1 s
1) of this inhibition is among the fastest protein–protein reactions described. The first step of the process depends on the presence of a free LBS and active site in the plasmin molecule and can be divided into two parts: a reaction between the LBS (mainly kringles 1–3) in the plasmin A chain and the plasmin(ogen)-binding site in the C-terminal part of AP (complimentary LBS), and subsequently a reaction between the active site Ser741 in the B-chain of plasmin with the reactive site peptide bond in AP (Arg376–Met377). A covalent bond is formed between the active site seryl residue in plasmin and the reactive site arginyl residue in the inhibitor (Figure 2). Plasmin and AP thus form a stoichiometric 1:1 complex of about 140 kDa. A nondisulfide-bonded peptide (8 kDa) is released concomitantly with complex formation. Upon disulfide bond reduction, the complex is dissociated in two parts: an intact plasmin A chain (60 kDa), and a stable complex between the plasmin B chain and cleaved AP (80 kDa). In plasma, the reaction rate between plasmin and AP may be reduced by the presence of proteins such as histidine-rich glycoprotein or fibrinogen, which interact with the LBS of plasmin(ogen). The half-life of plasmin molecules on the fibrin surface, which are

protected from inhibition by AP because their LBS and active site are occupied, is two to three orders of magnitude longer than that of free plasmin. Gene Structure

The gene for human AP, located on chromosome 17p13, is approximately 16 kb and contains 10 exons. The N-terminal region of the protein, comprising the fibrin cross-linking site is encoded by exon IV, whereas both the reactive site and the plasminogen-binding site in the C-terminal region are encoded by exon X. A ‘TATA box’ sequence is located 17 nucleotides upstream from the proposed transcription initiation site. The 50 -flanking region contains multiple ‘GC box’ and ‘CCAAT box’-like sequences.

Regulation of Production and Activity The concentration of AP in normal human plasma is about 7 mg dl
1 (about 1 mM); it is produced in the liver and kidney. Its in vivo half-life is 2.6 days, whereas the plasmin–AP complex disappears from plasma with a half-life of about half a day; both are cleared via the liver. The inhibitor in normal plasma is heterogeneous and consists of functionally active and inactive material. Complete activation of the plasminogen present in normal plasma converts only about 70% of the AP antigen into a complex with plasmin; 30% of the inhibitor is functionally inactive or slower reacting. These two forms of the inhibitor differ in their binding to plasminogen. The form that does not bind remains an active plasmin inhibitor but reacts much more slowly with plasmin; it lacks a 26-residue peptide from the C-terminal end that contains the plasminogen-binding site. The plasminogen-binding form of AP is primarily synthesized and becomes partly converted to the nonplasminogen-binding form in the circulating blood.

Biological Functions Phenotypic analysis of AP gene-deficient mice, generated by homologous recombination in embryonic stem cells, revealed a functional role for AP in several biological processes. In vivo studies revealed that these mice have an enhanced endogenous fibrinolytic capacity without overt bleeding. This is reflected by a higher spontaneous lysis rate of experimental pulmonary emboli, by a reduced fibrin deposition in the kidneys following challenge with endotoxin, by more limited photochemically induced arterial thrombosis, and by reduced infarct size following induction of focal cerebral ischemia by ligation of the left middle cerebral artery. In a vascular injury restenosis model,

506 PROTEINASE INHIBITORS / Alpha-2 Antiplasmin

AP deficiency has no significant effect on smooth muscle cell migration and neointima formation. The absence of a bleeding phenotype in these mice, in contrast to man, may reflect the fact that the coagulation system adequately prevents bleeding if the fibrinolytic system is not dramatically challenged. In man, rare cases of congenital AP deficiency and dysfunctional AP as well as acquired deficiencies have been reported. The first case of congenital homozygous AP deficiency was described in a patient who presented with a hemorrhagic diathesis. Several cases of heterozygosity have been described with no or only mild bleeding symptoms. The AP levels in all heterozygotes described thus far are consistently between 40% and 60% of normal. Antigen and activity levels usually correspond well, suggesting that the deficiency is due to decreased synthesis of a normal AP molecule. The bleeding tendency in these patients may be due to premature lysis of hemostatic plugs, because in the absence of AP, the half-life of plasmin molecules generated on the fibrin surface is considerably prolonged. Different molecular defects have been identified in AP, such as a trinucleotide deletion in exon VII leading to deletion of Glu137 (AP Okinawa), or an insertion of a cytidine nucleotide in exon X leading to a shift in the reading frame of the mRNA resulting in deletion of the C-terminal 12 amino acids of native AP and replacement with 178 unrelated amino acids (AP Nara). These mutations may lead to the deficiency by affecting the folding of the protein into the native configuration and thereby blocking its intracellular transport from the endoplasmatic reticulum to the Golgi complex. Plasma levels of AP in a normal population range between 80% and 120% of that in pooled human reference plasma. The level is significantly decreased in liver cirrhosis and in several other liver diseases, which may be an important factor in the increased fibrinolytic activity observed in liver cirrhosis. Decreased levels of AP have also been observed in patients with disseminated intravascular coagulation or with some forms of renal disease. AP levels may be significantly reduced in patients undergoing thrombolytic therapy as a result of systemic activation of the fibrinolytic system, in particular associated with the use of non-fibrin-specific thrombolytic agents. An abnormal AP (AP Enschede) associated with a serious bleeding tendency was found in two siblings in a Dutch family. These individuals had 3% of normal functional activity and 100% of normal antigen levels. The ability of the abnormal AP to reversibly bind plasmin or plasminogen is not affected, but it is converted from an inhibitor of plasmin to a substrate.

The molecular defect consists of the insertion of an extra alanine residue (GCG insertion) 7 to 10 positions on the N-terminal side of the P1 residue (Arg376).

a2-Antiplasmin in Respiratory Diseases Studies in the 1970s and 1980s indicated that microemboli in the pulmonary circulation sometimes persist longer than normal, resulting in the delayed microembolism syndrome with characteristic pathophysiological changes in the lungs. This delayed dissolution of microemboli may be caused by inhibition of the fibrinolytic system, mediated by AP. This is confirmed by a later finding that injection of AP into rats with intravascular coagulation in the lungs delayed the elimination of fibrin. Intra-alveolar fibrin deposition (hyaline membrane formation) is a hallmark of many acute inflammatory lung diseases. This may be beneficial in the gas exchange area by sealing leakage sites (e.g., at sites of denuded alveolar epithelium) and by providing a matrix for wound repair. However, it may also be harmful if abundant and persistent, by compromising endothelial monolayer integrity. In addition, surfactant components may be incorporated into polymerizing fibrin with loss of surface activity and alveolar instability, as well as altered mechanical properties and reduced fibrinolytic capacity. Besides favoring alveolar collapse and shunt-flow these events may play a pathogenetic role in the rapid onset of lung fibrosis in acute and widespread inflammatory lung injury. Abundant deposition of bronchoalveolar fibrin and fibronectin occurs during the exudative phase of the adult respiratory distress syndrome (ARDS), promoting hyaline membrane formation and alveolar fibrosis. Several studies in ARDS patients have reported depressed fibrinolytic activities due to decreased uPA activity and enhanced PAI-1 and AP levels in the bronchoalveolar compartments. In addition, procoagulant activity is enhanced. Pronounced disturbances of the alveolar hemostatic balance also occur in patients with severe pneumonia, comparable to those seen in ARDS triggered by nonpulmonary underlying events. A marked increase in procoagulant activity (mainly through the tissue factor pathway) is accompanied by decreased fibrinolytic activity in the alveolar lining layer with decreased uPA (patients with pneumonia) and increased PAI-1 and AP levels (patients with pneumonia receiving respiratory therapy). This shift in hemostatic balance, favoring fibrin formation, is a consistent feature of inflammatory lung injury, whether triggered by nonpulmonary systemic events or by primary lung infection.

PROTEINASE INHIBITORS / Antichymotrypsin

It has not been investigated whether reduction of AP levels may represent a treatment modality for patients with respiratory diseases. To our knowledge no pharmacologic agents have been described that allow specific inhibition of AP in vivo. The most rational approach to reduce AP activity would be to infuse plasmin moieties. However, in view of the high plasma concentration of AP, this would require large amounts of plasmin, a protease with broad substrate specificity. Whereas high-dose plasmin activity may be hazardous in itself, its use would also require stringent monitoring of AP activity levels, since excess plasmin may compromise the hemostatic system by degrading several plasma proteins. Therefore, it seems unlikely that this will represent a potential treatment option. See also: Acute Respiratory Distress Syndrome. Fibrinolysis: Overview; Plasminogen Activator and Plasmin.

Further Reading Aoki N (1990) Molecular genetics of alpha 2 plasmin inhibitor. Advances in Experimental Medicine and Biology 281: 195–200. Aoki N, Sumi Y, Miura O, and Hirosawa S (1993) Human alpha 2-plasmin inhibitor. Methods in Enzymology 223: 185–197. Bertozzi P, Astedt B, Zenzius L, et al. (1990) Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. New England Journal of Medicine 322: 890–897. Favier R, Aoki N, and de Moerloose P (2001) Congenital alpha(2)plasmin inhibitor deficiencies: a review. British Journal of Haematology 114: 4–10. Gu¨nther A, Mosavi P, Heinemann S, et al. (2000) Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 161: 454–462. Hirosawa S, Nakamura Y, Miura O, et al. (1988) Organization of the human alpha2-plasmin inhibitor gene. Proceedings of the National Academy of Sciences USA 85: 6836–6840. Holmes WE, Nelles L, Lijnen HR, and Collen D (1987) Primary structure of human alpha2-antiplasmin, a serine protease inhibitor (serpin). Journal of Biological Chemistry 262: 1659–1664. Idell S, Koenig KB, Fair DS, et al. (1991) Serial abnormalities of fibrin turnover in evolving adult respiratory distress syndrome. American Journal of Physiology 261: L240–L248. Koie K, Ogata K, Kamiya T, et al. (1978) Alpha2-plasmin-inhibitor deficiency (Miyasato disease). Lancet 2: 1334–1336. Lijnen HR and Collen D (1989) Congenital and acquired deficiencies of components of the fibrinolytic system and their relation to bleeding or thrombosis. Fibrinolysis 3: 67–77. Lijnen HR and Collen D (1995) Mechanisms of physiological fibrinolysis. Baillie`re’s Clinical Haematology 8: 277–290. Lijnen HR, Okada K, Matsuo O, et al. (1999) Alpha2-antiplasmin gene deficiency is associated with enhanced fibrinolytic potential without overt bleeding. Blood 93: 2274–2281. Wiman B and Collen D (1977) Purification and characterization of human antiplasmin, the fast-acting plasmin inhibitor in plasma. European Journal of Biochemistry 8: 19–26.

507

Wiman B and Collen D (1978) Molecular mechanism of physiological fibrinolysis. Nature 272: 549–550. Wiman B and Collen D (1979) On the mechanism of the reaction between human alpha2-antiplasmin and plasmin. Journal of Biological Chemistry 254: 9291–9297.

Antichymotrypsin N Kalsheker, K Morgan, and S Chappell, University of Nottingham, Nottingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Alpha-1-antichymotrypsin, also referred to as SERPIN A3, is a major plasma serine proteinase inhibitor. Its main physiological target is neutrophil cathepsin G, an enzyme that plays a key role in the killing of bacteria by neutrophils. The protein also plays a key role as a molecular chaperone for Ab peptides, derived from amyloid precursor protein in the brain and is a constituent of the senile plaques found in the brains of patients with Alzheimer’s disease. The protein behaves as an acute-phase reactant and plasma concentrations can increase about fourfold during inflammation. Genetic deficiency of alpha-1-antichymotrypsin has been reported and may result in altered lung function. The protein provides a natural defense against uninhibited proteolytic enzymes and this may be particularly important in the lung, an organ that is vulnerable to proteolytic damage. Alpha-1-antichymotrypsin also has an important role in controlling the inflammatory process in the lung where several serine proteinases are known to be both proinflammatory and can disturb the structural integrity of the lung.

Introduction Alpha-1-antichymotrypsin (ACT) is a plasma serine proteinase inhibitor (also referred to as SERPIN A3). It is a glycoprotein and its main target is chymotrypsin-like serine proteinases. It is particularly effective against the neutrophil enzyme cathepsin G, and its physiological role is to control the activity of this proteinase.

Protein Structure and Biological Function ACT has a molecular mass of between 55 and 66 kDa. The three-dimensional structure is in keeping with that of other serpins. It consists of three beta-sheets and nine alpha-helices. The interaction with target protease occurs at the reactive center loop (RCL) at the C-terminus. The RCL acts as bait for a target proteinase. The proteinase docks onto the RCL, which corresponds to residues 342 to 367, denoted P17-P90 , following the nomenclature of Schechter and Berger, and cleavage of the leucineserine bond, located at position 358–359, results in a

508 PROTEINASE INHIBITORS / Antichymotrypsin

major conformational change. The loop inserts into a space in the A-sheet as a strand, and the proteinase then moves to the opposite pole of the molecule. During this translocation process, the catalytic site of the serine proteinase is distorted so that it is rendered inactive. The inhibitor-proteinase complex is then rapidly removed from the circulation by a receptor system that belongs to the low-density lipoprotein receptor family and the tightest binding has been shown for low-density lipoprotein receptor associated protein 2 (Figure 1). A number of other proteinases are inhibited by ACT, including mast cell chymases and lymphocyte cell surface proteins; ACT has been shown to modulate lymphocyte activity by loss of both killing and antibody-dependent cytolytic activity. Cathepsin G, the main target of ACT, has been shown to be involved in the conversion of angiotensin I to angiotensin II, although the main role of the enzyme is related to bacterial killing and tissue remodeling. Another unusual property of ACT is its ability to bind DNA. Three basic lysine residues at positions 212–214 are critical for this and probably relate to the electronic charge provided by basic amino acid residues. The RCL is susceptible to proteolysis by a variety of microbial and mammalian proteinases. Most of these enzymes cause rapid inactivation of ACT as an inhibitor by cleavage of single sites, often between P5 lysine and P0 leucine. After cleavage at these sites, the molecule becomes a potent chemoattractant. Furthermore, the cleaved ACT has been shown to have a slow clearance underscoring its role in chemotaxis. A key function of neutrophils is to provide a defense against bacterial infection by a variety of mechanisms including superoxide generation and release of powerful proteinases. It has also been shown that ACT inhibits neutrophil superoxide generation and may play an important part in modulating neutrophil function. Also, cathepsin G is particularly effective against Gram-negative bacteria and it also stimulates airway gland secretion. In 1988, Abraham and coworkers reported that ACT was a significant component of the senile plaques associated with Alzheimer’s disease. The protein binds Ab1-42 peptide, comparable in specificity and stability to a protease–proteinase inhibitor interaction. The peptide is derived from amyloid precursor protein and ACT acts as a molecular chaperone that can stimulate amyloid fibril formation. The ACT-Ab complex is also probably cleared by the same receptor system that handles the inhibitor–protease complex described above. In 2002, Parmar and colleagues suggested that polymers have chemotactic activity. Thus, the ability of serpins to polymerize may have important implications for respiratory disease.

Figure 1 Model of the covalent protease. The protease is shown in gray and ACT is shown in yellow. Initial docking of the protease is followed by partial insertion of the protease and the P1–P10 bond remains intact. This is followed by an acylenzyme complex and full insertion of the protease. This is accompanied by movement of the protease to the opposite pole of ACT and distortion of the active site of the protease. Reproduced from O’Malley KM, Barry S, and Cooperman BS (2001) Formation of the covalent chymotrypsin–antichymotrypsin complex involves no large-scale movement of the enzyme. Journal of Biological Chemistry 276: 6631–6637, with permission from The American Society for Biochemistry & Molecular Biology.

The ACT Gene and Regulation The ACT gene is located on chromosome 14q32.1, is approximately 12 000 bp in length, and is organized into five exons and four introns. It is about 250 kb away from the alpha-1-antitrypsin gene and is in a cluster of genes that have arisen by gene duplication. The major site of synthesis is the liver, but other tissues also express the protein, including the heart, lung, kidney, brain, and prostate. In the lung, both

PROTEINASE INHIBITORS / Antichymotrypsin 57 bp

Exon size Intron size

649 bp 2.2 kb

I

509

268 bp 151 bp 442 bp 3.4 kb 1.6 kb 0.7 kb

II

III

IV

V

TSS (+1)

−125 STAT A

−117 −95

−87 −29 STAT B

−23 Promoter

Proximal enhancer region

13213

−13202

NF-
B

−12985

−12979

AP-1

−12831 NF-
B

Distal enhancer region Figure 2 Diagrammatic representation of the ACT gene structure. Exons are shown as boxes. The transcription start size (TSS) is designated þ 1. Regulatory regions are numbered relative to the TSS (not to scale). The elements marked STAT A and STAT B are on the sites at which oncostatin-M exerts its effects. The transcription factors that bind to the distal enhancer are shown.

alveolar macrophages and airway epithelia have been shown to synthesize ACT. The normal reference range for plasma concentrations of ACT is between 0.3 and 0.6 g l
1. During inflammation the concentrations can increase up to fourfold in about 8 h. This increase is mediated by cytokines such as interleukin-6 (IL-6) and oncostatin-M (OSM). The regions of the gene that contribute to expression are highlighted in Figure 2. In lung epithelial cells, OSM, IL-1, and dexamethasone have been shown to stimulate ACT production, both individually and, more dramatically, in combination between about three- to sixfold, depending on the cell type. OSM is the most potent stimulus in bronchial epithelial cells and induces expression by activating pathways that involve signal transducer and activator of transcription (STAT) elements, located at
125 to
127 bp and
96 to
87 bp away from the transcription initiation site identified by Kordula et al. in 1998. An additional element, located in a 413 bp long fragment, approximately 13 kb upstream from the transcription initiation site, has been shown to be involved in the IL-1 and tumor necrosis factor (TNF) alpha response. Located within this fragment are three transcription factor-binding sites, two nuclear factor kappa B (NF-kB) sites at
13 213 to
13 202 bp

and
12 831 to
12 820 bp, and an activating protein A (AP-1) site at
12 985 to
12 979 bp. In the liver cell line, Hep G2, IL-1, and TNF-a had a minimal effect, suggesting that there may be significant tissue-specific differences in response to cytokines.

Association with Disease The most important risk factor for the development of chronic obstructive pulmonary disease (COPD) is tobacco smoking. However, only 10–20% of heavy smokers develop symptomatic COPD. Based on the observations of alpha-1-antitrypsin deficiency, and its association with COPD, this has led to the protease–antiprotease imbalance theory of the causation of disease where inhibited proteinase contributes to lung damage. In 1990, Lindmark et al. described in a Swedish population heterozygotes for partial ACT deficiency occurring at a frequency of about 1 in 200–300 of the population. Some of these individuals had evidence of high residual volumes and total lung capacity, though these did not appear to give rise to serious pulmonary disease. This is analogous to the situation occurring in heterozygotes for alpha-1-antitrypsin deficiency. In 1993, Poller et al. reported an association of leucine 55-to-proline substitution, and a proline 229-to-alanine substitution in ACT with

510 PROTEINASE INHIBITORS / Antichymotrypsin

Proinflammatory proteinases

Anti-inflammatory inhibitors

Neutrophil cathepsin G

Alpha-1-antichymotrypsin

Chymase (activates interleukin-1)

Alpha-1-antichymotrypsin

Neutrophil elastase

Alpha-1-antitrypsin

Neutrophil proteinase 3 (activates interleukin-1 and tumor necrosis factor)

Modified inhibitors (chemotaxis) Recruitment of neutrophils

Figure 3 Balance of inflammation in the lung.

COPD. This association has been challenged. Other polymorphisms have been reported, including a polymorphism at
15 of the ACT signal peptide, where either alanine or threonine occurs at this residue. This polymorphism is in linkage disequilibrium with a promoter polymorphism that has been shown to exert a modest functional effect in the liver and in astrocytes. However, there are marked cell-specific differences in the expression of the promoter polymorphism, certainly in neuronal tissue, suggesting that expression can vary markedly, depending on the tissues being studied. One small study by Ishii et al. in 2000 reported that homozygotes for the alanine in the signal sequence have an odds ratio of 2.7 (1.2– 6.2) for COPD. This study should be interpreted with caution as only a small sample size was used, and was probably underpowered to detect significant differences. To assess the role of ACT in COPD, larger sample sizes are required and a more thorough analysis of polymorphic variants of the gene needs to be undertaken.

This may influence the half-life of the protein. Serine proteinases such as cathepsin G have broad specificity, and are capable of degrading structural elements such as elastin and collagen type III and IV under physiological conditions. They play a key role in tissue remodeling in addition to their role in bacterial killing. In addition, such enzymes have the potential to generate proinflammatory signals. Therefore, inhibition proteolytic activity is essential for both maintenance of structural integrity and control of inflammation. This is particularly important in the lung, an organ that is susceptible to the harmful effects of proteolysis (Figure 3).

Biological Role of ACT in the Lung

See also: Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-6. Oncostatin M. Transcription Factors: NF-kB and Ikb.

ACT is synthesized locally in the lung and both alveolar macrophages and human lung epithelial cells have been shown to produce it. It has been suggested that ACT in lung secretions is not effective as an inhibitor of chymotrypsin-like enzymes as it only retains about 15% of its inhibitory activity. This may reflect cleaved or alternative forms of the molecule in which the RCL has been lost. It is likely that local production controls the activity of target proteinases in the lung and probably contributes to a local anti-inflammatory effect. It has been suggested that there is a modified pattern of glycosylation of ACT produced in bronchial epithelial cells compared with that produced by the liver and the unglycosylated form is identical to that produced in the liver.

Conclusion ACT is an important modulator of proteinase activity and more is being learnt about its role at specific sites – from its role as a molecular chaperone for Ab peptides in the brain to its anti-inflammatory role in the lung. These insights should identify pathological mechanisms for disease causation.

Further Reading Abraham CR, Selkoe DJ, and Potter H (1988) Immunochemical identification of the serine protease inhibitor alpha-1-antichymotrypsin in the brain amyloid deposits of Alzheimer’s disease. Cell 52: 487–501. Baumann H and Gauldie J (1994) The acute phase response. Immunology Today 15: 74–80. Berman G, Afford SC, Burnett D, and Stockley RA (1986) Alpha1-antichymotrypsin in lung secretions is not an effective proteinase inhibitor. Journal of Biological Chemistry 261: 14095– 14099. Cichy J, Potempa J, Chawla RK, and Travis J (1995) Regulation of a-1-antichymotrypsin synthesis in cells of epithelial origin. FEBS Letters 359: 262–266.

PROTEINASE INHIBITORS / Cystatins 511 Eriksson S, Janclauskene S, and Lannfelt L (1995) Alpha-1-antichymotrypsin regulates Alzheimer b-amyloid peptide fibril formation. Proceedings of the National Academy of Sciences, USA 92: 2313–2317. Hudig D, Haverty T, Fulcher C, and Redelman J (1981) Inhibition of human natural cytotoxicity by macromolecular antiproteases. Journal of Immunology 126: 1569–1574. Ishii T, Matsuse T, Teramoto S, et al. (2000) Association between alpha-1-antichymotrypsin polymorphism and chronic obstructive pulmonary disease. European Journal of Clinical Investigation 30: 543–548. Kalsheker NA (1996) Alpha-1-antichymotrypsin. International Journal of Biochemistry and Cell Biology 28: 961–964. Kordula T, Rydel RE, Brigham EF, et al. (1998) Oncostatin-M and the interleukin-6 and soluble interleukin-6 receptor complex regulates alpha-antichymotrypsin express in human cortical astrocytes. Journal of Biological Chemistry 273: 4112–4118. Lindmark BE, Arborelius M, and Eriksson S (1990) Pulmonary function in middle-aged women with heterozygous deficiency of the serine protease inhibitor a-1-antichymotrypsin. American Review of Respiratory Diseases 141: 884–888. O’Malley KM, Barry S, and Cooperman BS (2001) Formation of the covalent chymotrypsin–antichymotrypsin complex involves no large-scale movement of the enzyme. Journal of Biological Chemistry 276: 6631–6637. Parmar JS, Mahadeva R, Reed BJ, et al. (2002) Polymers of alpha1-antichymotrypsin are chemotactic for human neutrophils – a new paradigm for the pathogenesis of emphysema. American Journal of Respiratory Cell and Molecular Biology 26: 723–730. Poller W, Faber J-P, Weidinger S, et al. (1993) A leucine to proline substitution causes a defective a-1-antichymotrypsin allele associated with familial obstructive lung disease. Genomics 17: 740–743. Poller W, Willnow TE, Hilpert J, and Herz J (1995) Differential recognition of a-1-antichymotrypsin–Cathepsin G complexes by the low density lipoprotein receptor-related protein. Journal of Biological Chemistry 270: 2841–2845. Reeves EP, Lu H, Jacobs HL, et al. (2002) Killing activity in neutrophils is mediated through activation of proteases by kt flux. Nature 416: 291–297. Rollini P and Fournier K (1997) A 370 kb cosmid configuration of the serpin gene cluster on human chromosome 14q32.1: molecular linkage of the genes encoding alpha-1-antichymotrypsin, protein C inhibitor, kallistatin, alpha-1-antitrypsin and corticosteroid binding globulin. Genomics 46: 409–415.

Cystatins P A Pemberton, Arriva Pharmaceuticals, Inc., Alameda, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The cystatins comprise a superfamily of proteins related primarily by virtue of DNA and amino acid sequence homology. The superfamily consists so far of four distinct types of molecules ranging from the simpler low-molecular-weight type I and II cystatins, which function primarily to inhibit lysosomal cysteine proteinases (CPs), to the higher-molecular-weight type III and IV cystatins, which possess additional latent functions expressed only during episodes of injury and inflammation, or have evolved entirely novel inhibitory functions.

The role cystatins play in respiratory diseases such as asthma and COPD is poorly understood. However, they do modulate the immune response by acting directly on neutrophils, macrophages, and antigen presenting cells. It is also clear that they do not function independently of other proteolytic pathways involved in remodeling of the lung. Limited proteolysis inactivates cystatins allowing lysosomal CP activity to directly contribute to lung tissue degradation and also liberates kinins which signal through G-protein-coupled receptors to cause both constriction and dilation of the bronchioles, pain via stimulation of sensory nerves, mucus secretion, cough, and edema.

Introduction The human cystatins comprise a superfamily of potent protein-based inhibitors of cysteine proteinases (CPs). It is one of the many protein proteinase inhibitor superfamilies that are involved in regulating mammalian homeostasis, including the serpins (e.g., a1-antitrypsin; AAT) and low-molecular-weight kunitz (e.g., tissue factor pathway inhibitor; TFPI), and kazal-type (e.g., pancreatic secretory trypsin inhibitory; PSTI) inhibitors. Current knowledge of their function suggests that they primarily serve to inhibit the activity of lysosomal proteinases that may be released during normal or pathological cellular or tissue remodeling events. The first cystatin described was isolated from chicken egg white in 1963 and found to exhibit potent inhibitory properties against the CPs papain and ficin. The name ‘cystatin’ was proposed by Barrett et al. in 1963 and later used to describe homologous proteins in the same superfamily. The first full sequence of a human cystatin was that of cystatin C. There are more than a dozen human cystatins all with different properties, unique distribution patterns, and functions. These have been grouped into four main cystatin types on the basis of DNA and protein sequence homology and over the last few years the superfamily has expanded to include additional CP inhibitors, molecules that have no CP inhibitory activity, and yet others that have evolved functions unrelated to CP inhibition.

The Cystatin Superfamily Type I Cystatins

Type I cystatins are intracellular and present in the cytosol of many different cell types. They are typically 100 amino acids long and lack disulfide bonds. There are two human cystatins called ‘stefins’ A and B to stress their difference from other cystatin superfamily members, but they do contain a general structure similar to the ‘cystatin-fold’ of other cystatins and similar CP inhibitory activity. In evolutionary

512 PROTEINASE INHIBITORS / Cystatins

terms, stefins A and B are closely related and form a distinct subgroup. Type II Cystatins

Type II cystatins are typically 120–125 residues long and contain two disulfide bonds. They are translated with a secretory peptide leader sequence and are considered extracellular but can also be found intracellularly. They are broadly distributed and can be found in most body fluids. Mammalian type II cystatins all present two disulfide bridges at the C-terminal end of the sequence with 10–20 residues between the cysteines. Significant diversity in the type II superfamily members arises from the existence of multigene families encoding many different proteins (Table 1) and by polymorphisms affecting the coding sequence and function of the protein. Several diseases are associated with functional deficiencies or aggregation states of certain type II cystatins. The ‘classical’ type II cystatins C, D, S, SA, and SN are 450% identical at the protein sequence level. In addition, several posttranslational modifications are found in the members of this family. They may also be glycosylated or phosphorylated. Examples of these are cystatins E/M (glycosylated on N108) and cystatins S and SN (consensus phosphorylation sites at S2 and S98, respectively). Cystatin S has been isolated from nasal and bronchoalveolar (BAL) fluids with varying states of phosphorylation, but the significance of this is currently unknown.

that possesses both direct and indirect actions in the airways including bronchoconstriction and bronchodilation, stimulation of cholinergic and sensory nerves, increased mucus secretion, and cough and edema resulting from promotion of microvascular leakage. Its activities are mediated primarily via the B1 and B2 G-protein-coupled receptors. Type IV Cystatins

The type IV cystatins are a small family of abundant fetal and bone glycoproteins known as the fetuins. The two related human members of this family are a2 Heremans Schmid glycoprotein (a2-HS glycoprotein) and histidine-rich glycoprotein (HRG). The fetuins are N- and O-glycosylated and phoshorylated. The N-terminal region consists of two tandem type II cystatin domains followed by a C-terminal region comprised of a histidine-rich domain between two proline-rich domains. The N- and C-terminal regions are linked by a disulfide bond. a2-HS has a structure similar to HRG except that it lacks the histidine-rich tandem repeat. Unlike the cystatin domains in the kininogens, the fetuins are devoid of CP inhibitor activity and consistent with this, they lack the conserved structural motifs responsible for CP inhibition. Surprisingly, orthologs have been found in snake venom that lack CP inhibitory activity but possess metalloproteinase inhibitory functions.

Structure: Function of Cystatins Type III Cystatins

These are multidomain proteins first described as kinin precursor proteins or kininogens. There are two types of human kininogens: high- and low-molecular-weight kininogens. These proteins are of high-molecular mass (60–120 kDa) and present three tandemly repeated type-2 like cystatin domains (D1, D2, and D3) with a total of eight disulfide bridges (six conserved and two additional at the beginning of cystatin domains D2 and D3). The D2 and D3 domains possess CP inhibitory activity similar to the type II cystatins. They are glycosylated proteins but the glycosylation sites are not present in the cystatin domains. Kininogens are expressed intravascularly and are found in blood plasma. However, in addition to their function as CP inhibitors, the kininogens serve as substrates for a diverse group of serine proteinases collectively termed the kininogenases. The kininogenases liberate the kinin family of inflammatory peptides from the parent molecules. The kinins consist primarily of bradykinin (BK) and kallidin (lysyl bradykinin). BK is a nine amino acid peptide released during inflammation and tissue injury

The alignment of cystatin sequences has identified three functional regions that have been conserved for more than a billion years of evolution and are responsible for the CP inhibitory activity of the superfamily: a glycine (G) residue in the N-terminal region of the molecule, a glutamine (Q) – X – valine (V) – X – glycine (G) motif (QXVXG: the ‘cystatin motif’) in one hairpin loop (see later), and a proline (P) – tryptophan (W) motif in a second hairpin loop. These regions form a surface on the cystatin molecule that can dock and bind into the enzymatically active site(s) of papain-like CPs. The classic example of a type II cystatin is cystatin C, for which the X-ray crystallographic structure of the chicken protein has been solved (Figure 1). The main feature of the structure is a five-stranded b-sheet wrapped around a five turn a-helix commonly referred to as the ‘cystatin fold’. The N-terminal 10 residues are disordered and flexible and the conserved G11 residue is present at the N-terminus of the five turn a-helix. The QXVXG motif is found on a hairpin loop located between bstrands B and C and the PW motif on a hairpin loop between b-strands D and E. Collectively, these three

Table 1 Cystatin genes, selected family members, and functions Cystatin type

Members

Common name(s)

Chromosomal Location/ (gene name)

Location

Function

Disease association

I

A

3cen q21

Intracellular, skin, blood

B

Stefin A, (epidermal SH-protease inhibitor) Stefin B, (CPI-B)

21

Intracellular, broad tissue distribution

Cysteine proteinase inhibitor (CPI) CPI

Progressive myoclonus epilepsy

C

Post-g-globulin

20p11.21 (CST3)

Widespread tissue/body fluids Saliva, tears Epidermal keratinocytes, sweat glands Intra- and extra-cellular, hematopoietic cells Saliva, tears, urine

II

D E/M F

20p11.21 (CST5 ) 11q13 (CST6) Leukocystatin

S SN SA III

IV

20p11.21 (CST7 ) 20p11.21 (CST4 ) 20p11.21 (CST1) 20p11.21 (CST2)

L-kininogen

a2-CPI

H-kininogen

a1-CPI

Fetuins

a2-HS glycoprotein HRG

3q26-qter

Blood, body fluids

3q27

Fetal, bone

CPI CPI CPI

Hereditary cystatin C amyloid angiopathy (HCCAA) Type 2 harlequin icthyosis Inflammatory lung disorders

CPI, regulators of vascular permeability, bronchoconstriction

Inflammatory lung disorders

514 PROTEINASE INHIBITORS / Cystatins

are some of the most potent naturally occurring mammalian inflammatory agents. All the three functions are critical for the maintenance of respiratory health and, in the settings of respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD), all the three contribute to the acute and chronic nature of these diseases. However, with the exception of the proinflammatory functions of the type III cystatins (kininogens), the contribution of cystatin superfamily members to pulmonary health and disease is poorly understood. Cystatins: Inhibitory Functions and Interaction with Other Proteolytic Systems

Figure 1 Three-dimensional structure of chicken cystatin C. A five-turn a-helix (green) is in the center of the structure. The conserved G11 residue is highlighted in yellow at the aminoteminal end of the a-helix. The conserved QXVXG motif is on a hairpin loop between b-sheet strands B and C. The conserved PW motif is on a hairpin loop between b-sheet strands D and E.

regions form a wedge-shaped edge complementary to the active site of the CP with many side-chain interactions. Each domain interacts with the target proteinase independently and binding of cystatins to CPs may or may not result in conformational changes in either protein. For example, cystatin binding to papain does not induce any conformational change in either protein whereas binding to cathepsin B involves the initial displacement of a loop occluding the active site to allow subsequent tight binding to occur. Cystatins differ considerably in their ability to displace the loop of cathepsin B. In most cases, the affinity of the cystatins for their respective target CPs is very high with inhibitory constants (Ki’s) in the nanomolar (nM) range but in several instances, where values may be in the micromolar (mM) range, effective inhibition may result from high local concentrations of the cystatins. Examples of this are the S-like cystatins which exist in high concentrations in saliva and tears.

Cystatins and Respiratory Health Cystatins serve at least three functions in respiratory health and disease: they can directly inhibit endogenous or exogenous CPs; they can modulate the activity of the immune system against inhaled bacteria and viruses and their degradation products (kinins)

A well-established mechanism for the loss of lung function observed during the progression of emphysema is the enlargement of the pulmonary airspaces due to an imbalance in degradative proteases and their respective inhibitors (see Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General). The CPs almost certainly contribute either directly or indirectly to this degradation, in particular, lysosomal cathepsins (cat) B, H, K, L, and S (see Cysteine Proteases, Cathepsins). Increased concentrations of cat L have been detected in the BAL fluids of patients with emphysema and alveolar macrophages from patients with COPD secrete more CP activity than macrophages from normal smokers or nonsmokers despite the fact that cystatin C concentrations are also increased. Direct inhibition of CP activity by the cystatins (primarily type II) in normal lung tissue likely contributes to the protection of the elastic lung tissue by directly inhibiting these CPs. The cystatins and CPs do not operate independently of other degradative systems (and their respective inhibitors) but participate in feedback systems that can produce profound changes in proteolytic activity referred to as the proteolytic burst (Figure 2). For example, several matrix metalloelastases, produced by inflammatory cells or activated airway epithelium, can inactivate AAT. Bronchial epithelial cells secrete procathepsin B and cystatin C, both of which are substrates for neutrophil elastase. Procathepsin B is activated by neutrophil elastase while human cystatin C is inactivated by cleavage between amino acids G11 and G12. The latter observation may explain why active cat L has been found in the BAL fluid of COPD patients despite the presence of elevated cystatin C levels. Cystatin C is also produced by monocytes and macrophages and its release is downregulated by proinflammatory lipopolysaccharide (LPS) and interferon gamma (IFN-g), which coincidentally increase

PROTEINASE INHIBITORS / Cystatins 515

Inflammatory stimulus (genetic/environmental (e.g., pollution))

Activated airway epithelium

Cytokines (e.g., IFN-
, IL-8)

Neutrophil

↑ Neutrophil elastase

α1AT

SLPI

Monocyte/macrophage

↑ Matrix metalloproteases

–

–

TIMPs

↑ Cathepsins

–

Cystatins

Proteolysis of lung tissue (loss of lung function) Figure 2 Interactions of inflammatory pathway proteases and inhibitors. IFN-g, interferon gamma; IL-8, interleukin 8; a1AT, alpha 1-antitrypsin; TIMPS, tissue inhibitors of metalloproteases; SLPI, secretory leukocyte protease inhibitor.

the expression of cat B, D, H, L, and S. In addition, cat B, L, and S can inactivate secretory leukocyte protease inhibitor (SLPI). Endogenous cystatins are not able to inhibit elastolytic CPs produced by bacterial pathogens such as Staphylococcus aureus or Clostridium histolyticum, but other proteases, such as V8 protease produced by S. aureus, may contribute to an increased elastolytic burden in the lung by inactivating AAT directly. In addition, some potent inhaled allergens possess cystatin inhibitory activity (e.g., cat Fel D3) or CP activity (e.g., dust mite Der P1) and these may have direct effects on lung epithelium and immune surveillance cells of the lung. Immunomodulatory Functions of Cystatins

Cystatins have a wide range of effects in and on the immune cells present in the pulmonary space. Cystatin C is chemotactic for neutrophils, yet inhibits superoxide production and neutrophil-mediated phagocytosis. It also modulates macrophage responses to

IFN-g by increasing production of nitric oxide sixto eightfold via a mechanism independent of its CP inhibitory activity; however, it also increases the production of TNF-a and IL-10. CPs have essential functions in antigen presenting cells (APCs) and cystatin C also plays an important role in modulating major histocompatibility complex (MHC) class II-mediated antigen presentation in peripheral dendritic cells by controlling cat S-mediated degradation of the invariant chain (Ii). This processing prevents targeting of the MHC class II molecules to the lysosomes for degradation. During maturation of APCs in the lymphoid tissue, endosomal cat S activity increases due to a decrease in the levels of cystatin C. Cathepsins K and F can also degrade Ii and cat K is found in bronchial epithelial cells that can serve as nonprofessional APCs. In contrast, cat F’s expression is restricted to hematopoietic cells making it a prime candidate for a role in immunomodulation in these cells. Finally, some members of the human cystatin superfamily (e.g., cystatin S) have potent bactericidal

516 PROTEINASE INHIBITORS / Cystatins

Environmental antigens/allergens

Respiratory glandular epithelium

Monocytes Neutrophils macrophages

IgE-mediated mast cell activation

Cytokines

Kininogenases (tKal, pKal, cat L, tryptase/neutrophil elastase)

Histamine Proteases: (e.g., tryptase, chymase)

Type III cystatins (HK/LK) Bronchoconstriction Kinins (bradykinin/kallidin)

Mucus secretion Pain Cough Edema

Figure 3 Generation of proinflammatory kinins from type III cystatins. tKal, tissue kallikrein; pKal, plasma kallikrein; cat L, cathepsin L; HK, high-molecular-weight kininogen; LK, low-molecular-weight kininogen.

activity unrelated to CP inhibitory activity which resides in specific peptide sequences present in the structure. Others (e.g.,C, D, and S) are able to block the replication of certain viruses. Cystatin C is a potent inhibitor of herpes simplex virus (HSV)-1, whereas cystatins C and D both inhibit coronavirus replication in human lung cells. The likely mechanism of action involves cellular uptake followed by inhibition of the host or viral CPs required for viral replication. Generation and Function of Proinflammatory Kinins from Type III Cystatins

Perhaps the best understood role for cystatins in respiratory health comes from our current understanding of how BK and kallidin are released from precursor kininogens (type III cystatins) and the multiple direct and indirect effects they have on the respiratory system. Under inflammatory conditions, there are multiple kininogenases that could contribute to kinin generation in the lung (Figure 3). Plasma kallikrein (pKal), tissue kallikrein (tKal), cat L, and a mixture of neutrophil elastase and mast cell tryptase are all able to liberate kinins from kininogens. In the case of highmolecular kininogen, degradation by pKal creates a kinin-free two-chain disulfide-linked molecule containing a heavy chain and a light chain that retains CP inhibitory activity.

tKal has been identified as the major kininogenase of the airway and cleaves both HK and low-mole cular-weight kininogen to yield lysyl-bradykinin (kallidin). In asthmatic airways, the underlying glandular epithelium releases tKal that contributes to the intial phase of kinin generation but the recruitment of activated monocytes, neutrophils, and alveolar macrophages contributes to the late increases in tKal that are associated with the development of airway hyper-responsiveness (AHR). Activated mast cells, macrophages, and neutrophils also release the tryptase, cat L, and elastase that contribute to kinin generation. It has been suggested that this mechanism also contributes to the etiology of other chronic inflammatory conditions such as chronic bronchitis. The generation of kinins by pKal may represent a more acute inflammatory response such as observed during acute pneumonia. Kinins cause bronchoconstriction in asthmatic subjects when given by inhalation or intravenously. Their effects and mechanism of action are described in more detail in Kinins and Neuropeptides: Bradykinin.

Conclusions The cystatins comprise a superfamily of proteins related primarily by virtue of DNA and amino acid

PROTEINASE INHIBITORS / Secretory Leukoprotease Inhibitor and Elafin 517

sequence homology. The superfamily consists so far, of four distinct types of molecules ranging from the simpler low- molecular-weight type I and II cystatins, which function primarily to inhibit lysosomal CPs, to the higher-molecular-weight type III and IV cystatins, which possess additional latent functions expressed only during episodes of injury and inflammation, or have evolved entirely novel inhibitory functions. The role cystatins play in respiratory diseases such as asthma and COPD is poorly understood. However, they do modulate the immune response by acting directly on neutrophils, macrophages, and APCs. It is also clear that they do not function independently of other proteolytic pathways involved in remodeling of the lung. Limited proteolysis inactivates cystatins allowing lysosomal CP activity to directly contribute to lung tissue degradation and also liberates kinins which signal through G-protein-coupled receptors to cause both constriction and dilation of the bronchioles, pain via stimulation of sensory nerves, mucus secretion, cough, and edema. See also: Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General. Cysteine Proteases, Cathepsins. Kinins and Neuropeptides: Bradykinin.

Kaufman HF (2003) Interaction of environmental allergens with airway epithelium as a key component of asthma. Current Allergy and Asthma Reports 3(2): 101–118. Leung-Tack J, Tavera C, Gensac MC, Martinez J, and Colle A (1990) Modulation of phagocytosis-associated respiratory burst by human cystatin C: role of the N-terminal tetra-peptide Lys–Pro–Pro–Arg. Experimental Cell Research 188: 16–22. Leung-Tack J, Tavera C, Martinez J, and Colle A (1990) Neutrophil chemotactic activity is modulated by human cystatin C, an inhibitor of cysteine proteinases. Inflammation 14: 247–258. Margis R, Reis EM, and Villeret V (1998) Structural and phylogenetic relationships among plant and animal cystatins. Archives of Biochemistry and Biophysics 359: 24–30. Pierre P and Mellman I (1998) Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 93: 1135–1145. Rawlings ND and Barrett AJ (1990) Evolution of proteins of the cystatin superfamily. Journal of Molecular Evolution 30: 60–71.

Secretory Leukoprotease Inhibitor and Elafin T D Tetley, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Further Reading Barrett AJ, Rawlings ND, Davies ME, et al. (1986) Cysteine proteinase inhibitors of the cystatin superfamily. In: Barrett AJ and Salvesen G (eds.) Proteinase Inhibitors, pp. 515–569. New York: Elsevier. Bobek LA and Levine MJ (1992) Cystatins – inhibitors of cysteine proteinases. Critical Reviews in Oral Biology and Medicine 3: 307–332. Burnett D, Abrahamson M, Devalia JL, et al. (1995) Synthesis and secretion of procathepsin B and cystatin C by human bronchial epithelial cells in vitro: modulation of cathepsin B activity by neutrophil elastase. Archives of Biochemistry and Biophysics 317: 305–310. Buttle DJ, Burnett D, and Abrahamson M (1990) Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation. Scandinavian Journal of Clinical and Laboratory Investigation 50: 509–516. Churg A and Wright JL (2005) Proteases and emphysema. Current Opinion in Pulmonary Medicine 11: 153–159. Collins AR and Grubb A (1991) Inhibitory effects of recombinant human cystatin C on human coronaviruses. Antimicrobial Agents and Chemotherapy 35: 2444–2446. Dickinson DP (2002) Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease. Critical Reviews in Oral Biology and Medicine 13: 238–275. Dickinson DP (2002) Salivary (SD-type) cystatins: over one billion years in the making but to what purpose? Critical Reviews in Oral Biology and Medicine 13(6): 485–508. Hall JM (1992) Bradykinin receptors: pharmacological properties and biological roles. Pharmacology and Therapeutics 56: 131–190.

Secretory leukoprotease inhibitor (SLPI) and elafin are acidstable, low-molecular-weight antiproteinases that are produced by the goblet cells, Clara cells, and alveolar type II cells in the pulmonary epithelium, and are also present in macrophages, while neutrophils contain SLPI. SLPI inhibits neutrophil elastase, cathepsin G, trypsin, chymotrypsin, and chymase. Elafin inhibits neutrophil elastase and proteinase-3. SLPI is 11.7 kDa; elafin (6 kDa) is a cleavage product of preelafin (also called trappin-2), which is 9.9 kDa. The inhibitory site of both inhibitors resides in the C-terminal four disulfide whey acidic protein (WAP) domain, which has 40% homology, and they belong to the WAP family of proteins located on chromosome 20q12–13. The N-terminal WAP domain of pre-elafin contains a transglutaminase substrate domain that enables the molecule to become tethered to cell surfaces and matrix proteins; proteolytic cleavage releases the C-terminal 6 kDa inhibitory domain. SLPI is also found in close association with elastic tissue, possibly reflecting its cationic properties. SLPI and elafin have antimicrobial activity against Gram-positive and Gram-negative bacteria, while SLPI has also been shown to have antiviral and antifungal properties. In addition, SLPI and elafin are immunomodulatory, interacting directly with lipopolysaccharide to subdue its inflammatory activity, and inhibiting release of mediators from inflammatory cells. SLPI also plays a significant role in wound healing, partly by preventing proteolytic activation of proinflammatory mediators. SLPI and elafin levels and activity change during lung diseases (e.g., chronic obstructive pulmonary disease, acute respiratory distress syndrome, and pneumonia), reflecting the degree of inflammation, proteolytic load, and oxidative stress. The possibility of SLPI and elafin therapy is under active investigation.

518 PROTEINASE INHIBITORS / Secretory Leukoprotease Inhibitor and Elafin

Introduction Secretory leukoproteinase inhibitor (SLPI; also known as antileukoprotease or mucus proteinase inhibitor) and elafin (also known as elastase-specific inhibitor and skin-derived antileukoprotease, SKALP) are low-molecular-weight, cationic, nonglycosylated, acid-stable, serine proteinase inhibitors that are synthesized and released at mucosal surfaces and within the skin, and are also important in host defense and tissue repair (Figure 1). Both antiproteases are potent, reversible inhibitors of neutrophil elastase; in addition, SLPI inhibits cathepsin G, trypsin, chymotrypsin and chymase, whereas elafin inhibits proteinase-3.

Structure and Function SLPI

SLPI consists of 107 amino acids with a molecular weight of 11.7 kDa and has two highly homologous cysteine-rich domains, each of which contains eight cysteine residues that form four disulfide bridges. These four disulfide core whey acidic protein (WAP) domains are so named after the original observation of the WAP motif in whey acidic protein in milk whey of lactating mice. SLPI is one of a series of small proteins which contain the same WAP motif, or repeated WAP domains, that have been located on chromosome 20q12–13. The protease inhibitory activity of SLPI is located in the C-terminal region, at residues Leu72-Met73. Within the lung, SLPI is synthesized and secreted by epithelial secretory cells throughout the lower respiratory tract, including the tracheobronchial goblet cells, Clara cells, and

alveolar epithelial type II cells, as well as the serous cells of the submucosal glands. SLPI is produced constitutively and is released apically into the epithelial lining liquid at high concentrations; it is also released basally and has been found in close association with elastic tissue, possibly reflecting its cationic properties, and, unlike alpha-1 proteinase inhibitor, is a very effective inhibitor of neutrophil elastase bound to elastin. In addition, SLPI is present in monocytes, macrophages, and neutrophils. Elafin

Elafin was originally found to have a molecular weight of 6.0 kDa, following isolation from skin and lung secretions. It consists of 57 amino acids, with a four disulfide core WAP domain which also has 40% homology with the C-terminal WAP region of SLPI. Like other WAP proteins, the elafin gene is located on chromosome 20q12–13. When the gene was subsequently cloned, the protein was found to be larger: B12 kDa and 117 amino acids inclusive of the signal peptide, 95 amino acids and 9.9 kDa exclusive of the signal peptide, due to the presence of an N-terminal domain that is unlike that of SLPI, or any other members of the WAP family. Instead, it contains a transglutaminase substrate domain. Proteins containing both the WAP domain and the transglutaminase substrate domain have now been brought together under the acronym trappin (transglutaminase substrate and WAP domain containing protein) for the trappin gene family. Elafin is the C-terminal region of trappin-2, being the second trappin to be discovered. Trappin-2 refers to the 117 amino acid molecule and is also called pre-elafin. The transglutaminase domain contains five glutamine and

Interstitial pre-elafin Elafin Antiproteolytic TNF Microbes LPS

Pre-elafin Antimicrobial

IL-1


Elafin SLPI

NE

Immunomodulatory Wound healing

SLPI Interstitial SLPI Figure 1 The sources of SLPI and elafin: the epithelium, macrophages, and neutrophils. Interleukin-1 beta (IL-1b) and tumor necrosis factor (TNF) (released by macrophages), serine proteinases (released by neutrophils; NE, neutrophil elastase), and microbial agents, such as lipopolysaccharide (LPS), stimulate SLPI and elafin release into the airway lumen and into the interstitium, where they exhibit antiproteolytic, antimicrobial, immunomodulatory, and wound healing properties.

PROTEINASE INHIBITORS / Secretory Leukoprotease Inhibitor and Elafin 519

lysine residue repeats (GQDPVK) which act as acyl % donor and acceptor sites,% rendering the protein susceptible to cross-linking by tissue transglutaminase; this property has resulted in the use of the term cementoin for the N-terminal region of pre-elafin. Thus, once secreted, the molecule can become tethered to extracellular matrix and cell surfaces and operate as an anchored protein. This then restricts the activity of pre-elafin to specific tissue sites and is likely to be an important mechanism in controlling site-specific activity of elastin. However, proteolytic release of the 57 amino acid C-terminal elafin molecule allows the protease inhibitory region to transude into other tissue compartments. Again, this may occur under specific circumstances to enable elafin to function in alternative lung compartments. Elafin is synthesized and secreted by tracheobronchial epithelium, Clara cells, and alveolar type II epithelial cells, and is present in macrophages. It is also present in lung-lining liquid, but usually at much lower concentrations than SLPI (B10% of SLPI levels). Unlike SLPI, it is not produced at high levels constitutively.

Regulation of SLPI and Elafin Synthesis SLPI is synthesized by lung epithelial cell lines and primary human lung epithelial cells in vitro at relatively high levels; in contrast, constitutive levels of elafin mRNA and protein are generally much lower. The expression of both SLPI and elafin can be stimulated in pulmonary epithelial cell lines and human primary bronchial and alveolar epithelial cells by early response proinflammatory stimuli such as tumor necrosis factor alpha (TNF-a) and interleukin 1 beta (IL-1b) via nuclear factor kappa B (NF-kB). Furthermore, while corticosteroids alone have little effect on SLPI and elafin, they synergize with TNF-a and IL-1b to further stimulate SLPI and elafin synthesis. Bacterial LPS and inactivated Pseudomonas aeruginosa can also induce SLPI and elafin synthesis by epithelial cells in vitro. Thus, exposure of pulmonary epithelial cells in vivo to proinflammatory cytokines and bacterial components likely upregulates pulmonary antiproteinase defense, possibly in preparation for the increased release of inflammatory cell proteases. Interestingly, neutrophil elastase has been shown to stimulate elafin and SLPI mRNA expression in airway epithelial cells in vitro, suggesting feedback control on excessive release of neutrophil elastase. Such feedback control mechanisms may be common during neutrophilic lung inflammation; both proteins have been shown to be altered in bronchoalveolar lavage (BAL) fluid during inflammation and may be differentially expressed, thus altering the efficacy of the antiproteinase screen

(see below). Few studies have been performed on the regulation of elafin expression by primary cells.

Antiproteinase Function SLPI and elafin were originally named for their ability to inhibit neutrophil elastase, although they are also important inhibitors of mast cell proteinases. Neutrophil elastase is a potent enzyme that impacts on numerous processes. For example, it degrades elastin and other connective tissue proteins, it is ciliostatic and a secretagogue and likely contributes to excessive mucus production and retention in chronic obstructive pulmonary disease (COPD), it can stimulate cytokine synthesis as well as activating and inactivating cytokines, it cleaves cell-surface receptors, such as CD14, and it activates complement. Many of the cleavage products have chemotactic activity and so can amplify inflammation. Furthermore, neutrophil elastase can amplify proteolytic potential by activating latent matrix metalloproteinases (MMPs) and inactivating their inhibitors. Lung injury following intratracheal neutrophil elastase into experimental animals is prevented by pretreatment either with pre-elafin (but not elafin) or with the antiproteolytic region of SLPI, illustrating the importance of these proteins in controlling neutrophil elastase-induced inflammation. Thus, neutrophil elastase activity is normally strictly regulated to achieve optimal activity in response to infection. Because SLPI, pre-elafin, and elafin can access substrate-bound and free enzyme, in the interstitium and lumen, as well as being reversible, they compliment the activity of alpha-1 antitrypsin (54 kDa, glycosylated, the major circulating serine proteinase inhibitor), which irreversibly binds to and inactivates free elastase prior to clearance via the blood. Thus, SLPI and elafin work in concert with alpha-1 antitrypsin to ensure complete inhibition and clearance of neutrophil elastase and other serine proteinases from inflamed tissue.

Antimicrobial Activity SLPI and elafin proteins have antimicrobial properties similar to defensins, which appear to be unrelated to their antiprotease activity, and which have been related to the N-terminal domain of SLPI and to both the N- and C-terminals of elafin. In vitro, SLPI has antibacterial activity towards Gram-positive (e.g., Staphylococcus aureus, Listeria monocytogenes) and Gram-negative (e.g., Escherichia coli) bacteria. Elafin is also active in vitro against S. aureus and P. aeruginosa. SLPI has been shown in vitro to block the infection of peripheral blood monocytes, primary T cells, and macrophages by

520 PROTEINASE INHIBITORS / Secretory Leukoprotease Inhibitor and Elafin

human immunodeficiency virus-1 (HIV-1), possibly by binding to annexin II at the cell surface, recently proposed to be a receptor for HIV-1, or by blocking chemokine receptors CXCR4 or CCR5. Interestingly, SLPI is not very active against other retroviruses, but has activity against other types of virus, including Sendai virus and influenza virus. SLPI also has antifungal properties, inhibiting the ability of Aspergillus fumigatus and Candida albicans to trigger release of proinflammatory cytokines from human epithelial cell lines. Overexpression of elafin reduces lung injury following P. aeruginosa infection in a mouse model, which may reflect specific microbicidal activity and/or be related to other anti-inflammatory mechanisms (see below).

Role of SLPI and Elafin in Immunity The role of SLPI and elafin in the immune response is currently under intensive investigation. In an endotoxic shock model, SLPI-deficient (SLPI / ) mice were more susceptible to lipopolysaccharide (LPS) exposure, which led to higher mortality than that in wild-type mice, and also resulted in higher macrophage IL-6 and increased NF-kB expression. In vivo and in vitro investigations following augmentation of SLPI indicate that one mechanism of action is downregulation of NF-kB, by inhibiting IkappaBalpha/beta degradation and upregulation of IkappaBalpha. SLPI also prevents activation of IL-1 receptor associated kinase (IRAK), a Toll-like receptor 4 (TLR4) adaptor molecule required for cellular signaling following TLR4 activation by LPS. Thus, downregulation of NF-kB and other intracellular molecules known to be involved in the inflammatory response is likely one of the mechanisms by which SLPI inhibits release of proinflammatory mediators (e.g., prostanoids, MMPs, and TNF) from lung cells in vivo. Another anti-inflammatory mechanism may be to prevent the interaction and biological reactivity of the infective agent with cellular targets. For example, SLPI binds to LPS. Interestingly, LPS triggers release of SLPI from pulmonary epithelial cells and macrophages, possibly to control excessive LPS activity (see below). These, and similar studies, illustrate an anti-inflammatory and immunomodulatory role for SLPI. Intranasal administration of engineered human pre-elafin to mice prior to intranasal LPS inhibited inflammatory neutrophil influx, two neutrophil chemokines (macrophage inflammatory protein-2 (MIP-2) and KC, murine homologs of IL-8), as well as downregulating expression of IL-1, suggesting significant anti-inflammatory mechanisms for elafin. However, adenovirus-mediated overexpression of the full-length human pre-elafin gene in mice (who do

not have an elafin gene equivalent) resulted in increased pulmonary airway inflammatory cell influx and elevated TNF-a and MIP-2 in BAL following LPS challenge. It is suggested that this apparent proinflammatory response may be an important acute defense mechanism, triggering the innate immune response system to combat bacterial infection, as has been proposed for some defensins. Since mice do not normally express elafin, it is difficult to appreciate the influence this itself may have, particularly in the context of cellular responses following elafin binding to LPS (see below). Another putative mechanism of action of SLPI and elafin in host defense involves binding to LPS and intercepting its interaction with target cells. As LPS is a rapid, potent activator of the innate immune system, its activity needs to be carefully modulated to elicit an appropriate response. The presence, in respiratory secretions, of constitutively high quantities of SLPI, and not insignificant levels of elafin, release of which can be further stimulated by endogenous and exogenous agents, is probably crucial during microbial infection with Gram-negative bacteria as both proteins bind LPS. Very small quantities of LPS evoke strong systemic responses which need to be tightly regulated. Macrophages transfected with SLPI that overexpress the inhibitor respond to LPS, but are less sensitive, showing a subdued response. SLPI has been shown to bind to LPS and CD14–LPS to prevent cell-surface TLR4 binding and macrophage activation. This is thought to be an important feedback control mechanism to modulate the intensity of the cellular response. More recently, elafin has also been demonstrated to bind to the lipid A core of LPS. Production of TNF-a by macrophages was inhibited by LPS–elafin complexes only in the presence of serum. However, if the experiment was carried out in a serum-free environment, then production of TNF-a increased. This fits with the findings using transgenic elafin mouse models and it is postulated that, within a serum-free environment, such as the lung lumen, elafin facilitates LPS to initiate an immune response in the absence of large quantities of LPS-binding protein. However, systemically, binding of LPS by elafin (which, if present at high levels, likely reflects acute tissue injury) would subdue an overt immune response.

Role of SLPI in Wound Healing The expression of SLPI is elevated during cutaneous wound healing in humans. Absence of SLPI leads to delayed healing due to reduced matrix production and exaggerated inflammatory response. Wound

PROTEINASE INHIBITORS / Secretory Leukoprotease Inhibitor and Elafin 521

healing in SLPI-deficient mice is impaired. In this model, there is increased inflammatory cell influx with elevated elastase activity, increased NK-kB activation, as well as an increase in levels of active transforming growth factor beta (TGF-b). It is suggested that in the absence of SLPI, there is greater macrophage activation, release of proteolytic enzymes, and cleavage and activation of latent TGF-b. Together, activated TGF-b and chemotactic peptides, generated during matrix turnover, stimulate a cycle of leukocyte recruitment during the remodeling process and delay the healing process. Thus, there appears to be a complex interaction between SLPI and the growth factor TGF-b that closely regulates connective tissue synthesis and repair processes. In addition, proepithelins are growth factors, released by epithelial cells, that stimulate re-epithelialization and inhibit proinflammatory chemokine release from epithelium; they are processed to generate epithelin which has opposite effects, stimulating IL-8 release and inhibiting epithelial growth. SLPI forms complexes with proepithelins which prevents elastolytic conversion of anti-inflammatory proepithelins to proinflammatory epithelin. In SLPI-null mice, application of proepithelins reverses the defect in wound healing, supporting the suggestion that SLPI enables re-epithelialization during wound healing by preventing proteolytic degradation of proepithelins. In in vitro studies of human lung fibroblasts, SLPI stimulates hepatocyte growth factor (HGF; also called scatter factor), which regulates mitogenesis and morphogenesis and is therefore relevant to normal tissue development and remodeling. SLPI has also been shown to inhibit fibroblast-mediated scar formation in a collagen gel model in vitro. The ability of SLPI to promote wound healing has been ascribed to a number of qualities, including its antiproteolytic activity and autocrine growth factor activity. Together with its antimicrobial and antiinflammatory properties, SLPI is clearly an important multifunctional mediator of wound healing.

SLPI and Elafin in Respiratory Disease Uncontrolled/elevated serine proteinases have been implicated in the pathology of a number of pulmonary diseases, including COPD, cystic fibrosis, bronchiectasis, acute respiratory distress syndrome (ARDS), bronchiolitis obliterans, bacterial pneumonia, asthma, and fibrosis. Abnormal SLPI and elafin expression and/or activity leading to uncontrolled serine proteinase activity may be a factor. SLPI is the major serine proteinase inhibitor in conducting airway secretions (between 50% and 90% of the total), reflecting synthesis and release by goblet cells and

serous cells in the bronchial glands. In the small airways, bronchioles, and alveolae, the Clara cells and type II epithelial cells also produce SLPI, although it forms relatively less of the total serine proteinase inhibitory screen (approximately 20% of the total). Elafin is synthesized by the same cells, but its contribution to the antiproteinase screen is usually relatively low since it is not released at high levels constitutively, but production may be triggered by external and endogenous acute stimuli, such as bacteria and IL-1b. Clearly, the levels and activity of SLPI and elafin during pulmonary disease will reflect both the changes in secretory cell profile (e.g., cell death, proliferation, and migration) and regulation of inhibitor expression by local mediators. The relative contribution of leukocyte-derived SLPI and elafin to lung antiprotease defense is less clear. Most studies of the role of SLPI and elafin in lung disease have measured levels in BAL fluid and sputum and there are no data regarding interstitial levels or upregulation of mRNA expression in individual cells. Secretions from subjects with COPD contain high levels of SLPI, probably reflecting increased numbers of SLPI-expressing epithelial cells in large and small airways, and possibly also because of increased release by leukocytes. However, during exacerbations, if uninhibited, active neutrophil elastase is present in lung secretions, SLPI is reduced. Furthermore, epithelial SLPI expression is reduced in areas of epithelial damage or epithelial metaplasia. Although a number of studies show that there is an apparent compensatory increase in pulmonary SLPI in alpha-1 antitrypsin deficiency, in alpha-1 antitrypsin-deficient subjects with COPD, SLPI has been shown to be compromised, possibly for the same reasons as alpha-1 antitrypsin-sufficient subjects, that is, increased neutrophil elastase and epithelial damage and/ or metaplasia. There is little information regarding elafin expression during COPD. There are fewer studies of SLPI and elafin in other lung diseases. In respiratory distress situations, SLPI and elafin are significantly elevated in BAL fluid from those at risk of developing ARDS compared to healthy subjects. In those who go on to develop ARDS, SLPI, but not elafin, levels increase further, to above the levels for those at risk of developing ARDS. Furthermore, SLPI concentrations in BAL fluid correlate with multiorgan failure score in patients with acute lung injury. SLPI is also increased in BAL fluid during pneumonia. However, despite increased levels of these locally produced inhibitors, antiprotease activity in lung secretions is usually reduced, particularly during severe disease, due to oxidation, complexing with elastase or degradation. In contrast, the level of SLPI is reduced in BAL fluid

522 PROTEINASE INHIBITORS / Secretory Leukoprotease Inhibitor and Elafin

collected from patients with bronchiolitis obliterans syndrome (after heart–lung transplantation). Although elafin has not been measured in lung secretions from subjects with pneumonia and bronchiolitis obliterans, it has been shown to be markedly elevated in BAL fluid obtained from subjects with active farmer’s lung, supporting its role as an acuteresponse antiprotease, but, interestingly, remained elevated in those who had recovered.

Alpha-1-Antitrypsin Deficiency; Emphysema, General. Clara Cells. Defensins. Endotoxins. Epithelial Cells: Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens. Gene Regulation. Human Immunodeficiency Virus. Interleukins: IL-1 and IL-18. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia. Leukocytes: Neutrophils; Pulmonary Macrophages. Serine Proteinases. Toll-Like Receptors. Transcription Factors: NF-kB and Ikb. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Viruses of the Lung.

SLPI and Elafin Augmentation Therapy SLPI and pre-elafin are not glycosylated, and recombinant forms are identical to the native proteins and therefore have therapeutic potential. As outlined above, the advantage of these inhibitors over alpha-1 antitrypsin is that their physicochemical properties enhance localization to the interstitium, where they can inhibit matrix-bound proteases. Whether SLPI is administered via inhalation, intratracheally, intravenously, or intraperitoneally, it is very effective in animal models, including acute lung injury, bleomycininduced fibrosis, and protease- and allergen-induced airway constriction and hyperreactivity. Furthermore, in an ovine model, aerosolized SLPI caused an increase in glutathione levels in lung lining liquid, enhancing antioxidant protection. There is minimal metabolism of SLPI in the lung and it remains active following inhalation of the aerosolized recombinant protein. However, studies of healthy subjects and cystic fibrosis patients show that its half-life in lunglining liquid is short, requiring treatment twice daily. Nevertheless, the antiproteinase activity increases, while IL-8 levels decrease in BAL fluid from SLPI-treated individuals. A difficulty with inhalation therapy is delivery of the drug to diseased/affected regions. This problem is particularly relevant for conditions involving mucous hypersecretion and airway remodeling, such as cystic fibrosis and emphysema. There are no similar studies of elafin. An alternative to inhalation therapy is gene therapy. In animal models, overexpression of intratracheally administered adenoviral elafin gene confers protection against P. aeruginosa-induced lung injury and S. aureus-induced inflammation. Although these studies suggest that this approach to augmenting pulmonary antiproteases might be useful, it is still in its infancy. There are no studies in humans. Furthermore, in the face of ongoing pulmonary disease, the problems associated with optimal delivery and expression of the protein are significant. See also: Acute Respiratory Distress Syndrome. Antiviral Agents. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview; Emphysema,

Further Reading Aarbiou J, van Schadewijk A, Stolk J, et al. (2004) Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways. Inflammation Research 53: 230–238. Ashcroft GS, Lei K, Jin W, Longenecker G, et al. (2000) Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing. Nature Medicine 6: 1147– 1153. Chughtai B and O’Riordan TG (2004) Potential role of inhibitors of neutrophil elastase in treating diseases of the airway. Journal of Aerosol Medicine 17: 289–298. Clauss A, Lilja H, and Lundwall A (2002) A locus on human chromosome 20 contains several genes expressing protease inhibitor domains with homology to whey acidic protein. Biochemical Journal 368: 233–242. Doumas S, Kolokotronis A, and Stefanopoulos P (2005) Antiinflammatory and antimicrobial roles of secretory leukocyte protease inhibitor. Infection and Immunity 73: 1271–1274. Greene C, Taggart C, Lowe G, et al. (2003) Local impairment of anti-neutrophil elastase capacity in community-acquired pneumonia. Journal of Infectious Diseases 188: 769–776. Henriksen PA, Hitt M, Xing Z, et al. (2004) Adenoviral gene delivery of elafin and secretory leukocyte protease inhibitor attenuates NF-kappa B-dependent inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli. Journal of Immunology 172: 4535–4544. Hiemstra PS (2002) Novel roles of protease inhibitors in infection and inflammation. Biochemical Society Transactions 30: 116– 120. McMicheal JW, Roghanian A, Jiang L, Ramage R, and Sallenave JM (2005) The antimicrobial antiproteinase elafin binds to lipolysaccharide and modulates macrophage responses. American Journal of Respiratory Cell and Molecular Biology 32: 443–452. Molhuizen HOF, Alkemade HAC, Zeeuwen PLJM, et al. (1993) SKALP/elafin: an elastase inhibitor from cultured human keratinocytes: purification, cDNA sequence, and evidence for transglutaminase cross-linking. Journal of Biological Chemistry 268: 12028–12032. Sallenave JM (2002) Antimicrobial activity of antiproteinases. Biochemical Society Transactions 30: 111–115. Schalkwijk J, Wiedow O, and Hirose XY (1999) The trappin gene family: proteins defined by an N-terminal transglutaminase substrate domain and a C-terminal four-disulphide core. Biochemical Journal 340: 569–577. Taggart CC, Greene CM, McElvaney NG, and O’Neill S (2002) Secretory leucoprotease inhibitor prevents lipopolysaccharideinduced IkappaBalpha degradation without affecting phosphorylation or ubiquitination. Journal of Biological Chemistry 277: 33648–33653.

PROTEINASE-ACTIVATED RECEPTORS 523

PROTEINASE-ACTIVATED RECEPTORS R C Chambers, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The discovery of the proteinase-activated receptors (PARs) resulted in the establishment of a new paradigm for ligand– receptor interaction mechanisms leading to cellular signaling in that proteolysis rather than classical ligand binding activates these receptors. The PARs were originally regarded as cellular signaling receptors for the coagulation serine proteinase thrombin. However, the cloning of additional PARs led to the identification of other proteinase activators, including trypsin, mast cell tryptase, and more recently nonserine proteinases such as MMP-1. PARs are widely distributed in the human lung, and recent evidence indicates a role for PARs in the pathophysiology of lung inflammatory and fibrotic responses.

Introduction Proteinase-activated receptors (PARs) belong to the family of seven transmembrane domain G-proteincoupled receptors and derive their name from their unique mechanism of activation. Unlike other Gprotein-coupled receptors, which are activated by direct ligand binding, activation of the PARs involves the proteolytic unmasking of a cryptic ligand that is already tethered to the receptor. Proteolysis is mediated by certain proteinases, of which thrombin and upstream proteinases of the extrinsic pathway of coagulation, as well as trypsin and tryptase are the most well recognized. The human gene for PAR1 (formerly known as the thrombin receptor) was discovered in 1991 by Shaun Coughlin and colleagues at the University of California–San Francisco at the end of an intense research effort aimed at identifying the cellular mechanism by which thrombin mediates platelet aggregation. The same year also saw the cloning of the hamster PAR1 gene by Van Obberghen-Schilling and coworkers in Strasbourg. Following the discovery of the thrombin receptor and its unique activation mechanism, it appeared possible that other receptors with a similar mechanism of activation might exist. For example, platelets obtained from PAR1 null mice surviving to adulthood still responded strongly to thrombin. The search for additional thrombin receptors culminated in the discovery of two further thrombin-sensitive receptors, PAR3 and PAR4. Genomic screening led to the identification of a further receptor, PAR2, with a similar mechanism of activation by the more broadspectrum proteinase trypsin but not thrombin.

Structure The four PAR genes are between 3.5 and 3.7 kb long and share a similar two-exon structure encoding around 400 amino acids (Table 1). Human PAR1, PAR2, and PAR3 cluster together on band q13 on chromosome 5, suggesting that they may have arisen by gene duplication from a single ancestral gene. In contrast, PAR4 is located separately at position p12 on chromosome 19. The sequence homology between human PARs is between 27% and 33%; with the greatest difference noted between PAR4 and the other three PARs in terms of both the N- and C-termini and the cleavage site. Comparison of amino acid sequence alignment between species revealed that the PARs are highly conserved between humans and mice and that homologs of these genes are present in amphibians. The predicted protein structure of the PARs share several features with classical seven transmembrane G-protein domain-linked receptors with their signature configuration consisting of seven helical hydrophobic transmembrane regions that in turn give rise to three intra- and three extracellular loops, a C-terminal intracellular tail, and a long N-terminal extracellular domain (Figure 1).

Regulation of Production and Activity Activation of the PARs is mediated via limited proteolysis of the N-terminus resulting in the cleavage of around 40 amino acids (Figure 1 and Table 1). This leads to the unmasking of a tethered ligand sequence that interacts with the second extracellular loop of the receptor to induce a conformational change allowing the receptor to interact and signal via heterotrimeric G-proteins, which in turn trigger a variety of downstream signal transduction pathways. There is little doubt that thrombin is an important physiological activator of PAR1, PAR3, and PAR4. The upstream coagulation proteinase, factor Xa, as well as the more potent tissue factor–factor VIIa–factor Xa complexes, activate both PAR1 and PAR2, depending on cell type. PAR1, PAR2, and PAR4 can also be activated by trypsin, whereas mast cell tryptase is thought to be a physiological activator of PAR2. A number of other proteinase activators have been described and are listed in Table 1. These include nonserine proteinases, such as the matrix metalloproteinase MMP-1. Recently, nonendogenous proteinases, including proteinases released by house dust mites and certain bacteria, have also been

Table 1 Human proteinase-activated receptor expression and pharmacology No. of amino acids

High-affinityactivating proteinases

Low-affinityactivating proteinases

Tethered ligand sequence

Activating peptides

Nonpeptide antagonists

Inactivating proteinases

Tissue expression

Cell types

PAR1

425

Thrombin

Trypsin, TF/FVIIa/ FXa, granzyme A, plasmin, trypsin IV, MMP-1

R41mSFLLRN

SFLLRNNH2, TFLLRNH2

RWJ56110, RWJ58259

Cathepsin G, neutrophil proteinase-3, elastase, chymase, Der p1

Airways, blood, brain, bone, breast, cardiovascular system, endometrium, immune system, intestine, lung parenchyma, lymph node, nervous system, skin

PAR2

397

Trypsin, tryptase, trypsin II, trypsin IV

Matriptase/ MTSP1, TF/FVIIa/ FXa, proteinase-3, Der p1, Der p3, Der p9

R34mSLIGKV

SLIGKVNH2, SFLLRNNH2

None to date

Elastase, chymase

Airways, blood, brain, cardiovascular system, GI tract, immune system, intestine, nervous system, pancreas, skin, testes, urogenital tract, eye

PAR3

374

Thrombin

Trypsin, Factor Xa

K38mTFRGAP

None known

–

Cathepsin G

Immune system

PAR4

385

Thrombin, trypsin

Cathepsin G,

R47mGYPGQV

GYPGQVNH2, AYPGKFNH2

YD-3

Unknown

Airway, blood, cardiovascular system

Astrocytes, epithelial cells, endothelial cells, fibroblasts, hematopoietic progenitor cells, keratinocytes, macrophages, mast cells, natural killer cells, neuronal cells, platelets, smooth muscle cells, T cells Endothelial cells, eosinophils, epithelial cells, fibroblasts, keratinocytes, mast cells, macrophages, monocytes, neuronal cells, neutrophils, platelets, smooth muscle cells, T cells Megakaryocytes of the bone marrow, platelets, T cells Epithelial cells, smooth muscle cells, endothelial cells, platelets, fibroblasts

Der p1, 3, and 9, house dust mite Dermatophagoides pteronyssinus proteinase 1, 3, and 9. MMP-1, matrix metalloproteinase-1. MT-SP1, Membrane-type serine protease 1. NH2, amide. Letters denote amino acid sequences in one letter code; arrow denotes cleavage site.

PROTEINASE-ACTIVATED RECEPTORS 525 Thrombin

Cleavage

Tethered ligand

G


 

PAR-1 Cell signaling Figure 1 Mechanism of activation of proteinase-activated receptor by thrombin. (1) The cleavage of an extracellular fragment of the receptor by thrombin unmasks a tethered ligand that binds to the body of the receptor. (2) The resulting conformational change induces cell signaling via activation of heterotrimeric G-proteins. PAR, proteinase-activated receptor; Ga, b, g, G-protein subunits.

recently shown to activate PARs in vitro. However, confirmation for the importance of these enzymes in activating PARs in vivo is still lacking. Delineating the signaling pathways and cellular responses elicited by PAR activators has been greatly aided by the use of peptide agonists, which mimic the tethered ligand sequence unmasked following receptor cleavage. Current evidence suggests that PAR1, PAR2, and PAR4 act as signaling receptors, whereas PAR3 is thought to act as a thrombin-docking receptor for efficient presentation of the proteinase to PAR4 at low concentrations. Following PAR activation, signal transduction is mediated via heterotrimeric G-proteins. PAR1 is relatively promiscuous in its ability to couple to multiple G-proteins, including Go/i, Gq, and G12/13. This enables the receptor to mediate its pluripotent effects via various signaling pathways, including amongst others phospholipase c/protein kinase c (PLC/PKC), mitogen-activated protein-kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and nuclear factor kappa B (NF-kB) pathways. Fewer signaling studies have been performed for the other three PARs. Current evidence suggests that PAR2 interacts with Gq/11 and possibly Go/i. As mentioned above, PAR3 does not appear to signal, whereas PAR4 has been shown to activate Gi, G12/13, and Gq pathways. In terms of the regulation of PAR signaling, the mechanisms involved are similar to those employed by other members of the seven transmembrane domain G-protein-coupled receptors. This includes classical receptor desensitization by phosphorylation at the C-terminus followed by endocytosis and degradation. Cell responsiveness is re-established via the appearance of new receptors cycled to the cell surface from pre-existing intracellular stores and de novo protein synthesis. Several proinflammatory cytokines, as well as the activating proteinases themselves (e.g., thrombin), have been reported to induce PAR gene expression. Finally, PAR signaling may also be controlled by inactivation by certain proteinases as a result of proteolysis at nonactivating sites (Table 1).

Biological Function The clearest physiological role for the PARs is in the activation of platelets by thrombin, one of the key events involved in blood clotting. In humans, this response is mediated by PAR1 and PAR4, whereas murine platelet responses appear to involve binding to PAR3 and subsequent cleavage of PAR4. Therefore, there are important differences in receptor utilization between humans and mice and this needs to be borne in mind when extrapolating results obtained in experimental models to human physiology and pathophysiology. Intense research efforts into the role of PARs (in particular PAR1 and PAR2) in other cell types has revealed that these receptors influence a wide range of cellular responses via both direct effects and their ability to induce the synthesis and release of a host of secondary mediators, including growth factors, chemokines, lipid mediators, and potent proinflammatory cytokines. It is therefore not surprising that these receptors are capable of influencing a wide range of cellular responses, including promoting cellular proliferation, differentiation, cytoskeletal reorganization, apoptosis, migration, and extracellular matrix production. In vivo evidence for the importance of these responses in the regulation of inflammation, vascular tone, angiogenesis, tissue and blood vessel repair, immune responses, as well as cell invasion in both pathological and nonpathological conditions is rapidly accumulating.

PARs in Respiratory Disease Fibroproliferative Lung Disease

Interest in the role of PARs in fibrotic lung disease was fuelled by the observation that activation of the coagulation cascade is a characteristic feature of both chronic and acute lung injury. In the normal uninjured lung, the alveolar hemostatic balance is generally antithrombotic and profibrinolytic. However, in both acute lung injury and chronic fibrotic lung disease, there is good evidence that this balance is shifted in

526 PROTEINASE-ACTIVATED RECEPTORS

favor of increased procoagulant activity and decreased fibrinolytic capability. Extravascular intra-alveolar accumulation of fibrin, often evident as hyaline membranes, is commonly observed in the lungs of patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and chronic fibrotic lung disease. Levels of tissue factor, the initiator of the extrinsic coagulation pathway is highly upregulated in the lungs of patients with idiopathic pulmonary fibrosis, interstitial pneumonia associated with systemic sclerosis and in idiopathic bronchiolitis obliterans with organizing pneumonia. Bronchoalveolar lavage fluid (BALF) from patients with ARDS also contains tissue factor/factor VII/VIIa complexes and levels of active thrombin have been shown to be increased in the lungs of patients with pulmonary fibrosis associated with systemic sclerosis and in pulmonary fibrosis associated with chronic lung disease of prematurity. Thrombin BALF levels are also elevated in animal models of lung injury (e.g., bleomycin) and pharmacological inhibition of the coagulation cascade has been shown to be protective against lung collagen accumulation in these models. There is extensive in vitro evidence that activation of PAR1 by thrombin exerts both proinflammatory and profibrotic effects on a number of cell types present in the lung. For example, activation of PAR1 by either thrombin or factor Xa leads to the release of a host of secondary proinflammatory mediators, increases endothelial cell permeability, influences inflammatory cell trafficking, and promotes fibroblast proliferation and transformation into activated myofibroblasts, the main cell type responsible for extracellular matrix deposition in the fibrotic lung. PAR1 immunoreactivity is increased both in patients with pulmonary fibrosis and in the bleomycin model of this condition. This raises the possibility that the coagulation proteinases may contribute to the pathogenesis of these disorders, at least in part via PAR1-dependent pathways. Support for a pivotal role for PAR1 in this model was recently obtained for both the inflammatory and fibrotic phases of this injury model. Lung collagen accumulation in response to bleomycin injury is attenuated by up to 60% and is preceded by similar reductions in inflammatory cell recruitment and microvascular leak. This protection is associated with a reduction in a number of PAR1inducible genes, including the chemokine monocyte chemotactic protein factor 1 (MCP-1) and the profibrotic mediators connective tissue growth factor and transforming growth factor beta (TGF-b1). Antagonists are currently being developed as potent antithrombotic agents and may therefore prove useful for interfering with the cellular effects associated with excessive activation or recurrent

activation of the coagulation cascade in response to both acute and chronic lung injury. Asthma and Airway Remodeling

Thrombin levels are increased in BALF from patients with asthma and have recently been shown to correlate with levels of interleukin-5 (IL-5), TGF-b, and inflammatory cell numbers after segmental challenge in asthmatic patients. Several functional responses elicited by thrombin observed in in vitro and ex vivo studies might contribute to the pathology of this condition. Thrombin acts as a bronchoconstrictor, exerts mitogenic effects for airway smooth muscle cells and fibroblasts, and, as mentioned above, releases a host of proinflammatory cytokines by a number of cell types, including airway and alveolar epithelial cells. Current in vitro and ex vivo evidence suggests an important role for PAR1 in mediating a number of these effects but confirmation of the importance of this receptor in this disease setting in vivo is still lacking. In contrast, evidence for a role for PAR2 in this syndrome is rapidly accumulating. Mast cell tryptase, a known PAR2 activator, has been used as a marker of allergic inflammation for many years. Tryptase inhibitors have been shown to be effective in animal models and, albeit to a lesser extent, in clinical trials. PAR2 protein expression by epithelial cells is further increased in asthmatic patients. Experimental evidence suggests several mechanisms by which activation of PAR2 might contribute to this syndrome. Similar to PAR1, activation of PAR2 in vitro leads to the release of potent proinflammatory mediators from a number of cell types present in the airway, and may therefore contribute to airway inflammation. This is consistent with the observation that the early inflammatory response to allergen inhalation is attenuated in PAR2-deficient mice. These mice also produce lower serum IgE levels, so it is possible that PAR2 may also play a role in the sensitization process. Of interest, PAR2 has been reported to trigger dendritic cell development suggesting a mechanism by which this receptor may influence this process in mice. The recent finding that the house dust mite allergens Der p1 (cysteine proteinase), Der p3 (serine proteinase), and Der p9 (serine proteinase) induce the release of the proinflammatory cytokines IL-6 and IL-8 from respiratory epithelial cells in a PAR2-dependent manner suggests another mechanism by which this receptor may contribute to the asthma syndrome. Finally, trypsin and PAR2 activators also influence airway smooth muscle contraction and relaxation in vitro but current in vivo evidence suggests that this receptor exerts a predominantly bronchoprotective/bronchodilator effect. PAR2 is

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therefore emerging as a receptor that may exert deleterious effects by contributing to airway inflammatory responses but that may also play important roles in airway homeostatic mechanisms. This will need to be borne in mind when targeting PAR2 for therapeutic intervention in any respiratory condition. Chronic Obstructive Pulmonary Disease

Tryptase levels are detectable in patients with COPD and in smokers, and intratracheal administration of trypsin to rats reproduces some of the cardinal features of COPD. Expression of PAR2 is increased in airway vessels of patients with bronchitis, raising the possibility that PAR2 may also be important in this disease setting. See also: Asthma: Overview. Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12). Chronic Obstructive Pulmonary Disease: Overview. Coagulation Cascade: Overview; Factor VII; Factor X; Thrombin. Connective Tissue Growth Factor. G-Protein-Coupled Receptors. Interstitial Lung Disease: Overview. Pulmonary Effects of Systemic Disease. Signal Transduction. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Asokananthan N, Graham PT, Stewart DJ, et al. (2002) House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1. Journal of Immunology 169(8): 4572–4578. Chambers RC (2003) Proteinase-activated receptors and the pathophysiology of pulmonary fibrosis. Drug Development Research 60: 29–35.

Proteoglycans

Cocks TM, Fong B, Chow JM, et al. (1999) A protective role for protease-activated receptors in the airways. Nature 398: 156–160. Hernandez-Rodriguez NA, Cambrey AD, Harrison NK, et al. (1995) Role of thrombin in pulmonary fibrosis. Lancet 346: 1071–1073. Howell DCJ, Goldsack NR, Marshall RP, et al. (2001) Direct thrombin inhibition reduces lung collagen accumulation and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis. American Journal of Pathology 159: 1383–1395. Howell DCJ, Johns RH, Lasky JA, et al. Absence of proteinaseactivated receptor-1 signaling affords protection from bleomycin-induced lung inflammation and fibrosis. American Journal of Pathology 166: 1353–1365. Idell S (2003) Coagulation, fibrinolysis, and fibrin deposition in acute lung injury. Critical Care Medicine 31(supplement 4): S213–S220. Idell S, Gonzalez K, Bradford H, et al. (1987) Procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome. Contribution of tissue factor associated with factor VII. American Review of Respiratory Diseases 136: 1466–1474. Imokawa S, Sato A, Hayakawa H, et al. (1997) Tissue factor expression and fibrin deposition in the lungs of patients with idiopathic pulmonary fibrosis and systemic sclerosis. American Journal of Respiratory and Critical Care Medicine 156: 631–636. Knight DA, Lim S, Scaffidi AK, Roche N, Chung KF, Stewart GA, and Thompson PJ (2001) Protease-activated receptors in human airways: upregulation of PAR-2 in respiratory epithelium from patients with asthma. Journal of Allergy and Clinical Immunology 108: 797–803. Ossovskaya VS and Bunnett NW (2004) Protease-activated receptors: contribution to physiology and disease. Physiological Reviews 84: 579–621. Rasmussen UB, Vouret-Craviari V, Jallat S, et al. (1991) cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2 þ mobilization. FEBS Letters 288(1
2): 123–128. Schmidlin F, Amadesi S, Dabbagh K, et al. (2002) Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway. Journal of Immunology 169: 5315–5321. Vu TK, Hung DT, Wheaton VI, and Coughlin SR (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64(6): 1057–1068.

see Extracellular Matrix: Matrix Proteoglycans; Surface Proteoglycans.

PROTEOME G Westergren-Thorsson and G Marko-Varga, Lund University, Lund, Sweden J Malmstro¨m, Institute of Systems Biology, Seattle, WA, USA K Larsen, Lund University, Lund, Sweden & 2006 Elsevier Ltd. All rights reserved.

Abstract Proteomics can be defined as the studies of protein properties on a large scale to obtain a global view of biological processes at

the protein level. Essentially, proteomics requires protein separation and identification, and in many cases quantification. The cornerstones in proteomics are protein/peptide separation by gel electrophoresis and/or different chromatographic techniques, and identification by mass spectrometry followed by bioinformatic and biological interpretation of data. By combining these different separation techniques and mass spectrometry it is now possible to identify low abundant proteins with 10–1000 copies per cell. Today, substantial research efforts in proteome studies of the lung are focused on obtaining new diagnostic markers, as well as fingerprinting disease mechanisms.

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Introduction During the past 10 years, several new techniques such as cDNA microarray, yeast two-hybrid analysis, and mass spectrometry (MS) have been introduced that allow simultaneous high-throughput analysis of multiple mRNAs and proteins within the same sample. These technologies have received a great deal of attention and gradually begun to infiltrate biochemistry and cell biology laboratories. The term proteome was introduced for the first time in 1994 at the first Proteome meeting in Siena, Italy, and was used to describe the protein complement of a genome. Proteomics can be defined as ‘‘a large-scale study of protein properties, e.g., expression level, posttranscriptional modification

Proteomics

Separation

Bioinformatics

MS

Data analysis

Biological readouts Figure 1 The main components essential for proteome analysis.

and protein interaction, in order to obtain a global view of disease processes or cellular processes at the protein level.’’ Three strategies have had a strong impact in the field of biology: (1) the generation of protein–protein linkage maps; (2) the annotation of genomic DNA sequences by generation of MS/MS peptide sequences; and (3) the measurement of protein expression by quantitative methods. The data output from a typical proteomics experiment is huge and therefore computer-based data storage and analysis is required. Essentially, proteomics is based on protein separation, identification, and data analysis followed by biological readouts (Figure 1). Proteomics have a great potential to give rise to novel discoveries and to generate new testable hypotheses by choosing the appropriate study design. Therefore, different types of separation techniques as well as MS will be discussed in more detail below along with some recent proteome findings relevant for the lung.

Protein/Peptide Separation Originally, protein separation was performed by twodimensional gel electrophoresis (2-DE) as described by O’Farrel in 1976 and Klose in 1975, and was for many years the most efficient way of separating proteins in complex mixtures. 2-DE is dependent on protein separation by isoelectric focusing and molecular weight (Figure 2(a)). Spots of interest are excised and identified with peptide mass fingerprinting by matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI–TOF MS). By

5−100 kDa

pH 4−7 pI 3−10 NL Mol.wt 90−95 kDa A

5−100 kDa

pH 4−7

Mol.wt 90−95 kDa B

Control pI 4.5−5.5

TGF-


Control (a)

TGF-
(b)

Figure 2 Example of 2D gel electrophoresis on cell lysate from control and TGF-b-treated fibroblast cultures. (a) Solubilized cells were applied on 4.7–7 NL pI strips and separated on 14% acrylamide gels. The spots were visualized by silver staining. The enlarged area from the lower part of the gel shows two spots, which were affected by TGF-b treatment compared to the control. (b) The separation of proteins was compared between different pI intervals. Proteins of specific interest in a certain pH interval were better separated in a narrower pI interval such as 4.5–5.5 compared to 3–10.

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using image analysis, it is possible to quantify the relative protein expression and thereby enable monitoring of protein expression over time or allow comparison between cellular or clinical material (Figure 2(a)). By using zoom gel electrophoresis with a narrower pI interval such as 4.5–5.5 compared to 3–10, it is possible to get a better separation of proteins of specific interest (Figure 2(b)). Although 2-DE has been the method of choice for many years, it has a number of drawbacks regarding difficulties to automatize sensitivity and separation of large proteins, glycosylated proteins, and membrane proteins. Several of these limitations are avoided when chromatographic-based separation techniques are used. The coupling of high-pressure liquid chromatography (HPLC) with MS has proven to be an alternative method to 2-D gels. Examples of such separation techniques are two-dimensional (strong-cationic exchange (SCR)/reverse phase (RP)) or three-dimensional (SCR/avidin affinity/RP) chromatographic separations of peptide mixtures generated by tryptic digestion of protein samples.

Sample plate

Detector Laser

TOF Reflector (a) Accelerator Detector

Ion source

Q1

Q2

Reflector (b) Sample plate

Detector Laser

TOF1

TOF2

Reflector

Collision cell

Mass Spectrometry (MS) Important technological advances have occurred in the field of mass spectrometry. Research on the ionization principles used today was awarded the Nobel Prize in Chemistry in 2002. A mass spectrometer consists of an ion source, a mass analyzer that measures the mass-to-charge ratio (m/z) of the ionized analytes, and a detector that registers the number of ions at each m/z value. Today, the ionization methods of choice are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Both methods are commonly used to volatize and ionize the protein or peptides for mass spectrometric analysis (Figures 3(a) and 3(b)). In MALDI, the peptides are embedded in a crystal matrix and ionized by a laser pulse. In ESI, the ionization occurs in solution and therefore is mainly used when coupled to chromatographic liquid-based separation. There are four basic types of mass analyzers currently used in proteomics research: ion-trap, time of flight (TOF), quadruple, and Fourier transform ion cyclotron (FT-MS) analyzers. In proteome analysis, it is of particular importance that the mass analyzers are sensitive, have high resolution and mass accuracy, and that they can generate ion-rich mass spectra from peptide fragments (tandem MS/MS spectra). In tandem MS/MS, the charged peptides are separated in the first MS according to their m/z ratio to create a list of the most intense peptide peaks. In the second analysis, the instrument is adjusted to select only a specific m/z and direct this peptide into the collision cell. By

(c) Figure 3 Schematic pictures of two types of mass spectrometer. (a) A matrix-assisted laser desorption/ionization–time of flight (MALDI–TOF) mass spectrometer; (b) a quadruple (Q) TOF instrument with electrospray ionization (ESI).

using the appropriate collision energy, fragmentation occurs predominantly at the peptide bond, generating daughter ions representing a ladder of fragments, each of which differs by the mass of a single amino acid. MALDI-TOF-TOF instruments characterize the sequences of peptides by using a collision cell between the two mass analyzers (Figure 3(c)).

Combined Strategies of Protein Separation and Quantitative MS The developments in the field of mass spectrometry over the past years have resulted in the identification of more than 2000 expressed proteins and the mapping of posttranslational modifications. An important step was when Hunt and colleagues pioneered the use of LC-MS/MS for the analysis of complex peptide mixtures, which is today at the core of MS-based proteomics. The combination of several protein/peptide separation steps based on different analytical properties is capable of detecting proteins of very low abundance, although considerable effort is required and a sufficient amount of sample material must be available. Relative quantification is possible by introducing stable isotopes via metabolic labeling of amino acids

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through enzymatic transfer of 18O from water to peptides or via chemical reactions using isotopecoded affinity tags or similar reagents. The most promising and well-developed method is based on pairs of isotope-coded affinity tags (ICAT), developed by Gygi and co-workers in 1999. The ICAT reagent contains a biotin moiety and a linker region with nine deuterium or hydrogen atoms. The reagent labels cysteine-containing peptides, which are enriched by binding the biotin tag to streptavidin. The tag introduces a mass shift in one of the samples by 9 Da, which can then be analyzed by dual mass spectrometry in MS mode for quantification and in MS/MS mode for protein identification. The ratio of the signal intensities can then accurately indicate the abundance ratio for the two peptides. Stable isotope dilution LC-MS/MS is commonly used to detect changes in quantitative protein profiles accurately.

Bioinformatics A common denominator of all proteomic approaches is the large amount of data that needs to be collected, making data analysis a difficult task. To enable analysis of these data, a bioinformatics approach is necessary, the main problems of which are the management, storage, and visualization of data and to draw accurate biological conclusions from the data. Computer-based storage, organization, and annotation are therefore essential to process and analyze these large datasets, especially when combining multiple experiments. To extract and compile the data into a list of identified proteins there is a need to electronically store and search within the data and to develop the data using statistical principles. Storage and compilation of these redundant data sets can be accomplished by relational databases that store the independent data in separate tables and, upon request, combine the data at desired levels.

Gene Ontology Once the data is made electronically searchable, the next daunting task becomes apparent – to be able to draw biological conclusions from the data. Even though protein sequences are easily stored and analyzed in computers, the same is not true for protein function and protein structure. To alleviate this problem, a number of classifications have been developed. One classification scheme that has recently received much attention is the Gene Ontology Project in which biological function is described by a set of defined terms organized in a hierarchical structure. Of primary importance is to reduce the complexity of the data by narrowing the number of entries

through intelligent clustering of the data in hierarchical levels. The use of public databases and software tools is essential in this process. This enables correct categorizing of the dataset, highlighting protein groups that seem to give similar matches to the perturbed state. From this, new hypotheses can be postulated and addressed, either by additional proteomic experiments or by functional validation.

Application of Proteomics in Respiratory Diseases To date, most studies related to diseases of the lung have used different body fluids such as blood, urine, and nasal lavage samples, but the most interesting results have been obtained with broncheoalveolar lavage fluid (BALF) and pulmonary edema fluid. Using animal experiments where animals are subjected to various challenges also gives relevant information. In addition, specific cell systems linked to respiratory diseases have been analyzed with and without specific perturbations. A general approach on plasma and urine proteomes has given promising results comparing controls and patients with different lung cancers. Using 1-D and 2-D mLC-MS/MS on albumin and IgG-depleted serum did enable the reliable identification of more than 100 proteins, where 16 were specific for lung adenocarcinoma. However, to reach clinical significance a greater number of patients must be analyzed. In urine, general markers for lung cancer using 2-D gels and MALDI-TOF-MS/MS indicated that proteins such as CD59 glycoprotein precursor and transthyretin may be useful markers for cancer. The use of BALF is very informative concerning several disorders of the lung such as fibrosing interstitial lung diseases, asthma, and chronic obstructive pulmonary disease (COPD). BALF has a very complex structure as proteins originate from many different cells and tissue compartments. It is very important to optimize the sample preparation regarding concentration and removal of albumin. Most identified proteins obtained by 2D-gel electrophoreses and various MS-methods are serum proteins. Interestingly, a large group of identified nonserum proteins contains those involved in immunological responses and inflammation. Other groups contain tissue repair and proliferation proteins and another contains cytoskeletal proteins. The group consisting of antioxidant proteins is also prominent. This field of research is very promising and is expected to give a lot of new basic information concerning processes of the epithelium, underlying extracellular matrix, mucus-producing cells, macrophages, and other inflammatory cells in the bronchial trees. However, the complexity

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is so great that it is unlikely that individual proteins will be suitable as markers for disease processes. Rather, sets of proteins will be useful for providing information of physiological, pathophysiological, and clinical concern. All these studies indicate that the generation of adequate results with proteomics requires well-designed experiments and well-characterized sample materials. This is especially important with clinical material where the biological diversity is large. By combining various separation techniques and MS approaches different scientific questions can be answered. A good example of study design to obtain biomarkers of specific common cell types or differentiation of cells involved in the inflammatory process is to use a global system with 2-DE followed by MALDI-TOF; this is particularly successful in identifying highly abundant proteins (10 000 copies per cell) such as cytoskeletal, scavenger, metabolic, and adhesion proteins. This approach has been used by several groups, where factors such as endothelin, transforming growth factor beta (TGF-b), and platelet-derived growth factor (PDGF) have been added to human fibroblast cell cultures to mimic remodeling processes that occur in the lung in several respiratory diseases. Related identifications as previously described have also been made in studies on BALF or cell cultures established from lung biopsies from control patients with different lung disorders like COPD, idiopathic pulmonary fibrosis (IPF), and asthma. By using nongel-based quantitative proteomic techniques involving liquid chromatography and MS/MS and protein labeling with ICAT, it is possible to identify proteins present at low levels (a few copies per cell) such as transcription and splicing factors. By using this technology it has been possible to gain insights into the role of TGF-b in splicing of mRNA. TGF-b specifically regulates splicing factors that are involved in exon inclusion of EDA in fibronectin, which is required for the differentiation of a fibroblast into a more activated type of fibroblast called a myofibroblast. Another important protein that has been associated with remodeling processes of extracellular matrix in asthma is ADAM33, a disintegrin metalloprotease that is also spliced; TGF-b might have a regulatory role in the splicing process. Thanks to the methods described above, today we have over 2000 identities of the human pulmonary fibroblast proteome, of both constituently and regulatory type. The future task is to determine the proteome directly on small biopsies taken after bronchoscopy or surgery of patients with respiratory disorders. This could be achieved with laser capture

microdissection (LCM), where the proteome could be determined from histological preparations taken from biopsies by using lasers to cut out specific regions. However, only very abundant proteins have been identified by this approach so far. The most challenging part of proteomics may be to answer scientific questions that have not been asked as yet, which gives these methods a high potential to generate new discoveries and hypotheses. Studies of the proteome so far have expanded our view of protein expression. In view of the rapid development of the techniques described above, it is likely that in the future there will be new insights into the lung proteome and pathobiology of respiratory diseases, provided well-controlled clinical material is available. See also: Bronchoalveolar Lavage. Fibroblasts.

Further Reading Aebersold R and Goodlett DR (2001) Mass spectrometry in proteomics. Chemical Review 101: 269–295. Fehninger TE, Sato-Folatre JG, Malmstro¨m J, et al. (2004) Exploring the context of the lung protoeome within the airway mucosa following allergen challenge. Journal of the Proteome Research 3: 307–320. Fujii K, Nakano T, Kanazawa M, et al. Clinical-scale highthroughput human plasma proteome analysis: lung adenocarcinoma. Proteomics (in press). Gygi SP, Rist B, Gerber SA, et al. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Biotechnology 17: 994–999. Hirsch J, Hansen KC, Burlingame AL, and Matthay MA (2004) Proteomics: current techniques and potential applications to lung disease. American Journal of Physiology. Lung Cellular and Molecular Physiology 287: L1–L23. Krutchinsky AN, Kalkum M, and Chait BT (2001) Automatic identification of proteins with a MALDI–quadrupole ion trap mass spectrometer. Analytical Chemistry 73: 5066–5077. Malmstro¨m J, Larsen K, Hansson L, et al. (2002) Proteoglycan and proteome profiling of human pulmonary fibrotic tissue utilizing miniaturized sample preparation: a feasibility study. Proteomics 2(4): 394–404. Malmstro¨m J, Larsen K, Malmstro¨m L, et al. (2004) Proteome annotations and identifications of the human pulmonary fibroblast. Journal of Proteome Research 3: 525–537. Malmstro¨m J, Tufvesson E, Bjermer L, Marko-Varga G, and Westergren-Thorsson G (2004) Remodelling of connective tissue during pathological conditions in the lung. Respiratory Medicine 18: 1–8. Malmstro¨m L, Malmstro¨m J, Marko-Varga G, and WestergrenThorsson G (2002) Proteomic 2DE database for spot selection, automated annotation, and data analysis. Journal of Proteome Research 1: 135–138. Pandey A and Mann M (2000) Proteomics to study genes and genomes. Nature 405: 837–846. Plymoth A, Lofdahl CG, Ekberg-Jansson A, et al. (2003) Human bronchoalveolar lavage: biofluid analysis with special emphasis on sample preparation. Proteomics 3: 962–972.

532 PULMONARY ALVEOLAR MICROLITHIASIS Shiio Y, Donohoe S, Yi EC, et al. (2002) Quantitative proteomic analysis of Myc oncoprotein function. EMBO Journal 21: 5088–5096. Wattiez R and Falmagne P (2005) Proteomics of bronchoalveolar lavage fluid. Journal of Chromatography B. Analytical and Technological Biomedical Life Sciences 815: 169–178.

Relevant Website http://www.geneontology.org – Gene Ontology Project website where biological function is described by a set of defined terms organized in a hierarchical structure.

PULMONARY ALVEOLAR MICROLITHIASIS G Castellana and V Lamorgese, Unita` Operativa di Pneumologia, Bari, Italy & 2006 Elsevier Ltd. All rights reserved.

Abstract Pulmonary alveolar microlithiasis (PAM) is a rare chronic disease of unknown etiology and pathogenesis, characterized by the widespread presence in the alveoli of minute calcific deposits known as microliths. The deposits are probably due to an inherited defect with autosomal recessive transmission. The disease is present worldwide and a recent review of the literature counted 576 cases. It has been most frequently reported in Europe, followed by Asia. The nation with the highest number of reported cases is Turkey, followed by Italy in second place. Cases of PAM are defined as (1) ‘familial’ when two or three, or in exceptional cases even four or five siblings are affected, and (2) ‘sporadic’ when screening of the rest of the family yields negative results. In most cases patients have mild clinical symptoms, contrasting with the severe radiographic appearance: these are characteristic features that should raise the suspicion of PAM. High-resolution computed tomography (HRCT) has made it possible to define the extent and severity of the disease more precisely. Bronchoalveolar lavage (BAL) and transbronchial biopsy (TBB) confirm the presence of microliths in the alveoli. Symptomatic cases generally feature the presence of effort dyspnea, a cough, chest pain, asthenia, finger clubbing, crepitations, and a restrictive defect revealed by lung function assessment. Macroscopic findings include markedly increased lung weight. The disease is initially endoalveolar, then involvement of the interalveolar tissue begins to appear and subsequently progression is observed, with the development of pleural calcifications, emphysema bubbles, and ossification nodules. Treatment is at present only palliative. Steroids have proved to be inefficacious while the results of administration of sodium etidronate and the BAL technique are controversial. Affected individuals may undergo progression to end-stage disease requiring lung transplantation.

Introduction Pulmonary alveolar microlithiasis (PAM) is a rare idiopathic disease characterized by the diffusion in the alveoli of innumerable minute calculi called microliths (see Figure 1). It comes under the general heading of dysmetabolic elective thesaurotic pneumoalveolitis.

Figure 1 Chest radiograph of a patient affected by PAM, showing fine microliths with a diffuse, uniform spread obscuring the cardiac and diaphragmatic borders (sandstorm lung).

The first to draw a concise, precise macroscopic description of the disease was an Italian researcher, Malpighi, in 1686, who wrote: ‘‘In vesciculis pulmonum innumeri lapilli sunt.’’ Much later, in 1918, a Norwegian, Harbitz, provided an accurate autoptic and radiological description of a second case. The third case was reported by a German, Schildknecht, in 1932, and in 1933 a Hungarian pathologist, Puhr, coined the name of PAM for the disease. Another Italian, Mariani, highlighted the clinical, functional, and radiological features in 1947. The first to report multiple cases in a single family was Mickailov from Bulgaria, in 1954. Since then, numerous reports have been made of individual and familial case histories or of small series of patients with the disease. Sosman, a radiologist, published a definitive description of the disease in 1957 and the first world review of 45 cases of PAM, extended by Perosa and Ramunni to 74 cases in 1959. The most recent worldwide review counted 576 cases. PAM is present in all continents with no clear geographic or racial distribution, although it is most prevalent in Europe, followed by Asia, particularly

PULMONARY ALVEOLAR MICROLITHIASIS 533

civilian purposes (military service) or health reasons (a higher frequency of diseases with respiratory involvement). The exceptional recent finding of analogous microliths outside the lungs, in the seminal vesicles in two patients and in the testicles in another one, raises the suspicion that the underlying metabolic disorder could also be localized in the male genital apparatus. Although cases of PAM have been described in all age groups, they are most frequently discovered in patients under 40 years of age. The youngest reported cases were premature twins and two newborns, while the most aged elderly case involved an 80-year old. Figure 2 shows the percentage of world cases of PAM subdivided by continents and Figure 3 by age at diagnosis.

Asia Minor. The nation with the highest number of recorded cases is Turkey, followed in order of prevalence by Italy, the US, India, Russia, Germany, Spain, Japan. The percentage of males versus females affected by the disease appears to be practically identical. Cases of PAM are defined as (1) ‘familial’ when two, three, or exceptionally even four, five, or six family members are affected and (2) ‘sporadic’ when screening of the family yields negative results. Sporadic cases have been found to be predominant in the male sex, whereas familial cases seem to develop more commonly in the female members. However, the higher male incidence in sporadic cases could perhaps be ascribed to more frequent X-ray examinations performed in male patients for

40 35 30 25 20 15 10 5 0 Europe

Asia

North and Central America

Africa

South America

Oceania

Figure 2 Percentage of world cases of PAM subdivided by continent.

18 16 14 12 10 8 6 4 2 0 0–12

13–20

21–30

31–40

41–50

51–60

Figure 3 Percentage of world cases of PAM subdivided by age at diagnosis.

61–70

71–80 Unspecified

534 PULMONARY ALVEOLAR MICROLITHIASIS

The disease does not affect only humans and has been described in orangutans, dogs, sheep, cats, and nude mice.

Etiology and Pathogenesis The etiology and pathogenesis are still unknown. Among those postulated, the most generally accepted etiology is an inherited metabolic abnormality, limited to the alveolar surface, involving the enzyme carbonic anhydrase. This promotes alkalinity of the alveolar surface, and hence a precipitation of calcareous salts. Research has also been made to ascertain whether a defect in calcium regulation could be responsible for the disease, but the few studies of calcium metabolism conducted in patients with PAM were unable to provide confirmation of this hypothesis. In the past, a controversy arose between the ‘nature’ and ‘nurture’ theories. One school of thought supported a hereditary disease with autosomal recessive transmission, since in familial cases the heredity is of horizontal type, in the sense that it affects members of the same sibship. The other school supported an environmental etiology, according to which the disease might be due to inhalation of given substances, as has been clearly documented in some cases. Nowadays, the consensus is that the disease is of inherited type but that exogenous factors can facilitate the formation of the microliths in the lungs. In fact, numerous family histories have been documented: family cases account for 36.56% of the total PAM cases. Horizontal sibling affection is the norm, although in two family groups first cousins were involved. Exceptionally, horizontal and vertical sibship was reported in two family groups, one featuring five patients – two sisters and three children of one of them – and the other, a woman, her brother and his child. In several family cases the parents were cousins. Two important reports supporting the hereditary hypothesis have recently been published, describing two particular family groups, one in Italy and one in Turkey, in which cases of PAM were present in several generations.

Pathology At autopsy, macroscopic findings include markedly increased lung weight, reaching maximum values of about 5 kg. The lungs are described as dark red, with a rigid consistency and granular surface, or with whitish striations. They always feature a much greater density than normal, and sometimes have a wood-hard or stone-like consistency. They are

Figure 4 Electron microscopy image of a microlith.

difficult to incise, and the operation provokes a strident noise. The cut edge is dark red and shows a uniform dissemination of grayish, shiny granules the size of pinheads: it has the appearance of sandpaper or a surface covered by a fine layer of sand. Histological findings consist of the presence of circular calcific formations (microliths), with a diameter ranging from 30 to 500 mm, inside the alveoli. Their lamellar, concentric surface features a compact central portion surrounded by successive layers arranged concentrically like an onion skin. As much as 4/5 of the alveolar surface may be involved in the microlithic process (see Figure 4). Generally, there is one microlith per alveolus that takes the shape of the cavity without deforming it. Instead, larger microliths deform the alveolus and compress the interalveolar septa, which become atrophied. The size of the microliths depends on the time since formation; smaller elements at the start of their evolution do not contain calcium deposits. Larger microliths have a very irregular, striated shape, probably resulting from fusion of smaller elements. Initial cases show a surprisingly intact lung parenchyma: in fact, the disease is initially endoalveolar and then progression is observed, with the development of pleural calcifications, emphysema bubbles, and ossification nodules. Microliths have also been observed in the interalveolar tissue, bronchioles, and lymph nodes or encapsulated in a bone tissue mass. The interalveolar septa are described as normal in initial cases but thickened in advanced cases. Several reports of more complete histochemical studies have been made, attempting to explain the pathogenesis of the disease: they have shown that the central nucleus consists of protein, neutral

PULMONARY ALVEOLAR MICROLITHIASIS 535

mucoprotein, and acid mucopolysaccharides. Contrasting results on the chemical composition of the lamellae have been obtained in different studies. Instead, there is a general consensus that the calcium content accounts for 70% of the inorganic component, while the calcium salt is hydroxyapatite. Some traces of aluminum, magnesium, iron, and silicon have also been reported.

Radiology The radiological aspects of the disease have been amply described. With the most recent technologies (e.g., computed tomography (CT), high-resolution computed tomography (HRCT), and magnetic resonance imaging (MRI)), these aspects can now be studied in greater detail. Superficial assessment of the radiological findings in patients with PAM can elicit difficulties in differential diagnosis with other diseases associated with miliary dissemination such as tuberculosis, mycosis, sarcoidosis, hemosiderosis, pneumoconiosis, and amyloidosis, which can present with diffuse opacifications, albeit with more severe symptoms. Due to the intense calcifications present in PAM, 3–5 times stronger X-ray exposure than normal is needed to obtain good radiograms demonstrating the typical picture of the disease. The radiological aspects and their evolution can be subdivided into four phases. In the early stage of the disease, known as precalcific, the radiological aspects are not yet typical due to the small number and lesser calcification of the microliths. This PAM model may fail to be recognized, also because it is occasionally observed in asymptomatic children. The second phase already shows the typical radiological picture: the lungs appear ‘sandy’, featuring diffuse, scattered micronodules with a diameter less than 1 mm and the typical calcific opacity. The microliths are typically clear-cut, shiny, with a uniform size and distributed throughout the pulmonary zones, although there tends to be greater density in the medial and inferior regions. The overall appearance resembles that of sandpaper, but the outlines of the heart and diaphragm are still clearly visible. This second calcification model is generally present in cases discovered in childhood or adolescence. By the third phase the number and volume of the opacifications have increased. The picture has become more granular, nodular, and confused due to initial thickening of the interstitial weave, that partly masks the micronodules. In the medial and inferior fields, superimposed opacifications hide the outlines of the heart and diaphragm. This third radiological model is more often seen in young adults.

By the fourth phase, the number and size of the calcific deposits have grown still more, and there is intense calcification of the interstices and sometimes pleural serosa, making the lungs appear almost entirely opaque; there is an overall aspect of ‘white lungs’ due to the diffuse presence of calcification, although the apical regions may be partially spared. Apart from interstitial calcific fibrosis, there may be paraseptal emphysema, large bubbles, or air cysts in the superior lobes, as well as pneumothorax and zones of ossification. This fourth radiological model is generally seen in older adults, or in any case in advanced cases. The extent and severity of PAM generally depend on the patient’s age and the speed of progression of the disease. Serial controls do not necessarily reveal constant deterioration of the radiological picture and the disease may remain stationary for many years. HRCT is best able to reveal the sandpaper aspect, expressing alveolitis, and smaller calcific deposits. It can also show interstitial calcification along the bronchovascular fascia, in the interlobular septa and subpleural site, especially in the dorsal and inferior regions of the parenchyma. The diffuse presence of calcification in the different pulmonary structures can be visualized by HRCT, which can demonstrate the characteristic overall appearance of ‘shiny lung’ or ‘stony lung’ (see Figure 5). As the microliths become confluent, the typical alveolar aspect of the disease with patterns of pervious bronchi surrounded by microliths becomes more and more unmistakable. HRCT is a more powerful tool than radiography and conventional CT for assessing the extent, degree of evolution and severity of the disease, and especially involvement of the secondary lobule,

Figure 5 CT scan with diffuse, ground-glass opacities (stony lung) in all pulmonary fields, and faint calcific densities, sometimes confluent at the posterior and inferior subpleural regions.

536 PULMONARY ALVEOLAR MICROLITHIASIS

pulmonary fibrosis, and the presence of pleural calcification. Moreover, HRCT can help to identify initial cases, can monitor evolution of the disease, and can replace lung biopsy because the morphological appearance delineated by this examination is sufficient for a firm diagnosis of PAM to be made.

Symptoms and Clinical Course In the cases reported between 1950 and 1960, patients affected by PAM were observed only when conditions of respiratory insufficiency had already developed, and diagnosis was most often made at autopsy. Today, approximately 50% of cases are identified when few or no symptoms have yet developed, and the findings come as a surprise in subjects undergoing chest X-rays for other reasons: checkups for military service or employment, or to exclude tuberculosis, or else for preoperative assessment or work-up for other organ disease. The disease is then confirmed by the presence of microliths in the bronchoalveolar lavage (BAL), by transbronchial biopsy (TBB), CT, bone scintigraphy with Tc99. The best diagnostic schedule for PAM is the association of BAL and HRCT; the former can document the diagnosis, whereas the latter provides further information about the degree of inflammation and/or fibrosis or calcification of the interstices. This association avoids the need for TBB, a more invasive investigation burdened by a higher complication rate. Nevertheless, if there is already a known case in the family, standard radiography of the chest and HRCT is quite sufficient in any other family member with the suspicion of the disease. In view of the rarity of the disease, after discovery of a single case, it would be helpful to screen the whole family, in the hope that in very early cases the use of therapeutic BAL could yield more promising results, analogous to those obtained in alveolar proteinosis. Regular follow-up can monitor the clinical course, demonstrating the speed of progression of the disease. In symptomatic patients the most common complaints are dyspnea, followed by cough without sputum, chest pain, and asthenia. In advanced disease there is chronic cor pulmonale, respiratory failure, and finger clubbing. Owing to the discrepancy between the severe radiological picture, showing diffuse multiple lesions in both lungs, and the relatively minor clinical symptoms, it is often difficult to pinpoint the chronological order characterizing the first phases of accumulation of the calcific deposits in the alveoli, and the clinical picture at onset and evolution of the disease. Most of the descriptions of this disease have

been case reports or case series, or some isolated reports underlining particular features. However, in five cases reported in the literature it was possible to reconstruct the time of onset of the disease, thanks to the available documentation consisting of X-rays taken some years before onset, demonstrating absence of any radiological abnormalities. Thus, the disease was not present in these patients from birth, although there have been exceptional reports of PAM in newborns and fetuses. Nevertheless, in most cases it was not possible to establish how long before, in terms of months or years, the disease started to develop. Functional assessment has most frequently shown a restrictive syndrome, whereas in the few reported initial cases the spirometric findings were generally normal.

Therapy In many cases prognosis is good: a number of reports describe patients followed up for many years during which the clinical and radiographic pictures progressed very slowly. The longest-lived patient was followed up for 50 years after diagnosis. However, less frequently the disease can evolve rapidly leading to cor pulmonale and respiratory failure. Several attempts have been made to treat this disorder, but with unsatisfactory results. Steroids were ineffective while BAL removes microliths with smaller diameters than the caliber of the alveolar ducta, but is inefficacious against intra-alveolar microliths just one size larger. Conflicting results have also been described for sodium etidronate administration. Affected individuals may progress to endstage lung disease requiring lung transplantation. There are no data currently available as to whether the disease ever recurs in patients who underwent lung transplant for this condition, as follow-up of these patients has never been reported. We believe more research is needed to find a parallel between a calcium–phosphorus metabolism disorder of the lung and the etiology of the disease. Clearly, only after its etiology and pathogenesis have been fully established may effective treatment strategies be devised. See also: Bronchoalveolar Lavage. Extracellular Matrix: Collagens. Signs of Respiratory Disease: Clubbing and Hypertrophic Osteoarthropathy.

Further Reading Arslan A, Yalin T, Akan H, and Belet U (1996) Pulmonary alveolar microlithiasis associated with calcifications in the seminal vesicles. Journal of Belge Radiologie 79: 118–119.

PULMONARY CIRCULATION 537 Castellana G, Castellana R, Fanelli C, Lamorgese V, and Florio C (2003) La microlitiasi alveolare polmonare: decorso clinico e radiologico, convenzionale e HRCT, in tre casi. Ipotesi di classificazione radiologica della malattia. La Radiologia Medica 106: 160–168. Castellana G, Gentile M, Castellana R, Fiorente F, and Lamorgese V (2002) Pulmonary alveolar microlithiasis: clinical features, evolution of the phenotype, and review of the literature. American Journal of Medical Genetics 111: 220–224. Castellana G and Lamorgese V (1997) La microlitiasi endoalveolare polmonare. Caso clinico a sostegno dell’ipotesi ereditaria. Rassegna Di Patologia Dell’Apparato Respiratorio 12: 247–251. Castellana G and Lamorgese V (1998) La microlitiasi alveolare polmonare: rivisitazione della casistica italiana. Rassegna Di Patologia Dell’Apparato Respiratorio 13: 405–407. Castellana G and Lamorgese V (2003) Pulmonary alveolar microlithiasis. World cases and review of the literature. Respiration 70: 549–555. Chang YC, Yang PC, Luh KT, Tsang YM, and Su CT (1999) High-resolution computed tomography of pulmonary alveolar microlithiasis. Journal of Formosan Medical Association 98: 440–443. Chinachoti N and Tangchai P (1957) Pulmonary alveolar microlithiasis associated with inhalation of snuff in Thailand. Diseases of the Chest 32: 687–689.

Edelman JD, Bavaria J, Kiaser LR, et al. (1997) Bilateral sequential lung transplantation for pulmonary alveolar microlithiasis. Chest 112: 1140–1144. Mariotta S, Ricci A, Papale M, et al. (2004) Pulmonary alveolar microlithiasis: report on 576 cases published in the literature. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 21: 173–181. Moran CA, Hochholzer L, Hasleton PS, Johnson FB, and Koss MN (1997) Pulmonary alveolar microlithiasis. A clinicopathologic and chemical analysis of seven cases. Archives of Pathology & Laboratory Medicine 121: 607–611. Perosa L and Ramunni M (1959) La microlitiasi endoalveolare del polmone. Recenti Progressi in Medicina 26: 353–429. Senyigit A, Yaramis A, Gurkan F, et al. (2001) Pulmonary alveolar microlithiasis: a rare familial inheritance with report of six cases in a family. Respiration 68: 204–209. Sosman MC, Dodd GD, Jones WD, and Pillmore GU (1957) The familial occurrence of pulmonary alveolar microlithiasis. American Journal of Roentgenology 77: 947–1012. Sosman MC, Dodd GD, Jones WD, and Pillmore GU (2004) The familial occurrence of pulmonary alveolar microlithiasis. Lung Disease 21: 173–181. Ucan ES, Keyf AI, Aydilek R, et al. (1993) Pulmonary alveolar microlithiasis: review of Turkish reports. Thorax 48: 171–173.

PULMONARY CIRCULATION L A Shimoda, Johns Hopkins School of Medicine, Baltimore, MD, USA

Anatomy, Structure, Histology of the Pulmonary Circulation

& 2006 Elsevier Ltd. All rights reserved.

Abstract The pulmonary vasculature is unique, both in volume and function. The pulmonary circulation, a low-pressure vascular bed that accommodates the entire cardiac output, carries the mixed venous blood to the alveoli, where gas exchange occurs, and then back to the left heart for distribution of oxygenated blood to the rest of the tissues in the body. As compared with the systemic circulation, the pulmonary arteries have thinner walls with much less vascular smooth muscle. Moreover, in order to maintain the low pulmonary arterial pressure, normal pulmonary vascular resistance is approximately one-tenth that of the systemic circulation. Factors that control pulmonary blood flow include vascular structure, gravity, mechanical effects of breathing, and the influence of neural and humoral factors. A unique aspect of the pulmonary circulation is the pressor response to hypoxia, as the systemic circulation dilates in response to decreased oxygen concentrations. In addition to gas exchange, the pulmonary circulation also serves to filter the blood, removing microemboli, and participates in the metabolic regulation of a variety of vasoactive hormones. Several diseases can affect the function of the pulmonary circulation, including primary and secondary pulmonary hypertension, arteriovenous malformation, embolism, and fibrotic lung disease.

The pulmonary circulation encompasses the circuit by which deoxygenated blood from the right heart enters the lungs via the pulmonary arteries, is channeled through the alveolar/capillary units where the blood is oxygenated, and is returned to the left side of the heart by the pulmonary veins for distribution to the systemic circulation. The anatomy of the pulmonary circulation differs in several respects from that of the systemic circulation. Blood exits the right ventricle into the main pulmonary artery, the diameter of which is similar to that of the aorta, but it has thinner walls. During gestation and immediately following birth, the pulmonary artery is nearly identical to that of the aorta, but, postnatally, the elastic tissue gradually diminishes. The main pulmonary artery divides into the right and left main branches. Each main branch further divides to supply each lobe before entering the lung. Within the lung, each lobar artery subdivides into rather irregular branches corresponding to the bronchial tree. The close proximity of the pulmonary arteries and airways underscores the relationship

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between ventilation and perfusion that defines the normal function of the lung. The large pulmonary arteries (43000 mm in diameter) are classified as elastic, with the media comprised primarily of elastic fibers and some smooth muscle. As vascular diameter decreases these elastic arteries gradually give rise to vessels with increased smooth muscle content. In general, arteries between 3000 and 150 mm in diameter can be considered muscular arteries, but are still more thin walled than systemic arteries of the same diameter. Pulmonary arteries also exhibit a thin intima comprised of endothelial cells, collagen, and fibroblasts and a longitudinal elastic lamina, which allows for expansion during inspiration. The small arterioles have a nonuniform smooth muscle cell layer, giving way to the small nonmuscular preacinar arterioles, which are located proximal to terminal bronchi. At the alveoli, the terminal arterioles break into a network of pulmonary capillaries within the alveolar walls. The capillaries have a very thin wall (approximately 2 mm) consisting of a single layer of endothelial cells and contain most of the surface area of the pulmonary vasculature. Indeed, the maximal surface area of the capillary network is approximately 20 times that of the rest of the pulmonary circulation. Gas exchange between the alveolar gas and blood takes place within the pulmonary capillary bed after which the blood flows into venules, which are indistinguishable in structure from arterioles. However, while each small arteriole supplies a specific unit of respiratory tissue, the venules drain several portions of the lung. Venules do not follow the bronchial tree and unite to form the pulmonary veins, which conduct the oxygenated blood into the left ventricle. Although 99% of the lung blood flow passes through the pulmonary circulation, 1% is carried by the bronchial circulation, which supplies oxygenated blood from the systemic circulation to the lung. Similar to the pulmonary circulation, branching of the intrapulmonary bronchial arteries along the length of the bronchial tree results in a vast network of capillaries, which form extensive anastomoses with the pulmonary vasculature. Owing to these multiple connections with the pulmonary circulation, the deoxygenated bronchial venous blood drains via pulmonary veins to the left heart, producing an anatomic right-to-left shunt. The small pulmonary vessels, including the capillaries, can be subdivided into extra-alveolar or alveolar based on the pressures to which they are subjected. In addition to the intravascular pressure, vessels in the lung are exposed to alveolar pressure and pressure exerted by lung tissue connections. Both of these forces increase with lung inflation; however,

they act in opposite directions, with alveolar pressure directed inward and tissue pressure directed outward. The alveolar vessels are surrounded by alveolar pressure, due to localization within the septal wall. The extra-alveolar vessels are surrounded by septa and are typically contained in a sheath of connective tissue. In addition to the effects of alveolar pressure, the connection of these vessels to lung parenchyma subjects them to substantial tissue forces. These vessels include a subset of the pulmonary capillaries called the corner vessels, which are located in the alveolar parenchyma at the alveolar corners.

Pulmonary Circulation in Normal Lung Function Physiological Function

The pulmonary vasculature is unique, both in capacity and function. Responsible for several physiological functions, the primary function of the pulmonary circulation is exchange of gases, adding oxygen and removing carbon dioxide from mixed venous blood. Under normal conditions, gas exchange occurs primarily in the alveolar capillaries, where blood flow rapidly equilibrates with the alveolar air. The average transit time for red blood cells within the capillary network is approximately 0.5–1 s. Since the volume of the capillary bed is roughly equal to stroke volume, the entire capillary volume is exchanged with each heartbeat. In addition to gas exchange, the lung also filters the blood, preventing thrombi and other microemboli from entering the systemic circulation. Moreover, since the entire circulating blood flow passes through the lungs, the pulmonary circulation plays an important role in performing metabolic functions. For example, the pulmonary endothelium contains an abundance of angiotensin-converting enzyme, and is the major site in the body for conversion of angiotensin I to the active vasopressor angiotensin II (ANG II) and, conversely, for inactivation of the vasodilator bradykinin. Other functions include supplying nutrients to the alveoli and acting as a blood reservoir to transiently support left ventricle output during periods of decreased right ventricular output. Regulation of Blood Flow

Vascular resistance The pulmonary circulation is the only circulation in the body to conduct the entire cardiac output, and the only one in which the arteries carry oxygen poor blood. In utero, oxygen is delivered to the fetus via the placenta and the pulmonary circulation is a high-resistance circuit, with little blood flow. Perinatally, with the commencement of

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ventilation, pulmonary vascular resistance falls, and blood flow increases 10-fold. In the adult, normal pulmonary artery pressures are approximately 25 mmHg systolic and 10 mmHg diastolic, while pulmonary venous pressure is approximately 9 mmHg. Given the relationship between blood flow, pressure, and resistance, which by Ohm’s law is defined as: pulmonary arterial
venous pressure ¼ pulmonary blood flow=pulmonary vascular resistance and the large volume of blood flow that must be accommodated (approximately 5 l min
1 at rest), the low pulmonary arterial pressure required to maximize the efficiency of the right ventricle work load must be maintained by low pulmonary vascular resistance, which is approximately one-tenth that of the systemic circulation. In the normal lung, significant increases in cardiac output have very little effect on pulmonary artery pressure. Indeed, a rapid 30% change in volume in moving from lying to standing, or a doubling of blood flow during exercise, can be accommodated with little rise in pulmonary arterial pressure. In order to maintain this low-pressure system, increases in cardiac output must be balanced by a reduction in pulmonary vascular resistance (Figure 1). This is possible because the pulmonary vasculature is highly distensible and possesses a significant reserve capacity. Decreases in pulmonary vascular resistance in response to increases in blood flow are not mediated by alterations in vascular tone, but are due to two passive processes: recruitment and distension (Figure 2). With an increase in blood flow, pressure rises transiently and opens or recruits capillaries and other small vessels that had been closed during resting

conditions due to insufficient intravascular pressure. This increase in vascular pressure also causes distension or expansion of individual capillaries. Both of these processes reduce pulmonary vascular resistance and minimize any flow-induced increase in pulmonary vascular pressure. Lung volume The resistance of both the alveolar and extra-alveolar vessels is affected by alveolar volume. Since these vessels are exposed to different surrounding pressures, their resistance is differentially regulated by changes in lung volume, leading to a complex relationship between lung volume and pulmonary vascular resistance (Figure 3). The extraalveolar vessels are positioned such that they become enlarged with increased lung volume (i.e., during inspiration) due to the fall in pleural pressure, which results in increased transmural pressure, and the pull exerted by the alveolar septa. Conversely, the increase in alveolar volume during inspiration results in greater air pressure compared to vascular pressure

Distension

Recruitment

Pulmonary vascular resistance

Figure 2 Schematic demonstrating the effects of recruitment and distension on capillary flow.

Pulmonary vascular resistance

Total

Alveolar

Extra-alveolar

Mean pulmonary arterial pressure Figure 1 Diagram illustrating the inverse relationship between pulmonary arterial pressure and resistance.

Lung volume Figure 3 Variation in pulmonary vascular resistance as a function of lung volume.

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and stretches the alveolar walls, causing the pulmonary capillaries to be compressed and elongated, thus increasing the resistance of these vessels. During expiration, lung volume falls and the process is reversed. Below functional residual capacity (FRC), extra-alveolar resistance is high due to lack of tension in the parenchyma, while above FRC the alveolar vessels collapse and the net effect of alveolar and extra-alveolar changes is that pulmonary vascular resistance increases with lung inflation. Thus, pulmonary vascular resistance is lowest near FRC and progressively rises with both increasing and decreasing lung volume. Blood flow heterogeneity Within the lung, blood flow is not uniform. The distensibility, compressibility, and low intravascular pressure that characterize the pulmonary circulation cause pulmonary blood flow and pulmonary vascular resistance to be influenced by factors that are independent of vascular smooth muscle tone, including both gravitational and structural factors. For example, within a column of fluid, the weight of the fluid exerts a higher pressure at the bottom than near the top. Similarly, in upright individuals, the vertical height of the lung is approximately 24 cm (at FRC), and while alveolar pressure is fairly uniform throughout the lung, intravascular pressure increases in the bottom (dependent) portions of the lungs due to gravity-dependent hydrostatic effects. This pressure gradient causes progressive vascular distension, decreased resistance, and increased flow at the bottom (base) of the lung.

Based on the relationship between pulmonary arterial pressure (PPA), pulmonary venous pressure (PV), and alveolar pressure (Palv), the lung can be divided into three zones (Figure 4). In zone 1, hydrostatic effects cause both arterial and venous pressures to fall below alveolar pressure such that Palv4PPA4PV and the alveolar vessels are completely collapsed, restricting blood flow to only the corner vessels. Under zone 2 conditions, PPA4 Palv4PV and the alveolar vessels are partially collapsed and the driving force for flow is the pressure gradient between arterial and alveolar pressures (PPA–Palv). In zone 3, PPA4PV4Palv, all of the alveolar blood vessels are fully open, and blood flow is driven by the difference between pulmonary arterial and venous pressure (PPA
PV). In a seated or standing normal subject, zone 1 conditions are typically absent but, if present, will be found at the apex of the lung. Conversely, at the bottom of the lung, blood flow is greatest and zone 3 conditions are present, while zone 2 conditions can be found in the mid-lung. In addition to gravitational forces, the extent and location of the lung zones vary with body position. In a supine subject, zone 1, if present, will be in the ventral portion of the lungs while flow increases to the anatomically dependent (dorsal) regions of the lungs, resulting in zone 3. When a subject lies on their side, blood flow increases to the dependent lung. In certain cases, alveolar pressure may exceed vascular pressure in the nondependent portions of the lung. For example, zone 1 and 2 regions can be created and/or increased during hemorrhage or hypovolemia,

Zone 1 Palv>PPA>PV

PPA>Palv>PV

Height

Zone 2

Zone 3 PPA>PV>Palv

Blood flow Figure 4 Diagram illustrating the zone 3 model describing regional variation in pulmonary blood flow. PPA, arterial pressure; PV, venous pressure; Palv, alveolar pressure. Adapted from West JB, Dollery CT, and Naimark A (1964) Distribution of blood flow in isolated lung: relation to vascular and alveolar pressures. Journal of Applied Physiology 19: 713–724.

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which result in low intravascular pressures, or during positive pressure ventilation or forced expiration, where alveolar pressure is increased. Regional blood flow differences can exist even between alveoli with similar vertical position, indicating that blood flow heterogeneity is not entirely dependent on the effects of gravity. At the microvascular level, the branching nature of the pulmonary vascular tree results in structural heterogeneities within isogravitational planes, producing variations in local driving pressures and resistances. This heterogeneity in driving pressure creates gravity-independent differences in regional pulmonary blood flow. Regulation of Pulmonary Vascular Tone

Factors that influence vascular smooth muscle cell tone, including neural and circulating factors and oxygen concentration, are potent regulators of both pulmonary vasomotor responses and vascular caliber. Nervous control The pulmonary circulation is supplied with both sympathetic and parasympathetic innervation. In general, increased sympathetic activity leads to release of catecholamines (e.g., dopamine, norepinephrine, epinephrine, and neuropeptide Y) that cause vasoconstriction and an increase in pulmonary vascular resistance. Pulmonary arteries contain fewer cholinergic than adrenergic nerve fibers. Parasympathetic stimulation causes the release of acetylcholine and vasoactive intestinal polypeptide, which mediate vascular dilation and a decrease in pulmonary vascular resistance. The lung also contains nonadrenergic, noncholinergic nerves that can be excitatory (e-NANC) or inhibitory (i-NANC). Release of vasoactive intestinal peptide, calcitonin generelated peptide, substance P, and nitric oxide from i-NANC nerves mediates vasodilation, while the e-NANC nerves mediate vasoconstriction, although the neurotransmitter involved remains unclear. Curiously, in contrast to the systemic vasculature, there appears to be minimal nervous control in the pulmonary circulation with respect to basal vascular caliber. Moreover, while the existence and activity of e-NANC and i-NANC nerves have been demonstrated in vitro, regulation of tone in vivo by these nerves has not been demonstrated. However, stimulation of adrenergic nerves may modulate pulmonary vascular resistance and blood flow during exercise and cold exposure and may increase in regulatory contribution during pathological states, particularly during pulmonary edema and embolism. Humoral factors A number of humoral factors can participate in active regulation of pulmonary

vascular tone. Some of these factors are endogenous, derived from the vascular endothelium, while others are produced by circulating cells or in other vascular beds. If the changes in vasomotor tone induced by these factors are not uniform, significant redistribution of blood flow can occur. Vasodilators Factors that induce pulmonary vasodilation include nitric oxide and nitrites, adenosine, bradykinin, atrial natriuric factor (ANP), and the eicosanoids prostaglandin E1 (PGE1) and PGI2 (prostacyclin). Nitric oxide and PGI2 are produced by the endothelium and are the most studied of the pulmonary vasodilators. Both have a short half-life and are quickly metabolized. Thus, neither is suited for action as a circulating factor and instead, once released by endothelial cells, quickly diffuse to the underlying smooth muscle and cause relaxation via stimulation of cGMP and cAMP. Bradykinin, a product of the renin–angiotensin system, exerts its dilatory influence by stimulating nitric oxide release. Indeed, in vitro, removal of the endothelium results in a loss of dilation in response to bradykinin, with vasoconstriction observed in some cases due to activation of receptors on the smooth muscle. ANP is a peptide produced primarily by stretch of the right heart, as occurs with an increase in pulmonary artery pressure. Given that the pulmonary circulation is the first vascular bed to see ANP, it is not surprising that plasma levels of ANP are 30% greater in the pulmonary than systemic circulation. Additionally, pulmonary arteries appear to be significantly more sensitive to the vasodilatory effects of ANP than arteries from other vascular beds. Although all of the aforementioned factors have been demonstrated to produce changes in vasomotor tone, only PGI2 and nitric oxide appear to regulate basal pulmonary vascular tone in the normal lung. Under pathological conditions, however, modulation of tone may be more pronounced. Vasoconstrictors Pulmonary vasoconstriction is caused by serotonin, endothelin-1 (ET-1), ANG II, histamine, and prostaglandins. Several of these factors are derived from the vascular endothelium. For example, arachidonic acid, which is readily taken up in the pulmonary circulation, is metabolized into a number of vasoactive eicosanoids, including PGE2, PGF2a, thromboxane, and leukotrienes, all of which diffuse to the smooth muscle and cause contraction. The lung endothelium is also the primary site of metabolism of angiotensin I, with approximately 60– 80% of plasma angiotensin I converted to ANG II, the vasoactive form of the peptide, in a single pass through the pulmonary circulation. ET-1 is perhaps

542 PULMONARY CIRCULATION 2% O2

PPA (mmHg)

30 25 20 15 10

15

PPA

10 Pt

5 0

Hypoxia One of the most unique aspects of the pulmonary circulation is the hypoxic pressor response. Unlike the systemic vasculature, which dilates in response to hypoxia in order to increase blood flow and oxygen delivery to tissues, alveolar hypoxia causes profound pulmonary vasoconstriction. Pulmonary vascular resistance rapidly increases as oxygen tension decreases, beginning within 1– 2 min after a drop in oxygen levels and reaches maximal response within 5 min (Figure 5). Hypoxic pulmonary vasoconstriction is maintained for the duration of hypoxia, and rapidly reverses (within 1 min) with a return to normoxia. When hypoxia is localized, this mechanism is thought to divert blood flow from regions of the lung that are poorly ventilated, helping to maintain arterial oxygen tension. However, in chronic lung disease, where alveolar hypoxia is global and prolonged, pulmonary hypertension develops. The exact mechanism underlying the hypoxiainduced increase in pulmonary vascular tone is unknown. Several factors have been shown to modulate the response, including prostaglandins, ANG II, serotonin, and leukotrienes, although these have all been ruled out as mediators of the response. Hypoxia has a direct effect on pulmonary vascular smooth muscle, although the maximal contractile response requires alterations in the release of the endothelial cell-derived mediators nitric oxide (decreased) and ET-1 (increased). Hypoxic pulmonary vasoconstriction is not mediated through the autonomic nervous system, as the response can be observed in isolated, perfused lungs that lack nervous input. Although the large pulmonary arteries are capable of responding to hypoxia, it is generally accepted

5 min

(a)

P (mmHg)

the most potent endogenous vasoconstrictor in the lung. While ET-1 can elicit nitric oxide production and transient dilation when receptors on the endothelial cells are activated, its main action is vasoconstriction mediated by receptors on the smooth muscle cells. Synthesized in the pulmonary endothelium, ET-1 secretion has been shown to increase in response to shear stress and hypoxia. Indeed, enhanced ET-1 levels are believed to contribute to the development of pulmonary hypertension. Circulating cells are also an important source of vasoconstrictors that can act on the pulmonary circulation, including serotonin, a primary product of activated platelets, and histamine, produced by mast cell granules. While these factors do not appear to be involved in basal regulation of tone, vasoconstrictors can modulate tone under pathological conditions, such as in pulmonary hypertension induced by anorexic agents, where serotonin uptake is impaired.

10 s

(b) Figure 5 (a) Effect of hypoxia on pulmonary arterial pressure in an isolated perfused rat lung model. In this preparation, left atrial pressure is negative and changes in pulmonary arterial pressure reflect changes in pulmonary vascular resistance. (b) Expanded scale showing the effect of ventilation on pulmonary arterial pressure. PPA, arterial pressure; Pt, tracheal pressure. Courtesy of J T Sylvester.

that the main site of increased resistance during hypoxic pulmonary vasoconstriction is in the small, muscular arterioles. There is evidence that postcapillary hypoxic venoconstriction may also occur, although the magnitude of the contribution of these vessels to the increase in total pulmonary vascular resistance is uncertain. Our understanding of the pulmonary circulation has been greatly aided by evaluation of pulmonary function in a variety of species, including sheep, rats, dogs, cats, ferrets, mice, cattle, guinea pigs, goats, and rabbits. Despite the obvious size differences, the pulmonary circulation across species is quite similar. For example, all mammals respond to hypoxic challenge with HPV, although the degree of the response varies from minor (rabbits) to robust (cattle). In developing animal models of human disease, the most widely used is the mouse. Although the mouse appears to lack a bronchial circulation, major advantages of this model include small size and ease of maintenance, quick availability, and reduced genetic variability. Perhaps the most important advantage of murine models is the ability to alter gene expression, providing a powerful tool to explore the functional role of specific proteins in physiological and pathophysiological conditions.

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Pulmonary Circulation in Respiratory Diseases

obstructive sleep apnea, may also result in pulmonary hypertension.

Primary Pulmonary Hypertension

Interstitial Lung Disease

Pulmonary vascular disease may be primary or secondary to other disorders of the lung or other organs. Primary pulmonary hypertension (PPH), also known as idiopathic pulmonary arterial hypertension, is a disease of unknown etiology, although certain cases have been linked to appetite suppressants. PPH is characterized by an elevated resting pulmonary artery pressure that increases dramatically during exercise. Increased pulmonary vascular resistance, due in part to pulmonary arteriolar obstruction with hypertrophy of wall elements, necrotizing arteritis, and/or endothelial cell lesions increases pulmonary arterial pressure, causing right ventricular hypertrophy in the absence of any other cardiac abnormality. Although the underlying cause of PPH remains unclear, dysfunction of the pulmonary endothelium, which is a rich source of both vasodilators and vasoconstrictors, may contribute. In the normal lung, the balance of vasodilators to vasoconstrictors favors low pulmonary vascular resistance. However, with endothelial cell dysfunction, release of the vasodilators nitric oxide and PGI2 is impaired while release of the vasoconstrictors ET-1 and thromboxane may be augmented, resulting in a net vasoconstriction. In addition, excessive endothelial cell proliferation may obliterate the lumen of small arteries and alveolar vessels, further increasing vascular resistance.

Interstitial lung disease refers to a diverse group of diseases with the common feature of alterations in the alveolar interstitial space, most commonly septal destruction and fibrosis. While interstitial lung disease will produce fibrosis of the vessels, early in the disease process only a small part of the pulmonary arterial tree is involved, and hypoxemia during exercise, due to reduced diffusion capacity and/or ventilation–perfusion mismatch, is commonly the only alteration in lung function. As the disease progresses to later stages, resting hypoxemia may be observed along with subsequent pulmonary hypertension.

Secondary Pulmonary Hypertension

Secondary pulmonary hypertension and right heart failure are common complications of chronic lung diseases, including emphysema, chronic bronchitis, cystic fibrosis, and severe chronic asthma. In this case, elevated pulmonary arterial pressure is caused by a combination of hypoxic pulmonary vasoconstriction, polycythemia, alveolar hypercapnia and acidosis, and raised intra-alveolar pressure during expiration. Global hypoxemia is also a consequence of residence at high altitude. The decrease in vascular caliber associated with prolonged hypoxia is comprised of both a reversible and fixed component. The reversible component is due to sustained active contraction of the pulmonary vascular smooth muscle while the fixed component is due to structural remodeling of the pulmonary circulation, primarily increased muscularization of the small pulmonary arteries. In addition to chronic lung diseases that produce continuous alveolar hypoxia, in recent years it has become clear that prolonged exposure to intermittent hypoxic episodes, as occurs in

Pulmonary Edema

Pulmonary edema can occur as a consequence of heart failure or lung microvascular injury and leads to an increase in vascular resistance. With left ventricular failure, elevated diastolic pressure results in elevated pulmonary venous pressure, which interferes with normal capillary blood flow and increases capillary pressure. The accumulation of fluid in the interstitial compartment around the vessels can lead to impaired gas exchange or, more commonly, can exaggerate the effects of lung volume on vascular resistance and affect blood flow distribution. In more severe cases, when the interstitial spaces are filled, or when the endothelial lining of the lung microvasculature is injured, alveolar flooding can occur. In either case, excess fluid in the alveoli results in marked deterioration in gas exchange. In some cases, alterations in vasomotor tone can cause edema, independent of microvascular injury or cardiac function. For example, excessive vasoconstriction in response to hypoxia is believed to be the underlying cause of high-altitude pulmonary edema. Arteriovenous Malformation and Stenosis

A pulmonary arteriovenous malformation is a direct communication between a pulmonary artery and a pulmonary vein producing a right-to-left shunt. These malformations can occur as a consequence of liver dysfunction, genetic abnormalities, as with Osler– Weber–Rendu disease, or are idiopathic. Pulmonary arteriovenous malformations are not uncommon, and in one-third of cases, are multiple. In addition to pulmonary hypotension, capillary dilation and extensive shunting results in impaired gas exchange and hypoxemia. Pulmonary vascular stenoses, although rare, occur most commonly at the bifurcation of the main

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pulmonary artery. The increase in pulmonary vascular resistance caused by the narrowing of the artery results in right ventricular hypertension. Stenoses in the left heart valves lead to passive pulmonary venous hypertension. The elevation in venous pressure results in elevated capillary and arterial pressures. Pulmonary Embolism

Pulmonary embolism is perhaps the most common pulmonary vascular disease. Clots originate in the systemic veins, often in the deep veins of the lower extremities, and formation is augmented following injury, venous stress, and hypercoagulable states. The thrombi detach and become lodged in the pulmonary arterial circulation. Occasionally, the right side of the heart is a source of a pulmonary embolus. Given the large reserve capacity of the pulmonary capillaries, many thromboemboli can go undiagnosed, and resolve quickly. Massive blockages produce a functional decrease in cross-sectional area of the pulmonary circulation, resulting in a significant increase in pulmonary vascular resistance and elevated pulmonary arterial pressure. Subsequent right ventricular strain decreases cardiac output and, if severe, can result in death. In approximately one-tenth of patients, large thromboemboli cause local cessation of flow and ischemic necrosis of the lung parenchyma (pulmonary infarction). See also: Bronchial Circulation. Diffusion of Gases. Endothelial Cells and Endothelium. High Altitude, Physiology and Diseases. Hypoxia and Hypoxemia. Oxygen–Hemoglobin Dissociation Curve. Peripheral

Gas Exchange. Pulmonary Vascular Remodeling. Smooth Muscle Cells: Vascular. Ventilation: Uneven. Ventilation, Perfusion Matching.

Further Reading Bakhle YS and Vane JR (1974) Pharmacokinetic function of the pulmonary circulation. Physiological Reviews 54(4): 1007–1045. Crofton J and Douglas A (eds.) (1975) The pulmonary circulation. In: Respiratory Diseases, 2nd edn., pp. 34–37. Philadelphia: Lippincott Co. Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) (1997) The Lung: Scientific Foundations, 2nd edn., sect. 5. New York: Lippincott-Raven. Dawson CA (1984) Role of pulmonary vasomotion in physiology of the lung. Physiological Reviews 64(2): 544–616. Hlastala MP and Glenny RW (1999) Vascular structure determines pulmonary blood flow distribution. News in Physiological Sciences 14: 182–186. Keith IM (2000) The role of endogenous lung neuropeptides in regulation of the pulmonary circulation. Physiological Research 49(5): 519–537. McMurtry IF (1986) Humoral control. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, pp. 83–125. New York: Churchill-Livingstone. Nadel JF and Nadel JA (eds.) (1988) Textbook of Respiratory Medicine. Philadelphia: WB Saunders. Peacock AJ (ed.) (1966) Pulmonary Circulation. London: Chapman and Hall Medical. Sylvester JT and Brower RG (1990) Pulmonary blood flow. In: Stein JH (ed.) Internal Medicine, 3rd edn., pp. 583–586. Boston: Little, Brown and Co. Ward JPT and Aaronson PI (1999) Mechanisms of hypoxic pulmonary vasoconstriction: can anyone be right? Respiratory Physiology 115(3): 261–271. West JB, Dollery CT, and Naimark A (1964) Distribution of blood flow in isolated lung: relation to vascular and alveolar pressures. Journal of Applied Physiology 19: 713–724.

PULMONARY EDEMA M A Matthay and T E Quinn, University of California, San Francisco, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pulmonary edema refers to the abnormal collection of fluid in the extravascular spaces of the lung such as the interstitium and the alveoli. Its two main pathophysiologic mechanisms are increased hydrostatic forces within the lung microvasculature and increased microvascular permeability. Understanding the pathophysiology of pulmonary edema requires a firm understanding of normal lung fluid balance. The Starling equation, which describes the net flow of fluid across a semipermeable membrane, applies to the filtration of fluid from the pulmonary microvasculature into the pulmonary interstitium. Interstitial fluid is primarily removed by the lung lymphatic vessels, and alveolar

fluid is removed via active transport mechanisms. Pulmonary edema occurs because of either increased hydrostatic forces or increased vascular permeability which then causes an increase in fluid filtration sufficient to overwhelm fluid removal mechanisms. The treatment of hydrostatic pulmonary edema targets a reduction in pulmonary microvascular pressure with diuretics, vasodilators, and sometimes inotropic agents. The treatment of increased permeability pulmonary edema is mainly supportive. Mechanical ventilation of patients with increased permeability pulmonary edema should be performed with a low tidal volume, lung-protective strategy.

Introduction Pulmonary edema refers to the abnormal collection of fluid in the extravascular spaces of the lung such as the interstitium and the alveoli. Its two main

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pathophysiologic mechanisms are increased hydrostatic forces within the lung microvasculature and increased microvascular permeability. Pulmonary edema from either mechanism may lead to profoundly diminished lung function, respiratory distress, and even death.

Normal Lung Fluid Balance Starling Forces

Understanding the mechanisms of pulmonary edema formation requires a firm understanding of normal lung fluid balance. The Starling equation describes the net flow of fluid across a semipermeable membrane. Applied to the lung microvasculature, it can be written as follows: Qf ¼ K½ðPmv
Pi Þ
sðpmv
pi Þ where Qf is the net filtration of fluid from the lung microvasculature to the interstitium, K is the filtration coefficient or leakiness of the endothelium to water, Pmv is the pulmonary microvascular pressure, Pi is the pulmonary interstitial pressure, s is the protein reflection coefficient, pmv is the protein osmotic pressure in the microvasculature, and pi is the interstitial protein osmotic pressure. Under normal conditions, the microvascular endothelium is somewhat leaky to fluid, and the pulmonary interstitial pressure is slightly negative. On the other hand, the endothelium is quite impermeable to proteins with a reflection coefficient near 1. Thus, the fluid filtered from the micro vessels is low in protein compared to the fluid in the vascular space. On balance, there is normally a small net flow of fluid from the lung microvasculature to the interstitium. Lung Fluid Clearance and Protective Mechanisms

Interstitial fluid clearance As described above, fluid is filtered into the lung interstitium from the microvasculature under normal conditions. This fluid must be constantly removed to maintain homeostasis. The most important mechanism for lung fluid removal is the lung lymphatic system. The pulmonary interstitium is rich with lymphatic channels which absorb filtered fluid and protein and convey it along channels in the interlobular septa to the mediastinal lymphatic vessels. Another mechanism of fluid removal from the lung under normal conditions is the direct reabsorbtion of fluid into the pulmonary venules. Since the normal filtered fluid is low in protein (low pi) and the intravascular fluid is protein rich (high pmv), Starling forces along the venules, where intravascular hydrostatic pressure (Pmv) is normally low, will favor reabsorbing fluid. Two other pathways

of lung fluid clearance, which are less important under normal conditions, include filtration through the visceral pleura into the pleural space, and movement of fluid via the loose peribronchovascular connective tissue into the mediastinum. Under pathological conditions in which excess fluid is filtered into the pulmonary interstitium, the above described mechanisms become safety factors against the development of pulmonary edema and alveolar flooding. The lymphatic system’s rate of liquid clearance can increase up to 10 times normal when hydrostatic forces increase. Second, as increased fluid is filtered into the perimicrovascular space, pressure in that space (Pmv) increases and protein concentration falls (decreased pi). These changes in the perimicrovascular space oppose further fluid accumulation. Third, the loose peribronchovascular connective tissue has a high capacitance, allowing a large amount of fluid accumulation (up to 500 ml) before tissue pressure rises. Finally, if fluid accumulation in the pulmonary interstitium continues, pressure in that space will increase enough to allow filtration across the visceral pleura and the formation of a pleural effusion. A large amount of fluid can sequester in the pleural space before lung function is sufficiently diminished to impact gas exchange. Alveolar fluid clearance In the presence of interstitial pulmonary edema, the alveoli are protected from flooding by two main mechanisms. In contrast to the relatively leaky microvascular endothelium, the alveolar epithelium has tight junctions which render it quite impermeable to interstitial fluid and protein. However, if edema fluid accumulation exceeds the capacity of the interstitial clearance mechanisms for long enough, the alveolar epithelial barrier changes, and alveolar flooding occurs. If the edema is formed from increased hydrostatic pressure, the alveoli are flooded with edema fluid that has a low protein concentration relative to plasma. If the edema is caused by increased permeability (acute respiratory distress syndrome (ARDS)), the alveoli are flooded with protein-rich fluid. In either case, alveolar fluid clearance then becomes vital to the restoration of lung function. Alveolar fluid clearance occurs primarily by active sodium transport. The alveolar type II cells are probably responsible for most of the active sodium transport out of the alveolus. Sodium is transported into the type II cell via an amiloride-sensitive epithelial sodium channel (ENaC) on the apical surface. The oubain-sensitive Na–K ATPase on the basolateral membrane surface then transports the sodium out of the cell. As an osmotic gradient forms, water flows out of the alveolus, possibly via water channels

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called aquaporins. In this fashion, water is rapidly cleared from flooded alveoli.

Pulmonary Edema Pathogenesis Pulmonary edema forms from two main mechanisms, increased microvascular pressure (Pmv) and increased microvascular permeability. The most common reason for increased Pmv is elevated left atrial pressure (cardiogenic pulmonary edema). The most common reasons for elevated left atrial pressure include severe volume overload, left ventricular systolic dysfunction, left ventricular diastolic dysfunction, mitral valve disease, and aortic valve disease. A full discussion of all of the etiologies of cardiogenic pulmonary edema is beyond the scope of this article. Nevertheless, all of these disorders cause pulmonary edema via the same pathophysiologic mechanism, that is, increased microvascular pressure causing increased fluid filtration into the interstitium. If the resultant edema fluid accumulation is confined to the interstitium by the safety factors described above, then only mild, if any, symptoms will result. However, if increased microvascular pressure is severe or persists, then the defense mechanisms are overwhelmed, alveolar flooding ensues, and gas exchange is impaired. Because the capillary endothelium and alveolar epithelium remain intact, the edema fluid suctioned out of the airway has a low protein content compared with plasma (ratio of p0.65). Increased vascular permeability (increased K and decreased s from the Starling equation) is caused by damage to the endothelium and alveolar epithelium. When this occurs, both the interstitium and alveoli are flooded with protein-rich fluid (edema fluid to plasma protein concentration ratio 40.65). Because of the damage to the alveolar epithelium, alveolar fluid clearance is usually impaired. Gas exchange is usually significantly diminished. Increased permeability pulmonary edema is also known as acute lung injury, or, in its severest form, ARDS (see Acute Respiratory Distress Syndrome). There are numerous causes of acute lung injury, the most common of which are listed in Table 1. These causes of acute lung injury may be grouped into those that constitute a direct insult to the alveolar epithelium such as aspiration of gastric contents or pneumonia, and those that are an indirect insult to the alveolar epithelium such as sepsis. Some less common types of pulmonary edema include postobstructive pulmonary edema (see Upper Airway Obstruction), neurogenic pulmonary edema, and high-altitude pulmonary edema (HAPES) (see High Altitude, Physiology and Diseases). Postobstructive pulmonary edema occurs in the setting of

Table 1 Clinical disorders associated with acute lung injury Direct

Indirect

Pneumonia Aspiration Thoracic trauma Inhalation injury Fat emboli Near drowning Reperfusion after transplant or embolectomy

Sepsis Nonthoracic trauma Acute pancreatitis Drug overdose Transfusion associated (TRALI) Cardiopulmonary bypass Burns

an acute upper airway obstruction such as postanesthesia laryngospasm. A patient with an acutely obstructed upper airway will make very vigorous respiratory effort leading to markedly negative intrathoracic pressures, which are transmitted to the perimicrovascular interstitium (decreased Pi from the Starling equation). Also, large negative intrathoracic pressures increase both preload and afterload on the heart. The increased preload and afterload probably increase pulmonary microvascular pressure (Pmv). Thus, there are increased net hydrostatic forces, and edema forms. Neurogenic pulmonary edema is thought to occur because of the massive catecholamine surge produced after a neurologic event. This surge causes marked systemic vasoconstriction and redistribution of blood from the systemic to pulmonary circulation. The resulting increased pulmonary vascular pressures may produce a hydrostatic pulmonary edema (low protein) or lead to physical damage to the vascular walls. This latter scenario causes an increased permeability edema. Similarly, HAPES may result from severe, nonhomogeneous pulmonary vasoconstriction. The vasoconstricted areas shunt blood flow to other areas of the lung leading to increased Pmv in the perfused areas. Again, a low-protein hydrostatic edema may result, or there may be damage to the endothelium from the increased pressure causing high-protein, increased-permeability edema.

Diagnosis Hydrostatic Pulmonary Edema

The vast majority of cases of hydrostatic pulmonary edema are of cardiac origin. The diagnosis rests heavily on the history, physical examination, and chest radiography. Most patients with acute pulmonary edema of any cause will present with dyspnea in which case the history of present illness should focus on dyspnea severity, time of onset, pace of onset, and associated symptoms. Patients with acute cardiogenic pulmonary edema may have sudden, severe dyspnea. Because a significant number of these severely affected patients have pulmonary

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edema secondary to an acute coronary event, one should thoroughly question the patient or family about chest pain or angina equivalents. Patients may also give a history of recently worsening chronic congestive heart failure symptoms such as worsening dependent edema, orthopnea, and paroxysmal nocturnal dyspnea. A history of dietary indiscretion is common in patients with an acute exacerbation of chronic congestive heart failure. The past medical history should focus on prior history of coronary artery disease, valvular heart disease, hypertension, or cardiomyopathy. On physical examination, patients with acute cardiogenic pulmonary edema may be very anxious and sitting ‘bolt upright’ in bed. In the most severe cases, patients may develop cyanosis, the development of which signifies severe respiratory failure and impending death if not corrected quickly. Other signs include jugular venous distension, an S3 gallop on heart examination, pitting edema, a palpable liver edge, and ascites. The respiratory examination is characterized by the presence of wet rales, possible extending up to the apices of the lung. Patients may also exhibit the use of accessory respiratory muscles. Chest radiography is valuable in diagnosing pulmonary edema. In cardiogenic pulmonary edema, the heart silhouette is often enlarged. If only interstitial edema is present, there may be evidence of apical vascular engorgement (so-called vascular redistribution), septal or Kerley’s lines, and decreased definition of smaller blood vessels and bronchial structures (perivascular and peribronchial cuffing). When alveolar flooding occurs, confluent parenchymal opacities develop. In hydrostatic edema, the radiographic opacities often develop centrally first. In severe cases, there may be complete opacification bilaterally with air bronchograms. Chest radiography cannot reliably distinguish between hydrostatic pulmonary edema and increased permeability pulmonary edema (acute lung injury (ALI)). Several other diagnostic tests may be useful in patients with dyspnea or respiratory distress and suspected cardiogenic pulmonary edema. As mentioned

above, acute pulmonary edema is often associated with an acute coronary event, so an electrocardiogram should be performed in all patients with suspected acute cardiogenic pulmonary edema. Particular attention should be paid to electrocardiographic signs of ischemia or infarction such as ST segment elevation, severe ST segment depression, new Q waves, or a new left bundle branch block. Likewise, creatine phosphokinase-MB (CPK-MB) and troponin levels are useful in patients with suspected cardiogenic pulmonary edema to rule out myocardial infarction. Blood levels of B-type natriuretic peptide (BNP) are useful in emergency department patients with dyspnea and suspected cardiogenic pulmonary edema, however, their diagnostic accuracy in inpatients is unproven. Arterial blood gases are useful in assessing the severity of respiratory compromise. Interstitial pulmonary edema may be associated with normal or slightly reduced oxygenation (decreased PaO2) with a reduced PaCO2 from tachypnea. With alveolar flooding, significant intrapulmonary shunt develops, and a markedly reduced PaO2 will result if untreated. Echocardiography may be very helpful in determining the etiology of pulmonary edema. Normal echocardiographic structure and function argue strongly against pulmonary edema of cardiac origin. Finally, pulmonary artery catheterization may provide valuable information in patients with pulmonary edema and shock. Not only can normal pulmonary artery occlusion pressures exclude cardiogenic pulmonary edema, but the clinician can follow trends in the pulmonary artery catheter data to help guide fluid and vasopressor management. Increased Permeability Pulmonary Edema

Increased permeability pulmonary edema is also known as ALI or ARDS in its severest form. Currently, its diagnosis is based on a set of criteria as set forth by the American–European Consensus Conference on Acute Respiratory Distress Syndrome (see Table 2) (see Acute Respiratory Distress Syndrome). These criteria identify a patient population with

Table 2 Diagnostic criteria for acute lung injury (ALI) acute respiratory distress syndrome (ARDS) Oxygenation PaO2 =FiO2 p300 for acute lung injury

a

a

Chest radiograph

Left atrial pressure

Bilateral infiltrates on frontal chest radiograph

Pulmonary artery occlusion pressure p18 mmHg when measured or no clinical evidence of left atrial hypertension

For the diagnosis of acute respiratory distress syndrome, use PaO2 =FiO2 p200 with the same chest radiograph and left atrial pressure criteria. Adapted from Bernard GB, Artigas A, Brigham K, et al. (1994) The American–European Consensus Conference on ARDS: definitions, mechanisms, relevant outcomes, and clinical trial coordination. American Journal of Respiratory and Critical Care Medicine 149: 818–824.

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hypoxemia and bilateral infiltrates on chest radiograph whose condition cannot be explained by increased left atrial pressure (noncardiogenic). Based on these criteria, the most useful data in the diagnosis of acute lung injury are the history, chest radiograph, and arterial blood gases. The history in suspected ALI should focus on eliciting the presence of one of the common causative conditions (see Table 1). In postoperative patients, a thorough examination of the anesthesia record for blood products transfused or witnessed aspiration during induction or recovery is helpful. On lung examination, patients with ALI may have bilateral rales or evidence of consolidation, but these findings are non-specific. Thus, the physical examination in suspected ALI patients should be directed toward determining whether the patient’s edema can be explained by elevated left atrial pressure and whether the patient has one of the potential causes of ALI. The absence of any history or physical examination evidence for volume overload or congestive heart failure in a patient with pulmonary edema strongly suggests ALI. In addition, the patient’s abdomen, rectum, and skin should be meticulously examined for a potential source of sepsis. As indicated by the diagnostic criteria, the chest radiograph and arterial blood gases are the most useful diagnostic tests in ALI. The chest radiograph may show only bilateral interstitial edema, but most likely it will demonstrate areas of alveolar filling. More severe cases may show extensive consolidation of both lungs. Because pneumonia is the most common cause of ALI, there also may be focal consolidation with air bronchograms. By definition, arterial blood gas analysis will demonstrate significant hypoxia and intrapulmonary shunt. A respiratory alkalosis may be present early in the course of ALI due to hypoxic respiratory drive and/or sepsis, but later respiratory acidosis may develop from worsening lung compliance and increased dead space. In addition, hypoxia and sepsis may cause a metabolic acidosis. Other laboratory tests should be directed at potential causes of ALI. Because sepsis and pneumonia are the most common causes of ALI, cultures of blood, sputum (or airway aspirate), urine, wounds, and, if appropriate, cerebrospinal fluid should be obtained. Abdominal tenderness on examination should be evaluated with imaging studies and amylase and lipase levels. Other possible diagnostic studies in ALI include pulmonary artery catheterization and echocardiography. Both of these modalities can be useful in determining whether the pulmonary edema is due to a cardiogenic source. Pulmonary artery catheterization may also provide valuable diagnostic information about the etiology of shock states which frequently

accompany ALI. Another potential advantage of pulmonary artery catheterization is that the hemodynamic data may be useful in guiding fluid and vasopressor therapy. However, the benefit of routine use of pulmonary artery catheters in ALI patients is not well established, and this issue is the subject of an ongoing multicenter, randomized, controlled trial.

Management Cardiogenic Pulmonary Edema

Some patients with mild cardiogenic pulmonary edema can be treated and released from the emergency department with close follow-up. However, patients with new onset or severe symptoms, hypoxia, evidence of acute ischemia, or poor follow-up should be admitted to the hospital for treatment and monitoring. Indications for intensive care unit admission will vary with each facility’s capability, but patients with profound hypoxia, need for ventilatory support, or need for invasive monitoring should be admitted to a critical care unit. Pulse oximetry should be monitored early in all patients who present with dyspnea, and patients with significant hypoxia or at risk for worsening should have continuous pulse oximetry. Patients with a suspected ischemic cause of their pulmonary edema should have continuous electrocardiographic monitoring also. An arterial catheter should be placed in patients receiving nitroprusside for severe cardiogenic pulmonary edema with hypertension and patients receiving vasopressors for shock. Also, mechanically ventilated patients requiring frequent arterial blood gases may benefit from an arterial catheter. Pulmonary artery catheterization for hemodynamic monitoring may be useful in patients with severe cardiogenic pulmonary edema and shock, but its use is controversial. Supportive care for patients with cardiogenic pulmonary edema includes oxygen supplementation and support of ventilation if necessary. Noninvasive ventilation initiated early in patients with severe hypoxia or respiratory distress may prevent the need for mechanical ventilation. This modality may not be appropriate for patients with an acute coronary syndrome, since it may not reduce the work of breathing and myocardial oxygen demand as much as intubation and mechanical ventilation. Intubation and mechanical ventilation is required in patients whose hypoxia or respiratory distress is refractory to noninvasive ventilation or who require rapid reduction in work of breathing. Pharmacotherapy in cardiogenic pulmonary edema is aimed at reducing pulmonary microvascular pressure (Pmv from the Starling equation) and reducing

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dyspnea. If there is an underlying cause of the acute pulmonary edema such as acute myocardial infarction, it must be treated aggressively. The reduction in pulmonary microvascular pressure is primarily achieved by vasodilating medications and diuretics. Loop diuretics such as furosemide are the most commonly used diuretics in acute cardiogenic pulmonary edema because of their rapid onset and effectiveness in producing intravascular volume reduction. They even have a small, venodilating effect which may slightly decrease pulmonary microvascular pressure immediately. However, even with the rapid onset of diuresis provided by furosemide, patients with severe acute cardiogenic pulmonary edema will need other medications that work more quickly to reduce pulmonary microvascular pressure, namely vasodilating agents. In addition to its usefulness in relieving dyspnea, morphine has venodilating effects which act to increase peripheral blood pooling. This pooling reduces left ventricular end diastolic pressure, and thereby pulmonary microvascular pressure is reduced. Nitroglycerin and nitroprusside are two agents that provide immediate vasodilation and can be given as a titratable intravenous infusion. Nitroglycerin causes both arterial and venous dilation, although venodilation is the more profound effect. Its dilation of epicardial coronary arteries may also play a role in its antianginal effect. Nitroprusside has more prominent arterial dilating effects. Both of these agents produce a rapid reduction in cardiac preload and afterload with consequent reduction in pulmonary microvascular pressure. Finally, nesiritide is recombinant human B-type natriuretic peptide. Despite its name, its dominant clinical action seems to be vasodilation instead of natriuresis. Nesiritide has been evaluated for use in acute cardiogenic pulmonary edema, but there is little evidence that its increased cost compared with nitroglycerin is justified by increased benefit. When cardiogenic pulmonary edema is refractory to the usual measures described above, or when it is accompanied by cardiogenic shock, inotropic agents may provide short-term improvement. The currently available inotropes fall into two categories, b-adrenergic agonists and phosphodiesterase inhibitors. The most commonly used adrenergic agonist in cardiogenic pulmonary edema is dobutamine. It stimulates cardiac b1-adrenergic receptors causing increased inotropy and chronotropy. The chief side effects are tachycardia and increased ventricular ectopy. In addition, dobutamine may cause vasodilation and hypotension because of its stimulation of b2 receptors in vascular smooth muscle. Phosphodiesterase inhibitors, such as milrinone, produce their inotropic effect by increasing cardiac intracellular

cyclic AMP, which increases intracellular calcium. These agents also cause peripheral vasodilation and reduction in blood pressure, so blood pressure should be closely monitored upon initiation of milrinone. Like the adrenergic agonists, milrinone has been associated with increased ventricular ectopy. Levosimendan is a novel inotropic agent currently undergoing clinical trials. Its mechanism of action, calcium sensitization of cardiac myocytes, may avoid some of the adverse effects of adrenergic agonists and phosphodiesterase inhibitors. Overall, the use of inotropic agents in acute cardiogenic pulmonary edema has not been shown to improve mortality, and they are associated with potentially serious adverse effects. Inotropic agents should therefore not be routinely used in patients with acute cardiogenic pulmonary edema. Permeability Pulmonary Edema

For practical purposes, permeability pulmonary edema and ALI are the same. ARDS is the most severe form of ALI. The large majority of cases of ALI will be treated in intensive care units either because of the severity of the hypoxia and respiratory distress or because of the severity of the causative disorder such as sepsis. Pulse oximetry should be monitored frequently or continuously in ALI patients, because they are at high risk of respiratory decompensation. An arterial catheter for frequent blood gas analysis is helpful, especially in intubated patients or patients at high risk of intubation. The relative benefit of a central venous catheter versus a pulmonary artery catheter for central venous pressure monitoring is currently under study in a multicenter, randomized, clinical trial. Since there is no specific pharmacotherapy for ALI, the mainstays of management are treatment for the underlying condition and supportive care. Unless there is a clear noninfectious cause of ALI, most patients should be treated with broad-spectrum antibiotics intitially. Source control, such as drainage of abscesses, should be performed urgently. Supportive care usually includes intubation and mechanical ventilation. Importantly, the method of mechanical ventilation is the only specific treatment for ALI. A mechanical ventilation regimen with low tidal volume (6 ml kg
1 based on ideal body weight), plateau pressures of less than 30 cm water, and a moderate level of positive end-expiratory pressure has been shown to significantly reduce mortality in ALI patients (see Table 3). In attempting to reach these goals, a pH as low as 7.25 and PaO2 as low as 55 mmHg are usually tolerated. Thus, unless there is a contraindication to the permissive hypercapnea (such as elevated intracranial pressure) or permissive

550 PULMONARY EFFECTS OF SYSTEMIC DISEASE Table 3 Protocol for low tidal volume ventilation for acute lung injury Variables

Protocol

Ventilator mode Tidal volume

Volume assist control p6 ml kg
1 body weight predicted by height; may decrease to 4 ml kg
1 if needed to achieve plateau pressure goal p30 cmH2O 6–35 breath min
1 Adjust ventilator rate to achieve arterial pHX7:30 if possible. Adjust flow to achieve I : E 1 : 1.0–1.3. 55pPaO2 p80 mmHg or 88%pSpO2 p95% 0:3 0:4 0:4 0:5 0:5 0:6 0:7 0:7 0:7 0:8 0:9 0:9 0:9 1:0 1:0 1:0 5 5 8 8 10 10 10 12 14 14 14 16 18 18 22 24

Plateau pressure Ventilator set rate Arterial pH Inspiratory flow, I:E Oxygenation FiO2/PEEP (cmH2O)

Adapted with permission from Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. New England Journal of Medicine 342(18): 1302–1308. Copyright & 2000 Massachusetts Medical Society. All rights reserved.

hypoxemia (such as an acute coronary syndrome), all patients who meet the diagnostic criteria for ALI should be ventilated with the low tidal volume strategy. Nutritional support should be considered early, because many ALI patients will have a prolonged ventilator course. Current clinical trials in patients with ALI are investigating possible pharmacologic agents such as activated protein C and granulocyte– macrophage colon stimulating factor (GM-CSF). The value of reducing pulmonary microvascular pressure by fluid restriction and diuresis is also being investigated in a randomized clinical trial. See also: Acute Respiratory Distress Syndrome. High Altitude, Physiology and Diseases. Upper Airway Obstruction.

Further Reading Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. New England Journal of Medicine 342(18): 1302–1308. Bernard GB, Artigas A, Brigham K, et al. (1994) The American– European Consensus Conference on ARDS: definitions, mechanisms, relevant outcomes, and clinical trial coordination. American Journal of Respiratory and Critical Care Medicine 149: 818–824.

Brower RG, Lanken PN, MacIntyre N, et al. (2004) Higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome. New England Journal of Medicine 351(4): 327–336. Gehlbach BK and Geppert E (2004) The pulmonary manifestations of left heart failure. Chest 125: 669–682. Givertz MM, Colucci WS, and Braunwald E (2001) Clinical aspects of heart failure: high output heart failure; pulmonary edema. In: Braunwald E, Zipes DP, and Libby P (eds.) Heart Disease: A Textbook of Cardiovascular Medicine, pp. 534–561. Philadelphia: Saunders. Guyton AC and Lindsey AW (1959) Effect of left atrial pressure and decreased plasma protein concentration on the development of pulmonary edema. Circulation Research 7: 649–657. Matthay MA, Folkesson HG, and Clerici C (2002) Lung epithelial fluid transport and the resolution of pulmonary edema. Physiological Reviews 82(3): 569–600. Matthay MA and Sakuma T (2001) Pulmonary edema: formation and reabsorption. In: Scharf SM, Pinsky MR, and Magder S (eds.) Respiratory–Circulatory Interactions in Health and Disease, pp. 361–387. New York: Dekker. Sharma M and Teerlink JR (2004) A rational approach for the treatment of acute heart failure: current strategies and future options. Current Opinion in Cardiology 19: 254–263. Staub NC (1988) Lung liquid and protein exchange. Applied Cardiopulmonary Pathophysiology 2: 117–123. Taylor AE (1981) Capillary fluid filtration: starling forces and lymph flow. Circulation Research 49: 557–575. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342(18): 1334– 1349.

PULMONARY EFFECTS OF SYSTEMIC DISEASE O P Sharma, University of Southern California School of Medicine, Los Angeles, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pulmonary manifestations of systemic disorders are common. Almost all systemic diseases can involve the lung. In many illnesses, lung complications remain relatively benign, producing

few or no symptoms; such is the case with pleural effusions related to renal or hepatic failure. In others diseases, lung involvement becomes a nuisance but is manageable; this occurs in gastroesophageal reflux disease and arteriovenous malformations in Osler–Rendau–Weber disease. On the other hand, in certain diseases lung involvement progresses relentlessly, causing death or serious injury that influences the course of the primary illness; such is the case in severe pneumonia in a diabetic patient or portopulmonary hypertension in chronic liver failure.

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Introduction It is difficult to assemble all that is known of the pulmonary manifestations of the systemic disorders that afflict the human body. The normal lung is essential for the rest of the body, and almost all organs influence lung behavior. These interrelationships are often complex and difficult to understand. Nevertheless, the interactions need to be recognized, explored, and diagnosed. Only a select few pulmonary complications of systemic diseases are described here. This chapter provides an overview of the most common and frequent changes observed in chronic hepatic, gastrointestinal, renal, metabolic and endocrine, hematological, and cutaneous disorders. The occurrence of lung disease in multisystem connective tissue diseases and vasculitides is widely recognized and described elsewhere in this encyclopedia.

The Liver–Lung Interface The interests of the pulmonologist and the hepatologist overlap in many ways. Both acute and chronic liver diseases affect pulmonary circulation. The lungs and liver can be simultaneously involved in many infections, such as tuberculosis, brucellosis, Q fever, and histoplasmosis; granulomatous disease, such as sarcoidosis; and genetic anomalies, such as a1-antirypsin deficiency. Autoimmune diseases may involve both organs. A disease may start in the lung but be diagnosed because of hepatic involvement. For instance, cancer from the lung or the illness may start in the liver but be recognized because of the pleuropulmonary expression (e.g., amebiasis). Some of the common liver–lung relationships are described here (Table 1). Hyperventilation and Cirrhosis

Hyperventilation is a common respiratory manifestation of cirrhosis. Arterial hypoxemia may be the Table 1 Pulmonary complications of liver diseases Structure

Illness

Pulmonary circulation

Hyperventilation Hepatopulmonary syndrome Portopulmonary hypertension Bronchitis, bronchiectasis BOOP, LIP, NSIP, UIP Basal atelectasis Chest X-ray infiltrate Raised diaphragms Hydrothorax Chylothorax Thoracobiliary fistula

Lung parenchyma

Pleura

BOOP, bronchiolitis obliterans obstructive pneumonitis; LIP, lymphocytic interstitial pneumonitis; NSIP, non-specific interstitial pneumonitis; UIP, usual interstitial pneumonitis.

cause in some patients, but often the degree of hyperventilation is greater than one would expect based on the degree of arterial hypoxemia. Elevated ammonia levels may be responsible, but studies have been unable to demonstrate a consistent correlation between ammonia levels and arterial carbon dioxide tension in patients who are hyperventilating. The speculation here is that hyperventilation is caused by chronic interstitial lung edema related to decreased intravascular oncotic pressure. Hepatopulmonary Syndrome

Hepatopulmonary syndrome is a clinical triad of advanced liver disease, intrapulmonary vascular dilatations in the absence of a primary cardiopulmonary disease, and hypoxemia. Hypoxemia results from low ventilation–perfusion ratios in areas of precapillary/capillary dilatations and anatomic shunting through microvascular arteriovenous communications. A reduction in transfer factor is related to the thickening of the small veins and capillaries by a layer of collagen. The vasoactive substances that induce pulmonary vasodilatation are not precisely known, but the list includes nitric oxide (NO), endothelin-1, and arachidonic acid and its metabolites. Finger clubbing is frequent, and platypnea and orthodeoxia are common. Cyanosis and clubbing are common in patients with autoimmune hepatitis and long-standing cirrhosis. Portopulmonary Hypertension

As many as 20% of patients with advanced liver disease have a mean pulmonary artery pressure of more than 25 mmHg. Causes include hyperdynamic circulation, increased blood volume, and nonembolic pulmonary vasoconstriction/obliteration. The latter process is termed portopulmonary hypertension. Histological findings include medial and intimal hypertrophy, endothelial proliferation, and plexogenic/ fibrotic changes. These changes are indistinguishable from histological changes seen in patients with primary pulmonary hypertension. However, patients with portopulmonary hypertension have increased cardiac output (Table 2). Pleural Effusions

Pleural effusions, unilateral or bilateral, occur in approximately 10% of patients with advanced liver disease. These transudative effusions are frequently right-sided and rarely occur in the absence of ascites. Nevertheless, pleural effusion may develop in the patient without clinically evident ascites. Negative pleural space pressure is generated with inspiration draws in the ascitic fluid through small diaphragmatic

552 PULMONARY EFFECTS OF SYSTEMIC DISEASE Table 2 Diagnosis and management of hepatopulmonary syndrome and portopulmonary hypertension Feature

Hepatopulmonary syndrome

Portopulmonary hypertension

Diagnostic criteria

Chronic liver dysfunction Intrapulmonary vascular dilatation Hypoxemia No existing lung disease Diffusion–perfusion abnormality Spider angiomata

Mean PAP 425 PCWP o15 Portal hypertension No other cause of pulmonary hypertension Vasoconstriction Vascular thickening Vascular obliteration Vascular remodeling Dyspnea, syncope, chest pain Loud P2, TR murmur, hypoxemia Echocardiography, cardiac catheterization Treat pulmonary hypertension

Pathophysiology

Symptoms Signs Diagnostic studies Treatment

Dyspnea, platypnea Cyanosis, clubbing, orthodeoxia Echocardiography ( þ ) bubble study Liver transplant

Figure 1 Posteroanterior and lateral views of the chest showing extensive bullous emphysema in a 29-year-old Caucasian male with a1-antitrypsin deficiency.

defects called Lieberman’s pores. Patients with a mild to moderate amount of fluid have no symptoms, but dyspnea and hypoxemia are present in patients with massive pleural effusion. Aggressive diuretic therapy and salt restriction to reduce the volume of ascites constitute the medical treatment. Thoracentesis is usually ineffective. Pleurodesis is helpful in select cases but is not without risk. Transjugular intrahepatic portosystemic shunt greatly reduces the need for repeat thoracentases in symptomatic patients. Refractory hepatic hydrothorax due to uncontrollable ascites is an indication for liver transplantation. a1-Antitrypsin Deficiency

a1-Antitrypsin deficiency is synthesized in the rough endoplasmic reticulum of the liver. It comprises 80–90% of the serum a1-globulin and is an inhibitor

of trypsin and other proteases. The abnormal a1 synthesis evolves from a single-point gene mutation located on chromosome 14 (more than 75 abnormal a1 alleles exist), which is codominantly expressed in the hepatocytes. The spectrum of a1-antitrypsindeficient liver disease extends from liver failure and need for transplantation in childhood to asymptomatic individuals with no evidence of disease. Emphysema, the most common pulmonary manifestation of a1 deficiency, affects predominantly the lung bases (Figure 1). It is caused by the imbalance between the protective a1 protein and neutrophil elastase derived from leukocytes. Smoking accelerates the destructive process. Combined liver and lung manifestations of a1 deficiency in the same patient are rare. In select patients with progressive lung disease, replacement therapy with weekly intravenous a1 protein from pooled donors may slow the decline.

PULMONARY EFFECTS OF SYSTEMIC DISEASE 553 Table 3 Sarcoidosis and primary biliary cirrhosis: a comparison Feature

Sarcoidosis

Primary biliary cirrhosis

Sex Age (years) Pulmonary symptoms Chest X-ray Pruritus Jaundice Serum alkaline phosphatase Serum angiotensin-converting enzyme Mitochondrial antibody Kveim–Siltzbach test Bronchoalveolar lavage Liver biopsy Lung biopsy Prognosis

Almost equal 20–45 Yes Abnormal No No Raised Raised Normal Positive Lymphocytosis Solid, discrete granulomas Noncaseating granulomas Generally good

80% women Middle age No Normal Yes Yes Raised Raised Raised (98%) Negative Lymphocytosis Poorly formed granulomas No granulomas, lymphocytes Generally poor

The replacement therapy has no role in the treatment of liver disease. Liver transplantation for a1-deficient childhood liver disease has a 3-year survival rate of 83–100%.

usually asymptomatic. Occasionally, they may result in an increase in alkaline phosphatase. Rarely, massive granulomatous inflammation may lead to liver fibrosis.

Primary Biliary Cirrhosis and Interstitial Lung Disease

Pulmonary Complications of Hepatic Transplantation

Primary biliary cirrhosis (PBC) is a disease of unknown origin in which intrahepatic bile ducts are progressively destroyed. T helper cells and cytotoxic T cells play an important role in the pathogenesis. The true incidence of lung disease in PBC is very low. However, many observations point to the existence of subclinical or asymptomatic lung inflammation in these patients. In early stages of PBC, bronchoalveolar lavage may reveal lymphocytic alveolitis similar to that seen in patients with sarcoidosis and Crohn’s disease. Gas transfer studies are abnormal, particularly in patients who have Sjogren’s or CREST syndrome in association with PBC. Nevertheless, severe restrictive abnormalities in pure PBC patients are rare. Chest X-ray abnormalities include hilar adenopathy, nodules, and interstitial reticular opacities. Lung biopsy specimens in patients with associated Sjogren’s syndrome have shown giant cells. Occasionally, noncaseating granulomas resembling sarcoidosis have been described; however, there are many differences between PBC and sarcoidosis (Table 3).

Chronic pulmonary disease may be a contraindication to liver transplantation. Severe hypoxemia due to right-to-left shunting with an arterial oxygen pressure of less than 50 mmHg is an absolute contraindication. In the early postoperative days after transplantation, the right diaphragm is paralyzed and right lower lobe atelectasis is common. Pleural effusions occur in almost all patients, but only 18% require aspiration. Pulmonary edema can occur from excess fluids and blood products during the operation. Later in the course, complications include pneumonias caused by bacteria, fungi, viruses, as well as Legionella and Pneumocystis carinii. Many of these infections occur after discharge from the hospital in patients with well-functioning livers. Azathioprine and cyclosporine can cause interstitial pneumonitis and acute respiratory distress syndrome (ARDS), respectively.

Gastrointestinal System and the Lungs Gastroesophageal Reflux Disease

The Liver in Sarcoidosis

Granulomas are found in 4–10% of needle biopsies, and in 10% of these no cause is found even after extensive investigation. Hepatic granulomas are always part of a generalized disease process. Approximately two-thirds of patients with systemic sarcoidosis have granulomas in the liver. Thus, liver biopsy may offer the diagnosis when other less invasive techniques have failed. Hepatic granulomas in sarcoidosis are

Cough can be the sole presenting symptom of gastroesophageal reflux. It is caused by one of three mechanisms. Reflux of stomach contents may irritate the esophageal mucosa and initiate the cough reflex through vagal sensory pathways. Aspirated gastric material may irritate sensory receptors of the tracheobronchial tree. Lastly, stomach contents may reach the hypopharynx and larynx, irritating the afferent limb of the cough reflex without aspiration.

554 PULMONARY EFFECTS OF SYSTEMIC DISEASE

Diagnosis is certain only when the cough stops in response to antireflux therapy. Pulmonary Complications of Esophageal Sclerotherapy

Esophageal varices are injected acutely at the time of bleeding; the remaining varices are obliterated later in the course of the illness. Fever, chest pain, usually retrosternal and nonpleuritic, and dysphagia are frequent complications. Pleural effusions – right, left, or bilateral – are frequent in patients who have chest pain and have a large volume of sclerosant injected. The fluid is usually an exudate. Aspiration pneumonia is another complication. Rarely, ARDS may complicate esophageal sclerotherapy with sodium morrhuate. Bronchoesophageal fistula formation has been described after endoscopic sclerotherapy.

bronchiectasis. Biopsies of the lung tissue usually show thickening of the epithelium and basement membrane with inflammatory cell infiltration of the underlying connective tissue. On the other hand, in Crohn’s disease subclinical inflammatory lung disease is frequent. Bronchoalveolar lavage fluid from patients with Crohn’s disease has shown that lymphocytes predominate with increased T4 subset during active disease. Despite the presence of alveolar lymphocytosis, there have been only a few reported cases of otherwise unexplained lung disease in these patients. The mechanisms leading to alveolar lymphocytosis in Crohn’s disease and its relationship with sarcoidosis remain uncertain.

Endocrine and Metabolic Disease Diabetes Mellitus

Pancreatitis

The lungs may be involved in 50–70% of patients with acute pancreatitis. Abnormalities include asymptomatic reduction in arterial oxygenation, significant hypoxemia with a normal chest radiograph, non-specific opacities, pleural effusion, and ARDS. The latter occurs in approximately 15% of the patients with acute pancreatitis and carries a poor prognosis. The onset of pulmonary symptoms in the patient with acute pancreatitis portends a poor prognosis. Sixty percent of deaths from acute pancreatitis that occur during the first week are associated with respiratory failure. Only 25% of those who require mechanical ventilation survive. During and after the second week, pulmonary complications are usually the result of pancreatic infection or pseudocyst formation. Pleural effusions and ascites reflect the severity of the illness. Inflammatory Bowel Disease

Chronic airway inflammation, both bronchiectasis and purulent bronchitis, is common in inflammatory bowel disease, particularly in ulcerative colitis. Involvement of the small airways is unusual. Organizing pneumonia, non-specific interstitial pneumonitis, and eosinophilic pneumonias have been described in ulcerative colitis. The pulmonary manifestations may appear at any time during the course of the bowel disease, but they follow the clinical onset of inflammatory bowel disease in 80% of cases. The airway inflammation is unrelated to the activity of the bowel disease or therapy. Dyspnea, fever, cough, expectoration, and chest pain are common and non-specific symptoms. In early stages, the chest radiograph may be normal and pulmonary function testing may reveal no consistent abnormality. Effective therapy of ulcerative colitis may prevent the worsening of

Patients with diabetes mellitus have an increased incidence of acute and chronic pulmonary infections. Pneumococcal pneumonia occurs with higher frequency, but Staphylococcus aureus and Escherichia coli are also frequent. The mechanisms behind the increased susceptibility of a diabetic patient to these infections include impaired chemotactic, phagocytic, and bactericidal activities of the neutrophils. Hyperglycemia decreases intracellular bactericidal activity of lymphocytes. The diminished phagocytic ability of monocytes increases the risk of fungal infections, particularly mucormycosis. Tuberculosis reactivation is two or three times more common in malnourished diabetic patients. Tuberculosis in these patients tends to affect the lower lobes. Sixty percent of nonsmoking, non-insulin-dependent diabetics have a mild reduction of the elastic recoil of the lung and diffusing capacity. Abnormalities of airflow or gas distribution are uncommon; however, the vagus-mediated basal airway tone is altered. Ventilatory responses to hypoxia and hypercapnia are abnormal due to altered function of the peripheral and central chemoreceptors. Hyperthyroidism

Dyspnea is common in thyrotoxicosis. Its cause is unclear, and its severity varies. Increased oxygen consumption, increased carbon dioxide production, increased minute ventilation, decreased vital capacity, impaired diffusion capacity, low lung compliance, respiratory muscle weakness, and increased central ventilatory drive have all been implicated in causes of dyspnea. Hypoxic and hypercapnic ventilatory drives are increased in patients with thyrotoxicosis compared to healthy controls; treatment of the disease normalizes both. Thyrotoxicosis patients tend to have high-output left ventricular failure. An

PULMONARY EFFECTS OF SYSTEMIC DISEASE 555

enlarged gland can compress the trachea and cause wheezing and strider. Hyperactivity of the airways has also been observed. Hypothyroidism

Hypothyroidism decreases central ventilatory sensitivity to hypoxia and hypercapnia. These abnormalities return to normal after successful treatment of hypothyroidism. Severe hypothyroidism can lead to respiratory failure and coma. Unsuspected hypothyroidism is one of the frequently overlooked causes of failure to wean from mechanical ventilation. Myxedema severe enough to cause respiratory failure and sleep apnea is associated with a decrease in tendon reflexes. The airway obstruction may be worsened by an enlarged tongue or oropharyngeal narrowing due to mucopolysaccharide and protein deposits in the soft tissue. Myopathic changes in the muscles may rarely occur. Transudative pleural effusions, unilateral or bilateral, have been observed. Parathyroid Disease

Both hyperparathyroid and hypoparathyroid patients can have sufficient neuromuscular weakness causing restrictive pulmonary impairment. Chest X-ray in hyperparathyroid patients may reveal osteopenia and osteitis fibrocystica, particularly in the clavicles and scapula. Occasionally, soft tissue calcifications are present. Pituitary and Adrenal Glands

Patients with acromegaly, particularly men, have increased lung volumes without airway obstruction or air trapping. The diffusing capacity remains normal, indicating that lung enlargement results from an increase in size rather than in the number of alveoli. There is an increase in the frequency of sleep apnea in patients with excessive growth hormone secretion. It can be central or obstructive in origin. Reproductive System

The triad of pleural effusion, ascites, and ovarian fibroma is called Meigs–Salmon syndrome. The syndrome also occurs in association with other ovarian and pelvic neoplasms, including fibroma, coma, granulosa cell tumor, Brenner tumors, adenocarcinoma, and fibroma of the uterus. Ascites is due to fluid secretion by the tumor. The pleural effusion, predominantly right-sided, results from ascitic fluid seeping through pores in the diaphragm. The effusion is frequently a transudate. Occasionally, the effusion may be left-sided or bilateral. Removal of the pelvic tumor causes the disappearance of hydrothorax as well as ascites.

Renal Disorders and the Lungs Chronic Renal Disease

A number of pleuropulmonary abnormalities complicate the course of chronic renal disease. Pulmonary edema, pleural effusion, pleural thickening and calcification, and pulmonary artery atherosclerosis and hypertension are well-described manifestations of renal disease. Urinothorax is an unusual cause of pleural effusion. The pleural fluid is a transudate with a creatinine level higher than that of serum creatinine and a pHo7.30. It is caused by extravasation of urine from the retroperitoneal space into the thorax. Pulmonary Complications of Renal Dialysis

Transient hypoxemia can occur within 15 min of initiation of hemodialysis. It can sometimes be severe enough to exacerbate myocardial ischemia. The decrease in PaO2 is related to acetate in the bath that causes hypoventilation and increased oxygen consumption. Dialysate of carbon dioxide and bicarbonate causes hypocapnia with a lag in bicarbonate regeneration from acetate metabolism. High-bicarbonate baths cause metabolic alkalosis. The compensating hypoventilation results in hypercapnia, which can be prevented by decreasing the bicarbonate concentration in the bath. Hypoxia can also result from complement activation by a bioincompatible dialyzer membrane causing leukostasis and plugging of the pulmonary capillaries. Using synthetic membranes and a citrate anticoagulant can reduce this effect. In some cases, hypoxemia may persist after dialysis has ended or develop in the postdialysis period.

Hematological Diseases and Lung Disease Sickle Cell Anemia

The combination of pleuritic chest pain, fever, cough, and parenchymal infiltrates on chest radiograph constitutes the acute chest syndrome. Infectious pneumonia and sickling of the abnormal red blood cells, either singly or in combination, are usually responsible. Rib infarctions, which are commonly observed radiologically, may also play a role. In situ thrombosis leading to pulmonary infarction may also occur. Infants and children commonly present with symptoms of infection, whereas adults usually have chest pain. The involvement of other organs – causing hemiplegia, an altered mental status, renal failure, and petechiae – suggests fat embolism. Recurrent episodes of acute chest syndrome can cause chronic lung disease. Hypoxemia is frequent in acute chest syndrome. Although oxygen may reduce the red cell

556 PULMONARY EFFECTS OF SYSTEMIC DISEASE

sickling, it does not alter the duration of the event, presumably because the damage due to vaso-occlusion occurred before oxygen administration. High oxygen concentration, in excess of that necessary to maintain a safe level of saturation, has not been recommended because it may suppress erythropoiesis. However, the suppression of erythropoiesis in this situation is poorly defined. The hemoglobin level and reticulocyte count can decline in patients on oxygen therapy. Monitoring is advised, although it is believed that oxygen concentrations of less than 50% do no harm; however, discomfort can be associated with the administration of oxygen therapy. Bone Marrow Transplantation

Patients undergoing bone marrow transplantation after aggressive salvage chemoradiation therapy are at high risk to develop pulmonary complications. Within the first 100 days after transplantation, pulmonary edema from fluid overload or myocardial injury, diffuse alveolar hemorrhage from cyclosporine toxicity, and transfusion reactions are seen. ARDS may be caused by pneumonia and sepsis due to bacterial and fungal organisms. Interstitial pneumonitis due to Pneumocystis carinii or cytomegalovirus, chemotherapy, or pulmonary embolism and veno-occlusive disease can occur during this period. Late complications are bronchopneumonia from the aforementioned infections, fat embolism, venoocclusive disease, interstitial pneumonitis of a nonspecific nature, bronchiolitis obliterans, organizing pneumonia, lymphocytic interstitial pneumonitis, and graft-versus-host disease. Severe supraglottic airway obstruction due to Epstein–Barr virus lymphoproliferative disease has been reported in children.

Dermatological Disorders and Lung Disease There are many multisystem disorders that affect the skin and lungs. This section includes clinical syndromes in which skin involvement is frequent, familiar, and easily recognizable, whereas the associated pulmonary disease is relatively infrequent, often overlooked, and not uncommonly misdiagnosed. Telangiectasia

Hereditary hemorrhagic telangiectasia (Osler–Weber– Rendau disease) is an inherited autosomal-dominant familial disorder with mutations localized to chromosome 9q3. This gene encodes endoglin, the transforming growth factor beta binding protein. The classical triad of hereditary hemorrhagic telangiectasia consists of epistaxis, telangiectasia, and a positive family history. Symptoms and signs due to pulmonary

arteriovenous malformations (PAVMs) occur between the fourth and sixth decades of life and include dyspnea, cyanosis, hemoptysis, clubbing, and a bruit. Because most of the PAVMs are located in the lower lung fields, orthodeoxia is a feature in some patients. Platypnea or improvement in dyspnea in the flat position is due to decreased blood flow through the pulmonary arteriovenous communications. On chest radiograph, this malformation can appear as a small compact nodule or a ‘comma-like density’ with vascular connections to hilar structures (Figure 2(a)). Conventional tomography, magnetic resonance imaging, and angiography are helpful in delineating the atrioventricular malformation and its vascular connections (Figure 2(b)). Occasionally, a vascular tumor may have an appearance on computed tomography suggestive of a vascular malformation. The definitive therapy in symptomatic patients includes embolotherapy and resection. Ataxia–telangiectasia syndrome is a recessively transmitted triad of sinopulmonary infections, telangiectasia, and cerebellar atrophy. The illness appears in childhood with ataxia and lower respiratory infections; telangiectasias follow. Hypoplasia of the thymus causes in vivo and in vitro impairment of delayed-type hypersensitivity. Immunoglobulin synthesis is stunted. Death results from either respiratory failure or lymphoma. Nodules, Albinism, and Yellow Nails

The lungs are affected in approximately 10% of neurofibromatosis (Recklinghausen syndrome) patients. The pulmonary changes usually appear after adolescence and consist of intrathoracic neurofibromas, interstitial lung disease, and honeycombing. Dyspnea is the main symptom; crackles and finger clubbing are rare. Tuberous sclerosis results from a genetic abnormality in the development of mesodermal tissue. The classic triad of epilepsy, mental retardation, and adenoma sebaceum is diagnostic. Lung involvement consists of interstitial lung disease, honeycombing, and spontaneous pneumothorax. Women are more commonly affected than men, and the average age of onset of symptoms is 34 years. The average life expectancy after the appearance of symptoms is 5 years. Cor pulmonale and spontaneous pneumothorax are the causes of death. Albinism, pulmonary fibrosis, and platelet dysfunction occur in Hermansky–Pudlak and related syndromes. Ceroid lipofuschin inclusions, a sine qua non of the disease, are found in the reticuloendothelial system. The incidence of pulmonary involvement is twice as high in women as in men. Dry cough and

PULMONARY FIBROSIS 557

Figure 2 (a) Posteroanterior view of the chest showing nodular densities in both lungs in a 30-year-old woman with a history of hemoptysis. (b) Pulmonary angiogram in the same patient showing multiple arteriovenous malformations.

dyspnea first appear in the third and fourth decades. The disease may remain stable, progress slowly, or progress rapidly to end-stage fibrosis and death. Yellow nail syndrome is a triad of yellow nails, lymphedema, and pleural effusion. Other pulmonary complications are bronchiectasis, sinusitis, and lower respiratory infections. Impaired lymphatic drainage is the underlying abnormality. See also: Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency. Gastroesophageal Reflux. Interstitial Lung Disease: Overview. Neurofibromatosis. Pleural Effusions: Overview. Pulmonary Fibrosis. Sleep Apnea: Overview. Systemic Disease: Sarcoidosis; Sickle Cell Disease. Vascular Disease.

Further Reading Eikenberry M, Bartakova H, Defor T, et al. (2005) Natural history of pulmonary complications in children after bone marrow

transplantation. Biology of Blood & Marrow Transplantation 11(1): 56–64. Fuchizaki U, Miyamori H, Kitagawa S, et al. (2003) Hereditary haemorrhagic telangiectasia (Rendu–Osler–Weber disease). Lancet 362: 1490. Hattori H, Hattori C, Yonekura A, and Nishimura T (2003) Two cases of sleep apnea syndrome caused by primary hypothyroidism. Acta Oto-Laryngologica. Supplementum 550: 59–64. Ingbar DH (2000) The pulmonary system in thyrotoxicosis. In: Braverman LE and Utiger RD (eds.) Werner and Ingbar’s the Thyroid. A Fundamental and Clinical Text, 8th edn. Philadelphia: Lippincott Williams & Wilkins. Murray J (1992) Pulmonary Complications of Systemic Disease, Lung Biology in Health Disease, vol. 59. New York: Dekker. Nassar A, Ghobrial G, Romero C, et al. (2004) Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn’s disease. Lancet 363: 1039–1044. Saner F, Lang H, Fruhauf N, et al. (2003) Postoperative ICU management of liver transplantation patients. European Journal of Medical Research 8: 511–516. Sharma O (1988) Pulmonary manifestations of systemic disease. Seminars in Respiratory Medicine 9(3). Taylor C, Carter F, Poulose J, et al. (2004) Clinical presentation of acute chest syndrome in sickle cell disease. Postgraduate Medicine Journal 80(944): 346–349.

PULMONARY FIBROSIS D G Morris, Yale School of Medicine, New Haven, CT, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Fibrosis of the lung results from relentless replacement of normal elastic lung with inflexible scar. This is caused by environmental exposures, systemic inflammatory diseases, and by other

as yet unknown cellular events. It complicates a range of diseases including connective tissue diseases, sarcoidosis, and chronic hypersensitivity pneumonitis. It occurs without known cause in the disease idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis is largely untreatable, progressive, and causes substantial morbidity with an approximately 50% 3year mortality rate. The fundamental biological processes that have been implicated in the pathogenesis of pulmonary fibrosis are: (1) altered regulation of inflammation and cytokine imbalance; (2) altered regulation of apoptosis; (3) oxidant-mediated

558 PULMONARY FIBROSIS lung injury; (4) dysregulated wound repair and epithelial to mesenchymal transformation; (5) protease/antiprotease imbalance; and (6) altered eicosanoid homeostasis. This manuscript will review our current biological understanding of pulmonary fibrosis in the context of these six fundamental processes and provide a framework for the interpretation of future studies in both animal models and patients with disease. Continued focused scientific and therapeutic investigations are critical to progress in the treatment of these vexatious lung diseases.

Historical Overview Progressive scarring in the lung, or pulmonary fibrosis, has plagued mankind since antiquity (see Interstitial Lung Disease: Hypersensitivity Pneumonitis. Occupational Diseases: Hard Metal Diseases – Berylliosis and Others; Silicosis. Pneumonia: Fungal (Including Pathogens); Mycobacterial). Beginning in the late nineteenth century, the concept of chronic fibrotic lung disease emerged. As Sir William Osler presciently and succinctly noted in the first (1892) edition of his classic textbook The Principles and Practice of Medicine: ‘‘So diverse are the different forms [of chronic interstitial pneumonia or cirrhosis of the lung] and so varied the conditions under which this change occurs that a proper classification is extremely difficult.’’ This situation remains little changed today.

Biological Paradigms of Pulmonary Fibrosis Chronic alveolitis, or inflammation of the distal bronchoalveolar gas exchange unit, presages fibrosis in many clinical conditions. Inflammation associated with lung injury is also a major feature of the principal experimental model of pulmonary fibrosis, bleomycin-induced lung injury. Bleomycin, which is derived from the bacteria Streptomyces verticillus, is a chemotherapeutic agent that is used to treat a range of human malignancies. It causes pulmonary fibrosis, particularly after high cumulative dosing. In mice, when administered directly through the trachea, by prolonged subcutaneous infusion, or through repeated injections, bleomycin also causes lung injury followed by a stereotypical, strain-dependent pulmonary fibrosis. Although bleomycin-induced pulmonary fibrosis is the dominant experimental model for human pulmonary fibrosis, many investigators question its validity. This skepticism is based on the model’s dependence on both the development of lung injury and the necessity for inflammation prior to the development of fibrosis. These do not occur in all forms of human fibrotic lung disease. Also, the fibrosis of bleomycin-induced lung injury is centered on distal airways and within alveolar spaces whereas idiopathic pulmonary fibrosis in humans is peripheral,

subpleural, and interstitial. Nevertheless, the effects of drugs, biological agents such as antibodies, and genetic manipulations on the development of pulmonary fibrosis are almost universally tested in this experimental system because of its ease, reproducibility, and broad scientific acceptance. Indeed, the past decade has witnessed an explosion of genetic and pharmacologic data on bleomycininduced pulmonary fibrosis in rodent models
principally in genetically manipulated mice. The molecular pathways implicated are summarized in Table 1. An overview of the data suggests six general biological pathways that are pivotal to the development, maintenance, or resolution of experimental pulmonary fibrosis. These are: (1) cytokine imbalance and altered regulation of inflammation; (2) abnormal regulation of apoptosis; (3) oxidantmediated lung injury; (4) dysregulated wound repair and epithelial mesenchymal transformation; (5) protease/antiprotease imbalance, including altered homeostasis of the coagulation/anticoagulation system; and (6) altered eicosanoid metabolism. Genetic and pharmacologic studies in mice suggest that each of the components of these pathways is critical to the fibrotic phenotype that follows bleomycin administration because the alteration of any one is generally sufficient to prevent or exacerbate fibrosis and/or inflammation. The interactions among these pathways are complex, cell-type specific, and very poorly understood. Persistent Inflammation and Cytokine Imbalance

The response of the lung to acute injury apparently follows a stereotypical pattern. In acute lung injury produced by infectious, mechanical, or systemic inflammatory responses, normal endothelial and epithelial barriers to protein and solute flow are lost. This causes alveolar flooding, the formation of an acellular fibrin clot, and the elaboration of a provisional extracellular matrix. The injury causes both epithelial apoptosis and necrosis. Recent studies from genetically manipulated mice suggest that epithelial apoptosis may be both proinflammatory and profibrotic. This contrasts with the anti-inflammatory effect of lymphocyte apoptosis. Key cytokines, chemokines, and cell adhesion molecules, such as interleukin-1 beta, macrophage inflammatory protein (CXCL2), interferon gamma, and the leukocyte adhesion molecules L-selectin, and inflammatory cell adhesion molecule (ICAM), recruit macrophages, circulating monocytes, lymphocytes, and neutrophils. These cells constitute the acute inflammatory response at the site of injury. Following closely on the heels of this injury, the lung initiates the resolution

PULMONARY FIBROSIS 559 Table 1 Molecule

Functional class

Inferred fibrotic/inflammatory role

5-Lipoxygenase Angiotensin 1 receptor Bleomycin hydrolase Cathepsin K CCL6 (C10) CD44 (hyalurinic acid receptor) CD28 Cox2 CXCL2 (MIP2) CXCL10/IP10 Ccr2 CXCR3 Connective tissue growth factor (CTGF) Cytosolic phospholipase A (2) Decorin Endothelin 1 Endothelin receptor A, B Egr 1 Granulocyte-macrophage colony stimulating factor ICAM IL1b IL-10 IL-13 Integrin avb6 IFN-g

Paracrine mediator synthesis Cell signaling, vasoconstriction Cysteine protease Cysteine protease Chemokine ECM receptor Cell surface receptor, co-stimulatory Paracrine factor enzyme Chemokine Chemokine Chemokine receptor Chemokine receptor Cytokine Paracrine mediator synthesis Extracellular protein Cell signaling, ECM synthesis Cell signaling, ECM synthesis Cell signaling, apoptosis Cytokine

Proinflammatory/profibrotic Profibrotic Anti-inflammatory/antifibrotic Antifibrotic Proinflammatory/profibrotic Anti-inflammatory Proinflammatory/profibrotic Proinflammatory/antifibrotic Proinflammatory/profibrotic Antifibrotic Proinflammatory/profibrotic Antifibrotic Profibrotic Proinflammatory Antifibrotic Profibrotic Profibrotic Profibrotic Antifibrotic

Adhesion molecule Cytokine Cytokine Cytokine Cell surface receptor, TGF-b activation Cytokine

L-selectin Leukotriene C4 Leukotriene E4 Mast Cell Chymase MMP-7 (matrilysin) Relaxin TNF-a TGF-a Thrombin Tissue Plasminogen Activator (tPA) p38 MAPK PAI-1 PDGF Plasminogen Prostaglandin D2 synthase Prostaglandin E2 Smad 3 Smad 7 Superoxide dismutase Tissue inhibitor of metalloprotease TGF-b1 Urokinase-type plasminogen activator

Adhesion molecule Paracrine factor Paracrine factor Extracellular protease Metalloprotease Paracrine factor Cytokine Cytokine Clotting factor, paracrine factor Extracellular protease activator Cell signaling Extracellular protease inhibitor Cytokine Extracellular protease Paracrine factor synthesis Paracrine factor Intracellular signaling Intracellular signaling inhibitor Extracellular enzyme Protease inhibitor Cytokine Extracellular protease activator

Proinflammatory Proinflammatory Anti-inflammatory Proinflammatory/profibrotic Anti-inflammatory/profibrotic Proinflammatory/profibrotic (by LOF), antifibrotic (by GOF) Proinflammatory/profibrotic Profibrotic Profibrotic Antifibrotic Proinflammatory/profibrotic Antifibrotic Antifibrotic ( þ /
) Profibrotic Profibrotic Antifibrotic Profibrotic Profibrotic Profibrotic Antifibrotic Anti-inflammatory/antifibrotic Antifibrotic Profibrotic Antifibrotic Antifibrotic Antifibrotic Anti-inflammatory/profibrotic Antifibrotic

phase during which the amplification of the inflammatory response is arrested, wound repair processes are initiated and, ideally, lung structure is returned to normal. The pleiotropic cytokine transforming growth factor beta 1 (TGF-b1) is critical to the development of lung injury and the switch from inflammation to resolution. Failure to resolve fully lung inflammation and complete normal repair causes fibrosis. In humans

this is clearly demonstrated in patients with underlying connective tissue diseases or chronic lung infections with fungal or mycobacterial pathogens. Indeed, the pneumoconioses, sarcoidosis, chronic hypersensitivity pneumonitis, and connective tissue disease associated interstitial lung diseases are fibrotic lung diseases in which foci of fibrosis are closely associated with accumulated chronic inflammatory cells. In these states, interstitial lung

560 PULMONARY FIBROSIS

inflammation does not resolve normally but is evidently still sufficient to trigger a healing or fibrotic phase of repair. This progression of inflammation to fibrosis is also seen following acute lung injury in the late, or the so-called fibroproliferative, stage of acute respiratory distress syndrome. However, the role of inflammation as the primary cause of all fibrotic lung disease remains a matter of spirited debate. The principal evidence weighing against inflammation as the sole mover of fibrosis is the disease idiopathic pulmonary fibrosis, in which interstitial inflammation is sparse or even absent, and in which intense immunosuppressive therapy has been tried without substantial benefit (see Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis). As in humans, bleomycin causes intense inflammation in susceptible strains of mice prior to the development of pulmonary fibrosis. Therefore, the fact that many inflammatory mediators have been implicated in fibrotic lung disease in mice is perhaps not surprising (Table 1). Of all the cytokines and chemokines associated with pulmonary fibrosis, the most consistently identified and best validated target in mice is TGF-b1 (see Transforming Growth Factor Beta (TGF-b) Family of Molecules). TGF-b1 is a pleuripotent cytokine that, broadly speaking, has four principal functions in the postnatal lung: inhibiting epithelial growth and inducing epithelial apoptosis, promoting fibroblast proliferation, enhancing the synthesis of multiple collagen isoforms and elastin, and inducing an anti-inflammatory state by downregulating both activated macrophages and lymphocytes. Excessive TGF-b1 activity has been conclusively shown to produce lung fibrosis in a multitude of models, and the prevention of the activation of latent TGF-b1 prevents both acute lung injury and fibrosis in response to bleomycin. Interestingly, pharmacologic blockade of TGF-b1 activity after the initiation of fibrosis, while limiting damage, has not yet been shown to cause complete resolution of fibrosis and return to normal lung architecture. Whether this is due to persistent downstream activity in other signaling pathways that prevent resolution or incomplete blockade of TGF-b1 activity in vivo remains undetermined. TGF-b1 (as well as TGF-b2 and TGF-b3) is constitutively secreted in inactive form. TGF-b1 is kept inactive and closely associated with the extracellular matrix through a noncovalent association with latency-associated protein and latent TGF-b1 binding protein. The principal regulatory step controlling the in vivo effects of TGF-b1 is its activation. Genetic manipulations in mice that block TGF-b1 activation in vivo have been shown to be sufficient to block the development of pulmonary fibrosis following administration of bleomycin (see

Transforming Growth Factor Beta (TGF-b) Family of Molecules). Altered Cellular Apoptosis

The role of apoptosis in lung disease – particularly fibrotic lung disease – is complex and cell-type specific. Epithelial apoptosis is prominent in a number of lung diseases including acute lung injury, pulmonary emphysema, and lung cancer. It is gaining ascendance in pulmonary fibrosis as well. The regulation of fibroblast apoptosis, or more specifically apoptosis of the more differentiated and contractile fibroblast subtype, the myofibroblast, is also the focus of increased scrutiny. Recent data from genetically manipulated mice suggest that both Fas/Fas Ligand-mediated and non-Fas-mediated apoptotic pathways play important regulatory roles. Data from TGF-b1 transgenic mice have shown not only that overexpression of active TGF-b1 induces pulmonary fibrosis, but that blockade of the striking epithelial apoptosis induced by overexpression of this cytokine largely ameliorates the fibrotic phenotype. Immunohistochemical evidence of increased epithelial apoptosis has also been noted in the lungs of patients with pulmonary fibrosis. Other studies from primary fibroblasts isolated from patients with fibrotic lung disease suggest intrinsic differences in the apoptotic machinery in these cells leading to decreased apoptosis. Isolation of fibroblasts from these patients shows increased fibroblast survival in response to inflammatory stimuli (interleukin-6) rather than the more normal increase in fibroblast apoptosis after such stimulation. The apoptosis story differs too among lymphocytes. Interestingly, selective deletion of Fas expression in T lymphocytes leads to increased surface Fas Ligand (FasL) on activated T cells. Among other things, this results in increased peripheral apoptosis of lymphocytes and an accumulation of leukocytes within the lung, eventually leading to pulmonary fibrosis. There have been no systematic studies of lymphocyte homeostasis in the lungs of patients with pulmonary fibrosis. In summary, the predominance of current data suggests that increased epithelial apoptosis and decreased fibroblast apoptosis are characteristic of fibrotic lung disease. These may both be the result of increased cytokine activity, particularly TGF-b1, or the result of the alteration of a combination of cellspecific intra- and extracellular signaling events, perhaps as a response to alterations in the mechanics of the extracellular environment. The role of lymphocyte apoptosis in human disease is uncertain. Moreover, the role of apoptosis in general as the primary

PULMONARY FIBROSIS 561

initiating event or the consequence of a more proximal derangement remains unknown. No agents specifically targeting apoptosis are in clinical trials for pulmonary fibrosis. Oxidant-Mediated Lung Injury

Oxidant-mediated lung injury has been implicated in a wide variety of lung diseases including pulmonary emphysema, lung injury, and fibrosis. The activities of a number of extracellular proteins, proteases, and antiproteases are sensitive to the redox state in the extracellular space. These proteins transduce alterations in the redox state into cellular events such as altered intracellular signaling, activation or inactivation of cytokines, and matrix remodeling. The instruments of oxidant-mediated lung injury include superoxide anion, nitric oxide, hydrogen peroxide, and hypohalides. Many of these agents are produced endogenously as part of innate host defenses – particularly by the membrane-bound NADPH system of phagocytes, and by the actions of nitric oxide synthase. Inducible nitric oxide synthase 2 (NOS2A), in particular, is abundant in the lung and several resident lung cells dramatically induce its expression after stimulation by proinflammatory cytokines or endotoxin exposure. The lung has a well-developed mechanism for buffering oxidant stress under normal conditions. These chemical buffers include reduced glutathione, several serum proteins including albumin, cerruloplasmin, transferrin, vitamins C and E, as well as the enzymes superoxide dismutase and catalase. Glutathione is by far the most important antioxidant in the lung with concentrations in alveolar lining fluid that are over 100 times higher than circulating levels. The level of reduced glutathione, which is capable of scavenging oxidant molecules, is decreased by chronic ethanol ingestion, and chronic inflammation. However, the level of glutathione is also tightly regulated in vivo with much of the regulation presumed to occur in lung epithelial cells themselves. Substrate availability – particularly cysteine
and intracellular levels of the rate-limiting first synthetic enzyme glutamate cysteine ligase (GCL) are critical regulatory factors. GCL is a heterodimeric enzyme composed of a 73 kDa catalytic heavy-chain subunit (GCLC) and a 31 kDa modifier light-chain subunit (GCLM). Interestingly, TGF-b1 inhibits expression of the heavy-chain gene in immortalized respiratory epithelial cells and expression of both the heavy- and light-chain proteins is reduced in lung tissue from patients with idiopathic pulmonary fibrosis. Many studies have documented substantially lower levels of glutathione in the alveolar lining fluid

recovered from patients with pulmonary fibrosis. Some studies have suggested that use of oral N-acetylcysteine (NAC) effectively raises the levels of glutathione in the alveolar lining fluid and may also stabilize or improve lung function in idiopathic pulmonary fibrosis. In a recently completed and as yet unpublished trial in Europe (the Idiopathic Pulmonary Fibrosis International Group Exploring NAC I Annual; IFIGENIA), 1800 mg of NAC (600 mg po t.i.d.) reportedly improved vital capacity by 9% (po0.05) and the diffusing capacity for carbon monoxide by 24% (po0.005) compared to placebo after 1 year of treatment. Of note, all 155 patients in this randomized, placebo-controlled trial also received prednisone and azathioprine. Dysregulated Wound Repair and Epithelial Mesenchymal Transformation

Many authors have drawn an analogy between idiopathic pulmonary fibrosis and a nonhealing wound. The roles of appropriate epithelialization and epithelial-fibroblast interactions in limiting the progression of fibrosis have been of particular interest. Others have suggested that, under the particular and currently unknown tissue microenvironment that promotes ongoing fibrosis, epithelial cells undergo a fundamental phenotypic transition to become fibroblastic cells – a so-called epithelial to mesenchymal transformation. Such trans-differentiation of epithelial cells to mesenchymal cells has been well documented in chronic renal and liver fibrosis. Recently, recruitment of bone marrow-derived circulating collagen-producing CD34 þ cells called fibrocytes has also been implicated in experimental pulmonary fibrosis. In a series of elegant experiments, investigators fluorescently labeled bone marrow cells, which were engrafted into mice that did not express the tag. Following bleomycin injury, substantial numbers of fluorescently labeled fibroblasts were localized in areas of injury indicating the cells derived from transplanted marrow developed into scar tissue in the lung. Other investigators have shown that fibrocytes, circulating cells that express both the leukocyte marker CD34 and Col1, traffick to the lung following bleomycin-induced lung injury in response to CXCL12 (SDF-1), which is produced in the lung following injury. The role of these cells in chronic fibrotic lung disease or repair of acute lung injury in humans is unknown. Two molecular signaling pathways that have been implicated in this process of epithelial-mesenchymal cross-talk are sonic hedgehog, a secreted factor that is critical to early lung morphogenesis and that is also induced in response to lung injury, and Wnt

562 PULMONARY FIBROSIS

(pronounced ‘wint’), another secreted factor that is important in both epithelial migration and lung development. Expression of the protease matrilysin (MMP7) is induced by Wnt signaling. Matrilysin is also among the most induced genes in the lungs of patients with idiopathic pulmonary fibrosis based on global gene expression analysis. Matrilysin also plays a critical role in the regulation of the inflammatory response through its cleavage of syndecan 1 and the release of the neutrophil chemoattractant KC. Matrilysin-deficient mice have reduced neutrophil accumulation in the alveolar airspace and a delay in the development of pulmonary fibrosis following lung injury with bleomycin. This illustrates the tight interplay between parenchymal and inflammatory cell biology in vivo in response to injury and the complexity of dissecting the molecular unpinning experimentally. Another molecular pathway that has been implicated in mesenchymal cell proliferation and potentially epithelial to mesenchymal transformation in vivo is the endothelin-1 pathway. Endothelins are a family of three proteins (Edn1, Edn2, Edn3), which vary in size from 160 to 214 amino acids, that are broadly expressed and mediate contraction of vascular and intestinal smooth muscle through their actions on two G-protein-coupled receptors, endothelin receptors A and B. Endothelins play a critical role in the maintenance of vascular tone, cardiac remodeling, intestinal motility, and the maintenance of glomerular function. In humans, endothelin 1 – the best characterized of the endothelins – is most highly expressed in the lungs and cardiac myocytes. Constitutive transgenic overexpression of Edn1 in the lungs and kidneys of mice induces pulmonary fibrosis and glomerulosclerosis. As a result of these findings, a dual endothelin receptor blocker, bosentan, which is used in primary pulmonary hypertension, is now also used in clinical trials to assess for efficacy in pulmonary fibrosis in humans. Protease/Antiprotease Imbalance

Genes encoding proteases are among the most common in the human genome. They play critical regulatory roles in inflammation, tissue remodeling, cell death, and migration. In the extracellular space, proteases are particularly important in regulating the deposition and maintenance of extracellular matrix. Proteases are expressed by a wide range of cells and both their expression and secretion are tightly regulated. Beyond intracellular control mechanisms, protease activity is buffered by antiproteases. These molecules bind to and inhibit the proteolytic activity of proteases. Among the most important of these

protease inhibitors are alpha-1 antiprotease (alpha-1 antitrypsin), which is secreted by the liver and bathes the entire extracellular space; the tissue inhibitors of metalloproteases (TIMPs), which are produced locally and have particular substrate specificities; and the cystatins, which are inhibitors of cysteine proteases. Of these, the locally produced inhibitors TIMPs have attracted the most interest in the tissue level regulation of collagen and elastin deposition and removal. In general, deficiencies of these protease inhibitors are thought to result in a ‘profibrotic’ environment. Interestingly, another protease/antiprotease system, the thrombin/plasmin/plasmin-activator-inhibitor pathway, has also been implicated in pulmonary fibrosis in mice. The details of this pathway are outlined in Coagulation Cascade: Thrombin. In this system, an excessive amount of the TGF-b1inducible protease inhibitor plasminogen activator inhibitor-1 (PAI-1) inhibits the dissolution of provisional matrix by plasmin and therefore promotes fibrosis. Consistent with this hypothesis, mice deficient in the gene for PAI have reduced pulmonary fibrosis in response to bleomycin. Altered Eicosinoid Homeostasis

Eicosinoids are biologically active derivatives of arachidonic acid, which are liberated from cell membrane phospholipids by the action of phospholipase A2. Eicosinoids are produced in one form or another by every cell and are rapidly mobilized following calcium influx. There are three broad categories of eicosinoids: prostenoids, leukotrienes, and lipoxins. The principal prostanoids PGD2, PGF2a, PGE2, PGI2, and TxA2 (thromboxane) are derived from arachidonic acid through the enzymatic activity of cyclooxygenase-1 and -2. Leukotrienes, which are produced principally by inflammatory cells, result from the enzymatic activity of 5-lipoxygenase. Lipoxins are produced via 5-, 12-, and 15-lipoxygenases and are generally considered to be anti-inflammatory. All of the eicosinoids have a rapid onset of action and are quickly metabolized making them both ideal mediators of cell–cell communication. The topic of eicosinoid biology is more fully reviewed elsewhere (see Lipid Mediators: Overview; Leukotrienes; Prostanoids; Platelet-Activating Factors; Lipoxins). The role of eicosinoids in pulmonary fibrosis is complex and controversial. In general, experimental data in genetically manipulated mice support the notion that leukotrienes promote the development of fibrosis (since deletion of 5-LO, a key regulatory enzyme, is protective) and prostenoids prevent fibrosis (since deletion of cyclooxygenase-2 exacerbates

PULMONARY FIBROSIS 563

fibrosis). Elevated levels of 5-lipoxygenase metabolites have been demonstrated in both lavage fluid and tissue from patients with idiopathic pulmonary fibrosis, and alveolar macrophages are probably the principal source. The stimulus for this excessive 5-lipoxygenase activity, if any, is unknown. The principal prostenoid implicated in pulmonary fibrosis is PGE2. Reduced levels have been documented both in humans with fibrotic lung disease and in susceptible mouse strains following bleomycin-induced fibrosis – consistent with its presumed protective role. Substantial work remains to be done in this area, as the key regulatory events, cellular substrate, and postsignaling events – many of which may be celltype and cell-state dependent – remain poorly understood. Notably, both pharmacologic and dietary interventions alter the activity of this pathway making an improved understanding of this signaling

Normal alveolar structure

pathway in disease pathogenesis of particular immediate appeal. A single-center clinical trial is currently underway to assess the efficacy of a 5-LO inhibitor in human pulmonary fibrosis.

Moving Models from Mice to Man Despite the dramatic progress over the last 10 years in the understanding of critical molecular events underlying scarring in the lungs of mice following bleomycin, there has been frustratingly little progress in the area of therapeutics. Fortunately, if the initiation of clinical trials is any indication, pulmonary fibrosis has now caught the attention of the pharmaceutical industry and many new compounds are now in various stages of clinical development. Translating basic molecular insight into clinically useful pharmaceuticals has been hampered by several factors. These

Fibrosis

Air Pulmonary arterial blood

Pulmonary venous blood

Dense fibrosis

CXCR3 CCL6 Leukotrienes Cox2 PGE2

Type II alveolar epithelial cell

Alveolar capillaries

Collagen Fibronectin Vitronectin Hyaluronan

Repopulating stem cells

Alveolar macrophage

(a)

IL-1, IL-13 IFN-, IP-10 Alveolar O3 macrophage SOD GSH Latent TGF- Mra7
v6 Decorin Active TGF-

CD44

Epithelial to mesenchymal transformation AT1 ICAM thrmb ET1 L-selectin UPA tPA Myofibroblasts, PAI-1 fibrocytes Disruption of Egr 1 basement membrane Epithelial apoptosis CTGF PDGF

Type I alveolar epithelial cell

Mature collagen Basement membrane

Normal lung

(b)

Figure 1 Pulmonary fibrosis requires a complex, multicellular environment. (a) Normal lung architecture and function. The healthy lung is a delicate, reticular structure with close apposition of epithelial cells lining the airway and alveolar space. These cells are separated from underlying stroma by tight junctions between cells, and a continuous basement membrane composed of laminin, type IV collagen, entactin, and proteoglycans. The structural support of the lung, or stroma, contains blood vessels, structural cells such as fibroblasts that secrete elastin, collagen (fibrillar, nonfibrillar, and low molecular weight) and proteoglycans, and migratory leukocytes such as dendritic cells, lymphocytes and interstitial macrophages. (b) Molecular processes implicated in the fibrotic process; the role of epithelial denudation, disruption of the epithelial basement membrane, and activation of inflammatory and noninflammatory cells is emphasized. The net result of these processes is loss of functional gas exchange units through capillary drop-out, decreased alveolar compliance, and interstitial thickening (see Table 1 and text for abbreviations). Reproduced from Journal of Clinical Investigation, 2004, with permission.

564 PULMONARY FUNCTION TESTING IN INFANTS

include the relative infrequency of pulmonary fibrosis in comparison to other lung diseases such as asthma, lung cancer, and pulmonary infections. Further, the relatively indolent clinical course and lack of good surrogate markers of disease progression has led to a need for both prolonged trials and large patient numbers. Finally, idiopathic pulmonary fibrosis has only recently been clearly separated from the other myriad diseases that have fibrosis as a final common pathway. This separation of idiopathic pulmonary fibrosis from the other idiopathic interstitial pneumonias has had a sobering effect by highlighting both the grim survival (50% mortality on average at 3 years) and the poor response to conventional immunosuppressive therapies. Fortunately, as fundamental molecular insights have identified key molecular pathways, the pharmaceutical industry has developed more directed therapies. These include small molecule inhibitors, humanized monoclonal antibodies, and other targeted therapeutics. Progress in the future will depend on the ability to capitalize on these initial forays and to maintain the focus of both the scientific and private sectors on this devastating human disease (Figure 1). See also: Interstitial Lung Disease: Hypersensitivity Pneumonitis; Idiopathic Pulmonary Fibrosis. Lipid Mediators: Overview; Leukotrienes; Prostanoids; PlateletActivating Factors; Lipoxins. Occupational Diseases: Hard Metal Diseases – Berylliosis and Others; Silicosis. Pneumonia: Fungal (Including Pathogens); Mycobacterial. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Borzone G, Moreno R, Urrea R, et al. (2001) Bleomycin-induced chronic lung damage does not resemble human idiopathic pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine 163(7): 1648–1653.

Chapman HA (2004) Disorders of lung matrix remodeling. Journal of Clinical Investigation 113(2): 148–157. Hashimoto N, Jin H, Liu T, Chensue SW, and Phan SH (2004) Bone marrow-derived progenitor cells in pulmonary fibrosis. Journal of Clinical Investigation 113(2): 243–252. Jiang D, Liang J, Hodge J, et al. (2004) Regulation of pulmonary fibrosis by chemokine receptor CXCR3. Journal of Clinical Investigation 114(2): 291–299. Kaminski N, Allard JD, Pittet JF, et al. (2000) Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proceedings of the National Academy of Sciences 97(4): 1778– 1783. Kolb M, Margetts PJ, Anthony DC, Pitossi F, and Gauldie J (2001) Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. Journal of Clinical Investigation 107(12): 1529–1536. Kuhn C III, Boldt J, King TE Jr, et al. (1989) An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. American Review of Respiratory Diseases 140: 1693–1703. Lee CG, Cho SJ, Kang MJ, et al. (2004) Early growth response gene 1-mediated apoptosis is essential for transforming growth factor beta1-induced pulmonary fibrosis. Journal of Experimental Medicine 200(3): 377–389. Lynch JP III (2004) Idiopathic Pulmonary Fibrosis. New York: Dekker. Morris DG, Huang X, Kaminski N, et al. (2003) Loss of avb6 integrin-mediated TGFb activation causes Mmp12 dependent pulmonary emphysema. Nature 422(6928): 169–173. Phillips RJ, Burdick MD, Hong K, et al. (2004) Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. Journal of Clinical Investigation 114(3): 438–446. Selman M, King TE Jr, and Pardo A (2001) Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Annals of Internal Medicine 134: 136–151. Teder P, Vandivier RW, Jiang D, et al. (2002) Resolution of lung inflammation by CD44. Science 296(5565): 155–158. Thannickal VJ, Toews GB, White ES, Lynch JP, and Martinez FJ (2004) Mechanisms of pulmonary fibrosis. Annual Review of Medicine 55: 395–417. Zuo F, Kaminski N, Eugui E, et al. (2002) Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proceedings of the National Academy of Sciences 99(9): 6292–6297.

PULMONARY FUNCTION TESTING IN INFANTS J Stocks, Institute of Child Health, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Infant pulmonary function tests (IPFTs) can be used to assess lung volumes, forced expiratory maneuvers, and respiratory mechanics in healthy infants and those with respiratory disease. International guidelines for standardizing equipment and measurements have been published. Such measurements are more

complex and time consuming than in children and adults, requiring specially trained personnel and facilities. IPFTs are generally performed during sleep (which, beyond the neonatal period, generally requires light sedation), in the supine position, and while breathing through a face mask. There are marked differences in developmental respiratory physiology that must be considered when performing and interpreting results from infants, including the rapid lung and airway growth and the fact that infants are preferential nose breathers and have a highly compliant chest wall. Although IPFTs have occasionally been used in clinical management, their primary role has been in

PULMONARY FUNCTION TESTING IN INFANTS 565 epidemiological and clinical research, where they have been used to investigate early determinants of lung function (including the detrimental effects of maternal smoking, intrauterine growth retardation, and preterm delivery), the nature and severity of various respiratory diseases, airway reactivity, and response to therapeutic interventions, including different types of ventilatory management.

Introduction Measurement of pulmonary function is an integral component of respiratory physiology and clinical assessment of lung diseases in school-aged children and adults. Despite their heightened vulnerability to respiratory diseases, similar assessments in infants and young children were, until recently, restricted to specialized research establishments. This was largely due to the lack of suitable equipment and difficulties in performing such measurements in small, potentially uncooperative subjects. The realization that insults to the developing lung may have lifelong effects, and that much of the burden of respiratory disease in childhood and later life has its origins in infancy and fetal life, emphasized the need for sensitive and reliable methods of assessing respiratory function in this age group. During the past 10 years, there has been increasing commercial availability of equipment for assessing respiratory function of infants that, together with the publication of international guidelines for standardized methods of data collection and analysis, has facilitated more widespread use of these tests.

The Differences in Assessing Lung Function in Infants Compared to Older Subjects Developmental differences in respiratory physiology that impact both on measurements of infant lung function and on interpretation of results are summarized in Table 1. These include factors such as the highly compliant chest wall, the tendency of infants to modulate expiratory flows and timing to dynamically elevate functional residual capacity (FRC) above that determined by passive mechanisms, and preferential nose breathing during early life. Major differences in performing pulmonary function tests (PFTs) in children younger than 2 years of age are summarized in Table 2 and relate to the following: *

*

*

* *

Sleep state. Tests should generally be confined to periods of quiet sleep, with a regular breathing pattern. Sedation. Usually necessary beyond 1 month postnatal age, and most commonly achieved using an oral dose of chloral hydrate (50–100 mg kg
1). Ethical issues, including the need for fully informed (written) parental consent. Posture Duration of tests. Given the need for explanation of procedures to parents prior to assent, clinical examination, sedation, and subsequent interval

Table 1 Major differences in respiratory physiology in the infant compared to the adult Chest wall  Configuration (horizontally placed ribs – decreased efficiency)  High chest wall compliance resulting in (i) Low outward recoil of chest wall with limited distending pressure (ii) Reduced ‘passive’ FRC and tendency to atelectasis (iii) Airway closure during tidal breathing with increased tendency to peripheral airway obstruction (iv) Impaired gas exchange (ventilation: perfusion imbalance) (v) Increased work of breathing due to chest wall recession (paradoxical ribcage–abdominal movement) Preferential nose breathers  Assessments of resistance in infants include that of the nasal passages, with nasal resistance (Rn) comprising B50% total resistance (i) Diminished sensitivity for detecting lower airway disease or response to therapeutic interventions (ii) Infant PFTs must be deferred for at least 3 weeks following an upper respiratory infection (iii) Pharmacological challenges by aerosol (bronchodilators or constrictors) may be preferentially deposited in the nose Dynamic elevation of FRC  Expiratory flows and timing are modulated to maintain end expiratory lung volume (FRC) above that passively determined by the outward recoil of chest wall and inward recoil of lung, thereby partially counteracting effects of high chest wall compliance  This ‘dynamic elevation’ of FRC is achieved by combining a relatively short expiratory time (rapid respiratory rate) with prolongation of the expiratory time constant (achieved by laryngeal braking or postinspiratory diaphragmatic activity)  Transition to a more relaxed pattern of expiration occurs between 6 and 12 months of age Respiratory reflexes  Stronger vagally mediated, stretch receptor activity than in adults; infants have an active Hering–Breuer inflation reflex during tidal breathing, such that airway occlusion at end tidal inspiration evokes a brief expiratory pause; this allows measurements of passive respiratory mechanics to be obtained in infants

566 PULMONARY FUNCTION TESTING IN INFANTS Table 2 Major differences in measuring pulmonary function in infants compared to older subjects

Consciousness Sedation Cooperation Duration of test Equipment Airway attachment Airflow Posture Used in clinical management Personnel Facilities

Informed consent

Infant

Child or adult

Sleeping Yes (if 41 month) Passive Up to 3 h Limited commercial availability Face mask Nose breathing Supine Rarely Two highly trained staff, at least one with clinical (medical/nursing) qualifications Additional facilities for feeding, changing, settling to sleep required, in addition to full resuscitation equipment From parents

Awake None Active o1 h Widely available Mouthpiece Mouth breathing Seated or standing Frequently Respiratory technician or therapist

From subject (and/or parent)

(vi) incorporate suitable software for optimizing data collection, analysis, and quality control. *

*

Figure 1 Use of face mask with ring of silicone putty to effect airtight seal during infant lung function tests.

*

Safety issues. Infant PFTs require two fully trained staff with basic life-support skills, together with a hygienic and safe testing environment with full resuscitation facilities. Pulse oximetry is used for continuous monitoring throughout the testing session. Anthropometry. Although essential for all lung function tests, accurate measurements using a stadiometer for length are particularly crucial during infancy due to rapid somatic growth.

Tests Used to Assess Pulmonary Function in Infants

until onset of sleep, the entire testing session may take 3 or 4 h per infant, only 30–60 min of which will actually be spent on data collection. A similar amount of time per infant may be required when recruiting subjects for research studies. Equipment, which must

A wide range of techniques have been adapted for use in infants, the applicability of which according to age, clinical status, and measurement conditions is summarized in Table 3.

(i)

Many of the techniques for measuring lung volumes in older children and adults have been successfully adapted for use in infants and young children. Since infants cannot be instructed to perform special breathing maneuvers, lung volume measurements in this age group have primarily been those that can be undertaken during tidal breathing (i.e., functional residual capacity (FRC) using either plethysmographic or gas dilution/washout techniques). In recent years, it has become possible to obtain measurements over an extended volume range in specialized centers using what has become known as the ‘raised volume technique’.

(ii) (iii) (iv)

(v)

be miniaturized, with minimization of both dead space and resistance; be easily disinfected between every subject; provide rapid access to the infant at all times; be adapted for nose-breathing subjects, a mask with or without silicone putty generally being used to effect an airtight seal between the apparatus and the infant (Figure 1). Any air leak will invalidate all measurements; have appropriate frequency response and resolution characteristics in the presence of low signal:noise ratio and rapid respiratory rate; and

Assessment of Lung Volumes and Ventilation

PULMONARY FUNCTION TESTING IN INFANTS 567 Table 3 Applicability of infant respiratory function tests in specific situationsa Diagnosis/ situation

Neonatal ICU Ventilatedb Spontaneous breathing Unsedated: o1 month Sedated 1–24 months

Tidal breathing

RIP

Esophageal Occlusion Forced Forced MBW or gas Plethysmography manometry ðFRC þ Raw Þ techniques oscillation expirations dilution (RL , CL ) (RRS , CRS ) (HIT or LFOT) (tidal or (FRC þ VI) RVRTC)

þ þ

(þ) þ

þ þ

þ þ

(þ) (þ)

# (þ)

(þ) þ

NA (þ)

þ

þ

þ

þ

(þ)

þ

þ

þ

þ

þ

Rarely applicable

þ

(þ)

þ

þ

þ

a The choice of tests depends not only on available equipment and what is feasible but also on the underlying clinical or research question. b Leaks around tracheal tube with or without interactions between ventilated and spontaneous breaths may invalidate measurements in intubated infants. þ , useful; ( þ ), limited to specialized research application; NA, not applicable; #, only by negative forced deflation; RIP, respiratory inductance plethysmography; RL and CL, lung resistance and compliance, respectively; RRS and CRS, respiratory resistance and compliance, respectively; ICU, intensive care unit; HIT, high interrupter technique (airway wall mechanics); LFOT, low-frequency forced oscillation (partitioned tissue mechanics); RVRTC, raised volume rapid thoracoabdominal compression; MBW, multiple breath inert gas washout; FRC, functional residual capacity; VI, ventilation inhomogeneity; Raw, airway resistance.

Tidal Breathing Parameters

Tidal breathing parameters are measured while the infant breathes through a face mask and flowmeter. They have been used in clinical and research settings *

* *

*

* *

to determine tidal volume, breathing frequency, and minute ventilation; to investigate the control of breathing; as an integral part of sleep staging and to establish regularity of breathing pattern prior to assessment of lung volumes and mechanics; in combination with CO2, O2, exhaled NO, and SaO2 monitoring and other clinical information; as an indirect measure of airway mechanics; and to trigger equipment.

Attempts to quantify patterns of tidal flow–volume loops have resulted in numerical descriptors of the tidal flow pattern, such as the time to peak tidal expiratory flow as a ratio of total expiratory time (tPTEF:tE) (Figure 2). This index may be reduced in the presence of airway obstruction and has been shown to be a useful outcome measure in various epidemiological studies investigating early determinants of airway function. However, tPTEF:tE is only distantly related to airway function and, as with most tidal breathing parameters, conveys mixed information on the interaction between control of breathing and airway mechanics, thereby requiring cautious interpretation, especially within individuals. Assessments of Functional Residual Capacity

FRC can be measured using either plethysmography, which measures all gas within the thorax, including

that trapped behind closed airways, or a gas dilution or washout technique, wherein only the gas that communicates readily with the airway opening is measured. Both techniques have been used widely to assess lung volume in infancy during clinical and epidemiological research. The principles of plethysmography are identical for infants and older subjects (Figures 3 and 4) with the major difference being that measurements are made in the sleeping, supine infant and that the airway occlusion is held for approximately 8–10 s to allow the infant to make at least two complete respiratory efforts against the occlusion. Equipment that meets most of the published international specifications is now commercially available. The main advantages of this technique are the rapidity and repeatability with which measurements can be made (five repeat measures within o5 min) and the fact that it can detect hyperinflation and gas trapping in infants with airway obstruction. However, plethysmography is not suitable for bedside measurements in sick or small infants due to the relatively bulky equipment. Simultaneous measurements of airway resistance can be obtained if the respired gas can be maintained under body temperature and pressure saturated conditions using a heated rebreathing bag, although this additional application is usually limited to specialist physiology departments. Methods of assessing FRC by gas dilution or washout are also essentially the same in infants as in older subjects, and guidelines for standardized equipment and measurements have been published. Although closed-circuit helium dilution has been widely used in the past, there are difficulties in reducing circuit size sufficiently for use in infants, as

568 PULMONARY FUNCTION TESTING IN INFANTS Insp

Exp

Insp

Exp

Flow

Volume

PTIF VT

tPTIF

PTEF

tE

tI

tPTEF

t tot Time

(a)

(b)

Time

V PTEF

PTEF

TEF50

Flow

Exp Insp PTIF

0.75 (c)

TIF50

0.5 VT

0.25

0.1

Volume

Figure 2 Tidal breathing parameters: (a) and (b) time-based trace of tidal volume and (c) tidal flow–volume loop.

Vent

Tidal flow & volume

3-way valve

Rebreathing bag with servo-controlled heater and thermometer

Heated pneumotachometer

Calibration syringe

∆ V pleth

Pressure at airway opening

Controlled mechanical leak Reference chamber

Figure 3 Infant plethysmography.

well as challenges posed when attempting to maintain a constant volume in the face of varying oxygen consumption and CO2 production. Consequently, there has been increasing emphasis on the use of

washout techniques in infants and young children, using either the bias flow nitrogen (N2) washout technique, which is based on a mixing chamber technique, or, more recently, the multiple breath inert gas

PULMONARY FUNCTION TESTING IN INFANTS 569 Flow (ml s–1)

150

Volume (ml)

69

−150

−69

Pao (kPa)

0.5

−0.5 0

5

10

15

20

25

30

Time (s)

(a)

2.0 1.5

Pao (kPa)

1.0 0.5 0.0 −0.5

FRCp 0 = 221.6 ml FRCp 1 = 221.7 ml

−1.0

FRCp 2 = 235.8 ml −1.5 −2.0

Mean

−50

FRCp

−40

(b)

= 226.3 ml

−30

−20

−10

0

10

20

30

40

50

Box volume change (ml)

Figure 4 Plethysmographic assessments of FRC in an infant. (a) Time-based recording; (b) x–y plot of box volume vs. Pao during airway occlusion.

washout (MBW) technique. The latter measures breath-to-breath changes in the concentration of an inert gas during the washout process and provides information on both lung volume and ventilation efficiency. Gas Mixing Efficiency

Although described many years ago, in the past the MBW technique for assessing gas mixing efficiency or ventilation inhomogeneity was only used

intermittently in infants. During recent years, technological advances combined with increasing awareness that conventional measures of airway function may not detect early changes in peripheral airway function until lung disease is well established have led to a resurgence of interest in this field. The MBW is applicable to subjects of all ages, including bedside measurements in unsedated infants, because measurements are performed during spontaneous tidal breathing. The lung clearance index, a measure of ventilation inhomogeneity that is calculated from the

570 PULMONARY FUNCTION TESTING IN INFANTS

cumulative expired volume required to clear a tracer gas from the lungs, is a sensitive indicator of early airway disease in infants with cystic fibrosis.

Respiratory Mechanics: Resistance and Compliance Assessments of respiratory resistance and, particularly, compliance are performed relatively frequently in infants and young children compared to older subjects. This probably reflects the relative ease with which such measurements can be performed in early life as well as the fact that, in contrast to older subjects, changes in the elastic properties of the lung often dominate the underlying pathophysiology of lung disease in newborn infants. These techniques are performed during tidal breathing and require little cooperation from the infant other than breathing through a face mask. Esophageal manometry was once commonplace during assessments of lung function in infants, but it has generally been replaced by the less invasive assessments of passive respiratory mechanics using one of the occlusion techniques. Plethysmographic assessments of airway resistance have been used to study normal airway growth and development in relation to lung volume during the first year of life and to discriminate between healthy infants and those with respiratory disease or prior wheezing. Passive Respiratory Mechanics

Measurements of passive respiratory mechanics (compliance, resistance, and expiratory time constant) require complete relaxation of the respiratory muscles. Although this is only feasible in highly trained adults, such relaxation can be routinely induced in infants in whom the vagally mediated Hering–Breuer inflation reflex (HBIR) is active within the tidal volume range. The ‘occlusion technique’ for measuring passive respiratory mechanics is based on the ability to invoke the HBIR by performing brief intermittent airway occlusions during spontaneous tidal breathing (Figure 5). Activation of vagally mediated pulmonary stretch receptors when the airway is occluded above FRC leads to inhibition of inspiration and prolongation of expiratory time. Provided there is no respiratory muscle activity and rapid equilibration of pressures across the respiratory system during the occlusion (as shown by the presence of a pressure plateau), alveolar pressure, and hence the elastic recoil of the respiratory system, can be measured at the airway opening (Figure 6). By relating this recoil pressure either to the volume above the passively determined end expiratory volume at which the airway occlusion was performed or to the airflow occurring on release of the occlusion, the compliance and resistance of the respiratory system

Flow and volume

Pneumotachometer Shutter

Airway opening pressure

Face mask

Figure 5 The occlusion technique.

P

Flow

∆P

Pressure A Time ∆F

B

Compliance = ∆V/ ∆P Resistance = ∆P / ∆Flow Vx Volume

∆V Figure 6 Calculation of passive respiratory mechanisms.

can be measured. The major limitation of this technique, as with all methods that utilize intermittent airway occlusions, is that in the presence of severe airway obstruction, pressures may not equilibrate rapidly enough to allow accurate measurements at the airway opening. Furthermore, it assumes that the time constant of the respiratory system (which is the product of resistance and compliance) can be described by a single value, which is unlikely to be true in the presence of respiratory disease. Forced Oscillation Technique

The forced oscillation technique (FOT), in which the impedance of the respiratory system is measured by superimposing small-amplitude pressure oscillations on the respiratory system and measuring the resultant oscillatory flow, is another technique that has been adapted for use in infants and preschool children. Respiratory impedance describes the spectral (frequency domain) relationship between pressure and airflow throughout the respiratory cycle, providing a global measure of resistive, elastic (viscous), and inertial forces and the opportunity to investigate the frequency dependence of respiratory mechanics. In recent years, there have been two interesting modifications to the FOT that, although remaining

PULMONARY FUNCTION TESTING IN INFANTS 571

strictly in the research domain, are of potential future clinical significance. Depending on the frequency of the applied pressure wave, the resultant impedance contains different mechanical information. The response to very slow pressure oscillations (o2 Hz), during what has been referred to as the low-frequency forced oscillation technique, allows the noninvasive partitioning of lung function into airway and tissue parameters. This approach may be of particular interest for studies in preterm infants, in whom parenchymal disease is a major component of acute respiratory illness. In contrast, at very high frequencies, as are utilized in the high-frequency interrupter technique, the information derived is primarily related to airway wall mechanics, which is particularly important in wheezing disorders.

commercially available infant lung function equipment, however, has yet to be established. V0 maxFRC is significantly lower (by approximately 15%) in boys than in girls throughout infancy and in infants whose mothers smoked during pregnancy. Forced Expiration from Raised Lung Volume

Despite the popularity of the tidal RTC, its value when assessing either baseline airway function or bronchial responsiveness may be limited by the dependence of reported values of V0 maxFRC on the resting lung volume. The latter may not be stable in infants, particularly in the presence of disease or during intervention studies. During the 1990s, the tidal RTC

Forced Expiratory Maneuvers Partial Forced Expiratory Maneuvers

Large-bore tubing & valve Flowmeter

Inflatable jacket

Figure 8 Measurements of partial forced expiratory maneuvers in an infant.

500

400

V ′maxFRC = 300 ml s−1

300

Flow (ml s−1)

Infants cannot be instructed to perform forced expiratory maneuvers, but partial expiratory flow volume curves can be produced by wrapping a jacket around the chest and abdomen that is inflated at the end of a tidal inspiration to force expiration. The resultant changes in airflow (and hence volume) are recorded through a flowmeter attached to a face mask, through which the child breathes (Figures 7 and 8). This technique is often referred to as the ‘squeeze’ or tidal rapid thoracoabdominal compression (RTC) technique. Maximal expiratory flow at FRC (V0 maxFRC), which measures forced expiratory flows at low lung volumes (similar to FEF75 or FEF85 in older children), is the most commonly reported parameter derived from this technique (Figure 9). In the presence of airway disease, not only is V0 maxFRC reduced but also there are characteristic changes in the shape of the flow volume curve (Figure 10). Guidelines regarding data collection and analysis for the tidal RTC have been published by the European Respiratory Society/American Thoracic Society task force, as have sex-specific collated reference data. The validity of such reference equations for interpreting data collected with the new generation of

200

100 Pressure relief valve Compressed air 50 l tank

Figure 7 The tidal RTC (squeeze) technique for measuring partial forced expiratory maneuvers.

F=0

0

−100 40

20

0

−20

−40

Volume (ml) Figure 9 Calculation of V 0 maxFRC from a partial RTC maneuver.

572 PULMONARY FUNCTION TESTING IN INFANTS 200

200

V ′max FRC

100 Flow (ml s−1)

Flow (ml s−1)

100

0

−100

0

−100

40

20

0

40

−20

Volume (ml)

(a)

(b)

20

0

−20

Volume (ml)

Figure 10 Comparison of partial flow volume loops in: (a) healthy infant and (b) airway obstruction.

Pressure relief valve

Fresh gas flow

Insp Exp

Pneumotachometer Face mask

Pressure relief valve Compressed air

Y-piece Pressure port

Large-bore 3-way tap

Inflatable bladder Jacket

100 l tank

Figure 11 Forced expiratory maneuvers from raised lung volume.

technique was modified so that forced expiratory flows and volumes could be measured over an extended volume range in what became known as the ‘raised volume RTC or RVRTC technique’ (Figure 11). Similar to spirometric assessments in older subjects, the RVRTC technique allows the infant’s lungs to be inflated toward total lung capacity (TLC) before rapid inflation of the jacket initiates forced expiration from this elevated lung volume, with the maneuver ending when the infant reaches residual volume (Figure 12). Application of three–five augmented breaths to induce a respiratory pause before forcing expiration encourages the child to breathe out toward residual volume. The relationship between a tidal and raised volume forced expiratory flow volume curve is shown in Figure 13. The rapidity of lung emptying during early life generally precludes measurement of forced expired volume in 1 s (FEV1), with results of FEV0.5 being more commonly reported in infants.

Use in Clinical Practice Although there is little doubt about the potential value of infant lung function tests as a means of providing objective outcome measures in clinical or epidemiological research studies, their potential usefulness with respect to influencing clinical management within an individual infant remains far more debatable. Lung function tests at any age are rarely performed for diagnostic purposes but rather to monitor the nature and severity of respiratory disease or to assess the response to treatment. ‘Typical’ changes in selected lung function parameters according to diagnostic group are summarized in Table 4. It is generally agreed that no single lung function test will provide the answer and that a combination of tests is required, the results of which should be interpreted in light of other information regarding current clinical status and prior medical and social

PULMONARY FUNCTION TESTING IN INFANTS 573 Apnea Flow 500

Forced expiration Volume 250

Tidal breathing

Pj

Passive lung inflations Jacket inflation

2.5

Pao 2.5

10 s Figure 12 Time-based recording of forced expiratory maneuvers using the raised volume technique.

900

Flow (ml s−1)

600

300

0

–300 150

F=0

100

50

0

–50

Volume (ml) Figure 13 Overlay of tidal and raised volume FEFV curves.

history. Choice of technique also depends on the setting, with tests such as the raised volume technique requiring highly trained staff and sophisticated equipment (Table 3). The clinical usefulness of any technique depends not only on its ability to measure parameters that are relevant to the underlying pathophysiology and to discriminate between health and disease but also on within-subject repeatability both within and between test sessions. Although highly reproducible measurements of lung function can be made in infants during

the same test session, little is known about the ‘between-test repeatability’. Similar problems arise with respect to distinguishing the effects of disease from those of growth and development since the timeconsuming nature of the tests and need for sedation limit the numbers of healthy infants who can be studied. If appropriate equipment were to be miniaturized and/or incorporated into the ventilatory circuit so that continuous online monitoring of appropriate parameters could be undertaken, the major area in which infant lung function tests could influence clinical management in the future is probably the neonatal and pediatric intensive care unit. Interpretation of results will depend on knowledge of growth and development of the lung, particularly after extremely preterm delivery. There may also be a role for infant lung function testing with respect to longitudinal measurements from birth and throughout the preschool years in high-risk groups, such as those with persistent wheezing, cystic fibrosis, and chronic lung disease. The potential prognostic value of such tests within individual subjects is unknown since it is only during the past few years that more routine assessments of lung function have been possible throughout the preschool years, but such studies are currently under way.

Uses and Applications in Respiratory Research The major role for infant lung function testing remains firmly within the research arena, in which it has been extensively used to examine the early determinants of airway function and to investigate underlying pathophysiology and response to therapeutic interventions in a variety of respiratory diseases during

Table 4 Typical changes in pulmonary function in various respiratory diseases during infancya Diagnosis

Respiratory rate

Forced expiratory maneuver V 0 maxFRC (partial FEFV)

FEV0.5, FEF% (RVRTC)

Resistance

Compliance

FRCpleth

FRCgas

Comments

Typically small, stiff lungs during acute phases Airway obstruction as evidenced by m resistance and k flows becomes apparent with onset of chronic changes Gas trapping and hyperinflation rare unless tested during acute attack Marked hyperinflation with or without gas trapping; (FRCpleth
FRCgas); despite marked airway obstruction, V 0 maxFRC may appear normal due to elevated FRC Marked m in inspiratory resistance and flattening of inspiratory flow– volume loop, with k inspiratory flows. Nb sedation may be contraindicated if upper airway obstruction occurs Diminished FRC not always evident if dynamic elevation is within normal limits

Acute RDS

m

NA

NA

N

k

NA

k

CLDI (BPD)

m or N

k or N

k

m

k or N

N

k or N

Wheezing

m or N

k or N

k

m or N

N

m or N

N

Cystic fibrosis

m

k or N

k

m

N

mm or N

m or N

Upper airway obstruction

N

N

N

mm (especially during inspiration)

N

N

N

Pulmonary hypoplasia

mm

N

N

N or k

N for lung size, k for body size

k

k

a

Due to the wide range of normative data, values from individual infants with respiratory disease may fall within ‘normal’ limits, with the changes indicated here only becoming significant when groups of infants with similar conditions are studied. V 0 maxFRC, maximum expiratory flow at FRC; FEFV, forced expiratory flow volume curve; RVRTC, raised volume rapid thoracoabdominal compression technique; FEV0.5, forced expired volume in 0.5 s; FRCgas, FRC by gas dilution or washout; FRCpleth, plethysmographic FRC; N, normal; m, increased; k, decreased; NA, not applicable; CLDI, chronic lung disease of infancy; BPD, bronchopulmonary dysplasia; RDS, respiratory distress syndrome.

PULMONARY FUNCTION TESTING IN INFANTS 575 Table 5 Reasons for assessing pulmonary function in infants Epidemiological research * Influence of early life events (e.g., maternal smoking, preterm delivery, intrauterine growth retardation) or genotype (e.g., family history of asthma/atopy) on lung development and relative risk of developing respiratory disease * Determinants of lung function: body size, age, maturity, sex, ethnic group * Longitudinal studies from infancy to school age: tracking of lung function Methodological research * Development and validation of methods and equipment for assessing respiratory function in infants * Establishment of reference data and prediction equations * Assessment of within- and between-subject repeatability Clinical research * Early recognition of lung disease (e.g., cystic fibrosis) * Determination of type and severity of pulmonary defect * Long-term effects of neonatal illness or respiratory support * Assessment of bronchial responsiveness Clinical assessment Serial measures to monitor disease progression or response to therapy may be justified (i) In infants who present with unexplained tachypnea, hypoxia, cough, or respiratory distress in whom a definitive diagnosis is not apparent from physical examination (ii) In those with known respiratory disease of uncertain severity in whom there is need to justify management decisions (iii) In infants with severe, chronic obstructive lung disease who do not respond to an adequate clinical trial of combined corticosteroid and bronchodilator therapy (iv) To optimize ventilatory support in the intensive care unit (only feasible if appropriate expertise, equipment, and techniques available)

early life (Table 5). It is now realized that much of the burden of respiratory disease in childhood and later life has its origins in infancy and early childhood and that remarkable ‘tracking’ of lung function occurs from infancy throughout life. This emphasizes the need to prevent lung injury both before and after delivery, when lung growth is so rapid. In addition, despite advances in molecular biology, the effects of new diagnostic and therapeutic advances still need to be evaluated in vivo by employing objective physiological outcome measures such as those provided by infant PFTs. Furthermore, the increased survival of extremely preterm infants without a reduction in the prevalence of chronic lung disease has increased awareness of the need for a better understanding, of both lung growth and development, and the effect of different ventilatory strategies in order to minimize lung injury during this critical period. Clinical Research Applications

In addition to population-based studies, in which they have been used to investigate the effects of factors such as preterm delivery, sex, intrauterine growth retardation, family history of asthma, and maternal smoking in pregnancy, infant PFTs have been widely used in clinical research. V0 maxFRC has been shown to be lower in infants with recurrent wheeze, bronchiolitis, tracheomalacia, and those with a history of life-threatening events. Airway

function is reduced at an early stage in infants with cystic fibrosis, even in the absence of clinically recognized lower respiratory illness (LRI), which may have important potential implications for early interventions in cystic fibrosis. Although several reports have suggested that the raised volume technique may be a more sensitive means of discriminating changes in lung function in infants with respiratory disease than either tidal breathing parameters or V0 maxFRC, it should be noted that this technique is more complex to apply and that guidelines to standardize data collection and analysis are only just beginning to emerge. Bronchial Responsiveness

One of the most extensively studied clinical research areas in which infant lung function tests have been applied is that of bronchial responsiveness using both bronchial challenge and bronchodilation. There is no standardized approach to performing either type of intervention with respect to agent used, dosage, mode of delivery, outcome measures, or methods of analyzing and reporting results. This has resulted in considerable difficulties when attempting to elucidate age-related changes in airway responsiveness during early life. Interpretation of these studies is further complicated by the lack of information regarding intersubject, between-test repeatability in the same test session.

576 PULMONARY FUNCTION TESTING IN INFANTS

Despite these difficulties, and the generally poor response to bronchodilators during early life, there is convincing evidence that the airways are fully innervated and capable of responding to a range of challenges during both fetal and early postnatal life. The effectiveness of bronchodilators in wheezy infants remains controversial, reflecting the fact that in many infants who wheeze, the reduction in baseline airway function is not due to reversible bronchoconstriction but, rather, transient conditions associated with diminished airway patency. Applications during and Following Intensive Care

Numerous studies have attempted to use parameters derived from infant lung function tests to assess the effects of preterm delivery, neonatal lung disease, and ventilatory support. The most commonly used approaches in recent years have been assessments of passive respiratory mechanics and lung volumes. Specific difficulties in undertaking and interpreting measurements of infant lung function during intensive care include the *

*

*

* *

*

*

relative invasiveness of many techniques in clinically unstable infants; lack of sensitivity of respiratory mechanics to detect subtle changes within individuals due to the magnitude of tracheal tube resistance; confounding of results due to interactions between the ventilator and spontaneous breathing activity; leaks around the tracheal tube; heterogeneous nature of the population with respect to maturity, body size, and clinical severity; multitude of possible treatment modalities that infants may be exposed to; and inappropriate correction for body size.

It is becoming increasingly evident that preterm delivery, even in the absence of any initial respiratory disease, may have an adverse effect on subsequent lung growth and development that persists and may even worsen throughout the first years of life. Most studies of infants with chronic lung disease of infancy have suggested that lung volumes are low early in infancy but subsequently become normal or elevated. Such infants may also have reduced compliance and increased resistance during the first year of life. Infant PFTs have also been used as objective outcome measures to assess the effect of different types of ventilatory support, including extracorporeal membrane oxygenation and high-frequency oscillation during the neonatal period on subsequent lung growth and development.

Summary During the past 20 years, there has been enormous progress in the field of infant lung function testing with respect to the range of tests and equipment now available, their applications in research and clinical studies, and the degree of national and international collaboration. The major role for lung function testing in infants remains firmly within the research arena, in which it has been extensively used to examine the early determinants of airway function and to investigate underlying pathophysiology and response to therapeutic interventions in a variety of respiratory diseases during early life. In recent years, there has been increasing emphasis on developing techniques that can be used in unsedated infants and for those requiring ventilatory support. Future strategies need to encompass a multicenter, multidisciplinary, collaborative approach, with results from infant pulmonary function tests being integrated with those from other disciplines, including imaging, genetics, inflammation, and immunology. See also: Arterial Blood Gases. Asthma: Overview. Breathing: Breathing in the Newborn. Bronchiolitis. Bronchodilators: Beta Agonists. Bronchopulmonary Dysplasia. Cystic Fibrosis: Overview. Environmental Pollutants: Overview. Genetics: Overview. Infant Respiratory Distress Syndrome. Lung Development: Overview; Congenital Parenchymal Disorders; Congenital Vascular Disorders. Pediatric Pulmonary Diseases. Signs of Respiratory Disease: Breathing Patterns. Ventilation, Mechanical: Positive Pressure Ventilation.

Further Reading Frey U (2001) Clinical applications of infant lung function testing: does it contribute to clinical decision making? Paediatric Respiratory Reviews 2: 126–130. Frey U, Stocks J, Coates A, Sly P, and Bates J (2000) Standards for infant respiratory function testing: specifications for equipment used for infant pulmonary function testing. European Respiratory Journal 16: 731–740. Gappa M, Ranganathan S, and Stocks J (2001) Lung function testing in infants with cystic fibrosis: lessons from the past and future directions. Pediatric Pulmonology 32: 228–245. Godfrey S, Bar-Yishay E, Avital A, and Springer C (2003) What is the role of tests of lung function in the management of infants with lung disease? Pediatric Pulmonology 36(1): 1–9. Goldstein AB, Castile RG, Davis SD, et al. (2001) Bronchodilator responsiveness in normal infants and young children. American Journal of Respiratory and Critical Care Medicine 164(3): 447–454. Hoo AF, Stocks J, Lum S, et al. (2004) Development of lung function in early life: influence of birth weight in infants of nonsmokers. American Journal of Respiratory and Critical Care Medicine 170: 527–533.

PULMONARY THROMBOEMBOLISM / Deep Venous Thrombosis 577 Jones MH, Howard J, Davis S, Kisling J, and Tepper RS (2003) Sensitivity of spirometric measurements to detect airway obstruction in infants. American Journal of Respiratory and Critical Care Medicine 167(9): 1283–1286. Lum S, Stocks J, Castile R, et al. (2005) Raised volume forced expirations in infants: recommendations for current practice. European Respiratory Society/American Thoracic Society consensus statement. American Journal of Respiratory and Critical Care Medicine (in press). Ranganathan S, Stocks J, Dezateux C, and the London Collaborative Cystic Fibrosis Group (2004) The evolution of airway function in infancy and early childhood following clinical diagnosis of cystic fibrosis. American Journal of Respiratory and Critical Care Medicine 169: 928–933. Schibler A and Frey U (2002) Role of lung function testing in the management of mechanically ventilated infants. Archives of Disease in Childhood: Fetal and Neonatal Edition 87(1): F7–F10.

Pulmonary Hypertension

Stocks J (2005) Pulmonary function tests in infants and young children. In: Chernick V, et al. (eds.) Kendig’s Disorders of the Respiratory Tract in Children, 7th edn. New York: Elsevier. Stocks J and Dezateux C (2003) The effect of parental smoking on lung function and development during infancy. Respirology 8: 266–285. Stocks J and Hislop AA (2002) Structure and function of the respiratory system: developmental aspects and their relevance to aerosol therapy. In: Bisgaard H, O’Callaghan C, and Smaldone GC (eds.) Drug Delivery to the Lung: Clinical Aspects, pp. 47– 104. New York: Dekker. Stocks J, Sly PD, Tepper RS, and Morgan WJ (1996) Infant Respiratory Function Testing. New York: Wiley. Turner SW, Palmer LJ, Rye PJ, et al. (2004) The relationship between infant airway function, childhood airway responsiveness, and asthma. American Journal of Respiratory and Critical Care Medicine 169(8): 921–927.

see Vascular Disease.

PULMONARY THROMBOEMBOLISM Contents

Deep Venous Thrombosis Pulmonary Emboli and Pulmonary Infarcts

Deep Venous Thrombosis V F Tapson, Duke University Medical Center, Durham, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Deep venous thrombosis (DVT) is a common and under-recognized disorder that places patients at risk for potentially fatal pulmonary embolism as well as chronic, painful postthrombotic syndrome. Thrombosis can affect essentially any vein in the body, depending on the underlying injury or condition and can involve superficial or deep veins. Stasis, venous injury, and hypercoagulability (thrombophila) are the classic basic risk factors from which all other risks are derived. The history and physical examinations are notoriously insensitive and non-specific for both DVT and pulmonary embolism. Leg swelling, erythema, warmth, pain, and/or tenderness may be present, but these symptoms may represent other entities such as cellulitis or trauma, and clinically distinguishing these can be difficult, requiring further evaluation. Compression ultrasound is the firstline diagnostic test, although when clinical suspicion is low, a negative D-dimer is generally adequate to rule out DVT. Treatment options for acute DVT include anticoagulation with low molecular weight or standard heparin. Larger, more symptomatic DVT may be better served by catheter-directed thrombolytic therapy with careful attention to the risk. Inferior vena cava filter placement is indicated if there is bleeding or

substantial bleeding risk, or recurrence on adequate therapy. DVT is often but not always preventable but prophylaxis must be considered for all at-risk patients. Preventive methods include low molecular weight heparin, standard heparin, or pneumatic compression. Low molecular weight heparin has distinct advantages over standard heparin.

Background and Incidence Deep venous thrombosis (DVT) is a common and under-recognized disorder that places patients at risk for potentially fatal pulmonary embolism as well as chronic, painful postthrombotic syndrome. Venous thromboembolism (VTE) includes the spectrum of thrombosis and pulmonary embolism (PE). Venous thrombosis can affect essentially any vein in the body, depending on the underlying injury or condition. Occlusion of veins of the arms, legs, and pelvis as well as more central veins including the superior and inferior vena cava can be symptomatic or asymptomatic. Thrombosis in the deep veins of the legs can involve superficial veins or can be DVT. The most devastating complication of acute DVT is acute, fatal PE. This article focuses primarily on thrombosis of the extremities, especially the legs, for this reason. The disease is more common in the setting of specific predipositions,

578 PULMONARY THROMBOEMBOLISM / Deep Venous Thrombosis

but can be idiopathic. It will address the risk factors, diagnostic approach, prophylaxis for medical patients at risk, and therapeutic options for DVT. The incidence of DVT is unknown. Population studies of the overall age- and sex-adjusted annual incidence of VTE is 1 to 2 per 1000 people. A significant minority of these cases represent recurrent disease. However, the precise incidence is difficult to be certain of because DVT is often asymptomatic(even when it precedes fatal PE), and because the number of autopsies per year is so low. Thus, as many as two million per year may develop this disease in US with approximately 60 000 to 200 000 patients dying from acute PE.

Table 1 Risk factors for venous thromboembolisma

Pathophysiology

Genetic/molecular factors Antithrombin III deficiency Factor V Leiden (activated protein C resistance) Protein C deficiency Protein S deficiency Prothrombin gene (G20210A) defect Heparin cofactor 2 deficiency Dysfibrinogenemia Disorders of plasminogen Elevated factor VIII levels Elevated factor XI levels Hyperhomocysteinemia

Venous thrombi typically develop within a deep vein at a site of trauma and/or in the presence of sluggish blood flow, particularly in the valve cusps in the venous sinuses of the calf veins. Fibrin and platelets accumulate with propagation of the clot in the direction of blood flow. Endogenous fibrinolysis may result in a partial or complete thrombolysis. Recanalization of residual thrombus will render the vein narrow and irregular, with venous valvular incompetency. Collateral vessels may develop.

Risk Factors for DVT Recognizing the risk factors for DVT offers several potential benefits. Their presence, together with compatible symptoms and signs, helps one suspect DVT or PE as well as being critical in determining appropriate prophylaxis in patients at risk (Table 1). The role of their presence in lowering the diagnostic suspicion for DVT is our primary reason for emphasizing them here. Nearly 150 years ago, Rudolf Virchow, the German pathologist, described the triad of stasis, venous injury, and hypercoagulability and its association with the development of venous thrombosis. This simple concept is perhaps one of the most enduring themes in medicine today, with essentially every risk factor for DVT being derived from this triad. The risk of DVT is significant in both medical and surgical patients with most surgical scenarios entailing at least some risk. One of the most easily recognized DVT (and thus, PE) risk factors is major surgery, particularly orthopedic surgery. Based on venographic studies, performed on patients not receiving prophylaxis, the prevalence of DVT at 7–14 days after total hip replacement, total knee replacement, and hip fracture surgery is about 50–60%, with proximal DVT rates of about 25%, 15–20%, and 30%, respectively. The leg undergoing the procedure is most commonly

Clinical factors Age greater than 40b Prior history of venous thromboembolism Major surgical procedure/trauma Hip fracture Immobilization/paralysis Varicose veins Congestive heart failure Myocardial infarction Obesity Pregnancy/postpartum Oral contraceptive therapy Cerebrovascular accident Cancer Paroxysmal nocturnal hemoglobinemia Acute medical illness with restricted mobility

Acquired factors Antiphospholipid antibody syndrome Lupus anticoagulant Anticardiolipin antibodies Myeloproliferative disease Hyperhomocysteinemia a

The presence of risk factors may lower the threshold for considering the diagnosis of DVT or PE. b While the risk of VTE begins to increase at approximately age 40, a significant increase is generally not recognized until approximately age 70.

affected but in some cases the other leg may be too. Patients undergoing other major surgery are also at risk, and associated underlying disease such as cancer may compound this risk. The risk of DVT appears to be approximately 24% in those not receiving antithrombotic treatment, and about 55% in patients with lower limb paresis or paralysis. Hereditary and acquired thrombophilias increase the risk, but are often not known to the clinician when a patient presents with symptoms. Pregnancy and the postpartum period are the most common settings in which women below age 40 develop DVT or PE, and are associated with a fivefold increase in risk. In patients admitted for treatment for respiratory disease, congestive heart failure, or other medical illnesses such as infection or rheumatic disease, the incidence of VTE without prophylaxis may be 15% or higher.

PULMONARY THROMBOEMBOLISM / Deep Venous Thrombosis 579

Serious illness and multiple risk factors increase the incidence of DVT. The risk is high in critically ill patients because of underlying disease, immobility, and veno-invasive catheters and devices. Thus, the reduced mobility associated with medical illness imparts a significant risk for DVT. In addition, the relative immobilization associated with prolonged air travel places individuals at risk with the travel distance correlating with the risk of DVT. Appropriate preventive approaches will be discussed subsequently in the section on prophylaxis.

Clinical Manifestations The history and physical examination are notoriously insensitive and non-specific for both DVT and PE. While the symptomatic presentation of DVT depends to some degree on the extent of thrombosis, it is clear that very large thrombi may evolve and never result in DVT symptoms but present first as symptomatic or even fatal PE. Leg swelling, erythema, warmth, pain, and/or tenderness may be present, but may be symptoms of other entities such as cellulitis or trauma, and clinically distinguishing these can be difficult, requiring further evaluation (Table 2). Pain with dorsiflexion of the foot (Homans’ sign) may be present in the setting of DVT, but this finding is neither sensitive nor specific. The differential diagnosis includes cellulitis, musculoskeletal pain, trauma, a ruptured Baker’s cyst, or asymmetric edema unrelated to DVT. Symptoms and signs of PE may be present, with the most common being dyspnea, pleuritic chest pain, hemoptysis, tachypnea, and tachcardia (see Pulmonary Thromboembolism: Deep Venous Thrombosis).

Diagnosis Blood Tests

If DVT is accompanied by acute PE, hypoxemia may be present. Some individuals, particularly young patients without underlying lung disease, may have a Table 2 Common symptoms and signs of acute deep venous thrombosisa Pain Cramping Tenderness Swelling Warmth Homans’ sign Erythema Palpable venous cord a

Symptoms and signs, including Homans’ sign, are notoriously non-sensitive and non-specific. Deep venous thrombosis can be present and even extensive without symptoms or signs. Symptoms of acute PE may be simultaneously present.

normal PaO2, and in rare cases, a normal alveolar– arterial oxygen difference. Plasma tests of circulating D-dimer (a specific derivative of cross-linked fibrin), in patients with acute PE have been extensively evaluated. A normal enzyme-linked immunosorbent assay (ELISA) appears sensitive in excluding PE, particularly when the clinical suspicion is relatively low. A number of D-dimer assays are available, and the sensitivity and specificity of these assays vary. A positive D-dimer test means that DVT or PE is possible, but the lack of specificity dramatically limits this result. This tenet makes the use of D-dimer very limited in hospitalized patients in whom infection, cancer, trauma, and other disease states are common, and frequently associated with a positive assay. These tests can now be done more quickly with very high sensitivity. A positive Ddimer is not proof of DVT and/or PE, and while the sensitivity of these tests are quite good, a strong clinical suspicion should not be ignored. Radiographic Studies

Venography has been the gold-standard test for DVT for decades, and while it is still deemed the most sensitive test, it is rarely done now. With the advent of ultrasound, a diagnostic test that is 490% sensitive in the setting of symptomatic DVT, the use of venography has become extremely uncommon. Ultrasonography is the first-line diagnostic test for suspected DVT in most settings. While color Doppler signals are examined, and a clot may be visualized, the most sensitive portion of the examination is compression of the vessels. If they do not compress, regardless of whether clot is seen, it is highly likely that there is venous thrombosis. The non-compressibility of leg vessels is the most specific sign for DVT (Figure 1). One drawback of ultrasound is that lack of compressibility by itself, does not distinguish new from old DVT. Magnetic resonance imaging (MRI) has proven extremely sensitive for both acute and chronic DVT, although it is generally not necessary (Figure 2). Above the knee, it is likely as sensitive or even more sensitive than venography. It is suitable to consider MRI in the setting of suspected DVT when severe edema, trauma, or a plaster cast or other device prevents the effective use of ultrasound. A major limitation of ultrasound is its reduced sensitivity in the setting of asymptomatic DVT. Thus, it is not generally used as a screening test. Diagnostic testing for suspected acute DVT is presented in Table 3.

Treatment Treatment options for acute DVT are the same as for acute PE and include anticoagulation with

580 PULMONARY THROMBOEMBOLISM / Deep Venous Thrombosis

low-molecular-weight heparin (LMWH) or standard heparin, thrombolytic therapy, and inferior vena cava (IVC) filter placement. Each approach has specific indications as well as advantages and disadvantages. Thrombolytic therapy has traditionally been used less often for acute DVT than for PE but increased interest in local thrombolytic delivery (directly into the involved vein) has led to increased research and application in the clinical setting. Heparin and LMWH

Figure 1 Ultrasound image of deep venous thrombosis of the common femoral vein. Compression is being applied and the vein does not flatten. There are no echoes suggesting thrombosis; it is lack of compression that is diagnostic.

Figure 2 Magnetic resonance image of deep venous thrombosis involving the left femoral vein.

The primary anticoagulants used to treat acute DVT and/or PE include unfractionated heparin and LMWH. These substances exert a prompt antithrombotic effect by accelerating the action of antithrombin III, preventing thrombus extension. Although they do not directly dissolve thrombus or emboli, they allow the fibrinolytic system to proceed unopposed and more readily reduce the size of the thromboembolic burden. While thrombus growth can be prevented, early recurrence can sometimes develop even in the setting of therapeutic anticoagulation. When DVT is diagnosed (or strongly suspected), anticoagulation should be immediately instituted unless contraindications are present. Confirmatory diagnostic testing should be arranged as soon as possible. LMWH should be considered first, based on the clear advantages (Table 4). Among the advantages of LMWH over standard heparin are the greater bioavailability of the LMWHs and more predictable dosing. In addition, the latter anticoagulants can be subcutaneously administered once or twice daily even at therapeutic doses and do not require monitoring of the activated partial thromboplastin time (APTT). Intravenous LMWH is never required

Table 3 Diagnostic testing for acute deep venous thrombosis Procedure

Major advantages

Major disadvantages

Compression ultrasound

Rapid, sensitive, noninvasive Least expensive imaging test Portable

Contrast venography

Very sensitive/specific Most accurate for calf DVT Very sensitive/specific Can combine with PE evaluation

Not sensitive for asymptomatic DVT Less sensitive for new vs. old clot Less sensitive for calf, pelvic clot Casts, severe obesity/edema may lower sensitivity Invasive Can (rarely) cause phlebitis More confining, claustrophobia Metal may contraindicate Much slower than CT More expensive than ultrasound Contrast/radiation

a

Magnetic resonance imaging

Computed tomography (CT) D-dimer

a

Sensitive, less data Easily combined with PE evaluation Sensitive (if ELISA) Inexpensive

Currently is first-line test in most settings.

Non-specific Should be used only if clinical suspicion relatively low

PULMONARY THROMBOEMBOLISM / Deep Venous Thrombosis 581 Table 4 Potential advantages of low-molecular-weight heparins over unfractionated heparin

Table 5 Initiation of LMWH for therapy of acute DVT (and/or PE)

Similar or superior efficacy Similar or superior safety Superior bioavailability Subcutaneous administrationa Once or twice daily dosing No laboratory monitoringb Less phlebotomy Lower incidence of heparin-induced thrombocytopenia Earlier ambulation Home therapy in certain patient subsets

Determine appropriateness of outpatient therapya Begin LMWH by subcutaneous administrationb Determine whether monitoring needed (extremes of weight, renal insufficiency, pregnancy) Warfarin from day 1; initial dose 5–10 mg, adjust according to INR Check platelet count between days 3 and 5; HIT is unusual but can occurc Stop LMWH after X5 days of combined therapy, and when INR is X2.0 for two consecutive days Anticoagulate with warfarin for X3 months (goal INR 2.0–3.0)d

a

a

For either prophylaxis or treatment. No monitoring needed for either prophylaxis or treatment. In a few specific ‘treatment’ settings such a renal insufficiency if renal functions changing or is severely abnormal, or if the body weight o40 kg, or 4150 kg, measuring antifactor Xa levels is recommended to aid in dosing.

b

in VTE. In addition, LMWHs have a more profound effect in inhibiting clotting factor Xa relative to thrombin. The reduced frequency of heparin-induced thrombocytopenia (HIT) with LMWH relative to unfractionated heparin is a very compelling reason to use LMWH instead of the latter whenever possible. Because of efficacy, safety, and convenience compared with standard heparin, these drugs are replacing it in many settings. A major advance in therapy over the last decade has been the move to outpatient therapy made feasible by the LMWH preparations (Table 5). When standard, unfractionated, intravenous heparin is initiated, the APTT should be followed at 6 h intervals until it is consistently in the therapeutic range of 1.5–2.0 times control values. Heparin is administered as an intravenous bolus of 5000 units followed by a maintenance dose of at least 30 000– 40 000 units per 24 h by continuous infusion. The lower dose is administered if the patient is considered at high risk for bleeding. Another recommended option has been an 80 units kg 1 bolus followed by 18 units kg 1 hour 1. This aggressive approach decreases the risk of subtherapeutic anticoagulation. It is possible that early initiation of warfarin, before therapeutic heparin or LMWH may intensify hypercoagulability and increase the clot burden due to the short half-life of anticoagulation factors that are also inhibited by warfarin. Adequately depleting clotting factors take approximately 4 to 5 days. Thus, at least five days of intravenous heparin or LMWH is generally recommended. Heparin should be maintained at a therapeutic level until two consecutive therapeutic international normalized ratio (INR) values of 2.0– 3.0 have been documented at least 24 h apart. Documented proximal DVT or PE should be treated for

Potential outpatients should be medically stable without severely symptomatic DVT. They should be compliant, capable of self-administration (or have a family member or visiting nurse for administration), at low risk of bleeding, and reimbursement should be addressed. b Enoxaparin (Lovenox) and tinzaparin (Innohep) are the two LMWHs that are FDA-approved for treatment of DVT. Data/indication for outpatient therapy is with enoxaparin. While LMWH preparations are sometimes used for patients presenting with PE in United States, and while clinical trials support this use, the FDA-approvals read ‘established DVT with or without PE’. c Heparin-induced thrombocytopenia. d Duration of warfarin therapy should be at least 6–12 months in patients with idiopathic DVT. LMWH, low-molecular-weight heparin, DVT, deep venous thrombosis, PE, pulmonary embolism, INR, international normalized ratio.

3–6 months. Treatment over a more extended interval is appropriate when significant risk factors persist, when thromboembolism is idiopathic, or when previous episodes of VTE have been documented. Oral Therapy

Warfarin remains the only FDA-approved oral drug for DVT. It inhibits the vitamin K-dependent coagulation factors II, VII, IX, and X. Although a prolongation of the prothrombin time (PT) can begin 5–7 h after drug administration due to the short halflife of factor VII, warfarin’s ability to fully exert its effect can take more than 72 h. Warfarin and LMWH or heparin can be started on the same day, but concomitant administration of these drugs for 4 or 5 days is recommended. Bleeding related to warfarin therapy increases with intensity and duration of therapy. Warfarin-induced skin necrosis is a rare but serious complication mandating immediate cessation of the drug. It is related, at least in some patients, to protein C or S deficiency. Warfarin crosses the placenta and may cause fetal malformations if used during pregnancy. The INR should be kept between 2.0 and 3.0. Newer oral drugs with rapid onset, and lack of significant drug and food interactions are being investigated. While heparin and LMWH work indirectly, requiring antithrombin III as a cofactor,

582 PULMONARY THROMBOEMBOLISM / Deep Venous Thrombosis

direct thrombin inhibitors have several advantages over heparin, including efficacy against fibrin clotbound thrombin. Lack of need for INR monitoring is among the advantages over warfarin. Complications of Anticoagulation

Bleeding is the major complication of anticoagulation. The rates of major bleeding in recent trials using heparin by continuous infusion or high-dose subcutaneous injection are o5%. HIT typically develops five or more days after the initiation of heparin therapy, occurring in about 5% of patients. The platelet count is usually o100 000 but may be higher. A significant drop without the latter criterion may still be seen in HIT; absolute platelet counts may be deceiving. The syndrome is caused by heparin-dependent IgG antibodies that activate platelets via their Fc receptors. The formation of heparin-dependent IgG antibodies and the risk of thrombocytopenia is lower with LMWH than with standard heparin. Testing for HIT (platelet aggregation and/or ELISA) should be performed in suspected HIT but heparin should be stopped without waiting for results. Both argatroban and lepirudin have been FDA-approved for use in the setting of VTE with HIT. Vena Cava Interruption

If a patient cannot be anticoagulated, IVC filter placement can be performed to prevent lower extremity thrombi from embolizing to the lungs. The primary indications for filter placement include contraindications to anticoagulation, significant bleeding complications during anticoagulation, and recurrent DVT or PE while on adequate therapy. A number of filter designs exist. These devices are effective and complications including insertion-related problems and migration are unusual. Temporary filters are being placed increasingly in patients in whom the risk of bleeding appears short term. Most of these devices can be removed up to 2 weeks later, and some may remain in place even longer, with subsequent removal.

commonly used systemically for acute massive PE. Tenecteplase has been studied extensively in acute myocardial infarction, and would be expected to be effective in VTE, but far less data are available about this agent. Catheter-directed techniques are being used increasingly for acute DVT. Such aggressive therapy with thrombolytics may reduce the frequency of postphlebitic syndrome. As with anticoagulants, hemorrhage is the primary adverse effect associated with thrombolytic therapy. Invasive procedures should be minimized as much as possible. The most devastating complication associated with systemic thrombolytic therapy is the development of intracranial hemorrhage which occurs in o1% of patients. Retroperitoneal hemorrhage may result from a vascular puncture above the inguinal ligament and may be life-threatening. The primary contraindications to thrombolytic therapy include active bleeding, surgery within the previous one to two weeks (depending on specific procedure), or any intracranial pathology or previous intracranial surgery. Catheter-directed thrombolysis appears to be safer, faster, and more effective in removing an acute iliofemoral venous thrombus than previous thrombolytic methods. This technique may result in a reduction in frequency of postthrombotic syndrome.

Postthrombotic Syndrome Postthrombotic (postphlebitic) syndrome is characterized by leg pain, edema, and other signs of venous insufficiency. Leg ulceration may eventually develop as a result of prolonged venous hypertension. Approximately 30% of patients with VTE develop this often debilitating disease. The risk of developing this problem depends on the speed of vein recanalization and development of ipsilateral recurrent events, but not necessarily the extent of thrombosis. Fitted compression stockings are the mainstay of therapy, which may decrease the risk of postthrombotic syndrome by 50%.

Thrombolytic Therapy

Prevention

Thrombolytic agents activate plasminogen to form plasmin which then results in fibrinolysis as well as fibrinogenolysis. These agents can dramatically accelerate clot lysis in acute PE and DVT. The use of thrombolytic therapy in patients with proximal occlusive DVT associated with significant swelling and symptoms is increasing. Clinical trials have culminated in the approval of streptokinase, urokinase, and recombinant tissue-type plasminogen activator (tPA) for the treatment of VTE but these are more

Measures to prevent VTE appear to be grossly underutilized. A substantial reduction in the incidence of DVT can be achieved when patients at risk receive appropriate prophylaxis. For example, the risk of DVT after total hip or knee replacement is 50% or greater without prophylaxis. The superiority of LMWH over unfractionated heparin is evidenced in the clinical trials evaluating exceptionally high-risk patients such as those above, as well as spinal cord injury and trauma patients. Unfractionated heparin

PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts 583

is clearly less effective in these settings. In hospitalized general medical patients, anticoagulant prophylaxis should always be strongly considered as the rate of DVT, based upon a venographic endpoint, is as high as 15% in patients receiving placebo. The rate of DVT, including proximal DVT is statistically significantly lower when enoxaparin is administered compared with placebo. It appears that either LMWH (enoxaparin 40 mg subcutaneously or dalteparin at 5000 units once daily) or subcutaneous heparin (5000 units every 8 h) is adequate for medical patient prophylaxis; again, LMWH has clear advantages. Pneumatic compression should be used when anticoagulant prophylaxis is contraindicated. See also: Anticoagulants. Coagulation Cascade: Overview; Fibrinogen and Fibrin; Thrombin. Fibrinolysis: Overview. Pulmonary Thromboembolism: Pulmonary Emboli and Pulmonary Infarcts. Thrombolytic Therapy.

Further Reading Bates SM and Ginsberg JS (2004) Clinical practice. Treatment of deep vein thrombosis. New England Journal of Medicine 351: 268–277. Geerts WH, Pineo GF, Heit JA, et al. (2004) Prevention of venous thromboembolism. The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 126: 338S– 400S. Gelfand EV, Piazza G, and Goldhaber SZ (2003) Venous thromboembolism guidebook, 4th edn. Critical Pathways in Cardiology: A Journal of Evidence-Based Medicine 2(4): 247–265. Goldhaber SZ and Tapson VF (2004) A prospective registry of 5,451 patients with ultrasound confirmed deep vein thrombosis. American Journal of Cardiology 93: 259–262. Heit JA, Silverstein MD, Mohr DN, et al. (1999) Predictors of survival after deep vein thrombosis and pulmonary embolism: a population-based, cohort study. Archives of Internal Medicine 159: 445–453. Kearon C (2003) Natural history of venous thromboembolism. Circulation 107: I22–I30. Kearon C (2004) Long term management of patients after venous thromboembolism. Circulation. Silverstein MD, Heit JA, Mohr DN, et al. (1998) Trends in the incidence of deep vein thrombosis and pulmonary embolism: a 25-year population-based study. Archives of Internal Medicine 158: 585–593. Tapson VF (2002) Venous thromboembolism. In: Topol E (ed.) Textbook of Cardiovascular Medicine, pp. 667–684. Philadelphia, PA: Lippincott–Raven. Tapson VF, Carroll BA, Davidson BL, et al. (1999) The diagnostic approach to acute venous thromboembolism. Clinical Practice Guideline. American Thoracic Society. American Journal of Respiratory and Critical Care Medicine 160: 1043–1066. von Virchow R (1846) Weitere Untersuchungen ueber die Verstopfung der Lungenarterien und ihre Folge. Traube’s Beitraege exp path u Physiol, Berlin 2: 21–31. Watson LI and Armon MP (2004) Thrombolysis for acute deep vein thrombosis. Cochrane Database of Systematic Reviews 4: CD002783.

Pulmonary Emboli and Pulmonary Infarcts V F Tapson, Duke University Medical Center, Durham, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Background and Incidence Pulmonary embolism (PE) occurs when blood clots (thrombi), usually from the deep veins of the legs, travel to the lungs, causing a spectrum of potential consequences, including dyspnea, chest pain, hypoxemia, and sometimes, death. Patients may present with symptoms of either deep vein thrombosis (DVT) or PE, or both, and these two entities represent a continuum of one disease known as pulmonary (or venous) thromboembolism. Pulmonary embolism probably accounts for 100 000 to 200 000 deaths per year in the United States. While some patients who die from acute PE may have an underlying terminal illness, it is clear that this disease is responsible for death in a considerable number of patients with an otherwise good prognosis. Autopsy studies have repeatedly documented the high frequency with which PE has gone unsuspected and thus, undetected. Pulmonary thromboembolism occurs worldwide, and is usually associated with specific risk factors although in many circumstances the disease appears idiopathic (Table 1). A crucial point is that DVT, and therefore PE, are often preventable. Furthermore, prophylaxis continues to be underutilized. The incidence of venous thromboembolism (VTE) is high in hospitalized patients and both surgical as well as medical patients are at risk. Our discussion will be limited to pulmonary thromboembolism and will not include nonthrombotic entities such as fat embolism.

Pathophysiology One or more components of Virchow’s triad (stasis, hypercoagulability, and venous injury), described more than 150 years ago, are present in nearly all patients. The risk increases with age. Idiopathic thromboembolism likely involves an underlying prothrombotic state that has not been characterized. Deep vein thrombi frequently originate in the calf veins and propagate proximally to the popliteal vein or above before embolizing. Thrombosis developing in the axillary–subclavian veins due to the presence of a central venous catheter, particularly in patients with malignant disease, as well as in those with effort-induced upper extremity thrombosis may result in PE as well.

584 PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts Table 1 Risk factors for pulmonary thromboembolism Clinical factors Age greater than 40 (more significant after age 70) Prior history of pulmonary thromboembolism Major surgical procedure/trauma Hip fracture Immobilization/paralysis Varicose veins Congestive heart failure Myocardial infarction Other acute medical illnesses Obesity Pregnancy/postpartum Oral contraceptive therapy Cerebrovascular accident Cancer Antiphospholipid antibody syndrome (including lupus anticoagulant) Genetic/molecular factors Anti-thrombin III deficiency Factor V Leiden (activated protein C resistance) Protein C deficiency Protein S deficiency Prothrombin gene (G20210A) defect Dysfibrinogenemia Disorders of plasminogen Elevated factor VIII levels Elevated factor XI levels Hyperhomocysteinemia Combined genetic mutations

The impact of an embolic event depends upon the extent of reduction of the cross-sectional area of the pulmonary arterial bed as well as upon the presence or absence of concomitant cardiopulmonary disease. In the setting of massive PE, cardiac output is diminished but may be sustained as the mean right atrial pressure increases. In the absence of underlying cardiopulmonary disease, obstruction of 25% to 30% of the vascular bed by emboli is associated with a rise in pulmonary artery pressure; hypoxemia also contributes to vasoconstriction and a further rise in pulmonary artery pressure. More than 50% obstruction of the pulmonary arterial bed is usually present before there is substantial elevation of the mean pulmonary artery pressure. Because the right ventricle is not accustomed to pumping against a high pressure, it may ultimately fail resulting in hypotension and often death, if not treated. Patients with underlying cardiopulmonary disease often experience a more substantial deterioration in cardiac output than normal individuals in the setting of massive PE.

Clinical Manifestations Unfortunately, the history and physical examination are notoriously insensitive and non-specific for both

Table 2 Symptoms of acute pulmonary embolism

Dyspnea Pleuritic chest pain Cough Leg pain Hemoptysis Palpitations Wheezing Angina-like pain

All patients (N ¼ 383)

No previous cardiopulmonary disease (N ¼ 117)

78% 59% 43% 27% 16% 13% 14% 6%

73% 66% 37% 26% 13% 10% 9% 4%

The symptoms listed above were based upon data from the Prospective Investigation of Pulmonary Embolism (PIOPED) study and modified from tables presented in Stein PD (ed.) (1996) Pulmonary Embolism. Baltimore: Williams and Wilkins.

DVT and PE. Patients with DVT often have no erythema, warmth, pain, swelling, or tenderness. When these are present, they may represent other entities such as cellulitis, though further evaluation may be appropriate. Pain with dorsiflexion of the foot (Homans’ sign) may be present in the setting of DVT but this finding is neither sensitive nor specific. The most common symptom of acute PE is a sudden onset of dyspnea. Pleuritic chest pain and hemoptysis occur more commonly when small, peripheral emboli obstruct very distal vessels, causing pulmonary infarction. Palpitations, anxiety, cough, and lightheadedness are among the non-specific symptoms of acute PE but these may have a number of other potential causes, contributing to diagnostic confusion. Syncope indicates more massive embolism. Pulmonary embolism should be considered whenever unexplained dyspnea, syncope, hypotension, or hypoxemia are present. The first indication of acute PE may be sudden death. Tachypnea and tachycardia are the most common signs of PE but are also nonspecific. Other possible physical findings include fever, wheezing, rales, a pleural rub, a loud pulmonic component of the second heart sound, a right-sided fourth heart sound, and a right ventricular lift. Symptoms and signs consistent with PE (Tables 2 and 3) should be particularly heeded in the setting of risk factors for VTE such as concomitant malignancy, immobility, or the postoperative state.

Diagnosis Patients with pulmonary thromboembolism may present with symptoms or signs of DVT or PE or both. When patients present with suspected DVT, (e.g., calf pain and/or swelling) a leg study, generally compression ultrasound, should be pursued unless

PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts 585 Table 3 Signs of acute pulmonary embolism

Tachypnea (20/min) Crackles Tachycardia (4100/min) Leg swelling Loud P2a DVTb Wheezes Diaphoresis Temperature (X38.5) Pleural rub Fourth heart sound Third heart sound Cyanosis Homans’ sign Right ventricular lift

All patients (N ¼ 383)

No previous cardiopulmonary disease (N ¼ 117)

73%

70%

55% 30%

51% 30%

31% 23% 15% 11% 10% 7%

28% 23% 11% 5% 11% 7%

4%

3% 24% 3% 1% 4% 4%

5% 3% 3%

a

P2, pulmonic component of second heart sound. DVT, deep venous thrombosis. The clinical signs listed above were based upon data from the Prospective Investigation of Pulmonary Embolism (PIOPED) study and modified from tables presented in Stein PD (ed.) (1996) Pulmonary Embolism. Baltimore: Williams and Wilkins. b

there is a clear, alternative explanation. In the setting of dyspnea and/or chest pain, the differential diagnosis is broad, and may include a flare of asthma or chronic obstructive lung disease, pneumothorax, pneumonia, anxiety with hyperventilation, heart failure, angina or myocardial infarction, musculoskeletal pain, rib fracture, pericarditis, pleuritis from connective tissue disease, herpes zoster, intrathoracic cancer, and occasionally intra-abdominal process. The presence of obvious risk factors for VTE, such as prolonged immobility, trauma, recent surgery, medical illness with reduced mobility, cancer, pregnancy, myocardial infarction, recent prolonged travel, or previous thromboembolism in the setting of compatible symptoms and signs should prompt consideration of this entity. It is important to realize that acute PE can be superimposed upon another underlying cardiopulmonary disease, upon which new or worsening symptoms are sometimes blamed. The use of clinical probability scores based upon simple clinical parameters such as presence of tachycardia, hemoptysis, and the presence or absence of risk factors such as cancer have been used to help exclude PE. While these scoring systems have been used in clinical trials, they have not been widely employed in clinical practice.

Blood Tests

Hypoxemia is common in acute PE. Some individuals, particularly young patients without underlying lung disease, may have a normal arterial oxygen tension (PaO2) and even rarely a normal alveolar– arterial difference. A sudden decrease in the PaO2 or oxygen saturation in a patient unable to communicate an accurate history (e.g., a demented or mechanically ventilated patient) suggests the possibility of acute PE. The diagnostic utility of plasma measurements of circulating D-dimer (a specific derivative of crosslinked fibrin) in patients with acute DVT and PE has been extensively evaluated. A normal enzymelinked immunosorbent assay (ELISA) appears sensitive in excluding PE, particularly when the clinical suspicion is relatively low. There are a number of D-dimer assays available, and the sensitivity and specificity of these assays vary. A positive D-dimer test means that DVT or PE is possible, but it is by no means proof. While both cardiac troponin T and troponin I levels have been found to be elevated in acute PE, they are not sensitive enough to rule out PE without additional diagnostic testing. Brain natriuretic peptide levels have also been found to be elevated in acute PE but may be elevated in other settings in which right or left ventricular stretch occurs.

Electrocardiography and Chest Radiography

Electrocardiographic findings, which are present in the majority of patients with acute PE, are nonspecific but these abnormalities, including ST-segment abnormalities, T-wave changes, and left or right axis deviation are common. Only one-third of patients with massive or submassive emboli have manifestations of acute cor pulmonale such as the S1 Q3 T3 pattern, right bundle branch block, P-wave pulmonale, or right axis deviation. The utility of electrocardiography in suspected acute PE is best characterized by its ability to establish or exclude alternative diagnoses, such as acute myocardial infarction. The chest radiograph is often abnormal in patients with acute PE, but as with electrocardiography, it is nearly always non-specific. Common radiographic findings include atelectasis, pleural effusion, pulmonary infiltrates, and mild elevation of a hemidiaphragm. Classic findings of pulmonary infarction such as Hampton’s hump or decreased vascularity (Westermark’s sign) are suggestive of the diagnosis, but are infrequent.

586 PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts Specific Imaging Studies

Specific imaging studies are required for the definitive diagnosis of acute PE. A diagnostic algorithm for suspected acute PE is presented in Figure 1. While pulmonary angiography remains the gold-standard test for suspected acute PE, it is rarely necessary. The ventilation–perfusion (VQ) scan was previously the most common diagnostic test utilized when PE was suspected, but over the past decade, spiral (helical) computed tomography (CT) scanning has replaced it at many centers. A normal VQ scan (or perfusion scan alone) rules out the diagnosis with a high enough degree of certainty so that further diagnostic

evaluation is almost never necessary. Unfortunately, low or intermediate probability (nondiagnostic) scans are commonly found with PE which requires further evaluation. The diagnosis of PE should be aggressively pursued even when the lung scan is low or intermediate probability if the clinical setting suggests the diagnosis, particularly when clinical probability is high. Stable patients with suspected acute PE, nondiagnostic lung scans and adequate cardiopulmonary reserve (absence of hypotension or severe hypoxemia), may undergo noninvasive lower extremity testing in an attempt to diagnose DVT. A positive compression

Suspected acute pulmonary embolisma

Clinical suspicion moderate to high

VQ normal; PE ruled out

Clinical suspicion low

(+) CT scan or VQ

scanb

D-dimer (−)

CT normala VQ scan HPc or CT (+) No further testing needed; PE proven US of legs

US (+) (treat)

No further studies (PE ruled out)

CT or VQ nondiagnosticd

US (−)

Pulmonary arteriogram (+) = treat (−) = no treatmenta

Abbreviations: PE, pulmonary embolism; VQ, ventilation-perfusion; CT, computed tomography; US, compression ultrasound; HP, high probability. a Level

of clinical suspicion may affect algorithm. For example, based upon outcome studies, additional studies after a normal CT may not always be necessary.

b The

optimal scenario for a VQ scan is patient with clear chest radiograph and no underlying cardiopulmonary disease. Patients with significant renal insufficiency should undergo a VQ scan instead of CT. Chest CT can be performed together with CT venography which may enhance sensitivity. c In

setting of prior PE, a HP VQ scan may reflect the previous PE. In this case, prior VQ scans should be reviewed whenever possible. d If

VQ scan nondiagnostic, a CT scan can also be considered, instead of angiography.

Figure 1 The algorithm for evaluating suspected acute pulmonary embolism depends to some degree on the particular resources available. A primary difference among algorithms is whether computed tomography or ventilation–perfusion scanning is utilized. In some institutions, ultrasound is utilized prior to a chest imaging study and, if positive, eliminates the need for the latter.

PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts 587

ultrasound may present the opportunity to treat without further testing. If the ultrasound is negative, additional testing is needed. Magnetic resonance imaging of the lower extremities and/or lungs may also be useful after a nondiagnostic lung scan if the medical facility has experience with this technique. Spiral CT scanning has been found to be approximately 70–80% sensitive, and greater than 90% specific. This test has the greatest sensitivity for emboli in the main, lobar, or segmental pulmonary arteries. The specificity for clot in these vessels is excellent. For subsegmental emboli, spiral CT appears less accurate, although the importance of emboli this size has been questioned. An advantage of spiral CT over VQ scanning and arteriography includes the ability to define nonvascular structures such as lymphadenopathy, lung tumors, emphysema, and other parenchymal abnormalities as well as pleural and pericardial disease. Another advantage of spiral CT over other diagnostic methods is the rapidity with which a study can be performed. A computed tomography scan demonstrating a very large PE is shown in Figure 2. Potential disadvantages of CT include the fact that it is not portable at present, and because of the need for intravenous contrast, patients with significant renal insufficiency cannot be scanned without risk of renal failure. In summary, with suspected PE, if the clinical probability is deemed low, and a sensitive D-dimer test is negative, no further testing is needed. If the clinical probability is not low, either a chest CT scan or VQ scan is performed. Additional testing such as with

Figure 2 This patient presented with sudden onset severe dyspnea 4 days after undergoing total knee replacement. Spiral computed tomography imaging reveals a large filling defect in the left main pulmonary artery (arrow), diagnostic of acute pulmonary embolism.

compression ultrasound may be needed. Finally, the use of CT venography performed at the same time as chest CT and with the same intravenous contrast bolus, may enhance the sensitivity by identifying DVT even if PE cannot be seen. Echocardiography

Echocardiography, which can often be obtained more rapidly than either lung scanning or pulmonary arteriography may reveal findings which strongly support hemodynamically significant pulmonary embolism. Imaging or Doppler abnormalities of right ventricular size or function may suggest the diagnosis. Unfortunately, because these patients often have underlying cardiopulmonary disease such as chronic obstructive lung disease, neither right ventricular dilation nor hypokinesis can be reliably used even as indirect evidence of PE in such settings.

Treatment Options for treatment of acute DVT and PE include anticoagulation with low-molecular-weight heparin (LMWH) or standard heparin, thrombolytic therapy, and inferior vena cava filter placement. Massive PE is occasionally treated with surgical embolectomy. Each approach has specific indications as well as advantages and disadvantages. Heparin and LMWH

The primary anticoagulants used to treat acute DVT and/or PE include unfractionated heparin and LMWH. These substances exert a prompt antithrombotic effect by accelerating the action of antithrombin III, preventing thrombus extension. Although they do not directly dissolve thrombus or emboli, they allow the fibrinolytic system to proceed unopposed and more readily reduce the size of the thromboembolic burden. There are substantial advantages of LMWH preparations over unfractionated heparin. When DVT or PE are diagnosed (or strongly suspected), anticoagulation should be immediately instituted unless contraindications are present. Confirmatory diagnostic testing should be undertaken as soon as possible. When standard, unfractionated, intravenous heparin is initiated, the activated partial thromboplastin time (APTT) should be followed at 6 h intervals until it is consistently in the therapeutic range of 1.5–2.0 times control values. Achieving a therapeutic APTT within 24 h after the onset of treatment of PE has been shown to reduce the recurrence rate. Heparin is administered as an intravenous bolus of 5000 units followed by a maintenance dose

588 PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts

by continuous infusion of at least 30 000 units per 24 h. Alternatively, a weight-adjusted regimen of 80 units/kg bolus followed by 18 units/kg/h is administered. This aggressive approach decreases the risk of subtherapeutic anticoagulation. At least five days of heparin or LMWH is generally recommended. Warfarin is initiated after the parenteral anticoagulant has been started (generally within 24 h). The parenteral drug should be maintained at a therapeutic level until two consecutive therapeutic international normalized ratio (INR) values of 2.0–3.0 have been documented at least 24 h apart. The LMWH preparations have tremendous advantages over unfractionated heparin and have dramatically changed treatment of thromboembolic disease. Among the differences between these two substances is the greater bioavailability of the LMWHs and more predictable dosing. The latter anticoagulants are subcutaneously administered once or twice daily even at therapeutic doses and do not require monitoring of the APTT. Intravenous LMWH is never required in VTE. In addition, LMWHs have a more profound effect in inhibiting clotting factor Xa relative to thrombin. The reduced frequency of heparin-induced thrombocytopenia with LMWH relative to unfractionated heparin is a very compelling reason to use LMWH instead of the latter whenever possible. Because of efficacy, safety, and convenience, compared with standard heparin, these drugs are replacing standard heparin in most settings. Anti-factor Xa levels appear reasonable to monitor in certain settings such as in morbidly obese patients, very small patients (o40 kg), pregnant patients, and those with renal insufficiency. Because these drugs are renally metabolized, the dose must be changed when the creatinine clearance is less than 30 ml per min. With severe renal insufficiency, standard heparin should be considered. There is no clear agreement on a weight limit above which LMWH should not be used, but some feel that an upper limit of approximately 150 kg is reasonable, with intravenous standard heparin being used in larger patients. It is unnecessary to monitor other patients with antifactor Xa levels. In the United States, at the present time, two LMWH preparations (enoxaparin and tinzaparin) are FDA-approved for use to treat patients presenting with DVT with or without acute PE. Finally, an ‘ultra-LMWH’, fondaparinux, has been studied in patients with acute DVT and PE and has proven effective for these indications. It should be noted that the prophylactic doses of these agents differ from the doses used for treating active disease. The characteristics of LMWHs compared with standard

Table 4 A comparison of LMWH with unfractionated heparin Characteristic

UFH

LMWH

Mean molecular weight Protein binding Anti-Xa activity Anti-IIa activity Administration (treatment) Subcutaneous Administration (prophylaxis) Subcutaneous Monitoring during treatment Outpatient therapy Incidence of HIT Reversibility with protamine

12 000–15 000 Substantial Substantial Substantial Intravenous

4000–6000 Minimala Substantial Minimal

Subcutaneous

APTT every 6 h Difficult 3–5% Complete

None in most settingsb Simplified o1% Partial

a

This implies significantly superior bioavailability of LMWH relative to UFH. b LMWH requires a dose change and sometimes monitoring in renal insufficiency (creatinine clearance o30 ml min
1), significant obesity (4150 kg), very small patients (o40 kg), and pregnant patients. Anti-Xa levels are followed, and not the activated partial thromboplastin time. For one LMWH (enoxaparin), the therapeutic dose is reduced from the usual recommended dose of 1 mg kg
1 q12 h, to 1 mg kg
1 qd when the creatinine clearance is o30 ml min
1, rendering monitoring unnecessary unless there are dynamic changes in renal function. Similarly, in this setting, the prophylactic dose of 40 mg qd is reduced to 30 mg qd. No such recommendations can be made for the other preparations. LMWH, low-molecular-weight heparin; UFH, unfractionated heparin; APTT, activated partial thromboplastin time; HIT, heparininduced thrombocytopenia.

unfractionated heparin are shown in Table 4. The approach to therapy utilizing these drugs is shown in Table 5. Documented proximal DVT or PE should be treated for 3–6 months. Treatment over a more extended interval is appropriate when significant risk factors persist, when thromboembolism is idiopathic, or when previous episodes of VTE have been documented. Bleeding is the major complication of anticoagulation. The rates of major bleeding in recent trials using heparin by continuous infusion or highdose subcutaneous injection are less than 5%. Direct Thrombin Inhibitors

Clinical studies suggest that more than 90% of patients with clinical heprin-induced thrombocytopenia have a platelet count fall of more than 50% during their heparin treatment. The syndrome is caused by heparin-dependent IgG antibodies that activate platelets via their Fc receptors. If a patient is placed on heparin for VTE and the platelet count progressively decreases to at least 50% of

PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts 589 Table 5 Initiation of LMWH for therapy of acute pulmonary thromboembolism Begin LMWH by subcutaneous administrationa Determine whether monitoring needed (extremes of weight, renal insufficiency, pregnancy) Consider outpatient therapy in appropriateb Warfarin from day 1; initial dose 5–10 mg, adjust according to INR Check platelet count between days 3 and 5c Stop LMWH after X5 days of combined therapy, and when INR is X2.0 for two consecutive days Anticoagulate with warfarin for X3 months (goal INR 2.0–3.0)d a

Enoxaparin, tinzaparin, and fondaparinux, are the preparations that are FDA-approved for treatment of DVT. Enoxaparin is approved for outpatient therapy in appropriate settings. Fondaparinux is approved for both inpatient DVT and PE. While enoxaparin and tinzaparin are sometimes used for patients presenting with PE in the United States, and clinical trials too support this use, the FDA-approvals read ‘‘established DVT with or without PE.’’ b Potential outpatients should be medically stable without severely symptomatic DVT. They should be compliant, capable of self-administration (or have a family member or visiting nurse for administration), at low risk of bleeding, and reimbursement should be addressed. c Heparin-induced thrombocytopenia occurs more commonly with unfractionated heparin than with LMWH. d The duration of warfarin therapy should be at least 6–12 months in patients with idiopathic venous thromboembolism or in those with persisting risk factors. When deemed safe, indefinite anticoagulation should be considered in these patients. LMWH, low-molecular-weight heparin; DVT, deep venous thrombosis; PE, pulmonary embolism; INR, international normalized ratio.

the baseline value or to less than 150 000 mm
3 heparin therapy should be discontinued. The formation of heparin-dependent IgG antibodies and the risk of thrombocytopenia is lower with LMWH than with standard heparin. Both argatroban and lepirudin have been FDAapproved for use in the setting of VTE with heparininduced thrombocytopenia. The half-life of argatroban is 45 min, but is prolonged in patients with hepatic dysfunction. Lepirudin is excreted by the kidneys so the dosage must be reduced in renal insufficiency. There is no antidote for either drug at present. Warfarin-induced skin necrosis is a rare but serious complication occurring in the setting of heparin-induced thrombocytopenia. Vena Cava Interruption

If a patient cannot be anticoagulated, inferior vena cava (IVC) filter placement can be performed to prevent lower extremity thrombi from embolizing to the lungs. The primary indications for filter placement include contraindications to anticoagulation, recurrent embolism while on adequate therapy, and

significant bleeding complications during anticoagulation. Filters are sometimes placed in the setting of massive PE when it is believed that any further emboli might be lethal, particularly if thrombolytic therapy is contraindicated. These devices are effective and complications, including insertion-related problems and migration, are unusual. More recently, retrievable filters are being placed in patients in whom the risk of bleeding appears short-term. Most of these devices can be removed up to two weeks later, and some may remain in place even longer with subsequent removal. Thrombolytic Therapy and Pulmonary Embolectomy

Thrombolytic agents activate plasminogen to form plasmin which then results in fibrinolysis as well as fibrinogenolysis. These agents can dramatically accelerate clot lysis in acute PE (and DVT) and such an approach was first documented more than several decades ago. Clinical trials have culminated in the approval of streptokinase, urokinase, and recombinant tissue-type plasminogen activator (tPA) for the treatment of massive PE. Urokinase is no longer available. For the past several decades, the clearly accepted recommendation to use intravenous thrombolytic therapy has been for PE with hemodynamic instability (hypotension). Other potential indications include PE associated with severely compromised oxygenation, and/or with echocardiographic evidence of right ventricular dysfunction without hypotension. More direct techniques, such as catheter-directed administration of intraembolic thrombolytic therapy, have been utilized in small clinical studies but the data is inadequate to formulate recommendations. The use of catheter-directed thrombolytic therapy in patients with proximal occlusive DVT associated with significant swelling and symptoms is increasing. Hemorrhage is the primary adverse effect associated with thrombolytic therapy with the most devastating complication being intracranial hemorrhage which occurs in approximately 1% of patients in clinical trials. The primary contraindications to thrombolytic therapy include active bleeding, surgery within the previous one to two weeks (depending on the specific procedure), or intracranial pathology. When patients appear to be at extraordinary risk of rapid death from PE, clinical judgment should be individualized with regard to contraindications. Pulmonary embolectomy may be appropriate in patients with massive embolism who cannot receive thrombolytic therapy.

590 PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts

Prognosis In the International Cooperative Pulmonary Embolism Registry of 2454 patients, all consecutive patients with a diagnosis of PE were included. PE was found to be the principal cause of death. The 3month mortality was 17.5%. Mean 1-month mortality rates of treated and untreated PE have been estimated at 5–8% and 30%, respectively. While a small percentage of patients with acute PE will ultimately develop chronic dyspnea and hypoxemia due to chronic thromboembolic pulmonary hypertension, most patients who survive the acute episode have no long-term pulmonary sequelae. However, chronic leg pain and swelling from DVT (postphlebitic syndrome) may result in significant morbidity.

Prevention Measures to prevent VTE appear to be grossly underutilized. A substantial reduction in the incidence of DVT can be achieved when patients at risk receive appropriate prophylaxis. For example, the risk of DVT after total hip or knee replacement is 50% or greater without prophylaxis. The superiority of LMWH over unfractionated heparin has been demonstrated in these settings, as well as in other highrisk settings such as acute spinal cord injury. In hospitalized general medical patients, anticoagulant prophylaxis should always be strongly considered as the rate of DVT, based upon a venographic endpoint, is as high as 15% in patients receiving placebo. Two LMWHs (enoxaparin and dalteparin) are FDA-approved for medical patient prophylaxis in the United States. Subcutaneous heparin administered as 5000 units every 8 h has been studied more recently and extensively than 5000 units every 12 h in the medical patient setting, and if standard heparin is used, this regimen may be the more efficacious one. The advantages of LMWH, including a reduction in heparin-induced thrombocytopenia, have resulted in increased use of these agents. Intermittent pneumatic compression devices should be utilized when pharmacologic prophylaxis is contraindicated. Both methods combined would be reasonable in patients deemed to be at exceptionally high risk, but an additional reduction in risk in such patients has not been well substantiated. Each hospitalized patient should be assessed for the need for such prophylactic measures and all hospitals should strongly consider formulating their own written guidelines for each particular clinical setting, based upon the available medical literature.

See also: Anticoagulants. Coagulation Cascade: Overview; Fibrinogen and Fibrin; Thrombin. Pulmonary Thromboembolism: Deep Venous Thrombosis.

Further Reading Ahearn GS and Bounameaux H (2000) The role of the D-dimer in the diagnosis of venous thromboembolism. Seminars in Respiratory and Critical Care Medicine 21: 521–536. Bratzler DW, Raskob GE, Murray CK, Bumpus LJ, and Piatt DS (1998) Underuse of venous thromboembolism prophylaxis for general surgery patients. Physician practices in the community hospital setting. Archives of Internal Medicine 158: 1909–1912. Dolovich LR, Ginsberg JS, Douketis JD, Holbrook AM, and Gillian C (2000) A meta-analysis comparing low molecular weight heparins with unfractionated heparin in the treatment of venous thromboembolism. Archives of Internal Medicine 160: 181–188. Geerts WH, Pineo GF, Heit JA, et al. (2001) Prevention of venous thromboembolism. Seventh American College of Chest Physicians Consensus Conference on Antithrombotic Therapy. Chest 126: 338S–400S. Goldhaber SZ and Tapson VF (2004) A prospective registry of 5,451 patients with ultrasound confirmed deep vein thrombosis. American Journal of Cardiology 93: 259–262. Kakkar VV, Howe CT, Nicolaides AN, et al. (1970) Deep vein thrombosis of the legs: is there a ‘high risk’ group? American Journal of Surgery 120: 527–530. Konstantinides S, Geibel A, Heusel G, Heinrich F, and Kasper W (2002) Heparin plus alteplase compared with heparin alone in patients with submassive pulmonary embolism. The New England Journal of Medicine 347: 1143–1150. Levine M, Gent M, Hirsh J, et al. (1996) A comparison of low molecular-weight-heparin administered primarily at home with unfractionated heparin administered in the hospital for proximal deep vein thrombosis. The New England Journal of Medicine 334: 677–681. Merli G, Spiro T, Olsson C-G, et al. (2001) Subcutaneous enoxaparin once or twice daily compared with intravenous unfractionated heparin for treatment of venous thromboembolic disease. Annals of Internal Medicine 134: 191–202. Perrier A, Roy PM, Sanchez O, et al. (2005) Multidetector-row computed tomography in suspected pulmonary embolism. New England Journal of Medicine 352: 1760–1768. Samama MM, Cohen AT, Darmon J-Y, et al. (1999) A comparison of enoxaparin with placebo for the prevention of venous thromboembolism in acutely ill medical patients. New England Journal of Medicine 341: 793–800. Stein PD (ed.) (1996) Pulmonary Embolism. Baltimore: Williams and Wilkins. Tapson VF, Carroll BA, Davidson BL, et al. (1999) The diagnostic approach to acute venous thromboembolism. Clinical practice guideline. American Thoracic Society. American Journal of Respiratory and Critical Care Medicine 160: 1043–1066. The PIOPED investigators (1990) Value of the ventilation/perfusion scan in acute pulmonary embolism. Results of the prospective investigation of pulmonary embolism diagnosis. Journal of the American Medical Association 263: 2753–2759. Wells PS, Anderson DR, Rodger M, et al. (2001) Excluding pulmonary embolism at the bedside without diagnostic imaging: management of patients with suspected pulmonary embolism presenting to the emergency department by using a simple clinical model and D-dimer. Annals of Internal Medicine 135: 98–107.

PULMONARY VASCULAR REMODELING 591

PULMONARY VASCULAR REMODELING I F McMurtry and S A Gebb, University of Colorado Health Sciences Center, Denver, CO, USA P L Jones, University of Pennsylvania, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Sustained vasoconstriction and vascular remodeling, comprised of adventitial and medial thickening of conduit and muscular pulmonary arteries, as well as muscularization of pulmonary arterioles, occurs in various forms of moderate pulmonary hypertension. By comparison, the severe idiopathic, familial, or secondary pulmonary arterial hypertension (PAH) that causes significant morbidity and mortality via right heart failure also involves a spectrum of constrictive and obliterative neointimal lesions. The pathogenesis of these neointimal lesions is poorly understood. This article addresses the controversy of vasoconstriction versus cell proliferation and matrix remodeling in the pathogenesis of PAH, recent studies of new rat models of severe PAH, the potential role of hemodynamic forces in the pulmonary arteriopathy, and possible links between genetic disruption of bone morphogenetic protein receptor type II signaling and development of PAH.

Introduction The normal adult pulmonary circulation is a lowresistance, low-pressure, high-flow system, and the pulmonary arteries (PAs) are correspondingly thin walled and highly distensible. However, there are several distinct clinical disorders and at least three genetic mutations that are associated with the development of pulmonary arterial hypertension (PAH), accompanied by pulmonary vascular wall remodeling and reduced vascular distensibility (Table 1). Despite differences in the underlying pathogenic mechanisms, pulmonary vascular remodeling, comprising cell- and matrix protein-dependent reorganization and thickening of the adventitial and medial layers of the vascular wall, is common to most forms of pulmonary hypertension. Whether idiopathic (primary), familial, or secondary to some other disease, severe PAH is characterized by adventitial and medial hypertrophy, neointimal hyperplasia and fibrosis, and complex neointimal lesions that constrict and obliterate the lumen of medium and small pulmonary arteries (Figure 1). The resultant increases in pulmonary vascular resistance and PA stiffness cause pressure and volume overloading of the right ventricle, which can lead to right heart failure and death.

Table 1 Diagnostic classification of pulmonary hypertension Pulmonary arterial hypertension (PAH) Idiopathic PAH Familial PAH PAH Related to: Collagen vascular disease Congenital systemic to pulmonary shunts Portal hypertension HIV infection Drugs and toxins Other (glycogen storage disease, Gaucher’s disease, hereditary hemorrhagic telangiectasia, hemoglobinopathies, myeloproliferative disorders, splenectomy) PAH associated with significant venous or capillary involvement Pulmonary veno-occlusive disease Pulmonary capillary hemagiomatosis Persistent pulmonary hypertension of the newborn Pulmonary venous hypertension Left-sided atrial or ventricular heart disease Left-sided valvular heart disease Pulmonary hypertension associated with lung disease and/or hypoxemia Chronic obstructive pulmonary disease Intersitial lung disease Sleep disordered breathing Alveolar hypoventilation disorders Chronic exposure to high altitude Developmental abnormalities Pulmonary hypertension due to chronic thrombotic and/or embolic disease Thromboembolic obstruction of proximal pulmonary arteries Thromboembolic obstruction of distal pulmonary arteries Pulmonary embolism (tumor, parasites, foreign material) Miscellaneous Sarcoidosis, histiocytosis X, lymphangiomatosis, compression of pulmonary vessels (adenopathy, tumor, fibrosing mediastinitis) Modified from Simonneau G, Galie N, Rubin LJ, et al. (2004) Clinical classification of pulmonary hypertension. Journal of the American College of Cardiology 43: 5S–12S.

The minority of adult PAH patients who show significant pulmonary vascular responsiveness to acute vasodilator testing, that is, a reduction of both mean PA pressure and pulmonary vascular resistance of at least 20% are treated with anticoagulants and Ca2 þ channel blockers, while those who appear to have a nonreactive or ‘fixed’ pulmonary vascular bed receive anticoagulants and prostacyclin (PGI2) analogs or endothelin 1 (ET-1) receptor blockers. These treatments improve exercise capacity and prolong

592 PULMONARY VASCULAR REMODELING

(a)

(b)

(c)

(d)

(e)

(f) ∗

(g)

(i)

(h)

(j)

(k)

Figure 1 Main histopathological features of arteriopathy in severe PAH (see original paper for details, sections were stained with Verhoeff–van Gieson unless specified). Medial hypertrophy: (a) pre-acinar pulmonary artery,
80; (b) intra-acinar artery,
600. Concentric laminar intimal thickening: (c) intra-acinar artery,
500; (d) pre-acinar artery. The vessel was decorated with anti-smooth muscle actin antibodies (SMA), revealing the intimal thickening (white arrow) to be composed of SMA-positive cells,
150. Eccentric (e)
100 and concentric nonlaminar (f)
100 intimal thickening of pre-acinar arteries. Plexiform lesions: (g) intra-acinar artery decorated with SMA showing SMA-negative endothelial cells (arrow) surrounded by a rim of SMA-positive cells (rusty color),
380; (h) pre-acinar artery adjacent to a plexiform lesion (arrow) and dilation lesions (shown by asterisk)
60. (i) Dilation lesions (arrows),
40; (j) colanderlike lesion, HE
400; (k) lymphomonocytic arteritis, HE,
300. Reproduced from Pietra GG, Capron F, Stewart S, et al. (2004) Pathologic assessment of vasculopathies in pulmonary hypertension. Journal of the American College of Cardiology 43: 25S–32S, with permission from The American College of Cardiology Foundation.

life, but do not cure PAH. Thus, new insights into the pathogenesis of the arteriopathy are needed. This article highlights the controversy of vasoconstriction versus vascular cell proliferation and

structural remodeling in the pathogenesis of PAH, recent studies describing new rodent models of severe PAH, the potential role of hemodynamic forces in the pulmonary arteriopathy, and the possible

PULMONARY VASCULAR REMODELING 593

mechanistic links between genetic disruption of bone morphogenetic protein receptor type II (BMPRII) signaling and development of PAH.

Vasoconstriction versus Proliferation Sustained pulmonary vasoconstriction has historically been considered a key component of the pathogenesis of PAH. Classic histopathological studies suggested that pulmonary vasoconstriction, medial hypertrophy of muscular arteries, and muscularization of arterioles preceded the development of neointimal thickening and plexiform lesions. Subsequent biochemical and molecular evidence in hypertensive lungs and PAs of an imbalance in vasoconstrictor signals (increased ET-1, thromboxane, serotonin, and angiotensin II, and decreased smooth muscle cell (SMC) hyperpolarizing K þ channel activity) over vasodilator signals (deficient nitric oxide (NO), PGI2, and vasoactive intestinal peptide, and increased phosphodiesterase activity) supported the concept that vasoconstriction was an important pathogenic factor in PAH, especially in the early stages of the disease. Yet, in addition to regulating vascular tone, these same vasoactive signals also regulate vascular cell growth, and the current view of many investigators of adult PAH is that the increased pulmonary vascular resistance is due largely, if not solely, to structural luminal narrowing and obliteration, and the disease should be considered one of uncontrolled vascular cell proliferation, deficient apoptosis, and excessive matrix protein deposition, rather than of inappropriate vasoconstriction. This argument, and the evidence for it, can be found in several recent papers, commentaries, and reviews. Because most adult PAH patients respond poorly, if at all, to acute administration of pulmonary vasodilators, such as PGI2 analogs, adenosine, and NO, it can be argued that by the time they are diagnosed and treated the vasoconstrictor component has disappeared (or that it was never involved), and the increased vascular resistance is due to structural luminal narrowing and obliteration of small distal PAs. Another possibility, however, is that the most effective vasodilator has not yet been tested. For example, while acute administration of a Ca2 þ channel blocker does not reduce sustained pulmonary hypertension in chronically hypoxic rats, a Rhokinase inhibitor, which can ‘activate’ myosin light chain phosphatase and thereby reverse increased Ca2 þ sensitivity of the contractile apparatus of hypertensive vascular smooth muscle (Figure 2), nearly normalizes the pressure and resistance. RhoA/Rho kinase signaling, which is increased in hypertensive arteries, is a convergence point in the signaling

pathways of several different G-protein-coupled receptors, and can theoretically mediate the response to multiple simultaneous vasoconstrictor stimuli. Thus, not all vasodilators are created equal, and it would be interesting to investigate if an inhaled Rhokinase inhibitor is any more effective than inhaled PGI2 or NO in eliciting selective pulmonary vasodilation in severe PAH. Whether or not vasoconstriction initiates the process in all cases, it is clearly involved in the pathogenesis of PAH in some patients, and appears to be more prevalent in children than adults. Moreover, even adult patients whose PAH has been successfully reversed by long-term intravenous epoprostenol (PGI2 analog) have to be maintained on oral vasodilators to prevent recurrence of the disease. Thus, it is premature to dismiss the importance of vasoconstriction, and perhaps a more accurate view of the pathogenesis of most forms of PAH is that it involves both vasoconstriction and an imbalance in vascular cell proliferation/apoptosis and matrix catabolism and deposition. Regardless, the key clinical question is whether simply lowering pulmonary arterial pressure with an effective vasodilator will reverse the obliterative neointimal and plexiform lesions of severe PAH, or does the therapy also have to more directly disrupt the autocrine or paracrine production of growth factors and inflammatory cytokines and/or the activation of intracellular signaling pathways responsible for the increased cell proliferation, survival, migration, and matrix protein deposition? In this regard, it is noteworthy that inhibition of Rho-kinase activity prevents induction of SMC tenascin-C (TN-C), a pro-proliferative matrix protein that is induced in experimental and clinical PA lesions.

Animal Models Most studies of the mechanisms of hypertensive pulmonary vascular remodeling are in animal models. The two most commonly used are hypoxia- and monocrotaline-induced pulmonary hypertension in rats, although transgenic mice are increasingly being used. While the rat models have provided important groundwork for the current use of Ca2 þ channel blockers, PGI2 analogs, ET-1 receptor antagonists, inhaled NO, and phosphodiesterase inhibitors in the clinical treatment of PAH, and continue to provide new insights into the myriad cellular and molecular mechanisms involved in regulation of pulmonary vascular tone and structure, it should be emphasized that neither develops the obstructive neointimal lesions found in severe PAH. In fact, if the hypertensive lungs of chronically hypoxic or monocrotaline-injected rats

594 PULMONARY VASCULAR REMODELING Stimulus (ET-1, 5-HT, TXA2, thrombin, S1P, etc.) PLC


GEF

GPCR

Ca2+

Rho-GT

No, statins

Active

GTP Rho-GDP Inactive IP3 Relaxation MLCK

Ca2+ Ca2+

Ca2+

Actin

P MBS

Ca2+ Ca2+

Ca2+

P Ca CaM MLCK Ca

P CPI-17

MLCP

P MLCP

Active

Inactive

P Myosin

P Myosin Ca2+

Y-27632 fasudil

Rho-kinase

Myosin

Ca2+ CaM Ca2+

Ca2+

SR

Myosin

Actin Contraction

Figure 2 RhoA/Rho kinase-mediated Ca2 þ sensitization of vascular smooth muscle cell contraction. Numerous G-protein-coupled receptor (GPCR)-dependent vasoconstrictors, including endothelin-1 (ET-1), serotonin (5-HT), thromboxane (TXA2), thrombin, and sphingosine 1-phosphate (S1P), lead to stimulation of guanine nucleotide exchange factors (GEF) and activation of the small GTPase RhoA, which binds to and activates Rho-kinase. Activated Rho-kinase, in turn, phosphorylates the myosin-binding subunit (MBS) of myosin light chain phosphatase (MLCP) and/or the MLCP inhibitory protein CPI-17, which inhibits MLCP and thereby increases phosphorylation of MLC and augments contraction at a given level of cytosolic [Ca2 þ ] and Ca2 þ -calmodulin (Ca2 þ -CaM)-mediated activation of MLC kinase (MLCK). Sustained RhoA/Rho kinase-mediated vasoconstriction is reversed by the Rho-kinase inhibitors Y27632 and fasudil (HA-1077) and by inhibition of RhoA activation by nitric oxide (NO) or statins. (PLC-b, phospholipase C beta; IP3, inositol-1, 4, 5-trisphosphate; SR, sarcoplasmic reticulum; GAP, GTPase activating protein).

are vasodilated and fixed under positive intravascular pressure, it is observed that the adventitial and medial thickening of small muscular PAs causes little, if any, reduction in lumen area. Thus, it appears that much of the muscular PA medial hypertrophy and luminal narrowing typically reported in hypoxia- and monocrotaline-induced hypertensive rat lungs fixed without maximal vasodilation is due to vasoconstriction (vasospasm), rather than to structural inward remodeling of the vascular wall. Recent studies showing marked reversal of established pulmonary hypertension in either chronically hypoxic or monocrotaline-injected rats by acute administration of Rho-kinase inhibitors, such as Y27632 and fasudil, supports the idea that sustained vasoconstriction, rather than structural medial thickening of either muscular PAs or neomuscularized pulmonary arterioles, is a major cause of increased pulmonary vascular resistance in these models. This is not to say that the remodeling plays no role in the

increased resistance, but rather that its apparent contribution is exaggerated in PAs that are not vasodilated before fixation. It is noteworthy that in studies of vascular remodeling in systemic hypertension, it has long been appreciated that the direct contribution of vascular structure to luminal narrowing needs to be determined under conditions of complete lack of vascular tone, that is, under maximal vasodilation. Variations of the standard monocrotaline-injected and chronically hypoxic rat models have recently been developed as an attempt to more closely mimic the obstructive multicell-type intimal lesions found in patients with severe PAH. One such model is the leftlung pneumonectomized plus monocrotaline-injected rat that develops smooth muscle a-actin-positive, but endothelial cell (EC) CD31-negative, neointimal lesions in the small peripheral PAs/arterioles of the right lung. It has been speculated that the combination of monocrotaline pyrrole-induced EC injury

PULMONARY VASCULAR REMODELING 595

and increased blood flow (shear stress) is responsible for the lesions, but the role of increased blood flow and shear stress is unclear (see below). Histological examination has suggested more than 50% occlusion of some arteries by medial hypertrophy and neointimal hyperplasia. Treatment of these rats with the pleiotrophic agent simvastatin reverses the severity of PA medial hypertrophy, neointimal lesions, and hypertension. This was associated with downregulation of lung tissue expression of various inflammatory genes and upregulation of eNOS and the cell-cycle inhibitor p27Kip1, and with decreased proliferation and increased apoptosis of cells in the media and neointima. Monocrotaline has also recently been found to cause severe PAH and development of neointimal lesions in the small distal PAs of ET-1 type B (ETB) receptor-deficient rats. The neointimal lesions express high levels of the proangiogenic factor vascular endothelial growth factor (VEGF) and the proliferative matrix protein TN-C, and also contain proliferating cells that express EC and SMC markers. Cardiac output was very low in the pulmonary hypertensive ETB receptor-deficient rats; so increased blood flow was apparently not a factor in sustaining the expression of pro-proliferative molecular signals and the neointimal hyperplasia. Another recent rat model combines VEGFR-II inhibition by the receptor tyrosine kinase inhibitor SU5416 (semaxinib) with exposure to chronic hypoxia to cause severe PAH associated with development of occlusive PA neointimal lesions, which resemble plexiform lesions. The development of pulmonary arteriopathy is accompanied by markers of increased EC death and attenuated by treatment with a nonspecific caspase inhibitor. Thus, it has been proposed the neointimal lesions are initiated by EC death followed by selection and exuberant overgrowth of an apoptosis-resistant EC phenotype, as has also been suggested to occur in some PAH patients. While all three rat models of severe PAH develop occlusive neointimal lesions in small distal PAs, there are apparent differences in the proliferative cell phenotypes involved, that is, SMCs in the pneumonectomy plus monocrotaline model, cells expressing smooth muscle and EC markers in the ETB receptordeficient plus monocrotaline model, and ECs in the SU5416 plus hypoxia model. Explanations of these differences in cell phenotype are not obvious, but possibly relate to inconsistencies in detection methods for the chosen cell-specific antigens, or to differences in the way in which the lung injury and hypertension are induced. Interestingly, formation of neointimal lesions in all three models apparently requires a combination of severe EC injury

(monocrotaline pyrrole or SU5416) and some other abnormality (pneumonectomy, ETB receptor deficiency, or hypoxia). It is intriguing that the differences in neointimal cell phenotype in these rat models are reminiscent of the varied cell phenotypes, for example, ECs, modified SMCs, fibroblasts, and myofibroblasts, that have been described in the neointimal and plexiform lesions of human PAH. If these phenotypic differences are real, instead of being artifacts of tissue preparation or antibody specificity, they could reflect differences in the combination of pathogenic factors causing the PAH in different patients. Whereas the presence of PA neointimal lesions in the three rat models is certain, the relative roles of vasoconstriction and structural encroachment in the apparent luminal occlusion are not. In these studies none of the lungs was maximally vasodilated during fixation. Preliminary experiments by M Oka and colleagues in the CVP research laboratory show significant pulmonary vasodilation by acute intravenous administration of the Rho-kinase inhibitor fasudil in the pneumonectomy plus monocrotaline and SU5416 plus hypoxia models (the ETB receptor model has not yet been tested). This suggests the severe PAH is not due solely to vascular structural remodeling but also importantly involves sustained Rho-kinase-mediated pulmonary vasoconstriction (Figure 2). Interestingly, in addition to its anti-inflammatory and antiproliferative effects, simvastatin, which reverses the pneumonectomy plus monocrotaline PAH, might also inhibit vasoconstriction by both inhibiting the activation of RhoA/Rho-kinase signaling and increasing the bioavailability of NO. Despite the drawbacks of using animal models to study the mechanisms and potential therapies of human diseases, studies of these rat models will hopefully provide new insights into the pathogenesis and treatment of severe PAH in humans. Possibly, these systems can be used to establish the properties of the neointimal cells and determine if and by what mechanisms they originate from the adventitia, media, or intima of the arterial wall, or even from circulating progenitor cells. Chronic treatment of rats with Rho-kinase inhibitors attenuates development of hypoxic pulmonary hypertension and reverses established monocrotaline-induced pulmonary hypertension, and it will be important to determine their effectiveness in the neointimal lesion models.

Hemodynamic Forces Hemodynamic forces, that is, pulsatile blood pressure (circumferential wall stress), hydrostatic pressure, and blood flow (wall shear stress), are considered

596 PULMONARY VASCULAR REMODELING

major regulators of EC and SMC function and arterial wall thickness and luminal diameter. These biomechanical forces interact in complex ways with biophysical elements and numerous circulating and locally generated chemical signals to induce various types of adaptive and maladaptive arterial wall remodeling, including hypertrophic, eutrophic, or hypotrophic changes in medial mass, inward or outward changes in structural luminal diameter, and neointima formation. Interestingly, there is evidence in systemic arteries that hypertensive remodeling of the media that reduces luminal area does not necessarily involve cell growth or an increase in wall material, that is, the inward remodeling can be eutrophic as a consequence of rearrangement of existing cells and matrix proteins. In addition, studies of systemic resistance arteries in organ culture show that ET-1induced SMC contraction can, independently of changes in pressure and flow, cause eutrophic inward structural remodeling of the vascular wall, a response that may involve signals transmitted directly from activated SMCs to the matrix via b3-integrins. There is a lively debate among investigators in this area as to whether or not these structural changes have functional consequences with respect to the hypertension, but it is apparent that the contractile phenotype of the SMCs is well maintained, which differs from the dedifferentiation that occurs at sites of vascular injury and neointimal formation. Similarly, sustained vasoconstriction and an adaptive vascular remodeling leading to increased medial muscularization may occur in moderate pulmonary hypertension, but what then triggers and sustains development of severe PAH and the maladaptive neointimal pathology? It is well established in the systemic circulation that whereas arteries exposed to normal or high levels of blood flow and laminar shear stress are relatively free of disease, neointimal and atherosclerotic lesions tend to develop in areas where shear stress is low or disturbed (turbulent, oscillatory), such as at arterial branch ostia and bifurcations. So, despite universal exposure of arteries to environmentally induced risk factors, local hemodynamic forces affect neointimal pathology. Numerous in vitro and in vivo studies indicate that the protective effect of laminar shear stress is mediated largely by EC function. Laminar fluid shear stress maintains normal EC function by stimulating production of NO and PGI2 and expression of several other antioxidant, anti-inflammatory, antithrombotic, antiproliferative, and antiapoptotic proteins. In contrast, low or disturbed shear stress has the opposite effect and promotes endothelial oxidative stress and dysfunction, which can lead to increased vascular permeability to bloodborne protein and lipid mediators, adhesion and

activation of platelets, and infiltration of inflammatory cells. These events, in turn, generate a multitude of factors (reactive oxygen species, cytokines, chemokines, and growth factors) that increase EC turnover, activate matrix metalloproteinases, disrupt the elastic lamina, and stimulate medial and/or adventitial SMC/myofibroblast proliferation, migration, and matrix protein production resulting in neointimal formation. Compelling studies by Passerini and colleagues comparing gene expression in porcine aortic ECs isolated from disease-free areas of laminar versus disturbed flow suggest that while disturbed flow alone does not cause neointimal pathology, it apparently induces a proinflammatory phenotype that ‘primes’ the ECs for adverse responses to additional risk factors. Conversely, in mouse models of no or low flow, such as that created by ligation of an upstream artery, neointimal thickening occurs in the absence of other perturbation. Thus, low or disturbed flow-promoted neointimal thickening is a complex, multifactorial process that appears to be initiated by endothelial dysfunction, plus recent studies in various strains of rats and mice indicate that the severity of vascular remodeling is genetically determined. An important unresolved question concerning the pathogenesis of PAH is what roles are played by blood pressure and flow. Are the neointimal thickening and plexiform lesions causes or consequences of the pulmonary hypertension, or both? Medial and intimal thickening can apparently occur independently of pulmonary hypertension in localized PAs, but does development of the diffuse neointimal thickening of PAH depend on increases in PA pressure and/or flow? Congenital left to right cardiac shunts can lead to the development of severe PAH and neointimal lesions. While the hypertension and arteriopathy are generally ascribed to effects of increased blood flow and shear stress, greater consideration needs to be given to the effects of the high PA pressure. This is especially true in cases of large ventricular septal defects or a large patent ductus arteriosus where systemic arterial pressure can be transmitted into the pulmonary circulation. Animal studies indicate that systemic artery to PA shunts, which increase both pressure and flow in the downstream PAs, can induce severe PAH and formation of neointimal lesions, whereas large arteriovenous shunts, which increase cardiac output and pulmonary blood flow, but not pressure, cause no or only modest pulmonary hypertension and vascular remodeling. In young pigs, for example, anastomosis of left lower lung lobe artery to aorta exposes PAs to high flow and pressure and leads to medial thickening and

PULMONARY VASCULAR REMODELING 597

intimal fibrosis with subsequent development of plexiform and dilatation lesions. Although the cause-and-effect relationship is unclear, it is interesting that at the time neointimal lesions are most severe, shunt flow, but not pressure, is markedly reduced. Similarly, in calves with a systemic to left PA shunt, those with the lowest shunt flow at 10 weeks had the most severe small PA obliteration. These observations are similar to those in the rat models of PA neointimal formation discussed above in which the neointimal lesions coexist with low pulmonary blood flow as an apparent consequence of cardiac failure. Thus, whatever the role of increased blood flow in initiating the hypertensive process, the neointimal lesions are evidently sustained during low flow. In fact, recent work indicates that increased pulmonary blood flow via an aortocaval fistula actually attenuates development of pulmonary hypertension and neointimal hyperplasia in the pneumonectomy plus monocrotaline rat. Collectively, these observations suggest that high pulmonary arterial pressure is a major factor in inducing formation of PA neointimal lesions. High flow alone is apparently not sufficient, and based on the inverse relationship between flow and neointimal formation observed in systemic arteries, the question arises as to whether low flow might actually promote initiation or progression of the lesions. The role of shear stress in the arteriopathy of PAH is poorly understood. However, it can be speculated that adverse effects of disturbed flow on EC function might explain the tendency of obliterative PA neointimal lesions to develop at sites distal to vessel bifurcations (Figure 3) and the ostia of supernumerary arteries. If high blood pressure is, in fact, both cause and consequence of neointimal thickening in PAH, then an interesting possible pathogenic scenario is that it promotes the neointimal pathology by inducing vascular inflammation. Inflammatory-induced endothelial dysfunction is believed to play an important role in vascular diseases, including PAH, but a key question is what induces the inflammation? In some cases, the inflammation is due to systemic immunological disorders or infections, such as those that occur in connective tissue diseases or HIV infection. However, some patients with idiopathic PAH show evidence of autoimmunity and/or systemic inflammation with no identifiable cause. Similar observations have been made in patients with systemic hypertension, and a current hypothesis is that high systolic pulse pressure causes a chronic low-grade oxidative stress and inflammation of the endothelium, possibly via increased cyclic wall stress, activation of local EC angiotensin II and

Figure 3 In severe PAH, concentric-obliterative and/or plexiform lesions show a tendency to develop at sites just distal to bifurcations of small- to medium-sized muscular PAs. In this section stained with FVIII (see original paper for details), the cells lining the multiple lumina of the plexiform lesions stained positive for FVIII. Dilatation lesions can be seen within and adjacent to the plexiform lesions (arrows). Reprinted from American Journal of Pathology, 1999, 155: 411–419, with permission from the American Society for Investigative Pathology.

ET-1 signaling, and increased NAD(P)H oxidasedependent production of reactive oxygen species. Perhaps similar mechanisms are operative in the pathogenesis of PAH.

BMPRII Signaling: A Missing Link? The recent discovery that heterozygous germline mutations in the BMPRII gene (BMPR2) are associated with B50% of familial and B20% of idiopathic PAH may provide important clues to the relative roles of vasoconstriction and vascular remodeling in PAH. Before considering this possibility, it is important to outline bone morphogenetic protein (BMP) signaling in normal cells. BMPs are developmentally regulated, TGF-b-related polypeptides that exert diverse cellular effects, depending upon cell type, context, and differentiation state. Briefly, BMPs (including BMP2, 4, 6, and 7) stimulate heterodimerization and activation of two types of BMP receptors (i.e., I and II) and initiate phosphorylation of the Smad proteins (1, 5, and 8), which combine with the common mediator Smad4. Upon translocation to the nucleus, the Smad complex binds target genes to activate or repress their transcription. Thus, it is tenable to hypothesize that whereas wild type BMPRII signaling suppresses pulmonary vascular cell proliferation and PA remodeling, haploinsufficiency generated by heterozygous mutations impairs this type of control. Indeed, BMP2, 4, and 7 do suppress normal main PA SMC proliferation,

598 PULMONARY VASCULAR REMODELING

and this response is lost in cells isolated from idiopathic PAH patients. Nevertheless, this ‘one-hit’ hypothesis is clearly insufficient, since it fails to explain why subpopulations of normal cultured peripheral PA SMCs actually proliferate in response to BMPs, and why PAH in individuals with BMPR2 mutations may take many years to develop, or never develop at all. A possible explanation is suggested by the recent finding that heterozygous BMPR2-mutant mice exhibit pulmonary hypertension and muscularization of distal PAs only after challenged with a leukotriene-induced inflammatory stress. Therefore, BMPR2 mutations may predispose PA SMCs to react abnormally to secondary genetic or extrinsic factors that are experienced over time in a cell-type and site-specific manner. In other words, development of PAH, even in patients with BMPR2 mutations, requires multiple ‘hits’. In addition, whereas the development of pulmonary hypertension in the inflammatory-stressed heterozygous BMPR2-mutant mice was immediate, the muscularization of PAs was delayed. This emphasizes that increased pulmonary vasoconstriction should not be ignored as a possible initiating factor in the PAH associated with BMPR2 haploinsufficiency. In terms of gene structure and function, a variety of heterozygous, inactivating mutations have been found in all 13 coding exons and the flanking intronic sequences of BMPR2. It is believed that these diverse mutations may generate a modified receptor that is deficient in ligand binding (due to mutations in the extracellular domain) or kinase activity (due to mutations in cytoplasmic domains), thereby subverting the normal ability of wild type BMPRII to promote EC regeneration and survival on the one hand, while suppressing SMC proliferation on the other. These functions appear to be mediated by Smad signaling, at least in SMCs. Although it is conceivable that any one of the mutations could result in deficient BMPRII signaling, it is unclear whether different mutations stimulate or repress the same or different signal transduction pathways and target genes in PAH. Recent work by K Ihida–Stansbury in our group shows that PA SMCs harboring ligandbinding domain BMPR2 mutations express higher levels of extracellular signal-regulated kinase (ERK)1/2 activity than those bearing kinase domain mutations, despite the fact that both cell types overexpress similar levels of the SMC mitogen, TN-C. Thus, it is likely that specific signaling pathways are fine-tuned by distinct BMPRII domains. In support of this, there is evidence that the wild type BMPRII cytoplasmic tail domain binds to and, depending on cell type, either inhibits or activates LIM kinase 1, which is a key regulator of the actin cytoskeleton via

the F-actin depolymerizing factor cofilin. Since a subset of familial PAH patients possess BMPR2 mutations that truncate the LIM kinase 1-interacting tail domain, and because cytoskeletal actin dynamics control MAP kinase activity, gene expression, and contraction in SMCs and ECs, it is conceivable that ligand binding and cytoplasmic domain mutations activate or repress different downstream signaling pathways that converge to control common mediators associated with pulmonary vasoconstriction and/or vascular remodeling. Additional studies on BMPRII signaling are clearly needed, and these will hopefully answer several outstanding questions, including: why do BMPR2 mutations affect pulmonary vascular cells, whereas systemic vascular cells bearing the same genetic abnormality are seemingly unaffected, why do normal ECs and SMCs isolated from different PA segments respond differently to BMPs, which target genes or signaling molecules are activated or inhibited by BMPR2 mutations, if one or more of the mutations promotes vasoconstriction, do they do so by increasing SMC contractility or the balance of vasoconstrictors to vasodilators, and do pulmonary hemodynamic forces modulate the function of wild type and mutated BMPRIIs? Eagerly awaited answers to these questions will eventually provide new insights into how BMPRII signaling influences pulmonary vascular tone and structure. See also: Neonatal Circulation. Smooth Muscle Cells: Vascular. Vascular Disease.

Further Reading Boudreau NJ and Jones PL (1999) Extracellular matrix and integrin signalling: the shape of things to come. Biochemical Journal 339: 481–488. Bund SJ and Lee RMKW (2003) Arterial structural changes in hypertension: a consideration of methodology, terminology and functional consequence. Journal of Vascular Research 40: 547–557. Cool CD, Stewart JS, Werahera P, et al. (1999) Three-dimensional reconstruction of pulmonary arteries in plexiform pulmonary hypertension using cell-specific markers. Evidence for a dynamic and heterogeneous process of pulmonary endothelial cell growth. American Journal of Pathology 155: 411–419. Dorfmuller P, Perros F, Balabanian K, and Humbert M (2003) Inflammation in pulmonary arterial hypertension. European Respiratory Journal 22: 358–363. Humbert M, Morrell NW, Archer SL, et al. (2004) Cellular and molecular pathobiology of pulmonary arterial hypertension. Journal of the American College of Cardiology 43: 13S–24S. Jeffery TK and Morrell NW (2002) Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension. Progress in Cardiovascular Diseases 45: 173–202. Malek AM, Alper SL, and Izumo S (1999) Hemodynamic shear stress and its role in atherosclerosis. Journal of the American Medical Association 282: 2035–2042.

PULMONARY VASCULAR REMODELING 599 Mandegar M, Fung YCB, Huang W, et al. (2004) Cellular and molecular mechanisms of pulmonary vascular remodeling: role in the development of pulmonary hypertension. Microvascular Research 68: 75–103. Newman JH, Fanburg BL, Archer SL, et al. (2004) Pulmonary arterial hypertension: future directions: report of a National Heart, Lung and Blood Institute/Office of Rare Diseases workshop. Circulation 109: 2947–2952. Newman JH, Trembath RC, Morse JA, et al. (2004) Genetic basis of pulmonary arterial hypertension: current understanding and future directions. Journal of the American College of Cardiology 43: 33S–39S. Pietra GG, Capron F, Stewart S, et al. (2004) Pathologic assessment of vasculopathies in pulmonary hypertension. Journal of the American College of Cardiology 43: 25S–32S. Shimokawa H and Takeshita A (2005) Rho-kinase is an important therapeutic target in cardiovascular medicine. Arteriosclerosis, Thrombosis, and Vascular Biology 25: 1767–1775.

Simonneau G, Galie N, Rubin LJ, et al. (2004) Clinical classification of pulmonary hypertension. Journal of the American College of Cardiology 43: 5S–12S. Stenmark KR, Davie NJ, Reeves JT, and Frid MG (2005) Hypoxia, leukocytes, and the pulmonary circulation. Journal of Applied Physiology 98: 715–721. Stewart DJ (2005) Bone morphogenetic protein receptor-2 and pulmonary arterial hypertension: unraveling a riddle inside an enigma? Circulation Research 96: 1033–1035. van Suylen RJ, Smits JFM, and Daemen MJAP (1998) Pulmonary artery remodeling differs in hypoxia- and monocrotaline-induced pulmonary hypertension. American Journal of Respiratory and Critical Care Medicine 157: 1423–1428. Voelkel NF and Cool C (2004) Pathology of pulmonary hypertension. Cardiology Clinics 22: 343–351. Widlitz A and Barst RJ (2003) Pulmonary arterial hypertension in children. European Respiratory Journal 21: 155–176.

R RADIATION-INDUCED PULMONARY DISEASE G D Rosen and D S Dube, Stanford University Medical Center, Stanford, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract This article discusses the natural history, clinical epidemiology, diagnosis, and treatment of radiation-induced lung diseases (RILDs). Furthermore, we review current experimental developments and assess potential areas for future investigation. Radiation therapy (RT) is a key component of the approach to the treatment of thoracic neoplasms. The use of radiation for the treatment of cancer exploits the preferential cytotoxicity of radiation to cells with high mitotic index. By the same principle, non-neoplastic cells with high mitotic index are also vulnerable to the deleterious effects of radiation. As a consequence, RT to the lung culminates in significant injury to normal cells with high mitotic index such as vascular endothelial cells and alveolar type II pneumocytes. The clinical manifestations of radiation-induced lung injury fall into two categories, acute radiation pneumonitis (RP) and radiation-induced lung fibrosis (RIF) which is a late sequelae of RT. Epidemiological analyses reveal that radiationmediated lung injury is amplified by many factors, such as radiation dose–lung volume relationships, concomitant use of chemotherapy, and the abrupt withdrawal of previously instituted steroid therapy. The cornerstone of the management of RILDs is prevention that is accomplished through surveillance of lung function, prompt evaluation of respiratory symptoms, and the early institution of steroids upon making a diagnosis of RP.

Introduction The discovery of X-rays by Wilhelm Roentgen in 1895 heralded the origin of radiation oncology. Shortly after the discovery of X-ray radiation, the first use of radiation for the treatment of cancer was reported. The development of the cathode ray tube and subsequently cobalt-60 teletherapy and linear accelerators revolutionalized the generation of Xrays for cancer treatment. Groover and co-workers first reported in 1922 that side effects of radiation therapy (RT) were radiation pneumonitis (RP) and pulmonary fibrosis (see Pulmonary Fibrosis). In 1928, Antoine Lacassagne observed that a short duration of the interval between radiation treatments, high radiation doses, low voltage, and opposing fields were associated with radiation injury. A rise in the number of cases of radiation-induced lung diseases (RILDs) can result from several

factors: (1) an increase in the incidence and prevalence of neoplasms as a result of the growing number of elderly patients; (2) use of RT as part of the conditioning regimen prior to bone marrow transplantation; and (3) increase in the number of immunocompromised hosts, in whom malignancies are more prevalent (see Table 1 for a list of RILDs). Both, the demand to improve the efficacy of RT and the need to limit lung injury, have spurred technical modifications in the delivery of RT. At present, the use of RT is preceded by rigorous planning that encompasses accurate tumor localization through the use of ultrasound (US), magnetic resonance imaging (MRI), and computer tomography (CT) (see Lung Imaging). The future of RT will, in part, focus on the reduction of side effects through targeting of RT to tumor but not normal cells and enhancing cytocidal effects of lower doses of RT through the use of radio-sensitizers and genetic engineering as well as the antagonism of endogenous mediators of RILDs.

Etiology Genetic Factors

The lack of stereotyped uniformity in the incidence of RILD among recipients of RT suggests that genetic factors may be crucial to the development of radiation lung injury. Investigators have identified that the loss of heterozygosity at the mannose 6-phosphate insulinlike growth factor-2 receptor (M6P/IGF2R) leads to elevated transforming growth factor beta (TGF-b) levels (see Transforming Growth Factor Beta (TGF-b) Family of Molecules). They proposed that the elevated TGF-b levels enhance the susceptibility of the surrounding tissues toward the development of RP. The repertoire of potential genes that are likely to be involved in the immune response to radiation injury is immense. Moreover, the interaction of potential genes is likely to be very complex but at present the precise role of genetic factors is not well understood. Host Factors

A variety of patient factors – age, sex, tobacco use, tumor site, and pre-RT pulmonary function tests

602 RADIATION-INDUCED PULMONARY DISEASE Table 1 Radiation-induced lung diseases

Table 2 Risk factors for radiation-induced lung disease

Parenchymal Acute Radiation-induced pneumonitis Diffuse alveolar damage Infectious sequel due to immunosuppression Late effects Pulmonary fibrosis Propensity to infection due to anatomical distorting effects of radiation

Patient factors Age Female sex (small lung volumes) Genetic susceptibility

Vascular Pulmonary hypertension Superior vena caval syndrome Secondary pulmonary venous hypertension from cardiomyopathy Pulmonary vessel thrombosis Fatal pulmonary hemorrhage (FPH) Airway disease Radiation-induced bronchitis (RIB) Tracheal stenosis Bronchiolitis obliterans Bronchiectasis Airway hyperreactivity Pleural diseases Pleural fibrosis Pneumothorax Broncho-pleural fistula Chylothorax Thoracic cage disease Radionecrosis of vertebrae, ribs Thoracic cage cutaneous contracture Neurological Phrenic nerve palsy Chronic pain syndromes Spinal cord damage Neoplasms Secondary malignancy Miscellaneous Recurrent cough Exercise-induced dyspnea Mediastinal fibrosis

(PFTs) as well as the Karnofsky performance status (Table 2) – have been proposed to impact the emergence of RP. The role of each factor alone is unclear because some of the studies exploring their significance reveal different dosing regimens which is the most important factor in the development of RP. It is clear that underlying pretreatment patient conditions that reduce total lung volume favor the development of RP. A retrospective study of 84 patients with small or non-small cell lung cancer (NSCLC) evaluated the role of gender, age, surgery, chemotherapy, chronic obstructive pulmonary disease (COPD), and performance status. The study found only COPD and the concomitant use of mitomycin as independent risk factors which are associated with the emergence of

Dosimetric variables V20, MLD Radiation fraction 42.67 Gy Medication Concurrent use of chemotherapy during cancer therapy Withdraw of corticosteroids

RP. Another study that assessed the influence of patient-specific factors on RT-induced reduction in pulmonary perfusion employed a multivariate analysis to analyze the effect of tobacco history, pre-RT diffusion capacity, TGF-b, chemotherapy exposure, disease type, and mean lung dose. The study found a trend but it was not statistically significant toward increased radiation sensitivity among patients who were nonsmokers and receiving radiation doses greater than 40 Gy. The authors concluded that patient-specific factors have a minimal effect on RTinduced reduction in regional lung perfusion. A study using CT scans among patients with limited small cell lung cancer (SCLC) that was designed to evaluate the magnitude of individual variation in the severity of lung fibrosis following treatment with chemoradiation, found significant patient-to-patient heterogeneity in patients at risk for developing radiationinduced fibrosis. The role of age in the development of RP is conflicting: some studies have failed to identify an association between age and the emergence of RP while others have reported an association. As alluded to previously, the propensity to develop RT may be more related to pretreatment lung volume than age. Dosimetric Factors

Several investigators have shown that dosimetric factors are the best predictors of clinically manifest RP following external-beam RT for a variety of neoplasms (see Table 2 and Radiotherapy). The dosimetric factors that have been evaluated include mean lung dose (MLD), V20, V30 (defined as the percentage of the total lung volume receiving greater than 20 or 30 Gy of radiation, respectively), and radiation dose per fraction. A prospective study of 96 patients who underwent three-dimensional conformal radiotherapy (3D-CRT) for NSCLC observed a threshold effect for the emergence of RP at radiation doses of 20–40 Gy. The study also noted that the MLD, V20, V30, and age were predictive of RP following RT.

RADIATION-INDUCED PULMONARY DISEASE 603

71 patients chemoradiation

382 patients RT alone

100 6/7

Incidence of RP (%)

80

2/2

60 6/12 40

6/29

20

11/50

14/82

2/23 10/95

2/14

0 0

20

40

60

V20 (%) Figure 1 Incidence of radiation pneumonitis (RP) in relation to V20 and effects of concurrent chemoradiation. Reproduced from Tsujino K, Hirota S, Endo M, et al. (2003) Predictive value of dose–volume histogram parameters for predicting radiation pneumonitis after concurrent chemoradiation for lung cancer. International Journal of Radiation Oncology, Biology, Physics 55(1): 110–115, with permission from Elsevier.

A univariate and multivariate analysis of patient- and treatment-related factors among lung cancer patients treated with conventional fractionated radiotherapy and concurrent chemoradiation identified V20 as the best predictor of the incidence and grade of the ensuing RP post-RT. The study also demonstrated that concurrent use of chemotherapy increases the incidence of RP (Figure 1). A study designed to correlate dose–volume histogram factors to clinically manifest RP reported that all dosimetric factors (V30, MLD, normal tissue complication probability (NTCD)) correlated with the emergence of RP. However, an analysis of factors such as age, irradiation of the lower lung field, and low pre-RT pulmonary function demonstrated no correlation with the emergence of RP. An intriguing observation is that the incidence of RP may be lower among smokers. Chemotherapy

The concurrent use of chemotherapy with radiation in regimes that exploit the radiosensitizing effects of drugs such as taxanes has been associated with the potentiation of the pneumotoxic effects of radiation. The compounding effects of chemotherapy on pneumonitis appear to be independent of the mechanisms of action of individual chemotherapeutic agents although some agents such as bleomycin are known to be radiosensitizers. Moreover, there is no clear dose–effect relationship between chemotherapy

Table 3 Selected chemotherapy regimes that potentiate radiation lung toxicity Doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) Bleomycin, adriamycin, cyclophosphamide, and vincristine Paclitaxel

and the emergence and extent of RP. A study of 13 SCLC cases treated with bleomycin, adriamycin, cyclophosphamide, and vincristine in addition to radiotherapy noted severe pulmonary fibrosis in five cases, two of which were fatal. In a study of 41 patients with breast cancer who were treated with RT concurrently with chemotherapy, the regimes that included paclitaxel showed that RP developed at a rate of 14.6%. In comparison, patients treated with RT and chemotherapy without paclitaxel developed RP at the rate of 1.1% (95% confidence interval ¼ 0.2–2.3%, po0.0001). Additional chemotherapeutic drugs that have been associated with RP are shown in Table 3. Pathology

Murine models of RP strongly suggest that injury to pulmonary capillary endothelial cells and type II pneumocytes mediates the pathogenesis of RP (Figure 2) (see Endothelial Cells and Endothelium). Within an hour of RT, quantitative and qualitative

604 RADIATION-INDUCED PULMONARY DISEASE

Radiation therapy with 137Cs or 60Co collimated to the appropriate lung sections

Mouse strain, e.g., C 57BL/6 with or without genetic manipulation and fed ad libitum and housed in germ free environment

Histopathological analysis Early RP (0–8 weeks) • type II pneumocyte injury followed by proliferation • inflammation and endothelial cell damage • fibrosis Late RP (after 12 weeks) • persistent free radical damage of endothelium and fibrosis

Analysis of outcomes • respiratory distress • survival

Bronchoalveolar lavage for cytokines, e.g., IL-6, TGF-
, and surfactant proteins

Analysis of gene expression • supra oxide dismutase • TGF-
and other cytokine genes • surfactant genes

Mathematical modeling of the spatial effects of radiation, e.g., dose–volume relationships

Figure 2 Murine radiation pneumonitis (RP) model. Reproduced from Hynes RO (2002) Integrins: bi-directional, allosteric, signaling machines. Cell 110(6): 673–687, with permission from Elsevier.

changes are observed in type II pneumocytes that include reduced intracytoplasmic lamellar bodies followed by the release of surfactant. These changes are followed by a loss of type II cells and the synthesis of less viscous surfactant. A compensatory lamellar body repletion and type II pneumocyte proliferation are observed 4–12 weeks after RT. These changes occur simultaneously with a florid inflammatory cellular infiltrate into the alveolar septae. Inflammatory cells such as mast cells, plasma cells, fibroblasts, macrophages, and polymorphonuclear cells populate the infiltrating cells. Overall, the inflammatory picture usually resolves by 6–9 months although continued type II pneumocyte and arterial smooth muscle proliferation, and free-radical-induced endothelial and type II pneumocyte damage can lead to extensive collagen deposition, that is, radiationinduced lung fibrosis (RIF). The histopathological features of RP and RIF are shown in Figure 2.

Clinical Features Incidence and Prevalence

The observation that not all radiologically evident RP is associated with symptomatic disease leads to higher prevalence and survival statistics when radiological criteria are used in isolation to diagnose RP. In contrast, clinical criteria for diagnosing RP yield lower prevalence rates. The incidence of RP following external-beam RT varies from 1% to 34%. The incidence of RP depends on the type of tumor owing to technical variation in irradiation strategies, but the variability in dosimetry is still the largest factor that determines the risk of developing RP (Table 3). RP is uncommon in breast cancer when RT is restricted to the thoracic wall following mastectomy or after breast-conserving surgery. However, the inclusion of the supraclavicular fossa, axilla, and lung apex increases the incidence of pneumonitis from 1.4% to 3.9%. Incorporation of all

RADIATION-INDUCED PULMONARY DISEASE 605

lymph nodes above the diaphragm, lung apices, and axilla bilaterally in the irradiation field for the treatment of Hodgkin’s lymphoma is associated with less than 5% risk of RP. Compared with RT for lung cancer, smaller areas of lung are irradiated in Hodgkin’s lymphoma. As a result, long-term follow-up of patients treated with RT for lymphoma shows only clinically insignificant pulmonary function changes in diffusing capacity of the lung for carbon monoxide (DLCO) and total lung capacity. In a study of 590 patients with stage 1A-IIIB Hodgkin’s disease, RP was observed in 3% of the patients receiving radiation alone while whole lung irradiation was associated with a 15% risk; the concurrent use of chemotherapy elevated the risk to 11%. A prospective evaluation of 96 patients with NSCLC who were treated with 3D-CRT reported an incidence of grade 1 RP to be 44% at 6 weeks while grade 2 or greater RP was evident in 7.8%. Mean lung dose, V20, V40, and age greater than 60 were predictive of RP. As we discussed previously, estimates of the prevalence of RIF are challenging owing to the variability in the diagnostic criteria, diagnostic methods, and the difficulty in excluding pre-existing lung injury. A prospective study, using CT scans, of 24 patients with advanced NSCLC treated with chemoradiation was undertaken to evaluate the incidence of RIF. Using the European Organization for Research and Treatment of CancerRadiation Therapy Oncology Group Scale, the investigators found the prevalence of RIF grades 1, 2, and 3 to be 14%, 33%, and 19%, respectively. Japanese Clinical Oncology Group trials for lung cancer observed 29 deaths out of 1176 (2.5%) that resulted from RT after a follow-up of four and a half years. Clinical Features of Radiation Pneumonitis

An inconstant relationship has been reported between radiological RILDs and clinically manifest RP. On occasion, clinically evident RP may precede radiographic changes. The spectrum of RP ranges from selflimiting radiological disease or evanescent dyspnea to respiratory failure that culminates in mechanical ventilatory support or rapid demise. RP manifests itself clinically 4–12 weeks after lung irradiation. Dyspnea is the most prominent and frequently the initial manifestation of RP and can be quantified using dyspnea scores (DSs) such as the Abratt or Borg scale. The Abratt scale defines a DS of 1 as dyspnea during brisk ambulation on a flat surface or while walking up an incline. A DS of 2 is dyspnea on ambulating distances greater than 100 m while a DS of 3 denotes failure to ambulate distances greater than 100 m. Patients with a DS of 4 are dyspneic at rest. A prospective

evaluation of lung function among patients irradiated for lung cancer observed a DS of 1 in 50% of the cases, while the remaining cases had a score of 2. At a 6-month follow-up, the DS increased from 1 to 2 in half of the cases. A DS of 3 was observed in approximately 5% of the cases. Cough is the second commonest manifestation of RP. Characteristically, the cough is nonproductive. In some cases, the cough becomes productive with pink frothy sputum but frank hemoptysis is rare in RP. Occasionally, systemic symptoms with ill-defined global malaise and fever occur together with dyspnea. A study of 29 patients with RP revealed the prevalence of symptoms to be dyspnea (93%), cough (58%), and fever (7%). Physical evaluation may be unrevealing. Classic auscultatory findings of RP include rales in the irradiated zone. However, RP has been reported in pulmonary locations outside the zone of irradiation and these findings may thus exist outside the irradiated lung zones. Regional RP may manifest as a focal consolidation. Also, pleural rubs may be evident in cases of radiation-induced pleuritis. A variety of conditions with a similar temporal appearance to RIP may mimic pneumonitis in the peri-irradiation period and include infectious pneumonitis, drug-induced pneumonitis, pulmonary alveolar hemorrhage, and less commonly, residual tumor. Also, underlying lung disease may confound an accurate diagnosis of RP. Clinical Features of Radiation-Induced Lung Fibrosis

RIF is characterized by an incipient pleuroparenchymal interstitial injury that ensues 6–24 months after RT. All patients who have received RT are at risk for developing RIF irrespective of whether they had evidence of RP. The clinical manifestations of RIF are diverse and patients with mild focal fibrosis may be asymptomatic. In contrast, patients with large areas of lung irradiation may present with a DS greater than 2 that can evolve into dyspnea at rest. Late cardiovascular and pulmonary complications accrue from the effects of RT on the vasculature. Advanced cases of RIF have been associated with delayed effects on the vasculature including thrombo-embolic disease, superior vena cava syndrome, and secondary pulmonary hypertension and cor pulmonale. The physical evaluation often reveals varying degrees of dyspnea and cutaneous effects of RT on the thoracic cavity may be evident on visual inspection. For example, the patient may show a deviated trachea and radiation-induced skeletal deformities. Chest excursion may be diminished along with inspiratory rales which is associated with a restrictive

606 RADIATION-INDUCED PULMONARY DISEASE

process. Regional air flow may also be diminished in some settings so PFTs may reveal a mixed obstructive and restrictive picture. Pulmonary hypertension is characterized by a loud pulmonic heart sound, pulmonic and tricuspid regurgitation, elevated jugular venous pressure, engorged liver, and bilateral leg edema. Superimposed complications accruing from fibrosis and the altered lung function and anatomy include bronchiectasis and recurrent pneumonia.

Diagnostic Evaluation Chest X-Rays

The chest X-ray (CXR) is useful in the surveillance of patients following RT. It also allows the speedy diagnosis of RP, in addition to assisting in the establishment of alternative diagnoses. The clinical utility is enhanced by the fact that it is inexpensive and can be deployed rapidly. Classic radiographic features of RP include a diffuse hazy opacification that evolves into a patchy appearance. In some settings, the abnormalities coalesce over time into a uniformly radiodense appearance that mirrors the configuration of the irradiated field. More frequently, these changes are transient but in other cases they progress to frank RIF. RIF may be detected by serial radiographs beginning at 6 months after RT and initial manifestations include patchy and linear opacities. These changes culminate in reticular parenchymal opacities, the progression of which leads to lung-volume reduction. Pleural adhesions to the thoracic wall, pericardium, or the diaphragm can occur. Radiographic appearances vary depending on the method and location of irradiation. The employment of oblique radiation beams in the treatment of breast cancer, for example, is associated with atypical distribution of interstitial opacities. Apical opacities are encountered with the use of supraclavicular portals. Peripheral reticular opacities result from tangential beam portals (as observed in breast cancer) and paramediastinal opacities are encountered following irradiation of a mediastinal tumor. Computer Tomography

CT permits the early detection of subtle parenchymal changes that may not be evident on routine chest radiographs. Moreover, the superior parenchymal definition seen with high-resolution computer tomography (HRCT) enables the earlier detection of consolidation, subtle pulmonary parenchymal changes, and endobronchial occlusion that may herald tumor recrudescence. Characteristic CT appearances of RP include nonhomogeneous opacities and alveolar filling defects. Homogeneous reticular ground glass

opacities are encountered more often in RIF. Ground glass opacities are observed in both RP and RIF and can progress into reticular opacities. Over time there is associated volume loss, and pleural and diaphragmatic thickening. In three-dimensional confocal radiotherapy, lung injury may adopt unusual radiographic configuration which reflects the field and mode of radiation delivery. A study looking at 87 CT scans in 17 patients noted CT abnormalities in 15 of the 17 cases within 16 weeks of RT. In three out of the 15 cases, there were no changes indicative of RP evident at 16 weeks. In three cases, CXR changes became apparent much later than 1, 5, and 8 weeks respectively. In nine cases, the radiological features of lung injury were detected simultaneously by either CT or CXR. This same study reported air bronchograms (25%), loss of lung volume (15%), and pleural thickening as changes complicating RT. Adjuncts to CT scans have included gallium scintigraphy but its value is limited by low specificity and sensitivity. Bronchoscopy

The use of bronchoscopy in the evaluation of RP is limited. Most patients receiving RT are also immunocompromised; consequently, the emergence of parenchymal opacities on CXR necessitates bronchoscopy to exclude alternative etiologies such as infections, or diffuse alveolar damage. In RP, inspection of the airways may show hyperemia and mucosal engorgement. Bronchoalveolar lavage fluid (BALF) from irradiated patients compared to that of nonirradiated patients with lung cancer showed that total lymphocyte and eosinophil counts were higher in irradiated lungs. Moreover, human leukocyte antigen DR (HLADR)-positive CD4 þ T cells and HLADR-positive CD8 þ T cells as well as intercellular adhesion molecule (ICAM)-1-positive T cells are markedly increased. An interesting observation from this study was that the emergence of ICAM-1 positive cells correlated with the interval between initiation of RT and emergence of RP. However, other investigators have found no difference in the lymphocytic content between irradiated breast cancer cases that develop RP and those that do not. Pulmonary Function Tests

PFTs in lung cancer patients treated with RT and followed for a mean of 38 months noted that RT caused a decline of forced vital capacity (FVC) (89% of subjects), forced expiratory volume in 1 s (FEV1)(89%), and DLCO (90%). These variables returned to baseline at 1 year (105%, 100%, and 90% of subjects, respectively). Nevertheless, the study further observed that a progressive decline

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followed the initial improvement phase. The study was however limited by its size. De Jaeger and coworkers evaluated the effect of RT on pulmonary function post-RT in 82 cases of NSCLC and correlated it to radiation dose, tumor regression, and changes in lung perfusion as measured by single photon emission CT (SPECT) lung perfusion scan. They estimated the post-RT perfusion using dose distribution and a dose–effect relation for loco-regional lung perfusion to calculate a predicted perfusion reduction (PPR). Reperfusion was defined as the difference between the baseline perfusion and the PPR. Tumor regression was associated with an improvement in FEV1 but a decline in DLCO, while the PPR did not correlate with improvement of FEV1 and FVC. Overall, RT causes restrictive ventilatory defects with diminution in FVC and FEV1 due to reduced compliance caused by fibrosis. The changes in FVC and FEV1 are variable and rather limited in their utility to follow the effects of RT. In contrast, the assessment of the alveolar functional integrity by DLCO is more sensitive for detecting RILDs. Serological Markers

No single serologic marker is a consistent predictor of risk for development of RP. Consequently, a search for a reliable marker for RP is ongoing. Markers that have been described as showing a positive correlation with RP include TGF-b, interleukin (IL)-1-a and IL-6. The clinical significance of these findings has, however, been questioned by others. The cytokeratin 19

fragment, a marker of epithelial cell damage, mucinlike high molecular weight glycoprotein (KL-6/MUC1), intercellular adhesion molecule-1 (ICAM-1), type III procollagen N-terminal peptide (P-III-P), laminin P1, and surfactant protein A and D have been shown to significantly correlate with RF. However, the clinical utility of these markers remains to be validated. Pathogenesis

The leading theories of the mechanism of RILDs invoke the role of free radicals and cytokines. Figure 3 shows a schematic representation of the putative mechanisms of RP and RIF. Free Radicals

Radiation causes structural modification of DNA, lipids, and proteins through peroxidation, aldehyde reactions, reactive oxygen species, and activates cytokine cascades that mediate processes that impair the structural integrity of pneumocytes and endothelial cells. Also, Cottier and co-workers showed that desferroxamine levels are elevated in patients who are likely to develop RP following RT. One group of investigators has observed that smokers undergoing RT for esophageal and breast cancer are less prone to RP possibly because glutathione (GSH) levels are known to be elevated in smokers compared to nonsmokers. Since GSH functions as a scavenger for singlet oxygen and superoxides, it may abrogate the proinflammatory effects of RT (76). Hashimura and co-workers have observed that LTC4 and LTD4 as

Radiation

DNA damage

Free radicals Reactive oxygen species Hydroxyperoxides

Inflammatory mediators

Radiation pneumonitis

Endogenous antioxidant mechanisms counteract the deleterious effects of free radicals Figure 3 Hypothetical mechanisms of radiation-induced pneumonitis.

Protein and lipid peroxidation

608 RADIATION-INDUCED PULMONARY DISEASE

well as glutathione peroxidase are acutely upregulated in the irradiated mouse lung and remain persistently elevated 24 h postradiation and that the second generation antihistamine, azelastine, and coenzyme – Q, both free radical scavengers ameliorated these changes. Further evidence supporting the theory that antioxidants protect against RP has been provided by experiments that demonstrate that the overexpression of manganese superoxide dismutase (MnSOD) in a transgenic murine model or the exogenous administration of the iNOS inhibitor L-NAME ameliorates the deleterious effects of RT. Cytokines

Both in vitro and clinical observations of the irradiated lung support the immunological basis of RILDs. As reviewed above, RT induces the generation of reactive oxygen species that is thought to initiate and propagate the pathological manifestations of RP. It has been shown that irradiated mice upregulate the chemokine macrophage chemoattractant protein 1 (CCL2), macrophage-derived chemokines (CCL22), thymus-derived and activation-derived chemokines (CCL17), and the CCR-4 receptors on alveolar lymphocytes and macrophages (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)) (Figure 4). Both CCL22 and CCL17 are the natural ligands for CCR-4 and assist in the recruitment of proinflammatory cells and also regulate the proportion of Th2/Th1 lymphocytes. An analysis of the BAL from irradiated lungs reveals elevated TGF-b, IL-6, and IL-1 whose levels appear to correlate with the extent of RP (see Interleukins: IL-1 and IL-18; IL-6). TGF-b plays a pivotal role in the pathogenesis of other fibrotic lung diseases such as idiopathic pulmonary fibrosis where TGF-b mediates the differentiation of lung fibroblasts into myofibroblasts (see Transforming Growth Factor Beta (TGF-b) Family of Molecules. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis) (Figure 5). The myofibroblast elaborates cellular matrix, for example, elastin and collagen, and produces TGF-b. IL-6 is pleiotropic cytokine that regulates multiple pro-inflammatory cytokines and is crucial for lymphocyte development. The role of TGF-b in RP is supported by the observation that the administration of a recombinant TGF-b1 type II receptor (TGF-bRII) gene, which inhibits TGF-b activation, in a mouse model of RP attenuates histological evidence of radiation-induced lung injury. The administration of halofuginone, an inhibitor of TGFb, was shown to reduce the colocalization of TGF-b with the tight junction protein Zo-1 and also reduces radiation-induced lung injury. This is interesting because the localization of TGF-b to Zo-1 is essential

Figure 4 Acute radiation pneumonitis showing alveolar injury with edematous thickened alveolar septa, atypical alveolar lining cells, and loose fibroblastic foci (H&E 200
). Permission of Gerald Berry, M.D., Department of Pathology, Stanford University Medical School.

Figure 5 Chronic radiation pneumonitis characterized by mixed interstitial fibrosis and fibroblastic foci, patchy mononuclear inflammatory cell pneumonitis, foamy macrophages within distorted airway lumens, and fibrointimal thickening of muscular pulmonary arteries (H&E 200
). Permission of Gerald Berry, M.D., Department of Pathology, Stanford University Medical School.

for the epithelial to mesenchymal transition suggesting that TGF-b-mediated epithelial–mesenchymal transition may play a role in radiation-induced lung injury. In summary, radiation induces chemokines and cytokines which promote the expression of proinflammatory and profibrotic mediators in RP.

Management and Current Therapy Steroids

Steroids are the mainstay for the treatment of RP (see Corticosteroids: Therapy). Up to 80% of RP cases have a dramatic clinical and radiological response to steroids. Supportive evidence for the use of steroids

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accrues from clinical experience and in vitro data. The employment of steroids in the treatment of experimental mouse models of RP demonstrates that steroids delay the emergence of RP, attenuate the endothelial damage, and reduce the inflammatory cellular infiltrate. Notably, the effects of steroids on RP do not affect the emergence of RIF but the discontinuation of steroids in mice with experimental RP leads to accelerated mortality. Human data supporting the use of steroids are lacking. On the whole, the discontinuation of steroids in human subjects leads to worsening of RP but there is no data to support the prophylactic use of corticosteroids to prevent the disease. The duration of steroid therapy is subjective but is on the order of several weeks accompanied by a slow taper. There are also sporadic reports showing cyclosporine is of benefit in steroid-refractory radiation pneumonitis. Angiotensin converting enzyme (ACE) inhibition has been suggested as a protective strategy against RP based on limited animal data. However, there is no sufficient human data to support the use of ACE inhibitors for protection against RP. In summary, these observations support the use of steroids in clinically symptomatic RP, although the empiric use of steroids in asymptomatic patients’ post-RT is not recommended. Novel Therapeutic Targets

Ongoing research to identify novel methods to mitigate the adverse effects of RT focuses on reducing total radiation doses and blocking molecular mediators of RP. Technological innovations have dominated the impetus to reduce the unintended lung injury. However, there is promise that targeting RT specifically to the tumor may be improved with DNAbased technology to activate proapoptotic genes such as tumor necrosis factor-related apoptosis-inducing ligand (Apo-2L) (TRAIL), which preferentially activates apoptotic pathways in tumor versus normal cells. Additional strategies are focusing on limiting damage from RT-induced free radicals by increasing scavenger activity through activation of manganese superoxide dismutase or administration of antioxidants (see Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants). Other potential strategies include the antagonism of mediators of inflammation such as blocking TGF-b, chemokines, and IL-6. See also: Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12). Corticosteroids: Therapy. Endothelial Cells and Endothelium. Interleukins: IL-1 and IL-18; IL-6.

Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Lung Imaging. Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants. Pulmonary Fibrosis. Radiotherapy. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Abratt RP, Morgan GW, Silvestri G, and Willcox P (2004) Pulmonary complications of radiation therapy. Clinics in Chest Medicine 25(1): 167–177. Chen Y, Williams J, Ding I, et al. (2002) Radiation pneumonitis and early circulatory cytokine markers. Seminars in Radiation Oncology 12(supplement 1): 26–33. Epperly M, Bray J, Kraeger S, et al. (1998) Prevention of late effects of irradiation lung damage by manganese superoxide dismutase gene therapy. Gene Therapy 5: 196–208. Garipagaoglu M, Munley MT, Hollis D, et al. (1999) The effect of patient-specific factors on radiation-induced regional lung injury. International Journal of Radiation Oncology, Biology, Physics 45(2): 331–338. Geara FB, Komaki R, Tucker SL, Travis EL, and Cox JD (1998) Factors influencing the development of lung fibrosis after chemoradiation for small cell carcinoma of the lung: evidence for inherent interindividual variation. International Journal of Radiation Oncology, Biology, Physics 41(2): 279–286. Groover TA, Christie AC, and Merrit EA (1922) Observations on the use of the copper filter in the treatment of deep-seated malignancies. Southern Medical Journal 15: 440–444. Gross NJ (1977) Pulmonary effects of radiation therapy. Annals of Internal Medicine 86: 81–92. Hynes RO (2002) Integrins: bi-directional, allosteric, signaling machines. Cell 110(6): 673–687. Kong FM, Anscher MS, Sporn TA, et al. (2001) Loss of heterozygosity at the mannose 6-phosphate insulin-like growth factor 2 receptor (M6P/IGF2R) locus predisposes patients to radiation-induced lung injury. International Journal of Radiation Oncology, Biology, Physics 4991: 35–41. Movsas B, Raffin TA, Epstein AH, and Link CJ Jr (1997) Pulmonary radiation injury. Chest 111: 1061–1076. Muraoka T, Bandoh S, Fujita J, et al. (2002) Corticosteroid refractory radiation pneumonitis that remarkably responded to cyclosporin A. Internal Medicine 41(9): 730–733. Park KJ, Chung JY, Chun MS, and Suh JH (2000) Radiationinduced lung disease and the impact of radiation methods on imaging features. Radiographics 20(1): 83–98. Rancati T, Ceresoli GL, Gagliardi G, Schipani S, and Cattaneo GM (2003) Factors predicting radiation pneumonitis in lung cancer patients: a retrospective study. Radiotherapy and Oncology 67(3): 275–283. Rosiello RA and Merrill WW (1990) Radiation-induced lung injury. Clinics in Chest Medicine 11(1): 65–71. Tsujino K, Hirota S, Endo M, et al. (2003) Predictive value of dose–volume histogram parameters for predicting radiation pneumonitis after concurrent chemoradiation for lung cancer. International Journal of Radiation Oncology, Biology, Physics 55(1): 110–115. Wang LW, Fu XL, Clough R, et al. (2000) Can angiotensin-converting enzyme inhibitors protect against symptomatic radiation pneumonitis? Radiation Research 153(4): 405–410.

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RADIOTHERAPY P Van Houtte, Institut Jules Bordet, Brussels, Belgium & 2006 Elsevier Ltd. All rights reserved.

Abstract Radiotherapy uses the properties of ionizing radiation to treat mainly cancers. It was introduced soon after the discovery of Xrays by Roentgen and radium by Marie Curie at the end of the nineteenth century. Over the years, the technique has evolved due to major technical developments, due to better machines, powerful computers, and a better knowledge of the interaction between radiation and matter. Nowadays, it remains a major component in the treatment of lung cancer and is a major part in the multidisciplinary management of this disease.

History and General Principles Very soon after their discovery, X-rays (discovered by Roentgen in 1895) and radium (discovered by Marie Curie in 1898) were used to treat cancer; the first report was published as early as 1898. Nowadays, the practice of radiotherapy requires a knowledge of the disease treated, the physics of the radiation, and the effect of radiation on normal tissues and tumors. The progress made during the last decades in radiation oncology results from major improvements in technical equipment and from a better understanding of the interaction between radiation and tumors which have led to the development of new treatment modalities. The type of radiation used in clinical practice is called ionizing radiation. During absorption in tissues, such radiation produces the ejection of an orbital electron, creating an ion. This will be the starting point of a cascade of events leading ultimately to the destruction of the cells either by inducing apoptosis or by causing a loss of their proliferative capacity (the cells will die during a subsequent intermitotic phase). This explains the delay between the radiation and the later clinical signs and symptoms occurring days, weeks, or months after the treatment. Different types of radiation are available: electromagnetic waves characterized by their wavelength or frequency (their energy is directly proportional to the frequency and inversely proportional to the wavelength) and particle beams, mainly electrons (protons and carbon ions are only used in a few places worldwide). The following considerations apply to radiotherapy practice: the dose, the volume, and the time effects. There is a relation between the total dose and the volume. This applies to both the tumor and

normal tissues. The radiation dose is necessary to control an increase of a tumor with respect to its size, volume, or the number of cells present. To control with a high probability a 3 cm epithelial tumor, doses in excess of 65 Gy are necessary. Larger tumors need higher total doses. In contrast, normal tissue tolerance is inversely proportional to the volume irradiated. Timing is another issue: increasing the duration of the treatment will lead to a loss of efficacy due to the risk of inducing an active repopulation within the tumor: several studies of lung cancers have demonstrated a negative impact of treatment breaks or prolonging the duration of the treatment. Before 1951, most treatment units available were X-ray machines producing only photon beams with limited penetration; the orthovoltage units operated in the range of 200 to 300 kVp. In the 1950s, the development of supervoltage units (60Co and 137Cs teletherapy machines, betatron, and later linear accelerators) completely modified the radiation practice by offering machines more efficient both in terms of beam energy available and in accuracy. The linear accelerator uses high-frequency electromagnetic waves to accelerate electrons to high energy through a microwave accelerator structure; it can produce photon or electron beams of different energies. The introduction of computed treatment planning based on data generated by computed tomography (CT) has dramatically changed the practice since the end of the 1970s, allowing a dose distribution to be established first in two and later in three dimensions: there is an optimization process to deliver the doses to the tumor while protecting the normal tissue. The modern radiation treatment is a complex multistep procedure requiring the cooperation and skill of different professionals (physicians involved in the different imaging procedures, nurses, technologists, physicists, dosimetrists). The initial step requires a precise clinical evaluation of the patient and the extent and nature of the disease. The clinical evaluation is based on all sources of information available: clinical examination, imaging procedures such as CT, magnetic resonance imaging (MRI), and radiographs. This must provide a precise staging of the tumor and sufficient information to take the decision to treat or not the patient. After the treatment decision, the different target volumes must be defined: the palpable or visible extent of the tumor represents the gross tumor volume. A margin has to be added around this volume to include direct

RADIOTHERAPY

subclinical spread representing the clinical target volume. Additional volume may be added to cover the possible regional lymph nodes. For radiation treatment planning, margins have to be added to the clinical target volume to take into account the variation in size and position of tissues during treatment (patient movement, breathing during treatment) and the possible variation in the daily set-up; this represents the planning target volume. Furthermore, the radiation oncologist must define the different critical structures. The next step is the treatment planning. The goal is to individualize the treatment to the patient needs and to obtain ideally a complete description of the radiation (Figures 1 and 2). Another important step is to provide a proper immobilization which can be made with foam, plastic, or plaster and a marking technique to ensure the daily reproducibility of the treatment.

611

The daily radiation treatment aims to deliver the radiation in the condition defined by the treatment planning and to reproduce it several times: this includes treatment parameters (field sizes, beam angles, beam energy, wedge filters), the patient’s position, and the quality of the radiotherapy unit. Several tools are useful to reduce the risk of errors and improve the accuracy of the treatment: a routine machine calibration, a system verifying the individual parameters for each patient, a portal imaging system giving online the position of the beam inside the patient. Nowadays, intensity modulated radiation therapy is available: modern linear accelerators are equipped with multileaf collimators which allow shaping of the beam while the leaf may be modified during the radiation leading to a difference in radiation intensity. The last technological development is fourdimensional radiation taking into account the time factor: for lung cancer, there is always a possible displacement of the tumor due to the patient’s breathing or heart movements; the gating technique allows irradiation during a specific phase of the breathing cycle while the tracking technique follows the displacement of the tumor.

Uses in Respiratory Medicine

Figure 1 Dose distribution for an inoperable stage I non-small cell lung cancer treated with a fields technique delivering 65 Gy to the tumor while limiting the dose to the lungs, the spinal cord, and the heart.

Indications for radiotherapy for lung cancer depend on several factors related to the tumor (extension, pathology, symptoms) and the host (the patient’s performance status and other diseases, and his attitude). Several textbooks are available and only a short summary will be presented on the principal indications as well as their limits and new approaches. Furthermore, management of lung cancer is more and more a multidisciplinary effort. Non-Small Cell Lung Cancer

Figure 2 PET CT of right lung tumor with partial atelectasia and lymph node involvement. The PET image allowed to modify the radiation field to include only the area of FDG-uptake and differentiating the tumor from the simple atelectasia.

Surgery remains the cornerstone in terms of cure for early lung cancer, but only fewer than one-third of patients are candidates for a surgical resection, usually presenting with state I to IIIa disease. Patients with more extensive but still localized disease within the chest (some stage IIIa and mainly stage IIIb) and those not candidates for a surgical resection due to medical contraindication are often treated with radiation. What have we learned over the last three decades? Local control and survival is closely related not only to the tumor extent and the patient’s status (weight loss, performance status) but also on the radiation treatment (total dose, fractionation, and quality). By increasing the dose from 40 to 60–65 Gy, doses used in most trials, the local control and survival was improved but the results were still very dismal with

612 RADIOTHERAPY

Figure 3 Simulation film for a left lung tumor with positive lymph nodes within the aortopulmonary arc and with the position of the different leafs from a multileaf collimator (a) and a portal image taken before treatment to check the accuracy of the beam position before treatment (b).

figures as low as 20% for local control. This is easily explained by the relation between tumor size and dose: most tumors are larger than 3 cm requiring higher total doses. Several approaches were and are under investigation: combined modalities, modifying the fractionation, and increasing the physical dose using the three- or four-dimensional approaches (Figure 3). In the 1980s, it was strongly believed that the best way of combining drugs and radiation was to start with the drugs: amongst the theoretical advantages, induction chemotherapy might also improve the results of chest irradiation due to a better oxygenation of the tumor or to a possible protection of normal tissue in case of response. Indeed, a trial observed an improvement in long-term survival after two cycles of cisplatine and vinblastine followed by chest irradiation (60 Gy with daily 2 Gy fractions). This was confirmed by additional trials and a meta-analysis. Nevertheless, this survival benefit was mainly due to a reduction in distant metastases and not to a better locoregional control. A concurrent approach is becoming more popular. The main concern is certainly an increase in acute hematological and nonhematological toxicity especially esophagitis but it is possible to take advantage of the radiosensitizing properties of drugs. Several reported trials all point in the same direction, with an improved 2 year survival rate, around 35% compared to less than 30% for the sequential approach. Fewer local relapses were observed. The problem is certainly the increase in acute esophagitis, a temporary side effect which leads very rarely to permanent esophageal stenosis. There are also many unsolved questions: the best sequence (induction followed by a concurrent approach, a concurrent approach followed by an adjuvant schedule), the place of a

maintenance chemotherapy, the drugs to be associated with cisplatine or carboplatine, the drug administration (single or multiple administration), and of course the radiation technique itself. In an attempt to improve the efficacy of radiation, classical or accelerated hyperfractionated schedules have been developed to increase the biological dose and to overcome the issue of repopulation. Indeed, for epithelial tumors, there is a risk of inducing a tumor proliferation after 3 to 4 weeks leading to a loss of efficacy. Furthermore, the tolerance of normal tissue is directly influenced by the dose delivered by each fraction which is not the case for the tumor: bigger fractions increase the risk of late effects. The CHART schedule (54 Gy delivered with three daily fractions of 1.5 Gy over 12 consecutive days including the weekend) was clearly superior to 60 Gy in 6 weeks with daily 2 Gy per fraction; the survival benefit was due to an improvement in local control. Another approach is certainly to use the modern tools available which allow to escalate the dose above 70 Gy by reducing the irradiated volume; studies are on going. Combining radiation or chemoradiotherapy with surgery is another alternative. Preoperative radiotherapy is currently only advocated for superior sulcus tumor. Postoperative radiotherapy has no place in case of a complete resection with clear margins except for some selected stage IIIa (nodal extracapsular spread) or stage IIIb. The place of surgery for a clinical proved stage IIIa disease (with positive mediastinal lymph node) remains controversial after an induction program: survival is in the same range for patients operated after chemoradiotherapy or not. Small endobronchial lesions may be treated with endobronchial brachytherapy. The technique requires the introduction of a thin flexible catheter during a

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flexible fiberbronchoscopy. Then the bronchial volume to be treated is determined as well as the dose required. After the dose distribution study, the treatment is performed by inserting the radiation source in the right configuration using an afterloading machine. The radiation takes usually 10–20 min while the whole procedure takes less than 2 h. Treatment is well tolerated and widely applicable even on outpatient basis. Small Cell Lung Cancer

This disease is characterized by a rapid proliferation and early dissemination. Chemotherapy is the basis of the treatment. In the curative treatment of small cell lung cancer, there are two indications for radiation: chest radiotherapy and prophylactic cranial irradiation. For limited disease, locoregional failures after only chemotherapy were very common and chest irradiation was added to the drugs. The classical approach includes the delivery of cisplatine etoposide concurrently with chest irradiation using either one or two fractions per day. Early administration, during the first cycles of chemotherapy, is usually preferable rather than delaying the radiation to the end of the chemotherapy. This can lead to a 5 year survival of around 20% to 25% in selected groups. Nowadays, studies are evaluating new drugs as well as higher total radiation dose similar to the one used for nonsmall cell lung cancer due to the still high rate of local failure (between 30% and 50%). Brain relapse is a common pattern of failure with a figure as high as 50% at 2 years. Prophylactic cranial irradiation has been showed to decrease the relapse rate by a factor of 2 with dose between 20 and 30 Gy. This has led to some improvement in survival for patients in complete remission. The process is nowadays advocated for patients with limited disease in complete remission but should not be given concurrently with chemotherapy to avoid the risk of toxicity. Palliative Radiotherapy

Radiation is often used to relieve the patients of symptoms due to the primary tumors (superior vena cava syndrome, chest pain, hemoptysis) or due to distant metastases (spinal cord compression, bone or brain metastases). Usually, the treatment delivers a few fractions with larger doses with the aim of only relieving the symptoms, which is achieved in more than two-thirds of patients. Brain metastases require special care: in some selected cases, radiosurgery may be advocated for single or a few small lesions. If the primary lesion may be or is under control and there is no other distant metastasis, this treatment may even

Figure 4 The ciberknife allowing to treat in stereotactic condition.

be curative with 5 year survival (Figure 4). Endobronchial brachytherapy may also be an alternative in case of endobronchial disease or compression. See also: Bronchoscopy, General and Interventional. Tumors, Malignant: Overview; Bronchogenic Carcinoma; Chemotherapeutic Agents.

Further Reading Arriagada R, Le Pe´choux C, and Pignon JP (2003) Resected nonsmall cell lung cancer: need for adjuvant lymph node treatment? From hope to reality. Lung Cancer 42: S57–S64. Auperin A, Arriagada R, Pignon JP, et al. (1999) Prophylactic cranial irradiation for patients with small-cell lung cancer in complete remission. New England Journal of Medicine 341: 476–484. Bradley J, Graham MV, Winter K, et al. (2005) Toxicity and outcome results RTOG 9311: a phase I-II dose-escalation study using three-dimensional conformal radiotherapy in patients with inoperable non-small-cell lung carcinoma. International Journal of Radiation Oncology, Biology, Physics 61: 318–328. Chang JY, Liu HH, and Komaki R (2005) Intensity modulated radiation therapy and proton radiotherapy for non-small cell lung cancer. Current Oncology Reports 7: 255–259. Farray D, Mirkovic N, and Albain K (2005) Multimodality therapy for stage III non-small cell lung cancer. Journal of Clinical Oncology 23: 3257–3269.

614 REACTIVE AIRWAYS DYSFUNCTION SYNDROME Grills IS, Yan D, Martinez AA, et al. (2003) Potential for reduced toxicity and dose escalation in the treatment of inoperable nonsmall-cell lung cancer: a comparison of intensity-modulated radiation therapy (IMRT) 3D conformal radiation, and elective nodal irradiation. International Journal of Radiation Oncology, Biology, Physics 57: 875–890. Hazard LJ and Sause WT (2004) Combined modality therapy for unresectable non-small cell lung cancer. In: Sculier JP and Fry WA (eds.) Malignant Tumors of the Lung, pp. 237–252. New York: Springer. Lee SW, Choi EK, Park HJ, et al. (2003) Stereotactic body frame based fractionated radiosurgery on consecutive days for primary or metastic tumors in the lung. Lung Ca 40: 309–315. Mornex F (2004) Non-small cell lung cancer. Seminars in Radiation Oncology 14(4). Saunders M, Dische S, Barrett A, et al. (1999) Continuous hyperfractionated, accelerated radiotherapy (CHART) versus conventional radiotherapy in non-small cell lung cancer: mature data from the randomized multicentre trial. Radiotherapy and Oncology 52: 137–148.

Steel G (2002) Basic Clinical Radiobiology. London: Arnold. Taulelle M, Vincent P, Chauvet B, Garcia R, and Reboul F (1999) Endobronchial brachytherapy. In: Brambilla C and Brambilla E (eds.) Lung Tumors: Fundamental Biology and Clinical Management, pp. 537–552. New York: Dekker. Van Houtte P (2001) The role of radiotherapy and the value of combined treatment in lung cancer. European Journal of Cancer 37(supplement 7): 91–98. Van Houtte P and Mornex F (2001) Radiotherapy of nonsmall cell and small cell lung cancer. In: Spiro SG (ed.) Lung Cancer, pp. 190–218. Hudders Field: European Respiratory Society. Van Houtte P, Roelandts M, Cappello M, and Mornex F (2004) Small cell lung cancer: limited disease. In: Sculier JP and Fry WA (eds.) Malignant Tumors of the Lung, pp. 277–286. New York: Springer. Van Limbergen E and Po¨tter R (2002) Bronchus cancer. In: Gerbaulet A, Po¨tter R, Mazeron JJ, Meertens H, and Van Limbergen E (eds.) The GEC ESTRO Handbook of Brachytherapy, pp. 545–560. Leuven: ESTRO.

REACTIVE AIRWAYS DYSFUNCTION SYNDROME S M Tarlo, University of Toronto, Toronto, ON, Canada C A Redlich, Yale University, New Haven, CT, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Reactive airways dysfunction syndrome (RADS) refers to asthma that begins after exposure to a very high level of respiratory irritant (usually accidental). The initial criteria included the onset of asthma symptoms within 24 h of a single such exposure, absence of preceding lung disease, objective evidence of asthma (spirometry showing a significant bronchodilator response, or methacholine or histamine challenge showing significant airway hyperresponsiveness), and continuation of asthma symptoms for at least three months. The criteria for this syndrome have been variably expanded to include multiple irritant exposures, less massive levels of exposure, a more delayed onset of symptoms after exposure, or a shorter duration of symptoms, termed ‘irritant-induced asthma’. RADS is considered a subset of this more inclusive, but less well defined entity. The diagnosis of RADS is largely circumstantial, relying on the history of exposure, absence of documented preceding airway disease, and objective documentation of asthma. Airway pathology does not clearly distinguish this syndrome from other asthma. Therapy is the same as for other types of asthma including corticosteroids and bronchodilators, as well as evaluation of workplace exposures. The outcome varies: symptoms and pulmonary function changes can clear within months but may persist for years.

Introduction The term reactive airways dysfunction syndrome (RADS) was initially used by Brooks and colleagues in 1985 to describe asthma that begins after exposure to a very high level of respiratory irritant (usually

accidental). The initial criteria (Table 1) included the onset of asthma symptoms within 24 h of such exposure, absence of preceding lung disease, objective evidence of asthma (spirometry showing a significant bronchodilator response, or methacholine or histamine challenge showing significant airway hyperresponsiveness), and continuation of asthma symptoms for at least three months. Subsequently, the criteria for this syndrome have been expanded to include multiple and less extreme exposures, a more delayed onset of symptoms after exposure, or a shorter Table 1 Criteria for diagnosis of RADSa *

* *

*

Onset of asthma symptoms within 24 h of exposure to a high level of a respiratory irritant agent (usually severe enough to require medical attention at the time) Persistence of symptoms for at least 12 weeks Objective evidence of asthma: airway hyperresponsiveness on histamine or methacholine challenge, or airflow limitation with significant bronchodilator responsiveness (at least 12% increase in FEV1) No previously documented evidence of asthma or other chronic lung disease

a Criteria for irritant-induced asthma are less stringent and may include those with symptom onset up to 7 days or longer after the acute exposure, duration of symptoms less than 12 weeks after onset, more than one single massive exposure, or less than a massive exposure. FEV, forced expiratory volume. Adapted from Tarlo SM and Chan-Yeung M (2005) Occupational asthma. In: Rosenstock L, Cullen MR, Redlich CA, and Brodkin D (eds.) Clinical Occupational and Environmental Medicine, 2nd edn. Philadelphia, PA: Saunders, with permission from Elsevier.

REACTIVE AIRWAYS DYSFUNCTION SYNDROME 615 Table 2 Some causes of irritant-induced occupational asthma Agents (with high-level exposures)

Exposure example

Volatile di-isocyanates, e.g., TDI

Spills in polurethane foam manufacture and other diisocyanate manufacturing/ using companies Paper mills Hospital workers, metal platers Accidental mixing of bleach and ammonia while cleaning Industrial settings, firefighters World Trade Center collapse with exposed firefighters, other aid workers, and building occupants Silo gas in farm workers Welders Police officers Paint sprayers Pesticide use

Chlorine spills (puffs) Acid spills, e.g., acetic acid Hypochlorite fumes Chemical fires Calcium oxide

Nitrogen oxides Welding fumes Tear gas Spray paint Metam sodium

Adapted from Tarlo SM and Chan-Yeung M (2005) Occupational asthma. In: Rosenstock L, Cullen MR, Redlich CA, and Brodkin D (eds.) Clinical Occupational and Environmental Medicine, 2nd edn. Philadelphia, PA: Saunders, with permission from Elsevier.

duration of symptoms, termed ‘irritant-induced asthma’. Irritant-induced asthma has been used to include both RADS and patients who fit this broader more inclusive description. Since the diagnosis of RADS is largely circumstantial (relying on the exposure assessment, absence of documented preceding airway disease, and objective documentation of asthma), the certainty of diagnosis is greatest when the strictest criteria are used. Irritant-induced asthma has also been termed ‘occupational asthma without latency’ or ‘nonsensitizer asthma’ to differentiate it from immune-mediated or sensitizer-induced occupational asthma. Irritant-induced asthma, or the more narrowly defined RADS, has been reported following high-level exposure to various respiratory irritant gases, fumes, and chemicals including sulfur dioxide, di-isocyanates perchloroethylene, chlorine, phosphoric acid, welding fumes diethylaminoethanol, nitrogen tetroxide, and alkaline dust exposure from the World Trade Center collapse (Table 2).

Epidemiology The incidence of RADS or irritant-induced asthma in the general population is unknown. In different studies, in groups with accidental exposure to a high concentration of a respiratory irritant agent, irritant-induced asthma has been reported in variable percentages of those exposed (1–20%), likely related to the diagnostic criteria used, extent and nature of the exposure, as well as host factors. Among those who develop RADS-like

symptoms following a high acute exposure, a variable but substantial number (up to 60%) can have persistent airway hyperresponsiveness. Limited compensation or clinical data, which is influenced by referral, reporting and other factors, have found a variable percentage of occupational asthma cases attributed to irritants (o5% to B25%), with a lower prevalence if more restrictive RADS criteria are used. Of great interest is the possibility that chronic or recurrent exposure to lower concentrations of respiratory irritants might also lead to asthma, as this type of exposure is much more common than acute high exposures. It is accepted that such exposures can exacerbate asthma in those with pre-existing asthma. Whether such exposures can cause new-onset asthma remains unclear. However, increasingly, epidemiologic associations are reported between chronic exposure to respiratory irritants and increased risk of asthma in several settings including textile workers, cleaners, janitors, and pulp workers exposed to chlorine or ozone. Risk factors for RADS or irritant-induced asthma have not been well defined, although the dose of exposure is clearly important. For example, a doseresponse relationship was found in hospital laboratory workers accidentally exposed to acetic acid, the greatest prevalence of airway hyperreactivity and symptoms being in the highest exposure group. Host susceptibility factors are also involved, as the syndrome occurs in only a minority of those with similar exposure. Several reports have included a large proportion of smokers and atopics, which may be risk factors. Although the identified presence of underlying airway disease is an exclusion criterion for RADS, it is possible that those with RADS and a previous smoking or atopic history may have had underlying subclinical airway disease, or there may be unidentified genetic or other predisposing factors, perhaps analogous to the genetic predisposition demonstrated to endotoxin. The airway responsiveness to moderate or relatively low concentrations of inhaled irritants (as illustrated by challenges with methacholine, histamine, mannitol, and other agents) is greater in asthmatics, atopic individuals without asthma, those with chronic obstructive lung disease, and in normal subjects following a respiratory viral infection. However, the possible role of these as predisposing factors for RADS or irritant-induced asthma is unknown. While it is clear clinically that respiratory irritant exposure will aggravate asthma, there is clear information as to the effect in asthmatics of exposures such as those that would cause RADS in nonasthmatics. There is no information to indicate whether the airway response under the same exposure conditions would be the same or greater, or whether the concurrent use

616 REACTIVE AIRWAYS DYSFUNCTION SYNDROME

of medications such as inhaled corticosteroids by those with previous asthma may ameliorate some of the effects. It may be expected that those with asthma might have earlier symptoms in an accidental exposure area and might therefore leave the area sooner; no study has clearly addressed this.

Pathogenesis and Pathology The underlying mechanisms of RADS are not well understood. The airway injury and inflammatory response following an acute irritant respiratory exposure most commonly gets resolved. However, in some cases the airway injury may result in persistent changes such as airway remodeling and chronic inflammation as with other asthma. It has been postulated that inhalation of a high concentration of an irritant substance injures the bronchial epithelium, with subsequent sloughing of the epithelium, neurogenic inflammation, airway edema, release of inflammatory mediators, growth factors and chemokines, recruitment and/or activation of mast and other inflammatory cells, and myofibroblast activation, leading to chronic airway inflammation, subepithelial fibrosis, airway hyperresponsiveness, and asthma symptoms. The mechanisms, pathways, and responses likely resemble those involved in the pathogenesis of chronic asthma due to other causes, i.e., epithelial injury, activation of the epithelial-mesenchymal trophic unit, airway remodeling, and chronic inflammation, as described by Holgate and others (Figure 1). Airway pathology does not clearly distinguish RADS from other causes of asthma. Reported biopsies show denuded epithelium, airway inflammation, and subepithelial fibrosis. In some reports, subepithelial fibrosis is more prominent with less eosinophilia and inflammatory infiltrate than in atopic asthma. However, findings have been limited

to small numbers of patients at variable time periods following the acute exposure and have not been consistent. Therefore, there is only limited information about the pathology of RADS to date.

Clinical Features and Diagnosis The typical RADS presentation is the development of new asthmatic symptoms such as cough, wheezing, and shortness of breath following acute exposure to a high level of irritant accidentally released in the workplace, which persist for at least three months after the initial exposure. Objective support for asthma is documented by a significant bronchodilator response on spirometry or a positive histamine or methacholine challenge. Once RADS exists, nonspecific environmental stimuli such as cigarette smoke, household cleaners, diesel exhaust, or cold air can precipitate symptoms. Acute irritant exposures capable of causing RADS can exacerbate preexisting asthma, and are also associated with several nonasthmatic disorders that can have overlapping symptoms including rhinitis, vocal cord dysfunction, and increased cough sensitivity, complicating diagnosis and management. These nonasthmatic conditions can be distinguished from RADS as they do not cause airway hyperresponsiveness, but they can coexist with RADS. The differential diagnosis of RADS or irritant-induced asthma includes: 1. Induction of new-onset asthma by a high level of respiratory irritant that also has the capacity to be a respiratory sensitizer (e.g., di-isocyanates), producing irritant-induced occupational asthma and concurrent sensitization to that agent. 2. Underlying airway hyperresponsiveness or underlying asthma with aggravation by the irritant exposure, triggering symptoms. This is the main differential diagnosis of RADS/irritant-induced

Irritant stimulus Epithelial and neuronal damage + inflammatory mediators Release profibrogenic growth factors, e.g., TGF-


Inflammation: activation of neutrophils, ± eosinophils, ± mast cells, ± T lymphocytes; release IL-4, IL-13, other mediators

Repair epithelium via EGF, other mediators − symptoms resolve

Chronic inflammation, myofibroblast activation and airway remodeling Airway hyperresponsiveness − chronic asthma manifestations Figure 1 Theoretical model of pathogenesis of RADS/Irritant-induced asthma. TGF, transforming growth factor; EGF, epithelial growth factor; IL, interleukin.

REACTIVE AIRWAYS DYSFUNCTION SYNDROME 617

asthma. This diagnosis may be evident in a patient who has known underlying asthma, but can be hard to distinguish from new-onset asthma in a patient who has a history of previous occasional mild asthma-like symptoms but no previous diagnosis of asthma. 3. Irritant effects mimicking asthma in a patient with coincidental underlying airway hyperresponsiveness, e.g., following an accidental high level of exposure to an irritant, there may be recurrent episodes of vocal cord dysfunction, hyperventilation/panic attacks, or rhinitis, which may mimic asthma. Distinguishing between these possible diagnoses is assisted by detailing the history, obtaining as much objective information as possible about the exposure agents and levels of exposure (obtained from the workplace), and reviewing previous medical records relating to respiratory status prior to the exposure and following the implicated exposure, including emergency medical treatment records. Industrial hygiene data documenting the high exposure typically are not available, as the events are frequently sporadic or accidental. Further investigations may be needed to assess the likelihood of other diagnoses such as vocal cord dysfunction or sinusitis. There is no gold standard or specific test to confirm the diagnosis of RADS or irritant-induced asthma, and thus the diagnosis depends largely on the history and ruling out other possible disorders.

Animal Models Several attempts to develop an animal model of RADS/IIA have been unsatisfactory to date, although Demnati and co-workers have reported an increase in lung resistance and methacholine responsiveness for up to one month in rats exposed to very high levels of chlorine (1500 ppm for 5 min), but no airway inflammation. Conversely, Klonne and coworkers showed no adverse response to chlorine in a 1-year inhalational toxicity study in rhesus monkeys. A report of chlorine-induced changes in airways of mice found airway epithelial cell loss as well as alveolar changes. There was associated airway hyperresponsiveness, which was prevented by inhibiting inducible NO synthase.

Management, Current Therapy, and Outcome Therapy is as for other asthma: treatment for acute exacerbations includes systemic corticosteroids and bronchodilators; chronic management includes

environmental control measures, inhaled corticosteroids, and bronchodilators. Treatment of acute high irritant exposures preceding the onset of RADS is the same as for other acute inhalational exposures, including general supportive care and evaluation of the airways and lung parenchyma. Corticosteroids are frequently given in an acute setting, with the hope of reducing airway inflammation and promoting resolution, but there is little data demonstrating beneficial effect. When an accidental exposure occurs in a workplace setting, occupational hygiene investigation of the conditions leading to the exposure is necessary to prevent recurrence. The affected workers may then be able to return to the same work if their asthma is adequately controlled. However, with some asthmatics, even moderate irritant exposures in the workplace may be sufficient to aggravate asthma. Subjects with non-specific airway hyperresponsiveness can develop reactions to levels of irritants that do not provoke symptoms in healthy subjects. Respiratory protective devices may be helpful for short-term exposures or the worker may need to relocate to a cleaner environment. When the initial exposure has been to an agent, which can also be a respiratory sensitizer (e.g., a di-isocyanate spill), the worker may have been sensitized at the time of the accidental exposure; subsequent exposures to moderate or low concentrations of that agent would then trigger asthma exacerbations. This should be assessed by careful monitoring of such workers on return to work, with serial peak flow or spirometry monitoring and serial measures of airway responsiveness. There can be discordance between symptoms and lung function tests, especially since high irritant exposures can also result in disorders such as rhinitis, vocal cord dysfunction, increased cough sensitivity, and chemical sensitivity or panic disorders, which may symptomatically be difficult to distinguish from asthma, and which may not respond to asthma treatment. For example, some firefighters developed disabling cough without airway hyperresponsiveness following exposure to World Trade Center dust. The natural history of RADS or irritant-induced asthma is not well defined. Limited case series and reports of subjects with RADS have shown that airway hyperreactivity can persist for years after an acute exposure in a substantial but variable proportion of subjects. Socioeconomic impact can be significant and may not correlate with pulmonary function measures. The determinants of resolution or progression are poorly defined. See also: Asthma: Overview; Extrinsic/Intrinsic. Occupational Diseases: Inhalation Injury, Chemical.

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Further Reading Alberts WM and do Pico GA (1996) Reactive airways dysfunction syndrome. Chest 109: 1618–1626. Arbour NC, Lorenz E, Schutte BC, et al. (2000) TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nature Genetics 25: 187–191. Balmes JR (2002) Occupational airways diseases from chronic low-level exposures to irritants. Clinics in Chest Medicine 23: 727–735. Balmes J, Becklake M, Blanc P, et al. (2003) American Thoracic Society Statement: Occupational contribution to the burden of airway disease. American Journal of Respiratory and Critical Care Medicine 167: 787–797. Banouch GI, Alleyne D, Sanchez R, et al. (2003) Persistent hyperreactivity and reactive airways dysfunction in firefighters at the World Trade Centre. American Journal of Respiratory and Critical Care Medicine 168: 54–62. Brooks SM, Hammad Y, Richards I, Giovinco-Barbas J, and Jenkins K (1998) The spectrum of irritant-induced asthma: sudden and not-so-sudden onset and the role of allergy. Chest 113: 42–49. Brooks SM, Weiss MA, and Bernstein IL (1985) Reactive airways dysfunction syndrome (RADS). Persistent asthma syndrome after high level irritant exposures. Chest 88: 376–384. Cullinen P and Newman Taylor AJ (1998) Acute toxic chemical exposures. In: Banks DE and Parker JE (eds.) Occupational

Lung Diseases: An International Perspective, pp. 453–463. London: Chapman and Hall Medical. Holgate ST, Holloway J, Wilson S, et al. (2004) Epithelial-mesenchymal communication in the pathogenesis of chronic asthma. Proceedings of the American Thoracic Society 1: 93–98. Kern DG (1991) Outbreak of the reactive airways dysfunction syndrome after a spill of glacial acetic acid. American Review of Respiratory Diseases 144: 1058–1064. Tarlo SM (1989) Broder I. Irritant-induced occupational asthma. Chest 96: 297–300. Tarlo SM (2000) Workplace respiratory irritants and asthma. Occupational Medicine: State of the Art Reviews 15: 471–483. Tarlo SM (2003) Workplace irritant exposures: do they produce true occupational asthma? Annals of Allergy, Asthma and Immunology 90(supplement 2): 19–23. Tarlo SM and Chan-Yeung M (2005) Occupational asthma. In: Rosenstock L, Cullen MR, Redlich CA, and Brodkin D (eds.) Clinical Occupational and Environmental Medicine, 2nd edn. Philadelphia: Elsevier, W.B. Saunders. Vandenplas O, Toren K, and Blanc PD (2003) Health and socioeconomic impact of work-related asthma. European Respiratory Journal 22: 689–697. Zock JP, Kogevinas M, Sunyer J, et al. (2001) Asthma risk, cleaning activities and use of specific cleaning products among Spanish indoor cleaners. Scandinavian Journal of Work and Environmental Health 27: 76–81.

REFLEXES FROM THE LUNGS AND CHEST WALL D R McCrimmon and G F Alheid, Northwestern University, Chicago, IL, USA E J Zuperku, Medical College of Wisconsin, Milwaukee, WI, USA

secretion. Chest wall receptors can reflexively alter motor drive to the diaphragm but have relatively little influence on respiratory muscle activation during eupnea.

& 2006 Elsevier Ltd. All rights reserved.

Introduction Abstract A variety of mechanical and chemical sensory receptors in the lungs and chest wall regulate breathing pattern and defend the respiratory system. Ascribing specific reflex responses to activation of particular receptors has been complicated by the difficulty in accessing and selectively activating receptor subtypes. Receptors within the lower airways and lungs are classified based on whether the sensory afferent fibers are myelinated or unmyelinated. Their axons primarily course in the vagus nerves. Receptors with myelinated axons include the slowly and rapidly adapting pulmonary stretch receptors (SARs and RARs). SARs respond to lung inflation and mediate the Breuer–Hering inspiratory inhibiting and expiratory facilitating reflexes. Activation of RARs by inhaled irritants evokes airway protective reflexes including a rapid breathing frequency, bronchoconstriction, and mucus secretion. These receptors also elicit sighs in response to a decrease in airway compliance or a reduction in lung volume below the normal resting volume. Cough is produced by a subset of receptors with small myelinated axons. Activation of receptors with unmyelinated fibers (C-fibers) also produces airway protective reflexes consisting of a shallow rapid breathing pattern, bronchoconstriction, and mucus

The airways, lungs, and chest wall are endowed with a variety of mechanical and chemical sensory receptors that regulate breathing pattern and defend the respiratory system. The receptors of the lower airways and lungs have been classified into two main types based on whether the sensory afferent fibers are myelinated or unmyelinated. For both types of fibers the vagus is the primary pathway to the central nervous system where they terminate in the nucleus of the solitary tract (NTS) within the medulla. Receptors are further subtyped according to their stimulus response characteristics and anatomical locations. Receptors with myelinated axons are the slowly and rapidly adapting pulmonary stretch receptors (SARs and RARs) while those with unmyelinated axons include pulmonary and bronchial C-fiber endings and neuroepithelial bodies (NEBs). Each of the receptor types can contribute to more than one type of reflex response. This results

REFLEXES FROM THE LUNGS AND CHEST WALL 619

in part from the varied location of the receptive fields (e.g., intrapulmonary, extrapulmonary, or extrathoracic airways) and from coactivation of multiple receptor types. In addition, ascribing specific reflex responses to activation of particular receptors has been complicated by the difficulty in accessing and selectively activating receptors deep within the airways.

NAmb

Slowly Adapting Pulmonary Stretch Receptors SARs play a role in controlling breathing pattern, airway smooth muscle tone, systemic vascular resistance, and heart rate. They are located in airway smooth muscle and are stimulated by stretch during lung inflation (Figure 1(a)). SARs behave as if they are

RAR

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Glu

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VRC

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C-fiber SP, CGRP, NKA Calbindin

NTS

Ach VIP

Ach VIP

Glu Phr

IML

Glu

DRG

PG

JG

JG

Ach Phr n.

A

(To diaphragm)

SM (a)

NG

NEB

NE

V

V

A VIP NO

SP CGRP

Ach

NG

AG

AG

VIP NO SM

Ach

Ach

(b)

Figure 1 Schematic diagram of pulmonary reflex pathways. (a) Myelinated slowly (SAR, orange) and rapidly (RAR, red) adapting receptor afferent pathways are depicted relative to a section of bronchiole pseudostratified columnar epithelium. SARs are located in airway smooth muscle (SM) and are stimulated by stretch during lung inflation. Sensory endings of RARs are located in airway epithelium where they respond to the inhalation of effective irritants. They are also postulated to be located in proximity to bronchial venules where they may be activated by edema. SAR and RAR neurons are found in the nodose (NG) and jugular ganglia (JG) of the vagus nerve and synapse in the nucleus of the solitary tract (NTS). Airway efferents originate mainly from cholinergic (Ach, blue) parasympathetic motoneurons in the nucleus ambiguus (NAmb). These synapse on cholinergic (blue) and noncholinergic neurons (magenta) in local airway ganglia (AG) which in turn target airway smooth muscle. Additional efferent targets include mucus secreting glands (at the level of the trachea), mucus secreting epithelial cells (e.g., goblet cells; not shown). Sensory information reaching the NTS alters activity of parasympathetic vagal motoneurons in the nucleus ambiguus and premotor neurons in the ventral respiratory column (VRC) of the medulla. Alterations in respiratory rate and pattern are effected by projections of inspiratory premotor neurons to phrenic motoneurons (Phr) in the cervical spinal cord. The latter innervate the diaphragm via the phrenic nerve (Phr n.). Expiratory premotor neurons in the caudal ventrolateral medulla also project to abdominal and intercostal motoneurons in the thoracic spinal cord (not shown). A, arteriole; Glu, glutamate; V, venule. (b) Unmyelinated C-fiber pulmonary afferents (green) and afferents to neuroepithelial bodies (NEB) (red, green) are depicted on the same diagram as in (a). C-fibers project diffusely throughout the parenchyma of the lung including the airway epithelial (mucosal) and submucosal layers, and in the vicinity of pulmonary blood vessels. Damage or irritation of the C-fibers and/or the surrounding lung tissue may lead to (antidromic) local release of neuropeptides from C-fiber sensory ending (i.e., the ‘axon reflex’); this appears to include extravasation from small venules. The axon reflex has been postulated to contribute to airway hyperresponsivity. Vagal calbindin-positive myelinated axons (red) and unmyelinated spinal afferents (green) contribute to the innervation of neuroepithelial bodies (NEB) which are potential pulmonary oxygen sensors. Vasoactive intestinal peptide (VIP) and nitric oxide synthase (NO) positive efferent axons originating in airway ganglia also target NEBs (connection not shown). CGRP, calcitonin gene related peptide; DRG, dorsal root ganglion; NE, norepinephrine; NKA, neurokinin A; PG, paravertebral ganglia; SP, substance P.

620 REFLEXES FROM THE LUNGS AND CHEST WALL

organized in series with airway smooth muscle as they are also stimulated by smooth muscle contraction. SAR Response Characteristics

While thought to relay information about lung volume, the discharge patterns of SARs are more closely related to transpulmonary pressure. Their discharge frequency displays a modest dynamic component but only partially adapts (o30% after 2 s) when lung inflation is held constant (Figure 2). A considerable proportion of SARs (between 27% and 60% in the different species studied) are active at end expiratory volume (i.e., at the functional residual capacity (FRC)), whereas others are recruited only during inspiration.

B–H inspiratory inhibitory reflex. SAR activity during the expiratory phase lengthens expiratory duration (TE) and is known as the B–H expiratory facilitatory reflex. The resulting combination of breathing frequency and VT results in a breathing pattern that tends to minimize the work of breathing while maintaining appropriate alveolar ventilation. Removal of SAR input produces a slow deep pattern accompanied with a higher amount of elastic work. The B–H reflex is active at normal resting tidal volumes in all mammals that have been examined, with the exception of humans where it is only activated when tidal volume exceeds 2–3 times normal. However, the B–H reflex in preterm and neonatal infants is very robust, especially during active sleep (REM), where end inspiratory occlusion of the airways prolongs TE by over 400%.

Breuer–Hering Reflexes SARs mediate the Breuer–Hering (B–H) inhibitory reflexes, which modify the breathing pattern. During inspiration, lung inflation increases SAR discharge frequency, which reflexively shortens the duration of the inspiratory phase (TI) and thereby reduces tidal volume (VT). This aspect of the reflex is known as the

Control of Expiratory Airflow and End Expired Volume

SARs appear to play a role in the control of expiratory flow rate. One aspect of this control involves the combined input of SARs located in the lower airways and those in the trachea. Together these

PT

SAR

PT

RAR

Figure 2 Responses to sustained inflation of the lungs in an anesthetized cat of individual slowly adapting (upper pair of traces) and rapidly adapting (lower pair of traces) afferent fibers teased from the vagus nerve. The ventilatory pump was stopped in expiration just before start of each record. The upper traces in each pair indicate intratracheal pressure (increased pressure upwards), lower traces show action potentials. The line at the bottom marks time in 100 and 500 ms intervals. Reproduced from Knowlton GC and Larrabee G (1946) A unitary analysis of pulmonary volume receptors. American Journal of Physiology 147: 100–114, used with permission from The American Physiological Society.

REFLEXES FROM THE LUNGS AND CHEST WALL 621

receptors mediate a compensatory motor control process that retards expiratory airflow by neurally mediated increases in laryngeal resistance and in diaphragm activity during the initial portion of expiration, along with decreases in abdominal muscle activity. This ‘expiratory braking’ reduces the rate of lung deflation, thereby increasing the duration of pulmonary stretch receptor discharge with a resultant increase in TE. SARs also act to increase the efficiency of breathing by controlling the expiratory musculature. This is mediated by modulating the activity of expiratory premotor neurons in the medulla. Starting at transpulmonary pressure levels well below those at FRC, SARs excite expiratory premotor neurons. However, as transpulmonary pressure increases above FRC levels, the expiratory premotor neurons are progressively inhibited, and their discharge is abolished at high transpulmonary pressure levels. This volume/ pressure-dependent biphasic activation–inhibition pattern is relayed via spinal motoneurons to expiratory internal intercostal and abdominal muscles. At end inspiration, lung volume and lung recoil force are high and the requirement for expiratory muscle activity is less. However, as lung volume and SAR activity decrease, the gradual reduction of inhibition allows greater expiratory muscle activity, thereby tending to preserve a constant expulsive force in the face of the declining lung recoil (Figure 3).

relationship between dead space and airway resistance. Smooth muscle control of local airflow resistance is important for the proper distribution of airflow to the various lung compartments. For example, body position alters vertical gradients in regional lung volume, ventilation, and perfusion. The resulting mismatches in ventilation and perfusion can be reduced by the reflex control of airway smooth muscle. Respiratory Sinus Arrhythmia

SARs also play a role in the production of respiratory sinus arrhythmia. Heart rate increases during inspiration and decreases during expiration. The exact relationship between heart rate and the phase of respiration depends on respiratory frequency and tidal volume. The greatest effect occurs at respiratory frequencies of 5–6 breaths min 1 and diminishes as the frequency is increased. While the central nervous system produces sinus arrhythmia, activation of SARs contributes to its magnitude. Within the medulla, the effect is to increase heart rate by inhibiting cardiac vagal motoneurons that innervate the atrial sinus node and produce slowing. This reflex helps stabilize cardiac output by allowing heart rate to increase during the reduction in left ventricular stroke volume that occurs during inspiration from a reduction in left ventricular filling.

Rapidly Adapting Pulmonary Stretch Receptors

An increase in SAR activity also relaxes airway smooth muscle by reducing parasympathetic tone to the airway. This negative feedback optimizes the reciprocal

Rapidly adapting receptors (RARs) adapt rapidly to a maintained mechanical stimulus, yet their response to a chemical one is often long lasting. They

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20

Increased expiratory airflow resistance

0

Neuron (spikes s–1)

Phrenic

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Figure 3 Activation of slowly adapting receptors by increases in airway pressure (PT), secondary to an increase in expiratory airway resistance (indicated by horizontal bar) in a dog. Increased resistance slowed the centrally generated breathing frequency as indicated by a reduction in burst frequency on motor output to the diaphragm (phrenic nerve) and increased the firing rate (spike s 1) of an expiratory bulbospinal premotor neuron. Reproduced from Bajic J, Zuperku EJ, Tonkovic-Capin M, and Hopp FA (1992) Expiratory bulbospinal neurons of dogs. I. Control of discharge patterns by pulmonary stretch receptors. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 262: R1075–R1086, used with permission from The American Physiological Society.

622 REFLEXES FROM THE LUNGS AND CHEST WALL

are responsible for a range of powerful respiratory reflexes, especially cough, augmented breaths, hyperpnoea, and constriction of the trachea and bronchi. Other reflexes from lung RARs include airway mucus secretion and closure of the larynx. Location

RARs are located in airways from the nose to smaller bronchi. However, only a few RARs have been found in the smaller bronchi and none are in alveoli. The greater concentration of RARs near the carina and in the hilar airways may be optimized for defense mechanisms against inhaled irritants. RAR sensory terminals form branching arrays of nonmyelinated fibers arranged circumferentially in the airway epithelium and submucosa (Figure 1(a)). Response Characteristics

During quiet breathing, RARs are not very active, but when active their discharge is irregular both in duration and in its timing within the respiratory cycle (Figures 2 and 4). However, RAR activity is readily recruited by inhalation of effective irritants, rapid changes in airway volume, and intraluminal mechanical stimuli, or by the release of inflammatory or immunological mediators. RARs are heterogeneous. Transpulmonary pressure is an important factor in their activation. In addition, RARs are stimulated by pulmonary edema as well as a variety of irritants

such as cigarette smoke, nicotine, and ammonia, or by intravenous injections of other chemicals such as bradykinin, substance P, histamine, and serotonin. They are sensitized by airway smooth muscle contraction and by decreases in lung compliance. The reflexes induced by RAR activation depend on the characteristics of individual receptors and their site in the airways. Activation of epipharyngeal RARs elicits a series of augmented inspirations (the aspiration reflex) while those in the bronchi can produce an increase in respiratory rate or an augmented expiratory effort or a full cough (see below). Stimulation of a subset of laryngeal RARs produces a cough. The removal of inhaled irritants is further facilitated by RAR-induced bronchoconstriction and increased mucus secretion. The resulting greater airflow velocity and turbulence increase the probability of inhaled irritants becoming entrapped in the mucus and transported back toward the mouth. Cough Reflex

The cough is an airway defense mechanism consisting of a large inspiratory effort followed by a forced, rapid expiratory effort and bronchoconstriction. Together these create high gas velocities that expel airway mucus in which foreign particles are entrapped. Reflex increases in mucus production aid this process. Coughs are evoked in response to a variety of mechanical or chemical stimuli that are likely to activate different sensory pathways. While a primary role in

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(b)

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Figure 4 Activation of an initially inactive rapidly adapting receptor by progressive reduction in dynamic lung compliance (Cdyn; b–d) in a dog. (a) Initially with compliance maximal, the receptor was silent. (b–d) Progressively lowered compliance after positive end expiratory pressure (PEEP) was removed for 5, 10, and 20 ventilatory cycles, respectively; in each case activity was recorded 1 min after PEEP restored. (b) Cdyn reduced by 23%, receptor stimulated but did not fire in all ventilation cycles. (c) Cdyn reduced by 35%. (d) Cdyn reduced by 41%. AP, action potentials; PT, tracheal pressure. Reproduced from Jonzon A, Pisarri TE, Coleridge JC, and Coleridge HM (1986) Rapidly adapting receptor activity in dogs is inversely related to lung compliance. Journal of Applied Physiology 61: 1980–1987, used with permission from The American Physiological Society.

REFLEXES FROM THE LUNGS AND CHEST WALL 623

the triggering of cough has usually been assigned to RARs, recent research has suggested that coughs may be triggered by a subset of polymodal Ad-fibers (cough receptors). Like RARs in general, these Adfibers have cell bodies in the superior or nodose ganglia of the vagus nerve. Also like RARs, they are responsive to mechanical and acid stimuli but are unresponsive to capsaicin, bradykinin, or airway stretch. C-fibers have also been ascribed a role in the production or facilitation of cough. This is considered below with C-fiber reflexes.

respond to a variety of inflammatory agents (e.g., capsaicin, histamine, and bradykinin) (Figure 5) as well as inhaled irritants (e.g., cigarette smoke, ozone, sulfur dioxide, ammonia, acrolein). However, not all receptors are stimulated by any given agent, thus suggesting the presence of functional subgroups. C-fibers with axons in the vagus nerve synthesize a number of neuroactive substances including substance P, neurokinin A, and CGRP. In response to prolonged or intense stimulation, these neuropeptides are released from sensory terminals in a local or

Augmented Breath (Sigh Reflex)

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RARs are responsible for the occasional deep spontaneous inspirations shown by all mammals at rest. As lung compliance decreases during quiet breathing, the sensitivity of RARs increases until a central nervous threshold is reached and an augmented breath results that reverses the decrease in lung compliance and prevents atelectasis. Sighs can also be provoked by lung deflations below FRC, by intravenous injections of substance P, serotonin or histamine, or by inhalation of ammonia, all of which are known to stimulate both RARs and C-fiber receptors; however, selective stimulants of C-fiber receptors do not cause augmented breaths.

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Unmyelinated (C-) fibers constitute the most abundant airway afferents with endings distributed throughout the entire respiratory tract, from large airways to the lung parenchyma. Their endings are located superficially in airway mucosa where they form a network of substance P and calcitonin gene related peptide (CGRP) containing fibers (Figure 1(b)). Most pulmonary C-fiber afferents reach the central nervous system through the vagus nerves from cell bodies in the jugular and nodose ganglia. Others, however, have cell bodies in dorsal root ganglia with fibers coursing through sympathetic nerves to reach the spinal cord. Lung C-fibers are difficult to selectively activate and record, and this has impeded the task of unequivocally establishing the pathophysiological roles of these afferents. C-fibers are frequently divided into two populations based on their accessibility to neuroactive agents administered into the bronchial (bronchial C-fibers) or pulmonary (pulmonary C-fibers) circulations. The two groups differ with respect to their apparent sensitivities to several chemicals including bradykinin and prostaglandins. Nevertheless, the reflex changes in breathing pattern elicited by their activation appear to be the same. Both C-fiber groups

C-fiber

C-Fibers

150

0

Figure 5 C-fiber afferent and arterial pressure responses to injection of the C-fiber stimulant capsaicin into the pulmonary circulation (upper traces). While not as sensitive as SAR and RAR to lung inflation C-fibers can be activated by larger lung inflations (lower traces). Top traces: pulmonary C-fiber with an ending in the right upper lobe of an anesthetized open-chest rat. Middle traces: airway pressure (PT). Lower traces: arterial blood pressure (BP). Reproduced from Ho C-Y, Gu Q, Lin YS, and Lee L-Y (2001) Sensitivity of vagal afferent endings to chemical irritants in the rat lung. Respiratory Physiology 127: 113–124, with permission from Elsevier.

624 REFLEXES FROM THE LUNGS AND CHEST WALL

‘axon’ reflex. The resulting actions on a variety of tissues and cells (airway and vascular smooth muscles, cholinergic ganglia, mast cells, and mucous glands) produce pronounced local effects including bronchoconstriction, extravasation of macromolecules, and airway edema. Stimulation of bronchopulmonary C-fibers activates several reflex responses with similarities to those elicited by RAR activation. These include a rapid, shallow breathing pattern. Although C-fibers usually demonstrate little spontaneous activity, removing their input to the central nervous system results in a slower, deeper breathing pattern, consistent with a small tonic influence on breathing pattern. When activation is produced by an intravenous injection of, for example, capsaicin there is frequently an initial apnea preceding the rapid, shallow pattern. However, the apnea is likely a response to the initial experimentally induced massive C-fiber discharge, with the shallow rapid respiratory pattern representing the more usual response to a more slowly developing pathophysiological activation. A role in cough production has also been ascribed to C-fibers because several stimuli that stimulate cough (e.g., citric acid, capsaicin, and bradykinin) are also known to activate C-fibers. Furthermore, depletion of airway C-fibers by capsaicin pretreatment abolishes citric acid-induced cough in unanesthetized animals while cough in response to mechanical stimulation of the airway persists. Pharmacological blockade of tachykinin receptors also reduces cough in response to a number of stimuli, including cigarette smoke and bronchospasm in guinea pigs. However, since direct C-fiber stimulation does not induce cough and can even inhibit it, the role of these fibers in cough remains unclear. C-fiber stimulation may contribute to cough by an indirect effect through a local axon reflex where there is C-fiber retrograde release of neuropeptides at the sensory endings in response to the various irritants described above. These activate receptors on nearby RAR (polymodal Ad) fibers as well as produce edema and inflammation. C-fiber reflexes also include broncho- and laryngoconstriction. In dogs and cats bronchoconstriction appears to be reflex mediated via pathways within the central nervous system, as blockade of ganglionic transmission with atropine prevents the reflex, while in guinea pigs, the reflex is atropine insensitive but attenuated by antagonists of neurokinin receptors, suggesting a contribution by the local axon reflex. In humans, capsaicin inhalation results in a relatively small centrally mediated bronchoconstriction which may be masked by concomitant activation of a nonadrenergic, noncholinergic (NANC) bronchodilator pathway.

C-fiber activation also produces edema, mucus secretion, airway mucosal vasodilation, bradycardia, and hypotension. Contributing to the C-fiber-induced edema is a reflex increase in bronchial circulation that occurs despite a vagally mediated systemic hypotension. The increase in blood flow is focused on the irritated region containing the activated C-fibers and is largely due to an inhibition of sympathetic outflow. The reflex response appears to be centrally mediated and includes activation of vagal efferents. Activation of C-fibers has also been associated with burning and choking sensations in the airways and upper chest.

Neuroepithelial Bodies Pulmonary neuroendocrine cells are scattered throughout the epithelium of the upper and lower airways. Within intrapulmonary airways neuroendocrine cells are often organized into clusters termed neuroepithelial bodies (NEBs). NEBs receive extensive innervation from vagal fibers as well as from dorsal root ganglion neurons and from local neurons with cell bodies in airway ganglia (Figure 1(b)). As with other neuroendocrine cells, they contain dense core vesicles that enclose a variety of neuroactive molecules including biogenic amines, especially serotonin, and several peptides including gastrin-releasing peptide, CGRP, enkephalin, somatostatin, cholecystokinin, and substance P. The contributions of NEBs to pulmonary homeostasis have yet to be established; based in large part on their resemblance to carotid body chemoreceptors, they are postulated to function as airway chemoreceptors. Whatever their functional role, it may vary with developmental age. They are among the first cells to mature in the fetal lung and they may consequently contribute to lung development. Immediately postnatally, a time when carotid chemoreceptors are relatively insensitive to hypoxia, it is suggested they provide chemosensory drive to breathing. In maturity NEBs may become more relevant for control of airway and/or pulmonary arteriolar tone.

Reflexes Originating from the Chest Wall Afferent activity from muscle spindles and tendon organs in intercostal and abdominal muscles influences the motor activity of neighboring synergist and antagonistic muscles as well as the diaphragm via spinal segmental pathways. In addition, intercostal muscle vibration, chest compression, or intercostal stretch can reduce or terminate inspiratory motor activity via supraspinal pathways. This latter effect is due to the activation of the tendon organs. These

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afferents do not influence the quiet breathing pattern but when the respiratory system is loaded or stressed, for example, during exercise, tendon organ afferents can modulate rate and depth of breathing. However, this effect is much smaller than those mediated by the lung receptors. The diaphragm contains few muscle spindles. Tendon organs, although relatively sparse, outnumber the spindles. Their afferent fibers course in the phrenic nerve and electrical stimulation of phrenic afferents inhibits phrenic efferent activity via segmental and supraspinal connections. It is generally accepted that phrenic afferents have little influence on respiratory muscle activation during eupneic breathing. However, there is emerging evidence that when these afferents are activated by mechanical stimulation they can reflexively alter efferent drive to the diaphragm. This may be due to sudden changes in lung volume, as occurs in going from the supine to the upright posture, or in response to chemical stimuli.

Lung Reflexes in Disease Since discharge patterns of the SARs are greatly influenced by their anatomical location within the tracheobronchial tree and the orientation of their receptor endings in airway wall structures, disease processes that alter airway caliber and tone and/or lung mechanics affect the SARs and the reflexes they mediate. The sensitivity of SARs to normal stimuli can be increased by bronchoconstriction, airway obstruction, or decreases in lung compliance. For example, an increase in lung collagen associated with pulmonary fibrosis results in a decrease in lung volume and compliance, and a reduction in diffusing capacity that can result in hypoxia and hypercapnia. These alterations are usually associated with a rapid, shallow breathing at rest and during exercise, and the sensation of dyspnea. The volume sensitivity of SARs in an experimentally induced pulmonary fibrosis animal model is significantly increased during inspiration, and SAR activity during the deflation phase of expiration is significantly reduced. These two effects acting via the B–H reflex would shorten inspiratory and expiratory durations, and account, at least in part, for the observed rapid, shallow breathing patterns. The activities of RARs and C-fiber afferents also appear to be altered in various pulmonary disease states, including pulmonary fibrosis. Pulmonary C-fibers are activated in a variety of pathologic conditions including pulmonary congestion and edema. These receptors have been referred to as ‘juxtapulmonary capillary receptors’ because of their sensitivity to an increase in interstitial fluid volume or pressure resulting from an increase in

pulmonary vascular pressure or fluid filtration across pulmonary capillaries. Pulmonary embolism and inflammation also strongly excite C-fibers as a result of the local release of chemical mediators such as serotonin and cyclooxygenase metabolites. RAR and C-fiber receptor sensitivity can be increased in airway disease as well as by exposure to ozone, histamine, and prostaglandins. It has been suggested that this sensitization may occur in disease states and underlie bronchial hyperresponsiveness. Interestingly, viral infection of the airways can lead to a transformation in RAR phenotype in which substance P and neurokinin A are detected within RAR cell bodies in the nodose ganglion where they are not normally present. Assuming these peptides are transported to the sensory terminals in the airways they may contribute to neurogenic inflammation of the airways and hypersensitive cough through the axon reflex. See also: Mucus. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Symptoms of Respiratory Disease: Cough and Other Symptoms. Ventilation: Overview; Control.

Further Reading Adriaensen D and Brouns I (2003) Functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors. Anatomical Record A 270A: 25–40. Bajic J, Zuperku EJ, Tonkovic-Capin M, and Hopp FA (1992) Expiratory bulbospinal neurons of dogs. 1. Control of discharge patterns by pulmonary stretch receptors. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 262: R1075–R1086. Barnes PJ (2001) Neurogenic inflammation in the airways. Respiratory Physiology 125: 145–154. Bolser DC, Lindsey BG, and Shannon R (1987) Medullary inspiratory activity: influence of intercostal tendon organs and muscle spindle endings. Journal of Applied Physiology 62: 1046–1056. Canning BJ, Mazzone SB, Meeker SN, et al. (2004) Identification of the tracheal and laryngeal afferent neurones mediating cough in anaesthetized guinea-pigs. Journal of Physiology (London) 557: 543–558. Coleridge HM and Coleridge JCG (1986) Reflexes evoked from tracheobronchial tree and lungs. In: Fishman AP (ed.) Handbook of Physiology: The Respiratory System, pp. 395–429. Bethesda: American Physiological Society. Daly MB (1997) Effects of respiration on the cardiovascular system. In: Huang CL-H, Dolphin AC, Green R, and Spyer KM (eds.) Peripheral Arterial Chemoreceptors and Respiratory– Cardiovascular Integration, pp. 182–224. New York: Oxford University Press. Ho C-Y, Gu Q, Lin YS, and Lee L-Y (2001) Sensitivity of vagal afferent endings to chemical irritants in the rat lung. Respiratory Physiology 127: 113–124. Iscoe S (1998) Control of abdominal muscles. Progress in Neurobiology 56: 433–506. Jonzon A, Pisarri TE, Coleridge JC, and Coleridge HM (1986) Rapidly adapting receptor activity in dogs is inversely related

626 RELAPSING POLYCHONDRITIS to lung compliance. Journal of Applied Physiology 61: 1980–1987. Jordan D (2001) Central nervous pathways and control of the airways. Respiratory Physiology 125: 67–81. Knowlton GC and Larrabee G (1946) A unitary analysis of pulmonary volume receptors. American Journal of Physiology 147: 100–114. Lee L-Y, Lin YS, Gu Q, Chung E, and Ho C-Y (2003) Functional morphology and physiological properties of bronchopulmonary C-fiber afferents. Anatomical Record A 270A: 17–24. Lee L-Y and Pisarri TE (2001) Afferent properties and reflex functions of bronchopulmonary C-fibers. Respiratoy Physiology 125: 47–65. Reynolds SM, Mackenzie AJ, Spina D, and Page CP (2004) The pharmacology of cough. Trends in Pharmacological Sciences 25: 569–576. Sant’Ambrogio G and Sant’Ambrogio FB (1991) Reflexes from the airway, lung, chest wall, and limbs. In: Crystal RG, West JB, et al. (eds.) The Lung: Scientific Foundations, pp. 1383–1395. New York: Raven Press. Schelegle ES and Green JF (2001) An overview of the anatomy and physiology of slowly adapting pulmonary stretch receptors. Respiratory Physiology 125: 17–31. Tonkovic-Capin M, Zuperku EJ, Stuth EA, et al. (2000) Effect of central CO2 drive on lung inflation responses of expiratory bulbospinal neurons in dogs. American Journal of Physiology 279: R1606–R1618. Widdicombe J (2003) Functional morphology and physiology of pulmonary rapidly adapting receptors (RARs). Anatomical Record A 270: 2–10.

Glossary Axon reflex – activation of a sensory receptor results in orthodromic conduction of the action potential toward the central nervous system, and antidromic conduction along peripheral branches of the

axon. The action potential can produce release of neuroactive agents from retrogradely invaded terminals Breuer–Hering reflex (B–H) – reflex changes in breathing pattern elicited by activation of SAR. Activation during inspiration shortens inspiratory duration and reduces tidal volume. Activation by maintained inflation during expiration results in expiratory prolongation Functional residual capacity (FRC) – volume of air in the lungs at the end of a normal expiration Non-adrenergic, non-cholinergic (NANC) – airway nerve fibers that release any of a variety of transmitter substances but do not release either norepinephrine or acetylcholine Neuroepithelial bodies (NEBs) – collection of endocrine cells in airway epithelium that contain dense core vesicles and a variety of neuroactive substances. They are extensively innervated by vagal fibers and fibers arising in dorsal root ganglia. They are responsive to hypoxia but their physiological role requires clarification Rapidly adapting pulmonary stretch receptor (RAR) – receptor in epithelium and subepithelial layers of airways with myelinated axon responsive to inhaled irritants, large lung inflation Slowly adapting pulmonary stretch receptor (SAR) – receptor in airway smooth muscle with myelinated axon that is responsive to lung inflation

RELAPSING POLYCHONDRITIS A Mehta and J C Yataco, Cleveland Clinic Foundation, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Relapsing polychondritis is a rare, chronic, episodic, and progressive inflammatory disease of unknown cause. It is characterized by inflammation and destruction of cartilaginous structures found in ears, nose, joints, and the tracheobronchial tree. Relapsing polychondritis can also affect other proteoglycan-rich structures, such as the eye, kidneys, heart, blood vessels, and inner ear, or cause systemic symptoms such as fever, lethargy, and weight loss. The exact etiology and pathogenesis of relapsing polychondritis remains unknown, but evidence suggest that it is an immunologically mediated disease. The most common manifestations include auricular and nasal chondritis, arthritis, episcleritis, and inflammation of cartilaginous tissues along the

laryngotracheal tree. The correct diagnosis is usually delayed in most patients due to its rarity and its multiple clinical features. No exact data exist on the incidence, prevalence, and mortality of relapsing polychondritis. The most common natural course of relapsing polychondritis is fluctuating but progressive with episodic flare-ups of inflammation that can eventually lead to significant dysfunction of the involved organs. Corticosteroids have been shown to be the most effective treatment, but their side effects prevent any long-term therapy. Other immunosuppressing agents, such as dapsone, methotrexate, azathioprine, and cyclophosphamide, have also been used.

Introduction Relapsing polychondritis is a rare, chronic, episodic, and progressive inflammatory disease in which different cartilaginous structures found in ears, nose,

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joints, and the tracheobronchial tree are mainly affected. Relapsing polychondritis can also affect other proteoglycan-rich structures, such as the eye, kidneys, heart, blood vessels, and inner ear, or cause systemic symptoms such as fever, lethargy, and weight loss. The correct diagnosis is usually delayed in most patients due to its rarity and its multiple clinical features. No exact data exist on the incidence, prevalence, and mortality of relapsing polychondritis. The incidence in Rochester, Minnesota, is estimated to be 3.5 per 1 million population per year. The most common natural course of relapsing polychondritis is fluctuating but progressive with episodic flare-ups of inflammation that can eventually lead to significant dysfunction of the involved organs. Corticosteroids have been shown to be the most effective treatment, but their side effects prevent any long-term therapy. Other drugs, such as dapsone, methotrexate, azathioprine, and cyclophosphamide, have also been used.

Etiology and Pathogenesis The exact etiology and pathogenesis of relapsing polychondritis remains unknown. The role of the immune system in the pathogenesis of relapsing polychondritis has been supported by different lines of evidence: 1. Serum autoantibodies against collagen type II (present in cartilage in large amounts), as well as types IX and XI, have been found in some patients with relapsing polychondritis. 2. Cell-mediated immune responses directed toward cartilage components have been demonstrated by some investigators. 3. Immunofluorescence studies of cartilage have shown granular IgG, IgA, IgM, and C3 deposits at the junction of fibrous and cartilaginous tissue, suggesting the presence of immune complexes. 4. An association with HLA-DR4 antigen has been reported more frequently in patients with relapsing polychondritis compared to controls. 5. Tissue infiltration composed of plasma cells, lymphocytes, and polymorphonuclear leukocytes has been well described. 6. The frequent association of relapsing polychondritis with other autoimmune diseases and the response to immunosuppressive agents support the theory of autoimmune pathogenesis.

Pathology The initial pathologic change is a loss of basophilic staining, followed by cellular infiltrates of

lymphocytes, plasma cells, and neutrophils at the chondrodermal junction in the affected cartilaginous sites. The lymphocytosis is a CD4 helper T cells dominate process. IgG and C3 may now be seen throughout the matrix. Finally, the chondrocytes become vacuolated and pyknotic, and they are replaced by fibroblastic granulation tissue that completely disrupts the tissue architecture. Focal regions of calcification and bone formation may be present. The ocular histopathology in patients with relapsing polychondritis includes mononuclear inflammatory cells, plasma cells, and even true vasculitis.

Clinical Features Relapsing polychondritis usually develops between the ages of 40 and 60 years, but the disease has been described in almost all ages. Both genders are affected with the same frequency, but most cases involve white patients. Since relapsing polychondritis is uncommon and can affect multiple organs and systems at different times, there is usually a significant mean delay of 2.9 years until diagnosis is established. Relapsing polychondritis should be considered a syndrome that can be primary or secondary, depending on its association with other entities. Up to 37% of patients have an associated condition, such as a hematologic disorder, connective tissue disease, vasculitis, dermatologic disorder, or other autoimmune disorder (Table 1). Auricular Chondritis, Vestibular Dysfunction, and Nasal Chondritis

Auricular chondritis is the most frequent presenting manifestation in patients with relapsing polychondritis. Patients present with external ear pain and redness and swelling of the cartilaginous portion of the ear with sparing of the lobule (Figure 1). The involvement of the external ear usually occurs in a relapsing pattern persisting for several weeks or resolving spontaneously after a few days. After several episodes or after a severe single one, the cartilaginous pinna becomes floppy or deformed. The external auditory canal and Eustachian tube are frequently involved also, with edema and collapse causing subsequent conductive hearing loss and otitis media. Sensorineural hearing loss with or without symptoms of vestibular dysfunction (dizziness, ataxia, nausea, and vomiting) can occur presumably from vasculitis in branches of the internal auditory artery. Nasal chondritis develops suddenly, is very painful, and the inflammation can destroy the distal part of the nasal cartilage leading to a saddle nose deformity (Figure 2).

628 RELAPSING POLYCHONDRITIS Table 1 Diseases associated with relapsing polychondritis Hematologic diseases Myelodysplastic syndromes Pernicious anemia Hematologic malignancies Collagen vascular diseases Rheumatoid arthritis Systemic lupus erythematosus Reiter’s syndrome Seronegative spondylarthropathies Scleroderma Sjo¨gren syndrome Systemic vasculitides Wegener’s granulomatosis Polyarteritis nodosa Behc¸et disease Churg–Strauss syndrome Other diseases Autoimmune thyroid disease Myasthenia gravis Inflammatory bowel disease Primary biliary cirrhosis Vitiligo Lichen planus Panniculitis Mixed cryoglobulinemia

Figure 1 Significant deformity of the external ear due to severe cartilaginous inflammation.

Otorhinolaryngeal Manifestations

More than 50% of patients develop chondritis of the laryngotracheal cartilages and may present with anterior neck pain, cough, hoarseness, dyspnea, wheezing, or choking. Acute upper airway obstruction may develop due to inflammation and edema of the glottic, laryngeal, or subglottic area. Some patients may require an emergency tracheostomy even before there is an established diagnosis.

Pulmonary Manifestations

Involvement of the respiratory tract (trachea and firstto-second order bronchi) is the presenting feature in up to 26% of patients with relapsing polychondritis. Acute swelling of the tracheobronchial tree can lead to localized or diffuse obstruction of the airway tract. The damaged tracheal and bronchial rings may lose their rigidity and collapse, allowing the development of dynamic obstruction. Thus, pulmonary function tests with flow volume loops should be performed in all patients, even in those without respiratory symptoms. Spirometry shows obstructive ventilatory impairment with reduction in expiratory flow and maximal voluntary ventilation. Other findings seen in patients with relapsing polychondritis include tracheal/bronchial stenosis due to granulation tissue,

Figure 2 Saddle nose deformity due to severe cartilaginous inflammation.

calcification of the airway walls, and obstructive bronchiectasis. Chest radiographic findings are usually not sensitive or specific but include pneumonia, atelectasis, calcifications in the airway wall, and prominence of

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proliferation, but other pathologies, such as IgA nephropathy, segmental necrotizing crescentic glomerulonephritis, and tubulointerstitial nephritis, have also been described. Cardiovascular Manifestations

Figure 3 Inflammatory destruction of the distal tracheal rings. Reproduced from Becker H and Kayser K (1991) Atlas of Bronchoscopy, p. 27. St Louis: Mosby, with permission.

the thoracic aorta. Computed tomography (CT) of the chest is more useful in revealing details of the tracheobronchial tree (e.g., narrowing of the lumen and calcifications or thickening of the tracheal/bronchial wall). Bronchoscopic exam of the airway confirms the diagnosis, showing erythema, edema, deformation, and collapse of the tracheobronchial tree (Figure 3). In addition, biopsies of the cartilage can be obtained through bronchoscopy. It is important to remember that bronchoscopy is less accurate in evaluating the functional status of the airway compared to pulmonary function testing. Bronchoscopy may also precipitate further respiratory distress since it can induce trauma in an airway suffering active inflammation. Musculoskeletal Manifestations

Arthritis is the second most common presenting symptom and is present in up to 85% of patients with relapsing polychondritis. When relapsing polychondritis is not associated with any other collagen vascular disease, the arthritis is nonerosive, asymmetric, seronegative, and can affect all synovial joints. Metacarpophalangeal, interphalangeal, and knee joints are the most commonly affected. Renal Manifestations

Renal disease occurs in 10–22% of patients with relapsing polychondritis, especially in cases associated with vasculitis or other collagen vascular diseases. The most common renal pathology is mesangial

Aortic regurgitation is the most frequent form of cardiovascular involvement in relapsing polychondritis, occurring in approximately 4–10% of patients. Ascending aortitis extending to the valve ring is responsible for aortic regurgitation and explains the common association with aortic aneurysms. Other forms of cardiovascular involvement include abdominal aortic aneurysms, myocarditis, pericarditis, coronary vasculitis, impairment of the conduction system, thrombophlebitis, and arterial thrombosis. These thrombotic complications have been explained by the presence of vasculitis or an associated antiphospholipid syndrome. Dermatologic Manifestations

Skin involvement in the absence of an associated condition occurs in approximately 35% of cases. The most common manifestations are oral aphthous ulcers, followed by macules, papules, nodules, and livedo reticularis. The most common histologic diagnosis in these lesions is leukocytoclastic vasculitis, with other histologic patterns including neutrophilic dermatosis, thrombotic occlusion of dermal vessels, and even granulomatous vasculitis. Patients with an associated myelodysplastic syndrome commonly have dermatologic involvement. Elderly patients with relapsing polychondritis and skin involvement should be monitored for the development of a myelodysplastic syndrome. Neurologic Manifestations

Central and peripheral nervous system involvement occurs in only 3% of patients. The cranial nerves are most commonly affected, but seizures, headache, hemiplegia, encephalopathy, and cerebral aneurysms have been described. Ocular Manifestations

Ocular disease occurs eventually in up to 60% of patients with relapsing polychondritis. Most of these patients tend to develop multiple systemic manifestations. Episcleritis and scleritis are the most common manifestations, usually in parallel with inflammation of the nose and joints (Figure 4). Other types of ocular involvement are uveitis, keratitis, keratoconjunctivitis sicca, central retinal vein occlusion, and ischemic optic neuropathy.

630 RELAPSING POLYCHONDRITIS Table 2 Diagnostic criteria for relapsing polychondritis McAdam et al. (1976) criteriaa Bilateral auricular chondritis Nasal chondritis Nonerosive, seronegative inflammatory polyarthritis Ocular inflammation (conjunctivitis, keratitis, scleritis and/or episcleritis, uveitis) Respiratory tract chondritis (laryngeal and/or tracheal cartilage) Cochlear and/or vestibular dysfunction (neurosensory hearing loss, tinnitus, and vertigo)

Figure 4 Episcleritis is a common manifestation of relapsing polychondritis. Reproduced from Forbes and Jackson (2002) Color Atlas and Text of Clinical Medicine, p. 128. St Louis: Mosby, with permission.

Michet et al. (1986) criteriab Proven inflammation in two of three cartilaginous sites: auricular, nasal, or laryngotracheal Proven inflammation in one of three cartilaginous sites: auricular, nasal, or laryngotracheal; plus two other clinical signs, including ocular inflammation, vestibular dysfunction, seronegative arthritis, and hearing loss a

Three criteria are the minimum required to confirm the diagnosis. b One diagnostic criteria is enough to confirm the diagnosis.

Diagnosis Although relapsing polychondritis seems to be easy to diagnose when it presents with its most typical symptoms, the correct diagnosis in many cases is delayed due to its rarity and multiple possible presenting features. McAdam and co-workers were the first to propose diagnostic criteria using the most common clinical features, requiring at least three out of six features to confirm the diagnosis (Table 2). Michet and colleagues modified these diagnostic criteria, requiring chondritis in two of three sites (auricular, nasal, or laryngotracheal) or one of those sites and two other signs, including ocular inflammation, vestibular dysfunction, seronegative arthritis, and hearing loss. In most patients, it is not necessary to biopsy the affected cartilaginous sites. No specific laboratory test exists for the diagnosis of relapsing polychondritis. During acute exacerbations, it is possible to observe elevated erythrocyte sedimentation rate, anemia of chronic disease, leukocytosis, thrombocytosis, and hypergammaglobulinemia. Serum antibodies to type II collagen have been detected in 20–50% of patients. However, the use of these antibodies is limited due to a relatively low specificity. Other serologic tests, such as rheumatoid factor, antinuclear antibodies, antineutrophil cytoplasmic antibodies, and complement levels, are helpful if other associated conditions are present simultaneously. Other ancillary tests, such as urinalysis, creatinine, echocardiography, CT chest, pulmonary function tests, and skin biopsy, can be helpful to assess the organs and systems affected by the disease. Some autoimmune disorders can overlap clinical features or involve multiple organs, as is the case for relapsing polychondritis. Wegener’s granulomatosis

and polyarteritis nodosa can cause ocular inflammation, polyarthitis, and cochlear and vestibular symptoms. However, these two entities have not been reported to cause chondritis and usually affect the lung parenchyma, which is not seen in relapsing polychondritis. Rheumatoid arthritis can also cause ocular inflammation, vasculitis, and multiple organ involvement. However, the joint involvement of rheumatoid arthritis is usually symmetric and erosive as opposed to the nonerosive arthritis seen in relapsing polychondritis. Vasculitis of medium and large vessels, such as the polyarteritis nodosa and Takayasu’s arteritis, may mimic the cardiovascular involvement seen in relapsing polychondritis with development of aneurysms or arteritis with or without thrombosis.

Management and Therapy There is no standard medical therapy for relapsing polychondritis due to its rarity, wide variety of presentations, and unpredictable course. Therapeutic guidelines are based on reports of treatment success from small series of patients or isolated cases. Arthritis or mild chondritis of nose and ears can be treated with the nonsteroidal anti-inflammatory drugs dapsone or colchicine. However, systemic corticosteroids are the traditional agents used in acute exacerbations. Prednisone at a high dose of 1 mg kg 1 day 1 is used for cases with ocular involvement, laryngeal inflammation, sensorineural hearing loss, vestibular symptoms, and vascular and renal involvement. Although steroids may resolve acute inflammation of the cartilage and decrease the frequency and

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degree of the exacerbations, they have no effect on the overall progression of the disease and do not alter the risk of multisystem organ involvement or the structural damage to cartilaginous organs. When an acute exacerbation goes into remission, the high doses of steroids can be gradually tapered to moderate maintenance doses. In patients with chronic active inflammation that requires high-dose steroids, the use of methotrexate or azathioprine may allow a reduction of the corticosteroid dose. In cases of severe end-organ damage, such as ocular, pulmonary, cardiac, or renal involvement, pulses of oral or intravenous cyclophosphamide can be used. Cyclosporine A has also been used with success in refractory cases to other agents. Infliximab has been tried in resistant relapsing polychondritis and associated scleritis. The respiratory problems caused by relapsing polychondritis can present suddenly and become very difficult to manage. Adliff and colleagues reported the use of nasal CPAP as a pneumatic splint for the tracheobronchial tree, preventing airway collapse and obtaining temporary control of airway patency. For sudden and severe upper airway obstruction, a tracheostomy can be life-saving. Despite a significant risk, endotracheal intubation may be needed, preferably with a small endotracheal tube because of the reduced glottic diameter. Airway stents, montgomery T tubes, and self-expanding metallic stents have been used in patients with collapse or refractory stenosis of the airway. They are able to gain airway patency but are associated with many potential problems, including erosion of the stent through the trachea, sudden asphyxia from stent displacement, aspiration pneumonia, development of granulation tissue or mucosal ulceration, and retention of airway secretions. Most patients with relapsing polychondritis have a fluctuating but slowly progressive course with unpredictable severity and site of involvement by the inflammatory process. In a 6-year follow-up study from the Mayo Clinic, 86% of patients were reported to have intermittent manifestations with a median of five episodes. The majority of patients develop some disability, depending on the organs involved and the severity of the disease (deafness, impaired vision, speech problems, and cardiopulmonary problems). In terms of mortality, Trentham and co-workers in 1998 reported a 94% survival rate with an average disease duration of 8 years. This number represents an improvement compared to the 55% survival rate at 10 years reported by McAdam et al. in 1976. The improved survival has been explained by improved medical and surgical management of the respiratory and cardiovascular complications. The most common

causes of death are infections, vasculitis, and malignancies. Pulmonary infections are of particular importance. They are severe and complicated due to the use of corticosteroid therapy and airway stricture/collapse, which impair the defense and clearing mechanisms of the lung. See also: Bronchomalacia and Tracheomalacia. Bronchoscopy, General and Interventional. Corticosteroids: Therapy. Extracellular Matrix: Collagens. Immunoglobulins. Leukocytes: T cells. Pulmonary Effects of Systemic Disease. Upper Airway Obstruction.

Further Reading Becker H and Kayser K (1991) Atlas of Bronchoscopy, p. 27. St Louis: Mosby. Crockford MP and Kerr IH (1988) Relapsing polychondritis. Clinical Radiology 39: 386–390. Del Rosso A, Petix NR, Pratesi M, and Bini A (1997) Cardiovascular involvement in relapsing polychondritis. Seminars in Arthritis and Rheumatism 26: 840–844. Ebringer R, Rook G, Swana GT, Bottazzo GF, and Doniach D (1981) Autoantibodies to cartilage and type II collagen in relapsing polychondritis and other rheumatic diseases. Annals of the Rheumatic Diseases 40: 473–479. Eng J and Sabanathan S (1991) Airway complications in relapsing polychondritis. Annals of Thoracic Surgery 51: 686–692. Forbes and Jackson, Color Atlas and Text of Clinical Medicine, p. 128. St Louis: Mosby. Hebbar M, Brouillard M, Wattel E, et al. (1995) Association of myelodysplastic syndrome and relapsing polychondritis: further evidence. Leukemia 9: 731–733. Kent PD, Michet CJ Jr, and Luthra HS (2004) Relapsing polychondritis. Current Opinion in Rheumatology 16: 56–61. Krell WS, Staats BA, and Hyatt RE (1986) Pulmonary function in relapsing polychondritis. American Review of Respiratory Disease 133: 1120–1123. Lee-Chiong TL Jr (1998) Pulmonary manifestations of ankylosing spondylitis and relapsing polychondritis. Clinics in Chest Medicine 19: 747–757. Letko E, Zafirakis P, Baltatzis S, et al. (2002) Relapsing polychondritis: a clinical review. Seminars in Arthritis and Rheumatism 31: 384–395. McAdam LP, O’Hanlan MA, Bluestone R, and Pearson CM (1976) Relapsing polychondritis: prospective study of 23 patients and a review of the literature. Medicine (Baltimore) 55: 193–215. Michet CJ Jr, McKenna CH, Luthra HS, and O’Fallon WM (1986) Relapsing polychondritis. Survival and predictive role of early disease manifestations. Annals of Internal Medicine 104: 74–78. Molina JF and Espinoza LR (2000) Relapsing polychondritis. Bailliere’s Best Practice & Research: Clinical Rheumatology 14: 97–109. Park J, Gowin KM, and Schumacher HR Jr (1996) Steroid sparing effect of methotrexate in relapsing polychondritis. Journal of Rheumatology 23: 937–938. Tillie-Leblond I, Wallaert B, Leblond D, et al. (1998) Respiratory involvement in relapsing polychondritis. Clinical, functional, endoscopic, and radiographic evaluations. Medicine (Baltimore) 77: 168–176. Trentham DE and Le CH (1998) Relapsing polychondritis. Annals of Internal Medicine 129: 114–122.

632 RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER C J Jolley and J Moxham, King’s College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Respiratory muscles are skeletal muscles whose function is to pump air in and out of the lungs. The chest wall muscles, diaphragm, and other muscles, including abdominal muscles, neck muscles, and upper limb muscles, may be broadly divided into inspiratory or expiratory muscles, according to whether their actions increase or decrease the capacity of the thoracic cage. The recruitment of individual muscles also depends on whether ventilatory requirements are high or low. Respiratory muscles share the common structural, physiological, and biochemical features of skeletal muscles, but are adapted to meet their unique task of being able to be active throughout life. These features determine their ability to generate contractile force, which is translated into inspiratory or expiratory pressure, and their resistance to fatigue. An imbalance between the load on the respiratory muscles and their capacity, resulting in breathlessness and ultimately respiratory failure, is a consequence of obstructive and restrictive respiratory disorders, and neuromuscular disease.

Anatomy, Histology and Structure Respiratory muscles are skeletal muscles whose function is to move air in and out of the lungs, through their actions on the thoracic cage. Respiratory movements have been studied throughout medical history, from Hippocrates to the present day. Advances in respiratory physiology, histochemical techniques, genetics, and cell physiology have recently provided further insights into the functional anatomy of the respiratory muscle pump. Anatomy

The chest wall is made up of the vertebral column posteriorly, sternum and costal cartilages anteriorly, and the ribs and intercostal spaces laterally. It comprises the lateral boundaries of the thoracic cavity, which communicates superiorly with the neck through the thoracic inlet, and is separated from the abdomen inferiorly by the diaphragm muscle. Although the respiratory muscles are conventionally divided into inspiratory and expiratory groups, some overlap exists. Generally, muscles that elevate the ribs increase both the lateral and dorsoventral diameters of the ribcage, reduce intrathoracic pressure, and are inspiratory. Muscles that lower the ribs are usually expiratory.

Inspiratory muscles Diaphragm The diaphragm is a dome-shaped structure. The muscular portions of the left and right hemidiaphragm are inserted into a mobile central tendon. Direct and indirect evidence, from observations made in humans, that the muscular portion of each hemidiaphragm is made up of two embryologically and anatomically distinct components has been supported by electrophysiological studies in mammalian quadrupeds (Figure 1). 1. The costal diaphragm is derived from myoblasts in the body wall. It arises from the deep surfaces of the lower six ribs and costal cartilages, and runs directly upwards, parallel to and against the inner surface of the ribcage, forming the zone of apposition (ZAP) before inserting into the central tendon. The ZAP contributes one-third of the area of the ribcage at end expiration, less during inspiration. 2. The crural (lumbar) component of the diaphragm is derived from the mesentery of the esophagus. The left and right crura arise from the sides of the bodies of the first two (left), and first three (right), lumbar vertebrae and intervertebral discs, and from the medial and lateral arcuate ligaments. These are thickened upper margins of fascia, which extend from the second to the first lumbar vertebra, and from the first lumbar vertebra to the lower border of the twelfth rib, respectively. The median arcuate ligament connects their fibrous medial borders, crossing over the anterior border of the surface of the aorta. Some fibers of the right crus pass up to the left, forming a sphincteric sling around the esophagus. Innervation Phrenic nerves, arising from the third, fourth, and fifth cervical nerve roots supply motor innervation to each hemidiaphragm. Despite earlier electrophysiological studies suggesting that the costal and crural parts had separate segmental innervation, topographical tracer studies now suggest that this is mixed at both a spinal level and within the phrenic motor nucleus. Supraspinal control of the diaphragm is a combination of phasic automatic brainstem activity and cortical input. Transcranial magnetic stimulation of hemiplegic stroke patients has demonstrated that each hemidiaphragm is separately represented in the contralateral motor cortex.

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER 633

Last rib

h arc al t os

La t. l um bo

c

Xiphoid process

Opening for lesser splanchnic nerve

Figure 1 Diaphragm as viewed from below, showing the origins of the costal and crural muscular components, and the central tendon. Reproduced from Gray H (1918) Anatomy of the Human Body. Philadelphia: Lea & Febiger.

Chest wall muscles Intercostal muscles The external intercostal and parasternal muscles are inspiratory. The internal intercostal and transversus thoracis are expiratory muscles, but are described here together with other muscles in the anatomical group. The thin muscle layers span the intercostal spaces, overlapping, from superficial to deep (Figures 2 and 3). 1. Muscle fibers of the external intercostals run downward and forward, from the tubercles of the rib above to the costal cartilage of the rib below. Anteriorly, they are replaced by a fibrous aponeurosis, the anterior intercostal membrane, which continues to the sternum. Muscle mass also decreases caudally. 2. Muscle fibers of the internal intercostals run downward and backward, from the sternocostal junction of the rib above, to near the tubercle of

the rib below, replaced posteriorly by a fibrous aponeurosis, the posterior intercostal membrane. Muscle mass in humans shows no definite rostrocaudal gradient. Internal intercostal muscles between the costochondral junction and the sternum are called the parasternal intercostals. They do not extend below the fifth interspace in humans. 3. Transversus thoracis arises from either side of the lower third of the posterior surface of the body of the sternum, posterior surface of the xiphoid process, and the sternal ends of the costal cartilages of the lower three or four ribs. Its fibers diverge upward and laterally, inserting into the lower borders and inner surfaces of the costal cartilages of the second, third, fourth, fifth, and sixth ribs. The lowest fibers of this muscle are directed horizontally, continuous with those of the transversus abdominis muscle, the intermediate fibers obliquely, and the highest almost vertically (Figure 3).

634 RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER

on nd Te

Sternum

ct. majo r of pe

Ulna

Radius

Figure 2 Chest wall, pectoral, and upper limb muscles. Muscle fibers of the external intercostals run downward and forward, and muscle fibers of the internal intercostals run downward and backward. Reproduced from Gray H (1918) Anatomy of the Human Body. Philadelphia: Lea & Febiger.

Levatores costarum There are 12 pairs of muscles. They arise from the tip of the transverse process of C7 and T1–T11 vertebrae, and insert into the rib below. Serrati muscles Serratus posterior superior arises from a thin and broad aponeurosis from the lower part of the ligamentum nuchae, the spinous processes of the seventh cervical and upper two or three thoracic vertebrae, and the supraspinal ligament. Directed downward and laterally, it is inserted into the upper borders of the second, third, fourth, and fifth ribs, a little beyond their angles. Serratus posterior inferior arises from a thin aponeurosis from the spinous processes of the lower two thoracic and upper two or three lumbar vertebrae, and the supraspinal ligament. It passes obliquely

upward and laterally, dividing into four flat digitations, which are inserted just beyond the angles of the lower four ribs, along their lower borders. It lowers and fixes the ribs. Innervation The intercostal muscles and serrati are supplied by the intercostal nerves, which are the anterior rami of the first eleven thoracic spinal nerves. Levatores costarum muscles are supplied by the posterior rami of the thoracic spinal nerves. Abdominal muscles In functional terms, the abdominal muscles are also an important part of the chest wall, but are mostly expiratory, and are described below. Neck muscles The scalenes comprise three muscle heads that run from the transverse processes of the

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER 635 1

2

3

4

5

6

Internal intercostal muscle

Sternal origin of diaphragm Figure 3 Inner (posterior) surface of the sternum and ribcage, showing the anatomical relationship between the internal intercostals, transversus thoracis, transversus abdominis, and diaphragm. The lowest fibers of transversus thoracis are continuous with those of transversus abdominis. Reproduced from Gray H (1918) Anatomy of the Human Body. Philadelphia: Lea & Febiger.

lower five cervical vertebrae to the upper surface of the first two ribs. The sternomastoids run from the mastoid process to the ventral surface of the manubrium sterni and the medial third of the clavicle (Figure 4). Innervation The scalenes are supplied by branches from the second to the seventh cervical nerves. The sternomastoids are supplied by the spinal accessory nerve and the anterior rami of the second and third cervical nerves. The latter are believed to be proprioceptive. Expiratory muscles Muscles of the ventrolateral abdominal wall These are the most important expiratory muscles (Figure 5). The rectus abdominis arises from the sternum, and the fifth, sixth, and seventh costal cartilages, running caudally to insert into the pubis. It is enclosed in sheaths formed by the aponeuroses of the three muscles situated laterally. These are, from superficial to deep: 1. the external oblique muscle, extending from the external surface of the lower eight ribs to the iliac crest, the inguinal ligament, and the linea alba;

2. the internal oblique muscle, extending from the iliac crest and inguinal ligament, diverging superiorly to insert into the costal margin and an aponeurosis contributing to the rectus sheath; and 3. the transversus abdominis muscle that arises from the inner surface of the lower six ribs, and runs circumferentially to terminate in the rectus sheath. Innervation The abdominal muscles are supplied by the lower six thoracic nerves. The internal and external obliques, and transversus abdominis muscles are also supplied by the iliohypogastric and ilioinguinal nerves. Internal intercostal and transversus thoracis muscles See ‘Intercostal muscles’. Serratus posterior inferior See ‘Serrati muscles’. This may assist forced expiration. Muscles connecting the upper limb and chest wall Pectoral muscles may act as expiratory muscles

636 RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER

Mastoid process

Levator scapulae Splenius capitis Sternomastoid Scalenus anterior Scalenus medius 1st rib

Clavicle

Sternal origin of sternomastoid Clavicular origin of sternomastoid

Figure 4 Neck muscles. The scalenes and sternomastoid muscles act as inspiratory muscles by elevating the upper ribcage. Scalenus posterior (not shown) may be absent, or blended with scalenus medius. Reproduced from Gray H (1918) Anatomy of the Human Body. Philadelphia: Lea & Febiger.

during forced expiration if the shoulder girdle is fixed (Figure 2). Histology and Structure

Overview of skeletal muscle structure The fundamental unit of skeletal muscle is the motor unit, which is a single motor neuron plus the group of muscle fibers it supplies. Respiratory muscles are therefore made up of thousands of skeletal muscle fibers, bound together with nerves and blood vessels by connective tissue. Motor neuron cell bodies are located in the anterior horn of the spinal cord. Muscle fibers Skeletal muscle fibers are multinucleate cells containing striated, thread-like myofibrils, which run the entire length of the muscle fiber and can be seen by light microscopy. They also contain mitochondria, and have an extensive sarcoplasmic reticulum (SR) which stores calcium. The outer cell membrane (sarcolemma) has extensions that project deep into the fiber to the SR, which surrounds the myofibrils. These are the transverse (t)-tubules.

Voltage-sensitive calcium release channels dihydropyridine receptor (DHPR) in t-tubular membranes, and isoforms of the calcium-sensitive calcium release channels, or ryanodine receptors, (RyR1 and RyR3), mediate calcium release from the SR during excitation–contraction coupling in skeletal muscle (Figure 6). Sarcomere structure Sarcomeres are the functional units of the muscle fiber. They contain thick (15 nm diameter) filaments made of the protein myosin, which hydrolyzes adenosine triphosphate (ATP), and thin (5 nm diameter) filaments, made mostly of the protein actin, in addition to the regulatory protein troponin, which binds Ca2 þ , and tropomyosin. Sarcomere shortening, described by the sliding filament model, causes muscle contraction. Fiber type classification Muscle fibers are classified on the basis of their physiological behavior as fast (or fatigable) or slow (fatigue-resistant) (largely an expression of aerobic enzyme activity and calcium metabolism) and by myosin heavy chain isoform into

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER 637

External intercostals

Internal intercostals

Rectus abdominis External oblique

Internal oblique

Transversus abdominis

Figure 5 Attachments of the intercostal muscles and abdominal muscles to the thoracic cage. In functional terms, the abdominal muscles are an important part of the chest wall. Reproduced from Zemlin E and Zemlin WR (1997) Study Guide/ Workbook to Accompany Speech and Hearing Science, 4th edn. Stipes Publishing Co., with permission.

Diaphragm muscle fibers have a smaller cross-sectional area than those in limb muscles but a similar number of capillary vessels. There is also an inverse relationship between aerobic enzyme activity and cross-sectional area. These functional adaptations likely improve oxygen diffusion and contribute to fatigue resistance. Muscle-fiber changes due to training and inactivity do not appear to happen in the same way in respiratory muscles as in limb muscles. General increases in the aerobic capacity of all respiratory muscle fiber types have been observed following whole-body endurance training in rats. Metabolic enzymes and myofibrillar proteins are also not strictly coupled as they are in limb muscles, and the response of aerobic metabolism to endurance training is less marked. Changes in respiratory muscle fiber characteristics are also responsible for increases in contractile strength during perinatal development, as embryonic and neonatal myosin are replaced by adult isoforms. Increases in fatigue-resistant (type 1) myosin from 10% in premature infants, to 25% at term, compared to 55% in adults, have been observed. Diaphragm contractility decreases with age. This is likely to be due to changes other than fiber type composition.

Respiratory Muscle Function in Normal Subjects Inspiration

types 1, 2A, 2B, and 2X. Type 2B is not expressed in human skeletal muscle. With only a few exceptions, each motor unit is composed of fibers of one type, determined mainly by the function of the muscle. Contractile activity can stimulate phenotype shifts between fiber types, depending on whether the stimulus is endurance- or resistance-training. In limb muscles, both endurance- and resistance-training result in transformation of fiber type from 2X to 2A. Transformation from type 1 to 2A has been observed following sprint training, and from 2A to 1 after endurance training. Immobilization shifts muscle fibers to the fast phenotype (Table 1). Fiber types in respiratory muscles The normal adult human diaphragm contains approximately 55% type 1 (slow) fibers, 21% fast oxidative (type 2A) fibers, and 24% fast glycolytic (type 2X) fibers. Fast-fiber types are larger, hence overall o50% is ‘slow’ myosin, and 40% is the 2A isoform. Human intercostal muscles contain slightly more slow-fibers than the diaphragm, approximately 60% in both internal and external intercostal muscles.

Action of the diaphragm accounts for about 70% of minute ventilation in normal humans. The pistonlike downward displacement of the central dome of the diaphragm increases the axial diameter of the thoracic cavity. Most of the diaphragm’s ability to expand the lower ribcage during inspiration is however due to the ‘insertional component’ of the costal diaphragm. The abdominal contents provide a fulcrum, which opposes further downward displacement of the central tendon, allowing elevation of the lower rib. Thirdly, downward displacement of the diaphragm also increases intra-abdominal pressure, causes the anterior abdominal wall to move outwards. This abdominal pressure is also transmitted to, and so expands, the lower ribcage through the ZAP. The effect size of this ‘appositional component’ of costal diaphragm action depends on the area of the ZAP and on the magnitude of abdominal pressure change, that in turn depends on abdominal compliance. The external intercostals, parasternal, and scalene muscles assist the diaphragm, and are active during resting breathing. Comparative studies with the dog suggest that the action of the scalenes, together with

638 RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER

(a)

Mitochondria

Z line

(b)

Z line

Sarcomere

Thick filament Thin filament Z line

H zone I band

I band (c)

A band

Figure 6 (a) Light micrograph of a single human diaphragm muscle fiber. Magnification
40. (b) Electron micrograph of human diaphragm muscle. Magnification
10 000. Sarcomeres, the functional units of skeletal muscle, extend from Z line to Z line. (c) Schematic diagram of the main contractile elements of a single sarcomere. The striated appearance seen in (a) is created by a pattern of alternating dark A bands (thick filaments containing myosin), and light I bands (containing actin, tropomyosin, and troponin) which are bisected by dense Z lines. Z lines anchor the sarcomeres, which extend from Z line to Z line. The H zone is the portion of the A band where the thick and thin bands do not overlap. Light and electron micrographs courtesy of A Moore and A Stubbings, Imperial College, London.

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER 639 Table 1 Skeletal muscle fiber classification according to myosin heavy chain (MyHC) isoform and functional properties MyHC isoform Maximum shortening velocity Myofibrillar ATPase activity Ca2 þ uptake in the sarcoplasmic reticulum Time course of muscle twitch Fatigue resistance Metabolism

Type 1 Slow Low Slow Slow High Oxidative

ribcage muscles, reduces ribcage distortion and therefore elastic work. The external and parasternal intercostals elevate the ribs, and so are inspiratory, and the internal intercostals lower the ribs, and are expiratory. As in the diaphragm, inspiratory drive is matched spatially to respiratory advantage. The inspiratory effect of the external intercostals is greatest in the dorsal portion of the rostral interspace, decreasing downwards and anteriorly, and also decreasing toward the lowest interspaces in the parasternals. During resting breathing, activity appears to be limited to the parasternal intercostals and the external intercostals in the dorsal portion of the more rostral interspaces. In humans, inspiratory drive to the external intercostals appears to be greater than that to the parasternals in the same (rostral) interspace. In forced inspiration, serratus posterior superior elevates the ribs, and serratus posterior inferior draws the lower ribs downward and backward, thus elongating the thorax; and fixes the lower ribs. The sternomastoids act by elevating the upper ribcage. Abdominal muscle recruitment also assists the diaphragm through the ZAP (see above). Expiration

In normal subjects at rest, expiration is passive, but when ventilatory requirements rise, as in exercise, or with disease, the expiratory muscles become active. The abdominal muscles are the most important expiratory muscles. They increase intra-abdominal pressure, displacing the diaphragm into the thorax, and increasing intrathoracic pressure. Expiratory electromyogram (EMG) activity of the internal intercostals has been recorded during resting breathing, but only in the ventrolateral portions of the caudal interspaces, where they have the greatest expiratory mechanical advantage. The transversus thoracis muscle is only active during forced expiration. Its theoretical expiratory effect is limited by its small muscle mass. The pectoral muscles can be recruited to aid forced expiration if the shoulder girdle is fixed. Physiological Determinants of Force (Pressure) Generation

Contraction results in either increased tension or muscle shortening, depending on whether the ends of

Type 2A Fast High High Fast Intermediate Oxidative gycolytic

Type 2X Very fast Very high Very high Fast Low Gycolytic

the muscle are fixed, or not. The force generated by muscle contraction is also related to the number of fibers stimulated, the frequency of stimulation, and length of the muscle at the time of stimulation. Force generation therefore depends on the force–length, force–frequency, and force–velocity relationships, and all are relevant to respiratory muscle function and ventilation. The force produced by the inspiratory muscles is greatest at low lung volumes, and that of the expiratory muscles at high lung volumes. Mean twitch transdiaphragmatic pressure decreases at 3.5 cmH2O l  1 at volumes above normal predicted thoracic gas volume in chronic obstructive pulmonary disease (COPD). Muscle shortening causes a disproportionately large reduction in force generated by stimulation at low frequencies, resulting in the force-generating capacity of the diaphragm being disproportionately diminished in response to physiologic firing frequencies (10–20 Hz). As inspiratory flow rises, the capacity of the respiratory muscles to generate tension is reduced, reflecting the force–velocity relationship. Measurement of Respiratory Muscle Activity

Volitional and nonvolitional tests of respiratory muscle strength are summarized in Figure 7. Pressure swings per unit time (pressure–time product) and normalized to maximum volitional efforts (tension– time index) during respiration are used to assess respiratory muscle workload, relative to capacity. Quantification of spontaneous EMG activity is also of value in the assessment of neural drive, and compound muscle action potential (CMAP) responses to electrical or magnetic phrenic nerve stimulation aid diagnosis of neuromuscular disorders.

Respiratory Muscles in Respiratory Disease Load–Capacity Balance

The clinical significance of respiratory muscle weakness depends on the degree of weakness and the load imposed on the respiratory system. When the respiratory system is otherwise normal, muscle weakness must be severe before breathlessness or respiratory

640 RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER Inspiratory muscle tests

Units

Normal values

Nonvolitional Bilateral TwPdi Right unilateral TwPdi Left unilateral TwPdi

cmH2O cmH2O cmH2O

>18 >7 >8

Volitional Sniff pdi Sniff poes Sniff nasal pressure Mouth inspiratory pressure

cmH2O cmH2O cmH2O cmH2O

>80 >60 >60 (F) or >70 (M) >60 (F) or >80 (M)

cmH2O

>15

Expiratory muscle tests Nonvolitional TwT10 Volitional Mouth expiratory pressure Cough gastric pressure

cmH2O cmH2O

>60 (F) or >80 (M) >100 (F) or >120 (M)

Diaphragm EMG (esophageal) Right phrenic nerve latency Right CMAP amplitude Left phrenic nerve latency Left CMAP amplitude

ms mV ms mV

6–8.0 ms >1.0 mV 6.5–8.5 ms >1.0 mV

Pulmonary function Dynamic compliance Vital capacity (lying) Vital capacity (seated) Vital capacity postural drop KCO PaO2 PaCO2 Bicarbonate pH

l cmH2O −1 l l %

0.1–0.3

kPa kPa HCO3

>11 4.5–6.0 21–28 7.35–7.45

<20

Figure 7 Example of respiratory muscle function test proforma. TwPdi, twitch transdiaphragmatic pressure; Sniff Pdi, sniff transdiaphragmatic pressure; Sniff Poes, sniff esophageal pressure; TwT10, twitch gastric pressure response to T10 nerve root stimulation; CMAP, compound muscle action potential; M, male; F, female; EMG, electromyogram; KCO, gas transfer coefficient.

failure result. However, the load on the respiratory muscles may be increased because of increased ventilatory requirements to ensure adequate oxygen delivery, or because the pressure changes required to achieve a given minute ventilation are increased. In these situations, lesser degrees of respiratory muscle weakness may cause breathlessness and respiratory failure (Figure 8). Respiratory Muscles in Obstructive Lung Diseases

Chronic obstructive pulmonary disease Increased mechanical load COPD imposes an increased load on the respiratory system, both resistive and elastic, and intrathoracic pressure swings are often much higher than normal. Decreased capacity Hyperinflation It impairs the capacity of the respiratory muscles to generate negative intrathoracic

pressure because of the shortening of the diaphragm, and to a lesser extent, the chest wall muscles. Hyperinflation also decreases the zone of apposition. The ability of the diaphragm to generate transdiaphragmatic, and particularly a negative intrathoracic, pressure is reduced in COPD due to the mechanical disadvantage of the muscle as well as shortening. Dynamic hyperinflation and work of breathing during exercise in COPD are reduced by bronchodilator therapy, and surgical or bronchoscopic lung-volume reduction. Heliox (a low-density gas mixture of 79% He, 21% O2) and oxygen also reduce dynamic hyperinflation and improve exercise capacity, the latter by reducing respiratory rate and increasing the relative time spent in expiration. Abdominal expiratory muscle strength is well maintained in COPD. More than half of patients with severe COPD actively recruit their transverse abdominis during resting breathing, and most vigorously recruit the abdominal muscles during exercise.

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER 641

Cortex

Ventilatory drive

Hyperinflation with muscle shortening abnormal geometry

Capacity of respiratory muscles

Respiratory muscles

Load on respiratory muscles

Increased ventilation airways obstruction increased PEEPi stiff lungs/chest wall

Dyspnea ventilatory failure Figure 8 An imbalance between the load on, and capacity of, the respiratory muscles leads to breathlessness and respiratory failure.

However, expiratory flow limitation prevents the expiratory muscles from effectively reducing endexpiratory lung volume, and the usefulness of this recruitment is not clear. Altered neural respiratory drive Despite electrophysiological recordings from the diaphragm, scalenes and parasternal intercostals demonstrating that neural inspiratory drive to the respiratory muscles is increased in COPD, ventilatory failure remains the most common cause of death. The results of a recent study using transcranial magnetic stimulation to investigate the excitability of intracortical facilitatory circuits suggest that there is a ceiling effect in motor cortical output to the respiratory muscles in COPD patients. During exercise, however, neural activation of the diaphragm increases progressively and out of proportion to increases in minute ventilation and tidal swings in transdiaphragmatic pressure, which increase relatively modestly and plateau. Neuromechanical dissociation of this degree is likely to be one explanation for the disabling levels of exertional breathlessness experienced by these patients. COPD-related myopathy In contrast to the detrimental effects of peripheral muscle-wasting on exercise capacity and quality of life in COPD, respiratory muscles appear to undergo adaptive changes that reduce the likelihood of the development of fatigue. Patients with COPD do not develop diaphragmatic fatigue when exercising to exhaustion. Possible protective factors include increased diaphragmatic

mitochondrial content, increased proportion of slow fatigue-resistant muscle fibers, unimpeded blood flow to the diaphragm because of the limited rise in transdiaphragmatic pressure, and redistribution of cardiac output from the lower limb-exercising muscles to the respiratory muscles. The effect of glucocorticoids on respiratory muscle strength is unclear. Although glucocorticoids are implicated in the generalized myopathic syndrome, patients with larger respiratory workloads who do not receive glucocorticoids have been shown to have greater respiratory endurance than those that do, leading to the suggestion that glucocorticoids may counterbalance a ‘training effect’ of airway obstruction. Respiratory muscle training The results of the few randomized controlled trials including an adequate training stimulus (an intensity of at least 30% PImax) provide evidence to support the use of specific inspiratory muscle training (SIMT) to improve the dyspnea and exercise capacity of symptomatic COPD patients who have inspiratory muscle weakness. Overall there is a paucity of data regarding specific expiratory muscle training (SEMT). Both SIMT and SEMT have been shown to improve exercise capacity, but improvements in exercise performance in response to SIMT (either alone or with SEMT) appear to exceed those due to SEMT, with no benefit of combining the two. SIMT, but not SEMT, has been shown to improve exertional dyspnea. Maintenance training preserves the effects of inspiratory muscle training. SIMT, adequate to produce a training response, has been associated

642 RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER

with increases in both the proportion of slow fibers and the size of the fast fibers in the external intercostals. More well-designed randomized controlled trials are needed to evaluate the long-term impact of respiratory muscle training on functional and clinical outcomes, and to design more effective and efficient training programs. Asthma Increased load The respiratory muscles of asthmatics are often heavily loaded, resulting from airways obstruction, as in COPD. Postinspiratory activity of the diaphragm and ribcage inspiratory muscles may brake expiratory airflow. End expiratory lung volume is actively increased, and it has been suggested that this may minimize flow limitation during bronchoconstriction, but at the cost of breathing at higher lung volumes. Abdominal muscle recruitment enhances the capacity of the diaphragm to generate pressure through its ZAP, and may limit the deleterious effects of the load imposed by hyperinflation. The contribution of inspiratory muscle recruitment during exhalation to chronic hyperinflation is unclear. Decreased capacity As with COPD, hyperinflation reduces the capacity of the respiratory muscles in asthma. A recent study of patients presenting to Accident and Emergency with moderately severe acute asthma found only a minor reduction in inspiratory pressure-generating capacity, after taking the degree of hyperinflation into account. Factors associated with an increased risk of death from acute asthma include underestimation of the severity of airway narrowing, and reduced perception of dyspnea, suggesting that the drive to breathe may be reduced in this subgroup of asthmatics. Although there is no direct evidence for this, voluntary activation of the diaphragm has been shown to be reduced in about half of unselected asthmatic patients studied using phrenic nerve stimulation to assess the completeness of voluntary diaphragm activation. Cystic fibrosis Diaphragm and abdominal muscle bulk are not affected by the general muscle wasting, which suggests that there may be a training effect of cystic fibrosis on respiratory muscles. Twitch transdiaphragmatic pressure (TwPdi) has been shown to be 23% lower, but twitch gastric pressure 22% greater in patients with cystic fibrosis than in control subjects. Although the values of Pdi were not compared at similar lung volumes, the decrease in TwPdi was similar to the reduction reported in patients with COPD, muscle-shortening secondary to hyperinflation likely to be the most important mechanism.

Respiratory Muscles in Restrictive Lung Diseases

Altered lung and chest wall mechanics increase the mechanical load on the inspiratory muscles in both lung parenchymal and chest wall restrictive diseases. Chest wall compliance is inversely proportional to the Cobb angle in stable patients with scoliosis, and spinal deformity produces inefficient coupling between the respiratory muscles and chest wall, so that both maximal mouth inspiratory and expiratory pressures may be reduced. However, exercise capacity is not directly related to either respiratory muscle strength or Cobb angle. Respiratory muscle dysfunction may also be observed in patients with multisystem disease. Maximum inspiratory muscle pressure has been shown to be reduced in adult patients with rheumatoid arthritis, and children with juvenile chronic arthritis can exhibit inspiratory and expiratory muscle weakness, which may be due to the associated myopathy. Although a glucocorticoid-responsive phrenic nerve neuropathy has been reported in systemic lupus erythematosus (SLE), TwPdi has been shown to be normal in ‘shrinking lung syndrome’. Sarcoid neuropathy and myopathy can cause respiratory muscle weakness, but this rarely causes ventilatory failure. Respiratory muscle weakness due to neuromuscular disease may complicate a wide range of disorders (Table 2). Ventilatory failure is an important cause of morbidity and mortality in progressive neurological disorders, such as amyotrophic lateral sclerosis (ALS) Table 2 Neuromuscular disease causing respiratory muscle weakness Neurogenic Amyotrophic lateral sclerosis Polyneuropathy (e.g., Guillain–Barre´ syndrome) Surgical trauma of phrenic nerves Poliomyelitis Multiple sclerosis Traumatic tetraplegia Acute porphyria Neuralgic amyotrophy Neuromuscular junction Myasthenia gravis Lambert–Eaton syndrome Muscular Muscular atrophy Inflammatory myopathies Muscular dystrophy Myotonic dystrophy SLE Acid maltase deficiency Thyroid myopathy Biochemical abnormalities, e.g., acidosis Steroid therapy

RETINOL AND RETINOIC ACID 643

(see Neuromuscular Disease: Inherited Myopathies; Lower Motor Neuron Diseases; Myasthenia Gravis and Other Diseases of the Neuromuscular Junction; Acquired Myopathies; Peripheral Nerves; Upper Motor Neuron Diseases). Expiratory muscle weakness resulting in an impaired cough, leads to chest infections. Symptoms, clinical signs, and results of imaging and lung function tests may alert clinicians to the possibility of respiratory muscle weakness. A combination of respiratory muscle function tests, including magnetic (or electrical) stimulation of the phrenic nerves, is used to confirm the diagnosis of respiratory muscle, and specifically diaphragm weakness. Although unilateral hemidiaphragm paralysis is unlikely if diaphragm elevation is not present on the chest X-ray, isolated elevation of hemidiaphragm on chest X-ray is of little value in diagnosis of the condition. Evidence of sleep-disordered breathing and/or daytime hypercapnia should alert the clinician to the need for consideration of noninvasive ventilatory support. Approximately 5% of spinal cord-injured individuals suffer from respiratory muscle paralysis and require chronic mechanical ventilation. Diaphragm pacing using either phrenic nerve or laparascopically-placed intramuscular electrodes has been successfully achieved in ventilator-dependent tetraplegics with intact phrenic nerves, with marked improvements in quality of life. Diaphragm plication can improve ventilation in bilateral diaphragm paralysis. The respiratory muscles are therefore affected by a wide variety of both primary lung diseases and multisystem disorders. Understanding of their normal, and abnormal, function, allows an understanding of the pathophysiology of breathlessness and respiratory failure, and facilitates their treatment and palliation. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Cystic Fibrosis: Overview. Neuromuscular Disease: Acquired Myopathies.

Neurophysiology: Neuroanatomy. Reflexes from the Lungs and Chest Wall.

Further Reading American Thoracic Society/European Respiratory Society (2002) ATS/ERS Statement on respiratory muscle testing. American Journal of Respiratory and Critical Care Medicine 166(4): 518–624. Binazzi B, Lanini B, and Scano G (2004) Assessing respiratory drive and central motor pathway in humans: clinical implications. Lung 182(2): 91–100. Cooke R (2004) The sliding filament model: 1972–2004. The Journal of General Physiology 123(6): 643–656. De Troyer A, Kirkwood PA, and Wilson TA (2005) Respiratory action of the intercostal muscles. Physiological Reviews 85(2): 717–756. Gray H (1918) Anatomy of the Human Body. Philadelphia: Lea & Febiger. Green M and Moxham J (1985) The respiratory muscles. Clinical Science (London) 68(1): 1–10. Laghi F and Tobin MJ (2003) Disorders of the respiratory muscles. American Journal of Respiratory and Critical Care Medicine 168(1): 10–48. Man WD, Moxham J, and Polkey MI (2004) Magnetic stimulation for the measurement of respiratory and skeletal muscle function. European Respiratory Journal 24(5): 846–860. Moxham J, Morris AJ, Spiro SG, Edwards RH, and Green M (1981) Contractile properties and fatigue of the diaphragm in man. Thorax 36(3): 164–168. Orcozi-Levi M (2003) Structure and function of the respiratory muscles in patients with COPD: impairment or adaptation? The European Respiratory Journal Supplement 46: 41s–51s. Pickering M and Jones JF (2002) The diaphragm: two physiological muscles in one. Journal of Anatomy 201(4): 305–312. Polkey MI (2002) Muscle metabolism and exercise tolerance in COPD. Chest 121(5 supplement): 131S–135S. Polkey MI, Lyall RA, Moxham J, and Leigh PN (1999) Respiratory aspects of neurological disease. Journal of Neurology, Neurosurgery, and Psychiatry 66(1): 5–15. Polkey MI and Moxham J (2001) Clinical aspects of respiratory muscle dysfunction in the critically ill. Chest 119(3): 926– 939. Polla B, D’Antona G, Bottinelli R, and Reggiani C (2004) Respiratory muscle fibres: specialisation and plasticity. Thorax 59(9): 808–817. Zemlin E and Zemlin WR (1997) Study Guide/Workbook to Accompany Speech and Hearing Science, 4th edn: Stipes Publishing Co.

RETINOL AND RETINOIC ACID J S Koo, University of Texas, Houston, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Retinol, also known as vitamin A, is essential for life. Retinol in the human body is produced by a series of metabolic processes

that begin with dietary intake of b-carotene and retinyl esters. Retinoic acid (RA) is one of the most potent physiologic metabolites of retinol. Retinol and RA are essential for pre- and postnatal development of the lung and for the induction, maintenance, and regeneration of differentiated epithelial cells in the proximal and distal parts of the lung. These effects are mediated mainly through the nuclear receptors RA receptor and retinoid X receptor. RA-bound dimers of RA receptors and retinoid

644 RETINOL AND RETINOIC ACID X receptors transactivate RA target genes complexed with numerous transcriptional coactivators. Recent observations that exogeneous RA regenerates alveoli septum damaged by elastase in a rat model suggest the possibility of using retinoids or novel targets in the retinoid signaling pathways involved in the regeneration of the damaged alveoli to treat emphysema. Some natural and synthetic retinoids used to prevent or treat several types of tumors have been clinically effective. Better understanding of the biologic effects of retinoids and their target genes regulated differentially in normal and cancer cells will help researchers exploit the beneficial aspects of the retinoids for the prevention and treatment of cancers.

Introduction The discovery of the presence of an active ingredient in the diet that was later identified as vitamin A can be dated back to the ancient Egyptians, Greeks, and Assyrians, who treated night blindness with a diet containing animal liver. The actual discovery of vitamin A was made by McCollum in 1913, who extracted ‘fat-soluble factor A’ from butter fat, and that factor proved to be essential for growth and survival in experimental animals. This factor was later isolated from egg yolk fat and cod-liver oil and renamed ‘factor A’ and then vitamin A or retinol. Yellow pigment extracted from carrots was found to have similar effects on growth to ‘factor A’. This pigment, which is found in certain plants, was identified as b-carotene, and is now known to be a metabolic precursor of retinol in animals. The chemical structure of b-carotene and retinol was determined by Karrer in 1931. During the 1950s and 1960s, numerous investigators mapped the metabolic pathway of b-carotene and retinol and identified retinoic acid (RA) as one of the most potent active metabolites of retinol. In 1987, understanding of the biologic activity of RA was tremendously boosted after the simultaneous discovery of RA receptor (RAR) in the laboratories of Chambon and Evans.

Structure The International Union of Pure and Applied Chemistry–International Union of Biochemistry Joint Commission on Biochemical Nomenclature designated the compound (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6trimethylcyclohexen-1-yl)-2,4,6,8-nonatetraen-1-ol as retinol and its acid form as RA. Retinol analogs named as retinoids were defined as: ‘‘a class of compounds consisting of four isoprenoid units joined in a head-totail manner. All retinoids may be formally derived from a monocyclic parent compound containing five carbon–carbon double bonds and a functional group at the terminus of the acyclic portion.’’

Regulation of Production and Activity All retinol and RA in the body originate from the diet as carotenoids in vegetables and retinyl esters in animal fats and dairy products (Figure 1). Most of the absorbed carotenoids and retinyl esters are further metabolized into retinol by a series of enzymes in the enterocytes of intestinal mucosa. The retinol bound to retinol-binding protein is transferred through the blood to the liver and other tissues and stored as retinyl esters in specific cells in various tissues, such as stellate cells in the liver and lipid interstitial cells in the lung. In the cytoplasm of cells, the retinol is oxidized into retinal by retinol dehydrogenases and then further oxidized into retinoic acid by retinal dehydrogenases. Complex formation of retinol with cellular retinol-binding protein and of RA with cellular retinoic acid-binding protein facilitates the enzymatic processes. The all-trans form of RA is the most potent physiologically active retinol metabolite, and its isomer, 9-cis-RA, also has substantial biologic activity. RA is further metabolized into 4-hydroxyRA, 4-oxo-RA, and 18-hydroxy-RA, but the enzymes involved in these processes and the biologic role of these metabolites remain to be fully characterized. Recent evidence has indicated that a family of cytochrome P-450 enzymes, Cyp26, catalyzes this process. All of these retinol metabolites, including RA, are collectively called retinoids. In humans, the average plasma concentration of retinol and RA is 1 mM and 4–14 nM, respectively.

Biologic Function Retinol is essential for embryonic development and the genesis of many different organs, including the lung. Retinol deficiency during embryonic development results in abnormal development of the lung, and uncontrolled overproduction of retinol metabolites causes tetratogenesis. In prenatal lung development, ablation of the genes involved in RA production results in incomplete branching morphogenesis in animal models. Although the role of RA in postnatal lung development is less clear, vitamin A deprivation in pregnant rats during fetal lung development is known to result in blunt-end trachea and lung agenesis in some embryos. In addition, expression of retinoid-binding proteins and RAR is temporally and spatially regulated during lung development. Retinol and RA are also essential for the maintenance of the mucociliary epithelium in the conducting airways of the lung. Chronic vitamin A deficiency causes severe squamous metaplasia in the airways. The importance of retinoids in epithelial cell differentiation

RETINOL AND RETINOIC ACID 645


-Carotene (from plant diet)
-Carotenoid-15, 15′-dioxygenase

COOH

CHO

Retinal dehydrogenase

Retinal

Retinoic acid

Retinal dehydrogenase

CH2OH

Retinol (vitamin A)

Retinyl ester hydrolase

ARAT (aceyl-CoA:retinol acyltransferase) LRAT (lecithin:retinol acyltransferase)

OCO-(CH2)7CH3

Retinyl palmitate (from animal fat diet) Figure 1 Major metabolic pathways of RA synthesis.

was first recognized in 1925 by Wolbach and Howe, who reported the development of widespread squamous metaplastic changes in the mucous membranes in the respiratory, reproductive, and urinary tracts of vitamin A-deficient animals. Since then, numerous investigators have shown that adding retinol or retinoids to the diet of vitamin A-deficient animals leads to full restoration of the mucous phenotype from the metaplastic squamous phenotype in the airway and other organs. These in vitro and in vivo studies clearly established vitamin A as a critical factor in controlling mucociliary differentiation in the conducting airways. Recent in vitro studies using organotypically cultured primary bronchial epithelial cells, which closely resemble in vivo mucociliary bronchial epithelium, have shown that RA restores the mucous phenotype from the metaplastic squamous phenotype

caused by RA deficiency. The induction and restoration of the mucous phenotype in airway epithelial cells by RA is accompanied by the induction of the expression of RARb and several mucin genes, including MUC2, MUC5AC, and MUC5B, which are the major secretory mucins expressed in the airway mucosa. MUC5AC is expressed exclusively by surface epithelial cells and thus is used as a specific marker for mucous cells. RA also suppresses a specific squamous cell marker, cornifin a, during induction of mucous cell differentiation. Thus, RA may have two roles in maintaining mucociliary bronchial epithelium. However, whether RA simultaneously regulates mucous and squamous phenotypes or whether suppression of squamous metaplasia is just a consequence of the induction of mucous cells (or vice versa) is unknown.

646 RETINOL AND RETINOIC ACID

Receptors RA binds to its receptors, which are located in the cell nucleus and act as transcription factors on binding of RA. There are two distinct families of nuclear retinoid receptors, the RARs and the retinoid X receptors (RXRs). Each family consists of three isotypes (a, b, and g) that are encoded on separate genes in different chromosomes. For each isotype, at least two isoforms are expressed via different transcriptional initiation and alternative splicing. RARs form dimers with RXRs to recognize and regulate RA target genes by binding to the RA response element (RARE). RARE is typically composed of direct repeats of (A/G)G(G/T)TCA, which are usually separated by one or more non-specific nucleotides, in the promoters of target genes (Figure 2). RXRs can form dimers with RARs, with themselves, or with various other nuclear receptors, including thyroid hormone receptor, vitamin D receptor, and peroxisome proliferator-activated receptor. RARs are activated by alltrans-RA and its 9-cis isomer, whereas RXRs are activated only by 9-cis-RA. When the receptor dimers are not bound to RA, they assemble with nuclear repressors NcoR and SMRT and repress RA target genes. On binding of RA to the ligand-binding pocket of RARs, the

Retinol

receptor changes its conformation, dissociates suppressors, and recruits the nuclear receptor coactivator complex. Ligand-bound RAR/RXR heterodimers complexed with the coactivators facilitate recruitment of general transcription machinery to the promoter and initiate transcription of the RA target genes (Figure 2). Some of the RA target genes that contain classic RARE in their promoters encode proteins important in RA metabolism and signaling, such as cellular retinol-binding protein, cellular retinoic acid-binding protein, Cyp26, and RARb2. RARE is also found at the 30 side of RA target genes, as seen in the Hox family of homeobox genes. Specific biologic functions of retinoid receptors have been identified using mutant mice with single, double, or compound disruptions of the receptors. Homozygous mutant mice for individual isoforms of RARa1, -b2, or -g2 exhibit normal phenotype, whereas disruption of all RARa or RARg isoforms results in postnatal lethality and malformations. RARa/b2 knockout mice exhibit agenesis of the left lung and hypoplasia of the right lung. RXRa knockout mice show embryonic lethality, but RXRb knockout mice show normal phenotype except for defects in spermatogenesis. Double knockout mice and mice with compound mutations of RXRs show that RXRa is sufficient for RXR function.

RA

Retinal Cytoplasm Nucleus * In the absence of RA

* In the presence of RA RA Coactivators

Repressors RA RAR

RXR

RARE

Transcription Repression

RAR

X

RXR

Transcription Activation

RARE

(A/G)G(G/T)TCA n5(A/G)G(G/T)TCA Target genes

Embryonal development

Neonatal lung development

Lung morphogenesis

Lung epithelial differentiation and regeneration

Figure 2 A simplified model of retinol and RA in the regulation of genes involved in various biological functions.

RETINOL AND RETINOIC ACID 647

Retinoid Therapy for Respiratory Disease RA is critical for lung development and maintenance of lung structure. Exogeneous RA can induce regeneration of alveoli damaged by elastase in the adult rat lung, which is used as an experimental model for human emphysema. Although the mechanisms of RA-induced regeneration are not yet clear, studies have shown that RARb negatively and RARg positively affects the regeneration of lung alveoli. However, complete, partial, and no response have all been observed in alveoli regeneration by RA in experimental animal model systems. Currently, clinical trials are underway to determine the feasibility of retinoids in the treatment of emphysema (FORTE study). Several natural and synthetic retinoids have been shown to be effective in reversing premalignant lesions, arresting the malignant progression of several types of cancers, and preventing the development of second primary tumors. However, randomized clinical trials using a pharmacologic dose of b-carotene to prevent lung cancer have not shown beneficial effects and, in fact, showed harmful effects for individuals who have a high risk of lung cancer, such as heavy smokers or people who have been exposed to asbestos. In contrast, recent clinical trials showed that 3 months of 9-cis-RA treatment in former smokers significantly reduced metaplasia and restored RARb expression, which is often lost in squamous metaplasia or preneoplasia in the bronchial epithelium, and restoration of RARb expression indicates restoration of the normal epithelium. This result raises the possibility of using the chemopreventive properties of retinoids in former smokers. Moreover, studies of the status of RARb expression and the incidence of second primary lung cancer in former and current smokers showed that RARb is protective only in former smokers. Comprehensive understanding of retinoid biology in reversible and irreversible squamous metaplasia or preneoplasia of bronchial epithelial cells will facilitate the identification and development of better molecular targets for chemoprevention and chemotherapy.

See also: Chronic Obstructive Pulmonary Disease: Emphysema, General. Cilia and Mucociliary Clearance. Epithelial Cells: Type II Cells. Lung Development: Overview. Mucins. Mucus. Tumors, Malignant: Chemotherapeutic Agents.

Further Reading Altucci L and Gronemeyer H (2001) The promise of retinoids to fight against cancer. Nature Review of Cancer 1: 181–193. Bastien J and Rochette-Egly C (2004) Nuclear retinoid receptors and the transcription of retinoid-target genes. Gene 328: 1–16. Blaner WS and Olson JA (1994) Retinol and retinoic acid metabolism. In: Sporn MB, Roberts AB, and Goodman DS (eds.) The Retinoids: Biology, Chemistry, and Medicine, 2nd edn., pp. 229–255. New York: Raven Press. Chambon P (1996) A decade of molecular biology of retinoic acid receptors. FASEB Journal 10: 940–954. Chytil F (1996) Retinoids in lung development. FASEB Journal 10: 986–992. Gudas LJ, Sporn MB, and Roberts AB (1994) Cellular biology and biochemistry of the retinoids. In: Sporn MB, Roberts AB, and Goodman DS (eds.) The Retinoids: Biology, Chemistry, and Medicine, 2nd edn., pp. 443–520. New York: Raven Press. Maden M and Hind M (2004) Retinoic acid in alveolar development, maintenance and regeneration. Philosophical Transactions of the Royal Society of London, Series B: Biological Sciences 359: 799–808. Mangelsdorf DJ, Umesono K, and Evans RM (1994) The retinoid receptors. In: Sporn MB, Roberts AB, and Goodman DS (eds.) The Retinoids: Biology, Chemistry, and Medicine, 2nd edn., pp. 319–349. New York: Raven Press. Massaro D and Massaro GD (2003) Retinoids, alveolus formation, and alveolar deficiency: clinical implications. American Journal of Respiratory Cell and Molecular Biology 28: 271–274. Napoli JL (1999) Interactions of retinoid binding proteins and enzymes in retinoid metabolism. Biochimica et Biophysica Acta 1440: 139–162. Nettesheim P, Koo JS, and Gray T (2000) Regulation of differentiation of the tracheobronchial epithelium. Journal of Aerosol Medicine 13: 207–218. Ross SA, McCaffery PJ, Drager UC, and De Luca LM (2000) Retinoids in embryonal development. Physiological Reviews 80: 1021–1054. Soria JC, Kim ES, Fayette J, et al. (2003) Chemoprevention of lung cancer. Lancet Oncology 4: 659–669. Sun SY and Lotan R (2002) Retinoids and their receptors in cancer development and chemoprevention. Critical Reviews in Oncology/Hematology 41: 41–55. Wolf G (1996) A history of vitamin A and retinoids. FASEB Journal 10: 1102–1107.

S Sarcoidosis

see Systemic Disease: Sarcoidosis.

Scatter Factor

see Hepatocyte Growth (Scatter) Factor.

SERINE PROTEINASES C A Owen, Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Serine proteinases are a family of mammalian enzymes with a serine group at their active site. They include the leukocyte serine proteinases, neutrophil elastase, cathespin G, proteinase-3, plasminogen activators, granzymes, chymases, tryptases, and digestive enzymes such as trypsin. Leukocyte serine proteinases are generally synthesized by their bone marrow precursors, stored in intracellular granules, and released from cells when they are activated. Serine proteinases cleave various proteins, such as extracellular matrix proteins, coagulation and complement proteins, cytokines, and cell-surface receptors. They also activate latent matrix metalloproteinases (MMPs) and inactivate tissue inhibitors of MMPs. Serine proteinases kill bacteria and fungi, and they induce platelet activation, goblet cell secretion, and synthesis of cytokines by epithelial cells and phagocytes. Granzymes of cytotoxic lymphocytes kill virally infected cells and tumor cells. The activity of serine proteinases is controlled by serine proteinase inhibitors present in extracellular fluids. Serine proteinases likely play diverse roles in physiologic processes such as tissue repair; in regulating inflammation, coagulation, fibrinolysis, and complement activation; and in host defense. However, when their activity is excessive or inadequately controlled, they may play pathogenetic roles in acute and chronic lung diseases, including chronic obstructive pulmonary disease, cystic fibrosis, acute lung injury, and pulmonary fibrosis.

Introduction Serine proteinases are the largest class of mammalian proteinases. They are so called because they have a catalytically essential serine residue at their active sites. Serine proteinases are optimally active at neutral pH and play major roles in extracellular proteolysis. This class of proteinases includes digestive enzymes (trypsin and chymotrypsin), leukocyte proteinases, and many of the proteins involved in

coagulation, fibrinolysis, and complement activation (Table 1). Examples of serine proteinases discussed further in this article include the leukocyte serine proteinases neutrophil elastase (NE), cathepsin G (CG), and proteinase-3 (PR3), plasminogen activators (PAs), and granzymes of cytotoxic lymphocytes (CTLs). Mast cell chymases and tryptases, the kinin family of serine proteinases, and serine proteinases involved in the coagulation and complement cascades are reviewed in other articles.

Historical Aspects In 1888, Friedrich Von Muller showed that extracts of pus contain enzymes that digest coagulated proteins at neutral pH, which we now know to be substantially mediated by polymorphonuclear neutrophil (PMN)derived serine proteinases. NE and CG were identified and biochemically characterized in the 1960s and 1970s. PR3 and granzymes were identified in the 1980s. Subsequent in vitro studies of serine proteinases showed that they cleave a broad spectrum of proteins, including extracellular matrix (ECM), proinflammatory mediators, growth factors, surface receptors, and bacterial cell wall proteins, suggesting they play roles in tissue repair and remodeling, inflammation, coagulation, and host defense. Since the 1990s, mice genetically deficient in serine proteinases have been generated by gene targeting. Studies of these mice are providing new insights into their biologic substrates and their pathologic roles in the lung. Structure of Serine Proteinases

Serine proteinases have a high degree of structural homology and are thought to have diverged from a

2 SERINE PROTEINASES Table 1 Sources and biologic activities of serine proteinases Proteinase

Sources

Substrates/activities

Neutrophil elastase Cathepsin G Proteinase-3

PMNs P Monocytes

ECM proteins (elastin, basement membrane proteins), proteoglycans

Plasminogen activators (PAs) Urokinase-type PA Tissue-type PA

PMNs MNPs Endothelial cells

Plasma proteins (immunoglobulins, clotting factors, complement components) Cytokines and their receptors Growth factor activation (pro-TGF-a, latent TGF-b for NE) Inactivation of TIMPs (NE) Angiotensin I conversion to angiotensin II (CG) Receptors (CD2, CD4, CD8, complement receptors, macrophage phosphatidyl serine receptor, protease activated receptors) Bacterial and fungal killing Lymphocyte and platelet activation Cytokine/chemokine secretion by epithelial cells and MNPs Mucin MUC5A secretion by submucosal glands Goblet cell degranulation Myelomonocytic growth and differentiation (PR3) Autoantigen in Wegener’s granulomatosis (PR3)

Plasmin

Convert plasminogen to plasmin Cell adhesion and migration (uPAR bound uPA)

Fibrin, fibronectin, laminin, proteoglycans Activates latent proMMPs

Coagulation proteins (e.g., thrombin)

Plasma

Hemostasis

Complement components

Plasma

Complement activation

Chymases

Mast cells

ECM proteins, proteoglycans, angiotensin I, substance P Mucus secretion by submucosal glands Goblet cell degranulation

Tryptase

Mast cells

ECM proteins Activation of pro-MMP-3

Granzymes (GRZs)

CD8 þ T cells NK cells

Trypsin and chymotrypsin

GI tract

Apoptosis of virally infected cells and tumor cells Proteoglycans (GRZ B) Cytokine secretion by MNPs, fibroblasts, and epithelial cells (GRZ A) Digestion of proteins in the small intestine

CG, cathepsin G; ECM, extracellular matrix; GRZs, granzymes; MMP, matrix metalloproteinase; MNPs, mononuclear phagocytes; NE, neutrophil elastase; PAs, plasminogen activators; PMNs, polymorphonuclear neutrophils; P monocytes, proinflammatory monocytes; PR3, proteinase 3; TGF, transforming growth factor; TIMPs, tissue inhibitors of metalloproteinases; NK, natural killer.

primitive digestive enzyme through gene duplication and mutation. The genes for most serine proteinases have five exons and four introns. The active site serine residue of serine proteinases forms part of the active site ‘catalytic triad’ of Asp, His, and Ser residues. Each residue of this Asp–His–Ser catalytic triad is located at a similar position in the same separate exon in serine proteinases. These residues are widely separated in the primary sequences of the enzymes but are brought together at the active sites of their tertiary structures. The active site of serine proteinases forms a charge relay system transferring electrons from the carboxy group of Asp to the oxygen group of Ser that becomes a powerful

nucleophile attacking the carbonyl carbon atom of the target peptide bond in the substrate. NE, CG, and PR3

The genes for human NE and PR3 are located on chromosome 19 (19p13.3). CG is located (with granzymes B and H and mast cell chymase) on chromosome 14q11.2. NE, CG, and PR3 are single-chain glycoproteins having MrB29 kDa, and they are highly cationic (with CG being the most and PR3 the least positively charged) due to the presence of multiple arginine residues on their external surfaces. NE has 218 amino acids and two N-linked carbohydrate side

SERINE PROTEINASES 3

chains. CG has 235 amino acids, shares 37% sequence homology with NE, and has one potential N-linked glycosylation site. PR3 has 228 amino acids, shares 54% sequence homology with NE, and has two potential N-linked glycosylation sites. Plasminogen Activators

PAs cleave inactive plasminogen (which is abundant in extracellular fluids) to plasmin, a serine proteinase that degrades fibrin. There are two classes of PAs: human tissue type PA (tPA) and urokinase PA (uPA). The gene for human uPA is located on chromosome 8 and contains 14 exons coding for a 70 kDa protein. The gene for human uPA is located on chromosome 10 and has 11 exons coding for a 53 kDa protein. Both PAs have a signal peptide (which is removed during synthesis) and are secreted as single-chain enzymes. While single-chain tPA is active, single-chain uPA is inactive (pro-uPA). Cleavage of pro-uPA by plasmin, kallikrein, factor XIIa, or cathepsin B yields the disulfide-linked two-chain active uPA enzyme. These enzymes also differ in the structure and function of their noncatalytic domains. In tPA, the structural domains are encoded by separate exons, including an amino-terminal finger domain, a growth factor-like domain, two triple-disulfide structures called kringles, and the active serine catalytic site at the carboxy terminus. However, uPA lacks the finger domain and has only the first kringle domain. The growth factor-like domain of uPA directs its binding to its cell-surface receptor (uPAR) on leukocytes, whereas the finger and kringle domains of tPA allow it to bind to fibrin and other ECM proteins. Granzymes

These ‘granule-associated enzymes’ are contained within the granules of CTLs. Granzymes (GRZs) A, B, H, K, and M are expressed by human cells, and GRZ A–G, J, K, M, and N are found in rodents. Human GRZ A and B are encoded by genes on chromosome 5q11–q12 and 14q11–q12, respectively. GRZs are structurally related to chymotrypsin. They have the Asp–His–Ser catalytic triad and a high degree of structural homology, but they vary in size from 27 to 65 kDa and have very heterogeneous glycosylation.

bone marrow precursors and are stored at millimolar concentrations within the azurophil granules of PMN and in monocyte primary granules. The inactive precursor forms are activated by cleavage of an N-terminal propeptide by cathepsin C (dipeptidyl peptidase I), a cysteine proteinase localized within the same granules. PMNs are homogeneous in their content of NE, CG, and PR3, but only a subpopulation of circulating monocytes (B30%) contains NE, CG, and PR3. These monocytes have a PMN-like proinflammatory (P) phenotype. As these P monocytes mature into macrophages, their content of serine proteinases is lost and replaced by MMPs. However, macrophages bind and internalize serine proteinases released by PMNs and then release serine proteinases when macrophages are exposed to hypoxic conditions. NE, CG, and PR3 are freely released from PMNs and P monocytes when they are activated to degranulate by proinflammatory mediators and immune complexes. The released enzymes also bind in a catalytically active form to the external surface of the plasma membranes of PMNs and monocytes by a charge-dependent mechanism. Membrane-bound NE, CG, and PR3 on activated PMNs and monocytes have a similar spectrum of catalytic activity as the soluble forms of the proteinases and likely play roles in pericellular proteolysis. Plasminogen Activators

Tissue-type PA is synthesized by endothelial cells, and uPA is produced by inflammatory cells, endothelial cells, and fibroblasts. Urokinase-type PA is stored within PMN granules (80% in the specific granules and 20% in the gelatinase granules), but it is synthesized and secreted by activated mononuclear phagocytes, endothelial cells, and fibroblasts. Inflammatory cells and lung resident cells also express a cell-surface form of uPA that binds to high-affinity uPA receptors (uPARs) attached to their plasma membrane by a glycosyl–phosphatidyl–inositol anchor. The growth-factor-like domain near the amino terminus of uPA binds to uPAR on leukocytes and resident cells, leaving the catalytic domain of uPA at its carboxy terminus accessible to plasminogen. Granzymes

Production and Localization of Serine Proteinases NE, CG, and PR3

These enzymes are not transcriptionally regulated in circulating leukocytes. They are synthesized as inactive precursors by promyelocytic and promonocytic

GRZ expression is restricted to CTLs (CD8 þ T lymphocytes and NK cells), gD T cells, and thymocytes. They are stored as inactive zymogens in the lytic granules of CTLs and are activated by cathepsin C within these granules. Activation of CTLs by the binding of their T-cell receptor to antigen bound to major histocompatability complex (MHC) class I

4 SERINE PROTEINASES

molecules on target cells leads to rapid exocytosis of their lytic granules containing GRZs and apoptosis of the target cell.

Activities and Biologic Functions of Serine Proteinases NE, CG, and PR3

NE and PR3 preferentially cleave bonds that are C-terminal to small, hydrophobic residues (e.g., alanine, valine, or serine) at the P1 position. CG has a chymotrypsin-like catalytic activity, preferentially cleaving peptide bonds that are C-terminal to bulky aliphatic or aromatic residues such as phenylalanine at the P1 position. NE degrades a broad spectrum of ECM components. It also cleaves coagulation proteins, complement components, and immunoglobulins, inactivates inflammatory mediators, and removes receptors from lymphocytes and inflammatory cells (Table 1). This suggests that it plays roles in ECM remodeling and wound healing and also in dampening inflammation and immune responses. However, NE also plays critical roles in host defense against Gram-negative bacteria and fungi in mice since mice genetically deficient in NE by gene targeting (NE
/
mice) die from overwhelming Gramnegative bacterial and fungal sepsis after intravenous inoculation of these organisms. NE kills bacteria by cleaving the outer membrane proteins of the cell wall of Gram-negative bacteria. CG enhances the activity of HLE against elastin and converts angiotensin I (an inactive decapeptide found in plasma) to angiotensin II (an octapeptide that stimulates smooth muscle contraction and increases vascular permeability). CG has other activities that are similar to those of NE (Table 1). PR3 has a similar spectrum of catalytic activity as NE. PR3 also regulates growth and terminal differentiation of the myelomonocyte lineage in the bone marrow. NE, CG, and PR3 also have direct activities on various cells. They all potently stimulate goblet cell degranulation. NE stimulates production of mucin MUC5AC by airway epithelial cells by shedding and activating membrane-associated protransforming growth factor alpha (pro-TGF-a), which stimulates mucin MUC5AC production by epithelial cells by activating the EGF receptor (EGFR). NE and CG stimulate lymphocyte activation. NE impairs macrophage uptake and clearance of apoptotic PMNs in tissues by cleaving the macrophage recognition receptor for phosphatidyl (PS), which binds to PS expressed on the surface of apoptotic PMNs. CG is a potent inducer of platelet activation, aggregation,

and degranulation that is mediated by activation of protease-activated receptor-4 (PAR-4). PARs are seven transmembrane-spanning G-protein-coupled receptors that are activated by proteolysis. Proteinases such as CG and thrombin cleave the extracellular portion of PARs, generating a new amino terminus that binds intramolecularly to activate the receptor. NE and PR3 also stimulate the release of cytokines and chemokines from epithelial cells and mononuclear phagocytes. PR3 and NE released by PMN stimulate release of cytokines by epithelial cells by activating epithelial cell PAR-2. Plasminogen Activators

The substrate specificity of PAs is restricted to cleavage of plasminogen to generate active plasmin which degrades fibrin, an important process in wound healing. However, plasmin also degrades other matrix components and activates procollagenases, yielding active collagenases that degrade interstitial collagens. Thus, by generating plasmin, PAs indirectly degrade many components of the ECM. The cell-surface uPA/uPAR system was initially thought to function simply as a mechanism to concentrate fibrinolysis at cell surfaces during inflammation and wound healing. However, uPAR also functions in regulating cell adhesion and migration by forming complexes with other membrane proteins that mediate signal transduction to regulate cell function in a nonproteolytic manner. For example, uPA promotes binding of uPAR to b1 and b2 integrins in the cell membranes of inflammatory cells and endothelial cells to regulate cell adhesion and migration through integrin signaling during inflammation, wound healing, and angiogenesis. Granzymes

Activation of CTLs by binding of their T-cell receptor to antigen bound to MHC class I molecules leads to rapid exocytosis of their granules containing GRZs and perforin. Perforin alters the properties of the cell membrane of the target cells allowing entry of GRZs into the target cell, which initiates apoptosis of the target cells. GRZ B initiates caspase-dependent apoptosis by cleaving the Bcl-2 family member BID; cleaved BID then translocates to the mitochondria, leading to the release of cytochrome c and caspase activation. GRZ B’s unique preference for aspartic acid at the P1 site of target proteins also enables it to cleave and activate procaspases directly. GRZ A is the most abundantly expressed GRZ in CTLs. It has tryptic-like activity, cleaving substrates with a PI arginine or lysine. It acts synergistically with GRZ B, but in a caspase-independent and still to be

SERINE PROTEINASES 5

elucidated manner, to induce the typical cellular characteristics associated with apoptosis, including chromatin condensation, and mitochondrial membrane potential changes. GRZ A and B have other activities in vitro (Table 1).

Inhibitors of Serine Proteinases The activities of serine proteinases are controlled by serine proteinase inhibitors (including the serpin family), which comprise B10% of all plasma proteins (Table 2). The major plasma serpins (a1 proteinase inhibitor (a1-PI), a1-antichymotrypsin, a2-antiplasmin inhibitor, plasminogen activator inhibitors (PAIs), and a2-macroglobulin (a2-M)) are synthesized predominantly by hepatocytes and generally at much lower levels by endothelial and epithelial cells and inflammatory cells. Secretory leukocyte proteinase inhibitor (SLPI) and elafin are produced locally by epithelial cells of the respiratory tract. Serine proteinase inhibitors inactivate serine proteinases by forming 1:1 noncovalent complexes with the enzymes. PI-9 (an exclusively intracellular serine proteinase inhibitor found in the cytoplasm of lymphocytes and other leukocytes) is an efficient inhibitor of GRZ B and likely protects lymphocytes from endogenous GRZ B-mediated cell death (Table 2). Human GRZ A and B are not inhibited by a1-PI, a1-antichymotrypsin, elafin, or SLPI.

Membrane-bound NE, CG, PR3, and uPA on inflammatory cells differ from soluble forms of the proteinases in that they are substantially resistant to inhibition by these naturally occurring proteinase inhibitors. For example, PAI-1 and -2 are less efficient inhibitors of uPA bound to uPAR than soluble uPA. In addition, uPAR-bound uPA converts membranebound plasminogen to membrane-associated plasmin, which is resistant to inhibition by a2-plasmin inhibitor, a highly effective inhibitor of soluble plasmin.

Serine Proteinases in Respiratory Diseases Serine proteinases are thought to contribute to the pathogenesis of several acute and chronic lung diseases, including chronic obstructive pulmonary disease (COPD), acute lung injury, pulmonary fibrosis, Wegener’s granulomatosis, and cystic fibrosis. The following sections outline the roles of serine proteinases in initiating and amplifying lung inflammation and injury in several lung diseases. Figure 1 illustrates the mechanisms by which serine proteinase family members contribute to lung pathologies occurring in various acute and chronic lung diseases. Figure 1 also shows the diverse mechanisms by which serine proteinases amplify and perpetuate lung inflammation and injury. Figure 2 illustrates the pathogenetic roles of one serine proteinase (NE) in COPD. Chronic Obstructive Pulmonary Disease

Table 2 Inhibitors of serine proteinases Proteinase

Inhibitor

Main sources

Neutrophil elastase

a1-Proteinase inhibitor

Cathepsin G

a1-Antichymotrypsin (CG) a1-Macroglobulin SLPI (not PR3) Elafin Monocyte/neutrophil

Hepatocytes (epithelial cells, MNPs) Hepatocytes (epithelial cells) Hepatocytes Epithelial cells Epithelial cells Inflammatory cells (intracellular)

Proteinase 3

Plasminogen activators (PAs)

Granzymes

Proteinase inhibitor PAI-1

Endothelial cells, MNPs

PAI-2 Protease nexin I

MNPs Fibroblasts and platelets

PI-9

Lymphocytes (intracellular inhibitor)

MNPs, mononuclear phagocytes; CG, cathepsin G; SLPI, secretory leukocyte proteinase inhibitor; PAI, plasminogen activator inhibitor; PI-9, proteinase inhibitor-9.

The proteinase–antiproteinase hypothesis for the pathogenesis of pulmonary emphysema was formulated 40 years ago in response to two key observations. First, genetic deficiency of a1-PI (the cognate inhibitor of NE) is associated with early onset, severe panlobular emphysema. Second, instillation of papain (an elastase) into rat lungs results in airspace enlargement. It has been thought that an imbalance between leukocyte proteinases and their inhibitors leads to destruction of the ECM components of the alveolar walls (especially loss of elastin), resulting in the development of pulmonary emphysema. However, this is probably an oversimplification of the mechanisms underlying COPD in cigarette smokers without AAT deficiency since oxidative stress and apoptosis of lung structural cells are also thought to play pathogenetic roles, and COPD patients have lung pathologies other than airspace enlargement, including chronic inflammation in the airways and alveoli, mucus hypersecretion in the large airways, and subepithelial fibrosis in the small airways (Figure 2). There is evidence that serine proteinases contribute to several pathologies in COPD.

6 SERINE PROTEINASES Coagulation cascade

Coagulation (fibrin deposition) Plasmin generated by uPA or tPA

Lung injury

Recurrent bacterial/viral infections

Inadequate clearance of fibrin Fibrosis Inflammation

Fibrinolysis PS receptor cleavage & decreased PMN removal

Lymph

Impaired host defense

PMN

MNP

Complement activation

Repair

NE, CG, PR3 NE

GRZ NE, CG

Serine proteinases

NE, CG, PR3 plasmin

Cytokine & chemokine release and processing

NE, CG, PR3

NE, TK ProMMP activation or TIMP inactivation

MMPs

Epithelial cells

Structural cell injury

ECM fragments

NE, CG, PR3 Plasmin

ECM degradation

Airspace enlargement or AC barrier injury

Inadequate repair

NE, CG, PR3

Plasmin NE

Growth factor activation

Fibrosis

EGFR activation or Goblet cell degranulation

Cleavage of lymphocyte & PMN receptors & IgG

Mucus hyper-secretion

Lung disease

Figure 1 Pathogenetic roles of serine proteinases in lung pathologies. The initial injury could be chronic exposure to cigarette smoke, pulmonary or systemic bacterial or viral infections, acute inhalation of smoke, or injury in another organ (e.g., severe burns or acute pancreatitis leading to ARDS). Examples of serine proteinases that contribute to pathologies in various acute and chronic lung diseases are shown along with mechanisms by which serine proteinases amplify and perpetuate lung inflammation and injury. Lymph, lymphocyte; MNP, mononuclear phagocyte; PMN, polymorphonuclear neutrophil; ECM, extracellular matrix; EGFR, epithelial growth factor receptor; AC barrier injury, alveolar–capillary barrier injury; PS receptor, phosphatidyl serine recognition receptor on macrophages; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase-3; uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; GRZs, granzymes; TK, tissue kallikrein; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitors of MMPs.

Airspace Enlargement

The levels of NE, CG, PR3, and uPA are increased in lower respiratory tract secretions and cells of COPD patients. In addition, instillation of NE and PR3 into

rodent lungs leads to progressive airspace enlargement. Studies of mice deficient in individual proteinases by gene targeting in murine models of COPD are dissecting the mechanistic roles of proteinases in

SERINE PROTEINASES 7

PMN

Recurrent bacterial infection

MNP

Inflammation

Reduced ciliary beat Epithelial cells frequency Neutrophil elastase EGRF activation Goblet cell degranulation

TGF-
activation

TIMP inactivation MMPs

Mucin synthesis

Small airway fibrosis ECM degradation

Mucus hypersecretion

Cytokine & chemokine release and processing

Chemotactic ECM fragments

Airspace enlargement

COPD Figure 2 Potential mechanisms of NE-mediated lung injury in COPD. NE leads to airspace enlargement by degrading the ECM components (especially elastin) of the alveolar walls directly and indirectly by cleaving and inactivating TIMPs, thereby promoting MMPmediated ECM degradation in the lung. NE may promote chronic inflammation in the airways and airspaces of the lung in COPD by generating fragments of elastin that are chemotactic for inflammatory cells and by stimulating epithelial cells (and mononuclear phagocytes) to release cytokines and chemokines. NE may promote mucous hypersecretion in the airways in COPD by stimulating mucin secretion by submucosal glands and goblet cell degranulation in airway epithelium. NE has the capacity to contribute to fibrosis in the small airways by activating latent TGF-b.

pathologies in COPD. Mice deficient in MMP-12 (MMP-12
/
mice) are 100% protected from the development of airspace enlargement in a chronic smoke exposure model of COPD, indicating that MMP-12 plays a major role in this pathology in mice likely by degrading elastin in the alveolar walls, leading to loss of alveolar units. However, studies of NE
/
mice in this model have confirmed that NE also plays a major role in this process since NE
/
mice are 60% protected from airspace enlargement. NE likely contributes to airspace enlargement directly by degrading elastin and other ECM protein components of the alveolar walls (Figure 2). NE may also cause airspace enlargement by promoting PMN and monocyte recruitment into the lungs since the influx of these cells is impaired in NE
/
mice exposed to cigarette smoke. One mechanism by which NE (and MMP-12) may amplify lung inflammation and injury to the lung parenchyma is by degrading elastin since elastin fragments generated by these

proteinases are chemotactic for inflammatory cells (Figures 1 and 2). Thus, NE and MMP-12 may amplify destruction of the alveolar walls by increasing the lung burden of inflammatory cells and their serine proteinases and MMPs. NE may also promote MMP-12-mediated airspace enlargement indirectly by inactivating tissue inhibitors of MMPs (TIMPs) (Figures 1 and 2) since NE cleaves and inactivates TIMPs in vitro. NE
/
mice have higher whole lung levels of TIMP-1 than wild-type mice following chronic exposure to cigarette smoke, indicating that TIMP inactivation is an important activity for NE in this murine model of COPD. Pulmonary Emphysema in Patients with a1-PI Deficiency

a1-PI is a highly polymorphic protein and more than 70 variants have been identified. A common deficiency variant of a1-PI, PiZ, results from a single

8 SERINE PROTEINASES

amino acid substitution (342lysine to glutamic acid) causing loop sheet polymerization of Z a1-PI molecules, which tangle within the endoplasmic reticulum of the hepatocyte forming inclusion bodies. This defective secretion of a1-PI from hepatocytes in PiZ homozygous individuals results in plasma a1-PI levels B10–15% of normal. PiZ homozygous individuals have an increased risk of developing very severe, early onset panlobular emphysema if they smoke cigarettes. The severe lung injury in a1-PI-deficient individuals may be due to defective confinement of NE-mediated proteolytic activity and to the proinflammatory properties of the PiZ variant protein. Polymers of the Z protein also have proinflammatory properties. These polymers form spontaneously in the lungs of PiZ patients, where they stimulate recruitment and activation of PMN leading to increased PMN serine proteinase burden, which may promote loss of alveoli in a1-PI-deficient patients.

mice that have increased and decreased lung fibrosis, respectively, compared to control mice in the bleomycin model. PAI-1 may promote fibrosis by decreasing PA activity, leading to (1) decreased plasmin-mediated clearance of fibrin and (2) decreased plasmin-mediated activation of pro-MMPs, and thus decreased MMP-mediated removal of collagen and other ECM proteins deposited in the lung. This is supported by studies of transgenic mice with inducible, lung-specific overexpression of uPA which are protected against bleomycin-mediated pulmonary fibrosis. However, PAI-1 stimulates adhesion and migration of inflammatory cells and fibroblasts, so it may also regulate lung fibrotic responses by promoting recruitment of collagen-producing fibroblasts. NE has a profibrotic role in the lung since NE
/
mice are significantly protected from developing bleomycin-mediated lung fibrosis. NE may promote pulmonary fibrosis by activating latent TFG-b.

Airway Pathology

Serine proteinases may contribute to mucus hypersecretion in COPD patients (Figure 2). NE and tissue kallikrein (a serine proteinase expressed by inflammatory cells and submucosal glands) increase expression of MUC5AC (a major mucin protein) by activating the EGFR. Both proteinases shed and activate cell-surface-bound pro-TGF-a, releasing active TGF-a, a ligand for the EGFR (Figure 1). Soluble NE and CG and membrane-bound NE and CG on activated PMNs are potent stimulators of goblet cell degranulation leading to their release of mucus. NE also impairs mucociliary clearance by reducing beating frequency of cilia on airway epithelial cells. NEmediated mucus hypersecretion and impaired mucociliary clearance together promote recurrent bacterial infection in the airways and lung parenchyma, thereby amplifying lung inflammation and injury in COPD patients (Figure 2). Plasmin and NE also activate latent growth factors such as latent TGF-b in vitro and thus have the potential to induce fibroblasts to deposit interstitial collagens in the walls of the small airways in COPD patients (Figures 1 and 2).

Acute Respiratory Distress Syndrome

There are several lines of evidence that the PMN serine proteinases NE and CG play pathogenetic roles in the acute exudative phase of acute respiratory distress syndrome (ARDS). First, ARDS is characterized by sequestration of large numbers of PMNs in the lung microvascular circulation and alveolar space. Lung PMNs are activated in ARDS patients, and there are increased levels of NE in respiratory tract secretions of most ARDS patients. Second, infusion of NE or CG into the lungs of experimental animals leads to sequestration of PMNs in the pulmonary vasculature, with increased vascular permeability and progressive pulmonary failure. Third, NE and CG can directly injure both the extracellular matrix and epithelial and endothelial cell components of the alveolar capillary barrier in vitro. Fourth, delivery of synthetic inhibitors of NE to experimental animals with acute lung injury attenuates lung neutrophilia and pulmonary edema. Fifth, NE
/
mice are resistant to normally lethal doses of endotoxin, and mice lacking NE and CG are protected against endotoxin-mediated alveolar injury.

Pulmonary Fibrosis

PAs and their inhibitors and NE may play roles in regulating lung fibrotic responses to injury. Following lung injury, leakage of plasma proteins into the lung and activation of coagulation enzymes results in deposition of the provisional fibrin matrix (Figure 1). Failure to adequately remove this fibrin matrix due to abnormally increased PAI-1 levels may promote lung fibrosis. This concept is supported by studies of transgenic mice overexpressing PAI-1 and PAI-1
/


Wegener’s Granulomatosis

Wegener’s granulomatosis is a systemic, autoimmune vasculitis that commonly affects the kidneys and the upper respiratory tract. Circulating antineutrophil cytoplasmic autoantibodies (c-ANCAs) are an obligate feature of this disease. There are different types of ANCAs, which are distinguished by their patterns of immunofluorescence staining of ethanol-fixed PMNs (reflecting their differing antigenic targets),

SERINE PROTEINASES 9

causing different types of systemic vasculitides. Classical or c-ANCAs, which occur in B95% of patients with active Wegener’s granulomatosis, are directed against PR3 and play a direct role in vascular injury in this disease. Circulating PMNs and monocytes from patients with Wegener’s granulomatosis express PR3 on their cell surface, and c-ANCAs bind via their F(ab)2 component to membrane-bound PR3. Ligation of Fcg receptors on PMNs by the Fc component of c-ANCAs is a potent stimulus for leukocyte degranulation and activation of the respiratory burst. The proteinases and oxidants released by PMNs activated in this way likely cause the vascular inflammation and injury characteristic of this syndrome. Cystic Fibrosis and Bronchiectasis

High levels of active NE, CG, and PR3 occur in lung secretions from patients with cystic fibrosis and bronchiectasis, indicating that these enzymes have overwhelmed the proteinase inhibitor defenses of the airways. There is also a correlation between serine proteinase activity and disease severity in both diseases. These proteinases may contribute to pathologies in cystic fibrosis and bronchiectasis by degrading ECM proteins and injuring structural cells in the airway walls. NE, CG, and PR3 may also promote and amplify inflammation and airway injury in these diseases by several mechanisms. First, these serine proteinases interfere with local host defense by degrading immunoglobulins and complement components and by cleaving complement receptors on neutrophils and receptors on lymphocytes involved in immune responses (Table 1 and Figure 1). Second, NE, CG, and PR3 stimulate the release of cytokines and chemokines from airway epithelial cells and mononuclear phagocytes (Figure 1). Third, PR3 processes latent forms of TNF-a and IL-1b, generating active forms of these cytokines. Finally, NE hinders clearance of apoptotic PMNs in the airways of patients with cystic fibrosis and bronchiectasis by cleaving the PS recognition receptor from the surface of macrophages (Table 1). Thus, by promoting necrosis of inflammatory cells (and excessive release of their complement of NE, CG, PR3, and MMPs into the airways), rather than clearance of apoptotic PMNs, NE may further amplify airway inflammation and injury (Figure 1).

Therapeutic Considerations Serine proteinases have been strongly implicated in several acute and chronic lung diseases based on the presence of abnormally high levels of these

proteinases in lung samples from patients with these lung diseases, our knowledge of the activities of serine proteinases in vitro, and results from studies of animal models of various lung diseases. In particular, studies of gene-targeted mice in murine models of lung diseases are providing novel insights into the mechanisms underlying the roles of serine proteinases in several acute and chronic lung diseases. However, there is little information in the public domain about the efficacy of inhibitors of serine proteinases in the treatment of human lung diseases. In addition, much remains to be learned about the pathogenetic roles of serine proteinases in human lung diseases since it is not clear whether the results in animal models can be directly extrapolated to humans. Potential problems associated with inhibition of serine proteinases as a new treatment strategy for human lung diseases include the potential loss of serine proteinase activities that are beneficial to the host, such as interference with the antimicrobial and anti-inflammatory properties of NE. Nevertheless, clinical trials of new treatment strategies to inhibit serine proteinases in diseases in which they have been implicated are justified. See also: Chronic Obstructive Pulmonary Disease: Overview; Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General; Smoking Cessation. Coagulation Cascade: Overview; Antithrombin III; Factor V; Factor VII; Factor X; Fibrinogen and Fibrin; Intrinsic Factors; iuPA, tPA, uPAR; Thrombin; Tissue Factor. Fibrinolysis: Overview; Plasminogen Activator and Plasmin. Leukocytes: Neutrophils.

Further Reading Barrett AJ (1980) The many forms and functions of cellular proteinases. Federal Proceedings 39: 9–14. Belaaouaj A, Kim KS, and Shapiro SD (2000) Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. Science 289: 1185–1188. Janoff A, Sloan B, Weinbaum G, et al. (1977) Experimental emphysema induced with purified human neutrophil elastase. Tissue localization of the instilled protease. American Review of Respiratory Disease 115: 461–478. Kohri K, Ueki IF, and Nadel JA (2002) Neutrophil elastase induces mucin production by ligand-dependent epidermal growth factor receptor activation. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L531–L540. Lee WL and Downey GP (2001) Leukocyte elastase: physiological functions and role in acute lung injury. American Journal of Respiratory and Critical Care Medicine 164: 896–904. Lomas DA and Parfrey H (2004) Alpha1-antitrypsin deficiency. 4: molecular pathophysiology. Thorax 59: 529–535. Owen CA and Campbell EJ (1999) The cell biology of leukocytemediated proteolysis. Journal of Leukocyte Biology 65: 137–150. Owen CA, Campbell MA, Sannes PL, Boukedes SS, and Campbell EJ (1995) Cell-surface-bound elastase and cathepsin G on human neutrophils. A novel, non-oxidative mechanism by

10

SIGNAL TRANSDUCTION

which neutrophils focus and preserve catalytic activity of serine proteinases. Journal of Cell Biology 131: 775–789. Rao NV, Wehner NG, Marshall BC, et al. (1994) Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase. Journal of Biological Chemistry 266: 9540–9548. Shapiro SD, Goldstein NM, Houghton AM, et al. (2003) Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. American Journal of Pathology 163: 2329–2335. Smyth MJ, Kelly JM, Sutton VR, et al. (2001) Unlocking the secrets of cytotoxic granule proteins. Journal of Leukocyte Biology 70: 18–29.

Sturrock AB, Franklin KF, Rao G, et al. (1992) Structure, chromosomal assignment, and expression of the gene for proteinase-3. The Wegener’s granulomatosis autoantigen. Journal of Biological Chemistry 267: 21193–21199. Tkalcevic J, Novelli M, Phylactides M, et al. (2000) Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12: 201– 210. Vassalli J-D, Sappino AP, and Belin D (1991) The plasminogen activator/plasmin system. Journal of Clinical Investigation 88: 1067–1072.

SIGNAL TRANSDUCTION M Torres, The Saban Research Institute of Childrens Hospital, Los Angeles, CA, USA H J Forman, University of California, Merced, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Signal transduction refers to all biochemical processes by which cells translate extracellular signals originating from their environment into specific responses. During the past 50 years, intensive research uncovered the enzymes and molecules that participate in this process (i.e., receptors, second messengers, phospholipases, kinases, phosphatases, etc.) and delineated the mechanisms by which cells integrate multiple signals. Signal transduction is now thought to occur through tightly organized networks in which protein–protein interactions and reversible assembly of signaling complexes are controlled by a small number of modular domains. The challenge in the future will be to better understand how signal specificity is achieved. Such information is vital to the reawakening of ‘systems biology’. Signal transduction is critical in regulating the lung nonrespiratory functions. Secretion of mucins by bronchial epithelial cells and surfactant by type II alveolar epithelial cells, phagocytosis and the respiratory burst by alveolar macrophages, production of cytokines by various cells, replacement of various cell types by cell division, and differentiation are among processes that are highly regulated through signaling pathways in the lung. Aberrant signal transduction underlies pathological changes such as fibroblast proliferation and cancer.

changes in the activities of enzymes and ion channels, altering steady-state concentrations of metabolites or ions, and posttranslational modifications such as protein phosphorylation. Return to basal state does not necessarily imply reaction reversibility, such that a signaling protein rendered active by phosphorylation may not be inactivated by dephosphorylation but instead by its degradation. Nonetheless, it is the general rule that the steady state is maintained by enzymatic processes catalyzed by opposing enzymes (protein kinases/protein phosphatases, nucleotide cyclases/cyclic nucleotide phosphatases, etc.) and that signaling involves a temporary shift in the activity of one of the enzymes. Signal transduction also implies that the signal emanating from one molecule of stimulus is amplified through the involvement of many enzymes engaged in a chain of events referred to as signaling cascades or pathways. After initial recognition of the molecules participating in signaling, the challenge has been to understand the mechanisms by which they are organized in tightly regulated signaling cascades. This chapter focuses on the best characterized aspects of signal transduction.

Receptors Principles of Signal Transduction Cells receive clues from their environment comprising other cells, extracellular matrices, and extracellular fluids, and these interactions activate a tightly regulated sequence of processes that modulate cellular function. Thus, signal transduction refers to the network of intracellular biochemical reactions that convert extracellular signals into a specific response, such as growth, survival, differentiation, secretion, or motility. Signaling processes include transient

The first step in the initiation of intracellular signaling cascades is the specific recognition of a cell surface transmembrane protein by an extracellular molecule, such as hormone, neurotransmitter, or chemokine. This ‘key–lock’ interaction between the ligand or first messenger and the membrane protein or receptor triggers the activation of downstream signaling pathways to convey and amplify the message intracellularly. Many receptors have been identified and grouped into large families according to their structure. The heterotrimeric G-protein-coupled

SIGNAL TRANSDUCTION 11

can interact with specific effectors. Thus, G proteins function as molecular switches, cycling between the active (GTP) and inactive (GDP) states. Sixteen genes encode for Ga, and the profile of specific effector proteins they activate differs with the subfamilies: Gas activates adenylate cyclase, which is inhibited by Gai, whereas Gaq are activators of phospholipase Cb and Gao of calcium channels (Figure 1). This classification, however, does not reflect variations in effector within each subfamily, the promiscuity of some Ga, which can couple to more than one effector, or the recent discovery of novel effectors. These include Rap1GAP interacting with Ga0, Bruton’s tyrosine kinase with Gaq, and the cytoskeletal protein radixin with Ga13, whereas interaction with heat shock protein 90 is required for Ga12 downstream responses. Five and 12 genes encode for Gb and Gg, respectively. Initially thought to play a role in Ga stabilization, Gbg dimer in various combinations is now known to activate ion channels PLCb2 and adenylate cyclase II, which are also Ga effectors, whereas PKD1, PI3 K, Raf-1 kinase, and others are activated by mostly interacting with Gb. Thus, the significance of dimer composition in directing effector and receptor coupling remains to be determined, and the identification of additional effectors is forthcoming. Termination of G-protein activation involves hydrolysis of GTP by the intrinsic GTPase activity of Ga, accelerated by the interaction of Ga with GTPase-activating proteins (GAP), and

receptors (GPCRs) constitute the largest family in the human genome and are also referred to as serpentine or seven-transmembrane receptors because they are made of a single polypeptide chain that weaves in and out of the plasma membrane to form a heptahelical core structure embedded in the membrane lipid bilayer and several extracellular or cytoplasmic loops. The extracellular N-terminus and loops form the agonist binding pocket, whereas the cytoplasmic C-terminus regulates downstream signaling by recruiting signaling molecules such as scaffolds, kinases, and cytoskeletal proteins and signal termination through its phosphorylation. The cytoplasmic loops interact with one or more heterotrimeric GTP-binding proteins that activate various effectors, propagating the signal from the agonist. G proteins consist of three subunits, Ga and Gbg dimer, anchored at the plasma membrane and bound to GDP via Ga, a guanine nucleotide binding protein and GTPase. The hydrophobic membrane anchor is provided by Ga and Gg, which undergo posttranslational lipid modifications (myristoylation, palmitoylation, and isoprenylation) that may also differentially regulate localization (plasma membrane vs. intracellular organelles) and protein– protein interactions. Changes in conformation induce binding of the GDP-Ga/Gbg to the receptor intracellular loops, GDP releases from Ga, the limiting step in G-protein activation, and GTP exchange. GTP-Ga is then freed from Gbg, and both subunits

Stimulus 1 Membrane

Receptor

G

G
s

2a

G
s 3a

G GDP 2b

GDP GTP 6

Adenylate cyclase

GTP

G

Regulatory

3b

PKA Catalytic cAMP

ATP

4a

PPi 5

G

cAMP

4b

cAMP PKA Catalytic

7 Pi

Phosphodiesterase Cytosol

AMP Figure 1 Signaling through cAMP. Binding of a stimulus to a GPCR (1) causes Gas exchange of GDP for GTP (2a) and dissociation from Gbg (2b) and the GPCR. Active Gas binds to (3a) and activates (3b) adenylate cyclase, producing cAMP. cAMP binds to multiple sites on the PKA regulatory subunit (4a) causing its dissociation from the catalytic subunit, thereby activating PKA. GTP hydrolysis by Gas (5), reassociation of the three G proteins and GPRC (6), and hydrolysis of cAMP by phosphodiesterase (7) participate in turning off GPCR signaling.

12

SIGNAL TRANSDUCTION

reassociation of Ga with Gbg, which in fact controls the inactive state of the G proteins. The transduction cascade is shut off by receptor phosphorylation on several sites (desensitization) by GPCR kinases (GRK1–7), followed by binding of b-arrestin to the multiphosphorylated receptor to prevent receptor coupling to cognate G proteins. GPCR are then removed from the cell surface via internalization/ endocytosis mediated by clathrin-coated vesicles, caveolae, or uncoated vesicles, dephosphorylated, and rapidly recycled to the cell surface. Receptor tyrosine kinases (RTKs) bind growth factors such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGF), and represent another family of well-studied receptors composed of a single-pass transmembrane domain, a large extracellular domain, and an intracellular C-terminus that contains a functional tyrosine kinase domain. The human genome contains 58 putative RTK, and 20 subfamilies have been identified, grouped according to the structure of the extracellular and kinase domains. Binding of a growth factor to its receptor induces receptor dimer formation, either through a conformational change in the intracellular domain or because the ligand is a dimer and cross-links two receptors as for PDGF. Dimerization is a general mechanism for monomeric RTK activation and enables the auto/transphosphorylation and activation of the tyrosine kinases that phosphorylate the receptor on several tyrosines in the cytoplasmic domain and other substrates. Effectors are recruited to the receptor via protein–protein interactions and their activation triggers downstream signaling. The discovery of RTK revealed the importance of tyrosine phosphorylation and protein–protein interactions in signal transduction. Receptors and c-Src

Changes in tyrosine phosphorylation also arise from activation of nonreceptor tyrosine kinases (NR-TK) by activated RTK and GPCR. Among NR-TK, c-Src defines the largest family that includes Fyn, Yes, Blk, Fgr, Hck, Lck, and Lyn. Initially identified as a Rous sarcoma virus gene (v-Src) that induced tumors, the cellular counterpart, c-Src, was cloned and is the first recognized and best studied proto-oncogene; its structure encompasses four Src homology (SH1–4) domains. SH2 and SH3 domains have been found in many signaling proteins, including adapter proteins such as Grb2, SHC, and others, which have no known enzymatic activity but serve as ‘glue’ in the formation of large protein complexes or signalosomes during receptor activation. SH2 domains recognize a specific phosphotyrosine and its immediate

surrounding amino acids, whereas SH3 domains interact with proline-rich regions (PxxP). Both are involved in protein–protein interactions. Intramolecular interactions mediated by these domains regulate c-Src activity. Inactive c-Src is in a closed conformation that prevents substrate access due to binding of the SH2 domain to a phosphotyrosine in the C-terminus (Y530 in humans) and binding of the SH3 domain to a proline-rich region in the SH1 kinase domain. Activation requires opening of the configuration through Y530 dephosphorylation by a protein tyrosine phosphatase (PTP) and autophosphorylation of Y419 in the kinase domain. SH2-containing PTP SHP-1 and SHP-2 or PTP-1B, and others have been shown to dephosphorylate the C-terminus tyrosine residue. Focal adhesion kinase (FAK) and p130Cas can bind to the SH2 and SH3 domains, displacing the intramolecular interactions and opening the c-Src configuration. c-Src tyrosine kinase (Csk) phosphorylates Y530, inhibiting kinase activity. c-Src is also regulated by its subcellular localization, present at the perinuclear membrane when inactive and translocating upon activation to the cell periphery, where it attaches to the plasma membrane through its myristoylated SH4 domain. Activated c-Src phosphorylates many substrates such as FAK, adapter proteins, and transcription factors (TFs). Thus, c-Src is a critical component of a complex network of interacting proteins. Other NR-TKs include Janus kinases (JAKs), which are physically associated with cytokine receptors; FAK, which participate in signaling via integrin receptors bound to extracellular matrix proteins; and the calcium-dependent kinase Pyk2. Receptors and Transactivation

Although it is well accepted that ligand–receptor binding results in signaling, a novel mechanism of cross-talk between GPCR and RTK has been described in which GPCR agonists activate RTK without binding to RTK. Examples of cross-talk are abundant and are described as ‘transactivation’, but the mechanisms are still poorly defined. Receptors for lysophosphatidic acid (LPA), endothelin-1, and thrombin activate EGFR through activation of metalloproteinases of the ADAM family (a disintegrin and metalloprotease), which cleave and release the membrane-anchored pro-heparin binding (HB)-EGF that can then bind and activate EGFR, resulting in downstream signaling. EGF-like ligands, such as amphiregulin and transforming growth factor-a, are also cleaved by ADAM proteases and can transactivate EGFR. Nevertheless, the mechanisms regulating the shedding process are still largely unknown. Receptors for angiotensin II, VEGF, PDGF, NGF, and

SIGNAL TRANSDUCTION 13

IGF participate in transactivation with various GPCR via unknown mechanisms. Pathways activated downstream of the transactivated RTK are numerous and often include the MAP kinases ERK1 and ERK2, indicating that RTK and GPCR induce the activation of key regulatory pathways in an integrated and dynamic manner.

the most controversial, hydrogen peroxide. Thus, second messengers are quite diverse in structure, solubility, and chemical reactivity. Nonetheless, they all conform to the following: (1) Their concentration is transiently changed in response to stimulation, (2) they are enzymatically generated (or, in the case of calcium, released through channel opening), (3) they are enzymatically degraded (calcium is actively transported; Figure 2), and (4) their activity is mediated through binding to an effector (Table 1). Production and degradation of second messengers constantly occur, even in quiescent cells, and their steady-state concentration is usually not zero. Although ligand binding mostly induces an increase in the concentration of second messengers, a decrease in the rate of production that contributes to steady-state concentration can also be an essential signaling process. For example, activation of Gai results in a decrease in cAMP production below the basal state.

Second Messengers The concept of ‘second messenger’ was introduced after the discovery of cyclic AMP (cAMP), a small molecule transiently produced intracellularly that regulates the activity of a protein kinase (PKA) downstream of the agonist (first messenger) (Figure 1). Other second messengers include calcium, cyclic guanosine monophosphate, diacylglycerol, inositol 1,4,5-trisphosphate, phosphatidic acid, ceramide, nitric oxide, and the most recently recognized but also

Ca2+

Membrane Ca2+ pump

2a

O

G
q

O

C O

C

O

O

PIP2

CH2

CH

CH2

O

GTP

PLC

P O

−

O

OH

O

1

O P

C

O

O

CH2

CH

O

O −

O−

7b

C O

5b

PKC


CH2 OH

DAG

PKC
5a

H H H

−

H OH

H

O O

OH

H

O O

P O−

O

2b O− P

−

O

OH

P

O

O−

O−

O

Ca2+ IP3R

3

4

H H H

H

O−

H

IP3 OH O

OH

P O−

H

O

4

Ca2+

Ca2+

Cytosol 6a

er 2+ 7a Ca pump

m

Ca

PP2b 6b

PP2b

Figure 2 Signaling through IP3 and Ca2 þ . Binding of a stimulus to a GPRC and guanine nucleotide exchange activate Gaq binding to PLCb (1), which hydrolyzes PIP2 to DAG (2a) and IP3 (2b). IP3 binds to its receptor on the endoplasmic reticulum (ER) (3), stimulating Ca2 þ release into the cytosol (4). Ca2 þ increase (5a) allows translocation of PKCa to the membrane. DAG binds to PKCa and stabilizes the interaction with the membrane (5b), resulting in PKCa activation. Signaling by Ca2 þ is turned off by decreasing its cytosolic concentration through energy-dependent pumps that restore Ca2 þ in the ER (7a) or expel it from the cell (7b). Ca2 þ also binds to calmodulin, a protein that activates several enzymes, such as protein phosphatase 2b (PP2b) (6), calcium/calmodulin-dependent kinases (CAMK II), and nitric oxide synthase.

14

SIGNAL TRANSDUCTION

Table 1 Second messengers and their targetsa Second messenger

Targets

cAMP cGMP Calcium

PKA cGKla, cGKlb, cGKII cPKC: a, bI, bII, g CAMKII Calcineurin/PTP2B cPKC: a, bI, bII, g nPKC: d, e, Z, y IP3 receptors Proteins with PH, PX, or FYVE domains: PKB/AKT, PDK1, and others Many targets, not all proven

Diacylglycerol Inositol 1,4,5-triphosphate Phosphoinositides: PIP, PIP2, PIP3 Lipids: arachidonic acid, ceramide, sphingosine-1 phosphate, phosphatidic acid Nitric oxide Hydrogen peroxide

Guanylate cyclase Protein tyrosine phosphatases, Thioredoxin

a

Listed are the targets of well-established and novel second messengers. cGK, cyclic GMP-dependent kinase; CAMKL, calmodulin kinase; PH, Pleckstrin homology; PX, Phox homology.

Similarly, an increase in second messenger concentration does not always enhance the activity of the effector. For example, hydrogen peroxide affects several signaling pathways by transiently inactivating PTP. In addition, second messenger production causes nonlinear signaling as one receptor–ligand interaction results in a large increase in the number of second messenger molecules and amplification of the message. Finally, the effectiveness of second messengers is not only transient in duration due to transient deviation from the steady-state concentration but also often limited by distance. As a second messenger is produced, it is both enzymatically destroyed and diluted by diffusion; thus, the concentration for binding to the effector will decrease below the binding constant with distance. Furthermore, if the interaction with the effector requires multiple binding as in cAMP activation of PKA, the second messenger ability to alter the activity of its target decreases exponentially with distance. This underscores the importance of not only the temporal but also the spatial relationship between signaling participants and the need to better understand the subcellular localization of signaling molecules, which is facilitated by the introduction of real-time technology such as video microscopy and fluorescent reporters for enzymatic activity.

Protein Phosphorylation Kinases and Phosphatases

One of the most critical posttranslational modifications is protein phosphorylation, which regulates a

variety of biological processes by altering enzymatic activity or protein conformation. Phosphorylation is a dynamic process in that it is controlled by the activity of protein kinases that add a phosphate on a particular amino acid residue (Ser, Thr, or Tyr) and that of protein phosphatases that remove it. Stimulation of cells often transiently alters the ratio of protein kinase to phosphatase activity and up to 40% of cellular proteins may undergo phosphorylation/dephosphorylation at diverse stages of proliferation or differentiation. The well-studied serine/threonine kinases are classified as either dependent or independent of second messengers (Table 1). The cAMP-dependent kinase or PKA was the first identified, and characterization of the calcium- and lipid-dependent PKC that is activated by phorbol ester tumor promoters and of other kinases pointed to the importance of protein phosphorylation (Figure 2), further emphasized by the characterization of growth factor receptors as tyrosine kinases. The study of protein phosphatases has lagged behind that of kinases. The human genome encodes more than 300 serine/threonine kinases, but only a small number of serine/threonine phosphatases dephosphorylate a vast array of proteins. The catalytic subunits of the most abundant – PP1, PP2A, and PP2B – have been cloned, but the mechanisms by which only a few phosphatases can specifically counteract the activity of so many serine/threonine kinases are still largely undefined. Formation of mutually exclusive complexes between the catalytic subunit and numerous regulatory subunits may be a universal means by which serine/threonine phosphatases are so versatile. PTPs, broadly classified into transmembrane and cytosolic PTP, represent a larger group, and all contain in their catalytic domain a conserved cysteine that is essential for catalysis. PTP regulate the activity of various RTK, either positively or negatively. Mice lacking PTP-1B exhibited a tissue-specific increase in insulin sensitivity, suggesting a role for PTP-1B as a negative regulator of the insulin receptor. Mice with naturally inactivating mutation of SHP-1 (motheaten) demonstrated an increase in stem cell factor (SCF) receptor phosphorylation, suggesting that SHP-1 is a negative regulator for SCF signaling. Stimulation with EGF and PDGF induces the production by novel NADPH oxidases of hydrogen peroxide that can reversibly oxidize the catalytic cysteine thiolate of various PTP, thereby transiently inhibiting PTP activity and increasing signaling. These effects of hydrogen peroxide on thiol proteins have been referred to as redox signaling. Thus, PTP plays a critical role in various signaling pathways.

SIGNAL TRANSDUCTION 15 Mitogen-Activated Protein Kinases

The discovery of the mitogen-activated protein kinases (MAPK) in the mid-1980s gave an enormous boost to our understanding of the organization of signaling pathways, in part because of the unusual properties of MAPKs, which are activated through a phosphorylation cascade. In addition, the diversity of their substrates in the cytoplasm and in the nucleus has indicated the importance of these kinases in many physiological processes. MAPKs are serine/ threonine kinases that phosphorylate substrates in a proline-dependent manner (S/T-P). Their activation uncommonly requires phosphorylation of a threonine and a tyrosine residue within a TXY motif, targeted by dual-specificity kinases (MAP2K/MKK/ MEK). MAPKs are grouped into subfamilies, mostly defined by the TXY motif and the activating kinases specifically targeting this motif, and are termed extracellular signal-regulated kinases (ERK1 and ERK2; TEY), c-Jun N-terminus kinases (JNK1–3; TPY), p38 MAP kinases a–g (TGY), and the recently identified ERK5, which is activated by a separate MKK (MKK5) despite its TEY motif. ERK are mostly activated by mitogens, whereas JNK and p38 MAPK are activated by various stresses and cytokines. MAP2Ks are activated by serine/threonine MAP3K (Figure 3); thus, each MAPK subfamily is organized in a three-tier kinase module that is activated through various mechanisms as a function of Stimulus

GTPase

Growth factor

the module and the receptor. The JNK and p38 MAPK modules can be activated by kinases regulated by the small GTPase Rac; nevertheless, the pathway from the receptor to the modules is not well characterized (Figure 3). In contrast, activation of the ERK module by RTK is well established, and this was the first pathway for which the link from receptor to nucleus was recognized. Activated RTK phosphorylate several tyrosines in the cytoplasmic domain, one of which recruits Grb2 via its SH2 domain. Grb2 is in a complex via its SH3 domain with the guanine nucleotide exchange factor SOS, and recruitment of the complex to the receptor brings SOS close to the membrane and in proximity of its target, the small GTPase Ras, which becomes activated after GDP-to-GTP exchange. Alternatively, SHC binds to another phosphotyrosine via its SH2 and is phosphorylated by the receptor, creating a binding site for the SH2 of Grb2/SOS. Activated Ras recruits the MAP3K for the ERK module, Raf-1, through its Ras binding domain and Raf-1 becomes activated via a complex process requiring phosphorylation by several kinases, changes in conformation, dephosphorylation, and interaction with multiple proteins. Nevertheless, this is a simplified scheme because cells may express several isoforms of Ras and Raf that are differently activated and, in the case of Ras, have different subcellular localization. Activated ERK phosphorylate substrates in the cytosol, whereas a fraction of ERK moves to the nucleus to

Stress, cytokines, TNF, UV

Rac/CDC42

Ras

HPK,GCK

MAP3K

RAF

MAP2K

MEK1/2

Oxidative stress

PAK

ASK,TAK1, MLK,DLK, TPL2,MEKK1 LZK,MLTK

MKK7

MKK4

T

MLK,ASK1,TAK1, TPL1, MUCS

MEKK3

MKK3/6

MKK5

Y

MAPK

ERK1/2

JNK1,2,3

Transcription factors, others

TCFs, Elk1, etc.

Jun, ATF2, Elk1, Bcl2, p53, Tau, etc.

p38MAPK
−

ATF2, CREB CHOP, MEF2C

ERK5

MEF2C

Figure 3 The MAPK modules. MAPK are activated through a phosphorylation cascade or MAPK modules in which the MAPK is activated by a dual-specificity MAPK kinase (MAP2K), itself activated by a MAPK kinase kinase (MAP3K). Receptor activation of a module may involve the participation of small GTPases, such as Ras and Rac. The MAPK phosphorylate many substrates, including transcription factors. Data from Torres M and Forman HJ (2000) Nitric Oxide, Oxidative Stress and Signal Transduction. In: Ignarro LJ (ed.) Nitric Oxide Biology and Pathobiology, pp. 329–342. Academic Press.

16

SIGNAL TRANSDUCTION

phosphorylate several TFs, thereby regulating gene expression. The mechanisms for nuclear translocation are best studied with ERK2, which does not have a nuclear localization signal peptide. Dimerization and passive or energy-dependent mechanisms appear to regulate ERK2 translocation, whereas nuclear anchors may maintain ERK2 in the nucleus. MAPK and docking domains MAPKs have specific and common substrates but all phosphorylate substrates in a proline-directed manner, suggesting the existence of additional domains to determine specificity. Identified in many TFs, the ‘docking’ domains are grouped into two types and constitute a stretch of residues located both upstream and downstream of the S/T-P phosphorylation site. D domains, similar to the c-Jun delta domain necessary for JNK targeting, are characterized by a cluster of basic residues followed by an LxL motif and/or a hydrophobic triplet. DEF domains are short peptides containing the FxF motif. Both domains increase MAPK efficiency and appear to enable the discrimination between multiple phosphoacceptors. Interestingly, the amplitude and duration of the ERK signal, known to play a role in cell fate decisions (proliferation vs. differentiation), may be controlled by the presence of DEF domains that act as sensor to direct ERK to physiologically relevant sites and may also interpret differences in signal strength from changes in agonist concentration. In addition, similar domains regulating specificity (CD and ED domains) were found in the sequence of MAP kinase-interacting proteins (i.e., kinases, phosphatases, and scaffolds). Further docking domains will undoubtedly be identified in the future. MAPK and scaffold proteins Assembly of the MAPK cascades into a functional module may be

mediated through interaction with one member of the cascade or through protein scaffolds. The latter was established by studies in yeast in which the mating response is regulated by the scaffold Ste5p, which binds to Ste11p (MAP3K), Ste7p (MAP2K), and Fus3p, the yeast homolog of mammalian ERK. The family of mammalian JNK-interacting protein (JIP) scaffolds that assembles the JNK modules was later discovered, along with others (Table 2). A single module may utilize more than one scaffold protein, whereas one scaffold protein may have dual function, interacting with several MAPK modules (b-arrestins), or be specific for one single module (MEK partner-1 (MP-1) or kinase suppressor of Ras (KSR)). The stoichiometry between scaffold and module components is critical for proper function, as shown when overexpression of JIP alone resulted in JNK inhibition. Scaffolds may play additional roles besides maintaining specificity and increasing efficiency, such as regulating targeting of specific substrates for a MAPK module through their different subcellular localization. The most recently identified MAPK scaffold, mitogen-activated protein kinase organizer-1, (MORG-1), binds to several components of the ERK module and to MP-1 in an agonist-specific manner, suggesting that a similar modular scaffold system may also be involved in the JNK and p38 MAPK modules. Scaffold proteins are not limited to the MAPK pathways, and more than a decade ago, an anchoring protein was shown to be involved in cAMP signaling and to bind the regulatory subunit RIIa of PKA, thereby maintaining PKA in an inactive state. This family of more than 30 A-kinase anchor proteins (AKAP), which shares the ability to bind to RII or RI of PKA, can associate with other proteins. For example, AKAP79 also binds PKC and PP2b/calcineurin and GPCR, thereby regulating the interaction of many signaling proteins.

Table 2 MAPK scaffold proteinsa ERK

JNK/p38 MAPK

Scaffold

Module

Scaffold

Module

b-Arrestin 1,2 KSR

Raf-1, MEK1, ERK2 Raf, MEK1, MEK2, ERK1, ERK2, 14-3-, C-TAK1, Gbg MEK1, ERK1, p14 Raf-1, MEK1, ERK2 Ras, Raf-1 Ras, Raf MP-1, Raf-1

b-Arrestin 2 JIP-1/IB1

ASK1, MKK4, JNK3 MLK, MKK7, JNK, MPK7, and others

JIP-2/IB2 JIP-3/JSAP1 JIP-4/JLP POSH CrkII Filamin

JNK, MKK7, MLK, TIAM1, and others MLK, MKK7, MKK4, JNK, MEKK1 JNK, MEKK3, MKK4, p38MAPK JNK, MKK4, MKK7, MLK, Rac1 JNK, HPK1, MKK3, p130cas, paxillin Actin, MKK4, TRAF2

MP-1 MEKK1 CNK Sur-8 MORG-1 a

Scaffold proteins not only bind several components of the MAPK modules but also bind other signaling proteins, linking the MAPK modules to receptors or the cytoskeleton. KSR, kinase suppressor of Ras; MP-1, MEK partner-1; CNK, connector enhancer of KSR; MORG-1, mitogen-activated protein kinase organizer; JIP, JNK interacting protein; POSH, plenty of SH3s.

SIGNAL TRANSDUCTION 17

Thus, scaffold proteins may represent dynamic platforms that can orchestrate multiple signaling pathways.

Signaling to the Nucleus Activation of signaling pathways may result in regulation of gene expression, and TFs, which are proteins that bind at cis-acting regulatory DNA sequences, are essential in this process. These proteins, currently in the hundreds, are classified according to the structure of their DNA-binding domain – that is, homeodomain, forkhead, zinc finger, leucine zipper, and helix–loop–helix proteins. Often, the three latter types of proteins can heterodimerize, increasing the regulatory diversity. They also contain activating regions that interact with components of the basal transcription machinery either directly or indirectly via coregulators. These TF bind to specific sites in particular genes, in contrast to basal TFs that are required for the transcription initiation of all genes by RNA polymerase II and are designated as TFII A–J. The regulation of the activity of the TF is multilevel. One of the most obvious control points is the tissue- and time-restricted transcription of genes that encode for TF. Another means of regulation is phosphorylation, which has varied consequences on their function, such as control of intracellular location, protein levels, ability to bind DNA, and interaction with other proteins. Many TF have been well described and the properties of AP-1, ATF, BRN, FOX, NF-kB, OCT, and PU1 are discussed elsewhere in this book.

Building of Signaling Networks: From Protein Domains to Signalosomes and Interactomes The discovery of SH2 and SH3 domains and their role in regulating signaling pathways has triggered the search for additional interaction domains, which may constitute the building blocks of signaling pathways by controlling protein–protein interactions and protein–lipid binding. (Table 3). The current viewpoint is that signal transduction occurs through the dynamic assembly of oligomeric complexes or ‘signalosomes’ that is controlled by specific modular domains and results in highly organized signaling networks that ultimately regulate cellular function. Interaction domains may regulate specificity through domain competition, varying binding constants, multidomain interactions, and differences in subcellular localization. The findings that membrane microdomains, such as caveolae and lipid rafts, contain many

Table 3 Interaction domainsa SH2 PTB 14-3-3 WW WD40 FHA Bromo Chromo

p-Tyr p-Tyr p-Ser p-Ser p-Thr p-Thr Ac-Lys Met-Lys

SH3 EVH1 WW

PxxP FPPPP PPxY

PDZ SAM DED DD CARD PB1

PDZ SAM DED DD CARD PB1

PH FYVE PX FERM

PI4,5P2, PI3,4,5P3 PI3P PI3P PI4,5P2

a Listed are the best recognized interaction protein domains that bind phosphorylated, acetylated, or methylated peptides; prolinerich sequences; domains; and phospholipids.

signaling molecules and that signaling is impaired when they are disrupted highlight the importance of compartmentalization that will be better studied with the use of novel in vivo techniques. Until recently, biological systems were studied using a reductionist approach that proved successful at deciphering mechanisms but failed to capture the complexity and dynamics of biological systems. Completion of the Human Genome Project and studies of other species (genomics), which required the development of high-throughput technologies and greater computational capabilities, advances in protein characterization (proteomics), and studies of changes in cellular metabolic response (metabolomics), and gene expression (transcriptomics), brought back to the forefront the field of systems biology. This rapidly developing area of investigation combines ‘omics’ data obtained from genetic or environmental perturbations and computational models to achieve an integrative understanding of biological phenomena, with the ultimate goal of building a mathematical framework to correctly predict and modify the behavior of biological systems. Signal transduction is a critical component of systems biology, and already the mapping of protein– protein interactions or interactome in yeast and fruit fly has demonstrated the presence of major ‘hubs’ of highly connected proteins, giving some insights into the assembly of signalosomes and the organization of

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SIGNS OF RESPIRATORY DISEASE / Breathing Patterns

larger cellular networks that facilitate communication to rapidly respond to changing conditions. Further studies involving in vitro validation, measurements of domain binding affinity, studies in single cells, and real-time measurements will provide a better understanding of protein dynamics and are essential to the development of systems biology. Knowledge of the complete human interactome or whole body human systems is lacking, but systems biology has already been successfully applied to microbes and to biological processes such as glycosylation. The potential applications of systems biology are far reaching, and future advances in this field will significantly revolutionize our understanding of physiology and pathophysiology mechanisms, and our ability to devise novel and effective therapeutics. See also: G-Protein-Coupled Receptors. NADPH and NADPH Oxidase. Oncogenes and Proto-Oncogenes: Overview; RAS. Platelet-Derived Growth Factor. Transcription Factors: Overview; AP-1; ATF; Fox; NF-kB and Ikb; PU.1.

Further Reading Alonso A, Sasin J, Bottini N, et al. (2004) Protein tyrosine phosphatases in the human genome. Cell 117(6): 699–711. Ceulemans H and Bollen M (2004) Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Physiological Reviews 84(1): 1–39.

Forman HJ, Fukuto JM, and Torres M (2004) Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers. American Journal of Physiology: Cell Physiology 287(2): C246–C256. Garcia A, Cayla X, Guergnon J, et al. (2003) Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis. Biochimie 85(8): 721–726. Golub T, Wacha S, and Caroni P (2004) Spatial and temporal control of signaling through lipid rafts. Current Opinion in Neurobiology 14(5): 542–550. Levchenko A (2003) Dynamical and integrative cell signaling: challenges for the new biology. Biotechnology and Bioengineering 84(7): 773–782. Newton AC (2003) Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochemistry Journal 370(Pt 2): 361–371. Pawson T and Nash P (2003) Assembly of cell regulatory systems through protein interaction domains. Science 300(5618): 445–452. Torres M (2003) Mitogen-activated protein kinase pathways in redox signaling. Frontiers in Bioscience 8: D369–D391. Tsien RY (2003) Imagining imaging’s future. Nature Reviews: Molecular Cell Biology (supplement): SS16–SS21. Waters C, Pyne S, and Pyne NJ (2004) The role of G-protein coupled receptors and associated proteins in receptor tyrosine kinase signal transduction. Seminars in Cell and Developmental Biology 15(3): 309–323. Westerhoff HV and Palsson BO (2004) The evolution of molecular biology into systems biology. Nature Biotechnology 22(10): 1249–1252. Weston AD and Hood L (2004) Systems biology, proteomics, and the future of health care: toward predictive, preventative, and personalized medicine. Journal of Proteome Research 3(2): 179–196.

SIGNS OF RESPIRATORY DISEASE Contents

Breathing Patterns Clubbing and Hypertrophic Osteoarthropathy General Examination Lung Sounds

Breathing Patterns D C J Howell, Centre for Respiratory Research, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract A diverse range of breathing patterns exist which occur in the unaffected and diseased lung. Every breathing pattern, whether it be considered normal or abnormal is made up of four individual components. These are: the respiratory rate (frequency), respiratory depth (drive), mode (symmetry), and regularity

(rhythm). Each of these components can be differentially affected by physiological and pathological insults. Changes in breathing patterns are invariably accompanied by symptoms, the commonest being breathlessness (dyspnea). Abnormal breathing patterns can occur when the underlying lung is unaffected by disease including physiological examples, such as in vigorous exercise. Examples of pathological conditions which cause abnormal breathing patterns in the absence of chest disease include severe neurological compromise, such as in Cheyne–Stokes breathing or as a result of severe endogenous or exogenous metabolic acidosis, as in Kussmaul breathing. In the compromised lung, tachypnea and bradypnea are the most frequently observed abnormal breathing patterns as they so commonly accompany underlying chest pathology, but other

SIGNS OF RESPIRATORY DISEASE / Breathing Patterns 19 abnormal patterns of breathing including hypopnea and platypnea also occur. In addition, various conditions can affect the symmetry/mode of breathing globally, causing thoracic, abdominal, and paradoxical breathing, or have effects more specifically during inspiration or expiration, or as localized phenomena.

Breathing Patterns: Overview Assessing the pattern of breathing is an extremely useful way of diagnosing underlying pulmonary and also extrapulmonary pathology. A wealth of clinical information can be gleaned from careful clinical examination and it is one of the more poorly performed tasks that physicians undertake when carrying out a physical examination. Part of the problem with assessing breathing patterns in the healthy and diseased lung is appreciating what a normal breathing pattern actually is. This can be particularly difficult as in healthy individuals, the act of breathing is on the whole, an unconscious one. Furthermore, the normal breathing pattern is difficult to fully define, as changes are often subjective, meaning that there is much individual variability in what a patient would volunteer in terms of symptoms. For this reason, the diverse range of breathing patterns that have been described in the normal and diseased

Rate

Depth

lung are mainly based on objective clinical measurements made by experienced observers. Broadly speaking, four factors contribute to breathing patterns, including the respiratory rate (frequency), depth of breathing (drive), mode of breathing (symmetry), and regularity of breathing pattern (rhythm). Each of these entities can be affected by normal physiological events and disease processes, which contributes to the varied range of breathing patterns observed in health and disease (Figure 1). Perhaps the most informative parameters of any breathing pattern is the respiratory rate. This is correctly measured by timing the number of breaths in a full minute and the normal frequency, at rest, in a healthy adult, is 12–14 breaths per minute. Respiratory depth is more difficult to estimate clinically as movements of the chest and diaphragm, on which it is dependent, cannot be accurately measured visually. With practise it is possible to recognize degrees of overventilation (so-called ‘air-hunger’) and underventilation. In normal subjects, inspiration is effected by contraction of the intercostal muscles and diaphragm, while expiration is a passive process dependent on elastic recoil of the lungs. Any deviation from this normal mode/symmetry of breathing should be

Symmetry

Rhythm

Normal lung

Diseased lung

Abnormal breathing pattern

Abnormal breathing pattern

Tachypnea Bradypnea Cheyne–Stokes Biot's Apneustic Central hyperventilation Ataxic Kussmaul

Tachypnea Bradypnea Hypopnea Platypnea Thoracic: diaphragmatic irritation Abdominal: ankylosing spondylitis Paradoxical: diaphragm paralysis Inspiratory impairment: asthma, upper airway obstruction, obstructive sleep apnea Expiratory impairment: COPD Pursed-lip breathing: COPD Localized impairment: pleural disease

Figure 1 Examples of breathing patterns in normal and diseased lungs. COPD, chronic obstructive pulmonary disease.

20

SIGNS OF RESPIRATORY DISEASE / Breathing Patterns

carefully assessed as it will invariably be associated with underlying pathology. Various modes of breathing are associated with a range of diseases, which either affect inspiration or expiration or locally impair respiratory movement. Finally, the regularity of breathing can also be assessed which often provides further detail on the underlying pathological process affecting the lung. Changes in the factors that determine breathing patterns are often associated with symptoms, the commonest being breathlessness. It has been advocated that the term ‘breathlessness’ should be restricted to the sensation experienced by normal subjects and ‘dyspnea’ should be used to indicate breathlessness caused by disease. However, for clarity, in this article the word dyspnea is used interchangeably with the term breathlessness. Dyspnea literally means ‘difficulty breathing’ and can occur when there is increased demand to breathe, a problem with the cardiorespiratory apparatus, or a mixture of both. It is worth pointing out that when patients present with the extremely common breathing pattern, tachypnea (increased respiratory rate), they are often described as dyspneic. Dyspnea is a subjective sensation that can only be perceived by the individual experiencing it (i.e., a symptom) and should not be used to describe the clinical appearance of a patient (i.e., a sign). This explains why tachypneic patients often complain of dyspnea, but individuals with bradypnea (reduced respiratory rate) are as likely to report the same symptom.

Breathing Patterns in the Normal Lung As discussed above, the normal breathing pattern is somewhat difficult to define. Not surprisingly, normal breathing patterns predominate in normal lungs!

However, abnormal breathing patterns can occur when the ultrastructure of the lung is completely intact which gives us some insight into the overall control of breathing (which will not be discussed in detail here). Abnormal breathing patterns in the normal lung can be divided into physiological and pathological (Figure 2). A number of physiological causes of abnormal breathing patterns occur in the normal lung such as during vigorous exercise, in anxiety, and when body core temperature is raised and in many cases they may go unnoticed by the individual. However, at the extremes of these various causes, it is well recognized that respiratory frequency and depth are increased, leading to air-hunger and the sensation of breathlessness. In extreme cases of anxiety and some functional disorders, hyperventilation may occur which promotes increased carbon dioxide excretion, and the development of a metabolic alkalosis. This often leads to a further change in breathing pattern where patients complain of variable dyspnea, the sensation that it is easier to breathe in than out. Light-headedness, paresthesiae, tetany, chest pain, and a feeling of impending collapse can also accompany the breathing pattern changes. A number of specific breathing pattern abnormalities also occur in the normal lung, which accompany pathological conditions such as severe central nervous system and neuromuscular disease and also profound metabolic acidosis of both endogenous and exogenous etiologies. Cheyne–Stokes breathing pattern was described by Scottish and Irish physicians in the 1800s. The pattern displays an abnormal rhythm, which is regularly irregular and is associated with a progressive increase in depth and often frequency of breathing. The pattern occurs over a 15–60 s cycle in a

Normal lung

Normal breathing pattern

Asymptomatic individual

Diseased lung

Abnormal breathing pattern

Physiological: exercise, anxiety, raised body temperature, functional

Pathological: CNS disease, endogenous toxins, exogenous toxins

Normal breathing pattern

Patient with stable thoracic pathology

Abnormal breathing pattern

Asthma COPD Interstitial lung disease Thromboembolic disease Upper airway obstruction Obstructive sleep apnea

Figure 2 Interaction of breathing patterns with healthy and diseased lungs. CNS, central nervous system; COPD, chronic obstructive pulmonary disease.

SIGNS OF RESPIRATORY DISEASE / Breathing Patterns 21

crescendo–decrescendo manner and is characterized by apneic episodes. It is thought that the pattern arises due to a delay in the central medullary chemoreceptor center response to changes in arterial carbon dioxide induced by variations in ventilation. As a result, the respiratory center hunts for equilibrium, but always overshoots, producing the pattern described above. It can be normal in some individuals during sleep but is always abnormal when it occurs while the subject is awake. Causes include aging, obesity, congestive heart failure, and neurologic disorders, for example, meningitis, infarction, or pontine hemorrhage. Biot’s breathing pattern was described by a French physician and is a variant of Cheyne–Stokes. There is a succession of hyperpnea, hyperventilation, and apnea but the pattern is not regular and there is no crescendo–decrescendo phase. Causes include meningitis and medullary compression and it often precedes death. Ataxic (agonal) breathing pattern occurs after brainstem damage and consists of continuous irregular shifts of hyperventilation, hypoventilation, and apnea in no particular succession (unlike the Biot’s and Cheyne–Stokes patterns). Apneustic breathing pattern is characterized by deep inspiration, followed by a postinspiratory breath hold and rapid exhalation. It generally signifies the presence of brainstem lesions usually at the level of the pons. In the central hyperventilation breathing pattern, patients are very tachypneic and breathe extremely deeply, exhibiting profound air-hunger. This pattern is seen in midbrain/upper pontine lesions and is due to damage to the medullary respiratory center. It is a sinister pattern, described as fibrillation of the respiratory centers, which usually heralds impending death. The Kussmaul breathing pattern is caused by severe metabolic acidosis, which can complicate endogenous diseases such as diabetic ketoacidosis and uremia and also exogenous conditions such as salicylate poisoning. It arises due to excessive stimulation of the respiratory center and is manifest by air-hunger and in severe cases can disturb the level of consciousness. Kussmaul breathing has similarities to the central hyperventilation pattern, as both cause dramatic changes in respiratory depth. Although both are associated with increases in the frequency of respiration, the latter pattern tends to have far greater effects on this parameter. Finally, endogenous disorders such as hypothyroidism and exogenous toxins, particularly overindulgence of opiod narcotics, are frequently associated with bradypnea.

Breathing Patterns in the Diseased Lung It is worthwhile pointing out that the most common breathing pattern in the diseased lung is obviously normal breathing. However, specific primary respiratory conditions affect breathing patterns in characteristic ways (Figure 1). For clarity, these patterns will be discussed in relation to their effect on respiratory rate, depth, mode, and rhythm. The most common effect of respiratory disease on the breathing pattern is to alter the rate of breathing, and tachypnea is seen in virtually all chest conditions such as asthma, chronic obstructive airways disease (COPD), interstitial lung disease, pulmonary suppuration, and thromboembolic disease. Other respiratory conditions are characterized by bradypnea, such as in type II respiratory failure and Pickwickian syndrome. Tachypnea and bradypnea are intimately linked to corresponding increases in respiratory depth/drive but interestingly, the proposed mechanisms of increased respiratory drive vary between conditions. In asthma, it is thought that increased respiratory drive is related to a combination of central effects due to the drop in arterial carbon dioxide, the degree of bronchoconstriction, and also the overinflation of the lungs, which reduces the efficiency of the respiratory muscles, leading to a greater motor stimulus to breathe. In COPD, similar mechanisms exist, but in this condition, additional effects of internal positive airways pressure, increase the respiratory work required before inspiration can begin. Patients with COPD who retain the ability to increase their ventilatory drive are classically described as ‘pink puffers’. They also breathe with pursed lips, in an attempt to counteract the dynamic airways collapse that occurs in this disease. However, in a separate population of patients with COPD, classically called ‘blue bloaters’, respiratory drive is reduced (hypopnea), which occurs as they develop hypercapnia and type II respiratory failure. There is a degree of overlap between this form of COPD and Pickwickian syndrome/obesity hypoventilation syndrome, where patients often suffer from obstructive sleep apnea. In this pattern of breathing, as the name suggests, there are periods where there is no airflow at all, due to obstruction by the lax musculature of the upper airway. Although, it may be intuitive that all patients with type II respiratory failure become bradypneic, this is not the case. A proportion of patients with type II respiratory failure present purely with hypopnea, which can still lead to hypercarbia and somnolence, but the onset can be insidious, meaning that this important breathing pattern can be incorrectly diagnosed by inexperienced observers.

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SIGNS OF RESPIRATORY DISEASE / Clubbing and Hypertrophic Osteoarthropathy

In interstitial lung disease and thromboembolic disease, it appears that increased drive to breathe and tachypnea may be due to activation of lung ‘c’ fibers, although this is not universally accepted. However, it is also recognized that respiratory drive and air-hunger can be noticeably increased at rest in massive pulmonary embolism, in the complete absence of tachypnea. The effect of respiratory conditions on the mode/ symmetry of breathing patterns is best discussed by considering the effects globally, on inspiration and expiration, and locally. In terms of global effects on breathing patterns, thoracic-type breathing occurs when diaphragmatic movement is inhibited by pain caused by, for instance, peritonitis or where the diaphragm is restricted, as in gaseous distension of bowel obstruction, massive ovarian cysts, or pregnancy. If the breathing pattern is mainly abdominal, diseases such as ankylosing spondylitis, intercostal paralysis, or pleural pain may be responsible for the paucity of thoracic movement. If the diaphragm is completely paralyzed, paradoxical respiration occurs as the abdomen sucks inwards with inspiration, rather than pouching out. In severe emphysema and asthma, adequate ventilation is not met by normal inspiratory efforts. In addition to the effects on respiratory rate and depth described above, abnormal breathing symmetry occurs in inspiration, produced principally by contraction of cervical muscles including the sternomastoid, scalene, and trapezius groups. This causes the whole thoracic cage to effectively lift off the diaphragm in inspiration. A more aggressive and concerning respiratory pattern of a similar character develops in upper airways obstruction of the larynx or trachea, particularly if the onset is acute. A seesaw movement of the chest is accompanied by in-drawing of the suprasternal and supraclavicular fossae, the intercostal spaces, and epigastrium with each inspiratory attempt. In patients who have sustained double fractures of a series of ribs, the portion of the thoracic cage between the fractures becomes mobile. Along with the overlying soft tissue structures, the affected area gets sucked in with every inspiration in a pattern called paradoxical movement, which can lead to severe respiratory embarrassment. Abnormal expiratory patterns are less common but can occur in emphysema and asthma. They are due to contractions of the abdominal muscles and latissimus dorsi and are observed when the elastic recoil of the lung is insufficient to completely expel air from the alveoli. In the severest of cases, patients sit bolt upright and often grasp the back of a chair in an attempt to fix the shoulder girdle, so that the latissimus dorsi can assist the expiratory effort.

Localized changes in the mode of breathing are common. Any large pleural collection of fluid or air will lead to a regional reduction in movement of the affected area. Furthermore, large pleural effusions are also associated with platypnea (dyspnea in the erect position, e.g., on sitting up). Large areas of parenchymal disease can also lead to regional differences in breathing patterns but may be more subtle to detect clinically. On the whole, the rhythm of the breathing disorders described in the previous paragraphs tends to be regular unless the clinical scenarios in which they are present are extreme. When this occurs, patterns may become increasingly chaotic as the patient becomes more anxious. Observation of this type of breathing pattern is clearly very serious and the patient may require endotracheal intubation to terminate the problem. See also: Chemoreceptors: Central; Arterial. Signs of Respiratory Disease: Lung Sounds. Ventilation: Control.

Further Reading Albert R, Spiro S, and Jett J (2004) Comprehensive Respiratory Medicine. St Louis: Mosby. Brewis RAL (1998) Lecture Notes on Respiratory Disease. Oxford: Blackwell. Gibson JG, Geddes DM, Costabel U, Sterk PJ, and Corrin B (2003) Respiratory Medicine. Philadelphia: Saunders. Munro J and Edwards C (2000) Macleod’s Clinical Examination. Edinburgh: Churchill Livingstone. Talley NJ and O’Connor S (2001) Clinical Examination. Oxford: Blackwell. West JB (2003) Pulmonary Pathophysiology: The Essentials. Philadelphia: Lippincott Williams & Wilkins. West JB (2004) Respiratory Physiology: The Essentials. Philadelphia: Lippincott Williams & Wilkins.

Clubbing and Hypertrophic Osteoarthropathy D C J Howell, Centre for Respiratory Research, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Clubbing and hypertrophic pulmonary osteoarthropathy (HPOA) are two components of a clinical syndrome which is more accurately referred to as hypertrophic osteoarthropathy (HOA). HOA occurs as a rare familial primary form and a more common secondary form, which is always due to an underlying disease process. Secondary HOA is now recognized as the correct term for HPOA. The reason for this confusing

SIGNS OF RESPIRATORY DISEASE / Clubbing and Hypertrophic Osteoarthropathy

23

nomenclature is that HPOA was historically thought only to occur associated with pulmonary disease. However, this is now recognized to be incorrect since numerous diseases affecting other organ systems, such as congenital cyanotic heart disease, are also complicated by clubbing and secondary HOA. Despite the clinical phenomena of HOA being recognized since the time of Hippocrates, the exact pathophysiological events responsible for these changes remain unknown. Various hypotheses including the production of paraneoplastic hormones, intrapulmonary shunting of blood resulting in peripheral platelet degranulation and the associated elaboration of growth factors, and the reappearance of the embryonic claw due to reactivation of dormant genes have been suggested as plausible mechanisms for this condition, but none is universally accepted. Although pulmonary suppuration commonly gives rise to clubbing and secondary HOA, primary lung neoplasia is responsible for the majority of pulmonary cases of this syndrome, meaning that the manifestation of the clinical phenomena of this condition should be investigated with a great deal of suspicion. Although analgesic agents can treat the pain associated with this syndrome, unfortunately, there is no specific treatment for clubbing and secondary HOA other than eradication of the underlying disease process.

Clubbing and Hypertrophic Pulmonary Osteoarthropathy Hypertrophic pulmonary osteoarthropathy (HPOA) is part of a clinical syndrome called hypertrophic osteoarthropathy (HOA) of which clubbing is a central clinical feature. Other features of this syndrome include enlargement of extremities and painful swollen joints and the condition is characterized by periostosis of tubular bones (Figure 1). Clubbing and HOA probably represent different stages of the same disease process. HOA can be divided into primary and secondary causes. Primary HOA or pachydermoperiostosis is a rare familial autosomal dominant condition, which occurs without any underlying cause. Secondary HOA occurs in the majority of patients with this syndrome and is always associated with an underlying disease process. It is this secondary form of HOA that was historically described as HPOA since as the name suggests, it was initially thought only to complicate pulmonary disease. However, nonpulmonary causes have also been described including pleural, cardiac, abdominal, and miscellaneous (Table 1) such that the term HPOA is now obsolete and should be more accurately referred to as secondary HOA. Although secondary HOA complicating respiratory conditions is relatively rare, its identification is of paramount importance since the commonest association of this syndrome with pulmonary pathology is primary lung malignancy, more specifically, large cell and adenocarcinoma of the lung. Of note, clubbing is an important clinical feature of primary and secondary HOA, but cases of secondary HOA

Figure 1 Photograph of lower limbs of man with marked enlargement of both legs and swollen ankle joints consistent with hypertrophic osteoarthropathy. The toes are also clubbed. Reproduced from John B, Subhash H, and Thomas K (2004) Journal of the New Zealand Medical Association, 117: 1192, with permission.

occur without clubbing and vice versa. For clarity, the remainder of this article will concentrate on the pathophysiology and clinical presentation of clubbing and secondary HOA, paying particular attention to the pulmonary causes.

Pathophysiology of Clubbing and Secondary HOA As mentioned above, the pathophysiology of clubbing and secondary HOA are thought to be similar. Clubbing of the fingers was first described by Hippocrates, but over 2500 years on, the pathophysiology of the process is still unclear. Microscopically, clubbed fingers show excessive deposition of collagen and interstitial edema. Blood vessel walls are thickened due to perivascular infiltrates of lymphocytes and vascular hyperplasia. Furthermore, extensive arteriovenous anastomoses are observed in the nail bed. In secondary HOA, digital clubbing is usually present but the characteristic feature is painful bilateral periosteal new bone formation predominantly affecting the long bones and also the joints, which can be detected by plain X-ray (Figure 2). More specifically, subperiosteal new bone formation occurs along the distal diaphysis of tubular bones, progressing proximally over time. In early stages, excessive

24

SIGNS OF RESPIRATORY DISEASE / Clubbing and Hypertrophic Osteoarthropathy

connective tissue is deposited associated with subperiosteal edema, which elevates the periosteum. New osteoid matrix is subsequently laid down beneath the periosteum, which mineralizes and can eventually progress to form a cuff around distal long bones. These pathological changes are particularly prominent in the tibia but also occur in the metacarpus, Table 1 Causes of clubbing and secondary HOA Organ system

Disease

Pulmonary

Bronchogenic carcinoma/sarcoma/rib tumors Pulmonary fibrosis Cystic fibrosis Tuberculosis Bronchiectasis/lung abscess Emphysema Hodgkin’s disease Metastatic disease Bronchial carcinoid Blastomycosis Pneumocystis carinii infection Sarcoidosis Pulmonary alveolar microlithiasis Lipid pneumonia Pleural fibroma/mesothelioma

Pleural, diaphragmatic Cardiac

Abdominal

Miscellaneous

Cyanotic congenital heart disease Infective endocarditis Infected aortic or axillary artery grafts Portal or biliary cirrhosis Ulcerative colitis/Crohn’s disease Amoebic/bacillary dysentery Gastrointestinal tract polyposis Neoplasms Whipple’s disease Biliary atresia Carcinoma: nasopharyngeal, esophageal, breast, thymus, thyroid, renal Myelofibrosis Graves’ disease

metatarsus, fibula, radius, ulna, femur, humerus, and clavicle. In addition to the changes in the periosteum, hypertrophic and osteolytic changes occur in bones affected by this condition. Hypertrophic bony changes predominate in patients where clubbing occurs postpuberty and is particularly prevalent in patients with lung cancer. Acroosteolysis is more commonly seen when clubbing occurs in childhood and is therefore often observed in patients with cyanotic congenital heart disease. Finally, synovial involvement may also occur with secondary HOA in conjunction with subperiosteal changes. Subsynovial blood vessel thickening, hyperplasia of the lining layer, infiltration of synovium with inflammatory cells, and associated edema have all been described.

Hypotheses for the Pathophysiology of Clubbing and Secondary HOA A number of hypotheses for the pathophysiolgical changes of clubbing and secondary HOA have been postulated. The production of paraneoplastic ectopic hormones associated with clubbing and secondary HOA has been described in a number of studies and provides one of the more compelling mechanisms to explain the clinical features of this condition. For example, electron microscopic examination of primary lung tumors have revealed the existence of secretory granules containing ectopic growth hormone and other studies have revealed that levels of growth hormone releasing hormones are increased in patients with lung cancer. In addition to the theory that paraneoplastic hormones may explain the features of this condition, it has also been observed that the majority of pulmonary diseases complicated by clubbing and secondary HOA, such as lung malignancy, are associated with intrapulmonary shunting of blood. It is proposed

Figure 2 (a) X-ray showing linear periosteal reaction involving radius and ulna bilaterally. (b) X-ray showing exuberant periosteal reaction of bilateral tibia and fibula and generalized soft tissue involvement. Reproduced from John B, Subhash H, and Thomas K (2004) Journal of the New Zealand Medical Association 117: 1192, with permission.

SIGNS OF RESPIRATORY DISEASE / Clubbing and Hypertrophic Osteoarthropathy

that when arteriovenous shunting occurs in the lung, vasoactive cells such as platelets, which are normally fragmented in the pulmonary microvasculature, escape this inactivating event. Subsequently, they are able to reach the systemic circulation, and subsequently impact and degranulate at distal sites, such as fingers and toes. Release of growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-b) from platelet alpha granules are able to locally activate the endothelium promoting neovascular proliferation, osteoclastic activity, and connective tissue matrix synthesis, thereby initiating clubbing. In addition to TGF-b and PDGF, levels of other growth factors such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) have also been shown to be associated with clubbing and secondary HOA. However, more recently a number of conflicting reports have questioned the relevance of these growth factors in this condition. Levels of blood VEGF and HGF are extremely high in extensive skin infections and multiple myeloma, but neither condition is complicated by clubbing. Furthermore, in a study of emphysema, serum TGF-b

25

levels were equivalent in patients whether they were clubbed or not. In addition, intravenous infusion of PDGF in an animal model failed to reproduce features of clubbing. Finally, clubbing is not observed in patients with arterial thrombosis, a condition associated with the dramatic release of growth factors from activated platelets. It has therefore been suggested that other, as yet unidentified, cytokines may be responsible for clubbing and secondary HOA. Of further interest, it has been proposed that these cytokines may act as hormones and be involved in a mechanism involving the activation of dormant genes in the nail bed, surrounding fat, connective tissue, and skin, thereby stimulating organogenesis. The reason behind the interest in this hypothesis stems from the historical observation that clubbed and embryonic fingers have a number of features in common. Clubbing may therefore be the reappearance of an organ lost in evolution (atavism), only induced by the production of specific cytokines in various pathological situations (Figure 3). Further research into this interesting concept may yet help unravel the exact pathological events occurring in this condition.

Figure 3 (a) Hand and finger of 32 mm long embryo. (b) Hand and middle finger of 44 mm long embryo. (c) Hand and finger of adult patient with digital clubbing. The hand of the patient with clubbing shows no metacarpal pads, but the increase in soft tissue at the volar side of the fingertip resembles a digital pad. Furthermore, the nail of clubbed finger resembles the curved nail field of the human embryonic claw finger. Reproduced from Brouwers AA, et al. (2004) Clubbed fingers: the claws we lost? Medical Hypotheses 62: 321–324, with permission from Elsevier.

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SIGNS OF RESPIRATORY DISEASE / Clubbing and Hypertrophic Osteoarthropathy

Clinical Presentation of Clubbing and Secondary HOA in Respiratory Disease

Diagnostic Evaluation and Therapeutic Options for Clubbing and Secondary HOA

Although progress in elucidating the pathophysiology of clubbing and secondary HOA has been extremely slow, it has not prevented the clinical manifestations of these conditions becoming some of the most important physical signs complicating respiratory disease that medical students encounter when they commence training. Clubbing has been described as progressing through four separate phases. First, fluctuation and softening of the nail bed occur due to increased edema. Second, the normal 151 angle between the nail and cuticle is lost. Third, the convexity of the nails becomes accentuated producing the clubbed appearance of the fingertips. Finally, the nail becomes shiny, with disappearance of the normal skin creases. Topographically, clubbing can either be symmetrical, unilateral, or even unidigital. Some observers also classify clubbing into three color groups including normal, which is usually associated with a slowly evolving process such as a chronic low-grade inflammatory infectious disease. Clubbing associated with red skin color usually occurs in conjunction with a rapidly progressive condition such as pulmonary malignancy or a severe pulmonary infection such as miliary tuberculosis. Finally, clubbing associated with blue skin color is usually associated with congenital cyanotic cardiac conditions but can occur in any pulmonary condition where profound hypoxia may be present, such as end-stage pulmonary fibrosis. A number of clinical signs have also been described to demonstrate clubbing which will not be discussed in detail here but essentially all highlight the excessive matrix production that occurs in the nail bed and surrounding structures in this condition. Clubbing can present in a more insidious fashion than secondary HOA and as the onset can be slower, and not necessarily associated with pain, patients often fail to present until at an advanced stage of the underlying condition, which can be devastating. Secondary HOA usually presents with a painful arthropathy in long bones and joints, and may be complicated by the presence of large joint effusions. Dysesthesia (abnormal sensation) of fingers accompanied by sweating and stiffness of the hands are other documented presenting features. Although these rheumatic-type symptoms may precede the appearance of constitutional symptoms such as fatigue, fever, and weight loss, they too can herald the appearance of an underlying incurable pulmonary malignancy.

Investigation of clubbing and secondary HOA essentially depends on establishing an underlying cause based on systematic history and clinical examination. This is vital since approximately 90% of HOA cases have an underlying neoplastic or parainfectious etiology. The treatment of clubbing and secondary HOA is then dependent on the eradication of the underlying cause. Unfortunately, as emphasized earlier, in pulmonary malignancy, the appearance of clubbing and secondary HOA can indicate an advanced and untreatable stage of disease. However, there are well-described pulmonary causes of established clubbing and secondary HOA which have fully resolved following treatment of the underlying disease process, presumably due to removal of the bioactive agent responsible for the clinical changes. Examples include surgical resection of lung cancer, treatment of lung cancer with chemotherapy or radiation, and lung transplantation for cystic fibrosis and pulmonary fibrosis. Of interest, extrapulmonary causes of clubbing and secondary HOA have also been reversed following correction of congenital cyanotic heart defects, hepatic transplantation for biliary atresia, primary sclerosing cholangitis, Wilson’s disease and primary biliary cirrhosis, treatment of infective endocarditis, and surgical resection of renal metastases. Finally, a number of conventional analgesic agents have been used for the treatment of pain associated with secondary HOA with a varying degree of success and of interest, the antiosteoclastic agent palmidronate and the somatostatin analog octreotide, which is an inhibitor of growth hormone, have been shown to be of analgesic benefit in this condition. See also: Angiogenesis, Angiogenic Growth Factors and Development Factors. Bronchiolitis. Cystic Fibrosis: Overview. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Hepatocyte Growth (Scatter) Factor. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Mesothelioma, Malignant. Platelet-Derived Growth Factor. Platelets. Systemic Disease: Sarcoidosis. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumors, Malignant: Bronchogenic Carcinoma; Chemotherapeutic Agents; Hematological Malignancies; Lymphoma; Metastases from Lung Cancer; Bronchial Gland and Carcinoid Tumor; Rare Tumors; Carcinoma, Lymph Node Involvement. Vascular Endothelial Growth Factor.

SIGNS OF RESPIRATORY DISEASE / General Examination 27

Further Reading Atkinson S and Fox SB (2004) Vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF) play a central role in the pathogenesis of digital clubbing. Pathology 203(2): 721–728. Augarten A, Goldman R, Laufer J, et al. (2002) Reversal of digital clubbing after lung transplantation in cystic fibrosis patients: a clue to the pathogenesis of clubbing. Pediatric Pulmonology 34: 378–380. Brouwers AA, Vermeij-Keers C, van Zoelen EJ, and Gooren LJ (2004) Clubbed fingers: the claws we lost? Medical Hypotheses 62: 321–324. Dickinson CJ (1993) The aetiology of clubbing and hypertrophic osteoarthropathy. European Journal of Clinical Investigation 23: 330–338. Hippocrates (1849) The Book of Prognostics. In: Adams F (translator) The Genuine Works of Hippocrates. London: Sydenham Society. John B, Subhash H, and Thomas K (2004) Journal of the New Zealand Medical Association, 117: 1192. Martinez-Lavin M (1997) Hypertrophic osteoarthropathy. Current Opinion in Rheumatology 9(1): 83–86. Martinez-Lavin M, Matucci-Cerinic M, Jajic I, and Pineda C (1993) Hypertrophic osteoarthropathy: consensus on its definition, classification, assessment and diagnostic criteria. Journal of Rheumatology 20(8): 1386–1387. Menard H (2004) Hypertrophic osteoarthropathy. Pineda C, Fonseca C, and Martinez-Lavin M (1990) The spectrum of soft tissue and skeletal abnormalities of hypertrophic osteoarthropathy. Journal of Rheumatology 17: 626–632.

General Examination T E King Jr, San Francisco General Hospital, San Francisco, CA, USA A C Zamora, Hospital Universitario ‘Dr Jose E Gonzalez’, Monterrey Nuevo Leon, Mexico & 2006 Elsevier Ltd. All rights reserved.

Abstract This article describes the initial evaluation of a patient with new onset respiratory problem. A thorough medical history and physical examination are the keys in making a specific diagnosis or narrowing the differential diagnosis of most respiratory disorders. The common signs and symptoms of respiratory problems include dyspnea, cough, hemoptysis, wheezing, chest pain, and snoring.

Introduction Patients with respiratory problems commonly present for medical attention because of the development of dyspnea, cough, hemoptysis, wheezing, chest pain, or snoring. Occasionally, patients are asymptomatic and abnormalities are detected on routine health screening examination or through the evaluation of another unrelated medical condition. This

article describes the initial evaluation of a patient with new onset respiratory problem. We will focus on the interpretation of common presenting symptoms and physical findings. A thorough medical history and physical examination are the keys in making a specific diagnosis or narrowing the differential diagnosis.

Medical History The past medical history is important in defining and characterizing the patient’s illness. It is important to determine whether or not there are previous or concurrent nonrespiratory illnesses that could be a factor in causing or worsening the present clinical problem. Particular attention needs to be placed on the identification of occupational and environmental exposures, smoking history, medication history, and family history. It is critical that the clinician obtain and review any prior chest-imaging studies. Often this process uncovers risk factors for specific respiratory disorders, identifies the recurrence of a dormant condition, or uncovers a previously undiagnosed condition. The family history is occasionally helpful since familial associations or genetic transmission of lung diseases have been identified (e.g., asthma, COPD, cystic fibrosis, a-1 antiprotease deficiency, sarcoidosis). Life style, habits, and travel history are important areas to explore. For example, a history of travel to an endemic area for a particular disease (e.g., tuberculosis) might identify this as the cause of the patient’s illness. Finally, the family history might identify illnesses related to common exposures. A strict chronological listing of the patient’s entire lifelong employment must be sought, including specific duties and known exposures to dusts, gases, and chemicals. The degree of exposure, duration, latency of exposure, and the use of protective devices should be elicited. Review of the environment (home and work, including that of spouse, children, and friends), especially relating to pets, air conditioners, humidifiers, hot tubs, evaporative cooling systems (e.g., swamp coolers), etc., is valuable as well. Symptoms may diminish or disappear after the patient leaves the exposure for several days; similarly, symptoms reappear on returning to the exposure. Obtaining the occupational and environmental history can be quite difficult. Consequently, questionnaires are commonly given to patients that allow them to review their employment record, military service, hobbies, pets, or other potential exposures. The history of tobacco use is important since some diseases occur largely among current or former smokers (e.g., chronic obstructive pulmonary disease

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SIGNS OF RESPIRATORY DISEASE / General Examination

(COPD)) or among never or former smokers (sarcoidosis and hypersensitivity pneumonitis). Active smoking can lead to complications such as pulmonary hemorrhage in patients with Goodpasture’s syndrome. A detailed medication history is needed to exclude the possibility of drug-induced disease, including over-the-counter medications, oily nose drops, and supplements. Importantly, lung disease may occur weeks to years after the drug has been discontinued. Gender is important in lymphangioleiomyomatosis which occurs exclusively in premenopausal women. Also, interstitial lung disease (ILD) in the connective tissue diseases is more common in women; the exception is ILD in rheumatoid arthritis which is more common in men. Age is helpful given that the majority of patients with asthma, sarcoidosis, and connective tissue disease present between the ages of 20–40 years. Conversely, most patients with idiopathic pulmonary fibrosis are over age 60.

symptoms, the relationship of symptoms to activity, and any factors that may improve or exacerbate symptoms. In most cases, the primary problem is heart, lung, or neuromuscular abnormalities, which can be identified largely by the history and physical examination. The quality of breathing discomfort often provides tips to the underlying diagnosis; the absence of cigarette smoking is strongly against a diagnosis of COPD. The occupational history may lead to a diagnosis of asbestosis or hypersensitivity pneumonitis. The presence of reproducible events such as exposure to fumes or cold air is common with airways hyperreactivity. When developing a differential diagnosis, it is useful to distinguish respiratory systems dyspnea from cardiovascular dyspnea. It is not uncommon for a patient to have more than one problem contributing to the breathing discomfort. Diagnostic testing commonly follows to identify the specific nature of the disorder. Dyspnea is classified into the following categories:

Respiratory Symptoms

1. Acute dyspnea has a short list of causes, most of which are readily identified: asthma, pulmonary infection, pulmonary edema, pneumothorax, pulmonary embolus, metabolic acidosis, or acute respiratory distress syndrome (ARDS). 2. Orthopnea (dyspnea on recumbency) and nocturnal dyspnea suggest asthma, gastroesophageal reflux disease (GERD), left ventricular dysfunction, or obstructive sleep apnea. 3. Platypnea (dyspnea that worsens in the upright position) is a rare complaint associated with arteriovenous malformations at the lung bases or with hepatopulmonary syndrome, resulting in increased shunting and hypoxemia in the upright position (orthodeoxia). 4. Episodic dyspnea suggests congestive heart failure, asthma, acute or chronic bronchitis, or recurrent pulmonary emboli. 5. Chronic dyspnea is invariably progressive. Symptoms often first appear during exertion; patients learn to limit their activity to accommodate their diminished pulmonary reserve until dyspnea occurs with minimal activity or at rest. The most common causes of chronic dyspnea are asthma, chronic obstructive lung disease, ILD, and cardiomyopathy, but deconditioning is often a major contributing factor in patients with chronic lung disease.

The most common presenting symptoms are progressive breathlessness with exertion, cough (with or without hemoptysis), wheezing, chest pain, or snoring. These are discussed in detail in other articles so we will only provide a general overview. Dyspnea

Dyspnea is the term applied to sensations experienced by individuals who complain of uncomfortable respiratory sensations (see Signs of Respiratory Disease: Breathing Patterns). Dyspnea has been defined in several ways, for example, ‘difficult, labored, uncomfortable breathing’, an ‘awareness of respiratory distress’, ‘the sensation of feeling breathless or experiencing air hunger’, and ‘an uncomfortable sensation of breathing’. The sensation of dyspnea derives from interactions among multiple physiological and behavioral responses. It is the result of an imbalance between ventilatory demand and capacity due to increased work of breathing. Often, the patient has attributed the insidious onset of breathlessness to aging, deconditioning, obesity, or a recent upper respiratory tract illness. Some patients deny the presence of dyspnea even when questioned because they perform a limited amount of activity and so do not ‘experience’ any significant discomfort. Occasionally a spouse or friend brings the problem to their attention. It is important to determine the duration and extent of dyspnea, cough, and sputum production (if any). The dyspnea history should focus on onset and timing of symptoms, the patient’s position at onset of

The initial evaluation following the history and physical examination should include a complete blood count (to exclude anemia as a contributing factor to respiratory discomfort), renal function test, chest radiograph, standard spirometry, and noninvasive oximetry during ambulation at a normal pace

SIGNS OF RESPIRATORY DISEASE / General Examination 29

over 200 meters. The chest radiography may provide evidence of hyperinflation and bullous disease suggestive of obstructive lung disease, or change in interstitial markings consistent with inflammation or interstitial fluid. Abnormalities of heart size may indicate valvular heart disease or other cardiac dysfunction. Standard spirometry can distinguish patients with restrictive pulmonary disease from those with obstructive airway disease. Lung-volume measurements and diffusing capacity are generally reserved for patients in whom ILD is being considered, in those who have significant declines in oxygen saturation with exercise, or in those for whom there is a suspicion of ventilatory muscle weakness. Echocardiography is reserved for patients in whom chest radiography reveals the heart to be enlarged, or in whom the diagnosis of chronic thromboembolic disease or pulmonary hypertension is being considered. Computed tomography (CT) of the chest is valuable in two circumstances – when patient has crackles on physical examination or reduced lung volumes on pulmonary function test even if the radiography is normal, and for those who have oxygen desaturation with exercise and a low diffusing capacity (occult emphysema). Cardiopulmonary exercise testing is recommended if the etiology of a patient’s dyspnea remains unclear after the initial evaluation described above. This test allows to determine if the patient’s dyspnea is more likely due to cardiovascular or respiratory system abnormalities, or if it is due to deconditioning. Cough

Cough by definition is an explosive expiration that provides a normal protective mechanism for clearing the tracheobronchial tree of secretions and foreign material. Cough is often divided into three categories: acute (defined as lasting o3 weeks), subacute (lasting 3–8 weeks), and chronic (lasting 48 weeks). Coughing may be initiated either voluntarily or reflexively. As a defensive reflex, it has both afferent and efferent pathways. The afferent limb includes receptors within the sensory distribution of the trigeminal, glossopharyngeal, superior laryngeal, and vagus nerves. The efferent limb includes the recurrent laryngeal nerve and the spinal nerves. The cough starts with a deep inspiration followed by glottic closure, relaxation of the diaphragm, and muscle contraction against a closed glottis. The resulting markedly positive intrathoracic pressure causes narrowing of the trachea. Once the glottis opens, the large pressure differential between the airways and the atmosphere coupled with tracheal narrowing produces rapid flow rates through the trachea. The shearing forces that develop, aid in the elimination of mucus and foreign materials.

The duration of the cough at the time of presentation helps to determine the likely cause. Acute cough is most often associated with upper respiratory tract infections such as common cold, acute bacterial sinusitis, pertussis, exacerbations of COPD, allergic rhinitis, and rhinitis due to environmental irritants. Acute cough can be the presenting manifestation of pneumonia, left ventricular failure, or asthma especially in elderly patients, because classic signs and symptoms may be minimal or not present. Subacute cough often follows an upper respiratory tract infection and lasts for 3–8 weeks, the most common conditions are postinfectious cough, bacterial sinusitis, and asthma. Postinfectious cough may result from postnasal drip or clearing of the throat due to rhinitis, tracheobronchitis, or both, with or without transient bronchial hyperresponsiveness. When cough is subacute and is not associated with an obvious respiratory infection, then the patient should be evaluated in the same way as patients with chronic cough (see below). The main causes (B95% of cases) of chronic cough include postnasal drip syndrome from conditions of the nose and sinuses, asthma, gastroesophageal reflux disease, chronic bronchitis due to cigarette smoking or other irritants, bronchiectasis, or the use of drugs (especially an angiotensin-converting enzyme (ACE) inhibitor). Other causes of chronic cough include bronchogenic carcinoma, carcinomatosis, sarcoidosis, left ventricular failure, and aspiration due to pharyngeal dysfunction. The patient’s clinical history is often very helpful in arriving at the cause of the cough (if there are symptoms suggestive of a respiratory infection, if there is a seasonal cause of wheezing or postnasal drip, if the patient has heartburn or sensation of regurgitation, if there is sputum or dry cough, etc.) Focus should be on the smoking history, environmental exposures, and medication use (e.g., ACE inhibitors or b blockers). Figure 1 provides an algorithm for diagnosing chronic cough in immunocompetent adults from a Consensus Panel Report of the American College of Chest Physicians. Physical examination is necessary to find a nonpulmonary cause of cough such as heart failure or AIDS. Auscultation of the chest is useful to find stridor (upper airway disease), rhonchi, or expiratory wheezing (lower airway disease), inspiratory crackles (ILD, pneumonia, or pulmonary edema). In a patient without asthma, sputum studies with greater than 3% eosinophils on staining suggest the possibility of eosinophilic bronchitis. Sometimes other tests are needed, for example, bronchoprovocation with methacholine to demonstrate hyperreactivity and confirm asthma; high resolution computed tomography

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SIGNS OF RESPIRATORY DISEASE / General Examination

Cough gone

Chronic cough Stop ACEI

Hx PE

ACEI

Cough persists

Chest radiograph

Normal

Abnormal

Avoid irritant

Cough gone

Order according to likely clinical possibility

Abnormality may not be related to cough

Sputum cytology, HRCT scan, modified BaE, bronchoscopy, cardiac studies

Cough persists

Treat accordingly

Evaluate for three most common conditions singly in the following order, or in combination: 1. PNDS

Cough gone

2. Asthma

Cough persists

Cough gone

3. GERD

Consider postinfectious cough

Cough persists

Evaluate for uncommon conditions

Sputum tests, HRCT scan, modified BaE, bronchoscopy, cardiac studies

Cough gone

Cough persists

Reconsider adequacy of treatment regimens before considering habit or psychogenic cough Figure 1 Guidelines for evaluating chronic cough in immunocompetent adults. ACEI, angiotensin-converting enzyme inhibitor; BaE, barium esophagography; GERD, gastroesophageal reflux disease; HRCT, high-resolution computed tomography; HX, history; PE, physical examination; PNDS, postnasal drip syndrome. Reproduced from Irwin RS, Boulet LP, Cloutier MM, et al. (1998) A consensus panel report of the American College of Chest Physicians. Managing cough as a defense mechanism and as a symptom. Chest 114(2 supplement Managing): 133S–181S, with permission.

(HRCT) to confirm the presence of ILD, COPD, or bronchiectasis; and 24 h monitoring of esophageal pH to help identify silent gastroesophageal reflux disease.

Hemoptysis

The word ‘hemoptysis’ comes from the Greek ‘haima’ for ‘blood’ and ‘ptysis’ for ‘a spitting’ together meaning, ‘a spitting of blood’. The source

SIGNS OF RESPIRATORY DISEASE / General Examination 31

of the blood originates below the vocal cords. It is classified as massive when more than 200–600 ml in 24 h, or when the patient is hemodynamically significant or it threatens ventilation. The history should include questions regarding smoking habits, past respiratory infections (e.g., pneumonia or tuberculosis), bronchiectasis, history of deep venous thrombosis, bleeding disorders, anticoagulant therapy, and recent weight loss (Table 1). Physical examination should include careful inspection of the upper airway. Certain findings may be helpful in suggesting a diagnosis: a saddle nose deformity with rhinitis and septal perforation are signs of Wegener’s granulomatosis; oral or genital aphthous ulcerations, uveitis, or cutaneous nodules may be the clinical presentations in patients with Behc¸et’s disease; clubbing may be a sign of lung carcinoma or bronchiectasis. In the examination of the lungs, a pleural friction rub may suggest pulmonary embolism. Cardiac examination may demonstrate findings of cor pulmonale or mitral stenosis. Chest imaging studies may help to identify lung parenchymal pathologies. HRCT can demonstrate lesions that may not be visible in chest radiograph, such as bronchiectasis or a small bronchial carcinoma. When performed with contrast material, CT may detect pulmonary embolism, thoracic aneurysm, or arteriovenous malformation. Fiberoptic bronchoscopy is commonly performed, both for anatomic localization of the bleeding site and to exclude neoplasm. Flexible bronchoscopy can be performed at bedside, does not require general anesthesia, and is usually quicker than the rigid bronchoscopy. It also allows easy access to the upper lobes. The disadvantages are a limited suctioning capability and an inability to localize bleeding when the hemorrhage rate is rapid. The diagnosis yield increases when patients undergo bronchoscopy within 48 h of a bleeding episode. In general, bronchoscopy should be performed in patients who smoke, in those who are older than 40 years, and those who have bleeding that persists (even intermittently) for more than 2 weeks. Some authors have found bronchogenic carcinoma in as many as 15–20% of bronchoscopies in patients with normal chest radiography. Except for life-threatening situations, CT should be performed before bronchoscopy. Rigid bronchoscopy is less frequently used but has been found to be safe and well tolerated under local anesthesia and conscious sedation, when performed by experienced hands. It offers better suctioning capability, continuous airway control, and a larger lumen for introducing packing materials and clearing clots and debris from the airways of patients with active bleeding. Its disadvantages include a reduced

Table 1 Common causes of hemoptysis Cardiac Mitral stenosis Tricuspid endocarditis Drugs/toxins Anticoagulants Aspirin Crack cocaine Hematologic Coagulopathy Platelet dysfunction Thrombocytopenia Infection Fungal Lung abscess Mycetoma Necrotizing pneumonia Tuberculosis Iatrogenic Bronchoscopic Lung biopsy Swan-Ganz catheter use Neoplastic Bronchial adenoma Lung cancer Metastasic disease Pulmonary Bronchitis Bronchiectasis Pulmonary embolism Cryptogenic Foreign body Cryptogenic hemoptysis Systemic Goodpasture’s syndrome Idiopathic pulmonary hemosiderosis Systemic lupus Systemic vasculitides Traumatic Aortic aneurysm Chest trauma Fat embolism Ruptured bronchus Vascular Aortic aneurysm Arteriovenous malformation Pulmonary hypertension

range of visibility of upper lobe and the need for general anesthesia. Chest Pain

The evaluation of acute chest pain should begin with a clinical history that focuses on the characteristics of the pain, the time of onset, and the duration of symptoms, and an examination that emphasizes vital

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SIGNS OF RESPIRATORY DISEASE / General Examination

signs and cardiovascular status. An electrocardiogram should be obtained within 5 min after presentation, followed by a chest X-ray. The goal of the initial evaluation of the patient is to rule out coronary artery disease (CHD) and conditions that are life-threatening. Common causes of chest pain include muscle or skeletal chest pain, costochondritis, gastroesophageal reflux, and stable angina pectoris. Unstable angina was an uncommon cause in a recent large prospective study. Nonetheless, cardiac pain must be ruled out quickly. Quality of the pain. The patient with ischemia often denies feeling chest ‘pain’. The sensation is commonly described as squeezing, tightness, pressure, constriction, strangling, burning, heartburn, fullness in the chest, to band-like sensation, knot in the center of the chest, lump in the throat, ache, heavy weight on chest (elephant sitting on chest), like a bra too tight, and toothache (when there is radiation to the lower jaw). Some patients cannot qualify the nature of the discomfort but will place fist in the center of the chest (the ‘Levine sign’). Patients with a history of CHD tend to have the same quality of chest pain with recurrent ischemic episodes. Location of the pain. Unlike the muscular or pleuritic pain that can be located, ischemia is more diffuse and thus difficult to locate. Radiation. The radiation of the pain to right arm (2.9 likelihood ratio) or even toward both arms (7.1 likelihood ratio) is a predictor of acute myocardial infarction. Pain associated with acute cholecystitis radiates to the right shoulder. The pain of aortic dissection radiates toward the back and that of pericarditis toward one or both trapezes. Onset of the pain. The pain due to pneumothorax, aortic dissection, or acute pulmonary embolism is sudden; ischemic pain is gradual and increases with time, and pain of the musculoskeletal type has a very vague beginning. Provocation. If the chest pain increases when eating, we suspect a gastrointestinal cause or myocardial ischemia. Myocardial ischemia also increases by exercise, cold, and emotional stress. The pain that increases with the change of position suggests a musculoskeletal source. Pleuritic type of pain increases with deep breathing and when lying down. Other useful findings. Vital signs can be helpful, for example, a marked difference in blood pressure between the two arms suggests the presence of aortic dissection. Hyperestesia combined with a skin rash indicates herpes zoster. Chest auscultation for pleural or pericardial rub, signifies heart murmur, crackles, wheezes or signs of consolidation are useful in suggesting possible cause of the chest pain, for example, pericarditis, valvular heart disease, ILD, asthma, or

pneumonia respectively. Also, careful examination of the abdomen is important, with attention to the right superior quadrant and epigastrium. Wheezing

A wheeze is a continuous musical sound that lasts longer than 250 msec. Wheezing occurs during inspiration or expiration and originates from airways of any size; conversely, stridor is an inspiratory wheezing that is loudest over the central airways. The whistling sound (wheezing) occurs when a patient attempts to exhale through bronchial passages that are constricted or excreting excess mucus due to irritation, infection, or allergy. The more common symptoms are tightening in the chest and dyspnea. In evaluating patients with wheezing, it is important to be aware that ‘‘All that wheezes is not asthma; all that wheezes is obstruction.’’ Using the history, physical examination, lung function studies, and knowledge of the spectrum of differential diagnostic possibilities, especially those that have been shown to be the most common is how we can make the diagnosis (see Table 2). Snoring

Snoring is an inspiratory sound produced by vibration of the soft tissues of the upper airway during sleep. Snoring is a common symptom in the general population. However, snoring may indicate a more serious medical problem, such as obstructive sleep apnea (OSA) or the upper airway resistance syndrome (UARS). Some patients appear asymptomatic unless the clinician makes a systematic effort to elicit symptoms. Consequently, it is important to recognize whether clinical features suggest an underlying sleeprelated breathing disorder and if some form of objective testing is indicated. The physiology, clinical significance, and evaluation of snoring is discussed elsewhere in this text.

Physical Examination Examination of the patient with pulmonary disease includes inspection, palpation, percussion, and auscultation of the chest. Inspection. This includes the respiratory rate and rhythm, breathing pattern, as well as the depth and symmetry of lung expansion. Respiratory rate normal values are 12–14 breaths per min. Tachypnea is an increased rate of breathing. During normal breathing, the primary muscle of respiration is the diaphragm, but when the patient uses the intercostals and sternocleidomastoid muscles (accessory muscles), it indicates labored breathing. The chest normally expands

SIGNS OF RESPIRATORY DISEASE / General Examination 33 Table 2 Causes of wheezing based on anatomic site of obstruction Extrathoracic upper airway obstruction

Intrathoracic upper airway obstruction

Lower airway obstruction

Postnasal drip syndrome Vocal cord dysfunction Hypertrophied tonsils Epiglottitis Laryngeal edema Laryngostenosis Postextubation granuloma Retropharyngeal abscess Neoplasms Anaphylaxis Malignancy Obesity Klebsiella rhinoscleroma Mobile supraglottic soft tissue Relapsing polychondritis Laryngocele Abnormal arytenoid movement Vocal cord hematoma Bilateral vocal cord paralysis Cricoarytenoid arthritis Wegener’s granulomatosis

Tracheal stenosis Foreign body aspiration Benign airway tumors Malignancies Intrathoracic goiter Tracheobronchomegaly Acquired tracheomalacia Herpetic tracheobronchitis Right sided aortic arch

Asthma COPD Pulmonary edema Aspiration Pulmonary embolism Bronchiolitis Cystic fibrosis Carcinoid syndrome Bronchiectasis Lymphangitic carcinomatosis Parasitic infections

Reproduced with permission from Irwin RS, Diagnosis of wheezing illnesses other than asthma in adults. In: UpToDate, Rose (ed.) UpToDate, Wellesley, MA, 2005. Copyright & 2005 UpToDate, Inc. For more information visit http://www.uptodate.com.

Table 3 Extrapulmonary physical findings associated with lung diseases Physical findings

Associated conditions

Fever

Infections, eosinophilic pneumonia, drug reactions, cryptogenic organizing pneumonia, hypersensitivity pneumonitis, sarcoidosis, lymphoma, lymphangitic carcinoma

Skin changes Erythema nodosum Maculopapular rash Heliotrope rash Telangiectasia Raynaud phenomenon Cutaneous vasculitis Subcutaneous nodules Calcinosis Cafe´-au-lait spots Eye changes Uveitis Scleritis Keratoconjunctivitis sicca Albinism Salivary gland enlargement Peripheral lymphadenopathy Myositis; muscle weakness Arthritis

Sarcoidosis, connective tissue disease, Behc¸et syndrome, histoplasmosis, coccioioidomycosis Drug-induced diasease, amyloidosis, lipoidosis connective tissue disease, Gaucher disease Dermatomyositis Scleroderma Connective tissue disease (scleroderma) Systemic vasculitides, connective tissue disease Von Recklinghausen disease, rheumatoid arthritis Dermatomyositis, scleroderma Neurofibromatosis

Sarcoidosis, Behc¸et syndrome, ankylosing spondilitis Systemic vasculitis, systemic lupus erythematosus, scleroderma, sarcoidosis Lymphocytic interstitial pneumonia, Sjo¨gren syndrome Hermansky–Pudlak syndrome Sarcoidosis, Lymphocytic interstitial pneumonia, Sjo¨gren syndrome Sarcoidosis, lymphangitic carcinomatosis, Lymphocytic interstitial pneumonia, Sjo¨gren syndrome Connective tissue disease, sarcoidosis, drugs Connective tissue disease, vasculitis, sarcoidosis, Goodpasture syndrome

Reproduced from Schwarz MI, King TE Jr, and Raghu G (2003) Approach to the evaluation and diagnosis of interstitial lung disease. In: Schwarz MI and King TE (eds.) Interstitial Lung Diseases, 4th edn., p. 8. Hamilton, ON: BC Decker, with permission from BC Decker.

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SIGNS OF RESPIRATORY DISEASE / General Examination

simultaneously. Asymmetric expansion of the chest is usually due to an asymmetric process affecting the lungs, and suggests unilateral pleural or parenchyma problem. Visible abnormalities of the thoracic cage include kyphoscoliosis which can alter compliance of the thorax and cause dyspnea. Palpation. The trachea at the suprasternal notch, detects shift in the mediastinum. The symmetry of lung expansion can be assessed, confirming the findings observed by inspection. Vibration produced by spoken sounds is transmitted to the chest wall and is assessed by the presence or absence and symmetry of tactile fremitus, especially on the posterior chest wall. Transmission of vibration is decreased or absent if pleural liquid is interposed between the lung and chest wall; in contrast, transmitted vibration may increase over an area of underlying pulmonary consolidation. Percussion. Assess the resonance or dullness of the tissue underlying. The normal sound of underlying aircontaining lung is resonant. Dull areas correspond to lung consolidation or pleural effusion and hyperresonant areas suggest emphysema or pneumothorax. Auscultation. We listen for both the quality and intensity of the breath sounds and the presence of abnormal or adventitious sounds. Normal lung sounds heard over the periphery of the lung are called vesicular breath sounds, in which inspiration is louder and longer that expiration. Normal sounds heard over the suprasternal notch are called tracheal or bronchial lung sounds and have a hollow quality that tends to be louder on expiration. The breath sounds are diminished in intensity or absent if there is an endobronchial obstruction, or when there is fluid in the pleural space. When sound transmission is improved and has a more pronounced expiratory phase, a consolidated lung is present. Listening through the consolidated lung, the spoken sound (bronchophony) or the whispered sound (pectoriloquia) is part of the routine evaluation as is the presence of egophony, which is the sound of a spoken E that becomes more like an A. Adventitious sounds can be continuous or discontinuous. Continuous ones are divided into wheezes and rhonchi. Wheezes that are more prominent during expiration than inspiration, reflect the oscillation of airway walls that occurs when there is airflow limitation, as may be produced by bronchospasm, airway edema or collapse, or intraluminal obstruction by neoplasm or secretions. Rhonchi originate in the large airways when excessive secretions and abnormal airway collapsibility cause repetitive rupture of fluid films and frequently clear after cough. Discontinuous sounds are called crackles which are typically inspiratory, are produced by the sound created when alveoli and small airways open and

close with respiration. Fine crackles are heard in ILD or filling alveoli liquid as pulmonary edema. Coarse crackles result from air bubbling through fluid and are heard in pneumonia and late pulmonary edema. Appearance Normal

Clubbed

(a) Nail-fold angles C D

Normal B A

B

Clubbed A

C D

(b) Phalangeal depth ratio Normal DPD IPD

Clubbed DPD IPD

(c) Schamroth sign Normal

Clubbed

(d) Figure 2 Appearance on inspection for clubbing: (a) normal finger viewed from above and in profile, and the changes occurring in established clubbing, viewed from above and in profile. (b) The finger on the left demonstrates normal profile (ABC) and normal hyponychial (ABD) nail-fold angles of 1691 and 1831, respectively. The clubbed finger on the right shows increased profile and hyponychial nail-fold angles of 1911 and 2031, respectively. (c) Distal phalangeal finger depth (DPD)/interphalangeal finger depth (IPD) represents the phalangeal depth ratio. In normal fingers, the IPD is greater than the DPD. In clubbing, this relationship is reversed. (d) Schamroth sign: in the absence of clubbing, opposition of the index fingers nail-to-nail creates a diamond-shaped window (arrowhead). In clubbed fingers, the loss of the profile angle due to the increase in tissue at the nail bed causes obliteration of this space (arrowhead). Reproduced from Myers KA and Farquhar DR (2001) The rational clinical examination. Does this patient have clubbing? Journal of the American Medical Association 286: 341–347, with permission. Copyright & (2001) American Medical Association. All rights reserved.

SIGNS OF RESPIRATORY DISEASE / Lung Sounds 35 Extrapulmonary Signs of Pulmonary Disease

Table 3 lists the extrapulmonary physical findings associated with lung diseases. Digital clubbing There are structural changes at the base of the nails that include softening of the nail bed and loss of the normal 1501 angle between the nail and the cuticle (Figure 2). The distal part of the finger is enlarged in comparison with the proximal part. It may be a normal variant but more commonly is a sign of underlying pulmonary disease such as lung cancer, ILD, and chronic infections in the thorax such as bronchiectasias, lung abscess, and empyema. It is not commonly seen in COPD; if present, a complicating lung carcinoma may be present. Cor pulmonale In the mid or late stages of pulmonary diseases, findings of pulmonary hypertension (e.g., augmented P2, right-sided lift, and S3 gallop) and cor pulmonale may become evident. These can be primary manifestations of a connective tissue disorder (e.g., progressive systemic sclerosis) though. See also: Chronic Obstructive Pulmonary Disease: Smoking Cessation. Signs of Respiratory Disease: Breathing Patterns; Clubbing and Hypertrophic Osteoarthropathy. Sleep Apnea: Overview. Symptoms of Respiratory Disease: Cough and Other Symptoms; Dyspnea; Chest Pain.

Further Reading American Thoracic Society (1999) Dyspnea. Mechanisms, assessment, and management: a consensus statement. American Journal of Respiratory and Critical Care Medicine 159: 321–340. Braman SS and Corrao WM (1987) Cough: differential diagnosis and treatment. Clinics in Chest Medicine 8: 177–188. Cahill BC and Ingbar DH (1994) Massive hemoptysis. Assessment and management. Clinics in Chest Medicine 15: 147–167. Fitzgerald RS and Lahiri S (1986) Reflex response to chemoreceptor stimulation. In: Cherniack NS and Widdicombe JG (eds.) Handbook of Physiology, Section 3: The Respiratory System, Vol. 2. Control of Breathing, pp. 313–362. Bethesda: American Physiological Society. Irwin RS (2005) Diagnosis of wheezing illnesses other than asthma. In: Rose BD (ed.) UpToDate. Wellesley, MA: UpToDate. Irwin RS, Boulet LP, Cloutier MM, et al. (1998) Managing cough as a defense mechanism and as a symptom. A consensus panel report of the American College of Chest Physicians. Chest 114(2 supplement Managing): 133S–181S. Irwin RS and Madison JM (2000) The diagnosis and treatment of cough. New England Journal of Medicine 343: 1715–1721. Jean-Baptiste E (2000) Clinical assessment and management of massive hemoptysis. Critical Care Medicine 28(5): 1642–1647. Lee TH and Goldman L (2000) Evaluation of the patient with acute chest pain. New England Journal of Medicine 342: 1187–1195. Luce JM and Luce JA (2001) Perspectives on care at the close of life. Management of dyspnea in patients with far-advanced lung disease: ‘‘once I lose it, it’s kind of hard to catch it.’’ Journal of the American Medical Association 285: 1331–1337.

Manning HL and Schwartzstein RM (1995) Pathophysiology of dyspnea. New England Journal of Medicine 333: 1547–1553. Myers KA and Farquhar DR (2001) The rational clinical examination. Does this patient have clubbing? Journal of the American Medical Association 286: 341–347. O’Neil KM and Lazarus AA (1991) Hemoptysis. Indications for bronchoscopy. Archives of Internal Medicine 151: 171–174. Schwarz MI, King TE Jr, and Raghu G (2003) Approach to the evaluation and diagnosis of interstitial lung disease. In: Schwarz MI and King TE Jr (eds.) Interstitial Lung Diseases, 4th edn., p. 8. Hamilton, ON: BC Decker. Weinberger SE (2005) Etiology and evaluation of hemoptysis. In: Rose BD (ed.) UpToDate. Wellesley, MA: UpToDate.

Lung Sounds D C J Howell, Centre for Respiratory Research, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Sound has a number of vital characteristics including frequency, intensity, duration, and quality. These components determine the plethora of lung sounds that can be heard by the human ear. Historically, lung sounds were detected by placing the ear on the chest wall of the patient. However, with the discovery of the stethoscope, this practice was abandoned. Although much can be elucidated about underlying chest pathology by listening to a patient breathing while standing at the end of the bed, auscultation has added much to the clinical examination of patients with respiratory disease. Lung sounds can broadly be divided into breath sounds and voice sounds, and can be normal and abnormal. Abnormal lung sounds can be either abnormally transmitted by breath or voice, or may be adventitial (accidental). In this article, the range of various normal and abnormal sounds that the lung produces will be discussed and their pertinence to respiratory disease highlighted.

The Perception of Sound, the Stethoscope, and Auscultation Prior to discussing lung sounds in detail, it is prudent to elaborate on the nature of sound itself. Sounds consist of audible vibrations, which are created by alternating regions of compression and rarefaction of air. Sound has a number of important characteristics including frequency, intensity, duration, and quality. The frequency of sound reflects a measurement of the number of vibrations per unit time, in cycles per second, and is expressed in hertz (Hz). The large range of normal and abnormal lung sounds will be elaborated upon below but as examples, a sound producing a large amount of vibrations per unit time will be interpreted on clinical examination as a highpitched wheeze. Conversely, sounds with low vibrations per unit time are heard as low-pitched wheezes.

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SIGNS OF RESPIRATORY DISEASE / Lung Sounds

Sound intensity is more complicated and is determined by the initial energy-producing source, the amplitude of the vibrations, the distance the vibrations travel, and the material that the vibrations subsequently travel through. This explains why some lung sounds are perceived loudly, such as in the consolidated lung or softly, as in a lung full of emphysematous bullae. The duration of vibrations determines whether the ear of the clinical examiner discerns sound as long or short, such as in the prolonged expiratory wheeze of a patient suffering from chronic obstructive pulmonary disease (COPD). Sound quality is also called timbre and is dependent on the component frequencies that make up a particular sound. This allows the examiner to distinguish between different sounds produced in the chest such as the vesicular-breath sounds produced in normal lung tissue versus bronchial-breath sounds transmitted via consolidated lung. The majority of breath sounds are produced in a frequency range which the human ear is unable to interpret. This is because the normal ear has maximum sensitivity in the region of 1000–2000 Hz and the majority of normal breath sounds are produced below the 500 Hz range. Listening to lung sounds as a routine part of physical examination dates to the time of ancient physicians such as Hippocrates (about 400 BC). At that time, it was done by directly applying the ear to the patient’s chest. Sounds such as pleural friction rubs and splashing due to fluid in the chest cavity were described. This changed in 1816 when a French doctor called Rene Theophile Laennec used a novel technique to auscultate the chest which he described in the following quote, ‘‘I was consulted by a young woman laboring under general symptoms of diseased heart, and in whose case percussion and the application of the hand were of little avail on the account of the great degree of fatness. I rolled a quire of paper (24 sheets) into a kind of cylinder and applied one end of it to the region of the heart and the other to my eary .’’ He subsequently modified his technique by using a wooden cylindrical tube and published his full findings in 1819 (Figures 1(a) and 1(b)). Since then, many adaptations of the first stethoscope have taken place and now the modern instrument usually consists of two chest pieces, namely the bell and the diaphragm, tubing, binaural headset, and plastic ear pieces. Skilful auscultation with modern stethoscopes reveals much about underlying lung pathology. There is no substitute for experience in this discipline and many students have been amazed at what sounds are audible on using their stethoscope for the first time. Auscultation should be performed with the patient

Figure 1 (a) Laennec performing auscultation of the chest with the ear. (b) Laennec performing auscultation of the chest with an early stethoscope.

sitting upright in a quiet environment and comparison of the respective bronchopulmonary segments of each lung should be made through inspiration and expiration. The aim of auscultation is to compare the quality and intensity of voice and breath sounds in both lungs and to detect any added sounds. There remains controversy about whether the bell or diaphragm should be used to auscultate the chest. The bell amplifies low-frequency sounds in the range 40–115 Hz by up to 10 decibels and it is suggested that it should be used when listening above the clavicles. The diaphragm attenuates low-frequency sound and since the bell would amplify low-frequency noise from

SIGNS OF RESPIRATORY DISEASE / Lung Sounds 37

movement of the intercostal muscles and chest wall, most observers favor the diaphragm when auscultating the chest.

Lung Sounds Lung sounds can broadly be classified into breath sounds and voice sounds. The pathophysiology of normal breath and voice sounds will be elaborated upon in the immediate sections below. Following this, abnormal lung sounds will be discussed which are described as either abnormally transmitted breath or voice sounds, or adventitious (accidental). It is to be noted that adventitious sounds occur only in the diseased lung on top of normal breath sounds (Figure 2). Normal Breath Sounds

Normal breath sounds represent the most common lung sounds of all but, interestingly, the exact physiological processes responsible for producing them are still debated. Historically, it was thought that breath sounds arose in alveoli or so-called vesicles. This gave rise to the term vesicular breathing, which was thought to be synonymous with normal breathing. However, further experiments have shown that airflow within the alveoli is not responsible for the sounds associated with normal breath sounds. In fact, it is thought that turbulent airflow in lobar and segmental bronchi provides a more plausible explanation, but this is still controversial. Turbulence only occurs at a critical flow velocity and it has been shown that such flow occurs in the trachea and initial generations of bronchi. In more peripheral bronchi, laminar flow occurs which is silent. Normal breath sounds are also produced by vortices, which disrupt

laminar flow and occur in the region between segmental bronchi and the 15th generation of the airways. It is hypothesized that these physiological events give rise to four specific breath sounds which are considered normal when auscultation is performed. Normal vesicular This is a soft breath sound which has a longer inspiration than expiratory phase, heard predominantly over the periphery of the lung. It is this type of breath sound that initial observers thought was due to air movement in the alveoli, which is now known to be incorrect. Bronchial Normal bronchial breath sounds are loud and high-pitched. They have an inspiratory and expiratory phase with the expiratory phase usually being louder. Observers disagree about the best place to hear these sounds, some favoring the manubrium, others the trachea and larynx. Bronchial breath sounds in the periphery of the lung are abnormal and will be discussed below. Bronchovesicular These sounds have inspiratory and expiratory phases, which are approximately equal in length and, as the name suggests, are a mixture of bronchial and vesicular sounds. They can be heard anteriorly in the first and second intercostal spaces, between the scapulae and over all lung fields in very thin people and in children. The sounds created have been described as having a tubular quality or resembling that of blowing through a straw. Tracheal Tracheal sounds are very loud and highpitched. Since they are heard over the extrathoracic portion of the trachea, they are not usually

Lung sounds

Normal

Normal breath sounds

Normal voice sounds

Normal vesicular Bronchial Bronchovesicular Tracheal

Figure 2 Examples of lung sounds.

Abnormal

Abnormally transmitted

Breath sounds

Voice sounds

Bronchial breathing Cavernous Abnormally reduced sounds

Egophony Pectoriloquy Bronchophony

Adventitious Crackles (discontinuous) Wheezes (continuous) Pleural friction-rubs Miscellaneous

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auscultated but if performed, they are heard as harsh sounds as if air is being blown through a pipe. Normal Voice Sounds

Normal voice sounds are best heard over the trachea and large airways but less well peripherally. When a subject is speaking, sounds radiate out through the chest wall which can be discerned easily by auscultation with the stethoscope, although understanding individual words is hard. Normal speech is made up of a low-frequency sound produced by the oscillations of the vocal cords and amplified by overtones from resonance of the mouth, pharynx, and the paranasal sinuses. Low-frequency sounds of up to 200 Hz are transmitted well but higher-frequency sounds, such as those produced by vowels, are filtered out of the lung. This explains why understanding individual words with a stethoscope is very difficult and with whispering, nothing is usually heard at all. Abnormal Lung Sounds

As discussed above, abnormal lung sounds can be broadly divided into two major categories, namely first, abnormally transmitted lung sounds, which similarly to normal lung sounds can be subdivided into abnormal breath sounds and abnormal voice sounds, and second, adventitial (accidental) sounds. Adventitious sounds are never heard over normal lungs and are the sounds superimposed on normal vesicular breath sounds in the presence of disease. Abnormally Transmitted: Breath Sounds

Bronchial breath sounds Bronchial breath sounds arise when lung tissue between the large airways and chest wall becomes airless such as in the consolidated lung of pneumonia. Clinically they are loud sounds, similar to normal tracheal breathing and audible throughout inspiration and expiration. The expiratory phase is often prolonged with a short gap between inspiration and expiration. They are present because consolidated lung allows better transmission of sound than when the lungs are filled with air since there is virtually no loss of sound transmission by filtration. In addition to consolidation, bronchial breathing can also be heard in atelectasis and pulmonary fibrosis. Decreased or absent breath sounds Breath sounds are reduced or absent in conditions such as pleural effusion or pleural thickening where there is increased filtration of sound. This effect is also observed when the pleural cavity is filled with air, as in a pneumothorax. Severe emphysema and asthma

lead to hyperinflation of the lung and gas trapping such that breath sounds are often only faintly heard through the chest wall. These conditions, as well as complete occlusion of large airways and breathing patterns associated with pain, lead to reduced chest movement, which further diminishes breath sounds. Cavernous breath sounds Cavernous breath sounds are characteristically hollow, as heard when blowing over a bottle opening. These are perhaps a little more difficult to hear but are described in conjunction with large cavities in the lungs and with large pneumothoraces associated with bronchopleural fistulae. Abnormally Transmitted: Voice Sounds

Bronchophony Bronchophony arises when the lung between the stethoscope and trachea is airless and results in higher frequencies such as vowel sounds, which are normally filtered, becoming audible to the ear. Different observers have debated the consequence of this and whether speech can be detected clearly over an area of affected lung. The condition has a similar acoustic basis to that of bronchial breathing and therefore is present in the consolidated lung. Whispering pectoriloquy Whispering is produced without vocal chord oscillation and the sounds generated are produced by turbulent flow through the trachea, glottis, and pharynx below the threshold of detection by the human ear when listening with a stethoscope. When the lung is airless and consolidated as in pneumonia, high-pitched sounds produced by whispering become audible at auscultation. The sign of whispering pectoriloquy is confirmed if an observer can accurately hear whispered words when listening over a bronchopulmonary segment. Egophony Egophony is derived from the Greek word for the voice of a goat and is thus bleating in its character. As in bronchophony and whispering pectoriloquy, it occurs in consolidated, airless lung. It can also occur above a pleural effusion when there is associated collapse and atelectasis of the surrounding lung. Clinically, it is detected when an individual with either of these conditions says E, which is heard as A, when listening through a stethoscope. Adventitious (Accidental) Sounds

Adventitious sounds can be heard from the lungs themselves, or from other parts of the chest, such as pleura or pericardium. The nomenclature used to describe adventitious sounds is quite confusing as there have been numerous classifications and terminologies used over the years. Laennec initially

SIGNS OF RESPIRATORY DISEASE / Lung Sounds 39

coined the term rales for adventitial lung sounds and classified them into four groups: moist, mucous, sonorous, and sibilant. He also further defined these terms by producing an alternative heading for each description. Moist rales were described as crepitations and mucous rales gargling, and referred to short explosive sounds of high and low pitch, respectively. A sonorous rale was also described as snoring and was heard as a low-pitched continuous musical note. Sibilant or whistling rales included both long and short high-pitched musical sounds. He further modified his scheme by adding a fifth group called dry crepitant rales, or crackling. However, rale was not a word favored by all as it also had connotations with the sound produced by sputum in the airways of patients approaching death. For this reason, Laennec used the term rhonchus to describe such sounds. Over the years, separate classifications were proposed by Latham and subsequently by Robertson and Cooper, who divided adventitious lung sounds into crackles (discontinuous interrupted sounds) and wheezes (continuous sounds), which form the underlying basis of the classification used in this article, which also includes pleurally based and miscellaneous adventitial sounds. Discontinuous Adventitious Sounds

Crackles Crackles are described as short explosive, nonmusical sounds. Laennec described them as the sound heard when heating salt in a frying pan. Classically, crackles were thought to be due to bubbling of secretions in the airways. This is probably true for conditions where sputum in the major airways is a feature, for example, cystic fibrosis. However, in disorders such as pulmonary fibrosis, where sputum production is minimal, this obviously cannot apply. In this condition, it is therefore thought that crackles occur when there is explosive equalization of gas between two compartments of the lung, when an intervening closed portion suddenly opens. The clinical consequence of this observation is that during the initial phase of inspiration, when the fibrotic lung is deflated, many peripheral airways remain closed. As the lung expands with resultant increased elastic tension, airways reopen, producing a single crackle each time, the airways remaining open until inspiration is completed. Crackles can be further divided into a number of different subtypes. Early inspiratory/expiratory crackles classically occur in patients with severe airways’ obstruction. They tend to be produced in proximal and larger airways and are usually heard in lower lobes as low-pitched, scanty sounds, unchanged by cough or posture. Conditions in which

these types of crackles predominate include COPD and asthma. Late inspiratory crackles are characteristic of patients with restrictive lung disease such as pulmonary fibrosis and also in interstitial pulmonary edema. The sounds are more numerous than early inspiratory crackles, vary with patient position, and are heard mainly at the bases. The crackling usually starts in the latter part of inspiration and becomes more profuse towards the end of inspiration. Late inspiratory crackles are also heard in conditions where there is a delayed opening of the small airways, such as a resolving lobar pneumonia. A point to be noted is that inspiratory crackles heard in left ventricular failure have a different physiology from that of pulmonary fibrosis and are caused by the equalization of gas pressure after there has been delayed inspiratory opening of the small airways, narrowed by peribronchial edema fluid. Furthermore, lack of disappearance of crackles on change of posture in both pulmonary fibrosis and pulmonary edema indicates worsening degree of lung deflation and therefore increasing severity. Finally, crackles can also be subdivided into fine and coarse. Fine crackles are heard with pulmonary edema, pulmonary fibrosis, and pneumonia; they are predominantly inspiratory and described as above. Coarse crackles are usually heard at the beginning of expiration and are characteristic of bronchiectasis. Continuous Adventitious Sounds

Wheezes The exact definition of wheeze and its relationship with the word rhonchi has also been controversial over the years. Strictly speaking, wheeze is used for musical lung sounds which are heard at the mouth or at a distance from the patient, whereas rhonchi is reserved for musical sounds which can be heard through the chest wall with the aid of a stethoscope. However, practically, wheeze is the preferred term for both these sounds. Wheeze has a well-defined pitch which gives it a musical quality easily appreciated by the ear. The musical character is determined by the spectrum of frequencies that make up the sound and the lowest frequency (also called the fundamental) determines the pitch. The mechanism of wheeze is also debated but is thought to be similar to that produced by a vibrating reed of a wind instrument. Wheezes can also be subdivided into a number of categories. Fixed monophonic wheeze is a single musical note of constant pitch. This is produced by air passing at high speed through a narrow space caused by incomplete obstruction of a major or lobar bronchus and is often caused by tumors and foreign bodies. Monophonic wheeze can also be random as in

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asthma. In this condition, widespread airflow obstruction generated by bronchial spasm or mucosal swelling in the airways often gives rise to loud inspiratory and expiratory wheeze. Interestingly, the number of actual wheezes may not be as large as often thought by the observer when auscultating the chest. The sound generated is often produced by a few loud wheezes being widely transmitted to different points on the chest with varying intensity. Sequential inspiratory wheezes, an unusual type of monophonic wheeze, consist of a series of short musical sounds, each sound having a different pitch and loudness. They are heard only in inspiration and rarely overlap each other in time, which differentiates them from the sounds heard in asthma. They are produced by airways in deflated areas of the lung and therefore are normally heard in pulmonary fibrosis. Polyphonic wheeze is composed of several notes and generally heard in expiration. It is produced by compression of central bronchi and frequently heard in COPD and asthma, but can also be produced in a normal subject by a forced expiration, so the sign can be ambiguous. It is also worthy of note that paradoxical absence of wheeze may occur in severe asthma and COPD. For instance, in severe asthma, peripheral flow resistance is very high and dynamic compression of airways is displaced from the central bronchi towards the periphery of the bronchial tree as gas trapping occurs. After a specific point, the volume of air flowing through individual airways is so minute, they fail to oscillate and wheeze is absent. Stridor is another form of wheeze that has a loud musical sound of constant pitch. It is most prominently heard in inspiration and is different from a monophonic wheeze purely in its intensity. It is caused by rapid airflow due to obstruction in the upper respiratory tract, usually the trachea or larynx, and also has a mechanism analogous to that of a vibrating reed of a musical instrument. As the airway becomes more compromised, stridor can become expiratory as well, for instance, if a foreign body is aspirated or a tumor expands. Pleural Friction-Rub Adventitious Sounds

Usually, the visceral and parietal pleura rub together silently during breathing but this is not the case when the pleura are inflamed, coated in fibrinous exudates, or thickened by neoplastic invasion. In these cases, a number of sounds may be heard on auscultation including a sound described as a stringed instrument. However, more commonly, the rubbing sound produced resembles nonmusical sounds and has been compared to the creaking sound of old leather and described as a pleural friction-rub. The most

distinctive feature of this sound is that the sound heard in inspiration, is heard in the reverse sequence in expiration. Pleural rubs may be difficult to identify and may be difficult to distinguish from crepitations, except that the latter are more likely to be altered by coughing. Miscellaneous Adventitious Sounds

Mediastinal crunch (Hammans sign) This is a coarse crackling sound, synchronous with systole and frequently heard over the precordium when mediastinal emphysema is present, such as in patients suffering chest trauma. The sound is produced by air between the visceral and parietal pericardium crunching during myocardial contraction. A crunching sound can also be heard in a shallow left-sided pneumothorax where the lung and heart are separated by a narrow air space. If there is sudden displacement of trapped air in the mediastinal pleural space and/or the impact of the heart with the mediastinal lung surface, the sound may be elicited. Bronchial leak squeak This sound is a diagnostic sign in patients with bronchopleurocutaneous fistula. The sound is heard in the affected area of the chest when a sustained Valsalva maneuver is performed. The pitch of the sound increases as the size of the fistula becomes smaller, which can be used to monitor progress of closure. Inspiratory squawk On occasion, patients with pulmonary fibrosis and hypersensitivity pneumonitis have been described as producing a distinctive inspiratory musical sound called a squawk. It is thought that this is due to inflammation of the alveoli. It is said that squawks of allergic etiology, as in hypersensitivity pneumonitis, are of a higher pitch than nonallergic causes. Similar to the crackles produced in association with these diseases, the sound is thought to arise from opening of airways. Loose cough These are low-pitched rattling sounds, which arguably could be considered crackles. They are commonly heard in chronic smokers and provide a loose quality to the sound of coughing which has been compared to rattling of sputum in the large airways. They are indicative of widespread bronchial disease but can be heard in patients who do not produce sputum. The reason for this is that the mechanism of the sound production is the intermittent passage of boluses of gas through airways that are only lightly occluded, rather than sputum in the airways per se.

SIGNS OF RESPIRATORY DISEASE / Lung Sounds 41 Abnormal Lung Sounds Heard at the Mouth

Although this article has predominantly concentrated on lung sounds heard by auscultation, it is also worth remembering that much knowledge of underlying pathology, lung or otherwise, can be gained by standing next to a patient and listening to their breathing. The final paragraphs will briefly cover various sounds that can be heard at the mouth. Usually, breath sounds cannot be heard when over a foot away from a patient but on exercise, normal breath sounds will be heard as airflow becomes increasingly turbulent. Gasping is a sound which produces a sudden, deep inspiratory effort often associated with fright, submersion in cold water, or if the respiratory center is damaged. Sighing and yawning are slower, deeper inspirations which occur in all normal individuals. It is thought they arise in order to overcome alveolar cohesion forces. Kussmaul’s breathing produces a hissing sound which is generated by hyperventilation through opposed teeth, Often, the patient will not complain of breathlessness as the condition is associated with metabolic acidosis of nonpulmonary etiology. Snoring produces a loud inspiratory sound, that can be up to 100 decibels in severe cases. It is often benign in etiology but is also associated with obstructive sleep apnea and upper airway resistance syndrome. Panting respiration occurs in any pulmonary condition where lung compliance is poor as in pulmonary fibrosis or acute respiratory distress syndrome (ARDS). In these diseases, there is increased elastic work of breathing and in severe cases, panting may occur even at rest. Wheezing has been discussed above but it is to be noted that wheezes heard easily at the mouth and audible at a distance indicate that they are more likely to be from the large central airways whereas wheezes heard only via the chest wall indicate that they may have arisen from the peripheral airways. Whistles and grunts can be heard in patients with COPD and are due to breathing through closed lips and opposing vocal chords, respectively. In both maneuvers, the patient is trying to reduce dynamic airways’ collapse by applying auto-positive end expiratory pressure. Sniffles usually indicate upper respiratory tract infection and rattles, first described above as rales, are due to the sound of air passing though fluid, causing mucus secretions to vibrate in the airway through inspiration and expiration. They are also described as death rattles but similar crackles can be heard at the mouth in COPD when secretions are present in the upper airway. See also: Acute Respiratory Distress Syndrome. Asthma: Overview; Allergic Bronchopulmonary Asp-

ergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; ExerciseInduced; Extrinsic/Intrinsic. Atelectasis. Bronchiectasis. Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General; Smoking Cessation. Coagulation Cascade: Fibrinogen and Fibrin. Cystic Fibrosis: Overview. Interstitial Lung Disease: Overview. Alveolar Proteinosis; Amyloidosis; Cryptogenic Organizing Pneumonia; Hypersensitivity Pneumonitis; Idiopathic Pulmonary Fibrosis. Mesothelioma, Malignant. Pleural Effusions: Overview; Pleural Fluid, Transudate and Exudate; Pleural Fluid Analysis, Thoracentesis, Biopsy, and Chest Tube; Parapneumonic Effusion and Empyema; Malignant Pleural Effusions; Postsurgical Effusions; Pleural Fibrosis; Chylothorax, Psuedochylothorax, LAM, and Yellow Nail Syndrome; Hemothorax. Pneumonia: Atypical; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens); Nosocomial; Parasitic; Mycobacterial; Viral; The Immunocompromised Host. Pneumothorax. Pulmonary Fibrosis. Signs of Respiratory Disease: Breathing Patterns; Clubbing and Hypertrophic Osteoarthropathy; General Examination; Lung Sounds. Symptoms of Respiratory Disease: Cough and Other Symptoms. Tumors, Malignant: Bronchogenic Carcinoma; Chemotherapeutic Agents; Hematological Malignancies; Lymphoma; Metastases from Lung Cancer; Bronchial Gland and Carcinoid Tumor; Rare Tumors; Carcinoma, Lymph Node Involvement. Upper Airway Obstruction.

Further Reading Albert R, Spiro S, and Jett J (2004) Comprehensive Respiratory Medicine. St Louis: Mosby. Brewis RAL (1998) Lecture Notes on Respiratory Disease. Oxford: Blackwell. Gibson JG, Geddes DM, Costabel U, Sterk PJ, and Corrin B (2003) Respiratory Medicine. Philadelphia: Saunders. Gudmundsson G and Asmundsson T (2000) Lung Sounds: Chest Auscultation. Jain DG (2000) Understanding lung sounds. Journal Indian Academy of Clinical Medicine 1(2): 177–189. Lehrer S (2002) Understanding Lung Sounds, 3rd edn. Philadelphia: Saunders. Munro J and Edwards C (2000) Macleod’s Clinical Examination. Edinburgh: Churchill Livingstone. Talley NJ and O’Connor S (2001) Clinical Examination. Oxford: Blackwell.

Relevant Websites http://www.vh.org – Virtual Hospital is a digital health sciences library created in 1992 at the University of Iowa. http://www.indegene.com – Indegene is a leading global provider of knowledge-intensive services that span a full spectrum of integrated scientific promotions, medical education and business analytics solutions for pharmaceutical, medical devices, biotechnology and healthcare organizations.

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SLEEP APNEA / Overview

Silicosis

see Occupational Diseases: Silicosis.

SLEEP APNEA Contents

Overview Adult Children Continuous Positive Airway Pressure Therapy Drug Treatments Genetics of Sleep Apnea Oral Appliances Surgery for Sleep Apnea

Overview S D West and J R Stradling, Churchill Hospital, Oxford, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The number of patients diagnosed with obstructive sleep apnea/ hypopnea syndrome (OSAHS) is increasing, and therefore the requirement to provide sleep services for such patients is also increasing. In a cash-limited healthcare system it is important for sleep physicians to have a pragmatic and evidence-based approach to OSAHS to be able to diagnose symptomatic OSAHS accurately in conjunction with a sleep study; to identify those patients most likely to benefit from continuous positive airways pressure (CPAP) treatment; to identify those who would benefit from alternative treatment, such as a mandibular advancement device; and to determine the simplest and most effective way to initiate and continue CPAP therapy for the patient.

cycles are associated with profound autonomic and cardiovascular changes, with systolic blood pressure falls of often 450 mmHg due to the frustrated inspiratory efforts, and rises of 480 mmHg with each arousal. These may directly damage the arterial system, but the evidence is unclear. Sleep fragmentation caused by the recurrent arousals is thought to be the main cause of the daytime sleepiness associated with OSAHS. Population studies have estimated the prevalence of OSAHS to be 2–4% of adult men and 1–2% of women. It is still underdiagnosed, although awareness amongst health professionals and the general public is increasing. As the number of people, particularly in the Western world, who are overweight continues to rise, OSAHS will increase in prevalence with increasing demands on sleep services.

Investigation of OSAHS Introduction Obstructive sleep apnea/hypopnea syndrome (OSAHS) is a common disorder, in which upper airway resistance increases during sleep. This increased resistance is due to upper airway dilator muscle relaxation and airway narrowing, causing inspiratory flow limitation, snoring, hypoventilation, and ultimately complete obstruction, that is, apnea. In response to this obstruction, respiratory effort increases and pharyngeal dilator muscles are activated in order to defend the airway. If this fails to solve the problem, a return to the awake/semi-awake state occurs, and this finally terminates the apnea. This cycle is repeated, often hundreds of times each night. These

Sleep studies are performed primarily to diagnose and quantify the severity of sleep-disordered breathing. Along with a clinical assessment, they can also be used to evaluate whether a patient’s daytime symptoms of sleepiness are likely to be attributable to arousals and sleep fragmentation that result from an obstructed upper airway. Sleep studies have evolved over time as the understanding of the pathology of OSAHS has progressed, with different techniques of direct and indirect measurement being developed. The way in which OSAHS is investigated and diagnosed varies among centers. The minimum standards required to diagnose OSAHS are contentious. Traditionally

SLEEP APNEA / Overview 43

polysomnography with EEG/EMG/EOG channels has been used to diagnose sleep-disordered breathing, and to initiate its treatment. There is no evidence that this complicated and expensive system of diagnosis and treatment is any more valid than simplified systems. Sleep centers increasingly have to review their investigative techniques to determine the most appropriate way of providing a pragmatic and evidence-based sleep service, with accurate, efficient, and cost-effective diagnosis and treatment. It needs to be appreciated that there is a difference between what can be studied and what should be studied in practical terms. Crucially, it is important for countries to work together towards the evidence-based minimums that are required for the accurate diagnosis of OSAHS. The current main types of sleep study are discussed below. Polysomnography

Polysomnography was first used for the diagnosis of sleep disorders by neurologists and neurophysiologists, who were the first physicians to be involved in the investigation and diagnosis of the sleepy patient. Techniques used for the investigation of patients with neurological disorders were adapted for sleepy patients. Electrophysiological signals derived from the electroencephalogram (EEG), electro-oculogram (EOG), and electromyelogram (EMG) were used to determine sleep onset, sleep stage and duration, as well as sleep fragmentation, arousals and, more recently, microarousals. Simple signal transducers, such as oronasal thermistors and ribcage/abdominal mercury strain gauges, were added to document irregular breathing. The obstructive apneas were counted and the apnea index per hour of sleep invented and defined. Oximetry was added later. Polysomnography became established as the standard method of investigation of sleep patients, against which other techniques were judged. It has not, however, been properly validated as a tool to measure the actual pathological process resulting from reduced upper airway patency with sleep onset, and many of the variables measured may not be the best, or only, way to determine sleep, wakefulness, or arousals. There is no evidence that the diagnosis and management of OSAHS requires full polysomnography and it is not indicated for the vast majority of patients. The complexity and cost of polysomnography has limited its usefulness as the demands on sleep services increase; it is time-consuming and labor-intensive to set up and analyze, and is considerably more expensive than simpler techniques of monitoring. The equipment and the expertise required are not widely available.

Limited Sleep Studies

This term encompasses those sleep studies that do not record EEG/EOG/EMG. These simplified tests have evolved and been shown to be at least as good as polysomnography for the diagnosis of continuous positive airways pressure (CPAP)-responsive OSAHS. They can produce apnea–hypopnea indices, or respiratory disturbance indices, similar to those obtained by full polysomnography, but may also provide more sophisticated derivatives. Commonly, some or all of the following are monitored: oximetry, snoring, body movement, heart rate, oronasal airflow, chest and abdominal movements, and leg movements. Arousals are inferred from respiratory signals and autonomic indices such as rises in heart rate, blood pressure, or pulse transit time. Estimates of sleep onset, sleep duration, and awake time can be determined from body movements, measured with actigraphy, video, or other devices. Many, even more limited, sleep study systems that vary in the number of recorded channels have been devised for home use. These can be applied by the patient at home, following suitable instruction from the sleep technician or physician. Most commonly they do not include measures of EEG/EMG/EOG. They are relatively cheap and do not require patients to spend a night in a hospital bed. However, they can be difficult to set up correctly, particularly for people living on their own or who cannot follow instructions easily, and this may lead to sufficient data loss to preclude a confident diagnosis. Overnight Oximetry Alone

Portable recording oximeters are now often used to perform sleep studies, usually in the patient’s own home. Tracings of all-night oximetry and pulse rate are obtained (see Figure 1). Algorithms count the number of hypoxic dips and, by inference, respiratory events. In those patients with symptoms suggestive of OSAHS and a baseline SaO2 of greater than 92%, more than 15 44% SaO2 dips per hour allows a confident diagnosis of OSAHS. Recurrent pulse rate rises are usually indicative of recurrent arousal and help in the analysis. A visual review of the overnight tracings is always recommended, particularly for negative studies, to ensure the characteristic subtle SaO2 dipping of OSAHS is not missed (for example, when many of the SaO2 dips do not quite reach the 4% threshold for counting). Oximetry alone is clearly a limited study, but can be successfully used, with appropriate expertise, to identify severe cases of OSAHS (see Figure 2) thus allowing rapid referral for continuous positive airways pressure (CPAP) treatment.

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Minutes Figure 1 Normal overnight oximetry: normal baseline SaO2 with few dips only. Reproduced with permission from Chapman S, Robinson G, Stradling J, and West S (2005) Sleep apnoea. In: Oxford Handbook of Respiratory Medicine. Oxford University Press.

False-positive oximetry can occur with Cheyne– Stokes breathing, which also causes cyclical falls in SaO2 (e.g., heart failure, post stroke), and if there is a low awake baseline SaO2 (e.g., chronic obstructive pulmonary disease) where the nocturnal SaO2 oscillates considerably with only small actual PaO2 changes, due to the shape of the oxygen–hemoglobin dissociation curve. False-negative oximetry can occur in younger and thinner patients, who can have frequent apneas and arousals without detectable desaturations. Those with negative oximetry studies, but symptoms suggestive of OSAHS, should undergo further sleep study assessment. Oximetry, sound, video, chest and abdominal movements, body position, and leg movements are probably the most helpful conventional components of a sleep study to help diagnose OSAHS. Other Tests

Objective tests for sleepiness, such as the maintenance of wakefulness test (MWT) or multiple sleep latency test (MSLT) can be performed, but these are more typically used in research studies. The correlations between both subjective and objective measures of sleepiness and indices of respiratory events or

sleep disturbance on the sleep study tend to be poor. Furthermore, the correlation with actual daytime performance, such as driving accident rate, is even poorer.

Conditions Associated with OSAHS OSAHS with excessive daytime sleepiness has been recognized increasingly to be associated with certain clinical conditions, either from the direct effects of apneas/hypopneas and hypoxia or from the sleep fragmentation. OSAHS is now recognized to be a cause of systemic hypertension, independent of obesity. The hypertension is likely to be due to increased day-time and night-time sympathetic nervous system activity caused by either the hypoxia and sleep fragmentation or both. The hypertension improves following effective treatment of the OSAHS. By extension, OSAHS may therefore be indirectly or directly related to vascular disease, including coronary heart disease, arrhythmias, heart failure, transient ischaemic attack (TIAs), and stroke; although controlled interventional studies to determine this have not yet been performed. The association of OSAHS with insulin resistance, also independent of obesity, has been demonstrated in some studies, although the

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Figure 2 Severe OSA on overnight oximetry: frequent regular desaturations throughout the night. Reproduced with permission from Chapman S, Robinson G, Stradling J, and West S (2005) Sleep apnoea. In: Oxford Handbook of Respiratory Medicine. Oxford University Press.

mechanisms and the clinical relevance of this are still unclear. There is some evidence of cognitive dysfunction in people with untreated OSA (and possible residual decrements even after treatment), which may be due to nocturnal hypoxia or other effects of severe sleep fragmentation apart from sleepiness. This may be manifest as poor concentration, vigilance, or attention problems. It is well known that there is an increased risk of motor vehicle accidents in people with untreated OSA, which is up to seven times that in the general population. Both cognitive impairment and simulated steering performance (as a surrogate for driving ability) improve following therapeutic CPAP.

Treatment of OSAHS In view of the increasing numbers of patients with OSAHS, the following questions should be asked regarding the use of CPAP: who will benefit the most from CPAP and are there better options for some patients? CPAP treatment involves the delivery of air to the upper airway noninvasively, via a nose or full-face mask, at a pressure that maintains airway patency during sleep by means of ‘pneumatic splinting’. It is a

well-established and effective therapy for OSAHS and, by eradicating the recurrent airway obstruction during sleep with its associated repeated arousals and sleep disturbance, abolishes daytime sleepiness. CPAP remains the mainstay of treatment of people with significant symptomatic OSAHS. The best correlates of whether a patient is likely to have improved daytime sleepiness following a therapeutic CPAP trial are the number of 44% SaO2 dips per hour and the number of body movements (indicating arousals). There is considerable night-to-night variation in sleep study indices, which makes the use of thresholds to initiate treatment illogical. The sleep study needs to be interpreted to assess if there is sufficient evidence to explain the patient’s symptoms. If the severity of the OSAHS (and how much it may be affecting the patient) is unclear, then it is reasonable to undertake a diagnostic (as well as therapeutic) trial of CPAP, to determine whether the patient has sufficient clinical improvement to warrant continued treatment; giving rise to the concept of CPAPresponsive disease. The initiation of CPAP therapy is an area of current development. Nasal CPAP has traditionally been initiated in hospital with overnight pressure titration. The optimal pressure at which the patient’s apneas,

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hypopneas, snoring, and arousals are largely abolished is established, as determined by polysomnography. The patient is then sent home on a fixed CPAP pressure. Recently, more expensive CPAP machines, capable of automatically titrating airway pressure, have been devised that continually adjust airway pressure in order to maintain airway patency. Self-titrating CPAP is well tolerated and improves OSA and sleep architecture in a manner comparable to manually adjusted CPAP. A technician is not required to be present overnight during the overnight titration CPAP. Currently, these machines are too expensive compared to fixed pressure machines for most health services to use them routinely in the long term. An alternative method of determining the required CPAP pressure is to use an algorithm that predicts the required pressure depending on neck circumference (or body mass index) and oxygen desaturation (or apnea–hypopnea index). This approach has been found to give comparable pressures and patient outcomes to self-titrating CPAP machines, and saves significant costs. Weight loss should also be advised in those patients with upper body obesity and a body mass index of greater than 26. Mandibular advancement devices can be effective in treating OSAHS, mainly in those who have milder disease, retrognathia, postural OSAHS, or in those who are intolerant of CPAP; but individual benefit is largely unpredictable. Drugs to increase respiratory drive during sleep, to suppress the rapid eye movement sleep often associated with more severe OSA, or to activate preferentially the upper airway dilator muscles have all been tried but have not been found to be sufficiently effective in the treatment of OSAHS. At present, excessive daytime sleepiness with OSAHS is the main indication for treatment, and improvement of the sleepiness is the outcome used to measure the success of treatment. As the evidence for other conditions being associated with OSAHS increases (e.g., vascular disease, diabetes), it is debated whether patients with OSA, but without excessive daytime sleepiness, should receive CPAP treatment to modify their associated conditions. At present, there is not enough evidence to support or justify this. The small benefits in terms of blood pressure are more easily obtained with conventional antihypertensive therapy, which is supported by longer term data on vascular event rates. See also: Obesity. Sleep Apnea: Adult; Children; Continuous Positive Airway Pressure Therapy; Drug Treatments; Genetics of Sleep Apnea; Oral Appliances; Surgery for Sleep Apnea. Sleep Disorders: Overview; Hypoventilation.

Further Reading Bennett LS, Langford BA, Stradling JR, and Davies RJ (1998) Sleep fragmentation indices as predictors of daytime sleepiness and nCPAP responsiveness in obstructive sleep apnoea. American Journal of Respiratory and Critical Care Medicine 158: 778–786. Chapman S, Robinson G, Stradling J, and West S (2005) Sleep apnoea. In: Oxford Handbook of Respiratory Medicine. Oxford University Press. Choi S, Bennett LS, Mullins R, Davies RJ, and Stradling JR (2000) Which derivative from overnight oximetry best predicts symptomatic response to nCPAP in patients with obstructive sleep apnoea? Respiratory Medicine 94: 895–899. Davies CWH, Crosby JH, Mullins RL, et al. (2000) Case-control study of 24 hour ambulatory blood pressure in patients with obstructive sleep apnoea and normal matched control subjects. Thorax 55: 736–740. Ip MS, Lam B, Ng MM, et al. (2002) Obstructive sleep apnea is independently associated with insulin resistance. American Journal of Respiratory and Critical Care Medicine 165: 670–676. Jenkinson C, Davies RJ, Mullins R, and Stradling JR (1999) Comparison of therapeutic and subtherapeutic nasal continuous positive airways pressure for obstructive sleep apnoea: a randomised prospective parallel trial. Lancet 353: 2100–2105. Peppard PE, Young T, Palta M, and Skatrud J (2000) Prospective study of the association between sleep-disordered breathing and hypertension. New England Journal of Medicine 342: 1378–1384. Pepperell JCT, Ramdassingh-Dow S, Crosthwaite N, et al. (2002) Ambulatory blood pressure following therapeutic and sub-therapeutic nasal continuous positive airway pressure for obstructive sleep apnoea: a randomised prospective parallel trial. Lancet 359: 204–210. Punjabi NM, Sorkin JD, Katzel LI, et al. (2002) Sleep-disordered breathing and insulin resistance in middle-aged and overweight men. American Journal of Respiratory and Critical Care Medicine 165: 677–682. Schmidt-Nowara W, Lowe AA, Wiegand L, et al. (1995) Oral appliances for the treatment of snoring and obstructive sleep apnea: a review. Sleep 18: 541–547. Smith IE and Quinnell TG (2004) Pharmacotherapies for obstructive sleep apnea; where are we now? Drugs 64(13): 1385–1399. Stradling JR, Hardinge M, Paxton J, and Smith D (2004) Relative accuracy of algorithm-based prescription of nasal CPAP in OSA. Respiratory Medicine 98: 152–154. Stradling JR, Pepperell JCT, and Davies RJO (2001) Sleep apnoea and hypertension: proof at last? Thorax 56: 45–49. Ward Flemons W, Littner MR, Rowley JA, et al. (2003) Home diagnosis of sleep apnea: a systematic review of the literature. Chest 124: 1543–1579.

Adult W T McNicholas, St Vincent’s University Hospital, Dublin, Republic of Ireland & 2006 Elsevier Ltd. All rights reserved.

Abstract Obstructive sleep apnea syndrome (OSAS) affects around 2–4% of the adult population and is characterized by recurring episodes of upper airway (UA) obstruction during sleep. Obstructive apnea results from the inability of UA dilating muscles to

SLEEP APNEA / Adult 47 withstand the high collapsing forces generated in the pharynx during inspiration, usually as a result of UA narrowing. Apneas are typically terminated by brief arousals, which result in marked sleep fragmentation and account for the major daytime symptoms of excessive sleepiness and cognitive dysfunction found in these patients. OSAS is also an independent risk factor for cardiovascular disease, particularly hypertension. The diagnosis of OSAS is based on the combination of compatible clinical features and diagnostic sleep studies. While polysomnography represents the gold standard diagnostic test, these studies require major resources; limited sleep studies that focus on recording cardiorespiratory variables are preferred in many cases. The management of moderate and severe OSAS largely centers on nasal continuous positive airway pressure (CPAP), but milder cases can often be managed by conservative measures, including weight loss, alcohol avoidance, relief of nasal congestion, and positional measures to avoid the supine position. A mandibular advancement device is an alternative to CPAP in mild to moderate cases.

Table 1 Factors contributing to the development of obstructive sleep apnea General factors

Anthropometric (male sex, age, obesity) Genetic factors (e.g., reduced UA caliber) Drugs (ethanol, hypnotics)

Mechanical factors

UA narrowing Supine posture Increased UA resistance Increased UA compliance

Upper airway muscle function

Ineffective UA dilator muscle activity Impaired relationship of UA muscle and diaphragm contraction

Neural factors

Reduced chemical drives Diminished response to negative pressure Feedback from the lungs

Arousal

Impaired arousal responses Postapneic hyperventilation with subsequent reduction in respiratory drive

Introduction The obstructive sleep apnea syndrome (OSAS) is a clinical syndrome marked by recurring episodes of upper airway (UA) obstruction that lead to markedly reduced or absent airflow, referred to as hypopnea and apnea, respectively. These episodes are typically accompanied by loud snoring and hypoxemia, and are usually terminated by transient arousals that result in marked sleep fragmentation and diminished amounts of the deeper stages of sleep, particularly slow wave and rapid eye movement (REM) sleep. Patients with OSAS are usually unaware of such arousals, but the resulting deterioration in sleep quality contributes significantly to the prominent symptom of excessive daytime sleepiness. However, despite having these significant breathing abnormalities during sleep, most patients have no readily detectable respiratory abnormality while awake. This article will focus on the pathophysiology and clinical manifestations of the disorder, but will not address treatment, since this aspect is covered in other articles. The prevalence of OSAS has been variously estimated at between 1% and over 6% of the adult population. One comprehensive epidemiological study of prevalence in a general adult population in North America found that 9% of women and 24% of men had an apnea index (AI) greater than 5 h 1, but this estimate of prevalence fell to 2% of women and 4% of men when an AI 45 was combined with symptomatic daytime sleepiness. These findings underline the importance of not viewing OSAS in terms of AI or apnea/hypopnea index (AHI) alone.

Etiology The central feature of OSAS is recurring obstruction of the UA during sleep and factors predisposing

to such obstruction are outlined in Table 1. These factors represent anatomical or pathophysiological abnormalities that result in increased resistance and/or collapsibility of the UA. Anatomical factors generally result in UA narrowing whereas pathophysiological factors generally impair the efficiency of UA dilating muscle contraction. For example, obesity, which is probably the most common predisposing factor, results in UA narrowing by increased fat deposition in the pharyngeal walls and external compression by superficially located fat masses. Genetic factors also play a role and OSAS is more common in family members. In particular, relatives of nonobese OSAS patients have a smaller UA and a different craniofacial morphologic structure than matched controls. Apparently healthy offspring of patients with OSAS demonstrate impaired respiratory responses to resistive loading during sleep and the hypoxic ventilatory response may be blunted in families with affected members. The supine position predisposes to OSA by gravitational forces that result in posterior displacement of the tongue. Ethanol ingestion is well known to increase the frequency and duration of apneas. This effect results from a combination of a reduction in UA dilating muscle activity and a depressant effect on the reticular activating system, which impairs arousal. Similar effects have been reported with diazepam.

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Physiology and Pathophysiology Oropharyngeal patency is dependent on the action of dilator muscles that contract in a rhythmic fashion coordinated with each inspiration. The pharynx is subjected to collapse when the negative airway pressure generated by inspiratory activity of the diaphragm and intercostal muscles exceeds the force produced by these muscles. A narrowed UA is very common among sleep apnea patients, which leads to increased pharyngeal resistance with the generation of more negative pharyngeal pressures during inspiration (Figure 1). This narrowing requires an increase in pharyngeal dilator muscle contraction to maintain airway patency. OSAS patients demonstrate more forceful contraction of these muscles while awake than normal subjects but show a larger decrement in contractile forces during sleep, thus contributing to the development of obstructive apnea. Nonetheless, OSAS patients develop at least as forceful pharyngeal dilator muscle contraction during sleep as normal subjects, thus reinforcing the fact that UA obstruction is a result of an imbalance between pharyngeal collapsing forces and dilator muscle contraction rather than a primary deficiency in muscle contraction. However, the sustained increase in dilator muscle UA narrowing: Genetic factors Obesity/neck size Nasal obstruction ↓ FRC

↑ Upper airway resistance and collapsibility

Ineffective UA dilator activity: Sleep state UA reflexes UA narrowing

Obstructive apnea

Reduced respiratory drive

contraction in OSAS may predispose to fatigue of these muscles, which could aggravate the tendency to pharyngeal occlusion. The importance of negative intrapharyngeal pressure as a stimulus to dilator muscle contraction is reinforced by studies of the impact of nasal continuous positive airway pressure (CPAP) on pharyngeal muscle function. CPAP results in a marked decrement in both tonic and phasic contraction of the genioglossus muscle, which allows resting of these muscles and may contribute to the therapeutic benefit of CPAP. Local UA neuromuscular reflexes play a significant role in maintaining pharyngeal patency and there is evidence of defects in these reflexes in patients with OSAS.

Clinical Features Definition of Sleep Apnea Syndrome

The definition of OSAS has evolved over the past 3 decades from an exclusive focus on apnea frequency to a realization that hypopneas and symptom level can be equally important in assessing disease severity. An important development in the definition of OSAS was the report of a Working Group of the American Academy of Sleep Medicine published in 1999, which laid out the clinical criteria necessary for the diagnosis of a clinically significant sleep apnea syndrome and also proposed a grading of severity. In this report, a diagnosis of OSAS requires that the patient report excessive daytime sleepiness together with at least two other compatible symptoms that are not better explained by other factors in addition to the objective demonstration of at least five obstructed breathing events per hour of sleep during sleep studies. Clinical Assessment

When interviewing a patient with suspected OSAS it is highly desirable to interview their partner as well who can usually provide important additional information based on direct observation of the patient while asleep (Table 2). Nocturnal Symptoms

Arousal

Hyperventilation

Figure 1 Schematic diagram of the pathophysiology of obstructive apnea demonstrating the balance of factors contributing to pharyngeal obstruction. Narrowing of the upper airway increases pharyngeal resistance and overcomes the ability of dilating muscles to withstand the collapsing forces that develop during inspiration. Apnea is terminated by arousal but the associated postapneic hyperventilation may contribute to the development of further pharyngeal obstruction by a reduction in respiratory drive. FRC, functional residual capacity.

Snoring Almost all patients with OSAS snore, usually very loudly. Population surveys indicate that 25% of men and 15% of women habitually snore and the prevalence of habitual snoring increases progressively with age. These data indicate that, while most patients with OSAS snore, only a small proportion of those who habitually snore have OSAS. Witnessed apneas These have been shown to be a good diagnostic predictor of OSAS but do not help in predicting severity of the disorder.

SLEEP APNEA / Adult 49 Table 2 Clinical features of sleep apnea syndrome Night

Day

Snoring Witnessed apneas

Excessive sleepiness Impaired work/school performance Morning headache Impaired concentration Impaired memory Intellectual deterioration Depressive symptoms Heartburn

Restless sleep Recurrent arousals Nocturnal choking Insomnia Nocturia Enuresis (particularly children) Reduced libido/impotence Sweating Cardiac arrhythmias

Nocturnal choking Many patients with OSAS report waking at night with a choking sensation, which can be alarming and likely reflects outright wakening during an episode of obstructive apnea. The choking almost invariably passes within a few seconds of wakening. Insomnia While OSAS patients rarely have difficulty falling asleep, many report recurring wakenings during the night, which likely reflects the disturbing effect on sleep of recurring arousal. Other nocturnal symptoms Several other nocturnal symptoms may be reported by patients or their bed partner such as nocturia, enuresis, frequent arousals, diaphoresis, and impotence. Daytime Symptoms

Excessive daytime sleepiness Although sleep apnea is the most common cause of excessive daytime sleepiness (EDS), the severity of EDS and sleep apnea correlate poorly, which may reflect the fact that other disorders can cause EDS. It is important to distinguish fatigue from sleepiness in this context since many patients may confuse the two symptoms. Other daytime symptoms OSAS is reported to be associated with many symptoms other than EDS such as fatigue, memory impairment, poor concentration, intellectual deterioration, personality changes, morning nausea, morning headaches, heartburn, automatic behavior, and depression. Physical Characteristics/Examination

Obesity Obesity is very common in OSAS, particularly upper body obesity, and there is evidence that patients with OSAS often have fat necks. Craniofacial anatomy Anatomical factors that predispose to UA narrowing should be sought in

patients with suspected OSAS, such as retrognathia, micrognathia, tonsillar hypertrophy, macroglossia, and inferior displacement of the hyoid. However, the most common physical finding in patients with OSAS is a non-specific narrowing of the oropharyngeal airway, with or without an increase in soft tissue deposition. Hypertension The finding of hypertension in a patient with symptoms suggestive of OSAS increases the likelihood of the disorder. Combined Features

In clinical practice, the suspicion of OSAS is usually based on a combination of supportive features but it is generally not possible to predict the severity of the disorder based on clinical features, except at the extremes of the clinical spectrum. Thus, some form of objective monitoring during sleep is necessary to confirm and grade the severity of the disorder.

Diagnosis Overnight polysomnography represents the standard for the diagnosis of OSAS since this investigation provides detailed information on sleep state and respiratory and gas exchange abnormalities, in addition to a range of other variables including body position, heart rate and rhythm, and muscle tone and contraction. An example of an obstructive apnea is given in Figure 2. However, these studies are complex and require major resources since they are usually performed in a sleep laboratory under the direct supervision of a trained technician. Thus, it is common to encounter long waiting lists for diagnostic sleep studies in sleep centers throughout the world. The high prevalence figures for OSAS make it necessary to consider other simplified approaches to the diagnosis. The development of many limited diagnostic systems in recent years represents recognition of these logistic difficulties. Most of these limited systems focus on recording cardiorespiratory variables, without recording sleep state. These systems are particularly useful in the investigation of patients at the extremes of the clinical spectrum, namely to confirm the diagnosis in patients with relatively severe OSAS and to exclude a major degree of OSAS in loud snorers who do not have a clinical presentation suggestive of significant OSAS. Unfortunately, there is no uniformity among these limited systems and the only consistent variable common to these systems is oxygen saturation (SaO2 ). Detailed SaO2 analysis is often sufficient to diagnose OSAS in severe cases because of the characteristic pattern of repetitive desaturations.

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EEG EOG

ECG 10 s

Time

Ribcage Abdomen

Sum Figure 2 Polysomnographic record of an obstructive apnea during REM sleep. Initially there is normal nonobstructed breathing with ribcage and abdominal movements in phase. With the onset of apnea in REM sleep (indicated by*) there is paradoxical indrawing of the ribcage, with absent tidal ventilation as indicated by the flat sum tracing. As the apnea proceeds there is increased abdominal effort culminating in an arousal. This coincides with relief of UA obstruction followed by postapneic hyperventilation. EEG, electroencephalogram; EOG, electrooculogram.

However, more complete sleep studies are often necessary in patients with mild to moderate disease. The very large numbers of patients presenting for assessment of possible OSAS have also focused attention on the role of home-based sleep studies. While there are obvious advantages of such studies, particularly improved sleep quality and cost savings, there are also disadvantages. The lack of technician supervision means that leads that become dislodged are not replaced during the study, and consequently the likelihood of technically unsatisfactory studies is higher.

Morbidity and Mortality Related to Sleep Apnea Syndrome The morbidity of OSAS relates principally to the cardiovascular system. However, this population of patients also has a high incidence of other coexisting cardiovascular risk factors such as obesity, hyperlipidemia, increased age, male sex, smoking history, and excessive alcohol intake, which makes the identification of a clear independent association of OSAS with cardiovascular disease more difficult. Nonetheless, there is now convincing evidence from many studies of an independent association of OSAS with hypertension and this association has been reinforced by recent studies demonstrating a reduction in blood pressure levels with nasal CPAP therapy. A growing body of evidence also points to an independent link between OSAS and ischemic heart disease and stroke.

The mechanisms by which OSAS predisposes to cardiovascular disease are not fully understood but probably include elevated sympathetic drive secondary to recurrent hypoxias and arousals from sleep, in addition to lipid dysfunction, platelet and endothelial cell dysfunction, and insulin resistance. There is emerging evidence that the intermittent hypoxia (IH) that is characteristic of OSA particularly predisposes to the selective activation of proinflammatory transcription factors such as nuclear factor kappa B (NF-kB) with consequent increased production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-a), interleukin 6 (IL-6), and interleukin 8 (IL-8). These cytokines have been linked to the pathogenesis of atherosclerosis and hypertension. Recent randomized placebo controlled trials have demonstrated clear objective benefits of CPAP therapy on blood pressure levels. Apart from hypertension, there are limited data to support a significant impact of treatment on cardiovascular morbidity although there is emerging evidence of a reduction in death rate from cardiovascular disease among OSAS patients successfully treated with CPAP.

Association of Sleep Apnea with Road Traffic Accidents The relationship of OSAS to road traffic accidents has been recognized for many years. Various studies have demonstrated an increase in accident rate between 3 and 7 times that of the general population

SLEEP APNEA / Children 51 Table 3 Management options for OSAS General measures Weight optimization, alcohol avoidance, sleep hygiene, and positional measures while asleep can help considerably and should be recommended to all OSAS patients, regardless of severity Severe cases Nasal CPAP is the treatment of choice unless there is a surgically correctable anatomical lesion Moderately severe cases Nasal CPAP is the best initial option, but alternative therapies can be considered, such as an oral appliance or mandibular advancement surgery in selected patients, or where CPAP is poorly tolerated Mild cases General measures may help mild cases considerably and may be all that is required Oral appliances are particularly suited to patients with mild OSAS Surgery can be considered in carefully selected patients such as uvulopalatopharyngoplasty (UPPP), mandibular advancement, or nasal surgery where appropriate

among untreated OSAS patients, which falls to normal levels after successful therapy with CPAP. There is also evidence that occupations such as long-haul truck driving are particularly associated with an increased risk of accident, particularly where there is evidence of associated OSAS. The risk of accident calls into question the suitability of untreated OSAS patients to hold a driving license and several countries have introduced regulations in this regard. However, patients with OSAS who are successfully treated do not pose a significantly increased accident risk.

Management of OSAS The management of OSAS is dealt with in other articles and will not be discussed in detail here. Nasal CPAP represents the most widely employed intervention, particularly in moderate to severe cases. Surgery has a limited role to play in the management of OSAS unless there is a specific correctable anatomic lesion. A mandibular advancement device is best suited to mild to moderate cases. An overview of potential management strategies is given in Table 3.

sleep apnea in adults. American Journal of Respiratory and Critical Care Medicine 169(10): 1160–1163. Collop NA, Adkins D, and Phillips BA (2004) Gender differences in sleep and sleep-disordered breathing. Clinics in Chest Medicine 25(2): 257–268. Deegan PC and McNicholas WT (1998) Pathophysiology of obstructive sleep apnoea. In: McNicholas WT (ed.) Respiratory Disorders during Sleep: European Respiratory Monographs, vol. 3, pp. 28–62. Copenhagen: Munsgaard Press. Engleman HM and Douglas NJ (2004) Sleepiness, cognitive function, and quality of life in obstructive sleep apnoea/hypopnoea syndrome. Thorax 59(7): 618–622. Flemons WW (2002) Clinical practice. Obstructive sleep apnea. New England Journal of Medicine 347(7): 498–504. Fogel RB, Malhotra A, and White DP (2004) Pathophysiology of obstructive sleep apnoea/hypopnoea syndrome. Thorax 59(2): 159–163. Krieger J, McNicholas WT, Levy P, et al. (2002) Public health and medicolegal implications of sleep apnoea: report of an ERS task force. European Respiratory Journal 20(6): 1594–1609. Lattimore JD, Celermajer DS, and Wilcox I (2003) Obstructive sleep apnea and cardiovascular disease. Journal of the American College of Cardiology 41(9): 1429–1437. Lavie L (2003) Obstructive sleep apnoea syndrome – an oxidative stress disorder. Sleep Medicine Reviews 7(1): 35–51. McNicholas WT and Phillipson EA (eds.) (2002) Breathing Disorders in Sleep. London: Saunders. Redline S and Tishler PV (2000) The genetics of sleep apnea. Sleep Medicine Reviews 4(6): 583–602. Remmers JE, DeGrott WJ, Sauerland EK, and Anch AM (1978) Pathogenesis of upper airway occlusion during sleep. Journal of Applied Physiology 44(6): 931–938. Report of a Task Force of the American Academy of Sleep Medicine (1999) Sleep-related breathing disorders in adults: recommendations for syndrome definition and measurement techniques in clinical research. Sleep 22: 667–689. Series F (2002) Upper airway muscles awake and asleep. Sleep Medicine Reviews 6(3): 229–242. Shamsuzzaman AS, Gersh BJ, and Somers VK (2003) Obstructive sleep apnea: implications for cardiac and vascular disease. Journal of the American Medical Association 290: 1906–1914. Veasey SC (2003) Molecular and physiologic basis of obstructive sleep apnea. Clinics in Chest Medicine 24(2): 179–193. Young T, Palta M, Dempsey J, Skatrud J, and Weber SSB (1993) The occurrence of sleep-disordered breathing among middleaged adults. New England Journal of Medicine 328: 1230–1235.

Children L A D’Andrea, Children’s Hospital of Wisconsin, Milwaukee, WI, USA & 2006 Elsevier Ltd. All rights reserved.

See also: Sleep Apnea: Children. Sleep Disorders: Upper Airway Resistance Syndrome. Ventilation: Control.

Further Reading ATS/ACCP/AASM Taskforce Steering Committee (2004) Executive summary on the systematic review and practice parameters for portable monitoring in the investigation of suspected

Abstract Obstructive sleep apnea (OSA) is a medical problem that is being increasingly recognized in children. It occurs in 1–3% of children of all ages. The peak presentation is 2–8 years of age when the tonsils and adenoids are largest in relation to upper airway size. OSA is characterized by loud snoring, paradoxical respiratory efforts, and prolonged partial upper airway

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obstruction or intermittent complete pharyngeal obstruction that disrupts normal ventilation and sleep continuity. If unrecognized or untreated, OSA can lead to neurobehavioral, growth, and cardiovascular sequelae in childhood. Adenotonsillectomy is considered standard therapy, though some children benefit from noninvasive positive pressure ventilation (e.g., CPAP), weight reduction, or management of craniofacial abnormalities.

Introduction Obstructive sleep apnea (OSA) is a common sleep disorder that is being increasingly recognized in children. Adenotonsillar hypertrophy is the major risk factor in children. OSA is characterized by loud snoring and paradoxical respiratory efforts. OSA occurs when there is prolonged partial upper airway obstruction or intermittent complete pharyngeal obstruction. Events with complete obstruction of the upper airway are identified as obstructive apneas whereas events with partial obstruction are identified as hypopneas. The obstructive events are associated with arousals that disrupt normal ventilation and sleep continuity. If unrecognized or untreated, OSA can lead to neurobehavioral, growth, and cardiovascular sequelae. Treatment ranges from adenotonsillectomy to noninvasive positive pressure ventilation (e.g., continuous positive airway pressure, CPAP). Weight reduction or management of craniofacial abnormalities may also be of use.

Epidemiology Obstructive sleep apnea occurs in 1–3% of all children, in comparison to 9% of women and 24% of men. Habitual (nightly) snoring, always present in OSA, is estimated to occur in 6–9% of school-age children. There is no clear association between gender and OSA in children. While the incidence of OSA appears to be similar in prepubertal boys and girls, it has been suggested that the incidence and severity of sleep apnea is increased in young boys. After puberty, the incidence in boys increases, similar to the pattern seen in adults. African–American children and children with a family history of OSA may be at increased risk to develop sleep apnea.

Etiology Adenotonsillar hypertrophy is the most common risk factor for developing OSA in children. The peak presentation is 2–8 years of age when the tonsils and adenoids are largest in relation to upper airway size. Children with craniofacial abnormalities are at increased risk of developing OSA and may develop OSA at a younger age (Table 1). Craniofacial

Table 1 Medical conditions associated with OSA in children Craniofacial syndromes Midface hypoplasia Down’s syndrome Apert’s syndrome Crouzon syndrome Pfeiffer syndrome Treacher Collins syndrome Achondroplasia Freeman–Sheldon syndrome Mandibular hypoplasia Pierre Robin syndrome Velocardiofacial syndrome (VCF) Treacher Collins syndrome Nager’s syndrome Hallermann–Streiff syndrome Cerebrocostomandibular syndrome Macroglossia/glossoptosis Down’s syndrome Beckwith–Wiedemann syndrome Other Choanal atresia or stenosis Klippel–Feil syndrome Goldenhar syndrome Marfan syndrome Mucopolysaccharidosis (e.g., Hunter’s, Hurler’s) Wolf–Hirschhorn syndrome Neurological disorders Cerebral palsy Myasthenia gravis Mobius syndrome Arnold–Chiari malformation Muscular dystrophy Spinal muscular atrophy Myotonic dystrophies Brainstem tumor or injury Obesity Primary obesity Prader–Willi syndrome Miscellaneous disorders Hypothyroidism Sickle cell disease Laryngomalacia Airway papillomatosis Cystic hygroma Subglottic stenosis Gastroesophageal reflux Chronic rhinitis Nasal septal deviation Postoperative disorders Cleft lip or palate repair Velopharyngeal flap Chemical-induced disorders Sedative medications Antihistamines or decongestants Alcohol consumption

abnormalities include midface hypoplasia, micro-/retrognathia or mandibular hypoplasia, and glossoptosis. These malformations alter the anatomic airway resulting in problems with airflow. A third risk factor

SLEEP APNEA / Children 53

for developing OSA is neuromuscular weakness, which affects pharyngeal patency. This includes children with cerebral palsy with incoordination of upper airway muscles and children with muscular dystrophy where a selective weakness of upper airway muscles may cause OSA long before the development of respiratory failure from weakness of the ventilatory muscles (i.e., the diaphragm). Most children with OSA are of normal weight. However, some children present with failure to gain weight; others are overweight or morbidly obese. OSA in obese children may be caused when an increase in adipose tissue in the pharynx (pharyngeal fat pads), neck, and chest wall creates increased upper airway resistance and increased work of breathing. Altered central ventilatory drive may exacerbate problems in these children. In adults, the incidence of OSA correlates with degree of obesity, as well as gender and age. The more common conditions associated with OSA in children are listed in Table 1.

Pathology/Pathogenesis The severity of OSA is not proportional to adenotonsillar size, but results from a combination of adenotonsillar size, pharyngeal size, upper airway motor tone, and central ventilatory drive. The presence or absence of each of these factors results in loud snoring, paradoxical respiratory efforts, and prolonged partial upper airway obstruction or intermittent complete pharyngeal obstruction. The obstructive events are associated with arousals that disrupt normal ventilation and sleep continuity leading to neurobehavioral, growth, and cardiovascular sequelae.

Clinical Features Nocturnal Symptoms

The most common presenting symptom for OSA in children is loud snoring during sleep. The snoring is associated with labored breathing, retractions, paradoxical breathing, or episodes of increased respiratory efforts. Episodes are often followed by gasping, choking, or arousal. Snoring and labored breathing are reported in more than 96% of children with OSA. OSA is unlikely in the absence of habitual snoring, but many children who snore do not have sleep apnea. The intensity of snoring does not correlate with the severity of the upper airway obstruction. Most children with OSA have a respiratory pattern of obstructive hypoventilation with loud snoring and

paradoxical respiratory efforts, with gas-exchange abnormalities (i.e., hypoxemia and hypercarbia). Other nocturnal symptoms include restless sleep with frequent position changes. Children are often found sleeping with their mouth open and their necks hyperextended. They may prefer to sleep in the upright position or propped up with several pillows. In addition, children with OSA are often quite sweaty, which is probably related to increased work of breathing during sleep. Nocturnal enuresis is a variable finding. Children with OSA rarely become cyanotic. Daytime Symptoms

The child’s breathing is usually normal while awake, although children with severe OSA may have some difficulty breathing during the day. Daytime symptoms are usually secondary to adenotonsillar hypertrophy or chronic rhinitis. Non-specific daytime symptoms include mouth breathing, hyponasal speech, frequent upper respiratory tract infections, and difficulty swallowing. Children may complain of a morning headache, believed to be secondary to carbon dioxide retention. Excessive daytime somnolence (EDS) is uncommon in children with OSA, in comparison to adults with OSA. Symptoms of EDS in children include difficulty in waking in the morning, falling asleep in school, or increased napping in younger children. More commonly, children with OSA present with subtle neurobehavioral signs such as mood changes (e.g., irritability, impatience, low frustration level) or aggressive behavior. Children with OSA may struggle with symptoms of poor concentration, inattentiveness, and behavior problems similar to children diagnosed with attention deficit hyperactivity disorder (ADHD). One study has found that over 20% of frequent ‘snorers’ had high ADHD scores. Another study reported that 18% of 297 poorly performing children in first grade had symptoms of OSA. Mean grades improved in half of the children who underwent adenotonsillectomy but there were no changes in grades in those who did not undergo surgery. Physical Examination

On physical examination the child with OSA may appear to be entirely normal, delaying the diagnosis. Vital signs are typically normal although hypertension has been reported. Most adults with OSA are overweight. Children with OSA may be overweight; however, most are of normal weight and height. A subset of children with OSA may present with failure to gain weight.

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The HEENT exam is of utmost importance. Midface or mandibular hypoplasia should be looked for and ease or resistance of nasal airflow should be assessed. Nasal obstruction is suggested by a hyponasal voice and difficulty breathing comfortably through the nose with the mouth closed. The ‘Mickey Mouse’ test checks for nasal obstruction or degree of hyponasality, especially in younger children. Words with m’s, n’s, or s’s require nasal airflow to sound normal. A child with significant nasal obstruction sounds the same with and without nasal occlusion. Tonsillar hypertrophy may be present, although several studies show no relation between tonsil size and the presence of OSA. The size of the oropharynx should also be considered. Examination of the mouth includes the integrity of the hard and soft palate, length of the soft palate, size of the tongue, and position of the teeth and jaw. A narrow, high-arched hard palate, elongated soft palate, or shallow pharyngeal area may be associated with obstructive symptoms. Abnormal positioning of the teeth and jaw structures may be important risk factors for the development of OSA. A class II occlusion of the teeth (‘overbite’ of maxillary teeth or ‘underbite’ of mandibular teeth) may be associated with a hypoplastic or retrognathic mandible. Other less common physical findings include a pectus excavatum (indication of long-standing airway obstruction and increased work of breathing), systemic hypertension, pulmonary hypertension, or cor pulmonale, and even congestive heart failure. The neurological examination should focus on cranial nerve function, neuromuscular strength, and development. Sequelae

If unrecognized or untreated, OSA leads to neurobehavioral or cardiovascular sequelae in childhood. Sequelae may be mild and reversible, or may become severe and progressive if left untreated.

but is reported only anecdotally in children. Isolated diastolic hypertension in children with OSA has been reported. Adults with OSA may have a ‘brady-tachy’ pattern associated with the obstructive events. Studies in children reveal cardiac arrhythmias during obstructive events, albeit not on a consistent basis. A recent study found only modest changes in RR intervals, with no evidence of life-threatening arrhythmias or bradycardia during periods of severe, prolonged oxygen desaturations to o65%.

Diagnostic Evaluation Questionnaires

Several validated questionnaires may help physicians screen for the most common sleep problems in children. The pediatric sleep questionnaire (PSQ) screens for OSA in children aged 2–18 years. The children’s sleep habits questionnaire (CSHQ) screens for the most common medical and behavioral sleep problems in children aged 4–12 years, including sleep apnea. Nocturnal Monitoring

The American Thoracic Society published a consensus statement on standards and indications for cardiopulmonary sleep studies in children in 1996. OSA may not be diagnosed reliably by history alone. Overnight polysomnography is currently the gold standard for the diagnosis of OSA in children. Polysomnography consists of noninvasive overnight monitoring of sleep and breathing patterns and allows for the differentiation between ‘benign’ or primary snoring versus ‘pathologic’ snoring associated with OSA. It can be used to confirm the suspected diagnosis of OSA, assess the severity of the obstructive symptoms, and monitor the efficacy of treatment. Abbreviated Monitoring

Neurobehavioral sequelae Neurobehavioral sequelae are felt to be secondary to nocturnal hypoxemia and sleep fragmentation. Learning difficulties, including ADHD-like symptoms, are frequently identified in children with OSA. An unusually high prevalence of snoring was identified among a group of children designated as showing ‘mild’ ADHD. OSA is not more likely to occur among children with significant ADHD, but is prevalent among children with mild hyperactive behavior. Cardiovascular sequelae Systemic hypertension is the most chronic complication in adults with OSA,

Various ‘screening’ studies have been suggested as alternatives to polysomnography. These include overnight audiotaping, overnight oximetry studies, and nap studies. Audiotaping can document the presence of snoring, but is unable to reliably distinguish between primary snoring and snoring associated with sleep apnea. Overnight oximetry studies that document hypoxemia during sleep are used to identify children with symptoms of OSA who need further evaluation. A negative oximetry study cannot rule out sleep apnea. Nap studies are not believed to provide an effective screening evaluation for children with suspected OSA.

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Current Therapy OSA develops gradually. Most children with OSA have been symptomatic for some time prior to diagnosis and can wait for elective management (Table 2). However, rarely does a child present with severe hypoxemia, heart failure, respiratory failure, or altered level of consciousness. Emergency Management

In an emergency, a child with life-threatening OSA can be managed with a nasal/oral airway. More recently, the manufacture of small masks has meant that CPAP is now an option for pediatric patients. Endotracheal intubation is rarely required. Supplemental oxygen should be used with caution as it may depress ventilatory drive. Primary Management

Adenotonsillectomy is the first line and most common treatment for pediatric OSA in any child with adenotonsillar hypertrophy. It is also the initial treatment of OSA in children with other predisposing factors. The surgery is readily available, easily performed, and generally effective in resolving symptoms of sleep apnea. Removal of both tonsils and adenoids are recommended to avoid recurrence of symptoms, even if one or the other is the primary abnormality. Children with OSA require careful monitoring after surgery. Postoperative complications are more common in children less than 3 years, those with cerebral palsy or craniofacial abnormalities, and children found to

Table 2 Treatment of OSA in children Emergency management Oral or nasal airway Mask positive airway pressure, either continuous or bilevel Endotracheal intubation Supplemental oxygen, need to monitor ventilation Primary management Adenotonsillectomy Secondary management Mask positive airway pressure, either continuous or bilevel Management for selected cases Nasal decongestants or steroids Weight management program, including weight loss, exercise, and behavioral adjustments Gastric bypass surgery Oral appliances Maxillofacial surgery, including septoplasty, supraglottoplasty, uvulopalatopharyngoplasty, tongue wedge resection, or maxillomandibular reconstructive surgery Tracheotomy

have severe OSA on polysomnography. Complications of surgery include dehydration, hemorrhage, pain, respiratory distress, anesthetic complications, velopharyngeal incompetence, and stricture. Secondary Management

Mask positive airway pressure, either as continuous (CPAP) or bilevel (BiPAP) support, is the most common treatment of OSA in adults. CPAP is a noninvasive means to ‘stent’ open the upper airway during sleep. In children, CPAP can be used for preoperative stabilization of severe OSA or transiently in the postoperative period following an adenotonsillectomy. More commonly, CPAP is used for children with residual OSA following surgery. This includes children with morbid obesity who are attempting weight loss or children with craniofacial abnormalities awaiting facial growth or other surgical procedures. Management for Selected Cases

Children with OSA that is exacerbated by chronic rhinitis deserve optimum management of their allergic symptoms. Nasal decongestants or nasal steroids may be appropriate. Children with obesity-related OSA should be referred for a weight management program. Gastric bypass surgery is rarely performed in children. Oral appliances, such as mandibular advancing devices or tongue retainers, are occasionally used in adolescents in whom facial bone growth is complete. Children with OSA secondary to underlying craniofacial abnormalities may benefit from a variety of surgeries including nasal septoplasty, supraglottoplasty, uvulopalatopharyngoplasty, tongue wedge resection, or maxillomandibular reconstructive surgery. A tracheostomy is rarely required, except in the case of severe, life-threatening OSA. See also: Sleep Apnea: Overview; Adult; Continuous Positive Airway Pressure Therapy; Drug Treatments; Genetics of Sleep Apnea; Oral Appliances; Surgery for Sleep Apnea. Sleep Disorders: Overview; Central Apnea (Ondine’s Curse); Hypoventilation; Upper Airway Resistance Syndrome.

Further Reading Ali NJ, Pitson DJ, and Stradling JR (1993) Snoring, sleep disturbance, and behavior in 4–5 year olds. Archives of Disease in Childhood 68(2): 360–366. American Academy of Pediatrics (2002) Clinical practice guidelines: diagnosis and management of childhood obstructive sleep apnea syndrome. Pediatrics 109(4): 704–712. American Academy of Pediatrics (2002) Technical report: diagnosis and management of childhood obstructive sleep apnea syndrome. Pediatrics 109(4).

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American Thoracic Society (1996) Standards and indications for cardiopulmonary sleep studies in children. American Journal of Respiratory and Critical Care Medicine 153: 866–878. Carroll JL, McColley SA, Marcus CL, Curtis S, and Loughlin GM (1995) Inability of clinical history to distinguish primary snoring from obstructive sleep apnea syndrome in children. Chest 108(3): 610–618. Chervin RD, Archbold KH, Dillon JE, et al. (2002) Inattention, hyperactivity, and symptoms of sleep-disordered breathing. Pediatrics 109(3): 449–456. Chervin RD, Hedger K, Dillon JE, and Pituch KJ (2000) Pediatric sleep questionnaire (PSQ): validity and reliability of scales for sleep-disordered breathing, snoring, sleepiness, and behavioral problems. Sleep Medicine 1: 21–32. Gozal D (1998) Sleep-disordered breathing and school performance in children. Pediatrics 102(3): 616–620. Marcus CL (2000) Obstructive sleep apnea syndrome: differences between children and adults. Sleep 23(supplement 4): S140– S141. Marcus CL, Omlin KJ, Basinki DJ, et al. (1992) Normal polysomnographic values for children and adolescents. American Review of Respiratory Diseases 146: 1235–1239. O’Brien LM, Holbrook CR, Mervis CB, et al. (2003) Sleep and neurobehavioral characteristics of 5- to 7-year-old children with parentally reported symptoms of attention-deficit/hyperactivity disorder. Pediatrics 111(3): 554–563. Owens JA, Spirito A, and McGuinn M (2000) The children’s sleep habits questionnaire (CSHQ): psychometric properties of a survey instrument for school-aged children. Sleep 23(8): 1–9. Redline S, Tishler PV, Schluchter M, et al. (1999) Risk factors for sleep-disordered breathing in children: associations with obesity, race, and respiratory problems. American Journal of Respiratory and Critical Care Medicine 159(5 Pt 1): 1527–1532. Suen JS, Arnold JE, and Brooks LJ (1995) Adenotonsillectomy for treatment of obstructive sleep apnea in children. Archives of Otolaryngology and Head and Neck Surgery 121(5): 525–530.

Relevant Website http://www.pediatrics.org – Pediatrics, the official journal of the American Academy of Pediatrics, is available here.

Continuous Positive Airway Pressure Therapy M S Badr, Wayne State University School of Medicine, Detroit, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Obstructive sleep apnea is a fairly common condition with significant adverse health outcome. The treatment of choice is nasal continuous positive airway pressure (CPAP), which restores upper airway patency by increasing intraluminal pressure and hence acting as a ‘pneumatic splint’ maintaining patency throughout the entire upper airway. Determination of the optimal CPAP pressure requires ‘titration’ during polysomnography.

The criteria for initiating nasal CPAP therapy are continuously evolving. Nasal CPAP can be delivered as a CPAP, as bi-level positive pressure therapy (BPAP) or as an auto-titrating device. While these modalities improve comfort; there is no evidence that adherence with therapy is enhanced. An area of controversy is whether patients with mild disease would benefit from CPAP therapy. Effectiveness of therapy requires adherence to therapy; unfortunately, adherence with anal CPAP remains suboptimal. Intensive support may improve adherence with potential improvement in patient outcome.

Introduction Obstructive sleep apnea syndrome (OSAS) is a common disorder with significant adverse health consequences. Specifically, OSAS is associated with reduced quality of life, increased risk of motor vehicle accidents, systemic hypertension, and cardiac consequences. Epidemiologic studies suggest a prevalence of 2% of women and 4% of men. Continuous positive airway pressure (CPAP) is an effective therapy and is the most widely used modality in patients with clinically significant sleep-disordered breathing.

Mechanism of Action The upper airway is an extrathoracic conduit, void of structural support. Therefore, upper airway patency depends on upper airway caliber, upper airway muscle tone, and the transmural pressure across pharyngeal wall. The occurrence of upper airway obstruction indicates the development of a collapsing transmural pressure across a highly compliant pharyngeal wall. Nasal CPAP restores upper airway patency by increasing intraluminal pressure above the positive critical transmural pressure across the pharyngeal wall. This pressure is referred to as the ‘critical opening pressure’. Thus, nasal CPAP serves as a ‘pneumatic splint’ maintaining patency throughout the entire upper airway. To initiate nasal CPAP therapy, the appropriate ‘critical opening pressure’ should be determined. Treatment with an optimal pressure is of utmost importance. If subtherapeutic pressure is used, snoring, sleep fragmentation, and daytime sleepiness may persist. Conversely, excessive pressure could result in patient discomfort, sleep fragmentation, or development of central apnea. Therefore, this is accomplished in the sleep laboratory during a sleep study (polysomnography) by titrating the pressure level upward until resolution of respiratory events, oxyhemoglobin desaturation, and sleep fragmentation. Various body positions and both non-rapid-eyemovement (non-REM) and REM sleep should be studied to determine optimal CPAP pressure under these conditions.

SLEEP APNEA / Continuous Positive Airway Pressure Therapy 57

Who Should Be Treated with Nasal CPAP?

Which Device Is Best for My Patient?

The severity of sleep apnea influences the decision to initiate therapy with nasal CPAP. Most sleep laboratories use the combined number of apneas and hypopneas per hour of sleep (known as apnea/hypopnea index (AHI)) as the ‘metric’ of the severity. Current reimbursement (treatment) guidelines in the US require five apneas per hour of sleep. However, AHI is not an optimal ‘metric’ as mild disease (by AHI) may have significant adverse consequences. In fact, several studies have shown that the treatment of mild disease may be beneficial in terms of daytime function or alertness. Another limitation of AHI as a metric is the lack of standardization across different sleep laboratories. There are multiple operational definitions for hypopnea incorporating variable degrees of oxyhemoglobin desaturation, reduced effort, or subsequent arousal. Such variations and the use of different flow sensors contribute to the variation in AHI. Interestingly, AHI for sleep apnea is used in a manner analogous to blood pressure in systemic hypertension. However, this analogy is premature given the variations in the measurement methods, instrumentations, or determinants of adverse outcome.

CPAP Mode

Does CPAP Titration Require a Full-Night Polysomnography? Studies have shown that a split-night study can identify an effective pressure in 78% of patients. However, a significant discrepancy in final CPAP pressure occurs when titration time is less than 3 h. In general, a measurable proportion will require changes in the prescription or the interface. Nevertheless, split-night studies are often necessary to reduce wait time and expedite treatment. The concern that split-night studies may decrease compliance was not substantiated by empirical evidence. However, there is some evidence that compliance may be less than optimal following split-night studies in patients with mild disease.

Selection of the Optimal Interface CPAP interface is critical to the delivery of comfortable and effective CPAP pressure. Nasal masks are the most common type of CPAP interface. Oro-nasal, oral masks, or hybrid masks are available. Optimal fit is essential for patient comfort; poor fit may adversely affect compliance. Identifying the optimal interface requires patience, perseverance, and readiness for trial and error.

This is the default mode in most patients. The prescribed CPAP pressure refers to the end-expiratory pressure. Most patients can be managed effectively with nasal CPAP with an effective pressure ranging from 5 to 15 cmH2O. Most patients do not tolerate higher pressure, especially during expiration. Bi-Level Positive Pressure

Bi-level positive pressure (BPAP) devices allow for independent titration of the inspiratory and expiratory pressures delivered to the patient. The positive pressure required to maintain upper airway patency is higher during inspiration relative to expiration owing to the presence of negative intraluminal pressure during inspiration. BPAP devices are more comfortable, especially with high-pressure requirements. BPAP devices are indicated in patients who have difficulty exhaling against the positive pressure, especially those who require high-pressure levels. In addition, patients who have nocturnal ventilatory failure or hypoventilation are also candidates for BPAP therapy. In this group, the term noninvasive positive pressure ventilation (NIPPV) is preferable since the device serves as a ventilatory support and not for upper airway patency per se. Autotitrating Devices

The mask pressure changes throughout the night based on indirect indications of changing upper airway mechanics. The primary ‘signal’ driving the change varies among devices. Typically, third-party payers require intolerance to conventional CPAP or failure to determine optimal pressure for approval.

Humidification Positive pressure therapy requires high flow of ambient air to pass through the circuit. This may cause significant dryness of the nasal and pharyngeal mucosa. The result is mucosal edema, increased nasal resistance, diminished effectiveness, and lack of adherence with therapy. Humidity in the breathing circuit can be increased by cold (passover) or heated humidification. There is evidence that heated humidification decreases nasal resistance and increases patient comfort. Humidification can alleviate dryness, heated humidification, but not passover. Humidification may enhance compliance with nasal CPAP.

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Other data demonstrate improved comfort but no effect on long-term use.

Adherence with CPAP Therapy Two terms are often used to describe patient’s fidelity to a treatment plan. The first term is compliance, which indicates following directives. The second term is adherence, which indicates an active role for the patient. Although the terms are different, different studies use them interchangeably. Adherence with Chronic Therapy

Adherence to chronic therapy is suboptimal. For example, studies have shown that adherence with inhaled antiasthma therapy and oral antihypertensive is suboptimal in one-half of the patients. Interestingly, this is noted even in patients with chronic renal failure requiring hemodialysis; one-third of the patients missed treatments over a 6-month period.

In summary, adherence to nasal CPAP therapy is a critical component of the management of sleep apnea. The available literature does not provide firm guidelines on optimizing adherence. Intensive support is the key intervention to enhance patient adherence to CPAP therapy.

Summary Nasal CPAP is the treatment of choice for obstructive sleep apnea. Attention to detail is required to ensure that optimal mode, interface, and settings are provided for each patient. Despite its effectiveness, compliance with nasal CPAP therapy remains suboptimal. Intensive follow-up and support can improve compliance. See also: Sleep Apnea: Overview; Adult; Children; Drug Treatments; Genetics of Sleep Apnea; Oral Appliances; Surgery for Sleep Apnea. Sleep Disorders: Overview; Central Apnea (Ondine’s Curse).

Adherence to Nasal CPAP Therapy

Several prospective, objective studies have investigated patient compliance with nasal CPAP therapy for sleep apnea. There is a discrepancy between subjective and objective compliance. One-half of the patients are regular users and about one-half of the patients are intermittent users. The pattern of use is established by day 4 of treatment. How Can We Optimize Compliance/Adherence?

The ability to identify determinants of adherence is essential to improving patient outcome. Studies have focused on several pertinent variables. For example, there is no evidence that positive pressure modalities influence adherence to CPAP as there is no evidence of improved compliance with bi-level devices. In contrast, patients receiving ‘intensive support’ have a higher likelihood of CPAP usage than standard support patients do. Likewise, patients who are self-referred have greater CPAP usage than those whose referral is initiated by bed partner. Similarly, patients with subjective complaints of excessive daytime sleepiness, however, show greater hours of CPAP use versus those with minimal daytime complaints. Finally, conclusive evidence of improved compliance with auto-CPAP, bi-level, self-titration, and humidification is also lacking. There is some evidence that psychological/educational interventions improve CPAP usage (The Cochrane Database of Systematic Reviews 2005). A recent study has identified three variables that predict compliance: female gender, improvement in ESS scores with CPAP, and advanced age.

Further Reading Bleyer AJ, Hylander B, Sudo H, et al. (1999) An international study of patient compliance with hemodialysis. Journal of the American Medical Association 281: 1211–1213. Haniffa M, Lasserson T, and Smith I (2004) Interventions to improve compliance with continuous positive airway pressure for obstructive sleep apnoea. Cochrane Database of Systematic Reviews CD003531. Hoy CJ, Vennelle M, Kingshott RN, Engleman HM, and Douglas NJ (1999) Can intensive support improve continuous positive airway pressure use in patients with the sleep apnea/hypopnea syndrome? American Journal of Respiratory and Critical Care Medicine 159: 1096–1100. Kribbs NB, Pack AI, Kline LR, et al. (1993) Objective measurements of patterns of nasal CPAP use by patients with obstructive sleep apnea. American Review of Respiratory Disease 147: 887–895. Likar LL, Panciera TM, Erickson AD, and Rounds S (1997) Group education sessions and compliance with nasal CPAP therapy. Chest 111: 1273–1277. Massie CA, Hart RW, Peralez K, and Richards GN (1999) Effects of humidification on nasal symptoms and compliance in sleep apnea patients using continuous positive airway pressure. Chest 116: 403–408. McArdle N, Devereux G, Heidarnejad H, et al. (1999) Long-term use of CPAP therapy for sleep apnea-hypopnea syndrome. American Journal of Respiratory and Critical Care Medicine 159: 1108–1114. Reeves-Hoche MK, Hudgel DW, Meck R, et al. (1995) Continuous versus bi-level positive airway pressure for obstructive sleep apnea. American Journal of Respiratory and Critical Care Medicine 151: 443–449. Reeves-Hoche MK, Meck R, and Zwillich CW (1994) Nasal CPAP: an objective evaluation of patient compliance. American Journal of Respiratory and Critical Care Medicine 149: 149– 154. Richards GN, Cistulli PA, Ungar RG, Berthon-Jones M, and Sullivan CE (1996) Mouth leak with nasal continuous positive

SLEEP APNEA / Drug Treatments 59 airway pressure increases nasal airway resistance. American Journal of Respiratory and Critical Care Medicine 154: 182– 186. Sin DD, Mayers I, Man GC, and Pawluk L (2002) Long-term compliance rates to continuous positive airway pressure in obstructive sleep apnea: a population-based study. Chest 121: 430–435. Wiest GH, Harsch IA, Fuchs FS, et al. (2002) Initiation of CPAP therapy for OSA: does prophylactic humidification during CPAP pressure titration improve initial patient acceptance and comfort. Respiration 69: 406–412. Young T, Palta M, Dempsey J, et al. (1993) The occurrence of sleep-disordered breathing among middle-aged adults. New England Journal of Medicine 328: 1230–1235.

Drug Treatments D Auckley, Case Western Reserve University, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The conventional first-line treatments available for sleep apnea (continuous positive airway pressure therapy, oral appliances, and surgical intervention) are appropriate for people with symptomatic disease. The benefit of a drug treatment approach is one of convenience and ability to intervene at an earlier stage of the disease, or an alternative for those who cannot or will not accept or tolerate surgical or mechanical treatments. However, drug therapy at present is an uncertain option for the management of sleep apnea. Medications for sleep apnea can broadly be divided into the following functional categories: those that alter the upper airway neuromechanical properties, those that affect the control of respiration, and those that work as adjunctive therapy to minimize symptoms. Some drugs are known to act through more than one discrete pathway. While most attempts at drug therapy for sleep apnea have generally been unsuccessful, certain medications may have some use in specific clinical settings. In summary, convincing data for drug treatment as primary therapy is lacking, and additional therapy with one of the conventional sleep apnea treatments is often still the better option. Recent interest has focused on serotonin active agents as a treatment for obstructive sleep apnea. However, this class of medications is not yet approved for this indication.

Pathophysiology of SDB

Treatment options

Airway collapse

Nasal steroids∗

Symptoms of SDB

Modafenil∗

Abnormal control of breathing

Thyroid hormone∗ MPA∗ Theophylline Acetazolamide

Figure 1 Pathophysiology of and treatment options for sleep disordered breathing (SDB). If any agent is used as monotherapy for sleep apnea, then repeat objective testing is recommended (with the exception of modafinil). * Treatment options for OSA; w treatment options for CSA; MPA, medroxyprogesterone.

to either modify the airway, help facilitate ventilatory support, and/or to bypass the site of obstruction altogether (tracheotomy). While these therapies can be very successful for controlling or eliminating sleep apnea, each is hampered by limitations that impact their effectiveness in clinical practice. CPAP is problematic from a patient compliance standpoint. Oral appliances are effective in only a subset of patients with OSA and are also limited by suboptimal patient compliance. Surgical interventions can be highly effective in the right setting, but are generally restricted to OSA patients with surgically amenable anatomy who are willing to accept the risk of complications and the possibility of unacceptable long-term consequences. Thus, the use of a pill to treat sleep apnea seems highly desirable and remains a field of intense study. This article will review the current state of medical therapy for sleep apnea (Figure 1).

History Sleep apnea has been recognized as a significant clinical disorder for only the last three decades. As data accumulates linking multiple adverse outcomes to obstructive sleep apnea (OSA), the importance of treating this condition has become widely accepted. Likewise, central sleep apnea (CSA) is associated with worse outcomes in those with impaired cardiac function. Conventional therapies for sleep apnea include the use of pneumatic airway splints (continuous positive airway pressure (CPAP) devices), oral appliances to enlarge the upper airway, and surgery

Uses of Respiratory Medicine Medications Affecting Airway Neuromechanical Properties

Serotonin active agents Upper airway dilator muscle activity decreases during sleep, narrowing the airway. In the presence of abnormal pharyngeal anatomy, this results in repetitive airway collapse during sleep, or OSA. The neurotransmitter serotonin (also known as 5-HT) plays an integral role in maintaining patency of the upper airway by

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activating upper airway dilator muscles. As such, investigators have reasoned that increasing central serotonin levels may improve airway patency and eliminate OSA. Unfortunately, the physiology is not this simple as at least 14 different serotonin receptor subtypes have been identified and some of these, when stimulated, exhibit inhibitory effects on upper airway motor tone. This may in part explain why human trials of selective serotonin reuptake inhibitors (SSRIs) have failed to show a clinical benefit in OSA patients. It is likely that mixed serotonin receptor agonist–antagonists are needed to produce clinically significant changes. Preliminary work with mirtazapine, a 5-HT1 receptor agonist and 5-HT2 and 5-HT3 receptor antagonist, has shown promise in animal models of sleep apnea. Trials are underway to assess its effectiveness in humans with sleep apnea. It is hoped that ongoing work with serotonin and serotonin receptors will lead to better-targeted therapies. While this area holds great promise, serotonin active agents as monotherapy for OSA cannot be recommended at this time. Protriptyline Protriptyline is a nonsedating tricyclic antidepressant with REM sleep-suppressing properties. Like serotonin, protriptyline also stimulates the hypoglossal motor neurons to increase upper airway muscle activity. A number of investigators have studied protriptyline as a potential therapy for OSA. In sum, these studies have not shown clinically significant improvements in OSA (two studies found statistically significant reductions in the apnea–hypopnea index (AHI), though moderate to severe OSA persisted). In addition, the high frequency of intolerable anticholinergic side effects limits protriptyline’s clinical utility. Protriptyline should not be considered as a treatment option for OSA at this time. Topical nasal steroids Nasal airflow resistance may contribute significantly to the development of OSA. Available data suggests that sleep apnea is more common in individuals with chronic nasal congestion as compared to those without. Initial studies suggested that treatment of chronic nasal congestion with nasal steroids improved sleep, reduced daytime fatigue, and decreased sleepiness, though measures of sleep apnea were not specifically addressed. Only one placebo-controlled study has examined the impact of nasal steroids on parameters of OSA in adults with concomitant allergic rhinitis. This small study (23 patients total) showed an improvement in the AHI in treated patients, though the individual response to treatment was highly variable. The group on the whole decreased their AHI from 20 to 12 events per h, whereas the 13 patients entering the study meeting

criteria for OSA (AHI X 5, the remainder considered to be primary snorers) only reduced their AHI from 30 to 23. Of the five individuals who decreased their AHI to o5, considered an optimal response, three had an AHI between 5 and 10 on placebo. These data suggest that in patients with mild OSA and allergic rhinitis, nasal steroid therapy may have a role, but for more severe disease, additional conventional therapy will probably be required. Medications Generally Affecting the Control of Breathing

Hormonal therapy Both estrogen and progesterone have been studied as potential therapies for OSA. Medroxyprogesterone (MPA) is a recognized ventilatory stimulant, though it may exert some stimulatory effects on the upper airway motor neurons as well. By attenuating hypoventilation, MPA was hoped to reduce periodic breathing and improve sleep apnea. An early small, uncontrolled study found that MPA improved OSA in a subset of patients with baseline hypercapnia. Two subsequent placebo-controlled trials failed to show a treatment effect of MPA on OSA, though only four of the patients were known to be hypercapnic. Until larger randomized controlled trials of hypercapnic OSA patients are performed, MPA cannot routinely be recommended as a treatment option. Its role in treating patients with OSA and obesity hypoventilation syndrome is uncertain. An empiric trial could be considered in some individuals, provided objective follow-up testing is obtained. MPA has not been studied as a treatment for CSA. Estrogen has effects on upper airway musculature in addition to possible centrally mediated effects on respiration. Growing evidence suggests that the incidence of OSA in postmenopausal women increases and approaches that of age-matched men. It is of interest that in postmenopausal women on hormone replacement therapy (HRT), the incidence of OSA remains low. Despite these findings, no large randomized controlled trials of HRT in postmenopausal women with OSA have been reported to date. Two small (5 and 6 patients) nonrandomized, uncontrolled trials found significant improvements in the AHI following therapy with estrogen alone, though only one of the 11 subjects had normalization of their AHI. In contrast, another uncontrolled study of 15 postmenopausal women with moderate to severe OSA found little change in their AHI following treatment with estrogen. As might be expected from these findings, the majority of participants in all the studies did not perceive much subjective benefit in their sleep or daytime symptoms. Further study is

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needed before HRT can be recommended in postmenopausal women for the treatment of OSA. Theophylline Theophylline is a methylxanthine that inhibits adenosine, a central-acting ventilatory depressant, as well as enhances the ventilatory response to hypoxia and hypercapnia. Thus, it might be expected that theophylline may improve CSA more than OSA. In two blinded placebo-controlled studies it was found that theophylline significantly improved central apneas with the most pronounced effect seen in the setting of left ventricular dysfunction (central apneas decreased from 26 to 6 h 1 on average in 15 subjects studied). However, sleep remained poor in these individuals due to theophyllineinduced sleep fragmentation. OSA, on the other hand, failed to improve following therapeutic range dosing of theophylline in a number of controlled trials. Two studies comparing theophylline to CPAP therapy have found CPAP to be considerably more efficacious. Before considering theophylline for patients with left ventricular dysfunction and central sleep apnea, one must consider the narrow therapeutic window and potential toxic side effects of theophylline. In addition, the sleep disruption this medication induces may lead to persistence of sleeprelated symptoms and, consequently, poor compliance with therapy. Acetazolamide Acetazolamide, a carbonic anhydrase inhibitor, produces a metabolic acidosis and thus stimulates respiration. This drug has been examined as a treatment for both central and obstructive sleep apnea. Uncontrolled data from a small number of subjects suggests acetazolamide may dramatically reduce the number of central events (mean central AHI decreased from 54 to 12 and 26 to 7 in two different studies) in sleep as well as improve associated daytime symptoms. No randomized controlled trial has been performed to confirm these findings. With regard to OSA, both uncontrolled studies and a single randomized placebo-controlled trial suggest that the AHI variably improves following treatment; however, significant OSA generally persists. Side effects, including intolerable parasthesias and severe metabolic acidosis, can occur when acetazolamide is given at higher doses and may restrict its clinical utility. At present, acetazolamide can be considered for patients with CSA, though close clinical monitoring is warranted. Thyroid replacement Hypothyroidism depresses the ventilatory drive, can impair upper airway muscle function due to myopathy, and may narrow the upper airway secondary to mucopolysaccharide

deposition. Reversing these changes might be expected to improve sleep apnea in those found to be hypothyroid. Numerous small case series addressing this have yielded conflicting results as some show dramatic decreases in the AHI with thyroid replacement therapy and others show no effect. It is unclear if those failing to respond to irreversible functional or anatomic changes due to hypothyroidism or that other factors contributing to OSA risk (such as obesity or craniofacial abnormalities) may be playing a role. No randomized controlled trials addressing thyroid replacement in hypothyroid patients with OSA have been reported at this time. Based upon available data, it may be reasonable to utilize thyroid replacement as monotherapy for hypothyroid patients with mild OSA, but adjunctive conventional treatments should be considered for more severe OSA patients. All patients should undergo repeat evaluation of their sleep apnea once euthyroid status is achieved. Opioid antagonist The cerebral spinal fluid of individuals with OSA contains increased opioid levels that fall following successful treatment of the OSA. Through generalized cortical stimulation, opioid antagonists are thought to stimulate respiration. Controlled trials of continuous infusions of opioid antagonists (naloxone and doxapram) showed minor improvements in several parameters of OSA (oxygen saturation, length of apneas), though by and large these changes were of marginal clinical significance. Furthermore, these agents disturb sleep and require dosing via intravenous infusion, both undesirable qualities. Nicotine Nicotine stimulates respiratory drive by acting on central respiratory neurons. One uncontrolled trial found a decrease in the number of apneas in the first 2 h of sleep after subjects with OSA chewed nicotine gum at bedtime. However, a subsequent placebo-controlled study of transdermal nicotine found no significant improvement in the AHI or snoring intensity in a population of individuals with OSA and/or primary snoring. Nicotine was also noted to worsen sleep quality and induce intolerable gastrointestinal side effects in a number of study subjects. Benzodiazepines Benzodiazepines are not routinely recommended for patients with OSA due to concerns about depressing the arousal response, altering respiratory drive, and worsening airway collapse. It is of interest that a randomized placebo-controlled trial of the benzodiazepine receptor antagonist flumazenil found no benefit in patients with OSA, suggesting

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endogenous benzodiazepine receptor activation does not play a role in the pathophysiology of OSA. In contrast to concerns about decreased respiratory drive, some benzodiazepines have been shown to increase respiratory drive during sleep and therefore might improve CSA. The use of diazepam in a rat model of CSA found this to be the case. In humans, one small randomized placebo-controlled cross-over study of triazolam in idiopathic CSA demonstrated a reduction in the central apnea index and number of arousals with triazolam. Similar findings were noted in case series of subjects with periodic limb movements and CSA. Further study of benzodiazepines in CSA is warranted to clarify the specific patient populations who might stand to benefit from this therapy. Medications that Minimize Symptoms

Despite optimal treatment of OSA, daytime sleepiness may not completely resolve. The reason for this is not clear, but the persistence of these symptoms can adversely impact quality of life and potentially increase the risk of accidents. Therefore, therapies to further reduce daytime symptoms of OSA in those already on conventional treatment have been studied. Modafinil Modafinil is a central-acting wake-promoting agent that has no effect on respiratory events in sleep. It has now been examined in two relatively large randomized placebo-controlled trials of OSA patients with persistence of daytime symptoms despite adequate treatment with CPAP. These studies showed consistent improvements in both subjective and objective measures of sleepiness as well as improvements in tests of vigilance and measures of quality of life. A clinically insignificant decrease in CPAP usage occurred during one of the trials, though this remains a concern when one considers the use of stimulants outside of a clinical trial setting. Based upon the available data, modafinil can be considered as an adjunct to conventional therapies for patients with residual OSA-related daytime sleepiness, though close monitoring of CPAP usage is encouraged. Other stimulants have not been tested in this setting. Etanercept Serum tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6) are elevated in individuals with OSA, and have been proposed as mediators of the excessive daytime sleepiness that accompanies OSA. Etanercept neutralizes TNF-a and thus was tested in a small placebo-controlled pilot study of patients with OSA to assess its effects on OSA and OSA-related symptoms. While having a marginal impact on the AHI (decreased from 53 to

44, statistically significant but not clinically significant), a substantial improvement in an objective measure of sleepiness was seen. No subjective ratings of sleepiness or other quality of life measures were reported. Further study of this medication as an adjunctive therapy for persistent daytime symptoms in CPAP-treated OSA is warranted. Modification of Sleep Apnea by Other Treatments

Antihypertensive agents While attempting to determine the best antihypertensive agents for patients with OSA, investigators noted that the AHI decreased modestly following treatment with betablockers or ACE inhibitors. Changing sympathetic tone or baroreceptor activity were the proposed mechanisms for these changes. These findings then led to two randomized trials of antihypertensive agents (beta-blockers, ACE inhibitors, calcium channel blockers, and diuretics) as treatments for OSA. Placebos were not included in either study. While all agents reduced blood pressure, the impact on OSA was marginal at best. One study showed a decrease in the AHI (from 40 to 27, clinically insignificant) while the other found no change in pre- and posttreatment measures. In the study reporting subjective symptoms, no improvement was noted. Clonidine, a rapid eye movement (REM)-suppressing agent, has also been evaluated as a treatment for OSA. One small placebo-controlled trial found no improvement in the AHI or symptoms following treatment with clonidine. Collectively, these data suggest that antihypertensive agents should not be utilized as therapy for the treatment of OSA. Glutamate antagonist Attenuating the respiratory response to acute hypoxia could prevent the periodic breathing pattern that may precipitate sleep apnea in some individuals. Glutamate, a neurotransmitter that may be partly responsible for hypoxia-induced respiratory stimulation, seems a reasonable target to antagonize and hopefully improve sleep apnea. One small randomized placebo-controlled trial of sabeluzole, a glutamate antagonist, documented an improved oxygen desaturation index during sleep, but did not measure the AHI specifically. On the other hand, baclofen, a glutamate antagonist and GABA agonist, did not decrease the AHI in 10 patients with mild OSA in a randomized placebo-controlled trial. Larger controlled trials of pure glutamate antagonists seem to be indicated. See also: Sleep Apnea: Overview; Adult; Continuous Positive Airway Pressure Therapy; Oral Appliances; Surgery for Sleep Apnea. Sleep Disorders: Central Apnea (Ondine’s Curse); Upper Airway Resistance Syndrome.

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Further Reading Hudgel DW and Thanakitcharu S (1998) Pharmacologic treatment of sleep-disordered breathing. American Journal of Respiratory and Critical Care Medicine 158: 691–699. Javaheri ST, Parker J, Wexler L, et al. (1996) Effect of theophylline on sleep-disordered breathing in heart failure. New England Journal of Medicine 335: 562–567. Kiely JL, Nolan P, and McNicholas WT (2004) Intranasal corticosteroid therapy for obstructive sleep apnoea in patients with co-existing rhinitis. Thorax 59(1): 50–55. Kingshott RN, Vennelle M, Coleman EL, et al. (2001) Randomized, double-blind, placebo-controlled crossover trial of modafinil in the treatment of residual excessive daytime sleepiness in the sleep apnea/hypopnea syndrome. American Journal of Respiratory and Critical Care Medicine 163: 918–923. Magalang UJ and Mador MJ (2003) Behavioral and pharmacologic therapy of obstructive sleep apnea. Clinics in Chest Medicine 24: 343–353. Pack AI, Black JE, Schwartz JRL, and Matheson JK (2001) Modafinil as adjunct therapy for daytime sleepiness in obstructive sleep apnea. American Journal of Respiratory and Critical Care Medicine 164: 1675–1681. Smith I, Lasserson T, and Wright J (2003) Drug treatments for obstructive sleep apnoea. Cochrane Database of Systematic Reviews 2001(4), article CD 003002. Smith IE and Quinnell TG (2004) Pharmacotherapies for obstructive sleep apnoea: where are we now? Drugs 64(13): 1385– 1399. Veasey SC (2003) Serotonin agonists and antagonists in obstructive sleep apnea: therapeutic potential. American Journal of Respiratory Medicine 2(1): 21–29. Vgontzas AN, Zoumakis E, Lin HM, et al. (2004) Marked decrease in sleepiness in patients with sleep apnea by Etanercept, a tumor necrosis factor-alpha antagonist. Journal of Clinical Endocrinology and Metabolism 89(9): 4409–4413. Yap WS and Fleetham JA (2001) Central sleep apnea and hypoventilation syndrome. Current Treatment Options in Neurology 3: 51–56.

Genetics of Sleep Apnea S R Patel, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA S Redline, Case Western Reserve University, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Obstructive sleep apnea is a disorder that has a clear genetic component. A familial basis for the disorder is evidenced by the substantially increased risk for snoring or sleep apnea among relatives of affected individuals. Approximately one-third of the population variance in apnea severity, as measured by the apnea/hypopnea index, is explained by familial clustering. The pathways by which genetic predisposition may influence the development of sleep apnea are multiple. Obesity, craniofacial anatomy, and ventilatory control are all traits that are highly heritable, and each influences the risk of apnea development. The genetics of obesity has been most well studied, with dozens of candidate genes proposed to influence a person’s weight. Data

suggest that these obesity-defining loci explain only half of the genetic variance in sleep apnea. Thus, other mechanisms are also important. Work is under way to identify risk genes for sleep apnea, and several candidates such as APOE have emerged. Research has also begun to identify genetic loci that may modulate the physiologic effect of sleep apnea on the development of secondary disorders, such as sleepiness, hypertension, and cardiac disease.

Since a report in 1978 of three brothers with obstructive sleep apnea (OSA), it has become increasingly clear that this disease clusters within families. This suggests that genetic susceptibility plays an important role in apnea pathogenesis. Knowledge of sleep apnea genetics provides not only an opportunity to better understand an individual’s predisposition to develop OSA and its neuropsychiatric and cardiovascular consequences but also insight into the molecular pathways that, when dysregulated, produce OSA. The ability to predict individual risk will allow for more efficient prevention and screening programs while knowledge of pathophysiology may allow for novel treatment strategies that specifically target the molecular defects.

Sleep Apnea Phenotypes An important consideration in understanding the role of genetics in sleep-disordered breathing is to identify the most relevant phenotype. A feature of OSA shared by many other complex disorders is the lack of a standardized phenotypic definition of disease. OSA is typically defined as the presence of an apnea/hypopnea index (AHI) above a certain threshold (often 45 or 10 events per hour of sleep). Obstructive sleep apnea hypopnea syndrome (OSAHS) represents the combination of OSA with symptoms of sleepiness referable to the OSA. One study found familial aggregation of a phenotype that included symptoms of sleepiness to be greater than phenotypes defined purely on polysomnographic criteria. An important issue is whether heritability is greater for a phenotype based strictly on AHI level or one that uses associated symptoms, such as sleepiness or daytime dysfunction. In addition, the choice of a dichotomous phenotype, such as OSA or OSAHS, versus a continuous phenotype, such as AHI, need also be considered. Although continuous phenotypes typically provide more power for identifying genetic susceptibility loci and do not require arbitrary threshold cutoffs that may vary across subgroups (e.g., children vs. adults), they also make more assumptions regarding scaling (a doubling of AHI from 2 to 4 is of equivalent importance as one from 10 to 20) that may or may not be appropriate.

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Sleep Apnea Risk Factors Many of the established risk factors for sleep apnea have known genetic determinants (Figure 1). Genetic polymorphisms that predispose an individual to develop these intermediate traits would therefore be expected to also be important risk factors for OSA. Obesity

It has long been recognized that obesity is one of the most important risk factors for sleep apnea. Among middle-aged individuals in the United States, obesity increases OSA risk by 10- to 14-fold. Modest amounts of weight loss can substantially ameliorate OSA severity, and more substantive weight loss, as experienced after bariatric surgery, can reduce the frequency of sleep-related breathing disturbances to clinically insignificant levels in up to 80% of cases. There are multiple mechanisms by which obesity predisposes to OSA. Fat deposition in the neck predisposes the upper airway to collapse. Fat deposition in the torso increases work of breathing, which may produce hypoventilation. This mechanical effect also reduces resting lung volume, which in turn reduces parenchymal traction on the trachea, making the airway more collapsible. Finally, the metabolic effects of obesity on hormones such as leptin may have important effects on ventilatory drive. Weight has been clearly established to have a strong genetic component. Twin studies have estimated that approximately 70% of the variance in body mass index is explained by familial factors. Rare autosomal recessive mutations in key genes involved in appetite regulation produce severe childhood obesity. Mutations in the genes encoding leptin, leptin receptor, proopiomelanocortin (POMC), and melanocortin-4 receptor are examples of single-gene causes of obesity. Genetic background

Craniofacial structure Ventilatory control

Obesity

OSA

Neurocognitive sequelae Figure 1 Sleep apnea risk factors.

Cardiovascular sequelae

In most individuals, however, obesity is a polygenic disorder, with multiple genes, each with small to modest effects, together accounting for familial similarities. Many whole genome linkage scans for obesity phenotypes have been conducted, and dozens of candidate genes have been identified as potential risk factors. Among the strongest candidate polymorphisms are functional mutations in the ghrelin gene on chromosome 3 and the b2 adrenergic receptor on chromosome 5. In addition, strong evidence exists for susceptibility loci on chromosomes 4, 10, and 20 as well as a locus on chromosome 2 close to the POMC gene. Since central obesity, upper body obesity, and fat in the neck and upper airway have been argued to be more important risk factors for OSA than overall obesity, genetic polymorphisms that influence fat deposition patterns may also play an important role in defining OSA susceptibility. Central versus peripheral fat deposition patterns exhibit familial aggregation, with as much as 25–30% of variability in waist and hip circumference explained by heritable factors. Craniofacial Morphology

Anatomic features of the face or pharynx that narrow the upper airway will tend to predispose individuals to airway collapse during sleep. This applies to both bony and soft tissue structures surrounding the upper airway. Among the bony phenotypes associated with OSA are brachycephalic head form, reduced anterioposterior dimension of the cranial base, reduced nasion-sella-basion angle, inferiorly displaced hyoid, and mandibular retrognathia and micrognathia. Soft tissue predictors include elongated soft palate, macroglossia, and adenotonsillar hypertrophy. Both twin and family studies support a genetic basis for determining craniofacial phenotypes. The cephalic index (ratio of head width to head length) appears to be almost completely genetically determined. Other bony features predictive of OSA, such as the length of the cranial base and the nasion-sellabasion angle, are also significantly heritable. Several studies have compared upper airway dimensions in relatives of OSA probands. Compared to relatives of controls, relatives of apneics have both bony and soft tissue craniofacial features, such as retropositioned maxillae and mandibles, longer soft palates, larger tongues, and larger lateral pharyngeal walls, that predispose to OSA. Bony anatomic structures have been reported to explain a larger proportion of the variance in indices of OSA among Caucasians compared to African Americans, suggesting ethnic heterogeneity in genetically determined anatomic factors that influence OSA susceptibility.

SLEEP APNEA / Genetics of Sleep Apnea 65 Ventilatory Control

Abnormalities in ventilatory control may predispose to OSA by several mechanisms. In the presence of a blunted ventilatory drive, neural stimulation of the upper airway dilator muscles may become insufficient to maintain airway patency. Conversely, an overly robust ventilatory drive may lead to instability of breathing with alternating cycles of hyperventilation and hypoventilation. During the nadir of this cycling, neural output to the upper airway dilators may again fall below the threshold required to keep the airway open. In mouse models, the satiety-promoting hormone, leptin, has important effects on ventilatory drive. Mice homozygous for a knockout mutation in leptin hypoventilate and have no ventilatory response to hypercapnia. In humans, obesity is typically associated with large elevations in leptin levels. Individuals with OSA have further elevations in leptin compared to obesity-matched controls. Whether leptin influences ventilatory drive in humans is an active area of investigation. A number of studies have established a strong correlation in the ventilatory response to hypoxia between twins, with stronger correlations between monozygotic than dizygotic twins. A reduced hypoxic ventilatory response has been found in the siblings of patients with various pulmonary disorders. The response to hypercapnia, on the other hand, has not been found to consistently display familial aggregation. Thus, there is stronger evidence for a genetic basis for hypoxic than hypercapnic responses, the latter of which may be more driven by environmental factors. Several studies suggest that individuals from families with multiple members who have OSA may have a reduced respiratory response to hypoxia. Under the stressor of inspiratory resistive loading, differences between nonapneic individuals from OSA families and control families have also been identified. Both a reduced respiratory impedance and lowered tidal volumes have been reported in relatives of OSA probands in response to loading, suggesting that an inherited predisposition to apnea may exist that requires the appropriate stress to become manifest. Another example of the role of genetic determinants on ventilatory drive is congenital central hypoventilation syndrome (CCHS), a disorder characterized by a depressed respiratory control system. CCHS has a strong genetic component, being due in most cases to a mutation in one of a family of genes that regulate neural crest differentiation or migration. The majority of cases (50–98%) are due to mutations in PHOX2B,

which encodes a transcription factor important in neural differentiation. Mouse knockouts of this gene display a phenotype very similar to human CCHS. An expansion of a polyalanine tract in exon 3 of PHOX2B is the most commonly isolated mutation, and the number of additional repeats correlates with disease severity. Mutations in other genes important in neural crest development, including RET, GDNF, EDN3, and BDNF, are also associated with the CCHS phenotype. Sudden Infant Death Syndrome

Sudden infant death syndrome (SIDS) is a disorder in which cardiopulmonary collapse occurs within the first year of life. Although some cases of SIDS may be due to abuse or to cardiac abnormalities, a large proportion of SIDS is attributed to prolonged apnea. Abnormalities in ventilatory control and craniofacial morphology are postulated to be important in SIDS pathogenesis, and, as previously described, there is strong evidence for an important genetic component in both pathways. Similarly, SIDS and near-miss SIDS have been found to aggregate within families. The risk of SIDS in a sibling of a patient is 3- to 10fold greater than in controls and increases further with more than one affected sibling. Interestingly, SIDS and OSA have been found to cosegregate within families. In other words, SIDS is more common in families in which a member has OSA, and OSA is more common in families with a SIDS child. These data suggest that the two disorders may have shared genetic predisposition. Congenital Abnormalities

A large number of congenital disorders that have a genetic basis are commonly associated with OSA. Infants with the Pierre Robin sequence characterized by cleft palate, mandibular hypoplasia, and resulting glossoptosis often present with recurrent airway obstruction. This disorder can be a consequence of several genetic defects. Treacher–Collins syndrome is an autosomal dominant disorder characterized by micrognathia, cleft palate, hypoplastic zygomatic arches, and upper airway obstruction. The causal gene located on chromosome 5 plays an important role in branchial arch development. Down’s syndrome (trisomy 21) is another disorder commonly associated with OSA. Predisposing features to upper airway obstruction include brachycephaly, macroglossia, tonsillar hypertrophy, and obesity. Prader– Willi syndrome, caused by a deletion in a segment of the paternally inherited copy of chromosome 15, is characterized by OSA and hypoventilation. Besides severe central obesity, individuals with this syndrome

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have markedly blunted ventilatory responses to hypercapnia and hypoxia.

Familial Aggregation There is clear evidence that both snoring and OSA cluster within families. Several studies have found greater concordance for snoring among monozygotic than dizygotic twins. Further support for a genetic predisposition derives from research demonstrating a family history of snoring to be a strong and independent risk factor for snoring. Similar findings have been found in studies using polysomnography to diagnose sleep apnea. The prevalence of sleep apnea in first-degree relatives of OSA probands is 22–84%. The odds ratio for OSA in relatives of apneics compared to relatives of controls is elevated, with estimates ranging from 2 to 46. In addition, OSA risk increases with the number of relatives who also have OSA. The heritability (the percentage of the total variance explained by familial factors) of the AHI is 30–40% in both Caucasian and African American populations.

Candidate Genes Segregation analyses of apnea phenotypes suggest that OSA is an oligogenic disorder, with several loci contributing to the determination of the overall genetic risk. Linkage studies for OSA have only just begun. Based on the available data, there is suggestive evidence for linkage to AHI on chromosomes 2p and 19q in Caucasians and to 8q in African Americans. Of note, the areas of interest on 2p and 8q include regions linked to obesity phenotypes. The POMC locus lies in the region of interest on chromosome 2p and polymorphisms of this gene predict leptin levels. Neurons expressing POMC in the hypothalamus play a key role in energy homeostasis, and a-melanocyte-stimulating hormone, a peptide derived from POMC, has important satietyinducing properties. Severe mutations of POMC in humans as well as mice result in severe obesity phenotypes. Another locus of interest is APOE, which codes for apolipoprotein E. There are three common polymorphisms of this gene. The e4 allele is an established risk factor for both cardiovascular disease and Alzheimer’s dementia. It may also influence breathing during sleep. The APOE locus on 19q is at the site of a linkage peak for AHI. Several studies have found an association between the presence of the e4 allele and OSA. Other studies, however, have not reproduced this finding, suggesting potential heterogeneity of effect by age or ethnicity.

Consequences of Sleep Apnea OSA is a risk factor for the development of a range of neurocognitive, cardiovascular, and metabolic abnormalities, including daytime sleepiness, memory and learning impairments, depressed mood, hypertension, heart failure, stroke, and insulin resistance. Most, if not all, of these disorders have been established to have a genetic basis. For example, the risk of excessive daytime sleepiness increases with the number of sleepy relatives. The heritability of sleepiness as measured by the Epworth Sleepiness Scale is approximately 40%. Work is only now beginning to indicate how the stressors of hypoxemia, hypercapnia, and recurrent arousal from OSA may interact with individual genetic makeup to affect the risk of developing complications. To date, the majority of work on this gene-by-environment interaction has focused on hypertension and cardiac disease. Two polymorphisms, one in the haptoglobin gene and one in the ACE gene, have been reported to modulate the risk of developing cardiovascular complications in the setting of OSA. Further work is required to confirm these preliminary findings. Clearly, the ability to identify genes that determine susceptibility to the adverse consequences of OSA will not only provide insight into the pathophysiology connecting OSA with these disorders but also provide a method of individualizing treatment recommendations for asymptomatic patients. See also: Obesity. Sleep Apnea: Overview; Adult; Children. Sleep Disorders: Central Apnea (Ondine’s Curse); Hypoventilation; Upper Airway Resistance Syndrome. Sudden Infant Death Syndrome.

Further Reading Bassiri AG and Guilleminault C (2000) Clinical features and evaluation of obstructive sleep apnea–hypopnea syndrome. In: Kryger MH, Roth T, and Dement WC (eds.) Principles and Practices of Sleep Medicine, 3rd edn., pp. 869–878. New York: Saunders. Palmer LJ and Redline S (2003) Genomic approaches to understanding obstructive sleep apnea. Respiratory Physiology & Neurobiology 135: 187–205. Redline S and Tishler PV (2000) The genetics of sleep apnea. Sleep Medicine Reviews 4: 583–602. Redline S, Tishler PV, and Strohl KP (2002) The genetics of the obstructive sleep apnea hypopnea syndrome. In: Pack AI (ed.) Sleep Apnea: Pathogenesis, Diagnosis, and Treatment, pp. 235– 264. New York: Dekker. Taheri S and Mignot E (2002) The genetics of sleep disorders. Lancet: Neurology 1: 242–250. Tankersley CG (2003) Genetic aspects of breathing: on interactions between hypercapnia and hypoxia. Respiratory Physiology & Neurobiology 135: 167–178. Weese-Mayer DE and Berry-Kravis EM (2004) Genetics of congential central hypoventilation: lessons from a seemingly orphan

SLEEP APNEA / Oral Appliances 67 disease. American Journal of Respiratory and Critical Care Medicine 170: 16–21. Weil JV (2003) Variation in human ventilatory control – genetic influence on the hypoxic ventilatory response. Respiratory Physiology & Neurobiology 135: 239–246.

variety of synonyms for OAs: rather than oral, they may be called intraoral, dental, or mandibular; similarly, rather than an appliance, they may be called a device, splint, or prosthesis.

Relevant Website

Appliance Type

www.ncbi.nlm.nih.gov – Obstructive sleep apnea. In Online Mendelian Inheritance in Man (OMIM).

Oral Appliances J A Fleetham, Vancouver, BC, Canada & 2006 Elsevier Ltd. All rights reserved.

Abstract Oral appliances (OAs) are prescribed for the treatment of snoring and mild/moderate obstructive sleep apnea–hypopnea (OSAH). In general, OAs increase the size of the upper airway by advancing either the mandible or the tongue. Mandibular advancement OAs are the most widely used type of OAs. These require at least eight teeth in each of the maxillary and mandibular arches, and the degree of mandibular advancement can be specified and subsequently adjusted. There is increasing evidence that OAs improve sleepiness, blood pressure, and indices of sleep disordered breathing. However, continuous positive airway pressure (CPAP) has been shown to be more effective than OAs in improving sleepiness, health status, and indices of sleep disordered breathing and remains the primary treatment for OSAH. Current guidelines recommend OAs for selected patients with mild OSAH and normal daytime alertness. Furthermore, OAs are the best alternative treatment for patients with OSAH who are unwilling or unable to comply with CPAP therapy. OA therapy may also be indicated as an adjuvant to CPAP when the patient is away from home or without electrical power. OA therapy should be supervised by both medical and dental specialists with a major interest in the management of sleep disordered breathing. They should monitor patients following initiation of OA therapy to allow for appliance adjustment and change of OSAH symptoms and severity.

There are currently a large number of different OAs available for the treatment of OSAH. In general, OAs increase the size of the upper airway by advancing either the mandible or the tongue. There are other minor design differences in the OAs currently available that may also impact on their success and treatment compliance. Mandibular advancement OAs are most widely used and utilize traditional dental techniques to attach the appliance to one or both dental arches (see Figures 1 and 2). Construction usually requires dental impressions, and fabrication by a dental laboratory. Some appliances are available in a prefabricated form that can be molded to the patient’s teeth in an office setting. Some appliances restrict mouth opening by means of clasps, whereas others allow relatively unhindered movement. More recently, OAs have been developed with an adjustable hinge that allows progressive advancement of the mandible after initial construction until the optimal mandibular position is achieved. The amount of anterior–posterior mandibular movement and the time interval in which this can be changed varies considerably between patients. The other major type of OA available is the tongue retainer that keeps the

Introduction Obstructive sleep apnea–hypopnea (OSAH) is a common syndrome that is characterized by recurrent episodes of partial or complete upper airway obstruction during sleep. Currently, the primary treatment for OSAH is continuous positive airway pressure (CPAP). However, some patients are unable to tolerate and comply with CPAP on a long-term basis. Oral appliances (OAs) have been used for many years to correct upper airway obstruction. OAs are now widely prescribed for the treatment of snoring and mild/moderate OSAH, both as primary therapy and as an alternative for patients who are unwilling or unable to tolerate CPAP. There are a

Figure 1 Lateral views of a Klearway mandibular advancement oral appliance. Reproduced with permission of Dr A Lowe, University of British Columbia, Vancouver, BC, Canada.

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also suggested a specific effect on the retropalatal oropharynx. Almost all of these upper airway imaging studies have been performed during wakefulness, and it is not known whether the same changes occur during sleep.

Effectiveness

Figure 2 Posterior view of a Klearway mandibular advancement oral appliance which shows the screw mechanism that connects the mandibular and maxillary components and enables gradual advancement of the mandible. Reproduced with permission of Dr A Lowe, University of British Columbia, Vancouver, BC, Canada.

tongue in an anterior position during sleep by means of negative pressure in a soft plastic bulb. It fits over both the mandibular and maxillary arches and has a flange that fits between the lips and teeth, keeping the appliance anterior in the mouth. This appliance was one of the first to be developed and is available in both fabricated and prefabricated forms. It can be used in edentulous patients and is the OA of choice for patients with no teeth, limited anterior–posterior mandibular movement or a large tongue.

Mechanism of Action The majority of OAs are designed to maintain the mandible and/or tongue in a protruded posture during sleep, thereby preventing upper airway obstruction. Proposed mechanisms of action of OAs include increased upper airway size, decreased upper airway collapsibility, activation of upper airway dilator muscles, and stabilization of mandibular posture. Several different upper airway imaging techniques have been used to assess changes in upper airway size and function with OAs in patients with OSAH. These imaging techniques include cephalometry, computed tomography, magnetic resonance imaging, and videoendoscopy. Voluntary mandibular and tongue protrusion has been shown to increase upper airway size and alter upper airway shape. Several studies have demonstrated an increase in the anteroposterior diameter of the upper airway following OA insertion. This increase was predominantly in the oropharynx and hypopharynx, but some studies have

The treatment of OSAH should be individualized for each patient based on their age, severity of symptoms, comorbid disease, and etiology of upper airway obstruction. Patient occupation may also be a consideration if they are employed in a high-risk occupation such as a lorry or bus driver in whom effective treatment of daytime sleepiness is a matter of some urgency. Until recently, the majority of the data concerning the efficacy of OAs in the treatment of OSAH were from uncontrolled case series studies which were subject to study design issues such as regression to the mean, and selection and reporting bias. A variety of prospective randomized trials have now been performed to evaluate the efficacy, side effects, compliance, and preference of OA treatment in patients with OSAH. There is increasing evidence that OAs improve sleepiness, blood pressure, and indices of sleep disordered breathing over an inactive control. Additional well-designed, large-scale randomized controlled trials comparing active and control OAs are required to determine which groups of patients are most likely to benefit from OA treatment, how these patients can be identified, how much benefit can be achieved and with what cost, side effects, and complications. There are at least seven randomized controlled trials that have compared the efficacy and side effects of OA and CPAP. Although both CPAP and OA led to improvements in sleepiness, health status, and indices of sleep disordered breathing, the magnitude of improvement was significantly more with CPAP. Furthermore, it takes longer to obtain optimal treatment with an OA than CPAP, which can be a significant issue in patients with excessive daytime sleepiness. More patients were unwilling or unable to use an OA than CPAP. However, patients who responded to both treatments preferred the use of an OA over CPAP. OA design has been proposed as an important determinant of treatment success, and there have been at least five prospective comparative studies evaluating different OA designs. There were varying degrees of improvements in indices of sleep disordered breathing and side effects with different OAs. These differences may be related to the degree of vertical opening and mandibular advancement achieved with each OA. Finally, there is one longitudinal parallel group study comparing the effectiveness of OA to

SLEEP APNEA / Oral Appliances 69

uvulopalatopharyngoplasty in patients with mild to moderate OSAH over 4 years, which suggests that an OA was more effective than uvulopalatopharyngoplasty in improving indices of sleep disordered breathing. However, the significance of this finding is questionable, as there is no definitive evidence of the effectiveness of this type of corrective upper airway surgery. OA therapy may also be indicated as an adjuvant to nasal CPAP when the patient is away from home or without electrical power. OAs have also been used as combination therapy in patients who have had an unsuccessful response to uvulopalatopharyngoplasty. Adenotonsillectomy is the most common treatment for OSAH in children. There are some reports which indicate that OAs are effective and well tolerated in children with OSAH associated with oral malocclusion.

Predictors of Treatment Success The utility of any treatment recommendation for OA based on clinical features, OSAH severity, or upper airway anatomy requires prospective validation. It has been suggested that younger, less obese patients with OSAH occurring predominantly in the supine position may be more likely to obtain a successful response with an OA. Treatment success may be inversely related to pretreatment severity, but this relationship may just be a function of the definition of treatment success. Several upper airway skeletal and soft tissue measurements made from pretreatment lateral cephalometry have been shown to be associated with treatment success. These include a smaller or receding mandible, and small soft palate and tongue. Upper airway fluoroscopy has also been proposed as a technique to guide successful OA therapy. A hypopharyngeal site of obstruction may be associated with a better treatment outcome. However, there is considerable overlap between good and poor treatment response with all these variables.

Compliance Only self-reported treatment compliance data are available on OA therapy. Similar to CPAP masks, some patients remove their OA during sleep. Selfreported treatment compliance has been reported as high as 96% patients using OA on more than 75% nights, and 80% patients used OA for longer than 75% of each night. However, previous experience with nasal CPAP suggests that patient reports tend to overestimate actual use. Attempts to develop a covert OA compliance monitor have been fraught with technological difficulties and at present no objective OA compliance data are available.

Contraindications, Side Effects, and Cost Mandibular advancement OAs require at least eight teeth in each of the maxillary and mandibular arches. Furthermore, the patients should be able to advance their mandible by at least 5 mm without discomfort. Temporomandibular joint disease, bruxism, and advanced periodontal disease are all relative contraindications to mandibular advancement OA treatment. Excessive salivation, mouth dryness, and discomfort in the gums, teeth, or jaw are common side effects in the first month of OA therapy, but usually resolve with time. Over the long term, temporomandibular joint dysfunction and dental crown damage appear uncommon, but tooth movement can occur causing change in dental occlusion. OA adjustment can decrease side effects by reducing pressure on the anterior teeth and excessive mandibular advancement. The cost of OA therapy varies depending on the type of OA used and the extent and expertise of the dental supervision. Costs can equal or exceed those associated with nasal CPAP therapy when the costs of follow-up visits to adjust the OA are included. OAs usually remain effective for at least 5 years, but they can break and require either repair or replacement. Despite optimal adjustment and compliance, OAs may be ineffective or in some patients can increase OSAH severity. Consequently, it is important to follow these patients closely, especially if they have decreased alertness or comorbid disease.

Treatment Guidelines A Cochrane systematic review performed a variety of meta-analyses on data from 11 randomized controlled trials involving 488 patients available up to 2002 and found that there was insufficient evidence to recommend the use of OA as first-choice therapy for OSAH. It recommended that OA therapy should be restricted to patients with OSAH who are unwilling or unable to comply with CPAP therapy until there is more definitive evidence on their effectiveness. The Scottish Intercollegiate Guideline Network has recently published guidelines for the management of OSAH which have been endorsed by the British Thoracic Society. They recommended that OAs are an appropriate therapy for snorers and for patients with mild OSAH with normal daytime alertness and an alternative therapy for patients who are unable to tolerate CPAP. They also indicated that OA use should be monitored following initiation of therapy to allow for appliance adjustment and assessment of OSAH control and symptoms. Both the American Academy of Sleep Medicine and Academy of Dental Sleep Medicine have developed practice guidelines

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for OA therapy. These guidelines emphasize the importance of combined medical and dental evaluation prior to OA treatment. They recommended that OA therapy should be supervised by both medical and dental specialists with a major interest in the management of sleep disordered breathing. Patients should not have major periodontal disease and all dental restorations should be complete prior to OA therapy. Patients treated with OA require long-term dental follow-up to ensure adequate OA retention and comfort. OAs may require adjustment following any new dental restorations. Finally, it is important to monitor patients following initiation of OA therapy to allow for appliance adjustment and change of OSAH symptoms and severity. See also: Sleep Apnea: Overview; Drug Treatments; Surgery for Sleep Apnea. Sleep Disorders: Upper Airway Resistance Syndrome.

Millman RP, Rosenberg CL, Carlisle CC, et al. (1998) The efficacy of oral appliances in the treatment of persistent sleep apnea after uvulopalatopharyngoplasty. Chest 113: 992–996. Ryan CF, Love LL, Peat D, Fleetham JA, and Lowe AA (1999) Mandibular advancement oral appliance therapy for obstructive sleep apnea: effect on awake calibre of the velopharynx. Thorax 54: 972–977. Schmidt-Nowara W, Lowe A, Wiegand L, et al. (1995) Oral appliances for the treatment of snoring and obstructive sleep apnea: a review. Sleep 18: 501–510.

Relevant Website http://www.sign.ac.uk – Scottish Intercollegiate Guideline Network Management of obstructive sleep apnoea/hypopnoea syndrome in adults.

Surgery for Sleep Apnea P Le´vy, J L Pe´pin, G Bettega, and R Tamisier, University Hospital, Grenoble, France

Further Reading ASDA Standards of Practice Committee (1995) Practice parameters for the treatment of snoring and obstructive sleep apnea with oral appliances. Sleep 18: 511–513. Engleman HM, McDonald JP, Graham D, et al. (2002) Randomized crossover trial of two treatments for sleep apnea/hypopnea syndrome: continuous positive airway pressure and mandibular repositioning splint. American Journal of Respiratory and Critical Care Medicine 166(6): 855–859. Ferguson KA (2001) Oral appliance therapy for obstructive sleep apnea. Finally evidence you can sink your teeth into. American Journal of Respiratory and Critical Care Medicine 163: 1294– 1295. Ferguson KA and Lowe AA (2005) Oral appliances for sleep disordered breathing. In: Kryger MH, Roth T, and Dement WC (eds.) Principles and Practice of Sleep Medicine, 4th edn., pp. 1098–1108. Philadelphia: Saunders. Ferguson KA, Ono T, Lowe AA, et al. (1997) A short term controlled trial of an adjustable oral appliance for the treatment of mild to moderate obstructive sleep apnoea. Thorax 52: 362–368. Fleetham JA (2002) Oral devices for snoring and obstructive sleep apnea. In: McNicholas WT and Phillipson EA (eds.) Breathing Disorders in Sleep, pp. 157–163. London: Saunders. Gotsopoulos H, Chen C, Qian J, Mehta A, and Cistulli PA (2002) Oral appliance therapy improves symptoms in obstructive sleep apnoea syndrome. A randomised controlled trial. American Journal of Respiratory and Critical Care Medicine 166: 743– 748. Gotsopoulos H, Kelly JJ, and Cistulli PA (2004) Oral appliance therapy reduces blood pressure in obstructive sleep apnea. A randomized, controlled trial. Sleep 27(5): 934–941. Lim JC, Lasserson T, Fleetham JA, and Wright J (2003) Oral appliances for obstructive sleep apnoea. Cochrane Database of Systematic Reviews (4): CD004435. Mehta A, Qian J, Petocz P, Darendeliler A, and Cistulli PA (2001) A randomized, controlled study of a mandibular advancement splint for obstructive sleep apnea. American Journal of Respiratory and Critical Care Medicine 163: 1457–1461. Millman RP and Rosenberg CL (2002) Are oral appliances a substitute for nasal positive pressure? Thorax 57: 283–284.

& 2006 Elsevier Ltd. All rights reserved.

Abstract Surgical treatment of severe obstructive sleep apnea syndrome (OSAS) is currently generally considered as second line therapy after the reference treatment, nasal continuous positive airway pressure (CPAP). In upper airway (UA) resistance syndrome (UARS) and mild and moderate OSAS subjects, surgery is progressively being replaced by oral appliance therapy. This article discusses the rationale for surgical therapy. The surgical procedures for uvulopalatopharyngoplasty (UPPP), nasal surgery, tongue operations, and maxillofacial surgery are described. Young and lean subjects are probably the best candidates for surgery. The methods that appear at this time to be the most clinically useful to detect UA narrowing or collapse sites are cephalometry and fiberoptic endoscopy. However, satisfactory selection of good surgical candidates remains elusive. UPPP treatment response rate in OSAS varies from 5% to 46% depending on the presence or absence of retrolingual narrowing. Maxillomandibular advancement osteotomy (MMO) is an efficient surgical technique for the treatment of severe sleep apnea, particularly in young motivated and nonobese patients. Acute UA obstructions are the more frequent life-threatening complication occurring in the early postoperative procedure. These complications can be avoided by standardizing anesthetic procedures and perioperative protocols. In children obstructive sleep apnea is an adequate indication for adenotonsillectomy. There is need for regular re-evaluation of surgical results and adjustment of treatment at regular time intervals.

Introduction Upper airway (UA) instability is the final common pathway leading to recurrent episodes of UA obstruction during sleep (i.e., from snoring to apnea). UA instability results from abnormal airway structure and/or function. Thus, treatment is designed to

SLEEP APNEA / Surgery for Sleep Apnea 71

enlarge and/or stabilize the UA. In this context, several surgical options for the management of obstructive sleep apnea syndrome (OSAS) have been proposed. The purpose of this short article is: 1. to describe the rationale for surgical therapy; 2. to review results and complications of such treatments; and 3. to summarize rule management and follow-up.

Rationale for Surgical Therapy of OSAS

size, and/or redundant pharyngeal soft tissues. Common modifications are nasal abnormalities, retrognathia, and inferior position of the hyoid bone (Figures 2 and 3). The tongue and pharyngeal soft tissues are usually larger than in healthy subjects and these abnormalities are worsened by fat deposition and edema induced by vibration and/or repeated UA collapse. Finally, UA size is also dependent upon lung volume; a low lung volume reduces UA size in obese subjects. UA anatomical abnormalities explain the major part of the variance in the apnea þ hypopnea index (AHI) only in young and lean subjects. In obese and

The patency of the UA is critically dependent upon size, shape, and function of the pharyngeal airway (Figure 1).

Non-snoring patient

Abnormal UA Structure in OSAS Patients

Snoring patient Deviated septum

There is a susceptibility to UA narrowing during sleep as a result of anatomical factors such as craniofacial skeletal abnormalities, increase in tongue

Hypertrophic turbinates Long, low, thick palate Vertical pharyngeal fold

R

Large tonsil O H

Higher tongue base

PL

Figure 1 Anatomical narrowing of the UA in snorers and OSAS patients. Pharyngeal segmentation used: R, rhinopharynx; O, oropharynx; H, hypopharynx; PL, pharyngolarynx.

(a)

Figure 3 The more frequent modifications in UA structure in a snoring versus a non-snoring patient. Adapted from Krespi YP, Pearlman SJ, and Keidar A (1994) Laser-assisted uvula-palatoplasty for snoring. Journal of Otolaryngology 23: 328–334.

(b)

Figure 2 (a) Normal UA. (b) Apneic patient: note the increase in soft palate length and width. The posterior airway space is reduced. Retrognathism is often observed and the distance between the hyoid bone and the mandibular plane is increased.

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SLEEP APNEA / Surgery for Sleep Apnea

older patients high UA collapsibility, ventilatory instability, fragmented sleep, and UA muscle function abnormalities are predominant. The site of UA narrowing or collapse determine surgical technique Surgical intervention for OSAS includes several procedures, each of which is designed to increase the UA patency. The procedures are more specifically orientated to skeletal or soft tissue abnormalities. Thus, the identification of collapsing or stenotic site(s) seems critical in selecting the appropriate procedure. When a retrolingual narrowing or collapse is demonstrated, uvulopalatopharyngoplasty (UPPP) is contraindicated as only a 5% success rate is obtained in OSAS. In this situation enlarging the retrolingual segment must be considered. Although several methods have been proposed for detecting the UA collapse site, so far they have had limited clinical application. Cephalometry and fiber optic endoscopy are simple, clinically useful techniques and are the most widely available. Abnormal Airway Function in OSAS Patients

Most patients have obstruction in more than one anatomical region due to an abnormal UA collapsibility. Abnormal pharyngeal dilator muscle function and/or increased compliance of pharyngeal walls leads to increased pharyngeal collapsibility. During wakefulness, OSAS patients exhibit an augmented dilator muscle activity to maintain UA patency (i.e., neuromuscular compensatory mechanism). A decrease in muscle tone during sleep leads to UA collapse. Pharyngeal collapsibility can be estimated by pharyngeal critical pressure, also termed ‘Pcrit’. When the pressure surrounding the pharyngeal airway exceeds Pcrit, the airway will collapse. Pcrit is negative (producing an open airway) by a clear margin in normal individuals (around
15 cmH2O) and positive in patients with severe OSAS, which causes collapse of the pharynx. Shwartz et al. have demonstrated that the response to UPPP is determined by the magnitude of a decrease in pharyngeal collapsibility, that is, by the fall (more negative direction) in UA Pcrit. There is, however, another dimension to this issue. A reflex to negative pharyngeal pressure is one major component of pharyngeal collapsibility and this reflex sensation is impaired in OSAS. Impaired reflexes could contribute to an impaired Pcrit. This mechanism could modulate the response to surgery as the more UA sensation is impaired the less surgery efficacy would be expected. In summary, the characteristics of UA structure and function in OSAS allow us to propose the

following three issues to consider when looking at surgery as an option for the treatment of OSAS: 1. UA abnormalities are predictive of sleep disordered breathing severity only in young and lean subjects. These patients may be candidates for surgery. 2. A minimally invasive attempt should be made to detect the UA collapse site, by cephalometry and/ or fiber optic endoscopy. However, satisfactory selection of good surgical candidates remains elusive with this technique. 3. The issue of pharyngeal collapsibility and sensation before and after surgery is probably of importance. A high Pcrit and severe impairment of UA sensation may be relevant for predictive UA surgery results in OSAS.

Surgery, Results and Complications Nasal Surgery

The goal is to lower nasal resistances to avoid pharyngeal collapse by decreasing the inspiratory pressure gradient. Nasal-septal reconstruction, cauterization, polypectomy, and outfracture of turbinates are used alone or in combination. The nasal airway must be free to allow nasal continuous positive airway pressure (CPAP) use immediately after surgery. Few studies have examined the efficacy of these procedures alone; all included small numbers and reported partial or no benefit. A study by Se´ries et al. of patients with moderate OSAS undergoing nasal surgery showed a predictive value of normal UA dimensions at any level (assessed by cephalometry) for nasal surgery success in treating OSAS. This identifies patients in whom nasal surgery alone may be indicated in association with other surgical techniques. Moreover, improving nasal permeability may decrease nasal CPAP level requested and potentially improve tolerance. UPPP and Laser-Assisted Uvulopalatoplasty with or without Tonsillectomy

The objective of these surgical techniques is to enlarge the oropharyngeal airway and to reduce its collapsibility. The steps of UPPP are well described. Laser-assisted uvulopalatoplasty (LAUP) can be performed under local anesthesia as an outpatient surgical procedure, involving one to seven laser sessions. Sher et al. summarized that UPPP results in a metaanalysis. In OSAS patients, UPPP should be restricted to patients with moderate disease and suspected retropalatal narrowing. With these indications the success rate for surgery was 46% of patients. After the

SLEEP APNEA / Surgery for Sleep Apnea 73

procedure 11 of 101 and 14 of 135 subjects developed acute obstructions of the UA that required management by noninvasive ventilation using a mask, orotracheal tube placement, or tracheotomy. Hemorrhage was reported in 14% of procedures and required vessel ligature under general anesthesia in 14 patients. Only a few studies have reported on the use of LAUP for the treatment of OSAS. It should be pointed out that 2–23% of patients exhibited a worse AHI after undergoing LAUP. Postoperative pain usually occurs for 1–3 weeks after LAUP. Velopharyngeal insufficiency seems less frequent with LAUP than after classical UPPP, but conversely the risk of postoperative velopharyngeal stenosis appears to be higher. To date, in upper airway resistance syndrome (UARS) and mild to moderate OSAS patients the tendency is to use oral appliances rather than carry out surgery. In the absence of results from therapeutic trials comparing the use of surgery with use of oral appliances, it is the authors’ opinion that these surgical techniques should not be carried out in potential nasal CPAP patients, as by increasing mouth leaks UPPP may compromise nasal CPAP therapy and reduce the maximal level of pressure that can be tolerated. Tongue Operations

These procedures require a preoperative tracheotomy. Linguoplasty with midline laser glossectomy (MLG) consists of laser extirpation of a rectangular strip of the posterior half of the tongue. Lingualplasty differs from MLG in that additional tongue

(a)

(b)
1

resection is done posteriorly and laterally to the portion excised in MLG. Glossopexia is a combination of partial tongue resection and anterior suspension of the tongue. Finally, an isolated tongue base resection with hyoidoplasty by cervical approach has been proposed. A reduction by at least 50% of the AHI (o20 h
1) was obtained postoperatively in 42% of cases for MLG while a 77–80% reduction was achieved for lingualplasty. Postoperative bleeding was reported in less than 20% of cases, some requiring general anesthesia. Moderate odynophagia and dysphagia were commonly present for 2–3 weeks postsurgery. Forty-seven percent (7/15) of patients were considered to be cured after surgery with a postoperative AHI of o10 h
1. Maxillofacial Surgery

The Riley et al. designed step-by-step surgery tailored to the specific anatomical abnormalities encountered in each patient, while other groups proceeded directly to maxillomandibular advancement osteotomy (MMO) (Figure 4). Genioglossal advancement and hyoid myotomy and suspension (GAHM) with or without UPPP: phase 1 surgery Riley et al. reported a success rate (reduction of AHI by more than 50% to less than 20 h
1) of 61%. Success rate was mitigated in patient with severe preoperative AHI, BMI, and severe mandibular deficiency. However, Riley et al.’s results have not been reproduced. Other studies using phase 1 surgery have been unable to achieve success rates of more than 40%.

(c)

Figure 4 (a) Cephalometry after UPPP (AHI ¼ 80 h of sleep). (b) Cephalometry after GAHM: note the rectangular inferior sagittal osteotomy of the mandible is visible. The hyoid bone is placed anteriorly and inferiorly by attachment to the superior border of the thyroid cartilage. Note the small enlargement of the UA. The patient was not cured. (c) Cephalometry after MMO, bilateral retromolar sagittal split osteotomy, and maxillary advancement by Le Fort I osteotomy. Fixation in the new position is achieved by miniplates in the maxillar and bicortical mini-screws in the mandible (see on the cephalometric picture (c)). Note the large increase in UA size. There was a normalization of the AHI after this procedure.

74

SLEEP APNEA / Surgery for Sleep Apnea

Maxillomandibular advancement osteotomy Riley et al. reported on 91 patients who had phase 2 surgery either following unsuccessful phase 1 surgery or directly in subjects with skeletal deformities. The success rate was 97%. In 37 of 38 patients treated by 10 mm MMO, Hochban et al. found a postoperative AHI o10 h
1. This was achieved by MMO without secondary refinements in all but two of the patients. MMO is an effective surgical technique for the treatment of severe sleep apnea. The MMO has been reproduced in other centers in order to treat sleep apnea patient and appear to be sustained on a longterm basis. The rationale for the continued use of phase 1 surgery is based on the risk-benefit ratio. MMO with its potential morbidity may not be justified for cases of simple snoring, UARS, and even moderate sleep apnea syndrome. Local pain and nasal fluid reflux are the usual discomforts seen with UPPP in the immediate postoperative days. All patients experience transient chin nerve paraesthesia. Mandibular fracture of the anterior mandibular fragment is occasionally observed (2%), as is damage of the inferior alveolar nerve during its dissection. Mandibular distraction Mandibular distraction is a surgical procedure planned bilaterally in order to lengthen the mandibular body and also increase UA volume. It is one possible approach for treating OSAS in both adults and children. In a report of successful mandibular advancement in 12 children, the average increase in mandibular length was 28.24% and of UA volume was 71.92%. The apnea–hypopnea index fell from 21.374.7 to 1.470.8; oxygen saturation and OSAS symptoms were improved. In adults, mandibular distraction has been proposed and there are some encouraging results in mild OSAS with abnormal mandibular length. Tracheostomy

Tracheotomy was the first effective surgical treatment for OSAS; it involves bypassing the collapsible airways. Patients demonstrating a very severe OSAS with a high risk of associated cardiovascular morbidity or mortality and who do not tolerate nasal CPAP may be candidates for tracheostomy. Radiofrequency Volumetric Reduction of the Palate

Radiofrequency (RF) volumetric reduction is carried out under local anesthesia in an outpatient setting and is designed to shrink redundant tissue in the UA, including turbinates, uvula, palate, and base of

tongue. Powell et al. were the first to report on RF treatment of the palate in 22 subjects with moderate OSAS or UARS. The mean number of visits to the clinic for RF treatment was 3.6 per patient. Subjective snoring scores and Epworth sleepiness scale values improved after treatment. A mean reduction of the soft palate length of 5.5 mm was demonstrated by cephalometry. The width of the soft palate did not change significantly. A worsening trend in AHI, explained by edema, was noted 48–72 h after RF. Recently, several studies have been published showing a 50% reduction in the AHI on average, which was of benefit to younger and lean patients. This procedure seems to induce a decrease in UA compliance rather than an increase in UA volume. Tonsillectomy in Children

Adenoidectomy is a common procedure in children in Western countries and this is classically indicated in children exhibiting OSAS with adenotonsillar hypertrophy. Cognitive function and sleep structure have been found to be improved 4.071.0 month after tonsillectomy. However, Van Staaij et al., in a randomized study tonsillectomy versus watchful waiting, did not find any differences after a 24month follow-up. An assessment of recovery from the disease is needed after surgery, as children may not be completely cured by the procedure.

Obstructive Sleep Apnea Surgery and Follow-Up: Risk Management On the basis of a US national survey, Fairbanks et al. reported 16 deaths and seven near-deaths occurring in the early postoperative period. Post UPPP acute UA obstruction, the more frequently occurring life-threatening complication, is explained by the association between pharmacological sedation and postsurgical edema. This can be avoided by standardizing anesthetic procedures and perioperative protocols. Thus, if the patient has over 20 events per hour, nasal CPAP should be started at least 2 weeks before surgery to stabilize cardiovascular sequelae of OSAS and reduce UA edema. The patients should be questioned regarding previous intubation difficulties and fiber optic intubation should be performed if necessary. After surgery, the operating room staff should carry out extubation in patients who are fully awake. Patients should then be transferred to the intensive care unit (ICU) and immediately placed under nasal CPAP. Patients should have previously been made aware of the fact that they will be kept in ICU for 24 h. Sedative and opioid premedications should not

SLEEP APNEA / Surgery for Sleep Apnea 75

be used and intra- and postoperative use of opioids should be limited or avoided. All anesthetic drugs should be administered by cautious titration to desirable effect, preferably using short-acting drugs. When possible nonopioid analgesics or local anesthetics should be used for postoperative analgesia. Nocturnal oximetry should be used to monitor patients throughout the hospital stay. Patients must continue to use nasal CPAP until a polysomnographic recording has verified that surgery has been successful. Patients should undergo a follow-up evaluation to assess the presence of residual disease. This evaluation includes an objective measure of the presence and severity of obstructive sleep apnea and of the sleep disruption. Insufficient evidence exists as to how long any immediate postoperative improvement is likely to be maintained. Cases of relapse after successful operations have been described; therefore, long-term follow-up is recommended for surgically treated patients. Thus, we would recommend simplified screening at 6-month intervals with polysomnography if indicated.

Acknowledgments This research was supported by the scientific council of AGIRa`dom and the Comares. See also: Sleep Apnea: Continuous Positive Airway Pressure Therapy; Oral Appliances. Sleep Disorders: Upper Airway Resistance Syndrome.

Further Reading Bettega G, Pepin JL, Veale D, et al. (2000) Obstructive sleep apnea syndrome. Fifty-one consecutive patients treated by maxillofacial surgery. American Journal of Respiratory and Critical Care Medicine 162: 641–649. Boushra NN (1996) Anaesthetic management of patients with sleep apnoea syndrome. Canadian Journal of Anaesthesiology 43: 599–616. Carrera M, Barbe F, Sauleda J, et al. (1999) Patients with obstructive sleep apnea exhibit genioglossus dysfunction that is normalized after treatment with continuous positive airway pressure. American Journal of Respiratory and Critical Care Medicine 159: 1960–1966. Conradt R, Hochban W, Brandenburg U, Heitmann J, and Peter JH (1997) Long-term follow-up after surgical treatment of obstructive sleep apnoea by maxillomandibular advancement. European Respiratory Journal 10: 123–128. Esclamado RM, Glenn MG, McCulloch TM, and Cummings CW (1989) Perioperative complications and risk factors in the surgical treatment of obstructive sleep apnea syndrome. Laryngoscope 99: 1125–1129. Fairbanks DN (1990) Uvulopalatopharyngoplasty complications and avoidance strategies. Otolaryngology and Head and Neck Surgery 102: 239–245.

Gotsopoulos H, Kelly JJ, and Cistulli PA (2004) Oral appliance therapy reduces blood pressure in obstructive sleep apnea: a randomized, controlled trial. Sleep 27: 934–941. Haavisto L and Suonpaa J (1994) Complications of uvulopalatopharyngoplasty. Clinical Otolaryngology 19: 243–247. Hochban W, Conradt R, Brandenburg U, Heitmann J, and Peter JH (1997) Surgical maxillofacial treatment of obstructive sleep apnea. Plastic Reconstructive Surgery 99: 619–626, discussion 617–618 Johnson NT and Chinn J (1994) Uvulopalatopharyngoplasty and inferior sagittal mandibular osteotomy with genioglossus advancement for treatment of obstructive sleep apnea. Chest 105: 278–283. Krespi YP, Pearlman SJ, and Keidar A (1994) Laser-assisted uvulapalatoplasty for snoring. Journal of Otolaryngology 23: 328–334. Larsson LH, Carlsson-Nordlander B, and Svanborg E (1994) Fouryear follow-up after uvulopalatopharyngoplasty in 50 unselected patients with obstructive sleep apnea syndrome. Laryngoscope 104: 1362–1368. Marcus CL, Katz ES, Lutz J, et al. (2005) Upper airway dynamic responses in children with the obstructive sleep apnea syndrome. Pediatric Research 57: 99–107. Mitchell RB and Kelly J (2004) Outcome of adenotonsillectomy for severe obstructive sleep apnea in children. International Journal of Pediatric Otorhinolaryngology 68: 1375–1379. Montgomery-Downs HE, Crabtree VM, and Gozal D (2005) Cognition, sleep and respiration in at-risk children treated for obstructive sleep apnoea. European Respiratory Journal 25: 336–342. Mortimore IL, Bradley PA, Murray JA, and Douglas NJ (1996) Uvulopalatopharyngoplasty may compromise nasal CPAP therapy in sleep apnea syndrome. American Journal of Respiratory and Critical Care Medicine 154: 1759–1762. Pepin JL, Leger P, Veale D, et al. (1995) Side effects of nasal continuous positive airway pressure in sleep apnea syndrome. Study of 193 patients in two French sleep centers. Chest 107: 375–381. Pepin JL, Veale D, Mayer P, et al. (1996) Critical analysis of the results of surgery in the treatment of snoring, upper airway resistance syndrome (UARS), and obstructive sleep apnea (OSA). Sleep 19: S90–S100. Rachmiel A, Aizenbud D, Pillar G, Srouji S, and Peled M (2005) Bilateral mandibular distraction for patients with compromised airway analyzed by three-dimensional CT. International Journal of Oral and Maxillofacial Surgery 34: 9–18. Riley RW, Powell NB, and Guilleminault C (1990) Maxillofacial surgery and nasal CPAP. A comparison of treatment for obstructive sleep apnea syndrome. Chest 98: 1421–1425. Schwartz AR, Schubert N, Rothman W, et al. (1992) Effect of uvulopalatopharyngoplasty on upper airway collapsibility in obstructive sleep apnea. American Review of Respiratory Diseases 145: 527–532. Series F, St Pierre S, and Carrier G (1993) Surgical correction of nasal obstruction in the treatment of mild sleep apnoea: importance of cephalometry in predicting outcome. Thorax 48: 360– 363. Sher AE, Schechtman KB, and Piccirillo JF (1996) The efficacy of surgical modifications of the upper airway in adults with obstructive sleep apnea syndrome. Sleep 19: 156–177. van Staaij BK, van den Akker EH, Rovers MM, et al. (2004) Effectiveness of adenotonsillectomy in children with mild symptoms of throat infections or adenotonsillar hypertrophy: open, randomised controlled trial. British Medical Journal 329: 651.

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SLEEP DISORDERS Contents

Overview Central Apnea (Ondine’s Curse) Hypoventilation Upper Airway Resistance Syndrome

Overview C D Hanning, Leicester General Hospital, Leicester, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Sleep is a universal phenomenon which is influenced by or influences many diseases, including most respiratory disorders. Two distinct sleep states are recognized, slow wave and rapid eye movement sleep, characterized by different encephalographic waveforms, eye movements, and muscle tone. Respiratory drive, control, and mechanics are changed during sleep such that most respiratory conditions are worsened by sleep.

Introduction Wakefulness and sleep, or an activity/inactivity cycle, are universal phenomena of all living organisms, regulated by one or more clock genes. In higher animals, including mammals, sleep is more structured and includes both slow wave (SWS) and rapid eye movement (REM) sleep. The physiological changes associated with sleep, particularly involving the respiratory system, are major factors in disease processes. It is probably fair to state that every respiratory disease is affected by sleep or itself disrupts sleep. Many respiratory diseases, particularly those with associated hypoxemia, are at their worst during sleep, particularly REM sleep, and some, for example obstructive sleep apnea, occur only during sleep but have effects on wakefulness. An understanding of sleep and its physiological changes is thus essential for the management of respiratory disease.

Sleep Typically in an adult human at sleep onset, after initial drowsiness, SWS begins and deepens until, after about 20–40 min, deep SWS (stage 4) is achieved. SWS is characterized by progressive

slowing of the electroencephalogram (EEG) frequency and increase in its amplitude from light sleep (stage 1) to deep (stage 4). Slow rolling eye movements often accompany stage 1 sleep. The appearance of specific waveforms such as sleep spindles and K complexes mark the transition into stage 2 SWS which many regard as the first true sleep stage. EMG activity is decreased with respect to wakefulness but movement is possible and most of the parasomnias (sleepwalking, night terrors, etc.) occur in SWS. Arousal from sleep by external stimuli becomes progressively more difficult as SWS deepens. Although cortical neurons are firing synchronously, it is a mistake to imagine that the cortex is inactive in SWS. Arousal from auditory stimuli is much faster if the sound is recognizable, for example one’s name or the cry of a child. About 40–60 min after sleep onset, REM sleep succeeds SWS. Under normal circumstances, REM sleep is only entered through SWS. However, in patients with narcolepsy and in subjects deprived of REM sleep, REM sleep may be entered from wakefulness (sleep onset REM or SOREM). REM sleep is characterized by a high-frequency, low-amplitude EEG which is very similar to that of wakefulness. Electromyogram (EMG) activity is low, reflecting the hyperpolarization of the descending alpha motor neurons in the pons. There is effective paralysis of skeletal muscles associated with maintenance of posture, sparing the diaphragm and upper airway muscles but reducing activity in the accessory muscles of respiration (intercostal and strap muscles). Characteristic rapid movements of the eyes also occur. A brief period of wakefulness may follow the end of the REM period. The pattern of SWS followed by REM sleep and brief wakefulness is termed a sleep cycle and lasts about 90 min. The first two cycles generally contain the bulk of the deep SWS taken in a night. As the night progresses, the cycles become composed predominantly of light (stage 2) SWS and REM sleep.

SLEEP DISORDERS / Overview 77 Hypnogram Wake REM

Stages of sleep

1 2 3 4

1

2

3

4 5 Hours of sleep

6

7

8

Figure 1 Hypnogram recording the pattern of the stages of sleep during a night’s sleep in a young adult. Reproduced by kind permission of Dr M Mitler.

The architecture of a night’s sleep is usually displayed as a hypnogram (Figure 1).

Effects of Sleep on Respiratory Physiology The onset of sleep is accompanied by a reduction in minute ventilation with a concomitant small increase in PaCO2 (0.5 kPa, 3 mmHg) and reduction in arterial oxygenation (SaO2 ) of 1–2%. In the transition from wakefulness to sleep, the respiratory rhythm is unstable with short (o10 s) pauses and becomes more regular with the onset of stage 2 SWS. The pattern of breathing is less regular in REM sleep and the sleep stage can often be inferred from the respiratory record. The hypercarbic ventilatory response decreases in SWS and, to a greater degree, in REM sleep. Hypoxic ventilatory drive is also decreased in SWS and is almost absent in REM sleep. Arousal responses to airway obstruction are similarly impaired in deep SWS and REM sleep. During REM sleep the reduction in tone and activity in those muscles which stabilize the ribcage and assist in respiration results in changes in the distribution of ventilation within the lungs and a reduc’ ’ to perfusion (Q) tion in the ratio of ventilation (V) ’ particularly in subjects with impaired ribcage ’ Q), (V= mechanics such as those with kyphoscoliosis or emphysema, resulting in hypoxemia. The overall effect of these changes in ventilatory drive, arousal responses, and lung mechanics is that the effects of respiratory disease on oxygenation are at their greatest during sleep and particularly REM sleep. This is particularly seen in patients with chronic lung disease (e.g., Chronic Obstructive Pulmonary Disease: Overview) and, of course, obstructive sleep apnea (Sleep Apnea: Overview).

Sleep Disorders Sleep disorders are themselves common as are complaints of poor sleep. There are few psychiatric disorders which do not have some effect on sleep. The same can be said for a wide variety of physical disorders including asthma and chronic lung disease with associated cough and dyspnea. There is an interaction between chronic pain, from any cause, and sleep with pain worsening sleep and vice versa. A useful, clinical classification of disorders of sleep is into disorders of initiating and maintaining sleep (DIMS) and disorders of excessive sleepiness (DOES), which mirrors the usual clinical presentations of ‘difficulty getting to sleep or staying asleep’ and ‘falling asleep in inappropriate times and places’. DIMS includes insomnia and delayed sleep phase syndrome (DSPS) and advanced sleep phase syndrome (ASPS) while DOES includes conditions such as obstructive sleep apnea (OSAs), restless legs syndrome (RLS, Ekbom’s syndrome), and narcolepsy. Parasomnias are those conditions which are associated with sleep, the ‘things that go bump in the night’. They are subdivided into arousal disorders (sleepwalking and sleep terrors), sleep–wake transition disorders (rhythmic movement disorders, hypnic jerks, and sleeptalking) and REM sleep-related phenomena (nightmares, sleep paralysis, and REM sleep-behavior disorder). Disorders of sleep have been formally classified by the International Classification of Sleep Disorders (ICSD) into four categories: dyssomnias, parasomnias, those associated with medical and/or psychiatric disease, and a final group of proposed disorders whose existence as distinct clinical entities has been questioned. Dyssomnias are those conditions where sleep is disrupted or ineffective and is further divided

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SLEEP DISORDERS / Overview

into intrinsic conditions, including central (Sleep Disorders: Central Apnea (Ondine’s Curse)) and obstructive sleep apnea (Sleep Apnea: Overview), periodic limb movements, insomnia, and narcolepsy, extrinsic conditions which include altitude insomnia and hypnotic dependent disorders, and circadian conditions which include DSPS and ASPS, jet lag, and shift-work sleep disorders. The advantage of the ICSD is that it classifies disorders from a sleep mechanism perspective but it results in disorders with widely differing symptoms, such as insomnia and narcolepsy, being classified together.

Diagnosis and Assessment of Sleep Disorders The purpose of sleep is to enable wakefulness and vice versa. Any assessment of sleep thus necessarily requires assessment of wakefulness. The standard assessment of sleep is generally regarded as polysomnography (PSG). This requires the continuous recording, throughout an entire night’s sleep, of one or more EEG leads, a measure of muscle tone by EMG and eye movements by electrooculognum (EOG). Particularly if OSA is suspected, heart rate, chest and abdominal movement, airflow, and oxyhemoglobin saturation are recorded as well. Video recording using infrared-sensitive cameras is extremely useful too. Unless the sleep study is designed to differentiate between a parasomnia and sleep-related epilepsy, one or two EEG leads are generally deemed sufficient to allow the different sleep stages to be distinguished. Typically, C3 or C4 vertex to the contralateral A1 mastoid leads of the 10–20 EEG lead configuration are chosen which give an overall display of cortical activity sufficient for sleep staging. Traditionally, sleep staging has been done with a set of agreed rules (Rechtstaffen and Kales) based upon EEG morphology, frequency, and amplitude, together with EMG and EOG changes, which allocates each 30 s epoch of recording into one of the distinct stages discussed above, wakefulness or movement. The disadvantage of this method of analysis, which remains widely accepted, is that the epoch length may prevent the recognition of transient events such as the microarousals which may be found in patients with periodic limb movements or upper airway resistance syndrome (Sleep Disorders: Upper Airway Resistance Syndrome). Staging was initially performed manually with the expected inter- and intraobserver variation but computer analysis is increasingly used, based

upon Rechtstaffen and Kales rules. More recently, the use of neural networks has allowed different forms of analysis using shorter epochs and treating sleep more as a continuum rather than discrete stages. EMG activity may be recorded from submandibular electrodes or from a limb, typically tibialis anterior, the latter being preferable if periodic limb movements are sought rather than for sleep staging. ‘Traditional’ full PSG is time-consuming and requires a high level of technical expertise and admission to a dedicated facility. It is not surprising that a number of alternative approaches and sensors have been developed. Cardiorespiratory monitoring alone, often in a home setting, is widely used for the diagnosis of OSA and other sleep-related breathing disorders. Commonly, respiration is monitored both by airflow sensors at the mouth and nose and chest wall movement. The latter may be by strain gauges, thoracic impedance, or inductance belts. Oxygenation is measured by pulse oximetry which also gives a pulse waveform and heart rate. Body position and snoring noise are also commonly measured. The expense of multiparameter monitoring for the diagnosis and quantification of OSA has led to the development of a number of simpler screening tests. Oximetry alone is widely used and is effective at detecting severe OSA with associated severe intermittent hypoxemia. However, it is less effective where the degree of obstruction is mild and the rate of arousal so quick that significant hypoxemia does not develop. This may be particularly the case in children and young people with high baseline levels of oxygenation. Analysis of the heart rate pattern may aid the diagnosis (see below). Analysis of the pattern and frequency range of snoring has been used successfully to detect OSA as have devices which detect apnea and hypopnea from analysis of airflow patterns at the mouth and nose. Periodic limb movements (PLMs) may be recorded using simple strain gauge devices around the ankle which may be interfaced with some of the cardiorespiratory monitors mentioned above. Alternatively, the movements may be sensed by small, wristwatchsized devices known as actigraphs. These instruments were initially devised for long-term recording of sleep patterns in the assessment of insomnia or circadian rhythm disturbances such as DSPS. They are worn on the nondominant wrist and any movement exceeding a preset acceleration within a preset epoch is recorded. Periods of movement are assumed to indicate wakefulness and quiescence to indicate sleep. When worn on the foot or ankle, they will record PLMs which are distinguished by the software by their repetitive nature.

SLEEP DISORDERS / Overview 79 Arousals

Many sleep disorders are associated with recurrent arousals, including OSA and PLMs. These may be detected by changes in the EEG but as these may be difficult to detect and require complex and expensive analysis, alternatives have been sought. Markers of physiological arousal are widely used, including changes in heart rate and blood pressure. Pulse transit time (PTT), the time taken for the pulse wave to travel from the heart to the periphery, is inversely related to blood pressure and can be used as an indirect measure. PTT can be derived from the R wave of the electrocardiogram (ECG) and the pulse wave from a pulse oximeter. Body movement often accompanies an arousal which can be detected by movement sensors or from video images comparing one frame with the next. Both of these techniques compare well with EEG-derived measures of arousal. Wakefulness

Wakefulness, or its converse, the propensity to fall asleep, varies throughout the day and is dependent upon situation. The circadian pressure to sleep (process C) is greatest at about 01.00–03.00 with a secondary peak at about 13.00–15.00. Added to this is the homeostatic drive to sleep (process S) which increases with time from the end of the last sleep period. Sleepiness in the early afternoon or evening is expected and even welcome when in a bedroom but not when driving a car. Thus measures of wakefulness should take account of both time of day and situation. The Epworth sleepiness scale (ESS) is a widely used situational scale which correlates well with objective measures of sleepiness. It asks subjects to grade their likelihood, over the preceding week, of dozing or falling asleep on a 0–3 scale (never, slight, moderate, high) in eight situations. The scores are added to a maximum of 24, indicating severe sleepiness. The upper limit of normal is regarded as 9. The Stanford sleepiness scale asks subjects to rate their sleepiness at that particular time on a 7-point scale from 1 (fully alert) to 7 (sleep imminent). The multiple sleep latency test (MSLT) was the first objective measure of sleepiness described. After a full night’s PSG, subjects recline in bed in a quiet, dark room four times throughout the day and asked to fall asleep. Depending on the time of day, normal subjects will stay awake for most of the 20 min test period. Patients with severe OSA or narcolepsy will be asleep within a few minutes. It was soon argued that the MSLT was not a good measure as most sleepy subjects are trying to stay awake rather than

fall asleep. The multiple wakefulness test (MWT) follows the same principles except the patient is instructed to try to stay awake. In both the MSLT and MWT, a trained observer monitors the PSG waveforms and decides when sleep has supervened and stops the test. A simplified test has been described, the OSLER test, where the subject follows the same protocol as the MWT but is asked to press a button whenever a light flashes. The light flashes every 3 s and, if seven consecutive flashes are missed, the subject is deemed to be asleep. The test correlates well with the MWT. It is important, in all these tests, not to allow the subject to take naps between the test periods, to sleep for more than a few minutes during the test periods, or to use stimulants such as coffee. Vigilance tests such as those described above are useful in diagnosis and monitoring the response to treatment but have been criticized as bearing little relationship to ‘real world’ situations such as driving. Research studies have used driving simulators of varying degrees of similarity to normal driving and have shown decreased performance with increasing objective and subjective sleepiness. However, while there is good evidence of increased risk of driving accidents in subjects with severe OSA as a group, the presently available tests do not help much in disputes as to whether an individual should be permitted to drive or not. See also: Chronic Obstructive Pulmonary Disease: Overview. Sleep Apnea: Overview. Sleep Disorders: Central Apnea (Ondine’s Curse); Upper Airway Resistance Syndrome.

Further Reading Berry RB and Harding SM (2004) Sleep and medical disorders. Medical Clinics of North America 88: 679–705. Kryger MH, Ferber R, Dahl RE, and Sheldon SH (2005) Principles and Practice of Pediatric Sleep Medicine. New York: Saunders. Kushida CA, Littner MR, Morgenthaler T, et al. (2005) Practice parameters for the indications for polysomnography and related procedures: an update for 2005. Sleep 28: 499–521. Mohsenin V (2005) Sleep in chronic obstructive pulmonary disease. Seminars in Respiratory and Critical Care Medicine 26: 109–116. Roth T, Dement WC, and Kryger MH (eds.) (2005) Principles and Practice of Sleep Medicine. New York: Saunders. Shneerson J (2005) A Guide to Sleep and its Disorders. Oxford: Blackwell.

Relevant Websites http://www.aasmnet.org – American Academy of Sleep Medicine. http://www.sleephomepages.org – The Sleep Home Pages.

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SLEEP DISORDERS / Central Apnea (Ondine’s Curse)

Central Apnea (Ondine’s Curse) M T Naughton, Monash University, Melbourne, VIC, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Congenital central hypoventilation syndrome is a rare disorder of unknown cause. It is frequently referred to as Ondine’s curse, the mythical curse that prevented breathing whilst asleep. The disorder is in contrast to nonhypercapnic central sleep apnea related to high altitude, drugs (e.g., narcotics), or heart failure (Cheyne–Stokes respiration). The diagnosis is one of exclusion. Characteristically, the central sleep apnea of Ondine’s curse begins in the neonatal years, with hypoventilation being most prominent in non-REM sleep than in REM sleep. Whilst awake, ventilation is normal. There is no cardiac, pulmonary, or neuromuscular deficit. An association has been identified between Ondine’s curse and Hirschsprung’s disease, an autonomic nervous system disorder of the large bowel, indicating the possibility of a central autonomic dysfunction at the brainstem level where ventilation is controlled. Ventilatory responses to hypercapnia and hypoxia are absent. In adults, hypercapnic central sleep apnea is usually secondary to an underlying condition such as trauma, degenerative neurological disorder, syrinx, or syringobulbia. Treatment is supportive with ventilatory assistance provided during sleep, usually with positive airway pressure delivered either invasively (via a tracheostomy) or noninvasively (via a nasal or face mask). The duration of Ondine’s curse is quite variable, with reports of spontaneous resolution of ventilatory control after months or years of ventilatory support.

Introduction Ondine’s curse refers to congenital central alveolar hypoventilation. The name, Ondine’s curse, is based upon a mythical mermaid named Ondine, the daughter of Poseidon, god of the sea. Mermaids were known for their immortality, which they lost if they married a mortal. One day, Ondine fell in love with a knight, Hans, who she married, thus losing her immortality. As time passed, Hans tired of Ondine and left for another woman. Infuriated, Poseidon placed a curse upon him so that none of his automatic body functions would occur unless he consciously willed them to. The story ends as Hans is about to fall asleep, knowing that he will forget to breathe, thus signifying the importance of metabolic control of ventilation during sleep!

Background Sleep is a time of serious and potentially life-threatening disturbance to ventilatory drive and respiratory muscular activity. The drive to breathe changes when moving from wakefulness to the sleep state (see Ventilation: Control). In contrast to wakefulness when cortical and metabolic factors control ventilation,

during sleep ventilation is under purely metabolic control: mainly the prevailing arterial CO2 level sensed at the peripheral (and rapidly responsive) carotid body and at the central (and slowly responsive) medullary region of the brainstem (see Chemoreceptors: Central; Arterial). The central region consists of rhythmic respiratory neurons in the dorsal (nucleus tractus solitarius) and ventrolateral (nucleus ambiguus and retroambiguus) locations within the brainstem. Although the importance of metabolic drive to breathing becomes very important during sleep, the sensitivity of this drive is attenuated during sleep compared with wakefulness. The respiratory muscle pump also changes from wakefulness to sleep. With conscious state change from wakefulness to non-REM and then to REM sleep, there is a progressive loss of respiratory accessory and then intercostal muscle activity such that during REM sleep, the respiratory pump is dependent upon pure diaphragmatic effort. As a consequence of the above changes, central and obstructive sleep apnea may occur (Table 1). Apneas refer to complete cessation of ventilation for at least 10 s, and hypopneas refer to partial reductions in ventilation for at least 10 s that are sufficient to cause a transient fall in arterial oxygen saturation ðSaO2 Þ or an arousal from sleep. Hypoventilation is defined as a prolonged hypopnea, usually greater than 2 min, with an associated rise in transcutaneous PCO2 of usually 42 mmHg (see Sleep Apnea: Children). Central sleep apnea (CSA) is a disorder that encompasses reductions in ventilation (apneas and hypopneas) due to disorders of respiratory drive or failure of the respiratory pump (Table 2). In contrast, Table 1 Clinical features of obstructive sleep apnea History Loud snoring Witnessed apneas Difficult intubation or delayed recovery from anesthetic Systemic hypertension Ischemic heart disease Cerebrovascular disease Alcohol, cigarette, sedative use Daytime sleepiness Unexplained single vehicle motor vehicle accidents Examination Obesity Large neck circumference Small mouth Retro (or micro) ganthia Maxillary insufficiency High arched palate Tonsillar hypertrophy Nasal obstruction

SLEEP DISORDERS / Central Apnea (Ondine’s Curse) 81 Table 2 Classification of nonhypercapnic and hypercapnic central sleep apnea

Gender History of respiratory failure Cor pulmonale Polycythemia History of cardiac failure Muscle/nerve pathology Early morning headaches

Nonhypercapnic

Hypercapnic

Male No No No Yes No No

Equal Yes Yes Yes No Yes Yes

obstructive sleep apnea (OSA) occurs as a result of upper airway instability caused by selective inability to maintain upper airway patency resulting in apneas and hypopneas whilst respiratory drive and pump activity remain intact. Snoring, cyclic apneas, and hypopneas with vigorous respiratory efforts, large negative intrathoracic pressures and significant hypoxemia, and fragmented sleep accompany OSA. Central sleep apnea can be further divided into nonhypercapnic and hypercapnic varieties. The non-hypercapnic type of CSA is usually represented by 5–30 min episodes of periodic breathing, with ventilation–apnea cycle lengths of 30–90 s, mainly during stages 1 and 2 of non-REM sleep. Throughout sleep, increased minute ventilation (and associated hypocapnia) is observed during the ventilatory components of the periodic breathing. In addition hyperventilation may occur whilst awake at rest and during exercise, leading to reduced PaCO2 levels. Exaggerated ventilatory responses to hypercapnia and hypoxia have been observed. Prevailing arterial CO2 levels are periodically driven below the apnea threshold, resulting in periodic pauses to ventilate. In adults, this type of CSA is often due to advanced heart failure (when the term Cheyne–Stokes respiration is used), renal failure, narcotic drugs, or is of unknown cause (idiopathic nonhypercapnic CSA). In infants, episodic CSA may be due to prematurity and oxygen toxicity. The hypercapnic form of CSA (Table 3) is associated with noncyclical hypoventilation during sleep and, in more severe forms, whilst awake and asleep. It usually occurs in REM (when only the diaphragm is operational) and less commonly in non-REM sleep (when the diaphragm and intercostal muscles are operational). The etiology may be related to respiratory pump pathology (myopathy, electrolyte disorders such as low phosphate, chest wall deformity) or reduced pulmonary compliance (e.g., due to parenchymal disease as with cystic fibrosis and emphysema). Abnormalities of respiratory drive such as encephalitis, brainstem trauma, drugs (narcotics, sedatives), cerebrovascular event, syrinx, or syringobulbia may result in hypercapnic CSA. The term Ondine’s curse

Table 3 Causes of hypercapnic central sleep apnea Central Primary (idiopathic) Congenital central hypoventilation syndrome Secondary Encephalitis Cervical cordotomy Brainstem infarct Brainstem tumor Brainstem trauma Brainstem poliomyelitis Syrinx, syringobulbia Spina bifida (myelomeningocele) Multiple sclerosis Peripheral Muscle Myotonic dystrophy Muscular dystrophy Acid maltase deficiency Myopathy related to electrolytes Chest wall Distortion and resultant myopathy Kyphoscoliosis Hyperinflation Decreased compliance Lung parenchyma, e.g., pulmonary fibrosis Chest wall, e.g., ankylosing spondylitis Neural Neuromyopathies Amyotrophic lateral sclerosis Post polio syndrome Myasthenia gravis Trauma, e.g., cervical nerve root

Table 4 Diagnostic criteria for CCHS Onset of symptoms in the first year of life Hypoventilation asleep (non-REM worse than REM) Loss/impaired hypoxic and hypercapnic ventilatory responses Ancillary features Autonomic disturbance (temperature and heart rate control) Bulbar dysfunction (e.g., squint) Neural crest abnormality (Hirschsprung’s disease) Absence of a primary pulmonary, cardiac, or neurological condition

(congenital central hypoventilation syndrome, CCHS) is used when respiratory drive is impaired, usually of neonatal onset, resulting in hypercapnic CSA, for which no cause can be identified (Table 4). CCHS is an extremely rare condition (an estimated 300 cases reported worldwide) in which severe hypoventilation occurs during sleep; it is easily corrected by ventilatory support (either positive airway pressure or diaphragmatic pacing). The term Ondine’s curse was coined initially by Severinghuas and Mitchell in 1962 based upon three

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adult patients who had undergone high cervical/ brainstem surgery and could ventilate whilst awake but not asleep. The patients lacked ventilatory responsivity to CO2. In 1970, Mellins and colleagues described an infant with clinical features corresponding to Ondine’s curse. Although this case was different from the original cases of Severinghaus and Mitchell, the term Ondine’s curse gained wide acceptance to denote CCHS in children.

Clinical Features Characteristically, two groups have been identified. The first group is characterized by a neonatal onset, an absence of dyspnea, an absence of ventilatory responses to respiratory stimuli, and the absence of a causative anatomic lesion (e.g., lung, heart, endocrine, or brain disease). There is no gender predisposition. Some children present at birth, requiring ventilatory support for several months, before their inherent respiratory control matures and ventilatory support can be weaned. In the second group, CCHS appears insidiously over months with the onset of progressive hypoxemia and cor pulmonale. Differential diagnosis at this stage includes cyanotic heart disease. In milder cases, tachycardia, diaphoresis, and sleep-related hypoxemia occur. Other patients might present with unexplained apnea or an apparent life-threatening event. Some may die and be classified as sudden infant death syndrome. Approximately 20% of children with CCHS have Hirschsprung’s Disease (HD) and neural crest tumors such as ganglioneuroma, neuroblastoma, and ganglioneuroblastoma (see Neurophysiology: Neuroendocrine Cells). Clinical examination of suspected patients should include searching for signs of Hirschsprung’s disease (malabsorption, malnutrition), ocular disorder (squint, Figure 1), upper airway pathology, and gastroesophageal reflux, in addition to a thorough respiratory, cardiac, and neurological examination. The long-term outcome of children with CCHS has improved with the advent of long-term ventilatory

Figure 1 Photograph of a patient with divergent squint due to encephalitis involving brainstem.

support. Prior to ventilatory support, such children had severe neurocognitive deficits, stunted growth, cor pulmonale, and seizure activity. With the introduction of ventilatory support, such features have diminished; however, long-term mortality is dependent upon adequate, complication-free ventilatory support. Some neonates may require ventilatory support for a few months but with maturity can maintain ventilation independently. The current school of thought is that maturity of respiratory drive overcomes the need for long-term ventilatory support.

Etiology The cause of CCHS is unknown. A genetic basis has been suspected due to the following: (1) a neonatal onset; (2) a family history of CCHS in some patients; and (3) an increased prevalence in patients with Hirschsprung’s disease. Recent genetic studies in patients with CCHS have identified numerous specific gene mutations for the sites responsible for the receptor for tyrosine kinase, endothelin 3, glial-derived neurotrophic factor, brain-derived neurotrophic factor, human achaete-scute homolog-1, PHOX2 (a and b), GFRAI, bone morphogenic protein-2, HASH-1, and endothelin-converting enzyme-1. Mutation of the gene for Ret tyrosine kinase was studied because of its reported association with HD and because the gene is associated with development of the neural crest and parasympathetic nervous system. Unfortunately, only a few patients with CCHS have been found to have this gene mutation. A similar story exists for the gene for endothelin. The search for genetic abnormalities of respiratory drive has to date been unrewarding.

Diagnostic Studies Physiologically, patients with CCHS appear to have absent or reduced metabolic drive to ventilate. Ventilatory responses to hypoxia and hypercapnia appear to be impaired. Arousal from sleep in response to added CO2 appears to be impaired. As ventilation during non-REM sleep is under metabolic control, hypoventilation is most profound during this stage of sleep. In contrast, ventilation during wakefulness and REM sleep appears to be relatively stable, as additional nonmetabolic factors control ventilation. The results from one study indicated that peripheral, but not central, chemosensitivity was intact in CCHS patients who were able to ventilate adequately during wakefulness. Moreover during exercise, ventilation would increase with workload, but at a reduced response compared with normal individuals; in part due to mechanoreceptors in the extremities.

SLEEP DISORDERS / Central Apnea (Ondine’s Curse) 83 Se:101 Im:7

[H]

[A]

Study date:18/02/2.. Study time:17:58:44 MRN:

[P]

Table 5 Modes of ventilatory support Medical Almitrine Doxapram Acetazolamide Theophylline Ventilatory assistance Rocking bed Negative pressure Iron lung Cuirass Positive pressure Tracheostomy Face mask (noninvasive) Diaphragmatic pacing

[F]

C542 W1582

Figure 2 Sagittal T1 magnetic image of syringobulbia (illustrated within the rectangle).

Tests of autonomic function, such as heart rate variability, pupillary light reaction, and poor temperature control (decreased body temperature, sporadic bursts of profuse sweating with cool extremities, dysphagia) have been reported to be impaired in CCHS. Thus, the autonomic integration of peripheral and central chemosensitivity may be problematic. Other useful investigations include urinalysis for amino acids and organic acids (to exclude metabolic disorders), brain and brainstem magnetic resonance imaging or computed tomography (CT) scans (excluding brainstem pathology) (Figure 2), nerve conduction studies, echo cardiography (excluding cardiac shunt), and pulmonary investigations (arterial blood gas, chest X-ray, diaphragm fluoroscopy). Polysomnography in a well-equipped laboratory with sleep montage, transcutaneous PCO2 , respiratory effort, airflow, SpO2 , and ECG is required, and may need to be repeated every 3–4 months as the infant matures. Hypercapnic and hypoxic ventilatory responses should be considered.

Treatment Oxygen therapy to overcome hypoxemia secondary to hypoventilation may worsen hypercapnia via loss of the already impaired hypoxic drive (Table 5) (see Oxygen Therapy). Some research groups have argued that CCHS patients may have an absent hypoxic stimulus to start with, and that oxygen may have relatively little effect on worsening hypercapnia, and may have a potential therapeutic role via reversal of the hypoxia-induced central nervous system

Figure 3 Photograph of an iron lung.

depression. As a result oxygen therapy can be considered in CCHS, but cautious monitoring of CO2 levels and sleep quality should be undertaken. Pharmacological respiratory stimulants (doxapram, progesterone, almitrine, acetazolamide, theophylline) have not proved to be successful. Noninvasive ventilatory support has been the mainstay of therapy. Initially, external negative pressure support with devices placed over the chest or abdomen (‘cuirass’) or body (‘iron lung’) (Figure 3) was tried out. Unfortunately, these devices had the capacity to make the upper airway unstable during the negative chest inflating cycle of the respiratory cycle, often negating the therapeutic effects of ventilatory support. More recently, positive pressure ventilatory support via a tracheostomy (Figure 4) or ‘non-invasively’ via a nasal mask (Figure 5) has proven to be the most successful technique. Attention to the ventilator interface (tracheostomy or face), ventilator settings, ventilator alarms, and feeding dictate the overall survival of such patients (Figure 5). Duration of

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and social work. Such patients should be looked after by specialist centers familiar with the care of patients on long-term ventilatory support.

Conclusion

Figure 4 Positive pressure ventilatory support via a tracheostomy.

Ondine’s curse, or congenital central hypoventilation syndrome, is a diagnosis of exclusion in which ventilation is impaired during sleep with no identifiable cause. Onset is usually neonatal and treatment is supportive. Associations have been noted with neural crest disorders such as Hirschsprung’s disease, suggesting an inherited abnormality of autonomic control. Prognosis is not clear given the rarity of the condition however, respiratory control does appear to mature in many neonates who successfully wean from ventilatory support and lead normal independent lives. See also: Chemoreceptors: Central; Arterial. Neurophysiology: Neuroendocrine Cells. Oxygen Therapy. Sleep Apnea: Children. Ventilation: Control.

Further Reading

Figure 5 Photograph of a patient using noninvasive ventilatory support via a nasal mask.

ventilatory support dictates the mode of positive pressure ventilation: if o12 h per day is required then a face mask interface is satisfactory; if 412 h per day is needed, tracheostomy may be required. Percutaneous gastrostomy tubes, which allow for feeding whilst bypassing the upper airway, may be required in some patients in whom swallowing is problematic. Diaphragmatic pacing has been used in exceptional cases, but is limited by poor coordination between upper airway and respiratory pump musculature contraction. Moreover, it is costly and uncomfortable. It involves implanting a pacing electrode around the phrenic nerve, either in the cervical or high thoracic region. A stable chest wall, intact phrenic nerve, and long-term ventilation are required. Professional healthcare follow-up for families of patients with CCHS is paramount and requires the involvement of the following: neurology, intensive care, cardiology, radiology, ear, nose and throat, pulmonologist, gastroenterologist, sleep laboratory,

American Thoracic Society Statement (1999) Idiopathic congenital central hypoventilation syndrome. American Journal of Respiratory and Critical Care Medicine 160: 368–373. Bradley TD, McNicholas WT, Rutherford R, et al. (1986) Clinical and physiological heterogeneity of the central sleep apnea syndrome. American Review of Respiratory Diseases 134: 217–221. Bradley TD and Phillipson EA (1992) Central sleep apnea. Clinics in Chest Medicine 13: 493–505. Chervin RD and Guilleminault C (1994) Diaphragmatic pacing: review and reassessment. Sleep 17: 176–187. Fleming PJ, Cade D, Bryan MH, and Bryan AC (1980) Congenital central hypoventilation and sleep state. Pediatrics 66: 425–428. Gozal D (1998) Congenital central hypoventilation syndrome: an update. Pediatric Pulmonology 26: 273–282. Gozal D, Marcus CL, Davidson Ward SL, and Keens TG (1996) Ventilatory responses to passive leg motion in children with congenital central hypoventilation syndrome. American Journal of Respiratory and Critical Care Medicine 153: 761–768. Hunt CE (2001) Sudden infant death syndrome and other causes of infant mortality. American Journal of Respiratory and Critical Care Medicine 164: 346–357. Keens TG and Davidson Ward SL (2000) Syndrome affecting respiratory control during sleep. In: Loughlin GM, Carroll JL, and Marcus CL (eds.) Sleep and Breathing in Children: A Developmental Approach, Lung Biology in Health and Disease, vol. 147, pp. 525–554. New York: Dekker. McNicholas WT, Carter JL, Rutherford R, et al. (1982) Beneficial effects of oxygen in primary alveolar hypoventilation with central sleep apnea. American Review of Respiratory Diseases 125: 773–775. Mellins RB, Balfor HH Jr, Turino GM, and Winters RW (1970) Failure of automatic control of ventilation (Ondine’s Curse). Medicine 49: 487–504. Paton JY, Swaminathan S, Sargent CW, Hawksburn A, and Keens TG (1993) Ventilatory response to exercise in children with congenital central hypoventilation syndrome. American Review of Respiratory Diseases 147: 1185–1191.

SLEEP DISORDERS / Hypoventilation 85 Severinghaus J and Mitchell RA (1962) Ondine’s curse – failure of respiratory center automaticity while awake. Clinical Research 10: 122. Weese-Mayer DE and Berry-Kravis EM (2004) Genetics of congenital central hypoventilation syndrome. American Journal of Respiratory and Critical Care Medicine 170: 16–21.

Hypoventilation F Han, Beijing University, Beijing, People’s Republic of China & 2006 Elsevier Ltd. All rights reserved.

Abstract Alveolar hypoventilation, defined as a partial arterial CO2 pressure (PaCO2 ) above 45 mmHg, develops in patients with breathing control defects, respiratory neuromuscular diseases, chest wall deformity, or lung and airway disorders. The impact of sleep on ventilation and gas exchange in these patients can be dramatic and potentially life threatening, and often is undetected. Associated with hypercapnia is hypoxemia and sleep disturbance, leading to clinical features manifested primarily in central nervous system and cardiovascular effects. Symptoms usually include dyspnea, morning headache, and excessive daytime sleepiness. Clinical examination may be normal or uncover erythrocytosis, pulmonary hypertension, cor pulmonale, or respiratory failure. Lung function tests can generally distinguish among underlying chest diseases causing hypoventilation, and a polysomnogram is helpful to characterize the presence of and type of sleep disordered breathing, such as sleep apnea, hypopnea, and sleep hypoventilation. Optimum management includes treating the underlying condition(s) and providing, as appropriate, oxygen and/or noninvasive positive pressure ventilation (NPPV) applied via a nasal or facial mask. Administration of NPPV only during sleep to prevent sleep-related deterioration of ventilation often produces a dramatic improvement in daytime symptoms and blood gas levels in most cases.

Introduction Hypoventilation is defined as an increase in partial arterial CO2 pressure (PaCO2 ) to a level above 45 mmHg. The concomitant hypoxemia leads to clinical sequelae such as erythrocytosis, pulmonary hypertension, cor pulmonale, or respiratory failure, which is referred to as hypoventilation syndrome. Prevalence of hypoventilation is currently unknown. The idiopathic form is rare. However, almost all disorders that result in awake hypercapnia are complicated by sleep hypoventilation. A recent study showed that the prevalence of obesity hypoventilation syndrome (OHS) among hospitalized adults with BMI of more than 35 is as high as 31% and is associated with excess morbidity and mortality, but in the majority of cases the condition has not been recognized by cargivers.

Etiology and Pathogenesis Hypoventilation is generally a consequence of obstructive pulmonary disease but can also develop in patients with normal lungs. The underlying mechanisms involve a defect in the central or peripheral respiratory control system, the respiratory neuromuscular system, or the mechanical apparatus (Table 1). In most cases, more than one mechanism is responsible. Sleep has a profound effect on hypoventilation. Pre-existing hypercapnia and hypoxemia deteriorate during sleep, especially during the rapid eye movement (REM) sleep stage. In some, clinically significant hypoventilation not associated with distinct apneas and hypopneas develops only in sleep. When chemoresponsiveness is markedly reduced or absent, hypoventilation may develop in the absence of chest wall, neuromuscular, or lung disease. The attenuation of the respiratory drive can be inherited or acquired. Drug use, chronic hypoxia and hypercapnia exposure, or diseases affecting the brainstem or peripheral chemoreceptors can produce depressed chemoresponsiveness. Hypothyroidism lowers respiratory drive and is a frequently overlooked cause of hypoventilation. However, patients with primary alveolar hypoventilation (PAH) may have no known underlying disease. In such cases, responses to chemical stimuli are impaired, but patients can normalize the PaCO2 by voluntary hyperventilation while they are awake. During sleep, with the decrease of stimulus from the behavioral control system, there is a further reduction in ventilation, and in particular in nonrapid eye movement (NREM) sleep stages 3 and 4. The defect in these patients seems to be the inability to centrally integrate chemoreceptor signals. Congenital central hypoventilation syndrome (CCHS) is a rare disease of childhood. Mutations in the PHOX2B gene may have a role in the pathogenesis of CCHS. This pathway may operate in the pathophysiology of adult PAH as well. Patients with primary disorders of the spinal cord, respiratory nerves, and respiratory muscles develop hypoventilation because the neural output from the brainstem respiratory center cannot always fully compensate for the muscle weakness. Generally, marked hypoventilation does not occur unless the function of the diaphragm is significantly ( 80%) impaired. REM sleep is one potentially vulnerable time for hypoventilation in these patients as there is a generalized postural muscle atonia in accessory muscle like the sternocleidomastoid, and breathing becomes dependent solely on diaphragm activity. Therefore, the initial nocturnal hypoventilation and eventual daytime respiratory failure in almost all of the patients with neuromuscular diseases is first

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Table 1 Disorders affecting specific components of the respiratory system Disorder Central control depression Drugs Metabolic alkalosis Central alveolar hypoventilation Primary alveolar hypoventilation Chronic hypoxia/hypercapnia exposure Hypothyroidism

Affected components of respiratory system Narcotics, alcohol, barbiturates, benzodiazepines, anesthetics Encephalitis, trauma, hemorrhage, tumor, stroke, degeneration, demyelinating Genetics COPD, sleep disordered breathing, high altitude

Neuromuscular diseases Spinal cord injury Anterior horn cell diseases Peripheral neuropathy Myoneural junction disease Myopathy

Postpolio syndrome, amyotrophic lateral sclerosis Guillain–Barre´, diphtheria, phrenic nerve damage Myasthenia gravis, anticholinesterase poisoning, curare-like drugs, botulism Duchenne muscular dystrophy, polymyositis

Mechanical apparatus disorders Chest wall deformities Upper airway obstruction Lower airway and lung diseases

Kyphoscoliosis, fibrothorax, thoracoplasty, obesity hypoventilation Sleep apnea, goiter, epiglottitis, tracheal stenosis COPD, cystic fibrosis

evident during REM sleep. Factors contributing to the rate of progression of hypoventilation from isolated REM events to NREM sleep, and then frank daytime respiratory failure include the pattern of initial respiratory muscle weakness, the rate of progression of underlying disease, age, weight gain, and development of acute respiratory infections. Patients with diseases of the lungs, airways, or chest wall develop alveolar hypoventilation because of the increased work of breathing. The most common example is chronic obstructive pulmonary disease (COPD). In addition to a substantial decrease in pulmonary function, abnormalities in ventilatory control, reduced strength and endurance of the respiratory muscles, and the alterations in breathing pattern are all responsible for the development of CO2 retention. During sleep, patients with COPD may experience significant nocturnal O2 desaturation (NOD), either because of increased ventilation-perfusion matching or because of sleep-induced hypoventilation. Massive obesity represents a mechanical load to the respiratory system and reduces the compliance of the chest wall; however, weight is not the sole determinant of the occurrence of obesity hypoventilation. The majority of obese individuals maintain a normal PaCO2 level through a compensatory increase in respiratory drive. Only a small proportion with reduced chemoresponsiveness retains CO2. Hypoventilation can be improved purely by increasing respiratory drive without altering the mechanical properties of the respiratory system. Recent studies showed that leptin-deficient ob/ob mice demonstrate hypoventilation before the onset of marked obesity. Such animals have an

impaired hypercapnic ventilatory response during both wakefulness and sleep, and this abnormality exists before the development of obesity. Furthermore, leptin infusion reverses both hypoventilation and the hypercapnic response. In humans, serum leptin level is as good or a better predictor than percent body fat for the presence of hypercapnia. That sleep-disordered breathing plays a role in daytime hypoventilation has been suggested by the fact that obstructive sleep apnea occurs not only in most patients with OHS, but also in some with hypercapnia and mild obesity, and in many cases daytime hypoventilation resolves after effective treatment of OSA with continuous positive airway pressure (CPAP) during sleep. How a disorder that occurs during sleep eventually produces diurnal hypercapnia is not well defined. A key element might be that chronic intermittent hypoxia and hypercapnia and sleep deprivation interact to result in blunted diurnal respiratory control. This vicious cycle results in decrementing responsiveness of the respiratory centers, leading to daytime hypoventilation. Short-term CPAP treatment in hypercapnic patients with OSA will reset chemosensitivity.

Clinical Features The fundamental disturbance in all hypoventilation syndromes is an increase in PaCO2 and a decrease in PaO2 . As hypercapnia and hypoxia occur in combination, it is often difficult to distinguish which is the primary cause of the clinical presentations. The clinical features are manifested primarily in central nervous system and cardiovascular effects (Figure 1). In

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Primary events

Secondary pathophysiological changes

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Cyanosis, polycythemia Pulmonary hypertension Cor pulmonale Peripheral edema Morning headache Sleep disturbance Daytime hypersomnolence Confusion, fatigue

Figure 1 Pathophysiological changes and clinical features in patients with hypoventilation. Adapted from Philipson EA and Sluteky AS (2000) Hypoventilation and hyperventilation syndromes. In: Murray JF and Nadel JA (eds.) Text book of Respiratory Medicine, 3rd edn., pp. 2139–2152. Philadelphia: Saunders, with permission from Elsevier.

the early stage, patients with hypoventilation experience minimal, if any, respiratory discomfort. In many cases, sleep disturbance and the effects of sleep deprivation such as lethargy, confusion, morning headache, fatigue, and sleepiness dominate the clinical presentation. When hypoventilation progresses, dyspnea on exertion, followed by dyspnea at rest, is the most frequent symptom in patients with neuromuscular diseases or mechanical apparatus disorders. In contrast, patients with impaired chemoresponsiveness generally do not show dyspnea, and often first come to attention due to other clinical presentations. If hypercapnia and hypoxia become more evident, patients develop signs of cardiovascular decompensation, including pulmonary hypertension and right heart failure or neurocognitive dysfunction. Other clinical features are related to the specific underlying disease. For example, significant muscle weakness, impaired cough, and repeated lower respiratory tract infections may occur in the course of neuromuscular disorders. Diagnostic Evaluation

The evaluation of a patient with hypoventilation syndrome includes tests to determine the existence of

alveolar hypoventilation and measurements to identify the medical conditions causing hypoventilation (Figure 2). The key diagnostic finding for hypoventilation is an elevation of PaCO2 value, which is usually associated with hypoxemia. However, hypercapnia may not be detected in a single arterial blood gas analysis, as patients with PAH could hyperventilate voluntarily, thus reducing the PaCO2 level to normal, and hypercapnia occurs only during sleep in some patients with sleep hypoventilation syndrome. Further evidence indicating the presence of chronic hypoventilation includes an increase in plasma HCO3 concentration and ECG, chest X-ray, and echocardiography findings of pulmonary hypertension and right ventricular hypertrophy. An elevated hematocrit and hemoglobin may be present as a complication of severe hypoxemia. History and physical examination may initially suggest the underlying diseases causing hypoventilation, and detail the severity of complications. Using further pulmonary function tests it should be possible to localize the failed component of the respiratory system responsible for hypercapnia. Minute ventilation, occlusion pressure, and diaphragmatic electromyographic activity have been used as noninvasive measures of central respiratory drive. Impaired responses

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Ascertain the diagnosis of hypoventilation

PaCO2 > 45 on ABG pH and serum HCO3− change

Initial evaluation

History

Initial tests

Physical examination

Identify contributing factors COPD CHF Obstructive sleep apnea Hypothroidism Neuromuscular diseases Smoking Alcohol Medications

Measure BMI

Ascertain etiology

Examine upper airway

Chest X-ray

Signs of right heart failure Signs of left heart failure

TSH Assess end-organ effect

Musculoskeletal abnormalities Breathing pattern to assess diaphragm function Neurologic examination

EKG CBC Echocardiogram

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Polysomnogram Identify sleep apnea hypopnea Identify sleep hypoventilation

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Figure 2 Evaluation of patients with hypoventilation. ABG, analysis of blood gases; TSH, thyroid-stimulating hormone; EKG, electrocardiogram; CBC, complete blood count.

of these indexes to chemical stimuli during hypoxia and hypercapnia rebreathing exist in patients with central control defects or neuromuscular disorders; however, the former patients can hyperventilate on command and the latter cannot. Decrease of maximum inspiratory pressure (MIP) and maximum expiratory pressure (MEP) indicates a global weakness of respiratory muscles. If MIP is low, then diaphragmatic function should be assessed by measuring transdiaphragmatic pressure. Phrenic nerve conduction assesses the integrity of the nerve-muscle

unit. Spirometry helps characterize whether the hypoventilation resulted from a restrictive or obstructive ventilatory disorder. Neuromuscular disease or chest wall disorders produces restrictive patterns on spirometric testing, manifested by a reduction in vital capacity (VC) with a similar reduction in forced expiratory volume in 1 s (FEV1). In contrast, COPD patients have a typical obstructive pattern with marked reductions in FEV1 and forced vital capacity (FVC). An elevated alveolar-arterial PO2 difference [(A-a PO2 )] on blood gas

SLEEP DISORDERS / Hypoventilation 89

tests suggests a mechanical apparatus disorder, and patients with respiratory control or neuromuscular defects could maintain a normal A-a PO2 unless they have significant atelectasis. Patients with hypoventilation should receive a polysomnogram to establish whether sleep apnea and hypopnea are present. An

increase in PaCO2 during sleep of 10 mmHg from awake supine values and sustained arterial desaturation lasting up to several minutes during sleep not explained by apnea or hypopnea events may indicate sleep hypoventilation. Overnight monitoring of dynamic changes of transcutaneous CO2 and

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(b) Figure 3 A 62 year women with a BMI of 29 kg m 2 complained about daytime sleepiness and dyspnea. She has no history of smoking. Blood gas analysis showed PaCO2 of 58 mmHg and PO2 of 72 mmHg. Lung function test had no remarkable findings. MIP and MEP were in normal range. Nocturnal oximetry screening indicated the existence of sleep hypoventilation (a) without remarkable sleep apnea, and this was confirmed by PSG testing (b).

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nocturnal oxygen saturation by oximetry (Figure 3) is a useful screening test before a polysomnography (PSG) sleep study.

Treatment In addition to the treatment of an underlying disorder, therapeutic strategies for patients with chronic hypoventilaton syndrome aim at correcting the hypercapnia and its associated hypoxemia, which can be achieved by either increasing alveolar ventilation or giving supplemental oxygen. Oxygen Therapy

As long as pH is maintained at an acceptable level, chronic hypercapnia by itself generally has little immediate clinical consequence. The most serious consequence of hypoventilation is hypoxemia. The administration of supplemental oxygen may improve oxygenation, and prevent hypoxic sequelae. However, high concentrations of oxygen may worsen hypercapnia, potentially to a dangerously high level, and ventilatory support should be considered. This happens less in patients with defects in respiratory control than in patients with neuromuscular diseases and mechanical apparatus disorders. In patients with sleep hypoventilation, oxygen alone may prevent NOD, but often results in prolonged breathing disturbances and worse sleep quality, therefore worsening the daytime symptoms, such as morning headache. Respiratory Stimulants

Medications to improve ventilatory drive have been used with limited success in patients with alveolar hypoventilation. The most commonly used agent is medroxyprogesterone, which effectively lowers PaCO2 in patients with OHS, but does not appear to work in patients with OSA who do not have hypercapnia. Acetazolamide enhances respiratory drive by producing a metabolic acidosis. It may have a role in the treatment of a subgroup of patients with periodic breathing or idopathic central sleep apnea. Theophylline administration induces a significant reduction in the frequency of central sleep apnea (CSA) in patients with chronic heart failure (CHF), but has an adverse effect on sleep quality, and may increase cardiac arrhythmia. Assisted Ventilation

In patients with severe hypoventilation, mechanical ventilation support may be required. Negative pressure ventilation is the first form of noninvasive ventilation. This ventilation modality is generally

effective, but can induce upper airway obstruction in 50% of the patients. Currently positive pressure ventilation is most often the treatment of choice. Although it could be administered by way of tracheotomy, positive pressure ventilation is usually applied noninvasively via a nasal or facial mask. CPAP is effective to suppress sleep apneas, but may worsen hypercapnia in patients with respiratory muscle weakness because the patient exhales against a high mask pressure. Bilevel ventilation (BiPAP) with a lower expiratory pressure is more comfortable and better for correcting CO2 retention. Automatically adjusting the CPAP (Auto-CPAP) device has the same efficacy as CPAP on sleep apnea; however, it is not recommended to treat hypoventilation. Sometimes assisted ventilation on its own may be insufficient to correct hypoxemia, particularly during REM sleep; the use of supplemental oxygen has been advocated. In most cases, assisted ventilation is confined to sleep if possible, as administration of such treatment only during sleep often produces dramatic improvement of daytime symptoms as well as the daytime blood gas levels. Electrophrenic or Diaphragm Pacing

Phrenic nerve stimulation has been used as an alternative to long-term mechanical ventilation in patients with reduced respiratory drive, particularly in those with PAH. Diaphragm pacing with laproscopically inserted muscle electrodes has recently become available to support ventilation. Both procedures require a functionally intact nerve-diaphragm axis; lower motor neuron disease, phrenic neuropathy, and respiratory muscle myopathy are contraindications for this treatment. As occurs in negative pressure ventilation treatment, approximately 50% of patients undergoing phrenic or diaphragm pacing develop upper airway obstruction during sleep. See also: Sleep Apnea: Continuous Positive Airway Pressure Therapy; Drug Treatments. Sleep Disorders: Central Apnea (Ondine’s Curse).

Further Reading American Academy of Sleep Medicine Task Force (1999) The Report of an American Academy of Sleep Medicine Task Force sleep related breathing disorders in adults: recommendations for syndrome definition and measurement techniques in clinical research. Sleep 22: 667–689. Amiel J, Laudier B, Attie-Bitach T, et al. (2003) Polyalanine expansion and frame shift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome. Nature Genetics 33: 459–461. Krachman S and Criner GJ (1998) Hypoventilation syndromes. Clinics in Chest Medicine 19: 139–155.

SLEEP DISORDERS / Upper Airway Resistance Syndrome 91 Nowbar S, Burkart KM, Gonzales R, et al. (2004) Obesityassociated hypoventilation in hospitalized patients: prevalence, effects, and outcome. American Journal of Medicine 116: 1–7. O’Donnell CP, Tankersley CG, Polotsky VP, Schwartz AR, and Smith PL (2000) Leptin, obesity, and respiratory function. Respiration Physiology 119: 163–170. Phillipson EA (2003) Disorders of ventilation. In: Braunwald E (ed.) Harrison’s Principles of Internal Medicine, 15th edn, pp. 1517–1519. Boston: McGraw-Hill. Philipson EA and Sluteky AS (2000) Hypoventilation and hyperventilation syndromes. In: Murray JF and Nadel JA (eds.) Textbook of Respiratory Medicine, 3rd edn., pp. 2139–2152. Philadelphia: Saunders. Subramanian S and Strohl KP (1999) A management guideline for obesity-hypoventilation syndromes. Sleep Breath 3: 131–138. Weinberger SE, Schwartzstein RM, and Weiss JW (1989) Hypercapnia. New England Journal of Medicine 321: 1223–1231.

Upper Airway Resistance Syndrome P Le´vy, R Tamisier, and JL Pe´pin, University Hospital, Grenoble, France & 2006 Elsevier Ltd. All rights reserved.

Abstract Obstructive sleep apnea syndrome (OSAS) has been individualized as a major public health problem. Both its cardiovascular morbidity and symptoms motivate for an accurate diagnosis and appropriate therapeutics. The upper airway resistance syndrome (UARS) has been described because of the hypothesis that repetitive increases in respiratory efforts that are inducing arousals (RERA) might produce a significant disease with associated cardiovascular and cognitive morbidity. International classifications of sleep disorders in 1999 did not individualize UARS but RERA and in 2005 recommend that it be included as part of OSAS but not as a separate entity. In this article, the authors attempt to describe the specificity of this syndrome that may be relevant for both clinicians and researchers.

Since the obstructive sleep apnea syndrome (OSAS) has been earmarked as a major public health problem, there have been many efforts in defining and understanding this syndrome. Some evidence suggests that this disease is not limited to the patient exhibiting obstructive apnea but includes a continuum from snoring to OSAS that may be part of a group named sleep-disordered breathing. The upper airway resistance syndrome (UARS) was reported by Christian Guilleminault in 1993. This particular syndrome came into being because of the hypothesis that repetitive increases in respiratory efforts that are inducing arousals (RERA) might produce a significant disease with associated cardiovascular and cognitive morbidity. The definitions of sleep-disordered breathing made by the American Academy of Sleep

Medicine (AASM) Task force in 1999 did not include UARS as a syndrome but did define RERA. Recently several authors have discussed the existence of this syndrome and the morbidity that might be related to RERA. ICSDII published in 2005 is overall in accordance with the AASM task force report published in 1999. If baseline oxygen saturation is normal, events including an absence of oxygen desaturation despite a clear drop in inspiratory flow, increased respiratory effort and a brief change in sleep state or arousal, are defined as respiratory effort related arousals. The UARS is a proposed diagnostic classification for patients with RERA who do not have events that would meet definitions for apneas and hypopneas. However, these events are presumed to have the same pathophysiology as obstructive apneas and hypopneas (upper airway obstruction) and are believed to be as much of a risk factor for symptoms of unrefreshing sleep, daytime somnolence, and fatigue as frank apnea or hypopnea. Therefore, ICSD II recommends that they be included as part of OSA and not be considered as a separate entity.

Definition The occurrence of repetitive RERA during sleep defines the UARS. RERA is characterized by a progressive increase in respiratory effort; this may be assessed by direct measurement of esophageal pressure or by another marker of respiratory effort such as the change in pulse transit time (Figure 1). RERA may induce both cortical and autonomic arousal and potentially lead to cardiovascular activation. Respiratory flow, when using nasal cannula or a pneumotachograph, exhibits only qualitative change and is named inspiratory flow limitation. This is of interest since inspiratory flow limitation results from progressive increase in UA resistance and is a useful noninvasive method to detect RERA. The time sequence of these obstructive respiratory events is close to what occurs with apneas and hypopneas, but the duration may be longer. This should be distinguished from episodes of sustained stable flow limitation occurring during slow wave sleep. This late-flow limited aspect does not lead to repeated arousals and thus differs from RERA. For qualifying as an individual disease, UARS should meet the following criteria: *

*

*

First, to exhibit specific clinical and polysomnographic diagnostic criteria. Second, these specific criteria should not be found in the general population. Third, a direct relationship should be found between this syndrome and a specific morbidity.

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UARS Clinical Aspects Snoring is reported to be present in 41% of the general population between 26 and 50 years old; in this cluster of population, 15% exhibit an apnea/ hypopnea index (AHI) more than 15 events per hour of sleep. Snoring is reported in 71% of men and 11% of women exhibiting UARS. Diurnal sleepiness remains the main diagnostic criteria, which is often confounded with tiredness in the obese or women. Twenty percent of patients may report insomnia. All other OSAS clinical criteria may apply to UARS such as nocturia, nocturnal awakening, sleep inefficiency, and cognitive impairment. In accordance with this limitation, several authors argue about the fact that these symptoms are not specific enough compared to OSAS and are quite similar to those reported during chronic asthenia. Compared to OSAS, body mass index in UARS is traditionally reported to be less and around 25 kg m
2. Some craniofacial characteristics were reported as being specific of UARS. These patients exhibit the classical long face syndrome with a short and narrow chin and reduced mouth opening (Figure 1). There is classically a ‘click’ and a subluxation when opening the temporo mandibular articulation, which may be evidenced by palpation. The mandible is in back position; the palate is high and narrow. This syndrome may need to be considered in severely obese young patients as well. These authors reported a high prevalence of UARS (about 15% to

20% based on polysomnographic criteria). We found similar results in a population of severely obese patients who were referred to consultation without sleep complaints (Tamisier, unpublished data). The physiopathology of UARS in these patients may merit some discussion as the repetitive respiratory efforts related to arousal that they experienced might not be only a consequence of UA collapse but reflect the increase in respiratory work due to reduced thoraco-abdominal compliance. However, polysomnographic criteria are close to those used in young lean UARS patients. Finally, Guilleminault, Faul, and Stoohs have described a low resting arterial blood pressure or orthostatic intolerance as occurring in one-fifth of subjects with UARS.

Polysomnographic Specificity RERA and arousal detection are the key elements during sleep monitoring (Figure 2). The number of events may be underestimated when tools used during this monitoring are not appropriate. By definition, detection of RERA is based on accurate flow measurement using the nasal cannula or a pneumotachograph, and respiratory effort measurement using either esophageal pressure or pulse transit time. By using a nasal thermocouple to detect flow, a shift between hypopnea to RERA will occur and an authentic OSAS will be misdiagnosed to be a UARS.

Figure 1 Representative patient exhibiting a UARS, with classical craniofacial abnormalities – long face syndrome with a short and narrow chin and the mandible in back position.

SLEEP DISORDERS / Upper Airway Resistance Syndrome 93 SaO2

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Figure 2 Representative pattern during a sleep study, illustrating the recurrent occurrence of respiratory event related arousals (RERA) with a progressive flow limitated aspect on nasal canula which corresponds to an increase in respiratory effort (both esophageal pressure and PTT). The consequences of these events are both sleep fragmentation (frank autonomic arousal on the first and third events) and arterial blood pressure rises. Reproduced from Pe´pin JL, Dale D, Argod J, and Le´vy PA (2005/2006) Recommendations for practical use of pulse transit time as a tool for respiratory effort measurements and microarousal recognition. In: Chokoverty S, Bhatt M, and Thomas RJ (eds.) Atlas of Sleep Medicine, Chap. 11C, with permission from Elsevier.

This has been described by Montserrat as the ‘thermistance syndrome’. We investigated subjects presenting with SDB and very little oxygen desaturation, and so being either mild OSAS or UARS, using the reference tools (pneumotachograph and esophageal pressure). They exhibited mainly hypopneas but we found only about 5% of RERAs. This is in agreement with the American Academy of Sleep Medicine report. To our understanding RERA must be considered as specific respiratory events expressed by patients depending on their UA collapsibility. To this extent, it is logical to include RERA in the Respiratory Disturbance Index. According to our studies, there is little reason to individualize UARS as a specific syndrome in a clinical population.

UARS Pathophysiology The pharyngeal collapse depends on the balance between collapsible factors like anatomic modifications, pharyngeal collapsibility, and protective factors like UA dilating muscle activity and the protective pharyngeal reflex. The more the collapsibility, the more the respiratory event severity (from RERA to apnea). In a given patient, the severity of obstructive respiratory events is not related to

different respiratory effort magnitude as assessed by esophageal pressure crescendo, but rather to different UA collapsibility. As a consequence, depending on UA collapsibility and UA protective mechanisms, the patient exhibits a variable combination among the full spectrum of obstructive respiratory events. This was also illustrated by measuring critical pressure (Pcrit) during sleep. Critical pressures have been measured and found to be less negative in snorers compared to OSAS patients, and ranging in UARS between OSAS and normal,
4.072.1, 1.672.6, and
15.476.1 cmH2O, respectively. Using negative expiratory pressure (Figure 3), we found a trend for reduced UA collapsibility in UARS when compared to OSAS. As a consequence, when measuring UA resistance, UARS exhibits higher levels compared to OSAS at the same sleep stage. The ability to maintain UA patency with high level of resistance argues for a less negative Pcrit. This does not support a specific pathophysiology for UARS but rather suggests a relative UA collapsibility leading to flow limitation or upper airway resistance events by opposition with OSAS exhibiting severe UA collapsibility and thus apneas at the same level of respiratory effort (Figure 4). A relative conservation of UA sensitivity argues again to less collapsible airways than

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in OSAS. Two studies have demonstrated that UARS subjects exhibit UA sensitivity close to normal controls. As UA sensitivity is not impaired in UARS, this may argue for a conserved UA protective reflex.

UARS Outcomes As sleep fragmentation defines UARS, it is logical that the daytime excessive sleepiness experienced by these patients may be related to it. We are not aware of data measuring cognitive dysfunction in these patients. Graded arterial blood pressure rises consecutive to graded arousals in UARS have been described during sleep. This leads to the hypothesis that nonhypoxic respiratory obstructive events might induce cardiovascular stress, possibly associated with cardiovascular morbidity in UARS. Regarding cardiovascular morbidity, few data exist to relate UARS to cardiovascular stress despite the absence of hypoxia.

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Epidemiological and experimental data provide some support to the link between sleep quality and cardiovascular disease. Some authors have also suggested the contribution of respiratory effort in the pathophysiological link between UARS and cardiovascular disease. Overall, the degree of evidence to support a significant cardiovascular morbidity in UARS is very limited.

UARS Diagnosis UARS diagnosis is based on RERA and sleep-fragmentation detection. RERA detection requires a reliable respiratory efforts measure. In adults, only pharyngeal and/or esophageal pressure and variation of pulse transit time are reliable enough to be used in this way. An optimal evaluation of inspiratory flow may be used to detect consequence of RERA that is, inspiratory flow limitation. However, two limitations need to be mentioned. First, inspiratory flow limitation is an indirect consequence of respiratory effort and it may be less accurate in REM sleep. Second, reliability of analysis will require flow signal quality, which may depend both upon hardware and software. Sleep fragmentation may be quantified by direct arousal detection using electroencephalographic measures following the specific ASDA criteria or using autonomic tools like pulse transit time.

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Therapeutic data are few and no long-term follow up is available. Continuous positive airway pressure (CPAP) has been used as therapeutic test, but compliance to treatment was uncertain. However, since several studies have demonstrated an improvement

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Figure 4 Relative proportion of apnea–hypopnea (AHI) and high-resistance episodes (RERA index: HRI) indices in three groups of patients with UARS, moderate, and severe OSAS. In these patients the absolute number of respiratory events did not differ significantly; however, the amount of high collapsibility events (i.e., apnea and hypopnea) and low collapsibility events (i.e., RERA) depend upon the sleep disorder the patient exhibits. Patients with UARS exhibit predominantly RERA and hypopnea while severe OSA exhibits mostly apneas related to their UA collapsibility status. Reproduced from Tamisier R, Wuyam B, Nicolle I, et al. (2005) Awake flow limitation with negative expiratory pressure in sleep disordered breathing. Sleep Medicine 6: 205–213, with permission from Elseveir.

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in outcome in mild OSAS with CPAP, it may also be considered an appropriate treatment in UARS. As many patients exhibit maxillofacial abnormality, surgery appears logical in normalizing these. To our knowledge, no study is available for that specific indication. Also, no evaluation exists for surgery assisted by laser or radio frequency. Using oral appliance is a common-sense decision, with the advantage being that it is reversible whereas surgery is not. A large study of this indication with outcome evaluation is required, however.

Conclusion Although UARS prevalence in the general population remains to be studied, in sleep-disordered breathing, there is some evidence to categorize UARS as an individual entity but part of OSAS with a lesscollapsible UA. This may be particularly true in young nonobese subjects with craniofacial abnormality. However, this deserves particular attention of clinicians as it may explain significant symptoms that need to be treated. Several simplified tools are available to appropriately characterize these patients based on both respiratory efforts and sleep fragmentation.

Acknowledgment This research was supported by the scientific council of AGRIRadom and the Comares. See also: Signs of Respiratory Disease: General Examination. Sleep Apnea: Adult; Children; Surgery for Sleep Apnea. Sleep Disorders: Overview.

Further Reading (1992) EEG arousals: scoring rules and examples: a preliminary report from the Sleep Disorders Atlas Task Force of the American Sleep Disorders Association. Sleep 15: 173–184. (1999) Sleep-related breathing disorders in adults: recommendations for syndrome definition and measurement techniques in clinical research. The Report of an American Academy of Sleep Medicine Task Force. Sleep 22: 667–689. (2005) International Classification of Sleep Disorders, 2nd edn., Diagnostic and Coding Manual: American Academy of Sleep Medicine. Argod J, Pepin JL, and Levy P (1998) Differentiating obstructive and central sleep respiratory events through pulse transit time. American Journal of Respiratory and Critical Care Medicine 158: 1778–1783. Argod J, Pepin JL, Smith RP, and Levy P (2000) Comparison of esophageal pressure with pulse transit time as a measure of respiratory effort for scoring obstructive nonapneic respiratory events. American Journal of Respiratory and Critical Care Medicine 162: 87–93.

Ayappa I, Norman RG, Krieger AC, et al. (2000) Non-invasive detection of respiratory effort-related arousals (REras) by a nasal cannula/pressure transducer system. Sleep 23: 763–771. Cracowski C, Pepin JL, Wuyam B, and Levy P (2001) Characterization of obstructive nonapneic respiratory events in moderate sleep apnea syndrome. American Journal of Respiratory and Critical Care Medicine 164: 944–948. Dematteis M, Levy P, and Pepin JL (2005) A simple procedure for measuring pharyngeal sensitivity: a contribution to the diagnosis of sleep apnoea. Thorax 60: 418–426. Douglas NJ (2000) Upper airway resistance syndrome is not a distinct syndrome. American Journal of Respiratory and Critical Care Medicine 161: 1413–1416. Eaker ED, Pinsky J, and Castelli WP (1992) Myocardial infarction and coronary death among women: psychosocial predictors from a 20-year follow-up of women in the Framingham Study. American Journal of Epidemiology 135: 854–864. Exar EN and Collop NA (1999) The upper airway resistance syndrome. Chest 115: 1127–1139. Frey WC and Pilcher J (2003) Obstructive sleep-related breathing disorders in patients evaluated for bariatric surgery. Obesity Surgery 13: 676–683. Gleadhill IC, Schwartz AR, Schubert N, et al. (1991) Upper airway collapsibility in snorers and in patients with obstructive hypopnea and apnea. American Review of Respiratory Disease 143: 1300–1303. Gold AR, Marcus CL, Dipalo F, and Gold MS (2002) Upper airway collapsibility during sleep in upper airway resistance syndrome. Chest 121: 1531–1540. Guilleminault C, Black JE, and Palombini L (1999) High (or abnormal) upper airway resistance. Revue des Maladies Respiratoires 16: 173–180. Guilleminault C, Faul JL, and Stoohs R (2001) Sleep-disordered breathing and hypotension. American Journal of Respiratory and Critical Care Medicine 164: 1242–1247. Guilleminault C and Le´ger D (2005) Le syndrome des voies ae´riennes supe´rieures: pertinence clinique et physiopathologique. Revue des Maladies Respiratoires 22: 27–30. Guilleminault C, Li K, Chen NH, and Poyares D (2002) Two-point palatal discrimination in patients with upper airway resistance syndrome, obstructive sleep apnea syndrome, and normal control subjects. Chest 122: 866–870. Guilleminault C, Poyares D, Palombini L, et al. (2001) Variability of respiratory effort in relation to sleep stages in normal controls and upper airway resistance syndrome patients. Sleep Medicine 2: 397–405. Guilleminault C, Stoohs R, Clerk A, Cetel M, and Maistros P (1993) A cause of excessive daytime sleepiness. The upper airway resistance syndrome. Chest 104: 781–787. Guilleminault C, Stoohs R, Shiomi T, Kushida C, and Schnittger I (1996) Upper airway resistance syndrome, nocturnal blood pressure monitoring, and borderline hypertension. Chest 109: 901–908. Lofaso F, Goldenberg F, d’Ortho MP, Coste A, and Harf A (1998) Arterial blood pressure response to transient arousals from NREM sleep in nonapneic snorers with sleep fragmentation. Chest 113: 985–991. Loube DI and Andrada TF (1999) Comparison of respiratory polysomnographic parameters in matched cohorts of upper airway resistance and obstructive sleep apnea syndrome patients. Chest 115: 1519–1524. Meier-Ewert HK, Ridker PM, Rifai N, et al. (2004) Effect of sleep loss on C-reactive protein, an inflammatory marker of cardiovascular risk. Journal of the American College of Cardiology 43: 678–683.

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Montserrat JM and Badia JR (1999) Upper airway resistance syndrome. Sleep Medicine Reviews 3: 5–21. Pe´pin JL, Dale D, Argod J, and Le´vy PA (2005/2006) Recommendations for practical use of pulse transit time as a tool for respiratory effort measurements and microarousal recognition. In: Chokoverty S, Bhatt M, and Thomas RJ (eds.) Atlas of Sleep Medicine, Chap. 11C. Phillipson EA (1993) Sleep apnea – a major public health problem. New England Journal of Medicine 328: 1271–1273. Pitson DJ and Stradling JR (1998) Autonomic markers of arousal during sleep in patients undergoing investigation for obstructive sleep apnoea, their relationship to EEG arousals, respiratory events and subjective sleepiness. Journal of Sleep Research 7: 53–59. Poyares D, Guilleminault C, Rosa A, Ohayon M, and Koester U (2002) Arousal, EEG spectral power and pulse transit time in UARS and mild OSAS subjects. Clinical Neurophysiology 113: 1598–1606.

Stradling JR, Barbour C, Glennon J, Langford BA, and Crosby JH (2000) Which aspects of breathing during sleep influence the overnight fall of blood pressure in a community population? Thorax 55: 393–398. Strohl KP and Redline S (1996) Recognition of obstructive sleep apnea. American Journal of Respiratory and Critical Care Medicine 154: 279–289. Tamisier R, Pepin JL, Wuyam B, et al. (2000) Characterization of pharyngeal resistance during sleep in a spectrum of sleepdisordered breathing. Journal of Applied Physiology 89: 120–130. Tamisier R, Wuyam B, Nicolle I, et al. (2005) Awake flow limitation with negative expiratory pressure in sleep disordered breathing. Sleep Medicine 6: 205–213. Yoshida K (2002) Oral device therapy for the upper airway resistance syndrome patient. Journal of Prosthetic Dentistry 87: 427–430.

SMOOTH MUSCLE CELLS Contents

Airway Vascular

Airway

Introduction

S J Hirst, King’s College London, London, UK

Airway smooth muscle is the major contractile tissue of the airways in the lung. Its localization and arrangement within the airway wall appears optimized to effect changes in airway caliber through active force generation and cell shortening. However, like smooth muscle present in other hollow organs, airway smooth muscle retains a capacity for other functions, including growth, migration, and synthesis of a vast array of regulatory molecules, which are manifest during disease. Coordination of these varied functions allows airway caliber to be regulated both transiently via reversible muscle shortening and in the longer term by alterations in its content and synthetic activity, perhaps as part of the normal contractile and repair programming of airway smooth muscle. In disorders of the lung involving airway narrowing exemplified by asthma, this programming is disrupted and the function of airway smooth muscle becomes aberrant.

& 2006 Elsevier Ltd. All rights reserved.

Abstract Below the level of the main bronchus, airway smooth muscle encircles the entire airway in a roughly circular orientation and is the primary effector cell regulating bronchomotor tone. In the healthy adult airway, its physiological function appears vestigial. In contrast, in lung disease its role seems unequivocal, particularly in allergic asthma. Contemporary concepts center on the remarkable mechanical and functional plasticity of airway smooth muscle. The traditional view that contraction is its sole function has given way to a new paradigm in which noncontractile properties, such as growth and immunomodulation, contribute to development and perpetuation of airway hyperresponsiveness. Ideas of contraction have advanced from classical sliding filament theory to models that reflect the oscillatory nature of loads imposed on the smooth muscle in the airways. Cyclical interactions of myosin cross-bridges with actin filaments underpin the initial shortening, whereas dynamic reorganization and polymerization of these filaments ensures maintenance of contraction during nonstatic conditions. Aside from contraction, airway smooth muscle content is pathologically increased in some airway diseases. Underlying mechanisms of accumulation in asthma remain illusive but likely involve the phosphoinositide 30 -kinase and extracellular signalregulated kinase pathways. Additional pathological functions include its newly described secretory or immunomodulatory properties allowing the muscle to contribute to known alterations within the extracellular matrix in asthma, as well as to mucosal inflammation, by expression of cellular adhesion molecules and release of multiple chemokines and cytokines. Thus, an integrated paradigm for plasticity of function has emerged that encompasses both acute and long-term regulation of contraction as well as proliferation and immunomodulation as smooth muscle effector mechanisms in lung disease.

Normal Anatomy and Location

Airway smooth muscle was first described in the early nineteenth century by the German anatomist Franz Reisseisen, who, using a hand-held magnifier, reported its arrangement in bundles in the bronchial tree to the level of the membranous bronchioles, from where it was inferred to continue to the lung periphery. Later works established that airway smooth muscle extends from the cartilaginous airways of the trachea and bronchi to the terminal bronchioles, where the smooth muscle fibers of the smallest bronchi are referred to as Reisseisen’s muscles.

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The arrangement of airway smooth muscle varies according to its location in the airway tree. In the posterior aspect of the adult trachea it forms the trachealis, a unified layer of thick unbranched bundles from the larynx to the carina in which the parallel smooth muscle fibers are orientated perpendicular to the long axis of the trachea to span the gap between the ends of incomplete cartilaginous rings. The trachealis comprises more than 75% muscle, with connective tissue, fibroblasts, and neuronal structures comprising the major portion of the balance. Airway smooth muscle cells in the large segmental or lobar bronchi are similar to those in trachealis. Muscle bundles encase the bronchi in a roughly circular orientation but appear devoid of a specific anatomic arrangement where they branch and interdigitate with plates of cartilage and other connective tissue structures within the airway wall. More distally, in the peripheral noncartilaginous airways the smooth muscle bundles surround the airway in a helix–antihelix pattern, with extensive crisscrossing of fibers between bundles giving an overall geodesic arrangement (Figure 1). The helical pitch of these bundles varies with lung inflation and airway level but is in the range 713–301 in noncartilaginous airways. However, because of the regularity of crossover of fibers from one bundle to another, the magnitude of this obliquity may not result in physiologically important changes in airway length during bronchoconstriction. Although absolute amounts of airway smooth muscle are less in peripheral airways compared with proximal airways, the proportion of airway wall structure occupied by muscle is higher in periphery (15–25%) than in proximal airways (2–4%). Classically, smooth muscle is categorized into single unit, multiunit, or intermediate types based on the extent of innervation to individual muscle cells and on its capacity to generate spontaneous contractile responses and myogenic tone via propagation of membrane-depolarizing pulses. Resting airway smooth muscle from the large central airways including the trachealis displays multiunit or intermediate properties having few gap junctions and being devoid of spontaneous action-potential generation or myogenic tone, but individual smooth muscle cells are relatively sparsely innervated. However, in some experimental situations, the trachealis exhibits spontaneous action potentials and myogenic contractile activity, reminiscent of single-unit smooth muscle. Likewise, smooth muscle in the smaller bronchioles appears to be single unit, typified by spontaneous action-potential generation and myogenic tone, and it is less innervated and has more gap junctions than smooth muscle in the larger central airways.

(a)

n

n

(b) Actin filament

Nucleus

Myosin Dense filament Plasmalemma plaque

Dense body

(c) Figure 1 Morphological and ultrastructural features of airway smooth muscle showing (a) peribronchial view of a-actin-labeled bundles in developing airways and (b) general orientation of contractile filaments and dense bodies (arrowheads) along the long axes of individual cells. Intercellular coupling via dense plaques appears within the oval. The inset shows thick myosin filaments running toward and past the nucleus (n). The scale bar represents 100 mm in (a) and 1 mm in (b). (c) Schematic of (b) showing overall contractile filament architecture in an airway smooth muscle bundle. (a) Reproduced from Sparrow MM and Lamb JP (2003) Ontogeny of airway smooth muscle: structure, innervation, myogenesis and function in fetal lung. Respiratory Physiology & Neurobiology 137: 361–372, with permission from Elsevier. (b and c) Reproduced from Kuo KH and Seow CY (2004) Contractile filament architecture and force transmission in swine airway smooth muscle. Journal of Cell Science 117: 1503– 1511, with permission from The Company of Biologist, Ltd.

Ultrastructural Features

Airway smooth muscle bundles interconnect with numerous collagen-rich elastic fibers of the extracellular matrix to provide mechanical coupling between

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the muscle and surrounding tissues. Individual airway smooth muscle cells are fusiform (spindleshaped) and typically are 3–5 mm wide and can exceed 1 mm in length. The detection and identification of many ultrastructural features within these cells is dependent on orientation. Mechanical coupling between cells occurs via dense plaques and gap junctions as well as frequent small invaginations of membrane called caveolae. Each smooth muscle cell may contain more than 100 000 of these invaginated structures occupying as much as 30% of the plasma membrane. Characteristic of smooth muscle, the nucleus is elongated and centrally located within the cytoplasm and often appears crenulated at higher power (Figure 1(b) inset). Mitochondrial clusters and Golgi apparatus are perinuclear, appearing most often adjacent to each pole of the nucleus. The sarcoplasmic reticulum accounts for 2–6% of the cytoplasm. In mature smooth muscle, it is in intimate contact with pinocytic vesicles and caveolae at the plasma membrane. Evidence suggests that there are two types of sarcoplasmic reticulum in airway smooth muscle: superficial and deep. In three dimensions, it is likened to a hollow pancake, with its flat face adjacent to the plasma membrane in the case of superficial sarcoplasmic reticulum or perpendicular for the deep sarcoplasmic reticulum, where the opposite edge extends to the mitochondria. The superficial sarcoplasmic reticulum and its proximity to the plasma membrane may govern local calcium gradients or oscillations, whereas the deep sarcoplasmic reticulum may provide a direct conduit for calcium transport within the cell and perhaps ultimately regulation of mitochondrial calcium concentration. The major feature of any mature smooth muscle cell under the transmission electron microscope is the dense appearance of numerous parallel myofilaments in the cytoplasm running along the longitudinal axis (Figure 1). These comprise mainly smooth muscle myosin heavy-chain thick and smooth muscle a-actin thin filaments that form the backbone of the force-generating machinery, as well as desmin- and vimentin-containing intermediate filaments. Frequent cytoplasmic and membrane-associated dense bodies and dense plaques form anchor points for attachment of thin and intermediate filaments (Figure 1(c)). Dense plaques are synonymous with focal adhesions, sites of linkage between actin filament polymerization, and membrane-spanning b subunits of collagen ligating integrins. Other actin filaments appear to attach to the nuclear envelope, making it a force-transmitting structure (Figure 1(b)). The resultant mesh of thin and intermediate filaments connected through dense plaques also includes long hollow tubular structures at regular intervals, called microtubules,

composed of tubulin proteins. Together, these structures form much of the cytoskeleton scaffold giving the cell its shape. The free ends of a-actin filaments interdigitate with myosin filaments, and the crossbridge interactions formed allow the actin and myosin filaments to slide over one another in opposite directions during cross-bridge cycling, pulling the dense plaques closer and shortening the cell during contraction (Figure 1(c)). The parallel arrangement of actomyosin filaments via dense plaques is aligned with similar sites in adjacent cells to create a transcellular parallel alignment of myofilaments that may allow airway smooth muscle to shorten as a syncytium (Figure 1(c)). Research suggests that the ultrastructural arrangement and density of thick myosin filaments in airways smooth muscle is highly dynamic and increases with contractile agonist stimulation, possibly involving myosin polymerization and elongation of myosin filaments. Ontogeny and Myogenesis

Although substantial morphological similarities exist between smooth muscle of the airways and that residing in other hollow organs, its pattern of development in the airways differs from that of other smooth muscle tissues. During embryogenesis, airway smooth muscle appears before vascular smooth muscle and originates from mesenchymal cells during branching morphogenesis – a period of prolific branching that establishes the bronchial tree. Initiation of airway smooth muscle differentiation and its arrangement into bundles begins by induction of a thin ring of mesenchyme cells around the base of the epithelial bud immediately under the epithelium at sites of laminin basement membrane assembly. It is assumed, as with myogenesis elsewhere, that maturation involves transition of mesenchyme cells to smooth muscle myoblasts and then to immature smooth muscle cells followed by maturation. In addition to laminin, soluble factors, including transforming growth factor beta released by the epithelium, are also important in maturation progression. The maturation ring steadily widens as new bundles appear distally, such that maturation progresses from the terminal tubules to the trachea as the developing epithelial bud extends. Initially, bundles encircle the developing trachea, but this is progressively restricted to the dorsal wall to form the trachealis as the incomplete cartilage rings develop in the pseudoglandular stage. The first detected maturation and differentiation marker proteins are smooth muscle a-actin and calponin. Other markers follow, also in the early pseudoglandular stage, including smooth muscle myosin heavy chain, sm-22a, and desmin. The appearance of mature

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airway smooth muscle cells in developing airway walls is temporally linked to the generation of regular waves of spontaneous action potentials and rhythmic contractions and relaxations, similar to that in the developing gut. These propel liquid toward the periphery and cause marked pulsatile distensions of the epithelial buds and elongation of the mesenchyme cells therein. The signals driving airway smooth muscle myogenesis and differentiation are poorly defined, but observations suggest that contact with polymerized laminin in basement membranes and rhythmic mechanical stretch during branching morphogenesis are required via induction of serum response factor and its cofactor, myocardin, as well as other transcription factors. Mechanical forces exerted on airway smooth muscle by tethering of the lung parenchyma may also modulate airway smooth muscle differentiation via induction of transcription of smooth muscle contractile protein genes. Postnatally, airway smooth muscle undergoes slow graded shortenings in which action potentials are no longer required to support rhythmic contractions.

Airway Smooth Muscle Function in the Normal Lung Airway smooth muscle is exposed continuously to changing mechanical conditions during normal breathing and is periodically subjected to larger forces of expansion by intermittent deep breaths. However, no consensus has emerged on its precise function in the healthy adult airway. In fact, much of the physiology of ventilation and its regional distribution can be explained by regarding the airways as passive conducting tubes, with the respiratory skeletal muscles providing the power stroke for ventilation and the smooth muscle being superfluous in these events. Indeed, relaxation of airway smooth muscle in healthy subjects by inhalation of the b2adrenoceptor agonist, albuterol, does not result in improved lung spirometry or ventilation. Speculation on possible functions has centered on its contractile role and includes peristalsis to assist exhalation and mucus propulsion and also the regional distribution of airflow during ventilation and optimization of anatomical dead space volume for matching of alveolar ventilation with blood perfusion to reduce the work of breathing. Other possibilities include stabilization of the airway wall and enhancing the effectiveness of cough or ejection of foreign material. It has been proposed that peristaltic contractions of airway smooth muscle in fetal lung generate fluid pressure for fetal airway differentiation and branching. However, most explanations for the presence of airway smooth muscle in healthy adults have not been based

on experimental validation and do not reflect a modern understanding of human physiology. Thus, airway smooth muscle in healthy adult airways appears vestigial, perhaps similar to the appendix or wisdom teeth, having no identifiable function but when diseased it can be the cause of major medical complications. Proponents of its vestigial nature have suggested its removal by pharmacological targeting with a directed toxin or physicochemical disruption to provide a somewhat drastic if not effective treatment for lung disease in the future.

Airway Smooth Muscle in Respiratory Diseases There is no known pathological condition or appreciable physiological defect that results in loss of airway smooth muscle function. Instead, in stark contrast to its subtle importance in the healthy airway, its amplified function is only too apparent in lung disease. Understanding these processes, particularly pathways that regulate cell differentiation and intracellular signaling, has relied largely on the development of cell culture-based methods. Accumulating evidence from these and intact tissue systems from human or animal airways suggests that airway smooth muscle function is remarkably diverse. It can respond to external stimulation in a variety of ways – by contraction and relaxation, by altered growth, and by increased secretion. Such studies must be viewed in the context of model systems to inform the future design and development of improved methodologies to test the importance of airway smooth muscle to the cause or progression of lung disease. Inappropriate airway smooth muscle responses are hypothesized to contribute to various lung pathologies, including asthma, chronic obstructive pulmonary disease, chronic lung disease of immaturity (bronchopulmonary dysplasia), and the rare disorder lymphangioleiomyomatosis, which affects young women. Limited evidence also supports alterations in airway smooth muscle in sudden infant death syndrome. However, most research has centered on understanding amplified airway smooth muscle responses as a basis for the airway hyperresponsiveness in asthma and, to a lesser extent, in chronic obstructive pulmonary disease. Mechanical Plasticity of Contraction in Asthma

Moderate or excessive airway smooth muscle shortening is the unequivocal basis of asthmatic symptoms and disability, particularly during acute allergentriggered exacerbations. However, theories of smooth muscle contraction are currently under radical review.

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Previously, airway smooth muscle shortening leading to airway lumen narrowing in asthma was considered strictly within the confines of a static equilibrium of forces. New concepts have displaced much of the classical understanding of contraction to account for the continuously changing oscillatory stresses and strains exerted on airway smooth muscle in its fundamentally dynamic, nonequilibrium environment created by tidal loading during spontaneous breathing and intermittent deep breaths. Force generation and cell shortening depends critically on levels of cytosolic calcium and the interaction of smooth muscle a-actin with myosin myofilaments via cross-bridge cycling. A prerequisite for the latter is phosphorylation of 20 kDa myosin light-chains, which is driven by smooth muscle myosin light-chain kinase by the binding of free calcium to calmodulin and countered by myosin light-chain phosphatase (Figure 2). Airway spasmogens, such as acetylcholine, leukotriene D4, endothelin-1, thrombin, or histamine, released from either airway nerves or inflammatory cells activate cognate cell surface Ga-type G-protein-coupled receptors via phospholipase C to cause an increase in cytosolic calcium concentration and propagation of calcium

oscillations. Calcium influx derives from the extracellular space and from release via second messengers of intracellular calcium stores, principally the sarcoplasmic reticulum. Two major types of calciumrelease channels are postulated in airway smooth muscle sarcoplasmic reticulum: one activated by inositol 1,4,5-trisphosphate (IP3) and the other termed the ryanodine receptor (RyR). The initial phase of the biphasic calcium response derives primarily from IP3-dependent calcium release, whereas more sustained elevations are dependent more on calciuminduced calcium release via RyR channels. The frequency of calcium oscillations occurring via repetitive release from RyR channels increases with agonist concentration, resulting in an overall higher mean calcium concentration. Several proinflammatory (interleukin (IL)-1b and tumor necrosis factor (TNF)-a) and Th2 (IL-4, IL-5, and IL-13) cytokines in the asthmatic inflammatory environment may induce airway hyperresponsiveness by acting directly on airway smooth muscle to enhance agonist-evoked calcium signals and contractile responses and by diminishing relaxations evoked by b2-adrenoceptor activation. Enhanced responsiveness of airway smooth muscle after cytokine treatment appears dependent

Histamine, acetylcholine, leukotrienes, endothelin-1

R G
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SR RyR

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Smooth muscle cell Figure 2 Classical pathway leading to contraction development in airway smooth muscle. Activation of cell surface G-protein (G)coupled receptors (R) via phospholipase C (PLC) results in the release of stored calcium (Ca2 þ ) from the sarcoplasmic reticulum (SR) via inositol trisphosphate (IP3)- and ryanodine (RyR)-activated channels. Binding of Ca2 þ to calmodulin (CaM) results in activation of myosin light-chain kinase (MCLK) to phosphorylate (P) 20 kDa myosin light-chains (MLC20) for the initiation of actomyosin cross-bridge cycling and active force generation. MLC20 dephosphorylation by myosin light-chain (MLC) phosphatase reduces cross-bridge formation and force generation.

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filament lengthening alone can explain the observed slowing of muscle-shortening velocity that occurs when approaching the sustained phase of a single contraction without any need to postulate a change in the rate of cross-bridge cycling – previously known as the latch state. Other theories of mechanical plasticity operating during sustained length adaptation between contractions involve polymerization and elongation of thin smooth muscle a-actin-containing filaments following the activation and clustering of b1 integrin focal adhesions. A refinement of both these possibilities is the perturbed equilibrium model of myosin binding, in which the oscillatory strain on airway smooth muscle occurring with each lung inflation during breathing is transmitted directly to the myosin head to cause unending perturbations of optimal actin and myosin binding. The result is that the contractile machinery is normally kept off balance and is unable to achieve its full force-generating potential. Each of these possibilities may operate coincidentally and is integrated completely by considering airway smooth muscle as a glassy material in which, by cytoskeletal and contractile filament deformation and reorganization, the mechanical properties of muscle are regulated from a state of relative fluidity to the rigidity of a solid. The underlying signaling pathways responsible for mechanical plasticity and glassy behavior are only just beginning to be investigated,

on RyR channel activation, and components of the cyclic ADP ribose pathway including transcriptional activation of CD38 are implicated in the amplification. In contrast, attenuated relaxations to salbutamol and other b-adrenoceptor agonists by cytokines involve uncoupling of the b2-adrenoceptor. Perhaps the most significant development and radical departure from the classical sliding filament hypothesis is the recent emergence of mechanical plastic theory for the understanding of sustained airway smooth muscle contraction under nonequilibrium conditions. Its tenet is that although the rate and extent to which airway smooth muscle shortens in response to a spasmogen are linear functions of smooth muscle length, active force generation is independent of length. Plastic restructuring is envisaged when dynamic length changes occur between sequential contractile events or during a single contraction. On a molecular level, several dynamic models are proposed to explain airway smooth muscle mechanical adaptation during single or multiple stimulations. One of these is referred to as the series-to-parallel filament transition theory and involves the net addition in series of thick myosin-containing filaments to increase the number of parallel contractile elements when optimal force generation is exceeded. At shorter lengths, depolymerization of contractile elements occurs. A major attraction of this model is that myosin

Contraction/ length change

p38

HSP27 Elongation/reorganization of actomyosin filaments RhoA

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Figure 3 Mechanism of mechanical plasticity in airway smooth muscle. Polymerization of globular (G) to filamentous (F) actin is regulated by pathways requiring RhoA, p38 mitogen-activated protein (MAP) kinase, and heat-shock protein (HSP) 27. Polymerization of myosin monomers is regulated by RhoA and by RhoA kinase (ROCK). Adapted from Halayko AJ and Solway J (2001) Molecular mechanisms of phenotypic plasticity in smooth muscle cells. Journal of Applied Physiology 90: 358–368, used with permission from The American Physiological Society.

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and available information relates to airway smooth muscle from nonasthmatics. Initial findings implicate agonist-induced activation of Rho GTP-binding proteins such as RhoA and its downstream intermediates in polymerization of myosin thick filaments, whereas RhoA, p38 MAP kinase, focal adhesion components, and small heat-shock proteins such as HSP27 appear to be effectors of actin thin fiber polymerization (Figure 3). It remains unclear whether a definite intrinsic abnormality exists in the contractile machinery of airway smooth muscle from asthmatics to account for the enhanced bronchoconstriction in asthma. A leading study showed that the shortening ability of unloaded bronchial smooth muscle cells from human subjects with asthma is increased. Measurements also indicate that increased smooth muscle myosin light-chain kinase content, via increased actomyosin ATPase activity, may account for the enhanced contractility. Abnormalities may also be present in the mechanisms that limit bronchoconstriction. In particular, bronchoconstriction in asthmatics induced by spasmogens such as methacholine is not reversed by deep inspiration, unlike bronchoconstriction in healthy individuals. Two possibilities to explain these differences are being studied. One is that airway smooth muscle in asthmatics shortens faster and thus recontracts more quickly after deep inspiration than in healthy airways, with the result that any bronchodilating effect of deep inhalation does not persist in asthmatics. The second relates to differences in the plasticity (stretch without regaining its original configuration)–elasticity balance of airway smooth muscle from healthy or asthmatic individuals. This may result from loss of airway smooth muscle–lung parenchymal interdependence (due to airway wall structural changes in asthma) or, at the level of contractile filament function, from excessive elasticity of asthmatic muscle, thus limiting the effects of deep inspiration, perhaps because the glassy properties of healthy and asthmatic airway smooth muscle cells differ. Enhanced Growth of Airway Smooth Muscle in Lung Disease

Increased airway smooth muscle content in small and large airways is a characteristic finding in fatal and nonfatal severe asthma and is present in the small cartilaginous airways of preterm infants with chronic lung disease (bronchopulmonary dysplasia), particularly those who require artificial ventilation. There are limited reports of airway smooth muscle accumulation in cases of sudden infant death syndrome and in lymphangioleiomyomatosis in young women. Airway smooth muscle accumulation also occurs in

severe chronic obstructive pulmonary disease but only in patients in whom there is persistent airflow obstruction and not to the extent found in asthma. As with contraction, most of the information on airway smooth muscle accumulation relates specifically to asthma, in which the increase correlates directly with both the severity and duration of the disease. Although not proven directly, it is generally agreed from mathematical modeling of thickened large and small airways that accumulation of airway smooth muscle is the major component of the development of airway hyperresponsiveness in severe asthma. Histopathological studies have reported that accumulation of airway smooth muscle involves both hyperplastic and hypertrophic processes, although migration of mesenchymal muscle precursor cells to form the additional muscle bulk cannot be excluded and this may explain why so few studies have detected increased markers of proliferation in airway smooth muscle from asthmatics. Bronchial biopsy studies reveal increased airway smooth muscle content even in mild asthmatics, but as with earlier autopsy findings, no consensus exists for the Thrombin Tryptase

PDGF EGF

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Figure 4 Example of mitogens and key signal transduction mechanisms that regulate airway smooth muscle cell division. Mitogens acting predominantly through receptor tyrosine kinaselinked receptors (RTK) or G-protein-coupled receptors (GPCR) activate small GTPase p21ras proteins, which interact with downstream effectors. These include phosphoinositide 30 -kinase (PI3K) and extracellular signal regulated kinase (ERK). PI3K, via 3-phosphorylated phosphoinositides and other intermediates, activates the NADPH oxidase complex to stimulate cell division.

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importance of either hyperplasia or hypertrophy in the events leading to these changes. The mitogens responsible for increased airway smooth muscle growth in vivo are unknown, although several mediators and mitogenic intracellular signaling pathways have emerged as leading candidates using in vitro airway smooth muscle cell culture-based studies (Figure 4). Some, but not all, airway smooth muscle

mitogens also promote migration of airway smooth muscle in vitro, although whether airway smooth muscle cell migration occurs in the intact airway wall is uncertain. The majority of mitogens identified fall into two broad categories: polypeptide growth factors that activate receptors with intrinsic protein tyrosine kinase activity and those that act through receptors coupled to heterotrimeric G-proteins, which includes Bronchoprotective mediators

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Figure 5 (a) Cultured human airway smooth muscle cells produce cytokines, chemokines, inflammatory mediators, matrix-modifying enzymes, growth factors, and adhesion molecules. (b) Cross-section of an intermediate airway of a patient with asthma showing eotaxin immunoreactivity in smooth muscle cells (large arrows) and epithelium (small arrow). (c) High-power magnification of transverse section of a large airway in a patient with asthma, demonstrating eotaxin immunoreactivity in bundles of smooth muscle (arrows). (a) Reproduced from Johnson SR and Knox AJ (1997) Synthetic functions of airway smooth muscle in asthma. Trends in Pharmacological Sciences, 18: 288–292, with permission from Elsevier. (b and c) Reproduced from Ghaffar O, Hamid Q, Renzi PM, et al. (1999) Constitutive and cytokine-stimulated expression of eotaxin by human airway smooth muscle. American Journal of Respiratory and Critical Care Medicine 159: 1933–1942, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

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the thromboxanes, acetylcholine, endothelin-1, leukotriene D4, and inflammatory cell-derived proteases. Many of these are co-mitogens that synergize with polypeptide growth factors to promote accelerated growth in vitro. Major effector signaling pathways include activation of phosphoinositide 3-kinase and extracellular signal-regulated kinase as well as NADPH oxidase pathways (Figure 4). Phosphoinositide 30 -kinase activation may also be important in airway smooth muscle hypertrophic changes. Initial reports suggest that airway smooth muscle cells cultured from asthmatics have an increased ability to proliferate, and that the altered composition of the airway wall extracellular matrix environment that predominates in asthma amplifies growth while impairing the antiproliferative effect of glucocorticoids. These observations may provide a mechanism for steroid-resistant asthma and may inform the design of future therapies that target airway smooth muscle accumulation in lung disease. Immunomodulatory Role

Findings with regard to airway smooth muscle suggest that it also fulfills an immunomodulatory role in asthma through the production of multiple proinflammatory cytokines (IL-1b, IL-5, IL-6, and Phenotypic plasticity

Maturation

GM-CSF) and chemokines (IL-8, RANTES, eotaxin, MCP, TARC), polypeptide growth factors (PDGF, TGF-b, CTGF, VEGF, and c-kit ligand), interstitial extracellular matrix proteins (collagen, fibronectin, perlecan, laminin, and elastin), cell adhesion molecules (ICAM-1, VCAM-1, CD40, CD44, and integrins), cytokine (TNFR1/2, IL-1R, IL-4Ra, and IL-13Ra1/2) and chemokine receptors (CCR3), as well as co-stimulatory molecules (CD80/CD86) (Figure 5). Thus, available data support a role for airway smooth muscle in contributing to the known alterations within the extracellular matrix in asthma and to actively perpetuate airway mucosal inflammatory processes, including mast cell and leukocyte (T lymphocyte, neutrophil, and eosinophil) activation and recruitment. Orchestration of these events by airway smooth muscle may be especially relevant in the diseased lung, in which its content is increased, and initial findings with cultured airway smooth muscle cells derived from asthmatics suggest that these cells respond differently, both qualitatively and quantitatively. Moreover, the production of anti-inflammatory mediators by airway smooth muscle that exert a braking effect on local inflammation, such as prostaglandin E2, may be impaired in airway smooth muscle from asthmatics. Initial studies have revealed Mechanical plasticity

Contractile filament reorganization

Modulation • Length change • Contraction

Synthetic myocyte • Cell division & hypertrophy • Migration • ECM synthesis • Cytokine synthesis

Contractile myocyte • Contract • Cellular hypertrophy • Do not migrate • Cytokine/chemokine synthesis • ECM synthesis

Figure 6 An integrated paradigm for functional and phenotypic plasticity of airway smooth muscle. This schematic representation shows the association between phenotypic and mechanical plasticity in smooth muscle. Phenotypic plasticity results from reversible modulation and maturation of smooth muscle cells between a functionally synthetic and contractile state. This diversity is mainly controlled through regulated expression of specific genes in response to a number of factors, including proinflammatory stimuli, changes in extracellular matrix, and cell–cell interactions. Mechanical plasticity occurs in contractile myocytes as the result of subcellular reorganization of the contractile thick and thin filaments in response to acute or chronic muscle length change during contraction or due to stretch. Reproduced from Halayko AJ and Amrani Y (2003) Mechanisms of inflammation-mediated airway smooth muscle plasticity in airways remodeling in asthma. Respiratory Physiology & Neurobiology 137: 209–222, with permission from Elsevier.

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that as with other cell types, multiple transcription factors and signaling cascades operate in airway smooth muscle for chemokine and cytokine production. Regulation of these mediators occurs at both transcriptional and posttranscriptional levels, although transcription has been studied more in these cells.

Conclusion The traditional passive partner view in which airway smooth muscle is the major effector cell regulating bronchomotor tone in response to various neurotransmitters, proinflammatory mediators, and exogenous substances is likely outmoded. In its place, an integrated paradigm for plasticity of airway smooth muscle function in lung disease has emerged that encompasses both acute and long-term modulation of cell contraction, proliferation, and synthesis/ secretion of inflammatory mediators (Figure 6). Increasing evidence supports a principal role for airway smooth muscle as an effector in airway hyperresponsiveness not only through its increased contractile responses to bronchoconstrictors and attenuated responses to bronchodilators but also as a result of its increased content in the airway wall, its production of extracellular matrix proteins in the muscle layer, submucosa, and adventia, and its capacity to perpetuate local inflammation. See also: Adhesion, Cell–Matrix: Focal Contacts and Signaling; Integrins. Asthma: Overview. Bronchodilators: Beta Agonists. Bronchopulmonary Dysplasia. Chemokines. Chronic Obstructive Pulmonary Disease: Overview. Contractile Proteins. Cytoskeletal Proteins. Extracellular Matrix: Basement Membranes; Collagens; Matrix Proteoglycans. G-Protein-Coupled Receptors. Interstitial Lung Disease: Lymphangioleiomyomatosis. Ion Transport: Calcium Channels. Lung Anatomy (Including the Aging Lung). Lung Development: Overview. Neurophysiology: Neural Control of Airway Smooth Muscle. Signal Transduction. Smooth Muscle Cells: Vascular.

Further Reading Fabry B and Fredberg JF (2003) Remodeling of the airway smooth muscle cell: are we built of glass? Respiratory Physiology & Neurobiology 137: 109–124. Fernandes DJ, Mitchell RW, Lakser O, et al. (2003) Do inflammatory mediators influence the contribution of airway smooth muscle contraction to hyperresponsiveness in asthma? Journal of Applied Physiology 95: 844–853. Ghaffar O, Hamid Q, Renzi PM, et al. (1999) Constitutive and cytokine-stimulated expression of eotaxin by human airway smooth muscle. American Journal of Respiratory and Critical Care Medicine 159: 1933–1942.

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Gunst SJ, Tang DD, and Opazo Saez A (2003) Cytoskeletal remodeling of the airway smooth muscle cell: a mechanism for adaptation to mechanical forces in the lung. Respiratory Physiology & Neurobiology 137: 109–124. Halayko AJ and Amrani Y (2003) Mechanisms of inflammationmediated airway smooth muscle plasticity in airways remodeling in asthma. Respiratory Physiology & Neurobiology 137: 209–222. Halayko AJ and Solway J (2001) Molecular mechanisms of phenotypic plasticity in smooth muscle cells. Journal of Applied Physiology 90: 358–368. Hirst SJ (2003) Regulation of airway smooth muscle immunomodulatory cell function: role in asthma. Respiratory Physiology & Neurobiology 137: 309–326. Hirst SJ, Martin JG, Bonacci JV, et al. (2004) Proliferative aspects of airway smooth muscle. Journal of Allergy and Clinical Immunology 114: S2–S17. Howarth PH, Knox AJ, and Amrani Y (2004) Synthetic responses in airway smooth muscle. Journal of Allergy and Clinical Immunology 114: S32–S50. Johnson PR and Burgess JK (2004) Airway smooth muscle and fibroblasts in the pathogenesis of asthma. Current Allergy and Asthma Reports 4: 102–108. Johnson SR and Knox AJ (1997) Synthetic functions of airway smooth muscle in asthma. Trends in Pharmacological Sciences, 18: 288–292. Kuo KH, Herrera AM, and Seow CY (2003) Ultrastructure of airway smooth muscle. Respiratory Physiology & Neurobiology 137: 197–208. Kuo KH and Seow CY (2004) Contractile filament architecture and force transmission in swine airway smooth muscle. Journal of Cell Science 117: 1503–1511. Ma X, Cheng Z, Kong H, et al. (2002) Changes in biophysical and biochemical properties of single bronchial smooth muscle cells from asthmatic subjects. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L1181–L1189. Mitchell RW and Halayko AJ (1997) Airway smooth muscle structure. In: Barnes PJ, Grunstein MM, Leff AR, and Woolcock AJ (eds.) Asthma, pp. 733–758. Philadelphia: Lippincott-Raven. Mitzner W (2004) Airway smooth muscle: the appendix of the lung. American Journal of Respiratory and Critical Care Medicine 169: 787–790. Seow CY and Fredberg JJ (2001) Historical perspective on airway smooth muscle: the saga of a frustrated cell. Journal of Applied Physiology 91: 938–952. Sparrow MM and Lamb JP (2003) Ontogeny of airway smooth muscle: structure, innervation, myogenesis and function in fetal lung. Respiratory Physiology & Neurobiology 137: 361–372. Zhou L and Hershenson MB (2003) Mitogenic signaling pathways in airway smooth muscle. Respiratory Physiology & Neurobiology 137: 295–308.

Vascular D Bishop-Bailey and K E Swales, Queen Mary, University of London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Vascular smooth muscle cells (VSMCs) provide the structural integrity for blood vessel walls. These multifunctional cells exhibit a remarkable phenotypic plasticity, allowing rapid reversible

106 SMOOTH MUSCLE CELLS / Vascular changes in function in response to alterations in their local environment. Adult VSMCs orchestrate their major function of controlling the pulmonary blood flow by contracting and relaxing in response to local and systemic stimuli. In addition to their contractile function, during vasculogenesis, injury or disease, VSMCs are proliferative and synthetic. The number of cells with a more synthetic phenotype is enhanced during respiratory diseases, when the delicate balance between proliferation and apoptosis is tipped towards hyperproliferation. In addition, exaggerated VSMC responses to normal contractile stimuli and a reduction in vasodilator levels in pulmonary hypertension result in chronic vasoconstriction. Consequently, in pulmonary hypertension, VSMC hyperproliferation and disproportionate matrix deposition, collectively known as vascular remodeling, in conjunction with chronic vasoconstriction result in vessel lumen narrowing and increased blood pressure. In the past, pulmonary hypertension therapies focused on vasodilation. Now substantial research efforts are being directed at identifying novel strategies to prevent or reverse vascular remodeling.

Introduction Pulmonary blood vessel walls consist of three layers: the adventitia, the media, and the intima. Vascular smooth muscle cells (VSMCs) are the major cellular component of the tunica media in blood vessels, where they align mainly in a circumferential direction, contacting each other with simple appositional contacts and interdigitations. However, there are also longitudinal bundles of VSMCs in the pulmonary artery, in veins, and around orifices of blood vessel branches. The VSMCs have an elongated morphology with longitudinal striations that correspond to membrane dense bodies. The dense bodies contain a actinin and are attachment sites for actin filaments, forming the contractile structures of VSMCs. The cells are embedded within two major types of matrix: the basal lamina surrounding each smooth muscle cell and the largely collagen interstitial matrix filling the extracellular space. In culture VSMCs are generally spindle-shaped and grow with a hill and valley morphology. Origins

There is considerable heterogeneity in VSMC origin, both during development and in adult vasculature. In the embryo VSMCs have complex origins with putative smooth muscle progenitors of local, mesenchymal, and vascular derivation. Pulmonary arterial VSMCs originate from all these different smooth muscle cell progenitors. Initially as the pulmonary arteries develop, the VSMCs originate from the bronchial smooth muscle of the adjacent airways, resulting in one or two layers of tightly packed brick-shaped nonreplicating smooth muscle cells around the newly formed artery. By day 59 of human gestation these cell layers become surrounded by up to four loosely packed layers of

smooth muscle cells derived from the lung mesenchyme. Between days 98 and 140 of human gestation, ‘endothelial cells’ express the VSMC markers alpha smooth muscle (aSM) actin and CD31, indicating a third source of VSMC. Pulmonary veins are formed at sites away from the bronchi, so venous smooth muscle is believed to arise by vasculogenesis from mesenchymal progenitors with no contribution from airway smooth muscle.

VSMC Function in the Normal Lung VSMCs are multifunctional cells that exhibit a remarkable plasticity with the ability for rapid and reversible changes in phenotype in response to alterations in their local environment. The major function of adult smooth muscle is in regulation of vascular tone, which in the lung is essential for the control of pulmonary blood flow (Figure 1). The cells also provide structural integrity for vessel walls. In contrast, during vasculogenesis and injury or disease, the principal functions of VSMCs are proliferation and synthesis of extracellular matrix (ECM) components, chemotactic, and mitogenic proteins. Initially it was believed that there were two distinct subpopulations of VSMC representing each contractile and synthetic phenotype; however, it is likely that cells exist with phenotypes across the spectrum and there is plasticity. Regulation of Tone

In order to perform their contractile function VSMCs express a unique repertoire of contractile proteins, ion channels, and signaling molecules, and contain an expansive network of filaments and organelles. Many of the markers of differentiated VSMCs are components of contractile apparatus or contractile apparatus-associated proteins including aSM and gSM actin, SM myosin heavy chain, telokin, desmin, SM-calponin, SM-caldesmon, metavinculin, smoothelin A and B, and SM22a. The expression of many of these smooth muscle specific genes is regulated by serum response factor (SRF) in association with tissue or developmental specific coactivators in a combinatorial manner. Contraction and relaxation of VSMCs is dependent on intracellular free calcium homeostasis and the phosphorylation/dephosphorylation of regulatory myosin light chain (MLC20). An increase in intracellular calcium triggers muscle contraction through calcium binding to calmodulin, which activates the catalytic subunit of myosin light chain kinase. MLC kinase phosphorylates serine 19 of MLC20. This activates ATPase leading to the cycling of cross-bridges (myosin heads) along actin filaments to generate

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Proliferation

Chemotactic and mitogenic proteins Extracellular matrix

Activation of cell cycle

Actin–myosin interaction and cycling

VSMC Actin Intima MLC

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Media Adventitia Vessel structure

Figure 1 The key functions of VSMCs in the lung. Smooth muscle cells are the major component of the tunica media, maintaining the structural integrity of the vessel. Vascular smooth muscle maintains vascular tone by contraction or dilation, involving the cycling of myosin heads along actin cross-bridges to generate force and shortening. During vasculogenesis or injury and disease they proliferate and synthesize ECM, mitogenic, and chemotactic proteins.

force and shortening and thus contraction of the VSMCs. VSMCs can be stimulated or inhibited by many hormones, chemokines, and transmitters via intricate signal transduction mechanisms involving both voltage- and ligand-gated ion channels, the distribution and properties of which vary between large and small vessels. A major criterion for determining a fully differentiated smooth muscle cell is the ability of that cell to respond to contractile agonists by increasing intracellular Ca2 þ , activating myofilament apparatus and generating force.

the differentiation of smooth muscle cells. The primary source of synthetic VSMCs during injury and disease is pre-existing contractile smooth muscle cells that undergo transient and reversible phenotypic modulation. However, proliferation of pre-existing synthetic or proliferative smooth muscle cell subpopulations within the media, circulating bone marrow progenitors, or cells derived from the adventitia may also participate, especially after severe injury.

VSMCs in Respiratory Diseases Synthesis

In contrast, during development the major function of VSMCs is synthetic. They are the major source of ECM components in the blood vessel wall, including collagens, elastins, laminin, fibronectin, and the glycosaminoglycans and integrins that bind to the ECM. VSMCs also produce the angiogenic factor angiopoietin-1, which is essential for lung vascular development. Angiopoietin-1 recruits muscle cells to endothelial tubes by migration and mitogenesis, which stabilize the mature arterial structures. The synthetic phenotype is also observed during injury or disease and can exacerbate the disease process by altering the extracellular environment, which in turn influences

Changes or abnormalities in VSMC function, both contractile and proliferative, are major contributors to respiratory disease, such as pulmonary hypertension and pulmonary fibrosis. The study of these diseases has aided elucidation of the essential functions of pulmonary VSMC. It is well known that VSMCs are involved in mediating local inflammatory responses as a result of injury and disease, and pulmonary VSMCs are no exception. For example, VSMCs are involved in extensive ECM deposition and scarring in the chronic inflammatory–fibroproliferative disease pulmonary fibrosis. In asthma there is mucosal vasodilation due to the direct action of mediators on VSMCs. The vasodilation increases

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mucosal thickness and edema, narrowing the airways and increasing the rigidity of their walls. Vasodilation is also observed in the intra-acinar muscular arteries of patients with adult respiratory distress syndrome in conjunction with VSMC proliferation into normally nonmuscular small arteries. VSMC proliferation is a common feature of respiratory diseases including chronic obstructive pulmonary disease (COPD), plexogenic pulmonary arteriopathy, and emphysema, when VSMCs proliferate and deposit ECM in the intimas of pulmonary arteries resulting in decreased lumen size and an increased proportion of muscularized small arteries. The most serious chronic disorder of the pulmonary circulation is pulmonary arterial hypertension (PAH) in which the abnormal functions of VSMCs reflect their role in other respiratory diseases, and as such PAH is an excellent example to examine in more detail here. The abnormally high pulmonary arterial pressure that defines PAH is due to vasoconstriction, remodeling of the vessel wall, and thrombosis. The exact pathogenesis of PAH remains elusive but scientists postulate that an initial inflammatory response is

triggered by stimuli such as toxic, hypoxic, and shear stress that initiate an abnormal relentless release of vasoconstrictive chemicals leading to PAH progression. The most important aspect of PAH progression is the vascular remodeling, mainly in the mediumsized arteries and arterioles, as it leads to increased resistance within the vascular bed and thus raised pressure in the main pulmonary artery. Vascular remodeling involves proliferation of the predominant cell types within each layer of the blood vessel. In PAH the balance of pulmonary artery smooth muscle cell proliferation and apoptosis, important in maintaining the structural and functional integrity of the pulmonary vasculature, is tipped toward proliferation (Figure 2). The VSMCs penetrate the nonmuscular layers within the blood vessel, a process known as neomuscularization, and other cell types such as pericytes and intermediate cells differentiate into smooth muscle cells to increase VSMC numbers in the blood vessel wall. VSMCs are modulated to a more synthetic phenotype by cytokines. These produce a wide variety of inflammatory mediators that cause inflammation and a worsening of symptoms.

All-trans retinoic acid

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Phosphodiesterase BMPR

Interleukins

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-1

ietin

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TGF-1 Growth factors

Cytosolic Ca2+

MMPs Rho kinase

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Vasodilation apoptosis

Vasoconstriction proliferation Figure 2 The disruption of VSMC function in respiratory disease. In PAH, the balance is tipped toward VSMC hyperproliferation and chronic vasoconstriction by the overexpression of vasoconstrictors and mitogens and the impaired production of vasodilators and the attenuation of apoptosis. This leads to vascular remodeling and vessel lumen narrowing, resulting in a sustained elevation of vascular resistance. BMPR, bone morphogenic protein receptor; ET-1, endothelin 1; MMPs, matrix metalloproteinases; NO, nitric oxide; PPAR-g, peroxisome proliferator-activated receptor gamma; TGF-b1, transforming growth factor beta 1; VIP, vasoactive intestinal polypeptide.

SMOOTH MUSCLE CELLS / Vascular

They synthesize ECM components, which are deposited within the media and adventitia. This reduces arterial compliance and can lead to the development of neointimal lesions, whereby smooth muscle cells and ECM build up on the luminal side of the internal elastic lamina. The final manifestation of vascular remodeling is vessel lumen narrowing or obliteration due to medial hypertrophy by VSMC hyperproliferation and attenuated apoptosis. In addition, a sustained elevation of pulmonary vascular resistance results from the exaggerated VSMC responses to normal contractile stimuli and a reduction in vasodilator levels. This in turn is able to generate further remodeling by enhancing shear stress during exercise. Therefore, both the vasoconstrictive and vascular remodeling components of PAH are dependent on VSMC and each of these components will now be considered in terms of their mechanism and as putative therapeutic targets. Vasoconstriction

Sustained elevation of cytosolic calcium induces a chronic state of vasoconstriction. Elevated cytosolic calcium also contributes to smooth muscle cell hypertrophy and vascular remodeling by propelling quiescent VSMC into the cell cycle and increasing the expression and binding activity of the transcription factor activator protein 1 enhancing the transcription of genes involved in cell proliferation, apoptosis, and migration, including endothelin 1 (ET-1) and vascular endothelial growth factor (VEGF). As such calcium channel antagonists were considered promising oral medication for PAH, reducing both pulmonary arterial pressure and resistance in 25–30% of patients. Their mechanism of action is to relax and vasodilate specifically vasoconstricted vascular smooth muscle and to prevent VSMC proliferation. However, up to 75% of patients are nonresponsive and at risk of systemic lethality with these drugs. VSMCs express numerous voltage-gated potassium channels which on opening cause hyperpolarization of the cells and thus vasodilation. Abnormal expression or function of potassium channels contributes to excessive vasoconstriction in PAH. In addition, in the normal pulmonary artery, the endothelium plays an important role maintaining the quiescent population of VSMCs by producing many mediators of VSMC tone, proliferation, and migration. In PAH endothelial dysfunction means that production of vasodilators is impaired and vasoconstrictors that act on VSMC are overexpressed, which affects vascular tone. These areas are now becoming the focus of PAH therapeutic strategies.

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In the lungs of PAH patients, the potassium channel Kv1.5 is downregulated, which leads to VSMC depolarization, opening of calcium channels, raising of cytosolic calcium, and initiation of constriction. Novel therapeutic strategies to control vascular tone via potassium channels include drugs that augment Kv pathways by directly acting on VSMC to enhance channel expression and function, such as iptakalim, dichloroacetate, and sildenafil. In addition the loss of potassium channels leads to inhibition of the early markers of apoptosis, attenuating programmed cell death and promoting vascular hypertrophy. Thus, potassium channel openers as a therapeutic target may beneficially modulate vascular remodeling as well as vasoconstriction. Pulmonary vasodilators produced by endothelial cells and thus impaired during endothelial dysfunction include prostacyclin (PGI2), nitric oxide, and vasoactive intestinal peptide. PGI2 is decreased in endothelial cells in PAH patients, as is the expression of PGI2 synthase and endothelial nitric oxide synthase. In idiopathic PAH, production of the neuropeptide vasoactive intestinal peptide is reduced, while its receptors are upregulated in VSMCs, indicating a vasoactive intestinal peptide deficiency. PGI2 analogs mimic PGI2 to cause vasodilation and inhibit platelet aggregation. They also induce regression of disease in the pulmonary vascular endothelium and intimal smooth muscle layers. The first therapeutic PGI2 analog, epoprostenol, had the disadvantage of a 3–6 min half-life, in addition to adverse side effects including nausea, diarrhea, and jaw discomfort. Now more stable analogs or drugs that induce endogenous PGI2 production have been developed including the oral analog beraprost, an aerosolized form iloprost, and the increased half-life epropostenol analog treprostinil. Inhaled nitric oxide or vasoactive intestinal polypeptide (VIP) has a direct vasodilatory effect on the pulmonary vascular bed and may inhibit vascular remodeling by inhibiting the proliferation of VSMCs and the endothelium. Nitric oxide is difficult and expensive to administer and is only effective in a proportion of patients. So therapies utilizing nitric oxide-potentiating or donating drugs such as phosphodiesterase inhibitors or gene therapy for those patients with genetically reduced levels of nitric oxide synthase are becoming a therapeutic focus. Phosphodiesterases have a central role in regulating tissue levels of cyclic nucleotides including the breakdown of cAMP and cGMP. Non-specific phosphodiesterase inhibitors prevented the enhanced nitric oxide-dependent cGMP-mediated vasodilation in PAH but affected systemic blood pressure. To reduce systemic complications, the enzyme responsible for cGMP metabolism in the lung phosphodiesterase 5 was

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targeted. Phosphodiesterase 5 activity is enhanced in PAH. Specific phosphodiesterase 5 inhibitors including sildenafil, E4021, and E4010 attenuate pulmonary artery medial thickening and neomuscularization and reduce pulmonary artery pressure with no adverse effects on systemic blood pressure. An additional potent vasodilator secreted by both VSMCs and endothelial cells in high levels in the lung is adrenomedullin. Unlike other vasodilators, this peptide is increased in PAH, has antiproliferative effects on a distinct subpopulation of pulmonary artery smooth muscle cells, and inhibits the release of the vasoconstrictor ET-1. Adrenomedullin infusion in a PAH animal model reduced pulmonary medial hypertrophy and PAH. However, it remains to be seen whether therapeutic strategies targeting adrenomedullin and its receptors in humans will have long-term benefits. Vasoconstrictors acting on VSMCs in the lung and overexpressed in PAH include ET-1 and 5-hydroxytryptamine (serotonin) (5-HT). ET-1 is a major contributor to the imbalance of vasodilators and vasoconstrictors in PAH, being overexpressed in various etiologies of the disease. It acts through the ET-1 receptors A and B in pulmonary artery smooth muscle cells, generating a rapid increase in intracellular calcium, leading to depolarization and contraction. 5-HT acts via the 5-HT receptors 5-HT1A and 5HT1B, in large and small human pulmonary arteries, respectively, to mediate contraction of VSMCs. In PAH the major repository of 5-HT, the platelets, is depleted leading to a corresponding increase in circulating 5-HT levels and thus pulmonary vasoconstriction. Therapeutic strategies to combat the excessive vasoconstriction include ET-1 receptor antagonists and 5-HT reuptake inhibitors. Selective inhibitors of the 5-HT transporter protect against hypoxia-induced pulmonary hypertension by reducing vasoconstriction and pulmonary vascular muscularization in mice, indicating their potential therapeutic properties in humans. ET-1 receptor antagonists modulate the elevated ET-1 levels, reversing the vasoconstriction it generates and thus reducing pulmonary artery blood pressure and vascular remodeling. Several ETA receptor antagonists have been developed but all have a short half-life requiring intravenous administration. Bosentan, a combined ETA and ETB receptor antagonist, has been shown to be extremely beneficial, reducing vasoconstriction and pulmonary artery blood pressure in addition to preventing medial thickening, neomuscularization, adventitial thickening, and fibrosis. It has the advantage that it can be taken orally but its side effects are quite severe including hepatotoxicity, anemia, and birth defects. It has been suggested that in view of the

opposing effects of ET-1 and PGI2 on VSMCs that the combined use of an ET-1 antagonist and a PGI2 analog would be even more effective in the treatment of PAH. Other potential vasodilators under investigation include the Rho kinase inhibitors and thromboxane pathway antagonists. Rho kinase phosphorylates one subunit of MLC phosphatase, suppressing the activity of this enzyme responsible for dephosphorylating MLC20 and thus promoting VSMC contraction. Rho kinase also mediates 5-HT-induced VSMC proliferative effects. Inhalation of Rho kinase inhibitors was shown more effective at vasodilation than nitric oxide in laboratory investigations. Thromboxane A2 is a bioactive metabolite of arachidonic acid. It is produced by the action of thromboxane synthase on the prostaglandin endoperoxide H(2), which results from the enzymatic degradation of arachidonic acid by cyclooxygenases. Thromboxane is a potent inducer of vasoconstriction, bronchoconstriction, and platelet aggregation. Combined administration of a thromboxane synthase inhibitor and a thromboxane A2 receptor antagonist has been shown to attenuate pulmonary vasoconstriction in PAH but clinical trials of one combination (Terbogel) were halted due to leg pain. This inhibitor–antagonist combination also demonstrated antiplatelet and antithrombotic activities in laboratory studies and thus may reduce the need for coadministration of anticoagulant drugs such as warfarin and heparin to avoid PAH-generated thrombosis. This would be advantageous as these drugs are subject to a plethora of drug–drug interactions. Gene therapy has also been used successfully in the laboratory as an alternative means of controlling PAH, involving overexpression of vasodilator genes such as endothelial nitric oxide synthase, inducible nitric oxide synthase, PGI2 synthase, and VEGF. Many difficulties must be overcome before this approach can be applied in patients, which include pulmonary-specific delivery systems. Vascular Remodeling

Vascular remodeling is a critical adaptive feature for the maintenance of blood flow in vessels with thickening intimas. While it is clear that other cell types and in situ thrombosis are all involved in PAH, it is now agreed that pulmonary artery smooth muscle cell proliferation and to some extent vasoconstriction lead to the vascular remodeling processes that underlie severe PAH, and as such vascular remodeling has become the focus for the development of new therapies. Most stimuli that acutely enhance vasoconstriction and/or raise intracellular calcium levels, such as ET-1

SMOOTH MUSCLE CELLS / Vascular

and VIP, also stimulate VSMC proliferation and thus vascular remodeling. For example, ET-1 activates the p42/p44 isoforms of mitogen-activated protein kinase (MAPK) and induces the early growth response genes c-jun and c-fos. Many of the pulmonary vasodilators, such as PGI2, inhibit VSMC proliferation. Thus, an advantage of the majority of the vasodilator drugs previously described, including the potassium channel opener iptakalim and the calcium channel antagonist nifedipine, is their ability to inhibit VSMC mitogenesis, reducing medial thickening and neomuscularization. It has been proposed as mentioned earlier that inflammation and the associated increase in inflammatory cytokines, including interleukins and tumor necrosis factor (TNF), is an important element of PAH. Serum concentrations of interleukins 1 and 6 are increased in PAH, the former being mitogenic for pulmonary VSMCs. Growth factors including platelet-derived, basic fibroblast, insulin-like, and epidermal growth factor (EGF) have been reported to be increased in PAH and as such have been implicated in the vascular remodeling process due to their VSMC proproliferative effects. Transforming growth factor beta 1 (TGF-b1) enhances the cell proliferation of pulmonary artery smooth muscle cells derived from PAH patients in contrast to its growth inhibition of normal pulmonary artery smooth muscle cells and may increase production of ECM components including elastin and collagen. The TGF-b superfamily may also regulate the activity of other factors implicated in vascular remodeling such as ET-1. The TGF-b superfamily includes the bone morphogenic protein (BMP) system, which is key to the etiology of PAH. BMP signal transduction involves BMP ligand binding to the BMP receptors I and II (BMPR) to initiate the intracellular signaling processes involved in nuclear transcription and apoptosis. Heterozygous mutations in BMPR2 have been found in approximately 55% of familial idiopathic PAH patients and 30% of patients with sporadic idiopathic PAH, suggesting a role in predisposition for the disease. The expression of BMPR2 is also reduced in PAH cases with no identified BMPR2 mutation. Reduction of BMPR2 leads to overexpression of the antiapoptotic protein Bcl-2, thus contributing to VSMC proliferation. In addition, the antiproliferative effects of BMP2, 4, and 7 fail in vascular cells derived from idiopathic or familial PAH cases. That the BMP system is critical for all forms of PAH was confirmed by the finding that BMPR1A is severely reduced in the lungs of patients with various forms of acquired as well as primary nonfamilial PAH. BMPR1A is a transmembrane protein required to heterodimerize with BMPR2 for BMP signaling. The

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VSMC synthesized angiogen angiopoietin-1 is chronically upregulated in patients with PAH and it downregulates BMPR1A in pulmonary arteriolar endothelial cells. In addition upregulation of angiopoietin-1 production by VSMC and its receptor in human PAH have been found to correlate directly with the severity of the disease. Engineered overexpression of angiopoietin-1 in rat lung leads to the development of PAH due to smooth muscle cell hyperplasia, whereas treatment of PAH rat models with an antagonist to its receptor TIE2 prevented PAH. Angiopoietin-1 is not only mitogenic and chemotaxic for VSMCs but also stimulates pulmonary arteriolar endothelial cells to secrete the vasoconstrictor 5-HT. This has firmly established the cross-talk between the BMP system in endothelial cells and angiopoietin-1 from VSMCs and their importance in the generation of all types, familial or nonfamilial, primary or secondary, of PAH. Angiotensin II is another powerful growth factor and mitogen, as well as being a pulmonary vasoconstrictor, overexpressed in PAH. Inhibitors of angiotensin-converting enzymes (ACEs) such as captopril and cilazapril, which convert angiotensin I to angiotensin II, have been reported to be reduced in human PAH patients. These inhibit pulmonary vascular remodeling possibly by reducing cell proliferation and matrix deposition, thus attenuating medial thickening and neomuscularization. All ACE inhibitors tested have been shown to reduce pulmonary artery blood pressure but only by a small amount and thus are not suitable for treatment of acute PAH. This effect has also been observed by angiotensin receptor antagonists losartan and GR138950C. Ligands of the nuclear receptor peroxisome proliferator-activated receptor g (PPAR-g) have been shown to antagonize the actions of angiotensin II, lowering systemic blood pressure and correcting vascular structure and endothelial dysfunction in several rodent models of systemic hypertension. PPAR-g is highly expressed in pulmonary vascular smooth muscle and is downregulated in patients with PAH, which has been linked to BMPR mutation, highlighting this receptor as a potential therapeutic target in PAH as well. Other nuclear receptors, the retinoic acid receptors (RAR) and retinoid X receptors (RXR) are expressed in human pulmonary artery smooth muscle cell. The RAR activator all-trans retinoic acid is significantly reduced in patients with idiopathic PAH and its administration to cultured pulmonary artery smooth muscle cell suppressed serotonin induced growth by eliciting growth inhibitory signals. Studies on the systemic circulation suggest that retinoic acid may reduce vascular thickening and as such may be a useful treatment of PAH once its effects are further understood. There are many other nuclear

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receptors of little known function in the vascular system, such as the farnesoid X receptor, so the examples of PPAR and RAR suggest a promising untapped source of future drug targets for PAH. Vascular remodeling involves not only VSMC proliferation but also excessive ECM modulation. Proteolysis of the ECM is believed to be important in the pathobiology of pulmonary vascular disease. Elastin was found to be decreased in pulmonary arteries from patients with pulmonary vascular disease and rat hypoxic PAH models demonstrated a heightened activity of elastase in these vessels. VSMCs produce an endogenous vascular elastase in response to serum factors. Serine elastases can activate matrix metalloproteinases (MMPs) and repress tissue inhibitors of MMPs (TIMPs). VSMCs express collagenases MMP 1 and 3 and gelatinases MMP 2 and 9. MMP2 expression and activity are increased in experimental PAH. Both elastases and MMPs can degrade most components of the ECM including elastin and collagen. In addition to ECM remodeling, degradation of collagen leads to the ligation of b3-integrins which activate transcription of tenascin C, a glycoprotein that cooperates with growth factors such as EGF, resulting in VSMC proliferation. Serine elastase inhibitors have been shown to suppress the PAH disease process by inducing VSMC apoptosis. For example, SC-39026 and SC-37698 successfully inhibit neomuscularization and medial thickening, while ZD0892 and M249314 are more potent, completely reversing vascular remodeling, normalizing mean arterial blood pressure, and increasing survival. The activity of elastase can also be repressed by nitric oxide donors and 5-HT receptor inhibitors. While several studies concluded that MMPs contribute to PAH progression in animals, the effects of MMP inhibitors differ across models and so further investigation of these potential therapeutic targets is required. The consideration of vascular remodeling in addition to vasoconstriction has opened a new field of potential drug targets that avoid many of the systemic side effects encountered with vasodilator drugs. Other substances that have been shown to inhibit vascular remodeling include platelet-activating factor inhibitors, interleukin receptor antagonists, heme oxygenase inducers, ghrelin, calpain inhibitors, and neutral endopeptidase inhibitors. If the mechanisms of action and administration of these substances are elucidated, they represent new sources of therapeutic potential.

Summary Normal pulmonary VSMC function is essential for the maintenance of pulmonary blood flow. When the

tightly regulated balance between vasodilatory and vasoconstrictory mediators within the lung is disrupted and the balance between the contractive and synthetic phenotypes of VSMC is tipped to stimulate vascular remodeling, respiratory diseases such as PAH develop. With such complex pathobiology, further understanding of the mechanisms underlying these conditions will presumably lead to the development of novel therapeutic strategies in the future. It is likely that the most effective therapy will be drugs aimed at combating multiple factors within the disease process instead of a single target. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Cell: Vascular; Focal Contacts and Signaling; Integrins. Angiogenesis, Angiogenic Growth Factors and Development Factors. Anticoagulants. Apoptosis. Arteries and Veins. Asthma: Overview. Bronchial Circulation. Cell Cycle and Cell-Cycle Checkpoints. Chemokines. Chronic Obstructive Pulmonary Disease: Overview; Emphysema, General. Contractile Proteins. Cyclic Nucleotide Phosphodiesterases. Cytoskeletal Proteins. DrugInduced Pulmonary Disease. Endothelial Cells and Endothelium. Endothelins. Epidermal Growth Factors. Extracellular Matrix: Basement Membranes; Collagens; Surface Proteoglycans. Fibroblast Growth Factors. Insulin-Like Growth Factors. Ion Transport: Calcium Channels; Potassium Channels. Kinins and Neuropeptides: Vasoactive Intestinal Peptide. Matrix Metalloproteinase Inhibitors. Matrix Metalloproteinases. Peroxisome Proliferator-Activated Receptors. Platelet-Derived Growth Factor. Pulmonary Fibrosis. Pulmonary Vascular Remodeling. Retinol and Retinoic Acid. Serine Proteinases. Signal Transduction. Transcription Factors: Overview; AP-1. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Vascular Disease. Vascular Endothelial Growth Factor.

Further Reading Botney MD (1999) Role of hemodynamics in pulmonary vascular remodeling: implications for primary pulmonary hypertension. American Journal of Respiratory and Critical Care Medicine 159: 361–364. Cheever KH (2005) An overview of pulmonary arterial hypertension: risk, pathogenesis, clinical manifestations and management. Journal of Cardiovascular Nursing 20: 108–116. Du L, Sullivan CC, Chu D, et al. (2003) Signaling molecules in nonfamilial pulmonary hypertension. New England Journal of Medicine 348: 500–509. Eickelberg O, Yeager ME, and Grimminger F (2003) The tantalizing triplet of pulmonary hypertension: BMP receptors, serotonin receptors and angiopoietins. Cardiovascular Research 60: 465–467. Hall SM, Hislop AA, and Pierce CM (2000) Prenatal origins of human intrapulmonary arteries: formation and smooth muscle maturation. American Journal of Respiratory Cell and Molecular Biology 23: 194–203.

SPACE, RESPIRATORY SYSTEM 113 Humbert M, Morrell NW, Archer SL, et al. (2004) Cellular and molecular pathobiology of pulmonary arterial hypertension. Journal of the American College of Cardiology 43: 13S–24S. Jeffery TK and Wanstall JC (2001) Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension. Pharmacology and Therapeutics 92: 1–20. Liu C, Nath KA, Katusic ZS, and Caplice NM (2004) Smooth muscle progenitor cells in vascular disease. Trends in Cardiovascular Medicine 14: 288–293. Mandegar M, Fung YB, and Huang W (2004) Cellular and molecular mechanisms of pulmonary vascular remodeling: role in the development of pulmonary hypertension. Microvascular Research 68: 75–103.

Owens GK (1995) Regulation of differentiation of vascular smooth muscle cells. Physiology Reviews 75: 487–517. Pass SE and Dusing ML (2002) Current and emerging therapy for primary pulmonary hypertension. Annals of Pharmacotherapy 36: 1414–1422. Shanahan CM and Weissberg PL (1998) Smooth muscle cell heterogeneity: patterns of gene expression in vascular smooth muscle cells in vitro and in vivo. Atherosclerosis, Thrombosis and Vascular Biology 18: 333–338. Strange JW, Wharton J, Phillips PG, and Wilkins MR (2002) Recent insights into the pathogenesis and therapeutics of pulmonary hypertension. Clinical Science 102: 253–268.

SPACE, RESPIRATORY SYSTEM G K Prisk, University of California – San Diego, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The lung is extremely sensitive to gravity, which induces large regional differences in ventilation, blood flow, and gas exchange in normal subjects. Functional residual capacity is reduced in the absence of gravity, as is residual volume, although by different mechanisms. While the removal of gravity greatly reduces the unevenness of both ventilation and perfusion in the lung, there are only minor changes in the degree of ventilation–perfusion inequality in microgravity (mG), suggesting a mechanism by which gravity serves to match ventilation to perfusion, making for a more efficient lung in 1G than might otherwise be anticipated. Despite some early predictions to the contrary, the lung does not become edematous in mG, and there is no apparent disruption to gas exchange. mG reduces the hypoxic, but not the hypercapnic, ventilatory response. Studies convincingly show that sleep disturbances in mG are not a result of respiratoryrelated events, and that obstructive sleep apnea is principally caused by the gravitational effects on the upper airways. Aerosol deposition in the lung is reduced in mG. However, aerosols may be deposited more peripherally, which may place the lung at risk in the reduced gravity environments of the Moon and Mars, which are likely to have reactive dust species on their surfaces.

Gravity, the Lung, and Space Flight Although there is little, if any, structural difference between the top and bottom of the normal human lung, there are marked functional differences caused by the effects of gravity (Table 1). For example, the alveoli at the top of the lung are relatively overexpanded compared to the bottom of the lung because the weight of the dependent portions of the lung stretches the upper portions. As a consequence of this, ventilation is higher at the bottom of the lung, because the initial smaller volume there makes the

lung more readily able to expand in response to a given breathing effort. The pulmonary vascular system operates at a relatively low pressure compared to the systemic circulation and, as a consequence, hydrostatic effects strongly influence the vertical distribution of blood flow in the normal human lung, with much greater flows near the bases than the apices. Because the differences in perfusion are larger than those in ventilation, the ventilation–perfusion ratio is higher at the top than the bottom of the lung. Since it is the ventilation–perfusion ratio that determines gas exchange, regional differences in alveolar gas and effluent blood composition occur. Making Measurements in Microgravity

There are only two practical methods of achieving microgravity suitable for human experimentation: parabolic flight in aircraft and space flight. Parabolic flight provides only short periods of microgravity (typically B25 s), and these are usually sandwiched between periods of hypergravity. Any measurements made in parabolic flight are therefore subject to the effects that may ensue from this period of hypergravity. In contrast, spaceflight provides sustained microgravity (typically from about 1 week to more than 1 year at present) but flight opportunities are infrequent at best. Skylab provided the first real opportunity to conduct pulmonary function measurements in space, but ground simulation studies showed that some of the changes observed may have at least been partly due to the atmospheric conditions (5 psia, B345 kPa or B260 mmHg, 70% O2) in the vehicle rather than microgravity per se. In contrast, more recent spacecraft (the Space Shuttle, the now-defunct

114 SPACE, RESPIRATORY SYSTEM Table 1 The effect of microgravity on various aspects of lung function Aspect of lung function

Change with microgravity

Lung volumes and pulmonary mechanics Vital capacity Slight initial reduction early inflight, probably due to increased intrathoracic blood volume, but unchanged from 1G thereafter Functional residual capacity Reduced by B15%; intermediate between standing and supine in 1G Residual volume Reduced by B18%; probably due to elimination of apicobasal gradients in lung expansion Tidal volume Reduced by B15% Respiratory frequency Increased by B9% Forced expiratory flows Initial decrease in peak expiratory flow at the onset of mG, with a subsequent return to normal after 4–5 days; no change in flows at low lung volumes Chest and abdominal mechanics Increased abdominal compliance (B30%); decrease in EMG activity of the ribcage Pulmonary ventilation Airway closure Large-scale inhomogeneity Small-scale inhomogeneity Pulmonary perfusion and fluid balance Cardiac output Diffusing capacity Lung water Distribution of pulmonary perfusion

Still occurs at approximately the same absolute lung volume Greatly reduced in vital capacity breaths; largely unchanged in tidal volume breaths Persists, although with some changes in acinar conformation, probably due to changes in lung fluid balance

Initial increase of B35% at the onset of mG with subsequent decrease, but remaining above 1G levels; some suggestion of subsequent increase after more than 2 weeks in mG Increases by B28% and remains unaltered for duration of mG exposure due to increases in both pulmonary capillary blood volume and surface area available for gas exchange Unchanged at the onset of mG, but decreased after some days More uniform, probably due to abolition of top-to-bottom gradients in blood flow, but with persisting inhomogeneity

Pulmonary gas exchange Resting gas exchange Total ventilation Alveolar ventilation End-tidal gas concentrations

Unchanged O2 consumption and CO2 production Slightly decreased (B7%) Unchanged PO2 unchanged, PCO2 may be slightly increased probably due to increased environmental CO2

Inequality of ventilation-perfusion ratio

Evidence for reduction in top-to-bottom gradients, but persisting inequality in face of reductions in inequality of both ventilation and perfusion

Response to exercise Ventilatory response Cardiac response

Largely unchanged Reduced increase in cardiac output for a given O2 consumption requirement

Ventilatory control Isocapnic hypoxic ventilatory response Hypercapnic ventilatory response

Largely unchanged

Sleep-disordered breathing Apnea-hypopnea index Snoring Respiratory-related arousals

Significantly reduced Reduced Virtually eliminated

Aerosol deposition Magnitude Location

Reduced due to absence of sedimentation, but unexpectedly high for small particles Probably more peripheral

Extra-vehicular activity Ventilation-perfusion ratios

Unaltered 24 h after EVA compared to before (all measurements in mG)

Approximately halved; equivalent to that supine in 1G

Except where noted the comparison is made with the upright position in 1G.

Russian space station Mir, and the International Space Station (ISS)) have provided researchers with a normoxic, normobaric environment. Since 1983, the US Space Shuttle has, at times, carried the European built Spacelab in the cargo bay, and subsequently the

commercially built Spacehab. Shuttle flights are limited to missions of short duration (the maximum currently being about 17 days), and so necessarily deal with only acute phases of the adaptation to microgravity. Longer duration missions now occur on

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the ISS and prior to that on Mir, Salyut, and Skylab. However, populations available for study are small, and in experiments involving human subjects, the design must often be such that statistically viable results can be achieved using only an n of 4. Typically, in such experiments subjects act as their own control, with extensive preflight and postflight testing.

Normal Physiology in Microgravity Lung Volumes and Chest Wall Mechanics

A study of lung volumes in microgravity (mG) performed in Skylab showed a B10% decrease in vital capacity. However, ground controls using a comparable atmosphere showed a 3–5% reduction, suggesting a confounding effect of the hypobaric environment. During the 1991 flight of Spacelab Life Sciences-1 (SLS-1), vital capacity showed a B5% reduction early in flight (Flight Day 2, FD-2) compared to that standing in 1G. However, by FD-4 the reduction had been abolished. An early inflight increase in intrathoracic blood volume, with a subsequent reduction as plasma volume is reduced, seems the most plausible explanation for these results. Functional residual capacity (FRC) decreased by B15% in mG compared to that of the standing subject in 1G. This reduction was largely in line with predictions of a B10% decrease, primarily based on a cranial shift of the diaphragm and abdominal contents as gravity was removed (a potentially large effect that would decrease FRC), an outward movement of the ribcage, and an upward movement of the shoulder girdle (both small effects that would increase FRC). As expected, the FRC in mG was intermediate to that measured standing and supine in 1G. Residual volume (RV) is generally quite resistant to change. Transitions between upright and supine, and water immersion typically result in no significant decrease in RV. However, in sustained mG, RV was markedly decreased by B18% (310 ml) compared to standing and was also significantly below that measured supine. In 1G, when lung volume is reduced below FRC, airways closure begins in the more gravitationally dependent lung regions (because of distortion of the lung by its own weight), and this airways closure progresses up the lung until RV is reached, resulting in a large difference in regional RV between the top and bottom of the lung in 1G. However, in mG the apico-basal gradients in regional lung volume due to gravity are abolished, resulting in the regional volume of lung units being much more uniform, reducing RV. It is reasonable to expect that removal of gravity may result in some alterations in local airway stress, and thus in expiratory flow limitation. Studies in

parabolic flight showed effects consistent with an increase in intrathoracic blood volume, altering elastic recoil (a scooping out of the MEFV curve at low lung volumes) similar to changes seen in recumbency and immersion. However, in space flight there were no discernable changes in the shape of the MEFV curve at low lung volumes, suggesting little, if any, change in the behavior of the central and peripheral airways. The mechanical properties of the lung and chest wall have only been studied in parabolic flight. There is a headward displacement of the diaphragm with an inward displacement of the abdominal wall reducing lung volume, and an increase in abdominal wall compliance. However there is also a decrease in the EMG activity of the muscles that activate the upper portion of the ribcage. This may be part of the operational length compensation reflex in which as the length of the diaphragm increases, there is a compensatory decrease in the activation of the other inspiratory muscles keeping tidal volume constant. Pulmonary Ventilation

Vital capacity single breath nitrogen washout (SBW) tests showed marked reductions in the height of the terminal rise in N2, and in cardiogenic oscillations (both markers of large-scale ventilatory inhomogeneity), but a clear persistence nevertheless, suggesting a considerable contribution from nongravitational factors. The onset of airways closure occurred at the same absolute lung volume in 1G and mG, consistent with the suggestion of ‘patchy’ airways closure occurring at a similar absolute lung volume regardless of the gravitational distortion of the lung. Multiple breath washouts (MBWs) using near tidal volume breaths showed few significant changes between standing data in 1G and mG. This surprising result led to the conclusion that the primary determinants of large-scale ventilatory inhomogeneity during tidal breathing in the upright posture were not primarily gravitational in origin. While there was good reason to suspect that large-scale inhomogeneity should be subject to considerable alteration in mG, there was generally no consideration that small-scale inhomogeneity should be affected. The SBW performed in space flight confirmed previous observations from parabolic flight that phase III slope (predominately a marker of small-scale inhomogeneity) was only slightly reduced by the removal of gravity (B25%). Small-scale effects can be studied by performing SBWs in which trace quantities of helium (He, a rapidly diffusing gas) and sulfur hexafluoride (SF6, a slowly diffusing gas) are included in the inspired test gas. At the level of the acinus, diffusion takes over

116 SPACE, RESPIRATORY SYSTEM

from convection as the principal mechanism of gas transport, and it is the interaction between these different transport processes that generates the inhomogeneity of ventilation. In normal humans in 1G, SF6 exhibits a steeper phase III slope than does He because of the structure of the human acinus, and because the more rapid diffusion of He serves to more readily abolish any concentration gradients. In space flight, the He and SF6 slope became the same in mG, and the slope for SF6 actually became flatter than that for He following a breathhold, similar to that seen in heart-lung transplant recipients undergoing acute rejection episodes. In that case, conformational changes near the entrance of the acinus, probably as a result of acute inflammation, were the cause. However, in space flight the effect was rapidly reversible, as the phase III slope difference had returned to preflight values within 6–10 h of landing, and the effect was absent in tests performed in parabolic flight, suggesting that it takes some time to develop. Recent observations in steep (601) headdown tilt, which raises pulmonary capillary pressures, suggest a role of changes in the distribution of lung water on acinar conformation, but these conclusions are preliminary at best. Cardiac Output

Cardiac output rises by approximately 35% above preflight standing levels 24 h after the onset of mG, and then decreases subsequently. This is accompanied by a slight bradycardia and, as a result, stroke volume is increased by 60–70% early in flight, and also shows a subsequent decrease. However, some data suggest that cardiac output may rise again after B2 weeks in mG due to an increase in heart rate in the face of a constant stroke volume, but to date there have been no studies in mG of longer duration to support these observations. These increases in cardiac output occur in the face of significant decreases in cardiac filling pressures inferred from central venous pressure (CVP) measurements using catheters in the superior vena cava. Since the changes in CVP occur within seconds of the removal of gravity, it seems highly unlikely that there is any change in cardiac muscle performance per se. Thus, cardiac transmural pressure must have increased, presumably due to a decrease in extracardiac pressure, which is probably similar to pleural pressure. The fact that FRC decreases upon entry into mG (increasing in pleural pressure) suggests that local pressure changes must be considered when considering cardiac performance, although a clear understanding of the seemingly contradictory results is not yet available.

Diffusing Capacity and Lung Water

The single-breath diffusing capacity for carbon monoxide (DLco) rose substantially (28%) on exposure to mG and remained elevated over the course of a 9-day flight. This was a result of parallel increases in both pulmonary capillary blood volume (Vc, 28%) and membrane diffusing capacity (Dm, 27%). There was a rapid return to preflight conditions after return to 1G. The changes in DLco and its components were attributed to a more uniform filling of the pulmonary capillary bed with an attendant increase in the surface area available for gas exchange. The possibility of pulmonary edema formation in mG caused by a headward shift of fluid, and what was at the time a presumed increase in central venous pressure, had previously been suggested. If edema had occurred, a decrease in either DLco or Dm might have been expected early in flight with increases later as the edema resolved, but this was not the case. Pulmonary tissue volume measured using soluble gas uptake (which is sensitive to extravascular fluid in the lungs) was unchanged after 24 h of mG and was 20– 25% lower after 9 days, despite increases in thoracic blood volume, although these measurements are taken from only a few subjects. These results are consistent with observations of the low compliance of the pulmonary interstitium, which in the presence of a fall in central venous pressure would be expected to result in a gradual reduction in fluid in these tissues. Pulmonary Perfusion

There are no direct measurements of the distribution of pulmonary blood flow during space flight, principally because of the technical challenges of such measurements. Some imaging studies using injected radioactive microaggregates in humans or microspheres in animals have been performed in parabolic flight, and these show a clear influence of gravity, albeit with a considerable degree of nongravitational influence as well. A technique similar to a SBW test using evolved CO2 as the tracer gas has been used in parabolic flight and space flight to provide estimate inhomogeneity of perfusion. In this technique, the presence of cardiogenic oscillations provides evidence for differences in perfusion between areas of the lung proximal and distal to the heart, while the fall in CO2 after airways closure is evidence for a lower CO2 (and thus lower blood flow) in the upper part of the lung in 1G. In 1G, there are prominent cardiogenic oscillations, and a marked fall in CO2 towards the end of exhalation, consistent with the known vertical gradient of pulmonary blood flow. In parabolic flight, there was no terminal fall, and it was found

SPACE, RESPIRATORY SYSTEM 117

that the size of the cardiogenic oscillations depended strongly on gravity with small but measurable values in mG. In sustained mG, the size of the cardiogenic oscillations was decreased to a similar degree to that supine (B60% standing), while the terminal fall was absent, consistent with parabolic flight. Since airways closure still occurs in mG, the absence of a terminal fall of CO2 means that the blood flow in the regions of lung behind airways that close, and in the regions of lung behind airways that remain open, must be similar, consistent with the abolition of the top to bottom gradients in blood flow. While mG would be expected to abolish apicobasal differences in perfusion, it would not necessarily affect other nongravitational mechanisms of inhomogeneity. The persistence of cardiogenic oscillations in mG implies that residual inhomogeneity persists in the absence of gravity, and that these differences in blood flow must be between widely separated regions of the lung, since differences in blood flow on a very small scale would not be expected to generate cardiogenic oscillations at the mouth. Pulmonary Gas Exchange and Ventilation– Perfusion Ratio

Resting V’ O2 and V’ CO2 are unchanged by exposure to mG although there are some changes in the parameters associated with gas exchange. Tidal volume is decreased by B15%, and there is a compensatory increase in breathing frequency (9%). While the total ventilation is decreased by B7%, when the reduction in physiological deadspace resulting from the re’ is factored in, alveolar moval of areas of high V’ A =Q ventilation remains unchanged. The reasons for the selection of a different combination of tidal volume and breathing frequency are unclear. There was no evidence of significant changes in respiratory drive. Some flights have shown a small increase in end-tidal PCO2 (B2.0 Torr), but since there were no changes in end-tidal PO2 , this was probably due to the increased environmental CO2 levels in the spacecraft. There have been no direct measurements of ventilation–perfusion ratio distribution in mG. The equipment needed for imaging techniques have not been taken on space flights, and techniques such as the multiple inert gas elimination technique (MIGET) are impractical. However, by performing a controlled slow exhalation from TLC to RV following a period of air breathing the intrabreath respiratory exchange ratio can be calculated. Studies in dogs challenged with methacholine have shown that the slope of the intrabreath R plot with exhaled vol’ inequality ume is correlated with the degree of V’ A =Q determined by MIGET.

There were significant cardiogenic oscillations seen in the CO2 expirogram in mG, which is strong evidence for continued inter-regional differences in ’ There was also a marked reduction in V’ A =Q ’ V’ A =Q. range after the onset of airways closure, consistent with the idea that the top-to-bottom gradient in ’ had been abolished in mG. However, over V’ A =Q phase III of the prolonged expiration there was no ’ between standing and change in the range of V’ A =Q mG. This result was surprising given the prior observations that mG results in reduction in the topographical gradients of both ventilation and perfusion. Thus, it appears that at lung volumes above the onset of airways closure, the principal determinants of ’ inequality in normal subjects are not gravitaV’ A =Q tional in origin, and there is some evidence to suggest that gravity may actually impose some degree of matching of ventilation to perfusion in the normal lung. Exercise

The ventilatory response to exercise is largely unaffected by mG. Maximum oxygen consumption seems to be maintained in short-duration flights (9–14 days) suggesting no pulmonary limitation to exercise. However, there is an abrupt reduction in peak V’ O2 upon return from short-duration space flight of B22%. The V O2 response returns to preflight levels within 6–9 days, with the recovery in the first 2 days being extremely rapid, suggesting that adjustments to the circulating blood volume of the subjects after return are a major factor. During short-duration flights, the cardiac output increase with increasing V’ O2 is substantially lower than that measured either upright or supine on the ground preflight. No such data exist for long-duration space flight. The cause for these changes is unknown. Alterations in venous return that result from mG may be a factor. If O2 delivery is controlled in response to exercise, as opposed to cardiac output, then the lower circulating blood volume in mG and associated increase in [Hb] may result in lower cardiac output demands. Alternatively, sequestration of circulatory blood volume in a more evenly filled pulmonary circulation may be a factor. There may also be changes in the efficiency of the muscle pump returning blood to the thorax. Ventilatory Control

Microgravity results in a substantial reduction in the ventilatory response to hypoxia, but leaves the ventilatory response to CO2 essentially unchanged. The hypercapnic response measured in the absence of any hypoxic drive showed only a minor increase in the

118 SPACE, RESPIRATORY SYSTEM

slope of the rise in ventilation with CO2, with changes in the break point such that ventilation at a PCO2 of 60 mmHg was unchanged. In sharp contrast, the isocapnic hypoxic ventilatory response (CO2 B46 mmHg) approximately halved in mG. There was no change over the course of a 16-day flight, suggesting the response was likely the result of a mechanical change as opposed to some slower adaptation to mG. Importantly, the decrease in the hypoxic response in mG was similar to that seen in the same subjects when they were placed acutely into the supine position in 1G. Simultaneous measurements of the inspiratory pressure measured 100 ms after the unexpected occlusion of the inspiratory path (an independent measure of ventilatory drive) essentially mirrored the combined results of the hypercapnic and hypoxic responses. This showed that the changes were due to changes in the ventilatory response itself, and not the result of changes in the mechanical configuration of the respiratory system limiting ventilation. The likely cause of the change in the hypoxic response is an increase in blood pressure at the level of the carotid bodies. While systemic blood pressure is little changed by either the supine position or mG, in the upright position in 1G, blood pressure at the carotid bodies is lower than at heart level due to the hydrostatic pressure difference. This difference is abolished in both the supine position and in mG. Stimulation of the carotid baroreceptors results in an inhibition of the carotid chemoreceptor output, and this is thought to be the cause in this circumstance.

arousals per hour. Thus, mG significantly reduced sleep-disordered breathing, probably because the weight of the soft tissues of the pharynx is absent in mG, eliminating their tendency to fall back and obstruct the upper airway. Inhaled Aerosols

Long-term space flight represents a situation in which aerosol deposition may be an important health consideration. The potential for significant airborne particle loads is high since the environment is closed, and no sedimentation occurs. Microgravity provides for potentially high particle concentrations in the periphery of the lung, since particles that normally sediment will not be removed from the airways, leaving them available for transport to the alveolar regions. There have been no studies of inhaled aerosols in sustained mG to date but extensive studies have been performed in parabolic flight. While total deposition is greatly reduced in mG, there is an unexpectedly high deposition of small particles (0.5–1 mm), with this deposition being more peripheral. It seems that with small particles, nongravitational factors such as nonreversibility of flows, play a significant role in determining particle deposition. In future exploration missions, inhaled particles may be a significant factor. The dust on the surface of Mars is oxidative, due to the UV environment, and so reacts in the presence of water. This combined with the low gravity environment (B1/3G) may result in a disproportionate response of the pulmonary system to such dust.

Sleep

Sleep is reported to be poor in space flight. There are numerous potential causes, especially in short-term flights, including disruptions to circadian rhythms, noise, and other environmental factors. At this time the question of whether or not mG per se contributes to sleep disruption remains unclear. The record is however clearer with respect to sleep-disordered breathing. Full polysomnography performed on five subjects showed a significant reduction in the apnea-hypopnea index (AHI) from 8.3 h 1 preflight to 3.4 h 1 inflight, with one subject who showed mild sleep-disordered breathing preflight having a reduction in AHI from 22.7 h 1 to 9.7 h 1 inflight. There were concomitant reductions in the amount of time spent snoring. Preflight there were on average 18 arousals per hour, 5.5 of which could be attributed to respiratory events. Inflight, the number of arousals fell to 13.4 h 1 almost entirely as a consequence of a 70% reduction in the number of respiratory-related arousals, which fell to only 1.8

Extravehicular Activity

Both the shuttle and the ISS have a 14.7 psia (1013 kPa, 760 mmHg) 21% O2 environment. However, space suits operate at a very much lower pressure with a 100% O2 environment, in order to maintain suit flexibility and, thus, astronaut mobility. A direct transition from cabin pressure to suit pressure (US suit, 4.3 psia, B295 kPa, B220 mmHg; Russian suit, 5.9 psia, B405 kPa, B290 mmHg) would result in decompression sickness (DCS). Therefore, an extensive denitrogenation procedure must be performed before an extravehicular activity (EVA). To date, no astronaut has formally reported DCS during EVA, although at least one astronaut has informally reported symptoms in the knee on two occasions, Gemini X and Apollo 11, after depressurization to a 5.0 psia cabin pressure after several hours of prelaunch O2 prebreathe. In contrast, research subjects during prebreathe validation tests report DCS in substantial numbers. There are

SPACE, RESPIRATORY SYSTEM 119

Figure 1 Astronaut Donald Petit performing a pulmonary function experiment on board the US International Space Station in 2003. He is holding a breathing assembly in his right hand. Flows are measured by a pneumotachograph (blue component of the breathing assembly) and gas concentrations are measured by a mass spectrometer mounted in the rack in front of him. In his left hand is a large pneumotachograph for performing maximum expiratory flow-volume curves. Photo courtesy of NASA.

numerous reasons why DCS may not have been reported in EVA including the routine use of analgesics prior to EVA, masking of joint symptoms while working in uncomfortable space suits, repressurization at the completion of the EVA, and underreporting by the flight crew. It is however almost certain that venous gas emboli (VGE) are present in EVA. In a study of comparable prebreathes and decompressions to those used during EVA, more than 50% of the participants had detectable bubbles as indicated by Doppler ultrasound. The lungs act as a filter for the bubbles. During ascents from deep saturation dives these bubbles have ’ been shown to significantly alter the range of V’ A =Q in the lung as evidenced by the intrabreath measure’ presumably as a result of their emments of V’ A =Q, bolic effects. In a recent study on the ISS, astronauts ’ inperforming EVA measured the degree of V’ A =Q equality before and on the day following EVA. There ’ was no significant change in the degree of V’ A =Q inequality in the lungs of the crew members 24 h after EVA, suggesting that the current denitrogenation protocols are sufficiently protective to not cause long-term harm to the lungs of the EVA crew. Whether or not there is an acute effect of VGE resulting from EVA remains unknown.

Respiratory Diseases in Space Flight Essentially nothing is known about pulmonary diseases in space flight. While it is clear that some pulmonary diseases, for example, pneumonia and tuberculosis, have significant gravitational components, there are no reports of such problems in space

flight. The population that flies is typically in their lower middle-age, healthy, and of above average fitness; a circumstance that is unlikely to change until space flight becomes much more accessible than is currently the case (Figure 1). See also: Aerosols. Chemoreceptors: Central; Arterial. Diving. Environmental Pollutants: Overview; Particulate Matter, Ultrafine Particles. Exercise Physiology. Fluid Balance in the Lung. High Altitude, Physiology and Diseases. Hyperbaric Oxygen Therapy. Oxygen Toxicity. Particle Deposition in the Lung. Peripheral Gas Exchange. Pulmonary Circulation. Sleep Apnea: Overview. Sleep Disorders: Overview. Ventilation: Control.

Further Reading Estenne M, Paiva M, and Engel LA (1995) Gravity. In: Roussos C (ed.) The Thorax, pp. 1515–1539. New York: Dekker. Paiva M and Engel LA (1989) Gas mixing in the lung periphery. In: Chang HK and Paiva M (eds.) Respiratory Physiology (Lung Biology in Health and Disease Series), vol. 40, pp. 245–276. New York: Dekker. Prisk GK (2000) Invited review: microgravity and the lung. Journal of Applied Physiology 89: 385–396. Prisk GK, Paiva M, and West JB (2001) Gravity and the Lung: Lessons from Microgravity. New York: Dekker. West JB (1992) Life in space. Journal of Applied Physiology 72: 1623–1630. West JB, Elliott AR, Guy HJB, and Prisk GK (1997) Pulmonary function in space. JAMA 277: 1957–1961. West JB, Guy HJB, Elliott AR, and Prisk GK (1996) Respiratory system in microgravity. In: Fregly MJ and Blatteis CM (eds.) The Handbook of Physiology Section 4: Environmental Physiology, pp. 675–689. New York: Oxford University Press.

120 STEM CELLS

STEM CELLS C Cole, Princess Margaret Hospital, Perth, WA, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Adult stem cells have the capacity for self-renewal and terminal differentiation to one or more cell types. Lung stem cells include basal cells and mucus-secreting cells in the trachea, Clara cells in the bronchioles, and type II pneumocytes in the alveoli. Mesenchymal cells predominate in fetal lung development. In postnatal life the Clara cell is the most actively dividing cell in the tracheobronchial epithelium while type II pneumocytes are the progenitor cells for the alveolar epithelium. Under specific circumstances basal cells and pulmonary neuroendocrine cells may also proliferate and act as stem cells. All these putative stem cells have varied roles in normal lung repair and in disease. Mesenchymal progenitor cells and multipotential adult progenitor cells found in bone marrow differentiate into cells of endodermal, mesodermal, and ectodermal origin including lung fibroblasts. The finding of such heterogeneous stem cell populations in bone marrow and their presence in lung tissue may lead to stem cell manipulative and regenerative therapies for conditions such as idiopathic pulmonary fibrosis and cystic fibrosis transmembrane regulator gene therapy for cystic fibrosis.

Introduction Adult stem cells differentiate from pluripotent cells to more committed, tissue-specific cells. The majority of cells in adult tissues are differentiated but a number of stem cells are present in most tissues. Stem cells have the capacity to proliferate for self-renewal, to produce a large number of daughter cells, and to replace damaged or injured cells. Proliferation and replication are common to all stem cells but the number of daughter cells varies. In bone marrow, where there is a constant need to replenish blood cells, stem cells are relatively plentiful. In more stable tissues minimal numbers of dormant stem cells exist. Unipotent stem cells such as skin cells are capable of proliferation and differentiation along a single pathway while hematopoietic stem cells, mesenchymal stem cells, and intestinal stem cells are pluripotent and give rise to several different cell types. Once committed to a somatic cell lineage, these cells were considered incapable of differentiating to cells of a different embryonic lineage. However, the concept of uncommitted stem cells and stem cell plasticity has changed our understanding of stem cell commitment and introduced the theory that stem cells are affected by local environmental conditions.

Some postulate fusion of marrow-derived cells with recipient cells to explain the alteration in differentiation markers rather than true stem cell plasticity. The hematopoietic stem cell (HSC) is the most well characterized adult stem cell. Attempts to purify HSCs on the basis of cell surface markers such as CD34, CD38, and CD45, have defined other multipotential stem cell subsets present in bone marrow. Mesenchymal stem cells (MSCs) give rise to bone, muscle, neural cells, cartilage, and hematopoietic stromal cells; multipotential adult progenitor cells (MAPCs) generate cell lineages of mesodermal, endodermal, and ectodermal origin. The CD34 þ stem cells generate HSCs and blood vessel endothelial cells. The CD45þ ClqRpþ CD34þ /
cells generate HSCs and liver stem cells while MSCs and MAPCs are CD45
. The finding of such heterogeneous stem cell populations in bone marrow has led to the burgeoning field of stem cell manipulation for regenerative medical therapies. While the potential therapeutic use of embryonic stem cells has raised enormous public controversy, there are far fewer ethical concerns surrounding the use of adult stem cells. The clinical use of allogeneic (donor-derived) hematopoietic stem cell transplantation (HSCT) for acquired disorders such as aplastic anemia and the leukemias is well known. Allogeneic HSCT is also curative for congenital and hereditary disorders of hematopoietic origin including red cell disorders (thalassemia), lymphocyte disorders (the immune deficiency syndromes), neutrophil disorders, and macrophage/monocyte disorders such as the storage diseases and other metabolic or enzyme deficiencies. MAPCs proliferate extensively, seemingly without senescence, and are an ideal source for stem cell therapy. Clinical trials of allogeneic transplantation are now being assessed to determine if MSCs and MAPCs can be successfully transplanted to treat hereditary disorders such as osteogenesis imperfecta. Autologous bone marrow MSCs expanded in culture and embedded in collagen have been successfully transplanted into osteoarthritic joints. CD34 þ cells and MSCs differentiating into cardiac myocytes have been incorporated into the heart as therapy for myocardial infarction. Studies of sex-mismatched allogeneic bone marrow recipients have revealed donor-derived endodermal tissue cells such as hepatocytes and cholangiocytes. Bone marrow stem cells can be readily mobilized into the circulation using chemotherapeutic agents and/or cytokines and

STEM CELLS 121

peripheral blood stem cell (PBSC)-derived ectodermal and endodermal tissues have also been demonstrated in sex-mismatched transplantation recipients. The availability of PBSCs circumvents the need for bone marrow harvesting and allows for enrichment by apheresis techniques. Umbilical cord blood is also rich in HSCs and is ideally suited for banking.

Stem Cell Function in the Normal Lung The lung is composed of a conducting airway and a gas-exchange system with many different cell types including ciliated cells, mucus cells, basal cells, Clara cells, type I and II pneumocytes, and pulmonary neuroendocrine cells. Lung development and repair of injury require a balance between proliferation and differentiation of epithelium. The study of lung stem cells is frustrated by slow cell turnover and difficulty with stem cell isolation; however, several endogenous lung progenitors have been identified. In fetal lung, signals from mesenchymal cells play an important role in normal branching morphogenesis and pulmonary epithelial differentiation. Late in fetal development and in postnatal life, there is lineage restriction between bronchial and alveolar epithelial cells. The most distinctive cell type in the tracheobronchial epithelium, the ciliated cell, is a terminally differentiated cell largely incapable of cell division. Progenitor cells, such as Clara cells, must differentiate to the ciliated phenotype. The Clara cells of the lung are nonciliated secretory cells characterized by apical cytoplasmic projections and abundant endoplasmic reticulum and secretory granules. Found throughout the respiratory system, Clara cells are the most actively dividing cells in the prenatal and postnatal lung and are the progenitors of themselves and of the ciliated cells in the bronchioles. A subset of Clara cells is resistant to airway pollutants and shows multipotent cell differentiation. Clara cells produce the anti-inflammatory protein CC10 in abundance and numerous cytochrome enzymes to inactivate carcinogens. Under oncogenic stimulation, the Clara cell induces expression of the cyclindependent kinase inhibitors to reduce proliferation. This prevents tumorigenesis. However, if the oncogenic stimulus persists, this mechanism is insufficient and the lung epithelium is transformed. Basal cells and pulmonary neuroendocrine cells affect the growth and differentiation of lung epithelium and may be recruited to act as stem cells. Type I and type II pneumocytes make up the alveolar epithelium. Type I cells are flat with cytoplasmic projections and a protuberant nucleus. They do not divide. Type II cells are cuboidal, metabolically

active cells with abundant cytoplasmic organelles. They secrete surfactant, proliferate after injury, and act as progenitor cells for type I and type II pneumocytes by transdifferentiating to an intermediate morphology and then differentiating to the alveolar type I phenotype with cytoplasmic projections, prominent nucleus, and loss of lamellar bodies. Type II pneumocytes are the progenitors responsible for repair of injured distal lung. The neuroendocrine cells are frequent in the developing lung and play a major role in airway growth and development. In the adult lung they form o1% of epithelial cells usually present in neuroepithelial bodies. They are unlikely to be stem cells but secrete regulatory factors for epithelial cell renewal. To characterize pulmonary stem cells further, gene promotor analyses of the surfactant protein genes and the Clara cell protein CC10 have identified the NKX2 homeodomain gene thyroid transcription factor-1 (TTF-1) as critical for lung formation and epithelial cell differentiation. Expression of TTF-1 is increased in lung injury and repair. The lung maintains regulatory pathways to counteract transformation and oncogenesis. Indeed most small cell carcinomas and adenocarcinomas express TTF-1. In addition, fibroblast growth factor (FGF) signaling regulates these putative alveolar stem cells and FGF genes may provide therapeutic targets to induce lung repair and regeneration. The murine models of Krause et al. demonstrated that male bone marrowderived cells were capable of differentiation into bronchiolar epithelium and type II pneumocytes following transplantation into lethally irradiated female mice. Furthermore, mesenchymal stem cells appeared to differentiate into distal alveolar epithelial cells in bleomycin-treated mice.

Stem Cells in Respiratory Diseases Asthma and Allergy

Bronchial myofibroblasts represent an important therapeutic target for asthma because of their role in the genesis of subepithelial fibrosis in airway remodeling. CD34þ hematopoietic precursor cells of myeloid lineage have been found in acute allergic inflammation in allergic rhinitis, asthma, and eczema. Indeed, allergen exposure induces the accumulation of CD34þ fibroblasts that differentiate into myofibroblasts. Thus, hematopoiesis and migrational pathways of hematopoietic cells are targets for treatment with corticosteroids, leukotriene inhibitors, blockade of the eosinophil-active cytokine, interleukin-5, antihistamines and chemokine receptor antagonists, which may modulate progenitor cell influx.

122 STEM CELLS Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating disease for which no effective therapy exists. There is inexorable airspace obliteration and the mean time from diagnosis to death is 5 years. Alveolar type II pneumocyte injury and apoptosis is an important early feature in the pathogenesis of IPF. On pathological examination, there are areas of active fibrogenesis with fibroblast foci and extracellular matrix deposition. The number and appearance of fibroblastic foci on lung biopsy specimens can predict length of survival. Fibroblasts are assumed to arise from intrapulmonary cells, but circulating blood cells resembling fibroblasts are recruited to areas of lung injury. These cells migrate to sites of wound healing and serve as a source of fibroblasts and myofibroblasts that normally participate in the repair process. Mice models of pulmonary fibrosis, using bleomycininduced lung injury and radiation fibrosis models, have shown that the collagen expression in fibrotic lung is due mainly to bone marrow-derived precursor cells strongly suggesting a direct role for these cells in the pathogenesis of fibrosis. The influx of these bone marrow-derived cells into the lung suggests fibrosis is mediated by chemokines in a similar fashion to the recruitment of leukocytes in pulmonary inflammation. Fibroblasts express the chemokine receptors CXCR4 and CCR7 and migrate towards stromal cell-derived factor 1 and secondary lymphoid tissue chemokine. The source of these chemokines is likely to be local lung fibroblasts or endothelial cells as well as bone marrow-derived macrophages that precede the arrival of the fibrocytes into areas of fibrosis. Idiopathic pulmonary fibrosis is a disease of abnormal wound repair and remodeling rather than inflammation, and treatment should be directed at inhibiting the fibroproliferative response. Perhaps there is a role for allogeneic hemopoietic stem cell transplantation to change the phenotype of the marrow-derived fibroblasts.

Gene therapy aimed at hemopoietic stem cells has been successful in children with other single gene disorders. It is speculated that damaged lung attracts hemopoietic stem cells and in cystic fibrosis, inflammation and tissue injury occur early. However, retroviral vectors insert themselves randomly and insertional mutagenesis remains a risk (two patients treated for adenosine deaminase deficiency have developed leukemia). If only 10% of cells need to be corrected for the chloride defect in cystic fibrosis to be reversed, then the bone marrow-derived engraftment rate may be adequate. However, if the sodium defect requires correction, 100% of the cells need to be corrected and this is unlikely to occur from a bone marrowderived cell. Lung Cancer

Lung cancer patients often have multiple lesions at different stages of tumorigenesis with both stem cells and differentiated cells escaping repair and control mechanisms. Either a single common transformed stem cell gives rise to different tumor histologies, or multiple cells within a field are transformed. Cytogenetic studies of lung cancers frequently reveal abnormalities in the region of the cellcycle-regulating genes such as the cyclin-dependent kinase inhibitors. Autologous peripheral blood stem cell rescue to allow for increased chemotherapeutic intensity is used to treat numerous malignancies. HSCs are collected by peripheral blood apheresis following chemotherapy and cytokine stimulation. CD34þ cell selection reduces the chance of malignant contamination of the stored aliquot. In small cell lung cancer, such chemotherapeutic intensification with stem cell rescue is feasible despite grade 4 hematological toxicity. Respiratory Complications of HSCT

Cystic Fibrosis

Identifying lung stem cells would provide an ideal target for gene therapy providing the ability to transfer the cystic fibrosis transmembrane regulator (CFTR) gene once. Gene therapy by topical delivery to the airway of patients with cystic fibrosis has been disappointing since vectors are inefficient and mucous barriers prevent transfection of airway cells. Even if transfection does occur, the cells are terminally differentiated and recurrent treatment is required. Indeed the CFTR gene is predominantly expressed in the submucosal glands, which are largely inaccessible to topical gene therapy.

Approximately 50% of patients having HSCT will develop respiratory complications with 40% mortality. Both chemotherapy and radiation predispose to idiopathic pneumonitis in which no infectious causative agent can be found. Diffuse alveolar hemorrhage, characterized by severe thrombocytopenia, is also of obscure etiology and may coexist with bacterial, viral, or fungal pneumonia. Pulmonary veno-occlusive disease (VOD) presents with dyspnea, hypoxemia, pulmonary hypertension, and right heart failure. Like hepatic VOD, its pathogenesis is unknown although conditioning agents and viral infections are implicated.

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Patients receiving total body irradiation for bone marrow transplantation or lung irradiation for esophageal or lung cancer frequently develop pulmonary damage. Acute pneumonitis and progression to fibrosis is dependent on the radiation dose, fraction size, and volume of lung irradiated. Treatment of acute pneumonitis with corticosteroids may not prevent the fibrotic changes that occur 6–24 months later. Normal lung defense mechanisms to prevent infection include alveolar macrophages as well as the influx of neutrophils to eradicate bacteria and fungi. Cell-mediated immunity relies on adequate numbers of lymphocytes and dendritic cells to act as antigenpresenting cells. Post-transplantation, patients have marked pancytopenia and severe combined immune deficiency and are prone to respiratory infections. Neutrophil recovery occurs in 2–3 weeks but donorderived macrophages take 3–4 weeks to repopulate the lungs and may not function normally for 2–3 months. Lymphocytes reach normal numbers by 3 months post-transplantation but a return to normal

immunoglobulin levels and cell-mediated immune function may take 12 months. The presence of acute or chronic graft-versus-host disease further delays immune recovery. Useful investigations include chest radiography, bronchoscopy with bronchoalveolar lavage, lung biopsy, and polymerase chain reaction techniques to detect viral antigenemia. Therapy for bacterial pneumonia is frequently empiric and must include antipseudomonal antibiotics. Empiric antifungal therapy with amphotericin B is added for prolonged fever. Cytomegalovirus (CMV) causes 40% of pneumonias in HSCT and may occur in as many as 20% of all transplant patients. Prophylactic therapy with acyclovir and gancyclovir is recommended where the donor or recipient is CMV seropositive prior to transplantation. CMV-negative blood products should be given where possible. The addition of CMV hyperimmune globulin is more controversial. Invasive pulmonary aspergillosis or candida pneumonia occur in 5–10% of patients and require treatment with amphotericin B. The routine use of

Pluripotent stem cell

Bone marrow

Hemopoietic stem cell

Site of allogeneic hemopoietic stem cell transplant or autologous gene stem cell therapy

Multipotent adult progenitor cell Mesenchymal stem cell Possible mechanisms: 'transdifferentiation' or stem cell fusion

Lung stem cells

Tracheobronchial airway Basal cells, mucus-secreting cells, Clara cells, and pulmonary neuroendocrine cells

Alveolar epithelium Type I and type II pneumocytes

Site of local gene therapy Figure 1 Postulated models of lung epithelial cell derivation from bone marrow stem cells and possible sites of therapy.

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prophylaxis has made infection with Pneumocystis carinii relatively rare. Following engraftment, cotrimoxazole prophylaxis should be re-instituted and continued until immune function has returned to normal. The outcome of patients requiring transfer to the intensive care unit for progressive respiratory failure during transplantation is poor Figure 1. See also: Allergy: Allergic Rhinitis. Alveolar Hemorrhage. Asthma: Overview. Bronchoalveolar Lavage. Cell Cycle and Cell-Cycle Checkpoints. Clara cells. Colony Stimulating Factors. Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Defense Systems. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Fibroblast Growth Factors. Fibroblasts. Gene Regulation. Gene Therapy for Respiratory Diseases. Genetics: Overview. Immunoglobulins. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Keratinocyte Growth Factor. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; Monocytes; T-Cells; Pulmonary Macrophages. Lipid Mediators: Leukotrienes. Lung Development: Overview. Lung Imaging. Macrophage Inflammatory Protein. Macrophage Migration Inhibitor Factor. Neurophysiology: Neuroendocrine Cells. Pneumonia: Atypical; Community Acquired Pneumonia, Bacterial and other Common Pathogens; Viral; The Immunocompromised Host. Pulmonary Effects of Systemic Disease. Pulmonary Fibrosis. RadiationInduced Pulmonary Disease. Surfactant: Overview. Surgery: Transplantation. Transcription Factors: Overview; NF-kB and Ikb. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumors, Malignant: Overview. Ventilation: Overview.

Horowitz EM (2003) Stem cell plasticity: a new image of the bone marrow cell. Current Opinion in Pediatrics 15: 32–37. Kotton DN, Summer R, and Fine A (2004) Lung stem cells: new paradigms. Experimental Hematology 32: 340–343. Kreit JW (2000) Respiratory complications. In: Ball ED, Lister J, and Law P (eds.) Hematopoietic Stem Cell Therapy, pp. 563– 577. USA: Churchill Livingstone. Magdaleno SM, Barrish J, Finegold MJ, and DeMayo FJ (1998) Investigating stem cells in the lung (review). Advances in Pediatrics 45: 363–396. Martin-Rendon E and Watt SM (2003) Stem cell plasticity (review). British Journal of Haematology 122: 877–891. Mason RJ, Williams MC, Moses HL, Mohla S, and Berberich MA (1997) Stem cells in lung development, disease and therapy (review). American Journal of Respiratory Cell and Molecular Biology 16: 355–363. Pitt BR (2004) Stem cells in lung biology. American Journal of Physiology. Lung Cellular and Molecular Physiology 286: L621–L623. Tao H and Ma DDF (2003) Evidence for transdifferentiation of human bone marrow-derived stem cells: recent progress and controversies. Pathology 35: 6–13.

Glossary Hemopoietic stem cells (HSCs) – capable of differentiation into all blood elements and characterized by the expression of the cell surface marker CD34 Mesenchymal stem cells (MSCs) – capable of differentiating into osteoblasts, chondroblasts, fibroblasts, and adipocytes Multipotent adult progenitor cells (MAPCs) – differentiate into most somatic cell types including lung Stem cell – an undifferentiated cell capable of longterm self-renewal and multilineage differentiation

Further Reading Bishop AE (2004) Pulmonary epithelial stem cells. Cell Proliferation 37: 89–96. Hashimoto N, Jin H, Lui T, Chensue SW, and Phan SH (2004) Bone marrow-derived progenitor cells in pulmonary fibrosis. Journal of Clinical Investigation 113: 243–252.

Stem cell fusion – fusion of 2 different somatic cells to form a cell with 2 nuclei Transdifferentiation/stem cell plasticity – committed stem cell capable of differentiation into cells of a different tissue

STRESS DISTRIBUTION IN THE LUNG S J Lai-Fook, University of Kentucky, Lexington, KY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The mechanical properties of the lung parenchyma are described as a linear elastic continuum. Small deformations

are measured from a state of uniform inflation defined by the pressure–volume curve of the lung. Two material constants, the bulk and shear modulus, are measured by standard deformation tests. Stress–strain distribution in the lung is studied using methods of linear elasticity. Two general types of nonuniform lung deformation problems are considered. First, the lung within the thorax is acted on by forces that arise from the interaction among the lung, heart, and abdomen. Gravity produces a vertical gradient in transpulmonary pressure of a

STRESS DISTRIBUTION IN THE LUNG 125 magnitude that depends on body position. Second, the lung parenchyma interacts with internal structures such as blood vessels and bronchi. This interaction produces interstitial pressures surrounding the structures that change with lung inflation and vascular pressure. These pressures are related to the formation of interstitial fluid cuffs with edema formation that affects blood flow and airway resistance.

Introduction The spatial distribution of mechanical stress and strain in the lung relates to several areas of respiratory medicine and physiology including ventilation distribution, pulmonary circulation, lung fluid balance, and acceleration physiology. A nonuniform distribution in stress and strain in the normal lung is considered from two different perspectives. One is the overall stress distribution that results from the effect of gravity on the lung and its adjacent structures. Here the lung parenchyma is treated as an isotropic, homogeneous elastic continuum. Forces due to gravity acting on the lung and its adjacent structures, the heart, ribcage and abdomen, cause a gravity-dependent gradient in regional lung expansion. The other perspective involves local nonuniform stresses in the lung parenchyma that surrounds internal structures such as pulmonary blood vessels and bronchi. The study of stress–strain behavior requires knowledge of material properties of the lung parenchyma on a macroscopic scale. Here, forces per unit area (stress) and changes in displacement per unit length (strain) are averaged over several alveoli. For an elastic continuum, material properties are defined mathematically by stress–strain (constitutive) equations. These equations define elastic constants whose magnitudes depend on the material. On a microscopic scale of the dimension of an alveolus, forces within the alveolar septal wall are not uniform and the assumption of a continuum is not valid. Models of the lung behavior at the alveolar level are treated elsewhere. The topics of this article are arranged as follows. First, the material properties of lung parenchyma are described in terms of linear (infinitesimal) elasticity in which the elastic constants are valid for small deformations from uniform states defined by the lung pressure–volume behavior. Stress distribution in the lung under the effects of gravity in different body positions is described. This is followed by the interaction among blood vessels, bronchi, and the surrounding lung parenchyma. Some examples of nonuniform stress associated with diseased lung are discussed.

Small Deformation Method Mead and co-workers showed in 1970 that the pressure acting on any internal surface of a uniformly

inflated lung is equal to the pleural pressure acting on its outer boundary. They described nonuniform macroscopic strains from states of uniform inflation given by the lung pressure–volume behavior. This method of considering small nonuniform deformations superimposed on a large uniform deformation was the basis for applying linear elasticity theory to solving nonuniform deformation problems in the lung. The alternative method considers large deformations from the stress-free state near residual volume and requires relatively more complex material properties and analysis. The linear elasticity method has several advantages. First, two independent elastic constants required for linear elasticity are easily measured with simple tests on isolated lung lobes. Time-dependent hysteresis, viscoelastic and viscoplastic effects, often observed with large volume inflations, are negligibly small when only small deformations are imposed. The assumptions of isotropy and homogeneity for an elastic continuum are closely approximated during a uniform lung inflation. Deformation problems of the complex lung geometry are tractable using well-developed computational (finite-element) methods of linear elasticity. Static deformation experiments are easily designed to verify theoretical predictions. Measurements of small-amplitude stress waves predicted by the linear elasticity theory support the validity of the elastic constants.

Elastic Constants: Bulk Modulus, Shear Modulus, Young’s Modulus, and Poisson’s Ratio Two elastic constants, the bulk modulus and the shear modulus, are sufficient to describe the lung parenchyma as a linear elastic continuum. The bulk modulus (K) is a measure of the bulk stiffness of the lung undergoing a uniform inflation. It is obtained by taking the slope (dP/dV) of small pressure (P)volume (V) loops imposed at different lung inflation (transpulmonary, Ptp) pressures and normalizing by the lung volume: K ¼ dP/(dV/V). K is the reciprocal of the specific lung compliance (dV/V)/dP. The bulk modulus increases with lung inflation; its magnitude lies between three and six times the transpulmonary pressure (Figure 1). The shear modulus is a measure of the resistance of the lung in response to a change in shape. The shear modulus is measured by an indentation test on the surface of an inflated lung. The shear modulus also increases with lung inflation and has a magnitude of 0.7 times the transpulmonary pressure. The much smaller value for the shear modulus than for the bulk modulus indicates that for a given external load, the lung deforms more easily

126 STRESS DISTRIBUTION IN THE LUNG

Shear modulus (cmH2O)

20 60 40 20

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40 20

100

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0 0

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Figure 1 Effect of age and transpulmonary pressure (Ptp) on the bulk and shear moduli of human lungs. Note that both moduli increase with Ptp and age. Reproduced from Lai-Fook SJ and Hyatt RE (2000) Journal of Applied Physiology 89: 163–168, used with permission of The American Physiological Society.

with a change in shape than with a change in volume. Other elastic constants commonly used in structural mechanics can be computed from the bulk and shear moduli. The Young’s modulus, the ratio of the direct stress to the direct strain in a uniaxial deformation, has a magnitude of twice the transpulmonary pressure. The Poisson’s ratio is a measure of the ability of the lung to change volume when it is deformed. The Poisson’s ratio ranges from 0.35 to 0.45, higher than values (0.2–0.3) for structural metals but less than the value of 0.5 for incompressible rubber-like materials.

Determinants of Elastic Constants Lung elastic constants are nearly unique functions of transpulmonary pressure and are independent of the history of the lung pressure–volume maneuver; that is, invariant with respect to the volume arrived at on lung inflation or on lung deflation. The elastic constants are similar among species. Both the bulk and shear moduli increase with age (Figure 1), most likely due to changes in the lung tissue elastin content or in alveolar surface forces associated with an increased alveolar size with age. The increased stiffness of the

lung with age in adulthood is opposite to the behavior observed with maturation from the newborn to the juvenile stage of development. Lung stiffness also increases in diseases such as fibrosis. Both tissue elastic and surface forces contribute to the elastic moduli. Elastic forces arise from the elastic fibers in the alveolar septal walls and interstitium surrounding blood vessels and bronchi. Studies relating the microstructural to the macrostructural properties attribute the bulk and shear moduli to the tensile forces in the alveolar walls. The elastic constants of the liquid-filled lung are similar in magnitude to those of the air-filled lung. Thus, the effects of alveolar surface tension in the air-filled lung are offset by the changes in alveolar configuration in the liquid-filled lung. Surface forces are generated by pulmonary surfactant that acts on the alveolar air–liquid interface. Alveolar surface forces also serve to reduce the interstitial fluid pressure on the alveolar epithelium to values below the alveolar air pressure. This effect increases the transvascular pressure gradient for microvascular fluid filtration and reduces the storage capacity of the interstitium during edema formation. In the normal lung, alveolar surface forces are small at functional residual capacity and increase with lung inflation. However with lung disease, an increase in alveolar surface tension predisposes the lung to the development of edema.

Stress Waves in the Lung: Relation to the Elastic Constants A property of the lung described as an elastic continuum is its ability to transmit stress waves. Examples of stress waves of small amplitudes include seismic waves that are generated on the earth’s surface by earthquakes. Stress waves of large amplitude, shock waves, commonly occur in the lung during combat, contact sports, and automobile accidents. There are three basic types of stress waves: bulk or longitudinal waves that depend primarily on the bulk modulus, shear waves that depend on the shear modulus, and Rayleigh-type surface waves that propagate on the lung surface. The velocity of surface waves is nearly equal to that of shear waves. Because of the dependence on the elastic constants, the velocity of lung stress waves increases with lung inflation, and the velocities of longitudinal waves are greater than those of shear waves. The velocity of stress waves is inversely related to the square root of the lung density, and thus, decreases with increases in vascular volume or with the formation of pulmonary edema. In the isolated lung, the pleural membrane

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covering the surface of the lung limits the transmission of surface waves to frequencies below 70 cycles per second. The pleural membrane also contributes about 20% of the total lung static recoil at functional residual capacity. In the intact chest at functional residual capacity, the presence of the relatively stiff ribcage reduces the transmission of lung surface waves to frequencies below 5 cycles per second. Thus, the ribcage serves to isolate and protect the lung from external impact loads.

The vertical gradients in transpulmonary pressure predicted by the structural analyses agree with the vertical gradients in regional lung volume measured by computerized imaging and with the vertical gradients in pleural liquid pressure measured by rib capsules. The three types of studies support the general concept that pleural surface pressure is equal to pleural liquid pressure.

Stress Distribution around Blood Vessels

Studies in both humans and experimental animals have established that regional lung volume within the thorax varies along its vertical dimension. There is a vertical gradient in regional lung volume and in transpulmonary pressure that depends on body position. Studies in 1966 by Milic-Emili and co-workers using radioactive xenon gas in humans in the upright and supine body positions, showed a vertical transpulmonary pressure gradient of 0.3 cmH2O cm
1 height with the upper (nondependent) regions more expanded than the lower (dependent) regions. Studies in dogs using computerized radiographic imaging of lung markers and lung density showed that the vertical gradient measured in the supine position was abolished with body inversion to the prone position. Finite-element methods of structural mechanics have been used to study the contribution of the weight of the lung, heart, and abdomen on the vertical stress and regional volume distributions in the lung. The earliest study in 1972 by West and Matthew used a nonlinear stress–strain behavior for the lung parenchyma to show that gravity produced a vertical gradient in transpulmonary pressure of 0.2 cmH2O cm
1 height, a result expected for a fluid with the density of the lung. Subsequent finite-element analyses using linear elastic constants showed that in addition to the weight of the lung, the vertical gradient was in part caused by the weight of the heart and abdomen in conjunction with a compliant diaphragm. In the upright and supine body positions, the weight of the heart and abdomen increases the vertical gradient primarily by compressing the dependent lung regions. In the prone position, the heart is supported by the sternum and has little effect on regional lung volume. In the upright position, the weight of the abdomen produces a vertical extension of the lung and an increase in lung volume. The opposite effect occurs with body inversion to the headdown position, where the weight of the abdomen compresses the lung to near-residual volume and eliminates regional differences in lung volume.

The stress distribution surrounding large pulmonary blood vessels has received much attention because of its importance to blood flow and fluid accumulation in the lung. In early studies (1946), Macklin observed that lung inflation increased the volume of pulmonary blood vessels and concluded that the parenchyma exerts a distending stress on the outside of the vessel. Subsequent experiments attempted to quantify the distending surface stress, the perivascular pressure. The application of continuum mechanics to explain the pressure–diameter behavior of blood vessels measured radiographically in isolated dog lungs provided estimates for the perivascular pressure (Figure 2). In these studies, the blood vessel is assumed to be a thin-walled compliant tube embedded in a cylindrical hole surrounded by lung parenchyma. The pressure–diameter behavior of the hole is related to the shear modulus of the parenchyma. The 130 Du vs. Ptp 120 Vessel diameter (%)

Effect of Gravity on Regional Lung Volume

Vein Ptp 25 16 12 8 4

110 100 90 80

Ptp cmH2O

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25 16 12

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−10

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Vascular pressure (cmH2O) Figure 2 Vessel diameter measured radiographically vs. vascular pressure at several transpulmonary pressures (Ptp). Du is the diameter of a uniformly expanding hole in the lung parenchyma. De is the intrinsic pressure–diameter behavior of the vessel. Lines are theoretical predictions through the data points. Reproduced from Lai-Fook SJ (1979) Journal of Applied Physiology 46: 419–429, used with permission from The American Physiological Society.

128 STRESS DISTRIBUTION IN THE LUNG Transpulmonary pressure (cmH2O) 0

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upper lung regions having the lowest interstitial pressures. This vertical gradient in interstitial pressure partly offsets the vertical hydrostatic gradient in vascular pressure (1 cmH2O cm
1 height) and produces a more uniform transvascular pressure difference for microvascular fluid filtration throughout the height of the lung. The close relationship between surface and liquid pressures in the interstitial and pleural spaces results in vertical gradients in liquid pressure that are not hydrostatic. The difference between the vertical gradient in liquid pressure and the hydrostatic value is matched by a viscous flowinduced pressure loss caused by a relatively high fluid resistance in the interstitial and pleural spaces.

0

Effect of Lung Development

−15 30

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Figure 3 Perivascular surface pressure relative to pleural pressure vs. transpulmonary pressure and lung volume at several vascular pressures (Pv). Reproduced from Lai-Fook SJ (1979) Journal of Applied Physiology 46: 419–429, used with permission from The American Physiological Society.

intrinsic pressure–diameter of the vessel is matched to that of the parenchymal hole at different lung volumes and vascular pressures. This procedure produces estimates of the perivascular pressure as a function of transpulmonary and vascular pressures (Figure 3). Perivascular pressure is near the pleural pressure at functional residual capacity, and is reduced with lung inflation and with a decrease in vascular pressure.

Relationship between Perivascular Surface and Interstitial Fluid Pressures In subsequent studies, interstitial fluid pressure measured between the artery and bronchus at the lung hilum was similar to the perivascular pressure predicted from the stress analysis. This indicated an identity between perivascular interstitial fluid pressure and perivascular surface pressure. Thus, interstitial fluid pressure is a reflection of the forces in the structure adjacent to the interstitium. A similar principle is invoked for the identity between pleural surface pressure and pleural liquid pressure. The lower perivascular pressure at higher lung volumes in conjunction with the vertical transpulmonary pressure gradient in the intact chest produces a nonuniform regional distribution of interstitial pressure with the

Pulmonary blood vessels expand with the inflation of air-filled lungs because perivascular pressure is reduced below the pleural pressure. By contrast, the inflation of liquid-filled lungs increases the perivascular pressure above the pleural pressure towards the alveolar pressure through the formation of interstitial fluid cuffs. As applied to the liquid-filled fetal and newborn lungs, the increased perivascular pressure with lung inflation results in compression of the vascular volume and a reduction in the transvascular pressure gradient. During birth, the reduced transvascular pressure gradient ameliorates the colloid osmotic absorption of interstitial liquid by the vasculature and the active absorption of alveolar liquid across the alveolar epithelium. Interstitial fluid cuffs are cleared within a few hours after birth.

Relationship of Interstitial Compliance to Airway and Vascular Resistance An increase in interstitial volume with edema formation results in an increase in interstitial pressure. This behavior is measured by the interstitial compliance defined as the change in interstitial volume per unit change in interstitial pressure. The perivascular interstitium interposed between the vessel and the parenchyma has a compliance nearly equal to that of the surrounding parenchyma because the contribution of the vessel wall is relatively small. A similar behavior applies to the peribronchial interstitium. The compliance of the surrounding parenchyma is inversely related to the parenchymal shear modulus that increases with the transpulmonary pressure. At functional residual capacity, parenchymal and interstitial compliances are relatively high, and most of the interstitial expansion associated with the initial formation of pulmonary edema occurs by an

STRESS DISTRIBUTION IN THE LUNG 129

outward expansion of the parenchyma with little compression of the vessel and bronchus. Thus, the formation of perivascular and peribronchial fluid cuffs would have only a small effect on vascular and airway resistance. Accordingly, the large increases in airway and vascular resistances observed with pulmonary edema formation are attributed to the blockage of the airway lumen and the compression of microvessels by alveolar edema, respectively. Staub and co-workers in 1967 reported the sequence of water accumulation in the lung during the formation of pulmonary edema: interstitial cuff expansion followed by pericapillary edema and alveolar flooding.

Growth Rate of Interstitial Fluid Cuffs: Interstitial Fluid Resistance That the perivascular pressure is near the pleural pressure at low lung volumes and is reduced below the pleural pressure with lung inflation is partly the result of the parenchymal stiffness and partly the result of the mismatch between the intrinsic diameter of the vessel and the uniform expansion of the parenchyma. This mismatch is greater for the larger vessels and is less for smaller vessels in the lung periphery where the vessels approach the alveolar size. Under the latter conditions, the continuum theory is no longer valid; the perivascular pressure approaches the alveolar pressure and prevents the formation of perivascular fluid cuffs. This results in a longitudinal gradient in perivascular pressure that drives fluid away from the site of microvascular filtration effectively keeping the gas-exchanging regions dry. During edema formation, a similar longitudinal gradient occurs with the sequential formation of interstitial cuffs from small to large vessels. Interstitial compliance together with interstitial fluid resistance (inverse of hydraulic conductivity) determines the time constant for the growth of interstitial fluid cuffs. The interstitium surrounding extra-alveolar blood vessels and bronchi is distributed as in-series fluid-resistive elements with shunt volume capacitors. The growth of interstitial cuffs occurs initially around small extra-alveolar vessels proximal to the site of microvascular fluid exchange followed by expansion around larger vessels. In liquid-inflated lungs, the growth rate of interstitial cuffs increases with the inflation pressure and varies among species. Interstitial resistance to flow depends on a complex colloid osmotic interaction among the interstitial constituents. Interstitial resistance is reduced with albumin solution, with increased hydration, and with the enzymatic degradation of interstitial constituents such as hyaluronan, elastin, and collagen. Thus, the

degradation of these constituents is sometimes associated with lung disease involving edema, such as adult respiratory distress syndrome (ARDS)

Differences between Perivascular and Peribronchial Pressure With lung inflation, perivascular pressure is reduced below the pleural pressure, while peribronchial pressure remains near the pleural pressure. This difference in behavior is partly a result of the different pressures acting within the blood vessel and bronchus. As the lung inflates, the same pleural pressure inflates both the bronchus and the lung simultaneously and produces a nearly homogeneous expansion of the bronchus with the parenchyma. By contrast, with lung inflation, blood vessels do not expand homogeneously with the parenchyma partly because pulmonary vascular pressure generated by the right heart pump varies independently of the pleural pressure.

Interaction among the Airway, Artery, and Lung Parenchyma The enclosure of the artery and the bronchus within a common interstitial sac occurs in many species, including man. A finite-element analysis of this system consisting of two adjoining tubes surrounded by an elastic continuum shows nonuniform stress and strain distributions surrounding the tubes (Figure 4). Changes in vascular diameter due to changes in vascular pressure or in vascular smooth muscle tone produce changes in the perivascular pressure with little change in the mean peribronchial pressure. This results in a shape change of the bronchus with no change in bronchial cross-sectional area and airway resistance. Similarly, bronchoconstriction results in a shape change of the vessel with no change in vascular resistance. However, at the arterial–bronchial joint there is a stress concentration that results in a reduction in interstitial pressure equal to twice the change in the perivascular pressure. This stress concentration is associated with the perivascular air dissection caused by lung hyperinflation, a phenomenon noted by Macklin. With edema formation, the oval shapes of the artery and bronchus are reduced to circular shapes as the perivascular and peribronchial interstitial pressures increase and become uniform (Figure 5). At a low lung volume, this bronchial shape change is accompanied by a small reduction in bronchial diameter because bronchial compliance is relatively high. By contrast at a high lung volume, bronchial diameter remains constant because bronchial compliance is low. Thus, any change in airway

130 STRESS DISTRIBUTION IN THE LUNG Db2

5

3

Bronchial diameter (%)

Stress cmH2O

Pa cmH2O −20

105

4

2 1 0 −1 −2

Db2 95

25 10 −20

90

Db1

0

Ptp = 25 cmH2O

50

100

Pa cmH2O

Figure 4 Interaction among the bronchus, artery, and lung parenchyma in response to a reduced arterial pressure. (a) The radial stress (solid circles) and circumferential stress (open circles) distribution in parenchyma surrounding the bronchus and artery. Note the stress concentration at the arterial–bronchial joint is equal to twice the mean perivascular stress. (b) The oval shapes of the bronchus and artery (solid lines) compared to the initial undeformed circular shapes (dotted lines). Reproduced from Lai-Fook SJ and Kallok MJ (1982) Journal of Applied Physiology 52: 1000–1007, used with permission from The American Physiological Society.

resistance due to the small reduction in bronchial diameter with edema would diminish with lung inflation.

Effect of Bronchoconstriction During lung inflation, the bronchus expands homogeneously with the lung parenchyma so that the peribronchial pressure remains close to the pleural pressure at all lung volumes. However, under conditions of bronchoconstriction, peribronchial pressure is reduced below the pleural pressure because of the distending force of the surrounding lung parenchyma. Bronchoconstriction of peripheral airways also increases the tension of surrounding alveolar walls. This results in an increased parenchymal shear modulus and a further reduction in peribronchial pressure. The parenchymal support of the airway during airway constriction is greater at higher lung volumes with a larger parenchymal shear modulus.

Bronchial diameter (%)

(b)

200

Ptp = 6 cmH2O

−10

Artery

150

Weight gain (% initial weight)

105

Bronchus

A

25

(a)

−4

(a)

B

10 100

85

−3

Db1

100

10 Db2

95 10 −10

90

Db1 85 (b)

0

50

100

150

200

Weight gain (% initial weight)

Figure 5 Effect of edema on bronchial shape. Orthogonal bronchial diameters (Db1 and Db2) are plotted vs. lung water accumulation at different arterial pressures (Pa). Note that mean bronchial diameter does not change at a transpulmonary pressure (Ptp) of 25 cmH2O (a) but decreases by 6% at a Ptp of 6 cmH2O (b). Oval shape becomes circular (Db1 ¼ Db2) with edema. Reproduced from Lai-Fook et al. (1984) Respiration Physiology 55: 223
237, with permission from Elsevier.

With bronchoconstriction present with chronic obstructive pulmonary disease (COPD) or induced by an asthmatic attack, breathing at a higher lung volume results in a greater airway diameter and a reduced airway resistance.

Axial Distortion of Airways As the lung expands with breathing, the bronchus that is rigidly attached to the parenchyma through interstitial connections also expands in length. Bronchial lengthening occurs by axial forces produced by shear stresses in the lung parenchyma. In the lung periphery, bronchial stiffness along its length is relatively low so that the shear stresses in the lung

STRESS DISTRIBUTION IN THE LUNG 131

parenchyma are primarily responsible for the support of the load. The axial deformation of the bronchus is determined by the parenchyma, and with breathing, bronchial length changes in proportion to the cube root of the lung volume. A similar behavior applies to pulmonary blood vessels. However, at the lung hilum bronchial stiffness is increased so that axial loads are primarily supported by the bronchus and the result is a smaller shear stress in the lung parenchyma. Thus, the greater stiffness of the lobar bronchus at the hilum protects the parenchyma from damaging stresses and strain where the bronchus enters the lung.

Stress Concentrations in Disease High stress concentrations, similar to those in the perivascular interstitial space, occur in the lung parenchyma under conditions of lung hyperinflation and are associated with lung disease. The high transpulmonary pressure in the apical regions of the upright lung as a result of gravity has been suggested as the focal point of the formation of centrilobular emphysema. A similar reasoning would apply to the spontaneous pneumothoraces that are occasionally detected in tall males of slender build. Pneumothoraces have been observed in humans subjected to increased acceleration forces in the ventral-to-dorsal direction (supine position) in a centrifuge. Under this loading, lung expansion occurs in addition to the increased transpulmonary pressure gradient imposed by the increased acceleration. A similar increase in lung expansion is observed with acceleration loads in the cranial-to-caudal direction (upright position). In the diseased lung, collapsed atelectatic regions are focal points of high stress concentration. Inflation of the lung parenchyma surrounding a collapsed region produces abnormally high parenchymal stresses, similar in magnitude to those that are associated with lung tearing. See also: Alveolar Surface Mechanics. Alveolar Wall Micromechanics. Asthma: Overview. Breathing: Fetal Lung Liquid; First Breath. Chronic Obstructive Pulmonary Disease: Overview. Extracellular Matrix: Elastin and Microfibrils; Collagens. Fluid Balance in the Lung. Interstitial Lung Disease: Overview. Lung Anatomy (Including the Aging Lung). Pneumothorax. Pulmonary Circulation. Pulmonary Fibrosis. Ventilation: Overview.

Further Reading Farrell Epstein MA and Ligas JR (eds.) (1990) Respiratory Biomechanics: Engineering Analysis of Structure and Function. New York: Springer.

Ganesan S, Man CS, and Lai-Fook SJ (1997) Generation and detection of lung stress waves from the chest surface. Respiration Physiology 110: 19–32. Gunst SJ, Warner DO, Wilson TA, and Hyatt RE (1988) Parenchymal interdependence on airway response to methacholine in excised dog lobes. Journal of Applied Physiology 65: 2490– 2497. Houtz PK, Jones PD, Aronson NE, Richardson LM, and Lai-Fook SJ (2004) Effect of pancreatic and leukocyte elastase on hydraulic conductivity in lung interstitial segments. Journal of Applied Physiology 97: 2139–2147. Kimmel E, Kamm RD, and Shapiro AS (1980) A cellular model of lung elasticity. Journal of Biomechanical Engineering 102: 124–136. Lai-Fook SJ (1979) Journal of Applied Physiology 46: 419–429. Lai-Fook SJ (1989) Mechanics of pulmonary interstitium. In: Chang HK and Paiva M (eds.) Respiratory Physiology: An Analytical Approach. Series in Respiratory Physiology. Lung Biology in Health and Disease, pp. 343–370. New York: Dekker. Lai-Fook SJ (1993) Mechanical factors in lung liquid distribution. Annual Review of Physiology 55: 155–179. Lai-Fook SJ (1997) Mechanical support of airways. In: Crystal RG, West JB, Barnes PJ, Cherniak NS, and Weibel ER (eds.) The Lung: Scientific Foundations, 2nd edn., vol. 1, 1227–1234. New York: Raven Press. Lai-Fook SJ (1997) Stress distribution in the lung. In: Crystal RG, West JB, Barnes PJ, Cherniak NS, and Weibel ER (eds.) The Lung: Scientific Foundations, 2nd edn., vol. 1, 1177–1186. New York: Raven Press. Lai-Fook SJ (1998) Interstitial mechanics and fluid flow. Pathogenesis and treatment of pulmonary edema. In: Weir EK and Reeves JT (eds.) Pulmonary Edema, pp. 135–147. Armonk, NY: Futura Publishing. Lai-Fook SJ (2004) Pleural mechanics and fluid exchange. Physiological Reviews 84: 385–410. Lai-Fook SJ, Beck KC, Sutcliffe AM, and Donaldson JT (1984) Respiration Physiology 55: 223–237. Lai-Fook SJ and Hyatt RE (2000) Journal of Applied Physiology 89: 163–168. Lai-Fook SJ and Kallok MJ (1982) Journal of Applied Physiology 52: 1000–1007. Macklin CC (1946) Evidences of increase in the capacity of the pulmonary arteries and veins of dogs, cats and rabbits during inflation of the freshly excised lung. Reviews in Canadian Biology 5: 199–232. Milic-Emili J (1986) Static distribution of lung volume. In: Macklem PT and Mead J (eds.) Handbook of Physiology. Section 3: The Respiratory System, vol. 3. Mechanics of Breathing, part 2, pp. 561–574. Baltimore, MD: Williams and Wilkens. Mead J, Takishima T, and Leith D (1970) Stress distribution in lungs: a model of pulmonary elasticity. Journal of Applied Physiology 28: 596–608. Staub NC (1974) Pulmonary edema. Physiological Reviews 54: 678–811. Staub NC, Nagano H, and Pearce ML (1967) Pulmonary edema in dogs, especially the sequence of fluid accumulation in lungs. Journal of Applied Physiology 22: 227–240. West JB and Matthews FL (1972) Stresses, strains, and surface pressures in the lung caused by its weight. Journal of Applied Physiology 32: 332–345. Wilson TA (1989) The mechanics of lung parenchyma. In: Chang HK and Paiva M (eds.) Respiratory Physiology: An Analytical Approach. Series in Respiratory Physiology. Lung Biology in Health and Disease, pp. 317–342. New York: Dekker.

132 SUDDEN INFANT DEATH SYNDROME

SUDDEN INFANT DEATH SYNDROME R M Harper, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The sudden infant death syndrome (SIDS) occurs during sleep from unknown causes between the first and ninth month of age; victims show no pathological signs at autopsy which are sufficient to cause death. The prevalence ranges between 0.5 per 1000 and 6.0 per 1000 live births in different countries and ethnic groups, is higher in males, aboriginal and African–American populations, and in lower socioeconomic groups. The prone sleeping position is a substantial risk factor, as is maternal smoking prior to and following birth. Current hypotheses of pathogenesis include a failure to arouse from, or adequately process an airway obstruction, CO2 or hypoxic challenge, or to compensate for a sudden loss in blood pressure. These failures possibly result from deficient serotonergic or other neurotransmitter binding in brainstem areas mediating cardiovascular and respiratory control. Multiple polymorphisms, related to serotonin transporter and autonomic nervous system target genes, have been identified in SIDS victims, and may contribute to risk.

use during pregnancy, markedly affect risk for the syndrome. Other indications that infants who succumb are compromised well before the fatal event include disordered sleep-state organization, reduced respiratory influences on heart rate variation, altered numbers of arousals from sleep, a higher incidence of obstructed respiratory events, periods of tachycardia, and relatively fixed respiratory rates. Thus, infants who succumb are vulnerable from birth, and die during a critical developmental period, likely from exposure to an exogenous stressor. No simple genetic mechanism has been associated with the syndrome; however, several genes have been identified for which the distribution of polymorphisms differs in a high proportion of SIDS victims over control infants. Some of the polymorphisms relate to the serotonin transporter protein gene (5-HTT); others relate to development of the autonomic nervous system; the proportion of mutations may differ in particular ethnic groups. These polymorphisms may contribute to risk, which, combined with particular stress circumstances and age of the infant, result in a fatal outcome.

Introduction Sudden infant death syndrome (SIDS) is defined as the ‘‘sudden and unexpected death of an infant under one year of age, with onset of the lethal episode apparently occurring during sleep, that remains unexplained after a thorough investigation including performance of a complete autopsy, and review of the circumstances of death and the clinical history.’’ The reference to ‘sleep’ was added in 2004 to incorporate recent findings. The new SIDS definition contains three subcategories which include the classic definition, but recognizes variations in autopsy information, death scene data, age, developmental characteristics, and similarity of deaths of close relatives. An ‘unclassified sudden infant death’ category includes cases not meeting classic criteria, but for which alterative diagnoses of natural or unnatural conditions are unclear, or in which no autopsy was performed. Approximately 92% of cases occur within 21 and 270 days of life. The mechanisms by which sparing occurs very early in life, and after 270 days, are unknown.

Etiology The origins of SIDS have roots in fetal life, because certain prenatal exposures, such as maternal tobacco

Pathology/Epidemiology The incidence varies widely internationally; the prevalence is rarer in Asian populations and more common in aboriginal populations in several countries and African–American groups. A male preponderance in deaths (60%) exists, and socioeconomic issues contribute in a major way, as does a higher risk for infants of younger mothers. The incidence has declined remarkably since the early 1990s, when educational campaigns urging parents to place infants down to sleep in the supine, rather than prone position, were initiated. Over the past 15 years, deaths declined from 2 per 1000 live births to less than 0.5 in regions within the UK (2004); however, the rate currently is 5.5 in aboriginal tribes in New Zealand. A comparable declining pattern has emerged in the USA since initiation of a ‘Back to Sleep’ campaign; however, substantial variance in incidence rate stems from ethnic and socioeconomic issues.

Clinical Features SIDS cases typically are found following a period in which the infant was sleeping. In addition to the face-down sleeping position, a finding of an environment inducing high body temperature is common,

SUDDEN INFANT DEATH SYNDROME 133

either from head or body covering or from room interior heat. No pathology sufficient to cause death is found at autopsy, that is, the diagnosis is one of exclusion. Indications of mild respiratory infections are common, but these signs are also insufficient for a fatal outcome. Frothy, mucous, or pink fluid may be present in oral and nasal passages. The presence of blood in the oral–nasal fluid may be indicative of accidental or intentional asphyxiation, and thus does not lie within the classification of SIDS. Pulmonary congestion and edema are commonly found. In over 80–90% of cases, petechial hemorrhages involving the thymus, epicardium, and visceral pleural surfaces are present, and may be increased after cardiopulmonary resuscitation and with increasing age of the victim. The distribution of petechiae (supradiaphragmatic, but not on facial or conjunctival areas, or on external surfaces of the thoracic wall) is characteristic, but not diagnostic for SIDS, and, in some cases, is not found.

Pathogenesis The mechanisms underlying the fatal event remain unknown. Since breathing cessation or cardiovascular collapse occurs during sleep, some state-related process contributes to failure. That process may be a failure to arouse, thus losing protective forebrain and other brain influences on breathing or blood pressure control. The reorganization of neural processes controlling vital functions that occurs normally during sleep may depend on structures that are deficient in SIDS victims. A prevalent hypothesis (current in the mid-1970s), that periods of central apnea precede the event long before death, is not supported by current evidence. One research focus has been the possibility of inadequate sensing of increased CO2 or hypoxia, or a failure of transduction of these chemoreceptor signals to signal appropriate breathing output. The demonstration in SIDS victims of defects in neurotransmitter receptors in brain areas involved with chemoreceptor and cardiovascular regulation, especially serotonergic receptor binding in the caudal raphe and ventral medulla, has added support to this hypothesis. A fatal cardiac arrhythmia, with prolonged Q–T syndrome a primary suspect, has also been proposed. A third possibility is a blood pressure collapse, in a shock-like process, with blood pressure loss triggered by deep visceral pain, infection, upper airway stimulation from foreign fluid or regurgitate, or occurring spontaneously during rapid eye movement sleep. The occasional report of reduced neck muscle tone in later SIDS victims, a characteristic of reduced blood pressure, suggests the potential for

underlying inadequate blood pressure control. The conditions surrounding the fatal event, including profuse sweating prior to the fatal sequence, and indications of profound bradycardia in recordings during some events, together with evidence of neuropathologic damage in blood pressure control areas, provide evidence for possible failure – to compensate for loss of blood pressure. Other support for the hypotensive hypothesis includes the possibility that elevated body temperature found in some SIDS victims has led to vasodilatation, and thus lowered blood pressure, and that altered vestibular input in the prone body position impedes compensatory blood pressure recovery. The developmental expression of the syndrome corresponds with substantial changes in sleep-waking state organization, with an increasing proportion of quiet sleep during the first 6 months over the high prevalence of the rapid eye movement state in the newborn period. Rapid eye movement sleep is accompanied by major neural control changes in cardiovascular and respiratory muscle actions, as well as in sensory processing associated with that control. The neuropathologic findings in SIDS include excessive apoptosis of vestibular nuclei and maldevelopment of inferior olive areas which project to cerebellar cortex and deep nuclei. Cortical cerebellar structures show delayed maturation in SIDS cases and play critical roles in breathing and blood pressure control.

Animal Models No adequate animal model exists for SIDS. However, numerous examinations of prenatal exposure to nicotine, compromised fetal circulation and other developmental insults, as well as sleep effects on physiology have benefited from animal studies.

Management and Current Therapy Interventions to reduce the incidence of SIDS begin with prenatal counseling against the use of tobaccorelated products, since overwhelming evidence demonstrates that maternal smoking during pregnancy increases the risk of SIDS nearly fivefold. The evidence for postnatal tobacco exposure influence on risk is less clear, but that influence appears also to be substantial. Educational efforts to switch from prone sleeping to the supine position have substantially reduced the risk for SIDS in every country where those efforts have begun; an intermediate position, that is, placing the infant on its side to sleep, doubles the risk for SIDS, possibly because the infants may turn prone during sleep time. Placing an infant who routinely sleeps supine into the prone

134 SUDDEN INFANT DEATH SYNDROME

position (a circumstance that can easily occur when the infant is placed in the hands of a novel caregiver, e.g., a day care center) puts the infant at exceptionally high risk. The mechanisms underlying reduced risk from supine sleeping are unclear, but may relate to ensuring adequate airflow, temperature reduction (since heat dissipates rapidly from the face), or vestibular influences, because the prone position dampens vestibular influences on protective heart rate and breathing responses to blood pressure loss. Breastfeeding is usually associated with protection against SIDS, although this association is confounded with other issues, including socioeconomic factors. Pacifier use also reduces risk for SIDS, possibly as a consequence of ‘exercising’ brain areas controlling upper airway muscle action or initiating blood pressure changes that accompany suckling, thus providing the brain ‘practice’ for recovery from sudden blood pressure falls. A continuing concern has been the possibility that vaccinations, typically administered at peak risk periods for SIDS, may enhance the predisposition for risk; no evidence for such an association has been demonstrated, and some data suggest a small protective influence in favor of vaccinations. Attention should be directed to assuring a safe sleep environment by use of firm mattresses and care in selection of bedding materials; a high proportion of infants found dead have had their heads covered – so the use of soft materials with the potential to cover the head, leading to elevated temperatures or impeded air exchange, should be discouraged. Electronic monitors that detect apnea, bradycardia, or oxygen saturation are fraught with difficulties in false positive and negative indications of breathing or cardiovascular dysfunction, particularly when an infant is ill from other causes. Monitors have a disconcerting history of failure to perform or inadequacy to alert a caregiver during events that result in fatalities. False triggering of alarms from inadequate placement of physiological sensors or from arousals associated with mild illnesses often prompt disuse on occasions when the necessity for monitoring may be especially warranted. Monitors, however, can supply solace to concerned parents, and substantial progress in technology may emerge in the

Surface Mechanics

near future. Monitors that supply recordings of physiological signals during near-fatal or fatal events have provided invaluable information on potential mechanisms in SIDS. See also: Breathing: Breathing in the Newborn. Infant Respiratory Distress Syndrome. Neurophysiology: Neuroanatomy. Sleep Apnea: Genetics of Sleep Apnea.

Further Reading Byard RW and Krous HF (2003) Sudden infant death syndrome: overview and update. Pediatric and Developmental Pathology 6(2): 112–127. Byard RW and Krous HF (eds.) (2004) Sudden Infant Death Syndrome: Problems Progress and Possibilities. London: Arnold. Fleming P, Blair P, Bacon C, and Berry J (eds.) (2000) Sudden Unexpected Deaths in Infancy: The Cesdi Sudi Studies. London: The Stationery Office. Gaultier C, Amiel J, Dauger S, et al. (2004) Genetics and early disturbances of breathing control. Pediatric Research 55(5): 729–733. Harper RM (2000) Sudden infant death syndrome: a failure of compensatory cerebellar mechanisms? Pediatric Research 48: 140–142. Harper RM and Hoffman HJ (eds.) (1988) Sudden Infant Death Syndrome: Risk Factors and Basic Mechanisms. New York: PMA Publishing. Harper RM, Kinney HC, Fleming PJ, and Thach BT (2000) Sleep influences on homeostatic functions: implications for sudden infant death syndrome. Respiratory Physiology 119(2–3): 123– 132. Kinney HC, Filiano JJ, and Harper RM (1992) The neuropathology of the sudden infant death syndrome. Journal of Neuropathology and Experimental Neurology 51(2): 115–126. Krous HF, Beckwith JB, Byard RW, et al. (2004) Sudden infant death syndrome and unclassified sudden infant deaths: a definitional and diagnostic approach. Pediatrics 114(1): 234–238. Mitchell EA (2000) SIDS: facts and controversies. Medical Journal of Australia 173(4): 175–176. Weese-Mayer DE, Berry-Kravis EM, Maher BS, Silvestri JM, Curran ME, and Marazita ML (2003) Sudden infant death syndrome: association with a promoter polymorphism of the serotonin transporter gene. American Journal of Medical Genetics 117A: 268–274. Weese-Mayer DE, Berry-Kravis EM, Zhou L, et al. (2004) Sudden infant death syndrome: case-control frequency differences at genes pertinent to early autonomic nervous system embryologic development. Pediatric Research 56(3): 391–395. Willinger M, James LS, and Catz C (1991) Defining the sudden infant death syndrome (SIDS): deliberations of an expert panel convened by The National Institute of Child Health and Human Development. Pediatric Pathology 11: 677–684.

see Alveolar Surface Mechanics.

SURFACTANT / Overview 135

SURFACTANT Contents

Overview Surfactant Protein A (SP-A) Surfactant Proteins B and C (SP-B and SP-C) Surfactant Protein D (SP-D)

Overview R H Notter, P R Chess, and Z Wang, University of Rochester Medical Center, Rochester, NY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pulmonary surfactant, a mixture of lipids and proteins produced in the alveolar epithelial lining, is required for normal respiration. Active lung surfactant lowers the work of breathing, stabilizes alveolar inflation and deflation to optimize gas exchange, and reduces the driving force for pulmonary edema. These physiological effects are generated by the biophysical activity of lung surfactant, which lowers and varies surface tension at the alveolar interface during breathing. Biophysically-important constituents of surfactant include dipalmitoyl phosphatidylcholine (DPPC), a mix of other saturated and unsaturated phospholipids, and three surfactant proteins (SP)-A, SP-B, and SP-C. A fourth apoprotein, SP-D, does not participate in surfactant biophysics but contributes to pulmonary host defense. Severe disease results if lung surfactant is deficient or inactivated. Surfactant deficiency causes the respiratory distress syndrome (RDS) in premature infants, and surfactant dysfunction is an important contributor to clinical acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) that affect patients of all ages. One approach to treating diseases of surfactant deficiency and dysfunction is to administer exogenous surfactants by tracheal or bronchoscopic instillation. Exogenous surfactant replacement has been shown to be life saving in preventing and treating RDS in premature infants, and is now being extended to treat surfactant dysfunction in several forms of lung-injury-related acute respiratory failure. Combination therapies using exogenous surfactant plus interventions aimed at other aspects of the multifaceted pathology of inflammatory lung injury are also under study.

Introduction Much of the internal pulmonary surface (B1 m2 kg
1 body weight) is covered by a thin aqueous layer (hypophase) in direct contact with air, resulting in an extensive air–liquid interface with associated surface tension forces. The significant influence of these forces on the mechanics of breathing is apparent in the higher pressures needed to inflate lungs, or maintain them at fixed volume during deflation, with air compared to liquid. This difference,

initially reported by the German physiologist von Neergaard in 1929, reflects surface tension forces present in air-filled lungs but not in liquid-filled lungs. The crucial existence of alveolar surface-active agents (surfactants) to moderate these surface tension forces during breathing was subsequently demonstrated in the 1950s, along with the linkage between surfactant-deficiency and the respiratory distress syndrome (RDS or hyaline membrane disease) in premature infants. It also later became apparent that surfactant dysfunction was a major contributor to severe acute pulmonary injury syndromes (acute lung injury (ALI)/acute respiratory distress syndrome (ARDS)) in patients of all ages (Figure 1).

Composition and Production of Lung Surfactant Lung surfactant contains approximately 85–90% (by weight) phospholipid, 7–10% protein, and 4–7% neutral lipid (Table 1). Phosphatidylcholine (PC) comprises about 80% of total surfactant phospholipid, and dipalmitoyl phosphatidylcholine (DPPC) is the most prevalent single compound (40–50% of total PC). Other disaturated PC compounds include isomers of C14:0, C16:0 PC, plus smaller amounts of C14:0, C14:0 PC, and C16:0, C18:0 PC. Multiple unsaturated PC components including C16:0, C16:1 PC, and C16:0, C18:1 PC are also present. Other phospholipid classes in lung surfactant (PG, PI, PS, PE, Sph, Table 1) also contain a mix of disaturated and unsaturated compounds. The protein content of lung surfactant is much smaller than the lipid content, but is highly important functionally and comprises three biophysically active surfactant proteins (SP)-A, SP-B, and SP-C. In addition, SP-D, a fourth protein not implicated in biophysical function but important in host defense, is also found (Table 2). Lung surfactant also contains 4–7% neutral lipids, primarily cholesterol plus small amounts of cholesterol esters, diglycerides, and triglycerides (Table 1). Lung surfactant is synthesized, packaged, stored, secreted, and recycled by alveolar type II epithelial

136 SURFACTANT / Overview

Extensive alveolar liquid–air interface

Substantial pulmonary surface tension forces

Surfactant needed to lower and vary alveolar surface tension

Surfactant deficiency (prematurity or injury-induced)

Surfactant dysfunction (injury-induced)

Acute respiratory failure • ALI/ARDS • RDS Figure 1 Necessity for lung surfactant in respiration. Surface tension forces at the air–water interface in the alveoli significantly impact pulmonary mechanics and function. By lowering and varying surface tension during breathing, lung surfactant reduces the work of breathing, stabilizes alveolar inflation/deflation behavior, reduces edema formation, and facilitates gas exchange. Surfactant deficiency causes RDS in premature infants, and surfactant dysfunction is an important contributor to the pathophysiology of clinical ALI and ARDS in patients of all ages. ALI and ARDS can also have a component of surfactant deficiency as well as dysfunction. Reproduced from Notter RH, Finkelstein JN, and Holm BA (eds.) (2005) Lung Injury: Mechanisms, Pathophysiology, and Therapy, ch. 9. Boca Raton: Taylor and Francis.

Table 1 Composition of endogenous pulmonary surfactant 85–90% phospholipids 80% PC 40–50% DPPC 10–15% other disaturated PCs 35–45% unsaturated PCs 15% anionic phospholipids (PG, PI, PS) 5% other phospholipid classes (PE, Sph) 7–10% apoproteins SP-A SP-B SP-C SP-D (not involved in biophysical function) 4–7% neutral lipids Cholesterol Cholesterol esters, glycerides Values are representative averages in weight percent for surfactant lavaged (washed) from the lungs of normal animals of different species and ages. PC: phosphatidylcholine; PG: phosphatidylglycerol; PI: phosphatidylinositol; PS: phosphatidylserine; PE: phosphatidylethanolamine; Sph: sphingomyelin; SP: surfactant protein. Reproduced from Notter RH, Finkelstein JN, and Holm BA (eds.) (2005) Lung Injury: Mechanisms, Pathophysiology, and Therapy, ch. 9. Boca Raton: Taylor and Francis.

cells. Biophysically-complete surfactant is synthesized only in this cell type, although extra-alveolar sites of synthesis or expression also exist for surfactant proteins other than SP-C (e.g., in nonciliated bronchiolar epithelial cells or Clara cells). Whole

surfactant secreted from type II pneumocytes is distributed in the alveolar hypophase as heterogeneous phospholipid-rich aggregates with incorporated apoproteins. These aggregates vary in size from tens of nanometers to several microns, with larger forms generally having the greatest surface activity and apoprotein content. The final site of action of lung surfactant in terms of surface tension lowering is in an adsorbed surface film that is repetitively compressed and expanded at the alveolar air–water interface during breathing.

Functional Activities of Lung Surfactant The major functional actions of lung surfactant are biophysical and physiological, although several of the surfactant apoproteins also have important biological activity in host defense (details on the biological functions of individual surfactant proteins are given in Surfactant: Surfactant Protein A (SP-A); Surfactant Protein D (SP-D); and Surfactant Proteins B and C (SP-B and SP-C)). In addition, alveolar type II pneumocytes function not only as the primary cells of surfactant metabolism, but also as stem cells for the alveolar epithelium and as important participants in the innate inflammatory response and in other pulmonary biological processes. Emphasis here is on the physiological and biophysical actions of

SURFACTANT / Overview 137 Table 2 Molecular characteristics of the four lung surfactant proteins Surfactant protein (SP)

Selected structural and functional characteristics

SP-A

MW 26–38 kDa (monomer), 228 AA in length in humans Most abundant surfactant apoprotein, relatively hydrophilic in structure Acidic glycoprotein with multiple posttranslational isoforms C-type lectin and member of the collectin family of proteins Oligomerizes to an active octadecamer (six trimer subunits) Aggregates and orders lipids (Ca2þ -dependent) Necessary for tubular myelin formation (along with SP-B, Ca2þ ) Helps regulate surfactant metabolism (e.g., reuptake/recycling in type II cells) Active in host defense along with SP-D

SP-B

MW 8.5–9 kDa (monomer), 79 AA in length in humans (active peptide) Hydrophobic in overall nature, with 2–3 amphipathic helical regions Also contains multiple charged AA at neutral pH (e.g., 10 Arg/Lys residues) Forms dimers of presumptive functional significance in humans Interacts biophysically with both the headgroups and chains of phospholipids Necessary for tubular myelin formation (along with SP-A, Ca2þ ) Disrupts and fuses lipid bilayers, and promotes lipid insertion into surface films Enhances adsorption, film spreading, and dynamic surface activity of lipids Most active SP in increasing adsorption and overall dynamic surface activity

SP-C

MW 4.2 kDa (monomer), 35 AA in length in humans (active peptide) Most hydrophobic surfactant protein Forms dimers and other oligomers of uncertain functional significance Human form has two palmitoylated cysteine residues Contains only two charged amino acids at neutral pH (2 Arg/Lys residues) Primarily a-helical in structure, with a length that spans a lipid bilayer Interacts biophysically primarily with phospholipid fatty chains Disrupts and fuses lipid bilayers Enhances adsorption, film spreading, and surface activity of lipids

SP-D

MW 39–46 kDa (monomer), 355 AA in length in humans Has significant structural similarity to SP-A C-type lectin and member of collectin family of host-defense proteins Oligomerizes to a dodecamer (four trimer subunits) Not implicated in lung surfactant biophysics Active in host defense and may also participate in surfactant metabolism

Adapted from Notter RH, Finkelstein JN, and Holm BA (eds.) (2005) Lung Injury: Mechanisms, Pathophysiology, and Therapy, ch. 9. Boca Raton: Taylor and Francis.

pulmonary surfactant, which are crucial for normal breathing (Table 3). Physiological and Biophysical Functions of Surfactant in Normal Lungs

By lowering surface tension, surfactant reduces the nonflow component of respiratory work (increases quasistatic lung compliance). In addition, by lowering and varying surface tension as a function of alveolar radius (surface area) during breathing, surfactant reduces atelectasis and promotes more uniform inflation based on the Laplace equation for a sphere: DP ¼ 2s/R, where DP is the pressure drop across the alveolar air–liquid interface, s is surface tension, and R is radius (Figure 2). Similar stability arguments can be made for an alveolar interface of arbitrary curvature based on the more

Table 3 Physiological actions and surface properties of functional lung surfactant Physiological actions of functional lung surfactant Reduces the work of breathing (increases lung compliance) Increases alveolar stability against collapse during expiration (reduces atelectasis) Improves the uniformity of alveolar inflation during inspiration Reduces the hydrostatic driving force for pulmonary edema Biophysical properties of functional lung surfactant Adsorbs rapidly to the air–water interface Generates very low minimum surface tensions during dynamic film compression Varies surface tension with surface area during dynamic cycling Respreads effectively at the interface during successive breathing cycles See text for discussion. Adapted from Notter RH (2000) Lung Surfactants: Basic Science and Clinical Applications. New York: Dekker.

138 SURFACTANT / Overview Simplified view of lung surfactant action in an alveolus

∆P =

2
R Hypophase Surfactant film

Air

Conceptually When alveolar radius is small

the surfactant film is compressed and surface tension is small

When alveolar radius is large

the surfactant film is expanded and surface tension is larger

Result: The ratio of surface tension to radius is more uniform in each alveolus and throughout the lung, stabilizing P–V behavior from Laplace’s law. Figure 2 Schematic showing the effects of lung surfactant on pulmonary pressure–volume behavior based on the Laplace equation. The pressure drop (DP) necessary to maintain alveoli at equilibrium is proportional to surface tension (s) and inversely proportional to radius (R), i.e., DP ¼ 2s/R (Laplace’s law for a sphere). By lowering and varying surface tension as a function of alveolar size (radius), lung surfactant makes equilibrium pressures more equal in different sized alveoli. As a result, small airsacs resist collapse during expiration, and large alveoli do not overinflate during inspiration. Surfactant also greatly decreases the nonflow component of the work of breathing by a generalized lowering of surface tension forces throughout the alveolar network. See text for details.

comprehensive Young and Laplace equation, which incorporates the two principal radii of curvature R1 and R2 for an interface of general shape: DP ¼ s (1/R1 þ 1/R2). Alveolar stability in vivo is further enhanced by specialized connective tissue support fibers plus an interconnected network structure that allows airsacs sharing common septa to help each other resist collapse. However, the consequences of surfactant deficiency are strikingly apparent in premature infants with RDS, who exhibit alveolar collapse and overdistension, decreased lung volumes and compliance, intrapulmonary shunting with reduced arterial oxygenation, and diffuse pulmonary edema. Surfactant dysfunction leads to similar acute respiratory deficits in patients with lung injury pathology (ALI/ARDS). The physiological actions of lung surfactant depend on specific surface behaviors (Table 3). Since surfactant is secreted into the alveolar hypophase, it must adsorb to form an interfacial film. Whole surfactant lavaged (washed) from normal lungs rapidly adsorbs to equilibrium surface tensions of 22–23 mN m
1 at 371C at the air–water interface. Similar rapid adsorption is also observed for chloroform:methanol extracts of lavaged surfactant containing lipids plus SP-B/C. When adsorbed lung surfactant films are compressed at rapid physiological rates (e.g., 20 cycles min
1), they generate extremely low minimum surface tensions. Multiple

studies show that lavaged whole surfactant or surfactant extracts can lower surface tension to o1 mN m
1 under rapid dynamic compression at 371C, as well as vary surface tension with surface area. Lung surfactant films must also respread effectively at the air–water interface during cycling, that is, molecules lost from the surface film during compression must reintegrate with remaining film material during expansion. Respreading from filmassociated structures in the interfacial region, together with ongoing adsorption from the hypophase, ensures that sufficient surfactant is available in the film to lower surface tension effectively during successive breathing cycles. Lung surfactant lipids and proteins interact at the molecular level to achieve the set of surface behaviors in Table 3. Disaturated phospholipids such as DPPC form tightly packed, rigid films capable of extreme surface tension lowering during dynamic compression. The presence of DPPC and other disaturated phospholipids in lung surfactant not only facilitates surface tension lowering, but also helps to vary surface tension with area during cycling. However, rigid disaturated phospholipids such as DPPC do not adsorb readily into the interface or respread effectively in cycled films. Fluid unsaturated phospholipids in lung surfactant have a major impact in improving film respreading, and also increase adsorption relative to DPPC. Neutral lipids also

SURFACTANT / Overview 139

facilitate adsorption and respreading, but can be detrimental to dynamic surface tension lowering if present in excess amounts. SP-A, SP-B, and SP-C all have extensive molecular interactions with surfactant phospholipids, and are essential in facilitating adsorption. Surfactant proteins, particularly hydrophobic SP-B and SP-C, also enhance film respreading and aid in refining the surface film during compression to optimize dynamic surface tension lowering. SP-A functions biophysically as a large octadecamer containing six triplet monomers. SP-B and SP-C also form oligomers including dimers, although the activities of specific oligomeric versus monomeric forms of the hydrophobic proteins are not fully defined. SP-A in the presence of calcium acts to increase phospholipid aggregation and order, including the formation of tubular myelin, a distinctive three-dimensional network of intersecting phospholipid bilayers with incorporated apoproteins found in the aqueous phase microstructure of lung surfactant dispersions. SP-B is also required for tubular myelin formation. Tubular myelin and other large aggregate forms of lung surfactant are highly active in adsorbing to the air– water interface. SP-B and SP-C disrupt and fuse phospholipid bilayers consistent with an important role in surfactant adsorption, and SP-B directly promotes the insertion and mixing of phospholipids into surface films. The amphipathic structure of SP-B allows it to interact with both the headgroups and chains of phospholipids in films and bilayers, while the extreme hydrophobicity of SP-C limits its interactions largely to the hydrophobic fatty chain region. Multiple studies have shown that SP-B is more active than SP-C in enhancing adsorption and overall dynamic surface tension lowering, making this apoprotein a particularly important functional component of endogenous and exogenous lung surfactants. SP-B is also more active than SP-C in increasing the ability of exogenous lung surfactants to resist inactivation by plasma proteins and other inhibitors. Disruption of Surfactant Activity: Deficiency and Dysfunction

Surfactant deficiency refers to a decreased total amount of surfactant in the alveoli, while surfactant dysfunction (also called inhibition or inactivation) implies a decrease in surface activity. Surfactant deficiency and dysfunction are not independent, since surface activity is reduced if surfactant concentration is decreased. Nonetheless, understanding the relative degree of surfactant dysfunction versus deficiency in lung disease is helpful in mechanistic understanding. Surfactant deficiency is most often associated with

deficits in type II cell function, either because these cells are not fully developed as in premature infants or because they are altered during injury. In contrast, surfactant dysfunction typically arises from physicochemical interactions of alveolar surfactant or its essential components with injury-induced inhibitors, inflammatory enzymes, or reactive oxygen/nitrogen species. Alterations in type II cells or alveolar processing that deplete large surfactant aggregate subtypes or reduce their activity can also contribute to surfactant dysfunction. Substances that act biophysically to inhibit surfactant activity include plasma and blood proteins (e.g., albumin, fibrinogen, fibrin monomer, or hemoglobin), cellular or bacterial lipids and lysophospholipids, fluid free fatty acids, and inhaled or aspirated toxicants (e.g., meconium). Plasma and blood proteins reduce surface activity primarily by competitive adsorption and interfacial shielding. These large molecules have intrinsic surface activity, and adsorb to form a film that occupies the interface and limits the entry of lung surfactant components. In contrast, smaller molecules such as fluid free fatty acids and unsaturated cell membrane lipids act primarily by mixing with surfactant components in the interfacial film to compromise surface tension lowering during dynamic cycling. In addition, inhibitory free fatty acids and lysophospholipids also can damage the alveolocapillary membrane and induce edema containing other inhibitors such as plasma proteins. Lung surfactant dysfunction can also result from exposure to chemically acting inhibitors such as phospholipases, proteases, and reactive oxygen/ nitrogen species during inflammatory injury. Phospholipases and proteases not only reduce the concentration of functional surfactant components by chemical degradation, but also produce reaction products that cause further inhibition (e.g., phospholipases A1 and A2 generate free fatty acids and lysophospholipids when they chemically degrade phospholipids). Reactive oxygen and nitrogen species that can chemically alter surfactant lipids and proteins during inflammatory injury include superoxide anion ðOd
2 Þ, hydrogen peroxide (H2O2), hydroxyl radical ðd OHÞ, peroxynitrite (ONOO
), nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), and S-nitrosothiols. In addition, another important mechanism by which surfactant activity can be impaired during lung injury involves the selective depletion or alteration of large surfactant aggregate subtypes, which normally have greater surface activity and apoprotein content than small aggregates. Aggregate-related changes in surfactant can be caused by direct interactions with inhibitors in the

140 SURFACTANT / Overview

alveoli, impaired intra-alveolar processing, injury to type II cell reuptake and recycling pathways, or a combination of these factors. When inhibition of surface activity occurs, it is typically most pronounced at low surfactant concentration and becomes mitigated as the level of active surfactant increases. For example, in the presence of inhibitory blood proteins or lipids, lung surfactant at a low concentration of 0.5 mg phospholipid ml
1 may reach minimum surface tensions of only 15–25 mN m
1 during prolonged interfacial cycling in vitro. However, minimum surface tensions of o1 mN m
1 can be restored if surfactant concentration is raised to 2 mg phospholipid ml
1, even in the presence of larger inhibitor concentrations. This behavior provides a rationale for using exogenous surfactant therapy to overcome surfactant dysfunction in addition to surfactant deficiency in a clinical setting. Physiological Correlates of Surfactant Activity and Dysfunction

Decades of research have shown the importance of correlating data on lung surfactant composition, surface activity, and inhibition resistance with physiological assessments of pulmonary mechanics and function in intact lungs. Animal models used in surfactant research can be categorized as primarily involving surfactant deficiency (e.g., premature animals) or surfactant dysfunction (e.g., animals with acute inflammatory lung injury). Animal models of surfactant deficiency relate most directly to RDS, while models of surfactant dysfunction and inflammatory lung injury relate to ALI/ARDS. Within either category, models can be subdivided further based on their biological and physiological details or methods of lung injury induction. Research in animal models of prematurity was essential in elucidating the pulmonary activity of exogenous surfactants used in replacement therapy for RDS. Research in animal models of acute pulmonary injury with surfactant dysfunction has also provided physiological correlates supporting the use of exogenous surfactants in ALI/ARDS. The consensus of basic research indicates that clinical exogenous surfactants with the greatest surface tension lowering ability and resistance to inhibitor-induced dysfunction have the highest activity in reversing surfactant deficiency and dysfunction in animal lungs in vivo.

Lung Surfactant in Respiratory Diseases RDS in premature infants is the major disease of surfactant-deficiency worldwide. The incidence of

RDS increases as gestational age and birth weight decrease. RDS is most prevalent in infants born at o32 weeks gestation (term ¼ 40 weeks; prematurity p37 weeks), and is rare at 435 weeks gestation. In the year 2000, approximately 77 500 infants at o32 weeks gestation, and 219 000 infants at 32–35 weeks gestation, were born in the US. There were over 24 000 reported cases of RDS, with an overall incidence of 6 per 1000 live births. Aside from the degree of prematurity, the risk of RDS is increased by maternal diabetes, male sex, Caucasian race, and Caesarian section. RDS is caused by surfactant deficiency from type II cell immaturity, but lung injury and surfactant dysfunction can also enter its clinical course because of mechanical ventilation, hyperoxia, and complications of prematurity. However, surfactant dysfunction is more prominent in the acute respiratory failure associated with ALI/ARDS. ARDS was originally described primarily in adults, and was commonly referred to as the ‘adult’ respiratory distress syndrome. The now standard nomenclature of ‘acute’ respiratory distress syndrome emphasizes that this condition reflects severe acute pulmonary injury in patients of any age. The American–European Consensus Committee in 1994 defined clinical ARDS based on an acute onset, bilateral infiltrates on frontal chest radiograph, a ratio of arterial partial pressure of oxygen to inspired fraction of oxygen (PaO2/FiO2 ratio) of p200 mmHg, and a pulmonary capillary wedge pressure of p18 mmHg (if measured) or no evidence of left atrial hypertension. The Consensus Committee defined clinical ALI identically except for a less severe PaO2/FiO2 ratio of p300 mmHg (by definition all patients with ARDS also have ALI). The incidence of ALI/ARDS has been estimated at 20–60 cases per 105 persons per year in the US, with a substantial mortality of 30–50% despite sophisticated ventilatory support and intensive care. Examples of disease processes that can lead to ALI/ARDS include sepsis, severe trauma with shock, gastric aspiration, pneumonia, pulmonary contusion, head or burn injuries, meconium aspiration, near drowning, hyperoxia, radiation, pancreatitis, and many others. Multiple studies have documented surfactant abnormalities in bronchoalveolar lavage from patients with ALI/ARDS. An additional group of surfactant-related diseases involves a genetic deficiency in a functional surfactant protein. Such conditions are rare, affecting only one in tens of thousands of infants. The best known is hereditary SP-B deficiency, the major human form of which is a homozygous mutation in exon 4 of the SP-B gene at codon 121, referred to as 121ins2. Heterozygous infants do not have overt respiratory pathology. Because of the crucial functionality of

SURFACTANT / Overview 141

SP-B, the homozygous 121ins2 mutation is associated with decreased surfactant activity and lethal perinatal respiratory failure (an early alveolar proteinosis is also common). Reduced levels of surfactant phosphatidylglycerol and abnormalities in SP-C processing are also present. There is genetic heterogeneity in human SP-B deficiency, and some variants allow survival into childhood. Lung transplantation is the only current option for infants with severe forms of this condition, although gene therapy is under investigation. In contrast to SP-B deficiency, a congenital deficiency of SP-C is not associated with severe acute respiratory disease in humans or transgenic mice. Clinical Exogenous Surfactant Therapy

Surfactant therapy is conceptually straightforward: if endogenous surfactant is deficient or dysfunctional, then it can be replaced or supplemented by delivering exogenous surfactant to the alveoli. Such therapy is necessarily acute, and requires that the lungs eventually acquire or recover the ability to produce active endogenous surfactant. Although simple in concept, developing effective exogenous surfactant therapy for premature infants with RDS required substantial time. Initial studies using aerosolized DPPC to treat RDS in premature infants in the 1960s were unsuccessful, both because DPPC alone does not adequately replace lung surfactant and because alveolar delivery by aerosolization was inefficient. Two decades of subsequent basic research clarified the biophysical and physiological basis of surfactant function, and the life-saving benefits of improved exogenous surfactants in premature infants were firmly documented in the 1980s. Exogenous surfactants are dispersed in saline and delivered by intratracheal instillation through an endotracheal tube (or in some cases a bronchoscope) at doses of B100 mg kg
1 body weight. Despite having theoretical advantages, aerosol methods for delivering exogenous surfactants are not yet perfected. Surfactant therapy for ALI/ARDS is much less developed than in the case for RDS, but has a firm rationale from biophysical and animal studies. Biophysical research has documented that raising surfactant concentration can mitigate surfactant dysfunction in the presence of inhibitor substances, and multiple animal models of ALI/ARDS have been shown to respond favorably to exogenous surfactants (e.g., lung injury from hyperoxia, vagotomy, viral pneumonia, meconium aspiration, or administration of antilung serum, bacteria, endotoxin, or N-nitrosoN-methylurethane). Clinical studies have shown that exogenous surfactant therapy is efficacious in term

infants with meconium aspiration or pneumonia, and also can improve lung function and outcomes in children with ALI/ARDS. Surfactant therapy in adults with ARDS has had less success. By far the largest trial of surfactant therapy in adults with ARDS reported no clinical benefits following treatment with aerosolized Exosurf in 1996. However, aerosol delivery is less effective than instillation as noted, and Exosurf is now known to have low activity compared to animal-derived exogenous surfactants (see below). Many of the most active exogenous surfactant drugs have not been studied in detail in adults with ARDS. Moreover, the complex pathophysiology of ALI/ ARDS includes inflammation, vascular dysfunction, and multiorgan failure in addition to surfactant dysfunction. Exogenous surfactant may thus need to be used in a combined-modality approach with other therapies that target multiple aspects of pathology. Clinical Exogenous Surfactants

Clinical exogenous surfactants currently used to treat surfactant-related lung disease are listed in Table 4. These preparations are divided into three functionally relevant groups: (1) organic solvent extracts of lavaged endogenous lung surfactant from animals; (2) organic solvent extracts of processed animal lung tissue with or without additional synthetic additives; and (3) synthetic preparations not containing surfactant Table 4 Clinical exogenous surfactant drugs for treating diseases of lung surfactant-deficiency or -dysfunction 1. Organic solvent extracts of lavaged animal lung surfactant Infasurf (CLSE) bLES Alveofact 2. Supplemented or unsupplemented organic solvent extracts of processed animal lung tissue Survanta Surfactant-TA Curosurf 3. Synthetic exogenous lung surfactants Exosurf ALEC KL4 Recombinant SP-C surfactant See text for discussion. Basic research indicates that category 1 surfactants from animal lung lavage have close compositional analogy to endogenous surfactant and correspondingly high surface activity and physiological activity. Infasurf (ONY, Inc and Forest Laboratories), Survanta (Abbott/Ross Laboratories), and Curosurf (Chesi Farmaceutici and Dey Laboratories) are currently FDA-approved in the United States, and KL4 (Discovery Laboratories) is under clinical evaluation. Exosurf (Glaxo-Wellcome) is also FDA-approved, but is no longer used due to its low activity. Adapted from Notter RH (2000) Lung Surfactants: Basic Science and Clinical Applications, ch. 14. New York: Dekker.

142 SURFACTANT / Overview

material from animal lungs. Organic solvent extracts of lavaged alveolar surfactant in category 1 in principle contain all of the hydrophobic lipid and protein components of endogenous surfactant (compositional details do vary somewhat depending on specific production methods). Extracts of minced or homogenized lung tissue in category 2 are noted separately because they contain a higher content of tissue-derived substances, and require more extensive processing that can further alter native surfactant components. The synthetic surfactants in category 3 that have been most widely studied to date are Exosurf and artificial lung expanding compound (ALEC), although neither is now in widespread clinical use due to their low surface activity. Exosurf is a mixture of DPPC:hexadecanol:tyloxapol (1:0.11:0.075 by weight) and ALEC is a mixture of 7:3 DPPC:egg PG. Synthetic KL4 and recombinant SP-C surfactant are currently under clinical evaluation. Relative Activity and Inhibition Resistance of Exogenous Surfactant Drugs

The relative activity and efficacy of exogenous surfactant drugs are crucial for evaluating and optimizing therapy. Direct clinical comparison trials indicate that ‘natural’ surfactants from animal lungs (categories 1 and 2, Table 4) have significantly greater efficacy than protein-free Exosurf, and basic research indicates that the same is true for ALEC. The hydrophobic surfactant proteins are highly active, and substituting for them in synthetic surfactants is challenging. The surface and physiological activity of Exosurf is significantly increased by the addition of bovine SP-B/SP-C, demonstrating that its synthetic components do not adequately replace the active apoproteins. Animal-derived clinical surfactants also differ markedly in their activity characteristics. Laboratory research indicates that the surface activity, inhibition resistance, and effects in animal lungs of category 1 surfactants in Table 4 are greater than those of other current clinical surfactants. These surfactants have the closest compositional analogy to endogenous surfactant. The surface and physiological activities of Infasurf, for example, are substantially greater than Survanta in basic research and correlate directly with the content of SP-B in the two preparations. Survanta has a very low SP-B content by ELISA of only 0.044% by weight relative to phospholipid, while Infasurf has an SP-B content of 0.9% by weight that approaches that of lavaged whole surfactant. As described earlier, SP-B is the most active of the hydrophobic surfactant proteins in enhancing the adsorption and overall dynamic surface activity of phospholipids. The addition of SP-B

or synthetic SP-B peptides to Survanta significantly improves its activity toward that of Infasurf and whole surfactant. Future Exogenous Surfactant Development

Although several current animal-derived surfactants have significant activity, low toxicity, and relatively close analogy to alveolar surfactant, synthetic surfactants have potential advantages in manufacturing, formulation, ease of quality control, and broader production availability. Synthetic surfactants also have no risk of transmitting animal-derived pathogens such as prions. Examples of conceptual approaches being followed for new exogenous surfactants include: (1) combining human sequence recombinant apoproteins or related synthetic peptides with synthetic glycerophospholipids; (2) modifying current clinical exogenous surfactants with added recombinant apoproteins or synthetic peptides to increase activity; and (3) developing new exogenous surfactants for use in ALI/ARDS that have novel beneficial properties based on specific synthetic components. KL4 and recombinant SP-C surfactant are examples of the first of these approaches. Examples of the second approach include adding human sequence SP-A or SP-A peptides to enhance inhibition resistance in exogenous surfactants or adding hydrophobic surfactant apoproteins or peptides if these substances are lacking (e.g., the addition of SP-B to Survanta). An example of the third approach is the use of synthetic phospholipase-resistant diether phosphonolipids that have dynamic surface tension lowering ability comparable to DPPC, but greatly improved adsorption and respreading properties. Exogenous surfactant preparations containing such phosphonolipids combined with purified surfactant proteins or synthetic peptides have very high overall surface activity plus an ability to resist degradation by phospholipases in ALI/ARDS. See also: Acute Respiratory Distress Syndrome. Epithelial Cells: Type I Cells; Type II Cells. Surfactant: Surfactant Protein A (SP-A); Surfactant Proteins B and C (SP-B and SP-C); Surfactant Protein D (SP-D).

Further Reading Avery ME and Mead J (1959) Surface properties in relation to atelectasis and hyaline membrane disease. American Journal of Diseases of Children 97: 517–523. Bernard GR, Artigas A, Brigham KL, et al. (1994) The American– European consensus conference on ARDS: definitions, mechanisms, relevant outcomes, and clinical trial coordination. American Journal of Respiratory and Critical Care Medicine 149: 818–824.

SURFACTANT / Surfactant Protein A (SP-A) 143 Clements JA (1957) Surface tension of lung extracts. Proceedings of the Society for Experimental Biology and Medicine 95: 170–172. Hall SB, Venkitaraman AR, Whitsett JA, Holm BA, and Notter RH (1992) Importance of hydrophobic apoproteins as constituents of clinical exogenous surfactants. American Review of Respiratory Disease 145: 24–30. Holm BA, Wang Z, and Notter RH (1999) Multiple mechanisms of lung surfactant inhibition. Pediatric Research 46: 85–93. Nogee LM, Garnier G, Dietz HC, et al. (1994) A mutation in the surfactant protein B gene responsible for fatal neonatal respiratory disease in multiple kindreds. Journal of Clinical Investigation 93: 1860–1863. Notter RH (2000) Lung Surfactants: Basic Science and Clinical Applications, p. 444. New York: Dekker. Notter RH, Finkelstein JN, and Holm BA (eds.) (2005) Lung Injury: Mechanisms, Pathophysiology, and Therapy, chs 3, 9, 15, 19, p. 847. Boca Raton: Taylor and Francis. Notter RH and Wang Z (1997) Pulmonary surfactant: physical chemistry, physiology, and replacement. Reviews in Chemical Engineering 13: 1–118. Notter RH, Wang Z, Egan EA, and Holm BA (2002) Componentspecific surface and physiological activity in bovine-derived lung surfactants. Chemistry and Physics Lipids 114: 21–34. Pattle RE (1955) Properties, function, and origin of the alveolar lining layer. Nature 175: 1125–1126. Petty TL and Ashbaugh DG (1971) The adult respiratory distress syndrome. Clinical features, factors influencing prognosis and principles of management. Chest 60: 233–239. Seeger WC, Grube C, Gu¨nther A, and Schmidt R (1993) Surfactant inhibition by plasma proteins: differential sensitivity of various surfactant preparations. European Respiratory Journal 6: 971– 977. Wang Z, Schwan AL, Lairson LL, et al. (2003) Surface activity of a synthetic lung surfactant containing a phospholipase-resistant phosphonolipid. American Journal of Physiology: Lung Cellular and Molecular Physiology 285: L550–L559. Willson DF, Thomas NJ, Markovitz BP, et al. (2005) Effect of exogenous surfactant (calfactant) in pediatric acute lung injury: a randomized controlled trial. Journal of the American Medical Association 293: 470–476.

to lower surface tension. In addition, SP-A plays a major role in host defense of the lung: it is an essential component of innate immunity and helps to activate acquired immunity. SP-A can act either as a proinflammatory or anti-inflammatory agent, increasing or decreasing production of inflammatory cytokines by inflammatory cells to either enhance pathogen killing or suppress inflammation under basal conditions. Finally, it has been shown that SP-A levels are altered during a number of disease states including adult respiratory distress syndrome (ARDS) and cystic fibrosis. Concomitantly, exposure of SP-A to severe inflammation may cause oxidation or nitration of the protein and result in inactivation. Inactivation of SP-A during disease states may therefore cause increased susceptibility to secondary lung infections.

Introduction Pulmonary surfactant is a mixture of phospholipids, cholesterol, triacylglycerol, free fatty acids, and proteins produced by the lung alveolar type II (ATII) cells and secreted in the alveolar space. An essential function of the surfactant is the reduction of surface tension to prevent collapse of the alveoli during expiration. Four surfactant proteins have been identified: SP-A, SP-B, SP-C, and SP-D. SP-B and SP-C are hydrophobic proteins involved in the respreading of surfactant lipids during inhalation, while SP-A and SP-D are hydrophilic hybrid molecules that belong to the Ca2 þ -dependent animal lectin superfamily termed collectins. Collectins are carbohydratebinding proteins other than antibodies and enzymes that share the common structural characteristics of an N-terminal collagen-like domain connected to a Ca2 þ -dependent carbohydrate-recognition domain (CRD). SP-A and SP-D are expressed in the lungs of all mammals, while humans and rodents also express the serum collectin mannose-binding protein (MBP), and cattle express three serum lectins: conglutinin, CL-43, and CL-1.

Surfactant Protein A (SP-A) S Matalon and J M Hickman-Davis, University of Alabama, Birmingham, AL, USA J R Wright, Duke University Medical Center, Durham, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Surfactant protein A (SP-A), the most abundant apoprotein of pulmonary surfactant, is a hydrophilic hybrid molecule that belongs to the Ca2þ-dependent animal lectin superfamily termed collectins. Collectins are carbohydrate-binding proteins other than antibodies and enzymes that share the common structural characteristics of an N-terminal collagen-like domain connected to a Ca2þ-dependent carbohydrate-recognition domain. SP-A has multiple functions including participating in tubular myelin formation, regulating the recycling of surfactant lipids, and acting synergistically with the two lipophilic surfactant apoproteins

Structure Collectins are assembled as oligimers of trimers with SP-A and MBP able to assemble into hexamers of trimers. SP-A and MBP resemble the first complement protein component Clq in their arrangement of 18 CRDs with kinked collagen stalks. SP-D and conglutinin arrange their globular heads around collagen spokes as cruciform structures (Figure 1), and CL-43 has the simplest arrangement of a single unit consisting of three polypeptides. The collectins are ideally suited to the role of first-line defense in that they are widely distributed, capable of antigen recognition, and can discern self versus nonself. They recognize bacteria, fungi and viruses by binding mannose and N-acetylglucosamine residues on microbial cell walls.

144 SURFACTANT / Surfactant Protein A (SP-A)

SP-A

MBL

SP-D

Figure 1 Structure of collectins. SP-A and mannose-binding lectin (MBL) are arranged as a hexamer of trimers having 18 CRDs with kinked collagen stalks. SP-D is arranged with its globular heads around collagen spokes as a cruciform structure. Modified from Wright JR (2005) Immunoregulatory functions of surfactant proteins. Nature Reviews: Immunology 5: 58–68.

(a)

(b) (c)

AM

·O2 Bacteria

N

NO

SP-A Receptor

(f)

(d)

?

Lamellar body Surfactant Virus Rough LPS

F-




TN

(e)

Smooth LPS N

Nucleus

Figure 2 Functions of SP-A. (a) SP-A binds and causes agglutination of bacteria; interacts with AMs either specifically through a number of receptors (SPR210, mannose receptor, C1q receptor) to trigger (b) uptake of bacteria (Fc and CR1-mediated and receptorindependent uptake); (c) uptake and secretion of surfactant; (d) binding of virus and inhibition of viral hemagglutination; (e) binding of rough and smooth LPS to trigger cytokine release; and (f) increases the production of reactive oxygen and nitrogen species. LPS, lipopolysaccharide.

Regulation of Production and Activity SP-A, the first surfactant protein to be discovered, is the most abundant and is highly conserved among several species (human, dog, rabbit, rat, mouse). Human SP-A is encoded by two separate genes with several different alleles characterized for each gene. SP-A is thought to participate in three major physiologic processes: (1) regulation of surfactant homeostasis

associated with tubular myelin formation; (2) cooperation with SP-B and SP-C in lowering surface tension of surfactant monolayers, especially in pathological situations; and (3) non-specific innate immune responses of the lung (Figure 2). In the alveolar space, surfactant exists as tubular myelin with SP-A localized to the corners of this lattice structure. Consistent with a role for SP-A in the formation of tubular myelin, infants suffering from respiratory distress syndrome

SURFACTANT / Surfactant Protein A (SP-A) 145

have a deficiency of tubular myelin and SP-A, and transgenic mice lacking SP-A are also deficient in tubular myelin. SP-A binds to phospholipids in a Ca2þ independent manner and is thought to be important for enriching the surface film with these compounds. Likewise, SP-A is thought to regulate surfactant homeostasis due to its ability to bind specifically to ATII cells and inhibit the secretion of lipid. SP-A has also been proposed to act as an antioxidant and protect the lung against oxidative stress from pollutants and inflammation, although, subsequent studies have demonstrated that exposure of SP-A to oxidized lipids causes inactivation of the protein. Finally, SP-A has been shown to reduce the ability of plasma proteins to inhibit surfactant function. Disruption of the alveolar epithelial barrier during lung injury can result in leakage of blood proteins into the airspace and these proteins have been shown to inhibit surfactant function in vitro and in vivo. In vitro studies have shown that SP-A added to surfactant lipid prior to the addition of plasma proteins protects against inhibition of surfactant activity. Likewise, the surfactant isolated from SP-A deficient mice is more susceptible to inhibition by plasma proteins. Interestingly, SP-A-deficient mice have no apparent breeding or survival abnormalities when housed under pathogen-free conditions; however, SP-A may be more important for surfactant function when surfactant phospholipid levels are decreased, i.e., during toxic insult, infection, or early development.

Biological Function Innate Immunity

SP-A interacts with alveolar macrophages (AMs) in a highly specific manner through a cell surface SP-A receptor, as well as a variety of non-specific receptors including mannose binding, C1q, etc. SP-A has been shown to: (1) affect release of reactive oxygen species from AMs; (2) stimulate chemotaxis of monocytes; (3) enhance phagocytosis and killing of bacterial, viral, and fungal pathogens by AMs; (4) enhance FcgR- and ClqR-mediated phagocytosis in vitro; (5) bind to allergenic proteins from dust and fungi and inhibit histamine release; and (6) bind and stimulate removal of apoptotic neutrophils. Collectins in general, and SP-A in particular, recognize bacteria, fungi, and viruses by binding mannose and N-acetylglucosamine residues on microbial cell walls. For example, SP-A and SP-D bind glycoconjugates on influenza A virus and a heavily glycosylated glycoprotein with mannose-containing oligosaccharide chains on the surface of Pneumocystis carinii, enhancing their attachment and phagocytosis by AMs.

The consequence to the host of SP-A-pathogen binding varies with the cellular environment and the nature of the microbe. For example, SP-A acts as a ligand to facilitate attachment of Staphylococcus aureus to rabbit and human AMs but has no effect on Streptococcus pneumoniae type 25. Furthermore, increased association of SP-A-coated S. aureus with AMs appears to consist mainly of attachment without ingestion and killing. We observed that although human SP-A bound to mycoplasmas in a Ca2þ-dependent fashion, SP-A binding to mycoplasmas alone did not enhance their phagocytosis or killing by mouse AMs. The creation of SP-A deficient mice has placed a greater emphasis on the role of SP-A following infectious or toxic insult. SP-A knockout mice have increased inflammation and decreased ability to clear bacteria and viruses after infection with a number of pathogens including group B streptococcus, Mycoplasma pulmonis, Pseudomonas aeruginosa, Haemophilus influenzae, respiratory syncytial virus, adenovirus, and influenza A virus. However, these data should be interpreted carefully as recent data generated with congenic C57BL/6 SP-A(
/
) mice have demonstrated that mouse genetic background contributes significantly to inflammation in response to infection. Inflammatory or Anti-Inflammatory?

Results of studies in SP-A(
/
) mice have raised some interesting questions concerning the ability of SP-A to act as a proinflammatory or an anti-inflammatory agent. Previously, transgenic mice deficient for SP-A have been shown to develop more severe pulmonary lesions and have increased levels of proinflammatory cytokines as compared to wild-type mice after inoculation of their lungs with various pathogens. However, recent data with congenic C57BL/6 SP-A(
/
) mice have demonstrated decreased proinflammatory cytokine production and reactive oxygen-nitrogen species in response to Pneumocystis carinii and M. pulmonis infection. These latter results suggest that the response to SP-A in vivo may be modified according to mouse genotype or may be pathogen specific. Significant controversy exists over the ability of SP-A to act as a pro- or anti-inflammatory stimulus in the lung. Several lines of evidence show that SP-A either enhances or decreases the production of reactive nitrogen and inflammatory cytokines by immune cells. For example, SP-A enhanced the production of nitric oxide (NO) and NOS2 (iNOS) expression in rat AMs, previously activated with interferon g, but inhibited NO production and NOS2 expression by

146 SURFACTANT / Surfactant Protein A (SP-A)

lipopolysaccharide (LPS)-activated macrophages. SP-A may exert differential effects depending on the type of stimulus or pathogen presented to the cell. For example, SP-A reduced the production of NO by AMs in the presence of M. tuberculosis, and this was correlated with reduced killing of organisms. In contrast, SP-A enhanced the production of NO and killing of pathogens by monocyte-derived macrophages in the presence of bacillus Calmette-Guerin and by mouse AMs in the presence of M. pulmonis. Finally, SP-A downregulated NO production by AMs from normal humans but upregulated NO production by a significant fraction of AMs harvested from the lungs of patients with transplanted lungs, presumably due to the presence of lung inflammation. Recent studies from two independent laboratories have provided at least partial explanations for these conflicting results. Recent data demonstrate that SP-A decreases LPS-activation of nuclear factor kappa B (NF-kB) by increasing protein expression of inhibitor kappa B-alpha (IkB-a) via posttranscriptional mechanisms. Based on experimental data, it was proposed that the effects of SP-A are due to inhibition of the ubiquitin-proteasome pathway. Since this pathway is responsible for the degradation of a number of altered proteins, SP-A may also delay their clearance with unforeseen implications. Likewise, other studies propose that SP-A binds to inflammatory cells either by the carbohydrate (‘globular heads’) recognition domains or by their collagenous tails. Binding of the globular heads to SIRPa initiates a signaling cascade that blocks NF-kB and decreases proinflammatory mediator production. On the other hand, when the collectin globular heads are bound by pathogens, interaction of the collagen tails with calreticulin/CD91 results in activation of NF-kB and increased production of inflammatory mediators. The predictions of this model are supported by the results of other studies. For example, SP-A had differential effects on LPSinduced production of cytokines by U-937 cells, depending on whether a rough or smooth serotype of LPS was used. Interaction of SP-A with the rough form of LPS, to which SP-A binds, downregulated TNF-a production whereas SP-A upregulated TNF-a production induced by smooth LPS, which does not interact with SP-A’s globular head. Furthermore, lower levels of the stable metabolites of NO were observed in the BAL of uninfected C57BL/6 mice as compared to SP-A knockout mice, and yet significantly higher values of nitrite were present in wildtype mice following mycoplasma infection. In the context of the immune challenges faced by the lung, a Yin/Yang balance may be of benefit to the host. For example, in the normal lung, the collectins may downregulate production of inflammatory mediators

while enhancing the clearance of pathogens via opsonization (e.g., the Yin). However, when the lung is challenged by an aggressive insult such as a bacterial or viral infection, induction of an inflammatory response by surfactant proteins (e.g., the Yang) would aid in neutralization of the pathogen. Anti-Microbial Effects

SP-A has also been reported to have direct antimicrobial activity. SP-A was shown to inhibit the proliferation of Escherichia coli, Klebsiella pneumoniae, and Enterobacter aerogenes in the absence of macrophages and independent of bacterial aggregation. This antibacterial effect was attributed to the ability of SP-A to increase bacterial membrane permeability. SP-A was shown to similarly inhibit the growth of the fungus Histoplasma capsulatum. Likewise, mutation of the LPS region of Bordetella pertussis or B. bronchiseptica renders them susceptible to increased membrane permeability and cell death after exposure to SP-A. In contrast, another Gram-negative bacteria, P. aeruginosa, was recently shown to degrade SP-A and alter the large aggregate conversion of surfactant when incubated with the protein in the absence of inflammatory cells. These data suggest that the direct antimicrobial properties of SP-A may be pathogen specific or have limited application in vivo. Acquired Immunity

Research on the immune functions of SP-A have primarily concentrated on the structural characteristics of SP-A as a collectin and have focused on the innate immune system. This arm of the immune system consists of structural, cellular, and protein components that are capable of targeting, removing and/or killing invading organisms quickly after they invade the host. These processes are rapid, non-specific, and occur before the development of the acquired or adaptive immune response. However, recent evidence suggests that SP-A may modulate the adaptive immune response and thus alter the outcome of disease secondarily to the initial inflammatory response. Recent studies demonstrated that SP-A inhibited basal- and LPS-mediated expression of major histocompatibility complex class II and CD86 expression on immature dendritic cells. Dendritic cells are potent antigen-presenting cells capable of activating naive T cells and thus initiating adaptive immune responses. These studies also showed that SP-A caused a decrease in dendritic cell chemotaxis and an inhibition of allostimulation of T cells. Previously SP-A had been shown to inhibit lymphocyte proliferation and histamine release by eosinophils in response to antigen. Similarly, in vivo studies with

SURFACTANT / Surfactant Protein A (SP-A) 147

SP-A knockout mice have demonstrated alterations in B lymphocyte and activated T lymphocyte responses after infection with influenza A virus, indicating that SP-A is important for appropriate activation of the adaptive immune system in response to this pathogen. Likewise, SP-A-deficient mice have been used to show that basal endogenous SP-A and enhanced alveolar SP-A levels modulate donor T-celldependent immune responses and prolong survival after allogeneic bone marrow transplant. These studies have resulted in speculation concerning the ability of SP-A to be used as a therapeutic agent in the treatment of autoimmune diseases and organ transplantation.

demonstrated in vivo and in vitro. These effects are a result of cell- and pathogen-specific interactions and should be interpreted carefully given that variability of response has been shown to be connected with cell type, mouse genotype, pathogen, dose, route of infection, and time of sampling. In general, the understanding of pulmonary host defense mechanisms lags behind that of other systems because of the poor accessibility and complexity of the lung environment. If SP-A is of primary importance in maintaining surfactant function and protection against pathogens, it may be possible to develop practical therapies or to increase the lungs’ protective capacity through a better understanding of this essential protein.

SP-A in Respiratory Disease

Acknowledgments

Lung inflammation due to infection or exposure to oxidizing environments results in surfactant dysfunction and impaired gas exchange. It has been shown that SP-A levels are altered in a number of disease states including cystic fibrosis, adult respiratory distress syndrome (ARDS), pneumonia, and trauma. Additionally, it has been found that SP-A is oxidized or nitrated by the inflammatory response thus rendering it inactive. Results of previous in vitro studies indicate that nitrated SP-A loses its ability to enhance the adherence of Pneumocystis carinii to rat AMs. Also, nitration of human SP-A by peroxynitrite or tetranitromethane inhibited its lipid aggregation and mannose-binding activities. Finally, SP-A isolated from the lungs of lambs exposed to high concentrations of inhaled NO had decreased ability to aggregate lipids. Thus, nitration of SP-A may be one of the factors responsible for increased susceptibility of patients with ARDS to nosocomial infections. Interestingly, despite being present at high concentrations in the epithelial lining fluid of patients with ARDS, albumin was nitrated to a much lesser degree than SP-A. This observation supports the suggestion that SP-A may have some antioxidant properties that protect surfactant lipids, yet result in protein inactivation. In summary, the respiratory tract is exposed to a multitude of toxic and infectious agents on a regular basis and yet, in the healthy lung, disease is a relatively rare event. Within the alveolar lining fluid, SP-A provides one of the first lines of defense against invading organisms before specific immunity can develop. While SP-A has been identified in a wide variety of tissues throughout the body, significant levels of protein have only been identified in the lung. The ability of SP-A to effectively enhance surfactant function, mediate pathogen clearance, and alter the inflammatory response has been convincingly

This work was supported by the following grants: RR00149 (J.M.H.-D.), HL31197 and HL51173 (S.M.) and HL-30923, HL-51134, and HL-68072 (JRW). See also: Breathing: Breathing in the Newborn; Fetal Breathing; Fetal Lung Liquid; First Breath. Epithelial Cells: Type II Cells. Pneumonia: Overview and Epidemiology. Surfactant: Overview.

Further Reading Akiyama J, Hoffman A, Brown C, et al. (2002) Tissue distribution of surfactant proteins A and D in the mouse. Journal of Histochemistry and Cytochemistry 50: 993–996. Creuwels LA, van Golde LM, and Haagsman HP (1997) The pulmonary surfactant system: biochemical and clinical aspects. Lung 175: 1–39. Crouch E, Hartshorn K, and Ofek I (2000) Collectins and pulmonary innate immunity. Immunology Reviews 173: 52–65. Daniels CB and Orgeig S (2003) Pulmonary surfactant: the key to the evolution of air breathing. News in Physiological Sciences 18: 151–157. Epstein J, Eichbaum Q, Sheriff S, and Ezekowitz RA (1996) The collectins in innate immunity. Current Opinion in Immunology 8: 29–35. Hickman-Davis JM, Gibbs-Erwin J, Lindsey JR, and Matalon S (2004) Role of surfactant protein-A in nitric oxide production and mycoplasma killing in congenic C57BL/6 mice. American Journal of Respiratory Cell and Molecular Biology 30: 319–325. Hussain S (2004) Role of surfactant protein A in the innate host defense and autoimmunity. Autoimmunity 37: 125–130. Ikegami M (1998) Characteristics of surfactant from SPA-deficient mice. American Journal of Physiology 275: L247–L254. Khubchandani KR and Snyder JM (2001) Surfactant protein A (SP-A): the alveolus and beyond. FASEB Journal 15: 59–69. Korfhagen TR, Bruno MD, Ross GF, et al. (1996) Altered surfactant function and structure in SP-A gene targeted mice. Proceedings of the National Academy of Sciences 93: 9594–9599. Lawson PR and Reid KB (2000) The roles of surfactant proteins A and D in innate immunity. Immunology Reviews 173: 66–78.

148 SURFACTANT / Surfactant Proteins B and C (SP-B and SP-C) LeVine AM and Whitsett JA (2001) Pulmonary collectins and innate host defense of the lung. Microbes and Infection 3: 161–166. Matalon S and Wright JR (2004) Surfactant proteins and inflammation: the Yin and the Yang. American Journal of Respiratory Cell and Molecular Biology 31: 585–586. Wright JR (2004) Host defense functions of pulmonary surfactant. Biology of the Neonate 85: 326–332. Wright JR (2005) Immunoregulatory functions of surfactant proteins. Nature Reviews: Immunology 5: 58–68. Zhu S, Ware LB, Geiser T, Matthay MA, and Matalon S (2001) Increased levels of nitrate and surfactant protein a nitration in the pulmonary edema fluid of patients with acute lung injury. American Journal of Respiratory and Critical Care Medicine 163: 166–172.

Surfactant Proteins B and C (SP-B and SP-C) M F Beers and S Mulugeta, University of Pennsylvania School of Medicine, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Surfactant proteins B and C (SP-B and SP-C) are small hydrophobic proteins of pulmonary surfactant that are essential for lung function. Each is produced in alveolar type II (ATII) cells from larger precursor proteins (proproteins) that undergo multistep posttranslational processing to generate the mature secreted forms. SP-B and SP-C participate in the reduction of surface tension of the alveoli and prevent alveolar collapse at low lung volume by forming a surface-active film at the air–fluid interface. They also have critical roles in the regulation of intracellular processes necessary for the production of surfactant and for the maintenance of ATII cell function. Deficiency or abnormal protein expression due to SP-B and SP-C gene mutations has been linked to various acute and chronic lung diseases.

Introduction Surfactant proteins B and C (SP-B and SP-C) are small amphipathic proteins found in alveolar surfactant that are necessary for proper pulmonary function. Initially isolated from bronchoalveolar lavage, their primary sequences are highly hydrophobic. They play a critical role in alveolar surface tension reduction by enhancing formation of a surface-active phospholipid film at the air–fluid interface, thereby reducing the work of breathing and preventing alveolar collapse at end-expiration. SP-B and SP-C promote the formation of a functional surfactant monolayer by enhancing the adsorption and surface spreading of phospholipids and by maintaining an efficient interfacial film. These proteins are generated in alveolar type II (ATII) cells from precursor

proteins (proproteins) via multistep processing that starts in the endoplasmic reticulum (ER), continues through the Golgi complex, small vesicles, and multivesicular bodies before ending in the lamellar bodies (LBs). The bioactive, mature forms of SP-B and SP-C are stored in LBs pending secretion into the alveolar space. SP-B and SP-C may contribute to surfactant metabolism by enhancing phospholipid uptake by ATII cells. Similar to SP-A and phospholipids, SP-B and SP-C are endocytosed by ATII cells, then either directed to lysosomes for degradation or recycled back to LBs via multivesicular bodies for secretion.

Surfactant Protein B Structure

Human SP-B is encoded by an 11 kb, 11 exon gene (SFTPB) on chromosome 2p. Transcription of SFTPB results in a 2 kb mRNA encoding a 381 amino acid, 40 kDa preproprotein. Exons VI and VII encode the mature SP-B (Figure 1). SP-B mRNA is highly conserved among mammalian species. SP-B cDNA exhibits B67% cross-species homology throughout the preproprotein, and 80–85% homology within the mature protein domain. Although the precise tertiary conformation of SP-B is unknown, amino acid sequence analysis predicts five amphipathic helices (Figure 2). Cellular Localization

SFTPB is expressed in ATII cells and nonciliated bronchiolar epithelial cells (Clara cells). However, while SP-B mRNA, proprotein, proprotein intermediates, and mature protein are present in ATII cells, only SP-B mRNA and proprotein are detected in Clara cells. It is unclear whether or not SP-B expression in Clara cells is associated with surfactant function. Mature SP-B also localizes in alveolar macrophages. However, absence of detectable SP-B mRNA or proprotein in macrophages suggests that mature SP-B is taken up from the alveolar space via phagocytosis. Processing

SP-B entry into the secretory pathway is mediated by a 23 amino acid N-terminal signal peptide, which is cleaved in the ER. In its proprotein stage, the 79 amino acid mature peptide is flanked by the 177 amino acid N-terminal and 102 amino acid C-terminal propeptide domains, which are removed by sequential proteolytic cleavages during transit through the secretory pathway. Proprotein processing includes glycosylation (which increases the

SURFACTANT / Surfactant Proteins B and C (SP-B and SP-C) 149 Surfactant protein B

Surfactant protein C

2p12-p11.2 I II

III IV

V

VI

VII VIII

XI

I

11 kb

II

III

IV V

VI

2.8 kb

Gene ATG

2 kb

TGA

Signal peptide 1

40 kDa NH2 39 kDa

8p21

Chromosome IX X

129

24 NH2

201

279 311

ATG

mRNA

381 COOH

Preproprotein

381 COOH

Proprotein Posttranslational processing

25 kDa

9 kDa 8 kDa

Mature protein

(a)

1 NH2

24

TGA

58

197 COOH

0.9 kb

21 kDa

16 kDa 6 kDa 5 kDa 3.7 kDa

(b)

Figure 1 Gene and protein structures of SP-B and SP-C. (a) Surfactant protein B: the SP-B gene containing 11 exons maps to chromosome 2. The mRNA transcribed from exons I to X is translated to a 381 amino acid preproprotein. A 23 amino acid N-terminus signal peptide directs the preproprotein to the ER where it is cleaved to form the proprotein. Further processing of N- and C-terminal peptides produces the mature SP-B consisting of 79 amino acids (8 kDa). (b) Surfactant protein C: the SP-C gene located on chromosome 8 comprises 6 exons; parts of exons V and VI are untranslated. The gene is transcribed into a 0.9 kb mRNA product, which in turn is translated to a 197 amino acid (21 kDa) proprotein. During transit through the regulated secretory pathway, several N- and C-terminal proteolytic cleavages result in the release of the mature 35 amino acid bioactive form.

Cytosol

Surfactant protein B proprotein

Surfactant protein C proprotein

ER or vesicular lumen

Figure 2 Precursors of SP-B and SP-C. SP-B is a small hydrophobic protein of 8 kDa produced from proteolytic processing of a 381 amino acid preproprotein. Although the structure is not known, amino acid sequence analysis predicts that the proprotein consists of five a-helical amphipathic domains connected by intermittent extended strands. These hydrophobic domains are believed to interact with the head groups of phospholipids. SP-C is a smaller hydrophobic protein of 3.7 kDa also produced from proteolytic processing of a 197 amino acid integral membrane precursor protein. The proprotein is a bitopic protein with a type II orientation. Part of the N-terminal domain and the entire transmembrane domain make up the mature bioactive form.

proprotein molecular weight from 39 kDa to 42 kDa), modification of N-linked carbohydrates, and proteolytic cleavages giving rise to a 25 kDa intermediate and subsequently a 9–10 kDa intermediate.

The final N-terminal cleavage produces the mature, bioactive 8 kDa SP-B peptide (Figure 1). The mature peptide is secreted into the alveolar space as an 18 kDa disulfide-linked homodimer, which is exceptionally

150 SURFACTANT / Surfactant Proteins B and C (SP-B and SP-C)

hydrophobic due to residues composed predominantly of valine and leucine. This amphipathic domain (arranged so that one face is charged and the other is hydrophobic) of mature SP-B facilitates association with the surface alveolar surface film, primarily via interaction with negatively charged phospholipid head groups. Biological Function

As a member of the saposin-like family of peptides that interact with lipids, SP-B avidly associates with surfactant phospholipids. SP-B is critical both for the intracellular formation of LBs and for generation of a surface film in the extracellular space. In vitro, the interaction of SP-B with lipid vesicles causes lysis and fusion resulting in the formation of phospholipid sheets from vesicular lipids. This is believed to facilitate transformation of multivesicular bodies into the densely packed lamellae seen in LB ultrastructure images, as well as to promote formation of tubular myelin and surface film multilayers in the alveolar space. Furthermore, SP-B increases adsorption of lipid molecules into the surface film to enhance the surface tension-lowering properties of surfactant required for normal lung function. These properties make SP-B a critical component of exogenous surfactant preparations used to treat surfactant deficiency, specifically respiratory distress syndrome (RDS) in preterm infants. SP-B in Respiratory Diseases

Targeted disruption of SFTPB in vivo demonstrates the importance of SP-B to lipid homeostasis and surfactant function. In SP-B knockout mice, prenatal lung morphogenesis and surfactant phospholipid synthesis proceed normally. However, fatal respiratory failure develops postnatally. Electron micrographs of affected lungs reveal disruption of LB formation in ATII cells resulting in abnormal inclusions consisting of multivesicular bodies and disorganized lamellae demonstrating the importance of SP-B in organizing surfactant phospholipids into LBs. Moreover, LB biogenesis disruption results in abnormal processing of the SP-C proprotein leading to accumulation of a 6–10 kDa processed intermediate without generation of detectable mature SP-C, thus resulting in a phenotypic ‘double knockout’. Although rare, human SFTPB mutations have been identified with homozygosity for the mutation being associated with fatal neonatal respiratory failure that is unresponsive to standard therapies, including surfactant replacement. Infants with SFTPB mutations who do not produce SP-B develop progressive respiratory failure within 24–48 h following birth.

Many of the clinical features are identical to those seen in SP-B knockout mice including altered surfactant biophysical activity, disrupted LB biogenesis, and impaired SP-C biosynthesis. Without lung transplantation, most of these infants die of chronic respiratory failure despite rigorous ventilatory support. Relatives of these infants heterozygous for SP-B mutation do not have clinically apparent lung abnormalities; however, heterozygous SP-B knockout mice have subtle surfactant function abnormalities and increased hyperoxia susceptibility.

Surfactant Protein C Structure

The 6 exon, 2.4 kb gene encoding human SP-C (SFTPC) is on chromosome 8p (Figure 1). Exon I contains a 25 bp 50 untranslated region. Part of exon V and all of exon VI constitute a 30 untranslated region. Transcription of the five exons results in a 0.9 kb mRNA encoding the 197 amino acid 21 kDa proprotein. Exon II encodes the 35 amino acid mature bioactive SP-C protein. Although there is a fair degree of interspecies heterogeneity in the SP-C C-terminus, the majority of amino acids in the mature SP-C primary sequence and the flanking domain are highly conserved. The SP-C proprotein C-terminal flanking domain contains a BRICHOS domain, a highly structurally conserved domain of B100 amino acids found in several proteins linked to human disease. SP-C is distinct from other surfactant proteins in its cellular localization and structure. SP-C is the only surfactant protein expressed solely by ATII cells. It is the only known protein that is synthesized as an integral membrane proprotein and subsequently secreted as a luminal protein. The bitopic proprotein has a type II orientation (cytosolic N-terminus; luminal C-terminus) with an average angle of 241 with respect to the phospholipid bilayer (Figure 2). While the N-terminus contains positively charged hydrophilic residues, the transmembrane domain comprises a 23 residue polyvaline-rich alphahelical hydrophobic domain. Charged residues near the membrane phospholipid polar head groups anchor the proprotein and direct proper transmembrane orientation. Processing

The SP-C proprotein undergoes extensive posttranslational processing as it is trafficked through the regulated secretory pathway (Figure 1). Proprotein sorting involves oligomeric association of proprotein monomers mediated by the mature SP-C domain.

SURFACTANT / Surfactant Proteins B and C (SP-B and SP-C) 151

The proprotein is modified by palmitoylation of two N-terminal cysteine residues situated near the phospholipid membrane. Similar to SP-B, SP-C is processed by multistep cleavages beginning with removal of a C-terminal segment of B30 residues during propeptide transit from the ER to the Golgi apparatus. The resultant 16 kDa proprotein intermediate undergoes sequential C- and N-terminal cleavages producing 6 and 5 kDa intermediates. Finally, removal of a 9 amino acid N-terminal segment liberates the mature, active 3.7 kDa peptide, which includes part of the N-terminus and the 23 residue transmembrane domain. Biological Function

SP-B and SP-C share several functional similarities (Table 1). Like SP-B, SP-C accelerates surface film formation by enhancing phospholipid adsorption and spreading at the air–fluid interface. Palmitoylation of cysteine residues enables the peptide to move within or between lipid layers and facilitates formation of the multilayer films that line the alveolus. Functional redundancy between SP-B and SP-C is highlighted by restoration of lung function in surfactant depletion or lung injury models using a surfactant preparation containing either SP-B or SP-C. Unlike SP-B knockout mice, SP-C knockout mice survive and reproduce like wild-type littermates, Table 1 Structure, processing, and function of SP-B compared with SP-C

Structure Reduced (nonreduced) in kDa Primary translation product (kDa) Homology Amphipathic domain(s) Integral transmembrane domain Processing Preproprotein Proprotein Proteolytic cleavages Glycosylation Palmitoylation N-linked carbohydrate modification Function Lamellar body formation Phospholipid adsorption and surface spreading Monolayer formation Tubular myelin formation Vesicle fusion Enhancement of phospholipid uptake by ATII cells

SP-B

SP-C

8 (18) 45

3.7 (6–7) 21

Saposins Yes No

BRICHOS Yes Yes

Yes Yes 3 Yes No Yes

No Yes 4 No Yes No

likely due to the presence of SP-B. However, depending on strain, age, and environment, these mice eventually develop respiratory distress and severe progressive pneumonitis. SP-C in Respiratory Diseases

Several histological and clinical subtypes of interstitial lung diseases (ILDs) have been linked to SP-C protein deficiency and SFTPC mutations. Approximately 2% of neonatal lung disease cases are associated with SFTPC mutations. Abnormalities in SP-C expression have also been linked to ILD in older children and adults. ILD associated with SP-C mutations can be de novo or inherited (typically dominant inheritance with incomplete penetrance). Given variable age of onset and severity within families, modifier genes and/or environmental factors likely influence phenotypic expression. Both SFTPC missense and deletion mutations have been described in association with familial ILD. Many of the mutations are clustered within the proprotein BRICHOS domain resulting in proprotein misfolding, thereby leading to aberrant trafficking, targeting, and processing of the propeptide. To date, all reported affected individuals were heterozygous for SFTPC mutations but had no detectable mature SP-C in their lung tissue, suggesting a dominant-negative effect of the mutant allele on the normal allele. A dominantnegative mechanism is plausible given the self-associative property of SP-C proprotein during transit through the secretory pathway. Cells transfected with mutant SP-C isoforms also form perinuclear aggregates in a dominant-negative manner. This formation of perinuclear aggregates is consistent with aggresome formation and an inability to clear misfolded proteins resulting in toxic gain of function. In vitro experiments have suggested various pathologic mechanisms for these mutations including induction of ER stress, cytotoxicity, and caspase-3-mediated apoptosis. Taken together, these studies support the hypothesis that chronic accumulation of misfolded SP-C protein causes cell stress, cell injury, and death, and eventual tissue damage leading to ILD.

Therapeutic Perspectives Yes Yes

No Yes

Yes Yes Yes Yes

Yes No Yes Yes

The association of SFTPB and SFTPC mutations with pulmonary disease was recently recognized. The molecular mechanisms underlying their pathophysiology have not yet been completely defined. Currently, treatment of SP-B- and SP-C-related lung disease is limited to either general supportive measures, such as lung transplantation for SP-B deficiency and surfactant replacement for neonatal RDS, or the use of

152 SURFACTANT / Surfactant Protein D (SP-D)

general anti-inflammatory agents such as corticosteroids for ILD associated with SP-C mutations. Targeted therapies can be developed as the underlying molecular and cellular mechanisms are elucidated. For SP-B deficiency, molecular therapy aimed at replacing the mutant alleles with functional isoforms might correct the associated downstream events. Alternatively, further characterization of the role of SP-B in LB biogenesis and proSP-C processing may facilitate development of targeted therapies to correct these secondary phenotypic defects. In contrast, most alterations in the proSP-C sequence that produce ILD result in either misfolding or mistargeting of mutant protein triggering induction of intracellular aggregate formation, ER stress, or accumulation in subcellular compartments. The cellular events resulting from expression of mutant SP-C appear similar to those seen in chronic neurodegenerative diseases and a-1 antitrypsin deficiency, representing a new class of ‘conformational diseases’ caused by mutant protein aggregation in non-native conformations. Thus, molecular therapies based on correcting protein misfolding or deleterious cellular responses to these events represent therapeutic targets. Emerging small molecule therapies, such as molecular chaperones, protein stabilizers, and lysosomotropic agents, could have a role. In support of this notion, sodium 4-phenylbutyrate, which corrects mutant CFTR trafficking in vivo, has recently been used to block intracellular mutant SP-C aggregation in vitro. Similarly, hydroxychloroquine has shown therapeutic potential in the treatment of non-specific interstitial pneumonitis associated with mutations in SFTPC. Thus, although rare, disease resulting from SP-B and SP-C alterations represents an important paradigm for understanding broader concepts of parenchymal lung disease. Improved understanding of the molecular mechanisms of these diseases may help to define characteristics of lung biology that are conducive to therapeutic strategies. See also: Acute Respiratory Distress Syndrome. Alveolar Surface Mechanics. Alveolar Wall Micromechanics. Atelectasis. Breathing: Breathing in the Newborn; First Breath. Bronchoalveolar Lavage. Genetics: Overview; Gene Association Studies. Infant Respiratory Distress Syndrome. Interstitial Lung Disease: Alveolar Proteinosis; Amyloidosis; Idiopathic Pulmonary Fibrosis. Pediatric Pulmonary Diseases. Proteasomes and Ubiquitin. Pulmonary Fibrosis. Surfactant: Overview.

Further Reading Beers M (1998) Molecular processing and cellular metabolism of surfactant protein C. In: Rooney SA (ed.) Lung Surfactant: Cellular and Molecular Processing, pp. 93–124. Austin: RG Landes.

Beers MF and Mulugeta S (2005) Surfactant protein C biosynthesis and its emerging role in conformational lung disease. Annual Review of Physiology 67: 663–696. Cole FS (2003) Surfactant protein B: unambiguously necessary for adult pulmonary function. American Journal of Physiology. Lung Cellular and Molecular Physiology 285: L540–L542. Guttentag S, Robinson L, Zhang P, et al. (2003) Cysteine protease activity is required for surfactant protein B processing and lamellar body genesis. American Journal of Respiratory Cell and Molecular Biology 28: 69–79. Hawgood S (2004) Surfactant protein B: structure and function. Biology of the Neonate 85: 285–289. Johansson J, Weaver TE, and Tjernberg LO (2004) Proteolytic generation and aggregation of peptides from transmembrane regions: lung surfactant protein C and amyloid beta-peptide. Cellular and Molecular Life Science 61: 326–335. Lin S and Weaver TE (1998) Cellular and molecular processing and cellular metabolism of surfactant protein B. In: Rooney SA (ed.) Lung Surfactant: Cellular and Molecular Processing, pp. 75–92. Austin: RG Landes. Nogee LM (2002) Abnormal expression of surfactant protein C and lung disease. American Journal of Respiratory Cell and Molecular Cell Biology 26: 641–644. Nogee LM (2004) Alterations in SP-B and SP-C expression in neonatal lung disease. Annual Review of Physiology 66: 601–623. Nogee LM (2004) Genetic mechanisms of surfactant deficiency. Biology of the Neonate 85: 314–318. Solarin KO, Wang WJ, and Beers MF (2001) Synthesis and posttranslational processing of surfactant protein C. Pediatric Pathology and Molecular Medicine 10: 471–500. Weaver TE and Conkright JJ (2001) Function of surfactant proteins B and C. Annual Review of Physiology 63: 555–578. Whitsett JA, Nogee LM, Weaver TE, and Horowitz AD (1995) Human surfactant protein B: structure, function, regulation, and genetic disease. Physiological Reviews 75: 749–757. Whitsett JA and Weaver TE (2002) Hydrophobic surfactant proteins in lung function and disease. New England Journal of Medicine 347: 2141–2148. Yurdakok M (2004) Inherited disorders of neonatal lung diseases. Turkish Journal of Pediatrics 46: 105–114.

Surfactant Protein D (SP-D) E C Crouch, Washington University School of Medicine, St Louis, MO, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Surfactant-associated protein D (SP-D) is a collagenous carbohydrate binding glycoprotein (collectin) that plays important roles in the lung’s innate immune response to microbial and antigenic challenge. SP-D enhances the neutralization and/or clearance of a wide variety of microorganisms and microbial products, organic particulate antigens, and apoptotic cells, while inhibiting deleterious inflammatory and immune host responses. SP-D also contributes to the regulation of surfactant homeostasis, in part by reorganizing surfactant lipids, with resulting effects on epithelial uptake and turnover. Thus, it is likely that SP-D plays important roles in the pathogenesis of a wide variety of infectious, inflammatory, and immune-mediated lung disorders.

SURFACTANT / Surfactant Protein D (SP-D) 153

Introduction Surfactant-associated protein D (SP-D) was first identified as a collagenous glycoprotein (CP4) secreted into the culture medium of freshly isolated rat alveolar epithelial cells. Because a significant fraction of lung CP4 was isolated in association with surfactant, and because of its biochemical similarities to surfactant protein A (SP-A), it was designated a surfactant-associated protein, SP-D. Subsequent biochemical analysis of the purified lavage protein and sequencing of the cDNA demonstrated its structural relationship to SP-A and other C-type lectins. Although SP-D showed no effects on the biophysical properties of isolated surfactant, it was serendipitously observed to agglutinate strains of Escherichia coli used for cloning. Subsequent studies confirmed its ability to interact with a variety of Gram-negative bacteria and bind to the core oligosaccharide of bacterial lipopolysaccharides. The protein was also observed to bind to phagocytic cells. The recognition 7 aa

of SP-D as a surfactant-associated host defense protein served to emphasize the functional heterogeneity of molecules associated with pulmonary surfactant.

Structure Domain Structure

SP-D is a member of the collectin subfamily of C-type lectins. Like other collectins, it is assembled from multimers of trimeric subunits. Each trimeric subunit consists of a short N-terminal cross-linking domain, a triple helical collagenous domain, a trimeric coiled-coil linking or neck domain, and a Ctype lectin carbohydrate recognition domain (CRD) (Figure 1). Molecular Assembly

In addition to structural attributes shared with other collectins, SP-D has certain distinctive molecular

73 aa

SP-A 20 nm 25 aa

177 aa

SP-D 46 nm (a)

N

Triple helical collagen domain

NCRD

(b) Figure 1 Structure of SP-D. (a) Trimeric subunits of surfactant protein A (SP-A) and surfactant protein D (SP-D) are compared. Domain sizes are to approximate scale. The N-terminal cross-linking domain is at the left, and the trimeric neck þ carbohydrate recognition domain (NCRD) is at the right. Dodecamers form through the association and disulfide cross-linking of trimeric subunits. SP-D is distinguished from SP-A and most other C-type lectins by a long and uninterrupted triple helical collagen domain, which is modified by both N- and O-linked glycosylation. The molecular diagrams were adapted from original drawings kindly provided by Dr Uffe Holmskov. (b) Rotary shadowing images of recombinant rat SP-D. Two well-spread dodecamers, each consisting of four trimeric subunits (4
3 ¼ 12), are identified in the box on the right. A higher order assembly consisting of four dodecamers is shown in the box on the left. Each dodecamer (or stellate multimer) measures approximately 100 nm in maximum dimension. Note that dodecamers typically show two apposing pairs of arms. The ultrastructural images were prepared by Dr J Heuser using the quick-freeze deep-etch technique.

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CRD CRD Neck

Neck

Ca2+ (a)

(b)

Figure 2 Structure of the trimeric human neck þ CRD domain. The images were generated using the coordinates of the original crystal structure of human SP-D. The three constituent chains of each trimeric assembly are shown. The calcium ion implicated in carbohydrate recognition is shown in red. (a) A trimeric NCRD viewed perpendicular to the longitudinal axis of the neck and collagen domain. The relatively planar ligand-binding surface is on the right. (b) The same trimeric NCRD is viewed looking down the long axis of the molecule, from the C-terminal end, toward the neck. The calcium ions identify the primary carbohydrate binding sites, which are separated by ˚ (5 nm). Simultaneous binding of all three sites is required for high-affinity binding. B50 A

features. SP-D is assembled exclusively as homotrimers of approximately 43 kDa monomers. It has a long and uninterrupted collagen domain, much longer than the corresponding collagenous domains of SP-A or MBL. SP-D also has a propensity to form X-shaped tetramers, or dodecamers (4
3 ¼ 12 chains). The four trimeric arms of each dodecamer are usually asymmetrically arrayed about the N-terminal hub, with two apposing pairs of rod-like arms that determine the spatial distribution of the globular C-terminal lectin domains (Figure 1(b)). Dodecamers are assembled through the N-terminal association and disulfide-mediated cross-linking of trimeric subunits. Higher order multimers appear to consist of highly ordered, stellate assemblies of dodecamers associated at their N-termini. Although dodecamers predominate in many preparations of SP-D, the proportions of various multimeric forms vary among species and different individuals.

Posttranslational Modifications

Most known SP-Ds are characterized by a single site of N-linked glycosylation near the N-terminal end of the collagen domain, corresponding to asparagine-70 in human SP-D. The only known exception is porcine SP-D, which has a second site of N-linked glycosylation within the lectin domain. Many of the proline and lysine residues in the collagen domain are hydroxylated, and most of the hydroxylysine residues are O-glycosylated with glucosyl-galactose.

Structure of the Lectin Domain

Crystallographic and biochemical studies have shown that the trimerization of CRDs is mediated by the 25 amino acid coiled-coil neck domain (Figure 2). The trimeric CRDs of an SP-D subunit constitute a relatively planar binding surface, perpendicular to the longitudinal axis of each subunit. Each CRD contains at least two calcium ions, and one of these ions coordinates directly with saccharides at the carbohydrate-binding site. Hydrophilic residues that flank the shallow carbohydrate-binding groove appear to ‘fine tune’ saccharide-binding preferences and, at least in part, account for species differences in ligand preferences.

Regulation of Production and Activity Sites of Synthesis and Accumulation

Lung SP-D is primarily synthesized by alveolar type II and bronchiolar epithelial cells. Parenchymal production of SP-D begins during mid-fetal life. However, at least in small animals, production increases dramatically near the time of birth. SP-D is synthesized with a signal peptide, which is removed prior to secretion to yield the mature protein. The secretion of SP-D by type II pneumocytes occurs via a constitutive pathway, independent of lamellar body secretion. However, it is also stored within the secretory granules of nonciliated bronchiolar cells. Total SP-D concentrations in human lavage are quite variable, but are usually estimated to be in the

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range of 0.5–1 mg ml  1. Both environmental (e.g., cigarette smoking) and genetic factors likely contribute to this variability. Most in vitro biological effects of SP-D occur in the range of 1–5 mg ml  1, which are similar to or lower than those levels estimated to occur in vivo. Alveolar concentrations have been calculated to be as high as 66 mg ml  1, and local concentrations near sites of granule exocytosis in the distal airways could be much higher. Extrapulmonary Sites of Synthesis and Accumulation

Although the lung appears to be the major organ site of SP-D production, it is expressed by epithelial cells in many other tissues in contact with the environment. SP-D is also present in the blood. The primary cellular source of blood SP-D has not been definitively established. Serum levels of immunoreactive SP-D are increased following various types of acute and chronic lung injury and in allergic patients. The SP-D Gene and Collectin Locus and Regulation of Production

Human surfactant protein D is encoded as a single gene (SFTP4) within a ‘collectin locus’ on the long arm of chromosome 10, at 10q22.2-23.1. This locus also includes the genes for surfactant protein A and serum mannose-binding protein. SP-D is constantly secreted by lung epithelial cells. Nevertheless, basal expression is rapidly increased following many types of experimental lung injury, and in response to microbial or antigenic challenge. The structure of the 50 -regulatory region of the SP-D gene is consistent with regulation by various environmental or immune stimuli. For example, the promoter contains a conserved AP-1 element and multiple CCAT enhancer-binding proteins (C/EBP isoforms), elements, and predicted binding sites for STAT proteins. It has recently been shown that NFATc3 (nuclear factor of activated T cells) and thyroid transcription factor 1 can synergistically activate the murine SP-D promoter. SP-D production is also increased in response to interleukin-4 (IL-4) and glucocorticoids. Degradation

Both alveolar macrophages and type II epithelial cells can internalize and degrade SP-D. However, their contributions to normal physiological turnover of SP-D remain undefined. It is likely that there is significant ongoing ‘consumption’ of SP-D, associated with internalization and or mucociliary clearance of foreign particles, and with the epithelial uptake of pulmonary surfactant.

Biological Function SP-D is a classical pattern-recognition molecule (Table 1). The ability of SP-D to recognize molecular patterns associated with many pathogens and complex particulate antigens is determined at several levels of protein structure. The capacity to recognize diverse ligands is influenced by the ligand-binding specificity of the individual CRDs, the spatial separation and orientation of the ligand-binding sites within each trimeric CRD, and the number and spatial distribution of trimeric CRDs. Interactions with Carbohydrate and Lipid Ligands

SP-D can bind to a variety of purified saccharides and glycoconjugates, including glycoproteins, lipoglycans, and glycolipids. All presently characterized SP-D molecules recognize glucose and maltose. However, there are significant species differences in binding affinity and relative saccharide preferences. This translates to differences in the recognition of complex polysaccharides and other glycoconjugates, such as mannans and glucans derived from fungal cell walls, and glycoconjugates associated with viral envelope proteins, in various binding assays.

Table 1 Potential functions of SP-D suggested by in vitro and in vivo studies Antimicrobial Enhanced neutralization/clearance of respiratory pathogens Opsonization and killing of some organisms Decreased internalization of some intracellular pathogens Enhanced microbial agglutination with increased physical or cellular clearance Enhanced inflammatory response to collectin/microbial complexes Direct antibacterial or antifungal activity Anti-inflammatory Regulation of inflammatory response to microorganisms and microbial products ‘Neutralization’ of LPS/altered presentation of LPS to host cells Enhanced uptake of apoptotic cells Modulation of pulmonary oxidant metabolism and metalloproteinase expression Immunomodulatory Decreased proliferative response of T lymphocytes to various mitogens Enhanced clearance of inhaled organic particulate antigens Altered presentation of specific organic antigens to specifically sensitized cells Enhanced antigen presentation by dendritic cells Altered signaling via Toll-like receptors Surfactant homeostasis Spatial organization of subfractions of surfactant lipid Alterations in epithelial uptake of surfactant lipids

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SP-D also binds to certain lipid molecules, particularly phosphatidylinositol and glucosyl-ceramide, which are minor lipid components of pulmonary surfactant. Lipid interactions are probably also important for binding to surfactant, and interactions with various lipid-containing components of microbial cell walls, such as bacterial lipopolysaccharides. Lastly, SP-D can bind to short oligonucleotides and DNA.

mediated through binding to specific cell surface glycoconjugates. For example, SP-D binds to various biologically active components derived from bacterial cell walls, such as Gram-negative lipopolysaccharides, Gram-positive lipoteichoic acids and peptidoglycans, and lipoarabinomannans of mycobacteria.

Mechanism of Ligand Binding

SP-D binds to a variety of complex organic antigens implicated in pulmonary hypersensitivity, and can modify cellular responses to these antigens. For example, SP-D shows CRD-dependent binding to major glycoprotein allergens associated with the cell walls of Aspergillus fumigatus, and can modulate the immune response to this organism in vivo. SP-D also shows CRD-dependent binding to glycoproteins associated with dust mite antigen, and certain pollens. SP-D can also enhance bacterial antigen presentation by immature bone marrow-derived dendritic cells.

The SP-D CRD has been shown to interact with the terminal 3- and 4-OH groups of the nonreducing glucose of maltose, similar to the binding orientation of mannose. However, there is evidence that binding can also be influenced by interactions with nonterminal sugars. The site and mechanisms of lipid binding are much less certain, and more than one mechanism could be involved. Basic subregions of the CRD probably contribute to the binding of various negatively charged ligands. Interactions with Microorganisms and Components of Microbial Cell Walls

SP-D binds to nearly all classes of microbial pathogens (Table 2). These interactions are primarily

Table 2 Pulmonary pathogens recognized by SP-D in vitroa Viruses Influenza A virus Respiratory syncytial virus (RSV) Human immunodeficiency virus (HIV) Fungi Aspergillus fumigatus Pneumocystis carinii Cryptococcus neoformans Candida albicans Histoplasma capsulatum Blastomyces dermatitidis Mycobacteria Mycobacterium tuberculosis Mycobacterium avium intracellulare Bacteria Gram-negative bacteria, e.g., Pseudomonas aeruginosa, Klebsiella pneumoniae Gram-positive bacteria, e.g., Streptococcus pneumoniae Mycoplasma pneumoniae Chlamydia pneumoniae Parasites Schistosoma mansoni a Listed approximately in order of number of relevant publications. At least some of these interactions are strain dependent or influenced by phenotypic variation in capsule expression and/or the biochemical structure of cell wall glycoconjugates.

Interactions with Organic Particulate Antigens

Models of SP-D Deficiency

Models of SP-D deficiency have proven invaluable in defining aspects of SP-D function (Table 3). SP-D null mice develop a profound alveolar lipidosis. They also show activation of alveolar macrophages, an increased oxidant burden, increased levels of active metalloproteinases, decreased clearance of apoptotic cells, and emphysema. SP-D-deficient mice also show an expansion of peribronchial lymphoid tissue, and increased numbers of T cells with an activated phenotype. The SP-D null phenotype can be fully rescued by targeting wild-type rat SP-D cDNA to alveolar type II cells. Although surfactant synthesis by type II cells and surfactant uptake by macrophages are normal in deficient animals, normal postnatal developmental changes in surfactant pools and metabolism do not occur. Significantly, SP-D null animals show abnormal type II uptake and catabolism of both small and large aggregate forms of surfactant. Despite obvious complexities of the SP-D null phenotype, there is evidence that SP-D-deficiency leads to abnormal cellular and cytokine responses to microbial and antigenic challenge. SP-D-deficient animals show particularly conspicuous defects in the clearance of respiratory viruses, including respiratory syncytial virus (RSV) and influenza A. These defects can be rescued using exogenous SP-D, or by targeting of rat SP-D cDNA to the respiratory epithelium. In general, SP-D null mice show augmented inflammatory or oxidant responses to microbial challenge or to microbial products such as endotoxin.

SURFACTANT / Surfactant Protein D (SP-D) 157 Table 3 Abnormalities in SP-D-deficient mice Structural Anatomic  Alveolar lipidosis with abnormal ultrastructure of airspace surfactant  Acquired emphysema Inflammatory  Multifocal accumulations of activated foamy macrophages  Multifocal chronic inflammation with expansion of peribronchial lymphoid tissue  Increased activated lymphocytes and activated macrophages in lavage  Increased levels of metalloproteinases (MMP-2, MMP-9, MMP12)  Increased macrophage oxidant production  Increased tissue levels of lipid peroxides and reactive carbonyls  Increased accumulation of apoptotic cells Functional Host defense  Deficient neutralization and clearance of SP-D-sensitive strains of influenza A  Deficient clearance of Pneumocystis carinii  Deficient uptake of some bacteria by alveolar macrophages  Enhanced inflammatory and/or oxidant responses to microbial challenge Immune regulation  Deficient clearance of endotoxin and apoptotic cells  Abnormal immune responses to antigenic challenge

Receptors and Interactions with Host Cells SP-D interacts with at least two classes of receptors or binding molecules on host cells. These include glycoconjugates reactive with the lectin domain, and membrane proteins that interact with the SP-D molecule. Nevertheless, no specialized protein receptor for SP-D has yet been identified, and the relevant signaling pathways are poorly understood. There is some evidence that SP-D can directly or indirectly modulate signaling through interactions with membrane and soluble CD14, and probably with other components of Toll-like receptor signaling pathways. In addition, SP-D and SP-D/ligand complexes might differentially modulate anti-inflammatory and proinflammatory signaling pathways in macrophages via SIRPa and calreticulin/CD91, respectively. In some situations, specific receptors are clearly not necessary to modulate the cellular function of SPD. For example, SP-D-mediated aggregation of viruses can alter the presentation of the virions to their sialylated cellular receptors on phagocytes. There are a variety of known cellular consequences of SP-D binding to microorganisms. These include: enhanced uptake with increased or decreased killing, decreased phagocytic uptake and killing, and

enhanced oxidant responses of the phagocyte to the bound organism. Some of these activities are attributable to direct effects of the protein on the phagocyte, whereas other effects involve prior recognition of the organism. Enhanced cellular uptake can involve classical opsonization with utilization of a collectin receptor, possibly calreticulin CD91 or microbial aggregation with altered interactions between microbial receptors and their binding sites on phagocytic cells. It should be recognized that SP-D can also exert direct cytotoxic effects on some bacterial and fungal organisms, independent of host effector cells, probably resulting from increases in membrane permeability.

SP-D in Respiratory Diseases Acquired Alterations in SP-D Levels or Activity

The levels of airspace SP-D are modestly decreased in the setting of cigarette smoking, and often markedly decreased in ARDS and cystic fibrosis. Although the mechanisms have not been elucidated, degradation and increased clearance can be increased in the setting of acute inflammation, and both inflammatory cells and certain microorganisms can degrade lung SP-D in vitro. Polymorphic Variants

Certain allelic variants in SP-D structure are associated with differences in the levels of secreted protein, and certain variations in coding sequence are also associated with differences in assembly and biological activity. For example, the SP-D Thr11 polymorphism, which is associated with the preferential secretion of trimeric molecules, is linked with an increased risk of tuberculosis, while the Met11 allele, which is associated with an increased proportion of secreted multimers, is associated with an increased risk of severe RSV infection. For the reasons indicated above, differently assembled forms could differ in their propensity to engage anti-inflammatory or proinflammatory signaling pathways in phagocytes and other host defense cells. Involvement of SP-D in Specific Diseases: Selected Examples

Influenza A infection SP-D binds to high mannose sugars associated with the viral hemagglutinin and neuraminidase. SP-D aggregates specific strains of influenza A virus (IAV), inhibits activity of hemagglutinin (the viral receptor for host cells), inhibits the activity of the neuraminidase (which is required for

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effective viral replication), increases neutrophil uptake of virus, and inhibits infectivity in vitro and in vivo. As indicated above, SP-D deficiency is associated with decreased clearance of SP-D-sensitive strains of virus, heightened inflammation, and increased mortality. SP-D also decreases the inactivation of the neutrophil respiratory burst to influenza A virus infection, possibly contributing to bacterial superinfection. Aspergillus infection and hypersensitivity SP-D binds to Aspergillus fumigatus glycoproteins and can enhance uptake and killing of fungal organisms by neutrophils and macrophages. Recent studies have shown that human trimeric neck þ CRD domains can protect mice against a lethal challenge of this organism. These molecules can also decrease eosinophilia and other allergic manifestations of fungal hypersensitivity in mice. Although the levels of SP-D can be increased or decreased at various time points following allergenic challenge, it is possible that decreased SP-D-dependent clearance of fungi contributes to hypersensitivity to fungal antigens and the pathogenesis of allergic bronchopulmonary aspergillosis (ABPA). Bacterial pneumonia SP-D binds to cell wall glycoconjugates of most classes of bacterial organisms and can enhance uptake and killing of some of these microorganisms in vitro. Although decreased uptake of certain bacteria was observed in SP-D-deficient mice, there is still little evidence for a protective role in vivo. Gram-negative capsular polysaccharides are not usually recognized by SP-D. As a result, interactions of SP-D with typically encapsulated pathogens are largely restricted to unencapsulated phase variants. Thus, its protective effects might be restricted to very specific stages of the infectious process. Furthermore, interactions with unencapsulated Gram-negative bacteria are highly dependent on the structure and length of the O-antigen domain of LPS. Epidemiological studies indicate that O-serotypes of Klebsiella that express nonreactive O-antigen are more frequently associated with pneumonia. Emphysema Smoking is associated with a reduction in lavage SP-D and SP-D-deficient mice develop emphysema. Thus, possible contributions of SP-D to emphysema merit careful examination. Abnormalities in the clearance of apoptotic cells might contribute to disease pathogenesis. See also: Acute Respiratory Distress Syndrome. Allergy: Allergic Reactions. Antiviral Agents. Apoptosis. Asthma: Overview; Allergic Bronchopulmonary

Aspergillosis. Bronchoalveolar Lavage. Chronic Obstructive Pulmonary Disease: Emphysema, General. Clara Cells. Collectins. Cystic Fibrosis: Overview. Dendritic Cells. Dust Mite. Endotoxins. Epithelial Cells: Type II Cells. Gene Regulation. Leukocytes: Neutrophils; Pulmonary Macrophages. Matrix Metalloproteinases. Oxidants and Antioxidants: Oxidants. Pneumonia: Overview and Epidemiology; Atypical; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens); Nosocomial; Parasitic; Mycobacterial; Viral; The Immunocompromised Host. Serine Proteinases. Surfactant: Overview; Surfactant Protein A (SP-A); Surfactant Protein D (SPD). Transcription Factors: AP-1. Transgenic Models.

Further Reading Clark H and Reid KB (2002) Structural requirements for SP-D function in vitro and in vivo: therapeutic potential of recombinant SP-D. Immunobiology 205: 619–631. Crouch E and Wright JR (2001) Surfactant proteins A and D and pulmonary host defense. Annual Review of Physiology 63: 521– 554. Crouch EC and Whitsett JA (2002) Diverse roles of lung collectins in pulmonary innate immunity. In: Ezekowitz AR and Hoffman JA (eds.) Innate Immunity, pp. 205–229. Rotowa, NJ: Humana Press. Gardai SJ, Xiao Q, Dickinson M, et al. (2003) By binding SIRPalpha or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation. Cell 115: 13–23. Hawgood S and Poulain FR (2001) The pulmonary collectins and surfactant metabolism. Annual Review of Physiology 63: 495–519. LeVine AM, Whitsett JA, Hartshorn KL, Crouch EC, and Korfhagen TR (2001) Surfactant protein D enhances clearance of influenza A virus from the lung in vivo. Journal of Immunology 167: 5868–5873. Madsen J, Kliem A, Tornoe I, et al. (2000) Localization of lung surfactant protein D (SP-D) on mucosal surfaces in human tissues. Journal of Immunology 164: 5866–5870. Sano H and Kuroki Y (2005) The lung collectins, SP-A and SP-D, modulate pulmonary innate immunity. Molecular Immunology 42: 279–287. Shrive AK, Tharia HA, Strong P, et al. (2003) High-resolution structural insights into ligand binding and immune cell recognition by human lung surfactant protein D. Journal of Molecular Biology 331: 509–523. Zhang L, Ikegami M, Dey CR, Korfhagen TR, and Whitsett JA (2002) Reversibility of pulmonary abnormalities by conditional replacement of surfactant protein D (SP-D) in vivo. Journal of Biological Chemistry 277: 38709–38713.

Glossary Carbohydrate recognition domain (CRD) – the carbohydrate binding domain of a lectin Collectin – a term used to describe C-type lectins that contain one or more collagenous domains C-type lectin – a subtype of mammalian lectin characterized by the presence of a C-type, usually calcium-dependent, sugar binding domain

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Dodecamer – a common mature form of surfactant protein D that is characterized by four trimeric subunits linked at their amino termini Pattern recognition molecule (PRM) – a molecule that can recognized pathogen-associated molecular patterns Phosphatidylinositol – a minor phospholipid component of pulmonary surfactant and various membranes. PI is the major surfactant-associated ligand of SP-D

Surfactant-associated proteins – proteins associated with the lipid components of pulmonary surfactant. This includes two groups of proteins (1) hydrophobic proteins, SP-B and SP-C, and (2) the hydrophilic proteins, SP-A and SP-D. Although all four proteins can be isolated in association with surfactant, only SP-B is required for surfactant function in vivo Trimer – the minimum functional subunit of a collectin molecule. The association of three monomers is maintained by the contiguous triple helical collagen domain and a trimeric neck domain

SURGERY Contents

Pectus Carinatum, Poland’s Syndrome, Cleft Sternum, and Acquired Restrictive Thoracic Dystrophy Lung Volume Reduction Surgery Transplantation

Pectus Carinatum, Poland’s Syndrome, Cleft Sternum, and Acquired Restrictive Thoracic Dystrophy F Robicsek and A A Fokin, Carolinas Medical Center, Charlotte, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract There are a variety of congenital and acquired deformities of the chest wall, including anomalies such as pectus carinatum, Poland’s syndrome, cleft sternum, and acquired restrictive thoracic dystrophy. All of these may cause respiratory impairment. Most are of congenital origin, manifest early in life, and progress during growth. Although most chest wall deformities occur sporadically, familial cases may also occur. The deformities may also be a part of a syndrome and/or connective tissue disorder. Contrary to congenital anomalies, acquired restrictive thoracic dystrophy occurs as a result of damage to the rib growth centers and/or owing to the suturing of perichondrium behind the sternum in young patients during repair of pectus excavatum. The severity of the respiratory impairment caused by these anomalies is related to the degree of the deformity and may be explained by a reduced thoracic volume, restricted mobility of the thorax, paradoxical movements of the chest wall and mediastinal structures, and concomitant cardiac abnormalities. The deformities may also leave the internal thoracic organs unprotected and may have significant psychologic implications. The treatment of anterior chest deformities consists primarily of timely surgical repair, which both corrects the deformity and normalizes further growth of the thoracic cage.

The most common deformities of the anterior chest wall are pectus excavatum, pectus carinatum, Poland’s syndrome, sternal clefts, and acquired restrictive thoracic dystrophy. All of these may cause respiratory impairment. This article describes the previously mentioned conditions except pectus excavatum (Figures 1 and 2), which is discussed separately.

Pectus Carinatum In pectus carinatum, there is an abnormal prominence of the sternum and/or adjacent costal cartilages. Second to pectus excavatum, it is the most common chest wall anomaly and accounts for approximately 5% of all anterior chest wall deformities. The condition is present in up to 1.7% of schoolage students. Males are affected two to four times more often than females. In most instances, pectus carinatum occurs sporadically; however, familial incidence has also been reported. Pectus carinatum may also be a part of a syndrome and/or connective tissue disorder. The mechanism is thought to be the overgrowth of the costal cartilages with secondary forward displacement of the sternum. Carinatum deformities may be divided into three main groups: keel chest, lateral pectus carinatum, and Pouter pigeon breast. Keel chest or chondrosternal prominence is the most common variety of pectus carinatum and is characterized by forward displacement of the lower third of the sternum with maximum prominence at the sternoxiphoidal junction.

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Figure 1 Normal chest: cross-sectional, lateral, and anterior views. The size of the thorax and the position of the heart and diaphragm are normal.

The condition is also known as pyramidal chest or sternal kyphosis. Sternal ossification in pectus carinatum is normal. The angle of Louis (the angle formed by the manubrium and the body of the sternum) approaches 180o. Most patients are usually asthenic. The deformity is also often associated with bilateral depressions of the lower costal cartilages, which may be fairly deep and may significantly reduce the thoracic volume (Figure 3). On lateral radiographs, the anteroposterior diameter of the chest is increased, and the retrosternal

Figure 2 Pectus excavatum: cross-sectional, lateral, and anterior views. Anterioposterior diameter is reduced. The heart is shifted to the left.

space is extended. The elongated cardiac silhouette is positioned in the mid-mediastinum. Lateral pectus carinatum is characterized by distinct unilateral protrusion of the costal cartilages. Often, there is concomitant rotation of the sternum toward the opposite side. This condition is always asymmetrical. Clinical Features

Discomfort during sleep in the prone position or irritation from local trauma to the protrusion are the

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substantially reduce the thoracic volume and lead to exercise intolerance. Decreased pulmonary reserve, caused by impaired respiratory movements of the thorax, may also increase residual air and reduce vital capacity. Respiratory insufficiency, associated with cor pulmonale, may be seen in patients with a combination of severe kyphosis and pectus carinatum. The protruding deformity, which may be difficult to hide under clothing, may have psychological consequences. In cases of lateral pectus carinatum, even moderate asymmetric prominence will make the chest unsightly. To conceal the protrusion, patients hold themselves slightly bent, which leads to an abnormal posture and further aggravates the deformity. Treatment

Figure 3 Pectus carinatum: cross-sectional, lateral, and anterior views. Protrusion at the xiphosternal junction. Lateral depressions reduce thoracic volume. The heart is located in mid-mediastinum.

most frequent patient complaints. Moderate dyspnea, fatigue, asthmatic breathing, and nondescript chest pain are present in approximately one-third of these patients. More severe respiratory symptoms are rare. Severe bilateral chest wall depression may

Various nonoperative methods, such as physiotherapy, plaster of paris, and external compression, have been applied but have proven uniformly ineffective. The aim of surgical intervention is multifold: to correct the unsightly deformity, allow normal growth of the thoracic cage, prevent or treat pulmonary dysfunction, and alleviate psychological problems. We prefer to perform surgery before children reach school age, thus preventing psychological trauma and pathological posture. Also, at an early age the surgical correction is both easier to perform and yields better results. The operation includes subperichondreal resection of the deformed costal cartilages. To ensure growth and regeneration, care is taken not to damage the growth plates at the junction of the cartilage and the bony rib. If the protrusion of the cartilages is asymmetrical, it is corrected by uneven bilateral resections. A transverse sternotomy is performed at the beginning of the abnormal forward curve. The sternum is bent at the level of the sternotomy, pressed posteriorly, and brought into a corrected position. In cases in which the sternum is elongated, the xiphoid is temporarily detached, 3 or 4 cm of the lower end of the sternum is resected, and then the xiphoid is reattached. The operation is completed by suturing the mobilized sternal attachments of the pectoralis muscles together presternally. In cases of lateral pectus carinatum, the skin incision is made directly over the protrusion, and the procedure is limited to subperichondrial resection of the deformed cartilages. An epidural catheter may be placed preoperatively with its tip positioned at the level of the second or third thoracic vertebra. Epidural analgesia not only effectively reduces the pain but also lowers the necessary dosage of medication. Pectus carinatum is often associated with kyphosis. In such cases, the surgery should be planned

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jointly by thoracic and orthopedic surgeons. In general, simultaneous operations on the anterior chest wall and the spine should be avoided.

Pouter Pigeon Breast Pouter pigeon breast is characterized by a protrusion of the manubriosternal junction and the adjacent ribs and is accompanied by premature sternal ossification. The condition is also known as arcuate pectus carinatum, chondromanubrial prominence with chondrosternal depression, and Currarino–Silverman syndrome. Most often, it occurs sporadically; however, familial cases have also been reported. It may also be a part of a syndrome such as Noonan’s or Turner’s. In our experience, the deformity was evenly distributed among males and females. Clinical Features

The main clinical feature of this deformity is the protrusion of the junction of the manubrium and the sternal body with a reduction in the angle of Louis. The normal angle of Louis is between 1751 and 1451, although in severe cases it may be decreased to as little as 1101. The adjacent costal cartilages, usually from the second to the fifth, also protrude. All patients with pouter pigeon breast, regardless of age, have a completely ossified manubriosternal joint, normally presenting as synchondrosis (Figure 4). Fusion of the sutures between the four sternal segments also occurs prematurely and may be confirmed by lateral radiographs. Depression of the lower third of the sternal body is present in approximately one-third of patients, and for this reason it may be mistaken for pectus excavatum, despite being noticeably different in other aspects. The condition is usually symmetrical. Several cardiovascular abnormalities are associated with premature sternal ossification, the most common being ventricular septal defect. The incidence of cardiac disease in patients with pouter pigeon breast and abnormal ossification at the sternum is estimated to be 18–55%. This makes the search for occult cardiac lesions advisable in all patients with this deformity. Individuals with pouter pigeon breast exhibit restricted compliance of the ribcage and the manubriosternal junction, which are thought to be physiologically necessary for optimal respiratory function. The entire chest may appear to be restrained in the position of inspiration. Due to the appearance and particularly high location of this deformity on the anterior chest wall, patients are reluctant to wear a deep de´colletage.

Figure 4 Pouter pigeon breast: cross-sectional, lateral, and anterior views. Protrusion at the angle of Louis. Abnormal ossification of the sternum.

Treatment

When planning the timing of surgical correction, the following should be considered: the degree of the deformity, the existence of depression of the lower part of the sternum, yearly progression of the deformity, associated cardiac and respiratory abnormalities, and whether or not the deformity is part of a syndrome. Our preferred age for repair is between the ages of 5 and 7 years.

SURGERY / Congenital and Acquired Deformities 163

If the deformity consists only of protrusion at the angle of Louis, the operation may be limited to ‘shaving off’ the prominent manubriosternal junction and resecting subperichondrially the second and third costal cartilages. Depression of the sternal body, if present, requires additional intervention. In these cases, the sternum should be freed by resecting the costal cartilages bilaterally, detaching the intercostal bundles, and dissecting the loose mediastinal tissues posteriorly. A transverse wedge osteotomy is then performed at the angle of Louis and at the level of the mesosternal depression. The axis of the sternum is corrected by breaking it at both osteotomy sites. The corrected position is then supported using a ‘hammock’ of Marlex mesh placed under the sternum with its edges sutured to the ends of the resected costal cartilages and to the xiphoid with nonabsorbable sutures. The pectoralis muscles are united presternally.

Poland’s Syndrome Poland’s syndrome or unilateral pectoral aplasia– dysdactylia syndrome is a rare congenital anomaly characterized by unilateral brachysyndactyly; hypoplasia or the absence of the breast and/or nipple; hypoplasia of the subcutaneous tissue; the absence of the costosternal portion of the pectoralis major muscle; lack of the pectoralis minor muscle; aplasia or deformity of the costal cartilages or ribs 2, 3, and 4 or 3, 4, and 5; and alopecia of the axillary and mammary region (Figure 5). The degree and extent of various components of the syndrome vary significantly, and rarely does a single individual manifest all its features. The anomaly was first described in 1826 by L M Lallemand and in 1841 by Alfred Poland. The estimated incidence of Poland’s syndrome is 1 in 30 000 live births. Males are affected more frequently than females, by 3:1 or 2:1. The right side of the body is involved in 60–75% of cases. Poland’s syndrome is a nongenetic congenital abnormality with a low risk of recurrence (o1%) in the same family. Familial transmission has been reported in approximately 25 cases and may be due to an autosomal dominant gene with low penetrance. The pathogenesis of Poland’s syndrome remains inconclusive. The dominant view is that it is caused by disturbed subclavian artery blood supply. Hypoplasia of the internal thoracic artery causes the absence of the sternocostal portion of the pectoralis major muscle, whereas hypoplasia of branches of the brachial artery will lead to hand abnormalities. Clinical Features

Because the main feature of Poland’s syndrome is the absence of the pectoralis muscles combined

Figure 5 Poland’s syndrome: cross-sectional, lateral, and anterior views. Deficiency of the muscles and ribs, and hand malformation.

sometimes with hypoplasia of the latissimus dorsi, external oblique, or serratus anterior muscles, one may expect some degree of functional impairment of the shoulder. However, this seldom occurs. Ipsilateral depression of the chest wall is caused by hypoplasia of the second to fourth or third to fifth ribs. Aplasia of the anterior portions of one to three ribs with more severe lateral depression may occur in up to 11% of patients. The sternum may rotate toward the involved side. Breast involvement ranges from mild hypoplasia to complete absence (amastia).

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Dextrocardia in patients with left-sided Poland’s syndrome has been documented in 19 patients and was combined with rib defects but was not associated with cardiovascular anomalies. Lung herniation occurs in approximately 8% of cases. Conventional anteroposterior radiography usually demonstrates a unilateral hyperlucent lung. Other causes of respiratory impairment observed in patients with Poland’s syndrome include bullous emphysema, spontaneous pneumothorax, and congenital diaphragmatic hernia. Hand involvement has been reported in 2.5–56% of patients and varies from mild shortness of the middle phalanges with cutaneous webbing to the complete absence of the hand. In turn, in patients with syndactyly, approximately 10% have Poland’s syndrome. There are well-known associations between Poland’s syndrome and Mo¨bius syndrome, Sprengel’s deformity, Klippel–Feil syndrome, and unilateral renal anomalies. Poland’s syndrome may also be associated with leukemia, non-Hodgkin’s lymphoma, leiomyosarcoma, and cervical cancer. Invasive ductal carcinoma in the hypoplastic breast has also been reported; therefore, patients with Poland’s syndrome should be carefully monitored for early detection of breast cancer.

Cleft Sternum Cleft sternum, also known as bifid sternum or sternal fissure, is a partial or complete separation of the sternal halves (Figure 6). It is caused by failed ventral midline fusion of the sternal bars, which occurs during the first 3 months of gestation. Females with cleft sternum outnumber males, especially in cases in which the cleft is accompanied by a combination of supraumbilical raphe and facial hemangiomas.

Treatment

Surgical intervention in Poland’s syndrome is indicated for the following reasons: the possibility of progression of the ipsilateral concave chest deformity, lack of adequate protection of the heart and lung, paradoxical movement of the chest wall, and aplasia or hypoplasia of the breast and pectoral muscles. In patients with aplasia of the ribs and lung hernia, unilateral ventilation of the opposite lung utilizing a double-lumen endotracheal tube is recommended to prevent pulmonary injury during surgical repair. The surgical treatment of Poland’s syndrome, depending on the age and sex of the patient, may be carried out in either one or two stages. It consists of stabilization and/or reconstruction of the chest wall with simultaneous augmentation mammoplasty in females. The latter involves insertion of a breast prosthesis beneath a pedicled musculocutaneous flap of the latissimus dorsi. In adults, the correction is usually done in a single stage, whereas in children the procedure is usually carried out in two steps. First, the depression of the chest is corrected using bone grafts and/or a synthetic mesh patch. Later, in adulthood, a second procedure consisting of latissimus dorsi muscle transposition and/or mammoplasty is carried out if necessary.

Figure 6 Cleft sternum: cross-sectional, lateral, and anterior views. Protrusion of the unprotected heart through defect in the sternal bone.

SURGERY / Congenital and Acquired Deformities 165 Clinical Features

Most often, the anomaly presents as a concave defect of the sternum. The defect paradoxically deepens upon inspiration and bulges with expiration, coughing, or the Valsalva maneuver. The shape of the defect varies from a narrow ‘U’ to a broad ‘V’, with the intraclavicular distance being 2–6 cm. In more severe cases, the pulsation of the heart is seen through the thin and sometimes ulcerated skin. Herniation of the lung may occur at the upper edge of the defect. A midline, pigmented raphe reaching from the xiphoid process to the navel is common. The presence of an omphalocele or an umbilical hernia, as well as cardiac anomalies such as ventricular septal defect or tetralogy of Fallot, may also complicate sternal clefts. Often, there is an association with craniofacial hemangiomas. There are four main types of sternal clefts. Superior sternal cleft, the most common type of this malformation, involves the manubrium and the upper part of the sternum and usually extends down to the fourth intercostal space. The position of the heart is normal, and the pericardium, pleura, and diaphragm are intact. Subtotal sternal cleft includes the manubrium and most of the sternal body, leaving only a narrow cartilaginous bridge at the xiphoid process. Inferior sternal cleft usually occurs in Cantrell’s pentalogy, which also includes an omphalocele or omphalocele-like abdominal defect, an anterior diaphragmatic defect, and pericardial–peritoneal communication. Cantrell’s pentalogy is also often associated with various intracardiac anomalies, such as atrial or ventricular septal defects, tetralogy of Fallot, or left ventricular diverticulum. In cases of total sternal cleft, the two halves are completely separated. Wide diastasis of the rectus abdominis muscle is common. Clinical symptoms of sternal clefts may be induced by abnormal changes in intrathoracic pressure, displacement of the cardiovascular structures, and impaired venous return. Dyspnea, cyanosis, arrhythmias, and other circulatory difficulties may be present. A chest wall defect may also lead to reduced air exchange and a loss of cough strength. Tracheal hemangioma may cause bleeding during endotracheal intubation. Diagnosis of sternal cleft is usually made at birth. Prenatal diagnosis is also feasible in the last weeks of gestation by ultrasonography. Establishment of the diagnosis should initiate a thorough search for associated cardiovascular anomalies as well as other midline structure malformations. In children with faciocutaneous vascular lesions, an angiographic examination of the vertebrobasilar system is

recommended. Congenital aortic arch abnormalities (aneurysm and coarctation) should be ruled out, especially in patients with hemangiomatosis and supraumbilical raphe. On chest radiograms, superior mediastinal widening with an increased distance between sternal ends of the clavicles is noticeable. Treatment

Surgical intervention for sternal clefts is indicated by a lack of bony protection that makes the heart and great vessels vulnerable. Paradoxical respiratory movements of the chest may induce dyspnea and predispose to recurrent respiratory infections. Impairment of the venous return affects cardiac function. The presence of a dermopericardial connection may lead to pericardial infection. The appearance of a protruding heart is frightening for both parents and patients. The possibility of enlargement of the defect over time will make its correction more difficult. In surgical planning, the age of the patient, the type of the defect, and any accompanying anomalies of the heart should be considered. Operative correction should preferably be carried out during the neonatal period, when the sternal halves are elastic and may be sutured together without tension and the thoracic cavity will likely accommodate the viscera. The presence of ulcerated and/or infected skin or a skin-topericardium sinus should hasten the time of surgical intervention. Simultaneous repair of cardiac and aortic malformations is feasible. At an older age, surgical repair is more difficult because the chest wall becomes rigid and requires complex methods of correction. In the course of surgical repair, the midline raphe or ulcerated skin is excised. The pericardium is separated from the skin and the sternal bands. The periosteum is incised on the anterior aspect of the sternal halves, turned inward, and sutured together. Notching of the sternal bars or a V-shaped sternotomy may ease their alignment. In older patients, oblique sliding chondrotomies may be performed to further facilitate approximation. If the cephalad diastasis is wide, it may require the detachment and mobilization of the sternoclavicular junctions. The sternal bars are then united with nonabsorbable sutures. Uniting the sternothyroid, sternohyoid, and sternocleidomastoid muscles from both sides at the base of the neck will prevent lung herniation. In cases of subtotal sternal clefts, the cartilaginous bridge holding the halves apart should be resected before the halves are united. In cases of total cleft, the trimmed edges of the sternal halves are sutured together. Existing umbilical hernias and rectus muscle diastases should be

166 SURGERY / Congenital and Acquired Deformities

repaired simultaneously. Reconstruction of the defect, especially if it is wide or in adults, may require an autologous bone graft or the application of a synthetic mesh patch. The first postoperative day is most critical due to the possibility of compression of the heart caused by acute reduction of the mediastinal space. This makes careful monitoring of the patient’s cardiorespiratory status mandatory.

Acquired Restrictive Thoracic Dystrophy Acquired restrictive thoracic dystrophy (ARTD), also called acquired Jeune’s syndrome or restrictive lung disease, is caused by an inappropriate technique applied in the course of pectus excavatum repair. The condition develops after complete removal of the growth centers of the ribs (located at the costochondral junctions) during the repair of congenital pectus excavatum in 3- and 4-year-old patients. Another factor may be the substernal suturing of the lower ribs for posterior sternal support during surgical correction of pectus excavatum. Both factors contribute to the reduced growth capacity of the thoracic cage in a young patient. Clinical Features

Patients with ARTD have an extremely narrow torso and an immobile, deformed chest (Figure 7). Consequently, the dynamics of the thoracic movements are substantially impaired. The patients have dyspnea and limited exercise tolerance of various degrees. Pulmonary function studies confirm restrictive pulmonary disorder with a decrease in both vital capacity and forced expiratory volume. Computed tomography scans usually reveal osseous or cartilaginous growth behind the sternum. Patients with ARTD, due to an obvious reduction in thorax dimensions, have severe physiological consequences that increase with age. Treatment

The key to the management of ARTD is not treatment but prophylaxis. Excessive costal cartilage extirpation during the original pectus excavatum repair should never be done. The synovial sternocostal joints should also be preserved to reduce postoperative morbidity and to maintain anterior thoracic wall mobility. Suturing of the perichondrium behind the sternum for posterior support should be avoided in young patients. Attempts of surgical palliation of ARTD, in addition to the correction of a recurrent concavity, have included elaborate interventions designed to elevate

Figure 7 Acquired restrictive thoracic dystrophy: cross-sectional, lateral, and anterior views. Reduced, restricted, and deformed thorax. Cartilaginous tissue behind the sternum. Lowlying diaphragm.

the sternum and expand the thorax with the help of implanted metal or bone struts. See also: Chest Wall Abnormalities. Pectus Excavatum. Respiratory Muscles, Chest Wall, Diaphragm, and Other.

Further Reading Bavinck JNB and Weaver DD (1986) Subclavian artery supply disruption sequence: hypothesis of a vascular etiology for

SURGERY / Lung Volume Reduction Surgery 167 Poland, Klippel-Feil, and Mo¨bius anomalies. American Journal of Medical Genetics 23: 903–918. Bove´ T, Goldstein JP, Viart P, and Duevaert RT (1997) Combined repair of upper sternal cleft and tetralogy of Fallot in an infant. Annals of Thoracic Surgery 64: 561–562. Currarino G and Silverman FN (1958) Premature obliteration of the sternal sutures and pigeon breast deformity. Radiology 79: 532–540. Fokin AA (2000a) Pouter pigeon breast. Chest Surgery Clinics of North America 10(2): 377–391. Fokin AA (2000b) Cleft sternum and sternal foramen. Chest Surgery Clinics of North America 10(2): 261–276. Fokin AA and Robicsek F (2002) Poland’s syndrome revisited. Annals of Thoracic Surgery 74(6): 2218–2225. Fonkalsrud EW (2004) Open repair of pectus excavatum with minimal cartilage resection. Annals of Surgery 240(2): 231–235. Haller JA Jr, Colombani PM, Humphries CT, et al. (1996) Chest wall constriction after too extensive and too early operations for pectus excavatum. Annals of Thoracic Surgery 61(6): 1618–1624. Ravitch MM (1977) Congenital Deformities of the Chest Wall and Their Operative Correction. Philadelphia: Saunders. Robicsek F (2000) Surgical treatment of pectus carinatum. Chest Surgery Clinics of North America 10(2): 357–376. Robicsek F and Fokin AA (1999) Surgical correction of pectus excavatum and carinatum. Journal of Cardiovascular Surgery 40(5): 725–731. Robicsek F and Fokin AA (2004) How not to do it: restrictive thoracic dystrophy after pectus excavatum repair. Interactive Cardiovascular and Thoracic Surgery 3(4): 566–568. Robicsek F, Sanger PW, Taylor FH, and Thomas MJ (1963) The surgical treatment of chondrosternal prominence (pectus carinatum). Journal of Thoracic and Cardiovascular Surgery 45: 691–701. Shamberger RC (2000) Cardiopulmonary effects of anterior chest wall deformities. Chest Surgery Clinics of North America 10(2): 245–252. Shamberger RC and Hendren WH III (2002) Congenital deformities of the chest wall and sternum. In: Pearson FG, Cooper JD, Deslauriers J, et al. (eds.) Thoracic Surgery, pp. 1351–1373. Philadelphia: Churchill Livingstone.

Lung Volume Reduction Surgery S J Mentzer, Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract In appropriately selected patients, surgery can play a useful role in the treatment of patients with emphysema. The removal of poorly functioning lung tissue – a procedure commonly referred to as lung volume reduction surgery (LVRS) – can result in a significant improvement in perceived shortness of breath and quality of life as well as objective measures of expiratory flow and exercise tolerance.

History The potential utility of removing poorly functioning lung tissue has been recognized for more than 50

years. Nissen treated giant bullous lung disease by excising the bulla and surrounding lung tissue. Monaldi decompressed tuberculosis cavities with the use of intracavitary drainage and suction. This technique was later modified by Avery and Venn to treat bullous emphysema. More recent attempts to treat bullous disease of the lung have used minimally invasive techniques to remove poorly functioning lung tissue. In all of these approaches, the goal of surgery is to improve lung function by recruiting previously compressed or underinflated portions of the lung. In the 1950s, Brantigan expanded the indications for surgical resection to patients with generalized emphysema. He suggested that emphysema created a condition in which 7 l of lung was stuffed into a 5 l chest cavity. By tailoring the lung volume to the chest cavity, Brantigan argued that airways would be tethered open and expiratory flows would improve. Despite evidence that respiratory mechanics were improved, the perioperative mortality of 16% was heavily criticized. In 1994, Cooper reintroduced Brantigan’s concept of lung volume reduction surgery (LVRS) in a report of 120 patients with heterogeneous emphysema. Cooper et al. had a 30 day mortality rate of 2.5% with significant improvements in subjective and objective measures of respiratory function. To evaluate the role of surgery in emphysema therapy, the National Emphysema Treatment Trial (NETT), supported by the National Heart, Lung, and Blood Institute (NHLBI) and the Center for Medicare and Medicaid Service (CMS), supported the first multicenter clinical trial. NETT was designed to determine the role, safety, and effectiveness of bilateral LVRS in the treatment of emphysema. Between January 1998 and July 2002, 3777 patients were evaluated and 1218 were selected for the trial. All participants initially received 6–10 weeks of medical therapy, including education, counseling, and exercise training. After this the patients were randomized to either surgical LVRS or maintenance medical therapy. A preliminary result of the NETT, published in 2001, identified a group of patients with a significant early mortality. These high-risk patients were characterized by a low forced expiratory volume in 1 s (FEV1) (o20% predicted) and either a homogeneous distribution of the emphysema on chest computed tomography (CT) scanning or a low carbon monoxide diffusion capacity (DLCO) (o20% predicted). The remainder of the clinical trial excluded this high-risk group. In 2003, the first full NETT results were reported. The effects of LVRS on the many follow-up measures varied widely. The overall NETT study population demonstrated no significant mortality benefit for LVRS over medical therapy. In contrast, more

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patients in the surgical group than in the medical group improved in function, symptoms, and quality of life. For example, 15% of those treated with surgery had more than a 10 W improvement in exercise capacity at 2 years compared to 3% for those treated with medical therapy alone. The non-high-risk survivors in the surgery group initially improved their scores (e.g., spirometry, exercise capacity, quality of life, and dyspnea); however, over the 2 years of follow-up, the elevated average scores gradually declined to nearly pre-LVRS values. In contrast, the functional ability in participants who received medical therapy without surgery consistently worsened over 2 years with functional levels falling below the levels documented prior to randomization. These results not only highlighted the progressive functional decline of patients with emphysema, but underscored the importance of a nonsurgical control group in therapeutic trials. A controversial result of the NETT was derived from subgroup analysis. Because the analysis was not specified at the outset of the trial, the validity of such an analysis is questionable; nonetheless, the original NETT report identified several factors with potential benefit for LVRS. First, patients with an apical predominance of their emphysema appeared to have a functional benefit from LVRS. Second, patients with low exercise capacity appeared to have not only a functional benefit, but a mortality benefit from LVRS. Moreover, the two risk factors were additive; that is, patients with apical predominant emphysema and low exercise capacity demonstrated an improvement in both function and mortality after LVRS.

Uses in Respiratory Medicine LVRS is based on surgical principles that can be applied to a continuum of disease from a single giant bulla to relatively diffuse emphysema. Patients with bullae can be classified into those patients with normal underlying lungs and those patients with structurally abnormal lungs. In patients with relatively normal underlying lungs, the bullae become the dominant focus of therapy. Bullous Disease

Bullae are air-filled spaces within the lung parenchyma that form as a result of the destruction of alveolar tissue. The bulla often reflects the trabecular architecture of the destroyed alveolar septa. Distinguished from bullae, blebs are subpleural collections of air within layers of the visceral pleura. Blebs appear to develop as a result of alveolar rupture. Blebs are most commonly found at the apex of the lung and are the commonest cause of spontaneous pneumothorax.

The presentation of patients with a giant bulla can vary from a completely asymptomatic radiographic finding to rapidly progressive shortness of breath. Patients with rapid symptomatic progression will often have coincident enlargement of a bulla. Rarely, the rapid expansion of the bulla may produce significant lung compression with secondary right heart failure. Bulla-induced right heart failure is the only semiemergent indication for bullectomy. More commonly, patients with a giant bulla have only mild shortness of breath. In this setting, the primary indication for bullectomy is the presence of compressed or recruitable lung function. The presence of potentially recruitable lung function can be radiographically identified by a mediastinal shift or ipsilateral atelectasis. Historically, angiography has been used to document vascular compression. More recently, chest CT scan evidence of parenchymal compression combined with even modest limitation of expiratory air flow would be an indication for a minimally invasive surgical bullectomy. Using current surgical techniques, a routine thoracoscopic bullectomy can be performed with an operative mortality less than 1% and a hospital stay of only a few days. Secondary indications for bullectomy include an elevated risk of pneumothorax and lung abscess. The risk of pneumothorax is dependent upon a variety of structural and functional properties of the bulla that are difficult to assess radiographically. In general, the risk of pneumothorax appears to increase in patients with bullous lung disease, and significantly increases in patients with a previous pneumothorax. A history of intense pleuritis also may be consistent with a previous subclinical pneumothorax. Frequent air travel is also a relative indication for bullectomy. Patients with a bulla of any size should not scuba dive. Similarly, the risk of lung abscess is difficult to assess prior to the first episode of infection. When a lung abscess has developed, the treatment is percutaneous drainage of the abscess, antibiotic treatment of the underlying infection, and improved bronchial toilet. When the lung abscess has been completely drained and the compliance of the surrounding lung has improved, elective bullectomy is indicated to prevent recurrence. The resolution of the surrounding pneumonitis, however, may take several months. Emphysema

The American Thoracic Society definition of emphysema is ‘‘alveolar wall destruction with irreversible enlargement of the air spaces distal to the terminal bronchioles and without evidence of fibrosis.’’ Similar to isolated bullous disease, heterogeneity in parenchymal destruction can lead to compression or underinflation of

SURGERY / Lung Volume Reduction Surgery 169 Table 1 Guidelinesa for LVRS History and physical examination

Radiographic

Pulmonary function testing

Arterial blood gas level Exercise a

History and examination consistent with emphysema No smoking for at least 4 months p10 mg day 1 prednisone (or equivalent) Chest CT scan evidence of bilateral emphysema with apical predominance Forced expiratory volume in 1 s (FEV1)415% and p45% predicted Total lung capacity (TLC)X100% predicted Residual volume (RV)X150% predicted carbon monoxide diffusion capacity (DLCO)420% predicted PCO2 p60 mmHg PO2 X45 mmHg on room air Postrehabilitation 6-min walk of X200 m

Relative guidelines based on the NETT and available trials.

other portions of the lung. The removal of these poorly functioning regions may result in an improvement in overall respiratory function. The clinical characteristics of patients likely to benefit from LVRS have been empirically defined (Table 1). A poorly understood observation is that the response to LVRS depends upon the location of the heterogeneity. The patients with apical predominance of their emphysema generally have a greater sustained improvement in FEV1 than patients with basilar emphysema. Most patients with predominant emphysema at the base of their lung have alpha-1 antitrypsin deficiency. Whether this different response to LVRS is due to the geometric differences between the apex and the base of the lung, or structurally distinct parenchyma as a result of the alpha-1 antitrypsin deficiency, is unknown. Emphysema patients who have functional disability sufficiently severe to consider LVRS typically have FEV1 measurements of 20–45% of predicted values. The expiratory airflow obstruction measured by conventional spirometry, however, can reflect either floppy or narrowed small airways. The destroyed and floppy airways might be expected to benefit from the tethering effect of LVRS. In contrast, the fixed and narrowed small airways would likely be limiting regardless of the potential tethering effect of LVRS. Consistent with small airway limitation, it has been shown that high inspiratory resistance is a negative predictor for LVRS. Although the measurement of inspiratory resistance may be impractical in many settings, an elevated inspiratory resistance tends to be correlated with clinical characteristics such as frequent exacerbations,

productive sputum, and the inability to be weaned from steroid therapy. Measurement of DLCO may also help identify patients unlikely to benefit from LVRS. An unexpectedly high DLCO may suggest that small airways disease is a greater limitation than is the destruction of alveolar–capillary units. Because of functional limitations associated with emphysema, preoperative assessment of cardiac function is challenging. Routine transthoracic echocardiography is useful to assess left-ventricular ejection fraction and pulmonary artery pressures. If the lung hyperinflation precludes adequate transthoracic visualization of the heart, a dobutamine–radionuclide cardiac scan may be useful to exclude ischemic heart disease. Right heart catheterization may be necessary to exclude pulmonary hypertension. Although numeric guidelines for the upper limit of acceptable pulmonary artery pressures remain elusive, most assessments of pulmonary artery pressure underestimate exercise-associated pulmonary hypertension. For this practical reason, any indication of pulmonary hypertension should be considered a relative contraindication to LVRS. A ventilation/perfusion (V/Q) scan may be useful to identify areas of hypoperfusion and poor lung function. The V/Q scan generally correlates with chest CT scan destruction of the lung parenchyma. Preoperative pulmonary rehabilitation is a critical component of surgical outcomes. The major mortality risk after LVRS is pneumonia. Positive pressure ventilation, especially after differential lung ventilation, results in regional microatelectasis. The trauma of surgery decreases lung compliance, further compromising lung ventilation. In addition, chest wall movements are often limited because of pain and chest tubes. To offset these inevitable consequences of LVRS, sustained ambulation in the early postoperative period is necessary to increase lung volumes, improve airway clearance, and minimize the risk of pneumonia. In addition to the physical benefits, pulmonary rehabilitation is an opportunity to educate patients about their expectations for LVRS. It is particularly useful to remind patients that supplemental oxygen is typically required after LVRS and that an ongoing exercise program is important to prolong the functional benefits of the surgery.

Surgical Technique All patients undergoing LVRS should receive an epidural catheter for postoperative pain control, and also for intraoperative management. The regional effects of epidural analgesia facilitate the use of an intravenous anesthetic approach, avoiding the unpredictable clearance of inhalational anesthetics.

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LVRS requires the placement of a double lumen endotracheal tube for differential lung ventilation. Even patients with severe emphysema will tolerate single lung ventilation during the procedure provided they do not have pulmonary hypertension and dynamic hyperinflation is avoided. Because of diminished elastic recoil, emphysematous lungs are slow to deflate even without positive airway pressure. The rate of deflation can be used intraoperatively to distinguish areas of the lung with preserved elastic recoil from regions with more destroyed emphysematous parenchyma. LVRS can be performed thoracoscopically or through a median sternotomy. Although initially there were debates regarding the efficacy of the approaches, it is now clear that the two approaches produce comparable results. An advantage of thoracoscopy is that it provides the option of a unilateral procedure (Figure 1). The advantages of a unilateral procedure is that there is substantially less surgical trauma and moderately improved postoperative oxygenation. A potential disadvantage of a unilateral lung reduction is that a staged reduction requires a second general anesthetic and postoperative recovery.

Unilateral LVRS is a particularly appealing solution for patients with asymmetric disease. The lung can be reduced by a variety of surgical techniques. Laser reduction has largely been abandoned because of thermal injury to the lung and an unacceptable incidence of late pneumothoraces. Most LVRS procedures are currently being performed using staples reinforced with a synthetic or tissue buttress. In most patients with apical predominant emphysema, the destroyed and slow-deflating apical portions of the lung are resected. This typically comprises 50–60% of the upper lobe volume. The contour of the upper lobe is preserved to facilitate pleural apposition at the apex of the chest. The goal of the surgery is to sufficiently reduce the hypercompliant portions of the lung so that pleural pressures are decreased and the underinflated portions of the lung are recruited. Balancing this goal is the need to achieve pleural apposition and minimize pleural air collections. Pleural apposition is critical to controlling postoperative air-leaks. Chest tubes are typically placed at the apex and base of the chest to drain pleural air, facilitate lung expansion, and encourage pleural apposition.

Figure 1 Chest imaging performed prior to surgery (a, c) and 6 months after a right-sided LVRS procedure (b, d). Resection of the apical portions of the right upper lobe resulted in elevation of the right hemidiaphragm (b) and cephalad expansion of the right lung (d).

SURGERY / Transplantation

Alternatives to surgical LVRS have focused on endobronchial approaches to treating emphysema. There are currently three endobronchial treatment strategies. Two of these approaches are designed to reduce lung volumes. Endobronchial valves are oneway valves placed in the airway to encourage distal atelectasis. Another endobronchial lung reduction approach involves the instillation of bioactive agents into target regions of the lung. The bioactive agents induce atelectasis and subsequent scarring to prevent re-expansion. A conceptually different approach is based upon the construction of endobronchial holes that connect the segmental airways to the more destroyed portions of the lung. The endobronchial holes are created by balloon dilatation of the intervening airway wall and lung parenchyma followed by placement of a deployable stent. Multiple stents are placed to bypass obstructed air passages and improve expiratory flow. Clinical trials are currently under way to assess the potential value of these endobronchial treatment strategies. See also: Atelectasis. Chronic Obstructive Pulmonary Disease: Emphysema, General. Lung Abscess. Pneumothorax. Symptoms of Respiratory Disease: Dyspnea.

Further Reading Brantigan OC, Mueller E, and Kress MB (1959) A surgical approach to pulmonary emphysema. American Review of Respiratory Disease 80: 194–206. Brewer KK, Sakai H, Alencar AM, et al. (2003) Lung and alveolar wall elastic and hysteretic behavior in rats: effects of in vivo elastase treatment. Journal of Applied Physiology 95(5): 1926–1936. Cooper JD, Trulock EP, Triantafillou AN, et al. (1995) Bilateral pneumectomy (volume reduction) for chronic obstructive pulmonary disease. Journal of Thoracic and Cardiovascular Surgery 109: 106–116. Drazen JM and Epstein AM (2003) Guidance concerning surgery for emphysema. New England Journal of Medicine 348: 2134–2136. Fishman A, Martinez F, Naunheim K, et al. (2003) A randomized trial comparing lung-volume-reduction surgery with medical therapy for severe emphysema. New England Journal of Medicine 348: 2059–2073. Head JR and Avery EF (1949) Intracavitary suction (Monaldi) in the treatment of emphysematous bullae and blebs. Journal of Thoracic Surgery 18: 761–776. Ingenito EP, Loring SH, Moy ML, et al. (2001) Interpreting improvement in expiratory flows after lung volume reduction surgery in terms of flow limitation theory. American Journal of Respiratory and Critical Care Medicine 163: 1074–1080. Keller CA (2003) Pathophysiology and classification of emphysema. Chest Surgery Clinics of North America 13(4): 589–613. Monaldi V (1947) Endocavitary aspiration: its practical applications. Tubercle 28: 223–228. Moy ML, Ingenito EP, Mentzer SJ, et al. (1999) Health-related quality of life improves following pulmonary rehabilitation and lung volume reduction surgery. Chest 115: 383–389.

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National Emphysema Treatment Trial (2001) Patients at high risk of death after lung-volume-reduction surgery. New England Journal of Medicine 345: 1075–1083. Nissen R (1945) Reconstructive operation of air cysts of lung. Rocky Mountain Medical Journal 28: 223–228. Ramsey SD, Berry K, Etzioni R, et al. (2003) Cost effectiveness of lung-volume-reduction surgery for patients with severe emphysema. New England Journal of Medicine 348: 2092–2102. Toma TP, Hopkinson NS, Polkey MI, and Geddes DM (2004) Endobronchial volume reduction: a myth or a marvel? Seminars in Respiratory and Critical Care Medicine 25: 399–404. Venn GE, Williams PR, and Goldstraw P (1988) Intracavity drainage for bullous, emphysematous lung disease: experience with the Brompton technique. Thorax 43(12): 998–1002.

Transplantation E P Trulock and R R Hachem, Washington University School of Medicine, St Louis, MO, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Lung transplantation has emerged over the past 20 years as a successful treatment option for a variety of end-stage lung diseases. The first human lung transplantation procedure was performed in 1963, but a limited number of procedures were carried out over the next 20 years because of poor outcomes. During this time period, the main complication was impaired bronchial anastomotic healing, and dehiscence was a common cause of death after the first postoperative week. In the early 1980s, when surgical techniques were refined and ciclosporin first became available, lung transplantation became a viable treatment option for carefully selected recipients suffering from a variety of end-stage lung diseases. Since then, activity has grown rapidly and, currently, approximately 1600 lung transplant procedures are performed annually worldwide. However, long-term results remain disappointing, and the 5-year survival rate is approximately 45%. Chronic rejection is the leading cause of death after the first year, and common complications including infections, hypertension, renal insufficiency, and diabetes mellitus that significantly impact the recipient’s quality of life. Nonetheless, in appropriately selected recipients, transplantation prolongs survival and improves quality of life.

History The first human lung transplantation procedure was performed in 1963, but the result was disappointing. The indication for the procedure was lung cancer obstructing the left mainstem bronchus resulting in chronic atelectasis and infection of the distal lung. The recipient was debilitated, malnourished, and suffered from glomerulonephritis, in addition to lung cancer, before transplantation, and died just 18 days after transplantation because of renal failure and

172 SURGERY / Transplantation

malnutrition. Nonetheless, this case demonstrated that lung transplantation was technically feasible and that the transplanted lung could function appropriately physiologically. Furthermore, the available immunosuppressives at the time, azathioprine and prednisolone, appeared sufficient to prevent rejection for a short period of time. However, over the ensuing 15 years, only approximately 40 transplants were performed, and the majority of recipients died perioperatively due to bronchial anastomotic complications. In 1981, heart–lung transplantation was performed successfully for patients with pulmonary vascular disease. The advent of ciclosporin and refinement in surgical techniques improved bronchial anastomotic healing, and in 1983, single lung transplantation was successfully performed for patients with pulmonary fibrosis. This was followed by successful double lung transplantation for patients with severe chronic obstructive pulmonary disease (COPD) in 1986. These successes led to a resurgence of lung transplantation as a viable treatment option for patients with a variety of end-stage lung diseases,

and activity grew rapidly in the late 1980s and early 1990s reaching a level of approximately 1600 procedures performed annually worldwide in recent years.

Uses in Respiratory Medicine Indications

The indications for lung transplantation have spanned the spectrum of end-stage lung diseases. COPD has been the most common indication for transplantation, accounting for 40% of procedures in recent years; other common indications include idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), emphysema due to alpha-1 antitrypsin deficiency, and primary pulmonary hypertension (PPH) (Figure 1). Less common indications include sarcoidosis, bronchiectasis, and lymphangioleiomyomatosis. Over the past decade, the percentage of transplant recipients with PPH has steadily declined from 13% in 1990 to 4.5% in 2002, as medical therapy improved survival and functional

Other 9% Sarcoidosis 3% Bronchiectasis 2% PH 5%

COPD 39%

A1E 9%

CF 16%

IPF 17% Figure 1 Indications for adult lung transplantation. COPD, chronic obstructive pulmonary disease; IPF, idiopathic pulmonary fibrosis; CF, cystic fibrosis; A1E, alpha-1 antitrypsin deficiency-associated emphysema; PH, pulmonary hypertension. Adapted from the International Society for Heart and Lung Transplantation Registry Report – 2003.

SURGERY / Transplantation

capacity. In contrast, the proportion of recipients with COPD and alpha-1 antitrypsin deficiency has increased from 21% to almost 50%, while the proportion of patients with IPF and CF undergoing transplantation has remained fairly stable over the past 10 years.

173

Table 1 General guidelines for recipient selection Severe clinical and physiological impairments Absence of significant improvement with optimal medical therapy Poor prognosis; life expectancy p2 years Ambulatory physical status Acceptable nutritional status Acceptable psychosocial profile and support system

Recipient Selection

The primary goal of transplantation is prolonging survival, but the prospect of improving the quality of life is often the motivation for many patients even if the survival advantage itself is marginal. Transplantation should be considered for patients with severe functional and physiological impairments and a poor prognosis despite optimal medical therapy in the absence of significant comorbidities. General guidelines for recipient selection are summarized in Table 1. Typically, appropriate candidates have severe lung disease but are otherwise in good health. Thus, absolute contraindications include significant extrapulmonary organ dysfunction (such as renal insufficiency, liver disease, and cardiac disease), acute critical illness, HIV infection, chronic viral hepatitis, malignancy (other than basal-cell or squamous-cell skin cancer), and an irresolvable psychiatric illness. Airway infection with Burkholderia cepacia has been a contraindication at some centers because the risk of serious infection with B. cepacia after transplantation has limited the long-term survival among patients with CF infected with this organism. In addition, because extrapulmonary comorbidities increase the risk of complications and are often exacerbated after transplantation, they constitute relative contraindications. Common problems include hypertension, diabetes mellitus, hyperlipidemia, obesity, and osteoporosis. Disease-specific guidelines for referral for transplantation are summarized in Table 2. These criteria are derived from prognostic data that are intended to identify patients with a poor prognosis over the next 2–3 years since the average waiting time has been in that range at most centers in the US. Under the current lung allocation system in the US, donor lungs are assigned to recipients based on position in the waiting list, after matching for body size and blood group compatibility. However, because of an increased mortality while waiting among certain patient groups, especially those with IPF and CF, the allocation system will change in 2005 such that medical urgency and expected outcome will become the primary determinants of donor organ allotment, after matching for body size and blood type. How this will affect referral patterns remains uncertain since early referral will become less important when

Table 2 Disease-specific guidelines for referral for lung transplantation Chronic obstructive pulmonary disease Postbronchodilator FEV1 o25% predicted PaCO2 X55 mmHg Pulmonary hypertension Idiopathic pulmonary fibrosis Vital capacity or total lung capacity o60–70% predicted Pulmonary hypertension Resting or exercise-induced hypoxemia Progressive disease despite medical therapy Cystic fibrosis FEV1 p30% predicted PaCO2 450 mmHg Pulmonary hypertension Rapidly declining lung function or clinical course, pulmonary cachexia Primary pulmonary hypertension New York Heart Association functional class III or IV Right atrial pressure 415 mmHg Mean pulmonary artery pressure 455 mmHg Cardiac index o2 l min
1 m
2 Failure of optimal medical therapy FEV1, forced expiratory volume in 1 s; PaCO2 , arterial partial pressure of carbon dioxide.

waiting time becomes an insignificant determinant of organ allocation. Transplant Procedure

Bilateral transplantation is necessary for patients with bronchiectasis because of the risk of spill-over infection from the native lung. While single lung transplantation was performed successfully for patients with PPH in the early days of lung transplantation, the postoperative course was often precarious and even mild complications in the allograft resulted in severe hypoxemia because of ventilation–perfusion mismatching. Thus, the majority of patients with PPH undergo bilateral transplantation in the current era. Severe right ventricular dysfunction associated with PPH does not necessitate heart–lung transplantation as right ventricular function recovers when pulmonary vascular resistance normalizes after transplantation. However, patients with Eisenmenger’s syndrome due to complex congenital cardiac anomalies that are not amenable to surgical

174 SURGERY / Transplantation

correction at the time of lung transplantation require heart–lung transplantation. Both bilateral and single-lung transplantation are acceptable alternatives for patients with COPD, alpha-1 antitrypsin deficiency, and IPF. However, bilateral transplantation provides more reserve lung function as a safeguard against future inevitable complications, and bilateral recipients have had superior long-term survival. Nonetheless, single-lung transplantation is a shorter operation with less perioperative complications than bilateral transplantation. Thus, it has been an attractive option for some recipients.

Post-Transplant Management and Complications Management

After transplantation, patients are maintained on an immunosuppression regimen consisting of a calcineurin inhibitor (ciclosporin or tacrolimus), a purine synthesis antagonist (azathioprine or mycophenolate mofetil), and corticosteroids. Since sirolimus has become available, it has been substituted for the purine synthesis antagonist occasionally and for the calcineurin inhibitor less often. Corticosteroid withdrawal has been rare; 5% of recipients reported to the International Society for Heart and Lung Transplantation Registry Report were managed with a corticosteroid-free regimen 5 years after transplantation. Overall, there is no consensus about the optimal immunosuppressive regimen, and the actual drug combinations are evenly divided among the available agents from each class. Prophylaxis for Pneumocystis carinii pneumonia is standard, and prophylaxis against cytomegalovirus (CMV) infection is used in many protocols depending on donor and recipient serostatus. Surveillance protocols differ among centers. Lab tests, chest X-ray, and spirometry are followed closely to detect complications early. The use of surveillance bronchoscopy with bronchoalveolar lavage and transbronchial biopsies to detect occult rejection has been controversial. While most centers adhere to a surveillance bronchoscopy protocol, others have reported satisfactory long-term results without surveillance bronchoscopy. Nonetheless, it is a widely held belief that bronchoscopy is the principal, and often only, diagnostic test necessary to evaluate allograft dysfunction. Complications

Shortly after transplantation, primary graft dysfunction, or ischemia–reperfusion injury, is not

uncommon. It is a form of acute lung injury with increased vascular permeability and diffuse alveolar damage. However, hyperacute rejection and venous anastomotic complications can have similar presentations and should be excluded. The severity of early graft dysfunction is highly variable, ranging from asymptomatic radiographic infiltrates to severe lung injury requiring a high level of critical care support. Treatment is the conventional supportive management for acute lung injury, but inhaled nitric oxide and extracorporeal membrane oxygenation have been used successfully for severe cases. The majority of recipients recover, but graft failure remains a leading cause of early death. Other early complications are similar to the perioperative complications seen after any thoracic surgery, and include bacterial pneumonia, atrial fibrillation, and phrenic nerve injury. Because the bronchial circulation is disrupted at the time of harvesting the donor lung, the bronchial mucosa of the transplanted lung is vulnerable to ischemic injury, which can result in stricture formation or malacia. Other airway complications include anastomotic dehiscence and web formation. A variety of treatments have been performed successfully including dilations, laser therapy, and stenting. Acute rejection has been one of the most common complications after lung transplantation. In fact, most recipients will have at least one episode of acute rejection, and the majority of episodes occur in the first year after transplantation. Acute rejection represents the recipient’s immune response to the allograft and is characterized by arteriolar lymphocytic inflammation. Transbronchial biopsy remains the gold standard for the diagnosis of acute rejection because clinical and radiographic findings are nonspecific. The Lung Rejection Study Group devised a grading system for acute rejection based on the extent and severity of perivascular inflammatory cell infiltration (A grade) and airway inflammation (B grade) (Table 3). An example of mild acute rejection (grade A2) is shown in Figure 2, and an example of severe acute rejection (grade A4) in Figure 3. Perivascular infiltrates remain the determining factor for grading acute rejection, but airway inflammation is noted histologically and recognized as a potential precursor of obliterative bronchiolitis. The significance of airway inflammation is confounded by difficulties distinguishing infectious from noninfectious causes. Acute rejection is typically treated with a 3-day course of high-dose corticosteroids and often augmentation of the maintenance immunosuppressive regimen. Chronic rejection has been the primary obstacle to improved long-term outcomes after lung transplantation. Unfortunately, 50% of recipients develop

SURGERY / Transplantation

175

Table 3 Grading of acute rejection Grade

Characteristics

Perivascular inflammation A0 (none) No perivascular infiltrates A1 (minimal) Scattered, infrequent perivascular infiltrates; not obvious on low power A2 (mild) Frequent perivascular infiltrates; endothelialitis; may expand the adventitia A3 (moderate) Obvious on low power; cells extend into the interstitium A4 (severe) Diffuse infiltration; necrotizing vasculitis, parenchymal necrosis Airway inflammation Bx (unknown) B0 (none) B1 (minimal) B2 (mild) B3 (moderate) B4 (severe)

Ungradeable No airway inflammation Infrequent infiltration of the airway submucosa Airway cuffing with inflammatory infiltrate; no epithelial damage Dense airway cuffing; epithelial infiltration and necrosis Epithelial necrosis, ulceration; fibrinopurulent exudates within airways

Figure 3 Severe acute rejection (grade A4). There is diffuse mononuclear cell infiltration, vasculitis, and parenchymal necrosis. Magnification 20.

Figure 4 Obliterative bronchiolitis (transbronchial biopsy). Fibroproliferative tissue narrows the bronchiole’s lumen with a mild degree of inflammatory cell infiltration of the peribronchiolar tissue. Magnification 10.

Figure 2 Mild acute rejection (grade A2). Perivascular infiltrates surround a small vessel and expand the adventitia. Magnification 20.

chronic rejection within 3 years of transplantation, and chronic rejection remains the leading cause of death after the first year. The histologic hallmark of chronic rejection is obliterative bronchiolitis (OB). This is a fibroproliferative scarring process that narrows the lumen of terminal and respiratory bronchioles (Figure 4). The pathogenesis of OB has not been elucidated, but the current paradigm proposes that airway inflammation and epithelial cell injury result in an exuberant and disordered repair process that leads to chronic remodeling of the airway and

narrowing of the lumen. Lymphocytic bronchitis and bronchiolitis may be the earliest histologic evidence of evolving OB. This airway inflammation may lead to epithelial cell injury and necrosis, which results in the migration of fibroblasts and myofibroblasts and the deposition of loose granulation tissue into the airway wall, narrowing its lumen. Over time this granulation tissue may develop into dense collagenous scars that obliterate the airway lumen almost completely. Histologic confirmation of OB is difficult because of the patchy nature of the disease and the paucity of bronchioles in transbronchial biopsy specimens. Thus, bronchiolitis obliterans syndrome (BOS), defined by changes in lung function, serves as its clinical surrogate. A persistent 20% decrease in FEV1 is defined as BOS stage 1, and progressive stages are

176 SURGERY / Transplantation Table 4 Classification of BOS Stage

Definition

BOS 0

FEV1 490% of baseline and FEF25–75% 475% of baseline FEV1 ¼ 81–90% of baseline or FEF25–75% p75% of baseline FEV1 ¼ 66–80% of baseline FEV1 ¼ 51–65% of baseline FEV1p50% of baseline

BOS 0-p BOS 1 BOS 2 BOS 3

defined according to the magnitude of decrease from the baseline FEV1 (Table 4). A new stage was recently proposed to detect early changes in lung function that might foretell the onset of BOS stage 1; stage 0-p (potential BOS) was defined as a 10% decrease in FEV1 or a 25% decrease in FEF25–75%. BOS is typically treated with augmented immunosuppression but the response to treatment has generally been disappointing. Therapy can stabilize lung function, but the median survival after the onset of BOS is 3 years. Infection is a common complication and unfortunately remains one of the leading causes of death across all time periods. The lung allograft is particularly vulnerable to infections in part because of the suppressed immune system, but also because the cough reflex is diminished, mucociliary clearance is impaired, and the allograft is in constant contact with the environment. Bacterial bronchitis and pneumonia are common, Pseudomonas aeruginosa frequently being the culprit. CMV is the most frequent viral infection after transplantation, and the risk of infection depends on donor and recipient serologic status. CMV can cause gastroenteritis, colitis, or hepatitis, but viremia and pneumonitis are the most common manifestations. Most episodes occur in the first year and treatment with ganciclovir is effective. Airway colonization with Aspergillus species and other fungi are common, but invasive disease with these organisms is less frequent.

Outcomes Recently, annual survival at 1, 3, 5, and 10 years after transplantation has been approximately 75%, 60%, 47%, and 20%, respectively. Unfortunately, current survival statistics are not substantially better than 10 years ago. The leading causes of death after the first year have been infections and chronic rejection. When survival is compared among various subgroups, younger recipients tend to have a better long-term survival. The impact of the underlying diagnosis on survival is most pronounced in the first

few months, as it reflects the perioperative mortality related to the complexity of the operation for different diseases. Thereafter, survival is impacted mostly by the development of the various post-transplant complications. Functional capacity and quality of life consistently improve after transplantation. However, not surprisingly, the development of various complications, especially chronic rejection, hampers the early improvement in quality of life. Nonetheless, despite a modest improvement in survival, the prospect of improving quality of life is the primary motive for transplantation for many patients. See also: Acute Respiratory Distress Syndrome. Bronchiectasis. Bronchiolitis. Bronchoalveolar Lavage. Bronchoscopy, General and Interventional. Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General. Corticosteroids: Therapy. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Gastroesophageal Reflux. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Nitric Oxide and Nitrogen Oxides. Occupational Diseases: Overview. Pneumonia: Overview and Epidemiology; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Nosocomial; The Immunocompromised Host. Pulmonary Fibrosis. Vascular Disease.

Further Reading Arcasoy SM and Kotloff RM (1999) Lung transplantation. New England Journal of Medicine 340(14): 1081–1091. Banner NR, Polak JM, and Yacoub MH (eds.) (2003) Lung Transplantation. Cambridge, UK: Cambridge University Press. Chakinala MM and Trulock EP (2003) Acute allograft rejection after lung transplantation: diagnosis and therapy. Chest Surgery Clinics of North America 13: 525–542. Estenne M, Maurer JR, Boehler A, et al. (2002) Bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria. Journal of Heart and Lung Transplantation 21: 297–310. Ginns LC and Wain JC (1999) Lung transplantation. In: Ginns LC, Cosimi AB, and Morris PJ (eds.) Transplantation, pp. 490–550. Malden, MA: Blackwell Science. Levine SM (2004) A survey of clinical practice of lung transplantation in North America. Chest 125: 1224–1238. Maurer JR, Frost AE, Estenne M, et al. (1998) International guidelines for the selection of lung transplant candidates. Journal of Heart and Lung Transplantation 17: 703–709. Trulock EP (1997) Lung transplantation. American Journal of Respiratory and Critical Care Medicine 155(3): 789–818. Trulock EP, Edwards LB, Taylor DO, et al. (2003) The Registry of the International Heart and Lung Transplantation: twentieth official adult lung and heart–lung transplant report – 2003. Journal of Heart and Lung Transplantation 22: 625–635. Yousem SA, Berry GJ, Cagle PT, et al. (1996) Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: lung rejection study group. Journal of Heart and Lung Transplantation 15: 1–15.

SYMPTOMS OF RESPIRATORY DISEASE / Cough and Other Symptoms 177

SYMPTOMS OF RESPIRATORY DISEASE Contents

Cough and Other Symptoms Dyspnea Chest Pain

Cough and Other Symptoms U G Lalloo, University of KwaZulu-Natal, Durban, South Africa & 2006 Elsevier Ltd. All rights reserved.

Abstract Cough is a common respiratory symptom. The respiratory system is a complex, integrated system with central and peripheral inputs to regulate breathing. Other symptoms arising from the respiratory system include wheeze, dyspnea, and chest pain. The pathophysiology of cough is not fully understood. Cough-sensitive nerves are present throughout the respiratory tract but most abundant in the large airways. Most of the cough afferents are carried in the vagus nerve and connect to the putative cough center in the medulla and is under voluntary and involuntary control. A number of neuropeptides and prostaglandins mediate cough. Chronic dry cough is a common problem and is due to a number of conditions. Most of these conditions are associated with sensitization of the cough reflex assessed by challenge testing in the laboratory with capsaicin, citric acid, and/or chloridedeficient solutions. Patients in whom the cause is not apparent following careful clinical evaluation, a postnasal drip syndrome, cough-variant asthma, eosinophilic bronchitis, and gastroesophageal reflux disease account for the majority.

Introduction The respiratory system is a complex and integrated system. It is comprised of: 1. the central control center situated in the medulla oblongata with afferent inputs from the cortex and periphery, efferents to the respiratory tract and chemoreceptors; 2. the upper respiratory tract – extends from the nose to the vocal cords; and 3. the thorax which is comprised of the cage (skin, soft tissue, muscle, sternum, ribs, vertebra, scapula, muscle, and neurovascular innervation) and the lower respiratory tract (tracheobronchial tree and lungs). It is apparent therefore that abnormalities in any of the components of the system can cause symptoms, and due acknowledgment of the integrated and complex system will help to localize the site. For example,

dyspnea may be due to metabolic acidosis without any disorder in the thorax. The thorax also accommodates the heart and great vessels, esophagus, and thoracic spinal cord with its connections, and nervous system tracts such as the sympathetic tracts and conditions affecting these structures give rise to symptoms that need to be discriminated from disease of the respiratory system. The respiratory tract has a limited response to injury and also has a limited constellation of symptoms that include cough (with or without sputum production), dyspnea, wheeze, and chest pain and tend to localize abnormality to the lower respiratory tract whilst sneezing, nasal itch, rhinorrhoea, sore throat and pharynx, hoarseness, facial pain, and cough arise from abnormality in the upper respiratory tract. Cough, dyspnea, wheeze, and chest pain may also reflect abnormality in the cardiovascular system and careful evaluation of the symptom complex will help to localize the condition to the respiratory or cardiovascular system. Extrathoracic symptoms may arise as a consequence of disease in the respiratory system. These include fever and night sweats (respiratory tract infection/inflammation), weight loss, lassitude, swelling of the body (cor pulmonale), headaches (hypercapnia), tremors (hypoxia), confusion (hypercapnia and/or hypoxia), painful joints (clubbing and osteoarthropathy), and paraneoplastic syndromes from lung malignancies. Expectoration of phlegm or mucus hypersecretion is invariably associated with a cough, but occasionally subjects may not cough but report excess mucus production. The latter has been observed in some epidemiological studies of respiratory symptoms. This article will review cough.

Cough Cough is a common symptom, significantly associated with smoking in epidemiologic studies, and is one of the commonest symptoms presenting in clinical practice. It may reflect disease within and outside the lower respiratory tract and may be the first sign of respiratory disease. It is a protective and

178 SYMPTOMS OF RESPIRATORY DISEASE / Cough and Other Symptoms

defensive reflex that is vital to maintain the integrity of the respiratory tract. It is a complex, highly conserved reflex and is subject to both voluntary and involuntary control. It functions chiefly to remove secretions from the respiratory tract and removal of substances inhaled or aspirated.

where there is loss of the explosive character (paralysis of the vocal cord/s), ‘brassy’ cough characterized by a metallic sound (compression of the central airways by a mediastinal mass such as an aortic aneurysm), and a ‘barking’ or ‘honking’ cough in functional disorders.

The Human Cough Reflex

Cough receptors No specific cough receptor has been identified. An extensive plexus of sensory nerve fibers situated in the airway epithelium is believed to serve as a network of cough receptors and mediate cough, although the evidence for this is still controversial. There are three types of afferent nerve endings in the tracheobronchial tree that may serve as cough receptors: rapidly adapting pulmonary stretch or irritant receptors (RARs), bronchial C-fibers, and pulmonary C-fibers. These fibers stain for sensory neuropeptides (tachykinins) such substance P, neurokinin, and calcitonin gene-related peptide (CGRP). These substances likely mediate neurogenic inflammation upon stimulation and set up a local axon reflex that results in the activation of afferent sensory nerves that connect to the cough center, which is depicted in Figure 1. The ferret and mouse, which has no cough reflex, lacks these intraepithelial nerves. The RARs are small-diameter A-d myelinated fibers, and the bronchial and pulmonary C-fibers are nonmyelinated. Cough-sensitive nerves/receptors are present throughout the tracheobronchial tree, lung parenchyma, and in extrathoracic sites such as the ear. Contrast medium introduced into a bronchopleural fistula only caused cough when it reached the subsegmental airways supporting the view that coughsensitive receptors are mainly in the large airways. Acid reflux from the stomach into the lower esophagus causes cough by the activation of a local esophageal–tracheobronchial reflex. Table 1 is a summary of the sites of cough receptors or coughsensitive nerve fibers, fiber subtype, and their afferent nervous connections. RARs are mainly located at the larynx and carina, which are the most sensitive cough sites and respond to touch and pressure. There is considerable controversy about the role of the nonmyelinated C-fibers. A simple view is that the bronchial C-fibers cause cough when stimulated, whereas the pulmonary C-fibers inhibit cough in humans. This is plausible because stimulation of cough deep within the pulmonary parenchyma will not clear particles, as airflow is negligible at these sites. Further evidence for this assertion is that 5-hydroxytryptamine which excites pulmonary C-fibers in animals inhibits cough in humans when administered systemically. However, this does not preclude a central site of action for

The human cough reflex is poorly understood and it is therefore not surprising that there are no effective cough suppressants for use in clinical practice. It is difficult to study cough in humans, and much of the information relating to the physiology of the human cough reflex is extrapolated from studies in animal models such as the guinea pig and cat. The central cough center is a ‘black box’ as its precise location and action in relation to the breathing center is undefined because it is difficult to investigate the central cough center in conscious animals. Mechanics of Cough

Once the sensation to cough is perceived, there is a coordinated outflow of signals from the putative cough center in the posterior aspect of the medulla to the inspiratory center. This results in inhalation of a relatively large volume of air followed by transient closure of the glottis, which facilitates the generation of large intrathoracic pressures proportional to the volume of air (explained by the Frank–Starling mechanism). This is followed by rapid expiratory flow once the glottis opens. Airflows 4600 l min
1 can be generated. Dynamic compression of the central airways due to the high pleural pressures enhances flow and removal of mucus attached to the airway walls. Glottic closure is not essential to cough. Recent physiological studies indicate that smooth muscle contraction previously believed to be integral to coughing is also not necessary. Mechanical stimulation of the larynx results in an immediate expiratory effort. Ineffective cough occurs when: 1. 2. 3. 4.

there is respiratory muscle weakness, mucus rheology is altered (becomes tenacious), consciousness level is depressed, vocal cord paralysis occurs (glottic closure is not achieved), and 5. mucociliary dysfunction exists (the mucociliary escalator system is required to transport secretions to the more central airways where cough is most effective in expelling mucus). The mechanical force of coughing also generates a loud sound, and characteristic alterations of the sound may occur in clinical practice: ‘bovine’ cough

SYMPTOMS OF RESPIRATORY DISEASE / Cough and Other Symptoms 179 Touch pressure

Neutrophilic inflammation

Epithelial damage

Mucus secretion

Eosinophilic inflammation Sensory nerve activation

Release of bradykinin substance P, neurokinins, C-GRP

}

Bradykinin prostaglandins

Nonmyelinated bronchial C-fibers Smooth muscle constriction

Myelinated rapidly adapting/irritant receptors Neutrophilic inflammation

Eosinophilic inflammation

Figure 1 Proposed mechanisms of cough in the large airways. Sensitization is evidenced by proliferation of airway sensory nerves (C-fibers and RARs), neutrophilic and eosinophilic inflammation, and increase in bradykinin, substance P and neurokinin, and CGRP (calcitonin gene-related peptide). Lables indicate various factors that stimulate cough in the airways. Adapted from Widdicombe JC (1995) Neurophysiology of the cough reflex. European Respiratory Journal 8: 1193–1202.

Table 1 Site of cough receptors and afferent cough fibers with their nervous connections to the CNS Site of cough receptors

Afferent fiber typesa

Nerves connecting to CNS

External auditory canal Sinuses, face, pharynxb Larynx Tracheobronchial tree Pulmonary parenchyma Lower esophagus Diaphragmb

Rapidly adapting receptors (RARs) RARs RARs and C-fibers RARs and C-fibers C-fibers (J-receptors) Esophageal-tracheobronchial reflex RARs

Auricular branch (Arnold’s nerve) of the vagus Branches of glossopharyngeal and trigeminal Superior laryngeal nerves of the vagus Branches of the vagus Branches of the vagus Branches of the vagus Phrenic nerve

a b

All except pulmonary C-fibers (J-receptors) are stimulatory. Most physiologists dispute the presence of cough receptors at these sites.

5-hydroxytryptamine. Cough due to interstitial lung disease in the absence of airway pathology may be due to destruction of the inhibitory pulmonary Cfibers. There is evidence in cats that stimulation of bronchial C-fibers also inhibits cough. Systemic administration of capsaicin in guinea pigs preferentially destroys C-fibers and results in a diminished cough response to inhaled tussive stimuli such as citric acid and capsaicin. Data from single C-fiber preparations of guinea pig trachea demonstrate that these fibers are stimulatory. Stimulation of the cough-sensitive nerves is thought to result in neurogenic inflammation mediated by the release of sensory neuropeptides that cause vasodilatation, mucus secretion, and local edema. This is the theoretical basis for the sensitization

of the cough reflex in many clinical conditions described later. Impulses travel through the A-d and bronchial C-fibers in the vagus nerve and reach the central nervous system where the signal is processed to produce the complex cough response. The mechanism of cough in the respiratory tract is illustrated in Figure 1. Afferent pathways and central control of cough The cough receptors connect to the cough center in the medulla mainly via the vagal afferent pathways as shown in Table 1. There are some clinical data to suggest that the glossopharyngeal, trigeminal, and phrenic nerves may also subserve cough receptors. There is also significant input from the higher centers as cough can be voluntarily suppressed

180 SYMPTOMS OF RESPIRATORY DISEASE / Cough and Other Symptoms

and occasionally manifest as a functional disorder. The chemical mediation of cough in the central nervous system is also uncertain. Limited experimental evidence proposes a role for serotoninergic (5-hydroxytryptamine-1), glutaminergic, and Nmethyl-D-aspartate receptors. Opioids are believed to suppress cough via central m2 opioid receptors based on studies in mice. They are unsuitable in clinical practice because they also cause depression of consciousness via k1 opioid receptors. Dextromethorphan, which is an opioid analog, has weak antitussive action in humans. Tussive Stimuli

Bronchoconstriction was considered a prerequisite for coughing. Whilst brochoconstriction stimulates cough, cough can occur independent of bronchoconstriction. Cough is stimulated by numerous exogenous and endogenous substances, which have contributed to our understanding the pathophysiology of the cough reflex. This has been exploited to test the sensitivity of the reflex in disease states and effect of antitussive agents. Capsaicin Capsaicin, the active ingredient of capsicum fruit, is a potent tussive agent in animals and humans when inhaled in low concentrations. It stimulates the bronchial C-fibers to produce cough. Capsaicin is used to assess cough reflex sensitivity very much like the metacholine challenge test, as it is reproducible under well-controlled laboratory conditions. Change in sensitivity to this challenge has been used to document response to treatment of cough due to common etiologies such as asthma, gastroesophageal reflux disease (GERD) and postnasal drip syndrome (PNDS). Capsaicin is a powerful tussive agent and no currently registered antitussive agent has been able to significantly suppress its tussive effects in clinical medicine. Interestingly, capsaicin (vanilloid) receptors have been identified in neuronal and non-neuronal tissue in humans. A capsaicin receptor antagonist, capsazepine, has been synthesized and shown to suppress capsaicin-induced cough in guinea pigs. Citric acid Citric acid was recognized as a tussive agent about 50 years ago. In humans, it is subject to short-term tachyphylaxis when inhaled. It mediates its effect through its low pH and mediates its action by a similar mechanism of action as capsaicin in guinea pigs. Iso-osmotic chloride deficient solutions and noniso-osmotic solutions Inhalation of iso-osmotic

solutions deficient in chloride such as sodium bicarbonate induces cough by stimulating A-d and C-fibers. This is inhibited by furosemide in healthy volunteers and proposes a role for Na þ –K þ –2Cl
cotransport mechanisms in cough. Inhalation of distilled water, on the other hand, is a powerful tussive and bronchoconstrictor agent suggesting that there are different pathways for cough and bronchoconstriction. Prostaglandins Inhalation of prostaglandin F2a and E2 causes tracheal irritation and cough and enhances the cough response to capsaicin suggesting that they may be involved in sensitization of the cough reflex. They stimulate both A-d and C-fibers in experimental animals. Bradykinins and tachykinins Bradykinin and substance P cause cough in patients with asthma and upper airway infection but not consistently in healthy volunteers. However, tachykinin antagonists inhibit cough caused by citric acid and cigarette smoke. This may be the mechanism of angiotensin enzyme inhibitor cough, because the degradation of endogenous bradykinin and tachykinins is inhibited. Sensitization of the Cough Reflex

The increased cough seen in a number of clinical conditions associated with cough may be due to sensitization of the cough reflex. Cough challenge studies support this hypothesis. Women have a more sensitive cough reflex in contrast to their male counterparts as observed in cough challenge studies in healthy volunteers. Patients with cough due to increased secretions do not, in general, demonstrate increased sensitivity of the cough reflex to inhalation of capsaicin, citric acid, or chloride deficient solutions. The cough is due to stimulation of the receptors by the airway secretions. Numerous conditions associated with a dry or nonproductive cough are associated with a hypersensitive cough reflex in response to several stimuli. The mechanism of sensitization includes epithelial damage with exposure of the sensory nerves, inflammation, increased airway bradykinin, tachykinins and prostaglandins, and proliferation of airway sensory nerves (Figure 1). Evidence for this is seen in positive staining of bronchial biopsies for these substances, increased neutrophils in induced sputum, and the observation of increased nerve density in patients with idiopathic chronic dry cough. Bronchoconstriction per se can also stimulate cough (Figure 1).

SYMPTOMS OF RESPIRATORY DISEASE / Cough and Other Symptoms 181 Clinical Phenotypes of Cough

The definition of a chronic cough is not standardized in the literature. In general, a cough is chronic if it is present for longer than 3 weeks. It is probably not cost effective to embark on costly investigations before 6 weeks, as postinfectious cough, for example, may take longer than 3 weeks to resolve. Chronic cough has a variety of etiologies. An algorithm that requires a clinical assessment of disease probability in order to determine whether to treat or investigate further has been applied with success and is cost effective. Such a systematic approach applied together with an understanding of the pathophysiology of the cough reflex will result in a diagnosis in over 95% of patients. Treatment of the underlying cause will result in resolution or improvement in the majority. Two or more conditions may coexist in some patients, thus complicating the clinical picture. Assessment of the severity of cough relies on assessment of patients’ perceptions. A questionnaire called the Leicester Cough Questionnaire (LCQ) is an example of a tool to assess and monitor cough severity. Only few research laboratories have facilities to measure the cough reflex sensitivity and are therefore not a practical means to assess cough. Cough may also be recorded using an ambulatory recorder but this remains a research tool. When the cough is productive as in chronic bronchitis and bronchiectasis, the cough reflex is usually not sensitized. The main clinical challenge relates to patients with a chronic dry cough, usually with a sensitized cough reflex. Often, these patients complain of a tickling sensation in the throat. There are no effective antitussive agents that effectively suppress the cough reflex; therefore, identification and treatment of the etiology is important. Most antitussives provide some relief by a demulcent effect in the pharynx, and menthol-containing cough mixtures appear to mediate their effect by cooling of the pharynx or some central gating mechanism of the cough reflex. A chronic cough is distressing and can lead to significant social and physical morbidity such as urinary incontinence, fracture of ribs, syncope and muscle cramps, and therefore warrants systematic investigation and management. Smoking Chronic cough in smokers may be due to a number of factors: mucus hypersecretion, bronchoconstriction, chemical irritation from constituents of cigarette smoke, airway inflammation, and bronchial malignancy. It has been recently demonstrated that smokers with no evidence of respiratory disease have a diminished cough reflex sensitivity to capsaicin. This intriguing observation implies that at least in a

subpopulation of smokers, smoking is tolerated because of a diminished sensitivity (acquired or constitutive), which allows them to continue smoking until above mechanisms begin to operate. Angiotensin converting enzyme inhibitor cough Angiotensin converting enzyme inhibitor (ACEi) cough is a clinical enigma. Between 5% and 20% of patients develop a cough within few days to several months of commencing an ACEi. It is a class effect. The cough also disappears rapidly in the majority after discontinuation of the drug. It may be dose related. A sensitized cough reflex has been demonstrated in these patients and in guinea pigs treated with captopril. The cough is most likely due to inhibition of breakdown of bradykinin and tachykinins and provides evidence for the role of these agents in chronic cough. A genetic predisposition has also been proposed. The cough may subside following treatment with sodium chromoglycate, a chromone with tachykinin antagonistic properties. Postnasal drip syndrome, chronic rhinitis, and sinusitis This group of related conditions is the commonest cause of a chronic dry cough, particularly where the cause is not evident following clinical evaluation. Thus, empirical treatment for a PNDS is recommended if a detailed clinical evaluation, chest radiology, and spirometry reveal no obvious cause. In its obvious form, PNDS presents with symptoms of secretions in the back of the throat and frequent need to clear the throat. Sometimes, vocal cord swelling is seen at laryngoscopy. Cough is due to secretions in the larynx and inflammation due to the ‘soup’ of mediators in the nasal secretions. Often, patients have no symptoms of PNDS but respond well to empirical treatment. Subjects with PNDS have been shown to have a sensitized cough reflex on challenge, which improves after effective treatment. Asthma Cough is a common symptom of asthma and usually improves following effective treatment of asthma. The cough is due to several factors usually operating not only in concert but also independently: increased mucus secretion, bronchoconstriction, neurogenic inflammation in the airway, and damage to the airway epithelium. Interestingly, asthmatics without cough do not have a sensitized cough reflex on challenge testing. In some patients, cough is the sole symptom of asthma without clinical or spirometric evidence of airflow limitation (cough variant asthma). These patients have a sensitized cough reflex, and airway hyperresponsiveness is demonstrable with metacholine or histamine challenge testing, and there is a

182 SYMPTOMS OF RESPIRATORY DISEASE / Dyspnea

good response to bronchodilator and/or corticosteroid treatment. The cough may persist in some asthmatics despite optimal control of asthma, and these patients may have increased neurogenic inflammation in the airways that is resistant to corticosteroids. Gastroesophageal reflux disease Like PNDS, patients with GERD and cough frequently do not have overt symptoms of acid reflux. If the cause of the cough is not obvious following careful clinical evaluation, chest radiology, and spirometry, and there is no response to empiric treatment for PNDS, then it is prudent to investigate for GERD (ambulatory esophageal pH monitoring, upper gastrointestinal endoscopy). In the absence of facilities to investigate, empiric treatment with proton pump inhibitors is indicated. The mechanism of cough is activation of a local esophago-tracheobronchial neural reflex by the aspiration of gastric acid into the lower esophagus. This reflex is stimulated by infusion of acid into esophagus (Bernheim test), which is blocked, by prior lignocaine infusion and inhalation of ipratropium bromide. In some patients reflux of gastric acid up to the pharynx may account for the cough. There is evidence of sensitization of the cough reflex with challenge testing in GERD, which also improves with effective antireflux treatment. Eosinophilic bronchitis The availability of noninvasive methods to investigate lung disease such as induced sputum and evaluation of the cellular constituents of induced sputum has resulted in the recognition of patients with a chronic dry cough with evidence of eosinophilic infiltration of the airway. These patients have increased eosinophils in induced sputum, without evidence of asthma and normal metacholine/histamine challenge testing. Cough with eosinophilic bronchitis responds to corticosteroids. Idiopathic cough There is a subgroup of patients who have a chronic dry cough and a sensitized cough reflex but no identifiable etiology despite intensive investigation and empiric treatment for common causes listed above. This group is classified as idiopathic cough. Studies in some cohorts have shown increased CGRP and vasoactive inflammatory peptide immunoreactive sensory nerves in biopsies of their airways. It is unclear whether these findings are the cause of the cough or the effect of chronic persistent coughing. A functional, habit, or psychogenic cough is rare but should be considered, particularly in the setting of an emotional or psychological problem. These

patients appear to make a throat clearing sound and the cough may be honking or barking in character. See also: Allergy: Allergic Rhinitis. Asthma: Overview. Pediatric Pulmonary Diseases. Reflexes from the Lungs and Chest Wall. Signs of Respiratory Disease: General Examination.

Further Reading Chung KF (1997) Cough. In: Crystal RG, West JB, and Barnes PJ (eds.) Scientific Foundations, pp. 2309–2319. Philadelphia: Lippincott–Raven Publishers. Dicpinigaitis PV (2004) Cough 4: cough in asthma and eosinophilic bronchitis. Thorax 59: 71–72. Fontana GA and Pistolesi M (2003) Cough 3: chronic cough and gastro-oesophageal reflux. Thorax 58: 1092–1095. Fox AJ, Lalloo UG, Belvisi MG, et al. (1996) Bradykinin-evoked sensitization of airway sensory nerves: a mechanism for ACEinhibitor cough. Nature Medicine 7: 814–817. Irwin RS (1998) Managing cough as a defense mechanism and as a symptom: consensus panel report of the American College of Chest Physicians. Chest 114: 133S–179S. Johnson D and Osborn LM (1991) Cough variant asthma: a review of the clinical literature. Journal of Asthma 28: 85–90. Karlsson J-A, Sant’Ambrogio G, and Widdicombe JG (1988) Afferent neural pathways in cough and reflex bronchoconstriction. Journal of Applied Physiology 65: 1007–1023. Lalloo UG (2003) The cough reflex and the ‘Healthy Smoker’. Chest 123: 660–662. Lalloo UG, Barnes PJ, and Chung KF (1996) Pathophysiology and clinical presentations of cough. Journal of Allergy and Clinical Immunology 98: S91–S96. Lalloo UG, Lim S, DuBois R, et al. (1998) Increased sensitivity of the cough reflex in progressive systemic sclerosis patients with interstitial lung disease. European Respiratory Journal 11: 702–705. O’Connell F, Thomas VE, Pride NB, et al. (1994) Capsaicin cough decreases with successful treatment of cough. American Journal of Respiratory and Critical Care Medicine 150: 347–380. Piirila¨ P and Sovija¨rvi ARA (1995) Objective assessment of cough. European Respiratory Journal 8: 1949–1956. Widdicombe JC (1995) Neurophysiology of the cough reflex. European Respiratory Journal 8: 1193–1202.

Dyspnea U G Lalloo, University of KwaZulu-Natal, Durban, South Africa & 2006 Elsevier Ltd. All rights reserved.

Abstract Dyspnea is a common symptom of pulmonary disease but also reflects cardiac and extrathoracic disorders such as anemia, abdominal distension, metabolic acidosis, pregnancy, and psychogenic disorders. It is simply defined as the uncomfortable awareness of breathing and the need to increase the respiratory effort. Respiration is coordinated in the respiratory center in the medulla, which receives afferent inputs from receptors in the chest wall and muscles, pulmonary capillary and interstitium,

SYMPTOMS OF RESPIRATORY DISEASE / Dyspnea 183 airways, central and peripheral chemoreceptors, and sensory receptors in the face and pharynx. There is simultaneous discharge from the respiratory center to the ventilatory muscles and the cortex. Thus, dyspnea may occur when there is a dissociation between the ventilatory output and expected response. A careful history based on good communication with the patient will unmask the mechanism and possible cause of the breathing difficulty. Dyspnea severity may be classified using a standardized classification system. Treatment of the underlying cause will resolve the symptom. Frequently, treatment of dyspnea, based on the pathophysiology is required.

Introduction Dyspnea is a complex, subjective, and common symptom used by clinicians to define the respiratory distress with which patients frequently manifest. It primarily reflects disease in the respiratory and cardiovascular system but may also indicate hormonal, hematologic, metabolic, and psychogenic disorders. It has a significant impact on the quality of life due to functional impairment and consequent disability. An understanding of the complex mechanisms underlying dyspnea should aid in identifying the origin of the symptom, and lead to appropriate investigations and management. Often the underlying condition is irreversible and the management of the symptom is important. The neural pathways underlying dyspnea are not well understood. Advances in understanding dyspnea relate to understanding dyspnea and related terms to facilitate communication with patients, classification systems for more objective assessment of dyspnea severity, defining the role of pulmonary and chest-wall receptors, and localization of regions in the brain responsible for processing respiratory sensations facilitated by positron emission tomography technology.

Definitions Dyspnea may be simply defined as an uncomfortable awareness of breathing and the need to increase the respiratory effort. This simple definition belies the complex nature of dyspnea which is underscored by the American Thoracic Society consensus statement definition as ‘‘a term used to characterize a subjective experience of breathing discomfort that consists of qualitatively distinct sensations that vary in intensity. The experience derives from interactions among multiple physiological, social, and environmental factors, and may induce secondary physiological and behavioural responses.’’ Given the subjective nature of the symptom, it is not surprising that a remarkable difference in perception occurs between individuals for the same physiological aberration.

The commonest presentation of dyspnea in clinical practice is effort-induced dyspnea. Many patients with effort-induced dyspnea due to left ventricular dysfunction may also have associated orthopnea and paroxysmal nocturnal dyspnea, which usually signifies more severe disease. Orthopnea is dyspnea upon assuming a recumbent position, and paroxysmal nocturnal dyspnea is dyspnea following a period of recumbency when the subject is suddenly awakened by shortness of breath and has to gasp for air. These latter two symptoms may also occur in respiratory diseases. Patients with severe chronic obstructive pulmonary disease (COPD) or asthma may have orthopnea due to difficulty in breathing when lying down, but the etiology is usually clinically evident. Asthma may manifest with nocturnal exacerbations and paroxysmal nocturnal dyspnea may be the sole manifestation of the disease. Platypnea is a rare symptom of shortness of breath upon assuming the standing position and relieved when lying down. It occurs as a manifestation of the hepatopulmonary syndrome and is frequently accompanied by orthodeoxia, which is oxygen desaturation upon assuming the standing position, and is associated with intrapulmonary vascular disorders noted in patients with advanced liver disease. Dyspnea presents as a common symptom in patients from diverse cultural, socioenvironmental, and educational backgrounds and it is therefore not surprising that there are many ‘dyspnea equivalents’ and the attending physician must become familiar with those in his/her practice settings. The physician should obtain an explanation about what precisely is the cause of the respiratory difficulty. In some instances, chest pain may be a surrogate symptom of dyspnea and obtaining a description from the patient may unmask the real symptom. Descriptors of dyspnea also frequently reflect the underlying pathophysiological abnormality and good communication with patients helps to understand the symptom and unmask the likely cause. Some examples of these are as follows: Chest tightness/constriction. It is noted in asthmatic patients. The sensation of dyspnea is mediated by the airway smooth muscle rapidly adapting receptors and airway irritant receptors. It is blocked by airway anesthesia and vagal inhibition. Work/effort. The sensation of an increased command to ventilatory muscles and the increased force of contraction accounts for breathing difficulty. Unrewarded inspiration. Motor output from the respiratory center is expected to lead to a specific

184 SYMPTOMS OF RESPIRATORY DISEASE / Dyspnea

increase in lung volume, but the latter may be inhibited by poor lung compliance as seen in interstitial lung disorders. This is mediated by mechanoreceptors such as muscle spindles and Golgi tendon organs. Rapid breathing. When inspiration is impeded by loading inspiratory muscles by, for example, external thoracic restriction, it leads to rapid shallow respiration and accounts for the breathing difficulty. Air hunger. Respiration is controlled by arterial carbon dioxide partial pressure (PaCO2) and thus hypercapnia drives respiration. The powerful urge to breathe is readily recognized with breathholding and is mediated by a rise in PaCO2. It is proposed that discharges from cortical centers give rise to a sense of breathing effort whereas that from medullary centers cause air hunger. Air hunger accounts for the dyspnea reported by patients with acidosis.

Causes of Dyspnea Dyspnea may be acute or chronic and is a common manifestation of cardiopulmonary disease. The causes of dyspnea with proposed mechanisms are presented in Table 1. Many of the causes will have coexisting hypoxia that may contribute, albeit unpredictably, to the breathlessness. COPD and asthma account for a large proportion of patients who present with chronic dyspnea due to respiratory disease. Acute exacerbations manifest with acute dyspnea or acute worsening of chronic dyspnea. Other respiratory causes include diffuse parenchymal lung diseases, neuromuscular disorders, lung malignancy, chronic pleural disease, and chronic infections such as tuberculosis, fungal infections, and AIDSrelated pneumocystis pneumonia. Acute dyspnea occurs in pulmonary embolism, infectious diseases such as pneumonia, acute bronchitis, pneumothorax,

Table 1 Causes of dyspnea and proposed mechanisms Respiratory diseases Acute Pneumonia (including acute interstitial lung disease, pneumocystis pneumonia) Acute pulmonary embolism Acute bronchitis Pneumothorax Pleural effusion Chronic COPD

Asthma Interstitial lung disease Lung cancer Neuromuscular disease involving respiratory muscles 9 Cardiac diseases > > = Myocardial ischemia ðangina pectoris and infarctionÞ Acute cardiac failure > > ; Chronic cardiac failure Miscellaneous Anemia

Pregnancy Abdominal distension (severe ascites and large masses) Metabolic acidosis Anxiety Sighing dyspnea

Proposed mechanism Stimulation of intrapulmonary juxtacapillary (J) receptors Stimulation of intrapulmonary juxtacapillary (J) receptors, or right atrial receptors Stimulation of airway irritant receptors, increased resistance Lung and chest-wall mechanoreceptors Lung and chest-wall mechanoreceptors

Hypoxia and hypercapnia – stimulation of chemoreceptors Compression of airways – stimulation of airway irritant receptors Increased sense of effort Muscle weakness Dynamic hyperinflation – stimulation of lung and chest-wall mechanoreceptors Stimulation of airway irritant receptors, increased resistance Stimulation of intrapulmonary juxtacapillary (J) receptors Combination of several factors depending on site and mechanical effect of tumors Hypercapnia leading to pH change Pulmonary venous congestion stimulation of intrapulmonary juxtacapillary (J) receptors

Increased pulmonary venous pressures and stimulation of intrapulmonary juxtacapillary (J) receptors Lactate accumulation in muscles Progesterone mediated increase in ventilation followed by pressure of gravid uterus with advancing pregnancy Splinting of diaphragm with inspiratory loading – stimulation of muscle sensors and lung and chest-wall mechanoreceptors Changes in pH and blood gases – stimulation of chemoreceptors Cortical overlay Cortical overlay

SYMPTOMS OF RESPIRATORY DISEASE / Dyspnea 185

pleural effusion (related to rate of accumulation), and acute exacerbations of COPD and asthma. Cardiac disorders are also important causes of dyspnea and must be considered in the differential diagnosis. Noncardiopulmonary disorders such as severe abdominal distension (due to ascites and large masses) and metabolic acidosis may present with dyspnea. Pregnancy and anemia also cause dyspnea. Psychogenic conditions such as sighing dyspnea are important, given the subjective nature of the symptom, and that respiration is subject to voluntary cortical control. Sighing dyspnea is characteristic and patients present with paroxysmal episodes of shallow breathing, usually at rest, and complain of inability to take a full breath. It is sometimes associated with organic disorders such as coronary artery disease, acute left ventricular failure and asthma, which must be excluded before labeling it psychogenic.

Respiration is a unique vital function in that it is under both autonomic and voluntary control. The efferent and afferent pathways and the central controller mechanisms of respiration are depicted in Figure 1.

Maintenance and control of respiration requires an intact feedback loop consisting of central input into the medullary respiratory controller that modulates a coordinated discharge to the voluntary muscles of respiration to produce expansion of the chest wall and inflate the lung during inspiration and to deflate it during active expiration. Tidal expiration is a passive process. In conditions associated with airflow limitation in the small airways there is difficulty in breathing out and the patient perceives this as dyspnea. Normally, respiration is set to maintain arterial partial pressure of carbon dioxide (PaCO2), and arterial partial pressure of oxygen (PaO2), and acid–base homeostasis. The respiratory center plays an important role in acid–base homeostasis by modulating PaCO2. The determinant of the level of respiration is the PaCO2 and the PaO2, which mediates their effects via central chemoreceptors in the medulla and the peripheral chemoreceptors in the carotid and aortic bodies. Lung inflation is sensed by the pulmonary stretch receptors. Airflow and increase in bronchial smooth muscle tone is sensed by bronchial irritant receptors. Increase in pulmonary interstitial and capillary pressure is sensed by pulmonary C-fibers. Sensory input from these receptors/sites is transmitted to the brain

Efferent signals

Afferent signals

Pathophysiological Mechanisms of Dyspnea

Motor cortex

Effort

Sensory cortex Effort ?

Chemoreceptors Air hunger Brain stem Upper airway

Upper airway

Chest tightness

Ventilatory muscles

Chest wall

Figure 1 Efferent and afferent signals that contribute to the sensation of dyspnea. There is evidence that the sense of respiratory effort arises from a signal transmitted from the motor cortex to the sensory cortex simultaneously with the outgoing motor command to the ventilatory muscles. The motor output of the brainstem may also contribute to the sense of effort, as shown by the arrow from the brainstem to the sensory cortex. The sense of air hunger arises, in part, from increased respiratory activity within the brainstem, and the sensation of chest tightness probably results from stimulation of vagal-irritant receptor. While afferent information from airway, lung, and chest-wall receptors probably passes through the brainstem before reaching the sensory cortex, the dashed lines indicate uncertainty about whether some afferents bypass the brainstem and project directly to the sensory cortex. Reproduced from Manning HL and Schwartztein RM (1995) Pathophysiology of dyspnea. New England Journal of Medicine 333: 1547–1553, with permission.

186 SYMPTOMS OF RESPIRATORY DISEASE / Dyspnea

stem via the vagus nerve. There are sensory receptors within muscles and tendons such as the muscle spindles and Golgi tendon organs that transmit afferent impulses and form the basis of local spinal and supraspinal reflexes. A simple explanation for dyspnea is that the symptom results from a dissociation or mismatch between central respiratory motor activity and afferent information from receptors in the chest wall, lung, and airways. This means that dyspnea is reported when respiratory drive is increased or when the respiratory system is subject to a mechanical load. For example, a given efferent output to the respiratory muscles will occur based on the perceived need. This should result in an anticipated change in lung volume and chest-wall expansion. Such perception is derived from experience. The desired change in muscle length tension, lung volume, and flow of air will occur under normal circumstances. Should this fail to occur due to neuromuscular disease, inspiratory or expiratory loading, change in lung compliance, or resistance to airflow, or psychogenic (cortical factors), then dyspnea is experienced. Impulses from the afferent pathways simultaneously transmit signals to higher cortical centers so that the subject may become aware of the altered motor activity independent of increased rate, depth, and force of contraction of respiratory muscles and lung compliance. This is referred to as the respiratory motor command corollary discharge. As explained, any dissociation may be perceived as breathing difficulty. One is well aware of the overwhelming urge to breathe following breath holding and the duration of breathhold can be increased with training, indicating the ability of higher cortical centers to override sensations, albeit with inherent protective properties. Thus, the perception of breathing, associated difficulty related to inspiratory and expiratory loading, airway caliber and flow limitation, pulmonary interstitial and capillary pressures are subject to voluntary modulation. Personal factors (genetic and experiential), cultural factors, socioenvironmental factors, and education all contribute to dyspnea perception. In certain circumstances, failure to perceive changes can have catastrophic consequences. It is proposed, based on impaired perception of induced hypoxia and hypercapnia in patients surviving near-fatal asthma, that blunted perception of dyspnea and reduced chemosensitivity may have predisposed patients to such attacks. Likewise, the classic ‘bluebloater’ syndrome of chronic bronchitis may be due to poor chemosensitivity and a blunted respiratory drive and lack of dyspnea, whereas the ‘pink-puffer’ syndrome is associated with a heightened respiratory

drive leading to increased work of breathing and persistent and frequently disabling dyspnea. Sensation of the Respiratory Effort

Ventilation is stimulated by a coordinated output to the voluntary muscles of respiration of which the diaphragm is the main muscle. This efferent output is accompanied by a simultaneous activation of the sensory cortex from the respiratory center in the brainstem. It is referred to as the respiratory motor command corollary discharge. This is borne out by the observation that lifting a heavy load results in a sensation of excessive effort not observed with lifting a light load. A similar sensation may occur in the setting of muscle fatigue, motor weakness or paralysis, or breathing at higher lung volumes. The latter involves expanding the lungs at a suboptimal position on the pressure–volume curve of the lung and explains in part the dyspnea in COPD. Experimental evidence suggests that the sensation of effort is modulated by level of PaCO2. There is more breathlessness when PaCO2 is elevated. Chemoreceptors

Chemoreceptor sensations arise from hypercapnia, hypoxia, and acidosis. It was originally believed that hypercapnia did not cause dyspnea based on early observations in a patient, who was studied following paralysis with curare, and a quadriplegic patient. Recent studies have shown that hypercapnia and hypoxia cause dyspnea that is chemically mediated and independent of respiratory muscle activity. A study in ventilator-dependent quadriplegia patients with absent inspiratory muscle function experienced air hunger when the PaCO2 was elevated. This was confirmed in healthy volunteers who received mechanical ventilation after paralysis with neuromuscular blocking agents and developed severe air hunger when the PaCO2 was elevated. Hypercapnia stimulates the central chemoreceptors in the medulla through changes in pH (hydrogen ion concentration). This also explains the deep sighing respirations (Kussmaul’s respiration) observed in metabolic acidosis such as in diabetic ketoacidosis and renal failure. Chronic hypercapnia results in compensatory/adaptive changes that include renal retention of bicarbonate to normalize blood pH, and therefore may not cause such severe air hunger. In chronic hypercapnia, respiration is controlled predominantly by hypoxic drive. The role of hypoxia in respiratory stimulation and perception of breathlessness is less well understood, although intuitively one would expect hypoxia to cause remarkable breathlessness. Studies in healthy

SYMPTOMS OF RESPIRATORY DISEASE / Dyspnea 187

volunteers showed that dyspnea was less when breathing hyperoxic air mixtures for the same level of exercise compared to normoxic and hypoxic air mixtures. The mechanism of breathlessness induced by hypoxic air mixtures is unclear but may occur by direct stimulation of the chemoreceptors. Many patients with hypoxia are not breathless. Correction of hypoxia does not necessarily resolve the breathlessness unless the underlying cause is addressed. Hypoxia associated with high altitude also stimulates chemoreceptors to cause breathlessness. Increase in skeletal muscle lactate during exercise may serve as a stimulus for respiration and explain the dyspnea associated with deconditioning. Contributing factors include age, malnutrition, and underlying cardiopulmonary disease. Mechanoreceptors

Receptors in the upper airways, lungs, and chest-wall modulate the sensation of dyspnea, or respiratory inadequacy for a given respiratory stimulus. Many subjects experience relief from dyspnea when the face is exposed to a current of air by sitting in front of a fan or open window. It is proposed that receptors on the face and upper airways modify the sensation. Breathing cold air also modifies dyspnea by stimulation of receptors in the upper airways. Receptors in the lung transmit sensations of ventilation and ventilatory responses centrally. These include the pulmonary airway stretch receptors (respond to pulmonary inflation), irritant receptors in the airway epithelium (respond to mechanical and chemical stimuli), and pulmonary C-fibers (J-receptors) in the alveolar wall and blood vessels (respond to interstitial congestion). These receptors transmit information via the vagus nerve and are responsible for shaping the pattern of breathing and mediate the sensation of dyspnea. Vagal block in healthy volunteers results in increased breathhold and decreases breathlessness in some patients. Bronchoconstriction is a powerful stimulus for chest tightness and is mediated via vagal irritant receptors in the airways. On the other hand, application of inspiratory resistive loads to breathing of similar intensity results in less perception of chest tightness. Stimulation of pulmonary stretch receptors appears to ameliorate the sensation of dyspnea. Chest-wall receptors are present in joints, tendons, and muscles of the chest wall and transmit information to the brain. Sensations from the ribcage contribute to propioception and kinaesthesia. Constraining respiration below the normal spontaneously adopted level produces a sensation of air hunger, even when blood gases are maintained within a

normal range. Vibration of the upper ribcage resulted in reduction of this unpleasant sensation highlighting the contribution of sensory input from chest-wall receptors. Clinical Evaluation of Dyspnea

The steps in the evaluation of dyspnea involve appreciation of the patient’s symptoms and understanding what the patient perceives as the source of the difficulty in breathing. It is vital to recognize patients with acute onset, severe, life-threatening difficulty in breathing that warrant emergency resuscitation and care. A careful history and clinical examination coupled with a few basic investigations such as a chest radiograph, spirometry, electrocardiograph, and arterial blood gases will elucidate the source of the symptom in the majority of the cases. In the balance, more intensive investigations prompted by clues from the evaluation will be required. It is often necessary to quantify dyspnea objectively, to classify disease severity, and establish a baseline to assess change with intervention. Numerous systems of objective evaluation have been derived. There is no gold standard against which to evaluate the sensitivity and specificity of each system. The best standard against which dyspnea classification can be evaluated is cardiopulmonary exercise testing. Several standardized systems of dyspnea classification in clinical practice are summarized in Table 2. Each one has particular strengths in specific clinical settings. For example, the New York Heart Association Classification system has been derived for cardiac disease; the Borg scale is useful for COPD and the transitional dyspnea index may be of value to measure changes with medical interventions. The simplest pulmonary dyspnea scale is the British Medical Research Council Breathlessness score and is comparable to the New York Heart Association Classification for cardiac dyspnea. The 6 min walk test is a simple exercise test against which dyspnea is measured and could be regarded as the poor man’s exercise test. It is combined with a dyspnea evaluation scale to assess baseline cardiopulmonary status and response to interventions.

Management of Dyspnea Diagnosis of the underlying disorder and appropriate treatment will result in resolution of dyspnea. In many cases, the underlying disease is incurable and management of the symptom of dyspnea is important. Treatment of dyspnea is difficult. The clinical rationale is based on the pathophysiological

188 SYMPTOMS OF RESPIRATORY DISEASE / Dyspnea Table 2 Clinically validated standardized dyspnea scales available to objectively evaluate dyspnea due to cardiopulmonary disease Dyspnea scale

Method of evaluation

Borg scale

Subject driven

Borge: effort to breathe

Scalea

Limitations and advantages Categoric scale does not allow gradation between discrete numbers selected by subjects. Easy to administer

Vertical scale labeled with corresponding verbal expressions of progressively increasing symptom intensity. Analogue scale

0–10

Operator dependent on subjects symptom evaluation of level of effort causing symptoms (dyspnea, fatigue, palpitations, or angina) Subject driven

I–IV

Based on subject perception and response to five statements Subject driven

0–5

Consists of 50 items with 76 responses and assesses Symptoms, activities, and impacts Subject driven

0–100

Includes dyspnea, fatigue, emotional function, and mastery: uses a seven-pointlikert scale. 0–20 for each component Subject driven

5–20

Horizontal scale in, e.g., 0–10 cm and subject asked to select point from no dyspnea ‘0’ to severe ‘10’

0–10

Subject and operator driven

BDI:

Categoric scale. Measures baseline and changes with intervention.

BDI ¼ 3 components viz. functional impairment, magnitude of task needed to evoke dyspnea, and magnitude of effort to evoke dyspnea. TDI ¼ compares changes from BDI in each category: 3 to 0 to þ 3

12–0

More complex but is a dynamic measure. Includes three components.

Borgd: respiratory discomfort New York Heart Association Classification British MRC Breathlessness score

ST George’s respiratory questionnaire

Chronic respiratory questionnaire

Visual analog scale

Baseline dyspnea index/transitional dyspnea index (BDI/TDI)

a

Categoric scale does not allow gradation between categories. Designed for subjects with cardiac disease. Insensitive to small changes Categoric scale does not allow gradation between categories. Designed for COPD. Easy to administer

Categoric scale. Wider range of scores but is complex and assesses several respiratory symptoms including cough. Designed for COPD. Complex.

Categoric scale. Designed for COPD. Complex. Cumulative score ranges from 20–140 if all components assessed

Prepared by each laboratory. Subject to interlaboratory variation. Continuous scale and may be supplemented with visual pictures to prompt subjects perception.

TDI: þ 9 to
9

Presented in scores ranging from no dyspnea (best score) to severe dyspnea (worst score).

mechanisms of dyspnea. These include: 1. Reducing ventilatory demand: (a) appropriate nutrition may reduce CO2 production exercise conditioning improves muscle strength; (b) supplemental O2 will reduce hypoxic drive; (c) opiates and anxiolytics reduce central respiratory drive;

(d) bronchodilator treatment to reduce airway irritant impulses and improve lung expansion and emptying; (e) altering afferent sensory impulses with air draughts (fans), vibration of chest wall stimulates sensory receptors on the face and chest wall; and (f) psychological counseling alters cortical perception of dyspnea.

SYMPTOMS OF RESPIRATORY DISEASE / Chest Pain

2. Improving lung mechanics: (a) lung volume reduction surgery in COPD; (b) continuous positive airway pressure pursed lip breathing in COPD, altered ventilator settings in mechanically ventilated patients; (c) physical positioning sitting forward with COPD to improve breathing mechanics; inspiratory muscle training; (d) nutrition to improve muscle bulk; and (e) reducing dynamic hyperinflation in COPD. Multiple interventions are necessary and best results are obtained within a systematic pulmonary rehabilitation program. See also: Asthma: Overview. Chemoreceptors: Central; Arterial. Chronic Obstructive Pulmonary Disease: Overview. Hypoxia and Hypoxemia.

Further Reading American Thoracic Society (1999) Dyspnea: mechanisms, assessment and management – a consensus statement. American Journal of Respiratory and Critical Care Medicine 159: 321–340. Banzett RB and Lansing RW (1996) Respiratory sensations arising from pulmonary and chemoreceptor afferents. In: Adams L and Guz A (eds.) Respiratory Sensations, pp. 155–180. New York: Dekker. Butani L and O’Connell EJ (1997) Functional respiratory disorders. Annals of Allergy, Asthma & Immunology 79: 91–99. Jones PW, Quirk FH, and Baveystock CM (1991) The St George’s respiratory questionnaire. Respiratory Medicine 85S: 25–31. Kikuchi Y, Okabe S, Tamura G, et al. (1994) Chemosensitivity and perception of dyspnea in patients with a history of near-fatal asthma. New England Journal of Medicine 330: 1329–1334. Mahler DA, Weinberg DH, Wells CK, and Feinstein AR (1984) The measurement of dyspnea: contents, interobserver agreement, and physiologic correlates of two new clinical indexes. Chest 85: 751–758. Manning HL and Schwartzstein RM (1995) Pathophysiology of dyspnea. New England Journal of Medicine 333: 1547–1553. NRC, Committee on the Aetiology of Chronic Bronchitis (1960) Standardised questionnaire on respiratory symptoms. British Medical Journal 2: 1665. Scano G, Stendardi L, and Grazzini M (2005) Understanding dyspnea by its language. European Respiratory Journal 25: 380–385.

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practical knowledge of the anatomy of the chest, and pathophysiology and epidemiology of chest pain is important in evaluating this symptom in clinical practice. Chest pain may be mediated by somatosensory nerves in which case it is well localized or mediated by autonomic nerves in which case it is more ill-defined, poorly localized and more likely to be referred or radiate. It is important to exclude the causes of chest pain that cause anxiety and those that represent serious clinical problems based on the history, risk factors, and index of suspicion. These include myocardial ischemia and infarction, dissecting aneurysm of the aorta, tension pneumothorax, pericardial disease with cardiac tamponade, and large pleural effusions, where urgent investigations and intervention may be required.

Introduction Chest pain is a common symptom and may arise from numerous structures within the thorax or may be referred to the chest from remote sites. Frequently ischemic pain of cardiac origin is referred to the neck, arms, and epigastrium. Chest pain is usually benign but can be serious and distressing to the patient and clinician and may signal an impending catastrophe. The complex etiologies of chest pain warrant a systematic approach. There are three approaches to the evaluation of chest pain: 1. an anatomical approach based on knowledge of the structures within the thorax and other tissue that may cause chest pain, and associated pathology, 2. a functional approach based on the sensory innervation of the structures likely to give rise to chest pain and the mechanism of pain, and 3. an epidemiological approach based on the important etiologies determined by demography, geography, and pathology. A synthesis of the above approaches in clinical practice should lead to an appropriate diagnosis and consequent management in most patients. Primarycare settings demand a cost-effective, pragmatic, yet comprehensive approach to chest pain. In most patients, the correct diagnosis would be achieved with a thorough history, detailed physical examination, and aid of targeted special investigations.

Chest Pain

An Anatomical Approach to Chest Pain

U G Lalloo and A Ambaram, University of KwaZuluNatal, Durban, South Africa

Figure 1 is an illustration of the anatomical structures in the thorax that can cause chest pain. These may be conveniently divided into:

& 2006 Elsevier Ltd. All rights reserved.

Abstract Chest pain is a common clinical condition. The large number of causes of chest pain reflect the complex structures within and adjacent to the chest that can give rise to this symptom. A

1. structures that make up the chest wall: (a) skin and subcutaneous tissue, (b) bone – ribs, sternum, clavicle, vertebrae (and spinal cord),

190 SYMPTOMS OF RESPIRATORY DISEASE / Chest Pain

Esophagus Larynx Visceral pleura Pulmonary vessels

Parietal pleura

Aorta Bronchi Ribs Heart Diaphragm Liver

Intercostal muscles Spleen Central portion of diaphragm

Figure 1 A diagrammatic representation of the anatomy of the thorax indicating the structures that may cause chest pain. Blue labels represent structures that cause chest pain via somatosensory nerves, red labels represent structures that cause pain mediated by autonomic nerves and green labels represent structures that cause pain referred to the chest and are mediated by autonomic nerves. Illustration with permission & Leslie Laurien, 2006.

(c) skeletal muscles, including intercostal muscles and diaphragm, (d) parietal pleura and parietal pericardium, and (e) somatosensory and autonomic nerves including nerve roots 2. chest contents: (a) heart and great vessels, (b esophagus, (c) thymus, and (d) lymph nodes 3. structures outside the chest: (a) liver, (b) gall bladder, and (c) spleen It is notable that the lung parenchyma, most of the tracheobronchial tree, and the visceral pleura and visceral pericardium do not have pain receptors. Disorders within these tissues result in pain if the pathology extends to involve pain-sensitive structures. For example, pneumonia causes chest pain if the inflammation involves the adjacent parietal pleura; endobronchial and parenchymal tumors cause chest pain if the growth causes pressure symptoms or invades pain-sensitive structures. This may explain why pulmonary tumors have a long asymptomatic period. Early detection of pathology in these pain-insensitive structures is facilitated by other symptoms such as cough, dyspnea, and wheeze, and constitutional symptoms such as fever, malaise, and loss of weight.

A Functional Approach Based on Somatosensory and Autonomic Innervation and Mechanism of Pain

Both somatosensory and autonomic nerves mediate pain. The pain-sensitive structures that may cause chest pain can therefore be classified as follows: 1. Those that cause somatosensory pain. These are characterized by tissue that is innervated by somatosensory nerve fibers that travel to the cortex via the posterior nerve roots and the spinothalamic tracts in the spinal cord. The pain is well localized and usually of a sharp, burning, or cutting nature. This includes the components of the chest wall, the parietal pleura, parietal pericardium, bony thorax (including vertebrae), upper and middle esophagus, large airways, and spinal dura mater. Pain from the parietal pleura and pericardium is also well localized and aggravated by movement of the pleural surfaces over each other as occurs with respiration and cough, and is termed pleuritic. 2. Those that are innervated by autonomic nerve fibers. Pain from these sites are poorly localized and frequently referred, and is dull, ill defined, and/or compressive in nature, and may radiate. These arise from the heart and great vessels. Structures outside the chest include liver, gall bladder, and spleen and may give rise to pain in the shoulder region or shoulder tip. The classic autonomic pain of cardiac ischemia is described in detail later.

Mechanism of Chest Pain Chest pain, like pain arising from any anatomical structure, is complex in nature. The pain differs

SYMPTOMS OF RESPIRATORY DISEASE / Chest Pain

remarkably depending on the site of origin, type of pain receptors, and whether somatosensory or autonomic nerve fibers mediate it. Somatosensory Pain

The anatomical sites of somatosensory chest pain have been described above. The pain receptors are nociceptors. They are known to exist in muscle, joints, and skin. Each nociceptor has selective sensitivity to mechanical (muscle-fiber stretching), chemical (including lactic acid), and thermal stimuli. Whether visceral nociceptors exist is uncertain although it is clear that visceral afferents possess many of the characteristics of nociceptors. This results in electrical stimuli that are transmitted via the sensory nerve fibers that are myelinated and enter the spinal cord via the posterior nerve roots and cross over to the contralateral side of the cord and complex to form the spinothalamic tract. The spinothalamic tract travels in the medial lemniscus to the ventrobasal complex of the thalamus where they synapse. Sensory impulses are integrated in the sensory cortex resulting in the perception of pain and subsequent motor reflex response. Central mechanisms of pain and its modulation appear to be similar for somatic and visceral origin of pain. Intercostal nerves are formed from the ventral and dorsal rami of thoracic spine segments T1–T12. They supply the general sensory innervation to the skin and parietal pleura. They also supply the motor innervation to intercostal muscles and carry the postganglionic sympathetic nerve fibers. Intercostal nerves 2–6 are confined to the thorax, whilst the first nerve contributes to the brachial plexus as well as the first intercostal space. Nerves 7–12 leave the intercostal spaces after innervating them and continue to innervate muscles, skin, and parietal peritoneum of the anterior abdominal wall. Autonomic Pain

All viscera receive dual innervation. Vagal afferent fibers innervate organs of the thoracic cavity with cell bodies in the nodose (mainly) and jugular (less commonly) ganglia as well as by spinal afferent fibers. Thus, in contrast to somatic input to the central nervous system (CNS) which has a single spinal destination, input from organs in the thoracic cavity arrives at two locations, the brainstem (nucleus tractus solitarii) and thoracic spinal cord. Therefore, potential exists for the interaction of the above inputs. In addition, there is an as yet poorly understood intrinsic nervous system within some organ systems in the thorax such as esophagus and heart. These cell

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bodies are located within the organ wall or in ganglia within epicardial fat. Thus in addition to the dual inputs, there is potential interaction between the extrinsic and intrinsic systems. Further differences between somatic and visceral innervation relate to density of innervation and pattern of termination. In general, there are disproportionately less visceral afferent fibers than somatic ones although visceral afferents have many more terminal swellings (synapses) than somatic nociceptor terminals. The visceral afferents are spread over several spinal cord segments. This may explain the loss of spatial discrimination of visceral pain. Pain is not a simple sensation, and neither the neurobiological nor the functional component of the sensory channels is fixed and immutable. Rather, from the level of nociceptor to the supraspinal sites of integration, the nervous system is characterized by its dynamic response to tissue insult. Some subjects may have an exaggerated perception of pain to a stimulus. This is seen in conditions such as postherpetic neuralgia (shingles) and is termed hyperalgesia. Hyperalgesia is an enhanced response to a stimulus that is normally noxious. Primary hyperalgesia refers to enhanced sensitivity to stimuli applied to a site of injury and secondary hyperalgesia refers to enhanced sensitivity to uninjured tissue adjacent to site of injury. The mechanisms are sensitization of nociceptors in primary hyperalgesia and increased excitability of central (spinal) neurons in secondary hyperalgesia. This change in excitability is believed to arise principally through the action of N-methyl-D-aspartate (NMDA) receptors, but contributions by AMPA (alpha-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid), kainate, and substance P are also likely. Epidemiological and Pathological Approach to Pain

This approach requires knowledge of the causes of disease within the population from which the patient comes. Generally good epidemiological data is scant from developing regions, but generally speaking, infectious causes will predominate in these regions, whereas noninflammatory and functional conditions will be common in developed regions. In a study from the US by Klinkman and co-workers, 60% of chest pain in primary-care settings was nonorganic, that is, not of cardiac, gastrointestinal, or pulmonary origin. Thirty percent of all chest pain was musculoskeletal, just less than half of those were costochondritis, and reflux esophagitis accounted for 13%. Stable angina pectoris accounted for 11% of all chest pain and only 1.5% was caused by unstable angina.

192 SYMPTOMS OF RESPIRATORY DISEASE / Chest Pain Table 1 Anatomical and pathological classification of causes of chest pain Chest wall

Airway Pleuropericardial

Vascular

Cardiovascular

Mediastinal Gastrointestinal

Miscellaneous

Skin diseases, muscular strain, intercostal myositis, postherpetic neuralgia and acute herpes zoster (shingles), Coxsackie B infection (Bornholm’s disease – ‘epidemic pleurodynia’), thoracic nerve infiltration or compression, rib fracture or metastases, costochondritis, slipping rib syndrome, vertebral disease, rheumatic disease (fibromyalgia or soft tissue rheumatism) and nonrheumatic disease Tracheitis, central bronchial carcinoma, foreign body, postintubation Pleurisy/serositis, pleural effusion, pleural tumors, pericarditis and postpericardiotomy syndrome, postmyocardial infarction syndrome (Dressler’s syndrome), pneumothorax, pneumonia, mesothelioma, metastatic carcinoma Aortic dissection, aortic aneurysm Pulmonary hypertension from embolism, vasculitis and primary pulmonary hypertension Myocardial ischemia or infarction, myocarditis, pulmonary stenosis, aortic stenosis, aortic aneurysm (including dissection and rupture) Mediastinitis, mediastinal adenopathy Reflux esophagitis, esophageal hyperalgesia, esophageal spasm, esophageal motility disorders, achalasia, and esophageal rupture (Boerhawe’s syndrome) Psychogenic pain and referred pain (from subphrenic pathology)

adapting stretch receptors in the conducting airways, myelinated axons that lead from rapidly adapting irritant (cough) receptors, and unmyelinated axons that connect an extensive network of C fibers that have been divided into pulmonary (J receptors) and bronchial C fibers. The parietal pleura that lines the interior of the ribcage and outer portion of each hemi-diaphragm is innervated by neighboring intercostal nerves and pain is localized to the cutaneous distribution of these nerves. The parietal pleura that lines the central portion of each diaphragm is innervated by fibers that travel with the phrenic nerves. When this portion of the diaphragm is stimulated, it results in referred shoulder or neck pain on the same side. The reason is that the diaphragm migrates embyronically from its cervical origin (cervical spinal cord segments 3, 4, and 5) to its final position in the thorax. Pleuritic pain tends to be largely limited to the affected region rather than diffuse. Pain is aggravated by each inspiration, so patients become aware of breathing and may experience dyspnea. The rapidity of onset of pleural pain may give a clue to its etiology. Rapid onset is associated with traumatic injury and spontaneous pneumothorax. A slower onset of minutes to hours heralds development of community-acquired pneumonia. Recurrent acute pleuritic pain is a feature of familial Mediterranean fever. Gradual onset suggests chronic illness such as malignancy or tuberculosis. Sometimes the pleural pain resolves when a sizable effusion develops, because the inflamed pleural surfaces are no longer apposed. Pulmonary Hypertension

An anatomical pathological classification of chest pain is presented in Table 1.

Clinical Phenotypes of Chest Pain Pleuropulmonary Disorders

The lung parenchyma and visceral pleura are considered to be insensitive to ordinary painful stimuli but pain can arise from stimulation of mucosa of trachea and main bronchi. The visceral pleura are not innervated by nociceptors, but inflammatory processes in the periphery of the lung may involve adjacent structures, as mentioned previously. Autonomic afferent fibers that transmit pain sensations from pleuropulmonary structures are anatomically associated with both sympathetic and parasympathetic fibers but those that travel in the vagus nerve are far more important. These include myelinated axons that carry impulses from slowly

The pain of pulmonary hypertension may be a crushing or constricting substernal pain which may radiate to the arms or neck. This pain is not particularly common but may occur in acute and chronic cases of pulmonary hypertension and is poorly understood. In acute situations such as massive pulmonary embolism, the pain may be due to sudden distension of the main pulmonary artery with subsequent stimulation of mechanoreceptors. In primary pulmonary hypertension, the pain, which occurs in about 50% of patients, may be provoked by right ventricular ischemia or by compression of the coronary artery by the dilated pulmonary trunk. Tracheobronchitis

Pain of tracheal origin is felt in the midline anteriorly from the larynx to xiphoid cartilage, and from either main bronchus in the anterior chest near the sternum. It occurs in the presence of viral or bacterial tracheobronchitis or, less often, with malignancy.

SYMPTOMS OF RESPIRATORY DISEASE / Chest Pain

Induced tracheal pain is abolished by vagal blockade or vagotomy. Disorders of Chest Wall

Inflammation or trauma of the joints, muscles, cartilages, bones, and fascia of the thoracic cage is a common cause of chest pain. Fibromyalgia and other rheumatological disorders can cause chest pain that may mimic that of myocardial ischemia. Ankylosing spondylitis may involve the ribcage. Rare causes of pain arising from the chest wall include chronic inflammation of the xiphoid process (xiphodynia) and superficial (migratory) thrombophlebitis of the chest wall (Mondor’s syndrome). Injuries to ribs and muscles of chest wall are common causes of chest pain and the etiology is evident from the history. Sometimes, there may be a delay in recognition after apparent minor trauma. Metastatic malignancy may present with spontaneous rib fractures. Severe coughing can result in rib fractures and result in localized chest pain. Costochondritis is due to inflammation of the cartilaginous insertion of the ribs into the sternum and may involve multiple ribs, commonly on one side. It may cause considerable diagnostic confusion. Its etiology is unknown, but it may be hereditary, viral, posttraumatic, associated with fibromyalgia, arthritis, or inflammatory bowel disease. Tietze’s syndrome is idiopathic and similar to costochondritis except that it manifests with a nonsuppurative swelling at these sites, especially the second rib, and always affects the same sites. The pain is usually localized and sharp but can mimic angina when it is dull and aching in nature. It lasts from hours to weeks. The diagnostic feature is point tenderness over one or more costal cartilages. Epidemic pleurodynia or Bornholm’s disease is an acute infectious febrile disorder caused by a group B Coxsackievirus. It manifests with severe epigastric or thoracic pain and is most common in children. Clinical onset is with severe, frequently intermittent, often pleuritic pain in the epigastrium or lower anterior chest, with fever and often headache, sore throat, and malaise. Local tenderness, hyperesthesia, muscle swelling, and myalgias of the trunk and extremities may occur. The disease usually subsides in 2–4 days, but relapse may occur within a few days and symptoms may continue or recur for several weeks. Up to 5% of the cases are complicated by aseptic meningitis, orchitis, and, less commonly, fibrinous pleuritis and pericarditis. Diagnosis is obvious during an epidemic. However, in sporadic cases or in the early stages of an epidemic, the disease may be mistaken for spontaneous pneumothorax, acute

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appendicitis, pancreatitis, costochondritis, a perforated viscus, an influenza-like respiratory infection, or myocardial infarction (MI). Laboratory diagnosis consists of demonstrating a rise in specific neutralizing-antibody titers or isolating the virus on a throat or stool culture. Prognosis is good in uncomplicated cases, although a few deaths have been reported. Treatment is symptomatic. Neuritis/Radiculitis

Pain due to neuritis or radiculitis that originates from disorders of the dorsocervical spine is frequently perceived as chest pain. It presents as a lancinating electric shock-like pain over dermatomes of the respective intercostal nerves. In some patients, the diagnosis becomes obvious after 2–3 days when the vesicular rash due to herpes zoster (shingles) erupts. Myocardial Ischemia

One of the most important and dreaded causes of chest pain that instils fear and anxiety amongst patients is the pain of myocardial ischemia, most frequently angina pectoris, which may portend a MI. Whilst this chapter deals with respiratory causes of chest pain, it is imperative to exclude myocardial ischemia as the source of chest pain. Often, reassuring patients is all that may be required. The clinician has a difficult task as the pain is often atypical and there is always a concern that myocardial ischemia may be overlooked, which can lead to over-investigation. In its classic form, angina pectoris is defined as a discomfort in the chest and/or adjacent area associated with myocardial ischemia but without myocardial necrosis. It is important to recognize that angina means tightening, not pain. Thus, the discomfort of angina often is described not as pain but rather as an unpleasant sensation; ‘pressing’, ‘squeezing’, ‘strangling’, ‘constricting’, ‘bursting’, and ‘burning’ are some of the adjectives commonly used to describe this sensation. ‘A band across the chest’, ‘a weight in the center of the chest’, and ‘a vice tightening around the chest’ are other frequent descriptors. It is characteristic of angina pectoris that the intensity of effort required to incite it may vary from day-to-day and throughout the day in the same patient, but often a careful history will uncover explanations for this, such as meals ingested, weather, emotions, and the like. A history of prolonged severe anginal chest discomfort accompanied by profound fatigue often signifies acute MI. Sometimes the patient may describe the mid-chest as the site of the shortness of breath, as an ‘anginal equivalent’. There are numerous descriptors for angina referred to as ‘anginal equivalents’. These

194 SYMPTOMS OF RESPIRATORY DISEASE / Chest Pain

include the mid-chest as a site of shortness of breath, discomfort in the radiation sites such as the ulnar aspect of the left arm and forearm, lower jaw, teeth, neck, or shoulders, and the development of gas and belching, nausea, ‘indigestion’, dizziness, and diaphoresis. When angina radiates to the arms, it is often described as a ‘painful heaviness’. Anginal equivalents above the mandible or below the umbilicus are quite uncommon. Embryologically, the heart is a midline viscus; thus, cardiac ischemia produces symptoms that are characteristically felt substernally or across both sides of the chest. Some patients complain of discomfort only to the left or, less commonly, only to the right of the midline. Features that make myocardial ischemia unlikely include: (1) pain or discomfort localized to the skin or superficial structures and reproduced by localized pressure suggesting origin from the chest wall; (2) if the patient can point directly to the site of discomfort with the tip of a finger, and if the site is quite small (o3 cm in diameter); and (3) pain that is localized to the region of or under the left nipple, or one that radiates to the right lower chest in origin and may be functional, or due to costochondritis, gaseous distension of the stomach, or the splenic flexure syndrome. Characteristic radiation of chest pain due to myocardial ischemia to the arm, neck, jaw, and epigastrium may also occur in pericarditis and disorders of the cervical spine. Dissection of the aorta or enlargement of an aortic aneurysm usually produces pain in the back in addition to the front of the chest. Angina pectoris is relatively brief, usually lasting from 2 to 10 min. However, if the pain is very brief (i.e., a momentary, lancinating, sharp pain, or other discomfort that lasts less than 15 s), angina can usually be excluded; such a short duration points instead to musculoskeletal pain, pain due to hiatal hernia, or functional pain. Pain that is otherwise typical of angina but that lasts for more than 10 min, occurs at rest, or changes in pattern is typical of unstable angina. Chest pain lasting for hours may be seen with acute MI, pericarditis, aortic dissection, musculoskeletal disease, herpes zoster, and anxiety. Prinzmetal’s (variant) angina characteristically occurs at rest and may or may not be affected by exertion; however, classical angina, although most often precipitated by effort, may progress to unstable angina, which is characterized by ischemic discomfort at rest. Pericarditis

There is very little information on the sensory innervation of the pericardium. There may be pain

receptors in the diaphragmatic portion of the parietal pericardium which are innervated by sensory axons that travel in the phrenic nerves. Stimulation of these fibers may result in referred pain to the upper margins of the trapezius. This is the characteristic of pericardial pain and is typically relieved by sitting up or leaning forward. Aortic Valve Disease

Angina-type pain may occur in two-thirds of patients with severe aortic valvular stenosis. The cause of this pain is unknown but may be related to high systolic and diastolic wall stresses and reduced coronary flow reserve that follows severe aortic stenosis. A similar type of pain may also accompany severe aortic regurgitation less commonly and is explained by reduced coronary blood flow. Dissection of the Aorta

The pain of dissection is usually sudden and extremely severe at the onset of dissection. The pain is characteristically tearing or crushing and spreads widely as the dissection extends from its origin. A careful history will identify this pattern and lead to appropriate emergency management. Associated features include autonomic symptoms of sweating, nausea, and vomiting. Gastrointestinal Disorders

A variety of gastrointestinal tract disorders account for 10–30% of cases of chest pain in primary care. Pain from esophageal disease may be commoner than previously thought and may coexist or contribute to the pain of coronary artery disease. Nociceptors have not been identified clearly in the esophagus but low- and high-threshold mechanoreceptors, chemoreceptors, and thermoreceptors are thought to mediate pain sensation when stimulated. Esophageal pain is usually referred to midline structures but may radiate to the arms and also be palliated by nitrates making diagnostic distinction between esophageal pain and coronary ischemia difficult. Generally, pain that lasts for longer than an hour with associated odynophagia or dysphagia suggests an esophageal origin. Hyperalgesia, as mentioned previously, may be an important mechanism of pain amplification in esophageal disease. Other gastrointestinal causes of chest pain include cholecystitis, peptic ulcer disease, and acute pancreatitis and motility disorders. These give rise to referred pain and are mediated by autonomic nerves. In addition, disease in the liver (liver abscesses that abut onto the diaphragm) and splenic disease can

SYMPTOMS OF RESPIRATORY DISEASE / Chest Pain

manifest with chest pain, usually referred to the shoulder region. Psychogenic Chest Pain

The origin of chest pain may be obscure in many patients despite exhaustive investigations in many patients. It may account for 30–50% of chest pain presenting to primary care in the US. Many of these have underlying psychosocial factors responsible for the chest pain. Neurocirculatory asthenia (‘irritable heart’) is a condition which includes atypical angina, dyspnea, and weakness in its manifestation. There is also a strong association between chest pain and anxiety disorders. The mechanism may be abnormality in visceral pain perception rather than stimuli from viscera. Hyperalgesia may play a role. The diagnosis becomes difficult because patients with genuine cardiac chest pain may also demonstrate neuropsychiatric symptoms at presentation. Therefore the diagnosis of psychogenic chest pain is one of exclusion.

A Practical Approach to Chest Pain Given the complexity of chest pain, it is difficult to create a practical algorithm for the approach to chest pain in primary care. Patients who are at risk for serious causes of chest pain, based on the characteristics of the pain and the known risk factors, may require emergency management. MI must be considered in any adult patient presenting with recent onset chest pain, especially if combined with any risk factors and features on history or physical examination to suggest cardiovascular instability. They should be referred to an emergency facility without delay. A defibrillator should be readily available. Intravenous access and supplemental oxygen should be given. If myocardial ischemia is suspected, 150 mg of aspirin should be given. A 12 lead electrocardiogram and blood for troponin T and/or cardiac enzymes should be obtained if facilities permit. Conditions such as a dissecting aortic aneurysm, pneumothorax, massive pleural effusion, severe pneumonia, esophageal rupture, and cardiac tamponade present dramatically and would be readily evident from the history and examination, combined

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with a high index of suspicion. Where possible and indicated, an urgent chest radiograph and/or computerized tomographic scan of the chest, and arterial blood gas estimation should be obtained. Targeted investigations, referral, and appropriate management should follow. Trauma is usually evident clinically. Assessment of vital signs and stabilization of patients is mandatory. Once these serious causes have been excluded, the clinician may then consider the less sinister causes of chest pain and investigate and manage as dictated from an anatomical, pathophysiologic, and epidemiologic approach. See also: Chest Wall Abnormalities. Mediastinal Masses. Mediastinitis, Acute and Chronic. Pleural Effusions: Overview. Pneumothorax. Vascular Disease.

Further Reading Braunwald E, Libby P, and Zipes DP (eds.) (2004) Braunwald’s heart disease. A Textbook of Cardiovascular Medicine, 7th edn., sec. 7, pp. 1129–1139. Saunders. Cervero F (1994) Sensory innervation of the viscera: peripheral basis of visceral pain. Physiological Reviews 74: 95–138. Eslick GD (2004) Noncardiac chest pain: epidemiology, natural history, health care seeking, and quality of life. Gastroenterology Clinics of North America 33: 1–23. Ginsburg GS (1996) The evaluation of chest pain in women. New England Journal of Medicine 333: 1311–1315. Klinkman MS (1994) Episodes of care for chest pain: a preliminary report from MIRNET. Journal of Family Practice 38: 345. Manning HL and Scheartzstein RM (1995) Efferent and afferent signals that contribute to the sensation of dyspnea. New England Journal of Medicine 333: 1548. Meisel JL (2005) Differential diagnosis of chest pain. Uptodate 13.1. Murray JF, and Nadel JA (2000) Textbook of Respiratory Medicine, 3rd edn., pp. 567–582. Saunders. Sugiura Y, and Tonosaki Y (1995) Spinal Organization of Unmyelinated Visceral Afferent Fibers in Comparison with Somatic Afferent Fibers, Progress in Pain Research and Management Series Vol. 5, pp. 41–59. Seattle: IASP Press.

Relevant Website www.uptodate.com – UpToDate is specifically designed to answer the clinical questions that arise in daily practice and to do so quickly and easily so that it can be used right at the print of case.

196 SYSTEMIC DISEASE / Sarcoidosis

SYSTEMIC DISEASE Contents

Sarcoidosis Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome Langerhans’ Cell Histiocytosis (Histiocytosis X) Sickle Cell Disease Eosinophilic Lung Diseases

Sarcoidosis C Agostini, Universita` degli Studi di Padova, Padua, Italy M Maffessanti, Universita` degli Studi di Trieste, Trieste, Italy G Semenzato, Universita` degli Studi di Padova, Padua, Italy & 2006 Elsevier Ltd. All rights reserved.

Abstract Sarcoidosis is a multisystem disorder of unknown cause(s) most commonly affecting young adults, and frequently presenting with hilar lymphadenopathy, pulmonary infiltration, and ocular and skin lesions. The diagnosis is established most securely when well-recognized clinicoradiographic findings are supported by histological evidence of widespread noncaseating epithelioid cell granulomas in more than one system. Because of the multiorgan involvement, sarcoidosis can present to clinicians in almost every specialty, but most often to chest physicians, opthalmologists, and dermatologists. The course and prognosis correlate with the mode of onset. An acute onset is usually indicative of a self-limited course of spontaneous

resolution whereas an insidious onset may be followed by relentless, progressive pulmonary fibrosis. Current therapy uses both anti-inflammatory agents (e.g., steroids) and immunosuppressive drugs (e.g., methotrexate).

Introduction Sarcoidosis is a multisystem granulomatous disorder which needs a synthesis of clinical features, histology, radiology, biochemical changes, and immunological data to distinguish this disease from non-specific sarcoid-tissue reactions. There are two different types of sarcoidosis (Table 1). Acute sarcoidosis (Loefgren syndrome) has an abrupt start with acute inflammatory lesions, bilateral hilar lymph nodes enlargement, high-grade fever, and erythema nodosum. These patients show a high incidence of spontaneous remission within two years, a good response to steroids and other anti-inflammatory drugs, and a good prognosis. In fact, the skin lesions subside within a month of onset, and the hilar adenopathy regresses within

Table 1 Main differences between acute and chronic sarcoidosis Features

Acute sarcoidosis

Chronic sarcoidosis

Course Age (years) Onset Prognosis Histology Chest X-ray Bronchoalveolar lavage Gallium scan Serum ACE levels Skin Eye Parotids Lymphadenopathy Splenomegaly Facial palsy Bone cystis Heart Calcium metabolism Resolution Recurrence after therapy

Transient 30 Abrupt Good Epithelioid/giant cells Hilar adenopathy T-cell alveolitis Positive Increased Erythema nodosum macuolopapular rashes Acute iritis, conjunctivitis conjunctival nodules Transient enlargement Transient Transient Transient No Arrhythmias Hypercalcemia, calciuria Frequently spontaneous Rare

Persistent 40 Insidious Fair Hyaline fibrosis Lung infiltration, fibrosis Normal, neutrophil alveolitis Positive Normal Lupus pernio plaques, ulcers, scar involvement, keloids Chronic uveitis, glaucoma, keratoconjunctivitis sicca Rarely persistent enlargement Rarely persistent Rarely persistent Rarely persistent Yes Cor pulmonale Nephrocalcinosis Rare Frequent

SYSTEMIC DISEASE / Sarcoidosis 197

one year; recurrences are rare. Chronic sarcoidosis has an insidious onset, grumbling relapsing course, fibrotic clinical and histological pictures in the lung, and a poor prognosis despite treatment with ultimate chronic respiratory failure. Sarcoidosis may be expected to resolve in more than 50% of patients, but resolution is less likely in older patients and those with accompanying extrathoracic disease, particularly bone and skin lesions. In the practical management of sarcoidosis, the disease would remain a relatively benign and unimportant disease but for the development of irreversible fibrosis in the lungs and eyes, and nephrocalcinosis. Thus, in assessing the prognosis, it is important to evaluate the degree of functional failure of lungs, heart, eyes, and kidneys.

Etiology The etiology of sarcoidosis is unknown. The seasonal peak, which occurs during spring months, has been described in several countries suggesting that an environmental factor may be associated with an increase in sarcoidosis incidence in spring. Cases of clustering have been also described indicating that individuals exposed to patients with sarcoidosis may be at higher risk of disease development, possibly due to a transmissible microbial agent. Several studies have attempted to isolate infective causative agents from sarcoid tissues. Various pathogens have been cultured from the serum, skin lesions, or lymph nodes of sarcoid patients (Table 2); however, their presence seems to be largely coincidental rather than causal. More convincing data have been provided by groups showing mycobacterial DNA or RNA in samples from patients with sarcoidosis by molecular biological techniques including hybridization techniques and amplification by PCR. Nonetheless, it should be noted that the presence of mycobacterial DNA in sarcoidosis tissue varies considerably and that some series of patients were completely negative, even when highly sensitive methodologies were utilized. There is

Table 2 Tentative list of infective agents that have been claimed as causative agents of sarcoidosis Bacterial microorganisms

Viral microorganisms

Aspergillus sp. Borrelia burgdorferi Chlamydia pneumoniae Mycobacteria Nocardia sp. Propionbacteria Rickettsia helvetica Yersinia enterocolitica

Epstein–Barr virus Hepatitis C virus Herpes simplex virus Human herpes virus type 8 Parainfluenza viruses Retroviral particles Rubella viruses

also evidence obtained by Japanese researchers suggesting that Propionibacterium acnes, a bacterial commensal which causes a granulomatous reaction when injected experimentally into sensitized rats and rabbits, may be involved in the development of the disease, at least in some geographical areas.

Genetic Factors Epidemiological studies have reported differences in the occurrence and course of sarcoidosis among races and populations. The disease is more frequent in developed countries and in some ethnic groups. For instance, white Scandinavians have the highest tendency along with African-Americans; AmericanHispanics seem to have a greater incidence than Caucasians for this disease. Moreover, the possibility of family clustering is generally accepted; individuals who have a family member with sarcoidosis show an increased risk of disease compared with the general population. Furthermore, it is also clear that the occurrence of familial sarcoidosis is not restricted to individual races or ethnic groups. Thus, a shared determinant (either genetic or environmental) seems to be operating in familial sarcoidosis. Many polymorphic genes have been suggested to contribute to genetic susceptibility to sarcoidosis (Table 2). Contribution to disease pathogenesis has been evaluated for genes encoding HLA products (classical and nonclassical), chemokine receptors, angiotensin converting enzyme, vitamin D3 hydroxylase, T-cell receptor genes, erythrocyte complement receptor 1, Gm and Km immunoglobulin, and complement receptor 1. Recent data have suggested that sarcoidosis is associated with a truncating splice site mutation in BTNL2 gene suggesting that this could favor dysregulated Th cell activation.

Pathology Sarcoid granuloma is the end result of a complex interplay of invading antigen, prolonged antigenemia, macrophage presentation, CD4 Th1 T-cell response, B cell hyperactivity, circulating immune complexes, and numerous biological mediators in the production of sarcoid granulomas in all organs and systems. Additional factors are HLA affinity, hormones, ethnic background, and possible enzyme defects. It is important to point out that sarcoid granuloma does not differ morphologically or immunohistologically from granulomas due to other causes. Its central core is made up of a number of monocyte/macrophages at various states of activation and differentiation, epithelioid cells, and multinucleated giant cells; furthermore, a number of CD4 T lymphocytes and

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plasma cells surround the central core of the sarcoid granuloma. The periphery of the granuloma contains a large number of antigen-presenting, interdigitating macrophages associated with CD8 T suppressor– cytotoxic lymphocytes. Caseous necrosis is not a common feature in sarcoidosis even if a minor degree of fibrinoid-type necrosis is demonstrated in the center of larger granulomas. Interestingly, adjacent granulomas may show different phases of development and resolution and can be next to areas of advanced fibrosis. Finally, the involvement of blood vessels is a frequent finding in tissues involved by granulomatous lesions. Consequently, perivascular distribution and destruction of vascular elastic tissue by granulomas are common.

infection. Pleurisy, pleural effusion, and clubbing are so infrequent that their presence raises the probability of another diagnosis. Spontaneous pneumothorax is a rare complication of the late stage of chronic fibrotic sarcoidosis; it also raises the possibility of another granulomatous lung disease, namely Langerhans’ granulomatosis. Radiologic Findings

The international statement on sarcoidosis recommends that a conventional chest radiograph represents the basis for staging, and that a grading system from 0 to IV must be used: Stage 0: Clear chest radiograph Stage 1: Hilar lymphadenopathy Stage 2: Hilar lymphadenopathy and pulmonary infiltration Stage 3: Pulmonary infiltration without hilar lymphadenopathy.

Clinical Features Sarcoidosis presents to chest physicians, dermatologists, and ophthalmologists predominantly in the 20 to 50 years age group and is much less frequent in children and the elderly. It has an equal sex distribution, except for erythema nodosum and lupus pernio which are far more frequent in women. The features are more florid in the black community with more severe uveitis, widespread skin lesions, nasal crusting, and more frequently, lacrimal gland enlargement. It is most frequent in the black population of the United States and the West Indies. Erythema nodosum and hilar lymphadenopathy (Lo¨fgren’s syndrome) are commonly recognized in women aged about 25 years, generally during pregnancy or lactation. The general examination of the patient must be thorough and should include slit-lamp examination of the eyes, chest radiography, and measurements of 24 h urine calcium. The clinical diagnosis then needs to be confirmed by histology, followed by assessment of the activity of the disease.

Intrathoracic Sarcoidosis All types of intrathoracic sarcoidosis may be expected to resolve in 60% of patients, but resolution is less likely in older patients and in those with accompanying extrathoracic disease, particularly bone and skin lesions. By contrast, the combination of pulmonary and cutaneous sarcoidosis constitutes the hallmark of chronicity, presumably because longstanding fibrosis has supervened. Despite a paucity of symptoms, there may be widespread radiographic changes. Wheezing or bronchospasm point to segmental bronchial narrowing. Breathlessness suggests chronic fibrotic disease. Hemoptysis also indicates this late irreversible stage with possible cavitation and/or secondary Aspergillus

This staging is simple and, most important, it is recognized and understood throughout the world. Some centers make refinements, such as adding a still more advanced Stage 4 in which there is considerable fibrosis, distortion of the architecture of the lungs and perhaps cavitation, and evidence of complicating cor pulmonale. The differential diagnosis of intrathoracic disease varies with the stage (Table 3). Table 3 Different diagnosis of intrathoracic sarcoidosis by chest X-ray Stage

Type

Differential diagnosis

1

Hilar adenopathy

2

Hilar adenopathy and pulmonary infiltration

3 and 4

Diffuse pulmonary infiltration

Tuberculosis, Hodgkin’s and non-Hodgkin’s disease, leukemia, enlarged pulmonary arteries, metastatic localization of cancers Tuberculosis, pneumoconiosis, lymphangitic carcinomatosis, alveolar cell carcinoma Langerhans’ granulomatosis, idiopathic hemosiderosis As for stage 2 Hypersensitivity pneumonitis, chronic beryllium disease, other diseases associated with fibrosing alveolitis, systemic sclerosis, Sjogren’s syndrome, rheumatoid arthritis, idiopathic pulmonary fibrosis, other granulomatoses, carcinomatosis, irradiation fibrosis, schistosomiasis, alveolar proteinosis

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Conventional chest X-ray radiography should be complemented by computed tomography(CT) scanning which is helpful in clarifying atypical manifestations and in determining areas of activity indicated by ground-glass attenuations (Figure 1). CT abnormalities correlate well with pulmonary function tests. Pulmonary Function Tests

Pulmonary sarcoidosis is characterized by a restrictive defect, an impaired transfer factor and a lowered co-efficient (KCO). The response of KCO to exercise is a sensitive index of abnormal function for it reveals impairment even in the presence of an apparently normal chest radiograph. Impairment depends on the size and number of pulmonary granulomas and the degree of alveolitis. With increasing pulmonary infiltration, there is a reduction of vital capacity, airway resistance, maximum mid-expiratory flow, and arterial oxygen tension on exercise. Airflow

Figure 1 HRCT aspects of pulmonary sarcoidosis.

obstruction in small airways is commonly noted and reduction may be due to loss of elastic recoil. Airway hyperreactivity may be demonstrated by bronchoprovocation testing with methacholine challenge; such hyperreactive patients have more wheezing and coughing, more airway obstruction, smaller vital capacity, and lower diffusing capacity for carbon monoxide. An obstructive defect may be evident by flow–volume loops even in the presence of a normal chest radiograph. Bronchoscopy, Transbronchial Biopsy, and Bronchoalveolar Lavage

Biopsy specimens from the mucosa or from the parenchyma by transbronchial technique may be obtained using a fiber bronchoscope. A bronchoalveolar lavage (BAL) CD4/CD8 ratio 43.5 suggests the presence of a sarcoidosis but it is not diagnostic. BAL is used in some centers to assess the pulmonary activity of the disease during the follow-up.

200 SYSTEMIC DISEASE / Sarcoidosis Additional Techniques

Skin Lesions

If the intrathoracic lymph node enlargement is unilateral, mediastinoscopy should be considered to rule out a malignant lymphoma. Since biopsy specimens for histologic examination can be obtained from the salivary glands, conjunctiva, lymph nodes, spleen, and liver, the use of video-assisted thoracoscopy or thoracotomy to obtain biopsy specimens is usually limited to a selected number of cases where it is mandatory for the diagnosis of the pulmonary involvement to initiate the treatment.

A variety of skin lesions is seen in combination with intrathoracic disease, bone cysts, and uveitis.

Sarcoidosis of the Upper Respiratory Tract Sarcoidosis of the upper respiratory tract (SURT) occurs in only about 6% of patients with sarcoidosis. It is twice as frequent in women and presents at the age of about 35 years. It involves nasal mucosa with nasal septal perforation, the pharynx, larynx, and with nasal bone involvement. It is associated with chronic fibrotic disease in the lungs, eyes, parotids, and with lupus pernio. It has an insidious onset, progressive fibrotic course, and chest X-ray resolution is infrequent. SURT must be differentiated from other granulomatoses affecting the upper respiratory tract, including tuberculosis, leprosy, and Wegener’s granulomatosis.

Ocular Involvement Sarcoidosis involves both anterior and posterior segments of the eye, so clinical presentations include anterior and posterior uveitis; choroidoretinitis; periphlebitis retinae; retinal, macular, and optic nerve edema; retinal hemorrhage; and rarely, neovascularization (Table 4).

Erythema Nodosum

Erythema nodosum presents with red, hot, tender symmetrical lesions on the shins, calves and knees; buttocks and arms are less often involved. It is usually associated with polyarthralgia, bilateral hilar lymphadenopathy, and acute iritis. Its common association in women with pregnancy, lactation, and a gynecological disease suggests that hormonal cofactors are important pathogenetic players. Lupus Pernio

Lupus pernio is a chronic persistent violaceous lesion which usually affects nose, cheeks, and ears; it is more frequently seen in women. Usually associated with chronic fibrotic sarcoidosis, it progresses indolently over the years. Nail Dystrophy

Nail dystrophy is infrequent. It is most often seen in persons with lupus pernio and bone cysts; it reflects chronic sarcoidosis. Other Skin Lesions

Other skin lesions include persistent plaques, maculopapular eruptions, and keloids. Previously atrophic scars may be involved by sarcoidosis and become livid and purple, proclaiming a flare-up of sarcoidosis.

Neurosarcoidosis Neurosarcoidosis occurs in about 4–7% of patients. Facial palsy is the most frequent presentation, either alone or with other cranial nerve palsies, or with

Table 4 Ocular involvement in sarcoidosis Manifestation

Features

Anterior uveitis

This frequent ocular finding may be subdivided into acute or chronic depending on its onset, course, and clinical accompaniment Due to granulomas involving the canal of Schlemm. Rare since the introduction of steroid therapy Abrupt onset with pain, photophobia, blurred vision, and reddening of the eye Insidious onset, prolonged course, and fibrosis leading to secondary glaucoma and cataract formation Incidence underestimated because it is often obscured by a turbid anterior chamber Frequent in women and associated with facial palsy, pain, and hyperalgesia of the trunk Bilateral non-specific conjunctivitis associated with erythema nodosum and hilar adenopathy is a manifestation of acute sarcoidosis Often feature of acute sarcoidosis may be associated with erythema nodosum and hilar adenopathy Typical of chronic, fibrotic, longstanding sarcoidosis A biopsy of a minor salivary gland may allow to distinguish enlargement due to other causes

Glaucoma Acute iridocyclitis Chronic iridocyclitis Posterior uveitis Papilloedema Conjunctival involvement Scleritis Keratoconjunctivitis sicca Lacrimal gland involvement

SYSTEMIC DISEASE / Sarcoidosis 201

papilloedema. Other features include peripheral neuropathy, myopathy, meningitis, space-occupying brain lesions, epilepsy, cerebellar ataxia, hypopituitarism, and diabetes insipidus.

Myocardial Sarcoidosis Myocardial sarcoidosis should be considered if a sarcoid patient develops bundle branch block, arrhythmias, congestive cardiac failure, pericarditis, or clinical evidence of cardiomyopathy. Sudden death without previous evidence of a heart lesion occurs particularly in patients over 40 years of age. Holter monitoring is crucial when myocardial sarcoidosis is suspected. Magnetic resonance imaging, two-dimensional echocardiogram, myocardial perfusion imaging, and cardiac positron emission tomography may be helpful in disclosing myocardial sarcoidosis. These techniques may localize the site of a multifocal inflammatory or infiltrative heart disease, allowing a more accurately directed endomyocardial biopsy.

sarcoidosis. In the majority of cases, renal calculi at presentation represent a marker of chronic disease.

Sarcoidosis in Pregnancy Sarcoidosis is not a contraindication to pregnancy. It often improves during pregnancy. The fetus is unaffected, and normal delivery can be expected. Chest radiography is contraindicated. Special attention should be paid to calcium metabolism that may be abnormal in pregnancy.

Childhood Sarcoidosis Sarcoidosis is infrequent in childhood. When it does occur, lesions can occur in any tissue or organ. The most common physical findings include peripheral lymphadenopathy, uveitis or iritis, skin and bone lesions, and liver or spleen enlargement.

Pathogenesis

Hepatic granulomas are found in two-thirds of aspiration liver biopsies when this procedure is used for confirming the diagnosis of sarcoidosis. Despite this, portal hypertension, intrahepatic cholestasis, or liver fibrosis are extremely rare complications in sarcoid patients. Although sarcoidosis may affect almost every tissue of the alimentary tract, the gastrointestinal involvement is rarely symptomatic. The pancreas is the least frequently involved abdominal organ and the sarcoid process may mimic pancreatic neoplasia. Two-thirds of these patients experience abdominal pains.

Since in hypersensitivity reaction, the granuloma structure is aimed at containing the dissemination of inciting agents, it is to be expected that the inflammatory response spontaneously clears once etiologic factors are isolated. However, this paradigm is not true in patients with sarcoidosis. Evidence suggests that in some patients the continuous formation of immune granulomas is a reflection of the dysregulation of mechanisms controlling T-cell homeostasis. In the presence of a chronic antigenic stimulation, macrophage–T-cell interaction perpetuates the local inflammatory processes and the continual formation of new granulomas. In turn, in the lung the invasion of sarcoid granuloma alters the permeability of type I cells causing alveolar and interstitial edema and derangement of alveolar structures. This leads to physiologic repair reactions by parenchymal cells which try to restore and re-establish normal alveolar structures. The repair processes lead to an exaggerated production of collagen associated with fibroblast migration and proliferation, and an abnormal increase in the extracellular matrix with derangement of alveolar structures and resultant fibrosis. With continued activity of the inflammatory cells, fibrosis becomes more widespread and involves the vasculature.

Renal Involvement

Experimental Models

Sarcoidosis may cause renal disease producing a granulomatous interstitial nephritis or, as a consequence of abnormal calcium metabolism, leads to nephrocalcinosis and urolithiasis. Renal calculi may be an unusual but possible primary manifestation of

The Kveim–Siltzbach (KS) skin test consists of the intradermal injection of a heat-sterilized extract obtained from splenic tissues of individuals with sarcoidosis. The cutaneous reaction mimics the naturally occurring sarcoid granuloma and its positivity

Bone Involvement Sarcoidosis of bone most often involves hands or feet, and rarely, temporal or frontal bones or the hard palate. It is associated with soft tissue swelling, joint stiffness, and pain. There is no correlation between bone involvement and abnormal calcium metabolism.

Hepatic, Pancreatic and Alimentary Tract Involvement

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has been used as a tool to confirm the diagnosis of sarcoidosis. After the injection of the extract, a subcutaneous lymphocytic infiltrate sustained by CD4þ helper-related cells develops. Within 3 weeks a granuloma is formed, which shows morphological and immunological features of the sarcoid nodule; the central area contains particulate KS material and aggregates of monocyte–macrophages, and is surrounded by a number of CD4 þ cells. The KS reaction has been also utilized as an in vivo model for studying the nature of the causative agent of sarcoidosis but despite extensive research efforts, all attempts to identify transmissible microbial agents in the several different KS batches have been unsuccessful. When administered by intradermal injection, several factors produce sarcoid-like lesions both in humans and animals. On this basis, other models of granuloma formation have been taken into account. Granulomas resembling those obtained with KS extracts have been reproduced by utilizing chemicals; bacterial, fungal, and viral products; autologous lung cells; and neoplastic cells. These models have undoubtedly improved our understanding of the mechanisms of granuloma formation but have not been useful in definitively identifing the agent responsible for sarcoidosis.

Management and Current Therapy Steroids

Steroids significantly influence the natural history of sarcoidosis by preventing ocular fibrosis and blindness, pulmonary fibrosis and pulmonary hypertension, and overcoming abnormal calcium metabolism. In patients with an acute onset, as in Lo¨fgren’s syndrome, oral steroids are uncommonly required and should not be routinely used. If signs of aggressive disease develop or if sarcoidosis involves extrathoracic sites (neurosarcoidosis, renal involvement, eyes, etc.) steroid treatment is warranted. The recommended dose is 20–40 mg daily of prednisolone or its equivalent. In most patients the dose may be slowly tapered, but once initiated, treatment should be continued for at least 8 months–1 year. In fact, relapses may occur during early steroid tapering or after discontinuation of therapy. Only patients who clearly benefit from steroids should be treated. Multiple relapses during tapering indicate that a low maintenance dose of steroids may be warranted even after regression of the symptoms. Immunosuppressants

Methotrexate is an alternative drug for long-term therapy in sarcoidosis. In patients with pulmonary

sarcoidosis, the treatment with methotrexate causes a drop in the number of pulmonary CD4 cells and a consequent decrease in the CD4/CD8 ratio. The drug is also particularly helpul in the treatment of chronic skin lesions, including lupus pernio. Azathioprine may be usefully employed as an adjunctive agent in patients with severe or refractory sarcoidosis unresponsive to corticosteroids. Furthermore, recent data indicate that the isoxazol derivative leflunomide shows immunosuppressive efficacy similar to that of methotrexate in sarcoidosis. Infliximab, a chimeric monoclonal antibody against tumor necrosis factor, has shown to be effective for the treatment of cutaneous sarcoidosis. Transplantation

This therapeutic option is rarely necessary because early diagnosis and early immunosuppressive treatment usually stop the progression of the disease. Nonetheless, transplantation may represent the only form of effective treatment for some patients with the chronic end-stage fibrosis. Unfortunately, recurrences of the same disease process can occur in transplanted organs. See also: Bronchoalveolar Lavage. Chemokines, CXC: CXCL10 (IP-10). Corticosteroids: Therapy. Genetics: Overview. Interleukins: IL-1 and IL-18; IL-15. Interstitial Lung Disease: Overview. Leukocytes: T cells. Mediastinal Masses. Pulmonary Fibrosis. Signs of Respiratory Disease: Breathing Patterns; Clubbing and Hypertrophic Osteoarthropathy; General Examination; Lung Sounds. Surgery: Transplantation. Symptoms of Respiratory Disease: Dyspnea. Upper Airway Obstruction.

Further Reading Agostini C, Meneghin A, and Semenzato G (2002) T-lymphocytes and cytokines in sarcoidosis. Current Opinion in Pulmonary Medicine 8: 435–440. Agostini C, Semenzato G, and James DG (1997) Immunological, clinical and molecular aspects of sarcoidosis. Molecular Aspects of Medicine 18: 91–165. Baughman RP (2004) Pulmonary sarcoidosis. Clinics in Chest Medicine 25: 521–530. Baughman RP, Lower EE, and du Bois RM (2003) Sarcoidosis. Lancet 361: 1111–1118. Hoitsma E, Faber CG, Drent M, and Sharma OP (2004) Neurosarcoidosis: a clinical dilemma. Lancet Neurology 3: 397–407. Hunninghake GW, Costabel U, Ando M, et al. (1999) ATS/ERS/ WASOG statement on sarcoidosis. American Thoracic Society/ European Respiratory Society/World Association of sarcoidosis and other granulomatous disorders. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 16: 149–173. Luisetti M, Beretta A, and Casali L (2000) Genetic aspects in sarcoidosis. The European Respiratory Journal 16: 768–780. McGrath DS, Goh N, Foley PJ, and du Bois RM (2001) Sarcoidosis: genes and microbes – soil or seed? Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 18: 149–164.

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Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome H R Collard, University of California, San Francisco, CA, USA M I Schwarz, University of Colorado Health Sciences Center, Denver, CO, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Diffuse alveolar hemorrhage (DAH) represents a medical emergency. There are many causes of DAH, including Goodpasture’s syndrome, vasculitidies, and collagen vascular disease. An organized approach to diagnosis is paramount. In patients suspected of having DAH due to Goodpasture’s syndrome, serologic evaluation searching for the presence of anti-basement membrane antibodies should be performed. If the diagnosis remains in question, emergent kidney biopsy should be obtained to demonstrate linear deposition of antibody along the glomerular basement membrane visible by direct immunofluorescence. Treatment of Goodpasture’s syndrome involves the combination of corticosteroids, cyclophosphamide or azathioprine, and plasmapheresis.

Diffuse alveolar hemorrhage (DAH) is a medical emergency, often leading to acute respiratory failure. Despite advances in the identification and management of DAH, it remains a highly morbid condition with substantial mortality. DAH is caused by a diverse group of disorders, among the most common of which is Goodpasture’s syndrome. Pulmonologists must be able to quickly identify the presence of DAH, expeditiously investigate potential underlying causes, and decide on therapy. This article reviews the clinical presentation of DAH and provides an approach to the identification of DAH and its underlying cause, with special attention given to one of the most common causes of DAH, Goodpasture’s syndrome.

Definitions The term ‘diffuse alveolar hemorrhage’ refers to a distinct form of pulmonary hemorrhage originating from the pulmonary microcirculation, which includes the alveolar capillaries, arterioles, and venules. This definition distinguishes DAH from other causes of pulmonary hemorrhage, such as bronchiectasis, malignancy, or infection, in which hemorrhage often arises from the bronchial (systemic) circulation. Bleeding in these conditions can be brisk, quickly flooding the alveoli and mimicking the clinical and radiographic appearance of DAH. Goodpasture’s syndrome, or anti-basement membrane antibody (ABMA) disease, is a clinical syndrome characterized by the combination of DAH

and glomerulonephritis. In three-fourths of cases, the pulmonary and renal disease occur simultaneously, with the remainder split between isolated DAH and isolated glomerulonephritis. More than 90% of patients with Goodpasture’s syndrome have circulating serum ABMA (these antibodies are also commonly referred to as anti-glomerular basement membrane antibodies). The target of this antibody has been identified as the NC1 domain of the a3 chain of type IV collagen.

Etiology The most common causes of DAH are listed in Table 1. A review of 34 cases of DAH found that approximately one-third were associated with Wegener’s granulomatosis. Goodpasture’s syndrome was the second most common etiology, accounting for 13% of cases. Any source of injury to the alveolar microcirculation can theoretically cause alveolar hemorrhage. The injury may be primarily in the lung (e.g., pneumonia) or more generalized (e.g., vasculitis). Many cases of DAH are associated with a neutrophilic infiltration of the alveolar wall centering on capillaries and venules. This histopathologic observation is called ‘capillaritis’ and is most closely associated

Table 1 Causes of diffuse alveolar hemorrhage Vasculitidies Wegener’s granulomatosis Microscopic polyangiitis Isolated pulmonary capillaritisa Mixed cryoglobulinemia Bechet’s syndrome Henoch–Scho¨nlein purpura Pauci-immune glomerulonephritis Immunologic Goodpasture’s syndrome Connective tissue diseaseb Immune complex-associated glomerulonephritis Acute lung allograft rejection Primary anti-phospholipid antibody syndrome

a

Coagulation disorders Thrombotic thrombocytopenic purpura Idiopathic thrombocytopenic purpura Anticoagulants, antiplatelet agents, or thrombolytics Idiopathic Idiopathic pulmonary hemosiderosis Other Drugs/toxinsc Diffuse alveolar damage Mitral stenosis Pulmonary veno-occlusive disease Pulmonary capillary hemangiomatosis Lymphangioleiomyomatosis Tuberous sclerosis

Most cases are pauci-immune, and some are p-ANCA associated. b DAH most commonly occurs in systemic lupus erythematosus. c The most common drugs/toxins are propylthiouracil, phenytoin, mitomycin, penicillamine, sirolimus, methotrexate, nitrofurantoin, all-trans retinoic acid, trimellitic anhydride, and isocyanates. Reproduced from Collard HR and Schwarz MI (2004) Diffuse alveolar hemorrhage. Clinics in Chest Medicine 25: 583–592, with permission from Elsevier.

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with the systemic vasculitidies and collagen vascular diseases. Although Goodpasture’s syndrome may be associated with capillaritis, it generally causes socalled ‘bland hemorrhage’ (i.e., showing no evidence of inflammation on biopsy). The etiology of Goodpasture’s syndrome is unknown, but it is commonly associated with cigarette smoking, suggesting a potential causative role. Smoking likely increases the risk and severity of DAH by increasing alveolar permeability. The typical patient is a young male smoker, although older people, women, and nonsmokers can also be affected. Other associations include respiratory infections, such as influenza A2, and hydrocarbon products, such as petroleum, turpentine, and pesticides. There is a strong association with HLA-DRw2 and HLA-B7, which portend a poor prognosis.

lar filling due to hemorrhage. Inspiratory crackles are occasionally present and may be focal or diffuse. Clubbing is not present. Specific etiologies may be suggested by the presence of a diastolic murmur (mitral stenosis) or ocular, nasopharyngeal, and cutaneous evidence of vasculitis or collagen vascular disease. There are no physical findings specific to Goodpasture’s syndrome. Radiology

The chest radiograph is almost always abnormal in DAH (Figure 1). Diffuse alveolar opacities are typically seen, although they can be focal and asymmetric. Pleural effusions are rarely seen. Although computed tomography is potentially useful in the evaluation of hemoptysis and is commonly obtained in patients presenting with this complaint, it has little role in the further evaluation of DAH.

Clinical Evaluation There is nothing unique to the presentation of DAH in Goodpasture’s syndrome. Although demographics and urinary findings may suggest the diagnosis, it is only with further laboratory and histopathologic evaluation that the diagnosis is made. The following discussion is therefore relevant to DAH occurring in Goodpasture’s syndrome as well as to other causes. History

Laboratory Evaluation

The laboratory evaluation is where the underlying etiology of DAH often becomes apparent. The hemoglobin is decreased in most cases of DAH, and leukocytosis may be seen. In Goodpasture’s syndrome, the serum creatinine may be elevated and the urine sediment active (i.e., dysmorphic red blood cells and/ or red blood cell casts), suggesting a concomitant glomerulonephritis. Serum ABMA will commonly

The three most common presenting symptoms of DAH are hemoptysis, dyspnea, and cough. These are generally acute in onset, but occasionally they are subacute and recurrent. Many cases will not have this classic clinical presentation. Importantly, hemoptysis is not universally present at the time of initial evaluation, even in severe cases. Dyspnea is generally a result of ventilation–perfusion mismatch secondary to alveolar filling, although rarely anemia may contribute. Depending on the etiology of DAH, fever and signs and symptoms referring to a systemic vasculitis or collagen vascular disease may be present. Occasionally, complaints of decreased urine output or hematuria may be reported, suggesting a concomitant glomerulonephritis and raising the possibility of Goodpasture’s syndrome, systemic vasculitis, or collagen vascular disease (particularly systemic lupus erythematosis). As noted previously, youth, male gender, and current cigarette smoking are common findings in patients with Goodpasture’s syndrome. Most cases occur in men in their 20s and 30s. Physical Examination

The physical examination is generally non-specific in DAH. The cardiac and pulmonary examinations are often normal, even in the setting of substantial alveo-

Figure 1 Chest radiograph of a patient with DAH. The chest radiograph is almost always abnormal in DAH. Diffuse alveolar infiltrates are typically seen, although they can be focal and asymmetric. Pleural effusions are rarely seen.

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(90%) be present. Serum anti-neutrophil cytoplasmic antibodies (ANCA) or serum markers of collagen vascular disease may be present and suggest alternative etiologies. Pulmonary function testing is often not feasible given the severity of illness in many DAH patients. If performed, normal or mildly restricted patterns may be observed. In acute hemorrhage, the carbon monoxide diffusing capacity may be elevated, and this can be used to follow patients for evidence of recurrent hemorrhage once clinically stabilized.

alveolar basement membrane is generally visible by direct immunofluorescence of lung biopsy tissue. In other conditions, such as vasculitis and collagen vascular disease, capillaritis may be present. Typical histopathologic features of capillaritis include fibrin thrombi occluding capillaries in the alveolar septae, fibrinoid necrosis of the capillary walls, and interstitial accumulation of fragmented neutrophils and nuclear dust adjacent to alveolar capillaries. Capillaritis has been described in Goodpasture’s syndrome but is extremely rare. Kidney Biopsy

Histopathology Lung Biopsy

Surgical lung biopsy is highly preferable to transbronchial biopsy for the histopathological diagnosis of DAH due to its larger sample size and architectural preservation (Figure 2). True alveolar hemorrhage must be distinguished from other causes of red blood cell accumulation in the alveolar space, most commonly bleeding into the lung at the time of biopsy. Alveolar hemorrhage usually demonstrates intra-alveolar fibrin, hemosiderin in the alveolar walls, and hemosiderin-laden alveolar macrophages. Hemosiderin requires at least 48 h to develop and, if present, is helpful in distinguishing DAH from surgical trauma. Areas of mild interstitial thickening and occasional associated organizing pneumonia or diffuse alveolar damage may be present. If Goodpasture’s syndrome is suspected and histopathology is required, kidney biopsy is generally preferred, but linear deposition of antibody along the

Figure 2 Surgical lung biopsy from a patient with DAH. Surgical lung biopsy in DAH usually demonstrates intra-alveolar fibrin, hemosiderin in the alveolar walls, and hemosiderin-laden alveolar macrophages. Typical histopathologic features of capillaritis include fibrin thrombi occluding capillaries in the alveolar septae, fibrinoid necrosis of the capillary walls, and interstitial accumulation of fragmented neutrophils and nuclear dust adjacent to alveolar capillaries (visible here).

In cases of Goodpasture’s syndrome without circulating ABMA, the diagnosis may require histopathologic confirmation. Kidney biopsy is generally preferable due to its lower morbidity and more consistent findings. As described previously for the lung, kidney biopsy in Goodpasture’s syndrome will demonstrate linear deposition of antibody along the glomerular basement membrane, visible by direct immunofluorescence (Figure 3). Even in the rare case of Goodpasture’s syndrome without clinically evident renal disease, kidney biopsy will still show the typical linear antibody deposition. Kidney biopsy is also useful for defining the presence and extent of glomerulonephritis in DAH associated with vasculitis, such as Wegener’s granulomatosis or microscopic polyangiitis. Diagnosis

A thoughtful and thorough approach to the diagnosis of DAH is critical to appropriate management. In cases of DAH presenting with dyspnea or cough, the differential diagnosis includes congestive heart failure, pneumonia, and acute presentations of other diffuse parenchymal lung diseases. The presence of hemoptysis narrows this differential diagnosis

Figure 3 Kidney biopsy from a patient with Goodpasture’s syndrome. Kidney biopsy in Goodpasture’s syndrome demonstrates linear deposition of antibody along the glomerular basement membrane visible by direct immunofluorescence.

206 SYSTEMIC DISEASE / Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome

significantly. In these patients, pseudohemoptysis (hemorrhage arising from the upper airway or gastrointestinal tract) must be excluded. Focal sources of pulmonary hemorrhage are common and must be considered (e.g., bronchitis, bronchiectasis, infection, and malignancy). As previously mentioned, most causes of hemoptysis can cause diffuse alveolar opacities if severe enough. History and Physical Examination

Initial evaluation should focus on the qualitative and quantitative assessment of symptoms, such as hemoptysis, cough, and dyspnea. Comorbidities such as collagen vascular disease or existing pulmonary disease should be noted. A detailed drug and occupational history should be taken. Careful evaluation of the eye, nasal septum, and skin should be performed to search for evidence of systemic vasculitis. Importantly, DAH may represent the primary manifestation of an underlying systemic vasculitis or collagen vascular disease, so the absence of historical or physical evidence should not eliminate these as possible etiologies. Diagnostic Studies

Initial diagnostic studies in patients suspected of having DAH should include chest computed tomography (because this may help identify alternative etiologies for pulmonary hemorrhage, such as bronchiectasis or bronchogenic cancer), complete blood count, and serum creatinine. Examination of the urinary sediment for evidence of glomerular injury cannot be overemphasized. In Goodpasture’s syndrome, an active urinary sediment will often be the first clue to the diagnosis. If Goodpasture’s syndrome is suspected, serum ABMA should be measured because their presence will confirm the diagnosis in up to 90% of patients. Serum ANCA, anti-nuclear, and anti-phospholipid antibodies should generally be sent along with ABMA in the evaluation of patients with DAH and evidence of concomitant glomerular disease.

rial and fungal cultures, and when clinically indicated, viral and Pneumocystis jiroveci studies should be added. Biopsy

Biopsy of the lung (usually via surgical biopsy) or kidney can be helpful in cases of DAH in which an underlying collagen vascular disease or vasculitis is suspected. In cases of suspected vasculitis, biopsy of the kidney shows focal segmental necrotizing glomerulonephritis. In Goodpasture’s syndrome, linear antibody deposition is seen. Current Therapy

DAH is a medical emergency and definitive treatment requires identification of the underlying etiology. Hospitalization in an intensive care unit is appropriate for all newly diagnosed or suspected cases of DAH. Although exsanguination from uncontrolled hemorrhage is much less common in DAH than in conditions such as bronchiectasis or invasive fungal infection, significant hemorrhage can occur and patients should be aggressively resuscitated. Furthermore, respiratory failure requiring intubation and mechanical ventilation is common and often precipitous. (A discussion of specific recommendations for the treatment of DAH due to the most common etiologies is beyond the scope of this article.) Aggressive treatment of Goodpasture’s syndrome involves the combination of corticosteroids, cyclophosphamide or azathioprine, and plasmapheresis. This combination of therapies has proven effective even in patents who have already developed renal failure from their disease. In cases of Goodpasture’s syndrome refractory to the previously mentioned therapies, responses to mycophenolate mofetil or anti-CD20 monoclonal antibody have been reported.

Prognosis Bronchoscopy

Bronchoscopy with bronchoalveolar lavage (BAL) is indicated in patients suspected of having DAH to both confirm the presence of true alveolar hemorrhage and exclude infection as an underlying etiology. An increasing number of red blood cells in sequential aliquots of bronchoalveolar lavagate from the same subsegmental location is considered diagnostic of DAH. In addition, quantitative scoring of the hemosiderin concentration of alveolar macrophages, although labor-intensive and time-consuming, may add to the diagnostic specificity of BAL. Bronchoalveolar lavage specimens should routinely be sent for bacte-

In Goodpasture’s syndrome, the majority of patients survive the initial presentation. A review of 29 cases of DAH due to Goodpasture’s syndrome showed the 2-year survival rate to be approximately 50%. The degree of glomerular involvement on kidney biopsy and the degree of clinical impairment of renal function both had prognostic value. If less than 30% of glomeruli were involved and renal function was relatively preserved, patients had improved responsiveness to therapy and improved survival. If more than 70% of glomeruli were involved and abnormal renal function was present, patients were unresponsive to therapy and had increased mortality.

SYSTEMIC DISEASE / Langerhans’ Cell Histiocytosis (Histiocytosis X) 207 See also: Alveolar Hemorrhage. Autoantibodies. Bronchiectasis. Bronchoalveolar Lavage. Granulomatosis: Wegener’s Disease.

Further Reading Arzoo K, Sadeghi S, and Liebman HA (2002) Treatment of refractory antibody mediated autoimmune disorders with an anti-CD20 monoclonal antibody (rituximab). Annals of Rheumatic Diseases 61(10): 922–924. Collard HR and Schwarz MI (2004) Diffuse alveolar hemorrhage. Clinics in Chest Medicine 25: 583–592. Donaghy M and Rees AJ (1983) Cigarette smoking and lung haemorrhage in glomerulonephritis caused by autoantibodies to glomerular basement membrane. Lancet 2(8364): 1390–1393. Fontenot AP and Schwarz MI (2003) Diffuse alveolar hemorrhage. In: Schwarz MI and King TE (eds.) Diffuse Lung Disease. 4th edn. New York: Decker. Fort J, Espinel E, Rogriquez JA, et al. (1984) Partial recovery of renal function in an oligoanuric patient affected with Goodpasture’s syndrome after treatment with steroids, immunosuppressives and plasmapheresis. Clinical Nephrology 22(4): 211–212. Garcia-Canton C, Toledo A, Palomar R, et al. (2000) Goodpasture’s syndrome treated with mycophenolate mofetil. Nephrology Dialysis Transplantation 15(6): 920–922. Levy JB, Turner AN, Rees AJ, and Pusey CD (2001) Long-term outcome of anti-glomerular basement membrane antibody disease treated with plasma exchange and immunosuppression. Annals of Internal Medicine 134(11): 1033–1042. Savage CO, Pusey CD, Bowman C, Rees AJ, and Lockwood CM (1986) Antiglomerular basement membrane antibody mediated disease in the British Isles 1980–4. British Medical Journal 292(6516): 301–304. Schwarz MI, Collard HR, and King TE Jr (2005) Diffuse alveolar hemorrhage and other rare infiltrative disorders. In: Murray JF, Nadel JA, Boushey HA, and Mason RJ (eds.) Textbook of Respiratory Medicine, 4th edn. Philadelphia: Saunders. Teague CA, Doak PB, Simpson IJ, Rainer SP, and Herdson PB (1978) Goodpasture’s syndrome: an analysis of 29 cases. Kidney International 13(6): 492–504. Travis WD, Colby TV, Lombard C, and Carpenter HA (1990) A clinicopathologic study of 34 cases of diffuse pulmonary hemorrhage with lung biopsy confirmation. American Journal of Surgical Pathology 14(12): 1112–1125. Zimmerman SW, Varanasi UR, and Hoff B (1979) Goodpasture’s syndrome with normal renal function. American Journal of Medicine 66(1): 163–171.

Langerhans’ Cell Histiocytosis (Histiocytosis X) S Harari and A Caminati, Unita` Operativa di Pneumologia, Milan, Italy & 2006 Elsevier Ltd. All rights reserved.

Abstract Langerhans’ cell histiocytosis (LCH) is a rare and pleiomorphic disorder of the bone marrow-derived histiocytes that may involve the skin, bone, bone marrow, liver, spleen, lungs, and lymph nodes. The pathogenesis and etiology of LCH remain

poorly understood. Whether LCH is a neoplastic disorder or a reactive process remains controversial. LCH is a disease with a broad spectrum of clinical presentations. All of the variants have in common the proliferation of cells that are morphologically, biochemically, and immunophenotypically indistinguishable from Langerhans’ cells. A definitive diagnosis of LCH can only be rendered when either there is demonstration of Birbeck granules on electron-microscopic study or there is the demonstration of CD1a expression with appropriate histological settings. The outcome and course of the disease are variable. LCH may in some cases regress on its own without treatment. In other situations, very minimal treatment will result in the resolution of symptoms and regression of the disease. The multisystem disease is treated with systemic chemotherapy but the treatment is still controversial.

Introduction The concept of histiocytosis X was proposed by Lichtenstein in 1953 and linked different clinical forms of the disease with the histological presence of eosinophilic granuloma. The definition of this group of diseases was based on morphological criteria detected by light microscopy and the term ‘histiocytosis X’ was proposed. This term embraces three major localized and generalized patterns of histiocytosis including a unifocal, multifocal, and disseminated variant. The unifocal variant (single system, single site) has previously been referred to as eosinophilic granuloma. This subtype commonly involves bone, lymph nodes, or lungs as a primary target. Children with localized disease tend to have bone involvement while adults have a greater propensity for lung involvement. The multifocal variant has been referred to in the past as Hand–Schu¨ller–Christian disease. This variant usually affects younger patients and involves several sites in one organ system (single system, multiple sites). The organ system involved varies from one patient to another. A classic triad is skull lesions, exophthalmos, and diabetes insipidus. Other bones, the oral cavity, skin, lymph nodes, brain, lungs, and liver represent other organ systems that may be affected in different patients. Multiple foci of disease will be found in the particular organ system affected for a given patient. The disseminated variant was previously referred to as Letterer–Sewe disease and affects multiple sites in multiple organ systems. This is a potentially fatal systemic disease in children under 3 years of age. Typical manifestations include multisystem involvement of bones and organs and may include persistent fevers, irritability, anorexia, failure to thrive, purpuric rush, superinfection, diarrhea, pancytopenia, and life-threatening sepsis. However, this concept of histiocytosis X is no longer used due to the imprecise definitions of these terms, the recognition that the proliferating cells are Langerhans’ cells, and the growing recognition that

208 SYSTEMIC DISEASE / Langerhans’ Cell Histiocytosis (Histiocytosis X)

there are profound differences in organ involvement, clinical course, and therapeutic response. The World Health Organization Committee on Histiocytic/Reticulum Cell Proliferations proposed that Langerhans’ cell histiocytosis (LCH) replaces the many previous terms for this disorder. In children, the diagnosis can be made from birth to adolescence with a peak frequency from the first year of life to 3 years. The annual incidence of LCH appears to be 3–5 cases per million children; the prevalence to be 1/50 000 with a male:female sex ratio of 2:1. A connection with solvent exposure in parents as well as perinatal infections has been suggested, but no increased incidence after viral epidemics exists. The epidemiology of the disease in adults is less well known. The largest series of adults were reported by lung specialists with the pulmonary localization of the disease apparently associated with heavy smoking. Pulmonary LCH is a rare disorder and it is seen in less than 5% of lung biopsies from patients with diffuse parenchymal lung diseases.

The favorable natural history in most cases, the high probability of survival for patients older than 2 years of age, and the failure to detect cytogenetic abnormalities support the idea that LCH is not a neoplastic process. However, the proliferation of LCs may be a physiologically appropriate but clinically pathologic response. Pulmonary LCH in adults is nonclonal in origin and appears more likely to represent a polyclonal, reactive disorder triggered by cigarette smoking. The finding of increased numbers of LCs in the bronchoalveolar lavage fluid of smokers, along with abnormal T-cell proliferative responses to tobacco glycoprotein in patients with pulmonary LCH, lends support to this hypothesis. Pulmonary LCH initially involves the lungs in a peribronchiolar fashion consistent with an inhaled antigen. Most investigators consider LCH to result from immunologic dysfunction since several cytokines are present at higher levels in the lesions than in normal surrounding tissue (‘cytokine storm’).

Etiology and Pathogenesis

Pathology

The etiology and pathogenesis of LCH are unknown and remain an enigma despite extensive research. Current theories suggest a role for environmental, infectious, immunologic, and genetic causes (Figure 1). Efforts to define a viral etiology or a neoplastic process have not been successful. Flow cytometric techniques suggest that Langerhans’ cells (LCs) from pulmonary, bone, and skin lesions are clonally expanded. However, a clonal proliferation does not necessarily mean that LCH is a neoplastic process.

Regardless of the clinical severity, the histopathology of LCH is generally uniform. The location and age of the lesion, to some extent, may influence the histopathology of the disease. Early in the course of the disease, lesions tend to be cellular and contain aggregates of LCs, macrophages, T cells, and giant histiocytes. Multinucleated giant cells are common; variable numbers of eosinophils are often present. In the lung, the lesions are centered on bronchioles and nodular infiltrates with stellate border extending into

Monoclonal origin? Smoking?

Cytogenetic abnormalities?

Gene rearrangements?

Cytokines?

Genetic traits?

Adhesion molecules?

↓ Apoptosis

Virus?

Langerhans' cell Reactive condition? Polyclonal expansion of LC? Figure 1 Possible mechanisms in the pathogenesis of LCH.

SYSTEMIC DISEASE / Langerhans’ Cell Histiocytosis (Histiocytosis X) 209

the surrounding interstitium are present. With the passage of time, cellularity and number of LCs are reduced and macrophages and fibrosis become prominent. The infiltrates have a tendency to destroy epithelial cells. Morphologically, LCs are 12 mm in diameter with moderately abundant lightly eosinophilic to eosinophilic cytoplasm and contain a medium size folded, indented, or lobulated nucleus with vesicular chromatin with one to three nucleoli and an elongated central groove producing a ‘coffee bean’ appearance. The identity of these cells as LCs must be confirmed by immunohistochemical staining with anti-CD1a monoclonal antibodies (Figure 2) or by the identification of Birbeck granules using electron microscopy (Figure 3). In the future, these methods will be replaced by the use of more specific antibodies that identify LCs, such as anti-Langerin antibodies. Although these cells also express the cytoplasmic

Figure 2 Intense positivity for antigen CD1a of Langerhans’ cell in transbronchial lung biopsy.

Figure 3 Birbeck granules, intracytoplasmic organelles, often of dumb-bell or tennis racquet form and with a pentalaminar structure are diagnostic for LCH (electron microscopy,
20 000). Reproduced from Harari S and Comel A (2001) Pulmonary Langerhans’ cell histiocystosis. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 18: 253–262, with permission.

S100 protein, this marker is present at the surface of other cell types, including neuroendocrine cells and some macrophages, and is therefore less specific.

Clinical Features LCH can be local and asymptomatic or can involve multiple organs and systems with significant symptomatology and consequences. The clinical manifestations depend on the site of the lesions and organs and systems involved and their functions. Thus, the clinical course of LCH ranges from spontaneous resolution to a chronic and sometimes lethal disease. The most frequent site of LCH in children is a lytic lesion of the skull; such lesions can occur in any bone of the body, with the sites most frequently involved being femur, orbit, ribs, and humerus. Patients with periorbital lesions often have proptosis because of the presence of a tumor mass behind the eye. In adult patients the primary site of bone involvement is jaw. Clinically, the lesions can be singular or multiple. Patients present with fracture or pain at the site of the lesion. Asymptomatic or painful involvement of vertebrae can occur and result in collapse. At times, lesions may cause a significant periosteal reaction. Extension to the adjacent tissues can produce symptomatology, which may be unrelated to the bone involvement. Diabetes insipidus (DI) and delayed puberty are observed in as many as 50% (with a usual range of 15–25%) of patients. Hypothalamic disease also may result in growth hormone deficiency and short stature. Cutaneous LCH is observed in up to 50% of patients with LCH (Figure 4). Rash is a common presentation, and skin lesions may be the only site of the disease or a part of systemic involvement. Infants may present with brown to purplish papules over any part of their body (Hashimoto–Pritzker disease); this manifestation is a benign one and the lesions disappear during the first year of life with no therapy. From infancy to adulthood a very different red papular rash occurs; skin infiltrates have a predilection for the midline of the trunk and the peripheral and flexural areas of skin. Skin infiltrates can be maculoerythematous, petechial xanthomatous, nodular papular, or nodular in appearance. Ulcerative lesions behind the ears, in the scalp, genitalia, or perianal region are especially troublesome, as they often are misdiagnosed as bacterial or fungal lesions. Seborrheic involvement of the scalp may be mistaken for prolonged ‘cradle cap’ in infants or a severe case of dandruff in older individuals. In the mouth, one can find gingival hypertrophy, ulcers of the soft or hard palate, buccal mucosa, or on the tongue and lips. Purulent otitis media may occur and may be difficult

210 SYSTEMIC DISEASE / Langerhans’ Cell Histiocytosis (Histiocytosis X)

to distinguish from infectious etiologies. Long-term sequelae, including deafness, have been reported. Pulmonary involvement is observed in 20–40% of patients and may result in respiratory symptoms or signs, such as cough, tachypnea, dyspnea, and pneumothorax (which can be bilateral or recurrent). Up to 25% of patients are asymptomatic at presentation. A male predominance exists. The majority of patients with pulmonary LCH are smokers who commonly acquire disease in the third or fourth decade of life. Physical examination reveals no abnormal signs in most patients. Results of pulmonary function studies are quite variable and may show normal, obstructive, restrictive, or mixed patterns. Chest high-resolution computed tomography (HRCT) findings vary depending on the stage of the disease. Cysts and/or nodules are present in the majority of patients. Early stage of pulmonary LCH is characterized by a centrilobular

pattern with ill-defined nodules distributed around small airways. Cystic changes appear later and vary in shape, size, and wall thickness. The HRCT appearance of cysts and nodules in an adult heavy smoker is virtually diagnostic of pulmonary LCH. Serial study of individual patients indicates that the lesions evolve as follows: nodules, cavitary nodules, thick-walled cysts, thin-walled cysts (Figures 5 and 6). These studies also demonstrate that whereas some lesions can regress (nodules, cavitary nodules), cystic lesions tend to remain stable or increase in size. Most important is the fact that other sites of LCH involvement (bone, skin, and pituitary gland) can be found in these patients. Pulmonary involvement is a ‘risk’ lesion and is a less frequent occurrence in children than in adults. Lymph node enlargement has been observed in approximately 30% of patients. Rarely, the nodes are symptomatic but, if massive in volume, may result in

Figure 4 (a) Purulent otitis and cutaneous LCH with erythematous, often crusted vesiculopustules. (b) Seborrheic involvement of the scalp in LCH.

Nodular lesion Cavitating nodules Thick-walled cystic lesion Thin-walled cystic lesion

Figure 5 Chest high-resolution computed tomography (HRCT) findings in a case of pulmonary LCH. The image shows the evolution of the lesions from nodules to thin-walled cysts.

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Figure 6 Chest high-resolution computed tomography (HRCT) findings (a) in a case of pulmonary LCH and gross findings (b) in explanted lung.

obstruction of, or damage to, the surrounding organs and tissues. Enlarged mediastinal nodes can mimic lymphoma or an infectious etiology. Accordingly, biopsy with culture and histologic examination is mandatory for this presentation. A few patients with diarrhea, malabsorption, or bleeding have been reported. Diagnosing gastrointestinal involvement with this disease is difficult because of intermittent involvement. Careful endoscopic examination with biopsies is needed. Liver involvement is characterized by elevated transaminases and, less commonly, increased bilirubin, clotting factor deficiencies, and hypoalbuminemia with ascites. Hematologic changes may be caused by marrow involvement or enlargement of the spleen.

Management and Current Therapy LCH may in some instances regress on its own without treatment. In other situations, very minimal treatment will result in the resolution of symptoms and regression of the disease. One must therefore try to balance the extent of medical intervention with the severity of the disease involvement. An isolated bone lesion can usually be effectively managed initially with surgical curettage or radiotherapy. Recurrent disease at the same site or a new lesion that appears in a different place may or may not require any intervention, depending upon their location and extent. Under such circumstances, some patients will benefit from the use of nonsteroidal anti-inflammatory agents or the local injection of steroids. Patients with only skin involvement, especially difficult ulcerative lesions of the scalp or inguinal region,

may respond well to topical nitrogen mustard, with minimal side effects and no evidence of late complications such as skin cancer. In pulmonary LCH, it is imperative that all patients stop smoking and improvement in patients with pulmonary LCH after smoking cessation has been reported in several cases. Steroids may help slowing or even stop the progression of lung LCH, but only in patients with nodular disease. For patients with more extensive disease, there is general agreement that systemic chemotherapy will be beneficial. The exact type of chemotherapy that is best has not been completely worked out, although several different drugs and combinations have been shown to give excellent results with relatively minimal side effects. Such chemotherapeutic agents include steroids (e.g., prednisone), vinblastine, vincristine, etoposide or VP-16, 6-mercaptopurine, and methotrexate. While all of these agents have demonstrated good activity in treating LCH, the very best schedule and combination are still being explored. For patients with refractory and/or progressive disease that is not responding to the ‘regular’ protocols, there are fewer very good treatments. The recent use of 2-chloro-20 deoxyadenosine (2CdA) has shown some promising early results. Bone marrow transplantation has also been tried for patients who have progressive disease despite receiving a variety of treatments. It is evident that in some children, bone marrow transplantation has been able to achieve a complete remission of disease. Patients with LCH have a higher than normal risk of developing secondary cancers. Leukemia (usually acute myeloid) and lymphoblastic lymphoma occurs after treatment. Solid tumors associated with

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LCH include retinoblastoma, brain tumors, hepatocellular carcinoma, and Ewing’s sarcoma. See also: CD1. Chronic Obstructive Pulmonary Disease: Smoking Cessation. Granulomatosis: Lymphomatoid Granulomatosis.

Sickle Cell Disease A Greenough and K Sylvester, King’s College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Further Reading Aguayo SA, King TE Jr, Waldron JA Jr, et al. (1990) Increased pulmonary neuroendocrine cells with bombesin-like immunoreactivity in adult patients with eosinophilic granuloma. Journal of Clinical Investigation 86: 838–844. Egeler RM, Neglia JP, Arico M, et al. (1998) The relation of Langerhans’ cell histiocytosis to acute leukaemia, lymphomas, and other solid tumors. The LCH-Malignancy Study Group of the Histiocyte Society. Hematology/Oncology Clinics of North America 12: 369–378. Egeler RM, Neglia JP, Puccetti DM, Brennan CA, and Nesbit ME (1993) Association of Langerhans’ cell histiocytosis with malignant neoplasms. Cancer 71: 865–873. Favara BE (1991) Langerhans’cell histiocytosis pathobiology and pathogenesis. Seminars in Oncology 18: 3–7. Favara BE, Feller AC, Pauli M, et al. (1997) Contemporary classification of histiocytic disorders. The WHO Committee On Histiocytic/Reticulum Cell Proliferations. Reclassification Working Group of the Histiocyte Society. Medical and Pediatric Oncology 29: 157–166. Gadner H, Grois N, Arico M, et al. (2001) A randomized trial of treatment for multisystem Langerhans’ cell histiocytosis. Journal of Pediatrics 138: 728–734. Harari S and Comel A (2001) Pulmonary Langerhans’ cell histiocytosis. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 18: 253–262. Howarth DM, Gilchrist GS, Mullan BP, et al. (1999) Langerhans’ cell histiocytosis: diagnosis, natural history, management, and outcome. Cancer 85: 2278–2290. Saven A, Foon KA, and Piro LD (1994) 2-Chlorodeoxyadenosineinduced complete remissions in Langerhans’-cell histiocytosis. Annals of Internal Medicine 121: 430–432. Travis WD, Borok Z, Roum JH, et al. (1993) Pulmonary Langerhans’ cell granulomatosis (histiocytosis X): a clinicopathologic study of 48 cases. American Journal of Surgery and Pathology 17: 971–986. Vassallo R, Ryu JH, Colby TV, Hartman T, and Limper AH (2000) Pulmonary Langerhans’-cell histiocytosis. New England Journal of Medicine 342: 1969–1978. Vassallo R, Ryu JH, Schroeder DR, Decker PA, and Limper AH (2002) Clinical outcomes of pulmonary Langerhans’-cell histiocytosis in adults. New England Journal of Medicine 346: 484–490. Willis B, Ablin A, Weinberg V, et al. (1996) Disease course and late sequelae of Langerhans’ cell histiocytosis: 25-year experience at the University of California, San Francisco. Journal of Clinical Oncology 14: 2073–2082. Willman CL, Busque L, Griffith BB, et al. (1994) Langerhans’-cell histiocytosis (histiocytosis X) – a clonal proliferative disease. New England Journal of Medicine 331: 154–160. Writing Group of the Histiocyte Society (1987) Histiocytosis syndromes in children. Lancet 1: 208
209. Yousem SA, Colby TV, Chen YY, Chen WG, and Weiss LM (2001) Pulmonary Langerhans’ cell histiocytosis: molecular analysis of clonality. American Journal of Surgery and Pathology 25: 630–636.

Sickle cell disease (SCD) is the most common inherited disorder affecting African/Caribbean populations. The pulmonary complications of SCD have a high morbidity and mortality. Acute chest syndrome (ACS) is the leading cause of death and recurrent ACS episodes are the major risk factor for sickle chronic lung disease (SCLD). Lung function abnormalities are present even in young children. Restrictive abnormalities become more prominent with increasing age and young adults with SCLD have restrictive lung disease, abnormal diffusing capacity, and hypoxemia. In those that develop pulmonary hypertension, the mortality is increased up to sevenfold, hence SCD adults should be screened for the development of pulmonary hypertension. SCD patients are susceptible to infection and should be given penicillin prophylaxis and appropriate immunizations. Regular transfusions are given to those who have developed complications. Hydroxyurea is effective and safe in severely affected adults, but further studies are required to elucidate its role in other patient groups. Bone marrow transplantation is considered for patients who have significant sickle-related complications. Gene therapy is a possible future direction for research.

Introduction The sickle cell diseases are disorders in which ‘sickling’ of erythrocytes produces clinical manifestations. In 1910, James Herrick was the first to observe sickled cells and, in 1923, it was demonstrated that the sickling phenomenon was inherited as an autosomal dominant trait. Subsequently, the abnormal composition of patients with ‘sickle cell’ hemoglobin (HbS) was highlighted. The sickle cell gene developed in Africa and Asia. The high prevalence of the gene for HbS in areas where malaria has been common suggests that individuals with sickle cell trait had an advantage against malaria. Indeed, although children with sickle cell trait are infected by Plasmodium falciparum, the parasite count remains low. HbS occurs most commonly in tropical Africa. The sickle cell trait has a frequency of approximately 8% in African-Americans and also occurs in the Middle East, Greece, and India. Sickle cell disease (SCD) is the most common inherited disorder affecting African and Caribbean populations. There are more than 200 million carriers of the sickle cell trait and, each year, between 200 000 and 250 000 children are born with SCD. Pulmonary complications, the acute chest syndrome (ACS) and sickle chronic lung disease (SCLD), are the main causes of morbidity and mortality in SCD patients. Fatal pulmonary complications occur in 20% of adults. Despite significant

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improvements in life expectancy in individuals with SCD, the median age of death for women is 48 years and for men 42 years. In this article the etiology, pathogenesis, management, and outcome of the pulmonary complications of SCD will be described.

Etiology Homozygous inheritance of the gene for HbS results in sickle cell anemia (HbSS), the most severe form of SCD. Other clinically important forms of SCD include sickle hemoglobin C disease (HbSC) and sickle beta-thalassemia (HbSbetathal). The sickle gene results in a point mutation and substitution of valine for glutamic acid in the beta-globin chain of the hemoglobin molecule to form HbS. When the deoxygenated HbS undergoes conformational changes, a hydrophobic region surrounding the valine site in the beta subunit is left exposed. Polymerization with other hemoglobin tetramers occurs with the formation of aggregates that distort the red blood cell membrane. Polymerization is dependent on the cellular concentration of HbS; the increased cellular hemoglobin concentration of dehydrated erythrocytes markedly enhances their tendency to polymerize and sickle. When a ‘sickle’ cell is exposed to a relatively hypoxic/acidic environment, the K þ Cl
cotransport is activated with loss of potassium and dehydration of the cell. Deoxygenation also increases intracellular free calcium and calcium-dependent dehydration takes place, as there is calcium-dependent movement of potassium (Gardos pathway). Partially or fully deoxygenated HbS, but not fully oxygenated HbS or fetal hemoglobin (HbF), can polymerize. The crystallized HbS molecules form a viscous solution within the erythrocyte. This stiffens the red blood cell and changes it from its normal biconcave shape to a sickle-shaped cell, which is less deformable and subject to hemolysis. These rigid cells can obstruct small blood vessels. Over time, cells that have sickled repeatedly become irreversibly sickled. Deoxygenation is maximal in the venous circulation and the sickled cells may cause extensive and progressive damage to the pulmonary vascular bed.

episode); only the latter will be discussed in this article which concentrates on the pulmonary problems arising from SCD. Acute Chest Syndrome

Acute chest syndrome (ACS) episodes are characterized by fever, chest pain, and respiratory symptoms including productive cough, wheeze, and dyspnea. The presenting symptoms and signs vary with age, fever and cough being more common in very young children and chest pain, shortness of breath, and hemoptysis being more prominent with advancing age. Essential to the diagnosis of an ACS is the presence of a new pulmonary opacity on the chest radiograph (Figure 1). The lower and middle lobes are more frequently affected than the upper lobes, although children are more likely than adults to have isolated upper lobe disease. ACS episodes occur more commonly in children than in adults; 50% of SCD children will have an episode prior to the age of 10 years. However, ACS is the most common cause of hospitalization in adults. Severe respiratory failure, necessitating mechanical ventilation, occurs in approximately 10–15% of affected patients. Recurrence is common, occurring in 80% of those who have had a prior episode and predisposes to chronic pulmonary disease, including pulmonary hypertension. Homozygosity for the SS genotype is a major risk factor for ACS and is found in approximately 80% of affected individuals. High hemoglobin levels predispose to vascular obstruction and increase the risk of complications, such as ACS, due to increased blood viscosity, poor blood flow, and hypoperfusion.

Clinical Features SCD patients have hemolytic anemia, but the newborn infant is protected by the high level of HbF in their erythrocytes. As the levels of HbF decline, the manifestations of SCD become apparent by 10–12 weeks of age. SCD patients have periods of stability punctuated with acute painful crises or vaso-occlusive crisis (VOC), characterized by severe pain involving the back, abdomen, joints, or chest (ACS

Figure 1 Chest radiograph in a 19-year-old patient. There is consolidation in the right lower lobe consistent with acute chest syndrome.

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The incidence of ACS is inversely proportional to the HbF level and the degree of anemia is directly proportional to the steady-state white blood count. High HbF levels inhibit HbS polymerization and this protects against ACS, but elevated leukocyte counts have a negative effect because the leukocytes release free radicals, elastase, proinflammatory mediators, and cytokines. The relative risk for ACS has been suggested to be more than eightfold for female carriers of certain endothelin nitric oxide (NO) synthase gene polymorphisms. The cause of ACS is unknown in a large number of cases; however, common known etiological factors include pulmonary fat embolism, hypoventilation, and infection. In approximately 10% of patients, an ACS is precipitated by a pulmonary fat embolism; affected patients have a more severe clinical course. Typically, the pulmonary signs and symptoms are preceded by bone pain; affected individuals may have systemic signs of a fat embolism, including changes in their mental state, thrombocytopenia, and petechiae. There are significant decreases in the hemoglobin and platelet counts and lipid-laden macrophages are found in fluid obtained by bronchoalveolar lavage. Infarction of the bone marrow with embolization of the fatty bone marrow to the lungs results in activation of pulmonary secretory phospholipase A2 (SPLA2). Markedly elevated levels of SPLA2 have been noted during ACS episodes. SPLA2 cleaves phospholipids and liberates free fatty acids, which cause acute pulmonary toxicity. Arachidonic acid is released, favoring vasoconstriction and oleic acid increases the upregulation of vascular cell adhesion molecule (VCAM-1). Infarction of the bony thorax may cause splinting, hypoventilation, and atelectasis, leading to hypoxia and intrapulmonary sickling. Opiods prescribed for the pain may suppress respiratory drive compounding the hypoventilation. Approximately 30% of ACS episodes are due to infection. In a multicenter study of ACS, 27 different pathogens were identified, but Chlamydia pneumoniae was the most frequent pathogen, followed by Mycoplasma pneumoniae and respiratory syncytial virus. There is a seasonal variation in the incidence of ACS in children, reflecting the occurrence of childhood respiratory infections. Parvovirus B10 has been associated with marrow necrosis and a particularly severe form of ACS. A proinflammatory state has been suggested to exist in SCD, which could predispose to ACS in response to a trigger such as infection. Evidence for this includes a leukocytosis in the absence of infection and reports of elevated cytokine levels, but such findings are not consistent. Several studies have demonstrated a significant association between ACS episodes and asthma in SCD children.

However, it is not clear from those reports whether the diagnosis of asthma predates the occurrence of ACS episodes. Sickle Chronic Lung Disease

Sickle cell lung disease (SCLD) may occur in 4% of SCD patients, but there is a lack of detailed epidemiological studies. The major risk factor for SCLD is recurrent episodes of ACS. SCLD is a progressive disease with an insidious onset progressing to endstage respiratory failure, characterized by hypoxemia, restrictive lung disease, and cor pulmonale and chest radiograph evidence of diffuse interstitial opacities. Pulmonary Hypertension

Over a third of affected adults develop pulmonary hypertension. Early diagnosis is essential as, although initially the patients may be asymptomatic, their condition progresses and they suffer worsening hypoxia and chest pain with impaired exercise tolerance. The pulmonary hypertension is characterized by progressive obliteration of the pulmonary vasculature. Possible causes of the pulmonary hypertension include chronic hypoxic stress causing irreversible remodeling of the pulmonary vasculature, recurrent pulmonary thromboembolism, sickle cell-related vasculopathy and pulmonary scarring from recurrent ACS episodes. A region of chromosome 2 has been identified as being involved in many cases of spontaneous primary pulmonary hypertension, but whether this influences the development of pulmonary hypertension in SCD merits further study. Lung Function Abnormalities

Children may suffer an ACS episode even in their first year after birth; not surprisingly then the lung function of even very young SCD children has been shown to differ significantly from that of healthy, ethnic matched children of similar age. Bronchial hyperreactivity (BHR) may be increased in SCD children, but this is not a universal finding; the relationship of BHR to the pulmonary complications of SCD requires further testing. Obstructive lung abnormalities are reported in children; restrictive abnormalities become more prominent with advancing age. Most adults with SCD develop abnormal pulmonary function characterized by restrictive lung disease, abnormal diffusing capacity and hypoxemia. Up to 40% of children and adolescents may suffer nocturnal desaturation episodes, possibly due to obstructive sleep apnea. It is clearly important to monitor SCD patients for such episodes, but certain methods of assessing oxygen saturation in SCD patients may be inaccurate. The value of oxygen saturation

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calculated by a blood analyzer assumes a normal P50 and hence yields falsely high levels in SCD patients. SCD patients have a higher P50 than nonaffected individuals, as oxygen is less tightly bound to HbS than HbA, they have higher levels of 2,3-diphosphoglycerate too. Pulse oximetry can also yield inaccurate results in SCD patients, as the fraction of carboxyhemoglobin (COHb) is increased in some patients with SCD and oximeters do not differentiate between COHb and oxyHb. In addition, the patterns of light absorption differ between HbS and HbA, and HbA is used to calibrate oximeters. The most accurate method to assess oxygen saturation is to use a CO-oximeter as the technique distinguishes between COHb and oxyHb, is unaffected by skin pigmentation, and does not assume a normal P50.

Laboratory Findings Patients with SCD have a normochromic, normocytic anemia with a steady-state hemoglobin level of between 5 and 11 g dl
1. The diagnosis of SCD is made by documenting the presence of sickle hemoglobin by electrophoresis. Chorionic villous sampling is used to obtain fetal DNA for diagnosis in the first trimester of pregnancy.

Pathogenesis The sickle erythrocytes carry adhesion factors (CD36 and very late activation antigen-4 (VLA-4)); those factors are present in progenitors in the bone

marrow, but are normally lost before the erythrocytes are released into the bloodstream. Hypoxia enhances adhesion of the red blood cells to the vascular endothelin, a process mediated by interaction of VLA-4 (the ligand for 1) and VCAM-1 expressed on endothelin cells. Expression of adhesion molecules on the endothelin also causes leukocyte adhesion. Cytokines (such as interleukin-1b and tumor necrosis factor alpha) and endothelin-1 upregulate endothelin VCAM-1. The levels of the inflammatory cytokines are increased in response to infection or the production of oxidants during reperfusion and are increased in ACS. The binding of the inflammatory mediators to the endothelin cells initiates the translocation of the transcription factor nuclear factor kappa B (NF-kB) from the cytoplasm to the nucleus, where it initiates the transcription of genes for adhesion molecules (Figure 2). During an ACS, soluble plasma VCAM-1 is markedly elevated, whereas NO and its metabolite levels are markedly decreased. Hypoxia inhibits NO production by decreasing protein levels of constitutive NO synthase in the endothelin and there is inactivation of NO by free radical species liberated from activated macrophages and leukocytes. Plasma hemoglobin levels are elevated in SCD patients; hemoglobin is a rapid and effective scavenger of NO and hence plasma hemoglobin limits NO bioavailability. The low NO levels enhance adhesion, as NO, under normal physiological circumstances, inhibits endothelin VCAM-1 upregulation. It also inhibits endothelin-1 production. In addition, there is a lack of inhibition of platelet

Infection/oxidants

↑ Inflammatory cytokines (IL-1
& TNF-) Translocation of transcription factor NF-B from the cytoplasm to the nucleus ↑ Transcription of genes for adhesion moleclues including VCAM-1 ↑ VCAM-1 ↑ Adhesion of erythrocytes to vascular endothelium

Release of vasoconstrictor metabolites such as thromboxane A2

↓ NO levels

Hypoxia, activated macrophages & leukocytes, ↑ plasma hemoglobin

Vascular occlusion

Inhibition of platelet activation

Figure 2 Pathogenesis of vascular occlusion in SCD.

↑ Endothelin-1 production

216 SYSTEMIC DISEASE / Sickle Cell Disease

activation and further potentiation of microvascular occlusion and the release of vasoconstrictor metabolites such as thromboxane A2. The adhesion of the erythrocytes to the vascular endothelin is enhanced and potentiated during a VOC, and this leads to direct endothelin injury. A potential mechanism for the vascular damage in ACS is the generation of oxygen-related radicals, such as superoxide, hydrogen peroxide, and peroxynitrite. Sickle red blood cells produce greater quantities of the radicals, which may reflect the greater autooxidation of HbS compared to HbA. Deficiencies in antioxidant enzyme systems (superoxide dismutase, catalyse, and glutathione peroxidase) in sickle red blood cells means that superoxide and hydrogen peroxide are removed less efficiently. The erythrocytes also adhere to each other. This may be potentiated by the hypercoagulable state found in patients with SCD, which promotes vascular obstruction. A variety of mechanisms have been postulated to explain hypercoagulable state, including low levels of proteins S and C, raised levels of tissue factors, and factor VIII. The sickle cells occlude vessels causing vascular injury, especially to organs with sluggish circulation such as the spleen and bone marrow and in atelectatic areas of the lung. Neutrophils are more adherent to endothelin cells in SCD patients and their numbers are increased in patients with sickle cell anemia; this has been associated with ACS episodes and stroke.

Animal Models Transgenic mouse models of SCD have been developed. Such transgenic constructs have generated results suggesting that the vascular system of sickle cell transgenic mice cannot increase NO output in response to a stimulus, as there is accelerated NO catabolism. Data has also been produced that supports the concept of an exaggerated response to an inflammatory challenge occurring in SCD. Murine models have also been used to assess potential therapies and it has been demonstrated that the sickling phenotype can be corrected by retroviral vector-mediated gene transfer into repopulating stem cells. In addition, results suggest that a nonionic surfactant, which appears to reduce blood viscosity and inhibit red cell endothelin adhesion, may prevent hypoxic lung injury. Sulfasalazine inhibits the transcription of NF-kB and has been shown to significantly reduce vessel wall endothelin expression of VCAM-1 in sickle transgenic mice.

Management and Current Therapy Patients with SCD are particularly susceptible to infection, as they have functional abnormalities in their

complement system. They also have functional asplenia, which puts them at risk of infection with encapsulated organisms such as Streptococcus pneumoniae. Penicillin prophylaxis from infancy is therefore recommended, but resistance is increasing and compliance can be poor. Pneumococcal immunization is used, but conventional polysaccharide vaccines can be ineffective in children up to 23 months of age, who are particularly at risk of infection, but conjugate pneumococcal vaccines have been shown to promote immunogenicity. SCD children should also be given Hemophilus influenzae vaccine. Children with SCD commonly require blood transfusions to manage complications including anemia, ACS, and splenic sequestration. Packed red cells are the preferred blood component, as leukocyte reduced units reduce the likelihood of alloimmunization, transfusion reactions, platelet refractoriness, and infection transmission. Up to 10% of adult SCD patients are hepatitis C positive and have liver damage. For those who have suffered major complications, regular erythrocyte transfusions are given to dilute the endogenous cells; regular transfusions also depress the bone marrow, resulting in a decreased production of the native erythrocytes with HbSS. Chronic transfusion to achieve an HbS level of less than 30% and a total hemoglobin level of less than 12 g dl
1 was associated in a randomized trial involving SCD children with abnormal transcranial Doppler ultrasonography results with a significant reduction in hospitalization rates. Alloimmunization has been reported to occur in between 5% and 36% of SCD patients who have had at least one blood transfusion. A serious complication of alloimmunization is the delayed hemolytic transfusion reaction/ hyperhemolysis; this occurs typically 1 week posttransfusion presenting with back, leg, or abdominal pain and hemoglobinuria. A number of therapies are given in SCD patients to increase HbF levels. Hydroxyurea, a ribonuclease reductase inhibitor, blocks the synthesis of DNA and, as a result of bone marrow suppression, raises the HbF level. In addition, hydroxyurea is an NO donor and has been shown to reduce the adhesion of sickle cells to the vascular endothelin in vitro by reducing VCAM-1 production. A randomized study was terminated prematurely, as hydroxyurea reduced the frequency of ACS episodes, hospitalization, and the need for transfusion. Hydoxyurea, however, may cause cytopenias and patients must be carefully monitored, especially early in the administration of therapy. The systematic review of studies to date concluded that hydroxyurea is effective and safe in adults severely affected by sickle cell anemia, but further studies are required to elucidate its role

SYSTEMIC DISEASE / Sickle Cell Disease 217

in other patient groups. HbF levels were shown to increase more when hydroxyurea was given with erythropoietin. Butyrate and decitabine also increase fetal hemoglobin levels; butyrate and hydroxyurea appear to work synergistically. Drugs to prevent dehydration of the sickle red blood cells, including the imidazole antimycotic clotrimazole (which inhibits the human erythrocyte Gardos channel) and magnesium (which inhibits K þ Cl
cotransport), are currently being studied. Stem cell transplantation in adults and children has been associated with no recurrence of painful crisis in those with stable engraftment. The best results (disease-free survival of 93%) are obtained in young children who have HLA-identical sibling donors and are transplanted early in the course of their disease. Overall, however, 10–20% of SCD patients reject their bone marrow and there is a transplantrelated mortality of 8%, and 6% suffer graft-versushost disease. Transplantation is usually considered for patients who have significant sickle-related complications. Gene therapy is a future direction. ACS Episodes

The treatment of ACS includes broad-spectrum antibiotics, including macrolides or quinolones to treat any atypical organisms. Oxygen therapy should be used to treat any hypoxemia; pulse oximeter correlation with arterial oxygen tensions may be poor and blood gas analysis should be undertaken in any patient suspected of being hypoxic. Patients should be carefully rehydrated and hydration should be limited to 1.5 times the maintenance fluid volume to avoid further impairment of lung function. Transfusion is indicated in patients with severe disease, multiorgan failure, and neurological abnormalities or cardiac disease. Packed red blood cell or exchange transfusion decrease the fraction of sickle hemoglobin and improve the oxygen-carrying capacity of the blood. In patients with severe anemia a simple transfusion is used, but if the patient has a relatively high hematocrit, then exchange transfusion should be undertaken so as to avoid causing increased blood viscosity. Analgesia should be given to control pain, but the amount limited to avoid respiratory depression; patient-controlled analgesia devices may reduce the risk of narcotic-induced hypoventilation. Routine use of incentive spirometry has been recommended to prevent atelectasis and ACS in SCD patients admitted to hospital with chest or bone pain; such management in a randomized trial was associated with a lower hospitalization rate. Pain and sedation, however, may limit cooperation with active breathing techniques and then continuous positive airway

pressure may be used if there is evidence of atelectasis on the chest radiograph or poor oxygen saturations in air. Assisted ventilation is required in a minority of patients, but some develop severe hypoxic respiratory failure; anecdotally, such patients have been successfully supported by highfrequency oscillation or extracorporeal membrane oxygenation. In small anecdotal series, inhaled NO (20–80 ppm) has been given to patients with ACS and pulmonary hypertension and has resulted in rapid and significant pulmonary vasodilation and improvement in oxygenation. Approximately 25% of patients wheeze during an ACS episode and may benefit from bronchodilator administration, but the effect on long-term outcome has not been investigated in randomized controlled trials. In children with mild to moderately severe ACS, dexamethasone administration was associated with a 40% reduction in the length of hospitalization, a shorter duration of supplementary oxygen requirement, and less need for analgesia. Such outcomes are biologically plausible, as corticosteroids have numerous effects on the inflammatory process, modulating endothelin cell adhesion molecule expression including VCAM-1, and have an inhibitory effect on phospholipase A2. However, it should be noted that a rebound effect was experienced, more dexamethasone-treated children requiring readmission, thus further trials are required to explore further the efficacy of this treatment. Sickle Chronic Lung Disease/Pulmonary Hypertension

Adult SCD patients should be screened for pulmonary hypertension with echocardiography. Longterm oxygen therapy should be given to affected patients with resting hypoxemia and, although the results of simple or exchange transfusion are disappointing, the combination of exchange transfusion with long-term supplementary oxygen can be beneficial. Chronic anticoagulation has been recommended, but the study demonstrating that this treatment improved survival was retrospective and nonrandomized. The role of vasodilators is less well tested and their use should be guided by the result of an acute vasodilator challenge. If there is a positive response the first option is a calcium channel blocker, but in those who do not respond, an endothelin receptor antagonist should be considered. If the disease is very advanced, then intravenous epoprostenal should be given. It has been demonstrated that arginase activity is increased in SCD patients with pulmonary hypertension; L-arginine, a nitrogen donor for the synthesis of NO, may be useful in affected

218 SYSTEMIC DISEASE / Eosinophilic Lung Diseases

patients. There is a high procedure-related mortality for lung transplantation in SCD patients. Sudden death in SCLD patients with pulmonary hypertension is common due to pulmonary thromboembolism, systemic hypotension, and cardiac arrhythmia. The mortality of patients with pulmonary hypertension is seven times greater than that of SCD patients without pulmonary hypertension. The mean survival of SCD patients with chronic lung disease and elevated pulmonary artery pressures can be as little as 2 years. See also: Adhesion, Cell–Cell: Vascular; Epithelial. Asthma: Acute Exacerbations. Bronchodilators: Beta Agonists. Corticosteroids: Therapy. Endothelins. Hemoglobin. Interleukins: IL-5. Nitric Oxide and Nitrogen Oxides. Oxidants and Antioxidants: Antioxidants, Enzymatic; Oxidants. Oxygen Therapy. Pediatric Pulmonary Diseases. Pneumonia: Atypical; Community Acquired Pneumonia, Bacterial and Other Common Pathogens. Symptoms of Respiratory Disease: Cough and Other Symptoms; Dyspnea; Chest Pain. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Vascular Disease.

Further Reading Atweh GF and Schechter AN (2001) Pharmacologic induction of fetal haemoglobin: raising the therapeutic bar in sickle cell disease. Current Opinion in Hematology 8: 123–130. Claster S and Vichinsky EP (2003) Managing sickle cell disease. British Medical Journal 327: 1151–1155. Gladwin MT, Sachdev V, Jison ML, et al. (2004) Pulmonary hypertension as a risk factor for death in patients with sickle cell disease. New England Journal of Medicine 350: 886–895. Greenough A (2004) Sickle cell disease – pulmonary complications and a proinflammatory state? American Journal of Respiratory and Critical Care Medicine 169: 663–664. Klings ES and Farber HW (2001) Role of free radicals in the pathogenesis of acute chest syndrome in sickle cell disease. Respiration Research 2: 280–285. Knight J, Murphy TM, and Browning I (1999) The lung in sickle cell disease. Pediatric Pulmonology 28: 205–216. Mak V and Davies SC (2003) The pulmonary physician in critical care. Illustrative case 6: acute chest syndrome of sickle cell anaemia. Thorax 58: 726–728. Miller ST, Wright E, Abboud M, et al. for the STOP Investigators (2001) Impact of chronic transfusion on incidence of pain and acute chest syndrome during the Stroke Prevention Trial (STOP) in sickle cell anaemia. Journal of Pediatrics 139: 785–789. Minter KR and Gladwin MT (2001) Pulmonary complications of sickle cell anaemia. A need for increased recognition, treatment and research. American Journal of Respiratory and Critical Care Medicine 164: 2016–2019. Platt OS (2000) The acute chest syndrome of sickle cell disease. New England Journal of Medicine 342(25): 1904–1907. Steinberg MR and Brugnara C (2003) Pathophysiological based approaches to treatment of sickle cell disease. Annual Review of Medicine 54: 89–112. Stuart MJ and Yamaja Setty BN (2001) Acute chest syndrome of sickle cell disease: new light on an old problem. Current Opinion in Hematology 8: 111–122.

Sylvester KP, Patey RA, Milligan P, et al. (2004) Pulmonary function abnormalities in children with sickle cell disease. Thorax 59: 67–70. Vichinsky EP, Neumayr LD, Earles AN, et al. for the National Acute Chest Syndrome Study Group (2000) Causes and outcomes of the acute chest syndrome in sickle cell disease. New England Journal of Medicine 342(25): 1855–1865. Walters MC, Nienhuis AW, and Vichinsky E (2002) Novel therapeutic approaches in sickle cell disease. Haematology 1: 10–34.

Eosinophilic Lung Diseases J P Lynch III, M C Fishbein, and R D Suh, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Eosinophilic lung diseases are a diverse group of disorders characterized by eosinophilic infiltration of the airways or lung parenchyma, with or without extrapulmonary involvement. Some pulmonary eosinophilic disorders are due to specific causes or exposures (e.g., parasitic, fungal, or mycobacterial infections; hematological or solid malignancies; connective tissue disorders; drugs; environmental exposures) whereas in others no cause or inciting factor is recognized. Eosinophils are proinflammatory and may cause tissue injury, but may also be helpful to eradicate parasites or specific infections. Irrespective of cause, clinical, pathologic, and radiographic features of eosinophilic syndromes overlap extensively. In this article, we discuss the salient clinical features and management of the following pulmonary eosinophilic syndromes: tropical pulmonary eosinophilia, a hypersensitivity response caused by parasites (helminths); acute eosinophilic pneumonia due to other infectious causes (e.g., mycobacteria, fungi, Pneumocystis carinii); idiopathic eosinophilic pneumonias, which comprise simple pulmonary eosinophilia, acute eosinophilic pneumonia, and chronic eosinophilic pneumonia; allergic angiitis and granulomatosis (Churg–Strauss syndrome); allergic bronchopulmonary aspergillosis; idiopathic hypereosinophilic syndrome; eosinophilia– myalgia syndrome; and toxic oil syndrome.

Introduction Eosinophilic lung diseases are a diverse group of disorders characterized by eosinophilic infiltration of the airways or lung parenchyma, with or without extrapulmonary involvement. Eosinophilic lung diseases were first described as ‘PIE’ syndromes, or pulmonary infiltrates with (blood) eosinophilia. Subsequently, pulmonary eosinophilic disorders without blood eosinophilia were recognized. Some pulmonary eosinophilic disorders are due to specific causes or exposures (e.g., parasitic, fungal, or mycobacterial infections; hematological or solid malignancies; connective tissue disorders; drugs; and environmental exposures) whereas in others no cause

SYSTEMIC DISEASE / Eosinophilic Lung Diseases 219

or inciting factor is recognized. Eosinophils are proinflammatory and may cause tissue injury, but may also be helpful in eradicating parasites or specific infections. Irrespective of cause, clinical, pathologic, and radiographic features of eosinophilic syndromes overlap extensively.

Pathogenesis of Eosinophilic Lesions Eosinophils are derived from myeloid progenitors in bone marrow, via the action of three hematopoietic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5. T-helper type 2 (Th2) lymphocytes orchestrate the recruitment of eosinophils (and other effector cells) by releasing cytokines such as IL-4, IL-5, and IL-13. IL-4 is required for class switching from immunoglobulin G (IgG) to IgE. IL-5, in association with IL-3 and GM-CSF, regulates eosinophilic proliferation. Following IgE-triggered activation, mast cells may promote eosinophilic inflammation of the airways by producing proinflammatory cytokines. In several types of eosinophilic pneumonias, elevations in serum IgE, activated CD4 T cells, CC chemokines (e.g., eotaxin, monocyte chemoattractant protein-1 (MCP-1), thymus- and activation-regulated chemokine (TARC), and CCR4), Th2 cytokines (e.g., IL-5, IL-13, IL-10), and interferon-g-inducing factor (IL-18) have been found, suggesting their importance in the pathogenesis of these disorders. The release of constituents of eosinophilic granules such as major basic protein (MBP), eosinophilic cationic protein (ECP), eosinophil-derived neurotoxins, reactive oxygen radicals, and proinflammatory cytokines may cause tissue injury. The life span of eosinophils in tissue is prolonged by IL-3, IL5, or GM-CSF, which inhibit apoptosis. In addition, tissue eosinophils can regulate their own survival through an autocrine pathway.

hypersensitivity response caused by the filarial worms Wuchereria bancrofti or Brugia malayi, which are endemic in India, Africa, South America, and South East Asia. With increasing travel and immigration, TPE may occur even in nonendemic regions. In this context, an erroneous diagnosis of asthma is often made. Laboratory findings include high levels of serum IgE and eosinophilia in blood and bronchoalveolar lavage fluid (BALF). Microfilariae can be found in lung, liver, or lymph nodes in affected patients. Treatment with diethylcarbamazine (6– 12 mg k
1 g
1 day
1 in three divided doses for 1–3 weeks) is highly effective. Untreated TPE can lead to progressive pulmonary fibrosis and respiratory failure. Other less common parasites that might evoke pulmonary eosinophilia include Schistosoma, Trichinella spiralis, Paragonimus westermani, Echinococcus granulosus, Dirofilaria immitis, Clonarichis sinensis, and Opisthorchis. Although parasitic infections are rare in the US, Strongyloides, Ascaris, Toxocara, and Ancylostoma may be implicated in eosinophilic lung disorders.

Acute Eosinophilic Pneumonia due to Other Infectious Causes Acute eosinophilic pneumonia (AEP) may complicate infections due to mycobacteria, fungi (e.g., Coccidioides immitis or Histoplasma capsulatum), or Pneumocystis jiroveci (formerly P. carinii). These causes should always be excluded before accepting the diagnosis of ‘idiopathic’ AEP. Idiopathic Eosinophilic Pneumonias

The idiopathic eosinophilic pneumonias include simple pulmonary eosinophilia, chronic eosinophilic pneumonia (CEP), and AEP.

Pulmonary Eosinophilia due to Specific Causes

Simple Pulmonary Eosinophila (Loeffler’s Syndrome)

Eosinophilic pneumonia may be a response to infection (most commonly helminthic), as a reaction to drugs, chemotherapy, or radiation therapy, or in the context of atopy, allergies, asthma, connective tissue disease, or malignancy. Given the space constraints of this article, our discussion is limited to a few specific forms of eosinophilic pneumonias (discussed below).

Simple pulmonary eosinophilia (also termed Loeffler’s syndrome) is characterized by migratory, patchy pulmonary opacities, peripheral blood eosinophilia, and few or no respiratory symptoms (Figure 1). The course is self limited. Given the mild and transient nature of this condition, treatment is not warranted. Idiopathic Chronic Eosinophilic Pneumonia

Pulmonary Eosinophilia due to Parasitic Infections

Worldwide, helminthic infections are the most common cause of pulmonary and blood eosinophilia. Tropical pulmonary eosinophilia (TPE) is a

CEP is a rare disorder of uncertain etiology characterized by cough, wheezing, migratory alveolar infiltrates, constitutional symptoms, and peripheral blood eosinophilia.

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necrosis or true vasculitis are not found. When vasculitis is present, allergic angiitis and granulomatosis (Churg–Strauss syndrome) (Figure 5) is more likely. Bronchial asthma, with smooth muscle hyperplasia and mucus-containing Charcot–Leyden crystals and eosinophils, may be present (Figure 6). Surgical lung biopsy is usually not necessary, since the diagnosis of CEP can usually be established by bronchoscopic techniques (e.g., transbronchial biopsies and/or BAL) provided clinical and radiographic features are consistent.

Figure 1 Simple pulmonary eosinophilia (Loeffler’s syndrome). (a) Posteroanterior (PA) chest radiograph demonstrates left upper lung airspace consolidation. (b) PA radiograph 2 weeks later shows changing or ‘fleeting’ areas of consolidation, now within both mid and left lower lung.

Etiology The cause is unknown, but a heightened allergic diathesis likely plays a contributory role. Histopathological features Lung biopsies in CEP demonstrate dense aggregates of eosinophils, histiocytes, and multinucleated giant cells within bronchioles and alveolar spaces and septa (Figures 2–4). Additional features include intra-alveolar eosinophilic abscesses, degenerating necrotic eosinophils, Charcot– Leyden crystals, alveolar macrophages containing eosinophilic fragments, foci of organizing pneumonia, scattered lymphocytes and plasma cells, and illdefined granulomatous inflammation. Well-formed granulomata are rare as are extensive fibrosis or parenchymal necrosis. Small and medium-sized arteries may be infiltrated by eosinophils, but fibrinoid

Clinical features Most common symptoms of CEP are cough (490%), dyspnea (490%), fever (475%), and weight loss (470%). The course is subacute or chronic, with symptoms developing over several weeks to months. Extrapulmonary involvement is lacking. Atopy or asthma antedate CEP in 450% of patients. CEP typically occurs in the 4th– 6th decade of life, but all ages can be affected. There is a 2:1 female:male predominance. The disease is less common in cigarette smokers. Chest radiographs reveal patchy, subpleural alveolar opacities, with a predilection for the upper lobes (Figures 7–10). The peripheral distribution of alveolar opacities, with central sparing, is evident in 50–70% of patients and is termed the ‘photographic negative of pulmonary edema’. Less common patterns include focal lobar consolidation or reticulonodular opacities. Pleural effusions are not found. Chest computed tomographic (CT) scans demonstrate peripheral consolidations, ground-glass opacities (GGO), and reticular opacities (mimicking cryptogenic organizing pneumonia (COP)) (Figure 9(b)). A striking feature of CEP is the rapidity of radiographic improvement (within hours to days) following institution of corticosteroid therapy (Figure 11). Pulmonary function tests may reveal restriction and/or obstruction, but are normal in a third of patients. BAL demonstrates marked eosinophilia (often 450%), often with degranulation and hypersegmentation of eosinophils; lymphocyte and neutrophil percentages are normal or mildly elevated. Elevations in the peripheral blood eosinophil counts and erythrocyte sedimentation rate (ESR) are noted in 480% of patients with CEP, and parallel the course of the disease. Serum IgE is increased in 50–75% of patients but severe elevations (42000 ng ml
1) are rare. Management and current therapy Corticosteroids are the cornerstone of therapy for CEP and are highly efficacious. Optimal dose and duration of therapy have not been studied in randomized trials. An initial dose of prednisone of 40–60 mg day
1 or equivalent is adequate; lower doses may be used in patients at

SYSTEMIC DISEASE / Eosinophilic Lung Diseases 221

Figure 2 Chronic eosinophilic pneumonia. Lung biopsy specimen demonstrating flooding of alveolar spaces and interstitium with bilobar eosinophilic leukocytes. Alveolar macrophages are laden with disintegrating eosinophils. There is no evidence of vasculitis or necrosis, and the lung architecture is preserved (hematoxylin–eosin (H&E) stain). Reproduced with permission from Lynch JP III and Flint AF (1984) Sorting out the eosinophilic pulmonary syndromes. Journal of Respiratory Diseases 5: 61–78.

Figure 3 Chronic eosinophilic pneumonia. Lung biopsy demonstrating numerous Touton-type multinucleated giant cells within alveolar spaces. Eosinophils are interspersed within the pulmonary interstitium and alveolar spaces (H&E). Reproduced with permission from Lynch JP III and Flint AF (1984) Sorting out the eosinophilic pulmonary syndromes. Journal of Respiratory Diseases 5: 61–78.

increased risk for side effects. Responses are usually dramatic (within 24–72 h). Relapses occur in 80% of patients as corticosteroids are tapered or discontinued. Some patients require long-term (sometimes indefinite) therapy with low-dose prednisone (e.g., 10–20 mg every other day) to maintain remissions.

Idiopathic Acute Eosinophilic Pneumonia

Idiopathic AEP, first described in 1989, is a rare acute febrile illness with cough, dyspnea, fever, diffuse infiltrates on chest radiographs, pronounced BAL eosinophilia (typically 425% eosinophils), and

222 SYSTEMIC DISEASE / Eosinophilic Lung Diseases

Figure 4 Chronic idiopathic eosinophilic pneumonia. (a) Note interstitial and alveolar infiltrates of eosinophils and red macrophages that have engulfed eosinophilic granules (H&E,  200).

Figure 5 Churg–Strauss syndrome. (a) Photomicrograph (lung biopsy): note the necrotizing arteritis with numerous eosinophils in the wall of, and around, a necrotic artery. (b) Extravascular granuloma with multinucleated giant cell (arrow) (H&E,  200).

M

∗

Figure 6 Bronchus in asthma: note hypertrophied smooth muscle wall (M), thickened basement membrane (asterisk), and dense infiltrate containing numerous eosinophils (H&E,  100).

hypoxemic respiratory failure, often requiring mechanical ventilatory support. Etiology

The cause is unknown.

Histopathology The histopathological features of AEP demonstrate extensive interstitial and intra-alveolar eosinophils resembling CEP but neutrophils, interalveolar septal edema, and fibrin deposition

SYSTEMIC DISEASE / Eosinophilic Lung Diseases 223

within alveolar spaces are more prominent in AEP. Hyaline membranes and diffuse alveolar damage (DAD) may also be present. Granulomata or vasculitis are not found in AEP. The diagnosis of AEP does not require surgical lung biopsy, as marked

BAL eosinophilia (often 450%) in the appropriate clinical context is sufficient to establish the diagnosis. Clinical features The onset of AEP is rapid, and most patients present within 1–7 days of onset of

Figure 8 Chronic eosinophilic pneumonia. PA chest radiograph from a 28-year-old woman with fever, cough, and wheezing demonstrates multiple, focal dense alveolar infiltrates in the upper lobes and axillary regions. Transbronchial lung biopsies demonstrated eosinophilic abscesses consistent with CEP. The infiltrates resolved following institution of corticosteroid therapy. Reproduced with permission from Lynch JP III and Keane M (2003) Current approaches to the treatment of parenchymal lung diseases. In: Spina D, Page CP, Metzger WJ, and O’Connor BJ (eds.) Drugs for the Treatment of Respiratory Diseases, pp. 247– 335. Cambridge: Cambridge University Press.

Figure 7 (a) Chronic eosinophilic pneumonia. Chest radiograph of a 26-year-old woman demonstrates a focal infiltrate in the apical region of the left upper lobe (arrow). She improved promptly with corticosteroid therapy. Reproduced with permission from: Lynch JP III and Flint AF (1984) Sorting out the eosinophilic pulmonary syndromes. Journal of Respiratory Diseases 5: 61– 78. (b) Chronic eosinophilic pneumonia. Chest radiograph from the same patient 5 months later, with three small focal alveolar infiltrates (arrows) in the right apex, right axillary region, and left axillary region. Note the peripheral, subpleural location of the infiltrates. Reproduced with permission from Lynch JP III and Flint AF (1984) Sorting out the eosinophilic pulmonary syndromes. Journal of Respiratory Diseases 5: 61–78. (c) Chronic eosinophilic pneumonia. Chest radiograph from the same patient during a relapse. Arrows depict the alveolar infiltrate with a distinct predilection for the subpleural region on the left and the left apex. There is a small nodular infiltrate in the right apex (arrow).

224 SYSTEMIC DISEASE / Eosinophilic Lung Diseases

Figure 10 Chronic eosinophilic pneumonia. PA chest radiograph demonstrates focal alveolar infiltrates in the left axillary and apical regions. Bronchoscopy demonstrated aggregates of eosinophils within alveolar spaces and interstitium consistent with CEP. Corticosteroid therapy led to prompt improvement. Reproduced from Shannon JJ and Lynch JP III (1995) Eosinophilic pulmonary syndromes. Clinical Pulmonary Medicine 2: 19–38, with permission from Lippincott Williams & Wilkins.

Figure 9 Idiopathic chronic eosinophilic pneumonia. (a) PA chest radiograph reveals dense airspace consolidation within the right greater than left upper lung. In addition, a small to moderate pneumothorax is seen on the right related to transbronchial lung biopsy. (b) Axial CT further demonstrates dense airspace consolidation within both upper lobes, favoring the lung periphery.

symptoms. However, the course may be subacute, evolving over 7–30 days. Acute hypoxemic respiratory failure ensues; mechanical ventilatory support is required in two-thirds of patients. The presentation of AEP resembles acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). However, extrapulmonary organ dysfunction or shock is not a feature of AEP. Chest pain, often pleuritic, occurs in 50–70% of patients and myalgias occur in 50%. There is a slight male predominance. Antecedent asthma is not a feature of AEP. Chest CT in AEP resembles pulmonary edema, with bilateral extensive alveolar opacities and interlobular septal thickening (i.e., Kerley B lines). The opacities lack the peripheral distribution characteristic of CEP. Pleural effusions are evident on CT in 70% of cases. BAL demonstrates striking increases in eosinophil number (often exceeding 50%). This feature

distinguishes AEP from other clinical syndromes such as ARDS or pneumonia, which exhibit marked neutrophilia with normal or low eosinophil counts. Modest elevations in lymphocytes and neutrophils are also typical of AEP. Eosinophils in CEP and AEP may be degranulated and possess multiple lobes. Peripheral blood eosinophilia is present in only 40– 65% of patients with AEP. Elevations in serum IgE may be striking. Pathogenesis Other causes of AEP include human immunodeficiency virus (HIV) infection, cigarette smoke, fireworks, exposure to noxious occupational agents, drugs, and microbial pathogens. Management and current therapy Idiopathic AEP is rare, and controlled therapeutic trials are lacking. However, corticosteroids are highly efficacious, with response rates approaching 100%. With severe cases, initial treatment with intravenous methylprednisolone (60–125 mg every 6 h) is advocated, until respiratory failure resolves. At that point, the corticosteroids are converted to oral prednisone. Responses to corticosteroids are usually dramatic with symptoms improving within hours to days of treatment; chest radiographs normalize within 1–4 weeks. Pulmonary function normalizes and late sequelae are rare. A brief course of corticosteroid therapy (6–12 weeks) is adequate, as late relapses are rare.

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characterized by blood and tissue eosinophilia, asthma, and a multisystemic necrotizing vasculitis. Etiology

The cause is unknown.

Histopathological features Histopathological features of CSS include a necrotizing eosinophilic and granulomatous vasculitis involving small vessels (capillaries, arterioles, venules); extravascular granulomas with pallisading histiocytes and multinucleated giant cells; eosinophilic infiltrates; and varying degrees of necrosis, hemorrhage, and fibrosis (Figures 5 and 12). The prominent eosinophilic and granulomatous components and propensity for small vessel involvement distinguish CSS from classic polyarteritis nodosa. Pulmonary involvement may include CEP, necrotizing vasculitis, or capillaritis (with alveolar hemorrhage).

Figure 11 (a) Chronic eosinophilic pneumonia. Chest radiograph demonstrating widespread but focal, dense alveolar opacities in a 28-year-old woman with fever, wheezing, and dyspnea. Transbronchial lung biopsies were compatible with CEP. The chest radiographs normalized within 3 weeks following institution of corticosteroid therapy. Reproduced from Shannon JJ and Lynch JP III (1995) Eosinophilic pulmonary syndromes. Clinical Pulmonary Medicine 2: 19–38, with permission from Lippincott Williams & Wilkins. (b) Chronic eosinophilic pneumonia. PA chest radiograph from a 47-year-old man showing confluent bilateral alveolar infiltrates. Peripheral blood eosinophil count was markedly elevated at 6600 per ml. Transbronchial lung biopsies showed extensive eosinophilic infiltration and consolidation of the alveolar spaces. Following institution of corticosteroid therapy, partial clearing of chest radiographs was noted within 36 h. Reproduced with permission from Lynch JP III and Flint AF (1984) Sorting out the eosinophilic pulmonary syndromes. Journal of Respiratory Diseases 5: 61–78.

Churg–Strauss Syndrome

Churg–Strauss syndrome (CSS), also termed allergic angiitis and granulomatosis, is a rare disease

Clinical manifestations Asthma is invariably present in CSS, and usually antedates the development of vasculitis by several years. Pulmonary opacities are present in 30–70% of patients and pleural effusions in 20–30%. Transient alveolar opacities, often in a peripheral distribution, are characteristic (Figure 13). In contrast to other granulomatous vasculitides, nodular densities and cavitation are unusual in CSS. Extrapulmonary sites of organ involvement include constitutional symptoms (90%); mononeuritis multiplex (63–92%); skin lesions (e.g., petechiae, purpura, ulcerations, miscellaneous) (50–70%); cardiac involvement (15–56%); central nervous system (CNS) involvement (8–27%); arthralgias or myalgias (30– 50%); systemic hypertension (50%); gastrointestinal (GI) tract (30–60%); renal failure (o5%); and ocular involvement (o5%). Prior to the availability of effective therapy, most deaths were ascribed to cardiac involvement (cardiac failure, pericarditis, coronary vasculitis, eosinophilic endocarditis). Typically, three phases of CSS can be identi fied. The prodromal period consists of an atopic or allergic diathesis (e.g., allergic rhinitis, nasal polyps, asthma), which antedates vasculitis by months to years. Later in the course of illness, blood and tissue eosinophilia (e.g., Loeffler’s syndrome, CEP, or eosinophilic gastroenteritis) develop. The vasculitic phase occurs as a late event, 10 or more years after the onset of allergic rhinitis and 3–5 years after the onset of asthma. Increasingly, severe and more frequent attacks of asthma are harbingers to the development of CSS. A shorter duration from the onset of asthma to the development of vasculitis is associated with a worse prognosis. Laboratory studies in CSS demonstrate elevated ESR and blood eosinophil counts in 480% during

226 SYSTEMIC DISEASE / Eosinophilic Lung Diseases

Figure 12 Churg–Strauss syndrome. Photomicrograph demonstrates necrotic lung tissue surrounded by a chronic inflammatory cell infiltrate, occasional giant cells, palisading histiocytes, and eosinophils (H&E,  165). Reproduced from Lynch JP III and Fantone JC III (1991) Other pulmonary granulomatous vasculitic syndromes. In: Lynch JP III and DeRemee R (eds.) Immunologically Mediated Pulmonary Diseases, pp. 302–321. Philadelphia, PA: JB Lippincott Company, with permission from Lippincott Williams & Wilkins.

acute exacerbations. Both the ESR and blood eosinophil counts correlate with disease activity. Serum IgE is elevated in 450% of patients. Circulating antineutrophil cytoplasmic antibodies (ANCA) (primarily p-ANCA) are present in 40–70% of patients with CSS.

rates exceeded 80% and 70%, respectively. Relapses occur in 20–40% as the corticosteroids or immunosuppressive therapy is tapered or discontinued. Longterm low-dose prednisone is usually required as maintenance therapy for chronic asthma. Allergic Bronchopulmonary Aspergillosis

Pathogenesis The immunopathogenesis of CSS involves a chronic allergic diathesis that evolves over time (often years). Th2 cells are pivotal to the early asthmatic phase and recruitment of eosinophils to tissues. During the later phases of the disease, circulating myeloperoxide (MPO)-ANCA antibodies likely play key contributory roles in the pathogenesis of glomerulonephritis and vasculitis. Possible contributory factors include repeated antigen stimulation, vaccination, and cysteinyl leukotriene receptor antagonists (LTRAs). Management and current therapy Corticosteroids are the cornerstone of therapy for CSS, and achieve remissions in 480% of patients. Immunosuppressive or cytotoxic agents are employed for more severe or steroid-recalcitrant cases or when unfavorable prognostic factors are present. Factors associated with a worse prognosis include older age (465 years), CNS involvement, cardiomyopathy, severe GI tract involvement, proteinuria (41 g day
1), and renal insufficiency. In several prospective studies employing various therapeutic regimens, 3- and 10-year survival

Allergic bronchopulmonary aspergillosis (ABPA) is a clinical syndrome that occurs in 2–8% of patients with chronic asthma or cystic fibrosis (CF). It is characterized by repetitive episodes of bronchospasm, mucus plugging, blood and tissue eosinophilia, and airflow obstruction. Etiology The immunopathogenesis of ABPA involves an exaggerated host response to endobronchial Aspergillus antigens. Eosinophils, basophils, mast cells, lymphocytes, mononuclear phagocytes, and humoral antibodies participate. The hyphae of Aspergillus fumigatus grow saprophytically in the bronchial lumens, and elicit a CD4 þ Th2 immune response characterized by production of the cytokines IL-4, IL-5, IL-13, and Aspergillus-specific and polyclonal IgE. Release of fungal proteases, hostderived proinflammatory cytokines (e.g., IL-8, IL-6, MCP-1), and mast cell degranulation results in persistent bronchial inflammation, central bronchiectasis, and ultimately pulmonary damage. These immunopathogenetic mechanisms are not unique to Aspergillus, because other fungi or molds may elicit

SYSTEMIC DISEASE / Eosinophilic Lung Diseases 227

identical hypersensitivity reactions (e.g., Candida, Helminthosporium, Curvularia, Dreschlera, and Steemphyllium species). Histopathological features Histopathological features of ABPA include dilated bronchi; endobronchial mucus; dense peribronchiolar cellular infiltrates composed of eosinophils, mast cells, and mononuclear cells; Charcot–Leyden crystals; Curschmann’s spirals; and degenerating eosinophils (Figure 14). Fungal hyphae may be observed within bronchial lumens or the inflammatory exudate. Vasculitis or extensive necrosis are not features of ABPA. The surrounding lung parenchyma may reveal CEP, organizing pneumonia, or foreign body giant cell reactions. Mucoid impaction of bronchi is characterized by filling of bronchiectatic bronchi and bronchioles with mucin, eosinophils, and inflammatory exudate. Lung biopsy is not necessary to make the diagnosis of ABPA, which is established by clinical and laboratory tests (discussed below).

Figure 13 Churg–Strauss syndrome. (a) PA radiograph shows ill-defined and faint consolidation within the right mid and upper lung. (b) Axial CT confirms and better delineates right upper lobe airspace consolidation. The left upper lobe contains ground-glass densities, consistent with both areas of lesser consolidation and air trapping.

Clinical manifestations In contrast to other eosinophilic lung diseases, the eosinophilic infiltration in ABPA is largely confined to the airways (bronchi and bronchioles). Consequently, the main symptoms of ABPA are difficult-to-treat or recurrent episodes of asthma. Major diagnostic criteria for ABPA include asthma, immediate cutaneous reaction to Aspergillus, precipitating IgG antibodies to A. fumigatus, proximal (central) bronchiectasis, chest radiographic infiltrates, blood eosinophilia, elevated serum total IgE, and increased A. fumigatus-specific IgE or IgG

Figure 14 Allergic bronchopulmonary aspergillosis. Mucus plug from bronchopulmonary aspergillosis: note numerous eosinophils and Charcot–Leyden crystals entrapped in mucus (H&E,  400). Inset shows typical features of Aspergillus fungal forms in mucus (methenamine silver stain,  400).

228 SYSTEMIC DISEASE / Eosinophilic Lung Diseases

antibodies. Immediate skin test reactivity and precipitating antibodies are sensitive but not specific for ABPA. Additional criteria required to establish the diagnosis of ABPA include airflow obstruction, elevations in serum IgE or Aspergillus-specific IgE, and chest radiographic infiltrates or bronchiectasis. Typical chest radiographic features include peribronchiolar thickening (most evident in the upper lobes), ring shadows, ‘tram lines’ (parallel linear shadows caused by inflamed bronchi), ‘finger-inglove’ sign (medium-sized bronchi filled with mucus), segmental consolidation, atelectasis, and mucoid impaction (Figure 15(a)). However, chest radiographs may be normal, even during acute flares of the disease. Chest CT scans reveal thickened and dilated bronchial walls, proximal bronchiectasis, centrilobular nodules, and mucus impaction (Figure 15(b)). Additional findings include expectoration of mucus plugs, sputum flecked with brown or black elements, sputum cultures yielding A. fumigatus, and sputum smears showing fungal hyphae or eosinophils. ABPA is characterized by acute flares with cough, wheezing, and airflow obstruction, which may resolve with corticosteroid therapy. Repetitive flares of ABPA, often evolving over years, may cause irreversible bronchial injury, central bronchiectasis, pulmonary dysfunction, pulmonary fibrosis, and emphysematous changes. Secondary complications include atypical mycobacteriosis, secondary infections, and cor pulmonale. With severe chronic disease, fatalities due to respiratory failure may occur. Extrapulmonary involvement does not occur. Serum IgE levels and blood eosinophil counts correlate with disease activity. During acute flares, marked elevations of serum IgE (4 2000 ng ml
1) are typical. Serum IgE levels fall in response to therapy, and serial studies are invaluable to assess disease activity. Management and current therapy Corticosteroids are the cornerstone of therapy for ABPA, and chronic corticosteroid therapy is often required. Initial treatment with prednisone 0.5–1.0 mg kg
1 day
1 (or equivalent) is usually adequate to achieve remissions. The corticosteroids are tapered to alternate day dosing over the next several weeks to months, with an attempt to control symptoms and maintain acceptable lung function. Inhaled corticosteroids have a modest steroid-sparing effect. Spirometry and serum IgE levels are helpful in following the disease. These parameters should be followed at least once monthly following an acute flare, with less frequent measurements once the disease is controlled. Poor control of ABPA leads to irreversible bronchiectasis, pulmonary fibrosis, and permanent airflow obstruction. Because chronic corticosteroid therapy

Figure 15 Allergic bronchopulmonary aspergillosis. (a) PA chest radiograph demonstrates tubular and branching densities within the right lung base. (b,c) Two contiguous axial CT images show branching of dilated mucus-impacted bronchi within the right lower lobe anterobasal segment and atelectasis and consolidation within the left lower lobe.

has cumulative side effects, it is not practical to aim for normal serum IgE levels. The extent and rate of corticosteroid taper needs to be gauged by clinical symptoms, chest radiographs, pulmonary function, serum IgE levels, and the presence or absence of adverse effects of corticosteroids. Relapses require

SYSTEMIC DISEASE / Eosinophilic Lung Diseases 229

retreatment with high-dose corticosteroids. Oral itraconazole (200 mg b.i.d.) may be efficacious as adjunctive therapy to reduce airway colonization with aspergilli, but is expensive. Idiopathic Hypereosinophilic Syndrome

Idiopathic hypereosinophilic syndrome (IHES) is a rare and complex chronic disorder characterized by severe hypereosinophilia (4 1500 per mm3) for 46 months, diffuse organ infiltration by mature eosinophils, anemia, and constitutional symptoms. Etiology The cause of IHES is unknown, but recent studies support segregating IHES into two major variants (myeloproliferative and lymphocytic). The myeloproliferative variant (m-HES) is associated with clonal proliferation of eosinophils; additional features found in some patients with m-HES include deletions in chromosome 4q12, a fusion gene encoding a protein displaying constitutive tyrosine kinase activity, and male predominance. Some patients with m-HES probably have chronic eosinophilic leukemia. By contrast, the lymphocytic variant (l-HES) is associated with abnormal clonal proliferation of activated T cells that produce IL-5 and IL-4; chromosomal abnormalities are present in some cases. Evolution of l-HES to T-cell lymphomas occurs in a minority of cases. m-HES and l-HES exhibit differences in prognosis and responsiveness to therapy. Histopathology Large quantities of eosinophils, eosinophilic metamyelocytes, and myelocytes are observed in bone marrow, peripheral blood, and affected tissues. In the myeloid variant (discussed below), bone marrow biopsies may show atypical, dysplastic mast cells and myelofibrosis. Fibrosis of affected tissues (e.g., heart) may be prominent. Clinical features The course and prognosis of IHES are strikingly heterogeneous. The heterogeneity in part reflects distinct hematological disorders involving either myeloid or lymphoid cells as the cause of IHES. The m-HES is more aggressive, often resistant to CS therapy, associated with an increased incidence of fibrotic complications, and may evolve to myeloid malignancy. By contrast, the l-HES is less aggressive and more often responsive to CS. Common presenting features of IHES include night sweats, anorexia, weight loss, pruritis, fever, and cough. Cardiac involvement (e.g., endocardial fibrosis, restrictive cardiomyopathy, valvular damage, and mural thrombus) is the major cause of morbidity and mortality in m-HES but is uncommon in l-HES. Features associated with m-HES include involvement

of liver or spleen (80%), nervous system (67%), thromboembolic disease (65%), lung (40%), and skin (40–60%). Other sites of eosinophilic infiltration include GI tract, kidneys, joints, and muscles. Among patients with l-HES, skin lesions (e.g., urticaria, angioedema, erythroderma) are invariably present. Other cardinal features of l-HES include eosinophilic infiltration of lung and the GI tract. Lung involvement in IHES may reveal marked BAL eosinophilia (470%), interstitial infiltrates and/or pleural effusions on chest radiographs, small pulmonary nodules, and focal GGO on CT. Peripheral blood eosinophilia is usually striking (30–70%) in both m-HES and l-HES. Serum IgE levels are markedly elevated in most patients with l-HES; this is variable with mHES. Serum levels of the chemokine TARC are often elevated in l-HES. Serum vitamin B12 levels and tryptase levels are often elevated with m-HES. Management and current therapy IHES is rare and controlled therapeutic studies are lacking. Treatment should be stratified based upon subtype (i.e., m-HES or l-HES). Therapy with corticosteroids alone is efficacious in a majority of patients with l-HES but not with m-HES. Since the 1980s, anecdotal successes have been cited with hydroxyurea, vinca alkaloids, chemotherapeutic agents, and a-interferon for IHES. Over the past 3 years, clinical successes were noted with the tyrosine kinase inhibito, imatinib-mesylate, which is now considered first-line therapy for m-HES. By contrast, monoclonal antibodies against IL-5 may be efficacious for l-HES, although rebound hypereosinophilia may occur. Leukopheresis may be necessary when blood eosinophil counts are exceptionally high (4100 000 mm3). Bone marrow or stem cell transplantation has been successful in refractory cases. Eosinophilia–Myalgia Syndrome

Eosinophilia–myalgia syndrome (EMS), initially described in New Mexico in 1989, is characterized by the abrupt onset of severe myalgias, eosinophilia, paresthesias, rash, constitutional symptoms, and multisystemic involvement. Etiology A relationship between ingestion of L-tryptophan and EMS was recognized, and the epidemic ceased after the Centers for Disease Control (CDC) recalled L-tryptophan-containing products. Clinical features Criteria for the diagnosis established by the CDC in Atlanta included blood eosinophil count 4 1000 per mm3, incapacitating myalgias, and no other identifiable etiology (e.g., parasitic reaction or drug hypersensitivity). Within a few weeks

230 SYSTEMIC DISEASE / Eosinophilic Lung Diseases

of being described, EMS assumed epidemic proportions in the US. One third of patients required hospitalization, and more than 30 patients died. Toxic Oil Syndrome

Toxic oil syndrome was described in the spring of 1991 as an epidemic illness in Spain characterized by blood eosinophilia, pulmonary infiltrates, and diverse musculoskeletal manifestations. Etiology Ingestion of contaminated rapeseed oil was implicated in June 1991, and the epidemic resolved quickly following withdrawal of the agent from the marketplace. See also: Asthma: Overview; Allergic Bronchopulmonary Aspergillosis. Colony Stimulating Factors. Interleukins: IL-1 and IL-18; IL-4; IL-5; IL-10; IL-13. Leukocytes: Eosinophils. Pneumonia: Mycobacterial. Pulmonary Fibrosis. Viruses of the Lung.

Further Reading Allen J, Magro CM, and King MA (2002) The eosinophilic pneumonias. Seminars in Respiratory and Critical Care Medicine 23: 127–134. Allen JN and Davis WB (1994) Eosinophilic lung diseases. American Journal of Respiratory and Critical Care Medicine 150: 1423–1438. Alonso-Ruiz A, Calabozo M, Perez-Ruiz F, and Mancebo L (1993) Toxic oil syndrome. A long-term follow-up of a cohort of 332 patients. Medicine (Baltimore) 72: 285–295. Arakawa H, Kurihara Y, Niimi H, et al. (2001) Bronchiolitis obliterans with organizing pneumonia versus chronic eosinophilic pneumonia: high-resolution CT findings in 81 patients. American Journal of Roentgenology 176: 1053–1058. Boggild AK, Keystone JS, and Kain KC (2004) Tropical pulmonary eosinophilia: a case series in a setting of nonendemicity. Clinical Infectious Diseases 39: 1123–1128. Cottin V, Frognier R, Monnot H, et al. (2004) Chronic eosinophilic pneumonia after radiation therapy for breast cancer. European Respiratory Journal 23: 9–13. Glazer CS, Cohen LB, and Schwarz MI (2001) Acute eosinophilic pneumonia in AIDS. Chest 120: 1732–1735. Guillevin L, Pagnoux C, and Mouthon L (2004) Churg-Strauss syndrome. Seminars in Respiratory and Critical Care Medicine 25: 535–545. Hertzman PA, Blevins WL, Mayer J, et al. (1990) Association of the eosinophilia-myalgia syndrome with the ingestion of tryptophan. New England Journal of Medicine 322: 869–873. Johkoh T, Muller NL, Akira M, et al. (2000) Eosinophilic lung diseases: diagnostic accuracy of thin-section CT in 111 patients. Radiology 216: 773–780.

King MA, Pope-Harman AL, Allen JN, Christoforidis GA, and Christoforidis AJ (1997) Acute eosinophilic pneumonia: radiologic and clinical features. Radiology 203: 715–719. Klion AD, Law MA, Noel P, et al. (2004) Safety and efficacy of the monoclonal anti-interleukin-5 antibody SCH55700 in the treatment of patients with hypereosinophilic syndrome. Blood 103: 2939–2941. Lynch JP III and Fantone JC III (1991) Other pulmonary granulomatous vasculitic syndromes. In: Lynch JP III and DeRemee R (eds.) Immunologically Mediated Pulmonary Diseases, pp. 302–321. Philadelphia, PA: JB Lippincott Company. Lynch JP III and Flint AF (1984) Sorting out the eosinophilic pulmonary syndromes. Journal of Respiratory Diseases 5: 61–78. Lynch JP III and Keane M (2003) Current approaches to the treatment of parenchymal lung diseases. In: Spina D, Page CP, Metzger WJ, and O’Connor BJ (eds.) Drugs for the Treatment of Respiratory Diseases, pp. 247–335. Cambridge: Cambridge University Press. Marchand E, Reynaud-Gaubert M, Lauque D, et al. (1998) Idiopathic chronic eosinophilic pneumonia. A clinical and followup study of 62 cases. The Groupe d’Etudes et de Recherche sur les Maladies ‘‘Orphelines’’ Pulmonaires (GERM‘‘O’’P). Medicine (Baltimore) 77: 299–312. Miyazaki E, Nureki S, Fukami T, et al. (2002) Elevated levels of thymus- and activation-regulated chemokine in bronchoalveolar lavage fluid from patients with eosinophilic pneumonia. American Journal of Respiratory and Critical Care Medicine 165: 1125–1131. Philit F, Etienne-Mastroianni B, Parrot A, et al. (2002) Idiopathic acute eosinophilic pneumonia: a study of 22 patients. American Journal of Respiratory and Critical Care Medicine 166(9): 1235–1239. Pope-Harman AL, Davis WB, Allen ED, Christoforidis AJ, and Allen JN (1996) Acute eosinophilic pneumonia. A summary of 15 cases and review of the literature. Medicine (Baltimore) 75: 334–342. Rothenberg ME (1998) Eosinophilia. New England Journal of Medicine 338: 1592–1600. Roufosse F, Cogan E, and Goldman M (2004) Recent advances in pathogenesis and management of hypereosinophilic syndromes. Allergy 59: 673–689. Shannon JJ and Lynch JP III (1995) Eosinophilic pulmonary syndromes. Clinical Pulmonary Medicine 2: 19–38. Shorr AF, Scoville SL, Cersovsky SB, et al. (2004) Acute eosinophilic pneumonia among US military personnel deployed in or near Iraq. JAMA 292(24): 2997–3005. Stevens DA, Moss RB, Kurup VP, et al. (2003) Allergic bronchopulmonary aspergillosis in cystic fibrosis – state of the art: Cystic Fibrosis Foundation Consensus Conference. Clinical Infectious Diseases 37(supplement 3): S225–S264. Stevens DA, Schwartz HJ, Lee JY, et al. (2000) A randomized trial of itraconazole in allergic bronchopulmonary aspergillosis. New England Journal of Medicine 342: 756–762. Tazelaar HD, Linz LJ, Colby TV, Myers JL, and Limper AH (1997) Acute eosinophilic pneumonia: histopathologic findings in nine patients. American Journal of Respiratory and Critical Care Medicine 155: 296–302.

T Tachykinins

see Kinins and Neuropeptides: Tachykinins.

Theophylline

see Bronchodilators: Theophylline.

THROMBOLYTIC THERAPY S Idell and S Shetty, University of Texas Health Center, Tyler, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Thrombolytic therapy, which involves the administration of agents designed to increase fibrinolytic capacity, has been utilized in respiratory medicine for more than 50 years. A variety of agents have been used to effect thrombolysis, including urokinase plasminogen activator (uPA) and streptokinase. Tissue plasminogen activator (tPA) has also been used to effect thrombolysis. Both uPA and tPA are endogenous fibrinolysins that are detectable in plasma and extravascular fluids that accumulate in the setting of inflammation. uPA is the major plasminogen activator present in bronchoalveolar lavage of normal subjects and accounts for the fibrinolytic activity that is detectable in these fluids. These agents are all characterized by a short half-life in plasma, and their effects are mitigated by inhibition attributable to the plasminogen activators, particularly plasminogen activator inhibitor-1, and by downstream antiplasmins. Thrombolytic therapy has been used to clear pathologic fibrin deposition in both extravascular and intravascular compartments. These agents are used to relieve intrapleural loculations associated with complicated parapneumonic pleural effusions and organizing hemothoraces. Thrombolytic therapy has also been used to treat pulmonary embolism associated with hemodynamic compromise. In both instances, the role of thrombolysis in pulmonary medicine continues to evolve based on recent and ongoing investigations.

Introduction Thrombolytic therapy – therapy that increases fibrin clearance – has been used in the practice of respiratory medicine for a number of years. More than half a century ago, Tillett and Sherry inferred that intrapleural organization in the setting of parapneumonic effusions or hemothoraces was initiated by intrapleural

fibrin deposition and that the process could be mitigated by the local administration of agents that would increase fibrinolysis. These investigators found that the intrapleural administration of either streptokinase or streptodornase effectively cleared pleural loculations associated with either parapneumonic effusions or hemothoraces. These observations supported the subsequent use of fibrinolytic agents to prevent intrapleural loculation and lung entrapment. A number of uncontrolled studies subsequently supported the administration of fibrinolytic agents to address intrapleural loculations. For example, in a study done in the 1970s, Bergh and colleagues used streptokinase to treat loculations in patients with hemothoraces or parapneumonic effusions. They reported that streptokinase increased lung expansion and drainage of pleural fluid. A number of other uncontrolled studies employed streptokinase or urokinase as interventional agents and affirmed these findings using drainage or the requirement for surgical intervention as endpoints. The relatively few randomized, controlled clinical trials that have been conducted likewise support the use of intrapleural fibrinolysins to relieve intrapleural loculations. In one such trial, intrapleural streptokinase administered in three daily doses facilitated pleural drainage and radiographic improvement of intrapleural loculations due to empyema. In this trial, 24 patients were randomized and received either intrapleural saline or streptokinase daily over 3 days. Only patients in the control group (n ¼ 3) required surgical intervention, and systemic fibrinolysis or bleeding did not occur in the streptokinase-treated patients. In another controlled trial, urokinase instillation was found to be superior to saline administration in patients with multiloculated effusions.

232 THROMBOLYTIC THERAPY

The use of thrombolytic therapy for pleural loculation has been challenged. In one study of patients with complicated pleural effusions or empyema, drainage was increased in patients treated with streptokinase but there was no difference in requirement for surgery, time to defervescence, and duration of hospitalization. This study raises questions about the broad efficacy of intrapleural fibrinolysis, a position that was initially offered by Cameron. A review of randomized trials of patients with complicated parapneumonic effusions or empyema who had not had prior surgical intervention cast further doubt on the efficacy of intrapleural fibrinolysins. A large multicenter clinical trial meeting compared the efficacy of intrapleural streptokinase or placebo in several hundred patients with complicated parapneumonic effusion or empyema and similarly found no benefit in terms of the same endpoints. Thrombolytic therapy has also been used for a number of years in the treatment of pulmonary emboli and in extensive ileofemoral thrombosis. Although thombolytic therapy has been shown to decrease the incidence of the postphlebitic syndrome in patients with proximal deep venous thrombosis, its application in these circumstances has been limited because of the risk of serious bleeding or intracranial hemorrhage. Based on a similar risk–benefit analysis, the use of thrombolytic therapy in pulmonary embolism remains the subject of ongoing debate. It is generally accepted that patients who present with acute pulmonary embolism associated with shock or who develop hemodynamic compromise based on pulmonary vascular obstruction should be treated with thrombolytic therapy or, alternatively, with pulmonary embolectomy. In a metaanalysis of all available randomized trials comparing thrombolytic therapy with acute pulmonary embolism, there was a significant reduction in recurrent embolism or death in trials that included patients with hemodynamic compromise but not in trials that excluded such patients. In this analysis, most of the trials revealed an increase in major bleeding, but the increment was not statistically significant. The risk of hemorrhage with thrombolytic therapy of patients with pulmonary embolus was increased in a prior meta-analysis. In patients with submassive pulmonary embolism and normal blood pressure, the use of thrombolytic therapy remains controversial. In a randomized, double-blind trial of thrombolytic therapy plus heparin versus heparin for patients with pulmonary embolism and pulmonary hypertension or right ventricular dysfunction without systemic hypotension or shock, the addition of thrombolytic therapy reduced the need for an escalation of therapy, including

repeat thrombolysis or infusion of pressors. Some authorities interpret these observations as supporting the application of thrombolytic therapy for these patients, whereas others continue to believe that thrombolytic therapy, with its associated risk of hemorrhage, should be reserved for patients with massive pulmonary embolism and hemodynamic compromise. A number of thrombolytic agents, including uPA, tPA, and streptokinase, have been used for intrapleural applications and to treat pulmonary embolism. When used in the recommended doses and at recommended dosage schedules, these agents appear to be of comparable efficacy. It appears that administration of any plasminogen activator can relieve pulmonary vascular obstruction in the setting of severe pulmonary embolus and that relatively short courses, delivered over a few hours, can reduce pulmonary vascular resistance. Interestingly, controlled trials have not shown any differences in the mortality of patients with pulmonary embolus who were treated with either heparin or plasminogen activators. However, the results may reflect the relatively small numbers of patients evaluated and lack of stratification for severity. Catastrophic events, including severe bleeding or intracranial hemorrhage, can occur with the use of any of the thrombolytic agents when used for the treatment of pulmonary embolus. Intrapleural administration is generally well tolerated. Intrapleural hemorrhage generally does not occur, and systemic fibrinolysis is not generally induced.

Chemical Structure of Thrombolytic Agents Streptokinase is a single-chain glycoprotein with a molecular weight of approximately 50 kDa. The molecule does not exhibit intrachain disulfide bonds. This plasminogen activator is exogenous, not normally found in the circulation. It was originally isolated from b-streptococci and was first characterized in the 1940s. Cells secrete uPA as a proenzyme known as singlechain or pro-urokinase, which has a molecular weight of approximately 54 kDa and can be activated by plasmin. The active forms of uPA include a high-molecular-weight, 54 kDa two-chain form and a low-molecular-weight form of approximately 33 kDa. Disulfide bonds connect the two chains of the high-molecular-weight form. uPA is an endogenous plasminogen activator that can be detected in circulating blood. A recombinant form has been reintroduced in clinical practice for the treatment of pulmonary embolus. uPA is mainly involved in

THROMBOLYTIC THERAPY 233 200 S C L P W N S E M C H I W V T L P L L K K I A A I S N Q A F G A W H R D K P K T R G V H H G D L Y S R R A P T R T I A Y S W N D Q P A E A Q K P Y S G R G N R R P L A S F Y E D A P A C L G R C I S G F Y S D R N A L G L G N E R F P V S C Q K Q G F H G P I P F N G A L H K I Y R C L S F W G L G L G H I H C C Q S N P L A N Q S D S H A K S D R D P N S T T Y P Q R C Y N C G L R T S V W L S C I I W C E Q A N C S S T PA L G V V C Y S E T R G R C S E V C C V A F R S E L F K S T G A G K Y S S P E Y C D Y S T D P R S E Q A A A V I G K S T S W T G R Y R D V L L E E E G P T Q L P D W T E C E L S G Q Q V D K G L F E I K C C L E G D N L C H G K M R V E V A A T E K Y P C Q C L I L A I F G E V V V P D L H L W G N S R F S R C K G P D Q P P I D F Y E G F V H I T D N S E Y F S D D L Q S S G D S F R E W G Q G G L R C S T Y Y G C Q S L L N I K Q C C K C T S Q V Q A V R A S R H L G P E S WQ L Y Q D V A V P D I L HV V H N S T L R E A R K N L C G H K T N Q D Y D L R A C T E V M G P G V N C S R T G Q N D K P M D T V W A R T Y V G R C R C P N G I S V S Y Q S

Kringle regions

G A

250

300

125

325

275

225

150

175

400

100

375

Growth factor domain

350

75

25

425

500

Finger domain

475

50

450

525

Serine protease part

COOH

NH2 Figure 1 Chemical structure of tPA. The primary structure of tPA is illustrated and molecular domains are indicated with solid lines. The plasmin cleavage site at Arg275-Ile276 is indicated by the arrow. Dark bars indicate disulfide bonds. Reproduced from Collen D and lojnen HR (1994) Fibrinolysis and the control of hemostasis. In: Stamatoyannopoulos GS, Nienuis AW, Majerus PW, and Vermus HV (eds.) The Molecular Basis of Blood Diseases, 2nd edn. Philadelphia: Saunders, with permission from Elsevier.

extravascular remodeling of transitional extravascular fibrin, as occurs in the setting of tissue injury or neoplasia, whereas intravascular fibrinolytic capacity is largely maintained by tPA. tPA is also a serine protease plasminogen activator. It is a glycosylated enzyme of molecular weight 68 kDa (Figure 1). Like uPA, tPA can be detected in plasma and in extravascular exudates following lung or pleural injuries. The recombinant form of the molecule that is used in clinical practice differs from the endogenous molecule and exhibits differences in the distribution of its component kringle domains.

Mode of Action Unlike uPA or tPA, streptokinase is not an enzyme, nor does it directly convert plasminogen to plasmin. Rather, streptokinase initially forms a complex with plasminogen, which is then capable of cleaving it at

uPA or tPA

SK Plasminogen

Arg560 − Val561 bond Plasmin

Figure 2 Mode of action of the plasminogen activators. uPA or tPA directly cleaves the Arg560–Val561 bond of the substrate plasminogen to generate plasmin. Streptokinase (SK) forms a complex with plasminogen that generates plasmin by cleavage of the same bond.

the Arg560–Val561 bond to generate plasmin from both complexed and free plasminogen (Figure 2). Interestingly, both uPA and tPA cleave plasminogen at this same bond to generate plasminogen. The activity of the streptokinase–plasminogen complexes appears to be influenced by a number of factors, including proteolytic cleavage of the streptokinase and differential activity of the fragments combined with

234 THROMBOLYTIC THERAPY SK, uPA, tPA PA inhibitors Plasminogen

Plasmin Antiplasmins Fibrin

FDPs Figure 3 Simplified schema of inhibitory control of the fibrinolytic system. With the administration of thrombolytic therapy, SK, uPA, and tPA all convert plasminogen to plasmin by proteolytic processing as illustrated in Figure 1. uPA and tPA are subject to inhibition by PA inhibitors, particularly PAI-1. Plasmin-mediated degradation of fibrin with generation of fibrin degradation products (FDPs) is subject to further inhibition by antiplasmins.

different forms of plasminogen and the interaction of the complexes with fibrin degradation products and inhibitors. Streptokinase is immunogenic and generates antibody formation and is likewise associated with febrile reactions. The plasminogen activators all have short half-lives and are cleared from the plasma within minutes. Whether given by the intravenous or the intrapleural route, uPA or tPA are subject to inhibition by the plasminogen activators, mainly plasminogen activator inhibitor-1. The fibrinolytic effects of these plasminogen activators, as well as that of streptokinase, are likewise subject to inhibition by antiplasmins (Figure 3). These features necessitate intravenous administration over intervals that vary according to the agent used. Differing dosage thrombolytic schedules have been used in the treatment of pulmonary embolism, as reviewed elsewhere. Many clinicians have become accustomed to using recombinant tPA in the setting of severe pulmonary embolism associated with hemodynamic compromise, based on familiarity with this agent. Recombinant tPA is generally administered at a dose of 100 mg, including a 10 mg initial bolus followed by administration of the balance of the dose by continuous infusion over 2 h. Most of the research concerning the use of local thrombolytic therapy for intrapleural loculations has involved the use of repeated intrapleural doses of either streptokinase or uPA. Typical daily doses for intrapleural applications have been 250 000 IU of streptokinase or 100 000 U of uPA.

Contraindications All systemically administered thrombolytic agents carry the risk of severe bleeding, of which intracranial

hemorrhage is the most feared complication. The risk of intracranial hemorrhage is approximately 1.9% in patients treated for pulmonary embolism, and the risk of major bleeding has been reported to be as high as 22%. These observations strongly speak to the need to carefully select patients most likely to benefit versus those most at risk for complications from systemic thrombolytic therapy. A number of contraindications have been identified and are listed as exclusion criteria by the American Heart Association. These exclusions include active internal bleeding, excluding menses within 21 days, or history of cerebrovascular, intracranial, or intraspinal event within 3 months. These intracerebral events specifically include stroke, arteriovenous malformations, neoplasm, aneurysm, cranial trauma, or surgery. Major trauma or surgery within 14 days, aortic dissection, and uncontrolled hypertension also represent exclusion criteria. Additional exclusion criteria are a known bleeding disorder, prolonged cardiopulmonary resuscitation with thoracic trauma, lumbar puncture within 7 days, and recent arterial puncture at a noncompressible site. For patients with a high risk of complications but who require aggressive intervention for pulmonary embolism and hemodynamic compromise, catheter-directed or surgical embolectomy may be considered. See also: Coagulation Cascade: Fibrinogen and Fibrin; iuPA, tPA, uPAR. Defensins. Extracellular Matrix: Degradation by Proteases. Fibrinolysis: Overview; Plasminogen Activator and Plasmin. Pleural Effusions: Parapneumonic Effusion and Empyema; Hemothorax. Proteinase Inhibitors: Alpha-2 Antiplasmin. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts.

Further Reading American Heart Association (2000) Handbook of Emergency Cardiovascular Care for Healthcare Providers. Dallas, TX: American Heart Association. Bell WR (2002) Present-day thrombolytic therapy: therapeutic agents – Pharmacokinetics and pharmacodynamics. Reviews in Cardiovascular Medicine 3(supplement 2): S34–S44. Bergh NP, Ekroth R, Larsson S, and Nagy P (1977) Intrapleural streptokinase in the treatment of haemothorax and empyema. Scandinavian Journal of Thoracic and Cardiovascular Surgery 11: 265–268. Bouros D, Schiza S, Tzanakis N, et al. (1999) Intrapleural urokinase versus normal saline in the treatment of complicated parapneumonic effusions and empyema. A randomized, doubleblind study. American Journal of Respiratory and Critical Care Medicine 159: 37–42. Cameron R (2000) Intra-pleural fibrinolytic therapy vs. conservative management in the treatment of parapneumonic effusions and empyema. Cochrane Database of Systematic Reviews CD002312.

TOLL-LIKE RECEPTORS 235 Chin NK and Lim TK (1997) Controlled trial of intrapleural streptokinase in the treatment of pleural empyema and complicated parapneumonic effusions. Chest 111: 275– 279. Collen D and Iojnen HR (1994) Fibrinolysis and the control of hemostasis. In: Stamatoyannopoulos GS, Nienuis AW, Majerus PW, and Vermus HV (eds.) The Molecular Basis of Blood Diseases, 2nd edn. Philadelphia: Saunders. Goldhaber SZ (2002) Modern treatment of pulmonary embolism. European Respiratory Journal Supplement 35: 22s–27s. Goldhaber SZ (2004) Pulmonary embolism. Lancet 363: 1295– 1305. Konstantinides S, Geibel A, Heusel G, Heinrich F, and Kasper W (2002) Heparin plus alteplase compared with heparin alone in patients with submassive pulmonary embolism. New England Journal of Medicine 347: 1143–1150. Marder VJ and Stewart D (2002) Towards safer thrombolytic therapy. Seminars in Hematology 39: 206–216.

Maskell NA, Davies CW, Nunn AJ, et al. (2005) First Multicenter Intrapleural Sepsis Trial (MIST1) Group, U.K. controlled trial of intrapleural streptokinase for pleural infection. New England Journal of Medicine 352(9): 865–874. Thabut G, Thabut D, Myers RP, et al. (2002) Thrombolytic therapy of pulmonary embolism: a meta-analysis. Journal of the American College of Cardiology 40: 1660–1667. Tillett WS and Sherry S (1949) The effect in patients of streptococcal fibrinolysin (streptokinase) and streptococcal deoxyribonuclease on fibrinous, purulent and sanguinous pleural exudations. Journal of Clinical Investigation 28: 173–190. Tillett WS, Sherry S, and Read CT (1951) The use of streptokinase–streptodornase in the treatment of postpneumonic empyema. Journal of Thoracic Surgery 21: 275–297. Wan S, Quinlan DJ, Agnelli G, and Eikelboom JW (2004) Thrombolysis compared with heparin for the initial treatment of pulmonary embolism. A meta-analysis of the randomized clinical trials. Circulation 110: 744–749.

TOLL-LIKE RECEPTORS I Sabroe, S K Dower, and M K B Whyte, University of Sheffield, Sheffield, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Toll-like receptors (TLRs) are members of the IL-1 receptor superfamily, and comprise a family of molecules that detect and facilitate responses to a broad range of bacterial and viral pathogens. Additional roles for these receptors in disease may include the detection of tissue damage at inflammatory sites. Genetic defects in TLR signaling pathways confirm their importance in controlling infections, and are associated with reduced risks of inflammatory diseases. TLR agonists exert powerful adjuvant effects, and are providing new treatments to enhance vaccine efficiency and desensitize allergic inflammation. TLR antagonists may also be useful anti-inflammatory treatments in established disease.

Introduction First described because of its role in fly development, conditional Toll mutants demonstrated a role for the Toll protein in fly immunity. The family of Toll-like receptors have become the subject of intense interest following the realization that they provide vital mechanisms allowing cells (immunological and nonimmunological) to detect and respond to both pathogens and endogenous signals of tissue damage. Two groups showed that mice resistant to the effects of bacterial endotoxin had defects in a mammalian homolog of Toll, named Toll-like receptor (TLR) 4, providing the identity of the long-sought-after signaling protein enabling responses to endotoxin. TLRs

comprise a large family (10 proteins in man, 11 in mouse). For most of these, evidence of an important role in host defense is accruing. Their enormous potential to alter patterns of sensitization and subsequent T-cell-driven responses has stimulated intense interest, and new therapeutic agents exploiting TLRs are already in clinical trials.

Structure TLRs are members of the IL-1R superfamily. Homology with the IL-1R resides in the intracellular regions involved in signaling. In particular, the receptors all possess the intracellular Toll/interleukin receptor (TIR) domain, which is crucial for signaling. Three regions within the TIR domain have been defined in receptor mutagenesis studies (principally of the type 1 IL-1R, but also of TLR2 and TLR4) that are of particular importance. Of these ‘boxes’, box 1 and box 2 are vital to signaling, and box 3 to cell surface expression. The TIR domain is also a feature of a family of adapter molecules, which associate with activated TLRs through TIR domain-mediated interactions and create the early signaling complexes. Dominant negative molecules that prevent TLR signaling can be readily created by point mutations (typically of a highly conserved proline residue within box 2) in either the TLRs or signaling adapters, emphasizing the crucial nature of this region of the molecule. The extracellular portions of the TLRs are characterized by leucine-rich repeat motifs, which are thought to be involved in ligand

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recognition (in contrast to the IL-1R, which has Ig-like domains on the extracellular face). The evidence to date is that TLRs signal as homodimers, though some TLR heterodimer pairs (most notably TLR1/2 and TLR2/6) can be formed to increase the range of potential agonists that can be recognized. Interaction of TLRs and their agonists is complex. For example, lipopolysaccharide (LPS, a major component of endotoxin, from the cell walls of Gram-negative bacteria) needs to be presented to TLR4 after interactions with a series of proteins including LPS-binding protein, CD14, and MD-2 (Figure 1). These interactions, probably resulting in monomeric LPS complexed to MD-2 as the final bioactive moiety, may be required to handle the hydrophobicity of large proportions of the LPS molecule. Other molecules, such as the integrin CD11b/ CD18, can also interact with LPS and TLR4 to promote signaling, and there is evidence that TLRs, once activated, form complexes with many proteins that may influence signaling in membrane microdomains such as lipid rafts. For other TLRs, agonist interactions occur in specific microenvironments, as

illustrated by signaling of bacterial DNA (CpG motifs), which occurs in acidified endosomes. Much work is still required for all the TLRs before a definitive understanding of the mechanisms of interaction of agonists and receptors is reached. Activation of distinct TLR homo- or heterodimers is associated with specific patterns of intracellular signaling and subsequent activation and cytokine generation. Specific patterns of signaling are mediated by interactions with adapters that show preferential TLR associations. The current list of adapters includes MyD88, Mal, TRIF, and TRAM; their specific roles are still being dissected, but coupling to type 1 interferon generation appears to be a particular feature of TRIF and TRAM, linking TLR3 and TLR4 activation to interferon-dependent signaling as well as direct activation of gene transcription. TLRs typically couple powerfully to classical proinflammatory signaling pathways such as mitogen-activated protein-kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. There is also probably selectivity between TLRs in induction of other signaling pathways (e.g., Ras, PI-3 kinase), but the details and consequences of these systems remain to be fully elucidated.

LBP LPS

Regulation of Production and Activity

MD-2 CD14

TLR4 dimer CpG DNA

Early signaling complex Uptake of CpG Activation of MAPKs and NF-
B TLR9 Early signaling complex

Gene transcription

Figure 1 TLR4 signaling requires transfer of LPS via CD14 to MD-2 and a TLR4 dimer. Signaling is initiated at the TIR domains of the receptors, resulting in formation of an early signaling complex involving proteins including the IRAKs and TRAF6. Downstream activation switches on gene transcription of pro-inflammatory cytokines by pathways including MAPKs and NF-kB. Signaling by CpG motifs uses TLR9, but in this case the CpGcontaining DNA must be taken up into the cell, and signaling proceeds from an acidified endosome.

Patterns of expression remain to be fully determined. Professional phagocytotic cells of the monocyte lineage (including dendritic cells and macrophages) typically constitutively express the widest range of TLRs, and many other leukocyte types including neutrophils, T cells, and T regulatory cells show selective TLR expression patterns. Many tissues, particularly those that interact with the external environment, express TLRs widely through epithelia and endothelia, facilitating rapid responses to infectious agents. Upregulation of these receptors at sites of sterile inflammation, in leukocytes and in tissue cells such as cardiac and vascular smooth muscle, suggests roles in noninfective inflammation. The regulation of expression is complex and varies from cell to cell: dendritic cells alter TLR expression as they mature (and TLR activation can stimulate such maturation); memory T cells can express TLRs not seen on naı¨ve cells; and activation of inflammatory leukocytes can result in induction or downregulation of surface expression. Additionally, there is intense regulation of signaling inside the cell. Repeated stimulation of cells with TLR agonists such as LPS typically induces a state of tolerance, limiting further responses to the same agonist. Tolerance can be mediated by regulation of expression of the TLRs themselves, signaling proteins, and a large panel of

TOLL-LIKE RECEPTORS 237

negative regulators of signaling that show selective expression between cell types. These complex regulatory processes certainly serve to control TLR activation in vivo, and provide potential therapeutic targets.

Biological Function TLR4 provides the mechanism by which LPS initiates signaling. There is also evidence that TLR4 may be involved in the recognition of a wide range of structurally dissimilar molecules, including pathogen-derived molecules (e.g., RSV coat protein) and molecules released or generated at sites of tissue damage (e.g., hyaluronan oligosaccharides, fibrinogen, transcription factors). Because of the exquisite sensitivity of TLR4 to LPS, exclusion of a contribution of contaminating LPS in responses to non-LPS mediators is challenging, and the exact and full range of TLR4 agonists remains to be clearly defined. TLR2 recognizes principally molecules released from Gram-positive organisms, including lipoproteins, and uses TLR1 or TLR6 as a heterodimeric partner to broaden the range of agonists it responds to. TLR5 responds to bacterial flagellin. TLR9 mediates responses to paired elements of cytosine (unmethylated) and guanine nucleotides (CpG motifs), which are features of bacterial DNA. TLRs 3, 7, and 8 are involved in an antiviral program, enabling responses to double-stranded (TLR3) and single-stranded (7/8) RNA. The function of TLR10 remains unclear. New TLR agonists are reported frequently. These are mostly derived from pathogenic microorganisms. The extraordinary ability of these receptors to mediate responses to widely different molecules has resulted in their being named ‘pattern recognition receptors’ (PRRs), responding to pathogen-associated molecular patterns (PAMPs). How dissimilar molecules initiate TLR-dependent signaling remains unknown, but is one of the most interesting and exciting properties of these receptors.

Toll-Like Receptors in Respiratory Diseases The major role of TLRs is clearly the detection and response to pathogens (Figure 2). Genetic studies of humans with polymorphic TLR alleles have associated TLR variants with altered risks of infections, and some mouse models of infectious disease show that TLRs can play vital roles in host defense. Their exact contribution remains to be determined, since whole microorganisms engage multiple PRRs and

signaling pathways, and not all infection models show nonredundant roles for TLRs. Overall, these receptors almost certainly form a central component of the recognition systems detecting pathogens, and rare human deficiencies in IRAK-4, a key early signaling protein in TLR and IL-1 signaling, are associated with devastating recurrent bacterial infections. Balancing a positive role for a strong TLR response to pathogens is increasing genetic information, suggesting that possession of TLR polymorphisms associated with reduced signaling may protect the individual to some degree against inflammatory diseases. TLR2 polymorphisms correlate with asthma risk in European farming populations, though TLR4 polymorphisms have failed to show significant associations with asthma risk or chronic obstructive pulmonary disease (COPD) severity. Leading on from this, the TLRs provide mechanisms that can underpin the observations encapsulated in the hygiene hypothesis. Increasing evidence that exposure to a wide range of microbial agonists, including LPS, can affect lifetime risks of developing Th2 disease suggests that TLR signaling is central to the development of appropriate T helper/T regulatory cell responses to common environmental allergens. In keeping with this, TLR biology is generating particular excitement in the field of immune adjuvant development. Stimulation of antigen-presenting cells (APCs) with TLR agonists typically results in Th1type cytokine generation, and presentation of antigens together with TLR agonists can generate profoundly Th1-biased T cell responses. This is particularly evident with agonists of TLR9, and conjugation of allergens to CpG-containing motifs is likely to offer a powerful new way to induce clinical desensitization of Th2-type T cell allergic disease. Agonists of other TLRs, such as TLR4 and perhaps TLR2, may also serve such roles, but here the situation is a little more complex, since animal models show that the dose and timing of administration of TLR agonists can have a major impact on the Th1/ Th2 balance of the resulting immunological response. Molecules based on lipid A (the biologically active portion of LPS) can be generated that show a range of agonistic and antagonistic actions at TLR4, and are thus likely to be associated with varying efficacies and advantages as immunoadjuvants. TLRbased immunoadjuvants are also likely to be useful in vaccine formulations, and potentially even in the induction of non-specific protective immunity at the mucosal surface. Complicating this, microbial-derived proinflammatory mediators such as LPS are as much a part of inhaled dusts as are allergens, and in these settings TLR activation is likely to aggravate inflammation.

238 TOLL-LIKE RECEPTORS Mechanism TLR4

TLR2/1 TLR2/6

Activation of neutrophilic & monocytic inflammation

LPS

LP LTAs Protective immunity

TLRs Viral 3, 7, 8 RNA

Septic shock

Activation AM/ epithelia

Asthma TLR activation

Th1/2 biasing

Cytokine production COPD ARDS

DC/T cell RSV TLR9 (CpG) TLR4 (LPS)

cep

tors

Bronchiolitis CpG TLR9

Ago

nist

TLR2 (LTA) TLR4

Th1

s/re

Th2

Clearance of infections

TLR4

TLR5

Consequence

Fgn

Figure 2 TLRs are central to the innate immune response, through their ability to recognize and mediate responses to a broad range of PAMPs. They are widely expressed in the lung, and are found on alveolar macrophages (AMs), endothelia, epithelia, neutrophils, monocytes, dendritic cells (DCs), and memory T cells. Expression on DC and T cells provides substantial opportunities to influence Th1/ 2 phenotypes, and in vivo, some TLR agonists seem to favor Th1 responses while others favor Th2 (LPS can enhance either memory T cell response, depending on the dose of LPS presented with allergen). Although this field of research is still very young, TLR signaling is thus implicated in a variety of processes influencing many respiratory diseases.

Given the additional ability of TLRs to sense tissue damage, it is also likely that therapeutic antagonism of these receptors may have specific clinical utility in inflammatory lung diseases. In support of this hypothesis, a TLR4 polymorphism associated with reduced function is protective against development of atherosclerosis in man, and may reduce risks of transplant rejection. In the treatment of diseases such as asthma, it is possible that TLR agonists may be useful in modifying allergen responses or risks of developing atopy, whilst TLR antagonists may reduce inflammation in established disease. Risks of rendering the individual susceptible to infections by TLR antagonism will need to be considered, but in the face of multiple immunological systems that are involved in detection and elimination of pathogens, TLR antagonism is likely to be clinically feasible.

The Toll-like receptors therefore provide a broad front on which to develop new treatments for human inflammatory lung disease. A number of therapies targeting TLRs are currently in clinical development. See also: Allergy: Overview. Asthma: Overview. Bronchiectasis. Bronchiolitis. CD11/18. CD14. Chronic Obstructive Pulmonary Disease: Overview. Chymase and Tryptase. Defense Systems. Endotoxins. Genetics: Overview. Leukocytes: Mast Cells and Basophils; Monocytes; T cells; Pulmonary Macrophages. Panbronchiolitis. Pneumonia: Overview and Epidemiology. Surfactant: Surfactant Protein D (SP-D).

Further Reading Akira S and Takeda K (2004) Toll-like receptor signalling. Nature Reviews: Immunology 4: 499–511.

TRACHEOSTOMY 239 Basu S and Fenton MJ (2004) Toll-like receptors: function and roles in lung disease. American Journal of Physiology: Lung Cellular and Molecular Physiology 286: L887–L892. Blander JM and Medzhitov R (2004) Regulation of phagosome maturation by signals from toll-like receptors. Science 304: 1014–1018. Eder W, Klimecki W, Yu L, et al. (2004) Toll-like receptor 2 as a major gene for asthma in children of European farmers. Journal of Allergy and Clinical Immunology 113: 482–488. Gordon S (2002) Pattern recognition receptors: doubling up for the innate immune response. Cell 111: 927–930. Hertzog PJ, O’Neill LA, and Hamilton JA (2003) The interferon in TLR signaling: more than just antiviral. Trends in Immunology 24: 534–539. Kiechl S, Lorenz E, Reindl M, et al. (2002) Toll-like receptor 4 polymorphisms and atherogenesis. New England Journal of Medicine 347: 185–192. Krieg AM (2002) CpG motifs in bacterial DNA and their immune effects. Annual Review of Immunology 20: 709–760.

Medvedev AE, Lentschat A, Kuhns DB, et al. (2003) Distinct mutations in IRAK-4 confer hyporesponsiveness to lipopolysaccharide and interleukin-1 in a patient with recurrent bacterial infections. Journal of Experimental Medicine 198: 521–531. O’Neill LA, Fitzgerald KA, and Bowie AG (2003) The Toll-IL-1 receptor adaptor family grows to five members. Trends in Immunology 24: 286–289. Poltorak A, He X, Smirnova I, et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in TLR4 gene. Science 282: 2085–2088. Sabroe I, Read RC, Whyte MKB, et al. (2003) Toll-like receptors in health and disease: complex questions remain. Journal of Immunology 171: 1630–1635. Tsan MF and Gao B (2004) Endogenous ligands of Toll-like receptors. Journal of Leukocyte Biology 76: 514–519. Ulevitch RJ (2004) Therapeutics targeting the innate immune system. Nature Reviews: Immunology 4: 512–520. Underhill DM (2003) Mini-review Toll-like receptors: networking for success. European Journal of Immunology 33: 1767–1775.

TRACHEOSTOMY Y L Colson and S Paul, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Tracheostomy is one of the most common and oldest surgical procedures performed. It provides direct access to the airway for ventilatory support, repeated bronchoscopic evaluation, management of secretions, and provides an airway in the setting of upper airway obstruction. The indications, technique, and complications associated with standard surgical and percutaneous approaches to tracheostomy are highlighted within this article, with special focus on diagnosis and management of the potentially lethal complication of a tracheoinominate fistula.

Introduction Tracheostomy has been utilized as a means to obtain access to the airway since ancient times. It is one of the oldest surgical procedures on record, with descriptions found in the sacred Hindu text Rig Veda dating back to 1500 BC. Tracheostomy has largely been utilized as an emergency procedure for treating acute upper airway obstruction, particularly during the diphtheria epidemics of the eighteenth and nineteenth centuries. More recently, tracheostomy is increasingly performed as an elective procedure airway in ventilator-dependent patients with acute or chronic respiratory failure, or in patients with sleep apnea or underlying pulmonary diseases that require either continuous or intermittent respiratory support. In general, a tracheostomy provides direct access

to the airway for ventilatory support, repeated bronchoscopic evaluation, management of secretions, and provides an airway in the setting of upper airway obstruction. The first modern description of tracheostomy was in 1909 by Chevalier Jackson and this approach remains the gold standard. More recent modifications to allow percutaneous tracheostomy placement have evolved, and in the correct patient population can offer an appropriate alternative surgical approach. Despite the noninvasive appearance of these percutaneous approaches, these methods are not to be used in emergency situations; rather a cricothyroidectomy should still be employed, due to the ease and rapidity with which airway access can be obtained in skilled hands using this approach. The remainder of this article will focus on the indications, available techniques, and the identification and treatment of complications associated with tracheostomy.

The Scientific Basis Anatomy

The adult trachea is approximately 10–11 cm in length, extending from the level of the sixth cervical vertebra to the fourth thoracic vertebra. Only approximately 5 cm of the trachea is located above the suprasternal notch. The trachea is elliptical in shape with the transverse diameter being greater than the anterioposterior diameter, secondary to 16–20 horseshoe-shaped cartilaginous rings that are located anteriorly. The posterior tracheal wall is known as the

240 TRACHEOSTOMY

membraneous trachea and consists of a fibrinous sheath with elements of smooth muscle, epithelium, and glandular tissue. The cricoid cartilage is the only complete ring in the airway and sits at the beginning of the trachea, with the cricothyroid membrane spanning the distance between the thyroid cartilage and the cricoid. Although the cricoid cartilage is often described as the first ring, it is technically a part of the larynx and is not considered a true tracheal ring. The superior surface of the cervical trachea is covered by the thyroid isthmus. Although usually above the site of tracheostomy placement, occasionally this gland must be divided to achieve adequate access to the trachea. The blood supply to the cervical trachea arises from the inferior thyroid vessels and associated branches. The lower trachea is supplied by branches from the bronchial arteries. The vessels approach the trachea laterally on each side forming a vascular network. Ideally, the tracheotomy is made between the second and third tracheal rings. It is important during the placement of a tracheostomy to note that the trachea is a mobile structure and moves with inspiration, such that the distal trachea may descend several centimeters into the thorax. Similarly, hyperextension of the neck can result in the intrathoracic portion of the trachea rising above the suprasternal notch, resulting in the placement of a tracheostomy that resides too low in the trachea when the head is returned to a neutral position.

Indications Tracheostomy is still indicated for the relief of airway obstruction, as occurs with sleep apnea, glottic stenosis, tumor, trauma, chronic laryngeal edema, or bilateral vocal cord paralysis. However, the most common indication for tracheostomy is to facilitate pulmonary toilet and ventilatory support for chronically ventilated patients. Tracheostomy has several advantages over prolonged translaryngeal intubation. They include: (1) decreased risk of vocal cord injury and subglottic stenosis, (2) improved patient comfort, (3) increased patient mobility, (4) decreased rates of sinusitis, (5) improved oral hygiene, (6) improved access to the airway for clearance of secretions and for bronchoscopy, and (7) the potential for speech with the aid of fenestrated devices. There continues to be much debate about the ideal timing of tracheostomy following intubation. In general, tracheostomy is a reasonable option for critically ill patients in whom extubation is likely to be delayed. In the not so distant past, when there were numerous complications associated with the use of rigid, low-volume, high-pressure endotracheal cuffs,

this had been deemed to be as short as 3 or 4 days. With the widespread use of low-pressure endotracheal cuffs, current complication rates secondary to strictures and ischemia have significantly decreased and translaryngeal intubation can be used for extended periods of up to 3 weeks without issue, particularly if the patient is considered at high risk for the placement of an operative tracheostomy. Although a relatively safe procedure, most patients are fairly ill at the time of tracheostomy accounting for a 4–10% morbidity but a o1% mortality. A variety of complications have been associated with tracheostomy placement including more limited problems such as localized infection, mucosal or incisional bleeding, granuloma formation at the stoma site, and swallowing dysfunction. However, more serious and long-term complications – such as tracheal ring rupture, tracheoinonimate fistula, tracheoesophageal fistula, tracheal stenosis, or tracheomalacia – some of which are life-threatening, can also occur. Therefore, the timing of tracheostomy must weigh the risk of complications from a tracheostomy with numerous factors including the probability of extubation, potential complications of multiple episodes of respiratory failure, and urgent reintubations, as well as the patient’s and family’s wishes. Timing should be individualized and thus no strict timelines should be mandated; however, prolonged intubation for 7–10 days without likely extubation over the ensuing week should prompt consideration of whether a tracheostomy would be of benefit in the care or ventilatory weaning of the patient. Contraindications for a tracheostomy include patients with high ventilatory requirements and poor oxygenation whereby the patient requires high levels of positive end expiratory pressure or oxygenation. This prohibits the patient from safely tolerating brief periods of apnea which are required for the exchange of the endotracheal tube for a tracheostomy tube. Other relative contraindications include bleeding disorders, hemodynamic instability, or anatomic restrictions such as an obese body habitus, elevated innominate artery, or short neck.

Tracheostomy Appliances The choice of the correct type of appliance requires knowledge of the appliance and features of the appliance appropriate for a particular patient. Important design information includes the inner and outer diameter, length, and presence or absence of a balloon cuff, inner cannula, or fenestrations. A tracheostomy should be large enough to provide adequate access for the desired purpose (i.e., ventilation and bronchoscopic pulmonary toilet) without

TRACHEOSTOMY 241

leading to stenosis associated with oversized cannulas. Small diameter cannulas result in increased airway resistance; therefore, smaller cannulas are often reserved for patients who do not require ventilation, and thus most often are placed without a balloon cuff. Appliance length is typically fixed but adjustable devices are available. The ideal length places the cannula 3–4 cm proximal to the carina. A fenestrated tracheostomy permits speech when the tracheostomy is capped and the fenestrations are open. Ventilation can be accomplished with a fenestrated tracheostomy, but the balloon must be inflated and an inner cannula without fenestrations is required to prevent loss of tidal volume through the oral cavity. However, fenestrations are often associated with increased granulation tissue within the airway and difficulties with suctioning; therefore, if the patient is to remain intubated and the fenestrations are not initially required, a nonfenestrated tracheostomy is the preferred initial choice with a plan for changing to a fenestrated tracheostomy when warranted. Alternatively, a one-way Pasey–Muir valve can be placed in order to permit speech with a nonfenestrated tracheostomy; however, failure to deflate the balloon cuff will result in the inability to exhale and the patient can succumb to respiratory failure if this is not identified. Given the number of variables available, careful thought can result in a tracheostomy tailored for specific indications and optimized for each individual patient.

Techniques Tracheostomy

Surgical tracheostomy is usually performed under general anesthesia in the operating room. With the patient in a supine position, the neck is mildly extended and a 2–3 cm transverse skin incision is made in the midline just above the suprasternal notch. The subcutaneous tissues and platysma are divided transversely; the superficial cervical fascia is divided transversely. Separation of the strap muscles at the median raphe reveals the thyroid isthmus and the underlying trachea. After choosing the appropriate appliance and sufficiently identifying the second and third tracheal rings, a small incision between these rings is made. Most surgeons will either divide the third ring or make a ‘Bjork flap’ to assure adequate access to the airway. Hemostasis must be obtained, but the risk of an airway fire mandates that electrocautery and FiO2 be minimized as much as possible during this portion of the operation. When all OR personnel are ready, ventilation is held and the endotracheal tube is slowly removed by the anesthesia team while

the surgical team observes the trachea through the tracheotomy. Once sufficient airway is visible, the tracheal opening can be enlarged gently with a tracheal dilator and the tracheostomy tube placed under direct vision. The cuff is inflated, respirations are resumed, and correct position within the airway is documented by CO2 return, bilateral chest elevation, and ideally bronchoscopy through the tracheostomy applicance. The tracheostomy is securely sutured to the skin and/or secured with a tie around the posterior neck to prevent loss of the airway in the immediate postoperative period. The endotracheal tube should not be removed from the upper trachea until correct positioning of the tracheostomy has been confirmed, as this allows the endotracheal tube to be advanced and the patient again ventilated if there is difficulty with insertion of the tracheostomy tube. Percutaneous tracheostomy techniques have recently increased in popularity due to the ability to perform the procedure at the bedside with reported economic savings secondary to exclusion of operating room costs. Recent studies comparing standard surgical techniques with dilational percutaneous tracheostomy suggest lower rates of peristomal bleeding and infection at a potential cost benefit. These procedures are best performed with direct bronchoscopic observation of serial dilations over a wire placed percutaneously into the trachea, after the endotracheal tube has been withdrawn above the first tracheal ring. The percutaneous tract, placed using the same landmarks as for a surgical tracheostomy, is serial dilated until a tracheostomy tube can be accommodated and this is slid over the guidewire and into position within the trachea. Some studies have reported increased complications with percutaneous techniques – such as inadvertent injury to the thyroid or other structures within the neck, puncture of the indwelling endotracheal tube, injury to the membranous trachea, or dilation of a false tract with placement of the tracheostomy tube outside of the trachea. Therefore, relative contraindications for this approach would include need for an urgent airway, obesity that prevents landmark identification, known abnormal anatomy such as a ‘high’ innominate artery, enlarged thyroid, or prior neck or tracheal surgery. Direct bronchoscopic guidance has decreased the incidence of many of these complications. It is likely that with proper patient selection and technique, percutaneous approaches are equivalent to standard surgical techniques. Cricothyroidotomy

Utilized predominantly in the emergency setting for rapid, safe airway access when translaryngeal

242 TRACHEOSTOMY

intubation has been unsuccessful or is not possible due to anatomy or trauma, cricothyroidotomy is performed through either a vertical or horizontal incision depending on patient anatomy, the familiarity of the practitioner with surgical anatomy of the neck, and prior experience with tracheostomy. In contrast to a tracheotomy, a cricothyroidotomy is made above the cricoid cartilage with passage through the cricothyroid membrane. The incision is gently dilated and a small endotracheal tube or tracheostomy tube can be placed through the cricothyroid membrane, taking care not to fracture the cricoid ring or place the tracheostomy in a false passage. Confirmation of accurate placement is made in exactly the same manner as discussed previously for tracheostomy. Cricothyroidotomy has been associated with an increased incidence of subglottic stenosis and thus, if continued ventilatory support is required, most surgeons advocate conversion to a standard tracheostomy in 2–3 days when the patient is stable.

pressure against the innominate artery to compress it against the sternum. This will result in compression of the inominate artery and tamponade the bleeding fistula on the way to the operating room where operative repair of the TIF can be performed. A tracheoesophageal fistula can also develop and typically presents with aspirated tube feeds in the setting of gastric dilation, or decreased tidal volumes when nasogastric suctioning is performed. Like TIF, tracheoesophageal fistula are also best diagnosed by bronchoscopy; however, these can sometimes be temporized by esophageal or airway stenting prior to definitive surgical repair. Other relatively minor complications of tracheostomy include local infection at the stoma site that usually resolves with antibiotic therapy, and tracheal stenosis which may resolve with dilation following decannulation or eventually require tracheal resection of the involved segment. See also: Sleep Apnea: Overview; Surgery for Sleep Apnea.

Complications Both percutaneous and standard tracheostomy techniques are associated with complications. Bleeding can occur from multiple sources. Minor bleeding can occur from small vessels at the site of the incision or from the tracheal mucosa. This bleeding is usually self-limited and can be controlled with minimal manual pressure. Bleeding which occurs 2–3 weeks following tracheostomy placement should suggest a possible tracheoinominate fistula (TIF), and appropriate diagnostic studies and surgical intervention are mandatory. Although TIF is rare, occurring in less than 1% of patients, it is lethal if not promptly treated. The first indication of a TIF is a minor ‘sentinel’ bleed. If the TIF is not recognized and treated immediately, massive hemorrhage and hemoptysis follows as the innominate artery bleeds into the trachea or out of the tracheotomy. Bronchoscopy is the best method to diagnose a suspected TIF, and if the suspicion is high, this should be performed in the operating room with a skilled anesthesiologist and cardiopulmonary bypass available. Other diagnostic tests such as computed tomography of the neck with intravenous contrast or magnetic resonance imaging can be useful, but may waste precious time that is better used in the operating room to repair the fistula. In the case of an actively bleeding TIF, the tracheal cuff should be quickly hyperinflated as an immediate temporizing maneuver, in an attempt to tamponade the fistula with the tracheal balloon. If this is unsuccessful, orotracheal intubation should be rapidly performed, followed by removal of the tracheal cannula with the insertion of a finger into the stoma to manually apply

Further Reading Angel LF and Simpson CB (2003) Comparison of surgical and percutaneous dilational tracheostomy. Clinics in Chest Medicine 24: 423–429. Bowen CP, Whitney LR, Truwit JD, Durbin CG, and Moore MM (2001) Comparison of safety and cost of percutaneous versus surgical tracheostomy. The American Surgeon 67: 54–60. deBoisblanc BP (2003) Percutaneous dilational tracheostomy techniques. Clinics in Chest Medicine 24: 399–407. Ernst A and Critchlow J (2003) Percutaneous tracheostomy – special considerations. Clinics in Chest Medicine 24: 409–412. Freeman BD, Isabella K, Cobb JP, et al. (2001) A prospective, randomized study comparing percutaneous with surgical tracheostomy in critically ill patients. Critical Care Medicine 29: 926–930. Freeman BD, Isabella K, Lin N, and Buchman TG (2000) A metaanalysis of prospective trials comparing percutaneous and surgical tracheostomy in critically ill patients. Chest 118: 1412–1418. Friedman Y, Fildes J, Mizock B, et al. (1996) Comparison of percutaneous and surgical tracheostomies. Chest 110: 480–485. Heffner JE (2003) Tracheotomy application and timing. Clinics in Chest Medicine 24: 389–398. Heffner JE and Hess D (2001) Tracheostomy management in the chronically ventilated patient. Clinics in Chest Medicine 22: 55–69. Kane TD, Rodriguez JL, and Luchette FA (1997) Early versus late tracheostomy in the trauma patient. Respiratory Care Clinics of North America 3: 1–20. Lewis RJ (1992) Tracheostomies. Indications, timing, and complications. Clinics in Chest Medicine 13: 137–149. Marsh HM, Gillespie DJ, and Baumgartner AE (1989) Timing of tracheostomy in the critically ill patient. Chest 96: 190–193. Rumbak MJ, Newton M, Truncale T, et al. (2004) A prospective, randomized, study comparing early percutaneous dilational tracheotomy to prolonged translaryngeal intubation (delayed tracheotomy) in critically ill medical patients. Critical Care Medicine 32: 1689–1694. Walts PA, Murthy SC, and DeCamp MM (2003) Techniques of surgical tracheostomy. Clinics in Chest Medicine 24: 413–422.

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TRANSCRIPTION FACTORS Contents

Overview AP-1 ATF Fox NF-jB and Ijb POU PU.1

Overview I M Adcock and K Ito, Imperial College London, London, UK G Caramori, University of Ferrara, Ferrara, Italy & 2006 Elsevier Ltd. All rights reserved.

Abstract The term ‘transcription factor’ refers to a large family of proteins, which exert transcriptional control via specific interactions with regulatory gene sequences. Here, we provide a summary of the different classes of the transcription factor divided according to their DNA-binding motifs. The modular structure of transcription factors and the presence of distinct interacting domains determine the ability of these factors to associate with each other and with coactivating/repressing proteins. By recruiting transcriptional coactivators, transcription factors can induce changes in chromatin structure enabling gene expression to occur. We use the activation of the Rel transcription factor NF-kB and the nuclear receptor GR as examples to indicate some of the intricacies and subtleties of DNA binding, chromatin remodeling, and transcription factor cross-talk. Finally, we review the evidence for the involvement of select transcription factors in allergic and inflammatory diseases of the lung, and how changes in the expression and/or activity of these factors may vary in disease and provide important targets for future drug development.

Description and Definition The production of messenger ribonucleic acid (mRNA) from deoxyribonucleic acid (DNA) (transcription) by a cell is strictly regulated. Transcriptional control results from the interaction between regulatory DNA sequences (promoters, enhancers, and silencers) and sequence-specific DNA-binding proteins. The binding of these proteins to DNA sequences results in an increase or a decrease in the transcription rate of the associated gene; thus, these proteins are known as transcription factors. Proteins recognize DNA in the same way as they recognize other proteins by forming a tertiary structure that is

compatible with the DNA sequence with which they must interact. After an initial contact is made, the protein and DNA come close enough together to form numerous atomic interactions. The atomic contacts between protein and DNA are varied, including hydrogen bonding, ionic interactions, and hydrophobic interactions. Activation of transcriptional enhancers appears to require the assembly of a highly specific three-dimensional nucleoprotein complex involving extensive protein–DNA and protein–protein interactions and the transcriptional efficiency of these complexes can be altered by subtle changes in the relative positions or orientations of proteins within the complex. The comprehensive understanding of protein–DNA interactions requires the isolation of these complexes and X-ray crystallographic studies. However, the study of these proteins has been simplified by the recognition of common functional and structural motifs within the many different proteins that recognize DNA (Table 1).

Table 1 Classification of transcription factors as structural families Domain

Factors containing domain

Helix–loop–helix

Homeodomain proteins POU factors, e.g., Oct-1, Oct-2, Pit-1, Unc-86

Zinc finger Cys-His Zn finger Cys-Cys Zn finger Basic element Lucine zipper Helix–loop–helix HTH þ ZIP b-Sheet

TFIIIA, Kruppel, Sp1 Nuclear hormone receptors, e.g., GR C/EBP, Fos, Jun, CREB, ATF2 MyoD1, E12, E47 Myc family proteins Rel proteins, NF-kB

The large number of DNA-binding proteins can be grouped together into a small number of families depending upon their common structural motifs.

244 TRANSCRIPTION FACTORS / Overview Generalized modular transcription factor DNA binding

AD-1

NLS-1 AD-2

Ligand-binding domain

NLS-2 AD-3

HSP binding Dimerization Nuclear translocation Transactivation/ cofactor association TF interaction

Figure 1 Modular structure of a generic transcription factor. The modular design of a transcription factor enables distinct regions of the protein to function, in isolation, as ligand-binding domains, dimerization domains, nuclear localization domains, and transactivation and transrepression (e.g., AP-1 and NF-kB interacting) domains. NLS, nuclear localization signal; AD-1/2/3, activating domain 1/2/3; TF-RE, transcription factor response element.

Functional Domains Transcription factors are generally composed of several domains possessing distinct functions such as DNA binding, transactivator/repressor binding, and appropriate ligand binding (Figure 1). This modular design with different functions in different domains is a common theme in eukaryotic transcription factors, where the DNA-binding domains are only a part of the intact proteins. This domain structure is conserved across species and can be interchanged or fused between transcription factors in experimental models, and often when this occurs in man in vivo, it can result in disease, for example, in acute promyelocytic leukemia where fusion proteins between the retinoic acid receptor alpha (RARa) and other transcription factors results in abnormal gene expression. In vitro, interchanging a glucocorticoid receptor ligand-binding domain with a similar domain in the RARa gene, for example, results in glucocorticoids switching on retinoic acid-regulated genes and retinoic acidmodulating glucocorticoid-sensitive gene expression.

Structural Motifs Helix–Turn–Helix

The helix–turn–helix (HTH) structure was the first DNA-recognition motif identified in which an a-helical region is followed by a sharp b-turn and then another a-helical region. The proteins bind as dimers, each monomer recognizing a half-site, and the approximate symmetry of the DNA-binding site is reflected in the symmetry of the complexes. The first a-helical motif lies across the major groove of DNA making hydrophobic interactions with the second helix, known as the recognition helix, which lies partly within the major groove and makes specific contact with DNA. The proteins are held in a stable ordered manner by an extensive array of hydrogen bonds. Mutations within this region allow dramatic changes in the specificity of DNA binding.

Examples of these HTH transcription factors include the Drosophila homeotic genes and a variety of mammalian proteins such as the Hox genes and thyroid transcription factor-1 (TTF-1). Although an isolated homeodomain can fold correctly and bind DNA with specificity similar to that of the intact protein, it seems likely that other regions of the protein modulate the precise DNA-binding specificity. Protein–protein interactions with other transcription factors and general coactivators/repressors may also have a role in modulating many homeodomain– DNA interactions and illustrate the important role that heterodimer formation can play in the regulation of gene expression. More recently, another class of regulatory protein has been identified in which the homeobox forms one part of the larger conserved domain known as the POU domain, so called because it includes the pituitary specific protein Pit-1, the octamer binding proteins Oct-1 and Oct-2, and the nematode gene unc-86 (Pit–Oct–Unc) domain to bind to DNA. The relative contribution of homeodomain and the POUspecific domain differs between the different proteins with the homeodomain being sufficient for sequencespecific binding of Pit-1, while both the homeodomain and the POU domain are necessary in the case of Oct-1. The high level of sequence similarity among different homeoboxes predicts that the target DNA sequences of many of these proteins will be similar or even identical, and indeed footprinting experiments have shown this to be the case. These observations pose the important problem as to how proteins with similar DNA-binding specificities execute different regulatory roles. The Leucine Zipper and the Basic DNA-Binding Domain (bZIP)

The leucine zipper element is found in many transcription factors throughout the animal kingdom,

TRANSCRIPTION FACTORS / Overview 245

such as the liver-specific transcription factor CCAAT/ enhancer binding protein (C/EBP) and the protooncogenes Myc, Fos, and Jun. In this structure, leucine residues occur every seven amino acids in an a-helical structure over 30–40 residues such that the leucines occur every two turns on the same side of the helix. The leucine zipper facilitates the dimerization of the protein by interdigitation of two leucinecontaining helices on different molecules and these residues form the buried subunit interface of the coiled-coil dimer. In turn, such dimerization results in the correct protein structure for DNA binding by the adjacent highly basic region that can interact directly with the acidic DNA. This B30 residue basic region is rich in arginines and lysines, but also contains other residues that are conserved throughout the family or in particular subfamilies. bZIP proteins are Y-shaped with the parallel leucine zipper region forming the stem of the Y, and the basic region forms a-helices that extend off like the arms of the Y. The N-terminal end of the twofold symmetric leucine zipper fits over the center of the DNA-binding site, and the helices from the basic region extend in opposite directions along the major grooves of the DNA. Transcription factors containing the basic DNA-binding domain can only bind to DNA once they have formed transcription factor dimers. Hence, factors containing the basic binding domain are further subgrouped according to the nature of the dimerization motif that they contain (Table 1). Leucine zipper proteins can form heterodimers, and these mixed dimers have important roles in the regulation of the biological activity of bZIP proteins. Both Fos and Jun proteins, the major components of the transcription factor activator protein-1 (AP-1) bind to palindromic sequences in DNA known as TRE sites [TGAC/GTCA], which mediate gene induction following phorbol ester treatment. Whilst Jun can bind to DNA as a homodimer, Fos is unable to homodimerize and cannot bind to DNA without the formation of a Fos–Jun heterodimer. This difference is due entirely to the differences in the leucine zippers of the two proteins since substitution of the Jun zipper region for that of the Fos zipper allows Fos homodimer formation and DNA binding through the basic region of Fos to occur. The requirement for dimer formation prior to DNA binding allows for regulatory control of AP-1 activity and thus gene expression. Heterodimers can also limit transcription factor activity: the transcription factor cAMP response element binding protein (CREB), a cAMP response regulator, is antagonized by formation of heterodimers with cAMP response element modulator (CREM). In addition, heterodimers may also acquire

new DNA-binding specificities and thus be targeted to different sites than homodimers. In other cases, heterodimer formation may allow for different combinations of activation and/or repression domains and thus change the regulatory properties of a molecule bound at a fixed DNA site. Although originally identified in leucine zippercontaining proteins, the basic DNA-binding domain has also been identified by homology comparisons in a number of transcriptional regulatory proteins, such as MyoD1. In this case the basic domain is associated with an adjacent region that can form a helix–loop– helix structure in which two amphipathic helices (containing all the charged residues on one side of the helix) are separated by an intervening nonhelical loop. This helix–loop–helix region acts in a manner analogous to that of the leucine zipper in that it allows protein dimerization and hence DNA binding by the adjacent basic DNA-binding domain. Members of the myc oncogene subfamily of DNA-binding proteins contain both a leucine zipper and a helix– loop–helix motif adjacent to the basic DNA region suggesting that proteins containing the basic region may form a related family comprised of three subgroups containing a leucine zipper, an HTH motif, or both (Table 1). b-Sheet Motifs

The Rel family of transcription factors are examples of DNA-binding proteins with b-sheet motifs. Generally, leucine-rich repeats are short-sequence motifs present in a number of proteins with diverse functions and cellular locations. All proteins containing these repeats are thought to be involved in protein– protein interactions. Leucine-rich repeats correspond to beta–alpha structural units arranged so that they form a parallel b-sheet with one surface exposed to the solvent, so that the protein acquires an unusual, nonglobular shape ideal for formation of specific protein–protein interactions. The X-ray crystallographic structure of some Rel proteins have been obtained and the Rel homology region (RHR) in the p50 nuclear factor kappa B (NFkB) subunit consists of two flexibly linked domains, each of which bears a remarkable similarity to an immunoglobulin domain. In its dimeric form bound to DNA, this RHR has the appearance of a butterfly. Although only the C-terminal domain contributes to the dimer interface, both domains are involved in important DNA interactions. The p50 dimer wraps itself around two-thirds of the cylindrical DNA tube making 38 contacts and leaving only the minor groove accessible. p50 interacts with DNA through loops on both the C-terminal dimerization domain

246 TRANSCRIPTION FACTORS / Overview

and the N-terminal domain. One of these loops in the N-terminal domain of p50, the recognition loop, reaches into the DNA and contacts the bases. The proximity of DNA-binding residues to the dimer interface makes for a remarkably seamless set of protein–protein and protein–DNA contacts. NF-kB binds to its consensus DNA sequence (50 GGGPuNNPyPyCC-30 ) with picomolar affinity compared to the nanomolar affinity of most other DNA-binding proteins. The wrapping of the DNA and the large number of contacts with DNA accounts for this extraordinary affinity. Zinc Fingers

Zinc (Zn) finger proteins are involved in many aspects of eukaryotic gene regulation and occur in proteins induced by differentiation and growth signals, in proto-oncogenes, in general transcription factors, and in regulatory genes of eukaryotic organisms. The DNA-binding region of these factors usually contains tandem repeats of the 30 residue zinc finger motif, Cys-X2/4-Cys-X12-His-X3–5-His. Therefore, each of these repeats contains two invariant pairs of cysteine and histidine residues, which coordinate a single atom of zinc and hold the antiparallel b-sheet and ahelices of the protein in a compact globular structure. This results in a finger-like structure that projects from the surface of the protein. The tips of these fingers make direct contact with the major groove of the DNA, alternate fingers binding on opposite sides of the helix. Examples of these types of transcription factors include Sp1, the GATA family, and the Drosophila Kruppel gene. The zinc atom is essential for DNA binding and the functioning of these transcription factors as mutation to remove zinc binding produces effects identical to gene deletion. Nuclear hormone receptors are an important family of regulatory proteins that includes the receptors for the hormones, retinoids, vitamin D, thyroid hormone, sex hormones (estrogens, progestins, and androgens), aldosterone, and glucocorticoids. These proteins contain separate regions for hormone binding, DNA binding, and for transcriptional activation. The DNA-binding domains contain two zinc atoms, which are each held in place by four cysteine residues. Therefore, this B70 amino acid region consists of two zinc fingers only as opposed to the multiple fingers (2–37) found in genes having Cys-His fingers and are probably not evolutionarily related. The DNA-binding domains from the glucocorticoid receptor and the estrogen receptor form two perpendicular a-helices held together by hydrophobic interactions and folded into a globular domain. The glucocorticoid receptor binds as a dimer. The first

helix of each subunit fits into the major groove, and side chains from this helix make contacts with the edges of the base pairs. The second major helix provides phosphate contacts with the DNA backbone and provides the dimerization interface.

Site-Specific Recognition The intracellular control and regulation of gene expression is mediated through complex interactions between specific transcription factors and DNA control elements. Transcription factors can achieve rapid target localization by initially binding to a nonspecific site on the DNA and then finding the specific site by one-dimensional diffusion along the DNA, by intersegment transfer, or both. Moreover, to overcome binding to the vast excess of non-specific DNA and to ensure occupancy at the target sequence, specific DNA-binding proteins must also distinguish between specific and non-specific DNA. The greater affinity of these proteins for specific sites provides the discrimination energy and thus determines their binding specificity. Recognition of the cognate binding sequence requires the formation of specific contacts between the protein and the DNA and is often accompanied by conformational changes in the protein, the DNA, or both. Many DNA-binding proteins, especially transcription regulatory proteins, induce DNA bending upon specific binding. The protein–DNA complex requires 10–20 hydrogen bonds between the side chains of amino acids and with the DNA backbone. Most of these contacts are made by a-helical regions within the protein motifs that fit into the major groove, interacting with specific bases, especially with purines, which are larger and offer more opportunities for hydrogen bonding. Folding and docking of the entire protein controls the meaning that any particular side chain has in site-specific recognition; furthermore, this recognition is a detailed structural process in which hydration may be important. Multiple DNA-binding domains are usually required for site-specific recognition. The same protein motifs, such as Zn fingers or bZIP, may be used more than once, as in binding by monomers and dimers, or when a single polypeptide contains tandem repeat motifs. Different motifs may also be used in the same transcriptional complex.

Role of Transcription Factors in Cell Regulation Cell growth, proliferation and differentiation are controlled largely by selective transcriptional modulation

TRANSCRIPTION FACTORS / Overview 247

of gene expression in response to extracellular stimuli. Much of this transcriptional control is governed by the action of sequence-specific transcription factors. In a similar manner, proinflammatory genes are stimulated following an inflammatory insult to the cell. Extracellular molecules interact with receptors on the cell surface, which sets in motion coordinated intracellular signaling cascades that cause the rapid transcriptional induction of selected genes. In the final steps of these cascades, many transcription factors are phosphorylated and bind to sites in the control regions of the required genes to stimulate their transcription. Interestingly, some transcription factors such as nuclear factor of activated T cells (NF-AT) are dephosphorylated during activation. This binding of the phosphorylated/dephosphorylated activator must then be communicated to the basal transcription machinery sitting some distance from the start site of transcription.

Proinflammatory Transcription Factors, Chromatin Modifications, and Gene Transcription Numerous transcription factors are activated during the inflammatory response including ubiquitous factors such as AP-1 and NF-kB, cell-selective factors including GATA3, NF-ATs, and Forkhead (FOX) proteins, and signal-selective factors such as signal transducers and activators of transcription (STATs), mothers against tetrapentaplegic (Smads), and C/EBP family members. In this overview we will discuss activation of NF-kB and the glucocorticoid receptor (GR) as an example of the general principles involved in transcription factor activation and signaling. NFkB is thought to be of paramount importance in asthmatic inflammation because it is activated by all the stimuli considered important in the inflammatory response to allergen exposure and, in addition, it is a major target for glucocorticoid action. NF-kB is ubiquitously expressed within cells, and it not only controls induction of inflammatory genes in its own right but also enhances the activity of other cell- and signal-specific transcription factors. NF-kB is activated by numerous extracellular stimuli, including cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-1b, viruses, some bacteria, and allergens. Activation of cell-surface receptors leads to phosphorylation of receptor-associated kinases, which in turn phosphorylate the inhibitors of NF-kB kinase (IKKs). Phosphorylation of IKKs results in phosphorylation of the NF-kB cytoplasmic inhibitor (IkBa), so that IkBa is targeted for proteosomal degradation. This degradation

precipitates the release of NF-kB from its inactive state, enabling nuclear translocation and binding to specific DNA response elements within the regulatory regions of responsive genes. NF-kB is predominantly composed of the p50/p65 heterodimer. Changes in p65 phosphorylation status can affect NF-kB function, for example, inactive p65 is nonphosphorylated and is associated predominantly with the repressor protein histone deacetylases 1 (HDAC1), whereas p65 is phosphorylated following IKK-2 stimulation and is able to bind to coactivator molecules such as CREB-binding protein (CBP) and the related 300 kDa protein p300 (p300/ CBP). Acetylation of p65 may also influence NF-kB function by affecting IkBa:p65 association and enhancing NF-kB activity. Chromatin Modifications and Gene Expression

DNA is tightly compacted around a protein core to form nucleosomes, which are further compacted to form chromatin. Each individual nucleosome consists of B146 bp DNA associated with an octamer of two molecules each of core histone proteins (H2A, H2B, H3, and H4). Since the early 1960s, it has been known that the expression and repression of genes is associated with alterations in chromatin structure by enzymatic modification of core histones. In the resting cell, DNA is tightly compacted around these basic core histones, excluding the binding of the enzyme RNA polymerase II, which activates the formation of mRNA. This conformation of the chromatin structure is described as closed and is associated with suppression of gene expression (Figure 2). The irregular 30 nm chromatin fiber is stabilized and further compacted by interactions between nucleosomes and linker histones such as H1. Histone H1 has long been regarded as a general repressor of transcription, but there are indications that it also has a role in transcriptional regulation. Here, histone

RNA polymerase II X 30 nm chromatin fiber Figure 2 In the resting cell, DNA (gray bands) is tightly compacted around core histones to form nucleosomes. These nucleosomes are further compacted to form a closed inaccessible structure, excluding the binding of the enzyme RNA polymerase II, which activates the formation of messenger RNA. This chromatin structure is associated with a lack of gene expression.

248 TRANSCRIPTION FACTORS / Overview

H1 acts as a ‘gate’ to nucleosomal DNA, preventing transcription factor DNA binding. Histone H1 removal is needed for opening factors to bind, thus making nucleosomal DNA accessible to further transcription factor binding and transcription. Phosphorylation of histone H1 may play an essential role in activation of gene transcription. Specific residues (lysines, arginines, and serines) within the N-terminal tails are capable of being posttranslationally modified by acetylation, methylation, ubiquitination, or phosphorylation, all of which have been implicated in the regulation of gene expression. Acetylation of the s-group on lysines reduces the charge of the histone residue and subsequently releases the tightly wound DNA, allowing the recruitment of further large protein complexes. A breakthrough in the discovery of the role of histone acetylation was the demonstration that transcriptional coactivators such as CBP and p300/CBPassociated factor (pCAF) have intrinsic histone acetyltransferase (HAT) activity, which is activated by the binding of transcription factors such as NF-kB. Increased gene transcription is therefore associated with an increase in histone acetylation, whereas hypoacetylation is correlated with reduced transcription or gene silencing. Histone acetylation is an active process whereby small changes in the activity of HATs or histone deacetylases (HDACs) can markedly affect the overall histone acetylase activity associated with inflammatory genes. Importantly, these changes in histone acetylation appear to be targeted toward regions of DNA associated with specific activator sites within the regulatory regions of induced inflammatory genes, although a global loosening of histone structure has also been proposed. Under resting conditions, less than half of the potential lysine residues available for acetylation are in fact acetylated, and these residues have a rapid turnover. This situation suggests that even small changes above or below the resting level are enough to lead to an activated chromatin state. This model predicts that changes in the ‘histone code’, the diverse range of histone tail posttranslational modifications such as acetylation, methylation, phosphorylation, and ubiquitination that are set and maintained by histone-modifying enzymes, must be translated into downstream events extremely rapidly. NF-jB Activation Induces Histone Acetylation

NF-kB can induce acetylation on both histone H3 and H4 in both a time- and concentration-dependent manner depending upon the stimulus and the cell type involved following the recruitment of a large coactivator complex that contains the HAT proteins

CBP, p300/CBP-associated factor (pCAF), and transcriptional intermediary factor-2 (TIF-2). IL-1b can also activate other pathways distinct from NF-kB that can impinge on NF-kB activation. These additional pathways, such as protein kinase C and nonreceptor tyrosine kinases, may enhance NF-kB activity, either by phosphorylating p65 and thereby enhancing cofactor recruitment or by phosphorylating NF-kB-associated cofactors.

Temporal Association of NF-jB with DNA, Cofactors, and Gene Induction The simplistic model described above does not explain all aspects of NF-kB action, however. Immediate early genes such as IkBa do indeed bind NF-kB to their promoters rapidly after lipopolysaccharide (LPS) stimulation, but within 10 min NF-kB dissociates from the IkBa promoter site and never reassociates. In contrast, NF-kB binds to its promoter sites in DNA for up to 2 h before dissociation in distinct sets of genes (such as manganese superoxide dismutase and macrophage inflammatory protein-2), in spite of stimulation by LPS at the same time. However, other NF-kB-regulated genes, such as the chemokines/cytokines RANTES (regulated on acti% secreted), mon% vation, normal T cell expressed and % % % % ocyte chemoattractant protein-1, and IL-6, do not show NF-kB binding to their promoters until 2 h after activation. NF-kB sites in the promoter regions of these genes are originally in a repressed chromatin environment that prevents NF-kB DNA binding and subsequent gene expression. These become accessible only after AP-1-mediated histone acetylation and subsequent alteration in the local nucleosomal structure (chromatin remodeling) (Figure 3). Thus, there are subtle changes in NF-kB DNA binding that are promoter context-dependent, precede coactivator recruitment, and are not detectable using conventional band shift and reporter gene assays. The question as to which aspect of chromatin changes occurs first, remodeling or modification, prior to gene induction is still a matter of intense debate. The question arises: how can NF-kB, or any other transcription factor, interact with its recognition site when DNA is compacted? NF-kB may bind to a kBRE within the linker DNA between nucleosomes or, alternatively, when the kB-RE core residues are facing outward from the DNA wrapped around histones. Binding to the kB-RE may then modify the local chromatin structure or cause local histone modifications altering NF-kB access to the promoter site. High-resolution mapping of GR interactions with a glucocorticoid-responsive promoter suggests that the GR not only reorganizes the chromatin

TRANSCRIPTION FACTORS / Overview 249 TF1

HATs txn

TF1 responsive promoter Modification/remodeling (a) TF1

TF2 X

No txn

SWI/SNF + ATP No txn X

Remodeling TF1 responsive promoter

TF1

HATs txn

Modification/remodeling

(b) Figure 3 Temporal activation of inflammatory genes. (a) Simple model of transcription factor (TF1) activation of immediate early response genes. Activation of TF1 by exogenous stimuli results in TF1 binding to TF1-response (RE)-sites in the regulatory regions of the immediate early response genes, subsequent recruitment of HATs, and chromatin modification, leading to chromatin remodeling and induction of gene transcription (txn). Often in the case of these genes, TF1 is rapidly ejected from DNA within 10 min. (b) A more complex model accounts for the fact that many genes do not show TF1 promoter binding for up to 2 h following stimulation of TF1 activation, and no transcription of these genes occurs during this time. Transcription requires binding of another TF, TF2, binding to its consensus site within the gene regulatory domain and subsequent ATP-dependent chromatin remodeling using the SWI/SNF complex to reveal the TF1 DNA-binding site. Once the regulatory regions are remodeled, TF1 is able to bind to DNA, recruit HATs, and drive gene expression.

immediately surrounding its binding site but can also have effects elsewhere, thereby enforcing a particular translational frame on the chromatin template and modifying the effects of other DNA-binding proteins. Ordered Recruitment of Coactivator Complexes

One other important question that also needs to be addressed is whether there is a specific order of recruitment of distinct coactivator factors to the activated DNA-bound NF-kB complex to enable gene transcription. Recent work examining androgen and thyroid hormone receptor gene activation shows that nuclear hormone receptors do not in themselves recruit all the cofactors required at the target promoters. Steroid receptor coactivators, recruited by receptors, can in turn recruit other coactivators such as p300/CBP, which can subsequently recruit SWI/ SNF (a large multisubunit protein complex) and mediator complexes. SWI/SNF enables chromatin remodeling to occur in an adenosine triphosphate (ATP)-dependent manner, but histone acetylation by p300/CBP facilitates the recruitment of SWI/SNF and mediator complexes. Thus, cofactor–cofactor interactions are essential for effective gene expression. The interactions do not have to occur sequentially, but histone acetylation can enhance the recruitment of large multiprotein complexes in a coordinated manner. However, again the story is found to be more complex. In some cases, such as the GR and possibly

p65, there is a ‘hit-and-run’ mechanism of action rather than a stable association with glucocorticoid response element (GRE). GR resides on DNA for less than 10 s before being ejected and replaced by another GR. This ejection may allow binding of additional regulatory factors that enhance gene transcription, such as HAT-containing complexes, and may also play a role in feedback regulation. Interestingly, in the absence of ATP and chromatin remodeling factors, the GR stably interacts with glucocorticoid-responsive genes.

Cross-Talk between Transcription Factors and Their Transduction Pathways The complexity of transcription factor activation pathways and their ability to engage in cross-talk may enable cells to overcome inhibition of one pathway and retain a capacity to activate specific inflammatory genes. Generally, it is necessary to have coordinate activation of several transcription factors in order to have maximal gene expression. This may explain how ubiquitous transcription factors may regulate specific genes in certain types of cells only. Activation of NF-kB leads to the coordinated induction of multiple inflammatory and immune genes. Whilst NF-kB is not the only transcription factor involved in regulation of these genes, it often appears to have a decisive role often in cooperation with other transcription factors such as AP-1 and C/EBP. The role of NF-kB should be seen as an amplifying

250 TRANSCRIPTION FACTORS / Overview

and perpetuating mechanism that will exaggerate the disease-specific inflammatory process through the coordinated activation of multiple inflammatory genes. The fact that many inflammatory genes, which are regulated by AP-1, NF-kB, and C/EBP, can be downregulated by glucocorticoids indicates the possible importance of cross-talk between these signal transduction pathways. For example, activation of the IL-8 promoter by NF-kB and C/EBP is inhibited by dexamethasone primarily via the NF-kB site. Conversely, activation of a GRE-dependent promoter by dexamethasone was inhibited by overexpression of p65. These and other studies show direct protein– protein interactions between NF-kB and GR, which prevent NF-kB transactivation and partly account for the anti-inflammatory properties of glucocorticoids. GR represses AP-1 transactivation using mechanisms similar to those used to inhibit NF-kB activation.

Transcription Factors in Lung Disease There has been a recent increase in the number of investigations of transcription factor activity and/or expression in human respiratory diseases to provide proof of concept and validation for murine disease models. Many transcription factors such as p53, TTF-1, early growth response-1 (Egr-1), STAT3, insulin-derived growth factor (IGF), and RARs are involved in the pathogenesis of lung carcinoma. In addition, NF-kB also appears to play a critical role in resistance to chemotherapy, inhibition of tumorigenesis, and the induction of anti-apoptotic genes. NF-kB has been implicated in most, if not all, inflammatory diseases and diseases of the lung are no exception. For example, NF-kB activity is enhanced in asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and mineral dust diseases. It has been suggested that cystic fibrosis transmembrane regulator (CFTR) acts as a pattern recognition receptor regulating NF-kB activity and the D508 mutation leads to constitutive NF-kB activation. Furthermore, hepatic nuclear factor (HNF)-1 confers tissue-selective expression of CFTR. In addition, there is evidence for increased expression of c-Fos in bronchial epithelial cells in asthmatic airways, and many of the stimuli relevant to asthma that activate NF-kB will also activate AP-1. In addition, enhanced activity of both NF-kB and AP-1 has been reported in the airways of severe therapyinsensitive asthmatic patients. In addition, a recent study showed a genetic association of asthma severity with the zinc finger transcription factor PHF11. Two transcription factors GATA-3 and T-bet appear to be critically important for Th2 and Th1

polarization, respectively, by acting both on regulated gene expression and by epigenetic control. For example, GATA-3 seems to be of particular importance in the differentiation of human Th2 cells and also the suppression of Th1 cytokine production by resetting the Th1 and Th2 gene loci. T-bet performs a similar function for Th1 differentiation and cytokine production. GATA-3 expression is increased in the peripheral venous blood T cells from atopic asthmatics and in induced sputum cells from asthmatic subjects particularly after allergen challenge and T-bet appears to be overexpressed in Th1 diseases. Understanding of the molecular mechanisms for Th1 and Th2 cytokine production developed in animal models predicts that altered expression of STAT4 and STAT6 would be found in allergic airway disease. Despite convincing data in disease models, this has not been confirmed in man. Interestingly, enhanced activation of STAT1 has been reported in the airways of patients with asthma and in subjects with cystic fibrosis. Protein inhibitor of activated STAT1 (PIAS1) has also been implicated as playing a role in the pathogenesis of cystic fibrosis. Sp1 is a ubiquitous transcription factor that binds to GCboxes and related motifs, which are frequently occurring DNA elements present in many promoters and enhancers. Modification of these sites within the IL-4, IL-10, and 5-lipoxygenase promoters has been associated with altered expression of these genes in distinct groups of asthmatic patients. The expression of the GR has been examined in several steroid-sensitive and -insensitive respiratory diseases. GRa expression has been reported to be lower in steroid-insensitive interstitial pulmonary fibrosis (IPF) than in steroid-sensitive diseases such as sarcoidosis and cryptogenic organizing pneumonia (COP). In contrast, no changes in GRa expression have been reported in severe therapy-insensitive asthma and determination of GRa expression in COPD has not yet been reported. Changes in the expression of GRb, a dominant negative form of GR, do not appear to correlate with steroid sensitivity in most lung diseases although this remains controversial for severe asthma. Furthermore, abnormal expression of other nuclear hormone receptors including peroxisome proliferating antigen receptor (PPAR)g and the vitamin D3 receptor (VD3R) has been reported in BAL macrophages and T cells in patients with sarcoidosis. Importantly, recent evidence suggests that a dysfunctional interaction between GR and C/EBPa in airway smooth muscle cells plays a role in their increased proliferation seen in the lower airways of asthmatics and might explain why this is not affected by treatment with inhaled corticosteroids.

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There is also evidence for activation of NF-kB in the bronchial epithelial cells of patients with COPD and in the sputum macrophages during COPD exacerbations. Finally, there is an increased number of activated STAT4þ cells in the bronchial biopsies of smokers with mild–moderate COPD compared with smokers with normal lung function. The presence of activated STAT4þ cells is significantly associated with the presence of IFN-gþ cells in both bronchial biopsies and bronchoalveolar lavage-derived lymphocytes and correlates with the degree of airflow limitation in smokers. Transforming growth factor beta (TGF-b) is postulated to play a major role in several lung diseases where airway remodeling has been implicated, such as asthma, COPD, and fibrosing lung disease. TGF-b stimulates a family of transcription factors known as Smads. The Smad family consists of the receptorregulated Smads (R-Smads), a common pathway Smad (co-Smad), and inhibitory Smads (anti-Smads). R-Smads include Smads 1, 2, 3, 5, 8, and 9 and are phosphorylated following TGF-b receptor binding. Smad4 is a co-Smad and is not phosphorylated by the TGF-b receptor binding. Anti-Smads include Smad6 and Smad7, which downregulate TGF-b receptor signaling. Recent reports suggest that the expression of the inhibitory SMADs 6 and 7 are significantly downregulated in COPD. In addition, abnormal Smad3 expression has been reported in pulmonary hypertension and cystic fibrosis. Fibrosis has also been associated with changes in hypoxia and specifically hypoxia-inducible factor (HIF)-1 and more general redox-sensitive transcription factors such as NF-kB and AP-1. Data from animal models have also implicated a role for factors such as Thy1 and NRF2, although their role in human disease awaits confirmation. In addition, aberrant epithelial cell regeneration characteristic of IPF has been associated with increased expression of p53, b-catenin/Wnt, and deltaNp63. Studies of transcription factor interactions may thus have therapeutic potential in the control of many lung diseases. Glucocorticoids are the most effective treatment for asthma and exert their anti-inflammatory effects largely by binding to transcription factors that have been activated by inflammatory cytokines. Other drugs that regulate the activity of specific transcription factors may also be developed in the future. The identification of enzyme targets, such as Jun kinases or IKKs, may also lead to the development of new drugs that are able to control oncogenesis and/or inflammation. Cell-specific transcription factors may be a more attractive target, as this may lead to the development of drugs with a reduced risk of adverse effects.

See also: Corticosteroids: Glucocorticoid Receptors. Transcription Factors: AP-1; ATF; Fox; NF-kB and Ikb; POU; PU.1.

Further Reading Andersen B and Rosenfeld MG (2001) POU domain factors in the neuroendocrine system: lessons from developmental biology provide insights into human disease. Endocrinology Review 22: 2–35. Barnes PJ and Adcock IM (2003) How do corticosteroids work in asthma? Annals of Internal Medicine 139: 359–370. Caramori G, Ito K, and Adcock IM (2004) Transcription factors in asthma and COPD. I Drugs 7: 764–770. Escoubet-Lozach L, Glass CK, and Wasserman SI (2002) The role of transcription factors in allergic inflammation. Journal of Allergy and Clinical Immunology 110: 553–564. Ghosh S and Karin M (2002) Missing pieces in the NF-kappaB puzzle. Cell 109(supplement): S81–S96. Groneberg DA, Witt H, Adcock IM, Hansen G, and Springer J (2004) Smads as intracellular mediators of airway inflammation. Experimental Lung Research 30: 223–250. Hermanson O, Glass CK, and Rosenfeld MG (2002) Nuclear receptor coregulators: multiple modes of modification. Trends in Endocrinology and Metabolism 13: 55–60. Latchman DS (1999) Eukaryotic Transcription Factors, 3rd edn. Oxford: Academic Press. Pabo CO and Sauer RT (1992) Transcription factors: structural families and principles of DNA recognition. Annual Review of Biochemistry 61: 1053–1095. Sharrocks AD (2001) The ETS-domain transcription factor family. Nature Review of Cellular and Molecular Biology 2: 827–837. Sirulnik A, Melnick A, Zelent A, and Licht JD (2003) Molecular pathogenesis of acute promyelocytic leukaemia and APL variants. Best Practice and Research in Clinical Haematology 16: 387–408. Urnov FD and Wolffe AP (2001) Chromatin remodeling and transcriptional activation: the cast (in order of appearance). Oncogene 20: 2991–3006. White RJ (2001) Gene Transcription: Mechanisms and Control. Oxford: Blackwell.

AP-1 E Shaulian, Hebrew University Medical School, Jerusalem, Israel & 2006 Elsevier Ltd. All rights reserved.

Abstract The AP-1 transcription factor is a dimeric transcription factor composed of proteins from several families including Jun, Fos, ATF, and Maf. AP-1 regulates various cell-autonomous as well as general physiological and pathological effects. This review describes the proteins making up AP-1, briefly mentions the diverse mechanisms by which the expression and activity of its components are regulated, and categorizes the major types of genes regulated by it. The experimental evidence implicating AP-1 in different processes such as proliferation, differentiation, apoptosis, migration and wound healing, malignant

252 TRANSCRIPTION FACTORS / AP-1 transformation, and angiogenesis are summarized also. The roles of AP-1 in disorders of the respiratory system are specified. Appropriate examples are provided to demonstrate AP-1 activity in the above-mentioned processes. The complexity of AP-1 research is emphasized as different AP-1 combinations may elicit different biological effects according to cell type, developmental stage, availability of interacting cofactors, and environmental conditions.

Composition and Structure Activating protein 1 (AP-1) is a dimeric transcription factor that binds specific sequences in the DNA and regulates the transcription of various genes. Historically, AP-1 activity was associated with neoplastic transformation; however, it is now thought that proteins composing the AP-1 are involved in diverse biological processes including regulation of proliferation, apoptosis, differentiation, immune responses, wound healing, and inflammation. In keeping with its diverse activities AP-1 is composed of proteins from several families whose common denominator is the possession of basic leucine zipper (bZIP) domains that are essential for dimerization and DNA binding. Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra1, and Fra2) subfamilies are the major AP-1 proteins. ATF proteins ATF2, LRF1/ATF3, B-ATF, JDP1, and JDP2 are also components of AP-1 and the last group of AP-1 proteins, which is less studied with respect to its activity, is the Maf subfamily, which includes c-Maf, MafB, MafA, MafG/F/K, and Nrl. The broad combinatorial possibilities provided by the large number of AP-1 proteins determine AP-1’s binding specificity, affinity, and consequently the spectrum of regulated genes. Jun proteins can homodimerize and heterodimerize whereas Fos proteins cannot homodimerize but can heterodimerize with Jun proteins to bind DNA. Jun–Fos heterodimers bind preferentially to a heptamer consensus sequence known as the TPA responsive element (TRE) 50 -TGA(C/G)TCA-30 , whereas Jun–ATF dimers bind with higher affinity to another consensus sequence known as the cyclic AMP responsive element (CRE) 50 -TGACGTCA-30 . In addition, AP-1 may bind diverse sites upon interaction with structurally unrelated proteins such as NFAT, or proteins from the Ets or Smad families. Therefore, in many natural promoters and enhancers of AP-1-regulated genes, the sequences to which AP-1 binds are often diverse.

proteins. AP-1 activity responds to a wide variety of extra- and intracellular stimuli. Interestingly, both mitogenic (serum and growth factors) and growtharresting stimuli such as environmental stress (UV, alkylating agents, and hypoxia) induce AP-1 activity. In addition, AP-1 is also induced by proinflammatory cytokines (tumor necrosis factor alpha (TNF-a) and interlukin-1 (IL-1)) during eruptions of immune responses. Serum, growth factors, and cytokines activate specific receptors on the cell membrane that initiate signaling cascades leading to activation of the mitogen-activated protein-kinase (MAPK) pathways. Exposure of cells to mitogenic signaling activates oncogenes such as Ha-Ras, which transduce AP-1 activating signals. Therefore, it is not surprising that deregulated expression or activity of numerous oncogenes results in induction of AP-1 activity. At least four MAPK pathways are involved in AP-1 induction. The ERKs are activated by mitogenic stimuli and contribute to c-fos induction, whereas environmental stress and proinflammatory cytokines activate the p38 kinases (a; b; g), the c-Jun N-terminal kinases 1, 2, 3 (JNKs), and ERK5. All three kinases phosphorylate and activate proteins involved in AP-1 induction. For example, following UV exposure, ATF2 phosphorylated by p38 and JNK dimerizes with c-Jun phosphorylated by JNK to induce transcription of c-jun in an autoregulatory fashion. Moreover, c-Jun phosphorylation by JNK also contributes to dramatic and persistent stabilization of the protein after exposure to UV (Figure 1).

Stress Growth factors ERK

The levels of expression of AP-1 proteins are tightly regulated at different levels including transcriptional rate, transcript stability, protein stability, posttranslational modifications, and interactions with other

p38

JNK

c-Jun c-Fos c-Jun ATF TRE site

Proliferation

AP-1 Regulation

JNK

CRE site

Stress responses

Figure 1 AP-1 activation pathways and composition determine its target genes. Activation of AP-1 by growth factors and stress activates the ERK and JNK pathway leading to c-Fos and c-Jun induction and regulation of genes involved in proliferation. Activation of AP-1 by environmental stress mediated by JNK and p38 activation results in regulation of stress-induced genes.

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Genes Regulated by AP-1 The overall number of AP-1-regulated genes is rather large. However, the number of genes that harbor AP-1 binding sites at their promoter and to which AP-1 binding in vivo has been demonstrated is lower. Furthermore, it is obvious that at any given time not all AP-1 sites are occupied and regulated. It is assumed that the ability to control gene expression depends on availability and activity of different AP-1 proteins and cofactors. The balance between AP-1 proteins may account for the differential effects of AP-1 proteins on transcription driven from the same gene. For example, c-Jun may activate transcription from certain genes whereas JunB represses it. However, overexpression of JunB in cells that do not express c-Jun can substitute for c-Jun in the regulation of genes whose expression is Jun/Fos dependent. The genes regulated by AP-1 proteins may be roughly divided into several functional groups (see Further Reading for further information). The first and most important group contains genes involved in cell-cycle control. It turns out that AP-1 proteins control the transcription of key factors in regulation of the cell cycle. One significant example is the regulation of cyclin D1 expression by c-Jun and c-Fos/ FosB, which bind its promoter and upregulate its transcription; furthermore, their deficiency reduces the basal expression of cyclin D1 and attenuates its induction by serum. Conversely, JunB represses cyclin D1 expression and increases the expression of p16INK4, a potent inhibitor of cyclin-dependent kinases (CDI). The Jun family also has a strong relationship with the p53 pathway. c-Jun binds to a divergent AP-1 element in the p53 promoter and represses its expression to allow cell-cycle progression. Furthermore, even when p53 levels are upregulated following exposure to UV radiation a prolonged expression of c-Jun prevents the induction of its target gene p21cip1, another CDI whose activity is required for growth arrest. c-Jun is not the only AP-1 protein functionally interacting with the p53 pathway. Mouse embryo fibroblasts (MEFs) deficient in junD express high levels of p19ARF, thus suggesting that JunD represses its expression. However, the proof for direct regulation of p19ARF is missing. The second group of genes regulated by AP-1 includes growth factors and growth factor receptors. The protein products of some of the genes in this group such as epidermal growth factor receptor (EGFR) are involved in proliferation, tissue organization, and transformation. The third and extremely important group of AP-1-regulated genes are those whose products are involved in pro- and antiapoptotic activities. Several Bcl proteins, which are

pivotal regulators of the apoptotic machinery, are regulated by AP-1. In addition, AP-1 proteins, especially c-Jun, also control the expression of upstream regulators of the death-receptor signaling pathway. However, it is of interest that whereas c-Jun increases the expression of the death ligand of Fas (FasL) in response to exposure to DNA-damaging agents it represses the expression of the Fas receptor in melanomas. The fourth group of AP-1-regulated genes are those involved in immune responses and inflammation. For example, the interferon b enhancer and TNF-a promoter contain a CRE site, which becomes occupied by ATF2-c-Jun dimers as part of a multiprotein enhanceosome complex in response to viral infection or other stimuli thereby inducing their expression. The fifth group of genes regulated by AP-1 are those coding for proteins involved in detoxification, which are regulated in response to xenobiotic and penolic antioxidants and genes involved in antioxidative defense and H2O2 production. AP-1 proteins also regulate the expression of matrix metalloproteases, which are involved in extracellular matrix remodeling. This process is important for wound healing and enables infiltration of components of the immune system to infected tissues. However, impairment of genetic constraints imposed on AP-1 activity during tumorigenesis results in deregulated expression of these genes leading to increased tumor invasiveness. The last group of AP-1 target genes includes those involved in angiogenesis.

Biological Activities Intensive in vitro studies of AP-1 activities provided initial information implicating AP-1 proteins in proliferation and transformation. The examination of genetically manipulated animals enabled a more rigorous examination of AP-1 functions in vivo, and demonstrated again the huge complexity of AP-1 activity. The activities of AP-1 in proliferation, apoptosis, migration and wound healing, differentiation and transformation are discussed below. Proliferation

The first indications for AP-1 involvement in proliferation came from experiments in which Jun and Fos proteins were inactivated by the introduction of antisense oligonucleotides or microinjection of specific antibodies to different cell lines. These experiments demonstrated requirement of Jun and Fos proteins for normal cell-cycle progression as their inactivation inhibits the DNA synthesis and prevents the entry of serum-starved cells into the cell cycle upon serum

254 TRANSCRIPTION FACTORS / AP-1

stimulation. However, examination of cells from knockout animals deficient in specific AP-1 proteins reveals differences between different AP-1 proteins. c-Jun is a positive regulator of proliferation under any circumstances. c-jun
/
MEFs proliferate extremely slowly, enter senescence after two passages in culture, and even after immortalization c-jun
/
fibroblasts proliferate slower than wt counterparts. Constitutive expression of c-Jun in these c-jun deficient fibroblasts enhances their proliferation rate, making them faster at proliferating than c-jun þ / þ fibroblasts. By contrast, junB-deficient MEFs proliferate normally and ectopic expression of JunB reduces their proliferation rate, suggesting that JunB is a negative regulator of proliferation. The effects of JunD on proliferation are the most obscure as it enhances proliferation and prevents premature senescence in MEFs but reduces the proliferation rate of immortalized fibroblasts. It was suggested that Jun proteins enhance passage from G1 into S phase of the cell cycle, but recently some data suggest that at least some of their effects take place also at the G2/M transition. Fos proteins also enhance proliferation but the effects of their inactivation are observed only when two Fos proteins, c-Fos and FosB, are inactivated or knocked out. Apoptosis

The involvement of AP-1 proteins in apoptosis emphasizes the great complexity of AP-1 activity, as it is dependent on cell context and developmental stage. AP-1 proteins and their upstream kinase JNK are major regulators of apoptosis of neuronal and lymphoid cells. Experiments performed in cells from the nervous system revealed that JNK activation followed by c-Jun phosphorylation, observed shortly after withdrawal of survival factors (NGF) from neurons, lead to neuronal cell death. Moreover, in addition to survival factor withdrawal, the repertoire of factors eliciting AP-1-dependent cell death is rapidly growing and includes exitotoxic stress, oxidative stress, b-amyloid exposure, DNA damage, and tissue remodeling. Knocking-out JNK3, a JNK isoform specifically expressed in the brain, or knocking-in a c-jun mutant in which the major JNK phosphorylation sites serines 63 and 73 were replaced by alanines render mice resistant to exitotoxic effects of kianic acid. However, in spite of the seemingly pro-apoptotic effects of the JNK/AP-1 pathway in the nervous system it is worth mentioning that AP-1 and JNK also elicit antiapoptotic effects. The morphology of brains of jnk1
/
jnk2
/
double knockout mice is abnormal due to deregulated apoptosis, which is low

in some regions but high in others. Apparently, the abnormality is only partially dependent on c-Jun as specific deficiency of it in mice brains does not result in gross abnormalities but leads to an increase in the number of certain neurons. JNK/AP-1 pathway is also considered to be a mediator of apoptosis caused by genotoxic agents. jnk1
/
jnk2
/
and c-jun
/
cells are less sensitive to UV-induced apoptosis. However, JNK may mediate part of its pro-apoptotic signaling independent of c-Jun by phosphorylating and activating several BH3-only Bcl2 family members. c-Jun itself regulates the expression of the pro-apoptotic genes FasL and Bim. The most obvious example of the antiapoptotic activity of AP-1 was obtained from c-jun-deficient mice, which die in uterus due to massive apoptosis of their livers. The protective effects of c-Jun in the liver are required for progression of liver cancer as they increase the tumor resistance to TNF-a-induced apoptosis. Similarly, livers of junD
/
mice are more sensitive to LPS-induced apoptosis. Cellular Migration and Wound Healing

The JNK/AP-1 response to stress and injury indicates a possible involvement in wound healing. Indeed, AP-1 proteins, especially c-Jun, are induced during liver hepatectomy, epidermal, or axonal injury. The induction may be mediated by a wound-induced increase in intracellular Ca þ and activation of JNK. It has also been reported that JNK is negatively regulated by high cell density, thereby is activated in cells residing in the border of the wound. The physiological importance of the induction was demonstrated by monitoring the healing process of organs from which c-Jun was specifically knocked out. These types of experiment revealed that in addition to its ability to support proliferation of hepatocytes after hepatectomy c-Jun is important for re-epithelialization of wounded skin, as in its absence the ability of the leading edge epidermis to migrate and invade fibrin clots is impaired. The effect of AP-1 on cellular migration was demonstrated in several in vitro studies and seems to play a role also in migration of endothelial cells, thus suggesting a possible role in angiogenesis. Differentiation

The most striking example of AP-1 effects in differentiation is the involvement of Fos proteins in bone development. Studies in mice overexpressing or deficient in AP-1 proteins demonstrated that c-Fos and Fra-1 elicit specific effects on the skeleton in genetically manipulated animals. Overexpression of c-Fos

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in an in vitro model of chondrogenesis inhibited the differentiation of chondrocytes. Overexpression in chimeric animals derived from embryonic stem (ES) cells into which c-fos transgene is introduced or in established c-fos transgenic mice expressing c-fos ubiquitously resulted in the development of chondrogenic tumors or osteogenic sarcomas, respectively. By contrast, Fra-1 transgenic mice develop osteosclerosis of the entire cytoskeleton, which is attributed to the excessive number of osteoblasts suggesting a positive role for Fra-1 in osteoblast differentiation. The effects of Fos proteins are not limited to osteoblasts as c-fos
/
mice are osteopetrotic due to the lack of osteoclasts; this suggests a major role for c-Fos in differentiation of osteoclasts. Fra-1 is dispensable for osteoclast differentiation but possibly contributes to the process in concert with c-fos, as it can partially rescue the osteopetrotic phenotype of c-fos
/
mice. Transformation

The ability of AP-1 proteins to regulate cell cycle and apoptosis has direct consequences on their oncogenic potential. A large number of in vitro studies demonstrated that c-Jun is a potent oncogene that can transform cells in concert with additional oncogenes such as Ha-ras. It was suggested that JunB and JunD antagonize c-Jun activity in transformation. Furthermore, in vivo studies that focused on several tissues including skin, liver, and bone demonstrated the significance of AP-1 in the development of cancer in these organs. However, in many cases AP-1 activity is important for tumor progression rather than initiation. Overexpression of ectopic Fos is a clear exception as it causes the appearance of osteosarcomas in 15% of the animals. Overexpression of c-Jun on the other hand does not increase tumorigenesis unless the animals are exposed to an additional carcinogenic stimulus. As skin is the major target for UV-derived genetic insults and UV also upregulates several AP-1 proteins, it was an obvious organ to use to explore the involvement of AP-1 proteins in transformation. Deletion of Fos or c-Jun or inactivation of AP-1 activity by ectopic expression of c-Jun dominant negative mutant, TAM67, in the skin, impairs the development of fully malignant tumors by chemical carcinogens. Furthermore, c-Jun may have an additional role in skin cancer by increasing the resistance of melanoma cells to UV by repressing the expression of the death receptor Fas. Involvement of AP-1 in cancer is not restricted to skin. Specific deletion of c-Jun in mouse livers results in inhibition of development of fully malignant

hepatocarcinomas. Apparently, in this case c-Jun increases the tumor survival to TNF-a-induced p53dependent apoptosis. Therefore, it is believed that c-Jun is required for tumor progression by increasing survival and maintaining proliferation. Indeed, these functions were found to be carried out by c-Jun and JunB in Hodgkin’s lymphomas, the only human cancer in which AP-1 proteins are frequently overexpressed. In addition to maintaining the proliferation capacity and increasing survival of the tumor cells themselves, c-Jun and JunD may also play roles in angiogenesis. Recent work in which introduction of DNAzyme targeting c-Jun expression into endothelial cells inhibited their proliferation, migration, invasion capacity, and tubule formation in vitro. Injection of the DNAzyme together with inoculation of tumor cells in mice reduced significantly the microvessel density in tumors and inhibited tumor growth by 60% suggesting a significant role for c-Jun enhancement of angiogenesis. These results support other findings demonstrating that endostatin, an endogenous angiogenic inhibitor, reduces the expression of both JNK and c-Jun in endothelial cells. However, it was recently suggested that JunD decreases tumorigenesis by reducing tumor angiogenesis. This process is a result of the ability of JunD to limit the production of reactive oxygen species, which in turn inhibit HIF-1a activation and VEGFA expression.

Role of AP-1 in Respiratory Diseases The development of pathological disorders in the respiratory system usually involves inflammation of the airway with accompanying infiltration of immune cells, mostly T cells into the airways. In addition, tissue remodeling modulates the thickness and composition of the airway walls, leading to decreased luminal diameter and function. In asthma, for example, the airway wall is infiltrated by mononuclear cells (mostly CD4 T cells) and eosinophils. Mast cells are degranulated, indicative of activation. CD4 Th2 cells are believed to initiate and perpetuate the disease. Levels of Th2 cytokines interleukin-4 (IL-4), IL-5, and IL-13 are increased in the bronchoalveolar lavage (BAL), BAL cells, and airway biopsies of asthmatics. IL-4 and IL-13 regulate isotype switching of naı¨ve B cells to IgE production and IL-5 controls growth and survival of eosinophils. Thus, they constitute a most significant factor in inflammation of the respiratory pathways. Two transcription factor families, nuclear factor kappa B (NF-kB) and the AP-1, regulate the expression of various cytokines involved in immune responses and

256 TRANSCRIPTION FACTORS / AP-1

inflammation. The contribution of AP-1 will be discussed here, while the activity of NF-kB will be discussed elsewhere. Increased activation and expression of AP-1 proteins has been demonstrated in the airways of asthmatic patients. AP-1 is capable of transcriptionally upregulating the expression of IL-4, IL-5, and IL-13, usually in cooperation with other transcription factors such as nuclear factor of activated T cells 1 (NFAT1), or GATA. IL-4 is a particularly good example of AP-1 regulation of cytokine expression since the molecular mechanism of its induction has been rigorously studied. The IL-4 promoter contains an AP-1/NFAT composite site that is occupied cooperatively by both NFAT1 and AP-1 proteins to elicit full activation of IL-4 transcription. Therefore, it is not surprising that an NFAT1 mutant that cannot interact with Fos:Jun dimers is impaired in its ability to regulate IL-4 expression. Moreover, the human IL-4 promoter exists in multiple allelic forms that exhibit distinct transcriptional activities in IL-4-positive T cells. Nucleotide polymorphisms leading to increased binding of transcription factors including NFAT and AP-1 and enhanced IL-4 transcription are associated with atopy, which predisposes individuals to develop asthma. Finally, inhibition of AP-1 by a small inhibitor molecule that targets AP-1 without affecting NFkB significantly reduces IL-4 levels as well as airway eosinophil infiltration, mucus hypersecretion, and edema in a mouse asthma model. The expression of IL-5 and IL-13 is also regulated by AP-1 proteins; nevertheless, IL-5 expression is regulated in a unique manner as its induction can occur independent of the induction of other cytokines and requires de novo synthesis of AP-1 proteins. AP-1 proteins are also involved in airway remodeling characterized by features such as epithelial injury, mucous gland hypertrophy, lumina reticularis thickening, airway smooth muscle hyperplasia, and angiogenesis. The expression of matrix metalloproteinases (MMPs) such as MMP-9 and MMP-3, which are elevated in asthmatics and affect both inflammation and tissue remodeling, is regulated by AP-1. In addition, the increase in proliferation of airway smooth muscle cells (ASMs) observed in asthmatic patients stems from integration of various signals that converge in the activation of p21Ras. p21Ras, in turn, activates ERK MAPK signaling that enhances c-Fos expression, which is followed by enhancement of expression of CyclinD1, a known AP-1 target. Thus, AP-1 might also contribute to an increase in airway smooth muscle mass (Figure 2). Given the involvement of AP-1 in respiratory diseases, it should be mentioned that major anti-inflammatory effects of corticosteroids are mediated by

Asthma

ASM proliferation MMPs ↑ Tissue remodeling ↑

AP-1 ↑

Th2 cytokines ↑ (IL-4, IL-3, IL-13) MMPs ↑ Inflammation ↑

Asthma enhancement & perpetuation Figure 2 AP-1 is involved in asthma development. AP-1 participates in both pathological features characterizing asthma development: enhancement of expression of Th2 cytokines and airway tissue remodeling.

interactions of activated corticosteroid receptors (GRs) with transcription factors such as AP-1 and NF-kB and abolishment of their proinflammatory effects. Increase in AP-1 levels or reduced ability of GRs to interact and repress AP-1 activity may, in certain cases, account for development of corticosteroid resistance.

Summary Despite the complexity involved in studying AP-1 the role of individual AP-1 proteins is starting to be elucidated and their significance in the development of different tissues observed. It seems that many AP-1 proteins play a role in regulation of proliferation, response to stress conditions, and inflammation. Frequently, the effects elicited by the same protein under different conditions are not identical. The aims of future research will be to understand the entire spectrum of AP-1 activities and the mechanisms that regulate it. By obtaining this knowledge the destructive effects of unregulated expression of AP-1 can be coped with more efficiently. See also: Apoptosis. Asthma: Overview. Interleukins: IL-4; IL-5; IL-13. Matrix Metalloproteinases. Oncogenes and Proto-Oncogenes: jun Oncogenes. Transcription Factors: NF-kB and Ikb. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Ameyar M, Wisniewska M, and Weitzman JB (2003) A role for AP-1 in apoptosis: the case for and against. Biochimie 85(8): 747–752. Angel P and Karin M (1991) The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochimica et Biophysica Acta 1072(2–3): 129–157.

TRANSCRIPTION FACTORS / ATF 257 Barnes PJ and Adcock IM (1998) Transcription factors and asthma. European Respiratory Journal 12(1): 221–234. Chinenov Y and Kerppola TK (2001) Close encounters of many kinds: Fos–Jun interactions that mediate transcription regulatory specificity. Oncogene 20(19): 2438–2452. Cohn L, Elias JA, Chupp GL, et al. (2004) Asthma: mechanisms of disease persistence and progression. Annual Review of Immunology 22: 789–815. Eferl R and Wagner EF (2003) AP-1: a double-edged sword in tumorigenesis. Nature Reviews: Cancer 3(11): 859–868. Florin L, Hummerich L, Dittrich BT, et al. (2004) Identification of novel AP-1 target genes in fibroblasts regulated during cutaneous wound healing. Oncogene 23(42): 7005–7017. Folkman J (2004) Angiogenesis and c-Jun. Journal of the National Cancer Institute 96(9): 644. Gerald D, Berra E, Frapart YM, et al. (2004) JunD reduces tumor angiogenesis by protecting cells from oxidative stress. Cell 118(6): 781–794. Rahman I (2003) Oxidative stress, chromatin remodeling and gene transcription in inflammation and chronic lung diseases. Journal of Biochemistry and Molecular Biology 36(1): 95–109. Raivich G, Bohatschek M, Da Costa C, et al. The AP-1 transcription factor c-Jun is required for efficient axonal regeneration. Neuron 43(1): 57–67. Shaulian E and Karin (2002) AP-1 as a regulator of cell life and death. Nature Cell Biology 4(5): E131–E136. Shaulian E, Schreiber M, Piu F, et al. (2000) The mammalian UV response: c-Jun induction is required for exit from p53-imposed growth arrest. Cell 103: 897–907. Wagner EF (2002) Functions of AP1 (Fos/Jun) in bone development. Annals of Rheumatic Diseases 61(supplement 2): ii40–ii42. Whitmarsh AJ and Davis RJ (1996) Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. Journal of Molecular Medicine 74(10): 589–607.

Introduction The term activating transcription factor (ATF) was first used in 1987 to refer to an activity that binds to the ATF consensus site TGACGT(C/A) (G/A) on the adenovirus early promoters E2, E3, and E4. cAMP responsive element binding protein (CREB) was named in the same year to refer to an activity that binds to the cAMP responsive element (CRE) ‘TGACGTCA’ on the somatostatin promoter. The fact that both these consensus binding sites had the same sequence but for two seemingly different promoter elements
one on viral promoters and the other on cellular promoters
generated much confusion initially. Later, the names ATF and CREB were used to refer to a group of bZip proteins that conform to the following criteria: (1) they bind to the consensus ATF/CRE sequence ‘TGACGTCA’ in vitro; and (2) they form homo- or heterodimers. Over the years, more than 10 different mammalian ATF/CREB cDNAs have been isolated and these can be divided into subgroups based on their amino acid similarity: the CREB/CREM, CREBP1 (commonly known as ATF2), ATF3, ATF4, ATF6, and B-ATF subgroups. Proteins within each subgroup share significant similarity both inside and outside the bZip domain. Proteins from different subgroups, however, do not share much similarity other than the bZip ‘motif’. Therefore, they should be considered as distinct proteins despite their common prefix (ATF or CREB). Table 1 lists the subgroups and their members. Because these cDNAs were isolated by a number of researchers with

ATF T Hai, D Lu, and C C Wolford, Ohio State University, Columbus, OH, USA

Table 1 The mammalian ATF/CREB family of transcription factors

& 2006 Elsevier Ltd. All rights reserved.

Subgroup

Members

Alternative names

CREB

CREB CREMa ATF1

CREB-1, CREB-327, ATF47

Abstract The mammalian activating transcription factor (ATF)/cAMP responsive element binding protein (CREB) family of transcription factors represents a group of basic region-leucine zipper (bZip) proteins that were originally defined in the late 1980s by two criteria: (1) they bind to the consensus ATF/CRE site ‘TGACGTCA’ in vitro; and (2) they form homo- or heterodimers. Over the years, more cDNAs encoding proteins that fulfill the above criteria have been isolated. The names of these cDNAs vary depending on the researchers who made the isolation and on the history of discovery, rather than the function of these proteins. Thus far, more than 50 bZip proteins have been identified. In this article, the classification of the ATF proteins is described and then they are compared with other bZip proteins. This is followed by a review of four ATF proteins: ATF2, ATF3, ATF4, and ATF6. Despite the diversity in the functions of these proteins, one common theme is their involvement in stress responses; their potential role in respiratory diseases is discussed.

CRE-BP1

CRE-BP1

ATF3

ATFa CREBPA ATF3 JDP2 ATF4

ATF4

ATF6 B-ATF a

ATF4L1 ATFx ATF6 CREB-RP B-ATF JDP1

ATF-43, TREB, TREB36, TCRATF1 ATF2, CREB-2, TCR-ATF2, mXBP, TREB, HB16 ATF7 LRF-1, LRG-21, CRG-5, TI-241 CREB2, TAXREB67, mATF4, C/ATF, mTR67 hATF5 ATF6a ATF6b, CREBL1, G13 p21SNFT, DNAJC12

The CREM gene also encodes a protein product ICER from an alternative intronic promoter.

258 TRANSCRIPTION FACTORS / ATF

different perspectives, the nomenclature in the literature for these proteins has been confusing. Not only are alternative names used to refer to the same protein, but also in some cases the same name is used to refer to different proteins. For example, ATF3, LRF-1, LRG-21, CRG-5, and TI-241 all refer to the same protein. However, CREB2 has been used to refer to three different proteins: an alternatively spliced CREB, CRE-BP1 (ATF2), and ATF4. Therefore, the only way to be sure of the identity of a given protein is to establish its amino acid sequence and not rely on its name alone. Although the term ‘activating transcription factor’ implies that these proteins activate transcription, some ATFs (such as ATF3) are transcriptional repressors. This misnomer reflects the prevailing concept in the mid-1980s that a given DNA site is recognized by a single protein. Since deletion of the ATF/CRE site from the corresponding promoters reduced their activity, it was deduced that the binding protein must be an activator. Now, it is clear that binding sites are recognized by multiple proteins. It is not surprising that some binding proteins are transcriptional activators whereas others are suppressors, allowing both positive and negative modulation of the promoters. In the following sections, ATF/CREB is compared with other bZip proteins and four ATF proteins (ATF2, ATF3, ATF4, and ATF6) are described with a focus on one aspect of their function
stress response.

ATF versus Other bZip Proteins Although the ATF/CREB proteins were originally defined by their ability to form heterodimers and to bind to the same consensus sequence, three observations indicate that these are arbitrary criteria and the distinction between ATF/CREB and other bZip proteins is blurred. First, overwhelming evidence, including a recent comprehensive analysis of bZip association by coil–coil array, indicates that members of the ATF/CREB family form selective heterodimers with other bZip proteins such as the AP-1, C/EBP, and Maf families of proteins. Second, the AP-1 and Maf consensus binding sites are identical (TGACTCA), and only differ by one nucleotide from the ATF/ CREB consensus binding site (TGACGTCA). Third, dendrogram analysis of the bZip region indicates that some ATF proteins are more closely related to bZip proteins without the ATF prefix than those with the prefix. One reason for this apparent discrepancy is that the original ATF clones (ATF1–ATF8) were isolated based on their ability to bind to the sequence TGACGTCA, not on their amino acid sequence

similarity. Therefore, the name ATF reflects the history of discovery, rather than the similarity between them. With the bZip DNA binding domain, these proteins have evolved to bind to a subset of sequences on the genome. However, by having other functional domains, they can interact with different proteins and exert a diversity of functions. The formation of heterodimers with other bZip proteins further expands their flexibility and versatility to regulate gene expression. One common theme of most bZip proteins identified thus far is their involvement in responding to extra- and/or intracellular stimulation. Four ATF proteins, ATF2, ATF3, ATF4, and ATF6, are reviewed below with a focus on their function in stress response.

ATFs in Stress Response ATF4 has been well documented to play a role in endoplasmic reticulum (ER) stress response. The ER is the organelle for proper folding and processing of proteins destined for plasma membrane or secretion. When unfolded or misfolded proteins accumulate in the ER, the cell initiates a complex and well-choreographed response – the ER stress response or unfolded protein response (UPR)
to transmit the signals from the ER to the nucleus and cytoplasm. One result of the ER stress response is activation of the eIF2a kinase PEK (also called PERK or EIF2AK3), which phosphorylates the translational initiation factor eIF2a. eIF2a phosphorylation attenuates the translation of most mRNAs to reduce the burden on the ER but at the same time increases the translation of specific mRNAs, including the ATF4 mRNA. ATF4, in turn, turns on the expression of many downstream genes, presumably to help the cell cope with the stress. The preferential upregulation of ATF4 protein synthesis by eIF2a phosphorylation puts ATF4 in a critical position in cellular stress response. Because other stress pathways, such as viral infection and nutrient deficiency, also lead to eIF2a phosphorylation, ATF4 serves as an integration point for various stress responses. ATF6 has also been well documented to play a role in ER stress response. It is synthesized as a precursor protein, which binds to the ER chaperone Bip/grp78 and localizes on the ER membrane. During ER stress, ATF6 dissociates from Bip/grp78 and translocates to the Golgi where it is cleaved by proteases to liberate its active N0 -terminal domain (ATF6N or p50). ATF6N is then translocated to the nucleus and upregulates its target genes, including those encoding ER chaperone proteins. Significantly, ATF6N can also downregulate gene expression: it binds to the sterol regulatory element-binding protein 2 (SREBP2) and

TRANSCRIPTION FACTORS / ATF 259

represses the target genes of SREBP2, resulting in the attenuation of the lipogenic effects of SREBP2. This action has significant physiological implications, since prolonged nutrient deprivation, such as amino acid or glucose deficiency, induces ER stress. By activating ATF6, the cells reduce the lipogenic effect of SREBP2, and thus save energy sources to withstand the stress. Therefore, ATF6 plays an important role in ER stress and in coordinating stress response with energy homeostasis. ATF2 is a ubiquitously expressed protein with the highest levels found in the brain. Upon exposure to a variety of stress signals, the mitogen-activated protein-kinase (MAPK) cascades including the p38 and JNK/SAPK pathways are activated, resulting in the dual phosphorylation of ATF2 at Thr69 and Thr71. The consequence of this dual phosphorylation is an increase in the half-life and transactivation activities of ATF2. Since p38 and JNK/SAPK are activated by many stress signals (such as genotoxic agents, inflammatory cytokines, UV irradiation, and osmotic stress), ATF2 is well recognized to play an important role in cellular stress response. Another recognized function of ATF2 is its involvement in oncogenesis. ATF2 is induced by growth-stimulating factors via a Ras- and p38-dependent pathway, and is thought to affect the function of oncoproteins, such as c-Jun, c-Myc, E1A, and Tax. As an example, ATF2 dimerizes with c-Jun and may affect Jun-dependent cellcycle progression, cell survival, and apoptosis. ATF3 is also a stress-inducible gene. Overwhelming evidence from many laboratories indicates that

ATF3 is induced by a variety of stress signals. However, it differs from the above three ATFs in its mode of induction: ATF2 is induced by posttranslational modification – phosphorylation by stress kinases; ATF4 is induced by translational regulation – increased translation of the ATF4 mRNA upon eIF2a phosphorylation; ATF6 is induced by posttranslational modification
proteolytic cleavage that allows subcellular translocation. ATF3, on the other hand, is induced at the mRNA level; its steady-state mRNA level is usually not detectable but greatly increases upon exposure of the cells to stress signals. Because of this feature, ATF3 has been identified in many screens using DNA microarray, a technique comparing the steady-state mRNA levels of cells under different conditions. One dramatic feature of ATF3 induction is that it is neither tissue specific nor stimulus specific. This ‘non-specificity’ is not unique to ATF3. Other genes such as Fos, Jun, and Erg-1 are also induced by a variety of signals in many different tissues. Therefore, the initial genome response to various seemingly unrelated signals appears to be the turning on of a set of common genes, irrespective of the nature of the signals or the type of cells. It suggests that the context of the cells would determine how cells ‘interpret’ the signals to give rise to a diversity of outcomes. Despite its well-documented induction, the functional consequence of ATF3 expression is not clear. Based on the current literature, we hypothesize the following. ATF3 modulates the cell cycle and cell death machinery. Under certain cellular contexts, it

Pathological state Genotoxic stress Ca2+ depletion

Viral infection Inflammation Injury

Nutrient depletion Oxidative stress

Toxins

Hypoxia ATF6 ER Stress kinase

NF-
B pathway

ATF4

ATF2/Jun

Nucleus

PEK

ATF3

ATF6N Golgi

and other transcriptional targets

Adaptive responses Maladaptation leads to the development of diseases Figure 1 A schematic diagram for ATF proteins in stress response and adaptation. Two features in the figure are not described in the text: the involvement of the NF-kB pathway in the induction of ATF3 by stress signals and the regulation of ATF3 gene expression by itself.

260 TRANSCRIPTION FACTORS / Fox

leads to tissue dysfunction and the development of diseases. In the context of already transformed cancerous cells, however, ATF3 promotes metastasis.

Potential Roles of ATFs in Respiratory Diseases In conclusion, all four ATF proteins reviewed above are involved in the adaptive responses to cope with stress conditions. Interestingly, ATF4 has been demonstrated to be required for the induction of ATF3 by ER stress and ATF2 has been shown to transactivate the ATF3 promoter, indicating interplays among these ATF proteins. Figure 1 shows a schematic diagram for ATF proteins in stress response. Since proper adaptation to stress conditions is critical to the function of cells, it is conceivable that these ATF proteins play an important role in the pathophysiology of many diseases. Indeed, some circumstantial evidence suggests a potential role for this family of proteins in pulmonary diseases. The surfactant protein A and B promoters contain ATF/CRE sites. Specifically, ATF2 and CREB were demonstrated to be induced in the intact lung tissue by metallic particulates, and ATF2 was shown to regulate the surfactant protein B promoter. Furthermore, knockout mice deficient in ATF2 display severe newborn respiratory distress syndrome, the major cause of neonatal morbidity and mortality in developed countries. Therefore, members of the ATF/CREB family of proteins are involved in various stress responses and may be key players in the pathogenesis of stress-associated lung diseases. See also: Oncogenes and Proto-Oncogenes: Overview; jun Oncogenes; MYC; RAS. Transcription Factors: Overview.

Further Reading Allen-Jennings AE, Hartman MG, Kociba GJ, and Hai T (2002) The roles of ATF3 in liver dysfunction and the regulation of phosphoenolpyruvate carboxykinase gene expression. Journal of Biological Chemistry 277: 20020–20025. Hai T and Hartman MG (2001) The molecular biology and nomenclature of the ATF/CREB family of transcription factors: ATF proteins and homeostasis. Gene 273: 1–11. Hai T, Wolfgang CD, Marsee DK, Allen AE, and Sivaprasad U (1999) ATF3 and stress responses. Gene Expression 7: 321–335. Harding HP, Novoa I, Zhang Y, et al. (2000) Regulated translation initiation controls stress-induced gene expression in mammalian cells. Molecular Cell 6: 1099–1108. Harding HP, Zhang Y, Zeng H, et al. (2003) An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Molecular Cell 11: 619–633. Hartman MG, Lu D, Kim ML, et al. (2004) Role for activating transcription factor 3 in stress-induced beta-cell apoptosis. Molecular and Cellular Biology 24: 5721–5732.

Jiang HY, Wek SA, McGrath BC, et al. (2004) Activating transcription factor 3 is integral to the eukaryotic initiation factor 2 kinase stress response. Molecular and Cellular Biology 24: 1365–1377. Maekawa T, Bernier F, Sato M, et al. (1999) Mouse ATF-2 null mutants display features of a severe type of meconium aspiration syndrome. Journal of Biological Chemistry 274: 17813–17819. Newman JRS and Keating AE (2003) Comprehensive identification of human bZIP interactions with coiled-coil arrays. Science 300: 2097–2101. Okamoto Y, Chaves A, Chen J, et al. (2001) Transgenic mice expressing ATF3 in the heart have conduction abnormalities and contractile dysfunction. American Journal of Pathology 159: 639–650. Shen J, Chen X, Hendershot L, and Prywes R (2002) ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Developmental Cell 3: 99–111. van Dam H and Castellazzi M (2001) Distinct roles of Jun: Fos and Jun: ATF dimers in oncogenesis. Oncogene 20: 2453– 2464. Zeng L, Lu M, Mori K, et al. (2004) ATF6 modulates SREBP2mediated lipogenesis. EMBO Journal 23: 950–958.

Fox V V Kalinichenko, University of Chicago, Chicago, IL, USA R H Costa, University of Illinois – Chicago, Chicago, IL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The Forkhead box (Fox) proteins are an extensive family of transcription factors, which share homology in the winged helix/forkhead DNA-binding domain. Mutations of the Fox genes elicit pronounced defects of cellular proliferation, differentiation, and metabolic homeostasis, leading to developmental abnormalities and human disease. In this review we will focus on Fox family members that contribute to lung development and disease. Deletion of the Foxa2 (HNF-3b) gene from respiratory epithelial cells is associated with airspace enlargement, goblet cell hyperplasia, and neutrophilic infiltration. Likewise, increased expression of Foxa2 in the distal respiratory epithelium caused a striking inhibition in branching morphogenesis and vasculogenesis of the lung. Foxj1 (HFH-4) deficient (
/
) mice display perinatal lethality due to defective differentiation of ciliated epithelial cells lining the pulmonary bronchioles and cerebral ventricles, leading to defects in lung function and hydrocephalus. Haploinsufficiency of the Foxf1 (HFH-8) gene causes fusion of the lung lobes and abnormal development of peripheral lung capillaries leading to perinatal pulmonary hemorrhage in 50% of the newborn Foxf1 þ /
mice that displayed an 80% reduction in lung expression levels of Foxf1. Earlier expression of the Foxm1b (HFH-11b) protein in transgenic mice following lung injury accelerates the onset of proliferation of different lung cell types. This partial list of genetic studies examining Fox proteins underscores the importance of this family in regulating genes involved in lung morphogenesis, respiratory diseases, and lung repair.

TRANSCRIPTION FACTORS / Fox

Introduction Drosophila homeotic forkhead and rodent hepatocyte nuclear factor 3 (HNF-3) proteins were the first members of this extensive family of transcription factors to be identified. These proteins share greater than 90% homology in the winged helix/forkhead DNA-binding domain sequences. The name ‘forkhead’ derives from two headlike structures in forkhead mutant Drosophila embryos that exhibited defects in the formation of anterior and posterior gut. Using polymerase chain reaction (PCR) amplification of rodent and human organ cDNA with primers made to conserve amino acid sequences in the winged helix DNA-binding domain, a number of new family members were isolated in the lung and other tissues. Recently, the winged helix/forkhead genes have been renamed the Forkhead box (Fox) family, and its 55 distinct mammalian members grouped into 17 subfamilies (designated as A–Q) with respect to their sequence homology within the winged helix/forkhead domain. Recent transgenic and gene knockout studies that facilitate our understanding of the role of Fox genes in lung development, function, and lung diseases are summarized below.

Structure Fox proteins are potent transcriptional activators, which contain an evolutionarily conserved 100 amino acid winged helix DNA-binding domain that allows these proteins to bind to their DNA targets as monomers. The Foxa proteins contain N-terminal and C-terminal transcriptional activation domains, which are critical for mediating protein interactions with other transcription factors and transcriptional coactivator proteins required to stimulate gene transcription (Figure 1). The Foxa C-terminal transcriptional activation domains contain the functionally important region II and III sequences, which are conserved in other Fox proteins. Interestingly, recent studies demonstrated that the region II sequences in the Foxa2 and Foxg1 C-terminal domain can associate with the human homolog of the Drosophila Groucho transcriptional repressor proteins, which suggests the possibility that Fox proteins may also function to inhibit transcription in cell types expressing Groucho proteins. A functional analysis of several Fox proteins demonstrated that the nuclear localization function resides at both ends of the winged helix DNA-binding domain. Fox proteins exhibit strong amino acid identity in the recognition helix 3 of the winged helix DNAbinding domain, but each Fox transcription factor is

IV/V TAD

Foxa1 14 Foxa2

93

Winged helix domain (NLS) I 100% 168 268

TAD 14

157

257 54%

45 1

466

361

TAD 458 376

TAD 275 421

39% 236

Foxp1

379

TAD 145 180 247

TAD 47% 47 110 119 219

Foxm1b

III TAD

94% 93

Foxf1 Foxj1

II

261

TAD 336 365

Zn TRD 36% 251 336 359 491 591

749

705

Figure 1 Functional protein domains of the lung Fox transcription factors. Schematically shown is the structural homology and functional protein domains of the six lung Forkhead/winged helix (Fox) transcription factors. Percent amino acid homology in DNAbinding domains (shown in green) is presented relative to Foxa1 protein. Fox DNA-binding domains contain the nuclear localization signal (NLS). Indicated on the Foxa proteins are their conserved N-terminal (purple) and C-terminal (white) transcriptional activation domains (TAD), latter of which contains conserved region II (red) and region III (orange). Also shown are the transcriptional activation domains (various colors) for Foxf1, Foxj1, Foxm1b, and Foxp1 proteins. TAD, transcriptional activation domain; TRD, transcriptional repressor domain; Zn, zinc-finger domain.

capable of interacting with distinct DNA-binding sites. Structure–function studies using chimeric winged helix recombinant proteins demonstrated that a 20 amino acid region adjacent to the recognition helix could alter DNA binding specificity and these amino acid sequences are the most divergent within the winged helix DNA-binding domain. Structural NMR studies have demonstrated that this divergent 20 amino acid region in the winged helix domain is involved in a distinct presentation of the conserved recognition helix into the major groove of DNA.

Expression, Biological Function, and Fox Target Genes Foxa Genes

Foxa1 and Foxa2 genes are coexpressed in epithelial cells of the bronchioles and peripheral lung (type II cells) (Table 1). The Foxa1 and Foxa2 proteins are functionally redundant because they share 94% homology in their DNA-binding domain, bind to nearly identical sequences, and will regulate transcription of similar target genes during lung morphogenesis (Figure 2). The Foxa1
/
mice exhibit normal lung development, but they display hypoglycemia due to

262 TRANSCRIPTION FACTORS / Fox Table 1 Expression patterns of Fox genes in the lung Gene

Mouse embryonic expression pattern

Adult expression pattern

Foxa1 Foxa2 Foxf1

Foregut endoderm and endoderm-derived epithelial cells of developing trachea and lung Splanchnic mesenchyme of foregut, mesenchyme of trachea and lung Ciliated epithelial cells of the proximal airways (trachea, bronchi, bronchioles), nose and paranasal sinuses Proliferation-specific expression pattern in all embryonic tissues Proximal and distal lung epithelium, lung mesenchyme Distal lung epithelium Proximal and distal lung epithelium

Bronchiolar and alveolar type II epithelial cells of the lung

Foxj1 Foxm1b Foxp1 Foxp2 Foxp4

Alveolar endothelial cells and smooth muscle surrounding pulmonary airways Ciliated bronchiolar epithelial cells Expression is reactivated following lung injury Vascular endothelial cells of the lung Type II epithelial cells of the lung Proximal and distal lung epithelium

Expression patterns were determined by immunohistochemistry and in situ hybridization of embryonic and adult mouse tissues. Only Fox expression in respiratory tissues is shown.

Foregut endoderm

Foxa2

Proximal epithelium precursors P

r

o

Foxa1 Foxa2

Splanchnic mesenchyme

Foxf1

Distal epithelium precursors

l

i

f

e

e

r

e

n

Foxj1

r

a

t

i

o

n

i

a

t

i

o

Foxm1 D

i

f

f

Ciliated cells Type II cells

Clara cells Nonciliated columnar cells

t

Endothelial cells Airway smooth muscle cells Vascular smooth muscle cells

Type I cells

Goblet cells

n

Foxf1

Fibroblasts

Foxa2 Figure 2 Schematic diagram showing the regulation of lung development by Fox transcription factors. Embryonic foregut endoderm and splanchnic mesenchyme undergo proliferation and differentiation to form the lung epithelial and mesenchymal cell lineages, respectively. The role of Fox genes in lung morphogenesis was determined by mouse genetic studies and shown with red arrows.

reduced pancreatic expression of glucagon, which is required to break down glycogen in the liver. Foxa2
/
embryos die in utero prior to lung development due to severe defects in the formation of embryonic endoderm, mesoderm, and ectoderm during mouse embryogenesis (Table 2). Mice with a conditional deletion of the Foxa2 allele in developing lung epithelial cells display airspace enlargement, goblet cell hyperplasia, increased mucin, and neutrophilic infiltration. Functional promoter studies demonstrated an important role for Foxa1 and Foxa2 proteins in regulating transcription of surfactant protein C (SPC), SPB, and Clara cell secretory protein (CCSP) genes required for bronchiolar and type II epithelial cell function. Foxa2 also regulates promoter expression of the Nkx homeodomain transcription factor TTF-1, which regulates transcription

of the surfactant protein genes. During lung development, Foxa2 protein is expressed at higher levels in epithelial cells lining the proximal airways and at lower levels in the distal type 2 epithelial cells. Transgenic SPC-Foxa2 mice were generated that express high levels of Foxa2 in the distal airway epithelial cells, which disrupted the normal decreasing gradient of Foxa2 in these cells during lung development. Increased expression of Foxa2 in the distal respiratory epithelium caused a striking inhibition in branching morphogenesis and vasculogenesis (development of new blood vessels) of the lung, coinciding with diminished expression of the cell adhesion E-cadherin protein and vascular endothelial growth factor (VEGF) in these cells. These transgenic mouse studies indicated that maintaining precise levels of Foxa2 is of critical importance in branching lung

TRANSCRIPTION FACTORS / Fox

263

Table 2 Lung phenotypes in Fox knockout and transgenic mice Gene

Phenotype of Null (
/
) mice

Phenotype of transgenic mice

Foxa2

Foxa2
/
embroys die in utero by 10 dpc due to defects in formation of node, notochord, and gut endoderm. Conditional Foxa2 deletion using SPC or CC10-driven Cre recombinase caused goblet cell hyperplasia and neutrophilic infiltration in the lung Foxf1
/
embroys die in utero by 8.5 dpc due to defects in extraembryonic mesoderm and alantois. Foxf1 þ /
mice exhibit fusion of lung lobes, defects in formation of lung capillaries and gall bladder abnormalities. 50% of Foxf1 þ /
mice die perinatally. Adult Foxf1 þ /
mice display defects in lung repair Foxj1
/
mice exhibit perinatal lethality due to lung dysfunction and hydrocephalus. Defective ciliogenesis in airway epithelial cells and brain ventricles.

Transgenic mice with SP-C-driven Foxa2 transgene display defects in respiratory epithelial cell differentiation, branching morphogenesis, and vasculogenesis, which was associated with decreased expression of E-cadherin and VEGF Not generated

Foxf1

Foxj1

Foxm1b

Foxm1b
/
mice die in utero by 17.5 dpc due to defects in hepatoblast and cardiomyocyte proliferation leading to accumulation of polyploid cells in the liver and heart. Defect in formation of intrahepatic bile ducts

Transgenic mice with SP-C-driven Foxj1 transgene display defects in pulmonary epithelial cell differentiation with atypical columnar cells lining the distal airways. Defect in expression of SP-B, SP-C, and CCSP Transgenic mice with Rosa26-driven Foxm1b transgene develop normally but display premature proliferation of lung epithelia, endothelial, and smooth muscle cells following butylated hydroxytoluene (BHT) lung injury

Lung phenotypes were observed in genetically altered transgenic (gain of function mutants) and knockout mice (loss of function mutants). dpc, days post coitum.

morphogenesis and epithelial differentiation. Although Foxa3 is also expressed in gut endoderm during embryonic development, Foxa3 is not expressed in the developing lung. Foxf Genes

Foxf genes are expressed in mesenchyme of the oral cavity, trachea, lung, intestine, and liver, and in the astrocytes of the brain. Foxf1
/
mice die prior to lung development due to defects in extraembryonic mesoderm and allantois (umbilical cord). Foxf1þ /
mice exhibit defects in branching lung morphogenesis and septation (formation of alveoli) and abnormal development of peripheral pulmonary capillaries leading to perinatal pulmonary hemorrhage, suggesting that wild-type levels of Foxf1 are necessary for normal lung development. Foxf1 regulates expression of genes critical for lung morphogenesis, mesenchyme proliferation, mesenchyme-epithelial signaling, and angiogenesis/vasculogenesis including: platelet derived growth factor (PDGF) receptors, which are required for alveolar structure formation; receptor tyrosine kinase Tie-1, platelet endothelial cell adhesion molecule-1 (Pecam-1), VEGF receptor 2 (Flk-1), and vascular endothelial cadherin, which are required for endothelial cell function and assembly of endothelial cells into functional blood vessels. In addition, Foxf1 regulates transcription of the hepatocyte growth factor (HGF) and bone morphogenetic protein-4 (BMP-4) genes, expression of which is

critical for mesenchyme-epithelial signaling and proximal-distal patterning of the lung, and the Notch-2 receptor, which is essential for cell differentiation and survival. Functional promoter studies suggest that Foxf1, which is constitutively expressed in alveolar endothelial cells and peribronchiolar smooth muscle cells, may participate in cell-typespecific activation of P-selectin in response to cytokines. Moreover, Foxf1 potentially regulates expression of cytokine and chemokine genes that are involved in recruiting inflammatory cells, including interleukin-8 (IL-8), IL-1a, monocyte chemoattractant protein-1 (MCP-1), and eotaxin. Although the related Foxf2 protein is also expressed in lung mesenchyme, Foxf2
/
mice do not display lung abnormalities, but die at birth due to cleft palate and nursing defects. Foxj Genes

Foxj1 (HFH-4) is expressed in ciliated epithelial cells during mouse embryogenesis; Foxj1
/
mice lacked staining for left-right dynein protein and 9 þ 2 microtubules (motile type of cilia) in proximal epithelial cells of the lung and in the ventricles of the brain. The Foxj1
/
mice displayed perinatal lethality because they were deficient in ciliated epithelial cells lining the proximal pulmonary bronchioles and ventricles of the brain, leading to defects in lung function and hydrocephalus. Foxj1 is also transiently expressed in the monociliated cells of the node, which

264 TRANSCRIPTION FACTORS / Fox

plays an important role in orchestrating gastrulation during formation of the embryonic germ layers. Although cilia were present on the Foxj1 deficient node cells, Foxj1 expression is required for left-right asymmetry of the internal organs. Foxj1
/
mice exhibit randomized situs inversus of their internal organs (internal organs display either left or right asymmetry). Transgenic mouse studies in which the Foxj1 protein was ectopically expressed in the distal respiratory epithelial cells using SPC promoter resulted in defects of lung branching morphogenesis. Moreover, the distal airways were lined with atypical cuboidal or columnar epithelial cells, which expressed TTF-1 and Foxa2, but these cells no longer expressed nonciliated epithelial marker genes including SP-C, SP-B, and CCSP. Ectopic expression of Foxj1 in developing mouse lung therefore promoted differentiation toward the ciliated epithelial cellular lineage and inhibited expression of nonciliated epithelial marker genes. Foxm1 Genes

The proliferation-specific Foxm1b (HFH-11, trident) protein is essential for normal embryonic development because Foxm1b
/
embryos exhibit severe defects in liver and heart development. Consistent with a role in mediating cell-cycle progression, Foxm1b
/
embryos display an abnormal polyploid phenotype in embryonic hepatocytes and cardiomyocytes, indicating that Foxm1b expression is required for progression of replicating cells into mitosis. Although Foxm1b is not expressed in adult lungs, lung injury reactivates Foxm1b expression in proliferating epithelial and endothelial cells during the period of lung repair. Furthermore, premature expression of the Foxm1b transgenic protein using ubiquitous Rosa26 promoter accelerates proliferation of different lung cell types following lung injury, including alveolar type II epithelial cells, bronchial epithelial and smooth muscle cells, and endothelial cells of pulmonary capillaries and arteries. This was associated with the earlier expression of the cell-cycle promoting genes and diminished protein levels of cyclin dependent kinase (Cdk) inhibitor p21Cip1, suggesting that increasing Foxm1b levels is an effective means to stimulate cellular proliferation during aging and in lung diseases such as emphysema. Foxo Genes

In contrast to the Foxm1b protein, the Foxo subfamily are negative regulators of the cellular proliferation and includes Foxo1 (FKHR), Foxo3a (FKHRL1), and Foxo4 (AFX1) proteins. Foxo

proteins are ubiquitously expressed and their activity is inhibited by the insulin/insulin-like growth factor 1 (IGF1) signaling pathway. Foxo proteins can be directly phosphorylated and inactivated by protein kinase B (PKB or c-Akt), which mediates their nuclear export to the cytoplasm. Foxo proteins stimulate expression of the p27Kip1 Cdk inhibitor and the retinoblastoma (RB)-related p130 proteins, which inhibits progression of cells into DNA replication. Furthermore, Foxo proteins regulate transcription of antiapoptotic (programed cell death) genes and therefore IGF1 signaling promotes cell survival by inhibiting activity of the Foxo proteins. Foxp Genes

Although most of the Fox proteins bind their target DNA as monomers, Foxp proteins bind to their DNA target sequence as dimers and form homodimers through a leucine zipper motif. Foxp genes are expressed in the lung, brain, and gut tissues during embryonic development. In developing lung, Foxp2 and Foxp4 expression is primarily restricted to pulmonary epithelial cells, whereas Foxp1 is observed in both epithelial and vascular endothelial cells of the lung. Mice carrying a loss-of-function mutation in Foxp3 (scurfy mice) display a fatal autoimmune disease due to hyperresponsive CD4 þ T cells. Mice that overexpress Foxp3 possess fewer mature T cells with reduced functional capabilities.

Fox Transcription Factors in Human Diseases Several mutations in Fox genes have been linked to human developmental disorders. For example, mutations in Foxc1, Foxc2, Foxe3, and Foxl2 cause various eye abnormalities. Mutations in Foxe1 and Foxn1 genes are linked to thyroid hypoplasia, cleft palate, and T-cell immunodeficiency. Foxp2 mutations are associated with speech and language disorders and Foxp3 deficiency causes polyendocrinopathy, immune deficiency, and enteropathy. Chromosomal translocations that involve Foxo proteins have been associated with human alveolar rhabdomyosarcoma and acute lymphoid leukemia. Based on recent mouse genetic studies, Fox proteins may provide new targets for clinical diagnosis and treatment of human lung malformations, lung cancer, and respiratory diseases. See also: Angiogenesis, Angiogenic Growth Factors and Development Factors. Epithelial Cells: Type II Cells. Gene Regulation. Lung Development: Overview. Surfactant: Surfactant Proteins B and C (SP-B and

TRANSCRIPTION FACTORS / NF-jB and Ijb 265 SP-C). Transcription Factors: Overview. Transgenic Models. Vascular Endothelial Growth Factor.

NF-jB and Ijb J P Mizgerd, Harvard School of Public Health, Boston, MA, USA

Further Reading Brody SL, Yan XH, Wuerffel MK, Song SK, and Shapiro SD (2000) Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice. American Journal of Respiratory Cell and Molecular Biology 23: 45–51. Burgering BM and Medema RH (2003) Decisions on life and death: FOXO Forkhead transcription factors are in command when PKB/Akt is off duty. Journal of Leukocyte Biology 73: 689–701. Carlsson P and Mahlapuu M (2002) Forkhead transcription factors: key players in development and metabolism. Developmental Biology 250: 1–23. Clevidence DE, Overdier DG, Tao W, et al. (1993) Identification of nine tissue-specific transcription factors of the hepatocyte nuclear factor 3/forkhead DNA-binding-domain family. Proceedings of the National Academy of Sciences, USA 90: 3948–3952. Costa RH, Kalinichenko VV, and Lim L (2001) Transcription factors in mouse lung development and function. American Journal of Physiology: Lung Cellular and Molecular Physiology 280: L823–L838. Kaestner KH, Knochel W, and Martinez DE (2000) Unified nomenclature for the winged helix/forkhead transcription factors. Genes and Development 14: 142–146. Kalinichenko VV, Gusarova GA, Tan Y, et al. (2003) Ubiquitous expression of the forkhead box M1B transgene accelerates proliferation of distinct pulmonary cell-types following lung injury. Journal of Biological Chemistry 278: 37888–37894. Kalinichenko VV, Lim L, Beer-Stoltz D, et al. (2001) Defects in pulmonary vasculature and perinatal lung hemorrhage in mice heterozygous null for the Forkhead box f1 transcription factor. Developmental Biology 235: 489–506. Kalinichenko VV, Major M, Wang X, et al. (2004) Forkhead box m1b transcription factor is essential for development of hepatocellular carcinomas and is negatively regulated by the p19ARF tumor suppressor. Genes and Development 18: 830–850. Korver W, Schilham MW, Moerer P, et al. (1998) Uncoupling of S phase and mitosis in cardiomyocytes and hepatocytes lacking the winged-helix transcription factor trident. Current Biology 8: 1327–1330. Mahlapuu M, Enerba¨ck S, and Carlsson P (2001) Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations. Development 128: 2397–2406. Shu W, Yang H, Zhang L, Lu MM, and Morrisey EE (2001) Characterization of a new subfamily of winged-helix/forkhead (Fox) genes that are expressed in the lung and act as transcriptional repressors. Journal of Biological Chemistry 276: 27488– 27497. Tichelaar JW, Lim L, Costa RH, and Whitsett JA (1999) HNF-3/ forkhead homologue-4 influences lung morphogenesis and respiratory epithelial cell differentiation in vivo. Developmental Biology 213: 405–417. Wan H, Kaestner KH, Ang SL, et al. (2004) Foxa2 regulates alveolarization and goblet cell hyperplasia. Development 131: 953–964. Zhou L, Dey CR, Wert SE, et al. (1997) Hepatocyte nuclear factor3b limits cellular diversity in the developing respiratory epithelium and alters lung morphogenesis in vivo. Developmental Dynamics 210: 305–314.

& 2006 Elsevier Ltd. All rights reserved.

Abstract Nuclear factor kappa B (NF-kB) transcription factors regulate gene expression directing inflammation and cellular survival. NF-kB complexes are homo- or heterodimers assembled from five structurally related proteins (RelA, RelB, c-Rel, p50, and p52). NF-kB is retained in the cytoplasm by physical interaction with inhibitor kappa B (IkB) proteins. Many cell stresses, including microbial stimuli and proinflammatory cytokines, activate signaling pathways that cause IkB proteins to be removed and degraded. NF-kB then accumulates in the nucleus, binds kB sequences in the DNA, recruits other nuclear proteins to chromatin, and regulates the expression of nearby genes, including those for cytokines, chemokines, anti-apoptotic proteins, immune effector proteins, and transcriptional regulatory proteins. NF-kB is activated in lung cells and leukocytes from patients with respiratory diseases, suggesting that NF-kB and IkB activities may contribute to their pathophysiology. In experimental settings, NF-kB is critical to pulmonary inflammation, respiratory infection, acute lung injury, asthma, and cancer. NF-kB function is an essential determinant of lung health and homeostasis.

Introduction Nuclear factor kappa B (NF-kB) was originally identified as a nuclear factor (NF) binding to a DNA sequence in the enhancer region of the gene for the k light chain in B cells (kB). Over the ensuing years, NF-kB was determined to be a family of transcription factors (Figure 1) that regulates the expression of many genes (in addition to k light chain) in many cells (in addition to B cells). In resting cells, NF-kB usually resides in the cytoplasm, bound to inhibitor proteins kappa B (IkB). Upon activation of cells by diverse stimuli, IkB proteins are degraded, allowing NF-kB dimers to accumulate in the nucleus, where they bind specific sequences (kB sites) in promoter regions and regulate gene transcription (Figure 2).

Structure There are five NF-kB protein subunits (Figure 1): RelA, RelB, c-Rel, p50, and p52. All five contain a stretch of 300 amino acids known as the Rel homology domain (RHD), which mediates dimerization (to other RHD-containing proteins), nuclear import (via a nuclear localization sequence, NLS), and DNA binding (to kB sites). Three of the NF-kB proteins (RelA, RelB, and c-Rel) also contain a transactivation domain-mediating transcriptional induction. IkB proteins (Figure 1) contain multiple copies of ankyrin

266 TRANSCRIPTION FACTORS / NF-jB and Ijb ReIA

(ReIA is also known as p65)

ReIB Legend

c-ReI

Protein

p50

Rel homology domain Transactivation domain

p105 p52

Ankyrin motif

p100

Cleavage site Serine targeted by I
B kinases (IKK)

I
B- I
B- I
B- Bcl-3

Figure 1 Constitutively expressed proteins of the NF-kB/IkB family. Domains that are biologically relevant and conserved across family members are depicted schematically.

Microbial factors (LPS, CpG DNA, etc.)

Cytokines (TNF, IL-1, etc.)

Receptors

IKK signalosome

NF-
B I
B

Nuclear proteins DNA

Regulation of gene transcription Chemokines (IL-8, MCP-1,etc.), adhesion molecules (ICAM-1, E-selectin, etc.), cytokines (TNF-, IL-6, etc.), enzymes (iNOS, COX2, etc.), antiapoptotic factors (c-IAP2, A1, etc.), transcription modifiers (I
B-, C/EBP-, etc.), and more. Figure 2 NF-kB and IkB control coordinated, integrated responses to stresses such as infection and injury.

motifs, 33 amino acid stretches that mediate protein– protein interactions. IkB proteins were so named because they bind to NF-kB dimers and inhibit NF-kB activity, typically by maintaining the dimers away from the DNA. Three IkB proteins that inhibit NF-kB are constitutively expressed (IkB-a, -b, and -e). Additional IkB proteins (not shown in figure) can inhibit NF-kB but require inducible expression. Precursor proteins to p50 and p52 (p105 and p100,

respectively) contain C-termini that are structurally and functionally analogous to IkB proteins (Figure 1), containing ankyrin repeats and inhibiting NF-kB activity. Bcl-3 is structurally analogous to the other IkB proteins, but functionally anomalous. Bcl-3 contains ankyrin motifs and binds NF-kB complexes (particularly homodimers of p50 or p52). However, Bcl-3 is a nuclear protein that does not interfere with DNA binding by NF-kB. Instead, Bcl-3 functions as

TRANSCRIPTION FACTORS / NF-jB and Ijb 267

a nuclear adapter protein, recruiting other sets of proteins (including transcription activating or repressing holocomplexes) to p50 and p52 homodimers bound to kB sites in the DNA.

Regulation of Production and Activity RelA and p50 are constitutively expressed in most nucleated cells. Expression of other NF-kB proteins is more restricted, and not clearly and consistently demonstrated. c-Rel is expressed in macrophages and lymphocytes. RelB and p52 are expressed in lymphoid tissues and B lymphocytes. Expression of RelB, c-Rel, and p105 is inducible, and regulated by NF-kB itself. Different dimers of NF-kB have unique expression patterns, mechanisms of regulation, and functional consequences. The most frequently observed (and studied) NF-kB complex is a heterodimer of RelA and p50, which is a strong transcriptional activator in most cases. IkB-a, -b, and -e are constitutively expressed in most tissues, including the lung. Bcl-3 is constitutively expressed in macrophages and lymphocytes. These and other IkB proteins can be induced by cell activation, often mediated by NF-kB transcription factors. NF-kB activity is prevented by IkB proteins. The IkB proteins inhibit NF-kB activity by at least two mechanisms. First, IkB proteins such as IkB-b can mask the NLS in the RHD when bound to NF-kB complexes, preventing the nuclear import of NF-kB. Second, IkB complexes such as IkB-a contain a nuclear export sequence that results in bound NF-kB complexes being actively exported from the nucleus to the cytoplasm. Diverse receptors (such as for cytokines or microbial molecules) and environmental stresses (such as reactive oxygen metabolites or ultraviolet irradiation) activate NF-kB, typically by removing the IkBmediated inhibition (Figure 2). IkB proteins that are phosphorylated on critical serine residues (Figure 1) become ubiquitinated on lysine residues (by btransducin repeat-containing protein) and then degraded (by the 26S proteasome). Similar to traditional IkB proteins, inhibition of NF-kB by p105 and p100 can be removed by phosphorylation, resulting in proteolysis of p105 and p100 to p50 and p52, respectively. Multiple kinases can phosphorylate IkB proteins. IkB kinase b (IKKb), within the multiprotein complex called the IKK signalosome, mediates this phosphorylation in response to many inflammatory stimuli. After liberation from IkB, NF-kB complexes are imported into the nucleus, likely mediated by karyopherins. NF-kB in the nucleus binds to kB sites

in the chromatin. By recruiting select nuclear proteins to the chromatin, NF-kB complexes regulate the transcription of nearby genes (Figure 2). One nuclear protein that may be recruited to the chromatin is the atypical IkB family member, Bcl-3. Other nuclear proteins interacting with NF-kB complexes include histone acetyl transferases, histone deacetylases, and transcription factors outside of the NF-kB family. NF-kB complexes are widely recognized as strong and important transcriptional activators, but evidence also supports roles for NF-kB in limiting or repressing gene transcription. Whether NF-kB complexes increase or decrease gene expression depends upon the nuclear proteins recruited to the chromatin, which in turn depends on signaling within the cell that causes posttranslational modifications such as kinase-mediated phosphorylations of NF-kB and other nuclear proteins. The gene for IkB-a is kB-regulated, and transcriptional activation by NF-kB induces IkB-a expression. Newly synthesized IkB-a can displace NF-kB from the DNA, facilitating its nuclear export and serving as a negative feedback loop. Posttranslational modifications of NF-kB proteins contribute to this regulation. For example, acetylated forms of RelA fail to bind IkB-a, and the removal of RelA-containing complexes from DNA by IkB-a requires deacetylation of RelA by histone deacetylase enzymes. Increased expression of other IkB proteins also contributes to terminating NF-kB activity. In addition, evidence is emerging that NF-kB proteins themselves are degraded by proteasomes and proteases, providing another mechanism for halting NF-kB activity.

Biological Function The function of NF-kB and IkB proteins is to regulate gene expression precisely. Many genes are regulated by NF-kB and IkB, including cytokines, chemokines, anti-apoptotic proteins, immune effector proteins, and transcriptional regulatory proteins (Figure 2). As such regulators, NF-kB and IkB are likely a means for reacting appropriately to a variety of stresses, including infection and injury. This regulation of gene expression is essential to homeostasis and health, as demonstrated by mice with targeted mutations in genes for NF-kB or IkB proteins. Preventing apoptosis is one important function of NF-kB. Null mutation of the gene for RelA is embryonic lethal. Cells cultured from RelA-deficient embryos are sensitive to apoptosis induced by tumor necrosis factor alpha (TNF-a), and crossing RelAdeficient mice into a mouse strain deficient in either TNF-a or TNF-a receptor 1 results in live births of mice without RelA. These data indicate that RelA is

268 TRANSCRIPTION FACTORS / NF-jB and Ijb

essential for preventing TNF-induced cell death during embryogenesis. Similar findings (gene interruption resulting in embryonic lethality that can be rescued by interrupting TNF-a signaling) are observed with IKK signalosome proteins IKKb and IKKg. NF-kB is also essential for preventing lethal infections. Although interrupting TNF-a signaling allows RelA-, IKKb-, or IKKg-deficient mice to survive to birth, these mice die prematurely (prior to reaching reproductive age). The mice display numerous infections, including bacterial pneumonias, and their life spans can be prolonged with antibiotics. Thus, signaling via the IKK signalosome and RelA is essential to fighting infection. RelB deficiency also results in premature lethality; the precise mechanisms remain speculative, but likely involve dysregulated immune function. Mice deficient in c-Rel, p50 (and p105), or p52 (and p100) do not demonstrate decreased life spans and are not observed to develop spontaneous diseases. However, deficiency of RelB or p52 impairs development of select immune cells and structures, and deficiency of c-Rel or p50 impairs innate and adaptive immune responses to select challenges. Thus, each of the NFkB proteins has unique roles that are essential to immune development and/or function. IkB-a deficiency results in widespread inflammation and neonatal lethality. This suggests that IkB-a is essential to preventing and/or terminating NF-kB activity. Targeted deletion of IkB-b or IkB-e is less remarkable. Interestingly, replacing the gene for IkB-a with that for IkB-b rescues mice from IkB-a deficiency, suggesting that differential gene regulation rather than differential biochemical activity is responsible for the unique role(s) of IkB-a.

NF-jB and IjB in Respiratory Diseases As regulators of immunity, inflammation, and cell survival, the NF-kB and IkB signaling axis is likely critical for many lung diseases. NF-kB activation is observed in lung and/or blood cells from patients with asthma, chronic obstructive pulmonary disease, pneumonia, acute respiratory distress syndrome, cystic fibrosis, and sarcoidosis. Exposures relevant to respiratory diseases activate NF-kB, including cigarette smoke, ozone, mineral dusts, ischemia/reperfusion, viruses, bacteria, and fungi. Interventions in respiratory medicine activate NF-kB as well, such as mechanical ventilation, oxygen therapy, lung transplantation, and cancer chemotherapy. Thus, NF-kB and IkB have many roles in pulmonary medicine. In mice, overexpression in the lungs of a constitutively active form of IKK is sufficient to induce

pulmonary inflammation. Transgenic mice with a dominant-negative IkB-a (preventing NF-kB activation by IKK) in the respiratory epithelial cells have diminished pulmonary inflammation induced by bacterial lipopolysaccharide (LPS) in the lungs. Thus, NF-kB activity is sufficient for pulmonary inflammation, and necessary in some settings. In many human primary or immortalized lung cells studied in vitro, activation of NF-kB is necessary and sufficient for the expression of proinflammatory genes, suggesting that NF-kB functions similarly across species. For such reasons, NF-kB inhibitors are actively pursued by the pharmaceutical industry as potential anti-inflammatory drugs. The actions of NF-kB in inhibiting apoptosis also have therapeutic implications. Interfering with NFkB can promote apoptosis, which may augment cancer treatment. Indeed, inhibiting NF-kB sensitizes lung cancer cells to chemotherapeutic agents in vitro. Dysregulated apoptosis may also contribute to noncancerous lung diseases, such as the acute respiratory distress syndrome and emphysema. Thus, modulating NF-kB activity as a means to influence cell survival represents another attractive therapeutic target for lung diseases. Although manipulating NF-kB is an enticing goal with therapeutic potential, it should be remembered that interfering with NF-kB can be disastrous to the host, as demonstrated by mice deficient in RelA, RelB, IkB-a, IKKb, or IKKg. Because of the complexity of this family of molecules, with distinct NF-kB and IkB proteins having unique roles, rationally directing and selectively targeting interventions will be imperative. Therefore, it is important to elucidate specific NF-kB functions in specific respiratory diseases. Bacterial stimuli in the lungs induce the nuclear translocation of RelA and p50. In response to bacterial LPS in the lungs, the deficiency of RelA (plus TNF receptor 1) decreases the expression of chemokines and adhesion molecules and the recruitment of neutrophils compared to either wild-type or TNF receptor 1-deficient mice. Mice deficient in RelA plus TNF receptor 1 are also predisposed to bacterial pneumonias. Thus, RelA is essential to inducing gene expression that recruits neutrophils to eliminate bacteria in the lungs. In contrast, p50 deficiency increases rather than decreases the expression of chemokines, adhesion molecules, and proinflammatory cytokines during bacterial pneumonia. Excessive inflammation in the lungs of p50-deficient mice exacerbates pulmonary edema and decreases epithelial barrier integrity, resulting in impaired blood–gas exchange and disseminated infection. Thus, p50 limits gene expression elicited by bacteria in the lungs,

TRANSCRIPTION FACTORS / POU 269

preventing inflammatory lung injury. Appropriate levels of gene expression during infection may require the balance of opposing actions from RelA and p50. Experimental allergic reactions in the mouse lung depend upon both c-Rel and p50. After antigen sensitization and airways challenge, mice deficient in c-Rel show decreased lung inflammation, decreased serum immunoglobulin E, and decreased airways hyperresponsiveness to methacholine challenge. Mice deficient in p50 show decreased differentiation of TH2 lymphocytes and decreased airways inflammation after antigen sensitization and challenge. These studies suggest that c-Rel and p50, particularly in lymphocytes, contribute to acquired immune responses relevant to asthma pathogenesis. Additional roles for specific NF-kB proteins in regulating gene expression, cell biology, organ function, system physiology, and disease pathogenesis must be better defined, emphasizing an important need for further research. NF-kB proteins are activated in many clinical settings and experimental models of disease, and NF-kB regulates the expression of a great number of genes, particularly in immune and apoptosis pathways. With greater levels of understanding, NF-kB and IkB hold tremendous promise as targets of pharmacological therapies for diverse lung diseases. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Cell: Epithelial. Allergy: Allergic Reactions. Apoptosis. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Emphysema, General. Colony Stimulating Factors. Cystic Fibrosis: Overview. Defense Systems. Dendritic Cells. Endothelial Cells and Endothelium. Endotoxins. Environmental Pollutants: Overview. Eotaxins. Epithelial Cells: Type II Cells. Fluid Balance in the Lung. Gene Regulation. G-Protein-Coupled Receptors. Human Immunodeficiency Virus. Immunoglobulins. Interstitial Lung Disease: Hypersensitivity Pneumonitis. Leukocytes: Eosinophils; Neutrophils; Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Platelet-Activating Factors. Macrophage Inflammatory Protein. Nadph and Nadph Oxidase. Occupational Diseases: Overview. Oncogenes and Proto-Oncogenes: Overview. Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants. Oxygen Toxicity. Permeability of the Blood–Gas Barrier. Peroxisome Proliferator-Activated Receptors. Pneumonia: Overview and Epidemiology; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Viral; The Immunocompromised Host. Proteasomes and Ubiquitin. Signal Transduction. Surgery: Transplantation. Systemic Disease: Sarcoidosis. Toll-Like Recept ors. Transcription Factors: Overview. Transgenic Models. Tumor Necrosis Factor Alpha (TNF-a).

Tumors, Malignant: Overview. Upper Respiratory Tract Infection. Ventilation, Mechanical: Positive Pressure Ventilation; Ventilator-Associated Pneumonia.

Further Reading Alcamo EA, Mizgerd JP, Horwitz BH, et al. (2001) Targeted mutation of tumor necrosis factor 1 rescues the RelA-deficient mouse and reveals a critical role for NF-kB in leukocyte recruitment. Journal of Immunology 167: 1592–1600. Das J, Chen CH, Yang LY, Cohn L, Ray P, and Ray A (2001) A critical role for NF-kB in GATA3 expression and T(H)2 differentiation in allergic airway inflammation. Nature Immunology 2: 45–50. Donovan CE, Mark DA, He HZ, et al. (1999) NF-kB/Rel transcription factors: c-Rel promotes airway hyperresponsiveness and allergic pulmonary inflammation. Journal of Immunology 163: 6827–6833. Hoffmann A, Leung TH, and Baltimore D (2003) Genetic analysis of NF-kB/Rel transcription factors defines functional specificities. EMBO Journal 22: 5530–5539. Kucharczak J, Simmons MJ, Fan Y, and Gelinas C (2003) To be, or not to be: NF-kB is the answer – role of Rel/NF-kB in the regulation of apoptosis. Oncogene 22: 8961–8982. Li Q and Verma IM (2002) NF-kB regulation in the immune system. Nature Reviews: Immunology 2: 725–734. Mizgerd JP, Lupa MM, Kogan MS, et al. (2003) Nuclear factor-kB p50 limits inflammation and prevents lung injury during Escherichia coli pneumonia. American Journal of Respiratory and Critical Care Medicine 168: 810–817. Mizgerd JP, Scott ML, Spieker MR, and Doerschuk CM (2002) Functions of IkB proteins in inflammatory responses to E. coli LPS in mouse lungs. American Journal of Respiratory Cell and Molecular Biology 27: 575–582. Moine P, McIntyre R, Schwartz MD, et al. (2000) NF-kB regulatory mechanisms in alveolar macrophages from patients with acute respiratory distress syndrome. Shock 13: 85–91. Orlowski RZ and Baldwin AS Jr (2002) NF-kB as a therapeutic target in cancer. Trends in Molecular Medicine 8: 385–389. Pahl HL (1999) Activators and target genes of Rel/NF-kB transcription factors. Oncogene 18: 6853–6866. Poynter ME, Irvin CG, and Janssen-Heininger YM (2003) A prominent role for airway epithelial NF-kB activation in lipopolysaccharide-induced airway inflammation. Journal of Immunology 170: 6257–6265. Sadikot RT, Han W, Everhart MB, et al. (2003) Selective IkB kinase expression in airway epithelium generates neutrophilic lung inflammation. Journal of Immunology 170: 1091–1098. Sen R and Baltimore D (1986) Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46: 705–716.

POU R A Sturm, Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract POU domain transcription factor proteins are critical regulators of many developmental processes during embryogenesis, are

270 TRANSCRIPTION FACTORS / POU prominent in the neural system where they are identified as BRN transcription factors, and can be reactivated during malignant transformation of cancerous tissue. This family of genes is characterized by a common 150–160 amino acid region encoding a bipartite DNA-binding structure, with POU-specific (POUS) and POU-homeodomain (POUH) subdomains joined by an unstructured linker region of variable length. This arrangement allows very flexible interaction with multiple degenerate, monomer, and dimer target recognition sites. Members of the POU transcription factor family interact by dimerizing not only with other POU members but also with other transcription factor classes to control gene expression from target promoter–enhancer sequences in a combinatorial fashion. A specific POU– SOX code permits specific members of each family to interact and assemble a functional protein complex able to promote or repress transcription. Small cell lung cancer cell lines display several neuroendocrine features and express the BRN2 POU transcription factor, which is also expressed in several malignant tissues, such as glioblastoma, neuroblastoma, Merkel cell carcinoma, and melanoma. In addition, neuroendocrine tumors of bronchial origin express high BRN3A levels thought to be associated with highly aggressive stages of disease. The critical importance of the presence and levels of POU transcription factors to cell fate determination demonstrate that they act as master regulators of cell lineage commitment.

domain containing chromosomal segments and one pseudogene. At present six POU protein subclasses (POU1F to POU6F) have been recognized in mammalian species using comparative genomic studies and grouped according to the extent of amino acid identity, linker length, and composition of the central DNA-binding segment, while the flanking regions of the protein family are generally unrelated.

POU Domain Structure and Gene Organization The POU DNA-binding structure consists of an N-terminal POU-specific (POUS) subdomain of 75 amino acids and a C-terminal POU-homeodomain (POUH) of 60 amino acids joined by a flexible linker of varying length ranging from 15 to 56 amino acid residues or greater (Figure 1(a)). These subdomains are able to form four and three alpha helices, respectively, with the linker being an unstructured

Introduction POU (pronounced as in pow-erful)-domain transcription factors constitute a highly conserved family of proteins that includes general, developmental, and tissue-specific regulators of many diverse cell types. Members of this family of DNA-binding proteins have been implicated in a broad range of biological processes from housekeeping gene functions, viral gene expression and DNA replication (OCT1), directing the expression of neuronal gene promoters (PIT1, BRN), the establishment of the immune system and antibody production (OCT2) to the programing of embryonic and adult stem cells (OCT4 and OCT6). The distinguishing motif of this transcription factor class was first described in 1988 based on the sequence similarity between the DNAbinding domains of four transcription factors: the mammalian factors PIT1, OCT1 and OCT2, and the Caenorhabditis elegans factor Unc-86 to give the acronym POU. The C-terminal region of this 150–160 amino acid stretch was shown to contain the previously characterized helix–turn–helix DNA-binding structure encoded in the homeobox (HOX) genes; however, the POU domain was distinguished by inclusion of a conserved N-terminal region linked via a segment of variable sequence. The bipartite nature of the POU motif suggested these two subdomains could act separately in DNA recognition, transcriptional activation, or functional interactions with other proteins. Annotation of the human genome sequencing project has allowed identification of 16 full POU

N

1

H-T-H 2 3 4 POUS

1 Linker

H-T-H 2 3 POUH

C

(a) C 1

POUS

3 2 1 4

POUH

POUH

2 3

ATGCAAAT TACGTTTA Linker

-N

POUS

(b) POUS

POUH

GCATNNTAAT CGTANNATTA

(c)

POUH

TAATGAGAT ATTACTCTA

POUS

Figure 1 Structure and DNA-binding properties of the POU domain. (a) Linear representation of the POU domain showing the numbered alpha-helical regions present in the POUS and POUH subdomains respectively joined by the unstructured Linker segment. (b) Schematic representations of the OCT1 POU domain bound to the consensus octamer motif. The POUS subdomain is shown in red with the POUH subdomain in blue with alpha-helices numbered as above; the Linker is shown as a dashed line indicating the flexible structure of this region. The arrows indicate the direction of the globular representation of each domain relative to the DNA. (c) The different arrangement and orientation adopted by the POU subdomains on alternative target sequences are shown.

TRANSCRIPTION FACTORS / POU 271

region. The OCT1 and OCT2 factors were first identified through their specific monomeric DNAbinding activity for the octamer consensus sequence 50 -ATGCAAAT-30 found as a regulatory element in a number of housekeeping, viral, and tissue-specific promoters. These OCT proteins were later found to be able to homo- and hetero-dimerize on specific DNA response elements such as the heptamer/octamer binding site present in the immunoglobulin gene promoter, the palindromic OCT recognition element (PORE) within the first intron of the osteopontin gene, and the more palindromic OCT recognition element (MORE) identified within the immunoglobulin heavy chain enhancer. The POU domain structure of a two-in-one DNA-binding domain allows protein binding to an assortment of target sites and the high degree of variation can make it difficult to recognize anything more than a weak consensus binding sequence. This variation is accommodated via the extent of conformational flexibility conferred by the unstructured POU domain linker, which has the ability to reorientate the subdomains to recognize different sites, and also confers the potential to cooperatively dimerize. NMR spectroscopy studies on several of the OCT proteins have been performed to examine the stereochemistry of the POU subdomains in isolation and in combination with synthetic oligonucleotide target recognition sites. In addition, the complete POU domains of OCT1, PIT1, and OCT4 in protein–DNA crystal complexes have also been examined by X-ray diffraction. From these studies, the POUS and POUH subdomains were found to be structurally autonomous and to have separate DNA sequence specificities. The OCT proteins bind monomerically to the octamer consensus sequence, with the crystal structure of the OCT1 POU domain bound to this sequence demonstrating that each subdomain binds to separate halves of the octamer motif on opposite faces of the DNA helix (Figure 1(b)). The POUS binds to 50 -ATGC and the POUH to AAAT-30 sequences, which can be considered as half sites. The crystal structure also revealed that the POUS and POUH domains both form helix–turn–helix structures, utilizing the second and third helix of each subdomain, with the third helix interacting with the major groove of the DNA. The POU domain proteins are able to adopt different arrangements on the DNA molecule due to the high flexibility of the central linker region joining the POU subdomains; in consequence, this allows for quite diverse target site recognition (Figure 1(c)). As can be seen with OCT1 protein–DNA binding, reversal in the orientation of the octamer recognition sequence half sites can occur through the different

orientation of the POUS and POUH domains. Moreover, introduction of extra bases into the recognition motif that separates the half sites also leads to differences in the positioning of the subdomains relative to each other when bound to DNA. Characterization of POU domain dimerization on PORE and MORE sites has shown that the sequence of DNA elements permissive for dimeric binding can vary the spatial arrangement of the POUS and POUH subdomains of each protein (Figure 2). This can, in turn, alter POU–POU protein interactions and further recruitment of other transcriptional co-regulators, which greatly increases the complexity of possible tertiary interactions. While highly homologous within the POU domain, members of this gene family have an ancient evolutionary history and are found distributed on several different chromosomes as opposed to the clustered arrangement of the HOX genes commonly found in mammalian genomes. There is no shared gene structure across all POU gene family members: some are large genes encoded by multiple exons whereas others are intronless. Though some members are obviously closely related with the POU domain split into similar multiple exons (OCT1/OCT2) with alternatively spliced transcripts able to produce several protein isoforms, others conversely are transcribed from a single coding exon (BRN1, BRN2, BRN4, OCT6) that has likely allowed ready gene duplication.

POU Gene Classification and Activities The neuronally expressed PIT1 protein defines, and is, the sole example of the POU1 class domain. Although it is expressed in the neural tube and pituitary during embryogenesis, it is restricted to the pituitary in adult tissues and is involved in the embryonic formation of this organ and cell fate specification. PIT1 is required for development of the somatotropes, lactotropes, and thyrotropes in the anterior pituitary gland; its mutations in humans cause combined pituitary hormone deficiency (CPHD). The ubiquitously expressed OCT1, more tissue-restricted lymphoid OCT2, and epithelial SKN1A proteins represent the POU2 type genes. The BRN1, BRN2, BRN4 (X-linked deafness DFN3), and OCT6 protein members, which are involved in neural, auditory, melanocytic, or Schwann cell development, comprise POU3, while BRN3A, BRN3B, and BRN3C (autosomal dominant deafness DFNA15) protein members, also involved in neural, auditory, and visual development, represent POU4 classes. POU5 domain genes are involved in early embryogenesis, spermatogenesis, and stem cell formation including OCT4 (also called OCT3) and SPRM1 proteins, with an

272 TRANSCRIPTION FACTORS / POU

POU

Dimer

POU

POU

S1

S2

H2

POU

H2

ATTTGAAATGCAAAT TAAACTTTACGTTTA

ATGCATATGCAT TACGTATACGTA

POU

POU

H1

POU

S2

H1

PORE-dimer

Germ cell tumors

Endoderm Mesoderm

POU

S1

MORE-dimer

150

Embryonic stem cell

OCT4 (%) level

Morula 50 POU

H

ATGCAAAT TACGTTTA

POU

POU

GCATNNTAAT CGTANNATTA

Monomer S

OCT-monomer 0

S

POU

H

Trophoectoderm

BRN-monomer Development, differentiation, tumorigenesis

Figure 2 POU protein levels, dimerization, and cell fate determination. The level of the OCT4 protein expressed in the morula stage of the embryo is shown relative to states of cellular differentiation that occur during morphogenesis and tumorigenesis. The distinct monomer and dimer configurations of the POU subdomains within the OCT, BRN, PORE, and MORE site complexes are shown with increasing and decreasing OCT4 protein levels. In the model presented, concentrations above 150% (dashed line) would favor POU domain dimerization leading to cellular differentiation and expression of genes for extra-embryonic endoderm and mesoderm cells, whereas below 50% (dashed line) the preferential monomeric interactions of the POU domain or its absence would lead to trophectoderm formation. Static expression of OCT4 in the inner cell mass of the embryo gives rise to embryonic stem cells with the special properties of continued self-renewal and totipotency, while high OCT4 levels are found in testicular germ cell tumors.

OCT3L gene not yet characterized. The POU6 class proteins BRN5 and RPF1 are expressed in the central nervous system and cells of the retina. These known expression patterns found for the POU protein family members are likely to expand once the activities of these transcription factors are further characterized in a range of body tissues.

Transcriptional Regulation by POU Proteins and Biological Function All cell types express the ubiquitous OCT1 factor, whereas the other POU family members each show more restricted expression. The diverse activities, regulation, and central importance of the POU family members to several basic cellular processes is exemplified by the roles played by the OCT1 protein. It is active in the specific S-phase transcriptional regulation of histone genes that are the building blocks of chromatin structure as well as the small nuclear RNA (snRNA) involved in RNA processing and transcriptional elongation, which are required by all cells and are particularly important for growing cells. OCT1 also participates in the activation of cell-specific

promoters, including the immune and nervous systems, usually in cooperation with cell-specific coactivators. The best characterized cell-specific promoters are those of the immunoglobulin heavy and light chains, in which the OCT1 and OCT2 POU domains are able to associate with a 256 amino acid proline-rich B cell-specific co-regulator OCAB (gene symbol POU2AF1). By interacting with OCT1 and OCT2, OCAB can be recruited to a subset of octamer sites where it serves a coactivator function for transcription. A strong transactivation domain in the C-terminus of the OCAB protein mediates this effect with the N-terminus containing the domain that interacts with the two POU subdomains by clamping them together as well as bridging the DNA within the octamer motif. The interaction of this coactivator is specific for OCT1 and OCT2 and does not occur with OCT4, OCT6, or PIT1. Through its ability to recognize the TAATGAGAT element (Figure 1(c)) present in herpes simplex virus (HSV) early genes, the OCT1 protein can be recruited to interact with host cell factors and the viral encoded VP16 transactivator protein to direct a complex regulating the transcriptional expression

TRANSCRIPTION FACTORS / POU 273

and further replication of the HSV viral genome. OCT1 has also been found to participate in adenoviral DNA replication through the interaction of the POU domain with adenovirus-encoded proteins involved in replication machinery. Thus, the POU domain of OCT1 has been found to be an attractive viral target because of the broad expression and the versatility of its DNA recognition; it remains possible that other POU family members will also be coopted to cognate sites by viral genomes in specific tissues. The critical importance of the presence and levels of POU transcription factors to cell fate determination is seen in the role played by the OCT4 protein in embryonic stem cell totipotency, pluripotency, differentiation, and testicular germ cell tumors (Figure 2). OCT4 gene activity is required during the very first stages of development and embryos that lack expression of this gene fail to survive. Levels between 50% and 150% of the endogenous OCT4 in embryonic stem cells are permissive for self-renewal and maintenance of totipotency. Differentiation and commitment of these cells to form all somatic lineages occurs at the blastocyst stage of development and is concurrent with OCT4 downregulation, with the protein reappearing in the inner cell mass that forms the primordial germ cells for the next generation. Loss of OCT4 causes the cells of the inner cell mass to differentiate into trophectoderm while twofold upregulation causes the stem cells to express genes involved in differentiation of the primitive endoderm and mesoderm. The relatively small window of protein levels in which POU transcription factors operate as critical determinants reprograming cell fate demonstrate that they act as master regulators of cell lineage commitment.

A POU–SOX Transcription Factor Code Members of the POU transcription factor family interact by dimerizing not only with other POU members but also with other transcription factor classes to control gene expression from target promoter-enhancer sequences in a combinatorial fashion. It is thought that both the POU and SOX families of transcription factors coevolved allowing acquisition of specific developmental codes during the evolution of multicellular body plans. Consistent with this, a number of studies have provided direct evidence for a specific combinatorial POU–SOX code, which permits specific members of each family to interact and assemble a functional protein complex able to promote or repress transcription. Heterodimerization between the OCT4 and SOX2 proteins provides the pre-eminent example of a POU–SOX partnership. The FGF4 gene enhancer is active in embryonic stem cells

but activation is only achieved upon cooperative binding of both OCT4 and SOX2 allowing the assembly of a ternary DNA–protein complex, which is disrupted by increasing the space between the two cognate binding sites. The synergistic effects of these two transcription factors are essential for the establishment of the first three lineages in the mammalian embryo.

Relevance of POU-Domain Factors in Lung Development and Neoplasia A range of specific transcription factors are expressed and interact to direct the formation and differentiation of the large number of cell lineages responsible for the morphogenic development of the lung. These cells must undergo extensive cell proliferation, branching, and alveolar saccule formation with pulmonary neuroendocrine (PNE) cells differentiating amongst the earliest in lung epithelial cell gestation. The PNE cells contain characteristic small densecored granules, which are secreted by exocytosis and include many bioactive substances including neurotransmitters, neuromodulators, growth factors, and mediators of vasoconstriction or dilation. It is known that the PNE cells are not uniform with respect to secretory content but it is not clear whether these differences in content represent different stable classes of PNE cells or merely different functional states of a single cell type. Small cell lung carcinomas (SCLCs) display prominent neuroendocrine characteristics and this has provided evidence that they may arise from PNE cells or PNE cell progenitors. What is clear is that SCLC cell lines propagated from tumors express the BRN2 POU transcription factor implicated in the development of neural and glial cell lineages, and which is also expressed in several malignant tissues, such as glioblastoma and neuroblastoma, Merkel cell carcinoma, and melanoma. In addition, the study of the BRN3A factor in neuroendocrine tumors of bronchial origin found high BRN3A expression to be associated with highly aggressive stages of disease. These data suggest that these POU proteins may exert an oncogenic action in neuroendocrine tumors of the lung and that the levels of these proteins indicate a poor prognosis. Understanding the structure–function, target sequence selectivity, and partner-protein interactions of the POU family in transcriptional regulation will provide valuable data on gene network organization modulating cell fate, pluripotency, stem cell plasticity, and oncogenic transformation relevant to all tissues including the lungs. See also: Stem Cells. Transcription Factors: Overview. Tumors, Malignant: Metastases from Lung Cancer.

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Further Reading Andersen B and Rosenfeld MG (2001) POU domain factors in the neuroendocrine system: lesson from developmental biology provides insights into human disease. Endocrine Reviews 22: 2–35. Dailey L and Basilico C (2001) Coevolution of HMG domains and homeodomains and the generation of transcriptional regulation by Sox/POU complexes. Journal of Cellular Physiology 186: 315–328. Herr W and Cleary MA (1995) The POU domain: versatility in transcriptional regulation by a flexible two-in-one DNA-binding domain. Genes and Development 9: 1679–1693. Herr W, Sturm RA, Clerc RG, et al. (1988) The POU domain: a large conserved region in the mammalian pit-1, oct-1, oct-2, and Caenorhabditis elegans unc-86 gene products. Genes and Development 2: 1513–1516. Pesce M and Scholer HR (2001) Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19: 271–278. Phillips K and Luisi B (2000) The virtuoso of versatility: POU proteins that flex to fit. Journal of Molecular Biology 302: 1023–1039. Remenyi A, Tomilin A, Scholer HR, and Wilmanns M (2002) Differential activity by DNA-induced quaternary structures of POU transcription factors. Biochemical Pharmacology 64: 979– 984. Ryan AK and Rosenfeld MG (1997) POU domain family values: flexibility, partnerships, and developmental codes. Genes and Development 11: 1207–1225. Scholer HR (1991) Octamania: the POU factors in murine development. Trends in Genetics 7: 323–329. Teitell MA (2003) OCA-B regulation of B-cell development and function. Trends in Immunology 24: 546–553. Wysocka J and Herr W (2003) The herpes simplex virus VP16induced complex: the makings of a regulatory switch. Trends in Biochemical Sciences 28: 294–304.

PU.1 C J Bachurski and B C Trapnell, Cincinnati Children’s Hospital, Cincinnati, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The PU.1/Spi-1 transcription factor is critical for hematopoiesis and functions as a master regulator of myeloid differentiation. PU.1 stimulates expression of myeloid growth factor receptors in myeloid progenitors and is a critical determinant of terminal differentiation of alveolar macrophages in which it stimulates surfactant catabolism and a variety of myeloid innate immune functions. PU.1 expression is under tight regulatory control during myeloid differentiation. Alveolar macrophage precursors are stimulated to terminally differentiate by granulocytemacrophage colony-stimulating factor (GM-CSF) in the lung, which acts locally to stimulate high PU.1 levels in macrophages. Absence of GM-CSF signaling in mice due to targeted disruption of the GM-CSF gene impairs surfactant catabolism by alveolar macrophages resulting in a lung disease histologically identical to the human disease idiopathic pulmonary alveolar proteinosis. This disorder in humans is specifically associated with neutralizing autoantibodies against GM-CSF that

completely neutralize GM-CSF bioactivity and reduce PU.1 expression in alveolar macrophages. Mutations in PU.1 are also associated with development of acute myelogenous leukemia. Mutations in the PU.1 binding sites of PU.1-stimulated genes are also associated with disease, for example, NADPH oxidase subunit gene mutations in chronic granulomatous disease. PU.1 has a critical role in blood and lung function in health and disease.

Introduction PU.1/Spi-1 is an ETS-family transcription factor that regulates myeloid and B-cell lineage development. PU.1 (purine-rich sequence DNA-binding protein 1) was originally cloned from a murine macrophage cDNA library and was immediately recognized as the protein product of Spi-1 (SFFV proviral insertion-1), initially identified as an oncogene involved in the malignant transformation of proerythroblasts in SFFV (spleen focus forming virus) infection-induced erythroleukemia. While high-level expression of PU.1 is required for macrophage differentiation, increased expression of PU.1 in proerythroblasts blocks erythroid differentiation and induces erythroleukemia. The assigned gene designation for PU.1/Spi-1 is Sfpi1. Targeted disruption of the Sfpi gene in mice blocks macrophage and B-lymphocyte development and delays T-lymphocyte development. PU.1 is not required for myeloid lineage commitment but promotes both proliferation and differentiation of myeloid progenitors. PU.1 expression is required for differentiated functions of alveolar macrophage including phagocytosis, bacterial cell killing, and efficient pulmonary surfactant uptake and metabolism. PU.1 is also expressed in nonhematopoietic tissues including the lung epithelium, bone osteoblasts, and prostate.

Structure The Sfpi1 gene is located on human chromosome 11p11.2 and contains 5 coding exons. The mouse homolog, which shares 85% sequence identity, is located on chromosome 2, band E3. PU.1 binds to DNA as a monomer and causes DNA bending. The DNA-binding ETS domain of PU.1 has a winged helix-turn-helix structure that is conserved between all ETS family members. Although all ETS family members share the approximately 85 amino acid ETS domain, family members differ in overall size and protein-domain structure. The ETS domain of PU.1 is C-terminal, like Ets1 and Ets2. The structure of PU.1 is shown in Figure 1. The tripartite domain structure is shown with specific areas of protein– protein interaction with other transcription factors and coactivators indicated below the diagram. The

TRANSCRIPTION FACTORS / PU.1 275

transactivation domain of PU.1 consists of an Nterminal acidic region followed by a glutamine-rich region (amino acids 75–93). This domain is the site of interaction with the retinoblastoma protein (Rb), the coactivator CBP/p300, and TATA box-binding basal transcription factor (TBP). PEST (P) proline, Transactivation domain Acidic

Q-rich

PEST DNA-binding ETS domain

N

Regulation of Production and Activity

C (1–74)

(75–100) (100–151)

Rb CBP/P300

TFIID

(152–264) 34

IRF family (PIP)

C-Jun C/EBP-
,- GATA-1,2 AML1

Figure 1 Structural organization of PU.1. Structural domains are indicated above the diagram, and amino acids are indicated in parentheses. Proteins known to interact directly with PU.1 are indicated below the binding domain. The N-terminal transactivation domain (amino acids 1–100) contains both acidic and glutamine-rich regions. The PEST domain binds to interferon regulatory factor (IRF) family members whereas diverse transcription factors compete for binding to the b3b4 region in the C-terminal DNA-binding ETS domain.

Surfactant homeostasis

(E) glutamic acid, (S) serine, and (T) threonine domains are thought to control protein stability and can target proteins for proteolytic degradation. The PEST domain of PU.1 is critical for myeloid development in vitro and is also the site of specific protein–protein interactions. Phosphorylation of Ser148 in the PEST domain of PU.1 is required for complex formation with the interferon regulatory factor (IRF) family of proteins (Pip and ICSBP).

The macrophage growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF) induce PU.1 expression in alveolar macrophage precursors to stimulate expression of maturation-associated genes. Granulocyte CSF (G-CSF) stimulates changes in macrophage adhesion and migration by induction of CD11b/CD18 (MAC1) expression via Stat3-mediated induction of PU.1 (Figure 2). PU.1 in turn regulates the expression of the receptors for these factors and induces its own expression in a self-stimulatory loop. The myeloidspecific promoter of PU.1 contains both octamer and ETS binding sites. PU.1 is highly expressed in

Innate immunity

Adaptive immunity

TNF-
GM-CSF

H2O2

IL-18 Surfactant

Th1

↑ PU.1

IFN-

IL-12 NK

Catabolism

T T

B

STAT3

Filopodia G-CSF

Cell adhesion M-CSF

Figure 2 Role of PU.1 in regulating immune and other functions of alveolar macrophages. GM-CSF present within the lungs is required for stimulation of PU.1 expression in alveolar macrophages, which in turn stimulates their terminal differentiation. Through this mechanism, PU.1 regulates surfactant homeostasis, innate immunity, and adaptive immunity in the lungs. Functions of alveolar macrophages regulated by PU.1 include surfactant catabolism, various alveolar macrophage effector and regulator functions, and molecular connections between innate and adaptive immunity. See text for details. T, T cell; B, B cell; NK, natural killer cell, Th1, T-helper-1 cell.

276 TRANSCRIPTION FACTORS / PU.1

alveolar macrophages of wild-type but not GM-CSF knockout mice. Local production of GM-CSF in the lung of GM-CSF knockout mice restores PU.1 expression and macrophage function. PU.1 interacts with other transcription factors and coactivators to enhance or repress gene expression during differentiation of hematopoetic cells. cAMP response element binding protein (CREB) interacts with PU.1 to induce expression of the common signaling b-chain (bc) of the IL-3, IL-5, and GM-CSF receptors in myeloid cells. PU.1 interaction with cJun induces M-CSF receptor expression and promotes monocyte differentiation. CCAAT enhancer binding protein alpha (C/EBPa) induces granulocyte differentiation of myeloid cells by interacting with the b3/b4 region of the PU.1 DNA-binding domain displacing c-JUN and functionally blocking PU.1 activity. GATA1 interacts with PU.1 and antagonizes its activity during normal erythroid cell maturation. The ability of ETS family members to bind DNA and activate transcription is also regulated by phosphorylation. PU.1 phosphorylation induces increased binding to DNA and activation of macrophage differentiation-specific genes including genes that comprise the phagocytic NADPH complex. LPS induces PU.1 phosphorylation at Ser148, and ras-PI3kinase dependent externally regulated kinase (AKT) at Ser41, and p38 at Ser142. In addition to its ability to bind DNA, PU.1 can bind to RNA and has been shown to effect mRNA splicing.

Biological Functions The transcription factor PU.1 recognizes and binds a purine-rich DNA sequence containing the core sequence 50 -GGAA/T-30 . PU.1 is required for normal blood cell development and is expressed in hematopoietic cells, including primitive CD34 þ cells, macrophages, B lymphocytes, neutrophils, mast cells, early erythroblasts, and osteoclasts. Important among its functions is regulation of expression of crucial myeloid genes including the receptors for MCSF, G-CSF, and GM-CSF. PU.1 expression increases during hematopoietic development as stem cells commit to the myeloid lineage. In contrast, PU.1 expression decreases inversely with GATA1 expression during erythroid lineage differentiation. Activation of PU.1/Spi-1 expression in proerythroblasts by insertional mutagenesis in SFFV-induced erythroleukemia blocks GATA-1 expression and blocks erythroid differentiation. Sfpi1 (PU.1) gene expression is tightly controlled during hematopoietic cell differentiation. The level of PU.1 expression appears to be involved in the regulation of differentiation into lymphocytes,

granulocytes, or macrophages. A low level of PU.1 expression in hematopoietic precursors is associated with B-cell lineage differentiation whereas a high level of PU.1 expression stimulates macrophage differentiation. PU.1 activates the transcription of many macrophage, neutrophil, and lymphoidspecific genes. Disruption of the Sfpi1 gene in mice results in delay of neutrophil development, and absence of monocytes/macrophages and osteoclasts. Lymphopoiesis is also affected with loss of B cells and delayed development of T cells, whereas other hematopoietic lineages are minimally affected including megacaryocytes and erythrocytes. PU.1 is a key dosage-dependent regulator of the development of several hematopoietic cell lineages. PU.1 is considered a ‘master’ transcription factor because it controls a large number of functionally diverse genes in early myeloid cells required for differentiated cell function. PU.1 regulates expression of cell-surface molecules including Ig receptors FcgRI (FcI/CD64) and FcgRIII (FcIIIA/CD16), integrins (CD11b/CD18, MAC1), complement receptors CR3, CR4, macrosialin, scavenger receptors I and II, mannose receptor, and receptors for GM-CSF, M-CSF, and G-CSF. M-CSF receptors are undetectable and G- and GM-CSF receptor expression is low in PU.1 knockout mice. PU.1 cooperates with other transcription factors including C/EBPa and AML1 to regulate expression of these receptors. PU.1 in association with CREB regulates expression of the common b-chain of the IL3, IL5, and GM-CSF receptors in myeloid cells. PU.1 induces expression of intracellular enzymes including NADPH oxidase subunits pg91phox and p47phox, and lysozyme. PU.1 is required for normal response to Toll-like receptor activation including production of TNF-a and cyclooxygenase-2 (Cox2, an enzyme that catalyzes production of prostaglandins) in response to bacterial endotoxin (LPS) challenge. PU.1 is also required for production of IL-18 and IL-2 by activated macrophages. Commensurate with the number of genes regulated by PU.1, many of which are involved in immune functions, PU.1 is a critical regulator of a number of innate and other immune functions in alveolar macrophages (Figure 2). These include: (1) expression of pathogen pattern-recognition receptors such as the mannose receptor, Toll-like receptors 2 and 4, scavenger and various cell-surface integrin molecules; (2) phagocytosis of both unopsonized and opsonized particles; (3) production of superoxide radicals; (4) microbial killing; (5) pathogen-stimulated secretion of proinflammatory cytokines, for example, tumor necrosis factor alpha (TNF-a), IL-12, and IL18; (6) cellular cytoskeletal organization and

TRANSCRIPTION FACTORS / PU.1 277

filopodia formation; and (7) cell adhesion. PU.1 also regulates important molecular connections between innate and adaptive immunity that modify specific adaptive immune responses to microbial pathogens following infection. Such molecular pathways include macrophage release of IL-12/IL18, which in turn stimulates interferon gamma (IFN-g) secretion by lymphocytes, and subsequent modulation of cellular and humoral immune responses (Figure 2). The sequential expression and activity of three ETS family members PU.1, Ets2, and Fli1 are pivotal for the regulation of macrophage development. PU.1 expression and activity is required early during myeloid development for proper expression of the appropriate growth factor receptors, the activation of which results in induction of increased levels of PU.1, Ets2 expression, and the activity of both Ets2 and Fli1. In mice, PU.1 is required for stimulation of a number of alveolar macrophage functions, including cell adhesion, pathogen receptor expression, phagocytosis, Fcg receptor signaling, bacterial killing, cytokine expression, and catabolism of surfactant lipids and protein (see below) (Figure 2). The number and diversity of functions stimulated concurrently in alveolar macrophages strongly suggests that PU.1 is required for terminal differentiation of these cells.

PU.1 in Respiratory Diseases A rapidly growing body of information has identified the critical role of PU.1 in alveolar macrophage ontogeny and development. Since these cells are the pulmonary representatives of the bone marrowderived mononuclear phagocyte system that plays a central role in lung homeostasis and host defense in health and disease, it is not surprising that molecular defects in PU.1 result in impairments with important clinical manifestations in the lungs. Because PU.1 functions at multiple points during macrophage development from precursors, molecular defects in PU.1 also have important clinical manifestations outside the lungs. These clinical manifestations will be reviewed briefly. Pulmonary Alveolar Proteinosis

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare disorder in which surfactant accumulates in the lung resulting in impaired gas exchange and respiratory insufficiency. Pulmonary surfactant is a mixture of lipids and proteins that plays a vital role in reducing surface tension on the alveolar walls thus preventing alveolar collapse and respiratory failure. Surfactant is secreted by the lung epithelium and is normally degraded by alveolar macrophages and by alveolar epithelial cells, which also recycle a portion of it.

Serendipitously, PAP was identified in mice deficient in GM-CSF. The cellular basis of this lung phenotype is impairment in the capacity for surfactant degradation by alveolar macrophages. This defect occurs because GM-CSF in the lungs is required to stimulate high levels of PU.1 in alveolar macrophages, which in turn stimulates terminal differentiation of alveolar macrophages. This PU.1-stimulated macrophage maturation process also includes acquisition of a number of innate immune functions (see Figure 2). These and other murine studies provided the first real clues about the pathogenesis of idiopathic PAP in humans and led to our current concept of PAP as an autoimmune disease directed at GM-CSF. Idiopathic PAP is specifically associated with high levels of neutralizing antiGM-CSF autoantibodies. The presence of these antibodies are probably critical to the pathogenesis of PAP because they eliminate GM-CSF signaling in these patients and are associated with impaired PU.1 levels in alveolar macrophages, which also have impaired innate immune functions similar to alveolar macrophages from GM-CSF-deficient mice. Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is a rare inherited phagocytic disorder caused by defective oxidative burst in neutrophils and monocytes. This disease is caused by mutations in any of the four genes that comprise the NADPH oxidase complex in phagocytic cells. The catalytic subunit of the NADPH oxidase complex is a heterodimer of gp91phox and p22phox. These genes are normally induced during myeloid cell differentiation and are highly expressed in macrophages and neutrophils. PU.1 activates gp91phox gene expression by binding to the ETS site (
57 to
50) in the human promoter. Single base pair mutations at
57,
55,
53, and
52 that interfere with PU.1 binding have been identified in a subset of patients with X chromosome-linked CGD. Failure to express the catalytic subunit of NADPH oxidase in phagocytic cells impairs their ability to kill pathogenic bacteria fungi and parasites. CGD patients have recurring life-threatening bacterial and fungal infections that require hospitalization including pneumonias and lung abscesses. Other Diseases Associated with PU.1 Abnormalities

Hematological diseases that alter either the number or function of alveolar macrophages have important clinical manifestations in the lungs. For example, acute myelogenous leukemia (AML) is a common leukemia characterized by accumulation of blast cells

278 TRANSFORMING GROWTH FACTOR BETA (TGF-b) FAMILY OF MOLECULES

(dysfunctional myeloid cells arrested at an early stage of differentiation) and is associated with clinical manifestations including increased susceptibility to lung infection and, rarely, development of PAP. Although AML is pathophysiologically diverse from a molecular standpoint, a variety of PU.1 gene mutations have been identified in a subset of AML patients. Mutations found in the DNA-binding domain of the PU.1 gene of AML patients cause reduced expression of known PU.1 target genes, supporting the concept that disruption of PU.1 function contributes to the block in myeloid differentiation found in AML patients. See also: Autoantibodies. Gene Regulation. Interstitial Lung Disease: Alveolar Proteinosis. Leukocytes: Pulmonary Macrophages. Surfactant: Overview. Toll-Like Receptors.

Further Reading Fisher RC and Scott EW (1998) Role of PU.1 in hematopoiesis. Stem Cells 16: 25–37.

Goebl MG (1990) The PU.1 transcription factor is the product of the putative oncogene Spi-1. Cell 61: 1165–1166. Li R, Pei H, and Watson DK (2000) Regulation of Ets function by protein–protein interactions. Oncogene 19(55): 6514– 6523. Lloberas J, Soler C, and Celada A (1999) The key role of PU.1/SPI1 in B cells, myeloid cells and macrophages. Immunology Today 20: 184–189. Mueller BU, Pabst T, Osato M, et al. (2002) Heterozygous PU.1 mutations are associated with acute myeloid leukemia. Blood 100: 998–1007. Sementchenko VI and Watson DK (2000) Ets target genes: past, present and future. Oncogene 19(55): 6533–6548. Tenen DG, Hromas R, Licht JD, and Zhang DE (1997) Transcription factors, normal myeloid development, and leukemia. Blood 90: 489–519. Trapnell BC and Whitsett JA (2002) GM-CSF regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense. Annual Reviews of Physiology 64: 775–802. Trapnell BC, Whitsett JA, and Nakata K (2003) Mechanisms of disease: pulmonary alveolar proteinosis. The New England Journal of Medicine 349: 2527–2539. Yordy JS and Muise-Helmericks RC (2000) Signal transduction and the Ets family of transcription factors. Oncogene 19(55): 6503–6513.

TRANSFORMING GROWTH FACTOR BETA (TGF-b) FAMILY OF MOLECULES R J McAnulty, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The transforming growth factor beta (TGF-b) family and activins are dimeric proteins that share structural and functional properties. They play important roles in lung development, homeostasis, and immune regulation. In the adult lung they are synthesized predominantly by epithelial cells, macrophages, lymphocytes, eosinophils, and mast cells. They mediate their effects through activation of serine threonine kinase receptors and the Smad signaling pathway. Their primary functions include tissue homeostasis, control of inflammation, and the regulation of wound repair through modulation of cell proliferation, adhesion, differentiation, extracellular matrix metabolism, and apoptosis. It is the dysregulation of these functions that associates them with the pathogenesis of respiratory diseases, including asthma, interstitial lung disease, chronic obstructive pulmonary disease, and infections where chronic inflammation and/or fibrosis are a feature.

Introduction The human transforming growth factor beta (TGF-b) superfamily consists of more than 35 structurally

related polypeptide factors including the TGF-b, activin, inhibin, bone morphogenetic protein, and growth and differentiation factor families. This article will focus on the TGF-b and activin families. The TGF-b family consists of three mammalian isoforms: TGF-b1, TGF-b2, and TGF-b3. TGF-b1 was discovered by several groups in the late 1970s and early 1980s and was first purified in 1983. Its name is derived from its ability to transform and induce anchorage-independent growth and colony formation of normal rat kidney fibroblasts in soft agar. The other homologs in the family were identified subsequently and TGF-b isoforms are now known to play important roles in regulating inflammation, cell growth, differentiation, migration, and apoptosis. The activins were first identified in 1986 as substances that stimulate follicle stimulating hormone secretion, although they have since been found to possess many other functions in common with TGF-b. There are currently four mammalian activin subunits, bA, bB, bC, and bE, which form activins by homo- and heterodimerization. Although all combinations are possible the most widely characterized are activins A, B, and AB. They are structurally

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related to the TGF-b family and share common receptor signaling pathways.

Structure Constituents of the TGF-b superfamily are dimeric polypeptides, which are synthesized as large precursor molecules with an N-terminal signal sequence and a prodomain of varying size, containing 7–9 cysteine residues in the C-terminal domain. All nine cysteine residues are conserved in TGF-bs and activins. The precursor protein is cleaved to release a mature C-terminal polypeptide, which in mammalian TGF-bs and activins consists of 112–116 amino acids, with a dimeric molecular mass of B25 kDa and an amino acid sequence identity of 71–76% between the three TGF-b isoforms and 50–70% between the activins (Figure 1). The TGF-bs and activins have similar crystal structures. The monomers form an outstretched hand-like structure with the heel of one of the monomers touching the outstretched fingers of the other and vice versa. Eight of the cysteine residues form four intrachain disulfide bonds to form the dimer, which is stabilized by the ninth cysteine forming an interchain disulfide bond. The TGF-b genes contain seven exons with conserved splice junctions, which is indicative of common ancestral gene duplication. Although there is considerable homology between TGF-b isoforms in the coding regions, each isoform has distinct 50 promoter and 30 untranslated regions providing isoformspecific regulation of transcription and translation. The activin genes have two or three exons, and the bC and bE subunit genes are also thought to have arisen by gene duplication. Details of the chromosomal location, mRNA, transcriptional control elements, and protein structure for TGF-bs and activins are shown in Figure 1.

Regulation of Production and Activity The cells synthesizing TGF-bs and activins are similar and include epithelial cells, macrophages, eosinophils, lymphocytes, mast cells, endothelial cells, smooth muscle cells, fibroblasts, and mesothelial cells. TGF-b is synthesized in a proform that dimerizes and is linked by disulfide bonds in C- and N-terminal positions (Figure 1). The N-terminal prodomain may also covalently bind to a latent TGF-b binding protein (LTBP) of which there are four types (LTBP-1 to -4) forming a large latent complex (LLC). The C-terminal mature TGF-b dimer is cleaved from the prodomain by a furin-like endopeptidase during secretion from the cell but is retained within the complex, bound to TGF-b by noncovalent

interactions and is termed the TGF-b latencyassociated protein (LAP). Binding of the TGF-b and LAP complex (called small latent TGF-b) to LTBP is necessary for efficient secretion and targets the LLC to the extracellular matrix where LTBP is covalently bound by transglutaminase. The proregion of activin is also essential for dimerization and secretion of biologically active molecules although, unlike TGF-b, the prodomain does not remain associated with the mature peptide after secretion. Disruption of the noncovalent interactions between LAP and TGF-b is required to release the mature active TGF-b dimer. There are several routes of activation including mechanisms involving proteolysis, thrombospondin, integrins, generation of reactive oxygen species, or acidic microenvironments. Plasmin may be the major mechanism of activation by endothelial and stromal cells, whereas thrombospondin appears to be important for the activation of matrix-bound TGF-b. avb6 and avb8 integrin-mediated activation is specific to epithelial cells, due to their restricted expression, and in addition, is specific to the activation of TGF-b1 and TGF-b3, as TGF-b2 lacks the necessary arginine– glycine–aspartic acid (RGD)-binding motif. LTBPs, the majority of which are secreted in an unbound form, are able to block TGF-b activation. There is limited sequence conservation between LAPs of the different TGF-b isoforms and this together with specific mechanisms of activation may, at least partly, be responsible for isoform and location-specific functions of TGF-bs.

Biological Function TGF-bs and activins have fundamental roles in development, tissue homeostasis, immunoregulation, and wound healing (Figure 2). Activins, TGF-bs, and their receptors are developmentally regulated temporally and spatially, and in the lung are involved in the control of branching morphogenesis. Upregulation of TGF-b1 or TGF-b2 inhibits, whilst inhibition of TGF-b or activin activity/signaling stimulates, branching. Gene deletion studies also demonstrate the importance of TGF-b and activin in the lung. TGF-b2 knockout mice die at birth exhibiting, among other developmental defects, collapsed distal and dilated conducting airways. Similarly, mice deficient in an inhibitor of activin activity, follistatin, fail to breathe and die shortly after birth. TGF-b3deficient mice have delayed pulmonary development and again die shortly after birth. In contrast, TGF-b1 knockout mice that survive to birth develop a progressive multifocal inflammation, which is particularly prominent in the lungs and fatal within 2–4

280 TRANSFORMING GROWTH FACTOR BETA (TGF-b) FAMILY OF MOLECULES

Gene characteristics Chromosomal location

mRNA (kb)

Transcriptional control elements

TGF-
1

19q13.1–q13.3

2.5

Sp-1 Ap-1 Egr-1 NF1 FSE2

TGF-
2

1q41

4.1, 5.1, 6.5

TATA box CRE

TGF-
3

14q23–q24

3.0

TATA box CRE

Activin
A

7p15–p14

1.7–6.4

TATA box CRE Sp-1 TRE TPA

Activin
B

2qcen–q13

3.8, 4.8

Sp-1 CRE

Activin
C

12q13.1

1.8, 2.1

–

Activin
E

12

2.7

–

Schematic representation of TGF-
and activin production Pre-pro monomeric protein

Signal peptide Prodomain TGF-
/activin Noncovalent interaction

S

S

S

S

S S

Cleaved dimer bound to prodomain by noncovalent interactions for secretion Active dimer

Protein characteristics Amino acids (n) Precursor

Mature

TGF-
1

390

112

TGF-
2

412

112

TGF-
3

412

112

Activin
A

426

116

Activin
B

407

115

Amino acid sequence homology (%)

71

Predominantly form homodimers

72 76

Activin
C Activin
E

64

352 350

116 114

51 44

53 48

Form homodimers and heterodimers

64

Figure 1 Gene and protein characteristics of the TGF-b and activin families.

weeks of birth. Interestingly, TGF-b1-deficient mice backcrossed onto a severe combined immunodeficient background show no inflammation and survive to adulthood, demonstrating that TGF-b1 is critically involved in limiting inflammation. Activin and TGF-b are potent immunoregulatory mediators, with pro- and anti-inflammatory effects.

They are highly expressed at sites of tissue injury and infection. TGF-bs attract inflammatory cells to sites of injury and activin stimulates production of proinflammatory mediators. However, they both inhibit B and T lymphocyte proliferation. TGF-b promotes the generation of regulatory T cells and inhibits survival of eosinophils, whilst activin A antagonizes the

TRANSFORMING GROWTH FACTOR BETA (TGF-b) FAMILY OF MOLECULES 281

Binding Proteins and Receptors

Regulation of lung development Regulation of inflammation Modulate cell proliferation TGF-


Promote cell adhesion

Activin

Regulate cell differentiation Regulation of extracellular matrix metabolism Regulation of apoptosis Figure 2 Common biological functions of TGF-b and activin.

effects of inflammatory mediators such as interleukin (IL)-1 and IL-6, indicating they have roles in the control of local and systemic inflammatory responses. TGF-bs and activins play important roles in tissue homeostasis and wound healing. They promote cell adhesion, differentiation of cells including lymphocytes, fibroblasts and smooth muscle cells, and regulate apoptosis of resident and inflammatory cells. In addition, they suppress the growth of epithelial cells, endothelial cells, and lymphocytes but have biphasic or stimulatory effects on mesenchymal cells such as fibroblasts and smooth muscle cells, with growth promotion at low concentrations and inhibition at higher concentrations. They stimulate the expression of extracellular matrix (ECM) proteins including fibronectin and collagen I. In addition, TGF-bs also stimulate expression of tenascin, thrombospondin, elastin, vitronectin, and proteoglycans. It also limits ECM degradation by modulating the expression, secretion, and activity of proteases and their inhibitors including matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases, plasmin, plasminogen activator, and plasminogen activator inhibitor-1, whereas activin stimulates MMP-2 expression. Although TGF-bs and activins appear to have broadly similar functions they do not necessarily overlap. The timing and degree of expression are often different developmentally, and in response to wound healing and inflammation. In addition, the activins are secreted as an active molecule, whereas TGF-bs are secreted in an inactive form and thus potentially play distinct but complimentary roles in the complex regulation of inflammation and wound healing.

TGF-bs and activins bind to a number of specific cell surface signaling receptors as well as a number of cell free- and cell surface-associated proteins that have no signaling capacity. Characteristic features of TGF-b superfamily signaling receptors include the presence of a toxin fold in the extracellular ligand-binding domain, a single transmembrane domain, and a serine/threonine kinase intracellular domain. Type I receptors are distinguished from type II receptors by the presence of a glycine-serine (GS)-rich juxtamembrane domain, which is critical for their activation by type II receptors. The TGF-bs and activins interact predominantly with activin receptor-like kinase5 (Alk5) and Alk4 type I receptors, respectively, but both can also interact with Alk1 and Alk2. One type II receptor, TbRII, has been identified for the TGF-bs, whilst two have been identified for the activin receptor II (ActRII) and ActRIIB. In order to signal, the ligand binds the constitutively active type II receptor, inducing a conformational change in the extracellular domain, which allows recruitment of type I receptors to form a tetrameric complex leading to serine phosphorylation of the type I receptor by the type II receptor. This results in a conformational change in the GS box, enabling ATP binding and phosphorylation of receptor R-Smads 2 or 3, followed by subsequent association of R-Smads with the common co-Smad 4 to form a heterotrimeric complex, which translocates to the nucleus and regulates transcriptional responses. Recruitment of alternative type I receptors can lead to different functional outcomes. There is also increasing evidence for alternative signaling via the mitogen-activated protein kinase, PI3 kinase, and protein kinase C pathways. Signaling is regulated by a number of transmembrane proteins. Betaglycan, also called the TGF-b type III receptor, is able to bind TGF-b1 and b2 whilst endoglin, a member of the glypican family of proteoglycans, binds TGF-b1, TGF-b3, and activin, and these are thought to be involved in presentation of the ligand to the type II receptor. In addition, the cytoplasmic tail of betaglycan interacts with TbRII enhancing signaling. Endoglin may have similar signal enhancing functions in conjunction with ActRII, since its intracellular domain has sequence similarity with betaglycan. Other proteins may also be required for the assembly of an active receptor complex and for recruitment of R-Smads to the receptor complex. For example, cripto, a member of the EGF-CFC family of GPI-linked membrane anchored proteins and SARA (Smad anchor for receptor activation) are required for assembly of active Alk4/ActRII and

282 TRANSFORMING GROWTH FACTOR BETA (TGF-b) FAMILY OF MOLECULES

Alk5/TbRII receptor complexes, respectively, and are required for recruitment of receptor Smads. Receptor activation is further regulated by competition for type I receptors between R-Smads and inhibitory Smad 7, which inhibits TGF-b and activin signaling. Inhibins can block activin activity by competing for the binding sites on the type II receptor and preventing recruitment of type I receptors. Furthermore, transmembrane proteins, such as nonmetastatic gene A protein, have extracellular domains with sequence similarity to type I receptors, and are thought to act as decoy receptors. Soluble forms of betaglycan, and possibly endoglin, exist that bind TGF-b and prevent binding to signaling receptors. Similarly, activins bind to soluble follistatin inhibiting its activity. Active TGF-b can also bind to a number of extracellular matrix proteins, including fibronectin, collagen IV, fibromodulin, decorin, and biglycan, which generally inhibit its activity. Receptor internalization and endosomal trafficking play roles in both, promoting human homolog of mothers against decapentaplegic (Smad) signaling and proteosomal degradation. Internalization of receptor/R-Smad/Smad anchor for receptor activation (SARA) complexes to endosomes containing early endosomal protein, EEA1, is necessary to propagate Smad phosphorylation and signal transduction, whereas ubiquitin ligases, Smad ubiquitin regulatory factor (Smurf) 1 and 2, complex with type I receptors and Smad 7, enhancing turnover of receptors and Smad 7.

TGF-b and Activin in Respiratory Disease

Studies from animal models using various strategies to modulate TGF-b and activin activity suggest that they have both protective and deleterious effects. TGF-b1 is thought to limit inflammation and airway hyperresponsiveness but promotes subepithelial deposition of ECM proteins in asthma. It is also thought to limit inflammation in other conditions including pneumonia and COPD. However, this function is considered to be deleterious in tuberculosis where its immunosuppressive effects may allow uncontrolled mycobacterial replication. TGF-b also plays fundamental roles in the development of fibrosis since overexpression or exogenous TGF-b induces airway and parenchymal fibrosis, and inhibition of TGF-b1 or TGF-b2 activity inhibits the development of airway remodeling and pulmonary fibrosis. Inhibition of TGF-b/activin signaling also prevents the development of pulmonary fibrosis, airway remodeling, and bronchiolitis obliterans but enhances antigen-induced airway inflammation and hyperreactivity. Numerous experimental approaches have been developed to modulate TGF-b and activin or their activity. These include: prevention of their synthesis by antisense oligonucleotides or small interfering ribonucleic acid (siRNA); inhibition of TGF-b activation; disruption of ligand–receptor interactions using antibodies, soluble receptors, natural inhibitory proteins and peptides, or prevention of receptor phosphorylation; and interference with Smad signaling by upregulation of Smad7. Of these, antibodies to TGFb1, TGF-b2, and TGF-b1-3, as well as antisense to TGF-b1 and TGF-b2 are currently in nonrespiratory clinical trials. In addition, several Alk5 receptor kinase inhibitors are in preclinical development.

TGF-b and activin are upregulated in situations associated with injury, repair, and inflammation. TGF-b has been implicated in many lung diseases, although most studies have concentrated on TGF-b1, which is increased in asthma, idiopathic pulmonary fibrosis (IPF), chronic lung disease of prematurity, sarcoidosis, chronic obstructive pulmonary disease (COPD), tuberculosis, brochiolitis obliterans, pneumonia, acute respiratory distress syndrome, pulmonary hypertension, and non-small cell lung cancer. There is also evidence for increased TGF-b2 in asthma, IPF, COPD, and pneumonia, and TGF-b3 in IPF. Studies of activin in lung disease are more restricted but activin A expression has been reported to be increased in IPF, asthma, and pulmonary hypertension. In addition, there is evidence for increased Alk5 expression in tuberculosis, and TGF-b/ activin signaling in asthma and COPD. Furthermore, TGF-b polymorphisms have been associated with asthma and COPD.

See also: Acute Respiratory Distress Syndrome. Apoptosis. Asthma: Overview. Bronchoalveolar Lavage. Bronchopulmonary Dysplasia. Chronic Obstructive Pulmonary Disease: Overview. Connective Tissue Growth Factor. Cytoskeletal Proteins. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes. Fibroblasts. Genetics: Overview. Interstitial Lung Disease: Overview. Leukocytes: Mast Cells and Basophils; Eosinophils; T cells; Pulmonary Macrophages. Lipid Mediators: Overview. Lung Development: Overview. Matrix Metalloproteinases. Matrix Metalloproteinase Inhibitors. Mesothelial Cells. Mesothelioma, Malignant. Myofibroblasts. Occupational Diseases: Overview. Oncogenes and Proto-Oncogenes: Overview. Pneumonia: Overview and Epidemiology. Progressive Systemic Sclerosis. Pulmonary Fibrosis. Radiation-Induced Pulmonary Disease. Serine Proteinases. Signal Transduction. Smooth Muscle Cells: Airway. Systemic Disease: Sarcoidosis. Transgenic Models.

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Further Reading Ball EMA and Risbridger GP (2001) Activins as regulators of branching morphogenesis. Developmental Biology 238: 1–12. Bartrum U and Speer CP (2004) The role of transforming growth factor b in lung development and disease. Chest 125: 754–765. Blobe GC, Schiemann WP, and Lodish HF (2000) Role of transforming growth factor b in human disease. New England Journal of Medicine 342: 1350–1358. Chen Y-G, Lui HM, Lin S-L, Lee JM, and Ying S-Y (2002) Regulation of cell proliferation, apoptosis, and carcinogenesis by activin. Experimental Biology and Medicine 227: 75–87. de Caestecker M (2004) The transforming growth factor-b superfamily of receptors. Cytokine and Growth Factor Reviews 15: 1–11. Derynck R and Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-b family signalling. Nature 425: 577–584. Gorelik L and Flavell RA (2002) Transforming growth factor-b in T-cell biology. Nature Reviews: Immunology 2: 46–53. Howell JE, McAnulty RJ (2006) TGF-b: its role in asthma and therapeutic potential. Current Drug Targets 7(Pt. 4).

Jones KL, de Kretser DM, Patella S, and Phillips DJ (2004) Activin A and follistatin in systemic inflammation. Molecular and Cellular Endocrinology 225: 119–125. Massague J (1998) TGF-b signal transduction. Annual Review of Biochemistry 67: 753–791. Moustakas A, Souchelnytskyi S, and Heldin C-H (2001) Smad regulation in TGF-b signal transduction. Journal of Cell Science 114: 4359–4369. Pangas SA and Woodruff TK (2000) Activin signal transduction pathways. Trends in Endocrinology and Metabolism 11: 309– 314. Saharinen J, Hyytiainen M, Taipale J, and Keski-Oja J (1999) Latent transforming growth factor-b binding proteins (LTBPs) – structural extracellular matrix proteins for targeting TGF-b action. Cytokine and Growth Factor Reviews 10: 99–117. Shi Y and Massague J (2003) Mechanisms of TGF-b signalling from cell membrane to the nucleus. Cell 113: 685–700. ten Dijke P and Hill CS (2004) New insights into TGF-b-Smad signalling. Trends in Biochemical Sciences 29: 265–273. Thompson TB, Cook RW, Chapman SC, Jardetzky TS, and Woodruff TK (2004) Beta A versus beta B: is it merely a matter of expression? Molecular and Cellular Endocrinology 225: 9–17.

TRANSGENIC MODELS C G Lee and J A Elias, Yale University School of Medicine, New Haven, CT, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Transgenic models are being used to define the in vivo effector function(s) of genes by enhancing the expression of individual proteins in a specific organ or tissues. The mouse has become the animal of choice for these studies mainly because of its welldefined genetic system and its extensively investigated immune, injury, inflammatory, and healing responses. To generate a transgenic mouse, a DNA construct is prepared that contains the target gene linked to a promotor that is designed to drive the expression of the transgene in the desired organ and/or tissue. The Clara cell 10-kDa secretory protein (CC10) or the surfactant protein C (SP-C) promoters have been successfully used to target gene expression specifically to the lung. Inducible transgenic systems, often using tetracycline, have also been used to allow the investigator to control the timing of transgene activation. A number of cytokines, growth factors, and other genes have been overexpressed in the lung. These transgenic mice have elegant phenotypes of inflammation and airway and parenchymal remodeling that simulate in many important ways the lesions and pathophysiology of a variety of diseases, including asthma, chronic obstructive lung disease, bronchopulmonary dysplasia, pulmonary fibrosis, and even lung cancer, providing valuable insights into the pathogenesis of human disorders.

Introduction Advancements in the field of molecular biotechnology have placed us squarely in the postgenomic era

and provided us with an impressive amount of genetic sequence information on humans and other animals. As a result of the federal and industrial human genome projects, we now know that there are approximately 40 000 protein-encoding genes in the human genome. However, the function(s) of the majority of these genes is still virtually unknown. As a result, characterization of the effector responses of these genes is the next major challenge for the biologic scientific community. This functional analysis will require a variety of scientific approaches. In keeping with the concept of ‘in vivo veritas’, the use of transgenic overexpression models has proven particularly powerful in this regard. The mouse has become the target animal of choice in generating these models. This preference is the result of our extensive amount of knowledge regarding the murine genetic system. It is also due to the extensive investigations of the immune, injury, inflammatory, and healing responses in murine systems. This provides great conveniences in designing and manipulating genes that one believes may be involved in human disease.

Generation of Overexpressing Transgenic Mice The first successful transgenic mice were generated in 1980. Since then, the use of transgenic animals in the study of biology and pathology has become a standard investigative tool. To generate transgenic mice,

284 TRANSGENIC MODELS

DNA encoding the desired transgene is isolated as a cDNA or a genomic fragment. It is then linked to a promoter that will successfully target it to the desired organ and/or cell type. When a cDNA is used, intronic sequences that enhance translational efficiency are added. If temporal regulation of transgene expression is desired, genetic materials that allow transgene activation to be externally controlled are also utilized. Once the construct is completed, male and female mice are mated, and fertilized eggs are washed out of the oviduct of the female mice. The completed construct is then inserted into these fertilized eggs by direct microinjection. The manipulated eggs are then placed in the uterus of a pseudopregnant female mouse (a female mouse that has been mated with a vasectomized male mouse). She then carries to term and delivers a litter of pups, some of which will have the transgene and some of which will not. Transgenic mice and transgene-negative littermate controls can be differentiated by genotyping with polymerase chain reactions or Southern blotting using DNA from tail biopsies from each pup. Thus, the investigator is able to compare the phenotypes of transgenic and littermate control mice that were born by the same mother, on the same day, exposed to the same environment, and that differ only in the transgene that was inserted.

Regulation of Transgene Expression To target transgenes to the lung, the Clara cell 10-kDa protein (CC10) and surfactant apoprotein C (SP-C) promoters are used most frequently. The PCC10

rtetR

former targets transgenes to airway Clara cells and, to a lesser extent, alveolar type II epithelial cells. In contrast, the SP-C promoter targets predominantly the alveolar epithelium. In early developmental overexpression systems these promoters are used to directly drive gene expression. Thus, in these systems, the transgene is activated, and during lung development the promoter is normally induced. In both cases, this results in transgene activation during in utero development and constitutive transgene expression thereafter. The standard lung-targeted overexpression transgenic approach has provided remarkable insights into pulmonary biology. It does, however, have a number of limitations. Specifically, the in utero activation confounds phenotypic interpretation by superimposing growth and/or development-related abnormalities on abnormalities caused by the gene product in the adult animal. It also limits the use of the system in the overexpression of genes that can cause fetal lethality by altering lung development. Lastly, this system is limited in its ability to model waxing and waning disease processes (e.g., asthma) and its ability to assess the natural history of transgene-induced phenotypic alterations. In an attempt to overcome these limitations, investigators have developed lung-specific overexpression systems in which transgene expression can be externally regulated. In general, these systems are based on the generation of mice with two transgenic constructs. A commonly used double transgenic system is illustrated in Figure 1. In this system, the first construct contains the CC10 promoter, the reverse tetracycline VP16 activator

rtTA

No Dox

Tet operator

PCMV

Transgene

No expression

Dox

Tet operator

PCMV

Transgene

Transgene

Figure 1 Schematic illustration of constructs for inducible expression of transgene based on tetracycline responsive regulatory system. The CC10 promotor drives reverse tetracycline-controlled transcriptional activator (rtTA) expression specifically in the lung. In this system, transgene is expressed only in the presence of doxycycline (Dox) after binding of rtTA at the tet-O-CMV promotor region of transgene construct.

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transactivator (rtTA), and human growth hormone (hGH) intronic, nuclear localization, and polyadenylation sequences. The rtTA is a fusion protein made up of a mutated tet operator-binding protein (tet-OBP) and the herpes virus VP-16 transactivator. The second construct contains a polymeric tetracycline operator (tet-O), minimal cytomegalovirus (CMV) promoter, the cytokine gene of interest, and hGH intronic, polyadenylation, and nuclear localization signals. In this system, the CC10 promoter directs the expression of rtTA to the lung. In the presence of doxycycline (dox) (which is added to the animal’s drinking water or provided via a subcutaneous pellet), rtTA binds, in trans, to the tet-O, and VP-16 (a powerful transcriptional activator) activates the transgene. In the absence of dox, rtTA binding does not occur or occurs only at a low level, and the transgene is not activated or is transcribed at a low level.

Transgenic Models of Th2 Inflammation and Remodeling in Asthma Asthma is a chronic inflammatory disorder characterized by airway obstruction, recurrent bronchospasm, and cough. Asthmatics also manifest airway hyperresponsiveness, an enhanced sensitivity to the bronchospasm-provoking effects of non-specific agents such as methacholine. The pathology of asthmatic airways is notable for eosinophil-, macrophage-, and lymphocyte-rich inflammation and varying degrees of airway remodeling with mucus metaplasia, subepithelial airway fibrosis, smooth muscle hypertrophy and hyperplasia, and angiogenesis. The current concept of asthma pathogenesis suggests that many of the clinical and pathologic features of asthma are the result of exaggerated T-helper (Th)-2type inflammation. To understand in vivo effects of each Th2 cytokine, lung-specific interleukin (IL)-4, IL-5, IL-9, IL-10, and IL-13 transgenic mice have been generated and characterized. In accord with the belief that Th2 cytokines play an important role in asthma pathogenesis, each has generated asthma-like tissue and/or physiologic alterations. Transgenic IL-4 caused lymphocytic and eosinophilic inflammation without airway hyperreactivity and airway wall remodeling. The modest nature of this response is in keeping with other studies that suggest that IL-4 is more active in the initiation rather than the effector phases of Th2 inflammation. Transgenic IL-5 caused more prominent responses, including peribronchial eosinophilic inflammation, goblet cell hyperplasia, subepithelial collagen deposition, and airway hyperresponsiveness to methacholine. Transgenic IL-9 also caused eosinophilic inflammation, mast cell

hyperplasia, and bronchial hyperreponsiveness. Interestingly, many of the effects of IL-9 were due to the ability of IL-9 to induce other Th2 cytokines, especially IL-13. The transgenic expression of IL-10 caused complex pulmonary alterations, where it inhibited lipopolysaccharide-induced inflammation and induced lymphocytic inflammation, goblet cell metaplasia, and subepithelial fibrosis. Interestingly, the IL-10-induced mucus response was also mediated by IL-10-induced IL-13. Among the most impressive asthma-like phenotypes were those caused by transgenic IL-13. In these mice, IL-13 engenders an eosinophil-, macrophage-, and lymphocyte-rich inflammatory response; airway fibrosis; mucus metaplasia; and airway hyperresponsiveness. These responses were not mediated by other Th2 cytokines, suggesting that IL-13 is a final common pathway in Th2 effector responses. By breeding these mice with mice with null mutations of other genes and treating these mice with chemical blockers or neutralizing antibodies, insights have been obtained into the mechanisms that mediate these IL-13induced responses. These studies have highlighted the important roles that chemokines and chemokine receptors, transforming growth factor beta (TGF-b1), proteases, and adenosine play in IL-13-induced alterations. It has been shown that the inducible overexpression of vascular endothelial growth factor (VEGF) in the lung also induces inflammation, mucus metaplasia, smooth muscle hyperplasia, airway hyperresponsiveness, and neoangiogenesis. These mice also had a defect in tolerance to inhaled antigen and enhanced numbers of activated pulmonary dendritic cells. Interestingly, VEGF was a weak stimulator of IL-13 in this setting. However, the mucus metaplasia was the only part of this impressive phenotype that was mediated by VEGF-induced IL-13 production. Taken together, it is clear that the transgenic modeling of Th2 cytokines and growth factors elegantly generates asthma and Th2 response-relevant phenotypes. These mice can then be utilized to generate hypotheses that can be evaluated in human tissues.

Transgenic Evaluations of Pulmonary Fibrotic Responses Exaggerated scarring is a cause of morbidity and/or mortality in a variety of pulmonary disorders. This is readily appreciated in idiopathic pulmonary fibrosis (IPF), a fatal progressive lung disease characterized by epithelial damage, fibroproliferative matrix deposition, enhanced collagen accumulation, and parenchymal remodeling. Unfortunately, the mechanisms that mediate the tissue responses in IPF are poorly

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understood. Growth factors such as TGF-b and platelet-derived growth factor (PDGF), which have the ability to directly stimulate fibroblast proliferation, myofibroblast differentiation, and extracellular matrix deposition, have been implicated in this and related disorders. To address these possibilities, the SP-C promoter has been used to overexpress human PBGD-B in the murine lung. These transgenic mice had focal pulmonary fibrosis, inflammation, and enlarged airspaces (emphysema), suggesting that PDGF by itself cannot account for all the alterations in IPF tissues. TGF-b is expressed in an exaggerated manner by a variety of cells, including macrophages and epithelial cells, in tissues from IPF patients. The important role that TGF-b plays in pulmonary fibrogenesis was experimentally supported in recent transgenic evaluations. These studies demonstrated that transgenic bioactive TGF-b1 causes epithelial injury that is followed by impressive airway and parenchymal fibrosis and myofibroblast accumulation (Figure 2). Interestingly, significant alveolar destruction and eventually honeycomb lung were also seen. These transgenic models are in vivo experimental systems in which investigators can characterize the molecular pathogenesis and reversibility of these responses in attempts to define new therapies for fibrotic disorders.

Transgenic Evaluations of Alveolar Remodeling and Chronic Obstructive Pulmonary Disease Chronic obstructive pulmonary disorder (COPD) represents one of the largest unmet medical needs in the world. In the US, it affects approximately 16 million people, accounts for 13% of hospitalizations, and is the fourth leading cause of death. The single most important factor in the development of emphysema in the Western world is cigarette smoke exposure, which causes chronic inflammation and destruction and dilatation of the terminal airspaces in susceptible individuals. A variety of models in rodents, dogs, guinea pigs, monkeys, and sheep have been used to elucidate the complex pathophysiology of COPD and other diseases associated with alveolar enlargement. With the advent of genetic engineering, transgenic mice with prominent alveolar alterations have been noted. The overexpression of PDGF-A, PDGF-B, tumor necrosis factor (TNF)-a, IL-11, TGF-b1, and IL-6 has been shown to cause alveolar enlargement. In many of these animals, the transgene interferes with normal lung development, making them models of potential import for disorders such as bronchopulmonary dysplasia. Interestingly, inducible overexpression of the Th2 cytokine IL-13 or the

(a)

(b) Figure 2 Representative lung histology of the TGF-b1 transgenic mice showing prominent airway fibrosis and alveolar remodeling (trichrome staining,  20). (a) Nontransgenic mice. (b) Transgenic mice. Reproduced from The Journal of Experimental Medicine, 2004, vol. 200(3), pp. 377–389, by copyright permission of The Rockefeller University Press.

Th1 cytokine interferon-g also produced prominent airspace enlargement. These alterations were seen in adult lungs, suggesting that this alveolar enlargement was due to alveolar destruction comparable to that seen in pulmonary emphysema. In both mice, transgene activation caused inflammation and prominent protease–antiprotease alterations. In accord with these findings, the overexpression of human interstitial collagenase (matrix metalloproteinase-1) also causes prominent airspace enlargement. The transgenic overexpression of placental growth factor driven by the phosphoglycerate kinase promotor has been shown to induce late-onset emphysema.

Transgenic Evaluations for Lung Cancer Lung cancer can be grossly divided into four major histologic subtypes: adenocarcinoma, squamous cell

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carcinoma, small cell carcinoma, and large cell carcinoma. Among these, adenocarcinoma is the most common type of lung cancer in the US. Although human studies provide insights into the molecular mechanisms involved in the progression of lung cancer, they do not allow the investigator to serially evaluate tumor progression in the setting of a defined genetic background. In addition, they do not allow the investigator to initiate experimental manipulations designed to define the molecular interactions that mediate disease progression. To address these limitations, transgenic models have been developed. Models of adenocarcinoma have been generated by targeting oncogenes using a variety of promoters, including the mammary tumor virus long terminal repeat (MMTV) promoter, immunoglobulin enhancer–simian virus 40 (SV40) fusion promotor, or human albumin promotor. The most successful example of the transgenic modeling of lung cancer is the overexpression of mutant forms of the p53 gene using the CC10 or SPC promoters. This intervention significantly increases the development of pulmonary adenocarcinomas. Insulin-like growth factor and the SV40 large T antigen (Ta) have also been used in transgenic systems to model certain types of lung cancer. With the introduction of inducible transgenic systems, the usefulness of transgenic mice in lung cancer research is now being extended from studies of carcinogenesis to the identification of markers of disease development and/or progression.

Limitations and Future Applications Transgenic mice provide impressive insights into the in vivo effector functions of specific genes. However, the data from these models should be interpreted with caution. This is the result of a number of features of the transgenic system, including the complexities of the timing of transgene expression as reviewed previously. This also includes the random nature of transgene integration, which by itself can generate genetic disruptions. These alterations can lead to phenotypes that are not mediated by the transgene in question. This issue, however, can be easily addressed by generating at least three independent lines of transgenic mice for each transgene that is being evaluated. If the same phenotype is seen in all the lines, it is safe to assume that the phenotype is transgene induced and not due to an insertion site artifact. Lastly, caution is needed because of important structural and physiologic differences between mice and humans. However, these differences should not be offsetting. Instead, transgenic mice should be

viewed as hypothesis generators for studies in human samples and tissues. They are also outstanding systems in which potential therapeutic agents can be screened and/or evaluated. See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Pulmonary Fibrosis.

Further Reading Elias JA, Geba GP, Tang W, et al. (1996) Transgenic modeling of cytokines in the investigation of pulmonary disease. Chest 109: 69S–73S. Elias JA, Lee CG, Zheng T, et al. (2003) New insights into the pathogenesis of asthma. Journal of Clinical Investigation 111: 291–297. Homer RJ and Elias JA (2005) Airway remodeling in asthma: therapeutic implications of mechanisms. Physiology (Bethesda) 20: 28–35. Kwak I, Tsai SY, and DeMayo FJ (2004) Genetically engineered mouse models for lung cancer. Annual Review of Physiology 66: 647–663. Lee CG, Cho SJ, Kang MJ, et al. (2004) Early growth response gene 1-mediated apoptosis is essential for transforming growth factor beta1-induced pulmonary fibrosis. Journal of Experimental Medicine 200: 377–389. Lee CG, Link H, Baluk P, et al. (2004) Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung. Nature Medicine 10: 1095–1103. Mahadeva R and Shapiro SD (2002) Chronic obstructive pulmonary disease 3: experimental animal models of pulmonary emphysema. Thorax 57: 908–914. Ramos-Barbon D, Ludwig MS, and Martin JG (2004) Airway remodeling: lessons from animal models. Clinical Reviews in Allergy & Immunology 27: 3–21. Vasquez YR and Spina D (2000) What have transgenic and knockout animals taught us about respiratory disease? Respiratory Research 1: 82–86. Vicencio AG, Lee CG, Cho SJ, et al. (2004) Conditional overexpression of bioactive transforming growth factor-beta1 in neonatal mouse lung: a new model for bronchopulmonary dysplasia? American Journal of Respiratory Cell and Molecular Biology 31: 650–656. Whitsett JA, Glasser SW, Tichelaar JW, et al. (2001) Transgenic models for study of lung morphogenesis and repair: Parker B. Francis lecture. Chest 120: 27S–30S. Zhao B, Magdaleno S, Chua S, et al. (2000) Transgenic mouse models for lung cancer. Experimental Lung Research 26: 567– 579. Zheng T, Zhu Z, Wang Z, et al. (2000) Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema. Journal of Clinical Investigation 106: 1081–1093. Zhu Z, Lee CG, Zheng T, et al. (2001) Airway inflammation and remodeling in asthma lessons from interleukin 11 and interleukin 13 transgenic mice. American Journal of Respiratory and Critical Care Medicine 164: S67–1370. Zhu Z, Zheng T, Lee CG, et al. (2002) Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling. Seminars in Cell and Developmental Biology 13: 121–128.

288 TRAUMA, CHEST / Bronchopleural Fistula

Transplantation

see Surgery: Transplantation.

TRAUMA, CHEST Contents

Bronchopleural Fistula Postpneumonectomy Syndrome Subcutaneous Emphysema

Bronchopleural Fistula S A Sahn and J T Heidecker, Medical University of South Carolina, Charleston, SC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Bronchopleural fistula is a persistent communication between the bronchi and pleural space for at least 10 days. It can be caused by blunt or penetrating trauma, blast injury, or barotrauma and usually persists because of an acute or chronic underlying lung disease. Signs may include crepitus, hypotension, and cardiovascular collapse with pneumomediastinum or pneumothorax with or without mediastinal shift on chest radiograph. The pathogenesis varies from direct lung injury from rib fractures, bronchial laceration, alveolar destruction, and airway stripping from blast injuries, to alveolar rupture with interstitial gas accumulation from shear forces in ventilated patients with acute lung injury. With rupture of the pulmonary interstitial air into the pleural space, a bronchopleural fistula will develop if there is impaired wound healing. Supportive therapy with lung protective ventilation (low tidal volumes and adequate positive end-expiratory pressure) fosters fistula healing by minimizing lung injury. High-frequency ventilatory techniques and independent lung ventilation may be used in refractory cases. Surgical oversewing or resection of blebs, bronchial stump repair, lobectomy, and pleurodesis may be used in selected cases. Bronchoscopic occlusion of the involved bronchial segment is an option in patients too ill for surgery.

Introduction Whether from blunt or penetrating trauma, blast injury, or barotrauma, a bronchopleural fistula (BPF) is arbitrarily defined as persistent communication from the bronchi to the pleural space for X10 days. Pneumothorax is often the preceding event in the development of a BPF; however, penetrating thoracic trauma, blunt thoracic trauma, blast injury, iatrogenesis, and barotrauma from mechanical ventilation uncommonly result in persistent BPFs. In a review of 1199 pneumothorax cases over 17 years (excluding barotrauma), BPF occurred in only 3% of cases. As

the number of patients requiring mechanical ventilation with acute respiratory distress syndrome (ARDS) increases, the incidence of BPF increases in these patients. The management of traumatic BPF can be problematic. Providing adequate tidal volume to mechanically ventilated patients may be a major problem. A persistent BPF can also lead to pleural space infection. A number of strategies for managing BPF have met with varying degrees of success. Ventilator strategies, closed-tube thoracostomy, bronchoscopic interventions, and surgical procedures have been used for treatment of persistent BPF. The choice of intervention depends upon the severity of illness, type of lung injury, and the familiarity of pulmonologists and surgeons with the available therapeutic modalities.

Etiology Although pneumothorax is common in penetrating trauma from war wounds, BPF was a rare complication occurring in only one of 235 patients. The incidence of BPF is less than expected because many patients with penetrating trauma will have lobectomy or pneumonectomy due to vascular injury and because most of these patients are young and have healthy lungs. Although blunt chest trauma from motor vehicle accident is a common occurrence, the development of BPF is not frequent, occurring in 24 of 515 (5%) in one series. Previously, BPF was a relatively common complication of lung resection surgery. However, its incidence has decreased dramatically with the advent of improved surgical techniques. BPF can be avoided in most surgical patients with proper initial postoperative management including clearance of secretions to avoid atelectasis and diligent monitoring of chest tube patency and placement. Long, large-diameter postoperative bronchial stumps predispose patients to postpulmonary resection BPF. Covering the stump with a skeletal muscle flap or omental tissue appears to decrease the

TRAUMA, CHEST / Bronchopleural Fistula 289

risk for development of BPF. In contrast, blast injury commonly results in BPF. In one series, 33% of patients who suffered blast injury developed BPF. The form of traumatic BPF that most clinicians will encounter is BPF due to barotrauma.

development of diffuse alveolar damage and giant emphysematous blebs. Patients with deceleration injuries or penetrating trauma may have proximal bronchial laceration or rupture with initially preserved pulmonary parenchymal architecture.

Pathology The pathologic features of BPF are usually a reflection of the initial cause of the injury, the disease state of the underlying lung, or reflect diffuse alveolar damage from acute lung injury. Underlying pathologic conditions include emphysematous blebs or bullae or pleural thickening. Acute lung injury as represented by diffuse alveolar damage pathologically leads to surfactant loss, flooding of alveoli with proteinaceous material, and regional alveolar collapse. Local areas of poor compliance predispose to the development of barotrauma leading to pneumothorax and subsequent BPF due to delayed lung healing. Blast injury has a similar pathology with

Clinical Features The diagnosis of BPF is straightforward when there is a persistent communication between the bronchi and pleural space for 10 or more days. Clinical signs include contralateral tracheal deviation, crepitus, hypotension, cardiovascular collapse, and persistent air leak from a chest tube. Radiographic evidence of a BPF may include a pneumothorax with or without mediastinal shift, lucency about the heart border indicating pneumomediastinum, a deep sulcus sign, or subcutaneous emphysema may be observed (see Figure 1). Some patients with blunt or penetrating trauma with proximal tracheobronchial injury may

Figure 1 Some radiographic presentations of bronchopleural fistula. (a) Deep sulcus sign in right hemithorax in supine radiograph. (b) Note right basilar lack of lung markings with contralateral mediastinal shift indicating tension pneumothorax. (c) Note lucency about the pericardium indicating pneumopericardium.

290 TRAUMA, CHEST / Bronchopleural Fistula

show atelectasis along the lateral chest wall with pneumothorax on chest radiograph.

Pathogenesis Pneumothorax and subsequent BPF from blunt chest trauma is often caused by rib fractures. However, BPF can occur in the absence of rib fractures as a result of tracheobronchial tear from deceleration injuries. A BPF rarely occurs (approximately 1%) as a result of degeneration of the bronchial stump following pulmonary resection. The pressure waves in blast injury cause widespread alveolar destruction and airway stripping, resulting in giant emphysematous blebs and air dissemination into the pleura and mediastinum. Further, these patients develop severe acute lung injury, which impairs healing of BPFs. Barotrauma, followed by iatrogenic pneumothorax from procedures, is the commonest cause of persistent BPF seen in the intensive care unit. Risk factors for BPF from barotrauma include pneumonia, COPD, lung cancer, and ARDS. Approximately 3– 15% of patients on mechanical ventilators will develop pneumothorax. Progression to BPF is rare in the absence of ARDS. Heffner and colleagues documented an incidence of recurrent pneumothorax in non-ARDS ventilated patients of 8% (2 of 26). In contrast, pneumothorax from barotrauma is a common complication of ARDS occurring in 10–50% of ARDS patients. In ARDS, there is pulmonary inflammation, increased vascular permeability, extravasation of proteinaceous fluid into alveoli, and decreased surfactant production. There is nonuniform injury leading to microatelectasis with shunt and ventilation/perfusion (v/q) mismatch. The lungs become increasingly stiff both globally and locally. This decreased lung compliance causes bronchioles and alveoli to repeatedly snap open and close. Shear forces may develop in areas between atelectatic and aerated areas causing further lung injury. These shear forces cause extravasation of air from the alveolar spaces into the pulmonary interstitium. The air tracks along the interstitium along collagen sheaths and ruptures into adjacent alveoli, mediastinal pleura, or visceral pleura leading to subpleural cysts and bullae, pneumomediastinum and subcutaneous emphysema, or pneumothorax, respectively. Figure 2 depicts the pathogenesis of pneumothorax from barotrauma. Flow through a BPF is directly proportional to the size of the fistula and inversely proportional to the compliance of the lungs. Thus, patients with ARDS and diffuse lung injury can develop BPF with flow rates greater than 10 l min
1. Often in these patients, increased pressures are required during mechanical

ventilation to maintain oxygenation. From the data in the ARDS network controlled trials, higher positive end-expiratory pressure (PEEP) is more predictive of the development of barotrauma than peak inspiratory pressure, plateau pressure, driving pressure, and mean airway pressure. Given that volutrauma has been shown to increase mortality in ARDS, PEEP as an independent predictor for barotrauma in ARDS probably reflects that patients needing high levels of PEEP have worse lung injury.

Animal Models Many models in mechanically ventilated animals describe the pathophysiologic process of acute lung injury that leads to pneumothorax and persistent BPF from barotrauma. Increased lung volumes from mechanical ventilation lead to increased vascular permeability in rat models. In rabbits with and without plaster chest binders, volutrauma as opposed to barotrauma was responsible for increased vascular permeability. It has also been demonstrated that PEEP decreases alveolar flooding from this permeability but does not decrease the pathologic process. Animal models have also demonstrated that there is alveolar interdependence, which allows inflation of one alveolus to augment inflation of others in its unit. However, in acute lung injury and high-volume ventilation, there is increased capillary permeability with regional flooding of alveoli causing breakdown of alveolar interdependence and thus increased regional shear forces leading to barotraumas. Further, the process of lung damage from surfactant loss has been shown to be dependent upon the presence of functional granulocyte cells. Surfactant loss in the absence of these cells has not been shown to lead to alveolar damage in animal models.

Management and Current Therapy Though the management of traumatic BPF includes thoracoplasty, decortication, and bronchial stump stapling, there is a trend toward nonsurgical treatment. Thus, pulmonologists and critical care physicians must understand the physiology of BPF and how to drain and limit air leaks through ventilator techniques, bronchoscopic maneuvers, and thoracostomy drainage systems. The initial management of BPF entails adequate pleural drainage and pleural apposition. Pleural apposition will not only hasten lung healing, but will also lessen the risk of pleural space infection. A thoracostomy tube must be of sufficient diameter to drain up to 16 l min
1 of air. Flow of air through a

TRAUMA, CHEST / Bronchopleural Fistula 291

High-volume lung ventilation Low-volume lung ventilation Regional hyperinflation

PEEP increases

PEEP decreases

Surfactant inactivation

Higher microvascular permeability

Atelectasis Increased filtration Pulmonary edema

Alveolar flooding PEEP decreases

PEEP decreases

Repeated opening and closing of alveoli

Leukocyte activation and margination

Decrease in recruited lung volume

Distal lung damage

Regional shear forces at junctures of aerated and atelectatic lung

Barotrauma

Rupture of alveoli

Gas escapes into alveolar interstitial space

PEEP increases

Air tracks along fibrous sheaths

Air ruptures into mediastinal pleura causing pneumomediastinum and subcutaneous emphysema

Air ruptures into mediastinal pleura causing pneumomediastinum and subcutaneous emphysema

Air ruptures into alveoli causing subpleural cyst or bulla

Figure 2 Pathogenesis of pneumothorax from barotraumas. Adapted from Dreyfuss D and Saumon G (1998) Ventilator-induced lung injury. Lessons from experimental studies. American Journal of Respiratory and Critical Care Medicine 157: 294–323.

chest tube is governed by the Fanning equation: v ¼ p2 r5 P=fl where r is radius, P is pressure, f is the Fanning friction factor for turbulent flow, and l is length of the tube. Thus, the diameter of the tube is the most

important determinant of flow. At
10 cmH2O, a 20 French (F) chest tube allows a flow rate of 15 l min
1. Smaller-bore chest tubes have been investigated. Flow rates of percutaneous chest tubes at suction of
20 cmH2O are shown in Table 1. For a nonventilated patient, a small-bore chest tube may provide sufficient drainage. However, for most BPFs

292 TRAUMA, CHEST / Bronchopleural Fistula Table 1 Flow rates for 11 commercially available percutaneous drainage catheters Device

Size (F)

Flow (l min
1)

Argyle thoracentesis Argyle pneumothorax Arrow thoracentesis Arrow pneumothorax Cook FSNS Cook 100 Cook 1600 Cook Wayne Arrow drainage (pigtail) Arrow drainage (straight) Cook 2400

8 8 8 8 8.5 9 16 14 14 14 24

2.6 5.5 3.4 4.9 6.5 6.4 14.8 12.8 16.8 16.8 28.1

F, French.

Table 2 Flow rates of commercially available drainage systems System

Maximal flow (l min
1) at
20 cmH2O

Emerson pump Pleur-Evac A 4000 Thora-Klex Sentinel seal

35.5 34.0 19.7 2.3

and all mechanical ventilators, a chest tube of at least 20 F diameter should be employed. A drainage system must be employed that can accommodate large flow rates. Four commercially produced drainage systems are listed in Table 2. The location of chest tube placement is important. Horizontal placement can result in a posterior position of the chest tube or even placement within the major fissure. This type of chest tube placement is an independent risk factor for development of recurrent pneumothorax in ARDS. Chest tube drainage is not without complications. In mechanically ventilated patients, a sizable percentage of minute ventilation may be shunted through the fistula. Air leaks comprising 25% of minute ventilation have been documented in ARDS patients. The negative pressure from suction may be associated with inappropriate ventilator cycling, delayed healing of the fistula, and infection in the pleural space. There are additional therapeutic modalities for managing BPF. Patients with BPF from blunt trauma, penetrating trauma, or as a complication post-pulmonary-resection can have proximal or distal fistulae. Occult BPF (i.e., not detected during resection) can rarely (1%) complicate pulmonary resection. Tracheobronchial injury from blunt or penetrating trauma also can be initially occult resulting in sepsis. Patients who develop BPF following pulmonary resection, blunt trauma, or penetrating trauma should have bronchoscopy to evaluate and isolate tracheobronchial injury.

For patients with low tracheal and bronchial injury, thoracostomy with repair is usually necessary with or without skeletal muscle or omental patch. Distal bronchial lesions may require resection of involved bronchopulmonary segments. Other options for proximal and distal BPF range from observation to thoracoplasty, pneumonectomy, creation of pneumoperitoneum to decrease thoracic volume and air leak, and video-assisted thoracoscopic surgery (VATS). VATS is often a diagnostic and therapeutic modality for patients with BPF. In patients with an expandable lung without large bulla, talc pleurodesis can be performed. Patients with blebs with collapsed lung or large bullae are treated with resection. Patients with pleural adhesions require lysis and those with pleural fibrosis require decortication. Patients who have severe pulmonary disease, tuberculosis, ARDS, or are debilitated are usually not candidates for surgical management. For these patients, medical management is the primary therapeutic option. An algorithm for surgical management of patients with BPF is shown in Figure 3. For many patients with BPF, the nature and degree of their underlying illness precludes surgical therapy. In mechanically ventilated patients, BPF unfortunately carries a high mortality rate (45–67%). For these patients, numerous less invasive approaches have been developed with varying degrees of success. These include ventilator management strategies, independent lung ventilation, bronchoscopic techniques, and pleurodesis. For the patient with BPF on mechanical ventilation, minimization of tidal volumes, respiratory rate, PEEP, and inspiratory time can decrease flow through the fistula, which improves the likelihood of healing. An intermittent mandatory ventilation mode (IMV) theoretically has advantage over assist control (AC) or pressure-regulated volume control (PRVC) in that the latter modes provide full breaths with each patient effort. However, the ARDS network trial showed a mortality benefit in ARDS with low lung volumes and assist control ventilation. Thus, in the setting of ARDS and BPF, lung protective ventilation must be balanced with efforts to minimize air leak through the fistula. Patients with ARDS can have markedly noncompliant lungs with considerable air leak. For patients with persistent air leak greater than 500 cc/breath, other modes of ventilation may be more successful. High-frequency ventilation (HFV) has been studied in animal models and in case series for management of BPF. In HFV, laminar flow is generated by inducing a low-volume jet stream of gas at high velocity. Gas is entrained behind this stream and pulled forward. Theoretically, gas volumes can be delivered at low pressures minimizing barotrauma. Bulk flow,

TRAUMA, CHEST / Bronchopleural Fistula 293

Patient with bronchopleural fistula

Is chest tube drainage sufficient? Yes

No Ensure tube patency Consider larger bore

Patient post-op or posttraumatic? Yes No

Resolution

Bronchoscopy shows proximal tear ?

Can patient tolerate pleuroscopy?

Yes

Can patient tolerate thoracotomy ?

No

Yes

No

Thoracotomy with bronchial repair

Conservative algorithm

Yes

Pleuroscopy

Fibrosis

Adhesions

Large tear, collapsed lung, or large bullae

No abnormalities or blebs with expandable lung

Decortication

Division of adhesions

Thoracotomy with bleb resection

Talc poudrage or slurry

Figure 3 Surgical algorithm for bronchopleural fistula management.

gas exchange through turbulent eddies, regional flow in areas of improved compliance, laminar flow, cardiogenic mixing, and molecular diffusion are theorized mechanisms of improved gas exchange on high-frequency ventilator modes. Parameters that can be altered include the respiratory rate (up to 300 breaths min
1) and the inspiratory/expiratory (I/E) time. HFV has been used successfully in management of BPF. A limitation of HFV is the tendency to induce air trapping; therefore, it should be used with caution in patients with bilateral lung disease. An alternative mode of ventilation is the highfrequency oscillatory ventilator (HFOV), which uses reciprocating diaphragms and allows an active inspiratory and expiratory process. The frequency of

oscillation, I/E ratio, tidal volumes (increased tidal volumes via increasing driving pressure and decreasing frequency of oscillation), and mean airway pressure can be controlled. HFOV has the theoretical advantage of improving ventilation and decreasing air trapping. There are case studies of successful treatment of BPF with HFOV. Selective intubation and conventional or highfrequency ventilation can be performed to minimize air leak provided the patient can oxygenate and ventilate adequately. Patients have been ventilated with independent lung ventilation through dual lumen endotracheal tubes. Independent lung ventilation has also been described through one endotracheal tube using a differential resistor valve. Other methods that

294 TRAUMA, CHEST / Bronchopleural Fistula

have been employed include adding some PEEP to the chest tube to decrease flow through the fistula and maintain minute volume to the patient with synchronized closure of the chest tube during inspiration. There are no large studies validating any of these methods. There can be considerable danger in using these methods and they require intense monitoring and experience. In both mechanically ventilated patients and those felt to be too ill for surgical intervention, bronchoscopy has emerged as a useful tool for some individuals with BPF. Bronchoscopy can be used to localize the site of the BPF by either occluding a subsegmental bronchus and observing cessation of air leak or by instillation of methylene blue into a subsegmental bronchus and observing its presence in the chest tube. Numerous agents can then be injected into the subsegmental bronchus or mucosa of the bronchus including fibrin glue, tissue glue, fibrin sealant, gel-foam, sclerosing agents, lead plugs, human

spongiosa, gelatin-sponge particles, balloons, bucrylate, autologous blood patches, and absolute alcohol. Use of a pulmonary artery catheter through the working channel of the bronchoscope has been used to both occlude the subsegmental bronchus (with the balloon of the catheter) as well as deliver the sclerosant (through the distal port) while under direct visualization. These methods have met with variable success. An algorithm for conservative management of BPF is shown in Figure 4. Patients with traumatic BPF present a difficult therapeutic challenge. Adequate chest tube diameter and function must be assured. For patients with bronchial tears, early surgical consideration is mandatory. In patients with distal fistulae, VATS and surgical management should be considered in suitable patients. If on a ventilator, lung protective ventilation should be employed if ARDS is present. Multiple ventilatory techniques are available to minimize the air leak and provide adequate ventilation

Patient with bronchopleural fistula

Ensure adequacy of chest tube drainage

Resolution Is patient on ventilator?

Yes

No

Minimize RR, PEEP, TV, inspiratory time, if ARDS use lung protective ventilation

Is the lung expanded? Yes

Resolution

Consider pleurodesis or bronchoscopic modalities

Can the patient maintain adequate ventilation ? Yes

Conservative management

No

Consider independent lung ventilation, HFOV, synchronized closure of chest tube, bronchoscopic techniques

Figure 4 Conservative management of BPF. RR, respiratory rate; TV, tidal volume.

No

Consider bronchoscopic modalities

TRAUMA, CHEST / Postpneumonectomy Syndrome 295

but close monitoring is mandatory. Broncoscopic therapy should be considered in patients too debilitated for surgical therapy. See also: Acute Respiratory Distress Syndrome. Chest Wall Abnormalities. Pneumothorax. Ventilation: Control.

Further Reading Asamura H, Kondo H, and Tsuchiya R (2000) Management of the bronchial stump in pulmonary resections: a review of 533 consecutive recent bronchial closures. European Journal of CardioThoracic Surgery 17: 106–110. Baumann MH and Sahn SA (1990) Medical management and therapy of bronchopleural fistulas in the mechanically ventilated patient. Chest 97: 721–728. Brower R, Ware LB, Berthiaume Y, et al. (1998) Treatment of ARDS. Chest 120: 1347–1367. Dreyfuss D and Saumon G (1998) Ventilator-induced lung injury. Lessons from experimental studies. American Journal of Respiratory and Critical Care Medicine 157: 294–323. Eisner MD, Thompson BT, Schoenfeld D, et al. (2002) Airway pressures and early barotrauma in patients with acute lung injury and acute respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 165: 978–982. Heffner JE, McDonald J, and Barbieri C (1995) Recurrent pneumothoraces in ventilated patients despite ipsilateral chest tubes. Chest 108: 1053–1058. Hood M (1989) Pulmonary and pleural complications of trauma. In: Hood M (ed.) Thoracic Trauma, pp. 360–366. Philadelphia: Saunders. Krishnan JA and Brower RG (2000) High-frequency ventilation for acute lung injury and ARDS. Chest 118: 795–807. Pierson DJ, Horton CA, and Bates PW (1986) Persistent bronchopleural air leak during mechanical ventilation. A review of 39 cases. Chest 90: 321–323. Pizov R, Oppenheim-Eden A, Matot I, et al. (1999) Blast lung injury from an explosion on a civilian bus. Chest 115: 165–172. The Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. New England Journal of Medicine 342: 1301–1308. Weissberg D and Refaely Y (2000) Pneumothorax: experience with 1,199 patients. Chest 117: 1279–1285.

right-sided pneumonectomies during childhood or early adult life. However, it can occur in patients at any age and can also be seen in patients who have undergone left-sided procedures. This syndrome develops as a result of severe anatomical changes that occur in the chest after pneumonectomy. Signs and symptoms include progressive dyspnea, wheezing, and recurrent episodes of pneumonia. Symptoms typically develop months to years after pneumonectomy. Pulmonary function tests, chest computed tomography, and bronchoscopy are helpful in confirming the diagnosis. Treatment is surgical, with excellent outcomes. Surgical correction of these anatomical changes results in resolution of symptoms, and good long-term survival.

Introduction Pneumonectomy, the surgical removal of one lung, is a procedure associated with significant morbidity. Expected complication rates can range between 40% and 60%. Many complications of pneumonectomy are not unique to this procedure, but are in fact common complications of any major surgery, for example, arrhythmias, myocardial infarction, thromboembolism, hypoxemia, and pneumonia. There are other complications that also are not unique to this procedure, but are more commonly associated with it, for example, empyema, bronchopleural fistula, esophagopleural fistula, chylothorax, intracardiac right-to-left shunt, cardiac herniation, and postoperative pulmonary edema. Finally, there is one complication truly unique to this surgical procedure, namely the postpneumonectomy syndrome. This is a condition resulting in large airway obstruction. It is a rare complication, and develops in probably less than 1% of the patients undergoing this procedure. The postpneumonectomy syndrome develops as a result of severe anatomical changes that occur in the postpneumonectomy thorax. Initially, this condition was called the right pneumonectomy syndrome, as it was initially reported to occur only after rightsided procedures. However, a few rare subsequent reports have described this syndrome occurring after left-sided procedures.

Etiology

Postpneumonectomy Syndrome S E Kopec and R S Irwin, University of Massachusetts Medical School, Worcester, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The postpneumonectomy syndrome is a rare complication of pneumonectomy that is typically seen several years after surgery. The classical presentation is in patients who have undergone

In order to understand how the postpneumonectomy syndrome develops, we will first review the typical changes that occur in the thorax after pneumonectomy. Within the first 24 h after surgery, the mediastinum starts to shift towards the pneumonectomy space, and the ipsilateral hemidiaphragm becomes slightly elevated. These changes are thought to occur due to the negative pressure that develops within the empty pleural space. Immediately after surgery, air within the postpneumonectomy space starts to be absorbed, and serosanguineous fluid begins to accumulate. As seen on serial chest radiographs, fluid

296 TRAUMA, CHEST / Postpneumonectomy Syndrome

Figure 1 Typical chest radiographic changes after pneumonectomy. (a) Immediately after left pneumonectomy, with the postpneumonectomy space empty and slight elevation of the ipsilateral hemidiaphragm. (b) Same patient, now 48 h after surgery. Fluid begins to fill the postpneumonectomy space after removal of the chest tube, and the mediastinum starts shifting towards the resected side. Reproduced from Kopec SE, Irwin RS, Umali-Torres CB, Balikian JP, and Conlan AA (1998) The postpneumonectomy state. Chest 114: 1158–1184, with permission.

typically accumulates at a rate of 1–2 rib spaces per day in the immediate postoperative period (Figure 1). After 2 weeks, 80–90% of the postpneumonectomy space is typically filled with fluid. Complete opacification of the postpneumonectomy space is seen on chest radiograph anywhere between 2 and 7 months after pneumonectomy. Over the course of several months to years, there is further shifting of the mediastinum and vital structures (heart, great vessels, and esophagus) towards the surgical side, further elevation of the hemidiaphragm, and hyperinflation of the remaining lung. With elevation of the hemidiaphragm, the liver, stomach, spleen, and large bowel can elevate into the thorax (Figure 2). Also during this time, there is narrowing of the ipsilateral intercostal spaces, and the development of mild scoliosis. The scoliosis is usually limited to the upper thoracic spine, and results in a 5–201 curvature,

Figure 2 Representative CT scans several years after pneumonectomy demonstrating the anatomical changes occurring in the thorax. (a) After left pneumonectomy, with the heart and mediastinal structures rotated to the left and posteriorly. RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; a, descending aorta: e, esophagus. (b) After right pneumonectomy, the liver (arrow) is elevated high in the thorax. Open arrow denotes fluid remaining in the postpneumonectomy space. Reproduced from Kopec SE, Irwin RS, Umali-Torres CB, Balikian JP, and Conlan AA (1998) The postpneumonectomy state. Chest 114: 1158–1184, with permission.

with the convexity of the spine towards the unoperated side. Symptoms associated with this degree of scoliosis have not been reported. Over time there is slow reabsorption of the fluid within the postpneumonectomy space. However, complete absorption of all fluid and obliteration of the postpneumonectomy space is uncommon, even 30–40 years after surgery. Autopsy studies have revealed that the postpneumonectomy space is completely obliterated in less than one-third of patients, with the remaining space containing various amounts of fluid. It is felt that this remaining fluid can serve as a nidus for infection in bacteremic patients, resulting in the development of a late postpneumonectomy empyema. The degree of shifting of the mediastinal structures and hyperinflation of the remaining lung depends, to

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some extent, on the age of the patient at the time of pneumonectomy. The younger the patient at the time of surgery, the more shifting and hyperinflation is likely to occur. The explanation for this is that younger patients have more elasticity and compliance of the mediastinal tissue and remaining lung, allowing for more shifting and hyperinflation. Women, for reasons not entirely clear, also tend to have more shifting and hyperinflation after a pneumonectomy than men do. It is the shifting of the vital structures and hyperinflation and herniation of the remaining lung that places the postpneumonectomy patient at risk for developing the postpneumonectomy syndrome.

Clinical Presentation Risk factors for developing the postpneumonectomy syndrome are age at the time of pneumonectomy, right-sided procedures, and female sex. Most of the cases of postpneumonectomy syndrome are seen in patients who have had a right pneumonectomy in childhood or early adulthood. However, this syndrome is also well described in patients who had lung removal during their late adulthood, as well as in patients who have undergone left-sided procedures. Onset of symptoms of postpneumonectomy syndrome can occur anywhere from a few months to over 35 years after surgery. Classic symptoms include progressive dyspnea, cough, and stridor. The narrowing of the bronchus can also result in the development of recurrent pneumonia and bronchiectasis. A history of recurrent pneumonias in a patient who has undergone a pneumonectomy, especially a rightsided procedure, should alert the physician to the possibility of this syndrome. If left untreated, postpneumonectomy syndrome can result in respiratory insufficiency, recurrent pneumonia, development of tracheobronchomalacia at the site of airway compression, and death. The diagnosis of postpneumonectomy syndrome should be entertained in any patient who develops the above symptoms and evidence of airflow obstruction on pulmonary function testing months to years after pneumonectomy. Spirometry may reveal a forced vital capacity (FVC) that is reduced, but higher than expected for a patient with one lung. This supports the finding of hyperinflation of the remaining lung. Forced expiratory volume in 1 s (FEV1)/FVC is usually reduced. Flow-volume loops may reveal a pattern of variable intrathoracic upper airway obstruction, suggesting the diagnosis. Compression of the airway is best confirmed with a chest computed tomography (CT) scan or with bronchoscopy. Bronchoscopy is particularly helpful in evaluating for the development

of tracheobronchomalacia at the site of compression, as well as evidence of purulent secretions trapped distal to the obstruction.

Pathophysiology This syndrome develops after a right-sided pneumonectomy when the mediastinal structures shift posteriorly and to the right, into the vacant pleural space. The left lung becomes severely hyperinflated and also herniates into the vacant pleural space. The rotation and shifting of the mediastinum coupled with hyperinflation of the remaining lung results in stretching and narrowing of the left mainstem bronchus (Figure 3). In addition, the stretched and narrowed bronchus can become compressed by the left pulmonary artery anteriorly, and the aorta or thoracic vertebrae posteriorly. In the rare patient who develops this syndrome after a left-sided pneumonectomy, severe shifting of the mediastinum and excess hyperinflation of the right lung are required. This results in stretching and narrowing of the distal trachea and right mainstem bronchus. Further compression of the distal trachea and right mainstem bronchus occurs between the mediastinum and thoracic vertebrae. It has also been reported that patients with right-sided aortic arches can develop postpneumonectomy syndrome after a left pneumonectomy when the stretched right mainstem bronchus is compressed posteriorly by the right-sided aorta.

Treatment Treatment of postpneumonectomy syndrome is surgical and is aimed at relieving the obstruction of the

Figure 3 Chest CT in a patient with postpneumonectomy syndrome after right pneumonectomy. Note the stretching and narrowing of the left mainstem bronchus (arrows), and severe hyperinflation of the left lung and shifting of the mediastinum. Reproduced from Kopec SE, Irwin RS, Umali-Torres CB, Balikian JP, and Conlan AA (1998) The postpneumonectomy state. Chest 114: 1158–1184, with permission.

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airway. Earlier surgical interventions to relieve the symptoms of postpneumonectomy syndrome were directed at the great vessels that were compressing the airway. Specifically, dividing the aortic arch, bypass grafts, and suspending the aorta from the posterior surface of the sternum were all attempted in an effort to relieve the compression of the airway. These procedures, however, had limited success. In patients who had airway compression from a vertebral body, attempts to remove part of the bone by surgery were also unsuccessful in the long-term management of this syndrome. In the early 1990s, Grillo and colleagues reported good success by repositioning of the mediastinum in patients with this condition. During this procedure, the mediastinum was repositioned in the center of the chest after being freed up from adhesions. To prevent re-shifting of the mediastinum, the postpneumonectomy space was filled with nonabsorbable materials. Material such as silicone or saline breast implants, testicular implants, and sulfur hexafluoride have been used. Today, saline-filled expandable breast implants are most frequently employed as they are felt to be the safest, and offer the advantage of easily being made larger if needed. This is of particular importance in the management of growing children with postpneumonectomy syndrome. Patients undergoing surgical re-positioning of the mediastinum with placement of an implant into the pleural space have had resolution of symptoms, and good long-term survival. Patients with underlying tracheobronchomalacia require an additional procedure after repositioning of the mediastinum, that is, treatment of the airway malacia. Placement of self-expanding metallic stents appears to be the most effective treatment for the tracheobronchomalacia. Resection of the damaged airway or placement of Silastic tubes has been associated with high mortality rates and poor outcomes. See also: Bronchomalacia and Tracheomalacia. Symptoms of Respiratory Disease: Cough and Other Symptoms.

Further Reading Deslauriers J and Faber LP (eds.) (1999) Pneumonectomy: Part 1. Chest Surgery Clinics of North America, pp. 267–500. Philadelphia: Saunders. Deslauriers J and Faber LP (eds.) (1999) Pneumonectomy: Part 2. Chest Surgery Clinics of North America, pp. 501–699. Philadelphia: Saunders. Grillo HC, Shepard JAO, Mathisen DJ, and Kanarek DJ (1992) Postpneumonectomy syndrome: diagnosis, management, and results. Annals of Thoracic Surgery 54: 638–651. Kopec SE, Irwin RS, Umali-Torres CB, Balikian JP, and Conlan AA (1998) The postpneumonectomy state. Chest 114: 1158–1184.

Mehran RJ and Deslauriers J (1999) Late complications: postpneumonectomy syndrome. Chest Surgery Clinics of North America 9: 655–673. Shepard JAO, Grillo HC, McLoud TC, Dedrick CG, and Spizarny DL (1986) Right-pneumonectomy syndrome: radiologic findings and CT correlation. Radiology 161: 661–664.

Subcutaneous Emphysema R C Hyzy and M R McClelland, University of Michigan, Ann Arbor, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Subcutaneous emphysema describes the presence of gas in subcutaneous tissue. It has several known causes, among them anaerobic infections, traumatic disruption of mucosal surfaces, and alveolar rupture. Diagnosis is characterized by the presence of crepitation on palpation over the affected area and radiographic evidence of gas in the subcutaneous tissues. The pathogenesis relates to the tracking of gas along fascial planes into the subcutaneous space of the neck, chest, or extremities. Therapy of subcutaneous emphysema itself is generally supportive and involves the treatment of the underlying condition, as subcutaneous emphysema is usually self-limited and benign. However, the appearance of subcutaneous emphysema often warrants a search for more serious underlying pathology such as pneumothorax, pneumomediastinum, or tracheobronchial or esophageal disruption.

Introduction Subcutaneous emphysema is a term used to describe the finding of gas within subcutaneous soft tissues, usually in the thorax or neck. The first known description dates back to the seventeenth century, when Louise Bourgeois, midwife to the Queen of France, described crepitations in patients associated with the extreme effort of childbirth. In the nineteenth century, Laennec provided a description in his Treatise on the Diseases of the Chest and Mediate Auscultation. Subcutaneous emphysema is a relatively common clinical finding associated with a broad range of disorders of varying degrees of severity, including trauma, surgery, tube thoracostomy placement, infection, and positive pressure ventilation. One study of 134 consecutive patients with chest tubes found 25 (18.6%) had subcutaneous emphysema. The incidence has not been described in any systematic way, presumably because subcutaneous emphysema is associated with such a diverse range of conditions and is typically a self-limited process of little direct clinical consequence. In general, knowledge of subcutaneous emphysema stems from individual case reports, small series, and clinician experience.

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Etiology Generally, the etiologies of subcutaneous emphysema can be divided into three major categories: infectious, particularly with anaerobic organisms; traumatic, both environmental and iatrogenic (from surgery or other procedures); and so-called spontaneous or pressure related (Table 1). Genetic factors or inheritied patient characteristics predisposing to subcutaneous emphysema per se are unknown, although both genetic and environmental factors contribute to some of the underlying potential causes of subcutaneous emphysema, such as congenital pulmonary blebs on the surface of the lung.

Pathology On gross examination, the presence of subcutaneous emphysema is confirmed by finding gas, usually air,

Table 1 Selected causes of subcutaneous emphysema and pneumomediastinum Infectious Retropharyngeal abscess Dental abscess Cervical Adenitis Tonsillitis Osteomyelitis Suppurative mediastinitis (usually results from esophageal rupture) Retroperitoneal infections Traumatic Fractures (facial bones, ribs) Direct penetrating trauma to face, oropharynx, or chest Blunt deceleration injuries Penetrating lung trauma Esophageal trauma or rupture Rupture of intra-abdominal or retroperitoneal viscera Iatrogenic Disruption of trachea or to the major airways (including endotracheal intubation) Dental procedures CPR or Heimlich maneuver Positive pressure ventilation Pulmonary function testing Incorrect assembly of anesthetic unit Bronchoscopy Tube thoracostomy placement Spontaneous Airways obstruction (particularly asthma) Mechanical obstruction (as by a tumor or foreign body) Smoking Valsalva (such as during childbirth, violent cough, emesis, straining at stool) Scuba diving Air travel, mountain climbing Vigorous exercise

within the subcutaneous tissue. Histologically, vacuolization within the subcutaneous tissue is seen. The presence of air in the subcutaneous tissue can be confirmed by opening tissues underwater. Such techniques have been recommended, for example, when performing autopsies in cases of barotrauma related to scuba diving.

Clinical Features Patients may complain of puffy face, neck, arms or chest, dyspnea, or awareness of a ‘crackling’ sensation under the skin. The hallmark physical examination sign of subcutaneous emphysema is crepitation on palpation of the affected area. It should not be painful. When associated with pain or erythema, the presence of subcutaneous emphysema should alert the clinician to the possibility of a serious underlying pathology such as infection. Regions of involvement vary according to the etiology of the subcutaneous emphysema. The site where the gas is first noted may help to identify the site of origin: air in the neck suggests antecedent pneumomediastinum or a rupture of the esophagus as the site of injury, whereas air in the flank or abdominal wall suggests the retroperitoneum as the site of origin. Air may be seen initially near a rib in the case of rib fracture. The most frequently affected sites include the subcutaneous skin overlying the neck and thorax. There is often overlap in areas of involvement due to anatomic considerations (see following section on pathogenesis). There may be marked distension of the skin due to entrained air, such that patients may be grossly disfigured. For example, subcutaneous emphysema involving the eyelids can become so severe as to cause the eyes to be completely shut. Air dissecting to the soft tissues of the larynx can produce hoarseness or a so-called hot potato voice. Gas in the retropharyngeal area may also give a nasal quality to the voice. Although fever and leukocytosis have been reported in isolated subcutaneous emphysema, such findings should raise suspicion for infection. Subcutaneous emphysema is frequently associated with pneumomediastinum, which is accompanied by stabbing precordial chest pain in as many as 80–90% of affected individuals. On examination of the anterior chest, the so-called Hamman’s crunch is reported in 50–80% of patients. This auscultatory finding consists of a crackling sound in the retrosternal location synchronous with the heartbeat. Radiographically, subcutaneous emphysema is appreciated as pockets of gas density within subcutaneous regions. This air frequently outlines anatomic

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structures such as the fascial planes of the chest and neck, the axilla, and the pectoralis muscles (Figures 1 and 2). The presence of subcutaneous emphysema on a chest radiograph should prompt a careful radiographic search for pneumothorax, pneumomediastinum, pneumoperitoneum, or fractures. Occasionally,

the presence of pneumomediastinum is uniquely evident on a lateral chest radiograph. Appropriate investigations for subcutaneous emphysema depend upon the clinical scenario. When infection is suspected, blood cultures and pertinent imaging should be initiated. Careful examination of the oral cavity, the source of many such infections, is mandatory. In cases of trauma, concern centers on the potential disruption of mucosal surfaces within one or more segments of the airways: nasal mucosa and paranasal sinuses; retropharynx and larynx; trachea and lower airways; or esophagus and lower gastrointestinal tract. Appropriate imaging may include X-rays or computed tomography (CT) scans of the chest or neck. When airways disruption is suspected, immediate bronchoscopy is warranted to evaluate for tracheobronchial rupture. Intraoperative bronchoscopy under general anesthesia may be advantageous, since bronchoscopy in an uncontrolled setting has the potential to worsen airway damage and compromise the airways. Intraoperative visualization allows for surgical correction immediately following diagnosis. Upper GI endoscopy is indicated if esophageal rupture is suspected as, for example, in Boerhaave’s syndrome.

Pathogenesis

Figure 1 Widespread subcutaneous emphysema in a 68-yearold woman who had undergone mechanical ventilation for acute respiratory distress syndrome (ARDS). Air may be seen tracking into the neck and highlighting the pectoralis muscles. Chest X-ray courtesy of E Kazerooni.

Figure 2 A 39-year-old woman 3 months status postsuperficial self-induced gunshot wound to the chest who developed subcutaneous emphysema associated with a chest wall abscess. Chest CT scan courtesy of E Kazerooni.

The respiratory and gastrointestinal tracts are closed systems surrounded by subcutaneous, prevertebral, visceral, and previsceral spaces (Figure 3). Air arising from a breach in the mucosal integrity of the respiratory or gastrointestinal tract can enter the visceral space and dissect along facial planes into the subcutaneous space, creating subcutaneous emphysema. Traumatic causes of subcutaneous emphysema are numerous. Blunt or penetrating trauma to the neck or chest can result in disruption of the tracheobronchial tree and tracking of air along fascial planes into the subcutaneous space. More than 80% of such injuries occur within 2–3 cm of the carina. Similarly, esophageal rupture can lead to subcutaneous emphysema as a result of penetrating trauma or from increased intrathoracic pressure as with protracted, severe vomiting. Iatrogenic causes of subcutaneous emphysema are not uncommon. Examples include dental procedures such as extractions or drilling of lower molar teeth. A number of case reports describe traumatic endotracheal intubation as a causative factor, particularly with the use of a rigid stylette extending beyond the tip of the endotracheal tube. Tracheobronchial rupture has also been reported during diagnostic bronchoscopy. Subcutaneous emphysema occurs frequently following tube thoracostomy placement, particularly in the setting

TRAUMA, CHEST / Subcutaneous Emphysema 301 Subcutaneous space

Visceral space

Prevertebral space

Previsceral space

C-7 Esophagus Trachea

Lung

T-2

Esophagus Trachea

T-5 Aorta Pulmonary artery Aorta

Superior vena cava Sternum

L-1 Aorta Inferior vena cava

Colon

Liver

Stomach

Pancreas

Figure 3 Soft-tissue compartments of the neck (level of C-7), thorax (levels of T-2 and T-5), and abdomen (level of L-1), demonstrating the continuity of the visceral space between regions and the adjacent nature of the subcutaneous space. Reproduced from Maunder RJ, Pierson DJ, and Hudson LD Subcutaneous and mediastinal emphysema: pathophysiology, diagnosis, and management. Archives of Internal Medicine, 1984, 144: 1447–1453, with permission. Copyrighted & (1984), American Medical Association, All rights reserved.

of: antecedent trauma; the presence of a bronchopleural fistula; the presence of large, bilateral pneumothoraces; the placement of chest tube outside of the operating room; or mechanical ventilation. More distally, air can be introduced into the subcutaneous space at the alveolar level in a nontraumatic fashion by alveolar disruption. This process requires a pressure gradient between the alveolar space and the lung interstitium. With alveolar rupture, air tracks along bronchovascular bundles within the bronchovascular sheaths, reaching the mediastinum through the visceral space, resulting in

pneumomediastinum. Subsequently, free air can dissect into the subcutaneous and retroperitoneal spaces, cervical soft tissue, or decompress into the pleural space, resulting in a pneumothorax. Most commonly, this form of nontraumatic subcutaneous emphysema results from positive pressure mechanical ventilation. Other etiologies include obstructed air flow during asthma exacerbations, viral-induced bronchiolitis in children, mechanical upper airways obstruction due to tumor or foreign body, or decompression sickness. Decompression sickness in scuba divers is the result of rapid ascent. As the pressure is

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rapidly decreased, the volume of gas in the lungs increases, causing gas to escape into the bronchovesicular sheath and dissect its way into the subcutaneous tissue. More serious problems from decompression sickness include pneumothorax and air embolism. In necrotizing infections, gas is produced within the subcutaneous tissue by anaerobic organisms. Subsequent dissection along facial planes can occur as outlined above. These infections tend to occur in tissues that are in proximity to mucosal surfaces covered by anaerobic bacteria or within tissue that has been damaged long enough to allow necrosis to develop. A broad range of bacterial species are capable of producing gas in subcutaneous tissue, including: anaerobes such as Clostridium, Bacteroides, peptostreptococci; and facultative anaerobes, such as Escherichia coli and Klebsiella species.

Management and Current Therapy In the vast majority of cases, the management of subcutaneous emphysema is supportive, since the problem is generally self-limiting. Of primary importance is treatment of the underlying condition. When subcutaneous emphysema has developed from barotrauma during mechanical ventilation, weaning and liberation from mechanical ventilation should be performed, if possible. If not clinically possible, attempts to favorably modify lung mechanics so as to decrease mean airway pressure should be performed. As clinically warranted, such measures may include the administration of bronchodilators, decreasing inspiratory time (by decreasing tidal volume and/or increasing the flow rate), and decreasing the amount of positive end expiratory pressure. Suspected infections should be treated aggressively with appropriate antimicrobials and, if indicated, surgical debridement. Traumatic sources of subcutaneous air should be sought and managed surgically, as warranted. Tracheobronchial disruptions generally require operative repair. Tube thoracostomy is warranted for pneumothorax, particularly if the patient is receiving mechanical ventilation. However, empiric insertion of a chest tube for subcutaneous emphysema is without clear benefit, and is not recommended unless a pneumothorax is also present. Other procedures that have been attempted include infraclavicular skin incisions, tracheostomy, and subcutaneous drains with or without suction. Such procedures have not been well studied, may be associated with complications such as infection, and are not recommended for the routine management of patients with subcutaneous emphysema, even if receiving mechanical ventilation.

Subcutaneous emphysema is usually a self-limiting, cosmetic problem. However, when severe, it can result in significant tissue distension and problems such as transient vision loss or dysphagia. It can also be a nursing problem, leading to difficulty in gaining intravenous access or caring for wounds. Rarely, severe complications have been reported from subcutaneous emphysema: airway compromise, respiratory failure, pacemaker malfunction, or cardiac tamponade physiology (in conjunction with pneumomediastinum). When associated with chest tube placement, the presence of subcutaneous emphysema is linked with an increased length of stay in the hospital and increased mortality compared to patients with chest tubes who did not develop subcutaneous emphysema. See also: Asthma: Overview. Bronchoscopy, General and Interventional. Chest Wall Abnormalities. Chronic Obstructive Pulmonary Disease: Overview. Diving. Lung Imaging. Mediastinitis, Acute and Chronic. Pneumothorax. Surfactant: Surfactant Protein D (SP-D). Tracheostomy.

Further Reading Beck PL, Heitman SJ, and Mody DH (2002) Simple construction of a subcutaneous catheter for treatment of severe subcutaneous emphysema. Chest 121(2): 647–649. Boyd AD (1989) Chest wall trauma. In: Hood RM, Boyd AD, and Culliford AT (eds.) Thoracic Trauma, pp. 101–132. Philadelphia: Saunders. Doweiko and Alter JP (1992) Subcutaneous emphysema: report of a case and review of the literature. Dermatology 184: 62–64. Jones PM, Hewer RD, Wolfenden HD, and Thomas PS (2001) Subcutaneous emphysema associated with chest tube drainage. Respirology 6: 87–89. Leitman BS, Birnbaum BA, and Naidich DP (1989) Radiologic evaluation of thoracic trauma. In: Hood RM, Boyd AD, and Culliford AT (eds.) Thoracic Trauma, pp. 67–100. Philadelphia: Saunders. Lomoschitz FM, Eisenhuber E, Linnau KF, et al. (2003) Imaging of chest trauma: radiological patterns of injury and diagnostic algorithms. European Journal of Radiology 48: 61–70. Macklin CC (1939) Transport of air along sheaths of pulmonic blood vessels from alveoli to mediastinum: clinical implications. Archives of Internal Medicine 64: 913–926. Macklin MT and Macklin CC (1944) Malignant interstitial emphysema of the lungs and mediastinum as an important occult complication in many respiratory diseases and other conditions: an interpretation of the clinical literature in the light of laboratory experiment. Medicine 23: 281–358. Maunder RJ, Pierson DJ, and Hudson LD (1984) Subcutaneous and mediastinal emphysema: pathophysiology, diagnosis, and management. Archives of Internal Medicine 144: 1447–1453. Parrish KL (2001) Sub-q emphysema: understanding the pathophysiology. Journal of Emergency Medical Services 26(3): 108–123.

TUBEROUS SCLEROSIS 303

Tryptase

see Chymase and Tryptase.

TUBEROUS SCLEROSIS E P Henske, Fox Chase Cancer Center, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Tuberous sclerosis complex (TSC) results from a germline mutation in either the TSC1 or TSC2 gene. It has autosomal dominant inheritance with 95% penetrance. Sixty percent of TSC patients have no prior family history of TSC and represent new germline mutations. The manifestations of TSC include seizures, mental retardation, autism; benign tumors of the brain, retina, kidney, heart, and skin; and pulmonary lymphangiomyomatosis (LAM). TSC has a wide range of phenotypic variability and many of the phenotypes are age or gender dependent. For example, seizures and kidney tumors occur in approximately 80% of individuals with TSC, mental retardation in approximately 50%, and LAM in approximately 30% (women only). This phenotypic variation occurs even within families carrying the same mutation. This variability and the high new mutation rate contribute to diagnostic uncertainty, especially in mild cases. Clinical genetic diagnostic assays are now available, but because of the size and complexity of the TSC1 and TSC2 genes, the absence of a mutation does not exclude a TSC diagnosis. Rapid advances regarding the biologic and biochemical pathways in which the TSC proteins participate have led to widespread optimism that specific targeted therapy for TSC patients will soon be available.

Introduction Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome in which the manifestations can include seizures, mental retardation, autism; benign tumors of the brain, retina, kidney, heart, and skin; and pulmonary lymphangiomyomatosis (LAM). Tuberous sclerosis was first described by Bourneville in 1880; he referred to the characteristic brain lesions as ‘tubers’ because of their potato-like consistency. The designation tuberous sclerosis complex is preferred to distinguish tuberous sclerosis from Tourette’s syndrome. TSC has autosomal dominant inheritance with 95% penetrance. Sixty percent of TSC patients have no prior family history of TSC and represent new germline mutations. TSC has a wide range of phenotypic variability. For example, seizures and kidney tumors occur in approximately 80% of individuals with TSC, mental handicap in approximately 50%, ‘shagreen’ skin patches in

approximately 30%, and LAM in approximately 30% (women only). This phenotypic variation is present even within families carrying the same germline mutation. The clinical variability and the high new mutation rate contribute to diagnostic uncertainty, especially in mild cases.

Diagnosis The diagnosis of TSC is primarily based on clinical, radiological, and histopathological findings. The spectrum and severity of the clinical manifestations vary widely and are age and gender dependent; thus, establishing a definitive diagnosis can be challenging. No single finding is considered pathognomonic. The diagnosis of TSC is based on a combination of major and minor features (Table 1). TSC patients can develop tumors in the brain, retina, skin, heart, and kidney, as well as pulmonary LAM. The tumors, which are almost always benign, are often referred to as hamartomas, reflecting their apparent origin from normal tissue components. If a diagnosis of TSC is suspected clinically, patients should undergo a detailed examination of the brain, eyes, heart, abdomen, and skin. Clinical genetic testing for TSC gene mutations is now available. However, there are significant limitations. Mutations are detected in 83% of patients with clinical TSC diagnoses. Therefore, the absence of a mutation does not exclude a TSC diagnosis. Among patients with clinical diagnoses of TSC, mutations are more often detected in TSC2 than in TSC1. It is not known whether this reflects a true difference in overall mutational susceptibility or a bias related to the increased severity of TSC2-linked disease. There is no clear evidence of a relationship between genotype and the likelihood of developing LAM or any of the other manifestations of TSC. Therefore, in a patient with a clear clinical diagnosis of TSC, identifying a specific mutation does not influence clinical management. Both genes are large (TSC1 has 21 coding exons spanning 50 kb on chromosome 9q34, and TSC2 has 41 exons spanning 40 kb on chromosome 16p13), and mutations are found in virtually all exons of both genes. The two most frequently detected mutations are both in

304 TUBEROUS SCLEROSIS Table 1 Revised diagnostic criteria for tuberous sclerosis complex (TSC) Major features Facial angiofibromas or forehead plaque Nontraumatic ungual or periungual fibroma Hypomelanotic macules (three or more) Shagreen patch (connective tissue nevus) Multiple retinal nodular hamartomas Cortical tubera Subependymal nodule Subependymal giant cell astrocytoma Cardiac rhabdomyoma, single or multiple Lymphangiomyomatosisb Renal angiomyolipomab Minor features Multiple, randomly distributed pits in dental enamel Hamartomatous rectal polypsc Bone cystsd Cerebral white matter radial migration linesa, d Gingival fibromas Nonrenal hamartomac Retinal achromic patch ‘Confetti’ skin lesions Multiple renal cystsc Definite TSC: Either two major features or one major feature plus two minor features Probable TSC: One major plus one minor feature Possible TSC: Either one major feature or two or more minor features a

When cerebral cortical dysplasia and cerebral white matter migration tracts occur together, they should be counted as one rather than two features of tuberous sclerosis. b When both lymphangiomyomatosis and renal angiomyolipomas are present, other features of tuberous sclerosis should be present before a definite diagnosis is assigned. c Histologic confirmation is suggested. d Radiographic confirmation is sufficient. Adapted from Roach ES, Gomez MR, and Northrup H (1998) Tuberous Sclerosis Complex Consensus Conference: revised clinical diagnostic criteria. Journal of Child Neurology 13: 624– 628, with permission from BC Decker.

TSC2: a missense change in exon 16 and an 18-base pair deletion in exon 40. Approximately 50% of the mutations are nonsense, splice-site, or missense changes, and 38% are small deletions or insertions. However, 12% of TSC2 mutations are large deletions that would be missed by most mutational screening approaches. Distinguishing between rare polymorphisms and disease-associated mutations can also be challenging. Central Nervous System

The clinical signs of involvement of the central nervous system in TSC can include seizures, autism, and mental handicap. Seizures, cerebral cortical malformations called tubers, and subependymal nodules

Figure 1 Facial angiofibromas (also called adenoma sebaceum), which are present in the majority of TSC patients. Photograph courtesy of S Roach, Wake Forest University, Baptist Medical Center.

occur in 80–90% of TSC patients. Importantly, many patients with TSC have normal intelligence. In general, there is a correlation between the extent of cortical lesions and neurological disease. However, patients can lack neurological symptoms despite the presence of typical radiological signs, such as subependymal calcifications and cortical tubers. Seizures frequently start within the first months of life with infantile spasms, but other types of epilepsy are also commonly observed. Subependymal giant cell astrocytomas occur in 10% of patients. Retinal hamartomas occur in approximately 25%. All of these manifestations occur in both TSC1- and TSC2-linked disease, but they tend to be severe more often in TSC2 patients. There is a clear correlation between TSC2 mutations and the severity of neurologic disease compared with TSC1 mutations. Skin

Facial angiofibromas (also called adenoma sebaceum) are found in approximately 80% of TSC patients (Figure 1) and tend to increase in severity during later childhood and adolescence. These highly vascular lesions are often present in a symmetrical pattern on the nose and cheeks. Ungual fibromas can occur on the nails of the hands or feet in

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approximately 25% of patients, increasing in frequency in adulthood. Hypomelanotic macules occur in 95% of TSC patients. However, hypomelanotic macules are relatively common in normal individuals, limiting their diagnostic specificity. Heart

Cardiac rhabdomyomas, which are frequently multifocal, occur in approximately 50% of TSC patients. These tumors have an extremely unusual natural history, developing during gestation and spontaneously regressing during infancy in most cases. Lung

The incidence of radiographic evidence of LAM among women with TSC is 26–39%, although many women with radiographic evidence of LAM have only mild clinical symptoms. In a Mayo Clinic series, LAM was the third-most frequent cause of TSC-related death after renal disease and brain tumors. Germline mutations in both TSC1 and TSC2 are associated with LAM. LAM is characterized by widespread pulmonary proliferation of abnormal smooth muscle cells and cystic changes within the lung parenchyma. LAM can also occur in women without TSC (sporadic LAM), often in association with angiomyolipomas. Multinodular multifocal pneumocyte hyperplasia occurs in both men and women with TSC. Kidney

The renal manifestations of TSC are unusual because they include both mesenchymal tumors (angiomyolipomas) and epithelial lesions and tumors (cysts, oncocytomas, and renal cell carcinomas). Angiomyolipomas are benign tumors composed of abnormal vessels, immature smooth muscle cells, and fat cells. Angiomyolipoma smooth muscle cells and LAM cells are identical at the histologic, immunohistologic, and electron microscopic levels. Both angiomyolipoma cells and LAM cells express estrogen and progesterone receptors and are immunoreactive with the melanoma antibody HMB-45. Most TSC patients have multiple, bilateral angiomyolipomas. Because of their abnormal vasculature (often containing aneurysms), spontaneous life-threatening bleeding is a significant problem. Renal cysts occur in approximately 25% of TSC patients. Those patients with TSC and polycystic kidneys often have large germline deletions within the TSC2 gene (sometimes including the adjacent polycystic kidney disease 1 gene, PKD1). Renal cell carcinomas in TSC patients occur less frequently than angiomyolipomas but have been reported in young children as well as adults with TSC.

Pathogenesis TSC is caused by germline mutations in either the TSC1 gene on chromosome 9q34 or the TSC2 gene on chromosome 16p13. The clinical features of TSC1- and TSC2-linked disease are very similar, although generally TSC1-linked disease tends to have milder clinical manifestations. TSC1 and TSC2 are classic tumor suppressor genes, fitting the Knudson ‘two hit’ model. In this model, a germline mutation inactivates one allele and a somatic mutation or somatic chromosomal deletion (often referred to as loss of heterozygosity) inactivates the remaining allele. Loss of heterozygosity in the TSC1 or TSC2 region occurs in most TSC-associated angiomyolipomas, rhabdomyomas, and microdissected LAM cells. TSC2 encodes tuberin, a 200 kDa protein with a domain near the carboxyl terminus containing GTPase activating protein (GAP) homology. GAP proteins convert members of the Ras superfamily from their active, GTP-bound state to their inactive, GDP-bound state. TSC1 encodes hamartin, a 140 kDa protein with no homology to tuberin. Tuberin and hamartin physically interact and function as a complex, consistent with the similar clinical features of TSC1- and TSC2-linked disease. The small GTPase Ras homolog enriched in brain (Rheb), which activates the mammalian target of rapamycin (mTOR) and inhibits B-Raf kinase, was identified as a key target of tuberin’s GAP domain. Rheb, like other Ras family members, cycles between an active GTP-bound and an inactive GDP-bound state. Tuberin converts Rheb-GTP to Rheb-GDP, thereby inactivating Rheb. Studies of Drosophila and mammalian cells have demonstrated that regulation of p70 S6 kinase (S6K) by the tuberin–hamartin complex is mediated by mTOR. mTOR and S6K are components of cellular pathways that integrate mitogenic signals and nutrient availability with protein synthesis. Hyperphosphorylation of S6K and/or its substrate ribosomal protein S6 was found in cells lacking hamartin from a murine TSC1 model, in cells lacking tuberin from the Eker rat TSC2 model, and in angiomyolipoma cells from TSC patients. These studies place hamartin and tuberin in the center of pathways through which cells integrate cues from the cell cycle, available energy status, and circulating growth factors in order to make decisions regarding proliferation and differentiation via mTOR. In addition to these Rheb-mediated functions, tuberin and/or hamartin have been shown to participate in other pathways, including vesicular trafficking, cellcycle regulation, steroid hormone function, and Rho signaling. Whether Rheb also mediates these pathways is not known.

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Some patients with the sporadic form of LAM have somatic inactivating TSC2 gene mutations in microdissected pulmonary LAM cells. These mutations are also present in renal angiomyolipomas but not in normal lung, kidney, or peripheral blood lymphocytes. Therefore, these data are consistent with a model in which LAM cells and angiomyolipoma cells have a common genetic origin. These findings have led to the hypothesis that pulmonary LAM results from the metastatic spread of histologically benign angiomyolipoma smooth muscle cells. Additional data supporting a metastatic model of LAM pathogenesis were obtained from sporadic LAM patients who underwent lung transplantation. The presence of the same TSC2 mutation in the pulmonary LAM, recurrent LAM, and in the lymph node LAM strongly supports the hypothesis that LAM cells migrate or metastasize in vivo, despite the fact that they are histologically benign. This places LAM in a small group of diseases, all of which affect women, resulting from the metastasis of histologically benign smooth muscle cells.

Animal Models Animal models of TSC include the Eker rat, which carries a TSC2 mutation, and knockout mouse models of TSC2 and TSC1. All of the rodent models are embryonic lethal in the homozygous state. In the kidney, heterozygotes develop cysts and carcinomas but not angiomyolipomas (which are the primary renal manifestations of human TSC) or LAM. TSC models have also been made in Drosophila melanogaster and Schizosaccharomyces pombe. The Drosophila models have been particularly important in elucidating TSC signaling pathways.

Therapy The most significant clinical management issues include intractable seizures, bleeding and pain from angiomyolipomas, and respiratory insufficiency from LAM. Seizures are managed with antiepileptic medications. Cortical resections for intractable seizures are occasionally required in children. Angiomyolipomas are often treated with embolectomy when they reach a size of 3 or 4 cm because of evidence that larger tumors are more likely to spontaneously bleed. The only treatment for end-stage LAM is lung transplantation. No specific systemic therapy or targeted therapy is available for TSC. However, advances in the TSC signaling pathways have led to the identification of several possible

targets, including mTOR and Rheb. Tuberin and hamartin inhibit mTOR. The therapeutic potential of the mTOR inhibitor rapamycin is being investigated. See also: Genetics: Overview. Interstitial Lung Disease: Lymphangioleiomyomatosis. Neurofibromatosis. Tumors, Benign.

Further Reading Bissler JJ and Kingswood JC (2004) Renal angiomyolipomata. Kidney International 66: 924–934. Carsillo T, Astrinidis A, and Henske EP (2000) Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proceedings of the National Academy of Science USA 97: 6085–6090. Dabora SL, Jozwiak S, Franz DN, et al. (2001) Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. American Journal of Human Genetics 68: 64– 80. Fingar DC and Blenis J (2004) Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23: 3151– 3171. Franz DN, Brody A, Meyer C, et al. (2001) Mutational and radiographic analysis of pulmonary disease consistent with lymphangioleiomyomatosis and micronodular pneumocyte hyperplasia in women with tuberous sclerosis. American Journal of Respiratory and Critical Care Medicine 164: 661–668. Gomez M, Sampson JR, and Whittemore VH (1999) Tuberous Sclerosis Complex. New York: Oxford University Press. Henske EP (2003) Metastasis of benign tumor cells in tuberous sclerosis complex. Genes, Chromosomes, and Cancer 38: 376– 381. Karbowniczek M, Astrinidis A, Balsara BR, et al. (2003) Recurrent lymphangiomyomatosis after transplantation: genetic analyses reveal a metastatic mechanism. American Journal of Respiratory and Critical Care Medicine 167: 976–982. Kwiatkowski DJ (2003) Tuberous sclerosis: from tubers to mTOR. Annals of Human Genetics 67: 87–96. Logginidou H, Ao X, Russo I, and Henske EP (2000) Frequent estrogen and progesterone receptor immunoreactivity in renal angiomyolipomas from women with pulmonary lymphangioleiomyomatosis. Chest 117: 25–30. Matsui K, Takeda K, Yu ZX, et al. (2000) Downregulation of estrogen and progesterone receptors in the abnormal smooth muscle cells in pulmonary lymphangioleiomyomatosis following therapy. An immunohistochemical study. American Journal of Respiratory and Critical Care Medicine 161: 1002–1009. Moss J, Avila NA, Barnes PM, et al. (2001) Prevalence and clinical characteristics of lymphangioleiomyomatosis (LAM) in patients with tuberous sclerosis complex. American Journal of Respiratory and Critical Care Medicine 164: 669–671. Roach ES, DiMario FJ, Kandt RS, and Northrup H (1999) Tuberous Sclerosis Consensus Conference: recommendations for diagnostic evaluation. National Tuberous Sclerosis Association. Journal of Child Neurology 14: 401–407. Roach ES, Gomez MR, and Northrup H (1998) Tuberous Sclerosis Complex Consensus Conference: revised clinical diagnostic criteria. Journal of Child Neurology 13: 624–628. Sullivan EJ (1998) Lymphangioleiomyomatosis: a review. Chest 114: 1689–1703.

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TUMOR NECROSIS FACTOR ALPHA (TNF-a) S J Levine, National Institutes of Health, Bethesda, MD, USA Published by Elsevier Ltd.

Abstract Tumor necrosis factor alpha (TNF, TNFSF2) is an important multifunctional cytokine that regulates inflammation, host defense, immune responses, and apoptosis. TNF exists as a membrane-bound 26 kDa, type II integral membrane protein, and a 17 kDa soluble homotrimeric TNF that is generated via proteolytic cleavage by TNF-a converting enzyme (TACE, ADAM17). Both the membrane-bound and soluble forms of TNF can interact with either the 55 kDa, type I tumor necrosis factor receptor (TNFRSF1A, TNFR1) or the 75 kDa, type II TNF receptor (TNFRSF1B, TNFR2). TNFR2, however, may be preferentially activated by membrane-bound TNF rather than soluble TNF, which may lead to qualitatively distinct TNF responses. The main biologic function of TNF is to induce inflammation via the upregulation of gene transcription, primarily through the NF-kB and AP-1 signaling pathways, which leads to the expression of a large number of genes. TNF plays an important role in innate immunity and host defense against bacterial, fungal, and parasitic pathogens and is particularly important for host defense against intracellular organisms, such as Mycobacterium tuberculosis. TNF has also been implicated in the pathogenesis of pulmonary diseases, such as idiopathic pulmonary fibrosis, emphysema, sarcoidosis, acute lung injury, bacterial pneumonia, and asthma.

Introduction Tumor necrosis factor alpha (TNF, TNFSF2) is the prototypical member of a large superfamily of at least 17 related cytokines that play key roles in regulating inflammation, host defense, adaptive immunity, apoptosis, autoimmunity, and organ development. Although the antitumor activity of TNF was initially recognized in the nineteenth century when tumor regression was observed in the setting of acute bacterial infection, it was not until 1984 that the protein was purified and the cDNA was cloned. TNF is recognized as an important mediator of inflammatory and immune responses as well as apoptosis. TNF also plays a central role in the pathogenesis of several inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease. Furthermore, the proinflammatory effects of TNF have served as the rationale behind the introduction of anti-TNF therapies for the treatment of rheumatoid arthritis and Crohn’s disease, which have included a recombinant soluble human TNFR2-Ig fusion protein and an anti-TNF monoclonal antibody.

Structure of the TNF Gene and Protein The TNFSF2 gene resides on human chromosome 6p21.3 and on murine chromosome 17, where it is preceded by the TNFSF1 gene that encodes lymphotoxin-a (LTA, LT, TNF-b). Human TNFSF2 is a single-copy gene of 3.6 kb that is composed of four exons and three introns. Expression of the TNFSF2 gene can be regulated at both the transcriptional and the translational level. The TNFSF2 promoter is complex and contains multiple transcription factor binding sites, including those for nuclear factor kappa B (NF-kB), activator protein 1 (AP-1), AP-2, specificity protein-1 (SP-1), Eq6-transformation specific (ETS), nuclear factor of activated T cells (NF-AT), activating transcription factor-2 (ATF-2), and Krox-24. A cyclic AMP response element (CRE), a purine-rich box (PU.1), and a MHC class II-like ‘Y box’ are also present. Translational regulation may also occur via the UA-rich sequence in the 30 untranslated region of human TNF mRNA, which may destabilize the transcript. The crystal structure for soluble TNF protein was initially solved in 1989 and has been obtained at high resolution. TNF exists as a compact trimer in the shape of a cone or bell that is composed of three monomers, which are intimately associated about a threefold axis of symmetry. Each of the TNF monomers exists as two elongated, antiparallel b-pleated sheets, each with eight, antiparallel b-strands that form a ‘jelly-roll’ topology. Further insights have been gained by analyses of the interaction between lymphotoxin and the extracellular domain of TNFR1, which forms a complex composed of three receptor molecules bound symmetrically to one lymphotoxin trimer. The receptors, which exist in a rod-shaped configuration, align along their long parallel axis and interact with the lateral grooves of the trimeric ligand. Although TNF receptors were thought to undergo ligand-dependent trimerization, pre-ligand-binding assembly domains have been identified in the extracellular domains of TNFR1 and TNFR2 that mediate specific ligand-independent assembly of receptor trimers.

Regulation of TNF Production and Activity Macrophages are a primary source of TNF production. TNF can also be produced by many cell types, including T and B cells, mast cells, fibroblasts, keratinocytes, osteoblasts, smooth muscle cells,

308 TUMOR NECROSIS FACTOR ALPHA (TNF-a)

Cellular sources of TNF

Mediators of increased TNF production Infection LPS Bacterial Viral Fungal Parasitic Inflammation IL-1 IL-2 IFN-
GM-CSF M-CSF TNF C5a Immune complexes Subtance P Injury

Macrophages T lymphocytes B lymphocytes Mast cells Eosinophils Epithelial cells Fibroblasts Smooth muscle cells Tumor cells

Disease pathogenesis Acute lung injury Asthma Emphysema Pneumonia Pulmonary fibrosis Sarcoidosis

TNF target genes & proteins Adhesion molecules Apoptosis CC10 Cytokines Chemokines Colony stimulating factors Enzymes Extracellular matrix Growth factors MHC class I and II Mucins Transcription factors Proteinases Proteinase inhibitors Receptors Signaling proteins

Figure 1 The role of TNF in respiratory disease.

endothelial cells, epithelial cells, and tumor cells (Figure 1). TNF production can be stimulated by a wide variety of mediators, including bacterial products (e.g., lipopolysaccharide), cytokines (e.g., IL-1, TNF, and interferon-g), viruses (e.g., HIV and influenza A), parasites (e.g., Plasmodium and Entamoeba), complement (e.g., C5a), immune complexes, phorbol ester, calcium ionophore, and UV irradiation. In particular, large quantities of TNF can be produced in response to bacterial lipopolysaccharide, which contributes to the pathogenesis of septic shock. Alternatively, suppressors of TNF production include cytokines (e.g., IL-4, IL-6, IL-10, IL-13, and TGF-b), cyclic AMP, cyclic nucleotide phosphodiesterase inhibitors (e.g., pentoxifylline and rolipram), cyclosporine A, dexamethasone, and viruses (e.g., Epstein– Barr virus). Human TNF exists in either membrane-bound or soluble forms. TNF is initially expressed as a 26 kDa, type II integral membrane protein that is arranged as a stable homotrimer. The 17 kDa soluble homotrimeric TNF is generated from the membranebound form via proteolytic cleavage between Ala 76 and Val 77 by TNF-a converting enzyme (TACE, ADAM17), which is a member of the ADAM (a

disintegrin and metalloprotease) family. ADAMs are a large family of type I transmembrane proteins that contain multiple functional domains, including a prodomain (that maintains the enzyme in an inactive state via a cysteine switch mechanism), a zinc-dependent catalytic domain, a disintegrin–cysteine-rich domain, an epidermal growth factor-like repeat, a transmembrane domain, and an intracytoplasmic tail. Both the membrane-bound and the soluble forms of TNF can interact with either the 55 kDa, type I tumor necrosis factor receptor (TNFRSF1A, TNFR1) or the 75 kDa, type II TNF receptor (TNFRSF1B, TNFR2). TNFR2, however, may be preferentially activated by membrane-bound TNF rather than soluble TNF, which may lead to qualitatively distinct TNF responses.

Biological Functions of TNF TNF is a multifunctional, proinflammatory cytokine that mediates pleiotropic biological functions, especially host defense. The broad spectrum of TNFmediated biological functions is a direct consequence of its ability to induce the expression of a large number of gene products. Genes or proteins that can

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be induced by TNF include transcription factors (e.g., c-fos and c-jun), cytokines (e.g., IL-1, TNF, IL-6, and IL-8), chemokines (e.g., eotaxin), growth factors (e.g., platelet-derived growth factor and granulocyte-macrophage colony-stimulating factor), adhesion molecules (e.g., ICAM-1, VCAM-1, and E-selectin), receptors (e.g., IL-2Ra and EGF receptor), complement (e.g., C3), major histocompatibility complex proteins (e.g., MHC class I and MHC class II), acute phase reactants (e.g., haptoglobin), and enzymes (e.g., nitric oxide synthase, manganese superoxide dismutase, and matrix metalloproteases). TNF was initially identified via its ability to induce cytotoxicity against tumor cell lines and to mediate tumor necrosis in certain animal models. TNF was also found to be identical to the protein cachectin, which mediates fever and muscle wasting in cancer patients and weight loss, anorexia, and lethality in mice. TNF has been identified as playing an important role in innate immunity and host defense against bacterial, fungal, and parasitic pathogens. In particular, TNF plays a key role in host defense against intracellular pathogens, such as Mycobacterium tuberculosis, Leishmania major, and Listeria monocytogenes. This is exemplified by the association between anti-TNF therapy and the reactivation of latent tuberculosis. Similarly, anti-TNF therapy has been associated with infections caused by Histoplasma capsulatum, Aspergillus fumigatus, and L. monocytogenes. Tissue macrophages and T lymphocytes produce TNF in response to invasion by intracellular pathogens, which then mediates cytokine and chemokine production and the subsequent recruitment of activated leukocytes to the site of infection. In later stages of infection, TNF enhances antigen presentation and T-cell costimulation, thereby promoting the induction of acquired immunity. Another important function of TNF is the induction of apoptosis, which typically occurs in the absence of concomitant signaling by NF-kB. This is exemplified by transgenic mice deficient in NF-kB signaling, which undergo embryonic lethality as a consequence of TNF-mediated liver cell apoptosis. The biologic role of TNF-mediated apoptosis, however, requires further clarification. Insights into the biological function of TNF have also come from studies utilizing transgenic models. TNF knockout (TNF
/
) mice develop normally and have no structural or morphological abnormalities. TNF
/
mice are highly susceptible to challenge with infectious agents (e.g., Candida albicans and L. monocytogenes), are resistant to lethality from minute doses of lipopolysaccharide following sensitization with D-galactosamine, demonstrate low toxicity to TNF, have defective granuloma

formation, do not form germinal centers after immunization, and display reduced immunoglobulin responses to thymus-dependent antigens. Furthermore, the function of macrophages, neutrophils, and T cells derived from TNF
/
mice is normal. TNF has been implicated in the pathogenesis of diabetes and may be an important mediator in obesity because TNF
/
mice are protected from obesityinduced insulin resistance. Insights into TNF function have also been derived from studies of transgenic mice lacking the two TNF receptors, TNFR1 and TNFR2. For example, TNF may play an important role in the regulation of central nervous system injury and function. TNF may have a neuroprotective function because neuronal damage secondary to ischemic and epileptic injury is exacerbated in TNFR1/TNFR2 double knockout mice. Similarly, in a model of experimental autoimmune encephalitis, TNF
/
mice develop severe autoimmune-mediated demyelination, which can be attenuated by TNF administration.

TNF Receptors and Signaling Pathways The main biologic function of TNF is to induce inflammation via the upregulation of gene transcription, primarily through the NF-kB and AP-1 signaling pathways. This is accomplished by signaling through two members of the TNF receptor superfamily, TNFR1 and TNFR2. TNFR1 is constitutively expressed by most cell types, whereas TNFR2 has a highly regulated pattern of cellular expression. Binding of TNF to TNFR1 mediates the translocation of TNFR1 to lipid rafts, where it recruits the adaptor protein, TNF receptor-associated death domain (TRADD), via a direct interaction with the TNFR1 death domain. TRADD then serves as an assembly platform to mediate the recruitment of other adaptor proteins, such as receptor-interacting protein (RIP) and TNFR-associated factor-2 (TRAF2), which in turn recruit I-kB kinases and thereby activate the NF-kB pathway (Figure 2). TNFR1 and RIP within the lipid rafts are then ubiquitinated, which leads to their degradation via the proteasome pathway. TNFR1 signaling through TRAF2 can also activate the c-Jun N-terminal kinase (JNK) pathway through the MAPK kinase, mitogenactivated protein kinase kinase-7 (MKK7), which results in the phosphorylation of c-Jun, with resultant increases in AP-1 activity. TNF signaling can also induce apoptosis, primarily via TNFR1-mediated signaling. TNF-mediated apoptosis requires the blockade of the NF-kB pathway because NF-kB activation induces the expression of several antiapoptotic genes, including c-FLIP

310 TUMOR NECROSIS FACTOR ALPHA (TNF-a)

TNF TNFR1 Plasma membrane Inter

naliz

TRADD TRAF2

ation

RIP1

MKK7

IKK- IKK-

JNK

IKK-


c-Jun

NF-B

TNF TNFR1

TNF receptosome

TRADD FADD Caspase 8

Inflammation

Apoptosis

Figure 2 TNFR1 signaling pathways. TNF binding to TNFR1 mediates the translocation of TNFR1 to lipid rafts, where it recruits the adaptor proteins TRADD, RIP1, and TRAF2, which can activate the NF-kB pathway via an I-kB kinase complex composed of I-kB kinase-a, I-kB kinase-b, and I-kB kinase-g (NEMO). Alternatively, TNFR1 signaling through TRAF2 can activate the c-Jun N-terminal kinase (JNK) pathway through the MAPK kinase, MKK7, which results in the phosphorylation of c-Jun, with resultant increases in AP-1 activity. Both the NF-kB and JNK pathways stimulate inflammation. Furthermore, activation of the NF-kB pathway inhibits apoptosis as a consequence of the increased expression of antiapoptotic genes (i.e., c-FLIP, cIAP1, and cIAP2). TNFR1 signaling can induce apoptosis in the absence of NF-kB activation. A model proposed that the formation of a TNFR1-associated death-inducing signaling complex (DISC) is dependent on receptor internalization within endocytic vesicles, termed TNF receptosomes. Internalized receptors recruit TRADD, FADD, and caspase-8 to form a TNFR1-associated DISC. This results in the rapid activation of caspase-8, which initiates apoptosis. TNF receptosomes can fuse with trans-Golgi vesicles to form multivesicular endosomes, allowing for the caspase8-mediated activation of acid sphingomyelinase and cathepsin D cascades, which also induce apoptosis via Bid cleavage and caspase-9 activation (not shown). Other signaling pathways that can be activated by TNF include the stress-activated protein kinase (SAPK) cascade, the p38 mitogen-activated protein kinase (MAPK) pathway, and the neutral sphingomyelinase/ceramide pathway (not shown).

(cellular FADD-like interleukin-1-converting enzyme-inhibitory protein) and the cellular inhibitor of apoptosis proteins (IAPs), cIAP1 and cIAP2. Two models have been proposed for TNF-mediated apoptosis. One model proposes two sequential signaling complexes, with the initial formation of a TNFR1, TRADD, RIP1, and TRAF2 signaling complex I at the plasma membrane, followed by a cytoplasmic complex II composed of TRADD, RIP1, FADD (Fas-associated death domain), and caspase-8 that mediates cell death in the absence of NF-kB activation. The second model proposes the liganddependent formation of a TNFR1-associated deathinducing signaling complex (DISC) that is dependent on receptor internalization within endocytic vesicles, termed TNF receptosomes. In this model, receptor internalization allows the recruitment of TRADD, FADD, and caspase-8 to form a TNFR1-associated DISC within TNF receptosomes (Figure 2). Caspase8 is rapidly activated and initiates apoptosis. TNF receptosomes then fuse with trans-Golgi vesicles to form multivesicular endosomes, allowing the

caspase-8-mediated activation of acid sphingomyelinase and cathepsin D cascades, which can induce apoptosis via Bid cleavage and caspase-9 activation. The adaptor protein FAN (factor associated with neutral Smase activation), via binding to a neutral sphingomyelinase domain in TNFR1, can also induce apoptosis via signaling through neutral sphingomyelinase and ceramide. TNFR2 does not contain intracytoplasmic death domains but can directly bind TRAF2, leading to the activation of the JKK/NF-kB and the JNK pathways. TNFR2-mediated TRAF2 degradation, however, may downregulate NF-kB activation and thereby promote apoptosis. Other signaling pathways that can be activated by TNF include the stress-activated protein kinase (SAPK) cascade and the p38 mitogen-activated protein kinase (MAPK) pathway.

TNF and Pulmonary Disease TNF has been implicated in the pathogenesis of a variety of diseases, including rheumatoid arthritis,

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inflammatory bowel disease, endotoxin-mediated septic shock, HIV infection, diabetes, graft-versushost disease, allograft rejection, and neoplastic disease. TNF may also play a role in the pathogenesis of pulmonary diseases, including pulmonary fibrosis, bacterial pneumonia, emphysema, sarcoidosis, acute lung injury, and asthma. Evidence of a role for TNF in pulmonary fibrosis derives from murine models in which overexpression of TNF in the lung produced a lymphocytic and fibrosing alveolitis that resembled human idiopathic pulmonary fibrosis. Similarly, TNF receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers. TNF has also been shown to mediate anti-inflammatory and antifibrotic effects in murine models of bleomycin-induced lung injury and fibrosis, consistent with a complex role for TNF in the pathogenesis of pulmonary fibrosis. TNF knockout mice display impaired bacterial killing and increased mortality, consistent with a role for TNF in host defense against bacterial pneumonia. TNF has also been implicated in the pathogenesis of cigarette smoke-induced emphysema because TNFmediated processes were responsible for 70% of the airspace enlargement in a murine model, which may be a consequence of enhanced neutrophil recruitment. Similarly, overexpression of TNF in murine type II alveolar epithelial cells has been associated with severe airspace enlargement and pulmonary hypertension. TNF may also contribute to weight loss and cachexia associated with severe chronic obstructive pulmonary disease. TNF has also been implicated in the pathogenesis of pulmonary sarcoidosis and the acute respiratory distress syndrome. Patients with progressive or severe sarcoidosis demonstrate increased spontaneous TNF production from alveolar macrophages, whereas elevated TNF release from alveolar macrophages may correlate with disease progression. TNF may play an important role in acute lung injury because TNF or TNFR1 knockout mice demonstrated attenuated lung neutrophil recruitment and microvascular permeability in a murine model of hemorrhage-induced acute lung injury. Lastly, although clinical evidence suggests a possible role for TNF in the pathogenesis of asthma, investigations utilizing murine models of allergic asthma have produced varied results. The results of future clinical trials will clarify whether a role exists for anti-TNF therapies in the treatment of these pulmonary diseases.

See also: Acute Respiratory Distress Syndrome. Apoptosis. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Human Immunodeficiency Virus. Leukocytes: Pulmonary Macrophages. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Mycobacterial. Pulmonary Fibrosis. Systemic Disease: Sarcoidosis.

Further Reading Aggarwal BB, Kohr WJ, et al. (1985) Human tumor necrosis factor. Production, purification, and characterization. Journal of Biological Chemistry 260(4): 2345–2354. Aggarwal BB, Samanta A, et al. (2001) TNFa. In: Oppenheim JJ and Feldmann M (eds.) Cytokine Reference: A Compendium of Cytokines and Other Mediators of Host Defense, pp. 413–434. San Diego: Academic Press. Bazzoni F and Beutler B (1996) The tumor necrosis factor ligand and receptor families. New England Journal of Medicine 334(26): 1717–1725. Chen G and Goeddel DV (2002) TNF-R1 signaling: a beautiful pathway. Science 296(5573): 1634–1635. Idriss HT and Naismith JH (2000) TNF alpha and the TNF receptor superfamily: structure–function relationship(s). Microscopy Research and Technique 50(3): 184–195. Legler DF, Micheau O, Doucey MA, Tschopp J, and Bron C (2003) Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalpha-mediated NF-kappaB activation. Immunity 18(5): 655–664. Locksley RM, Killeen N, et al. (2001) The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104(4): 487–501. Marino MW, Dunn A, et al. (1997) Characterization of tumor necrosis factor-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 94(15): 8093–8098. Micheau O and Tschopp J (2003) Induction of TNF receptor Imediated apoptosis via two sequential signaling complexes. Cell 114(2): 181–190. Pasparakis M, Alexopoulou L, et al. (1996) Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. Journal of Experimental Medicine 184(4): 1397–1411. Pennica D, Nedwin GE, et al. (1984) Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 312(5996): 724–729. Varfolomeev EE and Ashkenazi A (2004) Tumor necrosis factor: an apoptosis JuNKie? Cell 116(4): 491–497. Vilcek J and Lee TH (1991) Tumor necrosis factor. New insights into the molecular mechanisms of its multiple actions. Journal of Biological Chemistry 266(12): 7313–7316. Wajant H, Pfizenmaier K, et al. (2003) Tumor necrosis factor signaling. Cell Death and Differentiation 10(1): 45–65. Wallach D, Varfolomeev EE, et al. (1999) Tumor necrosis factor receptor and Fas signaling mechanisms. Annual Review of Immunology 17: 331–367.

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TUMORS, BENIGN D W Henderson and S Klebe, Flinders University, Adelaide, SA, Australia

combination of different anatomical sites (e.g., pleural and intrapulmonary solitary fibrous tumor).

& 2006 Elsevier Ltd. All rights reserved.

Abstract Benign tumors of the lung and pleura encompass a wide variety of epithelial and nonepithelial tumors that have an excellent prognosis in general. Many of these lesions are discovered incidentally in chest radiographs carried out for other reasons, most being asymptomatic at the time of presentation. Such benign tumors can occasionally produce symptoms related to bronchial obstruction (e.g., bronchial papillomas and mucous cell adenomas), or because of compression of lung parenchyma by sizable tumors (such as large solitary fibrous tumors of pleura/lung). Symptoms when present may include hemoptysis, cough or dyspnea, and other manifestations only rarely. Only exceptionally do benign lesions of the bronchus undergo malignant transformation (specifically the development of squamous cell carcinoma in tracheobronchial papillomatosis). In part, the importance of benign tumors of lung and pleura lies in the necessity to discriminate between them and malignant neoplasms of the respiratory tract.

Introduction The borderland between inflammation/hyperplasia and benign neoplasia, and between benign versus malignant neoplasia, is poorly delineated or controversial for many disorders of the lungs and pleura. Accordingly, the selection of disorders discussed in this article is to some extent arbitrary (e.g., minute meningothelioid nodules have been included whereas carcinoid tumorlets have not, partly because the latter are considered to represent reactive hyperplasias of neuroendocrine cells). The presenting clinical manifestations of benign tumors of the lung and pleura and their complications are largely dependent upon (i) the anatomical site where the lesions arise, and (ii) their size. Tumors arising within large airways can present with symptoms related to bronchial obstruction and/or hemoptysis, whereas those affecting the lung parenchyma or pleura are frequently discovered in radiographs taken for other reasons – unless they are large enough to produce compression of the lung with dyspnea, or attention is drawn to their existence by other factors, for example, the production of insulinlike growth factor (IGF) by some pleuropulmonary solitary fibrous tumors. Therefore, for the sake of convenience, the tumors discussed in this section are grouped according to whether they arise from large airways, lung parenchyma or the pleura, or a

Benign Tumors of Large Airways Solitary Squamous, Columnar, and Mixed Papillomas of Bronchus

Solitary bronchial squamous papillomas are rare lesions found mainly in middle-aged adults, and slightly more often in males than females. Solitary squamous papillomas of this type typically have histological appearances equivalent to papillomas found elsewhere, related to infection by human papilloma virus (HPV), and they can occur in association with HPV-related squamous papillomas of the larynx and upper aerodigestive tract. In small fiberoptic bronchial biopsies, discrimination between solitary squamous papilloma and well-differentiated squamous cell carcinoma can be problematic. Up to about 20% recur, and squamous cell carcinomas have been reported at the removal sites for these papillomas, suggesting that a few can transform into squamous carcinoma. Columnar papillomas are extremely rare lesions that comprise arborizing to club-shaped fronds covered by columnar epithelium (Figures 1 and 2) and, as the term implies, mixed papillomas represent a hybrid of columnar cells and squamous elements. Usually, these lesions can be managed by removal at endoscopy, and for those associated with malignant

Figure 1 Glandular papilloma of bronchus. The papilloma distends and occludes the bronchial lumen. The wall of the bronchus is depicted at right. Case contributed by Dr A S Pieterse, Adelaide, South Australia.

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Figure 2 Glandular papilloma of bronchus. Same case as Figure 1: the papillary processes are covered by mucin-producing columnar epithelium.

change, by surgical resection appropriate for conventional non-small cell carcinomas of lung. Multifocal Respiratory/Bronchial Papillomatosis

In cases of multifocal bronchial papillomatosis, HPV infection is typically acquired during vaginal delivery from an HPV-affected mother. Papillomatosis develops during early life, and children so affected typically develop oropharyngeal and/or laryngotracheal papillomatosis, which may thence spread to affect more distal airways. In some cases, the papillomatosis can extend into lung parenchyma, resulting in cavitating lesions for which the distinction between cavitary papillomatosis versus supervening malignant change with the development of a cavitary squamous cell carcinoma can be exceedingly difficult or impossible on either radiological grounds or on small biopsy or fine-needle aspiration samples. Children affected by multifocal tracheobronchial papillomatosis typically develop a combination of symptoms that can include hemoptysis, manifestations of bronchial obstruction with wheezing, and/or recurrent pneumonia. Benign Tumors of Bronchial Glands: Mucous Cell Adenoma and Bronchial Analogs of Benign Salivary Gland Adenomas

The expression ‘bronchial adenoma’ as a blanket term for indolent bronchial lesions has long outlived its usefulness; it was always beset with imprecision because it encompassed a group of indolent low-grade malignant neoplasms, including carcinoid tumors and adenoid cystic and low-grade mucoepidermoid carcinomas of bronchus. For bronchial lesions, the term ‘adenoma’ should be restricted nowadays to mucous cell adenoma of bronchus, papillary adenoma, and

pleomorphic adenoma (mixed tumor), with nomenclature specific for each neoplasm. Benign mucous gland adenoma (MGA), papillary adenoma, and pleomorphic adenoma of bronchus are extraordinarily rare neoplasms. For example, the textbook Practical Pulmonary Pathology published in 2005 refers to a study of over 3000 pulmonary tumors where there were no examples of MGA, and up to 1995, only 10 cases were found in the files of the Armed Forces Institute of Pathology in Washington, DC. MGAs can arise in any of the major or segmental bronchi and although some are asymptomatic, others can produce manifestations of bronchial obstruction. Characteristically, MGAs comprise microcystic collections of cuboidal to columnar mucus-producing epithelial cells. The main differential diagnosis for MGA is a lowgrade mucoepidermoid carcinoma, characterized by an admixture of squamous or intermediate cells, whereas MGAs consist exclusively of microcystic mucus-producing glandular structures. The very rare bronchial gland oncocytoma requires distinction from oncocytic carcinoid tumor and even metastatic tumors with oxyphilic cells. Benign Tumors of Nonepithelial Components of Bronchial Walls

Benign Schwannian or perineurial tumors of bronchi (Schwannoma, neurofibroma) are rare, but may be encountered in particular among patients with type I von Recklinghausens neurofibromatosis. Otherwise, these lesions are most likely to represent incidental findings at bronchoscopy and are sometimes contained in biopsy samples. Perhaps the most important benign neural tumor found in bronchi is the granular cell tumor (GCT, granular cell neurofibroma, Abrikossoff’s tumor). Again, some GCTs are asymptomatic and are found incidentally at bronchoscopy whereas others can be large enough to produce bronchial obstruction, cough, or hemoptysis. GCTs comprise uniform populations of rounded to fusiform cells with finely granular cytoplasm. Characteristically, these lesions have an immunophenotype of Schwannian differentiation, with positive labeling for S-100 proteins or myelin basic protein, and on electron microscopy they are characterized by myriad secondary lysosomes.

Benign Tumors Affecting Lung Parenchyma and Bronchi So-Called Localized Chondromatous Hamartoma

Although the great majority of localized chondromatous hamartomas (LCHs) arise within the peripheral

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Figure 3 Localized chondromatous hamartoma of lung. The lesion comprises lobulated collections of hyaline cartilage, together with lucent adipocytes and infolded respiratory mucosa. No calcification is present in this case.

lung parenchyma, the occurrence of these lesions in larger airways is well documented, where they can produce hemoptysis and/or signs and symptoms of bronchial obstruction. Although the term ‘hamartoma’ implies a nonneoplastic developmental disorder, there appears to be a consensus that LCH represents a benign connective tissue tumor that typically shows chondroid differentiation along with myxoid fibrous tissue and adipose tissue, and which entraps the epithelium of small airways as it enlarges (Figure 3). For peripheral LCHs, calcification within the cartilaginous tissue may produce a popcorn appearance that allows confident radiologic diagnosis. These lesions tend to shell out from the lung parenchyma quite easily at surgery. Although pediatric cases have been recorded, most LCHs are found in adults between the ages of about 40 and 60 years, and in some cases they can be seen to enlarge in serial chest radiographs. Most LCHs represent solitary lesions but they can be encountered as part of the lesional triad that includes pulmonary hamartoma, extra-adrenal paragangliomas, and gastric epithelioid stromal tumors (Carney’s triad). Benign tumors of smooth muscle in the lung are extremely rare. Lymphangioleiomyomatosis (LAM) has been excluded from this article, and although some authorities include benign metastasizing leiomyoma among the benign connective tissue tumors of bronchus/lung, these lesions are generally considered to represent indolent pulmonary metastases/ emboli from a low-grade uterine leiomyosarcoma that lacks cytological markers of malignancy. Lipoma of bronchus (Figure 4) is an extremely uncommon lesion, and requires distinction from a

Figure 4 Lipoma of bronchus. The lucent central zone of the tumor represents mature adipose tissue.

localized hamartoma that shows a predominance of adipocytic differentiation. Lipomas are similarly rare within the peripheral lung tissue. Glomus cell tumors and glomangiomyomas of bronchus/lung are even rarer, as are true hemangiomas of bronchus and lung. Mature (Differentiated) Teratoma

Primary intrapulmonary mature teratomas (dermoid cysts) are extremely rare tumors (Figure 5). These lesions must be distinguished on clinical grounds from metastatic germ-cell tumors with teratomatous elements, where the malignant components such as embryonal carcinoma have been ablated by chemotherapy.

Benign Tumors Arising in Lung Parenchyma Mucinous Cystadenoma of Lung

Mucinous cystadenoma of lung is an extraordinarily rare lesion, typically sharply demarcated from the surrounding peripheral lung parenchyma by a fibrous capsule that encloses locules of viscous mucinous material, the locules being lined by benign-appearing mucin-producing epithelial cells. Absence of invasion is a prerequisite for the diagnosis, and this tumor requires distinction from mucinous (colloid) adenocarcinoma of lung. Alveolar Adenoma

Benign alveolar adenoma of lung is a rare entity, most often discovered incidentally in chest radiographs taken for other reasons (Figure 6). These lesions are usually encountered in middle adult life, and in females more often than males. The

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Figure 6 Alveolar adenoma chest X-ray. Rounded and welldemarcated tumor located in the left lower zone of a 57-year-old woman. Figure 5 Mature teratoma (dermoid cyst) of bronchus/lung. This young woman presented with trichoptysis. The central zone of the teratoma comprises sebaceous material (yellow) and a tangled mass of hairs. The lesion is associated with bronchiectasis and areas of obstructive pneumonitis.

predominant localization of alveolar adenoma to the left lower lobe as seen in Figure 6 (about 60% of cases) is unexplained. These lesions represent well-demarcated but unencapsulated microcystic tumors that recapitulate the structure of the alveolar septum, with a lining of pneumocytes and variable amounts of underlying fibrovascular tissue (Figure 7). Typically, the lining epithelium displays reactivity for thyroid transcription factor-1 (TTF-1), surfactant apoprotein, cytokeratins, and carcinoembryonic antigen. The prognosis for these tumors following surgical resection is excellent. Lymphangioma is the main differential diagnosis and one case of alveolar adenoma has been reported as such in the literature. So-Called Sclerosing Hemangioma

Although the term ‘pneumocytoma’ has been proposed as an alternative to ‘sclerosing hemangioma’, the latter term is well entrenched (‘pneumocytoma’ would also invite confusion with alveolar adenoma). Typically, sclerosing hemangioma of lung is asymptomatic at the time of detection (only about 20% have respiratory symptoms that can include

Figure 7 Alveolar adenoma. The tumor comprises multiple microcystic spaces lined by alveolar epithelial cells that showed positive labeling for thyroid transcription factor-1. Small amounts of connective tissue are located beneath the epithelium, producing mimicry of alveolar septa. The intra-alveolar hemorrhage is artefactual.

cough, hemoptysis, or chest pain). Sclerosing hemangioma can arise in any of the lobes of lung, and involvement of large airways and pleura can occur but is rare (about 1% of cases). Sclerosing hemangiomas have been recorded from early childhood through to old age, and females predominate over males. The tumor seems to occur with particular frequency in East Asia, for reasons that are unexplained.

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Figure 8 Sclerosing hemangioma. Sclerotic tissue is evident in the right side of this field, together with papillary projections. More cellular tissue is evident elsewhere, with blood-filled cavernomatous spaces.

Figure 9 Benign clear-cell tumor (‘sugar’ tumor). A gaping blood vessel is present among the clear cells, near the center of this field, and some vessels have hyalinized walls (left). Gaping vessels with a staghorn pattern were more prominent in other areas of this tumor. On immunohistochemistry, the tumor showed positive labeling with/for S-100 proteins, HMB45, and melan A.

Usually, most sclerosing hemangiomas are less than 30 mm in diameter, and characteristically they comprise angiomatoid and hemorrhagic foci in association with stromal sclerosis, papillary structures covered by epithelioid cells (Figure 8), and other areas that consist of sheets of epithelioid cells. Although the genesis or pattern of differentiation for sclerosing hemangioma remained uncertain for many years, more recent studies reveal that the pattern of differentiation is that of embryonic alveolar epithelium (with positive labeling for TTF-1, although only the better-differentiated papillary areas usually show positive reactivity for surfactant-specific apoprotein). Benign Clear-Cell Tumor of Lung (‘Sugar’ Tumor)

First described by Liebow and Castleman in 1963, benign clear-cell tumor (CCT) of lung is a rare neoplasm, typically discovered in chest radiographs taken for other reasons. CCT usually comprises sheets of cells with eosinophilic to clear cytoplasm, with gaping blood vessels devoid of smooth muscle and which are often hyalinized (Figure 9) – a point of distinction from primary or secondary clear-cell carcinomas such as metastatic renal cell carcinoma. The histogenesis and pattern of differentiation in CCT remained elusive for many years, but recent studies indicate that these lesions show immunoreactivity for markers of melanocytic differentiation (such as HMB45 and melan A) and melanosomes have been found in some cases on electron microscopy (Figure 10). It has been proposed that CCT represents a member of the PEComas: a group of tumors related to perivascular epithelioid cells

Figure 10 Benign clear-cell tumor. One of many organelles resembling stage II melanosomes found in this case, showing the characteristic transverse periodicity. Same case as Figure 9.

(PECs), whose other members include LAM, micronodular pneumocyte hyperplasia in some patients with tuberous sclerosis, and angiomyolipoma of the kidney and other sites (which include lung on very rare occasions). Whether the PEComa concept will

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prove any more valid or useful than the neurocristopathies is open to doubt, and the normal cell counterpart for the PEComas is yet to be identified. Inflammatory Myofibroblastic Tumor

Inflammatory myofibroblastic tumor (IMFT) exemplifies the problem of delineation of the borderland between benign tumors of the lung and pleura versus inflammatory disorders and low-grade malignant lesions; the time-honored term ‘inflammatory pseudotumor’ and the alternative expression ‘plasma cell granuloma’ imply that these lesions are non-neoplastic. However, there appears to be an emerging consensus that the fibrohistiocytic subtype of IMFT represents a neoplastic lesion, whereas the plasma cell granuloma subtype probably represents a nonneoplastic lesion of unknown and perhaps variable causation. IMFT occurs with about equal frequency in both sexes, and from early childhood to late adult life, but with a peak incidence in middle adult life. Typically, patients present with cough, hemoptysis, dyspnea, or chest pain, while other lesions are asymptomatic at the time of detection. Typically, IMFT is a solitary sharply demarcated mass lesion (Figure 11), but multifocal lesions are well recognized. The tumors have diameters usually in the range of about 5– 300 mm, located within the lung parenchyma, but involvement of bronchial structures can occur, as can extension into pulmonary vessels. The fibrohistiocytic variant of IMFT also requires distinction from the cystic fibrohistiocytic tumor of lung (which in turn requires distinction from cystic change in pulmonary metastases from malignant fibrous histiocytomas of soft tissue). When IMFTs have been observed in serial chest radiographs before surgical resection, they have been

found to remain stable or increase slowly in size, and spontaneous resolution has been reported. However, other cases have been recorded that recur and progress, and in such cases the use of corticosteroids has been advocated. The recurrence rate for IMFT is about 25%, but substantially less than 1% metastasize. The dividing line between benign IMFT versus equivalent low-grade recurrent and malignant lesions remains ill-defined. Benign Meningeal Tumors and Tumor-Like Lesions of Lung

The vast majority of meningiomas occur along the neuraxis and less commonly in the skin, but primary intrapulmonary meningiomas are well recorded (the diagnosis requires exclusion of metastatic meningioma on clinical grounds). Primary meningiomas usually show meningothelial, transitional, or fibrous patterns indistinguishable from meningiomas found in the neuraxis. Although primary pulmonary meningiomas are exceedingly uncommon, the so-called minute meningothelioid nodules (MMTNs) are well recognized in the lung; in the past, these lesions were designated as minute pulmonary chemodectomas, but ultrastructural studies have demonstrated conclusively that these lesions are unrelated to chemoreceptor tissue and instead have interdigitating processes akin to neuraxial meningiomas. In general, MMTNs represent subclinical incidental findings in lung tissue resected for other reasons, but they are occasionally detectable in high-resolution CT (HRCT) scans, and the authors have encountered one such case where HRCT demonstrated multiple small nodules within the lungs, raising the differential diagnosis of small metastatic deposits. Typically, MMTNs comprise microscopic nodules of uniform-appearing epithelioid cells, closely associated with venules (Figure 12). In some instances, they are associated with cardiac failure or lung disorders of diverse type. They require no treatment, but they have been invoked as a potential precursor for primary pulmonary meningiomas (raising in turn the question of the histogenesis of MMTNs).

Benign Tumors That Can Affect Both Pleura and Lung Solitary Fibrous Tumor

Figure 11 Inflammatory myofibroblastic tumor (fibrohistiocytic type) in the lung of a child. The pale lesion is well demarcated from the surrounding lung parenchyma.

Although the great majority of solitary fibrous tumors (SFTs) affect the pleura and especially the visceral pleura, intrapulmonary SFTs are well described, as are the ones arising in sites outside

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Figure 13 Solitary fibrous tumor of pleura. Pedunculated lesion 30 mm in greatest diameter, attached to the visceral pleura. The pedicle is evident in the lower center of this photograph.

Figure 12 Minute meningothelioid nodule. Incidental finding in a resected lung lobe. The lesional cells are interstitial in location and are associated with small blood vessels.

pleuropulmonary tissues. In the past, SFTs were designated as fibrous mesotheliomas, inviting confusion with the sarcomatoid and desmoplastic varieties of malignant mesotheliomas, so the expression fibrous mesothelioma for these lesions should be abandoned. Typically, SFTs affecting the pleura or lung are asymptomatic and are found in chest radiographs taken for other reasons; however, when large, SFTs can produce compression of the lung, with dyspnea and cough, and on rare occasions SFTs can result in hypoglycemia related to the production of IGF by the tumor, or hypertrophic osteoarthropathy. SFTs affecting the pleura are uncommon, and it has been estimated that about two pleural mesotheliomas are encountered in everyday surgical pathology practice for every SFT. Typically, SFTs are found during adult life and they vary in size from about 10 mm to 350 mm. The most usual gross appearance for benign SFT is that of a pedunculated lesion localized to the visceral pleura (Figure 13). On histological examination, SFTs can show a variety of configurations, ranging from a so-called patternless pattern (Figure 14), to storiform, herringbone, and angiofibromatoid patterns. On immunohistochemistry, they are usually positive for CD34, bcl-2, and CD99, and they consistently lack cytokeratin expression (a point of distinction from most sarcomatoid mesotheliomas). A variety of cytogenetic aberrations has been recorded in SFTs, none of which appears to be specific. In the majority of cases, SFTs are benign, but malignant SFTs are also well recognized. The criteria for

Figure 14 Solitary fibrous tumor of pleura. So-called patternless pattern. The fibroblastoid cells are associated with bundles of collagen; they showed positive labeling for CD34 and bcl-2, without detectable cytokeratins.

discrimination between benign versus malignant SFTs remains somewhat unclear; in one study of over 220 SFTs, it was observed that no clear histological discriminator for the assessment of malignancy existed, and the criteria for malignancy in this study arbitrarily included nuclear atypia and pleomorphism, and more than four mitotic figures per 10 high-power fields, together with necrosis and hemorrhage. However, only a little over 50% of the lesions with these features pursued an aggressive course in terms of recurrence, metastasis, or both, and 45% were apparently cured by surgical resection. In another study of 50 cases, the authors felt that the most reliable indicators of malignancy were metastasis, invasion, size, and necrosis. However, as with gastrointestinal stromal tumors, such guidelines – although useful in many cases for the purposes of follow-up and management – have their limitations. The authors have encountered one case of malignant

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SFT devoid of nuclear atypia, mitotic activity, or necrosis, but which nonetheless invaded lung and pleura, with a massive recurrence following surgical resection, and death of the patient within 10 months of diagnosis. Although some authors suggest that all SFTs should be regarded as borderline tumors, it is probably safe to emphasize that small pedunculated SFTs attached to the visceral pleura are likely to be benign, with only a low risk of recurrence following complete surgical resection (including the base of the pedicle), whereas large sessile SFTs affecting the parietal pleura and especially those which show obvious necrosis, should be regarded as having a high potential for malignant behavior. Pleuropulmonary Thymoma

The occurrence of primary pleural thymomas is now well established, and rare cases can occur within the lung. One essential criterion for the diagnosis of such cases is exclusion of a primary thymic thymoma. The histological appearances of the pleuropulmonary tumors differ in no way from primary anterior mediastinal thymomas.

Benign Tumors of the Pleura Benign Adenomatoid Tumor of the Pleura

Benign adenomatoid tumors of the pleura are exceedingly rare. The histological appearances are essentially identical to those of extrapleural benign adenomatoid tumors affecting the genital tract. Typically, these rare lesions represent small incidental lesions found at thoracotomy carried out for other reasons. They require discrimination from malignant mesotheliomas of epithelioid type with a prominent adenomatoid pattern. Multicystic and Well-Differentiated Papillary Mesothelioma

In contrast to multicystic and well-differentiated papillary mesotheliomas (WDPMs) affecting the peritoneum, such lesions in the pleura are exceedingly rare, and we have encountered only a single case of a pleural multicystic mesothelioma (defined as such because of gross cystic spaces containing thin serous fluid as opposed to a microcystic architecture found in conventional malignant epithelioid mesotheliomas). In the peritoneum, solitary WDPMs are regarded as benign, whereas multifocal lesions of this type are sometimes considered to be borderline neoplasms with a protracted and indolent natural history. The occurrence of WDPM in the pleura is less well established than for the peritoneal counterpart, but

Figure 15 Calcifying fibrous tumor of pleura. Rounded calcospherites (psammoma bodies) are present within the hypocellular fibrocollagenous tissue. Case contributed by Dr G Elmberger, Stockholm, Sweden.

such lesions have been recorded. However, conventional malignant epithelioid mesotheliomas can have an extensive papillary architecture that may not be sampled by a small biopsy, so that accurate identification of these lesions in the pleura can be beset with uncertainty, and an aggressive clinical course would indicate this happenstance. Diagnosis of benign WDPM affecting the pleura preferably requires local removal of the entire lesion, which consists of delicate to club-shaped papillary processes covered by a single layer of cuboidalized mesothelial cells, with no evidence of invasion of the sublesional tissues. Calcifying Fibrous Tumor of the Pleura

Calcifying fibrous (pseudo-)tumor of the pleura is an exceedingly rare lesion described only in adults. CT scans have demonstrated well-demarcated and partly calcified nodular lesions affecting the pleura. These lesions comprise hypocellular benign-appearing fibrocollagenous tissue with rounded (psammoma-like) calcific bodies (Figure 15). Immunohistochemical studies have shown positive labeling for vimentin and CD34 positivity in scattered cells, along with actin and desmin, and a relationship to inflammatory myofibroblastic tumor has been invoked. Follow-up has been entirely benign. See also: Antiviral Agents. Mesothelioma, Malignant. Tuberous Sclerosis.

Further Reading Colby TV, Koss MN, and Travis WD (1995) Tumors of the Lower Respiratory Tract. Atlas of Tumor Pathology, 3rd Series, Fascicle 13. Washington, DC: American Registry of Pathology/ Armed Forces Institute of Pathology.

320 TUMORS, MALIGNANT / Overview Dail DH and Hammar SP (eds.) (1994) Pulmonary Pathology, 2nd edn. New York: Springer. Leslie KO and Wick MR (2005) Practical Pulmonary Pathology: A Diagnostic Approach. Philadelphia: Churchill Livingstone. Travis WD, Brambilla E, Mu¨ller-Hermelink HK, and Harris CC (eds.) (2004) Pathology and Genetics of Tumours of the Lung,

Pleura, Thymus and Heart. Lyon: International Agency for Research on Cancer. Travis WD, Colby TV, and Corrin B (1999). Histological Typing of Lung and Pleural Tumours, 3rd edn., World Health Organization International Histological Classification of Tumours. Berlin: Springer.

TUMORS, MALIGNANT Contents

Overview Bronchogenic Carcinoma Chemotherapeutic Agents Hematological Malignancies Lymphoma Metastases from Lung Cancer Bronchial Gland and Carcinoid Tumor Rare Tumors Carcinoma, Lymph Node Involvement

Overview K M Fong, P V Zimmerman, and R V Bowman, Prince Charles Hospital, The University of Queensland, Brisbane, QLD, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Although the most common malignancies of the lung are primary bronchogenic carcinomas and metastatic tumors, a wide range of rarer tumor types can occur in humans. Newer techniques to establish histological confirmation of malignancy include the use of ultrasound and fluorescence to enhance the precision of bronchoscopic biopsy. Needle aspiration of lung lesions can also be of high diagnostic yield. Certain tumor types can now be differentiated using new immunohistochemical markers, although tumor heterogeneity and sampling issues can occasionally influence the accuracy of the final diagnosis. The varying susceptibilities of cigarette smokers to developing bronchogenic carcinoma appear increasingly likely to be related to mutations at multiple genetic loci and their interactions. Recent advances in imaging techniques such as positron emission tomography, computed tomography densitometry, perfusion CT algorithms, and magnetic resonance imaging are increasing the accuracy of pretreatment staging and also improving the overall functional assessment of patients.

Introduction There are a large number of malignant tumors that affect the lungs, ranging from metastatic malignancies to the most common type, bronchogenic

carcinoma (see Tumors, Malignant: Bronchogenic Carcinoma). Bronchogenic cancers can also metastasize to the lungs, as well as other sites; this subject is covered elsewhere in this encyclopedia (see Tumors, Malignant: Metastases from Lung Cancer). Other primary cancers arising within the chest include tumors of lymphoid origin (see Tumors, Malignant: Lymphoma), vascular/blood origin (see Tumors, Malignant: Hematological Malignancies), and rare tumors (see Tumors, Malignant: Rare Tumors). Metastatic cancers can involve the lung parenchyma, the lymph nodes (see Tumors, Malignant: Carcinoma, Lymph Node Involvement), or other thoracic structures. Thus, the usual clinical challenge when faced with the possibility of cancer in the lung is to ascertain firstly the presence of malignancy, secondly the origin of the cancer, and finally the extent or stage of cancer so that therapy may be appropriately individualized. In future, this challenge may be magnified if low-dose helical computed tomography (CT) scanning is shown from randomized trial data to be effective in reducing lung cancer mortality. This technique has already been shown to be severalfold more sensitive than conventional chest radiography for detecting pulmonary nodules. Indeed, studies to date show that low-dose CT scanning can potentially detect a large number of pulmonary nodules in at-risk populations. Most of these pulmonary nodules turn out not to be malignant but require further evaluation using a variety of follow-up algorithms or invasive investigations.

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Etiology Bronchogenic carcinomas are estimated to be due to smoking in 85–90% of cases. Prior to the nineteenth century when smoking was less common and mostly in the form of cigars smoked by men, lung cancer was rare. An often-quoted anecdote is that in 1919, a medical student called Alton Oschner was invited to observe lung cancer surgery, it being a rare procedure. Indeed, another notable anecdote is the report of the first successful pneumonectomy being performed by Dr E A Graham of St Louis whose patient eventually outlived his surgeon, who himself succumbed to the disease. In the early twentieth century, smoking became more prevalent, particularly after World War I when cigarettes were widely available. As there is a time lag of about 20 years or more before the development of invasive lung cancer, it is not surprising that by the 1970s, lung cancer became the commonest cause of cancer deaths in males in some Western countries. Recently, lung cancer has become the commonest cause of cancer deaths in females. The lag between the sexes is explained by the temporal differences in smoking habits between men and women. Currently it is young females who represent an ongoing target population for tobacco control strategies as they continue to have relatively high rates of smoking. The epidemiology of lung cancer is further discussed elsewhere in this encyclopedia, including the role and interactions of asbestos and other lung carcinogens in nontobacco smoke-related lung cancer (see Tumors, Malignant: Bronchogenic Carcinoma). Although the risk of malignancy long after curative radiotherapy delivery has long been known, there is recent concern regarding the increasing use of medical radiation, particularly for diagnostic purposes, for example, CT scans. The issue of a genetic basis for lung cancer risk has long been debated since only a minority of smokers develop lung cancer and familial aggregation of lung cancer has been reported since the 1950s. Segregation analysis models range from Mendelian inheritance (codominant) to pure environmental models, although recent work provides evidence that multiple genetic factors, possibly multiple genetic loci and interactions, contribute to susceptibility and age-ofonset of primary lung cancer. Furthermore, a major susceptibility locus on 6q23-25 has been recently mapped through genome-wide linkage analysis. The lung is also a common destination for the metastatic spread of human malignancies. Pulmonary metastases may occur in 30–40% of cancer patients, although clearly the prevalence of metastases is a function of the natural history of the cancer, and the point in the clinical course at which investigation

for pulmonary involvement is undertaken. Even though pulmonary metastases are relatively common, they may not be clinically significant during life, and many are discovered only at postmortem.

Pathology and Pathogenesis The common classification of bronchogenic cancers into the small cell lung cancer (SCLC; previously also called oat-cell) and non-SCLC (NSCLC) subtypes has useful clinical implications for the diagnosis and treatment due to their differing characteristics (Table 1). Whilst the natural history of preinvasive squamous carcinomas has been relatively well studied due to their often proximal airway location and the ability to sample sequentially bronchial epithelium at bronchoscopy, much less is known about precursor lesions for the other subtypes. For squamous carcinogenesis, the multistep process affecting the bronchial epithelium includes basal cell hyperplasia, squamous metaplasia, dysplasia, and finally carcinoma-in-situ (CIS). Nonetheless, foci of ‘atypical adenomatous hyperplasia’, a bronchioloalveolar proliferation, are regarded as a precursor for peripheral adenocarcinoma and ‘diffuse idiopathic pulmonary neuroendocrine cell hyperplasia’, a rare condition in which the peripheral airways are involved diffusely by neuroendocrine cell hyperplasia and tumorlets, for peripheral typical carcinoids. Metastatic lung malignancies may arise from primaries within or external to the thoracic cavity. The process of lung metastases is complex and not fully understood, but includes tumor and host factors that modify the ability for micrometastases to adhere, invade, grow, cause angiogenesis, and avoid immune surveillance. Metastases may seed the lungs by several routes: hematogenous spread, lymphatic invasion, direct invasion, and possibly endobronchial dissemination. Certain tumors such as sarcomas and carcinomas of kidney, thyroid, breast, and lung appear particularly prone to disseminate to the lung by blood-borne spread. Lymphatic invasion by distant tumors reaching the large lymphatic channels, then the thoracic duct and pulmonary vasculature appears to explain the propensity for testicular tumors to spread to the lungs. Alternatively, local thoracic lymphadenopathic involvement (see Tumors, Malignant: Carcinoma, Lymph Node Involvement) may result in retrograde spread into pulmonary lymphatics, sometimes producing the clinical and radiological picture of lymphangitis carcinomatosis. Direct invasion of the lungs can occur from tumors arising in the mediastinum such as esophageal cancers, thymoma, lymphoma, or germ cell tumors; those arising

Table 1 The 1999 World Health Organization/International Association for the Study of Lung Cancer Histological Classification of Lung and Pleural Tumors 1.3.3.6. Variants 1 Epithelial tumors 1.3.3.6.1. Well-differentiated fetal adenocarcinoma 1.1. Benign 1.3.3.6.2. Mucinous (‘colloid’) adenocarcinoma 1.1.1. Papillomas 1.3.3.6.3. Mucinous cystadenocarcinoma 1.1.1.1. Squamous cell papilloma 1.3.3.6.4. Signet-ring adenocarcinoma Exophytic 1.3.3.6.5. Clear cell adenocarcinoma Inverted 1.3.4. Large cell carcinoma 1.1.1.2. Glandular papilloma Variants 1.1.1.3. Mixed squamous cell and glandular papilloma 1.3.4.1. Large cell neuroendocrine carcinoma 1.1.2. Adenomas 1.3.4.1.1. Combined large cell neuroendocrine 1.1.2.1. Alveolar adenoma carcinoma 1.1.2.2. Papillary adenoma 1.3.4.2. Basaloid carcinoma 1.1.2.3. Adenomas of salivary gland type 1.3.4.3. Lymphoepithelioma-like carcinoma Mucous gland adenoma 1.3.4.4. Clear cell carcinoma Pleomorphic adenoma 1.3.4.5. Large cell carcinoma with rhabdoid phenotype Others 1.3.5. Adenosquamous carcinoma 1.1.2.4. Mucinous cystadenoma 1.3.6. Carcinomas with pleomorphic, sarcomatoid, or 1.1.2.5. Others sarcomatous elements 1.2. Preinvasive lesions 1.3.6.1. Carcinomas with spindle and/or giant cells 1.2.1. Squamous dysplasia/carcinoma in situ 1.3.6.1.1. Pleomorphic carcinoma 1.2.2. Atypical adenomatous hyperplasia (AAH) 1.3.6.1.2. Spindle cell carcinoma 1.2.3. Diffuse idiopathic pulmonary neuroendocrine cell 1.3.6.1.3. Giant cell carcinoma hyperplasia (DIPNECH) 1.3.6.2. Carcinosarcoma 1.3. Malignant 1.3.6.3. Pulmonary blastoma 1.3.1. Squamous cell carcinoma (SCC) 1.3.6.4. Others Variants 1.3.7. Carcinoid tumor 1.3.1.1. Papillary 1.3.7.1. Typical carcinoid 1.3.1.2. Clear cell 1.3.7.2. Atypical carcinoid 1.3.1.3. Small cell 1.3.8. Carcinomas of salivary gland type 1.3.1.4. Basaloid 1.3.8.1. Mucoepidermoid carcinoma 1.3.2. Small cell carcinoma (SCLC) 1.3.8.2. Adenoid cystic carcinoma Variant 1.3.8.3. Others 1.3.2.1. Combined small cell carcinoma 1.3.9. Unclassified carcinoma 1.3.3. Adenocarcinoma 1.3.3.1. Acinar 2 Soft tissue tumors 1.3.3.2. Papillary 2.1. Localized fibrous tumor 1.3.3.3. Broncholoalveolar carcinoma 2.2. Epithelioid hemangioendothelioma 1.3.3.3.1. Nonmucinous (Clara/pneumocyte type II) 2.3. Pleuropulmonary blastoma 1.3.3.3.2. Mucinous 2.4. Chondroma 1.3.3.3.3. Mixed mucinous and nonmucinous or 2.5. Calcifying fibrous pseudotumor of the pleura intermediate cell type 2.6. Congenital peribronchial myofibroblastic tumor 1.3.3.4. Solid adenocarcinoma with mucin 2.7. Diffuse pulmonary lymphangiomatosis 1.3.3.5. Adenocarcinoma with mixed subtypes 2.8. Desmoplastic small round cell tumor

2.9. Other 3 Mesothelial tumors 3.1. Benign 3.1.1. Adenomatoid tumor 3.2. Malignant 3.2.1. Epithelioid mesothelioma 3.2.2. Sarcomatoid mesothelioma 3.2.2.1. Desmoplastic mesothelioma 3.2.3. Biphasic mesothelioma 3.2.4. Other 4 Miscellaneous tumors 4.1. Hamartoma 4.2. Sclerosing hemangioma 4.3. Clear cell tumor 4.4. Germ cell neoplasms 4.4.1. Teratoma, mature or immature 4.4.2. Malignant germ cell tumor 4.5. Thymona 4.6. Melanoma 4.7. Others 5 Lymphoproliferative disease 5.1. Lymphoid interstitial pneumonia 5.2. Nodular lymphoid hyperplasia 5.3. Low-grade marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue 5.4. Lymphomatoid granulomatosis 6 Secondary tumors 7 Unclassified tumors 8 Tumor-like lesions 8.1. Tumorlet 8.2. Multiple meningothelioid nodules 8.3. Langerhans cell histiocytosis 8.4. Inflammatory pseudotumor (inflammatory myofibroblastic tumor) 8.5. Organizing pneumonia 8.6. Amyloid tumor 8.7. Hyalinizing granuloma 8.8. Lymphangioleiomyomatosis 8.9. Multifocal micronodular pneumocyte hyperplasia 8.10. Endometriosis

Reproduced from Brambilla E, Travis WD, Colby TV, Corrin B, and Shimosato Y (2001) The New World Health Organization classification of lung tumors. European Respiratory Journal 18(6): 1059–1068, with permission from European Respiratory Society Journals Limited.

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in the chest wall such as sarcomas; and those arising in the abdominal cavity such as liver or gastric tumors.

Animal Models Humans appear to be one of the few species susceptible to developing spontaneous lung cancer. The initial histologic description 100 years ago of a papillary tumor in a mouse led to the idea of using animals as experimental tools for learning about lung cancer. Several types of animal models are used for experimental lung cancer research, including chemically induced lung tumors, transgenic mouse models, and human tumor xenografts. Chemical or carcinogen-induced lung tumors have been described in a variety of species, including dogs, cats, hamsters, mice, and ferrets. Transgenic mouse technology has proved useful in creating models of lung tumor development, for example, lung adenocarcinoma with mutant Kras transgene, as well as for testing experimental therapeutic approaches. In orthotopic models, human tumors are implanted into the appropriate organ of the animal, and have been described using endobronchial, intrathoracic, or intravenous injection of tumor cell suspensions and by surgical implantation of fresh tumor tissue. Thus, several lung cancer models are serving to increase our knowledge of lung cancer biology. Each has advantages and disadvantages and no single model is informative on the complexity of all aspects of lung cancer from carcinogenesis, proliferation, invasion, angiogenesis, and metastasis, to chemoprevention and therapeutic development.

Clinical Features The clinical features may be the same regardless of whether the lung cancer is a primary or secondary cancer. Symptoms clearly depend upon the local structures involved; patients commonly present with cough, hemoptysis, wheeze, obstructive infection or atelectasis, chest pain, and dyspnea. Particular symptoms such as stridor, dysphagia, facial edema from superior vena cava obstruction, or hoarseness due to recurrent laryngeal nerve involvement are a result of local damage to vital structures. Systemic symptoms such as anorexia or weight loss may also be present, and often indicate the presence of disseminated disease. Paraneoplastic syndromes occur in approximately 10% of patients with bronchogenic carcinoma and are not due to direct physical effects of the cancer, the best known perhaps being finger-clubbing and hypertrophic pulmonary osteoarthropathy.

Unlike bronchogenic cancers, which are frequently symptomatic, the majority of lung metastases are asymptomatic at the time of clinical detection. The clinical importance of lung metastases is highly dependent on the natural history of the originating cancer. When more than one site of cancer in the lung is evident, it may be difficult to decide whether the situation is one of satellite nodules, multiple primary lung cancers, or blood-borne metastases. Various models have been developed to guide clinical management in these circumstances. Clearly, when there is known cancer elsewhere, the probability of lung metastases is correspondingly higher although other conditions should always be considered in the differential diagnosis. Radiographic imaging may show a variety of abnormalities ranging from solitary pulmonary nodules (SPNs), multiple nodules, masses, pleural effusions, collapse/atelectasis, lymphadenopathy, or lymphangitis. Recent advances in nuclear medicine have led to the increasing use of metabolic imaging with positron emission tomography (PET) using 18Ffluoro-2-deoxy-glucose (FDG) where the glucose analog is highly taken up by certain malignancies including lung cancer. Despite high overall sensitivity and specificity of PET scans for lung cancer, imaging can usually only provide a guide to the likelihood of cancer, being neither diagnostic, nor able to determine the type of cancer. Consequently, the main diagnostic strategy is to obtain histological or cytological confirmation where possible. However, as lung cancer patients are often relatively older and have been smokers, there may be significant comorbidities precluding the use of invasive investigations. The common diagnostic tools include white light bronchoscopy, fine needle aspiration biopsy, mediastinoscopy, and rarely thoracotomy (open or videoassisted) or other biopsies. Modern techniques such as endobronchial ultrasound fluorescent bronchoscopy are likely to improve diagnostic capabilities. The safest technique likely to produce a histological diagnosis should be chosen, with consideration given to tumor factors (location, size, number, accessibility) and patient factors (fitness, comorbidities, age, concomitant lung and heart disease), as well as local expertise. Histology should be classified according to the World Health Organization/International Association for the Study of Lung Cancer, 1999 classification (Table 1). There are occasions, however, when accurate classification of morphology using the standard WHO classification may be difficult, even with the use of immunohistochemistry. Cytokeratin (CK) 7 and 20 are commonly used to help distinguish

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adenocarcinomas from various organs, and thyroid transcription factor 1 (TTF-1), a 38 kDa protein located primarily in type II pneumocytes and Clara cells, is present in a variety of lung tumors as well as thyroid carcinomas. Although pathological diagnosis can be complex due to intratumoral heterogeneity, mixed tumors, and sampling issues, classification of a particular tumor into one of the major subtypes is usually possible from small biopsies. In the future, it is likely that tumor classification by genetic and/or genomic approaches such as mutation analyses and microarray technology will be translated from the research to the clinical arena. The tumor molecular profile may also have implications for therapy (e.g., mutations predicting sensitivity to targeted therapies such as tyrosine kinase inhibitors) and prognosis (e.g., predictive gene panels). Once a diagnosis of bronchogenic cancer is made, the next management step is staging the extent of disease. In NSCLC, staging is carried out according to the current tumor node metastasis (TNM) schema (see Tumors, Malignant: Bronchogenic Carcinoma) with the aim of identifying patients who will benefit from surgery, radiation, chemotherapy, or multimodality treatment. In contrast, a simpler staging system (limited and extensive) is used for SCLC, which is essentially demarcated as to whether disease is confined to a single hemithorax. The usefulness of staging procedures depends on test characteristics, that is, sensitivity and specificity, and also on the potential of a staging test result to alter management. Tests that are useful for staging lung cancer include computed axial tomography (CAT) scans, PET scans, magnetic resonance imaging (MRI) scans, nuclear medicine bone scans, and even surgical biopsies. Participation of the radiologist and nuclear medicine physician in the multidisciplinary lung cancer team allows the greatest advantage to be taken of advanced imaging techniques such as PET, fusion CT–PET, CT densitometry, and perfusion CT in patient care. In management planning, physiological and functional assessment for performance status, lung function, and exercise capacity is essential to determine fitness for specific therapies. As chronic obstructive pulmonary disease (COPD) is frequent in smokers, diminished lung function is an important consideration, particularly in evaluation for treatments such as surgery and radiotherapy, which will further impair lung function. Advances in operative technique and perioperative care have reduced surgical morbidity and mortality considerably after pulmonary resection. In the past, only patients with normal or slightly impaired pulmonary function (forced expiratory volume in 1 s (FEV1) and diffusing capacity of the lung for carbon monoxide (DLCO) X80% predicted) and

no cardiovascular risk factors were considered suitable for a pneumonectomy. Now it is possible to predict postsurgical functional outcomes reasonably accurately in patients with abnormal lung function and other comorbidities using cardiopulmonary exercise testing and differential ventilation or perfusion lung scans. Lessons learned from tumor resection in the course of lung volume reduction, and evolving techniques including limited resection and minimally invasive surgery will challenge traditional concepts of surgical criteria further.

Management and Current Therapy The treatment of pulmonary malignancy hinges on the natural history and outcome from the primary tumor. The underlying principles include establishing a histological diagnosis where possible, followed by staging the extent of disease, and treatment planning with curative or palliative endpoints as appropriate to stage. Supportive and symptomatic therapy (potentially including radiotherapy and chemotherapy) are indicated where the disease is predicted to be incurable. In certain cases, where the primary tumor is controlled and with no other evidence of extrapulmonary disease, resection of a solitary metastasis may be considered in a suitably fit patient. The details of treatment for bronchogenic cancer are described elsewhere in this encyclopedia (see Tumors, Malignant: Bronchogenic Carcinoma). Briefly, for NSCLC, optimal treatment is surgery where appropriate, that is in those cases where the tumor is at an early stage and the patient is fit for resection. When disease is more advanced, technically unresectable, the patient is unfit, or the patient declines surgery, other modalities of treatment including radiotherapy, chemotherapy, supportive care, or a combination thereof are indicated. In comparison, SCLC, as a systemic disease, has the best outcome with systemic therapy (chemotherapy), which is combined with thoracic radiation therapy for limited stage disease, and with prophylactic cranial irradiation offered to those with complete responses. Provision of information, involvement of the patient and their carers, and consideration of patient preferences represent important aspects of both curative and palliative management of lung cancers.

Summary The most effective ‘management’ for lung cancer is its prevention by tobacco control, which would ultimately reduce the worldwide impact of this and other smoking-related diseases. In the meantime, clinicians continue to strive to optimize conventional

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therapies and outcomes for patients. Future challenges for lung cancer clinicians include optimizing strategies for clinical deployment of the new generation of technologies including early detection by helical low-dose CT scanning, functional imaging, and targeted biological therapies. For healthcare providers, the emerging challenge is how best to balance the costs and benefits of these differing technological developments. For scientists, it will be harnessing the clinical potential in the vast amount of biologic knowledge emerging from genomics and proteomics. See also: Tumors, Malignant: Bronchogenic Carcinoma; Chemotherapeutic Agents; Hematological Malignancies; Lymphoma; Metastases from Lung Cancer; Rare Tumors; Carcinoma, Lymph Node Involvement.

Further Reading Anonymous (2003) Diagnosis and management of lung cancer: ACCP Evidence-Based Guidelines. Chest 123 (1 supplement). Beasley MB, Brambilla E, and Travis WD (2005) The 2004 World Health Organization classification of lung tumors. Seminars in Roentgenology 40(2): 90–97. Brambilla E, Travis WD, Colby TV, Corrin B, and Shimosato Y (2001) The New World Health Organization Classification of Lung Tumors. European Respiratory Journal 18(6): 1059–1068. Fong KM, Sekido Y, Gazdar AF, and Minna JD (2003) Lung cancer 9: molecular biology of lung cancer: clinical implications. Thorax 58(10): 892–900. Leslie WT and Bonomi PD (2004) Novel treatments in non-small cell lung cancer. Hematology/Oncology Clinics of North America 18(1): 245–267. Liu J and Johnston MR (2002) Animal models for studying lung cancer and evaluation of novel intervention strategies. Surgical Oncology 11: 217–227. Matthay RA (ed.) (2002) Lung cancer. In: Clincs in Chest Medicine, vol. 23(1), pp. 1–282. Philadelphia, PA: Saunders. Elsevier Inc. Murray N, Salgia R, and Fossella FV (2004) Targeted molecules in small cell lung cancer. Seminars in Oncology 31(supplement 1): 106–111. Roth JA and Grammer SF (2004) Gene replacement therapy for non-small cell lung cancer: a review. Hematology/Oncology Clinics of North America 18(1): 215–229. Xu H, Spitz MR, Amos CI, and Shete S (2005) Complex segregation analysis reveals a multigene model for lung cancer. Human Genetics 116(1–2): 121–127.

Bronchogenic Carcinoma K M Fong, R V Bowman, and P V Zimmerman, Prince Charles Hospital, The University of Queensland, Brisbane, QLD, Australia

While death rates for all other major cancers have been stable or declining, the age-adjusted death rate due to lung cancer steadily increased in both men and women during the second half of the twentieth century. Public education and measures to control tobacco smoking have resulted in a steady decline in lung cancer deaths in men, and a stable rate in women in some Western countries during the past decade. The ultimate key to controlling the current prevalence and mortality of lung cancer lies in a global solution to tobacco smoking. Meanwhile, the impact of low dose CT screening in high-risk populations on lung cancer mortality is currently under evaluation. Secondary prevention strategies trialled during the latter part of last century did not reduce the incidence of lung cancer, and discovery of effective new agents for secondary prevention is a priority. Strategies to identify and treat very early disease such as carcinoma in situ and atypical adenomatous hyperplasia need to be more practical. Meanwhile refined clinical staging, reduced surgical morbidity and mortality, strategic application of chemotherapy, and definition of optimal combined modality therapy have contributed to improvements in clinical management and outcomes for individual patients. The outstanding issue in clinical lung cancer management remains the limited effectiveness of current agents and treatment modalities. Despite the development of new treatments targeting specific molecular vulnerabilities, a far more comprehensive understanding of the cell and molecular biology of lung cancer is still required before translational therapies can reach their full potential to make an impact on survival.

Introduction Bronchogenic carcinoma (lung cancer) is the highest ranking cause of cancer associated mortality in the world. Historically more prevalent in men, bronchogenic carcinoma is currently causing higher mortality among women living in several Western countries than breast or any other cancer. At present, it also has highest incidence among the major cancers affecting women in several Western countries. Non-small cell lung cancer accounts for approximately 80–85% of primary lung cancer and small cell cancer accounts for most of the remainder. The two types differ in both biological characteristics and clinical behavior. The International Agency for Research on Cancer (IARC) designates lung cancer as causally related to tobacco based upon the consistency, strength, temporal relationship, coherence, and specificity of the epidemiologic relationship between tobacco smoking and lung cancer. Compared with a twofold increased risk of breast cancer conferred by having an affected first degree relative, the relative risk of lung cancer in smokers is 20-fold the risk in nonsmokers – one of the highest associations between any agent and cancer.

& 2006 Elsevier Ltd. All rights reserved.

Epidemiology of Tobacco Use and Lung Cancer

Abstract

Seminal health reports by Wynder and Graham (USA) and Doll and Hill (UK) in 1950 linked smoking with lung cancer and in the early 1960s

Lung cancer is the most devastating, yet preventable, cause of premature cancer mortality and morbidity in the Western world.

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the Royal College of Physicians of London and the Surgeon General of the US issued reports that smoking causes lung cancer in men. Following these reports there was a steady decline in smoking rates in the US, UK, Canada, Western Europe and Australia, and during the past decade death rates from lung cancer in US males have decreased. Unfortunately, the next 20 years is predicted to see a transfer of the health burden of tobacco from rich to poor countries with only 15% of the world’s smokers living in rich countries by 2025. The number of people smoking regularly is predicted to increase from current 1.1 to 1.64 billion by the year 2025 and associated deaths to increase from 4.9 to 10 million per year with China becoming the leading country for tobaccorelated deaths. The lifetime risk of a smoker developing lung cancer is conditional upon smoking practices, competing tobacco-related causes of death, and genetic susceptibility factors. The Cancer Prevention Study II (CPS II) estimated this risk at a cumulative probability of 14.6% for males aged 485 years (compared with 1.6% for nonsmokers). For both men and women, the probability of dying between the ages of 35 and 69 from lung cancer increases with increasing age at the time of quitting smoking. For example, men who quit smoking after the age of 50 years have a 10% probability of dying before age 70 of lung cancer, compared with 1% probability for those who quit before the age of 40 years. Despite progress in reducing adult smoking rates to less than 25% in most Western countries, former smokers remain at risk, with more new lung cancer cases now occurring among former smokers than current smokers. Tobacco is a heterogeneous composition of thousands of chemicals including at least 60 listed carcinogens. During the conversion of tobacco carcinogens by cytochrome P450 and phase II enzymes to compounds with increased water solubility, DNA reactive intermediates are formed resulting in covalently bound DNA adducts. Cellular DNA repair systems can remove adducts, but those which escape repair persist in DNA and contribute to miscoding. Mutations in certain target genes have recently been shown to exhibit a tobacco-specific ‘signature’ consisting of characteristic patterns of DNA base changes that are not associated with other mutagenic stimuli – thus lending molecular evidence to the weight of epidemiologic evidence implicating tobacco as the most important cause of primary lung cancer.

Nontobacco Lung Carcinogenesis IARC, the International Union Against Cancer (UICC), and the US Department of Health and

Human Services have identified environmental tobacco smoke, asbestos, arsenic, bis(chloromethyl) ether, cadmium, chloromethyl methyl ether, chromium and hexavalent chromium, ionizing radiation (X-rays), gamma radiation, manmade mineral fibers, mustard gas, nickel, polycyclic aromatic hydrocarbons, radon decay products, silica, soots, sulfuric acid mists, tars, mineral oils, and vinyl chloride as established lung carcinogens. Collectively, these agents are responsible for less than 10% of all human lung cancer.

Genetic Susceptibility When adjusted for competing causes of death, risk estimates for lung cancer in lifetime smokers are considerably less than 100%, which has given rise to explorations of the constitutional susceptibility to lung cancer. The balance between metabolic activation and detoxification is variable between individuals, as is the ability to conduct repair, and these factors are likely to play a part in determining cancer risk. Because of the need to quantify and control for the overwhelming environmental factor of tobacco exposure which imposes a requirement for huge sample sizes and rigorous ascertainment methods, it has been difficult to reach definitive conclusions about the importance of individual biological susceptibility to lung cancer. The role of genetic polymorphisms with functional significance in genes encoding enzymes involved in carcinogen metabolic pathways such as CYP1A1 and the glutathione-stransferase families has been studied extensively with over 1000 scientific papers published. Only recently has clear evidence linking metabolic enzyme genotypes and specific adduct levels begun to emerge, for example, the demonstration of dependence of ( þ )-anti-BPDE-DNA adduct levels as a function of CYP1A1 and GSTM1 genotypes in smokers.

Gender, Race, and Familial Predisposition At least 10 case-control studies have reported that women who smoke have a higher relative risk of lung cancer than men. Because of the use of relative rather than absolute risk outcomes, lower background absolute risk of lung cancer in female nonsmokers, and possible systematic underreporting of smoking habits by women, drawing a firm conclusion of gender-based susceptibility from these studies is difficult. On the other hand, large cohort studies such as CPS II demonstrate similar or lower death rates from lung cancer in women compared with men adjusted for age and smoking strata. At present the epidemiological evidence in support of gender-based

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constitutional susceptibility to lung cancer is inconclusive. Similarly, confident conclusions regarding race susceptibility from differences in lung cancer incidence studies in African–Americans, Asians, and Caucasians is similarly confounded by differences in smoking preferences and habits, and variation in exposure to other lung carcinogens among different racial groups. The first report of familial lung cancer showed that risk of lung cancer for smoking relatives of patients with lung cancer was twice that of controls, while for nonsmoking relatives it was increased fourfold. At least three further studies have suggested that family history of lung cancer in a first degree relative is an independent risk factor for lung cancer. Relative risks are modest overall (OR 1.67–5), but higher amongst cases occurring at a relatively young age.

Pathology Invasive Carcinoma

There are three major histopathologic subtypes of non-small cell carcinoma: adenocarcinoma, squamous cell carcinoma, and large cell undifferentiated carcinoma, all strongly associated with tobacco smoking. Bronchoalveolar carcinoma is a distinctive minor subgroup which appears to be more frequent in women and not so strongly related to smoking. It accounts for less than 4% of lung cancers in the SEER database (1979–98). Adenocarcinoma is increasing as a proportion of the total, and squamous carcinoma decreasing over the past 20 years – a trend which may be attributable to factors such as the increasing use of filters and lower tar content in tobacco products during the latency period. Tissue sampling for histologic or cytologic examination is a prerequisite for appropriate management in order to confirm the diagnosis of lung cancer and to distinguish small cell cancer from non-small cell cancer. The latter is based upon distinctive cytological features and immunohistochemical evidence of neuroendocrine differentiation including expression of chromogranin A, synaptophysin, and neural cell adhesion molecule. There is currently no histological method of differentiating primary from secondary cancer in the lung with certainty, although thyroid transcription factor 1 is a useful immunohistochemical marker of primary lung adenocarcinoma. It is becoming increasingly important to distinguish adenocarcinoma from squamous cell carcinoma, and large cell carcinoma because of newly emerging information indicating that particular histologic subtypes may have specific therapeutic susceptibilities

(e.g., clinical responses to tyrosine kinase inhibitors appear to be largely restricted to adenocarcinomas). Preneoplastic Lesions

Since Auerbach described the morphology of preneoplastic bronchial lesions, the progression of hyperplasia–metaplasia–dysplasia–carcinoma in situ (CIS) has been proposed to precede invasive squamous carcinoma of lung. Autofluorescence bronchoscopy studies have demonstrated that approximately 9% of smokers have severe dysplastic lesions or CIS in the proximal bronchial tree. Sequential pathological sampling of CIS to determine natural history has demonstrated that most of these lesions progress to invasive carcinomas. On the other hand, most mild and moderate dysplastic lesions are stable or regress during longitudinal follow-up. The most recent World Health Organization classification of lung tumors recognizes both severe dysplasia and CIS as preneoplastic lesions and direct antecedents of invasive squamous cell carcinoma, and atypical adenomatous hyperplasia as an antecedent of adenocarcinoma.

Molecular Pathogenesis Numerous cellular functions are demonstrably abnormal in primary lung tumors including cell cycling and proliferation, apoptosis, telomere function, responses to growth factor signaling, adhesion, and angiogenesis. Each of these activities is subserved by one or more complex pathways of interacting proteins, and malfunction of any one of them is potentially important in lung carcinogenesis or progression. Lung cancer is characterized by a vast array of molecular abnormalities including point mutations, deletions, gene amplification, and abnormalities of gene expression mediated by aberrant promoter methylation affecting a large and expanding number of genes participating in these cellular functions. Molecular Biology of Precursor Lesions

In established tumors it is difficult to distinguish between molecular events arising as a result of the advanced genomic instability that accompanies invasive cancer and those which have played a role in tumor development. On the other hand, the molecular abnormalities of committed precursor lesions such as severe dysplasia and CIS are fewer in number, are not usually associated with gross genomic instability, and because they antecede the development of invasive malignancy they are more likely to be causal. Among the first molecular abnormalities to be described in preneoplastic lesions were allelic losses at chromosomal loci 3p, 5q, 9p, 8p, and 17p, which contribute

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to loss of function of genes which normally play some role in protecting cells from malignant transformation. These tumor suppressor genes include FHIT, APC and MCC, p16, p53, and others. p53

Mutations in p53 in expectorated bronchial epithelial cells of smokers have been demonstrated to precede clinical diagnosis of lung cancer by up to 12 months. In lung cancer, G–T transversions in p53 are more prevalent in smokers than nonsmokers, and the p53 mutational spectrum differs between lung cancer and other types of cancer which mainly contain G–A transitions, suggesting that p53 mutations are directly due to DNA damage by tobacco carcinogens, rather than to selection processes. The dysfunctional effects of p53 mutations disrupt cell-cycle checkpoints and remove a key mechanism of protecting genomic integrity during replication. p53 has the ability to either retard cell division to allow repair of damaged DNA during cycling, or to direct a series of events triggering apoptosis of the individual damaged cell in the interests of tissue integrity. Mutations resulting in loss of function of p53 are thus associated with accelerated accumulation of genomic abnormalities, and may be among the most critical events in lung carcinogenesis. Growth Receptor and Signaling Pathways

The ras family of signal transduction molecules is involved in transmission of growth signals through upstream tyrosine kinase receptor families such as EGFR. This pathway is strongly implicated in the biology of primary lung adenocarcinoma, with activating point mutations of Kras occurring in 20–30%. These mutations are most prevalent at codon 12 and in smokers they are typically of the G–T transversion type causing abnormal signaling through a phosphorylation-dependent downstream cascade resulting in autonomous growth independent of mitogen availability or receptor–ligand interaction. More recently, point mutations and small deletions of the EGFR tyrosine binding domain have been discovered in primary lung adenocarcinomas. EGFR mutations occur more frequently in the adenocarcinomas of ‘never smokers’ compared with smokers, women compared with men, and Asians compared with non-Asians. Although it has been suggested that tumor mutation status may determine response to treatment with tyrosine kinase inhibitors (TKIs), there is recent evidence that this is not the case. Efforts are now being directed towards identification of other factors that may predict TKI response, and towards establishing the functional integrity of other

members of this signaling pathway in lung adenocarcinoma. Cell-Cycle Regulation

Another major pathway implicated in lung cancer is the p16-Cyclin D1-CDK-pRB pathway, which controls G1-S phase progression through the cell cycle. Activation of this pathway results in phosphorylation of pRB which releases E2F, a transcription factor that promotes synthesis of protein components of replication machinery. Bi-allelic disruption of RB itself by mutation and deletion at 13q14 occurs in up to 90% of small cell lung cancer, but is not a common feature of non-small cell lung cancer. Members of retinoblastoma families with germline abnormalities of RB are prone to lung cancer, suggesting that its normal tumor suppressor function is protective, and implying a causal role for dysfunctional RB in lung cancer. Expression of another tumor suppressor protein in this pathway, p16, is lost in 30–50% of primary lung cancer. Loss of p16 through small deletions, point mutation, or gene silencing through promoter hypermethylation permits unchecked association between cyclin D1 and cyclindependent kinases and results in unrestricted entry to G1. The frequency of p16 hypermethylation increases in parallel with advancing morphological abnormality of bronchial epithelium, providing circumstantial evidence of a causal role in lung carcinogenesis through loss of ordered cell-cycle progression. A number of other genes are also silenced by this mechanism including DAPK, MGMT, FHIT, and RASSFIA with functional effects on apoptosis and other cell functions that could contribute to lung cancer development or progression. Telomere Function

There is evidence that many lung cancers have acquired the ability to maintain telomere length by reexpressing telomerase. The catalytic component of this enzyme complex is normally under strict transcriptional control in adult lung tissue so that cells eventually undergo senescence. Regaining the ability to replicate the specialized ends of chromosomes is associated with escape from telomere shortening and senescence, and acquisition of the potential for indefinite replication cycles. Aberrant expression of telomerase not only occurs in the majority of lung cancers but also in the nonmalignant bronchial epithelium of smokers. It is one of an increasing number of molecular abnormalities found to occur in nonmalignant tobacco-exposed respiratory epithelium that may be mechanistically involved in lung cancer.

TUMORS, MALIGNANT / Bronchogenic Carcinoma 329 Summary

At present the number and nature of critical events in human lung carcinogenesis remains unknown, and we are far from a complete understanding of how the disease develops and behaves. Nevertheless, continued research in molecular biology of lung cancer is fundamental to development of better tools for secondary prevention, screening, early diagnosis, prognosis, and treatment. Ground gained in elucidating molecular pathogenesis is already underpinning the development of new therapies, and helping in clinical selection of cases most likely to benefit from agents targeting specific molecular abnormalities in some lung cancers.

Prevention Primary prevention by avoidance of tobacco exposure would reduce the prevalence of lung cancer dramatically. In 1930, lung cancer was a rare disease for which the death rate was 4.9 per 100 000 men, whereas 60 years later in 1990 the age-adjusted death rate from lung cancer in men was 90 per 100 000. The only known effective secondary prevention for smokers is smoking cessation. Although risk in former smokers may not be as low as that in never smokers, smoking cessation is nevertheless associated with definite risk reduction. All other secondary prevention measures tested to date have failed to reduce risk. There is accumulating evidence that mutational DNA damage in respiratory epithelium persists after smoking cessation and that this has the effect of reducing the number of aging-related additional critical events required for lung cancer development in former smokers. Following the failure of retinoid-based secondary prevention strategies, a new wave of prevention agents based on updated biological understanding of human lung carcinogenesis is set to be tested.

Early Diagnosis Lung cancer symptoms (cough, hemoptysis, dyspnea, and pain) are due to an expanding mass located within a large airway or in proximity to major intrathoracic structures including pleura, pericardium, heart and great vessels, ribs, vertebrae, and intrathoracic nerves, and occur late in the biological course of disease. Patients presenting with symptoms have much poorer outcomes than those presenting with an asymptomatic radiologic abnormality. This fact has provided much of the impetus for earlier detection before the onset of symptoms. However, mass screening by chest X-ray and sputum cytology has not been

associated with improved mortality. While low dose spiral CT is clearly more sensitive in detecting lung cancers at a smaller size, a number of potential drawbacks to this mode of early detection are evident, including cost, and clinical penalties involved in distinguishing the large number of benign nodules concurrently detected. Other challenges common to all screening programs include selection of a population who will benefit most from screening, and resolution of issues surrounding overdiagnosis bias and competing cause mortality. The benefit of CT screening is currently under evaluation in two large trials: the international ELCAP trial, and the NCI sponsored National Lung Screening Trial, comparing CXR and LDCT. The diagnostic approach is based upon risk analysis (e.g., age, smoking), presenting symptoms, clinical signs, and radiologic characteristics. This allows an appraisal of whether or not a lesion is likely to be involving a proximal airway accessible by bronchoscopy. Symptoms such as wheeze, cough, and hemoptysis, signs of focal wheeze or absent breath sounds corresponding to a lung segment or lobe, or radiologic evidence of proximity, narrowing or obstruction to lobar or segmental airways are all indications for bronchoscopy during which washings, brushings, and biopsies can be performed under direct vision, or guided by image intensification or endobronchial ultrasound. Risk analysis is also an important component of developing a diagnostic approach to asymptomatic small peripheral nodules discovered by CT. A decision to undertake biopsy of such lesions will be strongly influenced by the prior likelihood that the lesion is malignant. Algorithms such as ‘SPN calculator’ are available to assess this prior likelihood based on patient factors and lesion characteristics. PET scan and CT observation over defined time periods to ascertain growth, stability, or regression are useful noninvasive means of informing the decision to undertake fine needle biopsy. Because of the false negative rate, a negative fine needle biopsy usually means that further clinical and radiologic follow-up is required. The general applicability of these diagnostic approaches is subject to factors that vary by location including the prevalence and underlying pathology of nodules in the local community, and availability of imaging and invasive biopsy techniques. Nonimaging screening based on biological methods of detecting early disease is fertile ground for research and development. Strategies include detection of tumor DNA, circulating tumor cells, autofluorescence bronchoscopic detection of dysplasia, and CIS, volatile compound analysis of breath condensate, detection of altered DNA content or chromatin

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arrangement in expectorated sputum, and refinement of biomarker panels targeting selected molecular abnormalities based on proteomics, gene expression profiling, or combinations of key individual mutational or loss of function events.

Clinical Behavior Lung cancer metastatic behavior is consistent with theories relating metastatic potential to tumor size, which predict that the likelihood of metastases commences at approximately 12 doublings when tumors are microscopic, increasing to approximately 40% likelihood at 25 doublings and tumor size of approximately 2 cm diameter. This is consistent with the frequency with which patients present with metastatic disease, and also with the failure rate of surgical treatment for early stage disease presumably attributable to the presence of undetected metastatic disease. It is also consistent with the relationship observed between survival and primary tumor size and provides a theoretical basis for the use of T (tumor) staging in clinical management. Lung cancer metastases occur by both hematogenous spread to extrathoracic organs and lymphatic spread to regional intrathoracic lymph nodes. The extent of lymph node metastases forms the basis of clinical and pathological N (node) staging, which guides management and predicts survival (see Table 1). In the current international staging system for non-small cell lung cancer N1 denotes involvement of ipsilateral intrapulmonary, peribronchial or hilar nodes (stations 10–14), N2 ipsilateral mediastinal nodes (stations 1–9), and N3 contralateral mediastinal or any supraclavicular or scalene nodes. Clinical node staging is based on imaging with CT or PET scanning. However because clinical N staging is inaccurate in approximately 40% of cases evaluated by CT scan alone, and 10% of cases evaluated by PET scan, confirmatory node sampling by mediastinoscopy is appropriate where a solitary abnormal node stations detected by imaging represents the only impediment to an otherwise curative management plan. The most common sites of hematogenous metastases from lung cancer are bones, brain, adrenal glands, and liver. Fewer than 1% of patients with extrathoracic metastatic disease to these sites survive 5 years.

Management Non-Small Cell Lung Cancer

Stage-I and -II diseases have the best outcomes when managed by complete surgical resection. Since

Table 1 Clinical staging of non-small cell lung cancer (AJCC/ UICC – 1997) and associated approximate 5-year survival by TNMa staging Stage

TNM subset

0 Ia Ib IIa IIb IIIa

Carcinoma in situ T1 N0 M0 T2 N0 M0 T1 N1 M0 T2 N1 M0 T3 N1 M0 T1-3 N2 M0 T4 any N M0 Any T N3 M0 M1

IIIb IV

5-year survival (%) 70 60 55 40 23 5 1

a

Tumor, node, metastasis. Key to staging symbols: T ¼ primary tumor; TX ¼ primary tumor cannot be assessed, or tumor cells found in sputum or bronchoscopic washings but not on radiography; T0 ¼ no evidence of primary tumor; Tis ¼ carcinoma in situ; T1 ¼ tumor o3 cm in size, surrounded by lung or visceral pleura; T2 ¼ tumor with any of the following features: 43 cm; involves the main bronchus; X2 cm distal to carina; invades the visceral pleura; associated with atelectasis or obstructive pneumonitis that extends to the hilum but does not involve the entire lung; T3 ¼ tumor of any size that invades any of the following: chest wall, diaphragm, mediastinal pleura, pericardium; or tumor in the main bronchus o2 cm from but not involving carina; or associated atelectasis or obstructive pneumonitis of the entire lung; T4 ¼ tumor of any size that involves any of the following: mediastinum, heart, great vessels, trachea, esophagus, vertebral body, carina; or tumor with a malignant effusion or with satellite tumor nodules within the same lobe of lung as the primary tumor; N ¼ lymph node; NX ¼ regional lymph nodes cannot be assessed; N0 ¼ no regional lymph node metastasis; N1 ¼ metastasis to ipsilateral peribronchial and/or ipsilateral hilar lymph nodes, and intrapulmonary nodes involved by direct extension of the tumor; N2 ¼ metastasis to ipsilateral mediastinal or subcarinal nodes; N3 ¼ metastasis to contralateral lymph nodes or to ipsilateral or contralateral scalene or supraclavicular nodes; M ¼ metastasis; MX ¼ presence of distant metastasis cannot be assessed; M0 ¼ no distant metastasis; M1 ¼ distant metastasis present. Adapted from AJCC/UICC, 1997.

survival for patients whose lung cancer is completely resected is still only 40–70%, methods of improving these results are currently under evaluation. Adjuvant chemotherapy has been studied in large with disparate results, resulting in uncertainty regarding its role. Meta-analyses of abstracted data from recent large randomized studies indicate overall improved survival for patients receiving postoperative adjuvant chemotherapy compared with those treated with surgery alone. The benefit is stronger in trials without stage-III patients (stage I and II only). These benefits are offset by a cost of potentially serious toxicity (grade 3 or 4) in o15% of patients, and toxicity-related deaths in less than 1% of patients. Randomized studies of surgery alone, neoadjuvant chemotherapy with surgery, and surgery with adjuvant chemotherapy are now in progress to establish the

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best means of achieving optimal outcomes for surgical patients. Adjuvant radiation therapy has been shown not to improve survival and to be possibly detrimental; however, its role in patients with mediastinal node involvement remains unclear. Stage-IIIa disease which includes larger, more proximal tumors with ipsilateral hilar nodes involvement and potentially resectable tumors with ipsilateral mediastinal nodes involved is best managed by multimodality therapy, but there is currently no clear evidence of superiority of platinum-based chemotherapy plus surgery over chemoradiation for this stage of disease. Stage-IIIb disease includes tumor involving structures that render it unresectable (T4) or contralateral mediastinal node involvement (N3). Chemoradiation is a reasonable vigorous approach for patients with good performance status, but many stage-IIIb patients are treated with palliative intent with the aim of controlling existing symptoms or delaying the onset of impending symptoms. Resection does not improve outcomes. Median 5-year survival is approximately 5%, with modest extension of median survival by a few months associated with chemotherapy. In patients with stage-IV disease, chemotherapy has a role in achieving symptom control, modest prolongation of survival, and improved quality of life. Symptoms due to lung cancer are among the most challenging oncological symptoms to palliate. Effective relief from symptoms of advanced lung cancer often requires flexible deployment of multiple strategic elements of palliative care including local and systemic anticancer therapy, medical management of dyspnea, cachexia, constipation, neuropathies, hypercalcaemia, and other complications of advanced malignant disease, as well as management of psychosocial adaptation to advancing disability and approaching death. Small Cell Lung Cancer

Compared with non-small cell lung cancer, small cell lung cancer is often more widespread when clinically diagnosed with more frequent involvement of both regional lymph nodes and extrathoracic sites. Management is planned using a two-tier system of limited and extensive stage disease determined by whether or not disease has spread beyond the thorax. Small cell lung cancer is sensitive to both chemotherapy and radiation therapy, and patients with both limited and extensive stage disease benefit from active treatment in terms of improved survival. Limited disease small cell cancer is potentially curable in a small proportion of patients represented by those with complete

responses to combination chemotherapy given concurrently with high-dose thoracic radiation. Such patients have a better chance of surviving 5 years if they also receive prophylactic cranial irradiation. Despite frequent initial therapeutic responses, longterm survival and cure rates for small cell lung cancer are poor.

Summary The late twentieth-century epidemic of lung cancer is now fortunately in decline in Western countries. Epidemiological studies have convincingly demonstrated its links to tobacco smoking as the major cause, with both rise and fall in prevalence closely parallel to tobacco use with a lag of several decades. Progress pinpointing therapeutic susceptibilities has been slow with much of the biology of lung cancer remaining enigma as we approach a new era of cancer therapeutics aimed at molecular targets. Unfortunately, with a possibly even more devastating wave of lung cancer predicted to occur in developing countries, Eastern Europe, and Asia, a better understanding of the molecular biology of this tumor is urgently needed to provide tools for effective secondary prevention, early diagnosis, and clinical treatment. See also: Chronic Obstructive Pulmonary Disease: Smoking Cessation. Tumors, Malignant: Overview; Metastases from Lung Cancer; Bronchial Gland and Carcinoid Tumor; Carcinoma, Lymph Node Involvement.

Further Reading (1986) IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans. In: Tobacco Smoking, vol. 38. Lyons: World Health Organisation, IARC. (2002) WHO atlas maps global tobacco epidemic. Public Health Report 117: 479. (2003) Cancer Facts and Figures 2003, Surveillance Research. American Cancer Society. Dragnev KH, Stover D, and Dmitrovsky E (2003) Lung cancer prevention: the guidelines. Chest 123: 60S–71S. Etzel CJ, Amos CI, and Spitz MR (2003) Risk for smoking-related cancer among relatives of lung cancer patients. Cancer Research 63: 8531–8535. Fong KM and Minna JD (2002) Molecular biology of lung cancer: clinical implications. Clinics in Chest Medicine 23: 83–101. Gould MK, Kuschner WG, Rydzak CE, et al. (2003) Test performance of positron emission tomography and computed tomography for mediastinal staging in patients with nonsmall-cell lung cancer: a meta-analysis. Annals of Internal Medicine 139: 879–892. Greenberg AK, Yee H, and Rom WN (2002) Preneoplastic lesions of the lung. Respiratory Research 3: 20. Henschke CI, Yankelevitz DF, McCauley DI, et al. (2003) Guidelines for the use of spiral computed tomography in screening for

332 TUMORS, MALIGNANT / Chemotherapeutic Agents lung cancer. The European Respiratory Journal Supplement 39: 45s–51s. Lam S, Lam B, and Petty TL (2001) Early detection for lung cancer. New tools for casefinding. Canadian Family Physician 47: 537–544. Mackay J (1998) The global tobacco epidemic. The next 25 years. Public Health Report 113: 14–21. Minna JD, Gazdar AF, Sprang SR, and Herz J (2004) Cancer. A bull’s eye for targeted lung cancer therapy. Science 304: 1458– 1461. Mountain CF (1997) Revisions in the International System for Staging Lung Cancer. Chest 111(6): 1710–1717. Phillips DH (2002) Smoking-related DNA and protein adducts in human tissues. Carcinogenesis 23: 1979–2004. Travis WD (2002) Pathology of lung cancer. Clinics in Chest Medicine 23: 65–81.

Chemotherapeutic Agents M J Edelman, University of Maryland, Baltimore, MD, USA E J Rupard, Walter Reed Army Medical Center, Washington, DC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Lung cancer is a common and deadly neoplasm, responsible for the majority of cancer deaths worldwide. Well-controlled clinical trials demonstrate that chemotherapy treatment offers a survival advantage in all stages of all types of lung cancer, including those which fit under the larger heading ‘non-small cell lung cancer’ (adenocarcinoma of the lung, squamous cell carcinoma, and large cell carcinoma) as well as small cell lung cancer. The benefits vary considerably depending upon the stage and performance status of the patient. In non-small cell lung cancer, which accounts for 85% of lung cancers, adjuvant chemotherapy has demonstrated a marked benefit, increasing cure rates by 10–15%. In locally advanced disease, chemotherapy has also demonstrated unequivocal benefit in improving survival when combined with either radiotherapy or surgery. In metastatic disease, benefits have been clearly demonstrated in terms of quality and length of life for both initial treatment as well as salvage therapies. However, the survival of these patients is usually brief and much work needs to be done in terms of both, developing new agents and better utilization of available drugs. Small cell lung cancer has historically been considered the more responsive of the lung cancer types, with a majority of patients demonstrating disease shrinkage. In limited disease the combination of chemotherapy with radiotherapy can result in cure in approximately 25% of patients. In extensive disease, the majority of patients will experience shrinkage of disease, frequently with substantial symptomatic benefit. Unfortunately, this response is frequently shortlived with rapid regrowth of disease. The progress of the last decade has removed a previously nihilistic attitude towards the drug treatment of lung cancer and led to a renaissance in lung cancer research. Numerous new agents and approaches are being explored including modifications of existing families of agents as well as development of new agents targeted on molecular pathways felt to be crucial to the survival and proliferation of this malignancy.

Introduction In 2004, an estimated 160 440 people in the US died of lung cancer. To put this into perspective, this figure is larger than the number of deaths expected for the four cancer types combined (colon, breast, lung, and pancreas). The trend has been relentless and upward since the 1930s, with a plateau and slight decline seen in the middle portion of the last decade. This decline is mostly attributable to a declining incidence rate due to improved efforts at public education about risk factors such as tobacco use. However, the last 10 years have been perhaps the most fruitful in the history of lung cancer research in terms of the development of new chemotherapeutic agents and the evidence-based extension of chemotherapy into new areas including the adjuvant treatment of resected disease and in combination with radiotherapy and/or surgery for locally advanced disease. The treatment of lung cancer revolves around three considerations: histologic type, stage, and physiologic status. There are two major histologic types of lung cancer, small cell and non-small cell lung cancer (NSCLC). While multiple subtypes exist, at this time this division is the only one of therapeutic importance. Stage is crucial for the determination of prognosis and selection of therapy. In NSCLC, staging depends upon the size and location (particularly invasion of critical structures) of the primary tumor; the presence, absence, and location of regional lymph node involvement; and the presence or absence of metastatic disease. Physiologic status depends upon both underlying comorbidities such as pre-existing pulmonary disease as well as the morbidity of cancer itself manifested through weight loss and diminished level of activity. Not surprisingly, patients with significant weight loss and decreased activity have substantially poorer outcomes for any stage of disease. Adjuvant chemotherapy for NSCLC is now clearly indicated for patients with resected stage Ib–IIIa disease on the basis of four randomized, multicenter clinical trials. These studies have demonstrated an absolute survival advantage of 5–15% over surgical resection alone and clearly demonstrate the ability of platinum-based chemotherapy to eradicate microscopic disease. In patients with unresected stage IIIa and selected stage IIIb patients, chemotherapy can clearly enhance the response and survival of patients over radiation or surgery alone. A fraction of patients (15–25%) can be rendered long-term survivors by this approach. In stage IV disease, chemotherapy has demonstrated unequivocal benefit in terms of survival and quality of life for patients with good performance status (ambulatory, and with minimal to moderate symptoms). There also appears to be

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benefit for patients with more significant symptomatology as well, though this remains controversial. Platinum-based (cisplatin or carboplatin) doublets are the cornerstone of therapy. The second agent can be vinorelbine, paclitaxel, docetaxel, gemcitabine, or irinotecan. Most studies have failed to demonstrate that any combination is superior to any other. Nonplatinum two-drug combinations may also be employed. As all patients with advanced disease will eventually demonstrate progressive disease, there is a concept of ‘lines of therapy’. First-line therapy is that administered initially and while it usually refers to chemotherapy for metastatic disease, may also refer to chemotherapy delivered as part of multimodality therapy for stage III or adjuvant treatment for stages I–III disease. Second-line treatment is that administered after documented progression, after having received initial chemotherapy. In addition to performance status, other determinants of outcome include sex, smoking status, and in the case of secondand third-line therapies, response and durability of response to first-line treatment. Patients with compromised performance status typically receive a single agent- or dose-attenuated combinations if they are treated. Small cell lung cancer (SCLC) is typically divided into limited disease defined as that ‘confined to the chest’ and ‘encompassed within a tolerable radiotherapy port’ or extensive disease. Current terminology would include patients with malignant effusions (pericardial or pleural) within the extensive category. Patients with limited disease are treated with chemoradiotherapy with a curative intent. Approximately 25% of patients with limited disease can be cured. Palliation of symptoms and extension of life with chemotherapy can be accomplished for patients with extensive disease, though few will become long-term survivors. Until recently, antineoplastics with activity in lung cancer had two broad targets, DNA and tubulin. Agents targeted synthesis (antimetabolites), the DNA molecule (platinum), or enzymes responsible for DNA function (topoisomerases). Antitubulin agents are generally thought to be those that interfere with tubulin polymerization (vinca alkaloids) or depolymerization (taxanes), though these divisions may be artificial. Current research in lung cancer therapeutics is focusing on ‘targeted therapies’, defined as drugs targeting specific receptors (e.g., epidermal growth factor receptor), pathways (e.g., ras), or processes (angiogenesis) that are abnormally expressed, activated, or triggered in cancer. In addition, new agents that disrupt the existing targets of DNA or tubulin are in development as well. Furthermore, rapidly improving technology is allowing

for individualization of therapy, based upon abnormalities peculiar to an individual cancer.

Chemotherapy Drugs Currently Used in Lung Cancer Antiangiogenesis

Bevacizumab (Avastins) Overview Bevacizumab is the most recent agent to enter the armamentarium of chemotherapy agents with activity against lung cancer. A randomized phase III trial evaluating the agent in combination with carboplatin/paclitaxel versus carboplatin/paclitaxel alone (ECOG 4599) demonstrated improvements in response, median, and landmark survival. The study was restricted to patients with nonsquamous histologies who did not have CNS metastases, require anticoagulants, or have a history of significant hemoptysis. Mechanism of action Bevacizumab is a recombinant humanized monoclonal antibody that binds to and inhibits the biological activity of human vascular endothelial growth factor. This may inhibit cancer by inhibiting neoangiogenesis. Recent work also indicates that it may result in decreased tumor interstitial pressure and enhance the delivery of chemotherapy agents to the tumor. Pharmacokinetics/metabolism Intravenous drug has an estimated half-life of 20 days. The metabolism of the agent is not well characterized. There is minimal hepatic or renal clearance. Current indications Bevacizumab is licensed for use in colorectal cancer. As noted above, it has now been demonstrated to have activity in NSCLC. In addition, activity has also been demonstrated in breast cancer. Toxicities Bevacizumab has significant potential toxicities relevant to the lung cancer population including hypertension, increased incidence of cardiovascular events, and the potential for delayed wound healing. Massive hemoptysis (frequently fatal) occurred in patients with squamous histology tumors in a preliminary trial and led to restriction of the drug to non-squamous histologies in the phase III study. Despite these problems, this agent has clearly demonstrated activity and is undergoing further evaluation in a variety of lung cancer settings including SCLC , as part of chemoradiotherapy and in the adjuvant setting.

334 TUMORS, MALIGNANT / Chemotherapeutic Agents Platinum Agents s

Cisplatin (CDDP, Platinol ) Overview Cisplatin is a potent, effective, antineoplastic agent which has been studied since the 1970s in patients with nearly all types of cancer. Despite its age and considerable toxicities, cisplatin still provides the backbone of treatment for a number of malignancies. Mechanism of action Cisplatin (and other platinum-based antineoplastics) works as an ‘atypical’ alkylator, binding and cross-linking DNA, which leads to DNA strand breakage during replication. Pharmacokinetics/metabolism Intravenous drug is rapidly distributed in tissue, with tissue half-life of 2-4 days. Cisplatin is eliminated by the kidneys, and is not extensively metabolized prior to excretion in the urine. Current indications Cisplatin has been used in nearly all solid and liquid tumors. It is FDA-approved for use in ovarian, testicular, and transitional cell cancer. Toxicities Nephrotoxicity and neurotoxicity are dose-limiting. The latter is often manifest as painful neuropathy or hearing loss. Myelosuppression is mild. Nausea and vomiting can be severe, though these respond well to the newer anti-nausea drugs (i.e., ondansetron and other 5-HT3 receptor blockers). Alopecia and cardiac toxicities are rare. Carboplatin (Paraplatins) Overview Carboplatin is a ‘second generation’ platinum compound, with a different and usually improved toxicity profile over that of cisplatin, and similar efficacy (though notably inferior in some trials, and significantly less studied than cisplatin). Its indications are similar to those for cisplatin, and its lack of substantial renal and ototoxicity makes it an attractive option for elderly patients and palliative regimens. Carboplatin is often used with paclitaxel in a doublet as a first-line treatment for unresectable/ metastatic NSCLC. Carboplatin is more myelotoxic than cisplatin. Mechanism of action Like cisplatin, carboplatin works as an ‘atypical’ alkylator, binding and crosslinking DNA, which leads to DNA strand breakage during replication. Pharmacokinetics/metabolism Intravenous carboplatin is rapidly cleared by the kidneys, with a 2.5 h

half-life. It is exclusively eliminated by the kidneys, and is excreted in the urine essentially unchanged. Current indications Carboplatin is used for most of the same solid cancer types as is cisplatin, including testicular, ovarian, lung, and head/neck. It is less often used in liquid tumors. FDA approval has been granted for ovarian cancer only. Toxicities Myelosuppression (particularly thrombocytopenia) is dose-limiting. Nausea and vomiting are generally mild, and respond to the 5-HT3 receptor blockers. Renal and neurological toxicities are rare. Antimetabolites

Gemcitabine (Gemzars) Overview Gemcitabine, a derivative of ARA-C (cytarabine), is a cytidine analog. The agent was initially approved for pancreatic cancer where it demonstrated an ability to improve both longevity and quality of life. It is a relatively mild antineoplastic agent with most toxicities very manageable, but still possessing considerable efficacy against its indicated diseases. It is often used palliatively as a single agent, with very little toxicity; when used in regimens with curative intent, it is usually either with radiation (for which it has proven an excellent sensitizer) or as part of combination chemotherapy (usually at higher, less frequent dosing schedules than those used for palliative care) and has generally more toxicity in these settings. Mechanism of action Gemcitabine is an antimetabolite which, acting as a nucleoside analog, intercalates itself into the DNA, thereby disrupting the replication process. Pharmacokinetics/metabolism After IV infusion, gemcitabine is rapidly distributed to tissues and processed by the body to inactive metabolites, which are cleared by the kidneys. Both the parent drug and metabolite are found in urine. Current indications In addition to its use in NSCLC, gemcitabine is also commonly used in pancreatic, breast, bladder, and ovarian cancer. Toxicities Dose-limiting myelosuppression (especially anemia, though generally mild with commonly used doses), nausea/vomiting (usually mild), and fever during administration. Uncommon responses include liver enzyme abnormalities, thrombocytosis, dyspnea, rash, CNS depression, and pneumonitis.

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Pemetrexed (Alimtas, LY231514) Overview This second-generation antifolate agent is different than methotrexate in that it successfully inhibits three ‘targeted’ enzymes, and thus is referred to as a multitargeted antifolate. Pemetrexed was approved for use in mesothelioma, followed by an indication as a second-line therapy in NSCLC in 2004. When compared with the other FDA-approved second-line NSCLC treatment, docetaxel, clinical parameters are comparable, while pemetrexed has reductions in many areas of toxicity. Mechanism of action Pemetrexed, like methotrexate, inhibits thymidilate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyl-transferase, all folate-dependent enzymes involved in the de novo biosynthesis of thymidine and purine nucleotides. Pharmacokinetics/metabolism Pemetrexed is not metabolized to any appreciable extent. It is primarily eliminated in the urine, with 70–90% of the dose recovered as unchanged parent drug within the first 24 h. The half-life is 3–5 h in patients with normal urinary function. Current indications Pemetrexed is currently FDAapproved in the US for use in mesothelioma which is not amenable to surgery, and as a second-line therapy for NSCLC. It is being studied in a number of other solid tumors. Toxicities A rapid decline in body stores of folate can be life-threatening in some cases, so it is necessary to supplement patients with folate prior to and during administration, along with B12 injections, 1000 mcg every 9 weeks. Myelosuppression in the form of neutropenia and/or anemia is common, but usually mild. Skin effects are common and can be severe (rash) but are preventable by dexamethasone administration 4 mg orally twice a day on the day prior to chemotherapy as well as the day of treatment and the day after. Nausea and vomiting are occasionally seen, as is diarrhea. Alopecia is occasional. Bilirubin elevations are common, but usually mild and self-limited. Pericarditis has been reported. Antitubulin Agents

Docetaxel (Taxoteres) Overview A semisynthetic relative of paclitaxel, which has been FDA-approved for second-line treatment of NSCLC, and has been studied as a doublet with carboplatin and cisplatin as well as in first-line treatment. Docetaxel has twofold greater affinity for the binding site on the beta-tubulin subunit as

compared with paclitaxel. Its indications are nearly identical with those of paclitaxel; docetaxel has proven to have superior efficacy in some studies in other cancers, though such improvement has not been definitively shown in lung cancer. Mechanism of action As with paclitaxel: inhibits microtubule disassembly, thereby blocking DNA synthesis. Pharmacokinetics/metabolism After IV infusion, paclitaxel is metabolized by the liver, and excreted via the biliary system. Terminal half-life is 18 h. Biliary excretion into feces is responsible for majority of the elimination of the drug. Current indications FDA-approved for metastatic breast cancer and second-line lung cancer treatment, but is also widely used as first-line treatment in both cancers, as well as in ovarian cancer. Toxicities Dose-limiting toxicity is myelosuppression, which is universal and can be severe. Alopecia is also expected. Unique to docetaxel (as compared to paclitaxel) is fluid retention, manifesting as peripheral edema and pleural effusions, and obviated by steroid pre-treatment. Elevated liver enzymes are common and reversible. Hypersensitivity reactions are less common than with paclitaxel, though still occur and may be severe, so that pre- and postmedication with steroids and antihistamines is required. Neuropathies and mucositis are common but mild, and loss or discoloration of fingernails is common. Paclitaxel (Taxols) Overview Paclitaxel was the earliest of the taxanes, a group of natural and semisynthetic antimicrotubule agents found to be efficacious in some solid tumors. The agent was initially identified in the mid1960s as part of the US National Cancer Institute (NCI) program of screening natural products for anticancer activity. In the 1980s a unique antitubulin mechanism, distinct from the vinca alkaloids, was identified and the drug entered clinical development. Initial approval was for ovarian cancer. It is part of a popular combination therapy with carboplatin, often used as first-line treatment in the US for metastatic/ unresectable NSCLC. Mechanism of action Paclitaxel inhibits disassembly of microtubules, thereby inducing apoptosis in dividing cells. Pharmacokinetics/metabolism After IV infusion, paclitaxel has a large volume of distribution and is

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metabolized by the liver; it is excreted primarily via the biliary system. Half-life is 7–20 h. Current indications In addition to its use in NSCLC, paclitaxel is used in breast, head/neck, and ovarian cancer. Toxicities Dose-limiting toxicity is myelosuppression, though this is lessened with weekly (as opposed to every-three-week) treatments. Mucositis is similarly decreased with weekly treatment. Important infusion-related toxicities include extravasation issues (drug is a vesicant and can therefore produce severe burns when extravasated) and a significant risk of anaphylaxis, which requires pre- and postmedication with dexamethasone and H2 blockers for each treatment. Liver enzyme tests are commonly transiently elevated during treatment, and must be carefully observed. Peripheral neuropathies are common and can be irreversible. Loss or discoloration of fingernails is common as are myopathies, which often require strong analgesics. Alopecia is expected and usually complete. Other toxicities include nausea/vomiting (usually mild), and fever during administration. Uncommon responses include diarrhea, and interstitial pneumonitis. Vinorelbine (Navelbines, VRL) Overview Vinorelbine is an antimicrotubule antineoplastic agent based on the vinca plant, and has been studied in both first- and second-line treatments of NSCLC. The vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine) are derivatives of the pink periwinkle plant. The first of these to be used in humans (vincristine and vinblastine) are not common components of modern anti-lung-cancer therapies, but are active in other diseases such as liquid tumors and germ-cell tumors. Vinorelbine has been FDAapproved for NSCLC in the US. Mechanism of action As with the taxanes, vinca alkaloids inhibit microtubule disassembly, thereby blocking DNA synthesis. However, they appear to have other, less-well-understood mechanisms of action including inhibition of purine synthesis, disruption of lipid metabolism, and others. Pharmacokinetics/metabolism Vinorelbine has a half-life of 1–2 days, and is excreted primarily via hepatic pathways, thus necessitating dose reduction for patients with hepatic dysfunction. There is a welldescribed interaction with all vinca alkaloids and methotrexate, causing elevated blood levels of the latter, and thus these two drugs should not be used concurrently.

Current indications FDA-approved for metastatic breast cancer and second-line lung cancer treatment, but is also widely used as first-line treatment in both cancers, as well as in ovarian cancer. Toxicities Dose-limiting toxicity is myelosuppression, which is universal but relatively brief (resolves in 1-2 weeks after administration). Vinorelbine shares with vincristine and vindesine a propensity for neurotoxicity, though to a lesser degree, with mild-tomoderate peripheral toxicity in up to one-third of patients (paresthesias, hyperesthesia), constipation in 30%, and paralytic ileus in 2–3%. Vindesine and vinorelbine have been compared in a clinical trial in NSCLC and the latter has been found to be significantly less neurotoxic. Nausea, vomiting, stomatitis, and (rarely) pancreatitis may occur. Because vinorelbine (like all vinca alkaloids) is a vesicant, local toxicity can occur, but is usually not severe. Chest pain and shortness of breath are uncommon, but the latter can be severe, and when accompanied with cough and interstitial infiltrates, should be treated with steroids. No known irreversible pulmonary toxicities have occurred with vinorelbine. Topoisomerase Inhibitors

Etoposide (VePesids, VP-16) Overview Etoposide is a semisynthetic derivative of podophyllotoxin, a substance found naturally in the mandrake plant. Also known as VP-16, this epipodophyllotoxin is used in SCLC and NSCLC, among many others. Most of the published trials utilize infusional etoposide, but an oral formulation is available as well. Etoposide is one of the most commonly used anticancer agents, as part of standard therapy for SCLC, leukemia/lymphoma, germ-cell tumors, and neuroblastoma. Mechanism of action Etoposide inhibits topoisomerase II, thereby halting DNA transcription and cell division. Pharmacokinetics/metabolism Half-life is 6–8 h, and the drug is hepatically metabolized, with 40% renal excretion. Current indications Etoposide is currently approved in the US as first-line treatment in SCLC (as a combination therapy with a platinum-based agent) and in refractory germ-cell tumors. Toxicities Dose-limiting toxicity is myelosuppression, including leukopenia and thrombocytopenia,

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with nadir at 2 weeks, recovery at 3 weeks. Nausea, vomiting, and anorexia are common, as is alopecia, and rash. Peripheral neurotoxicity and hepatotoxicity are rare.

Pharmacokinetics/metabolism Half-life is approximately 5–10 h, and the drug is eliminated by three mechanisms: urinary excretion, biliary excretion, and hepatic metabolism.

Topotecan (Hycamptins) Overview Topotecan is a camptothecin derivative used in refractory SCLC. There are both oral and intravenous formulations available.

Current indications Irinotecan has been studied in colorectal cancer, SCLC, NSCLC, leukemia, lymphoma, cervical, breast, gastric, prostate, and ovarian carcinoma. In the US, it is FDA-approved for firstand second-line treatment of metastatic colon cancer.

Mechanism of action Topotecan inhibits topoisomerase I, thereby halting DNA transcription. Pharmacokinetics/metabolism Half-life is approximately 3 h, and 50% of the administered drug is excreted in urine unchanged over 24 h after administration. Some biliary excretion occurs as well. Current indications Topotecan is currently approved in the US in two second-line settings: metastatic ovarian carcinoma, and chemosensitive smallcell carcinoma which has progressed at least 60 days after response to initial therapy. Toxicities Dose-limiting toxicity is myelosuppression, especially leukopenia, though anemia and thrombocytopenia have also been observed. Nausea, vomiting, and diarrhea are common, but mild. Alopecia is common. Elevated transaminases and/or bilirubin have been observed.

Toxicities Nearly all irinotecan patients have diarrhea, which comes in two forms: early-onset during infusion, and later-onset, approximately 12 h after treatment. The early-onset type involves a systemic cholinergic response to the irinotecan administration, with diarrhea, flushing, diaphoresis, cramping, and vomiting. This can be treated with atropine. The later-onset diarrhea can be severe and life-threatening, and can be successfully treated with loperamide given as an initial dose of 4 mg orally, followed by 2 mg every 2 h until all bowel movements cease for a 12 h period. Other toxicities include myelosuppression (predominantly neutropenia), mucositis, elevated transaminases, and (rarely) pulmonary toxicity manifest as reticulonodular infiltrate, shortness of breath, fever, and eosinophilia. Tyrosine Kinase Inhibitors

Irinotecan (Camptosars, CPT-11) Overview Irinotecan is a member of the camptothecin class, and is active in a number of human cancers, most notably colorectal cancer, where it forms part of the ‘FOLFIRI’ regimen (5-fluorouracil, leucovorin, irinotecan), a standard in spite of the very common and occasionally problematic side effect of severe diarrhea. Its FDA approval in the US is for colorectal cancer, but it has been approved in Japan for both SCLC and NSCLC. A phase III study in Japan demonstrated that irinotecan combined with cisplatin was superior to a standard regimen of cisplatin/etoposide. However, this result was not confirmed in a US trial. There may be significant differences in the activity and toxicity of this and related agents, based upon ethnicity.

Gefitinib (Iressas, ZD-1839) Overview Gefitinib is a selective inhibitor of the epidermal growth factor receptor (EGFR), a transmembrane molecule complex which plays a key role in the proliferation of NSCLC (along with breast, prostate, ovarian, and head/neck cancers). Initial clinical trials showed a response rate of 10% in a highly pre-treated population (progression after platinum-based therapy and second-line docetaxel). The agent was granted an accelerated approval for the ‘third-line’ therapy on NSCLC on the basis of response. Randomized trials, both alone and in combination with cytotoxic drugs, confirmed that the agent could result in responses; it failed,though, to demonstrate a statistically significant survival benefit. However, there appears to be subsets of patients who derive considerable benefit: Asians, women, nonsmokers, adenocarcinoma patients, and those with EGFR mutations.

Mechanism of action After metabolic activation to the active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin), irinotecan acts as an inhibitor of topoisomerase I, thereby preventing successful DNA transcription.

Mechanism of action In addition to its inhibition of the tyrosine kinase activity of EGFR, gefitinib may also inhibit the activity of other intracellular transmembrane tyrosine-specific protein kinases. EGFR activation is the first step in the initiation of

338 TUMORS, MALIGNANT / Chemotherapeutic Agents

a cascade of cellular reactions that result in increased cell division and influence other aspects of malignant progression of the tumor, including angiogenesis, metastasis, and an inhibition of apoptosis. Pharmacokinetics/metabolism Gefitinib is 60% available after oral administration and is widely distributed throughout the body. It is extensively metabolized in the liver. Over a 10-day period, approximately 86% of an orally administered dose is excreted in feces, with o4% of the dose in urine. After daily oral administration, steady-state plasma levels are reached in 10 days. Current indications Gefitinib is currently FDA -approved in the US for use in NSCLC as third-line treatment in patients who have failed platinum-based and docetaxel-based treatments. It is also being studied in a number of other solid tumors. Toxicities The most common side effects are rash (often acneiform) and diarrhea, which are both manifest in about half of the patients. Nausea, vomiting, and anorexia are less common (around 10%). ILD, which may be fatal, occurs in o1% of patients and seems to occur primarily in Japanese and other Asian ethnicities. Erlotinib (Tarcevas, OSI-774) Overview Erlotinib, like gefitinib, is an EGFR tyrosine kinase inhibitor used in molecular targeted therapy, introduced in clinical trials in the early 2000s and FDA-approved in NSCLC in 2004. Erlotinib was evaluated in a well-designed clinical trial, which enrolled 700-plus patients who had progressed after one or two courses of chemotherapy. Treatment was with erlotinib or placebo. Improved survival as well as improvement in cough, shortness of breath, and pain were demonstrated in erlotinib patients. Mechanism of action

See gefitinib.

Pharmacokinetics/metabolism Erlotinib is metabolized in the liver, with 83% of an orally administered dose excreted in feces, and 8% in urine. Half-life is about 36 h, with steady state achieved in approximately one week. Current indications Erlotinib is currently FDA-approved in the US for use in NSCLC in patients who have failed at least one prior first-line regimen. Toxicities The most common side effects are acneiform rash and diarrhea. There is a greater degree and intensity of the rash seen with erlotinib as opposed to

gefitinib due to a decision to develop the maximally tolerated dose of the drug. In contrast, gefitinib was developed based upon demonstration of EGFR inhibition in surrogate tissues. Nausea, vomiting, and anorexia are less common (around 10%). Hepatotoxicity has been seen, and appears to be readily reversible. ILD has been seen with erlotinib use, though in the phase III study, the 0.8% incidence was the same as placebo. It has been fatal in a minority of affected patients. Rifampin and ketoconazole both alter clearance of erlotinib, and concurrent use should be avoided. See also: Tumors, Malignant: Overview; Bronchogenic Carcinoma; Hematological Malignancies; Lymphoma; Metastases from Lung Cancer; Bronchial Gland and Carcinoid Tumor; Rare Tumors; Carcinoma, Lymph Node Involvement.

Further Reading Agra Y, Pelayo M, Sacristan M, et al. (2003) Chemotherapy versus best supportive care for extensive small cell lung cancer. Cochrane Database of Systematic Reviews CD001990. Albain KS, Crowley JJ, LeBlanc M, et al. (1991) Survival determinants in extensive-stage non-small cell lung cancer: the Southwest Oncology Group experience. American Journal of Clinical Oncology 9: 1618–1626. Chute JP, Chen T, Feigal E, et al. (1999) Twenty years of phase III trials for patients with extensive-stage small-cell lung cancer: perceptible progress. Journal of Clinical Oncology 17: 1794. Kris MG, Natale RB, and Herbst RS (2003) Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. Journal of the American Medical Association 290: 2149–2158. Noda K, Nishiwaki Y, Kawahara M, et al. (2002) Irinotecan plus cisplatin compared with etoposide plus cisplatin for extensive small-cell lung cancer. New England Journal of Medicine 346: 85. Roth BJ, Johnson DH, Einhorn LH, et al. (1992) Randomized study of cyclophosphamide, doxorubicin, and vincristine versus etoposide and cisplatin versus alternation of these two regimens in extensive small-cell lung cancer: a phase III trial of the Southeastern Cancer Study Group. Journal of Clinical Oncology 10: 282. Schiller JH, Harrington D, Belani CP, et al. (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. New England Journal of Medicine 346(2): 92–98. Sundstrom S, Bremnes RM, Kaasa S, et al. (2002) Cisplatin and etoposide regimen is superior to cyclophosphamide, epirubicin, and vincristine regimen in small-cell lung cancer: results from a randomized phase III trial with 5 years’ follow-up. Journal of Clinical Oncology 20: 4665. Videtic GM, Stitt LW, Dar AR, et al. (2003) Continued cigarette smoking by patients receiving concurrent chemoradiotherapy for limited-stage small-cell lung cancer is associated with decreased survival. Journal of Clinical Oncology 21(8): 1544– 1549. von Pawel J, Schiller JH, Sheperd FA, et al. (1999) Topotecan versus cyclophosphamide, doxorubicin, and vincristine for the treatment of recurrent small-cell lung cancer. Journal of Clinical Oncology 17: 658.

TUMORS, MALIGNANT / Hematological Malignancies 339

Hematological Malignancies K M Fong and P Wood, Prince Charles Hospital, The University of Queensland, Brisbane, QLD, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Most hematological malignancies involving the thorax are lymphomas either Hodgkin or non-Hodgkin in type. These usually present with mediastinal masses in patients in their third and fourth decade, and often reach large size before clinically detectable. Despite that they are often associated with a reasonable prognosis. Other hematological causes are much less common causes of thoracic disease and will be covered in this article. The disorders have been split into extrathoracic and thoracic malignancies. Multiple myeloma is the most common disorder in this group. It has an incidence of 5 per 100 000 population for Caucasians. It frequently presents with bone pain (often axial skeleton, clavicles, and ribs), hypercalcemia, and renal impairment. Its recognition is important for appropriate management to minimize complications and symptoms. Treatment usually consists of chemotherapy and/or radiotherapy. Newer immunomodulatory therapies will be discussed. Stem cell transplantation is appropriate for a minority of patients. Cutaneous malignancy may be evident on thoracic examination and should be considered in the workup. Finally, patients may present with hemorrhagic complications of hematological malignancy. Although these disorders are rare, they are easily excluded by careful laboratory and clinical evaluation.

Introduction Many diseases of the hematological system may present as a consequence of a thoracic referral. The diseases can be divided into extrathoracic and intrathoracic. Many disorders may affect the bony thorax. The most common hematological disorder is lymphoma, which is discussed in the article Tumors, Malignant: Lymphoma, and typically presents with mediastinal or hilar lymphadenopathy (Table 1).

Table 1 Hematological malignancies involving the thorax (excluding NHL and HD) Extrathoracic Myeloma/plasmacytoma Cutaneous lymphoma/leukemia cutis Myeloid sarcoma/chloroma Thoracic Plasmacytoma Myeloid sarcoma Posttransplant lymphoproliferative disorders (PTLD) Coagulopathy from dysproteinameia eg. Waldenstro¨m’s macroglobulinaemia or myeloma Amyloidosis

Multiple myeloma is a common hematological malignancy affecting 5 per 100 000 of the population. It typically presents in elderly patients with back pain, hypercalcemia, and renal impairment. Careful laboratory workup including biochemical analysis and serum and urine electrophoresis will usually identify these patients. It may often be confused with hypercalcemia of metastatic malignancy. Cutaneous lymphomas will often present to dermatologists but patients with advanced systemic disease may present with pleural or pericardial effusion (as in a recent case in our institution) and with circulating abnormal T cells. Parenchymal lung involvement can occur with a variety of hematological malignancies; these are often lymphoma but may occur with leukemia, particularly with monocytic subtypes or during transformation from chronic to acute leukemias. Coagulopathy may occur with hematological disorders, which may present with hemoptysis or other bleeding signs and symptoms. Laboratory investigation may demonstrate abnormalities in clotting tests suggesting factor deficiency or the presence of an inhibitor.

Multiple Myeloma Aetiology

There is no clear association between myeloma and radiation exposure. There is some evidence of environmental and occupational risk. This is particularly marked in farming and agriculture, and in paint, petrochemical, wood, metal, and rubber workers. Chronic antigenic stimulation has been postulated as a causative factor, but the only clear association described is with rheumatoid arthritis. Monoclonal gammopathy of undetermined significance (MGUS) is present at significantly increased rates in chronic hepatitis C (HCV) infection. Genetic factors appear to carry a strong effect in the development of multiple myeloma (MM). Murine models show strong predilections in C57BL/Ka compared to BALB/c mice where it is virtually unseen. There are strong racial effects with very low rates in Chinese and Japanese populations which do not change with migration to other countries. Several families have been reported with multiple family members involved. Pathology

In 1975 Salmon and Durie described a staging system (Table 2) based on the analysis of 71 patients to correlate clinical features with myeloma cell mass. It became the gold standard for myeloma staging. In 2003 a new International Staging System (ISS) was

340 TUMORS, MALIGNANT / Hematological Malignancies Table 2 The Durie–Salmon staging system Stage I Definition

All of the following

Stage II

Stage III

Neither stage I nor stage III

One or more of the following

1

Clinical features


Hemoglobin410 g dl
Calciumo12 mg dl  1 (3 mmol l  1)
p1 bone lesion
Low M component; IgG o5 g dl  1, IgAo3 g dl  1, Bence Jones protein o4 g dl  1

Myeloma cell mass (  1012 m  2)

o0.6


Hemoglobino8.5 g dl  1
Calcium412 g dl  1 (3 mmol l  1)
43 bone lesions
High M component; IgG 47 g dl  1, IgA 45 g dl  1, BJP 412 g dl  1

0.6–1.2

41.2

Subclassification (either A or B) A: creatinine o2 mg dl  1 (0.18 mmol l  1) B: creatinine X2 mg dl  1 Reproduced from British Medical Journal, 1975, vol. 36, pp. 842–854, with permission from the BMJ Publishing Group.

Table 3 International Staging System (ISS) for myeloma

Stage I Stage II

Stage III

Clinical parameters

Median survival (months)

b2Mo3.5 mg ml  1 and albumin X35 g l  1 b2Mo3.5 mg ml  1 and albumin o35 g l  1, or b2M 3.5–5.5 mg ml  1 b2MX5.5 mg ml  1

62 44

29

developed with a cohort of 11 171 cases (Table 3). By multivariate analysis, three groups were determined based on b2-microglobulin (b2M) and albumin level. The ISS compared to the Durie–Salmon staging provides a more powerful discrimination between groups and a more even distribution of patients within these groups. Classifications using genotypic data are currently in development. Clinical Features

Hematological disorders may occasionally affect the bony skeleton of the thorax. MM is the most common disease to do this. Local deposits of myeloma (plasmacytomata) commonly affect the ribs and clavicles; they also affect the skull, vertebral column, pelvis, and long bones. Plasmacytoma typically present as a large, firm, nonpainful mass in a rib or sternum. They may occur as a solitary plasmacytoma or as part of a diffuse myelomatous process. The differential diagnosis would be of metastatic malignancy from a nonhemopoietic primary malignancy.

Figure 1 Plain lateral X-ray showing typical ‘pepper-pot skull’ changes.

Skeletal survey may show the presence of numerous lytic lesions and the typical ‘pepper-pot skull’ (Figure 1). Technetium (Tm99) bone scans are only positive in 30% of patients, as the bony disease is predominantly osteoclastic rather than osteoblastic in nature. Sensitivity and specificity of 99mTcMIBI (Sestamibi) scintigraphy in detecting myeloma bone lesions are 92% and 90%, respectively. Magnetic resonance imaging (MRI) is also particularly sensitive but shows poor specificity for myeloma (Figure 2). The diagnosis can be confirmed either by biopsy of the bony lesion or by serum and urine electrophoresis

TUMORS, MALIGNANT / Hematological Malignancies 341 99mTc-MDP bone scan

27 May 2005 Dose: 890 MBQ L

R

M

P pelvis

RT lateral R

R

L

L

R

A pelvis

LT lateral

Anterior

L

Posterior

Figure 2 Bone scan in myeloma patient showing increased uptake in sternum, ribs, and right wrist.

(up to 20% of myelomas may be light chain secreting only), which will detect the characteristic M protein. Routine investigations may show a mild to moderate anemia and raised globulin level. Hypercalcemia may be present, requiring differentiation from primary hyperparathyroidism, secondary malignancy, or nonmetastatic hypercalcemia of malignancy (e.g., Ectopic PTH secretion) (Figure 3). Pathogenesis

The development of MM appears to be a multistep pathogenesis involving genetic changes within the malignant cell, changes in the marrow microenvironment which support tumor cell growth, and changes in the immune system which allow the proliferation of the malignant clone (Figure 4). Numeric chromosomal abnormalities are present in virtually all cases of MM. Comparative genomic hybridization (CGH) demonstrates unbalanced structural changes in most cases of MM. Recurrent chromosomal gains in 430% of cases of MM are seen on 1q, 3q, 9q, 11q, and 15q. The most common abnormalities of chromosomal loss affect 13q. It is thought that karyotypic complexity increases with tumor progression although this is not well documented. Primary immunoglobulin gene translocations are thought to occur early in the pathogenesis

Figure 3 CT scan showing involvement of the right maxillary sinus and right orbit with plasmacytoma.

of MM whilst secondary translocations occur with disease progression. Primary translocations are usually simple reciprocal translocations that juxtapose an oncogene with an immunoglobulin (Ig) gene enhancer.

342 TUMORS, MALIGNANT / Hematological Malignancies

Germinal center B cell

MGUS

Intramedullary myeloma

Extramedullary myeloma

Myeloma cell line

Primary Ig gene translocations Cyclin D1 and D3 FGFR3 and MMSET C-Maf, MAFB Karyotypic instability 13q deletions

Somatic mutations N-ras, K-ras, FGFR3 Secondary Ig gene translocations C-myc Others Figure 4 Development of MM.

Although the Ig partners are numerous, they fall into three main groups. The first group consists of cyclins D1 (11q13) and D3 (6p21). These occur in 20–25% of MM tumors. The second group is located on 4p16 and encode two proteins, MMSET and fibroblast growth factor receptor 3 (FGFR3), an oncogenic receptor tyrosine kinase. These translocations occur in 15% of tumors. The third group consists of two b-zip transcription factors: c-Maf (16q23) and MAFB (20q11). These occur in 10% of cases. Clinical studies from the Intergroupe Francophone du Myelome (IFM) using interphase fluorescent in situ hybridization (FISH) have demonstrated the prognostic significance of karyotypic abnormalities. Patients with t(4;14) translocations have a poor overall survival (23% at 80 months) and did not appear to achieve a lasting control to high-dose chemotherapy. In contrast, patients with t(11;14) translocations had a particularly good survival (805 at 80 months). These differences were independent of chromosome 13q deletions. Treatment and Prognosis

Melphalan and prednisolone have been the traditional therapies for many years. They produce an objective response in 50–60% of patients. More intensive chemotherapy regimens may produce a more rapid response and higher response rate (60% vs. 53% in a meta-analysis of 5000 patients) but do not demonstrate any additional effect on overall survival.

Vincristine, adriamycin, and dexamethasone (VAD) is typically used in relapsed or refractory disease or in younger patients as first line therapy prior to autologous stem cell transplant (ASCT). ASCT should be considered in patients under 65 years of age. A French Myeloma Group study of 200 patients under 65 years of age showed a significant benefit of ASCT compared to standard chemotherapy. Response rates were 81% versus 57%, with 5-year event-free survival (28% vs. 10%) and overall survival (52% vs. 12%) superior in the ASCT arm. Allogeneic transplantation is limited in MM mainly due to the high transplant related mortality of up to 30%. Newer agents have tried to target MM cell growth, tumor and BM stromal cell interactions, tumor survival, and immune surveillance. These agents include thalidomide and its more potent immunomodulatory analogs (iMiDs), the proteasome inhibitor Bortezomib and arsenic trioxide. Thalidomide has effects on tumor apoptosis, angiogenesis, and adhesion as well as augmenting cytotoxicity. Single agent thalidomide produces partial responses (450% reduction in paraprotein) in around 25% of patients with relapsed or refractory disease. Combination with other agents particularly dexamethasone increases response rates to around 50% in this patient group. Response rates in previously untreated patients are around 35% as a single agent and 65–70% in combination with dexamethasone. IMiDs (e.g., lenalidomide) have significantly enhanced efficacy compared to thalidomide in vitro but

TUMORS, MALIGNANT / Hematological Malignancies 343

with a reduced side effect profile. Early phase II studies in relapsed patients show similar response rates compared to thalidomide as a single agent but partial response rates of at least 85% when used in combination with dexamethasone. Bortezomib is a selective, reversible proteasome inhibitor. The proteasome is a multicatalytic complex responsible for the degradation of ubiquinated intracellular proteins. A recent phase III trial in relapsed patients comparing Bortezomib to high-dose dexamethasone was terminated at interim analysis due to a significant benefit in the Bortezomib arm. Bortezomib provided a significant 78% improvement in time to progression (TTP; 6.2 vs. 3.5 months). Response rates were also significantly better in the Bortezomib arm (38% vs. 18%). Phase I studies are currently underway evaluating a combination of Lenalidomide and Bortezomib.

Cutaneous Lymphoma and Leukemia Cutis Primary cutaneous lymphomas are predominantly T cell in type, and Mycosis fungoides (MF) and Sezary syndrome (SS) comprise approximately 65% of all cases. MF is characterized by epidermotrophic bandlike infiltrates of small to medium-sized T lymphocytes that are CD3 þ , CD4–, CD45RO þ , CD30–, and CD8–. The clinical course is indolent with slow progression over years from patches to more infiltrative plaques. The 5-year survival rate approximates 90%. Local therapy with radiotherapy or psoralens and ultraviolet A (PUVA) is usually adequate, with combination chemotherapy reserved for cases with nodal or visceral involvement. SS has been considered as the leukemic variant of MF, and is defined by the triad of erythroderma, generalized lymphadenopathy, and the presence of neoplastic T cells in the skin, lymph nodes and peripheral blood (SS). The disease is more aggressive than MF with a median survival of 3 years. B-cell lymphomas of follicle center type and diffuse large B-cell lymphoma may less frequently involve the skin and generally behave in an indolent manner. Leukemia Cutis

The cutaneous manifestations of leukemia include primary, specific skin lesions resulting from direct infiltration of the skin and subcutaneous tissues by leukemic cells. Examples are leukemia cutis, granulocytic sarcomas, and extramedullary tumor masses. The papulonodular lesions of leukemia cutis present as brown-red to violaceous, indurated dermal papules, plaques, or nodules. Early lesions may

be macular. Other rare clinical presentations of leukemia cutis include bullae, ulcerations, and erythroderma resulting from diffuse leukemic infiltration of the skin.

Lung Involvement with Acute and Chronic Leukemias Myeloid sarcomas (also known as granulocytic sarcomas, chloromas) are extramedullary masses of blasts or immature myeloid cells. The tumor mass may proceed or occur concurrently with acute or chronic myeloid leukemias, myeloproliferative disorders, or myelodysplasia. With the chronic disorders the sarcomas often occur at the time of progression of the disease to a more aggressive phase. The World Health Organization (WHO) now recognizes these tissue deposits as sufficient evidence to upgrade chronic myeloid leukemia from the chronic phase to the blastic phase of disease. There may be a long interval (months to years) between the occurrence of sarcomas and overt leukemia. The most common sites of occurrence are the skull, paranasal sinuses, sternum, ribs, vertebrae, and skin. Rarely, sarcomas may present in areas of invasive intervention, for example, at the site of insertion or subcutaneous tunnels of central venous catheters. They can be divided morphologically into three major types: blastic, immature, and differentiated. A less common monoblastic variant is also described. Myeloid sarcomas are more commonly associated with the AML types with maturation and t(8;21) (q22;q22), acute myelomonocytic leukemia (AMML) with inv(16) (p13q22) or t(16;16) (p13;q22). The monoblastic variant is commonly associated with translocations involving 11q23.

Coagulopathy and Hematological Malignancies Abnormalities of coagulation may be encountered during the routine workup prior to bronchoscopy or biopsy. Impaired hemostasis is most commonly present with Waldenstro¨m’s macroglobulinemia related to the high level of pentameric IgM interfering with normal coagulation factors (Table 4). Highlevel IgG or IgA paraproteins in myeloma may less commonly cause coagulopathy. The presence of high globulin levels in these patients is usually sufficient to suggest the diagnosis and warrant further investigation. Factor X inhibitors have been described with amyloidosis which classically present with periorbital bruising or postproctoscopic purpura.

344 TUMORS, MALIGNANT / Lymphoma Table 4 Coagulopathy in Waldenstro¨m’s macroglobulinaemia. 65-year-old male with a history of previous small cell lung cancer presenting with hemoptysis

INR Prothrombin time APTT Fibrinogen

19 Apr 2004

16 Nov 2004

1.4 15 54 2.3

1.1 12 32 3.2

Reference range 0.9–1.3 9–14 25–38 1.5–4.0

Note: abnormal profile at diagnosis with correction by chlorambucil therapy.

Acute leukemias (typically acute promyelocytic leukemia) may present with pulmonary embolism. Abnormalities of the full blood count and the laboratory parameters of DIC, for example, low fibrinogen, prolonged prothrombin time, activated partial thromboplastin time, and elevated fibrin degradation products, would usually be present. See also: Tumors, Malignant: Overview; Bronchogenic Carcinoma; Chemotherapeutic Agents; Lymphoma; Metastases from Lung Cancer; Bronchial Gland and Carcinoid Tumor; Rare Tumors; Carcinoma, Lymph Node Involvement.

Further Reading MacLennan ICM, Drayson M, and Dunn J (1975) British Medical Journal 36: 842–854. Richardson PG and Anderson KC (2004) Multiple Myeloma. London: Remedica Publishing. (2005) 10th International Myeloma Workshop. Haematologica/ The Haematology Journal, vol. 90 (S1), April 2005.

Relevant Websites www.hematology.org – American Society of Hematology Education Program Books. http://www.iarc.fr – WHO classification. Tumours of hemopoietic and lymphoid tissues. www.myeloma.org – International Myeloma Foundation.

Lymphoma R Ku¨ppers and E Tiacci, University of Duisburg – Essen, Essen, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract Lymphomas represent a heterogeneous group of about 30 distinct entities, whose overall incidence (about 15–20 new cases per 100 000 persons per year) has been increasing in the past 50 years. While etiology is largely unknown in most cases, various risk factors have been identified, including severe immune suppression, infection by certain viruses and bacteria, occupational

exposure to chemicals, and rare inherited lymphoproliferative disorders. A common presenting feature is a painless enlargement of superficial lymph nodes, but also systemic symptoms (fever, weight loss, night sweats) or signs due to tumor masses expanding in the mediastinum or the abdomen or infiltrating extrahematopoietic sites (e.g., gastrointestinal tract, skin, central nervous system) may occur. Besides a common disruption of the normal tissue architecture by infiltrating tumor cells, a broad spectrum of histological and immunohistological patterns is observed across the different types of lymphoma. The pathogenetic hallmark in many of them is represented by reciprocal chromosomal translocations leading to deregulated expression of protooncogenes or, less often, to generation of oncogenic fusion genes. Also chronic stimulation by microbial antigens (and, possibly, autoantigens) appears to play a role in the development of certain lymphomas. Variable combinations of chemotherapy, radiotherapy, immunotherapy, and antibiotic/antiviral therapy are currently used in the treatment of lymphomas.

Introduction B and T lymphocytes are specialized white blood cells that play important roles in immune responses. When such lymphocytes undergo malignant transformation, they give rise to lymphomas. Lymphomas occur at a frequency of about 15–20 new cases per 100 000 persons per year in the Western world. About 90% of them are of B-cell origin, the rest are T-cell malignancies. The first description of a lymphoma dates back to 1832, when Thomas Hodgkin described several cases of a tumor that was later named Hodgkin’s lymphoma (HL). Other types of lymphomas that were subsequently identified were grouped together and called non-Hodgkin’s lymphomas (NHLs). Nowadays, B30 different lymphoma entities are distinguished. Lymphomas can occur throughout the body, but predominant locations are lymph nodes, spleen, bone marrow, and peripheral blood. The various lymphomas differ not only in their pathology, but also in their clinical behavior.

Etiology While most lymphomas occur as sporadic tumors, there are a few inherited diseases that confer a highly increased risk of lymphoma development. One such disease is ataxia-telangiectasia, a rare neurodegenerative disorder caused by mutations in the ATM gene that plays an important role in the control of cell division after DNA damage. Lymphomas occur in B10% of affected children. The X-linked lymphoproliferative (XLP) disease is caused by mutations in the SH2D1a/SAP gene, which is involved in the regulation of T-cell activation. Affected boys cannot control infection by the ubiquitous Epstein–Barr virus (EBV), and EBV-associated B-cell lymphomas develop in 420% of patients. The autoimmune lymphoproliferative syndrome is also associated with

TUMORS, MALIGNANT / Lymphoma 345

an increased risk of NHLs (14-fold higher) and HL (50-fold higher); it is caused by inherited mutations in the CD95 gene, which codes for a surface receptor that controls apoptosis in lymphocytes and whose inactivation leads to deregulated lymphoproliferation. The important role of an intact immune system in controlling lymphocyte proliferation is obvious also from the increased risk of lymphoma in immunosuppressed patients. Transplant patients that are immunosuppressed have a highly increased risk for B-cell lymphomas. Since these are mostly EBV-positive, it is generally assumed that the suppression of T cells to prevent transplant rejection impairs the control of virus-driven B-cell proliferation that finally may give rise to B-cell lymphomas. Similarly, in AIDS (acquired immune deficiency syndrome) patients the immunosuppression caused by human immunodeficiency virus (HIV) causes a 60-fold increase in risk for lymphomas. As already indicated above, there is evidence for a viral etiology in several types of B-cell lymphomas. Besides transplant- and XLP-associated lymphomas, EBV is found in 490% of cases of endemic Burkitt’s lymphomas in Africa, and the tumor cells in HL are EBV-infected in B40% of cases. Human herpes virus-8 (HHV8) is found in all cases of the rare primary effusion lymphoma that mostly occurs in AIDS patients. Helicobacter pylori has been identified as an etiological factor in gastric B-cell lymphomas of the mucosa-associated lymphoid tissue (MALT) and, accordingly, elimination of H. pylori by antibiotics often leads to lymphoma regression.

Pathology Lymphomas are classified according to the World Health Organization scheme (Table 1), an updated version of the Revised European–American Classification of Lymphoid Neoplasms. In these classifications, tumor entities are defined (and diagnosed) by integrating a number of criteria: morphology, immunophenotype, cytogenetic, and molecular features, as well as clinical data. The relative importance of each criterion differs among diseases, with no single gold standard, though histological evaluation (by an experienced hematopathologist) of the involved organ through an excision biopsy is always important. Three major categories of lymphoma are recognized (Table 1): HL and, among NHLs, B-cell neoplasms and T-cell/NK-cell neoplasms. The latter two categories are further stratified into ‘precursor’ and ‘peripheral’ neoplasms, based on their derivation from the earliest (lymphoblastic) or the more

Table 1 Lymphoma classification according to the World Health Organization scheme Hodgkin’s lymphoma Classical Hodgkin’s lymphoma Morphological variants:a nodular sclerosis, mixed cellularity, lymphocyte depletion, lymphocyte rich Nodular lymphocyte-predominant Hodgkin’s lymphoma B-cell non-Hodgkin lymphomas/leukemias Precursor B-cell lymphoma/leukemia Precursor B-lymphoblastic lymphoma/leukemia Peripheral B-cell lymphomas/leukemias Diffuse large B-cell lymphoma Specific subtypes: mediastinal (thymic) large B-cell lymphoma, primary effusion lymphoma, intravascular large B-cell lymphoma Morphologic variants:a centroblastic, immunoblastic, T-cell/ histiocyte-rich, plasmablastic, anaplastic large B-cell, lymphomatoid granulomatosis type Follicular lymphoma Morphologic variants:a diffuse follicle center lymphoma Mantle cell lymphoma Splenic marginal zone B-cell lymphoma Nodal marginal zone-cell lymphoma Extranodal marginal zone B-cell lymphoma (MALTb lymphoma) Burkitt’s lymphoma/leukemia Subtypes: endemic, sporadic Lymphoplasmacytic lymphoma Plasma cell myeloma and plasmacytoma B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma B-cell prolymphocytic leukemia Hairy cell leukemia B-cell lymphoproliferative disorders with uncertain malignant potential Lymphomatoid granulomatosis Posttransplant polymorphic lymphoproliferative disorder T-cell and natural killer cell non-Hodgkin lymphomas/leukemias Precursor T-cell/natural killer cell lymphomas/leukemias Precursor T-lymphoblastic lymphoma/leukemia Blastic natural killer cell lymphomac Peripheral T-cell/natural killer cell lymphomas/leukemias Anaplastic large cell lymphoma, systemic Anaplastic large cell lymphoma, cutaneous Hepatosplenic T-cell lymphoma Angioimmunoblastic T-cell lymphoma Enteropathy-type T-cell lymphoma Extranodal natural killer cell/T-cell lymphoma, nasal type Subcutaneous panniculitis-like T-cell lymphoma Peripheral T-cell lymphoma, unspecified Mycosis fungoides and Sezary syndrome Adult T-cell leukemia/lymphoma T-cell prolymphocytic leukemia T-cell large granular lymphocytic leukemia Aggressive natural killer cell leukemia T-cell lymphoproliferative disorder with uncertain malignant potential Lymphomatoid papulosis a

Note that morphological variants are listed only for the most frequent lymphoma entities (Hodgkin’s lymphoma, diffuse large B-cell lymphoma, and follicular lymphoma). b MALT, mucosa-associated lymphoid tissue. c The assignment of this entity to the natural killer cell lineage is uncertain, and recent evidence rather indicates an origin from plasmacytoid dendritic cells.

346 TUMORS, MALIGNANT / Lymphoma

differentiated (mature) stages of normal lymphoid development, respectively. HL, which is of mature Bcell origin in nearly all cases, is further divided into classical HL (cHL) and nodular lymphocyte-predominant HL (NLPHL), with cHL being the commonest form (B95% of HLs). Both cHL and NLPHL are characterized by the fact that neoplastic cells represent o1–2% of the total cellularity in the involved tissue and are dispersed in a prominent inflammatory background (Figure 1(b)), whereas in NHLs the tumor population is conspicuous and usually infiltrates vast areas of the tissue (Figures 1(a), 1(c), and 1(d)). Although the tissue architecture is perturbed in almost all instances, a wide range of histological (Figure 1) and immunohistological patterns characterizes the morphologic and phenotypic spectrum of lymphomas. Diffuse large B-cell lymphomas (DLCLs), follicular lymphoma (FL), and cHL are the most frequent lymphomas, together accounting for about twothirds of all cases. Lymphomas primarily occurring in the thoracic cavity usually form a mediastinal mass (arising from the thymus and/or mediastinal lymph nodes), as in cHL, mediastinal large B-cell lymphoma, and T-cell lymphoblastic lymphoma/leukemia. Much less frequently, lymphomas localize primarily to the lung parenchyma (with MALT lymphomas accounting for most of such cases, see

below ‘Specific comments on primary pulmonary lymphomas’) or the pleura (as in primary effusion lymphoma).

Clinical Features Lymphomas mainly arise from lymph nodes (and spleen), and the presenting clinical feature in about two-thirds of patients is a painless swelling of a superficial lymph node (most frequently in the cervical, supraclavicular, axillary, or inguinal areas). Lymphomatous masses expanding in the mediastinum can present with cough, chest discomfort, dyspnea and, sometimes, superior vena cava syndrome, whereas retroperitoneal lymphadenopathy (unless extensive) is more often asymptomatic, and splenomegaly may be the only abdominal finding at physical examination. Systemic symptoms (called B symptoms) can also occur, including fever, night sweats, and unexplained weight loss. Lymphomas can also present at extranodal sites (sometimes as the only affected area), and, besides the frequent involvement of bone marrow and/or peripheral blood, extrahematopoietic organs commonly affected are gastrointestinal tract, skin, and central nervous system, with related signs and symptoms (e.g., dyspepsia, bowel obstruction, cutaneous lesions, headache, and neurological deficits) being possible presenting features.

(a)

(b)

(c)

(d)

Figure 1 Different histological features of lymphomas. (a) Burkitt’s lymphoma. Diffuse infiltration by monomorphic medium-sized lymphoma cells; some of these undergo apoptosis and the resulting nuclear debris is phagocytosed by intermingled benign macrophages (indicated by the arrow), producing a ‘moth-eaten’ appearance. Original magnification 400. (b) Classical Hodgkin’s lymphoma, mixed cellularity. The few neoplastic cells, called Reed–Sternberg cells if multinucleated (arrow) or Hodgkin’s cells if mononucleated (arrowhead), are very large and scattered in a benign cellular infiltrate composed of lymphocytes, eosinophils, plasma cells, and macrophages. Original magnification 200. (c) Follicular lymphoma. The lymph node architecture is packed with neoplastic follicles showing atypical features (e.g., frequent lack of mantle zones). Lymphoma cells are often observed also in the interfollicular areas (not shown). Original magnification 100. (d) Diffuse large B-cell lymphoma. Diffuse infiltration by large neoplastic cells, resembling either normal centroblasts (with peripheral nucleoli) or normal immunoblasts (with central nucleoli). Original magnification 400.

TUMORS, MALIGNANT / Lymphoma 347

The approach to initial evaluation of lymphoma patients is based on a detailed anamnesis (including comorbid illnesses and risk factors for lymphoma), physical examination (including superficial lymph Table 2 Ann Arbor staging classification Stagea

Descriptionb

I

Involvement of a single nodal (I) or extranodal (IE) site Involvement of two or more nodal sites located in the same side of the diaphragm (II), or localized involvement of an extranodal site and of one or more nodal sites located on the same side of the diaphragm (IIE) Involvement of nodal sites located on both sides of the diaphragm (III), which may be associated with spleen involvement (IIIS) or with localized involvement of an extranodal site (IIIE) or with both (IIISE) Disseminated or diffuse involvement of one or more distant extranodal sites with or without accompanying nodal involvement

II

III

IV

a Each of the four stages is further characterized as A or B (e.g., IA or IB) based on the absence or presence, respectively, of systemic symptoms (i.e., fever greater than 381C and/or night sweats and/or weight loss greater than 10% of body weight over the previous 6 months). b Note that for the purpose of staging, the spleen is considered as a lymph node site.

nodes, spleen, Waldeyer’s ring, and skin), staging studies (including computed tomography (CT) and positron emission tomography (PET) scans of the chest, abdomen and pelvis, and bone marrow biopsy and aspirate; see Table 2 for staging classification), and laboratory studies. The latter should include viral serology, blood cell count and smear, routine blood chemistry (including lactate dehydrogenase and uric acid, as indirect measurements of tumor burden and proliferation rate), and serum protein electrophoresis (to detect a potential lymphomaassociated monoclonal gammopathy).

Pathogenesis A hallmark of many lymphomas is reciprocal chromosomal translocations involving the immunoglobulin loci in B-cell lymphomas or the T-cell receptor loci in T-cell lymphomas (Table 3). As a consequence of these translocations, proto-oncogenes are brought under the control of these active loci, causing their deregulated, constitutive expression. Well known examples include the c-myc/Ig translocation in Burkitt’s lymphoma, the bcl-2/IgH translocation in FL, and the CCND1/IgH translocation in mantle cell lymphoma. Specific features of the breakpoints in these translocations suggest that they happen as

Table 3 Oncogenic events in human lymphomasa Lymphoma B-cell lymphomas Burkitt’s lymphoma Follicular lymphoma Mantle cell lymphoma Diffuse large B-cell lymphomas

B-cell chronic lymphocytic leukemia Primary effusion lymphoma MALT lymphoma T-cell lymphomas Anaplastic large cell lymphoma T-cell prolymphocytic leukemia Adult T-cell leukemia/lymphoma a

Chromosomal translocationsb

Other oncogenic eventsb

t(8;14), t(2;8), t(8;22); C-myc/Ig (100) t(14;18); bcl-2/IgH (90) t(11/14); CCND1/IgH (100) t(3;?); bcl-6/various (35)

EBVc (endemic: 90, sporadic: 30) p53 mutations (40)

t(11;18); API2/MALT1 (30) t(14;18); MALT1/IgH (15)

ATM deletion/mutation (40) Aberrant hypermutation of multiple proto-oncogenes (70) CD95 mutations (10–20) p53 mutations (30) ATM deletion/mutation (30) Human herpes virus-8 (100) EBVc (70) CD95 mutations (5–80)d

t(2;5); NPM/Alk (40–70) inv(14)/t(14;14); TCL1/TCRa/d (75)

ATM deletion/mutation (50) Human T-cell leukemia virus-1 (100)

The best characterized and most frequent oncogenic events are listed in this table. Numbers in parentheses indicate the frequency of the oncogenic events in percentage of cases affected. c EBV, Epstein–Barr virus. d Different frequencies observed in different subtypes of MALT lymphomas. b

348 TUMORS, MALIGNANT / Lymphoma

by-products of molecular processes acting at immunoglobulin or T-cell receptor loci during B-cell or T-cell development. In some instances, translocations do not involve these loci and play a pathogenetic role by producing fusion proteins (Table 3). Besides chromosomal translocations and viruses (see above), other transforming events have been implicated in lymphomagenesis (Table 3). Various lymphomas carry inactivating mutations in the tumor-suppressor gene p53, a main regulator of apoptosis, and mutations in the CD95 or ATM genes contribute to lymphomagenesis not only as inherited mutations (see above), but also occur somatically in a fraction of lymphomas (Table 3). In cHL, constitutive activity of the nuclear factor kappa B (NF-kB) transcription factor represents an important survival factor, and somatic mutations in the inhibitor of NFkB, inhibitor of kappa light chain gene enhancer in B cells, alpha (IkBa), or genomic amplifications of the NF-kB component c-Rel seem to contribute to the NF-kB activity. A specific situation is given in DLCLs, where the process of somatic hypermutation, which normally introduces mutations only into the variable regions of immunoglobulin genes, aberrantly targets multiple proto-oncogenes. The fact that 430 different types of lymphomas can be distinguished can, on the one hand, be attributed to the involvement of many different (combinations of) oncogenic events in the malignant transformation of lymphocytes that give rise to distinct diseases. On the other hand, different lymphomas may develop depending on the particular stage of differentiation at which a B or T lymphocyte is transformed. Indeed, most lymphomas have been shown to be related to germinal center B cells, whose apparent propensity toward malignant transformation may be due to the processes of somatic hypermutation and class-switch recombination that physiologically occur in these cells and bear an intrinsic risk for chromosomal translocations and other mutations.

Animal Models Animal models for lymphomas can be separated into three groups: those arising spontaneously, those induced by chemicals, and those developing in genetically modified animals. An example for the first group is lymphomas that occur at significant frequency in old mice of particular strains (e.g., the B10 strain), and that express the surface marker CD5. These lymphomas have been regarded as a model for human B-CLL (B-cell chronic lymphocytic leukemia), because this leukemia usually develops in humans over the age of 65 and is also CD5-positive.

The paradigm for chemical-induced lymphomas is plasma cell malignancies (plasmacytomas) developing in pristane-oil-treated mice. These plasmacytomas carry c-myc translocations, a situation reminiscent of human multiple myelomas, which also frequently show activating alterations of c-myc. A large number of transgenic mouse models have been generated to test the role of particular oncogenes in lymphoma development, and some of them closely resemble the human malignancy-carrying alteration of the respective gene (e.g., bcl-2 transgenic mice developing FLs and c-myc transgenic mice developing Burkitt’s lymphomas).

Management and Current Therapy Lymphomas are a heterogeneous group of cancers (see Table 1) and, although grossly subdivided into aggressive and indolent types, their clinical outcome is extremely variable, ranging, for example, in B-cell lymphomas from 480% long-term survival rate in HL to o15–20% in mantle cell lymphoma. Therapeutic strategies must be tailored to each entity with its associated prognostic factors, and, in some circumstances (such as low-burden indolent lymphomas), immediate treatment may not be necessary. In many lymphomas, the most important clinical variables that predict a worse outcome (and that are usually combined into integrated models, such as the International Prognostic Index – IPI) are older age and poor performance status of the patient, an advanced stage of the disease, and an abnormal lactate dehydrogenase value. The rapidity of response to induction therapy (evaluated by PET) is emerging as another clinical prognostic parameter in HL and aggressive NHLs, and new biological prognostic factors (e.g., germinal center versus nongerminal center molecular signature in DLCLs) are being introduced by genome-wide expression-profiling studies. Since the risk of systemic micrometastasis is high even when lymphomas apparently present as a localized disease, for many lymphomas the cornerstone of treatment is systemic chemotherapy (often in a multiagent fashion). Radiotherapy and immunotherapy play important integrating roles, and lymphomas associated with infectious agents often benefit from antibacterial/antiviral therapy. The principle underlying the currently used chemotherapeutic protocols (e.g., ABVD or BEACOPP for HL; CHOP or CHOPlike regimens for DLCLs) is to deliver with a precise dose intensity and time schedule, different drugs that have nonoverlapping toxicities toward normal cells and to which lymphoma cells are not crossresistant. Radiotherapy is an important adjunct to minimize chemotherapy duration and dose intensity (especially

TUMORS, MALIGNANT / Lymphoma 349

in limited-stage disease), to more efficiently target bulky sites of involvement and residual masses after chemotherapy, and to reach sanctuary sites not well penetrated by chemotherapy (e.g., testis and central nervous system). High-dose chemotherapy and/or radiotherapy followed by autologous (or, less often, allogeneic) hematopoietic stem cell transplantation is mostly reserved for patients not responding or relapsing after initial treatment. Passive immunotherapy, i.e., the delivery of monoclonal antibodies (as such, or conjugated to radionuclides or, less often, toxins) against antigens expressed only by B cells and/or T cells (e.g., CD20 or CD52), has recently allowed new advances in lymphoma treatment, by virtue of a different mechanism of action and a lower toxicity compared to chemotherapy and radiotherapy. Active immunotherapy (through vaccination with lymphoma-specific antigens, such as the immunoglobulin idiotype) has shown promising results (particularly in FL) and is currently under further evaluation.

Specific Comments on Primary Pulmonary Lymphomas Lymphomas primarily arising in the lung parenchyma and/or bronchi are extremely rare (B3–4% of extranodal lymphomas; o1% of primary pulmonary neoplasms). The most frequent type is MALT lymphoma, an indolent malignancy usually presenting in the sixth or seventh decade of age as an asymptomatic (or paucisymptomatic, with cough, mild dyspnea, or B symptoms sometimes being present) localized alveolar opacity (often with air bronchogram) at routine X-ray examination. CT scans frequently reveal multiplicity and bilaterality of the lesions, which are thought to arise from acquired bronchial MALT in the context of chronic stimulation by a self-antigen (rather than a microbial antigen, as happens in H. Pylori-associated gastric MALT lymphoma). Indeed, some patients have autoimmune disorders (e.g., Sjo¨gren’s syndrome) as accompanying disease, and they may show concomitant or subsequent involvement of other mucosal sites (salivary glands, ocular adnexa, gastrointestinal tract, upper aerodigestive tract). Another likely important pathogenetic event in pulmonary MALT lymphomagenesis is represented by the chromosomal translocation t(11;18), which is detected in about half of the cases and leads to the formation of the oncogenic fusion protein API2-MALT1. The diagnosis of pulmonary MALT lymphoma is reached through bronchoscopic or thoracoscopic biopsy, which typically shows an infiltration of neoplastic small, mature, ‘marginal

zone-like’ B cells surrounding reactive follicles and migrating to the bronchiolar epithelium (‘lymphoepithelial’ lesions). Therapeutic strategies range from watchful waiting to chemotherapy, radiotherapy, and surgical excision of localized nodules; despite the fact that relapses frequently occur, transformation to aggressive lymphoma is rare and long-term prognosis is good. A worse outcome is typical of two other (and rarer) types of primary pulmonary lymphoid disorders: aggressive B-cell DLCLs (which often develop from a previous or concurrent MALT lymphoma, or are associated with an underlying immune suppression) and lymphomatoid granulomatosis. The latter is a poorly understood B-cell lymphoproliferation of uncertain malignant potential that can involve other organs (e.g., skin and central nervous system) and usually presents with respiratory and systemic symptoms in patients with multiple nodular opacities tending to excavate. See also: Antiviral Agents. Apoptosis. Cell Cycle and Cell-Cycle Checkpoints. Corticosteroids: Therapy. Leukocytes: T cells. Lymphatic System. Mediastinal Masses. Oncogenes and Proto-Oncogenes: Overview; MYC. Pleural Effusions: Malignant Pleural Effusions. Radiation-Induced Pulmonary Disease. Radiotherapy. Surgery: Transplantation. Symptoms of Respiratory Disease: Cough and Other Symptoms; Dyspnea. Transgenic Models. Tumors, Malignant: Overview; Chemotherapeutic Agents; Hematological Malignancies; Rare Tumors.

Further Reading Cadranel J, Wislez M, and Antoine M (2002) Primary pulmonary lymphoma. European Respiratory Journal 2: 750–762. Cavalli F, Isaacson PG, Gascoyne RD, and Zucca E (2001) MALT lymphomas. Hematology 241–258. Fisher RI, Miller TP, and O’Connor OA (2004) Diffuse aggressive lymphoma. Hematology 221–236. Greer JP, Kinney MC, and Loughran TP Jr (2001) T cell and NK cell lymphoproliferative disorders. Hematology 259–281. Harris NL, Jaffe ES, Stein H, et al. (1994) A revised European– American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 84: 1361–1392. Jaffe ES, Harris NL, Stein H, and Vardiman J (eds.) (2001) Tumours of the Haematopoietic and Lymphoid Tissues. Lyon: IARC Press. Ku¨ppers R (2005) Pathogenetic mechanisms of B cell lymphomas. Nature Reviews: Cancer 5: 251–262. Mauch PM, Armitage JO, Coiffier B, Dalla-Favera R, and Harris NL (eds.) (2004) Non-Hodgkin’s Lymphomas. Philadelphia: Lippincott Williams & Wilkins. Mauch PM, Armitage JO, Diehl V, and Hoppe RT (eds.) (1999) Hodgkin’s Disease. Philadelphia: Lippincott Williams & Wilkins. Meyer RM, Ambinder RF, and Stroobants (2004) Hodgkin’s lymphoma: evolving concepts with implications for practice. Hematology 184–202.

350 TUMORS, MALIGNANT / Metastases from Lung Cancer Press OW, Leonard JP, Coiffier B, Levy R, and Timmerman J (2001) Immunotherapy of non-Hodgkin’s lymphomas. Hematology 221–240. Shaffer AL, Rosenwald A, and Staudt LM (2002) Lymphoid malignancies: the dark side of B-cell differentiation. Nature Reviews: Immunology 2: 920–932. Winter JN, Gascoyne RD, and Van Besien K (2004) Low-grade lymphoma. Hematology 203–220.

Metastases from Lung Cancer K M Fong, B E Clarke, and R V Bowman, Prince Charles Hospital, The University of Queensland, Brisbane, QLD, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Over half of the patients presenting with lung cancer have metastatic disease. The most common sites of metastatic involvement in lung cancer are regional lymph nodes, brain, bone, liver, and adrenal glands. The international staging system for lung cancer arose from recognition that the presence and extent of metastatic disease has a major impact on survival. N (node) and M (distant metastasis) staging is the most important determinant of treatment outcomes. Guidelines for management recommend algorithms to accurately determine the stage of disease in an individual patient, while balancing the financial cost associated with routinely performing staging investigations on all patients, and the morbidity costs of invasive investigations. Fortunately, technological advances in imaging modalities are providing increasingly reliable means of noninvasive staging for lung cancer. Metastatic disease from lung cancer presents tremendous clinical challenges in palliation of symptoms produced by expanding mass lesions, including pain, dyspnea, and loss of function. The potential of systemic therapy to control symptoms, improve quality of life, and extend survival in extensive stage lung cancer has been demonstrated in clinical trials of chemotherapy in patients with good performance status. Major challenges for the future include realizing a reduction in the proportion of patients presenting with metastatic disease through improved early detection, improving detection of micrometastases and understanding their significance, translating the promise of new biologically targeted therapies to control the development of metastasis, and developing new and better means of symptom palliation.

Clinical Features Lung cancer appears to be able to metastasize to most organs in the body. Indeed, in many cases, the poor outcome (12–14% overall, 5- year survival) is due to the late presentation of clinical lung cancer when local and/or distant metastases have already occurred. Consequently, apart from the local and paraneoplastic effects of lung cancer, symptoms may arise from these metastatic deposits, and will relate to the tumor infiltration, mass, or compressive effects. Perhaps one-third of lung cancer patients

present with symptoms as a result of distant metastases. Table 1 displays common sites of local and distant metastases associated with lung cancer. In addition, constitutional symptoms are frequent in metastatic lung cancer including anorexia, weight loss, lethargy, and fatigue. These do not reflect direct tumor involvement but indirectly indicate the presence of metastatic disease. Constitutional symptoms at presentation require evaluation for involvement of the common metastatic sites.

Clinical Evaluation Intrathoracic Metastases

Regional lymph nodes Lung cancers commonly metastasize or directly involve hilar and mediastinal lymph nodes (Figure 1) with clinical recognition of such involvement underpinning the assessment of the node (N) stage for non-small cell lung cancer (NSCLC). The characteristics of nodal metastases are discussed in detail in Tumors, Malignant: Carcinoma, Lymph Node Involvement. Sampling methods to establish metastatic involvement include imaging (CXR, computed tomography(CT), PET), transbronchial needle aspiration (TBNA), transthoracic needle aspiration (TTNA), endoscopic ultrasound-guided needle aspiration (EUS-NA), endobronchial ultrasound-guided needle aspiration (EBUS-NA), and mediastinoscopy. Selection of the appropriate diagnostic modality is dependent on the clinical pre-test probability of adenopathic disease, the patient’s fitness for investigation including comorbid illnesses, and the availability and performance characteristics of procedural options. Pulmonary metastases Satellite nodules, by definition within the same lobe as the index tumor, are quite common in lung cancer. While commonly found on pathological examination of resected lung, satellite nodules are also increasingly detected radiologically by sensitive CT- scanning techniques. Patients whose only evidence of metastatic spread is a satellite nodule(s) are staged M0. Contralateral lung nodules or nodules in a lobe different to the primary tumor are considered M1 disease on the basis of the differential survival outcomes between these two patient groups, and the fact that satellite nodules are resected or included in treatment fields for patients receiving curative therapy. Pleural invasion or metastasis Perhaps the commonest local complication of lung cancer causing major symptoms is pleural effusion due to either

TUMORS, MALIGNANT / Metastases from Lung Cancer 351 Table 1 Common lung cancer metastases

Locoregional

Distant

Site

Symptoms

Mechanism

Mediastinal nodes

Hoarseness Venous distension Upper body swelling Dyspnea, stridor

Recurrent laryngeal nerve compression Superior vena cava compression Major bronchus compression

Scalene or supraclavicular nodes

Discomfort

Pulmonary

Hemoptysis Pleural pain

Cardiac/pericardial

Dyspnea Pain

Tamponade Arryhthmias

Brain

Headaches Vomiting Visual disturbance Bowel/bladder dysfunction Focal weakness or sensory symptoms

Increased intracranial pressure

Seizures Mental state changes Gait disturbance Cranial nerve palsies Spinal cord

Back pain Radicular pain

Bowel/bladder dysfunction Parasthesiae Paraparesis, ataxia Bone

Pain

Liver

Abdominal pain or fullness, jaundice

Adrenal

Flank or loin pain Addisonian features

Figure 1 Lymph carcinoma.

node

metastases

from

bronchogenic

Deficits due to cortical or white matter involvement Cerebellar metastasis Cranial nerve compression Meningeal metastasis Extramedullary spinal cord compression (commonly extension from bony vertebral metastasis) Intramedullary metastases (rare) Corda equina compression Nerve root compression Periostial invasion Pathologic fractures

Rarely in bilateral disease

direct invasion or metastatic seeding. Patients may experience both chest pain and dyspnea. Malignant pleural effusion-causing dyspnea is managed by a staged approach; an initial needle aspiration to assess the degree of symptomatic benefit is useful in patients with malignant effusion due to primary lung cancer. If symptom palliation is good, either tube or thoracoscopic pleurodesis may be planned as definitive management. The staged approach, using simple aspiration initially to gauge the symptomatic benefit, is useful because of the frequent presence of ‘trapped’ lung (in which diseased pleura prevents lung re-expansion) and endobronchial obstruction preventing re-expansion of underlying lung. In these two circumstances, removing pleural fluid may be of limited benefit, and other means of palliating the symptoms need to be pursued. Pleural pain due to

352 TUMORS, MALIGNANT / Metastases from Lung Cancer

malignant involvement in lung cancer is often effectively palliated by radiotherapy in conjunction with appropriate analgesic management. Extrathoracic Metastases

The most common sites of distant lung cancer metastases are the brain, bone, liver, and adrenal glands. These sites are the focus of clinical algorithms for the distant metastases (M) staging of lung cancer. Since small cell lung cancer (SCLC) and possibly adenocarcinomas are more likely to metastasize than other NSCLC subtypes, patients with these primary subtypes warrant more vigorous staging investigations. Brain metastases Up to 25% of Stage IV NSCLC have brain metastasis as the sole clinical site of disease or, in addition, to metastases at other sites. The brain is also the first site of recurrence in approximately one-fifth of cases of recurrent lung cancer after resection. A small subset of stage IV NSCLC has a single brain metastasis as the only site of distant metastasis. As this group of patients may sometimes benefit from curative intent treatment, thorough exclusion of other sites of distant metastases is an essential initial step in their management. Detection modalities The commonest investigations for brain metastases are CT scans and magnetic resonance imaging (MRI). Whereas MRI is more sensitive than CT scan and detects more and smaller metastatic lesions, some studies have shown that this detection might not translate into meaningful differences in survival. Thus, either CT scan with contrast enhancement or MRI is an acceptable investigation for evaluation of patients for brain metastases. MRI may be useful where CT is technically limited such as posterior fossa imaging. Current PET technology using the glucose analog FDG is relatively insensitive for brain metastasis owing to high background uptake of glucose in the normal brain, making it difficult to detect focal areas of increased FDG uptake. Investigations to detect brain metastases should be performed for any NSCLC patients presenting with symptoms suggestive of such a lesion, and in those with constitutional symptoms where diagnosis of brain metastases would alter clinical management. Whilst the CT positivity rate is high in symptomatic lung cancer cases, some series report that brain lesions detected are sometimes ultimately identified as inflammatory or even incidental primary brain tumors. However, for asymptomatic patients without specific constitutional symptoms, pertinent abnormal physical signs, or biochemical abnormalities

to suggest other distant metastases, meta-analyses suggest that routine brain scanning is not necessarily indicated because of low yield. In NSCLC patients without clinical features of metastatic disease, the yield of MRI or CT brain scans is generally less than 10%. Patients with SCLC are staged according to a twostage, Veterans Administration Lung Cancer Study Group system, and classified as having limited-stage disease or extensive-stage disease. Limited-stage disease refers to tumor restricted to one hemithorax, that is, within a single radiation port, whereas extensive-stage disease is defined as the presence of metastatic disease. As SCLC tends to metastasize widely including brain, most investigators recommend assessment with either CT or MRI of the brain to fully stage the disease as management differs significantly for the two stages. Treatment Occasionally patients present with a solitary brain metastasis, where there is some evidence to suggest that resection followed by radiotherapy results in better local control and longer disease-free interval than radiotherapy alone. Stereotactic radiosurgery may also be an option in this circumstance. Clearly, investigations should be performed to ascertain the absence of multiple brain metastases as well as of extracranial disease. Cerebellar lesions may also require surgical intervention at times due to their tendency to cause obstructive hydrocephalus and symptomatic raised intracranial pressure. In addition, occasionally patients present with both an isolated brain metastasis and resectable lung cancer. For these synchronous presentations, where no other metastatic disease can be identified, resection of both may be considered in selected patients. For brain metastases otherwise, radiotherapy is generally indicated, particularly if there has been a clinical response to dexamethasone resulting from reduction of associated cerebral edema. Doses of whole brain radiotherapy ranging from 10 to 40 Gy appear similar in effectiveness for symptom palliation and time to progression, with shorter courses offering advantages of patient convenience and reduced linear accelerator usage. Symptomatic brain metastases carry a poor prognosis in the order of months. Steroids and radiotherapy offer some prolongation of life, symptom palliation, and improved quality of life. Spinal cord compression is regarded as a medical emergency and must be treated promptly by multidisciplinary management. Symptoms of spinal cord compression even in the absence of clinical signs should therefore be investigated. Diagnosis may be

TUMORS, MALIGNANT / Metastases from Lung Cancer 353

achieved by CT scan, but if available, MRI is the investigation of choice since it is capable of demonstrating the anatomical level or levels of compression. Steroids are generally used while awaiting definitive therapy to reduce the compressive effects of local edema. Because there are often coexistent widespread metastases when a diagnosis of spinal cord compression is made in lung cancer patients, survival expectation is often less than that associated with clinical recovery from surgical decompression. Radiotherapy is therefore the most practical approach to symptomatic cord compression in this setting.

MRI with chemical shift imaging detects the presence of fat-laden cells in adenomas where opposing fat and water signals can be manipulated to cause a signal drop-off between in-phase and out-of-phase imaging if the lesion is benign. This technique also has high sensitivity and specificity. FDG PET scan relies upon the high metabolic activity of adrenal metastases. Where available, this is an attractive option as it not only provides high sensitivity and specificity for the adrenal lesion but also noninvasively provides staging information on the mediastinum and other extrathoracic sites.

Adrenal metastases The detection of adrenal metastases from lung cancer, particularly NSCLC, is complicated by the frequent occurrence of benign adrenal lesions in the general population. The majority of CT-detected adrenal lesions, even in patients with known cancers, may be benign. Reports indicate that adrenal metastases occur in the order of 10% or fewer patients with NSCLC. In SCLC, metastases to the adrenals are more common and can be associated with other distant metastatic lesions. On the other hand, a normal CT does not exclude adrenal metastases in SCLC. Recommended lung cancer staging investigation includes imaging of the upper abdomen (liver and adrenals) with CT of the chest, so evaluation of an asymptomatic adrenal lesion, where a diagnosis of metastatic disease would impact on the management plan for the patient, is a frequent challenge.

Treatment There are case reports of resection of isolated adrenal metastases in NSCLC in highly selected patients. The overall 5-year survival ranges in these cases from 10–23%. The variability may relate to nodal involvement, histologic subtype, synchronous or metachronous presentation, and ipsilateral or contralateral location.

Diagnostic modalities There are no contrast CT imaging features such as size, contour, or heterogeneity that have high specificity for adrenal metastases. In view of this and the small but significant risk of morbidity associated with image-guided fine needle biopsy, special imaging techniques have been developed with improved accuracy in predicting that an adrenal lesion is benign or malignant. Noncontrast CT scan exploits the characteristic that fat-laden adenomas have lower attenuation values than malignant lesions. The sensitivity and specificity vary according to the level used to set the threshold to distinguish benign from malignant a specificity of 100% is achievable for benign disease at the cost of lower sensitivity. As most staging chest CTs involve contrast administration, this entails an additional scan. Contrast washout CT is based on the observation that, unlike metastases which have a rapid enhancement profile with slow washout, adenomas show only moderate enhancement and a rapid washout. Some series report high sensitivity and specificity for this technique.

Liver metastases Whilst liver metastases are common in lung cancer, liver test dysfunction is uncommon unless there is a significant metastatic tumor mass from numerous and large deposits. Since liver metastases are most often in the context of other metastatic disease, imaging and sampling of liver lesions is a less frequent clinical issue. Detection modalities Studies of liver metastases suggest that contrast may be beneficial for detecting disease, although some of this advantage may be obviated by the use of modern spiral multi-slice CTs. Small lesions, less than 5 mm in diameter, with low attenuation are usually regarded as benign, representing simple cysts which are common. Large, solid, or enhancing lesions are more likely to be malignant metastases and may require further assessment by ultrasound, MRI, or biopsy if the management plan is likely to be altered. PET scan again offers high specificity for liver metastases, while simultaneously providing an assessment of other metastatic sites. Treatment In general, the diagnosis of liver metastases is made in conjunction with detection of involvement in other metastatic sites and portends a guarded prognosis. Pain due to metastatic liver involvement can sometimes be problematic. Radiation therapy is limited by liver tolerance but can be used for focal lesions. Bone metastases Although the axial skeleton and long bones are most commonly involved, virtually any bone may be targeted by lung cancer metastases. The usual presentation is of bone pain which is

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present in up to a quarter of lung cancer patients at presentation, but of course may be due to inflammatory or degenerative conditions common in the age group of patients presenting with lung cancer. A small proportion of bony secondaries will lead to pathological fractures. A recent meta-analysis update of asymptomatic NSCLC patients suggested that where clinical evaluation (symptoms, examination, and routine blood tests) is negative, it is reasonable not to investigate for bony secondaries with a bone scan. However, there was heterogeneity in the negative predictive value, ranging from 0.7–1.0, possibly due to differences in reference standard used in included studies (histological confirmation was not always undertaken), and/or variations in stage and histological subtype of lung cancer. Also, studies using whole body PET scans were not included. There is persisting controversy regarding the need for diagnostic skeletal imaging in patients with negative clinical evaluation. The recommendation not to investigate in the absence of symptoms, signs, or abnormal biochemistry has recently been challenged by a prospective cohort study where one-quarter of skeletal metastases occurred in asymptomatic patients. Detection modalities Nuclear bone scans are more sensitive and specific for the detection of bony metastases than plain radiographic imaging or CT scan. However plain films, CT, or MRI are useful for confirming abnormal bone scan findings and excluding trauma or degenerative lesions (Figure 2). PET scan is emerging as a superior modality to isotope uptake bone scans. One study suggested sensitivity of 92% and specificity of 99% for PET compared to 50% and 92% for bone scans. Local availability and cost may be relevant factors in choice of test modality. Where a single bony lesion is noted on bone scan, further imaging by PET, CT, MRI, or occasionally even biopsy may be required to avoid misdiagnosis of a solitary benign bone lesion as a metastasis, particularly if the single lesion is the only evidence of advanced stage and the patient would be otherwise treated with curative intent. Treatment There are two aims in treatment of bony metastases – pain control and reduction of the risk of fracture in weight-bearing or long bones. Standard analgesia including narcotics should be provided to alleviate pain from bony metastases. Radiotherapy is an effective modality for the treatment of bony metastatic pain with a single 8 Gy fraction as efficacious as other radiotherapy schedules. Chemotherapy may also be an option for

Figure 2 MRI showing vertebral column involvement from bronchogenic carcinoma.

patients with multiple symptomatic bony metastases not controlled by simpler measures. Bisphosphonates inhibit osteoclastic bone resorption and are useful for hypercalcemia associated with malignancy. There is some emerging data that bisphosphonates may also improve pain from symptomatic bony metastases and reduce the number of skeletal events in solid cancers including lung cancer. Orthopedic intervention may be required in certain circumstances, including prophylactic fixation of long bones where there is a high risk of fracture from the secondary deposit, stabilization following pathological fractures. Consideration should be given to decompression of spinal cord or nerves þ /
vertebral stabilization for spinal cord involvement in the appropriate context where a reasonable survival is expected. Radiotherapy should be provided following internal fixation.

TUMORS, MALIGNANT / Metastases from Lung Cancer 355 Micrometastases from Lung Cancer

The natural history of lung cancer is such that at the time of clinical presentation, a lung cancer has already undergone much of its growth and its metastatic potential has already been established. Although prognosis is closely related to the presence or absence of overt local or distant metastases using current detection methodology, micrometastases may explain some of the variation in outcome observed in patients with the same stage of disease treated in the same manner. There is increasing data to support the notion that occult micrometastases can be detected prior to conventional clinical recognition using sophisticated techniques such as PCR, RT-PCR, flow cytometry, immunohistochemistry or immunocytochemistry with or without tumor-enrichment techniques to increase sensitivity, and automated laser detection methods are in development. In some studies, detection of occult micrometastases in bone marrow or lymph nodes is correlated with clinical outcomes (survival) in NSCLC. This approach highlights the possibility of sampling bias in the assessment of lymph nodes post resection. Routine histopathological examination has been calculated to underestimate the prevalence of nodal metastases; for example, about 1% chance of identifying a small (3-cell diameter) focus of malignancy has been suggested. The biological diversity in lymph node metastases is further discussed in Tumors, Malignant: Carcinoma, Lymph Node Involvement. Other sources of bodily fluids potentially suitable for micrometastasis detection include pleural fluid and blood. An unresolved issue is whether a few tumor cells, either in circulation or other body compartments, are sufficient to establish clinically important metastases. The logical progression of these studies is therefore to test if micrometastases detection allows (a) identification of tumors in asymptomatic patients (e.g., blood-test screening for lung cancer) or (b) in known lung cancer, identification of patients who would benefit from adjuvant systemic therapy.

Summary Metastases from lung cancer are unfortunately common and most often portend a poor prognosis. The clinical decision to investigate for distant metastases should reflect a balance between the need to accurately stage patients for treatment planning and the cost and risk of morbidity from invasive investigations. A recent updated meta-analysis suggests that as the negative predictive values of clinical evaluations for brain, abdominal, and bone metastases are

greater than 90%, routine imaging of asymptomatic NSCLC lung cancer patients may not be necessary. However, predictive values are dependent on disease prevalence in the population and thus prospective studies of various lung cancer populations are required to ensure the correct applicability of these data. In addition, whilst the yield may be low, a study from the Canadian Lung Oncology Group has suggested that in apparently operable NSCLCs, routine imaging with bone scan, CT of brain, chest, and upper abdomen may result in a reduced number of thoracotomies without a cure and a small reduction in overall cost. In SCLCs, metastatic disease is much more frequent and routine imaging is usually performed although costs can be contained by refraining from further investigation once extensive disease is confirmed and management will not be changed by identification of further sites of metastases. Another major issue for the future is development of better treatments for metastatic disease including biological and targeted therapies, and their effective use in conjunction with conventional therapies to improve the quality of life for patients with metastatic lung cancer. See also: Tumors, Malignant: Overview; Bronchogenic Carcinoma; Chemotherapeutic Agents; Hematological Malignancies; Lymphoma; Rare Tumors; Carcinoma, Lymph Node Involvement.

Further Reading Beckles MA, Spiro SG, Colice GL, and Rudd RM (2003) Initial evaluation of the patient with lung cancer: symptoms, signs, laboratory tests, and paraneoplastic syndromes (review). Chest 123(1 supplement): 97S–104S. Cote RJ, Hawes D, Chaiwun B, and Beattie EJ Jr (1998) Detection of occult metastases in lung carcinomas: progress and implications for lung cancer staging (review). Journal of Surgical Oncology 69(4): 265–274. Detterbeck FC, Jones DR, Kernstine KH, and Naunheim KS (2003) Lung cancer guidelines. Special treatment issues. Chest 123(1 supplement): 244S–258S. Guyatt GH (2001) The Canadian Lung Oncology Group. Investigating extrathoracic metastatic disease in patients with apparently operable lung cancer. Annals of Thoracic Surgery 71(2): 425–433; discussion 433–434. Hetzel M, Hetzel J, Arslandemir C, Nussle K, and Schirrmeister H (2004) Reliability of symptoms to determine use of bone scans to identify bone metastases in lung cancer: prospective study. British Medical Journal 328(7447): 1051–1052. Jiao X and Krasna MJ (2002) Clinical significance of micrometastasis in lung and esophageal cancer: a new paradigm in thoracic oncology (review). Annals of Thoracic Surgery 74(1): 278–284. McLoud TC (2002) Imaging techniques for diagnosis and staging of lung cancer (review). Clinics in Chest Medicine 23(1): 123–136. Pope RJ and Hansell DM (2003) Extra-thoracic staging of lung cancer (review). European Journal of Radiology 45(1): 31–38.

356 TUMORS, MALIGNANT / Bronchial Gland and Carcinoid Tumor Silvestri GA, Tanoue LT, Margolis ML, Barker J, and Detterbeck F (2003) The noninvasive staging of non-small cell lung cancer: the guidelines. Chest 123(1 supplement): 147S–156S. Simon GR and Wagner H (2003) Small cell lung cancer (review). Chest 123(1 supplement): 259S–271S. Toloza EM, Harpole L, Detterbeck F, and McCrory DC (2003) Invasive staging of non-small cell lung cancer: a review of the current evidence (review). Chest 123(1 supplement): 157S– 166S. Toloza EM, Harpole L, and McCrory DC (2003) Noninvasive staging of non-small cell lung cancer: a review of the current evidence (review). Chest 123(1 supplement): 137S–146S.

Bronchial Gland and Carcinoid Tumor P Macchiarini, G J Maria, and J R Ruiz, Heidehaus Hospital, Hannover, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract Malignant bronchial gland tumors are epithelial tumors that include the most frequent carcinoid tumors, and the less frequent carcinomas of the salivary glands, such as mucoepidermoid carcinoma and adenoid cystic carcinoma. Carcinoid tumors represent 1–2% of all lung tumors, and can be divided into typical and atypical. They are usually located in the major bronchi but approximately a third of the tumors are in the periphery of the lungs (segmental bronchi or beyond). They occur more in the right than the left lung, especially in the middle lobe. Most patients with pulmonary carcinoid are symptomatic, hemoptysis, cough, recurrent pulmonary infection, fever, or chest discomfort being the most common complaints. Paraneoplastic syndromes such as carcinoid syndrome or Cushing’s syndrome are infrequent. The preoperative diagnosis can be established by bronchoscopy and biopsy, but definitive diagnosis is always done after surgical resection. Immunoscintigraphy by somatostatin analogs can also aid in diagnosis. The treatment of choice is surgical resection, and prognosis is excellent in typical carcinoids but not so in atypical carcinoids. The role of radiotherapy and chemotherapy as part of multimodality treatment or palliation is still debated.

History There are two different types of malignant bronchial gland tumors, the most frequent pulmonary carcinoid tumors, and the less common carcinomas of the salivary glands, such as mucoepidermoid and adenoid cystic carcinomas. These last were described elsewhere (uncommon lung malignant tumors). Pulmonary carcinoids were first described in the early twentieth century and originally considered to be benign. In the past, carcinoid tumors were grouped with adenoid cystic carcinomas and mucoepidermoid carcinomas as ‘bronchial adenomas’ because of their relatively benign course, until they were included in a

spectrum of neuroendocrine tumors, ranging from low-grade malignant typical and intermediate atypical to high-grade large cell neuroendocrine carcinomas (LCNECs) and small cell lung carcinomas (SCLCs). The term ‘neuroendocrine’ was introduced with the finding that these cells are capable of synthesizing hormonal products and that several of them are identical to cells of the nervous system. The difference between typical and atypical carcinoids was first defined by the histological criteria of Arrigoni and co-workers and later modified by Travis and co-workers.

Epidemiology Male and female patients are almost equally distributed in the studies. However, in patients under the age of 50 years, carcinoids are seen almost twice as often in women than men. The mean age is approximately 47 years, but atypical carcinoid (AC) tumors usually occur in significantly older patients. Although this theory is speculative, this might be due to smoking as a risk factor in AC. This is supported by the finding that the frequency of molecular changes that are often seen in smoking-related lung tumors increases in the spectrum of neuroendocrine tumors, from AC (a few cases) to LCNEC (more frequently) to SCLC (very frequently). Several studies support the hypothesis that smoking may be a risk factor in the development of AC but not in patients with typical carcinoids (TC). The pulmonary carcinoids comprise 1–2% of all lung tumors. Most pulmonary carcinoids are confined to the main or lobar bronchi. Regional lymph node involvement is seen in 10%. The incidence of pulmonary neuroendocrine tumors, especially of TC, seems to be sharply increasing. It is unknown whether this is because of a true increase or because of a more accurate diagnosis with more advanced medical viewing techniques detecting asymptomatic carcinoids.

Carcinoid Tumors Pathology

In the classification of lung tumors by the World Health Organization and International Association for the Study of Lung Cancer, carcinoids are considered to belong to the neuroendocrine tumor spectrum. At one end of the spectrum are the low-grade typical carcinoids followed by the intermediate grade atypical carcinoids, whereas at the other extreme end of the spectrum are high-grade LCNECs and SCLCs. Typical carcinoids are composed of uniform cells with eosinophilic cytoplasm. Tumor cells have a

TUMORS, MALIGNANT / Bronchial Gland and Carcinoid Tumor 357

round centrally placed nuclei with finely dispersed chromatin (Figure 1). Most of these tumors (80%) are centrally located in the main stem bronchi, or in the lobar or segmental bronchi, and infrequently involve the main carina or trachea. They occur more (60%) in the right than in the left lung, especially in the middle lobe. Only 10–20% of these tumors present lymph node involvement. Most tumors are sessile. Because the bulk of the tumor is usually extraluminal, they have been called ‘iceberg tumors’. They penetrate the bronchial wall and may involve the pulmonary parenchyma and peribronchial lymph nodes. Atypical carcinoids represent approximately 10% of carcinoids. Tumor cells show pleomorphic, large, hyperchromatic nuclei, frequent mitoses, and foci of necrosis (Figure 2). In contrast with typical carcinoids, more than 50% of these tumors are located peripherally. The tumor behavior is aggressive and it

spreads to regional lymph nodes systemically in more than 50% of the cases. In some instances, it is difficult to differentiate this tumor from a small-cell carcinoma. The differentiation between typical and atypical carcinoids is clinically important and is based on the number of mitoses, the most accurate predictor of prognosis. Typical carcinoids present fewer than two mitoses per 2 mm2 of viable tumor (ten-high-power fields) with no necrosis. Atypical carcinoids present 2–10 mitoses per 2 mm2 and/or with foci of necrosis. Carcinoids contain many neurosecretory granules that are capable of synthesis, storage, and release of substances such as serotonin, histamine, prostaglandins, kallikrein, and dopamine. Among these substances, serotonin (5-hydroxytryptamine(5-HT)) is the one seen most frequently. Degradation of 5-HT results in 5-hydroxyindolacetic acid (5-HIAA). An indicator of 5-HT production in the body is 5-HIAA, that is excreted in urine. When released in the systemic circulation, 5-HT leads to a production of the so-called carcinoid syndrome. Atypical carcinoids present more often with nodal metastases and a higher recurrence rate and their prognosis is significantly worse compared with typical carcinoids. Distant metastases are more frequently found in liver, bone, brain, adrenal gland, and ovary. Pathogenesis

Figure 1 Typical carcinoid showing homogeneous nuclei in most of its cells. H&E 250.

The influence of smoking on the pathogenesis of carcinoids is still debated. It has been found that the frequency of molecular changes that are often seen in smoking-related lung tumors, increases in the spectrum of neuroendocrine tumors, from ACs in a few cases to LCNECs more frequently, to SCLCs very frequently. Genetic abnormalities have been seen in carcinoids. Molecular studies show differences between typical and atypical tumors, and also between atypical carcinoids and the other neuroendocrine tumors. Abnormalities in tumor-suppressor genes and oncogenes are seen more frequently in atypical carcinoids compared with typical carcinoids, but none of these have been shown to be useful preoperatively or intraoperatively in the selection of surgical management, and only the tumor stage and its histologic subtype are reliable factors for assessing aggressiveness. In carcinoid tumors DNA under-representation of 11q is frequent, whereas this is rare in other neuroendocrine tumors. In atypical carcinoids, there are often losses of 10q and 13q, and these can also be seen in LCNECs and SCLCs. Epidemiology

Figure 2 Atypical carcinoid demonstrating heterogeneous nuclei, with some pleomorphics cells. H&E 250.

Pulmonary carcinoids comprise 1–2% of all lung tumors. The incidence of these tumors seems to be

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increasing, perhaps because of a more accurate diagnosis with more advanced medical viewing techniques detecting asymptomatic carcinoids. Approximately 30% of all these tumors are detected at autopsy. The average age of patients with atypical carcinoids is 55 years, compared to 45 years for patients with typical carcinoids. Familial carcinoids of the lung are rare and associated with the syndrome of multiple endocrine neoplasia (MEN 1). MEN 1 is a familial tumor syndrome with an autosomal-dominant inheritance that is characterized by a high frequency of endocrine malignancies. Inactivation of the MEN 1 gene by mutation has been reported in both atypical (25%) and typical (67%) carcinoids, whereas these mutations are absent in SCLCs and exceptional in LCNECs. Clinical Features

The symptoms associated with carcinoids depend on their location (peripheral or central). Peripheral tumors are usually asymptomatic, and the diagnosis is established as the result of an incidental radiographic finding. Approximately, 50% of the patients present with a variety of pulmonary symptoms. The most common symptoms are hemoptysis, cough, recurrent pulmonary infection, fever, chest pain, unilateral wheezing, and shortness of breath. Bronchiectasis or chronic lung abscesses are found in patients with long-standing undiagnosed tumors which can lead to total destruction of the lung.

Pulmonary carcinoids have also been associated with other endocrine disorders such as increased ACTH production, ectopic production of growth hormone (GH)-releasing hormone, increased pigmentation from melanophore-stimulating hormone, inappropriate antidiuretic hormone secretion, and hypoglycemia.

Diagnostic Procedures Radiographic Studies

Frequently, a standard radiograph will detect no abnormality. CT scan can delimitate the endobronchial and parenchymal component of the tumor. Calcifications can be identified in 30% of the lesions (Figure 3). Homogeneous contrast enhancement of a typical carcinoid can be observed after intravenous bolus of contrast media. ACs usually show less contrast enhancement. Nuclear Scanning

Immunoscintigraphy by somatostatin analogs such as octeotride, lanreotride, and pentetreotide is potentially a useful diagnostic tool for neuroendocrine lung tumors, although its role has still not been fully clarified. This technique is based on the overexpression

Paraneoplastic Syndromes

Carcinoid syndrome The carcinoid syndrome is very rare in patients with pulmonary carcinoids (1–2%), and manifests only in patients with large primary tumors or hepatic metastases. Carcinoid syndrome is an entity consisting of cutaneous, cardiovascular, gastrointestinal, and respiratory manifestations, produced by the release of tumor vasoactive substances in the systemic circulation. Manipulation of the tumor during bronchoscopy, intubation, or surgery can release important concentrations of vasoactive substances into systemic circulation. In patients with carcinoid syndrome, the breakdown product of serotonin (5-HIAA) can be detected in blood or urine. Other Endocrinopathies

Cushing’s syndrome is the second most commonly observed paraneoplastic syndrome associated with carcinoids, being present in approximately 1% of patients. This is characterized by ectopic adrenocorticotropic hormone (ACTH) production, resulting in hypercortisolism.

Figure 3 CT scan showing calcifications in a typical carcinoid tumor of the middle lobe diagnosed 12 years earlier, presenting with heterogenous calcifications.

TUMORS, MALIGNANT / Bronchial Gland and Carcinoid Tumor 359

necessitate deeper biopsies than are necessary for other malignant bronchial neoplasms. With bronchoscopy, it is possible to do a biopsy to diagnose carcinoids in approximately 50% of the cases of central tumors, but is difficult to distinguish between typical and atypical types. In 10–15% of the cases, a misdiagnosis of SCLCs can occur. Some authors fear major hemorrhage with biopsy by fiberoptic bronchoscopy. Alternatively, biopsy samples can be taken in the operation room with a rigid bronchoscope while the patient is under general anesthesia. The use of the rigid bronchoscope has the advantage of not only enabling larger, more reliable biopsy samples, but also being a safer procedure, in case of bleeding. In peripheral tumors, diagnosis is more difficult, and can be obtained by fine needle aspiration cytology. Endobronchial Resection Figure 4 Typical carcinoid tumor in the origin of middle lobe bronchus seen by bronchoscopy.

of somatostatin receptors on carcinoids. Scintigraphic detection of pulmonary carcinoids with octeotride has a sensitivity of 86%, whereas combined iodine-131-metaiodobenzylguanidine and indium111-pentetreotride has a sensitivity of 95%. Because scintigraphy is also positive in many other tumors, granulomas, and autoimmune diseases, specificity is low. Also, this technique cannot be relied on to exclude a carcinoid tumor.

Endobronchial laser is considered only a palliative treatment because the majority of carcinoids spread extraluminally. It can be used as an alternative therapeutic option in patients not fit to undergo surgical resection. In highly selected subset of patients, however, some authors advocate endobronchial laser resection, with or without posterior photodynamic therapy as a definitive therapy. After resection, the bronchial wall can be screened with more accuracy for residual tumor and depth of tumor invasion, by means of the endobronchial ultrasonography.

Positron Emission Tomography

Surgery

When a solitary nodule is evaluated, use of positron emission tomography (PET) with 18-fluorodeoxyglucose (FDG) imaging often shows false–negative results with pulmonary carcinoids, because they are hypometabolic on FDG–PET.

Surgery offers the only chance of cure and is the treatment of choice. Despite the fact that these tumors are indeed malignant, their indolent growth has encouraged surgeons to accept a policy of conservative resection. Surgical lymph node staging is mandatory and regional lymphadenectomy is advised in all patients with positive nodes or atypical carcinoids. Carcinoids do not spread submucosally, and therefore a surgical bronchial margin as small as 5 mm is often considered adequate. The TNM staging system does not seem to be appropriate for these tumors given the fact that even in N1 and N2 patients, long-term survival has been reported, as high as 95% and 54% at 10 years for typical and atypical carcinoids, respectively. For typical carcinoids, bronchoplastic parenchyma-sparing surgery is the standard surgical procedure. Lobectomy (with or without bronchoplastic procedures) is the most common operation because most tumors occur in or near the origins of the lobar bronchi. Other surgical resections include bilobectomy, segmentectomy, wedge resection, or

Bronchoscopy

Bronchoscopy plays an important role in the diagnosis of carcinoids, because in the majority of the cases, the tumor is centrally located, and visible on endoscopic evaluation. Typical carcinoids appear as highly vascularized pink-to-purplish soft tumors covered by intact epithelium, making cytological washings or brushing almost always unrewarding, in contrast with biopsy specimens (Figure 4).

Management and Current Therapy Biopsy

Accurate identification of the tumor requires bronchial biopsy. These tumors present submucosally and

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bronchoplastic procedures. The indications for pneumonectomy are rare, and this operation should be avoided whenever possible. The presence of nodal disease does not preclude lobectomy or sleeve resection. Nevertheless, some researchers are of the opinion that in contrast to typical carcinoids, parenchyma-sparing surgery is not considered as suitable in atypical carcinoids. Therefore, the same surgical treatment as in non-small cell lung cancer is advocated. With an intraoperative diagnosis of AC, or if there is doubt during surgery about the diagnosis, the same treatment as in non-small cell lung cancer is advocated. Frozen-section examination of the margins is mandatory in every case. Long-term survival of typical carcinoids after complete surgical resection is excellent. Chemotherapy and Radiotherapy

In metastatic disease, chemotherapy results are discouraging. Cisplatin, etoposide, cyclophosphamide, doxiribucin, and vincristine have been used. Somatostatin analogs or metaiodobenzylguanidine has been utilized for incapacitating symptoms of the carcinoid syndrome. Octeotride is now registered for the treatment of the carcinoid syndrome and has a symptomatic and biochemical response rate of approximately 60% and 70%, respectively. Pulmonary carcinoids are resistant to irradiation. However, postoperative irradiation has been recommended for patients with atypical carcinoids and lymph nodes involvement, when complete surgical resection has been not possible or mediastinal lymph nodes are involved. Prognosis

Since the frequency of metastatic disease is very low, 5-year survivals after resection depend on the histological aggressiveness of the tumor but can be cured with local treatment options. Almost all typical carcinoid tumors with or without lymph node metastases are cured by adequate surgical treatment, 5-year survival rate being approximately 90%. Although atypical carcinoids spread frequently to regional nodes, their 5-year survival of about 60% is encouraging.

New Perspectives Several large clinical phase II trials are currently investigating the effects of 5-fluorouracil plus interferon a in advanced metastatic carcinoids and thalidomide, the rationale being that thalidomide may stop the growth of these tumors by stopping blood flow to the tumor. Radiofrequency ablation,

using a computed tomography-guided high-frequency electric current to kill tumor cells, is being tested in a phase II trial. Another area of research is the overexpression of somatostatin receptors in carcinoids. Somatostatin receptors can be used for both diagnosis and treatment. Treatment can be achieved by controlling symptoms with somatostatin analogs. Another treatment option could be labeling somatostatin analogs for somatostatin-targeted radiotherapy. However, the most beneficial therapeutic effects will probably come from the understanding of the genetics of these tumors, hoping that in near future, temporary or permanent restoration of gene expression may be feasible. See also: Keratin. Tumors, Malignant: Overview; Bronchogenic Carcinoma; Chemotherapeutic Agents; Hematological Malignancies; Lymphoma; Metastases from Lung Cancer; Rare Tumors; Carcinoma, Lymph Node Involvement.

Further Reading Arrigoni MG, Woolner LB, and Bernatz PE (1972) Atypical carcinoid tumors of the lung. The Journal of Thoracic and Cardiovascular Surgery 64: 413–421. Filosso PL, Rena O, Donati G, et al. (2002) Bronchial carcinoid tumors: surgical management and long-term outcome. The Journal of Thoracic Cardiovascular Surgery 123: 303–309. Ginsberg RJ (2000) Carcinoid tumors. In: Shields TW (ed.) General Thoracic Surgery, 5th edn., pp. 1493–1504. Philadelphia, PA: Lippincott Williams and Wilkins. Hofland LJ and Lamberts SWJ (2001) Somatostatin receptor subtype expression in human tumors. Annals of Oncology 12: S31– S36. Kosmidis PA (2004) Treatment of carcinoid of the lung. Current Opinion in Oncology 16: 146–149. Leotlela PD, Jauch A, Holtgreve-Grez H, and Thakker RV (2003) Genetics of neuroendocrine and carcinoid tumours. EndocrineRelated Cancer 10: 437–450. McMullan DM and Wood DE (2003) Pulmonary carcinoid tumors. Seminars in Thoracic and Cardiovascular Surgery 15: 289–300. Skuladottir H, Hirsch FR, Hansen HH, and Olsen JH (2002) Pulmonary neuroendocrine tumors: incidence and prognosis of histological subtypes. A population-based study in Denmark. Lung Cancer 37: 127–135. Terzi A, Lonardoni A, Feil B, et al. (1989) Bronchoplastic procedures for central carcinoid tumors: clinical experience. European Journal of Cardiothoracic Surgery 3: 288–229. Travis WD, Brambilla E, Muller-Hermelink HK, and Harris CC (eds.) (2004) World Health Organization Classification of Tumors. Pathology and Genetics of Tumors of the Lung, Pleura, Thymus and Heart, pp. 59–66. Lyon: IARC Press. Travis WD, Rush W, Flieder DB, et al. (1998) Survival analysis of 200 pulmonary neuroendocrine tumors with clarification of criteria for atypical carcinoid and its separation from typical carcinoid. The American Journal of Surgical Pathology 22: 934–944.

TUMORS, MALIGNANT / Rare Tumors 361

Rare Tumors

Table 1 Uncommon lung malignancies

P Macchiarini, Heidehaus Hospital, Hannover, Germany

Family

Subtype

Soft tissue sarcomas

Parenchymal and bronchial sarcomas Sarcomas of large vessel origin Sarcomas of small vessel origin

& 2006 Elsevier Ltd. All rights reserved.

Abstract Uncommon primary lung tumors represent less than 1% of all lung neoplasms, can be either benign or malignant, and have a broad spectrum of mesenchymal, epithelial, lymphoreticular, and vascular causes. Most frequently, they are soft-tissue sarcomas, carcinosarcomas, blastomas, or lymphomas, and have clinical features mimicking those of non-small cell lung cancer (NSCLC). The vast majority of the patients are usually symptomatic, most commonly with cough, dyspnea, chest pain, or hemoptysis, but a minority may present with more systemic compliants, such as wheezing, fatigue, weight loss, and fever. Although routine investigations parallel those made when diagnosing an NSCLC, uncommon lung malignancies are difficult to diagnose on the basis of imaging findings alone because such findings are non-specific in the majority of cases. Typically, final diagnosis is most commonly made during surgery. Their overall management should follow the guidelines of patients with NSCLC, with few exceptions. Prognosis is usually worse than in NSCLC.

Carcinosarcoma Pulmonary blastoma/ embryoma Lymphoma Ectopic tissue malignancies

Table 2 Uncommon primary lung soft tissue sarcomas Family

Subtype

Parenchymal and bronchial–endobronchial sarcomas

Fibrosarcoma Leiomyosarcoma Rhabdomyosarcoma Neurogenic sarcoma Chondrosarcoma Osteosarcoma Malignant mesenchymoma Liposarcoma Malignant fibrous hystiocytoma Pulmonary artery sarcomas

Primary Lung Tumors The most common rare primary malignant pulmonary neoplasms are soft-tissue sarcomas, carcinosarcomas, blastomas, lymphomas, and ectopic tissue tumors (Table 1). Soft-Tissue Sarcomas

These tumors have a frequency of 1 case per 500 primary lung cancers, and may be divided into parenchymal and bronchial–endobronchial tumors, and sarcomas of large or small vessels (Table 2). Parenchymal and bronchial or endobronchial tumors include malignant fibrous hystiocytoma, leiomyosarcomas, fibrosarcomas, rhabdomyosarcoma, neurogenic sarcoma, chondrosarcoma, osteosarcoma, malignant mesenchymoma, and liposarcoma. Malignant fibrous hystiocytoma represent the commonest primary malignant lung neoplasm of fibroblastic or fibrohistiocytic lineage. Histologically undistinguishable from its soft-tissue counterpart, it is characterized by an atypical spindle cell proliferation, marked cellular pleiomorphism with atypical giant cells, frequent mitosis, and collagen deposition. Usually observed in patients over 50 years, these tumors are very aggressive locally for which complete surgical excision followed by radiation or chemotherapy may be curative in selected patients. Stage of disease and incomplete resections are of poor prognosis.

Non-Hodgkin’s lymphoma Hodgkin’s disease Malignant melanoma, malignant teratoma, meningioma, choriocarcinoma, ependymoma, plasmacytoma

Sarcomas of large vessel origin Sarcomas of small vessel origin

Angiosarcomas Epithelioid hemangioendothelioma Hemangioendothelioma Hemangiopericytoma

Leiomyosarcomas are the second most frequent primary spindle cell sarcoma of the lung and unrelated to a smoking habitus. They may be well-, moderately, or poorly differentiated and occur more frequently in males who present in the fifth decade of life with 1–15 cm sized parenchymal or endobronchial (40% of cases) masses. Histological features are similar to the uterine and soft-tissue counterparts and differential diagnoses include fibrosarcoma, malignant schwannoma, and sarcomatoid carcinoma. Surgical resection yields 5-year survivals of 40–50% but these tumors are radiation- and chemoresistant. Prognosis is particularly poor for poorly differentiated lesions. Fibrosarcomas are characterized by an atypical spindle cell proliferation arranged in fascicles that adopt a herringbone pattern with evidence of interstitial collagen deposition. They may occur both endobronchially (more frequently in young women) and peripherally (more frequently in elderly) in the

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lung. Patients with bronchial or endobronchial lesions are symptomatic (cough, hemoptysis, and chest pain) and detected clinically at an earlier stage than intraparenchymal lesions. Surgical resection is the treatment of choice but life expectancy is less than 2 years because of recurrent disease. The value of adjuvant radiation or chemotherapy is unproven. Rhabdomyosarcomas are very rare spindle cellproliferating tumors occurring predominantly in children with other congenital abnormalities such as pulmonary (bronchogenic) cysts. They tend to be locally invasive, and despite surgery and adjuvant chemotherapy or radiation, survival is extremely poor and usually less than 2 years from diagnosis. Similarly, liposarcomas are extremely invasive local tumors with dismal prognosis. Possible pathogenetic factors for the development of primary liposarcoma include malignant degeneration of a pulmonary lipoma and pleuropulmonary asbestosis. Malignant mesenchymoma shares the histological components of osteo-, chondro-, and rhabdomyosarcomas. Chondrosarcomas and osteosarcomas represent cartilage- and bone-forming sarcomas, respectively. Less than 15 primary lung cases have been reported in the literature. Chondrosarcomas are usually welldifferentiated lesions developing most frequently in the trachea or major bronchi and tending to embolize into pulmonary vessels while peripheral lesions should be carefully evaluated to rule out parenchymal extension from primary chest wall tumors. Neurogenic intrapulmonary sarcomas usually occur within a diagnosis of neurofibromatosis as asymptomatic, well-defined nodules or as postobstructive changes secondary to endobronchial lesions. Their histological features include increased cellularity and mitotic rate, hyperchromasia, nuclear enlargement with predominant nucleoli, and necrotic areas. Treatment is surgical resection. Sarcomas of large vessels include a variety of intravascular tumors of fibrosarcoma, fibromyxosarcoma, and leiomyosarcoma cell type reported in the relevant literature under the designation of pulmonary artery sarcoma or outflow tract sarcoma. Patients are usually in their middle-age with a history of dyspnea, dull chest pain, cough, and hemoptysis, and the disease slowly progresses to a clinical picture mimicking patients with a chronic postembolic pulmonary hypertension and, ultimately, to right-sided heart failure. Most are diagnosed at autopsy and the survival after diagnosis is less than 1 year in the remaining patients. Although prolonged survival following surgical resection and adjuvant chemotherapy has been reported, the overall prognosis is fatal. Sarcomas of small vessels include angio sarcoma, epitheliod hemangioendothelioma, and

hemangioperycitoma. Usually occurring in young adults, angiosarcomas may present with chest pain, hemoptysis, pleural effusions and intrapulmonary hemorrhage, embolism, and infarction. Tumors usually present in a metastatic phase for which survival is expected to be less than 6 months. Epitheliod hemangioendotheliomas, previously called intravascular bronchioloalveolar tumors, are slow-growing tumors in the lung, occurring more frequently in women under the age of 30 years. They appear as multiple, well-circumscribed and small (usually o3 cm diameter) nodules in both lungs. Occasionally, a pleural effusion may be associated. These lesions are histologically characterized by polypoid tumor masses filling the lumen of alveolar spaces and peritumoral microvessel invasion. Differential diagnoses include metastatic germ tumors, metastatic chondrosarcomas or chondromas, multiple arteriovenous malformation, benign deposits of metastasizing leiomyomas, and malignant lymphoma. Patients are usually asymptomatic, or they may present with mild cough, dyspnea, chest pain, or weight loss. Some tumors behave in a benign fashion since they rarely metastasize and long-term survival has been sporadically reported. Others are highly malignant, especially in patients presenting intrapulmonary multicentric lesions, for which surgical resection is unfeasible, as well as adjuvant chemotherapy or radiation not effective. Most patients ultimately die from end-stage respiratory failure. Hemangiopericytomas usually originate from capillary pericytic cells, the so-called Zimmermann cells, and are most commonly located in soft tissues of the retroperitoneum. Only 5–10% of the hemangiopericitomas are primary lung tumors affecting men and women equally with the majority occurring between the fifth and sixth decade of life. The majority are asymptomatic and present lobulated wellcircumscribed, homogenous densities. The treatment is wide surgical resection, but most tumors relapse within 2 years, and chemotherapy and radiation are almost ineffective. Adverse prognostic factors include a size greater than 8 cm, pleural or bronchial invasion, angiolymphatic invasion, and more than three mitoses per 10 high-power fields. Carcinosarcomas

These tumors comprise 0.3% of all pulmonary neoplasms and are more often found in a proximal bronchus in heavy-smoking and male patients over the age of 50 years. They are composed of identifiable epithelial elements (of non-small cell lines) and a malignant mesenchymal component with features of rhabdomyosarcoma, leiomyosarcoma, chondrosarcoma, or

TUMORS, MALIGNANT / Rare Tumors 363

osteosarcoma. They are slow-growing tumors whose growth is principally endobronchial. The differential diagnosis may be broad because of the various cellular elements and growth patterns but is generally pulmonary blastoma and spindle cell carcinoma. Early lymph nodes and extrathoracic metastasis are often associated. Although surgical resection may be indicated, more than 80% of patients die within the first year after resection. Pulmonary Blastomas

Primary pulmonary blastomas are defined as tumors composed of both malignant mesenchymal and epithelial components that resemble the lung at 3 months gestation. These components make the pulmonary blastomas conceptually similar to the carcinoma from which it is differentiated by the fact that it forms gland-like structures that are lined by epithelial cells with a clear cytoplasm and that these glands are set in a primitive-looking stroma. These tumors have been divided into two classes, the well-differentiated fetal adenocarcinomas and biphasic blastoma. Only 150 cases have been reported in the literature and their origin still remains controversial. Tumors occur slightly more frequently in patients with previous history of smoking, more in women, and at an average age of 35 years at time of diagnosis. Most are asymptomatic. Histologically, the well-differentiated fetal adenocarcinomas are malignant glands composed of pseudostratified columnar epithelium and benign stroma; their mean diameter size is 4.5 cm. The biphasic blastomas have a mean size of 10 cm in diameter and histologically are malignant glands of either adult sarcomatous or embryonic mesenchyme. Differential diagnosis depends on the histological type, but primary adenocarcinoma of the lung is the most common differential diagnosis. Although these tumors are highly malignant, patients with well-differentiated fetal adenocarcinomas have a significant better 5-year survival than those with biphasic tumors. Surgery is the treatment of choice and chemotherapy and/or radiation therapy prolong late survival. Current terminological preferences are such that several formerly used terms, including spindle cell carcinoma, pulmonary blastoma, squamous cell carcinoma with pseudosarcomatous stroma, pseudosarcoma, and carcinosarcoma, are now encompassed by the more generic designation of sarcomatoid carcinoma. Pulmonary Lymphomas

Primary non-Hodgkin’s lymphoma (NHL) and Hodgkin’s disease (HD) comprise approximately 0.5% of all primary lung cancers.

Primary pulmonary NHLs occur in less than 0.5% of all NHLs, predominantly in the sixth decade of life with a male predominance. They are usually well-differentiated B-cell tumors and may occur in any of the deposits of lymphoid elements normally present in the lung, e.g., in the bronchial-associated lymphoid tissue (BALT), in the interstices of the lung parenchyma, and in the intrapulmonary or subpleural lymph nodes. BALT lymphomas (or small cell lymphocytic lymphomas) are proliferations of small lymphocytes with plasmacytoid features. One-third of the patients are asymptomatic, and pulmonary symptoms can occur in HIV-positive and AIDS patients as well. The chest radiographs may be normal or may reveal reticulonodular interstitial markings in the lower lobes; on CT scans the nodules are mostly in a peribronchial distribution. Treatment may be limited to surgery or additional radiation, chemotherapy, or both, if clinically indicated. Prognosis of BALT is excellent, with 5-year survivals of over 75%. Large cell (histiocytic) lymphomas are extremely uncommon, and can occur in patients with AIDS. Patients without AIDS are between the fifth and sixth decade of life with the tumor mostly located in the upper lobes and composed from cells with large nuclei and prominent nucleoli. Hilar nodes are always present. When feasible, surgery is indicated. In patients with widespread disease, chemotherapy is the treatment of choice; these tumors behave more aggressively and have a poorer prognosis. Primary pulmonary HD is thought to arise in lymphoid follicles or perihilar lymph nodes, and to define it as primary, most authors accept the following criteria: histological features of HD; restriction of the disease to the lung with no nodal involvement; and adequate clinical and/or pathological exclusion of disease at distant sites. According to this definition, the Ann Arbor staging system of patients with primary pulmonary HD would be IE (involvement of a single extranodal site) or IIE (localized involvement of an extranodal site and its contiguous lymph-node chain). Patients with primary pulmonary HD tend to be more symptomatic, with B symptoms (fever, night sweats, unexplained weight loss) occurring in more than 50% of cases. Radiographically, most patients show nodular or mass lesions in their lungs, either uni- or bilaterally. The most common histological types are the nodular sclerosing and the mixed cellularity forms of HD. In patients with stages IE or IIE, surgery plus radiation may be curative; the value of chemotherapy remains debatable. In contrast, patients with bilateral disease should be considered as having stage IV HD and therefore should receive chemotherapy. The prognosis of patients with primary pulmonary HD is worse in patients with B

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symptoms, involvement of more than one lobe or bilateral lung disease, or higher clinical stage. Other Primary Lymphoid Pulmonary Lesions

The current histologic criteria for the diagnostic differentiation of the truly benign lymphoid tumors of the lung like lymphocytic interstitial pneumonia (LIP), and pseudolymphoma (PSL) from primary pulmonary lymphocytic lymphomas (PPL) do not always allow for a clear distinction of LIP and PSL from PPL, and therefore LIP and PSL must be viewed as premalignant lesions. LIP is best considered a prelymphomatous state frequently associated with other immune disorders, and most patients are adult women with non-specific pulmonary symptoms, and typical radiographic features of diffuse bilateral lower lobe reticular infiltrates. The course of LIP varies ranging from spontaneous remissions to evolution to lymphomas. True PSLs are reactive lymphoid proliferations that manifest in the lung as one or several masses or as localized infiltrates, usually discovered in asymptomatic patients on routine chest X-ray. Microscopically, it is a lymphoid lesion with well-defined germinal centers separated by polyclonal plasma cells. Resection of PSLs is both diagnostic and curative. Lymphomatoid granulomatosis is a form of malignant lymphoma that primarily involves the lung and has a poor prognosis. It has a slight predominance in men, and the disease can also involve the skin, central nervous system, and sometimes upper respiratory tract. Usually, patients have small pulmonary nodules as atypical lymphoid infiltrates around the pulmonary blood vessels with necrosis. Primary pulmonary intravascular lymphomatosis has been reported in seven patients worldwide and represents a neoplastic proliferation of large atypical lymphoid cells within dilated pulmonary vessels; treatment is chemotherapy and radiation. Ectopic Tissue Neoplasms

These are among the intrapulmonary tumors and include malignant melanomas, teratomas, thymomas, meningiomas, choriocarcinomas, ependymomas, and plasmacytomas. Malignant melanomas or teratomas of the lung are nearly always metastatic in origin and true primary tumors are very rare. Melanocytes are not normally found in the lower respiratory tract. There is some speculation that these tumors may arise from areas of squamous metaplasia in which a certain number of epithelial cells have undergone differentiation toward melanocytes, a phenomenon which also explains the occurrence of pigmented squamous and

basal cell tumors in the skin. Suggestion of the primary occurrence in the lung is based on no previously resected pigmented skin lesion; no ocular tumor removed or evidence of a current or previous primary melanoma in any organ; solitary tumor in the surgical specimen from the lung; tumor morphology consistent with malignant melanoma involving the respiratory epithelium; junctional change, with invasion of intact bronchial mucosa by malignant melanoma cells; and full necropsy demonstrating absence of primary malignant melanoma elsewhere. Usually they are more endobronchial (lower trachea or main bronchus) than intraparenchymal lesions, occur more frequently in the fifth decade of life, and have a poorer long-term outcome than a primary bronchial carcinoma. Intrapulmonary teratomas are composed primarily of epithelial elements with squamous differentiation, similar to a dermoid cyst in other locations. Local recurrence rate following resection is high, and generally the prognosis is poor. Thymic primary lung tumors are defined as thymomas arising in an intrapulmonary location without an associated mediastinal component and derive from thymic epithelial cells; only 23 cases have been described in the literature. They appear as well-circumscribed intrapulmonary nodules measuring up to 10 cm in size, and occur either in the periphery or in the hilum and more frequently into the right lung. Patients are usually asymptomatic and rarely develop any of the diseases associated with thymomas. Histologically, thymomas display a lobulated, biphasic cell population composed of epithelial cells and mature lymphocytes surrounded by a fibrous septum, and the differential diagnosis includes malignant lymphomas, spindle cell tumors, and epithelia malignancies. Primary pulmonary thymomas are slow-growing tumors for which surgical resection can achieve long-term survival; radiation therapy is indicated in incompletely resected tumors. Extracranial meningiomas are rare neoplasms, especially in the lung where they arise from ectopic embryonic rests of arachnoid cells or from pleuripotential-subpleural mesenchyme. Only six cases of pulmonary meningioma have been described. Usually presenting with solitary masses without central nervous system lesions, they are composed of ovoid to spindle cells that form characteristic peritumoral meningioepithelial whorls. The differential diagnoses include metastatic meningiomas or carcinomas, carcinoid tumors, and spindle cell neoplasms. Treatment of choice is surgical resection with good long-term survival. Adjuvant chemotherapy or radiation are unjustifiable.

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Choriocarcinomas are extremely rare primary lung tumors, described in less than 25 cases. The majority of patients are men and present radiologically with solitary and ill-circumscribed lesions and clinically with shortness of breath, lethargy, weight loss, and hemoptysis. Serum levels of human chorionic gonadotropin (HCG) are usually elevated. Before accepting the diagnosis of primary choriocarcinoma, the presence of a testicular primary or a large-cell carcinoma of the lung with HCG-producing multinucleated cells must be ruled out. Histopathologically, these tumors display solid sheets of pleiomorphic cells with a dimorphic population of cytotrophoblasts and multinucleated syncytiotrophoblasts. Positive HCG immunohistochemical staining can be shown. Surgical resection is indicated but close follow-up is necessary to detect increases in serum HCG indicating the presence of micrometastasis for which systemic chemotherapy is necessary. Intrapulmonary ependymoma has been described only twice, occurring as an asymptomatic pulmonary nodule composed of pleomorphic spindle, round, and oval cells and abundant fibrillary, eosinophilic cytoplasm. Its origin and treatment options are unknown. Less than 30 cases of intrapulmonary plasmacytomas have been described. Usually asymptomatic, they occurred in patients of 42 years of age as discrete radiological masses composed histologically by a culture of monomorphic plasma cells in close apposition. They require differential diagnoses with large-cell lymphomas, neuroendocrine lung tumors, or plasma cell granulomas. Complete surgical resection is the treatment of choice although radiation therapy has been curative in selected cases. Since a small percentage of patients may develop systemic multiple myelomas, regular surveillance is recommended.

Tracheobronchial Gland Tumors The most frequent malignant tumors arising from submucosal glands of the trachea include the adenoid cystic and mucoepidermoid carcinomas. Adenoid cystic carcinoma is a rare salivary glandtype malignant neoplasm which arises from the bronchial gland. Although it is histologically indistinguishable from that found in salivary glands, it is indeed rare for a single patient to present with tumors, either synchronous or metachronous, both in the salivary gland and in the trachea. It is composed of a nest of small cells centered by basal membrane type of material. It is an infiltrating low-grade tumor. The speed of growth is low, and the clinical course is usually long. This tumor comprises less than 1% of all lung tumors, and no relationship has been discerned with cigarette-smoking or other known

carcinogenic factors. The airway tumor may occur at any level of the trachea, but seems to be more prevalent in the lower trachea and carina. It occurs much less often in main bronchi and very rarely distal to that. Multiple tumors are extremely rare. This tumor typically occurs in patients aged 40–50 years. The symptoms are usually due to proximal airway obstruction such as shortness of breath, cough, wheeze, and chest pain. Conventional tomography offers an excellent assessment of intraluminal length of gross tumor. CT scan is more accurate for demonstrating extraluminal tumor mass. Surgical resection is the best procedure to control localized lesions. However, because of their tendency to infiltrate along the airways, they are usually incompletely resected, and therefore prone to local recurrence. Despite almost uniformly excellent early response to external irradiation treatment, the tumors usually recur in o12 months if irradiation alone is used for treatment. Therefore, when the cancer is circumscribed, has not metastasized remotely, has not involved an excessive length of trachea, and has not invaded the mediastinum deeply, the best primary treatment is resection. For patients who cannot undergo surgery, palliation may be achieved by various endoscopic procedures including simple blunt resection, diathermy, cryotherapy, or laser photo-resection. Photodynamic therapy, brachytherapy, and endobronchial stents may also be applied. Radiotherapy should be considered in patients with an incomplete resection. Unfortunately, late local recurrence, even 15, 20, or more years after apparent cure by surgery and irradiation, occurs frequently and represents a poor prognostic factor. This tumor usually spreads after local recurrence to lungs, liver, brain, bone, spleen, kidney, and adrenal glands. Patients treated with surgical resection show 5-year survival rate of approximately 85%. Mucoepidermoid carcinoma is a rare tracheobronchial tumor, which affects the trachea less often than the bronchi. Because of its rarity, no large series has ever been published solely on airway mucoepidermoid tumors, and precise demographic data are lacking. It originates from the excretory ducts of the bronchial mucosa, and contains a variable admixture of mucus-secreting cells, squamous cells, and cells of intermediate type. Patients with mucoepidermoid carcinoma have an average age between 35 and 45 years. Fifty per cent of mucoepidermoid carcinomas occur in individuals less than 30 years, and form a significant proportion of pediatric endobronchial tumors. Signs and symptoms are related to the polypoid endobronchial growth of the tumor into the central airways. Most of these tumors arise beyond the carina, usually at

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the main stem bronchi. The extension into pulmonary parenchyma is unusual. Tumors are divided into low- and high-grade types according to their morphological and cytological features. This grading system follows that used for bronchial mucoepidermoid carcinomas and correlates with tumor behavior and disease outcome in tracheal tumors as well. Lowgrade tumors spread to regional lymph nodes by local growth in less than 5% of cases, and distant metastases are very infrequent. However, high-grade tumors involve nodes more frequently and may metastasize to liver, bones, adrenal glands, and brain. Surgical resection represents the treatment of choice for both, with low-grade showing a much better prognosis than high-grade tumors. See also: Granulomatosis: Lymphomatoid Granulomatosis. Oncogenes and Proto-Oncogenes: MYC. Tumors, Malignant: Overview; Lymphoma.

Further Reading Berho M, Moran CA, and Suster S (1995) Malignant mixed epithelial/mesenchymal neoplasms of the lung. Seminars in Diagnostic Pathology 12: 123–129. Canver CC and Voytovich MC (1996) Resection of an unsuspected primary pulmonary choriocarcinoma. Annals of Thoracic Surgery 61: 1249–1251. Cox JE (1997) Pulmonary artery sarcomas: a review of clinical and radiological features. Journal of Computer Assisted Tomography 21: 750–755. Crotty TB, Hooker RP, Swensen SJ, Scheithauer BW, and Myers JL (1992) Primary malignant ependymoma of the lung. Mayo Clinic Proceedings 67: 373–378. Habermann TM, Ryu JH, Inwards DJ, and Kurtin PJ (1999) Primary pulmonary lymphoma. Seminars in Oncology 26: 307– 315. Jennings TA, Axiotis CA, Kress Y, et al. (1990) Primary malignant melanoma of the lower respiratory tract. American Journal of Clinical Pathology 94: 649–655. Kakkar N, Vasishta RK, Banerjee AK, et al. (1996) Primary pulmonary malignant teratoma with yolk sac element associated with hematological neoplasia. Respiration 63: 52–54. Koss MN (1995) Pulmonary blastomas. Cancer Treatment and Research 72: 349–362. Koss MN, Hochholzer L, Moran CA, and Frizzera G (1998) Pulmonary plasmacytomas: a clinicopathologic and immunohistologic study of five cases. Annals of Diagnostic Pathology 2: 1–11. Macchiarini P (2002) Uncommon intrathoracic tumours. In: Souhami RL, Tannok I, Hohenberger P, and Horiot JC (eds.) Oxford Textbook of Oncology, 2nd edn., pp. 2165–2183. Oxford: Oxford University Press. Marchevsky A (1995) Lung tumors derived from ectopic tissues. Seminars in Diagnostic Pathology 12: 172–184. McCluggage WG and Bharucha H (1995) Primary pulmonary tumours of nerve sheath origin. Histopathology 26: 247–254. Suster S (1995) Primary sarcomas of the lung. Seminars in Diagnostic Pathology 12: 140–157. Van Kasteren ME, van der Wurff AA, Palmen FM, Dolman A, and Misere JF (1995) Epithelioid hemangioendothelioma of the

lung: clinical and pathological pitfalls. European Respiratory Journal 8: 1616–1619. Wick MR, Ritter JH, and Humphrey PA (1997) Sarcomatoid carcinomas of the lung: a clinicopathologic review. American Journal of Clinical Pathology 108: 40–53.

Carcinoma, Lymph Node Involvement K M Fong, M Windsor, R V Bowman, and E Duhig, Prince Charles Hospital, The University of Queensland, Brisbane, QLD, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Metastasis to mediastinal or hilar lymph nodes by cancer (adenopathic carcinoma) is among the commonest causes of intrathoracic node enlargement in adults. As it represents relatively advanced malignant disease, accurate diagnosis is important, but in this relatively inaccessible location, also challenging. Malignant node involvement by carcinoma must be distinguished from various primary mediastinal tumors including lymphoma, as well as from a wide variety of nonmalignant causes of lymphadenopathy and benign enlargements of mediastinal structures. A diagnostic approach based on knowledge of anatomy, radiology, test characteristics of clinical diagnostic procedures, and pathology is outlined in this article.

Introduction Malignant involvement of intrathoracic lymph nodes by carcinoma occurs as a result of direct extension or lymphatic seeding from tumors arising in other tissues. ‘Seeding’ is the process whereby malignant cells gain access to native lymphatics at the primary tumor site and are transported with lymph to draining node stations where, under certain conditions, they become resident, escape immunological defense systems, and proliferate. The most common primary tumor-causing secondary intrathoracic lymph node metastasis is bronchogenic carcinoma. Nodal involvement by bronchogenic carcinoma has important prognostic significance, and assessment for its presence or absence is an integral part of clinical management of primary lung cancer as outlined in Tumors, Malignant: Bronchogenic Carcinoma. While malignancy is the commonest cause of intrathoracic lymph node enlargement, the causes of radiologically abnormal mediastinal and hilar contours and structures are myriad, and a systematic approach to differential diagnosis is essential. A diagnostic strategy based upon anatomy, pathology, knowledge of lymphatic drainage routes, and common patterns of tumor invasion and metastasis is presented.

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Anatomy and Radiology Pulmonary Lymphatic Drainage

The hilum is composed of the pulmonary artery and its branches, adjacent airways, pulmonary veins, and lymph nodes. On plain film radiography, the visible component of the normal hilum is predominantly vascular, and normal hilar lymph nodes are usually not readily discernible. Abnormal enlargement of hilar lymph nodes becomes apparent as an altered contour or increase in radiodensity of the hilar shadows. Mediastinum

The mediastinum extends from the sternum to the vertebral column and lies between the pleural reflections in the median sagittal plane. The superior mediastinum lies above, and the lower mediastinum below the uppermost pericardium (approximately at the level of the angle of Louis). The lower mediastinum has three compartments anatomically distinguished by their relation to the pericardium: anterior mediastinum anterior to pericardium, middle mediastinum enclosed by the pericardium, and posterior mediastinum posterior to the pericardium. Considering the structural contents of each of the mediastinal compartments separately provides a useful basis for differential diagnosis of mediastinal abnormalities (Figure 1). The superior mediastinum contains thyroid gland, aortic arch and great vessels, proximal portions of

3

Superior

4 An ter ior

5 6

r

Hilum

2

rio Poste

Anatomical lymphatic drainage systems underpin patterns of lymph node metastasis observed in lung cancer and other cancers. The lungs are well supplied by an abundant network of lymphatics, accounting for the frequency of lymphatic permeation and lymph node metastasis. Superficial (subpleural) and deep (peribronchial) plexuses of lymphatic channels of the lungs join at the hilum and drain into mediastinal lymphatics through tracheobronchial and paratracheal nodes to the right and left bronchomediastinal trunks and then into the subclavian veins. The left bronchomediastinal trunk often drains into the thoracic duct. Because some lymph from the left lower lobe drains across the midline to the right bronchomediastinal trunk, left lower lobe lung cancers sometimes metastasize directly to contralateral mediastinal nodes. Apart from this, lymph drainage takes a predominantly ipsilateral route from distal to proximal through the mediastinum, hence the poorer prognostic significance of contralateral versus ipsilateral lymph node involvement in lung cancer staging.

1

Middle

7 8 9 10 11 12 L1

Figure 1 Mediastinal compartments.

the vagus, phrenic, and recurrent laryngeal nerves, esophagus, trachea, thoracic duct, and lymph nodes. The anterior mediastinum includes fat, ascending aorta, lymph nodes, internal mammary artery and vein, adjacent osseous structures (ribs and sternum), and thymus. The middle mediastinum contains the vagus nerve, recurrent laryngeal nerve, heart, proximal pulmonary arteries and veins, trachea and root of the bronchial tree and associated bronchial lymph nodes, and superior and inferior vena cava. The posterior mediastinum includes the descending aorta, adjacent osseous structures (the spine and ribs), nerves, roots, spinal cord, esophagus, azygous and hemiazygous veins, and some lymph nodes.

Differential Diagnosis of Mediastinal Masses The differential diagnosis of a radiologic abnormality in any particular compartment includes benign enlargement of a normal constituent of that compartment (e.g., retrosternal goiter causing enlargement of the superior mediastinum, bronchogenic cyst presenting as a mass in the middle mediastinum, and hiatus hernia or aneurysm of the descending aorta causing posterior mediastinal abnormality) and primary tumors arising in any normal constituent tissue (e.g., thymoma presenting as an anterior mediastinal mass, proximal bronchogenic carcinoma producing enlargement of the middle mediastinum, and neurofibroma causing posterior mediastinal mass).

368 TUMORS, MALIGNANT / Carcinoma, Lymph Node Involvement Hilar and Mediastinal Lymph Node Enlargement

Lymphoma

As lymph nodes are located in all compartments of the mediastinum and in both hilar regions, the first step in differential diagnosis of intrathoracic lymph node enlargement is to distinguish nodal enlargement from enlargement or mass lesions of the other structures sharing the particular mediastinal compartment. The differential diagnosis therefore includes a wide variety of nonmalignant and malignant conditions and is supported by using the anatomical strategy outlined in Table 1. A clinical diagnosis will be based not only on knowledge of the anatomy and patterns of lymphatic drainage in the thorax, but also on interpretation of radiologic features such as node radiodensity, calcification patterns, and lymph node contour preservation. Additional key considerations are the clinical context, presence and characteristics of concomitant pulmonary abnormalities, and differentiation between conditions causing diffuse versus focal node enlargement.

Primary lymph node tumors (lymphomas) may involve intrathoracic lymph nodes as the only site or as the initial site before extrathoracic disease is manifest. In general, primary thoracic lymphoma presents as a large confluent anterior mediastinal or hilar mass, but can also present as a diffuse lymph node enlargement, intrapulmonary mass, or diffuse lung involvement. Intrathoracic node involvement may occur with lymphomas of any type, but those that typically or predominantly involve intrathoracic nodal groups are primary mediastinal B-cell lymphomas arising in thymus, lymphoblastic (T cell) lymphomas, and Hodgkins lymphomas. Intrathoracic nodes are involved at presentation of non-Hodgkins lymphomas in up to 50% of cases. These can only be distinguished with certainty from adenopathic carcinoma

Nonmalignant Lymphadenopathy

Benign intrathoracic lymphadenopathy typically involves multiple nodal groups. Infections, granulomatous diseases, and silicosis are among the most common causes of nonmalignant bilateral hilar lymphadenopathy or diffuse mediastinal node enlargement (Table 2). However benign node enlargement may also occur in a single node or node region, commonly in the direct path of lymph drainage from a focal lung lesion such as an abscess or area of lung consolidation (see above: Pulmonary Lymphatic Drainage).

Table 2 Nonmalignant causes of mediastinal lymphadenopathy Tuberculosis Histoplasmosis Silicosis Sarcoidosis Coccidioidomycosis Paracoccidioidomycosis Infectious mononucleosis Sinus histocytosis with massive lymphadenopathy (SHML) Cat scratch disease Castleman disease Mycoplasma pneumonia Berylliosis Bronchiectasis and cystic fibrosis HIV infection CMV infection Drugs (e.g., phenytoin)

Table 1 Anatomical differential diagnosis of mediastinal masses Superior

Anterior

Middle

Posterior

Parathyroid adenoma Goitre, thyroid tumors

Parathyroid adenoma Goitre, thyroid tumors Teratoma Lymphoma Bronchogenic cyst Lymph node metastases Ascending aortic aneurysm Thymic tumors Lipoma

Teratoma Lymphoma Bronchogenic cyst Lymph node metastases Cardiac dilatation, aneurysm, tumor

Lymphoma Bronchogenic cyst Lymph node metastases Descending aortic aneurysm

Lymphoma

Pericardial cyst Angiomas Sarcomas Diaphragmatic hernia Hiatus hernia Achalasia Diverticula Gastroenteric cyst Neurogenic tumor Pheochromocytoma

TUMORS, MALIGNANT / Carcinoma, Lymph Node Involvement 369 Table 3 Primary mediastinal tumors Mediastinal compartment

Tumor classification

Superior

Parathyroid Thyroid

Anterior

Thymus

Germ-cell tumors

Mediastinal germ cell rests Embryonal or extraembryonal

Primary mediastinal lymphoma

Anterior/middle/posterior

Mesenchymal

Adipose

Lymphatic Blood vessels Fibroblastic Skeletal Muscle – striated Muscle – smooth Posterior

Neurogenic

Peripheral nerve

Sympathetic ganglia

Adenoma Adenoma Carcinoma Thymoma Thymic carcinoma Lymphoma Germ-cell tumors Thymic neuroendocrine carcinomas Seminoma (Nonseminomatous GCT) Embryonal carcinoma Teratoma Yolk sac tumor Choriocarcinoma Hodgkin’s lymphoma Large B-cell lymphoma Lymphoblastic lymphoma Lipoma Lipoblastoma Liposarcoma Lymphangiomatosis Haemangioma Haemangiopericytoma Fibrosarcoma Malignant fibrous histiocytoma Chondroma Chondrosarcoma osteosarcoma Rhabdomyoma Rhabdomyomsarcoma Leiomyoma Leiomyosarcoma Schwannoma Neurofibroma Malignant nerve-sheath tumors Neuroblastoma Ganglioneuroma Ganglioneuroblastoma

by pathological examination of biopsied or resected node tissue.

leiomyosarcoma, liposarcomas, and rhabdomyosarcoma) (Table 3).

Primary Tumors of Mediastinum

Malignant Lymph Node Involvement by Carcinoma

Other primary tumors of the mediastinum may arise from any of the tissues normally present in the mediastinal compartments, from fetal tissue remnants, or ectopic tissue located in the mediastinum. These include thyroid tumors, parathyroid adenomas, thymic tumors, mesothelioma, carcinoid tumors, a variety of neurogenic tumors (ganglioneuroblastoma, neuroblastoma, malignant peripheral nerve sheath tumors, primitive neurectodermal tumors), germ-cell tumors (teratomas, choriocarcinomas, seminomas, embryonal carcinomas, yolk sac tumors, and mixed germcell tumors), and sarcomas (hemangioendotheliomas,

Carcinoma does not arise primarily in lymph nodes, but involves them by direct extension from an adjacent expanding mass or by lymphatic metastasis. By far the most common tumor causing adenopathic carcinoma of intrathoracic nodes is bronchogenic carcinoma. However, metastases to intrathoracic nodes also arise from melanoma, colon cancer, breast, prostate, renal, thyroid, esophagus, and gonadal cancers. A common clinical problem is characterizing the site of origin of lymph node metastases, in particular differentiating bronchogenic

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carcinoma from other primary sites in patients with no detectable primary tumor. Lymph Node Metastases from Bronchogenic Carcinoma

Both small cell lung carcinoma (SCLC) and nonsmall cell lung carcinoma (NSCLC) including squamous cell carcinoma, adenocarcinoma, and large cell carcinoma and their variants, metastasize to regional and extrathoracic lymph nodes. In an autopsy series of patients with primary lung carcinoma, Spencer found involvement of the tracheobronchial lymph nodes in 70% of patients, intra-abdominal lymph nodes in 20%, cervical lymph nodes in 17%, retroperitoneal lymph nodes in 8%, axillary lymph nodes in 7%, pancreatic lymph nodes in 7%, and supraclavicular lymph node in 7%. Also, mediastinal lymph node involvement has been found in 26% of newly diagnosed lung cancer patients and extrathoracic metastases are found in 49%. Staging for NSCLC utilizes the tumor-node-metastasis (TNM) international staging system, which takes account of tumor size (T stage), regional lymph node involvement (N stage), and presence or absence of distant metastasis (M stage). Involvement of peribronchial and hilar lymph nodes (N1) indicates stage II disease while involvement of mediastinal lymph nodes indicates stage III disease: ipsilateral (N2) ¼ stage IIIa, contralateral (N3) ¼ stage IIIb disease. Clinical Presentation

Most mediastinal node metastases are asymptomatic; however, large node masses may involve adjacent vessels, nerves, airways, or esophagus by compression or invasion producing characteristic clinical presentations. Examples include superior vena caval obstruction from massive right paratracheal node metastases, recurrent laryngeal nerve palsy causing hoarseness resulting from aortopulmonary window node metastases, dysphagia from periesophageal node metastases, hemoptysis from erosion of extraluminal nodes into adjacent bronchus, and major airway compromise from subcarinal adenopathy. Malignant mediastinal lymphadenopathy from bronchogenic carcinoma is frequently associated with supraclavicular, scalene, or cervical node involvement. These nodes are frequently palpable and more accessible to biopsy than the intrathoracic nodes. Diagnosis and Minimally Invasive Staging

Diagnosis of lymph-node metastasis depends ultimately upon pathologic examination of the intrathoracic nodes involved. While radiologic enlargement of

nodes is helpful to show the location of the biopsy, radiologic enlargement per se is not sufficiently sensitive or specific for diagnosis of malignant nodes. Plain chest X-ray indicators of possible malignant mediastinal disease include distortion or enlargement of ‘border-forming’ lymph nodes, increased radiodensity of node regions, or displacement of structures from their normal positions. Classical examples include shelf-like widening of the paratracheal shadow, convexity of the aortopulmonary recess or of the azygous recess, splaying of the subcarinal angle, and increased hilar radiodensity. Much more comprehensive anatomical imaging of the intrathoracic nodes is provided by computerised tomography (CT) scan of thorax. Nodes of 1 cm or greater in their shortest axis are considered enlarged on CT; however, in lung cancer, CT-enlarged nodes by this criteria have only approximately 60% sensitivity and specificity for malignant node metastases. The preoperative accuracy of node staging in bronchogenic carcinoma has been considerably improved by the advent of positron emission tomography (PET) scanning which has sensitivity in excess of 90% and approximately 90% specificity. PET false-positive results occur when nodes are involved with inflammatory and granulomatous disease and do not contain tumor. Malignant tumors also vary in their uptake of fluorodeoxy glucose and PET positivity does not distinguish reliably between lymphoma or other primary mediastinal tumors, secondary malignancy, and bronchogenic carcinoma. Ultimately biopsy or node resection for pathological examination is the only means of certain diagnosis. Figure 2 shows an anatomical map of the mediastinal node stations used in determining N stage for lung cancer. In bronchogenic carcinoma, there is clear prognostic value in accurate mediastinal node staging as shown in Figure 3 which demonstrates the relationship between pathologic N stage and survival. NSCLC patients with completely resected N1 disease have 40% 5-year survival, while those with completely resected N2 disease have 20% 5-year survival. How accurate mediastinal node staging is best achieved remains debatable. Cervical mediastinoscopy provides the opportunity to sample lymph nodes from stations 1, 2 (paratracheal), 3, 4 (tracheobronchial), and 7 (subcarinal), although posterior nodes from station 7 lie behind the trachea and are difficult to reach. Mediastinoscopy is associated with a low incidence of both operative morbidity and false-negative results, and therefore should be performed by a surgeon with specific expertise. Anterior mediastinoscopy provides additional access to station 5 and 6 nodes. In some cases video-assisted thoracoscopy provides superior access and exposure

TUMORS, MALIGNANT / Carcinoma, Lymph Node Involvement 371

1L

1R

Innom V 2R

2L

Ao

4R 4L

PA

11 10 12

11

10

7

12

12

11

11 8 12

12 9 9 Inf. pulm.lig Figure 2 Mediastinal node stations. Used with the permission of the American Joint-Committee on Cancer (AJCC), Chicago, lL. The Original source for this material is the AJCC Cancer Staging Manual, 6th edn. (2002) published by Springer-Verlag, New York, www.springeronline.com.

to nodes on both sides of the mediastinum. Minimally invasive techniques of obtaining adequate pathological samples from mediastinal and hilar nodes are emerging as new staging tools. Ultrasound-guided transbronchial needle aspiration (EBUS) offers noninvasive sampling of stations 2, 3, 4, 7, 10, and 11, while the ultrasound-guided transesophageal (EUS) approach offers access to stations 4L, 5, 7, 8, and 9 which include the otherwise inaccessible posterior and inferior mediastinal nodes. Both procedures are able to provide histological confirmation of nodes identified as positive by CT or PET scan. At present, training and experience with the ultrasound-guided techniques are limited, performance characteristics are still being evaluated, and the potential for false negatives may be higher with needle sampling as compared with the amount

of lymph node tissue that can be removed at mediastinoscopy. Nevertheless, the combined (EBUS plus EUS) procedure presents as a potential successor to cervical mediastinoscopy in pre-operative staging. Percutaneous needle biopsy is also a potentially useful technique which can access most mediastinal node stations but with some risk of pneumothorax. Surgical Lymph-Node Staging

Lymph-node sampling in the course of resections of lung or mediastinal masses is important for prognosis and forms the basis of pathological staging in lung cancer. Nodes involved by carcinoma may be either enlarged and therefore potentially detectable by CT scan, or normal in size, often contained entirely within the lymph node capsule (intranodal metastasis

372 TUMORS, MALIGNANT / Carcinoma, Lymph Node Involvement Surgical-pathologic N

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Figure 3 Regional lymph node classification for lung cancer staging. Reproduced from Mountain CF and Dresler CM (1997) Chest 111(6): 1718–1723, with permission.

or micrometastasis). Micrometastases are not macroscopically visible and reliable detection may be difficult even with a systematic dissection by the pathologist unless serial sectioning or immunohistochemistry is also undertaken. Skip metastasis, in which mediastinal nodes are positive but intraparenchymal and hilar nodes in the path of lymphatic drainage from the primary tumor are not involved, occur in 20–30% of resected lung cancers. This may sometimes result from vascular anastomoses or obliteration of lymphatic vessels by inflammation or previous irradiation. There is considerable variation in outcomes for patients with completely resected pathological N2 disease, and some evidence to suggest that patients with micrometastatic deposits and skip metastases may have a better prognosis than other N2 cases. To reconcile these factors and evaluate the necessity for complete mediastinal lymph-node dissection in every surgical case, sentinel node-mapping using intraoperative 99mTc sulfur colloid intratumoral injection with detection of radioactivity at node stations has been evaluated recently. The idea is that provided the ‘correct’ node is examined by identifying the node station preferentially draining the tumor, dissection of this sentinel node station rather than all node stations makes closer pathological examination for micrometastatic involvement by serial sectioning and immunohistochemistry a more practical undertaking. While it is possible to identify a sentinel node in most

cases, and the strategy does enable detection of micrometastases that may otherwise be missed, sentinel node-mapping is not standard practice at present. New approaches to intra-operative node staging based upon detection of molecular properties of tumor metastases to hilar and mediastinal nodes are also under evaluation. Pathology

Pathological examination of a sampled or resected node will establish whether the pathology is malignant or benign, distinguish between lymphoma and metastatic carcinoma, determine the extent of capsular invasion and effacement of normal node structure, establish the histopathologic subtype of metastatic carcinoma and degree of differentiation, and finally ascertain the tissue of origin if possible. To assist in achieving all these goals, a variety of immunohistochemical methods may be employed. In intrathoracic node evaluation, cytokeratins and thyroid transcription factor 1 (TTF1) may be particularly useful in determining the origin of adenocarcinomas metastatic to nodes, but specific stains for melanoma, germ-cell tumor markers, and others are also valuable in arriving at a final pathological diagnosis. An example of TTF1 positive lymph-node metastasis in a patient without a clearly identified primary site is shown in Figure 4 indicating a high likelihood that the primary adenocarcinoma is of lung origin.

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offers useful palliation for many tumors that are radiation-sensitive. Airway or vascular stents are additional valuable adjunctive techniques for symptom palliation in this setting. See also: Tumors, Malignant: Overview; Bronchogenic Carcinoma; Chemotherapeutic Agents; Hematological Malignancies; Lymphoma; Metastases from Lung Cancer; Rare Tumors.

Further Reading

Figure 4 Lymph-node metastasis showing nuclear TTF1 immunoreactivity consistent with primary lung adenocarcinoma origin.

Management

There is conflicting evidence from three small randomized trials concerning the survival benefit of complete lymph-node dissection versus lymph-node sampling for patients with NSCLC. To resolve this issue, a large prospective randomized study of over 1000 patients (ACOSOG Z0030) was recently completed and results are now awaited. Management of mediastinal lymph-node metastasis is otherwise directed by the extent of nodal involvement, presence or absence of extrathoracic disease, and therapeutic susceptibilities of the particular tumor of origin. Ipsilateral mediastinal node disease is an important determinant of outcome in NSCLC and disease of this stage is, at present, optimally managed by a combined modality therapeutic approach, while contralateral node involvement portends a poor outcome for which surgery is not beneficial. (see Tumors, Malignant: Bronchogenic Carcinoma). There is no clear evidence that surgical resection of mediastinal lymph nodes involved in cancers of other primary sites is beneficial. For symptoms resulting from expanding malignant involvement of lymph nodes compromising vital structures such as major airways or vascular structures, radiation therapy

Type I and II Epithelial Cells

Balch CM, et al. (2002) Melanoma of the skin, In: Greene FL, Page DL, Fleming ID, et al. (eds.) AJCC Cancer Staging Manual, 6th edn., pp. 209–220. New York: Springer. Beckles MA, Spiro SG, Colice GL, and Rudd RM (2003) Initial evaluation of the patient with lung cancer: symptoms, signs, laboratory tests, and paraneoplastic syndromes (review). Chest 123(1 supplement): 97S–104S. Keller SM (2002) Complete mediastinal node dissection – does it make a difference? Lung Cancer 36: 7–8. Macchiarini P and Ostertag H (2004) Uncommon primary mediastinal tumours. Lancet Oncology 5: 107–118. MacDonald SL and Hansell DM (2003) Staging of non-small cell lung cancer: imaging of intrathoracic disease (review). European Journal of Radiology 45(1): 18–30. Mountain CF and Dresler CM (1997) Regional lymph node classification for lung cancer staging. Chest 111: 1718–1723. Rintoul RC, Schwarski KM, Murchison JT, et al. (2005) Endobronchial and endoscopic ultrasound-guided real-time fine needle aspiration for mediastinal staging. European Respiratory Journal 25: 416–421. Sharma A, Fidias P, Hayman LA, Loomis SL, Taber KH, and Aquino SL (2004) Patterns of lymphadenopathy in thoracic malignancies (review). Radiographics 24(2): 419–434. Silvestri GA, Tanoue LT, Margolis ML, Barker J, Detterbeck F, and American College of Chest Physicians (2003) The noninvasive staging of non-small cell lung cancer: the guidelines. Chest 123(1 supplement): 147S–156S. The Canadian Lung Oncology Group (2001) Investigating extrathoracic metastatic disease in patients with apparently operable lung cancer. Annals of Thoracic Surgery 71(2): 425–433; discussion 433–434. Toloza EM, Harpole L, Detterbeck F, and McCrory DC (2003) Invasive staging of non-small cell lung cancer: a review of the current evidence (review). Chest 123(1 supplement): 157S– 166S. Toloza EM, Harpole L, and McCrory DC (2003) Noninvasive staging of non-small cell lung cancer: a review of the current evidence (review). Chest 123(1 supplement): 137S–146S.

see Epithelial Cells: Type I Cells; Type II Cells.

U UPPER AIRWAY OBSTRUCTION S Kumar, Ipswich Hospital, Ipswich, UK R Salib, Southampton General Hospital, Southampton, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The upper airway extends from the nasal passages to the lower end of the cricoid cartilage (subglottis). The causes of upper airway obstruction can be classified in many ways, including pathological and anatomical. Symptoms and signs vary according to the site of obstruction and whether it is acute or chronic in nature. Common symptoms include nasal blockage, snoring, shortness of breath, coughing, and choking. The signs are dependent on the cause and can include stridor, abnormal voice, confusion, restlessness, cyanosis, use of accessory muscles, and suprasternal recession. Common causes include foreign bodies, inflammatory/infective conditions, and neoplastic disease. Diagnosis of the cause of the obstruction requires an accurate history and examination, including visualization of the upper airway from the nasal vestibules to the subglottic larynx using flexible fiber-optic endoscopy. In the acute situation, arterial blood gases may help to quantify the extent of the hypoxia and any associated acid–base imbalance. Radiological investigations, including plain neck/chest X-rays and computerized tomographic scans, can be useful in selected cases. Therapeutic options depend on the etiological agent and the acuteness of onset. The priority in acute obstruction should always be securing of the airway either medically or surgically followed by treatment of the underlying cause. In chronic cases, the emphasis is more on investigating and treating the underlying cause. Upper airway obstruction is a serious and potentially lifethreatening condition and as such requires prompt assessment and management.

Upper airway obstruction can vary from a simple nasal obstruction to life-threatening stridor and respiratory distress. The causes are varied, and most are treatable. This article considers the more common and important causes.

Nasal and Nasopharyngeal Congenital

Bilateral congenital choanal atresia It is one of the important causes of acute airway obstructions because neonates are near-obligate nasal breathers. The neonate presents with active accessory muscles of respiration and flaring of the ala nasi. There is pallor

and cyanosis, and after a few quick breaths the infant cries. The diagnosis is confirmed by nasoendoscopic examination. Computed tomography (CT) scanning is required to delineate the nature and thickness of obstruction. The first priority is inserting and maintaining an oral airway. Treatment is by surgical excision of either the membranous or the bony plate in the posterior choana by the transnasal or transpalatal approach. Encephalocele It is the herniation of meninges and brain outside the cranial vault. The internal or basal encephalocele is a mass of herniated neural tissue presenting as a unilateral nasal obstruction. Examination should reveal a pulsatile mass, which may be transilluminating. The Furstenberg test is positive. The key to diagnosis is a high index of suspicion. Magnetic resonance imaging (MRI)/CT scanning will help with the diagnosis and assessment of the bony defect. Treatment involves a combined neurosurgical and otolaryngologic approach. Gliomas Gliomas present with similar symptoms but do not possess an intracranial connection. They are connected via a fibrous stalk to the intracranial space. Diagnosis and treatment are similar to those for encephalocele. Inflammatory Conditions of the Nose

Rhinitis is an inflammation of the nose and nasal mucosa. It can be allergic, nonallergic, infective, granulomatous, and idiopathic. Allergic and nonallergic rhinitis Nasal obstruction is a common manifestation of both allergic and nonallergic rhinitis. Allergic rhinitis is one of the most common conditions affecting the nose. It can be perennial, seasonal, or occupational. The usual symptoms are runny nose, itchy nose, sneezing, nasal obstruction, and partial loss of sense of smell. Diagnosis is usually by history and clinical examination. Skin prick test and specific radioallergosorbent test may be required to rule out specific allergies. Treatment includes avoidance of known allergens, steroidal nasal sprays, mast cell stabilizers, antihistamines, and immunotherapy. In nonallergic rhinitis, the

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symptoms are similar but are usually of late onset and there is no family history. Seasonal variation is not seen, and the mucosa is often erythematous and inflamed. The nonallergic rhinitis with eosinophilia syndrome is characterized by perennial or seasonal nasal congestion, sneezing, or watery rhinorrhea. The trigger may include irritants such as perfumes or smoke. These patients have normal IgE levels and negative skin tests to allergens. Treatment is similar to that for allergic rhinitis, but immunotherapy is not helpful. Infective rhinitis Infections tend to produce an inflammatory response with increased mucosal secretions that lead to crusting and nasal obstruction. Viral rhinitis afflicts all age groups and tends to be characterized by clear watery rhinorrhea associated with sneezing and nasal obstruction. The rhinovirus, respiratory syncytial virus, parainfluenza, influenza, and adenoviruses are the common viruses. Treatment is symptomatic. Analgesics, antihistamines, and decongestants are frequently employed. Bacterial rhinitis is caused by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and, less frequently, group A Streptococcus species. Nasal obstruction, mucopurulent nasal discharge, facial pain, and vestibular crusting characterize bacterial rhinitis. Broad-spectrum antibiotics with analgesics, decongestants, and humidification are usually all that is required. Fungal infection causing airway obstruction is usually allergic fungal rhinosinusitis seen in atopic immunocompetent young adults. There are associated nasal polyps and asthma. Because of the potential for orbital and intracranial complications, treatment is instituted rapidly. Polypectomy and drainage of the sinuses are required. Antifungal therapy (itraconazole) and steroids are helpful. Nasal polyp Nasal polyposis is a chronic inflammatory disease of the upper airway characterized histologically by the infiltration of inflammatory cells such as eosinophils or neutrophils. The common presentations are nasal obstruction, rhinorrhea, sneezing, and anosmia. Ethmoidal polyps are usually seen in middle-aged patients. They are multiple, bilateral, smooth, pale in color, and insensitive to touch. Antrochoanal polyps are seen in younger patients and arise from the maxillary sinuses. Polyps are also seen in up to two-thirds of patients with cystic fibrosis. The polyps are probably a consequence of chronic inflammation; however, their specific etiology is unclear. Conservative treatment includes topical nasal steroids, decongestants, and antihistamines. In failed conservative treatment, simple nasal polypectomy or functional endoscopic sinus surgery with polypectomy combined with

medical therapy have successfully delayed the time to recurrence, decreased the need for surgery, and improved the underlying pulmonary status. Granulomatous Diseases

Rhinoscleroma It is a chronic granulomatous disease caused by Klebsiella rhinoscleromatis and involves the nose and other parts of the respiratory tract. It is endemic in Eastern Europe and Central America. The symptoms change as the disease progresses from the catarrhal stage to the atrophic stage and then to the granulomatous stage, where the patients have multiple granulomatous nodules leading to obstruction, which ends in the final fibrotic stage. The diagnosis is based on appropriate bacterial cultures and histologic demonstration of granulomas, fibrosis, and eosin staining Russell bodies. Streptomycin and tetracycline are effective antibiotics. Surgical resection of the scar tissue is required to relieve nasal obstruction. Wegener’s granulomatosis and midline nonhealing granulomatous These are a group of disorders of unknown etiology. They frequently present as an ulcerative, obstructive, or destructive lesion of the nasal cavity and paranasal sinuses. Diagnosis is based on biopsy of the involved tissues with demonstration of the vasculitis of the small vessels. Other histologic findings include necrotizing or non-necrotizing epitheloid granulomas, fibrinoid necrosis, and loss of elastic tissue. The midline granuloma is a more rapidly progressing form of disease and is sometimes referred to as atypical lymphoma, which shows some response to radiation. Wegener’s granuloma has been treated successfully with steroids and cyclophosphamide. Sarcoidosis It is an idiopathic granulomatous disease that can cause nasal obstruction. Typically, papules and nodules are seen in the nasal vestibule, with mucosal thickening in the anterior portion of the nasal cavity. The histologic findings include nodular granulomatous infiltrate composed of histiocytes, inflammatory multinucleated giant cells, and focal necrosis that are usually noncaseating. Systemic and intralesional corticosteroids are used for treatment. Tuberculosis It may occur primarily in the nose or as a manifestation of the involvement of the respiratory tract. The anterior half of the nose is commonly involved. Nasal obstruction, nasal discharge, and pain along with erythematous mucosal nodules are the usual symptoms. Cultures can demonstrate the causative organisms. Histologically, the tissue

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shows multiple epitheloid cells along with Langerhans giant cells, acid-fast bacilli, and regions of caseating necrosis. Antituberculous therapy with surgical management of any nasal scarring to relieve pain is the treatment. Nasal Trauma

The common causes are assaults, road traffic accidents, and sports injuries. The trauma may vary from simple nasal bone fracture to a combination of soft tissue injury, including septal hematoma, cerebrospinal fluid (CSF) leak, and maxillofacial fracture. Nose bleed, deformity, and nasal obstructions are the usual symptoms. The history and duration of the injury are noted. Tenderness, hematoma, and swelling can make assessment difficult. Simple fractures of the nose are reduced once the swelling has settled by manipulation of the nasal bones either digitally or by Walsham’s forceps. Complex soft tissue injuries and maxillofacial fractures require CT scanning. Maxillofacial fractures require open reduction. Septal hematoma should be managed by incision and drainage with quilting sutures to obliterate dead space. Small CSF leaks need no active management and stop spontaneously, but larger leaks may require surgical repair with temporalis fascia, fascia lata, or a mucosal flap from the nasal septum. Benign and Malignant Tumors of the Nose and Nasopharynx

Neoplasms can arise from contiguous areas of the nasal skin, vestibule, nasal cavity, nasopharynx, and oropharynx or from the paranasal sinuses. These tumors obstruct the nasal passage depending on the origin of the tumor and the direction of spread. Other symptoms are epistaxis, altered or loss of sense of smell, pain, deformity, and altered sensation due to involvement of the nerves (e.g., juvenile nasopharyngeal angiofibroma involving the maxillary division of the fifth nerve). Otalgia and hearing loss due to involvement of the eustachian tube are ominous signs of an underlying malignant tumor. A thorough history and complete examination of the nose, nasopharynx, and paranasal sinuses is necessary. Endoscopic examination using flexible fiber-optic or rigid endoscope may be required. CT or MRI of the nose and paranasal sinuses help the diagnosis and also provide additional information about the involvement of adjacent structures, distant spread, and vascularity. Angiograms may be necessary if a vascular lesion is suspected. Biopsy and histological diagnosis is necessary in most cases. The differential diagnoses of the causes of nose and nasopharyngeal tumors are detailed in Table 1. Surgery is the main modality of

Table 1 Causes of nasal and nasopharyngeal airway obstruction Congenital Bilateral posterior choanal atresia Maxillary hypoplasia (Crouzon’s syndrome) Albright syndrome Chordoma Glioma Hamartoma Meningocele, encephalocele Deviated nasal septum Granulomatous disease Sarcoidosis Wegener’s granulomatosis Midline granuloma Tuberculosis Rhinoscleroma Leprosy (lepromatous) Syphilis Trauma Septal hematoma Nasal fracture Frontoethmoidal fracture Benign tumors Epithelial Papilloma Mesodermal Nasopharyngeal angiofibroma Teratologic Dermoid Inflammatory Allergic Seasonal rhinitis Perennial rhinitis Occupational Nonallergic Nonallergic rhinitis with eosinophilia syndrome Infection Bacterial Viral Fungal Nasal polyps Ethmoidal polyp Antrochoanal Polyp Other polyps (aspirin sensitivity, cystic fibrosis) Others Atrophic rhinitis Vasomotor rhinitis Rhinitis medicamentosa Rhinitis sicca Intermediate tumors Inverted papilloma Malignant tumors Carcinoma Basal cell carcinoma Squamous cell Carcinoma Adenocarcinoma Nasopharyngeal Melanoma Sarcoma Rhabdomyosarcoma Fibrosarcoma Lymphoma Non-Hodgkin’s and Hodgkin’s disease Metastatic Renal cell carcinoma

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treatment for most of tumors. The approaches to the anterior and middle nasal cavities are unilateral and bilateral alotomy; lateral and total rhinotomy; and other approaches to the paranasal sinuses, such as external ethmoidectomy, medial maxillectomy, radical maxillectomy, and craniofacial resection. The approaches to the posterior nasal cavity are transpalatal, mandibular swing, transantral, transantroethmoid, and lefort osteotomy.

Laryngeal, Oropharyngeal, Hypopharyngeal, and Tracheal Congenital

Laryngomalacia Weakness and flabbiness of the laryngeal structure cause the collapse of the larynx. The aryepilottic folds, arytenoids, and epiglottis cause narrowing and collapse of the airway, giving rise to stridor. The stridor is usually inspiratory and high-pitched. It is noticed a few days after birth. The stridor is intermittent and appears only when the child is feeding and crying, and it is pronounced when sleeping, especially in the prone position. In most cases, laryngomalacia is asymptomatic, requiring no treatment. The condition can be diagnosed by direct examination of the laryngeal structures. A tubular omega-shaped epiglottis with a tendency to roll backwards, a flaccid medially prolapsing aryepilottic folds, and a prominent, elongated arytenoid cartilage usually indicate the diagnosis. Treatment is usually reassurance, but in rare cases in which the laryngomalacia is severe enough to cause feeding difficulties, intermittent respiratory distress, and delayed growth, excision of the redundant mucosa or laser division of the aryepiglottic fold have been suggested. Tracheostomy has been used very rarely to manage acute respiratory distress due to laryngomalacia (Table 2). Laryngeal atresia It is a rare lesion thought to arise from premature arrest of normal epithelial ingrowth into the larynx during the 14th to 17th stages of embryonic development. The child makes strong respiratory effort but cannot breathe or cry and does not have any stridor. Most children die unless an emergency tracheostomy is performed. The diagnosis is usually made at autopsy, but with the increasing use of ultrasonography, diagnosis has been made by noting enlarged edematous lungs, compressed fetal heart, severe ascites, and fetal hydrops. Treatment is usually tracheostomy. Laryngeal web It is a partial failure in canalization of the epithelial lamina between the vestibulotracheal

Table 2 Causes of laryngeal, oropharyngeal, hypopharyngeal, and tracheal airway obstruction Congenital Laryngomalacia Laryngeal atresia Saccular cyst Laryngocele Laryngeal web Vocal cord paralysis Subglottic stenosis Subglottic hemangioma Laryngotracheal cleft Tracheal agenesis Tracheal stenosis Tracheomalacia Vascular compression Benign tumors Laryngeal papilloma Recurrent respiratory papillomatosis Vocal cord polyp Malignant tumors Laryngeal carcinoma Hypopharyngeal carcinoma Oropharyngeal carcinoma Upper esophageal Thyroid carcinoma Infective and inflammatory Retropharygeal abscess Ludwig’s angina Angioedema of the soft palate and tongue Laryngeal edema Acute epiglottitis Acute laryngotracheobronchitis Acute laryngitis Diphtheria Trauma Thermal and chemical External blunt trauma Iatrogenic Foreign bodies in the larynx

canal above and the pharyngotracheal canal below, similar to the process in laryngeal atresia, in the fifth week of intrauterine life. The web can extend from the supraglottis to the subglottis but usually extends to the glottis. The web bands can vary from thin and membranous to thick and fibrotic. The presentation depends on the extent of the web, but it usually presents as hoarseness of voice/cry, stridor, and even respiratory distress. The diagnosis is usually by direct laryngoscopy. Treatment depends on the type and thickness of the web and varies from endoscopic excision in thin and membranous bands to tracheostomy with placement of a keel through a laryngofissure. Saccular cyst A saccular cyst is a mucus-filled dilatation of the mucus duct in the laryngeal/saccular appendage. The cysts cause bulging and distortion of

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the aryepiglottic fold, the false cords, and the laryngeal ventricle, depending on their size. The presentation can vary from no symptoms to stridor, which is primarily inspiratory. Dyspnea, apnea, and cyanosis may also be noted. CT scans of the neck provide useful information, but the definitive diagnosis is by direct laryngoscopy. Treatment is aimed at endolaryngeal excision, but if the cysts are large, marsupialization is done; very rarely, tracheostomy may be required if the respiratory distress becomes severe. Laryngocele Laryngocele is an air-filled dilatation of the ventricular sinus of Morgagni. Laryngoceles can be external if they extend beyond the limits of the thyroid cartilage, piercing the thyroid membrane. They can be asymptomatic but can also present as intermittent hoarseness of voice or respiratory distress, which worsens with straining or crying. Plain X-rays of the neck may demonstrate an air-filled sac, but the definitive diagnosis is made by direct laryngoscopy. No treatment is required in most cases, but symptomatic patients need marsupialization or deroofing of the laryngocele. Vocal cord paralysis Pediatric causes Pediatric causes can be congenital or acquired. Congenital lesions are frequently associated with hydrocephalus, meningomyelocele, Arnold–Chiari malformation, encephalocele, and cerebral agenesis. Acquired causes may be traumatic, most frequently due to stretching of the recurrent laryngeal nerve during delivery, or iatrogenic during surgery for management of bronchogenic cysts, tracheoesophageal fistula, or for ligation of patent ductus arteriosus. Infections such as encephalitis, poliomyelitis, diphtheria, tetanus, and syphilis, especially in Third World countries, can cause vocal cord paralysis. Tumors of the brain and spinal cord, although rare, can also cause paralysis. In meningomyelocele, Arnold–Chiari malformation, and hydrocephalus, decompression of the vagus nerve within 24 hours of birth has been shown to preserve the vocal cord function and recovery has been seen within 2 weeks. Airway distress and stridor are seen in bilateral abductor paralysis but can also be seen in unilateral recurrent laryngeal nerve paralysis when associated with respiratory exertion. The diagnosis is usually made by flexible laryngoscopy or by direct laryngoscopy. The airway is usually secured by intubation, followed by detailed assessment of the patient to ascertain the underlying cause of vocal cord paralysis. If no underlying cause is found and no recovery in vocal cord function is seen, tracheostomy is usually done to relieve the airway distress.

Adult causes Tumors (carcinoma of the nasopharynx, thyroid, bronchus, esophagus, lymphoma, and mediastenal metastasis) account for 25% of the causes. Surgical trauma and iatrogenic causes (thyroid, parathyroid, esophageal, pharyngeal pouch, left lung surgery, and carotid endarterectomy) account for 20%; idiopathic causes, mainly viral (herpes virus, Epstein–Barr virus, cytomegalovirus, and human immunodeficiency virus), account for 15%; neurological causes (multiple sclerosis, amyotrophic lateral sclerosis, and syringomyelia) account for 15%; and other causes, such as cardiovascular (aortic aneurysms), thrombosis of the subclavian vein, and various collagen vascular diseases, account for 5–10%. Symptoms include hoarseness of voice, difficulty breathing, stridor, aphonia, and aspiration, depending on the type and extent of paralysis. A detailed history must be obtained, and a complete head and neck and neurological examination, including cranial nerves, must be done. Laryngoscopy to evaluate the vocal cord mobility, position of the vocal process, symmetry of vocal cords, atrophy of vocal ligaments, glottic closure, flaccidity, and level of the match between the vocal process must be done. Investigations are recommended based on history and clinical findings rather than undertaking all possible investigations, which have low diagnostic yields. CT scan with contrast medium from the skull base to the aortic arch is recommended when there is no detectable lesion. MRI scan with gadolinium enhancement is recommended if vagal or other neuropathies are suspected or for neurodegenerative disorders, such as multiple sclerosis and Guillain–Barre syndrome. In patients with associated dysphagia, a rigid esophagoscopy or a barium swallow will give additional information. Treatment depends on the type of vocal cord paralysis, the underlying cause, and the impact of the disability on the quality of life of the patient. Speech therapy either alone or postoperatively is helpful. Phonosurgical techniques are the preferred treatment. The medialization techniques of vocal cords are injection of teflon, fat, or collagen; thyroplasty type I; arytenoid adduction; and reinnervation (ansa hypoglossi: recurrent laryngeal nerve). Commonly employed techniques for lateralization are laser cordectomy/arytenoidectomy, cordopexy with or without arytenoid removal, Woodman’s operation, and reinnervation. Subglottic stenosis Stenosis can be congenital or acquired. Stenosis in which there is no history of trauma or endotracheal intubation is termed congenital. Acquired causes may be blunt trauma, high tracheostomy, injury from prolonged intubation, and chemical or thermal injuries. Prolonged endotracheal

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intubation is the most common cause of acquired subglottic stenosis. A subglottic stenosis is considered to be present if the lumen in a full-term neonate is 3.5 mm in diameter or less. Subglottic stenosis varies in severity depending on the extent of narrowing, length of narrowing, and the involvement of the cartilage and soft tissues. In mild subglottic stenosis, patients may be asymptomatic until they develop an upper respiratory tract infection or the subglottis is traumatized by injury. The main symptoms relate to airway, voice, and feeding. In severe stenosis, stridor is the primary sign and is biphasic. Diagnosis is assisted by soft tissue radiography, but direct laryngoscopy and assessment of the thickness, the length of the stenosis, and involvement of the glottis using a Hopkins rod lens is the most important tool. Treatment is individualized. Mild stenosis requires expectant treatment, and some patients do grow out of the problem. In more severe cases, tracheostomy may be needed during early infancy, with normal growth allowing decannulation in 3–5 years. Others surgical techniques, such as anterior cricoid split, dilatation, endoscopic excision, laser excision, and open procedures with cartilage grafts, have been used to treat varying severity of the stenosis with significant outcome. Subglottic hemangioma Subglottic hemangiomas are hamartomas arising in the subglottis associated with other cutaneous hemangiomas. They usually present in early infancy, and most lesions manifest by 6 months of age. Symptoms can vary from dysphonia to stridor or respiratory distress. Radiographic examination will help with the diagnosis, but direct endoscopic examination of the larynx revealing a sessile, compressible, pink, red, or bluish-colored lesion is characteristic. Attempts to rule out possible hemangiomas in the mediastenum, abdomen, skeleton, and eyes should be made. Treatment may require immediate airway control either by intubation or by tracheostomy. A wide range of treatments, such as intralesional injection of corticosteroids, sclerosing agents, cryosurgery, external beam irradiation, gold seed irradiation, and laser excision, have been advocated. Laser excision is probably the treatment of choice in most centers. Laryngotracheal cleft Laryngotracheal cleft is an open communication between the laryngotracheal airway and the esophageal lumen. It is due to developmental failure of the tracheoesophageal septum during early fetal life. These conditions can be associated with other congenital anomalies, so attempts should be made to perform a complete examination. The patient usually presents with persistent aspiration,

but this can be accompanied by stridor, respiratory distress, and a toneless cry. The diagnosis is usually based on history and radiological examination, especially MRI, but the definitive diagnosis is by laryngoscopy and pharyngoesophagoscopy, on which the extent and the severity of the defect are visualized and assessed. Various surgical approaches have been suggested either by an external approach or by endoscopic repair of these clefts. Tracheal agenesis Complete or partial agenesis is incompatible with life. Agenesis of one main bronchus and its associated lung is survivable, but they are usually associated with other life-threatening congenital abnormalities. Tracheomalacia It is caused by weakness in tracheal wall due to softening of the supporting cartilage and the abnormal widening of the posterior wall. Patients usually present with biphasic stridor, wheezing, and coughing that can be harsh and barking in quality, and they may have dysphonia. Tracheomalacia has been divided into primary and secondary types. The primary types usually occur in premature infants and the secondary types due to an external compression such as innominate artery compression, vascular rings, cervical tumors, or bronchogenic cysts. The diagnosis is usually by history and endoscopic examination. Careful assessment must be made during endoscopic examination because tracheoesophageal fistulas have been associated with this condition, which can be missed. Most of the conditions resolve spontaneously, but in severe cases a tracheostomy may be necessary, with insertion of a long tube to cover the length of the tracheomalacia. Anterior tracheal wall suspension has been described. Tracheal stenosis Tracheal stenosis can be membranous webs (partial or complete), segmental stenosis of the trachea, or complete organ stenosis. Endotracheal intubation injuries, relapsing polychondritis, amyloidosis, and connective tissue disorders such as Hurler’s syndrome have been described to cause narrowing and stenosis. The symptoms include stridor, which is usually biphasic, but it can also present as acute airway distress. The webs can be treated by surgical excision. Tracheal stenosis has been treated by tracheoplasty using a costochondral cartilage graft. Infective and Inflammatory

Acute retropharyngeal abscess It is the collection of pus in the retropharyngeal space. Acute retropharyngeal abscess is seen in young children up to the

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age of 5 years due to suppuration in the retropharyngeal group of lymph nodes from an infection spreading from the upper respiratory tract or a foreign body in the oropharynx. The chronic abscess is due to tuberculosis of the cervical vertebra. The acute illness presents with the child feeling very unwell, associated with dysphagia and drooling of saliva, and the airway can be obstructed because of the abscess and also because of the development of laryngeal edema. The child holds the neck rigid and there is associated cervical lymphadenopathy. There is limitation of neck movements, torticollis, tenderness, and bulging of the posterior pharyngeal wall. X-rays of the lateral neck may show dilatation of the retropharyngeal space and sometimes fluid level. Treatment is by intravenous antibiotics and incision and drainage of the abscess. Tracheostomy is necessary if there is difficulty in securing a definitive airway. Diptheria It is a condition caused by Corynebacterium diptheriae. It is a potentially lethal condition, but because of vaccination it is rare in the Western world. However, it occurs in epidemics in underdeveloped countries, with a mortality rate of 10%. The disease spreads by droplet infection and can spread rapidly in crowded areas. The disease is localized to the pharynx, larynx, and nasal cavities but can involve the heart, kidneys, and nerves because of the secretion of the exotoxin. The characteristic feature of diphtheria is the presence of a pseudomembrane composed of fibrin, leukocytes, erythrocytes, respiratory cells, and dead microorganisms. The onset is abrupt, starting as sore throat, malaise, and low-grade fever. The pseudomembrane is grayish green and is surrounded by a deep red border, and any attempts to remove it result in bleeding. Symptoms include hoarseness of voice, respiratory distress, and a brassy cough. There is bilateral cervical lymphadenitis and progression of the pseudomembrane to the larynx necessitating acute airway management either by endotracheal intubation or by tracheostomy. Deaths are mainly due to myocarditis, arrhythmia, and cardiac failure. Neurological complications can occur gradually, with paralysis of the soft palate, diaphragm, external ocular muscles, and, occasionally, Guillain–Barre syndrome. Treatment consists of neutralization of the toxin by equine antitoxin (20 000–120 000 units), depending on the severity of illness, together with antibiotics usually of the penicillin group. Active immunization by injection of the toxoid in three doses has eliminated most of the incidences of this condition. Acute epiglottitis It is a life-threatening infection of the epiglottis. It is usually caused by Hemophilus

influenza type B but can be caused by non-type B H. influenza and bacteria such as Streptococcus pyogenes, Streptococcus pneumoniae, and Staphylococcus aureus. It affects children between 2 and 4 years of age and commonly during the winter months. Onset is very rapid, starting with sore throat and dysphagia that progresses to inspiratory stridor, and the patient becomes critically ill within 2–6 hours. The child appears ill, sitting up and leaning forward, and with drooling. The diagnosis is based purely on the history and clinical findings. Examination of the epiglottis is not recommended because of the risk of precipitation of the airway obstruction. X-ray lateral soft tissue of the neck may reveal a classical ‘thumb sign’ of the edematous epiglottis, but treatment should not be delayed to obtain radiographs. The child should be calmed and taken to a resuscitation room with facilities for intubation and tracheostomy. Treatment is directed at airway management and then providing antimicrobial and supportive care. After the child is anaesthetized by gaseous induction, the airway is secured with an endotracheal tube. Rigid bronchoscopy and tracheostomy set should be available in case there is failure of intubation. Once the endotracheal tube is in place, intravenous antibiotics and intravenous fluid infusions are started after taking blood and epiglottic samples for microbiology. Extubation is usually possible after 48–72 hours, at which time the edema has subsided to allow air leak around the endotracheal tube. Flexible laryngoscopy is a commonly employed technique to ensure resolution of the edema is complete before extubation (Figures 1 and 2). Laryngotracheobronchitis (croup) It is inflammation of the large parts of the respiratory tract, but it is maximally seen in the subglottis, affecting children between the ages of 3 months and 3 years.

Figure 1 Swollen and inflamed epiglottis obscuring most of the laryngeal inlet; only arytenoids and aryepiglottic folds are seen. Courtesy of A Hilger.

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Figure 3 Endoscopic picture of the larynx showing a chondroma of the thyroid cartilage. Courtesy of A Hilger.

Figure 2 Soft tissue X-ray of neck: lateral view showing a swollen epiglottis, the classical ‘thumb sign’. Courtesy of A Hilger.

Parainfluenza viruses (types I–III) are the most common pathogens, but it can be caused by influenza virus type A, respiratory syncytial virus, and rhinovirus. It is commonly preceded by an upper respiratory tract infection and can proceed to secondary bacterial infection. Cough and stridor are the most common presenting features. Stridor can be inspiratory to biphasic, and the cough is described as a barking cough. The child is usually restless, with flaring of ala nasae and development of intercostals recession with increasing distress. Anteroposterior radiography of the chest shows subglottic narrowing – the ‘steeple sign’. The child should be monitored regularly, for which numerous scoring systems have been devised. Most patients respond to medical and supportive therapy. Apart from reassurance, humidification, steroids, and nebulized adrenaline are sufficient to manage most patients; uncommonly, intubation is necessary to secure the airway. Neoplastic Lesions of the Larynx

Benign Squamous papillomatosis Papillomas occur in either a juvenile- or an adult-onset form. The juvenile-onset papilloma is also known as recurrent respiratory papillomatosis. The juvenile form occurs in children from birth to 5 years of age. The papillomas

are known to occur anywhere in the respiratory tract, but the glottis is the most common site. The papillomas are due to infection of the epithelial cells with human papilloma virus. The adult papillomas are usually single and arise from the glottis. Hoarse voice, abnormal cry, respiratory obstruction, and stridor are the usual symptoms. Endoscopic assessment and laser excisions are the recommended treatment. In recurrent cases requiring frequent laser treatment (every 1 or 2 months), a-interferon and isotretinion have shown significant responses. Juvenile papillomas can recur, whereas adult papillomas need to be followed up due to the risk of malignant change (Figures 3 and 4). Vocal polyp It is caused by accumulation of fluid in Reinke’s space with ballooning of the epithelium in front of the vocal cord. Rarely, a large polyp can cause respiratory obstruction. Microlaryngoscopic excision is the treatment of choice (Figure 5). Malignant Laryngeal tumors are usually cancers of epithelial origin, with squamous cell carcinoma being the most common; other forms of cancers are relatively rare. Laryngeal cancers cause airway obstruction by tumor extension into the glottis, by fixation of vocal cords, or by paralyzing the vocal cords through infiltration of the recurrent laryngeal nerves. The tumors are usually seen in middle-aged and elderly patients. Smoking and alcohol ingestion are important risk factors; however, irradiation and genetic and environmental factors are known to predispose patients to upper airway tumors. The symptoms are progressive dysphonia, dyspnea and stridor, neck swelling, and less commonly, dysphagia, pain,

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Figure 4 Endoscopic picture of the larynx showing recurrent respiratory papillomatosis. Courtesy of A Hilger.

Figure 5 Endoscopic picture of the larynx showing vocal cord polyp arising from the right vocal cord. Courtesy of A Hilger.

cough, and throat irritation. A detailed history, physical examination, and otolaryngological examination are necessary for a clinical diagnosis, but definitive diagnosis is only by histopathological examination of the tumor. CT of the neck, chest, and abdomen gives an accurate account of the extent of the disease and involvement of the laryngeal cartilages and adjacent structures, neck nodes, and distant metastasis. Treatment generally involves either surgery or radiotherapy or a combination of chemotherapy, radiotherapy, and surgery. The prognosis is good if the tumors are treated in the early stages of the disease (Figures 6 and 7). Trauma

Thermal injuries and inhalational injuries The common types of thermal injuries occur due to inhalation of hot gases or smoke in burning buildings

Figure 6 CT scan of the neck showing a laryngeal tumor compromising the airway with erosion of the thyroid cartilage and extension into the neck. Courtesy of A Hilger.

Figure 7 Tumor involving both the vocal cords and the anterior commissure of the larynx. Courtesy of A Hilger.

and, rarely, due to ingestion of hot liquids or foods. Inhalational injuries frequently cause severe and potentially life-threatening acute and long-term complications. The heat and the fumes produce intense mucosal injury, with sloughing of respiratory mucosa. If there is loss of the basal cell layer of the mucosa either due to inhalation or due to subsequent local trauma, delayed repair with granulation formation and potential airway stenosis occurs. The location of the injury due to inhalation depends on the size of the particles inhaled. Large particles mainly affect the trachea and the larynx, whereas small particles affect the alveoli. Injury due to ingestion of hot liquid or food is usually confined to the supraglottis and posterior larynx, and features may mimic infectious epiglottitis. Ingestion injury causes edema and airway obstruction, whereas inhalation injury can present with symptoms ranging from cough to stridor and respiratory arrest. There is

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controversy regarding the timing of intubation in such patients, but patients presenting with stridor, loss of consciousness, or massive burns need immediate intubation. If airway obstruction is not imminent, fiber-optic laryngoscopy and bronchoscopy can be performed and the airway assessed. There is controversy regarding the indications for tracheostomy in inhalational injuries, but the management of patients with inhalational injuries should be individualized. Edema secondary to the ingestion injury resolves rapidly, and decannulation or extubation can be accomplished in a short time. The management of long-term laryngeal and tracheal complications of inhalational injury should be focused on restoring the physiological function of the air as much as possible. Trauma to the larynx Laryngeal trauma can be due to blunt injury, such as in road traffic accidents, sports injuries, and assaults, whereas gunshot and knife wounds cause penetrating injuries. Most blunt injuries result in injury to the cartilaginous framework of the larynx, whereas penetrating injuries mainly involve soft tissue. Cartilaginous injuries are thyroid cartilage fracture, cricoid fracture, laryngotracheal separation, arytenoid dislocation, or a combination of these, whereas soft tissue injuries are usually hematomas or lacerations. Laryngeal trauma in children tends to be less severe due to the higher position of the larynx. Stridor and dysphonia suggest laryngeal trauma, but the symptoms can include pain, dyspnea, hemoptysis, and swelling. The initial management of the patient at the site of first contact should be to stabilize the cervical spine and establish an airway. In life-threatening airway compromise, intubation or tracheostomy should be done. Once the patient is stabilized, physical examination and assessment of the neck are done, including fiber-optic/rigid endoscopic examination of the larynx. Laryngeal CT scan is the most useful radiologic examination for evaluating laryngeal injury. After initial evaluation and management of airway, the treatment depends on the extent of the injury. Hematoma and edema are managed conservatively, but exposed cartilage, large mucosal lacerations, vocal cord immobility, dislocation of arytenoids, and any neck injury with airway obstruction require surgical intervention. Fractures require open reduction and internal fixation with or without stenting, whereas laryngotracheal separation or complex injuries require surgical repair or resection. Foreign body in the airway Foreign body obstruction of the larynx and trachea is less common and is seen in children between the ages of 1 and 3 years,

but it can also occur in adults, in whom the presentation is an acute respiratory distress due to accidental bolting of the food bolus in the larynx and pharynx. In adults, the presentation is acute distress, whereas hoarse cry, stridor, and excessive salivation suggest laryngeal foreign body in children, which can be associated with choking followed by bouts of coughing. A strong history is very suggestive of a foreign body in the airway. Wheeze, unexplained persistent respiratory symptoms that continue despite treatment, and persistent or recurrent lobar pneumonia suggest a bronchial foreign body warranting a diagnostic bronchoscopy. Examination should include assessment of the patient’s general health and a full otolaryngological assessment. Respiratory distress and cyanosis demand immediate action. Anteroposterior and lateral soft tissue neck and chest X-rays are helpful but should not delay an endoscopic assessment. Treatment is mainly to establish the airway. The Heimlich maneuver is life-saving in food bolus obstruction in adults. The rescuer stands behind the patient

Figure 8 X-ray of neck, chest, abdomen, and pelvis: anteroposterior view showing a radio-opaque foreign body (coin) in the upper aerodigestive tract (upper esophagus) of a pediatric patient. Courtesy of A Hilger.

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and places a clenched fist below the xiphisternum, followed by subdiaphragmatic, upward thrust producing a forced expiration/cough. If this method fails, then a cricothyrotomy is done. The cricothyroid membrane between the thyroid and cricoid cartilage is identified and cut with a knife and rotated 901 to maintain the airway. Any convenient tube is placed through the lumen until the patient is transferred to the hospital. Passing a minimum of three wide-bore (14-gauge) needles through the cricothyroid membrane can be an alternative method should there be an acute obstruction in a hospital setting. Definitive removal of the foreign body requires an experienced anesthetist, otolaryngologist, and appropriate equipment. Laryngeal foreign bodies are removed by direct laryngoscopy; rarely, a tracheostomy may be needed. Tracheal and bronchial foreign bodies are removed by rigid bronchoscopy (Figure 8). See also: Bronchomalacia and Tracheomalacia. Granulomatosis: Wegener’s Disease. Laryngitis and Pharyngitis. Systemic Disease: Sarcoidosis.

Further Reading Baines PB and Sarginson RE (2004) Upper airway obstruction. Hospital Medicine 65(2): 108–111. Cummings CW, Fredrickson JM, Harker LA, Krause CJ and Schuller DE (1986) Otolaryngology – Head and Neck Surgery, vol. 3, pp. 1895–1918. St Louis, MO: Mosby. Hammer J (2004) Acquired upper airway obstruction. Paediatric Respiratory Reviews 5(1): 25–33. Jafek BW and Stark AK (1996) ENT Secrets, Upper Air Obstruction, pp. 296–302. Philadelphia: Hanley & Belfus. Kerr AG (1997) Scott–Brown’s Otolaryngology, 6th edn., vol. 6. London: Arnold. Kimmelman CP (1989) Nasal obstruction due to benign and malignant neoplasms. Otolaryngologic Clinics of North America 22(2): 351–366. Kimmelman CP (1989) Rhinitis and nasal obstruction. Otolaryngologic Clinics of North America 22(2): 307–318. Roland NJ, McRae RDR, and McCombe AW (2001) Choanal atresia; epiglottitis. In: Key Topics in Otolaryngology and Head and Neck Surgery, 2nd edn. Oxford: Bios Scientific. Snow JB and Ballenger JJ (2002) Congenital anomalies of the larynx. In: Ballenger’s Otorhinolaryngology Head and Neck Surgery, 16th edn., pp. 1048–1072. New York: Decker. Sulica L and Myssiorek D (2004) Vocal fold paralysis. Otolaryngologic Clinics of North America 37(1): 121–138. Watkinson JC and Wilson JA (2000) Rehabilitation of speech and swallowing. In: Stell & Maran’s Text Book of Head and Neck Surgery, 4th edn., pp. 361–365. Oxford: Butterworth-Heinemann.

UPPER RESPIRATORY TRACT INFECTION T Heikkinen and O Ruuskanen, Turku University Hospital, Turku, Finland & 2006 Elsevier Ltd. All rights reserved.

Abstract Upper respiratory tract infection (URI) is a general term for a heterogeneous group of illnesses caused by numerous etiologic agents that affect the mucosal lining of the upper respiratory tract, including the middle-ear cavity and paranasal sinuses. URIs are primarily caused by viruses, rhinoviruses being the most common etiological agents. Respiratory viruses transmit easily via direct contact or aerosols. The incidence of URI is highest in children who suffer 6–8 infections per year. The main symptoms of URI are nasal blockage and discharge, sneezing, sore throat, and cough. Fever occurs variably, most commonly in children. Viral URIs often predispose to bacterial complications. Acute otitis media is the most common complication in children, whereas sinusitis and pneumonia are more frequent in adults and the elderly. The treatment of URI is mainly symptomatic because specific antivirals are available only for influenza viruses. Antibiotics have no efficacy for viral URI but are commonly used for treating acute otitis media and sinusitis. Most URIs are self-limited illnesses with an average duration of 7–10 days and an excellent prognosis.

Introduction Upper respiratory tract infection (URI), or ‘common cold’, is the most frequent illness in humans. The

main symptoms of URI are nasal stuffiness and discharge, sneezing, sore throat, and cough. The presence of low-grade fever is variable and more common in children than in adults. In spite of the benign nature of the illness, URIs cause a substantial economic burden on society in terms of medications, visits to physicians and other healthcare providers, and absenteeism. Young children suffer on average 6–8 and adults 2–4 URIs per year. The occurrence of URI displays a clear seasonal variation. The highest incidence rates are observed in the autumn and winter in temperate regions of the northern hemisphere. In tropical countries, most URIs occur during the rainy season. URIs are usually self-limited illnesses confined to the upper respiratory tract, but occasionally the viral infection spreads to adjacent organs, resulting in different clinical manifestations. Viral URIs may also predispose to bacterial complications. In children, the most common complication of URI is acute otitis media (AOM). The highest incidence of AOM occurs in children between 6 months and 2 years of age. By the age of 2 years, approximately 70% of children have experienced at least one episode of AOM. In adults and the elderly, the most frequent complications of URI are paranasal sinusitis and pneumonia.

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Sinusitis has been estimated to occur as a complication in 0.5–2% of URIs. Recent studies have demonstrated, however, that paranasal sinuses are frequently affected during URI, and most sinus abnormalities are not evidence of a true bacterial complication but instead are part of the natural history of URI.

detected by in situ hybridization in the epithelial cells of maxillary sinuses in adult patients with sinusitis. It is probable that with increasing interest in the etiology of sinusitis, our knowledge regarding the relative role of viruses in this condition will expand.

Pathology Etiology URIs have probably tormented mankind for thousands of years, but it was not until the middle of the last century that viruses were demonstrated as the etiologic agents of these illnesses. Influenza A virus was the first virus to be isolated in 1933. Since then, intensive research into the etiology of URIs during the 1950s and 1960s led to the discovery of several other groups of respiratory viruses, for example, adenoviruses, rhinoviruses, parainfluenza viruses, respiratory syncytial viruses, enteroviruses, and coronaviruses. During the past few years, the application of molecular techniques to viral detection has resulted in the discovery of several respiratory viruses that had remained unknown before the polymerase chain reaction (PCR) era, for example, human metapneumovirus and new coronaviruses. Although the viral etiology of URIs can be currently documented in over 90% of cases by the use of modern diagnostic techniques, it is probable that a number of viruses causing respiratory illnesses still remain to be identified. Among all respiratory viruses, rhinoviruses are the most common etiologic agents in URI. On an annual level, rhinoviruses are estimated to account for approximately 30–50% of all respiratory illnesses, but this percentage may rise to 80% during the autumn peak season. Recent studies have disclosed that at least in children the relative role of enteroviruses is greater than previously known. AOM is generally considered a bacterial complication of URI. However, bacterial pathogens – mainly Streptococcus pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis – can be isolated from the middle-ear fluid in only approximately 70% of cases. Intensive research into the etiology of AOM since the 1980s has produced strong evidence for a key role of viruses in the etiology of AOM. By the use of PCR-based techniques, viruses have been found in the middle-ear fluid in the majority of children with AOM, with or without bacteria. The bacterial pathogens found in sinusitis are much the same as those found in AOM. There is, however, growing evidence for an important role of viruses also in the etiology of sinusitis. Rhinovirus RNA has been detected in sinus aspirates even in the absence of bacteria. Rhinoviruses have also been

Owing to the great number of different viruses causing URIs, it is probable that the histopathologic changes during various viral infections will be markedly different. For example, influenza viruses are thought to have an extensive direct cytopathic effect on the respiratory epithelium, which leads to degeneration of epithelial cells and pseudometaplastic changes in the epithelium. On the other hand, during rhinovirus infection the number of infected cells in the upper respiratory epithelium is limited, and no histopathologic changes have been seen in nasal biopsy specimens from subjects infected with rhinoviruses. The absence of epithelial destruction during rhinovirus infections – the most frequent cause of URIs – suggests that the clinical symptoms of URI are not primarily caused by virus-induced epithelial injury but instead are caused by the inflammatory response of the host.

Clinical Features In most cases, the diagnosis of URI is obvious and the condition is usually self-diagnosed. The diagnosis may sometimes be delayed in infants and young children, especially when fever is the leading symptom during the early stage of the illness. The incubation period of URI varies considerably among different viruses, ranging from 12 h to 7 days. A typical illness starts with a sore throat that is shortly accompanied by nasal stuffiness, runny nose, sneezing, and cough. The soreness of the throat often disappears quickly, whereas the initial watery nasal discharge soon turns thicker and more purulent. Usually the severity of the symptoms increases rapidly and peaks within 2–3 days. The average duration of URI is 7–10 days, but in a number of patients some symptoms may still be present after 3 weeks. When AOM develops as a complication of URI, it is most frequently diagnosed on days 3 or 4 after the onset of upper respiratory symptoms. Most symptoms that are generally listed as associated with AOM overlap significantly with those of an uncomplicated URI. In clinical practice, earache is the only symptom that can be considered specific for AOM. However, the value of this symptom in suspecting AOM is severely limited by the fact that almost half of infants and young children with AOM either do

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not have earache or they cannot express it to their parents. The ultimate diagnosis of AOM requires inspection of the tympanic membrane by otoscopy. Analogous to symptoms of AOM, many symptoms that are conventionally thought to indicate sinusitis have been found to be as common in patients with radiologically normal sinuses. As sinuses are affected at least to some degree during most cases of URI, distinguishing between viral URI and bacterial sinusitis is a great challenge in clinical practice. In the absence of sinus puncture, facial pain (especially if unilateral), maxillary toothache, postnasal drip, and nasal obstruction or thick nasal discharge for more than 7–10 days are usually thought to be indicative of bacterial sinusitis. The value of detecting mucosal thickening in sinus radiographs or by ultrasound is questionable because the finding correlates poorly with bacterial etiology of the condition.

Pathogenesis The transmission of viruses occurs most often by direct contact with virus-containing secretions, followed by self-inoculation of the viruses in the anterior nasal mucosa or in the eye. Other routes of transmission include small-particle aerosols lingering in the air and direct hit by large-particle aerosols from an infected person. The primary ways of transmission as well as the detailed pathogenetic mechanisms differ among various respiratory viruses. For example, the primary site of replication of influenza viruses is in the trachea, whereas rhinovirus replication starts predominantly in the nasopharynx. The currently available evidence does not lend support to the popular belief that chilling or exposure to a cold environment has a role in the pathogenesis of URI. Respiratory viruses gain entrance to epithelial cells by binding to specific receptors on the cells. About 90% of rhinovirus serotypes use intercellular adhesion molecule-1 as their receptor. Viral replication starts rapidly inside the cells, and progeny viruses can be detected within 10 h of viral inoculation. However, not all infections lead to clinical illness. Viral infection of the nasal mucosa results in vasodilation and increased vascular permeability, which, in turn, cause nasal obstruction and discharge that are the hallmark symptoms of URI. Cholinergic stimulation leads to increased mucous gland secretion and sneezing. There is increasing evidence that the clinical symptoms of the patient are primarily caused by the host’s inflammatory response. Increased levels of several inflammatory mediators, for example, interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor, leukotrienes, kinins, histamine, and RANTES (regulated upon activation normally T-cell expressed and

secreted), have been found in nasal secretions from patients with URI. It has been reported that the concentrations of IL-6 and IL-8 in nasal secretions correlate with the severity of symptoms. The host response mechanisms during respiratory viral infections are, however, very complicated, consisting of a network of factors affecting each other in a timedependent manner, and it is probable that several essential factors still remain to be identified. Viral infection in the nasopharynx results in congestion of the nasopharyngeal mucosa. Congestion in and around the eustachian tube leads to dysfunction of the tube, which is considered the most important factor in the development of AOM. Inflammatory mediators play a key role in disruption of normal eustachian tube function although certain viruses, for example, influenza viruses, can also cause direct damage to the epithelial cells in the eustachian tube. Dysfunction of the eustachian tube results in formation of underpressure in the middle-ear cavity, decreased drainage of middle-ear secretions into the nasopharynx, and loss of protection of the middleear from nasopharyngeal secretions. Eventually, these events lead to microbial invasion of the middle-ear, host inflammatory response in the middle-ear mucosa, and clinical signs and symptoms of AOM. Scarce data are available on the exact pathogenic mechanisms involved in the development of sinusitis. It is likely that mucosal swelling around the narrow orifice of paranasal sinuses and viral infection of the sinus mucosa interfere with the normal cleansing mechanisms, which increases the possibility for secondary bacterial infection.

Animal Models Most of our knowledge about viral URIs is derived from extensive community follow-up studies and experimental infections in volunteers. However, animal studies have been of crucial importance for research into the role of viruses in the pathogenesis of AOM. Most experimental studies in this area have been performed in chinchillas. They are well suited for otitis media research because their middle-ear is easily accessible and otitis media can be produced by intranasal inoculation of different microbes, thus mimicking the sequence of events in humans.

Management and Current Therapy The treatment of URI is largely symptomatic and aimed at relieving the most unpleasant symptoms of the illness. Nasal stuffiness can be effectively reduced with intranasally or orally administered decongestants. First-generation (but not second-generation)

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antihistamines reduce sneezing and nasal discharge. This effect is probably more likely due to the anticholinergic rather than the antihistamine features of these drugs. Locally administered ipratropium has also been shown to reduce nasal discharge. Nonsteroidal anti-inflammatory drugs reduce fever and soreness of the throat. Cough medications are commonly used but their efficacy has not been demonstrated convincingly. Despite corticosteroids being potent anti-inflammatory agents, their intranasal or oral use has not resulted in any clinical benefits. The use of intranasal steroids in children during rhinovirus infection may even increase the risk of AOM. Data on the efficacy of zinc, vitamin C, or echinacea extracts in the treatment of URI are inconclusive. Although it is well known that antibiotics are not effective against viruses and that unnecessary use of antibiotics increases bacterial resistance, antibiotics are frequently used in the treatment of uncomplicated URIs. Specific antiviral treatments for respiratory viruses are currently available only for influenza viruses (oseltamivir, zanamivir, amantadine, and rimantadine). However, antiviral drugs for some other respiratory viruses (e.g., RSV, rhinoviruses, enteroviruses) are currently being developed. Although viruses play a key role in the development of AOM, bacteria are often found in samples of middle-ear fluid, and so AOM is usually treated with antibiotics. However, spontaneous resolution of the middle-ear effusion usually occurs within a couple of

weeks or months, which has initiated an intense debate on whether antibiotic treatment is necessary in all cases of AOM. Unfortunately, clinically useful indicators to help decide which patients would really benefit from antibiotics are not currently available. The same problem applies to the treatment of sinusitis, in which spontaneous resolution is also frequent and reliable diagnosis of bacterial sinusitis is extremely difficult without sinus puncture. See also: Antiviral Agents. Vaccinations: Bacterial, for Pneumonia. Viruses of the Lung.

Further Reading Cherry JD (1998) The common cold. In: Feigin RD and Cherry JD (eds.) Textbook of Pediatric Infectious Diseases, 4th edn., pp. 128–133. Philadelphia: Saunders. Heikkinen T and Chonmaitree T (2003) Importance of respiratory viruses in acute otitis media. Clinical Microbiology Reviews 16: 230–241. Heikkinen T and Ja¨rvinen A (2003) The common cold. Lancet 361: 51–59. Ison MG, Johnston SL, Openshaw P, Murphy B, and Hayden F (2004) Current research on respiratory viral infections: Fifth International Symposium. Antiviral Research 62: 75–110. Ruuskanen O and Hyypia¨ T (2005) Rhinovirus: is it really a relevant pathogen? In: Kimpen J and Ramilo O (eds.) The Microbe–Host Interface in Respiratory Tract Infections, pp. 291–317. Norfolk: Horizon Bioscience. Treanor JJ (2002) Respiratory infections. In: Richman DD, Whitley RJ, and Hayden FG (eds.) Clinical Virology, 2nd edn., pp. 7–26. Washington, DC: ASM Press.

V VACCINATIONS Contents

Bacterial, for Pneumonia Viral

Bacterial, for Pneumonia

Pneumococcal Pneumonia

N W Schluger, Columbia University College of Physicians and Surgeons, New York, NY, USA

Streptococcus pneumoniae remains the most commonly diagnosed cause of bacterial lower respiratory tract infection in all regions of the world. It is estimated, based on comprehensive epidemiologic studies performed from time to time, that as many as half of all cases of acute pneumonia are caused by S. pneumoniae. Invasive pneumococcal disease, in both children and adults, is extremely serious. In developing countries, children bear a substantial proportion of the morbidity and mortality due to S. pneumoniae, whereas in developed countries most serious infections caused by this pathogen occur among older adults. Pioneering studies done soon after the introduction of antibiotics indicated that early mortality from invasive pneumococcal disease was as high as 25%, and this is not affected by administration of antibiotics. In addition, over the past decade, resistance by S. pneumoniae to commonly used antibiotics such as penicillins and b-lactams is quite common. Although there is debate about the clinical ramifications of this resistance at the present time, there is no question that this issue will grow in importance as more and more antibiotics become more and more widely available. For these reasons, vaccination for pneumococcal disease seems to be an extremely attractive way to reduce the burden of this common illness.

& 2006 Elsevier Ltd. All rights reserved.

Abstract Bacterial respiratory infections are a major cause of morbidity and mortality around the world. The multiple serotypes of common bacterial pathogens have made vaccine development a challenge and have limited the effectiveness of vaccines for preventing serious illness in adults. However, very effective vaccines have been developed for the strains of Streptococcus pneumoniae, and Hemophilus influenzae which mainly affect children, and these vaccines have greatly decreased the incidence of serious invasive illness due to these pathogens in younger age groups. The commonly used vaccines against Bordatella pertussis have also had a profound impact on reducing morbidity and mortality from whooping cough, although it is recognized now that protection with this vaccine is not life-long, although clinical illness in previously vaccinated individuals is usually quite mild. The future refinement of these vaccines holds continued promise for prevention of even more deaths due to common respiratory pathogens.

Introduction Vaccines are among the greatest achievements of medical science, and there are many reasons to attempt to develop effective vaccines against acute respiratory illnesses: acute upper and lower respiratory tract infections are enormously common and important causes of morbidity and mortality around the world; for several pathogens, (early) mortality does not seem to be substantially affected by administration of antibiotics; and increasing antibiotic resistance around the world, along with a slower pace of new antibiotic development, increasingly limits treatment options and successes for these illness. This article will review the approaches to and efficacy of vaccination for three common upper and lower respiratory tract bacterial pathogens.

Biology of S. pneumoniae Relevant to Vaccine Development

S. pneumoniae is a Gram-positive coccus which usually grows in pairs and chains. It possesses a polysaccharide capsule which determines its serotype; there are at least 90 known serotypes of pneumococcus, although only a minority seem to be important in terms of causing human disease. The capsular polysaccharide provides the major antiphagocytic defense for the bacterium; the protein is encoded for

390 VACCINATIONS / Bacterial, for Pneumonia

by the galU gene, which, when knocked out, renders the pneumococcus essentially avirulent. The risk of invasive pneumococcal disease (bacteremia and meningitis) appears to vary among serotypes, and nasal or nasopharyngeal carriage may not necessarily predispose to invasive infection, as strains associated with carriage are often of serotypes felt to be less invasive. A recent meta-analysis suggested for example that serotypes 1, 5, and 7 were 60-fold more likely to cause invasive disease than types 5, 6A, and 15. In addition, this study detected an inverse correlation between the risk of carriage and the risk of invasive disease. Pneumococcal carriage is common. It is estimated that as many as 50% of the population have nasal or nasophayngeal carriage at any given time. However, invasive disease usually occurs within a few days of acquiring a new strain with a more virulent serotype. Risk factors for invasive pneumococcal disease include most strongly cigarette smoking, alcoholism, and extremes of age. Although persons with HIV infection appear in some studies to be at higher risk for invasive pneumococcal disease, this is not a consistent finding. Pneumococcal Vaccines

Polysaccharide vaccines The idea of using antibodies to treat or prevent invasive pneumococcal disease has existed for decades. Prior to the antibiotic era, pneumococcal isolates were used to raise serotypespecific serum in rabbits, which was then administered to patients. Descriptions of this are vividly recounted in Lewis Thomas’ wonderful collection of essays, The Youngest Science. A tetravalent polysaccharide vaccine was developed at roughly the same time as penicillin was introduced, and likely because of a sense that antibiotics alone would be enough to eliminate the pathogen as a leading cause of disease, further vaccine work was not pursued. In 1977 a vaccine directed against the 14 most common disease-causing serotypes of S. pneumoniae was introduced. This was followed in 1983 by the development of a 23-valent vaccine which in theory could provide protection against nearly 90% of the serotypes which are isolated from normally sterile sites. Since the introduction of the 14- and 23-valent polysaccharide vaccines, there has been substantial argument over their efficacy. Endpoints in studies of pneumococcal vaccine efficacy have included some which are somewhat difficult to quantitate exactly, such as the development of pneumococcal pneumonia (usually diagnosed with chest radiographs and sputum cultures), and some which are easier to define with precision, such as bacteremia and death. The

widespread and early use of antibiotics in patients with pneumonia have paradoxically made it more difficult to precisely determine vaccine efficacy, as a large proportion of pneumonia cases go without a specific etiologic diagnosis. Several independent meta-analyses and a recent Cochrane review have surveyed all the published experience with pneumococcal polysaccharide vaccines in adults, and these results will be summarized here. Overall, it has been difficult to demonstrate a reduction in either overall mortality or cases of pneumonia in almost any patient group on the basis of randomized trials of polysaccharide vaccines for S. pneumoniae. However, there does appear to be clear benefit of vaccination in reduction in cases of invasive pneumococcal disease, a more serious but less common form of the illnesses associated with this pathogen, in patients who have been vaccinated. This includes elderly patients with apparently normal host immunity. Although the authors of the Cochrane review conclude that the case for more widespread use of vaccination with against pneumococcus in adults is not proved, and not necessarily a strong one, most public health boards still recommend its use in elderly persons (over the age of 65) and persons with underlying heart or lung disease. Protein conjugate vaccines In contrast to polysaccharide vaccines in adults, protein conjugate vaccines against the pneumococcus show great promise for reducing serious illness in children. Polysaccharide vaccines are generally not thought to be suitable for use in children because children lack mature B lymphocytes necessary for a vigorous immune response to the vaccine. Children under the age of 2 years lack the ability to mount a vigorous response to capsular polysaccharide, which stimulates a T-cell-independent response. However, conjugation of capsular polysaccharide to a protein carrier converts a T-cell-independent response to a T-cell-dependent one which very young children are capable of mounting, and this is the approach exploited by protein conjugate vaccines, both for Hemophilus influenzae and S. pneumoniae. Studies examining the efficacy of the sevenvalent protein conjugate vaccine have been conducted using a variety of endpoints: invasive pneumococcal disease, radiographically confirmed pneumonia, and clinically diagnosed pneumonia. Overall efficacy for protection of invasive pneumococcal disease caused by serotypes covered by the vaccine has ranged from 73% to 94% in these studies. No protection is observed for serotypes not covered by the vaccine. The reduction of cases of radiographically confirmed pneumonia is estimated at 22%, although one must keep in mind that it is impossible to determine the

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bacteriologic cause, much less a specific serotype, of pneumonia based on a chest radiograph. Further evidence of the efficacy of the vaccine comes from epidemiologic studies which have demonstrated a substantial overall decrease in the number of cases of invasive pneumococcal disease in countries with widespread vaccine coverage, such as the USA. However, the decision for institute widespread vaccination of children with the protein conjugate vaccine depends on the prevalence of invasive pneumococcal disease and the specific serotypes responsible for it. Cost is a significant consideration as well. Of note, a nine-valent protein conjugate vaccine is currently in the late stages of clinical development.

Pertussis Bordatella pertussis, the causative agent of pertussis, or whooping cough, is highly contagious, particularly among young children, under the age of 5, and adults who lack immunity. The organism is a Gram-negative rod with particular affinity for the mucosal layer of the respiratory epithelium. The World Health Organization estimates that there are between 20 and 40 million cases of pertussis in the world each year, and between 200 000 and 400 000 deaths, primarily in very young children. Classically, the illness of pertussis is described as having a catarrhal stage consisting of mild upper respiratory tract symptoms and a paroxysmal stage characterized by severe coughing spasms, often with the characteristic whoop that gives the disease its name. In the convalescent phase there may be prolonged dry cough, and the illness generally wanes over a 6–10 week period. In children younger than 1 year of age, mortality associated with pertussis can be quite substantial. Original vaccines against pertussis, developed in the 1940s, were of the whole cell type. However, because of concerns about serious adverse events related to these whole cell vaccines, acellular pertussis vaccines were developed. Both vaccines are now included in the DPT series given to most children today. Acellular vaccines are quite a bit more costly. Currently about 80% of the world’s children receive pertussis vaccine, generally the whole cell type, as part of the DPT series. There is no question regarding the efficacy of whole cell and acellular pertussis vaccines. A variety of studies have demonstrated vaccine efficacy of 75–85%, although best efficacy with the acellular vaccine is seen with three- to five-component vaccines. Over the past several years, questions have been raised about the safety of pertussis vaccines, primarily by persons outside the medical community. There have been particular concerns about serious

neurological sequellae related to whole cell pertussis vaccines, leading to intensive study of this subject. Although fever and prolonged crying are more frequent after the administration of the whole cell vaccine, seizures and permanent neurological sequellae are not. However, because persistent but unfounded concerns about the safety of the whole cell vaccine caused many parents to forego vaccination for pertussis, leading to several mini-epidemics and considerable morbidity and mortality in young children, many countries have in fact switched to using the more expensive acellular vaccine. The World Health Organization endorses use of the acellular vaccine in countries where high-quality acellular vaccines are available and there is reluctance about the whole cell vaccine because of reactogenicity. Although pertussis vaccines have been extremely effective in reducing morbidity and mortality among young children, it has been increasingly recognized in the past several years that immunity may not be lifelong. Many published series of prolonged cough illness in young adults indicate that 15–20% of cough illness (syndromes manifest mainly as a dry cough of up to 3 weeks duration in otherwise young healthy persons) are due to pertussis, even in previously vaccinated persons. The diagnoses in these cases have been based on a combination of culture, serological testing, and nucleic acid amplification. Notably however, severe episodes of whooping cough have not been observed in previously vaccinated persons as a rule, so that the waning of immunity seems to be associated with only mild forms of the illness. Nonetheless, clinicians should not discount a diagnosis of pertussis in otherwise healthy adults with prolonged cough. The episodes are generally treatable with macrolides, and a good result can almost always be expected.

Hemophilus Influenzae Infections Type B H. influenzae Infections

Type B H. influenzae is responsible for nearly 3 million cases of serious illness, and many hundreds of thousands of deaths each year, mostly in children between the ages of 2 months and 5 years. The serious infections caused by H. influenzae include meningitis, pneumonia, septicemia, epiglottitis, cellulatis, and arthritis. In addition, H. influenzae is a common cause of otitis media in children. A first generation of vaccines against H. influenzae was developed in the 1980s, but those vaccines were not effective in children under 18 months of age, the age group in which many of the most severe infections occurred. This was felt to be because these

392 VACCINATIONS / Viral

first vaccines were similar to early pneumococcal vaccines, based on polysaccharides which were incapable of provoking a memory B-cell response. Because of the failure of these polysaccharide vaccines, protein conjugate H. influenzae vaccines were introduced, and have proved to be much more effective. Vaccines conjugated with at least four different carrier proteins have been developed: PRP-T, Hb-OC, PRP-OMP, and PRP-D. Of these, PRP-T seems to be the one which is most immunogenic and has become the most widely used vaccine. PRP-D is no longer used in children because of poor immunogencity. H. influenzae type B (Hib) vaccines may be administered separately or in combination with other vaccines. Carriage of type B H. influenzae is reduced in vaccinated individuals, and this reduction in carriage provides a degree of herd immunity, as nonvaccinated children have a lower risk of coming into contact with the organism if they are exposed mainly to vaccinated persons. On an epidemiological basis, since the introduction of conjugate Hib vaccines in developing countries, the incidence of serious infections due to type B H. influenzae has declined sharply. Overall, results of randomized trials demonstrate an 80% reduction in invasive disease caused by the pathogen. As noted above, this effect has been confirmed by epidemiologic surveillance which demonstrates the virtual elimination of invasive disease from type B H. influenzae in many parts of the USA. The vaccine is quite safe and is associated only with minor adverse effects. Infections due to Nontypable H. influenzae

In adults, most serious infections are caused by nontypable H. influenzae, and vaccinations against these pathogens have proved to be much more difficult. At present, although there are several candidate vaccines in preclinical trials, none has shown significant promise, and the reality of an effective vaccine for nontypable infections remains in the future. See also: Pneumonia: Overview and Epidemiology; Community Acquired Pneumonia, Bacterial and Other Common Pathogens. Upper Respiratory Tract Infection.

Further Reading Abraham-Van Parijs B (2004) Review of pneumococcal conjugate vaccine in adults: implications on clinical development. Vaccine 22: 1362–1371. Barenkamp SJ (2004) Rationale and prospects for a nontypable Haemophilus influenzae vaccine. The Pediatric Infectious Disease Journal 23: 461–462. Conaty S, Watson L, Dinnes J, and Waugh N (2004) The effectiveness of pneumococcal polysaccharide vaccines in adults: a systematic review of observational studies and comparison with

results from randomised controlled trials. Vaccine 22: 3214– 3224. Dear K, Holden J, Andrews R, and Tatham D (2003) Vaccines for preventing pneumococcal infection in adults. Cochrane Database of Systematic Reviews 4: CD000422. Finn A (2004) Bacterial polysaccharide-protein conjugate vaccines. British Medical Bulletin 70: 1. Jefferson T, Rudin M, and DiPietrantonj C (2003) Systematic review of the effects of pertussis vaccines in children. Vaccine 21: 2003–2014. Lucero MG, Dulalia VE, Parreno RN, et al. (2004) Pneumococcal conjugate vaccines for preventing vaccine-type invasive pneumococcal disease and pneumonia with consolidation on X-ray in children under two years of age. Cochrane Database of Systematic Reviews 4: CD004977. Mangtani P, Cutts F, and Hall AJ (2003) Efficacy of polysaccharide pneumococcal vaccine in adults in more developed countries: the state of the evidence. Lancet Infectious Diseases 3: 71–78. McVernon J, Mitchison NA, and Moxon ER (2004) T helper cells and efficacy of Haemophilus influenzae type b conjugate vaccination. Lancet Infectious Diseases 4: 40–43. Swingler G, Fransman D, and Hussey G (2003) Conjugate vaccines for preventing Haemophilus influenzae type b infections. Cochrane Database of Systematic Reviews 4: CD001729.

Viral W Olszewska, P J M Openshaw, and R Helson, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Vaccines are preparations of weakened or killed viruses or viral subunits that trigger specific protective immunity. Vaccination is the single most effective tool for preventing communicable disease, highlighted by the achievements of the smallpox and poliomyelitis eradication programs and the rise in infections for which no effective vaccine is available: human immunodeficiency virus, malaria, worms, and tuberculosis. World Health Organization initiatives target acute respiratory diseases in infancy and early childhood with novel immunization approaches. Effective vaccines are needed urgently for viral respiratory diseases, but progress has been disappointingly slow. Vaccine candidates often show reduced efficacy in infancy, in partially immune adults, the immunocompromised, and the elderly. However, several promising vaccines are in clinical trial, and it is likely that vaccines against respiratory syncytial virus and parainfluenza will be licensed within the next 5–10 years. Mucosally delivered influenza vaccine is now available, and novel adjuvants offer the prospect of better immunogenicity. Ideally, multivalent mucosal vaccines will be developed that provide protection against a spectrum of respiratory infections in specific target age groups.

History The ability to grow viruses in eggs and tissue culture has allowed the development of most of the live and killed viral vaccines in current use. Killed whole virus

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influenza vaccine was introduced in 1936, and vaccines against measles, mumps, and rubella came soon after World War II. These contributed substantially to the virtual eradication of these diseases from many industrialized countries. However, optimism was tempered by the trials of formalin-inactivated respiratory syncytial virus (RSV) and measles vaccines which lead to enhanced disease, with atypical measles rash or enhanced pneumonia, bronchiolitis, or bronchitis. This experience slowed progress and led to a more cautious approach in vaccine development. Recent history is also characterized by withdrawal of apparently effective vaccines because of unacceptable side effects (e.g., vaccines for rotavirus and Lyme disease). Oral live attenuated adenovirus vaccination had been used in US army recruits since the 1970s, eliminating the frequent epidemics at trainee camps. But in 1996, the manufacturer of this vaccine ceased production and outbreaks of adenoviral respiratory illness re-emerged in military settings. Developing a new vaccine for adenovirus infection is a very expensive exercise. The Burden of Disease

Viruses transmitted by the respiratory, gastrointestinal, and genital routes result in virtually all infections that propagate without the assistance of biting insects. Respiratory viruses are remarkably successful, causing very substantial morbidity and mortality in all parts of the world. About 200 serologically distinct viruses occupy this one ecological niche, but the most problematic pathogen changes with age: RSV, human metapneumovirus (hMPV), and parainfluenza are common in infancy, whereas rhinovirus and influenza predominate in older or elderly persons. The seasonality of many of these pathogens makes if difficult to plan efficient and economic health care for affected patients. Each year, influenza A causes 13 000–20 000 excess deaths in the UK. Approximately 90% of these deaths are in persons aged 65 years or older. Influenza deaths have increased substantially in the last two decades, in large part because of an aging and increasingly dense population. Rhinoviruses and coronaviruses cause most common colds, but relatively little direct mortality. Although sometimes considered a minor nuisance, they may infect the lower respiratory tract and cause exacerbations of chronic bronchitis and asthma. The effects of viral diseases during infancy are exacerbated by conditions such as prematurity, cardiac, or pulmonary diseases. RSV is the major cause of infantile hospitalization and infects about 65% of children in the first year of life, and parainfluenza

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accounts for approximately 20% of respiratory hospitalizations in children. Interestingly, parainfluenza virus 3 (PIV-3) peaks in late spring or summer, whereas peaks of PIV-1 and PIV-2 occur at one- or two-yearly intervals in the late autumn or early winter. Adenovirus causes 8–15% of all respiratory diseases in children younger than 5 years old. The emergence of new respiratory pathogens (e.g., severe acute respiratory syndrome (SARS) and avian influenza) to which we have little resistance, is a particular threat to human health and prosperity. Challenges and Opportunities in Respiratory Viral Vaccination

A global alliance in the area of respiratory vaccination has been supported by a $750 million donation from the Bill and Melinda Gates Foundation, but there are difficulties in developing vaccines that will work in target populations. In neonates, the presence of maternal antibodies and the nonmature immune system are major challenges and in the elderly immune responses tend to be relatively weak and ineffective. The rapid rate of viral mutation and the presence of different strains each year, together with the fact that infection with one strain offers no substantial immunity to another, provide the key challenges to the advancement of vaccine technology. An additional specific issue with emerging pathogens is the difficulty of predicting the viral strain and responding rapidly once it has been identified. For pandemic influenza, very large quantities of vaccine would be required in a short time to make significant impact in the face of a spreading epidemic that could kill a significant proportion of the world’s population in 4–6 months, the time required for vaccine production. Novel technology may assist in accelerating vaccine production. For example, cell culture could be more suitable than embryonated chicken eggs. However, the safety of growing large bulk stocks of lethal viruses for purification or inactivation is an additional concern. A major hurdle for RSV vaccine development is the possible enhancement of disease during subsequent natural infection. Formalin-inactivated RSV (FI-RSV) vaccines were tried in normal children in the 1960s, but vaccinees were not protected. Moreover, those that became naturally infected suffered an increased rate of hospitalization due to pneumonia, bronchiolitis, rhinitis, or bronchitis. This enhancement was clearly caused by an overexuberant immune response, but the exact reasons for the effect are still unclear. The full elucidation of mechanisms involved in vaccine-enhanced disease is crucial to the development of future RSV vaccines.

394 VACCINATIONS / Viral Ideal Vaccines

Uses in Respiratory Medicine

Any new vaccine must be completely safe. According to the ‘precautionary principle’, any possibility of side effects (even theoretical) can call a halt to development, or cause the vaccine to be withdrawn. This is especially true of vaccines for children, and for the prevention of so-called trivial diseases. The costs of developing a single candidate vaccine are growing at about 12% per year and now approach $1 billion; novel technologies and concepts are not expected to reduce the price significantly. On average, it now takes 15 years to make a new vaccine (Figure 1). Ideally, vaccines should confer long-lived protection after a single dose and should be easy to manufacture on a large scale. The final product should resist extremes of temperature and be convenient to store and use. They should also be available as combinations (to reduce the number of injections needed) and be effective by local (needle-free) administration. The licensing of vaccines should be global so as to bypass the need for costly local approval processes.

The most widely used and effective vaccines against respiratory viral infections are the inactivated purified subunit influenza vaccines. These are made from hemagglutinin and neuraminidase proteins, produced from the currently circulating influenza strains. The composition of these vaccines is decided each year according to the exact strains likely to cause winter influenza, determined the previous spring by expert international committees. The influenza isolates most likely to spread globally are selected, expanded by culture in embronated eggs and tested by limited immunogenicity study in human volunteers. The different vaccine preparations are not tested for efficacy in preventing influenza infection or symptoms. These vaccines are based on the assumption that induction of antibody is necessary and sufficient to protect against influenza. These normal vaccines have no efficacy against other strains of influenza, and are ineffective against emerging or mutant strains. A particular problem is that cross-species transmission of avian influenza (e.g., H5N1 or the 1918 Spanish influenza strains) is

Phase I • Tens of volunteers • Healthy adults (on no other medication) • Establishment of optimal dose • Primary evaluation of local and systemic reactions • Immediate toxicity, unanticipated side effects Phase II • Hundreds of volunteers • Match target population • Placebo controlled • Test both safety and effectiveness • Short-term safety Phase III • Thousands of volunteers • Long-term study (duration of protection) • Target population (e.g., taking other medicines) • Careful safety evaluation Phase IV • Vaccine approved for use • Postmarketing studies in very large number of patients to assess rare adverse events and efficacy Figure 1 Procedure of clinical testing of a potential vaccine.

VACCINATIONS / Viral

not prevented by these vaccines. An important priority is therefore to develop new methods of vaccination that extend the specificity of vaccines, giving at least some protection against newly emerging strains of influenza. Inactivated vaccines have been in use for more than 60 years, but tend to be associated with local side effects (Table 1). Live attenuated vaccines offer significant advantages, particularly when applied mucosally. They can be highly immunogenic, inducing good serum antibody levels, mucosal immunoglobulin A (IgA), and T cell responses. Cold-adapted live influenza vaccines have been widely evaluated in the US and Japan since 1975. Development continues, and a recent live attenuated, cold-adapted influenza virus vaccine was found to be 93% effective against influenza A (H3N2) in healthy children. For example, FluMist (MedImmune) is available for intranasal use in healthy persons aged 5–49 years. Paradoxically, it seems that protection can be seen in the absence of serum antibody under some circumstances, challenging the view that antibody is essential for protection.

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Attenuated cold-passaged RSV vaccines (cptsRSV 248 and cptsRSV 530) have entered clinical trial. However, some insufficient attenuations lead to otitis media. The more attenuated cptsRSV 248/404 appears to be safe, infectious, and immunogenic in seronegative children, only causing upper respiratory tract congestion in some young infants. Currently, methods of achieving full attenuation without the possibility of reversion to virulence are being tested, including deletion or insertion of entire genes. It is not clear whether genetically modified live viruses will be acceptable to the regulatory authorities and the public. Purified RSV glycoproteins have also been tested as vaccine candidates, and shown to induce neutralizing antibodies. They are effective in cotton rats and do not appear to enhance pulmonary pathology. Clinical trials have been performed with the F protein (PFP-1), purified by immunoaffinity chromatography from RSV-infected cell lysates. This was tested in seropositive children, inducing an eightfold increase in serum-neutralizing antibodies and a reduced risk of subsequent infection. Further studies in seronegative infants showed that multiple immunizations were

Table 1 Summary of common methods of vaccine preparation Active immunization

Explanation

Advantages

Disadvantages

Whole virus

Inactivated using physical or chemical means to destroy the integrity of viral particles Heterologous virus to induce milder/cross-protective disease

No residual pathogenicity No chance of reversion to wildtype infectious virus

Poor inducers of cellular immunity No long-lasting immunity Batch testing required to ensure no residual virus Expensive Examples of enhanced disease

Live attenuated (mutated) virus

Can be random or specifically site directed resulting in subclinical disease

Mimics natural infection, most efficacious induction of immunity Long-lasting immunity Less expensive to manufacture

Residual pathogenicity Possibility of reversion to wild-type virus Requires cold storage

Antigenic fragments or peptides

Purified antigen from the virus, usually present on surface of virus to which immune response is directed

Safer, as not whole virus

Industrial-scale production can be difficult and thus expensive (purification, stabilization)

DNA

Using plasmid vectors encoding protein of choice

Safe, simple Easily manufactured on a large scale Well defined, characterized, and controlled

Not very immunogenic, require large volumes to induce responses in humans

Genes cloned into living viral vector

Viruses can be engineered to introduce new genetic sequences from viral target

Directly infects target cells of the immune system Highly immunogenic

Prior immunity to carrier virus in target population can reduce immunogenicity Limits to size of insert without compromising replication of carrier virus

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required to achieve high antibody titers. A more highly purified preparation (PFP-2) has been tested in children suffering from bronchopulmonary dysplasia and shown to be safe. It is not yet clear if it is effective. Formalin-inactivated parainfluenza vaccine is also nonprotective against infection. Live-attenuated PIV3 and cold-adapted PIV-3 vaccine candidates are undergoing testing. For example, cp45 has been shown to be genetically stable, safe, and appropriately attenuated and immunogenic, even in infants as young as 1 month of age. Vaccines that are genetic hybrids of bovine PIV-3 with human coat proteins are being tested. A vaccine for parainfluenza has reached phase II in clinical trials, tested in 2 month old infants for safety, tolerability, infectivity, and immunogenicity. Emerging Respiratory Viruses

Human metapneumovirus Human metapneumovirus (hMPV) was discovered in 2001, but has been ubiquitous for at least 50 years and in all parts of the globe. It is now known to be the second most important cause of respiratory tract infections in young children. Clinical symptoms are similar to those of RSV and co-infection is sometimes seen, possibly leading to more severe disease. RSV and hMPV belong to the same family of viruses (Paramyxoviridae), and a

vaccine effective against both viruses would be highly desirable. Genetic hybrids of bovine PIV-3 expressing the fusion protein of hMPV are protective, immunogenic, and attenuated in African green monkeys, and warrant further evaluation in humans. Severe acute respiratory syndrome Severe acute respiratory syndrome (SARS) spread alarmingly in Southeast Asia in 2002–03. It was rapidly discovered that a coronavirus (SARS-CoV) was the cause, probably from wild-caught animals (e.g., via civet cats). The high mortality rate and potential for rapid spread gave great urgency to conventional (Table 2) and DNA vaccine initiatives (Table 3). A range of animal models has been used for testing, as no single model seems ideal. One particular concern is the possibility of enhancing disease (in a manner similar to that seen with formalin-inactivated RSV and feline peritonitis due to a coronavirus infection of cats), an effect already seen in murine models of SARS. Since SARS has now disappeared, it now seems unlikely that the opportunity will arise to test vaccine efficacy. Avian influenza A viruses A more worrying problem is the emergence of avian influenza A viruses (IAVs). These have spread from wild aquatic birds to domestic poultry, and cause sporadic infections in

Table 2 Viral-vectored vaccines against SARS Viral vector

Encoded antigen

Animal model

Route of immunization

Antibody response

T-cell response

Challenge?

Adenovirus (Ad5) MVA

S,M,N S

Macaques BALB/c mice

| (S) |

| (N)


No Yes

Attenuated parainfluenza virus (BHPIV-3)

S

African green monkeys

Intramuscular Intramuscular and intranasal Intranasal, intratracheal

|




Yes

Protective immunity Reduced viral titers

S, spike protein; N, nucleocapsid; M, membrane protein of SARS-CoV; MVA, modified vaccinia Ankara. Table 3 DNA vaccines against SARS Encoded antigen

Animal model

Route of immunization/ delivery method

Antibody response

T-cell response

Challenge?

Protective immunity

S

BALB/c mice

Intramuscular

|

|

Yes

N

C57BL/6 mice

Gene gun delivery

|

|

Yes, with rVV-N

Reduced viral titer Reduced viral titer

N N

C3H/He mice BALB/c mice

Intramuscular Intramuscular

|–CTL |–CTL

No No

M and N

SCID-PBL/hu mice

Intramuscular

| |–Dominant IgG2a


|–CTL induction and T-cell proliferation

No

S, spike protein; N, nucleocapsid; M, membrane protein of SARS-CoV; rVV-N, recombinant vaccinia virus expressing N protein of SARS-CoV; CTL, cytotoxic T lymphocytes.

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NA Modified virus harvested

H5N1

Candidate vaccine for testing

HA (1)

(6)

Embryonated chicken eggs

Transfection into Vero cells

(2)

RNA extraction

cDNA

PB2

(5)

NS

PB1

Basic amino acids at HA cleavage site replaced with avirulent sequence

PA

+

M

NP

Modified HA and NA cloned into expression vectors (3)

(4)

Figure 2 Molecular approach to production of influenza vaccine as discussed by Webby RJ et al. (1) H5N1 virus passaged in 10-dayold embryonated chicken eggs. (2) Total RNA is extracted and used to create a cDNA copy for polymerase chain reaction (PCR). (3) PCR used to amplify HA and NA genes using primers designed to modify specific sites in the sequence known to be associated with avirulent virus. (4) PCR products cloned into mammalian expression vector. (5) These plasmids and the six other plasmids expressing the remaining genes are transfected into Vero cells to reform the avirulent virus. (6) After 72 h of culture, cell supernatants are harvested and the virus extracted for testing. H, HA, hemaglutinin; N, NA, neuraminidase; NP, nucleoprotein; M, matrix protein; NS, non-structural protein; PB1, PB2, PA, RNA polymerase proteins.

humans. Initial reports came from Asia where H5N1 IAV was found to cause disease in chickens, big wild animals, and pet cats. Among 129 confirmed cases of H5N1 IAV infected humans, 60 were fatal by the time of writing (October 2005). Infections have been reported in the Netherlands (H7N7), the USA (H7N2), Hong Kong (H9N2), Canada (H7N3), and Egypt (H10N7), leading to fever, cough, and/or conjunctivitis progressing to respiratory failure and death in some infected persons. A major problem will be the ability to identify the correct subtype, scale up production, test vaccine stocks, and send out supplies of vaccine in time to have a significant impact on worldwide spread (Figure 2).

Vaccines for the Twenty-First Century Genetically Engineered Vaccines (Recombinant DNA Technology)

As previously described, it is now possible to genetically engineer not only DNA virus vectors, but to create novel RNA viruses by reverse genetics. It is therefore feasible to coexpress cytokines or chemokines

from the host to cause selective enhancement of certain types of immunity that may be protective and nonpathogenic, reducing reactivity of a vaccine and lessening the chances of disease augmentation. Mutations in spontaneous attenuated mutants can be identified and engineered, and intelligent knockout live vectors created. In the long term, it may be possible to understand the reasons why some pathogens evade immune recognition and persist. Once this is understood, vaccines that are able to modify immune responses to clear persistent infections may become feasible. ‘Naked DNA’ Vaccines

Injection of DNA that encodes viral antigens that confer protective immunity is able, under some conditions, to trigger protective immune responses (Figure 3). This surprising effect seems to be due to DNA uptake by antigen-presenting cells, and subsequent protein expression. Typically, the response includes cytotoxic T cells and in some cases good antibody responses. DNA has the advantage of stability and safety, but needs to be given in large quantities to achieve immunogenicity. There is

398 VACCINATIONS / Viral

Transfection of somatic cells (1)

(2)

APC (3) (5)

(4) CD8+ IL-2 Memory CTL pool

B cell CD4+ Memory B cell pool Th1 cells

IFN-
Figure 3 How DNA vaccination works. (1) DNA vaccine administered intramuscularly by injection or gene gun targets myocytes or keratinocytes. Once inside the target cell, the specified antigen is expressed using DNA replication material present. (2) APCs can be directly transfected with the plasmid, or the target cell acts as the APC directly. However, the majority of evidence exists for the secretion of antigen and phagocytosis/endocytosis by APCs for presentation via either the major histocompatibility complex (MHC) class I or class II pathways. (3) Presentation via MHC class I induces CD8 þ T cells to differentiate into effector and memory T cells, with the capacity to kill virus-infected cells. (4) Presentation via MHC class II induces CD4 þ T-cell activation. In turn, these cells, known as helper T (Th1) cells, function to promote B-cell antibody production and survival via CD40L–CD40 stimulation. This same interaction and interleukin (IL-2) secretion acts to help CD8 þ T-cell functions. CD4 þ T-cells also secrete a multitude of cytokines (e.g., interferon gamma, IFN-g) with consequent immunoregulatory effects. (5) A humoral response is also elicited following antigen encounter, which can lead to a pool of memory B cells as well as neutralizing antibody production.

currently much interest in finding ways to make lower quantities of DNA immunogenic with adjuvants, and to deliver DNA vaccines using gene guns or by mucosal administration. See also: Antiviral Agents. Human Immunodeficiency Virus. Pneumonia: Viral. Viruses of the Lung.

Further Reading Dudas RA and Karron RA (1998) Respiratory syncytial virus vaccines. Clinical Microbiology Reviews 11: 430–439. Fouchier RA, Rimmelzwaan GF, Kuiken T, and Osterhaus AD (2005) Newer respiratory virus infections: human metapneumovirus, avian influenza virus, and human coronaviruses. Current Opinion in Infectious Diseases 18: 141–146. Gonzalez IM, Karron RA, Eichelberger M, et al. (2000) Evaluation of the live attenuated cpts 248/404 RSV vaccine in combination with a subunit RSV vaccine (PFP-2) in healthy young and older adults. Vaccine 18: 1763–1772. Harper SA, Fukuda K, Cox NJ, and Bridges CB (2003) Using live attenuated influenza vaccine for prevention and control of

influenza: supplemental recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recommendations and Reports 52: 1–8. Ison MG, Johnston SL, Openshaw P, Murphy B, and Hayden F (2004) Current research on respiratory viral infections: Fifth International Symposium. Antiviral Research 62: 75–110. Karron RA, Belshe RB, Wright PF, et al. (2003) A live human parainfluenza type 3 virus vaccine is attenuated and immunogenic in young infants. Pediatric Infectious Disease Journal 22: 394–405. Olszewska W and Openshaw PJW (2004) Mucosal vaccination. In: Kaufmann SHE (ed.) Novel Vaccination Strategies, pp. 343– 363. Weinheim: Wiley. Olszewska W, Zambon M, and Openshaw PJM (2002) Development of vaccines against common colds. British Medical Bulletin 62: 99–111. Openshaw PJM and Tregoning JS (2005) Immune responses and disease enhancement during respiratory syncytial virus infection. Clinical Microbiology Reviews 18: 541–555. Stark CJ and Atreya C (2005) Molecular advances in the cell biology of SARS-CoV and current disease prevention strategies. Journal of Virology 2: 35–42. Stephenson I, Nicholson KG, Wood JM, Zambon MC, and Katz JM (2004) Confronting the avian influenza threat: vaccine

VASCULAR DISEASE 399 development for a potential pandemic. Lancet Infectious Diseases 4: 499–509. Tang RS, Mahmood K, Macphail M, et al. (2005) A host-range restricted parainfluenza virus type 3 (PIV3) expressing the human metapneumovirus (hMPV) fusion protein elicits protective immunity in African green monkeys. Vaccine 23: 1657–1667.

Webby RJ, Perez DR, Coleman S, et al. (2004) Responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines. Lancet 363: 1099–1103. Wood JM and Robertson JS (2004) From lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza. Nature Reviews: Microbiology 2: 842–847.

VASCULAR DISEASE I Adatia, University of California, San Francisco, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pulmonary arterial hypertension is defined as a mean pulmonary artery pressure above 25 or 30 mmHg with exercise. Pulmonary arterial hypertension may be idiopathic, familial, or associated with conditions such as congenital heart disease, liver disease and connective tissue disease. Pulmonary artery hypertension may also occur after ingestion of appetite suppressant drugs and toxic oils. The histological sequential changes are smooth muscle hypertrophy of the arterial wall, intimal proliferation, in situ thrombosis, small vessel occlusion, and the formation of plexiform lesions. The cross-sectional area of the pulmonary vascular bed is diminished severely by small vessel obliteration. The progressive and sustained elevation in pulmonary vascular resistance leads to right heart insufficiency. After the onset of symptoms, the clinical course is unremitting with progressive right heart failure until death. Until the last decade, conventional therapy was limited to digoxin, diuretics, calcium channel blockers, and warfarin anticoagulation. Median survival was 3.5 years. However, therapies now include prostacyclin and analogs delivered intravenously, subcutaneously or by inhalation, endothelin receptor blockers (bosentan), phosphodiesterase type 5 inhibitors (sildenafil), and continuous inhalation of nitric oxide gas. Pulmonary veno-occlusive disease is a form of pulmonary arterial hypertension associated with significant venous involvement. It is usually idiopathic but may be associated with malignant disease or treatment, congenital heart disease, and autoimmune disease. Pulmonary veno-occlusive disease is difficult to treat and pulmonary edema may be exacerbated by vasodilators. Early lung transplantation is the current treatment. Pulmonary arteriovenous malformations are characterized by abnormally large communications between the pulmonary arterial and venous systems. The physiological consequences are secondary to hypoxemia caused by the right to left shunt of desaturated pulmonary arterial blood into the pulmonary vein without traversing the pulmonary vascular alveolar interface. The etiology of pulmonary arteriovenous malformations may be idiopathic, or associated with hereditary hemorrhagic telangiectasia syndrome (Osler–Rendu–Weber syndrome), in patients with liver disease associated with portal hypertension. Pulmonary arteriovenous malformations complicate the course of patients with complex congenital heart disease who receive palliation with a superior cavopulmonary anastomosis. Treatment is by coil embolization of large malformations.

Pulmonary Arterial Hypertension Pulmonary arterial hypertension (PAH) is defined as a mean pulmonary artery pressure above 25 mmHg or 30 mmHg with exercise. PAH may be idiopathic, familial, or associated with other conditions such as congenital heart disease, liver disease, and connective tissue disease. It may also occur after ingestion of appetite suppressant drugs and toxic oils. The histological sequential changes are smooth muscle hypertrophy of the arterial wall, intimal proliferation, in situ thrombosis, small vessel occlusion, and the formation of plexiform lesions. The crosssectional area of the pulmonary vascular bed is diminished severely by small vessel obliteration. The progressive and sustained elevation in pulmonary vascular resistance leads to right heart insufficiency. After the onset of symptoms, the clinical course is unremitting with progressive right heart failure until death. Until the last decade, conventional therapy was limited to digoxin, diuretics, calcium channel blockers, and warfarin anticoagulation. Median survival was 3.5 years. However, therapies now include prostacyclin and analogs delivered intravenously, subcutaneously, or by inhalation, endothelin receptor blockers (bosentan), phosphodiesterase type 5 inhibitors (sildenafil), and continuous inhalation of nitric oxide (NO) gas. The early descriptions of pulmonary hypertensive disorders were histological. In 1891, E von Romberg described histological features of ‘pulmonary sclerosis’. In 1901, Dr A Ayerza described a syndrome of chronic cyanosis, dyspnea and polycythemia associated with pulmonary artery sclerosis. The etiology of the disease was obscure and obscured further by attributing it to syphilis. Brenner, in 1935, observed the disease to be a manifestation of right heart failure secondary to pulmonary disease. The introduction of cardiac catheterization allowed not only direct measurement of pulmonary pressures but also investigation of the functional state of the pulmonary vascular bed. In the late 1940s and 1950s hypoxic vasoconstriction

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and the effect of vasodilator drugs were described by von Euler, Cournand, Harris, Dresdale and Wood. In 1965, an epidemic of pulmonary hypertension was described and linked to the appetite suppressant drug aminorex. In 1973, the WHO convened a meeting to standardize clinical and pathological nomenclature. PAH was classified as primary or idiopathic and secondary. In 1981, the NIH registry was created to collect information of the clinical disease. In 1998 at the second WHO conference (Evian conference), a classification of pulmonary hypertension was put forth (Table 1). In 2003 (Venice conference), the classification was changed to reflect increased understanding of the disease, especially the genetic components (Table 2), pulmonary vascular disease associated with the congenital systemic to pulmonary shunts (Table 3) and the risk factors for pulmonary hypertension (Table 4). Etiology

The genetics of primary PAH Familial versus idiopathic sporadic disease The first report of the familial occurrence of primary pulmonary hypertension appears to have been in 1927. Clarke et al. described the clinical and autopsy findings of primary pulmonary hypertension in sisters (aged 5 and 8 years). They credited the suggestion of an ‘inherited etiological factor’ to Kidd in 1904. However, Dresdale et al. were the first to document familial transmission of the disease from one generation to the next. They described a woman and her son, who died from primary pulmonary hypertension aged 43 and 21 years old, respectively. The mother had, in addition, lost a brother (‘at a young age’) and sister (aged 31 years) from right heart failure, presumably due to primary pulmonary hypertension. These early clinical observations describe many of the now established features of the inheritance of familial primary pulmonary hypertension including vertical transmission, genetic anticipation, and the curious observation that within a family affected males are clinically symptomatic and die younger than females. Remarkable advances in the understanding of the genetics of primary pulmonary hypertension have emerged from the first clinical observations of familial pulmonary hypertension, 70 years ago, to the identification of the genetic mutations encoding bone morphogenetic protein receptor 2 (BMPR2). The incidence of familial primary pulmonary hypertension would appear to be 1 to 2 cases per million with 6% of the cases in the NIH Registry fulfilling the criteria for the disease, although subsequent observations suggest that this is an underestimate with at

Table 1 The Evian clinical classification 1. Pulmonary arterial hypertension 1.1 Primary pulmonary hypertension (a) Sporadic (b) Familial 1.2 Related to (a) Collagen vascular disease (b) Congenital systemic-to-pulmonary shunts (c) Portal hypertension (d) Human immunodeficiency virus infection (e) Drugs/toxins (1) Anorexigens (2) Other (f) Persistent pulmonary hypertension of the newborn (g) Other 2. Pulmonary venous hypertension 2.1 Left-sided atrial or ventricular heart disease 2.2 Left-sided valvular heart disease 2.3 Extrinsic compression of central pulmonary veins (a) Fibrosing mediastinitis (b) Adenopathy/tumors 2.4 Pulmonary veno-occlusive disease 2.5 Other 3. Pulmonary hypertension associated with disorders of the respiratory system or hypoxemia 3.1 Chronic obstructive pulmonary disease 3.2 Interstitial lung disease 3.3 Sleep-disordered breathing 3.4 Alveolar hypoventilation disorders 3.5 Chronic exposure to high altitude 3.6 Neonatal lung disease 3.7 Alveolar-capillary dysplasia 3.8 Other 4. Pulmonary hypertension caused by chronic thrombotic or embolic disease 4.1 Thromboembolic obstruction of proximal pulmonary arteries 4.2 Obstruction of distal pulmonary arteries (a) Pulmonary embolism (thrombus, tumor, ova, or parasites, foreign material) (b) In situ thrombosis (c) Sickle-cell disease 5. Pulmonary hypertension caused by disorders directly affecting the pulmonary vasculature 5.1 Inflammatory (a) Schistosomiasis (b) Sarcoidosis (c) Other 5.2 Pulmonary capillary hemangiomatosis Reproduced from Simonneau G, Galie N, Rubin LJ, et al. (2004) Clinical classification of pulmonary hypertension. Journal of the American College of Cardiology 43: 5S–12S, with permission from The American College of Cardiology Foundation.

least 50 affected families in the US. Familial primary pulmonary hypertension differs from nonfamilial or sporadic forms of the disease with an earlier diagnosis after the onset of symptoms. Familial primary pulmonary hypertension is otherwise clinically indistinguishable from the sporadic form with a female to male ratio of 2 : 1 in adults but not children in whom the ratio is 1.3 : 1.

VASCULAR DISEASE 401 Table 2 Revised clinical classification of pulmonary hypertension (Venice 2003)

Table 3 Guidelines for classification of congenital systemic-topulmonary shunts

1. Pulmonary arterial hypertension (PAH) 1.1. Idiopathic (IPAH) 1.2. Familial (FPAH) 1.3. Associated with (APAH) 1.3.1. Collagen vascular disease 1.3.2. Congenital systemic-to-pulmonary shunts 1.3.3. Portal hypertension 1.3.4. HIV infection 1.3.5. Drugs and toxins 1.3.6. Other (thyroid disorders, glycogen storage disease, Gaucher disease, hereditary hemorrhagic telangiectasia, hemoglobinopathies, myeloproliferative disorders, splenectomy) 1.4. Associated with significant venous or capillary involvement 1.4.1. Pulmonary veno-occlusive disease (PVOD) 1.4.2. Pulmonary capillary hemangiomatosis (PCH) 1.5. Persistent pulmonary hypertension of the newborn 2. Pulmonary hypertension with left heart disease 2.1. Left-sided atrial or ventricular heart disease 2.2. Left-sided valvular heart disease 3. Pulmonary hypertension associated with lung diseases and/or hypoxemia 3.1. Chronic obstructive pulmonary disease 3.2. Interstitial lung disease 3.3. Sleep-disordered breathing 3.4. Alveolar hypoventilation disorders 3.5. Chronic exposure to high altitude 3.6. Developmental abnormalities 4. Pulmonary hypertension due to chronic thrombotic and/or embolic disease 4.1. Thromboembolic obstruction of proximal pulmonary arteries 4.2. Thromboembolic obstruction of distal pulmonary arteries 4.3. Non-thrombotic pulmonary embolism (tumor, parasites, foreign material) 5. Miscellaneous Sarcoidosis, histiocytosis X, lymphangiomatosis, compression of pulmonary vessels (adenopathy, tumor, fibrosing mediastinitis)

1. Type Simple Atrial septal defect (ASD) Ventricular septal defect (VSD) Patent ductus arterious Total or partial unobstructed anomalous pulmonary venous return Combined Describe combination and define prevalent defect if any Complex Truncus arteriosus Single ventricle with unobstructed pulmonary blood flow Atrioventricular septal defects 2. Dimensions Small (ASDr2.0 cm and VSDr2.0 cm) Large (ASD42.0 cm and VSD42.0 cm) 3. Associated extracardiac abnormalities 4. Correction status Noncorrected Partially corrected (age) Corrected: spontaneously or surgically (age)

Main modifications to the previous Evian clinical classification are set in bold in table body. These include: idiopathic pulmonary hypertension instead of primary hypertension; some newly identified possible risk factors and associated conditions have been added in the APAH subgroup (glycogen storage disease), Gaucher disease, HHT, hemoglobinopathies, myeloproliferative disorders, splenectomy; another subgroup has been added in the PAH category: PAH associated with significant venous or capillary involvement (PVOD and PCH); the last group now termed ‘miscellaneous’ includes some conditions associated with pulmonary hypertension of various and multiple etiologies (histiocytosis X, lymphangiomatosis, compression of pulmonary vessels by adenopathy, tumor, fibrosing mediastinitis). Reproduced from Simonneau G, Galie N, Rubin LJ, et al. (2004) Clinical classification of pulmonary hypertension. Journal of the American College of Cardiology 43: 5S–12S, with permission from The American College of Cardiology Foundation.

Familial primary pulmonary hypertension is transmitted vertically and in one family as many as five successive generations have been affected. The mode of transmission excludes X linkage and is highly

Reproduced from Simonneau G, Galie N, Rubin LJ, et al. (2004) Clinical classification of pulmonary hypertension. Journal of the American College of Cardiology 43: 5S–12S, with permission from The American College of Cardiology Foundation.

suggestive of an autosomal dominant gene. However, despite fulfilling criteria for autosomal dominant transmission, not all offspring are affected and not all carriers of the gene manifest the disease. Autosomal dominance with variable and lower penetration in males may explain the female preponderance of adults who carry the gene and manifest the disease. Alternatively, the disease may affect males more severely at a younger age, thus limiting the males who survive to adulthood. In addition, fewer males are born to families who carry the gene, supposedly due to selective wastage of the male. The observation that within families with primary pulmonary hypertension successive generations are more severely affected at a younger age is termed genetic anticipation. Trinucleotide repeat amplification has been demonstrated in other diseases (e.g., fragile X syndrome), which show genetic anticipation, but not in primary pulmonary hypertension. Chromosome 2q33 and BMPR2 Independently, two groups have linked familial primary pulmonary hypertension with definitive ‘logarithm of odds’ (LOD) scores to chromosome 2q33. Asymptomatic carriers may demonstrate an abnormal pulmonary vascular response to exercise. Genetic testing offers the opportunity of close observation and early treatment if disease develops in families at risk. However,

402 VASCULAR DISEASE Table 4 Risk factors and associated conditions for PAH identified during the Evian meeting (1998) and classified according to the strength of evidence A. Drugs and toxins 1. Definite
Aminorex
Fenfluramine
Dexfenfluramine
Toxic rapeseed oil 2. Very likely
Amphetamines
L-tryptophan 3. Possible
Meta-amphetamines
Cocaine
Chemotherapeutic agents 4. Unlikely
Antidepressants
Oral contraceptives
Estrogen therapy
Cigarette smoking B. Demographic and medical conditions 1. Definite
Gender 2. Possible
Pregnancy
Systemic hypertension 3. Unlikely
Obesity C. Diseases 1. Definite
HIV infection 2. Very likely
Portal hypertension/liver disease
Collagen vascular diseases
Congenital systemic-pulmonary-cardiac shunts 3. Possible
Thyroid disorders Reproduced from Simonneau G, Galie N, Rubin LJ, et al. (2004) Clinical classification of pulmonary hypertension. Journal of the American College of Cardiology 43: 5S–12S, with permission from The American College of Cardiology Foundation.

more than just genotype is required for clinical disease to develop, and interaction with environmental or other genetic traits are important. Thus, identification of haplotype insufficiency may provoke more anxiety than benefit. Fifty percent of familial and 25% of sporadic cases of primary PAH are caused by mutations in the gene encoding BMPR2. Over 25 different mutations have been identified. A specific mutation is transmitted within a family. BMPR2 is part of the transforming growth factor beta (TGF-b) pathway. The TGF-b signaling pathway is important in maintaining vascular and endothelial cell integrity. TGF-b inhibits vascular endothelial cell proliferation in culture which may be pertinent to the observation that the plexiform lesions in patients with primary pulmonary hypertension represent abnormally proliferating

monoclonal endothelial cells. Mutations of a TGF-b type 1 receptor gene, activin-receptor-like kinase 1 (ALK1), and endoglin, an accessory protein of the TGF-b receptor complex, are present in hereditary hemorrhagic telangiectasia (HHT). Patients with HHT may develop pulmonary arteriovenous malformations but very rarely pulmonary hypertension. In fact patients with pulmonary arteriovenous malformations usually have low pulmonary artery pressures and resistances. However, 10 patients with HHT and pulmonary hypertension and mutations in ALK1 have been described. This suggests that mutations in the TGF-b pathway are associated with diverse vascular effects, including both abnormal dilatation, proliferation, and small vessel occlusion. Interestingly, pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension exhibit abnormal growth responses to TGF-b. Gene transfer of vascular endothelial growth factor (VEGF) attenuates monocrotaline-induced pulmonary hypertension mice and illustrates the potential impact of gene therapy. In summary, familial primary pulmonary hypertension is transmitted as an autosomal dominant gene with incomplete penetrance. From generation to generation, there is genetic anticipation and a preponderance of affected adult females with a reduction in male progeny. The discovery of the genetic mutations of BMPR2 on chromosome 2q33 in 25% of sporadic and 50% of families with primary pulmonary hypertension will focus research efforts toward early detection of disease in asymptomatic carriers, better understanding of the triggers that result in clinical disease in the genetically predisposed, and finally targeting the gene therapeutically. Pathology

The pathological changes of pulmonary artery hypertension whether familial, idiopathic, or associated with connective tissue disease (scleroderma), congenital heart disease, liver disease, or toxin may be indistinguishable. They include progressive changes with medial hypertrophy, intimal proliferation, obliteration of small vessels, and the formation of plexiform lesions. The latter are abnormalities in angiogenesis that may represent attempts at recanalization of obliterated vessels. In pulmonary artery hypertension associated with chronic thromboembolic disease, thrombosis is prominent. Two variants of PAH with different histological features are pulmonary veno-occlusive disease (PVOD) and pulmonary capillary hemangiomatosis.

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The histological features of PVOD are typified by complete or partial obliteration of small pulmonary veins and venules by intimal proliferation and the deposition of collagen and reticular fibers. The vein lumen may have the appearance of multiple septations that are thought to represent attempts at vein recanalization. Crescent-shaped lesions or irregular thick septa divide the vein lumen into compartments and may extend from pulmonary veins into bronchial veins. There may be secondary thrombosis of the vein. The small pulmonary veins are arterialized with medial hypertrophy. The extrapulmonary veins are usually normal, although large intrapulmonary veins may be affected. The paucity of normal intrapulmonary veins is a hallmark of the disease. The interlobular septa are markedly thickened containing congested lymphatics with many hemosiderin-laden macrophages. These are the histological correlates of Kerley B lines and thickened septa visible on chest X-ray and CT scan and evidence of postcapillary obstruction with passive congestion, focal hemorrhage, and hemosiderosis. Venous infarcts may occur adjacent to the interlobular septa. Pulmonary artery morphometry demonstrates marked medial hypertrophy in pre- and intra-acinar arteries. However, advanced pulmonary artery changes of fibrinoid necrosis, angiomatoid, or plexiform lesions are not found usually. Pulmonary capillary hemangiomatosis is characterized by localized capillary proliferation within the lung in which capillaries invade pulmonary interstitium, vessels, and even airways. Clinical features Most patients present with progressive exercise intolerance and dyspnea. Chest pain, right ventricular angina, syncope and sudden death, and cyanosis are also presentations. There may be a family history. Examination reveals evidence of right ventricular hypertrophy (RVH) with precordial bulge, accentuation of the second heart sound, which is often palpable, murmurs of tricuspid and pulmonary insufficiency. Secondary signs of right ventricular failure may be present with elevation in the jugular venous pulse (JVP), hepatomegaly, peripheral edema, pleural, and pericardial effusions. The electrocardiogram demonstrates RVH with strain and right atrial enlargement. The chest X-ray provides valuable evidence of pulmonary hypertension with cardiac enlargement and characteristic enlargement of the proximal pulmonary arteries with distal pruning. Pulmonary edema usually suggests pulmonary venous hypertension. Echocardiography is extremely useful both in the diagnostic workup and in sequential evaluation of the patient. Right-sided chamber enlargement is

prominent. The systolic pulmonary artery pressure may be estimated from the velocity of the tricuspid regurgitant jet and mean pulmonary artery pressure from the velocity of pulmonary insufficiency. The left heart may be compressed and ‘pancaked’ by the right ventricle and impair left ventricular filling in severe disease. The echocardiogram is also important to rule out associated causes of pulmonary hypertension such as cardiac shunts, anomalies of pulmonary venous return, and extraparenchymal pulmonary vein stenosis. In addition, left heart inflow and outflow anomalies such as cor triatriatum, mitral stenosis, and left heart failure can be diagnosed or excluded. CT scan is used to diagnose thromboembolic pulmonary vascular disease and PVOD, and to exclude parenchymal lung disease and mediastinal processes that may compress the pulmonary veins (Figure 1). MRI is increasingly used to provide not only images of the pulmonary arteries and veins but also pulmonary blood flow data. Nuclear ventilation–perfusion scans (VQ scan) may also be useful in the exclusion of pulmonary thromboembolic disease. The distance walked in 6 min is extremely useful as a prognostic indicator and to evaluate serial improvement or deterioration. Cardiac catheterization with acute vasodilator testing is used as the final diagnostic test and indicator of disease severity. Right ventricular function is an important determinant of survival and a right atrial pressure of o7.5 mmHg suggests a better outcome. Similarly, a low mixed venous saturation does not bode well (o63%). The assessment of pulmonary vascular resistance is not without difficulty – both theoretical and practical. The relationship between pressure and flow in the pulmonary circulation is complex. The resistance to flow is a function of the number, luminal area and length of the vessels, or the architecture of the vascular bed. In addition, pulmonary blood flow, left atrial pressure, pulmonary vascular tone, and critical closing pressure are important. A measure of resistance to flow through the lung can be provided for by analogy with that used in an electrical circuit. Pulmonary arteriolar resistance index (PARI) is calculated from PARI ¼ mean PAp  LAp=Qp where PAp is the mean pulmonary artery pressure (mmHg), LAp is left atrial pressure, and Qp is pulmonary blood flow in l min  1 m  2. The latter is usually indexed for body surface area in children. Resistance units are expressed as Wood’s units (Um2) or multiplied by 80 to convert the value to dyne s cm  5.

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Figure 1 High-resolution thin slice CT scans demonstrating a ground glass ‘mosaic’ pattern of uneven pulmonary edema with thickened interlobular septal lines and dilated lymphatics in histologically confirmed pulmonary veno-occlusive disease. Reproduced from Freedom RM, Yoo S-J, Mikhailian H, and Williams WG (eds.) (2003) The Natural and Modified History of Congenital Heart Disease, UK: Blackwell.

The response to vasodilator drugs is currently defined as follows: Responders. These experience a reduction in pulmonary vascular resistance (X20%) associated with reduction in mean pulmonary artery pressure (X20%) and no change or an increase in cardiac index. Nonresponders. The reduction in pulmonary vascular resistance is p20%, without significant reduction in mean pulmonary artery pressure. Unfavorable responders. Symptomatic hypotension (or mean blood pressure fall of 420%) with a reduction in cardiac index or an elevation in right atrial pressure. Other investigations. These should include pulmonary function testing, overnight oximetry, screen for connective tissue disease, HIV testing, complete blood count (CBC) and platelet count, liver function tests, antiphospholipid antibodies, clotting studies, uric acid, brain natriuretic peptide, and troponin levels. Lung biopsy may be indicated on occasion.

Management and Current Therapy

Patients with pulmonary artery hypertension should be referred to a specialist pulmonary hypertension clinic. Patients who respond to acute vasodilator testing may be candidates for high-dose calcium channel blockade. However, newer oral therapies are replacing the use of calcium channel blockers because a substantial number of patients do not respond favorably and may experience deterioration in right heart function. The drugs used to treat PAH include continuous infusions of prostacyclin, aerosolized or subcutaneously administered prostacyclin analogs, endothelin receptor blockers (bosentan), phosphodiesterase type 5 inhibitors (sildenafil), and inhaled domiciliary NO. They have been shown to improve exercise capacity and survival of patients with pulmonary hypertension. Combination therapy is likely to be used increasingly in the future. Medical therapy has a considerable impact with survival at 1, 2, and 3

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years of 88%, 76%, and 63% which is significantly better than 59%, 46%, and 35% compared to historical controls. Warfarin anticoagulation is recommended for most patients even in the absence of thromboembolic disease, and has been shown to improve survival. Adjunctive therapies with diuretics and digoxin may be indicated. Other Therapies

Atrial septostomy In selected cases, atrial septostomy may be beneficial and protect against syncope. Atrial shunting from right to left permits left heart filling at the expense of cyanosis in patients with right heart failure. Pulmonary endarterectomy Pulmonary endarterectomy is indicated for certain patients with chronic thromboembolic disease and yields good results in expert centers. Lung transplantation Lung transplantation is the last resort for patients who fail medical therapy for pulmonary vascular disease. However, the success of medical therapy suggests that listing should no longer occur as soon as the diagnosis is made but rather wait a period of drug therapy. However, serious consideration of lung transplantation should occur with the onset of right ventricular failure or New York Heart Association class IV status when mean survival time is o6 months. Absence of response to acute vasodilator therapy, suprasystemic pulmonary artery pressures, syncope, or low cardiac output are signals of severe diseases and should prompt careful assessment by the transplant team.

PAH Associated with Liver Disease Portopulmonary Hypertension

Pulmonary hypertension complicating liver disease is recognized increasingly, although the reported incidence varies from 0.7% of autopsies undertaken in patients with liver disease to 10% of a general population of patients. However, in patients referred for liver transplant the incidence is as high as 16%. Pathogenesis

The mechanism of porto-PAH is unclear. There is no relationship between the development of pulmonary hypertension and severity of the liver disease or synthetic function. Pulmonary hypertension seems to be more closely related to the degree of portal hypertension as discussed in the section on hepatopulmonary syndrome. Patients with liver disease have increased cardiac output and the increased shear

stress may lead to endothelial damage and subsequent vasoconstriction. The pulmonary vascular disease may progress with clinical radiographic and histological features indistinguishable from familial or idiopathic pulmonary hypertension. The presence of severe pulmonary hypertension has a marked impact on survival, with a median survival of 6 months. Five-year actuarial survival is reported between 10% and 50%. Treatment of the pulmonary hypertension follows the same algorithm as for idiopathic pulmonary hypertension with the exception of liver transplantation. Mild to moderate pulmonary hypertension due to an elevated cardiac output is reversible following liver transplantation, without significantly higher mortality. Patients with severe pulmonary hypertension have a high mortality both from complications of pulmonary hypertension and right heart dysfunction and from the deleterious effect that elevated hepatic venous pressure has on the new graft.

Pulmonary Hypertension Related to Drugs and Toxins Table 4 lists the drugs, which have been linked to the development of PAH. In the 1960s, a dramatic increase in the patients diagnosed with PAH was described in overweight European women. Exposure to aminorex and fenfluramine, both appetite suppressant drugs, was found to be responsible. The incidence of pulmonary hypertension in patients exposed to these drugs was 1–3%. The clinical manifestations and hemodynamic findings were indistinguishable from idiopathic PAH. Exposure time to anorectic drugs was 3 months to 8 years. Compared with 34% at 5 years for idiopathic pulmonary hypertension, survival rates appeared better than for idiopathic pulmonary hypertension with 75% at 5 years and 44% at 10 years. In some patients, the pulmonary vascular disease was reversible. However, in many it ran the relentless course typical of other forms of PAH. ‘Toxic Oil Syndrome’ due to Contaminated Rapeseed Oil

In early 1981, in Spain, an outbreak of disease with pulmonary symptoms and interstitial infiltrates on chest X-ray but failure to regress with antibiotics occurred. PAH developed in 20% of hospitalized patients 2–4 months after exposure. The disease clustered in families but did not affect infants. The responsible agent or vehicle for the epidemic was traced to consumption of contaminated rapeseed oil. After 8 years follow-up, 8% went on to develop PAH. Malignant pulmonary hypertension

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with plexiform lesions occurred in 2% and in 75% the disease regressed. However, in a cohort of patients with severe PAH secondary to ingestion of contaminated rapeseed oil, the mortality was 83% at 6 years. Interestingly, the male-to-female ratio was 4 : 1.

Pulmonary Veno-Occlusive Disease PVOD causes pulmonary hypertension with pulmonary edema. It is now classified as a form of PAH associated with significant venous involvement (Table 2). The patients are often extremely symptomatic at presentation and present a diagnostic dilemma. The disease may be mistaken for idiopathic PAH until lung biopsy or autopsy reveals the correct diagnosis. Pulmonary vascular resistance is elevated secondary to postcapillary idiopathic diffuse fibrous obstruction of the intraparenchymal pulmonary venules and veins. Brown and Heath first used the term PVOD in 1966 to describe the clinical syndrome of progressive dyspnea, pulmonary edema, pulmonary hypertension, and right heart failure without evidence of left heart disease. PVOD accounts for about 10% of patients evaluated for primary pulmonary hypertension. The disease is, therefore, rare with an estimated incidence of 0.1–0.2 cases per million. Reported cases have an equal sex ratio, in contrast to primary PAH. The median age at disease onset is 29 years (range 9 days to 68 years) and 30% of reported cases occur in patients o17 years of age. In children, the sex ratio is 1 : 1. Etiology

PVOD may occur in siblings and has been described in brothers and brother and sister, thus excluding a sex-linked transmission. PVOD has not been described, as yet, in successive generations. However, it has been described in one family with familial PAH and one case has been described with the BMPR2 mutation. PVOD and associated disease PVOD is usually idiopathic but may be associated with malignant disease or treatment, autoimmune disease, and congenital heart disease. Malignancy and PVOD PVOD has been associated with lymphomas, leukemia, cerebral gliomas, melanoma, lung cancer, and cervical and gastric carcinoma. It may also occur during chemotherapy (bleomycin, 1,3-bis(2-chloroethyl)-1 nitrosurea,

mitomycin C, gemcitabine), radiation therapy, and bone marrow transplantation. Autoimmune disease PVOD has been associated with the Felty’s syndrome, systemic lupus erythematosis, CREST variant of scleroderma syndrome, immune complex deposition, Raynaud’s phenomenon, following renal transplantation membranoproliferative glomerulonephritis, hepatic cirrhosis (due to chronic active hepatitis), and coeliac’s disease. Despite the association with autoimmune disease and positive serology, a direct link to immune-mediated vascular lesions is lacking. Associations with congenital heart disease PVOD has been associated rarely with heart disease other than patency of the foramen ovale. Other reported associations are with human immunodeficiency virus, oral contraceptive use, pregnancy, preceding viral infection, and toxoplasmosis. Clinical Features

The majority of patients present with dyspnea or syncope on exertion. The severity of symptoms is often out of keeping with the presenting clinical examination or resting hemodynamics. Other reported symptoms include orthopnea, paroxysmal nocturnal dyspnea, and chest pain. Hemoptysis is rare, although presentation as life-threatening pulmonary hemorrhage and sudden infant death are described. Physical examination reveals signs of pulmonary hypertension (loud, single, or palpable P2, murmur of tricuspid or pulmonary regurgitation). Cyanosis and clubbing are unusual features of primary pulmonary arteriolar hypertension and their presence may alert one to the diagnosis of PVOD. Chest X-ray The chest X-ray demonstrates signs of pulmonary edema in 70% of reported cases. Kerley B lines are characteristic and represent septal thickening seen by CT scan. The distribution of pulmonary edema may be either diffuse or patchy, and unlike mitral stenosis there is absence of upper-zone pulmonary vein dilation and lower-zone vasoconstriction. The right ventricle, right atrium, and central pulmonary arteries are enlarged, but the peripheral lung fields are not oligemic. Echocardiography Echocardiography is extremely useful in the diagnosis of all forms of pulmonary hypertension. However, there are no unique features of PVOD. Echocardiography can, however, rule out other causes of pulmonary hypertension with pulmonary edema such as extraparenchymal pulmonary

VASCULAR DISEASE 407

vein compression, ostial pulmonary vein stenosis, cor triatriatum, supramitral stenosing ring, mitral valve stenosis, left atrial myxoma, cardiomyopathy, and shunt lesions. Cardiac catheterization and angiography The pulmonary artery pressures are elevated. Right atrial pressures may be normal or elevated depending upon severity of disease and the presence of right heart failure. The characteristic hemodynamic feature is the variability of the pulmonary artery occlusion or ‘wedge’ pressure. The pressure may be high, low, or normal in the same patient depending on catheter position. The pulmonary artery ‘wedge’ pressure reflects left atrial pressure if there is an uninterrupted column of blood between pulmonary artery distal to the occluding balloon, or catheter tip, and the left atrium. PVOD is a patchy disease and wedge pressures will be normal, high, or low depending on the patency of the downstream pulmonary veins. In most cases of PVOD, it is difficult to make the diagnosis unequivocally by angiography alone. The described abnormalities include a delayed and patchy transit time of contrast from pulmonary artery to appearance in the left atrium. The pulmonary artery may, in extreme cases, remain well opacified even after contrast appears in the aorta. Pulmonary angiography may help to exclude chronic thromboembolic disease or peripheral pulmonary artery stenoses as a cause of proximal pulmonary artery. CT scan High-resolution chest CT scan differentiates PVOD from primary PAH. The CT scan appearances that have been described include diffuse multifocal groundglass opacities, a mosaic pattern of attenuation with patchy areas of increased or decreased attenuation due to regional differences in perfusion, smooth septal interlobular thickening, centrilobular nodules, mediastinal hilar adenopathy (Figure 1) (in the absence of malignant disease), and pleural effusions. Other CT findings, enlarged central pulmonary arteries with normal size airways, normal central pulmonary veins, right atrial and ventricular enlargement are present but do not help to differentiate pulmonary arterial occlusive disease from PVOD. However, demonstration of a normal left atrium and extrapulmonary veins excludes left heart inflow obstruction as a cause of pulmonary venous hypertension. Early disease may not show all of these features; it may be difficult to differentiate PVOD from infiltrative lung disease by CT scan. VQ scan Lung perfusion scans may be entirely normal in 60% of cases. Nuclear lung perfusion scan may demonstrate a segmental contour pattern: small

inhomogeneous subsegmental matched or mismatched defects, multiple mismatched segmental perfusion defects (which may be mistaken for thromboemboli) or non-specific soft fluffy inequalities of perfusion. Pulmonary function tests A severe impairment of single breath carbon monoxide diffusing capacity (DLCO), especially with normal lung volumes and spirometry, is an extremely useful test result in the differential diagnosis of primary pulmonary hypertension. DLCO has been reported in approximately 40 patients with PVOD with a median DLCO of 55% of predicted range (14–102%). In comparison, patients with arterial pulmonary hypertension have DLCO that is mildly reduced (60–70%) or normal. Patients with pulmonary fibrosis and pulmonary hypertension will demonstrate a marked reduction in vital and total lung capacity as well as in DLCO. Pulmonary function testing may yield mild restrictive, obstructive, or combined patterns in a minority of patients with PVOD. Treatment and Outcome

The survival of patients after the diagnosis of PVOD is poor. Survival is generally supposed to be o2 years from diagnosis. Pulmonary edema, which may be fatal, has been reported after prostacyclin infusion; nevertheless, symptomatic improvement and prolonged survival have been reported with prostacyclin, prazosin, dipyridamole, inhaled NO, and calcium channel blockers. PVOD related to cancer therapy may respond to steroids and warfarin. Most would recommend early listing for lung transplantation, a cautious trial of vasodilator therapy, anticoagulation, and perhaps a short course of steroids. A single prolonged response to azothioprine in a woman with associated autoimmune disease has been described.

PAH Associated with Congenital Heart Disease Eisenmenger’s Syndrome

PAH occurs in patients with an unrestrictive communication between the pulmonary and systemic circulations. The clinical manifestations of the shunt depend on the pulmonary vascular resistance. If the pulmonary vascular resistance is less than the systemic vascular resistance, the shunt is from left to right. In general, surgical intervention at this stage will halt the progression of the pulmonary vascular disease. As pulmonary vascular disease progresses, the shunt reverses and becomes predominantly right

408 VASCULAR DISEASE

to left. At this stage, the clinical symptoms are related to cyanosis (the Eisenmenger’s syndrome). In 1897, V Eisenmenger described the autopsy findings in a 32-year-old man with a large ventricular septal defect (VSD) and pulmonary hypertension. However, P Wood in 1958 defined the disease in a masterly account, which remains relevant to our understanding of the clinical syndrome today. P Wood used the term ‘Eisenmenger’s syndrome’ to describe patients with pulmonary hypertension at systemic level, due to a high pulmonary vascular resistance (4800 dyne s cm  5 or 10 Wood units m2) with a reversed or bidirectional shunt at aortopulmonary, ventricular, or atrial level . The term ‘Eisenmenger complex’ refers specifically to patients in whom the fundamental lesion is a VSD. At birth, the pulmonary artery pressure is close to the systemic pressure. Within the first hours and days of life, pulmonary artery pressure decreases rapidly. If successful transition of the pulmonary circulation occurs, the pulmonary artery mean pressure falls to 10–20 mmHg and is similar to adult levels in the first 3 weeks of life. The development of the pulmonary vascular bed is altered in neonates with a large left to right shunt and pulmonary hypertension and, if the defect remains uncorrected, can progress to obliterative pulmonary vascular obstructive disease. There is, however, great variability in this rate of progression. The type of congenital abnormality and the age of the patient are the two most important factors, which determine the onset of pulmonary vascular disease. Children with a large unrestrictive VSD, patent ductus arteriosus with both increased pulmonary flow and pressure develop pulmonary vascular changes more rapidly and should undergo correction in the first 3–12 months of life. Occasionally, severe pulmonary vascular disease may develop in association with a small congenital shunt lesion or continue to progress despite early correction. Childhood Eisenmenger’s syndrome is becoming increasingly rare in countries where early diagnosis and surgical correction of congenital heart disease is routine. However, in less fortunate countries, the incidence of pulmonary vascular disease that precludes surgical repair of a shunt lesion was 21%. Etiology

The endothelium situated between vascular smooth muscle and the vessel lumen is ideally located to influence vascular tone. Pulmonary endothelial cells when exposed to high shear stress from birth demonstrate structural abnormalities. Functional abnormalities are thought to precede morphological

damage raising the possibility that vasoactive mediators produced by the endothelium may contribute to the evolution of pulmonary vascular disease. The endothelium produces both vasodilators and vasoconstrictors and modulates the adhesion and aggregation of platelets. Thus, the changes of pulmonary hypertensive disease (including vasoconstriction, smooth muscle hypertrophy, and vessel obliteration) could be mediated through an imbalance in the production of endothelial-derived mediators subsequent to endothelial injury. Abnormalities in the balance of thromboxane, prostacyclin, endothelin, and NO have been described in patients with pulmonary vascular disease. In addition, genetic factors play a role in the development of pulmonary vascular disease associated with congenital heart disease. Pathology

The pathology of pulmonary vascular disease secondary to congenital heart disease is indistinguishable from that of familial and sporadic PAH. Except that the plexiform lesions of patients with familial primary pulmonary hypertension contain monoclonal proliferating endothelial cells in contrast to the polyclonal endothelial cell proliferation in pulmonary hypertension due to congenital cardiac shunts. There are two pathological classifications of pulmonary vascular disease associated with congenital heart disease. They were developed primarily to aid in diagnosis of disease that would predict a favorable response to surgical correction. Heath and Edwards classified the progression from grade 1 (medial hypertrophy with extension of smooth muscle into normally nonmuscular arteries) through to grade 6 (the stage of necrotizing arteritis with plexiform lesions). The more recent classification of Rabinovitch is as follows: grade A – the appearance of muscle more peripherally than usual with or without medial hypertrophy of normally muscular vessels; grade B – distal extension of muscle plus medial wall thickness 1.5–2 times normal (B mild) or more than two times normal (B severe); and grade C – grade B plus decreased density of peripheral arteries relative to alveoli with a o50% decrease in density of peripheral arteries being grade C mild and a reduction of 450% grade C severe. These grades have been correlated with both pulmonary artery wedge angiography and pulmonary artery pressure and resistance.

VASCULAR DISEASE 409 Clinical Features

The clinical features of Eisenmenger’s syndrome are related to cyanosis, polycythemia, right heart failure (exacerbated by atrioventricular valve and semilunar valve regurgitation), and pulmonary artery dilation. Patients with complex disease truncus arteriosus, atrioventricular septal defect, univentricular connection, and transposition are symptomatic at a younger age and have poorer outcome. Patients with trisomy 21 also fare less well. In general, symptoms are slowly progressive with age and become more prominent in late adolescence and adulthood. Effort intolerance is almost invariably present. Cyanosis is at first present with exercise but becomes constant as the disease progresses and reflects in most cases the severity of the right to left shunt. The mean arterial oxygen saturation is 83.677.7% at presentation. Clubbing is invariable once cyanosis becomes persistent and may progress to hypertrophic osteoarthropathy with arthralgia and joint effusions. Hematological abnormalities ensue from the hypoxemia, which results in erythrocytosis. Elevations in hemoglobin improve oxygen-carrying capacity and patients are not usually symptomatic from the hyperviscosity syndrome until the hemoglobin level rises above 18–20 g l  1. Symptoms attributed to hyperviscosity include headache, dizziness, and visual disturbance including rarely central retinal vein occlusion. Iron deficiency together with erythrocytosis aggravates the hyperviscosity and is a risk factor for cerebrovascular hemorrhage and stroke. Thrombocytopenia, prolonged bleeding times, abnormal prothrombin time and partial thromboplastin time with deficient clotting factors, and abnormal fibrinolysis have been described and contribute to a bleeding diathesis and prolonged bleeding after surgical and dental procedures. Hemoptysis (20.2%) may be due to intrapulmonary bleeding, ruptured bronchial artery, or rupture of a pulmonary artery aneurysm from progressive central pulmonary artery dilation. Pulmonary embolism and in situ thrombosis within dilated pulmonary arteries may also lead to hemoptysis (13.2%). Hyperuricemia is common due to increased production and decreased renal clearance of uric acid. Gout is less common affecting 13–23% of patients. Cholelithiasis and cholecystitis (15%) may occur from increased bilirubin in bile secretions secondary to high erythrocyte turnover. Renal dysfunction with proteinuria (65%) may progress to the nephrotic syndrome, and elevated serum creatinine is an independent risk factor for reduced survival.

Cerebral complications include stroke (7.9%), cerebral abscess (3.7%), and occur at a mean age of 31.4715.7 and 24.174.9 years, respectively. Arrhythmias include isolated supraventricular ectopics and ventricular ectopics (42%), atrial flutter, fibrillation (35.5%), and ventricular tachycardia (22%). Endocarditis (3.7%) may complicate outcome. Hoarseness and cough due to laryngeal nerve compression by dilated pulmonary arteries have been reported. Likewise, dilated pulmonary arteries may compress the left coronary artery with angina. Sudden death occurs in 30% of patients. Cardiac failure is more common in complex Eisenmenger syndrome and is due to AV valve, and /or semilunar valve stenosis and regurgitation. Survival of the patient with Eisenmenger’s syndrome is quite variable and risk factors include trisomy 21, complex underlying disease (worse for truncus, atrioventricular septal defect, univentricular hearts), anemia, pregnancy, sustained arryhthmias and elevated creatinine, and need for noncardiac surgery. In series that contain children, the average age at death is 29 years with a 56% probability of surviving 10 years for a 20-year-old patient, whereas in adult clinics median survival at 18 years is 53 years with average age at death of 37713.3 years. After diagnosis, survival is 98% at 1 year, 77% at 5 years, 58 % at 10 years. Patients with a simple VSD survive longer (42% at 25 years) with survival at 1, 2, and 3 years of 97%, 89%, and 77%. Others have reported that five-year survival for simple Eisenmenger VSD is 91%, with truncus 67% and 34% for patients with single ventricle. Factors associated with poor prognosis included syncope at presentation, right atrial mean pressure above 8 mmHg, a cardiac index less than 2.9, and arterial oxygen saturation below 85%. Quality of life as reflected by employment in adult patients is variable with 53–57% (nontrisomy 21) holding full-time jobs. However, only 30% with complex disease enjoyed full-time employment versus 89% with a VSD. Pregnancy In the 1960s, pregnancy in women with Eisenmenger’s syndrome carried 27% mortality. The risk of adverse outcome for mother and fetus remains high. The outcome of 84 pregnancies reported from six different centers were as follows: spontaneous abortion or stillbirth occurred in 25%, therapeutic abortion in 27%, premature or low birthweight baby in 26%, maternal death in 16%, and maternal deterioration (including stroke, postpartum hemorrhage, rapid decline in pulmonary vascular status) in 54%.

410 VASCULAR DISEASE Management and Current Therapy

Therapeutic options for Eisenmenger’s syndrome Despite remarkable advances in the understanding of pulmonary vascular disease and advances in treatment for primary PAH, patients with established Eisenmenger’s syndrome remain therapeutic orphans. However, demonstration of residual reactivity in these patients is cause for cautious optimism that they will be included in future studies. However, recent observations on the regression of advanced pulmonary vascular disease in animal models and the availability of oral therapies have stimulated renewed interest in devising treatment strategies for Eisenmenger’s syndrome. Patients with Eisenmenger’s syndrome should be followed at a center with special expertise. Oxygen therapy An early study, which was not randomized and included a heterogeneous group of patients, seemed to indicate that oxygen therapy was beneficial in the Eisenmenger’s syndrome. However, these results have not been confirmed in a more recent study. A 2-year randomized study of oxygen therapy in established Eisenmenger patients aged 20– 50 years and living at altitude failed to demonstrate improvement in exercise capacity or quality of life. The mean survival time was 20.7 months in both groups. The only factor associated with adverse outcome was a low mean corpuscular volume (MCV) underscoring the harm that may ensue from venesection and the importance of treating iron deficiency. Vasodilator therapy Prostacyclin, endothelin receptor blockers, and phosphodiesterase inhibition, nitric oxide therapy In general, most medical therapies that have been shown to be of benefit in primary, familial, or sporadic PAH have demonstrated promise in the treatment of Eisenmenger’s syndrome. However, the studies have been in smaller cohorts, and the results of larger trials are awaited. Short-term benefit has been reported with prostacyclin, endothelin receptor blockers and phosphodiesterase type 5 inhibitions, and NO therapy. Anticoagulation Routine anticoagulation is not recommended because of the increased risk of hemorrhage. However, certain patients at risk of thrombosis may be candidates. General medical care Proper attention to all the sequelae of cyanosis, cardiac failure, and including gout, arteriopathy, polycythemia, arryhthmias, dental care, and social needs are best accomplished by a

multidisciplinary team experienced in the management of these patients. Surgical Techniques

Pulmonary artery banding Although surgical techniques for established Eisenmenger’s syndrome with preparatory pulmonary artery banding and subsequent surgical closure of VSD have been revisited (following an initial report in 1971 by Azzolina and stimulated considerable debate), definitive follow-up data is awaited and this strategy remains limited to certain centers. Double patch VSD closure The use of a double patch to close VSDs with hinge and fenestration to permit right to left but not left to right shunting has been reported with considerable early success. Lung transplantation and heart and lung transplantation Lung transplantation alone, with concomitant cardiac repair, is possible for patients with ASD or PDA. Conversely for Eisenmenger VSD survival is better with heart lung transplant rather than lung transplantation and VSD closure. Heart and lung transplantation offers better survival than lung transplantation for most patients with Eisenmenger’s syndrome. Early mortality rates for adults after heart and lung transplantation are 16% for Eisenmenger’s syndrome versus 13% for nonEisenmenger. The 1-, 5-, and 10-year survival rates are 73%, 51%, and 28% for Eisenmenger compared with 74%, 48%, and 26% for non-Eisenmenger. Summary

Patients with Eisenmenger’s syndrome have few therapeutic options. The hope must lie in early diagnosis and repair of congenital heart disease with appropriate and timely postoperative use of therapeutic agents that show promise in primary PAH. The care of Eisenmenger patients should be cohorted in a clinic with experienced personnel who are willing to promote clinical trials of drugs that may improve the considerable morbidity associated with unrepaired and reversed central shunts.

Pulmonary Arteriovenous Malformations Pulmonary arteriovenous malformations are characterized by abnormally large communications between the pulmonary arterial and venous systems. The physiological consequences are secondary to hypoxemia caused by the right to left shunt of desaturated pulmonary arterial blood into the pulmonary

VASCULAR DISEASE 411

vein without traversing the pulmonary vascular alveolar interface.

a low systemic vascular resistance and high hemoglobin, which preserves mixed venous saturation.

Etiology

Echocardiography

The etiology of pulmonary arteriovenous malformations may be idiopathic, or associated with HHT syndrome (Osler–Rendu–Weber syndrome). This is an inherited disease with autosomal dominant transmission. Pulmonary arteriovenous malformations occur in patients with liver disease associated with portal hypertension and is known as the hepatopulmonary syndrome. Pulmonary arteriovenous malformations complicate the course of patients with complex congenital heart disease who receive palliation with a superior cavopulmonary anastomosis (also known as the Glenn shunt).

The diagnostic evaluation of unexplained hypoxemia should include an echocardiogram with a contrast injection or agitated saline injection into an upper body vein. Pulmonary arteriovenous malformations are characterized by the first return of contrast into the pulmonary veins often after the clearance of contrast from the right atrium, ventricle, and pulmonary artery. Immediate appearance of contrast in the left atrium, left ventricle, or great vessel suggests a right to left shunt through an atrial septal defect (ASD), VSD or patent ductus arteriosus, respectively, as the cause for hypoxemia. Isotope 99m technetium perfusion scan may be used diagnostically and to quantify the right to left shunt. Cardiac catheterization typically reveals that the pulmonary artery pressure (mean PAp 4–15 mmHg) and the pulmonary vascular resistance (0.33 Wood’s units m2) are low and with an elevated cardiac output. However, there is a subgroup of patients with pulmonary hypertension as discussed in the section on idiopathic pulmonary arterial hypertension. Angiography demonstrates the arteriovenous malformations and delineates feeding vessels. Treatment depends upon the underlying disorder. For patients with idiopathic pulmonary arteriovenous malformations or the HHT syndrome, the most effective treatment is transcatheter delivery of coils to embolize the feeding vessels. If the pulmonary arteriovenous malformations occur following the Glenn shunt, the most effective treatment is redirection of hepatic venous blood to the pulmonary artery. Liver transplantation offers the best therapy for the hepatopulmonary syndrome.

Pathology

Anatomically, pulmonary arteriovenous malformations may be of two types with dilation of pulmonary capillaries from 5–15 mm to 100 mm. This type of pulmonary arteriovenous malformation is typical of the hepatopulmonary syndrome. The second type is a macroscopic vascular sac of 3–50 mm, which represent coalescence of capillaries and venules. The majority of these malformations are found in HHT and idiopathic pulmonary arteriovenous malformations. They may also develop in patients who have undergone the Glenn operation or anastomosis of the superior caval vein to the pulmonary artery. Clinical Features

Patients with pulmonary arteriovenous malformations are hypoxemic and present with cyanosis, finger clubbing, and polycythemia. Cyanosis increases when the patient stands up. Pulmonary arteriovenous malformations are distributed more often in the lower lobes. In the standing position, a greater proportion of the cardiac output is distributed to the lower lobes and right to left shunt and hypoxemia increases when the patient stands up. This explains the interesting physical sign and useful screening test known as orthodeoxia or orthostatic hypoxemia. Arterial blood gas testing reveals not only a low PaO2 but also PaCO2 because of increased minute ventilation for any level of oxygen uptake. Formal measurement of the hypercapnic and isocapnic hypoxic ventilation curves reveals a normal response to hypoxia with an increased hypercapnic response. Pulmonary function tests are normal except for diffusion studies with a low carbon monoxide transfer factor and carbon monoxide transfer coefficient. Exercise capacity may be well preserved despite hypoxemia because of an increase in cardiac output,

Hereditary Hemorrhagic Telangiectasia

HHT is also known as the Osler–Rendu–Weber syndrome and accounts for 80% of patients with pulmonary arteriovenous malformations that occur without liver or congenital cardiac disease. 20% of cases are idiopathic. Rendu described the association between epistaxis and mucocutaneous angiomas in 1896. In 1901, Osler described the hereditary nature of the syndrome and Weber in 1907 described the triad. HHT is an autosomal dominant disorder; the gene for HHT has been mapped to chromosome 9q33 and is associated with endoglin, a nonsignaling receptor for TGF-b that is widely expressed on endothelium and close to BMPR2. The latter is responsible for

412 VASCULAR DISEASE

familial PAH and the location of the mutation may account for the rare and unusual patients with HHT and PAH. Most patients have epistaxis by the age of 12. Multiple telangiectasias occur on the skin, mucous membranes, gastrointestinal tract, brain, spine, and lung. The average age of presentation with pulmonary arteriovenous malformations is 40 years. The female to male (range 8–70 years) ratio is 1.6 : 1. Patients with pulmonary arteriovenous malformations present with dyspnea, hemorrhage, cyanosis, paradoxical embolus, finger clubbing, and a pulmonary bruit. Central nervous system complications occur in 40–55% with cerebral abscess in 9–20%, usually as a result of anerobic infection. The pulmonary arteriovenous malformations may be visible on chest X-ray with expert interpretation as small opacities with feeding vessels, but CT scan or angiography provides more precise visualization. Treatment of choice is transcatheter coil embolization. Follow-up should include endocarditis prophylaxis at times of risk because of the right to left shunt and predisposition to cerebral abscess. Screening of family members should be undertaken. Hepatopulmonary Syndrome

Hepatopulmonary syndrome occurs as a complication of liver cirrhosis and portal hypertension in as many as 15–20% of patients. However, it is increasingly recognized in acute and chronic hepatitis without portal hypertension, in prehepatic portal hypertension and in hepatic venous obstruction without cirrhosis. Pathogenesis

It is postulated from the experimental models of hepatopulmonary syndrome that hepatic endothelin-1 release stimulates pulmonary vascular endothelial NO synthase production through increased ETB receptors and subsequent vasodilation. Macrophages also produce NOS and CO from hemoxygenase and contribute to further vasodilation. Patients with liver disease dyspnea or desaturation or clubbing (note noninvasive pulse oximetry may overestimate oxygen saturation in patients with clubbing). Patients with liver disease may have multiple reasons for hypoxemia and, in these situations, contrast echocardiogram and technetium MAA scan may be required to elucidate the role of pulmonary arteriovenous malformations. Treatment

The most effective treatment is liver transplant. Pulmonary arteriovenous shunting may take up to

a year after transplant to resolve. The presence of hepatopulmonary syndrome adversely affects survival with only 41% surviving at 2.5 years. Although liver transplant may cure hepatopulmonary syndrome, the survival after liver transplant is only 71% at 1 year compared with 90–92% at 1 year without hepatopulmonary syndrome. Indeed, the strongest predictors of mortality after liver transplant are a PaO2 o50 mmHg plus macro-aggregated albumen shunt fraction of 20%. This suggests that early screening for pulmonary arteriovenous malformations in patients with liver disease is warranted. Other treatments used in small open uncontrolled trials or as case reports include methylene blue, aerosolized, N-nitro-L-arginine methyl ester, garlic powder, and aspirin. Almitrine, somatostatin, indomethacin, and plasma exchange have been ineffective.

Pulmonary Arteriovenous Malformations after the Superior Cavopulmonary Anastomosis or Glenn Shunt This operation consists of anastomosis of the superior vena cava directly into the pulmonary artery and is performed as the second stage of palliation for complex congenital disease without a subpulmonary ventricle. The inferior vena cava blood enters the atrium, the ventricle, and then the systemic circulation without traversing the pulmonary vascular bed. The etiology of the pulmonary arteriovenous malformations is related to the exclusion of hepatic venous blood from directly entering the pulmonary circulation. Arteriovenous malformations may be unilateral (if the Glenn shunt is unilateral) and regress if subsequent surgeries redirect the hepatic venous blood directly into the pulmonary circulation. Patients with associated lesions that include interruption of the hepatic inferior vena cava, polysplenia, or left atrial isomerism and heterotaxy syndromes are particularly prone to the development of pulmonary arteriovenous malformations following a superior cavopulmonary anastomosis. Treatment is the redirection of hepatic venous blood to the pulmonary artery and almost always results in resolution of the arteriovenous malformations over time. See also: Caveolins. Energy Metabolism. Fluid Balance in the Lung. Hypoxia and Hypoxemia. Pulmonary Effects of Systemic Disease. Smooth Muscle Cells: Vascular. Symptoms of Respiratory Disease: Chest Pain. Systemic Disease: Sarcoidosis; Sickle Cell Disease.

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Further Reading Badesch DB, McLaughlin VV, Delcroix M, et al. (2004) Prostanoid therapy for pulmonary arterial hypertension. Journal of the American College of Cardiology 43: 56S–61S. Barst RJ, McGoon M, Torbicki A, et al. (2004) Diagnosis and differential assessment of pulmonary arterial hypertension. Journal of the American College of Cardiology 43: 40S–47S. Channick RN, Sitbon O, Barst RJ, Manes A, and Rubin LJ (2004) Endothelin receptor antagonists in pulmonary arterial hypertension. Journal of the American College of Cardiology 43: 62S–67S. Dresdale DT, Schultz M, and Michtom RJ (1951) Primary pulmonary hypertension. American Journal of Medicine 11: 686–701. Freedom RM, Yoo S-J, Mikhailian H, and Williams WG (eds.) (2003) The Natural and Modified History of Congenital Heart Disease, UK: Blackwell. Gaines DI and Fallon MB (2004) Hepatopulmonary syndrome. Liver International 24: 397–401. Ghofrani HA, Pepke-Zaba J, Barbera JA, et al. (2004) Nitric oxide pathway and phosphodiesterase inhibitors in pulmonary arterial hypertension. Journal of the American College of Cardiology 43: 68S–72S. Hodgson CH, Burchell HB, Good CA, and Clagett OT (1959) Hereditary hemorrhagic telangiectasia and pulmonary arteriovenous fistula. New England Journal of Medicine 261: 941–943.

Hoeper MM, Krowka MJ, and Strassburg CP (2004) Portopulmonary hypertension and hepatopulmonary syndrome. Lancet 363: 1461–1468. Hughes JMB, Jackson JE, and Allison DJ (1996) Pulmonary arteriovenous malformations. In: Peacock AJ (ed.) Pulmonary Circulation, pp. 359–376. London: Chapman and Hall Medical. Humbert M, Morrell NW, Archer SL, et al. (2004) Cellular and molecular pathobiology of pulmonary arterial hypertension. Journal of the American College of Cardiology 43: 13S–24S. Newman JH, Trembath RC, Morse JA, et al. (2004) Genetic basis of pulmonary arterial hypertension: current understanding and future directions. Journal of the American College of Cardiology 433: 3S–39S. Pietra GG, Capron F, Stewart S, et al. (2004) Pathologic assessment of vasculopathies in pulmonary hypertension. Journal of the American College of Cardiology 43: 25S–32S. Rubin LJ and Rich S (1997) Primary Pulmonary Hypertension, pp. 1–358. New York: Dekker. Simonneau G, Galie N, Rubin LJ, et al. (2004) Clinical classification of pulmonary hypertension. Journal of the American College of Cardiology 43: 5S–12S. Wood P (1958) The Eisenmenger syndrome or pulmonary hypertension with reversed central shunt. British Medical Journal 2: 701–709, 755–762.

VASCULAR ENDOTHELIAL GROWTH FACTOR W M Maniscalco and C T D’Angio, University of Rochester, Rochester, NY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Vascular endothelial growth factor (VEGF or VEGF-A) is the prototype and best-described member of a family of at least six largely endothelial-specific, heparin-binding, angiogenic growth factors. VEGF family members are crucial to the normal development and maintenance of the vascular and lymphatic systems. VEGF stimulates endothelial cells to degrade extracellular matrix, proliferate, migrate, and form tubes, and acts as an endothelial cell survival factor. VEGF also increases vascular permeability, leading to its alternative name, vascular permeability factor. VEGF family members exert their effects through interactions with a family of three VEGF receptors and with two co-receptors known as neuropilins. Differing combinations of VEGF family members and receptors mediate tissue-specific effects. Expression of VEGF in the developing pulmonary epithelium is a major modulator of normal pulmonary vascular development during fetal life. Following a number of pulmonary insults, both local increases in VEGF production, which result in increased vascular leakage and edema, and an overall decrease in VEGF abundance, which coincides with microvascular endothelial cell loss, are associated with the development of acute lung injury. VEGF production by lung cancers is an important mechanism by which the cancers recruit new vasculature, allowing them to grow, invade neighboring tissue, and metastasize.

Introduction In 1983, Senger and colleagues partially purified a protein that markedly increased vascular permeability and named it vascular permeability factor (VPF). Ferrara and Henzel, in 1989, described an endothelial-cell-specific mitogen that they called vascular endothelial growth factor (VEGF). Subsequent complementary DNA cloning revealed that VPF and VEGF were the same factor. Since that time, the VEGF family has grown to include five related human proteins (VEGF-A, VEGF-B, VEGF-C, VEGFD, and placental growth factor or PlGF) and one viral protein (VEGF-E) that act through two receptor families to stimulate endothelial cell proliferation, migration and survival. This entry will focus primarily on VEGF-A, commonly known simply as VEGF, the best-described member of the family.

Structure The VEGF gene is located on human chromosome 6 (p12), has a coding region of about 14 kb, and contains eight exons and seven introns. VEGF undergoes alternative mRNA splicing of exons 6 and 7 to produce five protein isoforms varying from 121 to 206

414 VASCULAR ENDOTHELIAL GROWTH FACTOR Heparin-binding domain 1

2

3

4

5

6

VEGF206

1 2

3

4 5

VEGF189

1 2

3

4 5 6

7

6

7

7

8 Gene

8

8 mRNA splice variants

VEGF165

1 2

3

4 5

VEGF145

1 2

3

4 5 6 8

VEGF121

1 2

3

4 58

7

8

Figure 1 VEGF structure. The human VEGF gene contains eight exons and seven introns. Variable mRNA splicing results in five mRNA species, denoted by the length of the amino acid isoforms they encode. VEGF206, VEGF189, and VEGF145 have high affinity for heparin, whereas VEGF121 is nonheparin-binding and freely diffusible, and VEGF165 has intermediate properties. VEGF121 and VEGF165 are secreted, whereas the other isoforms are strongly cell associated.

amino acids, named by the number of amino acids they contain (Figure 1). VEGF121 and VEGF165 are the most commonly expressed isoforms in most cell types. VEGF121 lacks a heparin-binding domain and is soluble, while the other isoforms are heparin bound to varying degrees. The active forms of most members of the VEGF family are homodimers, although heterodimers have also been described.

Regulation of Production and Activity VEGF expression is regulated at the transcriptional and posttranscriptional levels. The VEGF gene promoter contains binding sites for multiple transcription factors, including Sp1, Stat3, AP-1, and hypoxia-inducible factor-1 (HIF-1). VEGF mRNA transcription is upregulated by a number of other growth factors, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor beta (TGF-b), and the cytokines tumor necrosis factor alpha (TNF-a), and interleukin-1 beta (IL-1b). The actions of the cytokines and growth factors appear to be transduced primarily through Sp1, Stat3, and AP-1. Mutations in several oncogenes, including ras and Wnt, also upregulate VEGF mRNA transcription. Hypoxia is a powerful stimulus for VEGF production. VEGF mRNA expression is increased by hypoxia and decreased by normoxia, an effect modulated largely through the hypoxia-sensing transcription factor HIF-1. Hyperoxia depresses VEGF mRNA expression to subnormoxic levels. Hypoxia also stabilizes VEGF mRNA through a protein known as

HuR, which binds to an AU-rich element of the 30 untranslated region (UTR) of VEGF mRNA and prevents its degradation. Other elements in both the 50 and 30 UTR are also necessary for maximum hypoxia-stimulated mRNA stability. Differential mRNA splicing allows for differing isoforms of VEGF to be expressed from a single gene under differing conditions in differing tissues. For instance, the ratio of soluble VEGF121 to less-soluble VEGF189 falls progressively during prenatal lung development, as the developing capillary network becomes more closely apposed to the airway epithelium and VEGF effects need to be mediated more locally. VEGF is expressed by a broad array of cell types, including epithelial cells, vascular smooth muscle cells, chondrocytes, hepatocytes, monocytes, macrophages, neutrophils, pituitary and pancreatic endocrine cells, and mesenchymal cells. Many tumor cells also express VEGF.

Biological Function VEGF plays an important role in the two major processes involved in vascular development: vasculogenesis (de novo development of new vascular tubes from endothelial cell precursors) and angiogenesis (formation of small vessels and capillaries by sprouting from existing larger vessels). VEGF stimulates both vascular and lymphatic endothelial cells to degrade extracellular matrix, proliferate, migrate, and form tubes, and acts as an endothelial cell survival factor. VEGF also potently increases vascular

VASCULAR ENDOTHELIAL GROWTH FACTOR 415

permeability. This effect appears to be mediated through the creation of fenestrations in the endothelium of small venules and capillaries. In the embryo, VEGF is crucial for hemangioblast differentiation, early hematopoiesis, vasculogenesis, and angiogenesis. Transgenic mouse embryos missing one or both VEGF alleles die early in gestation with defective vascularization, abnormal major blood vessels in the embryo, yolk sac and placenta, and decreased angiogenic sprouting. VEGF-deficient mouse embryos also have reduced numbers of nucleated red blood cells in the blood islands of the yolk sac. In embryos in which the VEGF signal cannot be detected due to an engineered receptor defect, hematopoietic and endothelial progenitor cells fail to proliferate and migrate, leading to lethal defects in both the vascular and hematologic systems. Conversely, even modest VEGF overexpression also leads to embryonic death, due to profound abnormalities in cardiac structural development including overvascularization. In early postnatal life, VEGF is important for the continued normal vascularization of organs. Renal glomerular development is heavily dependent on normal VEGF expression, as is normal bone growth. As postnatal development continues, VEGF expression in most tissues gradually falls. By adulthood, when most endothelial cells are quiescent, VEGF expression in most tissues is quite low. The exceptions are in sites of continued active angiogenesis, such as the ovaries, uterus, and skin. VEGF is also highly elevated during the proliferative phase of wound healing. Although initially thought to be endothelial specific, VEGF also affects a number of other cell types, including several bone-marrow-derived cells. It induces monocyte recruitment and activation. High concentrations of VEGF inhibit the development of dendritic and T cells from hematopoietic progenitor cells. VEGF has stimulatory effects on surfactant production by the alveolar type II epithelial cell. It also has direct effects on nervous tissue, affecting neurogenesis and neuronal maturation.

becomes steadily more closely apposed to the epithelium. VEGF continues to be expressed at high levels in the lung in early postnatal life, and type II alveolar epithelial cells express moderate levels of VEGF even in adulthood. Disruption of postnatal VEGF signaling by blocking its receptors in newborn animals inhibits alveolar development, resulting in fewer, larger alveoli. Lung vascular development is also impacted, with a reduction in arterial density, increased pulmonary artery wall thickness, and resultant pulmonary hypertension. These changes persist into adulthood.

Receptors Two major receptor classes mediate the effects of VEGF family members (Table 1). The VEGF receptors (VEGFRs) are a family of three closely related, membrane-spanning peptides containing seven extracellular immunoglobulin-like domains and two intracellular tyrosine kinases. VEGF interacts primarily with two of these receptors, VEGFR-1 (also known as flt-1) and VEGFR-2 (also known as flk-1 or KDR). Binding of VEGF to VEGFR-1 stimulates endothelial cell migration, and may mediate vascular organization. VEGFR-1 may also, in some cases, act as a ‘decoy’ receptor, by binding and sequestering VEGF away from VEGFR-2, which is felt to be the major mediator of the mitogenic, angiogenic, and vascular permeability effects of VEGF. VEGFR-2 activation stimulates endothelial cell differentiation, proliferation, and migration, and has been implicated in angioblast differentiation. VEGFR-3 (flt-4), expressed primarily in lymphatic endothelium, binds VEGF-C and VEGF-D. The cell surface proteins neuropilin (NRP)-1 and NRP-2 bind the VEGF165 isoform and several other VEGF family members. These proteins do not appear to act independently to transduce signal following binding to VEGF family members. NRP-1 appears to be a co-receptor for VEGFR-2, increasing the affinity of VEGF165 for VEGFR-2 10-fold. NRP-2 appears to fulfill the same function for VEGFR-3 on the lymphatic endothelium.

VEGF in Lung Development

VEGF is expressed abundantly in the developing lung of humans and animals, and is important in the genesis and maintenance of the developing pulmonary vasculature. The lung is one of the chief organs expressing VEGF mRNA in the midtrimester human fetus. In developing animals, lung VEGF expression increases throughout fetal life. Expression becomes progressively localized to the epithelial and subepithelial portions of advancing airways as the vasculature

Vascular Endothelial Growth Factor in Respiratory Diseases Both induction and inhibition of VEGF have been reported in lung diseases. The amount of VEGF present in any given tissue compartment, either within or outside the lung, arises from a complex interaction of many factors, including the stimulus for VEGF production, the degree of injury to VEGF-producing

416 VASCULAR ENDOTHELIAL GROWTH FACTOR Table 1 VEGF/receptor interactions Receptor

Predominant cell types expressing receptor

Ligands

Predominant actions

VEGFR-1

Vascular endothelium, monocytes, macrophages, pulmonary epithelial cells, renal mesangial cells

VEGF-A VEGF-B PIGF

‘Decoy’ receptor (?), endothelial cell proliferation and migration, cytokine and growth factor release, monocyte migration

VEGFR-2

Vascular endothelium

VEGF-A VEGF-C VEGF-D VEGF-E

Vascular endothelial cell proliferation, migration, survival, angiogenesis

VEGFR-3

Lymphatic endothelium

VEGF-C VEGF-D

Lymphatic endothelial cell proliferation, migration, survival, angiogenesis

NRP-1

Vascular endothelium, neurons

VEGF-A VEGF-B VEGF-E PIGF

Co-receptor for VEGFR-2

NRP-2

Lymphatic endothelium

VEGF-A VEGF-B VEGF-C VEGF-E PIGF

Co-receptor for VEGFR-3

cells, and the amount of VEGF efflux from and influx into the compartment. Acute Lung Injury

Insults to the lung from a variety of sources, including bacterial lipopolysaccharide (LPS), ischemiareperfusion, hyperoxia, toxicants, overdistention from mechanical ventilation, and acute respiratory distress syndrome (ARDS) produce similar patterns of acute lung injury. During the initial phase, there is an influx of inflammatory cells, epithelial and endothelial injury, and edema (Figure 2(a)). As the injury resolves, epithelial and endothelial integrity are restored. Many lung insults elicit falls in VEGF mRNA expression and protein production within hours. VEGFR-1 and VEGFR-2 mRNA expression also decreases during several of these injuries. These reductions in VEGF and its receptors parallel a profound loss of microvascular endothelial cells during injury. However, in some specific cases, increased levels of VEGF have also been described in lung injury. Pulmonary LPS exposure results in increased VEGF protein and mRNA levels at a time when capillary leakage is maximal, suggesting a role for VEGF in the increased vascular permeability of acute lung injury. Interestingly, exposure to bacteria, which contain LPS but also cause epithelial damage, results

in decreases in VEGF expression, potentially from destruction of VEGF-producing epithelial cells. VEGF levels in alveolar lavage fluid are elevated in animal models of hyperoxic lung injury, perhaps due to release of soluble isoforms from the extracellular matrix. During recovery from a variety of lung injuries, pulmonary VEGF levels are increased. In animals exposed to hyperoxia, type II alveolar epithelial cells express supranormal levels of VEGF if animals are allowed to recover from exposure. This increase in VEGF may be important to the re-establishment of vascular integrity and regrowth of the capillary network as the lung recovers. Newborn animals of many species are more resistant to hyperoxia than adults, but eventually succumb to oxygen-induced lung injury. Hyperoxiaexposed newborn rabbits exhibit a depression in pulmonary VEGF expression similar to the adult, albeit with a longer time frame. A similar decrease in lung VEGF expression is seen in animals delivered prematurely and exposed to mechanical ventilation. The lower levels of VEGF in these animals are associated with an arrest in the development of the pulmonary microcirculation. Bronchopulmonary dysplasia (BPD) is a chronic lung disease, characterized by disordered architecture, arrested alveolar development, and decreased vascularization, which arises largely in premature

VASCULAR ENDOTHELIAL GROWTH FACTOR 417

VEGF Local VEGF increase

Injury Type II cell

Capillary leak

Type I cell ? Alveolar macrophage

Capillaries

VEGF Recovery and repair

Overall VEGF decrease

(a)

Capillary regrowth

Endothelial death

VEGF

O2

O2

Central hypoxia

(b) Figure 2 VEGF function in respiratory diseases. Following many types of acute lung injury (a), VEGF expression falls rapidly, although localized VEGF protein levels may initially rise. The localized VEGF increases may increase vascular permeability, resulting in alveolar edema. The overall decrease in VEGF expression is associated with endothelial cell death. During recovery, VEGF expression rises and new capillary growth occurs. VEGF production by the hypoxic tissue at the center of lung cancers (b) is crucial to promoting neovascularization, which allows tumor growth.

infants who have suffered an acute lung injury. Several studies have evaluated levels of VEGF in lung fluid obtained from human premature newborns born at 23–33 weeks’ gestation. VEGF protein measured in lung fluid rises at several fold over the first 3–10 days after birth. However, infants destined to develop BPD have depressed lung fluid VEGF levels between days 4 and 7, when compared to those infants who go on recover without BPD. Strategies that seek to enhance VEGF expression in the injured lung may hold promise for protecting the lung from damage. Mice genetically engineered to overexpress the VEGF-stimulating cytokine IL-13 in the lung have increased pulmonary VEGF levels and are resistant to hyperoxic lung injury. Neutralization of VEGF in these animals eliminates the protection from hyperoxia.

Lung Cancer

Most human tumors studied to date, including carcinoma of the lung, express VEGF. VEGF is not necessary for the tumor cells to grow normally in vitro, but is required for tumor progression in vivo. Inhibition of VEGF prevents the tumor from recruiting a vascular supply, and limits its growth to the size that can be supported by diffusion of oxygen from surrounding tissues (Figure 2(b)). VEGF may also be necessary for tumor cells to access the vasculature and metastasize. Tumors also appear to recruit nearby stromal cells to produce VEGF, enhancing tumor-related angiogenesis. Non-small cell lung cancer (NSCLC) accounts for at least 75% of lung cancers, and responds poorly to traditional therapies. The tumor expresses VEGF,

418 VASCULITIS / Overview

and high levels of VEGF and a high density of microvessels within the tumor are associated with poorer prognosis. Specifically, tumors that have a high number of microvessels containing VEGF/VEGFR complexes at the advancing tumor front have the poorest prognosis. NSCLC has been the target of investigation for treatment with anti-VEGF compounds, including both antibodies and pharmacologic inhibitors. In early studies, addition of an antiVEGF antibody to standard therapy increased the response rate of NSCLC. See also: Acute Respiratory Distress Syndrome. Arteries and Veins. Bronchopulmonary Dysplasia. Endothelial Cells and Endothelium. Environmental Pollutants: Oxidant Gases. Epithelial Cells: Type II Cells. Fluid Balance in the Lung. Hypoxia and Hypoxemia. Interleukins: IL-13. Leukocytes: Monocytes. Lung Development: Overview. Lymphatic System. Occupational Diseases: Inhalation Injury, Chemical. Oxidants and Antioxidants: Oxidants. Oxygen Therapy. Oxygen Toxicity. Pulmonary Edema. Tumors, Malignant: Overview; Metastases from Lung Cancer.

Further Reading D’Angio CT and Maniscalco WM (2002) The role of vascular growth factors in hyperoxia-induced injury to the developing lung. Frontiers in Bioscience 7: 1609–1623.

Ferrara N (2004) Vascular endothelial growth factor: basic science and clinical progress. Endocrine Reviews 25: 581– 611. Ferrara N and Henzel WJ (1989) Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochemical and Biophysical Research Communications 161: 851–858. Hicklin DJ and Ellis LM (2005) Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. Journal of Clinical Oncology 23: 1011–1027. Kumar VH and Ryan RM (2004) Growth factors in the fetal and neonatal lung. Frontiers in Bioscience 9: 464–480. Mura M, dos Santos CC, Stewart D, et al. (2004) Vascular endothelial growth factor and related molecules in acute lung injury. Journal of Applied Physiology 97: 1605–1617. Pauling MH and Vu TH (2004) Mechanisms and regulation of lung vascular development. Current Topics in Developmental Biology 64: 73–99. Ribatti D (2005) The crucial role of vascular permeability factor/vascular endothelial growth factor in angiogenesis: a historical review. British Journal of Haematology 128: 303– 309. Senger DR, Galli SJ, Dvorak AM, et al. (1983) Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219: 983–985. Xie K, Wei D, Shi Q, et al. (2004) Constitutive and inducible expression and regulation of vascular endothelial growth factor. Cytokine & Growth Factor Reviews 15: 297–324. Zachary I (2005) Signal transduction in angiogenesis. EXS 94: 267–300. Zachary I and Gliki G (2001) Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family. Cardiovascular Research 49: 568–581.

VASCULITIS Contents

Overview Microscopic Polyangiitis

Overview S Homma, Toranomon Hospital, Tokyo, Japan & 2006 Elsevier Ltd. All rights reserved.

Abstract The pulmonary vasculitides represent a heterogeneous group of clinicopathological entities. Among these, Wegener’s granulomatosis (WG), Churg–Strauss syndrome, and microscopic polyangiitis (MPA) are associated with antineutrophil cytoplasmic antibodies which distinguish these conditions from other members of the pulmonary angiitis and granulomatosis group. Common complaints and signs of vasculitis include fatigue, weakness, fever, arthralgias, abdominal pain, hypertension, renal insufficiency (with an active urine sediment containing red and white cells and occasionally red cell casts), palpable

purpura, and neurologic dysfunction such as mononeuritis multiplex. Pathologically, vasculitis has been categorized in terms of the predominant sizes and types of the blood vessels most commonly affected among patients with the disorder. Therapy usually requires at least corticosteroid treatment, but depends upon the nature and severity of the vasculitis. Patients with rapidly progressive vasculitic diseases, such as WG or MPA, are likely to require combination therapy consisting of corticosteroids and a cytotoxic drug (usually cyclophosphamide).

Introduction The vasculitides are defined by the presence of leukocytes in the vessel wall with reactive intermediate damage to mural structures. Loss of vessel integrity leads to bleeding. Compromise of the lumen leads to

VASCULITIS / Overview 419 Table 1 Names and definitions of vasculitides Name Large vessel vasculitis Giant cell (temporal) arteritis

Takayasu’s arteritis Medium-sized vessel vasculitis Polyarteritis nodosa Kawasaki’s disease

Small vessel vasculitis Wegener’s granulomatosis

Churg–Strauss syndrome

Microscopic polyangiitis

Henoch–Scho¨nlein purpura

Essential cryoglobulinemic vasculitis

Cutaneous leukocytoclastic angiitis

Definition Granulomatous arteritis of the aorta and its major branches, with a predilection for the extracranial branches of the carotid artery. Often involves the temporal artery. Usually occurs in patients older than 50 and often is associated with polymyalgia rheumatica. Granulomatous inflammation of the aorta and its major branches. Usually occurs in patients younger than 50. Necrotizing inflammation of medium-sized or small arteries without glomerulonephritis or vasculitis in arterioles, capillaries, or venules. Arteritis involving large, medium-sized, and small arteries, and associated with mucocutaneous lymph node syndrome. Coronary arteries are often involved. Aorta and veins may be involved. Usually occurs in children. Granulomatous inflammation involving the respiratory tract, and necrotizing vasculitis affecting small- to medium-sized vessels (e.g., capillaries, venules, arterioles, and arteries). Necrotizing glomerulonephritis is common. Eosinophil-rich and granulomatous inflammation involving the respiratory tract, and necrotizing vasculitis affecting small to medium-sized vessels; associated with asthma and eosinophilia. Necrotizing vasculitis, with few or no immune deposits affecting small vessels (i.e., capillaries, venules, or arterioles). Necrotizing arteritis involving small- and medium-sized arteries may be present. Necrotizing glomerulonephritis is very common. Pulmonary capillaritis often occurs. Vasculitis, with IgA-dominant immune deposits, affecting small vessels (i.e., capillaries, venules, or arterioles). Typically involves skin, gut, and glomeruli, and is associated with arthralgias or arthritis. Vasculitis, with cryoglobulin immune deposits, affecting small vessels (i.e., capillaries, venules, or arterioles), and associated with cryoglobulins in serum. Skin and glomeruli are often involved. Isolated cutaneous leukocytoclastic angiitis without systemic vasculitis or glomerulonephritis.

Reproduced from Gross WL, Schnabel A, and Trabandt A (2000) New perspectives in pulmonary angiitis. From pulmonary angiitis and granulomatosis to ANCA associated vasculitis. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 17: 33–52, with permission.

tissue ischemia and necrosis. In general, affected vessels vary in size, type, and location in association with the specific vasculitic disorder. Vasculitis may occur as a primary process or may be secondary to another underlying disease (Table 1). Criteria for the classification of most of the major forms of vasculitis were established by the American College of Rheumatology (ACR) in 1990.

Etiology Pulmonary vasculitis defines a small group of idiopathic vasculitic syndromes that commonly affect the lung, a much larger number of vasculitic disorders that uncommonly affect the lung, and miscellaneous secondary conditions that cause pulmonary vascular inflammation (Table 2). The idiopathic vasculitic syndromes which commonly affect the lung are Wegener’s granulomatosis (WG), Churg–Strauss syndrome (CSS), and microscopic polyangiitis (MPA). Bronchocentric granulomatosis (BCG) and lymphomatoid granulomatosis have traditionally been grouped as pulmonary ‘angiitis and granulomatosis’;

however, neither of these entities is currently thought to be a vasculitic condition. BCG is a morphologic pattern of airway inflammation that occurs in a variety of conditions, especially infection. Lymphomatoid granulomatosis is now known to represent a lymphoproliferative disorder with prominent vascular involvement. In addition to the major idiopathic vasculitic lung disorders, there are some that uncommonly affect the lung. Necrotizing sarcoid granulomatosis was formerly regarded as one of the major vasculitic syndromes, but it is so rare that it is now grouped under syndromes that uncommonly affect the lung. Pulmonary vasculitis also occurs in a variety of miscellaneous systemic disorders, in diffuse pulmonary hemorrhagic syndromes, and in a variety of secondary or localized forms. The term benign lymphocytic angiitis and granulomatosis is not used since this may include low-grade examples of lymphomatoid granulomatosis. Disease Associations

Antineutrophil cytoplasmic antibodies (ANCA) are associated with many cases of WG, MPA, CSS,

420 VASCULITIS / Overview Table 2 Pulmonary vasculitis syndromes Idiopathic vasculitis syndromes which commonly affect the lung Wegener’s granulomatosis Churg–Strauss angiitis and granulomatosis Microscopic polyangiitis Idiopathic vasculitis syndromes which uncommonly affect the lung Necrotizing sarcoid granulomatosis Polyarteritis nodosa Takayasu’s arteritis Henoch–Sho¨nlein purpura Behc¸et syndrome Cryoglobulinemic vasculitis Hypocomplementemic vasculitis Idiopathic granulomatous arteritis Giant cell arteritis Disseminated visceral giant cell angiitis Miscellaneous systemic disorders Classic sarcoidosis Collagen vascular disease Inflammatory bowel disease Malignancy Diffuse pulmonary hemorrhage syndromes Secondary or localized vasculitis Pulmonary infection Bronchocentric granulomatosis Pulmonary hypertension Interstitial lung diseases Chronic eosinophilic pneumonia Langerhans cell histiocytosis Inflammatory pseudotumors Sequestration Embolic material (intravenous drug abuse) Drugs or toxic substances Transplantation Radiation Vascular involvement in lymphoproliferative disorders Angiocentric immunoproliferative lesion (lymphomatoid granulomatosis) Non-Hodgkin’s lymphoma Intravascular malignant lymphoma Reproduced from Travis WD, Colby TV, Koss MN, et al. (eds.) (2002) Non-Neoplastic Disorders of the Lower Respiratory Tract, p. 233. Washington, DC: Armed Forces Institute of Pathology and American Registry of Pathology, with permission.

‘renal-limited’ vasculitis (pauci-immune glomerulonephritis without evidence of extrarenal disease), and certain groups of pulmonary fibrosis and druginduced vasculitic syndromes. In these conditions, ANCA consistently have specificities for either proteinase 3 (PR3) or myeloperoxidase (MPO), but almost never for both. Pathology Classification

Classically, vasculitic syndromes have been categorized as follows by the predominant blood vessel

sizes and types most commonly affected among patients with the disorder. Large vessel vasculitis 1. Giant cell arteritis. Giant cell or temporal arteritis is a chronic vasculitis of large and medium-sized vessels. 2. Takayasu arteritis. Takayasu arteritis primarily affects the aorta and its primary branches. The inflammation may be localized to a portion of the thoracic or abdominal aorta and branches, or may involve the entire vessel. Medium-sized vessel vasculitis 1. Polyarteritis nodosa. Polyarteritis nodosa is a systemic necrotizing vasculitis that typically affects the small and medium-sized muscular arteries. 2. Kawasaki’s disease. Kawasaki’s disease is an arteritis of large, medium, and small arteries, particularly the coronary arteries. Small vessel vasculitis 1. Wegener’s granulomatosis. WG is a systemic vasculitis of the medium and small arteries, as well as the venules and arterioles. It typically produces granulomatous inflammation of the upper and lower respiratory tracts and necrotizing, pauci-immune glomerulonephritis in the kidneys. 2. Churg–Strauss arteritis. Churg–Strauss arteritis (also called allergic granulomatosis and angiitis) is a vasculitis of the medium- and small-sized muscular arteries, and is often found with vascular and extravascular granulomatosis. This vasculitis classically involves the arteries of the lung and skin, but may be generalized. 3. Microscopic polyangiitis. MPA is a vasculitis which primarily affects capillaries, venules, or arterioles. Involvement of small- and mediumsized arteries may also be present. 4. Hypersensitivity vasculitis. Hypersensitivity vasculitis includes several forms of vasculitis of small blood vessels. These include Henoch–Scho¨nlein purpura, mixed cryoglobulinemia, allergic vasculitis, and serum sickness.

Clinical Features The vasculitides are often serious and sometimes fatal diseases that require prompt recognition and therapy. Symptomatic involvement of affected organs may either occur in isolation or in combination with multiple organs. The distribution of affected organs

VASCULITIS / Overview 421

may suggest a particular vasculitic disorder, but significant overlap can be observed. Clinical Manifestations

The presence of vasculitis should be considered in patients who present with systemic symptoms in combination with evidence of single and/or multiorgan dysfunction. Although neither sensitive or specific, common complaints and signs of vasculitis include fatigue, weakness, fever, arthralgias, abdominal pain, hypertension, renal insufficiency (with an active urine sediment containing red and white cells and occasionally red cell casts), palpable purpura, and neurologic dysfunction such as mononeuritis multiplex. The combination of hemoptysis and renal involvement indicative of glomerulonephritis suggests a diagnosis of WG or MPA. However, certain other disorders, particularly antiglomerular basement membrane (anti-GBM) antibody disease, may yield an identical combination of findings. Laboratory Tests

A positive antinuclear antibody test suggests the presence of an underlying connective tissue disorder, particularly systemic lupus erythematosus. Low serum complement levels may be present in mixed cryoglobulinemia and lupus but not most other vasculitic disorders. ANCA serologies are useful in distinguishing MPA from classic polyarteritis nodosa (PAN), a vasculitis of medium-sized muscular arteries. Whereas nearly three quarters of patients with MPA are ANCA-positive, classic PAN is not associated with antibodies to either PR3 or MPO. WG is usually associated with PR3–ANCA. The strongest links between medications and ANCA-associated vasculitis are with propylthiouracil, hydralazine, and minocycline. Other drugs occasionally implicated include penicillamine, allopurinol, procainamide, carbimazole, thiamazole, clozapine, and phenytoin. Electromyogram

An electromyogram is useful if a systemic vasculitis is suspected and neuromuscular symptoms, such as a mononeuritis multiplex, are present. Tissue Biopsy

Biopsy examination of the most clinically involved tissue is essential for diagnosis. The chances of obtaining a diagnostic specimen are greatest when a specimen is taken from an involved portion of an affected organ and when the tissue sample is of adequate size.

Pathogenesis Three different pathogenic models of disease have been advanced to help explain why the lesions of a particular vasculitic syndrome are found only in specific vessels: (1) the distribution of the antigen responsible for the vasculitis determines the pattern of vessel involvement; (2) the recruitment and accumulation of the inflammatory infiltrate is determined by the endothelial cell phenotype, including the expression of adhesion molecules, the secretion of peptides and hormones, and the specific interaction with inflammatory cells; and (3) nonendothelial structures of the vessel wall are involved in controlling the inflammatory process. In addition to the endothelial cells, which provide co-stimulatory function, other cellular components serve as antigen-presenting cells and contribute proinflammatory mediators. Mechanisms of ANCA Production

The autoantibody response that produces ANCA is probably generated against newly exposed epitopes of the target autoantigen. Following the production of ANCA, the antibody response may generalize to the rest of the molecule or to other components of a macromolecular protein complex via the process of epitope spreading. With WG, these neoepitopes may arise at the sites of initial tissue injury. Since it is an antigen-driven process, the disease may heavily depend upon help from T cells.

Animal Models Two murine models of ANCA-associated vasculitis reveal that adoptive transfer of autoantibody alone is sufficient to induce a necrotizing vasculitis closely resembling human disease. Development of these models involved two types of genetically altered mice: the MPO knockout mouse, and the recombinase-activating gene 2 (RAG-2)-deficient mouse. The latter species lacks both T and B cells. These models provide the first in vivo evidence for the pathogenic potential of ANCA. In the first model, MPO knockout mice were initially immunized with mouse MPO, resulting in the formation of anti-MPO splenocytes and anti-MPO antibodies within immunized mice. RAG-2-deficient mice were subsequently injected with either anti-MPO splenocytes or control splenocytes which did not produce anti-MPO antibodies. RAG-2 mice that received anti-MPO splenocytes developed clinical features of ANCA-associated vasculitides, including crescentic glomerulonephritis and systemic necrotizing vasculitis. By comparison, RAG-2 mice that received nonMPO antibody-producing splenocytes displayed only a relatively mild immune complex glomerulonephritis.

422 VASCULITIS / Microscopic Polyangiitis

In the second disease model, RAG-2-deficient and wild-type mice were injected with anti-MPO or control immunoglobulins. Only mice receiving antiMPO antibodies developed a pauci-immune glomerulonephritis.

Management and Current Therapy Patients with systemic vasculitic involvement usually require at least corticosteroid therapy. As an example, corticosteroid therapy usually leads to remission in giant cell arteritis. In most patients, the dose can be reduced and eventually discontinued, but some require chronic therapy with low dose prednisone. By comparison, patients with rapidly progressive vasculitic diseases, such as WG or MPA, are likely to require combination therapy consisting of a cytotoxic drug (usually cyclophosphamide) and corticosteroids. Both oral or pulse cyclophosphamide have been utilized, based upon physician experience and disease severity; after 1 or 2 months of combination therapy, the corticosteroid dose may be reduced; therapy is usually continued for 6 to 12 months to diminish the risk of relapse. Azathioprine and methotrexate have been used in less severe forms of vasculitis and as maintenance therapy, after remission has been induced by cyclophosphamide. Fortunately, available treatments are helpful, particularly in the acute phase. However, during maintenance therapy with corticosteroids and immunosuppressive agents, the adverse effects of drugs and superimposed infections may assume increasing importance. Mortality data suggest that while early deaths in vasculitis are due to the active disease, late deaths may be due to the complications of therapy. The ability to specifically deplete B cells with anti-CD20 therapies may offer new insights into the pathophysiology of ANCA-associated vasculitis. The rationale as to why B cell depletion may be effective in ANCA-associated vasculitis is not clear, but possibilities include (1) the complete removal or substantial reduction of ANCA production, (2) diminution of the contribution of B cells to antigen presentation and cytokine production, and (3) the inhibition of B cell/T cell cross-talk. See also: Alveolar Hemorrhage. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome. Vasculitis: Microscopic Polyangiitis.

Further Reading Anonymous (2002) Pulmonary vasculitis. In: Travis WD, Colby TV, Koss MN, et al. (eds.) Non-Neoplastic Disorders of the Lower Respiratory Tract, pp. 233–264. Washington: Armed Forces Institute of Pathology and American Registry of Pathology.

Choi HK, Merkel PA, Walker AM, and Niles JL (2000) Drugassociated antineutrophil cytoplasmic antibody-positive vasculitis: prevalence among patients with high titers of antimyeloperoxidase antibodies. Arthritis and Rheumatism 43: 405–413. Collard HR and Schwarz MI (2004) Diffuse alveolar hemorrhage. Clinics in Chest Medicine 25: 583–592. Gross WL, Schnabel A, and Trabandt A (2000) New perspectives in pulmonary angiitis. From pulmonary angiitis and granulomatosis to ANCA associated vasculitis. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 17: 33–52. Guillevin L, Durand-Gasselin B, Cevallos R, et al. (1999) Microscopic polyangiitis. Arthritis and Rheumatism 42: 421–430. Gunton JE, Stiel J, Clifton-Bligh P, et al. (2000) Prevalence of positive anti-neutrophil cytoplasmic antibody (ANCa) in patients receiving anti-thyroid medication. European Journal of Endocrinology 142: 587–590. Homma S, Matsushita H, and Nakata K (2004) Pulmonary fibrosis in MPO–ANCA-associated vasculitides. Respirology 9: 190–196. Hunder GG, Arend WP, Block DA, et al. (1990) The American College of Rheumatology 1990 criteria for the classification of vasculitis. Arthritis and Rheumatism 33: 1065–1067. Jennette JC and Falk RJ (1997) Small-vessel vasculitis. New England Journal of Medicine 337: 1512–1523. Johnson RJ (1995) The mystery of the antineutrophil cytoplasmic antibodies. American Journal of Kidney Diseases 26: 57–61. Langford CA (1997) Chronic immunosuppressive therapy for systemic vasculitis. Current Opinion in Rheumatology 9: 41–47. Popa ER, Stegeman CA, and Bos NA (1999) Differential B- and Tcell activation in Wegener’s granulomatosis. Journal of Allergy and Clinical Immunology 103: 885–894. Seo P and Stone JH (2004) The antineutrophil cytoplasmic antibody-associated vasculitides. American Journal of Medicine 117: 39–50. Travis WD, Colby TV, Koss MN, et al. (eds.) (2002) Non-Neoplastic Disorders of the Lower Respiratory Tract, p. 233. Washington, DC: Armed Forces Institute of Pathology and American Registry of Pathology. Weyand CM and Goronzy JJ (1997) Multisystem interactions in the pathogenesis of vasculitis. Current Opinion in Rheumatology 9: 3–11. Xiao H, Heeringa P, Hu P, et al. (2002) Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. Journal of Clinical Investigation 110: 955–963.

Microscopic Polyangiitis U Specks and T Peikert, Mayo Clinic College of Medicine, Rochester, MN, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Microscopic polyangiitis (MPA) is a rare disease of unknown etiology. It is characterized by necrotizing inflammation of small blood vessels (arterioles, capillaries, and venules) in the absence of immune deposits and granulomatous inflammation. Antibodies against the content of azurophilic granules in neutrophilic granulocytes (antineutrophil cytoplasmic antibodies

VASCULITIS / Microscopic Polyangiitis 423 (ANCA)), most commonly targeting myeloperoxidase (MPOANCA), are present in the majority of patients. Although the etiology of MPA remains unknown, clinical observations and animal models suggest a causal association with MPO-ANCA. Clinically, MPA commonly presents with systemic symptoms, palpable purpura, renal dysfunction, pulmonary hemorrhage, and mononeuritis multiplex. If larger arteries are involved, patients may display disease manifestations historically attributed to panarteritis nodosa. Rapidly progressive glomerulonephritis and diffuse alveolar hemorrhage represent the most frequent life-threatening complications. The combination of glucocorticoids and cyclophosphamide is typically used for remission induction therapy. Despite aggressive therapy, relapses occur commonly and treatment-associated morbidity and mortality are frequently encountered.

Microscopic polyangiitis (MPA) is characterized by necrotizing inflammation involving the smallest blood vessels, namely arterioles, capillaries, and venules. It can involve almost any organ system but most commonly affects the kidneys, lungs, skin, and the peripheral nervous system. MPA is a rare disease, with an annual incidence of approximately 8 cases/million. It is slightly more common in men than in women. The age of onset is quite variable, with a wide range from childhood to advanced age. The highest incidence is seen during the fifth and sixth decades of life. After Kussmaul and Maier’s original description of ‘periarteritis nodosa’ in 1866, Davson and colleagues in 1948 coined the term ‘microscopic polyarteritis’ for patients with panarteritis nodosa associated with glomerulonephritis and small vessel vasculitis. In 1952, Zeek and colleagues published the first classification of necrotizing vasculitis. They proposed five categories: hypersensitivity angiitis, allergic granulomatous angiitis, rheumatic arteritis, periarteritis nodosa, and temporal arteritis. The term ‘periarteritis nodosa’ subsequently evolved into the pathologically more accurate term ‘polyarteritis nodosa’. In 1954, Churg and Goodman concluded that the microscopic form of polyarteritis nodosa is much more closely related to Wegener’s granulomatosis and Churg–Strauss syndrome than polyarteritis nodosa. During the following decades, no universally accepted classification was available and the terms hypersensitivity vasculitis, polyarteritis nodosa, and microscopic polyarteritis were used by different authors. In 1994, the Chapel Hill nomenclature for systemic vasculitis was published and is now widely accepted. MPA was defined as necrotizing small vessel (arterioles, capillaries, and venules) vasculitis associated with few immune deposits. Necrotizing vasculitis of small and medium-size arteries may coexist and it frequently presents with glomerulonephritis and pulmonary capillaritis associated with diffuse alveolar hemorrhage.

Etiology Although the cause of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitides including MPA remains unclear, a multifactorial etiology appears likely. Despite inconclusive results of studies investigating the association of major histocompatibility complexes, genetic factors are likely to contribute. Patients with ANCA-associated vasculitis have been shown to harbor polymorphisms of the Fcg receptor resulting in more effective IgG3 binding, leading to more pronounced neutrophil activation by ANCA. Furthermore, polymorphisms of the ANCA target antigen myeloperoxidase (MPO), the major proteinase inhibitor, a1 antitrypsin, and various cytokines have been described. There are rare familial cases and a family history of other autoimmune diseases is frequently present. Environmental factors such as exposure to certain medications, including propylthiouracil, penicillamine, and hydralazine, have been shown to induce MPA. In addition, ANCA-associated vasculitis has been linked in case–control studies to an environmental exposure to silica. In patients with Wegener’s granulomatosis, observations such as seasonal occurrence, high rate of staphylococcal carrier status, and beneficial responses to certain antimicrobial medications in patients and animal models raised the question of a causative infectious pathogen. In contrast, no clear link to an infectious etiology has been established for MPA.

Pathology The typical histological findings in MPA include segmental fibrinoid necrosis of small blood vessels and associated inflammatory infiltration. The acute phase is dominated by neutrophils. Karyorrhexis (leukocytoclasia) and accumulation of fibrin are frequently observed. Fibrinoid necrosis may weaken the vessel wall, resulting in perivascular hemorrhage and the formation of pseudoaneurysms. Associated hemorrhage accounts for common clinical manifestations such as purpura and alveolar hemorrhage. Involvement of small arteries may result in localized tissue ischemia manifesting as an elevation in liver enzymes, abdominal pain, pancreatitis, digital infarcts, and peripheral neuropathy. At the time of the tissue sampling, neutrophils have frequently been replaced by mononuclear leukocytes and collagen deposits have been substituted for fibrinoid necrosis. In advanced lesions, vascular sclerosis may be the only marker of prior vasculitis. Acute pulmonary involvement frequently presents with necrotizing capillaritis manifesting as alveolar

424 VASCULITIS / Microscopic Polyangiitis

hemorrhage. The presence of granulomatous inflammation in the lung or any other affected organ by definition excludes MPA and supports a diagnosis of Wegener’s granulomatosis or Churg–Strauss syndrome. Diffuse alveolar damage, interstitial fibrosis, and hemosiderin-laden macrophages are often found in chronic lesions and may be the only indicator of prior alveolar hemorrhage. Cutaneous involvement typically presents as palpable purpura. Inflammation of dermal venules causes clinical purpura, whereas the involvement of small arteries results in tender erythematous nodules and dermal ulcerations. These manifestations are clinically and pathologically indistinguishable from other disorders presenting with dermal small vessel vasculitis. The kidneys are the most common organs involved by MPA. Focal segmental necrotizing and crescentic glomerulonephritis are most frequently seen. The glomerular histopathology is identical in patients with MPA, Wegener’s granulomatosis, and Churg– Strauss syndrome. Necrotizing arteritis can affect the interlobar and arcuate arteries. In the acute phase, glomeruli with necrosis and crescents are frequently found. The characteristic absence of staining for immunoglobulins and complement resulted in the description ‘pauci-immune’ glomerulonephritis. In the chronic phase, focal or diffuse sclerosis may be the predominant histopathologic finding. It is potentially indistinguishable from other causes of chronic glomerulonephritis.

Clinical Features A gradual onset and non-specific disease manifestations, such as systemic symptoms, fever, and weight loss, frequently delay the diagnosis of MPA (Table 1). Clinical symptoms can involve almost any organ system. The most commonly affected organ is the kidney. Renal involvement presents most often with active urinary sediments with or without an elevation in serum creatinine caused by crescentic glomerulonephritis. Other commonly encountered disease manifestations include diffuse alveolar hemorrhage due to pulmonary capillaritis, palpable purpura caused by dermal leukocytoclastic vasculitis, and musculoskeletal complaints (e.g., arthralgias). MPA is the most frequent cause of pulmonary–renal syndrome. Sinusitis and asthma are rarely found in MPA and should lead to considerations of an alternative diagnosis (Figure 1). Recommended diagnostic tests include chest radiography, urine analysis, serum creatinine, liver function tests, erythrocyte sedimentation rate, C-reactive protein, ANCA serology, complete blood count, and

Table 1 Clinical symptoms of patients with microscopic polyangiitisa Symptoms

Average % of patients

Range between studies (n ¼ 235)

Systemic Renal involvement Skin manifestations Palpable purpura Pulmonary Diffuse alveolar hemorrhage Musculoskeletal Neurologic (peripheral) Neurologic (central) Gastrointestinal Ocular Ear, nose, and throat Sinusitis

77 92 57 41 38 34

73–79 79–100 35–62 41–44 25–55 n/a

73 32 14 44 30 26 4

72–74 14–58 0–40 30–56 28–30 20–30 1–9

a Summary of data reported by Serra et al., Savage et al., D’Agati et al., Adu et al., and Guillevin et al.

biopsy of affected organs for histologic evaluation. Additional laboratory tests aimed at the exclusion of alternative diagnoses should include hepatitis serology, determinations of complement levels and cryolobulins, as well as testing for antinuclear, antiglomerular basement membrane, and antiphospholipid antibodies. Since their original description in 1982, much has been learned about ANCA. ANCA testing by immunofluorescence and antigen-specific enzymelinked immunosorbent assays (ELISA) is necessary for the accurate assessment of ANCA status. In MPA, indirect immunofluorescence of ethanol fixed neutrophils is typically associated with a perinuclear staining pattern (P-ANCA). The target antigen has been identified as MPO, and MPO-specific ANCA are routinely identified using direct ELISA. The combination of P-ANCA by indirect immunofluorescence and MPO–ANCA by direct ELISA has a sensitivity of 68% and a specificity of 99% for the diagnosis of MPA. Occasionally, a cytoplasmatic staining pattern is identified during indirect immunofluorescence (C-ANCA) or ELISA identifies proteinase-3 (PR3ANCA) as the target antigen (15%). Changes in clinical presentation and the development of granulomatous inflammation may require the reclassification of some patients with MPA as Wegener’s granulomatosis. This underscores the close relationship and the dynamic nature of the subtypes of ANCA-associated vasculitis. During the course of the disease, the presence or absence of ANCA has been found to correlate with disease activity. Unfortunately, this correlation is not reliable enough to justify the initiation of aggressive immunosuppressive

VASCULITIS / Microscopic Polyangiitis 425

(c)

(a)

(e)

GS

GC ALV

(b)

(d)

(f)

Figure 1 Clinical and pathologic manifestations of microscopic polyangiitis. (a) Computerized tomography of the chest demonstration bilateral alveolar infiltrates and dense consolidation in a patient with diffuse alveolar hemorrhage. (b) Lung biopsy showing diffuse alveolar hemorrhage, alveolar filling with erythrocytes (ALV), and interstitial inflammation (arrow); hematoxylin & eosin (H&E) stain. Courtesy of Dr J L Myers. (c) Urine microscopy showing a red cell cast in a patient with glomerulonephritis. (d) Renal biopsy of focal segmental necrotizing glomerulonephritis, sclerosed glomerulus (GS), and crescent formation (GC); trichrome stain. (c and d) Courtesy of Dr F C Fervenza. (e) Palpable purpura. (f) Skin biopsy with leukocytoclastic vasculitis and inflammatory infiltrate of the vessel wall (short arrow) and the perivascular space (long arrow); H&E stain. Courtesy of Dr L E Gibson.

therapy, with all its associated risks, solely based on ANCA titers. In summary, the diagnosis of MPA can be made in patients presenting with typical clinical manifestations of small vessel vasculitis, positive P-ANCA/ MPO–ANCA, biopsies showing the presence of necrotizing vasculitis, or pauci-immune segmental necrotizing glomerulonephritis in whom other causes of small vessel vasculitis have been excluded. By definition, histopathological evidence or clinical features of granulomatous inflammation should be absent.

Pathogenesis The cause of ANCA-associated vasculitis including MPA remains unknown. Efforts to identify a pathogenetic microorganism or environmental exposure have failed. Since the description of ANCA and their association with small vessel vasculitis, our understanding of their role in the pathogenesis has advanced substantially. Although the mechanisms involved in

induction and maintenance of ANCA production remain unclear, ANCA are suspected to be actively involved in the pathogenesis of ANCA-associated vasculitis. Cytokine-activated neutrophils have been found to express ANCA target antigens on their cell surface. Interactions of ANCA with these target antigens and Fcg receptors on the surface of primed neutrophils are known to activate neutrophils by induction of an oxidative burst and promotion of degranulation. Furthermore, ANCA binding to monocytes is associated with enhanced cytokine production. Neutrophil degranulation is associated with the release of MPO and PR3 from neutrophils, which has been shown to result in endothelial cell injury and death. In addition, the interaction of ANCA and their target antigens on the cell surface of neutrophils induces apoptosis and causes perturbations of apoptotic clearance, which in turn may promote tissue necrosis. In Wegener’s granulomatosis, bacterial superantigens have been implicated in the polyclonal stimulation of T and B cells and, therefore, possibly

426 VASCULITIS / Microscopic Polyangiitis Infection Superantigen

LPS

PMN

Th

TNF- (B) Priming

(A)

(B1)

T


CD4+

IL-1 Monocyte

TCR Th

IL-8 (B3)

B cell

(G)

MCP-1 (C)

sIL-2R

Production of ANCA

Activation by ANCA PMN (B2)

IL-8 IL-1 (C)

PMN IL-8

ROS (D) MPO PR3 HLE

Adhesion induced by ANCA and cytokines

ELAM-1 ICAM-1 VCAM-1

Complement (F)

Binding of ANCA

Endothelial cell Apoptosis

(E)

Apoptosis

Figure 2 Proposed scheme outlining the pathogenic effects of ANCA leading to the development of vasculitis. Infections and other tissue injuries cause in an inflammatory response with associated cytokine release. These cytokines result in priming of neutrophils and monocytes (A), prompting the expression of ANCA target antigens on their surface (B). The display of target antigens promotes the binding of circulating ANCA, which recognize these antigens via their specific antigen-binding fragment (B1). Primed neutrophils and monocytes also express Fcg receptors and consequently are able to bind a variety of antibodies including ANCA to their surface (B2). Simultaneous binding of circulating antigen (i.e., released from activated inflammatory cells) (D) may cause cross-linking of Fcg receptors, initiating Fcg receptor engagement-dependent activation (B3). Furthermore, binding of ANCA to primed neutrophils and monocytes has been shown to induce the release of cytokines such as interleukin-8 (IL-8), IL-1b, monocyte chemotactic protein-1 (MCP-1), and other strong chemoattractants (C). Stimulation of neutrophils by ANCA also induces a respiratory burst with release of reactive oxygen species (ROS) and degranulation characterized by the release of human leukocyte elastase (HLE), proteinase-3 (PR3), and myeloperoxidase (MPO) (D). This can result in direct endothelial cell injury. Finally, stimulation by cytokines and ANCA activates endothelial cells, resulting in increased adherence of primed and activated neutrophils to their surface. Consequently, the release of cytotoxic neutrophil constituents can directly cause endothelial cell damage. Invoked mechanisms include direct cytotoxicity, apoptosis (E), as well as localized complement activation (F). The mechanisms by which ANCA production is triggered and perpetuated remain unclear. However, T cells are thought to play a significant role in mediating the production of ANCA by plasma cells that are derived from antigen-specific B cells (G). LPS, lipopolysaccharide; TNF-a, tumor necrosis factor alpha; TCR, T-cell receptor; SIL-2R, soluble interleukin-2 receptor; PMN, neutrophil; ELAM-1, endothelial-leukocyte adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1. Reproduced from Russell KA and Specks U (2001) Are antineutrophil cytoplasmic antibodies pathogenic? Rheumatic Disease Clinics of North America 27: 815–831, with permission from Elsevier.

contribute to the initiation and perpetuation of ANCA production. A role of bacterial superantigens in MPA is less well documented (Figure 2). These previous observations, the fact that patients with medication-induced ANCA production may develop small vessel vasculitis, and evidence derived from a murine model of MPO–ANCA-induced vasculitis support the notion that ANCA play a significant role in the pathogenesis of MPA. The precise mechanisms of initiation and maintenance of ANCA production and other potentially contributing mechanisms still need to be elucidated.

Animal Models Animal models are frequently utilized as surrogates for investigation of human diseases. The development

of a mouse model for vasculitis has been a rather challenging endeavor. A variety of models have been investigated but none were specific enough to allow firm conclusions. Xiao and colleagues provided convincing evidence that MPO–ANCA cause a disease similar to ANCAassociated vasculitis in mice. These investigators immunized MPO knockout mice (MPO
/
) with murine MPO to induce MPO–ANCA production. Immunoglobulins and splenocytes of MPO
/
mice immunized with either MPO or bovine serum albumin (BSA) were subsequently transferred to either immunodeficient Rag 2
/
mice (lacking functional T and B cells because of a recombinase activating gene deficiency) or wild-type mice. Transfer of splenocytes as well as transfer of immunoglobulins resulted in the development of necrotizing crescentic

VASCULITIS / Microscopic Polyangiitis 427

glomerulonephritis in all mice if the splenocytes originated from mice immunized with MPO. In contrast, there were only minimal changes if the splenocytes came from animals immunized with BSA. Furthermore, 30% of the mice that received either splenocytes or immunoglobulins from mice immunized with MPO developed alveolar hemorrhage. On the other hand, no disease developed in MPO-/- animals receiving anti-MPO splenocytes, confirming the importance of the antigen antibody interaction in the development of the disease.

Management and Therapy Clinical studies preceding the Chapel Hill consensus conference frequently combined panarteritis nodosa and microscopic polyangiitis and, therefore, evaluated a rather heterogeneous patient population. Prior to the utilization of immunosuppressive therapy, these diseases were associated with a very high mortality. The introduction of glucocorticoids resulted in an improvement in short-term survival from 13% to 48–57%. During the late 1970s and early 1980s, several investigators reported retrospective analyses suggesting that the combined use of glucocorticoids and cytotoxic drugs results in survival rates of approximately 75%. A landmark publication by Fauci and colleagues reported the survival of 14 of 17 patients with severe systemic necrotizing vasculitis after treatment with cyclophosphamide. During the past 20 years, a number of prospective controlled trials evaluating different treatment strategies were conducted by the French Vasculitis Study Group. Unfortunately, these trials included relatively few patients and were designed prior to the Chapel Hill classification. Therefore, these studies consist of patients with Churg–Strauss syndrome, panarteritis nodosa, panarteritis nodosa associated with hepatitis B infection, and microscopic polyangiitis. Regarding the management of microscopic polyangiitis, these studies suggest that patients with severe disease have a survival advantage when treated with cyclophosphamide in addition to glucocorticoids, whereas there was no such advantage in patients with mild disease. Furthermore, a 12-month course of cytotoxic therapy was found to be associated with fewer relapses compared to a 6-month course. Unfortunately, these survival benefits come with a price of high treatment-related morbidity and mortality, largely attributable to cyclophosphamide. This observation resulted in substantial efforts to decrease treatment-related toxicity and to develop new, more specific modes of therapy. Several investigators focused on potential benefits of intermittent administration of cyclophosphamide

compared to continuous therapy. The results of these studies are inconsistent, and superiority of this approach has not been demonstrated. In fact, in patients with Wegener’s granulomatosis, intermittent administration of cyclophosphamide was associated with an increased relapse rate. Alternatively, after remission induction, an early transition from cyclophosphamide to different immunosuppressive agents has been proposed. A randomized, controlled multicenter trial of patients with small vessel vasculitis (MPA and Wegener’s granulomatosis) determined that cyclophosphamide can be safely switched to azathioprine after the initial 3–6 months of therapy and induction of remission. Mortality and relapse rates were comparable in both treatment groups. Plasma exchange and intravenous IgG have been studied and found to be effective in certain patient populations, such as patients with diffuse alveolar hemorrhage and patients with advanced renal disease requiring hemodialysis. Furthermore, more specific biological therapies (e.g., the tumor necrosis factor-a antagonists and monoclonal antibodies directed against CD 20 (B cells)) are currently undergoing evaluation of their efficacy and safety in ANCA-associated vasculitis. Standard therapy for microscopic polyangiitis includes a combination of glucocorticoids and continuous oral cyclophosphamide for induction of remission. As remission is achieved, glucocorticoids are tapered and cyclophosphamide is replaced by azathioprine for remission maintenance. In order to prevent treatment-related complications, patients should receive trimethoprim/sulfamethoxazole or alternative regimens for pneumocystis prophylaxis and be prophylaxed for the development of osteoporosis with calcium, vitamin D supplementation, and bisphosphate therapy. See also: Alveolar Hemorrhage. Autoantibodies. Corticosteroids: Glucocorticoid Receptors; Therapy. Granulomatosis: Wegener’s Disease. Interstitial Lung Disease: Overview. Leukocytes: Eosinophils; Neutrophils; Monocytes. Serine Proteinases. Vasculitis: Overview.

Further Reading Booth AD, Pusey CD, and Jayne DR (2004) Renal vasculitis – an update in 2004. Nephrol Dial Transplant 19(9): 1964– 1968. Falk RJ and Jennette JC (1988) Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. New England Journal of Medicine 318: 1651–1657.

428 VENTILATION / Overview Fauci AS, Katz P, Haynes BF, et al. (1979) Cyclophosphamide therapy of severe systemic necrotizing vasculitis. New England Journal of Medicine 301: 235–238. Gayraud M, Gullevin L, and le Toumelin P (2001) Long-term follow-up of polyarteritis nodosa, microscopic polyangiitis, and Churg Strauss syndrome. Arthritis and Rheumatism 44: 666–675. Guillevin L, Durand-Gasselin B, Cevallos R, et al. (1999) Microscopic polyangiitis: clinical and laboratory findings in eighty-five patients. Arthritis and Rheumatism 42: 421–430. Harper L and Savage COS (2000) Pathogenesis of ANCAassociated systemic vasculitis. Journal of Pathology 190: 349–359. Jayne DR, Rasmussen N, Andrassy K, et al. (2003) A randomized trial of maintenance therapy for vasculitis associated with antineutrophil cytoplasmic antibodies. New England Journal of Medicine 349: 36–44. Jennette JC and Falk RJ (1997) Small-vessel vasculitis. New England Journal of Medicine 337: 1512–1523.

Vasoactive Intestinal Peptide (VIP)

Jennette JC, Falk RJ, Andrassy K, et al. (1994) Nomenclature of systemic vasculitides. Arthritis and Rheumatism 37: 187–192. Jennette JC, Thomas DB, and Falk RJ (2001) Microscopic polyangiitis (microscopic polyarteritis). Seminars in Diagnostic Pathology 18: 3–13. Lauque D, Cadranel J, Lazor R, et al. (2000) Microscopic polyangiitis with alveolar hemorrhage. A study of 29 cases and review of the literature. Medicine 79: 222–233. Russell KA and Specks U (2001) Are antineutrophil cytoplasmic antibodies pathogenic? Rheumatic Disease Clinics of North America 27: 815–831. Specks U (2000) Are animal models of vasculitis suitable tools? Current Opinion in Rheumatology 12: 11–19. Specks U (2001) Diffuse alveolar hemorrhage syndromes. Current Opinion in Rheumatology 13: 12–17. Xiao H, Heeringa P, Hu P, et al. (2002) Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. Journal of Clinical Investigation 110: 955–963.

see Kinins and Neuropeptides: Vasoactive Intestinal Peptide.

VENTILATION Contents

Overview Collateral Control Uneven

Overview M P Hlastala, University of Washington, Seattle, WA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract This article provides the basics for evaluation of ventilation and gas exchange. Symbols commonly used and basic gases present in air breathed into and out of the lung are described. The physical basis for the understanding of ventilation and gas exchange in the lungs is presented. Warming and humidification during inspiration are described. Ventilation-related concepts, such as alveolar ventilation and dead space ventilation are determined from the exchange properties of carbon dioxide and oxygen. The article provides the means for evaluation of the efficiency of a patient’s ventilation.

Introduction: Physics of Gases Gas Pressure

The total pressure exerted by the kinetic energy of all the molecules in the atmospheric mixture is

atmospheric or barometric pressure (PB). At sea level, atmospheric pressure will raise a column of mercury in an evacuated tube to a height of 760 mm (this is equivalent to 29.92 inches). PB decreases with increasing altitude. Pressure in the chest or lungs is often expressed relative to atmospheric pressure (Patm ¼ 0). Gauge pressure is the pressure relative to Patm and is also used when measuring blood pressure. Gas pressure in atmosphere, alveoli, and blood, absolute pressure terms are expressed as mmHg or torr (1 Torr ¼ 1 mmHg under standard gravitational conditions), a pleural pressure of
13 cmH2O or
10 mmHg (gauge) is equal to 750 mmHg (absolute) in absolute pressure terms. Atmospheric air is a mixture consisting of nitrogen 78.09%, oxygen 20.95%, argon 0.93%, and CO2 0.03%, with water vapor varying from 0% to 2% and diluting the other gases accordingly. For simplicity, we typically assume oxygen 21%, nitrogen 79%, ignore CO2, and water vapor varies with the degree of humidification.

VENTILATION / Overview 429 Partial Pressure

The pressure exerted by the kinetic energy of each individual gas in a mixture of gases is referred to as its partial pressure. A gas mixture in a sealed container will develop a pressure on the walls of the container by virtue of the collisions between the gas molecules and the container walls. The total pressure developed by the gas is a sum of the pressure developed by all the molecules of the mixture as they bounce off the container walls. The partial pressure of any component gas is the pressure developed by the molecules of that component acting alone. The partial pressure of a gas is a measure of its tendency to diffuse through either gas or fluid media. Dalton’s law describes a relationship that total pressure of a mixture of gases is equal to the sum of the partial pressures of each gas in the mixture. In the gas phase, partial pressure is proportional to concentration. The partial pressure of a gas is found by multiplying the fraction of the gas by the total pressure. For PO2 in air PO2 ¼ FO2  PB ¼ 0:21  760 mmHg ¼ 160 mmHg

½1

After humidification, partial pressure of inspired oxygen ðPI O2 Þ can be calculated as PI O2 ¼ FI O2  ðPB
PH2 O Þ ¼ 0:21  713 ¼ 150 mmHg

½3

PI N2 ¼ 0:79  713 ¼ 563 mmHg Inspired Air Warming and Humidification

The volume occupied by gas is directly proportional to the absolute temperature (Kelvin), inversely proportional to total pressure, and is affected by the amount of water vapor present. Exhaled gas collected in a spirometer or bag is saturated at ambient temperature and pressure (ATPS). Lung and ventilatory volumes are converted to body temperature and pressure, saturated (BTPS) conditions, an expansion of about 9–10%. Gas transfer quantities (V’ O2 , V’ CO2 , and diffusing capacity) are expressed at standard temperature (273 K), pressure (760 Torr), dry (STPD) conditions. Corrections can be made using the following V1  ðP1
P1H2 O Þ V2  ðP2
P2H2 O Þ ¼ T1 T2

½4

Total pressure is the sum of the partial pressures of individual gases in the lung

Ventilation: The Process PCO2 þ PO2 þ PN2 þ PH2 O ¼ PB

½2

Water Vapor

Water vapor behaves differently from the other respiratory gases. For a gas mixture in contact with liquid, the partial pressure of water vapor depends on temperature. Atmospheric gas is usually cooler than body temperature and is rarely 100% saturated. Inspired gas is warmed to body temperature and humidified to full saturation with water vapor in the respiratory system. At 371C water vapor has a partial pressure of 47 mmHg (water vapor pressure at saturation ¼ 17.5 mmHg at 201C, 47.0 mmHg at 371C, and 760 mmHg at 1001C). This pressure does not vary with changes in barometric pressure or changes in the other components in the gas mixture. If PB is 760 mmHg, and PH2 O is 47 mmHg, the difference, 713 mmHg, is the total partial pressure of the remaining inspired gases. Of this total, 21% is oxygen and 79% is nitrogen. The relative humidity is the actual water vapor partial pressure expressed as a fraction of the saturated vapor pressure at the same temperature. For example, at 201C, a PH2 O of 9.0 mmHg corresponds to a 51% (9.0/17.5) relative humidity.

Outside air must come into close proximity with blood within the lungs in order to transfer respiratory gases (oxygen and carbon dioxide) between blood and air in an efficient manner. Ventilation is the movement of outside fresh air to the alveoli for gas exchange and the subsequent exhalation of alveolar air to the environment.

Total Ventilation The total ventilation (minute volume) is the amount of air passing through the mouth during an exhalation and can be determined by collecting exhaled gas for a period of time (V’ E ¼ volume exhaled per minute). A tidal volume ðV’ T Þ is the volume of gas exhaled during one normal respiratory cycle and total ventilation is equal to the tidal volume multiplied by the breathing frequency (f) V’ E ¼ V’ T  f

½5

Alveolar Ventilation Gas exchange occurs in the alveolus when inspired air comes in close proximity to capillary blood. Not all inspired air reaches the alveoli to participate in

430 VENTILATION / Overview •

•

•

V1

VA

VD

•

VE

CO2

CO2

Figure 1 Schematic of dead space ventilation. Physiological dead space is composed of anatomical dead space plus alveolar dead space. Alveoli that are overventilated with respect to the amount of blood flow are a contribution to the alveolar dead space.

gas exchange (Figure 1). The inspired air first passes through the conducting airways: the nose, mouth, pharynx, larynx, trachea, bronchi, and bronchioles. Large airways ‘conduct’ air from the atmosphere to the alveoli. Because the conducting airways contain no alveoli, they do not participate in gas exchange. However, airways participate in the humidification and warming of inspired air. At the end of inspiration, the volume of air remaining in the conducting airways is called the anatomic dead space (because it does not participate in gas exchange). The effect of the conducting airways on ventilation and gas exchange can be considered in two ways. At the end of inspiration, humidified and warmed atmospheric air remains in these airways and leaves as the first gas out on the subsequent exhalation. At the end of expiration, alveolar gas (with CO2 added and O2 partially removed) fills the anatomic dead space and reenters the alveoli with the next breath. Thus a tidal breath may inspire 500 ml of air and will cause a 500 ml expansion of the alveolar volume followed by the expiration of 500 ml, but the volume of fresh air delivered to the alveoli and the volume of alveolar air exhaled to the atmosphere will each be less than 500 ml by the volume of the anatomic dead space. Any alveoli that are ventilated with air but not perfused with blood, do not participate in gas

exchange. The ventilation going to these alveoli also acts as dead space ventilation and is called alveolar dead space. Ventilation to areas of lung that have reduced perfusion behaves as if a portion were going to alveoli with normal perfusion and a portion to alveoli with no perfusion. This latter region is also part of the alveolar dead space. Dead space components are shown schematically in Figure 2. Alveolar Gas Composition

Carbon dioxide exchange The body’s CO2 production is eliminated almost completely by ventilation. V’ CO2 ¼ the volume of CO2 exhaled per minute (V’ E CO2 ) minus the volume of CO2 inhaled (usually a negligible amount). Because all the CO2 in the expired gas must have come from alveolar ventilation V’ CO2 ¼ V’ A  FA CO2

½6

where FA CO2 is the fraction of CO2 in the alveolar space. Rearranging and substitution of FA CO2 ¼

PA CO2 ðPB
47Þ

½7

VENTILATION / Overview 431

•

VD

anat

CO2

CO2

•

•

VA

VD

alv

Figure 2 Schematic of ventilation in the lung. Inspired ventilation splits between the dead space and the alveolar space, where it picks up CO2. With exhalation, ventilation from the dead space ðV’ D Þ and from the alveolar space ðV’ A Þ mix to form the total ventilation ðV’ E Þ.

provides the alveolar ventilation equation PA CO2 ¼

V’ CO2  ðPB
47Þ V’ A

½8

Alveolar PCO2 is directly related to the production of CO2 and inversely related to alveolar ventilation ðV’ A Þ. The body maintains a normal arterial and alveolar PCO2 of 35–40 mmHg by adjusting ventilation appropriately for the V’ CO2 determined by metabolic production. When using eqn [8], both V’ A and V’ CO2 must be expressed in the same units (i.e., either BTPS or STPD). Hyperventilation is ventilation in excess of metabolic needs. The alveolar ventilation equation indicates that PA CO2 less than normal is defined as alveolar hyperventilation. A PA CO2 greater than normal is defined as alveolar hypoventilation. Alveolar ventilation can be altered by changes in either total ventilation or dead space (wasted) ventilation ðV’ A ¼ V’ E
V’ D Þ. Any depression in central nervous system function that occurs may change V’ E ; for example, analgesics and sedatives can reduce V’ E . Any increase in V’ D will reduce V’ A unless V’ E increases accordingly. Many lung diseases increase physiologic

dead space, contributing to respiratory failure when the patient can no longer increase total ventilation. Alveolar PCO2 reflects a balance between the CO2 delivery to the alveoli ðV’ CO2 Þ and the CO2 elimination from the lungs by ventilation ðV’ E CO2 Þ. Production and excretion must be the same in a steady state. Under resting conditions V’ CO2 is relatively constant at approximately 200 ml min
1 for a normal-sized individual. With short-term hyperventilation CO2 will be exhaled at a greater rate than CO2 production; PA CO2 (and arterial PCO2 ) will fall. As PA CO2 falls, the CO2 exhaled per minute will decrease until it is again equal to CO2 production and a new steady state is established. During hypoventilation, the rate of CO2 exhalation initially falls, then PA CO2 (and arterial PCO2 ) rise until a new steady state is reached where excretion again equals production with less ventilation but each volume of gas leaving the alveoli carrying more CO2. This mechanism allows patients with severe lung disease and high work of breathing to excrete their CO2 production at less energy cost. Oxidation Energy necessary for life processes is produced by oxidation of carbohydrates, protein,

432 VENTILATION / Overview

and fats producing mainly CO2 and H2O as breakdown products. The ratio of metabolic CO2 production to the oxygen consumption ðV’ CO2 =V’ O2 Þ of the tissues is the respiratory quotient (RQ). When metabolizing carbohydrates the RQ ¼ 1:0 C6 H12 O6 þ 6O2 26CO2 þ 6H2 O RQ ¼

V’ CO2 6 ¼1 V’ O 6

½9 ½10

2

When metabolizing fats, RQ ¼ 0:7 and the RQ of protein has an average value of 0.8. The RQ for the entire body varies with the percentages of carbohydrate, fat, and protein being oxidized at any given time. The respiratory exchange ratio (R) relates the volume of CO2 eliminated to the lungs and the net volume of O2 taken up from the lungs. In a steady state, R must equal RQ, but R may vary transiently with factors other than metabolism. If an individual rapidly increases ventilation, R rises because CO2 is being ‘blown off’ from blood and tissue stores (but little O2 can be added). Oxygen exchange The alveolar O2 partial pressure also reflects a balance of two processes: O2 delivery to the alveoli by ventilation, and O2 removal from the alveoli by capillary blood. Oxygen delivery to alveoli is determined by ventilation (V’ A ) and the fraction of inspired O2 ðFI O2 Þ. Oxygen is carried away in exhaled air. Air leaving alveoli has the alveolar oxygen concentration ðFA O2 Þ. So the net O2 delivery is given by V’ A  ðFI O2
FA O2 Þ. Oxygen removal is governed by tissue O2 consumption, which varies with activity and disease. Under resting conditions, oxygen uptake is approximately 250 ml min
1 for an average-sized person. In a steady state the alveolar O2 level is not changing so these processes of delivery and removal are equal. The conservation of mass principle states that oxygen consumed ðV’ O2 Þ equals that removed from the alveolar ventilation (none is removed from dead space ventilation), thus V’ O2 ¼ V’ A ðFI O2
FA O2 Þ

V’ O2 ¼ ðPB
47Þ V’ A

PI O2
PA O2 ¼ PA CO2

½13

It will be useful in understanding arterial blood gas values in patients to estimate the PA O2 . From the discussion above we can see that if V’ CO2 ¼ V’ O2 , then (rearranging eqn [13]) PA O2 ¼ PI O2
PA CO2

½14

Since the arterial Pa CO2 , which we measure, is very close to the PA CO2 , we can rewrite this: PA CO2 ¼ PI O2
Pa CO2

½15

Usually V’ CO2 and V’ O2 are not identical but are closely linked and their ratio V’ CO2 =V’ O2 is the respiratory quotient or respiratory exchange ratio (R). The approximate relationship described in eqn [15] assumes that R ¼ 1, V’ CO2 ¼ V’ O2 , and V’ E ¼ V’ I . Normally, Ro1; V’ CO2 oV’ O2 , and V’ E oV’ I . A more complete form of this alveolar gas equation is   ð1
FI O2 Þ PA O2 ¼ PI O2
Pa CO2 FI O2 þ ½16 R When breathing 100% oxygen ðFI O2 ¼ 1Þ, Pa CO2 and PA O2 always equal PI O2 , for any R. For normal values (R ¼ 0:8; FI O2 ¼ 0:21; PI O2 ¼ 150 mm Hg; Pa CO2 ¼ 40 mm Hg), PA O2 turns out to be about 100 mm Hg   ð1
0:21Þ PA O2 ¼ 150
40 0:21 þ ¼ 102 mmHg 0:8

½11

Rearranging and converting fractions to partial pressures for inspired and alveolar oxygen, and multiplying both sides by (PB
47) gives PI O 2
PA O 2 ¼

PI O2 , V’ A and V’ O2 . If PI O2 and V’ O2 are constant and V’ A goes up (hyperventilation) then PA O2 must go up, and if V’ A goes down (hypoventilation) then PA O2 must go down. Since V’ CO2 is the metabolic product of the O2 consumption the quantities V’ CO2 and V’ O2 are linked to one another. If V’ CO2 and V’ O2 are identical ðR ¼ 1Þ, then eqns [8] and [12] are similar on the right and show that the fall in PO2 inspired to alveolar air is exactly the same as the rise in PCO2 from 0 to the alveolar level.

½12

This is analogous to the alveolar ventilation equation [8] for CO2. Alveolar PO2 is determined only by

This relationship is often misinterpreted as an indication that alveolar O2 is displaced by CO2. This is incorrect, as the removal of O2 and addition of CO2 proceed as independent processes in the lung. But since the normal RQ in the tissues is approximately equal to 1, the loss of O2 from the inspired air to the blood is approximately equal to the gain in CO2. Nitrogen exchange When Ra1; V’ CO2 aV’ O2 . Under these circumstances, the total amount of gas

VENTILATION / Overview 433

volume in the alveolus changes. Because the exchange of N2 is so small (because of nitrogen’s low blood solubility), the total number of N2 molecules in the alveolus remains unchanged. If the alveolar gas volume decreases (when Ro1), the relative fraction of N2 increases and PN2 increases. When R41, the opposite will occur and PN2 decreases. Under normal circumstances, if R ¼ 0:8, the alveolar PN2 (and alveolar end-capillary blood PN2 ) is greater than inspired PN2 (humidified) by about 10 mmHg. This difference occurs even though there is only minimal net nitrogen exchange. Alveolar PO2 and PCO2 are both changing during the respiratory cycle because ventilation is periodic while V’ O2 and V’ CO2 are relatively constant. During inspiration, fresh air is delivered to the alveolus causing PCO2 to decrease and PO2 to increase. During expiration, continuing gas exchange causes PCO2 to increase and PO2 to decrease. The alveolar gas equations predict constant values for PCO2 and PO2 . However, values actually fluctuate about the mean values by approximately 3 mmHg and 5 mmHg for PCO2 and PO2 , respectively with the respiratory cycle.

Dead Space Ventilation Anatomical Dead Space

The volume of the anatomical dead space in a normal adult male is about 150–180 ml (a commonly used approximation is that the VD in ml ¼ lean body weight in pounds). In a young normal individual the physiological dead space volume is only slightly greater than this or about 25–35% of an average tidal volume (referred to as the VD/VT ratio). The anatomic dead space is not fixed, but increases at higher lung volumes because the intrapulmonary airways increase in size along with the surrounding lung tissue during inspiration. Breathing with an increased tidal volume is associated with only a modest decrease in VD/VT ratio. With exercise tidal volume may increase to 2.5–3.0 l and VD/VT normally falls to 10–15%. A contributing factor is the increase in pulmonary blood flow that tends to reduce any poorly perfused alveolar dead space. At the other extreme it would seem that as tidal volume became small, approaching the anatomical dead space, alveolar ventilation should fall to zero and gas exchange would be impossible. However it has been demonstrated that gas exchange can be maintained even with tidal volumes equal to or smaller than the measured anatomical dead space if ventilation occurs at a high enough frequency. Some fresh gas reaches alveoli because of physical mixing induced by high breathing frequencies.

Physiological Dead Space

The physiological dead space is made up of anatomical and alveolar dead space VDphysiological ¼ VDanatomic þ VDalveolar

½17

The symbols VD and V’ D without further qualifiers are used herein to signify the physiological dead space and its ventilation. The physiological dead space (VD) and wasted ventilation ðV’ D Þ are usually expressed as a fraction of the tidal volume (VD/VT) and total ventilation ðV’ D =V’ E Þ, respectively. Physiological dead space may be increased with lung disease, due to an increase in the alveolar component. The volume of air that participates in gas exchange because it is in contact with perfused alveoli is the alveolar ventilation ðV’ A ¼ V’ E
V’ Dphysiological Þ. The alveolar ventilation is critical, as it determines the amount of air presented to alveoli into which CO2 can be added and from which O2 can be removed. The ventilation wasted by ventilating dead space includes that going to anatomical dead space plus that to unperfused alveoli and a portion of that to poorly perfused alveoli. These poorly perfused (or excessively ventilated) areas are considered as if they were made up of some perfect alveoli (normal gas exchange) and some unperfused alveoli. Thus the total physiological dead space is not an anatomically identifiable volume but a pseudo-volume that we obtain by calculation. It reflects the inefficiency of ventilation as it pertains to CO2 exchange. The total expired volume of air per minute is considered to come from two sources: ideal alveoli with PA CO2 ¼ Pa CO2 , and unperfused areas (conducting airways or alveolar dead space) with inspired PCO2 ¼ PI CO2 ¼ 0. The total expired volume of CO2 comes entirely from the effective (non-dead space) alveolar ventilation ðV’ E
V’ D Þ: V’ CO2 ¼ V’ E  FE CO2 ¼ ðV’ E
V’ D Þ  FA CO2 þ V’ D  0 VD ðFA CO2
FE CO2 Þ ¼ FA CO2 VT

½18 ½19

Multiplying top and bottom by (PB
47) converts fraction to partial pressure VD ðPA CO2
PE CO2 Þ ¼ PA CO2 VT

½20

There is no easy way to measure PACO2, but since ideal alveoli with PA CO2 ¼ Pa CO2 are assumed, the measured arterial blood gas value can be used. The

434 VENTILATION / Collateral

value of PE CO2 is obtained from a collection of expired gas VD ðPa CO2
PE CO2 Þ ¼ Pa CO2 VT

½21

This is spoken of as the VD/VT or wasted fraction of each tidal breath. Multiplying this fraction by the tidal volume or minute ventilation gives the volume of physiological dead space or wasted ventilation. Dead space has the effect of diluting the CO2 content of expired air below the alveolar level. Since the body needs to expire a certain volume of CO2 per minute the effect of a low PE CO2 requires more total ventilation to maintain homeostasis. In carrying out a measurement of physiological dead space, it must be remembered that the volume of air in mouthpiece, connections, and valve (mechanical dead space) will also contribute CO2-free air to the expired collection. See also: Alveolar Surface Mechanics. Alveolar Wall Micromechanics. Reflexes from the Lungs and Chest Wall. Ventilation: Collateral; Control. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation.

Further Reading ¨ ber die lungenathmung. Skandinavisches Archiv Bohr C (1891) U fu¨r Physiologie 2: 236–268. Enghoff H (1938) Volumen inefficax: Bemerkungen zur Frage des scha¨dlichen Raumes. Uppsala La¨karefoeren Fo¨rs 44: 191–218. Fowler WS (1948) Lung function studies. II. The respiratory dead space. American Journal of Physiology 154: 405–416. Hlastala MP (1996) Ventilation. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundations. 2nd ed., vol. 2, pp. 1209–1214. New York: Raven Press. Riley RL, Lilienthal JL Jr, Proemmel D, and Franke RE (1946) On the determination of the physiologically effective pressure of oxygen and carbon dioxide in alveolar air. American Journal of Physiology 147: 191–198. Severinghaus JW and Stupfel M (1957) Alveolar dead space as an index of distribution of blood flow in pulmonary capillaries. Journal of Applied Physiology 10: 335–348.

Collateral W Mitzner, Johns Hopkins University, Baltimore, MD, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract When an airway in the lung becomes obstructed, there exists the possibility for that obstructed region to get limited ventilation through pathways that are commonly called collateral channels.

Ventilation that results from such pathways is called collateral ventilation. Collateral ventilation may have functional roles in both normal and pathologic situations. Several different channels have been suggested as the site of such ventilation, with the alveolar pores of Kohn being most commonly cited. However, when considering the anatomy and physiology of such potential pathways, it is highly unlikely that these alveolar pores could provide sufficiently low resistance to account for the measured magnitude of collateral flow. Rather, the interbronchial channels of Martin, which exist at the level of the terminal bronchiole, are far more likely to be the primary site of collateral ventilation. These collateral channels exist in nearly all mammalian species. In humans, the collateral channels have been shown to be altered in lung pathology, particularly in both emphysema and asthma. Measurement of the collateral resistance may provide the only means of directly measuring the dimensions of most peripheral airways in the lung in living subjects.

Introduction The term collateral ventilation refers to ventilation of a region of lung through ‘collateral’ airways. In vernacular usage, the word collateral has several relevant definitions, including ‘accompanying, but secondary or subordinate’ and ‘corresponding in rank or function’. Nearly all reviews of collateral ventilation have emphasized this secondary nature, ignoring the corresponding nature of the collateral airways. This has led to a common notion that not only are the specific collateral airways responsible for collateral ventilation in some way different from the normal airways but also they take no part in normal ventilation. This is not the case. Collateral airways are in fact normal airways that are normally ventilated with each breath. However, the secondary nature of ventilation through these collateral airways can arise if and when a larger airway becomes obstructed. In this case, collateral ventilation to the obstructed region can now occur via increased flow through a limited number of normal airways. To provide a functional definition of collateral ventilation, one first must consider an anatomical region of lung parenchyma served by a given bronchus. Within this region ventilation normally occurs through that bronchus via the standard airway branching pattern from larger to smaller airways. Collateral ventilation to this given region then refers to any ventilation that arrives from a neighboring airway branching structure. Collateral ventilation can thus be defined at any level of airway branching. In pathologic conditions in which airway obstruction might occur at any airway size, collateral ventilation may thereby serve partially to bypass the obstruction. However, in the common practical measurement of collateral ventilation with a clinical bronchoscope or wedged catheter, the collateral communications are those that occur between distinct segmental bronchi.

VENTILATION / Collateral

Pathways for Collateral Flow There are three possible pathways for collateral ventilation: channels of Lambert, pores of Kohn, and channels of Martin. However, of these possible channels, the channels of Martin comprise the major functional pathway for collateral ventilation. The location and potential contribution of each of these are discussed separately. Channels of Lambert

In 1955, Lambert first described accessory bronchiole– alveolar communications in normal human and cat lungs. These channels consisted of epithelialized tubular communications through the bronchiolar wall to adjacent alveoli. The size of the channels varied from being practically closed to up to 30 mm in diameter. A later study suggested a potential role of these channels in the pathogenesis of pneumoconiosis, where it was observed that the Lambert channels and adjacent alveoli contained a high concentration of macrophages. The channels could be found associated with nearly every parabronchiolar accumulation. These channels of Lambert may be involved in the pathogenesis of pneumoconiosis, but there is little evidence showing how these channels relate to functional measurements of collateral flow. Although it is clear that these channels exist in many species, there is considerable doubt whether these channels can provide collateral ventilation when obstruction occurs at the level of segmental bronchi. Channels of Lambert never arise from segmental bronchi or allow communication between parenchymal regions served by different segmental bronchi. Thus, although the channels of Lambert might provide accessory collateral ventilation to adjacent alveoli, were obstruction to occur at a level smaller than terminal bronchioles (i.e., at the level where collateral ventilation is measured in experimental situations), the channels of Lambert would not otherwise be expected to play any significant role. Alveolar Pores (Pores of Kohn)

The functional description of alveolar pores dates back to the middle of the nineteenth century, and this early work is reviewed elsewhere. However, when one closely examines the anatomical and physiologic literature regarding these pores, it becomes fairly clear that alveolar pores are unlikely to be normal pathways of collateral ventilation. The key anatomical evidence is related to the size of the pores. The diameters reported by various investigators range typically from 2 to 30 mm, and there simply are not enough of these tiny high-resistance pathways to allow much flow. In addition, the pores are almost

435

certainly blocked by fluid in a living animal. The method of fixation plays a significant role in the size and appearance of the alveolar pores observed histologically. Since the alveolar wall is almost completely vascularized in the healthy lung, it is crucial to study lungs that have been fixed with the capillaries filled. When one views casts of the capillary network, it can be appreciated that normal pores, to the extent they are patent, could hardly be much larger than half a capillary diameter. In addition, these small intercapillary spaces in the alveolar wall are partially filled with structural fibers. The importance of fixing the lungs with the vessels filled cannot be overemphasized, but this is rarely done. In considering how much pressure it would take to open an alveolar pore that was filled with fluid, theoretical calculations show that it would require approximately 200 cmH2O. Such a high pressure would never occur in normal lungs. Thus, when either airway obstruction occurs in vivo or when collateral resistance is measured, the pressure differential that might be expected across a pore is generally less than 30 cmH2O. It is clear that in the normal lung the alveolar pores are not patent. The relative unimportance of pores of Kohn to functional collateral ventilation is demonstrated by the fact that pig does have alveolar pores of Kohn, despite there being a complete absence of measurable collateral ventilation. Further evidence of such pores being nonfunctional was supplied by studies in which pulmonary vasculature pressure was decreased. If one considered alveolar pores to be the collateral connections, then one would predict that decreases in capillary dimensions would tend to enlarge the pores in the intercapillary spaces, thereby decreasing the resistance. However, experimental results show just the opposite (i.e., collateral resistance increases). Channels of Martin

In 1966, Martin reported a painstaking histologic study of the interlobular septum in a dog lobe in which he first marked the collateral pathways by aerosolizing India ink and blowing this through a wedged catheter and the collateral pathways. Following fixation, 1600 serial sections (10 mm thick) were cut through the intersegmental plane, and he traced the track left by aerosolized India ink to outline the collateral airways. Martin showed connections at the level of the respiratory bronchiole and alveolar duct. This connection, at a level near the alveolar duct, was emphasized by Macklem in his 1971 review, in which he also noted that Martin seemed to have misinterpreted the histologic sections shown in his paper. However, subsequent schematic

436 VENTILATION / Collateral

drawings by most other investigators have led to the general impression that channels of Martin are simply short segments of bronchi connecting other bronchi. This impression is wrong. Quantitative analysis suggests that the channels of Martin by themselves have sufficiently low resistance to account for the observed degree of collateral ventilation. However, since Martin’s work was carried out in canine lungs, it is reasonable to ask if the communications described by him appear in other species, especially humans. Unfortunately, because of the difficulty of these anatomical studies, there is only limited evidence from other species. Nevertheless, such communications between different bronchi through the alveolar duct have been demonstrated by several investigators in human lungs. Boyden studied a wax reconstruction of the lung from a 6-year-old child and described the same kind of communication found by Martin. In addition, several casting studies with infusion of separate colors into adjacent segmental bronchi always showed one color passing into another, and the communicating airway was almost always at the level of a terminal respiratory bronchiole. In summary, the pathways described by Martin have sufficiently low resistance to account quantitatively for the degree of collateral ventilation measured as described in the next section. To the extent that some large alveolar pores might be patent at the segmental borders or that channels of Lambert might originate from larger bronchi, the quantitative contribution of such auxiliary communications to collateral ventilation is likely to be very small. One can therefore conclude that the primary pathways for collateral ventilation are normal airways that connect at the level of the terminal and respiratory bronchioles.

Measurement of Resistance through Collateral Pathways A simple method of measuring the resistance through collateral pathways was first described in 1970. This method consists of first wedging a small double lumen catheter or bronchoscope into an intralobar bronchus. A flow of gas is then passed through one lumen and the other lumen is used to measure pressure (Figure 1). At end expiration, the pressure in the trachea is atmospheric, and one can then divide the measured pressure by the known flow to calculate a resistance. Although many investigators have called this collateral resistance (Rcoll), it is clear from Figure 1 and the preceding discussion that the resistance includes the entire airway sequence from the

Pb

Figure 1 Schematic (not to scale) showing pathways measured with flow through a wedged catheter or bronchoscope. Arrows show flow path through connections at the level of the respiratory bronchiole. Nearly all of the resistance to flow occurs in these smallest airways, resulting in the pressure being measured at the tip of the catheter (Pb).

end of the wedged catheter down through the alveolar duct/respiratory bronchiole connections and back up the airways of the adjoining segment out to the trachea. Because most of the resistance to this flow occurs in the very smallest peripheral airways, the notation Rp is preferred for this resistance. Indeed, based on theoretical calculations, it can be shown that in the normal lung more than 90% of the resistance of this pathway would be in the respiratory bronchioles and alveolar ducts. Although this may not be true under all pathologic conditions, a simple calculation of resistance through single airways indicates that there would have to be extreme constriction for airways with baseline diameters larger than 0.5 mm to offer any substantial resistance. However, precisely where in the airways the resistance occurs in this technique, is not always certain since it will depend on the exact airway dimensions and the number of airways in parallel. It is worth noting that to the extent that Rp does measure the dimensions of the most terminal bronchioles, respiratory bronchioles, and alveolar ducts, these airways (in what is sometimes called ‘the silent zone’) cannot

VENTILATION / Collateral

be assessed by any other pulmonary function tests. Since all conventional measures of airway resistance in the whole lung can only reflect airways in which there is a pressure drop resulting from bulk flow, these measurements are completely insensitive to changes in these very peripheral airways. However, when Rp is measured, only a relatively few number of these peripheral airways are ventilated. This has the effect of increasing the rate of bulk gas flow much above normal, to the point at which there is now a measurable pressure drop. Again, it is important to emphasize that these ‘collateral’ airways are not different from normal airways. Without airway obstruction, they are normally ventilated with inspiratory and expiratory flows. They also show a similar sensitivity to pharmacologic challenges as normal small airways. Their uniqueness only arises from the fact that they can be supplied by two distinct larger bronchi. When measured with a wedged catheter, the flow may be transiently increased to obtain a resistive pressure drop, but this does not render these airways any less normal. There is also general agreement that increasing lung volume results in a substantial decrease in collateral resistance. This is a result of the increased outward acting forces of interdependence exerted on the airways of the surrounding lung and obstructed segment by the increased segment volume. Therefore, since the collateral airways behave exactly like normal bronchi with regard to the effect of lung inflation, this experimental approach thus provides a unique and probably the only means to assess the dimensional changes of very peripheral airways in vivo. Species Variation

Collateral ventilation has been measured in several species, including man, dog, horse, cat, rabbit, and ferret. The pig lacks any pathways for collateral flow that can be experimentally measured. Comparing the relative magnitude of collateral ventilation in the different species is difficult because it requires some form of normalization to compare for different size lungs. The collateral ventilation will clearly depend on how much of a lobe is wedged in addition to the extent of collateral airways. Considering the extremes, if the wedge occurs in a lobar bronchus of a completely independent lobe, the collateral ventilation through the wedge must be zero. At the other end, if one were to wedge at the level of a single alveolar duct, the collateral ventilation must also be zero. Thus, at intermediate levels, wedges progressing from the lobar bronchus down to an alveolar duct, the collateral ventilation must first increase and then decrease. The shape of such a plot of collateral

437

ventilation versus distance along the airway tree has not been well defined theoretically or experimentally. In one of the two studies that did address this important issue experimentally, the investigators found that moving the wedged catheter by an average of 2 cm had no effect on collateral resistance in sheep lungs. In contrast, in the other study, the investigators found that in dog lungs, advancing the wedged bronchoscope tip into the lobe a short distance resulted in a doubling of the collateral resistance. The reason for this discrepancy is unclear. Whether it relates to anatomical differences in the species studied or methodological or technical differences has not been determined. It has been speculated that the extent of collateral ventilation in different species is related to the degree of hypoxic pulmonary vasoconstriction. The teleological rationale presented for this speculation is that if an animal can compensate for airway obstruction with collateral ventilation, then a strong hypoxic pulmonary response is not required. At least for two animals with extreme variations of hypoxic vasoconstriction, the pig and the dog, this hypothesis would seem to hold. The dog constricts poorly to hypoxia and has low collateral resistance, whereas the pig has one of the strongest hypoxic vasoconstrictor with no collateral ventilation. However, this conjecture breaks down when one considers that the ferret also has a strong hypoxic vasoconstrictor response, but the collateral resistance is quite low.

Changes in Rp with Age and Pathology Collateral ventilation has been shown to increase with age in post-mortem and living human lungs. Reasons for this increase are unclear, but Rp is also known to increase with age in dogs, despite an increase in the size and number of alveolar pores. With such an increase in pore size, the only plausible interpretation is that the most peripheral airways likely narrow with age. However, not all species show this effect. For example, in sheep there is a significant decrease in the resistance to collateral flow with maturation. Reasons for these discrepant results regarding the effects of age on collateral ventilation are unclear, and thus this remains an unresolved controversy. There is limited information regarding changes in collateral resistance in lung disease. Several investigators have shown a substantially lower resistance to collateral flow in emphysematous lungs compared to normal lungs. A decrease in resistance to collateral flow might be expected in emphysema since with the destruction of alveolar walls there would not only be a progressive enlargement of the alveolar ducts but

438 VENTILATION / Control

also creation of new pathways through the missing walls. This concept has been supported by imaging results showing relatively large interlobar collateral pathways. The importance of collateral pathways also has impact on several new potential treatments for emphysema. One treatment actually involves the creation of new collateral channels (with physical implants) to allow emphysematous regions to empty more efficiently. Another method to empty emphysematous regions, which involves one-way valves placed in the airways, seems to be significantly improved by the presence of existing collateral channels. With regard to asthma, if collateral ventilation were to be beneficial in this disease with narrowed airways, such patients should have a lower resistance. However, direct measurements show just the opposite: asthmatic subjects have nearly an order of magnitude higher resistance to collateral flow than normal subjects. This striking separation between asthmatic and normal subjects suggests that the very small airways that comprise the Rp may reflect early pathologic changes in lung structure. This was further emphasized by the fact that the asthmatic subjects all had normal lung function as assessed with spirometry. It is thus possible that measurement of Rp might prove to be a sensitive method for the early detection of obstructive lung disease. Consistent with this conjecture are results showing that the airways responsible for Rp also contract more vigorously when stimulated with very dry air. See also: Ventilation: Overview; Control. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation; VentilatorAssociated Pneumonia.

Further Reading Boyden EA (1971) The structure of the pulmonary acinus in a child of six years and eight months. American Journal of Anatomy 132: 275–299. Hogg JC, Macklem PT, and Thurlbeck WM (1969) The resistance of collateral channels in excised human lungs. Journal of Clinical Investigation 48: 421–431. Hopkinson NS, Toma TP, Hansell DM, et al. (2005) Effect of bronchoscopic lung volume reduction on dynamic hyperinflation and exercise in emphysema. American Journal of Respiratory and Critical Care Medicine 171: 453–460. Kaminsky DA, Bates JH, and Irvin CG (2000) Effects of cool, dry air stimulation on peripheral lung mechanics in asthma. American Journal of Respiratory and Critical Care Medicine 162: 179–186. Kikuchi R, Kikuchi K, Hildebrandt J, et al. (1995) Dependence of collateral and small airway resistances of CO2 and volume in dog lobes. Respiration Physiology 100: 245–252. Lambert MW (1955) Accessory bronchioalveolar communications. Journal of Pathology and Bacteriology 70: 311–314. Lausberg HF, Chino K, Patterson GA, et al. (2003) Bronchial fenestration improves expiratory flow in emphysematous human lungs. Annals of Thoracic Surgery 75: 393–398.

Loosli C (1937) Interalveolar communications in normal and in pathologic mammalian lungs. Archives of Pathology 24: 743–776. Macklem PT (1971) Airway obstruction and collateral ventilation. Physiological Reviews 51: 368–436. Martin HB (1966) Respiratory bronchioles as the pathway for collateral ventilation. Journal of Applied Physiology 21: 1443–1447. Menkes H and Traystman RJ (1977) State of the art: collateral ventilation. American Review of Respiratory Diseases 116: 287–309. Mitzner W (1997) Collateral ventilation. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 1425–1435. New York: Lippincott-Raven. Salanitri J, Kalff V, Kelly M, et al. (2005) 133Xenon ventilation scintigraphy applied to bronchoscopic lung volume reduction techniques for emphysema: relevance of interlobar collaterals. Internal Medicine Journal 35: 97–103. Wagner EM, Liu MC, Weinmann GG, Permutt S, and Bleecker ER (1990) Peripheral lung resistance in normal and asthmatic subjects. American Review of Respiratory Disease 141: 584–588.

Control F L Powell, University of California – San Diego, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Physiological control of ventilation involves the reflex modulation of the fundamental respiratory rhythm, which is generated by neurons comprising a central pattern generator in the brain stem. Negative feedback reflexes that control breathing can be organized according to sensory system. Vagal sensory nerves in the airways and lungs respond to chemical or mechanical stimuli and cause reflex changes in breathing pattern, airway tone, and bronchial secretion. Arterial PCO2 is the most important stimulus to breathing in healthy humans, and the ventilatory response to arterial PCO2 is mediated by arterial (or peripheral) and central chemoreceptors. Arterial chemoreceptors also mediate the ventilatory response to changes in arterial PO2 and pH. Neural plasticity in the arterial chemoreflexes alters the response to different patterns and duration of stimuli, and it may play a role in chronic lung disease. The most common stimulus for increased ventilation in healthy subjects, exercise, cannot be explained by these negative feedback reflexes but involves a feed-forward system with central commands stimulating respiratory centers.

Introduction The body contains several physiologic control systems to maintain arterial pH, PCO2 , and PO2 within normal limits, to meet the oxygen demands of the tissues, to minimize the mechanical work of breathing, and to prevent lung injury by environmental agents. These systems control ventilation and typically involve reflex responses to stimulation of

VENTILATION / Control 439

chemoreceptors sensing arterial blood gases and pH, receptors in the lungs and airways, or receptors in skeletal muscles and the chest wall. However, the rhythmic drive to breathe occurs independently of these reflexes and is generated by neurons in the brainstem as described below. Hence, the physiological basis for the control of ventilation is the neurobiology of the generation of the respiratory rhythm and the reflexes that modulate this rhythm.

Respiratory Rhythm Generation Although breathing is similar to the heartbeat in terms of being automatic and continuous during sleep or general anesthesia, the skeletal muscles driving ventilation do not contract spontaneously like cardiac muscle does. Rhythmic breathing results from periodic activation of the ventilatory muscles by motor nerves from the central nervous system (CNS). A socalled central pattern generator, composed of networks of neurons, generates this basic respiratory rhythm. The central pattern generator is located in the medulla near other respiratory centers that integrate afferent information for ventilatory reflexes to fine-tune the motor output to ventilatory muscles (Figure 1). The central pattern generator for ventilation has been isolated in experimental animals to a very small region of the medulla called the pre-Bo¨tzinger complex. The respiratory rhythm can be measured from motor nuclei in an in vitro brain slice that contains

this complex. Killing a specific class of neurons in the pre-Bo¨tzinger complex, which have neurokinin 1 (NK-1) receptors that are responsive to the neurotransmitter substance P, causes an abnormal and unstable ventilatory pattern, resulting in severe arterial hypoxia and hypercapnia. However, the animals survive suggesting that other parts of the brain can compensate in part for the loss of the central pattern generator in the pre-Bo¨tzinger. Experiments on the pre-Bo¨tzinger in vitro have shown that the major inhibitory neurotransmitters GABA and glycine are not necessary for rhythm generation, which means reciprocal inhibition is not necessary for rhythm generation. Current evidence supports a hybrid network model, which incorporates pacemaker neurons and synaptic interactions that fine-tune the duration of, and transition between, inspiration and expiration. Glutamate is the major excitatory neurotransmitter within the central pattern generator, as well as between the rhythmgenerating neurons and bulbospinal (premotor) neurons, and at spinal (e.g., phrenic) motor neurons. Other Respiratory Centers

Other anatomical or functional groups of neurons involved in control of ventilation include the ventral and dorsal respiratory groups (VRG and DRG) in the medulla. The ventral respiratory group (VRG) is a column of neurons that fire action potentials in phase with respiration. It includes neurons depolarizing during inspiration (inspiratory, or I neurons) and

PRG IV Ventricle

VRG/pre-Bot

AP

AP

pre-Bot IX

NTS

VRG X

DRG

DRG

VRG

VRG

CC

Phrenic nucleus (a)

(b)

Figure 1 Respiratory centers in the brainstem. (a) Dorsal view of the pons and medulla with the cerebellum removed. (b) Transverse sections from three levels of the medulla. PRG, pontine respiratory group; pre-Bo¨t, pre-Bo¨tzinger complex, responsible for generating the normal respiratory rhythm; VRG, ventral respiratory group; DRG, dorsal respiratory group; IX and X, cranial nerves; AP, area postrema; NTS, nucleus tractus solitarius; CC, central canal.

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expiration (expiratory, or E neurons). The preBo¨tzinger is in the rostral VRG. The DRG contains mainly I neurons and is part of the nucleus of the solitary tract (or nucleus tractus solitarius, NTS). The afferent input from arterial chemoreceptors and lung mechanoreceptors synapses on neurons in the NTS. The pons includes a group of neurons in the pontine respiratory group (PRG), which are involved in the phase transition between inspiration and expiration, and the reflex effects of lung mechanoreceptors on ventilation. Apneusis (abnormally long inspiration) can occur if the pons is lesioned in humans. The Kolliker–Fuse nucleus is involved in protective reflexes for the airways (e.g., diving reflex) and the parabrachial complex is involved in plasticity of the Hering–Breuer reflex. Evidence for respiratory rhythm generators in the pons is still controversial.

Reflexes and Negative Feedback There are three components in reflex control systems. First, a sensory system detects and transmits information about the level of a physiologic variable in the body, called the physiologic stimulus. This sensory information is called afferent input, which codes stimulus intensity with frequency of action potentials. Second, central integration processes afferent input from multiple sensory systems and determines an appropriate response. Central integration of respiratory reflexes occurs in the medulla and pons, in close proximity to the areas that generate the respiratory rhythm. Third, efferent output occurs when a motor neuron or gland executes the appropriate response. All the reflexes considered in this article are examples of negative feedback control to maintain homeostasis. Negative feedback describes the effect of the response (efferent output) on the stimulus. For example, increased PaCO2 will stimulate chemoreceptors to cause a reflex increase in ventilation and this, in turn, decreases PaCO2 . Negative feedback reduces the original deviation in the stimulus from its normal level. In control systems terminology, the measured input from a regulated variable (PaCO2 ) is held constant by changes in the output of a controlled variable (ventilation). Reflexes are involuntary by definition, but notice that breathing can be changed voluntarily by neural commands for behaviors from higher centers in the CNS. For example, breathing may be changed during speech, which requires a voluntary command from the cortex. Afferent Sensory Systems

There are two main sensory systems conveying afferent information about the function of the respiratory

system: (1) chemoreceptors that sense changes in arterial blood gases; and (2) receptors in the lungs and airways. Note that receptor in this context refers to a specialized sensory nerve ending and not a neurotransmitter or drug receptor. Chemoreceptors monitor changes in arterial blood gases and cause reflex changes in ventilation that return arterial blood gases toward normal values. For example, increases in PaCO2 or decreases in PaCO2 stimulate reflex increases in ventilation. There are two main classes of chemoreceptors, central and arterial chemoreceptors, which are discussed in detail elsewhere. Receptors in the lungs and airways include vagal mechanoreceptors that monitor pressure and volume in the lungs and airways to provide afferent information about pulmonary mechanics. Generally, mechanoreceptors induce reflex changes in the rate and depth of breathing to fine-tune the pattern of breathing under different mechanical conditions and at different levels of ventilation. Other vagal afferents in the lungs include receptors that are sensitive to inhaled irritants or endogenous chemical stimuli (e.g., histamine). Sensory nerves from the lungs and airways cause reflex changes in ventilation as well as in airway smooth muscle tone and bronchial secretions to defend the lungs from environmental insult. Reflexes from the lungs and chest wall are covered elsewhere. Efferent Pathways

The effectors for ventilatory reflexes are respiratory skeletal muscles. Airway smooth muscle and bronchial secretory reflex responses are considered elsewhere. The primary efferent pathway for resting ventilation is the phrenic nerve to the diaphragm. Rhythmic contraction of the diaphragm causes inspiration while expiration at rest occurs from passive elastic recoil of the lung and chest wall. Figure 2 shows this pattern of activity for the phrenic nerve (lower trace). The rhythm from the central pattern generator is transmitted via multiple synapses to phrenic motor neurons at the third to fifth cervical levels of the spinal cord (C3–C5). Phrenic nerve activity reflects basic features of the central pattern generator, including (1) a sudden onset of inspiratory activity; (2) a ramp-like increase in activity during inspiration; and (3) a relatively sudden termination of activity at the onset of expiration. Low levels of phrenic activity at the onset of expiration (Figure 2) allow the diaphragm to smooth the transition to passive expiration. Additional ventilatory muscles may be recruited at high levels of stimulation. These include inspiratory

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of muscle spindles, primarily from intercostal muscles. In addition to the ventilatory muscle pathways described above, respiratory reflexes can stimulate responses from the autonomic nervous system.

VT Volume 0

Control of Arterial PO2 , PCO2 , and pH

Internal intercostal

External intercostal

Phrenic Inspiration Expiration

2s

Figure 2 Pattern of ventilatory motor nerve activity to an expiratory muscle (internal intercostal), and two inspiratory muscles (external intercostals and diaphragm). Adapted from Hlastala MP and Berger AJ (1996) Physiology of Respiration. Oxford: Oxford University Press.

and expiratory intercostal muscles that are innervated by spinal nerves from all levels of the thoracic spinal cord (T1–T11). The pattern of electrical activity in the external intercostal nerves is similar to that in the phrenic nerve, whereas the internal intercostals are activated during expiration (Figure 2). Expiration is passive at rest and ventilatory drive is elevated in Figure 2 to illustrate intercostal activity. Electrical activity in lower thoracic and upper lumbar spinal nerves, which innervate the abdominal expiratory muscles, is similar to the internal intercostal activity shown in Figure 2. The central pattern generator also sends efferent signals to upper airway muscles. An inspiratory pattern can be observed in the nerves to upper airway muscles in the larynx and pharynx, similar to that shown for the diaphragm in Figure 2. The upper airways also show reflex responses to mechanical stimuli of afferents in the upper airways. Quantitative and qualitative differences between the efferent responses in phrenic versus upper airway motor neuron pools can have important physiological consequences. For example, during sleep the inspiratory tone to upper airways muscles can decrease more than inspiratory drive to the diaphragm, leading to obstructive sleep apnea. The patterns of motor neuron discharge, which determine ventilatory flow profiles and tidal volume, are shaped by central integration of respiratory reflexes in the brainstem. However, some integration occurs in the spinal cord through g-efferent control

Arterial chemoreflexes monitor arterial blood gases as the output from gas exchange in the lungs and adjust ventilation to keep arterial blood gases within a normal range. Stimulating these chemoreceptors results in hyperventilation, which commonly refers to increased ventilation relative to resting levels. Conversely, withdrawing stimulation of these chemoreceptors can result in hypoventilation, where ventilation is less than normal and PaCO2 typically increases. Arterial PO2 , PCO2 , and pH are sensed directly by arterial chemoreceptors. PaCO2 is also sensed indirectly by chemoreceptors in the CNS. There is no strong evidence for mixed-venous or alveolar chemoreceptors that can affect breathing. Differences in the ventilatory response to PaO2 , PaCO2 , and pH can be explained by differences in the sensory mechanisms for each stimulus. For example, PaCO2 stimulates both central and arterial chemoreceptors but decreases in PaO2 or pHa only stimulate arterial chemoreceptors. Central chemoreceptors are located in the medulla and respond to changes in PaCO2 more slowly than arterial chemoreceptors. Arterial chemoreceptor is a generic term for both carotid body chemoreceptors and aortic body chemoreceptors, which are also referred to as peripheral chemoreceptors because they are located outside the central nervous system. Arterial chemoreceptors respond rapidly (within a breath) to changes in arterial PO2 , PCO2 , and pH. Mechanisms of chemotransduction and the physiology of central and arterial chemoreceptors are considered elsewhere. There is evidence that both central and arterial chemoreceptors have synaptic input to the central pattern generator, as well as to more distal respiratory centers in the medulla. The nucleus tractus solitarius is the site of the primary synapse from arterial chemoreceptors and an important site for integrating other afferent inputs to ventilation. Ventilatory Response to Arterial PCO2

In healthy resting humans, PaCO2 is the most important stimulus for ventilatory reflexes. Figure 3 shows the ventilatory response to changes in inspired CO2 for normal individuals and how the response varies between individuals. The hypercapnic ventilatory response is plotted as expired ventilation versus alveolar PCO2, which is a noninvasive measure of the

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40

V (l min−1)

32

Inspired CO2 = 6%

24

16

4%

2% 8

0%

0 35

45

40

50

PACO (mmHg) 2

Figure 3 Ventilatory response to increases in inspired CO2 plotted as a function of alveolar (E arterial) PCO2 . Solid line is mean response for population 7 s.d. Dashed lines demonstrate the large range of individual variability in normal subjects. Adapted from Kellogg RH (1964) Central chemical regulation of respiration. In: Fenn WO and Rahn H (eds.) Handbook of Physiology, section 3. Respiration, vol. I. Washington, DC: American Physiological Society.

actual physiologic stimulus, that is, arterial PCO2 . Ventilation approximately doubles when PaCO2 increases 5 mmHg above the control value and the average increase in ventilation is about 2 l/(min mmHg) over the physiologic range of 40–50 mmHg PaCO2 . The two dashed lines in Figure 3 show the large range in ventilatory response to CO2 between individuals, which may be under genetic control. The hypercapnic ventilatory response can be measured by the rebreathing method. A person breathes in and out of a bag filled with 7% CO2 and 93% O2, while continuously measuring ventilation and endexpired PCO2 (EPACO2 or PaCO2 ). Metabolic CO2, which is expired into the bag, progressively increases PCO2 in the bag, causing a progressive increase in PaCO2 and ventilation. High O2 levels in the bag eliminate any potential effects of hypoxia on ventilation. Both tidal volume and frequency increase with PaCO2 , but tidal volume reaches a maximum before frequency. Further increases in ventilation at high PaCO2 levels depend on increases in respiratory frequency and this is less effective than increasing tidal volume at increasing alveolar ventilation because of dead space. Hence, the hypercapnic ventilatory response is most efficient at low levels of PaCO2 , and larger increases in total ventilation are necessary at

high levels of PaCO2 when only frequency increases. This helps explain the increase in slope of the ventilatory response to CO2 at high of levels CO2 (Figure 3). It is difficult to decrease PaCO2 below normal without changing other ventilatory stimuli, but ventilation can decrease if this happens. Chemoreceptors are tonically active at normal PaCO2 levels so hypocapnia decreases chemoreceptor stimulation and ventilatory drive. However, ventilatory sensitivity to low levels of PaCO2 is relatively low, as shown by the low slope of the hypercapnic ventilatory response between 0% and 2% inspired CO2 (Figure 3). Ventilation may actually cease if PaCO2 is lowered enough to go below the apneic CO2 threshold. It is estimated that about two-thirds of the drive to breathe in normal resting humans is from chemoreceptor stimulation and the balance is from wakefulness, illustrating how mental state influences ventilation. The ventilatory response to CO2 is mainly a function of central chemoreceptor stimulation, which explains 75% of the total response. The rest of the response is explained by arterial chemoreceptors, which depends on stimulation by arterial PCO2 , as well as H þ from respiratory changes in arterial pH. The direct effect of PCO2 on arterial chemoreceptors probably explains less than 10% of the total ventilatory response. The independent effect of arterial pH on ventilation is described below. The time course of the ventilatory response to PaCO2 begins rapidly (within a few breaths of inhaling CO2) from stimulation of arterial chemoreceptors. However, it requires a few minutes for ventilation to reach a steady value after PaCO2 stabilizes at a new level. Ventilatory Response to Arterial pH

The ventilatory response to changes in arterial pH is the result of arterial chemoreceptor stimulation, and only the carotid bodies contribute to this response in humans. Patients without normal carotid body innervation do not have a response to metabolic changes in pHa. H þ cannot cross the blood–brain barrier, so a metabolic acidosis or alkalosis does not stimulate central chemoreceptors. The ventilatory response to physiologic changes in pHa of 70.1 pH units is very small (less than 10% of resting ventilation). Larger increases in pHa (to 7.6) also only cause small decreases in ventilation. However, larger decreases in pH have a stronger effect, so resting ventilation may double at pHa ¼ 7.2. In severe acidosis, ventilation increases even without the involvement of arterial chemoreceptors. Very large increases in arterial [H þ ] might allow some H þ to

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cross the blood–brain barrier, or this could represent a generalized response to stress. These reflexes drive respiratory compensation for metabolic acid–base disturbances. Ventilatory Response to Arterial PO2

The ventilatory response to PaO2 is called the hypoxic ventilatory response, and is notable for its nonlinearity. Figure 4 shows that increases in PaO2 have relatively little effect on ventilation, whereas decreases in PaO2 below about 60 mmHg cause large increases in ventilation. This contrasts with the effects of CO2 on ventilation, which are maximal when PaCO2 is near its normal value (Figure 3). The hypoxic ventilatory response is not large until PaO2 falls to a level at which O2–hemoglobin saturation starts decreasing significantly. Ventilation is a linear function of arterial O2 saturation but this is a coincidence and not a mechanistic explanation. Arterial chemoreceptors respond only to O2 partial pressure, and not O2 content or saturation. They are necessary for the hypoxic ventilatory response because patients without functional carotid bodies lack the response. Figure 4 illustrates the interactions between PaCO2 and PaCO2 as ventilatory stimuli. The lower response poikilocapnic (i.e., changing CO2) curve shows a normal ventilatory response to hypoxia where PaCO2 decreases as a result of hypoxic ventilatory stimulation.

Pulmonary ventilation (l min–1)

50 Hypercapnic PaCO = 48 2

25 Normocapnic PaCO2= 42

33 35 37

Poikilocapnic

42

60

140

= PaCO

2

0 20

100

PaO2 (mmHg) Figure 4 Ventilatory response to hypoxia as a function of alveolar (E arterial) PO2 . PaCO2 normally decreases when hypoxia stimulates ventilation (poikilocapnic curve) and the hypoxic ventilatory response is increased if PaCO2 is held constant (e.g., by adding inspired CO2, normocapnic curve). The response to combined hypoxia and hypercapnia is greater than the sum of the individual stimuli (hypercapnic curve). Adapted from Loeschke HH and Gertz KH (1958) Einfluss des O2-Druckes in der Einatmugsluft aur die Atemtatigkeit des Menschen, gepruft unter konstanthaltung des alveolaren CO2-Druckes. Pflugers Archivfu¨r die Gesamte Physiologie 267: 460–477.

This decreases ventilatory drive from central chemoreceptors. The net effect on ventilation is the result of stimulation by hypoxia and inhibition from decreased PaCO2 . In contrast, if PaCO2 is held constant during hypoxia, for example, by increasing inspired PCO2 , then the hypoxic ventilatory response is greater as shown on the normocapnic curve in Figure 4. The increase in ventilation between the poikilocapnic and normocapnic curves is explained by the removal of hypocapnic inhibition. Increasing PaCO2 above normal reveals a synergistic or multiplicative interaction between PaO2 and PaCO2 , that is, the combined effects of hypoxia and hypercapnia exceed the sum of the individual effects. The time course of the ventilatory response to changes in PaO2 begins within a single breath of breathing a low O2 mixture, as expected, given the rapid response of arterial chemoreceptors. However, the ventilatory response changes if hypoxemia persists, as described in the section on ‘Plasticity in ventilatory control’.

Ventilatory Response to Exercise The most common stimulus for increased ventilation is exercise. However, exercise hyperpnea, defined as the increase in ventilation during exercise, cannot be explained by the reflexes described above. Current theories for exercise hyperpnea involve a combination of feed-forward mechanisms, and feedback from the chemoreceptor and mechanoreceptor reflexes. Feed-forward mechanisms (also called central command) are neural inputs from higher centers in the brain that directly stimulate the respiratory centers. For example, signals from the motor cortex or hypothalamus to move a muscle can also stimulate ventilation. Feed-forward mechanisms have essentially no delay, and can explain the rapid increase in ventilation at the onset of exercise. The slower, secondary increase in ventilation is explained by neural mechanisms, such as second messenger systems activated by neurotransmitters, changes in intracellular calcium that stimulate neurons, and possibly changes in chemical stimuli to sensory nerves in muscle (e.g., potassium ions). Figure 5 shows how alveolar ventilation increases in perfect proportion to CO2 production, so PaCO2 is maintained at the normal resting value during submaximal steady-state exercise. This is why the term exercise hyperpnea is used; this is not hyperventilation because the increase in ventilation is appropriate for the increase in CO2 production. Because arterial blood gases do not change significantly, arterial chemoreceptors are not important for hyperpnea at submaximal levels of exercise. In fact, ventilation is

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normal during steady-state submaximal exercise in patients without carotid bodies. However, most animals except humans actually hyperventilate during exercise, which decreases PaCO2 and arterial chemoreceptor stimulation. Hence, arterial chemoreceptors limit the increase in ventilation and minimize the work of breathing in animals and may prevent hyperventilation in humans. Near maximal levels of exercise, lactic acid starts to appear in the blood and this stimulates arterial chemoreceptors. This explains the larger increase in ventilation above the lactate threshold (Figure 5). Ventilation increases in excess of CO2 production, decreasing PaCO2 and increasing PaO2 near maximum levels of exercise in normal subjects. The respiratory alkalosis helps compensate for the metabolic acidosis from lactic acid. Other afferent signals that may fine tune ventilation during exercise originate from mechanoreceptors in the heart and nonmyelinated sensory nerves in skeletal muscle. Mechanoreceptors from the lung and chest wall can adjust the pattern of breathing during exercise to minimize the work of breathing. Even arterial chemoreceptors can be stimulated by K þ released from muscle, norepinephrine released from the sympathetic nervous system, and larger oscillations of PaCO2 during the ventilatory cycle in exercise. However, the ventilatory response to moderate, steady-state exercise is normal without any of these afferent signals, so they are not necessary for exercise hyperpnea.

Plasticity in Ventilatory Control Plasticity in neural control systems is defined as a persistent change in structure or function based on prior experience. In ventilatory chemoreflexes, plasticity is a change in the stimulus–response relationships, for example, during chronic changes in arterial blood gases. This has been studied mainly in healthy subjects and it is not known if the same mechanisms occur in patients with chronic lung disease, chronic hypoxemia, and CO2 retention. Individual differences in plasticity may contribute to individual differences in the consequences of a given lung pathology because there is a genetic component to neural plasticity in control of ventilation in animals. Plasticity in the Hypoxic Ventilatory Response

The acute hypoxic ventilatory response is extremely rapid and ventilation increases within a single breath. However, if hypoxia persists, the response changes in magnitude and pattern (frequency vs. tidal volume), revealing several time domains of the hypoxic ventilatory response (Figure 6). For example, short-term potentiation of ventilation increases tidal volume while short-term depression of ventilation decreases breathing frequency during the first minute of isocapnic

STP

V STP

VT

STD

fR 5 min

(a)

VA (l min−1)

STD

1h V LTF

PaO2 (mmHg)

(b)

5 min

PaCO2 (mmHg)

VAH V

pHa

(c)

1

2

3

4

5

6

VCO (X resting) 2 Figure 5 Respiratory effects of exercise. Alveolar ventilation increases in proportion to CO2 production until very high levels of exercise and this maintains arterial blood gases at normal levels. At very high levels of exercise, lactic acid production decreases pH. This acidosis, and possibly other factors, stimulates hyperventilation so PaCO2 decreases and PaO2 increases.

HVD 15 min

Hours to days

HD Months to years

Figure 6 (a) After the acute hypoxic ventilatory response, short-term potentiation (STP) increases VT and short-term depression (STD) decreases fR; STP and STD persist for the first few minutes of normoxic recovery. (b) Intermittent hypoxia (5 min hypoxia followed by 5 min of normoxia for several cycles) results in long-term facilitation (LTF) of ventilation, persisting for 1 h after intermittent hypoxia. (c) Hypoxic ventilatory decline (HVD) causes V’ to ‘roll off’ during 5–15 min of hypoxia; ventilatory acclimatization to hypoxia (VAH) increases V’ during hours to days of sustained hypoxia but hypoxic desensitization (HD) decreases V’ during very long exposures.

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hypoxia in animals. If hypoxia persists for 5 min or more, ventilation decreases further, primarily by decreasing tidal volume. This hypoxic ventilatory decline occurs even when PaCO2 is held constant during hypoxic hyperventilation. These changes involve plasticity in the central integration of arterial chemoreceptor afferent information (neurotransmitter effects, secondary messengers, and cell signaling) and do not involve changes in stimulus–response relationships of arterial chemoreceptors. If hypoxia is sustained for hours to weeks, a timedependent increase in ventilation occurs that is called ventilatory acclimatization to hypoxia (Figure 6). This additional increase in ventilation, beyond the acute hypoxic ventilatory response, involves both an increase in the isocapnic hypoxic ventilatory response and an increase in ventilatory drive. The latter is observed as persistent hyperventilation after normoxia is restored. Two weeks are required for complete ventilatory acclimatization to hypoxia, although most of the change occurs during the first two days of hypoxia. The physiologic mechanism of ventilatory acclimatization to hypoxia involves both arterial chemoreceptors and the CNS. Carotid body chemoreceptors in animals become more sensitive to PaO2 during prolonged exposure to hypoxia, so afferent input increases for any given level of hypoxia. Renal compensation for the primary respiratory alkalosis with hyperventilation increases H þ stimulation of arterial chemoreceptors. Finally, CNS responsiveness to sensory input from arterial chemoreceptors increases during sustained hypoxia. If hypoxia is sustained for years, then the hypoxic ventilatory response decreases, at least until PaO2 reaches extremely low levels (Figure 6). Such hypoxic desensitization, or blunting of the hypoxic ventilatory response is observed in people who have lived at high altitude for years but may also include a genetic component in high-altitude natives. Hypoxic desensitization may reduce the work of breathing after other steps in the oxygen transport chain have adjusted sufficiently. The pattern of hypoxic exposure is also important and the effects of intermittent hypoxia are different from those of sustained hypoxia. For example, longterm facilitation of ventilation occurs when the carotid body is stimulated by several bouts of hypoxia interrupted with normoxia. After this pattern of stimulation is stopped, ventilation remains elevated for 15 min to 1 h (Figure 6). In animals, long-term facilitation of ventilation is also observed following intermittent electrical stimulation of the carotid sinus nerve and depends on a serotonergic mechanism at phrenic motor neurons (instead of involving the carotid body). Long-term facilitation may stabilize

ventilation during sleep when episodic hypoxia and hypercapnia are likely to occur. However, long-term facilitation of ventilation has not been observed in humans during wakefulness. Plasticity in Ventilatory Control of PaCO2

When PaCO2 is experimentally increased over hours to weeks, ventilation remains near the level measured during the first few minutes of exposure to CO2. This occurs despite a reduction in arterial chemoreceptor stimulation, as H þ decreases with renal compensation for the respiratory acidosis. Arterial chemoreceptors do not become more sensitive to PCO2 after chronic hypercapnia, in contrast to the effect of chronic hypoxia on carotid body O2-sensitivity. PaCO2 is decreased during chronic hypoxia. As described above, PaCO2 remains lower than control when normoxia is restored reflecting an increase in normoxic ventilatory drive. The changes in arterial pH and carotid body O2-sensitivity described above for chronic hypoxia are not sufficient to explain this persistent hyperventilation in normoxia. There are not sufficient changes in the pH of cerebrospinal fluid, which can affect central chemoreceptors, to explain the increased normoxic ventilatory drive either. Ventilatory CO2-sensitivity, in terms of the change in ventilation for a given change in PaCO2 , is not altered by chronic hypoxia but PaCO2 is regulated at a lower value. The mechanism for this change in PaCO2 ‘set point’ is not known. Plasticity with Chronic Changes in pH

Ventilation with chronic metabolic changes in pH is essentially unchanged from the acute response. For example, in a diabetic patient with long-term ketoacidosis, ventilation remains elevated despite pH returning towards normal, which reduces H þ stimulation of arterial chemoreceptors, and decreased PCO2 from respiratory compensation, which reduces stimulation of arterial and central chemoreceptors. Metabolic H þ changes in the blood are not sensed by the central chemoreceptors (see above) so the small arterial acidosis (from incomplete compensation) stimulates arterial chemoreceptors and provides the only known stimulus for increased ventilation. Ventilatory Chemoreflexes during Chronic Lung Disease

Some patients with chronic obstructive pulmonary disease (COPD) increase ventilation to maintain PaCO2 in the normal range despite significant ventilation–perfusion mismatch. However, other COPD patients have CO2 retention (chronic increases in PaCO2 ) and a decreased ventilatory response to CO2,

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although they can get PaCO2 in the normal range with voluntary hyperventilation. It is difficult to determine if this represents a time-dependent decrease in the responsiveness of chemoreceptors, a deficiency in the motor output from the respiratory centers, or reduced function of ventilatory muscles. Increasing airway resistance decreases the ventilatory response to PaCO2 in normal individuals, suggesting that patients with obstructive lung disease might retain CO2 to minimize the work of breathing. Ventilatory control in COPD patients is dominated by hypoxic stimulation of arterial chemoreceptors, in contrast to PaCO2 being the most important determinant of resting ventilation in healthy subjects. For example, if a patient with COPD has an acute illness that further impairs gas exchange (e.g., pneumonia), it is often counterproductive to administer supplemental oxygen. Supplemental oxygen can cause ventilation to decrease, presumably by relieving hypoxic stimulation of arterial chemoreceptors. This will increase PaCO2 but it does not increase ventilation as it would in normal subjects. The central chemoreceptors seem to be desensitized to increases in PaCO2 . See also: Acid–Base Balance. Arterial Blood Gases. Chemoreceptors: Central; Arterial. Chronic Obstructive Pulmonary Disease: Overview. Exercise Physiology. High Altitude, Physiology and Diseases. Neurophysiology: Neural Control of Airway Smooth Muscle; Neuroanatomy; Neurons and Neuromuscular Transmission. Oxygen Therapy. Reflexes from the Lungs and Chest Wall. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Sleep Apnea: Overview.

Further Reading Alheid GF, Milsom WK, and McCrimmon DR (2004) Pontine influences on breathing: an overview. Respiratory Physiology and Neurobiology 143: 105–114. Anthonisen NR and Cherniack RM (1981) Ventilatory control in lung disease. In: Hornbein TF (ed.) Regulation of Breathing, pp. 965–987. New York: Dekker. Bisgard GE and Neubauer JA (1995) Peripheral and central effects of hypoxia. In: Dempsey JA and Pack AI (eds.) Regulation of Breathing, pp. 617–618. New York: Dekker. Dempsey JA and Forster HV (1982) Mediation of ventilatory adaptations. Physiological Reviews 62(1): 262–346. Feldman JL, Mitchell GS, and Nattie EE (2003) Breathing: rhythmicity, plasticity, chemosensitivity. Annual Review of Neuroscience 26: 239–266. Hlastala MP and Berger AJ (1996) Physiology of Respiration. Oxford: Oxford University Press. Kellogg RH (1964) Central chemical regulation of respiration. In: Fenn WO and Rahn H (eds.) Handbook of Physiology, section 3, Respiration, vol. I. Washington, DC: American Physiological Society. Loeschke HH and Gertz KH (1958) Einfluss des O2-Druckes in des Einatmugsluft aur die Atemtatigkeit des Menschen, gepruft unt-

er konstanthaltung der alveolaren CO2-Druckes. Pflugers Archivfu¨r die Gesamte Physiologie 267: 460–477. Mitchell GS and Johnson SM (2003) Neuroplasticity in respiratory motor control. Journal of Applied Physiology 94: 358–374. Powell FL, Milsom WK, and Mitchell GS (1998) Time domains of the hypoxic ventilatory response. Respiratory Physiology 112: 123–134. Ramirez JM, Zuperku EJ, Alheid GF, et al. (2002) Respiratory rhythm generation: converging concepts from in vitro and in vivo approaches? Respiratory Physiology and Neurobiology 131: 43–56. Rupert JL and Hochachka PW (2001) The evidence for hereditary factors contributing to high altitude adaptation in Andean natives: a review. High Altitude Medicine and Biology 2: 235– 256. Smith CA, Dempsey JA, and Hornbein TF (2001) Control of breathing at high altitude. In: Hornbein TF and Schoene RB (eds.) High Altitude, pp. 139–173. New York: Dekker.

Uneven M P Hlastala, University of Washington, Seattle, WA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract This article deals with nonuniform ventilation in the lungs. The mechanisms governing ventilation heterogeneity are described. Both low-resolution and high-resolution methods used to quantify ventilation are presented. Finally, the fractal nature of ventilation is described.

Introduction The basic concepts of alveolar ventilation can be presented with the assumption of a homogeneous lung. In reality, ventilation is not uniformly distributed even in normal lungs and is particularly nonuniform in the presence of lung disease. The efficiency of the gas exchange function of the lung depends primarily on how well ventilation and perfusion are matched. In this article, the term ‘uneven’ may be expressed as nonuniform or heterogeneous. Because the lungs do not expand uniformly, alveolar ventilation (fresh air reaching the gas-exchanging alveolus) varies amongst the different regions of the lungs. Ventilation in any region depends on the resistance of the conducting airways and the compliance (C) (equal to the change in volume (V) divided by the change in pressure (P) of the parenchymal tissue): C ¼ DV=DP Figure 1 illustrates an imaginary two-compartment lung having two regions with differing resistance (R) and compliance. With inspiration these

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compartments inflate in a manner shown in Figure 2. Alveolar region 1 has a large compliance and a large resistance (narrower tube). Alveolar region 2 has a small compliance and a small resistance. If pleural pressure is decreased in a stepwise manner, the volume will increase in each alveolus in an exponential manner (Figure 2) with a time constant (t) equal to 1/RC. Over the initial phase of inspiration, more volume goes into region 2 than into region 1 because of the small resistance. However, later in the inspiration, more volume has gone into region 1. This nonuniformity is the source of sequential ventilation. Initially, ventilation goes primarily to the regions with smaller time constants. Later ventilation goes predominantly to the regions with larger time constants.

Small resistance

Large resistance

Regional Gradients of Ventilation Early studies described a vertical difference in the erect human for ventilation distribution. The upper regions of the lung were thought to be more distended because of the weight of the lung below. The upper lung alveoli were therefore less compliant because they were nearer to their maximum volume at functional residual capacity (FRC). The lessdistended lower alveoli were thought to receive more ventilation than the upper regions of the lungs. Figure 3 illustrates this concept with a schematic diagram of the lung and a compliance curve appropriate for the whole lung. At FRC, the pleural pressure surrounding the lung is more negative in the upper lung compared with the lower lung. This concept has been revised with the advent of higher-resolution methods for measuring ventilation (see below). The distribution of ventilation depends on posture and configuration of the chest wall and diaphragm. This is so because as the lung expands, the regional ventilation depends not only on the properties of the lung parenchyma, but also on the expansion of the chest wall and diaphragm, and movement of the abdomen. When comparing spontaneous breathing

2

Pleural pressure (FRC) − 10 cmH2O

1

Small compliance − 2.5 cmH2O

Large compliance

100

Figure 1 Schematic showing a hypothetical two-compartment lung. One compartment has a large compliance and is fed with an airway with large resistance. The second compartment has a small compliance and is fed with an airway with small resistance.

60

Region volume

40 Region 1

Volume (%)

80

20

Region 2 −10

Inspiration time Figure 2 Regional lung volume vs. inspiration time for two hypothetical lung compartments with differing time constants.

0 10 20 30 Transpulmonary pressure (cmH2O)

0 40

Figure 3 Pressure–volume relationship for the lung parenchyma. A region in the upper part of the lung starts with a more negative pressure at functional residual capacity (FRC) compared to a region at the bottom of the lung. During inhalation with an identical change in transpulmonary pressure, the lower region, lying on a steeper part of the pressure–volume curve, inspires a greater volume than the upper lung region.

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with mechanical ventilation, there are differences in the patterns of diaphragm motion caused by different sliding motions and differential rotations of upper and lower lobes and hence, a differing distribution of ventilation.

Low-Resolution Methods for Assessing Heterogeneity of Ventilation Phase III of Single-Breath Nitrogen Exhalation

An early means for assessing ventilation heterogeneity followed from the exhaled nitrogen provided after a single inspiration of 100% oxygen. Such a curve is shown in Figure 4. Phase I identifies the anatomic dead space. Air coming from the airways immediately after O2 inhalation has no nitrogen. Phase II represents the transition between air coming from the airways and air coming from the alveoli. Phase III represents alveolar emptying; the positive slope indicates uneven ventilation from various regions with differing ventilation to volume ratio. The slope of

25 Phase IV

Phase III FN2 (%)

Closing volume Phase II

Phase I

0 TLC

RV Volume (l)

Figure 4 Exhaled nitrogen partial pressure profile (from total lung capacity (TLC) to residual volume (RV)) after a full inspiration of 100% oxygen. Phase I represents air coming from the airways (dead space). Phase II represents air coming from the airways mixing with air from the alveoli. Phase III is air coming from the alveoli (mixing from all alveolar regions). The first part of phase III comes from alveoli with a greater proportion of ventilation (hence lower nitrogen fraction). Gradually phase III is dominated by air coming from alveoli with a poorer proportion of ventilation. The oscillations (called cardiac oscillations) in phase III are presumably due to fluctuations in nitrogen concentration caused by the periodic beating of the heart. Phase IV shows a marked increase in nitrogen concentration as lower regions in the lung start to close off airways causing a greater proportion of exhalation from the upper portions of the lung.

phase III is related to the degree of nonuniformity of ventilation and is often used as a means of quantifying ventilation heterogeneity. During inspiration, lower regions receive more oxygen per alveolus than upper regions. Phase IV represents interregional (including regions that are far apart) heterogeneity of ventilation. As the expiration proceeds and lung volume is reduced, the airways in some of the lower regions begin to close off. Subsequent exhalation contains air from the upper regions with a higher concentration. The volume of phase IV is called ‘closing volume’. Also seen in phase III are the oscillations of concentration, termed cardiogenic oscillations. The amplitude of the cardiogenic oscillations is thought to indicate the degree of intraregional heterogeneity of ventilation to volume ratio among neighboring regions. Multiple-Breath Nitrogen Washout: Interaction of Convection and Diffusion

An approach to quantifying uneven ventilation uses the multiple-breath N2 washout (MBNW) and the change of the phase III slope with subsequent breaths after switching to 100% oxygen breathing. One of the parameters measured for each breath is the normalized slope (Sn) of the alveolar plateau (phase III) of the N2 curve (slope divided by mean expiratory N2 concentration) as a function of cumulative expired volume. It reflects the ventilatory inhomogeneity persisting throughout the MBNW and is basically generated by two types of mechanisms, convectiondependent inhomogeneity (CDI) and diffusion- and convection-dependent inhomogeneity (DCDI). The convection-dependent mechanism is due to a flow sequence between unequally ventilated lung units, which may be situated in different lung regions (interregional CDI) or nearer to each other (intraregional CDI). The diffusion- and convection-dependent mechanism is due to interaction of diffusion and convection at branch points within an asymmetric structure and probably acts mainly at the acinar level. The Multiple Inert Gas Elimination Technique

The distribution of ventilation has also been quantified using the multiple inert gas elimination technique (MIGET). The method requires the infusion of six inert gases of varying blood solubility. The analysis of the retention (arterial partial pressure divided by mixed venous partial pressure) and excretion (mixed exhaled partial pressure divided by mixed venous partial pressure) for each of the six infused gases primarily yields a distribution of perfusion. The distribution of ventilation is secondarily determined

VENTILATION / Uneven 449

from the perfusion distribution with a potential resolution of three modes of ventilation. Here the ventilation heterogeneity is not expressed relative to lung volume, but rather relative to perfusion. Hence distributions are ventilation to low, intermediate, or ’ regions. high V’ A =Q An alternate approach to analyzing MBNW data using a similar analytic technique to that employed in MIGET is to assume that the regions with varying ventilation-to-volume ratio are arranged in parallel. The approach transforms washout data into a distribution of ventilation-to-volume ratio. This method yields a continuous distribution of ventilationto-volume with up to four distinct modes on the ventilation-to-volume scale. None of the methods mentioned above provides spatial information. They provide a global number indicating the degree of overall ventilation nonuniformity.

High-Resolution Methods for Assessing Heterogeneity of Ventilation In recent years, new methodology has been developed to allow the measurement of ventilation with a high degree of spatial resolution. These methods include use of positron emission tomography (PET), computed tomography (CT) using xenon (a radiodense gas) to enhance sensitivity, and fluorescent microspheres. Several studies have shown that ventilation is nonuniformly distributed in all lungs. PET is a noninvasive method for assessing both ventilation and perfusion heterogeneity. The PET

scanner measures the near-simultaneous arrival of two positrons emitted in opposite directions from a radioactive gas in the lungs. A common source is 13 N2 which can be infused via the venous circulation and exhaled in the breath. Thus, it is possible to noninvasively measure ventilation and perfusion within small regions of the lung. Xenon-enhanced computed tomography (Xe-CT) has been used to measure the spatial distribution of ventilation. This method takes advantage of the density of xenon being greater than that of air. Ventilation is measured from the recording of density during washin and washout of 60% xenon, balance oxygen. The method provides a noninvasive measure of the spatial distribution of uneven ventilation. Figure 5 shows the ventilation (measured using fluorescent microspheres) in the small (B2 cm3) regions in both a prone and a supine pig. There are some important observations in Figure 5. The ventilation within each region is designated by a dot. Ventilation is heterogeneous within a horizontal (isogravitational) plane, a finding not predicted by the earlier vertical concept of ventilation distribution (see above). A line of regression is shown using the horizontal variation in ventilation. These lines show that the mean ventilation is greatest in the bottom of the lung in the prone position, but greatest in the top of the lung in the supine position. However, the overall effect is quite weak when high-resolution data are acquired. The ventilation heterogeneity within any isogravitational plane is considerably greater than the heterogeneity suggested by the linear fit.

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Figure 5 Data obtained by injecting 15 mm fluorescent microspheres into the venous circulation of a pig. Data shown for both the supine and prone postures. The regression lines show that there is an overall increase in the prone position near the ventral (bottom) of the lungs. The supine posture shows the opposite with the average flow being greater near the top of the lungs. At any height from the bottom of the lung (isogravitational plane), there is considerable variation of ventilation. Adapted from Mure M, Domino KB, Lindahl SGE, et al. (2000) Regional ventilation–perfusion distribution is more uniform in the prone position. Journal of Applied Physiology 88: 1076– 1083, used with permission from The American Physiological Society.

450 VENTILATION, MECHANICAL / Negative Pressure Ventilation

Fractal Nature of Ventilation Distribution

Further Reading

The airways play a dominant role in the regional distribution of ventilation. This is caused by the slightly irregular branching pattern of the airways that is similar for each bifurcation generation. This results in a fractal structure to the pulmonary airways. Fractal structures are the most efficient way to fill a threedimensional volume. A fundamental characteristic of a fractal structure is that its basic form is replicated over a range of scales. This repetition is a result of efficient genetic coding. Another interesting feature of a fractal structure to the pulmonary airways is that while ventilation is heterogeneous, each region has a ventilation that is very similar in magnitude with its nearest (spatially) neighbors. A corollary is that ventilation in a region is quite different from the farthermost regions of lung. The net effect of sequential branching is to produce ventilation heterogeneity that can only be appreciated with high-resolution measurement.

Altemeier WA, McKinney S, and Glenny RW (2000) Fractal nature of regional ventilation distribution. Journal of Applied Physiology 888: 1551–1557. Anthonisen NR, Fleetham JA (1987) Ventilation: total, alveolar, and dead space. In: Handbook of Physiology, Section 3, The Respiratory System, vol. 4, Gas Exchange, pp. 113–129. Bethesda, MD: American Physiological Society. Lewis SM, Evans JW, and Jalowayski AA (1978) Continuous distributions of specific ventilation recovered from inert gas washout. Journal of Applied Physiology: Respiratory Environmental and Exercise Physiology 44: 416–423. Marcucci C, Nyhan D, and Simon BA (2001) Distribution of pulmonary ventilation using Xe-enhanced computed tomography in prone and supine dogs. Journal of Applied Physiology 90: 421–430. Mure M, Domino KB, Lindahl SGE, et al. (2000) Regional ventilation–perfusion distribution is more uniform in the prone position. Journal Applied Physiology 88: 1076–1083. Musch G, Layfield JDH, Harris RS, et al. (2002) Topographical distribution of pulmonary perfusion and ventilation, assessed by PET in supine and prone humans. Journal Applied Physiology 93: 1841–1851. Paiva M and Engel LA (1987) Theoretical studies of gas mixing and ventilation distribution in the lung. Physiological Reviews 67: 750–795. Pedley TJ, Sudlow MF, and Milic-Emili J (1972) A non-linear theory of the distribution of pulmonary ventilation. Respiratory Physiology 15: 1–38.

See also: Alveolar Wall Micromechanics. Dynamics of Gas Flow and Pressure Flow Relationships. Ventilation: Overview; Control. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation.

VENTILATION, MECHANICAL Contents

Negative Pressure Ventilation Noninvasive Ventilation Positive Pressure Ventilation Ventilator-Associated Pneumonia

Negative Pressure Ventilation M S Badr and J A Rowley, Wayne State University School of Medicine, Detroit, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract In negative pressure ventilation (NPV), the surface of the thorax is exposed to a subatmospheric pressure during inspiration. The subatmospheric pressure causes thoracic expansion and decrease in alveolar pressure, generating a gradient for air to move from the airway opening to the alveoli. In NPV, expiration occurs passively owing to the elastic recoil of the lung and chest wall. NPV was the mode of ventilation originally used for artificial ventilation and was widely used during the poliomyelitis epidemics of the mid-twentieth century. Its use is now limited to

a few centers, primarily in Europe. Physiological effects of NPV include improved gas exchange (decreased PaCO2 and increased PaO2 ) and improved pulmonary mechanics (resulting in decreased effort). Indications for NPV include acute exacerbations of COPD/acute hypercapnic respiratory failure and the chronic outpatient management of chronic respiratory failure in patients with neuromuscular disorders such as post-poliomyelitis syndrome and muscular dystrophy. Despite evidence that it can be an effective modality for the above indications, it is not widely used because of increased nursing needs in the acute hospital setting and problems with patient discomfort/compliance in the chronic setting.

History For ventilation to occur, a phasic pressure difference must be developed across the lung. This pressure

VENTILATION, MECHANICAL / Negative Pressure Ventilation

difference can be generated by either applying a positive pressure to the airway (positive pressure ventilation) or a negative pressure across the pleural space. In particular, in negative pressure ventilation (NPV), the surface of the thorax is exposed to a subatmospheric pressure during inspiration. The subatmospheric pressure causes thoracic expansion and decrease in alveolar pressure, generating a gradient for air to move from the airway opening to the alveoli. During expiration, the pressure surrounding the thorax becomes atmospheric (positive), establishing a gradient for air to move from the alveoli to the airway opening. In NPV, expiration occurs passively owing to the elastic recoil of the lung and chest wall. While positive pressure ventilation is most commonly used in ventilators today in pulmonary and critical care medicine, negative pressure ventilation is the older method of providing ventilatory support. The essential concept of a negative pressure ventilator was developed by Woillez in 1876 but was impractical as the negative pressure was generated by manually operated bellows. The first practical negative pressure ventilator was developed in the 1920s at Harvard University. Modifications of this ventilator were widely used during the poliomyelitis epidemics of the 1940s and 1950s. In the following decades, technological advances in positive pressure ventilation occurred, leading to a marked decrease in the clinical use of negative pressure ventilators. The traditional negative pressure ventilator is the tank ventilator or ‘iron lung’ (Figure 1). These ventilators consist of a cylindrical chamber that covers all the extracranial portions of the body. There are generally portholes for observing the patient, for introducing blood pressure cuffs and stethoscopes, and to allow nursing functions such as bathing. Because of the large chambers, tank ventilators are limited to

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hospital use. Alternatively, the negative pressure can be generated in a chamber that covers only the anterior surface of the thorax and upper abdomen. These chambers are either a solid (cuirass) shell or consist of a bodysuit wrapped over a rigid grid. These devices are lightweight and portable and the cuirass shell allows the patient to be ventilated in the sitting position. These devices were designed for long-term home use of negative pressure ventilation.

Uses in Respiratory Medicine Modes of Ventilation

Negative pressure ventilators have been designed to deliver negative pressure in three primary modes (Figure 2). Cyclical negative pressure ventilation (CNPV) generates a preset subatmospheric pressure during inspiration, with a passive expiration (external pressure is atmospheric). Since NPV is a pressurecycled ventilatory mode, the delivered tidal volume depends upon the compliance of the lung, with larger delivered volume with an increased lung compliance. Most CNPV ventilators work in both the control (all breaths are mandatory with the frequency set by the ventilator) and assist-control (patient spontaneously triggers breaths which are assisted by the generation of negative pressure) modes. Patient-generated breaths are generally triggered by either negative pressure (measured by a pressure transducer catheter) or flow (measured by a thermistor) at the nares. In continuous negative pressure (CNEP) ventilation, the patient breathes spontaneously while the ventilator provides a constant subatmospheric pressure throughout the respiratory cycle. In experimental animals, this mode has been found to have equivalent physiological effects as those observed with continuous positive end expiratory pressure. The third modality is negative pressure/CNEP, in which an increased negative pressure during inspiration is superimposed on the CNEP. A fourth uncommonly used modality is negative/ pressure ventilation, a combination of a negative pressure during inspiration and a positive pressure during expiration. This mode is believed to further increase the tidal volume of patients with chest wall disorders by decreasing the functional residual capacity that has been associated with cuirass ventilators. However, this modality is not widely used. Physiologic Effects of Negative Pressure Ventilation

Figure 1 A typical iron lung ventilator. Photo courtesy of A Malhotra.

Multiple studies have shown that NPV is effective at improving gas exchange in both, patients with acute and chronic respiratory failure. Studies have consistently shown a decreased PaCO2 and increased pH and

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452 VENTILATION, MECHANICAL / Negative Pressure Ventilation

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Time (s) Figure 2 Recordings of volume, flow, pleural pressure (Ppl), gastric pressure (Pga), transdiaphragmatic pressure (Pdi), and tank pressure (Ptank) in a patient with acute exacerbation of COPD during spontaneous breathing (SB), continuous negative expiratory pressure (CNEP), cyclical negative pressure ventilation (NPV), and the combination of CNEP and NPV (NPV–NEEP). The dashed lines indicate the level of zero in the flow, Ppl, Pga, and Pdi recordings. Reproduced from Gorini M, Corrado A, Villella G, et al. (2001) Physiologic effects of negative pressure ventilation in acute exacerbations of chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 163: 1614–1618, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

PaO2 with the use of negative pressure ventilation in acute exacerbations of COPD. In addition, long-term use of a negative pressure ventilator in patients with chronic hypercapnic respiratory failure is associated with sustained decreases in daytime PaCO2 . A recent study specifically studied the effects of different modalities of NPV on gas exchange in patients with an acute exacerbation of COPD. In particular, the group studied the effects of CNPV alone, CNEP alone, and CNPV/CNEP compared to spontaneous breathing (Figure 2). In summary, minute ventilation significantly increased secondary to an increased tidal volume with CNPV but was not significantly different in CNEP alone or when CNEP was added to CNVP. PaCO2 and pH improved slightly with CNEP and significantly with CNVP with no additional change when CNEP was added to the CNVP. The effect of NPV on pulmonary mechanics has also been studied. Studies have shown increases in maximum inspiratory pressure and maximum expiratory pressure with the use of NPV in acute COPD, indicating improved respiratory muscle strength with use of NPV. In the acute physiologic study discussed in the preceding paragraph, the transdiaphragmatic

pressure and the pressure–time product of the diaphragm, both indicators of respiratory muscle strength and pulmonary mechanics, improved progressively as the mode of ventilation was changed from CNEP to CNVP to CNVP/CNEP. Compared to positive pressure ventilation, which is associated with both, decreased venous return and left ventricular afterload, NPV is associated with increased venous return and left ventricular afterload. Because of these opposing effects, cardiac output has been reported as decreased, increased, and unchanged with NPV. The effect of NPV on cardiac output likely depends upon whether the negative pressure is delivered by a tank ventilator or by a shell ventilator. With the tank ventilator, the extrathoracic body is also exposed to the negative pressure, thereby maintaining a more normal gradient of pressure from the periphery to the right atrium, preventing the expected increase in venous return. With a shell ventilator, only the thorax is exposed to the negative pressure resulting in an increased pressure gradient from the periphery to the right atrium and increased venous return. Detailed study of the cardiovascular effects of NPV in dogs confirms these effects. In particular, positive pressure ventilation and NPV

VENTILATION, MECHANICAL / Negative Pressure Ventilation

delivered via a tank ventilator resulted in similar decreases in cardiac output while NPV delivered via a wrap ventilator resulted in an increased cardiac output compared to the other two modalities. Use of Negative Pressure Ventilation in Acute Hypercapnic Respiratory Failure

There have been several uncontrolled studies evaluating the efficacy of NPV in the management of acute hypercapnic respiratory failure. As noted above, physiologic effects of NPV in acute hypercapnic failure secondary to COPD include improved gas exchange (decreased PaCO2 and increased PO2 ) and improved pulmonary mechanics. Several retrospective series encompassing a large number of patients (range 100–2000 patients) have shown that NPV, administered via iron lung, can be successfully applied in acute respiratory failure secondary to both COPD and restrictive thoracic disorders. In general, in these studies, NPV was associated with a low complication rate, acute mortality of approximately 10%, and an observed long-term survival better than reported previously for conventional mechanical ventilation. NPV administered via iron lung has been compared to conventional invasive mechanical ventilation in two studies of patients with acute hypercapnic failure secondary to COPD. The first was a retrospective case-control study. In this comparison of 26 matched pairs, there was no difference in mortality or length of hospital stay between the two groups but NPV was associated with a shorter duration of ventilation (by approximately 80 h) and a decreased rate of infectious complications. More recently, a randomized study of 44 patients randomly assigned either NPV or conventional invasive mechanical ventilation has been published. There was no difference between the two groups with regard to gas exchange and mortality. However, NPV was associated with significantly increased ventilator-free days, decreased hospital length of stay, and decreased major complications such as pneumonia. These studies suggest that iron lung ventilation is as effective as invasive mechanical ventilation with regards to gas exchange and may be advantageous over invasive mechanical ventilation with regard to ventilator-free days and complications. NPV has also been compared to noninvasive positive pressure ventilation (NIPPV), which is widely used in intensive care units for the treatment of acute hypercapnic respiratory failure. In one retrospective case-control study of patients with acute exacerbation of COPD, there was a significantly decreased duration of mechanical ventilation and hospital stay

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in the NPV group with no difference in the need for conventional mechanical that has been reported in abstract form only. A recently published prospective but nonrandomized study compared patients with acute or chronic respiratory failure treated with either NPV or NIPPV. This study was unique as patients who failed the initial modality were switched to the alternative one prior to initiating conventional invasive mechanical ventilation. While the majority of patients had COPD, the study included patients with other etiologies for chronic respiratory failure that included neuromuscular disorders, chest wall disorders, and the obesity–hypoventilation syndrome. The study found that both modalities were equally effective in improving gas exchange and preventing invasive mechanical ventilation. There were similar hospital lengths of stay and mortality between the two groups. Therefore, the available evidence supports the use of NPV as a modality in the treatment of acute hypercapnic respiratory failure with evidence for decreased ventilatory time compared to conventional mechanical ventilation and similar gas exchange, prevention of intubation, and mortality compared to NIPPV. Despite this evidence, however, NPV is not widely used in the United States for this indication, primarily for nursing reasons (see below), and is only used by a few centers in Europe that have extensive experience with its use. Use of Negative Pressure Ventilation in Chronic Hypercapnic Respiratory Failure

There are multiple studies that have shown that negative pressure ventilation can be an effective modality for treating chronic hypercapnic respiratory failure secondary to neuromuscular disorders such as post-poliomyelitis syndrome, muscular dystrophy (particularly Duchenne’s type), and amyotrophic lateral sclerosis. However, it should be noted that these studies are primarily case cohort, nonrandomized studies with widely varying patient populations, different types of portable negative pressure ventilators, and length of study time (range 1–20 years). However, as noted previously, these studies show consistent and sustained (up to 5 years) improvement in the PaCO2 , PaO2 , and exercise tolerance during the day. Some of the other findings from these studies include decreased number of hospitalizations, improved sleep quality, and possibly improved longterm mortality. It should be noted that while the effectiveness of long-term NPV is supported in the literature, it has largely been supplanted by NIPPV, which has also been well-studied, provides equal benefits, and is

454 VENTILATION, MECHANICAL / Negative Pressure Ventilation

thought to be less cumbersome for patients and caregivers. A recent study compared the long-term results of NPV with NIPPV in a large group of outpatients with a variety of disorders but primarily post-poliomyelitis syndrome and Duchenne’s muscular dystrophy. This was a retrospective study and the groups treated with NPV and NIPPV were dissimilar in terms of age, diagnosis, and pulmonary function. Furthermore, the duration of mechanical ventilation was widely different (approximately 24 years in the NPV group and 3 years in the NIPPV group). However, several broad findings were observed. First, the proportion of patients that reported positive outcomes such as increased independence and ability to perform daytime activities, was greater in the NIPPV group. Second, there was an increased need for tracheostomy and hospitalization rates in the NPV group, primarily in the post-poliomyelitis patients; tracheostomy was primarily needed for progressive bulbar dysfunction. However, the tracheostomy and hospitalization rates may be more a function of the length of time of illness (which was longer in the NPV group) rather than the type of ventilation. This alternative explanation is supported by the observation that there was a high tracheostomy rate in patients with Duchenne’s muscular dystrophy treated with NIPPV. Third, despite the physician’s belief that NIPPV would be more comfortable for patients, for the group as a whole there were increased complaints regarding the patient/machine interface in the NIPPV which was associated with a higher rate of self-discontinuation. The authors conclude that NIPPV is the more favorable mode of nocturnal long-term ventilation. In summary, NPV can be effective for the long-term treatment of chronic hypercapnic failure in neuromuscular disorders; however at this time, it is probably best reserved as an alternative modality for patients who cannot tolerate NIPPV. Despite evidence that it may be an effective alternative modality in acute exacerbations of COPD, there is no evidence that negative pressure ventilation is effective in the long-term treatment of severe chronic obstructive pulmonary disease. Advantages, Contraindications, Side-Effects

The major advantage of negative pressure ventilation is avoidance of endotracheal intubation and its associated complications. In addition, there is preservation of physiological functions, such as speech, cough, swallowing, and feeding. The major disadvantage of negative pressure ventilation using a tank ventilator is the difficult nursing access to the patient. For the cuirass or body-wrap ventilators, there can

be complaints related to discomfort and inability to sleep. In addition, most medical and nursing staff is unfamiliar with this modality. Therefore, it is not widely used in the United States. Most recent studies on its use in the acute setting are from European centers. The side-effects of negative pressure ventilation have been primarily studied in the long-term homecare population. Frequently mentioned side-effects include poor compliance, upper airway collapse, poor sleep quality, and musculoskeletal (particularly, back) pain. There are several contraindications for the application of negative pressure ventilation. Because of the increased tendency for the upper airway to collapse, patients with obstructive sleep apnea or a neurologic disorder characterized by bulbar dysfunction, are not candidates for negative pressure ventilation. Negative pressure ventilation does not provide adequate protection of the upper airway and is contraindicated in unconscious patients for whom upper airway protection is warranted. Conditions that would prevent the adequate use of either the cuirass or body-wrap modalities are contraindications for the use of negative pressure ventilation and include severe obesity, kyphoscoliosis and other chest wall deformities, recent abdominal surgery, and claustrophobia. See also: Chronic Obstructive Pulmonary Disease: Overview; Emphysema, General. Neuromuscular Disease: Inherited Myopathies; Upper Motor Neuron Diseases. Ventilation, Mechanical: Noninvasive Ventilation; Positive Pressure Ventilation.

Further Reading Baydur A, Layne E, Arah H, et al. (2000) Long term non-invasive ventilation in the community for patients with musculoskeletal disorders: 46 year experience and review. Thorax 55: 4–11. Corrado A, Confalonieri M, Marchese S, et al. (2002) Iron lung vs mask ventilation in the treatment of acute on chronic respiratory failure in COPD patients. A multicenter study. Chest 121: 181–189. Corrado A, Ginanni R, Villella G, et al. (2004) Iron lung versus conventional mechanical ventilation in acute exacerbation of COPD. European Respiratory Journal 23: 419–424. Corrado A and Gorini M (2002) Negative-pressure ventilation: is there still a role? European Respiratory Journal 20: 187–197. Corrado A, Gorini M, Villella G, and DePaola E (1996) Negative pressure ventilation in the treatment of acute respiratory failure: an old noninvasive technique reconsidered. European Respiratory Journal 9: 1531–1544. Curran FJ and Colbert AP (1989) Ventilator management in Duchenne muscular dystrophy and postpoliomyelitis syndrome: twelve years’ experience. Archives of Physical Medicine and Rehabilitation 70: 180–185. Gorini M, Corrado A, Villella G, et al. (2001) Physiologic effects of negative pressure ventilation in acute exacerbations of

VENTILATION, MECHANICAL / Noninvasive Ventilation chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 163: 1614–1618. Gorini M, Ginanni R, Villela G, et al. (2004) Non-invasive negative and positive pressure ventilation in the treatment of acute on chronic respiratory failure. Intensive Care Medicine 30: 875–881. Jackson M, Kinnear W, King M, Hockley S, and Shneerson J (1993) The effects of five years of nocturnal cuirass-assisted ventilation in chest wall disease. European Respiratory Journal 6: 630–635. Levine S and Henson D (1994) Negative pressure ventilation. In: Tobin MJ (ed.) Principles and Practice of Mechanical Ventilation, pp. 393–411. New York: McGraw-Hill. Schiavina M and Fabiani A (1993) Intermittent negative pressure ventilation in patients with restrictive respiratory failure. Monaldi Archivces for Chest Disease 48: 169–175. Todisco T, Baglioni S, Eslami A, et al. (2004) Treatment of acute exacerbations of chronic respiratory failure. Integrated use of negative pressure ventilation and noninvasive positive pressure ventilation. Chest 125: 2217–2223.

Noninvasive Ventilation A S Slutsky and N D Ferguson, University of Toronto, Toronto, ON, Canada & 2006 Elsevier Ltd. All rights reserved.

Abstract The use of noninvasive ventilation (NIV) for the treatment of acute respiratory failure has expanded considerably over the past decade and a half. This therapy has the potential to limit complications of intubation, such as airway injury and ventilator-associated pneumonia. Ideally, it should be used in carefully selected patients who are cared for in a closely monitored setting such as an intensive care unit (ICU). The best evidence for the efficacy of NIV is in severe exacerbations of chronic obstructive pulmonary disease (COPD), where it has been shown to reduce the need for intubation and reduce hospital mortality. The other best-studied acute indication is cardiogenic pulmonary edema where noninvasive continuous positive airway pressure has been shown to reduce intubations and possibly mortality. Emerging indications for NIV include hypoxemic respiratory failure and the facilitation of weaning. The available evidence suggests that NIV is not useful in treating postextubation respiratory distress. NIV has emerged as a standard therapy that should be available in most modern ICUs. Future studies are still needed to help define its role in a number of areas including its use in non-COPD patients, and in the immediate postextubation phase.

History Noninvasive ventilation (NIV) is generally defined as any mode of ventilatory support that is provided without the use of an endotracheal or tracheostomy tube. As such, negative-pressure ventilation is technically a form of NIV; however, this topic will be addressed elsewhere in this encyclopedia, and

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throughout this article the term NIV will imply noninvasive positive-pressure ventilation. Instead of an invasive airway, NIV is delivered using a noninvasive interface, most commonly a tightly fitting mask covering the nose and mouth. The interface is connected to either a regular critical care ventilator or a portable ventilator specifically designed for NIV and inspiratory pressure assist is delivered; positive endexpiratory pressure (PEEP) is also typically provided. This same setup can also be used to provide simple continuous positive airway pressure (CPAP), which, despite not truly being a form of assisted ventilation (since no inspiratory assist is provided), can be very useful in the specific situations discussed below. NIV using positive pressure was first introduced and became widely used as a therapy for chronic respiratory failure in the early to mid 1980s. This was applied largely as nocturnal ventilation for neuromuscular diseases (such as Duchenne muscular dystrophy and myotonic dystrophy), and for restrictive deformities of the chest wall. In addition, CPAP was introduced as a treatment for obstructive sleep apnea around this time. These all remain important indications for NIV or CPAP, and the reader is referred to the ‘Further reading’ list for more information on these topics. This article will focus on the most recent application of NIV – its use to treat acute respiratory failure, both de novo disease and especially exacerbations of chronic respiratory disease. The first report of NIV to treat acute exacerbations of chronic obstructive pulmonary disease (COPD) was a case-series published in 1989 by Meduri and colleagues. Since that report, a number of randomized controlled trials have been conducted in COPD populations. In addition, the study of NIV in the acute setting has expanded to include cardiogenic pulmonary edema, hypoxemic respiratory failure, facilitation of weaning from invasive mechanical ventilation, and the treatment of postextubation respiratory distress. Each of these uses will be reviewed in more detail below.

Uses in Respiratory Medicine General Considerations

The use of NIV has increased substantially since its introduction in 1989. A large international observational study performed in 1998 found that 5% of ventilated patients were treated with NIV. This probably underestimates the prevalence of NIV use, as patients who received this therapy for less than 12 h were excluded from this study. The major rationale underlying the more widespread use of NIV is to reduce complications

456 VENTILATION, MECHANICAL / Noninvasive Ventilation Table 1 Contraindications to NIV Category

Specific contraindication

Clinical instability

Cardiac or respiratory arrest Unstable cardiac arrhythmia Hemodynamic instability (e.g., hypotension requiring inotropes)

Inability to protect airway

Decreased level of consciousness Copious or poorly handled pulmonary secretions Significant upper gastrointestinal bleeding

Upper airway compromise

Upper airway obstruction Facial trauma, fracture, or surgery

Other

Patient agitated and uncooperative Unable to adequately fit or seal interface

associated with invasive mechanical ventilation. These include airway injuries and problems occurring during intubation such as aspiration, hypotension, or increased intracranial pressure. Probably the most significant advantage of NIV, however, is the maintenance of intact upper airway defenses and the avoidance of ventilator-associated pneumonia. There are, however, contraindications to NIV, which are listed in Table 1. Primary intubation may be more appropriate in patients who are likely to require a prolonged duration of ventilation, and who require high levels of ventilatory pressures. In the acute setting NIV can be delivered using a variety of interfaces for connecting the patient with the ventilator. Full-face masks that seal tightly and cover both the nose and mouth are used most commonly. Nasal masks can sometimes be used but if a patient is in acute respiratory distress, involuntary mouth breathing and resultant air leak limit their effect. Some centers employ masks or helmets that apply positive pressure over a larger area and may decrease skin breakdown at vulnerable points such as the bridge of the nose, though these are not in widespread use. NIV can be applied using either the same critical care ventilators used for invasive ventilation, or with portable ventilators specifically designed for NIV. A detailed discussion regarding the ventilators themselves is outside the scope of this article, but the portable ventilators are typically smaller, more easily transported, and less expensive. The sophistication of the alarm system, monitoring capability, and oxygen blending capacity vary significantly across different portable systems. Finally, it is important to consider

the location in which acute NIV is administered. We believe that as a general rule, patients receiving NIV for acute respiratory failure require monitoring that is as close (if not closer) than those patients receiving invasive mechanical ventilation. This level of observation is needed because their airway is not protected or secured, and because rapid intervention (including intubation) may be needed if patients do not respond to NIV therapy. Therefore, although NIV may be initiated in a variety of locations, prompt arrangements should be made to transfer the patient to an intensive care unit or similar area where they can be carefully observed. Exacerbations of COPD

The most compelling evidence for the acute use of NIV is found in patients with acute exacerbations of COPD. Although providing positive pressure (particularly during expiration) may seem counterintuitive in patients with airflow obstruction, sound physiological rationale exists for its use. During an exacerbation of COPD, work of breathing may increase because of increased airway resistance, and because of dynamic hyperinflation with resultant reduced respiratory system compliance (see Figure 1). Pressure-assist during inspiration can directly off-load the respiratory muscles and minimize the work of breathing. PEEP, a phenomenon COPD patients often create for themselves through the use of purse-lip breathing, may be useful to stent open collapsible airways, thereby reducing gas trapping (Figure 1). In addition, in the setting of dynamic hyperinflation, set-PEEP on the ventilator may improve ventilator synchrony and reduce the work required to initiate a breath. At least 15 randomized controlled trials have been conducted examining the efficacy of NIV to prevent intubation and to reduce all-cause hospital mortality. The majority of these trials were small (only 1 included more than 100 patients) and their methodological quality varies significantly. Nevertheless, as demonstrated in two recent meta-analyses by Peter et al. and by Keenan et al. consistent and convincing results are observed across these studies. Pooled results show an absolute risk reduction of 28% for endotracheal intubation and a 10% absolute risk reduction in hospital mortality. Thus, NIV appears to be a highly effective therapy for patients with acute exacerbations of COPD. It is important, however, to recall the populations included in these studies. These were patients with fairly severe COPD exacerbations, but who did not require immediate intubation. Studies that only included patients with milder disease (defined as an initial pH of 47.30 or a control-group hospital mortality of o10%)

VENTILATION, MECHANICAL / Noninvasive Ventilation

457

COPD exacerbation

Airway resistance • Secretions • Bronchospasm • Inflammation

Dynamic hyperinflation

Diaphragmatic dysfunction • Flattening of diaphragm • Poor mechanical coupling • Oxygen cost of breathing

Respiratory system compliance Physiologic dead space

Resistive load

Elastic load

Dead space fraction (VD /VT) Minute ventilation load Work of breathing

VT and

respiratory rate

Hypercapnic respiratory failure Figure 1 Physiologic consequences of a COPD exacerbation. The numerous physiologic factors contributing to hypercapnic respiratory failure are illustrated beginning with increased airway resistance. NIV can interrupt this cascade by providing ventilatory assist to deal with the increased loads and diaphragmatic dysfunction, by preventing a decrease in tidal volume, and by reducing dynamic hyperinflation by stenting open collapsible small airways and by reducing tachypnea. VT, tidal volume; VD, dead space volume.

have not found any reductions in intubation rate or mortality with NIV. Thus, NIV is not indicated in every patient admitted to hospital with an exacerbation of COPD, but its use should be strongly considered in patients with severe exacerbations (with a low pH or judged to be at high risk of death) before they progress to the point of requiring endotracheal intubation. Cardiogenic Pulmonary Edema

Positive airway pressure is theoretically an ideal treatment for cardiogenic pulmonary edema. It not only reduces left ventricular preload and afterload, but does so in an easily titratable fashion with a rapid onset of action. However, current practice guidelines from specialist bodies do not recommend the routine

use of NIV for this syndrome. This is probably because available studies have shown a reduction in intubation rates but have not demonstrated differences in mortality. These studies, however, were all small and underpowered to detect important mortality differences, so the true impact of CPAP in severe cases of pulmonary edema is unknown. NIV in addition to CPAP for cardiogenic pulmonary edema has also been studied in a number of trials. Such randomized trial published in 1997 by Mehta and colleagues was the first to compare CPAP to bilevel NIV, and was stopped early because of concerns of harm with the increased rate of myocardial infarctions seen in the NIV group. Intubation rates, mortality, and other morbidity were not different between the groups, and it is unclear whether this observation occurred by chance alone because of

458 VENTILATION, MECHANICAL / Noninvasive Ventilation

baseline differences between the groups (more chest pain in the NIV group) or because there really was a harmful effect of NIV (possibly by excessively reducing preload and compromising coronary perfusion). More recently, randomized trials comparing NIV to standard therapy and NIV to CPAP to standard therapy have not demonstrated any harmful effects of NIV in terms of increased infarction risk, and one of these studies (by Park and colleagues) showed a reduction in early mortality and a trend toward reduced hospital mortality comparing NIV or CPAP with standard therapy. In summary, CPAP reduces the need for intubation in severe cases of cardiogenic pulmonary edema and may reduce mortality. There is little evidence to suggest that the addition of NIV to CPAP leads to further improvements in outcomes though this area remains understudied. Hypothesis-generating subgroup analyses suggest that NIV may be most effective in pulmonary edema patients who are hypercapnic, but this needs further study. Hypoxemic Respiratory Failure

Considerable debate exists regarding the appropriate indications for NIV versus primary intubation for patients with hypoxemic respiratory failure. This is a much more heterogeneous syndrome than exacerbations of COPD, and thus perhaps not unexpectedly trials of NIV in this population have shown mixed results. In addition, there is some concern that hypoxemic patients who fail a trial of NIV and require intubation may have worse outcomes than those who are intubated primarily, an observation not made in COPD patients. At least seven randomized trials of NIV in hypoxemic respiratory failure have been carried out. These were all small studies (o50 patients) and, with one exception, they were all conducted in a single center. Pooled results from another recent meta-analysis by Keenan and colleagues show a 23% absolute risk reduction for intubation, but no significant difference in hospital mortality. Significant heterogeneity was observed across study results. Several of these studies were conducted in specialized populations, including immunosuppressed patients and patients with respiratory failure postpneumonectomy. In both these populations a significant benefit of NIV was seen, perhaps because of the general poor prognoses of these patients when they require intubation and ventilation. Facilitation of Weaning from Invasive Mechanical Ventilation

Most patients with COPD intubated for acute exacerbations do not require this therapy for a

prolonged time and have durations of ventilation and weaning similar to other mechanically ventilated patients. A minority of these patients, however, does have difficulty separating from the ventilator and require a prolonged course of ventilation. NIV has been proposed as a bridge toward ventilator independence in these patients based on the hypothesis that NIV would diminish the risks of nosocomial infections and airway injury. There have been at least three published randomized trials that have assessed this question. The first study by Nava and colleagues assigned 50 ventilated COPD patients who had failed a trial of spontaneous breathing to either extubation and NIV, or to continued invasive ventilation. Patients assigned to NIV had a shorter length of stay in the intensive care unit (ICU), and had a trend toward improved 60-day mortality. These results were not confirmed in a subsequent study, but in the most recently published trial by Ferrer et al., patients who had failed spontaneous breathing trials on 3 consecutive days and were then extubated to NIV had shorter ICU and hospital lengths of stay and a 30% absolute reduction in mortality that was on the borderline of statistical significance. Overall, the evidence available to date suggests that NIV may be a useful adjunctive therapy in selected difficult-towean patients, likely with underlying chronic pulmonary abnormalities. This technique should be used only in centers that are familiar with the use of NIV. Patients who are extubated to NIV should continue to be closely monitored in an ICU setting until they are able to demonstrate ventilator independence for a reasonable period of time. Treatment of Postextubation Respiratory Failure

Patients who require reintubation have a higher mortality than those whose first extubation attempt is successful. While this may simply be a marker of severity of illness, extubation failure has been found to be an independent predictor of mortality and therefore treatments aimed at preventing reintubation have been hypothesized to improve survival in these patients. NIV has been evaluated in this regard in two recent randomized controlled trials. Both of these trials showed no difference in the rate of reintubation between patients treated with usual therapy versus NIV. In addition, the most recent study, a multicenter trial by Esteban and colleagues, was stopped early because of possible harm, with a higher ICU mortality rate observed in the NIV group. The most reasonable conclusion to draw from these studies is that NIV is not effective in preventing rein tubation in unselected patients who develop respiratory distress postextubation. It should be pointed

VENTILATION, MECHANICAL / Noninvasive Ventilation Table 2 Recommendations for NIV use Recommendation

Clinical condition

Use routinely unless a contraindication exists Use as an adjunctive therapy in some patients Use in some selected patients

Severe exacerbations of COPD

Use infrequently, only in a few selected patients Do not use routinely in most patients

459

Disease: Overview. Obesity. Oxygen Therapy. Pneumonia: Overview and Epidemiology. Sleep Apnea: Overview. Sleep Disorders: Overview.

Further Reading Cardiogenic pulmonary edema

Cardiogenic pulmonary edema Hypoxemic respiratory failure Immunocompromised patients Post lungresection Facilitation of weaning Difficult to wean patients with chronic pulmonary diseases Hypoxemic respiratory failure Acute lung injury Pneumonia Postextubation respiratory distress

out, however, that COPD patients made up only a small proportion of each study population and the extent to which these results apply to this and other specific populations is unknown.

Conclusions The study and use of NIV for the treatment of acute respiratory failure has expanded significantly over the past decade and a half. This therapy has the potential to limit complications of intubation and invasive ventilation, such as airway injury and ventilator-associated pneumonia. NIV should be available in most modern hospitals, but should only be used in carefully selected patients who are cared for in a closely monitored setting such as an ICU. Our general recommendations for when (and when not) to consider the use of NIV are shown in Table 2. At the present time NIV should be routinely considered in the care of patients with severe exacerbations of COPD. It should probably be used frequently in cardiogenic pulmonary edema (most likely in the form of CPAP), and in immuno-suppressed patients with hypoxemic respiratory failure. It should not be routinely used in unselected patients with postextubation respiratory distress. Further studies are needed to better define the role of this therapy in other forms of hypoxemic respiratory failure and as a tool to facilitate weaning from mechanical ventilation. See also: Arterial Blood Gases. Chest Wall Abnormalities. Chronic Obstructive Pulmonary

Anonymous (2001) International consensus conferences in intensive care medicine: noninvasive positive pressure ventilation in acute respiratory failure. American Journal of Respiratory and Critical Care Medicine 163(1): 283–291. Antonelli M, Conti G, Bufi M, et al. (2000) Noninvasive ventilation for treatment of acute respiratory failure in patients undergoing solid organ transplantation: a randomized trial. JAMA 283(2): 235–241. Brochard L, Mancebo J, Wysocki M, et al. (1995) Noninvasive ventilation for acute exacerbations of chronic obstructive pulmonary disease. New England Journal of Medicine 333(13): 817–822. Esteban A, Frutos-Vivar F, Ferguson ND, et al. (2004) Noninvasive positive-pressure ventilation for respiratory failure after extubation. New England Journal of Medicine 350(24): 2452–2460. Ferrer M, Esquinas A, Arancibia F, et al. (2003) Noninvasive ventilation during persistent weaning failure: a randomized controlled trial. American Journal of Respiratory and Critical Care Medicine 168(1): 70–76. Girault C, Daudenthun I, Chevron V, et al. (1999) Noninvasive ventilation as a systematic extubation and weaning technique in acute-on-chronic respiratory failure. American Journal of Respiratory and Critical Care Medicine 160: 86–92. Keenan SP, Powers C, McCormack DG, and Block G (2002) Noninvasive positive-pressure ventilation for postextubation respiratory distress: a randomized controlled trial. JAMA 287: 3238–3244. Keenan SP, Sinuff T, Cook DJ, and Hill NS (2003) Which patients with acute exacerbation of chronic obstructive pulmonary disease benefit from noninvasive positive-pressure ventilation? A systematic review of the literature. Annals of Internal Medicine 138(11): 861–870. Keenan SP, Sinuff T, Cook DJ, and Hill NS (2004) Does noninvasive positive pressure ventilation improve outcome in acute hypoxemic respiratory failure? A systematic review. Critical Care Medicine 32(12): 2516–2523. Liesching T, Kwok H, and Hill NS (2003) Acute applications of noninvasive positive pressure ventilation. Chest 124(2): 699–713. Mehta S and Hill NS (2001) Noninvasive ventilation. American Journal of Respiratory and Critical Care Medicine 163: 540–577. Nava S, Ambrosino N, Clini E, et al. (1998) Noninvasive mechanical ventilation in the weaning of patients with respiratory failure due to chronic obstructive pulmonary disease: a randomized, controlled trial. Annals of Internal Medicine 128: 721–728. Park M, Sangean MC, Volpe MS, et al. (2004) Randomized, prospective trial of oxygen, continuous positive airway pressure, and bilevel positive airway pressure by face mask in acute cardiogenic pulmonary edema. Critical Care Medicine 32(12): 2407– 2415. Peter JV, Moran JL, Phillips-Hughes J, and Warn D (2002) Noninvasive ventilation in acute respiratory failure – a meta-analysis update. Critical Care Medicine 30(3): 555–562. Plant PK, Owen JL, and Elliott MW (2000) Early use of non-invasive ventilation for acute exacerbations of chronic obstructive pulmonary disease on general respiratory wards: a multicentre randomised controlled trial. Lancet 355(9219): 1931–1935.

460 VENTILATION, MECHANICAL / Positive Pressure Ventilation

Positive Pressure Ventilation S E Lapinsky and A S Slutsky, University of Toronto, Toronto, ON, Canada & 2006 Elsevier Ltd. All rights reserved.

Abstract Positive pressure mechanical ventilation is used to support gas exchange during anesthesia and as a life-saving intervention in acute respiratory failure. Ventilators have evolved over the past 50 years from mechanical pressure or volume controlled machines to sophisticated computer-driven devices providing a variety of modes, allowing optimal interaction between patient and ventilator. The spectrum of ventilatory modes extends from controlled ventilation (with no response to patient effort) to support modes, which require a patient’s spontaneous breaths that trigger the ventilator. Increasing evidence has documented that inappropriately high pressures or volumes may cause or aggravate lung injury, as do low end-expiratory pressures, which permit repetitive opening and closing of alveolar regions. This has resulted in the development of lung protective protocols that incorporate pressure and volume limitation with adequate end-expiratory pressure. Mechanical ventilation can be discontinued when the underlying pulmonary condition has resolved, and when the patient is alert and the airways are patent. A daily trial of spontaneous breathing appears to be the most effective method to identify when a patient’s ventilatory support may be discontinued.

But that life may be restored to the animal, an opening must be attempted in the trunk of the trachea, into which a tube of reed or cane should be put; you will then blow into this, so that the lung may rise again and take air. – Andreas Vesalius, De Humani Corporis Fabrica (1543)

functionality, including more sophisticated modes and graphical displays.

Objectives of Mechanical Ventilation Mechanical ventilation is used to support gas exchange during general anesthesia and also as a lifesaving intervention to manage respiratory failure complicated by severe hypoxemia or hypercapnia. Ventilation can improve oxygenation by a direct effect on lung volumes and alveolar recruitment, as well as by the delivery of a high inspired oxygen fraction. Mechanical ventilation corrects hypercapnia and respiratory acidosis, although in some situations the high ventilatory pressures required to normalize PaCO2 may be excessive, and some degree of hypercapnia may be accepted. Ventilation also unloads the respiratory muscles, an important goal because the oxygen consumption of these muscles may account for up to 50% of total oxygen consumption in the patient with acute respiratory failure. As ventilatory support may result in significant complications, an important goal should be to minimize or prevent these adverse effects. In situations in which the patient has severe hypoxemia or cannot protect the airway, the decision to initiate mechanical ventilatory support may be straightforward. However, in some situations the decision requires clinical judgment, weighing risks against benefits. In this regard, there has been increasing evidence for the use of noninvasive ventilation in some patients with respiratory failure.

History

Modes of Ventilation

Positive pressure artificial ventilation was first described by Vesalius in 1543, using a reed introduced into the trachea of animals and cadavers. A published report of successful mouth-to-mouth resuscitation by Tossach in 1744 resulted in the development of bellows for positive pressure ventilatory support by Hales, which were used extensively throughout the late 1700s. Very little change occurred in this field until World War II, when advances in breathing apparatus for altitude aviation produced the technology required for mechanical ventilation as we know it today. A classic study by Lassen published in The Lancet in 1953 on the management of patients with paralytic polio clearly demonstrated the life-saving capabilities of mechanical ventilation. The pressurecontrolled Bird Mark 7 was developed in the 1950s, followed by the development of volume-controlled ventilators such as the Engstrom 100 and the Puritan Bennett MA1. The use of computers in mechanical ventilators since the 1980s has introduced additional

Ventilation may be achieved by delivering either a predetermined volume or a set pressure to the lungs, on a regular or a demand basis (Table 1). Volumecontrolled ventilation delivers a preset tidal volume with a pressure limit set as a safety feature. If this pressure level is reached, the pressure is released and a ventilator alarm sounds. Pressure-preset ventilation differs in that the delivered level of airway pressure is preset, with the tidal volume being determined by respiratory mechanics. The duration of delivery of the pressure is set as an inspiratory time or as an inspiratory:expiratory ratio and respiratory rate (Table 1). The patient–ventilator interaction involves a spectrum from controlled ventilation to ventilator-supported spontaneous patient efforts. Controlled ventilation, which is the most basic mode of ventilatory support, provides a set tidal volume and rate regardless of the patient’s effort (Figure 1). Significant asynchrony can occur between the patient’s

VENTILATION, MECHANICAL / Positive Pressure Ventilation

spontaneous efforts and the ventilator-generated breaths, which can be overcome by assisted modes in which the ventilator is triggered by the patient’s respiratory efforts. In the assist-control mode, the Table 1 Comparison of volume and pressure ventilation Volume preset

Pressure preset

Settings

Tidal volume Inspiratory flow rate Flow waveform

Airway pressure Inspiratory duration Inspiratory time or I:E ratio and frequency

Common settings

FiO2 Frequency PEEP Triggering

FiO2 Frequency PEEP Triggering

Usual modes

Control Assist-control SIMV

Control Assist-control Pressure support

Monitored variables

Airway pressure Auto-PEEP

Tidal volume Auto-PEEP

I:E, inspiratory:expiratory; PEEP, positive end-expiratory pressure; SIMV, synchronized intermittent mandatory ventilation.

461

ventilator supports every patient breath. A minimum rate is set, so that in the absence of patient efforts ventilation will continue. Synchronized intermittent mandatory ventilation (SIMV) offers a preset ventilator rate with the possibility of spontaneous breathing. Ventilator breaths are patient-triggered and therefore synchronized but are mandatory in the absence of patient effort. In contrast to assist-control, patient breaths in excess of the preset rate do not receive ventilatory assistance (Figure 1). Spontaneous breaths in this mode may require significant patient work due to circuit or demand-valve resistance. This mode has been used for weaning patients from ventilatory support, although other approaches are more effective. Pressure-support ventilation is a pressure-preset mode in which the ventilator supports all spontaneous efforts. The delivered pressure is triggered by the patient and is maintained until a specified decrease in inspiratory flow occurs (flow-cycling). The patient therefore determines both respiratory frequency and inspiratory time, with tidal volume being a function of the set level of pressure, patient effort, and pulmonary mechanics. This mode may reduce asynchrony and

Spontaneous

0 I

E Control

PEEP

Pressure

0

I

E

Assist-control

PEEP 0 Assisted

Assisted

Controlled SIMV

PEEP 0

Assisted

Spontaneous

Pressure assistcontrol

PEEP 0

Assisted

Assisted

Controlled

Pressure support

PEEP 0

I

E Time

Figure 1 Pressure-time tracings during spontaneous breathing and various modes of mechanical ventilation. In the assist-control and pressure assist-control tracings, the first two breaths are triggered, but the third breath is controlled because no spontaneous effort has occurred. During synchronized intermittent mandatory ventilation (SIMV), spontaneous breathing occurs between triggered ventilator breaths. The pressure-support profile demonstrates variation in duration of the inspiratory phase due to flow cycling, compared with the fixed inspiratory period of the time-cycled pressure-control mode. I, inspiratory phase; E, expiratory phase; CPAP, continuous positive airway pressure; PEEP, positive end-expiratory pressure.

462 VENTILATION, MECHANICAL / Positive Pressure Ventilation

optimize patient control over ventilation, and is a useful weaning mode. Pressure support requires spontaneous respiratory efforts and as it does not guarantee a minimum tidal volume, an apnea backup capability is essential. High-frequency oscillation (HFO) is an alternative mode of mechanical ventilation that has been developed to improve oxygenation and reduce ventilatorinduced lung injury. It allows delivery of a higher mean airway pressure (improving alveolar recruitment and oxygenation) without injurious high peak pressures. HFO is carried out at a frequency of 3–8 Hz (180–480 min 1), and frequency is adjusted according to PaCO2 . Lower frequencies often produce a higher tidal volume and lower PaCO2. During HFO, patients often require deep sedation or even neuromuscular blockade. There are no data demonstrating a clear outcome benefit over conventional ventilation.

Ventilator Settings Respiratory Rate

Depending on the mode, respiratory rate may be a function of the ventilator setting, spontaneous breathing, or a combination of these. Synchronization of spontaneous breaths with ventilator breaths is achieved by triggering the ventilator when it senses the patient initiating a spontaneous breath, either by a small change in airway pressure (e.g., 0.5 to 2 cmH2O) or by sensing inspiratory flow. Insensitive triggering systems may require significant effort from the patient; an oversensitive system may produce spontaneous ventilator cycling. The presence of residual positive alveolar pressure at the end of expiration (auto-PEEP) may inhibit triggering; the patient must create a negative pressure large enough to overcome this positive alveolar pressure before the ventilator senses a change in airway pressure. Flow Rates

The inspiratory flow rate and pattern of delivery (square-wave, sine-wave, or decelerating flow) are set during volume-controlled ventilation but are determined by patient effort and pulmonary mechanics in the pressure-preset modes. The flow rate setting must be sufficient to match the spontaneously breathing patient’s peak inspiratory demands, usually requiring from 40 to 100 l min 1 in the adult patient. While no significant benefit has been demonstrated for one flow pattern over another, a decelerating ramp inspiratory flow has the potential advantage of optimizing mean airway pressure and limiting peak pressure.

Cycling

The tidal inspiration is terminated when a preset volume (volume-control), time (pressure-control), or a flow rate (pressure-support) is achieved. Volume cycling delivers only the preset tidal volume, and additional patient effort does not produce further volume. In pressure-controlled ventilation the set airway pressure (and therefore flow) is maintained for a preset inspiratory time (time-cycling). Additional patient effort during this time produces an increased tidal volume. Pressure-support ventilation utilizes flow cycling, that is, airway pressure is maintained until inspiratory flow falls below a certain level (usually 25% of the peak inspiratory flow). Positive End-Expiratory Pressure

Positive end-expiratory pressure (PEEP) is used to improve oxygenation and to prevent ventilator-induced lung injury. The oxygenation benefit is a result of the increased mean airway pressure, lung volume recruitment, and the redistribution of lung water to the perivascular interstitial space. The most common methods for setting PEEP rely on optimizing a physiological endpoint such as arterial partial pressure of oxygen (PaO2 ), oxygen delivery, or lung compliance. Oxygen

Lung injury may result from an inspired oxygen concentration greater than 0.5 and the lowest inspired oxygen concentration producing an acceptable PaO2 should be selected. Other interventions to improve PaO2 include manipulating mean airway pressure or improving mixed venous oxygen saturation (by improving cardiac output).

Ventilatory Support of the Critically Ill Patient In the patient with acute respiratory distress syndrome (ARDS), mechanical ventilation with a low tidal volume (6 ml kg 1 predicted body weight) and plateau pressure less than 30 cmH2O improves outcome. Lung injury in ARDS is inhomogeneous, and the uninvolved lung should be protected from excessive inflation pressure. Low tidal volumes may produce alveolar hypoventilation and hypercapnia, which generally is well tolerated. In the patient with a poorly compliant chest wall (e.g., massive ascites, pleural effusions), higher airway pressures may be accepted. In the presence of inadequate PEEP, hypoxemia may persist and ARDS may deteriorate due to alveolar damage resulting from repeated opening and closing of alveoli. Application of a sustained inflation recruitment maneuver

VENTILATION, MECHANICAL / Positive Pressure Ventilation

(e.g., 40 cmH2O continuous positive airway pressure for 40 s) may improve oxygenation, particularly in early ARDS, but the impact on clinical outcomes is not clear. Patients ventilated for obstructive airways disease often demonstrate dynamic hyperinflation or autoPEEP due to incomplete exhalation at the end of each breath. This results in residual pressure (and volume) in the alveoli at end-expiration, which can cause barotrauma, hemodynamic collapse, and difficulty in triggering the ventilator. Auto-PEEP can be reduced by minimizing minute ventilation and maximizing expiratory time. In patients with severe airflow limitation, this may require controlled hypoventilation with permissive hypercapnia. The development of hemodynamic compromise from auto-PEEP should be anticipated and managed by reducing minute volume and by fluid administration. Auto-PEEP may increase the work of breathing required to trigger breaths; this effect can be overcome by applying extrinsic PEEP at a level similar to or less than the measured auto-PEEP.

Complications of Mechanical Ventilation Endotracheal Intubation

Intubation may produce complications related to trauma to the pharynx, glottis, and trachea during insertion of the tube or as a result of prolonged intubation. The endotracheal tube disrupts the coughing mechanism and the mucociliary escalator causing retention of secretions. While gross aspiration is prevented by a cuffed endotracheal tube, pharyngeal secretions do enter the trachea and increase the risk of pneumonia. Elevation of the head of the bed to 45 degrees may reduce the incidence of ventilator-associated pneumonia. Positive Pressure

High transpulmonary pressures (alveolar minus pleural pressure) and high tidal volumes can produce barotrauma, manifesting as pneumothorax, pneumomediastinum, or subcutaneous emphysema. Overdistension of the lung as well as repetitive opening and closing of alveoli can produce local lung injury, as well as systemic release of cytokines potentially resulting in multiorgan failure. PEEP may be beneficial by reducing the shear stresses associated with repeated opening and closing of alveoli. Positive pressure ventilation may reduce cardiac output and blood pressure by impairing venous return and right-ventricular filling, particularly in the volume-depleted patient.

463

Asynchrony

Patient–ventilator asynchrony, due to poor interaction between the set ventilation pattern and the patient’s natural breathing rhythm, can produce an increased work of breathing, hypoventilation, or barotrauma. It occurs most commonly with controlled ventilatory modes, with flow rates insufficient to meet patient needs, high pressure support levels, and in the presence of auto-PEEP. If ventilator adjustments do not correct patient–ventilator asynchrony, increased sedation or neuromuscular blockade may be required. Sedation and Paralysis

Sedation is usually necessary during mechanical ventilation to allow for patient comfort and to reduce patient–ventilator asynchrony. Sedative drugs may contribute to the hypotension that commonly accompanies positive pressure ventilation. Neuromuscular blockade may occasionally be required to immobilize the patient to facilitate ventilatory strategies. When spontaneous respiratory efforts are suppressed, inadvertent disconnection from the ventilator will rapidly produce hypoxemia. Immobilization can also lead to pressure necrosis of the skin, deep venous thrombosis, secretion retention, atrophy of the respiratory and peripheral muscles, and atelectasis. Prolonged weakness may occur after withdrawal of neuromuscular blockade. The depth of paralysis should be limited and neuromuscular blockade should be withdrawn periodically to assess the neuromuscular status.

Weaning and Extubation The decision as to when to discontinue mechanical ventilation is critical – early extubation may necessitate reintubation, while unnecessarily prolonging intubation increases costs and the risk of complications. A protocol to assess the patient’s ability to sustain spontaneous ventilation on a regular basis (e.g., every 24 h) can identify the optimal timing for extubation. The underlying respiratory problem that required ventilation and any subsequent complications (e.g., nosocomial pneumonia or atelectasis) must have resolved, the patient should be clinically stable and alert, and the respiratory system must be able to cope with the ventilatory workload. Various clinical measures are used to assess the likelihood of successful weaning, including the tidal volume, respiratory rate, spontaneous minute ventilation, vital capacity, and maximum inspiratory force, although these are insensitive. The patency of the upper airway and the patient’s ability to clear secretions must be assessed before extubation is considered.

464 VENTILATION, MECHANICAL / Ventilator-Associated Pneumonia

A trial of spontaneous respiration, which can be performed as a 1/2–2 h trial on a T-piece or low pressure support (5–10 cmH2O), is commonly used to identify patients suitable for extubation. The patient is monitored for signs such as tachypnea, tachycardia, oxygen desaturation, diaphoresis, or clinical fatigue. Recent studies comparing various methods of weaning demonstrated that weaning failure was less common and weaning duration was shorter when a daily trial of spontaneous breathing was used rather than gradual reduction in ventilatory support. See also: Acute Respiratory Distress Syndrome. Ventilation, Mechanical: Noninvasive Ventilation; Ventilator-Associated Pneumonia.

Further Reading Brower RG, Ware LB, Berthiaume Y, and Matthay MA (2001) Treatment of ARDS. Chest 120: 1347–1367. Dos Santos CC and Slutsky AS (2000) Invited review: mechanisms of ventilator-induced lung injury: a perspective. Journal of Applied Physiology 89: 1645–1655. MacIntyre NR, Cook DJ, Ely EW, et al. (2001) Evidence-based guidelines for weaning and discontinuing ventilatory support. Chest 120(supplement 6): 375S–395S. Marini JJ and Gattinoni L (2004) Ventilatory management of acute respiratory distress syndrome: a consensus of two. Critical Care Medicine 32: 250–252. Plant PK and Elliott MW (2003) Chronic obstructive pulmonary disease. 9: management of ventilatory failure in COPD. Thorax 58: 537–542. Plotz FB, Slutsky AS, van Vught AJ, and Heijnen CJ (2004) Ventilator-induced lung injury and multiple system organ failure: a critical review of facts and hypotheses. Intensive Care Medicine 30: 1865–1872. Singh JM and Stewart TE (2003) High-frequency oscillatory ventilation in adults with acute respiratory distress syndrome. Current Opinion in Critical Care 9: 28–32. The Acute Respiratory Distress Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. New England Journal of Medicine 342: 1301–1308. Tobin MJ (2001) Advances in mechanical ventilation. New England Journal of Medicine 344: 1986–1996.

Ventilator-Associated Pneumonia G Bellingan and A Al-Khafaji, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Ventilator-associated pneumonia (VAP) specifically refers to pneumonia developing in a mechanically ventilated patient more than 48 h after intubation or tracheostomy. The gold

standard for the diagnosis of VAP remains elusive; however, it has become clear that standardization of clinical definitions has harmonized the approach to this condition. VAP prolongs stay in the intensive care unit (ICU) and appears to be an independent determinant of mortality in critically ill patients. There are numerous well-described risk factors for developing VAP and data suggest that the application of management strategies aimed at shortening the duration of mechanical ventilation, prevention of aerodigestive tract colonization, and aspiration of contaminated secretions may prevent VAP and could result in improved patient outcomes. Treatment hinges on choosing an effective antibiotic from the outset and early stopping of antibiotics that are no longer needed.

Diagnosis As a gold standard the diagnosis of pneumonia should be confirmed histologically with inflammation involving a number of airspaces and confirmatory microbiological cultures. Similarly, positive cultures from blood or pleural fluid with corroborating clinical findings in the absence of other explanations are also taken as gold standard. It is seldom justified clinically however to take tissue histology given the invasive nature of lung biopsy and even of thoracocentesis. Similarly blood and pleural cultures are commonly negative and thus unhelpful. Clinical Scoring in Diagnosis

It is for these reasons that ventilator-associated pneumonia (VAP; defined as nosocomial pneumonia in patients on mechanical ventilation (via endotracheal tube or tracheostomy) for more than 48 h) has been diagnosed for many years by clinical criteria that include the appearance of a new or progressive pulmonary infiltrates on chest radiography, together with two or more of the following: fever (core temperature of more than 38.31C), leukocytosis (more than 10 000 mm–3) or leukopenia (less than 4000 mm–3), and purulent tracheobronchial secretions. These criteria are however known to be flawed as there are multiple noninfective causes both of radiological shadowing and of fever whilst tracheal aspirates are commonly simply colonized with bacteria. Even new pulmonary cavitation or a positive response to antibiotics may be misleading findings. Attempts have been made to improve the accuracy of clinical diagnosis, for example using the clinical pulmonary infection score (CPIS) first described by Pugin et al. in 1991 and modified by Singh and colleagues in 2002 and Brun–Buisson’s group in 2003. These scores include six variables, and scores of more than 6 are taken to suggest VAP (Table 1). Interestingly, these approaches score the actual level of oxygenation rather than reflect any recent deterioration in oxygenation which could reduce their applicability, especially for patients with adult respiratory

VENTILATION, MECHANICAL / Ventilator-Associated Pneumonia 465 Table 1 The modified clinical pulmonary infection score Points

0

1

2

Tracheal secretions Chest X-ray infiltrates Temperature (1C) Leukocytes count (per mm3) PaO2/FiO2 (mmHg) Microbiology

Rare No infiltrate 436.5 or o38.4 44000 or o11 000 4240 or ARDS Negative

Abundant Diffused 438.5 or o38.9 o4000 or 411 000

Abundant þ purulent Localized 439 or o36 4500 band forms o240 and no ARDS Positive

distress syndrome (ARDS). Overall the performance of such scores has been somewhat disappointing as, despite sensitivities of approximately 90%, their specificity may be less than 50%. Specificity can be improved by incorporation of retrospective criteria such as response to antibiotics or absence of alternative sources of sepsis although these cannot contribute to prospective clinical decision making. Brochoscopic Microbiological Diagnosis

Two bronchoscopic techniques have been introduced for the accurate diagnosis of VAP. The protected specimen brush (PSB) and bronchoalveolar lavage (BAL). PSB collects 0.001 ml of lower respiratory tract secretions using a plugged telescoping catheter brush and is designed to avoid contamination from saliva and tracheal secretions invariably encountered during fiberoptic bronchoscopy. It is technically more demanding both from the operator and from laboratory quantification aspects. In the absence of antibiotic treatment, PSB has a diagnostic threshold of 4103 CFU ml
1, whereas BAL, an unprotected technique, has a diagnostic threshold of 4105 CFU ml
1. Pooled results from a number of studies show that PSB has a sensitivity and specificity of approximately 90%, whereas BAL has a sensitivity reported to be between 53% and 93% and specificity of 60 up to 100% in the diagnosis of VAP. Detection of 45% of neutrophils or macrophages with intracellular organisms on a Wright– Giemsa stain of a smear of cytocentrifuged BAL fluid is also diagnostic of VAP. Post-mortem studies of such invasive sampling in animals and studies in humans demonstrated improved accuracy over clinical criteria alone, but the incidence of false positive and false negative rates remains significant in many (though not all) studies. Moreover, although there was an evident association between an increasing bacterial load in cultures of lung tissue and a greater degree of histologic damage, no quantitative threshold could be determined that would permit the separation of normal lung tissue from that showing bronchitis or pneumonia. Indeed, some argue that the presence of microorganisms even distal airways may represent colonization rather

than infection in some circumstances. These features, coupled with the technical aspects of quantitative cultures and potential morbidity from lavage, mean that although these methods certainly improve the accuracy of diagnosis of VAP, they have not been widely adopted. Nonbronchoscopic, Nondirected Mini-BAL in Diagnosis

Blind sampling using mini-BAL is far less invasive, cheaper and safer, and, as VAP is usually not focal, may be a valid approach as 85% or more of blind sampling will be from the right side. Such noninvasive approaches have been compared with invasive approaches (bronchoscopically retrieved samples of respiratory secretions) in four prospective studies evaluating the effects of their use on morbidity, the use of antimicrobial drugs, and mortality. One study comparing noninvasively retrieved qualitative cultures of tracheobronchial secretions with an invasive bronchoscopic approach involving quantitative cultures showed a significant advantage of the latter in terms of lower 14-day mortality, more rapid resolution of organ failure, and less antibiotic use. However, the three other studies, which used quantitative cultures with both invasive and noninvasive approaches, showed no difference between the two strategies. These studies had different designs, as well as some methodologic limitations, including an inappropriate selection of diagnostic techniques for comparison, insufficient control for previous antimicrobial treatment, inconsistent ways of managing antibiotic treatment in patients who had negative microbiologic assays, and insufficient power to detect clinically important differences among alternative strategies. Nevertheless, it is accepted that nonbronchoscopic, nondirected mini-BAL techniques have overall sensitivities and specificities of 60–100% and are comparable with bronchoscopic methods. Most clinicians still believe that, in the absence of local enthusiasts, the decision to treat should usually be based on simple clinical criteria or the CPIS, along with consideration of the short-term evolution of the clinical condition of the patient, after a vigorous

466 VENTILATION, MECHANICAL / Ventilator-Associated Pneumonia

search for noninfectious conditions that may mimic VAP and for alternative nonpulmonary sites of infection. With this approach it must be remembered that there remains a considerable proportion of patients for whom there is a discordance between the clinical diagnosis and the microbiologic results; in such patients, the risk of overtreatment has to be weighed against the risk of insufficient antimicrobial treatment.

Incidence VAP has been reported to be responsible for approximately half of the infections acquired in the ICU although these figures employed conventional clinical criteria alone with resultant over diagnosis. The incidence of VAP is now more accepted to be approximately 10%, a figure reported from studies using both PSB technique and those using stringent clinical diagnostic criteria alone. Epidemiologic investigations have shown that rates vary according to clinical population and type of critical care but are in the order of 7.5–34 per 1000 ventilator days. According to data from the National Nosocomial Infection Surveillance System, VAP occurs at a rate of 7.5 per 1000 ventilator days in medical ICUs and 13.6 per 1000 ventilator days in surgical ICUs. Viewed another way, crude VAP rates are 1–3% per day of mechanical ventilation, although some studies suggest the incidence is maximal between days 5 and 15 while others suggest a linear increase over the first 30 days. Crude mortality rates of 10–40% are accepted, with attributable mortality rates of between 5% and 27%, although these could be higher for organisms such as Pseudomonas or Acinetobacter. Certainly hospital length of stay and cost are both increased in patients who develop VAP.

Epidemiology and Etiology Infectious organisms that result in VAP are generally different from those that are associated with community-acquired pneumonia. Early onset pneumonia in mechanically ventilated patients (o4 days) may be caused by Streptococcus pneumonia or Moraxella catarrhalis and many do not typically consider these to represent true VAP. Gram-negative organisms, typically Pseudomonas aeruginosa, Enterobacter species, Klebsiella pneumoniae, and Acinetobacter species, are responsible for the majority of late-onset VAP (44 days of mechanical ventilation) and are commonly antibiotic resistant. Prolonged intubation and mechanical ventilation are almost invariably associated with colonization of the tracheo-bronchial

tree with multiple Gram-negative aerobes. It is important to recognize however that, despite positive cultures from tracheal aspirates, such organisms are mostly absent when ‘gold standard’ tissue samples are examined for VAP. Hence, tracheal aspirates commonly have a high sensitivity but very low specificity, significantly diminishing their utility in establishing the cause of VAP. The absence of Gramnegative bacteria in tracheal aspirates would however exclude them as a cause of VAP. Methicillin-resistant Staphylococcus aureus (MRSA) is assuming a greater prominence in certain countries, especially the UK, parts of Europe, Australia, and Brazil. In other countries including the Netherlands and Scandinavia, its incidence is vanishingly low; the reasons for these differences are unclear. Anaerobic bacteria seem to play a very limited role in VAP.

Risk Factors and Prevention A wide range of independent risk factors have been associated with VAP. These relate to the patients premorbid state, the severity of the injury or illness leading to mechanical ventilation, and to management strategies on the ICU (see Table 2). Table 2 Management strategies on the ICU Pre ICU admission

In ICU

Age over 60 years

H2 blockers7antacids Gastric colonization and pH

Premorbid state Low serum albumin Pulmonary disease COPD Neuromuscular disease

Supine head position Nasogastric tube

Initiating injury Trauma Severity of illness Hypotension Coma or impaired consciousness Large-volume gastric aspiration ARDS

Upper respiratory tract colonization Nasal intubation Sinusitis Reintubation Prolonged mechanical ventilation Subglottic suction Heated water humidification systems Positive end-expiratory pressure Frequent ventilator circuit changes

Surgery Transfusion of 44 units of blood products Season Winter

Intracranial pressure monitoring Prior antibiotic or no antibiotic therapy

VENTILATION, MECHANICAL / Ventilator-Associated Pneumonia 467

Colonization of the oropharynx and the stomach with potentially pathogenic organisms typically precedes the development of VAP, and it is generally believed that the pathogenesis of VAP involves microaspiration of oropharyngeal or gastric secretions contaminated with these organisms. Prevention of VAP has now become a fundamental issue and is promoted strongly, including as part of the ‘surviving sepsis’ campaign. As intubation and mechanical ventilation are prerequisites for the development of VAP, unnecessary intubation should be avoided at all times where noninvasive ventilation can be used as an alternative. This should be strongly considered as a number of studies report a real reduction in VAP, shorter ICU stay, and improved survival. Patients with acute exacerbation of chronic obstructive pulmonary disease, immunosuppressed patients with pulmonary infiltrates and respiratory failure, and even some patients with acute lung injury with hypoxic respiratory failure have been shown to benefit from trying to avoid mechanical ventilation. If intubation and mechanical ventilation are indicated, then strategies aimed at shortening the duration of mechanical ventilation should be implemented. Protocols aimed at limiting the administration of sedation and accelerated weaning from mechanical ventilation using either weaning protocols or early attempts at spontaneous breathing should be used. To successfully prevent the development of VAP and its subsequent complications, two general approaches are needed: prevention of aerodigestive tract colonization and prevention of aspiration of contaminated secretions. Prevention of Aerodigestive Tract Colonization

Avoidance of unnecessary antibiotics Prior antibiotic therapy is independently associated with VAP. The prolonged use of prophylactic antibiotics in trauma patients, although associated with the delayed onset of VAP, led to an increased incidence of pneumonia caused by antibiotic-resistant Gram-negatives and an increased overall incidence of nosocomial infections. Hence, unnecessary antibiotics should be avoided. One strategy designed specifically to minimize antimicrobial resistance while ensuring adequate empirical antimicrobial coverage is the practice of a scheduled switch of antibiotic class. Such an antibiotic rotation strategy has been associated with reductions in the occurrence of VAP, especially when preceded by emergence of resistance to the baseline antibiotic class. Overall, however, antibiotic rotation policies have been inconsistent in their efficacy, both on limiting development of resistance locally and on

occurrence of nosocomial infections including VAP. Practices focusing on antimicrobial heterogeneity without the application of other strategies aimed at preventing the emergence of antibiotic-resistant infections (e.g., short-course antibiotic therapy and appropriate hand disinfection) have the risk of increasing resistance to subsequently introduced antimicrobial drug classes. Selective decontamination of the digestive tract Short courses of prophylactic antibiotics may play a role in the prevention of VAP among specific highrisk populations including patients with severe head injury, coma, and high-risk surgical procedures. Selective decontamination of the digestive tract (SDD), which combines oropharyngeal decontamination with gastric decontamination and systemic administration of antimicrobials, has been shown in several meta-analyses to lead to significant reductions in the incidence of VAP and lower rates of hospital mortality, especially among surgical patients. This was confirmed in a recent study of SDD among surgical patients with a low prevalence of colonization with resistant bacteria (e.g., vancomycin-resistant enterococci, methicillin-resistant S. aureus (MRSA)). Here the authors found a statistically significant reduction in mortality rate without increasing overall colonization with resistant bacteria. Others have applied focused antimicrobial oropharyngeal decontamination only and have shown a reduction in nosocomial pneumonia, especially with MRSA infection. SDD carries the potential risk of promoting more widespread antibiotic resistance although these concerns do not seem to be borne out in practice thus far. Despite good evidence suggesting efficacy, SDD has not gained widespread acceptance as yet. Avoidance of unnecessary stress ulcer prophylaxis Both histamine antagonists and proton pump inhibitors are frequently used for stress ulcer prophylaxis. The risk of VAP is increased by decreasing intragastric acidity, which can result in greater gastric colonization with pathogenic bacteria. Additionally, gastric volumes may be increased with the administration of antacids promoting aspiration and the development of VAP. Sucralfate does not decrease intragastric acidity and does not increase gastric volume significantly but it is potentially less active for stress ulcer prophylaxis. The data comparing stress ulcer prophylaxis with sucralfate and histamine antagonists suggest a decreased incidence of VAP with sucralfate. Clinicians must weight the benefit of sucralfate in limiting the development of VAP against a potential decrease in protection against gastrointestinal bleeding compared with histamine antagonists.

468 VENTILATION, MECHANICAL / Ventilator-Associated Pneumonia Prevention of Aspiration of Contaminated Secretions

Semirecumbent positioning Among mechanically ventilated patients, supine positioning has been shown to be an independent risk factor for the development of VAP. Studies employing radiolabeled enteral feeding solutions in ventilated patients have demonstrated that aspiration of gastric contents occurs to a significantly greater degree when patients are in the supine position compared with the semirecumbent position. Additional benefit may be obtained from a reduction in bibasal atelectasis. One randomized trial demonstrated a threefold reduction in the incidence of VAP and a trend toward a reduced hospital mortality rate in patients treated in the semirecumbent position (451) compared with patients treated completely supine. Semirecumbent patient positioning is a low-cost, low-risk approach to preventing VAP and should be implemented in all eligible patients. Minimization of gastric distension Avoidance of gastric overdistention may reduce the occurrence of VAP and certain measures can be carried out to limit this, including reducing the use of narcotics and anticholinergic agents, monitoring gastric residual volumes following the administration of enteral feeding, using gastrointestinal motility agents, supplying enteral nutrition with smaller bore feeding tubes, and administering feeding solutions directly into the small bowel instead of the stomach. Small bowel compared with gastric feeding is associated with a reduction in gastroesophageal regurgitation, an increase in protein and calories delivery to patients, a shorter time to achieving the target dose of nutrition and reduction in VAP. However, no studies have demonstrated a mortality benefit when using small bowel feeding compared to gastric feeding. Endotracheal tube and ventilator circuitry Oral intubation significantly reduces nosocomial infection compared with nasal intubation; however, this may be through a reduction in sinusitis rather than VAP, sinusitis itself being associated with the development of VAP. Modified endotracheal tubes with a separate dorsal lumen allowing continuous aspiration of the subglottic secretions have been developed and evaluated. Four randomized, controlled studies all show a beneficial effect of continuous suctioning of subglottic secretions on the incidence of VAP, although none showed a corresponding effect on mortality rate, length of stay in the ICU, or even duration of mechanical ventilation.

Avoidance of ventilator circuit changes The importance of ventilator tubing in the development of pneumonia among mechanically ventilated patients has been extensively investigated. Studies suggest that heated water humidifiers may be associated with a greater incidence of nosocomial pneumonias than heat and moisture exchangers (HMEs). This is probably related to a reduction in condensate accumulation in the tubing with HMEs. A scheduled approach to the removal of tubing condensate, with its proper disposal, should be protocolized for heated water humidifiers. Trials examining the frequency with which circuitry should be changed have shown that neither the rate of bacterial contamination of inspiratory-phase gas nor the incidence of pneumonia was significantly altered when tubing was changed more frequently. Therefore, previous recommendations to change ventilator circuits routinely on the basis of duration of use should be abandoned in favor of a policy whereby circuitry (including HMEs) are changed between patients and otherwise when there is visual contamination with blood, vomit, or purulent secretions. Closed tracheal suction systems also probably offer an advantage although this was not confirmed in all studies. Other Approaches

Hand hygiene Transmission of the causative microorganisms of VAP to patients frequently occurs via healthcare personnel’s hands that become contaminated or transiently colonized with the microorganisms. Procedures such as tracheal suctioning and manipulation of ventilator circuits or endotracheal tubes increase the opportunity for cross-contamination with these pathogens. The risk of cross-contamination can be reduced by using aseptic techniques, closed circuit suction systems, sterile or disinfected equipment when appropriate and eliminating pathogens from the hands of personnel. The use of alcohol-based foams and lotions allows hand disinfection to occur more efficiently. The use of these methods has been advocated as a means to increase compliance among healthcare providers with hand disinfection before patient contacts. Appropriate intensive care unit staffing Nurse:patient staffing levels differ between countries and may influence the length of stay of patients in ICUs. In the US mean hours of nursing care have been reported at approximately 11 h. Importantly, increases in this are associated with a reduction in nosocomial pneumonia. In other countries such as the UK which have nearly a 1:1 nurse:patient ratio, the influence staffing on VAP is not clear.

VENTILATION, MECHANICAL / Ventilator-Associated Pneumonia 469

Oscillating beds Immobility in critically ill patients may lead to atelectasis and impaired clearance of bronchopulmonary secretions, thereby potentially increasing the risk for VAP. Although oscillating beds have no apparent benefit in general populations of medical patients, there is a good evidence that this practice may be effective in surgical patients or patients with neurologic problems. Use of oscillating beds in these select patient populations may be considered to prevent VAP; however, it is not clear how much their effect is simply on reduced atelectasis rather than specifically on prevention of VAP, especially when the difficulties in accurate diagnosis are considered.

should be linked with regular review which promotes stopping of antibiotics that are no longer needed as there is increasing evidence that short courses (8 days or even less) are just as effective as traditional 15 days of treatment and probably reduce resistance.

Biofilm prevention technology Bacteria settling on surfaces such as endotracheal tubes can form biofilms that may form a protective microenvironment. Certain bacteria such as Pseudomonas species appear to be more capable of forming such biofilms, especially in the presence of abnormal airway mucosa as exists in patients with cystic fibrosis. Bacterial biofilm formation can be prevented using surface coatings that impede bacterial adherence and also the use of nebulized antibiotics. None of these approaches has been subjected to clinical investigation.

See also: Acute Respiratory Distress Syndrome. Atelectasis. Bronchoalveolar Lavage. Bronchoscopy, General and Interventional. Pleural Effusions: Parapneumonic Effusion and Empyema. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Nosocomial. Ventilation, Mechanical: Positive Pressure Ventilation.

Avoidance of red blood cell transfusions The transfusion of packed red blood cells has been associated with serious nosocomial infections including VAP. In one study, transfusion of packed red blood cells was found to be an independent risk factor for the development of VAP, with a significant dose relationship. These data suggest that the unnecessary transfusion of packed red blood cells should be avoided in hospitalized patients in order to reduce the risk of VAP.

Treatment Clinical evidence suggests that inadequate and delayed initial antibiotic treatment of critically ill patients with suspected infection is associated with increased morbidity and mortality. On the other hand, overtreatment with antimicrobial drugs can have a substantial adverse effect on the prognosis because of adverse drug related events and the selection of resistant pathogens. Knowledge of local microbiological resistance patterns, close liaison between ICU staff and clinical microbiology, and prescribing according to individual patient characteristics all allow effective antibiotics to be chosen from the outset. It is reasonable to start with broad spectrum antibiotic and subsequently narrow the coverage when culture results are available. This

Conclusion Although diagnosis if VAP remains challenging, modern approaches now justifiably promote prevention, especially in protocolised approaches or ‘care bundles’. These principles, coupled with focused short course of antibiotics for treatment, remain the cornerstones in the management of VAP.

Further Reading American Thoracic Society (1996) Hospital-acquired pneumonia in adults: diagnosis, assessment of severity, initial antimicrobial therapy, and preventive strategies. American Journal of Respiratory and Critical Care Medicine 153: 1711–1725. Bellingan GJ (1999) Sputum sampling and bronchoalveolar lavage. In: Webb, Shapiro, Singer, and Suter (eds.) Oxford Textbook of Critical Care, pp. 1177–1179. Oxford: Oxford University Press. Bregeon F, Papazian L, Thomas P, et al. (2000) Diagnostic accuracy of protected catheter sampling in ventilator-associated bacterial pneumonia. European Respiratory Journal 16: 969– 975. Chastre J and Fagon J-Y (2002) Ventilator-associated pneumonia. American Journal of Respiratory and Critical Care Medicine 165: 867–903. Chastre J, Wolff M, Fagon JY, et al. (2003) Comparison of 8 vs 15 days of antibiotic therapy for ventilator-associated pneumonia in adults. Journal of the American Medical Association 290: 2588–2598. Collard HR, Saint S, and Matthay MA (2003) Prevention of ventilator-associated pneumonia: an evidence-based systematic review. Annals of Internal Medicine 138(6): 494–501. Georges H, Leroy O, Guery B, et al. (2000) Predisposing factors for nosocomial pneumonia in patients receiving mechanical ventilation and requiring tracheotomy. Chest 118: 767–774. Girou E, Schortgen F, Delclaux C, et al. (2000) Association of noninvasive ventilation with nosocomial infections and survival in critically ill patients. Journal of the American Medical Association 284: 2376–2378. Kollef MH (2004) Prevention of hospital-associated pneumonia and ventilator-associated pneumonia. Critical Care Medicine 32(6): 1396–1405. Micek ST, Ward S, Fraser VJ, and Kollef MH (2004) A randomized controlled trial of an antibiotic discontinuation policy for clinically suspected ventilator-associated pneumonia. Chest 125(5): 1600–1602.

470 VENTILATION, PERFUSION MATCHING Papazian L, Thomas P, Garbe L, et al. (1995) Bronchoscopic or blind sampling techniques for the diagnosis of ventilator-associated pneumonia. American Journal of Respiratory and Critical Care Medicine 152: 1982–1991. Rello J, Ollendorf DA, Oster G, et al. (2002) Epidemiology and outcomes of ventilator-associated pneumonia in a large US database. Chest 122: 2115–2121. Richards MJ, Edwards JR, Culver DH, et al. (1999) Nosocomial infections in medical intensive care units in the United States:

National Nosocomial Infections Surveillance System. Critical Care Medicine 27: 887–892. Torres A, Carlet J, Bouza E, et al. (2001) Ventilator-associated pneumonia. European task force on ventilator-associated pneumonia. European Respiratory Journal 17: 1034–1045. Vincent JL, Bihari DJ, Suter PM, et al. (1995) The prevalence of nosocomial infection in intensive care units in Europe: results of the European prevalence of infection in intensive care (EPIC) study. Journal of the American Medical Association 274: 639–644.

VENTILATION, PERFUSION MATCHING P D Wagner, University of California – San Diego, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The lungs exist primarily to exchange gases between the environment and the blood. This is accomplished by three linked processes: convective transport of air to the alveoli (ventilation), diffusion of gas molecules between alveolar gas and pulmonary capillary blood, and convective transport in blood by cardiac pumping (perfusion). The lung is unique because these processes are accurately described by simple mass balance considerations, ˙ ) is the which show that the ventilation/perfusion ratio (V˙A/Q ˙ is not ˙ A/Q key determinant of alveolar gas exchange. When the V ˙ inequality is the same everywhere throughout the lungs, V˙A/Q said to exist, causing hypoxemia, hypercapnia, and reduced O2 uptake and CO2 elimination. This compromises tissue metabolism, and may cause tissue hypoxia and acidosis. Clinical ˙ inequality is thus important, and uses three assessment of V˙A/Q measures: alveolar–arterial PO2 difference, physiological deadspace, and physiological shunt. The body has three principal ˙ inequality. First, ˙ A/Q ways of compensating for continuing V ˙ O2 and mixed venous PO2 falls and PCO2 rises, usually restoring V V˙CO2, but causing further hypoxemia and hypercapnia. Second, ventilation increases, stimulated partly by hypoxemia and hypercapnia. While usually effective in normalizing arterial PCO2 , PO2 often remains low. Third, cardiac output may rise, increasing mixed venous, and thus arterial, PO2 .

Description Basic Principles

The principal function of the lungs is the uptake of O2 from the atmosphere into the circulating blood, and the elimination of CO2 produced by metabolism. This requires three major gas transport processes: ventilation (see Ventilation: Overview) to bring inspired air to the alveoli, and alveolar gas to the atmosphere; diffusion (see Diffusion of Gases) to transfer gases across the alveolar–capillary blood–gas barrier; and blood flow (see Pulmonary Circulation) to transport gases in blood between the lungs and

body tissues. Other gases, including inhaled pollutants and anesthetics, are exchanged similarly. The same principles of gas exchange apply to all gases, whether they form chemical species in blood or are carried simply in physical solution, and for both uptake and elimination. Except for a few situations, the process of diffusion reaches equilibration. That is, alveolar and endcapillary partial pressures of gases are not significantly different. Here it will be considered that diffusion equilibration exists. Ventilation is tidal, and tidal volume is usually small (B500 ml) compared to alveolar volume (B3000 ml). Because anatomical deadspace (the volume of the conducting airways) is B150 ml, the amount of fresh air reaching alveoli with each breath is only 500–150 or B350 ml. Each breath thus ‘‘tops up’’ the alveolar volume by only about 10%. Respiratory frequency is B15 breaths min
1. This pattern of small but frequent breaths causes alveolar gas concentrations to vary minimally within each breath. They are therefore taken to be constant (under steady-state conditions). Despite pulsatile pulmonary blood flow, endcapillary and arterial blood gas concentrations are also taken to be constant in time. Because the amounts of inhaled gases depend on ventilation, and the amounts transported in the blood depend on blood flow, it is apparent that ventilation and blood flow critically affect the amounts of gas exchanged, and also the alveolar and blood concentrations and partial pressures. These concepts can be considered more concretely by describing gas exchange using the laws of mass balance. These laws acknowledge that all molecules of a gas removed from alveolar gas during gas uptake appear in the blood, while during gas elimination, all molecules removed from the blood appear in the alveolar gas. Ventilation, Perfusion, and Gas Exchange

Using O2 as the example, the laws of mass balance are now applied to steady-state gas exchange to

VENTILATION, PERFUSION MATCHING 471

quantify the relationships between ventilation, blood flow, and alveolar (and endcapillary) concentrations (and partial pressures). The volume of O2 inhaled per minute is the product of the total inspired alveolar ventilation (V˙I) and the fractional concentration of O2 in inspired gas (FIO2 ): ½1 Volume inhaled ¼V’ I  FIO2

law of partial pressures. V’ I and V’ A are almost identical, differing only when the amount of O2 taken up does not equal the amount of CO2 eliminated. Normally, V’ O2 is B300 ml min
1, V’ CO2 is B240 ml min
1. The difference, 60 ml min
1, is small in comparison to normal alveolar ventilation, which is usually 5–6 l min
1. While taking V’ I ¼ V’ A will introduce a small error, for ease of presentation, equality will be assumed so that

The volume of O2 exhaled per minute is the product of the total expired alveolar ventilation (V˙A) and the fractional concentration of O2 in alveolar gas (FAO2 ):

’ ¼ k  ½Cc0
CVO =½PIO
PAO  V’ A=Q 2 2 2 O2

Volume exhaled ¼V’ A  FAO2

½2

The difference between these must be the amount of O2 transferred from the atmosphere to the pulmonary capillary blood, that is, the V’ O2 : V’ O2 ¼ V’ I  FIO2
V’ A  FAO2

½3

We can apply identical considerations to the pulmonary circulation. The volume of O2 that leaves the capillaries en route to the left atrium per minute is ’ and the the product of pulmonary blood flow (Q) concentration of O2 in that blood (Cc0O2 ): ’  Cc0 Volume leaving ¼ Q O2

½4

However, the blood entering the pulmonary circulation carries O2 , and the volume of O2 in this venous ’ and blood entering per minute is the product of Q concentration of O2 in the venous blood: ’  CVO Volume entering ¼ Q 2

½5

The difference between the volumes of O2 leaving and entering the pulmonary circulation/min is the amount of O2 taken up from the alveolar gas (V’ O2 ): ’  Cc0
Q ’  CVO V’ O2 ¼ Q 2 O2

½6

Equations [3] and [6] both describe the amount/min of O2 taken up by the lungs (which in a steadystate equals tissue metabolic rate), and may thus be combined: ’  Cc0
Q ’  CVO V I  FIO2
V’ A  FAO2 ¼ Q 2 O2 Which may be rearranged to ’ ¼ ½Cc0
CVO =½ðV’ I=V’ AÞ  FIO
FAO  ½7 V’ A=Q 2 2 2 O2 or ’ ¼ k  ½Cc0
CVO =½ðV’ I=V’ AÞ V’ A=Q 2 O2  PIO2
PAO2 

½8

where k allows fractional concentrations (F) to be replaced by partial pressures (P) based on Dalton’s

½9

Now because alveolar and endcapillary PO2 values are taken to be the same (see above and Diffusion of Gases), Cc0O2 is the O2 concentration corresponding to the alveolar PO2, PAO2 . This equation therefore states: for a given composition of the venous blood (CVO2 ) and inspired gas (PIO2 ), the alveolar PO2 (PAO2 ) is uniquely determined by the ratio of venti’ lation to blood flow (V’ A=Q). ˙ In a region of lung having a low value of V˙A/Q (e.g., partial airway obstruction), PAO2 must be low. If airway obstruction is complete, V˙A is zero, and local PO2 equals that of venous blood. If the local ˙ ratio is high (e.g., partial vascular obstruction), V˙A/Q PAO2 will be high. If vascular obstruction is complete, ˙ is zero, and PAO2 must equal that of inspired gas. Q Identical principles may be applied to all other gases, and similar relationships will be obtained. For CO2 this becomes ’ ¼ k  ½CVCO
Cc0 =½PACO
PICO  V’ A=Q 2 2 2 O2

½10

˙ and PAO The unique relationship between V˙A/Q 2 ˙ ˙ (and between VA/Q and PACO2 ) is shown in Figure 1. Normal total alveolar ventilation and pulmonary blood flow are each about 5–6 l min
1. Therefore, ˙ ratio is B1.0. Figure 1 shows the overall lung V˙A/Q that PAO2 is B100 mmHg and PACO2 is B40 mmHg ˙ ratio. Hence, these values are those at that V˙A/Q expected in a perfectly homogeneous lung with a ˙ ratio. They are very close to what is normal V˙A/Q seen in young normal subjects, suggesting close matching of ventilation to blood flow throughout the many alveoli in their lungs.

Ventilation/Perfusion Inequality and Gas Exchange All normal subjects have what is called ventilation/ ˙ ) inequality. This is defined as the ˙ A/Q perfusion (V ˙ ratio is not the same everysituation where the V˙A/Q where throughout the lungs. It results from both gravitational and nongravitational influences on how both ventilation and blood flow are distributed (see Pulmonary Circulation. Ventilation: Overview).

472 VENTILATION, PERFUSION MATCHING 50 Alveolar P CO2 (mmHg)

Alveolar PO2 (mmHg)

140 120 100 O2

80 60 40 20 0 0.001

(a)

0.01

0.1

1

10

100

40 30 CO2

20 10 0 0.001

1000 (b)

Ventilation/perfusion ratio

0.01 0.1 1 10 100 Ventilation/perfusion ratio

1000

(c)

140 Alveolar PCO2 (mmHg)

Alveolar PO2 & P CO2 (mmHg)

The O2–CO2 diagram 50 O2

120 100 80 60 40

CO2

20 0 0.001

40 30 20 10 0

0.01

0.1 1 10 100 Ventilation/perfusion ratio

0

1000 (d)

20

40 60 80 100 120 140 160 Alveolar PO2 (mmHg)

˙ ) ratio, based on eqn [8]. When the V˙A/Q ˙ ratio is normal Figure 1 (a) Alveolar PO2 (P AO2 ) depends on the ventilation/perfusion (V˙A/Q ˙ is both very low and very high, P AO2 is insensitive to V˙A/Q ˙ . Part (b) shows the corresponding ˙ A/Q (B1), P AO2 ¼ 100 mmHg. When V relationship for P AO2 , and part (c) superimposes the two for comparison. Part (d) plots P ACO2 against P AO2 , creating the O2–CO2 ˙ . The left end of the line represents a V ˙ of 0, reflecting ˙ A/Q diagram, where each point on the line corresponds to a unique value of V˙A/Q ˙. ˙ A/Q mixed venous blood composition; the right end similarly reflects inspired gas and an infinitely high V

˙ inequality exists, gas exchange becomes ˙ A/Q When V inefficient. Specifically, arterial PO2 falls, arterial PCO2 rises, and pulmonary V’ O2 and V’ CO2 both fall, failing to meet the body’s metabolic needs to supply O2 and eliminate CO2. Unless V’ O2 and V’ CO2 are restored, either by removing the cause of inequality or by the compensatory mechanisms discussed below, tissue damage will ensue. ˙ inequality causes these changes is conHow V˙A/Q veniently illustrated in a simple model of inequality in a ‘two alveolus’ lung. Figure 2(a) depicts a perfect lung for reference, and two examples of inequality. In Figure 2(b) an inhaled foreign object has reduced ventilation in one alveolus by 90%; in Figure 2(c) a blood clot has reduced blood flow in one alveolus similarly by 90%. In both cases, this is the only perturbation. Thus, total alveolar ventilation and pulmonary blood flow remain normal, and the distribution of blood flow in Figure 2(b) and of ventilation in Figure 2(c) are unchanged from control. Moreover, venous blood composition is the same in all three cases. Therefore, these examples illustrate pure effects of inequality prior to any compensatory changes (described below) that would normally occur.

PAO2 and PACO2 in each alveolus in Figure 2 were obtained from the relationships in Figure 1. In ˙ ratio ˙ A/Q Figure 2(a), each alveolus has the same V of 1.0, and thus has the same PAO2 and PACO2 . As the expired gas streams from the two alveoli mix, overall exhaled PO2 must be the same as PAO2 in each alveolus, that is, 100 mmHg. So too, mixed exhaled PACO2 must be 40 mmHg. Turning to the vascular side, each ‘pulmonary vein’ carries blood at the same PO2 of 100 mmHg and PCO2 of 40 mmHg. Thus, mixed arterial blood must also have these values. Consequently, there is no difference between mixed alveolar and mixed arterial PO2 (or PCO2 ) in this perfect lung. V’ O2 is 300 ml min
1 and V’ CO2 240 ml min
1, numbers determined by inserting PAO2 ¼ 100 into eqn [3], and PACO2 ¼ 40 into the corresponding equation for CO2. In Figure 2(b), exemplifying partial airway obstruction, the same analysis is now applied, using Figure 1 to determine the alveolar PO2 and PCO2 for the two alveoli. With 90% reduction in ventilation of ˙ ratio becomes 0.1, while the left alveolus, its V˙A/Q redistribution of ventilation to the right alveolus ˙ ratio 1.6. PAO and PACO (Figure 1) makes its V˙A/Q 2 2

VENTILATION, PERFUSION MATCHING 473

VA (total) = 5.2

VA (total) = 5.2

100 (mixed exhaled PO ) 2 40 (mixed exhaled PCO )

Q (total) = 6.0 VO = 300 2 VCO = 240

115 (mixed exhaled PO ) 2 36 (mixed exhaled PCO )

Q (total) = 6.0 VO = 199 2 VCO = 213

2

2

2

2

VA : VA : 2.6 PAO : 2 PACO : 2

Q: VA /Q :

2.6

PAO : 2 PACO :

100 40

100 40

3 0.9

0.3 47 46

2

Q:

3 0.9 100 (mixed arterial PO2) 40 (mixed arterial PCO2)

(a)

VA/Q :

4.9 119 35

3

3

0.1

1.6 58 (mixed arterial PO ) 2 40 (mixed arterial PCO ) 2

(b)

V A (total) = 5.2

108 (mixed exhaled PO ) 2 31 (mixed exhaled PCO )

Q (total) = 6.0 VO

2

= 263

2

VCO = 182 2

VA :

3

PAO : 2 PACO :

141 17

2

Q: VA/Q:

3 75 44

0.3

5.7

10

0.53 75 (mixed arterial PO ) 2 42 (mixed arterial PCO ) 2

(c) Figure 2 (a) This shows a lung divided into two hypothetical alveoli, each receiving 50% of the total alveolar ventilation and perfusion. ˙ ratio, making the lung homogeneous. This lung exchanges normal amounts of O2 and CO2; arterial ˙ A/Q Each therefore has the same V ˙ tenfold. The ˙ A/Q PO2 and PCO2 are 100 and 40 mmHg, respectively. (b) Severe airway obstruction in one airway, reducing alveolar V result is arterial hypoxemia and hypercapnia, and reduced O2 uptake and CO2 elimination. (c) Severe pulmonary vascular obstruction, also causing hypoxemia, hypercapnia, and reduced gas exchange. P AO2 and P ACO2 for each alveolus in all three models are derived from eqns [8] and [10].

are shown. As the two alveoli exhale and their gas streams mix, the mixed exhaled PO2 (PEO2 ) must be the average of the PAO2 values from each, weighted by the amounts of exhaled gas coming from each. This is simply applying principles of mass conservation: PEO2 ¼ ½PAO2 ðleftÞ  V’ AðleftÞ þ PAO2 ðrightÞ  V’ AðrightÞ=V’ AðtotalÞ ¼ 115 mmHg

½11

Similar mixing of the endcapillary blood from the two alveoli produces a mixed arterial PO2 of 58 mmHg. Here, the mixing computation is done using endcapillary O2 concentrations, not partial pressures. The PO2 corresponding to this mixed arterial O2 concentration is then read from the oxygen–hemoglobin dissociation curve (see Oxygen–Hemoglobin Dissociation Curve).

When eqn [3] is used with the mixed expired PO2 above, V’ O2 for the entire lung is seen to be only 199 ml min
1. V’ CO2 is also reduced, to 213 ml min
1. ˙ inequality shows subThus, this model of V˙A/Q stantial arterial hypoxemia and slight hypercapnia (compared to A), and reduced ability to exchange both O2 (34% reduction in V’ O2 ) and CO2 (11% reduction in V’ CO2 ). In Figure 2(c), the model of pulmonary vascular obstruction, an identical analysis shows that mixed exhaled PO2 and PCO2 will be 108 and 31 mmHg respectively; mixed arterial PO2 and PCO2 will be 75 and 42 mmHg, respectively, and V’ O2 and V’ CO2 for the entire lung are both reduced, to 263 (12% reduction) and 182 (24% reduction) mmHg, respectively. The important result is that it does not matter ˙ inequality resides in whether the cause of the V˙A/Q the airways or the vasculature, hypoxemia and hypercapnia will occur, as will a reduction in the amount

474 VENTILATION, PERFUSION MATCHING

of both O2 and CO2 that the lungs can exchange. The absolute effects differ with the nature of the inequality, and in particular the model in Figure 2(b) affects O2 more than CO2 while the model in Figure 2(c) affects CO2 more than O2.

Compensation for Ventilation/Perfusion Inequality The reductions in V’ O2 and V’ CO2 described above prevent the lungs from meeting the tissue metabolic ˙ inequality is not corrected, ˙ A/Q requirements. If the V compensatory mechanisms must be brought into play to restore V’ O2 and V’ CO2 . Three principal mechanisms exist. The first is a fall in venous PO2 (and rise in venous PCO2 ). This happens immediately and requires no conscious effort or energy expenditure. It is effective because prior ˙ inequality, venous PO2 is high to developing V˙A/Q enough (B40 mmHg) to provide enough room for such a fall. This can usually restore the arterio venous O2 concentration difference and therefore V’ O2 (and V’ CO2 ). However, it causes a further fall in arterial PO2 (and rise in arterial PCO2 ). In the model in Figure 2(b), this mechanism can restore the V’ O2 and V’ CO2 of the entire lung. Venous PO2 would have to fall by about 10 mmHg and venous PCO2 would have to increase by about 5 mmHg. Arterial PO2 and PCO2 would now be 48 and 45 mmHg respectively, showing more hypoxemia and hypercapnia than before compensation occurred. In Figure 2(c), corresponding changes restore V’ O2 and V’ CO2 , resulting in venous PO2 and PCO2 values lower by about 2 and higher by about 16 mmHg, respectively. Arterial PO2 falls to 63 and PCO2 rises to 57 mmHg, respectively. While this primary, rapid, automatic process achieves the major objective of restoring both V’ O2 and V’ CO2 , the fall in arterial PO2 and rise in arterial PCO2 put the patient at greater risk of tissue hypoxia and acidosis than even the initial disturbance of ˙ relationships caused. ˙ A/Q V The second compensatory mechanism is an increase in ventilation. This mitigates arterial hypoxemia and hypercapnia, reducing tissue hypoxia and acidosis. The stimuli to increasing ventilation in the ˙ inequality (arterial hypoxemia, acido˙ A/Q face of V sis, and especially hypercapnia) are discussed in the article Ventilation: Control. Depending on the cause ˙ inequality, increased ventilation may not be ˙ A/Q of V possible, as for example when severe chest trauma is responsible. But when in the models of Figure 2 ventilation is progressively increased, arterial PO2 and PCO2 gradually improve. Since V’ O2 and V’ CO2 have already been restored by the first compensatory

mechanism, they remain normal as ventilation is raised. Figure 3 shows the effects of ventilatory compensation in the two models of Figure 2. Note that arterial PCO2 is much easier to normalize than is arterial PO2 , especially in the model of airway obstruc˙ ratios. This directly reflects tion with very low V˙A/Q the differences in the shapes and slopes of the O2 and CO2 dissociation curves in blood (see Carbon Dioxide. Oxygen–Hemoglobin Dissociation Curve). Because the curve for CO2 is nearly linear while that for O2 is quite nonlinear, arterial PCO2 is easier to normalize than is arterial PO2 . This difference in ability to restore arterial PO2 and PCO2 by increased ventilation often leads to a large increase in ventilation, sufficient to cause hypocapnia, while still not normalizing arterial PO2 (as the model suggests in Figure 3(b)). Figure 3 shows the progression of expected arterial blood gas values that would be anticipated stage by stage as these compensation processes occurred for the two models in Figure 2. It should be realized that while these stages are presented sequentially, in real life they would be occurring essentially together over a short time frame and would probably all be in place by the time a physician saw the patient. An important conclusion from analyzing this sequence of events is that just because a patient with ˙ inequality may present with hypoxemia but V˙A/Q not hypercapnia, it should not be thought that inequality affects only O2 and not CO2. In fact, some patterns of inequality, especially those in which areas ˙ ratio develop, can affect CO2 of very high V˙A/Q more than O2 (Figure 2(c)). The third way in which the body can compensate ˙ inequality is by increasing cardiac output. ˙ A/Q for V This is not infrequently seen in young asthmatic patients, and in the intensive care unit in patients who have high cardiac outputs from sepsis and other pathologies. What an increase in cardiac output allows is an increase in the venous PO2 . This can be deduced from eqn [6], otherwise known as the Fick principle. This equation shows that for a given metabolic rate ’ results in a (V’ O2 ), and increase in cardiac output (Q) reduction in the arteriovenous O2 concentration difference, and in turn a rise in venous PO2 . When low ˙ ratio regions exist, an increase in venous PO2 V˙A/Q will result in an increase in arterial PO2 . For example, in model B, increasing cardiac output from 6 to 10 l min
1 permits an increase in arterial PO2 from 48 to 55 mmHg. While this seems modest, arterial O2 saturation would rise from about 83% to almost 90%, significantly enhancing the arterial O2 concentration. While increases in either or both ventilation and cardiac output allow both arterial PO2 and PCO2 to move towards normalization, two points need

VENTILATION, PERFUSION MATCHING 475 50

110 Arterial PCO 2 (mmHg)

Arterial PO2 (mmHg)

100 90 80 70 60 50 40 30 Obstruction : Venous PO2 : Ventilation : O2 uptake :

45 40 35 30 25

Pre Post 41.6 41.6 32.5 32.3 32.2 32.1 32.0 31.9 31.8 5.2

5.2

300 199

Obstruction : Venous PCO2 :

7.5

Ventilation :

300 300 300 300 300 300 300

CO2 elimination :

5.2

5.8

6.0

6.3

6.5

7.0

5.2

5.2

5.2

5.8

6.0

6.3

240 213 240 240 240 240

6.5

7.0

7.5

240 240 240

(b) 110

60

100

55

Arterial PCO2 (mmHg)

Arterial PO2 (mmHg)

(a)

Pre Post 45.8 45.8 50.2 45.4 44.1 42.4 41.2 38.7 36.6

90 80 70 60 50

Ventilation : O2 uptake :

40 35 30 25

40 Obstruction : Venous PO2 :

50 45

Pre Post 41.6 41.6 39.8 41.3 41.2 40.8 40.4 39.5 38.7 5.2

5.2

5.2

300

263

300 300

6.0

7.0

7.5

8.0

Obstruction : Venous PCO2 : Ventilation :

9.0 10.0

300 300 300 300

300

(c)

CO2 elimination :

Pre Post 45.8 45.8 62.4 54.7 47.9 45.1 42.6 38.4 35.0 5.2

5.2

240 182

5.2

6.0

7.0

7.5

240 240 240 240

8.0

9.0 10.0

240 240 240

(d)

˙ inequality in the models of Figure 2, showing how arterial PO2 (a, c) and PCO2 (b, d) respond. Parts ˙ A/Q Figure 3 Compensation for V (a) and (b) reflect airway obstruction; parts (c) and (d) vascular obstruction. In each case, the second bar shows effects of inequality prior to compensation. The third bar shows restoration of V’ O2 and V’ CO2 by changes in mixed venous PO2 and PCO2 alone. The remaining bars show responses to increasing alveolar ventilation. See text for more details.

further consideration. First, increasing ventilation and cardiac output are both energy requiring, and patients with already compromised ventilatory or cardiac pumps may not be able to respond well. Second, in those patients who do respond with brisk increases in ventilation and/or cardiac output, the improvement in their arterial blood gases may lead the physician to underestimate the extent of the un˙ inequality. In other words, patients ˙ A/Q derlying V who show substantial increases in either ventilation or cardiac output and have nearly normal arterial ˙ inblood gases may still have considerable V˙A/Q equality, and should be managed with this in mind.

Assessment of Ventilation/Perfusion Inequality ˙ inequality is diffiThe detailed assessment of V˙A/Q cult, especially in clinical settings. Most often, the

lung is modeled as consisting of three virtual alveoli. One of these is unventilated, but perfused, and is labeled as a ‘shunt’. Another is ventilated but not perfused, and is labeled as a ‘deadspace’. The third is labeled the ‘ideal’ alveolus, and is both perfused and ˙ inequality ventilated. The objective in assessing V˙A/Q in this framework is to (1) apportion total ventilation between the ideal and deadspace alveoli on the one hand, and (2) apportion total cardiac output between the ideal and shunt alveoli on the other. This is a gross oversimplification of reality in almost all pa˙ tients, but does provide a quantitative index of V˙A/Q inequality that can be useful in guiding therapy. To apportion ventilation between the ideal and deadspace alveoli, one must measure both arterial PCO2 (PaCO2 ) and the PCO2 of mixed expired gas (PECO2 ). On the presumption that the deadspace alveolus contains no CO2 (because it is unperfused and therefore cannot get CO2 from the blood), while the

476 VENTILATION, PERFUSION MATCHING

PCO2 of the ideal alveolus equals that of the measured arterial blood, a conservation of mass analysis (much like that in eqn [11]) shows that the fraction of the total ventilation received by the deadspace alveolus (commonly referred to as VD/VT) comes to V D=V T ¼½PaCO2
PECO2 =PaCO2

½12

This is called the ‘physiological’ deadspace because it is measured using functional variables, and because it includes all causes of deadspace in a single outcome measure. Specifically, the normal contribution to deadspace from the conducting airways is a part of VD/VT, as are additional components caused by the ˙ ratio, if any. In presence of any regions of high V˙A/Q other words, VD/VT would be around 0.3 in a normal subject because the conducting airway volume is about 150 ml while the tidal volume is about 500 ml (150/500 ¼ 0.3). Any value of VD/VT higher than this would suggest the presence of alveolar regions of ˙ ratio. This conclusion must be accepted ˙ A/Q high V with caution however, since changes in tidal volume can have an obvious, significant effect on VD/VT since the numerator, conducting airway volume, is relatively constant as tidal volume changes. To overcome this limitation, multiplying VD/VT by measured tidal volume will yield an absolute value for VD per breath. This should be about 150 ml in a normal person irrespective of tidal volume, and so an increase above such a value is a better indication of the presence of ˙ ratio. In reality, conducting airareas of high V˙A/Q way volume varies with body size, and a commonly used estimate is about 1 ml per pound of body mass, correcting mass for obesity when significant. To estimate perfusion in the ‘shunt’ alveolus, a similar mass conservation approach is taken, but in this case one uses measures of O2 exchange. One must measure both arterial and venous (pulmonary arterial) O2 concentrations (CaO2 and CvO2 , respectively). The endcapillary O2 concentration in the ideal alveolus (Cc0O2 ) cannot be measured but is needed and is thus calculated from an estimate of its alveolar PO2. The equation that describes the perfusion of the shunt alveolus as a fraction of the total ˙ T) is cardiac output (QS/Q ’ T ¼ ½Cc0
CaO =½Cc0
CvO  QS=Q 2 2 O2 O2

½13

˙ T in normal subjects Unlike the case for VD/VT, QS/Q is essentially zero. Thus, any elevation (perhaps allowing 0.01–0.02 to encompass the range of normal responses) is abnormal. An important consideration in applying this equation arises when CvO2 is not in fact measured, but is assumed. As the equation shows, an error in CvO2 will give rise to an incorrect ˙ T. The second major consideration value for QS/Q

when using this equation relates to the meaning of ˙ T. In the case where there the value obtained as QS/Q is truly a pure shunt in the patient’s lung (e.g., in lobar consolidation, in atelectasis, or in the event of a right to left intracardiac communication), the value obtained accurately estimates the fraction of the cardiac output that is perfusing the abnormal pathway. However, in the relatively more common setting ˙ ratio are present rather where areas of low V˙A/Q ˙ T must be thought than true shunts, the value of QS/Q of as the fraction of the cardiac output that would flow through a hypothetical shunt pathway and lead to the same arterial PO2 as measured in the patient. Because alveolar (and thus endcapillary) PO2 in low ˙ regions is higher than in venous blood flowing V˙A/Q through a pure shunt pathway, the actual perfusion ˙ areas will always exceed that ˙ A/Q rate in low V obtained from eqn [13]. A special consideration relates to use of eqn [13] when FIO2 is raised, as is often the case in the ICU. ˙ The higher the FIO2 , the less will regions of low V˙A/Q ratio interfere with O2 exchange. In particular, when FIO2 is 1.0, each ventilated alveolus, no matter what ˙ ratio is, contains only O2, CO2, and water its V˙A/Q vapor as the resident N2 is eliminated. This means ˙ ratio, alveolar that even in regions of very low V˙A/Q PO2 will be several hundred mmHg, and allow full saturation of endcapillary blood. In practice, this ˙ T breathing pure O2 will be zero means that QS/Q ˙ inequality is present, unless there is also even if V˙A/Q ˙ inequala shunt. It also means that when both V˙A/Q ˙ T measity and shunt coexist in the same lungs, QS/Q ured while breathing air will exceed that measured ˙ T under while breathing pure O2. Measuring QS/Q both conditions therefore affords a way to separately ˙ inequality and shunt. assess the magnitude of V˙A/Q This well-established technique should be used with some caution, however. This is because by giving pure O2 to a patient, some alveoli that were ventilated, albeit poorly, on room air, may cease to be ventilated and collapse, thereby increasing the size of the shunt. Furthermore, increasing FIO2 may release hypoxic vasoconstriction in these hypoxic areas, raising their perfusion levels, and increasing the measured shunt fraction even more. ˙ inequality ˙ A/Q A third method for characterizing V is to determine the alveolar–arterial PO2 difference (AaPO2 ). As Figure 2 shows, the difference between mixed alveolar and arterial PO2 is normally close ˙ inequality increases alveoto zero. However, V˙A/Q lar and reduces arterial PO2 , causing AaPO2 to increase. While arterial PO2 is easily measured from a blood sample, mixed alveolar PO2 is difficult to measure, and is thus calculated from the alveolar gas equation. This equation uses eqn [3] above and the

VENTILATION, PERFUSION MATCHING 477

corresponding equation for CO2 to estimate alveolar PO2 (PAO2 ) as PAO2 ¼ PIO2
PACO2 =R þ PACO2  FIO2  ½ð1
RÞ=R

½14

A simpler version ignores the third term because it generally is only 1–2 mmHg: PAO2 ¼ PIO2
PACO2 =R

½15

where R is the ratio of V’ CO2 to V’ O2 (the respiratory exchange ratio). Then AaPO2 ¼ PAO2
PaO2

½16

Most often, one uses the measured arterial PCO2 (PaCO2 ) in place of the alveolar value, because they are generally quite similar. This means that the AaPO2 can be calculated from just the arterial PO2 and PCO2 , provided one knows PIO2 (inspired PO2 ) and R. PIO2 is usually easy to determine, but R is often assumed. It should be recognized that small errors in R can cause substantial errors in AaPO2 .

The final expression is AaPO2 ¼ PIO2
PaCO2 =R
PaO2

The Multiple Inert Gas Elimination Technique ˙ Another approach to assessing the amount of V˙A/Q inequality is to use what is known as the multiple inert gas elimination technique (MIGET) to measure directly the frequency distribution of ventilation/perfusion ratios throughout the lungs. This method makes use of the fact that the degree to which any particular inert gas is eliminated by the lungs from the body (after being introduced by either inhalation or intravenous infusion) depends on the distribution of ventilation and blood flow throughout the lungs. The degree of elimination also depends on the blood solubility of the gas, and it turns out that it is necessary to have about six or so gases of widely differing solubilities to obtain sufficient information from which to compute the distribution. The physiological basis of this method is the same mass balance principle discussed extensively above Asymptomatic asthma, .PaO2 = 79; FEV1 = normal

Normal subject 1.8

1.8

Normal VA /Q

1.2 0.9 0.6

0.0

No shunt

No low VA /Q

No high VA /Q

1.2 0.9 0.6 0.3

(a)

0.01 0.1 1 10 Ventilation/perfusion ratio

0

100 (b)

Severe, chronic asthma, .PaO2 = 53; FEV1 = 35% predicted 1.8

0.01 0.1 1 10 Ventilation/perfusion ratio

100

COPD: predominant emphysema

Normal VA /Q

Normal VA /Q

1.5 Low VA /Q

1.2 0.9 0.6 No shunt

No high VA /Q

Ventilation ( ), Blood flow ( )

Ventilation ( ), Blood flow ( )

No high VA /Q

1.8

1.5

1.2

0.6

0.0 0

0.01 0.1 1 10 Ventilation/perfusion ratio

100

High VA /Q

0.9

0.3

0.0 (c)

Low VA /Q No shunt

0.0 0

0.3

Normal VA /Q

1.5 Ventilation ( ), Blood flow ( )

Ventilation ( ), Blood flow ( )

1.5

0.3

½17

No shunt 0

(d)

Low VA /Q

0.01 0.1 1 10 Ventilation/perfusion ratio

100

˙ distribution showing how ventilation and blood flow are distributed with respect to V ˙ ratio throughout the lungs. The ˙ A/Q ˙ A/Q Figure 4 V normal distribution (a) is narrow and symmetrical; that in mild asthma (b) and severe asthma (c) contains normal areas and also areas of ˙ ratio, the latter due to partial airway obstruction; that in emphysema (d) has, in addition to normal regions, areas of very very low V˙A/Q ˙ probably caused by continued ventilation through poorly perfused ‘holes’ in the lung. high V˙A/Q

478 VESICULAR TRAFFICKING

for O2 and CO2, and shown in eqns [9] and [10]. From the measured pattern of elimination of the several gases, mathematical procedures are used to cal˙ ratios that best fit the culate the distribution of V˙A/Q elimination pattern over all six gases. The results are expressed as two separate but linked frequency distributions – one for ventilation and one for blood flow as in Figure 4. These distributions show how ventilation and blood flow are distributed along the ˙ ratio axis from essentially zero to essentially ˙ A/Q V infinity. Figure 4(a) shows the distribution in a young normal subject; Figure 4(b) exemplifies mild asthma, Figure 4(c) severe asthma, and Figure 4(d) emphysema. The normal subject shows a narrow distribu˙ of about 1, devoid of areas ˙ A/Q tion around a mean V ˙ ratio. The asthmatic lung ˙ of very low or high VA/Q ˙ ratio, contains substantial regions of very low V˙A/Q but does not have areas of abnormally high ratio. The ˙ areas in asthma presumably reflect partially low V˙A/Q obstructed airways, and may be seen even when forced expiratory volume in 1 s (FEV1) is normal or only slightly reduced. In contrast, the lung with ˙ emphysema shows abnormalities in the high V˙A/Q domain, likely the result of continued ventilation of poorly perfused ‘holes’ in the lung parenchyma. The MIGET provides the most complete picture ˙ inequality currently available that is usable of V˙A/Q in intact humans. It is a complex technique that demands considerable expertise and attention to detail, and remains a useful research tool in areas such as understanding the pathophysiology of, and

therapeutic responses to, new treatments for lung diseases. See also: Carbon Dioxide. Diffusion of Gases. Oxygen–Hemoglobin Dissociation Curve. Pulmonary Circulation. Ventilation: Overview; Control.

Further Reading Kelman GR (1968) Computer programs for the production of O2– CO2 diagrams. Respiration Physiology 4: 260–269. Rahn H and Fenn WO (1955) A Graphical Analysis of the Respiratory Gas Exchange. Washington, DC: American Physiological Society. Riley RL and Cournand A (1949) ‘‘Ideal’’ alveolar air and the analysis of ventilation/perfusion relationships in the lung. Journal of Applied Physiology 1: 825–847. Riley RL and Cournand A (1951) Analysis of factors affecting partial pressures of oxygen and carbon dioxide in gas and blood of lungs: theory. Journal of Applied Physiology 4: 77–101. Wagner PD (1978) Measurement of the distribution of ventilation/ perfusion ratios. In: Davies DG and Barnes CD (eds.) Regulation of Ventilation and Gas Exchange, pp. 217–260. New York: Academic Press. Wagner PD and West JB (1980) Ventilation–perfusion relationships. In: West JB (ed.) Ventilation, Blood Flow and Diffusion, pp. 219–262. New York: Academic Press. West JB (1969) Ventilation/perfusion inequality and overall gas exchange in computer models of the lung. Respiration Physiology 7: 88–110. West JB (1990) Ventilation/Blood Flow and Gas Exchange. Oxford and Philadelphia: Blackwell Scientific Publications and Lippincott. West JB and Wagner PD (2000) Ventilation, blood flow and gas exchange. In: Murray JF and Nadel JA (eds.) Textbook of Respiratory Medicine, pp. 55–89. Philadelphia, PA: Saunders.

VESICULAR TRAFFICKING N E Vlahakis, Mayo Clinic Rochester, Rochester, MN, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Vesicular trafficking is a continuous and dynamic process in all cells of the body, in which lipid vesicles move from the cell surface to intracellular lipid endosomes (endocytosis) or from organelles and endosomes to the cell surface (exocytosis). It functions as both a homeostatic process for maintenance of quiescent cell function and a stress response to ensure rapid protein synthesis, targeted intracellular transport, and expression of proteins on the cell surface or their release to the extracellular environment. Vesicles are primarily composed of lipid molecules that serve not only as a mechanical barrier to the perivesicular environment but also a physical support for their protein cargo during transport within the cell. Membrane lipids also provide a physical structure to prime vesicles for budding or fusion or to

modulate protein function and resultant transduction of cellular signaling. Respiratory epithelium is polarized and the apical and basolateral membranes are maintained by tightly controlled and membrane-specific vesicular trafficking pathways to maintain this polarized phenotype. Any defect in this transport machinery can result in inadequate responses to cellular stressors or respiratory disease, such as cystic fibrosis and interstitial lung disease. The vesicular trafficking process in type II alveolar pneumocytes has many unique features and is essential for surfactant secretion and reuptake. In addition, a rapid and targeted trafficking response is required for alveolar cells to add lipids to the cell surface to prevent cell rupture or to ‘patch’ cell breaks.

Description General Mechanisms of Vesicular Trafficking

Lipid vesicles originate in the endoplasmic reticulum (ER), budding from the ER as a means to distribute

VESICULAR TRAFFICKING 479

proteins to their designated cellular location. These transport vesicles are defined by their lipid composition and protein coats. For example, coat proteins I and II define vesicles for ER and Golgi transport and Rab GTPases for transport to other endosome compartments or the plasma membrane. Endocytosis is a process characterized by two general mechanisms: phagocytosis and pinocytosis (Figure 1). Phagocytosis is performed by specialized cells in the body such as macrophages (see Leukocytes: Pulmonary Macrophages). Pinocytosis is the uptake of smaller molecules with their specific receptors via specialized transport domains. The forms of pinocytosis (Figure 1) are characterized by the size and specific type of internalized molecule, the lipid and protein repertoire to effect endocytosis, and the size of the resultant endosome. Two specific mechanisms of pinocytosis known as clathrin-mediated and caveolin-mediated endocytosis represent the most well-characterized forms of endocytosis. Both mechanisms are characterized by cell surface invaginations or pits ‘coated’ by specific proteins, clathrin and caveolin, respectively. Formation of these coated pits requires protein–protein and lipid–protein interactions that specifically link the cytoplasmic domains of transmembrane receptors to the coat assembly apparatus. Exocytosis, like endocytosis, is facilitated by specific secretory vesicles that are identified by their lipid and protein composition. This process is important in all secretory cells, including the alveolar and airway epithelium. As a result of being at an air–liquid interface, respiratory epithelial cells are polarized; that is, they have specialized surface membrane compartments – apical and basal–lateral (Figure 2). To maintain the membrane-specific protein and lipid composition that defines its specific function, polarized epithelium has unique vesicle trafficking pathways. The unique role of each membrane compartment results from the function it subserves, such as protein biosynthesis for

cell expression, endocytosis followed by recycling to the respective membrane, and transcytosis (Figure 2). Synthesized proteins traffic from the ER to the Golgi and then through specific endosomes to their predefined membrane compartment. When endocytosed, these compartment-specific proteins enter early endosomes and are sorted either to late endosome, lysosomes, and degradation or recycling endosomes for return to their parent membrane, or enter the common endosome to undergo transcytosis. Transcytosis is a process by which proteins residing in one membrane (e.g., basolateral) are endocytosed, sorted to, and modified in common endosomes, and then transported to the opposite membrane (apical). The movement of vesicles is facilitated by the framework of the cytoskeleton and associated motor proteins and regulated by specific proteins in a temporospatial manner to maintain the apical and basolateral domains. After arrival at their target lipid compartment, membrane fusion via specific lipid–lipid and protein– protein interactions must occur for cargo delivery (Figure 3). As the vesicle approaches its target membrane, a multiprotein tether facilitates specific protein–protein binding between vesicle (v)-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) and target membrane (t)-SNARE proteins. Once docked, lipid–lipid interactions allow fusion of the lipid bilayers and secretion of the vesicular cargo. Localized areas of membrane with specific phospholipid composition (cholesterol) make up membrane ‘target patches’ or ‘lipid rafts’ that serve as both, a signal transduction function and as predefined and localized areas for vesicle fusion and delivery of specific protein cargo, such as the cell–cell junctional protein E-cadherin. Defects in the vesicular trafficking machinery are implicated in a number of respiratory diseases. Ten percent of all mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) involve a defect in apical sorting determinants resulting in

Pinocytosis Phagocytosis (particle-dependent) Clathrinmediated endocytosis (∼120 nm)

Caveolinmediated endocytosis (∼60 nm)

Clathrin- and caveolin-independent endocytosis (∼90 nm)

Figure 1 Mechanisms of endocytosis. This figure demonstrates the different mechanisms used by the cell to endocytose molecules. Each pathway is characterized by the size of the molecule internalized, the lipid and protein repertoire to effect endocytosis, and the size of the resultant endosome. Reproduced with permission from Conner SD and Schmid SL (2003) Regulated portals of entry into the cell. Nature 422(6927): 37–44.

480 VESICULAR TRAFFICKING Apical membrane

Apical early endosome Basolateral early endosome Apical recycling

BL transcytosis

Common endosome Apical recycling endosome

Ap EE

Ap RE

Late endosome LE

Lysosome

Ly

ing

ycl

c Re

BL EE

Golgi

Ap tr ansc ytosis

Lateral membrane

CE

Basal membrane Figure 2 Vesicular trafficking pathways within polarized epithelial cells. Specific trafficking pathways, composed of specific endosome compartments, serve to maintain the apical and basolateral membrane compartments. Each membrane compartment has early endosomes (Ap EE and BL EE) from which vesicles may recycle to the parent membrane, traffic to the lysosome (Ly) for degradation, or undergo transcytosis through the common endosome from the apical to basolateral membrane (orange then blue/orange then yellow/ orange arrows) and vice versa (yellow then yellow/blue then green/yellow arrows). Reproduced from Mostov KE et al. (2000) Polarized epithelial membrane traffic: conservation and plasticity. Current Opinion in Cell Biology 12: 483–490, with permission from Elsevier.

Budding

Tethering

Docking

Fusion GTP

GTP

t v NSFSNAP Separation Priming

t v

Recycling Figure 3 Mechanism of vesicle docking and membrane fusion. Initially, a vesicle buds from a ‘donor’ membrane and is transported toward its recipient membrane. In order to fuse with this target membrane, the vesicle must first be secured to it through a multiprotein tether and then docked through specific interactions of the v (vesicle)-SNARE and t (target)-SNARE and finally fuse with the target membrane. These SNARE proteins are then recycled after being dissociated by an NEM-sensitive factor (NSF) ATPase. Reproduced from Mellman I (2000) The road taken: part and future foundations of membrane traffic. Cell 100: 99–112, with permission from Elsevier.

misguided trafficking of CFTR. In genetically engineered mice, the null mutant of caveolin-2 results in interstitial fibrotic lung disease. Although not directly indicated in its pathogenesis, patients with lymphangioleiomyomatosis have mutations in the

tuberous sclerosis complex 2 gene encoding the Rab GTPase, tuberin, an activating protein important in the control of vesicular trafficking pathways in alveolar cells. Finally, in two uncommon diseases resulting from lipid enzyme deficiencies, Gaucher’s disease

VESICULAR TRAFFICKING 481

(glucocerebrosidase) and Nieman–Pick disease (sphingomyelinase), nodular interstitial lung disease results from lipid-laden macrophage accumulation in the lung interstitium and alveolar spaces. With this as a general background, the lung and its cellular components utilize vesicular trafficking for two unique processes. First, the cellular environment is characterized by a dynamic, cyclic, and continuous process of surfactant secretion, metabolism, and endocytosis. Second, due to respiration, alveolar cells are always under the influence of deforming forces and at risk of mechanical disruption. The role of vesicular trafficking in these processes is discussed later.

Surfactant The lung, and specifically the alveolar epithelium, is a secretory organ of the body. Alveolar type II cells (AEC II) continually produce, secrete, metabolize, and recycle surfactant. Surfactant is composed of phospholipids, mostly phosphatidylcholine and proteins A–D (see Surfactant: Surfactant Protein A (SP-A); Surfactant Proteins B and C (SP-B and SP-C); Surfactant Protein D (SP-D)). Their relative proportions vary with local alveolar conditions in both the normal and the pathologic state. Its key functions and physiology are to reduce alveolar surface tension during breathing and immunomodification (see Surfactant: Overview). The nature and control of vesicle and surfactant trafficking pathways in alveolar epithelium are poorly understood and underinvestigated. Current knowledge is derived from in vitro studies of primary cultured rat AEC II. Lamellar Bodies

The handling of surfactant is conducted by the specialized AEC II (see Epithelial Cells: Type II Cells). Lamellar bodies (LBs; see Surfactant: Overview) are specialized vesicles within AEC II (about 150 per cell) that store and secrete surfactant. Among vesicular bodies, these structures have distinct and unique characteristics. They are one of the largest secretory vesicles in mammalian cells, and they have many characteristics of lysosomes, which, unlike LBs, are not storage and secretion vesicles but rather serve a degradative function. The secretion of surfactant is stimulated by mechanical force, namely deformation of its underlying basement membrane by respiration. This serves to maintain an active (surface tension-reducing) surfactant layer. This mechanical stimulus and other bioactive stimuli serve as signals to initiate intracellular effectors of surfactant secretion, the most important of which are the

additive effects of intracellular calcium release and cyclic adenosine monophosphate. Secretion

Similar to other secretory cells, the process of secretion proceeds through three main phases: trafficking of vesicles to the cell surface, fusion with the plasma membrane, and release of vesicle contents. This three-step process depends on cytoskeletal actin, and secretion is enhanced when actin is in a depolymerized state. Compared to other secretory cell types such as neurons, AEC II are unique. First, they lack a predocked pool of vesicles ready to respond to secretory stimuli. Second, vesicle fusion events, initiated in a more typical time span of seconds to minutes, are maintained for 30 min or more. Finally, surfactant secretion is regulated in a unique manner by this ‘long-lasting’ LB–plasma membrane fusion pore, the neck of which changes in size as a function of the magnitude and duration of the stimulus. Reuptake

Like secretion, uptake of secreted surfactant demonstrates both constitutive and stimulated endocytic processes. Approximately 80% of surfactant lipid is endocytosed by the AEC II, of which 50% is recycled to the LBs and 50% to lysosomes. The remaining 20% of the surfactant is phagocytosed by macrophages. This mechanism serves to regulate surfactant pool size and recycle the constituents of surfactant. Reuptake of phospholipid and surfactant protein A is both clathrin and actin dependent, and once internalized, both traffic to the same early endosome.

Lung Deformation: Injury and Repair Adaptive Responses to Prevent Cell Injury

With each large tidal breath, a shape change is imposed on the lung that at the level of the alveolar cell translates into deformation of the underlying basement membrane. Since alveolar cells are adherent to their basement membrane, an increase in cell surface area results (Figure 4). Unless adaptive responses are employed to maintain a normal membrane tension, the cell’s plasma membrane will rupture; this is especially the case in the setting of mechanical ventilation when large volume changes are imposed on the lung. Clinically, cell rupture is illustrated by elevated lactate dehydrogenase in the setting of hemolytic anemia or creatinine kinase with myocardial infarction. Cells are able to ‘sense’ the increase in cell membrane tension or strain (fractional length change) and initiate adaptive responses, including remodeling of the cell’s cytoskeleton (stress-bearing elements of the

482 VESICULAR TRAFFICKING

A

Increasing strain amplitude or rate

B

C

D

E.1

E.2

Cell survival

Cell death

Figure 4 The response of alveolar cells to deforming mechanical forces. With deformation of the basement membrane, a deforming strain is applied to adherent alveolar cells. The cell utilizes a number of mechanisms (A–C) to prevent cell injury. As strain increases, plasma membrane ruffles unfold (A), then lipid molecules (red) within the membrane can yield (B), but intracellular vesicles (pink) must traffic to the plasma membrane (C). If these preventive measures fail or are overwhelmed, plasma membrane breaks occur (D) that result in cell death (E.2) unless further lipid trafficking is able to ‘patch’ (yellow vesicles) the injury (E.1).

cell) and lipid vesicle trafficking to the plasma membrane. Lipid trafficking as a means to prevent cell stress has been documented in many cells and appears to be an evolutionarily conserved phenomenon. Vesicle Trafficking Prevents and Repairs Membrane Breaks

A vesicle-trafficking response in response to mechanical forces has been demonstrated in alveolar epithelial cells both in vitro and in vivo (Figure 4). These vesicles are recruited from a ‘lipid reservoir’ and their transport is initiated in a rapid and repeatable manner once unfolding of cell surface projections (e.g., microvilli) is complete and the lytic tension of the plasma membrane (B25 mN m
1) is approached. With increases in plasma membrane tension, exocytosis is initiated, resulting in a net increase in surface membrane. Furthermore, when the mechanical stress is removed, there is a decrease in tension resulting in membrane retrieval (endocytosis). Deformation-induced lipid trafficking is energy dependent and requires an intact actin cytoskeleton and a plasma membrane rich in cholesterol. The vesicles that make up the alveolar epithelial cell lipid reservoir are not well characterized, but based on findings from other cell types, they likely involve lysosomes.

Even when lungs suffer relatively mild mechanical strain, for example, from interstitial pulmonary edema, the lipid microdomains of the lung cell surface membrane undergo a substantial reorganization that is accompanied by changes in both lipid composition and surface protein expression. These modifications induce many biologic responses, including transdifferentiation from the AEC II to AEC I epithelial phenotype. These different cell types possess different mechanical properties and, by inference, there are significant changes in the cells’ susceptibility to mechanical injury. This vesicle-trafficking response is available not only to prevent membrane breaks but also to repair them and thus is an essential mechanism of cell survival. It is a Ca2þ-dependent process in which an increase in cytosolic Ca2þ, consequent to the loss of the cell barrier, promotes exocytosis of vesicles near the wound site. If the wound size is larger (41 mm), Ca2þ transduces the coalescence of vesicular endomembranes that are transported along the cytoskeleton to ‘patch’ the wound site.

Conclusions Vesicular trafficking is an essential cell function, playing a central role in normal and stress-induced

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biologic processes. This article highlighted the integral role that vesicular trafficking plays in lung cell biology as it relates to maintaining efficient respiration through surfactant secretion and reuptake and the maintenance of an intact alveolar–capillary barrier through prevention of mechanical force-induced cell injury. Defects in this trafficking machinery can result in specific respiratory disease. See also: Epithelial Cells: Type II Cells. Leukocytes: Pulmonary Macrophages. Surfactant: Overview; Surfactant Protein A (SP-A); Surfactant Proteins B and C (SP-B and SP-C); Surfactant Protein D (SP-D).

Further Reading Bonifacino JS and Glick BS (2004) The mechanisms of vesicle budding and fusion. Cell 116(2): 153–166. Cohen AW, Hnasko R, Schubert W, and Lisanti MP (2004) Role of caveolae and caveolins in health and disease. Physiology Review 84(4): 1341–1379. Conner SD and Schmid SL (2003) Regulated portals of entry into the cell. Nature 422(6927): 37–44. Dietl P and Haller T (2005) Exocytosis of lung surfactant: from the secretory vesicle to the air–liquid interface. Annual Review of Physiology 67: 24.1–24.27. Janmey PA and Weitz DA (2004) Dealing with mechanics: mechanisms of force transduction in cells. Trends in Biochemical Science 29(7): 364–370.

McNeil PL and Steinhardt RA (2003) Plasma membrane disruption: repair, prevention, adaptation. Annual Review of Cell and Developmental Biology 19: 697–731. Mellman I (2000) The road taken: part and future foundations of membrane traffic. Cell 100: 99–112. Miaczynska M, Pelkmans L, and Zerial M (2004) Not just a sink: endosomes in control of signal transduction. Current Opinion in Cell Biology 16(4): 400–406. Mostov KE, et al. (2000) Polarized epithelial membrane traffic: conservation and plasticity. Current Opinion in Cell Biology 12: 483–490. Mostov K, Su T, and ter Beest M (2003) Polarized epithelial membrane traffic: conservation and plasticity. Nature Cell Biology 5(4): 287–293. Pfeffer S (2003) Membrane domains in the secretory and endocytic pathways. Cell 112(4): 507–517. Salaun C, James DJ, and Chamberlain LH (2004) Lipid rafts and the regulation of exocytosis. Traffic 5(4): 255–264. Spiliotis ET and Nelson WJ (2003) Spatial control of exocytosis. Current Opinion in Cell Biology 15(4): 430–437. Stein M, Wandinger-Ness A, and Roitbak T (2002) Altered trafficking and epithelial cell polarity in disease. Trends in Cell Biology 12(8): 374–381. Stein MP, Dong J, and Wandinger-Ness A (2003) Rab proteins and endocytic trafficking: potential targets for therapeutic intervention. Advanced Drug Delivery Reviews 55(11): 1421– 1437. Veldhuizen R and Possmayer F (2004) Phospholipid metabolism in lung surfactant. Subcellular Biochemistry 37: 359–388. Vlahakis NE and Hubmayr RD (2005) Cellular stress failure in ventilator injured lungs. American Journal of Respiratory and Critical Care Medicine 171: 1328–1342.

VIRUSES OF THE LUNG N G Papadopoulos and C L Skevaki, University of Athens, Athens, Greece & 2006 Elsevier Ltd. All rights reserved.

on avoidance of risk factors and vaccination when indicated. Treatment modalities include over-the-counter and non-specific remedies along with a small number of specific antiviral medications such as the influenza neuraminidase inhibitors.

Abstract Respiratory viruses include rhinoviruses and enteroviruses (Picornaviridae), influenza viruses (Orthomyxoviridae), parainfluenza, metapneumoviruses and respiratory syncytial viruses (Paramyxoviridae), coronaviruses (Coronaviridae), and several adenoviruses. With the exception of adenoviruses, all possess an RNA genome. They are usually transmitted by direct hand to surface to hand contact and/or aerosol inhalation, and replicate in both upper and lower airways. Cellular and humoral immunity are both activated in response to respiratory viral infections as well as neural pathways, which contribute to distant inflammatory effects. Respiratory viruses are responsible for a wide variety of clinical syndromes including the common cold, acute otitis media, laryngitis, sinusitis, pneumonia, bronchiolitis, influenza, and exacerbations of asthma and chronic obstructive pulmonary disease. Diagnosis of respiratory viral infections is primarily clinical and is further supported by laboratory techniques such as antigen detection, culture, serology and more recently nucleic acid detection. Preventive strategies are based

Structure and Taxonomy Human rhinoviruses (RVs), which probably represent the most abundant pathogenic microorganisms universally, are RNA viruses and belong to the family of Picornaviridae. The Picornaviridae also include the enteroviruses (polio, coxsackie, and echo viruses), which are more closely related to RVs, and the cardio- and aphtho-viruses, which do not infect the respiratory tract. Coxsackie virus A2, 10, 21, 24 and B2, 5 and echovirus type 1, 11, 19, 20, and 22 are the most frequently isolated enteroviral agents in upper respiratory tract infections. More than 100 serotypes of RVs are identified and numbered. They are divided into major (90%) and minor (10%) groups depending on their receptor; major RVs attach to the

484 VIRUSES OF THE LUNG

Figure 1 Rhinovirus (RV). The rhinovirus capsid is arranged in an icosahedron composed of 60 copies of each of three subunits (shown in red, blue, and yellow). Reproduced with permission from Dr N G Papadopoulos.

intercellular adhesion molecule-1, while low-density lipoprotein receptor is the cellular receptor of minor RVs. RVs consist of a 2 MDa single-stranded positive-sense genomic RNA surrounded by a nonenveloped capsid arranged in an icosahedron, 20–30 nm in diameter (Figure 1). VP1, 2, and 3 structural proteins of the virus are highly variable surface proteins, which interact with antiviral antibodies. VP4 is confined to the interior of the capsid and is closely associated with the viral RNA. Orthomyxoviridae’s major representatives are the influenza viruses (IFVs), which are grouped into three types: A, B, and C. IFVs are negative-stranded segmented RNA viruses. The A and B types are enveloped pleomorphic agents, with spherical and filamentous forms; IFV-C is structurally distinct. IFVs attach to sialic acid receptors on ciliated columnar epithelial cells in the tracheobronchial tree via their hemagglutinin (HA) protein (Figure 2). Following replication, the virion is released by enzymatic action of the neuraminidase (NA) surface protein. The latter, along with the HA protein of the virus, is regularly subjected to small changes, which are capable of producing viral strains causing annual epidemics. This phenomenon is called ‘antigenic drift’, while ‘antigenic shift’ is the process by which a sudden major change in the HA or NA proteins of IFV-A occurs due to genetic reassortment. The latter is responsible for influenza pandemics, which have occurred every 10–40 years. Paramyxoviridae include parainfluenza viruses (PIVs), the respiratory syncytial viruses (RSVs) as

Figure 2 Influenza virus (IFV). Schematic representation of an influenza virus. Hemagglutinin spikes (blue) radiate all over the surface and are interspersed by clusters of neuraminidase (yellow). The latter are embedded in the envelope’s lipid bilayer (orange), which in turn surrounds a layer of matrix protein, M1 (blue). The segmented RNA of the virus is located in the interior and encodes for eight distinct proteins. Reproduced with permission from Dr N G Papadopoulos.

well as the recently identified human metapneumovirus (hMPV). PIVs are grouped into subtypes 1 and 3, which belong to the paramyxovirus genus, and subtypes 2, 4a, and 4b, which belong to the rubella virus genus along with the mumps virus. PIVs are single-stranded negative-sense RNA viruses, 150–200 nm in diameter, with helical nucleocapsids. In contrast to RSVs, PIVs possess both HA and NA proteins, which attach to sialic acid receptors. RSVs are pleomorphic enveloped viruses, 120– 300 nm in diameter, with a nonsegmented negativesense RNA genome. There are two subtypes: A and B. The viral genome encodes for the glycosylated F (fusion) and G (attachment) proteins among others. The former contributes to the agent’s infectivity and virulence, while the latter is involved in virus–cell and cell–cell fusion, and exhibits high variability among different serogroups. MPVs are recently identified paramyxovirus-like pleomorphic agents. They are RNA viruses and two potential subgroups have been distinguished. Human coronaviruses (CVs) belong to the Coronaviridae and are enveloped, roughly spherical viruses, 100 nm in diameter, with 20 nm-long round surface projections. Their genome consists of a positive RNA strand. Most of the CVs studied so far are closely related to one of two reference strains, OC43 and 229E, which dominate in outbreaks alternately

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Figure 3 Adenovirus. Adenoviruses are nonenveloped DNA viruses surrounded by an icosahedral capsid from which a stalklike structure the fiber, protrudes. Reproduced with permission from Dr N G Papadopoulos.

from year to year. The former binds to MHC class I molecules, while the latter binds to human aminopeptidase N (CD13). SARS-CoV is another recently identified CV, which differs phylogenetically from all other known human CVs. Adenoviruses are nonenveloped single-stranded DNA viruses with a genome of 30–38 kbp that encodes 10 structural proteins. They are surrounded by a capsid of icosahedral symmetry, from which a stalk-like structure protrudes, called the fiber (Figure 3). The latter interacts with various cellular receptors, such as the MHC class I molecule and the coxsackie-adenovirus receptor. Over 50 serotypes and six subgenera (A–F) have been identified.

Mode of Infection RVs are transmitted by both, direct hand to surface to hand contact and aerosol inhalation, and more frequently, as with RSVs, through the eye or nose than through the mouth. Nasal epithelium represents the primary site of infection for these viruses. RVs replicate mainly in the nose, although replication takes place in the lower airways as well. IFVs are usually transmitted through inhalation of droplets, which initially attach to the mucous layer covering the tracheobronchial tree and replicate primarily in the lower respiratory tract. Epithelial infection by RVs has a ‘patchy’ distribution and is not overtly cytotoxic, in contrast to the majority of respiratory viruses, including IFVs, paramyxoviruses, and

adenoviruses, which cause extensive inflammation and epithelial shedding. Respiratory virus infection causes an increase in both vascular permeability mediated by vasoactive amines and glandular secretion under the influence of cholinergic reflexes and neuropeptides. Several cytokines and chemokines have also been implicated in virus-induced inflammation. Inflammatory cells further aggravate events by release of additional mediators. Humoral immunity is also activated in response to viral infections with production of serotype-specific antibodies. The nature of the response is associated with age, previous infection, and vaccination status of the host. Recovery from respiratory viral illness is usually achieved prior to the detection of specific antibody indicating that cellular and/or non-specific immune responses are primarily responsible for viral eradication. Nevertheless, antibodies are able to provide protection from secondary infections. Respiratory viruses not only induce a local inflammatory reaction at the level of infected epithelial cells but may also act at a distance through neuronal pathways. All parasympathetic, sympathetic, and nonadrenergic noncholinergic nervous supply of the respiratory tree may be affected by viral infections, suggesting an important neuroimmune interaction, which may contribute to the virus-mediated reactive airway disease (Figure 4).

Clinical Manifestations Respiratory viruses are related to various distinct as well as non-specific clinical presentations. There is considerable overlap between viruses and clinical presentations; although differences exist, all agents may cause any of several clinical syndromes (Table 1). Among these, the common cold is by far the commonest with an enormous socioeconomic impact. Clinical presentation ranges from asymptomatic to upper respiratory tract (URT) symptoms such as nasal congestion, rhinorrhea and nasopharyngeal irritation, lower respiratory tract symptoms such as cough, to systemic symptoms including general malaise, headache, and sleep impairment depending on the viral culprit and host. The incubation period is 24–48 h and symptoms usually last for 5–7 days. The common cold is a rather benign clinical entity, which may however be complicated by secondary bacterial infections, otitis media, sinusitis, pneumonia, and asthma exacerbations; death may occur in immunocompromised patients. Viral URT infection often results in impairment of the function of the eustachian tube, which predisposes to the development of acute otitis media. The

486 VIRUSES OF THE LUNG Respiratory viruses Immune response

Virus eradication

Vascular leakage + glandular secretion

Symptoms (rhinorrhea, cough, sneezing)

2 Replication Inflammatory mediators (chemokines, cytokines, bradykinins)

1 Entry

Neurogenic pathways

Figure 4 Pathogenesis of respiratory viral infections. Respiratory viruses enter the human body mainly through the nose. Thereafter, they replicate in nasopharyngeal epithelial cells, but also in the lower respiratory tract. Immune mediators such as cytokines, chemokines, and bradykinins will both elicit an ‘immune’ response leading to effective virus eradication and contribute to the development of associated symptomatology, through vascular leakage and increased glandular secretions. Neurogenic pathways are also involved in the development of clinical manifestations. Reproduced with permission from Dr N G Papadopoulos.

Table 1 Respiratory viruses in upper and lower respiratory disease syndromes

Rhinoviruses Influenza RSV Parainfluenza Coronaviruses Adenoviruses

Asthma

Bronchitis

Bronchiolitis

Common cold

Croup

Otitis/sinusitis

Pneumonia

*** ** * * ** *

* * * ** * *

* * *** *

*** * * * ** *

* * ** *** * *

** ** ** * * *

* ** * *

**

latter is one of the commonest infections among children and is very often attributed to viral etiology. Respiratory viruses may also predispose to nasopharyngeal bacterial colonization and alteration of the host’s immune response. Finally, viruses may be responsible for antibiotic treatment failure in cases of concurrent bacterial and viral infections. Likewise, respiratory viruses have been implicated in the development of sinusitis, either directly as an extension of URT infection or as a result of secondary bacterial infection. Laryngitis and laryngotracheobronchitis-croup have more frequently been associated with the parainfluenza virus group. Pneumonia in infants and young children is most commonly attributed to viruses. Disease burden is also significant among the elderly and the immunocompromised. RVs and RSVs are the agents most frequently isolated in viral pneumonias, although age, season, year, and other variations can be considerable. The presence of adenovirus is a risk factor for severe disease in infants. Influenza occurs in yearly winter epidemics and pandemics every 10–40 years resulting in enormous morbidity and mortality, especially among the very young, the elderly, and the immunocompromised. RSV is also the major pathogen implicated in acute bronchiolitis, which afflicts infants and is responsible for a great number of hospitalizations and deaths in

**

this age group. Host immune response skewed towards type 2 cytokine production seems to play a central role in the pathogenesis of the disease. Bronchiolitis is further associated with recurrent wheezing. The great majority of acute asthma exacerbations in children and a considerable proportion in adults is preceded by a viral URT infection; such exacerbations may often result in hospitalization. The most frequently isolated offending agent is RV, a fact that reflects the preponderance of the virus among common cold cases. Finally, exacerbations of chronic obstructive pulmonary disease may also have a viral etiology, most commonly due to RVs.

Diagnosis and Treatment The diagnosis of respiratory viral infections relies upon clinical criteria and is further supported by laboratory techniques such as direct viral, nucleic acid or antigen detection, culture, and serology. Direct detection is based on the identification of whole virus or inclusion bodies with electron or light microscopy, respectively. Viral antigens may also be detected by the use of immunofluorescence and enzyme immunoassays but an adequate viral load is required. Viral cultures usually demonstrate the biological effects of an agent, such as the cytopathic effect or hemagglutination, in cultured cells. Culture

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methods, however, have drawbacks due to their low sensitivity and extensive time length (days to weeks). Determination of specific viral antibodies in a host’s blood sample and comparison between acute and convalescent phases of respiratory illness is another diagnostic modality, which is mainly used for epidemiological purposes. Serological tests include immunoassays, complement fixation, and passive agglutination assays as well as hemagglutination inhibition. Polymerase chain reaction (PCR)-based techniques are being developed, replacing in several instances other techniques as they are rapid and sensitive, and may also provide an estimation of the number of viral copies present in a given biological sample (real-time PCR). Control of the transmission of respiratory infections may be partly achieved through adequate hygiene practices and avoidance of congregation and stress. The development of effective vaccines is hampered by the large number of viral serotypes; however, vaccines are available against IFVs, several RSV and PIV vaccine candidates are currently under clinical investigation, and oral live attenuated adenovirus vaccines have been successfully used in military recruitment centers in the past. Over-thecounter remedies for the alleviation and reduction of duration of common cold symptoms include vitamin C, zinc, and echinacea, although these agents are only moderately effective. Nasal decongestants, first generation antihistamines, anticholinergic nasal sprays, oral and intranasal a-adrenergic agonists, inhalation of humidified hot air at 42–441C, nonsteroidal anti-inflammatory drugs, and cromones have been proposed for symptomatic relief with moderate results. A combination of specific antivirals with one or more anti-inflammatory drugs may offer the ideal therapeutic approach since it would both inhibit viral replication and block inflammatory events, although cost and compliance are considerable obstacles.

Current Antimicrobial Therapy Unfortunately, there are only a limited number of antiviral agents available against respiratory viruses due to the frequent mutations resulting in the constant emergence of new and often resistant strains. Specific antiviral chemotherapy exists against IFVs in the form of the classical adamantanes (amantadine and rimantadine) as well as the new NA inhibitors, zanamivir (intranasal formulation) and oseltamivir. The former were only effective against IFV A, had significant adverse effects, and often resulted in the development of resistant strains, in contrast to the latter which are active against both A and B IFVs,

possess minimal adverse effects, and are currently indicated for both prophylactic and therapeutic purposes. Palivizumab is a monoclonal antibody against RSV, which may be administered prophylactically to infants at high risk for severe bronchiolitis; ribavirin is an antiviral agent currently less frequently used. The most effective compound against a variety of respiratory viruses and the resulting common cold is interferon alpha-2b, administered intranasally, before or shortly after exposure. However, due to its high cost, dosage frequency (six times daily), and side effects (mainly nasal irritation and bleeding) clinical application has been abandoned. Several other agents have been assessed for activity against rhinoviruses. Pleconaril is an oral agent, which binds to VP1, a capsid protein of Picornaviruses, and thus inhibits capsid function, viral attachment to cellular receptors, and viral uncoating. Ag7088 (ruprintivir) is a well-tolerated irreversible inhibitor of Picornavirus 3C protease. Reported adverse effects were usually restricted to blood-tinged mucus and nasal passage irritation. Both compounds are currently under clinical trials. See also: Antiviral Agents. Asthma: Acute Exacerbations. Bronchiectasis. Bronchiolitis. Chronic Obstructive Pulmonary Disease: Acute Exacerbations. Laryngitis and Pharyngitis. Panbronchiolitis. Pediatric Pulmonary Diseases. Pneumonia: Viral. Signs of Respiratory Disease: Breathing Patterns; Clubbing and Hypertrophic Osteoarthropathy; General Examination; Lung Sounds. Symptoms of Respiratory Disease: Cough and Other Symptoms. Upper Respiratory Tract Infection.

Further Reading Johnston SL and Papadopoulos NG (eds.) (2003) Respiratory Infections in Asthma and Allergy. New York: Dekker. McCracken GH (2000) Etiology and treatment of pneumonia. Pediatric Infectious Diseases Journal 19: 373–377. Mossad SB (1998) Treatment of the common cold. British Medical Journal 317: 33–36. Myint SH (1994) Human coronaviruses: a brief review. Reviews in Medical Virology 4: 35–46. Papadopoulos NG and Johnston SL (1999) The acute exacerbation of asthma: Pathogenesis. In: Holgate ST, Boushey HA, and Fabbri LM (eds.) Difficult Asthma, pp. 183–204. London: Martin Dunitz. Papadopoulos NG and Johnston SL (2001) Viral infections of the nose and lung. In Wallaert B and Chanez P (eds.) The Nose and Lung Diseases, pp. 79–100. European Respiratory Monographs. Papadopoulos NG and Johnston SL (2003) Rhinoviruses. In Zuckerman AJ, Banatvala JE, Griffiths P, Pattison JR and Schoub B (eds.) Principles and Practice of Clinical Virology, 5th edn. Chichester: John Wiley & Sons.

488 VIRUSES OF THE LUNG Papadopoulos NG, Xatzipsalti M, and Johnston SL The common cold. In: Torres, Ewig, Mandell, and Woodhead (eds.) Respiratory Infections. London: Arnold (in press). Phelan PD, Landau LI, and Olinsky A (1994) Respiratory Illness in Children, 4th edn. Oxford: Blackwell Scientific Publications. Rabella N, Rodriguez P, Labeaga R, et al. (1999) Conventional respiratory viruses recovered from immunocompromised

patients: clinical considerations. Clinical Infectious Diseases 28: 1043–1048. Stein RT, Sherrill D, Morgan WJ, et al. (1999) Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet 354: 541–545. Turner R (1997) Epidemiology, pathogenesis, and treatment of the common cold. Annals of Allergy, Asthma and Immunology 78: 531–539; quiz 9–40.